This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents  
►B ►C1 COMMISSION REGULATION (EC) No 440/2008  
of 30 May 2008  
laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament 
and of the Council on the Registration, Evaluation, Authorisation and Restriction of 
Chemicals (REACH)  
(Text with EEA relevance) ◄ 
(OJ L 142, 31.5.2008, p. 1)  
Amended by:  
Official Journal  
No page  date  
►M1 Commission Regulation (EC) No 761/2009 of 23 July 2009  L 220  1  24.8.2009  
►M2 Commission Regulation (EU) No 1152/2010 of 8 December 2010  L 324  13  9.12.2010  
►M3 Commission Regulation (EU) No 640/2012 of 6 July 2012  L 193  1  20.7.2012  
►M4 Commission Regulation (EU) No 260/2014 of 24 January 2014  L 81  1  19.3.2014  
Corrected by:  
►C1 Corrigendum, OJ L 143, 3.6.2008, p. 55 (440/2008)  2008R0440 — EN — 22.03.2014 — 004.001 — 1
▼C1 
COMMISSION REGULATION (EC) No 440/2008  
of 30 May 2008  
laying down test methods pursuant to Regulation (EC) 
No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of 
Chemicals (REACH)
 
(Text with EEA relevance)  
▼B 
THE COMMISSION OF THE EUROPEAN COMMUNITIES,  
Having regard to the Treaty establishing the European Community, 
Having regard to Regulation (EC) No 1907/2006 of 18 December 2006 
of the European Parliament and of the Council concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), estab ­
lishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC (
 1 ), and in particular Article 13(3) thereof, 
Whereas:  
(1) Pursuant to Regulation (EC) No 1907/2006, test methods are to 
be adopted at Community level for the purposes of tests on substances where such tests are required to generate information on intrinsic properties of substances.
 
(2) Council Directive 67/548/EEC of 27 June 1967 on the approximation 
of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances (
 2 ), 
laid down, in Annex V, methods for the determination of the physico- chemical properties, toxicity and ecotoxicity of substances and preparations. Annex V to Directive 67/548/EEC has been deleted by Directive 2006/121/EC of the European Parliament and of the Council with effect from 1 June 2008.
 
(3) The test methods contained in Annex V to Directive 67/548/EEC 
should be incorporated into this Regulation.  
(4) This Regulation does not exclude the use of other test methods, 
provided that their use is in accordance with Article 13(3) of 
Regulation 1907/2006.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 2  
( 1 ) OJ L 396, 30.12.2006, p. 1, as corrected by OJ L 136, 29.5.2007, p. 3. 
( 2 ) OJ 196, 16.8.1967, p. 1. Directive as last amended by Directive 2006/121/CE of 
the European Parliament and of the Council (OJ L 396, 30.12.2006, p. 850, as corrected by OJ L 136, 29.5.2007, p. 281).
 (5) The principles of replacement, reduction and refinement of the 
use of animals in procedures should be fully taken into account in the design of the test methods, in particular when appropriate validated methods become available to replace, reduce or refine animal testing.
 
(6) The provisions of this Regulation are in accordance with the 
opinion of the Committee established under Article 133 of Regu ­
lation (EC) No 1907/2006,  
HAS ADOPTED THIS REGULATION:  
Article 1  
The test methods to be applied for the purposes of Regulation 1907/2006/EC 
are set out in the Annex to this Regulation.  
Article 2  
The Commission shall review, where appropriate, the test methods 
contained in this Regulation with a view to replacing, reducing or refining testing on vertebrate animals.
 
Article 3  
All references to Annex V to Directive 67/548/EEC shall be construed 
as references to this Regulation.  
Article 4  
This Regulation shall enter into force on the day following its 
publication in the  Official Journal of the European Union . 
It shall apply from 1 June 2008.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 3
 
ANNEX  
PART A: METHODS FOR THE DETERMINATION OF PHYSICO- 
CHEMICAL PROPERTIES  
TABLE OF CONTENTS 
A.1. MELTING/FREEZING TEMPERATURE 
A.2. BOILING TEMPERATURE 
A.3. RELATIVE DENSITY A.4. VAPOUR PRESSURE A.5. SURFACE TENSION 
A.6. WATER SOLUBILITY 
A.8. PARTITION COEFFICIENT A.9. FLASH-POINT 
A.10. FLAMMABILITY (SOLIDS) 
A.11. FLAMMABILITY (GASES) A.12. FLAMMABILITY (CONTACT WITH WATER) 
A.13. PYROPHORIC PROPERTIES OF SOLIDS AND LIQUIDS 
A.14. EXPLOSIVE PROPERTIES A.15. AUTO-IGNITION TEMPERATURE (LIQUIDS AND GASES) A.16. RELATIVE SELF-IGNITION TEMPERATURE FOR SOLIDS 
A.17. OXIDISING PROPERTIES (SOLIDS) 
A.18. NUMBER — AVERAGE MOLECULAR WEIGHT AND 
MOLECULAR WEIGHT DISTRIBUTION OF POLYMERS 
A.19. LOW MOLECULAR WEIGHT CONTENT OF POLYMERS A.20. SOLUTION/EXTRACTION BEHAVIOUR OF POLYMERS IN 
WATER 
A.21. OXIDISING PROPERTIES (LIQUIDS) 
A.22. LENGTH WEIGHTED GEOMETRIC MEAN DIAMETER OF 
FIBRES
 
▼M4 
A.23. PARTITION COEFFICIENT (1-OCTANOL/WATER): 
SLOW-STIRRING METHOD  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 4
 
A.1. MELTING/FREEZING TEMPERATURE  
1. METHOD  
The majority of the methods described are based on the OECD Test 
Guideline (1). The fundamental principles are given in references (2) 
and (3).  
1.1. INTRODUCTION 
The methods and devices described are to be applied for the deter ­
mination of the melting temperature of substances, without any 
restriction with respect to their degree of purity. 
The selection of the method is dependent on the nature of the 
substance to be tested. In consequence the limiting factor will be according to, whether or not the substance can be pulverised easily, with difficulty, or not at all. 
For some substances, the determination of the freezing or solidifi ­
cation temperature is more appropriate and the standards for these 
determinations have also been included in this method. 
Where, due to the particular properties of the substance, none of the 
above parameters can be conveniently measured, a pour point may 
be appropriate.
 
1.2. DEFINITIONS AND UNITS 
The melting temperature is defined as the temperature at which the 
phase transition from solid to liquid state occurs at atmospheric 
pressure and this temperature ideally corresponds to the freezing temperature. 
As the phase transition of many substances takes place over a 
temperature range, it is often described as the melting range. 
Conversion of units (K to
 o C) 
t = T - 273,15 
t: Celsius temperature, degree Celsius (  o C) 
T: thermodynamic temperature, kelvin (K)  
1.3. REFERENCE SUBSTANCES 
Reference substances do not need to be employed in all cases when 
investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods. 
Some calibration substances are listed in the references (4).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 5
 
1.4. PRINCIPLE OF THE TEST METHOD 
The temperature (temperature range) of the phase transition from the 
solid to the liquid state or from the liquid to the solid state is determined. In practice while heating/cooling a sample of the test 
substance at atmospheric pressure the temperatures of the initial 
melting/freezing and the final stage of melting/freezing are deter ­
mined. Five types of methods are described, namely capillary method, hot stage methods, freezing temperature determinations, 
methods of thermal analysis, and determination of the pour point 
(as developed for petroleum oils). 
In certain cases, it may be convenient to measure the freezing 
temperature in place of the melting temperature.
 
1.4.1.  Capillary method  
1.4.1.1.  Melting temperature devices with liquid bath  
A small amount of the finely ground substance is placed in a 
capillary tube and packed tightly. The tube is heated, together with a thermometer, and the temperature rise is adjusted to less than about 
1 K/min during the actual melting. The initial and final melting 
temperatures are determined.
 
1.4.1.2.  Melting temperature devices with metal block  
As described under 1.4.1.1, except that the capillary tube and the 
thermometer are situated in a heated metal block, and can be observed through holes in the block.
 
1.4.1.3.  Photocell detection  
The sample in the capillary tube is heated automatically in a metal 
cylinder. A beam of light is directed through the substance, by way of a hole in the cylinder, to a precisely calibrated photocell. The 
optical properties of most substances change from opaque to trans ­
parent when they are melting. The intensity of light reaching the photocell increases and sends a stop signal to the digital indicator reading out the temperature of a platinum resistance thermometer 
located in the heating chamber. This method is not suitable for 
some highly coloured substances.
 
1.4.2.  Hot stages  
1.4.2.1.  Kofler hot bar  
The Kofler hot bar uses two pieces of metal of different thermal 
conductivity, heated electrically, with the bar designed so that the temperature gradient is almost linear along its length. The temperature of the hot bar can range from 283 to 573 K with a 
special temperature-reading device including a runner with a 
pointer and tab designed for the specific bar. In order to determine a melting temperature, the substance is laid, in a thin layer, directly on the surface of the hot bar. In a few seconds a sharp dividing line 
between the fluid and solid phase develops. The temperature at the 
dividing line is read by adjusting the pointer to rest at the line.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 6
 
1.4.2.2.  Melt microscope  
Several microscope hot stages are in use for the determination of 
melting temperatures with very small quantities of material. In most of the hot stages the temperature is measured with a sensitive 
thermocouple but sometimes mercury thermometers are used. A 
typical microscope hot stage melting temperature apparatus has a heating chamber which contains a metal plate upon which the sample is placed on a slide. The centre of the metal plate contains 
a hole permitting the entrance of light from the illuminating mirror 
of the microscope. When in use, the chamber is closed by a glass plate to exclude air from the sample area. 
The heating of the sample is regulated by a rheostat. For very precise 
measurements on optically anisotropic substances, polarised light may be used.
 
1.4.2.3.  Meniscus method  
This method is specifically used for polyamides. 
The temperature at which the displacement of a meniscus of silicone 
oil, enclosed between a hot stage and a cover-glass supported by the polyamide test specimen, is determined visually.
 
1.4.3.  Method to determine the freezing temperature  
The sample is placed in a special test tube and placed in an apparatus 
for the determination of the freezing temperature. The sample is 
stirred gently and continuously during cooling and the temperature 
is measured at suitable intervals. As soon as the temperature remains constant for a few readings this temperature (corrected for ther ­
mometer error) is recorded as the freezing temperature. 
Supercooling must be avoided by maintaining equilibrium between 
the solid and the liquid phases.
 
1.4.4.  Thermal analysis  
1.4.4.1  Differential thermal analysis (DTA)  
This technique records the difference in temperatures between the 
substance and a reference material as a function of temperature, 
while the substance and reference material are subjected to the same controlled temperature programme. When the sample undergoes a transition involving a change of enthalpy, that change 
is indicated by an endothermic (melting) or exothermic (freezing) 
departure from the base line of the temperature record.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 7
 
1.4.4.2  Differential scanning calorimetry (DSC)  
This technique records the difference in energy inputs into a 
substance and a reference material, as a function of temperature, while the substance and reference material are subjected to the 
same controlled temperature programme. This energy is the energy 
necessary to establish zero temperature difference between the substance and the reference material. When the sample undergoes a transition involving a change of enthalpy, that change is indicated 
by an endothermic (melting) or exothermic (freezing) departure from 
the base line of the heat flow record.
 
1.4.5.  Pour point  
This method was developed for use with petroleum oils and is 
suitable for use with oily substances with low melting temperatures. 
After preliminary heating, the sample is cooled at a specific rate and 
examined at intervals of 3 K for flow characteristics. The lowest temperature at which movement of the substance is observed is recorded as the pour point.
 
1.5. QUALITY CRITERIA 
The applicability and accuracy of the different methods used for the 
determination of the melting temperature/melting range are listed in the following table:
 
TABLE: APPLICABILITY OF THE METHODS  
A. Capillary methods  
Method of 
measurement Substances which 
can be pulverised Substances which 
are not readily 
pulverised Temperature 
range Estimated 
accuracy (  1 ) Existing standards  
Melting 
temperature devices with 
liquid bath yes only to a few 273 to 573 K ± 0,3 K JIS K 0064 
Melting 
temperature with metal block yes only to a few 293 to 
> 573 K ± 0,5 K ISO 1218 (E) 
Photocell 
detection yes several with 
appliance 
devices 253 to 573 K ± 0,5 K
 
( 1 ) Dependent on type of instrument and on degree of purity of the substance.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 8
 
B. Hot stages and freezing methods  
Method of 
measurement Substances which 
can be pulverised Substances which 
are not readily 
pulverised Temperature 
range Estimated 
accuracy (  1 ) Existing standards  
Kofler hot bar yes no 283 to > 
573 K ± 1K ANSI/ASTM 
D 3451-76 
Melt microscope yes only to a few 273 to > 
573 K ± 0,5 K DIN 53736 
Meniscus method no specifically for 
polyamides 293 to > 
573 K ± 0,5 K ISO 1218 (E) 
Freezing 
temperature yes yes 223 to 573 K ± 0,5 K e.g. BS 4695  
( 1 ) Dependent on type of instrument and on degree of purity of the substance  
C. Thermal analysis  
Method of 
measurement Substances which 
can be pulverised Substances which 
are not readily 
pulverised Temperature 
range Estimated 
accuracy (  1 ) Existing standards  
Differential 
thermal analysis yes yes 173 to 
1 273 K up to 600 K 
± 0,5 K up to 
1 273 K 
± 2,0 K ASTM E 537-76 
Differential 
scanning 
calorimetry yes yes 173 to 
1 273 K up to 600 K 
± 0,5 K up to 
1 273 K 
± 2,0 K ASTM E 537-76  
( 1 ) Dependent on type of instrument and on degree of purity of the substance  
D. Pour point  
Method of 
measurement Substances which 
can be pulverised Substances which 
are not readily 
pulverised Temperature 
range Estimated 
accuracy (  1 ) Existing standards  
Pour point for petroleum 
oils and oily 
substances for petroleum 
oils and oily 
substances 223 to 323 K ± 0,3 K ASTM D 97-66  
( 1 ) Dependent on type of instrument and on degree of purity of the substance  
1.6. DESCRIPTION OF THE METHODS 
The procedures of nearly all the test methods have been described in 
international and national standards (see Appendix 1).  
1.6.1.  Methods with capillary tube  
When subjected to a slow temperature rise, finely pulverised 
substances usually show the stages of melting shown in figure 1.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 9
 
Figure 1
  
During the determination of the melting temperature, the 
temperatures are recorded at the beginning of the melting and at the final stage.
 
1.6.1.1.  Melting temperature devices with liquid bath apparatus  
Figure 2 shows a type of standardised melting temperature apparatus 
made of glass (JIS K 0064); all specifications are in millimeters.  
Figure 2
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 10
 
Bath liquid:  
A suitable liquid should be chosen. The choice of the liquid depends 
upon the melting temperature to be determined, e.g. liquid paraffin 
for melting temperatures no higher than 473 K, silicone oil for melting temperatures no higher than 573 K. 
For melting temperatures above 523 K, a mixture consisting of three 
parts sulphuric acid and two parts potassium sulphate (in mass ratio) can be used. Suitable precautions should be taken if a mixture such 
as this is used.
 
Thermometer:  
Only those thermometers should be used which fulfil the 
requirements of the following or equivalent standards: 
ASTM E 1-71, DIN 12770, JIS K 8001.  
Procedure:  
The dry substance is finely pulverised in a mortar and is put into the 
capillary tube, fused at one end, so that the filling level is approxi ­
mately 3 mm after being tightly packed. To obtain a uniform packed sample, the capillary tube should be dropped from a height of 
approximately 700 mm through a glass tube vertically onto a 
watch glass. 
The filled capillary tube is placed in the bath so that the middle part 
of the mercury bulb of the thermometer touches the capillary tube at 
the part where the sample is located. Usually the capillary tube is introduced into the apparatus about 10 K below the melting 
temperature. 
The bath liquid is heated so that the temperature rise is approxi ­
mately 3 K/min. The liquid should be stirred. At about 10 K 
below the expected melting temperature the rate of temperature 
rise is adjusted to a maximum of 1 K/min.
 
Calculation:  
The calculation of the melting temperature is as follows: 
T = T  D + 0,00016 (T  D - T E ) n 
where: 
T = corrected melting temperature in K 
T D = temperature reading of thermometer D in K 
T E = temperature reading of thermometer E in K 
n = number of graduations of mercury thread on thermometer D 
at emergent stem.  
1.6.1.2.  Melting temperature devices with metal block  
Apparatus: 
This consists of:  
— a cylindrical metal block, the upper part of which is hollow and 
forms a chamber (see figure 3), 
— a metal plug, with two or more holes, allowing tubes to be 
mounted into the metal block,  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 11
 
— a heating system, for the metal block, provided for example by 
an electrical resistance enclosed in the block, 
— a rheostat for regulation of power input, if electric heating is 
used, 
— four windows of heat-resistant glass on the lateral walls of the 
chamber, diametrically disposed at right-angles to each other. In 
front of one of these windows is mounted an eye-piece for observing the capillary tube. The other three windows are used for illuminating the inside of the enclosure by means of lamps, 
— a capillary tube of heat-resistant glass closed at one end 
(see 1.6.1.1).
 
Thermometer:  
See standards mentioned in 1.6.1.1. Thermoelectrical measuring 
devices with comparable accuracy are also applicable.  
Figure 3
  
1.6.1.3.  Photocell detection 
Apparatus and procedure:  
The apparatus consists of a metal chamber with automated heating 
system. Three capillary are filled accordingly to 1.6.1.1 and placed in 
the oven.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 12
 
Several linear increases of temperature are available for calibrating 
the apparatus and the suitable temperature rise is electrically adjusted 
at a pre-selected constant and linear rate. recorders show the actual oven temperature and the temperature of the substance in the capillary tubes.
 
1.6.2.  Hot stages  
1.6.2.1.  Kofler hot bar  
See Appendix.  
1.6.2.2.  Melt microscope  
See Appendix.  
1.6.2.3.  Meniscus method (polyamides)  
See Appendix. 
The heating rate through the melting temperature should be less than 
1 K/min.  
1.6.3.  Methods for the determination of the freezing temperature  
See Appendix.  
1.6.4.  Thermal analysis  
1.6.4.1.  Differential thermal analysis  
See Appendix.  
1.6.4.2.  Differential scanning calorimetry  
See Appendix.  
1.6.5.  Determination of the pour point  
See Appendix.  
2. DATA  
A thermometer correction is necessary in some cases.  
3. REPORTING  
The test report shall, if possible, include the following information: 
— method used, 
— precise specification of the substance (identity and impurities) 
and preliminary purification step, if any, 
— an estimate of the accuracy. The mean of at least two measurements which are in the range of the 
estimated accuracy (see tables) is reported as the melting 
temperature.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 13
 
If the difference between the temperature at the beginning and at the 
final stage of melting is within the limits of the accuracy of the 
method, the temperature at the final stage of melting is taken as the melting temperature; otherwise the two temperatures are reported. 
If the substance decomposes or sublimes before the melting 
temperature is reached, the temperature at which the effect is 
observed shall be reported. 
All information and remarks relevant for the interpretation of results 
have to be reported, especially with regard to impurities and physical state of the substance.
 
4. REFERENCES  
(1) OECD, Paris, 1981, Test Guideline 102, Decision of the 
Council C(81) 30 final. 
(2) IUPAC, B. Le Neindre, B. Vodar, eds. Experimental thermody ­
namics, Butterworths, London 1975, vol. II, p. 803-834. 
(3) R. Weissberger ed.: Technique of organic Chemistry, Physical 
Methods of Organic Chemistry, 3rd ed., Interscience Publ., New York, 1959, vol. I, Part I, Chapter VII. 
(4) IUPAC, Physicochemical measurements: Catalogue of reference 
materials from national laboratories, Pure and applied chemistry, 
1976, vol. 48, p. 505-515.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 14
 
Appendix  
For additional technical details, the following standards may be consulted for 
example.  
1. Capillary methods  
1.1. Melting temperature devices with liquid bath 
ASTM E 324-69 Standard test method for relative initial 
and final melting points and the 
melting range of organic chemicals 
BS 4634 Method for the determination of 
melting point and/or melting range 
DIN 53181 Bestimmung des Schmelzintervalles 
von Harzen nach Kapillarverfarehn 
JIS K 00-64 Testing methods for melting point of 
chemical products  
1.2. Melting temperature devices with metal block 
DIN 53736 Visuelle Bestimmung der Schmelztem ­
peratur von teilkristallinen Kunststoffen 
ISO 1218 (E) Plastics — polyamides — deter ­
mination of ‘melting point’
 
2. Hot stages  
2.1. Kofler hot bar 
ANSI/ASTM D 3451-76 Standard recommended practices for 
testing polymeric powder coatings  
2.2. Melt microscope 
DIN 53736 Visuelle Bestimmung der Schmelztem ­
peratur von teilkristallinen Kunststoffen
 
2.3. Meniscus method (polyamides) 
ISO 1218 (E) Plastics — polyamides — deter ­
mination of ‘melting point’  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 15
 
ANSI/ASTM D 2133-66 Standard specification for acetal resin 
injection moulding and extrusion materials 
NF T 51-050 Résines de polyamides. Détermination 
du ‘point de fusion’ méthode du ménisque
 
3. Methods for the determination of the freezing temperature  
BS 4633 Method for the determination of cryst ­
allising point 
BS 4695 Method for Determination of Melting 
Point of petroleum wax (Cooling 
Curve) 
DIN 51421 Bestimmung des Gefrierpunktes von 
Flugkraftstoffen, Ottokraftstoffen und 
Motorenbenzolen 
ISO 2207 Cires de pétrole: détermination de la 
température de figeage 
DIN 53175 Bestimmung des Erstarrungspunktes 
von Fettsäuren 
NF T 60-114 Point de fusion des paraffines 
NF T 20-051 Méthode de détermination du point de 
cristallisation (point de congélation) 
ISO 1392 Method for the determination of the 
freezing point  
4. Thermal analysis  
4.1. Differential thermal analysis 
ASTM E 537-76 Standard method for assessing the 
thermal stability of chemicals by methods of differential thermal analysis 
ASTM E 473-85 Standard definitions of terms relating 
to thermal analysis
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 16
 
ASTM E 472-86 Standard practice for reporting thermo ­
analytical data 
DIN 51005 Thermische Analyse, Begriffe  
4.2. Differential scanning calorimetry 
ASTM E 537-76 Standard method for assessing the 
thermal stability of chemicals by 
methods of differential thermal analysis 
ASTM E 473-85 Standard definitions of terms relating 
to thermal analysis 
ASTM E 472-86 Standard practice for reporting thermo ­
analytical data 
DIN 51005 Thermische Analyse, Begriffe
 
5. Determination of the pour point  
NBN 52014 Echantillonnage et analyse des 
produits du pétrole: Point de trouble 
et point d'écoulement limite — 
Monsterneming en ontleding van aardolieproducten: Troebelingspunt en vloeipunt 
ASTM D 97-66 Standard test method for pour point of 
petroleum oils 
ISO 3016 Petroleum oils — Determination of 
pour point
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 17
 
A.2. BOILING TEMPERATURE  
1. METHOD  
The majority of the methods described are based on the OECD Test 
Guideline (1). The fundamental principles are given in references (2) 
and (3).  
1.1. INTRODUCTION 
The methods and devices described here can be applied to liquid and 
low melting substances, provided that these do not undergo chemical 
reaction below the boiling temperature (for example: auto-oxidation, 
rearrangement, degradation, etc.). The methods can be applied to pure and to impure liquid substances. 
Emphasis is put on the methods using photocell detection and 
thermal analysis, because these methods allow the determination of 
melting as well as boiling temperatures. Moreover, measurements can be performed automatically. 
The ‘dynamic method’ has the advantage that it can also be applied 
to the determination of the vapour pressure and it is not necessary to correct the boiling temperature to the normal pressure (101,325 kPa) because the normal pressure can be adjusted during the measurement 
by a manostat.  
Remarks:  
The influence of impurities on the determination of the boiling 
temperature depends greatly upon the nature of the impurity. When there are volatile impurities in the sample, which could affect the results, the substance may be purified.
 
1.2. DEFINITIONS AND UNITS 
The normal boiling temperature is defined as the temperature at 
which the vapour pressure of a liquid is 101,325 kPa. 
If the boiling temperature is not measured at normal atmospheric 
pressure, the temperature dependence of the vapour pressure can 
be described by the Clausius-Clapeyron equation:  
log p ¼ ΔH v 
2; 3RT þ const : 
where: 
p = the vapour pressure of the substance in pascals 
Δ H v = its heat of vaporisation in J mol  -1 
R = the universal molar gas constant = 8,314 J mol  -1 K -1 
T = thermodynamic temperature in K 
The boiling temperature is stated with regard to the ambient pressure 
during the measurement.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 18
 
Conversions  
Pressure (units: kPa) 100 kPa = 1 bar = 0,1 MPa 
(‘bar’ is still permissible but not recommended) 
133 Pa = 1 mm Hg = 1 Torr 
(the units ‘mm Hg’ and ‘Torr’ are not permitted) 
1 atm = standard atmosphere = 101 325 Pa 
(the unit ‘atm’ is not permitted) 
Temperature (units: K) 
t = T - 273,15 
t: Celsius temperature, degree Celsius (
 o C) 
T: thermodynamic temperature, kelvin (K)  
1.3. REFERENCE SUBSTANCES 
Reference substances do not need to be employed in all cases when 
investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods. 
Some calibration substances can be found in the methods listed in 
the Appendix.
 
1.4. PRINCIPLE OF THE TEST METHOD 
Five methods for the determination of the boiling temperature 
(boiling range) are based on the measurement of the boiling 
temperature, two others are based on thermal analysis.  
1.4.1.  Determination by use of the ebulliometer  
Ebulliometers were originally developed for the determination of the 
molecular weight by boiling temperature elevation, but they are also 
suited for exact boiling temperature measurements. A very simple apparatus is described in ASTM D 1120-72 (see Appendix). The liquid is heated in this apparatus under equilibrium conditions at 
atmospheric pressure until it is boiling.
 
1.4.2.  Dynamic method  
This method involves the measurement of the vapour recondensation 
temperature by means of an appropriate thermometer in the reflux 
while boiling. The pressure can be varied in this method.  
1.4.3.  Distillation method for boiling temperature  
This method involves distillation of the liquid and measurement of 
the vapour recondensation temperature and determination of the 
amount of distillate.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 19
 
1.4.4.  Method according to Siwoloboff  
A sample is heated in a sample tube, which is immersed in a liquid 
in a heat-bath. A fused capillary, containing an air bubble in the lower part, is dipped in the sample tube.
 
1.4.5.  Photocell detection  
Following the principle according to Siwoloboff, automatic photo- 
electrical measurement is made using rising bubbles.  
1.4.6.  Differential thermal analysis  
This technique records the difference in temperatures between the 
substance and a reference material as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. When the sample 
undergoes a transition involving a change of enthalpy, that change 
is indicated by an endothermic departure (boiling) from the base line of the temperature record.
 
1.4.7.  Differential scanning calorimetry  
This technique records the difference in energy inputs into a 
substance and a reference material as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. This energy is the energy 
necessary to establish zero temperature difference between the 
substance and the reference material. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic departure (boiling) from the base line of the heat 
flow record.
 
1.5. QUALITY CRITERIA 
The applicability and accuracy of the different methods used for 
the determination of the boiling temperature/boiling range are 
listed in table 1.  
Table 1:  
Comparison of the methods  
Method of measurement Estimated accuracy Existing standard  
Ebulliometer ± 1,4 K (up to 373 K) (  1 ) ( 2 ) 
± 2,5 K (up to 600 K) (  1 ) ( 2 ) ASTM D 1120-72 (  1 ) 
Dynamic method ± 0,5 K (up to 600 K) (  2 ) 
Distillation process (boiling range) ± 0,5 K (up to 600 K) ISO/R 918, DIN 53171, 
BS 4591/71 
According to Siwoloboff ± 2 K (up to 600 K) (  2 ) 
Photocell detection ± 0,3 K (up to 373 K) (  2 ) 
Differential thermal calorimetry ± 0,5 K (up to 600 K) 
± 2,0 K (up to 1 273 K) ASTM E 537-76 
Differential scanning calorimetry ± 0,5 K (up to 600 K) 
± 2,0 K (up to 1 273 K) ASTM E 537-76  
( 1 ) This accuracy is only valid for the simple device as for example described in ASTM D 1120-72; it can be improved 
with more sophisticated ebulliometer devices. 
( 2 ) Only valid for pure substances. The use in other circumstances should be justified.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 20
 
1.6. DESCRIPTION OF THE METHODS 
The procedures of some test methods have been described in inter ­
national and national standards (see Appendix).  
1.6.1.  Ebulliometer  
See Appendix.  
1.6.2.  Dynamic method  
See test method A.4 for the determination of the vapour pressure. 
The boiling temperature observed with an applied pressure of 
101,325 kPa is recorded.  
1.6.3.  Distillation process (boiling range)  
See Appendix.  
1.6.4.  Method according to Siwoloboff  
The sample is heated in a melting temperature apparatus in a sample 
tube, with a diameter of approximately 5 mm (figure 1). 
Figure 1 shows a type of standardised melting and boiling 
temperature apparatus (JIS K 0064) (made of glass, all specifications in millimetres).
 
Figure 1
  
A capillary tube (boiling capillary) which is fused about 1 cm above 
the lower end is placed in the sample tube. The level to which the test substance is added is such that the fused section of the capillary is below the surface of the liquid. The sample tube containing the 
boiling capillary is fastened either to the thermometer with a rubber 
band or is fixed with a support from the side (see figure 2).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 21
 
Figure 2  
Principle according to Siwoloboff  Figure 3  
Modified principle
  
The bath liquid is chosen according to boiling temperature. At 
temperatures up to 573 K, silicone oil can be used. Liquid paraffin may only be used up to 473 K. The heating of the bath liquid should be adjusted to a temperature rise of 3 K/min at first. The bath liquid 
must be stirred. At about 10 K below the expected boiling 
temperature, the heating is reduced so that the rate of temperature rise is less than 1 K/min. Upon approach of the boiling temperature, bubbles begin to emerge rapidly from the boiling capillary. 
The boiling temperature is that temperature when, on momentary 
cooling, the string of bubbles stops and fluid suddenly starts rising 
in the capillary. The corresponding thermometer reading is the boiling temperature of the substance. 
In the modified principle (figure 3) the boiling temperature is 
determined in a melting temperature capillary. It is stretched to a fine point about 2 cm in length (a) and a small amount of the sample 
is sucked up. The open end of the fine capillary is closed by melting, 
so that a small air bubble is located at the end. While heating in the melting temperature apparatus (b), the air bubble expands. The boiling temperature corresponds to the temperature at which the 
substance plug reaches the level of the surface of the bath liquid (c).
 
1.6.5.  Photocell detection  
The sample is heated in a capillary tube inside a heated metal block. 
A light beam is directed, via suitable holes in the block, through the 
substance onto a precisely calibrated photocell. 
During the increase of the sample temperature, single air bubbles 
emerge from the boiling capillary. When the boiling temperature is 
reached the number of bubbles increases greatly. This causes a 
change in the intensity of light, recorded by a photocell, and gives a stop signal to the indicator reading out the temperature of a platinum resistance thermometer located in the block. 
This method is especially useful because it allows determinations 
below room temperature down to 253,15 K (– 20
 o C) without any 
changes in the apparatus. The instrument merely has to be placed in 
a cooling bath.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 22
 
1.6.6.  Thermal analysis  
1.6.6.1.  Differential thermal analysis  
See Appendix.  
1.6.6.2.  Differential scanning calorimetry  
See Appendix.  
2. DATA  
At small deviations from the normal pressure (max. ± 5 kPa) the 
boiling temperatures are normalised to T  n by means of the following 
number-value equation by Sidney Young: 
T n = T + (f  T × Δp) 
where: 
Δp = (101,325 - p) [note sign] 
P = pressure measurement in kPa f
 T = rate of change of boiling temperature with pressure in K/kPa 
T = measured boiling temperature in K 
T n = boiling temperature corrected to normal pressure in K 
The temperature-correction factors, f  T , and equations for their 
approximation are included in the international and national standards mentioned above for many substances. 
For example, the DIN 53171 method mentions the following rough 
corrections for solvents included in paints:
 
Table 2:  
Temperature — corrections factors f  T 
Temperature T (K) Correction factor f  T (K/kPa)  
323,15 0,26 
348,15 0,28 
373,15 0,31 
398,15 0,33 
423,15 0,35 448,15 0,37 473,15 0,39 498,15 0,41 523,15 0,4 548,15 0,45 573,15 0,47
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 23
 
3. REPORTING  
The test report shall, if possible, include the following information: 
— method used, — precise specification of the substance (identity and impurities) 
and preliminary purification step, if any, 
— an estimate of the accuracy. 
The mean of at least two measurements which are in the range of the 
estimated accuracy (see table 1) is reported as the boiling temperature. 
The measured boiling temperatures and their mean shall be stated 
and the pressure(s) at which the measurements were made shall be 
reported in kPa. The pressure should preferably be close to normal 
atmospheric pressure. 
All information and remarks relevant for the interpretation of results 
have to be reported, especially with regard to impurities and physical 
state of the substance.
 
4. REFERENCES  
(1) OECD, Paris, 1981, Test Guideline 103, Decision of the 
Council C (81) 30 final. 
(2) IUPAC, B. Le Neindre, B. Vodar, editions. Experimental 
thermodynamics, Butterworths, London, 1975, vol. II. 
(3) R. Weissberger edition: Technique of organic chemistry, 
Physical methods of organic chemistry, Third Edition, Inter ­
science Publications, New York, 1959, vol. I, Part I, 
Chapter VIII.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 24
 
Appendix  
For additional technical details, the following standards may be consulted for 
example.  
1. Ebulliometer  
1.1. Melting temperature devices with liquid bath 
ASTM D 1120-72 Standard test method for boiling point of 
engine anti-freezes  
2. Distillation process (boiling range)  
ISO/R 918 Test Method for Distillation (Distillation 
Yield and Distillation Range) 
BS 4349/68 Method for determination of distillation of 
petroleum products 
BS 4591/71 Method for the determination of distil ­
lation characteristics 
DIN 53171 Losungsmittel für Anstrichstoffe, 
Bestimmung des Siedeverlaufes 
NF T 20-608 Distillation: détermination du rendement 
et de l'intervalle de distillation  
3. Differential thermal analysis and differential scanning 
calorimetry  
ASTM E 537-76 Standard method for assessing the thermal 
stability of chemicals by methods of 
differential thermal analysis 
ASTM E 473-85 Standard definitions of terms relating to 
thermal analysis 
ASTM E 472-86 Standard practice for reporting thermoana ­
lytical data 
DIN 51005 Thermische Analyse, Begriffe  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 25
 
A.3. RELATIVE DENSITY  
1. METHOD  
The methods described are based on the OECD Test Guideline (1). 
The fundamental principles are given in reference (2).  
1.1. INTRODUCTION 
The methods for determining relative density described are 
applicable to solid and to liquid substances, without any restriction 
in respect to their degree of purity. The various methods to be used are listed in table 1.
 
1.2. DEFINITIONS AND UNITS 
The relative density D  20 4 of solids or liquids is the ratio between the 
mass of a volume of substance to be examined, determined at 20  o C, 
and the mass of the same volume of water, determined at 4  o C. The 
relative density has no dimension. 
The density, ρ, of a substance is the quotient of the mass, m, and its 
volume, v. 
The density, ρ, is given, in SI units, in kg/m  3 . 
1.3. REFERENCE SUBSTANCES (1) (3) 
Reference substances do not need to be employed in all cases when 
investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow 
comparison with results from other methods.
 
1.4. PRINCIPLE OF THE METHODS 
Four classes of methods are used.  
1.4.1.  Buoyancy methods  
1.4.1.1.  Hydrometer  (for liquid substances) 
Sufficiently accurate and quick determinations of density may be 
obtained by the floating hydrometers, which allow the density of a liquid to be deduced from the depth of immersion by reading a graduated scale.
 
1.4.1.2.  Hydrostatic balance  (for liquid and solid substances) 
The difference between the weight of a test sample measured in air 
and in a suitable liquid (e.g. water) can be employed to determine its 
density. 
For solids, the measured density is only representative of the 
particular sample employed. For the determination of density of liquids, a body of known volume, v, is weighed first in air and 
then in the liquid.
 
1.4.1.3.  Immersed body method  (for liquid substances) (4) 
In this method, the density of a liquid is determined from the 
difference between the results of weighing the liquid before and 
after immersing a body of known volume in the test liquid.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 26
 
1.4.2.  Pycnometer methods  
For solids or liquids, pycnometers of various shapes and with known 
volumes may be employed. The density is calculated from the difference in weight between the full and empty pycnometer and 
its known volume.
 
1.4.3.  Air comparison pycnometer  (for solids) 
The density of a solid in any form can be measured at room 
temperature with the gas comparison pycnometer. The volume of a 
substance is measured in air or in an inert gas in a cylinder of 
variable calibrated volume. For the calculation of density one mass measurement is taken after concluding the volume measurement.
 
1.4.4.  Oscillating densitimeter  (5) (6) (7) 
The density of a liquid can be measured by an oscillating densi ­
timeter. A mechanical oscillator constructed in the form of a U-tube is vibrated at the resonance frequency of the oscillator which depends on its mass. Introducing a sample changes the resonance 
frequency of the oscillator. The apparatus has to be calibrated by two 
liquid substances of known densities. These substances should preferably be chosen such that their densities span the range to be measured.
 
1.5. QUALITY CRITERIA 
The applicability of the different methods used for the determination 
of the relative density is listed in the table.  
1.6. DESCRIPTION OF THE METHODS 
The standards given as examples, which are to be consulted for 
additional technical details, are attached in the Appendix. 
The tests have to be run at 20  o C, and at least two measurements 
performed.  
2. DATA  
See standards.  
3. REPORTING  
The test report shall, if possible, include the following information: 
— method used, 
— precise specification of the substance (identity and impurities) 
and preliminary purification step, if any. 
The relative density,  D 20 
4 , shall be reported as defined in 1.2, along 
with the physical state of the measured substance. 
All information and remarks relevant for the interpretation of results 
have to be reported, especially with regard to impurities and physical 
state of the substance.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 27
 
Table:  
Applicability of methods  
Method of measurement Density Maximum possible 
dynamic viscosity Existing Standards 
solid liquid  
1.4.1.1. Hydrometer yes 5 Pa s ISO 387, 
ISO 649-2, 
NF T 20-050 
1.4.1.2. Hydrostatic balance (a) solids yes ISO 1183 (A) (b) liquids yes 5 Pa s ISO 901 and 758 
1.4.1.3. Immersed body method yes 20 Pa s DIN 53217 
1.4.2. Pycnometer ISO 3507 
(a) solids yes ISO 1183(B), 
NF T 20-053 
(b) liquids yes 500 Pa s ISO 758 1.4.3. Air comparison 
pycnometer yes DIN 55990 Teil 3, 
DIN 53243 
1.4.4. Oscillating densitimer yes 5 Pa s
 
4. REFERENCES  
(1) OECD, Paris, 1981, Test Guideline 109, Decision of the 
Council C(81) 30 final. 
(2) R. Weissberger ed., Technique of Organic Chemistry, Physical 
Methods of Organic Chemistry, 3rd ed., Chapter IV, Inter ­
science Publ., New York, 1959, vol. I, Part 1. 
(3) IUPAC, Recommended reference materials for realization of 
physico-chemical properties, Pure and applied chemistry, 1976, vol. 48, p. 508. 
(4) Wagenbreth, H., Die Tauchkugel zur Bestimmung der Dichte 
von Flüssigkeiten, Technisches Messen tm, 1979, vol. II, 
p. 427-430. 
(5) Leopold, H., Die digitale Messung von Flüssigkeiten, 
Elektronik, 1970, vol. 19, p. 297-302. 
(6) Baumgarten, D., Füllmengenkontrolle bei vorgepackten Erzeug ­
nissen -Verfahren zur Dichtebestimmung bei flüssigen 
Produkten und ihre praktische Anwendung, Die Pharmazeu ­
tische Industrie, 1975, vol. 37, p. 717-726. 
(7) Riemann, J., Der Einsatz der digitalen Dichtemessung 
im Brauereilaboratorium, Brauwissenschaft, 1976, vol. 9, 
p. 253-255.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 28
 
Appendix  
For additional technical details, the following standards may be consulted for 
example.  
1. Buoyancy methods  
1.1. Hydrometer 
DIN 12790, ISO 387 Hydrometer; general instructions 
DIN 12791 Part I: Density hydrometers; construction, 
adjustment and use 
Part II: Density hydrometers; standardised 
sizes, designation 
Part III: Use and test 
ISO 649-2 Laboratory glassware: Density hydro ­
meters for general purpose 
NF T 20-050 Chemical products for industrial use — 
Determination of density of liquids — 
Areometric method 
DIN 12793 Laboratory glassware: range find hydro ­
meters  
1.2. Hydrostatic balance  
For solid substances  
ISO 1183 Method A: Methods for determining the 
density and relative density of plastics 
excluding cellular plastics 
NF T 20-049 Chemical products for industrial use — 
Determination of the density of solids 
other than powders and cellular products 
— Hydrostatic balance method 
ASTM-D-792 Specific gravity and density of plastics by 
displacement 
DIN 53479 Testing of plastics and elastomers; deter ­
mination of density  
For liquid substances  
ISO 901 ISO 758  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 29
 
DIN 51757 Testing of mineral oils and related 
materials; determination of density 
ASTM D 941-55, ASTM D 1296-67 and ASTM D 1481-62 
ASTM D 1298 Density, specific gravity or API gravity of 
crude petroleum and liquid petroleum products by hydrometer method 
BS 4714 Density, specific gravity or API gravity of 
crude petroleum and liquid petroleum 
products by hydrometer method
 
1.3. Immersed body method 
DIN 53217 Testing of paints, varnishes and similar 
coating materials; determination of 
density; immersed body method  
2. Pycnometer methods  
2.1. For liquid substances 
ISO 3507 Pycnometers 
ISO 758 Liquid chemical products; determination 
of density at 20  o C 
DIN 12797 Gay-Lussac pycnometer (for non-volatile 
liquids which are not too viscous) 
DIN 12798 Lipkin pycnometer (for liquids with 
a kinematic viscosity of less than 100 
10 -6 m 2 s -1 at 15  o C) 
DIN 12800 Sprengel pycnometer (for liquids as DIN 
12798) 
DIN 12801 Reischauer pycnometer (for liquids with 
a kinematic viscosity of less than 100. 
10 -6 m 2 s -1 at 20  o C, applicable in 
particular also to hydrocarbons and aqueous solutions as well as to liquids 
with higher vapour pressure, approxi ­
mately 1 bar at 90
 o C) ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 30
 
DIN 12806 Hubbard pycnometer (for viscous liquids 
of all types which do not have too high a vapour pressure, in particular also for 
paints, varnishes and bitumen) 
DIN 12807 Bingham pycnometer (for liquids, as in 
DIN 12801) 
DIN 12808 Jaulmes pycnometer (in particular for 
ethanol — water mixture) 
DIN 12809 Pycnometer with ground-in thermometer 
and capillary side tube (for liquids which are not too viscous) 
DIN 53217 Testing of paints, varnishes and similar 
products; determination of density by pycnometer 
DIN 51757 Point 7: Testing of mineral oils and 
related materials; determination of density 
ASTM D 297 Section 15: Rubber products — chemical 
analysis 
ASTM D 2111 Method C: Halogenated organic 
compounds 
BS 4699 Method for determination of specific 
gravity and density of petroleum 
products (graduated bicapillary pycnometer method) 
BS 5903 Method for determination of relative 
density and density of petroleum products by the capillary — stoppered pycnometer method 
NF T 20-053 Chemical products for industrial use — 
Determination of density of solids in powder and liquids — Pyknometric 
method
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 31
 
2.2. For solid substances 
ISO 1183 Method B: Methods for determining the 
density and relative density of plastics excluding cellular plastics 
NF T 20-053 Chemical products for industrial use — 
Determination of density of solids in 
powder and liquids — Pyknometric method 
DIN 19683 Determination of the density of soils
 
3. Air comparison pycnometer  
DIN 55990 Part 3: Prüfung von Anstrichstoffen und 
ähnlichen Beschichtungsstoffen; 
Pulverlack; Bestimmung der Dichte 
DIN 53243 Anstrichstoffe; chlorhaltige Polymere; 
Prüfung  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 32
 
A.4. VAPOUR PRESSURE  
1. METHOD  
This method is equivalent to OECD TG 104 (2004).  
1.1. INTRODUCTION 
This revised version of method A.4(1) includes one additional 
method; Effusion method: isothermal thermogravimetry, designed for substances with very low pressures (down to 10
 –10 Pa). In the 
light of needs for procedures, especially in relation to obtaining 
vapour pressure for substances with low vapour pressure, other procedures of this method are re-evaluated with respect to other applicability ranges. 
At the thermodynamic equilibrium the vapour pressure of a pure 
substance is a function of temperature only. The fundamental prin ­
ciples are described elsewhere (2)(3). 
No single measurement procedure is applicable to the entire range of 
vapour pressures from less than 10
 –10 to 10  5 Pa. Eight methods for 
measuring vapour pressure are included in this method which can be 
applied in different vapour pressure ranges. The various methods are compared as to application and measuring range in Table 1. The methods can only be applied for compounds that do not 
decompose under the conditions of the test. In cases where the 
experimental methods cannot be applied due to technical reasons, the vapour pressure can also be estimated, and a recommended estimation method is set out in the Appendix.
 
1.2. DEFINITIONS AND UNITS 
The vapour pressure of a substance is defined as the saturation 
pressure above a solid or liquid substance. 
The SI unit of pressure, which is the pascal (Pa), should be used. 
Other units which have been employed historically are given here ­
after, together with their conversion factors: 
1 Torr = 1 mm Hg = 1,333 × 10  2 Pa 
1 atmosphere = 1,013 × 10  5 Pa 
1 bar = 10  5 Pa 
The SI unit of temperature is the kelvin (K). The conversion 
of degrees Celsius to kelvin is according to the formula: 
T = t + 273,15 where, T is the kelvin or thermodynamic temperature and t is the 
Celsius temperature.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 33
 
Table 1  
Measuring method Substances Estimated repeat ­
ability Estimated repro ­
ducibility Recommended range 
Solid Liquid  
Dynamic method Low melting Yes up to 25 % 
1 to 5 % up to 25 % 
1 to 5 % 10 3 Pa 
to 2 × 10  3 Pa 
2 × 10  3 Pa 
to 10  5 Pa 
Static method Yes Yes 5 to 10 % 5 to 10 % 10 Pa to 10  5 Pa 
10 –2 Pa to 10  5 Pa (  1 ) 
Isoteniscope 
method Yes Yes 5 to 10 % 5 to 10 % 10  2 Pa to 10  5 Pa 
Effusion method: 
vapour pressure balance Yes Yes 5 to 20 % up to 50 % 10
 –3 to 1 Pa 
Effusion method: 
Knudsen cell Yes Yes 10 to 30 % — 10  –10 to 1 P 
Effusion method: 
isothermal thermo ­
gravimetry Yes Yes 5 to 30 % up to 50 % 10  –10 to 1 Pa 
Gas saturation 
method Yes Yes 10 to 30 % up to 50 % 10  –10 to 10  3 Pa 
Spinning rotor 
method Yes Yes 10 to 20 % — 10  –4 to 0,5 Pa  
( 1 ) When using a capacitance manometer  
1.3. PRINCIPLE OF THE TEST 
In general, the vapour pressure is determined at various temperatures. 
In a limited temperature range, the logarithm of the vapour pressure 
of a pure substance is a linear function of the inverse of the 
thermodynamic temperature according to the simplified Clapeyron- Clausius equation:
 
log p ¼ ΔHv 
2; 3RT þ constant  
where: 
p = the vapour pressure in pascals 
ΔHv = the heat of vaporisation in J mol  –1 
R = the universal gas constant, 8,314 J mol  –1 K –1 
T = the temperature in K  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 34
 
1.4. REFERENCE SUBSTANCES 
Reference substances do not need to be employed. They serve 
primarily to check the performance of a method from time to time as well as to allow comparison between results of different methods.
 
1.5. DESCRIPTION OF THE METHOD  
1.5.1.  Dynamic method (Cottrell’s method)  
1.5.1.1.  Principle  
The vapour pressure is determined by measuring the boiling 
temperature of the substance at various specified pressures between 
roughly 10  3 and 10  5 Pa. This method is also recommended for the 
determination of the boiling temperature. For that purpose it is useful up to 600 K. The boiling temperatures of liquids are approximately 0,1 °C higher at a depth of 3 to 4 cm than at the surface because of 
the hydrostatic pressure of the column of liquid. In Cottrell’s method 
(4) the thermometer is placed in the vapour above the surface of the liquid and the boiling liquid is made to pump itself continuously over the bulb of the thermometer. A thin layer of liquid which is 
in equilibrium with vapour at atmospheric pressure covers the bulb. 
The thermometer thus reads the true boiling point, without errors due to superheating or hydrostatic pressure. The pump originally employed by Cottrell is shown in figure 1. Tube A contains the 
boiling liquid. A platinum wire B sealed into the bottom facilitates 
uniform boiling. The side tube C leads to a condenser, and the sheath D prevents the cold condensate from reaching the ther ­
mometer E. When the liquid in A is boiling, bubbles and liquid 
trapped by the funnel are poured via the two arms of the pump F over the bulb of the thermometer.  
Figure 1
 Figure 2
  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 35
 
Cottrell pump (4) 
A: Thermocouple 
B: Vacuum buffer volume 
C: Pressure gauge D: Vacuum 
E: Measuring point 
F: Heating element c.a. 150 W
 
1.5.1.2.  Apparatus  
A very accurate apparatus, employing the Cottrell principle, is shown 
in figure 2. It consists of a tube with a boiling section in the lower 
part, a cooler in the middle part, and an outlet and flange in the 
upper part. The Cottrell pump is placed in the boiling section which is heated by means of an electrical cartridge. The temperature is measured by a jacketed thermocouple, or resistance thermometer 
inserting through the flange at the top. The outlet is connected to 
the pressure regulation system. The latter consists of a vacuum pump, a buffer volume, a manostat for admitting nitrogen for pressure regulation and manometer.
 
1.5.1.3.  Procedure  
The substance is placed in the boiling section. Problems may be 
encountered with non-powder solids but these can sometimes be solved by heating the cooling jacket. The apparatus is sealed at 
the flange and the substance degassed. Frothing substances cannot 
be measured using this method. 
The lowest desired pressure is then set and the heating is switched 
on. At the same time, the temperature sensor is connected to a recorder. 
Equilibrium is reached when a constant boiling temperature is 
recorded at constant pressure. Particular care must be taken to avoid bumping during boiling. In addition, complete condensation must occur on the cooler. When determining the vapour pressure of 
low melting solids, care should be taken to prevent the condenser 
from blocking. 
After recording this equilibrium point, a higher pressure is set. The 
process is continued in this manner until 10
 5 Pa has been reached 
(approximately 5 to 10 measuring points in all). As a check, equi ­
librium points must be repeated at decreasing pressures.  
1.5.2.  Static method  
1.5.2.1.  Principle  
In the static method (5), the vapour pressure at thermodynamic equi ­
librium is determined at a specified temperature. This method is suitable for substances and multicomponent liquids and solids in 
the range from 10
 –1 to 10  5 Pa and, provided care is taken, also in 
the range 1 to 10 Pa.  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 36
 
1.5.2.2.  Apparatus  
The equipment consists of a constant-temperature bath (precision of 
± 0,2 K), a container for the sample connected to a vacuum line, a manometer and a system to regulate the pressure. The sample 
chamber (figure 3a) is connected to the vacuum line via a valve 
and a differential manometer (U-tube containing a suitable manometer fluid) which serves as zero indicator. Mercury, silicones and phthalates are suitable for use in the differential mano ­
meter, depending on the pressure range and the chemical behaviour 
of the test substance. However, based on environmental concerns, the use of mercury should be avoided, if possible. The test substance 
must not dissolve noticeably in, or react with, the U-tube fluid. A 
pressure gauge can be used instead of a U-tube (figure 3b). For the manometer, mercury can be used in the range from normal pressure down to 10
 2 Pa, while silicone fluids and phthalates are suitable for use 
below 10  2 Pa down to 10 Pa. There are other pressure gauges which can 
be used below 10  2 Pa and heatable membrane capacity manometers can 
even be used at below 10  –1 Pa. The temperature is measured on the 
outside wall of the vessel containing the sample or in the vessel itself.  
1.5.2.3.  Procedure  
Using the apparatus as described in figure 3a, fill the U-tube with the 
chosen liquid, which must be degassed at an elevated temperature 
before readings are taken. The test substance is placed in the 
apparatus and degassed at reduced temperature. In the case of a multiple-component sample, the temperature should be low enough to ensure that the composition of the material is not altered. Equi ­
librium can be established more quickly by stirring. The sample can 
be cooled with liquid nitrogen or dry ice, but care should be taken to avoid condensation of air or pump-fluid. With the valve over the sample vessel open, suction is applied for several minutes to remove 
the air. If necessary, the degassing operation is repeated several 
times.  
Figure 3a
 Figure 3b
  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 37
 
When the sample is heated with the valve closed, the vapour 
pressure increases. This alters the equilibrium of the fluid in the 
U-tube. To compensate for this, nitrogen or air is admitted to the apparatus until the differential pressure indicator is at zero again. The pressure required for this can be read off the manometer or off an 
instrument of higher precision. This pressure corresponds to the 
vapour pressure of the substance at the temperature of the measurement. Using the apparatus described in figure 3b, the vapour pressure is read off directly. 
The vapour pressure is determined at suitably small temperature 
intervals (approximately 5 to 10 measuring points in all) up to the 
desired temperature maximum. 
Low-temperature readings must be repeated as a check. If the values 
obtained from the repeated readings do not coincide with the curve 
obtained for increasing temperature, this may be due to one of the 
following situations: 
(i) the sample still contains air (e.g. in the case of highly viscous 
materials) or low-boiling substances which is or are released during heating; 
(ii) the substance undergoes a chemical reaction in the temperature 
range investigated (e.g. decomposition, polymerisation).
 
1.5.3.  Isoteniscope Method  
1.5.3.1.  Principle  
The isoteniscope (6) is based on the principle of the static method. 
The method involves placing a sample in a bulb maintained at constant temperature and connected to a manometer and a vacuum 
pump. Impurities more volatile than the substance are removed 
by degassing at reduced pressure. The vapour pressure of the sample at selected temperatures is balanced by a known pressure of inert gas. The isoteniscope was developed to measure the 
vapour pressure of certain liquid hydrocarbons but it is appropriate 
for the investigation of solids as well. The method is usually not suitable for multicomponent systems. Results are subject to only 
slight errors for samples containing non-volatile impurities. The 
recommended range is 10
 2 to 10  5 Pa. 
1.5.3.2.  Apparatus  
An example of a measuring device is shown in figure 4. A complete 
description can be found in ASTM D 2879-86 (6).  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 38
 
1.5.3.3.  Procedure  
In the case of liquids, the substance itself serves as the fluid in the 
differential manometer. A quantity of the liquid, sufficient to fill the bulb and the short leg of the manometer, is put in the isoteniscope. The isoteniscope is attached to a vacuum system and evacuated, then 
filled by nitrogen. The evacuation and purge of the system is 
repeated twice to remove residual oxygen. The filled isoteniscope is placed in a horizontal position so that the sample spreads out into a thin layer in the sample bulb and manometer. The pressure of the 
system is reduced to 133 Pa and the sample is gently warmed until it 
just boils (removal of dissolved gases). The isoteniscope is then placed so that the sample returns to the bulb and fills the short leg of the manometer. The pressure is maintained at 133 Pa. The drawn- 
out tip of the sample bulb is heated with a small flame until the 
sample vapour released expands sufficiently to displace part of the sample from the upper part of the bulb and manometer arm into the manometer, creating a vapour-filled, nitrogen-free space. The 
isoteniscope is then placed in a constant temperature bath, and the 
pressure of the nitrogen is adjusted until it equals that of the sample. At the equilibrium, the pressure of the nitrogen equals the vapour pressure of the substance.
 
Figure 4
  
In the case of solids, and depending on the pressure and temperature 
ranges, manometer liquids such as silicon fluids or phthalates are 
used. The degassed manometer liquid is put in a bulge provided on 
the long arm of the isoteniscope. Then the solid to be investigated is placed in the sample bulb and is degassed at an elevated temperature. After that, the isoteniscope is inclined so that the manometer liquid 
can flow into the U-tube.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 39
 
1.5.4.  Effusion method: vapour pressure balance  (7) 
1.5.4.1.  Principle  
A sample of the test substance is heated in a small furnace and 
placed in an evacuated bell jar. The furnace is covered by a lid 
which carries small holes of known diameters. The vapour of the 
substance, escaping through one of the holes, is directed onto a balance pan of a highly sensitive balance which is also enclosed in the evacuated bell jar. In some designs the balance pan is 
surrounded by a refrigeration box, providing heat dissipation to the 
outside by thermal conduction, and is cooled by radiation so that the escaping vapour condenses on it. The momentum of the vapour jet 
acts as a force on the balance. The vapour pressure can be derived in 
two ways: directly from the force on the balance pan and also from the evaporation rate using the Hertz-Knudsen equation (2):
 
p ¼ G ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ  
2πRT Ü 10  3 
M s 
where: 
G = evaporation rate (kg s  –1 m –2 ) 
M = molar mass (g mol  –1 ) 
T = temperature (K) 
R = universal gas constant (J mol  –1 K –1 ) 
P = vapour pressure (Pa) 
The recommended range is 10  –3 to 1 Pa.  
1.5.4.2.  Apparatus  
The general principle of the apparatus is illustrated in figure 5.  
Figure 5
  
A: Base plate F: Refrigeration box and cooling bar 
B: Moving coil instrument G: Evaporator furnace 
C: Bell jar H: Dewar flask with liquid nitrogen D: Balance with scale pan I: Measurement of temperature of sample 
E: Vacuum measuring device J: Test Substance
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 40
 
1.5.5.  Effusion method: Knudsen cell  
1.5.5.1.  Principle  
The method is based on the estimation of the mass of test substance 
flowing out per unit of time of a Knudsen cell (8) in the form of vapour, through a micro-orifice under ultra-vacuum conditions. The mass of effused vapour can be obtained either by determining the 
loss of mass of the cell or by condensing the vapour at low 
temperature and determining the amount of volatilised substance using chromatography. The vapour pressure is calculated by applying the Hertz-Knudsen relation (see section 1.5.4.1) with 
correction factors that depend on parameters of the apparatus (9). 
The recommended range is 10
 –10 to 1 Pa (10)(11)(12)(13)(14).  
1.5.5.2.  Apparatus  
The general principle of the apparatus is illustrated in figure 6.  
Figure 6
  
1: Connection to vacuum 7: Threaded lid 
2: Wells from platinum resistance ther ­
mometer or temperature measurement and control 8: Butterfly nuts 
3: Lid for vacuum tank 9: Bolts 
4: O-ring 10: Stainless steel effusion cells 
5: Aluminum vacuum tank 11: Heater cartridge 6: Device for installing and removing the 
effusion cells
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 41
 
1.5.6.  Effusion method: isothermal thermogravimetry  
1.5.6.1.  Principle  
The method is based on the determination of accelerated evaporation 
rates for the test substance at elevated temperatures and ambient 
pressure using thermogravimetry (10)(15)(16)(17)(18)(19)(20). The 
evaporation rates v  T result from exposing the selected compound 
to a slowly flowing inert gas atmosphere, and monitoring the weight loss at defined isothermal temperatures T in Kelvin over 
appropriate periods of time. The vapour pressures p
 T are calculated 
from the v  T values by using the linear relationship between the 
logarithm of the vapour pressure and the logarithm of the evap ­
oration rate. If necessary, an extrapolation to temperatures of 20 
and 25 °C can be made by regression analysis of log p  T vs. 1/T. 
This method is suitable for substances with vapour pressures as low as 10
 –10 Pa (10  –12 mbar) and with purity as close as possible 
to 100 % to avoid the misinterpretation of measured weight losses.  
1.5.6.2.  Apparatus  
The general principle of the experimental set-up is shown in figure 7.  
Figure 7
  
The sample carrier plate, hanging on a microbalance in a temperature 
controlled chamber, is swept by a stream of dry nitrogen gas which 
carries the vaporised molecules of the test substance away. After 
leaving the chamber, the gas stream is purified by a sorption unit.  
1.5.6.3.  Procedure  
The test substance is applied to the surface of a roughened glass 
plate as a homogeneous layer. In the case of solids, the plate is 
wetted uniformly by a solution of the substance in a suitable solvent and dried in an inert atmosphere. For the measurement, the coated plate is hung into the thermogravimetric analyser and 
subsequently its weight loss is measured continuously as a 
function of time.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 42
 
The evaporation rate v  T at a definite temperature is calculated from 
the weight loss Δm of the sample plate by  
v T ¼ Δm 
F Ö t gcm  –2 h –1 Ê Ì  
where F is the surface area of the coated test substances, normally 
the surface area of the sample plate, and t is the time for weight loss 
Δm. 
The vapour pressure p  T is calculated on the basis of its function of 
evaporation rate v  T : 
Log p  T = C + D · log v  T 
where C and D are constants specific for the experimental 
arrangement used, depending on the diameter of the measurement 
chamber and on the gas flow rate. These constants must be 
determined once by measuring a set of compounds with known vapour pressure and regressing log p
 T vs. log v  T (11)(21)(22). 
The relationship between the vapour pressure p  T and the temperature 
T in Kelvin is given by 
Log p  T = A + B · 1/T 
where A and B are constants obtained by regressing log p  T vs. 1/T. 
With this equation, the vapour pressure can be calculated for any 
other temperature by extrapolation.  
1.5.7.  Gas saturation method  (23)  
1.5.7.1.  Principle  
Inert gas is passed, at room temperature and at a known flow rate, 
through or over a sample of the test substance, slowly enough to 
ensure saturation. Achieving saturation in the gas phase is of critical 
importance. The transported substance is trapped, generally using a sorbent, and its amount is determined. As an alternative to vapour trapping and subsequent analysis, in-train analytical techniques, like 
gas chromatography, may be used to determine quantitatively the 
amount of material transported. The vapour pressure is calculated on the assumption that the ideal gas law is obeyed and that the total pressure of a mixture of gases is equal to the sum of the 
pressures of the component gases. The partial pressure of the test 
substance, i.e. the vapour pressure, is calculated from the known total gas volume and from the weight of the material transported. 
The gas saturation procedure is applicable to solid or liquid 
substances. It can be used for vapour pressures down to 10
 –10 Pa 
(10)(11)(12)(13)(14). The method is most reliable for vapour 
pressures below 10  3 Pa. Above 10  3 Pa, the vapour pressures are 
generally overestimated, probably due to aerosol formation. Since the vapour pressure measurements are made at room temperature, 
the need to extrapolate data from high temperatures is not 
necessary and high temperature extrapolation, which can often cause serious errors, is avoided.
 
1.5.7.2.  Apparatus  
The procedure requires the use of a constant-temperature box. The 
sketch in figure 8 shows a box containing three solid and three liquid sample holders, which allow for the triplicate analysis of either a 
solid or a liquid sample. The temperature is controlled to ± 0,5 °C or 
better.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 43
 
Figure 8
  
In general, nitrogen is used as an inert carrier gas but, occasionally, 
another gas may be required (24). The carrier gas must be dry. The gas stream is split into 6 streams, controlled by needle valves 
(approximately 0,79 mm orifice), and flows into the box via 3,8 mm 
i.d. copper tubing. After temperature equilibration, the gas flows through the sample and the sorbent trap and exists from the box. 
Solid samples are loaded into 5 mm i.d. glass tubing between glass 
wool plugs (see Figure 9). Figure 10 shows a liquid sample holder and sorbent system. The most reproducible method for measuring the vapour pressure of liquids is to coat the liquid on glass beads or on 
an inert sorbent such as silica, and to pack the holder with these 
beads. As an alternative, the carrier gas may be made to pass a coarse frit and bubble through a column of the liquid test substance.  
Figure 9
 Figure 10
  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 44
 
The sorbent system contains a front and a backup sorbent section. At 
very low vapour pressures, only small amounts are retained by the 
sorbent and the adsorption on the glass wool and the glass tubing between the sample and the sorbent may be a serious problem. 
Traps cooled with solid CO
 2 are another efficient way for collecting 
the vaporised material. They do not cause any back pressure on the 
saturator column and it is also easy to quantitatively remove the 
trapped material.  
1.5.7.3.  Procedure  
The flow rate of the effluent carrier gas is measured at room 
temperature. The flow rate is checked frequently during the experiment to assure that there is an accurate value for the total volume of carrier gas. Continuous monitoring with a mass flow- 
meter is preferred. Saturation of the gas phase may require 
considerable contact time and hence quite low gas flow rates (25). 
At the end of the experiment, both the front and backup sorbent 
sections are analysed separately. The compound on each section is desorbed by adding a solvent. The resulting solutions are analysed 
quantitatively to determine the weight desorbed from each section. 
The choice of the analytical method (also the choice of sorbent and desorbing solvent) is dictated by the nature of the test material. The desorption efficiency is determined by injecting a known amount of 
sample onto the sorbent, desorbing it and analysing the amount 
recovered. It is important to check the desorption efficiency at or near the concentration of the sample under the test conditions. 
To assure that the carrier gas is saturated with the test substance, 
three different gas flow rates are used. If the calculated vapour 
pressure shows no dependence on flow rate, the gas is assumed to be saturated. 
The vapour pressure is calculated through the equation:
 
p ¼ W 
V Ü RT 
M 
where: 
p = vapour pressure (Pa) 
W = mass of evaporated test substance (g) V = volume of saturated gas (m
 3 ) 
R = universal gas constant 8,314 (J mol  –1 K –1 ) 
T = temperature (K) M = molar mass of test substance (g mol
 –1 ) 
Measured volumes must be corrected for pressure and temperature 
differences between the flow meter and the saturator.  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 45
 
1.5.8.  Spinning rotor  
1.5.8.1.  Principle  
This method uses a spinning rotor viscosity gauge, in which the 
measuring element is a small steel ball which, suspended in a magnetic field, is made to spin by rotating fields (26)(27)(28). 
Pick-up coils allow its spinning rate to be measured. When the 
ball has reached a given rotational speed, usually about 400 revol ­
utions per second, energising is stopped and deceleration, due to gas friction, takes place. The drop of rotational speed is measured as a 
function of time. The vapour pressure is deduced from the pressure- 
dependent slow-down of the steel ball. The recommended range is 10
 –4 to 0,5 Pa.  
1.5.8.2.  Apparatus  
A schematic drawing of the experimental set-up is shown in 
figure 11. The measuring head is placed in a constant-temperature enclosure, regulated within 0,1 °C. The sample container is placed in 
a separate enclosure, also regulated within 0,1 °C. All other parts of 
the set-up are kept at a higher temperature to prevent condensation. The whole apparatus is connected to a high-vacuum system.
 
Figure 11
  
2. DATA AND REPORTING  
2.1. DATA 
The vapour pressure from any of the preceding methods should be 
determined for at least two temperatures. Three or more are preferred 
in the range from 0 to 50 °C, in order to check the linearity of the 
vapour pressure curve. In case of Effusion method (Knudsen cell and isothermal thermogravimetry) and Gas saturation method, 120 to 150 °C is recommended for the measuring temperature range 
instead of 0 to 50 °C.
 
2.2. TEST REPORT 
The test report must include the following information: 
— method used,  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 46
 
— precise specification of the substance (identity and impurities) 
and preliminary purification step, if any, 
— at least two vapour pressure and temperature values — and 
preferably three or more — required in the range from 0 to 50 °C (or 120 to 150 °C), 
— at least one of the temperatures should be at or below 25 °C, if 
technically possible according to the chosen method, 
— all original data, 
— a log p versus 1/T curve, — an estimate of the vapour pressure at 20 or 25 °C. 
If a transition (change of state, decomposition) is observed, the 
following information should be noted: 
— nature of the change, 
— temperature at which the change occurs at atmospheric pressure, — vapour pressure at 10 and 20 °C below the transition temperature 
and 10 and 20 °C above this temperature (unless the transition is 
from solid to gas). 
All information and remarks relevant for the interpretation of results 
have to be reported, especially with regard to impurities and physical 
state of the substance.
 
3. LITERATURE  
(1) Official Journal of the European Communities  L 383 A, 
26-47 (1992). 
(2) Ambrose, D. (1975). Experimental Thermodynamics, Vol. II, 
Le Neindre, B., and Vodar, B., Eds., Butterworths, London. 
(3) Weissberger R., ed. (1959). Technique of Organic Chemistry, 
Physical Methods of Organic Chemistry, 3rd ed., Vol. I, Part I. 
Chapter IX, Interscience Publ., New York. 
(4) Glasstone, S. (1946). Textbook of Physical Chemistry, 2nd ed., 
Van Nostrand Company, New York. 
(5) NF T 20-048 AFNOR (September 1985). Chemical products 
for industrial use — Determination of vapour pressure of solids 
and liquids within a range from 10  –1 to 10  5 Pa — Static 
method. 
(6) ASTM D 2879-86, Standard test method for vapour pressure 
— temperature relationship and initial decomposition 
temperature of liquids by isoteniscope. 
(7) NF T 20-047 AFNOR (September 1985). Chemical products 
for industrial use — Determination of vapour pressure of solids 
and liquids within range from 10  –3 to 1 Pa — Vapour pressure 
balance method.  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 47
 
(8) Knudsen, M. (1909). Ann. Phys. Lpz., 29, 1979; (1911), 34, 593. 
(9) Ambrose, D., Lawrenson, I.J., Sprake, C.H.S. (1975). J. Chem. 
Thermodynamics 7, 1173. 
(10) Schmuckler, M.E., Barefoot, A.C., Kleier, D.A., Cobranchi, 
D.P. (2000), Vapor pressures of sulfonylurea herbicides; Pest 
Management Science  56, 521-532. 
(11) Tomlin, C.D.S. (ed.), The Pesticide Manual, Twelfth Edition 
(2000). 
(12) Friedrich, K., Stammbach, K., Gas chromatographic deter ­
mination of small vapour pressures determination of the vapour pressures of some triazine herbicides. J. Chromatog. 
16 (1964), 22-28. 
(13) Grayson, B.T., Fosbraey, L.A., Pesticide Science 16 (1982), 
269-278. 
(14) Rordorf, B.F., Prediction of vapor pressures, boiling points and 
enthalpies of fusion for twenty-nine halogenated dibenzo-p- dioxins, Thermochimia Acta 112 Issue 1 (1987), 117-122. 
(15) Gückel, W., Synnatschke, G., Ritttig, R., A Method for Deter ­
mining the Volatility of Active Ingredients Used in Plant Protection; Pesticide Science
 4 (1973) 137-147. 
(16) Gückel, W., Synnatschke, G., Ritttig, R., A Method for Deter ­
mining the Volatility of Active Ingredients Used in Plant Protection II. Application to Formulated Products; Pesticide Science 5 (1974) 393-400. 
(17) Gückel, W., Kaestel, R., Lewerenz, J., Synnatschke, G., A 
Method for Determining the Volatility of Active Ingredients Used in Plant Protection. Part III: The Temperature Rela ­
tionship between Vapour Pressure and Evaporation Rate; 
Pesticide Science 13 (1982) 161-168. 
(18) Gückel, W., Kaestel, R., Kroehl, T., Parg, A., Methods for 
Determining the Vapour Pressure of Active Ingredients Used 
in Crop Protection. Part IV: An Improved Thermogravimetric 
Determination Based on Evaporation Rate; Pesticide Science 45 (1995) 27-31. 
(19) Kroehl, T., Kaestel, R., Koenig, W., Ziegler, H., Koehle, H., 
Parg, A., Methods for Determining the Vapour Pressure of Active Ingredients Used in Crop Protection. Part V: Thermo ­
gravimetry Combined with Solid Phase MicroExtraction 
(SPME); Pesticide Science, 53 (1998) 300-310. 
(20) Tesconi, M., Yalkowsky, S.H., A Novel Thermogravimetric 
Method for Estimating the Saturated Vapor Pressure of Low- 
Volatility Compounds; Journal of Pharmaceutical Science 87 ­
(12) (1998) 1512-20. 
(21) Lide, D.R. (ed.), CRC Handbook of Chemistry and Physics, 
81st ed. (2000), Vapour Pressure in the Range — 25 °C 
to 150 °C. 
(22) Meister, R.T. (ed.), Farm Chemicals Handbook, Vol. 
88 (2002). 
(23) 40 CFR, 796. (1993). pp 148-153, Office of the Federal 
Register, Washington DC.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 48
 
(24) Rordorf B.F. (1985). Thermochimica Acta 85, 435. 
(25) Westcott et al. (1981). Environ. Sci. Technol. 15, 1375. (26) Messer G., Röhl, P., Grosse G., and Jitschin W. (1987). J. Vac. 
Sci. Technol. (A), 5(4), 2440. 
(27) Comsa G., Fremerey J.K., and Lindenau, B. (1980). J. Vac. 
Sci. Technol. 17(2), 642. 
(28) Fremerey, J.K. (1985). J. Vac. Sci. Technol. (A), 3(3), 1715.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 49
 
Appendix  
Estimation method  
INTRODUCTION 
Estimated values of the vapour pressure can be used: 
— for deciding which of the experimental methods is appropriate, — for providing an estimate or limit value in cases where the experimental 
method cannot be applied due to technical reasons.
 
ESTIMATION METHOD 
The vapour pressure of liquids and solids can be estimated by use of the 
modified Watson correlation (a). The only experimental data required is the normal boiling point. The method is applicable over the pressure range from 10
 5 Pa to 10  –5 Pa. 
Detailed information on the method is given in ‘Handbook of Chemical Property 
Estimation Methods’ (b). See also OECD Environmental Monograph No.67 (c).  
CALCULATION PROCEDURE 
The vapour pressure is calculated as follows:  
ln P vp ≈ ΔH vb 
ΔZ b RT b 1 – 3 – 2  T 
T b Í Î  m 
T 
T b – 2m 3 – 2  T 
T b Í Î  m – 1 
ln T 
T b 2 
6 6 4 3 
7 7 5
 
where: 
T = temperature of interest T
 b = normal boiling point 
P vp = vapour pressure at temperature T 
ΔH vb = heat of vaporisation 
ΔZ b = compressibility factor (estimated at 0,97) 
m = empirical factor depending on the physical state at the temperature of 
interest 
Further,  
ΔH vb 
T b ¼ K F ð8; 75 þ R ln T b Þ 
where, K  F is an empirical factor considering the polarity of the substance. For 
several compound types, K  F factors are listed in reference (b).  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 50
 
Quite often, data are available in which a boiling point at reduced pressure is 
given. In such a case, the vapour pressure is calculated as follows:  
ln P vp ≈ ln P 1 þ ΔH v1 
ΔZ b RT 1 1 – 3 – 2  T 
T 1 Í Î  m T 1 
T – 2m 3 – 2  T 
T 1 Í Î  m – 1 
ln T 
T 1 " #  
where, T  1 is the boiling point at the reduced pressure P  1 . 
REPORT 
When using the estimation method, the report shall include a comprehensive 
documentation of the calculation.  
LITERATURE 
(a) Watson, K.M. (1943). Ind. Eng. Chem, 35, 398. 
(b) Lyman, W.J., Reehl, W.F., Rosenblatt, D.H. (1982). Handbook of Chemical 
Property Estimation Methods, McGraw-Hill. 
(c) OECD Environmental Monograph No.67. Application of Structure-Activity 
Relationships to the Estimation of Properties Important in Exposure 
Assessment (1993).  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 51
 
A.5. SURFACE TENSION  
1. METHOD  
The methods described are based on the OECD Test Guideline (1). 
The fundamental principles are given in reference (2).  
1.1. INTRODUCTION 
The described methods are to be applied to the measurement of the 
surface tension of aqueous solutions. 
It is useful to have preliminary information on the water solubility, 
the structure, the hydrolysis properties and the critical concentration for micelles formation of the substance before performing these tests. 
The following methods are applicable to most chemical substances, 
without any restriction in respect to their degree of purity. 
The measurement of the surface tension by the ring tensiometer 
method is restricted to aqueous solutions with a dynamic viscosity of less than approximately 200 mPa s.
 
1.2. DEFINITIONS AND UNITS 
The free surface enthalpy per unit of surface area is referred to as 
surface tension. 
The surface tension is given as: N/m (SI unit) or mN/m (SI sub-unit) 1 N/m = 10
 3 dynes/cm 
1 mN/m = 1 dyne/cm in the obsolete cgs system  
1.3. REFERENCE SUBSTANCES 
Reference substances do not need to be employed in all cases when 
investigating a new substance. They should primarily serve to check 
the performance of the method from time to time and to allow 
comparison with results from other methods. 
Reference substances which cover a wide range of surface tensions 
are given in references 1 and 3.  
1.4. PRINCIPLE OF THE METHODS 
The methods are based on the measurement of the maximum force 
which is necessary to exert vertically, on a stirrup or a ring in contact 
with the surface of the liquid being examined placed in a measuring 
cup, in order to separate it from this surface, or on a plate, with an edge in contact with the surface, in order to draw up the film that has formed. 
Substances which are soluble in water at least at a concentration of 
1 mg/l are tested in aqueous solution at a single concentration.
 
1.5. QUALITY CRITERIA 
These methods are capable of greater precision than is likely to be 
required for environmental assessment.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 52
 
1.6. DESCRIPTION OF THE METHODS 
A solution of the substance is prepared in distilled water. The 
concentration of this solution should be 90 % of the saturation solubility of the substance in water; when this concentration 
exceeds 1 g/l, a concentration of 1 g/l is used for testing. Substances 
with water solubility lower than 1 mg/l need not be tested.
 
1.6.1.  Plate method  
See ISO 304 and NF T 73-060 (Surface active agents — deter ­
mination of surface tension by drawing up liquid films).  
1.6.2.  Stirrup method  
See ISO 304 and NF T 73-060 (Surface active agents — deter ­
mination of surface tension by drawing up liquid films).  
1.6.3.  Ring method  
See ISO 304 and NF T 73-060 (Surface active agents — deter ­
mination of surface tension by drawing up liquid films).  
1.6.4.  OECD harmonised ring method  
1.6.4.1.  Apparatus  
Commercially available tensiometers are adequate for this 
measurement. They consist of the following elements: 
— mobile sample table, 
— force measuring system, — measuring body (ring), 
— measurement vessel.
 
1.6.4.1.1. M o b i l e s a m p l e t a b l e 
The mobile sample table is used as a support for the temperature- 
controlled measurement vessel holding the liquid to be tested. 
Together with the force measuring system, it is mounted on a stand.  
1.6.4.1.2. F o r c e m e a s u r i n g s y s t e m 
The force measuring system (see figure) is located above the sample 
table. The error of the force measurement shall not exceed ± 10  -6 N, 
corresponding to an error limit of ± 0,1 mg in a mass measurement. In most cases, the measuring scale of commercially available tensio ­
meters is calibrated in mN/m so that the surface tension can be read 
directly in mN/m with an accuracy of 0,1 mN/m.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 53
 
1.6.4.1.3. M e a s u r i n g b o d y ( r i n g ) 
The ring is usually made of a platinum-iridium wire of about 0,4 mm 
thickness and a mean circumference of 60 mm. The wire ring is 
suspended horizontally from a metal pin and a wire mounting bracket to establish the connection to the force measuring system (see figure).
 
Figure  
Measuring body  
(All dimensions expressed in millimetres)
  
1.6.4.1.4. M e a s u r e m e n t v e s s e l 
The measurement vessel holding the test solution to be measured 
shall be a temperature-controlled glass vessel. It shall be designed so that during the measurement the temperature of the test solution liquid and the gas phase above its surface remains constant and 
that the sample cannot evaporate. Cylindrical glass vessels having 
an inside diameter of not less than 45 mm are acceptable.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 54
 
1.6.4.2.  Preparation of the apparatus  
1.6.4.2.1. C l e a n i n g 
Glass vessels shall be cleaned carefully. If necessary they shall be 
washed with hot chromo-sulphuric acid and subsequently with syrupy phosphoric acid (83 to 98 % by weight of H
 3 PO 4 ), thor­
oughly rinsed in tap water and finally washed with double-distilled 
water until a neutral reaction is obtained and subsequently dried or 
rinsed with part of the sample liquid to be measured. 
The ring shall first be rinsed thoroughly in water to remove any 
substances which are soluble in water, briefly immersed in chromo-sulphuric acid, washed in double-distilled water until a neutral reaction is obtained and finally heated briefly above a 
methanol flame.  
Note:  
Contamination by substances which are not dissolved or destroyed 
by chromo-sulphuric acid or phosphoric acid, such as silicones, shall be removed by means of a suitable organic solvent.
 
1.6.4.2.2. C a l i b r a t i o n o f t h e a p p a r a t u s 
The validation of the apparatus consists of verifying the zero point 
and adjusting it so that the indication of the instrument allows 
reliable determination in mN/m.  
Mounting:  
The apparatus shall be levelled, for instance by means of a spirit 
level on the tensiometer base, by adjusting the levelling screws in the base.
 
Zero point adjustment:  
After mounting the ring on the apparatus and prior to immersion in 
the liquid, the tensiometer indication shall be adjusted to zero and 
the ring checked for parallelism to the liquid surface. For this 
purpose, the liquid surface can be used as a mirror.  
Calibrations:  
The actual test calibration can be accomplished by means of either of 
two procedures: 
(a) Using a mass: procedure using riders of known mass between 
0,1 and 1,0 g placed on the ring. The calibration factor, Φ a by 
which all the instrument readings must be multiplied, shall be determined according to equation (1).
 
Φ a ¼ σ r 
σ a 
where:  
σ r ¼ mg 
2b (mN/m) 
m = mass of the rider (g) 
g = gravity acceleration (981 cm s  -2 at sea level) 
b = mean circumference of the ring (cm) σ
 a = reading of the tensiometer after placing the rider on the 
ring (mN/m).  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 55
 
(b) Using water: procedure using pure water whose surface tension 
at, for instance, 23  o C is equal to 72,3 mN/m. This procedure is 
accomplished faster than the weight calibration but there is always the danger that the surface tension of the water is falsified by traces of contamination by surfactants. 
The calibration factor, Φ
 b by which all the instrument readings 
shall be multiplied, shall be determined in accordance with the 
equation (2):  
Φ b ¼ σ o 
σ g 
where: 
σ o = value cited in the literature for the surface tension of 
water (mN/m) 
σ g = measured value of the surface tension of the water 
(mN/m) both at the same temperature.  
1.6.4.3.  Preparation of samples  
Aqueous solutions shall be prepared of the substances to be tested, 
using the required concentrations in water, and shall not contain any 
non-dissolved substances. 
The solution must be maintained at a constant temperature 
(± 0,5  o C). Since the surface tension of a solution in the 
measurement vessel alters over a period of time, several measurements shall be made at various times and a curve plotted 
showing surface tension as a function of time. When no further 
change occurs, a state of equilibrium has been reached. 
Dust and gaseous contamination by other substances interfere with 
the measurement. The work shall therefore be carried out under a protective cover.
 
1.6.5.  Test conditions  
The measurement shall be made at approximately 20  o C and shall be 
controlled to within ± 0,5  o C. 
1.6.6.  Performance of test  
The solutions to be measured shall be transferred to the carefully 
cleaned measurement vessel, taking care to avoid foaming, and 
subsequently the measurement vessel shall be placed onto the table of the test apparatus. The table-top with measurement vessel shall be raised until the ring is immersed below the surface of the solution to 
be measured. Subsequently, the table-top shall be lowered gradually 
and evenly (at a rate of approximately 0,5 cm/min) to detach the ring from the surface until the maximum force has been reached. The liquid layer attached to the ring must not separate from the ring. 
After completing the measurements, the ring shall be immersed 
below the surface again and the measurements repeated until a constant surface tension value is reached. The time from transferring the solution to the measurement vessel shall be recorded for each 
determination. Readings shall be taken at the maximum force 
required to detach the ring from the liquid surface.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 56
 
2. DATA  
In order to calculate the surface tension, the value read in mN/m on 
the apparatus shall be first multiplied by the calibration factor Φ a or 
Φ b (depending on the calibration procedure used). This will yield a 
value which applies only approximately and therefore requires 
correction. 
Harkins and Jordan (4) have empirically determined correction 
factors for surface-tension values measured by the ring method which are dependent on ring dimensions, the density of the liquid 
and its surface tension. 
Since it is laborious to determine the correction factor for each 
individual measurement from the Harkins and Jordan tables, in 
order to calculate the surface tension for aqueous solutions the simplified procedure of reading the corrected surface-tension values directly from the table may be used. (Interpolation shall be used for 
readings ranging between the tabular values.)
 
Table:  
Correction of the measured surface tension  
Only for aqueous solutions, ρ = 1 g/cm  3 
r = 9,55 mm (average ring radius) 
r = 0,185 mm (ring wire radius)  
Experimental Value (mN/m) Corrected Value (mN/m) 
Weight calibration (see 1.6.4.2.2(a)) Water calibration (see 1.6.4.2.2(b))  
20 16,9 18,1 
22 18,7 20,1 24 20,6 22,1 
26 22,4 24,1 
28 24,3 26,1 30 26,2 28,1 32 28,1 30,1 34 29,9 32,1 36 31,8 34,1 38 33,7 36,1 40 35,6 38,2 42 37,6 40,3 44 39,5 42,3 46 41,4 44,4 48 43,4 46,5 
50 45,3 48,6 
52 47,3 50,7 54 49,3 52,8 56 51,2 54,9
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 57
 
Experimental Value (mN/m) Corrected Value (mN/m) 
Weight calibration (see 1.6.4.2.2(a)) Water calibration (see 1.6.4.2.2(b))  
58 53,2 57,0 
60 55,2 59,1 
62 57,2 61,3 
64 59,2 63,4 66 61,2 65,5 68 63,2 67,7 70 65,2 69,9 72 67,2 72,0 74 69,2 — 76 71,2 — 78 73,2 — 
This table has been compiled on the basis of the Harkins-Jordan 
correction. It is similar to that in the DIN Standard (DIN 53914) 
for water and aqueous solutions (density ρ = 1 g/cm
 3 and is for a 
commercially available ring having the dimensions R = 9,55 mm (mean ring radius) and r = 0,185 mm (ring wire radius). The table 
provides corrected values for surface-tension measurements taken 
after calibration with weights or calibration with water. 
Alternatively, without the preceding calibration, the surface tension 
call can be calculated according to the following formula:
 
σ ¼ f Ü F 
4πR 
where: 
F = the force measured on the dynamometer at the breakpoint of 
the film 
R = the radius of the ring 
f = the correction factor (1)  
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, include the following information: 
— method used, — type of water or solution used, — precise specification of the substance (identity and impurities), — measurement results: surface tension (reading) stating both the 
individual readings and their arithmetic mean as well as the 
corrected mean (taking into consideration the equipment factor 
and the correction table),
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 58
 
— concentration of the solution, 
— test temperature, — age of solution used; in particular the time between preparation 
and measurement of the solution, 
— description of time dependence of surface tension after trans ­
ferring the solution to the measurement vessel, 
— all information and remarks relevant for the interpretation of 
results have to be reported, especially with regard to impurities 
and physical state of the substance.
 
3.2. INTERPRETATION OF RESULTS 
Considering that distilled water has a surface tension of 72,75 mN/m 
at 20  o C, substances showing a surface tension lower than 60 mN/m 
under the conditions of this method should be regarded as being surface-active materials.
 
4. REFERENCES  
(1) OECD, Paris, 1981, Test Guideline 115, Decision of the 
Council C(81) 30 final. 
(2) R. Weissberger ed.: Technique of Organic Chemistry, Physical 
Methods of Organic Chemistry, 3rd ed., Interscience Publ., New York, 1959, vol. I, Part I, Chapter XIV. 
(3) Pure Appl. Chem., 1976, vol. 48, p. 511. (4) Harkins, W.D., Jordan, H.F., J. Amer. Chem. Soc., 1930, 
vol. 52, p. 1751.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 59
 
A.6. WATER SOLUBILITY  
INTRODUCTION 
1. This Test Method is equivalent to OECD Test Guideline (TG) 105 (1995). 
This Test Method is a revised version of the original TG 105 which was adopted in 1981. There is no difference of substance between the current 
version and that from 1981. Mainly the format has been changed. The 
revision was based on the EU Test Method ‘Water Solubility’ (
 1 ). 
INITIAL CONSIDERATIONS 
2. The water solubility of a substance can be considerably affected by the 
presence of impurities. This Test Method addresses the determination of the solubility in water of essentially pure substances which are stable in 
water and not volatile. Before determining water solubility, it is useful to 
have some preliminary information on the test substance, like structural formula, vapour pressure, dissociation constant and hydrolysis as a function of pH. 
3. Two methods, the column elution method and the flask method which cover 
respectively solubilities below and above 10
 –2 g/l are described in this Test 
Method. A simple preliminary test is also described. It allows the deter ­
mination of approximately the appropriate amount of sample to be used in 
the final test, as well as the time necessary to achieve saturation.  
DEFINITIONS AND UNITS 
4. The water solubility of a substance is the saturation mass concentration of 
the substance in water at a given temperature. 
5. Water solubility is expressed in mass of solute per volume of solution. The 
SI unit is kg/m  3 but g/l may also be used.  
REFERENCE CHEMICALS 
6. Reference chemicals do not need to be employed when investigating a test 
substance.  
DESCRIPTION OF THE METHODS  
Test conditions  
7. The test is preferably run at 20 ± 0,5 ° C. The chosen temperature should be 
kept constant in all relevant parts of the equipment.  
Preliminary test  
8. In a stepwise procedure, increasing volumes of water are added at room 
temperature to approximately 0,1 g of the sample (solid test substances must 
be pulverized) in a 10 ml glass-stoppered measuring cylinder. After each 
addition of an amount of water, the mixture is shaken for 10 minutes and is visually checked for any undissolved parts of the sample. If, after addition of 10 ml of water, the sample or parts of it remain undissolved, the 
experiment is continued in a 100 ml measuring cylinder. The approximate 
solubility is given in Table 1 below under that volume of water in which
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 60
 
complete dissolution of the sample occurs. When the solubility is low, a 
long time may be required to dissolve a test substance and at least 24 hours 
should be allowed. If, after 24 hours, the test substance is still not dissolved, more time (up to 96 hours) should be allowed or further dilution should be attempted to ascertain whether the column elution method or flask method 
should be used.
 
Table 1  
ml of water for 0,1 g soluble 0,1 0,5 1 2 10 100 > 100 
approximate solubility in g/l > 1 000 1 000 to 
200 200 to 
100 100 to 50 50 to 10 10 to 1 < 1  
Column elution method  
Principle  
9. This method is based on the elution of a test substance with water from 
a micro-column which is charged with an inert support material, previously 
coated with an excess of the test substance (2). The water solubility is given by the mass concentration of the eluate when this has reached a plateau as a function of time.
 
Apparatus  
10. The apparatus consists of a microcolumn (Figure 1), maintained at constant 
temperature. It is connected either to a recirculating pump (Figure 2) or to a levelling vessel (Figure 3). The microcolumn contains an inert support held in place by a small plug of glasswool which also serves to filter out 
particles. Possible materials which can be employed for the support are 
glass beads, diatomaceous earth, or other inert materials. 
11. The microcolumn shown in Figure 1 is suitable for the set-up with recircu ­
lating pump. It has a head space providing for five bed volumes (discarded 
at the start of the experiment) and the volume of five samples (withdrawn 
for analysis during the experiment). Alternatively, the size can be reduced if water can be added to the system during the experiment to replace the initial 
five bed volumes removed with impurities. The column is connected with 
tubing made of an inert material to the recirculating pump, capable of delivering approximately 25 ml/h. The recirculating pump can be, for example, a peristaltic or membrane pump. Care must be taken that no 
contamination and/or adsorption occur with the tube material. 
12. A schematic arrangement using a levelling vessel is shown in Figure 3. In 
this arrangement the microcolumn is fitted with a one way stopcock. The connection to the levelling vessel consists of a ground glass joint and tubing 
made of an inert material. The flow rate from the levelling vessel should be 
approximately 25 ml/h.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 61
 
Figure 1
  
Dimensions in mm 
A. Connection for ground glass joint 
B. Headspace C. Interior 5 D. Exterior 19 
E. Plug of glass wool 
F. Stopcock
 
Figure 2
  
A. Atmospheric equilibration 
B. Flowmeter 
C. Microcolumn D. Thermostatically controlled circulating pump 
E. Recirculating pump 
F. Two-way valve for sampling
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 62
 
Figure 3
  
A. Levelling vessel (e.g. 2,5 litres chemical flask) 
B. Column 
C. Fraction accumulator 
D. Thermostat E. Teflon tubing F. Ground glass joint G. Water line (between thermostat and column, inner diameter approxi ­
mately 8 mm) 
13. Approximately 600 mg of support material is transferred to a 50 ml 
round-bottom flask. A suitable amount of test substance is dissolved in a volatile solvent of analytical reagent quality and an appropriate amount of 
this solution is added to the support material. The solvent is completely 
evaporated, e.g. using a rotary evaporator, as otherwise water saturation of the support will not be achieved during the elution step because of parti ­
tioning on the surface. The loaded support material is soaked for two hours 
in approximately 5 ml of water and the suspension is poured into the micro ­
column. Alternatively, dry loaded support material may be poured into the water-filled microcolumn and two hours are allowed for equilibrating. 
14. The loading of the support material may cause problems, leading to 
erroneous results, e.g. when the test substance is deposited as an oil. 
These problems should be examined and the details reported.
 
Procedure using a recirculating pump  
15. The flow through the column is started. It is recommended that a flow rate 
of approximately 25 ml/h, corresponding to 10 bed volumes per hour for the column described, be used. At least the first five bed volumes are discarded to remove water soluble impurities. Following this, the pump is allowed to 
run until equilibrium is established, as defined by five successive samples 
whose concentrations do not differ by more than ± 30 % in a random fashion. These samples should be separated from each other by time intervals corresponding to the passage of at least ten bed volumes. 
Depending on the analytical method used, it may be preferable to 
establish a concentration/time curve to show that equilibrium is reached.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 63
 
Procedure using a levelling vessel  
16. Successive eluate fractions should be collected and analysed by the chosen 
method. Fractions from the middle eluate range, where the concentrations are constant within ± 30 % in at least five consecutive fractions, are used to 
determine the solubility. 
17. Double distilled water is the preferred eluent. Deionized water with a resis ­
tivity above 10 megohms/cm and total organic carbon content below 0,01 % 
can also be used. 
18. Under both procedures, a second run is performed at half the flow rate of 
the first. If the results of the two runs are in agreement, the test is satis ­
factory. If the measured solubility is higher with the lower flow rate, then 
the halving of the flow rate must continue until two successive runs give the 
same solubility. 
19. Under both procedures, the fractions should be checked for the presence of 
colloidal matter by examination of the Tyndall effect. The presence of 
particles invalidates the test and the test should be repeated after 
improvement of the filtering action of the column. 
20. The pH of each sample should be measured, preferably by using special 
indicator strips.
 
Flask method  
Principle  
21. The test substance (solids must be pulverized) is dissolved in water at a 
temperature somewhat above the test temperature. When saturation is 
achieved, the mixture is cooled and kept at the test temperature. Alter ­
natively, and if it is assured by appropriate sampling that the saturation equilibrium is reached, the measurement can be performed directly at the test temperature. Subsequently, the mass concentration of the test substance 
in the aqueous solution, which must not contain any undissolved particles, is 
determined by a suitable analytical method (3).
 
Apparatus  
22. The following materials are needed: 
— normal laboratory glassware and instrumentation; — a device for the agitation of solutions under controlled constant 
temperature; 
— if required for emulsions, a centrifuge (preferably thermostated); and — analytical equipment.
 
Procedure  
23. The quantity of test substance necessary to saturate the desired volume of 
water is estimated from the preliminary test. About five times that quantity 
is weighed into each of three glass vessels fitted with glass stoppers (e.g. 
centrifuge tubes, flasks). A volume of water, chosen in function of the analytical method and solubility range, is added to each vessel. The vessels are tightly stoppered and then agitated at 30 °C. A shaking or 
stirring device capable of operating at constant temperature should be 
used, e.g. magnetic stirring in a thermostated water bath. After one day, one of the vessels is equilibrated for 24 hours at the test temperature with occasional shaking. The contents of the vessel are then centrifuged at the 
test temperature and the concentration of the test substance in the clear 
aqueous phase is determined by a suitable analytical method. The other
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 64
 
two flasks are treated similarly after initial equilibration at 30 °C for two 
and three days respectively. If the concentrations measured in at least the 
two last vessels do not differ by more than 15 %, the test is satisfactory. If the results from vessels 1, 2 and 3 show a tendency of increasing values, the whole test should be repeated using longer equilibration times. 
24. The test can also be performed without pre-incubation at 30 °C. In order to 
estimate the rate of establishment of the saturation equilibrium, samples are 
taken until the stirring time no longer influences the concentrations 
measured. 
25. The pH of each sample should be measured, preferably by using special 
indicator strips.
 
Analytical determinations  
26. A substance-specific method is preferred since small amounts of soluble 
impurities can cause large errors in the measured solubility. Examples of such methods are: gas or liquid chromatography, titration, photometry, volt ­
ametry.
 
DATA AND REPORTING  
Data  
Column elution method  
27. For each run, the mean value and standard deviation from at least five 
consecutive samples taken from the saturation plateau should be calculated. 
The mean values obtained from two tests with different flows should not differ by more than 30 %.
 
Flask method  
28. The individual results from each of the three flasks, which should not differ 
by more than 15 %, are averaged.  
Test Report  
Column elution method  
29. The test report must include the following information: 
— the results of the preliminary test — chemical identity and impurities (preliminary purification step, if any) — the concentrations, flow rates and pH for each sample — the means and standard deviations from at least five samples from the 
saturation plateau of each run 
— the average of at least two successive runs — the temperature of the water during the saturation process 
— the method of analysis 
— the nature of the support material — loading of the support material — solvent used — evidence of any chemical instability of the substance during the test — all information relevant for the interpretation of the results, in particular 
with regard to impurities and physical state of the test substance.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 65
 
Flask method  
30. The test report must include the following information: 
— the results of the preliminary test — chemical identity and impurities (preliminary purification step, if any) — the individual analytical determinations and the average where more than 
one value was determined for each flask 
— the pH of each sample 
— the average of the values for different flasks which were in agreement — the test temperature — the analytical method — evidence of any chemical instability of the substance during the test — all information relevant for the interpretation of the results, in particular 
with regard to impurities and physical state of the test substance.
 
LITERATURE:  
(1) Commission Directive 92/69/EEC of 31 July 1992 adapting to technical 
progress for the seventeenth time Council Directive 67/548/EEC on the 
approximation of laws, regulations and administrative provisions relating to 
the classification, packaging and labelling of dangerous substances (OJ L 383, 29.12.1992, p. 113). 
(2) NF T 20-045 (AFNOR) (September 1985). Chemical products for industrial 
use — Determination of water solubility of solids and liquids with low 
solubility — Column elution method. 
(3) NF T 20-046 (AFNOR) (September 1985). Chemical products for industrial 
use — Determination of water solubility of solids and liquids with high solubility — Flask method.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 66
 
A.8. PARTITION COEFFICIENT  
1. METHOD  
The ‘shake flask’ method described is based on the OECD Test 
Guideline (1).  
1.1. INTRODUCTION 
It is useful to have preliminary information on structural formula, 
dissociation constant, water solubility, hydrolysis, n-octanol solu ­
bility and surface tension of the substance to perform this test. 
Measurements should be made on ionisable substances only in their 
non-ionised form (free acid or free base) produced by the use of an 
appropriate buffer with a pH of at least one pH unit below (free acid) 
or above (free base) the pK. 
This test method includes two separate procedures: the shake flask 
method and high performance liquid chromatography (HPLC). The former is applicable when the log P
 ow value (see below for defini ­
tions) falls within the range - 2 to 4 and the latter within the range 0 
to 6. Before carrying out either of the experimental procedures a 
preliminary estimate of the partition coefficient should first be obtained. 
The shake-flask method applies only to essentially pure substances 
soluble in water and n-octanol. It is not applicable to surface active materials (for which a calculated value or an estimate based on the 
individual n-octanol and water solubilities should be provided). 
The HPLC method is not applicable to strong acids and bases, metal 
complexes, surface-active materials or substances which react with 
the eluent. For these materials, a calculated value or an estimate 
based on individual n-octanol and water solubilities should be provided. 
The HPLC method is less sensitive to the presence of impurities in 
the test compound than is the shake-flask method. Nevertheless, in some cases impurities can make the interpretation of the results 
difficult because peak assignment becomes uncertain. For mixtures 
which give an unresolved band, upper and lower limits of log P should be stated.
 
1.2. DEFINITION AND UNITS 
The partition coefficient (P) is defined as the ratio of the equilibrium 
concentrations (c  i ) of a dissolved substance in a two-phase system 
consisting of two largely immiscible solvents. In the case n-octanol and water:
 
P ow ¼ c nÄoctanol  
c water 
The partition coefficient (P) therefore is the quotient of two 
concentrations and is usually given in the form of its logarithm to 
base 10 (log P).  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 67
 
1.3. REFERENCE SUBSTANCES  
Shake-flask method  
Reference substances do not need to be employed in all cases when 
investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow 
comparison with results from other methods.
 
HPLC method  
In order to correlate the measured HPLC data of a compound with 
its P value, a calibration graph of log P versus chromatographic data 
using at least six reference points has to be established. It is for the 
user to select the appropriate reference substances. Whenever possible, at least one reference compound should have a P
 ow 
above that of the test substance, and another a P  ow below that of 
the test substance. For log P values less than 4, the calibration can be 
based on data obtained by the shake-flask method. For log P values greater than 4, the calibration can be based on validated literature values if these are in agreement with calculated values. For better 
accuracy, it is preferable to choose reference compounds which are 
structurally related to the test substance. 
Extensive lists of values of log P
 ow for many groups of chemicals 
are available (2)(3). If data on the partition coefficients of struc ­
turally related compounds are not available, then a more general 
calibration, established with other reference compounds, may be used. 
A list of recommended reference substances and their P
 ow values is 
given in Appendix 2.  
1.4. PRINCIPLE OF THE METHOD  
1.4.1.  Shake-flask method  
In order to determine a partition coefficient, equilibrium between all 
interacting components of the system must be achieved, and the 
concentrations of the substances dissolved in the two phases must 
be determined. A study of the literature on this subject indicates that several different techniques can be used to solve this problem, i.e. the thorough mixing of the two phases followed by their separation 
in order to determine the equilibrium concentration for the substance 
being examined.
 
1.4.2.  HPLC method  
HPLC is performed on analytical columns packed with a commer ­
cially available solid phase containing long hydrocarbon chains (e.g. C
 8 , C 18 ) chemically bound onto silica. Chemicals injected onto such 
a column move along it at different rates because of the 
different degrees of partitioning between the mobile phase and the hydrocarbon stationary phase. Mixtures of chemicals are eluted in order of their hydrophobicity, with water-soluble chemicals eluted 
first and oil-soluble chemicals last, in proportion to their hydro ­
carbon-water partition coefficient. This enables the relationship between the retention time on such a (reverse phase) column and the n-octanol/water partition coefficient to be established. The 
partition coefficient is deduced from the  capacity factor  k, given 
by the expression:
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 68
 
k ¼ t r Ä t o 
t o 
in which, t  r = retention time of the test substance, and t  o = average 
time a solvent molecule needs to pass through the column (dead- 
time). 
Quantitative analytical methods are not required and only the deter ­
mination of elution times is necessary.  
1.5. QUALITY CRITERIA  
1.5.1.  Repeatability  
Shake-flask method  
In order to assure the accuracy of the partition coefficient, duplicate 
determinations are to be made under three different test conditions, 
whereby the quantity of substance specified as well as the ratio of 
the solvent volumes may be varied. The determined values of the partition coefficient expressed as their common logarithms should fall within a range of ± 0,3 log units.
 
HPLC method  
In order to increase the confidence in the measurement, duplicate 
determinations must be made. The values of log P derived from 
individual measurements should fall within a range of ± 0,1 log 
units.  
1.5.2.  Sensitivity  
Shake-flask method  
The measuring range of the method is determined by the limit of 
detection of the analytical procedure. This should permit the 
assessment of values of log P  ow in the range of - 2 to 4 (occasionally 
when conditions apply, this range may be extended to log P  ow up 
to 5) when the concentration of the solute in either phase is not more than 0,01 mol per litre.
 
HPLC method  
The HPLC method enables partition coefficients to be estimated in 
the log P  ow range 0 to 6. 
Normally, the partition coefficient of a compound can be estimated 
to within ± l log unit of the shake-flask value. Typical correlations can be found in the literature (4)(5)(6)(7)(8). Higher accuracy can 
usually be achieved when correlation plots are based on structurally- 
related reference compounds (9).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 69
 
1.5.3.  Specificity  
Shake-flask method  
The Nernst Partition Law applies only at constant temperature, 
pressure and pH for dilute solutions. It strictly applies to a pure substance dispersed between two pure solvents. If several different 
solutes occur in one or both phases at the same time, this may affect 
the results. 
Dissociation or association of the dissolved molecules result in devi ­
ations from the Nernst Partition Law. Such deviations are indicated by the fact that the partition coefficient becomes dependent upon the 
concentration of the solution. 
Because of the multiple equilibria involved, this test method should 
not be applied to ionisable compounds without applying a correction. 
The use of buffer solutions in place of water should be considered for such compounds; the pH of the buffer should be at least 1 pH unit from the pKa of the substance and bearing in mind the relevance 
of this pH for the environment.
 
1.6. DESCRIPTION OF THE METHOD  
1.6.1.  Preliminary estimate of the partition coefficient  
The partition coefficient is estimated preferably by using a calcu ­
lation method (see Appendix 1), or where appropriate, from the ratio of the solubilities of the test substance ill the pure solvents (10).
 
1.6.2.  Shake-flask method  
1.6.2.1.  Preparation  
n-Octanol: the determination of the partition coefficient should be 
carried out with high purity analytical grade reagent. 
Water: water distilled or double distilled in glass or quartz apparatus 
should be employed. For ionisable compounds, buffer solutions in place of water should be used if justified.  
Note:  
Water taken directly from an ion exchanger should not be used.
 
1.6.2.1.1. P r e - s a t u r a t i o n o f t h e s o l v e n t s 
Before a partition coefficient is determined, the phases of the solvent 
system are mutually saturated by shaking at the temperature of the 
experiment. To do this, it is practical to shake two large stock bottles of high purity analytical grade n-octanol or water each with a sufficient quantity of the other solvent for 24 hours on a mechanical 
shaker and then to let them stand long enough to allow the phases to 
separate and to achieve a saturation state.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 70
 
1.6.2.1.2. P r e p a r a t i o n f o r t h e t e s t 
The entire volume of the two-phase system should nearly fill the test 
vessel. This will help prevent loss of material due to volatilisation. 
The volume ratio and quantities of substance to be used are fixed by the following: 
— the preliminary assessment of the partition coefficient (see 
above), 
— the minimum quantity of test substance required for the 
analytical procedure, and 
— the limitation of a maximum concentration in either phase of 
0,01 mol per litre. 
Three tests are carried out. In the first, the calculated volume ratio of 
n-octanol to water is used; in the second, this ratio is divided by two; 
and in the third, this ratio is multiplied by two (e.g. 1:1, 1:2, 2:1).
 
1.6.2.1.3. T e s t s u b s t a n c e 
A stock solution is prepared in n-octanol pre-saturated with water. 
The concentration of this stock solution should be precisely 
determined before it is employed in the determination of the 
partition coefficient. This solution should be stored under conditions which ensure its stability.
 
1.6.2.2.  Test conditions  
The test temperature should be kept constant (± 1  o C) and lie in the 
range of 20 to 25  o C. 
1.6.2.3.  Measurement procedure  
1.6.2.3.1. E s t a b l i s h m e n t o f t h e p a r t i t i o n e q u i l i b r i u m 
Duplicate test vessels containing the required, accurately measured 
amounts of the two solvents together with the necessary quantity of 
the stock solution should be prepared for each of the test conditions. 
The n-octanol phases should be measured by volume. The test 
vessels should either be placed in a suitable shaker or shaken by hand. When using a centrifuge tube, a recommended method is to 
rotate the tube quickly through 180
 o about its transverse axis so that 
any trapped air rises through the two phases. Experience has shown that 50 such rotations are usually sufficient for the establishment of the partition equilibrium. To be certain, 100 rotations in five minutes 
are recommended.
 
1.6.2.3.2. P h a s e s e p a r a t i o n 
When necessary, in order to separate the phases, centrifugation of the 
mixture should be carried out. This should be done in a laboratory centrifuge maintained at room temperature, or, if a non-temperature controlled centrifuge is used, the centrifuge tubes should be kept for 
equilibration at the test temperature for at least one hour before 
analysis.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 71
 
1.6.2.4.  Analysis  
For the determination of the partition coefficient, it is necessary to 
determine the concentrations of the test substance in both phases. This may be done by taking an aliquot of each of the two phases 
from each tube for each test condition and analyzing them by the 
chosen procedure. The total quantity of substance present in both phases should be calculated and compared with the quantity of the substance originally introduced. 
The aqueous phase should be sampled by a procedure that minimises 
the risk of including traces of n-octanol: a glass syringe with a 
removable needle can be used to sample the water phase. The 
syringe should initially be partially filled with air. Air should be gently expelled while inserting the needle through the n-octanol layer. An adequate volume of aqueous phase is withdrawn into the 
syringe. The syringe is quickly removed from the solution and the 
needle detached. The contents of the syringe may then be used as the aqueous sample. The concentration in the two separated phases should preferably be determined by a substance-specific method. 
Examples of analytical methods which may be appropriate are: 
— photometric methods, 
— gas chromatography, — high-performance liquid chromatography.
 
1.6.3.  HPLC method  
1.6.3.1.  Preparation  
Apparatus  
A liquid chromatograph, fitted with a pulse-free pump and a suitable 
detection device, is required. The use of an injection valve with injection loops is recommended. The presence of polar groups in the stationary phase may seriously impair the performance of the 
HPLC column. Therefore, stationary phases should have the 
minimal percentage of polar groups (11). Commercial micropar ­
ticulate reverse-phase packings or ready-packed columns can be used. A guard column may be positioned between the injection 
system and the analytical column.
 
Mobile phase  
HPLC grade methanol and HPLC grade water are used to prepare 
the eluting solvent, which is degassed before use. Isocratic elution should be employed. Methanol/water ratios with a minimum water content of 25 % should be used. Typically a 3:1 (v/v) methanol- 
water mixture is satisfactory for eluting compounds of log P 6 within 
an hour, at a flow rate of 1 ml/min. For compounds of high log P it may be necessary to shorten the elution time (and those of the reference compounds) by decreasing the polarity of the mobile 
phase or the column length. 
Substances with very low solubility in n-octanol tend to give 
abnormally low log P
 ow values with the HPLC method; the peaks 
of such compounds sometimes accompany the solvent front. This is probably due to the fact that the partitioning process is too slow to reach the equilibrium in the time normally taken by an HPLC separ ­
ation. Decreasing the flow rate and/or lowering the methanol/water 
ratio may then be effective to arrive at a reliable value.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 72
 
Test and reference compounds should be soluble in the mobile phase 
in sufficient concentrations to allow their detection. Only in excep ­
tional cases may additives be used with the methanol-water mixture, since additives will change the properties of the column. For chromatograms with additives it is mandatory to use a separate 
column of the same type. If methanol-water is not appropriate, 
other organic solvent-water mixtures call be used, e.g. ethanol- water or acetonitrile-water. 
The pH of the eluent is critical for ionisable compounds. It should be 
within the operating pH range of the column, which is usually between 2 and 8. Buffering is recommended. Care must be taken 
to avoid salt precipitation and column deterioration which occur with 
some organic phase/buffer mixtures. HPLC measurements with silica-based stationary phases above pH 8 are not advisable since the use of an alkaline, mobile phase may cause rapid deterioration in 
the performance of the column.
 
Solutes  
The reference compounds should be the purest available. 
Compounds to be used for test or calibration purposes are dissolved in the mobile phase if possible.
 
Test conditions  
The temperature during the measurements should not vary by more 
than ± 2 K.  
1.6.3.2.  Measurement  
Calculation of dead time t  o 
The dead time to can be determined by using either a homologous 
series (e.g. n-alkyl methyl ketones) or unretained organic compounds 
(e.g. thiourea or formamide). For calculating the dead time to by using a homologous series, a set of at least seven members of a homologous series is injected and the respective retention times 
are determined. The raw retention times t
 r (n c + 1) are plotted as a 
function of t  r(nc) and the intercept a and slope b of the regression 
equation: 
t r(n c + 1) = a + b t  r(n c ) 
are determined (n  c = number of carbon atoms). The dead time to is 
then given by: 
t o = a/(1 - b)  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 73
 
Calibration graph  
The next step is to construct a correlation plot of log k values versus 
log p for appropriate reference compounds. In practice, a set of between 5 and 10 standard reference compounds whose log p is around the expected range are injected simultaneously and the 
retention times are determined, preferably on a recording integrator 
linked to the detection system. The corresponding logarithms of the capacity factors, log k, are calculated and plotted as a function of the log p determined by the shake-flask method. The calibration is 
performed at regular intervals, at least once daily, so that possible 
changes in column performance can be allowed for.
 
Determination of the capacity factor of the test substance  
The test substance is injected in as small a quantity of mobile phase 
as possible. The retention time is determined (in duplicate), permitting the calculation of the capacity factor k. From the correlation graph of the reference compounds, the partition coef ­
ficient of the test substance can be interpolated. For very low and 
very high partition coefficients, extrapolation is necessary. In those cases particular care has to be taken of the confidence limits of the regression line.
 
2. DATA  
Shake-flask method  
The reliability of the determined values of P can be tested by 
comparison of the means of the duplicate determinations with the 
overall mean.  
3. REPORTING  
The test report shall, if possible, include the following information: 
— precise specification of the substance (identity and impurities), 
— when the methods are not applicable (e.g. surface active 
material), a calculated value or an estimate based on the indi ­
vidual n-octanol and water solubilities should be provided, 
— all information and remarks relevant for the interpretation of 
results, especially with regard to impurities and physical state 
of the substance.  
For shake-flask method:  
— the result of the preliminary estimation, if any, 
— temperature of the determination, — data on the analytical procedures used in determining concen ­
trations, 
— time and speed of centrifugation, if used,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 74
 
— the measured concentrations in both phases for each deter ­
mination (this means that a total of 12 concentrations will be 
reported), 
— the weight of the test substance, the volume of each phase 
employed in each test vessel and the total calculated amount of 
test substance present in each phase after equilibration, 
— the calculated values of the partition coefficient (P) and the mean 
should be reported for each set of test conditions as should the mean for all determinations. If there is a suggestion of concen ­
tration dependency of the partition coefficient, this should be 
noted in the report, 
— the standard deviation of individual P values about their mean 
should be reported, 
— the mean P from all determinations should also be expressed as 
its logarithm (base 10), 
— the calculated theoretical P
 ow when this value has been 
determined or when the measured value is > 10  4 , 
— pH of water used and of the aqueous phase during the 
experiment, 
— if buffers are used, justification for the use of buffers in place of 
water, composition, concentration and pH of the buffers, pH of 
the aqueous phase before and after the experiment.  
For HPLC method:  
— the result of the preliminary estimation, if any, 
— test and reference substances, and their purity, — temperature range of the determinations, 
— pH at which the determinations are made, 
— details of the analytical and guard column, mobile phase and 
means of detection, 
— retention data and literature log P values for reference 
compounds used in calibration, 
— details of fitted regression line (log k versus log P), — average retention data and interpolated log P value for the test 
compound, 
— description of equipment and operating conditions, — elution profiles, — quantities of test and references substances introduced in the 
column, 
— dead-time and how it was measured.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 75
 
4. REFERENCES  
(1) OECD, Paris, 1981, Test Guideline 107, Decision of the 
Council C(81) 30 final. 
(2) C. Hansch and A.J. Leo, Substituent Constants for Correlation 
Analysis in Chemistry and Biology, John Wiley, New York, 1979. 
(3) Log P and Parameter Database, A tool for the quantitative 
prediction of bioactivity (C. Hansch, chairman, A.J. Leo, dir.) — Available from Pomona College Medical Chemistry Project 1982, Pomona College, Claremont, California 91711. 
(4) L. Renberg, G. Sundström and K. Sundh-Nygärd, Chemo ­
sphere, 1980, vol. 80, p. 683. 
(5) H. Ellgehausen, C. D'Hondt and R. Fuerer, Pestic. Sci., 1981, 
vol. 12, p. 219. 
(6) B. McDuffie, Chemosphere, 1981, vol. 10, p. 73. 
(7) W.E. Hammers et al., J. Chromatogr., 1982, vol. 247, p. 1. 
(8) J.E. Haky and A.M. Young, J. Liq. Chromat., 1984, vol. 7, 
p. 675. 
(9) S. Fujisawa and E. Masuhara, J. Biomed. Mat. Res., 1981, 
vol. 15, p. 787. 
(10) O. Jubermann, Verteilen und Extrahieren, in Methoden der 
Organischen Chemie (Houben Weyl), Allgemeine Laboratori ­
umpraxis (edited by E. Muller), Georg Thieme Verlag, Stuttgart, 1958, Band I/1, p. 223-339. 
(11) R.F. Rekker and H.M. de Kort, Euro. J. Med. Chem., 1979, 
vol. 14, p. 479. 
(12) A. Leo, C. Hansch and D. Elkins, Partition coefficients and 
their uses. Chem. Rev., 1971, vol. 71, p. 525. 
(13) R.F. Rekker, The Hydrophobic Fragmental Constant, Elsevier, 
Amsterdam, 1977. 
(14) NF T 20-043 AFNOR (1985). Chemical products for industrial 
use — Determination of partition coefficient — Flask shaking method. 
(15) C.V. Eadsforth and P. Moser, Chemosphere, 1983, vol. 12, 
p. 1459. 
(16) A. Leo, C. Hansch and D. Elkins, Chem. Rev., 1971, vol. 71, 
p. 525. 
(17) C. Hansch, A. Leo, S.H. Unger, K.H. Kim, D. Nikaitani and 
E.J. Lien, J. Med. Chem., 1973, vol. 16, p. 1207. 
(18) W.B. Neely, D.R. Branson and G.E. Blau, Environ. Sci. 
Technol., 1974, vol. 8, p. 1113. 
(19) D.S. Brown and E.W. Flagg, J. Environ. Qual., 1981, vol. 10, 
p. 382. 
(20) J.K. Seydel and K.J. Schaper, Chemische Struktur und biol ­
ogische Aktivität von Wirkstoffen, Verlag Chemie, Weinheim, 
New York, 1979.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 76
 
(21) R. Franke, Theoretical Drug Design Methods, Elsevier, 
Amsterdam, 1984. 
(22) Y.C. Martin, Quantitative Drug Design, Marcel Dekker, 
New York, Base1, 1978. 
(23) N.S. Nirrlees, S.J. Noulton, C.T. Murphy, P.J. Taylor; J. Med. 
Chem., 1976, vol. 19, p. 615.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 77
 
Appendix 1  
Calculation/estimation methods  
INTRODUCTION  
A general introduction to calculation methods, data and examples are provided in 
the Handbook of Chemical Property Estimation Methods (a). 
Calculated values of P  ow can be used: 
— for deciding which of the experimental methods is appropriate (shake-flask 
range: log P  ow : - 2 to 4, HPLC range: log P  ow : 0 to 6), 
— for selecting the appropriate test conditions (e.g. reference substances for 
HPLC procedures, volume ratio n-octanol/water for shake flask method), 
— as a laboratory internal check on possible experimental errors, — for providing a P
 ow -estimate in cases where the experimental methods cannot 
be applied for technical reasons.  
ESTIMATION METHOD  
Preliminary estimate of the partition coefficient  
The value of the partition coefficient can be estimated by the use of the solu ­
bilities of the test substance in the pure solvents: For this:  
P estimate ¼ saturation c  nÄoctanol  
saturation c  water 
CALCULATION METHODS  
Principle of the calculation methods  
All calculation methods are based on the formal fragmentation of the molecule 
into suitable substructures for which reliable log P  ow -increments are known. The 
log P  ow of the whole molecule is then calculated as the sum of its corresponding 
fragment values plus the sum of correction terms for intramolecular interactions. 
Lists of fragment constants and correction terms ate available (b)(c)(d)(e);. Some 
are regularly updated (b).  
Quality criteria  
In general, the reliability of the calculation method decreases with increasing 
complexity of the compound under study. In the case of simple molecules 
with low molecular weight and one or two functional groups, a deviation of 0,1 to 0,3 log P
 ow units between the results of the different fragmentation 
methods and the measured value can be expected. In the case of more 
complex molecules the margin of error can be greater. This will depend on 
the reliability and availability of fragment constants, as well as on the ability to recognise intramolecular interactions (e.g. hydrogen bonds) and the correct use of the correction terms (less of a problem with the computer software CLOGP-3) 
(b). In the case of ionising compounds the correct consideration of the charge 
or degree of ionisation is important.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 78
 
Calculation procedures  
Hansch π-method  
The original hydrophobic substituent constant, π, introduced by Fujira et al. (f) is 
defined as: 
π x = log P  ow (PhX) - log P  ow (PhH) 
where P  ow (PhX) is the partition coefficient of an aromatic derivative and P  ow 
(PhH) that of the parent compound 
(e.g. π Cl = log P  ow (C 6 H 5 Cl) - log P  ow (C 6 H 6 ) = 2,84 - 2,13 = 0,71). 
According to its definition the π-method is applicable predominantly for aromatic 
substitution. π-values for a large number of substituents have been tabulated 
(b)(c)(d). They are used for the calculation of log P  ow for aromatic molecules 
or substructures.  
Rekker method  
According to Rekker (g) the log P  ow value is calculated as follows:  
log P  ow ¼ Σ i a i f i þ Σ j ðinteractious termsÞ 
where f  i represents the different molecular fragment constants and a  i the 
frequency of their occurrence in the molecule under investigation. The correction 
terms can be expressed as an integral multiple of one single constant C  m (so- 
called magic constant). The fragment constants f  i and C  m were determined from a 
list of 1 054 experimental P  ow values (825 compounds) using multiple regression 
analysis (c)(h). The determination of the interaction terms is carried out according 
to set rules described in the literature (e)(h)(i).  
Hansch-Leo method  
According to Hansch and Leo (c), the log P  ow value is calculated from:  
log P  ow ¼ Σ i a i f i þ Σ j b j F j 
where f  i represents the different molecular fragment constants, F  j the correction 
terms and a  i , b j the corresponding frequencies of occurrence. Derived from 
experimental P  ow values, a list of atomic and group fragmental values and a 
list of correction terms F  j (so-called factors) were determined by trial and 
error. The correction terms have been ordered into several different classes (a)(c). It is relatively complicated and time consuming to take into account all 
the rules and correction terms. Software packages have been developed (b).
 
Combined method  
The calculation of log P  ow of complex molecules can be considerably improved, 
if the molecule is dissected into larger substructures for which reliable log P  ow 
values are available, either from tables (b)(c) or from one's own measurements. 
Such fragments (e.g. heterocycles, anthraquinone, azobenzene) can then be combined with the Hansch π-values or with Rekker or Leo fragment constants.
 
Remarks  
(i) The calculation methods can only be applied to partly or fully ionised 
compounds when it is possible to take the necessary correction factors 
into account;  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 79
 
(ii) if intramolecular hydrogen bonds can be assumed, the corresponding 
correction terms (approx. + 0,6 to + 1,0 log P  ow units) have to be added 
(a). Indications for the presence of such bonds can be obtained from stereo models or spectroscopic data of the molecule; 
(iii) If several tautomeric forms are possible, the most likely form should be used 
as the basis of the calculation; 
(iv) the revisions of lists of fragment constants should be followed carefully.
 
Report  
When using calculation/estimation methods, the test report shall, if possible, 
include the following information: 
— description of the substance (mixture, impurities, etc.), — indication of any possible intramolecular hydrogen bonding, dissociation, 
charge and any other unusual effects (e.g. tautomerism), 
— description of the calculation method, — identification or supply of database, — peculiarities in the choice of fragments, — comprehensive documentation of the calculation.
 
LITERATURE  
(a) W.J. Lyman, W.F. Reehl and D.H. Rosenblatt (ed.), Handbook of Chemical 
Property Estimation Methods, McGraw-Hill, New York, 1983. 
(b) Pomona College, Medicinal Chemistry Project, Claremont, California 91711, 
USA, Log P Database and Med. Chem. Software (Program CLOGP-3). 
(c) C. Hansch, A.J. Leo, Substituent Constants for Correlation Analysis in 
Chemistry and Biology, John Wiley, New York, 1979. 
(d) A. Leo, C. Hansch, D. Elkins, Chem. Rev., 1971, vol. 71, p. 525. 
(e) R.F. Rekker, H.M. de Kort, Eur. J. Med. Chem. -Chill. Ther. 1979, vol. 14, 
p. 479. 
(f) T. Fujita, J. Iwasa and C. Hansch, J. Amer. Chem. Soc., 1964, vol. 86, 
p. 5175. 
(g) R.F. Rekker, The Hydrophobic Fragmental Constant, Pharmacochemistry 
Library, Elsevier, New York, 1977, vol. 1. 
(h) C.V. Eadsforth, P. Moser, Chemosphere, 1983, vol. 12, p. 1459. 
(i) R.A. Scherrer, ACS, American Chemical Society, Washington D.C., 1984, 
Symposium Series 255, p. 225.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 80
 
Appendix 2  
Recommended Reference Substances for the HLPC Method  
No Reference Substance log P  ow pKa 
1 2-Butanone 0,3 
2 4-Acetylpyridine 0,5 
3 Aniline 0,9 
4 Acetanilide 1,0 
5 Benzylalcohol 1,1 6 p-Methoxyphenol 1,3 pKa = 10,26 
7 Phenoxy acetic acid 1,4 pKa = 3,12 
8 Phenol 1,5 pKa = 9,92 
9 2,4-Dinitrophenol 1,5 pKa = 3,96 
10 Benzonitrile 1,6 11 Phenylacetonitrile 1,6 
12 4-Methylbenzyl alcohol 1,6 
13 Acetophenone 1,7 
14 2-Nitrophenol 1,8 pKa = 7,17 
15 3-Nitrobenzoic acid 1,8 pKa = 3,47 16 4-Chloraniline 1,8 pKa = 4,15 
17 Nitrobenzene 1,9 
18 Cinnamic alcohol 1,9 
19 Benzoic acid 1,9 pKa = 4,19 
20 p-Cresol 1,9 pKa = 10,17 21 Cinnamic acid 2,1 pKa = 3,89 cis 4,44 trans 
22 Anisole 2,1 
23 Methylbenzoate 2,1 
24 Benzene 2,1 
25 3-Methylbenzoic acid 2,4 pKa = 4,27 26 4-Chlorophenol 2,4 pKa = 9,1 
27 Trichloroethylene 2,4 
28 Atrazine 2,6 
29 Ethylbenzoate 2,6 
30 2,6-Dichlorobenzonitrile 2,6 31 3-Chlorobenzoic acid 2,7 pKa = 3,82 
32 Toluene 2,7 
33 1-Naphthol 2,7 pKa = 9,34 34 2,3-Dichloroaniline 2,8 
35 Chlorobenzene 2,8 
36 Allyl-phenylether 2,9 
37 Bromobenzene 3,0
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 81
 
No Reference Substance log P  ow pKa 
38 Ethylbenzene 3,2 
39 Benzophenone 3,2 
40 4-Phenylphenol 3,2 pKa = 9,54 
41 Thymol 3,3 42 1,4-Dichlorobenzene 3,4 43 Diphenylamine 3,4 pKa = 0,79 44 Naphthalene 3,6 45 Phenylbenzoate 3,6 
46 Isopropylbenzene 3,7 
47 2,4,6-Trichlorophenol 3,7 pKa = 6 48 Biphenyl 4,0 49 Benzylbenzoate 4,0 50 2,4-Dinitro-6 sec. butyophenol 4,1 51 1,2,4-Trichlorobenzene 4,2 52 Dodecanoic acid 4,2 53 Diphenylether 4,2 54 n-Butylbenzene 4,5 55 Phenanthrene 4,5 56 Fluoranthene 4,7 57 Dibenzyl 4,8 
58 2,6-Diphenylpyridine 4,9 
59 Triphenylamine 5,7 60 DDT 6,2 
Other reference substances of low log P
 ow 
1 Nicotinic acid - 0,07  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 82
 
A.9. FLASH-POINT  
1. METHOD  
1.1. INTRODUCTION 
It is useful to have preliminary information on the flammability of 
the substance before performing this test. The test procedure is 
applicable to liquid substances whose vapours can be ignited by ignition sources. The test methods listed in this text are only reliable for flash-point ranges which are specified in the individual 
methods. 
The possibility of chemical reactions between the substance and the 
sample holder should be considered when selecting the method to be 
used.
 
1.2. DEFINITIONS AND UNITS 
The flash-point is the lowest temperature, corrected to a pressure of 
101,325 kPa, at which a liquid evolves vapours, under the conditions 
defined in the test method, in such an amount that a flammable 
vapour/air mixture is produced in the test vessel. 
Units:  o C 
t = T - 273,15 
(t in  o C and T in K)  
1.3. REFERENCE SUBSTANCES 
Reference substances do not need to be employed in all cases when 
investigating a new substance. They should primarily serve to check 
the performance of the method from time to time and to allow 
comparison with results from other methods.  
1.4. PRINCIPLE OF THE METHOD 
The substance is placed in a test vessel and heated or cooled to the 
test temperature according to the procedure described in the indi ­
vidual test method. Ignition trials are carried out in order to ascertain 
whether or not the sample flashed at the test temperature.  
1.5. QUALITY CRITERIA  
1.5.1.  Repeatability  
The repeatability varies according to flash-point range and the test 
method used; maximum 2  o C. 
1.5.2.  Sensitivity  
The sensitivity depends on the test method used.  
1.5.3.  Specificity  
The specificity of some test methods is limited to certain flash-point 
ranges and subject to substance-related data (e.g. high viscosity).  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 83
 
1.6. DESCRIPTION OF THE METHOD  
1.6.1.  Preparations  
A sample of the test substance is placed in a test apparatus according 
to 1.6.3.1 and/or 1.6.3.2. 
For safety, it is recommended that a method utilising a small sample 
size, circa 2 cm  3 , be used for energetic or toxic substances.  
1.6.2.  Test conditions  
The apparatus should, as far as is consistent with safety, be placed in 
a draught-free position.  
1.6.3.  Performance of the test  
1.6.3.1.  Equilibrium method  
See ISO 1516, ISO 3680, ISO 1523, ISO 3679.  
1.6.3.2.  Non-equilibrium method  
Abel apparatus:  
See BS 2000 part 170, NF M07-011, NF T66-009.  
Abel-Pensky apparatus:  
See EN 57, DIN 51755 part 1 (for temperatures from 5 to 65  o C), 
DIN 51755 part 2 (for temperatures below 5  o C), NF M07-036.  
Tag apparatus:  
See ASTM D 56.  
Pensky-Martens apparatus:  
See ISO 2719, EN 11, DIN 51758, ASTM D 93, BS 2000-34, NF 
M07-019.  
Remarks:  
When the flash-point, determined by a non-equilibrium method in 
1.6.3.2, is found to be 0 ± 2  o C, 21 ± 2  o C or 55 ± 2  o C, it should be 
confirmed by an equilibrium method using the same apparatus. 
Only the methods which can give the temperature of the flash-point 
may be used for a notification. 
To determine the flash-point of viscous liquids (paints, gums and 
similar) containing solvents, only apparatus and test methods suitable for determining the flash-point of viscous liquids may be used. 
See ISO 3679, ISO 3680, ISO 1523, DIN 53213 part 1.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 84
 
2. DATA  
3. REPORTING  
The test report shall, if possible, include the following information: 
— the precise specification of the substance (identification and 
impurities), 
— the method used should be stated as well as any possible devi ­
ations, 
— the results and any additional remarks relevant for the interpre ­
tation of results.  
4. REFERENCES  
None.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 85
 
A.10.  FLAMMABILITY (SOLIDS)  
1. METHOD  
1.1. INTRODUCTION 
It is useful to have preliminary information on potentially explosive 
properties of the substance before performing this test. 
This test should only be applied to powdery, granular or paste-like 
substances. 
In order not to include all substances which can be ignited but only 
those which burn rapidly or those whose burning behaviour is in any way especially dangerous, only substances whose burning velocity exceeds a certain limiting value are considered to be highly flam ­
mable. 
It can be especially dangerous if incandescence propagates through a 
metal powder because of the difficulties in extinguishing a fire. Metal powders should be considered highly flammable if they 
support spread of incandescence throughout the mass within a 
specified time.
 
1.2. DEFINITION AND UNITS 
Burning time expressed in seconds.  
1.3. REFERENCE SUBSTANCES 
Not specified.  
1.4. PRINCIPLE OF THE METHOD 
The substance is formed into an unbroken strip or powder train about 
250 mm long and a preliminary screening test performed to determine if, on ignition by a gas flame, propagation by burning with flame or smouldering occurs. If propagation over 200 mm of 
the train occurs within a specified time then a full test programme to 
determine the burning rate is carried out.
 
1.5. QUALITY CRITERIA 
Not stated.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 86
 
1.6. DESCRIPTION OF METHOD  
1.6.1.  Preliminary screening test  
The substance is formed into an unbroken strip or powder train about 
250 mm long by 20 mm wide by 10 mm high on a non-combustible, 
non-porous and low heat-conducting base plate. A hot flame from a 
gas burner (minimum diameter 5 mm) is applied to one end of the powder train until the powder ignites or for a maximum of two minutes (five minutes for powders of metals or metal-alloys). It 
should be noted whether combustion propagates along 200 mm of 
the train within the 4 minutes test period (or 40 minutes for metal powders). If the substance does not ignite and propagate combustion 
either by burning with flame or smouldering along 200 mm of the 
powder train within the four minutes (or 40 minutes) test period, then the substance should not be considered as highly flammable and no further testing is required. If the substance propagates burning of 
a 200 mm length of the powder train in less than four minutes, or 
less than 40 minutes for metal powders, the procedure described below (point 1.6.2. and following) should be carried out.
 
1.6.2.  Burning rate test  
1.6.2.1.  Preparation  
Powdery or granular substances are loosely filled into a mould 
250 mm long with a triangular cross-section of inner height 
10 mm and width 20 mm. On both sides of the mould in a longi ­
tudinal direction two metal plates are mounted as lateral limitations which project 2 mm beyond the upper edge of the triangular cross section (figure). The mould is then dropped three times from a height 
of 2 cm onto a solid surface. If necessary the mould is then filled up 
again. The lateral limitations are then removed and the excess substance scraped off. A non-combustible, non-porous and low heat-conducting base plate is placed on top of the mould, the 
apparatus inverted and the mould removed. 
Paste-like substances are spread on a non-combustible, non-porous 
and low heat-conducting base plate in the form of a rope 250 mm in length with a cross section of about 1 cm
 2 . 
1.6.2.2.  Test conditions  
In the case a moisture-sensitive substance, the test should be carried 
out as quickly as possible after its removal from the container.  
1.6.2.3.  Performance of the test  
Arrange the pile across the draught in a fume cupboard. 
The air-speed should be sufficient to prevent fumes escaping into the 
laboratory and should not be varied during the test. A draught screen 
should be erected around the apparatus. 
A hot flame from a gas burner (minimum diameter of 5 mm) is used 
to ignite the pile at one end. When the pile has burned a distance of 
80 mm, the rate of burning over the next 100 mm is measured.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 87
 
The test is performed six times, using a clean cool plate each time, 
unless a positive result is observed earlier.  
2. DATA  
The burning time from the preliminary screening test (1.6.1) and the 
shortest burning time in up to six tests (1.6.2.3) are relevant for 
evaluation.  
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, include the following information: 
— the precise specification of the substance (identification and 
impurities), 
— a description of the substance to be tested, its physical state 
including moisture content, 
— results from the preliminary screening test and from the burning 
rate test if performed, 
— all additional remarks relevant to the interpretation of results.  
3.2. INTERPRETATION OF THE RESULT 
Powdery, granular or paste-1ike substances are to be considered as 
highly flammable when the time of burning in any tests carried out 
according to the test procedure described in 1.6.2 is less than 45 seconds. Powders of metals or metal-alloys are considered to be highly flammable when they can be ignited and the flame or 
the zone of reaction spreads over the whole sample in 10 minutes 
or less.
 
4. REFERENCES  
NF T 20-042 (September 85) Chemical products for industrial use. 
Determination of the flammability of solids.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 88
 
Appendix  
Figure  
Mould and accessories for the preparation of the pile  
(All dimensions in millimetres)
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 89
 
A.11.  FLAMMABILITY (GASES)  
1. METHOD  
1.1. INTRODUCTION 
This method allows a determination of whether gases mixed with air 
at room temperature (circa 20  o C) and atmospheric pressure are 
flammable and, if so, over what range of concentrations. Mixtures of increasing concentrations of the test gas with air are exposed to an electrical spark and it is observed whether ignition occurs.
 
1.2. DEFINITION AND UNITS 
The range of flammability is the range of concentration between the 
lower and the upper explosion limits. The lower and the upper explosion limits are those limits of concentration of the flammable gas in admixture with air at which propagation of a flame does not 
occur.
 
1.3. REFERENCE SUBSTANCES 
Not specified.  
1.4. PRINCIPLE OF THE METHOD 
The concentration of gas in air is increased step by step and the 
mixture is exposed at each stage to an electrical spark.  
1.5. QUALITY CRITERIA 
Not stated.  
1.6. DESCRIPTION OF THE METHOD  
1.6.1.  Apparatus  
The test vessel is an upright glass cylinder having a minimum inner 
diameter of 50 mm and a minimum height of 300 mm. The ignition 
electrodes are separated by a distance of 3 to 5 mm and are placed 
60 mm above the bottom of the cylinder. The cylinder is fitted with a pressure-release opening. The apparatus has to be shielded to restrict any explosion damage. 
A standing induction spark of 0,5 sec. duration, which is generated 
from a high voltage transformer with an output voltage of 10 to 15 kV (maximum of power input 300 W), is used as the 
ignition source. An example of a suitable apparatus is described in 
reference (2).
 
1.6.2.  Test conditions  
The test must be performed at room temperature (circa 20  o C). ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 90
 
1.6.3.  Performance of the test  
Using proportioning pumps, a known concentration of gas in air is 
introduced into the glass cylinder. A spark is passed through the mixture and it is observed whether or not a flame detaches itself 
from the ignition source and propagates independently. The gas 
concentration is varied in steps of 1 % vol. until ignition occurs as described above. 
If the chemical structure of the gas indicates that it would be non- 
flammable and the composition of the stoichiometric mixture with air 
can be calculated, then only mixtures in the range from 10 % less than the stoichiometric composition to 10 % greater than this composition need be tested in 1 % steps.
 
2. DATA  
The occurrence of flame propagation is the only relevant information 
data for the determination of this property.  
3. REPORTING  
The test report shall, if possible, include the following information: 
— the precise specification of the substance (identification and 
impurities), 
— a description, with dimensions, of the apparatus used, — the temperature at which the test was performed, — the tested concentrations and the results obtained, — the result of the test: non-flammable gas or highly flammable 
gas, 
— if it is concluded that the gas is non-flammable then the concen ­
tration range over which it was tested in 1 % steps should be stated, 
— all information and remarks relevant to the interpretation of 
results have to be reported.
 
4. REFERENCES  
(1) NF T 20-041 (September 85) Chemical products for industrial 
use. Determination of the flammability of gases. 
(2) W. Berthold, D. Conrad, T. Grewer, H. Grosse-Wortmann ‘Ent ­
wicklung einer Standard-Apparatur zur Messung von Explo ­
sionsgrenzen’. Chem.-Ing.- Tech. 1984, vo1. 56, 2, 126-127., T. Redeker und H. Schacke, p. 126-127.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 91
 
A.12.  FLAMMABILITY (CONTACT WITH WATER)  
1. METHOD  
1.1. INTRODUCTION 
This test method can be used to determine whether the reaction of a 
substance with water or damp air leads to the development of 
dangerous amounts of gas or gases which may be highly flammable. 
The test method can be applied to both solid and liquid substances. 
This method is not applicable to substances which spontaneously ignite when in contact with air.
 
1.2. DEFINITIONS AND UNITS 
Highly flammable: substances which, in contact with water or damp 
air, evolve highly flammable gases in dangerous quantities at a 
minimum rate of 1 litre/kg per hour.  
1.3. PRINCIPLE OF THE METHOD 
The substance is tested according to the step by step sequence 
described below; if ignition occurs at any step, no further testing 
is necessary. If it is known that the substance does not react violently with water then proceed to step 4 (1.3.4).
 
1.3.1.  Step 1  
The test substance is placed in a trough containing distilled water at 
20 o C and it is noted whether or not the evolved gas ignites.  
1.3.2.  Step 2  
The test substance is placed on a filter paper floating on the surface 
of a dish containing distilled water at 20  o C and it is noted whether 
or not the evolved gas ignites. The filter paper is merely to keep the 
substance in one place to increase the chances of ignition.  
1.3.3.  Step 3  
The test substance is made into a pile approximately 2 cm high and 
3 cm diameter. A few drops of water are added to the pile and it is 
noted whether or not the evolved gas ignites.  
1.3.4.  Step 4  
The test substance is mixed with distilled water at 20  o C and the rate 
of evolution of gas is measured over a period of seven hours, at one- hour intervals. If the rate of evolution is erratic, or is increasing, after seven hours, the measuring time should be extended to a maximum 
time of five days. The test may be stopped if the rate at any time 
exceeds 1 litre/kg per hour.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 92
 
1.4. REFERENCE SUBSTANCES 
Not specified.  
1.5. QUALITY CR1TERIA 
Not stated.  
1.6. DESCRIPTION OF METHODS  
1.6.1.  Step 1  
1.6.1.1.  Test conditions  
The test is performed at room temperature (circa 20  o C). 
1.6.1.2.  Performance of the test  
A small quantity (approximately 2 mm diameter) of the test 
substance should be placed in a trough containing distilled water. 
A note should be made of whether (i) any gas is evolved and (ii) if 
ignition of the gas occurs. If ignition of the gas occurs then no further testing of the substance is needed because the substance is regarded as hazardous.
 
1.6.2.  Step 2  
1.6.2.1.  Apparatus  
A filter-paper is floated flat on the surface of distilled water in any 
suitable vessel, e.g. a 100 mm diameter evaporating dish.  
1.6.2.2.  Test conditions  
The test is performed at room temperature (circa 20  o C). 
1.6.2.3.  Performance of the test  
A small quantity of the test substance (approximately 2 mm 
diameter) is placed onto the centre of the filter-paper. A note should be made of whether (i) any gas is evolved and (ii) if ignition of the gas occurs. If ignition of the gas occurs then no 
further testing of the substance is needed because the substance is 
regarded as hazardous.
 
1.6.3.  Step 3  
1.6.3.1.  Test conditions  
The test is performed at room temperature (circa 20  o C). 
1.6.3.2.  Performance of the test  
The test substance is made into a pile approximately 2 cm high and 
3 cm diameter with an indentation in the top. A few drops of water are added to the hollow and a note is made of whether (i) any gas is evolved and (ii) if ignition of the gas occurs. If ignition of the gas 
occurs then no further testing of the substance is needed because the 
substance is regarded as hazardous.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 93
 
1.6.4.  Step 4  
1.6.4.1.  Apparatus  
The apparatus is set up as shown in the figure.  
1.6.4.2.  Test conditions  
Inspect the container of the test substance for any powder < 500 μm 
(particle size). If the powder constitutes more than 1 % w/w of the 
total, or if the sample is friable, then the whole of the substance 
should be ground to a powder before testing to allow for a reduction in particle size during storage and handling; otherwise the substance is to be tested as received. The test should be performed at room 
temperature (circa 20
 o C) and atmospheric pressure.  
1.6.4.3.  Performance of the test  
10 to 20 ml of water are put into the dropping funnel of the 
apparatus and 10 g of substance are put in the conical flask. The volume of gas evolved can be measured by any suitable means. The tap of the dropping funnel is opened to let the water into the conical 
flask and a stop watch is started. The gas evolution is measured 
each hour during a seven hour period. If, during this period, the gas evolution is erratic, or if, at the end of this period, the rate of gas evolution is increasing, then measurements should be continued 
for up to five days. If, at any time of measurement, the rate of gas 
evolution exceeds 1 litre/kg per hour, the test can be discontinued. This test should be performed in triplicate. 
If the chemical identity of the gas is unknown, the gas should be 
analysed. When the gas contains highly flammable components and 
it is unknown whether the whole mixture is highly flammable, a 
mixture of the same composition has to be prepared and tested according to the method A.11.
 
2. DATA  
The substance is considered hazardous if: 
— spontaneous ignition takes place in any step of the test procedure, 
or 
— there is evolution of flammable gas at a rate greater than 1 
litre/kg of the substance per hour.  
3. REPORTING  
The test report shall, if possible, include the following information: 
— the precise specification of the substance (identification and 
impurities), 
— details of any initial preparation of the test substance,  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 94
 
— the results of the tests (steps 1, 2, 3 and 4), 
— the chemical identity of gas evolved, — the rate of evolution of gas if step 4 (1.6.4) is performed, 
— any additional remarks relevant to the interpretation of the 
results.
 
4. REFERENCES  
(1) Recommendations on the transport of dangerous goods, test and 
criteria, 1990, United Nations, New York. 
(2) NF T 20-040 (September 85) Chemical products for industrial 
use. Determination of the flammability of gases formed by the 
hydrolysis of solid and liquid products.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 95
 
Appendix  
Figure  
Apparatus
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 96
 
A.13.  PYROPHORIC PROPERTIES OF SOLIDS AND LIQUIDS  
1. METHOD  
1.1. INTRODUCTION 
The test procedure is applicable to solid or liquid substances, which, 
in small amounts, will ignite spontaneously a short time after coming 
into contact with air at room temperature (circa 20  o C). 
Substances which need to be exposed to air for hours or days at 
room temperature or at elevated temperatures before ignition occurs 
are not covered by this test method.  
1.2. DEFINITIONS AND UNITS 
Substances are considered to have pyrophoric properties if they 
ignite or cause charring under the conditions described in 1.6. 
The auto-flammability of liquids may also need to be tested using 
method A.15. Auto-ignition temperature (liquids and gases).  
1.3. REFERENCE SUBSTANCES 
Not specified.  
1.4. PRINCIPLE OF THE METHOD 
The substance, whether solid or liquid, is added to an inert carrier 
and brought into contact with air at ambient temperature for a period of five minutes. If liquid substances do not ignite then they are 
absorbed onto filter paper and exposed to air at ambient temperature 
(circa 20
 o C) for five minutes. If a solid or liquid ignites, or a liquid 
ignites or chars a filter paper, then the substance is considered to be pyrophoric.
 
1.5. QUALITY CRITERIA 
Repeatability: because of the importance in relation to safety, a 
single positive result is sufficient for the substance to be considered 
pyrophoric.  
1.6. DESCRIPTION OF THE TEST METHOD  
1.6.1.  Apparatus  
A porcelain cup of circa 10 cm diameter is filled with diatomaceous 
earth to a height of about 5 mm at room temperature (circa 20  o C). 
Note: Diatomaceous earth or any other comparable inert substance which is 
generally obtainable shall be taken as representative of soil onto which the test substance might be spilled in the event of an accident. 
Dry filter paper is required for testing liquids which do not ignite on 
contact with air when in contact with an inert carrier.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 97
 
1.6.2.  Performance of the test  
(a) Powdery solids  
1 to 2 cm  3 of the substance to be tested is poured from circa 1 m 
height onto a non-combustible surface and it is observed whether the 
substance ignites during dropping or within five minutes of settling. 
The test is performed six times unless ignition occurs;  
(b) liquids  
Circa 5 cm  3 of the liquid to be tested is poured into the prepared 
porcelain cup and it is observed whether the substance ignites within five minutes. 
If no ignition occurs in the six tests, perform the following tests: A 0,5 ml test sample is delivered from a syringe to an indented filter 
paper and it is observed whether ignition or charring of the filter 
paper occurs within five minutes of the liquid being added. The test is performed three times unless ignition or charring occurs.
 
2. DATA  
2.1. TREATMENT OF RESULTS 
Testing can be discontinued as soon as a positive result occurs in 
any of the tests.  
2.2. EVALUATION 
If the substance ignites within five minutes when added to an inert 
carrier and exposed to air, or a liquid substance chars or ignites a filter paper within five minutes when added and exposed to air, it is considered to be pyrophoric.
 
3. REPORTING  
The test report shall, if possible, include the following information: 
— the precise specification of the substance (identification and 
impurities), 
— the results of the tests, — any additional remark relevant to the interpretation of the results.
 
4. REFERENCES  
(1) NF T 20-039 (September 85) Chemical products for industrial 
use. Determination of the spontaneous flammability of solids and liquids. 
(2) Recommendations on the Transport of Dangerous Goods, Test 
and criteria, 1990, United Nations, New York.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 98
 
A.14.  EXPLOSIVE PROPERTIES  
1. METHOD  
1.1. INTRODUCTION 
The method provides a scheme of testing to determine whether a 
solid or a pasty substance presents a danger of explosion when 
submitted to the effect of a flame (thermal sensitivity), or to shock or friction (sensitivity to mechanical stimuli), and whether a liquid substance presents a danger of explosion when submitted to the 
effect of a flame or shock. 
The method comprises three parts: 
(a) a test of thermal sensitivity (1); 
(b) a test of mechanical sensitivity with respect to shock (1); (c) a test of mechanical sensitivity with respect to friction (1). The method yields data to assess the likelihood of initiating an 
explosion by means of certain common stimuli. The method is not intended to ascertain whether a substance is capable of exploding 
under any conditions. 
The method is appropriate for determining whether a substance will 
present a danger of explosion (thermal and mechanical sensitivity) under the particular conditions specified in the directive. It is based on a number of types of apparatus which are widely used inter ­
nationally (1) and which usually give meaningful results. It is 
recognised that the method is not definitive. Alternative apparatus to that specified may be used provided that it is internationally recognised and the results can be adequately correlated with those 
from the specified apparatus. 
The tests need not be performed when available thermodynamic 
information (e.g. heat of formation, heat of decomposition) and/or absence of certain reactive groups (2) in the structural formula estab ­
lishes beyond reasonable doubt that the substance is incapable of 
rapid decomposition with evolution of gases or release of heat (i.e. 
the material does not present any risk of explosion). A test of mech ­
anical sensitivity with respect to friction is not required for liquids.
 
1.2. DEFINITIONS AND UNITS 
Explosive: 
Substances which may explode under the effect of flame or which 
are sensitive to shock or friction in the specified apparatus (or are 
more mechanically sensitive than 1,3-dinitrobenzene in alternative 
apparatus).  
1.3. REFERENCE SUBSTANCES 
1,3-dinitrobenzene, technical crystalline product sieved to pass 
0,5 mm, for the friction and shock methods. 
Perhydro-1,3,5-trinitro-1,3,5-triazine (RDX, hexogen, cyclonite — 
CAS 121-82-4), recrystallised from aqueous cyclohexanone, wet- sieved through a 250 μm and retained on a 150 μm sieve and 
dried at 103 ± 2
 o C (for four hours) for the second series of 
friction and shock tests.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 99
 
1.4. PRINCIPLE OF THE METHOD 
Preliminary tests are necessary to establish safe conditions for the 
performance of the three tests of sensitivity.  
1.4.1.  Safety-in-handling tests (3)  
For safety reasons, before performing the main tests, very small 
samples (circa 10 mg) of the substance are subjected to heating without confinement in a gas flame, to shock in any convenient 
form of apparatus and to friction by the use of a mallet against an 
anvil or any form of friction machine. The objective is to ascertain if the substance is so sensitive and explosive that the prescribed sensi ­
tivity tests, particularly that of thermal sensitivity, should be 
performed with special precautions so as to avoid injury to the 
operator.
 
1.4.2.  Thermal sensitivity  
The method involves heating the substance in a steel tube, closed by 
orifice plates with differing diameters of hole, to determine whether the substance is liable to explode under conditions of intense heat and defined confinement.
 
1.4.3.  Mechanical sensitivity (shock)  
The method involves subjecting the substance to the shock from a 
specified mass dropped from a specified height.  
1.4.4.  Mechanical sensitivity (friction)  
The method involves subjecting solid or pasty substances to friction 
between standard surfaces under specified conditions of load and relative motion.
 
1.5. QUALITY CRITERIA 
Not stated.  
1.6. DESCRIPTION OF METHOD  
1.6.1.  Thermal sensitivity (effect of a flame)  
1.6.1.1.  Apparatus  
The apparatus consists of a non-reusable steel tube with its re-usable 
closing device (figure 1), installed in a heating and protective device. 
Each tube is deep-drawn from sheet steel (see Appendix) and has an 
internal diameter of 24 mm, a length of 75 mm and wall thickness of 0,5 mm. The tubes are flanged at the open end to enable them to be closed by the orifice plate assembly. This consists of a pressure- 
resistant orifice plate, with a central hole, secured firmly to a tube 
using a two-part screw joint (nut and threaded collar). The nut and threaded collar are made from chromium-manganese steel (see Appendix) which is spark-free up to 800
 o C. The orifice plates are 
6 mm thick, made from heat-resistant steel (see Appendix), and are 
available with a range of diameters of opening.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 100
 
1.6.1.2.  Test conditions  
Normally the substance is tested as received although in certain 
cases, e.g. if pressed, cast or otherwise condensed, it may be necessary to test the substance after crushing. 
For solids, the mass of material to be used in each test is determined 
using a two-stage dry run procedure. A tared tube is filled with 9 cm
 3 of substance and the substance tamped with 80 N force 
applied to the total cross-section of the tube. For reasons of safety 
or in cases where the physical form of the sample can be changed by 
compression other filling procedures may be used; e.g. if the substance is very friction sensitive then tamping is not appropriate. If the material is compressible then more is added and tamped until 
the tube is filled to 55 mm from the top. The total mass used to fill 
the tube to the 55 mm level is determined and two further incre ­
ments, each tamped with 80 N force, are added. Material is then either added with tamping, or taken out, as required, to leave the 
tube filled to a level 15 mm from the top. A second dry run is 
performed, starting with a tamped quantity of a third of the total mass found in the first dry run. Two more of these increments are added with 80 N tamping and the level of the substance in the tube 
adjusted to 15 mm from the top by addition or subtraction of 
material as required. The amount of solid determined in the second dry run is used for each trial; filling being performed in three equal amounts, each compressed to 9 cm
 3 by whatever force 
is necessary. (This may be facilitated by the use of spacing rings). 
Liquids and gels are loaded into the tube to a height of 60 mm 
taking particular care with gels to prevent the formation of voids. The threaded collar is slipped onto the tube from below, the appro ­
priate orifice plate is inserted and the nut tightened after applying 
some molybdenum disulphide based lubricant. It is essential to check 
that none of the substance is trapped between the flange and the plate, or in the threads. 
Heating is provided by propane taken from an industrial cylinder, 
fitted with a pressure regulator (60 to 70 mbar), through a meter and 
evenly distributed (as indicated by visual observation of the flames from the burners) by a manifold to four burners. The burners are located around the test chamber as shown in figure 1. The four 
burners have a combined consumption of about 3,2 litres of 
propane per minute. Alternative fuel gases and burners may be used but the heating rate must be as specified in figure 3. For all apparatus, the heating rate must be checked periodically using tubes 
filled with dibutyl phthalate as indicated in figure 3.
 
1.6.1.3.  Performance of the tests  
Each test is performed until either the tube is fragmented or the tube 
has been heated for five minutes. A test resulting in the fragmen ­
tation of the tube into three or more pieces, which in some cases may be connected to each other by narrow strips of metal as illus ­
trated in figure 2, is evaluated as giving an explosion. A test 
resulting in fewer fragments or no fragmentation is regarded as not 
giving an explosion.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 101
 
A series of three tests with a 6,0 mm diameter orifice plate is first 
performed and, if no explosions are obtained, a second series of 
three tests is performed with a 2,0 mm diameter orifice plate. If an explosion occurs during either test series then no further tests are required.
 
1.6.1.4.  Evaluation  
The test result is considered positive if an explosion occurs in either 
of the above series of tests.  
1.6.2.  Mechanical sensitivity (shock)  
1.6.2.1.  Apparatus (figure 4)  
The essential parts of a typical fall hammer apparatus are a cast steel 
block with base, anvil, column, guides, drop weights, release device and a sample holder. The steel anvil 100 mm (diameter) × 70 mm (height) is screwed to the top of a steel block 230 mm (length) × 
250 mm (width) × 200 mm (height) with a cast base 450 mm 
(length) × 450 mm (width) × 60 mm (height). A column, made from seamless drawn steel tube, is secured in a holder screwed on to the back of the steel block. Four screws anchor the apparatus to a 
solid concrete block 60 × 60 × 60 cm such that the guide rails are 
absolutely vertical and the drop weight falls freely. 5 and 10 kg weights, made from solid steel, are available for use. The striking head of each weight is of hardened steel, HRC 60 to 63, and has a 
minimum diameter of 25 mm. 
The sample under test is enclosed in a shock device consisting of 
two coaxial solid steel cylinders, one above the other, in a hollow 
cylindrical steel guide ring. The solid steel cylinders should be of 10 (- 0,003, - 0,005) mm diameter and 10 mm height and have polished surfaces, rounded edges (radius of curvature 0,5 mm) and a hardness 
of HRC 58 to 65. The hollow cylinder must have an external 
diameter of 16 mm, a polished bore of 10 (+ 0,005, + 0,010) mm and a height of 13 mm. The shock device is assembled on an inter ­
mediate anvil (26 mm diameter and 26 mm height) made of steel and 
centred by a ring with perforations to allow escape of fumes.
 
1.6.2.2.  Test conditions  
The sample volume should be 40 mm  3 , or a volume to suit any 
alternative apparatus. Solid substances should be tested in the dry 
state and prepared as follows: 
(a) powdered substances are sieved (sieve size 0,5 mm); all that has 
passed through the sieve is used for testing; 
(b) pressed, cast or otherwise condensed substances are broken into 
small pieces and sieved; the sieve fraction from 0,5 to 1 mm diameter is used for testing and should be representative of the 
original substance. 
Substances normally supplied as pastes should be tested in the dry 
state where possible or, in any case, following removal of the 
maximum possible amount of diluent. Liquid substances are tested 
with a 1 mm gap between the upper and lower steel cylinders.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 102
 
1.6.2.3.  Performance of the tests  
A series of six tests are performed dropping the 10 kg mass from 
0,40 m (40 J). If an explosion is obtained during the six tests at 40 J, a further series of six tests, dropping a 5 kg mass from 0,15 m 
(7,5 J), must be performed. In other apparatus, the sample is 
compared with the chosen reference substance using an established procedure (e.g. up-and-down technique etc.).
 
1.6.2.4.  Evaluation  
The test result is considered positive if an explosion (bursting into 
flame and/or a report is equivalent to explosion) occurs at least once 
in any of the tests with the specified shock apparatus or the sample is 
more sensitive than 1,3-dinitrobenzene or RDX in an alternative shock test.
 
1.6.3.  Mechanical sensitivity (friction)  
1.6.3.1.  Apparatus (figure 5)  
The friction apparatus consists of a cast steel base plate on which is 
mounted the friction device. This consists of a fixed porcelain peg 
and moving porcelain plate. The porcelain plate is held in a carriage which runs in two guides. The carriage is connected to an electric motor via a connecting rod, an eccentric cam and suitable gearing 
such that the porcelain plate is moved, once only, back and forth 
beneath the porcelain peg for a distance of 10 mm. The porcelain peg may be loaded with, for example, 120 or 360 newtons. 
The flat porcelain plates are made from white technical porcelain 
(roughness 9 to 32 μm) and have the dimensions 25 mm (length) 
× 25 mm (width) × 5 mm (height). The cylindrical porcelain peg is also made of white technical porcelain and is 15 mm long, has a diameter of 10 mm and roughened spherical end surfaces with a 
radius of curvature of 10 mm.
 
1.6.3.2.  Test conditions  
The sample volume should be 10 mm  3 or a volume to suit any 
alternative apparatus. 
Solid substances are tested in the dry state and prepared as follows: 
(a) powdered substances are sieved (sieve size 0,5 mm); all that has 
passed through the sieve is used for testing; 
(b) pressed, cast or otherwise condensed substances are broken into 
small pieces and sieved; the sieve fraction < 0,5 mm diameter is 
used for testing. 
Substances normally supplied as pastes should be tested in the dry 
state where possible. If the substance cannot be prepared in the dry state, the paste (following removal of the maximum possible amount 
of diluent) is tested as a 0,5 mm thick, 2 mm wide, 10 mm long 
film, prepared with a former.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 103
 
1.6.3.3.  Performance of the tests  
The porcelain peg is brought onto the sample under test and the load 
applied. When carrying out the test, the sponge marks of the porcelain plate must lie transversely to the direction of the 
movement. Care must be taken that the peg rests on the sample, 
that sufficient test material lies under the peg and also that the plate moves correctly under the peg. For pasty substances, a 0,5 mm thick gauge with a 2 × 10 mm slot is used to apply the 
substance to the plate. The porcelain plate has to move 10 mm 
forwards and backwards under the porcelain peg in a time of 0,44 seconds. Each part of the surface of the plate and peg must 
only be used once; the two ends of each peg will serve for two trials 
and the two surfaces of a plate will each serve for three trials. 
A series of six tests are performed with a 360 N loading. If a 
positive event is obtained during these six tests, a further series of 
six tests must be performed with a 120 N loading. In other apparatus, 
the sample is compared with the chosen reference substance using an established procedure (e.g. up-and-down technique, etc.).
 
1.6.3.4.  Evaluation  
The test result is considered positive if an explosion (crepitation 
and/or a report or bursting into flame are equivalent to explosion) 
occurs at least once in any of the tests with the specified friction 
apparatus or satisfies the equivalent criteria in an alternative friction test.
 
2. DATA  
In principle, a substance is considered to present a danger of 
explosion in the sense of the directive if a positive result is 
obtained in the thermal, shock or friction sensitivity test.  
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, include the following information: 
— identity, composition, purity, moisture content, etc. of the 
substance tested, 
— the physical form of the sample and whether or not it has been 
crushed, broken and/or sieved, 
— observations during the thermal sensitivity tests (e.g. sample 
mass, number of fragments, etc.), 
— observations during the mechanical sensitivity tests (e.g. 
formation of considerable amounts of smoke or complete decom ­
position without a report, flames, sparks, report, crepitation, etc.), 
— results of each type of test, — if alternative apparatus has been used, scientific justification as 
well as evidence of correlation between results obtained with 
specified apparatus and those obtained with equivalent 
apparatus must be given,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 104
 
— any useful comments such as reference to tests with similar 
products which might be relevant to a proper interpretation of 
the results, 
— all additional remarks relevant for the interpretation of the results.  
3.2. INTERPRETATION AND EVALUATION OF RESULTS 
The test report should mention any results which are considered 
false, anomalous or unrepresentative. If any of the results should be discounted, an explanation and the results of any alternative or 
supplementary testing should be given. Unless an anomalous result 
can be explained, it must be accepted at face value and used to classify the substance accordingly.
 
4. REFERENCES  
(1) Recommendations on the Transport of Dangerous Goods: Tests 
and criteria, 1990, United Nations, New York. 
(2) Bretherick, L., Handbook of Reactive Chemical Hazards, 4th 
edition, Butterworths, London, ISBN 0-750-60103-5, 1990. 
(3) Koenen, H., Ide, K.H. and Swart, K.H., Explosivstoffe, 1961, 
vol. 3, 6-13 and 30-42. 
(4) NF T 20-038 (September 85) Chemical products for industrial 
use — Determination of explosion risk.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 105
 
Appendix  
Example of material specification for thermal sensitivity test (see DIN 1623)  
(1) Tube: Material specification No 1.0336.505 g 
(2) Orifice plate: Material specification No 1.4873 (3) Threaded collar and nut: Material specification No 1.3817
 
Figure 1  
Thermal sensitivity test apparatus  
(all dimensions in millimetres)
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 106
 
Figure 2  
Thermal sensitivity test  
(example of fragmentation)
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 107
 
Figure 3  
Heating rate calibration for thermal sensitivity test
  
Temperature/time curve obtained on heating dibutyl phtalate (27 cm  3 ) in a closed (1,5 mm 
orifice plate) tube using a propane flow rate of 3,2 litre/minute. The temperature is measured 
with a 1 mm diameter stainless steel sheathed chromel/alumel thermocouple, placed centrally 
43 mm below the rim of the tube. The heating rate between 135  o C and 285  o C should be 
between 185 and 215 K/minute.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 108
 
Figure 4  
Shock test apparatus 
(all dimensions in millimetres)
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 109
 
Figure 4  
Continued
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 110
 
Figure 5  
Friction sensitivity apparatus
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 111
 
A.15.  AUTO-IGNITION TEMPERATURE (LIQUIDS AND GASES)  
1. METHOD  
1.1. INTRODUCTION 
Explosive substances and substances which ignite spontaneously in 
contact with air at ambient temperature should not be submitted to 
this test. The test procedure is applicable to gases, liquids and vapours which, in the presence of air, can be ignited by a hot surface. 
The auto-ignition temperature can be considerably reduced by the 
presence of catalytic impurities, by the surface material or by a 
higher volume of the test vessel.
 
1.2. DEFINITIONS AND UNITS 
The degree of auto-ignitability is expressed in terms of the auto- 
ignition temperature. The auto-ignition temperature is the lowest 
temperature at which the test substance will ignite when mixed 
with air under the conditions defined in the test method.  
1.3. REFERENCE SUBSTANCES 
Reference substances are cited in the standards (see 1.6.3). They 
should primarily serve to check the performance of the method 
from time to time and to allow comparison with results from other 
methods.  
1.4. PRINCIPLE OF THE METHOD 
The method determines the minimum temperature of the inner 
surface of an enclosure that will result in ignition of a gas, vapour 
or liquid injected into the enclosure.  
1.5. QUALITY CRITERIA 
The repeatability varies according to the range of auto-ignition 
temperatures and the test method used. 
The sensitivity and specificity depend on the test method used.  
1.6. DESCRIPTION OF THE METHOD  
1.6.1.  Apparatus  
The apparatus is described in the method referred to in 1.6.3.  
1.6.2.  Test conditions  
A sample of the test substance is tested according to the method 
referred to in 1.6.3.  
1.6.3.  Performance of the test  
See IEC 79-4, DIN 51794, ASTM-E 659-78, BS 4056, NF T 20-037.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 112
 
2. DATA  
Record the test-temperature, atmospheric pressure, quantity of 
sample used and time-1ag until ignition occurs.  
3. REPORTING  
The test report shall, if possible, include the following information: 
— the precise specification of the substance (identification and 
impurities), 
— the quantity of sample used, atmospheric pressure, — the apparatus used, — the results of measurements (test temperatures, results concerning 
ignition, corresponding time-lags), 
— all additional remarks relevant to the interpretation of results.
 
4. REFERENCES  
None.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 113
 
A.16.  RELATIVE SELF-IGNITION TEMPERATURE FOR SOLIDS  
1. METHOD  
1.1. INTRODUCTION 
Explosive substances and substances which ignite spontaneously in 
contact with air at ambient temperature should not be submitted to 
this test. 
The purpose of this test is to provide preliminary information on the 
auto-flammability of solid substances at elevated temperatures. 
If the heat developed either by a reaction of the substance with 
oxygen or by exothermic decomposition is not lost rapidly enough to the surroundings, self-heating leading to self-ignition occurs. Self- 
ignition therefore occurs when the rate of heat-production exceeds 
the rate of heat loss. 
The test procedure is useful as a preliminary screening test for solid 
substances. In view of the complex nature of the ignition and combustion of solids, the self-ignition temperature determined according to this test method should be used for comparison 
purposes only.
 
1.2. DEFINITIONS AND UNITS 
The self-ignition temperature as obtained by this method is the 
minimum ambient temperature expressed in  o C at which a certain 
volume of a substance will ignite under defined conditions.  
1.3. REFERENCE SUBSTANCE 
None.  
1.4. PRINCIPLE OF THE METHOD 
A certain volume of the substance under test is placed in an oven at 
room temperature; the temperature/time curve relating to conditions in the centre of the sample is recorded while the temperature of the 
oven is increased to 400
 o C, or to the melting point if lower, at a rate 
of 0,5  o C/min. For the purpose of this test, the temperature of the 
oven at which the sample temperature reaches 400  o C by self-heating 
is called the self-ignition temperature.  
1.5. QUALITY CRITERIA 
None.  
1.6. DESCRIPTION OF THE METHOD  
1.6.1.  Apparatus  
1.6.1.1.  Oven  
A temperature-programmed laboratory oven (volume about 2 litres) 
fitted with natural air circulation and explosion relief. In order to 
avoid a potential explosion risk, any decomposition gases must not 
be allowed to come into contact with the electric heating elements.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 114
 
1.6.1.2.  Wire mesh cube  
A piece of stainless steel wire mesh with 0,045 mm openings should 
be cut according to the pattern in figure 1. The mesh should be folded and secured with wire into an open-topped cube.
 
1.6.1.3.  Thermocouples  
Suitable thermocouples.  
1.6.1.4.  Recorder  
Any two-channel recorder calibrated from 0 to 600  o C or 
corresponding voltage.  
1.6.2.  Test conditions  
Substances are tested as received.  
1.6.3.  Performance of the test  
The cube is filled with the substance to be tested and is tapped 
gently, adding more of the substance until the cube is completely full. The cube is then suspended in the centre of the oven at room temperature. One thermocouple is placed at the centre of the cube 
and the other between the cube and the oven wall to record the oven 
temperature. 
The temperatures of the oven and sample are continuously recorded 
while the temperature of the oven is increased to 400
 o C, or to the 
melting point if lower, at a rate of 0,5  o C/min. 
When the substance ignites the sample thermocouple will show a 
very sharp temperature rise above the oven temperature.  
2. DATA  
The temperature of the oven at which the sample temperature 
reaches 400  o C by self-heating is relevant for evaluation (see 
figure 2).  
3. REPORTING  
The test report shall, if possible, include the following information: 
— a description of the substance to be tested, — the results of measurement including the temperature/time curve, — all additional remarks relevant for the interpretation of the results.
 
4. REFERENCES  
NF T 20-036 (September 85) Chemical products for industrial use. 
Determination of the relative temperature of the spontaneous flamm ­
ability of solids.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 115
 
Figure 1  
Pattern of 20 mm test cube
  
Figure 2  
Typical temperature/time curve
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 116
 
A.17.  OXIDISING PROPERTIES (SOLIDS)  
1. METHOD  
1.1. INTRODUCTION 
It is useful to have preliminary information on any potentially 
explosive properties of the substance before performing this test. 
This test is not applicable to liquids, gases, explosive or highly 
flammable substances, or organic peroxides. 
This test need not be performed when examination of the structural 
formula establishes beyond reasonable doubt that the substance is incapable of reacting exothermically with a combustible material. 
In order to ascertain if the test should be performed with special 
precautions, a preliminary test should be performed.
 
1.2. DEFINITION AND UNITS 
Burning time: reaction time, in seconds, taken for the reaction zone 
to travel along a pile, following the procedure described in 1.6. 
Burning rate: expressed in millimetres per second. Maximum burning rate: the highest value of the burning rates 
obtained with mixtures containing 10 to 90 % by weight of oxidiser.
 
1.3. REFERENCE SUBSTANCE 
Barium nitrate (analytical grade) is used as reference substance for 
the test and the preliminary test. 
The reference mixture is that mixture of barium nitrate with 
powdered cellulose, prepared according to 1.6, which has the maximum burning rate (usually a mixture with 60 % barium nitrate by weight).
 
1.4. PRINCIPLE OF THE METHOD 
A preliminary test is carried out in the interests of safety. No further 
testing is required when the preliminary test clearly indicates that the 
test substance has oxidising properties. When this is not the case, the 
substance should then be subject to the full test. 
In the full test, the substance to be tested and a defined combustible 
substance will be mixed in various ratios. Each mixture is then formed into a pile and the pile is ignited at one end. The 
maximum burning rate determined is compared with the maximum 
burning rate of the reference mixture.
 
1.5. QUALITY CRITERIA 
If required, any method of grinding and mixing is valid provided that 
the difference in the maximum rate of burning in the six separate 
tests differs from the arithmetic mean value by no more than 10 %.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 117
 
1.6. DESCRIPTION OF THE METHOD  
1.6.1.  Preparation  
1.6.1.1.  Test substance  
Reduce the test sample to a particle size < 0,125 mm using the 
following procedure: sieve the test substance, grind the remaining 
fraction, repeat the procedure until the whole test portion has passed the sieve. 
Any grinding and sieving method satisfying the quality criteria may 
be used. 
Before preparing the mixture the substance is dried at 105
 o C, until 
constant weight is obtained. If the decomposition temperature of the 
substance to be tested is below 105  o C, the substance has to be dried 
at a suitable lower temperature.  
1.6.1.2.  Combustible substance  
Powdered cellulose is used as a combustible substance. The cellulose 
should be a type used for thin-layer chromatography or column 
chromatography. A type with fibre-lengths of more than 85 % between 0,020 and 0,075 mm has proved to be suitable. The cellulose powder is passed through a sieve with a mesh-size of 
0,125 mm. The same batch of cellulose is to be used throughout 
the test. 
Before preparing the mixture, the powdered cellulose is dried at 
105
 o C until constant weight is obtained. 
If wood-meal is used in the preliminary test, then prepare a soft- 
wood wood-meal by collecting the portion which passes through a sieve mesh of 1,6 mm, mix thoroughly, then dry at 105
 o C for 
four hours in a layer not more than 25 mm thick. Cool and store 
in an air-tight container filled as full as practicable until required, preferably within 24 hours of drying.
 
1.6.1.3.  Ignition source  
A hot flame from a gas burner (minimum diameter 5 mm) should be 
used as the ignition source. If another ignition source is used (e.g. when testing in an inert atmosphere), the description and the justifi ­
cation should be reported.
 
1.6.2.  Performance of the test  
Note:  
Mixtures of oxidisers with cellulose or wood-meal must be treated as 
potentially explosive and handled with due care.  
1.6.2.1.  Preliminary test  
The dried substance is thoroughly mixed with the dried cellulose or 
wood-meal in the proportions 2 of test substance to 1 of cellulose or wood-meal by weight and the mixture is formed into a small cone- shaped pile of dimensions 3,5 cm (diameter of base) × 2,5 cm 
(height) by filling, without tamping, a cone-shaped former (e.g. a 
laboratory glass funnel with the stem plugged).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 118
 
The pile is placed on a cool, non-combustible, non-porous and low 
heat-conducting base plate. The test should be carried out in a fume 
cupboard as in 1.6.2.2. 
The ignition source is put in contact with the cone. The vigour and 
duration of the resultant reaction are observed and recorded. 
The substance is to be considered as oxidising if the reaction is 
vigorous. 
In any case where the result is open to doubt, it is then necessary to 
complete the full train test described below.  
1.6.2.2.  Train test  
Prepare oxidiser cellulose-mixtures containing 10 to 90 % weight of 
oxidiser in 10 % increments. For borderline cases, intermediate 
oxidiser cellulose mixtures should be used to obtain the maximum 
burning rate more precisely. 
The pile is formed by means of a mould. The mould is made of 
metal, has a length of 250 mm and a triangular cross-section with an 
inner height of 10 mm and an inner width of 20 mm. On both sides of the mould, in the longitudinal direction, two metal plates are mounted as lateral limitations which project 2 mm beyond the 
upper edge of the triangular cross-section (figure). This arrangement 
is loosely filled with a slight excess of mixture. After dropping the mould once from a height of 2 cm onto a solid surface, the remaining excess substance is scraped off with an obliquely posi ­
tioned sheet. The lateral limitations are removed and the remaining 
powder is smoothed, using a roller. A non-combustible, non-porous and low heat-conducting base plate is then placed on the top of the 
mould, the apparatus inverted and the mould removed. 
Arrange the pile across the draught in a fume cupboard. The air-speed should be sufficient to prevent fumes escaping into the 
laboratory and should not be varied during the test. A draught screen 
should be erected around the apparatus. 
Due to hygroscopicity of cellulose and of some substances to be 
tested, the test should be carried out as quickly as possible. 
Ignite one end of the pile by touching with the flame. 
Measure the time of reaction over a distance of 200 mm after the 
reaction zone has propagated an initial distance of 30 mm. 
The test is performed with the reference substance and at least once 
with each one of the range of mixtures of the test substance with 
cellulose. 
If the maximum burning rate is found to be significantly greater than 
that from the reference mixture, the test can be stopped; otherwise 
the test should be repeated five times for each of the three mixtures 
giving the fastest burning rate.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 119
 
If the result is suspected of being a false positive, then the test 
should be repeated using an inert substance with a similar particle 
size, such as kieselguhr, in place of cellulose. Alternatively, the test substance cellulose mixture, having the fastest burning rate, should be retested in an inert atmosphere (< 2 % v/v oxygen content).
 
2. DATA  
For safety reasons the maximum burning rate — not the mean value 
— shall be considered to be the characteristic oxidising property of 
the substance under test. 
The highest value of burning rate within a run of six tests of a given 
mixture is relevant for evaluation. 
Plot a graph of the highest value of burning rate for each mixture 
versus the oxidiser concentration. From the graph take the maximum burning rate. 
The six measured values of burning rate within a run obtained from 
the mixture with the maximum burning rate must not differ from the 
arithmetic mean value by more than 10 %; otherwise the methods of 
grinding and mixing must be improved. 
Compare the maximum burning rate obtained with the maximum 
burning rate of the reference mixture (see 1.3). 
If tests are conducted in an inert atmosphere, the maximum reaction 
rate is compared with that from the reference mixture in an inert 
atmosphere.
 
3. REPORT  
3.1. TEST REPORT 
The test report shall, if possible, include the following information: 
— the identity, composition, purity, moisture content etc. of the 
substance tested, 
— any treatment of the test sample (e.g. grinding, drying), — the ignition source used in the tests, — the results of measurements, — the mode of reaction (e.g. flash burning at the surface, burning 
through the whole mass, any information concerning the combustion products, etc.), 
— all additional remarks relevant for the interpretation of results, 
including a description of the vigour (flaming, sparking, fuming, 
slow smouldering, etc.) and approximate duration produced in 
the preliminary safety/screening test for both test and reference substance, 
— the results from tests with an inert substance, if any, — the results from tests in an inert atmosphere, if any.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 120
 
3.2. INTERPRETATION OF THE RESULT 
A substance is to be considered as an oxidising substance when: 
(a) in the preliminary test, there is a vigorous reaction; 
(b) in the full test, the maximum burning rate of the mixtures tested 
is higher than or equal to the maximum burning rate of the 
reference mixture of cellulose and barium nitrate. 
In order to avoid a false positive, the results obtained when testing 
the substance mixed with an inert material and/or when testing under an inert atmosphere should also be considered when interpreting the 
results.
 
4. REFERENCES  
NF T 20-035 (September 85) Chemical products for industrial use. 
Determination of the oxidising properties of solids.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 121
 
Appendix  
Figure  
Mould and accessories for the preparations of the pile  
(All dimensions in millimetres)
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 122
 
A.18.  NUMBER-AVERAGE MOLECULAR WEIGHT AND 
MOLECULAR WEIGHT DISTRIBUTION OF POLYMERS  
1. METHOD  
This Gel Permeation Chromatographic method is a replicate of the 
OECD TG 118 (1996). The fundamental principles and further 
technical information are given in reference (1).  
1.1. INTRODUCTION 
Since the properties of polymers are so varied, it is impossible to 
describe one single method setting out precisely the conditions for separation and evaluation which cover all eventualities and specifi ­
cities occurring in the separation of polymers. In particular, complex 
polymer systems are often not amenable to gel permeation chroma ­
tography (GPC). When GPC is not practicable, the molecular weight may be determined by means of other methods (see Appendix). In such cases, full details and justification should be given for the 
method used. 
The method described is based on DIN Standard 55672 (1). Detailed 
information about how to carry out the experiments and how to 
evaluate the data can be found in this DIN Standard. In case modi ­
fications of the experimental conditions are necessary, these changes must be justified. Other standards may be used, if fully referenced. 
The method described uses polystyrene samples of known polydis ­
persity for calibration and it may have to be modified to be suitable for certain polymers, e.g. water soluble and long-chain branched polymers.
 
1.2. DEFINITIONS AND UNITS 
The number-average molecular weight M  n and the weight average 
molecular weight M  w are determined using the following equations: 
M n ¼ Σ n 
i ¼ 1 H i 
Σ n 
i ¼ 1 H i=M i M w ¼ Σ n 
i ¼ 1 H i Ü M i 
Σ n 
i ¼ 1 H i 
where, 
H i is the level of the detector signal from the baseline for the 
retention volume V  i , 
M i is the molecular weight of the polymer fraction at the retention 
volume V  i , and 
n is the number of data points. The breadth of the molecular weight distribution, which is a measure 
of the dispersity of the system, is given by the ratio M
 w /M n . ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 123
 
1.3. REFERENCE SUBSTANCES 
Since GPC is a relative method, calibration must be undertaken. 
Narrowly distributed, linearly constructed polystyrene standards with known average molecular weights M
 n and M  w and a known 
molecular weight distribution are normally used for this. The cali ­
bration curve can only be used in the determination of the molecular weight of the unknown sample if the conditions for the separation of the sample and the standards have been selected in an identical 
manner. 
A determined relationship between the molecular weight and elution 
volume is only valid under the specific conditions of the particular 
experiment. The conditions include, above all, the temperature, the 
solvent (or solvent mixture), the chromatography conditions and the separation column or system of columns. 
The molecular weights of the sample determined in this way are 
relative values and are described as ‘polystyrene equivalent 
molecular weights’. This means that dependent on the structural and chemical differences between the sample and the standards, the molecular weights can deviate from the absolute values to a 
greater or a lesser degree. If other standards are used, e.g. poly ­
ethylene glycol, polyethylene oxide, polymethyl methacrylate, poly ­
acrylic acid, the reason should be stated.
 
1.4. PRINCIPLE OF THE TEST METHOD 
Both the molecular weight distribution of the sample and the average 
molecular weights (M  n , M  w ) can be determined using GPC. GPC is 
a special type of liquid chromatography in which the sample is separated according to the hydrodynamic volumes of the individual constituents (2). 
Separation is effected as the sample passes through a column which 
is filled with a porous material, typically an organic gel. Small molecules can penetrate the pores whereas large molecules are 
excluded. The path of the large molecules is thereby shorter and 
these are eluted first. The medium-sized molecules penetrate some of the pores and are eluted later. The smallest molecules, with a mean hydrodynamic radius smaller than the pores of the gel, can 
penetrate all of the pores. These are eluted last. 
In an ideal situation, the separation is governed entirely by the size 
of the molecular species, but in practice it is difficult to avoid at least some absorption effects interfering. Uneven column packing and 
dead volumes can worsen the situation (2). 
Detection is effected by, e.g. refractive index or UV-absorption, and 
yields a simple distribution curve. However, to attribute actual molecular weight values to the curve, it is necessary to calibrate 
the column by passing down polymers of known molecular weight 
and, ideally, of broadly similar structure e.g. various polystyrene standards. Typically a Gaussian curve results, sometimes distorted by a small tail to the low molecular weight side, the vertical axis 
indicating the quantity, by weight, of the various molecular weight 
species eluted, and the horizontal axis the log molecular weight.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 124
 
1.5. QUALITY CRITERIA 
The repeatability (Relative Standard Deviation: RSD) of the elution 
volume should be better than 0,3 %. The required repeatability of the analysis has to be ensured by correction via an internal standard if a 
chromatogram is evaluated time-dependently and does not 
correspond to the above mentioned criterion (1). The polydispersities are dependent on the molecular weights of the standards. In the case of polystyrene standards typical values are: 
M
 p < 2 000 M  w /M n < 1,20 
2 000 ≤ M p ≤ 10 6 M w /M n < 1,05 
M p > 10  6 M w /M n < 1,20 
(M p is the molecular weight of the standard at the peak maximum)  
1.6. DESCRIPTION OF THE TEST METHOD  
1.6.1.  Preparation of the standard polystyrene solutions  
The polystyrene standards are dissolved by careful mixing in the 
chosen eluent. The recommendations of the manufacturer must be taken into account in the preparation of the solutions. 
The concentrations of the standards chosen are dependent on various 
factors, e.g. injection volume, viscosity of the solution and sensitivity of the analytical detector. The maximum injection volume must be adapted to the length of the column, in order to avoid overloading. 
Typical injection volumes for analytical separations using GPC with 
a column of 30 cm × 7,8 mm are normally between 40 and 100 μl. 
Higher volumes are possible, but they should not exceed 250 μl. The 
optimal ratio between the injection volume and the concentration 
must be determined prior to the actual calibration of the column.
 
1.6.2.  Preparation of the sample solution  
In principle, the same requirements apply to the preparation of the 
sample solutions. The sample is dissolved in a suitable solvent, e.g. 
tetrahydrofuran (THF), by shaking carefully. Under no circumstances should it be dissolved using an ultrasonic bath. When necessary, the 
sample solution is purified via a membrane filter with a pore size of 
between 0,2 and 2 μm. 
The presence of undissolved particles must be recorded in the final 
report as these may be due to high molecular weight species. An appropriate method should be used to determine the percentage by 
weight of the undissolved particles. The solutions should be used 
within 24 hours.
 
1.6.3.  Apparatus  
— solvent reservoir, 
— degasser (where appropriate), — pump,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 125
 
— pulse dampener (where appropriate), 
— injection system, 
— chromatography columns, 
— detector, 
— flowmeter (where appropriate), 
— data recorder-processor, 
— waste vessel. 
It must be ensured that the GPC system is inert with regard to the 
utilised solvents (e.g. by the use of steel capillaries for THF solvent).  
1.6.4.  Injection and solvent delivery system  
A defined volume of the sample solution is loaded onto the column 
either using an auto-sampler or manually in a sharply defined zone. Withdrawing or depressing the plunger of the syringe too quickly, if done manually, can cause changes in the observed molecular weight 
distribution. The solvent-delivery system should, as far as possible, 
be pulsation-free ideally incorporating a pulse dampener. The flow rate is of the order of 1 ml/min.
 
1.6.5.  Column  
Depending on the sample, the polymer is characterised using either a 
simple column or several columns connected in sequence. A number 
of porous column materials with defined properties (e.g. pore size, exclusion limits) are commercially available. Selection of the separation gel or the length of the column is dependent on both 
the properties of the sample (hydrodynamic volumes, molecular 
weight distribution) and the specific conditions for separation such as solvent, temperature and flow rate (1)(2)(3).
 
1.6.6.  Theoretical plates  
The column or the combination of columns used for separation must 
be characterised by the number of theoretical plates. This involves, in the case of THF as elution solvent, loading a solution of ethyl benzene or other suitable non-polar solute onto a column of known 
length. The number of theoretical plates is given by the following 
equation: 
N ¼ 5; 54 Í
 V e 
W 1=2 Î2 
or N ¼ 16 V e 
W Í Î  2 
where, 
N = the number of theoretical plates 
V e = the elution volume at the peak maximum  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 126
 
W = the baseline peak width 
W 1/2 = the peak width at half height  
1.6.7.  Separation efficiency  
In addition to the number of theoretical plates, which is a quantity 
determining the bandwidth, a part is also played by the separation 
efficiency, this being determined by the steepness of the calibration 
curve. The separation efficiency of a column is obtained from the following relationship:
 
V e;M x Ä V e;ð10M  x Þ 
cross sectional area of the column ≥ 6; 0 cm 3 
cm 2 " #  
where, 
V e, M x = the elution volume for polystyrene with the molecular 
weight M  x 
V e,(10.M x) = the elution volume for polystyrene with a ten times 
greater molecular weight 
The resolution of the system is commonly defined as follows:  
R 1;2 ¼ 2 Ü V e1 Ä V e2 
W 1 þ W 2 Ü 1 
log 10 ðM 2=M 1 Þ 
where, 
V e1 , V e2 = the elution volumes of the two polystyrene standards at 
the peak maximum 
W 1 , W  2 = the peak widths at the base-line 
M 1 , M  2 = the molecular weights at the peak maximum (should 
differ by a factor of 10) 
The R-value for the column system should be greater than 1.7 (4).  
1.6.8.  Solvents  
All solvents must be of high purity (for THF purity of 99,5 % is 
used). The solvent reservoir (if necessary in an inert gas atmosphere) must be sufficiently large for the calibration of the column and several sample analyses. The solvent must be degassed before it is 
transported to the column via the pump.
 
1.6.9.  Temperature control  
The temperature of the critical internal components (injection loop, 
columns, detector and tubing) should be constant and consistent with 
the choice of solvent.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 127
 
1.6.10.  Detector  
The purpose of the detector is to record quantitatively the concen ­
tration of sample eluted from the column. In order to avoid unnecessary broadening of peaks the cuvette volume of the 
detector cell must be kept as small as possible. It should not be 
larger than 10 μl except for light scattering and viscosity detectors. 
Differential refractometry is usually used for detection. However, if required by the specific properties of the sample or the elution 
solvent, other types of detectors can be used, e.g. UV/VIS, IR, 
viscosity detectors, etc.
 
2. DATA AND REPORTING  
2.1. DATA 
The DIN Standard (1) should be referred to for the detailed 
evaluation criteria as well as for the requirements relating to the 
collecting and processing of data. 
For each sample, two independent experiments must be carried out. 
They have to be analysed individually. 
M n , M  w , M  w /M n and M  p must be provided for every measurement. It 
is necessary to indicate explicitly that the measured values are relative values equivalent to the molecular weights of the standard used. 
After determination of the retention volumes or the retention times 
(possibly corrected using an internal standard), log M
 p values (M  p 
being the peak maxima of the calibration standard) are plotted 
against one of those quantities. At least two calibration points are 
necessary per molecular weight decade, and at least five 
measurement points are required for the total curve, which should cover the estimated molecular weight of the sample. The low molecular weight end-point of the calibration curve is defined by 
n-hexyl benzene or another suitable non-polar solute. The number 
average and the weight-average molecular weights are generally determined by means of electronic data processing, based on the formulas of section 1.2. In case manual digitisation is used, ASTM 
D 3536-91 can be consulted (3). 
The distribution curve must be provided in the form of a table or as 
figure (differential frequency or sum percentages against log M). In the graphic representation, one molecular weight decade should be 
normally about 4 cm in width and the peak maximum should be 
about 8 cm in height. In the case of integral distribution curves the difference in the ordinate between 0 and 100 % should be about 10 cm.
 
2.2. TEST REPORT 
The test report must include the following information:  
2.2.1.  Test substance:  
— available information about test substance (identity, additives, 
impurities),  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 128
 
— description of the treatment of the sample, observations, 
problems.  
2.2.2.  Instrumentation:  
— reservoir of eluent, inert gas, degassing of the eluent, 
composition of the eluent, impurities, 
— pump, pulse dampener, injection system, — separation columns (manufacturer, all information about the char ­
acteristics of the columns, such as pore size, kind of separation 
material, etc., number, length and order of the columns used), 
— number of the theoretical plates of the column (or combination), 
separation efficiency (resolution of the system), 
— information on symmetry of the peaks, 
— column temperature, kind of temperature control, 
— detector (measurement principle, type, cuvette volume), — flowmeter if used (manufacturer, measurement principle), — system to record and process data (hardware and software).
 
2.2.3.  Calibration of the system:  
— detailed description of the method used to construct the cali ­
bration curve, 
— information about quality criteria for this method (e.g. correlation 
coefficient, error sum of squares, etc.), 
— information about all extrapolations, assumptions and approxi ­
mations made during the experimental procedure and the 
evaluation and processing of data, 
— all measurements used for constructing the calibration curve have 
to be documented in a table which includes the following information for each calibration point: 
— name of the sample, 
— manufacturer of the sample, — characteristic values of the standards M
 p , M  n , M  w , M  w /M n , as 
provided by the manufacturer or derived by subsequent 
measurements, together with details about the method of 
determination, 
— injection volume and injection concentration,  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 129
 
— M  p value used for calibration, 
— elution volume or corrected retention time measured at the 
peak maxima, 
— M  p calculated at the peak maximum, 
— percentage error of the calculated M  p and the calibration 
value.  
2.2.4.  Evaluation:  
— evaluation on a time basis: methods used to ensure the required 
reproducibility (method of correction, internal standard, etc.), 
— information about whether the evaluation was effected on the 
basis of the elution volume or the retention time, 
— information about the limits of the evaluation if a peak is not 
completely analysed, 
— description of smoothing methods, if used, — preparation and pre-treatment procedures of the sample, — the presence of undissolved particles, if any, — injection volume ( μl) and injection concentration (mg/ml), 
— observations indicating effects which lead to deviations from the 
ideal GPC profile, 
— detailed description of all modifications in the testing procedures, — details of the error ranges, — any other information and observations relevant for the interpre ­
tation of the results.
 
3. REFERENCES  
(1) DIN 55672(1995) Gelpermeationschromatographie (GPC) mit 
Tetrahydrofuran (THF) als Elutionsmittel, Teil 1. 
(2) Yau, W.W., Kirkland, J.J., and Bly, D.D. eds., (1979) Modern 
Size Exclusion Liquid Chromatography, J. Wiley and Sons. 
(3) ASTM D 3536-91, (1991). Standard Test Method for Molecular 
Weight Averages and Molecular Weight Distribution by Liquid Exclusion Chromatography (Gel Permeation Chromatography- 
GPC) American Society for Testing and Materials, Philadelphia, 
Pennsylvania. 
(4) ASTM D 5296-92, (1992) Standard Test Method for Molecular 
Weight Averages and Molecular Weight Distribution of Poly ­
styrene by High Performance Size-Exclusion Chromatography. 
American Society for Testing and Materials, Philadelphia, Penn ­
sylvania.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 130
 
Appendix  
Examples of other methods for determination of number average molecular 
weight (Mn) for polymers  
Gel permeation chromatography (GPC) is the preferred method for determination 
of M  n , especially when a set of standards are available, whose structure are 
comparable with the polymer structure. However, where there are practical 
difficulties in using GPC or there is already an expectation that the substance will fail a regulatory M
 n criterion (and which needs confirming), alternative 
methods are available, such as:  
1. Use of colligative properties  
1.1. Ebullioscopy/Cryoscopy 
involves measurement of boiling point elevation (ebullioscopy) or 
freezing point depression (cryoscopy) of a solvent, when the polymer is added. The method relies on the fact that the effect of 
the dissolved polymer on the boiling/freezing point of the liquid is 
dependent on the molecular weight of the polymer (1) (2). 
Applicability, M
 n < 20 000. 
1.2. Lowering of vapour pressure 
involves the measurement of the vapour pressure of a chosen 
reference liquid before and after the addition of known quantities of polymer (1) (2). 
Applicability, M
 n < 20 000 (theoretically; in practice however of 
limited value). 
1.3 Membrane osmometry 
relies on the principle of osmosis, i.e. the natural tendency of solvent 
molecules to pass through a semi-permeable membrane from a dilute 
to a concentrated solution to achieve equilibrium. In the test, the dilute solution is at zero concentration, whereas the concentrated solution contains the polymer. The effect of drawing solvent 
through the membrane causes a pressure differential that is 
dependent on the concentration and the molecular weight of the polymer (1) (3) (4). 
Applicability, M
 n between 20 000 - 200 000. 
1.4 Vapour phase osmometry 
involves comparison of the rate of evaporation of a pure solvent 
aerosol to at least three aerosols containing the polymer at different concentrations (1)(2)(4). 
Applicability, M
 n < 20 000.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 131
 
2. End-group analysis  
To use this method, knowledge of both the overall structure of the 
polymer and the nature of the chain terminating end groups is needed (which must be distinguishable from the main skeleton by, e.g. NMR or titration/derivatisation). The determination of the 
molecular concentration of the end groups present on the polymer 
can lead to a value for the molecular weight (7) (8) (9). 
Applicability, M
 n up to 50 000 (with decreasing reliability).  
3. References  
(1) Billmeyer, F.W. Jr., (1984) Textbook of Polymer Science, 3rd 
Edn., John Wiley, New York. 
(2) Glover, C.A., (1975) Absolute Colligative Property Methods. 
Chapter 4. In: Polymer Molecular Weights, Part I P.E. Slade, Jr. ed., Marcel Dekker, New York. 
(3) ASTM D 3750-79, (1979) Standard Practice for Determination 
of Number-Average Molecular Weight of Polymers by 
Membrane Osmometry. American Society for Testing and Materials, Philadelphia, Pennsylvania. 
(4) Coll, H. (1989) Membrane Osmometry. In: Determination 
of Molecular Weight, A.R. Cooper ed., J. Wiley and Sons, 
pp. 25-52. 
(5) ASTM 3592-77, (1977) Standard Recommended Practice for 
Determination of Molecular Weight by Vapour Pressure, American Society for Testing and Materials, Philadelphia, Penn ­
sylvania. 
(6) Morris, C.E.M., (1989) Vapour Pressure Osmometry. In: Deter ­
minationn of Molecular Weight, A.R. Cooper ed., John Wiley and Sons. 
(7) Schröder, E., Müller, G., and Arndt, K-F., (1989) Polymer Char ­
acterisation, Carl Hanser Verlag, Munich. 
(8) Garmon, R.G., (1975) End-Group Determinations, Chapter 3 In: 
Polymer Molecular Weights, Part I, P.E. Slade, Jr. ed., Marcel Dekker, New York. 
(9) Amiya, S., et al. (1990) Pure and Applied Chemistry, 62, 
2139-2146.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 132
 
A.19.  LOW MOLECULAR WEIGHT CONTENT OF POLYMERS  
1. METHOD  
This Gel Permeation Chromatographic method is a replicate of the 
OECD TG 119 (1996). The fundamental principles and further 
technical information are given in the references.  
1.1. INTRODUCTION 
Since the properties of polymers are so varied, it is impossible to 
describe one single method setting out precisely the conditions for 
separation and evaluation which cover all eventualities and specifi ­
cities occurring in the separation of polymers. In particular, complex polymer systems are often not amenable to gel permeation chroma ­
tography (GPC). When GPC is not practicable, the molecular weight 
may be determined by means of other methods (see Appendix). In such cases, full details and justification should be given for the method used. 
The method described is based on DIN Standard 55672 (1). Detailed 
information about how to carry out the experiments and how to evaluate the data can be found in this DIN Standard. In case modi ­
fications of the experimental conditions are necessary, these changes 
must be justified. Other standards may be used, if fully referenced. The method described uses polystyrene samples of known polydis ­
persity for calibration and it may have to be modified to be suitable 
for certain polymers, e.g. water soluble and long-chain branched polymers.
 
1.2. DEFINITIONS AND UNITS 
Low molecular weight is arbitrarily defined as a molecular weight 
below 1 000 dalton. 
The number-average molecular weight M  n and the weight average 
molecular weight M  w are determined using the following equations: 
M n ¼ Σ n 
i ¼ 1 H i 
Σ n 
i ¼ 1 H i=M i M w ¼ Σ n 
i ¼ 1 H i Ü M i 
Σ n 
i ¼ 1 H i 
where, 
H i = the level of the detector signal from the baseline for the 
retention volume V  i , 
M i = the molecular weight of the polymer fraction at the retention 
volume V  i , and n is the number of data points 
The breadth of the molecular weight distribution, which is a measure 
of the dispersity of the system, is given by the ratio M  w /M n . ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 133
 
1.3. REFERENCE SUBSTANCES 
Since GPC is a relative method, calibration must be undertaken. 
Narrowly distributed, linearly constructed polystyrene standards with known average molecular weights M
 n and M  w and a known 
molecular weight distribution are normally used for this. The cali ­
bration curve can only be used in the determination of the molecular weight of the unknown sample if the conditions for the separation of the sample and the standards have been selected in an identical 
manner. 
A determined relationship between the molecular weight and elution 
volume is only valid under the specific conditions of the particular 
experiment. The conditions include, above all, the temperature, the 
solvent (or solvent mixture), the chromatography conditions and the separation column or system of columns. 
The molecular weights of the sample determined in this way are 
relative values and are described as ‘polystyrene equivalent 
molecular weights’. This means that dependent on the structural and chemical differences between the sample and the standards, the molecular weights can deviate from the absolute values to a 
greater or a lesser degree. If other standards are used, e.g. poly ­
ethylene glycol, polyethylene oxide, polymethyl methacrylate, poly ­
acrylic acid, the reason should be stated.
 
1.4. PRINCIPLE OF THE TEST METHOD 
Both the molecular weight distribution of the sample and the average 
molecular weights (M  n , M  w ) can be determined using GPC. GPC is 
a special type of liquid chromatography in which the sample is separated according to the hydrodynamic volumes of the individual constituents (2). 
Separation is effected as the sample passes through a column which 
is filled with a porous material, typically an organic gel. Small molecules can penetrate the pores whereas large molecules are 
excluded. The path of the large molecules is thereby shorter and 
these are eluted first. The medium-sized molecules penetrate some of the pores and are eluted later. The smallest molecules, with a mean hydrodynamic radius smaller than the pores of the gel, can 
penetrate all of the pores. These are eluted last. 
In an ideal situation, the separation is governed entirely by the size 
of the molecular species, but in practice it is difficult to avoid at least some absorption effects interfering. Uneven column packing and 
dead volumes can worsen the situation (2). 
Detection is effected by e.g. refractive index or UV-absorption and 
yields a simple distribution curve. However, to attribute actual molecular weight values to the curve, it is necessary to calibrate 
the column by passing down polymers of known molecular weight 
and, ideally, of broadly similar structure, e.g. various polystyrene standards. Typically a Gaussian curve results, sometimes distorted by a small tail to the low molecular weight side, the vertical axis 
indicating the quantity, by weight, of the various molecular weight 
species eluted, and the horizontal axis the log molecular weight.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 134
 
The low molecular weight content is derived from this curve. The 
calculation can only be accurate if the low molecular weight species 
respond equivalently on a per mass basis to the polymer as a whole.  
1.5. QUALITY CRITERIA 
The repeatability (Relative Standard Deviation: RSD) of the elution 
volume should be better than 0,3 %. The required repeatability of the 
analysis has to be ensured by correction via an internal standard if a chromatogram is evaluated time-dependently and does not correspond to the above mentioned criterion (1). The polydispersities 
are dependent on the molecular weights of the standards. In the case 
of polystyrene standards typical values are: 
M
 p < 2 000 M  w /M n < 1,20 
2 000  < M  p < 10  6 M w /M n < 1,05 
M p > 10  6 M w /M n < 1,20 
(M p is the molecular weight of the standard at the peak maximum)  
1.6. DESCRIPTION OF THE TEST METHOD  
1.6.1.  Preparation of the standard polystyrene solutions  
The polystyrene standards are dissolved by careful mixing in the 
chosen eluent. The recommendations of the manufacturer must be 
taken into account in the preparation of the solutions. 
The concentrations of the standards chosen are dependent on various 
factors, e.g. injection volume, viscosity of the solution and sensitivity of the analytical detector. The maximum injection volume must be adapted to the length of the column, in order to avoid overloading. 
Typical injection volumes for analytical separations using GPC with 
a column of 30 cm × 7,8 mm are normally between 40 and 100 μl. 
Higher volumes are possible, but they should not exceed 250 μl. The 
optimal ratio between the injection volume and the concentration 
must be determined prior to the actual calibration of the column.
 
1.6.2.  Preparation of the sample solution  
In principle, the same requirements apply to the preparation of the 
sample solutions. The sample is dissolved in a suitable solvent, e.g. 
tetrahydrofuran (THF), by shaking carefully. Under no circumstances 
should it be dissolved using an ultrasonic bath. When necessary, the sample solution is purified via a membrane filter with a pore size of between 0,2 and 2 μm. 
The presence of undissolved particles must be recorded in the final 
report as these may be due to high molecular weight species. An appropriate method should be used to determine the percentage by 
weight of the undissolved particles. The solutions should be used 
within 24 hours.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 135
 
1.6.3.  Correction for content of impurities and additives  
Correction of the content of species of M < 1 000 for the 
contribution from non-polymer specific components present (e.g. impurities and/or additives) is usually necessary, unless the 
measured content is already < 1 %. This is achieved by direct 
analysis of the polymer solution or the GPC eluate. 
In cases where the eluate, after passage through the column, is too 
dilute for a further analysis it must be concentrated. It may be necessary to evaporate the eluate to dryness and dissolve it again. Concentration of the eluate must be effected under conditions which 
ensure that no changes occur in the eluate. The treatment of the 
eluate after the GPC step is dependent on the analytical method used for the quantitative determination.
 
1.6.4.  Apparatus  
GPC apparatus comprises the following components: 
— solvent reservoir, — degasser (where appropriate), 
— pump, 
— pulse dampener (where appropriate), 
— injection system, 
— chromatography columns, 
— detector, 
— flowmeter (where appropriate), 
— data recorder-processor, 
— waste vessel. It must be ensured that the GPC system is inert with regard to the 
utilised solvents (e.g. by the use of steel capillaries for THF solvent).
 
1.6.5.  Injection and solvent delivery system  
A defined volume of the sample solution is loaded onto the column 
either using an auto-sampler or manually in a sharply defined zone. 
Withdrawing or depressing the plunger of the syringe too quickly, if 
done manually, can cause changes in the observed molecular weight distribution. The solvent-delivery system should, as far as possible, be pulsation-free ideally incorporating a pulse dampener. The flow 
rate is of the order of 1 ml/min.
 
1.6.6.  Column  
Depending on the sample, the polymer is characterised using either a 
simple column or several columns connected in sequence. A number 
of porous column materials with defined properties (e.g. pore size, 
exclusion limits) are commercially available. Selection of the separation gel or the length of the column is dependent on both the properties of the sample (hydrodynamic volumes, molecular 
weight distribution) and the specific conditions for separation such 
as solvent, temperature and flow rate (1) (2) (3).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 136
 
1.6.7.  Theoretical plates  
The column or the combination of columns used for separation must 
be characterised by the number of theoretical plates. This involves, in the case of THF as elution solvent, loading a solution of ethyl 
benzene or other suitable non-polar solute onto a column of known 
length. The number of theoretical plates is given by the following equation: 
N ¼ 5; 54 Í
 V e 
W 1=2 Î2 
or N ¼ 16 V e 
W Í Î  2 
where, 
N = the number of theoretical plates 
V e = the elution volume at the peak maximum 
W = the baseline peak width W
 1/2 = the peak width at half height  
1.6.8.  Separation efficiency  
In addition to the number of theoretical plates, which is a quantity 
determining the bandwidth, a part is also played by the separation 
efficiency, this being determined by the steepness of the calibration 
curve. The separation efficiency of a column is obtained from the following relationship:
 
V e;M x Ä V e;ð10M  x Þ 
cross sectional area of the column ≥ 6; 0 cm 3 
cm 2 " #  
where, 
V e , M x = the elution volume for polystyrene with the molecular 
weight M  x 
V e,(10.M x) = the elution volume for polystyrene with a ten times 
greater molecular weight 
The resolution of the system is commonly defined as follows:  
R 1;2 ¼ 2 Ü V e1 Ä V e2 
W 1 þ W 2 Ü 1 
log 10 ðM 2=M 1 Þ 
where, 
V e1 , V e2 = the elution volumes of the two polystyrene standards at 
the peak maximum 
W 1 , W  2 = the peak widths at the base-1ine 
M 1 , M  2 = the molecular weights at the peak maximum (should 
differ by a factor of 10). 
The R-value for the column system should be greater than 1,7 (4).  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 137
 
1.6.9.  Solvents  
All solvents must be of high purity (for THF purity of 99,5 % is 
used). The solvent reservoir (if necessary in an inert gas atmosphere) must be sufficiently large for the calibration of the column and 
several sample analyses. The solvent must be degassed before it is 
transported to the column via the pump.
 
1.6.10.  Temperature control  
The temperature of the critical internal components (injection loop, 
columns, detector and tubing) should be constant and consistent with 
the choice of solvent.  
1.6.11.  Detector  
The purpose of the detector is to record quantitatively the concen ­
tration of sample eluted from the column. In order to avoid 
unnecessary broadening of peaks the cuvette volume of the detector cell must be kept as small as possible. It should not be 
larger than 10 μl except for light scattering and viscosity detectors. 
Differential refractometry is usually used for detection. However, if required by the specific properties of the sample or the elution solvent, other types of detectors can be used, e.g. UV/VIS, IR, 
viscosity detectors, etc.
 
2. DATA AND REPORTING  
2.1. DATA 
The DIN Standard (1) should be referred to for the detailed 
evaluation criteria as well as for the requirements relating to the 
collecting and processing of data. 
For each sample, two independent experiments must be carried out. 
They have to be analysed individually. In all cases it is essential to 
determine also data from blanks, treated under the same conditions as the sample. 
It is necessary to indicate explicitly that the measured values are 
relative values equivalent to the molecular weights of the standard used. 
After determination of the retention volumes or the retention times 
(possibly corrected using an internal standard), log M
 p values 
(M p being the peak maxima of the calibration standard) are plotted 
against one of those quantities. At least two calibration points are 
necessary per molecular weight decade, and at least five measurement points are required for the total curve, which should cover the estimated molecular weight of the sample. The low molecular weight end-point 
of the calibration curve is defined by n-hexyl benzene or another 
suitable non-polar solute. The portion of the curve corresponding to molecular weights below 1 000 is determined and corrected as necessary for impurities and additives. The elution curves are 
generally evaluated by means of electronic data processing. In case 
manual digitisation is used, ASTM D 3536-91 can be consulted (3).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 138
 
If any insoluble polymer is retained on the column, its molecular 
weight is likely to be higher than that of the soluble fraction, and if 
not considered would result in an overestimation of the low molecular weight content. Guidance for correcting the low molecular weight content for insoluble polymer is provided in the 
Appendix. 
The distribution curve must be provided in the form of a table or as 
figure (differential frequency or sum percentages against log M). In the 
graphic representation, one molecular weight decade should be 
normally about 4 cm in width and the peak maximum should be about 8 cm in height. In the case of integral distribution curves the 
difference in the ordinate between 0 and 100 % should be about 10 cm.
 
2.2. TEST REPORT 
The test report must include the following information:  
2.2.1.  Test substance:  
— available information about test substance (identity, additives, 
impurities), 
— description of the treatment of the sample, observations, 
problems.  
2.2.2.  Instrumentation:  
— reservoir of eluent, inert gas, degassing of the eluent, 
composition of the eluent, impurities, 
— pump, pulse dampener, injection system, 
— separation columns (manufacturer, all information about the char ­
acteristics of the columns, such as pore size, kind of separation 
material, etc., number, length and order of the columns used), 
— number of the theoretical plates of the column (or combination), 
separation efficiency (resolution of the system), 
— information on symmetry of the peaks, 
— column temperature, kind of temperature control, — detector (measurement principle, type, cuvette volume), 
— flowmeter if used (manufacturer, measurement principle), 
— system to record and process data (hardware and software).
 
2.2.3.  Calibration of the system:  
— detailed description of the method used to construct the cali ­
bration curve,  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 139
 
— information about quality criteria for this method (e.g. correlation 
coefficient, error sum of squares, etc.), 
— information about all extrapolations, assumptions and approxi ­
mations made during the experimental procedure and the evaluation and processing of data, 
— all measurements used for constructing the calibration curve have 
to be documented in a table which includes the following information for each calibration point: 
— name of the sample, 
— manufacturer of the sample, — characteristic values of the standards M
 p , M  n , M  w , M  w /M n , as 
provided by the manufacturer or derived by subsequent 
measurements, together with details about the method of determination, 
— injection volume and injection concentration, 
— M
 p value used for calibration, 
— elution volume or corrected retention time measured at the 
peak maxima, 
— M  p calculated at the peak maximum, 
— percentage error of the calculated M  p and the calibration 
value.  
2.2.4.  Information on the low molecular weight polymer content:  
— description of the methods used in the analysis and the way in 
which the experiments were conducted, 
— information about the percentage of the low molecular weight 
species content (w/w) related to the total sample, 
— information about impurities, additives and other non-polymer 
species in percentage by weight related to the total sample.  
2.2.5.  Evaluation:  
— evaluation on a time basis: all methods to ensure the required 
reproducibility (method of correction, internal standard etc.), 
— information about whether the evaluation was effected on the 
basis of the elution volume or the retention time, 
— information about the limits of the evaluation if a peak is not 
completely analysed, 
— description of smoothing methods, if used, 
— preparation and pre-treatment procedures of the sample, 
— the presence of undissolved particles, if any,  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 140
 
— injection volume ( μl) and injection concentration (mg/ml), 
— observations indicating effects which lead to deviations from the 
ideal GPC profile, 
— detailed description of all modifications in the testing procedures, — details of the error ranges, — any other information and observations relevant for the interpre ­
tation of the results.
 
3. REFERENCES  
(1) DIN 55672 (1995) Gelpermeationschromatographie (GPC) mit 
Tetrahydrofuran (THF) als Elutionsmittel, Teil 1. 
(2) Yau, W.W., Kirkland, J.J., and Bly, D.D. eds. (1979) Modern 
Size Exclusion Liquid Chromatography, J. Wiley and Sons. 
(3) ASTM D 3536-91, (1991) Standard Test method for Molecular 
Weight Averages and Molecular Weight Distribution by Liquid Exclusion Chromatography (Gel Permeation Chromatography- 
GPC). American Society for Testing and Materials, Philad ­
elphia, Pennsylvania. 
(4) ASTM D 5296-92, (1992) Standard Test method for Molecular 
Weight Averages and Molecular Weight Distribution of Poly ­
styrene by High Performance Size-Exclusion Chromatography. 
American Society for Testing and Materials, Philadelphia, Penn ­
sylvania.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 141
 
Appendix  
Guidance for correcting low molecular content for the presence of insoluble 
polymer  
When insoluble polymer is present in a sample, it results in mass loss during the 
GPC analysis. The insoluble polymer is irreversibly retained on the column or 
sample filter while the soluble portion of the sample passes through the column. 
In the case where the refractive index increment (dn/dc) of the polymer can be estimated or measured, one can estimate the sample mass lost on the column. In that case, one makes a correction using an external calibration with standard 
materials of known concentration and dn/dc to calibrate the response of the 
refractometer. In the example hereafter a poly(methyl methacrylate) (pMMA) standard is used. 
In the external calibration for analysis of acrylic polymers, a pMMA standard of 
known concentration in tetrahydrofuran, is analysed by GPC and the resulting 
data are used to find the refractometer constant according to the equation: 
K = R/(C × V × dn/dc) 
where: 
K = the refractometer constant (in microvolt second/ml), R = the response of the pMMA standard (in microvolt/second), C = the concentration of the pMMA standard (in mg/ml), 
V = the injection volume (in ml), and 
dn/dc = the refractive index increment for pMMA in tetrahydrofuran (in 
ml/mg). 
The following data are typical for a pMMA standard: R = 2 937 891 C = 1,07 mg/ml V = 0,1 ml dn/dc = 9 × 10
 -5 ml/mg 
The resulting K value, 3,05 × 10  11 is then used to calculate the theoretical 
detector response if 100 % of the polymer injected had eluted through the 
detector.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 142
 
A.20.  SOLUTION/EXTRACTION BEHAVIOUR OF POLYMERS IN 
WATER  
1. METHOD  
The method described is a replicate of the revised version of 
OECD TG 120 (1997). Further technical information is given in 
reference (1).  
1.1. INTRODUCTION 
For certain polymers, such as emulsion polymers, initial preparatory 
work may be necessary before the method set out hereafter can be 
used. The method is not applicable to liquid polymers and to 
polymers that react with water under the test conditions. 
When the method is not practical or not possible, the solution/ ­
extraction behaviour may be investigated by means of other methods. In such cases, full details and justification should be 
given for the method used.
 
1.2. REFERENCE SUBSTANCES 
None.  
1.3. PRINCIPLE OF THE TEST METHOD 
The solution/extraction behaviour of polymers in an aqueous 
medium is determined using the flask method (see A.6 Water Solu ­
bility, Flask method) with the modifications described below.  
1.4. QUALITY CRITERIA 
None.  
1.5. DESCRIPTION OF THE TEST METHOD  
1.5.1.  Equipment  
The following equipment is required for the method: 
— crushing device, e.g. grinder for the production of particles of 
known size, 
— apparatus for shaking with possibility of temperature control, 
— membrane filter system, 
— appropriate analytical equipment, — standardised sieves.
 
1.5.2.  Sample preparation  
A representative sample has first to be reduced to a particle size 
between 0,125 and 0,25 mm using appropriate sieves. Cooling may be required for the stability of the sample or for the grinding 
process. Materials of a rubbery nature can be crushed at liquid 
nitrogen temperature (1). 
If the required particle size fraction is not attainable, action should be 
taken to reduce the particle size as much as possible, and the result 
reported. In the report, it is necessary to indicate the way in which 
the crushed sample was stored prior to the test
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 143
 
1.5.3.  Procedure  
Three samples of 10 g of the test substance are weighed into each of 
three vessels fitted with glass stoppers and 1 000 ml of water is added to each vessel. If handling an amount of 10 g polymer 
proves impracticable, the next highest amount which can be 
handled should be used and the volume of water adjusted accord ­
ingly. 
The vessels are tightly stoppered and then agitated at 20
 o C. A 
shaking or stirring device capable of operating at constant temperature should be used. After a period of 24 hours, the 
content of each vessel is centrifuged or filtered and the concentration 
of polymer in the clear aqueous phase is determined by a suitable analytical method. If suitable analytical methods for the aqueous 
phase are not available, the total solubility/extractivity can be 
estimated from the dry weight of the filter residue or centrifuged precipitate. 
It is usually necessary to differentiate quantitatively between the 
impurities and additives on the one hand and the low molecular weight species on the other hand. In the case of gravimetric deter ­
mination, it is also important to perform a blank run using no test 
substance in order to account for residues arising from the experi ­
mental procedure. 
The solution/extraction behaviour of polymers in water at 37
 o C at 
pH 2 and pH 9 may be determined in the same way as described for the conduct of the experiment at 20
 o C. The pH values can be 
achieved by the addition of either suitable buffers or appropriate 
acids or bases such as hydrochloric acid, acetic acid, analytical grade sodium or potassium hydroxide or NH
 3 . 
Depending on the method of analysis used, one or two tests should 
be performed. When sufficiently specific methods are available for direct analysis of the aqueous phase for the polymer component, one 
test as described above should suffice. However, when such methods 
are not available and determination of the solution/extraction behaviour of the polymer is limited to indirect analysis by deter ­
mining only the total organic carbon content (TOC) of the 
aqueous extract, an additional test should be conducted. This 
additional test should also be done in triplicate, using ten times smaller polymer samples and the same amounts of water as those used in the first test.
 
1.5.4.  Analysis  
1.5.4.1.  Test conducted with one sample size  
Methods may be available for direct analysis of polymer components 
in the aqueous phase. Alternatively, indirect analysis of dissolved/ ­
extracted polymer components, by determining the total content of 
soluble parts and correcting for non polymer-specific components, could also be considered. 
Analysis of the aqueous phase for the total polymeric species is 
possible: 
either by a sufficiently sensitive method, e.g.: — TOC using persulphate or dichromate digestion to yield CO
 2 
followed by estimation by IR or chemical analysis,  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 144
 
— Atomic Absorption Spectrometry (AAS) or its Inductively 
Coupled Plasma (ICP) emission equivalent for silicon or metal 
containing polymers, 
— UV absorption or spectrofluorimetry for aryl polymers, — LC-MS for low molecular weight samples, or by vacuum evaporation to dryness of the aqueous extract and 
spectroscopic (IR, UV, etc.) or AAS/ICP analysis of the residue. 
If analysis of the aqueous phase as such is not practicable, the 
aqueous extract should be extracted with a water-immiscible organic solvent e.g. a chlorinated hydrocarbon. The solvent is then 
evaporated and the residue analysed as above for the notified 
polymer content. Any components in this residue which are identified as being impurities or additives are to be subtracted for the purpose of determining the degree of solution/extraction of the 
polymer itself. 
When relatively large quantities of such materials are present, it may 
be necessary to subject the residue to e.g. HPLC or GC analysis to differentiate the impurities from the monomer and monomer-derived species present so that the true content of the latter can be deter ­
mined. 
In some cases, simple evaporation of the organic solvent to dryness 
and weighing the dry residue may be sufficient.
 
1.5.4.2.  Test conducted with two different sample sizes  
All aqueous extracts are analysed for TOC. 
A gravimetric determination is performed on the undissolved/not 
extracted part of the sample. If, after centrifugation or filtering of the content of each vessel, polymer residues remain attached to the 
wall of the vessel, the vessel should be rinsed with the filtrate until 
the vessel is cleared from all visible residues. Following which, the filtrate is again centrifuged or filtered. The residues remaining on the filter or in the centrifuge tube are dried at 40
 o C under vacuum and 
weighed. Drying is continued until a constant weight is reached.  
2. DATA  
2.1. TEST CONDUCTED WITH ONE SAMPLE SIZE 
The individual results for each of the three flasks and the average 
values should be given and expressed in units of mass per volume of the solution (typically mg/l) or mass per mass of polymer sample (typically mg/g). Additionally, the weight loss of the sample (cal ­
culated as the weight of the solute divided by the weight of the 
initial sample) should also be given. The relative standard deviations (RSD) should be calculated. Individual figures should be given for the total substance (polymer + essential additives, etc.) and for the 
polymer only (i.e. after subtracting the contribution from such 
additives).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 145
 
2.2. TEST CONDUCTED WITH TWO DIFFERENT SAMPLE SIZES 
The individual TOC values of the aqueous extracts of the two trip ­
licate experiments and the average value for each experiment should be given expressed as units of mass per volume of solution (typically 
mgC/l), as well as in units of mass per weight of the initial sample 
(typically mgC/g). 
If there is no difference between the results at the high and the low 
sample/water ratios, this may indicate that all extractable components 
were indeed extracted. In such a case, direct analysis would normally 
not be necessary. 
The individual weights of the residues should be given and 
expressed in percentage of the initial weights of the samples. Averages should be calculated per experiment. The differences 
between 100 and the percentages found represent the percentages 
of soluble and extractable material in the original sample.
 
3. REPORTING  
3.1. TEST REPORT 
The test report must include the following information:  
3.1.1.  Test substance:  
— available information about test substance (identity, additives, 
impurities, content of low molecular weight species).  
3.1.2.  Experimental conditions:  
— description of the procedures used and experimental conditions, 
— description of the analytical and detection methods.  
3.1.3.  Results:  
— results of solubility/extractivity in mg/l; individual and mean 
values for the extraction tests in the various solutions, broken 
down in polymer content and impurities, additives, etc., 
— results of solubility/extractivity in mg/g of polymer, — TOC values of aqueous extracts, weight of the solute and 
calculated percentages, if measured, 
— the pH of each sample, 
— information about the blank values, 
— where necessary, references to the chemical instability of the test 
substance, during both the testing process and the analytical process, 
— all information which is important for the interpretation of the 
results.
 
4. REFERENCES  
(1) DIN 53733 (1976) Zerkleinerung von Kunststofferzeugnissen 
für Prüfzwecke.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 146
 
A.21.  OXIDISING PROPERTIES (LIQUIDS)  
1. METHOD  
1.1. INTRODUCTION 
This test method is designed to measure the potential for a liquid 
substance to increase the burning rate or burning intensity of a 
combustible substance, or to form a mixture with a combustible substance which spontaneously ignites, when the two are thoroughly mixed. It is based on the UN test for oxidising liquids (1) and is 
equivalent to it. However, as this method A.21 is primarily designed 
to satisfy the requirements of Regulation (EC) No 1907/2006, comparison with only one reference substance is required. Testing 
and comparison to additional reference substances may be necessary 
when the results of the test are expected to be used for other purposes. (
 1 ) 
This test need not be performed when examination of the structural 
formula establishes beyond reasonable doubt that the substance is 
incapable of reacting exothermically with a combustible material. 
It is useful to have preliminary information on any potential 
explosive properties of the substance before performing this test. 
This test is not applicable to solids, gases, explosive or highly 
flammable substances, or organic peroxides. 
This test may not need to be performed when results for the test 
substance in the UN test for oxidising liquids (1) are already 
available.  
1.2. DEFINITIONS AND UNITS 
Mean pressure rise time is the mean of the measured times for a 
mixture under test to produce a pressure rise from 690 kPa to 2 070 
kPa above atmospheric.  
1.3. REFERENCE SUBSTANCE 
65 % (w/w) aqueous nitric acid (analytical grade) is required as a 
reference substance. (  2 ) ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 147  
( 1 ) As, for example, in the framework of UN transport regulations. 
( 2 ) The acid should be titrated before testing to confirm its concentration.
 Optionally, if the experimenter foresees that the results of this test 
may eventually be used for other purposes (  1 ), testing of additional 
reference substances may also be appropriate. (  2 ) 
1.4. PRINCIPLE OF THE TEST METHOD 
The liquid to be tested is mixed in a 1 to 1 ratio, by mass, with 
fibrous cellulose and introduced into a pressure vessel. If during 
mixing or filling spontaneous ignition occurs, no further testing is necessary. 
If spontaneous ignition does not occur the full test is carried out. The 
mixture is heated in a pressure vessel and the mean time taken for the pressure to rise from 690 kPa to 2 070 kPa above atmospheric is determined. This is compared with the mean pressure rise time for 
the 1:1 mixture of the reference substance(s) and cellulose.
 
1.5. QUALITY CRITERIA 
In a series of five trials on a single substance no results should differ 
by more than 30 % from the arithmetic mean. Results that differ by 
more than 30 % from the mean should be discarded, the mixing and filling procedure improved and the testing repeated.
 
1.6. DESCRIPTION OF THE METHOD  
1.6.1.  Preparation  
1.6.1.1.  Combustible substance  
Dried, fibrous cellulose with a fibre length between 50 and 250 μm 
and a mean diameter of 25 μm (  3 ), is used as the combustible 
material. It is dried to constant weight in a layer not more than 
25 mm thick at 105  o C for four hours and kept in a desiccator, 
with desiccant, until cool and required for use. The water content of the dried cellulose should be less than 0,5 % by dry mass (
 4 ). If 
necessary, the drying time should be prolonged to achieve this. (  5 ) 
The same batch of cellulose is to be used throughout the test.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 148  
( 1 ) As, for example, in the framework of UN transport regulations. 
( 2 ) E.g.: 50 % (w/w) perchloric acid and 40 % (w/w) sodium chlorate are used in 
reference 1. 
( 3 ) E.g. Whatman Column Chromatographic Cellulose Powder CF 11, catalogue No 4021 050. 
( 4 ) Confirmed by, e.g. Karl-Fisher titration. 
( 5 ) Alternatively, this water content can also be achieved by, e.g. heating at 105  o C under 
vacuum for 24 h.
 1.6.1.2.  Apparatus  
1.6.1.2.1. P r e s s u r e v e s s e l 
A pressure vessel is required. The vessel consists of a cylindrical 
steel pressure vessel 89 mm in length and 60 mm in external diameter (see figure 1). Two flats are machined on opposite sides (reducing the cross-section of the vessel to 50 mm) to facilitate 
holding whilst fitting up the firing plug and vent plug. The vessel, 
which has a bore of 20 mm diameter is internally rebated at either end to a depth of 19 mm and threaded to accept 1'' British Standard Pipe (BSP) or metric equivalent. A pressure take-off, in the form of a 
side arm, is screwed into the curved face of the pressure vessel 
35 mm from one end and at 90
 o to the machined flats. The socket 
for this is bored to a depth of 12 mm and threaded to accept the 1/2" BSP (or metric equivalent) thread on the end of the side-arm. If 
necessary, an inert seal is fitted to ensure a gas-tight seal. The 
side-arm extends 55 mm beyond the pressure vessel body and has a bore of 6 mm. The end of the side-arm is rebated and threaded to accept a diaphragm type pressure transducer. Any pressure- 
measuring device may be used provided that it is not affected by 
the hot gases or the decomposition products and is capable of responding to rates of pressure rise of 690-2 070 kPa in not more than 5 ms. 
The end of the pressure vessel farthest from the side-arm is closed 
with a firing plug which is fitted with two electrodes, one insulated from, and the other earthed to, the plug body. The other end of the 
pressure vessel is closed by a bursting disk (bursting pressure 
approximately 2 200 kPa) held in place with a retaining plug which has a 20 mm bore. If necessary, an inert seal is used with the firing plug to ensure a gas-tight fit. A support stand (figure 2) 
holds the assembly in the correct attitude during use. This usually 
comprises a mild steel base plate measuring 235 mm × 184 mm × 6 mm and a 185 mm length of square hollow section (S.H.S.) 70 mm × 70 mm × 4 mm. 
A section is cut from each of two opposite sides at one end of the 
length of S.H.S. so that a structure having two flat sided legs surmounted by 86 mm length of intact box section results. The ends of these flat sides are cut to an angle of 60
 o to the horizontal 
and welded to the base plate. A slot measuring 22 mm wide × 
46 mm deep is machined in one side of the upper end of the base section such that when the pressure vessel assembly is lowered, firing plug end first, into the box section support, the side-arm is 
accommodated in the slot. A piece of steel 30 mm wide and 6 mm 
thick is welded to the lower internal face of the box section to act as a spacer. Two 7 mm thumb screws, tapped into the opposite face, serve to hold the pressure vessel firmly in place. Two 12 mm wide 
strips of 6 mm thick steel, welded to the side pieces abutting the 
base of the box section, support the pressure vessel from beneath.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 149
 
1.6.1.2.2. I g n i t i o n s y s t e m 
The ignition system consists of a 25 cm long Ni/Cr wire with a 
diameter 0,6 mm and a resistance of 3,85 ohm/m. The wire is 
wound, using a 5 mm diameter rod, in the shape of a coil and is attached to the firing plug electrodes. The coil should have one of the configurations shown in figure 3. The distance between the 
bottom of the vessel and the underside of the ignition coil should 
be 20 mm. If the electrodes are not adjustable, the ends of the ignition wire between the coil and the bottom of the vessel should be insulated by a ceramic sheath. The wire is heated by a constant 
current power supply able to deliver at least 10 A.
 
1.6.2.  Performance of the test  ( 1 ) 
The apparatus, assembled complete with pressure transducer and 
heating system but without the bursting disk in position, is supported firing plug end down. 2,5 g of the liquid to be tested is mixed with 2,5 g of dried cellulose in a glass beaker using a glass 
stirring rod (
 2 ). For safety, the mixing should be performed with a 
safety shield between the operator and mixture. If the mixture ignites during mixing or filling, no further testing is necessary. The mixture is added, in small portions with tapping, to the pressure vessel 
making sure that the mixture is packed around the ignition coil 
and is in good contact with it. It is important that the coil is not distorted during the packing process as this may lead to erroneous results (
 3 ). The bursting disk is placed in position and the retaining 
plug is screwed in tightly. The charged vessel is transferred to the 
firing support stand, bursting disk uppermost, which should be located in a suitable, armoured fume cupboard or firing cell. The power supply is connected to the external terminals of the firing plug 
and 10 A applied. The time between the start of mixing and 
switching on the power should not exceed 10 minutes. 
The signal produced by the pressure transducer is recorded on a 
suitable system which allows both evaluation and the generation of 
a permanent record of the time pressure profile obtained (e.g. a 
transient recorder coupled to a chart recorder). The mixture is heated until the bursting disk ruptures or until at least 60 s have elapsed. If the bursting disk does not rupture, the mixture should be 
allowed to cool before carefully dismantling the apparatus, taking 
precautions to allow for any pressurisation which may occur. Five trials are performed with the test substance and the reference substance(s). The time taken for the pressure to rise from 690 kPa 
to 2 070 kPa above atmospheric is noted. The mean pressure rise 
time is calculated. 
In some cases, substances may generate a pressure rise (too high or 
too low), caused by chemical reactions not characterising the 
oxidising properties of the substance. In these cases, it may be 
necessary to repeat the test with an inert substance, e.g. diatomite (kieselguhr), in place of the cellulose in order to clarify the nature of the reaction.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 150  
( 1 ) Mixtures of oxidisers with cellulose must be treated as potentially explosive and handled 
with due care. 
( 2 ) In practice this can be achieved by preparing a 1:1 mixture of the liquid to be tested and 
cellulose in a greater amount than needed for the trial and transferring 5 ± 0,1 g to the 
pressure vessel. The mixture is to be freshly prepared for each trial. 
( 3 ) In particular, contact between the adjacent turns of the coil must be avoided.
 2. DATA  
Pressure rise times for both the test substance and the reference 
substance(s). Pressure rise times for the tests with an inert substance, if performed.
 
2.1. TREATMENT OF RESULTS 
The mean pressure rise times for both the test substance and the 
reference substances(s) are calculated. 
The mean pressure rise time for the tests with an inert substance (if 
performed) is calculated. 
Some examples of results are shown in Table 1.  
Table 1  
Examples of results  ( a ) 
Substance (  b ) Mean pressure rise time for a 1:1 mixture with celulose 
(ms) 
Ammonium dichromate, saturated aqueous solution 20 800 
Calcium nitrate, saturated aqueous solution 6 700 Ferric nitrate, saturated aqueous solution 4 133 Lithium perchlorate, saturated aqueous solution 1 686 Magnesium perchlorate, saturated aqueous solution 777 
Nickel nitrate, saturated aqueous solution 6 250 
Nitric acid, 65 % 4 767 (
 c ) 
Perchloric acid, 50 % 121 (  c ) 
Perchloric acid, 55 % 59 Potassium nitrate, 30 % aqueous solution 26 690 
Silver nitrate, saturated aqueous solution (
 d ) 
Sodium chlorate, 40 % aqueous solution 2 555 (  c ) 
Sodium nitrate, 45 % aqueous solution 4 133  
Inert substance  
Water: cellulose (  d ) 
( a ) See reference (1) for classification under the UN transport scheme. 
( b ) Saturated solutions should be prepared at 20  o C. 
( c ) Mean value from interlaboratory comparative trials. 
( d ) Maximum pressure of 2 070 kPa not reached.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 151
 
3. REPORT  
3.1. TEST REPORT 
The test report should include the following information: 
— the identity, composition, purity, etc. of the substance tested, — the concentration of the test substance, 
— the drying procedure of the cellulose used, 
— the water content of the cellulose used, — the results of the measurements, — the results from tests with an inert substance, if any, 
— the calculated mean pressure rise times, 
— any deviations from this method and the reasons for them, — all additional information or remarks relevant to the interpretation 
of the results.
 
3.2. INTERPRETATION OF THE RESULTS (  1 ) 
The test results are assessed on the basis of: 
(a) whether the mixture of test substance and cellulose sponta ­
neously ignites; and 
(b) the comparison of the mean time taken for the pressure to 
rise from 690 kPa to 2 070 kPa with that of the reference 
substance(s). 
A liquid substance is to be considered as an oxidiser when: 
(a) a 1:1 mixture, by mass, of the substance and cellulose sponta ­
neously ignites; or 
(b) a 1:1 mixture, by mass, of the substance and cellulose exhibits a 
mean pressure rise time less than or equal to the mean pressure 
rise time of a 1:1 mixture, by mass, of 65 % (w/w) aqueous 
nitric acid and cellulose. 
In order to avoid a false positive result, if necessary, the results 
obtained when testing the substance with an inert material should also be considered when interpreting the results.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 152  
( 1 ) See reference 1 for interpretation of the results under the UN transport regulations using 
several reference substances.
 4. REFERENCES  
(1) Recommendations on the Transport of Dangerous Goods, 
Manual of Tests and Criteria. 3  rd revised edition. UN 
Publication No: ST/SG/AC.10/11/Rev. 3, 1999, page 342. Test 
O.2: Test for oxidising liquids.  
Figure 1  
Pressure vessel
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 153
 
Figure 2  
Support stand
  
Figure 3  
Ignition system
  
Note: either of these configurations may be used.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 154
 
A.22.  LENGTH WEIGHTED GEOMETRIC MEAN DIAMETER OF 
FIBRES  
1. METHOD  
1.1. INTRODUCTION 
This method describes a procedure to measure the Length Weighted 
Geometric Mean Diameter (LWGMD) of bulk Man Made Mineral 
Fibres (MMMF). As the LWGMD of the population will have a 95 % probability of being between the 95 % confidence levels (LWGMD ± two standard errors) of the sample, the value reported 
(the test value) will be the lower 95 % confidence limit of the 
sample (i.e. LWGMD — 2 standard errors). The method is based on an update (June 1994) of a draft HSE industry procedure agreed at a meeting between ECFIA and HSE at Chester on 26/9/93 and 
developed for and from a second inter-laboratory trial (1, 2). This 
measurement method can be used to characterise the fibre diameter of bulk substances or products containing MMMFs including refractory ceramic fibres (RCF), man-made vitreous fibres 
(MMVF), crystalline and polycrystalline fibres. 
Length weighting is a means of compensating for the effect on the 
diameter distribution caused by the breakage of long fibres when 
sampling or handling the material. Geometric statistics (geometric 
mean) are used to measure the size distribution of MMMF diameters because these diameters usually have size distributions that approximate to log normal. 
Measuring length as well as diameter is both tedious and time 
consuming but, if only those fibres that touch an infinitely thin line on a SEM field of view are measured, then the probability of 
selecting a given fibre is proportional to its length. As this takes care 
of the length in the length weighting calculations, the only measurement required is the diameter and the LWGMD-2SE can be calculated as described.
 
1.2. DEFINITIONS  
Particle : An object with a length to width ratio of less than 3:1.  
Fibre : An object with a length to with ratio (aspect ratio) of at 
least 3:1.  
1.3. SCOPE AND LIMITATIONS 
The method is designed to look at diameter distributions which have 
median diameters from 0,5 μm to 6 μm. Larger diameters can be 
measured by using lower SEM magnifications but the method will be increasingly limited for finer fibre distributions and a TEM (trans ­
mission electron microscope) measurement is recommended if the 
median diameter is below 0,5 μm.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 155
 
1.4. PRINCIPLE OF THE TEST METHOD 
A number of representative core samples are taken from the fibre 
blanket or from loose bulk fibre. The bulk fibres are reduced in length using a crushing procedure and a representative sub-sample 
dispersed in water. Aliquots are extracted and filtered through a 
0,2 μm pore size, polycarbonate filter and prepared for examination 
using scanning electron microscope (SEM) techniques. The fibre diameters are measured at a screen magnification of × 10 000 or 
greater (
 1 ) using a line intercept method to give an unbiased 
estimate of the median diameter. The lower 95 % confidence interval (based on a one sided test) is calculated to give an 
estimate of the lowest value of the geometric mean fibre diameter 
of the material.
 
1.5. DESCRIPTION OF THE TEST METHOD  
1.5.1.  Safety/precautions  
Personal exposure to airborne fibres should be minimised and a fume 
cupboard or glove box should be used for handling the dry fibres. 
Periodic personal exposure monitoring should be carried out to determine the effectiveness of the control methods. When handling MMMF’s disposable gloves should be worn to reduce skin irritation 
and to prevent cross-contamination.
 
1.5.2.  Apparatus/equipment  
— Press and dyes (capable of producing 10 MPa). 
— 0,2 μm pore size polycarbonate capillary pore filters (25 mm 
diameter). 
— 5 μm pore size cellulose ester membrane filter for use as a 
backing filter. 
— Glass filtration apparatus (or disposable filtration systems) to take 
25 mm diameter filters (e.g. Millipore glass microanalysis kit, 
type No XX10 025 00). 
— Freshly distilled water that has been filtered through a 0,2 μm 
pore size filter to remove micro-organisms. 
— Sputter coater with a gold or gold/palladium target. — Scanning electron microscope capable of resolving down 
to 10 nm and operating at × 10 000 magnification. 
— Miscellaneous: spatulas, type 24 scalpel blade, tweezers, SEM 
tubes, carbon glue or carbon adhesive tape, silver dag. 
— Ultrasonic probe or bench top ultrasonic bath. 
— Core sampler or cork borer, for taking core samples from 
MMMF blanket.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 156  
( 1 ) This magnification value is indicated for 3 μm fibres, for 6 μm fibres a magnification 
of × 5 000 may be more suitable.
 1.5.3.  Test Procedure  
1.5.3.1.  Sampling  
For blankets and bats a 25 mm core sampler or cork borer is used to 
take samples of the cross-section. These should be equally spaced 
across the width of a small length of the blanket or taken from 
random areas if long lengths of the blanket are available. The same equipment can be used to extract random samples from loose fibre. Six samples should be taken when possible, to reflect 
spatial variations in the bulk material. 
The six core samples should be crushed in a 50 mm diameter dye at 
10 MPa. The material is mixed with spatula and re-pressed at 
10 MPa. The material is then removed from the dye and stored in a sealed glass bottle.
 
1.5.3.2.  Sample Preparation  
If necessary, organic binder can be removed by placing the fibre 
inside a furnace at 450 °C for about one hour. 
Cone and quarter to subdivide the sample (this should be done inside 
a dust cupboard). 
Using a spatula, add a small amount (< 0,5 g) of sample to 100 ml 
of freshly distilled water that has been filtered through a 0,2 μm 
membrane filter (alternative sources of ultra pure water may be 
used if they are shown to be satisfactory). Disperse thoroughly by 
the use of an ultrasonic probe operated at 100 W power and tuned so that cavitation occurs. (If a probe is not available use the following method: repeatedly shake and invert for 30 seconds; ultrasonic in a 
bench top ultrasonic bath for five minutes; then repeatedly shake and 
invert for a further 30 seconds.) 
Immediately after dispersion of the fibre, remove a number of 
aliquots (e.g. three aliquots of 3, 6 and 10 ml) using a wide- 
mouthed pipette (2-5 ml capacity). 
Vacuum filter each aliquot through a 0,2 μm polycarbonate filter 
supported by a 5 μm pore MEC backing filter, using a 25 mm 
glass filter funnel with a cylindrical reservoir. Approximately 5 ml of filtered distilled water should be placed into the funnel and the aliquot slowly pipetted into the water holding the pipette tip below 
the meniscus. The pipette and the reservoir must be flushed thor ­
oughly after pipetting, as thin fibres have a tendency to be located more on the surface. 
Carefully remove the filter and separate it from the backing filter 
before placing it in a container to dry.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 157
 
Cut a quarter or half filter section of the filtered deposit with a type 
24 scalpel blade using a rocking action. Carefully attach the cut 
section to a SEM stub using a sticky carbon tab or carbon glue. Silver dag should be applied in at least three positions to improve the electrical contact at the edges of the filter and the stub. When the 
glue/silver dag is dry, sputter coat approximately 50 nm of gold or 
gold/palladium onto the surface of the deposit.
 
1.5.3.3.  SEM calibration and operation  
1.5.3.3.1. C a l i b r a t i o n 
The SEM calibration should be checked at least once a week (ideally 
once a day) using a certified calibration grid. The calibration should be checked against a certified standard and if the measured value 
(SEM) is not within ± 2 % of the certified value, then the SEM 
calibration must be adjusted and re-checked. 
The SEM should be capable of resolving at least a minimum 
visible diameter of 0,2 μm, using a real sample matrix, at a magnifi ­
cation of × 2 000.
 
1.5.3.3.2. O p e r a t i o n 
The SEM should be operated at 10 000 magnification (  1 ) using 
conditions that give good resolution with an acceptable image at slow scan rates of, for example, 5 seconds per frame. Although 
the operational requirements of different SEMs may vary, 
generally to obtain the best visibility and resolution, with relatively low atomic weight materials, accelerating voltages of 5-10 keV should be used with a small spot size setting and short working 
distance. As a linear traverse is being conducted, then a 0° tilt 
should be used to minimise re-focussing or, if the SEM has a eucentric stage, the eucentric working distance should be used. Lower magnification may be used if the material does not contain 
small (diameter) fibres and the fibre diameters are large (> 5 μm).
 
1.5.3.4.  Sizing  
1.5.3.4.1. L o w m a g n i f i c a t i o n e x a m i n a t i o n t o a s s e s s t h e 
s a m p l e 
Initially the sample should be examined at low magnification to look 
for evidence of clumping of large fibres and to assess the fibre density. In the event of excessive clumping it is recommended that 
a new sample is prepared. 
For statistical accuracy it is necessary to measure a minimum number 
of fibres and high fibre density may seem desirable as examining 
empty fields is time consuming and does not contribute to the analysis. However, if the filter is overloaded, it becomes difficult to measure all the measurable fibres and, because small fibres may 
be obscured by larger ones, they may be missed.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 158  
( 1 ) For 3 μm fibres, see previous note.
 Bias towards over estimating the LWGMD may result from fibre 
densities in excess of 150 fibres per millimetre of linear traverse. 
On the other hand, low fibre concentrations will increase the time of analysis and it is often cost effective to prepare a sample with a fibre density closer to the optimum than to persist with counts on low 
concentration filters. The optimum fibre density should give an 
average of about one or two countable fibre per fields of view at 5 000 magnification. Nevertheless the optimum density will depend on the size (diameter) of the fibres, so it is necessary that the 
operator uses some expert judgement in order to decide whether 
the fibre density is close to optimal or not.
 
1.5.3.4.2. L e n g t h w e i g h t i n g o f t h e f i b r e d i a m e t e r s 
Only those fibres that touch (or cross) an (infinitely) thin line drawn 
on the screen of the SEM are counted. For this reason a horizontal (or vertical) line is drawn across the centre of the screen. 
Alternatively a single point is placed at the centre of the screen and a 
continuous scan in one direction across the filter is initiated. Each 
fibre of aspect ratio grater than 3:1 touching or crossing this point 
has its diameter measured and recorded.
 
1.5.3.4.3. F i b r e s i z i n g 
It is recommended that a minimum of 300 fibres are measured. Each 
fibre is measured only once at the point of intersection with the line 
or point drawn on the image (or close to the point of intersection if 
the fibre edges are obscured). If fibres with non-uniform cross sections are encountered, a measurement representing the average diameter of the fibre should be used. Care should be taken in 
defining the edge and measuring the shortest distance between the 
fibre edges. Sizing may be done on line, or off-line on stored images or photographs. Semi-automated image measurement systems that download data directly into a spreadsheet are recommended, as 
they save time, eliminate transcription errors and calculations can 
be automated. 
The ends of long fibres should be checked at low magnification to 
ensure that they do not curl back into the measurement field of view and are only measured once.
 
2. DATA  
2.1. TREATMENT OF RESULTS 
Fibre diameters do not usually have a normal distribution. However, 
by performing a log transformation it is possible to obtain a 
distribution that approximates to normal. 
Calculate the arithmetic mean (mean lnD) and the standard deviation 
(SD  lnD ) of the log to base e values (lnD) of the n fibre diameters (D). 
mean lnD ¼ ΣlnD 
n (1) ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 159
 
SD lnD ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ  
Σ ðlnD – mean lnDÞ 2 
n – 1 s 
(2) 
The standard deviation is divided by the square root of the number 
of measurements (n) to obtain the standard error (SE  lnD ). 
SE lnD ¼ SD 
ﬃﬃﬃ n p (3) 
Subtract two times the standard error from the mean and calculate 
the exponential of this value (mean minus two standard errors) to 
give the geometric mean minus two geometric standard errors. 
LWGMD – 2SE ¼ e ðmean lnD – 2SE  lnd Þ (4) 
3. REPORTING  
TEST REPORT 
The test report should include at least the following information: 
— The value of LWGMD-2SE. — Any deviations and particularly those which may have an effect 
on the precision or accuracy of the results with appropriate 
justifications.
 
4. REFERENCES  
1. B. Tylee SOP MF 240. Health and Safety Executive, February 1999. 
2. G. Burdett and G. Revell. Development of a standard method to 
measure the length-weigthed geometric mean fibre diameter: Results of the Second inter-laboratory exchange. IR/L/MF/94/ ­
07. Project R42.75 HPD. Health and Safety Executive, 
Research and Laboratory Services Division, 1994.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 160
 
A.23.  PARTITION COEFFICIENT (1-OCTANOL/WATER): SLOW- 
STIRRING METHOD  
INTRODUCTION 
1. This Test Method is equivalent to OECD Test Guideline (TG) 123 (2006). 
1-octanol/water partition coefficient (P  OW ) values up to a log P  OW of 8,2 
have been accurately determined by the slow-stirring method (1). Therefore 
it is a suitable experimental approach for the direct determination of P  OW of 
highly hydrophobic substances. 
2. Other methods for the determination of the 1-octanol/water partition coef ­
ficient (P  OW ) are the ‘shake-flask’ method (2), and the determination of the 
P OW from reversed phase HPLC-retention behaviour (3). The ‘shake-flask’ 
method is prone to artifacts due to transfer of octanol micro-droplets into the aqueous phase. With increasing values of P
 OW the presence of these droplets 
in the aqueous phase leads to an increasing overestimation of the concen ­
tration of the test substance in the water. Therefore, its use is limited to 
substances with log P  OW < 4. The second method relies on solid data of 
directly determined P  OW values to calibrate the relationship between 
HPLC-retention behaviour and measured values of P  OW . A draft OECD 
guideline was available for determining 1-octanol/water partition coefficients 
of ionisable substances (4) but shall no longer be used. 
3. This Test Method has been developed in The Netherlands. The precision of 
the methods described here has been validated and optimized in a ring-test 
validation study in which 15 laboratories participated (5).  
INITIAL CONSIDERATIONS  
Significance and use  
4. For inert organic substances highly significant relationships have been found 
between 1-octanol/water partition coefficients (P  OW ) and their bioaccumu ­
lation in fish. Moreover, P  OW has been demonstrated to be correlated to fish 
toxicity as well as to sorption of chemicals to solids such as soils and sediments. An extensive overview of the relationships has been given in reference (6). 
5. A wide variety of relationships between the 1-octanol/water partition coef ­
ficient and other substance properties of relevance to environmental toxi ­
cology and chemistry have been established. As a consequence, the 1- octanol/water partition coefficient has evolved as a key parameter in the assessment of the environmental risk of chemicals as well as in the 
prediction of fate of chemicals in the environment.
 
Scope  
6. The slow-stirring experiment is thought to reduce the formation of 
micro-droplets from 1-octanol droplets in the water phase. As a 
consequence, overestimation of the aqueous concentration due to test substance molecules associated to such droplets does not occur. Therefore, the slow-stirring method is particularly suitable for the determination of P
 OW 
for substances with expected log P  OW values of 5 and higher, for which the 
shake-flask method (2) is prone to yield erroneous results.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 161
 
DEFINITION AND UNITS 
7. The partition coefficient of a substance between water and a lipophilic 
solvent (1-octanol) characterizes the equilibrium distribution of the chemical between the two phases. The partition coefficient between water 
and 1-octanol (P
 OW ) is defined as the ratio of the equilibrium concentrations 
of the test substance in 1-octanol saturated with water (C  O ) and water 
saturated with 1-octanol (C  W ). 
P OW ¼ C O=C W 
As a ratio of concentrations it is dimensionless. Most frequently it is given 
as the logarithm to the base 10 (log P  OW ). P  OW is temperature dependent 
and reported data should include the temperature of the measurement.  
PRINCIPLE OF THE METHOD 
8. In order to determine the partitioning coefficient, water, 1-octanol, and the 
test substance are equilibrated with each other at constant temperature. Then the concentrations of the test substance in the two phases are determined. 
9. The experimental difficulties associated with the formation of micro-droplets 
during the shake-flask experiment can be reduced in the slow-stirring experiment proposed here. In the slow-stirring experiment, water, 1- 
octanol and the test substance are equilibrated in a thermostated stirred 
reactor. Exchange between the phases is accelerated by stirring. The stirring introduces limited turbulence which enhances the exchange between 1-octanol and water without micro-droplets being formed (1).
 
APPLICABILITY OF THE TEST 
10. Since the presence of substances other than the test substance might 
influence the activity coefficient of the test substance, the test substance 
should be tested as a pure substance. The highest purity commercially available should be employed for the 1-octanol/water partition experiment. 
11. The present method applies to pure substances that do not dissociate or 
associate and that do not display significant interfacial activity. It can be applied to determine the 1-octanol/water partition ratio of such substances and of mixtures. When the method is used for mixtures, the 1-octanol/water 
partition ratios determined are conditional and depend on the chemical 
composition of the mixture tested and on the electrolyte composition employed as aqueous phase. Provided additional steps are taken, the method is also applicable to dissociating or associating compounds 
(paragraph 12). 
12. Due to the multiple equilibria in water and 1-octanol involved in the 1- 
octanol/water partitioning of dissociating substances such as organic acids 
and phenols, organic bases, and organometallic substances, the 1- 
octanol/water partition ratio is a conditional constant strongly dependent on electrolyte composition (7)(8). Determination of the 1-octanol/water partition ratio therefore requires that pH and electrolyte composition be 
controlled in the experiment and reported. Expert judgement has to be 
employed in the evaluation of these partition ratios. Using the value of dissociation constant(s), suitable pH-values need to be selected, such that a partitioning ratio is determined for each ionization state. Non-complexing 
buffers must be used when testing organometallic compounds (8). Taking 
the existing knowledge on the aqueous chemistry (complexation constants, dissociation constants) into account, the experimental conditions should be chosen in such a manner that the speciation of the test substance in the 
aqueous phase can be estimated. The ionic strength should be identical in all 
experiments by employing a background electrolyte.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 162
 
13. Difficulties in the test may arise in conducting the test for substances with 
low water solubility or high P  OW , due to the fact that the concentrations in 
the water become very low such that their accurate determination is difficult. This Test Method provides guidance on how to deal with this problem.
 
INFORMATION ON THE TEST SUBSTANCE 
14. Chemical reagents should be of analytical grade or of higher purity. The use 
of non-labelled test substances with known chemical composition and preferably at least 99 % purity, or of radiolabelled test substances with 
known chemical composition and radiochemical purity, is recommended. 
In the case of short half-life tracers, decay corrections should be applied. In the case of radiolabelled test substances, a chemical specific analytical method should be employed to ensure that the measured radioactivity is 
directly related to the test substance. 
15. An estimate of log P
 OW may be obtained by using commercially available 
software for estimation of log P  OW , or by using the ratio of the solubilities 
in both solvents. 
16. Before carrying out a slow-stirring experiment for determination of P  OW , the 
following information on the test substance should be available: 
(a) structural formula 
(b) suitable analytical methods for determination of the concentration of the 
substance in water and 1-octanol 
(c) dissociation constant(s) of ionisable substances (OECD Guideline 112 
(9)) 
(d) aqueous solubility (10) 
(e) abiotic hydrolysis (11) 
(f) ready biodegradability (12) 
(g) vapour pressure (13).  
DESCRIPTION OF THE METHOD  
Equipment and apparatus  
17. Standard laboratory equipment is required, in particular, the following: 
— magnetic stirrers and Teflon coated magnetic stir bars are employed to 
stir the water phase; 
— analytical instrumentation, suitable for determination of the concentration 
of the test substance at the expected concentrations; 
— stirring-vessel with a tap at the bottom. Dependent on the estimate of log 
P OW and the Limit of Detection (LOD) of the test compound, the use of 
a reaction vessel of the same geometry larger than one litre has to be 
considered, so that a sufficient volume of water can be obtained for 
chemical extraction and analysis. This will result in higher concen ­
trations in the water extract and thus a more reliable analytical deter ­
mination. A table giving estimates of the minimum volume needed, the 
LOD of the compound, its estimated log P  OW and its water solubility is 
given in Appendix 1. The table is based on the relationship between log P
 OW and the ratio between the solubilities in octanol and water, as 
presented by Pinsuwan  et al.  (14): 
log P  OW ¼ 0; 88 log SR þ 0; 41 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 163
 
where  
SR ¼ S oct=S w (in molarity); 
and the relationship given by Lyman (15) for predicting water solubility. 
Water solubilities calculated with the equation given in Appendix 1 must be seen as a first estimate. It should be noted that the user is free to generate an estimate of water solubility by means of any relationship 
that is considered to better represent the relationship between hydro ­
phobicity and solubility. For solid compounds, inclusion of melting point in the prediction of solubility is for instance recommended. In 
case a modified equation is used, it should be ascertained that the 
equation for calculation of solubility in octanol is still valid. A schematic drawing of a glass-jacketed stirring-vessel with a volume of ca. one litre is given in Appendix 2. The proportions of the vessel shown 
in Appendix 2 have proven favourable and should be maintained when 
apparatus of a different size is used; 
— a means for keeping the temperature constant during the slow-stirring 
experiment is essential. 
18. Vessels should be made from inert material such that adsorption to vessel 
surfaces is negligible.
 
Preparation of the test solutions  
19. The P  OW determination should be carried out with the highest purity 1- 
octanol that is commercially available (at least + 99 %). Purification of 1- 
octanol by extraction with acid, base and water and subsequent drying is 
recommended. In addition, distillation can be used to purify 1-octanol. Purified 1-octanol is to be used to prepare standard solutions of the test substances. Water to be used in the P
 OW determination should be glass or 
quartz distilled, or obtained from a purification system, or HPLC-grade 
water may be used. Filtration through a 0,22 μm filter is required for 
distilled water, and blanks should be included to check that no impurities are in the concentrated extracts that may interfere with the test substance. If 
a glass fibre filter is used, it should be cleaned by baking for at least 
three hours at 400 °C. 
20. Both solvents are mutually saturated prior to the experiment by equilibrating 
them in a sufficiently large vessel. This is accomplished by slow-stirring the two-phase system for two days. 
21. An appropriate concentration of test substance is selected and dissolved in 
1-octanol (saturated with water). The 1-octanol/water partition coefficient needs to be determined in dilute solutions in 1-octanol and water. 
Therefore the concentration of the test substance should not exceed 70 % 
of its solubility with a maximum concentration of 0,1 M in either phase (1). The 1-octanol solutions used for the experiment must be devoid of suspended solid test substance. 
22. The appropriate amount of test substance is dissolved in 1-octanol (saturated 
with water). If the estimate of log P
 OW exceeds five, care has to be taken 
that the 1-octanol solutions used for the experiment are devoid of suspended 
solid test substance. To that end, the following procedure for chemicals with an estimated value of log P
 OW > 5 is followed: 
— the test substance is dissolved in 1-octanol (saturated with water);  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 164
 
— the solution is given sufficient time for the suspended solid substance to 
settle out. During the settling period, the concentration of the test 
substance is monitored; 
— after the measured concentrations in the 1-octanol-solution have attained 
stable values, the stock solution is diluted with an appropriate volume of 
1-octanol; 
— the concentration of the diluted stock solution is measured. If the 
measured concentration is consistent with the dilution, the diluted stock solution can be employed in the slow-stirring experiment.
 
Extraction and analysis of samples  
23. A validated analytical method should be used for the assay of test substance. 
The investigators have to provide evidence that the concentrations in the 
water saturated 1-octanol as well as in the 1-octanol saturated water phase during the experiment are above the method limit of quantification of the analytical procedures employed. Analytical recoveries of the test substance 
from the water phase and from the 1-octanol phase need to be established 
prior to the experiment in those cases for which extraction methods are necessary. The analytical signal needs to be corrected for blanks and care should be taken that no carry-over of analyte from one sample to another 
can occur. 
24. Extraction of the water phase with an organic solvent and preconcentration 
of extract are likely to be required prior to analysis, due to rather low concentrations of hydrophobic test substances in the water phase. For the 
same reason it is necessary to reduce eventual blank concentrations. To that 
end, it is necessary to employ high purity solvents, preferably solvents for residue analysis. Moreover, working with carefully pre-cleaned (e.g. solvent washing or baking at elevated temperature) glassware can help to avoid 
cross-contamination. 
25. An estimate of log P
 OW may be obtained from an estimation program or by 
expert judgment. If the value is higher than six then blank corrections and analyte carry-over need to be monitored closely. Similarly, if the estimate of 
log P
 OW exceeds six, the use of a surrogate standard for recovery correction 
is mandatory, so that high preconcentration factors can be reached. A number of software programs for the estimation of log P
 OW are commer ­
cially available (  1 ), e.g. Clog P (16), KOWWIN (17), ProLogP (18) and 
ACD log P (19). Descriptions of the estimation approaches can be found in references (20-22). 
26. The limits of quantification (LOQ) for determination of the test substance in 
1-octanol and water are established using accepted methods. As a rule of 
thumb, the method limit of quantification can be determined as the concen ­
tration in water or 1-octanol that produces a signal to noise ratio of ten. A suitable extraction and pre-concentration method should be selected and 
analytical recoveries should also be specified. A suitable pre-concentration 
factor is selected in order to obtain a signal of the required size upon analytical determination.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 165  
( 1 ) This information is only given for the convenience of users. Other equivalent computer 
programmes may be used if they can be shown to produce the same results.
 27. On the basis of the parameters of the analytical method and the expected 
concentrations, an approximate sample size required for an accurate deter ­
mination of the compound concentration is determined. The use of water samples that are too small to obtain a sufficient analytical signal should be avoided. Also, the use of excessively large water samples should be 
avoided, since otherwise there might be too little water left for the 
minimum number of analyses required (n = 5). In Appendix 1, the minimum sample volume is indicated as a function of the vessel volume, the LOD of the test substance and the solubility of the test substance. 
28. Quantification of the test substances occurs by comparison with calibration 
curves of the respective compound. The concentrations in the samples analysed must be bracketed by concentrations of standards. 
29. For test substances with a log P
 OW estimate higher than six a surrogate 
standard has to be spiked to the water sample prior to extraction in order to register losses occurring during extraction and pre-concentration of the water samples. For accurate recovery correction, the surrogates must have prop ­
erties that are very close to, or identical with, those of the test substance. 
Preferably, (stable) isotopically-labelled analogues of the substances of interest (for example, perdeuterated or
 13 C-labelled) are used for this 
purpose. If the use of labelled stable isotopes, i.e.  13 C or  2 H, is not 
possible it should be demonstrated from reliable data in the LITERATURE 
that the physical-chemical properties of the surrogate are very close to those of the test substance. During liquid-liquid extraction of the water phase emulsions can form. They can be reduced by addition of salt and 
allowing the emulsion to settle overnight. Methods used for extracting 
and pre-concentrating the samples need to be reported. 
30. Samples withdrawn from the 1-octanol phase may, if necessary, be diluted 
with a suitable solvent prior to analysis. Moreover, the use of surrogate 
standards for recovery correction is recommended for substances for which the recovery experiments demonstrated a high degree of variation in the recovery experiments (relative standard deviation > 10 %). 
31. The details of the analytical method need to be reported. This includes the 
method of extraction, pre-concentration and dilution factors, instrument parameters, calibration routine, calibration range, analytical recovery of the test substance from water, addition of surrogate standards for recovery 
correction, blank values, detection limits and limits of quantification.
 
Performance of the Test  
Optimal 1-octanol/water volume ratios  
32. When choosing the water and 1-octanol volumes, the LOQ in 1-octanol and 
water, the pre-concentration factors applied to the water samples, the volumes sampled in 1-octanol and water, and the expected concentrations should be considered. For experimental reasons, the volume of 1-octanol in 
the slow-stirring system should be chosen such that the 1-octanol layer is 
sufficiently thick (> 0,5 cm) in order to allow for sampling of the 1-octanol phase without disturbing it. 
33. Typical phase ratios used for the determinations of compounds with log 
P
 OW of 4,5 and higher are 20 to 50 ml of 1-octanol and 950 to 980 ml 
of water in a one litre vessel.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 166
 
Test conditions  
34. During the test the reaction vessel is thermostated to reduce temperature 
variation to below 1 °C. The assay should be performed at 25 °C. 
35. The experimental system should be protected from daylight by either 
performing the experiment in a dark room or by covering the reaction vessel with aluminium foil. 
36. The experiment should be performed in a dust-free (as far as possible) 
environment. 
37. The 1-octanol-water system is stirred until equilibrium is attained. In a pilot 
experiment the length of the equilibration period is assessed by performing 
a slow-stirring experiment and sampling water and 1-octanol periodically. The sampling time points should be interspersed by a minimum period of five hours. 
38. Each P
 OW determination has to be performed employing at least three 
independent slow-stirring experiments.  
Determination of the equilibration time  
39. It is assumed that the equilibrium is achieved when a regression of the 1- 
octanol/water concentration ratio against time over a time span of four time 
points yields a slope that is not significantly different from zero at a p-level of 0,05. The minimum equilibration time is one day before sampling can be started. As a rule of thumb, sampling of substances with a log P
 OW estimate 
of less than five can take place during days two and three. The equilibration 
might have to be extended for more hydrophobic compounds. For a compound with log P
 OW of 8,23 (decachlorobiphenyl) 144 hours were 
sufficient for equilibration. Equilibrium is assessed by means of repeated 
sampling of a single vessel.  
Starting the experiment  
40. At the start of the experiment the reaction vessel is filled with 1-octanol- 
saturated water. Sufficient time should be allowed to reach the thermostated temperature. 
41. The desired amount of test substance (dissolved in the required volume of 1- 
octanol saturated with water) is carefully added to the reaction vessel. This is a crucial step in the experiment, since turbulent mixing of the two phases 
has to be avoided. To that end, the 1-octanol phase can be pipetted slowly 
against the wall of the experimental vessel, close to the water surface. It will subsequently flow along the glass wall and form a film above the water phase. The decantation of 1-octanol directly into the flask should always be 
avoided; drops of 1-octanol should not be allowed to fall directly into the 
water. 
42. After starting the stirring, the stirring rate should be increased slowly. If the 
stirring motors cannot be appropriately adjusted the use of a transformer 
should be considered. The stirring rate should be adjusted so that a vortex at the interface between water and 1-octanol of 0,5 to maximally 2,5 cm depth is created. The stirring rate should be reduced if the vortex depth of 2,5 cm 
is exceeded; otherwise micro-droplets may be formed from 1-octanol 
droplets in the water phase, leading to an overestimation of the concen ­
tration of the test substance in the water. The maximum stirring rate of 2,5 cm is recommended on the basis of the findings in the ring-test vali ­
dation study (5). It is a compromise between achieving a rapid rate of 
equilibration, while limiting the formation of 1-octanol micro-droplets.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 167
 
Sampling and Sample Treatment  
43. The stirrer should be turned off prior to sampling and the liquids should be 
allowed to stop moving. After sampling is completed, the stirrer is started again slowly, as described above, and then the stirring rate is increased 
gradually. 
44. The water phase is sampled from a stopcock at the bottom of the reaction 
vessel. Always discard the dead volume of water contained in the taps 
(approximately 5 ml in the vessel shown in the Appendix 2). The water 
in the taps is not stirred and therefore not in equilibrium with the bulk. Note the volume of the water samples, and make sure that the amount of test substance present in the discarded water is taken into account when setting 
up a mass balance. Evaporative losses should be minimized by allowing the 
water to flow quiescently into the separatory funnel, such that there is no disturbance of the water/1-octanol layer. 
45. 1-Octanol samples are obtained by withdrawing a small aliquot (ca. 100 μl) 
from the 1-octanol layer with a 100 microlitre all glass-metal syringe. Care should be taken not to disturb the boundary. The volume of the sampled liquid is recorded. A small aliquot is sufficient, since the 1-octanol sample 
will be diluted. 
46. Unnecessary sample transfer steps should be avoided. To that end the 
sample volume should be determined gravimetrically. In case of water 
samples this can be achieved by collecting the water sample in a separatory 
funnel that contains already the required volume of solvent.
 
DATA AND REPORTING 
47. According to the present Test Method, P  OW is determined by performing 
three slow-stirring experiments (three experimental units) with the 
compound under investigation employing identical conditions. The 
regression used to demonstrate attainment of equilibrium should be based on the results of at least four determinations of C
 O /C W at consecutive time 
points. This allows for calculating variance as a measure of the uncertainty 
of the average value obtained by each experimental unit. 
48. The P  OW can be characterized by the variance in the data obtained for each 
experimental unit. This information is employed to calculate the P  OW as the 
weighted average of the results of the individual experimental units. To do 
so, the inverse of the variance of the results of the experimental units is 
employed as weight. As a result, data with a large variation (expressed as the variance) and thus with lower reliability have less influence on the result 
than data with a low variance. 
49. Analogously, the weighted standard deviation is calculated. It characterizes 
the repeatability of the P
 OW measurement. A low value of the weighted 
standard deviation indicates that the P  OW determination was very repeatable 
within one laboratory. The formal statistical treatment of the data is outlined 
below.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 168
 
Treatment of the results  
Demonstration of attainment of equilibrium  
50. The logarithm of the ratio of the concentration of the test substance in 1- 
octanol and water (log (C  O /C w )) is calculated for each sampling time. 
Achievement of chemical equilibrium is demonstrated by plotting this ratio against time. A plateau in this plot that is based on at least four 
consecutive time points indicates that equilibrium has been attained, and 
that the compound is truly dissolved in 1-octanol. If not, the test needs to be continued until four successive time points yield a slope that is not significantly different from 0 at a p-level of 0,05, indicating that log 
C
 o /C w is independent of time.  
Log P  OW -calculation  
51. The value of log P  OW of the experimental unit is calculated as the weighted 
average value of log C  o /C w for the part of the curve of log C  o /C w vs. time, 
for which equilibrium has been demonstrated. The weighted average is 
calculated by weighting the data with the inverse of the variance so that the influence of the data on the final result is inversely proportional to the uncertainty in the data.
 
Average log P  OW 
52. The average value of log P  OW of different experimental units is calculated 
as the average of the results of the individual experimental units weighted with their respective variances. 
The calculation is performed as follows: 
log P
 OW ;Av ¼ ðΣw i Ü log P  OW ;i Þ Ü ðΣw i Þ –1 
where: 
log P  OW,i = the log P  OW value of the individual experimental unit i; 
log P  OW,Av  = the weighted average value of the individual log P  OW deter­
minations; 
w i = the statistical weight assigned to the log P  OW value of the 
experimental unit i. 
The reciprocal of the variance of log P  OW,i is employed as w  i 
(w i ¼ varðlog P  OW ;i Þ –1 ) 
53. The error of the average of log P  OW is estimated as the repeatability of log 
C o /C w determined during the equilibrium phase in the individual experi ­
mental units. It is expressed as the weighted standard deviation of log 
P OW,Av  (σ log Pow,Av  ) which in turn is a measure of the error associated 
with log P  OW,Av  . The weighted standard deviation can be computed from 
the weighted variance (var  log Pow,Av  ) as follows: 
var log Pow ;Av ¼ ðΣw i Ü ðlog P  OW ;i Ä log P  OW ;Av Þ 2 Þ Ü ðΣw i Ü ðn Ä 1ÞÞ –1 
σ log Pow ;Av ¼ ðvar log Pow ;Av Þ 0;5 
The symbol n stands for the number of experimental units.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 169
 
Test Report  
54. The test report should include the following information:  
Test substance:  
— common name, chemical name, CAS number, structural formula (indi ­
cating position of label when radiolabelled substance is used) and relevant physical-chemical properties (see paragraph 17) 
— purity (impurities) of test substance 
— label purity of labelled chemicals and molar activity (where appropriate) — the preliminary estimate of log P
 ow , as well as the method used to derive 
the value.  
Test conditions:  
— dates of the performance of the studies 
— temperature during the experiment 
— volumes of 1-octanol and water at the beginning of the test — volumes of withdrawn 1-octanol and water samples 
— volumes of 1-octanol and water remaining in the test vessels 
— description of the test vessels and stirring conditions (geometry of the 
stirring bar and of the test vessel, vortex height in mm, and when 
available: stirring rate) used 
— analytical methods used to determine the test substance and the method 
limit of quantification 
— sampling times 
— the aqueous phase pH and the buffers used, when pH is adjusted for 
ionizable molecules 
— number of replicates.  
Results:  
— repeatability and sensitivity of the analytical methods used 
— determined concentrations of the test substance in 1-octanol and water as 
a function of time 
— demonstration of mass balance 
— temperature and standard deviation or the range of temperature during 
the experiment 
— the regression of concentration ratio against time 
— the average value log P
 ow,Av  and its standard error 
— discussion and interpretation of the results  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 170
 
— examples of raw data figures of representative analysis (all raw data 
have to be stored in accordance with GLP standards), including 
recoveries of surrogates, and the number of levels used in the calibration (along with the criteria for the correlation coefficient of the calibration curve), and results of quality assurance/quality control (QA/QC) 
— when available: validation report of the assay procedure (to be indicated 
among references).
 
LITERATURE:  
(1) De Bruijn JHM, Busser F, Seinen W, Hermens J. (1989). Determination of 
octanol/water partition coefficients with the ‘slow-stirring’ method. Environ. 
Toxicol. Chem. 8: 499-512. 
(2) Chapter A.8 of this Annex, Partition Coefficient. (3) Chapter A.8 of this Annex, Partition Coefficient. (4) OECD (2000). OECD Draft Guideline for the Testing of Chemicals: 122 
Partition Coefficient (n-Octanol/Water): pH-Metric Method for Ionisable Substances. Paris. 
(5) Tolls J (2002). Partition Coefficient 1-Octanol/Water (Pow) Slow-Stirring 
Method for Highly Hydrophobic Chemicals, Validation Report. RIVM 
contract-Nrs 602730 M/602700/01. 
(6) Boethling RS, Mackay D (eds.) (2000). Handbook of property estimation 
methods for chemicals. Lewis Publishers Boca Raton, FL, USA. 
(7) Schwarzenbach RP, Gschwend PM, Imboden DM (1993). Environmental 
Organic Chemistry. Wiley, New York, NY. 
(8) Arnold CG, Widenhaupt A, David MM, Müller SR, Haderlein SB, Schwar ­
zenbach RP (1997). Aqueous speciation and 1-octanol-water partitioning of 
tributyl- and triphenyltin: effect of pH and ion composition.  Environ. Sci. 
Technol.  31: 2596-2602. 
(9) OECD (1981) OECD Guidelines for the Testing of Chemicals: 112 Disso ­
ciation Constants in Water. Paris. 
(10) Chapter A.6 of this Annex, Water Solubility. (11) Chapter C.7 of this Annex, Degradation – Abiotic Degradation Hydrolysis 
as a Function of pH. 
(12) Chapter C.4 — Part II – VII (Method A to F) of this Annex, Determination 
of ‘Ready’ Biodegradability. 
(13) Chapter A.4 of this Annex, Vapour Pressure. (14) Pinsuwan S, Li A and Yalkowsky S.H. (1995). Correlation of octanol/water 
solubility ratios and partition coefficients,  J. Chem. Eng. Data.  40: 623-626. 
(15) Lyman WJ (1990). Solubility in water. In: Handbook of Chemical Property 
Estimation Methods: Environmental Behavior of Organic Compounds, Lyman WJ, Reehl WF, Rosenblatt DH, Eds. American Chemical Society, 
Washington, DC, 2-1 to 2-52. 
(16) Leo A, Weininger D (1989). Medchem Software Manual. Daylight 
Chemical Information Systems, Irvine, CA. 
(17) Meylan W (1993). SRC-LOGKOW for Windows. SRC, Syracuse, N.Y. (18) Compudrug L (1992). ProLogP. Compudrug, Ltd, Budapest. (19) ACD. ACD logP; Advanced Chemistry Development: Toronto, Ontario 
M5H 3V9, Canada, 2001.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 171
 
(20) Lyman WJ (1990). Octanol/water partition coefficient. In Lyman WJ, Reehl 
WF, Rosenblatt DH, eds,  Handbook of chemical property estimation , 
American Chemical Society, Washington, D.C. 
(21) Rekker RF, de Kort HM (1979). The hydrophobic fragmental constant: An 
extension to a 1 000 data point set.  Eur. J. Med. Chem. Chim. Ther.  14: 
479-488. 
(22) Jübermann O (1958). Houben-Weyl, ed,  Methoden der Organischen 
Chemie : 386-390.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 172
 
Appendix 1  
Spreadsheet for computation of minimum volumes of water required for 
detection of test substances of different log P  OW values in aqueous phase  
Assumptions: 
— Maximum volume of individual aliquots = 10 % of total volume; 5 aliquots = 
50 % of total volume. 
— Concentrationoftestsubstances  ¼ 0; 7 Ü solubilityineitherphase. In case of 
lower concentrations, larger volumes would be required. 
— Volume used for LOD determination = 100 ml. 
— log P  ow vs. log S  w and log P  ow vs. SR (S  oct /S w ) are reasonable represen ­
tations of relationships for test substances.  
Estimation of S  w 
log P  ow Equation log S  w S w (mg/l)  
4 ð –Þ0; 922 Ü log P  ow þ 4; 184 0,496 3,133E+00 
4,5 ð –Þ0; 922 Ü log P  ow þ 4; 184 0,035 1,084E+00 
5 ð –Þ0; 922 Ü log P  ow þ 4; 184 –0,426 3,750E-01 
5,5 ð –Þ0; 922 Ü log P  ow þ 4; 184 –0,887 1,297E-01 
6 ð –Þ0; 922 Ü log P  ow þ 4; 184 –1,348 4,487E-02 
6,5 ð –Þ0; 922 Ü log P  ow þ 4; 184 ––1,809 1,552E-02 
7 ð –Þ0; 922 Ü log P  ow þ 4; 184 –2,270 5,370E-03 
7,5 ð –Þ0; 922 Ü log P  ow þ 4; 184 –2,731 1,858E-03 
8 ð –Þ0; 922 Ü log P  ow þ 4; 184 –3,192 6,427E-04  
Estimation of S  oct 
log P  ow Equation S  oct (mg/l)  
4 log P  ow ¼ 0; 88log SR þ 0; 41 3,763E+04 
4,5 log P  ow ¼ 0; 88log SR þ 0; 42 4,816E+04 
5 log P  ow ¼ 0; 88log SR þ 0; 43 6,165E+04 
5,5 log P  ow ¼ 0; 88log SR þ 0; 44 7,890E+04 
6 log P  ow ¼ 0; 88log SR þ 0; 45 1,010E+05 
6,5 log P  ow ¼ 0; 88log SR þ 0; 46 1,293E+05 
7 log P  ow ¼ 0; 88log SR þ 0; 47 1,654E+05 
7,5 log P  ow ¼ 0; 88log SR þ 0; 48 2,117E+05 
8 log P  ow ¼ 0; 88log SR þ 0; 49 2,710E+05  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 173
 
Total Mass test 
substance 
(mg) Mass  oct /Mass  water Mass  H2O 
(mg) Conc  H2O 
(mg/l) Mass  oct 
(mg) Conc  oct 
(mg/l)  
1 319 526 2,5017 2,6333 1 317 26 333 
1 686 1 664 1,0127 1,0660 1 685 33 709 2 158 5 263 0,4099 0,4315 2 157 43 149 2 762 16 644 0,1659 0,1747 2 762 55 230 
3 535 52 632 0,0672 0,0707 3 535 70 691 
4 524 1664 36 0,0272 0,0286 4 524 90 480 5 790 5263 16 0,0110 0,0116 5 790 115 807 7 411 1 664 357 0,0045 0,0047 7 411 148 223 9 486 5 263 158 0,0018 0,0019 9 486 189 713
 
Computation of volumes  
Minimum volume required for H  2 O phase at each LOD concentration  
log K  ow LOD (micrograms/l) → 0,001 0,01 0,10 1,00 10  
4 0,04 0,38 3,80 38 380 
4,5 0,09 0,94 9,38 94 938 
5 0,23 2,32 23,18 232 2 318 
5,5 0,57 5,73 57,26 573 5 726 
6 1,41 14,15 141 1 415 14 146 
6,5 3,50 34,95 350 3 495 34 950 
7 8,64 86,35 864 8 635 86 351 
7,5 21,33 213 2 133 21 335 213 346 
8 52,71 527 5 271 52 711 527 111 
Volume used 
for LOD (l) 0,1 
Key to Computations  
Represents < 10 % of total volume of aqueous phase, 1 litre equilibration vessel. 
Represents < 10 % of total volume of aqueous phase, 2 litre equilibration vessel. 
Represents < 10 % of total volume of aqueous phase, 5 litre equilibration vessel. 
Represents < 10 % of total volume of aqueous phase, 10 litre equilibration 
vessel. 
Exceeds 10 % of even the 10 liter equilibration vessel.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 174
 
Overview of volumes required, as a function of water solubility and Log P  ow 
Minimum volume required for H  2 O phase at each LOD concentration (ml)  
log P  ow S w (mg/l) LOD (micrograms/l) → 0,001 0,01 0,10 1,00 10  
4 10 0,01 0,12 1,19 11,90 118,99 
5 0,02 0,24 2,38 23,80 237,97 3 0,04 0,40 3,97 39,66 396,62 1 0,12 1,19 11,90 118,99 1 189,86 
4,5 5 0,02 0,20 2,03 20,34 203,37 
2 0,05 0,51 5,08 50,84 508,42 
1 0,10 1,02 10,17 101,68 1 016,83 
0,5 0,20 2,03 20,34 203,37 2 033,67 
5 1 0,09 0,87 8,69 86,90 869,01 
0,5 0,17 1,74 17,38 173,80 1 738,02 
0,375 0,23 2,32 23,18 231,75 2 317,53 
0,2 0,43 4,35 43,45 434,51 4 345,05 
5,5 0,4 0,19 1,86 18,57 185,68 1 856,79 
0,2 0,37 3,71 37,14 371,36 3 713,59 0,1 0,74 7,43 74,27 742,72 7 427,17 
0,05 1,49 14,85 148,54 1 485,43 14 854,35 
6 0,1 0,63 6,35 63,48 634,80 6 347,95 
0,05 1,27 12,70 126,96 1 269,59 12 695,91 
0,025 2,54 25,39 253,92 2 539,18 25 391,82 
0,0125 5,08 50,78 507,84 5 078,36 50 783,64 
6,5 0,025 2,17 21,70 217,02 2 170,25 21 702,46 
0,0125 4,34 43,40 434,05 4 340,49 43 404,93 
0,006 9,04 90,43 904,27 9 042,69 90 426,93 
0,003 18,09 180,85 1 808,54 18 085,39 180 853,86 
7 0,006 7,73 77,29 772,89 7 728,85 77 288,50 
0,003 15,46 154,58 1 545,77 15 457,70 154 577,01 
0,0015 23,19 231,87 2 318,66 23 186,55 231 865,51 
0,001 46,37 463,73 4 637,31 46 373,10 463 731,03 
7,5 0,002 19,82 198,18 1 981,77 19 817,73 198 177,33 
0,001 39,64 396,35 3 963,55 39 635,47 396 354,66 
0,0005 79,27 792,71 7 927,09 79 270,93 792 709,32
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 175
 
log P  ow S w (mg/l) LOD (micrograms/l) → 0,001 0,01 0,10 1,00 10  
0,00025 158,54 1 585,42 15 854,19 158 541,86 1 585 418,63 
8 0,001 33,88 338,77 3 387,68 33 876,77 338 767,72 
0,0005 67,75 677,54 6 775,35 67 753,54 677 535,44 
0,00025 135,51 1 355,07 13 550,71 135 507,09 1 355 070,89 
0,000125 271,01 2 710,14 27 101,42 271 014,18 2 710 141,77 
Volume used for LOD (l) 0,1  
Appendix 2  
An example of glass-jacketed test vessel for the slow-stirring experiment for 
determination of P  OW
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 176
 
PART B: METHODS FOR THE DETERMINATION OF TOXICITY AND 
OTHER HEALTH EFFECTS  
TABLE OF CONTENTS 
GENERAL INTRODUCTION 
B.1 bis. ACUTE ORAL TOXICITY — FIXED DOSE PROCEDURE B.1 tris. ACUTE ORAL TOXICITY — ACUTE TOXIC CLASS 
METHOD 
B.2. ACUTE INHALATION TOXICITY B.3. ACUTE TOXICITY (DERMAL) 
B.4. ACUTE TOXICITY: DERMAL IRRITATION/CORROSION 
B.5. ACUTE TOXICITY: EYE IRRITATION/CORROSION B.6. SKIN SENSITISATION 
B.7. REPEATED DOSE 28-DAY ORAL TOXICITY STUDY IN 
RODENTS 
B.8. SUBACUTE INHALATION TOXICITY: 28-DAY STUDY 
B.9. REPEATED DOSE (28 DAYS) TOXICITY (DERMAL) B.10. MUTAGENICITY —  IN VITRO  MAMMALIAN CHRO ­
MOSOME ABERRATION TEST 
B.11. MUTAGENICITY —  IN VIVO  MAMMALIAN BONE 
MARROW CHROMOSOME ABERRATION TEST 
B.12. MUTAGENICITY —  IN VIVO  MAMMALIAN 
ERYTHROCYTE MICRONUCLEUS TEST 
B.13/14. MUTAGENICITY: REVERSE MUTATION TEST USING 
BACTERIA 
B.15. MUTAGENICITY TESTING AND SCREENING FOR CARCI ­
NOGENICITY GENE MUTATION —  SACCHAROMYCES 
CEREVISIAE  
B.16. MITOTIC RECOMBINATION — SACCHAROMYCES 
CEREVISIAE 
B.17. MUTAGENICITY — IN VITRO MAMMALIAN CELL GENE 
MUTATION TEST 
B.18. DNA DAMAGE AND REPAIR — UNSCHEDULED DNA 
SYNTHESIS — MAMMALIAN CELLS  IN VITRO  
B.19. SISTER CHROMATID EXCHANGE ASSAY  IN VITRO  
B.20. SEX-LINKED RECESSIVE LETHAL TEST IN  DROSOPHILA 
MELANOGASTER
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 177
 
B.21.  IN VITRO  MAMMALIAN CELL TRANSFORMATION TESTS 
B.22. RODENT DOMINANT LETHAL TEST B.23. MAMMALIAN SPERMATOGONIAL CHROMOSOME ABER ­
RATION TEST 
B.24. MOUSE SPOT TEST B.25. MOUSE HERITABLE TRANSLOCATION B.26. SUB-CHRONIC ORAL TOXICITY TEST REPEATED DOSE 90 
— DAY ORAL TOXICITY STUDY IN RODENTS 
B.27. SUB-CHRONIC ORAL TOXICITY TEST REPEATED DOSE 90 
— DAY ORAL TOXICITY STUDY IN NON-RODENTS 
B.28. SUB-CHRONIC DERMAL TOXICITY STUDY 90-DAY 
REPEATED DERMAL DOSE STUDY USING RODENT SPECIES 
B.29. SUBCHRONIC INHALATION TOXICITY: 90-DAY STUDY B.30. CHRONIC TOXICITY STUDIES B.31. PRENATAL DEVELOPMENTAL TOXICITY STUDY B.32. CARCINOGENICITY STUDIES B.33. COMBINED CHRONIC TOXICITY/CARCINOGENICITY 
STUDIES 
B.34. ONE-GENERATION REPRODUCTION TOXICITY TEST B.35. TWO-GENERATION REPRODUCTION TOXICITY STUDY B.36. TOXICOKINETICS B.37. DELAYED NEUROTOXICITY OF ORGANOPHOSPHORUS 
SUBSTANCES FOLLOWING ACUTE EXPOSURE 
B.38. DELAYED NEUROTOXICITY OF ORGANOPHOSPHORUS 
SUBSTANCES 28 DAY REPEATED DOSE STUDY 
B.39. UNSCHEDULED DNA SYNTHESIS (UDS) TEST WITH 
MAMMALIAN LIVER CELLS  IN VIVO  
B.40.  IN VITRO  SKIN CORROSION: TRANSCUTANEOUS ELEC ­
TRICAL RESISTANCE TEST (TER) 
B.40 BIS.  IN VITRO  SKIN CORROSION: HUMAN SKIN MODEL TEST
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 178
 
B.41.  IN VITRO  3T3 NRU PHOTOTOXICITY TEST 
B.42. SKIN SENSITISATION: LOCAL LYMPH NODE ASSAY 
B.43. NEUROTOXICITY STUDY IN RODENTS 
B.44. SKIN ABSORPTION:  IN VIVO  METHOD 
B.45. SKIN ABSORPTION:  IN VITRO  METHOD 
B.46.  IN VITRO  SKIN IRRITATION: RECONSTRUCTED HUMAN 
EPIDERMIS TEST METHOD 
B.47. BOVINE CORNEAL OPACITY AND PERMEABILITY TEST 
METHOD FOR IDENTIFYING OCULAR CORROSIVES AND 
SEVERE IRRITANTS 
B.48. ISOLATED CHICKEN EYE TEST METHOD FOR IDEN ­
TIFYING OCULAR CORROSIVES AND SEVERE IRRITANTS 
B.49.  IN VITRO  MAMMALIAN CELL MICRONUCLEUS TEST 
B.50. SKIN SENSITISATION: LOCAL LYMPH NODE ASSAY: DA B.51. SKIN SENSITISATION: LOCAL LYMPH NODE ASSAY: 
BrdU-ELISA
 
▼M4 
B.52. ACUTE INHALATION TOXICITY — ACUTE TOXIC CLASS 
METHOD  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 179
 
GENERAL INTRODUCTION  
A. CHARACTERISATION OF THE TEST SUBSTANCE  
The composition of the test substance, including major impurities, 
and its relevant physico-chemical properties including stability, 
should be known prior to the initiation of any toxicity study. 
The physico-chemical properties of the test substance provide 
important information for the selection of the route of administration, 
the design of each particular study and the handling and storage of 
the test substance. 
The development of an analytical method for qualitative and quanti ­
tative determination of the test substance (including major impurities 
when possible) in the dosing medium and the biological material should precede the initiation of the study. 
All information relating to the identification, the physico-chemical 
properties, the purity, and behaviour of the test substance should be included in the test report.
 
B. ANIMAL CARE  
Stringent control of environmental conditions and proper animal care 
techniques are essential in toxicity testing.  
(i) Housing conditions  
The environmental conditions in the experimental animal rooms or 
enclosures should be appropriate to the test species. For rats, mice 
and guinea pigs, suitable conditions are a room temperature of 22  o C 
± 3  o C with a relative humidity of 30 to 70 %; for rabbits the 
temperature should be 20 ± 3  o C with a relative humidity of 30 to 70 %. 
Some experimental techniques are particularly sensitive to 
temperature effects and, in these cases, details of appropriate conditions are included in the description of the test method. In all investigations of toxic effects, the temperature and humidity should 
be monitored, recorded, and included in the final report of the study. 
Lighting should be artificial, the sequence being 12 hours light, 
12 hours dark. Details of the lighting pattern should be recorded 
and included in the final report of the study. 
Unless otherwise specified in the method, animals may be housed 
individually, or be caged in small groups of the same sex; for group 
caging, no more than five animals should be housed per cage. 
In reports of animal experiments, it is important to indicate the type 
of caging used and the number of animals housed in each cage both 
during exposure to the chemical and any subsequent observation 
period.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 180
 
(ii) Feeding conditions  
Diets should meet all the nutritional requirements of the species 
under test. Where test substances are administered to animals in their diet the nutritional value may be reduced by interaction 
between the substance and a dietary constituent. The possibility of 
such a reaction should be considered when interpreting the results of tests. Conventional laboratory diets may be used with an unlimited supply of drinking water. The choice of the diet may be influenced 
by the need to ensure a suitable admixture of a test substance when 
administered by this method. 
Dietary contaminants which are known to influence the toxicity 
should not be present in interfering concentrations.
 
C. ALTERNATIVE TESTING  
The European Union is committed to promoting the development 
and validation of alternative techniques which can provide the 
same level of information as current animal tests, but which use fewer animals, cause less suffering or avoid the use of animals completely. 
Such methods, as they become available, must be considered 
wherever possible for hazard characterisation and consequent clas ­
sification and labelling for intrinsic hazards and chemical safety assessment.
 
D. EVALUATION AND INTERPRETATION  
When tests are evaluated and interpreted, limitations in the extent to 
which the results of animal and  in vitro  studies can be extrapolated 
directly to man must be considered and therefore, evidence of 
adverse effects in humans, where available, may be used for confir ­
mation of testing results.  
E. LITERATURE REFERENCES  
Most of these methods are developed within the framework of the 
OECD programme for Testing Guidelines, and should be performed 
in conformity with the principles of Good Laboratory Practice, in 
order to ensure as wide as possible ‘mutual acceptance of data’. 
Additional information may be found in the references listed in the 
OECD guidelines and the relevant literature published elsewhere.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 181
 
B.1 bis.  ACUTE ORAL TOXICITY — FIXED DOSE PROCEDURE  
1. METHOD  
This test method is equivalent to OECD TG 420 (2001)  
1.1. INTRODUCTION 
Traditional methods for assessing acute toxicity use death of animals 
as an endpoint. In 1984, a new approach to acute toxicity testing was 
suggested by the British Toxicology Society based on the adminis ­
tration at a series of fixed dose levels (1). The approach avoided using death of animals as an endpoint, and relied instead on the observation of clear signs of toxicity at one of a series of fixed 
dose levels. Following UK (2) and international (3)  in vivo  validation 
studies the procedure was adopted as a testing method in 1992. Subsequently, the statistical properties of the Fixed Dose Procedure have been evaluated using mathematical models in a 
series of studies (4)(5)(6). Together, the  in vivo  and modelling 
studies have demonstrated that the procedure is reproducible, uses fewer animals and causes less suffering than the traditional methods and is able to rank substances in a similar manner to the other acute 
toxicity testing methods. 
Guidance on the selection of the most appropriate test method for a 
given purpose can be found in the Guidance Document on Acute Oral Toxicity Testing (7). This guidance document also contains additional information on the conduct and interpretation of Testing 
Method B.1 bis. 
It is a principle of the method that in the main study only moderately 
toxic doses are used, and that administration of doses that are 
expected to be lethal should be avoided. Also, doses that are known to cause marked pain and distress, due to corrosive or 
severely irritant actions, need not be administered. Moribund 
animals, or animals obviously in pain or showing signs of severe and enduring distress shall be humanely killed, and are considered in the interpretation of the test results in the same way as animals that 
died on test. Criteria for making the decision to kill moribund or 
severely suffering animals, and guidance on the recognition of predictable or impending death, are the subject of a separate Guidance Document (8). 
The method provides information on the hazardous properties and 
allows the substance to be ranked and classified according to the 
Globally Harmonised System (GHS) for the classification of 
chemicals which cause acute toxicity (9). 
The testing laboratory should consider all available information on 
the test substance prior to conducting the study. Such information will include the identity and chemical structure of the substance; its 
physico-chemical properties; the results of any other  in vitro  or in 
vivo toxicity tests on the substance; toxicological data on structurally 
related substances; and the anticipated use(s) of the substance. This information is necessary to satisfy all concerned that the test is 
relevant for the protection of human health, and will help in the 
selection of an appropriate starting dose.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 182
 
1.2. DEFINITIONS  
Acute oral toxicity:  refers to those adverse effects occurring 
following oral administration of a single dose of a substance or multiple doses given within 24 hours.  
Delayed death:  means that an animal does not die or appear 
moribund within 48 hours but dies later during the 14-day obser ­
vation period.  
Dose:  is the amount of test substance administered. Dose is 
expressed as weight of test substance per unit weight of test animal (e.g. mg/kg).  
Evident toxicity:  is a general term describing clear signs of toxicity 
following the administration of test substance (see (3) for examples) such that at the next highest fixed dose either severe pain and enduring signs of severe distress, moribund status (criteria are 
presented in the Humane Endpoints Guidance Document (8)), or 
probable mortality in most animals can be expected.  
GHS:  Globally Harmonised Classification System for Chemical 
Substances and Mixtures. A joint activity of OECD (human health 
and the environment), UN Committee of Experts on Transport of Dangerous Goods (physical-chemical properties) and ILO (hazard communication) and coordinated by the Interorganisation 
Programme for the Sound Management of Chemicals (IOMC).  
Impending death:  when moribund state or death is expected prior to 
the next planned time of observation. Signs indicative of this state in rodents could include convulsions, lateral position, recumbence and 
tremor. (See the Humane Endpoint Guidance Document (8) for more 
details).  
LD
 50 (median lethal dose):  is a statistically derived single dose of a 
substance that can be expected to cause death in 50 % of animals 
when administered by the oral route. The LD  50 value is expressed in 
terms of weight of test substance per unit weight of test animal (mg/kg).  
Limit dose:  refers to a dose at an upper limitation on testing (2 000 
or 5 000 mg/kg).  
Moribund status:  being in a state of dying or inability to survive, 
even if treated. (See the Humane Endpoint Guidance Document (8) 
for more details).  
Predictable death:  presence of clinical signs indicative of death at a 
known time in the future before the planned end of the experiment, 
for example: inability to reach water or food. (See the Humane 
Endpoint Guidance Document (8) for more details).
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1.3. PRINCIPLE OF THE TEST METHOD 
Groups of animals of a single sex are dosed in a stepwise procedure 
using the fixed doses of 5, 50, 300 and 2 000 mg/kg (exceptionally an additional fixed dose of 5 000 mg/kg may be considered, see 
Section 1.6.2). The initial dose level is selected on the basis of a 
sighting study as the dose expected to produce some signs of toxicity without causing severe toxic effects or mortality. Clinical signs and conditions associated with pain, suffering, and impending death, are 
described in detail in a separate OECD Guidance Document (8). 
Further groups of animals may be dosed at higher or lower fixed doses, depending on the presence or absence of signs of toxicity or 
mortality. This procedure continues until the dose causing evident 
toxicity or no more than one death is identified, or when no effects are seen at the highest dose or when deaths occur at the lowest dose.
 
1.4. DESCRIPTION OF THE TEST METHOD  
1.4.1.  Selection of animal species  
The preferred rodent species is the rat, although other rodent species 
may be used. Normally females are used (7). This is because 
literature surveys of conventional LD  50 tests show that usually 
there is little difference in sensitivity between the sexes, but in those cases where differences are observed, females are generally slightly more sensitive (10). However, if knowledge of the toxico ­
logical or toxicokinetic properties of structurally related chemicals 
indicates that males are likely to be more sensitive then this sex should be used. When the test is conducted in males, adequate justification should be provided. 
Healthy young adult animals of commonly used laboratory strains 
should be employed. Females should be nulliparous and non- 
pregnant. Each animal, at the commencement of its dosing, should be between eight and 12 weeks old and its weight should fall in an interval within ± 20 % of the mean weight of any previously dosed 
animals.
 
1.4.2.  Housing and feeding conditions  
The temperature of the experimental animal room should be 22  o C (± 
3 o C). Although the relative humidity should be at least 30 % and 
preferably not exceed 70 % other than during room cleaning the aim should be 50-60 %. Lighting should be artificial, the sequence being 
12 hours light, 12 hours dark. For feeding, conventional laboratory 
diets may be used with an unlimited supply of drinking water. Animals may be group-caged by dose, but the number of animals per cage must not interfere with clear observations of each animal.
 
1.4.3.  Preparation of animals  
The animals are randomly selected, marked to permit individual 
identification, and kept in their cages for at least five days prior to 
the start of dosing to allow for acclimatisation to the laboratory 
conditions.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 184
 
1.4.4.  Preparation of doses  
In general test substances should be administered in a constant 
volume over the range of doses to be tested by varying the concen ­
tration of the dosing preparation. Where a liquid end product or 
mixture is to be tested however, the use of the undiluted test 
substance, i.e. at a constant concentration, may be more relevant to the subsequent risk assessment of that substance, and is a requirement of some regulatory authorities. In either case, the 
maximum dose volume for administration must not be exceeded. 
The maximum volume of liquid that can be administered at one time depends on the size of the test animal. In rodents, the 
volume should not normally exceed 1ml /100 g of body weight: 
however in the case of aqueous solutions 2 ml/100 g body weight can be considered. With respect to the formulation of the dosing preparation, the use of an aqueous solution/suspension/emulsion is 
recommended wherever possible, followed in order of preference by 
a solution/suspension/emulsion in oil (e.g. corn oil) and then possibly solution in other vehicles. For vehicles other than water the toxicological characteristics of the vehicle should be known. 
Doses must be prepared shortly prior to administration unless the 
stability of the preparation over the period during which it will be used is known and shown to be acceptable.
 
1.5. PROCEDURE  
1.5.1.  Administration of doses  
The test substance is administered in a single dose by gavage using a 
stomach tube or a suitable intubation canula. In the unusual circum ­
stance that a single dose is not possible, the dose may be given in 
smaller fractions over a period not exceeding 24 hours. 
Animals should be fasted prior to dosing (e.g. with the rat, food but 
not water should be withheld over-night; with the mouse, food but not water should be withheld for three to four hours). Following the period of fasting, the animals should be weighed and the test 
substance administered. After the substance has been administered, 
food may be withheld for a further three to four hours in rats or one to two hours in mice. Where a dose is administered in fractions over a period of time, it may be necessary to provide the animals with 
food and water depending on the length of the period.
 
1.5.2.  Sighting study  
The purpose of the sighting study is to allow selection of the appro ­
priate starting dose for the main study. The test substance is admin ­
istered to single animals in a sequential manner following the flow ­
charts in Appendix 1. The sighting study is completed when a decision on the starting dose for the main study can be made (or 
if a death is seen at the lowest fixed dose). 
The starting dose for the sighting study is selected from the fixed 
dose levels of 5, 50, 300 and 2 000 mg/kg as a dose expected to produce evident toxicity based, when possible, on evidence from  in 
vivo and in vitro  data from the same chemical and from structurally 
related chemicals. In the absence of such information, the starting 
dose will be 300 mg/kg. 
A period of at least 24 hours will be allowed between the dosing of 
each animal. All animals should be observed for at least 14 days.
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Exceptionally, and only when justified by specific regulatory needs, 
the use of an additional upper fixed dose level of 5 000 mg/kg may 
be considered (see Appendix 3). For reasons of animal welfare concern, testing of animals in GHS Category 5 ranges (2 000- 5 000 mg/kg is discouraged and should only be considered when 
there is a strong likelihood that the results of such a test have a 
direct relevance for protecting human or animal health or the environment. 
In cases where an animal tested at the lowest fixed dose level 
(5 mg/kg) in the sighting study dies, the normal procedure is to terminate the study and assign the substance to GHS Category 1 
(as shown in Appendix 1). However, if further confirmation of the 
classification is required, an optional supplementary procedure may be conducted, as follows. A second animal is dosed at 5 mg/kg. If this second animal dies, then GHS Category 1 will be confirmed and 
the study will be immediately terminated. If the second animal 
survives, then a maximum of three additional animals will be dosed at 5 mg/kg. Because there will be a high risk of mortality, these animals should be dosed in a sequential manner to protect 
animal welfare. The time interval between dosing each animal 
should be sufficient to establish that the previous animal is likely to survive. If a second death occurs, the dosing sequence will be 
immediately terminated and no further animals will be dosed. 
Because the occurrence of a second death (irrespective of the number of animals tested at the time of termination) falls into outcome A (two or more deaths), the classification rule of 
Appendix 2 at the 5 mg/kg fixed dose is followed (Category 1 if 
there are two or more deaths or Category 2 if there is no more than one death). In addition, Appendix 4 gives guidance on the classifi ­
cation in the EU system until the new GHS is implemented.
 
1.5.3.  Main study  
1.5.3.1.  Numbers of animals and dose levels  
The action to be taken following testing at the starting dose level is 
indicated by the flowcharts in Appendix 2. One of three actions will 
be required; either stop testing and assign the appropriate hazard classification class, test at a higher fixed dose or test at a lower fixed dose. However, to protect animals, a dose level that caused 
death in the sighting study will not be revisited in the main study 
(see Appendix 2). Experience has shown that the most likely outcome at the starting dose level will be that the substance can be classified and no further testing will be necessary. 
A total of five animals of one sex will normally be used for each 
dose level investigated. The five animals will be made up of one 
animal from the sighting study dosed at the selected dose level together with an additional four animals (except, unusually, if a dose level used on the main study was not included in the 
sighting study). 
The time interval between dosing at each level is determined by the 
onset, duration, and severity of toxic signs. Treatment of animals at 
the next dose should be delayed until one is confident of survival of the previously dosed animals. A period of three or four days between dosing at each dose level is recommended, if needed, to allow for the 
observation of delayed toxicity. The time interval may be adjusted as 
appropriate, e.g. in case of inconclusive response.
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When the use of an upper fixed dose of 5 000 mg/kg is considered, 
the procedure outlined in Appendix 3 should be followed (see also 
section 1.6.2).  
1.5.3.2.  Limit test  
The limit test is primarily used in situations where the experimenter 
has information indicating that the test material is likely to be nontoxic, i.e., having toxicity only above regulatory limit doses. 
Information about the toxicity of the test material can be gained 
from knowledge about similar tested compounds or similar tested mixtures or products, taking into consideration the identity and percentage of components known to be of toxicological significance. 
In those situations where there is little or no information about its 
toxicity, or in which the test material is expected to be toxic, the main test should be performed. 
Using the normal procedure, a sighting study starting dose of 2 000 
mg/kg (or exceptionally 5 000 mg/kg) followed by dosing of a 
further four animals at this level serves as a limit test for this 
guideline.
 
1.6. OBSERVATIONS 
Animals are observed individually after dosing at least once during 
the first 30 minutes, periodically during the first 24 hours, with special attention given during the first four hours, and daily there ­
after, for a total of 14 days, except where they need to be removed 
from the study and humanely killed for animal welfare reasons or are found dead. However, the duration of observation should not be 
fixed rigidly. It should be determined by the toxic reactions, time 
of onset and length of recovery period, and may thus be extended when considered necessary. The times at which signs of toxicity appear and disappear are important, especially if there is a 
tendency for toxic signs to be delayed (11). All observations are 
systematically recorded, with individual records being maintained for each animal. 
Additional observations will be necessary if the animals continue to 
display signs of toxicity. Observations should include changes in 
skin and fur, eyes and mucous membranes, and also respiratory, 
circulatory, autonomic and central nervous systems, and soma ­
tomotor activity and behaviour pattern. Attention should be directed to observations of tremors, convulsions, salivation, diar ­
rhoea, lethargy, sleep and coma. The principles and criteria 
summarised in the Humane Endpoints Guidance Document should be taken into consideration (8). Animals found in a moribund condition and animals showing severe pain or enduring signs of 
severe distress should be humanely killed. When animals are killed 
for humane reasons or found dead, the time of death should be recorded as precisely as possible.
 
1.6.1.  Body weight  
Individual weights of animals should be determined shortly before 
the test substance is administered and at least weekly thereafter. 
Weight changes should be calculated and recorded. At the end of 
the test surviving animals are weighed and then humanely killed.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 187
 
1.6.2.  Pathology  
All test animals (including those that die during the test or are 
removed from the study for animal welfare reasons) should be subjected to gross necropsy. All gross pathological changes should 
be recorded for each animal. Microscopic examination of organs 
showing evidence of gross pathology in animals surviving 24 or more hours after the initial dosing may also be considered because it may yield useful information.
 
2. DATA  
Individual animal data should be provided. Additionally, all data 
should be summarised in tabular form, showing for each test 
group the number of animals used, the number of animals displaying signs of toxicity, the number of animals found dead during the test or killed for humane reasons, time of death of individual animals, a 
description and the time course of toxic effects and reversibility, and 
necropsy findings.
 
3. REPORTING  
3.1. TEST REPORT 
The test report must include the following information, as appro ­
priate: 
Test substance: — physical nature, purity, and, where relevant, physico-chemical 
properties (including isomerisation), 
— identification data, including CAS number. Vehicle (if appropriate): — justification for choice of vehicle, if other than water. Test animals: — species/strain used, — microbiological status of the animals, when known, 
— number, age and sex of animals (including, where appropriate, a 
rationale for use of males instead of females), 
— source, housing conditions, diet, etc. Test conditions: 
— details of test substance formulation, including details of the 
physical form of the material administered, 
— details of the administration of the test substance including 
dosing volumes and time of dosing, 
— details of food and water quality (including diet type/source, 
water source), 
— the rationale for the selection of the starting dose.
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Results: 
— tabulation of response data and dose level for each animal (i.e. 
animals showing signs of toxicity including mortality, nature, 
severity and duration of effects), 
— tabulation of body weight and body weight changes, — individual weights of animals at the day of dosing, in weekly 
intervals thereafter, and at time of death or sacrifice, 
— date and time of death if prior to scheduled sacrifice, — time course of onset of signs of toxicity and whether these were 
reversible for each animal, 
— necropsy findings and histopathological findings for each animal, 
if available. 
Discussion and interpretation of results. Conclusions.
 
4. REFERENCES  
(1) British Toxicology Society Working Party on Toxicity (1984) 
Special report: a new approach to the classification of 
substances and preparations on the basis of their acute 
toxicity. Human Toxicol., 3, p. 85-92. 
(2) Van den Heuvel, M.J., Dayan, A.D. and Shillaker, R.O (1987) 
Evaluation of the BTS approach to the testing of substances and preparations for their acute toxicity. Human Toxicol.‚ 6, 
p. 279-291. 
(3) Van den Heuvel, M.J., Clark, D.G., Fielder, R.J., Koundakjian, 
P.P., Oliver, G.J.A., Pelling, D., Tomlinson, N.J. and Walker, A.P (1990) The international validation of a fixed-dose 
procedure as an alternative to the classical LD
 50 test. Fd. 
Chem. Toxicol. 28, p. 469-482. 
(4) Whitehead, A. and Curnow, R.N (1992) Statistical evaluation 
of the fixed-dose procedure. Fd. Chem. Toxicol., 30, p. 313-324. 
(5) Stallard, N. and Whitehead, A (1995) Reducing numbers in the 
fixed-dose procedure. Human Exptl. Toxicol. 14, p. 315-323. 
(6) Stallard, N., Whitehead, A and Ridgeway, P. (2002) Statistical 
evaluation of the revised fixed dose procedure. Hum. Exp. Toxicol., 21, p. 183-196. 
(7) OECD (2001) Guidance Document on Acute Oral Toxicity 
Testing. Environmental Health and Safety Monograph Series 
on Testing and Assessment No 24. Paris 
(8) OECD (2000) Guidance Document on the Recognition, 
Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. Environ ­
mental Health and Safety Monograph Series on Testing and 
Assesment No 19.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 189
 
(9) OECD (1998) Harmonised Integrated Hazard Classification for 
Human Health and Environmental Effects of Chemical 
Substances as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals in November 1998, Part 2, p. 11 [http://webnet1.oecd.org/ ­
oecd/pages/home/displaygeneral/0,3380, EN-documents-521- 
14-no-24-no-0,FF.html]. 
(10) Lipnick, R.L., Cotruvo, J.A., Hill, R.N., Bruce, R.D., Stitzel, 
K.A., Walker, A.P., Chu, I., Goddard, M., Segal, L., Springer, J.A. and Myers, R.C (1995) Comparison of the Up-and-Down, 
Conventional LD
 50 , and Fixed-Dose Acute Toxicity 
Procedures. Fd. Chem. Toxicol. 33, p. 223-231. 
(11) Chan P.K and A. W Hayes (1994) Chapter 16 Acute Toxicity and 
Eye Irritation. In: Principles and Methods of Toxicology. 3rd Edition. A.W. Hayes, Editor. Raven Press Ltd. New York, USA.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 190
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 ▼B 
Appendix 1  
FLOW CHART FOR THE SIGHTING STUDY

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 ▼B

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 ▼B 
Appendix 2  
FLOW CHART FOR THE MAIN STUDY

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 ▼B

 
Appendix 3  
CRITERIA FOR CLASSIFICATION OF TEST SUBSTANCES WITH 
EXPECTED LD  50 VALUES EXCEEDING 2 000 MG/KG WITHOUT 
THE NEED FOR TESTING.  
Criteria for hazard Category 5 are intended to enable the identification of test 
substances which are of relatively low acute toxicity hazard but which, under 
certain circumstances may present a danger to vulnerable populations. These substances are anticipated to have an oral or dermal LD
 50 in the range of 
2 000-5 000 mg/kg or equivalent doses for other routes. Test substances could 
be classified in the hazard category defined by: 2 000 mg/kg < LD  50 < 5 000 
mg/kg (Category 5 in the GHS) in the following cases: 
(a) if directed to this category by any of the testing schemes of Appendix 2, 
based on mortality incidences 
(b) if reliable evidence is already available that indicates the LD  50 to be in the 
range of Category 5 values; or other animal studies or toxic effects in humans indicate a concern for human health of an acute nature; 
(c) through extrapolation, estimation or measurement of data if assignment to a 
more hazardous class is not warranted; and 
— reliable information is available indicating significant toxic effects in 
humans, or 
— any mortality is observed when tested up to Category 4 values by the oral 
route, or 
— where expert judgement confirms significant clinical signs of toxicity, 
when tested up to Category 4 values, except for diarrhoea, piloerection 
or an ungroomed appearance, or 
— where expert judgement confirms reliable information indicating the 
potential for significant acute effects from the other animal studies.
 
TESTING AT DOSES ABOVE 2 000 MG/KG  
Exceptionally, and only when justified by specific regulatory needs, the use of an 
additional upper fixed dose level of 5 000 mg/kg may be considered. Recog ­
nising the need to protect animal welfare, testing at 5 000 mg/kg is discouraged 
and should only be considered when there is a strong likelihood that the results of such a test would have a direct relevance for protecting animal or human health (9).
 
Sighting study  
The decision rules governing the sequential procedure presented in Appendix 1 
are extended to include a 5 000 mg/kg dose level. Thus, when a sighting study starting dose of 5 000 mg/kg is used outcome A (death) will require a second animal to be tested at 2 000 mg/kg; outcomes B and C (evident toxicity or no 
toxicity) will allow the selection of 5 000 mg/kg as the main study starting dose. 
Similarly, if a starting dose other than 5 000 mg/kg is used then testing will progress to 5 000 mg/kg in the event of outcomes B or C at 2 000 mg/kg; a subsequent 5 000 mg/kg outcome A will dictate a main study starting dose of 
2 000 mg/kg and outcomes B and C will dictate a main study starting dose of 
5 000 mg/kg.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 195
 
Main study  
The decision rules governing the sequential procedure presented in Appendix 2 
are extended to include a 5 000 mg/kg dose level. Thus, when a main study starting dose of 5 000 mg/kg is used, outcome A ( ≥ 2 deaths) will require the 
testing of a second group at 2 000 mg/kg; outcome B (evident toxicity and/or ≤ 1 
death) or C (no toxicity) will result in the substance being unclassified according 
to GHS. Similarly, if a starting dose other than 5 000 mg/kg is used then testing will progress to 5 000 mg/kg in the event of outcome C at 2 000 mg/kg; a subsequent 5 000 mg/kg outcome A will result in the substance being assigned 
to GHS Category 5 and outcomes B or C will lead to the substance being 
unclassified.
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 ▼B 
Appendix 4  
TEST METHOD B.1 bis  
Guidance on classification according to the EU scheme to cover the transition period until full implementation of the Globally Harmonised Classification System (GHS) (taken from 
reference (8))

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 ▼B

 
B.1 tris.  ACUTE ORAL TOXICITY — ACUTE TOXIC CLASS 
METHOD  
1. METHOD  
This test method is equivalent to OECD TG 423 (2001)  
1.1. INTRODUCTION 
The acute toxic class method (1) set out in this test is a stepwise 
procedure with the use of three animals of a single sex per step. Depending on the mortality and/or the moribund status of the 
animals, on average two to four steps may be necessary to allow 
judgement on the acute toxicity of the test substance. This procedure is reproducible, uses very few animals and is able to rank substances in a similar manner to the other acute toxicity testing methods. The acute 
toxic class method is based on biometric evaluations (2)(3)(4)(5) with 
fixed doses, adequately separated to enable a substance to be ranked for classification purposes and hazard assessment. The method as adopted in 1996 was extensively validated  in vivo  against LD
 50 data obtained 
from the literature, both nationally (6) and internationally (7). 
Guidance on the selection of the most appropriate test method for a 
given purpose can be found in the Guidance Document on Acute 
Oral Toxicity Testing (8). This Guidance Document also contains 
additional information on the conduct and interpretation of testing method B.1tris. 
Test substances, at doses that are known to cause marked pain and 
distress due to corrosive or severely irritant actions, need not be administered. Moribund animals, or animals obviously in pain or showing signs of severe and enduring distress shall be humanely 
killed, and are considered in the interpretation of the test results in 
the same way as animals that died on test. Criteria for making the decision to kill moribund or severely suffering animals, and guidance on the recognition of predictable or impending death, are the subject 
of a separate Guidance Document (9). 
The method uses pre-defined doses and the results allow a substance 
to be ranked and classified according to the Globally Harmonised 
System for the classification of chemicals which cause acute 
toxicity (10). 
In principle, the method is not intended to allow the calculation of a 
precise LD
 50 , but does allow for the determination of defined 
exposure ranges where lethality is expected since death of a proportion of the animals is still the major endpoint of this test. The method allows for the determination of an LD
 50 value only 
when at least two doses result in mortality higher than 0 % and 
lower than 100 %. The use of a selection of pre-defined doses, regardless of test substance, with classification explicitly tied to number of animals observed in different states improves the oppor ­
tunity for laboratory to laboratory reporting consistency and repeat ­
ability.
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The testing laboratory should consider all available information on 
the test substance prior to conducting the study. Such information 
will include the identity and chemical structure of the substance; its physico-chemical properties; the result of any other  in vivo  or in 
vitro  toxicity tests on the substance; toxicological data on the struc ­
turally related substances; and the anticipated use(s) of the substance. 
This information is necessary to satisfy all concerned that the test is relevant for the protection of human health and will help in the selection of the most appropriate starting dose.
 
1.2. DEFINITIONS  
Acute oral toxicity:  refers to those adverse effects occurring 
following oral administration of a single dose of a substance or multiple doses given within 24 hours.  
Delayed death:  means that an animal does not die or appear 
moribund within 48 hours but dies later during the 14-day obser ­
vation period.  
Dose:  is the amount of test substance administered. Dose is 
expressed as weight of test substance per unit weight of test 
animal (e.g. mg/kg).  
GHS:  Globally Harmonised Classification System for Chemical 
Substances and Mixtures. A joint activity of OECD (human health and the environment), UN Committee of Experts on Transport of Dangerous Goods (physical-chemical properties) and ILO (hazard 
communication) and coordinated by the Interorganisation 
Programme for the Sound Management of Chemicals (IOMC).  
Impending death:  when moribund state or death is expected prior to 
the next planned time of observation. Signs indicative of this state in rodents could include convulsions, lateral position, recumbence and tremor (See the Humane Endpoint Guidance Document (9) for more 
details).  
LD
 50 (median lethal oral dose):  is a statistically derived single dose 
of a substance that can be expected to cause death in 50 % of 
animals when administered by the oral route. The LD  50 value is 
expressed in terms of weight of test substance per unit weight of test animal (mg/kg).  
Limit dose:  refers to a dose at an upper limitation on testing (2 000 
or 5 000 mg/kg).  
Moribund status:  being in a state of dying or inability to survive, 
even if treated (See the Humane Endpoint Guidance Document (9) for more details).  
Predictable death:  presence of clinical signs indicative of death at a 
known time in the future before the planned end of the experiment; 
for example: inability to reach water or food. (See the Humane 
Endpoint Guidance Document (9) for more details).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 200
 
1.3. PRINCIPLE OF THE TEST 
It is the principle of the test that, based on a stepwise procedure with 
the use of a minimum number of animals per step, sufficient information is obtained on the acute toxicity of the test substance 
to enable its classification. The substance is administered orally to a 
group of experimental animals at one of the defined doses. The substance is tested using a stepwise procedure, each step using three animals of a single sex (normally females). Absence or 
presence of compound-related mortality of the animals dosed at 
one step will determine the next step, i.e.; 
— no further testing is needed, 
— dosing of three additional animals, with the same dose, 
— dosing of three additional animals at the next higher or the next 
lower dose level. 
Details of the test procedure are described in Appendix 1. The 
method will enable a judgement with respect to classifying the test 
substance to one of a series of toxicity classes defined by fixed LD
 50 
cut-off values.  
1.4. DESCRIPTION OF THE METHOD  
1.4.1.  Selection of animal species  
The preferred rodent species is the rat, although other rodent species 
may be used. Normally females are used (9). This is because literature surveys of conventional LD
 50 tests show that, although 
there is little difference in sensitivity between the sexes, in those 
cases where differences are observed females are generally slightly 
more sensitive (11). However if knowledge of the toxicological or toxicokinetic properties of structurally related chemicals indicates 
that males are likely to be more sensitive, then this sex should be 
used. When the test is conducted in males, adequate justification should be provided. 
Healthy young adult animals of commonly used laboratory strains 
should be employed. Females should be nulliparous and non- pregnant. Each animal, at the commencement of its dosing, should be between eight and 12 weeks old and its weight should fall in an 
interval within ± 20 % of the mean weight of any previously dosed 
animals.
 
1.4.2.  Housing and feeding conditions  
The temperature in the experimental animal room should be 22  o C (± 
3 o C). Although the relative humidity should be at least 30 % and 
preferably not exceed 70 % other than during room cleaning the aim 
should be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. 
Animals may be group-caged by dose, but the number of animals 
per cage must not interfere with clear observations of each animal.
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1.4.3.  Preparation of animals  
The animals are randomly selected, marked to permit individual 
identification, and kept in their cages for at least five days prior to dosing to allow for acclimatisation to the laboratory conditions.
 
1.4.4.  Preparation of doses  
In general, test substances should be administered in a constant 
volume over the range of doses to be tested by varying the concen ­
tration of the dosing preparation. Where a liquid end product or 
mixture is to be tested however, the use of the undiluted test substance, i.e. at a constant concentration, may be more relevant to the subsequent risk assessment of that substance, and is a 
requirement of some regulatory authorities. In either case, the 
maximum dose volume for administration must not be exceeded. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. In rodents, the 
volume should not normally exceed 1 ml/100 g of body weight: 
however in the case of aqueous solutions 2 ml/100 g body weight can be considered. With respect to the formulation of the dosing preparation, the use of an aqueous solution/suspension/emulsion is 
recommended wherever possible, followed in order of preference by 
a solution/suspension/emulsion in oil (e.g. corn oil) and then possibly solution in other vehicles. For vehicles other than water the toxicological characteristics of the vehicle should be known. 
Doses must be prepared shortly prior to administration unless the 
stability of the preparation over the period during which it will be used is known and shown to be acceptable.
 
1.5. PROCEDURE  
1.5.1.  Administration of doses  
The test substance is administered in a single dose by gavage using a 
stomach tube or a suitable intubation canula. In the unusual circum ­
stance that a single dose is not possible, the dose may be given in 
smaller fractions over a period not exceeding 24 hours. 
Animals should be fasted prior to dosing (e.g. with the rat, food but 
not water should be withheld overnight, with the mouse, food but not water should be withheld for three or four hours). Following the period of fasting, the animals should be weighed and the test 
substance administered. After the substance has been administered, 
food may be withheld for a further three or fours hours in rats or one or two hours in mice. Where a dose is administered in fractions over a period it may be necessary to provide the animals with food and 
water depending on the length of the period.
 
1.5.2.  Number of animals and dose levels  
Three animals are used for each step. The dose level to be used as 
the starting dose is selected from one of four fixed levels, 5, 50, 300 
and 2 000 mg/kg body weight. The starting dose level should be that 
which is most likely to produce mortality in some of the dosed animals. The flowcharts of Appendix 1 describe the procedure that should be followed for each of the starting doses. In addition, 
Appendix 4 gives guidance on the classification in the EU system 
until the new GHS is implemented.
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When available information suggests that mortality is unlikely at the 
highest starting dose level (2 000 mg/kg body weight), then a limit 
test should be conducted. When there is no information on a substance to be tested, for animal welfare reasons it is recommended to use the starting dose of 300 mg/kg body weight. 
The time interval between treatment groups is determined by the 
onset, duration, and severity of toxic signs. Treatment of animals at the next dose should be delayed until one is confident of survival of the previously dosed animals. 
Exceptionally, and only when justified by specific regulatory needs, 
the use of additional upper dose level of 5 000 mg/kg body weight may be considered (see Appendix 2). For reasons 
of animal welfare concern, testing of animals in GHS Category 
5 ranges (2 000-5 000 mg/kg) is discouraged and should only be considered when there is a strong likelihood that the results of such a test would have a direct relevance for protecting human or 
animal health or the environment.
 
1.5.3.  Limit test  
The limit test is primarily used in situations where the experimenter 
has information indicating that the test material is likely to be non- toxic, i.e., having toxicity only above regulatory limit doses. 
Information about the toxicity of the test material can be gained 
from knowledge about similar tested compounds or similar tested mixtures or products, taking into consideration the identity and percentage of components known to be of toxicological significance. 
In those situations where there is little or no information about its 
toxicity, or in which the test material is expected to be toxic, the main test should be performed. 
A limit test at one dose level of 2 000 mg/kg body weight may be 
carried out with six animals (three animals per step). Exceptionally a 
limit test at one dose level of 5 000 mg/kg may be carried out with three animals (see Appendix 2). If test substance-related mortality is produced, further testing at the next lower level may need to be 
carried out.
 
1.6. OBSERVATIONS 
Animals are observed individually after dosing at least once during 
the first 30 minutes, periodically during the first 24 hours, with special attention given during the first four hours, and daily there ­
after, for a total of 14 days, except where they need to be removed 
from the study and humanely killed for animal welfare reasons or are found dead. However, the duration of observation should not be fixed rigidly. It should be determined by the toxic reactions, time 
of onset and length of recovery period, and may thus be extended 
when considered necessary. The times at which signs of toxicity appear and disappear are important, especially if there is a tendency for toxic signs to be delayed (12). All observations are 
systematically recorded with individual records being maintained 
for each animal.
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Additional observations will be necessary if the animals continue to 
display signs of toxicity. Observations should include changes in 
skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and soma ­
tomotor activity and behaviour pattern. Attention should be 
directed to observations of tremors, convulsions, salivation, diar ­
rhoea, lethargy, sleep and coma. The principles and criteria summarised in the Humane Endpoints Guidance Document (9) should be taken into consideration. Animals found in a moribund 
condition and animals showing severe pain or enduring signs of 
severe distress should be humanely killed. When animals are killed for humane reasons or found dead, the time of death should be recorded as precisely as possible.
 
1.6.1.  Body weight  
Individual weights of animals should be determined shortly before 
the test substance is administered, and at least weekly thereafter. Weight changes should be calculated and recorded. At the end of the test surviving animals are weighed and humanely killed.
 
1.6.2.  Pathology  
All test animals (including those that die during the test or are 
removed from the study for animal welfare reasons) should be subjected to gross necropsy. All gross pathological changes should 
be recorded for each animal. Microscopic examination of organs 
showing evidence of gross pathology in animals surviving 24 or more hours may also be considered because it may yield useful information.
 
2. DATA  
Individual animal data should be provided. Additionally, all data 
should be summarised in tabular form, showing for each test group the number of animals used, the number of animals displaying 
signs of toxicity, the number of animals found dead during the test 
or killed for humane reasons, time of death of individual animals, a description and the time course of toxic effects and reversibility, and necropsy findings.
 
3. REPORTING  
3.1. Test report  
The test report must include the following information, as appro ­
priate: 
Test substance: 
— physical nature, purity, and, where relevant, physico-chemical 
properties (including isomerisation), 
— identification data, including CAS number. 
Vehicle (if appropriate): 
— justification for choice of vehicle, if other than water. 
Test animals: 
— species/strain used,  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 204
 
— microbiological status of the animals, when known, 
— number, age, and sex of animals (including, where appropriate, a 
rationale for the use of males instead of females), 
— source, housing conditions, diet, etc. 
Test conditions: 
— details of test substance formulation including details of the 
physical form of the material administered, 
— details of the administration of the test substance including 
dosing volumes and time of dosing, 
— details of food and water quality (including diet type/source, 
water source), 
— the rationale for the selection of the starting dose. 
Results: — tabulation of response data and dose level for each animal (i.e. 
animals showing signs of toxicity including mortality; nature, 
severity, and duration of effects), 
— tabulation of body weight and body weight changes, 
— individual weights of animals at the day of dosing, in weekly 
intervals thereafter, and at the time of death or sacrifice, 
— date and time of death if prior to scheduled sacrifice, 
— time course of onset of signs of toxicity, and whether these were 
reversible for each animal, 
— necropsy findings and histopathological findings for each animal, 
if available. 
Discussion and interpretation of results. 
Conclusions.
 
4. REFERENCES  
(1) Roll R., Höfer-Bosse Th. And Kayser D (1986) New 
Perspectives in Acute Toxicity Testing of Chemicals. 
Toxicol. Lett., Suppl. 31, p. 86. 
(2) Roll R., Riebschläger M., Mischke U. and Kayser D (1989) 
Neue Wege zur Bestimmung der akuten Toxizität von Chemi ­
kalien. Bundesgesundheitsblatt 32, p. 336-341. 
(3) Diener W., Sichha L., Mischke U., Kayser D. and Schlede E 
(1994) The Biometric Evaluation of the Acute-Toxic-Class 
Method (Oral). Arch. Toxicol. 68, p. 559-610. 
(4) Diener W., Mischke U., Kayser D. and Schlede E., (1995) The 
Biometric Evaluation of the OECD Modified Version of the 
Acute-Toxic-Class Method (Oral). Arch. Toxicol. 69, p. 729-734. 
(5) Diener W., and Schlede E., (1999) Acute Toxicity Class 
Methods: Alterations to LD/LC
 50 Tests. ALTEX 16, 
p. 129-134. 
(6) Schlede E., Mischke U., Roll R. and Kayser D., (1992). A 
National Validation Study of the Acute-Toxic- Class Method 
— An Alternative to the LD  50 Test. Arch. Toxicol. 66, 455-470.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 205
 
(7) Schlede E., Mischke U., Diener W. and Kayser D., (1994) The 
International Validation Study of the Acute-Toxic-Class 
Method (Oral). Arch. Toxicol. 69, p. 659-670. 
(8) OECD, (2001) Guidance Document on Acute Oral Toxicity 
Testing. Environmental Health and Safety Monograph Series on Testing and Assessment No 24. Paris. 
(9) OECD, (2000) Guidance Document on the Recognition, 
Assessment and Use of Clinical Signs as Humane Endpoints 
for Experimental Animals Used in Safety Evaluation. Environ ­
mental Health and Safety Monograph Series on Testing and Assessment No 19. 
(10) OECD, (1998) Harmonised Integrated Hazard Classification 
System For Human Health And Environmental Effects Of Chemical Substances as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working 
Party on Chemicals in November 1998, Part 2, p. 11 
[http://webnet1.oecd.org/oecd/pages/home/displaygeneral/0,3380, EN-documents-521-14-no-24-no-0,FF.html]. 
(11) Lipnick R. L., Cotruvo, J.A., Hill R. N., Bruce R. D., Stitzel 
K. A., Walker A. P., Chu I.; Goddard M., Segal L., Springer J. 
A. and Myers R. C. (1995) Comparison of the Up-and Down, 
Conventional LD
 50 , and Fixed Dose Acute Toxicity 
Procedures. Fd. Chem. Toxicol 33, p. 223-231. 
(12) Chan P.K. and A.W. Hayes. (1994). Chap. 16. Acute Toxicity 
and Eye Irritancy.  Principles and Methods of Toxicology . Third 
Edition. A.W. Hayes, Editor. Raven Press, Ltd., New York, USA.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 206
 
Appendix 1  
PROCEDURE TO BE FOLLOWED FOR EACH OF THE STARTING 
DOSES  
GENERAL REMARKS  
For each starting dose, the respective testing schemes as included in this 
Appendix outline the procedure to be followed. 
— Appendix 1 a: starting dose is 5 mg/kg bw, — Appendix 1 b: starting dose is 50 mg/kg bw, — Appendix 1 c: starting dose is: 300 mg/kg bw, — Apendix 1 d: starting dose is: 2 000 mg/kg bw. Depending on the number of humanely killed or dead animals, the test procedure 
follows the indicated arrows.
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 ▼B 
Appendix 1A  
TEST PROCEDURE WITH A STARTING DOSE OF 5 MG/KG BODY WEIGHT

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 ▼B 
Appendix 1B  
TEST PROCEDURE WITH A STARTING DOSE OF 50 MG/KG BODY WEIGHT

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 ▼B 
Appendix1C  
TEST PROCEDURE WITH A STARTING DOSE OF 300 MG/KG BODY WEIGHT

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 ▼B 
Appendix 1D  
TEST PROCEDURE WITH A STARTING DOSE OF 2 000 MG/KG BODY WEIGHT

 Appendix 2  
CRITERIA FOR CLASSIFICATION OF TEST SUBSTANCES WITH 
EXPECTED LD  50 VALUES EXCEEDING 2 000 MG/KG WITHOUT 
THE NEED FOR TESTING  
Criteria for hazard Category 5 are intended to enable the identification of test 
substances which are of relatively low acute toxicity hazard but which, under 
certain circumstances may present a danger to vulnerable populations. These substances are anticipated to have an oral or dermal LD
 50 in the range of 
2 000-5 000 mg/kg or equivalent doses for other routes. The test substance 
should be classified in the hazard category defined by: 2 000 mg/kg < LD  50 < 
5 000 mg/kg (Category 5 in the GHS) in the following cases: 
(a) If directed to this category by any of the testing schemes of Appendix 1a-1d, 
based on mortality incidences; 
(b) if reliable evidence is already available that indicates the LD  50 to be in the 
range of Category 5 values; or other animal studies or toxic effects in 
humans indicate a concern for human health of an acute nature; 
(c) through extrapolation, estimation or measurement of data if assignment to a 
more hazardous class is not warranted; and 
— reliable information is available indicating significant toxic effects in 
humans, or 
— any mortality is observed when tested up to Category 4 values by the oral 
route, or 
— where expert judgement confirms significant clinical signs of toxicity, 
when tested up to Category 4 values, except for diarrhoea, piloerection or an ungroomed appearance, or 
— where expert judgement confirms reliable information indicating the 
potential for significant acute effects from the other animal studies.
 
TESTING AT DOSES ABOVE 2 000 MG/KG  
Recognising the need to protect animal welfare, testing of animals in Category 5 
(5 000 mg/kg) ranges is discouraged and should only be considered when there is 
a strong likelihood that results of such a test have a direct relevance for 
protecting human or animal health (10). No further testing should be conducted at higher dose levels. 
When testing is required a dose of 5 000 mg/kg, only one step (i.e. three animals) 
is required. If the first animal dosed dies, then dosing proceeds at 2 000 mg/kg in 
accordance with the flowcharts in Appendix 1. If the first animal survives, two further animals are dosed. If only one of the three animals dies, the LD
 50 value is 
expected to exceed 5 000 mg/kg. If both animals die, then dosing proceeds at 
2 000 mg/kg.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 212
 
Appendix 3  
TEST METHOD B.1 tris: Guidance on classififcation according to EU scheme to cover the 
transition period until full implementation of the Globally Harmonised Classification System 
(GHS) (taken from reference (8))
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B.2. ACUTE INHALATION TOXICITY  
INTRODUCTION 
1. This Test Method is equivalent to OECD Test Guideline 403 (2009) (1). 
The original acute inhalation Test Guideline 403 (TG 403) was adopted in 1981. This revised Test Method B.2 (as equivalent to the revised TG 403) 
has been designed to be more flexible, to reduce animal usage, and to fulfil 
regulatory needs. The revised Test Method features two study types: a Traditional LC
 50 protocol and a Concentration × Time (C × t) protocol. 
Primary features of this Test Method are the ability to provide 
a concentration-response relationship ranging from non-lethal to lethal 
outcomes in order to derive a median lethal concentration (LC  50 ), 
non-lethal threshold concentration (e.g. LC01), and slope, and to identify possible sex susceptibility. The C × t protocol should be used when there is 
a specific regulatory or scientific need that calls for the testing of animals 
over multiple time durations, such as for purposes of emergency response planning [e.g. deriving Acute Exposure Guideline Levels (AEGL), Emergency Response Planning Guidelines (ERPG), or Acute Exposure 
Threshold Levels (AETL) values], or for land-use planning. 
2. Guidance on the conduct and interpretation of this Test Method studies can 
be found in the Guidance Document on Acute Inhalation Toxicity Testing (GD 39) (2). 
3. Definitions used in the context of this Test Method are provided at the end 
of this chapter and in GD 39 (2). 
4. This Test Method enables test chemical characterisation and quantitative risk 
assessment, and allows test chemicals to be ranked and classified according to Regulation (EC) No 1272/2008 (3). GD 39 (2) provides guidance in the 
selection of the appropriate Test Method for acute testing. When information 
on classification and labelling only is required, chapter B.52 of this Annex (4) is generally recommended [see GD 39 (2)]. This Test Method B.2 is not specifically intended for the testing of specialised materials, such 
as poorly soluble isometric or fibrous materials or manufactured nano ­
materials.
 
INITIAL CONSIDERATIONS 
5. Before considering testing in accordance with this Test Method all available 
information on the test chemical, including existing studies (e.g. chapter B.52 of this Annex (4)) whose data would support not doing additional testing should be considered by the testing laboratory in order to minimise animal 
usage. Information that may assist in the selection of the most appropriate 
species, strain, sex, mode of exposure and appropriate test concentrations include the identity, chemical structure, and physico-chemical properties of the test chemical; results of any  in vitro  or in vivo  toxicity tests; anticipated 
uses and potential for human exposure; available (Q)SAR data and toxico ­
logical data on structurally related substances [see GD 39 (2)].
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 217
 
6. Testing corrosive and/or irritating test chemicals at concentrations that are 
expected to cause severe pain and/or distress should be avoided to the extent 
possible. The corrosive/irritating potential should be evaluated by expert judgment using such evidence as human and animal experience (e.g. from repeat dose studies performed at non-corrosive/irritant concentrations), 
existing  in vitro  data (e.g. from chapters B.40, (5), B.40bis (6) of this 
Annex or OECD TG 435 (7)), pH values, information from similar substances or any other pertinent data, for the purpose of investigating whether further testing can be waived. For specific regulatory needs (e.g. 
for emergency planning purposes), this Test Method may be used for 
exposing animals to these materials because it provides the study director or principal investigator with control over the selection of target concen ­
trations. However, the targeted concentrations should not induce severe 
irritation/corrosive effects, yet sufficient to extend the 
concentration-response curve to levels that reach the regulatory and scientific objective of the test. These concentrations should be selected on a case-by-case basis and justification for concentration selection should be 
provided [see GD 39 (2)].
 
PRINCIPLE OF THE TEST 
7. This revised Test Method B.2 has been designed to obtain sufficient 
information on the acute toxicity of a test chemical to enable its classifi ­
cation and to provide lethality data ( e.g. LC  50 , LC  01 and slope) for one or 
both sexes as needed for quantitative risk assessments. This Test Method 
offers two methods. The first method is a traditional protocol in which groups of animals are exposed to a limit concentration (limit test) or a series of concentrations in a stepwise procedure for a predetermined 
duration of usually 4 hours. Other durations of exposure may apply to 
serve specific regulatory purposes. The second method is a (C × t) protocol in which groups of animals are exposed to one (limit concentration) or a series of multiple concentrations over multiple durations. 
8. Moribund animals or animals obviously in pain or showing signs of severe 
and enduring distress should be humanely killed and are considered in the interpretation of the test result in the same way as animals that died on test. 
Criteria for making the decision to kill moribund or severely suffering 
animals, and guidance on the recognition of predictable or impending death, are the subject of an OECD Guidance Document No 19 on Humane Endpoints (8).
 
DESCRIPTION OF THE METHOD  
Selection of animal species  
9. Healthy young adult animals of commonly used laboratory strains should be 
used. The preferred species is the rat and justification should be provided if 
other species are used.  
Preparation of animals  
10. Females should be nulliparous and non-pregnant. On the exposure day, 
animals should be young adults 8 to 12 weeks of age, and body weights should be within ± 20 % of the mean weight for each sex of any previously 
exposed animals of the same age. The animals are randomly selected and 
marked for individual identification. The animals are kept in their cages for at least 5 days prior to the start of the test to allow for acclimatisation to laboratory conditions. Animals should also be acclimatised to the test 
apparatus for a short period prior to testing, as this will lessen the stress 
caused by introduction to the new environment.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 218
 
Animal husbandry  
11. The temperature of the experimental animal maintenance room should be 22 
± 3 °C. The relative humidity should ideally be maintained in the range of 30 to 70 %, though this may not be possible when using water as a vehicle. 
Before and after exposures, animals generally should be caged in groups by 
sex and concentration, but the number of animals per cage should not interfere with clear observation of each animal and should minimise losses due to cannibalism and fighting. When animals are to be exposed 
nose-only, it may be necessary for them to be acclimated to the restraining 
tubes. The restraining tubes should not impose undue physical, thermal, or immobilisation stress on the animals. Restraint may affect physiological 
endpoints such as body temperature (hyperthermia) and/or respiratory 
minute volume. If generic data are available to show that no such changes occur to any appreciable extent, then pre-adaptation to the restraining tubes is not necessary. Animals exposed whole-body to an 
aerosol should be housed individually during exposure to prevent them 
from filtering the test aerosol through the fur of their cage mates. Conven ­
tional and certified laboratory diets may be used, except during exposure, accompanied with an unlimited supply of municipal drinking water. 
Lighting should be artificial, the sequence being 12 hours light/12 hours 
dark.
 
Inhalation chambers  
12. The nature of the test chemical and the objective of the test should be 
considered when selecting an inhalation chamber. The preferred mode of 
exposure is nose-only (which term includes head-only, nose-only or snout-only). Nose-only exposure is generally preferred for studies of liquid or solid aerosols and for vapours that may condense to form aerosols. Special 
objectives of the study may be better achieved by using a whole-body mode 
of exposure, but this should be justified in the study report. To ensure atmosphere stability when using a whole-body chamber, the total volume of the test animals should not exceed 5 % of the chamber volume. Principles 
of the nose-only and whole body exposure techniques and their particular 
advantages and disadvantages are described in GD 39 (2).
 
EXPOSURE CONDITIONS  
Administration of concentrations  
13. Nose-only exposures may be any duration up to 6 hours in rats. If mice are 
exposed nose-only, exposures generally should not exceed 4 hours. Justifi ­
cation should be provided if longer duration studies are needed [see GD 39 
(2)]. Animals exposed to aerosols in whole-body chambers should be housed individually to prevent ingestion of test chemical due to grooming of cage mates. Feed should be withheld during the exposure period. Water 
may be provided throughout a whole-body exposure. 
14. Animals are exposed to the test chemical as a gas, vapour, aerosol, or a 
mixture thereof. The physical state to be tested depends on the 
physico-chemical properties of the test chemical, the selected concentration, and/or the physical form most likely present during the handling and use of the test chemical. Hygroscopic and chemically reactive test chemicals should 
be tested under dry air conditions. Care should be taken to avoid generating 
explosive concentrations.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 219
 
Particle-size distribution  
15. Particle sizing should be performed for all aerosols and for vapours that may 
condense to form aerosols. To allow for exposure of all relevant regions of the respiratory tract, aerosols with mass median aerodynamic 
diameters (MMAD) ranging from 1 to 4 μm with a geometric standard 
deviation ( σ
 g ) in the range of 1,5 to 3,0 are recommended (2) (9) (10). 
Although a reasonable effort should be made to meet this standard, expert judgment should be provided if it cannot be achieved. For example, metal 
fumes may be smaller than this standard, and charged particles, fibres, and 
hygroscopic materials (which increase in size in the moist environment of the respiratory tract) may exceed this standard.
 
Test chemical preparation in a vehicle  
16. A vehicle may be used to generate an appropriate concentration and particle 
size of the test chemical in the atmosphere. As a rule, water should be given preference. Particulate material may be subjected to mechanical processes to achieve the required particle size distribution, however, care should be taken 
to not decompose or alter the test chemical. In cases where mechanical 
processes are believed to have altered test chemical composition (e.g. extreme temperatures from excessive milling due to friction), the composition of the test chemical should be verified analytically. Adequate 
care should be taken to not contaminate the test chemical. It is not necessary 
to test non-friable granular materials which are purposefully formulated to be un-inhalable. An attrition test should be used to demonstrate that respirable particles are not produced when the granular material is 
handled. If an attrition test produces respirable substances, an inhalation 
toxicity test should be performed.
 
Control animals  
17. A concurrent negative (air) control group is not necessary. When a vehicle 
other than water is used to assist in generating the test atmosphere, a vehicle 
control group should only be used when historical inhalation toxicity data are not available. If a toxicity study of a test chemical formulated in a vehicle reveals no toxicity, it follows that the vehicle is non-toxic at the 
concentration tested; thus, there is no need for a vehicle control.
 
MONITORING OF EXPOSURE CONDITIONS  
Chamber airflow  
18. The flow of air through the chamber should be carefully controlled, 
continuously monitored, and recorded at least hourly during each exposure. The monitoring of test atmosphere concentration (or stability) is an integral measurement of all dynamic parameters and provides an indirect 
means to control all relevant dynamic atmosphere generation parameters. 
Special consideration should be given to avoiding re-breathing in nose-only chambers in cases where airflow through the exposure system are inadequate to provide dynamic flow of test chemical atmosphere. 
There are prescribed methodologies that can be used to demonstrate that 
re-breathing does not occur under the selected operation conditions (2) (11). Oxygen concentration should be at least 19 % and carbon dioxide concen ­
tration should not exceed 1 %. If there is reason to believe that these 
standards cannot be met, oxygen and carbon dioxide concentrations 
should be measured.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 220
 
Chamber temperature and relative humidity  
19. Chamber temperature should be maintained at 22 ± 3 °C. Relative humidity 
in the animals’ breathing zone, for both nose-only and whole-body exposures, should be monitored and recorded at least three times for 
durations of up to 4 hrs, and hourly for shorter durations. The relative 
humidity should ideally be maintained in the range of 30 to 70 %, but this may either be unattainable ( e.g. when testing water based mixtures) 
or not measurable due to test chemical interference with the test method.
 
Test chemical: Nominal concentration  
20. Whenever feasible, the nominal exposure chamber concentration should be 
calculated and recorded. The nominal concentration is the mass of generated test chemical divided by the total volume of air passed through the chamber 
system. The nominal concentration is not used to characterise the animals’ 
exposure, but a comparison of the nominal concentration and the actual concentration gives an indication of the generation efficiency of the test 
system, and thus may be used to discover generation problems.
 
Test chemical: Actual concentration  
21. The actual concentration is the test chemical concentration at the animals’ 
breathing zone in an inhalation chamber. Actual concentrations can be 
obtained by specific methods (e.g. direct sampling, adsorptive or chemical 
reactive methods, and subsequent analytical characterisation) or by non-specific methods such as gravimetric filter analysis. The use of gravi ­
metric analysis is acceptable only for single component powder aerosols or 
aerosols of low volatility liquids and should be supported by appropriate 
pre-study test chemical-specific characterisations. Multi-component powder aerosol concentration may also be determined by gravimetric analysis. However, this requires analytical data which demonstrate that the 
composition of airborne material is similar to the starting material. If this 
information is not available, a reanalysis of the test chemical (ideally in its airborne state) at regular intervals during the course of the study may be necessary. For aerosolised agents that may evaporate or sublimate, it should 
be shown that all phases were collected by the method chosen. The target, 
nominal, and actual concentrations should be provided in the study report, but only actual concentrations are used in statistical analyses to calculate lethal concentration values. 
22. One lot of the test chemical should be used, if possible, and the test sample 
should be stored under conditions that maintain its purity, homogeneity, and 
stability. Prior to the start of the study, there should be a characterisation of 
the test chemical, including its purity and, if technically feasible, the identity, and quantities of identified contaminants and impurities. This can be demonstrated by, but is not limited to, the following data: retention time 
and relative peak area, molecular weight from mass spectroscopy or gas 
chromatography analyses, or other estimates. Although the test sample’s identity is not the responsibility of the test laboratory, it may be prudent for the test laboratory to confirm the sponsor’s characterisation at least in a 
limited way (e.g. colour, physical nature, etc.). 
23. The exposure atmosphere shall be held as constant as practicable and 
monitored continuously and/or intermittently depending on the method of 
analysis. When intermittent sampling is used, chamber atmosphere samples should be taken at least twice in a four hour study. If not feasible due to limited air flow rates or low concentrations, one sample may be collected 
over the entire exposure period. If marked sample-to-sample fluctuations 
occur, the next concentrations tested should use four samples per exposure.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 221
 
Individual chamber concentration samples should not deviate from the mean 
concentration by more than ± 10 % for gases and vapours or ± 20 % for 
liquid or solid aerosols. Time to chamber equilibration (t  95 ) should be 
calculated and recorded. The duration of an exposure spans the time that the test chemical is generated and this takes into account the times required 
to attain t
 95 . Guidance for estimating t  95 can be found in GD 39 (2). 
24. For very complex mixtures consisting of gases/vapours, and aerosols (e.g. 
combustion atmospheres and test chemicals propelled from purpose-driven 
end-use products/devices), each phase may behave differently in an inha ­
lation chamber so at least one indicator substance (analyte), normally the principal active substance in the mixture, of each phase (gas/vapour and 
aerosol) should be selected. When the test chemical is a mixture, the 
analytical concentration should be reported for the mixture and not just for the active substance or the component (analyte). Additional information regarding actual concentrations can be found in GD 39 (2).
 
Test chemical: Particle size distribution  
25. The particle size distribution of aerosols should be determined at least twice 
during each 4 hour exposure by using a cascade impactor or an alternative instrument such as an aerodynamic particle sizer. If equivalence of the 
results obtained by a cascade impactor or an alternative instrument can be 
shown, then the alternative instrument may be used throughout the study. A second device, such as a gravimetric filter or an impinger/gas bubbler, should be used in parallel to the primary instrument to confirm the 
collection efficiency of the primary instrument. The mass concentration 
obtained by particle size analysis should be within reasonable limits of the mass concentration obtained by filter analysis [see GD 39 (2)]. If equiv ­
alence can be demonstrated in the early phase of the study, then further 
confirmatory measurements may be omitted. For animal welfare reasons, 
measures should be taken to minimise inconclusive data which may lead to a need to repeat an exposure. Particle sizing should be performed for vapours if there is any possibility that vapour condensation may result in the 
formation of an aerosol, or if particles are detected in a vapour atmosphere 
with potential for mixed phases (see paragraph 15).
 
PROCEDURE 
26. Two study types are described below: the Traditional protocol, and the C × t 
protocol. Both protocols may include a sighting study, a main study, and/or 
a limit test (Traditional protocol) or testing at a limit concentration (C × t). If one sex is known to be more susceptible, the study director may choose to perform these studies using only the susceptible sex. If rodent species 
other than rats are exposed nose-only, maximum exposure durations may be 
adjusted to minimise species-specific distress. Before commencing, all available data should be considered in order to minimise animal usage. For example, data generated using chapter B.52 of this Annex (4) may 
eliminate the need for a sighting study, and may also demonstrate 
whether one sex is more susceptible [see GD 39 (2)].
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 222
 
TRADITIONAL PROTOCOL  
General considerations: Traditional protocol  
27. In a Traditional study, groups of animals are exposed to a test chemical for 
a fixed period of time (generally 4 hours) in either a nose-only or whole-body exposure chamber. Animals are exposed to either a limit concentration (limit test), or to at least three concentrations in a stepwise 
procedure (main study). A sighting study may precede a main study unless 
some information about the test chemical already exists, such as a previously performed B.52 study [see GD 39 (2)].
 
Sighting study: Traditional protocol  
28. A sighting study is used to estimate test chemical potency, identify sex 
differences in susceptibility, and assist in selecting exposure concentration levels for the main study or limit test. When selecting concentration levels 
for the sighting study, all available information should be used including 
available (Q)SAR data and data for similar chemicals. No more than three males and three females should be exposed at each concentration (3 animals/sex may be needed to establish a sex difference). A sighting 
study may consist of a single concentration, but more concentrations may 
be tested if necessary. A sighting study should not test so many animals and concentrations that it resembles a main study. A previously performed B.52 study (4) may be used instead of a sighting study [see GD 39 (2)].
 
Limit test: Traditional protocol  
29. A limit test is used when the test chemical is known or expected to be 
virtually non-toxic, i.e. eliciting a toxic response only above the regulatory 
limit concentration. In a limit test, a single group of three males and three 
females is exposed to the test chemical at a limit concentration. Information about the toxicity of the test chemical can be gained from knowledge about similar tested chemicals, taking into consideration the identity and 
percentage of components known to be of toxicological significance. In 
those situations where there is little or no information about its toxicity, or the test chemical is expected to be toxic, the main test should be performed. 
30. The selection of limit concentrations usually depends on regulatory require ­
ments. When Regulation (EC) No 1272/2008 is used, the limit concen ­
trations for gases, vapours, and aerosols are 20 000 ppm, 20 mg/l and 5 mg/l, respectively (or the maximum attainable concentration) (3). It can be technically challenging to generate limit concentrations of some test 
chemicals, especially as vapours and aerosols. When testing aerosols, the 
primary goal should be to achieve a respirable particle size (MMAD of 1- 4 μm). This is possible with most test chemicals at a concentration of 2 
mg/l. Aerosol testing at greater than 2 mg/l should only be attempted if a 
respirable particle size can be achieved [see GD 39 (2)]. Regulation (EC) 
No 1272/2008 discourages testing in excess of a limit concentration for animal welfare reasons (3). The limit concentration should only be considered when there is a strong likelihood that results of such a test 
would have direct relevance for protecting human health (3), and justifi ­
cation provided in the study report. In the case of potentially explosive test chemicals, care should be taken to avoid conditions favourable for an explosion. To avoid an unnecessary use of animals, a test run without 
animals should be conducted prior to the limit test to ensure that the 
chamber conditions for a limit test can be achieved.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 223
 
31. If mortality or moribundity is observed at the limit concentration, the results 
of the limit test can serve as a sighting study for further testing at other 
concentrations (see main study). If a test chemical’s physical or chemical properties make it impossible to attain a limit concentration, the maximum attainable concentration should be tested. If less than 50 % lethality occurs 
at the maximum attainable concentration, no further testing is necessary. If 
the limit concentration could not be attained, the study report should provide an explanation and supportive data. If the maximum attainable concentration of a vapour does not elicit toxicity, it may be necessary to generate the test 
chemical as a liquid aerosol.
 
Main study: Traditional protocol  
32. A main study is typically performed using five males and five females (or 5 
animals of the susceptible sex, if known) per concentration level, with at 
least three concentration levels. Sufficient concentration levels should be 
used to obtain a robust statistical analysis. The time interval between exposure groups is determined by the onset, duration, and severity of toxic signs. Exposure of animals at the next concentration level should be 
delayed until there is reasonable confidence of survival for previously tested 
animals. This allows the study director to adjust the target concentration for the next exposure group. Due to the dependence on sophisticated technol ­
ogies, this may not always be practical in inhalation studies, so the exposure 
of animals at the next concentration level should be based on previous 
experience and scientific judgement. GD 39 (2) should be consulted when testing mixtures.
 
CONCENTRATION × TIME (C × T) PROTOCOL  
General considerations: C × t protocol  
33. A step-wise C × t study may be considered as an alternative to a Traditional 
protocol when assessing inhalation toxicity (12) (13) (14). This approach 
allows animals to be exposed to a test chemical at several concentration levels and for multiple time durations. All testing is performed in a nose- only chamber (whole-body chambers are not practical for this protocol). A 
flow diagram in Appendix 1 illustrates this protocol. A simulation analysis 
has shown that the Traditional protocol and the C × t protocol are both capable of yielding robust LC
 50 values, but the C × t protocol is generally 
better at yielding robust LC  01 and LC  10 values (15). 
34. A simulation analysis has demonstrated that using two animals per C × t 
interval (one per sex using both sexes, or two of the more susceptible sex) 
may generally be adequate when testing 4 concentrations and 5 exposure 
durations in a main study. Under some circumstances, the study director may elect to use two rats per sex per C × t interval (15). Using 2 animals per sex per concentration and time point may reduce bias and variability of 
the estimates, increase the estimation success rate, and improve confidence 
interval coverage. However, in case of an insufficient close fit to the data for estimation (when using one animal per sex or two animals of the more susceptible sex) a 5th exposure concentration may also suffice. Further 
guidance on the number of animals and concentrations to be used in a C 
× t study can be found in GD 39 (2).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 224
 
Sighting study: C × t protocol  
35. A sighting study is used to estimate test chemical potency and to assist in 
selecting exposure concentration levels for the main study. A sighting study using up to three animals/sex/concentration [for details see Appendix III of 
GD 39 (2)] may be needed to choose an appropriate starting concentration 
for the main study and to minimise the number of animals used. It may be necessary to use three animals per sex to establish a sex difference. These animals should be exposed for a single duration, generally 240 min. The 
feasibility of generating adequate test atmospheres should be assessed during 
technical pre-tests without animals. It is generally not necessary to perform a sighting study if mortality data are available from a B.52 study (4). When 
selecting the initial target concentration in a B.2 study, the study director 
should consider the mortality patterns observed in any available B.52 studies (4) for both sexes and for all concentrations tested [see GD 39 (2)].
 
Initial Concentration: C × t protocol  
36. The initial concentration (Exposure Session I) (Appendix 1) will either be a 
limit concentration or a concentration selected by the study director based 
on the sighting study. Groups of 1 animal/sex are exposed to this concen ­
tration for multiple durations ( e.g. 15, 30, 60, 120, or 240 minutes), 
resulting in a total number of 10 animals (called Exposure Session I) (Appendix 1). 
37. The selection of limit concentrations usually depends on regulatory require ­
ments. When Regulation (EC) No 1272/2008 is used, the limit concen ­
trations for gases, vapours, and aerosols are 20 000 ppm, 20 mg/l and 5 mg/l, respectively (or the maximum attainable concentration) (3). It can be 
technically challenging to generate limit concentrations of some test 
chemicals, especially as vapours and aerosols. When testing aerosols, the goal should be to achieve a respirable particle size (i.e. an MMAD of 1- 4 μm) at a limit concentration of 2 mg/l. This is possible with most test 
chemicals. Aerosol testing at greater than 2 mg/l should only be attempted if 
a respirable particle size can be achieved [see GD 39 (2)]. Regulation (EC) No 1272/2008 discourages testing in excess of a limit concentration for animal welfare reasons (3). Testing in excess of the limit concentration 
should only be considered when there is a strong likelihood that results 
of such a test would have direct relevance for protecting human health (3), justification should be provided in the study report. In the case of potentially explosive test chemicals, care should be taken to avoid 
conditions favourable for an explosion. To avoid an unnecessary use of 
animals, a test run without animals should be conducted prior to testing at the initial concentration to ensure that the chamber conditions for this concentration can be achieved. 
38. If mortality or moribundity is observed at the initial concentration, the 
results at this concentration can serve as a starting point for further testing at other concentrations (see main study). When a test chemical’s physical or chemical properties make it impossible to attain a limit concen ­
tration, the maximum attainable concentration should be tested. If less than 
50 % lethality occurs at the maximum attainable concentration, no further testing is necessary. If the limit concentration could not be attained, the study report should provide an explanation and supportive data. If the 
maximum attainable concentration of a vapour does not elicit toxicity, it 
may be necessary to generate the test chemical as a liquid aerosol.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 225
 
Main study: C × t protocol  
39. The initial concentration (Exposure Session I) (Appendix 1) tested in the 
main study will either be a limit concentration or a concentration selected by the study director based on the sighting study. If mortality has been 
observed during or following Exposure Session I, the minimum exposure 
(C × t) which results in mortality will be taken as a guide to establish the concentration and periods of exposure for Exposure Session II. Each subsequent exposure session will depend on the previous session (see 
Appendix 1). 
40. For many test chemicals the results obtained at the initial concentration, 
together with three additional exposure sessions with a smaller time grid 
(i.e. the geometric spacing of exposure periods as indicated by the factor 
between successive periods, generally √2), will be sufficient to establish the 
C × t mortality relationship (15), but there may be some benefit to using a 5th exposure concentration [see Appendix 1 and GD 39 (2)]. For math ­
ematical treatment of results for the C × t protocol, see Appendix 1.
 
OBSERVATIONS 
41. The animals should be clinically observed frequently during the exposure 
period. Following exposure, clinical observations should be made at least twice on the day of exposure, or more frequently when indicated by the 
response of the animals to treatment, and at least once daily thereafter for a 
total of 14 days. The length of the observation period is not fixed, but should be determined by the nature and time of onset of clinical signs and length of the recovery period. The times at which signs of toxicity 
appear and disappear are important, especially if there is a tendency for 
signs of toxicity to be delayed. All observations are systematically recorded with individual records being maintained for each animal. Animals found in a moribund condition and animals showing severe pain 
and/or enduring signs of severe distress should be humanely killed for 
animal welfare reasons. Care should be taken when conducting examinations for clinical signs of toxicity that initial poor appearance and transient respiratory changes, resulting from the exposure procedure, are not 
mistaken for test chemical-related toxicity that would require premature 
killing of the animals. The principles and criteria summarised in the Guidance Document on Humane Endpoints (GD 19) should be taken into 
consideration (7). When animals are killed for humane reasons or found 
dead, the time of death should be recorded as precisely as possible. 
42. Cage-side observations should include changes in the skin and fur, eyes and 
mucous membranes, and also respiratory, circulatory, autonomic and central 
nervous systems, and somatomotor activity and behaviour patterns. When 
possible, any differentiation between local and systemic effects should be noted. Attention should be directed to observations of tremors, convulsions, 
salivation, diarrhoea, lethargy, sleep and coma. The measurement of rectal 
temperature may provide supportive evidence of reflex bradypnea or hypo/ hyperthermia related to treatment or confinement.
 
Body weights  
43. Individual animal weights should be recorded once during the acclimat ­
ization period, on the day of exposure prior to exposure (day 0), and at 
least on days 1, 3 and 7 (and weekly thereafter), and at the time of death or 
euthanasia if exceeding day 1. Body weight is recognised as a critical indicator of toxicity so animals exhibiting a sustained decrement of ≥ 
20 %, compared to pre-study values, should be closely monitored. 
Surviving animals are weighed and humanely killed at the end of the 
post-exposure period.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 226
 
Pathology  
44. All test animals, including those which die during the test or are euthanised 
and removed from the study for animal welfare reasons, should be subjected to gross necropsy. If necropsy cannot be performed immediately after a dead 
animal is discovered, the animal should be refrigerated (not frozen) at 
temperatures low enough to minimise autolysis. Necropsies should be performed as soon as possible, normally within a day or two. All gross pathological changes should be recorded for each animal with particular 
attention to any changes in the respiratory tract. 
45. Additional examinations included a priori by design may be considered to 
extend the interpretive value of the study, such as measuring lung weight of 
surviving rats, and/or providing evidence of irritation by microscopic exam ­
ination of the respiratory tract. Examined organs may also include those showing evidence of gross pathology in animals surviving 24 or more hours, and organs known or expected to be affected. Microscopic exam ­
ination of the entire respiratory tract may provide useful information for test 
chemicals that are reactive with water, such as acids and hygroscopic test chemicals.
 
DATA AND REPORTING  
Data  
46. Individual animal data on body weights and necropsy findings should be 
provided. Clinical observation data should be summarised in tabular form, showing for each test group the number of animals used, the number of 
animals displaying specific signs of toxicity, the number of animals found 
dead during the test or killed for humane reasons, time of death of indi ­
vidual animals, a description and time course of toxic effects and reversi ­
bility, and necropsy findings.
 
Test report  
47. The test report should include the following information, as appropriate:  
Test animals and husbandry  
— Description of caging conditions, including: number (or change in 
number) of animals per cage, bedding material, ambient temperature 
and relative humidity, photoperiod, and identification of diet 
— Species/strain used and justification for using a species other than the rat — Number, age and sex of animals 
— Method of randomisation 
— Details of food and water quality (including diet type/source, water 
source) 
— Description of any pre-test conditioning including diet, quarantine, and 
treatment for disease;
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 227
 
Test chemical  
— Physical nature, purity and, where relevant, physico-chemical properties 
(including isomerisation) 
— Identification data and Chemical Abstract Services (CAS) Registry 
Number, if known;  
Vehicle  
— Justification for use of vehicle and justification for choice of vehicle (if 
other than water) 
— Historical or concurrent data demonstrating that the vehicle does not 
interfere with the outcome of the study;  
Inhalation chamber  
— Description of the inhalation chamber including dimensions and volume — Source and description of equipment used for the exposure of animals as 
well as generation of atmosphere 
— Equipment for measuring temperature, humidity, particle-size, and actual 
concentration 
— Source of air and treatment of air supplied/extracted and system used for 
conditioning 
— Methods used for calibration of equipment to ensure a homogeneous test 
atmosphere 
— Pressure difference (positive or negative) — Exposure ports per chamber (nose-only); location of animals in the 
system (whole-body) 
— Temporal homogeneity/stability of test atmosphere — Location of temperature and humidity sensors and sampling of test 
atmosphere in the chamber 
— Air flow rates, air flow rate/exposure port (nose-only), or animal load/ 
chamber (whole-body) 
— Information about the equipment used to measure oxygen and carbon 
dioxide, if applicable 
— Time required to reach inhalation chamber equilibrium (t
 95 ) 
— Number of volume changes per hour — Metering devices (if applicable);  
Exposure data  
— Rationale for target concentration selection in the main study — Nominal concentrations (total mass of test chemical generated into the 
inhalation chamber divided by the volume of air passed through the chamber) 
— Actual test chemical concentrations collected from the animals’ 
breathing zone; for mixtures that produce heterogeneous physical 
forms (gases, vapours, aerosols), each may be analysed separately
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 228
 
— All air concentrations should be reported in units of mass (e.g. mg/l, 
mg/m  3 , etc.); units of volume ( e.g. ppm, ppb, etc.) may also be reported 
parenthetically 
— Particle size distribution, mass median aerodynamic diameter (MMAD), 
and geometric standard deviation ( σg), including their methods of calcu ­
lation. Individual particle size analyses should be reported;  
Test conditions  
— Details of test chemical preparation, including details of any procedures 
used to reduce the particle size of solid materials or to prepare solutions 
of the test chemical. In cases where mechanical processes may have altered test chemical composition, include the results of analyses to verify the composition of the test chemical 
— A description (preferably including a diagram) of the equipment used to 
generate the test atmosphere and to expose the animals to the test atmosphere 
— Details of the chemical analytical method used and method validation 
(including efficiency of recovery of test chemical from the sampling medium) 
— The rationale for the selection of test concentrations;  
Results  
— Tabulation of chamber temperature, humidity, and airflow 
— Tabulation of chamber nominal and actual concentration data 
— Tabulation of particle size data including analytical sample collection 
data, particle size distribution and calculations of the MMAD and σ
 g 
— Tabulation of response data and concentration level for each animal (i.e. 
animals showing signs of toxicity including mortality, nature, severity, time of onset and duration of effects) 
— Individual body weights of animals collected on study; date and time of 
death if prior to scheduled euthanasia, time course of onset of signs of toxicity and whether these were reversible for each animal 
— Necropsy findings and histopathological findings for each animal, if 
available 
— Lethality estimates (e.g. LC
 50 , LD  01 ) including 95 % confidence limits, 
and slope (if provided by the evaluation method) 
— Statistical relation, including estimate for the exponent n (C × t 
protocol). The name of the statistical software used should be provided;  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 229
 
Discussion and interpretation of results  
— Particular emphasis should be made to the description of methods used 
to meet this Test Method’s criteria, e.g. the limit concentration or the 
particle size 
— The respirability of particles in light of the overall findings should be 
addressed, especially if the particle-size criteria could not be met 
— An explanation should be provided if there was a need to humanely 
sacrifice animals in pain or showing signs of severe and enduring 
distress, based on the criteria in the OECD Guidance Document on Humane Endpoints (8) 
— If testing with chapter B.52 of this Annex (4) was discontinued in favour 
of this Test Method B.2, justifications should be provided 
— The consistency of methods used to determine nominal and actual 
concentrations, and the relation of actual concentration to nominal concentration should be included in the overall assessment of the study 
— The likely cause of death and predominant mode of action (systemic 
versus local) should be addressed.
 
LITERATURE:  
(1) OECD (2009). Acute Inhalation Toxicity Testing. OECD Guideline for 
Testing of Chemicals No 403, OECD, Paris. Available at: [http:// www.oecd.org/env/testguidelines] 
(2) OECD (2009). Guidance Document on Acute Inhalation Toxicity Testing. 
Environmental Health and Safety Monograph Series on Testing and 
Assessment No 39, OECD, Paris. Available at: [http://www.oecd.org/env/ 
testguidelines] 
(3) Regulation (EC) No 1272/2008 of the European Parliament and of the 
Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC and amending Regulation (EC) No 1907/2006 (OJ L 353, 
31.12.2008, p. 1). 
(4) Chapter B.52 of this Annex, Acute Inhalation Toxicity — Acute Toxic 
Class (ATC) Method. 
(5) Chapter B.40 of this Annex, In Vitro Skin Corrosion: Transcutaneous 
Electrical Resistance Test (TER). 
(6) Chapter B.40bis of this Annex, In Vitro Skin Corrosion: Human Skin 
Model Test. 
(7) OECD (2005), In Vitro Membrane Barrier Test Method For Skin 
Corrosion. OECD Guideline for Testing of Chemicals No 435, OECD, 
Paris. Available at: [http://www.oecd.org/env/testguidelines] 
(8) OECD (2000). Guidance Document on the Recognition, Assessment and 
Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. Environmental Health and Safety Monograph Series on Testing and Assessment No 19, OECD, Paris. Available at: 
[http://www.oecd.org/env/testguidelines] 
(9) SOT (1992). Technical Committee of the Inhalation Specialty Section, 
Society of Toxicology (SOT). Recommendations for the Conduct of 
Acute Inhalation Limit Tests. Fund. Appl. Toxicol. 18: 321-327. 
(10) Phalen RF (2009). Inhalation Studies: Foundations and Techniques. (2nd 
Edition) Informa Healthcare, New York.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 230
 
(11) Pauluhn J and Thiel A (2007). A Simple Approach to Validation of 
Directed-Flow Nose-Only Inhalation Chambers. J. Appl. Toxicol. 27: 
160-167. 
(12) Zwart JHE, Arts JM, ten Berge WF, Appelman LM (1992). Alternative 
Acute Inhalation Toxicity Testing by Determination of the Concentration-Time-Mortality Relationship: Experimental Comparison 
with Standard LC50 Testing. Reg. Toxicol. Pharmacol. 15: 278-290. 
(13) Zwart JHE, Arts JM, Klokman-Houweling ED, Schoen ED (1990). Deter ­
mination of Concentration-Time-Mortality Relationships to Replace LC50 Values. Inhal. Toxicol. 2: 105-117. 
(14) Ten Berge WF and Zwart A (1989). More Efficient Use of Animals in 
Acute Inhalation Toxicity Testing. J. Haz. Mat. 21: 65-71. 
(15) OECD (2009). Performance Assessment: Comparison of 403 and C × t 
Protocols via Simulation and for Selected Real Data Sets. Environmental Health and Safety Monograph Series on Testing and Assessment No 104, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines] 
(16) Finney DJ (1977). Probit Analysis, 3rd ed. Cambridge University Press, 
London/New York.
 
DEFINITION  
Test chemical:  Any substance or mixture tested using this Test Method.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 231
 
Appendix 1  
C × t Protocol  
1. A step-wise Concentration × Time (C × t) study may be considered as an 
alternative to the Traditional protocol for assessing inhalation toxicity (12) (13) (14). It should be performed preferentially when there is a specific regulatory or scientific need that calls for the testing of animals over 
multiple time durations such as for emergency response planning or land 
use planning. This approach usually begins with testing at a limit concen ­
tration (Exposure Session I) in which animals are exposed to a test chemical for five time durations (e.g. 15, 30, 60, 120 and 240 min) so that multiple 
durations of time will be obtained within one exposure session (see Figure 
1). When Regulation (EC) No 1272/2008 is used, the limit concentrations for gases, vapours, and aerosols are 20 000 ppm, 20 mg/l, and 5 mg/l, respectively. These levels may only be exceeded if there is a regulatory 
or scientific need for testing at these levels (see paragraph 37 in the B.2 
main text). 
2. In situations where there is little or no information about the toxicity of a 
test chemical, a sighting study should be performed in which groups of no more than 3 animals per sex are exposed to target concentrations selected by 
the study director, generally for 240 min. 
3. If a limit concentration is tested during Exposure Session I and less than 
50 % mortality is observed, no additional testing is needed. If there is a 
regulatory or scientific need to establish the concentration/time/response relationship at higher levels than the indicated limit concentration, the next exposure should be carried out at a higher level such as at two 
times the limit concentration (i.e. 2L in Figure 1). 
4. If toxicity is observed at the limit concentration, additional testing (main 
study) is necessary. These additional exposures are carried out either at 
lower concentrations (in Figure 1: Exposure Sessions II, III or IV') or at higher concentrations using shorter durations (in Figure 1: Exposure Session 
IV) using durations that are adapted and not as widely spaced. 
5. The test (initial concentration and additional concentrations) is carried out 
using 1 animal/sex per concentration/time point or with 2 animals of the 
more susceptible sex per concentration/time point. Under some circum ­
stances, the study director may elect to utilise 2 rats per sex per concen ­
tration/time point (or 4 animals of the susceptible sex per concentration/time 
point) (15). Using 2 animals per sex per concentration/time point generally 
reduces bias and variability of the estimates, increases the estimation success rate, and improves confidence interval coverage relative to the protocol as described here. Further details are provided in GD 39 (2). 
6. Ideally, each exposure session is carried out on one day. This gives the 
opportunity to delay the next exposure until there is reasonable confidence 
of survival, and it allows the study director to adjust the target concentration 
and durations for the next exposure session. It is advised to start each exposure session with the group that will be exposed the longest, e.g. the 240-min exposure group, followed by the 120 minute exposure group, and 
so on. If, for example, animals in the 240 minute group are dying after 90 
minutes or showing severe signs of toxicity (e.g. extreme changes in breathing pattern such as laboured breathing), it would not make sense to expose a group for 120 minutes because mortality would likely be 100 %. 
Thus the study director should select shorter exposure durations for that 
concentration (e.g. 90, 65, 45, 33 and 25 minutes).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 232
 
7. The chamber concentration should be measured frequently to determine the 
time-weighted-average concentration for each exposure duration. Whenever 
possible, the time of death for each animal (rather than the exposure duration) should be used in the statistical analysis. 
8. The results of the first four exposure sessions should be examined to 
identify a data gap in the concentration-time curve (see Figure 1). In case of an insufficient fit, an additional exposure (5th concentration) may be 
performed. Concentration and exposure durations for the 5th exposure 
should be chosen to cover this gap. 
9. All exposure sessions (including the first Exposure Session) will be used to 
calculate the concentration-time-response relationship using Statistical Analysis (16). If possible, for each C × t interval, the time-weighted average concentration and the duration of exposure until death (if death 
occurs during the exposure) should be used.
 
Figure 1  
Hypothetical illustration of a concentration-time-mortality relationship in 
rats
 
Open symbols = survivors; closed symbols = dead animals 
Triangles = females; circles = males Solid line = LC
 50 values (range 7,5-240 min) for males with n = 1 
Dashed line = LC  50 values (range 7,5-240 min) for females with n = 1 
Dotted lines = hypothetical LC  50 values line for males and females if n had 
been equal to 2 (12). 
Glossary 
Concentration: Time of exposure:
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 233
 
10. Below is an example of the stepwise procedure:  
Exposure Session I — Testing at the limit concentration (see Figure 1)  
— 1 animal/sex per concentration/time point; 10 animals in total (  a ) 
— Target concentration (  b ) = limit concentration. 
— Expose five groups of animals at this target concentration for durations 
of 15, 30, 60, 120 and 240 minutes, respectively.  
↓ 
Exposure Session II  ( c ) — Main Study  
— 1 animal/sex per concentration/time point; 10 animals in total. 
— Expose five groups of animals at a lower concentration (  d ) (1/2L) with 
slightly longer exposure durations (factor √2 spaced; see Figure 1).  
↓ 
Exposure Session III — Main Study  
— 1 animal/sex per concentration/time point; 10 animals total. 
— Expose five groups of animals at a lower concentration (  d ) (1/4L) with 
slightly longer exposure durations (factor √2 spaced; see Figure 1).  
↓ 
Exposure Session IV’ — Main Study  
— 1 animal/sex per concentration/time point; 10 animals total. 
— Expose five groups of animals at a lower concentration (  d ) (1/8L) with 
slightly longer exposure durations (factor √2 spaced; see Figure 1).  
↓ or ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 234  
( a ) If no sex susceptibility information is available, rats of both sexes will be used,  i.e. 1 
animal/sex per concentration. Based on existing information, or if it becomes apparent 
during this exposure session that one sex is more susceptible, 10 animals of the 
susceptible sex will be used (2 animals per concentration/time point) at each concen ­
tration level during subsequent testing. 
( b ) When Regulation (EC) No 1272/2008 is used, the limit concentrations for gases, 
vapours, and aerosols are 20 000 ppm, 20 mg/l, and 5 mg/l, respectively. In case of expected toxicity or based on the results of the sighting study, lower starting concen ­
trations should be chosen. In case of regulatory or scientific needs, higher concentrations 
may be used. 
(
 c ) Ideally, exposure of animals at the next concentration level should be delayed until there 
is reasonable confidence of survival for previously treated animals. This allows the study 
director to adjust the target concentration and durations for the next exposure session. 
( d ) The minimum dose (concentration x time) which resulted in mortality during testing at 
initial concentration (first exposure session) will be taken as a guide to establish the next 
combination of concentration and exposure durations. Typically, the concentration will 
be decreased two-fold (1/2L) and animals will be exposed over a new time range with a 
finer grid using a geometric division of exposure periods with a factor 1,4 ( √2; see 
reference 11) around the time according to the minimum lethal dose level (time x 
concentration) observed during the first exposure. In this figure (Figure 1), mortality 
in Exposure session I was first observed at 15 min; the durations during session II are 
therefore centred around 30 min, and are 15, 21 30, 42 and 60 min. After the first two 
exposures, it is strongly advised to plot the data in a similar figure as indicated above, 
and to check whether the relationship between concentration and time has an angle of 
45 degrees (n = 1) or if the concentration-time-response relationship is less steep (e.g. n 
= 2) or steeper ( e.g. n = 0,8). In the latter cases it is strongly advised to adapt the next 
concentrations and durations accordingly.
 Exposure Session IV — Main Study  
— 1 animal/sex per concentration/time point; 10 animals total. 
— Expose five groups of animals at a higher concentration (  e ) (2L) with 
slightly shorter exposure durations (factor √2 spaced; see Figure 1).  
Mathematical treatment of results for the C × t protocol  
11. A C × t procedure with 4 or 5 exposure concentrations and five durations 
will yield 20 or 25 data points, respectively. With these data points, the 
C × t relationship can be calculated using statistical analysis (16):  
Equation 1: 
ProbitðPÞ ¼ b 0 þ b 1 ln C þ b 2 ln t 
where C = concentration; t = exposure duration, or  
Equation 2: 
Response ¼ ƒðC n tÞ 
where  n ¼ b 1=b 2: 
Using equation 1, the LC  50 value can be calculated for a given time period 
(e.g. 4 hour, 1 hour, 30 minutes, or any time period within the range of time 
periods tested) using P = 5 (50 % response). Note that Haber’s rule is only applicable when n = 1. The LC
 01 can be calculated using P = 2,67.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 235  
( e ) In certain cases it may be necessary to increase the concentration (2L) over a new time 
range with a still finer grid using a geometric division of exposure periods with a factor 
1,4 (√2) around the time according to the minimum lethal concentration level observed 
during the first exposure. The minimum exposure duration should preferably exceed 5 
minutes; the maximum exposure duration should not exceed 8 hours.
 B.3. ACUTE TOXICITY (DERMAL)  
1. METHOD  
1.1. INTRODUCTION 
See General introduction Part B (A).  
1.2. DEFINITION 
See General introduction Part B (B).  
1.3. REFERENCE SUBSTANCES 
None.  
1.4. PRINCIPLE OF THE TEST METHOD 
The test substance is applied to the skin in graduated doses to several 
groups of experimental animals, one dose being used per group. Subsequently, observations of effects and deaths are made. Animals, which die during the test are necropsied and at the 
conclusion of the test surviving animals are necropsied. 
Animals showing severe and enduring signs of distress and pain may 
need to be humanely killed. Dosing test substances in a way known to cause marked pain and distress due to corrosive or irritating 
properties need not be carried out.
 
1.5. QUALITY CRITERIA 
None.  
1.6. DESCRIPTION OF THE TEST METHOD  
1.6.1.  Preparations  
The animals are kept in their experimental cages under the experi ­
mental housing and feeding conditions for at least five days prior to 
the experiment. Before the test, healthy young adult animals are 
randomised and assigned to the treatment groups. Approximately 24 hours before the test, fur should be removed by clipping or shaving from the dorsal area of the trunk of the animals. When 
clipping or shaving the fur, care must be taken to avoid abrading 
the skin which could alter its permeability. Not less than 10 % of the body surface should be clear for the application of the test substance. When testing solids, which may be pulverised if appropriate, the test 
substance should be moistened sufficiently with water or, where 
necessary, a suitable vehicle to ensure good contact with the skin. When a vehicle is used, the influence of the vehicle on penetration of skin by the test substance should be taken into account. Liquid 
test substances are generally used undiluted.
 
1.6.2.  Test conditions  
1.6.2.1.  Experimental animals  
The adult rat or rabbit may be used. Other species may be used but 
their use would require justification. Commonly used laboratory strains should be employed. For each sex, at the start of the test 
the range of weight variation in the animals used should not exceed 
± 20 % of the appropriate mean value.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 236
 
1.6.2.2.  Number and sex  
At least five animals are used at each dose level. They should all be 
of the same sex. If females are used, they should be nulliparous and non-pregnant. Where information is available demonstrating that a 
sex is markedly more sensitive, animals of this sex should be dosed.  
Note:  in acute toxicity tests with animals of a higher order than 
rodents, the use of smaller numbers should be considered. Doses 
should be carefully selected, and every effort should be made not 
to exceed moderately toxic doses. In such tests, administration of lethal doses of the test substance should be avoided.
 
1.6.2.3.  Dose levels  
These should be sufficient in number, at least three, and spaced 
appropriately to produce test groups with a range of toxic effects 
and mortality rates. Any irritant or corrosive effects should be taken into account when deciding on dose levels. The data should be sufficient to produce a dose/response curve and, where possible, 
permit an acceptable determination of the LD
 50 . 
1.6.2.4.  Limit test  
A limit test at one dose level of at least 2 000 mg/kg bodyweight 
may be carried out in a group of five male and five female animals, using the procedures described above. If compound-related mortality is produced, a full study may need to be considered.
 
1.6.2.5.  Observation period  
The observation period should be at least 14 days. However, the 
duration of observation should not be rigidly fixed. It should be determined by the toxic reactions, their rate of onset and the length of the recovery period; it may thus be extended when 
considered necessary. The time at which signs of toxicity appear 
and disappear, their duration and the time of death are important, especially if there is a tendency for deaths to be delayed.
 
1.6.3.  Procedure  
Animals should be caged individually. The test substance should be 
applied uniformly over an area, which is approximately 10 % of the 
total body surface area. With highly toxic substances the surface area 
covered may be less but as much of the area should be covered with a layer as thin and uniform as possible. 
Test substances should be held in contact with the skin with a porous 
gauze dressing and non-irritating tape throughout a 24-hour exposure 
period. The test site should be further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance. Restrainers may be used to 
prevent the ingestion of the test substance but complete immobili ­
sation is not a recommended method.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 237
 
At the end of the exposure period, residual test substance should be 
removed, where practicable, using water or some other appropriate 
method of cleansing the skin. 
Observations should be recorded systematically as they are made. 
Individual records should be maintained for each animal. Obser ­
vations should be made frequently during the first day. A careful clinical examination should be made at least once each working day, 
other observations should be made daily with appropriate actions 
taken to minimise loss of animals to the study, e.g. necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals. 
Observations should include changes in fur, treated skin, eyes and 
mucous membranes, and also respiratory, circulatory, autonomic and 
central nervous systems, and somatomotor activity and behaviour 
pattern. Particular attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The time of death must be recorded as precisely as 
possible. Animals that die during the test and those surviving at 
the termination of the test are subjected to necropsy. All gross path ­
ological changes should be recorded. Where indicated, tissues should be taken for histopathological examination.
 
Assessment of toxicity in the other sex  
After completion of the study in one sex, at least one group of five 
animals of the other sex is dosed to establish that animals of this sex are not markedly more sensitive to the test substance. The use of fewer animals may be justified in individual circumstances. Where 
adequate information is available to demonstrate that animals of the 
sex tested are markedly more sensitive, testing in animals of the other sex may be dispensed with.
 
2. DATA  
Data should be summarised in tabular form, showing for each test 
group the number of animals at the start of the test, time of death of individual animals, number of animals displaying other signs of 
toxicity, description of toxic effects and necropsy findings. Indi ­
vidual weights of animals should be determined and recorded shortly before the test substance is applied, weekly thereafter, and at death; changes in weight should be calculated and recorded when 
survival exceeds one day. Animals, which are humanely killed due 
to compound-related distress and pain are recorded as compound- related deaths. The LD
 50 should be determined by a recognised 
method. 
Data evaluation should include an evaluation of relationships, if any, 
between the animal's exposure to the test substance and the incidence 
and severity of all abnormalities, including behavioural and clinical 
abnormalities, gross lesions, body weight changes, mortality, and any other toxicological effects.
 
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, include the following information:  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 238
 
— species, strain, source, environmental conditions, diet, etc., 
— test conditions (including method of skin cleansing and type of 
dressing: occlusive or not occlusive), 
— dose levels (with vehicle, if used, and concentrations), — sex of animals dosed, — tabulation of response data by sex and dose level (i.e. number of 
animals that died or were killed during the test, number of animals showing signs of toxicity, number of animals exposed), 
— time of death after dosing, reasons and criteria used for humane 
killing of animals, 
— all observations, — LD
 50 value for the sex subjected to a full study, determined at 14 
days with the method of determination specified, 
— 95 % confidence interval for the LD  50 (where this can be 
provided), 
— dose/mortality curve and slope where permitted by the method of 
determination, 
— necropsy findings, — any histopathological findings, — results of any test on the other sex, — discussion of results (particular attention should be given to the 
effect that humane killing of animals during the test may have on 
the calculated LD
 50 value), 
— interpretation of the results.  
3.2. EVALUATION AND INTERPRETATION 
See General introduction Part B (D).  
4. REFERENCES  
See General introduction Part B (E).  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 239
 
B.4. ACUTE TOXICITY: DERMAL IRRITATION/CORROSION  
1. METHOD  
This method is equivalent to the OECD TG 404 (2002).  
1.1. INTRODUCTION 
In the preparation of this updated method special attention was given 
to possible improvements in relation to animal welfare concerns and 
to the evaluation of all existing information on the test substance in order to avoid unnecessary testing in laboratory animals. This method includes the recommendation that prior to undertaking the 
described  in vivo  test for corrosion/irritation of the substance, a 
weight-of-the-evidence analysis be performed on the existing relevant data. Where insufficient data are available, they can be developed through application of sequential testing (1). The testing 
strategy includes the performance of validated and accepted  in vitro  
tests and is provided as an Appendix to this method. In addition, where appropriate, the successive, instead of simultaneous, appli ­
cation of the three test patches to the animal in the initial  in vivo  
test is recommended. 
In the interest of both sound science and animal welfare,  in vivo  
testing should not be undertaken until all available data relevant to the potential dermal corrosivity/irritation of the substance have been 
evaluated in a weight-of-the-evidence analysis. Such data will 
include evidence from existing studies in humans and/or laboratory animals, evidence of corrosivity/irritation of one or more structurally related substances or mixtures of such substances, data demon ­
strating strong acidity or alkalinity of the substance (2)(3), and 
results from validated and accepted  in vitro  or ex vivo  tests (4)(5) ­
(5a). This analysis should decrease the need for  in vivo  testing for 
dermal corrosivity/irritation of substances for which sufficient 
evidence already exists from other studies as to those two endpoints. 
A preferred sequential testing strategy, which includes the 
performance of validated and accepted  in vitro  or ex vivo  tests for 
corrosion/irritation, is included as an Appendix to this Method. The strategy was developed at, and unanimously recommended by the 
participants of, an OECD workshop (6), and has been adopted as the 
recommended testing strategy in the Globally Harmonised System for the Classification of Chemical Substances (GHS) (7). It is recom ­
mended that this testing strategy be followed prior to undertaking  in 
vivo testing. For new substances it is the recommended a stepwise 
testing approach for developing scientifically sound data on the corrosivity/irritation of the substance. For existing substances with 
insufficient data on dermal corrosion/irritation, the strategy should be 
used to fill missing data gaps. The use of a different testing strategy or procedure, or a decision not to use a stepwise testing approach, should be justified. 
If a determination of corrosivity or irritation cannot be made using a 
weight-of-the-evidence analysis, consistent with the sequential 
testing strategy, an  in vivo  test should be considered (see Appendix).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 240
 
1.2. DEFINITIONS  
Dermal irritation:  is the production of reversible damage of the 
skin following the application of a test substance for up to four hours.  
Dermal corrosion : is the production of irreversible damage of the 
skin; namely, visible necrosis through the epidermis and into the dermis, following the application of a test substance for up to four hours. Corrosive reactions are typified by ulcers, bleeding, 
bloody scabs, and, by the end of observation at 14 days, by 
discoloration due to blanching of the skin, complete areas of alopecia, and scars. Histopathology should be considered to evaluate questionable lesions.
 
1.3. PRINCIPLE OF THE TEST METHOD 
The substance to be tested is applied in a single dose to the skin of 
an experimental animal; untreated skin areas of the test animal serve 
as the control. The degree of irritation/corrosion is read and scored at specified intervals and is further described in order to provide a complete evaluation of the effects. The duration of the study 
should be sufficient to evaluate the reversibility or irreversibility of 
the effects observed. 
Animals showing continuing signs of severe distress and/or pain at 
any stage of the test should be humanely killed, and the substance assessed accordingly. Criteria for making the decision to humanely 
kill moribund and severely suffering animals can be found in 
reference (8).
 
1.4. DESCRIPTION OF THE TEST METHOD  
1.4.1.  Preparation for the  in vivo  test  
1.4.1.1.  Selection of animal species  
The albino rabbit is the preferable laboratory animal and healthy 
young adult rabbits are used. A rationale for using other species 
should be provided.  
1.4.1.2.  Preparation of the animals  
Approximately 24 hours before the test, fur should be removed by 
closely clipping the dorsal area of the trunk of the animals. Care should be taken to avoid abrading the skin, and only animals with 
healthy, intact skin should be used. 
Some strains of rabbit have dense patches of hair that are more 
prominent at certain times of the year. Such areas of dense hair growth should not be used as test sites.
 
1.4.1.3.  Housing and feeding conditions  
Animals should be individually housed. The temperature of the 
experimental animal room should be 20  o C (± 3  o C) for rabbits. 
Although the relative humidity should be at least 30 % and 
preferably not exceed 70 %, other than during room cleaning, the aim should be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional 
laboratory diets may be used with an unrestricted supply of 
drinking water.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 241
 
1.4.2.  Test procedure  
1.4.2.1.  Application of the test substance  
The test substance should be applied to a small area (approximately 
6 cm  2 ) of skin and covered with a gauze patch, which is held in 
place with non-irritating tape. In cases in which direct application is 
not possible (e.g. liquids or some pastes), the test substance should first be applied to the gauze patch, which is then applied to the skin. The patch should be loosely held in contact with the skin by means 
of a suitable semi-occlusive dressing for the duration of the exposure 
period. If the test substance is applied to the patch, it should be attached to the skin in such a manner that there is good contact 
and uniform distribution of the substance on the skin. Access by 
the animal to the patch and ingestion or inhalation of the test substance should be prevented. 
Liquid test substances are generally used undiluted. When testing 
solids (which may be pulverised, if considered necessary), the test substance should be moistened with the smallest amount of water (or, where necessary, of another suitable vehicle) sufficient to ensure 
good skin contact. When vehicles other than water are used, the 
potential influence of the vehicle on irritation of the skin by the test substance should be minimal, if any. 
At the end of the exposure period, which is normally four hours, 
residual test substance should be removed, where practicable, using water or an appropriate solvent without altering the existing response or the integrity of the epidermis.
 
1.4.2.2.  Dose level  
A dose of 0,5 ml. of liquid or 0,5 g of solid or paste is applied to the 
test site.  
1.4.2.3.  Initial test ( in vivo  dermal irritation/corrosion test using one animal)  
It is strongly recommended that the  in vivo  test be performed initially 
using one animal, especially when the substance is suspected to have 
corrosion potential. This is in accordance with the sequential testing strategy (see Appendix 1). 
When a substance has been judged to be corrosive on the basis of a 
weight-of-the-evidence analysis, no further animal testing is needed. For most substances suspected of being corrosive, further  in vivo  
testing is normally not necessary. However, in those cases where 
additional data are felt warranted because of insufficient evidence, 
limited animal testing may be carried out using the following approach: up to three tests patches are applied sequentially to the animal. The first patch is removed after three minutes. If no serious 
skin reaction is observed, a second patch is applied and removed 
after one hour. If the observations at this stage indicate that exposure can humanely be allowed to extend to four hours, a third patch is applied and removed after four hours, and the response is graded. 
If a corrosive effect is observed after any of the three sequential 
exposures, the test is immediately terminated. If a corrosive effect is not observed after the last patch is removed, the animal is 
observed for 14 days, unless corrosion develops at an earlier time 
point.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 242
 
In those cases in which the test substance is not expected to produce 
corrosion but may be irritating, a single patch should be applied to 
one animal for four hours.  
1.4.2.4.  Confirmatory test ( in vivo  dermal irritation test with additional 
animals)  
If a corrosive effect is not observed in the initial test, the irritant or 
negative response should be confirmed using up to two additional animals, each with one patch, for an exposure period of four hours. If an irritant effect is observed in the initial test, the confirmatory test 
may be conducted in a sequential manner, or by exposing two 
additional animals simultaneously. In the exceptional case, in which the initial test is not conducted, two or three animals may be treated with a single patch, which is removed after four hours. 
When two animals are used, if both exhibit the same response, no 
further testing is needed. Otherwise, the third animal is also tested. Equivocal responses may need to be evaluated using additional animals.
 
1.4.2.5.  Observation period  
The duration of the observation period should be sufficient to 
evaluate fully the reversibility of the effects observed. However, the experiment should be terminated at any time that the animal 
shows continuing signs of severe pain or distress. To determine 
the reversibility of effects, the animals should be observed up to 14 days after removal of the patches. If reversibility is seen before 14 days, the experiment should be terminated at that time.
 
1.4.2.6.  Clinical observations and grading of skin reactions  
All animals should be examined for signs of erythema and oedema, 
and the responses scored at 60 minutes, and then at 24, 48 and 72 hours after patch removal. For the initial test in one animal, 
the test site is also examined immediately after the patch has been 
removed. Dermal reactions are graded and recorded according to the grades in the Table below. If there is damage to skin which cannot be identified as irritation or corrosion at 72 hours, observations may 
be needed until day 14 to determine the reversibility of the effects. In 
addition to the observation of irritation, all local toxic effects, such as defatting of the skin, and any systemic adverse effects (e.g. effects on clinical signs of toxicity and body weight), should be fully 
described and recorded. Histopathological examination should be 
considered to clarify equivocal responses. 
The grading of skin responses is necessarily subjective. To promote 
harmonisation in grading of skin response and to assist testing 
laboratories and those involved in making and interpreting the obser ­
vations, the personnel performing the observations need to be adequately trained in the scoring system used (see Table below). 
An illustrated guide for grading skin irritation and other lesions 
could be helpful (9).
 
2. DATA  
2.1. PRESENTATION OF RESULTS 
Study results should be summarised in tabular form in the final test 
report and should cover all items listed in section 3.1.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 243
 
2.2. EVALUATION OF RESULTS 
The dermal irritation scores should be evaluated in conjunction with 
the nature and severity of lesions, and their reversibility or lack of reversibility. The individual scores do not represent an absolute 
standard for the irritant properties of a material, as other effects of 
the test material are also evaluated. Instead, individual scores should be viewed as reference values, which need to be evaluated in combination with all other observations from the study. 
Reversibility of dermal lesions should be considered in evaluating 
irritant responses. When responses such as alopecia (limited area), 
hyperkeratosis, hyperplasia and scaling, persist to the end of the 14- day observation period, the test substance should be considered an irritant.
 
3. REPORTING  
3.1. TEST REPORT 
The test report must include the following information: 
Rationale for  in vivo  testing: weight-of-evidence analysis of pre- 
existing test data, including results from sequential testing strategy: 
— description of relevant data available from prior testing, — data derived at each stage of testing strategy, — description of  in vitro  tests performed, including details of 
procedures, results obtained with test/reference substances, 
— weight-of-the-evidence analysis for performing  in vivo  study. 
Test substance: 
— identification data (e.g. CAS number, source, purity, known 
impurities, lot number), 
— physical nature and physicochemical properties (e.g. pH, vola ­
tility, solubility, stability), 
— if mixture, composition and relative percentages of components. 
Vehicle: — identification, concentration (where appropriate), volume used, — justification for choice of vehicle. Test animals: 
— species/strain used, rationale for using animals other than albino 
rabbit, 
— number of animals of each sex, 
— individual animal weights at start and conclusion of test, 
— age at start of study, — source of animals, housing conditions, diet, etc.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 244
 
Test conditions: 
— technique of patch site preparation, — details of patch materials used and patching technique, 
— details of test substance preparation, application, and removal. 
Results: — tabulation of irritation/corrosion response scores for each animal 
at all time points measured, 
— descriptions of all lesions observed, 
— narrative description of nature and degree of irritation or 
corrosion observed, and any histopathological findings, 
— description of other adverse local (e.g. defatting of skin) and 
systemic effects in addition to dermal irritation or corrosion. 
— Discussion of results
 
4. REFERENCES  
(1) Barratt, M.D., Castell, J.V., Chamberlain, M., Combes, R.D., 
Dearden, J.C., Fentem, J.H., Gerner, I., Giuliani, A., Gray, T.J.B., Livingston, D.J., Provan, W.M., Rutten, F.A.J.J.L., 
Verhaar, H.J.M., Zbinden, P. (1995) The Integrated Use of 
Alternative Approaches for Predicting Toxic Hazard. ECVAM Workshop Report 8. ATLA 23, p. 410-429. 
(2) Young, J.R., How, M.J., Walker, A.P., Worth W.M.H. (1988) 
Classification as Corrosive or Irritant to Skin of Preparations Containing Acidic or Alkaline Substance Without Testing on 
Animals. Toxicollogy  In Vitro , 2, p. 19-26. 
(3) Worth, A.P., Fentem, J.H., Balls, M., Botham, P.A., Curren, 
R.D., Earl, L.K., Esdaile, D.J., Liebsch, M. (1998) Evaluation 
of the proposed OECD Testing Strategy for skin corrosion. 
ATLA 26, p. 709-720. 
(4) ECETOC (1990) Monograph No 15, ‘Skin Irritation’, European 
Chemical Industry, Ecology and Toxicology Centre, Brussels. 
(5) Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, 
R.D., Earl, L.K., Edsail, D.J., Holzhutter, H.G. and Liebsch, M. (1998) The ECVAM international validation study on in vitro tests for skin corrosivity. 2. Results and evaluation by the 
Management Team. Toxicology  In Vitro  12, p. 483-524. 
(5a) Testing Method B.40 Skin Corrosion. 
(6) OECD (1996) OECD Test Guidelines Programme: Final 
Report of the OECD Workshop on Harmonisation of Vali ­
dation and Acceptance Criteria for Alternative Toxicological 
Test Methods. Held in Solna, Sweden, 22-24 January 1996 (http://www.oecd1.org/ehs/test/background.htm). 
(7) OECD (1998) Harmonised Integrated Hazard Classification 
System for Human Health and Environmental Effects of Chemical Substances, as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on 
Chemicals, November 1998 (http://www.oecd1.org/ehs/Class/ ­
HCL6.htm).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 245
 
(8) OECD (2000). Guidance Document on the Recognition, 
Assessment and Use of Clinical Signs as Humane Endpoints 
for Experimental Animals Used in Safety Evaluation. OECD Environmental Health and Safety Publications. Series on Testing and Assessment No 19 (http://www.oecd1.org/ehs/test/ ­
monos.htm). 
(9) EPA (1990). Atlas of Dermal Lesions,  (20T-2004).  United 
States Environmental Protection Agency, Office of Pesticides and Toxic Substances, Washington, DC, August 1990. 
[Available from OECD Secretariat upon request].
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 246
 
Table I  
GRADING OF SKIN REACTIONS  
Erythema and Eschar formation  
No erythema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 
Very slight erythema (barely perceptible) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Well defined erythema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Moderate to severe erythema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Severe erythema (beef redness) to eschar formation preventing grading 
of erythema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4  
Maximum possible: 4
 
Oedema formation  
No oedema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 
Very slight oedema (barely perceptible) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 
Slight oedema (edges of area well defined by definite raising) . . . . . . . 2 Moderate oedema (raised approximately 1 mm) . . . . . . . . . . . . . . . . . . . . . 3 Severe oedema (raised more than 1 mm and extending beyond area of 
exposure) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4 
Maximum possible: 4 
Histopathological examination may be carried out to clarify equivocal responses.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 247
 
Appendix  
A Sequential Testing Strategy for Dermal Irritation and Corrosion  
GENERAL CONSIDERATIONS  
In the interest of sound science and animal welfare, it is important to avoid the 
unnecessary use of animals and to minimise any testing that is likely to produce 
severe responses in animals. All information on a substance relevant to its 
potential skin corrosivity/irritancy should be evaluated prior to considering  in 
vivo testing. Sufficient evidence may already exist to classify a test substance 
as to its dermal corrosion or irritation potential without the need to conduct 
testing in laboratory animals. Therefore, utilising a weight-of-the-evidence 
analysis and a sequential testing strategy, will minimise the need for  in vivo  
testing, especially if the substance is likely to produce severe reactions. 
It is recommended that a weight-of-the-evidence analysis be used to evaluate 
existing information regarding the skin irritation and corrosion of substances to 
determine whether additional studies, other than  in vivo  dermal studies, should be 
performed to help characterise such potential. Where further studies are needed, it is recommended that the sequential testing strategy be utilised to develop the 
relevant experimental data. For substances which have no testing history, the 
sequential testing strategy should be utilised to develop the data set needed to evaluate its dermal corrosion/irritation potential. The testing strategy described in this Appendix was developed at an OECD workshop (1) and was later affirmed 
and expanded in the Harmonised Integrated Hazard Classification System for 
Human Health and Environmental Effects of Chemical Substances, as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, in November 1998 (2). 
Although this sequential testing strategy is not an integral part of testing method 
B.4, it expresses the recommended approach for the determination of skin irritation/corrosion characteristics. This approach represents both best practice 
and an ethical benchmark for  in vivo  testing for skin irritation/corrosion. The 
testing method provides guidance for the conduct of the  in vivo  test and 
summarises the factors that should be addressed before initiating such a test. The strategy provides an approach for the evaluation of existing data on the 
skin irritation/corrosion properties of test substances and a tiered approach for 
the generation of relevant data on substances for which additional studies are needed, or for which no studies have been performed. It also recommends the performance of validated and accepted  in vitro  or ex vivo  tests for skin 
corrosion/irritation under specific circumstances.
 
DESCRIPTION OF THE EVALUATION AND TESTING STRATEGY  
Prior to undertaking tests as part of the sequential testing strategy (Figure), all 
available information should be evaluated to determine the need for  in vivo  skin 
testing. Although significant information might be gained from the evaluation of single parameters (e.g. extreme pH), the totality of existing information should be considered. All relevant data on the effects of the substance in question, or its 
analogues, should be evaluated in making a weight-of-the-evidence decision, and 
a rationale for the decision should be presented. Primary emphasis should be placed upon existing human and animal data on the substance, followed by the outcome of  in vitro  or ex vivo  testing.  In vivo  studies of corrosive substances 
should be avoided whenever possible. The factors considered in the testing 
strategy include:
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 248
 
Evaluation of existing human and animal data (Step 1).  Existing human data, e.g. 
clinical or occupational studies and case reports, and/or animal test data, e.g. 
from single or repeated dermal exposure toxicity studies, should be considered first, because they provide information directly related to effects on the skin. Substances with known irritancy or corrosivity, and those with clear evidence of 
non-corrosivity or non-irritancy, need not be tested in  in vivo  studies.  
Analysis of structure activity relationships (SAR) (Step 2).  The results of testing 
of structurally related substances should be considered, if available. When sufficient human and/or animal data are available on structurally related substances or mixtures of such substances to indicate their skin corrosion/ ­
irritancy potential, it can be presumed that the test substance being evaluated 
will produce the same responses. In those cases, the test substance may not need to be tested. Negative data from studies of structurally related substances or 
mixtures of such substances do not constitute sufficient evidence of non- 
corrosivity/non-irritancy of a substance under the sequential testing strategy. Validated and accepted SAR approaches should be used to identify both dermal corrosion and irritation potential.  
Physicochemical properties and chemical reactivity (Step 3).  Substances 
exhibiting pH extremes such as ≤ 2,0 and ≥ 11,5 may have strong local 
effects. If extreme pH is the basis for identifying a substance as corrosive to skin, then its acid/alkali reserve (or buffering capacity) may also be taken into consideration (3)(4). If the buffering capacity suggests that a substance may not 
be corrosive to the skin, then further testing should be undertaken to confirm this, 
preferably by the use of a validated and accepted  in vitro  or ex vivo  test (see 
steps 5 and 6).  
Dermal toxicity (Step 4).  If a chemical has proven to be very toxic by the dermal 
route, an  in vivo  dermal irritation/corrosion study may not be practicable because 
the amount of test substance normally applied could exceed the very toxic dose and, consequently result in the death or severe suffering of the animals. In addition, when dermal toxicity studies utilising albino rabbits have already 
been performed up to the limit dose level of 2 000 mg/kg body weight or 
higher, and no dermal irritation or corrosion has been seen, additional testing for skin irritation/corrosion may not be needed. A number of considerations should be borne in mind when evaluating acute dermal toxicity in previously 
performed studies. For example, reported information on dermal lesions may be 
incomplete. Testing and observations may have been made on a species other than the rabbit, and species may differ widely in sensitivity of their responses. 
Also the form of test substance applied to animals may not have been suitable for 
assessment of skin irritation/corrosion (e.g., dilution of substances for testing dermal toxicity (5). However, in those cases in which well-designed and conducted dermal toxicity studies have been performed in rabbits, negative 
findings may be considered sufficient evidence that the substance is not 
corrosive or irritating.  
Results from in vitro or ex vivo tests (Steps 5 and 6).  Substances that have 
demonstrated corrosive or severe irritant properties in a validated and accepted  
in vitro  or ex vivo  test (6)(7) designed for the assessment of these specific effects, 
need not be tested in animals. It can be presumed that such substances will 
produce similar severe effects  in vivo .
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 249
 
In vivo test in rabbits (Steps 7 and 8).  Should a weight-of the-evidence decision 
be made to conduct  in vivo  testing, it should begin with an initial test using one 
animal. If the results of this test indicate the substance to be corrosive to the skin, further testing should not be performed. If a corrosive effect is not observed in the initial test, the irritant or negative response should be confirmed using up to 
two additional animals for an exposure period of four hours. If an irritant effect is 
observed in the initial test, the confirmatory test may be conducted in a sequential manner, or by exposing the two additional animals simultaneously.
 
REFERENCES  
(1) OECD, (1996) Test Guidelines Programme: Final Report on the OECD 
Workshop on Harmonisation of Validation and Acceptance Criteria for Alternative Toxicological Test Methods. Held on Solna, Sweden, 22- 24 January 1996 (http://www1.oecd.org/ehs/test/background.htm). 
(2) OECD, (1998) Harmonised Integrated Hazard Classification System for 
Human Health and Environmental Effects of Chemical Substances, as 
endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, November 1998 (http://www1.oecd.org/ehs/ Class/HCL6.htm). 
(3) Worth, A.P., Fentem J.H., Balls M., Botham P.A., Curren R.D., Earl L.K., 
Esdaile D.J., Liebsch M., (1998). An Evaluation of the Proposed OECD Testing Strategy for Skin Corrosion. ATLA 26, p. 709-720. 
(4) Young, J.R., How, M.J., Walker, A.P., Worth, W.M.H., (1988). Classifi ­
cation as Corrosive or Irritant to Skin of Preparations Containing Acidic or 
Alkaline Substances, Without Testing on Animals. Toxicology  In Vitro , 
2(1), p. 19-26. 
(5) Patil, S.M., Patrick, E., Maibach, H.I. (1996) Animal, Human, and In 
Vitro Test Methods for Predicting Skin Irritation, in: Francis N. Marzulli and Howard I. Maibach (editors): Dermatotoxicology. Fifth Edition ISBN 
1-56032-356-6, Chapter 31, p. 411-436. 
(6) Testing Method B.40. (7) Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., 
Earl, L.K., Esdaile, D.J., Holzhutter, H.G. and Liebsch, M. (1998) The ECVAM international validation study on in vitro tests for skin 
corrosivity. 2. Results and evaluation by the Management Team. Toxicology  
In Vitro  12, p. 483-524.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 250
 
Figure  
TESTING AND EVALUATION STRATEGY FOR DERMAL IRRITATION/CORROSION
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 251
 
B.5. ACUTE TOXICITY: EYE IRRITATION/CORROSION  
1. METHOD  
This method is equivalent to the OECD TG 405 (2002)  
1.1. INTRODUCTION 
In the preparation of this updated method special attention was given 
to possible improvements through the evaluation of all existing 
information on the test substance in order to avoid unnecessary testing in laboratory animals and thereby address animal welfare concerns. This method includes the recommendation that prior to 
undertaking the described  in vivo  test for acute eye irritation/ ­
corrosion, a weight-of-the-evidence analysis be performed (1) on the existing relevant data. Where insufficient data are available, it is recommended that they be developed through application of 
sequential testing (2)(3). The testing strategy includes the 
performance of validated and accepted  in vitro  tests and is 
provided as an Appendix to the testing method. In addition, the use of an  in vivo  dermal irritation/corrosion test to predict eye 
corrosion prior to consideration of an  in vivo  eye test is recom ­
mended. 
In the interest of both sound science and animal welfare,  in vivo  
testing should not be considered until all available data relevant to the potential eye corrosivity/irritation of the substance has been 
evaluated in a weight-of-the-evidence analysis. Such data will 
include evidence from existing studies in humans and/or laboratory animals, evidence of corrosivity/irritation of one or more structurally 
related substances or mixtures of such substances, data demon ­
strating high acidity or alkalinity of the substance (4)(5), and results from validated and accepted  in vitro  or ex vivo  tests for 
skin corrosion and irritation (6)(6a). The studies may have been 
conducted prior to, or as a result of, a weight-of-the-evidence 
analysis. 
For certain substances, such an analysis may indicate the need for  in 
vivo studies of the ocular corrosion/irritation potential of the 
substance. In all such cases, before considering the use of the  in 
vivo eye test, preferably a study of the  in vivo  dermal effects of the 
substance should be conducted first and evaluated in accordance with testing method B.4 (7). The application of a weight-of-the-evidence analysis and the sequential testing strategy should decrease the need 
for in vivo  testing for eye corrosivity/irritation of substances for 
which sufficient evidence already exists from other studies. If a determination of eye corrosion or irritation potential cannot be made using the sequential testing strategy, even after the 
performance of an  in vivo  study of dermal corrosion and irritation, 
an in vivo  eye corrosion/irritation test may be performed.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 252
 
A preferred sequential testing strategy, which includes the 
performance of validated  in vitro  or ex vivo  tests for corrosion/ ­
irritation, is included in the Appendix to this testing method. The strategy was developed at, and unanimously recommended by the participants of, an OECD workshop (8), and has been adopted as the 
recommended testing strategy in the Globally Harmonised System 
for the Classification of Chemical Substances (GHS) (9). It is recom ­
mended that this testing strategy be followed prior to undertaking  in 
vivo testing. For new substances it is the recommended stepwise 
testing approach for developing scientifically sound data on the 
corrosivity/irritation of the substance. For existing substances with insufficient data on skin and eye corrosion/irritation, the strategy should be used to fill missing data gaps. The use of a different 
testing strategy or procedure, or the decision not to use a stepwise 
testing approach, should be justified.
 
1.2. DEFINITIONS  
Eye irritation:  is the production of changes in the eye following the 
application of a test substance to the anterior surface of the eye, 
which are fully reversible within 21 days of application.  
Eye corrosion:  is the production of tissue damage in the eye, or 
serious physical decay of vision, following application of a test 
substance to the anterior surface of the eye, which is not fully 
reversible within 21 days of application.  
1.3. PRINCIPLE OF THE TEST METHOD 
The substance to be tested is applied in a single dose to one of the 
eyes of the experimental animal; the untreated eye serves as the 
control. The degree of eye irritation/corrosion is evaluated by scoring lesions of conjunctiva, cornea, and iris, at specific intervals. Other effects in the eye and adverse systemic effects are also 
described to provide a complete evaluation of the effects. The 
duration of the study should be sufficient to evaluate the reversibility or irreversibility of the effects. 
Animals showing continuing signs of severe distress and/or pain at 
any stage of the test should be humanely killed, and the substance assessed accordingly. Criteria for making the decision to humanely kill moribund and severely suffering animals can be found in 
reference (10).
 
1.4. DESCRIPTION OF THE TEST METHOD  
1.4.1.  Preparation for the  in vivo  test  
1.4.1.1.  Selection of species  
The albino rabbit is the preferable laboratory animal, and healthy 
young adult animals are used. A rationale for using other strains or 
species should be provided.  
1.4.1.2.  Preparation of animals  
Both eyes of each experimental animal provisionally selected for 
testing should be examined within 24 hours before testing starts. 
Animals showing eye irritation, ocular defects, or pre-existing 
corneal injury should not be used.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 253
 
1.4.1.3.  Housing and feeding conditions  
Animals should be individually housed. The temperature of the 
experimental animal room should be 20  o C (± 3  o C) for rabbits. 
Although the relative humidity should be at least 30 % and 
preferably not exceed 70 %, other than during room cleaning, the 
aim should be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unrestricted supply of 
drinking water.
 
1.4.2.  Test procedure  
1.4.2.1.  Application of the test substance  
The test substance should be placed in the conjunctival sac of one 
eye of each animal after gently pulling the lower lid away from the 
eyeball. The lids are then gently held together for about one second in order to prevent loss of the material. The other eye, which remains untreated, serves as a control.
 
1.4.2.2.  Irrigation  
The eyes of the test animals should not be washed for at least 
24 hours following instillation of the test substance, except for solids (see Section 1.4.2.3.2), and in case of immediate corrosive 
or irritating effects. At 24 hours a washout may be used if 
considered appropriate. 
Use of a satellite group of animals to investigate the influence of 
washing is not recommended unless it is scientifically justified. If a 
satellite group is needed, two rabbits should be used. Conditions of washing should be carefully documented, e.g. time of washing; composition and temperature of wash solution; duration, volume, 
and velocity of application.
 
1.4.2.3.  Dose level  
1.4.2.3.1.  Testing of liquids  
For testing liquids, a dose of 0,1 ml is used. Pump sprays should not 
be used for instilling the substance directly into the eye. The liquid spray should be expelled and collected in a container prior to instilling 0,1 ml into the eye.
 
1.4.2.3.2.  Testing of solids  
When testing solids, pastes, and particulate substances, the amount 
used should have a volume of 0,1 ml or a weight of not more than 
100 mg. The test material should be ground to a fine dust. The 
volume of solid material should be measured after gently compacting it, e.g. by tapping the measuring container. If the solid test substance has not been removed from the eye of the test animal by physio ­
logical mechanisms at the first observation time point of one hour 
after treatment, the eye may be rinsed with saline or distilled water.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 254
 
1.4.2.3.3.  Testing of aerosols  
It is recommended that all pump sprays and aerosols be collected 
prior to instillation into the eye. The one exception is for substances 
in pressurised aerosol containers, which cannot be collected due to vaporisation. In such cases, the eye should be held open, and the test substance administered to the eye in a simple burst of about 
one second, from a distance of 10 cm directly in front of the eye. 
This distance may vary depending on the pressure of the spray and its contents. Care should be taken not to damage the eye from the pressure of the spray. In appropriate cases, there may be a need to 
evaluate the potential for ‘mechanical’ damage to the eye from the 
force of the spray. 
An estimate of the dose from an aerosol can be made by simulating 
the test as follows: the substance is sprayed on to weighing paper through an opening the size of a rabbit eye placed directly before the 
paper. The weight increase of the paper is used to approximate the 
amount sprayed into the eye. For volatile substances, the dose may be estimated by weighing a receiving container before and after removal of the test material.
 
1.4.2.4.  Initial test ( in vivo  eye irritation/corrosion test using one animal)  
As articulated in the sequential testing strategy (see Appendix 1), it 
is strongly recommended that the  in vivo  test be performed initially 
using one animal. 
If the results of this test indicate the substance to be corrosive or a 
severe irritant to the eye using the procedure described, further testing for ocular irritancy should not be performed.
 
1.4.2.5.  Local anaesthetics  
Local anaesthetics may be used on a case-by-case basis. If the 
weight-of-the-evidence analysis indicates that the substance has the 
potential to cause pain, or initial testing shows that a painful reaction 
will occur, a local anaesthetic may be used prior to instillation of the test substance. The type, concentration, and dose of the local anaes ­
thetic should be carefully selected to ensure that differences in 
reaction to the test substance will not result from its use. The 
control eye should be similarly anaesthetised.
 
1.4.2.6.  Confirmatory test ( in vivo  eye irritation test with additional animals)  
If a corrosive effect is not observed in the initial test, the irritant or 
negative response should be confirmed using up to two additional animals. If a severe irritant effect is observed in the initial test indicating a possible strong (irreversible) effect in the confirmatory 
testing, it is recommended that the confirmatory test be conducted in 
a sequential manner in one animal at a time, rather than exposing the two additional animals simultaneously. If the second animal reveals corrosive or severe irritant effects, the test is not continued. 
Additional animals may be needed to confirm weak or moderate 
irritant responses.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 255
 
1.4.2.7.  Observation period  
The duration of the observation period should be sufficient to 
evaluate fully the magnitude and reversibility of the effects observed. However, the experiment should be terminated at any 
time that the animal shows continuing signs of severe pain or 
distress (9). To determine reversibility of effects, the animals should be observed normally for 21 days post administration of the test substance. If reversibility is seen before 21 days, the 
experiment should be terminated at that time.
 
1.4.2.7.1.  Clinical observations and grading of eye reactions  
The eyes should be examined at one, 24, 48, and 72 hours after test 
substance application. Animals should be kept on test no longer than necessary once definitive information has been obtained. Animals showing continuing severe pain or distress should be humanely 
killed without delay, and the substance assessed accordingly. 
Animals with the following eye lesions post-instillation should be humanely killed: corneal perforation or significant corneal ulceration including staphyloma; blood in the anterior chamber of the eye; 
grade 4 corneal opacity which persists for 48 hours; absence of a 
light reflex (iridial response grade 2) which persists for 72 hours; ulceration of the conjunctival membrane; necrosis of the conjuctivae or nictitating membrane; or sloughing. This is because such lesions 
generally are not reversible 
Animals that do not develop ocular lesions may be terminated not 
earlier than three days post instillation. Animals with mild to 
moderate lesions should be observed until the lesions clear, or for 
21 days, at which time the study is terminated. Observations should be performed at seven, 14, and 21 days in order to determine the status of the lesions, and their reversibility or irreversibility. 
The grades of ocular reaction (conjunctivae, cornea and iris) should 
be recorded at each examination (Table I). Any other lesions in the 
eye (e.g. pannus, staining) or adverse systemic effects should also be 
reported. 
Examination of reactions can be facilitated by use of a binocular 
loupe, hand slit-lamp, biomicroscope, or other suitable device. 
After recording the observations at 24 hours, the eyes may be further examined with the aid of fluorescein. 
The grading of ocular responses is necessarily subjective. To 
promote harmonisation of grading of ocular response and to assist testing laboratories and those involved in making and interpreting the 
observations, the personnel performing the observations need to be 
adequately trained in the scoring system used.
 
2. DATA  
2.2. EVALUATION OF RESULTS 
The ocular irritation scores should be evaluated in conjunction with 
the nature and severity of lesions, and their reversibility or lack of 
reversibility. The individual scores do not represent an absolute standard for the irritant properties of a material, as other effects of the test material are also evaluated. Instead, individual scores should 
be viewed as reference values and are only meaningful when 
supported by a full description and evaluation of all observations.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 256
 
3. REPORTING  
3.1. TEST REPORT 
The test report must include the following information: 
Rationale for  in vivo  testing: weight-of-the-evidence analysis of pre- 
existing test data, including results from sequential testing strategy 
— description of relevant data available from prior testing, — data derived in each step of testing strategy, — description of  in vitro  tests performed, including details of 
procedures, results obtained with test/reference substances, 
— description of  in vivo  dermal irritation/corrosion study performed, 
including results obtained, 
— weight-of-the-evidence analysis for performing  in vivo  study. 
Test substance: — identification data (e.g. CAS number, source, purity, known 
impurities, lot number), 
— physical nature and physicochemical properties (e.g. pH, vola ­
tility, solubility, stability, reactivity with water), 
— in case of a mixture, composition and relative percentages of 
components, 
— if local anaesthetic is used, identification, purity, type, dose, and 
potential interaction with test substance. 
Vehicle: — identification, concentration (where appropriate), volume used, — justification for choice of vehicle. 
Test animals: 
— species/strain used, rationale for using animals other than albino 
rabbit, 
— age of each animal at start of study, — number of animals of each sex in test and control groups (if 
required), 
— individual animal weights at start and conclusion of test, — source, housing conditions, diet, etc. Results: — description of method used to score irritation at each observation 
time (e.g. hand slitlamp, biomicroscope, fluorescein),
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 257
 
— tabulation of irritant/corrosive response data for each animal at 
each observation time up to removal of each animal from the 
test, 
— narrative description of the degree and nature of irritation or 
corrosion observed, 
— description of any other lesions observed in the eye (e.g. vascu ­
larisation, pannus formation, adhesions, staining), 
— description of non-ocular local and systemic adverse effects, and 
histopathological findings, if any. 
Discussion of results.  
3.2. INTERPRETATION OF THE RESULTS 
Extrapolation of the results of eye irritation studies in laboratory 
animals to humans is valid only to a limited degree. In many cases the albino rabbit is more sensitive than humans to ocular irritants or corrosives. 
Care should be taken in the interpretation of data to exclude irritation 
resulting from secondary infection.
 
4. REFERENCES  
(1) Barratt, M.D., Castell, J.V., Chamberlain, M., Combes, R.D., 
Dearden, J.C., Fentem, J.H., Gerner, I., Giuliani, A., Gray, T.J.B., Livingston, D.J., Provan, W.M., Rutten, F.A.J.J.L., Verhaar, H.J.M., Zbinden, P. (1995) The Integrated Use of 
Alternative Approaches for Predicting Toxic Hazard. 
ECVAM Workshop Report 8. ATLA 23, p. 410-429. 
(2) de Silva, O., Cottin, M., Dami, N., Roguet, R., Catroux, P., 
Toufic, A., Sicard, C., Dossou, K.G., Gerner, I., Schlede, E., 
Spielmann, H., Gupta, K.C., Hill, R.N., (1997) Evaluation of 
Eye Irritation Potential: Statistical Analysis and Tier Testing Strategies. Food Chem. Toxicol 35, p. 159-164. 
(3) Worth A.P. and Fentem J.H., (1999) A general approach for 
evaluating stepwise testing strategies ATLA 27, p. 161-177 
(4) Young, J.R., How, M.J., Walker, A.P., Worth W.M.H., (1988) 
Classification as Corrosive or Irritant to Skin of Preparations 
Containing Acidic or Alkaline Substance Without Testing on Animals. Toxicollogy  In Vitro , 2, p. 19-26. 
(5) Neun, D.J. (1993) Effects of Alkalinity on the Eye Irritation 
Potential of Solutions Prepared at a Single pH. J. Toxicol. Cut. 
Ocular Toxicol. 12, p. 227-231. 
(6) Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, 
R.D., Earl, L.K., Edsaile, D.J., Holzhutter, H.G. and Liebsch, M. (1998) The ECVAM international validation study on in 
vitro tests for skin corrosivity. 2. Results and evaluation by the 
Management Team. Toxicology  In Vitro  12, p. 483-524. 
(6a) Testing Method B.40 Skin Corrosion. (7) Testing method B.4. Acute toxicity: dermal irritation/corrosion. (8) OECD, (1996) OECD Test Guidelines Programme: Final 
Report of the OECD Workshop on Harmonisation of Vali ­
dation and Acceptance Criteria for Alternative Toxicological 
Test Methods. Held in Solna, Sweden, 22-24 January 1996 
(http://www.oecd.org/ehs/test/background.htm).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 258
 
(9) OECD, (1998) Harmonised Integrated Hazard Classification 
System for Human Health and Environmental Effects of 
Chemical Substances, as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, November 1998 (http://www.oecd.org/ehs/Class/ ­
HCL6.htm). 
(10) OECD, (2000) Guidance Document on the Recognition, 
Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. OECD Environmental Health and Safety Publications. Series on 
Testing and Assessment No 19 (http://www.oecd.org/ehs/test/ 
monos.htm).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 259
 
Table I  
GRADING OF OCULAR LESIONS  
Cornea  
Opacity: degree of density (readings should be taken from most dense area) (*) 
No ulceration or opacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 
Scattered or diffuse areas of opacity (other than slight dulling of normal 
lustre); details of iris clearly visible . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 
Easily discernible translucent area; details of iris slightly obscured . . . 2 Nacrous area; no details of iris visible; size of pupil barely discernible 3 Opaque cornea; iris not discernible through the opacity . . . . . . . . . . . . . . 4 
Maximum possible: 4 
NOTES 
(*) The area of corneal opacity should be noted
 
Iris 
Normal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 
Markedly deepened rugae, congestion, swelling, moderate circum ­
corneal hyperaemia; or injection; iris reactive to light (a sluggish 
reaction is considered to be an effect) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 
Hemorrhage, gross destruction, or no reaction to light . . . . . . . . . . . . . . . 2 
Maximum possible: 2  
Conjunctivae  
Redness (refers to palpebral and bulbar conjunctivae; excluding cornea and iris) 
Normal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 
Some blood vessels hyperaemic (injected) . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Diffuse, crimson colour; individual vessels not easily discernible . . . . 2 Diffuse beefy red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 
Maximum possible: 3
 
Chemosis  
Swelling (refers to lids and/or nictating membranes) 
Normal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 
Some swelling above normal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Obvious swelling, with partial eversion of lids . . . . . . . . . . . . . . . . . . . . . . 2 Swelling, with lids about half closed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 
Swelling, with lids more than half closed . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 
Maximum possible: 4
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 260
 
Appendix  
A Sequential Testing Strategy for Eye Irritation and Corrosion  
GENERAL CONSIDERATIONS  
In the interests of sound science and animal welfare, it is important to avoid the 
unnecessary use of animals, and to minimise testing that is likely to produce 
severe responses in animals. All information on a substance relevant to its 
potential ocular irritation/corrosivity should be evaluated prior to considering  in 
vivo testing. Sufficient evidence may already exist to classify a test substance as 
to its eye irritation or corrosion potential without the need to conduct testing in 
laboratory animals. Therefore, utilising a weight-of-the-evidence analysis and 
sequential testing strategy will minimise the need for  in vivo  testing, especially 
if the substance is likely to produce severe reactions. 
It is recommended that a weight-of-the-evidence analysis be used to evaluate 
existing information pertaining to eye irritation and corrosion of substances and to determine whether additional studies, other than  in vivo  eye studies, 
should be performed to help characterise such potential. Where further studies 
are needed, it is recommended that the sequential testing strategy be utilised to 
develop the relevant experimental data. For substances which have no testing history, the sequential testing strategy should be utilised to develop the data needed to evaluate its eye corrosion/irritation. The testing strategy described in 
this Appendix was developed at an OECD workshop (1). It was subsequently 
affirmed and expanded in the Harmonised Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances, as endorsed by the 28th Joint Meeting of the Chemicals Committee and the 
Working Party on Chemicals, in November 1998 (2). 
Although this testing strategy is not an integrated part of testing method B.5, it 
expresses the recommended approach for the determination of eye irritation/ ­
corrosion properties. This approach represents both best practice and an ethical benchmark for  in vivo  testing for eye irritation/corrosion. The testing method 
provides guidance for the conduct of the  in vivo  test and summarises the 
factors that should be addressed before considering such a test. The sequential 
testing strategy provides a weight-of-the-evidence approach for the evaluation of existing data on the eye irritation/corrosion properties of substances and a tiered approach for the generation of relevant data on substances for which additional 
studies are needed or for which no studies have been performed. The strategy 
includes the performance first of validated and accepted  in vitro  or ex vivo  tests 
and then of testing method B.4 skin irritation/corrosion studies under specific circumstances (3)(4).
 
DESCRIPTION OF THE STEPWISE TESTING STRATEGY  
Prior to undertaking tests as part of the sequential testing strategy (Figure), all 
available information should be evaluated to determine the need for  in vivo  eye 
testing. Although significant information might be gained from the evaluation of single parameters (e.g., extreme pH), the totality of existing information should be assessed. All relevant data on the effects of the substance in question, and its 
structural analogues, should be evaluated in making a weight-of-the-evidence 
decision, and a rationale for the decision should be presented. Primary emphasis should be placed upon existing human and animal data on the substance, followed by the outcome of  in vitro  or ex vivo  testing.  In vivo  
studies of corrosive substances should be avoided whenever possible. The 
factors considered in the testing strategy include:
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 261
 
Evaluation of existing human and animal data (Step 1).  Existing human data, e.g. 
clinical and occupational studies, and case reports, and/or animal test data from 
ocular studies should be considered first, because they provide information directly related to effects on the eyes. Thereafter, available data from human and/or animal studies investigating dermal corrosion/irritation should be 
evaluated. Substances with known corrosivity or severe irritancy to the eye 
should not be instilled into the eyes of animals, nor should substances showing corrosive or irritant effects to the skin; such substances should be considered to be corrosive and/or irritating to the eyes as well. Substances 
with sufficient evidence of non-corrosivity and non-irritancy from previously 
performed ocular studies should also not be tested in  in vivo  eye studies.  
Analysis of structure activity relationships (SAR) (Step 2).  The results of testing 
of structurally related chemicals should be considered, if available. When 
sufficient human and/or animal data are available on structurally related substances or mixtures of such substances to indicate their eye corrrosion/ ­
irritancy potential, it can be presumed that the test substance will produce the 
same responses. In those cases, the substance may not need to be tested. 
Negative data from studies of structurally related substances or mixtures of such substances do not constitute sufficient evidence of non-corrosivity/non- irritancy of a substance under the sequential testing strategy. Validated and 
accepted SAR approaches should be used to identify the corrosion and irritation 
potential for both dermal and ocular effects.  
Physicochemical properties and chemical reactivity (Step 3).  Substances 
exhibiting pH extremes such as ≤ 2,0 or ≥ 11,5 may have strong local effects. 
If extreme pH is the basis for identifying a substance as corrosive or irritant to the eye, then its acid/alkaline reserve (buffering capacity) may also be taken into consideration (5)(6). If the buffering capacity suggests that a substance may
 not 
be corrosive to the eye, then further testing should be undertaken to confirm this, 
preferably by the use of a validated and accepted  in vitro  or ex vivo  test (see 
Section step 5 and 6).  
Consideration of other existing information (Step 4).  All available information on 
systemic toxicity via the dermal route should be evaluated at this stage. The acute 
dermal toxicity of the test substance should also be considered. If the test substance has been shown to be very toxic by the dermal route, it may not need to be tested in 
the eye. Although there is not necessarily a relationship between acute dermal 
toxicity and eye irritation/corrosion, it can be assumed that if an agent is very toxic via the dermal route, it will also exhibit high toxicity when instilled into the eye. Such data may also be considered between Steps 2 and 3.  
Results from in vitro or ex vivo tests (Steps 5 and 6).  Substances that have 
demonstrated corrosive or severe irritant properties in an  in vitro  or ex vivo  
test (7)(8) that has been validated and accepted for the assessment specifically of eye or skin corrosivity/irritation, need not be tested in animals. It can be 
presumed that such substances will produce similar severe effects  in vivo . If 
validated and accepted  in vitro/ex vivo  tests are not available, one should 
bypass Steps 5 and 6 and proceed directly to Step 7.  
Assessment of in vivo dermal irritancy or corrosivity of the substance (Step 7).  
When insufficient evidence exists with which to perform a conclusive weight-of- the-evidence analysis of the potential eye irritation/corrosivity of a substance based upon data from the studies listed above, the  in vivo  skin irritation/corrosion 
potential should be evaluated first, using testing method B.4 (4) and its accom ­
panying Appendix (9). If the substance is shown to produce corrosion or severe skin irritation, it should be considered to be a corrosive eye irritant unless other information supports an alternative conclusion. Thus, an  in vivo  eye test would 
not need to be performed. If the substance is not corrosive or severely irritating 
to the skin, an  in vivo  eye test should be performed.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 262
 
In vivo  test in rabbits (Steps 8 and 9):  in vivo  ocular testing should begin with an 
initial test using one animal. If the results of this test indicate the substance to be 
a severe irritant or corrosive to the eyes, further testing should not be performed. If that test does not reveal any corrosive or severe irritant effects, a confirmatory test is conducted with two additional animals.
 
REFERENCES  
(1) OECD, (1996) OECD Test Guidelines Programme: Final Report of the 
OECD Workshop on Harmonisation of Validation and Acceptance Criteria for Alternative Toxicological Test Methods. Held in Solna, Sweden, 22- 
24 January 1996 (http://www.oecd.org/ehs/test/background.htm). 
(2) OECD, (1998) Harmonised Integrated Hazard Classification System for 
Human Health and Environmental Effects of Chemical Substances, as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, November 1998 (http://www.oecd.org/ehs/ 
Class/HCL6.htm). 
(3) Worth, A.P. and Fentem J.H., (1999). A General Approach for Evaluating 
Stepwise Testing Strategies. ATLA
 27, p. 161-177. 
(4) Testing method B.4. Acute Toxicity: dermal irritation/corrosion. (5) Young, J.R., How, M.J., Walker, A.P., Worth W.M.H., (1988) Classification 
as Corrosive or Irritant to Skin of Preparations Containing Acidic or 
Alkaline Substance Without Testing on Animals. Toxicolohy  In Vitro ,
 27, 
p. 19-26. 
(6) Neun, D.J., (1993) Effects of Alkalinity on the Eye Irritation Potential of 
Solutions Prepared at a Single pH. J. Toxicol. Cut. Ocular Toxicol.  12, 
p. 227-231. 
(7) Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., Earl, 
L.K., Edsail, D.J., Holzhutter, H.G. and Liebsch, M., (1998) The ECVAM international validation study on  in vitro  tests for skin corrosivity. 
2. Results and evaluation by the Management Team. Toxicology  In Vitro  12, 
p. 483-524. 
(8) Testing Method B.40 Skin Corrosion. (9) Appendix to Testing method B.4: A Sequential Testing Strategy for Skin 
Irritation and Corrosion.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 263
 
Figure  
TESTING AND EVALUATION STRATEGY FOR EYE IRRITATION/CORROSION
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 264
 
▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 265
 
B.6. SKIN SENSITISATION  
1. METHOD  
1.1. INTRODUCTION  
Remarks:  
The sensitivity and ability of tests to detect potential human skin 
sensitisers are considered important in a classification system for 
toxicity relevant to public health. 
There is no single test method which will adequately identify all 
substances with a potential for sensitising human skin and which 
is relevant for all substances. 
Factors such as the physical characteristics of a substance, including 
its ability to penetrate the skin, must be considered in the selection of 
a test. 
Two types of tests using guinea pigs have been developed: the 
adjuvant-type tests, in which an allergic state is potentiated by dissolving or suspending the test substance in Freunds Complete Adjuvant (FCA), and the non-adjuvant tests. 
Adjuvant-type tests are likely to be more accurate in predicting a 
probable skin sensitising effect of a substance in humans than those methods not employing Freunds Complete Adjuvant and are thus the 
preferred methods. 
The Guinea-Pig Maximisation Test (GPMT) is a widely used 
adjuvant-type test. Although several other methods can be used to 
detect the potential of a substance to provoke skin sensitisation reaction, the GPMT is considered to be the preferred adjuvant tech ­
nique. 
With many chemical classes, non-adjuvant tests (the preferred one 
being the Buehler test) are considered to be less sensitive. 
In certain cases there may be good reasons for choosing the Buehler 
test involving topical application rather than the intradermal injection used in the Guinea-Pig Maximisation Test. Scientific justification 
should be given when the Buehler test is used. 
The Guinea-Pig Maximisation Test (GPMT) and the Buehler test are 
described in this method. Other methods may be used provided that 
they are well-validated and scientific justification is given. 
If a positive result is seen in a recognised screening test, a test 
substance may be designated as a potential sensitiser, and it may 
not be necessary to conduct a further guinea pig test. However, if a negative result is seen in such a test, the guinea pig test must be conducted using the procedure described in this tes method. 
See also General introduction Part B.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 266
 
1.2. DEFINITIONS  
Skin sensitisation : (allergic contact dermatitis) is an immuno ­
logically mediated cutaneous reaction to a substance. In the human, the responses may be characterised by pruritis, erythema, 
oedema, papules, vesicles, bullae or a combination of these. In 
other species the reactions may differ and only erythema and oedema may be seen.  
Induction exposure : an experimental exposure of a subject to a test 
substance with the intention of inducing a hypersensitive state.  
Induction period : a period of at least one week following an 
induction exposure during which a hypersensitive state may be developed.  
Challenge exposure : an experimental exposure of a previously 
treated subject to a test substance following an induction period, to determine if the subject reacts in a hypersensitive manner.
 
1.3. REFERENCE SUBSTANCES 
The sensitivity and reliability of the experimental technique used 
should be assessed every six months by use of substances, which 
are known to have mild-to-moderate skin sensitisation properties. 
In a properly conducted test, a response of at least 30 % in an 
adjuvant test and at least 15 % in a non-adjuvant test should be 
expected for mild/moderate sensitisers. 
The following substances are preferred.  
CAS numbers EINECS numbers EINECS names Common names  
101-86-0 202-983-3 α-hexylcinnamaldehyde α-hexylcinnamaldehyde 
149-30-4 205-736-8 Benzothiazole-2-thiol (mercap ­
tobenzothiazole) kaptax 
94-09-7 202-303-5 Benzocaine norcaine 
There may be circumstances where, given adequate justification 
other control substances meeting the above criteria may be used.  
1.4. PRINCIPLE OF THE TEST METHOD 
The test animals are initially exposed to the test substance by intra ­
dermal injections and/or epidermal application (induction exposure). 
Following a rest period of 10 to 14 days (induction period), during which an immune response may develop, the animals are exposed to a challenge dose. The extent and degree of skin reaction to the 
challenge exposure in the test animals is compared with that demon ­
strated by control animals which undergo sham treatment during induction and receive the challenge exposure.
 
1.5. DESCRIPTION OF THE TEST METHODS 
If removal of the test substance is considered necessary, this should 
be achieved using water or an appropriate solvent without altering 
the existing response or the integrity of the epidermis.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 267
 
1.5.1.  Guinea-Pig Maximisation Test (GPMT)  
1.5.1.1. P r e p a r a t i o n s 
Healthy young adult albino guinea pigs are acclimatised to the 
laboratory conditions for at least five days prior to the test. Before 
the test, animals are randomised and assigned to the treatment 
groups. Removal of hair is by clipping, shaving or possibly by chemical depilation, depending on the test method used. Care should be taken to avoid abrading the skin. The animals are 
weighed before the test commences and at the end of the test.
 
1.5.1.2. T e s t c o n d i t i o n s  
1.5.1.2.1. Test animals 
Commonly used laboratory strains of albino guinea-pigs are used.  
1.5.1.2.2. Number and sex 
Male and/or female animals can be used. If females are used, they 
should be nulliparous and non-pregnant. 
A minimum of 10 animals is used in the treatment group and at least 
five animals in the control group. When fewer than 20 test and 10 control guinea pigs have been used, and it is not possible to conclude that the test substance is a sensitiser, testing in additional 
animals to give a total of at least 20 test and 10 control animals is 
strongly recommended.
 
1.5.1.2.3. Dose levels 
The concentration of the test substance used for each induction 
exposure should be well-tolerated systemically and should be the highest to cause mild-to-moderate skin irritation. The concentration 
used for the challenge exposure should be the highest non-irritant 
dose. The appropriate concentrations should be determined from a pilot study using two or three animals, if other information is not available. Consideration should be given to the use of FCA-treated 
animals for this purpose.
 
1.5.1.3. P r o c e d u r e  
1.5.1.3.1. Induction 
Day 0-treated group 
Three pairs of intradermal injections of 0,1 ml volume are given in 
the shoulder region which is cleared of hair so that one of each pair 
lies on each side of the midline. 
Injection 1: a 1:1 mixture (v/v) FCA/water or physiological saline. 
Injection 2: the test substance in an appropriate vehicle at the 
selected concentration. 
Injection 3: the test substance at the selected concentration 
formulated in a 1:1 mixture (v/v) FCA/water or physiological saline. 
In injection 3, water soluble substances are dissolved in the aqueous 
phase prior to mixing with FCA. Liposoluble or insoluble substances are suspended in FCA prior to combining with the aqueous phase. 
The final concentration of test substance shall be equal to that used 
in injection 2.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 268
 
Injections 1 and 2 are given close to each other and nearest the head, 
while 3 is given towards the caudal part of the test area. 
Day 0-control group 
Three pairs of intradermal injections of 0,1 ml volume are given in 
the same sites as in the treated animals. 
Injection 1: a 1:1 mixture (v/v) FCA/water or physiological saline. Injection 2: the undiluted vehicle. Injection 3: a 50 % w/v formulation of the vehicle in a 1:1 mixture 
(v/v) FCA/water or physiological saline. 
Day 5-7-treated and control groups Approximately 24 hours before the topical induction application, if 
the substance is not a skin irritant, the test area, after close-clipping and/or shaving is treated with 0,5 ml of 10 % sodium lauryl sulphate 
in vaseline, in order to create a local irritation. 
Day 6-8-treated group The test area is again cleared of hair. A filter paper (2 × 4 cm) is 
fully-loaded with test substance in a suitable vehicle and applied to the test area and held in contact by an occlusive dressing for 48 hours. The choice of the vehicle should be justified. Solids are 
finely pulverised and incorporated in a suitable vehicle. Liquids can 
be applied undiluted, if appropriate. 
Day 6-8-control group The test area is again cleared of hair. The vehicle only is applied in a 
similar manner to the test area and held in contact by an occlusive dressing for 48 hours.
 
1.5.1.3.2. Challenge 
Day 20-22-treated and control groups 
The flanks of treated and control animals are cleared of hair. A patch 
or chamber loaded with the test substance is applied to one flank of the animals and, when relevant, a patch or chamber loaded with the vehicle only may also be applied to the other flank. The patches are 
held in contact by an occlusive dressing for 24 hours.
 
1.5.1.3.3. Observation and Grading: treated and control groups 
— approximately 21 hours after removing the patch the challenge 
area is cleaned and closely-clipped and/or shaved and depilated if necessary; 
— approximately three hours later (approximately 48 hours from the 
start of the challenge application) the skin reaction is observed 
and recorded according to the grades shown in the Appendix; 
— approximately 24 hours after this observation a second obser ­
vation (72 hours) is made and once again recorded. 
Blind reading of test and control animals is encouraged. If it is necessary to clarify the results obtained in the first challenge, 
a second challenge (i.e. a rechallenge), where appropriate with a new control group, should be considered approximately one week after 
the first one. A rechallenge may also be performed on the original 
control group.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 269
 
All skin reactions and any unusual findings, including systemic 
reactions, resulting from induction and challenge procedures should 
be observed and recorded according to the grading scale of Magnusson/Kligman (See Appendix). Other procedures, e.g. histo ­
pathological examination, the measurement of skin fold thickness, 
may be carried out to clarify doubtful reactions.
 
1.5.2.  Buehler test  
1.5.2.1. P r e p a r a t i o n s 
Healthy young adult albino guinea-pigs are acclimatised to the 
laboratory conditions for at least five days prior to the test. Before the test, animals are randomised and assigned to the treatment 
groups. Removal of hair is by clipping, shaving or possibly by 
chemical depilation, depending on the test method used. Care should be taken to avoid abrading the skin. The animals are weighed before the test commences and at the end of the test.
 
1.5.2.2. T e s t c o n d i t i o n s  
1.5.2.2.1. Test animals 
Commonly used laboratory strains of albino guinea-pigs are used.  
1.5.2.2.2. Number and sex 
Male and/or female animals can be used. If females are used, they 
should be nulliparous and non-pregnant. 
A minimum of 20 animals is used in the treatment group and at least 
10 animals in the control group.  
1.5.2.2.3. Dose levels 
The concentration of test substance used for each induction exposure 
should be the highest possible to produce a mild but not excessive 
irritation. The concentration used for the challenge exposure should 
be the highest non-irritating dose. If necessary, the appropriate concentration can be determined from a pilot study using two or three animals. 
For water soluble test materials, it is appropriate to use water or a 
dilute non-irritating solution of surfactant as the vehicle. For other test materials 80 % ethanol/water is preferred for induction and acetone for challenge.
 
1.5.2.3. P r o c e d u r e  
1.5.2.3.1. Induction 
Day 0-treated group 
One flank is cleared of hair (closely-clipped). The test patch system 
should be fully loaded with test substance in a suitable vehicle (the 
choice of the vehicle should be justified; liquid test substances can be applied undiluted, if appropriate). 
The test patch system is applied to the test area and held in contact 
with the skin by an occlusive patch or chamber and a suitable 
dressing for six hours.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 270
 
The test patch system must be occlusive. A cotton pad is appropriate 
and can be circular or square, but should approximate 4-6 cm  2 . 
Restraint using an appropriate restrainer is preferred to assure occlusion. If wrapping is used, additional exposures may be required. 
Day 0-control group One flank is cleared of hair (closely-clipped). The vehicle only is 
applied in a similar manner to that used for the treated group. The test patch system is held in contact with the skin by an occlusive 
patch or chamber and a suitable dressing for six hours. If it can be 
demonstrated that a sham control group is not necessary, a naive control group may be used. 
Days 6-8 and 13-15-treated and control group The same application as on day 0 is carried out on the same test area 
(cleared of hair if necessary) of the same flank on day 6-8, and again on day 13-15.
 
1.5.2.3.2. Challenge 
Day 27-29-treated and control group 
The untreated flank of treated and control animals is cleared of hair 
(closely-clipped). An occlusive patch or chamber containing the 
appropriate amount of test substance is applied, at the maximum 
non-irritant concentration, to the posterior untreated flank of treated and control animals. 
When relevant, an occlusive patch or chamber with vehicle only is 
also applied to the anterior untreated flank of both treated and 
control animals. The patches or chambers are held in contact by a 
suitable dressing for six hours.
 
1.5.2.3.3. Observation and grading 
— approximately 21 hours after removing the patch the challenge 
area is cleared of hair, 
— approximately three hours later (approximately 30 hours after 
application of the challenge patch) the skin reactions are 
observed and recorded according to the grades shown in the 
Appendix, 
— approximately 24 hours after the 30 hour observation (approxi ­
mately 54 hours after application of the challenge patch) skin reactions are again observed and recorded. 
Blind reading of the test and control animals is encouraged. If it is necessary to clarify the results obtained in the first challenge, 
a second challenge (i.e. a rechallenge), where appropriate with a new control group, should be considered approximately one week after 
the first one. A rechallenge may also be performed on the original 
control group.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 271
 
All skin reactions and any unusual findings, including systemic 
reactions, resulting from induction and challenge procedures should 
be observed and recorded according to the Magnusson/Kligman grading scale (See Appendix). Other procedures, e.g. histopath ­
ological examination, the measurement of skin fold thickness, may 
be carried out to clarify doubtful reactions.
 
2. DATA (GPMT and Buehler test)  
Data should be summarised in tabular form, showing for each animal 
the skin reactions at each observation.  
3. REPORTING (GPMT and Buehler test)  
If a screening assay is performed before the guinea pig test the 
description or reference of the test (e.g. Mouse Ear Swelling Test 
(MEST)), including details of the procedure, must be given together with results obtained with the test and reference substances.
 
Test report (GMPT and Buehler test)  
The test report shall, if possible, include the following information: 
Test animals: — strain of guinea-pig used, — number, age and sex of animals, — source, housing conditions, diet, etc., — individual weights of animals at the start of the test. Test conditions: — technique of patch site preparation, 
— details of patch materials used and patching technique, 
— result of pilot study with conclusion on induction and challenge 
concentrations to be used in the test, 
— details of test substance preparation, application and removal, 
— justification for choice of vehicle, — vehicle and test substance concentrations used for induction and 
challenge exposures and the total amount of substance applied 
for induction and challenge. 
Results: 
— a summary of the results of the latest sensitivity and reliability 
check (see 1.3) including information on substance, concentration 
and vehicle used,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 272
 
— on each animal including grading system, 
— narrative description of the nature and degree effects observed, — any histopathological findings. 
Discussion of results. 
Conclusions.
 
4. REFERENCES  
This method is analogous to OECD TG 406.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 273
 
Appendix  
TABLE  
Magnusson/Kligman grading scale for the evaluation of challenge patch test 
reactions  
0 = no visible change 
1 = discrete or patchy erythema 
2 = moderate and confluent erythema 3 = intense erythema and swelling
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 274
 
B.7. REPEATED DOSE 28-DAY ORAL TOXICITY STUDY IN 
RODENTS  
INTRODUCTION 
1. This Test Method is equivalent to OECD Test Guideline 407 (2008). The 
original Test Guideline 407 was adopted in 1981. In 1995 a revised version 
was adopted, to obtain additional information from the animal used in the 
study, in particular on neurotoxicity and immunotoxicity. 
2. In 1998, the OECD initiated a high-priority activity, to revise existing Test 
Guidelines and to develop new Test Guidelines for the screening and testing 
of potential endocrine disruptors (8). One element of the activity was to update the existing OECD guideline for ‘repeated dose 28-day oral toxicity study in rodents’ (TG 407) by parameters suitable to detect endocrine 
activity of test chemicals. This procedure underwent an extensive inter ­
national program to test for the relevance and practicability of the additional parameters, the performance of these parameters for chemicals with (anti)oestrogenic, (anti)androgenic, and (anti)thyroid activity, the intra- and 
inter-laboratory reproducibility, and the interference of the new parameters 
with those required by the prior TG 407. The large amount of data thereby obtained has been compiled and evaluated in detail in a comprehensive OECD report (9). This updated Test Method B.7 (as equivalent to TG 
407) is the outcome of the experience and results gained during the inter ­
national test program. This Test Method allows certain endocrine mediated effects to be put into context with other toxicological effects.
 
INITIAL CONSIDERATIONS AND LIMITATIONS 
3. In the assessment and evaluation of the toxic characteristics of a chemical, 
the determination of oral toxicity using repeated doses may be carried out after initial information on toxicity has been obtained by acute toxicity testing. This Test Method is intended to investigate effects on a very 
broad variety of potential targets of toxicity. It provides information on 
the possible health hazards likely to arise from repeated exposure over a relatively limited period of time, including effects on the nervous, immune and endocrine systems. Regarding these particular endpoints, it should 
identify chemicals with neurotoxic potential, which may warrant further 
in-depth investigation of this aspect, and chemicals that interfere with thyroid physiology. It may also provide data on chemicals that affect the male and/or female reproductive organs in young adult animals and may 
give an indication of immunological effects. 
4. The results from this Test Method B.7 should be used for hazard identifi ­
cation and risk assessment. The results obtained by the endocrine related 
parameters should be seen in the context of the ‘OECD Conceptual 
Framework for Testing and Assessment of Endocrine Disrupting Chemicals’ (11). The method comprises the basic repeated dose toxicity study that may be used for chemicals on which a 90-day study is not warranted (e.g. when 
the production volume does not exceed certain limits) or as a preliminary to 
a long-term study. The duration of exposure should be 28 days.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 275
 
5. The international program conducted on the validation of parameters 
suitable to potentially detect endocrine activity of a test chemical showed 
that the quality of data obtained by this Test Method B.7 will depend much on the experience of the test laboratory. This relates specifically to the histopathological determination of cyclic changes in the female reproductive 
organs and to the weight determination of the small hormone dependent 
organs which are difficult to dissect. Guidance on histopathology has been developed (19). It is available on the OECD public website on Test Guide ­
lines. It is intended to assist pathologists in their examinations and help 
increase the sensitivity of the assay. A variety of parameters were found 
to be indicative of endocrine-related toxicity and have been incorporated in the Test Method. Parameters for which insufficient data were available to prove usefulness or which showed only weak evidence in the validation 
programme of their ability to help in detection of endocrine disrupters are 
proposed as optional endpoints (see Appendix 2). 
6. On the basis of data generated in the validation process, it must be 
emphasised that the sensitivity of this assay is not sufficient to identify 
all substances with (anti)androgenic or (anti)oestrogenic modes of action 
(9). The Test Method is not performed in a life-stage that is most sensitive to endocrine disruption. The Test Method nevertheless, during the validation process identified substances weakly and strongly affecting 
thyroid function, and strong and moderate endocrine active substances 
acting through oestrogen or androgen receptors, but in most cases failed to identify endocrine active substances that weakly affect oestrogen or androgen receptors. Thus it cannot be described as a screening assay for 
endocrine activity. 
7. Consequently, the lack of effects related to these modes of action can not be 
taken as evidence for the lack of effects on the endocrine system. Regarding 
endocrine mediated effects, substance characterisation should not therefore 
be based on the results of this Test Method alone but should be used in a weight of evidence approach incorporating all existing data on a chemical to characterise potential endocrine activity. For this reason, regulatory decision 
making on endocrine activity (substance characterisation) should be a 
broadly based approach, not solely reliant on results from application of this test method. 
8. It is acknowledged that all animal-based procedures will conform to local 
standards of animal care; the descriptions of care and treatment set forth 
below are minimal performance standards, and will be superseded by local regulations where more stringent. Further guidance of the humane treatment of animals is given by the OECD (14). 
9. Definitions used are given in Appendix 1.
 
PRINCIPLE OF THE TEST 
10. The test chemical is orally administered daily in graduated doses to several 
groups of experimental animals, one dose level per group for a period of 28 days. During the period of administration the animals are observed closely, each day for signs of toxicity. Animals that die or are euthanised during the 
test are necropsied and at the conclusion of the test surviving animals are 
euthanised and necropsied. A 28 day study provides information on the
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 276
 
effects of repeated oral exposure and can indicate the need for further longer 
term studies. It can also provide information on the selection of concen ­
trations for longer term studies. The data derived from using the Test Method should allow for the characterisation of the test chemical toxicity, for an indication of the dose response relationship and the determination of 
the No-Observed Adverse Effect Level (NOAEL).
 
DESCRIPTION OF THE METHOD  
Selection of animal species  
11. The preferred rodent species is the rat, although other rodent species may be 
used. If the parameters specified within this Test Method B.7 are inves ­
tigated in another rodent species a detailed justification should be given. 
Although it is biologically plausible that other species should respond to toxicants in a similar manner to the rat, the use of smaller species may result in increased variability due to technical challenges of dissecting smaller 
organs. In the international validation program for the detection of 
endocrine disrupters, the rat was the only species used. Young healthy adult animals of commonly used laboratory strains should be employed. Females should be nulliparous and non pregnant. Dosing should begin as 
soon as feasible after weaning, and, in any case, before the animals are nine 
weeks old. At the commencement of the study the weight variation of animals used should be minimal and not exceed ± 20 % of the mean weight of each sex. When a repeated oral dose is conducted as a preliminary 
to a longer-term study, it is preferable that animals from the same strain and 
source should be used in both studies.
 
Housing and feeding  
12. All procedures should conform to local standards of laboratory animal care. 
The temperature in the experimental animal room should be 22 °C (± 3 °C). Although the relative humidity should be at least 30 % and preferably not to exceed 70 % other than during room cleaning, the aim should be 50-60 %. 
Lighting should be artificial, the photoperiod being 12 hours light, 12 hours 
dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. The choice of diet may be influenced by the need to ensure a suitable admixture of a test chemical when admin ­
istered by this method. Animals should be group housed in small groups of 
the same sex; animals may be housed individually if scientifically justified. For group caging, no more than five animals should be housed per cage. 
13. The feed should be regularly analysed for contaminants. A sample of the 
diet should be retained until finalisation of the report.
 
Preparation of animals  
14. Healthy young adult animals are randomly assigned to the control and 
treatment groups. Cages should be arranged in such a way that possible 
effects due to cage placement are minimised. The animals are identified uniquely and kept in their cages for at least five days prior to the start of the treatment study to allow for acclimatisation to the laboratory conditions.
 
Preparation of doses  
15. The test chemical is administered by gavage or via the diet or drinking 
water. The method of oral administration is dependent on the purpose of 
the study, and the physical/chemical/toxico-kinetic properties of the test 
chemical.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 277
 
16. Where necessary, the test chemical is dissolved or suspended in a suitable 
vehicle. It is recommended that, wherever possible, the use of an aqueous 
solution/suspension be considered first, followed by consideration of a solution/suspension in oil (e.g. corn oil) and then by possible solution in other vehicles. For vehicles other than water the toxic characteristics of the 
vehicle must be known. The stability of the test chemical in the vehicle 
should be determined.
 
PROCEDURE  
Number and sex of animals  
17. At least 10 animals (five female and five male) should be used at each dose 
level. If interim euthanasia are planned, the number should be increased by the number of animals scheduled to be euthanised before the completion of the study. Consideration should be given to an additional satellite group of 
ten animals (five per sex) in the control and in the top dose group for 
observation of reversibility, persistence, or delayed occurrence of toxic effects, for at least 14 days post treatment.
 
Dosage  
18. Generally, at least three test groups and a control group should be used, but 
if from assessment of other data, no effects would be expected at a dose of 
1 000 mg/kg bw/d, a limit test may be performed. If there are no suitable 
data available, a range finding study (animals of the same strain and source) may be performed to aid the determination of the doses to be used. Except for treatment with the test chemical, animals in the control group should be 
handled in an identical manner to the test group subjects. If a vehicle is used 
in administering the test chemical, the control group should receive the vehicle in the highest volume used. 
19. Dose levels should be selected taking into account any existing toxicity and 
(toxico-) kinetic data available for the test chemical or related chemicals. The highest dose level should be chosen with the aim of inducing toxic effects but not death or severe suffering. Thereafter, a descending sequence 
of dose levels should be selected with a view to demonstrating any dosage 
related response and no-observed-adverse effects at the lowest dose level (NOAEL). Two to four fold intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is 
often preferable to using very large intervals (e.g. more than a factor of 10) 
between dosages. 
20. In the presence of observed general toxicity (e.g. reduced body weight, liver, 
heart, lung or kidney effects, etc.) or other changes that may not be toxic 
responses (e.g. reduced food intake, liver enlargement), observed effects on 
immune, neurological or endocrine sensitive endpoints should be interpreted with caution.
 
Limit test  
21. If a test at one dose level of at least 1 000 mg/kg body weight/day or, for 
dietary or drinking water administration, an equivalent percentage in the 
diet, or drinking water (based upon body weight determinations), using 
the procedures described for this study, produces no observable toxic effects and if toxicity would not be expected based upon data from struc ­
turally related chemicals, then a full study using three dose levels may not 
be considered necessary. The limit test applies except when human exposure 
indicates the need for a higher dose level to be used.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 278
 
Administration of doses  
22. The animals are dosed with test chemical daily 7 days each week for a 
period of 28 days. When the test chemical is administered by gavage, this should be done in a single dose to the animals using a stomach tube or a 
suitable intubation cannula. The maximum volume of liquid that can be 
administered at one time depends on the size of the test animal. The volume should not exceed 1 ml/100 g body weight except in the case of aqueous solutions where 2 ml/100 g body weight may be used. Except for 
irritating or corrosive chemicals, which will normally reveal exacerbated 
effects with higher concentrations, variability in test volume should be minimized by adjusting the concentration to ensure a constant volume at 
all dose levels. 
23. For chemicals administered via the diet or drinking water it is important to 
ensure that the quantities of the test chemical involved do not interfere with 
normal nutrition or water balance. When the test chemical is administered in the diet either a constant dietary concentration (ppm) or a constant dose level in terms of the animals’ body weight may be used; the alternative used 
must be specified. For a chemical administered by gavage, the dose should 
be given at similar times each day, and adjusted as necessary to maintain a constant dose level in terms of animal body weight. Where a repeated dose study is used as a preliminary to a long term study, a similar diet should be 
used in both studies.
 
Observations  
24. The observation period should be 28 days. Animals in a satellite group 
scheduled for follow-up observations should be kept for at least 14 days 
without treatment to detect delayed occurrence, or persistence of, or 
recovery from toxic effects. 
25. General clinical observations should be made at least once a day, preferably 
at the same time(s) each day and considering the peak period of anticipated effects after dosing. The health condition of the animals should be recorded. 
At least twice daily, all animals are observed for morbidity and mortality. 
26. Once before the first exposure (to allow for within-subject comparisons), 
and at least once a week thereafter, detailed clinical observations should be 
made in all animals. These observations should be made outside the home cage in a standard arena and preferably at the same time of day on each occasion. They should be carefully recorded, preferably using scoring 
systems, explicitly defined by the testing laboratory. Effort should be 
made to ensure that variations in the test conditions are minimal and that observations are preferably conducted by observers unaware of the treatment. Signs noted should include, but not be limited to, changes in 
skin, fur, eyes, mucous membranes, occurrence of secretions and excretions 
and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. 
excessive grooming, repetitive circling) or bizarre behaviour (e.g. 
self-mutilation, walking backwards) should also be recorded (2). 
27. In the fourth exposure week sensory reactivity to stimuli of different types 
(2) (e.g. auditory, visual and proprioceptive stimuli) (3)(4)(5), assessment of grip strength (6) and motor activity assessment (7) should be conducted. Further details of the procedures that could be followed are given in the 
respective references. However, alternative procedures than those referenced 
could be used.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 279
 
28. Functional observations conducted in the fourth exposure week may be 
omitted when the study is conducted as a preliminary study to a subsequent 
subchronic (90-day) study. In that case, the functional observations should be included in this follow-up study. On the other hand, the availability of data on functional observations from the repeated dose study may enhance 
the ability to select dose levels for a subsequent subchronic study. 
29. As an exception, functional observations may also be omitted for groups 
that otherwise reveal signs of toxicity to an extent that would significantly interfere with the functional test performance. 
30. At necropsy, the oestrus cycle of all females could be determined (optional) 
by taking vaginal smears. These observations will provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in 
histological evaluation of estrogen sensitive tissues [see guidance on histo ­
pathology (19)].
 
Body weight and food/water consumption  
31. All animals should be weighed at least once a week. Measurements of food 
consumption should be made at least weekly. If the test chemical is admin ­
istered via the drinking water, water consumption should also be measured 
at least weekly.  
Haematology  
32. The following haematological examinations should be made at the end of 
the test period: haematocrit, haemoglobin concentrations, erythrocyte count, reticulocytes, total and differential leucocyte count, platelet count and a 
measure of blood clotting time/potential. Other determinations that should 
be carried out, if the test chemical or its putative metabolites have or are suspected to have oxidising properties include methaemoglobin concen ­
tration and Heinz bodies. 
33. Blood samples should be taken from a named site just prior to or as part of 
the procedure for euthanasia of the animals, and stored under appropriate conditions. Animals should be fasted overnight prior to euthanasia (
 1 ). 
Clinical biochemistry  
34. Clinical biochemistry determinations to investigate major toxic effects in 
tissues and, specifically, effects on kidney and liver, should be performed 
on blood samples obtained of all animals just prior to or as part of the procedure for euthanasia of the animals (apart from those found moribund and/or euthanised prior to the termination of the study). Investigations of 
plasma or serum shall include sodium, potassium, glucose, total cholesterol, 
urea, creatinine, total protein and albumin, at least two enzymes indicative of hepatocellular effects (such as alanin aminotransferase, aspartate amino ­
transferase, alkaline phosphatase, γ-glutamyl trans-peptidase and glutamate 
dehydrogenase), and bile acids. Measurements of additional enzymes (of 
hepatic or other origin) and bilirubin may provide useful information under certain circumstances. 
35. Optionally, the following urinalysis determinations could be performed 
during the last week of the study using timed urine volume collection; appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 280  
( 1 ) For a number of measurements in serum and plasma, most notably for glucose, overnight 
fasting would be preferable. The major reason for this preference is that the increased 
variability which would inevitably result from non-fasting, would tend to mask more 
subtle effects and make interpretation difficult. On the other hand, however, overnight 
fasting may interfere with the general metabolism of the animals and, particularly in 
feeding studies, may disturb the daily exposure to the test chemical. If overnight fasting 
is adopted, clinical biochemical determinations should be performed after the conduct of 
functional observations in week 4 of the study.
 36. In addition, studies to investigate plasma or serum markers of general tissue 
damage should be considered. Other determinations that should be carried 
out, if the known properties of the test chemical may, or are suspected to, affect related metabolic profiles include calcium, phosphate, triglycerides, specific hormones, and cholinesterase. These need to be identified for 
chemicals in certain classes or on a case-by-case basis. 
37. Although in the international evaluation of the endocrine related endpoints a 
clear advantage for the determination of thyroid hormones (T3, T4) and 
TSH could not be demonstrated, it may be helpful to retain plasma or 
serum samples to measure T3, T4 and TSH (optional) if there is an indi ­
cation for an effect on the pituitary-thyroid axis. These samples may be frozen at – 20° for storage. The following factors may influence the varia ­
bility and the absolute concentrations of the hormone determinations: 
— time of sacrifice because of diurnal variation of hormone concentrations 
— method of sacrifice to avoid undue stress to the animals that may affect 
hormone concentrations 
— test kits for hormone determinations that may differ by their standard 
curves. 
Definitive identification of thyroid-active chemicals is more reliable by 
histopathological analysis rather than hormone levels. 
38. Plasma samples specifically intended for hormone determination should be 
obtained at a comparable time of the day. It is recommended that 
consideration should be given to T3, T4 and TSH determinations triggered based upon alterations of thyroid histopathology. The numerical 
values obtained when analysing hormone concentrations differ with various 
commercial assay kits. Consequently, it may not be possible to provide performance criteria based upon uniform historical data. Alternatively, laboratories should strive to keep control coefficients of variation below 
25 for T3 and T4 and below 35 for TSH. All concentrations are to be 
recorded in ng/ml. 
39. If historical baseline data are inadequate, consideration should be given to 
determination of haematological and clinical biochemistry variables before 
dosing commences or preferably in a set of animals not included in the experimental groups.
 
PATHOLOGY  
Gross necropsy  
40. All animals in the study shall be subjected to a full, detailed gross necropsy 
which includes careful examination of the external surface of the body, all 
orifices, and the cranial, thoracic and abdominal cavities and their contents. 
The liver,  kidneys,  adrenals,  testes,  epididymides,  prostate + seminal  
vesicles with coagulating glands as a whole,  thymus,  spleen,  brain and  
heart of all animals (apart from those found moribund and/or euthanised 
prior to the termination of the study) should be trimmed of any adherent 
tissue, as appropriate, and their wet weight taken as soon as possible after dissection to avoid drying. Care must be exercised when trimming the prostate complex to avoid puncture of the fluid filled seminal vesicles. 
Alternatively, seminal vesicles and prostate may be trimmed and weighed 
after fixation.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 281
 
41. In addition, two other tissues could be optionally weighed as soon as 
possible after dissection, to avoid drying: paired ovaries (wet weight) and 
uterus, including cervix (guidance on removal and preparation of the uterine tissues for weight measurement is provided in OECD TG 440 (18)). 
42. The thyroid weight (optional) could be determined after fixation. Trimming 
should also be done very carefully and only after fixation to avoid tissue 
damage. Tissue damage could compromise histopathology analysis. 
43. The following tissues should be preserved in the most appropriate fixation 
medium for both the type of tissue and the intended subsequent histopath ­
ological examination (see paragraph 47): all
 gross lesions,  brain (represen ­
tative regions including cerebrum, cerebellum and pons),  spinal cord,  eye,  
stomach,  small and large intestines (including Peyer’s patches),  liver,  
kidneys,  adrenals,  spleen,  heart,  thymus,  thyroid,  trachea and  lungs (pre ­
served by inflation with fixative and then immersion),  gonads (testis and  
ovaries),  accessory sex organs (uterus and cervix, epididymides, prostate + 
seminal vesicles with coagulating glands),  vagina,  urinary bladder,  lymph  
nodes [besides the most proximal draining node another lymph node should 
be taken according to the laboratory’s experience (15)],  peripheral nerve 
(sciatic or tibial) preferably in close proximity to the muscle,  skeletal  
muscle and  bone, with  bone marrow (section or, alternatively, a fresh 
mounted bone marrow aspirate). It is recommended that testes be fixed 
by immersion in Bouin’s or modified Davidson’s fixative (16) (17). The tunica albuginea must be gently and shallowly punctured at the both poles of the organ with a needle to permit rapid penetration of the fixative. The 
clinical and other findings may suggest the need to examine additional 
tissues. Also any organs considered likely to be target organs based on the known properties of the test chemical should be preserved. 
44. The following tissues may give valuable indication for endocrine-related 
effects: Gonads (ovaries and testes), accessory sex organs (uterus including cervix, epididymides, seminal vesicles with coagulation glands, dorsolateral and ventral prostate), vagina, pituitary, male mammary gland, 
the thyroid and adrenal gland. Changes in male mammary glands have not 
been sufficiently documented but this parameter may be very sensitive to substances with oestrogenic action. Observation of organs/tissues that are not listed in paragraph 43 is optional (see Appendix 2). 
45. The Guidance on histopathology (19) details extra information on dissection, 
fixation, sectioning and histopathology of endocrine tissues. 
46. In the international test program some evidence was obtained that subtle 
endocrine effects by chemicals with a low potency for affecting sex 
hormone homeostasis may be identified by disturbance of the synchroni ­
sation of the oestrus cycle in different tissues and not so much by frank histopathological alterations in female sex organs. Although no definitive 
proof was obtained for such effects, it is recommended that evidence of 
possible asynchrony of the oestrus cycle should be taken into account in interpretation of the histopathology of the ovaries (follicular, thecal, and granulosa cells), uterus, cervix and vagina. If assessed, the stage of cycle 
as determined by vaginal smears could be included in this comparison as 
well.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 282
 
Histopathology  
47. Full histopathology should be carried out on the preserved organs and 
tissues of all animals in the control and high dose groups. These examin ­
ations should be extended to animals of all other dosage groups, if 
treatment-related changes are observed in the high dose group. 
48. All gross lesions shall be examined. 49. When a satellite group is used, histopathology should be performed on 
tissues and organs identified as showing effects in the treated groups.
 
DATA AND REPORTING  
Data  
50. Individual data should be provided. Additionally, all data should be 
summarised in tabular form showing for each test group the number of 
animals at the start of the test, the number of animals found dead during 
the test or euthanised for humane reasons and the time of any death or euthanasia, the number showing signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration, and severity of any 
toxic effects, the number of animals showing lesions, the type of lesions, 
their severity and the percentage of animals displaying each type of lesion. 
51. When possible, numerical results should be evaluated by an appropriate and 
generally acceptable statistical method. Comparisons of the effect along a dose range should avoid the use of multiple t-tests. The statistical methods 
should be selected during the design of the study. 
52. For quality control it is proposed that historical control data are collected 
and that for numerical data coefficients of variation are calculated, especially 
for the parameters linked with endocrine disrupter detection. These data can be used for comparison purposes when actual studies are evaluated.
 
Test report  
53. The test report must include the following information:  
Test chemical:  
— physical nature, purity and physicochemical properties; — identification data.  
Vehicle (if appropriate):  
— justification for choice of vehicle, if other than water.  
Test animals:  
— species/strain used; — number, age and sex of animals; — source, housing conditions, diet, etc.; — individual weights of animals at the start of the test. — justification for species if not rat  
Test conditions:  
— rationale for dose level selection; — details of test chemical formulation/diet preparation, achieved concen ­
tration, stability and homogeneity of the preparation;
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 283
 
— details of the administration of the test chemical; 
— conversion from diet/drinking water test chemical concentration (ppm) to 
the actual dose (mg/kg body weight/day), if applicable; 
— details of food and water quality.  
Optional endpoints investigated  
— list of optional endpoints investigated  
Results:  
— body weight/body weight changes; — food consumption, and water consumption, if applicable; — toxic response data by sex and dose level, including signs of toxicity; — nature, severity and duration of clinical observations (whether reversible 
or not); 
— sensory activity, grip strength and motor activity assessments; — haematological tests with relevant base-line values; — clinical biochemistry tests with relevant base-line values; — body weight at euthanasia and organ weight data; — necropsy findings; — a detailed description of all histopathological findings; — absorption data if available; — statistical treatment of results, where appropriate.  
Discussion of results Conclusions
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 284
 
Appendix 1  
DEFINITIONS 
Androgenicity  is the capability of a chemical to act like a natural androgenic 
hormone (e.g. testosterone) in a mammalian organism.  
Antiandrogenicity  is the capability of a chemical to suppress the action of a 
natural androgenic hormone (e.g. testosterone) in a mammalian organism.  
Antioestrogenicity  is the capability of a chemical to suppress the action of a 
natural oestrogenic hormone (e.g. oestradiol 17ß) in a mammalian organism.  
Antithyroid activity  is the capability of a chemical to suppress the action of a 
natural thyroid hormone (e.g. T  3 ) in a mammalian organism.  
Dosage  is a general term comprising of dose, its frequency and the duration of 
dosing.  
Dose  is the amount of test chemical administered. The dose is expressed as 
weight of test chemical per unit body weight of test animal per day (e.g. 
mg/kg body weight/day), or as a constant dietary concentration.  
Evident toxicity  is a general term describing clear signs of toxicity following 
administration of test chemical. These should be sufficient for hazard assessment and should be such that an increase in the dose administered can be expected to 
result in the development of severe toxic signs and probable mortality.  
NOAEL  is the abbreviation for no-observed-adverse-effect level. This is the 
highest dose level where no adverse treatment-related findings are observed due to treatment.  
Oestrogenicity  is the capability of a chemical to act like a natural oestrogenic 
hormone (e.g. oestradiol 17ß) in a mammalian organism.  
Test chemical : Any substance or mixture tested using this Test Method.  
Thyroid activity  is the capability of a chemical to act like a natural thyroid 
hormone (e.g. T
 3 ) in a mammalian organism.  
Validation  is a scientific process designed to characterise the operational 
requirements and limitations of a test method and to demonstrate its reliability 
and relevance for a particular purpose.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 285
 
Appendix 2  
Endpoints recommended for the detection of endocrine disrupters (EDs) in 
this Test Method B.7  
Mandatory endpoints Optional endpoints 
Weight  
— Testes 
— Epididymides — Adrenals 
— Prostate + seminal vesicles with 
coagulating glands — Ovaries 
— Uterus, including cervix — Thyroid
 
Histopathology  
— Gonads: 
— Testes and — Ovaries 
— Accessory sex organs: 
— Epididymides, 
— Prostate + seminal vesicle with 
coagulating glands 
— Uterus, including cervix 
— Adrenal 
— Thyroid — Vagina — Vaginal smears 
— Male mammary glands — Pituitary
 
Hormones measurement  
— Circulating levels of T3, T4 
— Circulating levels of TSH  
LITERATURE:  
(1) OECD (Paris, 1992). Chairman’s Report of the Meeting of the ad hoc 
Working Group of Experts on Systemic Short-term and (Delayed) Neur ­
otoxicity. 
(2) IPCS (1986). Principles and Methods for the Assessment of Neurotoxicity 
Associated with Exposure to Chemicals. Environmental Health Criteria Document No 60. 
(3) Tupper DE, Wallace RB (1980). Utility of the Neurologic Examination in 
Rats. Acta Neurobiol. Exp. 40: 999-1003. 
(4) Gad SC (1982). A Neuromuscular Screen for Use in Industrial Toxicology. 
J. Toxicol Environ. Health 9: 691-704. 
(5) Moser VC, McDaniel KM, Phillips PM (1991). Rat Strain and Stock 
Comparisons Using a Functional Observational Battery: Baseline Values and Effects of Amitraz. Toxicol. Appl. Pharmacol. 108: 267-283. 
(6) Meyer OA, Tilson HA, Byrd WC, Riley MT (1979). A Method for the 
Routine Assessment of Fore- and Hindlimb Grip Strength of Rats and 
Mice. Neurobehav. Toxicol. 1: 233-236.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 286
 
(7) Crofton KM, Howard JL, Moser VC, Gill MW, Reiter LW, Tilson HA, 
MacPhail RC (1991). Interlaboratory Comparison of Motor Activity 
Experiments: Implication for Neurotoxicological Assessments. Neur ­
otoxicol. Teratol. 13: 599-609. 
(8) OECD (1998). Report of the First Meeting of the OECD Endocrine 
Disrupter Testing and Assessment (EDTA) Task Force, 10th-11 March 
1998, ENV/MC/CHEM/RA(98)5. 
(9) OECD. (2006). Report of the Validation of the Updated Test Guideline 
407: Repeat Dose 28-day Oral Toxicity Study in Laboratory Rats. Series on Testing and Assessment No 59, ENV/JM/MONO(2006)26. 
(10) OECD (2002). Detailed Review Paper on the Appraisal of Test Methods 
for Sex Hormone Disrupting Chemicals. Series on Testing and Assessment 
No 21, ENV/JM/MONO(2002)8. 
(11) OECD (2012).Conceptual Framework for Testing and Assessment of 
Endocrine Disrupting Chemicals. http://www.oecd.org/document/58/ 0,3343,fr_2649_37407_2348794_1_1_1_37407,00.htm l 
(12) OECD (2006). Final Summary report of the meeting of the Validation 
Management Group for mammalian testing. ENV/JM/TG/EDTA/M(2006)2. 
(13) OECD. Draft Summary record of the meeting of the Task Force on 
Endocrine Disrupters Testing and Assessment. ENV/JM/TG/ EDTA/M(2006)3. 
(14) OECD (2000). Guidance document on the recognition, assessment and use 
of clinical signs as humane endpoints for experimental animals used in 
safety evaluation. Series on Testing and Assessment No 19. ENV/JM/ MONO(2000)7. 
(15) Haley P, Perry R, Ennulat D, Frame S, Johnson C, Lapointe J-M, Nyska A, 
Snyder PW, Walker D, Walter G (2005). STP Position Paper: Best Practice 
Guideline for the Routine Pathology Evaluation of the Immune System. Toxicol Pathol 33: 404-407. 
(16) Hess RA, Moore BJ (1993). Histological Methods for the Evaluation of the 
Testis. In: Methods in Reproductive Toxicology, Chapin RE and Heindel JJ 
(eds). Academic Press: San Diego, CA, pp. 52-85. 
(17) Latendresse JR, Warbrittion AR, Jonassen H, Creasy DM.(2002) Fixation 
of testes and eyes using a modified Davidson’s fluid: comparison with Bouin’s fluid and conventional Davidson’s fluid. Toxicol. Pathol. 30, 
524-533. 
(18) OECD (2007). OECD Guideline for Testing of Chemicals No 440: Uterot ­
rophic Bioassay in Rodents: A short-term screening test for oestrogenic properties. 
(19) OECD (2009). Guidance Document 106 on Histologic evaluation of 
Endocrine and Reproductive Tests in Rodents ENV/JM/Mono(2009)11.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 287
 
B.8. SUBACUTE INHALATION TOXICITY: 28-DAY STUDY  
SUMMARY 
This revised Test Method B.8 has been designed to fully characterise test 
chemical toxicity by the inhalation route following repeated exposure for a 
limited period of time (28 days), and to provide data for quantitative inhalation 
risk assessments. Groups of at least 5 male and 5 female rodents are exposed 6 hours per day for 28 days to a) the test chemical at three or more concentration levels, b) filtered air (negative control), and/or c) the vehicle (vehicle control). 
Animals are generally exposed 5 days per week but exposure for 7 days per 
week is also allowed. Males and females are always tested, but they may be exposed at different concentration levels if it is known that one sex is more susceptible to a given test chemical. This method allows the study director the 
flexibility to include satellite (reversibility) groups, bronchoalveolar 
lavage (BAL), neurologic tests, and additional clinical pathology and histopath ­
ological evaluations in order to better characterise the toxicity of a test chemical.
 
INTRODUCTION 
1. This Test Method is equivalent to OECD Test Guideline 412 (2009). The 
original subacute inhalation Test Guideline 412 (TG 412) was adopted in 
1981 (1). This Test Method B.8 (as equivalent to the revised TG 412) has been updated to reflect the state of science and to meet current and future regulatory needs. 
2. This method enables the characterisation of adverse effects following 
repeated daily inhalation exposure to a test chemical for 28 days. The 
data derived from 28-day sub-acute inhalation toxicity studies can be used 
for quantitative risk assessments [if not followed by a 90-day subchronic inhalation toxicity study (Chapter B.29 of this Annex)]. The data can also provide information on the selection of concentrations for longer term 
studies such as the 90-day subchronic inhalation toxicity study. This test 
method is not specifically intended for the testing of nanomaterials. Defi ­
nitions used in the context of this Test Method are provided at the end of this chapter and in the Guidance Document 39 (2).
 
INITIAL CONSIDERATIONS 
3. All available information on the test chemical should be considered by the 
testing laboratory prior to conducting the study in order to enhance the quality of the study and minimize animal usage. Information that will assist in the selection of appropriate test concentrations might include the 
identity, chemical structure, and physico-chemical properties of the test 
chemical; results of any  in vitro  or in vivo  toxicity tests; anticipated 
use(s) and potential for human exposure; available (Q)SAR data and toxi ­
cological data on structurally related chemicals; and data derived from acute 
inhalation toxicity testing. If neurotoxicity is expected or is observed in the 
course of the study, the study director may choose to include appropriate evaluations such as a functional observational battery (FOB) and measurement of motor activity. Although the timing of exposures relative 
to specific examinations may be critical, the performance of these additional 
activities should not interfere with the basic study design.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 288
 
4. Dilutions of corrosive or irritating test chemicals may be tested at concen ­
trations that will yield the desired degree of toxicity [refer to GD 39 (2)]. 
When exposing animals to these materials, the targeted concentrations should be low enough to not cause marked pain and distress, yet sufficient to extend the concentration-response curve to levels that reach the regulatory 
and scientific objective of the test. These concentrations should be selected 
on a case-by-case basis, preferably based upon an adequately designed range-finding study that provides information regarding the critical endpoint, any irritation threshold, and the time of onset (see paragraphs 
11-13). The justification for concentration selection should be provided. 
5. Moribund animals or animals obviously in pain or showing signs of severe 
and enduring distress should be humanely killed. Moribund animals are considered in the same way as animals that die on test. Criteria for making the decision to kill moribund or severely suffering animals, and 
guidance on the recognition of predictable or impending death, are the 
subject of an OECD Guidance Document on Humane Endpoints (3).
 
DESCRIPTION OF THE METHOD  
Selection of Animal Species  
6. Healthy young adult rodents of commonly used laboratory strains should be 
employed. The preferred species is the rat. Justification should be provided 
if other species are used.  
Preparation of Animals  
7. Females should be nulliparous and non-pregnant. On the day of randomis ­
ation, animals should be young adults 7 to 9 weeks of age. Body weights should be within ± 20 % of the mean weight for each sex. The animals are 
randomly selected, marked for individual identification, and kept in their 
cages for at least 5 days prior to the start of the test to allow for acclimati ­
sation to laboratory conditions.
 
Animal Husbandry  
8. Animals should be individually identified, if possible with subcutaneous 
transponders, to facilitate observations and avoid confusion. The temperature of the experimental animal maintenance room should be 22 ± 3 °C. The 
relative humidity should ideally be maintained in the range of 30 to 70 %, 
though this may not be possible when using water as a vehicle. Before and after exposures, animals generally should be caged in groups by sex and concentration, but the number of animals per cage should not interfere with 
clear observation of each animal and should minimise losses due to canni ­
balism and fighting. When animals are to be exposed nose-only, it may be necessary for them to be acclimated to the restraining tubes. The restraining tubes should not impose undue physical, thermal, or immobilisation stress 
on the animals. Restraint may affect physiological endpoints such as body 
temperature (hyperthermia) and/or respiratory minute volume. If generic data are available to show that no such changes occur to any appreciable extent, then pre-adaptation to the restraining tubes is not necessary. Animals 
exposed whole-body to an aerosol should be housed individually during 
exposure to prevent them from filtering the test aerosol through the fur of their cage mates. Conventional and certified laboratory diets may be used, except during exposure, accompanied with an unlimited supply of municipal 
drinking water. Lighting should be artificial, the sequence being 12 hours 
light/12 hours dark.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 289
 
Inhalation Chambers  
9. The nature of the test chemical and the object of the test should be considered 
when selecting an inhalation chamber. The preferred mode of exposure is nose-only (which term includes head-only, nose-only, or snout-only). 
Nose-only exposure is generally preferred for studies of liquid or solid 
aerosols and for vapours that may condense to form aerosols. Special objectives of the study may be better achieved by using a whole-body mode of exposure, but this should be justified in the study report. To 
ensure atmosphere stability when using a whole-body chamber, the total 
‘volume’ of the test animals should not exceed 5 % of the chamber volume. Principles of the nose-only and whole-body exposure techniques 
and their particular advantages and disadvantages are addressed in GD 39 (2).
 
TOXICITY STUDIES  
Limit Concentrations  
10. Unlike with acute studies, there are no defined limit concentrations in 28- 
day sub-acute inhalation toxicity studies. The maximum concentration tested 
should consider: (1) the maximum attainable concentration, (2) the ‘worst case’ human exposure level, (3) the need to maintain an adequate oxygen 
supply, and/or (4) animal welfare considerations. In the absence of 
data-based limits, the acute limits of the Regulation (EC) No 1272/2008 (13) may be used (i.e. up to a maximum concentration of 5 mg/l for aerosols, 20 mg/l for vapours and 20 000 ppm for gases); refer to GD 39 
(2). Justification should be provided if it is necessary to exceed these limits 
when testing gases or highly volatile test chemicals (e.g. refrigerants). The limit concentration should elicit unequivocal toxicity without causing undue stress to the animals or affecting their longevity (3).
 
Range-Finding Study  
11. Before commencing with the main study, it may be necessary to perform 
a range-finding study. A range-finding study is more comprehensive than a sighting study because it is not limited to concentration selection. Knowledge learned from a range-finding study can lead to a successful 
main study. A range-finding study may, for example, provide technical 
information regarding analytical methods, particle sizing, discovery of toxic mechanisms, clinical pathology and histopathological data, and esti ­
mations of what may be NOAEL and MTC concentrations in a main study. 
The study director may choose to use the range-finding study to identify the 
threshold of respiratory tract irritation (e.g. with histopathology of the respiratory tract, pulmonary function testing, or bronchoalveolar lavage), the upper concentration which is tolerated without undue stress to the 
animals, and the parameters that will best characterise a test chemical’s 
toxicity. 
12. A range-finding study may consist of one or more concentration levels. 
No more than three males and three females should be exposed at each 
concentration level. A range-finding study should last a minimum of 5 days and generally no more than 14 days. The rationale for the selection of concentrations for the main study should be provided in the study report. 
The objective of the main study is to demonstrate a concentration-response 
relationship based on what is anticipated to be the most sensitive endpoint. The low concentration should ideally be a no-observed-adverse effect concentration while the high concentration should elicit unequivocal 
toxicity without causing undue stress to the animals or affecting their 
longevity (3).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 290
 
13. When selecting concentration levels for the range-finding study, all available 
information should be considered including structure-activity relationships 
and data for similar chemicals (see paragraph 3). A range-finding study may verify/refute what are considered to be the most sensitive mechanistically based endpoints, e.g. cholinesterase inhibition by organophosphates, methae ­
moglobin formation by erythrocytotoxic agents, thyroidal hormones (T
 3 , T 4 ) 
for thyrotoxicants, protein, LDH, or neutrophils in brochoalveolar lavage for innocuous poorly soluble particles or pulmonary irritant aerosols.
 
Main Study  
14. The main sub-acute toxicity study generally consists of three concentration 
levels, and also concurrent negative (air) and/or vehicle controls as needed 
(see paragraph 17). All available data should be utilised to aid selection of 
appropriate exposure levels, including the results of systemic toxicity studies, metabolism and kinetics (particular emphasis should be given to avoiding high concentration levels which saturate kinetic processes). Each 
test group contains at least 10 rodents (5 male and 5 female) that are 
exposed to the test chemical for 6 hours per day on a 5 day per week basis for a period of 4 weeks (total study duration of 28 days). Animals may also be exposed 7 days per week (e.g. when testing inhaled phar ­
maceuticals). If one sex is known to be more susceptible to a given test 
chemical, the sexes may be exposed at different concentration levels in order to optimise the concentration-response as described in paragraph 15. If rodent species other than rats are exposed nose-only, maximum exposure 
durations may be adjusted to minimise species-specific distress. A rationale 
should be provided when using an exposure duration less than 6 hours/day, or when it is necessary to conduct a long duration (e.g. 22 hours/day) whole-body exposure study [refer to GD 39 (2)]. Feed should be withheld 
during the exposure period unless exposure exceeds 6 hours. Water may be 
provided throughout a whole-body exposure. 
15. The target concentrations selected should identify the target organ(s) and 
demonstrate a clear concentration-response: 
— The high concentration level should result in toxic effects but not cause 
lingering signs or lethality which would prevent a meaningful evalu ­
ation. 
— The intermediate concentration level(s) should be spaced to produce a 
gradation of toxic effects between that of the low and high concen ­
tration. 
— The low concentration level should produce little or no evidence of 
toxicity.
 
Satellite (Reversibility) Study  
16. A satellite (reversibility) study may be used to observe reversibility, 
persistence, or delayed occurrence of toxicity for a post-treatment period 
of an appropriate length, but no less than 14 days. Satellite (reversibility) 
groups consist of five males and five females exposed contemporaneously with the experimental animals in the main study. Satellite (reversibility) study groups should be exposed to the test chemical at the highest concen ­
tration level and there should be concurrent air and/or vehicle controls as 
needed (see paragraph 17).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 291
 
Control Animals  
17. Concurrent negative (air) control animals should be handled in a manner 
identical to the test group animals except that they are exposed to filtered air rather than test chemical. When water or another substance is used to assist 
in generating the test atmosphere, a vehicle control group, instead of a 
negative (air) control group, should be included in the study. Water should be used as the vehicle whenever possible. When water is used as the vehicle, the control animals should be exposed to air with the same 
relative humidity as the exposed groups. The selection of a suitable vehicle 
should be based on an appropriately conducted pre-study or historical data. If a vehicle’s toxicity is not well known, the study director may choose to 
use both a negative (air) control and a vehicle control, but this is strongly 
discouraged. If historical data reveal that a vehicle is non-toxic, then there is no need for a negative (air) control group and only a vehicle control should be used. If a pre-study of a test chemical formulated in a vehicle reveals no 
toxicity, it follows that the vehicle is non-toxic at the concentration tested 
and this vehicle control should be used.
 
EXPOSURE CONDITIONS  
Administration of Concentrations  
18. Animals are exposed to the test chemical as a gas, vapour, aerosol, or a 
mixture thereof. The physical state to be tested depends on the physico-chemical properties of the test chemical, the selected concentration, 
and/or the physical form most likely present during the handling and use of 
the test chemical. Hygroscopic and chemically reactive test chemicals should be tested under dry air conditions. Care should be taken to avoid generating explosive concentrations. Particulate material may be subjected to mech ­
anical processes to decrease the particle size. Further guidance is provided 
in GD 39 (2).
 
Particle-Size Distribution  
19. Particle sizing should be performed for all aerosols and for vapours that may 
condense to form aerosols. To allow for exposure of all relevant regions of 
the respiratory tract, aerosols with mass median aerodynamic 
diameters (MMAD) ranging from 1 to 3 μm with a geometric standard 
deviation ( σ g ) in the range of 1,5 to 3,0 are recommended (4). Although 
a reasonable effort should be made to meet this standard, expert judgement 
should be provided if it cannot be achieved. For example, metal fume particles may be smaller than this standard, and charged particles and fibres may exceed it.
 
Test chemical Preparation in a Vehicle  
20. Ideally, the test chemical should be tested without a vehicle. If it is 
necessary to use a vehicle to generate an appropriate test chemical concen ­
tration and particle size, water should be given preference. Whenever a test chemical is dissolved in a vehicle, its stability should be demonstrated.
 
MONITORING OF EXPOSURE CONDITIONS  
Chamber Airflow  
21. The flow of air through the exposure chamber should be carefully 
controlled, continuously monitored, and recorded at least hourly during 
each exposure. The real-time monitoring of the test atmosphere concen ­
tration (or temporal stability) is an integral measurement of all dynamic parameters and provides an indirect means to control all relevant dynamic 
inhalation parameters. If the concentration is monitored real-time, the 
frequency of measurement of air flows may be reduced to one single
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 292
 
measurement per exposure per day. Special consideration should be given to 
avoiding re-breathing in nose-only chambers. Oxygen concentration should 
be at least 19 % and carbon dioxide concentration should not exceed 1 %. If there is reason to believe that this standard cannot be met, oxygen and carbon dioxide concentrations should be measured. If measurements on 
the first day of exposure show that these gases are at proper levels, no 
further measurements should be necessary.
 
Chamber Temperature and Relative Humidity  
22. Chamber temperature should be maintained at 22 ± 3 °C. Relative humidity 
in the animals’ breathing zone, for both nose-only and whole-body exposures, should be monitored continuously and recorded hourly during each exposure where possible. The relative humidity should preferably be 
maintained in the range of 30 to 70 %, but this may either be unattainable 
(e.g. when testing water based mixtures) or not measurable due to test chemical interference with the Test Method.
 
Test chemical: Nominal Concentration  
23. Whenever feasible, the nominal exposure chamber concentration should be 
calculated and recorded. The nominal concentration is the mass of generated test chemical divided by the total volume of air passed through the inha ­
lation chamber system. The nominal concentration is not used to char ­
acterise the animals’ exposure, but a comparison of the nominal concen ­
tration and the actual concentration gives an indication of the generation efficiency of the test system, and thus may be used to discover generation 
problems.
 
Test chemical: Actual Concentration  
24. The actual concentration is the test chemical concentration as sampled at the 
animals’ breathing zone in an inhalation chamber. Actual concentrations can be obtained either by specific methods (e.g. direct sampling, adsorptive or chemical reactive methods, and subsequent analytical characterisation) or by 
non-specific methods such as gravimetric filter analysis. The use of gravi ­
metric analysis is acceptable only for single component powder aerosols or aerosols of low volatility liquids and should be supported by appropriate pre-study test chemical-specific characterisations. Multi-component powder 
aerosol concentration may also be determined by gravimetric analysis. 
However, this requires analytical data which demonstrate that the composition of airborne material is similar to the starting material. If this information is not available, a reanalysis of the test chemical (ideally in its 
airborne state) at regular intervals during the course of the study may be 
necessary. For aerosolised agents that may evaporate or sublimate, it should be shown that all phases were collected by the method chosen. 
25. One lot of the test chemical should be used throughout the duration of the 
study, if possible, and the test sample should be stored under conditions that 
maintain its purity, homogeneity, and stability. Prior to the start of the study, 
there should be a characterisation of the test chemical including its purity and, if technically feasible, the identity, and quantities of identified contaminants and impurities. This can be demonstrated but is not limited 
by the following data: retention time and relative peak area, molecular 
weight from mass spectroscopy or gas chromatography analyses, or other estimates. Although the test sample’s identity is not the responsibility of the test laboratory, it may be prudent for the test laboratory to confirm the 
sponsor’s characterisation at least in a limited way (e.g. colour, physical 
nature, etc.).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 293
 
26. The exposure atmosphere should be held as constant as practicable. A 
real-time monitoring device, such as an aerosol photometer for aerosols or 
a total hydrocarbon analyser for vapours may be used to demonstrate the stability of the exposure conditions. Actual chamber concentration should be measured at least 3 times during each exposure day for each exposure level. 
If not feasible due to limited air flow rates or low concentrations, one 
sample per exposure period is acceptable. Ideally, this sample should then be collected over the entire exposure period. Individual chamber concen ­
tration samples should deviate from the mean chamber concentration by no 
more than ± 10 % for gases and vapours, and by no more than ± 20 % for 
liquid or solid aerosols. Time to attain chamber equilibration (t
 95 ) should be 
calculated and reported. The duration of an exposure spans the time that the test chemical is generated. This takes into account the times required to 
attain chamber equilibration (t
 95 ) and decay. Guidance for estimating t  95 
can be found in GD 39 (2). 
27. For very complex mixtures consisting of gases/vapours and aerosols (e.g. 
combustion atmospheres and test chemicals propelled from purpose-driven end-use products/devices), each phase may behave differently in an inha ­
lation chamber. Therefore, at least one indicator substance (analyte), 
normally the principal active substance in the mixture, of each phase (gas/ 
vapour and aerosol) should be selected. When the test chemical is a mixture, the analytical concentration should be reported for the total mixture, and not just for the active ingredient or the indicator substance (analyte). Additional 
information regarding actual concentrations can be found in GD 39 (2).
 
Test chemical: Particle Size Distribution  
28. The particle size distribution of aerosols should be determined at least 
weekly for each concentration level by using a cascade impactor or an 
alternative instrument, such as an aerodynamic particle sizer (APS). If 
equivalence of the results obtained by a cascade impactor and the alternative instrument can be shown, then the alternative instrument may be used throughout the study. 
29. A second device, such as a gravimetric filter or an impinger/gas bubbler, 
should be used in parallel to the primary instrument to confirm the 
collection efficiency of the primary instrument. The mass concentration obtained by particle size analysis should be within reasonable limits of the mass concentration obtained by filter analysis [see GD 39 (2)]. If equiv ­
alence can be demonstrated at all concentrations tested in the early phase of 
the study, then further confirmatory measurements may be omitted. For the sake of animal welfare, measures should be taken to minimise inconclusive data which may lead to a need to repeat a study. 
30. Particle sizing should be performed for vapours if there is any possibility 
that vapour condensation may result in the formation of an aerosol, or if 
particles are detected in a vapour atmosphere with potential for mixed 
phases.
 
OBSERVATIONS 
31. The animals should be clinically observed before, during and after the 
exposure period. More frequent observations may be indicated depending 
on the response of the animals during exposure. When animal observation is hindered by the use of animal restraint tubes, poorly lit whole body chambers, or opaque atmospheres, animals should be carefully observed 
after exposure. Observations before the next day’s exposure can assess 
any reversibility or exacerbation of toxic effects.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 294
 
32. All observations are recorded with individual records being maintained for 
each animal. When animals are killed for humane reasons or found dead, the 
time of death should be recorded as precisely as possible. 
33. Cage-side observations should include changes in the skin and fur, eyes, and 
mucous membranes; changes in the respiratory and circulatory systems, 
changes in the nervous system, and changes in somatomotor activity and 
behaviour patterns. Attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep, and coma. The measurement of rectal temperatures may provide supportive evidence of 
reflex bradypnea or hypo/hyperthermia related to treatment or confinement. 
Additional assessments may be included in the study protocol such as kinetics, biomonitoring, lung function, retention of poorly soluble materials that accumulate in lung tissue, and behavioural changes.
 
BODY WEIGHTS 
34. Individual animal weights should be recorded shortly before the first 
exposure (day 0), twice weekly thereafter (for example: on Fridays and Mondays to demonstrate recovery over an exposure-free weekend or at a time interval to allow assessment of systemic toxicity), and at the time of 
death or euthanasia. If there are no effects in the first 2 weeks, body weights 
may be measured weekly for the remainder of the study. Satellite (reversi ­
bility) animals (if used) should continue to be weighed weekly throughout the recovery period. At study termination, all animals should be weighed 
shortly before sacrifice to allow for an unbiased calculated of organ to body 
weight ratios.
 
FOOD AND WATER CONSUMPTION 
35. Food consumption should be measured weekly. Water consumption may 
also be measured.  
CLINICAL PATHOLOGY 
36. Clinical pathology assessments should be made for all animals, including 
control and satellite (reversibility) animals, when they are sacrificed. The 
time interval between the end of exposure and blood collection should be recorded, particularly when the reconstitution of the addressed endpoint is rapid. Sampling following the end of exposure is indicated for those 
parameters with a short plasma half-time (e.g. COHb, CHE, and MetHb). 
37. Table 1 lists the clinical pathology parameters that are generally required for 
all toxicology studies. Urinalysis is not required on a routine basis, but may be performed when deemed useful based on expected or observed toxicity. 
The study director may choose to assess additional parameters in order to 
better characterise a test chemical’s toxicity (e.g. cholinesterase, lipids, hormones, acid/base balance, methaemoglobin or Heinz bodies, creatine kinase, myeloid/erythroid ratio, troponins, arterial blood gases, lactate dehy ­
drogenase, sorbitol dehydrogenase, glutamate dehydrogenase, and gamma 
glutamyl transpeptidase).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 295
 
Table 1  
Standard Clinical Pathology Parameters  
Haematology  
Erythrocyte count 
Haematocrit 
Haemoglobin concentration Mean corpuscular haemoglobin Mean corpuscular volume 
Mean corpuscular haemoglobin 
concentration 
Reticulocytes Total leukocyte count 
Differential leukocyte count 
Platelet count Clotting potential (select one): — Prothrombin time 
— Clotting time 
— Partial thromboplastin time
 
Clinical Chemistry  
Glucose (*) 
Total cholesterol Triglycerides 
Blood urea nitrogen 
Total bilirubin Creatinine Total protein 
Albumin 
Globulin Alanine aminotransferase 
Aspartate aminotransferase Alkaline phosphatase 
Potassium 
Sodium Calcium Phosphorus 
Chloride
 
Urinalysis (optional)  
Appearance (colour and turbidity) 
Volume 
Specific gravity or osmolality pH Total protein 
Glucose 
Blood/blood cells
 
(*) Because a lengthy fasting period can introduce bias in glucose measurements for 
the treated versus control animals, the study director should determine whether it 
is appropriate to fast the animals. If a fasting period is used, it should be appro ­
priate to the species used; for the rat this may be 16 h (overnight fasting). 
Determination of fasting glucose may be carried out after overnight fasting 
during the last exposure week, or after overnight fasting prior to necropsy (in 
the latter case together with all other clinical pathology parameters).  
38. When there is evidence that the lower respiratory tract (i.e., the alveoli) is 
the primary site of deposition and retention, then bronchoalveolar lavage (BAL) may be the technique of choice to quantitatively analyse hypothesis-based dose-effect parameters focusing on alveolitis, pulmonary 
inflammation, and phospholipidosis. This allows for dose-response and 
time-course changes of alveolar injury to be suitably probed. The BAL fluid may be analysed for total and differential leukocyte counts, total protein, and lactate dehydrogenase. Other parameters that may be considered 
are those indicative of lysosomal injury, phospholipidosis, fibrosis, and 
irritant or allergic inflammation which may include the determination of pro-inflammatory cytokines/chemokines. BAL measurements generally complement the results from histopathology examinations but cannot 
replace them. Guidance on how to perform lung lavage can be found in 
GD 39 (2).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 296
 
GROSS PATHOLOGY AND ORGAN WEIGHTS 
39. All test animals, including those which die during the test or are removed 
from the study for animal welfare reasons, should be subjected to complete exsanguination (if feasible) and gross necropsy. The time between the end 
of each animal’s last exposure and their sacrifice should be recorded. If a 
necropsy cannot be performed immediately after a dead animal is discovered, the animal should be refrigerated (not frozen) at a temperature low enough to minimise autolysis. Necropsies should be performed as soon 
as possible, normally within a day or two. All gross pathological changes 
should be recorded for each animal with particular attention to any changes in the respiratory tract. 
40. Table 2 lists the organs and tissues that should be preserved in a suitable 
medium during gross necropsy for histopathological examination. The pres ­
ervation of the [bracketed] organs and tissues and any other organs and tissues is at the discretion of the study director. The  bolded  organs 
should be trimmed and weighed wet as soon as possible after dissection 
to avoid drying. The thyroid and epididymides should only be weighed if needed because trimming artefacts may hinder histopathological evaluation. Tissues and organs should be fixed in 10 % buffered formalin or another 
suitable fixative as soon as necropsy is performed, and no less than 24- 
48 hours prior to trimming depending on the fixative to be used.
 
Table 2  
Organs and Tissues Preserved During Gross Necropsy 
Adrenals  
Bone marrow (and/or fresh aspirate)  
Brain  (including sections of cerebrum, 
cerebellum, and medulla/pons) 
[Eyes (retina, optic nerve) and eyelids]  
Heart 
Kidneys  
Larynx (3 levels, 1 level to include the base 
of the epiglottis)  
Liver 
Lung  (all lobes at one level, including main 
bronchi) 
Lymph nodes from the hilar region of the 
lung, especially for poorly soluble particulate test chemicals, For more in depth examin ­
ations and/or studies with immunological 
focus, additional lymph nodes may be 
considered, e.g. those from the mediastinal, cervical/submandibular and/or auricular regions. 
Nasopharyngeal tissues (at least 4 levels; 1 
level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue(NALT) 
Oesophagus 
[Olfactory bulb] 
Ovaries Seminal vesicles 
Spinal cord (cervical, mid-thoracic, and 
lumbar)  
Spleen  
Stomach  
Testes Thymus  
Thyroid 
Trachea (at least 2 levels including 1 longi ­
tudinal section through the carina and 1 transverse section) 
[Urinary bladder] 
Uterus All gross lesions
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 297
 
41. The lungs should be removed intact, weighed, and instilled with a suitable 
fixative at a pressure of 20-30 cm of water to ensure that lung structure is 
maintained (5). Sections should be collected for all lobes at one level, including main bronchi, but if lung lavage is performed, the unlavaged lobe should be sectioned at three levels (not serial sections). 
42. At least 4 levels of the nasopharyngeal tissues should be examined, one of 
which should include the nasopharyngeal duct, (5, 6, 7, 8, 9) to allow 
adequate examination of the squamous, transitional (non-ciliated respir ­
atory), respiratory (ciliated respiratory) and olfactory epithelium, and the draining lymphatic tissue (NALT; 10, 11). Three levels of the larynx should be examined, and one of these levels should include the base of 
the epiglottis (12). At least two levels of the trachea should be examined 
including one longitudinal section through the carina of the bifurcation of the extrapulmonary bronchi and one transverse section.
 
HISTOPATHOLOGY 
43. A histopathological evaluation of all the organs and tissues listed in Table 2 
should be performed for the control and high concentration groups, and for 
all animals which die or are sacrificed during the study. Particular attention should be paid to the respiratory tract, target organs, and gross lesions. The organs and tissues that have lesions in the high concentration group should 
be examined in all groups. The study director may choose to perform 
histopathological evaluations for additional groups to demonstrate a clear concentration response. When a satellite (reversibility) group is used, histo ­
pathological evaluation should be performed for all tissues and organs 
identified as showing effects in the treated groups. If there are excessive early deaths or other problems in the high exposure group that compromise the significance of the data, the next lower concentration should be 
examined histopathologically. An attempt should be made to correlate 
gross observations with microscopic findings.
 
DATA AND REPORTING  
Data  
44. Individual animal data on body weights, food consumption, clinical 
pathology, gross pathology, organ weights, and histopathology should be provided. Clinical observation data should be summarised in tabular form 
showing for each test group the number of animals used, the number of 
animals displaying specific signs of toxicity, the number of animals found dead during the test or killed for humane reasons, time of death of indi ­
vidual animals, a description and time course of toxic effects and reversi ­
bility, and necropsy findings. All results, quantitative and incidental, should 
be evaluated by an appropriate statistical method. Any generally accepted statistical method may be used and the statistical methods should be selected during the design of the study.
 
Test Report  
45. The test report should include the following information, as appropriate:  
Test animals and husbandry  
— Description of caging conditions, including: number (or change in 
number) of animals per cage, bedding material, ambient temperature 
and relative humidity, photoperiod, and identification of diet.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 298
 
— Species/strain used and justification for using a species other than the 
rat. Source and historical data may be provided, if they are from animals 
exposed under similar exposure, housing, and fasting conditions. 
— Number, age, and sex of animals. — Method of randomisation. — Description of any pre-test conditioning including diet, quarantine, and 
treatment for disease.  
Test chemical  
— Physical nature, purity, and, where relevant, physico-chemical properties 
(including isomerisation). 
— Identification data and Chemical Abstract Services (CAS) Registry 
Number, if known.  
Vehicle  
— Justification for use of vehicle and justification for choice of vehicle (if 
other than water). 
— Historical or concurrent data demonstrating that the vehicle does not 
interfere with the outcome of the study.  
Inhalation chamber  
— Detailed description of the inhalation chamber including volume and a 
diagram. 
— Source and description of equipment used for the exposure of animals as 
well as generation of the atmosphere. 
— Equipment for measuring temperature, humidity, particle-size, and actual 
concentration. 
— Source of air and system used for conditioning. 
— Methods used for calibration of equipment to ensure a homogeneous test 
atmosphere. 
— Pressure difference (positive or negative). — Exposure ports per chamber (nose-only); location of animals in the 
chamber (whole-body). 
— Stability of the test atmosphere. 
— Location of temperature and humidity sensors and sampling of test 
atmosphere in the chamber. 
— Treatment of air supplied/extracted. — Air flow rates, air flow rate/exposure port (nose-only), or animal load/ 
chamber (whole-body). 
— Time to inhalation chamber equilibrium (t95). 
— Number of volume changes per hour. 
— Metering devices (if applicable).  
Exposure data  
— Rationale for target concentration selection in the main study.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 299
 
— Nominal concentrations (total mass of test chemical generated into the 
inhalation chamber divided by the volume of air passed through the 
chamber). 
— Actual test chemical concentrations collected from the animals’ 
breathing zone; for mixtures that produce heterogeneous physical forms (gases, vapours, aerosols), each may be analysed separately. 
— All air concentrations should be reported in units of mass (mg/l mg/m
 3 , 
etc.) rather than in units of volume (ppm, ppb, etc.). 
— Particle size distribution, mass median aerodynamic diameter (MMAD), 
and geometric standard deviation ( σ g ), including their methods of calcu ­
lation. Individual particle size analyses should be reported.  
Test conditions  
— Details of test chemical preparation, including details of any procedures 
used to reduce the particle size of solids or to prepare solutions of the test chemical. 
— A description (preferably including a diagram) of the equipment used to 
generate the test atmosphere and to expose the animals to the test atmos ­
phere. 
— Details of the equipment used to monitor chamber temperature, 
humidity, and chamber airflow (i.e. development of a calibration curve). 
— Details of the equipment used to collect samples for determination of 
chamber concentration and particle size distribution. 
— Details of the chemical analytical method used and method validation 
(including efficiency of recovery of test chemical from the sampling 
medium). 
— Method of randomisation in assigning animals to test and control groups. — Details of food and water quality (including diet type/source, water 
source). 
— The rationale for the selection of test concentrations.  
Results  
— Tabulation of chamber temperature, humidity, and airflow. — Tabulation of chamber nominal and actual concentration data. — Tabulation of particle size data including analytical sample collection 
data, particle size distribution, and calculations of the MMAD and σ
 g . 
— Tabulation of response data and concentration level for each animal (i.e. 
animals showing signs of toxicity including mortality, nature, severity, 
time of onset, and duration of effects).  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 300
 
— Tabulation of individual animal weights. 
— Tabulation of food consumption 
— Tabulation of clinical pathology data 
— Necropsy findings and histopathological findings for each animal, if 
available. 
— Tabulation of any other parameters measured  
Discussion and interpretation of results  
— Particular emphasis should be made to the description of methods used 
to meet the criteria of this Test Method, e.g. the limit concentration or 
the particle size. 
— The respirability of particles in light of the overall findings should be 
addressed, especially if the particle-size criteria could not be met. 
— The consistency of methods used to determine nominal and actual 
concentrations, and the relation of actual concentration to nominal concentration should be included in the overall assessment of the study. 
— The likely cause of death and predominant mode of action (systemic 
versus local) should be addressed. 
— An explanation should be provided if there was a need to humanely 
sacrifice animals in pain or showing signs of severe and enduring distress, based on the criteria in the OECD Guidance Document on Humane Endpoints (3). 
— The target organ(s) should be identified. 
— The NOAEL and LOAEL should be determined.
 
LITERATURE:  
(1) OECD (1981). Subchronic Inhalation Toxicity Testing, Original Test 
Guideline No 412, Environment Directorate, OECD, Paris. 
(2) OECD (2009). Guidance Document on Acute Inhalation Toxicity Testing, 
Environmental Health and Safety Monograph Series on Testing and 
Assessment No 39, ENV/JM/MONO(2009)28, OECD, Paris. 
(3) OECD (2000). Guidance Document on the Recognition, Assessment and 
Use of Clinical Signs as Humane Endpoints for Experimental Animals 
Used in Safety Evaluation, Environmental Health and Safety Monograph 
Series on Testing and Assessment No 19, ENV/JM/MONO(2000)7, OECD, Paris. 
(4) Whalan JE and Redden JC (1994). Interim Policy for Particle Size and 
Limit Concentration Issues in Inhalation Toxicity Studies. Office of Pesticide Programs, United States Environmental Protection Agency. 
(5) Dungworth DL, Tyler WS, Plopper CE (1985). Morphological Methods for 
Gross and Microscopic Pathology (Chapter 9) in Toxicology of Inhaled Material, Witschi, H.P. and Brain, J.D. (eds), Springer Verlag Heidelberg, pp. 229-258. 
(6) Young JT (1981). Histopathological examination of the rat nasal cavity. 
Fundam. Appl. Toxicol. 1: 309-312. 
(7) Harkema JR (1990). Comparative pathology of the nasal mucosa in 
laboratory animals exposed to inhaled irritants. Environ. Health Perspect. 
85: 231-238.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 301
 
(8) Woutersen RA, Garderen-Hoetmer A, van Slootweg PJ, Feron VJ (1994). 
Upper respiratory tract carcinogenesis in experimental animals and in 
humans. In: Waalkes MP and Ward JM (eds) Carcinogenesis. Target Organ Toxicology Series, Raven Press, New York, 215-263. 
(9) Mery S, Gross EA, Joyner DR, Godo M, Morgan KT (1994). Nasal 
diagrams: A tool for recording the distribution of nasal lesions in rats 
and mice. Toxicol. Pathol. 22: 353-372. 
(10) Kuper CF, Koornstra PJ, Hameleers DMH, Biewenga J, Spit BJ, 
Duijvestijn AM, Breda Vriesman van PJC, Sminia T (1992). The role of nasopharyngeal lymphoid tissue. Immunol. Today 13: 219-224. 
(11) Kuper CF, Arts JHE, Feron VJ (2003). Toxicity to nasal-associated 
lymphoid tissue. Toxicol. Lett. 140-141: 281-285. 
(12) Lewis DJ (1981). Mitotic Indices of Rat Laryngeal Epithelia. Journal of 
Anatomy 132(3): 419-428. 
(13) Regulation (EC) No 1272/2008 of the European Parliament and of the 
Council of 16 December 2008 on classification, labelling and packaging 
of substances and mixtures, amending and repealing Directives 67/548/EEC 
and 1999/45/EC, and amending Regulation (EC) No 1907/2006 (OJ L 353, 31.12.2008, p. 1).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 302
 
Appendix 1  
DEFINITION  
Test chemical:  Any substance or mixture tested using this Test Method.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 303
 
B.9. REPEATED DOSE (28 DAYS) TOXICITY (DERMAL)  
1. METHOD  
1.1. INTRODUCTION 
See General introduction Part B (A).  
1.2. DEFINITIONS 
See General introduction Part B (B).  
1.3. REFERENCE SUBSTANCES 
None.  
1.4. PRINCIPLE OF THE TEST METHOD 
The test substance is applied daily to the skin in graduated doses to 
several groups of experimental animals, one dose per group, for a period of 28 days. During the period of application, the animals are observed daily to detect signs of toxicity. Animals, which die during 
the test, are necropsied and at the conclusion of the test surviving 
animals are necropsied.
 
1.5. QUALITY CRITERIA 
None.  
1.6. DESCRIPTION OF THE TEST METHOD  
1.6.1.  Preparations  
The animals are kept under the experimental housing and feeding 
conditions for at least five days prior to the test. Before the test, healthy young animals are randomised and assigned to the treatment and control groups. Shortly before testing, fur is clipped from the 
dorsal area of the trunk of the test animals. Shaving may be 
employed but it should be carried out approximately 24 hours before the test. Repeat clipping or shaving is usually needed at approximately weekly intervals. When clipping or shaving the fur, 
care must be taken to avoid abrading the skin. Not less than 10 % of 
the body surface area should be clear for the application of the test substance. The weight of the animal should be taken into account when deciding on the area to be cleared and on the dimensions of 
the covering. When testing solids, which may be pulverised if appro ­
priate, the test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle to ensure good contact with 
the skin. Liquid test substances are generally used undiluted. Daily 
application on a five to seven-day per week basis is used.
 
1.6.2.  Test conditions  
1.6.2.1.  Experimental animals  
The adult rat, rabbit or guinea-pig may be used. Other species may 
be used but their use would require justification.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 304
 
At the commencement of the study, the range of weight variation in 
the animals used should not exceed ± 20 % of the appropriate mean 
value.  
1.6.2.2.  Number and sex  
At least 10 animals (five female and five male) with healthy skin 
should be used at each dose level. The females should be nulliparous 
and non-pregnant. If interim sacrifices are planned, the numbers 
should be increased by the number of animals scheduled to be sacrificed before the completion of the study. In addition, a satellite group of 10 animals (five animals per sex) may be treated 
with the high dose level for 28 days and observed for reversibility, 
persistence, or delayed occurrence of toxic effects for 14 days post- treatment. A satellite group of 10 control animals (five animals per sex) is also used.
 
1.6.2.3.  Dose levels  
At least three dose levels are required with a control or a vehicle 
control if a vehicle is used. The exposure period should be at least six hours per day. The application of the test substance should be made at similar times each day, and adjusted at intervals (weekly or 
bi-weekly) to maintain a constant dose level in terms of animal 
body-weight. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the 
test group subjects. Where a vehicle is used to facilitate dosing, the 
vehicle control group should be dosed in the same way as the treated groups, and receive the same amount as that received by the highest dose level group. The highest dose level should result in toxic effects 
but produce no, or few, fatalities. The lowest dose level should not 
produce any evidence or toxicity. Where there is a usable estimation of human exposure, the lowest level should exceed this. Ideally, the intermediate dose level should produce minimal observable toxic 
effects. If more than one intermediate dose is used the dose levels 
should be spaced to produce a gradation of toxic effects. In the low and intermediate groups and in the controls, the incidence of fatalities should be low in order to permit a meaningful evaluation 
of the results. 
If application of the test substance produces severe skin irritation, the 
concentrations should be reduced and this may result in a reduction 
in, or absence of, other toxic effects at the high dose level. Moreover 
if the skin has been badly damaged it may be necessary to terminate the study and undertake a new study at lower concentrations.
 
1.6.2.4.  Limit test  
If a preliminary study at a dose level of 1 000 mg/kg, or a higher 
dose level related to possible human exposure where this is known, 
produces no toxic effects, further testing may not be considered necessary.
 
1.6.2.5.  Observation period  
The experimental animals should be observed daily for signs of 
toxicity. The time of death and the time at which signs of toxicity 
appear and disappear should be recorded.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 305
 
1.6.3.  Procedure  
Animals should be caged individually. The animals are treated with 
the test substance, ideally on seven days per week, for a period of 28 days. Animals in any satellite groups scheduled for follow-up obser ­
vations should be kept for a further 14 days without treatment to 
detect recovery from or persistence of toxic effects. Exposure time should be at least six hours per day. 
The test substance should be applied uniformly over an area, which 
is approximately 10 % of the total body surface area. With highly 
toxic substances, the surface area covered may be less but as much 
of the area as possible should be covered with as thin and uniform a layer as possible. 
During exposure the test substance is held in contact with the skin 
with porous gauze dressing and non-irritating tape. The test site should be further covered in a suitable manner to retain the gauze 
dressing and test substance and ensure that the animals cannot ingest 
the test substance. Restrainers may be used to prevent the ingestion of the test substance but complete immobilisation is not a recom ­
mended method. As an alternative a ‘collar protective device’ may 
be used. 
At the end of the exposure period, residual test substance should be 
removed, where practicable, using water or some other appropriate 
method of cleansing the skin. 
All the animals should be observed daily and signs of toxicity 
recorded including the time of onset, their degree and duration. Observations should include changes in skin and fur, eyes and mucous membranes as well as respiratory, circulatory, autonomic 
and central nervous systems, somatomotor activity and behaviour 
pattern. Measurements should be made weekly of the animals' weight. It is also recommended that food consumption is measured weekly. Regular observation of the animals is necessary to ensure 
that animals are not lost from the study due to causes such as canni ­
balism, autolysis of tissues or misplacement. At the end of the study period, all survivors in the non-satellite treatment groups are 
necropsied. Moribund animals and animals in severe distress or 
pain should be removed when noticed, humanely killed and necropsied. 
The following examinations shall be made at the end of the test on 
all animals including the controls: 
(1) haematology, including at least haematocrit, haemoglobin 
concentration, erythrocyte count, total and differential leucocyte count, and a measure of clotting potential; 
(2) clinical blood biochemistry including at least one parameter of 
liver and kidney function: serum alanine aminotransferase 
(formerly known as glutamic pyruvic transaminase), serum 
aspartate aminotransferase (formerly known as glutamic oxaloacetic transaminase), urea nitrogen, albumin, blood creatinine, total bilirubin and total serum protein; 
Other determinations which may be necessary for an adequate toxi ­
cological evaluation include calcium, phosphorus, chloride, sodium, 
potassium, fasting glucose, analysis of lipids, hormones, acid/base 
balance, methaemoglobin and cholinesterase activity.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 306
 
Additional clinical biochemistry may be employed, where necessary, 
to extend the investigation of observed effects.  
1.6.4.  Gross necropsy  
All animals in the study should be subjected to a full gross necropsy. 
At least the liver, kidneys, adrenals, and testes should be weighed wet as soon as possible after dissection, to avoid drying. Organs and 
tissues, i.e. normal and treated skin, liver, kidney, spleen, testes, 
adrenals, heart, and target organs (that is those organs showing gross lesions or changes in size) should be preserved in a suitable 
medium for possible future histopathological examination.
 
1.6.5.  Histopathological examination  
In the high dose group and in the control group, histological exam ­
ination should be performed on the preserved organs and tissues. Organs and tissues showing defects attributable to the test 
substance at the highest dosage level should be examined in all 
lower-dosage groups. Animals in the satellite group should be examined histologically with particular emphasis on those organs and tissues identified as showing effects in the other treated groups.
 
2. DATA  
Data should be summarised in tabular form, showing for each test 
group the number of animals at the start of the test and the number of animals displaying each type of lesion. 
All observed results should be evaluated by an appropriate statistical 
method. Any recognised statistical method may be used.
 
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, include the following information: 
— animal data (species, strain, source, environmental conditions, 
diet, etc.), 
— test conditions (including the type of dressing: occlusive or not- 
occlusive), 
— dose levels (including vehicle, if used) and concentrations, 
— no-effect level, where possible, 
— toxic response data by sex and dose, 
— time of death during the study or whether animals survived to 
termination, 
— toxic or other effects, — the time of observation of each abnormal sign and its subsequent 
course, 
— food and body-weight data, 
— haematological tests employed and results,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 307
 
— clinical biochemistry tests employed and results, 
— necropsy findings, — a detailed description of all histopathological findings, 
— statistical treatment of results where possible, 
— discussion of the results, — interpretation of the results.
 
3.2. EVALUATION AND INTERPRETATION 
See General introduction Part B (D).  
4. REFERENCES  
See General introduction Part B (E).  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 308
 
B.10.  MUTAGENICITY —  IN VITRO  MAMMALIAN CHRO ­
MOSOME ABERRATION TEST  
1. METHOD  
This method is a replicate of the OECD TG 473,  In Vitro  
Mammalian Chromosome Aberration Test (1997).  
1.1. INTRODUCTION 
The purpose of the  in vitro  chromosomal aberration test is to identify 
agents that cause structural chromosome aberrations in cultured mammalian cells (1)(2)(3). Structural aberrations may be of two 
types, chromosome or chromatid. With the majority of chemical 
mutagens, induced aberrations are of the chromatid type, but chro ­
mosome-type aberrations also occur. An increase in polyploidy may indicate that a chemical has the potential to induce numerical aber ­
rations. However, this method is not designed to measure numerical 
aberrations and is not routinely used for that purpose. Chromosome mutations and related events are the cause of many human genetic diseases and there is substantial evidence that chromosome mutations 
and related events causing alterations in oncogenes and tumour- 
suppressor genes of somatic cells are involved in cancer induction in humans and experimental animals. 
The in vitro  chromosome aberration test may employ cultures of 
established cell lines, cell strains or primary cell cultures. The cells used are selected on the basis of growth ability in culture, stability of the karyotype, chromosome number, chromosome diversity and 
spontaneous frequency of chromosome aberrations. 
Tests conducted  in vitro  generally require the use of an exogenous 
source of metabolic activation. This metabolic activation system cannot mimic entirely the mammalian  in vivo  conditions. Care 
should be taken to avoid conditions which would lead to positive 
results which do not reflect intrinsic mutagenicity and may arise from changes in pH, osmolality or high levels of cytotoxicity (4)(5). 
This test is used to screen for possible mammalian mutagens and 
carcinogens. Many compounds that are positive in this test are mammalian carcinogens; however, there is not a perfect correlation between this test and carcinogenicity. Correlation is dependent on 
chemical class and there is increasing evidence that there are carci ­
nogens that are not detected by this test because they appear to act through mechanisms other than direct DNA damage. 
See also General introduction Part B.
 
1.2. DEFINITIONS  
Chromatid-type aberration:  structural chromosome damage 
expressed as breakage of single chromatids or breakage and reunion between chromatids.  
Chromosome-type aberration:  structural chromosome damage 
expressed as breakage, or breakage and reunion, of both chromatids 
at an identical site.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 309
 
Endoreduplication:  a process in which after an S period of DNA 
replication, the nucleus does not go into mitosis but starts another S 
period. The result is chromosomes with four, eight, 16, …chromatids.  
Gap:  an achromatic lesion smaller than the width of one chromatid, 
and with minimum misalignment of the chromatids).  
Mitotic index:  the ratio of cells in metaphase divided by the total 
number of cells observed in a population of cells; an indication of 
the degree of proliferation of that population.  
Numerical aberration:  a change in the number of chromosomes 
from the normal number characteristic of the cells utilised.  
Polyploidy:  a multiple of the haploid chromosome number ( n) other 
than the diploid number (i.e. 3 n, 4n and so on).  
Structural aberration:  a change in chromosome structure detectable 
by microscopic examination of the metaphase stage of cell division, observed as deletions and fragments, intrachanges or interchanges.
 
1.3. PRINCIPLE OF THE TEST METHOD 
Cell cultures are exposed to the test substance both with and without 
metabolic activation. At predetermined intervals after exposure of 
cell cultures to the test substance, they are treated with a meta ­
phase-arresting substance (e.g. Colcemid® or colchicine), harvested, stained and metaphase cells are analysed microscopically for the presence of chromosome aberrations.
 
1.4. DESCRIPTION OF THE TEST METHOD  
1.4.1.  Preparations  
1.4.1.1.  Cells  
A variety of cell lines, strains or primary cell cultures, including 
human cells, may be used (e.g. Chinese hamster fibroblasts, human or other mammalian peripheral blood lymphocytes).
 
1.4.1.2.  Media and culture conditions  
Appropriate culture media and incubation conditions (culture vessels, 
CO 2 concentration, temperature and humidity) should be used in 
maintaining cultures. Established cell lines and strains should be 
checked routinely for stability in the modal chromosome number 
and the absence of mycoplasma contamination and should not be used if contaminated. The normal cell cycle time for the cells and culture conditions used should be known.
 
1.4.1.3.  Preparation of cultures  
Established cell lines and strains: cells are propagated from stock 
cultures, seeded in culture medium at a density such that the cultures 
will not reach confluency before the time of harvest, and incubated 
at 37  o C. ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 310
 
Lymphocytes: whole blood treated with an anti-coagulant (e.g. 
heparin) or separated lymphocytes obtained from healthy subjects 
are added to the culture medium containing a mitogen (e.g. phyto ­
haemagglutinin) and incubated at 37  o C. 
1.4.1.4.  Metabolic activation  
Cells should be exposed to the test substance both in the presence 
and absence of an appropriate metabolic activation system. The most 
commonly used system is a cofactor-supplemented post-mito ­
chondrial fraction (S9) prepared from the livers of rodents treated with enzyme-inducing agents such as: Aroclor 1254 (6)(7)(8)(9), or a mixture of phenobarbitone and ß–naphthoflavone (10)(11)(12). 
The post-mitochondrial fraction is usually used at concentrations in 
the range from 1-10 % v/v in the final test medium. The condition of a metabolic activation system may depend upon the class of 
chemical being tested. In some cases it may be appropriate to 
utilise more than one concentration of post-mitochondrial fraction. 
A number of developments, including the construction of genetically 
engineered cell lines expressing specific activating enzymes, may 
provide the potential for endogenous activation. The choice of the 
cell lines used should be scientifically justified (e.g. by the relevance of the cytochrome P450 isoenzyme for the metabolism of the test substance).
 
1.4.1.5.  Test substance/Preparation  
Solid test substances should be dissolved or suspended in appropriate 
solvents or vehicles and diluted if appropriate prior to treatment of the cells. Liquid test substances may be added directly to the test systems and/or diluted prior to treatment. Fresh preparations of the 
test substance should be employed unless stability data demonstrate 
the acceptability of storage.
 
1.4.2.  Test conditions  
1.4.2.1.  Solvent/vehicle  
The solvent/vehicle should not be suspected of chemical reaction 
with the test substance and should be compatible with the survival 
of the cells and the S9 activity. If other than well-known solvent/ ­
vehicles are used, their inclusion should be supported by data indi ­
cating their compatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle be considered first. When 
testing water-unstable substances, the organic solvents used should 
be free of water. Water can be removed by adding a molecular sieve.
 
1.4.2.2.  Exposure concentrations  
Among the criteria to be considered when determining the highest 
concentration are cytotoxicity, solubility in the test system and 
changes in pH or osmolality. 
Cytotoxicity should be determined with and without metabolic 
activation in the main experiment using an appropriate indication of cell integrity and growth, such as degree of confluency, viable 
cell counts, or mitotic index. It may be useful to determine cytot ­
oxicity and solubility in a preliminary experiment.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 311
 
At least three analysable concentrations should be used. Where cytot ­
oxicity occurs, these concentrations should cover a range from the 
maximum to little or no toxicity; this will usually mean that the concentrations should be separated by no more than a factor between 2 and √10. At the time of harvesting, the highest concen ­
tration should show a significant reduction in degree of confluency, 
cell count or mitotic index, (all greater than 50 %). The mitotic index is only an indirect measure of cytotoxic/cytostatic effects and depends on the time after treatment. However, the mitotic index is 
acceptable for suspension cultures in which other toxicity 
measurements may be cumbersome and impractical. Information on cell cycle kinetics, such as average generation time (AGT), could be used as supplementary information. AGT, however, is an overall 
average that does not always reveal the existence of delayed 
subpopulations, and even slight increases in average generation time can be associated with very substantial delay in the time of optimal yield of aberrations. 
For relatively non-cytotoxic substances, the maximum test concen ­
tration should be 5 μl/ml, 5 mg/ml or 0,01 M, whichever is the 
lowest. 
For relatively insoluble substances that are not toxic at concen ­
trations lower than the insoluble concentration, the highest dose 
used should be a concentration above the limit of solubility in the 
final culture medium at the end of the treatment period. In some cases (e.g. when toxicity occurs only at higher than the lowest insoluble concentration) it is advisable to test at more than one 
concentration with visible precipitation. It may be useful to assess 
solubility at the beginning and the end of the treatment, as solubility can change during the course of exposure in the test system due to 
presence of cells, S9, serum etc. Insolubility can be detected by 
using the unaided eye. The precipitate should not interfere with the scoring.
 
1.4.2.3.  Negative and positive controls  
Concurrent positive and negative (solvent or vehicle) controls, both 
with and without metabolic activation, should be included in each 
experiment. When metabolic activation is used, the positive control 
chemical should be the one that requires activation to give a mutagenic response. 
Positive controls should employ a known clastogen at exposure 
levels expected to give a reproducible and detectable increase over 
background, which demonstrates the sensitivity of the test system. 
Positive control concentrations should be chosen so that the effects 
are clear but do not immediately reveal the identity of the coded slides to the reader. Examples of positive control substances include:
 
Metabolic Activation condition Substance CAS No EINECS No  
Absence of exogenous 
metabolic Activation Methyl methanesulphonate 66-27-3 200-625-0 
Ethyl methanesulphonate 62-50-0 200-536-7  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 312
 
Metabolic Activation condition Substance CAS No EINECS No  
Ethyl nitrosourea 759-73-9 212-072-2 
Mitomycin C 50-07-7 200-008-6 
4-Nitroquinoline- N-oxide 56-57-5 200-281-1 
Presence of exogenous 
metabolic Activation Benzo[ a]pyrene 50-32-8 200-028-5 
Cyclophosphamide 
Cyclophosphamide 
monohydrate 50-18-0 
6055-19-2 200-015-4 
Other appropriate positive control substances may be used. The use 
of chemical class-related positive control chemicals should be considered, when available. 
Negative controls, consisting of solvent or vehicle alone in the 
treatment medium, and treated in the same way as the treatment cultures, should be included for every harvest time. In addition, 
untreated controls should also be used unless there are historical 
control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent.
 
1.4.3.  Procedure  
1.4.3.1.  Treatment with the test substance  
Proliferating cells are treated with the test substance in the presence 
and absence of a metabolic activation system. Treatment of lymp ­
hocytes should commence at about 48 hours after mitogenic stimu ­
lation. 
1.4.3.2. Duplicate cultures should normally be used at each concentration, 
and are strongly recommended for negative/solvent control cultures. Where minimal variation between duplicate cultures can be demon ­
strated (13)(14), from historical data, it may be acceptable for single 
cultures to be used at each concentration. 
Gaseous or volatile substances should be tested by appropriate 
methods, such as in sealed culture vessels (15)(16).
 
1.4.3.3.  Culture harvest time  
In the first experiment, cells should be exposed to the test substance, 
both with and without metabolic activation, for three to six hours, 
and sampled at a time equivalent to about 1,5 normal cell cycle 
length after the beginning of treatment (12). If this protocol gives negative results both with and without activation, an additional experiment without activation should be done, with continuous 
treatment until sampling at a time equivalent to about 1,5 normal 
cell cycle lengths. Certain chemicals may be more readily detected by treatment/sampling times longer than 1,5 cycle lengths. Negative results with metabolic activation need to be confirmed on a case-by- 
case basis. In those cases where confirmation of negative results is 
not considered necessary, justification should be provided.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 313
 
1.4.3.4.  Chromosome preparation  
Cell cultures are treated with Colcemid® or colchicine usually for 
one to three hours prior to harvesting. Each cell culture is harvested and processed separately for the preparation of chromosomes. Chro ­
mosome preparation involves hypotonic treatment of the cells, 
fixation and staining.
 
1.4.3.5.  Analysis  
All slides, including those of positive and negative controls, should 
be independently coded before microscopic analysis. Since fixation procedures often result in the breakage of a proportion of metaphase 
cells with loss of chromosomes, the cells scored should therefore 
contain a number of centromeres equal to the modal number ± 2 for all cell types. At least 200 well spread metaphases should be scored per concentration and control, equally divided amongst the 
duplicates, if applicable. This number can be reduced when high 
number of aberrations is observed. 
Though the purpose of the test is to detect structural chromosome 
aberrations, it is important to record polyploidy and endoredupli ­
cation when these events are seen.
 
2. DATA  
2.1. TREATMENT OF RESULTS 
The experimental unit is the cell, and therefore the percentage of 
cells with structural chromosome aberration(s) should be evaluated. 
Different types of structural chromosome aberrations should be listed 
with their numbers and frequencies for experimental and control cultures. Gaps are recorded separately and reported but generally not included in the total aberration frequency. 
Concurrent measures of cytotoxicity for all treated and negative 
control cultures in the main aberration experiments should also be 
recorded. 
Individual culture data should be provided. Additionally, all data 
should be summarised in tabular form. 
There is no requirement for verification of a clear positive response. 
Equivocal results should be clarified by further testing preferably using modification of experimental conditions. The need to 
confirm negative results has been discussed in 
1.4.3.3. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments. Study parameters that might be modified include the concentration 
spacing and the metabolic activation conditions.
 
2.2. EVALUATION AND INTERPRETATION OF RESULTS 
There are several criteria for determining a positive result, such as a 
concentration-related increase or a reproducible increase in the 
number of cells with chromosome aberrations. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results (3)(13). Statistical 
significance should not be the only determining factor for a 
positive response.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 314
 
An increase in the number of polyploid cells may indicate that the 
test substance has the potential to inhibit mitotic processes and to 
induce numerical chromosome aberrations. An increase in the number of cells with endoreduplicated chromosomes may indicate that the test substance has the potential to inhibit cell cycle 
progression (17)(18). 
A test substance for which the results do not meet the above criteria 
is considered non-mutagenic in this system. 
Although most experiments will give clearly positive or negative 
results, in rare cases the data set will preclude making a definite 
judgement about the activity of the test substance. Results may 
remain equivocal or questionable regardless of the number of times the experiment is repeated. 
Positive results from the  in vitro  chromosome aberration test indicate 
that the test substance induces structural chromosome aberrations in cultured mammalian somatic cells. Negative results indicate that, 
under the test conditions, the test substance does not induce chro ­
mosome aberrations in cultured mammalian somatic cells.
 
3. REPORTING  
TEST REPORT 
The test report must include the following information: 
Solvent/Vehicle: — justification for choice of vehicle, 
— solubility and stability of the test substance in solvent/vehicle, if 
known. 
Cells: 
— type and source of cells, — karyotype features and suitability of the cell type used, — absence of mycoplasma, if applicable, 
— information on cell cycle length, 
— sex of blood donors, whole blood or separated lymphocytes, 
mitogen used, 
— number of passages, if applicable, — methods for maintenance of cell culture, if applicable, 
— modal number of chromosomes. 
Test conditions: — identity of metaphase arresting substance, its concentration and 
duration of cell exposure, 
— rationale for selection of concentrations and number of cultures 
including, e.g. cytotoxicity data and solubility limitations, if available, 
— composition of media, CO
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— concentration of test substance, 
— volume of vehicle and test substance added, 
— incubation temperature, — incubation time, — duration of treatment, 
— cell density at seeding, if appropriate, 
— type and composition of metabolic activation system, including 
acceptability criteria, 
— positive and negative controls, 
— methods of slide preparation, 
— criteria for scoring aberrations, — number of metaphases analysed, 
— methods for the measurements of toxicity, 
— criteria for considering studies as positive, negative or equivocal. Results: — signs of toxicity, e.g. degree of confluency, cell cycle data, cell 
counts, mitotic index, 
— signs of precipitation, — data on pH and osmolality of the treatment medium, if deter ­
mined, 
— definition for aberrations, including gaps, — number of cells with chromosome aberrations and type of chro ­
mosome aberrations given separately for each treated and control 
culture, 
— changes in ploidy if seen, — dose-response relationship, where possible, — statistical analyses, if any, 
— concurrent negative (solvent/vehicle) and positive control data, 
— historical negative (solvent/vehicle) and positive control data, 
with ranges, means and standard deviations. 
Discussion of results. 
Conclusions.
 
4. REFERENCES  
(1) Evans, H. J., (1976) Cytological Methods for Detecting 
Chemical Mutagens. In: Chemical mutagens, Principles and Methods for their Detection, Vol. 4, Hollaender, A. (ed) 
Plenum Press, New York and London, p. 1-29. 
(2) Ishidate, M. Jr. and Sofuni, T., (1985). The  In Vitro  Chro­
mosomal Aberration Test Using Chinese Hamster Lung (CHL) Fibroblast Cells in Culture. In: Progress in Mutation 
Research, Vol. 5, Ashby, J. et al., (Eds) Elsevier Science 
Publishers, Amsterdam-New York-Oxford, p. 427-432.
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(3) Galloway, S.M., Armstrong, M.J., Reuben, C., Colman, S., 
Brown, B., Cannon, C., Bloom, A.D., Nakamura, F., Ahmed, 
M., Duk, S., Rimpo, J., Margolin, G.H., Resnick, MA, Anderson, G. and Zeiger, E., (1978). Chromosome aberration and sister chromatic exchanges in Chinese hamster ovary cells: 
Evaluation of 108 chemicals. Environs. Molec. Mutagen 10 
(suppl. 10), p. 1-175. 
(4) Scott, D., Galloway, S.M., Marshall, R.R., Ishidate, M.Jr., 
Brusick, D., Ashby, J. and Myhr, B.C., (1991) Genotoxicity under Extreme Culture Conditions. A report from ICPEMC Task Group 9. Mutation Res, 257, p. 147-204. 
(5) Morita, T., Nagaki, T., Fukuda, I. and Okumura, K., (1992). 
Clastogenicity of low pH to Various Cultured Mammalian 
Cells. Mutation Res., 268, p. 297-305. 
(6) Ames, B.N., McCann, J. and Yamasaki, E., (1975). Methods 
for Detecting Carcinogens and Mutagens with the Salmonella/ ­
Mammalian Microsome Mutagenicity Test. Mutation Res., 31, p. 347-364. 
(7) Maron, D.M. and Ames, B.N., (1983) Revised Methods for the 
Salmonella Mutagenicity Test. Mutation Res., 113, p. 173-215. 
(8) Natarajan, A.T., Tates, A.D., van Buul, P.P.W., Meijers, M. 
and de Vogel, N., (1976) Cytogenetic Effects of Mutagen/ ­
Carcinogens after Activation in a Microsomal System in Vitro, I. Induction of Chromosome Aberrations and Sister Chromatid Exchange by Diethylnitrosamine (DEN) and 
Dimethylnitrosamine (DMN) in CHO Cells in the Presence 
of Rat-Liver Microsomes. Mutation Res., 37, p. 83-90. 
(9) Matsuoka, A., Hayashi, M. and Ishidate, M. Jr., (1979) Chro ­
mosomal Aberration Tests on 29 Chemicals Combined with S9 Mix In Vitro . Mutation Res., 66, p. 277-290. 
(10) Elliot, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., 
Gibson, G.G., Mackay, J.M. and Wolf, R.C., (1992). Report of 
UK Environmental Mutagen Society Working Party. Alter ­
natives to Aroclor 1254-induced S9 in  In Vitro  Genotoxicity 
Assays. Mutagenesis, 7, p. 175-177. 
(11) Matsushima, T., Sawamura, M., Hara, K. and Sugimura, T., 
(1976) A Safe Substitute for Polychlorinated Biphenyls as an Inducer of Metabolic Activation Systems. In: de Serres, F.J., 
Fouts, J.R., Bend, J.R. and Philpot, R.M. (eds)  In Vitro  
Metabolic Activation in Mutagenesis Testing, Elsevier, North-Holland, p. 85-88. 
(12) Galloway, S.M., Aardema, M.J., Ishidate, M.Jr., Ivett, J.L., 
Kirkland, D.J., Morita, T., Mosesso, P., Sofuni, T., (1994) 
Report from Working Group on in  In Vitro  Tests for Chro ­
mosomal Aberrations. Mutation Res., 312, p. 241-261. 
(13) Richardson, C., Williams, D.A., Allen, J.A., Amphlett, G., 
Chanter, D.O. and Phillips, B., (1989) Analysis of Data from  
In Vitro  Cytogenetic Assays. In: Statistical Evaluation of Muta ­
genicity Test Data. Kirkland, D.J., (ed) Cambridge University 
Press, Cambridge, p. 141-154. 
(14) Soper, K.A. and Galloway, S.M., (1994) Replicate Flasks are 
not Necessary for  In Vitro  Chromosome Aberration Assays in 
CHO Cells. Mutation Res., 312, p. 139-149. 
(15) Krahn, D.F., Barsky, F.C. and McCooey, K.T., (1982) 
CHO/HGPRT Mutation Assay: Evaluation of Gases and Volatile Liquids. In: Tice, R.R., Costa, D.L., Schaich, K.M. 
(eds). Genotoxic Effects of Airborne Agents. New York, 
Plenum, p. 91-103.
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(16) Zamora, P.O., Benson, J.M., Li, A.P. and Brooks, A.L., (1983) 
Evaluation of an Exposure System Using Cells Grown on 
Collagen Gels for Detecting Highly Volatile Mutagens in the CHO/HGPRT Mutation Assay. Environmental Mutagenesis, 5, p. 795-801. 
(17) Locke-Huhle, C., (1983) Endoreduplication in Chinese hamster 
cells during alpha-radiation induced G2 arrest. Mutation Res., 
119, p. 403-413. 
(18) Huang, Y., Change, C. and Trosko, J.E., (1983) Aphidicolin- 
induced endoreduplication in Chinese hamster cells. Cancer Res., 43, p. 1362-1364.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 318
 
B.11.  MUTAGENICITY —  IN VIVO  MAMMALIAN BONE MARROW 
CHROMOSOME ABERRATION TEST  
1. METHOD  
This method is a replicate of the OECD TG 475, Mammalian Bone 
Marrow Chromosome Aberration Test (1997).  
1.1. INTRODUCTION 
The mammalian  in vivo  chromosome aberration test is used for the 
detection of structural chromosome aberrations induced by the test substance to the bone marrow cells of animals, usually rodents (1) ­
(2)(3)(4). Structural chromosome aberrations may be of two types, 
chromosome or chromatid. An increase in polyploidy may indicate that a chemical has the potential to induce numerical aberrations. With the majority of chemical mutagens, induced aberrations are 
of the chromatid-type, but chromosome-type aberrations also occur. 
Chromosome mutations and related events are the cause of many human genetic diseases and there is substantial evidence that chro ­
mosome mutations and related events causing alterations in 
oncogenes and tumour-suppressor genes are involved in cancer in 
humans and experimental systems. 
Rodents are routinely used in this test. Bone marrow is the target 
tissue in this test, since it is a highly vascularised tissue, and it 
contains a population of rapidly cycling cells that can be readily 
isolated and processed. Other species and target tissues are not the subject of this method. 
This chromosome aberration test is especially relevant to assessing 
mutagenic hazard in that it allows consideration of factors of  in vivo  
metabolism, pharmacokinetics and DNA-repair processes although 
these may vary among species and among tissues. An  in vivo  test 
is also useful for further investigation of a mutagenic effect detected 
by in vitro  test. 
If there is evidence that the test substance, or a reactive metabolite, 
will not reach the target tissue, it is not appropriate to use this test. 
See also General introduction Part B.
 
1.2. DEFINITIONS  
Chromatid-type aberration:  structural chromosome damage 
expressed as breakage of single chromatids or breakage and 
reunion between chromatids.  
Chromosome-type aberration:  structural chromosome damage 
expressed as breakage, or breakage and reunion, of both chromatids at an identical site.  
Endoreduplication:  a process in which after an S period of DNA 
replication, the nucleus does not go into mitosis but starts another S period. The result is chromosomes with four, eight, 16, ...chromatids.  
Gap:  an achromatic lesion smaller than the width of one chromatid, 
and with minimum misalignment of the chromatid(s).  
Numerical aberration:  a change in the number of chromosomes 
from the normal number characteristic of the cells utilised.
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Polyploidy:  a multiple of the haploid chromosome number ( n) other 
than the diploid number (i.e. 3 n, 4n and so on).  
Structural aberration:  a change in chromosome structure detectable 
by microscopic examination of the metaphase stage of cell division, observed as deletions and fragments, intrachanges or interchanges.
 
1.3. PRINCIPLE OF THE TEST METHOD 
Animals are exposed to the test substance by an appropriate route of 
exposure and are sacrificed at appropriate times after treatment. Prior 
to sacrifice, animals are treated with a metaphase arresting agent (e.g. Colcemid® or colchicine). Chromosome preparations are then made from the bone marrow cells and stained, and metaphase cells 
are analysed for chromosome aberrations.
 
1.4. DESCRIPTION OF THE TEST METHOD  
1.4.1.  Preparations  
1.4.1.1.  Selection of animal species  
Rats, mice and Chinese hamsters are commonly used, although any 
appropriate mammalian species may be used. Commonly used 
laboratory strains of young healthy adult animals should be 
employed. At the commencement of the study, the weight variation of animals should be minimal and not exceed ± 20 % of 
the mean weight of each sex.
 
1.4.1.2.  Housing and feeding conditions  
General conditions referred in the General introduction to Part B are 
applied although the aim for humidity should be 50-60 %.  
1.4.1.3.  Preparation of the animals  
Healthy young adult animals are randomly assigned to the control 
and treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimised. The animals 
are identified uniquely. The animals are acclimated to the laboratory 
conditions for at least five days.
 
1.4.1.4.  Preparation of doses  
Solid test substances should be dissolved or suspended in appropriate 
solvents or vehicles and diluted, if appropriate, prior to dosing of the 
animals. Liquid test substances may be dosed directly or diluted 
prior to dosing. Fresh preparations of the test substance should be employed unless stability data demonstrate the acceptability of storage.
 
1.4.2.  Test conditions  
1.4.2.1.  Solvent/Vehicle  
The solvent/vehicle should not produce toxic effects at the dose 
levels used, and should not be suspected of chemical reaction with the test substance. If other than well-known solvents/vehicles are used, their inclusion should be supported with data indicating their 
compatibility. It is recommended that wherever possible, the use of 
an aqueous solvent/vehicle should be considered first.
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1.4.2.2.  Controls  
Concurrent positive and negative (solvent/vehicle) controls should be 
included for each sex in each test. Except for treatment with the test substance, animals in the control groups should be handled in an 
identical manner to the animals in the treated groups. 
Positive controls should produce structural aberrations  in vivo  at 
exposure levels expected to give a detectable increase over back ­
ground. Positive control doses should be chosen so that the effects are clear but do not immediately reveal the identity of the coded 
slides to the reader. It is acceptable that the positive control be 
administered by a route different from the test substance and sampled at only a single time. The use of chemical class related 
positive control chemicals may be considered, when available. 
Examples of positive control substances include:
 
Substance CAS No EINECS No  
Ethyl methanesulphonate 62-50-0 200-536-7 
Ethyl nitrosourea 759-73-9 212-072-2 Mitomycin C 50-07-7 200-008-6 Cyclophosphamide 
Cyclophosphamide monohydrate 50-18-0 
6055-19-2 200-015-4 
Triethylenemelamine 51-18-3 200-083-5 
Negative controls, treated with solvent or vehicle alone, and 
otherwise treated in the same way as the treatment groups, should be included for every sampling time, unless acceptable inter-animal 
variability and frequencies of cells with chromosome aberrations are 
available from historical control data. If single sampling is applied for negative controls, the most appropriate time is the first sampling time. In addition, untreated controls should also be used unless there 
are historical or published control data demonstrating that no 
deleterious or mutagenic effects are induced by the chosen solvent/ ­
vehicle.
 
1.5. PROCEDURE  
1.5.1.  Number and sex of animals  
Each treated and control group must include at least five analysable 
animals per sex. If at the time of the study there are data available 
from studies in the same species and using the same route of exposure that demonstrate that there are no substantial differences in toxicity between sexes, then testing in a single sex will be suffi ­
cient. Where human exposure to chemicals may be sex-specific, as 
for example with some pharmaceutical agents, the test should be performed with animals of the appropriate sex.
 
1.5.2.  Treatment schedule  
Test substances are preferably administered as a single treatment. 
Test substances may also be administered as a split dose, i.e. two treatments on the same day separated by no more than a few hours, 
to facilitate administering a large volume of material. Other dose 
regimens should be scientifically justified.
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Samples should be taken at two separate times following treatment 
on one day. For rodents, the first sampling interval is 1,5 normal cell 
cycle length (the latter being normally 12-18 hr) following treatment. Since the time required for uptake and metabolism of the test substance as well as its effect on cell cycle kinetics can affect the 
optimum time for chromosome aberration detection, a later sample 
collection 24 hr after the first sample time is recommended. If dose regimens of more than one day are used, one sampling time at 1,5 normal cell cycle lengths after the final treatment should be used. 
Prior to sacrifice, animals are injected intraperitoneally with an 
appropriate dose of a metaphase arresting agent (e.g. Colcemid® or colchicine). Animals are sampled at an appropriate interval there ­
after. For mice this interval is approximately three to five hours; for 
Chinese hamsters this interval is approximately four to five hours. 
Cells are harvested from the bone marrow and analysed for chro ­
mosome aberrations.
 
1.5.3.  Dose levels  
If a range finding study is performed because there are no suitable 
data available, it should be performed in the same laboratory, using 
the same species, strain, sex and treatment regimen to be used in the main study (5). If there is toxicity, three dose levels are used for the first sampling time. These dose levels should cover a range from the 
maximum to little or no toxicity. At the later sampling time only the 
highest dose needs to be used. The highest dose is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality. 
Substances with specific biological activities at low non-toxic doses 
(such as hormones and mitogens) may be exceptions to the dose- setting criteria and should be evaluated on a case-by-case basis. The highest dose may also be defined as a dose that produces some 
indication of toxicity in the bone marrow (e.g. greater than 50 % 
reduction in mitotic index).
 
1.5.4.  Limit test  
If a test at one dose level of at least 2 000 mg/kg body weight using 
a single treatment, or as two treatments on the same day, produces 
no observable toxic effects, and if genotoxicity would not be 
expected based on data from structurally related substances, then a full study using three dose levels may not be considered necessary. For studies of a longer duration, the limit dose is 2 000 mg/kg/body 
weight/day for treatment up to 14 days, and 1 000 mg/kg/body 
weight/day for treatment longer than 14 days. Expected human exposure may indicate the need for a higher dose level to be used in the limit test.
 
1.5.5.  Administration of doses  
The test substance is usually administered by gavage using a 
stomach tube or a suitable intubation cannula, or by intraperitoneal 
injection. Other routes of exposure may be acceptable where they can be justified. The maximum volume of liquid that can be admin ­
istered by gavage or injection at one time depends on the size of the 
test animal. The volume should not exceed 2 ml/100 g body weight. 
The use of volumes higher than these must be justified. Except for irritating or corrosive substances which will normally reveal exacerbated effects with higher concentrations, variability in test 
volume should be minimised by adjusting the concentration to 
ensure a constant volume at all dose levels.
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1.5.6.  Chromosome preparation  
Immediately after sacrifice, bone marrow is obtained, exposed to 
hypotonic solution and fixed. The cells are then spread on slides and stained.
 
1.5.7.  Analysis  
The mitotic index should be determined as a measure of cytotoxicity 
in at least 1 000 cells per animal for all treated animals (including 
positive controls) and untreated negative control animals. 
At least 100 cells should be analysed for each animal. This number 
could be reduced when high numbers of aberrations are observed. All slides, including those of positive and negative controls, should 
be independently coded before microscopic analysis. Since slide 
preparation procedures often result in the breakage of a proportion of metaphases with loss of chromosomes, the cells scored should therefore contain a number of centromeres equal to the number 2n ± 2.
 
2. DATA  
2.1. TREATMENT OF RESULTS 
Individual animal data should be presented in tabular form. The 
experimental unit is the animal. For each animal the number of cells scored, the number of aberrations per cell and the percentage of cells with structural chromosome aberration(s) should be 
evaluated. Different types of structural chromosome aberrations 
should be listed with their numbers and frequencies for treated and control groups. Gaps are recorded separately and reported but generally not included in the total aberration frequency. If there is 
no evidence for a difference in response between the sexes, the data 
from both sexes may be combined for statistical analysis.
 
2.2. EVALUATION AND INTERPRETATION OF RESULTS 
There are several criteria for determining a positive result, such as a 
dose-related increase in the relative number of cells with chromosome aberrations or a clear increase in the number of cells with aberrations in 
a single dose group at a single sampling time. Biological relevance of 
the results should be considered first. Statistical methods may be used as an aid in evaluating the test results (6). Statistical significance should not be the only determining factor for a positive response. Equivocal 
results should be clarified by further testing preferably using a 
modification of experimental conditions. 
An increase in polyploidy may indicate that the test substance has 
the potential to induce numerical chromosome aberrations. An increase in endoreduplication may indicate that the test substance 
has the potential to inhibit cell cycle progression (7)(8). 
A test substance for which the results do not meet the above criteria 
is considered non-mutagenic in this test.
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Although most experiments will give clearly positive or negative 
results, in rare cases the data set will preclude making a definite 
judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of experiments performed. 
Positive results from the  in vivo  chromosome aberration test indicate 
that a substance induces chromosome aberrations in the bone marrow 
of the species tested. Negative results indicate that, under the test conditions, the test substance does not induce chromosome aber ­
rations in the bone marrow of the species tested. 
The likelihood that the test substance or its metabolites reach the 
general circulation or specifically the target tissue (e.g. systemic 
toxicity) should be discussed.
 
3. REPORTING  
3.1. TEST REPORT 
The test report must include the following information: 
Solvent/Vehicle: — justification for choice of vehicle, — solubility and stability of the test substance in solvent/vehicle, if 
known, 
Test animals: — species/strain used, — number, age and sex of animals, — source, housing conditions, diet, etc., — individual weight of the animals at the start of the test, including 
body weight range, mean and standard deviation for each group, 
Test conditions: — positive and negative (vehicle/solvent) controls, — data from range-finding study, if conducted, — rationale for dose level selection, — details of test substance preparation, — details of the administration of the test substance, — rationale for route of administration, — methods for verifying that the test substance reached the general 
circulation or target tissue, if applicable, 
— conversion from diet/drinking water test substance concentration 
(ppm) to the actual dose (mg/kg body weight/day), if applicable, 
— details of food and water quality, — detailed description of treatment and sampling schedules, — methods for measurements of toxicity,
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— identity of metaphase arresting substance, its concentration and 
duration of treatment, 
— methods of slide preparation, — criteria for scoring aberrations, — number of cells analysed per animal, 
— criteria for considering studies as positive, negative or equivocal. 
Results: — signs of toxicity, — mitotic index, 
— type and number of aberrations, given separately for each animal, 
— total number of aberrations per group with means and standard 
deviations, 
— number of cells with aberrations per group with means and 
standard deviations, 
— changes in ploidy, if seen, — dose-response relationship, where possible, 
— statistical analyses, if any, 
— concurrent negative control data, — historical negative control data with ranges, means and standard 
deviations, 
— concurrent positive control data. 
Discussion of the results. Conclusions.
 
4. REFERENCES  
(1) Adler, I.D., (1984) Cytogenetic Tests in Mammals. In: Mutage ­
nicity Testing: a Practical Approach. S. Venitt and J.M. Parry 
(Eds). IRL Press, Oxford, Washington D.C., p. 275-306. 
(2) Preston, R.J., Dean, B.J., Galloway, S., Holden, H., McFee, 
A.F. and Shelby, M. (1987). Mammalian  In Vivo  Cytogenetic 
Assays: Analysis of Chromosome Aberrations in Bone Marrow Cells. Mutation Res., 189, p. 157-165. 
(3) Richold, M., Chandley, A., Ashby, J., Gatehouse, D.G., 
Bootman, J. and Henderson, L., (1990)  In Vivo  Cytogenetic 
Assays. In: D.J. Kirkland (Ed.) Basic Mutagenicity Tests, UKEMS Recommended Procedures. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Report. Part I revised. 
Cambridge University Press, Cambridge, New York, Port 
Chester, Melbourne, Sydney, p. 115-141. 
(4) Tice, R.R., Hayashi, M., MacGregor, J.T., Anderson, D., 
Blakey, D.H., Holden, H.E., Kirsch-Volders, M., Oleson Jr., 
F.B., Pacchierotti, F., Preston, R.J., Romagna, F., Shimada, 
H., Sutou, S. and Vannier, B., (1994) Report from the Working Group on the  In Vivo  Mammalian Bone Marrow Chro ­
mosomal Aberration Test. Mutation Res., 312, p. 305-312. 
(5) Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, J., 
Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B., Kirkland, D.J. and Richold, M. (1992). Report of British Toxicology Society/UK Environmental Mutagen 
Society Working Group: Dose setting in  In vivo  Mutagenicity 
Assays. Mutagenesis, 7, p. 313-319.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 325
 
(6) Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., 
Clare, G., Ferguson, R., Richold, M., Papworth, D.G. and 
Savage, J.R.K. (1989). Statistical Analysis of  In Vivo  Cyto­
genetic Assays. In: UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Report Part III. Statistical Evaluation of 
Mutagenicity Test Data. D.J. Kirkland, (Ed.) Cambridge 
University Press, Cambridge. p. 184-232. 
(7) Locke-Huhle, C., (1983) Endoreduplication in Chinese hamster 
cells during alpha-radiation induced G2 arrest. Mutation Res. 119, p. 403-413. 
(8) Huang, Y., Change, C. and Trosko, J.E., (1983) Aphidicolin- 
induced endoreduplication in Chinese hamster cells. Cancer 
Res., 43, p. 1362-1364.
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B.12.  MUTAGENICITY —  IN VIVO  MAMMALIAN ERYTHROCYTE 
MICRONUCLEUS TEST  
1. METHOD  
This method is a replicate of the OECD TG 474, Mammalian 
Erythrocyte Micronucleus Test (1997).  
1.1. INTRODUCTION 
The mammalian  in vivo  micronucleus test is used for the detection of 
damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts by analysis of erythrocytes as 
sampled in bone marrow and/or peripheral blood cells of animals, 
usually rodents. 
The purpose of the micronucleus test is to identify substances that 
cause cytogenetic damage, which results in the formation of micro ­
nuclei containing lagging chromosome fragments or whole chromo ­
somes. 
When a bone marrow erythroblast develops into a polychromatic 
erythrocyte, the main nucleus is extruded; any micronucleus that 
has been formed may remain behind in the otherwise anucleated 
cytoplasm. Visualisation of micronuclei is facilitated in these cells because they lack a main nucleus. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an 
indication of induced chromosome damage. 
The bone marrow of rodents is routinely used in this test since 
polychromatic erythrocytes are produced in that tissue. The measurement of micronucleated immature (polychromatic) 
erythrocytes in peripheral blood is equally acceptable in any 
species in which the inability of the spleen to remove micronucleated erythrocytes has been demonstrated, or which has shown an adequate sensitivity to detect agents that cause structural or numerical chro ­
mosome aberrations. Micronuclei can be distinguished by a number 
of criteria. These include identification of the presence or absence of a kinetochore or centromeric DNA in the micronuclei. The frequency of micronucleated immature (polychromatic) erythrocytes is the 
principal endpoint. The number of mature (normochromatic) 
erythrocytes in the peripheral blood that contain micronuclei among a given number of mature erythrocytes can also be used as the endpoint of the assay when animals are treated continuously for 
four weeks or more. 
This mammalian  in vivo  micronucleus test is especially relevant to 
assessing mutagenic hazard in that it allows consideration of factors of in vivo  metabolism, pharmacokinetics and DNA-repair processes 
although these may vary among species, among tissues and among 
genetic endpoints. An  in vivo  assay is also useful for further inves ­
tigation of a mutagenic effect detected by an  in vitro  system. 
If there is evidence that the test substance, or a reactive metabolite, 
will not reach the target tissue, it is not appropriate to use this test. 
See also General introduction Part B.
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1.2. DEFINITIONS  
Centromere (Kinetochore):  region(s) of a chromosome with which 
spindle fibers are associated during cell division, allowing orderly movement of daughter chromosomes to the poles of the daughter 
cells.  
Micronuclei:  small nuclei, separate from and additional to the main 
nuclei of cells, produced during telophase of mitosis (meiosis) by 
lagging chromosome fragments or whole chromosomes.  
Normochromatic erythrocyte:  mature erythrocyte that lacks 
ribosomes and can be distinguished from immature, polychromatic 
erythrocytes by stains selective for ribosomes.  
Polychromatic erythrocyte:  immature erythrocyte, in an inter ­
mediate stage of development, that still contains ribosomes and therefore can be distinguished from mature, normochromatic erythrocytes by stains selective for ribosomes.
 
1.3. PRINCIPLE OF THE TEST METHOD 
Animals are exposed to the test substance by an appropriate route. If 
bone marrow is used, the animals are sacrificed at appropriate times after treatment, the bone marrow extracted, and preparations made and stained (1)(2)(3)(4)(5)(6)(7). When peripheral blood is used, the 
blood is collected at appropriate times after treatment and smear 
preparations are made and stained (4)(8)(9)(10). For studies with peripheral blood, as little time as possible should elapse between 
the last exposure and cell harvest. Preparations are analysed for 
the presence of micronuclei.
 
1.4. DESCRIPTION OF THE TEST METHOD  
1.4.1.  Preparations  
1.4.1.1.  Selection of animal species  
Mice or rats are recommended if bone marrow is used, although any 
appropriate mammalian species may be used. When peripheral blood is used, mice are recommended. However, any appropriate mammalian species may be used provided it is a species in which 
the spleen does not remove micronucleated erythrocytes or a species, 
which has shown an adequate sensitivity to detect agents that cause structural or numerical chromosome aberrations. Commonly used 
laboratory strains of young healthy animals should be employed. 
At the commencement of the study, the weight variation of animals should be minimal and not exceed ±20 % of the mean weight of each sex.
 
1.4.1.2.  Housing and feeding conditions  
General conditions referred in the General introduction to Part B are 
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1.4.1.3.  Preparation of the animals  
Healthy young adult animals are randomly assigned to the control 
and treatment groups. The animals are identified uniquely. The animals are acclimated to the laboratory conditions for at least five 
days. Cages should be arranged in such a way that possible effects 
due to cage placement are minimised.
 
1.4.1.4.  Preparation of doses  
Solid test substances should be dissolved or suspended in appropriate 
solvents or vehicles and diluted, if appropriate, prior to dosing of the animals. Liquid test substances may be dosed directly or diluted prior to dosing. Fresh preparations of the test substance should be 
employed unless stability data demonstrate the acceptability of 
storage.
 
1.4.2.  Test conditions  
1.4.2.1.  Solvent/Vehicle  
The solvent/vehicle should not produce toxic effects at the dose 
levels used, and should not be suspected of chemical reaction with the test substance. If other than well-known solvents/vehicles are used, their inclusion should be supported with reference data indi ­
cating their compatibility. It is recommended that wherever possible, 
the use of an aqueous solvent/vehicle should be considered first.
 
1.4.2.2.  Controls  
Concurrent positive and negative (solvent/vehicle) controls should be 
included for each sex in each test. Except for treatment with the test 
substance, animals in the control groups should be handled in an identical manner to animals of the treatment groups. 
Positive controls should produce micronuclei  in vivo  at exposure 
levels expected to give a detectable increase over background. Positive control doses should be chosen so that the effects are 
clear but do not immediately reveal the identity of the coded 
slides to the reader. It is acceptable that the positive control be administered by a route different from the test substance and sampled at only a single time. In addition, the use of chemical 
class-related positive control chemicals may be considered, when 
available. Examples of positive control substances include:
 
Substance CAS No EINECS No  
Ethyl methanesulphonate 62-50-0 200-536-7 
N-ethyl-N-nitrosourea 759-73-9 212-072-2 Mitomycin C 50-07-7 200-008-6 Cyclophosphamide 
Cyclophosphamide monohydrate 50-18-0 
6055-19-2 200-015-4 
Triethylenemelamine 51-18-3 200-083-5
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 329
 
Negative controls, treated with solvent or vehicle alone, and 
otherwise treated in the same way as the treatment groups should 
be included for every sampling time, unless acceptable inter-animal variability and frequencies of cells with micronuclei are demon ­
strated by historical control data. If single sampling is applied for 
negative controls, the most appropriate time is the first sampling 
time. In addition, untreated controls should also be used unless there are historical or published control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent/ ­
vehicle. 
If peripheral blood is used, a pre-treatment sample may also be 
acceptable as a concurrent negative control, but only in the short 
peripheral blood studies (e.g. 1-3 treatment(s)) when the resulting data are in the expected range for the historical control.
 
1.5. PROCEDURE  
1.5.1.  Number and sex of animals  
Each treated and control group must include at least five analysable 
animals per sex (11). If at the time of the study there are data 
available from studies in the same species and using the same route of exposure that demonstrate that there are no substantial differences between sexes in toxicity, then testing in a single sex 
will be sufficient. Where human exposure to chemicals may be sex- 
specific, as for example with some pharmaceutical agents, the test should be performed with animals of the appropriate sex.
 
1.5.2.  Treatment schedule  
No standard treatment schedule (i.e. one, two or more treatments at 
24 h intervals) can be recommended. The samples from extended dose regimens are acceptable as long as a positive effect has been 
demonstrated for this study or, for a negative study, as long as 
toxicity has been demonstrated or the limit dose has been used, and dosing continued until the time of sampling. Test substances may also be administered as a split dose, i.e., two treatments on 
the same day separated by no more than a few hours, to facilitate 
administering a large volume of material. 
The test may be performed in two ways: 
(a) animals are treated with the test substance once. Samples of bone 
marrow are taken at least twice, starting not earlier than 24 hours after treatment, but not extending beyond 48 hours after treatment with appropriate intervals between samples. The use 
of sampling times earlier than 24 hours after treatment should be 
justified. Samples of peripheral blood are taken at least twice, starting not earlier than 36 hours after treatment, with appropriate intervals following the first sample, but not extending beyond 
72 hours. When a positive response is recognised at one 
sampling time, additional sampling is not required.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 330
 
(b) if two or more daily treatments are used (e.g. two or more 
treatments at 24 hour intervals), samples should be collected 
once between 18 and 24 hours following the final treatment for the bone marrow and once between 36 and 48 hours following the final treatment for the peripheral blood (12); 
Other sampling times may be used in addition, when relevant.
 
1.5.3.  Dose levels  
If a range finding study is performed because there are no suitable 
data available, it should be performed in the same laboratory, using 
the same species, strain, sex, and treatment regimen to be used in the main study (13). If there is toxicity, three dose levels are used for the first sampling time. These dose levels should cover a range from the 
maximum to little or no toxicity. At the later sampling time only the 
highest dose needs to be used. The highest dose is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality. 
Substances with specific biological activities at low non-toxic doses 
(such as hormones and mitogens) may be exceptions to the dose- setting criteria and should be evaluated on a case-by-case basis. The highest dose may also be defined as a dose that produces some 
indication of toxicity in the bone marrow (e.g. a reduction in the 
proportion of immature erythrocytes among total erythrocytes in the bone marrow or peripheral blood).
 
1.5.4.  Limit test  
If a test at one dose level of at least 2 000 mg/kg body weight using 
a single treatment, or as two treatments on the same day, produces 
no observable toxic effects, and if genotoxicity would not be 
expected based upon data from structurally related substances, then a full study using three dose levels may not be considered necessary. For studies of a longer duration, the limit dose is 2 000 mg/kg/body 
weight/day for treatment up to 14 days, and 1 000 mg/kg/body 
weight/day for treatment longer than 14 days. Expected human exposure may indicate the need for a higher dose level to be used in the limit test.
 
1.5.5.  Administration of doses  
The test substance is usually administered by gavage using a 
stomach tube or a suitable intubation cannula, or by intraperitoneal 
injection. Other routes of exposure may be acceptable where they can be justified. The maximum volume of liquid that can be admin ­
istered by gavage or injection at one time depends on the size of the 
test animal. The volume should not exceed 2 ml/100 g body weight. 
The use of volumes higher than these must be justified. Except for irritating or corrosive substances, which will normally reveal exacerbated effects with higher concentrations, variability in test 
volume should be minimised by adjusting the concentration to 
ensure a constant volume at all dose levels.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 331
 
1.5.6.  Bone marrow/blood preparation  
Bone marrow cells are usually obtained from the femurs or tibias 
immediately following sacrifice. Commonly, cells are removed from femurs or tibias, prepared and stained using established methods. 
Peripheral blood is obtained from the tail vein or other appropriate 
blood vessel. Blood cells are immediately stained supravitally (8) ­
(9)(10) or smear preparations are made and then stained. The use of a DNA specific stain (e.g. acridine orange (14) or Hoechst 33258 
plus pyronin-Y (15)) can eliminate some of the artifacts associated 
with using a non DNA specific stain. This advantage does not preclude the use of conventional stains (e.g. Giemsa). Additional 
systems (e.g. cellulose columns to remove nucleated cells (16)) 
can also be used provided that these systems have been shown to adequately work for micronucleus preparation in the laboratory.
 
1.5.7.  Analysis  
The proportion of immature among total (immature + mature) 
erythrocytes is determined for each animal by counting a total of 
at least 200 erythrocytes for bone marrow and 1 000 erythrocytes for 
peripheral blood (17). All slides, including those of positive and negative controls, should be independently coded before microscopic analysis. At least 2 000 immature erythrocytes per animal are scored 
for the incidence of micronucleated immature erythrocytes. 
Additional information may be obtained by scoring mature erythrocytes for micronuclei. When analysing slides, the proportion of immature erythrocytes among total erythrocytes should not be less 
than 20 % of the control value. When animals are treated 
continuously for four weeks or more, at least 2 000 mature erythrocytes per animal can also be scored for the incidence of micronuclei. Systems for automated analysis (image analysis and 
cell suspensions flow cytometry) are acceptable alternatives to 
manual evaluation if appropriately justified and validated.
 
2. DATA  
2.1. TREATMENT OF RESULTS 
Individual animal data should be presented in tabular form. The 
experimental unit is the animal. The number of immature 
erythrocytes scored, the number of micronucleated immature erythro ­
cytes, and the number of immature among total erythrocytes should be listed separately for each animal analysed. When animals are treated continuously for four weeks or more, the data on mature 
erythrocytes should also be given if it is collected. The proportion 
of immature among total erythrocytes and, if considered applicable, the percentage of micronucleated erythrocytes is given for each animal. If there is no evidence for a difference in response 
between the sexes, the data from both sexes may be combined for 
statistical analysis.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 332
 
2.2. EVALUATION AND INTERPRETATION OF RESULTS 
There are several criteria for determining a positive result, such as a 
dose-related increase in the number of micronucleated cells or a clear increase in the number of micronucleated cells in a single dose group 
at a single sampling time. Biological relevance of the results should 
be considered first. Statistical methods may be used as an aid in evaluating the test results (18)(19). Statistical significance should not be the only determining factor for a positive response. 
Equivocal results should be clarified by further testing preferably 
using a modification of experimental conditions. 
A test substance for which the results do not meet the above criteria 
is considered non-mutagenic in this test. 
Although most experiments will give clearly positive or negative 
results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of 
times the experiment is repeated. 
Positive results in the micronucleus test indicate that the substance 
induces micronuclei which are the result of chromosomal damage or 
damage to the mitotic apparatus in the erythroblasts of the test species. Negative results indicate that, under the test conditions, the test substance does not produce micronuclei in the immature 
erythrocytes of the test species. 
The likelihood that the test substance or its metabolites reach the 
general circulation or specifically the target tissue (e.g. systemic 
toxicity) should be discussed.
 
3. REPORTING  
TEST REPORT 
The test report should include the following information: 
Solvent/Vehicle: 
— justification for choice of vehicle, 
— solubility and stability of the test substance in solvent/vehicle, if 
known. 
Test animals: — species/strain used, 
— number, age and sex of animals, 
— source, housing conditions, diet, etc., — individual weight of the animals at the start of the test, including 
body weight range, mean and standard deviation for each group. 
Test conditions: 
— positive and negative (vehicle/solvent) control data, 
— data from range-finding study, if conducted,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 333
 
— rationale for dose level selection, 
— details of test substance preparation, 
— details of the administration of the test substance, — rationale for route of administration, — methods for verifying that the test substance reached the general 
circulation or target tissue, if applicable, 
— conversion from diet/drinking water test substance concentration 
(ppm) to the actual dose (mg/kg body weight/day), if applicable, 
— details of food and water quality, 
— detailed description of treatment and sampling schedules, 
— methods of slide preparation, — methods for measurements of toxicity, 
— criteria for scoring micronucleated immature erythrocytes, 
— number of cells analysed per animal, — criteria for considering studies as positive, negative or equivocal. Results: 
— signs of toxicity, 
— proportion of immature erythrocytes among total erythrocytes, — number of micronucleated immature erythrocytes, given 
separately for each animal, 
— mean ± standard deviation of micronucleated immature 
erythrocytes per group, 
— dose-response relationship, where possible, 
— statistical analyses and methods applied, 
— concurrent and historical negative control data, — concurrent positive control data. — Discussion of the results. 
— Conclusions.
 
4. REFERENCES  
(1) Heddle, J.A., (1973) A Rapid  In Vivo  Test for Chromosomal 
Damage, Mutation Res., 18, p. 187-190. 
(2) Schmid, W., (1975) The Micronucleus Test, Mutation Res., 31, 
p. 9-15. 
(3) Heddle, J.A., Salamone, M.F., Hite, M., Kirkhart, B., 
Mavournin, K., MacGregor, J.G. and Newell, G.W. (1983). The Induction of Micronuclei as a Measure of Genotoxicity. 
Mutation Res., 123, p. 61-118. 
(4) Mavournin, K.H., Blakey, D.H., Cimino, M.C., Salamone, 
M.F. and Heddle, J.A., (1990) The  In Vivo  Micronucleus 
Assay in Mammalian Bone Marrow and Peripheral Blood. A 
report of the U.S. Environmental Protection Agency Gene-Tox 
Program, Mutation Res., 239, p. 29-80.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 334
 
(5) MacGregor, J.T., Schlegel, R. Choy, W.N., and Wehr, C.M., 
(1983) Micronuclei in Circulating Erythrocytes: A Rapid 
Screen for Chromosomal Damage During Routine Toxicity Testing in Mice. Iin: ‘Developments in Science and Practice of Toxicology’, Ed. A.W. Hayes, R.C. Schnell and T.S. Miya, 
Elsevier, Amsterdam, p. 555-558. 
(6) MacGregor, J.T., Heddle, J.A. Hite, M., Margolin, G.H., 
Ramel, C., Salamone, M.F., Tice, R.R., and Wild, D., (1987) 
Guidelines for the Conduct of Micronucleus Assays in Mammalian Bone Marrow Erytrocytes. Mutation Res., 189, p. 103-112. 
(7) MacGregor, J.T., Wehr, C.M., Henika, P.R., and Shelby, M.E., 
(1990) The  in vivo  Erythrocyte Micronucleus Test: 
Measurement at Steady State Increases Assay Efficiency and 
Permits Integration with Toxicity Studies. Fund. Appl. Toxicol., 14, p. 513-522. 
(8) Hayashi, M., Morita, T., Kodama, Y., Sofuni, T. and Ishidate, 
M. Jr., (1990) The Micronucleus Assay with Mouse Peripheral Blood Reticulocytes Using Acridine Orange-Coated Slides. 
Mutation Res., 245, p. 245-249. 
(9) The Collaborative Study Group for the Micronucleus Test, 
(1992) Micronucleus Test with Mouse Peripheral Blood 
Erythrocytes by Acridine Orange Supravital Staining: The Summary Report of the 5th Collaborative Study by CSGMT/ ­
JEMMS. MMS. Mutation Res., p. 278, 83-98. 
(10) The Collaborative Study Group for the Micronucleus Test 
(CSGMT/JEMMS. MMS: The Mammalian Mutagenesis 
Study Group of the Environmental Mutagen Society of 
Japan), (1995) Protocol recommended for the short-term mouse peripheral blood micronucleus test. Mutagenesis, 10, p. 153-159. 
(11) Hayashi, M., Tice, R.R., MacGregor, J.T., Anderson, D., 
Blackey, D.H., Kirsch-Volders, M., Oleson, Jr. F. B., Pacchie ­
rotti, F., Romagna, F., Shimada, H., Sutou, S. and Vannier, B., 
(1994)  In Vivo  Rodent Erythrocyte Micronucleus Assay. 
Mutation Res., 312, p. 293-304. 
(12) Higashikuni, N. and Sutou, S., (1995) An optimal, generalised 
sampling time of 30 ± 6 h after double dosing in the mouse peripheral blood micronucleus test. Mutagenesis, 10, 
p. 313-319. 
(13) Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, J., 
Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, 
D.B., Kirkland, D.J. and Rochold, M., (1992) Report of British Toxicology Society/UK Environmental Mutagen Society Working Group: Dose Setting in  In Vivo  Mutagenicity 
Assays. Mutagenesis, 7, p. 313-319. 
(14) Hayashi, M., Sofuni, T. and Ishidate, M. Jr., (1983) An Appli ­
cation of Acridine Orange Fluorescent Staining to the Micro ­
nucleus Test. Mutation Res., 120, p. 241-247. 
(15) MacGregor, J.T., Wehr, C.M. and Langlois, R.G., (1983) A 
Simple Fluorescent Staining Procedure for Micronuclei and RNA in Erythrocytes Using Hoechst 33258 and Pyronin Y. Mutation Res., 120, p. 269-275. 
(16) Romagna, F. and Staniforth, C.D., (1989) The automated bone 
marrow micronucleus test. Mutation Res., 213, p. 91-104. 
(17) Gollapudi, B. and McFadden, L.G., (1995) Sample size for the 
estimation of polychromatic to normochromatic erythrocyte 
ratio in the bone marrow micronucleus test. Mutation Res., 
347, p. 97-99.
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(18) Richold, M., Ashby, J., Bootman, J., Chandley, A., Gatehouse, 
D.G. and Henderson, L., (1990)  In Vivo  Cytogenetics Assay. 
In: D.J. Kirkland (Ed.) Basic Mutagenicity tests. UKEMS Recommended Procedures. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Report, Part I, revised. 
Cambridge University Press, Cambridge, New York, Port 
Chester, Melbourne, Sydney, p. 115-141. 
(19) Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., 
Clare, G., Ferguson, R., Richold, M., Papworth, D.G. and Savage, J.R.K., (1989) Statistical Analysis of  In Vivo  Cyto­
genetic Assays. In: D.J. Kirkland (Ed.) Statistical Evaluation 
of Mutagenicity Test Data. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Report, Part III. Cambridge University Press, Cambridge, New York, Port 
Chester, Melbourne, Sydney, p. 184-232.
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B.13/14.  MUTAGENICITY: REVERSE MUTATION TEST USING 
BACTERIA  
1. METHOD  
This method is a replicate of the OECD TG 471, Bacterial Reverse 
Mutation Test (1997).  
1.1. INTRODUCTION 
The bacterial reverse mutation test uses amino-acid requiring strains 
of Salmonella typhimurium  and  Escherichia coli  to detect point 
mutations, which involve substitution, addition or deletion of one or a few DNA base pairs (1)(2)(3). The principle of this bacterial reverse mutation test is that it detects mutations, which revert 
mutations present in the test strains and restore the functional capa ­
bility of the bacteria to synthesise an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino-acid required by the parent test strain. 
Point mutations are the cause of many human genetic diseases and 
there is substantial evidence that point mutations in oncogenes and 
tumour-suppressor genes of somatic cells are involved in tumour 
formation in humans and experimental animals. The bacterial reverse mutation test is rapid, inexpensive and relatively easy to perform. Many of the test strains have several features that make 
them more sensitive for the detection of mutations including 
responsive DNA sequences at the reversion sites, increased cell permeability to large molecules and elimination of DNA repair systems or enhancement of error-prone DNA repair processes. The 
specificity of the test strains can provide some useful information on 
the types of mutations that are induced by genotoxic agents. A very large data base of results for a wide variety of structures is available for bacterial reverse mutation tests and well-established method ­
ologies have been developed for testing chemicals with different 
physico-chemical properties, including volatile compounds. 
See also General introduction Part B.
 
1.2. DEFINITIONS  
A reverse mutation test  in either  Salmonella typhimurium  or 
Escherichia coli  detects mutation in an amino-acid requiring strain 
(histidine or tryptophan, respectively) to produce a strain inde ­
pendent of an outside supply of amino-acid.  
Base pair substitution mutagens  are agents that cause a base 
change in DNA. In a reversion test this change may occur at the site of the original mutation, or at a second site in the bacterial 
genome.  
Frameshift mutagens  are agents that cause the addition or deletion 
of one or more base pairs in the DNA, thus changing the reading 
frame in the RNA.
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1.3. INITIAL CONSIDERATIONS 
The bacterial reverse mutation test utilises prokaryotic cells, which 
differ from mammalian cells in such factors as uptake, metabolism, chromosome structure and DNA repair processes. Tests conducted  in 
vitro  generally require the use of an exogenous source of metabolic 
activation.  In vitro  metabolic activation systems cannot mimic 
entirely the mammalian  in vivo  conditions. The test therefore does 
not provide direct information on the mutagenic and carcinogenic 
potency of a substance in mammals. 
The bacterial reverse mutation test is commonly employed as an 
initial screen for genotoxic activity and, in particular, for point mutation-inducing activity. An extensive database has demonstrated that many chemicals that are positive in this test also exhibit 
mutagenic activity in other tests. There are examples of mutagenic 
agents, which are not detected by this test; reasons for these short ­
comings can be ascribed to the specific nature of the endpoint detected, differences in metabolic activation, or differences in bioa ­
vailability. On the other hand, factors, which enhance the sensitivity 
of the bacterial reverse mutation test can lead to an overestimation of mutagenic activity. 
The bacterial reverse mutation test may not be appropriate for the 
evaluation of certain classes of chemicals, for example highly bacte ­
ricidal compounds (e.g. certain antibiotics) and those which are 
thought (or known) to interfere specifically with the mammalian 
cell replication system (e.g. some topoisomerase inhibitors and some nucleoside analogues). In such cases, mammalian mutation tests may be more appropriate. 
Although many compounds that are positive in this test are 
mammalian carcinogens, the correlation is not absolute. It is 
dependent on chemical class and there are carcinogens that are not 
detected by this test because they act through other, non-genotoxic, mechanisms or mechanisms absent in bacterial cells.
 
1.4. PRINCIPLE OF THE TEST METHOD 
Suspensions of bacterial cells are exposed to the test substance in the 
presence and in the absence of an exogenous metabolic activation system. In the plate incorporation method, these suspensions are 
mixed with an overlay agar and plated immediately onto minimal 
medium. In the preincubation method, the treatment mixture is incubated and then mixed with an overlay agar before plating onto minimal medium. For both techniques, after two or three days of 
incubation, revertant colonies are counted and compared to the 
number of spontaneous revertant colonies on solvent control plates. 
Several procedures for performing the bacterial reverse mutation 
test have been described. Among those commonly used are the plate incorporation method (1)(2)(3)(4), the preincubation method (2)(3)(5)(6)(7)(8), the fluctuation method (9)(10), and the 
suspension method (11). Modifications for the testing of gases or 
vapours have been described (12).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 338
 
The procedures described in the method pertain primarily to the plate 
incorporation and preincubation methods. Either of them is 
acceptable for conducting experiments both with and without metabolic activation. Some substances may be detected more effi ­
ciently using the preincubation method. These substances belong to 
chemical classes that include short chain aliphatic nitrosamines, 
divalent metals, aldehydes, azo-dyes and diazo compounds, pyrol ­
lizidine alkaloids, allyl compounds and nitro compounds (3). It is also recognised that certain classes of mutagens are not always 
detected using standard procedures such as the plate incorporation 
method or preincubation method. These should be regarded as ‘special cases’ and it is strongly recommended that alternative procedures should be used for their detection. The following 
‘special cases’ could be identified (together with examples of 
procedures that could be used for their detection): azo-dyes and diazo compounds (3)(5)(6)(13), gases and volatile chemicals (12) ­
(14)(15)(16) and glycosides (17)(18). A deviation from the standard 
procedure needs to be scientifically justified.
 
1.5. DESCRIPTION OF THE TEST METHOD  
1.5.1.  Preparations  
1.5.1.1.  Bacteria  
Fresh cultures of bacteria should be grown up to the late exponential 
or early stationary phase of growth (approximately 10  9 cells per ml). 
Cultures in late stationary phase should not be used. It is essential 
that the cultures used in the experiment contain a high titre of viable 
bacteria. The titre may be demonstrated either from historical control data on growth curves, or in each assay through the determination of viable cell numbers by a plating experiment. 
The recommended incubation temperature is 37
 o C. 
At least five strains of bacteria should be used. These should include 
four strains of  S. typhimurium  (TA 1535; TA 1537 or TA97a or TA97; 
TA98; and TA100) that have been shown to be reliable and repro ­
ducibly responsive between laboratories. These four  S. typhimurium  
strains have GC base pairs at the primary reversion site and it is known 
that may not detect certain oxidising mutagens, cross-linking agents 
and hydrazines. Such substances may be detected by  E. coli  WP2 
strains or  S. typhimurium  TA102 (19), which have an AT base pair 
at the primary reversion site. Therefore the recommended combination 
of strains is: 
— S. typhimurium  TA1535, and 
— S. typhimurium  TA1537 or TA97 or TA97a, and 
— S. typhimurium  TA98, and 
— S. typhimurium  TA100, and 
— E. coli  WP2 uvrA, or  E. coli  WP2 uvrA (pKM101), or  S. typhi­
murium  TA102. 
In order to detect cross-linking mutagens it may be preferable to 
include TA102 or to add a DNA repair-proficient strain of  E. coli  
[e.g. E. coli  WP2 or  E. coli  WP2 (pKM101)]  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 339
 
Established procedures for stock culture preparation, marker verifi ­
cation and storage should be used. The amino-acid requirement for 
growth should be demonstrated for each frozen stock culture prep ­
aration (histidine for  S. typhimurium  strains, and tryptophan for  E. 
coli strains). Other phenotypic characteristics should be similarly 
checked, namely: the presence or absence of R-factor plasmids 
where appropriate [i.e. ampicillin resistance in strains TA98, TA100 and TA97a or TA97, WP2 uvrA and WP2 uvrA (pKM101), and ampicillin + tetracycline resistance in strain 
TA102]; the presence of characteristic mutations (i.e. rfa mutation 
in S. typhimurium  through sensitivity to crystal violet, and uvrA 
mutation in  E. coli  or uvrB mutation in  S. typhimurium,  through 
sensitivity to ultraviolet light) (2)(3). The strains should also yield 
spontaneous revertant colony plate counts within the frequency 
ranges expected from the laboratory's historical control data and preferably within the range reported in the literature.
 
1.5.1.2.  Medium  
An appropriate minimal agar (e.g. containing Vogel-Bonner minimal 
medium E and glucose), and an overlay agar containing histidine and biotin or tryptophan to allow for a few cell divisions, is used (1) ­
(2)(9).
 
1.5.1.3.  Metabolic activation  
Bacteria should be exposed to the test substance both in the presence 
and absence of an appropriate metabolic activation system. The most 
commonly used system is a cofactor-supplemented post-mito ­
chondrial fraction (S9) prepared from the livers of rodents treated with enzyme-inducing agents such as Aroclor 1254 (1)(2) or a 
combination of Phenobarbitone and ß-naphthoflavone (18)(20)(21). 
The post-mitochondrial fraction is usually used at concentrations in the range from 5 to 30 % v/v in the S9-mix. The choice and condition of a metabolic activation system may depend upon the 
class of chemical being tested. In some cases, it may be appropriate 
to utilise more than one concentration of post-mitochondrial fraction. For azo-dyes and diazo-compounds, using a reductive metabolic activation system may be more appropriate (6)(13).
 
1.5.1.4.  Test substance/Preparation  
Solid test substances should be dissolved or suspended in appropriate 
solvents or vehicles and diluted if appropriate prior to treatment of 
the bacteria. Liquid test substances may be added directly to the test 
systems and/or diluted prior to treatment. Fresh preparations should be employed unless stability data demonstrate the acceptability of storage. 
The solvent/vehicle should not be suspected of chemical reaction 
with the test substance and should be compatible with the survival 
of the bacteria and the S9 activity (22). If other than well-known 
solvent/vehicles are used, their inclusion should be supported by data indicating their compatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle be considered first. 
When testing water-unstable substances, the organic solvents used 
should be free of water.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 340
 
1.5.2.  Test conditions  
1.5.2.1.  Test strains (see 1.5.1.1)  
1.5.2.2.  Exposure concentration  
Amongst the criteria to be taken into consideration when determining 
the highest amount of the test substance to be used are the cytot ­
oxicity and the solubility in the final treatment mixture. 
It may be useful to determine toxicity and insolubility in a 
preliminary experiment. Cytotoxicity may be detected by a 
reduction in the number of revertant colonies, a clearing or diminution of the background lawn, or the degree of survival of treated cultures. The cytotoxicity of a substance may be altered in 
the presence of metabolic activation systems. Insolubility should be 
assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye. 
The recommended maximum test concentration for soluble non- 
cytotoxic substances is 5 mg/plate or 5 μl/plate. For non-cytotoxic 
substances that are not soluble at 5 mg/plate or 5 μl/plate, one or 
more concentrations tested should be insoluble in the final treatment 
mixture. Test substances that are cytotoxic already below 5 mg/plate 
or 5 μl/plate should be tested up to a cytotoxic concentration. The 
precipitate should not interfere with the scoring. 
At least five different analysable concentrations of the test substance 
should be used with approximately half log (i.e. √10) intervals 
between test points for an initial experiment. Smaller intervals may be appropriate when a concentration-response is being investigated. Testing above the concentration of 5 mg/plate or 5 μl/plate may be 
considered when evaluating substances containing substantial 
amounts of potentially mutagenic impurities.
 
1.5.2.3.  Negative and positive controls  
Concurrent strain-specific positive and negative (solvent or vehicle) 
controls, both with and without metabolic activation, should be 
included in each assay. Positive control concentrations that demon ­
strate the effective performance of each assay should be selected. 
For assays employing a metabolic activation system, the positive 
control reference substance(s) should be selected on the basis of 
the type of bacteria strains used. 
The following substances are examples of suitable positive controls 
for assays with metabolic activation:  
CA numbers EINECS numbers Names  
781-43-1 212-308-4 9,10-dimethylanthracene 
57-97-6 200-359-5 7,12-dimethylbenz[ a]anthracene 
50-32-8 200-028-5 benzo[a]pyrene 
613-13-8 210-330-9 2-aminoanthracene  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 341
 
CA numbers EINECS numbers Names  
50-18-0 cyclophosphamide 
6055-19-2 200-015-4 cyclophosphamide monohydrate 
The following substance is a suitable positive control for the 
reductive metabolic activation method:  
CA numbers EINECS numbers Names  
573-58-0 209-358-4 Congo Red 
2-Aminoanthracene should not be used as the sole indicator of the 
efficacy of the S9-mix. If 2-aminoanthracene is used, each batch of 
S9 should also be characterised with a mutagen that requires 
metabolic activation by microsomal enzymes, e.g. benzo[a]pyrene, dimethylbenzanthracene. 
The following substances are examples of strain-specific positive 
controls for assays performed without exogenous metabolic 
activation system:
 
CAS numbers EINECS numbers Names Strain  
26628-22-8 247-852-1 Sodium azide TA 1535 and TA 100 
607-57-8 210-138-5 2-nitrofluorene TA 98 
90-45-9 201-995-6 9-aminoacridine TA 1537, TA 97 and TA 97a 
17070-45-0 241-129-4 ICR 191 TA 1537, TA 97 and TA 97a 
80-15-9 201-254-7 Cumene hydroperoxide TA 102 
50-07-7 200-008-6 Mitomycin C WP2 uvrA and TA102 
70-25-7 200-730-1 N-ethyl-N-nitro-N-nitro ­
soguanidine WP2, WP2uvrA and 
WP2uvrA(pKM101) 
56-57-5 200-281-1 4-nitroquinoline-1-oxide WP2, WP2uvrA and 
WP2uvrA(pKM101) 
3688-53-7 Furylfuramide (AF2) plasmid-containing strains 
Other appropriate positive control reference substances may be used. 
The use of chemical class-related positive control chemicals should 
be considered, when available. 
Negative controls, consisting of solvent or vehicle alone, without test 
substance, and otherwise treated in the same way as the treatment groups, should be included. In addition, untreated controls should also be used unless there are historical control data demonstrating 
that no deleterious or mutagenic effects are induced by the chosen 
solvent.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 342
 
1.5.3.  Procedure  
For the plate incorporation method (1)(2)(3)(4), without metabolic 
activation, usually 0,05 ml or 0,1 ml of the test solutions, 0,1 ml of fresh bacterial culture (containing approximately 10
 8 viable cells) 
and 0,5 ml of sterile buffer are mixed with 2,0 ml of overlay agar. 
For the assay with metabolic activation, usually 0,5 ml of metabolic activation mixture containing an adequate amount of post-mito ­
chondrial fraction (in the range from 5 to 30 % v/v in the 
metabolic activation mixture) are mixed with the overlay agar (2,0 
ml), together with the bacteria and test substance/test solution. The contents of each tube are mixed and poured over the surface of a 
minimal agar plate. The overlay agar is allowed to solidify before 
incubation. 
For the preincubation method (2)(3)(5)(6), the test substance/test 
solution is preincubated with the test strain (containing approxi ­
mately 10
 8 viable cells) and sterile buffer or the metabolic activation 
system (0,5 ml) usually for 20 min. or more at 30-37  o C prior to 
mixing with the overlay agar and pouring onto the surface of a minimal agar plate. Usually, 0,05 or 0,1 ml of test substance/test solution, 0,1 ml of bacteria, and 0,5 ml of S9-mix or sterile buffer 
are mixed with 2,0 ml of overlay agar. Tubes should be aerated 
during pre-incubation by using a shaker. 
For an adequate estimate of variation, triplicate plating should be 
used at each dose level. The use of duplicate plating is acceptable 
when scientifically justified. The occasional loss of a plate does not 
necessarily invalidate the assay. 
Gaseous or volatile substances should be tested by appropriate 
methods, such as in sealed vessels (12)(14)(15)(16).
 
1.5.4.  Incubation  
All plates in a given assay should be incubated at 37  o C for 48- 
72 hours. After the incubation period, the number of revertant 
colonies per plate is counted.  
2. DATA  
2.1. TREATMENT OF RESULTS 
Data should be presented as the number of revertant colonies per 
plate. The number of revertant colonies on both negative (solvent control, and untreated control if used) and positive control plates 
should also be given. Individual plate counts, the mean number of 
revertant colonies per plate and the standard deviation should be presented for the test substance and positive and negative (untreated and/or solvent) controls. 
There is no requirement for verification of a clear positive response. 
Equivocal results should be clarified by further testing preferably 
using a modification of experimental conditions. Negative results need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, 
justification should be provided. Modification of study parameters to 
extend the range of conditions assessed should be considered in follow-up experiments. Study parameters that might be modified include the concentration spacing, the method of treatment (plate- 
incorporation or liquid pre-incubation), and metabolic activation 
conditions.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 343
 
2.2. EVALUATION AND INTERPRETATION OF RESULTS 
There are several criteria for determining a positive result, such as a 
concentration-related increase over the range tested and/or a repro ­
ducible increase at one or more concentrations in the number of 
revertant colonies per plate in at least one strain with or without 
metabolic activation system (23). Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results (24). However, statistical 
significance should not be the only determining factor for a 
positive response. 
A test substance for which the results do not meet the above criteria 
is considered non-mutagenic in this test. 
Although most experiments will give clearly positive or negative 
results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may 
remain equivocal or questionable regardless of the number of 
times the experiment is repeated. 
Positive results from the bacterial reverse mutation test indicate that 
the substance induces point mutations by base substitutions or frameshifts in the genome of either  Salmonella typhimurium  and/or  
Escherichia coli.  Negative results indicate that under the test 
conditions, the test substance is not mutagenic in the tested species.
 
3. REPORTING  
TEST REPORT 
The test report must include the following information: 
Solvent/Vehicle: — justification for choice of solvent/vehicle, — solubility and stability of the test substance in solvent/vehicle, if 
known. 
Strains: 
— strains used, 
— number of cells per culture, — strain characteristics. Test conditions: — amount of test substance per plate (mg/plate or μl/plate) with 
rationale for selection of dose and number of plates per concen ­
tration, 
— media used, — type and composition of metabolic activation system, including 
acceptability criteria, 
— treatment procedures. Results: 
— signs of toxicity, 
— signs of precipitation, — individual plate counts,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 344
 
— the mean number of revertant colonies per plate and standard 
deviation, 
— dose-response relationship, where possible, — statistical analyses, if any, — concurrent negative (solvent/vehicle) and positive control data, 
with ranges, means and standard deviations, 
— historical negative (solvent/vehicle) and positive control data 
with ranges, means and standard deviations. 
Discussion of results. Conclusions.
 
4. REFERENCES  
(1) Ames, B.N., McCann, J. and Yamasaki E., (1975) Methods for 
Detecting Carcinogens and Mutagens with the Salmonella/ ­
Mammalian-Microsome Mutagenicity Test. Mutation Res., 
31, p. 347-364. 
(2) Maron, D.M. and Ames, B.N., (1983) Revised Methods for the 
Salmonella Mutagenicity Test. Mutation Res., 113, p. 173-215. 
(3) Gatehouse, D., Haworth, S., Cebula, T., Gocke, E., Kier, L., 
Matsushima, T., Melcion, C., Nohmi, T., Venitt, S. and Zeiger, 
E., (1994) Recommendations for the Performance of Bacterial 
Mutation Assays. Mutation Res., 312, p. 217-233. 
(4) Kier, L.D., Brusick D.J., Auletta, A.E., Von Halle, E.S., 
Brown, M.M., Simmon, V.F., Dunkel, V., McCann, J., Mortelmans, K., Prival, M., Rao, T.K. and Ray V., (1986) 
The Salmonella typhimurium /Mammalian Microsomal Assay: 
A Report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Res., 168, p. 69-240. 
(5) Yahagi, T., Degawa, M., Seino, Y.Y., Matsushima, T., Nagao, 
M., Sugimura, T. and Hashimoto, Y., (1975) Mutagenicity of 
Carcinogen Azo Dyes and their Derivatives, Cancer Letters, 1, 
p. 91-96. 
(6) Matsushima, M., Sugimura, T., Nagao, M., Yahagi, T., Shirai, 
A. and Sawamura, M., (1980) Factors Modulating Mutage ­
nicity Microbial Tests. In: Short-term Test Systems for 
Detecting Carcinogens. Ed. Norpoth K.H. and Garner, R.C., Springer, Berlin-Heidelberg-New York. p. 273-285. 
(7) Gatehouse, D.G., Rowland, I.R., Wilcox, P., Callender, R.D. 
and Foster, R., (1980) Bacterial Mutation Assays. In: Basic 
Mutagenicity Tests: UKEMS Part 1 Revised. Ed. D.J. 
Kirkland, Cambridge University Press, p. 13-61. 
(8) Aeschacher, H.U., Wolleb, U. and Porchet, L., (1987) Liquid 
Preincubation Mutagenicity Test for Foods. J. Food Safety, 8, p. 167-177. 
(9) Green, M.H.L., Muriel, W.J. and Bridges, B.A., (1976) Use of 
a simplified fluctuation test to detect low levels of mutagens. 
Mutation Res., 38, p. 33-42. 
(10) Hubbard, S.A., Green, M.H.L., Gatehouse, D. and J.W. 
Bridges (1984) The Fluctuation Test in Bacteria. In: Handbook of Mutagenicity Test Procedures. 2nd Edition. Ed. 
Kilbey, B.J., Legator, M., Nichols, W. and Ramel, C., Elsevier, 
Amsterdam-New York-Oxford, p. 141-161.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 345
 
(11) Thompson, E.D. and Melampy, P.J., (1981) An Examination 
of the Quantitative Suspension Assay for Mutagenesis with 
Strains of  Salmonella typhimurium . Environmental Muta ­
genesis, 3, p. 453-465. 
(12) Araki, A., Noguchi, T., Kato, F. and T. Matsushima (1994) 
Improved Method for Mutagenicity Testing of Gaseous 
Compounds by Using a Gas Sampling Bag. Mutation Res., 307, p. 335-344. 
(13) Prival, M.J., Bell, S.J., Mitchell, V.D., Reipert, M.D. and 
Vaughn, V.L., (1984) Mutagenicity of Benzidine and Benzidine-Congener Dyes and Selected Monoazo Dyes in a Modified Salmonella Assay. Mutation Res., 136, p. 33-47. 
(14) Zeiger, E., Anderson, B.E., Haworth, S., Lawlor, T. and 
Mortelmans, K., (1992) Salmonella Mutagenicity Tests. V. Results from the Testing of 311 Chemicals. Environ. Mol. Mutagen., 19, p. 2-141. 
(15) Simmon, V., Kauhanen, K. and Tardiff, R.G., (1977) 
Mutagenic Activity of Chemicals Identified in Drinking Water. In Progress in Genetic Toxicology, D. Scott, B. 
Bridges and F. Sobels (Eds.) Elsevier, Amsterdam, p. 249-258. 
(16) Hughes, T.J., Simmons, D.M., Monteith, I.G. and Claxton, 
L.D., (1987) Vaporisation Technique to Measure Mutagenic Activity of Volatile Organic Chemicals in the Ames/ ­
Salmonella Assay. Environmental Mutagenesis, 9, p. 421-441. 
(17) Matsushima, T., Matsumoto, A., Shirai, M., Sawamura, M., 
and Sugimura, T., (1979) Mutagenicity of the Naturally 
Occurring Carcinogen Cycasin and Synthetic Methylazoxy 
Methane Conjugates in  Salmonella typhimurium . Cancer 
Res., 39, p. 3780-3782. 
(18) Tamura, G., Gold, C., Ferro-Luzzi, A. and Ames, B.N., (1980) 
Fecalase: A Model for Activation of Dietary Glycosides to 
Mutagens by Intestinal Flora. Proc. Natl. Acad. Sci. USA, 77, p. 4961-4965. 
(19) Wilcox, P., Naidoo, A., Wedd, D.J. and Gatehouse, D.G., 
(1990) Comparison of  Salmonella typhimurium  TA 102 
with Escherichia coli  WP2 Tester strains. Mutagenesis, 5, 
p. 285-291. 
(20) Matsushima, T., Sawamura, M., Hara, K. and Sugimura, T., 
(1976) A Safe Substitute for Polychlorinated Biphenyls as an Inducer or Metabolic Activation Systems. In: ‘ In vitro  
metabolic Activation in Mutagenesis Testing’ Eds. F.J. de 
Serres et al. Elsevier, North Holland, p. 85-88. 
(21) Elliot, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., 
Gibson, G.G., Mackay, J.M. and Wolf, R.C., (1992) Alter ­
natives to Aroclor 1254-induced S9 in  in vitro  Genotoxicity 
Assays. Mutagenesis, 7, p. 175-177. 
(22) Maron, D., Katzenellenbogen, J. and Ames, B.N., (1981) 
Compatibility of Organic Solvents with the Salmonella/ ­
Microsome Test. Mutation Res., p. 88343-350. 
(23) Claxton, L.D., Allen, J., Auletta, A., Mortelmans, K., 
Nestmann, E. and Zeiger, E., (1987) Guide for the  Salmonella 
typhimurium /Mammalian Microsome Tests for Bacterial Muta ­
genicity. Mutation Res. 189, p. 83-91.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 346
 
(24) Mahon, G.A.T., Green, M.H.L., Middleton, B., Mitchell, I., 
Robinson, W.D. and Tweats, D.J., (1989) Analysis of Data 
from Microbial Colony Assays. In: UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Part II. Statistical Evaluation of Mutagenicity Test Data. Ed. Kirkland, D.J., 
Cambridge University Press, p. 28-65.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 347
 
B.15.  MUTAGENICITY TESTING AND SCREENING FOR CARCI ­
NOGENICITY GENE MUTATION —  SACCHAROMYCES 
CEREVISIAE  
1. METHOD  
1.1. INTRODUCTION 
See General introduction Part B.  
1.2. DEFINITION 
See General introduction Part B.  
1.3. REFERENCE SUBSTANCES 
None.  
1.4. PRINCIPLE OF THE TEST METHOD 
A variety of haploid and diploid strains of the yeast  Saccharomyces 
cerevisiae  can be used to measure the production of gene mutations 
induced by chemical agents with and without metabolic activation. 
Forward mutation systems in haploid strains, such as the 
measurement of mutation from red, adenine-requiring mutants 
(ade-1, ade -2) to double adenine-requiring white mutants and 
selective systems such as the induction of resistance to canavnaine 
and cycloheximide, have been utilised. 
The most extensively validated reverse mutation system involves the 
use of the haploid strain XV 185-14C which carries the ochre nonsense mutations  ade 2-1,  arg 4-17,  lys 1-1 and  trp 5-48, which 
are reversible by base substitution mutagens that induce site specific 
mutations or ochre suppressor mutations. XV 185-14C also carries 
the his 1-7 marker, a missense mutation reverted mainly by second 
site mutations, and the marker  hom  3-10 which is reverted by 
frameshift mutagens. 
In diploid strains of  S. cerevisiae  the only extensively used strain is 
D
 7 which is homozygous for  ilv 1-92.  
1.5. QUALITY CRITERIA 
None.  
1.6. DESCRIPTION OF THE TEST METHOD  
Preparations  
Solutions of test chemicals and control should be prepared just prior 
to testing, using an appropriate vehicle. In the case of organic 
compounds, which are not water soluble, not more than a 2 % solution v/v of organic solvents such as ethanol, acetone or dimethyl ­
sulphoxide (DMSO) should be used. The final concentration of the 
vehicle should not significantly affect cell viability and growth char ­
acertistics.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 348
 
M e t a b o l i c a c t i v a t i o n 
Cells should be exposed to test chemicals both in the presence and 
absence of an appropriate exogenous metabolic activation system. 
The most commonly used system is a co-factor supplemented post- 
mitochondrial fraction from the livers of rodents pre-treated with 
enzyme inducing agents. The use of other species, tissues, post-mito ­
chondrial fractions, or procedures may also be appropriate for metabolic activation.
 
Test conditions  
T e s t e r s t r a i n s 
The haploid strain XV 185-14C and the diploid strain D  7 are the 
most used in gene mutation studies. Other strains may also be appro ­
priate.  
M e d i a 
Appropriate culture media are used for the determination of survival 
and mutant numbers.  
U s e o f n e g a t i v e a n d p o s i t i v e c o n t r o l s 
Positive, untreated and solvent controls should be performed concur ­
rently. Appropriate positive control chemicals should be used for 
each specific mutational endpoint.  
E x p o s u r e c o n c e n t r a t i o n 
At least five adequately spaced concentrations of the test substance 
should be used. For toxic substances, the highest concentration tested should not reduce survival below 5 to 10 %. Relatively water- 
insoluble substances should be tested up to their limit of solubility, 
using appropriate procedures. For freely water-soluble non-toxic substances, the upper concentration should be determined on a case by case basis.
 
I n c u b a t i o n c o n d i t i o n s 
The plates are incubated four to seven days at 28 to 30  o C in the 
dark.  
S p o n t a n e o u s m u t a t i o n f r e q u e n c i e s 
Sub-cultures should be used with spontaneous mutation frequencies 
within the accepted normal range.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 349
 
N u m b e r o f r e p l i c a t e s 
At least three replicate plates should be used per concentration for 
the assay of prototrophs produced by gene mutation and for cell viability. In the case of experiments using markers such as  hom  3- 
10 with a low mutation rate, the number of plates used must be 
increased to provide statistically relevant data.
 
Procedure  
Treatment of  S. cerevisiae  strains is usually performed in a liquid test 
procedure involving either stationary or growing cells. Initial 
experiments should be carried out on growing cells: 1-5 × 10  7 
cells/ml are exposed to the test chemical for up to 18 hours at 28 
to 37  o C with shaking; an adequate amount of metabolic activation 
system is added during treatment when appropriate. At the end of the 
treatment, cells are centrifuged, washed and seeded upon an appro ­
priate culture medium. After incubation, plates are scored for survival and the induction of gene mutation. If the first experiment is negative, then a second experiment should be carried out using 
stationary phase cells. If the first experiment is positive it is 
confirmed in an appropriate independent experiment.
 
2. DATA  
Data should be presented in tabular form indicating the number of 
colonies counted, number of mutants, survival and mutant frequency. All results should be confirmed in an independent experiment. The 
dara should be evaluated using appropriate statistical methods.
 
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, contain the following information: 
— strain used, — test conditions: stationary phase or growing cells, compositions 
of media, incubation temperature and duration, metabolic activation system, 
— treatment conditions: exposure levels, procedure and duration of 
treatment, treatment temperature, positive and negative controls, 
— number of colonies counted, number of mutants, survival and 
mutant frequency, dose/response relationship if applicable, stat ­
istical evaluation of data, 
— discussion of results, 
— interpretation of results.
 
3.2. EVALUATION AND INTERPRETATION 
See General introduction Part B.  
4. REFERENCES  
See General introduction Part B.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 350
 
B.16.  MITOTIC RECOMBINATION —  SACCHAROMYCES 
CEREVISIAE  
1. METHOD  
1.1. INTRODUCTION 
See General introduction Part B.  
1.2. DEFINITION 
See General introduction Part B.  
1.3. REFERENCE SUBSTANCES 
None.  
1.4. PRINCIPLE OF THE TEST METHOD 
Mitotic recombination in  Saccharomyces cerevisiae  can be detected 
between genes (or more generally between a gene and its centromere) and within genes. The former event is called mitotic crossing-over and generates reciprocal products whereas the latter 
event is most frequently non-reciprocal and is called gene 
conversion. Crossing-over is generally assayed by the production of recessive homozygous colonies or sectors produced in a heterozygous strain, whereas gene conversion is assayed by the 
production of prototrophic revertants produced in an auxotrophic 
heteroallelic strain carrying two different defective alleles of the same gene. The most commonly used strains for the detection of mitotic gene conversion are D
 4 (heteroallelic at  ade 2 and  trp 5) D  7 
(heteroallelic at  trp 5) BZ  34 (heteroallelic at  arg 4) and JDl (hetero ­
allelic at  his 4 and  trp 5). Mitotic crossing-over producing red and 
pink homozygous sectors can be assayed in D  5 or in D  7 (which also 
measures mitotic gene conversion and reverse mutation at  ilv 1-92) 
both strains being heteroallelic for complementing alleles of  ade 2. 
1.5. QUALITY CRITERIA 
None.  
1.6. DESCRIPTION OF THE TEST METHOD  
Preparations  
Solutions of test chemicals and control or reference compounds 
should be prepared just prior to testing, using an appropriate 
vehicle. With organic compounds that are water insoluble not more than a 2 % solution v/v of organic solvents such as ethanol, acetone or dimethylsulphoxide (DMSO) should be used. The final 
concentration of the vehicle should not significantly affect cell 
viability and growth characteristics.
 
M e t a b o l i c a c t i v a t i o n 
Cells should be exposed to test chemicals both in the presence and 
absence of an appropriate exogenous metabolic activation system. 
The system most commonly used is a co-factor supplemented post- mitochondrial fraction from the livers of rodents pre-treated with enzyme inducing agents. The use of other species, tissues, post-mito ­
chondrial fractions, or procedures may also be appropriate for 
metabolic activation.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 351
 
Test conditions  
T e s t e r s t r a i n s 
The most frequently used strains are the diploids D  4 , D  5 , D  7 and 
JD1. The use of other strains may be appropriate.  
M e d i a 
Appropriate culture media are used for the determination of survival 
and the frequency of mitotic recombination.  
U s e o f n e g a t i v e a n d p o s i t i v e c o n t r o l s 
Positive, untreated and solvent controls should be performed concur ­
rently. Appropriate positive control chemicals should be used for 
each specific recombination endpoint.  
E x p o s u r e c o n c e n t r a t i o n s 
At least five adequately spaced concentrations of the test substance 
should be used. Among the factors to be taken into consideration are cytotoxicity and solubility. The lowest concentration must have no effect on celf viability. For toxic chemicals, the highest concentration 
tested should not reduce survival below 5 to 10 %. Relatively water- 
insoluble chemicals should be tested up to the limit of solubility using appropriate procedures. For freely water-soluble non-toxic substances the upper concentration should be determined on a case 
by case basis. 
Cells may be exposed to test chemicals in either the stationary phase 
or during growth for periods of up to 18 hours. However, for long 
treatment times cultures should be microscopically inspected for 
spore formation, the presence of which invalidates the test.
 
I n c u b a t i o n c o n d i t i o n s 
The plates are incubated in the dark for four to seven days at 28 to 
30 o C. Plates used for the assay of red and pink homozygous sectors 
produced by mitotic crossing-over should be kept in a refrigerator 
(about 4  o C) for a further one to two days before scoring to allow for 
the development of the appropriate pigmented colonies.  
S p o n t a n e o u s m i t o t i c r e c o m b i n a t i o n f r e q u e n c i e s 
Sub-cultures should be used with spontaneous mitotic recombination 
mutation frequencies within the accepted normal range.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 352
 
N u m b e r o f r e p l i c a t e s 
A minimum of three replicate plates should be used per concen ­
tration for the assay of prototrophs produced by mitotic gene conversion and for viability. In the case of the assay of recessive homozygosis produced by mitotic crossing-over, the plate number 
should be increased to provide an adequate number of colonies.
 
Procedures  
Treatment of  S. cerevisiae  strains is usually performed in a liquid test 
procedure involving either stationary or growing cells. Initial 
experiments should be done on growing cells. 1-5 × 10  7 celles/ml 
are exposed to the test chemical for up to 18 hours at 28 to 37  o C 
with shaking; an adequate amount of metabolic activation system is added during treatment when appropriate. 
At the end of the treatment, cells are centrifuged, washed and seeded 
upon appropriate culutre medium. After incubation plates are scored 
for survival and the induction of mitotic recombination. 
If the first experiment is negative, then a second experiment should 
be carried out using stationary phase cells. If the first experiment is positive it is confirmed in an independent experiment.
 
2. DATA  
Data should be presented in tabular form indicating the number of 
colonies counted, the number of recombinants, survival and the 
frequency of recombinants. 
Results should be confirmed in an independent experiment. The data should be evaluated using appropriate statistical methods.
 
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, contain the following information: 
— strain used, — test conditions: stationary phase or growing cells, composition of 
media, incubation temperature and duration, metabolic activation 
system, 
— treatment conditions: exposure concentration, procedure and 
duration of treatment, treatment temperature, positive and negative controls, 
— number of colonies counted, number of recombinants; survival 
and recombination frequency, dose/response relationship if appli ­
cable, statistical evaluation of data, 
— discussion of the results, — interpretation of the results.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 353
 
3.2. EVALUATION AND INTERPRETATION 
See General introduction Part B.  
4. REFERENCES  
See General introduction Part B.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 354
 
B.17.  MUTAGENICITY —  IN VITRO  MAMMALIAN CELL GENE 
MUTATION TEST  
1. METHOD  
This method is a replicate of the OECD TG 476,  In Vitro  
Mammalian Cell Gene Mutation Test (1997).  
1.1. INTRODUCTION 
The in vitro  mammalian cell gene mutation test can be used to detect 
gene mutations induced by chemical substances. Suitable cell lines 
include L5178Y mouse lymphoma cells, the CHO, CHO-AS52 and 
V79 lines of Chinese hamster cells, and TK6 human lymphoblastoid cells (1). In these cell lines the most commonly-used genetic endpoints measure mutation at thymidine kinase (TK) and hypoxan ­
thine-guanine phosphoribosyl transferase (HPRT), and a transgene of 
xanthine-guanine phosphoribosyl transferase (XPRT). The TK, HPRT and XPRT mutation tests detect different spectra of genetic events. The autosomal location of TK and XPRT may allow the 
detection of genetic events (e.g. large deletions) not detected at the 
HPRT locus on X-chromosomes (2)(3)(4)(5)(6). 
In the  in vitro  mammalian cell gene mutation test, cultures of estab ­
lished cell lines or cell strains can be used. The cells used are selected on the basis of growth ability in culture and stability of the spontaneous mutation frequency. 
Tests conducted  in vitro  generally require the use of an exogenous 
source of metabolic activation. This metabolic activation system cannot mimic entirely the mammalian  in vivo  conditions. Care 
should be taken to avoid conditions, which would lead to results 
not reflecting intrinsic mutagenicity. Positive results, which do not reflect intrinsic mutagenicity may arise from changes in pH, osmo ­
lality or high levels of cytotoxicity (7). 
This test is used to screen for possible mammalian mutagens and 
carcinogens. Many compounds that are positive in this test are 
mammalian carcinogens; however, there is not a perfect correlation 
between this test and carcinogenicity. Correlation is dependent on chemical class and there is increasing evidence that there are carci ­
nogens that are not detected by this test because they appear to act 
through other, non genotoxic mechanisms or mechanisms absent in 
bacterial cells (6). 
See also General introduction Part B.
 
1.2. DEFINITIONS  
Forward mutation:  a gene mutation from the parental type to the 
mutant form which gives rise to an alteration or a loss of the enzymatic activity of the function of the encoded protein.  
Base pair substitution mutagens:  substances, which cause substi ­
tution of one or several base pairs in the DNA.  
Frameshift mutagens:  Substances, which cause the addition or 
deletion of single or multiple base pairs in the DNA molecule.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 355
 
Phenotypic expression time:  a period during which unaltered gene 
products are depleted from newly mutated cells.  
Mutant frequency:  the number of mutant cells observed divided by 
the number of viable cells.  
Relative total growth:  increase in cell number over time compared 
to a control population of cells; calculated as the product of suspension growth relative to the negative control times cloning efficiency relative to negative control.  
Relative suspension growth:  increase in cell number over the 
expression period relative to the negative control.  
Viability:  the cloning efficiency of the treated cells at the time of 
plating in selective conditions after the expression period.  
Survival:  the cloning efficiency of the treated cells when plated at 
the end of the treatment period; survival is usually expressed in 
relation to the survival of the control cell population.
 
1.3. PRINCIPLE OF THE TEST METHOD 
Cells deficient in thymidine kinase (TK) due to the mutation 
TK +/- -> TK  -/- are resistant to the cytotoxic effects of the pyrimidine 
analogue trifluorothymidine (TFT). Thymidine kinase proficient cells 
are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus mutant cells are able to proliferate in the presence of TFT, whereas normal cells, 
which contain thymidine kinase, are not. Similarly, cells deficient 
in HPRT or XPRT are selected by resistance to 6-thioguanine (TG) or 8-azaguanine (AG). The properties of the test substance should be considered carefully if a base analogue or a compound related to the 
selective agent is tested in any of the mammalian cell gene mutation 
tests. For example, any suspected selective toxicity by the test substance for mutant and non-mutant cells should be investigated. Thus, performance of the selection system/agent must be confirmed 
when testing chemicals structurally related to the selective agent (8). 
Cells in suspension or monolayer culture are exposed to the test 
substance, both with and without metabolic activation, for a suitable 
period of time and subcultured to determine cytotoxicity and to allow 
phenotypic expression prior to mutant selection (9)(10)(11)(12)(13). Cytotoxicity is usually determined by measuring the relative cloning efficiency (survival) or relative total growth of the cultures after the 
treatment period. The treated cultures are maintained in growth 
medium for a sufficient period of time, characteristic of each selected locus and cell type, to allow near-optimal phenotypic expression of induced mutations. Mutant frequency is determined by seeding 
known numbers of cells in medium containing the selective agent to 
detect mutant cells and in medium without selective agent to determine the cloning efficiency (viability). After a suitable incubation time, colonies are counted. The mutant frequency is derived from the 
number of mutant colonies in selective medium and the number of 
colonies in non-selective medium.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 356
 
1.4. DESCRIPTION OF THE TEST METHOD  
1.4.1.  Preparations  
1.4.1.1.  Cells  
A variety of cell types are available for use in this test including 
subclones of L5178Y, CHO, CHO-AS52, V79 or TK6 cells. Cell 
types used in this test should have a demonstrated sensitivity to chemical mutagens, a high cloning efficiency and a stable spon ­
taneous mutant frequency. Cells should be checked for mycoplasma 
contamination and should not be used if contaminated. 
The test should be designed to have a predetermined sensitivity and 
power. The number of cells, cultures and concentrations of test substance used should reflect these defined parameters (14). The 
minimal number of viable cells surviving treatment and used at 
each stage in the test should be based on the spontaneous mutation frequency. A general guide is to use a cell number, which is at least 10 times the inverse of the spontaneous mutation 
frequency. However, it is recommended to utilise at least 10
 6 cells. 
Adequate historical data on the cell system used should be available to indicate consistent performance of the test.
 
1.4.1.2.  Media and culture conditions  
Appropriate culture media, and incubation conditions (culture 
vessels, temperature, CO  2 concentration, and humidity) should be 
used. Media should be chosen according to the selective systems 
and cell type used in the test. It is particularly important that 
culture conditions should be chosen that ensure optimal growth of cells during the expression period and colony forming ability of both mutant and non-mutant cells.
 
1.4.1.3.  Preparation of cultures  
Cell are propagated from stock cultures, seeded in culture medium 
and incubated at 37  o C. Prior to use in this test, cultures may need to 
be cleansed of pre-existing mutant cells.  
1.4.1.4.  Metabolic activation  
Cells should be exposed to the test substance both in the presence 
and absence of an appropriate metabolic activation system. The most commonly used system is a cofactor-supplemented post-mito ­
chondrial fraction (S9) prepared from the livers of rodents treated 
with enzyme-inducing agents such as Aroclor 1254 (15)(16)(17)(18) 
or a combination of phenobarbitone and ß–naphthoflavone (19)(20). 
The post-mitochondrial fraction is usually used at concentrations in 
the range from 1-10 % v/v in the final test medium. The choice and condition of a metabolic activation system may depend upon the 
class of chemical being tested. In some cases it may be appropriate 
to utilise more than one concentration of post-mitochondrial fraction.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 357
 
A number of developments, including the construction of genetically 
engineered cell lines expressing specific activating enzymes, may 
provide the potential for endogenous activation. The choice of the cell lines used should be scientifically justified (e.g. by the relevance of the cytochrome P450 isoenzyme for the metabolism of the test 
substance).
 
1.4.1.5.  Test substance/Preparation  
Solid test substances should be dissolved or suspended in appropriate 
solvents or vehicles and diluted if appropriate prior to treatment of 
the cells. Liquid test substances may be added directly to the test systems and/or diluted prior to treatment. Fresh preparations of the test substance should be employed unless stability data demonstrate 
the acceptability of storage.
 
1.4.2.  Test conditions  
1.4.2.1.  Solvent/Vehicle  
The solvent/vehicle should not be suspected of chemical reaction 
with the test substance and should be compatible with the survival of the cells and the S9 activity. If other than well-known solvent/ ­
vehicles are used, their inclusion should be supported by data indi ­
cating their compatibility. It is recommended that wherever possible, 
the use of an aqueous solvent/vehicle be considered first. When testing water-unstable substances, the organic solvents used should be free of water. Water can be removed by adding a molecular sieve.
 
1.4.2.2.  Exposure concentrations  
Among the criteria to be considered when determining the highest 
concentration are cytotoxicity, solubility in the test system and 
changes in pH or osmolality. 
Cytotoxicity should be determined with and without metabolic 
activation in the main experiment using an appropriate indication of cell integrity and growth, such as relative cloning efficiency (sur ­
vival) or relative total growth. It may be useful to determine cytot ­
oxicity and solubility in a preliminary experiment. 
At least four analysable concentrations should be used. Where there 
is cytotoxicity, these concentrations should cover a range from the maximum to little or no toxicity; this will usually mean that the concentration levels should be separated by no more than a factor 
between 2 and √10. If the maximum concentration is based on cytot ­
oxicity then it should result in approximately 10-20 % (but not less than 10 %) relative survival (relative cloning efficiency) or relative total growth. For relatively non-cytotoxic substances, the maximum 
test concentration should be 5 mg/ml 5 μl/ml, or 0,01 M, whichever 
is the lowest.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 358
 
Relatively insoluble substances should be tested up to or beyond 
their limit of solubility under culture conditions. Evidence of inso ­
lubility should be determined in the final treatment medium to which cells are exposed. It may be useful to assess solubility at the beginning and the end of the treatment, as solubility can change 
during the course of exposure in the test system due to presence 
of cells, S9, serum, etc. Insolubility can be detected by using the unaided eye. The precipitate should not interfere with the scoring.
 
1.4.2.3.  Controls  
Concurrent positive and negative (solvent or vehicle) controls, both 
with and without metabolic activation should be included in each 
experiment. When metabolic activation is used, the positive control chemical should be the one that requires activation to give a mutagenic response. 
Examples of positive control substances include:
 
Metabolic activation 
condition Locus Substance CAS No EINECS No  
Absence of 
exogenous 
metabolic 
activation HPRT Ethyl methanesulphonate 62-50-0 200-536-7 
Ethyl nitrosourea 759-73-9 212-072-2 
TK (small and 
large 
colonies) Methyl methanesulphonate 66-27-3 200-625-0 
XPRT Ethyl methanesulphonate 62-50-0 200-536-7 
Ethyl nitrosourea 759-73-9 212-072-2 
Presence of 
exogenous metabolic 
activation HPRT 3-Methylcholanthrene 56-49-5 200-276-4 
N-Nitrosodimethylamine 62-75-9 200-549-8 7,12-Dimethylbenzanthracene 57-97-6 200-359-5 
TK (small and 
large 
colonies) Cyclophosphamide 50-18-0 200-015-4 
Cyclophosphamide monohydrate 6055-19-2 Benzo[ a]pyrene 50-32-8 200-028-5 
3-Methylcholanthrene 56-49-5 200-276-5 
XPRT N-Nitrosodimethylamine (for high 
levels of S-9) 62-75-9 200-549-8 
Benzo[ a]pyrene 50-32-8 200-028-5 
Other appropriate positive control reference substances may be used, 
e.g. if a laboratory has a historical data base on 5-Bromo 2'- deoxyuridine (CAS n. 59-14-3, EINECS n. 200-415-9), this reference substance could be used as well. The use of chemical 
class-related positive control chemicals should be considered, when 
available.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 359
 
Negative controls, consisting of solvent or vehicle alone in the 
treatment medium, and treated in the same way as the treatment 
groups, should be included. In addition, untreated controls should also be used unless there are historical control data demonstrating that no deleterious or mutagenic effects are induced by the chosen 
solvent.
 
1.4.3.  Procedure  
1.4.3.1.  Treatment with the test substance  
Proliferating cells should be exposed to the test substance both with 
and without metabolic activation. Exposure should be for a suitable 
period of time (usually three to six hours is effective). Exposure time may be extended over one or more cell cycles. 
Either duplicate or single treated cultures may be used at each 
concentration tested. When single cultures are used, the number of concentrations should be increased to ensure an adequate number of 
cultures for analysis (e.g. at least eight analysable concentrations). 
Duplicate negative (solvent) control cultures should be used. 
Gaseous or volatile substances should be tested by appropriate 
methods, such as in sealed culture vessels (21)(22).
 
1.4.3.2.  Measurement of survival, viability and mutant frequency  
At the end of the exposure period, cells are washed and cultured to 
determine survival and to allow for expression of the mutant 
phenotype. Measurement of cytotoxicity by determining the relative cloning efficiency (survival) or relative total growth of the cultures is usually initiated after the treatment period. 
Each locus has a defined minimum time requirement to allow near 
optimal phenotypic expression of newly induced mutants (HPRT and XPRT require at least six to eight days, and TK at least two days). 
Cells are grown in medium with and without selective agent(s) for 
determination of numbers of mutants and cloning efficiency, respect ­
ively. The measurement of viability (used to calculate mutant 
frequency) is initiated at the end of the expression time by plating 
in non-selective medium. 
If the test substance is positive in the L5178Y TK
 +/- test, colony 
sizing should be performed on at least one of the test cultures (the 
highest positive concentration) and on the negative and positive controls. If the test substance is negative in the L5178Y TK
 +/- test, 
colony sizing should be performed on the negative and positive 
controls. In studies using TK6TK  +/- , colony sizing may also be 
performed.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 360
 
2. DATA  
2.1. TREATMENT OF RESULTS 
Data should include cytotoxicity and viability determination, colony 
counts and mutant frequencies for the treated and control cultures. In 
the case of a positive response in the L5178Y TK  +/- test, colonies are 
scored using the criteria of small and large colonies on at least one concentration of the test substance (highest positive concentration) and on the negative and positive control. The molecular and cyto ­
genetic nature of both large and small colony mutants has been 
explored in detail (23)(24). In the TK
 +/- test, colonies are scored 
using the criteria of normal growth (large) and slow growth 
(small) colonies (25). Mutant cells that have suffered the most 
extensive genetic damage have prolonged doubling times and thus form small colonies. This damage typically ranges in scale from the losses of the entire gene to karyotypically visible chromosome aber ­
rations. The induction of small colony mutants has been associated 
with chemicals that induce gross chromosome aberrations (26). Less seriously affected mutant cells grow at rates similar to the parental cells and form large colonies. 
Survival (relative cloning efficiencies) or relative total growth should 
be given. Mutant frequency should be expressed as number of 
mutant cells per number of surviving cells. 
Individual culture data should be provided. Additionally, all data 
should be summarised in tabular form. 
There is no requirement for verification of a clear positive response. 
Equivocal results should be clarified by further testing preferably 
using modification of experimental conditions. Negative results 
need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, justification should be provided. Modification of study parameters to 
extend the range of conditions assessed should be considered in 
follow-up experiments for either equivocal or negative results. Study parameters that might be modified include the concentration spacing and the metabolic activation conditions.
 
2.2. EVALUATION AND INTERPRETATION OF RESULTS 
There are several criteria for determining a positive result, such as a 
concentration-related increase or a reproducible increase in mutant 
frequency. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining 
factor for a positive response. 
A test substance for which the results do not meet the above criteria 
is considered non-mutagenic in this system.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 361
 
Although most studies will give clearly positive or negative results, 
in rare cases the data set will preclude making a definite judgement 
about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated. 
Positive results from the  in vitro  mammalian cell gene mutation test 
indicate that the test substance induces gene mutations in the cultured mammalian cells used. A positive concentration response 
that is reproducible is most meaningful. Negative results indicate 
that, under the test conditions, the test substance does not induce gene mutations in the cultured mammalian cells used.
 
3. REPORTING  
TEST REPORT 
The test report must include the following information: 
Solvent/Vehicle: 
— justification for choice of vehicle/solvent, 
— solubility and stability of the test substance in solvent/vehicle, if 
known, 
Cells: 
— type and source of cells, 
— number of cell cultures, 
— number of cell passages, if applicable, 
— methods for maintenance of cell culture, if applicable, 
— absence of mycoplasma. 
Test conditions: 
— rationale for selection of concentrations and number of cultures 
including, e.g. cytotoxicity data and solubility limitations, if 
available, 
— composition of media, CO  2 concentration, 
— concentration of test substance, 
— volume of vehicle and test substance added, 
— incubation temperature, — incubation time, 
— duration of treatment, 
— cell density during treatment, 
— type and composition of metabolic activation system, including 
acceptability criteria, 
— positive and negative controls,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 362
 
— length of expression period (including number of cells seeded, 
and subcultures and feeding schedules, if appropriate), 
— selective agents, — criteria for considering tests as positive, negative or equivocal, 
— methods used to enumerate numbers of viable and mutant cells. 
— definition of colonies of which size and type are considered 
(including criteria for ‘small’ and ‘large’ colonies, as appro ­
priate). 
Results: 
— signs of toxicity, 
— signs of precipitation, 
— data on pH and osmolality during the exposure to the test 
substance, if determined, 
— colony size if scored for at least negative and positive controls, 
— laboratory's adequacy to detect small colony mutants with the 
L5178Y TK+/- system, where appropriate, 
— dose-response relationship, where possible, 
— statistical analyses, if any, 
— concurrent negative (solvent/vehicle) and positive control data, 
— historical negative (solvent/vehicle) and positive control data 
with ranges, means and standard deviations, 
— mutant frequency. 
Discussion of results. 
Conclusions.
 
4. REFERENCES  
(1) Moore, M.M., DeMarini, D.M., DeSerres, F.J. and Tindall, 
K.R. (Eds.), (1987) Banbury Report 28: Mammalian Cell 
Mutagenesis, Cold Spring Harbor Laboratory, New York. 
(2) Chu, E.H.Y. and Malling, H.V., (1968) Mammalian Cell 
Genetics. II. Chemical Induction of Specific Locus Mutations 
in Chinese Hamster Cells  In Vitro , Proc. Natl. Acad. Sci., 
USA, 61, p. 1306-1312. 
(3) Liber, H.L. and Thilly, W.G., (1982) Mutation Assay at the 
Thymidine Kinase Locus in Diploid Human Lymphoblasts. 
Mutation Res., 94, p. 467-485. 
(4) Moore, M.M., Harington-Brock, K., Doerr, C.L. and Dearfield, 
K.L., (1989) Differential Mutant Quantitation at the Mouse 
Lymphoma TK and CHO HGPRT Loci. Mutagenesis, 4, 
p. 394-403.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 363
 
(5) Aaron, C.S. and Stankowski, Jr.L.F., (1989) Comparison of the 
AS52/XPRT and the CHO/HPRT Assays: Evaluation of Six 
Drug Candidates. Mutation Res., 223, p. 121-128. 
(6) Aaron, C.S., Bolcsfoldi, G., Glatt, H.R., Moore, M., Nishi, Y., 
Stankowski, Jr.L.F., Theiss, J. and Thompson, E., (1994) Mammalian Cell Gene Mutation Assays Working Group Report. Report of the International Workshop on Standard ­
isation of Genotoxicity Test Procedures. Mutation Res., 312, 
p. 235-239. 
(7) Scott, D., Galloway, S.M., Marshall, R.R., Ishidate, M., 
Brusick, D., Ashby, J. and Myhr, B.C., (1991) Genotoxicity Under Extreme Culture Conditions. A report from ICPEMC Task Group 9. Mutation Res., 257, p. 147-204. 
(8) Clive, D., McCuen, R., Spector, J.F.S., Piper, C. and 
Mavournin, K.H., (1983) Specific Gene Mutations in 
L5178Y Cells in Culture. A Report of the U.S. Environmental 
Protection Agency Gene-Tox Program. Mutation Res., 115, p. 225-251. 
(9) Li, A.P., Gupta, R.S., Heflich, R.H. and Wasson, J.S., (1988) 
A Review and Analysis of the Chinese Hamster Ovary/ 
Hypoxanthine Guanine Phosphoribosyl Transferase System to 
Determine the Mutagenicity of Chemical Agents: A Report of Phase III of the U.S. Environmental Protection Agency Gene- Tox Program. Mutation Res., 196, p. 17-36. 
(10) Li, A.P., Carver, J.H., Choy, W.N., Hsie, A.W., Gupta, R.S., 
Loveday, K.S., O'Neill, J.P., Riddle, J.C., Stankowski, L.F. Jr. 
and Yang, L.L., (1987) A Guide for the Performance of the 
Chinese Hamster Ovary Cell/Hypoxanthine-Guanine Phos ­
phoribosyl Transferase Gene Mutation Assay. Mutation Res., 189, p. 135-141. 
(11) Liber, H.L., Yandell, D.W and Little, J.B., (1989) A 
Comparison of Mutation Induction at the TK and HPRT 
Loci in Human Lymphoblastoid Cells: Quantitative Differences 
are Due to an Additional Class of Mutations at the Autosomal TK Locus. Mutation Res., 216, p. 9-17. 
(12) Stankowski, L.F. Jr., Tindall, K.R. and Hsie, A.W., (1986) 
Quantitative and Molecular Analyses of Ethyl Methanosul ­
phonate -and ICR 191- Induced Molecular Analyses of Ethyl 
Methanosulphonate -and ICR 191-Induced Mutation in AS52 
Cells. Mutation Res., 160, p. 133-147. 
(13) Turner, N.T., Batson, A.G. and Clive, D., (1984) Procedures 
for the L5178Y/TK
 +/- — TK  +/- Mouse Lymphoma Cell Muta ­
genicity Assay. In: Kilbey, B.J. et al (eds.) Handbook of Muta ­
genicity Test Procedures, Elsevier Science Publishers, New 
York, p. 239-268. 
(14) Arlett, C.F., Smith, D.M., Clarke, G.M., Green, M.H.L., Cole, 
J., McGregor, D.B. and Asquith, J.C., (1989) Mammalian Cell Gene Mutation Assays Based upon Colony Formation. In: Statistical Evaluation of Mutagenicity Test Data, Kirkland, 
D.J., Ed., Cambridge University Press, p. 66-101. 
(15) Abbondandolo, A., Bonatti, S., Corti, G., Fiorio, R., Loprieno, 
N. and Mazzaccaro, A., (1977) Induction of 6-Thioguanine- 
Resistant Mutants in V79 Chinese Hamster Cells by Mouse- Liver Microsome-Activated Dimethylnitrosamine. Mutation Res., 46, p. 365-373. 
(16) Ames, B.N., McCann, J. and Yamasaki, E., (1975) Methods 
for Detecting Carcinogens and Mutagens with the Salmonella/ 
Mammalian-Microsome Mutagenicity Test. Mutation Res., 31, 
p. 347-364.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 364
 
(17) Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G. and 
Brown M.M.M., (1979) Validation and Characterisation of 
the L5178Y/TK  +/- Mouse Lymphoma Mutagen Assay 
System. Mutat. Res., 59, p. 61-108. 
(18) Maron, D.M. and Ames, B.N., (1983) Revised Methods for the 
Salmonella Mutagenicity Test. Mutation Res., 113, p. 173-215. 
(19) Elliott, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., 
Gibson, G.G., Mackay, J.M. and Wolf, R.C., (1992) Alter ­
natives to Aroclor 1254-Induced S9 in  In Vitro  Genotoxicity 
Assays. Mutagenesis, 7, p. 175-177. 
(20) Matsushima, T., Sawamura, M., Hara, K. and Sugimura, T., 
(1976) A Safe Substitute for Polychlorinated Biphenyls as an 
Inducer of Metabolic Activation Systems. In:  In vitro  
Metabolic Activation in Mutagenesis Testing, de Serres, F.J., Fouts, J.R., Bend, J.R. and Philpot, R.M. (eds), Elsevier, 
North-Holland, p. 85-88. 
(21) Krahn, D.F., Barsky, F.C. and McCooey, K.T., (1982) 
CHO/HGPRT Mutation Assay: Evaluation of Gases and Volatile Liquids. In: Tice, R.R., Costa, D.L., Schaich, K.M. (eds). Genotoxic Effects of Airborne Agents. New York, 
Plenum, p. 91-103. 
(22) Zamora, P.O., Benson, J.M., Li, A.P. and Brooks, A.L., (1983) 
Evaluation of an Exposure System Using Cells Grown on Collagen Gels for Detecting Highly Volatile Mutagens in the 
CHO/HGPRT Mutation Assay. Environmental Mutagenesis, 5, 
p. 795-801. 
(23) Applegate, M.L., Moore, M.M., Broder, C.B., Burrell, A. and 
Hozier, J.C., (1990) Molecular Dissection of Mutations at the Heterozygous Thymidine Kinase Locus in Mouse Lymphoma 
Cells. Proc. Natl. Acad. Sci. USA, 87, p. 51-55. 
(24) Moore, M.M., Clive, D., Hozier, J.C., Howard, B.E., Batson, 
A.G., Turner, N.T. and Sawyer, J., (1985) Analysis of Trifluor ­
othymidine-Resistant (TFT
 r ) Mutants of L5178Y/TK  +/- Mouse 
Lymphoma Cells. Mutation Res., 151, p. 161-174. 
(25) Yandell, D.W., Dryja, T.P. and Little, J.B., (1990) Molecular 
Genetic Analysis of Recessive Mutations at a Heterozygous Autosomal Locus in Human Cells. Mutation Res., 229, p. 89-102. 
(26) Moore, M.M. and Doerr, C.L., (1990) Comparison of Chro ­
mosome Aberration Frequency and Small-Colony TK- 
Deficient Mutant Frequency in L5178Y/TK
 +/- - 3.7.2C 
Mouse Lymphoma Cells. Mutagenesis, 5, p. 609-614.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 365
 
B.18.  DNA DAMAGE AND REPAIR — UNSCHEDULED DNA 
SYNTHESIS — MAMMALIAN CELLS  IN VITRO  
1. METHOD  
1.1. INTRODUCTION 
See General introduction Part B.  
1.2. DEFINITION 
See General introduction Part B.  
1.3. REFERENCE SUBSTANCES 
None.  
1.4. PRINCIPLE OF THE TEST METHOD 
The Unscheduled DNA Synthesis (UDS) test measures the DNA 
repair synthesis after excision and removal of a stretch of DNA containing the region of damage induced by chemical and physical 
agents. The test is based on the incorporation of tritium labelled 
thymidine (
 3 H-TdR) into the DNA of mammalian cells, which are 
not in the S phase of the cell cycle. The uptake of  3 H-TdR may be 
determined by autoradiography or by liquid scintillation counting 
(LSC) of DNA from the treated cells. Mammalian cells in culture, 
unless primary rat hepatocytes are used, are treated with the test agent with and without an exogenous metabolic activation system. UDS may also be measured in  in vivo  systems.
 
1.5. QUALITY CRITERIA 
None.  
1.6. DESCRIPTION OF THE TEST METHOD  
Preparations  
Test chemicals and control or reference substances should be 
prepared in growth medium or dissolved or suspended in appropriate vehicles and then further diluted in growth medium for use in the assay. The final concentration of the vehicle should not affect cell 
viability. 
Primary cultures of rat hepatocytes, human lymphocytes or estab ­
lished cell lines (e.g. human diploid fibroblasts) may be used in the 
assay. 
Cells should be exposed to the test chemical both in the presence 
and absence of an appropriate metabolic activation system.
 
Test conditions  
N u m b e r o f c u l t u r e s 
At least two cell cultures for autoradiography and six cultures (or 
less if scientifically justified) for LSC UDS determinations are 
necessary for each experimental point.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 366
 
U s e o f n e g a t i v e a n d p o s i t i v e c o n t r o l s 
Concurrent positive and negative (untreated and/or vehicle) controls 
with and without metabolic activation should be included in each experiment. 
Examples of positive controls for the rat hepatocyte assay include 
7,12- dimethylbenzathracene (7,12- DMBA) or 2-acetylamino ­
fluorene (2-AAF). In the case of established cell lines 4-nitroqui ­
noline-N-oxide (4-NQO) is an example of a positive control for both the autoradiographic and LSC assays performed without 
metabolic activation; N-dimethylnitrosamine is an example of a 
positive control compound when metabolic activation systems are used.
 
E x p o s u r e c o n c e n t r a t i o n s 
Multiple concentrations of the test substance over a range adequate 
to define the response should be used. The highest concentration should elicit some cytotoxic effects. Relatively water-insoluble compounds should be tested up to the limit of solubility. For 
freely water-soluble non-toxic chemicals, the upper test chemical 
concentration should be determined on a case-by-case basis.
 
C e l l s 
Appropriate growth media, CO  2 concentration, temperature and 
humidity should be used in maintaining cultures. Established cell lines should be periodically checked for  Mycoplasma  contamination.
 
M e t a b o l i c a c t i v a t i o n 
A metabolic activation system is not used with primary hepatocyte 
cultures. Established cell lines and lymphocytes are exposed to test substance both in the presence and absence of an appropriate 
metabolic activation system.
 
Procedure  
P r e p a r a t i o n o f c u l t u r e s 
Established cell lines are generated from stock cultures (e.g. by 
trypsinisation or by shaking off), seeded in culture vessels at appro ­
priate density, and incubated at 37  o C. 
Short-term cultures of rat hepatocytes are established by allowing 
freshly dissociated hepatocytes in an appropriate medium to attach 
themselves to the growing surface. 
Human lymphocyte cultures are set up using appropriate techniques.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 367
 
T r e a t m e n t o f t h e c u l t u r e s w i t h t h e t e s t s u b s t a n c e  
Primary rat hepatocytes 
Freshly isolated rat hepatocytes are treated with the test substance in 
a medium containing  3 H-TdR for an appropriate length of time. At 
the end of the treatment period, medium should be drained off the 
cells, which are then rinsed fixed and dried. Slides should be dipped 
in autoradiographic emulsion (alternative stripping film may be used), exposed, developed, stained and counted.
 
Established cell lines and lymphocytes 
Autoradiographic techniques: cell cultures are exposed to the test 
substance for appropriate durations followed by treatment with  3 H- 
TdR. The times will be governed by the nature of the substance, the activity of metabolising systems and the type of cells. To detect the 
peak of UDS,
 3 H-TdR should be added either simultaneously with 
the test subsrance or within a few minutes after exposure to the test substance. The choice between these two procedures will be influenced by possible interactions between test substance and
 3 H- 
TdR. In order to discriminate between UDS and semi-conservative 
DNA replication, the latter can be inhibited, for example, by the use of an arginine-deficient medium, low serum content or by hydro ­
xyurea in the culture medium. 
LSC measurements of UDS: prior to treatment with test substance, 
entry of cells into S-phase should be blocked as described above; cells should then be exposed to test chemical as described for auto ­
radiography. At the end of the incubation period, DNA should be 
extracted from the cells and the total DNA content, and the extent of
 
3 H-TdR, incorporation determined. 
It should be noted that, where human lymphocytes are used in the 
above techniques, the suppression of semi-conservative DNA repli ­
cation is unnecessary in unstimulated cultures.  
Analysis  
A u t o r a d i o g r a p h i c d e t e r m i n a t i o n s  
In determining UDS in cells in culture, S-phase nuclei are not 
counted. At least 50 cells per concentration should be counted. Slides should be coded before counting. Several widely separated random fields should be counted on each slide. The amount of
 
3 H-TdR incorporation in the cytoplasm should be determined by 
counting three nucleus-sized areas in the cyptoplasm of each cell counted.
 
L S C d e t e r m i n a t i o n s 
An adequate number of cultures should be used at each concen ­
tration and in the controls in LSC UDS determinations.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 368
 
All results should be confirmed in an independent experiment.  
2. DATA  
Data should be presented in tabular form.  
2.1. AUTORADIOGRAPHIC DETERMINATIONS 
The extent of  3 H-TdR incorporation in the cytoplasm and the 
number of grains found over the cell nucleus should be recorded 
separately. 
Mean, median and mode may be used to describe the distribution of 
the extent of  3 H-TdR incorporation in the cytoplasm and the number 
of grains per nucleus.  
2.2. LSC DETERMINATIONS 
For LSC determinations,  3 H-TdR incorporation should be reported as 
dpm/μg DNA. The mean dpm/ μg DNA with standard deviation may 
be used to describe the distribution of incorporation. 
Data should be evaluated using appropriate statistical methods.  
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, contain the following information: 
— cells used, density and passage number at time of treatment, 
number of cell cultures, 
— methods used for maintenance of cell cultures including medium, 
temperature and CO  2 concentration, 
— test substance, vehicle, concentrations and rationale for selection 
of concentrations used in the assay, 
— details of metabolic activation systems, 
— treatment schedule, 
— positive and negative controls, — autoradiographic technique used, 
— procedures used to block entry of cells into S-phase, 
— procedures used for DNA extraction and determination of total 
DNA content in LSC determination, 
— dose/response relationship, where possible, — statistical evaluation, 
— discussion of results, 
— interpretation of results.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 369
 
3.2. VALUATION AND INTERPRETATION 
See General introduction Part B.  
4. REFERENCES  
See General introduction Part B.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 370
 
B.19.  SISTER CHROMATID EXCHANGE ASSAY  IN VITRO  
1. METHOD  
1.1. INTRODUCTION 
See General introduction Part B.  
1.2. DEFINITION 
See General introduction Part B.  
1.3. REFERENCE SUBSTANCES 
None.  
1.4. PRINCIPLE OF THE TEST METHOD 
The Sister Chromatid Exchange (SCE) assay is a short-term test for 
the detection of reciprocal exchanges of DNA between two sister 
chromatids of a duplicating chromosome. SCEs represent the inter ­
change of DNA replication products at apparently homologous loci. The exchange process presumably involves DNA breakage and reunion, although little is known about its molecular basis. 
Detection of SCEs requires some means of differentially labelling 
sister chromatids and this can be achieved by incorporation of bromodeoxyuridine (BrdU) into chromosomal DNA for two cell cycles. 
Mammalian cells  in vitro  are exposed to the test chemical with and 
without a mammalian exogenous metabolic activation system, if 
appropriate, and cultured for two rounds of replication in BrdU- containing medium. After treatment with a spindle inhibitor (e.g. colchicine) to accumulate cells in a metaphase-like stage of mitosis 
(c-metaphase), cells are harvested and chromosome preparations are 
made.
 
1.5. QUALITY CRITERIA 
None.  
1.6. DESCRIPTION OF THE TEST METHOD  
1.6.1.  Preparations  
— Primary cultures, (human lymphocytes) or established cell lines 
(e.g. Chinese hamster ovary cells) may be used in the assay. Cell lines should be checked for  Mycoplasma  contamination, 
— appropriate culture media and incubation conditions (e.g. 
temperature, culture vessels, CO
 2 concentration and humidity) 
should be used, 
— test substances may be prepared in culture media or dissolved or 
suspended in appropriate vehicles prior to treatment of the cells. The final concentration of a vehicle in the culture system should 
not significantly affect, cell viability or growth rate and effects 
on SCE frequency should be monitored by a solvent control,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 371
 
— cells should be exposed to the test substance both in the presence 
and absence of an exogenous mammalian metabolic activation 
system. Alternatively, where cell types with intrinsic metabolic activity are used, the rate and nature of the activity should be appropriate to the chemical class being tested.
 
1.6.2.  Test conditions  
N u m b e r o f c u l t u r e s 
At least duplicate cultures should be used for each experimental 
point.  
U s e o f n e g a t i v e a n d p o s i t i v e c o n t r o l s 
Positive controls, using both a direct acting compound and a 
compound requiring metabolic activation should be included in 
each experiment; a vehicle control should also be used. 
The following are examples of substances which might be used as 
positive controls: 
— direct acting compound: 
— ethylmethanesulphonate, 
— indirect acting compound: 
— cyclophosphamide. 
When appropriate, an additional positive control of the same 
chemical class as the chemical under test may be included.  
E x p o s u r e c o n c e n t r a t i o n s 
At least three adequately spaced concentrations of the test substance 
should be used. The highest concentration should give rise to a significant toxic effect but must still allow adequate cell replication to occur. Relatively water-insoluble substances should be tested up 
to the limit of solubility using appropriate procedures. For freely 
water-soluble non-toxic substances the upper test substance concen ­
tration should be determined on a case-by-case basis.
 
1.6.3.  Procedure  
P r e p a r a t i o n o f c u l t u r e s 
Established cell lines are generated from stock cultures (e.g. by 
trypsinisation or by shaking off), seeded in culture vessels at appro ­
priate density and incubated at 37  o C. For monolayer cultures, the 
number of cells per culture vessel should be adjusted so that the cultures are not much more than 50 % confluent at the time of harvest. Alternatively, cells may be used in suspension culture. 
Human lymphocyte cultures are set up from heparinized blood, 
using appropriate techniques, and incubated at 37
 o C. ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 372
 
T r e a t m e n t 
Cells in an exponential stage of growth are exposed to the test 
substance for a suitable period of time; in most cases one to two hours may be effective, but the treatment time may be extended up to two complete cell cycles in certain cases. Cells 
without sufficient instrinsic metabolic activity should be exposed 
to the test chemical in the presence and absence of an appropriate metabolic activation system. At the end of the exposure period, cells are washed free of test substance and cultured for two rounds of 
replication in the presence of BrdU. As an alternative procedure cells 
may be exposed simultaneously to the test chemical and BrdU for the complete culture time of two cell cycles. 
Human lymphocyte cultures are treated while they are in a semisyn ­
chronous condition. 
Cells are analysed in their second post-treatment division, ensuring 
that the most sensitive cell cycle stages have been exposed to the chemical. All cultures to which BrdU is added should be handled in darkness or in dim light from incandescent lamps up to the 
harvesting of cells in order to minimise photolysis of BrdU- 
containing DNA.
 
H a r v e s t i n g o f c e l l s 
Cell cultures are treated with a spindle inhibitor (e.g. colchicine) one 
to four hours prior to harvesting. Each culture is harvested and processed separately for the preparation of chromosomes.
 
C h r o m o s o m e p r e p a r a t i o n a n d s t a i n i n g 
Chromosome preparations are made by standard cytogenetic tech ­
niques. Staining of slides to show SCEs can be performed by several techniques, (e.g. the fluorescence plus Giemsa method).
 
A n a l y s i s 
The number of cells analysed should be based on the spontaneous 
control frequency of SCE. Usually, at least 25 well-spread meta ­
phases per culture are analysed for SCEs. Slides are coded before 
analysis. In human lymphocytes only metaphases containing 46 
centromeres are analysed. In established cell lines only metaphases containing ± 2 centromeres of the modal number are analysed. It should be stated whether or not centromeric switch of label is, scored 
as an SCE. The results should be confirmed in an independent 
experiment.
 
2. DATA  
Data should be presented in tabular form. The number of SCEs for 
each metaphase and the number of SCEs per chromosome for each 
metaphase should be listed separately for all treated and control 
cultures. 
The data should be evaluated using appropriate statistical methods.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 373
 
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, contain the following information: 
— cells used, methods of maintenance of cell culture, — test conditions: composition of media, CO
 2 concentration, 
concentration of test substance, vehicle used, incubation 
temperature, treatment time, spindle inhibitor used, its concen ­
tration and the duration of treatment with it, type of mammalian activation system used, positive and negative controls, 
— number of cell cultures per experimental point, 
— details of the technique used for slide preparation, — number of metaphases analysed (data given separately for each 
culture), 
— mean number of SCE per cell and per chromosome (data given 
separately for each culture), 
— criteria for scoring SCE, — rationale for dose selection, 
— dose-response relationship, if applicable, 
— statistical evaluation, — discussion of results, — interpretation of results.
 
3.2. EVALUATION AND INTERPRETATION 
See General introduction Part B.  
4. REFERENCES  
See General introduction Part B.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 374
 
B.20.  SEX-LINKED RECESSIVE LETHAL TEST IN  DROSOPHILA 
MELANOGASTER  
1. METHOD  
1.1. INTRODUCTION 
See General introduction Part B.  
1.2. DEFINITION 
See General introduction Part B.  
1.3. REFERENCE SUBSTANCES 
None.  
1.4. PRINCIPLES OF THE TEST METHOD 
The sex-linked recessive lethal (SLRL) test using  Drosophila 
melanogaster  detects the occurrence of mutations, both point 
mutations and small deletions, in the germ line of the insect. This 
test is a forward mutation assay capable of screening for mutations at about 800 loci on the X-chromosome; this represents about 80 % of all X-chromosal loci. The X-chromosome represents approximately 
one-fifth of the entire haploid genome. 
Mutations in the X-chromosome of  Drosophila melanogaster  are 
phenotypically expressed in males carrying the mutant gene. When the mutation is lethal in the hemizygous condition, its presence is 
inferred from the absence of one class of male offspring out of the 
two that are normally produced by a heterozygous female. The SLRL test takes advantage of these facts by means of specially marked and arranged chromosomes.
 
1.5. QUALITY CRITERIA 
None.  
1.6. DESCRIPTION OF THE TEST METHOD  
Preparations  
S t o c k s 
Males of a well-defined wild-type stock and females of the Muller-5 
stock may be used. Other appropriately marked female stocks with multiple inverted X-chromosomes may also be used.
 
T e s t s u b s t a n c e 
Test substances should be dissolved in water. Substances, which are 
insoluble in water may be dissolved or suspended in appropriate vehicles (e.g. a mixture of ethanol and Tween-60 or 80), then 
diluted in water or saline prior to administration. Dimethylsulphoxide 
(DMSO) should be avoided as a vehicle.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 375
 
N u m b e r o f a n i m a l s 
The test should be designed with a predetermined sensitivity and 
power. The spontaneous mutant frequency observed in the appro ­
priate control will influence strongly the number of treated chro ­
mosomes that must be analysed.  
R o u t e o f a d m i n i s t r a t i o n 
Exposure may be oral, by injection or by exposure to gases or 
vapours. Feeding of the test substance may be done in sugar solution. When necessary, substances may be dissolved in a 0,7 % NaCl solution and injected into the thorax or abdomen.
 
U s e o f n e g a t i v e a n d p o s i t i v e c o n t r o l s 
Negative (vehicle) and positive controls should be included. 
However, if appropriate laboratory historical control data are available, no concurrent controls are needed.
 
E x p o s u r e l e v e l s 
Three exposure levels should be used. For a preliminary assessment 
one exposure level of the test substance may be used, that exposure level being either the maximum tolerated concentration or that producing some indication of toxicity. For non-toxic substances 
exposure to the maximum practicable concentration should be used.
 
P r o c e d u r e 
Wild-type males (three to five days old) are treated with the test 
substance and mated individually to an excess of virgin females from 
the Muller-5 stock or from another appropriately marked (with 
multiple inverted X-chromosomes) stock. The females are replaced with fresh virgins every two to three days to cover the entire germ cell cycle. The offspring of these females are scored for lethal effects 
corresponding to the effects on mature sperm, mid or late-stage 
spermatids, early spermatids, spermatocytes and spermatogonia at the time of treatment. 
Heterozygous F
 1 females from the above crosses are allowed to mate 
individually (i.e. one female per vial) with their brothers. In the F  2 
generation, each culture is scored for the absence of wild-type males. 
If a culture appears to have arisen from an F  1 female carrying a 
lethal in the parental X-chromosome (i.e. no males with the treated chromosome are observed) daughters of that female with the same 
genotype should be tested to ascertain whether the lethality is 
repeated in the next generation.
 
2. DATA  
Data should be tabulated to show the number of X-chromosomes 
tested, the number of non-fertile males and the number of lethal chromosomes at each exposure concentration and for each mating period for each male treated. Numbers of clusters of different sizes 
per male should be reported. These results should be confirmed in a 
separate experiment.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 376
 
Appropriate statistical methods should be used in evaluation sex- 
linked recessive lethal tests. Clustering of recessive lethals orig ­
inating from one male should be considered and evaluated in an appropriate statistical manner.
 
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, contain the following information: 
— stock: Drosophila stocks or strains used, age of insects, number 
of males treated, number of sterile males, number of F2 cultures 
established, number of F2 cultures without progeny, number of chromosomes carrying a lethal detected at each germ cell stage, 
— criteria for establishing the size of treated groups, — test conditions. detailed description of treatment and sampling 
schedule, exposure levels, toxicity data, negative (solvent) and 
positive controls, if appropriate, 
— criteria for scoring lethal mutations, — exposure/effect relationship where possible, — evaluation of data, — discussion of results, — interpretation of results.
 
3.2. EVALUATION AND INTERPRETATION 
See General introduction Part B.  
4. REFERENCES  
See General introduction Part B.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 377
 
B.21.  IN VITRO  MAMMALIAN CELL TRANSFORMATION TESTS  
1. METHOD  
1.1. INTRODUCTION 
See General introduction Part B.  
1.2. DEFINITION 
See General introduction Part B.  
1.3. REFERENCE SUBSTANCES 
None.  
1.4. PRINCIPLE OF THE TEST METHOD 
Mammalian cell culture systems may be used to detect phenotypic 
changes in vitro induced by chemical substances associated with 
malignant transformation  in vivo . Widely used cells include 
C3H10T  1/2 , 3T3, SHE, Fischer rat and the tests rely on changes in 
cell morphology, focus formation or changes in anchorage dependence in semi-solid agar. Less widely used systems exist 
which detect other physiological or morphological changes in cells 
following exposure to carcinogenic chemicals. None of the  in vitro  
test endpoints has an established mechanistic link with cancer. Some of the test systems are capable of detecting tumour promotors. Cytot ­
oxicity may be determined by measuring the effect of the test 
material on colony-forming abilities (cloning efficiency) or growth rates of the cultures. The measurement of cytotoxicity is to establish that exposure to the test chemical has been toxicologically relevant 
but cannot be used to calculate transformation: frequency in all 
assays since some may involve prolonged incubation and/or replating.
 
1.5. QUALITY CRITERIA 
None.  
1.6. DESCRIPTION OF THE TEST METHOD  
Preparations  
C e l l s 
A variety of cell lines or primary cells are available depending on the 
transformation test being used. The investigator must ensure that the 
cells in the test being performed exhibit the appropriate phenotypic change after exposure to known carcinogens and that the test, in the investigator's laboratory, is of proven and documented validity and 
reliability.
 
M e d i u m 
Media and experimental conditions should be used that are appro ­
priate to the transformation assay in use.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 378
 
T e s t s u b s t a n c e 
Test substances may be prepared in culture media or dissolved or 
suspended in appropriate vehicles prior to treatment of the cells. The final concentration of the vehicle in the culture system should not affect cell viability, growth rate or transformation incidence.
 
M e t a b o l i c a c t i v a t i o n 
Cells should be exposed to the test substance both in the presence 
and absence of an appropriate metabolic activation system. Alter ­
natively, when cell types are used that possess intrinsic metabolic activity, the nature of the activity should be known to be appropriate to the chemical class being tested.
 
Test conditions  
U s e o f n e g a t i v e a n d p o s i t i v e c o n t r o l s 
Positive controls, using both a direct-acting compound and a 
compound requiring metabolic activation should be included in each experiment; a negative (vehicle) control should also be used. 
The following are examples of substances, which might be used as 
positive controls: 
— Direct-acting chemicals: 
— Ethylmethanesulphonate, 
— β-propiolactone, 
— Compounds requiring metabolic activation: 
— 2-acetylaminofluorene, 
— 4-dimethylaminoazobenzene, 
— 7,12-dimethylbenzanthracene. 
When appropriate, an additional positive control of the same 
chemical class as the compound under test should be included.
 
E x p o s u r e c o n c e n t r a t i o n s 
Several concentrations of the test substance should be used. These 
concentrations should yield a concentration-related toxic effect, the highest concentration producing a low level of survival and the 
survival in the lowest concentration being approximately the same 
as that in the negative control. Relatively water-insoluble substances should be tested up to the limit of solubility using appropriate procedures. For freely water-soluble non-toxic substances the upper 
test substance concentration should be determined on a case-by-case 
basis.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 379
 
Procedure  
Cells should be exposed for a suitable period of time depending on 
the test system in use, and this may involve re-dosing accompanied by a change of medium (and if necessary, fresh metabolic activation mixture) if exposure is prolonged. Cells without sufficient intrinsic 
metabolic activity should be exposed to the test substance in the 
presence and absence of an appropriate metabolic activation system. At the end of the exposure period, cells are washed free of test substance and cultured under conditions appropriate for the 
appearance of the transformed phenotype being monitored and the 
incidence of transformation determined. All results are confirmed in an independent experiment.
 
2. DATA  
Data should be presented in tabular form and may take a variety of 
forms according to the assay being used e.g. plate counts, positive 
plates or numbers of transformed cells. Where appropriate, survival 
should be expressed as a percentage of control levels and trans ­
formation frequency expressed as the number of transformants per number of survivors. Data should be evaluated using appropriate 
statistical methods.
 
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, contain the following information: 
— cell type used, number of cell cultures, methods for maintenance 
of cell cultures, 
— test conditions: concentration of test substance, vehicle used, 
incubation time, duration and frequency of treatment, cell 
density during treatment, type of exogenous metabolic activation 
system used, positive and negative controls, specification of phenotype being monitored, selective system used (if appro ­
priate), rational for dose selection, 
— method used to enumerate viable and transformed cells, 
— statistical evaluation, — discussion of results, — interpretation of results.
 
3.2. EVALUATION AND INTERPRETATION 
See General introduction Part B.  
4. REFERENCES  
See General introduction Part B.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 380
 
B.22.  RODENT DOMINANT LETHAL TEST  
1. METHOD  
1.1. INTRODUCTION 
See General introduction Part B.  
1.2. DEFINITION 
See General introduction Part B.  
1.3. REFERENCE SUBSTANCES 
None.  
1.4. PRINCIPLE OF THE TEST METHOD 
Dominant lethal effects cause embryonic or foetal death. Induction of 
dominant lethals by exposure to a chemical substance indicates that 
the substance has affected germinal tissue of the test species. It is generally accepted that dominant lethals are due to chromosomal damage (structural and numerical anomalies). Embryonic death if 
females are treated may also be the result of toxic effects. 
Generally, male animals are exposed to the test compound and mated 
to untreated virgin females. The various germ cell stages can be tested separately by the use of sequential mating intervals. The increase of dead implants per female in the treated group over the 
dead implants per female in the control group reflects the post- 
implantational loss. Pre-implantational loss can be estimated based on corpora lutea counts or by comparing the total implants per female in treated and control groups. The total dominant lethal 
effect is the sum of pre- and post-implantational loss. The calculation 
of the total dominant lethal effect is based on comparison of the live implants per female in the test group to the live implants per female in the control group. A reduction in the number of implants at 
certain intervals may be the result of cell killing (i.e. of sperma ­
tocytes and/or spermatogonia).
 
1.5. QUALITY CRITERIA 
None.  
1.6. DESCRIPTION OF THE TEST METHOD  
Preparations  
When possible, test substances should be dissolved or suspended in 
isotonic saline. Chemicals insoluble in water may be dissolved or suspended in appropriate vehicles. The vehicle used should neither 
interfere with the test chemical nor produce toxic effects. Fresh 
preparations of the test chemical should be employed.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 381
 
Test conditions  
R o u t e o f a d m i n i s t r a t i o n 
The test compound should generally be administered only once. 
Based on toxicological information a repeated treatment schedule can be employed. The usual routes of administration are oral intu ­
bation or intraperitoneal injection. Other routes of administration 
may be appropriate.
 
E x p e r i m e n t a l a n i m a l s 
Rats or mice are recommended as the test species. Healthy fully 
sexually mature animals are randomised and assigned to treatment 
and control groups.  
N u m b e r a n d s e x 
An adequate number of treated males should be used, taking into 
account the spontaneous variation of the biological character being 
evaluated. The number chosen should be based on the pre- 
determined sensitivity of detection and power of significance. For example in a typical test, the number of males in each dose group should be sufficient to provide between 30 and 50 pregnant females 
per mating interval.
 
U s e o f n e g a t i v e a n d p o s i t i v e c o n t r o l s 
Generally concurrent positive and negative (vehicle) controls should 
be included in each experiment. When acceptable positive control 
results are available from experiments conducted recently in the 
same laboratory these results can be used instead of a concurrent positive control. Positive control substances should be used at an appropriate low dose (e.g. MMS, intraperitoneally, at 10 mg/ ­
kilogram) to demonstrate the test sensitivity.
 
D o s e l e v e l s 
Normally, three dose levels should be used. The high dose should 
produce signs of toxicity or reduced fertility in the treated animals. In certain cases a single high dose level may be sufficient.
 
L i m i t t e s t 
Non-toxic substances should be tested at 5 g/kilogram on a single 
administration or at 1 g/kilogram/day on repeated administration.  
Procedure  
Several treatment schedules are available. Single administration of 
the test substance is the most widely used. Other treatment schedules 
may be used. 
Individual males are mated sequentially to one or two untreated 
virgin females at appropriate intervals after treatment. Females should be left with the males for at least the duration of one 
oestrous cycle or until mating has occurred as determined by the 
presence of sperm in the vagina or by the presence of a vaginal plug.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 382
 
The number of matings following treatment is governed by the 
treatment schedule and should ensure that all germ cell stages are 
sampled after treatment. 
Females are sacrificed in the second half of pregnancy and uterine 
contents are examined to determine the number of dead and live 
implants. The ovaries may be examined to determine the number of corpora lutea.
 
2. DATA  
Data should be tabulated to show the number of males, the number 
of pregnant females, and the number of non-pregnant females. Results of each mating, including the identity of each male and 
female, should be reported individually. For each female, week of 
mating, dose level received by the males, the frequencies of live implants and of dead implants should be recorded. 
The calculation of the total dominant lethal effect is based on 
comparison of the live implants per female in the test group to the live implants per female in the control group. The ratio of dead to live implants from the treated group compared to the same ratio from 
the control group is analysed to indicate the post-implantation loss. 
If the data are recorded as early and late deaths, the tables should 
make that clear. If pre-implantation loss is estimated, it should be 
reported. Pre-implantation loss can be calculated as a discrepancy between the number of corpora lutea and the number of implants or as a reduction in the average number of implants per uterus in 
comparison with control matings. 
Data are evaluated using appropriate statistical methods.
 
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, contain the following information: 
— species, strain, age and weights of animals used, number of 
animals of each sex in experimental and control groups, 
— test substance, vehicle, dose levels tested and rationale for dose 
selection, negarive and positive controls, toxicity data, 
— route and treatment schedule, 
— mating schedule, 
— method used to determine that mating has occurred, — time of sacrifice, 
— criteria for scoring dominant lethals, 
— dose/response relationship, if applicable,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 383
 
— statistical evaluation, 
— discussion of results, — interpretation of results.
 
3.2. EVALUATION AND INTERPRETATION 
See General introduction Part B.  
4. REFERENCES  
See General introduction Part B.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 384
 
B.23.  MAMMALIAN SPERMATOGONIAL CHROMOSOME 
ABERRATION TEST  
1. METHOD  
This method is a replicate of the OECD TG 483, Mammalian Sper ­
matogonial Chromosome Aberration Test (1997).  
1.1. INTRODUCTION 
The purpose of the  in vivo  mammalian spermatogonial chromosome 
aberration test is to identify those substances that cause structural chro ­
mosome aberrations in mammalian spermatogonial cells (1)(2)(3)(4)(5). Structural aberrations may be of two types, chromosome or chromatid. With the majority of chemical mutagens, induced aberrations are of the 
chromatid type, but chromosome-type aberrations also occur. This 
method is not designed to measure numerical aberrations and is not routinely used for that purpose. Chromosome mutations and related events are the cause of many human genetic diseases. 
This test measures chromosome events in spermatogonial germ cells 
and is, therefore, expected to be predictive of induction of inheritable 
mutations in germ cells. 
Rodents are routinely used in this test. This  in vivo  cytogenetic test 
detects chromosome aberrations in spermatogonial mitoses. Other target cells are not the subject of this method. 
To detect chromatid-type aberrations in spermatogonial cells, the first 
mitotic cell division following treatment should be examined before 
these lesions are lost in subsequent cell divisions. Additional 
information from treated spermatogonial stem cells can be obtained by meiotic chromosome analysis for chromosome-type aberrations at diakinesis-metaphase I when the treated cells become spermatocytes. 
This in vivo  test is designed to investigate whether somatic cell 
mutagens are also active in germ cells. In addition, the spermato ­
gonial test is relevant to assessing mutagenicity hazard in that it 
allows consideration of factors of  in vivo  metabolism, pharmacoki ­
netics and DNA-repair processes. 
A number of generations of spermatogonia are present in the testis 
with a spectrum of sensitivity to chemical treatment. Thus, the aber ­
rations detected represent an aggregate response of treated sperma ­
togonial cell populations, with the more numerous differentiated spermatogonial cells predominating. Depending on their position within the testis, different generations of spermatogonia may or 
may not be exposed to the general circulation, because of the 
physical and physiological Sertoli cell barrier and the blood-testis barrier. 
If there is evidence that the test substance, or a reactive metabolite, 
will not reach the target tissue, it is not appropriate to use this test. 
See also General introduction Part B.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 385
 
1.2. DEFINITIONS  
Chromatid-type aberration:  structural chromosome damage 
expressed as breakage of single chromatids or breakage and reunion between chromatids.  
Chromosome-type aberration:  structural chromosome damage 
expressed as breakage, or breakage and reunion, of both chromatids 
at an identical site.  
Gap:  an achromatic lesion smaller than the width of one chromatid, 
and with minimum misalignment of the chromatids.  
Numerical aberration:  a change in the number of chromosomes 
from the normal number characteristic of the animals utilised.  
Polyploidy:  a multiple of the haploid chromosome number ( n) other 
than the diploid number (i.e. 3 n, 4n and so on).  
Structural aberration:  a change in chromosome structure detectable 
by microscopic examination of the metaphase stage of cell division, observed as deletions, intrachanges or interchanges.
 
1.3. PRINCIPLE OF THE TEST METHOD 
Animals are exposed to the test substance by an appropriate route of 
exposure and are sacrificed at appropriate times after treatment. Prior 
to sacrifice, animals are treated with a metaphase-arresting substance 
(e.g. Colcemid® or colchicine). Chromosome preparations are then made from germ cells and stained, and metaphase cells are analysed for chromosome aberrations.
 
1.4. DESCRIPTION OF THE TEST METHOD  
1.4.1.  Preparations  
1.4.1.1.  Selection of animal species  
Male Chinese hamsters and mice are commonly used. However, 
males of other appropriate mammalian species may be used. Commonly used laboratory strains of young healthy adult animals should be employed. At the commencement of the study the weight 
variation of animals should be minimal and not exceed ± 20 % of 
the mean weight.
 
1.4.1.2.  Housing and feeding conditions  
General conditions referred in the General introduction to Part B are 
applied although the aim for humidity should be 50-60 %.  
1.4.1.3.  Preparation of the animals  
Healthy young adult males are randomly assigned to the control and 
treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimised. The animals 
are identified uniquely. The animals are acclimated to the laboratory 
conditions for at least five days prior to the start of the study.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 386
 
1.4.1.4.  Preparation of doses  
Solid test substances should be dissolved or suspended in appropriate 
solvents or vehicles and diluted, if appropriate, prior to dosing of the animals. Liquid test substances may be dosed directly or diluted 
prior to dosing. Fresh preparations of the test substance should be 
employed unless stability data demonstrate the acceptability of storage.
 
1.4.2.  Test conditions  
1.4.2.1.  Solvent/Vehicle  
The solvent/vehicle should not produce toxic effects at the dose 
levels used and should not be suspected of chemical reaction with 
the test substance. If other than well-known solvents/vehicles are used, their inclusion should be supported by data indicating their compatibility. It is recommended that wherever possible, the use of 
an aqueous solvent/vehicle should be considered first.
 
1.4.2.2.  Controls  
Concurrent positive and negative (solvent/vehicle) controls should be 
included in each test. Except for treatment with the test substance, animals in the control groups should be handled in an identical 
manner to animals in the treated groups. 
Positive controls should produce structural chromosome aberrations  
in vivo  in spermatogonial cells when administered at exposure levels 
expected to give a detectable increase over background. 
Positive control doses should be chosen so that the effects are clear 
but do not immediately reveal the identity of the coded slides to the 
reader. It is acceptable that the positive control be administered by a 
route different from the test substance and sampled at only a single time. In addition, the use of chemical class-related positive control 
chemicals may be considered, when available. Examples of positive 
control substances include:
 
Substance CAS No Einecs No  
Cyclophosphamide 
Cyclophosphamide monohydrate 50-18-0 
6055-19-2 200-015-4 
Cyclohexylamine 108-91-8 203-629-0 
Mitomycin C 50-07-7 200-008-6 
Monomeric acrylamide 79-06-1 201-173-7 
Triethylenemelamine 51-18-3 200-083-5 
Negative controls, treated with solvent or vehicle alone, and 
otherwise treated in the same way as the treatment groups, should 
be included for every sampling time, unless acceptable inter-animal 
variability and frequency of cells with chromosome aberrations are demonstrated by historical control data. In addition, untreated controls should also be used unless there are historical or 
published control data demonstrating that no deleterious or 
mutagenic effects are induced by the chosen solvent/vehicle.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 387
 
1.5. PROCEDURE  
1.5.1.  Number of animals  
Each treated and control group must include at least five analysable 
males.  
1.5.2.  Treatment schedule  
Test substances are preferably administered once or twice (i.e. as a 
single treatment or as two treatments). Test substances may also be 
administered as a split dose, i.e. two treatments on the same day separated by no more than a few hours, to facilitate administering a large volume of material. Other dose regimens should be scien ­
tifically justified. 
In the highest dose group two sampling times after treatment are 
used. Since cell cycle kinetics can be influenced by the test substance, one early and one late sampling time are used around 24 and 48 hours after treatment. For doses other than the highest 
dose, a sampling time of 24 hours or 1,5 cell cycle length after 
treatment should be taken, unless another sampling time is known to be more appropriate for detection of effects (6). 
In addition, other sampling times may be used. For example in the 
case of chemicals, which may induce chromosome lagging, or may 
exert S-independent effects, earlier sampling times may be appro ­
priate (1). 
The appropriateness of a repeated treatment schedule needs to be 
identified on a case-by-case basis. Following a repeated treatment schedule the animals should then be sacrificed 24 hours (1,5 cell 
cycle length) after the last treatment. Additional sampling times may 
be used where appropriate. 
Prior to sacrifice, animals are injected intraperitoneally with an 
appropriate dose of a metaphase arresting substance (e.g. Colcemid® or colchicine). Animals are sampled at an appropriate 
interval thereafter. For mice this interval is approximately three to 
five hours, for Chinese hamsters this interval is approximately four to five hours.
 
1.5.3.  Dose levels  
If a range finding study is performed because there are no suitable 
data available, it should be performed in the same laboratory, using the same species, strain and treatment regimen to be used in the main 
study (7). If there is toxicity, three dose levels are used for the first 
sampling time. These dose levels should cover a range from the maximum to little or no toxicity. At the later sampling time only the highest dose needs to be used. The highest dose is defined as the 
dose producing signs of toxicity such that higher dose levels, based 
on the same dosing regimen, would be expected to produce lethality.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 388
 
Substances with specific biological activities at low non-toxic doses 
(such as hormones and mitogens) may be exceptions to the dose- 
setting criteria and should be evaluated on a case-by-case basis. The highest dose may also be defined as a dose that produces some indication of toxicity in the spermatogonial cells (e.g. a reduction 
in the ratio of spermatogonial mitoses to first and second meiotic 
metaphases; this reduction should not exceed 50 %).
 
1.5.4.  Limit test  
If a test at one dose level of at least 2000 mg/kg body weight/day 
using a single treatment, or as two treatments on the same day, produces no observable toxic effects, and if genotoxicity would 
not be expected based upon data from structurally related substances, 
then a full study using three dose levels may not be considered necessary. Expected human exposure may indicate the need for a higher dose level to be used in the limit test.
 
1.5.5.  Administration of doses  
The test substance is usually administered by gavage using a 
stomach tube or a suitable intubation cannula, or by intraperitoneal injection. Other routes of exposure may be acceptable where they can be justified. The maximum volume of liquid that can be admin ­
istered by gavage or injection at one time depends on the size of the 
test animal. The volume should not exceed 2 ml/100 g body weight. The use of volumes higher than these must be justified. Except for irritating or corrosive substances, which will normally reveal 
exacerbated effects with higher concentrations, variability in test 
volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels.
 
1.5.6.  Chromosome preparation  
Immediately after sacrifice, cell suspensions are obtained from one or 
both testes, exposed to hypotonic solution and fixed. The cells are 
then spread on slides and stained.  
1.5.7.  Analysis  
For each animal at least 100 well-spread metaphase should be 
analysed (i.e. a minimum of 500 metaphases per group). This number could be reduced when high numbers of aberrations are 
observed. All slides, including those of positive and negative 
controls, should be independently coded before microscopic analysis. Since fixation procedures often result in the breakage of a proportion of metaphases with loss of chromosomes, the cells 
scored should contain a number of centromeres equal to the 
number 2n ± 2.
 
2. DATA  
2.1. TREATMENT OF RESULTS 
Individual animal data should be presented in a tabular form. The 
experimental unit is the animal. For each individual animal the 
number of cells with structural chromosome aberrations and the 
number of chromosome aberrations per cell should be evaluated. Different types of structural chromosome aberrations should be listed with their numbers and frequencies for treated and control 
groups. Gaps are recorded separately and reported but generally 
not included in the total aberration frequency.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 389
 
If mitosis as well as meiosis is observed, the ratio of spermatogonial 
mitoses to first and second meiotic metaphases should be determined 
as a measure of cytotoxicity for all treated and negative control animals in a total sample of 100 dividing cells per animal to establish a possible cytotoxic effect. If only mitosis is observed, 
the mitosis index should be determined in at least 1 000 cells for 
each animal.
 
2.2. EVALUATION AND INTERPRETATION OF RESULTS 
There are several criteria for determining a positive result, such as a 
dose-related increase in the relative number of cells with chro ­
mosome aberrations or a clear increase in the number of cells with 
aberrations in a single dose at a single sampling time. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results (8). 
Statistical significance should not be the only determining factor for 
a positive response. Equivocal results should be clarified by further testing preferably using a modification of experimental conditions. 
A test substance for which the results do not meet the above criteria 
is considered non-mutagenic in this test. 
Although most experiments will give clearly positive or negative 
results, in rare cases the data set will preclude making a definite 
judgement about the activity of the test substance. Results may 
remain equivocal or questionable regardless of the number of times the experiment is repeated. 
Positive results from the  in vivo  spermatogonial chromosome aber ­
ration test indicate that the test substance induces structural chro ­
mosome aberrations in the germ cells of the species tested. Negative results indicate that, under the test conditions, the test substance does not induce chromosome aberrations in the germ cells of the species 
tested. 
The likelihood that the test substance or its metabolites reach the 
target tissue should be discussed.
 
3. REPORTING  
TEST REPORT 
The test report must include the following information: 
Solvent/Vehicle: 
— justification for choice of vehicle, 
— solubility and stability of the test substance in solvent/vehicle, if 
known. 
Test animals: 
— species/strain used, 
— number and age of animals, — source, housing conditions, diet, etc., 
— individual weight of the animals at the start of the test, including 
body weight range, mean and standard deviation for each group.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 390
 
Test conditions: 
— data from range finding study, if conducted, 
— rationale for dose level selection, 
— rationale for route of administration, 
— details of test substance preparation, 
— details of the administration of the test substance, 
— rationale for sacrifice times, — conversion from diet/drinking water test substance concentration 
(ppm) to the actual dose (mg/kg body weight/day), if applicable. 
— details of food and water quality, 
— detailed description of treatment and sampling schedules, 
— methods for measurement of toxicity, 
— identity of metaphase arresting substance, its concentration and 
duration of treatment, 
— methods of slide preparation, 
— criteria for scoring aberrations, 
— number of cells analysed per animal, 
— criteria for considering studies as positive, negative or equivocal. 
Results: 
— signs of toxicity, 
— mitotic index, — ratio of spermatogonial mitoses cells to first and second meiotic 
metaphases, 
— type and number of aberrations, given separately for each animal, 
— total number of aberrations per group, 
— number of cells with aberrations per group, — dose-response relationship, if possible, 
— statistical analyses, if any, 
— concurrent negative control data, 
— historical negative control data with ranges, means and standard 
deviations, 
— concurrent positive control data, 
— changes in ploidy, if seen. Discussion of results. 
Conclusions.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 391
 
4. REFERENCES  
(1) Adler, I.D., (1986) Clastogenic Potential in Mouse Sperma ­
togonia of Chemical Mutagens Related to their Cell-Cycle Spec ­
ifications. In: Genetic Toxicology of Environmental Chemicals, 
Part B: Genetic Effects and Applied Mutagenesis, Ramel, C., 
Lambert, B. and Magnusson, J. (Eds.) Liss, New York, p. 477-484. 
(2) Adler, I.D., (1984) Cytogenetic tests in Mammals. In: Mutage ­
nicity Testing: a Practical Approach. Ed. S. Venitt and J.M. 
Parry. IRL Press, Oxford, Washington DC, p. 275-306. 
(3) Evans, E.P., Breckon, G. and Ford, C.E., (1964) An Air-Drying 
Method for Meiotic Preparations from Mammalian Testes. Cyto ­
genetics and Cell Genetics, 3, p. 289-294. 
(4) Richold, M., Ashby, J., Chandley, A., Gatehouse, D.G. and 
Henderson, L., (1990)  In Vivo  Cytogenetic Assays, In: D.J. 
Kirkland (Ed.) Basic Mutagenicity Tests, UKEMS Recom ­
mended Procedures. UKEMS Subcommittee on Guidelines for 
Mutagenicity Testing. Report. Part I revised. Cambridge 
University Press, Cambridge, New York, Port Chester, Melbourne, Sydney, p. 115-141. 
(5) Yamamoto, K. and Kikuchi, Y., (1978) A New Method for 
Preparation of Mammalian Spermatogonial Chromosomes. 
Mutation Res., 52, p. 207-209. 
(6) Adler, I.D., Shelby M.D., Bootman, J., Favor, J., Generoso, W., 
Pacchierotti, F., Shibuya, T. and Tanaka N., (1994) International Workshop on Standardisation of Genotoxicity Test Procedures. 
Summary Report of the Working Group on Mammalian Germ 
Cell Tests. Mutation Res., 312, p. 313-318. 
(7) Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, J., 
Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B., Kirkland, D.J. and Richold, M., (1992) Report of 
British Toxicology Society/UK Environmental Mutagen 
Society Working group: Dose setting in  In Vivo  Mutagenicity 
Assays. Mutagenesis, 7, p. 313-319. 
(8) Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., 
Clare, G., Ferguson, R., Richold, M., Papworth, D.G. and 
Savage, J.R.K., (1989) Statistical Analysis of  In Vivo  Cyto­
genetic Assays In: D.J. Kirkland (Ed.) Statistical Evaluation of Mutagenicity Test Data. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing, report, Part III. Cambridge University 
Press, Cambridge, New York, Port Chester, Melbourne, Sydney, 
p. 184-232.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 392
 
B.24.  MOUSE SPOT TEST  
1. METHOD  
1.1. INTRODUCTION 
See General introduction Part B.  
1.2. DEFINITION 
See General introduction Part B.  
1.3. REFERENCE SUBSTANCES 
None.  
1.4. PRINCIPLE OF THE TEST METHOD 
This is an  in vivo  test in mice in which developing embryos are 
exposed to the chemicals. The target cells in the developing embryos are melanoblasts, and the target genes are those which control the 
pigmentation of the coat hairs. The developing embryos are 
heterozygous for a number of these coat colour genes. A mutation in, or loss of (by a variety of genetic events), the dominant allele of such a gene in a melanoblast results in the expression of the 
recessive phenotype in its descendant cells, constituting a spot of 
changed colour in the coat of the resulting mouse. The number of offspring with these spots, mutations, are scored and their frequency is compared with that among offspring resulting from embryos 
treated with the solvent only. The mouse spot test detects 
presumed somatic mutations in foetal cells.
 
1.5. QUALITY CRITERIA 
None.  
1.6. DESCRIPTION OF THE TEST METHOD  
Preparations  
When possible, test substances are dissolved or suspended in isotonic 
saline. Chemicals insoluble in water are dissolved or suspended in 
appropriate vehicles. The vehicle used should neither interfere with the test chemical nor produce toxic effects. Fresh preparations of the test chemical should be used.
 
E x p e r i m e n t a l a n i m a l s 
Mice of the T strain (nonagouti, a/a; chinchilla, pink eye, c  ch p/c ch p; 
brown, b/b; dilute, short ear, d se/d se; piebald spotting, s/s) are 
mated either with the HT strain (pallid, nonagouti, brachypody, pa a bp/pa a bp; leaden fuzzy, ln fz/ln fz; pearl pe/pe) or with C57BL (nonagouti, a/a). Other appropriate crosses such as between NMRI 
(nonagouti, a/a; albino, c/c) and DBA (nonagouti, a/a; brown, b/b; 
dilute d/d) may be used provided they produce nonagouti offspring.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 393
 
N u m b e r a n d s e x 
Sufficient pregnant females are treated to provide an appropriate 
number of surviving offspring for each dose level used. The appro ­
priate sample size in governed by the number of spots observed in the treated mice and the scale of the control data. A negative result is 
acceptable only when at least 300 offspring from females treated 
with the highest dose have been scored.
 
U s e o f n e g a t i v e a n d p o s i t i v e c o n t r o l s 
Concurrent control data from mice treated with the vehicle only 
(negative controls) should be available. Historical control data 
from the same laboratory may be pooled to increase the sensitivity 
of the test provided they are homogeneous. Positive control data recently obtained in the same laboratory from treatment with a chemical known to show mutagenicity by this test should be 
available if no mutagenicity of the test chemical is detected.
 
R o u t e o f a d m i n i s t r a t i o n 
The usual routes of administration are oral intubation or intraperi ­
toneal injection of the pregnant females. Treatment by inhalation or other routes of administration are used when appropriate.
 
D o s e l e v e l s 
At least two dose levels are used including one showing signs of 
toxicity or reduced litter size. For non-toxic chemicals exposure to the maximum practicable dose should be used.
 
Procedure  
A single treatment is normally given on day 8,9 or 10 of pregnancy, 
counting as day 1 the day on which the vaginal plug is first 
dbserved. These days correspond to 7,25, 8,25 and 9,25 days after conception. Successive treatments over these days may be used.
 
A n a l y s i s 
The offspring are coded and scored for spots between three and four 
weeks after birth. Three classes of spots are distinguished: 
(a) white spots within 5 mm of the mid-ventralline which are 
presumed to result from cell killing (WMVS); 
(b) yellow, agouti-like, spots associated with mammae, genitalia, 
throat, axillary and inguinal areas and on the mid-forehead, 
which are presumed to result from misdifferentiation (MDS); and 
(c) pigmented and white spots randomly distributed on the coat 
which are presumed to result from somatic mutations (RS).  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 394
 
All three classes are scored but only the last, RS, is of genetic 
relevance. Problems of distinguishing between MDS and RS may 
be solved by fluorescence microscopy of sample hairs. 
Obvious gross morphological abnormalities of the offspring should 
be noted.  
2. DATA  
The data are presented as the total number of offspring scored and 
the number having one or more presumed somatic mutation spots. 
Treatment and negative control data are compared by appropriate 
methods. Data are also presented on a per-litter basis.  
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, contain the following information: 
— the strains used in the cross, — the number of pregnant females in the experimental and control 
groups, 
— the average litter size in the experimental and control groups at 
birth and at weaning, 
— the dose level(s) of the test chemical, — the solvent used, — the day of pregnancy of which treatment was given, — the route of treatment, — the total number of offspring scored, and the number with 
WMVS, MDS and RS in the experimental and control groups, 
— gross morphological abnormalities, — dose/response relationship of RS when possible, — statistical evaluation, — discussion of results, — interpretation of results.
 
3.2. EVALUATION AND INTERPRETATION 
See General introduction Part B.  
4. REFERENCES  
See General introduction Part B.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 395
 
B.25.  MOUSE HERITABLE TRANSLOCATION  
1. METHOD  
1.1. INTRODUCTION 
See General introduction Part B.  
1.2. DEFINITION 
See General introduction Part B.  
1.3. REFERENCE SUBSTANCES 
None.  
1.4. PRINCIPLE OF THE TEST METHOD 
The mouse heritable translocation test detects structural and 
numerical chromosome changes in mammalian germ cells as recovered in first generation progeny. The types of chromosome changes detected are reciprocal translocations and, if female 
progeny are included, X-chromosome loss. Carriers of translocations 
and XO-females show reduced fertility which is used to select F
 1 
progeny for cytogenetic analysis. Complete sterility is caused by 
certain types of translocations (X-autosome and c-t type). Trans ­
locations are cytogenetically observed in meiotic cells at diakinesis- 
metaphase I of male individuals, either F  1 males or male offspring of 
F 1 females. The XO-females are cytogenetically identified by the 
presence of only 39 chromosomes in bone marrow mitoses.  
1.5. QUALITY CRITERIA 
None.  
1.6. DESCRIPTION OF THE TEST METHOD  
Preparations  
The test chemicals are dissolved in isotonic saline. If insoluble they 
are dissolved or suspended in appropriate vehicles. Freshly prepared 
solutions of the test compound are employed. If a vehicle is used to facilitate dosing, it must not interfere with the test compound or 
produce toxic effects.
 
R o u t e o f a d m i n i s t r a t i o n 
Routes of administration are usually oral intubation or intraperitoneal 
injection. Other routes of administration may be appropriate.  
E x p e r i m e n t a l a n i m a l s 
For the ease of breeding and cytological verification these 
experiments are performed with mice. No specific mouse strain is 
required. However, the average litter-size of the strain should be 
greater than eight and be relatively constant. 
Healthy sexually mature animals are used.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 396
 
N u m b e r o f a n i m a l s 
The number of animals necessary depends upon the spontaneous 
translocation frequency and the minimal rate of induction required for a positive result. 
The test is usually performed by analyses of male F
 1 progeny. At 
least 500 male F  1 progeny should be tested per dose group. If female 
F 1 progeny are included, 300 males and 300 females are required.  
U s e o f n e g a t i v e a n d p o s i t i v e c o n t r o l s 
Adequate control data, derived from concurrent and historic control 
should be available. When acceptable positive control results are 
available from experiments conducted recently in the same laboratory these results can be used instead of a concurrent positive control.
 
D o s e l e v e l s 
One dose level is tested, usually the highest dose associated with the 
production of minimal toxic effects, but without affecting repro ­
ductive behaviour or survival. To establish a dose/response rela ­
tionship two additional lower doses are required. For non-toxic 
chemicals exposure to the maximum practicable dose should be used.
 
Procedure  
T r e a t m e n t a n d m a t i n g 
Two treatment schedules are available. Single administration of the 
test substance is most widely used. Administration of the test 
substance on seven days per week for 35 days may also be used. 
The number of matings following treatment is governed by the treatment schedule and should ensure that all treated germ cell stages are sampled. At the end of the mating period females are 
caged individually. When females give birth, the date, litter size 
and sex of progeny are recorded. All male progeny are weaned and all female progeny are discarded unless they are included in the experiment.
 
T e s t i n g f o r t r a n s l o c a t i o n h e t e r o z y g o s i t y 
One of two possible methods is used: 
— fertility testing of F  1 progeny and subsequent verification of 
possible translocation carriers by cytogenetic analysis, 
— cytogenetic analysis of all male F  1 progeny without prior 
selection by fertility testing. 
(a) Fertility testing 
Reduced fertility of an F  l individual can be established by litter 
size observation and/or analysis of uterine contents of female 
mates. 
Criteria for determining normal and reduced fertility must be 
established for the mouse strain used.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 397
 
Litter size observation: F  1 males to be tested are caged indi ­
vidually with females either from the same experiment or from 
the colony. Cages are inspected daily beginning 18 days after mating. Litter size and sex of the F
 2 progeny are recorded at birth 
and litters are discarded thereafter. If female F  1 progeny are 
tested the F  2 progeny of small litters are kept for further 
testing. Female translocation carriers are verified by cytogenetic analysis of a translocation in any of their male offspring. XO- females are recognised by the change in sex ratio among their 
progeny from 1:1 to 1:2 males  versus  females. In a sequential 
procedure, normal F
 l animals are eliminated from further testing 
if the first F  2 litter reaches or exceeds a predetermined normal 
value, otherwise a second or third F  2 litter is observed. 
F 1 animals that cannot be classified as normal after observation 
of up to three F  2 litters are either tested further by analysis of 
uterine contents of female mates or directly subjected to cyto ­
genetic analysis. 
Analysis of uterine contents: the reduction in litter size of trans ­
location carriers is due to embryonic death so that a high number of dead implants is indicative of the presence of a translocation in the animal under test. F
 1 males to be tested are mated to two 
to three females each. Conception is established by daily 
inspection for vaginal plugs in the morning. Females are sacrificed 14 to 16 days later and living and dead implants in their uteri are recorded. 
(b) Cytogenetic analysis 
Testes preparations are made by the air-drying technique. Trans ­
location carriers are identified by the presence of multivalent configurations at diakinesis-metaphase I in primary spermato ­
cytes. Observation of at least two cells with multivalent 
association constitutes the required evidence that the tested animal is a translocation carrier. 
If no breeding selection has been performed all F
 1 males are 
inspected cytogenetically. A minimum of 25 diakinesis- metaphase I cells per male must be scored microscopically. Examination of mitotic metaphases, in spermatogonia or bone- 
marrow, is required in F
 1 males with small testes and meiotic 
breakdown before diakinesis or from F  1 female XO suspects. The 
presence of an unusually long and/or short chromosome in each of 10 cells is evidence for a particular male sterile translocation 
(c-t type). Some X-autosome translocations that cause male 
sterility may only be identified by banding analysis of mitotic chromosomes. The presence of 39 chromosomes in all of 10 mitoses is evidence for an XO condition in a female.
 
2. DATA  
Data are presented in tabular form. 
The mean litter size and sex ratio from parental matings at birth and 
weaning are reported for each mating interval.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 398
 
For fertility assessment of F  1 animals, the mean litter size of all 
normal matings and the individual litter sizes of F  1 translocation 
carriers are presented. For analysis of uterine contents, the mean number of living and dead implants of normal matings and the individual numbers of living and dead implants for each mating of 
F
 1 translocation carriers are reported. 
For cytogenetic analysis of diakinesis-metaphase I, the numbers of 
types of multivalent configurations and the total number of cells are 
listed for each translocation carrier. 
For sterile F  1 individuals, the total number of matings and the 
duration of the mating period are reported. Testes weights and cyto ­
genetic analysis details are given. 
For XO females, the mean litter size, sex ratio of F  1 progeny and 
cytogenetic analysis results are reported. 
Where possible F  1 translocation carriers are preselected by fertility 
tests, the tables have to include information on how many of these were confirmed translocation heterozygotes. 
Data from negative controls and the positive control experiments are 
reported.
 
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, contain the following information: 
— strain of mice, age of animals, weights of treated animals, 
— numbers of parental animals of each sex in experimental and 
control groups, 
— test conditions, detailed description of treatment, dose levels, 
solvents, mating schedule, 
— number and sex of offspring per female, number and sex of 
offspring raised for translocation analysis, 
— time and criteria of translocation analysis, 
— number and detailed description of translocation carriers, 
including breeding data and uterine content data, if applicable; 
— cytogenetic procedures and details of microscopic analysis, 
preferably with pictures, 
— statistical evaluation, 
— discussion of results, 
— interpretation of results.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 399
 
3.2. EVALUATION AND INTERPRETATION 
See General introduction Part B.  
4. REFERENCES  
See General introduction Part B.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 400
 
B.26.  SUB-CHRONIC ORAL TOXICITY TESTREPEATED DOSE 90 
– DAY ORAL TOXICITY STUDY IN RODENTS  
1. METHOD  
This sub-chronic oral toxicity test method is a replicate of the OECD 
TG 408 (1998).  
1.1. INTRODUCTION 
In the assessment and evaluation of the toxic characteristics of a 
chemical, the determination of sub-chronic oral toxicity using 
repeated doses may be carried out after initial information on 
toxicity has been obtained from acute or repeated dose 28-day toxicity tests. The 90-day study provides information on the possible health hazards likely to arise from repeated exposure over 
a prolonged period of time covering post-weaning maturation and 
growth well into adulthood. The study will provide information on the major toxic effects, indicate target organs and the possibility of 
accumulation, and can provide an estimate of a no-observed-adverse- 
effect level of exposure which can be used in selecting dose levels for chronic studies and for establishing safety criteria for human exposure. 
The method places additional emphasis on neurological endpoints 
and gives an indication of immunological and reproductive effects. The need for careful clinical observations of the animals, so as to 
obtain as much information as possible, is also stressed. This study 
should allow for the identification of chemicals with the potential to cause neurotoxic, immunological or reproductive organ effects, which may warrant further in-depth investigation. 
See also General introduction Part B.
 
1.2. DEFINITIONS  
Dose:  is the amount of test substance administered. Dose is 
expressed as weight (g, mg) or as weight of test substance per 
unit weight of test animal (e.g. mg/kg), or as constant dietary concentrations (ppm).  
Dosage:  is a general term comprising of dose, its frequency and the 
duration of dosing.  
NOAEL:  is the abbreviation for no-observed-adverse-effect level 
and is the highest dose level where no adverse treatment-related findings are observed.
 
1.3. PRINCIPLE OF THE TEST METHOD 
The test substance is orally administered daily in graduated doses to 
several groups of experimental animals, one dose level per group for a period of 90 days. During the period of administration the animals are observed closely for signs of toxicity. Animals, which die or are 
killed during the test are necropsied and, at the conclusion of the 
test, surviving animals are also killed and necropsied.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 401
 
1.4. DESCRIPTION OF THE TEST METHOD  
1.4.1.  Preparations of animals  
Healthy animals, which have been acclimated to laboratory 
conditions for at least five days and have not been subjected to 
previous experimental procedures, should be used. The test animals 
should be characterised as to species, strain, source, sex, weight and/or age. Animals should be randomly assigned to the control and treatment groups. Cages should be arranged in such a way 
that possible effects due to cage placement are minimised. Each 
animal should be assigned a unique identification number.
 
1.4.2.  Preparations of doses  
The test substance is administered by gavage or via the diet or 
drinking water. The method of oral administration is dependent on 
the purpose of the study, and the physical/chemical properties of the 
test material. 
Where necessary, the test substance is dissolved or suspended in a 
suitable vehicle. It is recommended that, wherever possible, the use 
of an aqueous solution/suspension be considered first, followed by 
consideration of a solution/emulsion in oil (e.g. corn oil) and then by possible solution in other vehicles. For vehicles other than water the toxic characteristics of the vehicle must be known. The stability of 
the test substance under the conditions of administration should be 
determined.
 
1.4.3.  Test conditions  
1.4.3.1.  Experimental animals  
The preferred species is the rat, although other rodent species, e.g. 
the mouse, may be used. Commonly used laboratory strains of 
young healthy adult animals should be employed. The females 
should be nulliparous and non-pregnant. Dosing should begin as soon as possible after weaning and, in any case, before the animals are nine weeks old. At the commencement of the study 
the weight variation of animals used should be minimal and not 
exceed ± 20 % of the mean weight of each sex. Where the study is conducted as a preliminary to a long term chronic toxicity study, animals from the same strain and source should be used in both 
studies.
 
1.4.3.2.  Number and sex  
At least 20 animals (10 female and 10 male) should be used at each 
dose level. If interim kills are planned, the number should be increased by the number of animals scheduled to be killed before the completion of the study. Based on previous knowledge of the 
chemical or a close analogue, consideration should be given to 
including an additional satellite group of ten animals (five per sex) in the control and in the top dose group for observation, after the treatment period, of reversibility or persistence of any toxic effects. 
The duration of this post-treatment period should be fixed appro ­
priately with regard to the effects observed.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 402
 
1.4.3.3.  Dose levels  
At least three dose levels and a concurrent control shall be used, 
except where a limit test is conducted (see 1.4.3.4). Dose levels may be based on the results of repeated dose or range finding studies and 
should take into account any existing toxicological and toxicokinetic 
data available for the test substance or related materials. Unless limited by the physical-chemical nature or biological effects of the test substance, the highest dose level should be chosen with the aim 
to induce toxicity but not death or severe suffering. A descending 
sequence of dose levels should be selected with a view to demon ­
strating any dosage related response and a no-observed-adverse- 
effect level (NOAEL) at the lowest dose level. Two to four-fold 
intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g. more than a factor of about 6-10) 
between dosages. 
The control group shall be an untreated group or a vehicle-control 
group if a vehicle is used in administering the test substance. Except for treatment with the test substance, animals in the control group 
should be handled in an identical manner to those in the test groups. 
If a vehicle is used, the control group shall receive the vehicle in the highest volume used. If a test substance is administered in the diet, and causes reduced dietary intake, then a pair-fed control group may 
be useful in distinguishing between reductions due to palatability or 
toxicological alterations in the test model. 
Consideration should be given to the following characteristics of the 
vehicle and other additives, as appropriate: effects on the absorption, 
distribution, metabolism, or retention of the test substance; effects on the chemical properties of the test substance which may alter its toxic characteristics; and effects on the food or water consumption 
or the nutritional status of the animals.
 
1.4.3.4.  Limit test  
If a test at one dose level, equivalent to at least 1 000 mg/kg body 
weight/day, using the procedures described for this study, produces no-observed-adverse-effects and if toxicity would not be expected 
based upon data from structurally related substances, then a full 
study using three dose levels may not be considered necessary. The limit test applies except when human exposure indicates the need for a higher dose level to be used.
 
1.5. PROCEDURE  
1.5.1.  Administration of doses  
The animals are dosed with the test substance daily seven days each 
week for a period of 90 days. Any other dosing regime, e.g. five days per week, needs to be justified. When the test substance is 
administered by gavage, this should be done in a single dose to 
the animals using a stomach tube or a suitable intubation cannula. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. The volume should not 
exceed 1 ml/100 g body weight, except in the case of aqueous 
solutions where 2 ml/100 g body weight may be used. Except for irritating or corrosive substances, which will normally reveal exacerbated effects with higher concentrations, variability in test 
volume should be minimised by adjusting the concentration to 
ensure a constant volume at all dose levels.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 403
 
For substances administered via the diet or drinking water it is 
important to ensure that the quantities of the test substance 
involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (ppm) or a constant dose level in 
terms of the animal's body weight may be used; the alternative 
used must be specified. For a substance administered by gavage, the dose should be given at similar times each day, and adjusted as necessary to maintain a constant dose level in terms of animal 
body weight. Where a 90-day study is used as a preliminary to a 
long term chronic toxicity study, a similar diet should be used in both studies.
 
1.5.2.  Observations  
The observation period should be at least 90 days. Animals in a 
satellite group scheduled for follow-up observations should be kept 
for an appropriate period without treatment to detect persistence of, 
or recovery from toxic effects. 
General clinical observations should be made at least once a day, 
preferably at the same time(s) each day, taking into consideration the peak period of anticipated effects after dosing. The clinical condition 
of the animals should be recorded. At least twice daily, usually at the 
beginning and end of each day, all animals are inspected for signs of morbidity and mortality. 
At least once prior to the first exposure (to allow for within-subject 
comparisons), and once a week thereafter, detailed clinical obser ­
vations should be made in all animals. These observations should be made outside the home cage, preferably in a standard arena and at similar times on each occasion. They should be carefully recorded, 
preferably using scoring systems, explicitly defined by the testing 
laboratory. Effort should be made to ensure that variations in the observation conditions are minimal. Signs noted should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, 
occurrence of secretions and excretions and autonomic activity (e.g. 
lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive 
grooming, repetitive circling) or bizarre behaviour (e.g. self-muti ­
lation, walking backwards) should also be recorded (1). 
Ophthalmological examination, using an ophthalmoscope or 
equivalent suitable equipment, should be made prior to the adminis ­
tration of the test substance and at the termination of the study, 
preferably in all animals but at least in the high dose and control groups. If changes in the eyes are detected all animals should be examined. 
Towards the end of the exposure period and in any case not earlier 
than in week 11, sensory reactivity to stimuli of different types (1) (e.g. auditory, visual and proprioceptive stimuli) (2), (3), (4), assessment of grip strength (5) and motor activity assessment (6) 
should be conducted. Further details of the procedures that could 
be followed are given in the respective references. However, alter ­
native procedures than those referenced could also be used. 
Functional observations conducted towards the end of the study may 
be omitted when data on functional observations are available from 
other studies and the daily clinical observations did not reveal any 
functional deficits.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 404
 
Exceptionally, functional observations may also be omitted for 
groups that otherwise reveal signs of toxicity to an extent that 
would significantly interfere with the functional test performance.  
1.5.2.1.  Body weight and food/water consumption  
All animals should be weighed at least once a week. Measurements 
of food consumption should be made at least weekly. If the test 
substance is administered via the drinking water, water consumption 
should also be measured at least weekly. Water consumption may also be considered for dietary or gavage studies during which drinking activity may be altered.
 
1.5.2.2.  Haematology and clinical biochemistry  
Blood samples should be taken from a named site and stored, if 
applicable, under appropriate conditions. At the end of the test period, samples are collected just prior to or as part of the procedure for killing the animals. 
The following haematological examinations should be made at the 
end of the test period and when any interim blood samples may have been collected: haematocrit, haemoglobin concentration, erythrocyte 
count, total and differential leukocyte count, platelet count and a 
measure of blood clotting time/potential. 
Clinical biochemistry determinations to investigate major toxic 
effects in tissues and, specifically, effects on kidney and liver, 
should be performed on blood samples obtained from each animal 
just prior to or as part of the procedure for killing the animals (apart from those found moribund and/or intercurrently killed). In a similar manner to haematological investigations, interim sampling for 
clinical biochemical tests may be performed. Overnight fasting of 
the animals prior to blood sampling is recommended (
 1 ). Deter­
minations in plasma or serum should include sodium, potassium, 
glucose, total cholesterol, urea, blood urea nitrogen, creatinine, 
total protein and albumin, and more than two enzymes indicative of hepatocellular effects (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl 
transpeptidase, and sorbitol dehydrogenase). Measurements of 
additional enzymes (of hepatic or other origin) and bile acids, which may provide useful information under certain circumstances, may also be included. 
Optionally, the following urinalysis determinations could be 
performed during the last week of the study using timed urine volume collection: appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells. 
In addition, studies to investigate serum markers of general tissue 
damage should be considered. Other determinations that should be carried out if the known properties of the test substance may, or are 
suspected to, affect related metabolic profiles include calcium, phos ­
phorus, fasting triglycerides, specific hormones, methaemoglobin and cholinesterase. These need to be identified for chemicals in certain classes or on a case-by-case basis.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 405  
( 1 ) For a number of measurements in serum and plasma, most notably for glucose, overnight 
fasting would be preferable. The major reason for this preference is that the increased 
variability which would inevitably result from non-fasting, would tend to mask more 
subtle effects and make interpretation difficult. On the other hand, however, overnight 
fasting may interfere with the general metabolism of the animals and, particularly in 
feeding studies, may disturb the daily exposure to the test substance. If overnight fasting 
is adopted, clinical biochemical determinations should be performed after the conduct of 
functional observations of the study.
 Overall, there is a need for a flexible approach, depending on the 
species and the observed and/or expected effect from a given 
substance. 
If historical baseline data are inadequate, consideration should be 
given as to whether haematological and clinical biochemistry 
variables need to be determined before dosing commences; it is 
generally not recommended that this data be generated before treatment (7).
 
1.5.2.3.  Gross necropsy  
All animals in the study shall be subjected to a full, detailed gross 
necropsy which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The liver, kidneys, adrenals, testes, 
epididymides, uterus, ovaries, thymus, spleen, brain and heart of 
all animals (apart from those found moribund and/or intercurrently killed) should be trimmed of any adherent tissue, as appropriate, and their wet weight taken as soon as possible after dissection to avoid 
drying. 
The following tissues should be preserved in the most appropriate 
fixation medium for both the type of tissue and the intended 
subsequent histopathological examination: all gross lesions, brain 
(representative regions including cerebrum, cerebellum and medulla/ ­
pons), spinal cord (at three levels: cervical, mid-thoracic and lumbar), pituitary, thyroid, parathyroid, thymus, oesophagus, 
salivary glands, stomach, small and large intestines (including 
Peyer's patches), liver, pancreas, kidneys, adrenals, spleen, heart, trachea and lungs (preserved by inflation with fixative and then immersion), aorta, gonads, uterus, accessory sex organs, female 
mammary gland, prostate, urinary bladder, gall bladder (mouse), 
lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of adminis ­
tration to cover systemic effects), peripheral nerve (sciatic or tibial) 
preferably in close proximity to the muscle, a section of bone 
marrow (and/or a fresh bone marrow aspirate), skin and eyes (if changes were observed during ophthalmological examinations). The clinical and other findings may suggest the need to examine 
additional tissues. Also any organs considered likely to be target 
organs based on the known properties of the test substance should be preserved.
 
1.5.2.4.  Histopathology  
Full histopathology should be carried out on the preserved organs 
and tissues of all animals in the control and high dose groups. These examinations should be extended to animals of all other dosage groups, if treatment-related changes are observed in the high dose 
group. 
All gross lesions should be examined. When a satellite group is used, histopathology should be performed 
on tissues and organs identified as showing effects in the treated 
groups.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 406
 
2. DATA AND REPORTING  
2.1. DATA 
Individual data should be provided. Additionally, all data should be 
summarised in tabular form showing for each test group the number 
of animals at the start of the test, the number of animals found dead 
during the test or killed for humane reasons and the time of any death or humane kill, the number showing signs of toxicity, a description of the signs of toxicity observed, including time of 
onset, duration, and severity of any toxic effects, the number of 
animals showing lesions, the type of lesions and the percentage of animals displaying each type of lesion. 
When applicable, numerical results should be evaluated by an appro ­
priate and generally acceptable statistical method. The statistical 
methods and the data to be analysed should be selected during the design of the study.
 
2.2. TEST REPORT 
The test report must include the following information:  
2.2.1.  Test substance:  
— physical nature, purity and physico-chemical properties, 
— identification data, 
— vehicle (if appropriate): justification for choice of vehicle, if 
other than water.  
2.2.2.  Test species:  
— species and strain used, 
— number, age and sex of animals, — source, housing conditions, diet etc., 
— individual weights of animals at the start of the test.
 
2.2.3.  Test conditions:  
— rationale for dose level selection, 
— details of test substance formulation/diet preparation, achieved 
concentration, stability and homogeneity of the preparation, 
— details of the administration of the test substance, 
— actual doses (mg/kg body weight/day), and conversion factor 
from diet/drinking water test substance concentration (ppm) to the actual dose, if applicable, 
— details of food and water quality.
 
2.2.4.  Results:  
— body weight and body weight changes, 
— food consumption, and water consumption, if applicable, 
— toxic response data by sex and dose level, including signs of 
toxicity,  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 407
 
— nature, severity and duration of clinical observations (whether 
reversible or not), 
— results of ophthalmological examination, — sensory activity, grip strength and motor activity assessments 
(when available), 
— haematological tests with relevant base-line values, — clinical biochemistry tests with relevant base-line values, — terminal body weight, organ weights and organ/body weight 
ratios, 
— necropsy findings, 
— a detailed description of all histopathological findings, — absorption data if available, — statistical treatment of results, where appropriate, Discussion of results. Conclusions.
 
3. REFERENCES  
(1) IPCS (1986) Principles and Methods for the Assessment of 
Neurotoxicity Associated with Exposure to Chemicals. Environ ­
mental Health Criteria Document No 60. 
(2) Tupper, D.E., Wallace, R.B., (1980) Utility of the Neurologic 
Examination in Rats. Acta Neurobiol. Exp., 40, p. 999-1003. 
(3) Gad, S.C., (1982) A Neuromuscular Screen for Use in Industrial 
Toxicology. J.Toxicol. Environ. Health, 9, p. 691-704. 
(4) Moser, V.C., Mc Daniel, K.M., Phillips, P.M., (1991) Rat Strain 
and Stock Comparisons Using a Functional Observational 
Battery: Baseline Values and Effects of Amitraz. Toxicol. Appl. Pharmacol., 108, p. 267-283. 
(5) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T., (1979) A 
Method for the Routine Assesment of Fore- and Hind- limb 
grip Strength of Rats and Mice. Neurobehav. Toxivol., 1, 
p. 233-236. 
(6) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Reiter 
L.W., Tilson H.A., MacPhail R.C., (1991) Interlaboratory Comparison of Motor Activity Experiments: Implication for 
Neurotoxicological Assessments. Neurotoxicol. Teratol., 13, 
p. 599-609. 
(7) Weingand K., Brown G., Hall R.  et al ., (1996) ‘Harmonisation 
of Animal Clinic Pathology Testing in Toxicity and Safety 
Studies’, Fundam. & Appl. Toxicol., 29, p. 198-201.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 408
 
B.27.  SUB-CHRONIC ORAL TOXICITY TEST REPEATED DOSE 90- 
DAY ORAL TOXICITY STUDY IN NON-RODENTS  
1. METHOD  
This sub-chronic oral toxicity test method is a replicate of the OECD 
TG 409 (1998).  
1.1. INTRODUCTION 
In the assessment and evaluation of the toxic characteristics of a 
chemical, the determination of sub-chronic oral toxicity using repeated doses may be carried out after initial information on 
toxicity has been obtained from acute or repeated dose 28-day 
toxicity tests. The 90-day study provides information on the possible health hazards likely to arise from repeated exposure over a period of rapid growth and into young adulthood. The study will 
provide information on the major toxic effects, indicate target organs 
and the possibility of accumulation, and can provide an estimate of a no-observed-adverse-effect level of exposure which can be used in selecting dose levels for chronic studies and for establishing safety 
criteria for human exposure. 
The test method allows for the identification in non-rodent species of 
adverse effects of chemical exposure and should only be used: 
— where effects observed in other studies indicate a need for 
clarification/characterisation in a second, non-rodent species, or 
— where toxicokinetic studies indicate that the use of a specific 
non-rodent species is the most relevant choice of laboratory 
animal, or 
— where other specific reasons justify the use of a non-rodent 
species. 
See also General introduction Part B.
 
1.2. DEFINITIONS  
Dose:  is the amount of test substance administered. Dose is 
expressed as weight (g, mg) or as weight of test substance per 
unit weight of test animal (e.g. mg/kg), or as constant dietary concentrations (ppm).  
Dosage:  is a general term comprising of dose, its frequency and the 
duration of dosing.  
NOAEL:  is the abbreviation for no-observed-adverse-effect level 
and is the highest dose level where no adverse treatment-related 
findings are observed.
 
1.3. PRINCIPLE OF THE TEST METHOD 
The test substance is orally administered daily in graduated doses to 
several groups of experimental animals, one dose level per group for a period of 90 days. During the period of administration the animals are observed closely for signs of toxicity. Animals, which die or are 
killed during the test are necropsied and at the conclusion of the test 
surviving animals are also killed and necropsied.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 409
 
1.4. DESCRIPTION OF THE TEST METHOD  
1.4.1.  Selection of animal species  
The commonly used non-rodent species is the dog, which should be 
of a defined breed; the beagle is frequently used. Other species, e.g. 
swine, mini-pigs, may also be used. Primates are not recommended 
and their use should be justified. Young, healthy animals should be employed, and in the case of the dog, dosing should begin preferably at four to six months and not later than nine months of age. Where 
the study is conducted as a preliminary to a long-term chronic 
toxicity study, the same species/breed should be used in both studies.
 
1.4.2.  Preparation of animals  
Healthy young animals, which have been acclimated to laboratory 
conditions and have not been subjected to previous experimental 
procedures, should be used. The duration of acclimatisation will 
depend upon the selected test species and their source. At least five days for dogs or purpose bred swine from a resident colony and at least two weeks for these animals if from external sources are 
recommended. The test animals should be characterised as to 
species, strain, source, sex, weight and/or age. Animals should be randomly assigned to the control and treatment groups. Cages should be arranged in such a way that possible effects due to cage 
placement are minimised. Each animal should be assigned a 
unique identification number.
 
1.4.3.  Preparations of doses  
The test substance may be administered in the diet or in the drinking 
water, by gavage or in capsules. The method of oral administration is 
dependent on the purpose of the study, and the physical-chemical 
properties of the test material. 
Where necessary, the test substance is dissolved or suspended in a 
suitable vehicle. It is recommended that, wherever possible, the use 
of an aqueous solution/suspension be considered first, followed by 
consideration of a solution/emulsion in oil (e.g. corn oil) and then by possible solution in other vehicles. For vehicles other than water the toxic characteristics of the vehicle must be known. The stability of 
the test substance under the conditions of administration should be 
determined.
 
1.5. PROCEDURE  
1.5.1.  Number and sex of animals  
At least eight animals (four female and four male) should be used at 
each dose level. If interim kills are planned, the number should be 
increased by the number of animals scheduled to be killed before the 
completion of the study. The number of animals at the termination of the study must be adequate for a meaningful evaluation of toxic effects. Based on previous knowledge of the substance or a close 
analogue, consideration should be given to including an additional 
satellite group of eight animals (four per sex) in control and in top dose group for observation after the treatment period of reversibility or persistence of any toxic effects. The duration of this post- 
treatment period should be fixed appropriately with regard to the 
effects observed.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 410
 
1.5.2.  Dosage  
At least three dose levels and a concurrent control shall be used, 
except where a limit test is conducted (see 1.5.3). Dose levels may be based on the results of repeated dose or range finding studies and 
should take into account any existing toxicological and toxicokinetic 
data available for the test compound or related materials. Unless limited by the physical-chemical nature or biological effects of the test substance, the highest dose level should be chosen with the aim 
to induce toxicity but not death or severe suffering. A descending 
sequence of dose levels should be selected with a view to demon ­
strating any dosage related response and a no-observed-adverse- 
effect level (NOAEL) at the lowest dose level. Two to fourfold 
intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g. more than a factor of about 6–10) 
between dosages. 
The control group shall be an untreated group or a vehicle-control 
group if a vehicle is used in administering the test substance. Except 
for treatment with the test substance, animals in the control group 
should be handled in an identical manner to those in the test groups. If a vehicle is used, the control group shall receive the vehicle in the highest volume used. If a test substance is administered in the diet, 
and causes reduced dietary intake, then a pair-fed control group may 
be useful in distinguishing between reductions due to palatability or toxicological alterations in the test model. 
Consideration should be given to the following characteristics of the 
vehicle and other additives, as appropriate: effects on the absorption, distribution, metabolism, or retention of the test substance; effects on the chemical properties of the test substance which may alter its 
toxic characteristics; and effects on the food or water consumption 
or the nutritional status of the animals.
 
1.5.3.  Limit test  
If a test at one dose level, equivalent to at least 1 000 mg/kg body 
weight/day, using the procedures described for this study, produces 
no-observed-adverse-effects and if toxicity would not be expected based upon data from structurally related substances, then a full study using three dose levels may not be considered necessary. 
The limit test applies except when human exposure indicates the 
need for a higher dose level to be used.
 
1.5.4.  Administration of doses  
The animals are dosed with the test substance daily seven days each 
week for a period of 90 days. Any other dosing regime, e.g. five 
days per week, needs to be justified. When the test substance is 
administered by gavage, this should be done in a single dose to the animals using a stomach tube or a suitable intubation cannula. The maximum volume of liquid that can be administered at one time 
depends on the size of the test animal. Normally the volume should 
be kept as low as possible. Except for irritating or corrosive substances which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be 
minimised by adjusting the concentration to ensure a constant 
volume at all dose levels.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 411
 
For substances administered via the diet or drinking water it is 
important to ensure that the quantities of the test substance 
involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (ppm) or a constant dose level in 
terms of the animal's body weight may be used; any alternative 
used must be specified. For a substance administered by gavage or by capsule, the dose should be given at similar times each day, and adjusted as necessary to maintain a constant dose level in terms of 
animal body weight. Where the 90 day study is used as a preliminary 
to a long term chronic toxicity study, a similar diet should be used in both studies.
 
1.5.5.  Observations  
The observation period should be at least 90 days. Animals in a 
satellite group scheduled for follow-up observations should be kept for an appropriate period without treatment to detect persistence of, 
or recovery from toxic effects. 
General clinical observations should be made at least once a day, 
preferably at the same time(s) each day, taking into consideration the 
peak period of anticipated effects after dosing. The clinical condition of the animals should be recorded. At least twice daily, usually at the beginning and end of each day, all animals should be inspected for 
signs of morbidity and mortality. 
At least once prior to the first exposure (to allow for within-subject 
comparisons), and once a week thereafter, detailed clinical obser ­
vations should be made in all animals. These observations should be made, where practical outside the home cage in a standard arena and preferably at similar times on each occasion. Effort should be 
made to ensure that variations in the observation conditions are 
minimal. Signs of toxicity should be carefully recorded, including time of onset, degree and duration. Observations should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, 
occurrence of secretions and excretions and autonomic activity (e.g. 
lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive 
grooming, repetitive circling) or any bizarre behaviour should also be 
recorded. 
Ophthalmological examination, using an ophthalmoscope or 
equivalent suitable equipment, should be made prior to the adminis ­
tration of the test substance and at the termination of the study, 
preferably in all animals but at least in the high dose and control 
groups. If treatment related changes in the eyes are detected all animals should be examined.
 
1.5.5.1.  Body weight and food/water consumption  
All animals should be weighed at least once a week. Measurements 
of food consumption should be made at least weekly. If the test substance is administered via the drinking water, water consumption should also be measured at least weekly. Water consumption may 
also be considered for dietary or gavage studies during which 
drinking activity may be altered.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 412
 
1.5.5.2.  Haematology and clinical biochemistry  
Blood samples should be taken from a named site and stored, if 
applicable, under appropriate conditions. At the end of the test period, samples are collected just prior to or as part of the 
procedure for killing the animals. 
Haematology, including haematocrit, haemoglobin concentration, 
erythrocyte count, total and differential leukocyte count, platelet 
count and a measure of clotting potential such as clotting time, 
prothrombin time, or thromboplastin time should be investigated at the start of the study, then either at monthly intervals or midway through the test period and finally at the end of the test period. 
Clinical biochemistry determinations to investigate major toxic 
effects in tissues and, specifically, effects on kidney and liver, should be performed on blood samples obtained from all animals 
at the start, then either at monthly intervals or midway through the 
test and finally at the end of the test period. Test areas, which should be considered are electrolyte balance, carbohydrate metabolism, and 
liver and kidney function. The selection of specific tests will be 
influenced by observations on the mode of action of the test substance. Animals should be fasted for a period appropriate to the species prior to blood sampling. Suggested determinations include 
calcium, phosphorus, chloride, sodium, potassium, fasting glucose, 
alanine aminotransferase, aspartate aminotransferase, ornithine decar ­
boxylase, gamma glutamyl transpeptidase, urea nitrogen, albumin, blood creatinine, total bilirubin and total serum protein measure ­
ments. 
Urinalysis determinations should be performed at least at the start, 
then midway and finally at the end of the study using timed urine 
volume collection. Urinalysis determinations include appearance, 
volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells. Additional parameters may be employed where necessary to extend the investigation of observed effect(s). 
In addition, studies to investigate markers of general tissue damage 
should be considered. Other determinations, which may be necessary for an adequate toxicological evaluation include analyses of lipids, 
hormones, acid/base balance, methaemoglobin, and cholinesterase 
inhibition. Additional clinical biochemistry may be employed where necessary to extend the investigation of observed effects. These need to be identified for chemicals in certain classes or on 
a case-by-case basis. 
Overall, there is a need for a flexible approach, depending on the 
species and the observed and/or expected effect from a given 
substance.
 
1.5.5.3.  Gross necropsy  
All animals in the study shall be subjected to a full, detailed gross 
necropsy which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal 
cavities and their contents. The liver with gall bladder, kidneys, 
adrenals, testes, epididymides, ovaries, uterus, thyroid (with parathy ­
roids), thymus, spleen, brain and heart of all animals (apart from those found moribund and/or inter-currently killed) should be 
trimmed of any adherent tissue, as appropriate, and their wet 
weight taken as soon as possible after dissection to avoid drying.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 413
 
The following tissues should be preserved in the most appropriate 
fixation medium for both the type of tissue and the intended 
subsequent histopathological examination: all gross lesions, brain (representative regions including cerebrum, cerebellum and medulla/ ­
pons), spinal cord (at three levels: cervical, mid-thoracic and 
lumbar), pituitary, eyes, thyroid, parathyroid, thymus, oesophagus, 
salivary glands, stomach, small and large intestines (including Peyer's patches), liver, gall bladder, pancreas, kidneys, adrenals, spleen, heart, trachea and lungs, aorta, gonads, uterus, accessory 
sex organs, female mammary gland, prostate, urinary bladder, 
lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of adminis ­
tration to cover systemic effects), peripheral nerve (sciatic or tibial) 
preferably in close proximity to the muscle, a section of bone 
marrow (and/or a fresh bone marrow aspirate) and skin. The clinical and other findings may suggest the need to examine additional tissues. Also any organs considered likely to be target 
organs based on the known properties of the test substance should 
be preserved.
 
1.5.5.4.  Histopathology  
Full histopathology should be carried out on the preserved organs 
and tissues in at least all animals in control and high dose group. The 
examination should be extended to animals of all other dosage 
groups, if treatment-related changes are observed in the high dose group. 
All gross lesions should be examined. 
When a satellite group is used, histopathology should be performed 
on tissues and organs identified as showing effects in the treated 
groups.
 
2. DATA AND REPORTING  
2.1. DATA 
Individual data should be provided. Additionally, all data should be 
summarised in tabular form showing for each test group the number 
of animals at the start of the test, the number of animals found dead during the test or killed for humane reasons and the time of any death or humane kill, the number showing signs of toxicity, a 
description of the signs of toxicity observed, including time of 
onset, duration, and severity of any toxic effects, the number of animals showing lesions, the type of lesions and the percentage of animals displaying each type of lesion. 
When applicable, numerical results should be evaluated by an appro ­
priate and generally acceptable statistical method. The statistical methods and the data to be analysed should be selected during the 
design of the study.
 
2.2. TEST REPORT 
The test report must include the following information:  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 414
 
2.2.1.  Test substance:  
— physical nature, purity and physico-chemical properties, 
— identification data, — vehicle (if appropriate): justification for choice of vehicle, if 
other than water.
 
2.2.2.  Test species:  
— species and strain used, 
— number, age and sex of animals, 
— source, housing conditions, diet etc., 
— individual weights of animals at the start of the test.  
2.2.3.  Test conditions:  
— rationale for dose level selection, 
— details of test substance formulation/diet preparation, achieved 
concentration, stability and homogeneity of the preparation, 
— details of the administration of the test substance, — actual doses (mg/kg body weight/day), and conversion factor 
from diet/drinking water test substance concentration (ppm) to 
the actual dose, if applicable, 
— details of food and water quality.
 
2.2.4.  Results:  
— body weight/body weight changes, 
— food consumption, and water consumption, if applicable, — toxic response data by sex and dose level, including signs of 
toxicity, 
— nature, severity and duration of clinical observations (whether 
reversible or not), 
— ophthalmological examination, 
— haematological tests with relevant base-line values, — clinical biochemistry tests with relevant base-line values, — terminal body weight, organ weights and organ/body weight 
ratios, 
— necropsy findings, — a detailed description of all histopathological findings, — absorption data if available, — statistical treatment of results, where appropriate. 
Discussion of results. 
Conclusions.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 415
 
B.28.  SUB-CHRONIC DERMAL TOXICITY STUDY 90-DAY 
REPEATED DERMAL DOSE STUDY USING RODENT 
SPECIES  
1. METHOD  
1.1. INTRODUCTION 
See General introduction Part B.  
1.2. DEFINITIONS 
See General introduction Part B.  
1.3. REFERENCE SUBSTANCES 
None.  
1.4. PRINCIPLE OF THE TEST METHOD 
The test substance is applied daily to the skin in graduated doses to 
several groups of experimental animals, one dose per group for a 
period of 90 days. During the period of application the animals are observed daily to detect signs of toxicity. Animals, which die during the test are necropsied, and at the conclusion of the test surviving 
animals are necropsied.
 
1.5. QUALITY CRITERIA 
None.  
1.6. DESCRIPTION OF THE TEST METHOD  
1.6.1.  Preparations  
The animals are kept under the experimental housing and feeding 
conditions for at least five days prior to the test. Before the test healthy young animals are randomised and assigned to the treated 
and control groups. Shortly before testing fur is clipped from the 
dorsal area of the trunk of the test animals. Shaving may be employed but it should be carried out approximately 24 hours before the test. Repeat clipping or shaving is usually needed at 
approximately weekly intervals. When clipping or shaving the fur, 
care must be taken to avoid abrading the skin. Not less than 10 % of the body surface area should be clear for the application of the test substance. The weight of the animal should be taken into account 
when deciding on the area to be cleared and on the dimensions of 
the covering. When testing solids, which may be pulverised if appro ­
priate, the test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle to ensure good contact with 
the skin. Liquid test substances are generally used undiluted. Daily 
application on a five to seven-day per week basis is used.
 
1.6.2.  Test conditions  
1.6.2.1. E x p e r i m e n t a l a n i m a l s 
The adult rat, rabbit or guinea pig may be used. Other species may 
be used but their use would require justification. At the commencement of the test the range of the weight variation should be ± 20 % of the mean weight. Where a sub-chronic dermal study is 
conducted as a preliminary to a long-term study, the same species 
and strain should be used in both studies.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 416
 
1.6.2.2. N u m b e r a n d s e x 
At least 20 animals (10 female and 10 male) with healthy skin 
should be used at each dose level. The females should be nulliparous and non-pregnant. If interim sacrifices are planned the number 
should be increased by the number of animals scheduled to be 
sacrificed before the completion of the study. In addition, a satellite group of 20 animals (10 animals per sex) may be treated with the high-dose level for 90 days and observed for reversibility, 
persistence, or delayed occurrence of toxic effects for 28 days post- 
treatment.
 
1.6.2.3. D o s e l e v e l s 
At least three dose levels are required with a controle or a vehicle 
control if a vehicle is used. The exposure period should be at least six hours per day. The application of the test substance should be made at similar times each day, and the amount of substance applied 
adjusted at intervals (weekly or bi-weekly) to maintain a constant 
dose level in terms of animal body weight. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the test group subjects. Where a vehicle is 
used to facilitate dosing, the vehicle control group should be dosed 
in the same way as the treated groups, and receive the same amount as that received by the highest dose level group. The highest dose level should result in toxic effects but produce no, or few, fatalities. 
The lowest dose level should not produce any evidence of toxicity. 
Where there is a usable estimation of human exposure the lowest level should exceed this. Ideally, the intermediate dose level should 
produce minimal observable toxic effects. If more than one inter ­
mediate dose is used, the dose levels should be spaced to produce a gradation of toxic effects. In the low and intermediate groups, and in the controls, the incidence of fatalities should be low, in order to 
permit a meaningful evaluation of the results. 
If application of the test substance produces severe skin irritation the 
concentrations should be reduced and this may result in a reduction 
in, or absence of, other toxic effects al: the high-dose level. If the skin has been badly damaged it may be necessary to terminate the study and undertake a new study at lower concentrations.
 
1.6.3. L i m i t t e s t 
If a preliminary study at a dose level of 1 000 mg/kilograms, or a 
higher dose level related to possible human exposure where this is 
known, produces no toxic effects, further testing may not be 
considered necessary.  
1.6.4. O b s e r v a t i o n p e r i o d 
The experimental animals should be observed daily for signs of 
toxicity. The time of death and the time at which signs of toxicity 
appear and disappear should be recorded.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 417
 
1.6.5. P r o c e d u r e 
Animals should be caged individually. The animals are treated with 
the test substance, ideally on seven days per week, for a period of 90 days. 
Animals in any satellite groups scheduled for follow-up observations 
should be kept for a further 28 days without treatment to detect recovery from, or persistence of, toxic effects. Exposure time should be six hours per day. 
The test substance should be applied uniformly over an area, which 
is approximately 10 % of the total body surface area. With highly toxic substances, the surface area covered may be less but as much 
of the area should be covered with as thin and uniform a film as 
possible. 
During exposure the test substance is held in contact with the skin 
with a porous gauze dressing and non-irritating tape. The test site 
should be further covered in a suitable manner to retain the gauze 
dressing and test substance and ensure that the animals cannot ingest the test substance. Restrainers may be used to prevent the ingestion of the test substance but complete immobilisation is not a recom ­
mended method. 
At the end of the exposure period, residual test substance should be 
removed where practicable using water or some other appropriate method of cleansing the skin. 
All the animals should be observed daily and signs of toxicity 
recorded, including the time of onset, their degree and duration. Cageside observations should include changes in skin and fur, eyes and mucous membranes, as well as respiratory, circulatory, 
autonomic and central nervous systems, somatomotor activity and 
behavior pattern. Measurements should be made of food consumption weekly and the animals weighed weekly. Regular observations of the animals are necessary to ensure that animals 
are not lost from the study due to causes such as cannibalism, 
autolysis of tissues or misplacement. At the end of the study period all survivors in the non-satellite treatment groups are necropsied. Moribund animals should be removed and necropsied 
when noticed. 
The following examinations are customarily made on all animals 
including the controls: 
(a) ophthalmological examination, using an ophthalmoscope or 
equivalent suitable equipment, should be made prior to 
exposure to the test substance and at the termination of the study, preferably in all animals but at least in the high-dose and control groups. If changes in the eyes are detected all 
animals should be examined. 
(b) haematology, including haematocrit, haemoglobin concentration, 
erythrocyte count, total and differential leucocyte count, and a measure of clotting potential, such as clotting time, prothrombin 
time, thromboplastin time, or platelet count, should be inves ­
tigated at the end of the test period.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 418
 
(c) clinical biochemistry determination on blood should be carried 
out at the end of the test period. Test areas, which are considered 
appropriate to all studies are electrolyte balance, carbohydrate metabolism, liver and kidney function. The selection of specific tests will be influenced by observations on the mode 
of action of the substance. Suggested determinations are calcium, 
phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species), sercum glutamic pyruvic transaminase (
 1 ), serum glutamic oxaloacetic 
transaminase (  2 ), ornithine decarboxylase, gamma glutamyl trans ­
peptidase, urea nitrogen, albumin, blood creatinine, total bilirubin and total serum protein measurements. Other deter ­
minations which may be necessary for an adequate toxicological 
evaluation include analyses of lipids, hormones, acid/base 
balance, methaemoglobin and choliensterase activity. Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed effects. 
(d) urinalysis is not required on a routine basis but only when there 
is an indication based on expected or observed toxicity. 
If historical baseline data are inadequate, consideration should be 
given to determination of haem a to logical and clinical biochemistry 
parameters before dosing commences.
 
G r o s s n e c r o p s y 
All animals should be subjected to a full gross necropsy which 
includes examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The liver, kidneys, adrenals and testes must be weighed 
wet as soon as possible after dissection to avoid drying. The 
following organs and tissues should be preserved in a suitable medium for possible future histopathological examination: all gross lesions, brain — including sections of medulla/pons, cerebellar 
cortex and cerebral cortex, pituitary, thyroid/parathyroid, any 
thymic tissue, (trachea), lungs, heart, aorta, salivary glands, liver, spleen, kidneys, adrenals, pancreas, gonads, uterus, accessory genital organs, gall bladder (if present), oesophagus, stomach, 
duodenum, jejunum, ileum, caecum, colon, rectum, urinary 
bladder, representative lymph node, (female mammary gland), (thigh musculature), peripheral nerve, (eyes), (sternum with bone marrow), (femur — including articular surface), (spinal cord at 
three levels — cervical, mid-thoracic and lumbar), and (exorbital 
lachrymal glands). The tissues mentioned between brackets need only be examined if indicated by signs of toxicity or target organ involvement.
 
H i s t o p a t h o l o g i c a l e x a m i n a t i o n 
(a) Full histopathology should be carried out on normal and treated 
skin and on organs and tissues of animals in the control and 
high-dose groups. 
(b) all gross lesions should be examined.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 419  
( 1 ) Now known as serum alanine aminotransferase. 
( 2 ) Now known as serum aspartate aminotransferase.
 (c) target organs in other dose groups should be examined. 
(d) where rats are used, lungs of animals in the low- and inter ­
mediate-dose groups should be subjected to histopathological examination for evidence of infection, since this provides a 
convenient assessment of the state of health of the animals. 
Further histopathological examination may not be required routinely on the animals in these groups, but must always be carried out in organs, which show evidence of lesions in the 
high-dose group. 
(e) when a satellite group is used, histopathology should be 
performed on tissues and organs identified as showing effects 
in the other treated groups.
 
2. DATA  
Data should be summarised in tabular form, showing for each test 
group the number of animals at the start of the test, the number of 
animals showing lesions, the type of lesions and the percentage of animals displaying each type of lesion. Results should be evaluated by an appropriate statistical method. Any recognised statistical 
method may be used.
 
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, contain the following information: 
— species, strain, source, environmental conditions, diet, 
— test conditions, — dose levels (including vehicle, if used) and concentrations, 
— toxic response data by sex and dose, 
— no-effect level, where possible, — time of death during the study or whether animals survived to 
termination, 
— description of toxic or other effects, 
— the time of observation of each abnormal sign and its subsequent 
course, 
— food and bodyweight data, — ophthalmological findings, 
— haematological tests employed and all results, 
— clinical biochemistry tests employed and all results (including 
results of any urinalysis), 
— necropsy findings,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 420
 
— a detailed description of all histopathological findings, 
— statistical treatment of results where possible, — discussion of the results, 
— interpretation of the results.
 
3.2. EVALUATION AND INTERPRETATION 
See General introduction Part B.  
4. REFERENCES  
See General introduction Part B.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 421
 
B.29.  SUBCHRONIC INHALATION TOXICITY: 90-DAY STUDY  
SUMMARY 
This revised Test Method B.29 has been designed to fully characterise test 
chemical toxicity by the inhalation route for a subchronic duration (90 days), and to provide robust data for quantitative inhalation risk assessments. Groups of 
10 male and 10 female rodents are exposed 6 hours per day during a 90 day (13 
week) period to a) the test chemical at three or more concentration levels, b) filtered air (negative control), and/or c) the vehicle (vehicle control). Animals are generally exposed 5 days per week but exposure for 7 days per week is also 
allowed. Males and females are always tested, but they may be exposed at 
different concentration levels if it is known that one sex is more susceptible to a given test chemical. This method allows the study director the flexibility to include satellite (reversibility) groups, interim sacrifices, bronchoalveolar 
lavage (BAL), neurologic tests, and additional clinical pathology and histopath ­
ological evaluations in order to better characterise the toxicity of a test chemical.
 
INTRODUCTION 
1. This Test Method is equivalent to OECD Test Guideline 413 (2009). The 
original subchronic inhalation Test Guideline 413 (TG 413) was adopted in 
1981 (1). This Test Method B.29 (as equivalent to the revised TG 413 
(2009)) has been updated to reflect the state of the science and to meet current and future regulatory needs. 
2. Subchronic inhalation toxicity studies are primarily used to derive regulatory 
concentrations for assessing worker risk in occupation settings. They are also used to assess human residential, transportation, and environmental risk. 
This method enables the characterisation of adverse effects following 
repeated daily inhalation exposure to a test chemical for 90 days (approxi ­
mately 10 % of the lifespan of a rat). The data derived from subchronic inhalation toxicity studies can be used for quantitative risk assessments and 
for the selection of concentrations for chronic studies. This test method is 
not specifically intended for the testing of nanomaterials. Definitions used in the context of this Test Method are provided at the end of this chapter and in the Guidance Document (GD) 39 (2).
 
INITIAL CONSIDERATIONS 
3. All available information on the test chemical should be considered by the 
testing laboratory prior to conducting the study in order to enhance the quality of the study and minimise animal usage. Information that will assist in the selection of appropriate test concentrations might include the 
identity, chemical structure, and physico-chemical properties of the test 
chemical; results of any  in vitro  or in vivo  toxicity tests; anticipated 
use(s) and potential for human exposure; available (Q)SAR data and toxi ­
cological data on structurally related chemicals; and data derived from other 
repeated exposure studies. If neurotoxicity is expected or is observed in the 
course of the study, the study director may choose to include appropriate evaluations such as a functional observational battery (FOB) and measurement of motor activity. Although the timing of exposures relative 
to specific examinations may be critical, the performance of these additional 
activities should not interfere with the basic study design.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 422
 
4. Dilutions of corrosive or irritating test chemicals may be tested at concen ­
trations that will yield the desired degree of toxicity. Please refer to GD 39 
(2) for further information. When exposing animals to these materials, the targeted concentrations should be low enough to not cause marked pain and distress, yet sufficient to extend the concentration-response curve to levels 
that reach the regulatory and scientific objective of the test. These concen ­
trations should be selected on a case-by-case basis, preferably based upon an adequately designed range-finding study that provides information regarding the critical endpoint, any irritation threshold, and the time of onset (see 
paragraphs 11-13). The justification for concentration selection should be 
provided. 
5. Moribund animals or animals obviously in pain or showing signs of severe 
and enduring distress should be humanely killed. Moribund animals are considered in the same way as animals that die on test. Criteria for making the decision to kill moribund or severely suffering animals, and 
guidance on the recognition of predictable or impending death, are the 
subject of an OECD Guidance Document on Humane Endpoints (3).
 
DESCRIPTION OF THE METHOD  
Selection of Animal Species  
6. Healthy young adult rodents of commonly used laboratory strains should be 
employed. The preferred species is the rat. Justification should be provided 
if other species are used.  
Preparation of Animals  
7. Females should be nulliparous and non-pregnant. On the day of randomis ­
ation, animals should be young adults 7 to 9 weeks of age. Body weights 
should be within ± 20 % of the mean weight for each sex. The animals are 
randomly selected, marked for individual identification, and kept in their cages for at least 5 days prior to the start of the test to allow for acclimat ­
ization to laboratory conditions.
 
Animal Husbandry  
8. Animals should be individually identified, preferably with subcutaneous 
transponders, to facilitate observations and avoid confusion. The temperature of the experimental animal maintenance room should be 22 ± 3 °C. The 
relative humidity should ideally be maintained in the range of 30 to 70 %, 
though this may not be possible when using water as a vehicle. Before and after exposures, animals generally should be caged in groups by sex and concentration, but the number of animals per cage should not interfere with 
clear observation of each animal and should minimise losses due to canni ­
balism and fighting. When animals are to be exposed nose-only, it may be necessary for them to be acclimated to the restraining tubes. The restraining tubes should not impose undue physical, thermal, or immobilisation stress 
on the animals. Restraint may affect physiological endpoints such as body 
temperature (hyperthermia) and/or respiratory minute volume. If generic data are available to show that no such changes occur to any appreciable extent, then pre-adaptation to the restraining tubes is not necessary. Animals 
exposed whole-body to an aerosol should be housed individually during 
exposure to prevent them from filtering the test aerosol through the fur of their cage mates. Conventional and certified laboratory diets may be used, except during exposure, accompanied with an unlimited supply of municipal 
drinking water. Lighting should be artificial, the sequence being 12 hours 
light/12 hours dark.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 423
 
Inhalation Chambers  
9. The nature of the test chemical and the object of the test should be considered 
when selecting an inhalation chamber. The preferred mode of exposure is nose-only (which term includes head-only, nose-only, or snout-only). 
Nose-only exposure is generally preferred for studies of liquid or solid 
aerosols and for vapours that may condense to form aerosols. Special objectives of the study may be better achieved by using a whole-body mode of exposure, but this should be justified in the study report. To 
ensure atmosphere stability when using a whole-body chamber, the total 
volume of the test animals should not exceed 5 % of the chamber volume. Principles of the nose-only and whole body exposure techniques and their 
particular advantages and disadvantages are addressed in GD 39 (2).
 
TOXICITY STUDIES  
Limit Concentrations  
10. Unlike with acute studies, there are no defined limit concentrations in 
subchronic inhalation toxicity studies. The maximum concentration tested 
should consider: 1) the maximum attainable concentration, 2) the ‘worst case’ human exposure level, 3) the need to maintain an adequate oxygen 
supply, and/or 4) animal welfare considerations. In the absence of 
data-based limits, the acute limits of Regulation (EC) No 1272/2008 (13) may be used (i.e. up to a maximum concentration of 5 mg/l for aerosols, 20 mg/l for vapours, and 20 000 ppm for gases); refer to GD 39 (2). Justifi ­
cation should be provided if it is necessary to exceed these limits when 
testing gases or highly volatile test chemicals (e.g. refrigerants). The limit concentration should elicit unequivocal toxicity without causing undue stress to the animals or affecting their longevity (3).
 
Range-Finding Study  
11. Before commencing with the main study, it is generally necessary to 
perform a range-finding study. A range-finding study is more compre ­
hensive than a sighting study because it is not limited to concentration selection. Knowledge learned from a range-finding study can lead to a 
successful main study. A range-finding study may, for example, provide 
technical information regarding analytical methods, particle sizing, discovery of toxic mechanisms, clinical pathology and histopathological data, and estimations of what may be NOAEL and MTC concentrations 
in a main study. The study director may choose to use the range-finding 
study to identify the threshold of respiratory tract irritation (e.g. with histo ­
pathology of the respiratory tract, pulmonary function testing, or bronchoal ­
veolar lavage), the upper concentration which is tolerated without undue 
stress to the animals, and the parameters that will best characterise a test 
chemical’s toxicity. 
12. A range-finding study may consist of one or more concentration levels. 
Depending on the endpoints chosen, three to six males and three to six 
females should be exposed at each concentration level. A range-finding study should last a minimum of 5 days and generally no more than 28 days. The rationale for the selection of concentrations for the main study 
should be provided in the study report. The objective of the main study is to 
demonstrate a concentration-response relationship based on what is anticipated to be the most sensitive endpoint. The low concentration should ideally be a no-observed-adverse effect concentration while the 
high concentration should elicit unequivocal toxicity without causing 
undue stress to the animals or affecting their longevity (3).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 424
 
13. When selecting concentration levels for the range-finding study, all available 
information should be considered including structure-activity relationships 
and data for similar chemicals (see paragraph 3). A range-finding study may verify/refute what are considered to be the most sensitive mechanistically based endpoints, e.g. cholinesterase inhibition by organophosphates, methae ­
moglobin formation by erythrocytotoxic agents, thyroidal hormones (T
 3 , T 4 ) 
for thyrotoxicants, protein, LDH, or neutrophils in bronchoalveolar lavage for innocuous poorly soluble particles or pulmonary irritant aerosols.
 
Main Study  
14. The main subchronic toxicity study generally consists of three concentration 
levels, and also concurrent negative (air) and/or vehicle controls as needed (see paragraph 18). All available data should be utilised to aid selection of 
appropriate exposure levels, including the results of systemic toxicity 
studies, metabolism and kinetics (particular emphasis should be given to avoiding high concentration levels which saturate kinetic processes). Each 
test group contains 10 male and 10 female rodents that are exposed to the 
test chemical for 6 hours per day on a 5 day per week basis for a period of 13 weeks (total study duration of at least 90 days). Animals may also be exposed 7 days per week (e.g. when testing inhaled pharmaceuticals). If one 
sex is known to be more susceptible to a given test chemical, the sexes may 
be exposed at different concentration levels in order to optimise the concentration-response as described in paragraph 15. If rodent species other than rats are exposed nose-only, maximum exposure durations may 
be adjusted to minimise species-specific distress. A rationale should be 
provided when using an exposure duration less than 6 hours/day, or when it is necessary to conduct a long duration (e.g. 22 hours/day) whole-body exposure study (refer to GD 39) (2). Feed should be withheld during the 
exposure period unless exposure exceeds 6 hours. Water may be provided 
throughout a whole-body exposure. 
15. The target concentrations selected should identify the target organ(s) and 
demonstrate a clear concentration-response: 
— The high concentration level should result in toxic effects but not cause 
lingering signs or lethality which would prevent a meaningful evalu ­
ation. 
— The intermediate concentration level(s) should be spaced to produce a 
gradation of toxic effects between that of the low and high concen ­
tration. 
— The low concentration level should produce little or no evidence of 
toxicity.
 
Interim Sacrifices  
16. If interim sacrifices are planned, the number of animals at each exposure 
level should be increased by the number to be sacrificed before study 
completion. The rationale for using interim sacrifices should be provided, 
and statistical analyses should properly account for them.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 425
 
Satellite (Reversibility) Study  
17. A satellite (reversibility) study may be used to observe reversibility, 
persistence, or delayed occurrence of toxicity for a post-treatment period of an appropriate length, but no less than 14 days. Satellite (reversibility) 
groups consist of 10 males and 10 females exposed contemporaneously with 
the experimental animals in the main study. Satellite (reversibility) study groups should be exposed to the test chemical at the highest concentration level and there should be concurrent air and/or vehicle controls as needed 
(see paragraph 18).
 
Control Animals  
18. Concurrent negative (air) control animals should be handled in a manner 
identical to the test group animals except that they are exposed to filtered air rather than test chemical. When water or another substance is used to assist 
in generating the test atmosphere, a vehicle control group, instead of a 
negative (air) control group, should be included in the study. Water should be used as the vehicle whenever possible. When water is used as the vehicle, the control animals should be exposed to air with the same 
relative humidity as the exposed groups. The selection of a suitable vehicle 
should be based on an appropriately conducted pre-study or historical data. If a vehicle’s toxicity is not well known, the study director may choose to 
use both a negative (air) control and a vehicle control, but this is strongly 
discouraged. If historical data reveal that a vehicle is non-toxic, then there is no need for a negative (air) control group and only a vehicle control should be used. If a pre-study of a test chemical formulated in a vehicle reveals no 
toxicity, it follows that the vehicle is non-toxic at the concentration tested 
and this vehicle control should be used.
 
EXPOSURE CONDITIONS  
Administration of Concentrations  
19. Animals are exposed to the test chemical as a gas, vapour, aerosol, or a 
mixture thereof. The physical state to be tested depends on the physico-chemical properties of the test chemical, the selected concentrations, and/or the physical form most likely present during the handling and use of 
the test chemical. Hygroscopic and chemically reactive test chemicals should 
be tested under dry air conditions. Care should be taken to avoid generating explosive concentrations. Particulate materials may be subjected to mech ­
anical processes to decrease the particle size. Further guidance is provided in 
GD 39 (2).
 
Particle-Size Distribution  
20. Particle sizing should be performed for all aerosols and for vapours that may 
condense to form aerosols. To allow for exposure of all relevant regions of the respiratory tract, aerosols with mass median aerodynamic 
diameters (MMAD) ranging from 1 to 3 μm with a geometric standard 
deviation ( σ
 g ) in the range of 1,5 to 3,0 are recommended (4). Although 
a reasonable effort should be made to meet this standard, expert judgement should be provided if it cannot be achieved. For example, metal fume 
particles will be smaller than this standard, and charged particles and 
fibres may exceed it.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 426
 
Test chemical Preparation in a Vehicle  
21. Ideally, the test chemical should be tested without a vehicle. If it is 
necessary to use a vehicle to generate an appropriate test chemical concen ­
tration and particle size, water should be given preference. Whenever a test 
chemical is dissolved in a vehicle, its stability should be demonstrated.  
MONITORING OF EXPOSURE CONDITIONS  
Chamber Airflow  
22. The flow of air through the exposure chamber should be carefully 
controlled, continuously monitored, and recorded at least hourly during each exposure. The real-time monitoring of the test atmosphere concen ­
tration (or temporal stability) is an integral measurement of all dynamic 
parameters and provides an indirect means to control all relevant dynamic inhalation parameters. If the concentration is monitored real-time, the frequency of measurement of air flows may be reduced to one single 
measurement per exposure per day. Special consideration should be given 
to avoiding rebreathing in nose-only chambers. Oxygen concentration should be at least 19 % and carbon dioxide concentration should not 
exceed 1 %. If there is reason to believe that this standard cannot be met, 
oxygen and carbon dioxide concentrations should be measured. If measurements on the first day of exposure show that these gases are at proper levels, no further measurements should be necessary.
 
Chamber Temperature and Relative Humidity  
23. Chamber temperature should be maintained at 22 ± 3 °C. Relative humidity 
in the animals’ breathing zone, for both nose-only and whole-body exposures, should be monitored continuously and recorded hourly during 
each exposure where possible. The relative humidity should preferably be 
maintained in the range of 30 to 70 %, but this may either be unattainable (e.g. when testing water based mixtures) or not measurable due to test chemical interference with the Test Method.
 
Test chemical: Nominal Concentration  
24. Whenever feasible, the nominal exposure chamber concentration should be 
calculated and recorded. The nominal concentration is the mass of generated test chemical divided by the total volume of air passed through the inha ­
lation chamber system. The nominal concentration is not used to char ­
acterise the animals’ exposure, but a comparison of the nominal concen ­
tration and the actual concentration gives an indication of the generation efficiency of the test system, and thus may be used to discover generation 
problems.
 
Test chemical: Actual Concentration  
25. The actual concentration is the test chemical concentration as sampled at the 
animals’ breathing zone in an inhalation chamber. Actual concentrations can 
be obtained either by specific methods (e.g. direct sampling, adsorptive or 
chemical reactive methods, and subsequent analytical characterisation) or by non-specific methods such as gravimetric filter analysis. The use of gravi ­
metric analysis is acceptable only for single component powder aerosols or 
aerosols of low volatility liquids and should be supported by appropriate 
pre-study test chemical-specific characterisations. Multi-component powder
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 427
 
aerosol concentration may also be determined by gravimetric analysis. 
However, this requires analytical data which demonstrate that the 
composition of airborne material is similar to the starting material. If this information is not available, a reanalysis of the test chemical (ideally in its airborne state) at regular intervals during the course of the study may be 
necessary. For aerosolised agents that may evaporate or sublimate, it should 
be shown that all phases were collected by the method chosen. 
26. One lot of the test chemical should be used throughout the duration of the 
study, if possible, and the test sample should be stored under conditions that maintain its purity, homogeneity, and stability. Prior to the start of the study, 
there should be a characterisation of the test chemical, including its purity 
and, if technically feasible, the identity, and quantities of identified contaminants and impurities. This can be demonstrated by, but is not limited to, the following data: retention time and relative peak area, 
molecular weight from mass spectroscopy or gas chromatography 
analyses, or other estimates. Although the test sample’s identity is not the responsibility of the test laboratory, it may be prudent for the test laboratory to confirm the sponsor’s characterisation at least in a limited way (e.g. 
colour, physical nature, etc.). 
27. The exposure atmosphere should be held as constant as practicable. A 
real-time monitoring device, such as an aerosol photometer for aerosols or a total hydrocarbon analyser for vapours, may be used to demonstrate the stability of the exposure conditions. Actual chamber concentration should be 
measured at least 3 times during each exposure day for each exposure level. 
If not feasible due to limited air flow rates or low concentrations, one sample per exposure period is acceptable. Ideally, this sample should then be collected over the entire exposure period. Individual chamber concen ­
tration samples should deviate from the mean chamber concentration by no 
more than ± 10 % for gases and vapours, and by no more than ± 20 % for liquid or solid aerosols. Time to attain chamber equilibration (t
 95 ) should be 
calculated and reported. The duration of an exposure spans the time that the 
test chemical is generated. This takes into account the times required to 
attain chamber equilibration (t  95 ) and decay. Guidance for estimating t  95 
can be found in GD 39 (2). 
28. For very complex mixtures consisting of gases/vapours and aerosols (e.g. 
combustion atmospheres and test chemicals propelled from purpose-driven 
end-use products/devices), each phase may behave differently in an inha ­
lation chamber. Therefore, at least one indicator substance (analyte), normally the principal active ingredient in the mixture, of each phase (gas/ vapour and aerosol) should be selected. When the test chemical is a mixture, 
the analytical concentration should be reported for the total mixture, and not 
just for the active ingredient or the indicator substance (analyte). Additional information regarding actual concentrations can be found in GD 39 (2).
 
Test chemical: Particle Size Distribution  
29. The particle size distribution of aerosols should be determined at least 
weekly for each concentration level by using a cascade impactor or an alternative instrument such as an aerodynamic particle sizer (APS). If equiv ­
alence of the results obtained by a cascade impactor and the alternative 
instrument can be shown, then the alternative instrument may be used 
throughout the study.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 428
 
30. A second device, such as a gravimetric filter or an impinger/gas bubbler, 
should be used in parallel to the primary instrument to confirm the 
collection efficiency of the primary instrument. The mass concentration obtained by particle size analysis should be within reasonable limits of the mass concentration obtained by filter analysis [see GD 39 (2)]. If equiv ­
alence can be demonstrated at all concentrations tested in the early phase of 
the study, then further confirmatory measurements may be omitted. For the sake of animal welfare, measures should be taken to minimise inconclusive data which may lead to a need to repeat a study. 
31. Particle sizing should be performed for vapours if there is any possibility 
that vapour condensation may result in the formation of an aerosol, or if particles are detected in a vapour atmosphere with potential for mixed phases.
 
OBSERVATIONS 
32. The animals should be clinically observed before, during, and after the 
exposure period. More frequent observations may be indicated depending 
on the response of the animals during exposure. When animal observation is hindered by the use of animal restraint tubes, poorly lit whole body chambers, or opaque atmospheres, animals should be carefully observed 
after exposure. Observations before the next day’s exposure can assess 
any reversibility or exacerbation of toxic effects. 
33. All observations are recorded with individual records being maintained for 
each animal. When animals are killed for humane reasons or found dead, the 
time of death should be recorded as precisely as possible. 
34. Cage-side observations should include changes in the skin and fur, eyes, and 
mucous membranes; changes in the respiratory and circulatory systems; 
changes in the nervous system; and changes in somatomotor activity and 
behaviour patterns. Attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep, and coma. The measurement of rectal temperatures may provide supportive evidence of 
reflex bradypnea or hypo/hyperthermia related to treatment or confinement. 
Additional assessments may be included in the study protocol such as kinetics, biomonitoring, lung function, retention of poorly soluble materials that accumulate in lung tissue, and behavioural changes.
 
BODY WEIGHTS 
35. Individual animal weights should be recorded shortly before the first 
exposure (day 0), twice weekly thereafter (for example: on Fridays and 
Mondays to demonstrate recovery over an exposure-free weekend, or at a time interval to allow assessment of systemic toxicity), and at the time of death or euthanasia. If there are no effects in the first 4 weeks, body weights 
may be measured weekly for the remainder of the study. Satellite (reversi ­
bility) animals (if used) should continue to be weighed weekly throughout the recovery period. At study termination, all animals should be weighed shortly before sacrifice to allow for an unbiased calculated of organ to body 
weight ratios.
 
FOOD AND WATER CONSUMPTION 
36. Food consumption should be measured weekly. Water consumption may 
also be measured.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 429
 
CLINICAL PATHOLOGY 
37. Clinical pathology assessments should be made for all animals, including 
controls and satellite (reversibility) animals, when they are sacrificed. The time interval between the end of exposure and blood collection should be 
recorded, particularly when the reconstitution of the addressed endpoint is 
rapid. Sampling following the end of exposure is indicated for those parameters with a short plasma half-time (e.g. COHb, CHE, and MetHb). 
38. Table 1 lists the clinical pathology parameters that are generally required for 
all toxicology studies. Urinalysis is not required on a routine basis, but may 
be performed when deemed useful based on expected or observed toxicity. The study director may choose to assess additional parameters in order to better characterise a test chemical’s toxicity (e.g. cholinesterase, lipids, 
hormones, acid/base balance, methaemoglobin or Heinz bodies, creatine 
kinase, myeloid/erythroid ratio, troponins, arterial blood gases, lactate dehy ­
drogenase, sorbital dehydrogenase, glutamate dehydrogenase, and gamma glutamyl transpeptidase).
 
Table 1  
Standard Clinical Pathology Parameters  
Haematology  
Erythrocyte count 
Haematocrit 
Haemoglobin concentration Mean corpuscular haemoglobin Mean corpuscular volume 
Mean corpuscular haemoglobin 
concentration 
Reticulocytes Total leukocyte count 
Differential leukocyte count 
Platelet count Clotting potential (select one): — Prothrombin time 
— Clotting time 
— Partial thromboplastin time
 
Clinical Chemistry  
Glucose (*) 
Total cholesterol Triglycerides 
Blood urea nitrogen 
Total bilirubin Creatinine Total protein 
Albumin 
Globulin Alanine aminotransferase 
Aspartate aminotransferase Alkaline phosphatase 
Potassium 
Sodium Calcium Phosphorus 
Chloride
 
Urinalysis (optional)  
Appearance (colour and turbidity) 
Volume 
Specific gravity or osmolality pH Total protein 
Glucose 
Blood/blood cells
 
(*) Because a lengthy fasting period can introduce bias in glucose measurements for 
the treated versus control animals, the study director should determine whether it 
is appropriate to fast the animals. If a fasting period is used, it should be appro ­
priate to the species used; for the rat this may be 16 h (overnight fasting). 
Determination of fasting glucose may be carried out after overnight fasting 
during the last exposure week, or after overnight fasting prior to necropsy (in 
the latter case together with all other clinical pathology parameters).  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 430
 
39. When there is evidence that the lower respiratory tract (i.e. the alveoli) is the 
primary site of deposition and retention, then bronchoalveolar lavage (BAL) 
may be the technique of choice to quantitatively analyse hypothesis-based dose-effect parameters focusing on alveolitis, pulmonary inflammation, and phospholipidosis. This allows for dose-response and time-course changes of 
alveolar injury to be suitably probed. The BAL fluid may be analysed for 
total and differential leukocyte counts, total protein, and lactate dehy ­
drogenase. Other parameters that may be considered are those indicative of lysosomal injury, phospholipidosis, fibrosis, and irritant or allergic 
inflammation which may include the determination of pro-inflammatory 
cytokines/chemokines. BAL measurements generally complement the results from histopathology examinations but cannot replace them. Guidance on how to perform lung lavage can be found in GD 39 (2).
 
OPHTHALMOLOGICAL EXAMINATION 
40. Using an ophthalmoscope or an equivalent device, ophthalmological exam ­
inations of the fundus, refractive media, iris, and conjunctivae should be 
performed for all animals prior to the administration of the test chemical, and for all high concentration and control groups at termination. If changes 
in the eyes are detected, all animals in the other groups should be examined 
including the satellite (reversibility) group.
 
GROSS PATHOLOGY AND ORGAN WEIGHTS 
41. All test animals, including those which die during the test or are removed 
from the study for animal welfare reasons, should be subjected to complete exsanguination (if feasible) and gross necropsy. The time between the end 
of each animal’s last exposure and its sacrifice should be recorded. If a 
necropsy cannot be performed immediately after a dead animal is discovered, the animal should be refrigerated (not frozen) at a temperature low enough to minimise autolysis. Necropsies should be performed as soon 
as possible, normally within a day or two. All gross pathological changes 
should be recorded for each animal with particular attention to any changes in the respiratory tract. 
42. Table 2 lists the organs and tissues that should be preserved in a suitable 
medium during gross necropsy for histopathological examination. The pres ­
ervation of the  [bracketed ] organs and tissues and any other organs and 
tissues is at the discretion of the study director. The  bolded  organs 
should be trimmed and weighed wet as soon as possible after dissection 
to avoid drying. The thyroid and epididymides should only be weighed if needed because trimming artefacts may hinder histopathological evaluation. Tissues and organs should be fixed in 10 % buffered formalin or another 
suitable fixative as soon as necropsy is performed, and no less than 24- 
48 hours prior to trimming depending on the fixative to be used.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 431
 
Table 2  
Organs and Tissues Preserved During Gross Necropsy 
Adrenals  
Aorta 
Bone marrow (and/or fresh aspirate)  
Brain  (including sections of cerebrum, 
cerebellum, and medulla/pons) 
Caecum Colon 
Duodenum 
[Epididymides ] 
[Eyes (retina, optic nerve) and eyelids] 
Femur and stifle joint 
Gallbladder (where present) [Harderian glands]  
Heart  
Ileum Jejunum  
Kidneys  
[Lacrimal glands (extraorbital)] Larynx (3 levels including the base of the 
epiglottis)  
Liver Lung  (all lobes at one level, including main 
bronchi) 
Lymph nodes from the hilar region of the 
lung, especially for poorly soluble particulate test chemicals. For more in depth examin ­
ations and/or studies with immunological 
focus, additional lymph nodes may be considered, e.g. those from the mediastinal, cervical/submandibular and/or auricular 
regions. 
Lymph nodes (distal from the portal-of-entry) Mammary gland (female) 
Muscle (thigh) 
Nasopharyngeal tissues (at least 4 levels; 1 
level to include the nasopharyngeal duct and the Nasal Associated Lymphoid 
Tissue (NALT)) Oesophagus 
[Olfactory bulb]  
Ovaries  
Pancreas Parathyroids Peripheral nerve (sciatic or tibial, preferably 
close to muscle) 
Pituitary 
Prostate Rectum Salivary glands Seminal vesicles Skin 
Spinal cord (cervical, mid-thoracic, and 
lumbar)  
Spleen  
Sternum Stomach Teeth  
Testes 
Thymus Thyroids  
[Tongue] Trachea (at least 2 levels including 1 longi ­
tudinal section through the carina and 1 
transverse section) 
[Ureter] [Urethra] Urinary bladder  
Uterus  
Target organs 
All gross lesions and masses
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 432
 
43. The lungs should be removed intact, weighed, and instilled with a suitable 
fixative at a pressure of 20-30 cm of water to ensure that lung structure is 
maintained (5). Sections should be collected for all lobes at one level, including main bronchi, but if lung lavage is performed, the unlavaged lobe should be sectioned at three levels (not serial sections). 
44. At least 4 levels of the nasopharyngeal tissues should be examined, one of 
which should include the nasopharyngeal duct (5) (6) (7) (8) (9) to allow 
adequate examination of the squamous, transitional (non-ciliated respir ­
atory), respiratory (ciliated respiratory) and olfactory epithelium, and the draining lymphatic tissue (NALT) (10) (11). Three levels of the larynx should be examined, and one of these levels should include the base of 
the epiglottis (12). At least two levels of the trachea should be examined 
including one longitudinal section through the carina of the bifurcation of the extrapulmonary bronchi and one transverse section.
 
HISTOPATHOLOGY 
45. A histopathological evaluation of all the organs and tissues listed in Table 2 
should be performed for the control and high concentration groups, and for 
all animals which die or are sacrificed during the study. Particular attention should be paid to the respiratory tract, target organs, and gross lesions. The organs and tissues that have lesions in the high concentration group should 
be examined in all groups. The study director may choose to perform 
histopathological evaluations for additional groups to demonstrate a clear concentration response. When a satellite (reversibility) group is used, histo ­
pathological evaluation should be performed for all tissues and organs 
identified as showing effects in the treated groups. If there are excessive early deaths or other problems in the high exposure group that compromise the significance of the data, the next lower concentration should be 
examined histopathologically. An attempt should be made to correlate 
gross observations with microscopic findings.
 
DATA AND REPORTING  
Data  
46. Individual animal data on body weights, food consumption, clinical 
pathology, gross pathology, organ weights, and histopathology should be provided. Clinical observation data should be summarised in tabular form 
showing for each test group the number of animals used, the number of 
animals displaying specific signs of toxicity, the number of animals found dead during the test or killed for humane reasons, time of death of indi ­
vidual animals, a description and time course of toxic effects and reversi ­
bility, and necropsy findings. All results, quantitative and incidental, should 
be evaluated by an appropriate statistical method. Any generally accepted statistical method may be used and the statistical methods should be selected during the design of the study.
 
Test Report  
47. The test report should include the following information, as appropriate:  
Test animals and husbandry  
— Description of caging conditions, including: number (or change in 
number) of animals per cage, bedding material, ambient temperature 
and relative humidity, photoperiod, and identification of diet.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 433
 
— Species/strain used and justification for using a species other than the 
rat. Source and historical data may be provided, if they are for animals 
exposed under similar exposure, housing, and fasting conditions. 
— Number, age, and sex of animals. — Method of randomisation. — Description of any pre-test conditioning including diet, quarantine, and 
treatment for disease.  
Test chemical  
— Physical nature, purity, and, where relevant, physico-chemical properties 
(including isomerisation). 
— Identification data and Chemical Abstract Services (CAS) Registry 
Number, if known.  
Vehicle  
— Justification for use of vehicle and justification for choice of vehicle (if 
other than water). 
— Historical or concurrent data demonstrating that the vehicle does not 
interfere with the outcome of the study.  
Inhalation chamber  
— Detailed description of the inhalation chamber including volume and a 
diagram. 
— Source and description of equipment used for the exposure of animals as 
well as generation of atmosphere. 
— Equipment for measuring temperature, humidity, particle-size, and actual 
concentration. 
— Source of air and system used for conditioning. 
— Methods used for calibration of equipment to ensure a homogeneous test 
atmosphere. 
— Pressure difference (positive or negative). — Exposure ports per chamber (nose-only); location of animals in the 
chamber (whole-body). 
— Stability of the test atmosphere. 
— Location of temperature and humidity sensors and sampling of test 
atmosphere in the chamber. 
— Treatment of air supplied/extracted. — Air flow rates, air flow rate/exposure port (nose-only), or animal load/ 
chamber (whole-body). 
— Time to inhalation chamber equilibrium (t95). 
— Number of volume changes per hour. 
— Metering devices (if applicable).  
Exposure data  
— Rationale for target concentration selection in the main study.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 434
 
— Nominal concentrations (total mass of test chemical generated into the 
inhalation chamber divided by the volume of air passed through the 
chamber). 
— Actual test chemical concentrations collected from the animals’ 
breathing zone; for mixtures that produce heterogeneous physical forms (gases, vapours, aerosols), each may be analysed separately. 
— All air concentrations should be reported in units of mass (mg/l, mg/m
 3 , 
etc.) rather than in units of volume (ppm, ppb, etc.). 
— Particle size distribution, mass median aerodynamic diameter (MMAD), 
and geometric standard deviation ( σ g ), including their methods of calcu ­
lation. Individual particle size analyses should be reported.  
Test conditions  
— Details of test chemical preparation, including details of any procedures 
used to reduce the particle size of solid materials or to prepare solutions of the test chemical. 
— A description (preferably including a diagram) of the equipment used to 
generate the test atmosphere and to expose the animals to the test atmos ­
phere. 
— Details of the equipment used to monitor chamber temperature, 
humidity, and chamber airflow (i.e. development of a calibration curve). 
— Details of the equipment used to collect samples for determination of 
chamber concentration and particle size distribution. 
— Details of the chemical analytical method used and method validation 
(including efficiency of recovery of test chemical from the sampling 
medium). 
— Method of randomisation in assigning animals to test and control groups. — Details of food and water quality (including diet type/source, water 
source). 
— The rationale for the selection of test concentrations.  
Results  
— Tabulation of chamber temperature, humidity, and airflow. — Tabulation of chamber nominal and actual concentration data. — Tabulation of particle size data including analytical sample collection 
data, particle size distribution, and calculations of the MMAD and σ
 g . 
— Tabulation of response data and concentration level for each animal (i.e. 
animals showing signs of toxicity including mortality, nature, severity, 
time of onset, and duration of effects).  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 435
 
— Tabulation of individual animal weights. 
— Tabulation of food consumption 
— Tabulation of clinical pathology data 
— Necropsy findings and histopathological findings for each animal, if 
available.  
Discussion and interpretation of results  
— Particular emphasis should be made to the description of methods used 
to meet the criteria of this Test Method, e.g. the limit concentration or the particle size. 
— The respirability of particles in light of the overall findings should be 
addressed, especially if the particle-size criteria could not be met. 
— The consistency of methods used to determine nominal and actual 
concentrations, and the relation of actual concentration to nominal concentration should be included in the overall assessment of the study. 
— The likely cause of death and predominant mode of action (systemic 
versus local) should be addressed. 
— An explanation should be provided if there was a need to humanely 
sacrifice animals in pain or showing signs of severe and enduring distress, based on the criteria in the OECD Guidance Document on 
Humane Endpoints (3). 
— The target organ(s) should be identified. — The NOAEL and LOAEL should be determined.
 
LITERATURE:  
(1) OECD (1981).  Subchronic Inhalation Toxicity Testing , Original Test 
Guideline No 413, Environment Directorate, OECD, Paris. 
(2) OECD (2009).  Guidance Document on Acute Inhalation Toxicity Testing , 
Environmental Health and Safety Monograph Series on Testing and 
Assessment No 39, ENV/JM/MONO(2009)28, OECD, Paris. 
(3) OECD (2000). Guidance Document on the Recognition, Assessment and 
Use of Clinical Signs as Humane Endpoints for Experimental Animals 
Used in Safety Evaluation, Environmental Health and Safety Monograph 
Series on Testing and Assessment No 19, ENV/JM/MONO(2000)7, OECD, Paris. 
(4) Whalan.E and Redden JC (1994). Interim Policy for Particle Size and Limit 
Concentration Issues in Inhalation Toxicity Studies. Office of Pesticide 
Programs, United States Environmental Protection Agency. 
(5) Dungworth DL, Tyler WS, Plopper CE (1985). Morphological Methods for 
Gross and Microscopic Pathology (Chapter 9) in  Toxicology of Inhaled 
Material , Witschi, H.P. and Brain, J.D. (eds), Springer Verlag Heidelberg, 
pp. 229-258. 
(6) Young JT (1981). Histopathological examination of the rat nasal cavity. 
Fundam. Appl. Toxicol. 1: 309-312. 
(7) Harkema JR (1990). Comparative pathology of the nasal mucosa in 
laboratory animals exposed to inhaled irritants. Environ. Health Perspect. 
85: 231-238.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 436
 
(8) Woutersen RA, Garderen-Hoetmer A, van Slootweg PJ, Feron VJ (1994). 
Upper respiratory tract carcinogenesis in experimental animals and in 
humans. In: Waalkes MP and Ward JM (eds) Carcinogenesis. Target Organ Toxicology Series, Raven Press, New York, 215-263. 
(9) Mery S, Gross EA, Joyner DR, Godo M, Morgan KT (1994). Nasal 
diagrams: A tool for recording the distribution of nasal lesions in rats 
and mice. Toxicol. Pathol. 22: 353-372. 
(10) Kuper CF, Koornstra PJ, Hameleers DMH, Biewenga J, Spit BJ, 
Duijvestijn AM, Breda Vriesman van PJC, Sminia T (1992). The role of nasopharyngeal lymphoid tissue. Immunol. Today 13: 219-224. 
(11) Kuper CF, Arts JHE, Feron VJ (2003). Toxicity to nasal-associated 
lymphoid tissue. Toxicol. Lett. 140-141: 281-285. 
(12) Lewis DJ (1981). Mitotic Indices of Rat Laryngeal Epithelia. Journal of 
Anatomy 132(3): 419-428. 
(13) Regulation (EC) No 1272/2008 of the European Parliament and of the 
Council of 16 December 2008 on classification, labelling and packaging 
of substances and mixtures, amending and repealing Directives 67/548/EEC 
and 1999/45/EC, and amending Regulation (EC) No 1907/2006 (OJ L 353, 31.12.2008, p. 1).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 437
 
Appendix 1  
DEFINITION  
Test chemical:  Any substance or mixture tested using this Test Method.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 438
 
B.30.  CHRONIC TOXICITY STUDIES  
INTRODUCTION 
1. This Test Method is equivalent to OECD Test Guideline (TG) 452 (2009). 
The original TG 452 was adopted in 1981. Development of this revised Test 
Method B.30 was considered necessary in order to reflect recent devel ­
opments in the field of animal welfare and regulatory requirements (1) (2) (3) (4). The updating of this Test Method B.30 has been carried out in parallel with revisions of Chapter B.32 of this Annex, Carcinogenicity 
Studies, and Chapter B.33 of this Annex, Combined Chronic Toxicity/Car ­
cinogenicity studies, with the objective of obtaining additional information from the animals used in the study and providing further detail on dose selection. This Test Method is designed to be used in the testing of a broad 
range of chemicals, including pesticides and industrial chemicals. 
2. The majority of chronic toxicity studies are carried out in rodent species, 
and this Test Method is intended therefore to apply primarily to studies carried out in these species. Should such studies be required in 
non-rodent species, the principles and procedures outlined in this Test 
Method, together with those outlined in Chapter B.27 of this Annex, Repeated Dose 90-day Oral Toxicity Study in Non-Rodents (5), may also be applied, with appropriate modifications, as outlined in the OECD 
Guidance Document No 116 on the Design and Conduct of Chronic 
Toxicity and Carcinogenicity Studies (6). 
3. The three main routes of administration used in chronic toxicity studies are 
oral, dermal and inhalation. The choice of the route of administration 
depends on the physical and chemical characteristics of the test chemical and the predominant route of exposure of humans. Additional information on choice of route of exposure is provided in the OECD Guidance 
Document No 116 (6). 
4. This Test Method focuses on exposure via the oral route, the route most 
commonly used in chronic toxicity studies. While long–term chronic toxicity studies involving exposure via the dermal or inhalation routes may also be 
necessary for human health risk assessment and/or may be required under 
certain regulatory regimes, both routes of exposure involve considerable technical complexity. Such studies will need to be designed on a case-by- case basis, although the Test Method outlined here for the assessment and 
evaluation of chronic toxicity by oral administration could form the basis of 
a protocol for inhalation and/or dermal studies, with respect to recommen ­
dations for treatment periods, clinical and pathology parameters, etc. OECD Guidance is available on the administration of test chemicals by the inha ­
lation (6) (7) and dermal routes (6). Chapter B.8 of this Annex (8) and 
Chapter B.29 of this Annex (9), together with the OECD Guidance Document on acute inhalation testing (7), should be specifically consulted in the design of longer term studies involving exposure via the inhalation 
route. Chapter B.9 of this Annex (10) should be consulted in the case of 
testing carried out by the dermal route.
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5. The chronic toxicity study provides information on the possible health 
hazards likely to arise from repeated exposure over a considerable part of 
the lifespan of the species used. The study will provide information on the toxic effects of the test chemical; indicate target organs and the possibility of accumulation. It can also provide an estimate of the no-observed-adverse 
effect level which can be used for establishing safety criteria for human 
exposure. The need for careful clinical observations of the animals, so as to obtain as much information as possible, is also stressed. 
6. The objectives of studies covered by this Test Method include: 
— The identification of the chronic toxicity of a test chemical; 
— The identification of target organs; 
— Characterisation of the dose-response relationship; — Identification of a no-observed-adverse-effect level (NOAEL) or point of 
departure for establishment of a Benchmark Dose (BMD); 
— The prediction of chronic toxicity effects at human exposure levels; — Provision of data to test hypotheses regarding mode of action (6).
 
INITIAL CONSIDERATIONS 
7. In the assessment and evaluation of the toxicological characteristics of a test 
chemical, all available information on the test chemical should be 
considered by the testing laboratory prior to conducting the study, in 
order to focus the design of the study to more efficiently test for chronic toxicity potential and to minimize animal usage. Information that will assist in the study design includes the identity, chemical structure, and 
physico-chemical properties of the test chemical; any information on the 
mode of action; results of any  in vitro  or in vivo  toxicity tests; anticipated 
use(s) and potential for human exposure; available (Q)SAR data and toxi ­
cological data on structurally-related chemicals; available toxicokinetic data 
(single dose and also repeat dose kinetics where available) and data derived 
from other repeated exposure studies. The determination of chronic toxicity should only be carried out after initial information on toxicity has been obtained from repeated dose 28-day and/or 90-day toxicity tests. A 
phased testing approach to chronic toxicity testing should be considered 
as part of the overall assessment of the potential adverse health effects of a particular test chemical (11) (12) (13) (14). 
8. The statistical methods most appropriate for the analysis of results, given the 
experimental design and objectives, should be established before commencing the study. Issues to consider include whether the statistics 
should include adjustment for survival and analysis in the event of 
premature termination of one or more groups. Guidance on the appropriate statistical analyses and key references to internationally accepted statistical methods are given in Guidance Document No 116 (6), and also in Guidance 
Document No 35 on the analysis and evaluation of chronic toxicity and 
carcinogenicity studies (15).
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9. In conducting a chronic toxicity study, the guiding principles and consider ­
ations outlined in the OECD Guidance Document No 19 on the recognition, 
assessment, and use of clinical signs as humane endpoints for experimental animals used in safety evaluation (16), in particular paragraph 62 thereof, should always be followed. This paragraph states that ‘ In studies involving 
repeated dosing, when an animal shows clinical signs that are progressive, 
leading to further deterioration in condition, an informed decision as to whether or not to humanely kill the animal should be made. The decision should include consideration as to the value of the information to be gained 
from the continued maintenance of that animal on study relative to its 
overall condition. If a decision is made to leave the animal on test, the frequency of observations should be increased, as needed. It may also be possible, without adversely affecting the purpose of the test, to temporarily 
stop dosing if it will relieve the pain or distress, or reduce the test dose. ’ 
10. Detailed guidance on and discussion of the principles of dose selection for 
chronic toxicity and carcinogenicity studies can be found in Guidance Document No 116 (6), as well as two International Life Sciences Institute publications (17) (18). The core dose selection strategy is dependent on the 
primary objective or objectives of the study (paragraph 6). In selecting 
appropriate dose levels, a balance should be achieved between hazard screening on the one hand and characterisation of low-dose responses and their relevance on the other. This is particularly relevant in the situation 
where a combined chronic toxicity and carcinogenicity study (Chapter B.33 
of this Annex) is to be carried out (paragraph 11). 
11. Consideration should be given to carrying out a combined chronic toxicity 
and carcinogenicity study (Chapter B.33 of this Annex), rather than separate execution of a chronic toxicity study (this Test Method B.30) and carcino ­
genicity study (Chapter B.32 of this Annex). The combined test provides 
greater efficiency in terms of time and cost compared to conducting two separate studies, without compromising the quality of the data in either the chronic phase or the carcinogenicity phase. Careful consideration should 
however be given to the principles of dose selection (paragraphs 9 and 
20-25) when undertaking a combined chronic toxicity and carcinogenicity study (Chapter B.33 of this Annex), and it is also recognised that separate studies may be required under certain regulatory frameworks. 
12. Definitions used in the context of this Test Method can be found at the end 
of this chapter and in the Guidance Document No 116 (6).
 
PRINCIPLE OF THE TEST 
13. The test chemical is administered daily in graduated doses to several groups 
of experimental animals, normally for a period of 12 months, although 
longer or shorter durations may also be chosen depending on regulatory requirements (see paragraph 33). This duration is chosen to be sufficiently long to allow any effects of cumulative toxicity to become manifest, without 
the confounding effects of geriatric changes. Deviations from exposure 
duration of 12 months should be justified, particularly in the case of shorter durations. The test chemical is normally administered by the oral route although testing by the inhalation or dermal route may also be appro ­
priate. The study design may also include one or more interim kills, e.g. at 3 
and 6 months, and additional groups of animals may be included to accom ­
modate this (see paragraph 19). During the period of administration the animals are observed closely for signs of toxicity. Animals which die or 
are killed during the test are necropsied and, at the conclusion of the test, 
surviving animals are killed and necropsied.
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DESCRIPTION OF THE METHOD  
Selection of animal species  
14. This Test Method primarily covers assessment and evaluation of chronic 
toxicity in rodents (see paragraph 2) although it is recognised that similar studies in non-rodents may be required under certain regulatory regimes. The choice of species should be justified. The design and conduct of chronic 
toxicity studies in non-rodent species, when required, should be based on 
the principles outlined in this Test Method together with those in Chapter B.27 of this Annex, Repeated Dose 90-day Oral Toxicity Study in Non-Rodents (5). Additional information on choice of species and strain 
is provided in Guidance Document No 116 (6). 
15. In this Test Method, the preferred rodent species is the rat, although other 
rodent species, e.g. the mouse, may be used. Rats and mice have been 
preferred experimental models because of their relatively short life span, their widespread use in pharmacological and toxicological studies, their susceptibility to tumour induction, and the availability of sufficiently char ­
acterised strains. As a consequence of these characteristics, a large amount 
of information is available on their physiology and pathology. Young healthy adult animals of commonly used laboratory strains should be employed. The chronic toxicity study should be carried out in animals 
from the same strain and source as those used in preliminary toxicity 
study(ies) of shorter duration. The females should be nulliparous and non-pregnant.
 
Housing and feeding conditions  
16. Animals may be housed individually, or be caged in small groups of the 
same sex; individual housing should be considered only if scientifically justified (19) (20) (21). Cages should be arranged in such a way that possible effects due to cage placement are minimised. The temperature in 
the experimental animal room should be 22 °C (± 3 °C). Although the 
relative humidity should be at least 30 % and preferably not exceed 70 % other than during room cleaning, the aim should be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For 
feeding, conventional laboratory diets may be used with an unlimited supply 
of drinking water. The diet should meet all the nutritional requirements of the species tested and the content of dietary contaminants including but not limited to pesticide residues, persistent organic pollutants, phytoestrogens, 
heavy metals and mycotoxins, that might influence the outcome of the test, 
should be as low as possible. Analytical information on the nutrient and dietary contaminant levels should be generated periodically, at least at the 
beginning of the study and when there is a change in the batch used, and 
should be included in the final report. Analytical information on the drinking water used in the study should similarly be provided. The choice of diet may be influenced by the need to ensure a suitable admixture of a 
test chemical and to meet the nutritional requirements of the animals when 
the test chemical is administered by the dietary route.
 
Preparation of animals  
17. Healthy animals, which have been acclimated to laboratory conditions for at 
least 7 days and have not been subjected to previous experimental 
procedures, should be used. In the case of rodents, dosing of the animals 
should begin as soon as possible after weaning and acclimatisation and preferably before the animals are 8 weeks old. The test animals should be characterised as to species, strain, source, sex, weight and age. At the 
commencement of the study, the weight variation for each sex of animals 
used should be minimal and not exceed ± 20 % of the mean weight of all
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the animals within the study, separately for each sex. Animals should be 
randomly assigned to the control and treatment groups. After randomisation, 
there should be no significant differences in mean body weights between groups within each sex. If there are statistically significant differences, then the randomisation step should be repeated, if possible. Each animal should 
be assigned a unique identification number, and permanently marked with 
this number by tattooing, microchip implant, or other suitable method.
 
PROCEDURE  
Number and sex of animals  
18. Both sexes should be used. A sufficient number of animals should be used 
so that at the end of the study enough animals in every group are available for thorough biological and statistical evaluation. For rodents, at least 20 animals per sex per group should normally be used at each dose level, while 
for non-rodents a minimum of 4 per sex per group is recommended. In 
studies involving mice, additional animals may be needed in each dose group to conduct all required haematological determinations.
 
Provision for interim kills, satellite groups and sentinel animals  
19. The study may make provision for interim kills (at least 10 animals/sex/ 
group), e.g. at 6 months, to provide information on progression of toxico ­
logical changes and mechanistic information, if scientifically justified. Where such information is already available from previous repeat dose toxicity studies on the test chemical, interim kills may not be scientifically 
justified. Satellite groups may also be included to monitor the reversibility 
of any toxicological changes induced by the test chemical under investi ­
gation; these will normally be restricted to the highest dose level of the study plus control. An additional group of sentinel animals (typically 5 
animals per sex) may also be included for monitoring of disease status, if 
necessary, during the study (22). If interim kills or inclusion of satellite or sentinel groups are planned, the number of animals included in the study design should be increased by the number of animals scheduled to be killed 
before the completion of the study. These animals should normally undergo 
the same observations, including body weight, food/water consumption, haematological and clinical biochemistry measurements and pathological 
investigations as the animals in the chronic toxicity phase of the main 
study, although provision may also be made (in the interim kill groups) for measurements to be restricted to specific, key measures such as neur ­
otoxicity or immunotoxicity.
 
Dose groups and dosage  
20. Guidance on all aspects of dose selection and dose level spacing is provided 
in Guidance Document No 116 (6). At least three dose levels and a 
concurrent control should be used, except where a limit test is conducted (see paragraph 27). Dose levels will generally be based on the results of shorter-term repeated dose or range finding studies and should take into 
account any existing toxicological and toxicokinetic data available for the 
test chemical or related chemicals.
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21. Unless limited by the physical-chemical nature or biological effects of the 
test chemical, the highest dose level should normally be chosen to identify 
the principal target organs and toxic effects while avoiding suffering, severe toxicity, morbidity, or death. While taking into account the factors outlined in paragraph 22 below, the highest dose level should be chosen to elicit 
evidence of toxicity, as evidenced by, for example, depression of body 
weight gain (approximately 10 %). 
22. However, dependent on the objectives of the study (see paragraph 6), a top 
dose lower than the dose providing evidence of toxicity may be chosen, e.g. 
if a dose elicits an adverse effect of concern that nonetheless has little 
impact on lifespan or body weight. The top dose should not exceed 1 000 mg/kg body weight/day (limit dose, see paragraph 27). 
23. Dose levels and dose level spacing may be selected to establish a dose- 
response and a NOAEL or other intended outcome of the study, e.g. a BMD (see paragraph 25) at the lowest dose level. Factors that should be considered in the placement of lower doses include the expected slope of 
the dose–response curve, the doses at which important changes may occur in 
metabolism or mode of toxic action, where a threshold is expected, or where a point of departure for low-dose extrapolation is expected. 
24. The dose level spacing selected will depend on the characteristics of the test 
chemical, and cannot be prescribed in this Test Method, but two to four fold 
intervals frequently provide good test performance when used for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g. more than a factor of about 6-10) between 
dosages. In general the use of factors greater than 10 should be avoided, and 
should be justified if used. 
25. As outlined further in Guidance Document No 116 (6), points to be 
considered in dose selection include: 
— Known or suspected nonlinearities or inflection points in the dose–re ­
sponse; 
— Toxicokinetics, and dose ranges where metabolic induction, saturation, 
or nonlinearity between external and internal doses does or does not occur; 
— Precursor lesions, markers of effect, or indicators of the operation of key 
underlying biological processes; 
— Key (or suspected) aspects of mode of action, such as doses at which 
cytotoxicity begins to arise, hormone levels are perturbed, homeostatic 
mechanisms are overwhelmed, etc.; 
— Regions of the dose–response curve where particularly robust estimation 
is needed, e.g. in the range of the anticipated BMD or a suspected 
threshold; 
— Consideration of anticipated human exposure levels.
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26. The control group shall be an untreated group or a vehicle-control group if a 
vehicle is used in administering the test chemical. Except for treatment with 
the test chemical, animals in the control group should be handled in an identical manner to those in the test groups. If a vehicle is used, the control group shall receive the vehicle in the highest volume used among 
the dose groups. If a test chemical is administered in the diet, and causes 
significantly reduced dietary intake due to the reduced palatability of the diet, an additional pair-fed control group may be useful, to serve as a more suitable control. 
27. If it can be anticipated, based on information from preliminary studies, that 
a test at one dose level, equivalent to at least 1 000 mg/kg body weight/day, using the procedures described for this study, is unlikely to produce adverse effects and if toxicity would not be expected based upon data from struc ­
turally related chemicals, then a full study using three dose levels may not 
be considered necessary. A limit of 1 000 mg/kg body weight/day may apply except when human exposure indicates the need for a higher dose level to be used.
 
Preparation of doses and administration of test chemical  
28. The test chemical is normally administered orally, via the diet or drinking 
water, or by gavage. Additional information on routes and methods of administration is provided in Guidance Document No 116 (6). The route and method of administration is dependent on the purpose of the study, the 
physical/chemical properties of the test chemical, its bioavailability and the 
predominant route and method of exposure of humans. A rationale should be provided for the chosen route and method of administration. In the interests of animal welfare, oral gavage should normally be selected only 
for those agents for which this route and method of administration 
reasonably represent potential human exposure (e.g. pharmaceuticals). For dietary or environmental chemicals including pesticides, administration is typically via the diet or drinking water. However, for some scenarios, e.g. 
occupational exposure, administration via other routes may be more appro ­
priate. 
29. Where necessary, the test chemical is dissolved or suspended in a suitable 
vehicle. Consideration should be given to the following characteristics of the 
vehicle and other additives, as appropriate: effects on the absorption, distribution, metabolism, or retention of the test chemical; effects on the chemical properties of the test chemical which may alter its toxic character ­
istics; and effects on the food or water consumption or the nutritional status 
of the animals. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/emulsion in oil (e.g. corn oil) and then by possible solution in 
other vehicles. For vehicles other than water, the toxic characteristics of the 
vehicle should be known. Information should be available on the stability of the test chemical and the homogeneity of dosing solutions or diets (as appropriate) under the conditions of administration (e.g. diet). 
30. For chemicals administered via the diet or drinking water it is important to 
ensure that the quantities of the test chemical involved do not interfere with normal nutrition or water balance. In long-term toxicity studies using dietary administration, the concentration of the test chemical in the feed should not 
normally exceed an upper limit of 5 % of the total diet, in order to avoid 
nutritional imbalances. When the test chemical is administered in the diet, either a constant dietary concentration (mg/kg diet or ppm) or a constant dose level in terms of the animal’s body weight (mg/kg body weight), 
calculated on a weekly basis, may be used. The alternative used should 
be specified.
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31. In the case of oral administration, the animals are dosed with the test 
chemical daily (seven days each week), normally for a period of 12 
months (see also paragraph 33), although a longer duration may be required depending on regulatory requirements. Any other dosing regime, e.g. five days per week, needs to be justified. In the case of dermal adminis ­
tration, animals are normally treated with the test chemical for at least 
6 hours per day, 7 days per week, as specified in Chapter B.9 of this Annex (10), for a period of 12 months. Exposure by the inhalation route is carried out for 6 hours per day, 7 days per week, but exposure for 5 days 
per week may also be used, if justified. The period of exposure will 
normally be for a period of 12 months. If rodent species other than rats are exposed nose-only, maximum exposure durations may be adjusted to minimise species-specific distress. A rationale should be provided when 
using an exposure duration of less than 6 hours per day. See also Chapter 
B.8 of this Annex (8). 
32. When the test chemical is administered by gavage to the animals this should 
be done using a stomach tube or a suitable intubation cannula, at similar times each day. Normally a single dose will be administered once daily, 
where for example a chemical is a local irritant, it may be possible to 
maintain the daily dose-rate by administering it as a split dose (twice a day). The maximum volume of liquid that can be administered at one time depends on the size of the test animal. The volume should be kept 
as low as practical, and should not normally exceed 1 ml/100 g body weight 
for rodents (22). Variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels. Potentially corrosive or irritant chemicals are the exception, and need to be 
diluted to avoid severe local effects. Testing at concentrations that are likely 
to be corrosive or irritant to the gastrointestinal tract should be avoided.
 
Duration of study  
33. While this Test Method primarily is designed as a 12 month chronic toxicity 
study, the study design also allows for and can be applied to either shorter (e.g. 6 or 9 months) or longer (e.g. 18 or 24 months) duration studies, depending on the requirements of particular regulatory regimes or for 
specific mechanistic purposes. Deviations from an exposure duration of 12 
months should be justified, particularly in the case of shorter durations. Satellite groups included to monitor the reversibility of any toxicological changes induced by the test chemical under investigation should be main ­
tained without dosing for a period not less than 4 weeks and not more than 
one third of the total study duration after cessation of exposure. Further guidance, including consideration of survival in the study, is provided in Guidance Document No 116 (6).
 
OBSERVATIONS 
34. All animals should be checked for morbidity or mortality, usually at the 
beginning and end of each day, including at weekends and holidays. General clinical observations should be made at least once a day, preferably 
at the same time(s) each day, taking into consideration the peak period of 
anticipated effects after dosing in the case of gavage administration.
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35. Detailed clinical observations should be made on all animals at least once 
prior to the first exposure (to allow for within-subject comparisons), at the 
end of the first week of the study and monthly thereafter. The protocol for observations should be arranged such that variations between individual observers are minimised and independent of test group. These observations 
should be made outside the home cage, preferably in a standard arena and at 
similar times on each occasion. They should be carefully recorded, preferably using scoring systems, explicitly defined by the testing laboratory. Efforts should be made to ensure that variations in the obser ­
vation conditions are minimal. Signs noted should include, but not be 
limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloe ­
rection, pupil size, and unusual respiratory pattern). Changes in gait, 
posture and response to handling as well as the presence of clonic or 
tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) should also be recorded (24). 
36. Ophthalmological examination, using an ophthalmoscope or other suitable 
equipment, should be carried out on all animals prior to the first adminis ­
tration of the test chemical. At the termination of the study, this examination 
should be preferably conducted in all animals but at least in the high dose and control groups. If treatment-related changes in the eyes are detected, all 
animals should be examined. If structural analysis or other information 
suggests ocular toxicity, then the frequency of ocular examination should be increased. 
37. For chemicals where previous repeated dose 28-day and/or 90-day toxicity 
tests indicated the potential to cause neurotoxic effects, sensory reactivity to stimuli of different types (24) (e.g. auditory, visual and proprioceptive 
stimuli) (25), (26), (27), assessment of grip strength (28) and motor 
activity assessment (29) may optionally be conducted before commencement of the study and at 3 month periods after study initiation up to and including 12 months, as well as at study termination (if longer than 12 months). 
Further details of the procedures that could be followed are given in the 
respective references. However, alternative procedures than those referenced could also be used. 
38. For chemicals where previous repeated dose 28-day and/or 90-day toxicity 
tests indicated the potential to cause immunotoxic effects, further investi ­
gations of this endpoint may optionally be conducted at termination.
 
Body weight, food/water consumption and food efficiency  
39. All animals should be weighed at the start of treatment, at least once a week 
for the first 13 weeks, and at least monthly thereafter. Measurements of food 
consumption and food efficiency should be made at least weekly for the first 
13 weeks and at least monthly thereafter. Water consumption should be measured at least weekly for the first 13 weeks and at least monthly thereafter when the chemical is administered in drinking water. Water 
consumption measurements should also be considered for studies in which 
drinking activity is altered.
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Haematology and clinical biochemistry  
40. In studies involving rodents, haematological examinations should be carried 
out in at least 10 male and 10 female animals per group, at 3, 6, and 12 months, as well as at study termination (if longer than 12 months), using the 
same animals throughout. In mice, satellite animals may be needed in order 
to conduct all required haematological determinations (see paragraph 18). In non-rodent studies, samples will be taken from smaller numbers of animals (e.g. 4 animals per sex and per group in dog studies), at interim sampling 
times and at termination as described for rodents. Measurements at 3 
months, either in rodents or non-rodents, need not be conducted if no effect was seen on haematological parameters in a previous 90 day study 
carried out at comparable dose levels. Blood samples should be taken from a 
named site, for example by cardiac puncture or from the retro-orbital sinus, under anaesthesia. 
41. The following list of parameters should be investigated (30): Total and 
differential leukocyte count, erythrocyte count, platelet count, haemoglobin 
concentration, haematocrit (packed cell volume), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), prothrombin time, and activated partial 
thromboplastin time. Other hematology parameters such as Heinz bodies or 
other atypical erythrocyte morphology or methaemoglobin may be measured as appropriate depending on the toxicity of the test chemical. Overall, a flexible approach should be adopted, depending on the observed and/or 
expected effect from a given test chemical. If the test chemical has an 
effect on the haematopoietic system, reticulocyte counts and bone marrow cytology may also be indicated, although these need not be routinely conducted. 
42. Clinical biochemistry determinations to investigate major toxic effects in 
tissues and, specifically, effects on kidney and liver, should be performed on blood samples obtained from at least 10 male and 10 female animals per 
group at the same time intervals as specified for the haematological inves ­
tigations, using the same animals throughout. In mice, satellite animals may be needed in order to conduct all required clinical biochemistry deter ­
minations. In non-rodent studies, samples will be taken from smaller 
numbers of animals (e.g. 4 animals per sex and per group in dog 
studies), at interim sampling times and at termination as described for rodents. Measurements at 3 months, either in rodents or non-rodents, need not be conducted if no effect was seen on clinical biochemistry parameters 
in a previous 90 day study carried out at comparable dose levels. Overnight 
fasting of the animals (with the exception of mice) prior to blood sampling is recommended The following list of parameters should be investigated (30): glucose, urea (urea nitrogen), creatinine, total protein, albumin, 
calcium, sodium, potassium, total cholesterol, at least two appropriate tests 
for hepatocellular evaluation (alanine aminotransferase, aspartate amino ­
transferase, glutamate dehydrogenase, total bile acids) (31), and at least two appropriate tests for hepatobiliary evaluation (alkaline phosphatase, 
gamma glutamyl transferase, 5’-nucleotidase, total bilirubin, total bile 
acids) (31). Other clinical chemistry parameters such as fasting triglycerides, specific hormones and cholinesterase may be measured as appropriate, depending on the toxicity of the test chemical. Overall, there is a need 
for a flexible approach, depending on the observed and/or expected effect 
from a given test chemical.
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43. Urinalysis determinations should be performed on at least 10 male and 10 
female animals per group on samples collected at the same intervals as for 
haematology and clinical chemistry. Measurements at 3 months need not be conducted if no effect was seen on urinalysis in a previous 90 day study carried out at comparable dose levels. The following list of parameters was 
included in an expert recommendation on clinical pathology studies (30): 
appearance, volume, osmolality or specific gravity, pH, total protein, and glucose. Other determinations include ketone, urobilinogen, bilirubin, and occult blood. Further parameters may be employed where necessary to 
extend the investigation of observed effect(s). 
44. It is generally considered that baseline haematological and clinical 
biochemistry variables are needed before treatment for dog studies, but need not be determined in rodent studies (30). However, if historical 
baseline data (see paragraph 50) are inadequate, consideration should be 
given to generating such data.
 
Pathology  
Gross necropsy  
45. All animals in the study shall normally be subjected to a full, detailed gross 
necropsy which includes careful examination of the external surface of the 
body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. However provision may also be made (in the interim kill or satellite groups) for measurements to be restricted to specific, key 
measures such as neurotoxicity or immunotoxicity (see paragraph 19). 
These animals need not be subjected to necropsy and the subsequent procedures described in the following paragraphs. Sentinel animals may require necropsy on a case-by-case basis, at the discretion of the study 
director. 
46. Organ weights should be collected from all animals, other than those 
excluded by the latter part of paragraph 45. The adrenals, brain, epididy ­
mides, heart, kidneys, liver, ovaries, spleen, testes, thyroid (weighed 
post-fixation, with parathyroids), and uterus of all animals (apart from 
those found moribund and/or intercurrently killed) should be trimmed of any adherent tissue, as appropriate, and their wet weight taken as soon as possible after dissection to prevent drying. In a study using mice, weighing 
of the adrenal glands is optional. 
47. The following tissues should be preserved in the most appropriate fixation 
medium for both the type of tissue and the intended subsequent histopath ­
ological examination (32) (tissues in square brackets are optional): 
all gross lesions heart pancreas stomach (forestomach, 
glandular stomach) 
adrenal gland ileum parathyroid gland [teeth] 
aorta jejunum peripheral nerve testis brain (including sections of 
cerebrum, cerebellum, and medulla/pons) kidney pituitary thymus 
caecum lacrimal gland (exorbital) prostate thyroid 
cervix liver rectum [tongue]
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coagulating gland lung salivary gland trachea 
colon lymph nodes (both 
superficial and deep) seminal vesicle urinary bladder 
duodenum mammary gland 
(obligatory for females 
and, if visibly dissectable, from males) skeletal muscle uterus (including cervix) 
epididymis [upper respiratory tract, 
including nose, turbi ­
nates, and paranasal sinuses] skin [ureter] 
eye (including retina) oesophagus spinal cord (at three 
levels: cervical, 
mid-thoracic, and lumbar) [urethra] 
[femur with joint] [olfactory bulb] spleen vagina 
gall bladder (for species 
other than rat) ovary [sternum], section of bone marrow and/ 
or a fresh bone marrow aspirate 
Harderian gland 
In the case of paired organs, e.g. kidney, adrenal, both organs should be 
preserved. The clinical and other findings may suggest the need to examine 
additional tissues. Also any organs considered likely to be target organs based on the known properties of the test chemical should be preserved. In studies involving the dermal route of administration, the list of organs as 
set out for the oral route should be preserved, and specific sampling and 
preservation of the skin from the site of application is essential. In inhalation studies, the list of preserved and examined tissues from the respiratory tract should follow the recommendations of Chapters B.8 of this Annex (8) and 
Chapter B.29 of this Annex (9). For other organs/tissues (and in addition to 
the specifically preserved tissues from the respiratory tract) the list of organs as set out for the oral route should be examined.
 
Histopathology  
48. Guidance is available on best practices in the conduct of toxicological 
pathology studies (32). The minimum histopathological examinations should be: 
— all tissues from the high dose and control groups; 
— all tissues from animals dying or killed during the study; 
— all tissues showing macroscopic abnormalities; — target tissues, or tissues which showed treatment-related changes in the 
high dose group, from all animals in all other dose groups; 
— in the case of paired organs, e.g. kidney, adrenal, both organs should be 
examined.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 450
 
DATA AND REPORTING  
Data  
49. Individual animal data should be provided for all parameters evaluated. 
Additionally, all data should be summarised in tabular form showing for each test group the number of animals at the start of the test, the number of animals found dead during the test or killed for humane reasons and the 
time of any death or humane kill, the number showing signs of toxicity, a 
description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects, the number of animals showing lesions, the type of lesions and the percentage of animals 
displaying each type of lesion. Summary data tables should provide the 
means and standard deviations (for continuous test data) of animals showing toxic effects or lesions, in addition to the grading of lesions. 
50. Historical control data may be valuable in the interpretation of the results of 
the study, e.g. in the case when there are indications that the data provided 
by the concurrent controls are substantially out of line when compared to 
recent data from control animals from the same test facility/colony. Historical control data, if evaluated, should be submitted from the same laboratory and relate to animals of the same age and strain generated 
during the five years preceding the study in question. 
51. When applicable, numerical results should be evaluated by an appropriate 
and generally acceptable statistical method. The statistical methods and the 
data to be analysed should be selected during the design of the study (paragraph 8). Selection should make provision for survival adjustments, if needed.
 
Test Report  
52. The test report should include the following information:  
Test chemical:  
— physical nature, purity, and physicochemical properties; 
— identification data; 
— source of chemical; — batch number; — certificate of chemical analysis  
Vehicle (if appropriate):  
— justification for choice of vehicle (if other than water).  
Test animals:  
— species/strain used and justification for choice made; — number, age, and sex of animals at start of test;
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 451
 
— source, housing conditions, diet, etc.; 
— individual weights of animals at the start of the test.  
Test conditions:  
— rationale for route of administration and dose selection; 
— when applicable, the statistical methods used to analyse the data; — details of test chemical formulation/diet preparation; 
— analytical data on achieved concentration, stability and homogeneity of 
the preparation; 
— route of administration and details of the administration of the test 
chemical; 
— for inhalation studies, whether nose only or whole body; 
— actual doses (mg/kg body weight/day), and conversion factor from diet/ 
drinking water test chemical concentration (mg/kg or ppm) to the actual dose, if applicable; 
— details of food and water quality.  
Results (summary tabulated data and individual animal data should be 
presented):  
— survival data; — body weight/body weight changes; 
— food consumption, calculations of food efficiency, if made, and water 
consumption if applicable; 
— toxic response data by sex and dose level, including signs of toxicity; — nature, incidence (and, if scored, severity), and duration of clinical 
observations ((whether transitory or permanent); 
— ophthalmological examination; 
— haematological tests; 
— clinical biochemistry tests; — urinalysis tests; — outcome of any investigations of neurotoxicity or immunotoxicity; 
— terminal body weight; 
— organ weights (and their ratios, if applicable); — necropsy findings; 
— a detailed description of all treatment-related histopathological findings; 
— absorption data if available;
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 452
 
Statistical treatment of results, as appropriate 
Discussion of results including:  
— Dose: response relationships 
— Consideration of any mode of action information — Discussion of any modelling approaches 
— BMD, NOAEL or LOAEL determination 
— Historical control data 
— Relevance for humans  
Conclusions
 
LITERATURE:  
(1) OECD (1995). Report of the Consultation Meeting on Sub-chronic and 
Chronic Toxicity/Carcinogenicity Testing (Rome, 1995), internal working 
document, Environment Directorate, OECD, Paris. 
(2) Combes RD, Gaunt I, Balls M (2004). A Scientific and Animal Welfare 
Assessment of the OECD Health Effects Test Guidelines for the Safety 
Testing of Chemicals under the European Union REACH System. ATLA 
32: 163-208. 
(3) Barlow SM, Greig JB, Bridges JW  et al.  (2002). Hazard identification by 
methods of animal-based toxicology. Food. Chem. Toxicol. 40, 145-191. 
(4) Chhabra RS, Bucher JR, Wolfe M, Portier C (2003). Toxicity character ­
ization of environmental chemicals by the US National Toxicology 
Programme: an overview. Int. J. Hyg. Environ. Health 206: 437-445. 
(5) Chapter B.27 of this Annex, Sub-chronic Oral Toxicity Test Repeated Dose 
90-day Oral Toxicity Study in Non-Rodents. 
(6) OECD (2012). Guidance Document on the Design and Conduct of Chronic 
Toxicity and Carcinogenicity Studies, Supporting Test Guidelines 451, 452 
and 453 — Second edition. Series on Testing and Assessment No 116, available on the OECD public website for Test Guideline at www.oecd.org/ env/testguidelines. 
(7) OECD (2009). Guidance Document on Acute Inhalation Toxicity Testing, 
Series on Testing and Assessment No 39, ENV/JM/MONO(2009)28, OECD, Paris. 
(8) Chapter B.8 of this Annex, Subacute Inhalation Toxicity: 28-Day Study. (9) Chapter B.29 of this Annex, Subchronic Inhalation Toxicity: 90-Day Study. 
(10) Chapter B.9 of this Annex, Repeated Dose (28 Days) Toxicity (Dermal). 
(11) Carmichael NG, Barton HA, Boobis AR  et al.  (2006). Agricultural 
Chemical Safety Assessment: A Multisector Approach to the Modern ­
ization of Human Safety Requirements. Critical Reviews in Toxicology 36: 1-7. 
(12) Barton HA, Pastoor TP, Baetcke T  et al.  (2006). The Acquisition and 
Application of Absorption, Distribution, Metabolism, and 
Excretion (ADME) Data in Agricultural Chemical Safety Assessments. 
Critical Reviews in Toxicology 36: 9-35. 
(13) Doe JE, Boobis AR, Blacker A  et al.  (2006). A Tiered Approach to 
Systemic Toxicity Testing for Agricultural Chemical Safety Assessment. 
Critical Reviews in Toxicology 36: 37-68.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 453
 
(14) Cooper RL, Lamb JS, Barlow SM  et al . (2006). A Tiered Approach to Life 
Stages Testing for Agricultural Chemical Safety Assessment. Critical 
Reviews in Toxicology 36: 69-98. 
(15) OECD (2002). Guidance Notes for Analysis and Evaluation of Chronic 
Toxicity and Carcinogenicity Studies, Series on Testing and Assessment 
No 35 and Series on Pesticides No 14, ENV/JM/MONO(2002)19, OECD, Paris. 
(16) OECD (2000). Guidance Document on the recognition, assessment, and use 
of clinical signs as humane endpoints for experimental animals used in safety evaluation, No 19, ENV/JM/MONO(2000)7, OECD, Paris. 
(17) Rhomberg LR, Baetcke K, Blancato J, Bus J, Cohen S, Conolly R, Dixit R, 
Doe J, Ekelman K, Fenner-Crisp P, Harvey P, Hattis D, Jacobs A, Jacobson-Kram D, Lewandowski T, Liteplo R, Pelkonen O, Rice J, Somers D, Turturro A, West W, Olin S (2007). Issues in the Design and 
Interpretation of Chronic Toxicity and Carcinogenicity Studies in Rodents: 
Approaches to Dose Selection Crit Rev. Toxicol. 37 (9): 729 - 837. 
(18) ILSI (International Life Sciences Institute) (1997). Principles for the 
Selection of Doses in Chronic Rodent Bioassays. Foran JA (Ed.). ILSI Press, Washington, DC. 
(19) Directive 2010/63/EU of the European parliament and of the Council of 
22 September 2010 on the protection of animals used for scientific purposes (OJ L 276, 20.10.2010, p. 33). 
(20) National Research Council, 1985. Guide for the care and use of laboratory 
animals. NIH Publication No 86-23. Washington D.C., US. Dept. of Health and Human Services. 
(21) GV-SOLAS (Society for Laboratory Animal Science, Gesellschaft für 
Versuchstierkunde, 1988). Publication on the Planning and Structure of Animal Facilities for Institutes Performing Animal Experiments. ISBN 3- 906255-04-2. 
(22) GV-SOLAS (Society for Laboratory Animal Science, Gesellschaft für 
Versuchstierkunde, 2006). Microbiological monitoring of laboratory 
animals in various housing systems. 
(23) Diehl K-H, Hull R, Morton D, Pfister R, Rabemampianina Y, Smith D, 
Vidal J-M, van de Vorstenbosch C. 2001. A good practice guide to the 
administration of substances and removal of blood, including routes and 
volumes. Journal of Applied Toxicology 21:15-23. 
(24) IPCS (1986). Principles and Methods for the Assessment of Neurotoxicity 
Associated with Exposure to Chemicals. Environmental Health Criteria 
Document No 60. 
(25) Tupper DE, Wallace RB (1980). Utility of the Neurologic Examination in 
Rats. Acta Neurobiol. Exp. 40: 999-1003. 
(26) Gad SC (1982). A Neuromuscular Screen for Use in Industrial Toxicology. 
J. Toxicol.Environ. Health 9: 691-704. 
(27) Moser VC, McDaniel KM, Phillips PM (1991). Rat Strain and Stock 
Comparisons Using a Functional Observational Battery: Baseline Values 
and Effects of Amitraz. Toxicol. Appl. Pharmacol. 108: 267-283. 
(28) Meyer OA, Tilson HA, Byrd WC, Riley MT (1979). A Method for the 
RoutineAssessment of Fore- and Hind-limb Grip Strength of Rats and 
Mice. Neurobehav. Toxicol. 1: 233-236. 
(29) Crofton KM, Howard JL, Moser VC, Gill MW, Reiter LW, Tilson HA, 
MacPhail RC (1991). Interlaboratory Comparison of Motor Activity 
Experiments: Implication for Neurotoxicological Assessments. Neur ­
otoxicol. Teratol. 13: 599-609.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 454
 
(30) Weingand K, Brown G, Hall R  et al.  (1996). Harmonisation of Animal 
Clinical Pathology Testing in Toxicity and Safety Studies. Fundam. & 
Appl. Toxicol. 29: 198-201. 
(31) EMEA (draft) document ‘Non-clinical guideline on drug-induced hepatot ­
oxicity’ (Doc. Ref. EMEA/CHMP/SWP/a50115/2006). 
(32) Crissman JW, Goodman DG, Hildebrandt PK et al. (2004). Best Practices 
Guideline: Toxicological Histopathology. Toxicologic Pathology 32: 126- 
131. ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 455
 
Appendix 1  
DEFINITION  
Test chemical:  Any substance or mixture tested using this Test Method.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 456
 
B.31.  PRENATAL DEVELOPMENTAL TOXICITY STUDY  
1. METHOD  
This method is a replicate of OECD TG 414 (2001).  
1.1. INTRODUCTION 
This method for developmental toxicity testing is designed to 
provide general information concerning the effects of prenatal exposure on the pregnant test animal and on the developing 
organism in utero; this may include assessment of maternal effects 
as well as death, structural abnormalities, or altered growth in the foetus. Functional deficits, although an important part of devel ­
opment, are not an integral part of this test method. They may be 
tested for in a separate study or as an adjunct to this study using the 
test method for developmental neurotoxicity. For information on testing for functional deficiencies and other postnatal effects the 
test method for the two-generation reproductive toxicity study and 
the developmental neurotoxicity study should be consulted as appro ­
priate. 
This test method may require specific adaptation in individual cases 
on the basis of specific knowledge on e.g. physicochemical or toxi ­
cological properties of the test substance. Such adaptation is 
acceptable, when convincing scientific evidence suggests that the 
adaptation will lead to a more informative test. In such a case, this scientific evidence should be carefully documented in the study report.
 
1.2. DEFINITIONS  
Developmental toxicology:  the study of adverse effects on the 
developing organism that may result from exposure prior to 
conception, during prenatal development, or postnatally to the time 
of sexual maturation. The major manifestations of developmental toxicity include 1) death of the organism, 2) structural abnormality, 3) altered growth, and 4) functional deficiency. Developmental toxi ­
cology was formerly often referred to as teratology.  
Adverse effect:  any treatment-related alteration from baseline that 
diminishes an organism's ability to survive, reproduce or adapt to the environment. Concerning developmental toxicology, taken in its widest sense it includes any effect which interferes with normal 
development of the conceptus, both before and after birth.  
Altered growth:  an alteration in offspring organ or body weight or 
size. 
Alterations (anomalies):  structural alterations in development that 
include both malformations and variations (28).  
Malformation/Major abnormality:  structural change considered 
detrimental to the animal (may also be lethal) and is usually rare.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 457
 
Variation/Minor abnormality:  structural change considered to have 
little or no detrimental effect on the animal; may be transient and 
may occur relatively frequently in the control population.  
Conceptus:  the sum of derivatives of a fertilised ovum at any stage 
of development from fertilisation until birth including the extra- 
embryonic membranes as well as the embryo or foetus.  
Implantation (nidation):  attachment of the blastocyst to the 
epithelial lining of the uterus, including its penetration through the uterine epithelium, and its embedding in the endometrium.  
Embryo:  the early or developing stage of any organism, especially 
the developing product of fertilisation of an egg after the long axis appears and until all major structures are present.  
Embryotoxicity:  detrimental to the normal structure, development, 
growth, and/or viability of an embryo.  
Foetus:  the unborn offspring in the post-embryonic period.  
Foetotoxicity:  detrimental to the normal structure, development, 
growth, and/or viability of a foetus.  
Abortion:  the premature expulsion from the uterus of the products 
of conception: of the embryo or of a nonviable foetus.  
Resorption:  a conceptus which, having implanted in the uterus, 
subsequently died and is being, or has been resorbed.  
Early resorption:  evidence of implantation without recognisable 
embryo/foetus  
Late resorption:  dead embryo or foetus with external degenerative 
changes  
NOAEL:  abbreviation for no-observed-adverse-effect level and is 
the highest dose or exposure level where no adverse treatment- related findings are observed.
 
1.3. REFERENCE SUBSTANCE 
None.  
1.4. PRINCIPLE OF THE TEST METHOD 
Normally, the test substance is administered to pregnant animals at 
least from implantation to one day prior to the day of scheduled kill, 
which should be as close as possible to the normal day of delivery without risking loss of data resulting from early delivery. The test method is not intended to examine solely the period of organo ­
genesis, (e.g. days 5-15 in the rodent, and days 6-18 in the rabbit) 
but also effects from preimplantation, when appropriate, through the entire period of gestation to the day before caesarean section. Shortly before caesarean section, the females are killed, the uterine contents 
are examined, and the foetuses are evaluated for externally visible 
anomalies and for soft tissue and skeletal changes.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 458
 
1.5. DESCRIPTION OF THE TEST METHOD  
1.5.1.  Selection of animal species  
It is recommended that testing be performed in the most relevant 
species, and that laboratory species and strains which are commonly 
used in prenatal developmental toxicity testing be employed. The 
preferred rodent species is the rat and the preferred non-rodent species is the rabbit. Justification should be provided if another species is used.
 
1.5.2.  Housing and feeding conditions  
The temperature in the experimental animal room should be 22  o C (± 
3 o ) for rodents and 18  o C (± 3  o ) for rabbits. Although the relative 
humidity should be at least 30 % and preferably not exceed 70 % 
other than during room cleaning, the aim should be 50-60 %. 
Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. 
Mating procedures should be carried out in cages suitable for the 
purpose. While individual housing of mated animals is preferred, 
group housing in small numbers is also acceptable.
 
1.5.3.  Preparation of the animals  
Healthy animals, which have been acclimated to laboratory 
conditions for at least five days and have not been subjected to previous experimental procedures, should be used. The test animals 
should be characterised as to species, strain, source, sex, weight 
and/or age. The animals of all test groups should, as nearly as prac ­
ticable, be of uniform weight and age. Young adult nulliparous female animals should be used at each dose level. The females 
should be mated with males of the same species and strain, and 
the mating of siblings should be avoided. For rodents day 0 of gestation is the day on which a vaginal plug and/or sperm are observed; for rabbits day 0 is usually the day of coitus or of artificial 
insemination, if this technique is used. Mated females should be 
assigned in an unbiased manner to the control and treatment groups. Cages should be arranged in such a way that possible 
effects due to cage placement are minimised. Each animal should 
be assigned a unique identification number. Mated females should be assigned in an unbiased manner to the control and treatment groups, and if the females are mated in batches, the animals in each batch 
should be evenly distributed across the groups. Similarly, females 
inseminated by the same male should be evenly distributed across the groups.
 
1.6. PROCEDURE  
1.6.1.  Number and sex of animals  
Each test and control group should contain a sufficient number of 
females to result in approximately 20 female animals with implan ­
tation sites at necropsy. Groups with fewer than 16 animals with implantation sites may be inappropriate. Maternal mortality does 
not necessarily invalidate the study providing it does not exceed 
approximately 10 %.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 459
 
1.6.2.  Preparation of doses  
If a vehicle or other additive is used to facilitate dosing, 
consideration should be given to the following characteristics: effects on the absorption, distribution, metabolism, and retention or 
excretion of the test substance; effects on the chemical properties of 
the test substance which may alter its toxic characteristics; and effects on the food or water consumption or the nutritional status of the animals. The vehicle should neither be developmentally toxic 
nor have effects on reproduction.
 
1.6.3.  Dosage  
Normally, the test substance should be administered daily from 
implantation (e.g. day 5 post mating) to the day prior to scheduled 
caesarean section. If preliminary studies, when available, do not 
indicate a high potential for preimplantation loss, treatment may be extended to include the entire period of gestation, from mating to the day prior to scheduled kill. It is well known that inappropriate 
handling or stress during pregnancy can result in prenatal loss. To 
guard against prenatal loss from factors which are not treatment related, unnecessary handling of pregnant animals as well as stress from outside factors such as noise should be avoided. 
At least three dose levels and a concurrent control should be used. 
Healthy animals should be assigned in an unbiased manner to the control and treatment groups. The dose levels should be spaced to 
produce a gradation of toxic effects. Unless limited by the physical/ ­
chemical nature or biological properties of the test substance, the highest dose should be chosen with the aim to induce some devel ­
opmental and/or maternal toxicity (clinical signs or a decrease in 
body weight) but not death or severe suffering. At least one inter ­
mediate dose level should produce minimal observable toxic effects. The lowest dose level should not produce any evidence of either maternal or developmental toxicity. A descending sequence of 
dose levels should be selected with a view to demonstrating any 
dosage-related response and no-observed-adverse-effect level (NOAEL). Two- to four-fold intervals are frequently optimal for setting the descending dose levels, and the addition of a fourth test 
group is often preferable to using very large intervals (e.g. more than 
a factor of 10) between dosages. Although establishment of a maternal NOAEL is the goal, studies which do not establish such a level may also be acceptable (1). 
Dose levels should be selected taking into account any existing 
toxicity data as well as additional information on metabolism and toxicokinetics of the test substance or related materials. This 
information will also assist in demonstrating the adequacy of the 
dosing regimen. 
A concurrent control group should be used. This group should be a 
sham-treated control group or a vehicle-control group if a vehicle is 
used in administering the test substance. All groups should be 
administered the same volume of either test substance or vehicle. Animals in the control group(s) should be handled in an identical manner to test group animals. Vehicle control groups should receive 
the vehicle in the highest amount used (as in the lowest treatment 
group).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 460
 
1.6.4.  Limit test  
If a test at one dose level of at least 1 000 mg/kg body weight/day 
by oral administration, using the procedures described for this study, produces no observable toxicity in either pregnant animals or their 
progeny and if an effect would not be expected based upon existing 
data (e.g. from structurally and/or metabolically related compounds), then a full study using three dose levels may not be considered necessary. Expected human exposure may indicate the need for a 
higher oral dose level to be used in the limit test. For other types of 
administration, such as inhalation or dermal application, the physico- chemical properties of the test substance often may indicate and limit 
the maximum attainable level of exposure (for example, dermal 
application should not cause severe local toxicity).
 
1.6.5.  Administration of doses  
The test substance or vehicle is usually administered orally by intu ­
bation. If another route of administration is used, the tester should 
provide justification and reasoning for its selection, and appropriate 
modifications may be necessary (2)(3)(4). The test substance should be administered at approximately the same time each day. 
The dose to individual animals should normally be based on the 
most recent individual body weight determination. However, caution should be exercised when adjusting the dose during the 
last trimester of pregnancy. Existing data should be used for dose 
selection to prevent excess maternal toxicity. However, if excess toxicity is noted in the treated dams, those animals should be humanely killed. If several pregnant animals show signs of excess 
toxicity, consideration should be given to terminating that dose 
group. When the substance is administered by gavage, this should preferably be given as a single dose to the animals using a stomach tube or a suitable intubation canula. The maximum volume of liquid 
that can be administered at one time depends on the size of the test 
animal. The volume should not exceed 1 ml/100 g body weight, except in the case of aqueous solutions where 2 ml/100 g body weight may be used. When corn oil is used as a vehicle, the 
volume should not exceed 0.4 ml/100 g body weight. Variability 
in test volume should be minimised by adjusting the concentrations to ensure a constant volume across all dose levels.
 
1.6.6.  Observations of the dams  
Clinical observations should be made and recorded at least once a 
day, preferably at the same time(s) each day taking into consideration the peak period of anticipated effects after dosing. The condition of the animals should be recorded including mortality, 
moribundity, pertinent behavioural changes, and all signs of overt 
toxicity.
 
1.6.7.  Body weight and food consumption  
Animals should be weighed on day 0 of gestation or no later than 
day 3 of gestation if time-mated animals are supplied by an outside 
breeder, on the first day of dosing, at least every three days during 
the dosing period and on the day of scheduled kill.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 461
 
Food consumption should be recorded at three-day intervals and 
should coincide with days of body weight determination.  
1.6.8.  Post-mortem examination  
Females should be killed one day prior to the expected day of 
delivery. Females showing signs of abortion or premature delivery prior to scheduled kill should be killed and subjected to a thorough macroscopic examination. 
At the time of termination or death during the study, the dam should 
be examined macroscopically for any structural abnormalities or pathological changes. Evaluation of the dams during caesarean section and subsequent foetal analyses should be conducted 
preferably without knowledge of treatment group in order to 
minimise bias.
 
1.6.9.  Examination of uterine contents  
Immediately after termination or as soon as possible after death, the 
uteri should be removed and the pregnancy status of the animals 
ascertained. Uteri that appear non gravid should be further 
examined (e.g. by ammonium sulphide staining for rodents and Salewski staining or a suitable alternative method for rabbits) to confirm the non-pregnant status (5). 
Gravid uteri including the cervix should be weighed. Gravid uterine 
weights should not be obtained from animals found dead during the study. 
The number of corpora lutea should be determined for pregnant 
animals. 
The uterine contents should be examined for numbers of embryonic 
or foetal deaths and viable foetuses. The degree of resorption should be described in order to estimate the relative time of death of the 
conceptus (see Section 1.2).
 
1.6.10.  Examination of foetuses  
The sex and body weight of each foetus should be determined. 
Each foetus should be examined for external alterations (6). Foetuses should be examined for skeletal and soft tissue 
alterations (e.g. variations and malformations or anomalies) 
(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). 
Categorisation of foetal alterations is preferable but not required. When categorisation is done, the criteria for defining each category should be clearly stated. Particular attention should be paid to the reproductive tract 
which should be examined for signs of altered development. 
For rodents, approximately one-half of each litter should be prepared 
and examined for skeletal alterations. The remainder should be prepared and examined for soft tissue alterations, using accepted or 
appropriate serial sectioning methods or careful gross dissection 
techniques.
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For non-rodents, e.g. rabbits, all foetuses should be examined 
for both soft tissue and skeletal alterations. The bodies of these 
foetuses are evaluated by careful dissection for soft tissue alterations, which may include procedures to further evaluate internal cardiac structure (25). The heads of one-half of the foetuses examined in 
this manner should be removed and processed for evaluation of soft 
tissue alterations (including eyes, brain, nasal passages and tongue), using standard serial sectioning methods (26) or an equally sensitive method. The bodies of these foetuses and the remaining intact 
foetuses should be processed and examined for skeletal alterations, 
utilising the same methods as described for rodents.
 
2. DATA  
2.1. TREATMENT OF RESULTS 
Data shall be reported individually for the dams as well as for their 
offspring and summarised in tabular form, showing for each test 
group and each generation the number of animals at the start of the test, the number of animals found dead during the test or killed for humane reasons, the time of any death or humane kill, 
the number of pregnant females, the number of animals showing 
signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects, the types of embryo/foetal observations, and all relevant litter data. 
Numerical results should be evaluated by an appropriate statistical 
method using the litter as the unit for data analysis. A generally accepted statistical method should be used; the statistical methods should be selected as part of the design of the study and should be 
justified. Data from animals that do not survive to the scheduled kill 
should also be reported. These data may be included in group means where relevant. Relevance of the data obtained from such animals, and therefore inclusion or exclusion from any group mean(s), should 
be justified and judged on an individual basis.
 
2.2. EVALUATION OF RESULTS 
The findings of the Prenatal Developmental Toxicity Study should 
be evaluated in terms of the observed effects. The evaluation will include the following information: 
— maternal and embryo/foetal test results, including the evaluation 
of the relationship, or lack thereof, between the exposure of the animals to the test substance and the incidence and severity of all findings, 
— criteria used for categorising foetal external, soft tissue, and 
skeletal alterations if categorisation has been done,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 463
 
— when appropriate, historical control data to enhance interpretation 
of study results, 
— the numbers used in calculating all percentages or indices, — adequate statistical analysis of the study findings, when appro ­
priate, which should include sufficient information on the method 
of analysis, so that an independent reviewer/statistician can re- evaluate and reconstruct the analysis. 
In any study which demonstrates the absence of any toxic effects, 
further investigations to establish absorption and bioavailability of 
the test substance should be considered.
 
2.3. INTERPRETATION OF RESULTS 
A prenatal developmental toxicity study will provide information on 
the effects of repeated exposure to a substance during pregnancy on 
the dams and on the intrauterine development of their progeny. The 
results of the study should be interpreted in conjunction with the findings from subchronic, reproduction, toxicokinetic and other studies. Since emphasis is placed both on general toxicity in terms 
of maternal toxicity and on developmental toxicity endpoints, the 
results of the study will allow to a certain extent for the discrimi ­
nation between developmental effects occurring in the absence of 
general toxicity and those which are only induced at levels that 
are also toxic to the maternal animal (27).
 
3. REPORTING  
3.1. TEST REPORT 
The test report must include the following specific information: 
Test substance: 
— physical nature and, where relevant, physiochemical properties, — identification including CAS number if known/established, 
— purity. 
Vehicle (if appropriate): 
— justification for choice of vehicle, if other than water. 
Test animals: — species and strain used, 
— number and age of animals, 
— source, housing conditions, diet, etc., 
— individual weights of animals at the start of the test. 
Test conditions: — rationale for dose level selection, 
— details of test substance formulation/diet preparation, achieved 
concentration, stability and homogeneity of the preparation,
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— details of the administration of the test substance, 
— conversion from diet/drinking water test substance concentration 
(ppm) to the actual dose (mg/kg body weight/day), if applicable, 
— environmental conditions, 
— details of food and water quality. 
Results: 
Maternal toxic response data by dose, including but not limited to: 
— the number of animals at the start of the test, the number of 
animals surviving, the number pregnant, and the number 
aborting, number of animals delivering early, 
— day of death during the study or whether animals survived to 
termination, 
— data from animals that do not survive to the scheduled kill should 
be reported but not included in the inter-group statistical comparisons, 
— day of observation of each abnormal clinical sign and its 
subsequent course, 
— body weight, body weight change and gravid uterine weight, 
including, optionally, body weight change corrected for gravid uterine weight, 
— food consumption and, if measured, water consumption, 
— necropsy findings, including uterine weight, — NOAEL values for maternal and developmental effects should be 
reported. 
Developmental endpoints by dose for litters with implants, including: 
— number of corpora lutea, 
— number of implantations, number and percent of live and dead 
foetuses and resorptions, 
— number and percent of pre- and post-implantation losses. 
Developmental endpoints by dose for litters with live foetuses, 
including: 
— number and percent of live offspring, 
— sex ratio, 
— foetal body weight, preferably by sex and with sexes combined, 
— external, soft tissue, and skeletal malformations and other 
relevant alterations, 
— criteria for categorisation if appropriate,
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— total number and percent of foetuses and litters with any external, 
soft tissue, or skeletal alteration, as well as the types and inci ­
dences of individual anomalies and other relevant alterations. 
Discussion of results. 
Conclusions.  
4. REFERENCES  
(1) Kavlock R.J. et al., (1996) A Simulation Study of the Influence 
of Study Design on the Estimation of Benchmark Doses for 
Developmental Toxicity.  Risk Analysis  16; p. 399-410. 
(2) Kimmel, C.A. and Francis, E.Z., (1990) Proceedings of the 
Workshop on the Acceptability and Interpretation of Dermal 
Developmental Toxicity Studies.  Fundamental and Applied 
Toxicology  14; p. 386-398. 
(3) Wong, B.A., et al., (1997) Developing Specialised Inhalation 
Exposure Systems to Address Toxicological Problems.  CIIT 
Activities  17; p. 1-8. 
(4) US Environmental Protection Agency, (1985) Subpart E- 
Specific Organ/Tissue Toxicity, 40 CFR 798.4350: Inhalation 
Developmental Toxicity Study. 
(5) Salewski, E., (1964) Faerbermethode zum Makroskopischen 
Nachweis von Implantations Stellen am Uterusder Ratte.  
Naunyn-Schmeidebergs Archiv fur Pharmakologie und Experi ­
mentelle Pathologie , 247, 367. 
(6) Edwards, J.A., (1968) The external Development of the Rabbit 
and Rat Embryo. In  Advances in Teratology . D.H.M. Woolam 
(ed.) Vol. 3. Academic Press, NY. 
(7) Inouye, M. (1976) Differential Staining of Cartilage and Bone 
in Fetal Mouse Skeleton by Alcian Blue and Alizarin Red S.  
Congenital Anomalies  16, p. 171-173. 
(8) Igarashi, E. et al., (1992) Frequency Of Spontaneous Axial 
Skeletal Variations Detected by the Double Staining Tech ­
niquefor Ossified and Cartilaginous Skeleton in Rat Foetuses.  
Congenital Anomalies  32, p. 381-391. 
(9) Kimmel, C.A. et al. (1993) Skeletal Development Following 
Heat Exposure in the Rat.  Teratology , 47 p.229-242. 
(10) Marr, M.C. et al. (1988) Comparison of Single and Double 
Staining for Evaluation of Skeletal Development: The Effects of Ethylene Glycol (EG) in CD Rats.  Teratology  37; 476. 
(11) Barrow, M.V. and Taylor, W.J. (1969) A Rapid Method for 
Detecting Malformations in Rat Foetuses.  Journal of 
Morphology , 127, p. 291-306. 
(12) Fritz, H. (1974) Prenatal Ossification in Rabbits ss Indicative 
of Foetal Maturity.  Teratology , 11 p. 313-320. 
(13) Gibson, J.P. et al. (1966) Use of the Rabbit in Teratogenicity 
Studies.  Toxicology and Applied Pharmacology , 9, p. 398-408. 
(14) Kimmel, C.A. and Wilson, J.G. (1973) Skeletal Deviation in 
Rats: Malformations or Variations?  Teratology , 8, p. 309-316. 
(15) Marr, M.C. et al. (1992) Developmental Stages of the CD 
(Sprague-Dawley) Rat Skeleton after Maternal Exposure to 
Ethylene Glycol.  Teratology , 46, p. 169-181.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 466
 
(16) Monie, I.W. et al. (1965) Dissection Procedures for Rat 
Foetuses Permitting Alizarin Red Staining of Skeleton and 
Histological Study of Viscera.  Supplement to Teratology 
Workshop Manual , p. 163-173. 
(17) Spark, C. and Dawson, A.B. (1928) The Order and Time of 
appearance of Centers of Ossification in the Fore and Hind 
Limbs of the Albino Rat, with Special Reference to the 
Possible Influence of the Sex Factor.  American Journal of 
Anatomy , 41, p. 411-445. 
(18) Staples, R.E. and Schnell, V.L. (1964) Refinements in Rapid 
Clearing Technique in the KOH-Alizarin Red S Method for 
Fetal Bone.  Stain Technology , 39, p. 61-63. 
(19) Strong, R.M. (1928) The Order Time and Rate of Ossification 
of the Albino Rat (Mus Norvegicus Albinus) Skeleton.  American Journal of Anatomy , 36, p. 313-355. 
(20) Stuckhardt, J.L. and Poppe, S.M. (1984) Fresh Visceral Exam ­
ination of Rat and Rabbit Foetuses Used in Teratogenicity 
Testing.  Teratogenesis, Carcinogenesis, and Mutagenesis , 4, 
p. 181-188. 
(21) Walker, D.G. and Wirtschafter, Z.T. (1957)  The Genesis of the 
Rat Skeleton . Thomas, Springfield, IL. 
(22) Wilson, J.G. (1965) Embryological Considerations in Tera ­
tology. In  Teratology: Principles and Techniques,  Wilson 
J.G. and Warkany J. (eds). University of Chicago, Chicago, IL, p. 251-277. 
(23) Wilson, J.G. and Fraser, F.C. (eds). (1977)  Handbook of Tera ­
tology , Vol. 4. Plenum, NY. 
(24) Varnagy, L. (1980) Use of Recent Fetal Bone Staining Tech ­
niques in the Evaluation of Pesticide Teratogenicity.  Acta Vet. 
Acad. Sci. Hung , 28, p. 233-239. 
(25) Staples, R.E. (1974) Detection of visceral Alterations in 
Mammalian Foetuses.  Teratology , 9, p. 37-38. 
(26) Van Julsingha, E.B. and C.G. Bennett (1977) A Dissecting 
Procedure for the Detection of Anomalies in the Rabbit Foetal Head. In:  Methods in Prenatal Toxicology  Neubert, 
D., Merker, H.J. and Kwasigroch, T.E. (eds.). University of 
Chicago, Chicago, IL, p. 126-144. 
(27) US Environmental Protection Agency (1991) Guidelines for 
Developmental Toxicity Risk Assessment.  Federal Register , 
56, p. 63798-63826. 
(28) Wise, D.L. et al. (1997) Terminology of Developmental 
Abnormalities in Common Laboratory Mammals (Version 1) 
Teratology, 55, p. 249-292.
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B.32.  CARCINOGENICITY STUDIES  
INTRODUCTION 
1. This Test Method is equivalent to OECD Test Guideline (TG) 451 (2009). 
The original TG 451 on Carcinogenicity Studies was adopted in 1981. Development of this revised Test Method B.32 was considered necessary, 
in order to reflect recent developments in the field of animal welfare and 
regulatory requirements (2) (3) (4) (5) (6). The updating of this Test Method B.32 has been carried out in parallel with revisions of Chapter B.30 of this Annex, Chronic Toxicity Studies, and Chapter B.33, of this Annex, 
Combined Chronic Toxicity\Carcinogenicity Studies, and with the 
objective of obtaining additional information from the animals used in the study and providing further detail on dose selection. This Test Method B.32 is designed to be used in the testing of a broad range of chemicals, 
including pesticides and industrial chemicals. It should be noted however 
that some details and requirements may differ for pharmaceuticals (see International Conference on Harmonisation (ICH) Guidance S1B on Testing for Carcinogenicity of Pharmaceuticals). 
2. The majority of carcinogenicity studies are carried out in rodent species, and 
this Test Method is intended therefore to apply primarily to studies carried out in these species. Should such studies be required in non-rodent species, 
the principles and procedures outlined in this Test Method together with 
those outlined in Chapter B.27 of this Annex, Repeated Dose 90-day Oral Toxicity Study in Non-Rodents (6), should be applied, with appropriate modifications. Further guidance is available in the OECD Guidance 
Document No 116 on the Design and Conduct of Chronic Toxicity and 
Carcinogenicity Studies (7). 
3. The three main routes of administration used in carcinogenicity studies are 
oral, dermal and inhalation. The choice of the route of administration depends 
on the physical and chemical characteristics of the test chemical and the predominant route of exposure of humans. Additional information on choice of route of exposure is provided in Guidance Document No 116 (7). 
4. This Test Method focuses on exposure via the oral route, the route most 
commonly used in carcinogenicity studies. While carcinogenicity studies involving exposure via the dermal or inhalation routes may also be 
necessary for human health risk assessment and/or may be required under 
certain regulatory regimes, both routes of exposure involve considerable technical complexity. Such studies will need to be designed on a case-by- case basis, although the Test Method outlined here for the assessment and 
evaluation of carcinogenicity by oral administration could form the basis of 
a protocol for inhalation and/or dermal studies, with respect to recommen ­
dations for treatment periods, clinical and pathology parameters, etc. OECD Guidance is available on the administration of test chemicals by the dermal 
(7), and inhalation routes (7) (8). Chapter B.8 of this Annex (9) and Chapter 
B.29 of this Annex (10), together with the OECD Guidance Document on acute inhalation testing (8), should be specifically consulted in the design of longer term studies involving exposure via the inhalation route. Chapter B.9 
of this Annex (11) should be consulted in the case of testing carried out by 
the dermal route.
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5. The carcinogenicity study provides information on the possible health 
hazards likely to arise from repeated exposure for a period lasting up to 
the entire lifespan of the species used. The study will provide information on the toxic effects of the test chemical including potential carcinogenicity, and may indicate target organs and the possibility of accumulation. It can 
provide an estimate of the no-observed-adverse effect level for toxic effects 
and, in the case of non-genotoxic carcinogens, for tumour responses, which can be used for establishing safety criteria for human exposure. The need for careful clinical observations of the animals, so as to obtain as much 
information as possible, is also stressed. 
6. The objectives of carcinogenicity studies covered by this Test Method 
include: 
— The identification of the carcinogenic properties of a test chemical, 
resulting in an increased incidence of neoplasms, increased proportion of malignant neoplasms or a reduction in the time to appearance of 
neoplasms, compared with concurrent control groups; 
— The identification of target organ(s) of carcinogenicity; 
— The identification of the time to appearance of neoplasms; 
— Characterisation of the tumour dose-response relationship; — Identification of a no-observed-adverse-effect level (NOAEL) or point of 
departure for establishment of a Benchmark Dose (BMD); 
— Extrapolation of carcinogenic effects to low dose human exposure 
levels; 
— Provision of data to test hypotheses regarding mode of action (2) (7) 
(12) (13) (14) (15).
 
INITIAL CONSIDERATIONS 
7. In the assessment and evaluation of the potential carcinogenicity of a test 
chemical, all available information on the test chemical should be 
considered by the testing laboratory prior to conducting the study, in order to focus the design of the study to more efficiently test for carci ­
nogenic potential and to minimise animal usage. Information on, and 
consideration of, the mode of action of a suspected carcinogen (2) (7) 
(12) (13) (14) (15) is particularly important, since the optimal design may differ depending on whether the test chemical is a known or suspected genotoxic carcinogen. Further guidance on mode of action considerations 
can be found in Guidance Document No 116 (7). 
8. Information that will assist in the study design includes the identity, 
chemical structure, and physico-chemical properties of the test chemical; 
results of any  in vitro  or in vivo  toxicity tests including genotoxicity tests; 
anticipated use(s) and potential for human exposure; available (Q)SAR data, mutagenicity/genotoxicity, carcinogenicity and other toxicological data on structurally-related chemicals; available toxicokinetic data (single dose and 
also repeat dose kinetics where available) and data derived from other 
repeated exposure studies. Assessment of carcinogenicity should be
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carried out after initial information on toxicity has been obtained from 
repeated dose 28-day and/or 90-day toxicity tests. Short-term cancer 
initiation-promotion tests could also provide useful information. A phased testing approach to carcinogenicity testing should be considered as part of the overall assessment of the potential adverse health effects of a particular 
test chemical (16) (17) (18) (19). 
9. The statistical methods most appropriate for the analysis of results, given the 
experimental design and objectives, should be established before 
commencing the study. Issues to consider include whether the statistics should include adjustment for survival, analysis of cumulative tumour risks relative to survival duration, analysis of the time to tumour and 
analysis in the event of premature termination of one or more groups. 
Guidance on the appropriate statistical analyses and key references to inter ­
nationally accepted statistical methods are given in Guidance Document 
No 116 (7), and also in Guidance Document No 35 on the analysis and 
evaluation of chronic toxicity and carcinogenicity studies (20). 
10. In conducting a carcinogenicity study, the guiding principles and consider ­
ations outlined in the OECD Guidance Document No 19 on the recognition, assessment, and use of clinical signs as humane endpoints for experimental animals used in safety evaluation (21), in particular paragraph 62 thereof, 
should always be followed. This paragraph states that ‘ In studies involving 
repeated dosing, when an animal shows clinical signs that are progressive, leading to further deterioration in condition, an informed decision as to whether or not to humanely kill the animal should be made. The decision 
should include consideration as to the value of the information to be gained 
from the continued maintenance of that animal on study relative to its overall condition. If a decision is made to leave the animal on test, the 
frequency of observations should be increased, as needed. It may also be 
possible, without adversely affecting the purpose of the test, to temporarily stop dosing if it will relieve the pain or distress, or reduce the test dose. ’ 
11. Detailed guidance on and discussion of the principles of dose selection for 
chronic toxicity and carcinogenicity studies can be found in Guidance Document No 116 (7) as well as two International Life Sciences Institute 
publications (22) (23). The core dose selection strategy is dependent on the 
primary objective or objectives of the study (paragraph 6). In selecting appropriate dose levels, a balance should be achieved between hazard screening on the one hand and characterisation of low-dose responses and 
their relevance on the other. This is particularly relevant in the situation 
where a combined chronic toxicity and carcinogenicity study (Chapter B.33 of this Annex) is to be carried out (paragraph 12). 
12. Consideration should be given to carrying out a combined chronic toxicity 
and carcinogenicity study (Chapter B.33 of this Annex), rather than separate 
execution of a chronic toxicity study (Chapter B.30 of this Annex) and 
carcinogenicity study (this Test Method B.32). The combined test provides greater efficiency in terms of time and cost compared to conducting two separate studies, without compromising the quality of the 
data in either the chronic phase or the carcinogenicity phase. Careful 
consideration should however be given to the principles of dose selection (paragraphs 11 and 22-25) when undertaking a combined chronic toxicity and carcinogenicity study (Chapter B.33 of this Annex), and it is also 
recognised that separate studies may be required under certain regulatory 
frameworks.
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13. Definitions used in the context of this Test Method can be found at the end 
of this chapter and in the Guidance Document No 116 (7).  
PRINCIPLE OF THE TEST 
14. The test chemical is administered daily in graduated doses to several groups 
of test animals for the majority of their life span, normally by the oral route. 
Testing by the inhalation or dermal route may also be appropriate. The animals are observed closely for signs of toxicity and for the development of neoplastic lesions. Animals which die or are killed during the test are 
necropsied and, at the conclusion of the test, surviving animals are killed 
and necropsied.
 
DESCRIPTION OF METHOD  
Selection of animal species  
15. This Test Method primarily covers assessment and evaluation of carcinoge ­
nicity in rodents (paragraph 2). The use of non-rodent species may be 
considered when available data suggest that they are more relevant for the prediction of health effects in humans. The choice of species should be justified. The preferred rodent species is the rat, although other rodent 
species, e.g. the mouse, may be used. Although the use of the mouse in 
carcinogenicity testing may have limited utility (24) (25) (26), under some current regulatory programmes carcinogenicity testing in the mouse is still required unless it is determined that such a study is not scientifically 
necessary. Rats and mice have been preferred experimental models 
because of their relatively short life span, their widespread use in phar ­
macological and toxicological studies, their susceptibility to tumour induction, and the availability of sufficiently characterised strains. As a 
consequence of these characteristics, a large amount of information is 
available on their physiology and pathology. Additional information on choice of species and strain is provided in Guidance Document No 116 (7). 
16. Young healthy adult animals of commonly used laboratory strains should be 
employed. The carcinogenicity study should preferably be carried out in 
animals from the same strain and source as those used in preliminary toxicity study(ies) of shorter duration although, if animals from this strain and source are known to present problems in achieving the normally 
accepted criteria of survival for long-term studies [see Guidance 
Document No 116 (7)], consideration should be given to using a strain of animal that has an acceptable survival rate for the long-term study. The females should be nulliparous and non-pregnant.
 
Housing and feeding  
17. Animals may be housed individually, or be caged in small groups of the 
same sex; individual housing should be considered only if scientifically 
justified (27) (28) (29). Cages should be arranged in such a way that 
possible effects due to cage placement are minimised. The temperature in the experimental animal room should be 22 °C (± 3 °C). Although the relative humidity should be at least 30 % and preferably not exceed 70 % 
other than during room cleaning, the aim should be 50-60 %. Lighting 
should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. The diet should meet all the nutritional requirements of 
the species tested and the content of dietary contaminants, including but not 
limited to pesticide residues, persistent organic pollutants, phytoestrogens,
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heavy metals and mycotoxins, that might influence the outcome of the test, 
should be as low as possible. Analytical information on the nutrient and 
dietary contaminant levels should be generated periodically, at least at the beginning of the study and when there is a change in the batch used, and should be included in the final report. Analytical information on the 
drinking water used in the study should similarly be provided. The choice 
of diet may be influenced by the need to ensure a suitable admixture of a test chemical and to meet the nutritional requirements of the animals when the test chemical is administered by the dietary route.
 
Preparation of animals  
18. Healthy animals, which have been acclimated to laboratory conditions for at 
least 7 days and have not been subjected to previous experimental procedures, should be used. In the case of rodents, dosing of the animals should begin as soon as possible after weaning and acclimatisation and 
preferably before the animals are 8 weeks old. The test animals should be 
characterised as to species, strain, source, sex, weight and age. At the commencement of the study, the weight variation for each sex of animal used should be minimal and not exceed ± 20 % of the mean weight of all 
the animals within the study, separately for each sex. Animals should be 
randomly assigned to the control and treatment groups. After randomisation, there should be no significant differences in mean body weights between groups within each sex. If there are statistically significant differences, then 
the randomisation step should be repeated, if possible. Each animal should 
be assigned a unique identification number, and permanently marked with this number by tattooing, microchip implant, or other suitable method.
 
PROCEDURE  
Number and sex of animals  
19. Both sexes should be used. A sufficient number of animals should be used 
so that a thorough biological and statistical evaluation is possible. Each dose 
group and concurrent control group should therefore contain at least 50 
animals of each sex. Depending on the aim of the study, it may be possible to increase the statistical power of the key estimates by differen ­
tially allocating animals unequally to the various dose groups, with more 
than 50 animals in the low dose groups; e.g. to estimate the carcinogenic 
potential at low doses. However it should be recognised that a moderate increase in group size will provide relatively little increase in statistical power of the study. Further information on statistical design of the study 
and choice of dose levels to maximise statistical power is provided in 
Guidance Document No 116 (7).
 
Provision for interim kills and satellite (sentinel) groups  
20. The study may make provision for interim kills, e.g. at 12 months, to 
provide information on progression of neoplastic changes and mechanistic 
information, if scientifically justified. Where such information is already 
available from previous repeat dose toxicity studies on the test chemical, interim kills may not be scientifically justified. If interim kills are included in the study design, the number of animals in each dose group scheduled for 
an interim kill will normally be 10 animals per sex, and the total number of 
animals included in the study design should be increased by the number of animals scheduled to be killed before the completion of the study. An additional group of sentinel animals (typically 5 animals per sex) may be 
included for monitoring of disease status, if necessary, during the study (30). 
Further guidance is provided in Guidance Document No 116 (7).
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Dose groups and dosage  
21. Guidance on all aspects of dose selection and dose level spacing is provided 
in Guidance Document No 116 (7). At least three dose levels and a concurrent control should be used. Dose levels will generally be based on 
the results of shorter-term repeated dose or range finding studies and should 
take into account any existing toxicological and toxicokinetic data available for the test chemical or related chemicals. 
22. Unless limited by the physical-chemical nature or biological effects of the 
test chemical, the highest dose level should be chosen to identify the 
principal target organs and toxic effects while avoiding suffering, severe 
toxicity, morbidity, or death. While taking into account the factors outlined in paragraph 23 below, the highest dose level should normally be chosen to elicit evidence of toxicity, as evidenced by, for example, 
depression of body weight gain (approximately 10 %). However, 
dependent on the objectives of the study (see paragraph 6), a top dose lower than the dose providing evidence of toxicity may be chosen, e.g. if a dose elicits an adverse effect of concern that nonetheless has little impact 
on lifespan or body weight. 
23. Dose levels and dose level spacing may be selected to establish a dose- 
response and, depending on the mode of action of the test chemical, a NOAEL or other intended outcome of the study, e.g. a BMD (see paragraph 25) at the lowest dose level. Factors that should be considered 
in the placement of lower doses include the expected slope of the dose–re ­
sponse curve, the doses at which important changes may occur in metabolism or mode of toxic action, where a threshold is expected, or where a point of departure for low-dose extrapolation is expected. 
24. The dose level spacing selected will depend on the characteristics of the test 
chemical, and cannot be prescribed in this Test Method, but two to four fold 
intervals frequently provide good test performance for setting the descending 
dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g. more than a factor of about 6-10) between dosages. 
In general, the use of factors greater than 10 should be avoided, and should 
be justified if used. 
25. As discussed further in Guidance Document No 116 (7), points to be 
considered in dose selection include: 
— Known or suspected nonlinearities or inflection points in the dose–re ­
sponse; 
— Toxicokinetics, and dose ranges where metabolic induction, saturation, 
or nonlinearity between external and internal doses does or does not occur; 
— Precursor lesions, markers of effect, or indicators of the operation of key 
underlying biological processes; 
— Key (or suspected) aspects of mode of action, such as doses at which 
cytotoxicity begins to arise, hormone levels are perturbed, homeostatic 
mechanisms are overwhelmed, etc.;
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— Regions of the dose–response curve where particularly robust estimation 
is needed, e.g. in the range of the anticipated BMD or a suspected 
threshold; 
— Consideration of anticipated human exposure levels. 
26. The control group shall be an untreated group or a vehicle-control group if a 
vehicle is used in administering the test chemical. Except for treatment with 
the test chemical, animals in the control group should be handled in an identical manner to those in the test groups. If a vehicle is used, the control group shall receive the vehicle in the highest volume used among 
the dose groups. If a test chemical is administered in the diet, and causes 
significantly reduced dietary intake due to the reduced palatability of the diet, an additional pair-fed control group may be useful, to serve as a more suitable control.
 
Preparation of doses and administration of test chemical  
27. The test chemical is normally administered orally, via the diet or drinking 
water, or by gavage. Additional information on routes and methods of administration is provided in Guidance Document No 116 (7). The route 
and method of administration is dependent on the purpose of the study, the 
physical-chemical properties of the test chemical, its bioavailability and the predominant route and method of exposure of humans. A rationale should be provided for the chosen route and method of administration. In the 
interest of animal welfare, oral gavage should normally be selected only 
for those agents, for which this route and method of administration reasonably represent potential human exposure (e.g. pharmaceuticals). For dietary or environmental chemicals including pesticides, administration is 
typically via the diet or drinking water. However, for some scenarios, e.g. 
occupational exposure, administration via other routes may be more appro ­
priate. 
28. Where necessary, the test chemical is dissolved or suspended in a suitable 
vehicle. Consideration should be given to the following characteristics of the vehicle and other additives, as appropriate: effects on the absorption, 
distribution, metabolism, or retention of the test chemical; effects on the 
chemical properties of the test chemical which may alter its toxic character ­
istics; and effects on the food or water consumption or the nutritional status of the animals. It is recommended that, wherever possible, the use of an 
aqueous solution/suspension be considered first, followed by consideration 
of a solution/emulsion in oil (e.g. corn oil) and then by possible solution in other vehicles. For vehicles other than water, the toxic characteristics of the vehicle should be known. Information should be available on the stability of 
the test chemical and the homogeneity of dosing solutions or diets (as 
appropriate) under the conditions of administration (e.g. diet). 
29. For chemicals administered via the diet or drinking water it is important to 
ensure that the quantities of the test chemical involved do not interfere with normal nutrition or water balance. In long-term toxicity studies using dietary administration, the concentration of the test chemical in the feed should not 
normally exceed an upper limit of 5 % of the total diet, in order to avoid 
nutritional imbalances. When the test chemical is administered in the diet, either a constant dietary concentration (mg/kg diet or ppm) or a constant dose level in terms of the animal’s body weight (mg/kg body weight), 
calculated on a weekly basis, may be used. The alternative used should 
be specified.
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30. In the case of oral administration, the animals are dosed with the test 
chemical daily (seven days per week), normally for a period of 24 
months for rodents (see also paragraph 32). Any other dosing regime, e.g. five days per week, needs to be justified. In the case of dermal adminis ­
tration, animals are normally treated with the test chemical for at least 
6 hours per day, 7 days per week, as specified in Chapter B.9 of this 
Annex (11), for a period of 24 months. Exposure by the inhalation route is carried out for 6 hours per day, 7 days per week, but exposure for 5 days per week may also be used, if justified. The period of exposure will 
normally be for a period of 24 months. If rodent species other than rats 
are exposed nose-only, maximum exposure durations may be adjusted to minimise species-specific distress. A rationale should be provided when using an exposure duration of less than 6 hours per day. See also Chapter 
B.8 of this Annex (9). 
31. When the test chemical is administered by gavage to the animals, this 
should be done using a stomach tube or a suitable intubation cannula, at 
similar times each day. Normally a single dose will be administered once 
daily; where for example a chemical is a local irritant, it may be possible to maintain the daily dose-rate by administering it as a split dose (twice a day). The maximum volume of liquid that can be administered at one time 
depends on the size of the test animal. The volume should be kept as 
low as practical, and should not normally exceed 1 ml/100g body weight for rodents (31). Variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels. 
Potentially corrosive or irritant chemicals are the exception, and need to be 
diluted to avoid severe local effects. Testing at concentrations that are likely to be corrosive or irritant to the gastrointestinal tract should be avoided.
 
Duration of study  
32. The duration of the study will normally be 24 months for rodents, repre ­
senting the majority of the normal life span of the animals to be used. 
Shorter or longer study durations may be used, dependent on the lifespan of the strain of the animal species in the study, but should be justified. For specific strains of mice, e.g. AKR/J, C3H/J or C57BL/6J strains a duration 
of 18 months may be more appropriate. The following provides some 
guidance on duration, termination of the study and survival; further guidance, including consideration of the acceptability of a negative carcino ­
genicity relative to survival in the study, is provided in the OECD Guidance 
Document No 116 on the Design and Conduct of Chronic Toxicity and 
Carcinogenicity Studies (7). 
— Termination of the study should be considered when the number of 
survivors in the lower dose groups or the control group falls below 25 
per cent. 
— In the case where only the high dose group dies prematurely due to 
toxicity, this should not trigger termination of the study. 
— Survival of each sex should be considered separately. — The study should not be extended beyond the point when the data 
available from the study are no longer sufficient to enable a statistically 
valid evaluation to be made.
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OBSERVATIONS 
33. All animals should be checked for morbidity or mortality, usually at the 
beginning and the end of each day, including at weekends and holidays. Animals should additionally be checked once a day for specific signs of 
toxicological relevance, taking into consideration the peak period of 
anticipated effects after dosing in the case of gavage administration. Particular attention should be paid to tumour development; and the time of tumour onset, location, dimensions, appearance, and progression of 
each grossly visible or palpable tumour should be recorded.
 
Body weight, food/water consumption and food efficiency  
34. All animals should be weighed at the start of treatment, at least once a week 
for the first 13 weeks and at least monthly thereafter. Measurements of food consumption and food efficiency should be made at least weekly for the first 13 weeks and at least monthly thereafter. Water consumption should be 
measured at least weekly for the first 13 weeks and at least monthly 
thereafter when the test chemical is administered in drinking water. Water consumption measurements should also be considered for studies in which drinking activity is altered.
 
Haematology, clinical biochemistry and other measurements  
35. In order to maximise the information obtained from the study, especially for 
mode of action considerations, blood samples may be taken for haematology 
and clinical biochemistry, and this at the discretion of the study director. 
Urinalysis may also be appropriate. Further guidance on the value of taking such samples as part of a carcinogenicity study is provided in Guidance Document No 116 (7). If considered appropriate, blood sampling for haema ­
tological and clinical chemistry determinations and urinalysis may be 
conducted as part of an interim kill (paragraph 20) and at study termination on a minimum of 10 animals per sex per group. Blood samples should be taken from a named site, for example by cardiac puncture or from the 
retro-orbital sinus under anaesthesia, and stored, if applicable, under appro ­
priate conditions. Blood smears may also be prepared for examination, particularly if bone marrow appears to be the target organ, although the value of such examination for the assessment of carcinogenic/oncogenic 
potential has been questioned (32).
 
PATHOLOGY  
Gross necropsy  
36. All animals in the study except sentinel animals (see paragraph 20) and 
other satellite animals should be subjected to a full, detailed gross 
necropsy which includes careful examination of the external surface of 
the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. Sentinel animals and other satellite animals may require necropsy on a case-by-case basis, at the discretion of the study director. 
Organ weights are not normally part of a carcinogenesis study, since 
geriatric changes and, at later stages, the development of tumours confounds the usefulness of organ weight data. They may, however, be critical to performing a weight of evidence evaluation and especially for 
mode of action considerations. If they are part of a satellite study, they 
should be collected at no later than one year after initiation of the study.
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37. The following tissues should be preserved in the most appropriate fixation 
medium for both the type of tissue and the intended subsequent histopath ­
ological examination (33) (tissues in square brackets are optional): 
all gross lesions heart pancreas stomach (forestomach, 
glandular stomach) 
adrenal gland ileum parathyroid gland [teeth] 
aorta jejunum peripheral nerve testis 
brain (including sections of 
cerebrum, cerebellum, and 
medulla/pons) kidney pituitary thymus 
caecum lacrimal gland (exorbital) prostate thyroid 
cervix liver rectum [tongue] 
coagulating gland lung salivary gland trachea 
colon lymph nodes (both 
superficial and deep) seminal vesicle urinary bladder 
duodenum mammary gland 
(obligatory for females 
and, if visibly dissectable, from males) skeletal muscle uterus (including cervix) 
epididymis [upper respiratory tract, 
including nose, turbi ­
nates, and paranasal 
sinuses] skin [ureter] 
eye (including retina) oesophagus spinal cord (at three 
levels: cervical, mid-thoracic, and 
lumbar) [urethra] 
[femur with joint] [olfactory bulb] spleen vagina 
gall bladder (for species 
other than rat) ovary [sternum], section of bone marrow and/ 
or a fresh bone marrow 
aspirate 
Harderian gland 
In the case of paired organs, e.g. kidney, adrenal, both organs should be 
preserved. The clinical and other findings may suggest the need to examine 
additional tissues. Also, any organs considered likely to be target organs 
based on the known properties of the test chemical should be preserved. In studies involving the dermal route of administration, the list of organs as set out for the oral route should be preserved, and specific sampling and pres ­
ervation of the skin from the site of application is essential. In inhalation 
studies, the list of preserved and examined tissues from the respiratory tract should follow the recommendations of Chapters B.8 and B.29 of this Annex. For other organs/tissues (and in addition to the specifically 
preserved tissues from the respiratory tract) the list of organs as set out 
for the oral route should be examined.
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Histopathology  
38. Guidance is available on best practices in the conduct of toxicological 
pathology studies (33). The minimum tissues examined should be: 
— All tissues from the high dose and control groups; 
— All tissues of animals dying or killed during the study; 
— All tissues showing macroscopic abnormalities including tumours; — When treatment-related histopathological changes are observed in the 
high dose group, those same tissues are to be examined from all 
animals in all other dose groups; 
— In the case of paired organs, e.g. kidney, adrenal, both organs should be 
examined.
 
DATA AND REPORTING  
Data  
39. Individual animal data should be provided for all parameters evaluated. 
Additionally, all data should be summarised in tabular form showing for each test group the number of animals at the start of the test, the number of animals found dead during the test or killed for humane reasons and the 
time of any death or humane kill, the number showing signs of toxicity, a 
description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects, the number of animals showing lesions, the type of lesions and the percentage of animals 
displaying each type of lesion. Summary data tables should provide the 
means and standard deviations (for continuous test data) of animals showing toxic effects or lesions, in addition to the grading of lesions. 
40. Historical control data may be valuable in the interpretation of the results of 
the study, e.g. in the case when there are indications that the data provided 
by the concurrent controls are substantially out of line when compared to recent data from control animals from the same test facility/colony. 
Historical control data, if evaluated, should be submitted from the same 
laboratory and relate to animals of the same age and strain generated during the five years preceding the study in question. 
41. When applicable, numerical results should be evaluated by an appropriate 
and generally acceptable statistical method. The statistical methods and the 
data to be analysed should be selected during the design of the study 
(paragraph 9). Selection should make provision for survival adjustments, if needed.
 
Test report  
42. The test report should include the following information:  
Test chemical:  
— physical nature, purity, and physicochemical properties; — identification data;
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— source of chemical; 
— batch number; — certificate of chemical analysis;  
Vehicle (if appropriate):  
— justification for choice of vehicle (if other than water);  
Test animals:  
— species/strain used and justification for choice made; — number, age, and sex of animals at start of test; — source, housing conditions, diet, etc.; — individual weights of animals at the start of the test;  
Test conditions:  
— rationale for route of administration and dose selection; — when applicable, the statistical methods used to analyse the data; — details of test chemical formulation/diet preparation. — analytical data on achieved concentration, stability and homogeneity of 
the preparation; 
— route of administration and details of the administration of the test 
chemical; 
— for inhalation studies, whether nose only or whole body; — actual doses (mg/kg body weight/day), and conversion factor from diet/ 
drinking water test chemical concentration (mg/kg or ppm) to the actual 
dose, if applicable; 
— details of food and water quality;  
Results (summary tabulated data and individual animal data should be 
presented) 
General  
— survival data; — body weight/body weight changes; — food consumption, calculations of food efficiency, if made, and water 
consumption, if applicable; 
— toxicokinetic data (if available); — opthalmoscopy (if available); — haematology (if available); — clinical chemistry (if available);
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 479
 
Clinical findings  
— Signs of toxicity; — Incidence (and, if scored, severity) of any abnormality; — Nature, severity, and duration of clinical observations (whether transitory 
or permanent);  
Necropsy data  
— Terminal body weight; — Organ weights and their ratios, if applicable; — Necropsy findings; Incidence and severity of abnormalities;  
Histopathology  
— Non neoplastic histopathological findings,; 
— Neoplastic histopathological findings; 
— Correlation between gross and microscopic findings; — Detailed description of all treatment-related histopathological findings 
including severity gradings; 
— Report of any peer review of slides;  
Statistical treatment of results, as appropriate Discussion of results including  
— Discussion of any modelling approaches; — Dose-response relationships; — Historical control data; — Consideration of any mode of action information; — BMD, NOAEL or LOAEL determination; — Relevance for humans;  
Conclusions
 
LITERATURE:  
(1) OECD (1995). Report of the Consultation Meeting on Sub-chronic and 
Chronic Toxicity/Carcinogenicity Testing (Rome, 1995), internal working 
document, Environment Directorate, OECD, Paris. 
(2) EPA (2005). Guidelines for Carcinogen Risk Assessment Risk Assessment 
Forum U.S. Environmental Protection Agency Washington, DC. 
(3) Combes RD, Gaunt, I, Balls M (2004). A Scientific and Animal Welfare 
Assessment of the OECD Health Effects Test Guidelines for the Safety Testing of Chemicals under the European Union REACH System. ATLA 32: 163-208. 
(4) Barlow SM, Greig JB, Bridges JW et al (2002). Hazard identification by 
methods of animal-based toxicology. Food. Chem. Toxicol. 40: 145-191. 
(5) Chhabra RS, Bucher JR, Wolfe M, Portier C (2003). Toxicity character ­
ization of environmental chemicals by the US National Toxicology 
Programme: an overview. Int. J. Hyg. Environ. Health 206: 437-445.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 480
 
(6) Chapter B.27 of this Annex, Sub-chronic Oral Toxicity Test Repeated Dose 
90-day Oral Toxicity Study in Non-Rodents. 
(7) OECD (2012). Guidance Document on the Design and Conduct of Chronic 
Toxicity and Carcinogenicity Studies, Supporting Test Guidelines 451, 452 
and 453 — Second edition. Series on Testing and Assessment No 116, 
available on the OECD public website for Test Guideline at www.oecd.org/ env/testguidelines. 
(8) OECD (2009). Guidance Document on Acute Inhalation Toxicity Testing, 
Series on Testing and Assessment No 39, ENV/JM/MONO(2009)28, OECD, Paris. 
(9) Chapter B.8 of this Annex, Subacute Inhalation Toxicity: 28-Day Study. 
(10) Chapter B.29 of this Annex, Subchronic Inhalation Toxicity: 90-Day Study. 
(11) Chapter B.9 of this Annex, Repeated Dose (28 Days) Toxicity (Dermal). 
(12) Boobis AR, Cohen SM, Dellarco V, McGregor D, Meek ME, Vickers C, 
Willcocks D, Farland W (2006). IPCS Framework for analyzing the 
Relevance of a Cancer Mode of Action for Humans. Crit. Rev. in 
Toxicol, 36: 793-801. 
(13) Cohen SM, Meek ME, Klaunig JE, Patton DE, and Fenner-Crisp PA 
(2003). The human relevance of information on carcinogenic Modes of Action: An Overview. Crit. Rev. Toxicol. 33: 581-589. 
(14) Holsapple MP, Pitot HC, Cohen SN, Boobis AR, Klaunig JE, Pastoor T, 
Dellarco VL, Dragan YP (2006). Mode of Action in Relevance of Rodent Liver Tumors to Human Cancer Risk. Toxicol. Sci. 89: 51-56. 
(15) Meek EM, Bucher JR, Cohen SM, Dellarco V, Hill RN, 
Lehman-McKemmon LD, Longfellow DG, Pastoor T, Seed J, Patton DE 
(2003). A Framework for Human Relevance analysis of Information on 
Carcinogenic Modes of Action. Crit. Rev. Toxicol. 33: 591-653. 
(16) Carmichael NG, Barton HA, Boobis AR et al (2006). Agricultural 
Chemical Safety Assessment: A Multisector Approach to the Modern ­
ization of Human Safety Requirements. Critical Reviews in Toxicology 36: 1-7. 
(17) Barton HA, Pastoor TP, Baetcke T et al (2006). The Acquisition and 
Application of Absorption, Distribution, Metabolism, and Excretion (ADME) Data in Agricultural Chemical Safety Assessments. 
Critical Reviews in Toxicology 36: 9-35. 
(18) Doe JE, Boobis AR, Blacker A et al (2006). A Tiered Approach to 
Systemic Toxicity Testing for Agricultural Chemical Safety Assessment. 
Critical Reviews in Toxicology 36: 37-68. 
(19) Cooper RL, Lamb JS, Barlow SM et al (2006). A Tiered Approach to Life 
Stages Testing for Agricultural Chemical Safety Assessment. Critical Reviews in Toxicology 36: 69-98. 
(20) OECD (2002). Guidance Notes for Analysis and Evaluation of Chronic 
Toxicity and Carcinogenicity Studies, Series on Testing and Assessment No 35 and Series on Pesticides No 14, ENV/JM/MONO(2002)19, OECD, Paris. 
(21) OECD (2000). Guidance Document on the recognition, assessment, and use 
of clinical signs as humane endpoints for experimental animals used in 
safety evaluation, Series on Testing and Assessment No 19, ENV/JM/ 
MONO(2000)7, OECD, Paris.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 481
 
(22) Rhomberg LR, Baetcke K, Blancato J, Bus J, Cohen S, Conolly R, Dixit R, 
Doe J, Ekelman K, Fenner-Crisp P, Harvey P, Hattis D, Jacobs A, 
Jacobson-Kram D, Lewandowski T, Liteplo R, Pelkonen O, Rice J, Somers D, Turturro A, West, W, Olin S(2007). Issues in the Design and Interpretation of Chronic Toxicity and Carcinogenicity Studies in Rodents: 
Approaches to Dose Selection Crit Rev. Toxicol. 37 (9): 729 – 837. 
(23) ILSI (International Life Sciences Institute) (1997). Principles for the 
Selection of Doses in Chronic Rodent Bioassays. Foran JA (Ed.). ILSI Press, Washington, DC. 
(24) Griffiths SA, Parkinson C, McAuslane JAN and Lumley CE (1994). The 
utility of the second rodent species in the carcinogenicity testing of phar ­
maceuticals. The Toxicologist 14(1):214. 
(25) Usui T, Griffiths SA and Lumley CE (1996). The utility of the mouse for 
the assessment of the carcinogenic potential of pharmaceuticals. In D’Arcy POF & Harron DWG (eds). Proceedings of the Third International 
Conference on Harmonisation. Queen’s University Press, Belfast. pp 279- 
284. 
(26) Carmichael NG, Enzmann H, Pate I, Waechter F (1997). The Significance 
of Mouse Liver Tumor Formation for Carcinogenic Risk Assessment: Results and Conclusions from a Survey of 10 Years of Testing by the 
Agrochemical Industry. Environ Health Perspect. 105:1196-1203. 
(27) Directive 2010/63/EU of the European Parliament and of the Council of 
22 September 2010 on the protection of animals used for scientific purposes (OJ L 276, 20.10.2010, p. 33). 
(28) National Research Council, 1985. Guide for the care and use of laboratory 
animals. NIH Publication No 86-23. Washington, D.C., US Dept. of Health 
and Human Services. 
(29) GV-SOLAS (Society for Laboratory Animal Science, Gesellschaft für 
Versuchstierkunde, 1988). Publication on the Planning and Structure of Animal Facilities for Institutes Performing Animal Experiments. ISBN 3- 
906255-04-2. 
(30) GV-SOLAS (Society for Laboratory Animal Science, Gesellschaft für 
Versuchstierkunde, 2006). Microbiological monitoring of laboratory animals in various housing systems. 
(31) Diehl K-H, Hull R, Morton D, Pfister R, Rabemampianina Y, Smith D, 
Vidal J-M, van de Vorstenbosch C. (2001). A good practice guide to the 
administration of substances and removal of blood, including routes and volumes. Journal of Applied Toxicology 21:15-23. 
(32) Weingand K,  et al.  (1996). Harmonization of Animal Clinical Pathology 
Testing in Toxicity and Safety Studies. Fund. Appl. Toxicol. 29: 198-201. 
(33) Crissman J, Goodman D, Hildebrandt P,  et al.  (2004). Best Practices 
Guideline: Toxicological Histopathology. Toxicologic Pathology 32: 126- 131.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 482
 
Appendix 1  
DEFINITION  
Test chemical:  Any substance or mixture tested using this Test Method.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 483
 
B.33.  COMBINED CHRONIC TOXICITY/CARCINOGENICITY 
STUDIES  
INTRODUCTION 
1. This Test Method is equivalent to OECD Test Guideline (TG) 453 (2009). 
The original TG 453 was adopted in 1981. Development of this updated 
Test Method B.33 was considered necessary, in order to reflect recent developments in the field of animal welfare and regulatory requirements (1) (2) (3) (4) (5). The updating of this Test Method B.33 has been 
carried out in parallel with revisions of Chapter B.32 of this Annex, Carci ­
nogenicity Studies, and Chapter B.30 of this Annex, Chronic Toxicity Studies, with the objective of obtaining additional information from the animals used in the study and providing further detail on dose selection. 
This Test Method is designed to be used in the testing of a broad range of 
chemicals, including pesticides and industrial chemicals. It should be noted however that some details and requirements may differ for pharmaceuticals [see International Conference on Harmonisation (ICH) Guidance S1B on 
Testing for Carcinogenicity of Pharmaceuticals]. 
2. The majority of chronic toxicity and carcinogenicity studies are carried out 
in rodent species and this Test Method is intended therefore to apply primarily to studies carried out in these species. Should such studies be 
required in non-rodent species, the principles and procedures outlined may 
also be applied, with appropriate modifications, together with those outlined in Chapter B.27 of this Annex, Repeated Dose 90-day Oral Toxicity Study in Non-Rodents (6), as outlined in the OECD Guidance Document No 116 
on the Design and Conduct of Chronic Toxicity and Carcinogenicity 
Studies (7). 
3. The three main routes of administration used in chronic toxicity/carcinoge ­
nicity studies are oral, dermal and inhalation. The choice of the route of 
administration depends on the physical and chemical characteristics of the 
test chemical and the predominant route of exposure of humans. Additional information on choice of route of exposure is provided in Guidance Document No 116 (7). 
4. This Test Method focuses on exposure via the oral route, the route most 
commonly used in chronic toxicity and carcinogenicity studies. While long–term studies involving exposure via the dermal or inhalation routes may also be necessary for human health risk assessment and/or may be 
required under certain regulatory regimes, both routes of exposure involve 
considerable technical complexity. Such studies will need to be designed on a case-by-case basis, although the Test Method outlined here for the assessment and evaluation of chronic toxicity and carcinogenicity by oral 
administration could form the basis of a protocol for inhalation and/or 
dermal studies, with respect to recommendations for treatment periods, clinical and pathology parameters, etc. OECD Guidance is available on the administration of test chemicals by the inhalation (7) (8) and dermal 
routes (7). Chapter B.8 of this Annex (9) and Chapter B.29 of this 
Annex (10), together with the OECD Guidance Document on acute inha ­
lation testing (8), should be specifically consulted in the design of longer term studies involving exposure via the inhalation route. Chapter B.9 of this 
Annex (11) should be consulted in the case of testing carried out by the 
dermal route.
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5. The combined chronic toxicity/carcinogenicity study provides information 
on the possible health hazards likely to arise from repeated exposure for a 
period lasting up to the entire lifespan of the species used. The study will provide information on the toxic effects of the test chemical, including potential carcinogenicity, indicate target organs and the possibility of 
accumulation. It can provide an estimate of the no-observed-adverse effect 
level for toxic effects and, in the case of non-genotoxic carcinogens, for tumour responses, which can be used for establishing safety criteria for human exposure. The need for careful clinical observations of the 
animals, so as to obtain as much information as possible, is also stressed. 
6. The objectives of chronic toxicity/carcinogenicity studies covered by this 
Test Method include: 
— The identification of the carcinogenic properties of a test chemical, 
resulting in an increased incidence of neoplasms, increased proportion of malignant neoplasms or a reduction in the time to appearance of 
neoplasms, compared with concurrent control groups; 
— The identification of the time to appearance of neoplasms; — The identification of the chronic toxicity of the test chemical; 
— The identification of target organ(s) of chronic toxicity and carcinoge ­
nicity, 
— Characterisation of the dose:response relationship, 
— Identification of a no-observed-adverse-effect level (NOAEL) or point of 
departure for establishment of a Benchmark Dose (BMD), 
— Extrapolation of carcinogenic effects to low dose human exposure levels, 
— Prediction of chronic toxicity effects at human exposure levels, 
— Provision of data to test hypotheses regarding mode of action (2) (7) 
(12) (13) (14) (15).
 
INITIAL CONSIDERATIONS 
7. In the assessment and evaluation of the potential carcinogenicity and chronic 
toxicity of a test chemical, all available information on the test chemical 
should be considered by the testing laboratory prior to conducting the study, 
in order to focus the design of the study to more efficiently test for its toxicological properties and to minimise animal usage. Information on, and consideration of, the mode of action of a suspected carcinogen (2) (7) (12) 
(13) (14) (15) is particularly important, since the optimal design may differ 
depending on whether the test chemical is a known or suspected genotoxic carcinogen. Further guidance on mode of action considerations can be found in Guidance Document No 116 (7). 
8. Information that will assist in the study design includes the identity, 
chemical structure, and physico-chemical properties of the test chemical; any information on the mode of action; results of any  in vitro  or in vivo  
toxicity tests including genotoxicity tests; anticipated use(s) and potential for 
human exposure; available (Q)SAR data, mutagenicity/genotoxicity, carci ­
nogenicity and other toxicological data on structurally-related chemicals; available toxicokinetic data (single dose and also repeat dose kinetics 
where available) and data derived from other repeated exposure studies. 
The determination of chronic toxicity/carcinogenicity should only be
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carried out after initial information on toxicity has been obtained from 
repeated dose 28-day and/or 90-day toxicity tests. Short-tem cancer 
initiation-promotion tests could also provide useful information. A phased testing approach to carcinogenicity testing should be considered as part of the overall assessment of the potential adverse health effects of a particular 
test chemical (16) (17) (18) (19). 
9. The statistical methods most appropriate for the analysis of results, given the 
experimental design and objectives, should be established before commencing the study. Issues to consider include whether the statistics should include adjustment for survival, analysis of cumulative tumour 
risks relative to survival duration, analysis of the time to tumour and 
analysis in the event of premature termination of one or more groups. Guidance on the appropriate statistical analyses and key references to inter ­
nationally accepted statistical methods are given in Guidance Document 
No 116 (7), and also in Guidance Document No 35 on the analysis and 
evaluation of chronic toxicity and carcinogenicity studies (20). 
10. In conducting a carcinogenicity study, the guiding principles and consider ­
ations outlined in the OECD Guidance Document on the recognition, assessment, and use of clinical signs as humane endpoints for experimental 
animals used in safety evaluation (21), in particular paragraph 62 thereof, 
should always be followed. This paragraph states that ‘ In studies involving 
repeated dosing, when an animal shows clinical signs that are progressive, leading to further deterioration in condition, an informed decision as to 
whether or not to humanely kill the animal should be made. The decision 
should include consideration as to the value of the information to be gained from the continued maintenance of that animal on study relative to its overall condition. If a decision is made to leave the animal on test, the 
frequency of observations should be increased, as needed. It may also be 
possible, without adversely affecting the purpose of the test, to temporarily stop dosing if it will relieve the pain or distress, or reduce the test dose. ’ 
11. Detailed guidance on and discussion of the principles of dose selection for 
chronic toxicity and carcinogenicity studies can be found in Guidance 
Document No 116 (7), as well as two International Life Sciences Institute 
publications (22) (23). The core dose selection strategy is dependent on the primary objective or objectives of the study (paragraph 6). In selecting appropriate dose levels, a balance should be achieved between hazard 
screening on the one hand and characterisation of low-dose responses and 
their relevance on the other. This is particularly relevant in the case of this combined chronic toxicity and carcinogenicity study. 
12. Consideration should be given to carrying out this combined chronic 
toxicity and carcinogenicity study, rather than separate execution of a 
chronic toxicity study (Chapter B.30 of this Annex) and carcinogenicity 
study (Chapter B.32 of this Annex). The combined test provides greater efficiency in terms of time and cost, and some reduction in animal use, compared to conducting two separate studies, without compromising the 
quality of the data in either the chronic phase or the carcinogenicity 
phase. Careful consideration should however be given to the principles of dose selection (paragraphs 11 and 22-26) when undertaking a combined chronic toxicity and carcinogenicity study, and it is also recognised that 
separate studies may be required under certain regulatory frameworks. 
Further guidance on the design of the combined chronic toxicity and carci ­
nogenicity study in order to achieve maximum efficiency of the study in terms of possibilities for reduction in numbers of animals used as well as via 
the streamlining of the various experimental procedures can be found in 
Guidance Document No 116 (7).
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13. Definitions used in the context of this Test Method can be found at the end 
of this chapter and in Guidance Document No 116 (7).  
PRINCIPLE OF THE TEST 
14. The study design consists of two parallel phases, a chronic phase and a 
carcinogenicity phase (for duration see paragraphs 34 and 35, respectively). The test chemical is normally administered by the oral route although testing 
by the inhalation or dermal route may also be appropriate. For the chronic 
phase, the test chemical is administered daily in graduated doses to several groups of test animals, one dose level per group, normally for a period of 12 months, although longer or shorter durations may also be chosen depending 
on regulatory requirements (see paragraph 34). This duration is chosen to be 
sufficiently long to allow any effects of cumulative toxicity to become manifest, without the confounding effects of geriatric changes. The study design may also include one or more interim kills, e.g. at 3 and 6 months, 
and additional groups of animals may be included to accommodate this (see 
paragraph 20). For the carcinogenicity phase, the test chemical is admin ­
istered daily to several groups of test animals for a major portion of their life 
span. The animals in both phases are observed closely for signs of toxicity 
and for the development of neoplastic lesions. Animals which die or are killed during the test are necropsied and, at the conclusion of the test, surviving animals are killed and necropsied.
 
DESCRIPTION OF THE METHOD  
Selection of animal species  
15. This Test Method primarily covers assessment and evaluation of chronic 
toxicity and carcinogenicity in rodents (paragraph 2). The use of non-rodent 
species may be considered when available data suggest that they are more relevant for the prediction of health effects in humans. The choice of species should be justified. The preferred rodent species is the rat, although other 
rodent species, e.g. the mouse, may be used. Although the use of the mouse 
in carcinogenicity testing may have limited utility (24) (25) (26), under some current regulatory programmes carcinogenicity testing in the mouse is still required unless it is determined that such a study is not scientifically 
necessary. Rats and mice have been preferred experimental models because 
of their relatively short life span, their widespread use in pharmacological and toxicological studies, their susceptibility to tumour induction, and the availability of sufficiently characterised strains. As a consequence of these 
characteristics, a large amount of information is available on their 
physiology and pathology. The design and conduct of chronic toxicity/car ­
cinogenicity studies in non-rodent species, when required, should be based on the principles outlined in this Test Method together with those in Chapter 
B.27 of this Annex, Repeated Dose 90-day Oral Toxicity Study in 
Non-Rodents (6). Additional information on choice of species and strain is provided in Guidance Document No 116 (7). 
16. Young healthy adult animals of commonly used laboratory strains should be 
employed. The combined chronic toxicity/carcinogenicity study should be carried out in animals from the same strain and source as those used in 
preliminary toxicity study(ies) of shorter duration, although, if animals from 
this strain and source are known to present problems in achieving the normally accepted criteria of survival for long-term studies [see Guidance Document No 116 (7)], consideration should be given to using a strain of 
animal that has a acceptable survival rate for the long-term study. The 
females should be nulliparous and non-pregnant.
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Housing and feeding conditions  
17. Animals may be housed individually, or be caged in small groups of the 
same sex; individual housing should be considered only if scientifically justified (27) (28) (29). Cages should be arranged in such a way that 
possible effects due to cage placement are minimised. The temperature in 
the experimental animal room should be 22 °C (± 3 °C). Although the relative humidity should be at least 30 % and preferably not exceed 70 % other than during room cleaning, the aim should be 50-60 %. Lighting 
should be artificial, the sequence being 12 hours light, 12 hours dark. For 
feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. The diet should meet all the nutritional requirements of 
the species tested and the content of dietary contaminants, including but not 
limited to pesticide residues, persistent organic pollutants, phytoestrogens, heavy metals and mycotoxins, that might influence the outcome of the test, should be as low as possible. Analytical information on the nutrient and 
dietary contaminant levels should be generated periodically, at least at the 
beginning of the study and when there is a change in the batch used, and should be included in the final report. Analytical information on the drinking water used in the study should similarly be provided. The choice 
of diet may be influenced by the need to ensure a suitable admixture of a 
test chemical and to meet the nutritional requirements of the animals when the test chemical is administered by the dietary route.
 
Preparation of animals  
18. Healthy animals, which have been acclimated to laboratory conditions for at 
least 7 days and have not been subjected to previous experimental 
procedures, should be used. In the case of rodents, dosing of the animals 
should begin as soon as possible after weaning and acclimatisation and preferably before the animals are 8 weeks old. The test animals should be 
characterised as to species, strain, source, sex, weight and age. At the 
commencement of the study, the weight variation for each sex of animals used should be minimal and not exceed ± 20 % of the mean weight of all the animals within the study, separately for each sex. Animals should be 
randomly assigned to the control and treatment groups. After randomisation, 
there should be no significant differences in mean body weights between groups within each sex. If there are statistically significant differences, then the randomisation step should be repeated, if possible. Each animal should 
be assigned a unique identification number, and permanently marked with 
this number by tattooing, microchip implant, or other suitable method.
 
PROCEDURE  
Number and sex of animals  
19. Both sexes should be used. A sufficient number of animals should be used 
so that a thorough biological and statistical evaluation is possible. For rodents, each dose group (as outlined in paragraph 22) and concurrent control group intended for the carcinogenicity phase of the study should 
therefore contain at least 50 animals of each sex. Depending on the aim of 
the study, it may be possible to increase the statistical power of the key estimates by differentially allocating animals unequally to the various dose groups, with more than 50 animals in the low dose groups, e.g. to estimate 
the carcinogenic potential in low doses. However it should be recognised 
that a moderate increase in group size will provide relatively little increase in statistical power of the study. Each dose group (as outlined in paragraph 22) and concurrent control group intended for the chronic toxicity phase of 
the study should contain at least 10 animals of each sex, in the case of 
rodents. It should be noted that this number is lower than in the chronic
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 488
 
toxicity study (Chapter B.30 of this Annex). The interpretation of the data 
from the reduced number of animals per group in the chronic toxicity phase 
of this combined study will however be supported by the data from the larger number of animals in the carcinogenicity phase of the study. In studies involving mice, additional animals may be needed in each dose 
group of the chronic toxicity phase, to conduct all required haematological 
determinations. Further information on statistical design of the study and choice of dose levels to maximise statistical power is provided in Guidance Document No 116 (7).
 
Provision for interim kills, satellite group and sentinel animals  
20. The study may make provision for interim kills, e.g. at 6 months for the 
chronic toxicity phase, to provide information on progression of 
non-neoplastic changes and mechanistic information, if scientifically justified. Where such information is already available from previous repeat dose toxicity studies on the test chemical, interim kills may not be 
scientifically justified. The animals used in the chronic toxicity phase of the 
study, normally of 12 months duration (paragraph 34) provide interim kill data for the carcinogenicity phase of the study, thus achieving a reduction in the number of animals used overall. Satellite groups may also be included in 
the chronic toxicity phase of the study, to monitor the reversibility of any 
toxicological changes induced by the test chemical under investigation. These may be restricted to the highest dose level of the study plus control. An additional group of sentinel animals (typically 5 animals per 
sex) may be included for monitoring of disease status, if necessary, during 
the study (30). Further guidance on study design to include interim kills, satellite and sentinel animals, while minimising the number of animals used overall is provided in Guidance Document No 116 (7). 
21. If satellite animals and/or interim kills are included in the study design, the 
number of animals in each dose group included for this purpose will normally be 10 animals per sex, and the total number of animals included 
in the study design should be increased by the number of animals scheduled 
to be killed before the completion of the study. Interim kill and satellite animals should normally undergo the same observations, including body weight, food/water consumption, haematological and clinical biochemistry 
measurements and pathological investigations as the animals in the chronic 
toxicity phase of the main study, although provision may also be made (in the interim kill groups) for measurements to be restricted to specific, key measures such as neurotoxicity or immunotoxicity.
 
Dose groups and dosage  
22. Guidance on all aspects of dose selection and dose level spacing is provided 
in Guidance Document No 116 (7). At least three dose levels and a 
concurrent control should be used, for both the chronic and carcinogenicity phases. Dose levels will generally be based on the results of shorter-term repeated dose or range finding studies and should take into account any 
existing toxicological and toxicokinetic data available for the test chemical 
or related chemicals.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 489
 
23. For the chronic toxicity phase of the study, a full study using three dose 
levels may not be considered necessary, if it can be anticipated that a test at 
one dose level, equivalent to at least 1 000 mg/kg body weight/day, is unlikely to produce adverse effects. This should be based on information from preliminary studies and a consideration that toxicity would not be 
expected, based upon data from structurally related chemicals. A limit of 
1 000 mg/kg body weight/day may apply except when human exposure indicates the need for a higher dose level to be used. 
24. Unless limited by the physical-chemical nature or biological effects of the 
test chemical, the highest dose level should be chosen to identify the 
principal target organs and toxic effects while avoiding suffering, severe toxicity, morbidity, or death. The highest dose level should be normally chosen to elicit evidence of toxicity, as evidenced by, for example, 
depression of body weight gain (approximately 10 %). However, 
dependent on the objectives of the study (see paragraph 6), a top dose lower than the dose providing evidence of toxicity may be chosen, e.g. if a dose elicits an adverse effect of concern, which nonetheless has little 
impact on lifespan or body weight. 
25. Dose levels and dose level spacing may be selected to establish a dose- 
response and, depending on the mode of action of the test chemical, a 
NOAEL or other intended outcome of the study, e.g. a BMD (see 
paragraph 27). Factors that should be considered in the placement of lower doses include the expected slope of the dose–response curve, the doses at which important changes may occur in metabolism or mode of 
toxic action, where a threshold is expected, or where a point of departure for 
low-dose extrapolation is expected. In conducting a combined carcinogenic ­
ity/chronic toxicity study, the primary objective will be to obtain information for carcinogenicity risk assessment purposes, and information 
on chronic toxicity will normally be a subsidiary objective. This should be 
borne in mind when selecting dose levels and dose level spacing for the study. 
26. The dose level spacing selected will depend on the objectives of the study 
and the characteristics of the test chemical, and cannot be prescribed in 
detail in this Test Method, but two to four fold intervals frequently provide good test performance when used for setting the descending dose levels and addition of a fourth test group is often preferable to using very 
large intervals (e.g. more than a factor of about 6-10) between dosages. In 
general the use of factors greater than 10 should be avoided, and should be justified if used. 
27. As outlined further in Guidance Document No 116 (7), points to be 
considered in dose selection include: 
— Known or suspected nonlinearities or inflection points in the dose–re ­
sponse; 
— Toxicokinetics, and dose ranges where metabolic induction, saturation, 
or nonlinearity between external and internal doses does or does not occur; 
— Precursor lesions, markers of effect, or indicators of the operation of key 
underlying biological processes;
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 490
 
— Key (or suspected) aspects of mode of action, such as doses at which 
cytotoxicity begins to arise, hormone levels are perturbed, homeostatic 
mechanisms are overwhelmed, etc.; 
— Regions of the dose–response curve where particularly robust estimation 
is needed, e.g. in the range of the anticipated BMD or a suspected threshold; 
— Consideration of anticipated human exposure levels, especially in the 
choice of mid and low doses. 
28. The control group shall be an untreated group or a vehicle-control group if a 
vehicle is used in administering the test chemical. Except for treatment with the test chemical, animals in the control group should be handled in an identical manner to those in the test groups. If a vehicle is used, the 
control group shall receive the vehicle in the highest volume used among 
the dose groups. If a test chemical is administered in the diet, and causes significantly reduced dietary intake due to the reduced palatability of the diet, an additional pair-fed control group may be useful, to serve as a more 
suitable control.
 
Preparation of doses and administration of test chemical  
29. The test chemical is normally administered orally, via the diet or drinking 
water, or by gavage. Additional information on routes and methods of 
administration is provided in Guidance Document No 116 (7). The route and method of administration is dependent on the purpose of the study, the physical/chemical properties of the test chemical, its bioavailability, and the 
predominant route and method of exposure of humans. A rationale should 
be provided for the chosen route and method of administration. In the interests of animal welfare, oral gavage should normally be selected only for those agents for which this route and method of administration 
reasonably represent potential human exposure (e.g. pharmaceuticals). For 
dietary or environmental chemicals including pesticides, administration is typically via the diet or drinking water. However, for some scenarios, e.g. occupational exposure, administration via other routes may be more appro ­
priate. 
30. Where necessary, the test chemical is dissolved or suspended in a suitable 
vehicle. Consideration should be given to the following characteristics of the vehicle and other additives, as appropriate: effects on the absorption, distribution, metabolism, or retention of the test chemical; effects on the 
chemical properties of the test chemical which may alter its toxic character ­
istics; and effects on the food or water consumption or the nutritional status of the animals. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first, followed by consideration 
of a solution/emulsion in oil (e.g. corn oil) and then by possible solution in 
other vehicles. For vehicles other than water, the toxic characteristics of the vehicle should be known. Information should be available on the stability of the test chemical and the homogeneity of dosing solutions or diets (as 
appropriate) under the conditions of administration (e.g. diet). 
31. For chemicals administered via the diet or drinking water it is important to 
ensure that the quantities of the test chemical involved do not interfere with normal nutrition or water balance. In long-term toxicity studies using dietary administration, the concentration of the test chemical in the feed should not 
normally exceed an upper limit of 5 % of the total diet, in order to avoid 
nutritional imbalances. When the test chemical is administered in the diet, either a constant dietary concentration (mg/kg diet or ppm), or a constant dose level in terms of the animal’s body weight (mg/kg body weight), 
calculated on a weekly basis, may be used. The alternative used should 
be specified.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 491
 
32. In the case of oral administration, the animals are dosed with the test 
chemical daily (seven days each week) for a period of 12 months 
(chronic phase) or 24 months (carcinogenicity phase), see also paragraphs 33 and 34. Any other dosing regime, e.g. five days per week, needs to be justified.. In the case of dermal administration, animals are normally treated 
with the test chemical for at least 6 hours per day, 7 days per week, as 
specified in Chapter B.9 of this Annex (11), for a period of 12 months (chronic phase) or 24 months (carcinogenicity phase). Exposure by the inhalation route is carried out for 6 hours per day, 7 days per week, but 
exposure for 5 days per week may also be used, if justified. The period of 
exposure will normally be for a period of 12 months (chronic phase) or 24 months (carcinogenicity phase). If rodent species other than rats are exposed nose-only, maximum exposure durations may be adjusted to minimise 
species-specific distress. A rationale should be provided when using an 
exposure duration of less than 6 hours per day. See also Chapter B.8 of this Annex (9). 
33. When the test chemical is administered by gavage to the animals this should 
be done using a stomach tube or a suitable intubation cannula, at similar times each day. Normally a single dose will be administered once daily, where for example a chemical is a local irritant, it may be possible to 
maintain the daily dose-rate by administering it as a split dose (twice a 
day). The maximum volume of liquid that can be administered at one time depends on the size of the test animal. The volume should be kept as low as practical, and should not normally exceed 1 ml/100g body weight 
for rodents (31). Variability in test volume should be minimised by 
adjusting the concentration to ensure a constant volume at all dose levels. Potentially corrosive or irritant chemicals are the exception, and need to be diluted to avoid severe local effects. Testing at concentrations that are likely 
to be corrosive or irritant to the gastrointestinal tract should be avoided.
 
Duration of study  
34. The period of dosing and duration of the chronic phase of this study is 
normally 12 months, although the study design also allows for and can be 
applied to either shorter (e.g. 6 or 9 months) or longer (e.g. 18 or 24 
months) duration studies, depending on the requirements of particular regu ­
latory regimes or for specific mechanistic purposes. Deviations from an exposure duration of 12 months should be justified, particularly in the 
case of shorter durations. All dose groups allocated to this phase will be 
terminated at the designated time for evaluation of chronic toxicity and non-neoplastic pathology. Satellite groups included to monitor the reversi ­
bility of any toxicological changes induced by the test chemical under 
investigation should be maintained without dosing for a period not less 
than 4 weeks and not more than one third of the total study duration after cessation of exposure. 
35. The duration of the carcinogenicity phase of this study will normally be 24 
months for rodents, representing the majority of the normal life span of the animals to be used. Shorter or longer study durations may be used, dependent on the lifespan of the strain of the animal species in the study, 
but should be justified. For specific strains of mice, e.g. AKR/J, C3H/J or 
C57BL/6J strains a duration of 18 months may be more appropriate. The following provides some guidance on duration, termination of the study and survival; further guidance, including consideration of the acceptability of a 
negative carcinogenicity study relative to survival in the study, is provided 
in Guidance Document No 116 (7):
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 492
 
— Termination of the study should be considered when the number of 
survivors in the lower dose groups or the control group falls below 25 
per cent. 
— In the case where only the high dose group dies prematurely due to 
toxicity, this should not trigger termination of the study. 
— Survival of each sex should be considered separately. 
— The study should not be extended beyond the point when the data 
available from the study are no longer sufficient to enable a statistically valid evaluation to be made.
 
OBSERVATIONS (CHRONIC TOXICITY PHASE) 
36. All animals should be checked for morbidity or mortality, usually at the 
beginning and end of each day, including at weekends and holidays. 
General clinical observations should be made at least once a day, preferably at the same time(s) each day, taking into consideration the peak period of anticipated effects after dosing in the case of gavage administration. 
37. Detailed clinical observations should be made on all animals at least once 
prior to the first exposure (to allow for within-subject comparisons), at the end of the first week of the study and monthly thereafter. The protocol for 
observations should be arranged such that variations between individual 
observers are minimised and independent of test group. These observations should be made outside the home cage, preferably in a standard arena and at similar times on each occasion. They should be carefully recorded, 
preferably using scoring systems, explicitly defined by the testing 
laboratory. Efforts should be made to ensure that variations in the obser ­
vation conditions are minimal. Signs noted should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of 
secretions and excretions and autonomic activity (e.g. lacrimation, piloe ­
rection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic move ­
ments, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre 
behaviour (e.g. self-mutilation, walking backwards) should also be 
recorded (32). 
38. Ophthalmological examination, using an ophthalmoscope or other suitable 
equipment, should be carried out on all animals prior to the first adminis ­
tration of the test chemical. At the termination of the study, this examination should be preferably conducted in all animals but at least in the high dose and control groups. If treatment-related changes in the eyes are detected, all 
animals should be examined. If structural analysis or other information 
suggests ocular toxicity, then the frequency of ocular examination should be increased. 
39. For chemicals where previous repeated dose 28-day and/or 90-day toxicity 
tests indicated the potential to cause neurotoxic effects, sensory reactivity to stimuli of different types (32) (e.g. auditory, visual and proprioceptive stimuli) (33) (34) (35), assessment of grip strength (36) and motor 
activity assessment (37) may optionally be conducted before commencement 
of the study and at 3 month periods after study initiation up to and including 12 months, as well as at study termination (if longer than 12 months). Further details of the procedures that could be followed are given in the 
respective references. However, alternative procedures than those referenced 
could also be used.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 493
 
40. For chemicals where previous repeated dose 28-day and/or 90-day toxicity 
tests indicated the potential to cause immunotoxic effects, further investi ­
gations of this endpoint may optionally be conducted at termination.  
Body weight, food/water consumption and food efficiency  
41. All animals should be weighed at the start of treatment, at least once a week 
for the first 13 weeks and at least monthly thereafter. Measurements of food consumption and food efficiency should be made at least weekly for the first 
13 weeks and at least monthly thereafter. Water consumption should be 
measured at least weekly for the first 13 weeks and at least monthly thereafter when the test chemical is administered in drinking water. Water consumption measurements should also be considered for studies in which 
drinking activity is altered.
 
Haematology and clinical biochemistry  
42. In studies involving rodents, haematological examinations should be carried 
out on all study animals (10 male and 10 female animals per group) at 3, 6, and 12 months, as well as at study termination (if longer than 12 months). In mice, satellite animals may be needed in order to conduct all required 
haematological determinations (see paragraph 19). In non-rodent studies, 
samples will be taken from smaller numbers of animals (e.g. 4 animals per sex and per group in dog studies), at interim sampling times and at termination as described for rodents. Measurements at 3 months, either in 
rodents or non-rodents, need not be conducted if no effect was seen on 
haematological parameters in a previous 90 day study carried out at comparable dose levels. Blood samples should be taken from a named site, for example by cardiac puncture or from the retro-orbital sinus, 
under anaesthesia. 
43. The following list of parameters should be investigated (38): total and 
differential leukocyte count, erythrocyte count, platelet count, haemoglobin 
concentration, haematocrit (packed cell volume), mean corpuscular 
volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), prothrombin time, and activated partial thromboplastin time. Other hematology parameters such as Heinz bodies or 
other atypical erythrocyte morphology or methaemoglobin may be measured 
as appropriate depending on the toxicity of the test chemical. Overall, a flexible approach should be adopted, depending on the observed and/or expected effect from a given test chemical. If the test chemical has an 
effect on the haematopoietic system, reticulocyte counts and bone marrow 
cytology may also be indicated, although these need not be routinely conducted. 
44. Clinical biochemistry determinations to investigate major toxic effects in 
tissues and, specifically, effects on kidney and liver, should be performed 
on blood samples obtained from all study animals (10 male and 10 female animals per group), at the same time intervals as specified for the haema ­
tological investigations. In mice, satellite animals may be needed in order to 
conduct all required clinical biochemistry determinations. In non-rodent 
studies, samples will be taken from smaller numbers of animals (e.g. 4 animals per sex and per group in dog studies), at interim sampling times and at termination as described for rodents. Measurements at 3 months, 
either in rodents or non-rodents, need not be conducted if no effect was 
seen on clinical biochemistry parameters in a previous 90 day study carried
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 494
 
out at comparable dose levels. Overnight fasting of the animals (with the 
exception of mice) prior to blood sampling is recommended (  1 ). The 
following list of parameters should be investigated (38): glucose, urea (urea nitrogen), creatinine, total protein, albumin, calcium, sodium, potassium, total cholesterol, at least two appropriate tests for hepatocellular 
evaluation (alanine aminotransferase, aspartate aminotransferase, glutamate 
dehydrogenase, total bile acids) (39), and at least two appropriate tests for hepatobiliary evaluation (alkaline phosphatase, gamma glutamyl transferase, 5’-nucleotidase, total bilirubin, total bile acids) (39). Other clinical chemistry 
parameters such as fasting triglycerides, specific hormones and cholines ­
terase may be measured as appropriate, depending on the toxicity of the test chemical. Overall, there is a need for a flexible approach, depending on the observed and/or expected effect from a given test chemical. 
45. Urinalysis determinations should be performed on all study animals (10 
male and 10 female animals per group), on samples collected at the same intervals as for haematology and clinical chemistry. Measurements at 3 months need not be conducted if no effect was seen on urinalysis in a 
previous 90 day study carried out at comparable dose levels. The 
following list of parameters was included in an expert recommendation on clinical pathology studies (38): appearance, volume, osmolality or specific gravity, pH, total protein, and glucose. Other determinations include ketone, 
urobilinogen, bilirubin, and occult blood. Further parameters may be 
employed where necessary to extend the investigation of observed effect(s). 
46. It is generally considered that baseline haematological and clinical 
biochemistry variables need be determined before treatment for dog 
studies, but need not be determined in rodent studies (38). However, if historical baseline data (see paragraph 58) are inadequate, consideration should be given to generating such data.
 
PATHOLOGY  
Gross necropsy  
47. All animals in the study shall be normally subjected to a full, detailed gross 
necropsy which includes careful examination of the external surface of the 
body, all orifices, and the cranial, thoracic and abdominal cavities and their 
contents. However provision may also be made (in the interim kill or satellite groups) for measurements to be restricted to specific, key measures such as neurotoxicity or immunotoxicity (see paragraph 21). 
These animals need not be subjected to necropsy and the subsequent 
procedures described in the following paragraphs. Sentinel animals may require necropsy on a case-by-case basis, at the discretion of the study director.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 495  
( 1 ) For a number of measurements in serum and plasma, most notably for glucose, overnight 
fasting is preferable. The major reason for this preference is that the increased variability 
which would inevitably result from non-fasting, would tend to mask more subtle effects 
and make interpretation difficult. However it should be noted that overnight fasting may 
interfere with the general metabolism of the animals and, particularly in feeding studies, 
may disrupt the daily exposure to the test chemical. All animals should be assessed in the 
same physiological condition and preferably detailed or neurological assessments should 
therefore be scheduled for a different day than clinical biochemistry sampling.
 48. Organ weights should be collected from all animals, other than those 
excluded by the latter part of paragraph 47. The adrenals, brain, epididy ­
mides, heart, kidneys, liver, ovaries, spleen, testes, thyroid (weighed post-fixation, with parathyroids), and uterus of all animals (apart from those found moribund and/or intercurrently killed) should be trimmed of 
any adherent tissue, as appropriate, and their wet weight taken as soon as 
possible after dissection to prevent drying. 
49. The following tissues should be preserved in the most appropriate fixation 
medium for both the type of tissue and the intended subsequent histopath ­
ological examination (40) (tissues in square brackets are optional): 
all gross lesions heart pancreas stomach (forestomach, 
glandular stomach) 
adrenal gland ileum parathyroid gland [teeth] 
aorta jejunum peripheral nerve testis 
brain (including sections of 
cerebrum, cerebellum, and 
medulla/pons) kidney pituitary thymus 
caecum lacrimal gland (exorbital) prostate thyroid 
cervix liver rectum [tongue] 
coagulating gland lung salivary gland trachea 
colon lymph nodes (both 
superficial and deep) seminal vesicle urinary bladder 
duodenum mammary gland 
(obligatory for females 
and, if visibly 
dissectable, from males) skeletal muscle uterus (including cervix) 
epididymis [upper respiratory tract, 
including nose, turbi ­
nates, and paranasal sinuses] skin [ureter] 
eye (including retina) oesophagus spinal cord (at three 
levels: cervical, mid-thoracic, and lumbar) [urethra] 
[femur with joint] [olfactory bulb] spleen vagina 
gall bladder (for species 
other than rat) ovary [sternum], section of bone marrow and/ 
or a fresh bone marrow 
aspirate 
Harderian gland
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In the case of paired organs, e.g. kidney, adrenal, both organs should be 
preserved. The clinical and other findings may suggest the need to examine 
additional tissues. Also any organs considered likely to be target organs based on the known properties of the test chemical should be preserved. In studies involving the dermal route of administration, the list of organs as 
set out for the oral route should be examined, and specific sampling and 
preservation of the skin from the site of application is necessary. In inha ­
lation studies, the list of preserved and examined tissues from the respiratory tract should follow the recommendations of Chapters B.8 of this Annex (9) 
and Chapter B.29 of this Annex (10). For other organs/tissues (and in 
addition to the specifically preserved tissues from the respiratory tract) the list of organs as set out for the oral route should be examined.
 
Histopathology  
50. Guidance is available on best practices in the conduct of toxicological 
pathology studies (40). The minimum histopathological examinations should be: 
— all tissues from the high dose and control groups; — all tissues from animals dying or killed during the study; — all tissues showing macroscopic abnormalities; — target tissues, or tissues which showed treatment-related changes in the 
high dose group, from all animals in all other dose groups, 
— in the case of paired organs, e.g. kidney, adrenal, both organs should be 
examined.
 
OBSERVATIONS (CARCINOGENICITY PHASE) 
51. All animals should be checked for morbidity or mortality, usually at the 
beginning and the end of each day, including at weekends and holidays. Animals should additionally be checked once a day for specific signs of toxicological relevance. In the case of gavage studies, animals should be 
checked in the period immediately following dosing. Particular attention 
should be paid to tumour development; and the time of tumour onset, location, dimensions, appearance, and progression of each grossly visible or palpable tumour should be recorded. 
52. All animals should be weighed at the start of treatment, at least once a week 
for the first 13 weeks and at least monthly thereafter. Measurements of food 
consumption and food efficiency should be made at least weekly for the first 
13 weeks and at least monthly thereafter. Water consumption should be measured at least weekly for the first 13 weeks and at least monthly thereafter when the test chemical is administered in drinking water. Water 
consumption measurements should also be considered for studies in which 
drinking activity is altered.
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Haematology, clinical biochemistry and other measurements  
53. In order to maximise the information obtained from the study, especially for 
mode of action considerations, blood samples may be taken for haematology and clinical biochemistry, although this is at the discretion of the study 
director. Urinalysis may also be appropriate. Data on the animals used in 
the chronic toxicity phase of the study, normally of 12 months duration (paragraph 34) will provide information on these parameters. Further guidance on the value of taking such samples as part of a carcinogenicity 
study is provided in Guidance Document No 116 (7). If blood samples are 
taken, these should be collected at the end of the test period, just prior to or as part of the procedure for killing the animals. They should be taken from a 
named site, for example by cardiac puncture or from the retro-orbital sinus, 
under anaesthesia. Blood smears may also be prepared for examination, particularly if bone marrow appears to be the target organ, although the value of such examination of blood smears in the carcinogenicity phase for 
the assessment of carcinogenic/oncogenic potential has been 
questioned (38).
 
PATHOLOGY  
Gross necropsy  
54. All animals in the study except sentinel animals and other satellite animals 
(see paragraph 20) shall be subjected to a full, detailed gross necropsy 
which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. Sentinel animals and other satellite animals may require 
necropsy on a case-by-case basis, at the discretion of the study director. 
Organ weights are not normally part of a carcinogenesis study, since geriatric changes and, at later stages, the development of tumours confounds the usefulness of organ weight data. They may, however, be 
critical to performing a weight of evidence evaluation and especially for 
mode of action considerations. If they are part of a satellite study, they should be collected at no later than one year after initiation of the study. 
55. The following tissues should be preserved in the most appropriate fixation 
medium for both the type of tissue and the intended subsequent histopath ­
ological examination (40) (tissues in square brackets are optional): 
all gross lesions heart pancreas stomach (forestomach, 
glandular stomach) 
adrenal gland ileum parathyroid gland [teeth] 
aorta jejunum peripheral nerve testis brain (including sections of 
cerebrum, cerebellum, and 
medulla/pons) kidney pituitary thymus 
caecum lacrimal gland (exorbital) prostate thyroid 
cervix liver rectum [tongue] coagulating gland lung salivary gland trachea colon lymph nodes (both 
superficial and deep) seminal vesicle urinary bladder
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 498
 
duodenum mammary gland 
(obligatory for females 
and, if visibly dissectable, from males) skeletal muscle uterus (including cervix) 
epididymis [upper respiratory tract, 
including nose, turbi ­
nates, and paranasal 
sinuses] skin [ureter] 
eye (including retina) oesophagus spinal cord (at three 
levels: cervical, mid-thoracic, and lumbar) [urethra] 
[femur with joint] [olfactory bulb] spleen vagina 
gall bladder (for species 
other than rat) ovary [sternum], section of bone marrow and/ 
or a fresh bone marrow 
aspirate 
Harderian gland 
In the case of paired organs, e.g. kidney, adrenal, both organs should be 
preserved. The clinical and other findings may suggest the need to examine 
additional tissues. Also, any organs considered likely to be target organs based on the known properties of the test chemical should be preserved. In studies involving the dermal route of administration, the list of organs as set 
out for the oral route should be examined, and specific sampling and pres ­
ervation of the skin from the site of application is necessary. In inhalation studies, the list of preserved and examined tissues from the respiratory tract should follow the recommendations of Chapters B.8 of this Annex (8) and 
Chapter B.29 of this Annex (9). For other organs/tissues (and in addition to 
the specifically preserved tissues from the respiratory tract) the list of organs as set out for the oral route should be examined.
 
Histopathology  
56. Guidance is available on best practices in the conduct of toxicological 
pathology studies (40). The minimum tissues examined should be: 
— All tissues from the high dose and control groups; 
— All tissues of animals dying or killed during the study; 
— All tissues showing macroscopic abnormalities including tumours; 
— When treatment-related histopathological changes are observed in the 
high dose group, those same tissues are to be examined from all animals in all other dose groups, 
— In the case of paired organs, e.g. kidney, adrenal, both organs should be 
examined.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 499
 
DATA AND REPORTING (CARCINOGENICITY AND CHRONIC 
TOXICITY)  
Data  
57. Individual animal data should be provided for all parameters evaluated. 
Additionally, all data should be summarised in tabular form showing for each test group the number of animals at the start of the test, the number of 
animals found dead during the test or killed for humane reasons and the 
time of any death or humane kill, the number showing signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects, the number of animals 
showing lesions, the type of lesions and the percentage of animals 
displaying each type of lesion. Summary data tables should provide the means and standard deviations (for continuous test data) of animals showing toxic effects or lesions, in addition to the grading of lesions. 
58. Historical control data may be valuable in the interpretation of the results of 
the study, e.g, in the case when there are indications that the data provided 
by the concurrent controls are substantially out of line when compared to 
recent data from control animals from the same test facility/colony. Historical control data, if evaluated, should be submitted from the same laboratory, relate to animals of the same age and strain, generated during 
the five years preceding the study in question. 
59. When applicable, numerical results should be evaluated by an appropriate 
and generally acceptable statistical method. The statistical methods and the 
data to be analysed should be selected during the design of the study 
(paragraph 9). Selection should make provision for survival adjustments, if needed. 
60. The test report should include the following information:  
Test chemical:  
— physical nature, purity, and physicochemical properties; 
— identification data; 
— source of chemical; 
— batch number; — certificate of chemical analysis.  
Vehicle (if appropriate):  
— justification for choice of vehicle (if other than water).  
Test animals:  
— species/strain used and justification for choice made; 
— number, age, and sex of animals at start of test; 
— source, housing conditions, diet, etc.; 
— individual weights of animals at the start of the test.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 500
 
Test conditions:  
— rationale for route of administration and dose selection; — when applicable, the statistical methods used to analyse the data; — details of test chemical formulation/diet preparation; 
— analytical data on achieved concentration, stability and homogeneity of 
the preparation; 
— route of administration and details of the administration of the test 
chemical; 
— for inhalation studies, whether nose only or whole body; — actual doses (mg/kg body weight/day), and conversion factor from diet/ 
drinking water test chemical concentration (mg/kg or ppm) to the actual 
dose, if applicable; 
— details of food and water quality.  
Results (summary tabulated data and individual animal data should be 
presented): 
General  
— Survival data; — Body weight/body weight changes; — Food consumption, calculations of food efficiency, if made, and water 
consumption if applicable; 
— Toxicokinetic data if available; — Opthalmoscopy (if available) — Haematology (if available) — Clinical chemistry (if available)  
Clinical findings  
— Signs of toxicity; — Incidence (and, if scored, severity) of any abnormality; — Nature, severity, and duration of clinical observations (whether transitory 
or permanent);  
Necropsy data  
— Terminal body weight; — Organ weights and their ratios, if applicable; — Necropsy findings; Incidence and severity of abnormalities.  
Histopathology  
— Non neoplastic histopathological findings, — Neoplastic histopathological findings,
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 501
 
— Correlation between gross and microscopic findings 
— Detailed description of all treatment-related histopathological findings 
including severity gradings; 
— Report of any peer review of slides  
Statistical treatment of results, as appropriate Discussion of results including:  
— Discussion of any modelling approaches 
— Dose:response relationships 
— Historical control data — Consideration of any mode of action information — BMD, NOAEL or LOAEL determination — Relevance for humans  
Conclusions
 
LITERATURE:  
(1) OECD (1995). Report of the Consultation Meeting on Sub-chronic and 
Chronic Toxicity/Carcinogenicity Testing (Rome, 1995), internal working 
document, Environment Directorate, OECD, Paris. 
(2) EPA (2005). Guidelines for Carcinogen Risk Assessment Risk Assessment 
Forum U.S. Environmental Protection Agency Washington, DC. 
(3) Combes RD, Gaunt I, Balls M (2004). A Scientific and Animal Welfare 
Assessment of the OECD Health Effects Test Guidelines for the Safety 
Testing of Chemicals under the European Union REACH System. ATLA 32: 163-208 
(4) Barlow SM, Greig JB, Bridges JW et al (2002). Hazard identification by 
methods of animal-based toxicology. Food. Chem. Toxicol. 40: 145-191 
(5) Chhabra RS, Bucher JR, Wolfe M, Portier C (2003). Toxicity character ­
ization of environmental chemicals by the US National Toxicology Programme: an overview. Int. J. Hyg. Environ. Health 206: 437-445 
(6) Chapter B.27 of this Annex, Sub-Chronic Oral Toxicity Test Repeated 
Dose 90 — Day Oral Toxicity Study In Non-Rodents. 
(7) OECD (2012). Guidance Document on the Design and Conduct of Chronic 
Toxicity and Carcinogenicity Studies, Supporting Test Guidelines 451, 452 and 453 — Second edition. Series on Testing and Assessment No 116, 
available on the OECD public website for Test Guideline at www.oecd.org/ 
env/testguidelines. 
(8) OECD (2009). Guidance Document on Acute Inhalation Toxicity Testing. 
Series on Testing and Assessment No 39, ENV/JM/MONO(2009)28, OECD, Paris. 
(9) Chapter B.8 of this Annex. Subacute Inhalation Toxicity: 28-Day Study. 
(10) Chapter B.29 of this Annex, Subchronic Inhalation Toxicity: 90-Day Study. (11) Chapter B.9 of this Annex, Repeated Dose (28 Days) Toxicity (Dermal).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 502
 
(12) Boobis AR, Cohen SM, Dellarco V, McGregor D, Meek ME, Vickers C, 
Willcocks D, Farland W (2006). IPCS Framework for analyzing the 
Relevance of a Cancer Mode of Action for Humans. Crit. Rev. in Toxicol, 36: 793-801. 
(13) Cohen SM, Meek ME, Klaunig JE, Patton DE, Fenner-Crisp PA (2003). 
The human relevance of information on carcinogenic Modes of Action: An 
Overview. Crit. Rev. Toxicol. 33: 581-589. 
(14) Holsapple MP, Pitot HC, Cohen SN, Boobis AR, Klaunig JE, Pastoor T, 
Dellarco VL, Dragan YP (2006). Mode of Action in Relevance of Rodent Liver Tumors to Human Cancer Risk. Toxicol. Sci. 89: 51-56. 
(15) Meek EM, Bucher JR, Cohen SM, Dellarco V, Hill RN, 
Lehman-McKemmon LD, Longfellow DG, Pastoor T, Seed J, Patton DE 
(2003). A Framework for Human Relevance analysis of Information on Carcinogenic Modes of Action. Crit. Rev. Toxicol. 33: 591-653. 
(16) Carmichael NG, Barton HA, Boobis AR  et al.  (2006). Agricultural 
Chemical Safety Assessment: A Multisector Approach to the Modern ­
ization of Human Safety Requirements. Crit. Rev. Toxicol. 36, 1-7. 
(17) Barton HA, Pastoor TP, Baetcke T  et al.  (2006). The Acquisition and 
Application of Absorption, Distribution, Metabolism, and Excretion (ADME) Data in Agricultural Chemical Safety Assessments. 
Crit. Rev. Toxicol. 36: 9-35. 
(18) Doe JE, Boobis AR, Blacker A  et al . (2006). A Tiered Approach to 
Systemic Toxicity Testing for Agricultural Chemical Safety Assessment. Crit. Rev. Toxicol. 36: 37-68. 
(19) Cooper RL, Lamb JS, Barlow SM  et al.  (2006). A Tiered Approach to Life 
Stages Testing for Agricultural Chemical Safety Assessment. Crit. Rev. 
Toxicol. 36: 69-98. 
(20) OECD (2002). Guidance Notes for Analysis and Evaluation of Chronic 
Toxicity and Carcinogenicity Studies, Series on Testing and Assessment No 35 and Series on Pesticides No 14, ENV/JM/MONO(2002)19, OECD, 
Paris. 
(21) OECD (2000). Guidance Document on the recognition, assessment, and use 
of clinical signs as humane endpoints for experimental animals used in safety evaluation, Series on Testing and Assessment No 19, ENV/JM/ MONO(2000)7, OECD, Paris. 
(22) Rhomberg LR, Baetcke K, Blancato J, Bus J, Cohen S, Conolly R, Dixit R, 
Doe J, Ekelman K, Fenner-Crisp P, Harvey P, Hattis D, Jacobs A, Jacobson-Kram D, Lewandowski T, Liteplo R, Pelkonen O, Rice J, Somers D, Turturro A, West W, Olin S (2007). Issues in the Design and 
Interpretation of Chronic Toxicity and Carcinogenicity Studies in Rodents: 
Approaches to Dose Selection Crit Rev. Toxicol. 37 (9): 729 – 837. 
(23) ILSI (International Life Sciences Institute) (1997). Principles for the 
Selection of Doses in Chronic Rodent Bioassays. Foran JA (Ed.). ILSI Press, Washington, DC. 
(24) Griffiths SA, Parkinson C, McAuslane JAN and Lumley CE (1994). The 
utility of the second rodent species in the carcinogenicity testing of phar ­
maceuticals. The Toxicologist 14(1):214. 
(25) Usui T, Griffiths SA and Lumley CE (1996). The utility of the mouse for 
the assessment of the carcinogenic potential of pharmaceuticals. In D’Arcy 
POF & Harron DWG (eds). Proceedings of the Third International 
Conference on Harmonisation. Queen’s University Press, Belfast. pp 279- 284. 
(26) Carmichael NG, Enzmann H, Pate I, Waechter F (1997). The Significance 
of Mouse Liver Tumor Formation for Carcinogenic Risk Assessment: 
Results and Conclusions from a Survey of 10 Years of Testing by the 
Agrochemical Industry. Environ Health Perspect 105:1196-1203.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 503
 
(27) Directive 2010/63/EU of the European parliament and of the Council of 
22 September 2010 on the protection of animals used for scientific 
purposes (OJ L 276, 20.10.2010, p. 33). 
(28) National Research Council, 1985. Guide for the care and use of laboratory 
animals. NIH Publication No 86-23. Washington D.C., US. Dept. of Health and Human Services. 
(29) GV-SOLAS (Society for Laboratory Animal Science, Gesellschaft für 
Versuchstierkunde, December, 1989). Publication on the Planning and 
Structure of Animal Facilities for Institutes Performing Animal Experi ­
ments. ISBN 3-906255-06-9. 
(30) GV-SOLAS (Society for Laboratory Animal Science, Gesellschaft für 
Versuchstierkunde, 2006). Microbiological monitoring of laboratory 
animals in various housing systems. 
(31) Diehl K-H, Hull R, Morton D, Pfister R, Rabemampianina Y, Smith D, 
Vidal J-M, van de Vorstenbosch C. (2001). A good practice guide to the administration of substances and removal of blood, including routes and 
volumes. Journal of Applied Toxicology, 21: 15-23. 
(32) IPCS (1986). Principles and Methods for the Assessment of Neurotoxicity 
Associated with Exposure to Chemicals. Environmental Health Criteria Document No 60. 
(33) Tupper DE, Wallace RB (1980). Utility of the Neurologic Examination in 
Rats. Acta Neurobiol. Exp. 40: 999-1003. 
(34) Gad SC (1982). A Neuromuscular Screen for Use in Industrial Toxicology. 
J. Toxicol.Environ. Health 9: 691-704. 
(35) Moser VC, McDaniel KM, Phillips PM (1991). Rat Strain and Stock 
Comparisons Using a Functional Observational Battery: Baseline Values 
and Effects of Amitraz. Toxicol. Appl. Pharmacol. 108: 267-283. 
(36) Meyer OA, Tilson HA, Byrd WC, Riley MT (1979). A Method for the 
RoutineAssessment of Fore- and Hind-limb Grip Strength of Rats and Mice. Neurobehav. Toxicol. 1: 233-236. 
(37) Crofton KM, Howard JL, Moser VC, Gill MW, Reiter LW, Tilson HA, 
MacPhail RC (1991). Interlaboratory Comparison of Motor Activity 
Experiments: Implication for Neurotoxicological Assessments. Neur ­
otoxicol. Teratol. 13: 599-609. 
(38) Weingand K, Brown G, Hall R  et al . (1996). Harmonisation of Animal 
Clinical Pathology Testing in Toxicity and Safety Studies. Fundam. & 
Appl. Toxicol. 29: 198-201. 
(39) EMEA (draft) document ‘Non-clinical guideline on drug-induced hepatot ­
oxicity’ (Doc. Ref. EMEA/CHMP/SWP/a50115/2006). 
(40) Crissman JW, Goodman DG, Hildebrandt PK  et al.  (2004). Best Practices 
Guideline: Toxicological Histopathology. Toxicologic Pathology 32: 126-131.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 504
 
Appendix 1  
DEFINITION  
Test chemical:  Any substance or mixture tested using this Test Method.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 505
 
B.34.  ONE-GENERATION REPRODUCTION TOXICITY TEST  
1. METHOD  
1.1. INTRODUCTION 
See General introduction Part B.  
1.2. DEFINITIONS 
See General introduction Part B.  
1.3. REFERENCE SUBSTANCES 
None.  
1.4. PRINCIPLE OF THE TEST METHOD 
The test substance is administered in graduated doses to several 
groups of males and females. Males should be dosed during growth and for at least one complete spermatogenic cycle (approxi ­
mately 56 days in the mouse and 70 days in the rat) in order to elicit 
any adverse effects on spermatogenesis by the test substance. 
Females of the parental (P) generation should be dosed for at least 
two complete oestrous cycles in order to e adverse effects on oestrus 
by the test substance. The animals are then mated. The test substance 
is administered to both sexes during the mating period and thereafter only to females during pregnancy and for the duration of the nursing period. For administration by inhalation the method will require 
modification.
 
1.5. QUALITY CRITERIA 
None.  
1.6. DESCRIPTION OF THE TEST METHOD  
1.6.1.  Preparations  
Before the test, healthy young adult animals are randomised and 
assigned to the treated and control groups. The animals are kept under the experimental housing and feeding conditions for at least five days prior to the test. It is recommended that the test substance 
be administered in the diet or drinking water. Other routes of admin ­
istration are also acceptable. All animals should be dosed by the same method during the appropriate experimental period. If a vehicle or other additives are used to facilitate dosing, they should 
be known not to produce toxic effects. Dosing should be on a seven- 
day per week basis.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 506
 
1.6.2. E x p e r i m e n t a l a n i m a l s  
Selection of species 
The rat or mouse are the preferred species. Healthy animals, not 
subjected to previous experimental procedures, should be used. Strains with low fecundity should not be used. The test animals 
should be characterized as to species, strain, sex, weight and/or age. 
For an adequate assessment of fertility, both males and females 
should be studied. All test and control animals should be weaned 
before dosing begins.
 
Number and sex 
Each treated and control group should contain a sufficient number of 
animals to yield about 20 pregnant females at or near term. 
The objective is to produce enough pregnancies and offspring to 
assure a meaningful evaluation of the potential of the substance to 
affect fertility, pregnancy and maternal behaviour in P generation 
animals and suckling, growth and development of the F  1 offspring 
from conception to weaning.  
1.6.3. T e s t c o n d i t i o n s 
Food and water should be provided ad libitum. Near parturition, 
pregnant females should be caged separately in delivery or maternity cages and may be provided with nesting materials.
 
1.6.3.1. D o s e l e v e l s 
At least three treated groups and a control group should be used. If a 
vehicle is used in administering the test substance, the control group should receive the vehicle in the highest volume used. If a test substance causes reduced dietary intake or utilisation, then the use 
of a paired fed control group may be considered necessary. Ideally, 
unless limited by the physical/chemical nature or biological effects of the test substance, the highest dose level should induce toxicity but not mortality in the parental (P) animals. The intermediate dose(s) 
should induce minimal toxic effects attributable to the test substance, 
and the low dose should not induce any observable adverse effects on the parents or offspring. When administered by gavage or capsule the dosage given to each animal should be based on the individual 
animal's body weight and adjusted weekly for changes in body 
weight. For females during pregnancy, dosages may be based on the body weight at day 0 or 6 of the pregnancy, if desired.
 
1.6.3.2. L i m i t t e s t 
In the case of substances of low toxicity, if a dose level of at least 
1 000 mg/kilogram produces no evidence of interference with repro ­
ductive performance, studies at other dose levels may not be considered necessary. If a preliminary study at the high-dose level, with definite evidence of maternal toxicity, shows no adverse effects 
on fertility, studies at other dose levels may not be considered 
necessary.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 507
 
1.6.3.3.  Performance of the test  
E x p e r i m e n t a l s c h e d u l e s 
Daily dosing of the parental (P) males should begin when they are 
about five to nine weeks of age, after they have been weaned and acclimatised for at least five days. In rats, dosing is continued for 10 
weeks prior to the mating period (for mice, eight weeks). Males 
should be killed and examined either at the end of the mating period or, alternatively, males may be retained on the test diet for the possible production of a second litter and should be killed and 
examined at some time before the end of the study. For parental (P) 
females dosing should begin after at least five days of acclimati ­
sation and continue for at least two weeks prior to mating. Daily dosing of the p females should continue throughout the three-week 
mating period, pregnancy and up to the weaning of the F
 l offspring. 
Consideration should be given to modification of the dosing schedule based on other available information on the test substance, such as induction of metabolism or bioaccumulation.
 
M a t i n g p r o c e d u r e 
Either 1:1 (one male to one female) or 1:2 (one male to two females) 
mating may be used in reproduction toxicity studies. 
Based on 1:1 mating, one female should be placed with the same 
male until pregnancy occurs or three weeks have elapsed. Each morning the females should be examined for presence of sperm or vaginal plugs. Day 0 of pregnancy is defined as the day a vaginal 
plug or sperm is found. 
Those pairs that fail to mate should be evaluated to determine the 
cause of the apparent infertility. 
This may involve such procedures as providing additional oppor ­
tunities to mate with other proven sires or dams, microscopic exam ­
ination of the reproductive organs, and examination of the oestrous cycle or spermatogenesis.
 
L i t t e r s i z e s 
Animals dosed during the fertility study are allowed to litter 
normally and rear their progency to the stage of weaning without 
standardisation of litters. 
Where standardisation is done, the following procedure is suggested. 
Between day 1 and day 4 after birth, the size of each litter may be 
adjusted by eliminating extra pups by selection to yield, as nearly as 
possible, four males and four females per litter. 
Whenever the number of male or female pups prevents having four 
of each sex per litter, partial adjustment (for example, five males and 
three females) is acceptable. Adjustments are not applicable for 
litters of less than eight pups.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 508
 
1.6.4. O b s e r v a t i o n s 
Throughout the test period, each animal should be observed at least 
once daily. Pertinent behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity, including mortality, 
should be recorded. During pre-mating and mating periods, food 
consumption may be measured daily. After parturition and during lactation, food consumption measurements (and water consumption measurements when the test substance is administered in the 
drinking water) should be made on the same day as the weighing 
of the litter. P males and females should be weighed on the first day of dosing and weekly thereafter. These observations should be 
reported individually for each adult animal. 
The duration of gestation should be calculated from day 0 of preg ­
nancy. Each litter should be examined as soon as possible after 
delivery to establish the number and sex of pups, still births, live births and the presence of gross anomalies. 
Dead pups and pups sacrificed at day 4 should be preserved and 
studied for possible defects. Live pups should be counted and litters weighed on the morning after birth and on days 4 and 7 and weekly 
thereafter until the termination of the study, when animals should be 
weighed individually. 
Physical or behavioural abnormalities observed in the dams or 
offspring should be recorded.
 
1.6.5. P a t h o l o g y  
1.6.5.1. N e c r o p s y 
At the time of sacrifice or death during the study the animals of the 
P generation should be examined macroscopically for any structural abnormalities or pathological changes, with special attention being 
paid to the organs of the reproductive system. Dead or moribund 
pups should be examined for defects.
 
1.6.5.2. H i s t o p a t h o l o g y 
The ovaries, uterus, cervix, vagina, testes, epididymes, seminal 
vesicles, prostate, coagulating gland, pituitary gland and target 
organ(s) of all P animals should be preserved for microscopic exam ­
ination. In the event that these organs have not been examined in other multiple-dose studies, they should be microscopically 
examined in all high-dose and control animals and animals which 
die during the study where practicable. 
Organs showing abnormalities in these animals should then be 
examined in all other P animals. In these instances, microscopic examination should be made of all tissues showing gross patho ­
logical changes. As suggested under mating procedures, reproductive 
organs of animals suspected of infertility may be subjected to micro ­
scopic examination.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 509
 
2. DATA  
Data may be summarised in tabular form, showing for each test 
group the number of animals at the start of the test, the number of fertile males, the number of pregnant females, the types of changes 
and the percentage of animals displaying each type of change. 
When possible, numerical results should be evaluated by an appro ­
priate statistical method. Any generally accepted statistical method may be used.
 
3. REPORTING  
TEST REPORT 
The test report shall, if possible, contain the following information: 
— species/strain used, — toxic response data by sex and dose, including fertility, gestation 
and viability, 
— time of death during the study or whether animals survived to 
time of scheduled sacrifice or to termination of the study, 
— table presenting the weights of each litter, the mean: pup weights 
and the individual weights of the pups at termination, 
— toxic or other effects on reproduction, offspring and postnatal 
growth, 
— the day of observation of each abnormal sign and its subsequent 
course, 
— bodyweight data for P animals, — necropsy findings, — a detailed description of all microscopic findings, — statistical treatment of results, where appropriate, — discussion of the results, — interpretation of the results.
 
3.2. EVALUATION AND INTERPRETATION 
See General introduction Part B.  
4. REFERENCES  
See General introduction Part B.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 510
 
B.35.  TWO-GENERATION REPRODUCTION TOXICITY STUDY  
1. METHOD  
This method is a replicate of the OECD TG 416 (2001).  
1.1. INTRODUCTION 
This method for two-generation reproduction testing is designed to 
provide general information concerning the effects of a test substance 
on the integrity and performance of the male and female repro ­
ductive systems, including gonadal function, the oestrus cycle, mating behaviour, conception, gestation, parturition, lactation, and 
weaning, and the growth and development of the offspring. The 
study may also provide information about the effects of the test substance on neonatal morbidity, mortality, and preliminary data on prenatal and postnatal developmental toxicity and serve as a 
guide for subsequent tests. In addition to studying growth and devel ­
opment of the F1 generation, this test method is also intended to assess the integrity and performance of the male and female repro ­
ductive systems as well as growth and development of the F2 gener ­
ation. For further information on developmental toxicity and func ­
tional deficiencies, either additional study segments can be incor ­
porated into this protocol, consulting the methods for developmental toxicity and/or developmental neurotoxicity as appropriate, or these 
endpoints could be studied in separate studies, using the appropriate 
test methods.
 
1.2. PRINCIPLE OF THE TEST METHOD 
The test substance is administered in graduated doses to several 
groups of males and females. Males of the P generation should be dosed during growth and for at least one complete spermatogenetic 
cycle (approximately 56 days in the mouse and 70 days in the rat) in 
order to elicit any adverse effects on spermatogenesis. Effects on sperm are determined by a number of sperm parameters (e.g. sperm morphology and motility) and in tissue preparation and 
detailed histopathology. If data on spermatogenesis are available 
from a previous repeated dose study of sufficient duration, e.g. a 90-day study, males of the P generation need not be included in the evaluation. It is recommended, however, that samples or digital 
recordings of sperm of the P generation are saved, to enable later 
evaluation. Females of the P generation should be dosed during growth and for several complete oestrus cycles in order to detect 
any adverse effects on oestrus cycle normality by the test substance. 
The test substance is administered to parental (P) animals during their mating, during the resulting pregnancies, and through the weaning of their F1 offspring. At weaning the administration of 
the substance is continued to F1 offspring during their growth into 
adulthood, mating and production of an F2 generation, until the F2 generation is weaned. 
Clinical observations and pathological examinations are performed 
on all animals for signs of toxicity with special emphasis on effects 
on the integrity and performance of the male and female repro ­
ductive systems and on the growth and development of the offspring.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 511
 
1.3. DESCRIPTION OF THE TEST METHOD  
1.3.1.  Selection of animal species  
The rat is the preferred species for testing. If other species are used, 
justification should be given and appropriate modifications will be 
necessary. Strains with low fecundity or well-known high incidence 
of developmental defects should not be used. At the commencement of the study, the weight variation of animals used should be minimal and not exceed 20 % of the mean weight of each sex.
 
1.3.2.  Housing and feeding conditions  
The temperature in the experimental animal room should be 22  o C (± 3  o ). 
Although the relative humidity should be at least 30 % and preferably not exceed 70 % other than during room cleaning, the aim should be 
50-60 %. Lighting should be artificial, the sequence being 12 hours 
light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. The choice of diet may be influenced by the need to ensure a suitable admixture of a test 
substance when administered by this method. 
Animals may be housed individually or be caged in small groups of 
the same sex. Mating procedures should be carried out in cages 
suitable for the purpose. After evidence of copulation, mated 
females shall be single-caged in delivery or maternity cages. Mated rats may also be kept in small groups and separated one or two days prior to parturition. Mated animals shall be provided with 
appropriate and defined nesting materials when parturition is near.
 
1.3.3.  Preparation of animals  
Healthy young animals, which have been acclimated to laboratory 
conditions for at least five days and have not been subjected to 
previous experimental procedures, should be used. The test animals 
should be characterised as to species, strain, source, sex, weight and/or age. Any sibling relationships among the animals should be known so that mating of siblings is avoided. The animals should be 
randomly assigned to the control and treated groups (stratification by 
body weight is recommended). Cages should be arranged in such a way that possible effects due to cage placement are minimised. Each animal should be assigned a unique identification number. For the P 
generation, this should be done before dosing starts. For the F1 
generation, this should be done at weaning for animals selected for mating. Records indicating the litter of origin should be maintained for all selected F1 animals. In addition, individual identification of 
pups as soon after birth as possible is recommended when individual 
weighing of pups or any functional tests are considered. 
Parental (P) animals shall be about five to nine weeks old at the start 
of dosing. The animals of all test groups shall, as nearly as prac ­
ticable, be of uniform weight and age.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 512
 
1.4. PROCEDURE  
1.4.1.  Number and sex of animals  
Each test and control group should contain a sufficient number of 
animals to yield preferably not less than 20 pregnant females at or 
near parturition. For substances that cause undesirable treatment 
related effects (e.g. sterility, excessive toxicity at the high dose) this may not be possible. The objective is to produce enough preg ­
nancies to assure a meaningful evaluation of the potential of the 
substance to affect fertility, pregnancy and maternal behaviour and 
suckling, growth and development of the F1 offspring from conception to maturity, and the development of their offspring 
(F2) to weaning. Therefore, failure to achieve the desired number 
of pregnant animals (i.e. 20) does not necessarily invalidate the study and should be evaluated on a case-by-case basis.
 
1.4.2.  Preparation of doses  
It is recommended that the test substance be administered orally (by 
diet, drinking water or gavage) unless another route of administration (e.g. dermal or inhalation) is considered more appropriate. 
Where necessary, the test substance is dissolved or suspended in a 
suitable vehicle. It is recommended that, wherever possible, the use 
of an aqueous solution/suspension be considered first, followed by 
consideration of a solution/emulsion in oil (e.g. corn oil) and then by possible solution in other vehicles. For vehicles other than water, the toxic characteristics of the vehicle must be known. The stability of 
the test substance in the vehicle should be determined.
 
1.4.3.  Dosage  
At least three dose levels and a concurrent control shall be used. 
Unless limited by the physical-chemical nature or biological effects 
of the test substance, the highest dose level should be chosen with the aim to induce toxicity but not death or severe suffering. In case of unexpected mortality, studies with a mortality rate of less than 
approximately 10 % in the parental (P) animals would normally still 
be acceptable. A descending sequence of dose levels should be selected with a view to demonstrating any dosage related effect and no-observed-adverse-effects levels (NOAEL). Two to four fold 
intervals are frequently optimal for setting the descending dose levels 
and addition of a fourth test group is often preferable to using very large intervals (e.g. more than a factor of 10) between dosages. For the dietary studies the dose interval should be not more than three 
fold. Dose levels should be selected taking into account any existing 
toxicity data, especially results from repeated dose studies. Any available information on metabolism and kinetics of the test compound or related materials should also be considered. In 
addition, this information will also assist in demonstrating the 
adequacy of the dosing regimen.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 513
 
The control group shall be an untreated group or a vehicle-control 
group if a vehicle is used in administering the test substance. Except 
for treatment with the test substance, animals in the control group should be handled in an identical manner to the test group subjects. If a vehicle is used, the control group shall receive the vehicle in the 
highest volume used. If a test substance is administered in the diet, 
and causes reduced dietary intake or utilisation, then the use of a pair-fed control group may be considered necessary. Alternatively data from controlled studies designed to evaluate the effects of 
decreased food consumption on reproductive parameters may be 
used in lieu of a concurrent pair-fed control group. 
Consideration should be given to the following characteristics of 
vehicle and other additives: effects on the absorption, distribution, 
metabolism, or retention of the test substance; effects on the 
chemical properties of the test substance which may alter its toxic characteristics; and effects on the food or water consumption or the 
nutritional status of the animals.
 
1.4.4.  Limit test  
If an oral study at one dose level of at least 1 000 mg/kg body 
weight/day or, for dietary or drinking water administration, an equivalent percentage in the diet or drinking water using the 
procedures described for this study, produces no observable toxic 
effects in either parental animals or their offspring and if toxicity would not be expected based upon data from structurally and/or metabolically related compounds, then a full study using several 
dose levels may not be considered necessary. The limit test applies 
except when human exposure indicates the need for a higher oral dose level to be used. For other types of administration, such as inhalation or dermal application, the physical-chemical properties 
of the test substance, such as solubility, often may indicate and 
limit the maximum attainable level of exposure.
 
1.4.5.  Administration of doses  
The animals should be dosed with the test substance on a 7-days per 
week basis. The oral route of administration (diet, drinking water, or 
gavage) is preferred. If another route of administration is used, 
justification shall be provided, and appropriate modifications may be necessary. All animals shall be dosed by the same method during the appropriate experimental period. When the test 
substance is administered by gavage, this should be done using a 
stomach tube. The volume of liquid administered at one time should not exceed 1 ml/100 g body weight (0,4 ml/100 g body weight is the maximum for corn oil), except in the case of aqueous solutions 
where 2 ml/100 g body weight may be used. Except for irritant or 
corrosive substances, which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting the concentration to ensure a constant 
volume at all dose levels. In gavage studies, the pups will 
normally only receive test substance indirectly through the milk, until direct dosing commences for them at weaning. In diet or drinking water studies, the pups will additionally receive test 
substance directly when they commence eating for themselves 
during the last week of the lactation period.
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For substances administered via the diet or drinking water, it is 
important to ensure that the quantities of the test substance 
involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (ppm) or a constant dose level in 
terms of the body weight of the animal may be used; the alternative 
used must be specified. For a substance administered by gavage, the dose should be given at similar times each day, and adjusted at least weekly to maintain a constant dose level in terms of animal body 
weight. Information regarding placental distribution should be 
considered when adjusting the gavage dose based on weight.
 
1.4.6.  Experimental schedules  
Daily dosing of the parental (P) males and females shall begin when 
they are five to nine weeks old. Daily dosing of the F1 males and females shall begin at weaning; it should be kept in mind that in cases of test substance administration via diet or drinking water, 
direct exposure of the F1 pups to the test substance may already 
occur during the lactation period. For both sexes (P and F1), dosing shall be continued for at least 10 weeks before the mating period. Dosing is continued in both sexes during the two week mating 
period. Males should be humanely killed and examined when they 
are no longer needed for assessment of reproductive effects. For parental (P) females, dosing should continue throughout pregnancy 
and up to the weaning of the F1 offspring. Consideration should be 
given to modifications in the dosing schedule based on available information on the test substance, including existing toxicity data, induction of metabolism or bioaccumulation. The dose to each 
animal should normally be based on the most recent individual 
body weight determination. However, caution should be exercised when adjusting the dose during the last trimester of pregnancy. 
Treatment of the P and F1 males and females shall continue until 
termination. All P and F1 adult males and females should be 
humanely killed when they are no longer needed for assessment of 
reproductive effects. F1 offspring not selected for mating and all F2 offspring should be humanely killed after weaning.
 
1.4.7.  Mating procedure  
1.4.7.1.  Parental (P) mating  
For each mating, each female shall be placed with a single male 
from the same dose level (1:1 mating) until copulation occurs or twq 
weeks have elapsed. Each day, the females shall be examined for 
presence of sperm or vaginal plugs. Day 0 of pregnancy is defined as the day a vaginal plug or sperm are found. In case pairing is unsuc ­
cessful, re-mating of females with proven males of the same group 
could be considered. Mating pairs should be clearly identified in the 
data. Mating of siblings should be avoided.
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1.4.7.2.  F1 mating  
For mating the F1 offspring, at least one male and one female should 
be selected at weaning from each litter for mating with other pups of the same dose level but different litter, to produce the F2 generation. 
Selection of pups from each litter should be random when no 
significant differences are observed in body weight or appearance between the litter mates. In case these differences are observed, the best representatives of each litter should be selected. Pragmatically, 
this is best done on a body weight basis but it may be more appro ­
priate on the basis of appearance. The F1 offspring should not be mated until they have attained full sexual maturity. 
Pairs without progeny should be evaluated to determine the apparent 
cause of the infertility. This may involve such procedures as 
additional opportunities to mate with other proven sires or dams, 
microscopic examination of the reproductive organs, and exam ­
ination of the oestrous cycles or spermatogenesis.
 
1.4.7.3.  Second mating  
In certain instances, such as treatment-related alterations in litter size 
or the observation of an equivocal effect in the first mating, it is recommended that the P or F1 adults be remated to produce a second 
litter. It is recommended to remate females or males, which have not 
produced a litter with proven breeders of the opposite sex. If production of a second litter is deemed necessary in either gener ­
ation, animals should be remated approximately one week after 
weaning of the last litter.
 
1.4.7.4.  Litter size  
Animals shall be allowed to litter normally and rear their offspring to 
weaning. Standardisation of litter sizes is optional. When standard ­
isation is done, the method used should be described in detail.  
1.5. OBSERVATIONS  
1.5.1.  Clinical observations  
A general clinical observation should be made each day and, and in 
the case of gavage dosing its timing should take into account the anticipated peak period of effects after dosing. Behavioural changes, 
signs of difficult or prolonged parturition and all signs of toxicity 
should be recorded. An additional, more detailed examination of each animal should be conducted on at least a weekly basis and could conveniently be performed on an occasion when the animal 
is weighed. Twice daily, during the weekend once daily when appro ­
priate, all animals should be observed for morbidity and mortality.
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1.5.2.  Body weight and food/water consumption of parent animals  
Parental animals (P and Fl) shall be weighed on the first day of 
dosing and at least weekly thereafter. Parental females (P and F1) shall be weighed at a minimum on gestation days 0, 7, 14, and 20 or 
21, and during lactation on the same days as the weighing of litters 
and on the day the animals are killed. These observations should be reported individually for each adult animal. During the premating and gestation periods food consumption shall be measured weekly 
at a minimum. Water consumption shall be measured weekly at a 
minimum if the test substance is administered in the water.
 
1.5.3.  Oestrus cycle  
Estrous cycle length and normality are evaluated in P and F1 females 
by vaginal smears prior to mating, and optionally during mating, 
until evidence of mating is found. When obtaining vaginal/cervical 
cells, care should be taken to avoid disturbance of mucosa and subsequently, the induction of pseudopregnancy (1).
 
1.5.4.  Sperm parameters  
For all P and F1 males at termination, testis and epididymis weight 
shall be recorded and one of each organ reserved for histopath ­
ological examination (see Section 1.5.7, 1.5.8.1). Of a subset of at 
least 10 males of each group of P and F1 males, the remaining testes and epididymides should be used for enumeration of homogeni ­
sation-resistant spermatids and cauda epididymal sperm reserves, 
respectively. For this same subset of males, sperm from the cauda 
epididymides or vas deferens should be collected for evaluation of sperm motility and sperm morphology. If treatment-related effects are observed or when there is evidence from other studies of 
possible effects on spermatogenesis, sperm evaluation should be 
conducted in all males in each dose group; otherwise enumeration may be restricted to control and high-dose P and F1 males. 
The total number of homogenisation-resistant testicular spermatids 
and cauda epididymal sperm should be enumerated (2)(3). Cauda 
sperm reserves can be derived from the concentration and volume of sperm in the suspension used to complete the qualitative evalu ­
ations, and the number of sperm recovered by subsequent mincing 
and/or homogenising of the remaining cauda tissue. Enumeration 
should be performed on the selected subset of males of all dose groups immediately after killing the animals unless video or digital recordings are made, or unless the specimens are freezed and 
analysed later. In these instances, the controls and high dose group 
may be analysed first. If no treatment-related effects (e.g. effects on sperm count, motility, or morphology) are seen the other dose groups need not be analysed. When treatment-related effects are noted in the 
high-dose group, then the lower dose groups should also be 
evaluated.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 517
 
Epididymal (or ductus deferens) sperm motility should be evaluated 
or video taped immediately after sacrifice. Sperm should be 
recovered while minimising damage, and diluted for motility analysis using acceptable methods (4). The percentage of progressively motile sperm should be determined either subjectively 
of objectively. When computer-assisted motion analysis is performed 
(5)(6)(7)(8)(9)(10) the derivation of progressive motility relies on user-defined thresholds for average path velocity and straightness or linear index. If samples are videotaped (11) or the images are 
otherwise recorded at the time of necropsy, subsequent analysis of 
only control and high-dose P and F1 males may be performed unless treatment-related effects are observed; in that case, the lower dose groups should also be evaluated. In the absence of a video or digital 
image, all samples in all treatment groups should be analysed at 
necropsy. 
A morphological evaluation of an epididymal (or  vas deferens ) 
sperm sample should be performed. Sperm (at least 200 per 
sample) should be examined as fixed, wet preparations (12) and 
classified as either normal or abnormal. Examples of morphologic sperm abnormalities would include fusion, isolated heads, and misshapen heads and/or tails. Evaluation should be performed on 
the selected subset of males of all dose groups either immediately 
after killing the animals, or, based on the video or digital recordings, at a later time. Smears, once fixed, can also be read at a later time. In these instances, the controls and high dose group may be analysed 
first. If no treatment-related effects (e.g. effects on sperm 
morphology) are seen the other dose groups need not be analysed. When treatment-related effects are noted in the high-dose group, then the lower dose groups should also be evaluated. 
If any of the above sperm evaluation parameters have already been 
examined as part of a systemic toxicity study of at least 90 days, they need not necessarily be repeated in the two-generation study. It is recommended, however, that samples or digital recordings of 
sperm of the P generation are saved, to enable later evaluation, if 
necessary.
 
1.5.5.  Offspring  
Each litter should be examined as soon as possible after delivery 
(lactation day 0) to establish the number and sex of pups, stillbirths, live births, and the presence of gross anomalies. Pups found dead on 
day 0, if not macerated, should preferably be examined for possible 
defects and cause of death and preserved. Live pups should be counted and weighed individually at birth (lactation day 0) or on day 1, and on regular weigh days thereafter, e.g. on days 4, 7, 14, 
and 21 of lactation. Physical or behavioural abnormalities observed 
in the dams or offspring should be recorded.
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Physical development of the offspring should be recorded mainly by 
body weight gain. Other physical parameters (e.g. ear and eye 
opening, tooth eruption, hair growth) may give supplementary information, but these data should preferably be evaluated in the context of data on sexual maturation (e.g. age and body weight at 
vaginal opening or balano-preputial separation) (13). Functional 
investigations (e.g. motor activity, sensory function, reflex ontogeny) of the F1 offspring before and/or after weaning, particularly those related to sexual maturation, are recommended if 
such investigations are not included in separate studies. The age of 
vaginal opening and preputial separation should be determined for F1 weanlings selected for mating. Anogenital distance should be measured at postnatal day 0 in F2 pups if triggered by alterations 
in F1 sex ratio or timing of sexual maturation. 
Functional observations may be omitted in groups that otherwise 
reveal clear signs of adverse effects (e.g. significant decrease in weight gain, etc.). If functional investigations are made, they should not be done on pups selected for mating.
 
1.5.6.  Gross necropsy  
At the time of termination or death during the study, all parental 
animals (P and F1), all pups with external abnormalities or clinical signs, as well as one randomly selected pup/sex/litter from both the F1 and F2 generation, shall be examined macroscopically for any 
structural abnormalities or pathological changes. Special attention 
should be paid to the organs of the reproductive system. Pups that are humanely killed in a moribund condition and dead pups, when not macerated, should be examined for possible defects and/or cause 
of death and preserved. 
The uteri of all primiparous females should be examined, in a 
manner which does not compromise histopathological evaluation, for the presence and number of implantation sites.
 
1.5.7.  Organ weights  
At the time of termination, body weight and the weight of the 
following organs of all P and F1 parental animals shall be 
determined (paired organs should be weighed individually): 
— uterus, ovaries, — testes, epididymides (total and cauda), — prostate, — seminal vesicles with coagulating glands and their fluids and 
prostate (as one unit), 
— brain, liver, kidneys, spleen, pituitary, thyroid and adrenal glands 
and known target organs. 
Terminal body weights should be determined for F1 and F2 pups 
that are selected for necropsy. The following organs from the one 
randomly selected pup/sex/litter (see Section 1.5.6) shall be weighed: 
Brain, spleen and thymus.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 519
 
Gross necropsy and organ weight results should be assessed in 
context with observations made in other repeated dose studies, 
when feasible.  
1.5.8.  Histopathology  
1.5.8.1.  Parental animals  
The following organs and tissues of parental (P and F1) animals, or 
representative samples thereof, shall be fixed and stored in a suitable medium for histopathological examination. 
— Vagina, uterus with cervix, and ovaries (preserved in appropriate 
fixative), 
— one testis (preserved in Bouin's or comparable fixative), one 
epididymis, seminal vesicles, prostate, and coagulating gland, 
— previously identified target organ(s) from all P and F1 animals 
selected for mating. 
Full histopathology of the preserved organs and tissues listed above 
should be performed for all high dose and control P and F1 animals selected for mating. Examination of the ovaries of the P animals is 
optional. Organs demonstrating treatment-related changes should also 
be examined in the low- and mid-dose groups to aid in the eluci ­
dation of the NOAEL. Additionally, reproductive organs of the low- and mid-dose animals suspected of reduced fertility, e.g. those that 
failed to mate, conceive, sire, or deliver healthy offspring, or for 
which oestrus cyclicity or sperm number, motility, or morphology were affected, should be subjected to histopathological evaluation. All gross lesions such as atrophy or tumours shall be examined. 
Detailed testicular histopathological examination (e.g. using Bouin's 
fixative, paraffin embedding and transverse sections of 4-5 μm 
thickness) should be conducted in order to identify treatment- 
related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of sperma ­
togenic cells into the lumen (14). Examination of the intact 
epididymis should include the caput, corpus, and cauda, which can 
be accomplished by evaluation of a longitudinal section. The epididymis should be evaluated for leukocyte infiltration, change in prevalence of cell types, aberrant cell types, and phagocytosis 
of sperm. PAS and haematoxylin staining may be used for exam ­
ination of the male reproductive organs. 
The postlactational ovary should contain primordial and growing 
follicles as well as the large corpora lutea of lactation. Histopath ­
ological examination should detect qualitative depletion of the primordial follicle population. A quantitative evaluation of primordial 
follicles should be conducted for F1 females; the number of animals, 
ovarian section selection, and section sample size should be statistically appropriate for the evaluation procedure used. Examination should include enumeration of the number of primordial follicles, which can 
be combined with small growing follicles, for comparison of treated 
and control ovaries (15)(16)(17)(18)(19).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 520
 
1.5.8.2.  Weanlings  
Grossly abnormal tissue and target organs from all pups with 
external abnormalities or clinical signs, as well as from the one randomly selected pup/sex/litter from both the F1 and F2 generation 
which have not been selected for mating, shall be fixed and stored in 
a suitable medium for histopathological examination. Full histopath ­
ological characterisation of preserved tissue should be performed with special emphasis on the organs of the reproductive system.
 
2. DATA  
2.1. TREATMENT OF RESULTS 
Data shall be reported individually and summarised in tabular form, 
showing for each test group and each generation the number of 
animals at the start of the test, the number of animals found dead during the test or killed for humane reasons, the time of any death or humane kill, the number of fertile animals, the number of pregnant 
females, the number of animals showing signs of toxicity, a 
description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects, the types of parental and offspring observations, the types of histopathological 
changes, and all relevant litter data. 
Numerical results should be evaluated by an appropriate, generally 
accepted statistical method; the statistical methods should be selected as part of the design of the study and should be justified. Dose- response statistical models may be useful for analysing data. The 
report should include sufficient information on the method of 
analysis and the computer program employed, so that an independent reviewer/statistician can re-evaluate and reconstruct the analysis.
 
2.2. EVALUATION OF RESULTS 
The findings of this two-generation reproduction toxicity study 
should be evaluated in terms of the observed effects including necropsy and microscopic findings. The evaluation will include the 
relationship, or lack thereof, between the dose of the test substance 
and the presence or absence, incidence and severity of abnormalities, including gross lesions, identified target organs, affected fertility, clinical abnormalities, affected reproductive and litter performance, 
body weight changes, effects on mortality and any other toxic 
effects. The physico-chemical properties of the test substance, and when available, toxicokinetics data should be taken into consideration when evaluating test results. 
A properly conducted reproduction toxicity test should provide a 
satisfactory estimation of a no-effect level and an understanding of 
adverse effects on reproduction, parturition, lactation, postnatal 
development including growth and sexual development.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 521
 
2.3. INTERPRETATION OF RESULTS 
A two-generation reproduction toxicity study will provide 
information on the effects of repeated exposure to a substance during all phases of the reproductive cycle. In particular, the study 
provides information on the reproductive parameters, and on devel ­
opment, growth, maturation and survival of offspring. The results of the study should be interpreted in conjunction with the findings from subchronic, prenatal developmental and toxicokinetic and other 
available studies. The results of this study can be used in 
assessing the need for further testing of a chemical. Extrapolation of the results of the study to man is valid to a limited degree. They 
are best used to provide information on no-effect-levels and 
permissible human exposure (20)(21)(22)(23).
 
3. REPORTING  
3.1. TEST REPORT 
The test report must include the following information: 
Test substance: 
— physical nature and, where relevant, physicochemical properties, — identification data, 
— purity. 
Vehicle (if appropriate): — ustification for choice of vehicle if other than water.  
Test animals:  
— species/strain used, — number, age and sex of animals, 
— source, housing conditions, diet, nesting materials, etc., 
— individual weights of animals at the start of the test.  
Test conditions:  
— rationale for dose level selection, 
— details of test substance formulation/diet preparation, achieved 
concentrations, 
— stability and homogeneity of the preparation, 
— details of the administration of the test substance, 
— conversion from diet/drinking water test substance concentration 
(ppm) to the achieved dose (mg/kg body weight/day), if appli ­
cable, 
— details of food and water quality.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 522
 
Results: 
— food consumption, and water consumption if available, food effi ­
ciency (body weight gain per gram of food consumed), and test material consumption for P and F1 animals, except for the period of cohabitation and for at least the last third of lactation, 
— absorption data (if available), 
— body weight data for P and F1 animals selected for mating, 
— litter and pup weight data, 
— body weight at sacrifice and absolute and relative organ weight 
data for the parental animals, 
— nature, severity and duration of clinical observations (whether 
reversible or not), 
— time of death during the study or whether animals survived to 
termination, 
— toxic response data by sex and dose, including indices of mating, 
fertility, gestation, birth, viability, and lactation; the report should indicate the numbers used in calculating these indices, 
— toxic or other effects on reproduction, offspring, post-natal 
growth, etc., 
— necropsy findings, 
— detailed description of all histopathological findings, — number of P and F1 females cycling normally and cycle length, 
— total cauda epididymal sperm number, percent progressively 
motile sperm, percent morphologically normal sperm, and percent of sperm with each identified abnormality, 
— time-to-mating, including the number of days until mating, 
— gestation length, — number of implantations, corpora lutea, litter size, 
— number of live births and post-implantation loss, 
— number of pups with grossly visible abnormalities, if determined 
the number of runts should be reported, 
— data on physical landmarks in pups and other post natal devel ­
opmental data, physical landmarks evaluated should be justified, 
— data on functional observations in pups and adults, as applicable, 
— statistical treatment of results, where appropriate.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 523
 
Discussion of results. 
Conclusions, including NOAEL values for maternal and offspring 
effects.  
4. REFERENCES  
(1) Sadleir, R.M.F.S., (1979) Cycles and Seasons, In:  Repro­
duction in Mammals:  I. Germ Cells and Fertilisation, C.R. 
Auston and R.V. Short (eds.), Cambridge, New York. 
(2) Gray, L.E. et al., (1989) A Dose-Response Analysis of Metho ­
xychlor-Induced Alterations of Reproductive Development and 
Function in the Rat.  Fundamental and Applied Toxicology , 12, 
p. 92-108. 
(3) Robb, G.W. et al., (1978). Daily Sperm Production and 
Epididymal Sperm Reserves of Pubertal and Adult Rats.  
Journal of Reproduction and Fertility  54:103-107. 
(4) Klinefelter, G.R. et al., (1991) The Method of Sperm 
Collection Significantly Influences Sperm Motion Parameters Following Ethane Dimethanesulfonate Administration in the 
Rat. Reproductive Toxicology , 5, p. 39-44. 
(5) Seed, J. et al., (1996). Methods for Assessing Sperm Motility, 
Morphology, and Counts in the Rat, Rabbit, and Dog: a 
Consensus Report.  Reproductive Toxicology , 10(3), 
p. 237-244. 
(6) Chapin, R.E. et al., (1992) Methods for Assessing Rat Sperm 
Motility.  Reproductive Toxicology , 6, p. 267-273 
(7) Klinefelter, G.R. et al., (1992) Direct Effects of Ethane Dime ­
thanesulphonate on Epididymal Function in Adult Rats: an  In 
Vitro  Demonstration.  Journal of Andrology , 13, p. 409-421. 
(8) Slott, V.L. et al., (1991) Rat Sperm Motility Analysis: Method ­
ologic Considerations.  Reproductive Toxicology , 5, p. 449-458. 
(9) Slott, V.L. and Perreault, S.D., (1993) Computer-Assisted 
Sperm Analysis of Rodent Epididymal Sperm Motility Using 
the Hamilton-Thorn Motility Analyzer. In:  Methods in Toxi ­
cology , Part A., Academic, Orlando, Florida, p. 319-333. 
(10) Toth, G.P. et al., (1989) The Automated Analysis of Rat 
Sperm Motility Following Subchronic Epichlorhydrin Admin ­
istration: Methodologic and Statistical Considerations. Journal of Andrology, 10, p. 401-415. 
(11) Working, P.K. and M. Hurtt, (1987) Computerised Video ­
micrographic Analysis of Rat Sperm Motility. Journal of Andrology, 8, p. 330-337. 
(12) Linder, R.E. et al., (1992) Endpoints of Spermatoxicity in the 
Rat After Short Duration Exposures to Fourteen Reproductive Toxicants. Reproductive Toxicology, 6, p. 491-505. 
(13) Korenbrot, C.C. et al., (1977) Preputial Separation as an 
External Sign of Pubertal Development in the Male Rat. Biological Reproduction, 17, p. 298-303. 
(14) Russell, L.D. et al., (1990) Histological and Histopathological 
Evaluation of the Testis, Cache River Press, Clearwater, Florida. 
(15) Heindel, J.J. and R.E. Chapin, (eds.) (1993) Part B. Female 
Reproductive Systems, Methods in Toxicology, Academic, Orlando, Florida. 
(16) Heindel, J.J. et al., (1989) Histological Assessment of Ovarian 
Follicle Number in Mice As a Screen of Ovarian Toxicity. In: Growth Factors and the Ovary, A.N. Hirshfield (ed.), Plenum, 
New York, p. 421-426. 
(17) Manson, J.M. and Y.J. Kang, (1989) Test Methods for 
Assessing Female Reproductive and Developmental Toxi ­
cology. In: Principles and Methods of Toxicology, A.W. 
Hayes (ed.), Raven, New York.
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(18) Smith, B.J. et al., (1991) Comparison of Random and Serial 
Sections in Assessment of Ovarian Toxicity. Reproductive 
Toxicology, 5, p. 379-383. 
(19) Heindel, J.J., (1999) Oocyte Quantitation and Ovarian Histology. 
In: An Evaluation and Interpretation of Reproductive Endpoints for Human Health Risk Assessment, G. Daston,. and C.A. 
Kimmel, (eds.), ILSI Press, Washington, DC. 
(20) Thomas, J. A., (1991) Toxic Responses of the Reproductive 
System. In: Casarett and Doull's Toxicology, M.O. Amdur, J. Doull, and C.D. Klaassen (eds.), Pergamon, New York. 
(21) Zenick, H. and E.D. Clegg, (1989) Assessment of Male Repro ­
ductive Toxicity: A Risk Assessment Approach. In: Principles 
and Methods of Toxicology, A.W. Hayes (ed.), Raven Press, New York. 
(22) Palmer, A.K., (1981) In: Developmental Toxicology, Kimmel, 
C.A. and J. Buelke-Sam (eds.), Raven Press, New York. 
(23) Palmer, A.K., (1978) In Handbook of Teratology, Vol. 4, J.G. 
Wilson and F.C. Fraser (eds.), Plenum Press, New York.
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B.36.  TOXICOKINETICS  
INTRODUCTION 
1. This Test Method is equivalent to OECD TG 417 (2010). Studies examining 
the toxicokinetics (TK) of a test chemical are conducted to obtain adequate information on its absorption, distribution, biotransformation (i.e. metab ­
olism) and excretion, to aid in relating concentration or dose to the 
observed toxicity, and to aid in understanding its mechanism of toxicity. TK may help to understand the toxicology studies by demonstrating that the test animals are systemically exposed to the test chemical and by revealing 
which are the circulating moieties (parent chemical/metabolites). Basic TK 
parameters determined from these studies will also provide information on the potential for accumulation of the test chemical in tissues and/or organs and the potential for induction of biotransformation as a result of exposure 
to the test chemical. 
2. TK data can contribute to the assessment of the adequacy and relevance of 
animal toxicity data for extrapolation to human hazard and/or risk assessment. Additionally, toxicokinetic studies may provide useful information for determining dose levels for toxicity studies (linear vs. 
non-linear kinetics), route of administration effects, bioavailability, and 
issues related to study design. Certain types of TK data can be used in physiologically based toxicokinetic (PBTK) model development. 
3. There are important uses for metabolite/TK data such as suggesting possible 
toxicities and modes of action and their relation to dose level and route of 
exposure. In addition, metabolism data can provide information useful for assessing the toxicological significance of exposures to exogenously produced metabolites of the test chemical. 
4. Adequate toxicokinetic data will be helpful to support the further accepta ­
bility and applicability of quantitative structure-activity relationships, 
read-across or grouping approaches in the safety evaluation of chemicals. Kinetics data may also be used to evaluate the toxicological relevance of other studies (e.g.  in vivo /in vitro ). 
5. Unless another route of administration is mentioned (see in particular para ­
graphs 74-78), this Test Method is applicable to oral administration of the test chemical.
 
INITIAL CONSIDERATIONS 
6. Regulatory systems have different requirements and needs regarding the 
measurement of endpoints and parameters related to toxicokinetics for different classes of chemicals (e.g. pesticides, biocides, industrial chemicals). 
Unlike most Test Methods this Test Method describes toxicokinetics testing, 
which involves multiple measurements and endpoints. In the future, several new Test Methods, and/or guidance document(s), may be developed to describe each endpoint separately and in more detail. In the case of this 
Test Method, which tests or assessments are conducted is specified by the 
requirements and/or needs of each regulatory system.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 526
 
7. There are numerous studies that might be performed to evaluate the TK 
behaviour of a test chemical for regulatory purposes. However, depending 
on particular regulatory needs or situations, not all of these possible studies may be necessary for the evaluation of a test chemical. Flexibility, taking into consideration the characteristics of the test chemical being investigated, 
is needed in the design of toxicokinetic studies. In some cases, only a 
certain set of questions may need to be explored in order to address test chemical-associated hazard and risk concerns. In some situations, TK data can be collected as part of the evaluation in other toxicology studies. For 
other situations, additional and/or more extensive TK studies may be 
necessary, depending on regulatory needs and/or if new questions arise as part of test chemical evaluation. 
8. All available information on the test chemical and relevant metabolites and 
analogues should be considered by the testing laboratory prior to conducting the study in order to enhance study quality and avoid unnecessary animal use. This could include data from other relevant Test Methods ( in vivo  
studies,  in vitro  studies, and/or  in silico  evaluations). Physicochemical prop ­
erties, such as octanol-water partition coefficient (expressed as log P
 OW ), 
pKa, water solubility, vapour pressure, and molecular weight of a chemical 
may be useful for study planning and interpretation of results. They can be 
determined using appropriate methods as described in the relevant Test Methods.
 
LIMITATIONS 
9. This Test Method is not designed to address special circumstances, such as 
the pregnant or lactating animal and offspring, or to evaluate potential 
residues in exposed food-producing animals. However, the data obtained from a B.36 study can provide background information to guide the design of specific studies for these investigations. This Test Method is 
not intended for the testing of nanomaterials. A report on preliminary 
review of OECD Test Guidelines for their applicability to nanomaterials indicates that TG 417 (equivalent to this Test Method B.36) may not apply to nanomaterials (1).
 
DEFINITIONS 
10. Definitions used for the purpose of this Test Method are provided in 
Appendix.  
ANIMAL WELFARE CONSIDERATIONS 
11. Guidance on humane treatment of animals is available in OECD Guidance 
Document (GD) 19 (2). It is recommended that OECD GD 19 be consulted 
for all  in vivo  and in vitro  studies described in this Test Method.  
DESCRIPTION OF THE METHODS  
Pilot Studies  
12. The use of pilot studies is recommended and encouraged for the selection of 
experimental parameters for the toxicokinetics studies (e.g. metabolism, mass balance, analytical procedures, dose-finding, exhalation of CO
 2 , 
etc.). Characterisation of some of these parameters may not necessitate the 
use of radiolabelled chemicals.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 527
 
Animal Selection  
Species  
13. The animal species (and strain) used for TK testing should preferably be the 
same as that used in other toxicological studies performed with the test chemical of interest. Normally, the rat should be used as it has been used extensively for toxicological studies. The use of other or additional species 
may be warranted if critical toxicology studies demonstrate evidence of 
significant toxicity in these species or if their toxicity/toxicokinetics is shown to be more relevant to humans. Justification should be provided for the selection of the animal species and its strain. 
14. Unless mentioned otherwise, this Test Method refers to the rat as the test 
species. Certain aspects of the method might have to be modified for the use of other test species.
 
Age and Strain  
15. Young healthy adult animals (normally 6-12 weeks at the time of dosing) 
should be used (see also paragraphs 13 and 14). Justification should be provided for the use of animals that are not young adults. All animals should be of similar age at the outset of the study. The weight variation 
of individual animals should not exceed ± 20 % of the mean weight of the 
test group. Ideally, the strain used should be the same as that used in deriving the toxicological database for the test chemical.
 
Number and Sex of Animals  
16. A minimum of four animals of one sex should be used for each dose tested. 
Justification should be provided for the sex of the animals used. The use of 
both sexes (four males and four females) should be considered if there is evidence to support significant sex-related differences in toxicity.
 
Housing and feeding conditions  
17. Animals should generally be housed individually during the testing period. 
Group housing might be justified in special circumstances. Lighting should 
be artificial, the sequence being 12 h light/12 h dark. The temperature of the experimental animal room should be 22 °C (± 3 °C) and the relative humidity 30-70 %. For feeding, conventional laboratory diets may be 
used with an unlimited supply of drinking water.
 
Test Chemical  
18. A radiolabelled test chemical using  14 C should be used for all mass balance 
and metabolite identification aspects of the study; however, if it can be demonstrated that: 
— mass balance and metabolite identification can be adequately evaluated 
using the unlabelled test chemical, 
— the analytical specificity and sensitivity of the method used with 
non-radioactive test chemical is equal to or greater than that which 
could be obtained with the radiolabelled test chemical,
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 528
 
then a radiolabelled test chemical does not need to be used. Furthermore, 
other radioactive and stable isotopes may be used, particularly if the element 
is responsible for or is a part of the toxic portion of the test chemical. If possible, the radiolabel should be located in a core portion of the molecule which is metabolically stable (it is not exchangeable, is not removed meta ­
bolically as CO
 2 , and does not become part of the one-carbon pool of the 
organism). Labelling of multiple sites or specific regions of the molecule may be necessary to follow the metabolic fate of the test chemical. 
19. The radiolabelled and non-radiolabelled test chemicals should be analysed 
using appropriate methods to establish purity and identity. The radio-purity 
of the radioactive test chemical should be the highest attainable for a particular test chemical (ideally it should be greater than 95 %) and reasonable effort should be made to identify impurities present at or 
above 2 %. The purity, along with the identity and proportion of any 
impurities which have been identified, should be reported. Individual regu ­
latory programmes may choose to provide additional guidance to assist in the definition and specifications of test chemicals composed of mixtures and 
methods for determination of purity.
 
Dose Selection  
Pilot Study  
20. Usually a single oral dose is sufficient for the pilot study. The dose should 
be non-toxic, but high enough to allow for metabolite identification in 
excreta (and plasma, if appropriate) as well as to meet the stated purpose of the pilot study as noted in paragraph 12 of this Test Method.
 
Main Studies  
21. For the main studies, a minimum of two doses is preferred since information 
gathered from at least two dose groups may aid in dose setting in other 
toxicity studies, and help in the dose-response assessment of already 
available toxicity tests. 
22. Where two doses are administered, both doses should be high enough to 
allow for metabolite identification in excreta (and plasma, if appropriate). 
Information from available toxicity data should be considered for dose selection. If information is not available (e.g. from acute oral toxicity studies recording clinical signs of toxicity, or from repeated dose toxicity 
studies) a value for the higher dose that is below the LD
 50 (oral and dermal 
routes) or LC  50 (inhalation route) estimate or below the lower value of the 
acute toxicity range estimate may be considered. The lower dose should be some fraction of the higher dose. 
23. If only one dose level is investigated, ideally the dose should be high 
enough to allow for metabolite identification in excreta (and plasma, if appropriate), while not producing apparent toxicity. A rationale should be provided as to why no second dose level has been included. 
24. If the effect of dose on kinetic processes needs to be established, two doses 
may not be sufficient and at least one dose should be high enough so as to saturate these processes. If the area under the plasma concentration-time curve (AUC) is not linear between two dose levels used in the main 
study, this is a strong indication that saturation of one or more of the 
kinetic processes is occurring somewhere between the two dose levels.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 529
 
25. For test chemicals of low toxicity, a maximum dose of 1 000 mg/kg body 
weight (oral and dermal routes) should be used (if administration is by the 
inhalation route, refer to Chapter B.2 of this Annex for guidance; typically this dose would not exceed 2 mg/l). Chemical-specific considerations may necessitate a higher dose depending on regulatory needs. Dose selection 
should always be justified. 
26. Single dose toxicokinetic and tissue distribution data may be adequate to 
determine the potential for accumulation and/or persistence. However in 
some circumstances repeated dose administration may be needed (i) to 
address more fully the potential for accumulation and/or persistence or changes in TK (i.e. for instance, enzyme induction and inhibition), or (ii) as required by the applicable regulatory system. In studies involving 
repeated dosing, while repeated low dose administration is usually sufficient, 
under certain circumstances repeated high dose administration may also be necessary (see also paragraph 57).
 
Administration of Test Chemical  
27. The test chemical should be dissolved or suspended homogeneously in the 
same vehicle employed for the other oral gavage toxicity studies performed 
with the test chemical, if such vehicle information is available. Rationale for the choice of vehicle should be provided. The choice of the vehicle and the volume of dosing should be considered in the design of the study. The 
customary method of administration is by gavage; however, administration 
by gelatine capsule or as a dietary mixture may be advantageous in specific situations (in both cases, justification should be given). Verification of the actual dose administered to each animal should be provided. 
28. The maximum volume of liquid to be administered by oral gavage at one 
time depends on the size of the test animals, the type of dose vehicle, and 
whether or not feed is withheld prior to administration of the test chemical. 
The rationale for administering or restricting food prior to dosing should be provided. Normally the volume should be kept as low as practical for either aqueous or non-aqueous vehicles. Dose volumes should not normally exceed 
10 ml/kg body weight for rodents. Volumes of vehicles used for more 
lipophilic test chemicals might start at 4 ml/kg body weight. For repeated dosing, when daily fasting would be contraindicated, lower dose volumes (e.g. 2-4 ml/kg body weight) should be considered. Where possible, 
consideration may be given to the use of a dose volume consistent with 
that administered in other oral gavage studies for a test chemical. 
29. Intravenous (IV) administration of the test chemical and measurement of the 
test chemical in blood and/or excreta may be used to establish bioavail ­
ability or relative oral absorption. For the IV study, a single dose (usually equivalent to but not to exceed the lower oral dose – see dose selection) of test chemical is administered using an appropriate vehicle. This material 
should be administered in a suitable volume (e.g. 1 ml/kg bw) at the 
chosen site of administration to at least four animals of the appropriate sex (both sexes might be used, if warranted, see paragraph 16). A fully dissolved or suspended dose preparation is necessary for IV administration 
of the test chemical. The vehicle for IV administration should not interfere 
with blood integrity or blood flow. If the test chemical is infused, the
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 530
 
infusion rate should be reported and standardised between animals, provided 
an infusion pump is used. Anaesthesia should be used if one cannulates the 
jugular vein (for administration of test chemical and/or collection of blood) or if one uses the femoral artery for administration. Due consideration should be given to the type of anaesthesia as it may have effects on toxi ­
cokinetics. Animals should be allowed to recover adequately before admin ­
istration of the test chemical plus the vehicle. 
30. Other routes of administration, such as dermal and inhalation, (see para ­
graphs 74-78) may be applicable for certain test chemicals, considering their physico-chemical properties and the expected human use or exposure.
 
Measurements  
Mass Balance  
31. Mass balance is determined by summation of the percent of the administered 
(radioactive) dose excreted in urine, faeces, and expired air, and the percent present in tissues, residual carcass, and cage wash (see paragraph 46). Generally, total recoveries of administered test chemical (radioactivity) in 
the order of > 90 % are considered to be adequate.
 
Absorption  
32. An initial estimation of absorption can be achieved by excluding the 
percentage of dose in the gastro-intestinal (GI) tract and/or faeces from 
the mass balance determination. For the calculation of percent absorption, see paragraph 33. For investigation of excreta, see paragraphs 44-49. If the exact extent of absorption following oral dosing cannot be established from 
mass balance studies (e.g. where greater than 20 % of the administered dose 
is present in faeces), further investigations might be necessary. These studies could comprise either 1) oral administration of test chemical and measurement of test chemical in bile or 2) oral and IV administration of 
test chemical and measurement of net test chemical present in urine plus 
expired air plus carcass by each of the two routes. In either study design, measurement of radioactivity is conducted as a surrogate method for chemical-specific analysis of test chemical plus metabolites. 
33. If a biliary excretion study is undertaken, the oral route of administration is 
typically used. In this study, the bile ducts of at least four animals of the 
appropriate sex (or of both sexes, if warranted) should be cannulated and a single dose of the test chemical should be administered. Following adminis ­
tration of the test chemical, excretion of radioactivity/test chemical in bile 
should be monitored as long as necessary to estimate the percentage of the administered dose that is excreted via this route, which can be used to directly calculate the extent of oral absorption, as follows: 
Percent absorption ¼ ðamount in bile þ urine þ expired air þ 
carcass without GI tract contents Þ=amount administered  Ü 100 
34. With some classes of test chemical, direct secretion of the absorbed dose 
can occur across intestinal membranes. In such cases the measurement of % dose in faeces following an oral dose in the bile duct cannulated rat is not considered to be representative of the unabsorbed dose. It is recommended 
that where intestinal secretion is thought to occur then the % dose absorbed 
be based on the absorption calculated from a comparison of the excretion following the oral versus IV route (intact or bile duct cannulated rat) (see paragraph 35). It is also recommended that where quantification of the 
intestinal secretion is considered necessary, excretion in the bile duct 
cannulated rat following IV dose administration be measured.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 531
 
Bioavailability  
35. Bioavailability can be determined from plasma/blood kinetics of the oral and 
IV groups, as described in paragraphs 50-52, by specific chemical analysis of the test chemical and/or relevant metabolite(s), therefore not requiring 
radiolabelled test chemical. The calculation of bioavailability (F) of the test 
chemical or relevant metabolite(s) can then be made as follows: 
F ¼ ðAUC
 exp=AUC  IV Þ Ü ðDose  IV=Dose  exp Þ 
where AUC is the area under the plasma concentration-time curve, and exp 
is the experimental route (oral, dermal or via inhalation). 
36. For use in risk assessment of systemic effects, bioavailability of the toxic 
component is in general preferred over the percent absorption when 
comparing systemic concentrations from animal studies with analogous 
biomonitoring data from worker exposure studies. The situation may become more complex if doses are in the non-linear range so it is important that toxicokinetic screening determines doses in the linear range.
 
Tissue Distribution  
37. Knowledge of tissue distribution of a test chemical and/or its metabolites is 
important for the identification of target tissues, and understanding of the 
underlying mechanisms of toxicity, and in order to get information on the potential for test chemical and metabolite accumulation and persistence. The percent of the total (radioactive) dose in tissues as well as residual carcass 
should at a minimum be measured at the termination of the excretion 
experiment (e.g. typically up to 7 days post dose or less depending on test chemical specific behaviour). When no test chemical is detected in tissues at study termination (e.g. because the test chemical might have 
been eliminated before study termination due to a short half-life), care 
should be taken in order to prevent misinterpretation of the data. In this type of situation, tissue distribution should be investigated at the time of test chemical (and/or metabolite) peak plasma/blood concentration (T
 max ) or 
peak rate of urinary excretion, as appropriate (see paragraph 38). 
Furthermore, tissue collection at additional time points may be needed to determine tissue distribution of the test chemical and/or its metabolites, to evaluate time dependency (if appropriate), to aid in establishing mass 
balance, and/or as required by a competent authority. Tissues that should 
be collected include liver, fat, GI tract, kidney, spleen, whole blood, residual carcass, target organ tissues and any other tissues (e.g. thyroid, erythrocytes, reproductive organs, skin, eye (particularly in pigmented animals) of 
potential significance in the toxicological evaluation of the test chemical. 
Analysis of additional tissues at the same time points should be considered to maximise utilisation of animals and in the event that target organ toxicity is observed in sub-chronic or chronic toxicity studies. The (radioactive) 
residue concentration and tissue-to-plasma (blood) ratios should also be 
reported. 
38. The evaluation of tissue distribution at additional time points such as the 
time of peak plasma/blood concentration (e.g. T
 max ) or the peak rate of 
urinary excretion, obtained from the respective plasma/blood kinetic or 
excretion experiments, may also be needed or required by a competent authority. This information can be useful for understanding toxicity and the potential for test chemical and metabolite accumulation and persistence. 
Justification for sample selection should be provided; samples for analysis 
generally should be the same as those above (see paragraph 37).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 532
 
39. Quantification of radioactivity for tissue distribution studies can be 
performed using organ dissection, homogenisation, combustion and/or solu ­
bilisation, followed by liquid scintillation counting (LSC) of trapped residues. Certain techniques, currently at various stages of development, e.g. Quantitative whole-body autoradiography and receptor microscopic 
autoradiography, may prove useful in determining the distribution of a 
test chemical in organs and/or tissues (3) (4). 
40. For routes of exposure other than oral, specific tissues should be collected 
and analysed, such as lungs in inhalation studies and skin in dermal studies. 
See paragraphs 74-78.
 
Metabolism  
41. Excreta (and plasma, if appropriate) should be collected for identification 
and quantitation of unchanged test chemical and metabolites as described under paragraphs 44-49. Pooling of excreta to facilitate metabolite identifi ­
cation within a given dose group is acceptable. Profiling of metabolites from 
each time period is recommended. However, if lack of sample and/or radio ­
activity precludes this, pooling of urine and faeces across several time points is acceptable but pooling across sexes or doses is not acceptable. Appro ­
priate qualitative and quantitative methods should be used to assay urine, 
faeces, expired radioactivity from treated animals, and bile if appropriate. 
42. Reasonable efforts should be made to identify all metabolites present at 5 % 
or greater of the administered dose and to provide a metabolic scheme for the test chemical. Test chemicals which have been characterised in excreta 
as comprising 5 % or greater of the administered dose should be identified. 
Identification refers to the exact structural determination of components. Typically, identification is accomplished either by co-chromatography of the metabolite with known standards using two dissimilar systems or by 
techniques capable of positive structural identification such as mass spec ­
trometry, nuclear magnetic resonance (NMR), etc. In the case of co-chromatography, chromatographic techniques utilising the same stationary phase with two different solvent systems are not considered to 
be an adequate two-method verification of metabolite identity, since the 
methods are not independent. Identification by co-chromatography should be obtained using two dissimilar, analytically independent systems such as reverse and normal phase thin layer chromatography (TLC) and high 
performance liquid chromatography (HPLC). Provided that the chromato ­
graphic separation is of suitable quality, then additional confirmation by spectroscopic means is not necessary. Alternatively, unambiguous identifi ­
cation can also be obtained using methods providing structural information 
such as: liquid chromatography/mass spectrometry (LC-MS), or liquid chro ­
matography/tandem mass spectrometry (LC-MS/MS), gas chroma ­
tography/mass spectrometry (GC-MS), and NMR spectrometry. 
43. If identification of metabolites at 5 % or greater of the administered dose is 
not possible, a justification/explanation should be provided in the final 
report. It might be appropriate to identify metabolites representing less than 5 % of the administered dose to gain a better understanding of the metabolic pathway for hazard and/or risk assessment of the test chemical. 
Structural confirmation should be provided whenever possible. This may 
include profiling in plasma or blood or other tissues.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 533
 
Excretion  
44. The rate and extent of excretion of the administered dose should be 
determined by measuring the percent recovered (radioactive) dose from urine, faeces and expired air. These data will also assist in establishing 
mass balance. The quantities of test chemical (radioactivity) eliminated in 
the urine, faeces, and expired air should be determined at appropriate time intervals (see paragraphs 47-49). Repeated dose experiments should be properly designed to allow for collection of excretion data to meet the 
objectives described in the paragraph 26. This will allow for comparison 
to single dose experiments. 
45. If a pilot study has shown that no significant amount of test chemical 
(radioactivity) (according to paragraph 49) is excreted in expired air, then expired air does not need to be collected in the definitive study. 
46. Each animal is to be placed in a separate metabolic unit for collection of 
excreta (urine, faeces and expired air). At the end of each collection period (see paragraphs 47-49), the metabolic units should be rinsed with appro ­
priate solvent (this is known as the ‘cage wash’) to ensure maximum 
recovery of the test chemical (radioactivity). Collection of excreta should be terminated at 7 days, or after at least 90 % of the administered dose has been recovered, whichever occurs first. 
47. The total quantities of test chemical (radioactivity) in urine are to be 
determined for at least two time points on day 1 of collection, one of 
which should be at 24 h post dosing, and daily thereafter until study 
termination. The selection of more than two sampling points on day one (e.g. at 6, 12 and 24 h) is encouraged. The results of pilot studies should be analysed for information on alternate or additional time points for collection. 
A rationale should be provided for the collection schedules. 
48. The total quantities of test chemical (radioactivity) in faeces should be 
determined on a daily basis beginning at 24 h post-dosing until study 
termination, unless pilot studies suggest alternate or additional time points for collection. A rationale should be provided for alternative collection schedules. 
49. The collection of expired CO
 2 and other volatile materials may be discon ­
tinued in a given study experiment when less than 1 % of the administered 
dose is found in the exhaled air during a 24-h collection period.  
Time Course Studies  
Plasma/Blood Kinetics  
50. The purpose of these studies is to obtain estimates of basic TK parameters 
[e.g. C  max , T max , half-life (t  1/2 ), AUC] for the test chemical. These studies 
may be conducted at one dose or, more likely, at two or more doses. Dose 
setting should be determined by the nature of the experiment and/or the 
issue being addressed. Kinetic data may be needed to resolve issues such as test chemical bioavailability and/or to clarify the effect of dose on clearance (e.g. to clarify whether clearance is saturated in a dose-dependent fashion). 
51. For these studies a minimum of four animals of one sex per dose group 
should be used. Justification should be provided for the sex of the animals used. The use of both sexes (four males and four females) should be 
considered if there is evidence to support significant sex-related differences 
in toxicity.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 534
 
52. Following administration of the test chemical (radiolabelled), blood samples 
should be obtained from each animal at suitable time points using appro ­
priate sampling methodology. The volume and number of blood samples which can be obtained per animal might be limited by potential effects of repeated sampling on animal health/physiology and/or the sensitivity of the 
analytical method. Samples should be analysed for each individual animal. 
In some circumstances (e.g. metabolite characterisation), it might be necessary to pool samples from more than one animal. Pooled samples should be clearly identified and an explanation for pooling provided. If a 
radiolabelled test chemical is used, analysis of total radioactivity present 
might be adequate. If so, total radioactivity should be analyzed in whole blood and plasma or plasma and red blood cells to allow calculation of the blood/plasma ratio. In other circumstances, more specific investigations 
requiring the identification of parent compound and/or metabolites, or to 
assess protein binding might be necessary.
 
Other Tissue Kinetics  
53. The purpose of these studies is to obtain time course information to address 
questions related to issues such as toxic mode of action, bioaccumulation 
and bio-persistence via determination of levels of test chemical in various 
tissues. The selection of tissues and the number of time points evaluated will depend on the issue to be addressed and the toxicological database for the test chemical. The design of these additional tissue kinetics studies should 
take into account information gathered as described in paragraphs 37-40. 
These studies might involve single or repeated dosing. A detailed rationale for the approach used should be provided. 
54. Reasons for performing other tissue kinetic studies might include: 
— Evidence of extended blood half-life, suggesting possible accumulation 
of test chemical in various tissues, or 
— interest in seeing if a steady state level has been achieved in specific 
tissues (e.g. in repeated dosing studies, even though an apparent blood 
steady state level of test chemical may have been achieved, there may be interest in ascertaining that a steady state level has also been attained in target tissues). 
55. For these types of time-course studies, an appropriate oral dose of test 
chemical should be administered to a minimum of four animals per dose per time point and the time course of distribution monitored in selected 
tissues. Only one sex may be used, unless gender specific toxicity is 
observed. Whether total radioactivity or parent chemical and/or metabolites are analysed will also depend on the issue being addressed. Assessment of tissue distribution should be made using appropriate techniques.
 
Enzyme Induction/Inhibition  
56. Studies addressing the possible effects of enzyme induction/inhibition or 
biotransformation of test chemical under study may be needed under one or more of the following cases: 
(1) Available evidence indicates a relationship between biotransformation of 
test chemical and enhanced toxicity; 
(2) The available toxicity data indicate a non-linear relationship between 
dose and metabolism;
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 535
 
(3) The results of metabolite identification studies show identification of a 
potentially toxic metabolite that might have been produced by an 
enzyme pathway induced by the test chemical; 
(4) In explaining effects which are postulated to be linked to enzyme 
induction phenomena; 
(5) If toxicologically significant alterations in the metabolic profile of the 
test chemical are observed through either  in vitro  or in vivo  experiments 
with different species or conditions, characterisation of the enzyme(s) 
involved may be needed (e.g. Phase I enzymes such as isoenzymes of the Cytochrome P450-dependent mono-oxygenase system, Phase II enzymes such as isoenzymes of sulfotransferase or uridine diphosphate 
glucuronosyl transferase, or any other relevant enzymes). This 
information might be used to evaluate the pertinence of species to species extrapolations. 
57. Appropriate study protocols to evaluate test chemical related changes in TK, 
suitably validated and justified should be used. Example study designs 
consist of repeated dosing with unlabelled test chemical, followed by a single radiolabelled dose on day 14, or repeated dosing with radiolabelled test chemical and sampling at days 1, 7 and 14 for determination of meta ­
bolite profiles. Repeated dosing with radiolabelled test chemical may also 
provide information on bioaccumulation (see paragraph 26).
 
SUPPLEMENTAL APPROACHES 
58. Supplemental approaches beyond the  in vivo  experiments described in this 
Test Method may provide useful information on the absorption, distribution, metabolism or elimination of a test chemical in certain species.
 
Use of  in vitro  information  
59. Several questions concerning the metabolism of the test chemical may be 
addressed in  in vitro  studies using appropriate test systems. Freshly isolated 
or cultured hepatocytes and subcellular fractions (e.g. microsomes and 
cytosol or S9 fraction) from liver may be used to study possible metabolites. 
Local metabolism in the target organ, e.g. lung, may be of interest for risk assessment. For these purposes, microsomal fractions of target tissues may be useful. Studies with microsomes may be useful to address potential 
gender and life-stage differences and characterise enzyme parameters (K
 m 
and V  max ) which can aid in the assessment of dose dependency of 
metabolism in relation to exposure levels. In addition microsomes may be useful to identify the specific microsomal enzymes involved in the 
metabolism of the test chemical which can be relevant in species extra ­
polation (see also paragraph 56). The potential for induction of biotrans ­
formation can also be examined by using liver subcellular fractions (e.g. microsomes and cytosol) of animals pre-treated with the test chemical of 
interest,  in vitro  via hepatocyte induction studies or from specific cell lines 
expressing relevant enzymes. In certain circumstances and under appropriate conditions, subcellular fractions coming from human tissues might be considered for use in determining potential species differences in biotrans ­
formation. The results from  in vitro  investigations may also have utility in 
the development of PBTK models (5).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 536
 
60. In vitro  dermal absorption studies may provide supplemental information to 
characterise absorption (6). 
61. Primary cell cultures from liver cells and fresh tissue slices may be used to 
address similar questions as with liver microsomes. In certain cases, it may be possible to answer specific questions using cell lines with defined 
expression of the relevant enzyme or engineered cell lines. In certain 
cases, it may be useful to study the inhibition and induction of specific cytochrome P450 isozymes (e.g. CYP1A1, 2E1, 1A2, and others) and/or phase II enzymes by the parent compound using  in vitro  studies. 
Information obtained may have utility for similarly structured compounds.
 
Use of Toxicokinetic Data from Toxicity Studies as Complementary 
Information  
62. Analysis of blood, tissue and/or excreta samples obtained during the conduct 
of any other toxicity studies can provide data on bioavailability, changes in plasma concentration in time (AUC, C
 max ), bioaccumulation potential, 
clearance rates, and gender or life-stage changes in metabolism and kinetics. 
63. Consideration of the study design can be used to answer questions relating 
to: saturation of absorption, biotransformation or excretion pathways at higher dose levels; the operation of new metabolic pathways at higher 
doses and the limitation of toxic metabolites to higher doses. 
64. Other hazard assessment considerations could include issues such as: 
— Age-related sensitivity due to differences in the status of the blood-brain 
barrier, the kidney and/or detoxification capacities; 
— Sub-population sensitivity due to differences in biotransformation 
capacities or other TK differences; 
— Extent of exposure of the foetus by transplacental transfer of chemicals 
or of the newborn through lactation.
 
Use of Toxicokinetic Modelling  
65. Toxicokinetic models may have utility for various aspects of hazard and risk 
assessment as for example in the prediction of systemic exposure and 
internal tissue dose. Furthermore specific questions on mode of action 
may be addressed, and these models can provide a basis for extrapolation across species, routes of exposure, dosing patterns, and for human risk assessment. Data useful for developing PBTK models for a test chemical 
in any given species include (1) partition coefficients, (2) biochemical 
constants and physiological parameters, (3) route-specific absorption parameters and 4)  in vivo  kinetic data for model evaluation [e.g. clearance 
parameters for relevant (> 10 %) excretion pathways, K
 m and V  max for 
metabolism]. The experimental data used in model development should be 
generated with scientifically sound methods and the model results validated. Test chemical- and species-specific parameters such as absorption rates, blood-tissue partitioning and metabolic rate constants are often determined 
to facilitate development of non-compartmental or physiologically-based 
models (7).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 537
 
DATA AND REPORTING 
66. It is recommended that the study report include a table of contents.  
Body of the Report  
67. The body of the report should include information covered by this Test 
Method organised into sections and paragraphs as follows:  
Summary  
68. This section of the study report should include a summary of the study 
design and a description of methods used. It should also highlight the key 
findings regarding mass balance, the nature and magnitude of metabolites, tissue residue, rate of clearance, bioaccumulation potential, sex differences, etc. The summary should be presented in sufficient detail to permit 
evaluation of the findings.
 
Introduction  
69. This section of the report should include the study objectives, rationale and 
design, as well as, appropriate references and any background history.  
Materials and Methods  
70. This section of the report should include detailed descriptions of all 
pertinent information including: 
(a) Test Chemical 
This subsection should include identification of the test chemical: 
chemical name, molecular structure, qualitative and quantitative deter ­
mination of its chemical composition, chemical purity and whenever 
possible, type and quantities of any impurities. It should also include 
information on physical/chemical properties including physical state, colour, gross solubility and/or partition coefficient, stability, and if appropriate, corrosivity. If applicable, information on isomers should 
be provided. If the test chemical is radiolabelled, information on the 
following should be included in this subsection: the type of radio ­
nuclide, position of label, specific activity, and radiochemical purity. 
The type or description of any vehicle, diluents, suspending agents, and 
emulsifiers or other materials used in administering the test chemical 
should be stated. 
(b) Test Animals 
This subsection should include information on the test animals, 
including selection and justification for species, strain, and age at 
study initiation, sex as well as body weight, health status, and animal 
husbandry. 
(c) Methods 
This subsection should include details of the study design and 
methodology used. It should include a description of: 
(1) Justification for any modification of route of exposure and 
exposure conditions, if applicable;
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 538
 
(2) Justification for selection of dose levels; 
(3) Description of pilot studies used in the experimental design of the 
follow-up studies, if applicable. Pilot study supporting data should be submitted; 
(4) How the dosing solution was prepared and the type of solvent or 
vehicle, if any, used; 
(5) Number of treatment groups and number of animals per group; 
(6) Dosage levels and volume (and specific activity of the dose when 
radioactivity is used); 
(7) Route(s) and methods of administration; 
(8) Frequency of dosing; (9) Fasting period (if used); 
(10) Total radioactivity per animal; 
(11) Animal handling; 
(12) Sample collection and handling; 
(13) Analytical methods used for separation, quantitation and identifi ­
cation of metabolites; 
(14) Limit of detection for the employed methods; (15) Other experimental measurements and procedures employed 
(including validation of methods for metabolite analysis). 
(d) Statistical Analysis 
If statistical analysis is used to analyse the study findings, then sufficient 
information on the method of analysis and the computer program employed should be included, so that an independent reviewer/statis ­
tician can re-evaluate and reconstruct the analysis. 
In the case of systems modelling studies such as PBTK, presentation of 
models should include a full description of the model to allow inde ­
pendent reconstruction and validation of the model (see paragraph 65 
and Appendix: Definitions).
 
Results  
71. All data should be summarised and tabulated with appropriate statistical 
evaluation and described in the text of this section. Radioactivity counting data should be summarised and presented as appropriate for the study, 
typically as microgram or milligram equivalents per mass of sample, 
although other units may be used. This section should include graphic illus ­
trations of the findings, reproduction of representative chromatographic and spectrometric data, metabolite identification/quantification and proposed 
metabolic pathways including molecular structure of metabolites. In 
addition the following information is to be included in this section, if applicable: 
(1) Quantity and percent recovery of radioactivity in urine, faeces, expired 
air, and urine and faeces cage wash. 
— For dermal studies, also include data on test chemical recovery from 
treated skin, skin washes, and residual radioactivity in the skin 
covering apparatus and metabolic unit as well as results of the 
dermal washing study. For further discussion, see paragraphs 74-77.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 539
 
— For inhalation studies, also include data on recovery of test chemical 
from lungs and nasal tissues (8). For further discussion, see 
paragraph 78. 
(2) Tissue distribution reported as percent of administered dose and concen ­
tration (microgram equivalents per gram of tissue), and tissue-to-blood or tissue-to-plasma ratios; 
(3) Material balance developed from each study involving the assay of body 
tissues and excreta; 
(4) Plasma concentrations and toxicokinetic parameters (bioavailability, 
AUC, Cmax, Tmax, clearance, half-life) after administration by the relevant route(s) of exposure; 
(5) Rate and extent of absorption of the test chemical after administration 
by the relevant route(s) of exposure; 
(6) Quantities of the test chemical and metabolites (reported as percent of 
the administered dose) collected in excreta; 
(7) Reference to appendix data which contain individual animal data for all 
measurement endpoints (e.g. dose administration, percent recovery, concentrations, TK parameters, etc.); 
(8) A figure with the proposed metabolic pathways and the molecular 
structures of the metabolites.
 
Discussion and Conclusions  
72. In this section the author(s) should: 
(1) Provide a proposed metabolic pathway based on the results of the 
metabolism and disposition of the test chemical; 
(2) Discuss any potential species and sex differences regarding the 
disposition and/or biotransformation of the test chemical; 
(3) Tabulate and discuss the identification and magnitude of metabolites, 
rates of clearance, bioaccumulation potential, and level of tissue residues 
of parent, and/or metabolite(s), as well as possible dose-dependent 
changes in TK parameters, as appropriate; 
(4) Integrate into this section any relevant TK data obtained in the course of 
conducting toxicity studies; 
(5) Provide a concise conclusion that can be supported by the findings of 
the study; 
(6) Add Sections (as needed or appropriate). 
73. Additional sections should be used to include supporting bibliographic 
information, tables, figures, appendices, etc.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 540
 
ALTERNATIVE ROUTES OF EXPOSURE  
Dermal  
Dermal Treatment  
74. This section provides specific information on the investigation of the toxi ­
cokinetics of the test chemical by the dermal route. For dermal absorption, 
chapter B.44 of this Annex [Skin absorption:  in vivo  method (9)] should be 
consulted. For other endpoints such as distribution and metabolism, this Test Method B.36 can be used. One or more dose levels for the test chemical 
should be used in the dermal treatment. The test chemical (e.g. neat, diluted 
or formulated material containing the test chemical which is applied to the skin) should be the same (or a realistic surrogate) as that to which humans or other potential target species might be exposed. The dose level(s) should 
be selected in accordance with paragraphs 20-26 of this Test Method. 
Factors that could be taken into consideration in dermal dose selection include expected human exposure and/or doses at which toxicity was observed in other dermal toxicity studies. The dermal dose(s) should be 
dissolved, if necessary, in a suitable vehicle and applied in a volume 
adequate to deliver the doses. Shortly before testing, fur should be clipped from the dorsal area of the trunk of the test animals. Shaving may be employed, but it should be carried out approximately 24 h before 
the test. When clipping or shaving the fur, care should be taken to avoid 
abrading the skin, which could alter its permeability. Approximately 10 % of the body surface should be cleared for application of the test chemical. With highly toxic chemicals, the surface area covered may be less than 
approximately 10 %, but as much of the area as possible is to be covered 
with a thin and uniform film. The same treatment surface area should be used for all dermal test groups. The dosed areas are to be protected with a suitable covering which is secured in place. The animals should be housed 
separately. 
75. A dermal washing study should be conducted to assess the amount of the 
applied dose of the test chemical that may be removed from the skin by 
washing the treated skin area with a mild soap and water. This study can also aid in establishing mass balance when the test chemical is administered by the dermal route. For this dermal washing study, a single dose of the test 
chemical should be applied to two animals. Dose level selection is in 
accordance with paragraph 23 of this Test Method (also see paragraph 76 for discussion of skin contact time). The amounts of test chemical recovered in the washes should be determined to assess the effectiveness of removal of 
the test chemical by the washing procedure. 
76. Unless precluded by corrosiveness, the test chemical should be applied and 
kept on the skin for a minimum of 6 h. At the time of removal of the 
covering, the treated area should be washed following the procedure as 
outlined in the dermal washing study (see paragraph 75). Both covering and the washes should be analysed for residual test chemical. At the termination of the studies, each animal should be humanely killed in 
accordance with (2), and the treated skin removed. An appropriate 
section of treated skin should be analysed to determine residual test chemical (radioactivity). 
77. For the toxicokinetic assessment of pharmaceuticals, different procedures, in 
accordance with the appropriate regulatory system, may be needed.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 541
 
Inhalation  
78. A single concentration (or more if needed) of test chemical should be used. 
The concentration(s) should be selected in accordance with paragraphs 20- 26 of this Test Method. Inhalation treatments are to be conducted using a 
‘nose-cone’ or ‘head-only’ apparatus to prevent absorption by alternate 
routes of exposure (8). If other inhalation exposure conditions are used, justification for the modification should be documented. The duration of exposure by inhalation should be defined; a typical exposure is 4-6 h.
 
LITERATURE:  
(1) OECD (2009). Preliminary Review of OECD Test Guidelines for their 
Applicability to Manufactured Nanomaterials, Series on the Safety of 
Manufactured Nanomaterials No 15, ENV/JM/MONO(2009)21, OECD, Paris. 
(2) OECD (2000). Guidance Document on Recognition, Assessment and Use 
of Clinical Signs as Humane Endpoints for Experimental Animals Used in 
Safety Evaluation; Environmental Health and Safety Publications, Series on 
Testing and Assessment No 19, ENV/JM/MONO(2000), OECD, Paris. 
(3) Solon E G, Kraus L (2002). Quantitative whole-body autoradiography in 
the pharmaceutical industry; Survey results on study design, methods, and 
regulatory compliance, J Pharm and Tox Methods 46: 73-81. 
(4) Stumpf WE (2005). Drug localization and targeting with receptor micro ­
scopic autoradiography. J. Pharmacological and Toxicological Methods 51: 25-40. 
(5) Loizou G, Spendiff M, Barton HA, Bessems J, Bois FY, d’Yvoire MB, 
Buist H, Clewell HJ 3rd, Meek B, Gundert-Remy U, Goerlitz G, Schmitt 
W. (2008). Development of good modelling practice for physiologically 
based pharmacokinetic models for use in risk assessment: The first steps. Regulatory Toxicology and Pharmacology 50: 400 – 411. 
(6) Chapter B.45 of this Annex, Skin Absorption: In Vitro Method. (7) IPCS (2010). Characterization and application of Physiologically-Based 
Pharmacokinetic Models in Risk Assessment. IPCS Harmonization 
Project Document No 9. Geneva, World Health Organization, International Programme on Chemical Safety. 
(8) OECD (2009). Guidance Document on Acute Inhalation Toxicity Testing, 
Series on Testing and Assessment No 39, ENV/JM/MONO(2009)28, 
OECD, Paris. 
(9) Chapter B.44 of this Annex, Skin Absorption: In Vivo Method. 
(10) Barton HA,  et al.  (2006). The Acquisition and Application of Absorption, 
Distribution, Metabolism, and Excretion (ADME) Data in Agricultural Chemical Safety Assessments, Critical Reviews in Toxicology 36: 9-35. 
(11) Gibaldi M and Perrier D, (1982), Pharmacokinetics, 2nd edition, Marcel 
Dekker, Inc., New York.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 542
 
Appendix  
DEFINITIONS  
Absorption : Process(es) of uptake of chemicals into or across tissues. Absorption 
refers to parent compound and all its metabolites. Not to be confused with 
‘bioavailability’.  
Accumulation  (Bioaccumulation): Increase of the amount of a test chemical over 
time within tissues (usually fatty tissues, following repeated exposure); if the 
input of a test chemical into the body is greater than the rate at which it is 
eliminated, the organism accumulates the test chemical and toxic concentrations of a test chemical might be achieved.  
ADME : Acronym for ‘Absorption, Distribution, Metabolism, and Excretion’.  
AUC : (Area under the plasma concentration-time curve): Area under the curve in 
a plot of concentration of test chemical in plasma over time. It represents the 
total amount of test chemical absorbed by the body within a predetermined 
period of time. Under linear conditions, the AUC (from time zero to infinity) is proportional to the total amount of a test chemical absorbed by the body, irrespective of the rate of absorption.  
Autoradiography : (Whole-body autoradiography): Used to determine quali ­
tatively and/or quantitatively the tissue localisation of a radioactive test 
chemical, this technique uses X-ray film or more recently digital phosphor ­
imaging to visualize radioactively labelled molecules or fragments of molecules by recording the radiation emitted within the object under study. 
Quantitative whole-body autoradiography, compared to organ dissection, may 
have some advantages for the evaluation of test chemical distribution and the assessment of overall recovery and resolution of radioactive material in tissues. One significant advantage, for example, is it can be used in a pigmented animal 
model to assess possible association of the test chemical with melanin, which can 
bind certain molecules. However, while it may provide convenient whole body overviews of the high-capacity-low-affinity binding sites, this technique might be limited in recognising specific target sites such as receptor-binding sites where 
relatively high-resolution and high-sensitivity are needed for detection. When 
autoradiography is used, experiments intended to determine mass balance of administered compound should be conducted as a separate group or in a 
separate study from the tissue distribution experiment, where all excreta (which 
may also include expired air) and whole carcasses are homogenised and assayed by liquid scintillation counting.  
Biliary excretion : Excretion via the bile ducts.  
Bioaccumulation : See ‘Accumulation’.  
Bioavailability : Fraction of an administered dose that reaches the systemic circu ­
lation or is made available at the site of physiological activity. Usually, bioa ­
vailability of a test chemical refers to the parent compound, but it could refer to 
its metabolite. It considers only one chemical form.  Nota Bene : bioavailability 
and absorption are not the same. The difference between e.g. oral absorption (i.e. presence in gut wall and portal circulation) and bioavailability (i.e. presence in 
systemic blood and in tissues) can arise from chemical degradation due to gut 
wall metabolism or efflux transport back to the intestinal lumen or presystemic metabolism in the liver, among other factors (10). Bioavailability of the toxic component (parent compound or a metabolite) is a critical parameter in human 
risk assessment (high-to-low dose extrapolation, route-to-route extrapolation) for 
derivation of an internal value from the external NOAEL or BMD (applied dose). For liver effects upon oral administration, it is the oral absorption that suffices. However, for every effect other than at the portal of entry, it is the bioavailability 
that is in general a more reliable parameter for further use in risk assessment, not 
the absorption.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 543
 
Biopersistence : See ‘Persistence’.  
Biotransformation : (Usually enzymatic) chemical conversion of a test chemical 
of interest into a different chemical within the body. Synonymous with ‘meta ­
bolism’.  
C max : Either maximal (peak) concentration in blood (plasma/serum) after admin ­
istration or maximal (peak) excretion (in urine or faeces) after administration.  
Clearance rate : Quantitative measure of the rate at which a test chemical is 
removed from the blood, plasma or a certain tissue per unit time.  
Compartment : Structural or biochemical portion (or unit) of a body, tissue or 
cell, that is separate from the rest.  
Detoxification pathways : Series of steps leading to the elimination of toxic 
chemicals from the body, either by metabolic change or excretion.  
Distribution : Dispersal of a test chemical and its derivatives throughout an 
organism.  
Enzymes/Isozymes : Proteins that catalyse chemical reactions. Isozymes are 
enzymes that catalyse similar chemical reactions but differ in their amino acid sequence.  
Enzymatic Parameters : K
 m : Michaelis constant and V  max : maximum velocity.  
Excretion : Process(es) by which an administered test chemical and/or its meta ­
bolites are removed from the body.  
Exogenously : Introduced from or produced outside the organism or system.  
Extrapolation : Inference of one or more unknown values on the basis of that 
which is known or has been observed.  
Half-life (t  1/2 ): The time taken for the concentration of the test chemical to 
decrease by one-half in a compartment. It typically refers to plasma concentration or the amount of the test chemical in the whole body.  
Induction/Enzyme induction : Enzyme synthesis in response to an environ ­
mental stimulus or inducer molecule.  
Linearity/linear kinetics : A process is linear in terms of kinetics when all 
transfer rates between compartments are proportional to the amounts or concen ­
trations present, i.e. first order. Consequently, clearance and distribution volumes 
are constant, as well as half-lives. The concentrations achieved are proportional to the dosing rate (exposure), and accumulation is more easily predictable. Linearity/Non-linearity can be assessed by comparing the relevant parameters, 
e.g. AUC, after different doses or after single and repeated exposure. Lack of 
dose dependency may be indicative of saturation of enzymes involved in the metabolism of the compound, an increase of AUC after repeated exposure as compared to single exposure may be an indication for inhibition of metabolism 
and a decrease in AUC may be an indication for induction of metabolism [see 
also (11)].  
Mass balance : Accounting of test chemical entering and leaving the system.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 544
 
Material balance : See ‘mass balance’.  
Mechanism (Mode) of toxicity/Mechanism (Mode) of action : Mechanism of 
action refers to specific biochemical interactions through which a test chemical 
produces its effect. Mode of action refers to more general pathways leading to 
the toxicity of a test chemical.  
Metabolism : Synonymous with ‘biotransformation’.  
Metabolites : Products of metabolism or metabolic processes.  
Oral Absorption : The percentage of the dose of test chemical absorbed from the 
site of administration (i.e. GI tract). This critical parameter can be used to understand the fraction of the administered test chemical that reaches the 
portal vein, and subsequently the liver.  
Partition coefficient : Also known as the distribution coefficient, it is a measure 
of the differential solubility of a chemical in two solvents.  
Peak blood (plasma/serum) levels : Maximal (peak) blood (plasma/serum) 
concentration after administration (see also ‘C
 max ’). 
Persistence (biopersistence) : Long-term presence of a chemical (in a biological 
system) due to resistance to degradation/elimination.  
Read-across : The endpoint information for one or more chemicals is used to 
make a prediction of the endpoint for the target chemical.  
Receptor Microscopic Autoradiography  (or Receptor Microautoradiography): 
This technique may be used to probe xenobiotic interaction with specific tissue 
sites or cell populations as for instance in receptor binding or specific mode of action studies that may require high-resolution and high sensitivity which may not be feasible with other techniques such as whole-body autoradiography.  
Route of administration  (oral, IV, dermal, inhalation, etc.): Refers to the means 
by which chemicals are administered to the body (e.g. orally by gavage, orally by 
diet, dermal, by inhalation, intravenously, etc.).  
Saturation : State whereby one or more of the kinetic (e.g. absorption, 
metabolism or clearance) process(es) are at a maximum (read ‘saturated’).  
Sensitivity : Capability of a method or instrument to discriminate between 
measurement responses representing different levels of a variable of interest.  
Steady-state blood (plasma) levels : Non-equilibrium state of an open system in 
which all forces acting on the system are exactly counter-balanced by opposing 
forces, in such a manner that all its components are stationary in concentration although matter is flowing through the system.  
Systems Modelling  (Physiologically-based Toxicokinetic, Pharmacokinetic- 
based, Physiologically-based Pharmacokinetic, Biologically-based, etc.): Abstract model that uses mathematical language to describe the behaviour of a 
system.  
Target tissue : Tissue in which a principal adverse effect of a toxicant is mani ­
fested.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 545
 
Test chemical : Any chemical or mixture tested using this Test Method.  
Tissue distribution : Reversible movement of a test chemical from one location 
in the body to another. Tissue distribution can be studied by organ dissection, 
homogenisation, combustion and liquid scintillation counting or by qualitative and/or quantitative whole body autoradiography. The former is useful to obtain concentration and percent of recovery from tissues and remaining carcass of the 
same animals, but may lack resolution for all tissues and may have less than 
ideal overall recovery (< 90 %). See definition for the latter above.  
T
 max : Time to reach C  max . 
Toxicokinetics  (Pharmacokinetics): Study of the absorption, distribution, metab ­
olism, and excretion of chemicals over time.  
Validation of models : Process of assessing the adequacy of a model to 
consistently describe the available toxicokinetic data. Models may be evaluated 
via statistical and visual comparison of model predictions with experimental values against a common independent variable (e.g. time). The extent of evaluation should be justified in relation to the intended use of the model.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 546
 
B.37.  DELAYED NEUROTOXICITY OF ORGANOPHOSPHORUS 
SUBSTANCES FOLLOWING ACUTE EXPOSURE  
1. METHOD  
1.1. INTRODUCTION 
In the assessment and evaluation of the toxic effects of substances, it 
is important to consider the potential of certain classes of substances to cause specific types of neurotoxicity that might not be detected in other toxicity studies. Certain organophosphorus substances have 
been observed to cause delayed neurotoxicity and should be 
considered as candidates for evaluation.  
In vitro  screening tests could be employed to identify those 
substances which may cause delayed polyneuropathy; however, negative findings from  in vitro  studies do not provide evidence 
that the test substance is not a neurotoxicant. 
See General introduction Part B.
 
1.2. DEFINITIONS 
Organophopsphorus substances include uncharged organophosphorus 
esters, thioesters or anhydrides of organophosphoric, organophos ­
phonic or organophosphoramidic acids or of related phosphorothioic, 
phosphonothioic or phosphorothioamidic acids, or other substances that may cause the delayed neurotoxicity sometimes seen in this class of substances. 
Delayed neurotoxicity is a syndrome associated with prolonged 
delayed onset of ataxia, distal axonopathies in spinal cord and peripheral nerve, and inhibition and aging of neuropathy target 
esterase (NTE) in neural tissue.
 
1.3. REFERENCE SUBSTANCES 
A reference substance may be tested with a positive control group as 
a means of demonstrating that under the laboratory test conditions, the response of the tested species has not changed significantly. 
An example of a widely used neurotoxicant is tri-o-tolyl phosphate 
(CAS 78-30-8, Einecs 201-103-5, CAS nomenclature: phosphoric acid, tris(2-methylphenyl)ester), also known as tris-o-cresylphos ­
phate.
 
1.4. PRINCIPLE OF THE TEST METHOD 
The test substance is administered orally in a single dose to domestic 
hens which have been protected from acute cholinergic effects, when appropriate. The animals are observed for 21 days for behavioural 
abnormalities, ataxia, and paralysis. Biochemical measurements, in 
particular neuropathy target esterase inhibition (NTE), are undertaken on hens randomly selected from each group, normally 24 and 48 hours after dosing. Twenty-one days after exposure, the 
remainder of the hens are killed and histopathological examination 
of selected neural tissues is undertaken.
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1.5. DESCRIPTION OF THE TEST METHOD  
1.5.1.  Preparations  
Healthy young adult hens free from interfering viral diseases and 
medication and without abnormalities of gait should be randomised 
and assigned to treatment and control groups and acclimatised to the 
laboratory conditions for at least five days prior to the start of the study. 
Cages or enclosures which are large enough to permit free mobility 
of the hens, and easy observation of gait should be used. 
Dosing with the test substance should normally be by the oral route 
using gavage, gelatine capsules, or a comparable method. Liquids may be given undiluted or dissolved in an appropriate vehicle such 
as corn oil; solids should be dissolved if possible since large doses of 
solids in gelatine capsules may not be absorbed efficiently. For non- aqueous vehicles the toxic characteristics of the vehicle should be known, and if not known should be determined before the test.
 
1.5.2.  Test conditions  
1.5.2.1.  Test animals  
The young adult domestic laying hen ( Gallus gallus domestícus ), 
aged eight to 12 months, is recommended. Standard size breeds and strains should be employed and the hens normally should have been reared under conditions which permitted free mobility.
 
1.5.2.2.  Number and sex  
In addition to the treatment group, both a vehicle control group and a 
positive control group should be used. The vehicle control group 
should be treated in a manner identical to the treatment group, except that administration of the test substance is omitted. 
Sufficient number of hens should be utilised in each group of birds 
so that at least six birds can be killed for biochemical determination 
(three at each of two time points) and six can survive the 21-day observation period for pathology. 
The positive control group may be run concurrently or be a recent 
historical control group. It should contain at least six hens, treated 
with a known delayed neurotoxicant, three hens for biochemistry and 
three hens for pathology. Periodic updating of historical data is recommended. New positive control data should be developed when some essential element (e.g. strain, feed, housing conditions) 
of the conduct of the test has been changed by the performing 
laboratory.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 548
 
1.5.2.3.  Dose levels  
A preliminary study using an appropriate number of hens and dose 
levels groups should be performed to establish the level to be used in the main study. Some lethality is typically necessary in this 
preliminary study to define an adequate main study dose. 
However, to prevent death due to acute cholinergic effects, atropine or another protective agent, known to not interfere with delayed neurotoxic responses, may be used. A variety of test 
methods may be used to estimate the maximum non-lethal dose of 
test substances (See method B.1 bis). Historical data in the hen or 
other toxicological information may also be helpful in dose selection. 
The dose level of the test substance in the main study should be as 
high as possible taking into account the results of the preliminary 
dose selection study and the upper limit dose of 2 000 mg/kg body weight. Any mortality which might occur should not interfere with the survival of sufficient animals for biochemistry (six) and histology 
(six) at 21 days. Atropine or another protective agent, known to not 
interfere with delayed neurotoxic responses, should be used to prevent death due to acute cholinergic effects.
 
1.5.2.4.  Limit test  
If a test at a dose level of at least 2 000 mg/kg body weight/day, 
using the procedures described for this study, produces no 
observable toxic effects and if toxicity would not be expected 
based upon data from structurally related substances, then a study using a higher dose may not be considered necessary. The limit test 
applies except when human exposure indicates the need for a higher 
dose level to be used.
 
1.5.3.  Observation period  
Observation period should be 21 days.  
1.5.4.  Procedure  
After administration of a protective agent to prevent death due to 
acute cholinergic effect, the test substance is administered in a single dose.
 
General observation 
Observations should start immediately after exposure. All hens 
should be carefully observed several times during the first two days and thereafter at least once daily for a period of 21 days or 
until scheduled kill. All signs of toxicity should be recorded, 
including the time of onset, type, severity and duration of behav ­
ioural abnormalities. Ataxia should be measured on an ordinal grading scale consisting of at least four levels, and paralysis 
should be noted. At least twice a week the hens selected for 
pathology should be taken outside the cages and subjected to a period of forced motor activity, such as ladder climbing, in order to facilitate the observation of minimal toxic effects. Moribund 
animals and animals in severe distress or pain should be removed 
when noticed, humanely killed and necropsied.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 549
 
Body weight 
All hens should be weighed just prior to administration of the test 
substance and at least once a week thereafter.  
Biochemistry 
Six hens randomly selected from each of the treatment and vehicle 
control groups, and three hens from the positive control group (when 
this group is run concurrently), should be killed within a few days 
after dosing, and the brain and lumbar spinal cord prepared and assayed for neuropathy target esterase inhibition activity. In addition, it may also be useful to prepare and assay sciatic nerve 
tissue for neuropathy target esterase inhibition activity. Normally, 
three birds of the control and each treatment group are killed after 24 hours and three at 48 hours, whereas the three hens of the positive controls should be killed at 24 hours. If observation of 
clinical signs of intoxication (this can often be assessed by obser ­
vation of the time of onset of cholinergic signs) indicates that the toxic agent may be disposed of very slowly then it may be preferable to sample tissue from three birds at each of two times between 24 
and as late as 72 hours after dosing. 
Analyses of acetylcholinesterase (AChE) may also be performed on 
these samples, if deemed appropriate. However, spontaneous reacti ­
vation of AChE may occur  in vivo , and so lead to underestimation of 
the potency of the substance as an AChE inhibitor.
 
Gross necropsy 
Gross necropsy of all animals (scheduled killed and killed when 
moribund) should include observation of the appearance of the brain and spinal cord.
 
Histopathological examination 
Neural tissue from animals surviving the observation period and not 
used for biochemical studies should be subjected to microscopic examination. Tissues should be fixed  in situ,  using perfusion tech ­
niques. Sections should include cerebellum (mid-longitudinal level), 
medulla oblongata, spinal cord, and peripheral nerves. The spinal 
cord sections should be taken from the upper cervical segment, the mid-thoracic and the lumbo-sacral regions. Sections of the distal region of the tibial nerve and its branches to the gastrocnemial 
muscle and of the sciatic nerve should be taken. Sections should 
be stained with appropriate myelin and axon-specific stains.
 
2. DATA  
Negative results on the endpoints selected in this method (biochem ­
istry, histopathology and behavioural observation) would not normally require further testing for delayed neurotoxicity. 
Equivocal or inconclusive results for these endpoints may require 
further evaluation. 
Individual data should be provided. Additionally, all data should be 
summarised in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, behavioural or biochemical effects, the types and severity of 
these lesions or effects, and the percentage of animals displaying 
each type and severity of lesion or effect.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 550
 
The findings of this study should be evaluated in terms of the inci ­
dence, severity, and correlation of behavioural, biochemical and 
histopathological effects and any other observed effects in the treated and control groups. 
Numerical results should be evaluated by appropriate and generally 
acceptable statistical methods. The statistical methods used should be 
selected during the design of the study.
 
3. REPORTING  
TEST REPORT 
The test report shall, if possible, include the following information: 
3.1. Test animals: 
— strain used, 
— number and age of animals, — source, housing conditions, etc., — individual weights of animals at the start of the test. 
3.2. Test conditions: 
— details of test substance preparation, stability and homogeneity, 
where appropriate, 
— justification for choice of vehicle, — details of the administration of the test substance, 
— details of food and water quality, 
— rationale for dose selection, — specification of doses administered, including details of the 
vehicle, volume and physical form of the material administered, 
— identity and details of the administration of any protective agent. 
3.3. Results: 
— body weight data, — toxic response data by group, including mortality, — nature, severity and duration of clinic observations (whether 
reversible or not), 
— a detailed description of biochemical methods and findings, — necropsy findings, — a detailed description of all histopathological findings, — statistical treatment of results, where appropriate. Discussion of results. Conclusions.
 
4. REFERENCES  
This method is analogius to OECD TG 418.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 551
 
B.38.  DELAYED NEUROTOXICITY OF ORGANOPHOSPHORUS 
SUBSTANCES 28-DAY REPEATED DOSE STUDY  
1. METHOD  
1.1. INTRODUCTION 
In the assessment and evaluation of the toxic effects of substances, it 
is important to consider the potential of certain classes of substances to cause specific types of neurotoxicity that might not be detected in other toxicity studies. Certain organophosphorus substances have 
been observed to cause delayed neurotoxicity and should be 
considered as candidates for evaluation.  
In vitro  screening tests could be employed to identify those 
substances which may cause delayed polyneuropathy; however, 
negative findings from  in vitro  studies do not provide evidence 
that the test substance is not a neurotoxicant. 
This 28-day delayed neurotoxicity test provides information on 
possible health hazards likely to arise from repeated exposures over a limited period of time. It will provide information on dose 
response and can provide an estimate of a no-observed-adverse effect 
level, which can be of use for establishing safety criteria for exposure. 
See also General introduction Part B.
 
1.2. DEFINITIONS 
Organophosphorus substances include uncharged organophosphorus 
esters, thioesters or anhydrides of organophosphoric, organophos ­
phonic or organophosphoramidic acids or of related phosphorothioic, 
phosphonothioic or phosphorothioamidic acids or other substances that may cause the delayed neurotoxicity sometimes seen in this class of substances. 
Delayed neurotoxicity is a syndrome associated with prolonged 
delayed onset of ataxia, distal axonopathies in spinal cord and 
peripheral nerve, and inhibition and ageing of neuropathy target esterase (NTE) in neural tissue.
 
1.3. PRINCIPLE OF THE TEST METHOD 
Daily doses of the test substance are administered orally to domestic 
hens for 28 days. The animals are observed at least daily for behav ­
ioural abnormalities, ataxia and paralysis until 14 days after the last 
dose. Biochemical measurements, in particular neuropathy target 
esterase inhibition (NTE), are undertaken, on hens randomly selected from each group, normally 24 and 48 hours after the last dose. Two weeks after the last dose, the remainder of the hens are 
killed and histopathological examination of selected neural tissues is 
undertaken.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 552
 
1.4. DESCRIPTION OF THE TEST METHOD  
1.4.1.  Preparations  
Healthy young adult hens free from interfering viral diseases and 
medication, and without abnormalities of gait should be randomised 
and assigned to treatment and control groups and acclimatised to the 
laboratory conditions for at least five days prior to the start of the study. 
Cages or enclosures which are large enough to permit free mobility 
of the hens and easy observation of gait should be used. 
Oral dosing each day, seven days per week, should be carried out, 
preferably by gavage or administration of gelatine capsules. Liquids may be given undiluted or dissolved in an appropriate vehicle such as corn oil; solids should be dissolved if possible since large doses of 
solids in gelatine capsules may not be absorbed efficiently. For non- 
aqueous vehicles the toxic characteristics of the vehicle should be known, and if not known should be determined before the test.
 
1.4.2.  Test conditions  
1.4.2.1.  Test animals  
The young adult domestic laying hen ( Gallus gallus domesticus ), 
aged eight to 12 months, is recommended. Standard size, breeds 
and strains should be employed and the hens normally should 
have been reared under conditions which permitted free mobility.  
1.4.2.2.  Number and sex  
Generally at least three treatment groups and a vehicle control group 
should be used. The vehicle control group should be treated in a manner identical to the treatment group, except that administration of 
the test substance is omitted. 
Sufficient number of hens should be utilised in each group of birds 
so that at least six birds can be killed for biochemical determinations (three at each of two timepoints) and six birds can survive the 14- day post-treatment observation period for pathology.
 
1.4.2.3.  Dose levels  
Dose levels should be selected taking into account the results from 
an acute test on delayed neurotoxicity and any other existing toxicity 
or kinetic data available for the test compound. The highest dose 
level should be chosen with the aim of inducing toxic effects, preferably delayed neurotoxicity, but not death nor obvious suffering. Thereafter, a descending sequence of dose levels should 
be selected with a view to demonstrate any dose-related response and 
no-observed-adverse effects at the lowest dose level.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 553
 
1.4.2.4.  Limit test  
If a test at a dose level of at least 1 000 mg/kg body weight/day, 
using the procedures described for this study, produces no observable toxic effects and if toxicity would not be expected 
based upon data from structurally related substances, then a study 
using a higher dose may not be considered necessary. The limit test applies except when expected human exposure indicates the need for a higher dose level to be used.
 
1.4.2.5.  Observation period  
All the animals should be observed at least daily during the exposure 
period and 14 days after, unless scheduled necropsy.  
1.4.3.  Procedure  
Animals are dosed with the test substance on seven days per week 
for a period of 28 days.  
General observations  
Observations should start immediately after treatment begins. All 
hens should be carefully observed at least once daily on each of 
the 28 days of treatment, and for 14 days after dosing or until scheduled kill. All signs of toxicity should be recorded including their time of onset, type, severity and duration. Observations 
should include, but not be limited to, behavioural abnormalities. 
Ataxia should be measured on an ordinal grading scale consisting of at least four levels, and paralysis should be noted. At least twice a week the hens should be taken outside the cages and subjected to a 
period of forced motor activity, such as ladder climbing, in order to 
facilitate the observation of minimal toxic effects. Moribund animals in severe distress or pain should be removed when noticed, humanely killed and necropsied.
 
Body weight  
All hens should be weighed just prior to the first administration of 
the test substance and at least once a week thereafter.  
Biochemistry  
Six hens randomly selected from each of the treatment and vehicle 
control groups should be killed within a few days after the last dose, and the brain and lumbar spinal cord prepared and assayed for neur ­
opathy target esterase (NTE) inhibition activity. In addition, it may 
also be useful to prepare and assay sciatic nerve tissue for neur ­
opathy target esterase (NTE) inhibition activity. Normally, three birds of the control and each treatment group are killed after 
24 hours and three at 48 hours after the last dose. If data from the 
acute study or other studies (e.g. toxicokinetics) indicate that other times of killing after final dosing are preferable then these times should be used and the rationale documented. 
Analyses of acetylcholinesterase (AChE) may also be performed on 
these samples, if deemed appropriate. However, spontaneous reacti ­
vation of AChE may occur  in vivo,  and so lead to underestimation of 
the potency of the substance as an AChE inhibitor.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 554
 
Gross necropsy  
Gross necropsy of all animals (scheduled killed and killed when 
moribund) should include observation of the appearance of the brain and spinal cord.
 
Histopathological examination  
Neural tissue from animals surviving the observation period and not 
used for biochemical studies should be subjected to microscopic 
examination. Tissues should be fixed  in situ,  using perfusion tech ­
niques. Sections should include cerebellum (mid longitudinal level), medulla oblongata, spinal cord and peripheral nerves. The spinal 
cord sections should be taken from the upper cervical segment, the 
mid-thoracic and the lumbo-sacral regions. Sections of the distal region of the tibial nerve and its branches to the gastrocnemial muscle and of the sciatic nerve should be taken. Sections should 
be stained with appropriate myelin and axon-specific stains. Initially, 
microscopic examination should be carried out on the preserved tissues of all animals in the control and high dose group. When there is evidence of effects in the high dose group, microscopic 
examination should also be carried out in hens from the intermediate 
and low dose groups.
 
2. DATA  
Negative results on the endpoints selected in this method (biochem ­
istry, histopathology and behavioural observation) would not normally require further testing for delayed neurotoxicity. Equivocal or inconclusive results for these endpoints may require 
further evaluation. 
Individual data should be provided. Additionally, all data should be 
summarised in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, behavioural or biochemical effects, the types and severity of 
these lesions or effects, and the percentage of animals displaying 
each type and severity of lesion or effect. 
The findings of this study should be evaluated in terms of the inci ­
dence, severity, and correlation of behavioural, biochemical and histopathological effects and any other observed effects in each of 
the treated and control groups. 
Numerical results should be evaluated by appropriate and generally 
acceptable statistical methods. The statistical methods should be 
selected during the design of the study.
 
3. REPORTING  
TEST REPORT 
The test report shall, if possible, include the following information: 
3.1. Test animals: 
— strain used, — number and age of animals, — source, housing conditions, etc., — individual weights of animals at the start of the test.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 555
 
3.2. Test conditions: 
— details of test substance preparation, stability and homogeneity, 
where appropriate, 
— justification for choice of vehicle, — details of the administration of the test substance, — details of food and water quality, — rationale for dose selection, — specification of doses administered, including details of the 
vehicle, volume and physical form of the material administered, 
— rationale for choosing other times for biochemical determination, 
if other than 24 and 48 h. 
3.3. Results: 
— body weight data, — toxic response data by dose level, including mortality, — no-observed adverse effect level, — nature, severity and duration of clinic observations (whether 
reversible or not), 
— a detailed description of biochemical methods and findings, 
— necropsy findings, 
— a detailed description of all histopathological findings, — statistical treatment of results, where appropriate. Discussion of results. Conclusions.
 
4. REFERENCES  
This method is analogous to OECD TG 419.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 556
 
B.39.  UNSCHEDULED DNA SYNTHESIS (UDS) TEST WITH 
MAMMALIAN LIVER CELLS IN VIVO  
1. METHOD  
This method is a replicate of the OECD TG 486, Unscheduled DNA 
Synthesis (UDS) Test with Mammalian Liver Cells  In Vivo  (1997).  
1.1. INTRODUCTION 
The purpose of the unscheduled DNA Synthesis (UDS) test with 
mammalian liver cells i n vivo  is to identify test substances that 
induce DNA repair in liver cells of treated animals (see 1,2,3,4). 
This in vivo  test provides a method for investigating genotoxic 
effects of chemicals in the liver. The end-point measured is indicative of DNA damage and subsequent repair in liver cells. 
The liver is usually the major site of metabolism of absorbed 
compounds. It is thus an appropriate site to measure DNA damage  
in vivo . 
If there is evidence that the test substance will not reach the target 
tissue, it is not appropriate to use this test. 
The end-point of unscheduled DNA synthesis (UDS) is measured by 
determining the uptake of labelled nucleosides in cells that are not 
undergoing scheduled (S-phase) DNA synthesis. The most widely used technique is the determination of the uptake of tritium- labelled thymidine (
 3 H-TdR) by autoradiography. Rat livers are 
preferably used for  in vivo  UDS tests. Tissues other than the livers 
may be used, but are not the subject of this method. 
The detection of a UDS response is dependent on the number of 
DNA bases excised and replaced at the site of the damage. 
Therefore, the UDS test is particularly valuable to detect substance-induced ‘longpatch repair’ (20-30 bases). In contrast, ‘shortpatch repair’ (1-3 bases) is detected with much lower sensi ­
tivity. Furthermore, mutagenic events may result because of non- 
repair, misrepair or misreplication of DNA lesions. The extent of the UDS response gives no indication of the fidelity of the repair process. In addition, it is possible that a mutagen reacts with DNA 
but the DNA damage is not repaired via an excision repair process. 
The lack of specific information on mutagenic activity provided by the UDS test is compensated for by the potential sensitivity of this endpoint because it is measured in the whole genome. 
See also General introduction Part B.
 
1.2. DEFINITIONS  
Cells in repair:  a net nuclear grain (NNG) higher than a preset 
value, to be justified at the laboratory conducting the test.  
Net nuclear grains (NNG):  quantitative measure for UDS activity 
of cells in autoradiographic UDS tests, calculated by subtracting the average number of cytoplasmic grains in nucleus-equivalent cyto ­
plasmic areas (CG) from the number of nuclear grains (NG): NNG 
= NG - CG. NNG counts are calculated for individual cells and then 
pooled for cells in a culture, in parallel cultures, etc.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 557
 
Unscheduled DNA Synthesis (UDS):  DNA repair synthesis after 
excision and removal of a stretch of DNA containing a region of 
damage induced by chemical substances or physical agents.  
1.3. PRINCIPLE OF THE TEST METHOD 
The UDS test with mammalian liver cells  in vivo  indicates DNA 
repair synthesis after excision and removal of a stretch of DNA containing a region of damage induced by chemical substances or 
physical agents. The test is usually based on the incorporation of
 3 H- 
TdR into the DNA of liver cells which have a low frequency of cells in the S-phase of the cell cycle. The uptake of
 3 H-TdR is usually 
determined by autoradiography, since this technique is not as 
susceptible to interference from S-phrase cells as, for example, liquid scintillation counting.
 
1.4. DESCRIPTION OF THE METHOD  
1.4.1.  Preparations  
1.4.1.1.  Selection of animal species  
Rats are commonly used, although any appropriate mammalian 
species may be used. Commonly used laboratory strains of young healthy adult animals should be employed. At the commencement of 
the study the weight variation of animals should be minimal and not 
exceed ± 20 % of the mean weight for each sex.
 
1.4.1.2.  Housing and feeding conditions  
General conditions referred in the General introduction to Part B are 
applied although the aim for humidity should be 50-60 %.  
1.4.1.3.  Preparation of the animals  
Healthy young adult animals are randomly assigned to the control 
and treatment groups. Cages should be arranged in such a way that 
possible effects due to cage placement are minimised. The animals are identified uniquely and kept in their cages for at least five days prior to the start of the study to allow for acclimatisation to the 
laboratory conditions.
 
1.4.1.4.  Test substance/Preparation  
Solid test substances should be dissolved or suspended in appropriate 
solvents or vehicles and diluted, if appropriate, prior to dosing of the animals. Liquid test substances may be dosed directly or diluted 
prior to dosing. Fresh preparations of the test substance should be 
employed unless stability data demonstrate the acceptability of storage.
 
1.4.2.  Test conditions  
1.4.2.1.  Solvent/Vehicle  
The solvent/vehicle should not produce toxic effects at the dose 
levels used, and should not be suspected of chemical reaction with the test substance. If other than well-known solvents/vehicles are used, their inclusion should be supported with data indicating their 
compatibility. It is recommended that wherever possible, the use of 
an aqueous solvent/vehicle should be considered first.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 558
 
1.4.2.2.  Controls  
Concurrent positive and negative controls (solvent/vehicle) should be 
included in each independently performed part of the experiment. Except for treatment with the test substance, animals in the control 
group should be handled in an identical manner to the animals in the 
treated groups. 
Positive controls should be substances known to produce UDS when 
administered at exposure levels expected to give a detectable increase over background. Positive controls needing metabolic activation should be used at doses eliciting a moderate response 
(4). The doses may be chosen so that the effects are clear but do 
not immediately reveal the identity of the coded slides to the reader. Examples of positive control substances include:
 
Sampling Times Substance CAS No EINECS No  
Early sampling times (2-4 hours) N-Nitrosodimethylamine 62-75-9 200-249-8 
Late sampling times (12-16 hours) N-2-Fluorenylacetamide (2-AAF) 53-96-3 200-188-6 
Other appropriate positive control substances may be used. It is 
acceptable that the positive control should be administered by a 
route different from the test substance.  
1.5. PROCEDURE  
1.5.1.  Number and sex of animals  
An adequate number of animals should be used, to take account of 
natural biological variation in test response. The number of animals 
should be at least three analysable animals per group. Where a significant historical database has been accumulated, only one or two animals are required for the concurrent negative and positive 
control groups. 
If at the time of the study there are data available from studies in the 
same species and using the same route of exposure that demonstrate 
that there are no substantial differences in toxicity between sexes, then testing in a single sex, preferably males, will be sufficient. Where human exposure to chemicals may be sex-specific, as for 
example with some pharmaceutical agents, the test should be 
performed with animals of the appropriate sex.
 
1.5.2.  Treatment schedule  
Test substances are generally administered as a single treatment.  
1.5.3.  Dose levels  
Normally, at least two dose levels are used. The highest dose is 
defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected 
to produce lethality. In general, the lower dose should be 50 % to 
25 % of the high dose.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 559
 
Substances with specific biological activities at low non-toxic doses 
(such as hormones and mitogens) may be exceptions to the dose- 
setting criteria and should be evaluated on a case-by-case basis. If a range finding study is performed because there are no suitable data available, it should be performed in the same laboratory, using the 
same species, strain, sex, and treatment regimen to be used in the 
main study. 
The highest dose may also be defined as a dose that produces some 
indication of toxicity in the liver (e.g. pyknotic nuclei).
 
1.5.4.  Limit test  
If a test at one dose level of at least 2 000 mg/kg body weight, 
applied in a single treatment, or in two treatments on the same day, produces no observable toxic effects, and if genotoxicity would not be expected, based upon data from structurally related 
substances, then a full study may not be necessary. Expected 
human exposure may indicate the need for a higher dose level to be used in the limit test.
 
1.5.5.  Administration of doses  
The test substance is usually administered by gavage using a 
stomach tube or a suitable intubation cannula. Other routes of 
exposure may be acceptable where they can be justified. However, the intraperitoneal route is not recommended as it could expose the liver directly to the test substance rather than via the circulatory 
system. The maximum volume of liquid that can be administered 
by gavage or injection at one time depends on the size of the test animal. The volume should not exceed 2 ml/100 g body weight. The use of volumes higher than these must be justified. Except for irri ­
tating or corrosive substances, which will normally reveal 
exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels.
 
1.5.6.  Preparation of liver cells  
Liver cell are prepared from treated animals normally 12-16 hours 
after dosing. An additional earlier sampling time (normally two to four hours post-treatment) is generally necessary unless there is a clear positive response at 12-16 hours. However, alternative 
sampling times may be used when justified on the basis of toxi ­
cokinetic data. 
Short-term cultures of mammalian liver cells are usually established 
by perfusing the liver  in situ  with collagenase and allowing freshly 
dissociated liver cells to attach themselves to a suitable surface. 
Liver cells from negative control animals should have a viability (5) of at least 50 %.
 
1.5.7.  Determination of UDS  
Freshly isolated mammalian liver cells are incubated usually with 
medium containing  3 H-TdR for an appropriate length of time, e.g. 
3-8 hours. At the end of the incubation period, medium should be removed from the cells, which may then be incubated with medium containing excess unlabelled thymidine to diminish unincorporated 
radioactivity (‘cold chase’). The cells are then rinsed, fixed and 
dried. For more prolonged incubation times, cold chase may not be necessary. Slides are dipped in autoradiographic emulsion, exposed in the dark (e.g. refrigerated for 7-14 days), developed, 
stained, and exposed silver grains are counted. Two to three slides 
are prepared from each animal.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 560
 
1.5.8.  Analysis  
The slide preparations should contain sufficient cells of normal 
morphology to permit a meaningful assessment of UDS. Preparations are examined microscopically for signs of overt cytotoxicity (e.g. 
pyknosis, reduced levels of radiolabelling). 
Slides should be coded before grain counting. Normally 100 cells are 
scored from each animal from at least two slides; the scoring of less than 100 cells/animal should be justified. Grain counts are not scored for S-phase nuclei, but the proportion of S-phase cells may be 
recorded. 
The amount of
 3 H-TdR incorporation in the nuclei and the cytoplasm 
of morphologically normal cells, as evidenced by the deposition of silver grains, should be determined by suitable methods. 
Grain counts are determined over the nuclei (nuclear grains, NG) and 
nucleus equivalent areas over the cytoplasm (cytoplasmic grains, 
CG). CG counts are measured by either taking the most heavily labelled area of cytoplasm, or by taking an average of two to three random cytoplasmic grain counts adjacent to the nucleus. 
Other counting methods (e.g. whole cell counting) may be used if 
they can be justified (6).
 
2. DATA  
2.1. TREATMENT OF RESULTS 
Individual slide and animal data should be provided. Additionally, all 
data should be summarised in tabular form. Net nuclear grain (NNG) counts should be calculated for each cell, for each animal and for 
each dose and time by subtracting CG counts from NG counts. If 
‘cells in repair’ are counted, the criteria for defining ‘cells in repair’ should be justified and based on historical or concurrent negative control data. Numerical results may be evaluated by statistical 
methods. If used, statistical tests should be selected and justified 
prior to conducting the study.
 
2.2. EVALUATION AND INTERPRETATION OF RESULTS 
Examples of criteria for positive/negative responses include: 
positive (i) NNG values above a pre-set threshold which 
is justified on the basis of laboratory historical 
data; or 
(ii) NNG values significantly greater than 
concurrent control; 
negative (i) NNG values within/below historical control 
threshold; or 
(ii) NNG values not significantly greater than 
concurrent control.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 561
 
The biological relevance of data should be considered: i.e. 
parameters such as inter-animal variation, dose-response relationship 
and cytotoxicity should be taken into account. Statistical methods may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor 
for a positive response. 
Although most experiments will give clearly positive or negative 
results, in rare cases the data set will preclude making a definite 
judgement about the activity of the test substance. Results may 
remain equivocal or questionable regardless of the number of times the experiment is repeated. 
A positive result from the UDS test with mammalian liver cells  in 
vivo indicate that a test substance induces DNA damage in 
mammalian liver cells  in vivo  that can be repaired by unscheduled 
DNA synthesis  in vitro . A negative result indicates that, under the 
test conditions, the test substance does not induce DNA damage that is detectable by this test. 
The likelihood that the test substance reaches the general circulation 
or specifically the target tissue (e.g. systemic toxicity) should be discussed.
 
3. REPORTING  
TEST REPORT 
The test report must include the following information: 
Solvent/Vehicle: 
— justification for choice of vehicle, 
— solubility and stability of the test substance in solvent/vehicle, if 
known. 
Test animals: — species/strain used, 
— number, age and sex of animals, 
— source, housing conditions, diet, etc., — individual weight of the animals at the start of the test, including 
body weight range, mean and standard deviation for each group, 
Test conditions: 
— positive and negative vehicle/solvent controls, 
— data from range-finding study, if conducted, — rationale for dose level selection, — details of test substance preparation, 
— details of the administration of the test substance, 
— rationale for route of administration, — methods for verifying that test agent reached the general circu ­
lation or target tissue, if applicable,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 562
 
— conversion from diet/drinking water test substance concentration 
(ppm) to the actual dose (mg/kg body weight/day), if applicable, 
— details of food and water quality, 
— detailed description of treatment and sampling schedules, — methods for measurement of toxicity, — method of liver cell preparation and culture, — autoradiographic technique used, — number of slides prepared and numbers of cells scored, — evaluation criteria, 
— criteria for considering studies as positive, negative or equivocal, 
Results: — individual slide, animal and group mean values for nuclear 
grains, cytoplasmic grains, and net nuclear grains, 
— dose-response relationship, if available, — statistical evaluation if any, — signs of toxicity, — concurrent negative (solvent/vehicle) and positive control data, — historical negative (solvent/vehicle) and positive control data 
with range, means and standard deviations, 
— number of ‘cells in repair’ if determined, — number of S-phase cells if determined, — viability of the cells. Discussion of results. 
Conclusions.
 
4. REFERENCES  
(1) Ashby, J., Lefevre, P.A., Burlinson, B. and Penman, M.G., 
(1985) An Assessment of the  In Vivo  Rat Hepatocyte DNA 
Repair Assay. Mutation Res., 156, p. 1-18. 
(2) Butterworth, B.E., Ashby, J., Bermudez, E., Casciano, D., 
Mirsalis, J., Probst, G. and Williams, G., (1987) A Protocol and Guide for the  In Vivo  Rat Hepatocyte DNA-Repair Assay. 
Mutation Res., 189, p. 123-133. 
(3) Kennelly, J.C., Waters, R., Ashby, J., Lefevre, P.A., Burlinson, 
B., Benford, D.J., Dean, S.W. and Mitchell, I. de G., (1993) In 
Vivo Rat Liver UDS Assay. In: Kirkland D.J. and Fox M., 
(Eds) Supplementary Mutagenicity Tests: UKEM Recom ­
mended Procedures. UKEMS Subcommittee on Guidelines for Mutagenicity Testing. Report. Part II revised. Cambridge 
University Press, Cambridge, New York, Port Chester, 
Melbourne, Sydney, p. 52-77.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 563
 
(4) Madle, S., Dean, S.W., Andrae, U., Brambilla, G., Burlinson, 
B., Doolittle, D.J., Furihata, C., Hertner, T., McQueen, C.A. and 
Mori, H., (1993) Recommendations for the Performance of UDS Tests  In Vitro  and In Vivo . Mutation Res., 312, p. 263-285. 
(5) Fautz, R., Hussain, B., Efstathiou, E. and Hechenberger-Freudl, 
C., (1993) Assessment of the Relation Between the Initial 
Viability and the Attachment of Freshly Isolated Rat Hepa ­
tocytes Used for the  In Vivo/In Vitro  DNA Repair Assay 
(UDS). Mutation Res., 291, p. 21-27. 
(6) Mirsalis, J.C., Tyson, C.K. and Butterworth, B.E., (1982) 
Detection of Genotoxic Carcinogens in the  In Vivo/In Vitro  
Hepatocyte DNA Repair Assay. Environ. Mutagen, 4, 
p. 553-562.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 564
 
B.40.  IN VITRO  SKIN CORROSION: TRANSCUTANEOUS 
ELECTRICAL RESISTANCE TEST (TER)  
1. METHOD  
This testing method is equivalent to the OECD TG 430 (2004).  
1.1. INTRODUCTION 
Skin corrosion refers to the production of irreversible tissue damage 
in the skin following the application of a test material (as defined by the Globally Harmonised System for the Classification and Labelling 
of Chemical Substances and Mixtures (GHS)) (1). This method 
provides a procedure by which the assessment of corrosivity is not carried out in live animals. 
The assessment of skin corrosivity has typically involved the use of 
laboratory animals (2). Concern for the pain and suffering of animals involved with this procedure has been addressed in the revision of testing method B.4 that allows for the determination of skin 
corrosion by using alternative,  in vitro,  methods, avoiding pain and 
suffering. 
A first step towards defining alternative tests that could be used for 
skin corrosivity testing for regulatory purposes was the conduct of 
prevalidation studies (3). Following this, a formal validation study of  
in vitro  methods for assessing skin corrosion (4)(5) was conducted 
(6)(7)(8). The outcome of these studies and other published literature 
led to the recommendation that the following tests could be used for 
the assessment of in vivo skin corrosivity (9)(10)(11): the human skin model test (see tesing method B.40 bis) and the transcutaneous 
electrical resistance test (this method). 
A validation study and other published studies have reported that the 
rat skin transcutaneous electrical resistance (TER) assay (12)(13) is able to reliably discriminate between known skin corrosives and non- 
corrosives (5)(9). 
The test described in this method allows the identification of 
corrosive chemical substances and mixtures. It further enables the 
identification of non-corrosive substances and mixtures when 
supported by a weight of evidence determination using other existing information (e.g. pH, structure-activity-relationships, human and/or animal data) (1)(2)(11)(14). It does not provide 
information on skin irritation, nor does it allow the sub-categori ­
sation of corrosive substances as permitted in the Globally Harmonised Classification System (GHS) (1). 
For a full evaluation of local skin effects after a single dermal 
exposure, it is recommended to follow the sequential testing strategy as appended to testing method B.4 (2) and provided in the Globally Harmonised System (1). This testing strategy includes 
the conduct of  in vitro  tests for skin corrosion (as described in this 
method) and skin irritation before considering testing in live animals.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 565
 
1.2. DEFINITIONS  
Skin corrosion  in vivo : is the production of irreversible damage of 
the skin: namely, visible necrosis through the epidermis and into the dermis, following the application of a test subsance for up to 
four hours. Corrosive reactions are typified by ulcers, bleeding, 
bloody scabs, and, by the end of the observation at 14 days, by discolouration due to blanching of the skin, complete areas of alopecia, and scars. Histopathology should be considered to 
evaluate questionable lesions.  
Transcutaneous Electrical Resistance (TER):  is a measure of the 
electrical impendance of the skin, as a resistance value in kilo Ohms. 
A simple and robust method of assessing barrier function by recording the passage of ions through the skin using a Wheatstone bridge apparatus.
 
1.3. REFERENCE SUBSTANCES  
Table 1  
Reference chemicals  
Name EINECS No CAS No  
1,2-Diaminopropane 201-155-9 78-90-0 Severely corrosive 
Acrylic Acid 201-177-9 79-10-7 Severely Corrosive 2-tert. Butylphenol 201-807-2 88-18-6 Corrosive Potassium hydroxide (10 %) 215-181-3 1310-58-3 Corrosive Sulfuric acid (10 %) 231-639-5 7664-93-9 Corrosive Octanoic acid (caprylic acid) 204-677-5 124-07-02 Corrosive 4-Amino-1,2,4-triazole 209-533-5 584-13-4 Not corrosive Eugenol 202-589-1 97-53-0 Not corrosive Phenethyl bromide 203-130-8 103-63-9 Not corrosive Tetrachloroethylene 204-825-9 27-18-4 Not corrosive Isostearic acid 250-178-0 30399-84-9 Not corrosive 4-(Methylthio)-benzaldehyde 222-365-7 3446-89-7 Not corrosive 
Most of the chemicals listed are taken from the list of chemicals 
selected for the ECVAM international validation study (4). Their selection is based on the following criteria: 
(i) equal number of corrosive and non-corrosive substances;
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 566
 
(ii) commercially available substances covering most of the relevant 
chemical classes; 
(iii) inclusion of severely corrosive as well as less corrosive 
substances in order to enable discrimination based on corrosive potency; 
(iv) choice of chemicals that can be handled in a laboratory without 
posing other serious hazards than corrosivity.
 
1.4. PRINCIPLE OF THE TEST METHOD 
The test material is applied for up to 24 hours to the epidermal 
surfaces of skin discs in a two-compartment test system in which 
the skin discs function as the separation between the compartments. 
The skin discs are taken from humanely killed rats aged 28-30 days. Corrosive materials are identified by their ability to produce a loss of normal stratum corneum integrity and barrier function, which is 
measured as a reduction in the TER below a threshold level (12). 
For rat TER, a cut-off value of 5 k Ώ has been selected based on 
extensive data for a wide range of chemicals where the vast majority 
of values were either clearly well above (often > 10 k Ω), or well 
below (often < 3 k Ω) this value (12). Generally, materials which are 
non-corrosive in animals but are irritating or non-irritating do not reduce the TER below this cut-off value. Furthermore, use of other 
skin preparations or other equipment may alter the cut-off value, 
necessitating further validation. 
A dye-binding step is incorporated into the test procedure for confir ­
mation testing of positive results in the TER including values around 5 kΩ. The dye-binding step determines if the increase in ionic 
permeability is due to physical destruction of the stratum corneum. 
The TER method utilising rat skin has shown to be predictive of  in 
vivo corrosivity in the rabbit assessed under Testing Method B.4 (2). 
It should be noted that the  in vivo  rabbit test is highly conservative 
with respect to skin corrosivity and skin irritation when compared 
with the human skin patch test (15).
 
1.5. DESCRIPTION OF THE TEST METHOD  
1.5.1.  Animals  
Rats are the species of choice because the sensitivity of their skin to 
chemicals in this test has been previously demonstrated (10). The age (when the skin is collected) and strain of the rat is particularly 
important to ensure that the hair follicles are in the dormant phase 
before adult hair growth begins.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 567
 
The dorsal and flank hair from young, approximately 22 day-old, 
male or female rats (Wistar-derived or a comparable strain), is 
carefully removed with small clippers. Then, the animals are washed by careful wiping, whilst submerging the clipped area in antibiotic solution (containing, for example, streptomycin, penicillin, 
chloramphenicol, and amphotericin, at concentrations effective in 
inhibiting bacterial growth). Animals are washed with antibiotics again on the third or fourth day after the first wash and are used within three days of the second wash, when the stratum corneum has 
recovered from the hair removal.
 
1.5.2.  Preparation of the skin discs  
Animals are humanely killed when 28-30 days old; this age is 
critical. The dorso-lateral skin of each animal is then removed and stripped of excess subcutaneous fat by carefully peeling it away from 
the skin. Skin discs, with a diameter of approximately 20 mm each, 
are removed. The skin may be stored before disks are used where it is shown that positive and negative control data are equivalent to that obtained with fresh skin. 
Each skin disc is placed over one of the ends of a PTFE (polytetra ­
fluoroethylene) tube, ensuring that the epidermal surface is in contact with the tube. A rubber ‘O’ ring is press-fitted over the end of the tube to hold the skin in place and excess tissue is trimmed away. 
Tube and ‘O’ ring dimensions are shown in Figure 2. The rubber ‘O’ 
ring is then carefully sealed to the end of the PTFE tube with petroleum jelly. The tube is supported by a spring clip inside a receptor chamber containing MgSO
 4 solution (154 mM) (Figure 1). 
The skin disc should be fully submerged in the MgSO  4 solution. As 
many as 10-15 skin discs can be obtained from a single rat skin. 
Before testing begins, the electrical resistance of two skin discs is 
measured as a quality control procedure for each animal skin. Both 
discs should give resistance values greater than 10 k Ω for the 
remainder of the discs to be used for the test. If the resistance value is less than 10 k Ω, the remaining discs from that skin 
should be discarded.
 
1.5.3.  Application of the test and control substances  
Concurrent positive and negative controls should be used for each 
study to ensure adequate performance of the experimental model. Skin discs from a single animal should be used. The suggested 
positive and negative control substances are 10 M hydrochloric 
acid and distilled water, respectively. 
Liquid test substances (150 μL) are applied uniformly to the 
epidermal surface inside the tube. When testing solid materials, a 
sufficient amount of the solid is applied evenly to the disc to ensure 
that the whole surface of the epidermis is covered. Deionised water (150 μL) is added on top of the solid and the tube is gently agitated. 
In order to achieve maximum contact with the skin, solids may need 
to be warmed to 30
 o C to melt or soften the test substance, or ground 
to produce a granular material or powder.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 568
 
Three skin discs are used for each test and control substance. Test 
substances are applied for 24 hours at 20-23  o C. The test substance 
is removed by washing with a jet of tap water at up to 30  o C until no 
further material can be removed.  
1.5.4.  TER measurements  
The skin impedance is measured as TER is measured by using a 
low-voltage, alternating current Wheatstone databridge (13). General specifications of the bridge are 1-3 Volt operating voltage, a sinus or rectangular shaped alternating current of 50 – 1 000 Hz, and a 
measuring range of at least 0,1 – 30 k Ω. The databridge used in 
the validation study measureds inductance, capacitance and resistance up to values of 2 000 H, 2 000 μF, and 2 M Ω, respectively 
at frequencies of 100 Hz or 1 kHz, using series or parallel values. 
For the purposes of the TER corrosivity assay measurements are 
recorded in resistance, at a frequency of 100 Hz and using series values. Prior to measuring the electrical resistance, the surface tension of the skin is reduced by adding a sufficient volume of 
70 % ethanol to cover the epidermis. After a few seconds, the 
ethanol is removed from the tube and the tissue is then hydrated by the addition of 3 mL MgSO
 4 solution (154 mM). The databridge 
electrodes are placed on either side of the skin disc to measure the 
resistance in k Ω/skin disc (Figure 1). Electrode dimensions and the 
length of the electrode exposed below the crocodile clips are shown in Figure 2. The clip attached to the inner electrode is rested on the top of the PTFE tube during resistance measurement to ensure 
that a consistent length of electrode is submerged in the MgSO
 4 
solution. The outer electrode is positioned inside the receptor 
chamber so that it rests on the bottom of the chamber. The distance between the spring clip and the bottom of the PTFE 
tube is maintained as a constant (Figure 2), because this distance 
affects the resistance value obtained. Consequently, the distance between the inner electrode and the skin disc should be constant and minimal (1-2 mm). 
If the measured resistance value is greater than 20 k Ω, this may be 
due to the remains of the test substance coating the epidermal surface 
of the skin disc. Further removal of this coating can be attempted, 
for example, by sealing the PTFE tube with a gloved thumb and shaking it for approximately 10 seconds; the MgSO
 4 solution is 
discarded and the resistance measurement is repeated with fresh 
MgSO  4 . 
The properties and dimensions of the test apparatus and the experi ­
mental procedure used may influence the TER values obtained. The 
5 kΩ corrosive threshold was developed from data obtained with the 
specific apparatus and procedure described in this method. Different threshold and control values may apply if the test conditions are 
altered or a different apparatus is used. Therefore, it is necessary 
to calibrate the methodology and resistance threshold values by testing a series of reference standards chosen from the chemicals used in the validation study (4)(5), or from similar chemical 
classes to the chemicals being investigated. A set of suitable 
reference chemicals is shown in Table 1.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 569
 
1.5.5.  Dye binding methods  
Exposure of certain non-corrosive materials can result in a reduction 
of resistance below the cut-off of 5 k Ω allowing the passage of ions 
through the stratum corneum, thereby reducing the electrical 
resistance (5). For example, neutral organics and chemicals that 
have surface-active properties (including detergents, emulsifiers and other surfactants) can remove skin lipids making the barrier more permeable to ions. Thus, if the TER values of test substances are less 
than or around 5 k Ω in the absence of visual damage, an assessment 
of dye penetration should be carried out on the control and treated tissues to determine if the TER values obtained were the result 
of increased skin permeability, or skin corrosion (3)(5). In case 
of the latter where the stratum corneum is disrupted, the dye sulforhodamine B, when applied to the skin surface rapidly penetrates and stains the underlying tissue. This particular dye is 
stable to a wide range of chemicals and is not affected by the 
extraction procedure described below.
 
1.5.5.1.  Sulforhodamine B dye application and removal  
Following TER assessment, the magnesium sulfate is discarded from 
the tube and the skin is carefully examined for obvious damage. If there is no obvious major damage, Sulforhodamine B dye (Acid Red 52; C.I. 45100; EINECS Number 222-529-8; CAS number 3520-42-1), 150 μL 
of a 10 % (w/v) dilution in distilled water, is applied to the epidermal 
surface of each skin disc for two hours. These skin discs are then washed with tap water at up to room temperature for approximately 10 seconds to remove any excess/unbound dye. Each skin disc is 
carefully removed from the PTFE tube and placed in a vial (e.g. a 
20 mL glass scintillation vial) containing deionised water (8 mL). The vials are agitated gently for five minutes to remove any additional unbound dye. This rinsing procedure is then repeated, after which the 
skin discs are removed and placed into vials containing 5 ml of 30 % 
(w/v) sodium dodecyl sulphate (SDS) in distilled water and are incubated overnight at 60
 o C. 
After incubation, each skin disc is removed and discarded and the 
remaining solution is centrifuged for eight minutes at 21  o C (relative 
centrifugal force ~175 × g). A 1 ml sample of the supernatant is 
diluted 1 in 5 (v/v) [i.e. 1 mL + 4 mL] with 30 % (w/v) SDS in distilled water. The optical density (OD) of the solution is measured at 565 nm.
 
1.5.5.2.  Calculation of dye content  
The sulforhodamine B dye content per disc is calculated from the 
OD values (5) (sulforhodamine B dye molar extinction coefficient at 
565 nm = 8,7 × 10  4 ; molecular weight = 580). The dye content is 
determined for each skin disc by the use of an appropriate calibration curve and a mean dye content is then calculated for the replicates.
 
2. DATA  
Resistance values (k Ω) and mean dye content values ( μg/disc), where 
appropriate, for the test material, as well as for positive and negative controls should be reported in tabular form (individual trial data and 
means ± S.D.), including data for replicates/repeat experiments, 
mean and individual values.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 570
 
2.1. INTERPRETATION OF RESULTS 
The mean TER results are accepted if the concurrent positive and 
negative control values fall within the acceptable ranges for the method in the testing laboratory. The acceptable resistance ranges 
for the methodology and apparatus described above are given in the 
following table:
 
Control Substance Resistance 
range (kΩ) 
Positive 10M Hydrochloric acid 0,5-1,0 
Negative Distilled water 10-25 
The mean dye binding results are accepted on condition that 
concurrent control values fall within the acceptable ranges for the 
method. Suggested acceptable dye content ranges for the control substances for the methodology and apparatus described above are given below:
 
Control Substance Dye content 
range (μg/disc)  
Positive 10M Hydrochloric acid 40-100 
Negative Distilled water 15-35 
The test substance is considered to be non-corrosive to skin: 
(i) if the mean TER value obtained for the test substance is greater 
than 5 kΩ; or 
(ii) the mean TER value is less than or equal to 5 k Ω; and 
— the skin disc is showing no obvious damage, and — the mean disc dye content is well below the mean disc dye 
content of the 10M HCl positive control obtained concur ­
rently. 
The test substance is considered to be corrosive to skin: 
(i) if the mean TER value is less than or equal to 5 k Ω and the skin 
disk is obviously damaged; or 
(ii) the mean TER value is less than or equal to 5 k Ω; and 
— the skin disc is showing no obvious damage, but — the mean disc dye content is greater than or equal to the 
mean disc dye content of the 10M HCl positive control 
obtained concurrently.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 571
 
3. REPORTING  
3.1. TEST REPORT 
The test report must include the following information: 
Test and control substances: 
— chemical name(s) such as IUPAC or CAS name and CAS 
number, if known, 
— purity and composition of the substance or preparation (in 
percentage(s) by weight) and physical natur, 
— physico-chemical properties such as physical state, pH, stability, 
water solubility, relevant to the conduct of the study, 
— treatment of the test/control substances prior to testing, if 
applicable (e.g. warming, grinding), 
— stability, if known. 
Test animals: 
— strain and sex used, 
— age of the animals when used as donor animals, 
— source, housing condition, diet, etc, — details of the skin preparation. 
Test conditions: 
— calibration curves for test apparatus, — calibration curves for dye binding test performance, 
— details of the test procedure used for TER measurements, 
— details of the test procedure used for the dye binding assessment; 
if appropriate, 
— description of any modification of the test procedures, 
— description of evaluation criteria used. 
Results: 
— tabulation of data from the TER and dye binding assay (if appro ­
priate) for individual animals and individual skin samples; 
— description of any effects observed. 
Discussion of the results. Conclusions.
 
4. REFERENCES  
(1) OECD, (2001) Harmonised Integrated Classification System 
for Human Health and Environmental Hazards of Chemical Substances and Mixtures. OECD Series on Testing and Assessment Number 33. ENV/JM/MONO(2001)6, Paris. http:/ ­
/www.olis.oecd.org/olis/2001doc.nsf/LinkTo/env-jm- 
mono(2001)6.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 572
 
(2) Testing Method B.4. Acute Toxicity: Dermal Irritation/Corrosion. 
(3) Botham, P.A., Chamberlain, M., Barratt, M.D., Curren, R.D., 
Esdaile, D.J., Gardner, J.R., Gordon, V.C., Hildebrand, B., Lewis, R.W., Liebsch, M., Logemann, P., Osborne, R., Ponec, M., Regnier, J.F., Steiling, W., Walker, A.P., and 
Balls, M., (1995) A prevalidation study on  in vitro  skin 
corrosivity testing. The report and recommendations of ECVAM Workshop 6. ATLA 23, p. 219-255. 
(4) Barratt, M.D., Brantom, P.G., Fentem, J.H., Gerner, I., Walker, 
A.P., and Worth, A.P. (1998). The ECVAM international vali ­
dation study on  in vitro  tests for skin corrosivity. 1. Selection 
and distribution of the test chemicals. Toxicology.  In Vitro  12, 
p. 471-482. 
(5) Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, 
R.D., Earl, L.K., Esdaile, D.J., Holzhütter, H.-G., and Liebsch, M., (1998) The ECVAM international validation study on  in 
vitro  tests for skin corrosivity. 2. Results and evaluation by the 
Management Team. Toxicology.  In Vitro  12, p. 483- 524. 
(6) OECD, (1996) Final Report of the OECD Workshop on 
Harmonisation of Validation and Acceptance Criteria for Alter ­
native Toxicological Test Methods, p. 62. 
(7) Balls, M., Blaauboer, B.J., Fentem. J.H., Bruner. L., Combes, 
R.D., Ekwall, B., Fielder. R.J., Guillouzo, A., Lewis, R.W., Lovell, D.P., Reinhardt, C.A., Repetto, G., Sladowski. D., Spielmann, H., and Zucco, F., (1995) Practical aspects of the 
validation of toxicity test procedures. The report and recom ­
mendations of ECVAM workshops. ATLA 23, p. 129-147. 
(8) ICCVAM, (Interagency Coordinating Committee on the 
Validation of Alternative Methods)., (1997) Validation 
and Regulatory Acceptance of Toxicological Test Methods. NIH Publication No 97-3981. National Institute of Environmental Health Sciences, Research Triangle 
Park, NC, USA. http://iccvam.niehs.nih.gov/docs/guidelines/ 
validate.pdf. 
(9) ECVAM, (1998) ECVAM News & Views. ATLA 26, 
275-280. 
(10) ICCVAM (Interagency Coordinating Committee on the Vali ­
dation of Alternative Methods)., (2002) ICCVAM evaluation of EpiDerm
 TM , EPISKIN  TM (EPI-200), and the Rat Skin 
Transcutaneous Electrical Resistance (TER) assay: In Vitro 
test methods for assessing dermal corrosivity potential of 
chemicals. NIH Publication No 02-4502. National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods, National Institute of Environmental 
Health Sciences, Research Triangle Park, NC, USA. 
http://iccvam.niehs.nih.gov/methods/epiddocs/epis_brd.pdf. 
(11) OECD (2002) Extended Expert Consultation Meeting on The 
In Vitro Skin Corrosion Test Guideline Proposal, Berlin, 
1st–2nd November 2001, Secretariat's Final Summary Report, 
27
 th March 2002, OECD ENV/EHS, available upon request 
from the Secretariat. 
(12) Oliver, G.J.A., Pemberton, M.A., and Rhodes, C., (1986) An  
in vitro  skin corrosivity test -modificationsand validation. Fd. 
Chem. Toxicol. 24, 507-512.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 573
 
(13) Botham, P.A., Hall, T.J., Dennett, R., McCall, J.C., Basketter, 
D.A., Whittle, E., Cheeseman, M., Esdaile, D.J., and Gardner, 
J., (1992) The skin corrosivity test  in vitro:  results of an inter ­
laboratory trial. Toxic.  In Vitro  6, p. 191-194. 
(14) Worth A.P., Fentem J.H., Balls, M., Botham, P.A., Curren, 
R.D., Earl, L.K., Esdaile D.J., Liebsch, M., (1998) An 
Evaluation of the Proposed OECD Testing Strategy for Skin 
Corrosion. ATLA 26, p. 709-720 
(15) Basketter, D.A., Chamberlain, M., Griffiths, H.A., Rowson, 
M., Whittle, E., York, M., (1997) The classification of skin irritants by human patch test. Fd. Chem. Toxicol. 35, 
p. 845-852. 
(16) Oliver G.J.A, Pemberton M.A and Rhodes C., (1988) An In 
Vitro model for identifying skin-corrosive chemicals. I. Initial Validation. Toxicology  In Vitro . 2, p. 7-17.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 574
 
Figure 1  
Apparatus for the rat skin TER assay
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 575
 
Figure 2  
Dimensions of the polytetrafluoroethylene (PFTE) and receptor tubes and 
electrodes used
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 576
 
Critical factors of the apparatus shown above:  
— the inner diameter of the PTFE tube, — the length of the electrodes relative to the PTFE tube and 
receptor tube, such that the skin disc is not touched by the 
electrodes and that a standard length of electrode is in contact with the MgSO
 4 solution, 
— the amount of MgSO  4 solution in the receptor tube should give a 
depth of liquid, relative to the level in the PFTE tube, as shown 
in Figure 1, 
— the skin disk should be fixed well enough to the PFTE tube, such 
that the electrical resistance is a true measure of the skin prop ­
erties.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 577
 
B.40 BIS.  IN VITRO  SKIN CORROSION: HUMAN SKIN MODEL 
TEST  
1. METHOD  
This testing method is equivalent to the OECD TG 431 (2004).  
1.1. INTRODUCTION 
Skin corrosion refers to the production of irreversible tissue damage 
in the skin following the application of a test material [as defined by 
the Globally Harmonised System for the Classification and Labelling of Chemical Substances and Mixtures (GHS)] (1). This Testing Method does not require the use of live animals or animal tissue 
for the assessment of skin Corrosivity. 
The assessment of skin corrosivity has typically involved the use of 
laboratory animals (2). Concern for the pain and suffering involved with this procedure has been addressed in the revision of testing 
method B.4 that allows for the determination of skin corrosion by 
using alternative,  in vitro , methods, avoiding pain and suffering of 
animals. 
A first step towards defining alternative tests that could be used for 
skin corrosivity testing for regulatory purposes was the conduct of 
prevalidation studies (3). Following this, a formal, validation study 
of in vitro  methods for assessing skin corrosion (4)(5) was conducted 
(6)(7)(8). The outcome of these studies and other published literature (9) led to the recommendation that the following tests could be used 
for the assessment of the  in vivo  skin corrosivity (10)(11)12)(13): the 
human skin model test (this method) and the transcutaneous elec ­
trical resistance test (see testing method B.40). 
Validation studies have reported that tests employing human skin 
models (3)(4)(5)(9) are able to reliably discriminate between 
known skin corrosives and non-corrosives. The test protocol may also provide an indication of the distinction between severe and less severe skin corrosives. 
The test described in this method allows the identification of 
corrosive chemical substances and mixtures. It further allows the 
identification of non-corrosive substances and mixtures when supported by a weight of evidence determination using other existing information (e.g. pH, structure-activity relationships, 
human and/or animal data) (1)(2)(13)(14). It does not normally 
provide adequate information on skin irritation, nor does it allow the subcategorisation of corrosive substances as permitted in the Globally Harmonised Classification System (GHS) (1). 
For a full evaluation of local skin effects after single dermal 
exposure, it is recommended to follow the sequential testing 
strategy as appended to testing method B.4 (2) and provided in the Globally Harmonised System (GHS) (1). This testing strategy includes the conduct of  in vitro  tests for skin corrosion (as 
described in this method) and skin irritation before considering 
testing in live animals.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 578
 
1.2. DEFINITIONS  
Skin corrosion  in vivo : is the production of irreversible damage of 
the skin: namely, visible necrosis through the epidermis and into the dermis, following the application of a test subsance for up to 
four hours. Corrosive reactions are typified by ulcers, bleeding, 
bloody scabs, and, by the end of the observation at 14 days, by discolouration due to blanching of the skin, complete areas of alopecia, and scars. Histopathology should be considered to 
evaluate questionable lesions.  
Cell viability:  parameter measuring total activity of a cell population 
(e.g. ability of cellular mitochondrial dehidrogenases to reduce the vital dye MTT), which, depending on the end point measured and 
the test design used, correlates with the total number and/or vitality 
of the cells.
 
1.3. REFERENCE SUBSTANCES  
Table 1  
Reference chemicals  
Name EINECS No CAS No  
1,2-Diaminopropane 201-155-9 78-90-0 Severely corrosive 
Acrylic Acid 201-177-9 79-10-7 Severely corrosive 
2-tert. Butylphenol 201-807-2 88-18-6 Corrosive Potassium hydroxide (10 %) 215-181-3 1310-58-3 Corrosive Sulfuric acid (10 %) 231-639-5 7664-93-9 Corrosive Octanoic acid (caprylic acid) 204-677-5 124-07-02 Corrosive 4-Amino-1,2,4-triazole 209-533-5 584-13-4 Not corrosive 
Eugenol 202-589-1 97-53-0 Not corrosive 
Phenethyl bromide 203-130-8 103-63-9 Not corrosive Tetrachloroethylene 204-825-9 27-18-4 Not Corrosive Isostearic acid 250-178-0 30399-84-9 Not corrosive 4-(Methylthio)-benzaldehyde 222-365-7 3446-89-7 Not corrosive 
Most of the chemicals listed are taken from the list of chemicals 
selected for the ECVAM international validation study (4). Their selection is based on the following criteria: 
(i) equal number of corrosive and non-corrosive substances; 
(ii) commercially available substances covering most of the relevant 
chemical classes; 
(iii) inclusion of severely corrosive as well as less corrosive 
substances in order to enable discrimination based on 
corrosive potency;
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 579
 
(iv) choice of chemicals that can be handled in a laboratory without 
posing other serious hazards than corrosivity.  
1.4. PRINCIPLE OF THE TEST METHOD 
The test material is applied topically to a three-dimensional human 
skin model, comprising at least a reconstructed epidermis with a 
functional stratum corneum. Corrosive materials are identified by their ability to produce a decrease in cell viability (as determined, for example, by using the MTT reduction assay (15)) below defined 
threshold levels at specified exposure periods. The principle of the 
human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the underlying cell layers.
 
1.4.1.  Procedure  
1.4.1.1.  Human skin models  
Human skin models can be constructed or obtained commercially 
(e.g. the EpiDerm  TM and EPISKIN  TM models) (16)(17)(18)(19) or 
be developed or constructed in the testing laboratory (20)(21). It is recognised that the use of human skin is subject to national and international ethical considerations and conditions. Any new model 
should be validated (at least to the extent described under 1.4.1.1.2). 
Human skin models used for this test must comply with the following:
 
1.4.1.1.1. G e n e r a l m o d e l c o n d i t i o n s : 
Human keratinocytes should be used to construct the epithelium. 
Multiple layers of viable epithelial cells should be present under a 
functional stratum corneum. The skin model may also have a stromal component layer. Stratum corneum should be multi-layered with the necessary lipid profile to produce a functional barrier with robustness 
to resist rapid penetration of cytotoxic markers. The containment 
properties of the model should prevent passage of material around the stratum corneum to the viable tissue. Passage of test chemicals around the stratum corneum will lead to poor modeling of the 
exposure to skin. The skin model should be free of contamination 
with bacteria (including mycoplasma) or fungi.
 
1.4.1.1.2. F u n c t i o n a l m o d e l c o n d i t i o n s : 
The magnitude of viability is usually quantified by using MTT or the 
other metabolically converted vital dyes. In these cases the optical 
density (OD) of the extracted (solubilised) dye from the negative 
control tissue should be at least 20 fold greater than the OD of the extraction solvent alone (for an overview, see (22)). The negative control tissue should be stable in culture (provide similar 
viability measurements) for the duration of the test exposure period. 
The stratum corneum should be sufficiently robust to resist the rapid penetration of certain cytotoxic marker chemicals (e.g. 1 % Triton X- 100). This property can be estimated by the exposure time required 
to reduce cell viability by 50 % (ET
 50 ) (e.g. for the EpiDerm  TM and 
EPISKIN  TM models this is > 2 hours). The tissue should demonstrate 
reproductivity over time and preferably between laboratories. Moreover it should be capable of predicting the corrosive potential 
of the reference chemicals (see Table 1) when used in the testing 
protocol selected.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 580
 
1.4.1.2.  Application of the test and control substances  
Two tissue replicates are used for each treatment (exposure time), 
including controls. For liquid materials, sufficient test substance must be applied to uniformly cover the skin surface: a minimum of 25 
μL/cm
 2 should be used. For solid materials, sufficient test substance 
must be applied evenly to cover the skin, and it should be moistened with deionised or distilled water to ensure good contact with the skin. Where appropriate, solids should be ground to a powder 
before application. The application method should be appropriate 
for the test substance (see e.g. reference 5). At the end of the exposure period, the test material must be carefully washed from 
the skin surface with an appropriate buffer, or 0,9 % NaCl. 
Concurrent positive and negative controls should be used for each 
study to ensure adequate performance of the experimental model. The suggested positive control substances are glacial acetic acid or 8N KOH. The suggested negative controls are 0,9 % NaCl or water.
 
1.4.1.3.  Cell viability measurements  
Only quantitative, validated, methods can be used to measure cell 
viability. Furthermore, the measure of viability must be compatible 
with use in a three-dimensional tissue construct. Non-specific dye 
binding must not interfere with the viability measurement. Protein binding dyes and those which do not undergo metabolic conversion (e.g. neutral red) are therefore not appropriate. The most frequently 
used assay is MTT (3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetra ­
zolium bromide, Thiazolyl blue: EINECS number 206-069-5, CAS number 298-93-1)) reduction, which has been shown to give accurate and reproducible results (5) but others may be used. The skin sample 
is placed in an MTT solution of appropriate concentration (e.g. 0,3- 
1 mg/mL) at appropriate incubation temperature for three hours. The precipitated blue formazan product is then extracted using a solvent 
(isopropanol), and the concentration of the formazan is measured by 
determining the OD at wavelength between 540 and 595 nm. 
Chemical action by the test material on the vital dye may mimic that 
of cellular metabolism leading to a false estimate of viability. This has been shown to happen when such a test material is not 
completely removed from the skin by rinsing (9). If the test 
material directly acts on the vital dye, additional controls should be used to detect and correct for the test substances interference with the viability measurement (9)(23).
 
2. DATA  
For each tissue, OD values and calculated percentage cell viability 
data for the test material, positive and negative controls, should be 
reported in tabular form, including data from replicate repeat 
experiments as appropriate, mean and individual values.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 581
 
2.1. INTERPRETATION OF RESULTS 
The OD values obtained for each test sample can be used to 
calculate a percentage viability relative to the negative control, which is arbitrarily set at 100 %. The cut-off percentage cell 
viability value distinguishing corrosive from non-corrosive test 
materials (or discriminating between different, corrosive classes), or the statistical procedure(s) used to evaluate the results and identify corrosive materials, must be clearly defined and documented, and be 
shown to be appropriate. In general, these cut-off values are estab ­
lished during test optimisation, tested during a prevalidation phase, and confirmed in a validation study. As an example, the prediction 
of Corrosivity associated with the EpiDerm
 TM model is (9): 
The test substance is considered to be corrosive to skin: 
(i) if the viability after three minutes exposure is less than 50 %; or 
(ii) if the viability after three minutes exposure is greater than or 
equal to 50 % and the viability after 1 hour exposure is less than 
15 %. 
The test substance is considered to be non-corrosive to skin: (i) if the viability after three minutes exposure is greater than or 
equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 %.
 
3. REPORTING  
3.1. TEST REPORT 
The test report must include the following information: 
Test and control substance: — chemical names(s) such as IUPAC or CAS name and CAS 
number, if known, 
— purity and composition of the substance or preparation (in 
percentage(s) by weight), 
— physico-chemical properties such as physical state, pH, stability, 
water solubility relevant to the conduct of the study, 
— treatment of the test/control substances prior to testing, if 
applicable (e.g. warming, grinding), 
— stability, if known. Justification of the skin model and protocol used. Test conditions: — cell system used, — calibration information for measuring device used for measuring 
cell viability (e.g. Spectrophotometer),
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 582
 
— complete supporting information for the specific skin model used 
including its validity, 
— details of the test procedure used, — test doses used, 
— description of any modifications of the test procedure, 
— reference to historical data of the model, — description of evaluation criteria used. 
Results: 
— tabulation of data from individual test samples, — description of other effects observed. 
Discussion of the results. 
Conclusion.
 
4. REFERENCES  
(1) OECD (2001) Harmonised Integrated Classification System for 
Human Health and Environmental Hazards of Chemical 
Substances and Mixtures. OECD Series on Testing and Assessment Number 33. ENV/JM/MONO(2001)6, Paris. http://www.olis.oecd.org/olis/2001doc.nsf/LinkTo/env-jm- 
mono(2001)6. 
(2) Testing Method B.4. Acute Toxicity: Dermal Irritation/ 
Corrosion. 
(3) Botham, P.A., Chamberlain, M., Barratt, M.D., Curren, R.D., 
Esdaile, D.J., Gardner, J.R., Gordon, V.C., Hildebrand, B., Lewis, R.W., Liebsch, M., Logemann, P., Osborne, R., Ponec, M., Regnier, J.F., Steiling, W., Walker, A.P., and 
Balls, M., (1995) A prevalidation study on  in vitro  skin 
corrosivity testing. The report recommendations of ECVAM Workshop 6 ATLA 23, p. 219-255. 
(4) Barratt, M.D., Brantom, P.G., Fentem, J.H., Gerner, I., Walker, 
A.P., and Worth, A.P. (1998). The ECVAM international vali ­
dation study on  in vitro  tests for skin corrosivity. 1. Selection 
and distribution of the test chemicals. Toxicology In Vitro 12, p. 471-482. 
(5) Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, 
R.D., Earl, L.K., Esdaile, D.J., Holzhutter, H.G. and Liebsch, M., (1998) The ECVAM international validation study on  in 
vitro  tests for skin corrosivity. 2. Results and evaluation by the 
Management Team. Toxicology In Vitro 12, p. 483-524. 
(6) OECD, (1996) Final Report of the OECD Workshop on 
Harmonisation of Validation and Acceptance Criteria for Alter ­
native Toxicological Test Methods, p. 62. 
(7) Balls, M., Blaauboer, B.J., Fentem, J.H., Bruner, L., Combes, 
R.D., Ekwall, B., Fielder, R.J., Guillouzo, A., Lewis, R.W., 
Lovell, D.P., Reinhardt, C.A., Repetto, G., Sladowski, D., 
Spielmann, H., and Zucco, F., (1995) Practical aspects of the validation of toxicity test procedures. Test report and recom ­
mendations of ECVAM workshops. ATLA 23, p. 129-147. 
(8) ICCVAM (Interagency Coordinating Committee on the Vali ­
dation of Alternative Methods)., (1997) Validation and Regu ­
latory Acceptance of Toxicological Test Methods. NIH Publication No 97-3981. National Institute of Environmental 
Health Sciences, Research Triangle Park, NC, USA. 
http://iccvam.niehs.nih.gov/docs/guidelines/validate.pdf.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 583
 
(9) Liebsch, M., Traue, D., Barrabas, C., Spielmann, H., Uphill, 
P., Wilkins, S., McPherson, J.P., Wiemann, C., Kaufmann, T., 
Remmele, M. and Holzhutter, H.G., (2000) The ECVAM prevalidation study on the use of EpiDerm for skin Corrosivity testing. ATLA 28, p. 371-401. 
(10) ECVAM, (1998) ECVAM News & Views. ATLA 26, 
p. 275-280. 
(11) ECVAM, (2000) ECVAM News & Views. ATLA 28, 
p. 365-67. 
(12) ICCVAM (Interagency Coordinating Committee on the Vali ­
dation of Alternative Methods)., (2002) ICCVAM evaluation of EpiDerm
 TM , EPISKIN  TM (EPI-200), and the Rat Skin 
Transcutaneous Electrical Resistance (TER) assay: In Vitro 
test methods for assessing dermal corrosivity potential of chemicals. NIH Publication No 02-4502. National Toxicology 
Program Interagency Center for the Evaluation of Alternative 
Toxicological Methods, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA. http://iccvam.niehs.nih.gov/methods/epiddocs/epis_brd.pdf. 
(13) OECD, (2002) Extended Expert Consultation Meeting on The 
In Vitro Skin Corrosion Test Guideline Proposal, Berlin, 
1st–2nd November 2001, Secretariat's Final Summary Report, 27
 th March 2002, OECD ENV/EHS, available upon request 
from the Secretariat 
(14) Worth, A.P., Fentem, J.H., Balls, M., Botham, P.A., Curren, 
R.D., Earl, L.K., Esdaile, D.J., Liebsch, M., (1998) An 
Evaluation of the Proposed OECD Testing Strategy for Skin Corrosion. ATLA 26, p. 709-720. 
(15) Mosmann, T., (1983) Rapid colorimetric assay for cellular 
growth and survival: application to proliferation and cytot ­
oxicity asssays. J.Immunol. Meth. 65, p. 55-63. 
(16) Cannon, C.L., Neal, P.J., Southee, J.A., Kubilus, J., and 
Klausner, M., 1994. New epidermal model for dermal irritancy testing. Toxicology  In Vitro  8, p. 889-891. 
(17) Ponec, M., Boelsma, E., Weerheim, A., Mulder, A., Boutwstra, 
J., and Mommaas, M., 2000. Lipid and ultrastructural char ­
acterisation of reconstructed skin models. International Journal of Pharmaceutics. 203, p. 211-225. 
(18) Tinois, E., Gaetani, Q., Gayraud, B., Dupont, D., Rougier, A., 
Pouradier, D.X., (1994) The Episkin model: Successful recon ­
struction of human epidermis in vitro. In  In vitro  Skin Toxi ­
cology. Edited by A Rougier, AM Goldberg and HI Maibach, p. 133-140 
(19) Tinois E, Tiollier J, Gaucherand M, Dumas H, Tardy M, 
Thivolet J (1991).  In vitro  and post–transplantation differ ­
entiation of human keratinocytes grown on the human type IV collagen film of a bilayered dermal substitute. Experimental Cell Research 193, p. 310-319 
(20) Parentau, N.L., Bilbo, P., Molte, C.J., Mason, V.S., and 
Rosenberg, H., (1992) The organotypic culture of human 
skin keratinocytes and fibroblasts to achieve form and function. Cytotechnology 9, p. 163-171. 
(21) Wilkins, L.M., Watson, S.R., Prosky, S.J., Meunier, S.F., 
Parentau, N.L., (1994) Development of a bilayered living 
skin construct for clinical applications. Biotechnology and 
Bioengineering 43/8, p. 747-756.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 584
 
(22) Marshall, N.J., Goodwin, C.J., Holt, S.J., (1995) A critical 
assessment of the use of microculture tetrazolium assays to 
measure cell growth and function. Growth Regulation 5, p. 69-84. 
(23) Fentem, J.H., Briggs, D., Chesne’, C., Elliot, G.R., Harbell, 
J.W., Heylings, J.R., Portes, P., Rouget, R., and van de Sandt, 
J.J.M., and Botham, P.A., (2001) A prevalidation study on  in 
vitro  tests for acute skin irritation: results and evaluation by the 
Management Team. Toxicology  In Vitro  15, p. 57-93.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 585
 
B.41.  IN VITRO  3T3 NRU PHOTOTOXICITY TEST  
1. METHOD  
This method is equivalent to OECD TG 432 (2004).  
1.1. INTRODUCTION 
Phototoxicity is defined as a toxic response from a substance applied 
to the body which is either elicited or increased (apparent at lower 
dose levels) after subsequent exposure to light, or that is induced by skin irradiation after systemic administration of a substance. 
The in vitro  3T3 NRU phototoxicity test is used to identify the 
phototoxic potential of a test substance induced by the excited chemical after exposure to light. The test evaluates photo-cytot ­
oxicity by the relative reduction in viability of cells exposed to the 
chemical in the presence versus absence of light. Substances 
identified by this test are likely to be phototoxic  in vivo  following 
systemic application and distribution to the skin, or after topical application. 
Many types of chemicals have been reported to induce phototoxic 
effects (1)(2)(3)(4). Their common feature is their ability to absorb light energy within the sunlight range. According to the first law of photochemistry (Grotthaus-Draper Law), photoreaction requires 
sufficient absorption of light quanta. Thus, before biological testing 
is considered, a UV/vis absorption spectrum of the test chemical must be determined according to OECD Test Guideline 101. It has been suggested that if the molar extinction/absorption coefficient is 
less than 10 litre × mol
 -1 × cm  -1 the chemical is unlikely to be 
photoreactive. Such chemical may not need to be tested in the  in 
vitro  3T3 NRU phototoxicity test or any other biological test for 
adverse photochemical effects (1)(5). See also Appendix 1. 
The reliability and relevance of the  in vitro  3T3 NRU phototoxicity 
test was recently evaluated (6)(7)(8) (9). The  in vitro  3T3 NRU 
phototoxicity test was shown to be predictive of acute phototoxicity 
effects in animals and humans  in vivo . The test is not designed to 
predict other adverse effects that may arise from combined action of a chemical and light, e.g. it does not address photogenotoxicity, photoallergy, or photocarcinogenicity, nor does it allow an 
assessment of phototoxic potency. In addition, the test has not 
been designed to address indirect mechanisms of phototoxicity, effects of metabolites of the test substance, or effects of mixtures. 
Whereas the use of metabolising systems is a general requirement for 
all in vitro  tests for the prediction of genotoxic and carcinogenic 
potential, up to now, in the case of phototoxicology, there are only rare examples where metabolic transformation is needed for the chemical to act as a phototoxin  in vivo  or in vitro . Thus, it is 
neither considered necessary nor scientifically justified for the 
present test to be performed with a metabolic activation system.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 586
 
1.2. DEFINITIONS  
Irradiance:  the intensity of ultraviolet (UV) or visible light incident 
on a surface, measured in W/m  2 or mW/cm  2 . 
Dose of light:  the quantity ( = intensity × time) of ultraviolet (UV) or 
visible radiation incident on a surface, expressed in Joules (= W × s) 
per surface area, e.g. J/m  2 or J/cm  2 . 
UV light wavebands:  the designations recommended by the CIE 
(Commission Internationale de L'Eclairage) are: UVA (315- 
400 nm), UVB (280-315 nm) and UVC (100-280 nm). Other desig ­
nations are also used; the division between UVB and UVA is often placed at 320 nm, and the UVA may be divided into UV-A1 and 
UV-A2 with a division made at about 340 nm.  
Cell viability:  parameter measuring total activity of a cell population 
(e.g. uptake of the vital dye Neutral Red into cellular lysosomes), 
which, depending on the endpoint measured and the test design used, correlates with the total number and/or vitality of the cells.  
Relative cell viability:  cell viability expressed in relation of solvent 
(negative) controls which have been taken through the whole test procedure (either +Irr or -Irr) but not treated with test chemical.  
PIF (Photo-Irritation-Factor):  factor generated by comparing two 
equally effective cytotoxic concentrations (IC
 50 ) of the test chemical 
obtained in the absence (-Irr) and in the presence (+Irr) of a non- 
cytotoxic irradiation with UVA/vis light.  
IC 50 : the concentration of the test chemical by which the cell 
viability is reduced by 50 %.  
MPE (Mean-Photo-Effect):  measurement derived from math ­
ematical analysis of the concentration response curves obtained in the absence (-Irr) and in the presence (+Irr) of a non-cytotoxic irradiation with UVA/vis light.  
Phototoxicity:  acute toxic response that is elicited after the first 
exposure of skin to certain chemicals and subsequent exposure to 
light, or that is induced similarly by skin irradiation after systemic administration of a chemical.
 
1.3. PRINCIPLE OF THE TEST METHOD 
The in vitro  3T3 NRU phototoxicity test is based on a comparison of 
the cytotoxicity of a chemical when tested in the presence and in the absence of exposure to a non-cytotoxic dose of simulated solar light. Cytotoxicity in this test is expressed as a concentration-dependent 
reduction of the uptake of the vital dye Neutral Red when measured 
24 hours after treatment with the test chemical and irradiation (10). NR is a weak cationic dye that readily penetrates cell membranes by non-diffusion, accumulating intracellulary in lysosomes. Alterations 
of the surface of the sensitive lysosomal membrane lead to lysosomal 
fragility and other changes that gradually become irreversible. Such changes brought about by the action of xenobiotics result in a decreased uptake and binding of NR. It is thus possible to 
distinguish between viable, damaged or dead cells, which is the 
basis of this test.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 587
 
Balb/c 3T3 cells are maintained in culture for 24 h for formation of 
monolayers. Two 96-well plates per test chemical are pre-incubated 
with eight different concentrations of the test substance for 1 h. Thereafter one of the two plates is exposed to the highest non- cytotoxic irradiation dose whereas the other plate is kept in the 
dark. In both plates the treatment medium is then replaced by 
culture medium and after another 24 h of incubation cell viability is determined by Neutral Red uptake. Cell viability is expressed as percentage of untreated solvent controls and is calculated for each 
test concentration. To predict the phototoxic potential, the concen ­
tration responses obtained in the presence and in the absence of irradiation are compared, usually at the IC
 50 level, i.e., the concen ­
tration reducing cell viability to 50 % compared to the untreated 
controls.  
1.4. DESCRIPTION OF THE TEST METHOD  
1.4.1.  Preparations  
1.4.1.1.  Cells  
A permanent mouse fibroblast cell line, Balb/c 3T3, clone 31, either 
from the American Type Culture Collection (ATCC), Manassas, VA, USA, or from the European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, was used in the validation study, and 
therefore is recommended to obtain from a well qualified cell 
depository. Other cells or cell lines may be used with the same test procedure if culture conditions are adapted to the specific needs of the cells, but equivalency must be demonstrated. 
Cells should be checked regularly for the absence of mycoplasma 
contamination and only used if none is found (11). 
It is important that UV sensitivity of the cells is checked regularly 
according to the quality control procedure described in this method. 
Because the UVA sensitivity of cells may increase with the number 
of passages, Balb/c 3T3 cells of the lowest obtainable passage number, preferably less than 100, should be used. (See Section 1.4.2.2.2 and Appendix 2).
 
1.4.1.2.  Media and culture conditions  
Appropriate culture media and incubation conditions should be used 
for routine cell passage and during the test procedure, e.g. for Balb/c 3T3 cells these are DMEM (Dulbecco's Modified Eagle's Medium) 
supplemented with 10 % new-born calf serum, 4 mM glutamine, 
penicillin (100 IU), and streptomycin (100 μg/mL), and humidified 
incubation at 37
 o C, 5-7,5 % CO  2 depending on the buffer (See 
Section 1.4.1.4, second paragraph.). It is particularly important that 
cell culture conditions assure a cell cycle time within the normal 
historical range of the cells or cell line used.  
1.4.1.3.  Preparation of cultures  
Cells from frozen stock cultures are seeded in culture medium at an 
appropriate density and subcultured at least once before they are 
used in the  in vitro  3T3 NRU phototoxicity test.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 588
 
Cells used for the phototoxicity test are seeded in culture medium at 
the appropriate density so that cultures will not reach confluence by 
the end of the test, i.e., when cell viability is determined 48 h after seeding of the cells. For Balb/c 3T3 cells grown in 96-well plates, the recommended cell seeding density is 1 × 10
 4 cells per well. 
For each test chemical cells are seeded identically in two separate 
96-well plates, which are then taken concurrently through the entire 
test procedure under identical culture conditions except for the time period where one of the plates is irradiated (+Irr) and the other one is kept in the dark (-Irr).
 
1.4.1.4.  Preparation of test substance  
Test substances must be prepared fresh, immediately prior to use 
unless data demonstrate their stability in storage. It is recommended 
that all chemical handling and the initial treatment of cells be performed under light conditions that would avoid photoactivation or degradation of the test substance prior to irradiation. 
Test chemicals shall be dissolved in buffered salt solutions, e.g. 
Earle's Balanced Salt Solution (EBSS), or other physiologically 
balanced buffer solutions, which must be free from protein components, light absorbing components (e.g. pH-indicator colours and vitamins) to avoid interference during irradiation. Since during 
irradiation cells are kept for about 50 minutes outside of the CO
 2 
incubator, care has to be taken to avoid alkalisation. If weak buffers 
like EBSS are used this can be achieved by incubating the cells at 7,5 % CO
 2 . If the cells are incubated at 5 % CO  2 only, a stronger 
buffer should be selected. 
Test chemicals of limited solubility in water should be dissolved in 
an appropriate solvent. If a solvent is used it must be present at a constant volume in all cultures, i.e. in the negative (solvent) controls 
as well as in all concentrations of the test chemical, and be noncy ­
totoxic at that concentration. Test chemical concentrations should be selected so as to avoid precipitate or cloudy solutions. 
Dimethylsulphoxide (DMSO) and ethanol (ETOH) are the recom ­
mended solvents. Other solvents of low cytotoxicity may be appro ­
priate. Prior to use, all solvents should be assessed for specific 
properties, e.g. reaction with the test chemical, quenching of the phototoxic effect, radical scavenging properties and/or chemical stability in the solvent. 
Vortex mixing and/or sonication and/or warming to appropriate 
temperatures may be used to aid solubilisation unless this would 
affect the stability of the test chemical.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 589
 
1.4.1.5.  Irradiation conditions  
1.4.1.5.1. L i g h t s o u r c e 
The choice of an appropriate light source and filters is a crucial 
factor in phototoxicity testing. Light of the UVA and visible regions is usually associated with phototoxic reactions  in vivo  
(3)(12), whereas generally UVB is of less relevance but is highly 
cytotoxic; the cytotoxicity increases 1 000-fold as the wavelength 
goes from 313 to 280 nm (13). Criteria for the choice of an appro ­
priate light source must include the requirement that the light source emits wavelengths absorbed by the test chemical (absorption 
spectrum) and that the dose of light (achievable in a reasonable 
exposure time) should be sufficient for the detection of known photocytotoxic chemicals. Furthermore, the wavelengths and doses employed should not be unduly deleterious to the test system, e.g. 
the emission of heat (infrared region). 
Simulation of sunlight with solar simulators is considered the 
optimal artificial light source. The irradiation power distribution of the filtered solar simulator should be close to that of outdoor daylight given in (14). Both, Xenon arcs and (doped) mercury- 
metal halide arcs are used as solar simulators (15). The latter has 
the advantage of emitting less heat and being cheaper, but the match to sunlight is less perfect compared to that of xenon arcs. Because all solar simulators emit significant quantities of UVB they should be 
suitably filtered to attenuate the highly cytotoxic UVB wavelengths. 
Because cell culture plastic materials contain UV stabilisers the spectrum should be measured through the same type of 96-well 
plate lid as will be used in the assay. Irrespective of measures 
taken to attenuate parts of the spectrum by filtering or by unavoidable filter effects of the equipment the spectrum recorded below these filters should not deviate from standardised outdoor 
daylight (14). An example of the spectral irradiance distribution of 
the filtered solar simulator used in the validation study of the  in vitro  
3T3 NRU phototoxicity test is given in (8)(16). See also Appendix 2 Figure 1.
 
1.4.1.5.2. D o s i m e t r y 
The intensity of light (irradiance) should be regularly checked before 
each phototoxicity test using a suitable broadband UV-meter. The intensity should be measured through the same type of 96-well plate lid as will be used in the assay. The UV-meter must have been 
calibrated to the source. The performance of the UV-meter should 
be checked, and for this purpose the use of a second, reference UV- meter of the same type and identical calibration is recommended. Ideally, at greater intervals, a spectroradiometer should be used to 
measure the spectral irradiance of the filtered light source and to 
check the calibration of the broadband UV-meter. 
A dose of 5 J/cm
 2 (as measured in the UVA range) was determined 
to be non-cytotoxic to Balb/c 3T3 cells and sufficiently potent to excite chemicals to elicit phototoxic reactions, (6) (17) e.g. to 
achieve 5 J/cm
 2 within a time period of 50 min, irradiance was 
adjusted to 1,7 mW/cm  2 . See Appendix 2 Figure 2. If another cell 
line or a different light source are used, the irradiation dose may have to be calibrated so that a dose regimen can be selected that is 
not deleterious to the cells but sufficient to excite standard photo ­
toxins. The time of light exposure is calculated in the following way:
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 590
 
tðminÞ ¼ irradiation dose ðJ=cm 2 Þ Ü 1000 
irradiance ðmW =cm 2 Þ Ü 60 (1 J = 1 Wsec)  
1.4.2.  Test conditions  
1.4.2.1.  Test substance concentrations  
The ranges of concentrations of a chemical tested in the presence 
(+Irr) and in the absence (-Irr) of light should be adequately determined in dose range-finding experiments. It may be useful to assess solubility initially and at 60 min (or whatever treatment 
time is to be used), as solubility can change during time or during 
the course of exposure. To avoid toxicity induced by improper culture conditions or by highly acidic or alkaline chemicals, the pH of the cell cultures with added test chemical should be in the 
range 6,5 - 7,8. 
The highest concentration of the test substance should be within 
physiological test conditions, e.g. osmotic and pH stress should be avoided. Depending on the test chemical, it may be necessary to 
consider other physico-chemical properties as factors limiting the 
highest test concentration. For relatively insoluble substances that are not toxic at concentrations up to the saturation point the highest achievable concentration should be tested. In general, 
precipitation of the test chemical at any of the test concentrations 
should be avoided. The maximum concentration of a test substance should not exceed 1 000 μg/mL; osmolarity should not exceed 
10 mmolar. A geometric dilution series of eight test substance 
concentrations with a constant dilution factor should be used (See 
Section 2.1, second paragraph). 
If there is information (from a range finding experiment) that the test 
chemical is not cytotoxic up to the limit concentration in the dark 
experiment (-Irr), but is highly cytotoxic when irradiated (+Irr), the 
concentration ranges to be selected for the (+Irr) experiment may differ from those selected for the (-Irr) experiment to fulfill the requirement of adequate data quality.
 
1.4.2.2.  Controls  
1.4.2.2.1. R a d i a t i o n s e n s i t i v i t y o f t h e c e l l s , e s t a b l i s h i n g 
o f h i s t o r i c a l d a t a : 
Cells should be checked regularly (about every fifth passage) for 
sensitivity to the light source by assessing their viability following 
exposure to increasing doses of irradiation. Several doses of irradi ­
ation, including levels substantially greater than those used for the 3T3 NRU Phototoxicity test should be used in this assessment. These doses are easiest quantitated by measurements of UV parts 
of the light source. Cells are seeded at the density used in the  in vitro  
3T3 NRU phototoxicity test and irradiated the next day. Cell viability is then determined one day later using Neutral Red uptake. It should be demonstrated that the resulting highest non- 
cytotoxic dose (e.g. in the validation study: 5 J/cm
 2 [UVA]) was 
sufficient to classify the reference chemicals (Table 1) correctly.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 591
 
1.4.2.2.2. R a d i a t i o n s e n s i t i v i t y , c h e c k o f c u r r e n t t e s t : 
The test meets the quality criteria if the irradiated negative/solvent 
controls show a viability of more than 80 % when compared with 
non-irradiated negative/solvent.  
1.4.2.2.3. V i a b i l i t y o f s o l v e n t c o n t r o l s : 
The absolute optical density (OD  540 NRU  ) of the Neutral Red 
extracted from the solvent controls indicates whether the 1×10  4 
cells seeded per well have grown with a normal doubling time 
during the two days of the assay. A test meets the acceptance criteria if the mean OD
 540 NRU  of the untreated controls is ≥ 0,4 
(i.e. approximately 20 times the background solvent absorbance).  
1.4.2.2.4. P o s i t i v e c o n t r o l : 
A known phototoxic chemical shall be tested concurrently with each  
in vitro  3T3 NRU phototoxicity test. Chlorpromazine (CPZ) is 
recommended. For CPZ tested with the standard protocol in the  in 
vitro  3T3 NRU phototoxicity test, the following test acceptance 
criteria were defined: CPZ irradiated (+Irr): IC  50 = 0,1 to 2,0 
μg/ml, CPZ non-irradiated (-Irr): IC  50 = 7,0 to 90,0 μg/mL. The 
Photo Irritation Factor (PIF), should be > 6. The historical 
performance of the positive control should be monitored. 
Other phototoxic chemicals, suitable for the chemical class or solu ­
bility characteristics of the chemical being evaluated, may be used as the concurrent positive controls in place of chlorpromazine.
 
1.4.3.  Test procedure (6)(7)(8)(16)(17):  
1.4.3.1.  1st day:  
Dispense 100 μL culture medium into the peripheral wells of a 96- 
well tissue culture microtiter plate (= blanks). In the remaining wells, 
dispense 100 μL of a cell suspension of 1×10  5 cells/mL in culture 
medium (= 1×10  4 cells/well). Two plates should be prepared for 
each series of individual test substance concentrations, and for the 
solvent and positive controls. 
Incubate cells for 24 h (See Section 1.4.1.2) until they form a half 
confluent monolayer. This incubation period allows for cell recovery, adherence, and exponential growth.
 
1.4.3.2.  2nd day:  
After incubation, decant culture medium from the cells and wash 
carefully with 150 μL of the buffered solution used for incubation. 
Add 100 μL of the buffer containing the appropriate concentration of 
test chemical or solvent (solvent control). Apply eight different concentrations of the test chemical. Incubate cells with the test 
substance in the dark for 60 minutes (See Section 1.4.1.2 and 
1.4.1.4 second paragraph). 
From the two plates prepared for each series of test substance 
concentrations and the controls, one is selected, generally at random, for the determination of cytotoxicity (-Irr) (i.e., the control 
plate), and one (the treatment plate) for the determination of 
photocytotoxicity (+Irr).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 592
 
To perform the +Irr exposure, irradiate the cells at room temperature 
for 50 minutes through the lid of the 96-well plate with the highest 
dose of radiation that is non-cytotoxic (see also Appendix 2). Keep non-irradiated plates (-Irr) at room temperature in a dark box for 50 min (= light exposure time). 
Decant test solution and carefully wash twice with 150 μL of the 
buffered solution used for incubation, but not containing the test 
material. Replace the buffer with culture medium and incubate 
(See Section 1.4.1.2.) overnight (18-22 h).
 
1.4.3.3.  3rd day:  
1.4.3.3.1. M i c r o s c o p i c e v a l u a t i o n 
Cells should be examined for growth, morphology, and integrity of 
the monolayer using a phase contrast microscope. Changes in cell 
morphology and effects on cell growth should be recorded.  
1.4.3.3.2. N e u t r a l R e d u p t a k e t e s t 
Wash the cells with 150 μL of the pre-warmed buffer. Remove the 
washing solution by gentle tapping. Add 100 μL of a 50 μg/mL 
Neutral Red (NR) (3-amino-7-dimethylamino-2-methylphenazine 
hydrochloride, EINECS number 209-035-8; CAS number 553-24-2; C.I. 50040) in medium without serum (16) and incubate as described in paragraph 1.4.1.2., for 3 h. After incubation, remove the NR 
medium, and wash cells with 150 μL of the buffer. Decant and 
remove excess buffer by blotting or centrifugation. 
Add exactly 150 μL NR desorb solution (freshly prepared 49 parts 
water + 50 parts ethanol + 1 part acetic acid). 
Shake the microtiter plate gently on a microtiter plate shaker for 
10 min until NR has been extracted from the cells and has formed a homogeneous solution. 
Measure the optical density of the NR extract at 540 nm in a spec ­
trophotometer, using blanks as a reference. Save data in an appro ­
priate electronic file format for subsequent analysis.
 
2. DATA  
2.1. QUALITY AND QUANTITY OF DATA 
The test data should allow a meaningful analysis of the concen ­
tration-response obtained in the presence and in the absence of irradiation, and if possible the concentration of test chemical by 
which cell viability is reduced to 50 % (IC
 50 ). If cytotoxicity is 
found, both the concentration range and the intercept of individual concentrations shall be set in a way to allow the fit of a curve to the experimental data. 
For both clearly positive and clearly negative results (See Section 
2.3, first paragraph), the primary experiment, supported by one or 
more preliminary dose range-finding experiment(s), may be suffi ­
cient.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 593
 
Equivocal, borderline, or unclear results should be clarified by 
further testing (see also section 2.4, second paragraph). In such 
cases, modification of experimental conditions should be considered. Experimental conditions that might be modified include the concen ­
tration range or spacing, the pre-incubation time, and the irradiation- 
exposure time. A shorter exposure time may be appropriate for 
water-unstable chemicals.
 
2.2. EVALUATION OF RESULTS 
To enable evaluation of the data, a Photo-Irritation-Factor (PIF) or 
Mean Photo Effect (MPE) may be calculated. 
For the calculation of the measures of photocytotoxicity (see below) 
the set of discrete concentration-response values has to be 
approximated by an appropriate continuous concentration-response 
curve (model). Fitting of the curve to the data is commonly performed by a non-linear regression method (18). To assess the influence of data variability on the fitted curve a bootstrap 
procedure is recommended. 
A Photo-Irritation-Factor (PIF) is calculated using the following 
formula:
 
PIF ¼ IC 50 ðÄ IrrÞ 
IC 50 ðþ IrrÞ 
If an IC  50 in the presence or absence of light cannot be calculated, a 
PIF cannot be determined for the test material. The mean photo effect (MPE) is based on comparison of the complete concen ­
tration-response curves (19). It is defined as the weighted average 
across a representative set of photo effect values
 
MPE ¼ Σ 
i ¼ 1 n 
w i PE c i 
Σ 
i ¼ 1 n 
w i 
The photo effect PE  c at any concentration C is defined as the product 
of the response effect RE  c and the dose effect DE  c i.e. PE  c = RE  c × 
DE c . The response effect RE  c is the difference between the responses 
observed in the absence and presence of light, i.e. RE  c = R  c (-Irr) - 
R c (+Irr). The dose-effect is given by  
DE  c ¼ C=C ä Ä 1 
C=C ä þ 1 Æ Æ Æ Æ Æ Æ Æ Æ
 
where C* represents the equivalence concentration, i.e. the concen ­
tration at which the +Irr response equals the –Irr response at concen ­
tration C. If C* cannot be determined because the response values of 
the +Irr curve are systematically higher or lower than R  C (-Irr) the 
dose effect is set to 1. The weighting factors w  i are given by the 
highest response value, i.e. w  i = MAX {R  i (+Irr), R  i (-Irr) }. The 
concentration grid C  i is chosen such that the same number of points 
falls into each of the concentration intervals defined by the concen ­
tration values used in the experiment. The calculation of MPE is restricted to the maximum concentration value at which at least 
one of the two curves still exhibits a response value of at 
least 10 %. If this maximum concentration is higher than the highest concentration used in the +Irr experiment the residual part of the +Irr curve is set to the response value ‘0’. Depending on 
whether the MPE value is larger than a properly chosen cut-off 
value (MPE
 c = 0,15) or not, the chemical is classified as phototoxic.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 594
 
A software package for the calculation of the PIF and MPE is 
available from (20).  
2.3. INTERPRETATION OF RESULTS 
Based on the validation study (8), a test substance with a PIF < 2 or 
an MPE < 0,1 predicts: ‘no phototoxicity’. A PIF > 2 and < 5 or an MPE > 0,1 and < 0,15 predicts: ‘probable phototoxicity’; and a PIF 
> 5 or an MPE > 0,15 predicts: ‘phototoxicity’. 
For any laboratory initially establishing this assay, the reference 
materials listed in Table 1 should be tested prior to the testing of 
test substances for phototoxic assessment. PIF or MPE values should 
be close to the values mentioned in Table 1.
 
Table 1  
Chemical name EINECS No CAS No PIF MPE Absorption 
peak Solvent (  1 ) 
Amiodarone 
HCL 243-293-2 [19774-82-4] > 3,25 0,2-0,54 242 nm 
300 nm 
(shoulder) ethanol 
Choloropro ­
mazine HCL 200-701-3 [69-09-0] > 14,4 0,33-0,63 309 nm ethanol 
Norfloxacin 274-614-4 [70458-96-7] > 71,6 0,34-0,90 316 nm acetonitrile 
Anthracene 204-371-1 [120-12-7] > 18,5 0,19-0,81 356 nm acetonitrile Protoporphyrin 
IX, Disodium 256-815-9 [50865-01-5] > 45,3 0,54-0,74 402 nm ethanol 
L-Histidine [7006-35-1] no PIF 0,05-0,10 211 nm water 
Hexacholo ­
rophene 200-733-8 [70-30-4] 1,1-1,7 0,00-0,05 299 nm 
317 nm 
(shoulder) ethanol 
Sodium lauryl 
sulphate 205-788-1 [151-21-3] 1,0-1,9 0,00-0,05 no 
absorption water
 
( 1 ) Solvent used for measuring absorption.  
2.4. INTERPRETATION OF DATA 
If phototoxic effects are observed only at the highest test concen ­
tration, (especially for water soluble test chemicals) additional considerations may be necessary for assessment of hazard. These may include data on skin absorption, and accumulation of the 
chemical in the skin and/or data from other tests, e.g. testing of 
the chemical in  in vitro  animal or human skin, or skin models.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 595
 
If no toxicity is demonstrated (+Irr and -Irr), and if poor solubility 
limited the concentrations that could be tested, then the compatibility 
of the test substance with the assay may be questioned and confirmatory testing should be considered using, e.g. another model.
 
3. REPORTING  
TEST REPORT 
The test report must include at least the following information: 
Test substance: 
— identification data, common generic names and IUPAC and CAS 
number, if known, 
— physical nature and purity, — physicochemical properties relevant to conduct of the study, 
— UV/vis absorption spectrum, 
— stability and photostability, if known. Solvent: — justification for choice of solvent, — solubility of the test chemical in solvent, — percentage of solvent present in treatment medium. Cells: — type and source of cells, — absence of mycoplasma, — cell passage number, if known, — Radiation sensitivity of cells, determined with the irradiation 
equipment used in the  in vitro  3T3 NRU phototoxicity test. 
Test conditions (1);  incubation before and after treatment:  
— type and composition of culture medium, — incubation conditions (CO
 2 concentration; temperature; 
humidity), 
— duration of incubation (pre-treatment; post-treatment). Test conditions (2);  treatment with the chemical:  
— rationale for selection of concentrations of the test chemical used 
in the presence and in the absence of irradiation, 
— in case of limited solubility of the test chemical and absence of 
cytotoxicity: rationale for the highest concentration tested, 
— type and composition of treatment medium (buffered salt 
solution), 
— duration of the chemical treatment. Test conditions (3);  irradiation:  
— rationale for selection of the light source used,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 596
 
— manufacturer and type of light source and radiometer, 
— spectral irradiance characteristics of the light source, 
— transmission and absorption characteristics of the filter(s) used, 
— characteristics of the radiometer and details on its calibration, — distance of the light source from the test system, — UVA irradiance at this distance, expressed in mW/cm
 2 , 
— duration of the UV/vis light exposure, 
— UVA dose (irradiance × time), expressed in J/cm  2 , 
— temperature of cell cultures during irradiation and cell cultures 
concurrently kept in the dark. 
Test conditions (4);  Neutral Red viability test:  
— composition of Neutral Red treatment medium, 
— duration of Neutral Red incubation, — incubation conditions (CO
 2 concentration; temperature; 
humidity), 
— Neutral Red extraction conditions (extractant; duration), 
— wavelength used for spectrophotometric reading of Neutral Red 
optical density, 
— second wavelength (reference), if used, — content of spectrophotometer blank, if used. 
Results: 
— cell viability obtained at each concentration of the test chemical, 
expressed in percent viability of mean, concurrent solvent controls, 
— concentration response curves (test chemical concentration vs. 
relative cell viability) obtained in concurrent +Irr and -Irr experi ­
ments, 
— analysis of the concentration-response curves: if possible, 
computation/calculation of IC
 50 (+Irr) and IC  50 (-Irr), 
— comparison of the two concentration response curves obtained in 
the presence and in the absence of irradiation, either by calcu ­
lation of the Photo-Irritation-Factor (PIF), or by calculation of the 
Mean-Photo-Effect (MPE), 
— test acceptance criteria; concurrent solvent control: — absolute viability (optical density of Neutral Red extract) of 
irradiated and non-irradiated cells, 
— historic negative and solvent control data; means and standard 
deviations, 
— test acceptance criteria; concurrent positive control,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 597
 
— IC  50 (+Irr) and IC  50 (-Irr) and PIF/MPE of positive control 
chemical, 
— historic positive control chemical data: IC  50 (+Irr) and IC  50 (-Irr) 
and PIF/MPE; means and standard deviations. 
Discussion of the results. Conclusions.
 
4. REFERENCES  
(1) Lovell W.W., (1993) A scheme for  in vitro  screening of 
substances for photoallergenic potential. Toxicology  In Vitro  
7, p. 95-102. 
(2) Santamaria, L. and Prino, G., (1972) List of the photodynamic 
substances. In ‘Research Progress in Organic, Biological and 
Medicinal Chemistry’ Vol. 3 part 1. North Holland Publishing Co. Amsterdam. p. XI-XXXV. 
(3) Spielmann, H., Lovell, W.W., Hölzle, E., Johnson, B.E., 
Maurer, T., Miranda, M.A., Pape, W.J.W., Sapora, O., and Sladowski, D., (1994)  In vitro  phototoxicity testing: The 
report and recommendations of ECVAM Workshop 
2. ATLA, 22, p. 314-348. 
(4) Spikes, J.D., (1989) Photosensitisation. In ‘The science of 
Photobiology’ Edited by K.C. Smith. Plenum Press, New 
York. 2nd edition, p. 79-110. 
(5) OECD, (1997) Environmental Health and Safety Publications, 
Series on Testing and Assessment No 7 ‘Guidance Document On Direct Phototransformation Of Chemicals In Water’ Environment Directorate, OECD, Paris. 
(6) Spielmann, H., Balls, M., Döring, B., Holzhütter, H.G., 
Kalweit, S., Klecak, G., L'Eplattenier, H., Liebsch, M., Lovell, W.W., Maurer, T., Moldenhauer. F. Moore. L., Pape, 
W., Pfannbecker, U., Potthast, J., De Silva, O., Steiling, W., 
and Willshaw, A., (1994) EEC/COLIPA project on  in vitro  
phototoxicity testing: First results obtained with a Balb/c 3T3 cell phototoxicity assay. Toxic. In Vitro 8, p. 793-796. 
(7) Anon, (1998) Statement on the scientific validity of the 3T3 
NRU PT test (an  in vitro  test for phototoxicity), European 
Commission, Joint Research Centre: ECVAM and DGXI/E/2, 
3 November 1997, ATLA, 26, p. 7-8. 
(8) Spielmann, H., Balls, M., Dupuis, J., Pape, W.J.W., Pecho ­
vitch, G. De Silva, O., Holzhütter, H.G., Clothier, R., 
Desolle, P., Gerberick, F., Liebsch, M., Lovell, W.W., Maurer, T., Pfannenbecker, U., Potthast, J. M., Csato, M., 
Sladowski, D., Steiling, W., and Brantom, P., (1998) The inter ­
national EU/COLIPA  In vitro  phototoxicity validation study: 
results of phase II (blind trial), part 1: the 3T3 NRU photo ­
toxicity test. Toxicology  In Vitro  12, p. 305-327. 
(9) OECD, (2002) Extended Expert Consultation Meeting on The 
In Vitro 3T3 NRU Phototoxicity Test Guideline Proposal, Berlin, 30
 th -31 th October 2001, Secretariat's Final Summary 
Report, 15  th March 2002, OECD ENV/EHS, available upon 
request from the Secretariat. 
(10) Borenfreund, E., and Puerner, J.A., (1985) Toxicity deter ­
mination  in vitro  by morphological alterations and neutral 
red absorption. Toxicology Lett., 24, p. 119-124. 
(11) Hay, R.J., (1988) The seed stock concept and quality control 
for cell lines. Analytical Biochemistry 171, p. 225-237.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 598
 
(12) Lambert L.A, Warner W.G., and Kornhauser A., (1996) 
Animal models for phototoxicity testing. In ‘Dermatot ­
oxicology’, edited by F.N. Marzulli and H.I. Maibach. Taylor & Francis, Washington DC. 5th Edition, p. 515-530. 
(13) Tyrrell R.M., Pidoux M., (1987) Action spectra for human skin 
cells: estimates of the relative cytotoxicity of the middle ultra ­
violet, near ultraviolet and violet regions of sunlight on 
epidermal keratinocytes. Cancer Res., 47, p. 1825-1829. 
(14) ISO 10977., (1993) Photography — Processed photographic 
colour films and paper prints — Methods for measuring image stability. 
(15) Sunscreen Testing (UV.B) TECHNICALREPORT, CIE, Inter ­
national Commission on Illumnation, Publication No 90, Vienna, 1993, ISBN 3 900 734 275 
(16) ZEBET/ECVAM/COLIPA — Standard Operating Procedure:  
In Vitro  3T3 NRU Phototoxicity Test. Final Version, 7 
September, 1998. p. 18. 
(17) Spielmann, H., Balls, M., Dupuis, J., Pape, W.J.W., De Silva, 
O., Holzhütter, H.G., Gerberick, F., Liebsch, M., Lovell, W.W., and Pfannenbecker, U., (1998) A study on UV filter 
chemicals from Annex VII of the European Union  Directive 
76/768/EEC,  in the  in vitro  3T3 NRU phototoxicity test. 
ATLA 26, p. 679-708. 
(18) Holzhütter, H.G., and Quedenau, J., (1995) Mathematical 
modeling of cellular responses to external signals. J. Biol. 
Systems 3, p. 127-138. 
(19) Holzhütter, H.G., (1997) A general measure of  in vitro  photo­
toxicity derived from pairs of dose-response curves and its use for predicting the  in vivo  phototoxicity of chemicals. ATLA, 
25, p. 445-462. 
(20) http://www.oecd.org/document/55/ 
0,2340,en_2649_34377_2349687_1_1_1_1,00.html
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 599
 
Appendix 1  
Role of the 3T3 NRU PT in a sequential approach to the phototoxicity testing of chemicals
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 600
 
Appendix 2  
Figure 1  
Spectral power distribution of a filtered solar simulator
  
(see Section 1.4.1.5, second paragraph) 
Figure 1 gives an example of an acceptable spectral irradiance distribution of a filtered solar 
simulator. It is from the doped metal halide source used in the validation trial of the 3T3 NRU 
PT (6)(8)(17). The effect of two different filters and the additional filtering effect of the lid of a 96- 
well cell culture plate are shown. The H2 filter was only used with test systems that can tolerate a higher amount of UVB (skin model test and red blood cell photo-haemolysis test). In the 3T3 NRU- PT the H1 filter was used. The figure shows that additional filtering effect of the plate lid is mainly 
observed in the UVB range, still leaving enough UVB in the irradiation spectrum to excite 
chemicals typically absorbing in the UVB range, like Amiodarone (see Table 1).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 601
 
Figure 2  
Irradiation sensivity of Balb/c 3T3 cells (as measured in the UVA range)  
Cell viability (% Neutral Red uptake of dark controls)
  
(see Sections 1.4.1.5.2 second paragraph; 1.4.2.2.1, 1.4.2.2.2) 
Sensitivity of Balb/c 3T3 cells to irradiation with the solar simulator used in the validation trial 
of the 3T3NRU-phototoxicity test, as measured in the UVA range. Figure shows the results obtained in seven different laboratories in the pre-validation study (1). While the two curves 
with open symbols were obtained with aged cells (high number of passages), that had to be 
replaced by new cell stocks the curves with bold symbols show cells with acceptable irradiation tolerance. 
From these data the highest non-cytotoxic irradiation dose of 5 J/cm
 2 was derived (vertical dashed 
line). The horizontal dashed line shows in addition the maximum acceptable irradiation effect given 
in paragraph 1.4.2.2.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 602
 
B.42.  SKIN SENSITISATION: LOCAL LYMPH NODE ASSAY  
INTRODUCTION 
1. OECD Guidelines for the Testing of Chemicals and EU Test Methods based 
on them are periodically reviewed in light of scientific progress, changing regulatory needs, and animal welfare considerations. The original Test Method (TM) for the determination of skin sensitisation in the mouse, 
the Local Lymph Node Assay (LLNA; OECD Test Guideline 429; 
Chapter B.42 of this Annex) was adopted previously (1). The details of the validation of the LLNA and a review of the associated work have been published (2) (3) (4) (5) (6) (7) (8) (9) (10) (11). The updated LLNA is 
based on the evaluation of experience and scientific data (12). This is the 
second TM to be designed for assessing skin sensitisation potential of chemicals (substances and mixtures) in animals. The other TM ( i.e. 
OECD Test Guideline 406; Chapter B.6 of this Annex) utilises guinea 
pig tests, notably the guinea pig maximisation test and the Buehler test 
(13). The LLNA provides advantages over B.6 and OECD Test Guideline 406 (13) with regard to animal welfare. This updated LLNA TM includes a set of Performance Standards (PS) (Appendix 1) that can be used to 
evaluate the validation status of new and/or modified test methods that 
are functionally and mechanistically similar to the LLNA, in accordance with the principles of OECD Guidance Document No 34 (14). 
2. The LLNA studies the induction phase of skin sensitisation and provides 
quantitative data suitable for dose-response assessment. It should be noted that the mild/moderate sensitisers which are recommended as suitable 
positive control chemicals (PC) for guinea pig test methods ( i.e. B.6; 
OECD Test Guideline 406) (13) are also appropriate for use with the LLNA (6) (8) (15). A reduced LLNA (rLLNA) approach, which could use up to 40 % fewer animals is also described as an option in this TM 
(16) (17) (18). The rLLNA may be used when there is a regulatory need to 
confirm a negative prediction of skin sensitising potential, provided there is adherence to all other LLNA protocol specifications, as described in this TM. Prediction of a negative outcome should be made based on all 
available information as described in paragraph 4. Before applying the 
rLLNA approach, clear justifications and scientific rationale for its use should be provided. If, against expectations, a positive or equivocal result is obtained in the rLLNA, additional testing may be needed in order to 
interpret or clarify the finding. The rLLNA should not be used for the 
hazard identification of skin sensitising test substances when dose-response information is needed such as sub-categorisation for Regulation (EC) No 1272/2008 on classification, labelling and packaging 
of substances and mixtures and UN Globally Harmonised System of Clas ­
sification and Labelling of Chemicals.
 
DEFINITIONS 
3. Definitions used are provided in Appendix 2.  
INITIAL CONSIDERATIONS AND LIMITATIONS 
4. The LLNA provides an alternative method for identifying potential skin 
sensitising chemicals. This does not necessarily imply that in all instances 
the LLNA should be used in place of guinea pig tests ( i.e. B.6; OECD Test 
Guideline 406) (13), but rather that the assay is of equal merit and may be employed as an alternative in which positive and negative results generally no longer require further confirmation. The testing laboratory should 
consider all available information on the test substance prior to conducting 
the study. Such information will include the identity and chemical structure
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 603
 
of the test substance; its physicochemical properties; the results of any other  
in vitro  or in vivo  toxicity tests on the test substance; and toxicological data 
on structurally related chemicals. This information should be considered in order to determine whether the LLNA is appropriate for the substance (given the incompatibility of limited types of chemicals with the LLNA 
— see paragraph 5) and to aid in dose selection. 
5. The LLNA is an  in vivo  method and, as a consequence, will not eliminate 
the use of animals in the assessment of allergic contact sensitising activity. 
It has, however, the potential to reduce the number of animals required for this purpose. Moreover, the LLNA offers a substantial refinement (less pain and distress) of the way in which animals are used for allergic contact 
sensitisation testing. The LLNA is based upon consideration of immuno ­
logical events stimulated by chemicals during the induction phase of sensiti ­
sation. Unlike guinea pig tests ( i.e. B.6; OECD Test Guideline 406) (13) the 
LLNA does not require that challenge-induced dermal hypersensitivity 
reactions be elicited. Furthermore, the LLNA does not require the use of 
an adjuvant, as is the case for the guinea pig maximisation test (13). Thus, the LLNA reduces animal pain and distress. Despite the advantages of the LLNA over B.6 and OECD Test Guideline 406, it should be recognised that 
there are certain limitations that may necessitate the use of B.6 or OECD 
Test Guideline 406 (13) ( e.g. false negative findings in the LLNA with 
certain metals, false positive findings with certain skin irritants (such as some surfactant type chemicals) (19) (20), or solubility of the test 
substance). In addition, chemical classes or substances containing functional 
groups shown to act as potential confounders (21) may necessitate the use of guinea pig tests ( i.e. B.6; OECD Test Guideline 406) (13). Further, based 
on the limited validation database, which consisted primarily of pesticide 
formulations, the LLNA is more likely than the guinea pig test to yield a 
positive result for these types of test substances (22). However, when testing formulations, one could consider including similar substances with known results as benchmark substances to demonstrate that the LLNA is 
functioning properly (see paragraph 16). Other than such identified limi ­
tations, the LLNA should be applicable for testing any substances unless there are properties associated with these substances that may interfere with 
the accuracy of the LLNA.
 
PRINCIPLE OF THE TEST 
6. The basic principle underlying the LLNA is that sensitisers induce prolif ­
eration of lymphocytes in the lymph nodes draining the site of test 
substance application. This proliferation is proportional to the dose and to the potency of the applied allergen and provides a simple means of obtaining a quantitative measurement of sensitisation. Proliferation is 
measured by comparing the mean proliferation in each test group to the 
mean proliferation in the vehicle treated control (VC) group. The ratio of the mean proliferation in each treated group to that in the concurrent VC group, termed the Stimulation Index (SI), is determined, and should be ≥ 3 
before classification of the test substance as a potential skin sensitiser is 
warranted. The procedures described here are based on the use of  in vivo  
radioactive labelling to measure an increased number of proliferating cells in the draining auricular lymph nodes. However, other endpoints for 
assessment of the number of proliferating cells may be employed 
provided the PS requirements are fully met (Appendix 1).
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 604
 
DESCRIPTION OF THE ASSAY  
Selection of animal species  
7. The mouse is the species of choice for this test. Young adult female mice of 
CBA/Ca or CBA/J strain, which are nulliparous and non-pregnant, are used. 
At the start of the study, animals should be between 8-12 weeks old, and the weight variation of the animals should be minimal and not exceed 20 % of the mean weight. Alternatively, other strains and males may be used 
when sufficient data are generated to demonstrate that significant strain 
and/or gender-specific differences in the LLNA response do not exist.
 
Housing and feeding conditions  
8. Mice should be group-housed (23), unless adequate scientific rationale for 
housing mice individually is provided. The temperature of the experimental 
animal room should be 22 ± 3 °C. Although the relative humidity should be 
at least 30 % and preferably not exceed 70 %, other than during room cleaning, the aim should be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional 
laboratory diets may be used with an unlimited supply of drinking water.
 
Preparation of animals  
9. The animals are randomly selected, marked to permit individual identifi ­
cation (but not by any form of ear marking), and kept in their cages for at least five days prior to the start of dosing to allow for acclimatisation to the laboratory conditions. Prior to the start of treatment all animals are 
examined to ensure that they have no observable skin lesions.
 
Preparation of dosing solutions  
10. Solid chemicals should be dissolved or suspended in solvents/vehicles and 
diluted, if appropriate, prior to application to an ear of the mice. Liquid chemicals may be applied neat or diluted prior to dosing. Insoluble chemicals, such as those generally seen in medical devices, should be 
subjected to an exaggerated extraction in an appropriate solvent to reveal 
all extractable constituents for testing prior to application to an ear of the mice. Test substances should be prepared daily unless stability data demon ­
strate the acceptability of storage.
 
Reliability check  
11. Positive control chemicals (PC) are used to demonstrate appropriate 
performance of the assay by responding with adequate and reproducible 
sensitivity as a sensitising test substance for which the magnitude of the response is well characterised. Inclusion of a concurrent PC is recom ­
mended because it demonstrates competency of the laboratory to 
successfully conduct each assay and allows for an assessment of intra- 
and inter-laboratory reproducibility and comparability. A PC for each study is also required by some regulatory authorities and therefore users are encouraged to consult the relevant authorities prior to conducting the 
LLNA. Accordingly, the routine use of a concurrent PC is encouraged to 
avoid the need for additional animal testing to meet such requirements that might arise from the use of a periodic PC (see paragraph 12). The PC should produce a positive LLNA response at an exposure level expected 
to give an increase in the SI > 3 over the negative control (NC) group. The 
PC dose should be chosen such that it does not cause excessive skin
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 605
 
irritation or systemic toxicity and the induction is reproducible but 
not excessive ( i.e. a SI > 20 would be excessive). Preferred PC are 
25 % hexyl cinnamic aldehyde (Chemical Abstracts Service (CAS) No 101-86-0) in acetone: olive oil (4:1, v/v) and 5 % mercaptobenzothiazole (CAS No 149-30-4) in  N,N -dimethylformamide (see Appendix 1, Table 1). 
There may be circumstances in which, given adequate justification, other PC, 
meeting the above criteria, may be used. 
12. While inclusion of a concurrent PC group is recommended, there may be 
situations in which periodic testing ( i.e. at intervals ≤ 6 months) of the PC 
may be adequate for laboratories that conduct the LLNA regularly ( i.e. 
conduct the LLNA at a frequency of no less than once per month) and have an established historical PC database that demonstrates the labora ­
tory’s ability to obtain reproducible and accurate results with PCs. 
Adequate proficiency with the LLNA can be successfully demonstrated by generating consistent positive results with the PC in at least 10 inde ­
pendent tests conducted within a reasonable period of time ( i.e. less than 
one year). 
13. A concurrent PC group should always be included when there is a 
procedural change to the LLNA ( e.g. change in trained personnel, change 
in test method materials and/or reagents, change in test method equipment, change in source of test animals), and such changes should be documented in laboratory reports. Consideration should be given to the impact of these 
changes on the adequacy of the previously established historical database in 
determining the necessity for establishing a new historical database to document consistency in the PC results. 
14. Investigators should be aware that the decision to conduct a PC study on a 
periodic basis instead of concurrently has ramifications on the adequacy and acceptability of negative study results generated without a concurrent PC during the interval between each periodic PC study. For example, if a false 
negative result is obtained in the periodic PC study, negative test substance 
results obtained in the interval between the last acceptable periodic PC study and the unacceptable periodic PC study may be questioned. Impli ­
cations of these outcomes should be carefully considered when determining 
whether to include concurrent PCs or to only conduct periodic PCs. 
Consideration should also be given to using fewer animals in the concurrent PC group when this is scientifically justified and if the laboratory demon ­
strates, based on laboratory-specific historical data, that fewer mice can be 
used (12). 
15. Although the PC should be tested in the vehicle that is known to elicit a 
consistent response ( e.g. acetone: olive oil; 4:1, v/v), there may be certain 
regulatory situations in which testing in a non-standard vehicle (clinically/ chemically relevant formulation) will also be necessary (24). If the concurrent PC is tested in a different vehicle than the test substance, then 
a separate VC for the concurrent PC should be included. 
16. In instances where test substances of a specific chemical class or range of 
responses are being evaluated, benchmark substances may also be useful to 
demonstrate that the test method is functioning properly for detecting the skin sensitisation potential of these types of test substances. Appropriate benchmark substances should have the following properties: 
— structural and functional similarity to the class of the test substance 
being tested; 
— known physical/chemical characteristics; 
— supporting data from the LLNA; 
— supporting data from other animal models and/or from humans.
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TEST PROCEDURE  
Number of animals and dose levels  
17. A minimum of four animals is used per dose group, with a minimum of 
three concentrations of the test substance, plus a concurrent NC group 
treated only with the vehicle for the test substance, and a PC (concurrent or recent, based on laboratory policy in considering paragraphs 11-15). Testing multiple doses of the PC should be considered, especially when 
testing the PC on an intermittent basis. Except for absence of treatment with 
the test substance, animals in the control groups should be handled and treated in a manner identical to that of animals in the treatment groups. 
18. Dose and vehicle selection should be based on the recommendations given 
in references (3) and (5). Consecutive doses are normally selected from an appropriate concentration series such as 100 %, 50 %, 25 %, 10 %, 5 %, 2,5 %, 1 %, 0,5 %, etc. Adequate scientific rationale should accompany the 
selection of the concentration series used. All existing toxicological 
information ( e.g. acute toxicity and dermal irritation) and structural and 
physicochemical information on the test substance of interest (and/or struc ­
turally related substances) should be considered where available, in 
selecting the three consecutive concentrations so that the highest concen ­
tration maximises exposure while avoiding systemic toxicity and/or excessive local skin irritation (3) (25). In the absence of such information, an initial pre-screen test may be necessary (see paragraphs 21-24). 
19. The vehicle should not interfere with or bias the test result and should be 
selected on the basis of maximising the solubility in order to obtain the highest concentration achievable while producing a solution/suspension 
suitable for application of the test substance. Recommended vehicles are 
acetone: olive oil (4:1, v/v),  N,N -dimethylformamide, methyl ethyl ketone, 
propylene glycol, and dimethyl sulphoxide (19) but others may be used if sufficient scientific rationale is provided. In certain situations it may be 
necessary to use a clinically relevant solvent or the commercial formulation 
in which the test substance is marketed as an additional control. Particular care should be taken to ensure that hydrophilic test substances are incor ­
porated into a vehicle system, which wets the skin and does not 
immediately run off, by incorporation of appropriate solubilisers ( e.g. 1 % 
Pluronic® L92). Thus, wholly aqueous vehicles are to be avoided. 
20. The processing of lymph nodes from individual mice allows for the 
assessment of inter-animal variability and a statistical comparison of the 
difference between test substance and VC group measurements (see 
paragraph 35). In addition, evaluating the possibility of reducing the number of mice in the PC group is feasible when individual animal data are collected (12). Further, some regulatory authorities require the collection 
of individual animal data. Nonetheless, pooled animal data may be 
considered acceptable by some regulatory authorities and in such situations, users may have the option of collecting either individual or pooled animal data.
 
Pre-screen test  
21. In the absence of information to determine the highest dose to be tested (see 
paragraph 18), a pre-screen test should be performed in order to define the appropriate dose level to test in the LLNA. The purpose of the pre-screen 
test is to provide guidance for selecting the maximum dose level to use in 
the main LLNA study, where information on the concentration that induces systemic toxicity (see paragraph 24) and/or excessive local skin irritation (see paragraph 23) is not available. The maximum dose level tested should 
be 100 % of the test substance for liquids or the maximum possible concen ­
tration for solids or suspensions.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 607
 
22. The pre-screen test is conducted under conditions identical to the main 
LLNA study, except there is no assessment of lymph node proliferation 
and fewer animals per dose group can be used. One or two animals per dose group are suggested. All mice will be observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body 
weights are recorded pre-test and prior to termination (Day 6). Both ears of 
each mouse are observed for erythema and scored using Table 1 (25). Ear thickness measurements are taken using a thickness gauge ( e.g. digital 
micrometer or Peacock Dial thickness gauge) on Day 1 (pre-dose), Day 3 
(approximately 48 hours after the first dose), and Day 6. Additionally, on 
Day 6, ear thickness could be determined by ear punch weight deter ­
minations, which should be performed after the animals are humanely killed. Excessive local skin irritation is indicated by an erythema score ≥ 
3 and/or an increase in ear thickness of ≥ 25 % on any day of measurement 
(26) (27). The highest dose selected for the main LLNA study will be the next lower dose in the pre-screen concentration series (see paragraph 18) that does not induce systemic toxicity and/or excessive local skin irritation.
 
Table 1  
Erythema Scores  
Observation Score  
No erythema 0 
Very slight erythema (barely perceptible) 1 Well-defined erythema 2 
Moderate to severe erythema 3 
Severe erythema (beet redness) to eschar formation preventing 
grading of erythema 4 
23. In addition to a 25 % increase in ear thickness (26) (27), a statistically 
significant increase in ear thickness in the treated mice compared to control mice has also been used to identify irritants in the LLNA (28) (29) (30) (31) (32) (33) (34). However, while statistically significant 
increases can occur when ear thickness is less than 25 % they have not 
been associated specifically with excessive irritation (30) (32) (33) (34). 
24. The following clinical observations may indicate systemic toxicity (35) (36) 
when used as part of an integrated assessment and therefore may indicate the maximum dose level to use in the main LLNA: changes in nervous 
system function ( e.g. pilo-erection, ataxia, tremors, and convulsions); 
changes in behaviour ( e.g. aggressiveness, change in grooming activity, 
marked change in activity level); changes in respiratory patterns ( i.e. 
changes in frequency and intensity of breathing such as dyspnea, gasping, 
and rales), and changes in food and water consumption. In addition, signs 
of lethargy and/or unresponsiveness and any clinical signs of more than slight or momentary pain and distress, or a > 5 % reduction in body weight from Day 1 to Day 6, and mortality should be considered in the evaluation. 
Moribund animals or animals obviously in pain or showing signs of severe 
and enduring distress should be humanely killed (37).
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 608
 
Main study experimental schedule  
25. The experimental schedule of the assay is as follows: 
— Day 1:  Individually identify and record the weight of each animal and 
any clinical observation. Apply 25 μL of the appropriate dilution of the 
test substance, the vehicle alone, or the PC (concurrent or recent, based 
on laboratory policy in considering paragraphs 11-15), to the dorsum of 
each ear. 
— Days 2 and 3:  Repeat the application procedure carried out on Day 1. 
— Days 4 and 5:  No treatment. 
— Day 6:  Record the weight of each animal. Inject 250 μL of sterile 
phosphate-buffered saline (PBS) containing 20 μCi (7,4 × 10  5 Bq) of 
tritiated (  3 H)-methyl thymidine into all test and control mice via the tail 
vein. Alternatively, inject 250 μL sterile PBS containing 2 μCi (7,4 × 
10 4 Bq) of  125 I-iododeoxyuridine and 10  –5 M fluorodeoxyuridine into all 
mice via the tail vein. Five hours (5 h) later, humanely kill the animals. 
Excise the draining auricular lymph nodes from each mouse ear and process together in PBS for each animal (individual animal approach); 
alternatively excise and pool the lymph nodes from each ear in PBS for 
each treatment group (pooled treatment group approach). Details and diagrams of the lymph node identification and dissection can be found in reference (12). To further monitor the local skin response in the main 
study, additional parameters such as scoring of ear erythema or ear 
thickness measurements (obtained either by using a thickness gauge, or ear punch weight determinations at necropsy) may be included in the study protocol.
 
Preparation of cell suspensions  
26. A single-cell suspension of lymph node cells (LNC) excised bilaterally 
using the individual animal approach or alternatively, the pooled treatment group approach is prepared by gentle mechanical disaggregation 
through 200 micron-mesh stainless steel gauze or another acceptable 
technique for generating a single-cell suspension. The LNC are washed twice with an excess of PBS and the DNA is precipitated with 5 % trich ­
loroacetic acid (TCA) at 4 °C for 18h (3). Pellets are either resuspended in 
1 mL TCA and transferred to scintillation vials containing 10 mL of scin ­
tillation fluid for
 3 H-counting, or transferred directly to gamma counting 
tubes for  125 I-counting.  
Determination of cellular proliferation (incorporated radioactivity)  
27. Incorporation of  3 H-methyl thymidine is measured by β-scintillation counting as 
disintegrations per minute (DPM). Incorporation of  125 I-iododeoxyuridine is 
measured by  125 I-counting and also is expressed as DPM. Depending on the 
approach used, the incorporation is expressed as DPM/mouse (individual animal 
approach) or DPM/treatment group (pooled treatment group approach).  
Reduced LLNA  
28. In certain situations, when there is a regulatory need to confirm a negative 
prediction of skin sensitising potential, an optional rLLNA protocol (16) 
(17) (18) using fewer animals may be used, provided there is adherence to all other LLNA protocol specifications in this TM. Before applying the rLLNA approach, clear justifications and scientific rationale for its use 
should be provided. If a positive or equivocal result is obtained, additional 
testing may be needed in order to interpret or clarify the finding.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 609
 
29. The reduction in number of dose groups is the only difference between the 
LLNA and the rLLNA test method protocols and for this reason the rLLNA 
does not provide dose-response information. Therefore, the rLLNA should not be used when dose-response information is needed. Like the multi-dose LLNA, the test substance concentration evaluated in the rLLNA should be 
the maximum concentration that does not induce overt systemic toxicity 
and/or excessive local skin irritation in the mouse (see paragraph 18).
 
OBSERVATIONS  
Clinical observations  
30. Each mouse should be carefully observed at least once daily for any clinical 
signs, either of local irritation at the application site or of systemic toxicity. All observations are systematically recorded with records being maintained 
for each mouse. Monitoring plans should include criteria to promptly 
identify those mice exhibiting systemic toxicity, excessive local skin irri ­
tation, or corrosion of skin for euthanasia (37).
 
Body weights  
31. As stated in paragraph 25, individual animal body weights should be 
measured at the start of the test and at the scheduled humane kill.  
CALCULATION OF RESULTS 
32. Results for each treatment group are expressed as the SI. When using the 
individual animal approach, the SI is derived by dividing the mean 
DPM/mouse within each test substance group, and the PC group, by the mean DPM/mouse for the solvent/VC group. The average SI for the VCs is then one. When using the pooled treatment group approach, the SI is 
obtained by dividing the pooled radioactive incorporation for each 
treatment group by the incorporation of the pooled VC group; this yields a mean SI. 
33. The decision process regards a result as positive when SI ≥ 3. However, the 
strength of the dose-response, the statistical significance and the consistency 
of the solvent/vehicle and PC responses may also be used when determining 
whether a borderline result is declared positive (4) (5) (6). 
34. If it is necessary to clarify the results obtained, consideration should be 
given to various properties of the test substance, including whether it has a structural relationship to known skin sensitisers, whether it causes excessive 
local skin irritation in the mouse, and the nature of the dose-response 
relationship seen. These and other considerations are discussed in detail elsewhere (7). 
35. Collecting radioactivity data at the level of the individual mouse will enable 
a statistical analysis for presence and degree of dose-response relationship in the data. Any statistical assessment could include an evaluation of the 
dose-response relationship as well as suitably adjusted comparisons of test 
groups ( e.g. pair-wise dosed group versus concurrent VC comparisons). 
Statistical analyses may include,  e.g. linear regression or William’s test to 
assess dose-response trends, and Dunnett’s test for pair-wise comparisons. 
In choosing an appropriate method of statistical analysis, the investigator 
should maintain an awareness of possible inequalities of variances and other related problems that may necessitate a data transformation or a non-para ­
metric statistical analysis. In any case the investigator may need to carry out 
SI calculations and statistical analyses with and without certain data points 
(sometimes called ‘outliers’).
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 610
 
DATA AND REPORTING  
Data  
36. Data should be summarised in tabular form. When using the individual 
animal approach, show the individual animal DPM values, the group 
mean DPM/animal, its associated error term ( e.g. SD, SEM), and the 
mean SI for each dose group compared against the concurrent VC group. When using the pooled treatment group approach, show the mean/median 
DPM and the mean SI for each dose group compared against the concurrent 
VC group.
 
Test report  
37. The test report should contain the following information: 
Test and control substances: 
— identification data ( e.g. CAS and EC numbers, if available; source; 
purity; known impurities; lot number); 
— physical nature and physicochemical properties ( e.g. volatility, stability, 
solubility); 
— if mixture, composition and relative percentages of components; 
Solvent/vehicle: 
— identification data (purity; concentration, where appropriate; volume 
used); 
— justification for choice of vehicle; Test animals: — source of CBA mice; — microbiological status of the animals, when known; 
— number and age of animals; 
— source of animals, housing conditions, diet, etc.; Test conditions: — details of test substance preparation and application; — justification for dose selection (including results from pre-screen test, if 
conducted); 
— vehicle and test substance concentrations used, and total amount of test 
substance applied; 
— details of food and water quality (including diet type/source, water 
source); 
— details of treatment and sampling schedules; — methods for measurement of toxicity; 
— criteria for considering studies as positive or negative; 
— details of any protocol deviations and an explanation on how the 
deviation affects the study design and results; 
Reliability check: — summary of results of latest reliability check, including information on 
test substance, concentration and vehicle used; 
— concurrent and/or historical PC and concurrent NC data for testing 
laboratory;
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 611
 
— if a concurrent PC was not included, the date and laboratory report 
for the most recent periodic PC and a report detailing the historical 
PC data for the laboratory justifying the basis for not conducting a concurrent PC; 
Results: — individual weights of mice at start of dosing and at scheduled kill; as 
well as mean and associated error term ( e.g. SD, SEM) for each 
treatment group; 
— time course of onset and signs of toxicity, including dermal irritation at 
site of administration, if any, for each animal; 
— a table of individual mouse (individual animal approach) or mean/ 
median (pooled treatment group approach) DPM values and SI values for each treatment group; 
— mean and associated error term ( e.g. SD, SEM) for DPM/mouse for 
each treatment group and the results of outlier analysis for each 
treatment group when using the individual animal approach; 
— calculated SI and an appropriate measure of variability that takes into 
account the inter-animal variability in both the test substance and control groups when using the individual animal approach; 
— dose-response relationship; — statistical analyses, where appropriate; Discussion of results: — a brief commentary on the results, the dose-response analysis, and stat ­
istical analyses, where appropriate, with a conclusion as to whether the 
test substance should be considered a skin sensitiser.
 
LITERATURE 
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H.J., Lehmann, P. and Vohr, V.W. (1998), An integrated model for the 
differentiation of chemical-induced allergic and irritant skin reactions,  
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(30) Woolhiser, M.R., Hayes, B.B. and Meade, B.J. (1998), A combined murine 
local lymph node and irritancy assay to predict sensitisation and irritancy potential of chemicals,  Toxicol. Meth. , 8, 245-256. 
(31) Hayes, B.B. and Meade, B.J. (1999), Contact sensitivity to selected acrylate 
compounds in B6C3F1 mice: relative potency, cross reactivity, and 
comparison of test methods,  Drug. Chem. Toxicol. , 22, 491-506. 
(32) Ehling, G., Hecht, M., Heusener, A., Huesler, J., Gamer, A.O., van 
Loveren, H., Maurer, T., Riecke, K., Ullmann, L., Ulrich, P., Vandebriel, 
R. and Vohr, H.W. (2005), A European inter-laboratory validation of alter ­
native endpoints of the murine local lymph node assay: first round.  Toxicol. , 
212, 60-68. 
(33) Vohr, H.W. and Ahr, H.J. (2005), The local lymph node assay being too 
sensitive?  Arch. Toxicol. , 79, 721-728. 
(34) Patterson, R.M., Noga, E. and Germolec, D. (2007), Lack of evidence for 
contact sensitisation by Pfiesteria extract,  Environ. Health Perspect. , 115, 
1023-1028. 
(35) OECD (1987),  Acute Dermal Toxicity , OECD Guideline for Testing of 
Chemicals No 402, Paris, France. Available at: [http://www.oecd.org/env/ 
testguidelines]
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(36) ICCVAM (2009), Report on the ICCVAM-NICEATM/ECVAM/JaCVAM 
Scientific Workshop on Acute Chemical Safety Testing: Advancing In Vitro 
Approaches and Humane Endpoints for Systemic Toxicity Evaluations. Research Triangle Park, NC: National Institute of Environmental Health Sciences, Available at: [http://iccvam.niehs.nih.gov/methods/acutetox/ 
Tox_workshop.htm] 
(37) OECD (2000),  Guidance Document on the Recognition, Assessment and 
Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation , Environmental Health and Safety Monograph, 
Series on Testing and Assessment No 19, ENV/JM/MONO(2000)7, OECD, 
Paris. Available at: [http://www.oecd.org/env/testguidelines]
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 615
 
Appendix 1  
Performance standards for assessment of proposed similar or modified llna 
test methods for skin sensitisation  
INTRODUCTION 
1. The purpose of Performance Standards (PS) is to communicate the basis by 
which new test methods, both proprietary ( i.e. copyrighted, trademarked, 
registered) and non-proprietary can be determined to have sufficient accuracy and reliability for specific testing purposes. These PS, based on validated and accepted test methods, can be used to evaluate the reliability 
and accuracy of other similar methods (colloquially referred to as ‘me-too’ 
tests) that are based on similar scientific principles and measure or predict the same biological or toxic effect (14). 
2. Prior to adoption of modified methods ( i.e. proposed potential 
improvements to an approved test method), there should be an evaluation 
to determine the effect of the proposed changes on the test’s performance and the extent to which such changes affect the information available for 
the other components of the validation process. Depending on the number 
and nature of the proposed changes, the generated data and supporting documentation for those changes, they should either be subjected to the same validation process as described for a new test, or, if appropriate, to 
a limited assessment of reliability and relevance using established PS (14). 
3. Similar or modified methods proposed for use under this TM should be 
evaluated to determine their reliability and accuracy using chemicals repre ­
senting the full range of the LLNA scores. To avoid unwarranted animal 
use, it is strongly recommended that model developers consult the appro ­
priate authorities before starting validation studies in accordance with the PS and guidance provided in this TM. 
4. These PS are based on the US-ICCVAM, EC-ECVAM and 
Japanese-JaCVAM harmonised PS (12), for evaluating the validity of similar or modified versions of the LLNA. The PS consists of essential test method components, recommended reference chemicals and standards 
for accuracy and reliability that the proposed method should meet or 
exceed.
 
I. Essential test method components  
5. To ensure that a similar or modified LLNA method is functionally and 
mechanistically analogous to the LLNA and measures the same biological 
effect, the following components should be included in the test method protocol: 
— The test substance should be applied topically to both ears of the mouse; 
— Lymphocyte proliferation should be measured in the lymph nodes 
draining from the site of test substance application; 
— Lymphocyte proliferation should be measured during the induction 
phase of skin sensitisation;
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— For test substances, the highest dose selected should be the maximum 
concentration that does not induce systemic toxicity and/or excessive 
local skin irritation in the mouse. For positive reference chemicals, the highest dose should be at least as high as the LLNA EC3 values of the corresponding reference chemicals (see Table 1) without producing 
systemic toxicity and/or excessive local skin irritation in the mouse; 
— A concurrent VC should be included in each study and, where appro ­
priate, a concurrent PC should also be used; 
— A minimum of four animals per dose group should be used; — Either individual or pooled animal data may be collected. If any of these criteria are not met, then these PS cannot be used for 
validation of the similar or modified method.
 
II. Minimum list of reference chemicals  
6. The US-ICCVAM, EC-ECVAM and Japanese-JaCVAM harmonised PS 
(12) identified 18 minimum reference chemicals that should be used and four optional reference chemicals ( i.e. substances that produced either false 
positive or false negative results in the LLNA, when compared to human 
and guinea pig results (B.6, or OECD Test Guideline 406) (13), and 
therefore provide the opportunity to demonstrate equal to or better performance than the LLNA) that are included in the LLNA PS. The selection criteria for identifying these chemicals were: 
— The list of reference chemicals represented the types of substances 
typically tested for skin sensitisation potential and the range of responses that the LLNA is capable of measuring or predicting; 
— The substances had well-defined chemical structures; — LLNA data from guinea pig tests ( i.e. B.6; OECD Test Guideline 406) 
(13) and (where possible) data from humans were available for each 
substance; and 
— The substances were readily available from a commercial source. The recommended reference chemicals are listed in Table 1. Studies using 
the proposed reference chemicals should be evaluated in the vehicle with which they are listed in Table 1. In situations where a listed substance may 
not be available, other substances that meet the selection criteria mentioned 
may be used, with adequate justification.
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Table 1  
Recommended Reference chemicals for the LLNA PS.  
Number Chemicals (  1 ) CAS No Form Veh (  2 ) EC3 % (  3 ) N (  4 ) 0,5x-2,0x EC3 Actual EC3 
Range LLNA vs GP LLNA vs 
Human  
1 5-Chloro-2-methyl-4-isothiazolin-3-one (CMI)/2- 
methyl-4-isothiazolin-3-one (MI) (  5 ) 26172-55-4/ 
2682-20-4 Liq DMF 0,009 1 0,0045-0,018 NC +/+ +/+ 
2 DNCB 97-00-7 Sol AOO 0,049 15 0,025-0,099 0,02-0,094 +/+ +/+ 
3 4-Phenylenediamine 106-50-3 Sol AOO 0,11 6 0,055-0,22 0,07-0,16 +/+ +/+ 
4 Cobalt chloride 7646-79-9 Sol DMSO 0,6 2 0,3-1,2 0,4-0,8 +/+ +/+ 5 Isoeugenol 97-54-1 Liq AOO 1,5 47 0,77-3,1 0,5-3,3 +/+ +/+ 6 2-Mercaptobenzothiazole 149-30-4 Sol DMF 1,7 1 0,85-3,4 NC +/+ +/+ 7 Citral 5392-40-5 Liq AOO 9,2 6 4,6-18,3 5,1-13 +/+ +/+ 8 HCA 101-86-0 Liq AOO 9,7 21 4,8-19,5 4,4-14,7 +/+ +/+ 
9 Eugenol 97-53-0 Liq AOO 10,1 11 5,05-20,2 4,9-15 +/+ +/+ 
10 Phenyl benzoate 93-99-2 Sol AOO 13,6 3 6,8-27,2 1,2-20 +/+ +/+ 11 Cinnamic alcohol 104-54-1 Sol AOO 21 1 10,5-42 NC +/+ +/+ 12 Imidazolidinyl urea 39236-46-9 Sol DMF 24 1 12-48 NC +/+ +/+ 13 Methyl methacrylate 80-62-6 Liq AOO 90 1 45-100 NC +/+ +/+ 14 Chlorobenzene 108-90-7 Liq AOO 25 1 NA NA –/– –/ (*) 15 Isopropanol 67-63-0 Liq AOO 50 1 NA NA –/– –/+
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Number Chemicals (  1 ) CAS No Form Veh (  2 ) EC3 % (  3 ) N (  4 ) 0,5x-2,0x EC3 Actual EC3 
Range LLNA vs GP LLNA vs 
Human  
16 Lactic acid 50-21-5 Liq DMSO 25 1 NA NA –/– –/ (*) 
17 Methyl salicylate 119-36-8 Liq AOO 20 9 NA NA –/– –/– 18 Salicylic acid 69-72-7 Sol AOO 25 1 NA NA –/– –/– 
Optional Substances to Demonstrate Improved Performance Relative to the LLNA 
19 Sodium lauryl sulphate 151-21-3 Sol DMF 8,1 5 4,05-16,2 1,5-17,1 +/– +/– 20 Ethylene glycol dimethacrylate 97-90-5 Liq MEK 28 1 14-56 NC +/– +/+ 21 Xylene 1330-20-7 Liq AOO 95,8 1 47,9-100 NC +/ (*) +/– 22 Nickel chloride 7718-54-9 Sol DMSO 5 2 NA NA –/+ –/+
 
Abbreviations: AOO = acetone: olive oil (4:1, v/v); CAS No = Chemical Abstracts Service Number; DMF =  N,N -dimethylformamide; DMSO = dimethyl sulfoxide; DNCB = 2,4-dinitrochlorobenzene; EC3 = estimated 
concentration needed to produce a stimulation index of 3; GP = guinea pig test result ( i.e. B.6 or OECD Test Guideline 406) (13); HCA = hexyl cinnamic aldehyde; Liq = liquid; LLNA = murine local lymph node assay result  
(i.e. B.42 or OECD Test Guideline 429) (1); MEK = methyl ethyl ketone; NA = not applicable since stimulation index < 3; NC = not cal culated since data was obtained from a single study; Sol = solid; Veh = test vehicle. 
(*) Presumed to be a non-sensitiser in humans based on the fact that no clinical patch test results were located, it is not inc luded as a patch test kit allergen, and no case reports of human sensitisation were located. 
(**) GP data not available. 
( 1 ) Chemicals should be prepared daily unless stability data demonstrate the acceptability of storage. 
( 2 ) Because of the potential impact of different vehicles on the performance of the LLNA, the recommended vehicle for each refere nce chemical should be used (24) (32). 
( 3 ) Mean value where more than one EC3 value was available. For negative substances ( i.e. with stimulation index < 3, the highest concentration tested is provided). 
( 4 ) Number of LLNA studies from which data were obtained. 
( 5 ) Commercially available as Kathon CG (CAS No 55965-84-9), which is a 3:1 mixture of CMI and MI. The relative concentrations of  each component range from 1,1 % to 1,25 % (CMI) and 0,3 % to 0,45 % (MI). The 
inactive components are magnesium salts (21,5 % to 24 %) and copper nitrate (0,15 % to 0,17 %), with the remaining formulation 74 % to 77 % water. Kathon CG is readily available through Sigma-Aldrich and Rohm and 
Haas (now Dow Chemical Corporation).
 III. Defined reliability and accuracy standards  
7. The accuracy of a similar or modified LLNA method should meet or exceed 
that of the LLNA PS when it is evaluated using the 18 minimum reference 
chemicals that should be used. The new or modified method should result in the correct classification based on a ‘yes/no’ decision. However, the new or modified method might not correctly classify all of the minimum 
reference chemicals that should be used. If, for example, one of the weak 
sensitisers were misclassified, a rationale for the misclassification and appropriate additional data ( e.g. test results that provide correct classifi ­
cations for other substances with physical, chemical, and sensitising prop ­
erties similar to those of the misclassified reference chemical) could be 
considered to demonstrate equivalent performance. Under such circum ­
stances, the validation status of the new or modified LLNA test method would be evaluated on a case-by-case basis.
 
Intra-laboratory reproducibility  
8. To determine intra-laboratory reproducibility, a new or modified LLNA 
method should be assessed using a sensitising substance that is well char ­
acterised in the LLNA. Therefore, the LLNA PS are based on the variability of results from repeated tests of hexyl cinnamic aldehyde (HCA). To assess 
intra-laboratory reliability, threshold estimated concentration (ECt) values 
for HCA should be derived on four separate occasions with at least one week between tests. Acceptable intra-laboratory reproducibility is indicated by a laboratory’s ability to obtain, in each HCA test, ECt values between 
5 % and 20 %, which represents the range of 0,5-2,0 times the mean EC3 
specified for HCA (10 %) in the LLNA (see Table 1).
 
Inter-laboratory reproducibility  
9. Inter-laboratory reproducibility of a new or modified LLNA method should 
be assessed using two sensitising substances that are well characterised in 
the LLNA. The LLNA PS are based on the variability of results from tests 
of HCA and 2,4-dinitrochlorobenzene (DNCB) in different laboratories. ECt values should be derived independently from a single study conducted in at least three separate laboratories. To demonstrate acceptable inter-laboratory 
reproducibility, each laboratory should obtain ECt values of 5 % to 20 % 
for HCA and 0,025 % to 0,1 % for DNCB, which represents the range of 0,5-2,0 times the mean EC3 concentrations specified for HCA (10 %) and DNCB (0,05 %), respectively, in the LLNA (see Table 1).
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Appendix 2  
Definitions  
Accuracy:  The closeness of agreement between test method results and accepted 
reference values. It is a measure of test method performance and one aspect of relevance. The term is often used interchangeably with ‘concordance’ to mean 
the proportion of correct outcomes of a test method (14).  
Benchmark substance:  A sensitising or non-sensitising substance used as a 
standard for comparison to a test substance. A benchmark substance should 
have the following properties: (i) consistent and reliable source(s); (ii) structural 
and functional similarity to the class of substances being tested; (iii) known physicochemical characteristics; (iv) supporting data on known effects; and (v) known potency in the range of the desired response.  
Estimated concentration threshold (ECt):  Estimated concentration of a test 
substance needed to produce a stimulation index that is indicative of a positive response.  
Estimated concentration three (EC3):  Estimated concentration of a test substance 
needed to produce a stimulation index of three.  
False negative:  A test substance incorrectly identified as negative or non-active 
by a test method, when in fact it is positive or active.  
False positive:  A test substance incorrectly identified as positive or active by a 
test, when in fact it is negative or non-active.  
Hazard:  The potential for an adverse health or ecological effect. The adverse 
effect is manifested only if there is an exposure of sufficient level.  
Inter-laboratory reproducibility:  A measure of the extent to which different 
qualified laboratories, using the same protocol and testing the same test substances, can produce qualitatively and quantitatively similar results. 
Inter-laboratory reproducibility is determined during the pre-validation and vali ­
dation processes, and indicates the extent to which a test can be successfully transferred between laboratories, also referred to as between-laboratory reproduci ­
bility (14).  
Intra-laboratory reproducibility:  A determination of the extent that qualified 
people within the same laboratory can successfully replicate results using a specific protocol at different times. Also referred to as within-laboratory repro ­
ducibility (14).  
Me-too test:  A colloquial expression for a test method that is structurally and 
functionally similar to a validated and accepted reference test method. Such a test method would be a candidate for catch-up validation. Interchangeably used with 
similar test method (14).  
Outlier:  An outlier is an observation that is markedly different from other values 
in a random sample from a population.  
Performance standards (PS):  Standards, based on a validated test method, that 
provide a basis for evaluating the comparability of a proposed test method that is functionally and mechanistically similar. Included are: (i) essential test method components; (ii) a minimum list of Reference Chemicals selected from among the 
chemicals used to demonstrate the acceptable performance of the validated test 
method; and (iii) the similar levels of accuracy and reliability, based on what was obtained for the validated test method, that the proposed test method should demon ­
strate when evaluated using the minimum list of Reference Chemicals (14).  
Proprietary test method:  A test method for which manufacture and distribution is 
restricted by patents, copyrights, trademarks, etc.
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Quality assurance:  A management process by which adherence to laboratory 
testing standards, requirements, and record keeping procedures, and the 
accuracy of data transfer, are assessed by individuals who are independent from those performing the testing.  
Reference chemicals:  Chemicals selected for use in the validation process, for 
which responses in the  in vitro  or in vivo  reference test system or the species of 
interest are already known. These chemicals should be representative of the 
classes of chemicals for which the test method is expected to be used, and should represent the full range of responses that may be expected from the chemicals for which it may be used, from strong, to weak, to negative. 
Different sets of reference chemicals may be required for the different stages 
of the validation process, and for different test methods and test uses (14).  
Relevance:  Description of relationship of the test to the effect of interest and 
whether it is meaningful and useful for a particular purpose. It is the extent to which the test correctly measures or predicts the biological effect of interest. 
Relevance incorporates consideration of the accuracy (concordance) of a test 
method (14).  
Reliability:  Measures of the extent that a test method can be performed repro ­
ducibly within and between laboratories over time, when performed using the 
same protocol. It is assessed by calculating intra- and inter-laboratory reproduci ­
bility (14).  
Skin sensitisation:  An immunological process that results when a susceptible 
individual is exposed topically to an inducing chemical allergen, which provokes a cutaneous immune response that can lead to the development of 
contact sensitisation.  
Stimulation Index (SI):  A value calculated to assess the skin sensitisation 
potential of a test substance that is the ratio of the proliferation in treated groups to that in the concurrent vehicle control group.  
Test substance (also referred to as test chemical):  Any substance or mixture 
tested using this TM.  
Validated test method:  A test method for which validation studies have been 
completed to determine the relevance (including accuracy) and reliability for a specific purpose. It is important to note that a validated test method may not have 
sufficient performance in terms of accuracy and reliability to be found acceptable 
for the proposed purpose (14).
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B.43.  NEUROTOXICITY STUDY IN RODENTS  
1. METHOD  
This method is equivalent of OECD TG 424 (1997). 
This test method has been designed to obtain the information 
necessary to confirm or to further characterise the potential neur ­
otoxicity of chemicals in adult animals. It can either be combined with existing test methods for repeated dose toxicity studies or to be 
carried out as a separate study. It is recommended that the OECD 
Guidance Document on Neurotoxicity Testing Strategies and Methods (1) be consulted to assist in the design of studies based on this test method. This is particularly important when modifi ­
cations of the observations and test procedures as recommended 
for routine use of this method are considered. The guidance document has been prepared to facilitate the selection of other test procedures for use in specific circumstances. 
The assessment of developmental neurotoxicity is not the subject of 
this method.
 
1.1. INTRODUCTION 
In the assessment and evaluation of the toxic characteristics of 
chemicals, it is important to consider the potential for neurotoxic 
effects. Already the test method for repeated dose systemic toxicity 
includes observations that screen for potential neurotoxicity. This test method can be used to design a study to obtain further information on, or to confirm, the neurotoxic effects observed in the repeated 
dose systemic toxicity studies. However, consideration of the 
potential neurotoxicity of certain classes of chemicals may suggest that they may be more appropriately evaluated using this Method without prior indications of the potential neurotoxicity from repeated 
dose systemic toxicity studies. Such considerations include, for 
example: 
— observation of neurological signs or neuropathological lesions in 
toxicity studies other than repeated dose systemic toxicity 
studies, or 
— structural relationship or other information linking them to 
known neurotoxicants. 
In addition there may be other instances when use of this test method 
is appropriate; for further details see (1). 
This method has been developed so that it can be tailored to meet 
particular needs to confirm the specific histopathological and behav ­
ioural neurotoxicity of a chemical as well as provide a characteri ­
sation and quantification of the neurotoxic responses.
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In the past, neurotoxicity was equated with neuropathy involving 
neuropathological lesions or neurological dysfunctions, such as 
seizure, paralysis or tremor. Although neuropathy is an important manifestation of neurotoxicity, it is now clear that there are many other signs of nervous system toxicity (e.g. loss of motor co-ordi ­
nation, sensory deficits, learning and memory dysfunctions) that may 
not be reflected in neuropathy or other types of studies. 
This neurotoxicity test method is designed to detect major neurobe ­
havioural and neuropathological effects in adult rodents. While 
behavioural effects, even in the absence of morphological changes, 
can reflect an adverse impact on the organism, not all behavioural changes are specific to the nervous system. Therefore, any changes observed should be evaluated in conjunction with correlative histo ­
pathological, haematological or biochemical data as well as data on 
other types of systemic toxicity. The testing called for in this method to provide a characterisation and quantification of the neurotoxic responses includes specific histopathological and behavioural 
procedures that may be further supported by electrophysiological 
and/or biochemical investigations (1)(2)(3)(4). 
Neurotoxicants may act on a number of targets within the nervous 
system and by a variety of mechanisms. Since no single array of 
tests is capable of thoroughly assessing the neurotoxic potential of all substances, it may be necessary to utilise other  in vivo  or in vitro  
tests specific to the type of neurotoxicity observed or anticipated. 
This test method can also be used, in conjunction with the guidance 
set out in the OECD Guidance Document on Neurotoxicity Testing Strategies and Methods (1) to design studies intended to further 
characterise or increase the sensitivity of the dose-response quantifi ­
cation in order or better estimate a no-observed-adverse effect level or to substantiate known or suspected hazards of the chemical. For example, studies may be designed to identify and evaluate the neur ­
otoxic mechanism(s) or supplement the data already available from 
the use of basic neurobehavioural and neuropathological observation procedures. Such studies need not replicate data that would be generated from the use of the standard procedures recommended in 
this Method, if such data are already available and are not considered 
necessary for the interpretation of the results of the study. 
This neurotoxicity study, when used alone or in combination, 
provides information that can: 
— identify whether the nervous system is permanently or reversibly 
affected by the chemical tested; 
— contribute to the characterisation of the nervous system alter ­
ations associated with exposure to the chemical, and to under ­
standing the underlying mechanism.
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— determine dose-and time-response relationships in order to 
estimate a no-observed-adverse-effect level (which can be used 
to establish safety criteria for the chemical). 
This test method uses oral administration of the test substance. Other 
routes of administration (e.g. dermal or inhalation) may be more 
appropriate, and may require modification of the procedures recom ­
mended. Considerations of the choice of the route of administration depend on the human exposure profile and available toxicological or kinetic information.
 
1.2. DEFINITIONS  
Adverse effect:  is any treatment-related alteration from baseline that 
diminishes an organism's ability to survive, reproduce or adapt to the environment.  
Dose:  is the amount of test substance administered. Dose is 
expressed as weight (g, mg) or as weight of test substance per 
unit weight of the test animal (e.g. mg/Kg), or as constant dietary concentrations (ppm).  
Dosage:  is a general term comprising of dose, its frequency and the 
duration of dosing.  
Neurotoxicity:  is an adverse change in the structure or function of 
the nervous system that results from exposure to a chemical, 
biological or physical agent.  
Neurotoxicant:  is any chemical, biological or physical agent having 
the potential to cause neurotoxicity.  
NOAEL:  is the abbreviation for no-observed-adverse effect level 
and is the highest dose level where no adverse treatment-related 
findings are observed.
 
1.3. PRINCIPLE OF THE TEST METHOD 
The test chemical is administered by the oral route across a range of 
doses to several groups of laboratory rodents. Repeated doses are 
normally required, and the dosing regimen may be 28 days, 
subchronic (90 days) or chronic (1 year or longer). The procedures set out in this test method may also be used for an acute neur ­
otoxicity study. The animals are tested to allow the detection or 
the characterisation of behavioural and/or neurological abnormalities. 
A range of behaviours that could be affected by neurotoxicants is assessed during each observation period. At the end of the test, a subset of animals of each sex from each group are perfused  in situ  
and sections of the brain, spinal cord, and peripheral nerves are 
prepared and examined. 
When the study is conducted as a stand-alone study to screen for 
neurotoxicity or to characterise neurotoxic effects, the animals in 
each group not used for perfusion and subsequent histopathology 
(see Table 1) can be used for specific neurobehavioural, neuropath ­
ological, neurochemical or electrophysiological procedures that may supplement the data obtained from the standard examinations 
required by this method (1). These supplemental procedures can be 
particularly useful when empirical observations or anticipated effects indicate a specific type or target of a chemical's neurotoxicity. Alter ­
natively, the remaining animals can be used for evaluations such as 
those called for in test methods for repeated dose toxicity studies in 
rodents.
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When the procedures of this test method are combined with those of 
other test methods, a sufficient number of animals is needed to 
satisfy the requirements for the observations of both studies.  
1.4. DESCRIPTION OF THE TEST METHOD  
1.4.1.  Selection of animal species  
The preferred rodent species is the rat, although other rodent species, 
with justification, may be used. Commonly used laboratory strains of young adult healthy animals should be employed. The females should be nulliparous and non-pregnant. Dosing should normally 
begin as soon as possible after weaning, preferably not later than 
when animals are six weeks, and, in any case, before the animals are nine weeks age. However, when this study is combined with other studies this age requirement may need adjustment. At the 
commencement of the study the weight variation of animals used 
should not exceed ± 20 % of the mean weight of each sex. Where a repeated dose study of short duration is conducted as a preliminary to a long term study, animals from the same strain and source should 
be used in both studies.
 
1.4.2.  Housing and feeding conditions  
The temperature in the experimental animal room should be 22 °C 
(± 3 °C). Although the relative humidity should be at least 30 % and 
preferably not exceed 70 % other than during room cleaning, the aim 
should be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. Loud intermittent noise should be kept 
to a minimum. For feeding, conventional laboratory diets may be 
used with an unlimited supply of drinking water. The choice of diet may be influenced by the need to ensure a suitable admixture of a test substance when administered by this method. Animals may be 
housed individually, or be caged in small groups of the same sex.
 
1.4.3.  Preparation of animals  
Healthy young animals are randomly assigned to the treatment and 
control groups. Cages should be arranged in such a way that possible 
effects due to cage placement are minimised. The animals are 
identified uniquely and kept in their cages for at least (5) five days prior the start of the study to allow for acclimatisation to the laboratory conditions.
 
1.4.4.  Route of administration and preparation of doses  
This test method specifically addresses the oral administration of the 
test substance. Oral administration may be by gavage, in the diet, in drinking water or by capsules. Other routes of administration (e.g. 
dermal or inhalation) can be used but may require modification of 
the procedures recommended. Considerations of the choice of the route of administration depend on the human exposure profile and available toxicological or kinetic information. The rationale for 
choosing the route of administration as well as resulting modifi ­
cations to the procedures of this test method should be indicated.
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Where necessary, the test substance may be dissolved or suspended 
in a suitable vehicle. It is recommended that the use of an aqueous 
solution/suspension be considered first, followed by consideration of a solution/suspension in oil (e.g. corn oil) and then by possible solution/suspension in other vehicle. The toxic characteristics of 
the vehicle must be known. In addition, consideration should be 
given to the following characteristics of the vehicle: effects of the vehicle on absorption, distribution, metabolism, or retention of the test substance which may alter its toxic characteristics; and effects on 
the food or water consumption or the nutritional status of the 
animals.
 
1.5. PROCEDURES  
1.5.1.  Number and sex animals  
When the study is conducted as a separate study, at least 20 animals 
(10 females and 10 males) should be used in each dose and control group for the evaluation of detailed clinical and functional observa ­
tions. At least five males and five females, selected from these 
10 males and 10 females, should be perfused  in situ  and used for 
detailed neurohistopathology at the end of the study. In cases where only a limited number of animals in a given dose group are observed for signs of neurotoxic effects, consideration should be given to the 
inclusion of these animals in those selected for perfusion. When the 
study is conducted in combination with a repeated dose toxicity study, adequate numbers of animals should be used to meet the objectives of both studies. The minimum numbers of animals per 
group for various combinations of studies are given in Table 1. If 
interim kills or recovery groups for observation of reversibility, persistence or delayed occurrence of toxic effects post treatment are planned or when supplemental observations are considered, 
then the number of animals should be increased to ensure that the 
number of animals required for observation and histopathology are available.
 
1.5.2.  Treatment and control group  
At least three dose groups and a control group should generally be 
used, but if from the assessment of other data, no effects would be expected at a repeated dose of 1 000 mg/kg body weight/day, a limit 
test may be performed. If there are no suitable data available, a range 
finding study may be performed to aid in the determination of the doses to be used. Except for treatment with the test substance, animals in the control group should be handled in an identical 
manner to the test group subjects. If a vehicle is used in adminis ­
tering the test substance, the control group should receive the vehicle at the highest volume used.
 
1.5.3.  Reliability check  
The laboratory performing the study should present data demon ­
strating its capability to carry out the study and the sensitivity of the procedures used. Such data should provide evidence of the ability to detect and quantify, as appropriate, changes in the different end 
points recommended for observation, such as autonomic signs, 
sensory reactivity, limb grip strength and motor activity. Information on chemicals that cause different types of neurotoxic responses and could be used as positive control substances can be found in 
references 2 to 9. Historical data may be used if the essential 
aspects of the experimental procedures remain the same. Periodic updating of historical data is recommended. New data that demon ­
strate the continuing sensitivity of the procedures should be 
developed when some essential element of the conduct of the test 
or procedures has been changed by the performing laboratory.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 627
 
1.5.4.  Dose selection  
Dose levels should be selected by taking into account any previously 
observed toxicity and kinetic data available for the test compound or related materials. The highest dose level should be chosen with the 
aim of inducing neurotoxic effects or clear systemic toxic effects. 
Thereafter, a descending sequence of dose levels should be selected with a view to demonstrating any dose-related response and no- observed-adverse effect (NOAEL) at the lowest dose level. In prin ­
ciple, dose levels should be set so that primary toxic effects on the 
nervous system can be distinguished from effects related to systemic toxicity. Two to three intervals are frequently optimum and addition 
of a fourth test group is often preferable to using very large intervals 
(e.g. more than a factor of 10) between dosages. Where there is a reasonable estimation of human exposure this should also be taken into account.
 
1.5.5.  Limit test  
If a study at one dose level of at least 1 000 mg/kg body weight/day, 
using the procedures described, produces no observable neurotoxic effects and if toxicity would not be expected based upon data from structurally related compounds, then a full study using three dose 
levels may not be considered necessary. Expected human exposure 
may indicate the need for a higher oral dose level to be used in the limit test. For other types of administration, such as inhalation or dermal application, the physical chemical properties of the test 
substance often may dictate the maximum attainable level of 
exposure. For the conduct of an oral acute study, the dose for a limit test should be at least 2 000 mg/kg.
 
1.5.6.  Administration of doses  
The animals are dosed with the test substance daily, seven days each 
week, for a period at least 28 days; use of a five-day dosing regime 
or a shorter exposure period needs to be justified. When the test substance is administered by gavage, this should be done in a single dose using a stomach tube or a suitable intubation cannula. 
The maximum volume of a liquid that can be administered at one 
time depends on the size of the test animals. The volume should not exceed 1 ml/100 g body weight. However in the case of aqueous solutions, the use of up to 2 ml/100 g body weight can be 
considered. Except for irritating or corrosive substances, which will 
normally reveal exacerbated effects with higher concentrations, varia ­
bility in test volume should be minimised by adjusting the concen ­
tration to ensure a constant volume at all dose levels. 
For substances administered via the diet or drinking water, it is 
important to ensure that the quantities of the test substance involved do not interfere with normal nutrition or water balance. 
When the test substance is administered in the diet either a 
constant dietary concentration (ppm) or a constant dose level in terms of the animals' body weight may be used; the alternative used must be specified. For a substance administered by gavage, 
the dose should be given at similar times each day, and adjusted 
as necessary to maintain a constant dose level in terms of animal body weight. Where a repeat dose study is used as a preliminary to a long term study, a similar diet should be used in both studies. For 
acute studies, if a single dose is not possible, the dose may be given 
in smaller fractions over a period not exceeding 24 hours.
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1.6. OBSERVATION  
1.6.1.  Frequency of observations and tests  
In repeated dose studies, the observation period should cover the 
dosage period. In acute studies, 14-day post-treatment period 
should be observed. For animals in satellite groups which are kept 
without exposure during a post-treatment period, observations should cover this period as well. 
Observations should be made with sufficient frequency to maximise 
the probability of detection of any behavioural and/or neurological 
abnormalities. Observations should be made preferably at the same 
times each day with consideration given to the peak period of anticipated effects after dosing. The frequency of clinical obser ­
vations and functional tests is summarised in Table 2. If kinetic or 
other data generated from previous studies indicates the need to use 
different time points for observations, tests or post-observation periods, an alternative schedule should be adopted in order to achieve maximum information. The rationale for changes to the 
schedule should be provided.
 
1.6.1.1.  Observations of general health condition and mortality/morbidity  
All animals should be carefully observed at least once daily with 
respect to their health condition as well as at least twice daily for morbidity and mortality.
 
1.6.1.2.  Detailed clinical observations  
Detailed clinical observations should be made on all animals selected 
for this purpose (see Table 1) once before the first exposure (to allow for within-subject comparisons) and at different intervals thereafter, 
dependant on the duration of the study (see Table 2). Detailed 
clinical observations on satellite recovery groups should be made at the end of the recovery period. Detailed clinical observations should be made outside the home cage in a standard arena. They 
should be carefully recorded using scoring systems that include 
criteria or scoring scales for each measurement in the observations. The criteria or scales used should be explicitly defined by the testing laboratory. Effort should be made to ensure that variations in the test 
conditions are minimal (not systematically related to treatment) and 
that observations are conducted by trained observers unaware of the actual treatment. 
It is recommended that the observations be carried out in a structured 
fashion in which well-defined criteria (including the definition of the 
normal ‘range’) are systematically applied to each animal at each 
observation time. The ‘normal range’ should be adequately docu ­
mented. All observed signs should be recorded. Whenever feasible, the magnitude of the observed signs should also be recorded. 
Clinical observations should include, but not be limited to, 
changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern and/or mouth 
breathing, any unusual signs of urination or defecation, and 
discoloured urine).
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Any unusual responses with respect to body position, activity level 
(e.g. decreased or increased exploration of the standard arena) and 
co-ordination of movement should also be noted. Changes in gait (e.g. waddling, ataxia), posture (e.g. hunched-back) and reactivity to handling, placing or other environmental stimuli, as well as the 
presence of clonic or tonic movements, convulsions or tremors, 
stereotypes (e.g. excessive grooming, unusual head movements, repetitive circling) or bizarre behaviour (e.g. biting or excessive licking, self mutilation, walking backwards, vocalisation) or 
aggression should be recorded.
 
1.6.1.3.  Functional tests  
Similar to the detailed clinical observations, functional tests should 
also be conducted once prior to exposure and frequently thereafter in 
all animals selected for this purpose (see Table 1). The frequency of 
functional testing is also dependent on the study duration (see Table 2). In addition to the observation periods as set out in Table 2, functional observations on satellite recovery groups 
should also be made as close as possible to the terminal kill. Func ­
tional tests should include sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli (5)(6)(7)), assessment of limb grip strength (8) and assessment of motor activity 
(9). Motor activity should be measured with an automated device 
capable of detecting both decreases and increases in activity. If another defined system is used it should be quantitative and its sensitivity and reliability should be demonstrated. Each device 
should be tested to ensure reliability across time and consistency 
between devices. Further details of the procedures that can be followed are given in the respective references. If there are no data (e.g. structure-activity, epidemiological data, other toxicology 
studies) to indicate the potential neurotoxic effects, the inclusion of 
more specialised tests of sensory and motor function or learning and memory to examine these possible effects in greater details should be considered. More information on more specialised tests and their use 
is provided in (1). 
Exceptionally, animals that reveal signs of toxicity to an extent that 
would significantly interfere with the functional test may be omitted 
from that test. Justification for the elimination of animals from a functional test should be provided.
 
1.6.2.  Body weight and food/water consumption  
For studies up to 90 days duration, all animals should be weighed at 
least once a week and measurements should be made of food consumption (water consumption, when the test substance is admin ­
istered by that medium) at least weekly. For long term studies, all 
animals should be weighed at least once at week for the first 13 
weeks and at least once every four weeks thereafter. Measurements should be made of food consumption (water consumption, when the test substance is administered by that medium) at least weekly for 
the first 13 weeks and then at approximately three-month intervals 
unless the health status or body weight changes dictate otherwise.
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1.6.3.  Ophthalmology  
For studies longer than 28 days duration, ophthalmologic examin ­
ation, using an ophthalmoscope or an equivalent suitable instrument, should be made prior to the administration of the test substance and 
at the termination of the study, preferably on all animals, but at least 
on animals in the high dose and control groups. If changes in the eyes are detected or, if clinical signs indicate the need, all animals should be examined. For long term studies, an ophthalmologic exam ­
ination should also be carried out at 13 weeks. Ophthalmologic 
examinations need not to be conducted if this data is already available from others studies of similar duration and at similar 
dose levels.
 
1.6.4.  Haematology and clinical biochemistry  
When the neurotoxicity study is carried out in combination with a 
repeated dose systemic toxicity study, haematological examinations 
and clinical biochemistry determinations should be carried out as set out in the respective method of the systemic toxicity study. Collection of samples should be carried out in such a way that 
any potential effects on neurobehaviour are minimised.
 
1.6.5.  Histopathology  
The neuropathological examination should be designed to complement 
and extend the observations made during the  in vivo  phase of the study. 
Tissues from at least five animals/sex/group (see Table 1 and next paragraph) should be fixed  in situ , using generally recognised 
perfusion and fixation techniques (see reference 3, chapter 5 and 
reference 4, chapter 50). Any observable gross changes should be 
recorded. When the study is conducted as a stand-alone study screen for neurotoxicity or to characterise neurotoxic effects, the remainder of the animals may be used either for specific neurobehavioural (10)(11), 
neuropathological (10)(11)(12)(13), neurochemical (10)(11)(14)(15) or 
electrophysiological (10)(11)(16)(17) procedures that may supplement the procedures and examinations described here, or to increase the number of subjects examined for histophatology. These supplementary 
procedures are of particular use when empirical observations 
or anticipated effects indicate a specific type or target of neurotoxicity (2)(3). Alternatively, the remainder of the animals can also be used for routine pathological evaluations as described in 
Method for repeated dose studies. 
A general staining procedure, such as haematoxylin and eosin 
(H&E), should be performed on all tissue specimens embedded in paraffin and microscopic examination should be carried out. If signs of peripheral neuropathy are observed or suspected, plastic- 
embedded samples of peripheral nerve tissue should be examined. 
Clinical signs may also suggest additional sites for examination or the use of special staining procedures. Guidance on additional sites to be examined can be found in (3)(4). Appropriate special stains to 
demonstrate specific types of pathological change may also be 
helpful (18).
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Representative sections of the central and peripheral nervous system 
should be examined histologically (see reference 3, chapter 5 and 
reference 4, chapter 50). The areas examined should normally include: the forebrain, the centre of the cerebrum, including a section through the hippocampus, the midbrain, the cerebellum, the 
pons, the medulla oblongata, the eye with optic nerve and retina, the 
spinal cord at the cervical and lumbar swellings, the dorsal root ganglia, the dorsal and ventral root fibres, the proximal sciatic nerve, the proximal tibial nerve (at the knee) and the tibial nerve 
calf muscle branches. The spinal cord and peripheral nerve sections 
should include both cross or transverse and longitudinal sections. Attention should be given to the vasculature of the nervous system. A sample of skeletal muscle, particularly calf muscle, 
should also be examined. Special attention should be paid to sites 
with cellular and fibre structure and pattern in the CNS and PNS known to be particularly affected by neurotoxicants. 
Guidance on neurophatological alterations that typically result from 
toxicant exposure can be found in the references (3)(4). A stepwise examination of tissue samples is recommended in which sections 
from the high dose group are first compared with those of the 
control group. If no neurophatological alterations are observed in the samples from these groups, subsequent analysis is not required. If neuropathological alterations are observed in the high dose group, 
sample from each of the potentially affected tissues from the inter ­
mediate and low dose groups should then be coded and examined sequentially. 
If any evidence of neuropathological alterations is found in the quali ­
tative examination, then a second examination should be performed 
on all regions of the nervous system showing these alterations. 
Sections from all dose groups from each of the potentially affected regions should be coded and examined at random without knowledge of the code. The frequency and severity of each lesion should be 
recorded. After all regions from all dose groups have been rated, the 
code can be broken and statistical analysis performed to evaluate dose-response relationships. Examples of different degrees of severity of each lesion should be described. 
The neuropathological findings should be evaluated in the context of 
behavioural observations and measurements, as well as other data 
from preceding and concurrent systemic toxicity studies of the test 
substance.
 
2. DATA  
2.1. TREATMENT OF RESULTS 
Individual data should be provided. Additionally, all data should be 
summarised in tabular form showing for each test or control group the number of animals at the start of the test, the number of animals 
found dead during the test or killed for humane reasons and the time 
of any death or humane kill, the number showing signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration, type and severity of any toxic effects, the number of 
animals showing lesions, including the type and severity of the 
lesion(s).
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2.2. EVALUATION AND INTERPRETATION OF RESULTS 
The findings of the study should be evaluated in terms of the inci ­
dence, severity and correlation of neurobehavioural and neuropath ­
ological effects (neurochemical or electrophysiological effects as well 
if supplementary examinations are included) and any other adverse 
effects observed. When possible, numerical results should be evaluated by an appropriate and generally acceptable statistical method. The statistical methods should be selected during the 
design of the study.
 
3. REPORTING  
3.1. TEST REPORT 
the test report must include the following information: 
Test substance: — physical nature (including isomerism, purity and physicochemical 
properties), 
— identification data. 
Vehicle (if appropriate): — justification for choice of vehicle. Test animals: — species/strain used, — number, age and sex of animals, — source, housing conditions, acclimatisation, diet, etc, — individual weights of animals at the start of the test. 
Test conditions: 
— details of test substance formulation/diet preparation, achieved 
concentration, stability and homogeneity of the preparation, 
— specification of the doses administered, including details of the 
vehicle, volume and physical form of the material administered, 
— details of the administration of the test substance, — rationale for dose levels selected, 
— rationale for the route and duration of the exposure, 
— conversion from diet/drinking water test substance concentration 
(ppm) to the actual dose (mg/kg body weight/day), if applicable, 
— details of the food and water quality. Observation and test procedures: — details of the assignment of animals in each group to the 
perfusion subgroups, 
— details of scoring systems, including criteria and scoring scales 
for each measurement in the detailed clinical observations,
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— details on the functional tests for sensory reactivity to stimuli of 
different modalities (e.g. auditory, visual and proprioceptive), for 
assessment of limb grip strength, for motor activity assessment (including details of automated devices for detecting activity), and other procedures used, 
— details of ophthalmologic examinations and, if appropriate, 
haematological examinations and clinical biochemistry tests 
with relevant base-line values, 
— details for specific neurobehavioural, neuropathological, neur ­
ochemical or electrophysiological procedures. 
Results: 
— body weight/body weight changes including body weight at kill, 
— food consumption and water consumption, as appropriate, — toxic response data by sex and dose level, including signs of 
toxicity or mortality, 
— nature, severity and duration (time of onset and subsequent 
course) of the detailed clinical observations (whether reversible 
or not), 
— a detailed description of all functional test results, 
— necropsy findings, 
— a detailed description of all neurobehavioural, neuropathological, 
and neurochemical or electrophysiological findings, if available, 
— absorption and metabolism data, if available, 
— statistical treatment of results, where appropriate. Discussion of results: — dose response information; — relationship of any other toxic effects to a conclusion about the 
neurotoxic potential of the test chemical; 
— no-observed-adverse effect level. Conclusions: — a specific statement of the overall neurotoxicity of the test 
chemical is encouraged.
 
4. REFERENCES  
(1) OECD Giudance Document on Neurotoxicity Testing 
Strategies and Test Methods. OECD, Paris, In Preparation. 
(2) Test Guideline for a Developmental Neurotoxicity Study, 
OECD Guidelines for the Testing of Chemicals. In preparation. 
(3) World Health Organisation (WHO) (1986) Environmental 
Health Criteria document 60: Principles and Methods for the Assessment of Neurotoxicity associated with Exposure to 
Chemicals. 
(4) Spencer, P.S. and Schaumburg, H.H. (1980) Experimental and 
Clinical Neurotoxicology. Eds. Spencer, P.S. and Schaumburg, 
H.H. eds. Williams and Wilkins, Baltimore/London.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 634
 
(5) Tupper, D.E. and Wallace, R.B., (1980) Utility of the Neur ­
ological Examination in Rats. Acta Neurobiol. Exp., 40, 
p. 999-1003. 
(6) Gad, S.C., (1982) A Neuromuscular Screen for Use in 
Industrial Toxicology. J. Toxicol. Environ. Health, 9, p. 691-704. 
(7) Moser, V.C., McDaniel, K.M. and Phillips, P.M., (1991) Rat 
Strain and Stock Comparisons Using a Functional Observa ­
tional Battery: Baseline Values and Effects of amitraz. Toxic. Appl. Pharmacol., 108, p. 267-283. 
(8) Meyer, O.A., Tilson, H.A., Byrd, W.C. and Riley, M.T., 
(1979) A Method for the Routine Assessment of Fore- and 
Hind- limb Grip Strength of Rats and Mice. Neurobehav. Toxicol., 1, p. 233-236. 
(9) Crofton, K.M., Haward, J.L., Moser, V.C., Gill, M.W., Reirer, 
L.W., Tilson, H.A. and MacPhail, R.C. (1991) Interlaboratory 
Comparison of Motor Activity Experiments: Implication for 
Neurotoxicological Assessments. Neurotoxicol. Teratol., 13, p. 599-609. 
(10) Tilson, H.A., and Mitchell, C.L. eds., (1992) Neurotoxicology 
Target Organ Toxicology Series. Raven Press, New York. 
(11) Chang, L.W., ed., (1995) Principles of Neurotoxicology. 
Marcel Dekker, New York. 
(12) Broxup, B., (1991) Neuopathology as a screen for Neur ­
otoxicity Assessment. J. Amer. Coll. Toxicol., 10, p. 689-695. 
(13) Moser, V.C., Anthony, D.C., Sette, W.F. and MacPhail, R.C., 
(1992) Comparison of Subchronic Neurotoxicity of 2-Hydro ­
xyethyl Acrylate and Acrylamide in Rats. Fund. Appl.Toxicol., 18, p. 343-352. 
(14) O'Callaghan, J.P. (1988). Neurotypic and Gliotypic Proteins as 
Biochemical Markers of Neurotoxicity. Eurotoxicol. Teratol., 
10, p. 445-452. 
(15) O'Callaghan J.P. and Miller, D.B., (1988) Acute Exposure of 
the Neonatal Rat to Triethyltin Results in Persistent Changes in Neurotypic and Gliotypic Proteins. J. Pharmacol. Exp. Ther., 
244, p. 368-378. 
(16) Fox. D.A., Lowndes, H.E. and Birkamper, G.G., (1982) Elec ­
trophysiological Techniques in Neurotoxicology. In: Nervous System Toxicology. Mitchell, C.L. ed. Raven Press, New York, p. 299-335. 
(17) Johnson, B.L., (1980) Electrophysiological Methods in neur ­
otoxicity Testing. In: Experimental and Clinical Neurotox ­
icology. Spencer, P.S. and Schaumburg, H.H. eds., Williams and Wilkins Co.,. Baltimore/London, p. 726-742. 
(18) Bancroft, J.D. and Steven A., (1990) Theory and Pratice of 
Histological Techniques. Chapter 17, Neuropathological Tech ­
niques. Lowe, James and Cox, Gordon eds. Churchill Living ­
stone.
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Table 1  
Minimum numbers of animals needed per group when the neurotoxicity study is conducted separately or in 
combination whit studies  
NEUROTOXICITY STUDY CONDUCTED AS: 
Separate study Combined study with 
the 28-day study Combined study with 
the 90-day study Combined study with 
the chronic toxicity 
study  
Total number of animals per 
group 10 males and 
10 females 10 males and 
10 females 15 males and 
15 females 25 males and 
25 females 
Number of animals selected 
for functional testing including detailed clinical observations 10 males and 
10 females 10 males and 
10 females 10 males and 
10 females 10 males and 
10 females 
Number of animals selected 
per perfusion  in situ  and 
neurohistopathology 5 males and 
5 females 5 males and 
5 females 5 males and 
5 females 5 males and 
5 females 
Number of animals selected 
for repeated dose/sub ­
chronic/chronic toxicity observations, haematology, 
clinical biochemistry, histo ­
pathology, etc. as indicate in the respective  Guidelines  5 males and 
5 females 10 males † and 10 
females † 20 males † and 
20 females † 
Supplemental observations, 
as appropriate 5 males and 5 
females
 
† Includes five animals selected for functional testing and detailed clinical observations as part of the neurotoxicity study.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 636
 
Table 2  
Frequency of clinical observation and functional tests  
Type of observations Study duration 
Acute 28-day 90-day Chronic  
In all animals General health 
condition daily daily daily daily 
Mortality/mor ­
bidity Twice daily Twice daily Twice daily Twice daily 
In animals selected 
for functional observations Detailed clinical 
observations — prior to first 
exposure 
— within 
8 hours of 
dosing at 
estimate time of peak effect 
— at day 7 and 
14 after dosing — prior to first 
exposure 
— once weekly 
thereafter — prior to first 
exposure 
— once during 
the first 
or second 
week of exposure 
— monthly 
thereafter — prior to first 
exposure 
— once at the 
end of the 
first month of 
exposure 
— every three 
months 
thereafter 
Functional tests — prior to first 
exposure 
— within 
8 hours of dosing at estimate 
time of peak 
effect 
— at day 7 and 
14 after 
dosing — prior to first 
exposure 
— during the 
fourth week of treatment as close as 
possible to 
the end of the exposure period — prior to first 
exposure 
— once during 
the first or second week of 
exposure 
— monthly 
thereafter — prior to first 
exposure 
— once at the 
end of the first month of exposure 
— every three 
months thereafter
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B.44.  SKIN ABSORPTION:  IN VIVO  METHOD  
1. METHOD  
This testing method is equivalent to the OECD TG 427 (2004).  
1.1. INTRODUCTION 
Exposure to many chemicals occurs mainly  via the skin whilst the 
majority of toxicological studies performed in laboratory animals use the oral route of administration. The  in vivo  percutaneous absorption 
study set out in this guideline provides the linkage necessary to 
extrapolate from oral studies when making safety assessments 
following dermal exposure. 
A substance must cross a large number of cell layers of the skin 
before it can reach the circulation. The rate-determining layer for most substances is the  stratum corneum  consisting of dead cells. 
Permeability through the skin depends both on the lipophilicity of 
the chemical and the thickness of the outer layer of epidermis, as 
well on factors such as molecular weight and concentration of the substance. In general, the skin of rats and rabbits is more permeable than that of humans, whereas the skin permeability of guinea pigs 
and monkeys is more similar to that of humans. 
The methods for measuring percutaneous absorption can be divided 
into two categories;  in vivo  and  in vitro .The in vivo  method is 
capable of providing good information, in various laboratory species, on skin absorption. More recently  in vitro  methods have 
been developed. These utilise transport across full or partial 
thickness animal or human skin to a fluid reservoir. The  in vitro  
method is described in a separate testing method (1). It is recom ­
mended that the OECD Guidance Document for the Conduct of Skin Absorption Studies (2) be consulted to assist in the selection of the 
most appropriate method in the given situation, as it provides more 
details on the suitability of both  in vivo  and in vitro methods. 
The in vivo  method, described in this method, allows the deter ­
mination of the penetration of the test substance through the skin into the systemic compartment. The technique has been widely used for many years (3)(4)(5)(6)(7). Although  in vitro  percutaneous 
absorption studies may in many cases be appropriate there may be 
situations in which only an  in vivo  study can provide the necessary 
data. 
Advantages of the  in vivo  method are that it uses a physiologically 
and metabolically intact system, uses a species common to many toxicity studies and can be modified for use with other species. The disadvantages are the use of live animals, the need for radio ­
labelled material to facilitate reliable results, difficulties in deter ­
mining the early absorption phase and the differences in permeability of the preferred species (rat) and human skin. Animal skin is generally more permeable and therefore may overestimate human 
percutaneous absorption (6)(8)(9). Caustic/corrosive substances 
should not be tested in live animals.
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1.2. DEFINITIONS  
Unabsorbed dose:  represents that washed from the skin surface after 
exposure and any present on the non-occlusive cover, including any dose shown to volatilise from the skin during exposure.  
Absorbed dose ( in vivo ): comprises that present in urine, cage wash, 
faeces, expired air (if measured), blood, tissues (if collected) and the 
remaining carcass, following removal of application site skin.  
Absorbable dose:  represents that present on or in the skin following 
washing.
 
1.3. PRINCIPLE OF THE TEST METHOD 
The test substance, preferably radiolabelled, is applied to the clipped 
skin of animals at one or more appropriate dose levels in the form of a representative in-use preparation. The test preparation is allowed to remain in contact with the skin for a fixed period of time under a 
suitable cover (non-occlusive, semi-occlusive, or occlusive) to 
prevent ingestion of the test preparation. At the end of the exposure time the cover is removed and the skin is cleaned with an appropriate cleansing agent, the cover and the cleansing materials 
are retained for analysis and a fresh cover applied. The animals are 
housed prior to, during and after the exposure period in individual metabolism cages and the excreta and expired air over these periods are collected for analysis. The collection of expired air can be 
omitted when there is sufficient information that little or no 
volatile radioactive metabolite is formed. Each study will normally involve several groups of animals that will be exposed to the test preparation. One group will be killed at the end of the exposure 
period. Other groups will be killed at scheduled time intervals 
thereafter (2). At the end of the sampling time the remaining animals are killed, blood is collected for analysis, the application site removed for analysis and the carcass is analysed for any unexcreted material. 
The samples are assayed by appropriate means and the degree of 
percutaneous absorption is estimated (6)(8)(9).
 
1.4. DESCRIPTION OF THE METHOD  
1.4.1.  Selection of animal species  
The rat is the most commonly used species, but hairless strains 
and species having skin absorption rates more similar to those of human, can also be used (3)(6)(7)(8)(9). Young adult healthy 
animals of a single sex (with males as the default sex) of 
commonly used laboratory strains should be employed. At the commencement of the study, the weight variation of animals used should not exceed ± 20 % of the mean weight. As an example, male 
rats of 200 g – 250 g are suitable, particularly in the upper half of 
this range.
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1.4.2.  Number and sex of animals  
A group of at least four animals of one sex should be used for each 
test preparation and each scheduled termination time. Each group of animals will be killed after different time intervals, for example at 
the end of the exposure period (typically 6 or 24 hours) and 
subsequent occasions (e.g. 48 and 72 hours). If there are data available that demonstrate substantial differences in dermal toxicity between males and females, the more sensitive sex should be chosen. 
If there are no such data, then either gender can be used.
 
1.4.3.  Housing and feeding conditions  
The temperature in the experimental animal room should be 22  o C 
(± 3  o C). Although the relative humidity should be at least 30 % and 
preferably not exceed 70 % other than during room cleaning, the aim should be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory 
diets may be used and should be freely available together with an 
unlimited supply of drinking water. During the study, and preferably also during the acclimatisation, the animals are individually housed in metabolism cages. Since food and water spillage would 
compromise the results, the probability of such events should be 
minimised.
 
1.4.4.  Preparation of animals  
The animals are marked to permit individual identification and kept 
in their cages for at least five days prior to the start of the study to 
allow for acclimatisation to the laboratory conditions. 
Following the acclimatisation period, and approximately 24 hours 
prior to dosing, each animal will have an area of skin in the region of the shoulders and the back clipped. The permeation prop ­
erties of damaged skin are different from intact skin and care should 
be taken to avoid abrading the skin. Following the clipping and 
approximately 24 hours before the test substance is applied to the skin, (See Section 1.4.7) the skin surface should be wiped with acetone to remove sebum. An additional soap and water wash is 
not recommended because any soap residue might promote test 
substance absorption. The area must be large enough to allow reliable calculation of the absorbed amount of test chemical per cm
 2 skin, preferably at least 10 cm  2 . This area is practicable 
with rats of 200-250 g bodyweight. After preparation, the animals 
are returned to metabolism cages.  
1.4.5.  Test substance  
The test substance is the entity whose penetration characteristics are 
to be studied. Ideally, the test substance should be radiolabelled.  
1.4.6.  Test preparation  
The test substance preparation (e.g. neat, diluted, or formulated 
material containing the test chemical which is applied to the skin) 
should be the same (or realistic surrogate) as that to which humans 
or other potential target species may be exposed. Any variations from the ‘in-use’ preparation must be justified. Where necessary, the test substance is dissolved or suspended in a suitable vehicle. 
For vehicles other than water the absorption characteristics and 
potential interaction with the test substance should be known.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 640
 
1.4.7.  Application to the skin  
An application site of a specific surface area is defined on the skin 
surface. A known amount of the test preparation is then evenly applied to the site. This amount should normally mimic potential 
human exposure, typically 1-5 mg/cm
 2 for a solid or up to 10 
μl/cm  2 for liquids. Any other quantities should be justified by the 
expected use conditions, the study objectives or physical character ­
istics of the test preparation. Following application, the treated site 
must be protected from grooming. An example of a typical device is 
shown in Figure 1. Normally, the application site will be protected by a non-occlusive cover (e.g. a permeable nylon gauze cover). 
However, for infinite applications the application site should be 
occluded. In case of evaporation of semivolatile test substances reduces the recovery rate of the test substance to an unacceptable extend (see also section 1.4.10, first paragraph), it is necessary to 
trap the evaporated substance in a charcoal filter covering the appli ­
cation device (see Figure 1). It is important that any device does not damage the skin, nor absorb or react with the test preparation. The animals are returned to individual metabolism cages in order to 
collect excreta.
 
1.4.8.  Duration of exposure and sampling  
The duration of exposure is the time interval between application and 
removal of test preparation by skin washing. A relevant exposure period (typically 6 or 24 hours) should be used, based on the expected human exposure duration. Following the exposure period, 
the animals are maintained in the metabolism cages until the 
scheduled termination. The animals should be observed for signs of toxicity/abnormal reactions at regular intervals for the entire duration of the study. At the end of the exposure period the 
treated skin should be observed for visible signs of irritation. 
The metabolism cages should permit separate collection of urine and 
faeces throughout the study. They should also allow collection of
 
14 C-carbon dioxide and volatile  14 C-carbon compounds, which 
should be analysed when produced in quantity (> 5 %). The urine, 
faeces and trap fluids (e.g.  14 C-carbon dioxide and volatile  14 C- 
compounds) should be individually collected from each group at each sampling time. If there is sufficient information that little or no volatile radioactive metabolite is formed, open cages can be used. 
Excreta are collected during the exposure period, up to 24 hours after 
the initial skin contact and then daily until the end of the experiment. Whilst three excreta collection intervals will normally be sufficient, the envisaged purpose of the test preparation or existing kinetic data 
may suggest more appropriate or additional time points for study. 
At the end of the exposure period the protective device is removed 
from each animal and retained separately for analysis. The treated skin of all animals should be washed at least three times with cleansing agent using suitable swabs. Care must be taken to avoid 
contaminating other parts of the body. The cleansing agent should be 
representative of normal hygiene practice, e.g. aqueous soap solution. Finally, the skin should be dried. All swabs and washings must be retained for analysis. A fresh cover should be 
applied to protect the treated site of those animals forming later 
time point groups prior to their return to individual cages.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 641
 
1.4.9.  Terminal procedures  
For each group, the individual animals should be killed at the 
scheduled time and blood collected for analysis. The protective device or cover should be removed for analysis. The skin from the 
application site and a similar area of non-dosed, clipped skin should 
be removed from each animal for separate analysis. The application site may be fractioned to separate the  stratum corneum  from the 
underlying epidermis to provide more information on the test 
chemical disposition. The determination of this disposition over a 
time course after the exposure period should provide some indication of the fate of any test chemical in the  stratum corneum . To facilitate 
skin fractionation (following the final skin wash and killing the 
animal) each protective cover is removed. The application site skin, with annular ring of surrounding skin, is excised from the rat and pinned on a board. A strip of adhesive tape is applied to the skin 
surface using gentle pressure and the tape removed together with part 
of the  stratum corneum . Successive strips of tape are applied until 
the tape no longer adheres to the skin surface, when all of the  
stratum corneum  has been removed. For each animal, all the tape 
strips may be combined in a single container to which a tissue 
digestant is added to solubilise the  stratum corneum . Any potential 
target tissues may be removed for separate measurement before the residual carcass is analysed for absorbed carcass dose. The carcasses 
of the individual animals should be retained for analysis. Usually 
analysis of the total content will be sufficient. Target organs may be removed for separate analysis (if indicated by other studies). Urine present in the bladder at scheduled kill should be added to the 
previous urine collection. After collection of the excreta from 
metabolism cages at the time scheduled kill, the cages and their traps should be washed with an appropriate solvent. Other potentially contaminated equipment should likewise be analysed.
 
1.4.10.  Analysis  
In all studies adequate recovery (i.e. mean of 100 ± 10 % of the 
radioactivity) should be achieved. Recoveries outside this range must 
be justified. The amount of the administered dose in each sample should be analysed by suitably validated procedures. 
Statistical considerations should include a measure of variance for 
the replicates for each application.
 
2. DATA  
The following measurements should be made for each animal, at 
each sampling time for the test chemical and/or metabolites. In 
addition to individual data, data grouped according to sampling 
times should be reported as means. 
— quantity associated with the protective appliances, — quantity that can be dislodged from the skin, — quantity in/on skin that cannot be washed from the skin,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 642
 
— quantity in the sampled blood, 
— quantity in the excreta and expired air (if appropriate), — quantity remaining in the carcass and any organs removed for 
separate analysis. 
The quantity of test substance and/or metabolites in the excreta, 
expired air, blood and in the carcass will allow determination of the total amount absorbed at each time point. A calculation of the amount of test chemical absorbed per cm
 2 of skin exposed to the test 
substance over the exposure period can also be obtained.  
3. REPORTING  
3.1. TEST REPORT 
The test report must include the requirements stipulated in the 
protocol, including a justification for the test system used and 
should comprise the following: 
test substance: 
— identification data (e.g. CAS number, if available, source, purity 
(radiochemical purity), known impurities, lot number), 
— physical nature, physicochemical properties (e.g. pH, volatility, 
solubility, stability, molecular weight and log P  ow ). 
Test preparation: — formulation and justification of use, 
— details of the test preparation, amount applied, achieved concen ­
tration, vehicle, stability and homogeneity. 
Test animal: 
— species/strain used, — number, age and sex of animals, 
— source of animals, housing conditions, diets, etc., 
— individual animal weights at start of test. Test conditions: 
— details of the administration of the test preparation (site of appli ­
cation, assay methods, occlusion/non-occlusion, volume, 
extraction, detection), 
— details of food and water quality. 
Results: 
— any signs of toxicity, — tabulated absorption data (expressed as rate, amount or percen ­
tage),
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 643
 
— overall recoveries of the experiment, 
— interpretation of the results, comparison with any available data 
on percutaneous absorption of the test compound. 
Discussion of the results. Conclusions.
 
4. REFERENCES  
(1) Testing Method B.45. Skin Absorption:  In vitro  Method. 
(2) OECD (2002). Guidance Document for the Conduct of Skin 
Absorption Studies. OECD, Paris. 
(3) ECETOC, (1993) Percutaneous Absorption. European Centre 
for Ecotoxicology and Toxicology of Chemicals, Monograph 
No 20. 
(4) Zendzian R.P. (1989) Skin Penetration Method suggested for 
Environmental Protection Agency Requirements.  J. Am. Coll. 
Toxicol . 8(5), p. 829-835. 
(5) Kemppainen, B.W., Reifenrath WG (1990) Methods for skin 
absorption. CRC Press Boca Raton, FL, USA. 
(6) EPA, (1992) Dermal Exposure Assessment: Principles and 
Applications. Exposure Assessment Group, Office of Health and Environmental Assessment. 
(7) EPA, (1998) Health Effects Test Guidelines, OPPTS 870-7600, 
Dermal Penetration. Office of Prevention, Pedsticides and Toxic 
Substances. 
(8) Bronaugh, R.L., Wester, R.C., Bucks, D., Maibach H.I. and 
Sarason, R., (1990) In vivo percutaneous absorption of fragrance ingredients in reshus monkeys and humans.  Fd. 
Chem. Toxic.  28, p. 369-373. 
(9) Feldman, R.J. and Maibach, H.I., (1970) Absorption of some 
organic compounds through the skin in man.  J. Invest Dermatol.  
54, p. 399-404.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 644
 
Figure 1  
An example of a design of a typical device used to define and protect dermal application site 
during  in vivo  percutaneous absorption studies
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 645
 
B.45.  SKIN ABSORPTION:  IN VITRO  METHOD  
1. METHOD  
This testing method is equivalent to the OECD TG 428 (2004).  
1.1. INTRODUCTION 
This method has been designed to provide information on absorption 
of a test substance applied to excised skin. It can either be combined with the method for skin absorption:  in vivo  method (1), or be 
conducted separately. It is recommended that the OECD Guidance 
Document for the Conduct of Skin Absorption Studies (2) be 
consulted to assist in the design of studies based on this method. The Guidance Document has been prepared to facilitate the selection of appropriate  in vitro  procedures for use in specific circumstances, 
to ensure the reliability of results obtained by this method. 
The methods for measuring skin absorption and dermal delivery can 
be divided into two categories:  in vivo  and in vitr o. In vivo  methods 
on skin absorption are well established and provide pharmacokinetic information in a range of animal species. An  in vivo  method is 
separately described in another testing method (1).  In vitro  
methods have also been used for many years to measure skin 
absorption. Although formal validation studies of the  in vitro  
methods covered by this testing method have not been performed, OECD experts agreed in 1999 that there was sufficient data 
evaluated to support the  in vitro  method (3). Further details that 
substantiate this support, including a significant number of direct comparisons of  in vitro  and  in vivo  methods, are provided with 
the Guidance Document (2). There are a number of monographs 
that review this topic and provide detailed background on the use 
of an  in vitro  method (4)(5)(6)(7)(8)(9)(10)(11)(12).  In vitro  methods 
measure the diffusion of chemicals into and across skin to a fluid reservoir and can utilise non-viable skin to measure diffusion only, 
or fresh, metabolically active skin to simultaneously measure 
diffusion and skin metabolism. Such methods have found particular use as a screen for comparing delivery of chemicals into and through skin from different formulations and can also provide useful models 
for the assessment of percutaneous absorption in humans. 
The in vitro  method may not be applicable for all situations and classes 
of chemicals. It may be possible to use the  in vitro  test method for an 
initial qualitative evaluation of skin penetration. In certain cases, it may be necessary to follow this up with  in vivo  data. The guidance 
document (2) should be consulted for further elaboration of situations 
where the  in vitro  method would be suitable. Additional detailed 
information to support the decision is provided in (3). 
This method presents general principles for measuring dermal 
absorption and delivery of a test substance using excised skin. 
Skin from many mammalian species, including humans, can be used. The permeability properties of skin are maintained after excision from the body because the principal diffusion barrier is 
the non-viable  stratum corneu m; active transport of chemicals 
through the skin has not been identified. The skin has been shown to have the capability to metabolise some chemicals during percut ­
aneous absorption (6), but this process is not rate limiting in terms of 
actual absorbed dose, although it may affect the nature of the 
material entering the bloodstream.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 646
 
1.2. DEFINITIONS  
Unabsorbed dose:  represents that washed from the skin surface after 
exposure and any present on the non-occlusive cover, including any dose shown to volatilise from the skin during exposure.  
Absorbed dose ( in vitro ): mass of test substance reaching the 
receptor fluid or systemic circulation within a specified period of 
time.  
Absorbable dose ( in vitro ): represents that present on or in the skin 
following washing.
 
1.3. PRINCIPLE OF THE TEST METHOD 
The test substance, which may be radiolabelled, is applied to the 
surface of a skin sample separating the two chambers of a diffusion cell. The chemical remains on the skin for a specified time under 
specified conditions, before removal by an appropriate cleansing 
procedure. The receptor fluid is sampled at time points throughout the experiment and analysed for the test chemical and/or metabolites. 
When metabolically active systems are used, metabolites of the test 
chemical may be analysed by appropriate methods. At the end of the experiment the distribution of the test chemical and its metabolites 
are quantified, when appropriate. 
Using appropriate conditions, which are described in this method and 
the guidance document (2), absorption of a test substance during a 
given time period is measured by analysis of the receptor fluid and the treated skin. The test substance remaining in the skin should be considered as absorbed unless it can be demonstrated that absorption 
can be determined from receptor fluid values alone. Analysis of the 
other components (material washed off the skin and remaining within the skin layers) allows for further data evaluation, including total test substance disposition and percentage recovery. 
To demonstrate the performance and reliability of the test system in 
the performing laboratory, the results for relevant reference chemicals should be available and in agreement with published 
literature for the method used. This requirement could be met by 
testing an appropriate reference substance (preferably of a lipo ­
philicity close to the test substance) concurrently with the test substance or by providing adequate historical data for a number of 
reference substances of different lipophilicity (e.g. caffeine, benzoic 
acid, and testosterone).
 
1.4. DESCRIPTION OF THE TEST METHOD  
1.4.1.  Diffusion cell  
A diffusion cell consists of a donor chamber and a receptor chamber 
between which the skin is positioned (an example of a typical design is provided in Figure 1). The cell should provide a good seal around the skin, enable easy sampling and good mixing of the receptor 
solution in contact with the underside of the skin, and good 
temperature control of the cell and its contents. Static and flow- through diffusion cells are both acceptable. Normally, donor chambers are left unoccluded during exposure to a finite dose of a 
test preparation. However, for infinite applications and certain 
scenarios for finite doses, the donor chambers may be occluded.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 647
 
1.4.2.  Receptor fluid  
The use of a physiologically conducive receptor fluid is preferred 
although others may also be used provided that they are justified. The precise composition of the receptor fluid should be provided. 
Adequate solubility of the test chemical in the receptor fluid should 
be demonstrated so that it does not act as a barrier to absorption. In addition, the receptor fluid should not affect skin preparation integrity. In a flow-through system, the rate of flow must not 
hinder diffusion of a test substance into the receptor fluid. In a 
static cell system, the fluid should be continuously stirred and sampled regularly. If metabolism is being studied, the receptor 
fluid must support skin viability throughout the experiment.
 
1.4.3.  Skin preparations  
Skin from human or animal sources can be used. It is recognised that 
the use of human skin is subject to national and international ethical considerations and conditions. Although viable skin is preferred, non-viable skin can also be used provided that the integrity of the 
skin can be demonstrated. Either epidermal membranes (enzymically, 
heat or chemically separated) or split thickness skin (typically 200- 400 μm thick) prepared with a dermatome, are acceptable. Full 
thickness skin may be used but excessive thickness (approximately 
> 1 mm) should be avoided unless specifically required for deter ­
mination of the test chemical in layers of the skin. The selection of species, anatomical site and preparative technique must be justified. Acceptable data from a minimum of four replicates per test prep ­
aration are required.
 
1.4.4.  Skin preparation integrity  
It is essential that the skin is properly prepared. Inappropriate 
handling may result in damage to the  stratum corneu m, hence the 
integrity of the prepared skin must be checked. When skin 
metabolism is being investigated, freshly excised skin should be 
used as soon as possible, and under conditions known to support metabolic activity. As a general guidance, freshly excised skin should be used within 24 hours, but the acceptable storage period 
may vary depending on the enzyme system involved in metaboli ­
sation and storage temperatures (13). When skin preparations have been stored prior to use, evidence should be presented to show that barrier function is maintained.
 
1.4.5.  Test substance  
The test substance is the entity whose penetration characteristics are 
to be studied. Ideally, the test substance should be radiolabelled.  
1.4.6.  Test preparation  
The test substance preparation (e.g. neat, diluted or formulated 
material containing the test substance which is applied to the skin) should be the same (or a realistic surrogate) as that to which humans 
or other potential target species may be exposed. Any variation from 
the ‘in-use’ preparation must be justified.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 648
 
1.4.7.  Test substances concentrations and formulations  
Normally more than one concentration of the test substance is used 
spanning the upper of potential human exposures. Likewise, testing a range of typical formulations should be considered.
 
1.4.8.  Application to the skin  
Under normal conditions of human exposure to chemicals, finite 
doses are usually encountered. Therefore, an application that 
mimics human exposure, normally 1-5 mg/cm  2 of skin for a solid 
and up to10 μl/cm  2 for liquids, should be used. The quantity should 
be justified by the expected use conditions, the study objectives or physical characteristics of the test preparation. For example, appli ­
cations to the skin surface may be infinite, where large volumes per 
unit area are applied.
 
1.4.9.  Temperature  
The passive diffusion of chemicals (and therefore their skin 
absorption) is affected by temperature. The diffusion chamber and skin should be maintained at a constant temperature close to normal skin temperature of 32 ± 1
 o C. Different cell designs will require 
different water bath or heated block temperatures to ensure that the 
receptor/skin is at its physiological norm. Humidity should preferably be between 30 and 70 %.
 
1.4.10.  Duration of exposure and sampling  
Skin exposure to the test preparation may be for the entire duration 
of the experiment or for shorter times (i.e., to mimic a specific type of human exposure). The skin should be washed of excess test 
preparation with a relevant cleansing agent, and the rinses 
collected for analysis. The removal procedure of the test preparation will depend on the expected use condition, and should be justified. A period of sampling of 24 hours is normally required to allow for 
adequate characterisation of the absorption profile. Since skin 
integrity may start to deteriorate beyond 24 hours, sampling times should not normally exceed 24 hours. For test substances that penetrate the skin rapidly this may not be necessary but, for test 
substances that penetrate slowly, longer times may be required. 
Sampling frequency of the receptor fluid should allow the absorption profile of the test substance to be presented graphically.
 
1.4.11.  Terminal procedures  
All components of the test system should be analysed and recovery 
is to be determined. This includes the donor chamber, the skin surface rinsing, the skin preparation and the receptor fluid/chamber. 
In some cases, the skin may be fractionated into the exposed area of 
skin and area of skin under the cell flange, and into  stratum 
corneu m, epidermis and dermis fractions, for separate analysis.
 
1.4.12.  Analysis  
In all studies adequate recovery should be achieved (the aim should 
be a mean of 100 ± 10 % of the radioactivity and any deviation should be justified). The amount of test substance in the receptor 
fluid, skin preparation, skin surface washings and apparatus rinse 
should be analysed, using a suitable t echnique.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 649
 
2. DATA  
The analysis of receptor fluid, the distribution of the test substance 
chemical in the test system and the absorption profile with time, should be presented. When finite dose conditions of exposure are 
used, the quantity washed from the skin, the quantity associated with 
the skin (and in the different skin layers if analysed) and the amount present in the receptor fluid (rate, and amount or percentage of applied dose) should be calculated. Skin absorption may 
sometimes be expressed using receptor fluid data alone. However, 
when the test substance remains in the skin at the end of the study, it may need to be included in the total amount absorbed (see paragraph 
66 in reference (3)). When infinite dose conditions of exposure are 
used the data may permit the calculation of a permeability constant (Kp). Under the latter conditions, the percentage absorbed is not relevant.
 
3. REPORTING  
3.1. TEST REPORT 
The test report must include the requirements stipulated in the 
protocol, including a justification for the test system used and 
should, comprise the following: 
test substance: 
— physical nature, physicochemical properties (at least molecular 
weight and log P  ow ), purity (radiochemical purity), 
— identification information (e.g. batch number), 
— solubility in receptor fluid. Test preparation: — formulation and justification of use, — homogeneity. 
Test conditions: 
— sources and site of skin, method of preparation, storage 
conditions prior to use, any pre-treatment (cleaning, antibiotic 
treatments, etc.), skin integrity measurements, metabolic stat us, 
justification of use, 
— cell design, receptor fluid composition, receptor fluid flow rate or 
sampling times and procedures, 
— details of application of test preparation and quantification of 
dose applied, 
— duration of exposure, — details of removal of test preparation from the skin, e.g. skin 
rinsing, 
— details of analysis of skin and any fractionation techniques 
employed to demonstrate skin distribution,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 650
 
— cell and equipment washing procedures, 
— assay methods, extraction techniques, limits of detection and 
analytical method validation. 
Results: — overall recoveries of the experiment (Applied dose ≡ Skin 
washings + Skin + Receptor fluid + Cell washings), 
— tabulation of individual cell recoveries in each compartment, — absorption profile, — tabulated absorption data (expressed as rate, amount or percen ­
tage). 
Discussion of results. 
Conclusions.
 
4. REFERENCES  
(1) Testing Method B.44. Skin Absorption:  In Vivo  Method. 
(2) OECD, (2002) Guidance Document for the Conduct of Skin 
Absorption Studies. OECD, Paris. 
(3) OECD, (2000) Report of the Meeting of the OECD Extended 
Steering Committee for Percutaneous Absorption Testing, Annex 1 to ENV/JM/TG(2000)5. OECD, Paris. 
(4) Kemppainen B.W. and Reifenrath W.G., (1990) Methods for 
skin absorption. CRC Press, Boca Raton. 
(5) Bronaugh R.L. and Collier, S.W., (1991) Protocol for In vitro 
Percutaneous Absorption Studies, in  In vitro Percutaneous 
Absorption: Principles, Fundamentals and Applications , RL 
Bronaugh and HI Maibach, Eds., CRC Press, Boca Raton, p. 237-241. 
(6) Bronaugh R.L. and Maibach H.I., (1991)  In vitro Percutaneous 
Absorption: Principles, Fundamentals and Application s. CRC 
Press, Boca Raton. 
(7) European Centre for Ecotoxicology and Toxicology of 
Chemicals, (1993) Monograph No 20, Percutaneous 
Absorption, ECETOC, Brussels. 
(8) Diembeck W, Beck H, Benech-Kieffer F, Courtellemont P, 
Dupuis J, Lovell W, Paye M, Spengler J, Steiling W., (1999) Test Guidelines for  In Vitro  Assessment of Dermal 
Absorption and Percutaneous Penetration of Cosmetic 
Ingredients, Fd Chem Tox, 37, p. 191-205. 
(9) Recommended Protocol for  In vitro  Percutaneous Absorption 
Rate Studies (1996). US Federal Register, Vol. 61, No 65. 
(10) Howes, D., Guy, R., Hadgraft, J., Heylings, J.R.  et al.  (1996). 
Methods for assessing Percutaneous absorption. ECVAM 
Workshop Report ATLA 24, 81 R10.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 651
 
(11) Schaefer, H. and Redelmeier, T.E., (1996). Skin barrier: prin ­
ciples of percutaneous absorption. Karger, Basel. 
(12) Roberts, M.S. and Walters, K.A., (1998). Dermal absorption 
and toxicity assessment. Marcel Dekker, New York. 
(13) Jewell, C., Heylings, J.R., Clowes, H.M. and Williams, F.M. 
(2000). Percutaneous absorption and metabolism of dinitro ­
chlorobenzene in vitro. Arch Toxicol 74, p. 356-365.  
Figure 1  
An example of a typical design of a static diffusion cell for  in vitro  percutaneous 
absorption studies
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B.46.  IN VITRO  SKIN IRRITATION: RECONSTRUCTED HUMAN 
EPIDERMIS TEST METHOD  
INTRODUCTION 
1. Skin irritation refers to the production of reversible damage to the skin 
following the application of a test chemical for up to 4 hours (as defined 
by the United Nations (UN) Globally Harmonised System of Classification and Labelling of Chemicals (GHS) and Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on 
classification, labelling and packaging of substances and mixtures (1) (3)). 
This Test Method (TM) provides an  in vitro  procedure that may be used for 
the hazard identification of irritant chemicals (substances and mixtures) in accordance with UN GHS and EU CLP Category 2 (1) (2) (3). In the EU 
and other regions, that have not adopted the optional UN GHS Category 3 
(mild irritants), this TM can also be used to identify non-classified chemicals, i.e. UN GHS and EU CLP ‘No Category’ (1) (3). This TM 
may be used to determine the skin irritancy of chemicals as a stand-alone 
replacement test for  in vivo  skin irritation testing within a tiered testing 
strategy (4 and Chapter B.4 in this Annex). 
2. The assessment of skin irritation has typically involved the use of laboratory 
animals (OECD Test Guideline 404; Chapter B.4 in this Annex) (4). In relation to animal welfare concerns, B.4 was revised in 2004 allowing for the determination of skin corrosion/irritation by applying a tiered testing 
strategy, using validated  in vitro  or ex vivo  test methods, thus avoiding pain 
and suffering of animals. Three validated  in vitro  test methods have been 
adopted as OECD Test Guidelines 430, 431 and 435 (5) (6) (7) and two of them as Chapters B.40 and B.40a of this Annex, to be used for the 
corrosivity part of the tiered testing strategy of B.4 or OECD Test 
Guideline 404 (4). 
3. This TM addresses the human health endpoint skin irritation. It is based on 
reconstructed human epidermis (RhE), which in its overall design (the use of human derived non-transformed epidermis keratinocytes as cell source and use of representative tissue and cytoarchitecture) closely mimics the 
biochemical and physiological properties of the upper parts of the human 
skin, i.e. the epidermis. This TM also includes a set of Performance Standards (PS) (Appendix 2) for the assessment of similar and modified RhE-based methods developed by EC-ECVAM (8), in accordance with the 
principles of OECD Guidance Document No 34 (9). 
4. There are three validated methods that adhere to this TM. Prevalidation, 
optimisation and validation studies have been completed for an  in vitro  
method (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20), using a 
RhE model, commercially available as EpiSkin
 TM (designated the 
Validated Reference Method — VRM). Two other commercially available  
in vitro  skin irritation RhE methods have shown similar results to the VRM 
according to PS-based validation (21), and these are the EpiDerm  TM SIT 
(EPI-200) and the SkinEthic  TM RHE methods (22).  ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 653
 
5. Before a proposed similar or modified  in vitro  RhE method other than 
the VRM, EpiDerm  TM SIT (EPI-200) or SkinEthic  TM RHE methods can 
be used for regulatory purposes, its reliability, relevance (accuracy), and limitations for its proposed use should be determined in order to ensure that it can be regarded as similar to that of the VRM, in accordance with 
the requirements of the PS set out in this TM (Appendix 2). Moreover, it 
is recommended to consult the OECD Explanatory Background Document on  in vitro  skin irritation testing before developing and vali ­
dating a similar or modified  in vitro  RhE method and submitting it for 
regulatory adoption (23).
 
DEFINITIONS 
6. Definitions used are provided in Appendix 1.  
INITIAL CONSIDERATIONS AND LIMITATIONS 
7. A limitation of the TM, as demonstrated by the validation study (16), is that 
it does not allow the classification of chemicals to the optional UN GHS 
Category 3 (mild irritants) (1). When used as a partial replacement test, follow-up  in vivo  testing may be required to fully characterise skin irritation 
potential (4 and Chapter B.4 of this Annex). It is recognised that the use of 
human skin is subject to national and international ethical considerations 
and conditions. 
8. This TM addresses the  in vitro  skin irritation component of the tiered 
testing strategy of B.4 (OECD Test Guideline 404) on dermal corrosion/ irritation (4). While this TM does not provide adequate information on skin corrosion, it should be noted that B.40a (OECD Test Guideline 431) on 
skin corrosion is based on the same RhE test system, though using another 
protocol (Chapter B.40a). This method is based on RhE-models using human keratinocytes, which therefore represent  in vitro  the target organ 
of the species of interest. It moreover directly covers the initial step of 
the inflammatory cascade/mechanism of action (cell damage and tissue 
damage resulting in localised trauma) that occurs during irritation  in vivo . 
A wide range of chemicals has been tested in the validation underlying this TM and the empirical database of the validation study amounted to 58 
chemicals in total (16) (18) (23). This is applicable to solids, liquids, 
semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water. Whenever possible, solids should be ground to a fine powder before application; no other 
pre-treatment of the sample is required. Gases and aerosols have not been 
assessed yet in a validation study (24). While it is conceivable that these can be tested using RhE technology, the current TM does not allow testing of gases and aerosols. It should also be noted that highly coloured 
chemicals may interfere with the cell viability measurements and need the 
use of adapted controls for corrections (see paragraphs 24-26). 
9. A single testing run composed of three replicate tissues should be sufficient 
for a test chemical when the classification is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent viability equal to 50 ± 5 %, a second run should be 
considered, as well as a third one in case of discordant results between the 
first two runs.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 654
 
PRINCIPLE OF THE TEST 
10. The test chemical is applied topically to a three-dimensional RhE model, 
comprised of non-transformed human-derived epidermal keratinocytes, 
which have been cultured to form a multilayered, highly differentiated model of the human  epidermis . It consists of organised basal, spinous 
and granular layers, and a multilayered  stratum corneum  containing inter ­
cellular lamellar lipid layers representing main lipid classes analogous to 
those found  in vivo . 
11. Chemical-induced skin irritation, manifested by erythema and oedema, is 
the result of a cascade of events beginning with penetration of the  stratum 
corneum  and damage to the underlying layers of keratinocytes. The dying 
keratinocytes release mediators that begin the inflammatory cascade which acts on the cells in the  dermis , particularly the stromal and endothelial cells. 
It is the dilatation and increased permeability of the endothelial cells that 
produce the observed erythema and oedema (24). The RhE-based methods measure the initiating events in the cascade. 
12. Cell viability in RhE models is measured by enzymatic conversion of the 
vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue); CAS number 298-93-1 into a blue formazan salt that is quantitatively measured after extraction from tissues (25). 
Irritant chemicals are identified by their ability to decrease cell viability 
below defined threshold levels ( i.e. ≤ 50 %, for UN GHS/EU CLP 
Category 2). Depending on the regulatory framework in which the results of this TM are used, chemicals that produce cell viabilities above the 
defined threshold level, may be considered non-irritants ( i.e. > 50 %, 
No Category).
 
DEMONSTRATION OF PROFICIENCY 
13. Prior to routine use of any of the three validated methods that adhere to this 
TM, laboratories should demonstrate technical proficiency, using the ten Reference Chemicals listed in Table 1. For similar methods developed under this TM or for modifications of any of the three validated 
methods, the PS requirements described in Appendix 2 of this TM 
should be met prior to using the method for regulatory testing. 
14. As part of the proficiency exercise, it is recommended that the user verifies 
the barrier properties of the tissues after receipt as specified by the RhE 
model producer. This is particularly important if tissues are shipped over long distance/time periods. Once a method has been successfully established and proficiency in its use has been demonstrated, such verification will not 
be necessary on a routine basis. However, when using a method routinely, it 
is recommended to continue to assess the barrier properties in regular intervals.
 
Table 1  
Reference Chemicals  ( 1 ) 
Chemical CAS NR  In vivo  score (  2 ) Physical state UN GHS/EU CLP 
Category  
naphthalene acetic acid 86-87-3 0 Solid No Cat. 
isopropanol 67-63-0 0,3 Liquid No Cat.  ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 655
 
Chemical CAS NR  In vivo  score (  2 ) Physical state UN GHS/EU CLP 
Category  
methyl stearate 112-61-8 1 Solid No Cat. 
heptyl butyrate 5870-93-9 1,7 Liquid No Cat. 
(Optional Cat. 
3) ( 3 ), ( 4 ) 
hexyl salicylate 6259-76-3 2 Liquid No Cat. 
(Optional Cat. 
3) ( 3 ), ( 4 ) 
cyclamen aldehyde 103-95-7 2,3 Liquid Cat. 2 1-bromohexane 111-25-1 2,7 Liquid Cat. 2 
potassium hydroxide (5 % aq.) 1310-58-3 3 Liquid Cat. 2 
1-methyl-3-phenyl-1-piperazine 5271-27-2 3,3 Solid Cat. 2 Heptanal 111-71-7 3,4 Liquid Cat. 2
 
( 1 ) These Reference Chemicals are a subset of the Reference Chemicals used in the validation study. 
( 2 ) In vivo  score in accordance with B.4 and OECD Test Guideline 404 (4). 
( 3 ) Under this Test Method, the UN GHS optional Category 3 (mild irritants) (1) is considered as No Category. 
( 4 ) The UN GHS optional Category3 is not applicable under the EU CLP.  
PROCEDURE 
15. The following is a description of the components and procedures of a RhE 
method for skin irritation assessment. A RhE model should be recon ­
structed, and can be in-house-prepared or obtained commercially. Standard Operating Procedures (SOPs) for the EpiSkin
 TM , EpiDerm  TM 
SIT (EPI-200) and SkinEthic  TM RHE are available (26) (27) (28). Testing 
should be performed according to the following:  
RhE Test Method Components  
General conditions  
16. Non-transformed human keratinocytes should be used to reconstruct the 
epithelium. Multiple layers of viable epithelial cells (basal layer,  stratum 
spinosum, stratum granulosum ) should be present under a functional  
stratum corneum . Stratum corneum  should be multilayered containing the 
essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of cytotoxic marker chemicals,  e.g.  sodium 
dodecyl sulphate (SDS) or Triton X-100. The barrier function should be 
demonstrated and may be assessed either by determination of the concen ­
tration at which a marker chemical reduces the viability of the tissues by 50 % (IC
 50 ) after a fixed exposure time, or by determination of the 
exposure time required to reduce cell viability by 50 % (ET  50 ) upon appli ­
cation of the marker chemical at a specified, fixed concentration. The containment properties of the RhE model should prevent the passage of material around the  stratum corneum  to the viable tissue, which would lead 
to poor modelling of skin exposure. The RhE model should be free of 
contamination by bacteria, viruses, mycoplasma, or fungi.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 656
 
Functional conditions  
Viability 
17. The assay used for determining the magnitude of viability is the MTT-assay 
(25). The RhE model users should ensure that each batch of the RhE model 
used meets defined criteria for the negative control (NC). The optical density (OD) of the extraction solvent alone should be sufficiently small,  
i.e. OD< 0,1. An acceptability range (upper and lower limit) for the 
negative control OD values (in the Skin Irritation Test Method conditions) 
are established by the RhE model developer/supplier, and the acceptability ranges for the 3 validated methods are given in Table 2. It should be documented that the tissues treated with NC are stable in culture (provide 
similar viability measurements) for the duration of the test exposure period.
 
Table 2  
Acceptability ranges for negative control OD values  
Lower acceptance limit Upper acceptance limit  
EpiSkin  TM (SM) ≥ 0,6 ≤ 1,5 
EpiDerm  TM SIT (EPI-200) ≥ 1,0 ≤ 2,5 
SkinEthic  TM RHE ≥ 1,2 ≤ 2,5 
Barrier function 
18. The  stratum corneum  and its lipid composition should be sufficient to resist 
the rapid penetration of cytotoxic marker chemicals,  e.g.  SDS or Triton 
X-100, as estimated by IC  50 or ET  50 (Table 3).  
Morphology 
19. Histological examination of the RhE model should be performed demon ­
strating human  epidermis -like structure (including multilayered  stratum 
corneum ). 
Reproducibility 
20. The results of the positive control chemical (PC) and negative controls (NC) 
of the test method should demonstrate reproducibility over time.  
Quality control (QC) 
21. The RhE model developer/supplier should ensure and demonstrate that each 
batch of the RhE model used meets defined production release criteria, 
among which those for  viability  (paragraph 17),  barrier function  (paragraph 
18) and  morphology  (paragraph 19) are the most relevant. These data 
should be provided to the method users, so that they are able to include 
this information in the test report. An acceptability range (upper and lower 
limit) for the IC  50 or the ET  50 should be established by the RhE model 
developer/supplier (or investigator when using an in-house model). Only results produced with qualified tissues can be accepted for reliable 
prediction of irritation classification. As an example, the acceptability 
ranges for the three validated methods are given in Table 3.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 657
 
Table 3  
Examples of QC batch release criteria  
Lower acceptance limit Upper acceptance limit  
EpiSkin  TM (SM) 
(18 hours treatment with SDS) (26) IC 50 = 1,0 mg/ml IC  50 = 3,0 mg/ml 
EpiDerm  TM SIT (EPI-200) 
(1 % Triton X-100) (27) ET 50 = 4,8 hr ET  50 = 8,7 hr 
SkinEthic  TM RHE 
(1 % Triton X-100) (28) ET 50 = 4,0 hr ET  50 = 9,0 hr  
Application of the Test and Control Chemicals  
22. At least three replicates should be used for each test chemical and for the 
controls in each run. For liquids as well as solids, sufficient amount of test chemical should be applied to uniformly cover the  epidermis  surface while 
avoiding an infinite dose,  i.e. a minimum of 25 μL/cm
 2 or 25 mg/cm  2 
should be used. For solids, the  epidermis  surface should be moistened 
with deionised or distilled water before application, to improve contact between the test chemical and the  epidermis  surface. Whenever possible, 
solids should be tested as a fine powder. At the end of the exposure period, 
the test chemical should be carefully washed from the  epidermis  surface 
with aqueous buffer, or 0,9 % NaCl. Depending on which of the three validated RhE methods is used, the exposure period varies between 15 and 60 minutes, and the incubation temperature between 20 and 37 °C. 
These exposure periods and temperatures are optimised for each RhE 
method and represent the different intrinsic properties of the methods, for details, see the Standard Operating Procedures (SOPs) for the methods (26) (27) (28). 
23. Concurrent NC and PC should be used in each run to demonstrate that 
viability (with the NC), barrier function and resulting tissue sensitivity (with 
the PC) of the tissues are within a defined historical acceptance range. The 
suggested PC is 5 % aqueous SDS. The suggested NC chemicals are water or phosphate buffered saline (PBS).
 
Cell Viability Measurements  
24. The most important element of the test procedure is that viability 
measurements are not performed immediately after the exposure to the test chemicals, but after a sufficiently long post-treatment incubation 
period of the rinsed tissues in fresh medium. This period allows both for 
recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects. The test optimisation phase (11) (12) (13) (14) (15) demonstrated 
that a 42 hours post-treatment incubation period was optimal. 
25. The MTT assay is a validated quantitative method which should be used to 
measure cell viability under this TM. It is compatible with use in a three- 
dimensional tissue construct. The tissue sample is placed in MTT solution 
of appropriate concentration ( e.g. 0,3-1 mg/mL) for 3 hours. The 
precipitated blue formazan product is then extracted from the tissue using a solvent ( e.g. isopropanol, acidic isopropanol), and the concentration of 
formazan is measured by determining the OD at 570 nm using a filter band 
pass of maximum ± 30 nm.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 658
 
26. Optical properties of the test chemical or its chemical action on the MTT 
may interfere with the assay leading to a false estimate of viability (because 
the test chemical may prevent or reverse the colour generation as well as cause it). This may occur when a specific test chemical is not completely removed from the tissue by rinsing or when it penetrates the  epidermis . If a 
test chemical acts directly on the MTT (MTT-reducer), is naturally 
coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test chemical interference with the viability measurement technique. Detailed description of how to correct 
direct MTT reduction and interferences by colouring agents is available 
in the SOPs for the three validated methods (26) (27) (28).
 
Acceptability Criteria  
27. For each method using valid RhE model batches (see paragraph 21), tissues 
treated with the NC should exhibit OD reflecting the quality of the tissues that followed shipment, receipt steps and all protocol processes. Control OD values should not be below historically established boundaries. Similarly, 
tissues treated with the PC,  i.e. 5 % aqueous SDS, should reflect their 
ability to respond to an irritant chemical under the conditions of the TM(26) (27) (28). Associated and appropriate measures of variability between tissue replicates should be defined ( e.g. if standard 
deviations (SD) are used they should be within the 1-sided 95 % 
tolerance interval calculated from historical data; for the VRM SD < 18 %).
 
Interpretation of Results and Prediction Model  
28. The OD values obtained with each test chemical can be used to calculate 
the percentage of viability normalised to NC, which is set to 100 %. The cut-off value of percentage cell viability distinguishing irritant from non-classified test chemicals and the statistical procedure(s) used to 
evaluate the results and identify irritant chemicals should be clearly 
defined, documented, and proven to be appropriate. The cut-off values for the prediction of irritation are given below: 
— the test chemical is considered to be irritant to skin in accordance with 
UN GHS/EU CLP Category 2 if the tissue viability after exposure and post-treatment incubation is less than or equal ( ≤) to 50 %. 
— depending on the regulatory framework in which the results of this TM 
are used, the test chemical may be considered to be non-irritant to skin 
in accordance with UN GHS/EU CLP No Category if the tissue viability 
after exposure and post-treatment incubation is more than (>) 50 %.
 
DATA AND REPORTING  
Data  
29. For each run, data from individual replicate tissues ( e.g. OD values and 
calculated percentage cell viability data for each test chemical, including classification) should be reported in tabular form, including data from repeat experiments as appropriate. In addition means ± SD for each run should be 
reported. Observed interactions with MTT reagent and coloured test 
chemicals should be reported for each tested chemical.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 659
 
Test Report  
30. The test report should include the following information: 
Test and Control Chemicals: 
— Chemical name(s) such as CAS name and number, EC name and 
number, if known; 
— Purity and composition of the chemical (in percentage(s) by weight); — Physical/chemical properties relevant to the conduct of the study ( e.g. 
physical state, stability, volatility, pH and water solubility if known); 
— Treatment of the test/control chemicals prior to testing, if applicable 
(e.g. warming, grinding); 
— Storage conditions; 
Justification of the RhE model and protocol used 
Test Conditions: 
— Cell system used; — Complete supporting information for the specific RhE model used 
including its performance. This should include, but is not limited to; 
i) viability 
ii) barrier function 
iii) morphology 
iv) reproducibility and predictivity 
v) Quality controls (QC) of the model 
— Details of the test procedure used; 
— Test doses used, duration of exposure and post treatment incubation 
period; 
— Description of any modifications of the test procedure; 
— Reference to historical data of the model. This should include, but is not 
limited to: 
i) acceptability of the QC data with reference to historical batch data 
ii) acceptability of the positive and negative control values with 
reference to positive and negative control means and ranges 
— Description of evaluation criteria used including the justification for the 
selection of the cut-off point(s) for the prediction model; 
— Reference to historical control data;
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 660
 
Results: 
— Tabulation of data from individual test chemicals for each run and each 
replicate measurement; 
— Indication of controls used for direct MTT-reducers and/or colouring 
test chemicals; 
— Description of other effects observed; Discussion of the results 
Conclusion
 
LITERATURE 
(1) UN (2009), United Nations Globally Harmonised System of Classification 
and Labelling of Chemicals (GHS), Third revised edition, UN New York 
and Geneva. Available at: [http://www.unece.org/trans/danger/publi/ghs/ 
ghs_rev03/03files_e.html] 
(2) EC-ECVAM (2009), Statement on the ‘Performance under UN GHS of 
three  in vitro  assays for skin irritation testing and the adaptation of the 
Reference Chemicals and Defined Accuracy Values of the ECVAM skin 
irritation Performance Standards’, issued by the ECVAM Scientific Advisory Committee (ESAC30), 9 April 2009. Available at: [http:// ecvam.jrc.ec.europa.eu] 
(3) Regulation (EC) No 1272/2008 of the European Parliament and of the 
Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC 
and 1999/45/EC, and amending Regulation (EC) No 1907/2006. OJ L 353, 
31.12.2008, p. 1. 
(4) OECD (2004), Acute Dermal Irritation/Corrosion, OECD Guideline for the 
Testing of Chemicals No 404, OECD, Paris. Available at: [http:// 
www.oecd.org/env/testguidelines] 
(5) OECD (2004),  In Vitro  Skin Corrosion: Transcutaneous Electrical 
Resistance (TER), OECD Guideline for the Testing of Chemicals 
No 430, OECD, Paris. Available at: [http://www.oecd.org/env/testguide ­
lines] 
(6) OECD (2004),  In Vitro  Skin Corrosion: Human Skin Model Test, OECD 
Guideline for the Testing of Chemicals No 431, OECD, Paris. Available at: 
[http://www.oecd.org/env/testguidelines] 
(7) OECD (2006),  In Vitro  Membrane Barrier Test Method for Skin Corrosion, 
OECD Guideline for the Testing of Chemicals No 435, OECD, Paris. 
Available at: [http://www.oecd.org/env/testguidelines] 
(8) EC-ECVAM (2009), Performance Standards for  in vitro  skin irritation test 
methods based on Reconstructed human Epidermis (RhE)? Available at: 
[http://ecvam.jrc.ec.europa.eu] 
(9) OECD (2005), Guidance Document on the Validation and International 
Acceptance of New or Updated Test Methods for Hazard Assessment, OECD Series on Testing and Assessment No 34, OECD, Paris. Available 
at: [http://www.oecd.org/env/testguidelines] 
(10) Fentem, J.H., Briggs, D., Chesné, C., Elliot, G.R., Harbell, J.W., Heylings, 
J.R., Portes, P., Roguet, R., van de Sandt, J.J. M. and Botham, P. (2001), A 
prevalidation study on  in vitro  tests for acute skin irritation, Results and 
evaluation by the Management Team, Toxicol. in Vitro 15, 57-93. 
(11) Portes, P., Grandidier, M.-H., Cohen, C. and Roguet, R. (2002), Refinement 
of the EPISKIN protocol for the assessment of acute skin irritation of 
chemicals: follow-up to the ECVAM prevalidation study, Toxicol. in 
Vitro 16, 765-770.
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(12) Kandárová, H., Liebsch, M., Genschow, E., Gerner, I., Traue, D., Slawik, 
B. and Spielmann, H. (2004), Optimisation of the EpiDerm test protocol for 
the upcoming ECVAM validation study on  in vitro  skin irritation tests, 
ALTEX 21, 107-114. 
(13) Kandárová, H., Liebsch, M., Gerner, I., Schmidt, E., Genschow, E., Traue, 
D. and Spielmann, H. (2005), The EpiDerm test protocol for the upcoming 
ECVAM validation study on  in vitro  skin irritation tests — An assessment 
of the performance of the optimised test, ATLA 33, 351-367. 
(14) Cotovio, J., Grandidier, M.-H., Portes, P., Roguet, R. and Rubinsteen, G. 
(2005), The  in vitro  acute skin irritation of chemicals: optimisation of the 
EPISKIN prediction model within the framework of the ECVAM validation process, ATLA 33, 329-349. 
(15) Zuang, V., Balls, M., Botham, P.A., Coquette, A., Corsini, E., Curren, R.D., 
Elliot, G.R., Fentem, J.H., Heylings, J.R., Liebsch, M., Medina, J., Roguet, R., van De Sandt, J.J.M., Wiemann, C. and Worth, A. (2002), Follow-up to the ECVAM prevalidation study on  in vitro  tests for acute skin irritation, 
The European Centre for the Validation of Alternative Methods Skin Irri ­
tation Task Force report 2, ATLA 30, 109-129. 
(16) Spielmann, H., mailto:Hoffmann, S., Liebsch, M., Botham, P., Fentem, J., 
Eskes, C., Roguet, R., Cotovio, J., Cole, T., Worth, A., Heylings, J., Jones, 
P., Robles, C., Kandárová, H., mailto:Gamer, A., Remmele, M., Curren, R., Raabe, H., Cockshott, A., Gerner, I. and Zuang, V. (2007), The ECVAM international validation study on  in vitro  tests for acute skin irritation: 
Report on the validity of the EPISKIN and EpiDerm assays and on the 
skin integrity function test, ATLA 35, 559-601. 
(17) Hoffmann, S. (2006), ECVAM skin irritation validation study phase II: 
Analysis of the primary endpoint MTT and the secondary endpoint IL1- α. 
Available at: [http://ecvam.jrc.ec.europa.eu] 
(18) Eskes, C., Cole, T., Hoffmann, S., Worth, A., Cockshott, A., Gerner, I. and 
Zuang, V. (2007), The ECVAM international validation study on  in vitro  
tests for acute skin irritation: selection of test chemicals, ATLA 35, 603- 
619. 
(19) Cotovio, J., Grandidier, M.-H., Lelièvre, D., Roguet, R., 
Tinois-Tessonneaud, E. and Leclaire, J. (2007),  In vitro  acute skin 
irritancy of chemicals using the validated EPISKIN model in a tiered strategy — Results and performances with 184 cosmetic ingredients, AATEX, 14, 351-358. 
(20) EC-ECVAM (2007), Statement on the validity of  in vitro  tests for skin 
irritation, issued by the ECVAM Scientific Advisory Committee (ESAC26), 27 April 2007. Available at: [http://ecvam.jrc.ec.europa.eu] 
(21) EC-ECVAM (2007), Performance Standards for applying human skin 
models to  in vitro  skin irritation testing. Available at: [http:// 
ecvam.jrc.ec.europa.eu] 
(22) EC-ECVAM (2008), Statement on the scientific validity of  in vitro  tests for 
skin irritation testing, issued by the ECVAM Scientific Advisory Committee 
(ESAC29), 5 November 2008. Available at: [http://ecvam.jrc.ec.europa.eu] 
(23) OECD (2010), Explanatory background document to the OECD draft Test 
Guideline on  in vitro  skin irritation testing. OECD Series on Testing and 
Assessment, No 137, OECD, Paris. Available at: [http://www.oecd.org/ document/24/0,3746,en_2649_34377 _47858904_1_1_1_1,00.html] 
(24) Welss, T., Basketter, D.A. and Schröder, K.R. (2004),  In vitro  skin irri ­
tation: fact and future. State of the art review of mechanisms and models, 
Toxicol. in Vitro 18, 231-243.
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(25) Mosmann, T. (1983), Rapid colorimetric assay for cellular growth and 
survival: application to proliferation and cytotoxicity assays, J. Immunol. 
Methods 65, 55-63. 
(26) EpiSkin  TM SOP, Version 1.8 (February 2009), ECVAM Skin Irritation 
Validation Study: Validation of the EpiSkin  TM test method 15 
min-42 hours for the prediction of acute skin irritation of chemicals. 
Available at: [http://ecvam.jrc.ec.europa.eu] 
(27) EpiDerm  TM SOP, Version 7.0 (Revised March 2009), Protocol for:  In vitro  
EpiDerm  TM skin irritation test (EPI-200-SIT), For use with MatTek Corpor ­
ation’s reconstructed human epidermal model EpiDerm (EPI-200). Available at: [http://ecvam.jrc.ec.europa.eu] 
(28) SkinEthic
 TM RHE SOP, Version 2.0 (February 2009), SkinEthic skin irri ­
tation test-42a test method for the prediction of acute skin irritation of chemicals: 42 minutes application + 42 hours post-incubation. Available at: [http://ecvam.jrc.ec.europa.eu] 
(29) Harvell, J.D., Lamminstausta, K., and Maibach, H.I. (1995), Irritant contact 
dermatitis, In: Practical Contact Dermatitis, pp 7-18, (Ed. Guin J. D.). Mc 
Graw-Hill, New York. 
(30) Commission Directive 2001/59/EC of 6 August 2001 adapting to technical 
progress for the 28th time Council Directive 67/548/EEC on the approxi ­
mation of laws, regulations and administrative provisions relating to the 
classification, packaging and labelling of dangerous substances, OJ L 225, 21.8.2001, p. 1. 
(31) Basketter, D.A., York, M., McFadden, J.P. and Robinson, M.K. (2004), 
Determination of skin irritation potential in the human 4-h patch test. 
Contact Dermatitis 51, 1-4. 
(32) Jirova, D., Liebsch, M., Basketter, D., Spiller, E., Kejlova, K., Bendova, H., 
Marriott, M. and Kandarova, H. (2007), Comparison of human skin irri ­
tation and photo-irritation patch test data with cellular  in vitro  assays and 
animal  in vivo  data, ALTEX, 14, 359-365. 
(33) Jírová, D., Basketter, D., Liebsch, M., Bendová, H., Kejlová, K., Marriott, 
M. and Kandárová, H. (2010), Comparison of human skin irritation patch test data with  in vitro  skin irritation assays and animal data, Contact 
Dermatitis, 62, 109-116.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 663
 
Appendix 1  
Definitions  
Accuracy:  The closeness of agreement between test method results and accepted 
reference values. It is a measure of test method performance and one aspect of relevance. The term is often used interchangeably with ‘concordance’ to mean 
the proportion of correct outcomes of a test method (9).  
Cell viability:  Parameter measuring total activity of a cell population  e.g.  as 
ability of cellular mitochondrial dehydrogenases to reduce the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue), which depending on the endpoint measured and the test design used, correlates 
with the total number and/or vitality of living cells.  
Concordance:  This is a measure of test method performance for test methods that 
give a categorical result, and is one aspect of relevance. The term is used inter ­
changeably with accuracy, and is defined as the proportion of all chemicals tested that are correctly classified as positive or negative. (9).  
ET
 50 : Can be estimated by determination of the exposure time required to reduce 
cell viability by 50 % upon application of the marker chemical at a specified, fixed concentration, see also IC
 50 . 
EU CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the 
Council of 16 December 2008 on classification, labelling and packaging of 
Substances and Mixtures):  Implements in the European Union (EU) the UN 
GHS system for the classification and labelling of chemicals (substances and mixtures) (3).  
GHS (Globally Harmonised System of Classification and Labelling of Chemicals 
by the United Nations (UN)):  A system proposing the classification of chemicals 
(substances and mixtures) according to standardised types and levels of physical, 
health and environmental hazards, and addressing corresponding communication elements, such as pictograms, signal words, hazard statements, precautionary statements and safety data sheets, so that to convey information on their 
adverse effects with a view to protect people (including employers, workers, 
transporters, consumers and emergency responders) and the environment (1).  
IC
 50 : Can be estimated by determination of the concentration at which a marker 
chemical reduces the viability of the tissues by 50 % (IC  50 ) after a fixed 
exposure time, see also ET  50 . 
Infinite dose:  Amount of test chemical applied to the  epidermis  exceeding the 
amount required to completely and uniformly cover the  epidermis  surface.  
Me-too test:  A colloquial expression for a test method that is structurally and 
functionally similar to a validated and accepted reference test method. Such a test 
method would be a candidate for catch-up validation. Interchangeably used with similar test method (9).  
Performance standards (PS):  Standards, based on a validated test method, that 
provide a basis for evaluating the comparability of a proposed test method that is mechanistically and functionally similar. Included are; (i) essential test method 
components; (ii) a minimum list of Reference Chemicals selected from among 
the chemicals used to demonstrate the acceptable performance of the validated test method; and (iii) the comparable levels of accuracy and reliability, based on what was obtained for the validated test method, that the proposed test method 
should demonstrate when evaluated using the minimum list of Reference 
Chemicals (9).
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 664
 
Reference chemicals:  Chemicals selected for use in the validation process, for 
which responses in the  in vitro  or in vivo  reference test system or the species of 
interest are already known. These chemicals should be representative of the classes of chemicals for which the test method is expected to be used, and should represent the full range of responses that may be expected from the 
chemicals for which it may be used, from strong, to weak, to negative. 
Different sets of reference chemicals may be required for the different stages of the validation process, and for different test methods and test uses (9).  
Relevance:  Description of relationship of the test to the effect of interest and 
whether it is meaningful and useful for a particular purpose. It is the extent to 
which the test correctly measures or predicts the biological effect of interest. 
Relevance incorporates consideration of the accuracy (concordance) of a test method (9).  
Reliability:  Measures of the extent that a test method can be performed repro ­
ducibly within and between laboratories over time, when performed using the 
same protocol. It is assessed by calculating intra- and inter-laboratory reproduci ­
bility (9).  
Replacement test:  A test which is designed to substitute for a test that is in 
routine use and accepted for hazard identification and/or risk assessment, and 
which has been determined to provide equivalent or improved protection of 
human or animal health or the environment, as applicable, compared to the accepted test, for all possible testing situations and chemicals (9).  
Sensitivity:  The proportion of all positive/active test chemicals that are correctly 
classified by the test. It is a measure of accuracy for a test method that produces 
categorical results, and is an important consideration in assessing the relevance of 
a test method (9).  
Skin irritation:  The production of reversible damage to the skin following the 
application of a test chemical for up to 4 hours. Skin irritation is a locally arising, 
non-immunogenic reaction, which appears shortly after stimulation (29). Its main 
characteristic is its reversible nature involving inflammatory reactions and most of the clinical characteristic signs of irritation (erythema, oedema, itching and pain) related to an inflammatory process.  
Specificity:  The proportion of all negative/inactive test chemicals that are 
correctly classified by the test. It is a measure of accuracy for a test method 
that produces categorical results and is an important consideration in assessing the relevance of a test method (9).  
Tiered testing strategy:  Testing which uses test methods in a sequential manner; 
the test methods selected in each succeeding level are decided based on the 
results in the previous level of testing (9).  
Test chemical (also referred to as test substance):  Any substance or mixture 
tested using this TM.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 665
 
Appendix 2  
Performance Standards for assessment of proposed similar or modified  
in vitro  reconstructed human  epidermis  (RhE) methods for skin irritation  
INTRODUCTION 
1. The purpose of Performance Standards (PS) is to communicate the basis by 
which new methods, both proprietary ( i.e. copyrighted, trademarked, regis ­
tered) and non-proprietary can be determined to have sufficient accuracy 
and reliability for specific testing purposes. These PS, based on validated and accepted methods, can be used to evaluate the reliability and accuracy of other analogous methods (colloquially referred to as ‘me-too’ tests) that 
are based on similar scientific principles and measure or predict the same 
biological or toxic effect (9). 
2. Prior to adoption of modified methods,  i.e.  proposed potential 
improvements to an approved method, there should be an evaluation to determine the effect of the proposed changes on the test’s performance 
and the extent to which such changes affect the information available for 
the other components of the validation process. Depending on the number and nature of the proposed changes, the generated data and supporting documentation for those changes, they should either be subjected to the 
same validation process as described for a new test, or, if appropriate, to 
a limited assessment of reliability and relevance using established PS (9). 
3. Similar (me-too) or modified methods of any of the three validated methods 
(EpiSkin™ (Validated Reference Method — VRM), EpiDerm™ SIT (EPI- 200) and SkinEthic™ RHE) proposed for use under this TM should be 
evaluated to determine their reliability and accuracy using chemicals repre ­
senting the full range of the Draize irritancy scores. When evaluated using the 20 recommended Reference Chemicals of the PS (Table 1), the proposed similar or modified methods should have reliability and 
accuracy values which are comparable or better than those derived from 
the VRM (Table 2) (2) (16). The reliability and accuracy values that should be achieved are provided in paragraphs 8 to 12 of this Appendix. Non-classified (UN GHS/EU CLP No Category) and classified (UN 
GHS/EU CLP Category 2) (1) chemicals, representing different chemical 
classes are included, so that the reliability and accuracy (sensitivity, specificity and overall accuracy) of the proposed method can be compared to that of the VRM. The reliability of the method, as well as 
its ability to correctly identify UN GHS/EU CLP Category 2 irritant 
chemicals and, depending on the regulatory framework for which data are produced, also its ability to correctly identify UN GHS/EU CLP No Category chemicals (should be determined prior to its use for testing 
new test chemicals. 
4. These PS are based on the EC-ECVAM PS (8), updated according to the 
UN GHS and EU CLP systems on classification and labelling (1) (3). The original PS were defined after the completion of the validation study (21) and were based on the EU classification system as laid down in 
Commission Directive 2001/59/EC of 6 August 2001 adapting to 
technical progress for the 28th time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous 
substances (
 1 ). Due to the adoption of the UN GHS system for classification  ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 666  
( 1 ) OJ L 225, 21.8.2001, p. 1.
 and labelling in EU (EU CLP) (3), which took place between the finali ­
sation of the validation study and the completion of this TM, the PS have 
been updated (8). This update concerns mainly changes (i), in the set of the PS Reference Chemicals; and (ii), the defined reliability and accuracy values (2) (23).
 
PERFORMANCE STANDARDS FOR  IN VITRO  RhE TEST METHODS FOR 
SKIN IRRITATION 
5. The PS comprises the following three elements (9): 
I) Essential Test Method Components 
II) Minimum List of Reference Chemicals 
III) Defined Reliability and Accuracy Values  
I) Essential Test Method Components  
6. These consist of essential structural, functional, and procedural elements of 
a validated method that should be included in the protocol of a proposed, mechanistically and functionally similar or modified method. These 
components include unique characteristics of the method, critical procedural 
details, and quality control measures. Adherence to essential test method components will help to assure that a similar or modified proposed method 
is based on the same concepts as the corresponding VRM (9). The essential 
test method components are described in detail in paragraphs 16 to 21 of the TM and testing should be performed according to the following: 
— The general conditions (paragraph 16) 
— The functional conditions, which include: 
— viability (paragraph 17); 
— barrier function (paragraph 18); 
— morphology (paragraph 19); 
— reproducibility (paragraph 20); and, 
— quality control (paragraph 21)
 
II) Minimum List of Reference Chemicals  
7. Reference Chemicals are used to determine if the reliability and accuracy of 
a proposed similar or modified method, proven to be structurally and func ­
tionally sufficiently similar to the VRM, or representing a minor modifi ­
cation of one of the three validated methods, are comparable or better than 
those of the VRM (2) (8) (16) (23). The 20 recommended Reference 
Chemicals listed in Table 1 include chemicals representing different chemical classes ( i.e. chemical categories based on functional groups), 
and are representative of the full range of Draize irritancy scores (from 
non-irritant to strong irritant). The chemicals included in this list 
comprise 10 UN GHS/EU CLP Category 2 chemicals and 10 non-categorised chemicals, of which 3 are optional UN GHS Category 3 chemicals. Under this Test Method, the optional Category 3 is considered as 
No Category. The chemicals listed in Table 1 are selected from the 
chemicals used in the optimisation phase that followed prevalidation and in the validation study of the VRM, with regard to chemical functionality and physical state (14) (18). These Reference Chemicals represent the 
minimum number of chemicals that should be used to evaluate the 
accuracy and reliability of a proposed similar or modified method, but
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 667
 
should not be used for the development of new methods. In situations 
where a listed chemical is unavailable, other chemicals for which 
adequate  in vivo  reference data are available could be used, primarily 
from the chemicals used in the optimisation phase following prevalidation or the validation study of the VRM. If desired, additional chemicals repre ­
senting other chemical classes and for which adequate  in vivo  reference data 
are available may be added to the minimum list of Reference Chemicals to further evaluate the accuracy of the proposed method.
 
Table 1  
Minimum List of Reference Chemicals for Determination of Accuracy and Reliability Values for Similar or 
Modified RhE Skin Irritation Methods  ( 1 ) 
Chemical CAS Number Physical state  In vivo  score VRM  in vitro  
Cat. UN GHS/EU CLP  in 
vivo Cat. 
1-bromo-4-chlorobutane 6940-78-9 Liquid 0 Cat. 2 No Cat. 
diethyl phthalate 84-66-2 Liquid 0 No Cat. No Cat. 
naphthalene acetic acid 86-87-3 Solid 0 No Cat. No Cat. allyl phenoxy-acetate 7493-74-5 Liquid 0,3 No Cat. No Cat. isopropanol 67-63-0 Liquid 0,3 No Cat. No Cat. 4-methyl-thio-benzaldehyde 3446-89-7 Liquid 1 Cat. 2 No Cat. methyl stearate 112-61-8 Solid 1 No Cat. No Cat. heptyl butyrate 5870-93-9 Liquid 1,7 No Cat. No Cat. hexyl salicylate 6259-76-3 Liquid 2 No Cat. No Cat. Cinnamaldehyde 104-55-2 Liquid 2 Cat. 2 No Cat. 
(Optional Cat. 
3) (
 3 ) 
1-decanol  ( 2 ) 112-30-1 Liquid 2,3 Cat. 2 Cat. 2  
cyclamen aldehyde 103-95-7 Liquid 2,3 Cat. 2 Cat. 2 1-bromohexane 111-25-1 Liquid 2,7 Cat. 2 Cat. 2 2-chloromethyl-3,5-dimethyl- 
4-methoxypyridine HCl 86604-75-3 Solid 2,7 Cat. 2 Cat. 2  
di-n-propyl disulphide  (
 2 ) 629-19-6 Liquid 3 No Cat. Cat. 2  
potassium hydroxide (5 % aq.) 1310-58-3 Liquid 3 Cat. 2 Cat. 2 
benzenethiol, 5-(1,1- 
dimethylethyl)-2-methyl 7340-90-1 Liquid 3,3 Cat. 2 Cat. 2 
1-methyl-3-phenyl-1- 
piperazine 5271-27-2 Solid 3,3 Cat. 2 Cat. 2  ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 668
 
Chemical CAS Number Physical state  In vivo  score VRM  in vitro  
Cat. UN GHS/EU CLP  in 
vivo Cat. 
Heptanal 111-71-7 Liquid 3,4 Cat. 2 Cat. 2 
Tetrachloroethylene 127-18-4 Liquid 4 Cat. 2 Cat. 2  
( 1 ) The chemical selection is based on the following criteria: (i) the chemicals are commercially available; (ii) they are repres entative of 
the full range of Draize irritancy scores (from non-irritant to strong irritant); (iii) they have a well-defined chemical struc ture; (iv) 
they are representative of the chemical functionality used in the validation process; and (v) they are not associated with an e xtremely 
toxic profile ( e.g. carcinogenic or toxic to the reproductive system) and they are not associated with prohibitive disposal costs. 
( 2 ) Chemicals that are irritant in the rabbit but for which there is reliable evidence that they are non-irritant in humans (31) (32) (33). 
( 3 ) Under the UN GHS, not in the EU CLP  
III) Defined Reliability and Accuracy Values  
8. For purposes of establishing the reliability and relevance of proposed 
similar or modified methods to be transferred between laboratories, all 20 Reference Chemicals in Table 1 should be tested in at least three labora ­
tories. However, if the proposed method is to be used in a single laboratory 
only, multi-laboratory testing will not be required for validation. It is 
however essential that such validation studies are independently assessed by internationally recognised validation bodies, in agreement with inter ­
national guidelines (9). In each laboratory, all 20 Reference Chemicals 
should be tested in three independent runs performed with different tissue 
batches and at sufficiently spaced time points. Each run should consist of a minimum of three concurrently tested tissue replicates for each included test chemical, NC and PC. 
9. The calculation of the reliability and accuracy values of the proposed 
method should be done considering all four criteria below together, 
ensuring that the values for reliability and relevance are calculated in a predefined and consistent manner: 
1. Only the data of runs from complete run sequences qualify for the 
calculation of the method within, and between-laboratory variability 
and predictive capacity (accuracy). 
2. The final classification for each Reference Chemicals in each partici ­
pating laboratory should be obtained by using the mean value of viability over the different runs of a complete run sequence. 
3. Only the data obtained for chemicals that have complete run sequences 
in all participating laboratories qualify for the calculation of the method 
between-laboratory variability. 
4. The calculation of the accuracy values should be done on the basis of the 
individual laboratory predictions obtained for the 20 Reference 
Chemicals by the different participating laboratories. 
In this context, a  run sequence  consists of three independent runs from one 
laboratory for one test chemical. A  complete run sequence  is a run 
sequence from one laboratory for one test chemical where all three runs 
are valid. This means that any single invalid run invalidates an entire run 
sequence of three runs.
 
Within-laboratory reproducibility  
10. An assessment of within-laboratory reproducibility should show a 
concordance of classifications (UN GHS/EU CLP Category 2 and No Category) obtained in different, independent test runs of the 20 
Reference Chemicals within one single laboratory equal or higher ( ≥) 
than 90 %.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 669
 
Between-laboratory reproducibility  
11. An assessment of between-laboratory reproducibility is not essential if the 
proposed method is to be used in a single laboratory only. For methods to 
be transferred between laboratories, the concordance of classifications (UN GHS/EU CLP Category 2 and No Category) obtained in different, inde ­
pendent test runs of the 20 Reference Chemicals between preferentially a 
minimum of three laboratories should be equal or higher ( ≥) than 80 %.
 
Predictive capacity (accuracy)  
12. The accuracy (sensitivity, specificity and overall accuracy) of the proposed 
similar or modified method should be comparable or better to that of the 
VRM, taking into consideration additional information relating to relevance 
in the species of interest (Table 2). The sensitivity should be equal or higher (≥) than 80 % (2) (8) (23). However, a further specific restriction applies to 
the sensitivity of the proposed  in vitro  method inasmuch as only two  in vivo  
Category 2 chemicals,  1-decanol  and  di-n-propyl disulphide , may be 
misclassified as No Category by more than one participating laboratory. The specificity should be equal or higher ( ≥) than 70 % (2) (8) (23). 
There is no further restriction with regard to the specificity of the 
proposed  in vitro  method,  i.e. any participating laboratory may misclassify 
any in vivo  No Category chemical as long as the final specificity of the test 
method is within the acceptable range. The overall accuracy should be equal 
or higher ( ≥) than 75 % (2) (8) (23). Although the sensitivity of the VRM 
calculated for the 20 Reference Chemicals listed in Table 1 is equal to 90 %, the defined minimum sensitivity value required for any similar or modified method to be considered valid is set at 80 % since both  1-decanol  
(a borderline chemical) and  di-n-propyl disulphide  (a false negative of the 
VRM) are known to be non-irritant in humans (31) (32) (33), although being identified as irritants in the rabbit test. Since RhE models are based on cells of human origin, they may predict these chemicals as non-irritant 
(UN GHS/EU CLP No Category).
 
Table 2  
Required predictive values for sensitivity, specificity and overall accuracy for any similar or 
modified method to be considered valid  
Sensitivity Specificity Overall Accuracy  
≥ 80 % ≥ 70 % ≥ 75 %  
Study Acceptance Criteria  
13. It is possible that one or several tests pertaining to one or more test 
chemicals does/do not meet the test acceptance criteria for the test and 
control chemicals or is/are not acceptable for other reasons. To complement 
missing data, for each test chemical a maximum number of two additional tests is admissible (‘retesting’). More precisely, since in case of retesting also PC and NC have to be concurrently tested, a maximum number of two 
additional runs may be conducted for each test chemical. 
14. It is conceivable that even after retesting, the minimum number of three 
valid runs required for each tested chemical is not obtained for every 
Reference Chemical in every participating laboratory, leading to an 
incomplete data matrix. In such cases the following three criteria should all be met in order to consider the datasets acceptable: 
1. All 20 Reference Chemicals should have at least one complete run 
sequence.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 670
 
2. In each of at least three participating laboratories, a minimum of 85 % of 
the run sequences need to be complete (for 20 chemicals;  i.e. 3 invalid 
run sequences are allowed in a single laboratory). 
3. A minimum of 90 % of all possible run sequences from at least three 
laboratories need to be complete (for 20 chemicals tested in 3 labora ­
tories;  i.e. 6 invalid run sequences are allowed in total).  ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 671
 
B. 47.  BOVINE CORNEAL OPACITY AND PERMEABILITY TEST 
METHOD FOR IDENTIFYING OCULAR CORROSIVES AND SEVERE IRRITANTS
 
INTRODUCTION 
1. The Bovine Corneal Opacity and Permeability (BCOP) test method is an  in 
vitro  test method that can be used, under certain circumstances and with 
specific limitations, to classify substances and mixtures as ‘ocular corrosives and severe irritants’ (1) (2) (3). For the purpose of this test method, severe 
irritants are defined as those that induce ocular lesions that persist in the 
rabbit for at least 21 days after administration. While it is not considered valid as a complete replacement for the  in vivo  rabbit eye test, the BCOP is 
recommended for use as part of a tiered-testing strategy for regulatory clas ­
sification and labelling within a specific applicability domain (4) (5). Test 
substances and mixtures (6) can be classified as ocular corrosives or severe irritants without further testing in rabbits. A substance that tests negative would need to be tested in rabbits using a sequential testing strategy, as 
outlined in OECD Test Guideline 405 (7) (chapter B. 5 of this Annex). 
2. The purpose of this test method is to describe the procedures used to 
evaluate the potential ocular corrosivity or severe irritancy of a test 
substance as measured by its ability to induce opacity and increased permea ­
bility in an isolated bovine cornea. Toxic effects to the cornea are measured by: (i) decreased light transmission (opacity), and (ii) increased passage of 
sodium fluorescein dye (permeability). The opacity and permeability 
assessments of the cornea following exposure to a test substance are combined to derive an  In Vitro  Irritancy Score (IVIS), which is used to 
classify the irritancy level of the test substance. 
3. Ocular irritants that induce lesions that resolve in less than 21 days and 
non-irritants have also been tested using the BCOP test method. However, 
the accuracy and reliability of the BCOP test method for substances in these 
categories have not been formally evaluated. 
4. Definitions are provided in Appendix 1.
 
INITIAL CONSIDERATIONS AND LIMITATIONS 
5. This test method is based on the Interagency Coordinating Committee on the 
Validation of Alternative Methods (ICCVAM) BCOP test method protocol 
(8), which was developed following an international validation study (4)(5)(9), with contributions from the European Centre for the Validation of Alternative Methods (ECVAM) and the Japanese Center for the Vali ­
dation of Alternative Methods (JaCVAM). The protocol is based on 
information obtained from the Institute for In Vitro Sciences (IIVS) and INVITTOX Protocol 124 (10), which represents the protocol used for the European Community-sponsored prevalidation study of the BCOP assay 
conducted in 1997-1998. Both of these protocols are based on the BCOP 
assay methodology first reported by Gautheron  et al.  (11).
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 672
 
6. The identified limitations for this test method are based on the high false 
positive rates for alcohols and ketones and the high false negative rate for 
solids observed in the validation database (see paragraph 44) (5). When substances within these chemical and physical classes are excluded from the database, the accuracy of BCOP across the EU, EPA, and GHS classifi ­
cation systems is substantially improved (5). Based on the purpose of this 
assay ( i.e., to identify ocular corrosives/severe irritants only), false negative 
rates are not critical since such substances would be subsequently tested in rabbits or with other adequately validated  in vitro  tests, depending on regu ­
latory requirements, using a sequential testing strategy in a weight of 
evidence approach. Furthermore, the current validation database did not allow for an adequate evaluation of some chemical or product classes (e.g., mixtures). However, investigators could consider using this test 
method for all types of test material (including mixtures), whereby a 
positive result could be accepted as indicative of an ocular corrosive or severe irritant response. However, positive results obtained with alcohols or ketones should be interpreted cautiously due to risk of over-prediction. 
7. All procedures with bovine eyes and bovine corneas should follow the 
testing facility’s applicable regulations and procedures for handling 
animal-derived materials, which include, but are not limited to, tissues and tissue fluids. Universal laboratory precautions are recommended (12). 
8. A limitation of the test method is that, although it takes into account some of 
the ocular effects evaluated in the rabbit ocular irritancy test method and to 
some degree their severity, it does not consider conjunctival and iridal injuries. Also, although the reversibility of corneal lesions cannot be evaluated  per se  in the BCOP assay, it has been proposed, based on 
rabbit eye studies, that an assessment of the initial depth of corneal injury 
can be used to distinguish between irreversible and reversible effects (13). Finally, the BCOP does not allow for an assessment of the potential for systemic toxicity associated with ocular exposure. 
9. Efforts are ongoing to further characterize the usefulness and limitations of 
the BCOP assay for identifying non-severe irritants and non-irritants (see also paragraph 45). Users are also encouraged to provide specimens and/or data to validation organizations for a formal evaluation of possible future 
uses of the BCOP test method, including for the identification of non-severe 
irritants and non-irritants. 
10. For any laboratory initially establishing this assay, the proficiency chemicals 
provided in Appendix 2 should be used. A laboratory can use these chemicals to demonstrate their technical competence in performing the 
BCOP test method prior to submitting BCOP assay data for regulatory 
hazard classification purposes.
 
PRINCIPLE OF THE TEST 
11. The BCOP test method is an organotypic model that provides short-term 
maintenance of normal physiological and biochemical function of the bovine 
cornea  in vitro . In this test method, damage by the test substance is assessed 
by quantitative measurements of changes in corneal opacity and permeability with an opacitometer and a visible light spectrophotometer, respectively. Both measurements are used to calculate an IVIS, which is used to assign 
an in vitro  irritancy hazard classification category for prediction of the  in 
vivo ocular irritation potential of a test substance (see Decision criteria).
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 673
 
12. The BCOP test method uses isolated corneas from the eyes of freshly 
slaughtered cattle. Corneal opacity is measured quantitatively as the 
amount of light transmission through the cornea. Permeability is measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior 
chamber. Test substances are applied to the epithelial surface of the 
cornea by addition to the anterior chamber of the corneal holder. Appendix 3 provides a description and a diagram of a corneal holder used in the BCOP. Corneal holders can be obtained commercially from different 
sources or can be constructed.
 
Source and Age of Bovine Eyes and Selection of Animal Species  
13. Cattle sent to slaughterhouses are typically killed either for human 
consumption or for other commercial uses. Only healthy animals considered suitable for entry into the human food chain are used as a source of corneas 
for use in the BCOP. Because cattle have a wide range of weights, 
depending on breed, age, and sex, there is no recommended weight for the animal at the time of slaughter. 
14. Variations in corneal dimensions can result when using eyes from animals of 
different ages. Corneas with a horizontal diameter > 30,5 mm and central 
corneal thickness (CCT) values ≥ 1 100 μm are generally obtained from 
cattle older than eight years, while those with a horizontal diameter < 28,5 mm and CCT < 900 μm are generally obtained from cattle less 
than five years old (14). For this reason, eyes from cattle greater than 60 
months old are not typically used. Eyes from cattle less than 12 months of 
age have not traditionally been used since the eyes are still developing and the corneal thickness and corneal diameter are considerably smaller than that 
reported for eyes from adult cattle. However, the use of corneas from young 
animals ( i.e., 6 to 12 months old) is permissible since there are some 
advantages, such as increased availability, a narrow age range, and decreased hazards related to potential worker exposure to Bovine Spon ­
giform Encephalopathy (15). As further evaluation of the effect of corneal 
size or thickness on responsiveness to corrosive and irritant substances would be useful, users are encouraged to report the estimated age and/or weight of the animals providing the corneas used in a study.
 
Collection and Transport of Eyes to the Laboratory  
15. Eyes are collected by slaughterhouse employees. To minimize mechanical 
and other types of damage to the eyes, the eyes should be enucleated as soon as possible after death. To prevent exposure of the eyes to potentially irritant substances, the slaughterhouse employees should not use detergent when 
rinsing the head of the animal. 
16. Eyes should be immersed completely in Hanks’ Balanced Salt 
Solution (HBSS) in a suitably sized container, and transported to the laboratory in such a manner as to minimize deterioration and/or bacterial contamination. Because the eyes are collected during the slaughter process, 
they might be exposed to blood and other biological substances, including 
bacteria and other microorganisms. Therefore, it is important to ensure that the risk of contamination is minimized ( e.g., by keeping the container 
containing the eyes on wet ice, by adding antibiotics to the HBSS used to 
store the eyes during transport [ e.g., penicillin at 100 IU/mL and strep ­
tomycin at 100 μg/mL]).
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 674
 
17. The time interval between collection of the eyes and use of corneas in the 
BCOP should be minimized (typically collected and used on the same day) 
and should be demonstrated to not compromise the assay results. These results are based on the selection criteria for the eyes, as well as the positive and negative control responses. All eyes used in the assay should 
be from the same group of eyes collected on a specific day.
 
Selection Criteria for Eyes Used in the BCOP  
18. The eyes, once they arrive at the laboratory, are carefully examined for 
defects including increased opacity, scratches, and neovascularisation. Only 
corneas from eyes free of such defects are to be used. 
19. The quality of each cornea is also evaluated at later steps in the assay. 
Corneas that have an opacity greater than seven opacity units (NOTE: the opacitometer should be calibrated with opacity standards that are used to 
establish the opacity units, see Appendix 3) after an initial one hour equili ­
bration period are to be discarded. 
20. Each treatment group (test substance, concurrent negative and positive 
controls) consists of a minimum of three eyes. Three corneas should be 
used for the negative control corneas in the BCOP assay. Since all corneas are excised from the whole globe, and mounted in the corneal chambers, there is the potential for artefacts from handling upon individual 
corneal opacity and permeability values (including negative control). 
Furthermore, the opacity and permeability values from the negative control corneas are used to correct the test article and positive control-treated corneal opacity and permeability values in the IVIS calculations.
 
PROCEDURE  
Preparation of the Eyes  
21. Corneas free of defects are dissected with a 2 to 3 mm rim of sclera 
remaining to assist in subsequent handling, with care taken to avoid 
damage to the corneal epithelium and endothelium. Isolated corneas are 
mounted in specially designed corneal holders that consist of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. Both chambers are filled to excess with 
pre-warmed Eagle's Minimum Essential Medium (EMEM) (posterior 
chamber first), ensuring that no bubbles are formed. The device is then equilibrated at 32 ± 1 °C for at least one hour to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to 
the extent possible (the approximate temperature of the corneal surface  in 
vivo is 32 °C). 
22. Following the equilibration period, fresh pre-warmed EMEM is added to 
both chambers and baseline opacity readings are taken for each cornea. 
Any corneas that show macroscopic tissue damage ( e.g., scratches, pigmen ­
tation, neovascularisation) or an opacity > 7 opacity units are discarded. The mean opacity of all equilibrated corneas is calculated. A minimum of three corneas with opacity values close to the median value for all corneas are 
selected as negative (or solvent) control corneas. The remaining corneas are 
then distributed into treatment and positive control groups.
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 675
 
23. Because the heat capacity of water is higher than that of air, water provides 
more stable temperature conditions for incubation. Therefore, the use a water 
bath for maintaining the corneal holder and its contents at 32 ± 1 °C is recommended. However, air incubators might also be used, assuming precaution to maintain temperature stability ( e.g., by pre-warming of 
holders and media).
 
Application of the Test Substance  
24. Two different treatment protocols are used, one for liquids and surfactants 
(solids or liquids), and one for non-surfactant solids. 
25. Liquids are tested undiluted, while surfactants are tested at a concentration of 
10 % w/v in a 0,9 % sodium chloride solution, distilled water, or other 
solvent that has been demonstrated to have no adverse effects on the test system. Semi-solids, creams, and waxes are typically tested as liquids. Appropriate justification should be provided for alternative dilution concen ­
trations. Corneas are exposed to liquids and surfactants for 10 minutes. Use 
of other exposure times should be accompanied by adequate scientific rationale. 
26. Non-surfactant solids are typically tested as solutions or suspensions at 20 % 
concentration in a 0,9 % sodium chloride solution, distilled water, or other 
solvent that has been demonstrated to have no adverse effects on the test system. In certain circumstances and with proper scientific justification, 
solids may also be tested neat by direct application onto the corneal 
surface using the open chamber method (see paragraph 29). Corneas are exposed to solids for four hours, but as with liquids and surfactants, alter ­
native exposure times may be used with appropriate scientific rationale. 
27. Different treatment methods can be used, depending on the physical nature 
and chemical characteristics ( e.g., solids, liquids, viscous vs. non-viscous 
liquids) of the test substance. The critical factor is ensuring that the test substance adequately covers the epithelial surface and that it is adequately 
removed during the rinsing steps. A closed-chamber method is typically used 
for non-viscous to slightly viscous liquid test substances, while an open-chamber method is typically used for semi-viscous and viscous liquid test substances and for neat solids. 
28. In the closed-chamber method, sufficient test substance (750 μL) to cover 
the epithelial side of the cornea is introduced into the anterior chamber through the dosing holes on the top surface of the chamber, and the holes are subsequently sealed with the chamber plugs during the exposure. It is 
important to ensure that each cornea is exposed to a test substance for the 
appropriate time interval. 
29. In the open-chamber method, the window-locking ring and glass window 
from the anterior chamber are removed prior to treatment. The control or test 
substance (750 μL, or enough test substance to completely cover the cornea) 
is applied directly to the epithelial surface of the cornea using a micropipette. If a test substance is difficult to pipette, the test substance can be pressure-loaded into a positive displacement pipette to aid in dosing. The 
pipette tip of the positive displacement pipette is inserted into the dispensing 
tip of the syringe so that the material can be loaded into the displacement tip under pressure. Simultaneously, the syringe plunger is depressed as the pipette piston is drawn upwards. If air bubbles appear in the pipette tip, 
the test article is removed (expelled) and the process repeated until the tip is 
filled without air bubbles. If necessary, a normal syringe (without a needle) can be used since it permits measuring an accurate volume of test substance and an easier application to the epithelial surface of the cornea. After dosing, 
the glass window is replaced on the anterior chamber to recreate a closed 
system.
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 676
 
Post-Exposure Incubation  
30. After the exposure period, the test substance, the negative control, or the 
positive control substance is removed from the anterior chamber and the 
epithelium washed at least three times (or until no visual evidence of test substance can be observed) with EMEM (containing phenol red). Phenol red-containing medium is used for rinsing since a colour change in the 
phenol red may be monitored to determine the effectiveness of rinsing 
acidic or alkaline materials. The corneas are washed more than three times if the phenol red is still discoloured (yellow or purple), or the test substance is still visible. Once the medium is free of test substance, the corneas are 
given a final rinse with EMEM (without phenol red). The EMEM (without 
phenol red) is used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. The anterior chamber is then refilled with fresh EMEM without phenol red. 
31. For liquids or surfactants, after rinsing, the corneas are incubated for an 
additional two hours at 32 ± 1 °C. Longer post-exposure time may be 
useful in certain circumstances and could be considered on a case-by-case 
basis. Corneas treated with solids are rinsed thoroughly at the end of the four-hour exposure period, but do not require further incubation. 
32. At the end of the post-exposure incubation period for liquids and surfactants 
and at the end of the four-hour exposure period for non-surfactant solids, the opacity and permeability of each cornea are recorded. Also, each cornea is 
observed visually and pertinent observations recorded (e.g., tissue peeling, 
residual test substance, non-uniform opacity patterns). These observations could be important as they may be reflected by variations in the opaci ­
tometer readings.
 
Control Substances  
33. Concurrent negative or solvent/vehicle controls and positive controls are 
included in each experiment. 
34. When testing a liquid substance at 100 %, a concurrent negative control 
(e.g., 0,9 % sodium chloride solution or distilled water) is included in the 
BCOP test method so that nonspecific changes in the test system can be detected and to provide a baseline for the assay endpoints. It also ensures that the assay conditions do not inappropriately result in an irritant response. 
35. When testing a diluted liquid, surfactant, or solid, a concurrent solvent/ 
vehicle control group is included in the BCOP test method so that 
nonspecific changes in the test system can be detected and to provide a 
baseline for the assay endpoints. Only a solvent/vehicle that has been demonstrated to have no adverse effects on the test system can be used. 
36. A known ocular irritant is included as a concurrent positive control in each 
experiment to verify that an appropriate response is induced. As the BCOP assay is being used in this test method to identify corrosive or severe 
irritants, ideally the positive control should be a reference substance that 
induces a severe response in this test method. However, to ensure that variability in the positive control response across time can be assessed, the magnitude of irritant response should not be excessive. 
37. Examples of positive controls for liquid test substances are dimethyl ­
formamide or 1 % sodium hydroxide. An example of a positive control 
for solid test substances is 20 % (weight to volume) imidazole in 0,9 % 
sodium chloride solution.
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 677
 
38. Benchmark substances are useful for evaluating the ocular irritancy potential 
of unknown chemicals of a specific chemical or product class, or for 
evaluating the relative irritancy potential of an ocular irritant within a specific range of irritant responses.
 
Endpoints Measured  
39. Opacity is determined by the amount of light transmission through the 
cornea. Corneal opacity is measured quantitatively with the aid of an opaci ­
tometer, resulting in opacity values measured on a continuous scale. 
40. Permeability is determined by the amount of sodium fluorescein dye that 
penetrates all corneal cell layers ( i.e., the epithelium on the outer cornea 
surface through the endothelium on the inner cornea surface). 1 mL sodium fluorescein solution (4 or 5 mg/mL when testing liquids and surfactants or non-surfactant solids, respectively) is added to the anterior chamber of the 
corneal holder, which interfaces with the epithelial side of the cornea, while 
the posterior chamber, which interfaces with the endothelial side of the cornea, is filled with fresh EMEM. The holder is then incubated in a hori ­
zontal position for 90 ± 5 min at 32 ± 1 °C. The amount of sodium fluor ­
escein that crosses into the posterior chamber is quantitatively measured with 
the aid of UV/VIS spectrophotometry. Spectrophotometric measurements evaluated at 490 nm are recorded as optical density (OD
 490 ) or absorbance 
values, which are measured on a continuous scale. The fluorescein permea ­
bility values are determined using OD  490 values based upon a visible light 
spectrophotometer using a standard 1 cm path length. 
41. Alternatively, a 96-well microtiter plate reader may be used provided that; (i) 
the linear range of the plate reader for determining fluorescein OD  490 values 
can be established; and (ii), the correct volume of fluorescein samples are used in the 96-well plate to result in OD
 490 values equivalent to the standard 
1 cm path length (this could require a completely full well [usually 
360 mL]).  
DATA AND REPORTING  
Data Evaluation  
42. Once the opacity and mean permeability (OD  490 ) values have been corrected 
for background opacity and the negative control permeability OD  490 values, 
the mean opacity and permeability OD  490 values for each treatment group 
should be combined in an empirically-derived formula to calculate an  in 
vitro  irritancy score (IVIS) for each treatment group as follows: 
IVIS = mean opacity value + (15 × mean permeability OD  490 value) 
Sina et al . (16) reported that this formula was derived during in-house and 
inter-laboratory studies. The data generated for a series of 36 compounds in a multi-laboratory study were subjected to a multivariate analysis to 
determine the equation of best fit between  in vivo  and  in vitro  data. This 
analysis was performed by scientists at two separate companies, who derived nearly identical equations. 
43. The opacity and permeability values should also be evaluated independently 
to determine whether a test substance induced corrosivity or severe irritation 
through only one of the two endpoints (see Decision Criteria).
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 678
 
Decision Criteria  
44. A substance that induces an IVIS ≥ 55,1 is defined as a corrosive or severe 
irritant. As stated in paragraph 1, if the test substance is not identified as an 
ocular corrosive or severe irritant, additional testing should be conducted for classification and labelling purposes. The BCOP test method has an overall accuracy of 79 % (113/143) to 81 % (119/147), a false positive rate of 19 % 
(20/103) to 21 % (22/103), and a false negative rate of 16 % (7/43) to 25 % 
(10/40), when compared to  in vivo  rabbit eye test method data classified 
according to the EPA (1), EU (2), or GHS (3) classification systems. When substances within certain chemical ( i.e., alcohols, ketones) or physical ( i.e., 
solids) classes are excluded from the database, the accuracy of BCOP across 
the EU, EPA, and GHS classification systems ranges from 87 % (72/83) to 92 % (78/85), the false positive rates range from 12 % (7/58) to 16 % (9/56), and the false negative rates range from 0 % (0/27) to 12 % (3/26). 
45. Even if an ocular corrosive or severe irritant classification is not obtained for 
a test substance, BCOP data can be useful, in conjunction with test data from the in vivo  rabbit eye test or from an adequately validated  in vitro  test, to 
further evaluate the usefulness and limitations of the BCOP test method for 
identifying non-severe irritants and non-irritants (a Guidance Document on the use of  in vitro  ocular toxicity test methods is under development).
 
Study Acceptance Criteria  
46. A test is considered acceptable if the positive control gives an IVIS that falls 
within two standard deviations of the current historical mean, which is to be 
updated at least every three months, or each time an acceptable test is conducted in laboratories where tests are conducted infrequently ( i.e., less 
than once a month). The negative or solvent/vehicle control responses should 
result in opacity and permeability values that are less than the established 
upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control.
 
Test Report  
47. The test report should include the following information, if relevant to the 
conduct of the study:  
Test and Control Substances  
Chemical name(s) such as the structural name used by the Chemical 
Abstracts Service (CAS), followed by other names, if known; 
The CAS Registry Number (RN), if known; Purity and composition of the substance or mixture (in percentage(s) by 
weight), to the extent this information is available; 
Physicochemical properties such as physical state, volatility, pH, stability, 
chemical class, water solubility relevant to the conduct of the study; 
Treatment of the test/control substances prior to testing, if applicable ( e.g., 
warming, grinding); 
Stability, if known.
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 679
 
Information Concerning the Sponsor and the Test Facility  
Name and address of the sponsor, test facility and study director; 
Identification of the source of the eyes ( i.e., the facility from which they 
were collected); 
Storage and transport conditions of eyes ( e.g., date and time of eye 
collection, time interval prior to initiating testing, transport media and temperature conditions, any antibiotics used); 
If available, specific characteristics of the animals from which the eyes were 
collected ( e.g., age, sex, weight of the donor animal).
 
Justification of the Test Method and Protocol Used  
Test Method Integrity  
The procedure used to ensure the integrity ( i.e., accuracy and reliability) of 
the test method over time ( e.g., periodic testing of proficiency substances, 
use of historical negative and positive control data).  
Criteria for an Acceptable Test  
Acceptable concurrent positive and negative control ranges based on 
historical data; 
If applicable, acceptable concurrent benchmark control ranges based on 
historical data.  
Test Conditions  
Description of test system used; 
Type of corneal holder used; 
Calibration information for devices used for measuring opacity and permea ­
bility ( e.g., opacitometer and spectrophotometer); 
Information on the bovine corneas used, including statements regarding their 
quality; 
Details of test procedure used; 
Test substance concentration(s) used; 
Description of any modifications of the test procedure; 
Reference to historical data of the model ( e.g., negative and positive 
controls, proficiency substances, benchmark substances); 
Description of evaluation criteria used.  
Results  
Tabulation of data from individual test samples ( e.g., opacity and OD  490 
values and calculated IVIS for the test substance and the positive, 
negative, and benchmark controls [if included], reported in tabular form, including data from replicate repeat experiments as appropriate, and means ± the standard deviation for each experiment); 
Description of other effects observed.
 
Discussion of the Results  
Conclusion  
LITERATURE 
(1) U.S. EPA (1996). Label Review Manual: 2nd Edition. EPA737-B-96- 
001. Washington, DC: U.S. Environmental Protection Agency.  ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 680
 
(2) Regulation (EC) No 1272/2008 of the European Parliament and of the 
Council of 16 December 2008 on classification, labelling and packaging 
of substances and mixtures, amending and repealing Directive 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006. OJ L 353, 31.12.2008, p. 1. 
(3) UN (2007). Globally Harmonized System of Classification and Labelling 
of Chemicals (GHS). Second revised edition, New York & Geneva: United Nations Publications, 2007. Available: 
[http://www.unece.org/trans/danger/publi/ghs/ghs_rev02/02files_e.html] 
(4) ESAC (2007). Statement on the conclusion of the ICCVAM retro ­
spective study on organotypic  in vitro  assays as screening tests to 
identify potential ocular corrosives and severe eye irritants. Available: 
[http://ecvam.jrc.it/index.htm] 
(5) ICCVAM (2007). Test Method Evaluation Report -  In Vitro  Ocular 
Toxicity Test Methods for Identifying Ocular Severe Irritants and 
Corrosives. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative 
Toxicological Methods (NICEATM). NIH Publication No: 07-4517. 
Available: 
[http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ocu_tmer.htm] 
(6) Regulation (EC) No 1907/2006 of the European Parliament and of the 
Council of 18 December 2006 concerning the Registration, Evaluation, 
Authorisation and Restriction of Chemicals (REACH), establishing a 
European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission 
Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC 
and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC. OJ L 396, 30.12.2006, p. 1. 
(7) OECD (2002). Test Guideline 405. OECD Guideline for Testing of 
Chemicals. Acute eye irritation/corrosion. Available: 
[http://www.oecd.org/document/40/ 
0,2340,en_2649_34377_37051368_1_1_1_1,00.htm l] 
(8) ICCVAM (2007). ICCVAM Recommended BCOP Test Method 
Protocol. In: ICCVAM Test Method Evaluation Report -  In Vitro  
Ocular Toxicity Test Methods for Identifying Ocular Severe Irritants and Corrosives. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Toxicology 
Program (NTP) Interagency Center for the Evaluation of Alternative 
Toxicological Methods (NICEATM). NIH Publication No: 07-4517. Available: 
[http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ocu_tmer.htm] 
(9) ICCVAM. (2006). Current Status of  In Vitro  Test Methods for Iden ­
tifying Ocular Corrosives and Severe Irritants: Bovine Corneal Opacity 
and Permeability Test Method. NIH Publication No: 06-4512. Research Triangle Park: National Toxicology Program. Available: 
[http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ocu_brd_ice.htm] 
(10) INVITTOX (1999). Protocol 124: Bovine Corneal Opacity and Permea ­
bility Assay – SOP of Microbiological Associates Ltd. Ispra, Italy: 
European Centre for the Validation of Alternative Methods (ECVAM).
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 681
 
(11) Gautheron, P., Dukic, M., Alix, D. and Sina, J.F. (1992). Bovine corneal 
opacity and permeability test: An  in vitro  assay of ocular irritancy.  
Fundam. Appl. Toxicol.  18:442-449. 
(12) Siegel, J.D., Rhinehart, E., Jackson, M., Chiarello, L., and the Healthcare 
Infection Control Practices Advisory Committee (2007). Guideline for Isolation Precautions: Preventing Transmission of Infectious Agents in 
Healthcare Settings. Available: 
[http://www.cdc.gov/ncidod/dhqp/pdf]. 
(13) Maurer, J.K., Parker, R.D. and Jester, J.V. (2002). Extent of corneal 
injury as the mechanistic basis for ocular irritation: key findings and recommendations for the development of alternative assays.  Reg. Tox. 
Pharmacol.  36:106-117. 
(14) Doughty, M.J., Petrou, S. and Macmillan, H. (1995). Anatomy and 
morphology of the cornea of bovine eyes from a slaughterhouse.  Can. 
J. Zool.  73:2159-2165. 
(15) Collee, J. and Bradley, R. (1997). BSE: A decade on - Part I. The Lancet 
349: 636-641. 
(16) Sina, J.F., Galer, D.M., Sussman, R.S., Gautheron, P.D., Sargent, E.V., 
Leong, B., Shah, P.V., Curren, R.D., and Miller, K. (1995). A collab ­
orative evaluation of seven alternatives to the Draize eye irritation test using pharmaceutical intermediates.  Fundam Appl Toxicol  26:20-31. 
(17) ICCVAM (2006). Background review document, Current Status of In 
Vitro Test Methods for Identifying Ocular Corrosives and Severe Irri ­
tants: Bovine Corneal Opacity and Permeability (BCOP) Test Method. Available: 
[http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ocu_brd_bcop.htm] 
(18) ICCVAM (2006). Background review document, Current Status of In 
Vitro Test Methods for Identifying Ocular Corrosives and Severe Irri ­
tants: Isolated Chicken Eye (ICE) Test Method. Available: 
[http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ocu_brd_bcop.htm]
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 682
 
Appendix 1  
DEFINITIONS  
Accuracy:  The closeness of agreement between test method results and accepted 
reference values. It is a measure of test method performance and one aspect of 
‘relevance’. The term is often used interchangeably with ‘concordance’, to mean the proportion of correct outcomes of a test method.  
Benchmark substance:  A substance used as a standard for comparison to a test 
substance. A benchmark substance should have the following properties; (i) a 
consistent and reliable source(s); (ii) structural and functional similarity to the class of substances being tested; (iii) known physical/chemical characteristics; (iv) supporting data on known effects, and (v) known potency in the range of 
the desired response.  
Cornea:  The transparent part of the front of the eyeball that covers the iris and 
pupil and admits light to the interior.  
Corneal opacity:  Measurement of the extent of opaqueness of the cornea 
following exposure to a test substance. Increased corneal opacity is indicative 
of damage to the cornea. Opacity can be evaluated subjectively as done in the Draize rabbit eye test, or objectively with an instrument such as an ‘opacito ­
meter’.  
Corneal permeability:  Quantitative measurement of damage to the corneal 
epithelium by a determination of the amount of sodium fluorescein dye that passes through all corneal cell layers.  
EPA Category 1:  Corrosive (irreversible destruction of ocular tissue) or corneal 
involvement or irritation persisting for more than 21 days (1).  
EU Category R41:  Production of tissue damage in the eye, or serious physical 
decay of vision, following application of a test substance to the anterior surface 
of the eye, which is not fully reversible within 21 days of application (2).  
False negative rate:  The proportion of all positive substances falsely identified 
by a test method as negative. It is one indicator of test method performance.  
False positive rate:  The proportion of all negative substances that are falsely 
identified by a test method as positive. It is one indicator of test method 
performance.  
GHS (Globally Harmonized System of Classification and Labelling of 
Chemicals):  A system proposing the classification of chemicals (substances 
and mixtures) according to standardized types and levels of physical, health 
and environmental hazards, and addressing corresponding communication elements, such as pictograms, signal words, hazard statements, precautionary statements and safety data sheets, so that to convey information on their 
adverse effects with a view to protect people (including employers, workers, 
transporters, consumers and emergency responders) and the environment (3).
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 683
 
GHS Category 1:  Production of tissue damage in the eye, or serious physical 
decay of vision, following application of a test substance to the anterior surface 
of the eye, which is not fully reversible within 21 days of application (3).  
Hazard:  Inherent property of an agent or situation having the potential to cause 
adverse effects when an organism, system or (sub) population is exposed to that agent.  
In Vitro  Irritancy Score (IVIS):  An empirically-derived formula used in the 
BCOP assay whereby the mean opacity and mean permeability values for each 
treatment group are combined into a single  in vitro  score for each treatment 
group. The  IVIS  = mean opacity value + (15 × mean permeability value).  
Negative control:  An untreated replicate containing all components of a test 
system. This sample is processed with test substance-treated samples and other 
control samples to determine whether the solvent interacts with the test system.  
Non-irritant:  Substances that are not classified as EPA Category I, II, or III; EU 
Category R41 or R36; or GHS Category 1, 2A, or 2B ocular irritants.  
Ocular corrosive:  (a) A substance that causes irreversible tissue damage to the 
eye; (b) Substances that are classified as GHS Category 1, EPA Category I, or 
EU Category R41 ocular irritants (1) (2) (3).  
Ocular irritant:  (a) A substance that produces a reversible change in the eye 
following application to the anterior surface of the eye; (b) Substances that are classified as EPA Category II or III, EU Category R36, or GHS Category 2A or 
2B ocular irritants (1) (2) (3).  
Ocular severe irritant:  (a) A substance that causes tissue damage in the eye 
following application to the anterior surface of the eye that does not resolve within 21 days of application or causes serious physical decay of vision; (b) Substances that are classified as GHS Category 1, EPA Category I, or EU 
Category R41 ocular irritants (1) (2) (3).  
Opacitometer:  An instrument used to measure ‘corneal opacity’ by quanti ­
tatively evaluating light transmission through the cornea. The typical instrument has two compartments, each with its own light source and photocell. One 
compartment is used for the treated cornea, while the other is used to calibrate 
and zero the instrument. Light from a halogen lamp is sent through a control compartment (empty chamber without windows or liquid) to a photocell and compared to the light sent through the experimental compartment, which 
houses the chamber containing the cornea, to a photocell. The difference in 
light transmission from the photocells is compared and a numeric opacity value is presented on a digital display.  
Positive control:  A replicate containing all components of a test system and 
treated with a substance known to induce a positive response. To ensure that 
variability in the positive control response across time can be assessed, the 
magnitude of the severe response should not be excessive.
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 684
 
Reliability:  Measures of the extent that a test method can be performed repro ­
ducibly within and between laboratories over time, when performed using the 
same protocol. It is assessed by calculating intra- and inter-laboratory reproduci ­
bility and intra-laboratory repeatability.  
Solvent/vehicle control:  An untreated sample containing all components of a test 
system, including the solvent or vehicle that is processed with the test 
substance-treated and other control samples to establish the baseline response 
for the samples treated with the test substance dissolved in the same solvent or vehicle. When tested with a concurrent negative control, this sample also demonstrates whether the solvent or vehicle interacts with the test system.  
Tiered testing:  A stepwise testing strategy where all existing information on a 
test substance is reviewed, in a specified order, using a weight of evidence 
process at each tier to determine if sufficient information is available for a hazard classification decision, prior to progression to the next tier. If the irritancy potential of a test substance can be assigned based on the existing 
information, no additional testing is required. If the irritancy potential of a test 
substance cannot be assigned based on the existing information, a step-wise sequential animal testing procedure is performed until an unequivocal classifi ­
cation can be made.  
Validated test method:  A test method for which validation studies have been 
completed to determine the relevance (including accuracy) and reliability for a specific purpose. It is important to note that a validated test method may not have sufficient performance in terms of accuracy and reliability to be found acceptable 
for the proposed purpose.  
Weight-of-evidence:  The process of considering the strengths and weaknesses of 
various pieces of information in reaching and supporting a conclusion concerning the hazard potential of a substance.
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 685
 
Appendix 2  
Proficiency substances for the BCOP test method  
Prior to routine use of a test method that adheres to this test method, laboratories 
may wish to demonstrate technical proficiency by correctly identifying the ocular 
corrosivity classification of the 10 substances recommended in Table 1. These substances were selected to represent the range of responses for local eye irri ­
tation/corrosion, which is based on results in the  in vivo  rabbit eye test (TG 405) 
(i.e., Categories 1, 2A, 2B, or Not Classified and Labelled according to the UN 
GHS (3) (7). However, considering the validated usefulness of these assays ( i.e., 
to identify ocular corrosives/severe irritants only), there are only two test outcomes for classification purposes (corrosive/severe irritant or 
non-corrosive/non-severe irritant) to demonstrate proficiency. Other selection 
criteria were that substances are commercially available, there are high quality  
in vivo  reference data available, and there are high quality data from the two  in 
vitro  methods for which Test Guidelines are being developed. For this reason, 
irritant substances were selected from the ICCVAM recommended list of 122 
reference substances for the validation of  in vitro  ocular toxicity test methods 
(see Appendix H: ICCVAM Recommended Reference Substances) (5). Reference 
data are available in the ICCVAM Background Review Documents for BCOP 
and Isolated Chicken Eye (ICE) test method (17) (18).
 
Table 1  
Recommended substances for demonstrating technical proficiency with BCOP  
Substance CASRN Chemical Class (  1 ) Physical 
Form  In Vivo  Clas­
sification (  2 ) In Vitro  Classification (  3 ) 
Benzalkonium 
chloride (5 %) 8001-54-5 Onium 
compound Liquid Category 1 Corrosive/Severe Irritant 
Chlorhexidine 55-56-1 Amine, Amidine Solid Category 1 Corrosive/Severe Irritant 
Dibenzoyl-L- 
tartaric acid 2743-38-6 Carboxylic acid, 
Ester Solid Category 1 Corrosive/Severe Irritant 
Imidazole 288-32-4 Heterocyclic Solid Category 1 Corrosive/Severe Irritant 
Trichloroacetic 
acid (30 %) 76-03-9 Carboxylic Acid Liquid Category 1 Corrosive/Severe Irritant 
2,6-Dichlorobenz- 
oyl chloride 4659-45-4 Acyl halide Liquid Category 2A Non corrosive/Non severe 
irritant 
Ethyl-2-methyl ­
aceto-acetate 609-14-3 Ketone, Ester Liquid Category 2B Non corrosive/Non severe 
irritant 
Ammonium nitrate 6484-52-2 Inorganic salt Solid Category 2A Non corrosive/Non severe 
irritant  ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 686
 
Substance CASRN Chemical Class (  1 ) Physical 
Form  In Vivo  Clas­
sification (  2 ) In Vitro  Classification (  3 ) 
Glycerol 56-81-5 Alcohol Liquid Not 
Labelled Non corrosive/Non severe 
irritant 
n-Hexane 110-54-3 Hydrocarbon 
(acyclic) Liquid Not Labeled Noncorrosive/Non severe 
irritant  
Abbreviations: CASRN = Chemical Abstracts Service Registry Number 
( 1 ) Chemical classes were assigned to each test substance using a standard classification scheme, based on the National Library o f 
Medicine Medical Subject Headings (MeSH) classification system (available at http://www.nlm.nih.gov/mesh). 
( 2 ) Based on results from the  in vivo  rabbit eye test (OECD TG 405) and using the UN GHS (3)(7). 
( 3 ) Based on results in BCOP and ICE.  ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 687
 
Appendix 3  
THE BCOP CORNEAL HOLDER 
1. The BCOP corneal holders are made of an inert material ( e.g., polypropy ­
lene). The holders are comprised of two halves (an anterior and posterior 
chamber), and have two similar cylindrical internal chambers. Each chamber holds a volume of 5 mL and terminates in a glass window, through which opacity measurements are recorded. Each of the inner chambers is 1,7 cm in 
diameter and 2,2 cm in depth (
 1 ). An o-ring located on the posterior chamber 
is used to prevent leaks. The corneas are placed endothelial side down on the o-ring of the posterior chambers and the anterior chambers are placed on the epithelial side of the corneas. The chambers are maintained in place by three 
stainless screws located on the outer edges of the chamber. The end of each 
chamber houses a glass window which can be removed for easy access to the cornea. An o-ring is also located between the glass window and the chamber to prevent leaks. Two holes on the top of each chamber permit 
introduction and removal of medium and test compounds. They are closed 
with rubber caps during the treatment and incubation periods.
 
Glossary 
Glass disc: 
PTFE-O-ring: Refill: Hanger: Cap: Nut: O-ring: Posterior compartment: Anterior compartment: Fixing screws:
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 688  
( 1 ) The dimensions provided are based on a corneal holder that is used for cows ranging in 
age from 12 to 60 months old. In the event that animals 6 to 12 months are being used, 
the holder would instead need to be designed such that each chamber holds a volume of 
4 mL, and each of the inner chambers is 1,5 cm in diameter and 2,2 cm in depth. With 
any newly designed corneal holder, it is very important that the ratio of exposed corneal 
surface area to posterior chamber volume should be the same as the ratio in the tradi ­
tional corneal holder. This is necessary to assure that permeability values are correctly 
determined for the calculation of the IVIS by the proposed formula.
 THE OPACITOMETER 
2. The opacitometer is a light transmission measuring device. Light from a 
halogen lamp is sent through a control compartment (empty chamber 
without windows or liquid) to a photocell and compared to the light sent through the experimental compartment, which houses the chamber containing the cornea, to a photocell. The difference in light transmission 
from the photocells is compared and a numeric opacity value is presented on 
a digital display. The opacity units are established. 
3. The opacitometer should provide a linear response through a range of 
opacity readings covering the cut-offs used for the different classifications described by the Prediction Model ( i.e., up to the cut-off determining 
corrosiveness/severe irritancy). To ensure linear and accurate readings up 
to 75-80 opacity units, it is necessary to calibrate the opacitometer using a series of calibrators. Calibrators (opaque sheets of polyester) are placed into the calibration chamber (a corneal chamber designed to hold the calibrators) 
and read on the opacitometer. The calibration chamber is designed to hold 
the calibrators at approximately the same distance between the light and photocell that the corneas would be placed during the opacity measurements. 
The opacitometer is first calibrated to 0 opacity units using the calibration 
chamber without a calibrator. Three different calibrators are then placed into the calibration chamber one by one and the opacities are measured. Cali ­
brators 1, 2 and 3 should result in opacity readings equal to their set values 
of 75, 150, and 225 opacity units, respectively, ± 5 %.
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 689
B. 48.  ISOLATED CHICKEN EYE TEST METHOD FOR IDENTIFYING 
OCULAR CORROSIVES AND SEVERE IRRITANTS  
INTRODUCTION 
1. The Isolated Chicken Eye (ICE) test method is an  in vitro  test method that can 
be used, under certain circumstances and with specific limitations, to classify substances and mixtures as ocular corrosives and severe irritants, (1) (2) (3). For the purpose of this test method, severe irritants are defined as those that 
induce ocular lesions that persist in the rabbit for at least 21 days after adminis ­
tration. While it is not considered valid as a complete replacement for the  in vivo  
rabbit eye test, the ICE is recommended for use as part of a tiered testing strategy for regulatory classification and labelling within a specific applicability 
domain (4) (5). Test substances and mixtures (6) that are positive in this assay 
can be classified as ocular corrosives or severe irritants without further testing in rabbits. A substance that tests negative would need to be tested in rabbits using a sequential testing strategy, as outlined in OECD Test Guideline 405 (7) 
(chapter B. 5 of this Annex). 
2. The purpose of this test method is to describe the procedures used to 
evaluate the potential ocular corrosivity or severe irritancy of a test substance as measured by its ability to induce toxicity in an enucleated 
chicken eye. Toxic effects to the cornea are measured by (i) a qualitative 
assessment of opacity, (ii) a qualitative assessment of damage to epithelium based on application of fluorescein to the eye (fluorescein retention), (iii) a quantitative measurement of increased thickness (swelling), and (iv) a quali ­
tative evaluation of macroscopic morphological damage to the surface. The 
corneal opacity, swelling, and damage assessments following exposure to a test substance are assessed individually and then combined to derive an Eye Irritancy Classification. 
3. Ocular irritants that induce lesions that resolve in less than 21 days and 
non-irritants have also been tested using the ICE test method. However, the accuracy and reliability of the ICE test method for substances in these categories have not been formally evaluated. 
4. Definitions are provided in Appendix 1.
 
INITIAL CONSIDERATIONS AND LIMITATIONS 
5. This test method is based on the Interagency Coordinating Committee on the 
Validation of Alternative Methods (ICCVAM) ICE test method protocol (8), which was developed following an international validation study (4) (5) (9), 
with contributions from the European Centre for the Validation of Alter ­
native Methods, the Japanese Center for the Validation of Alternative Methods, and TNO Quality of Life Department of Toxicology and Applied Pharmacology (Netherlands). The protocol is based on information 
obtained from published protocols, as well as the current protocol used by 
TNO (10) (11) (12) (13) (14).
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 690
 
6. The identified limitations for this method are based upon the false positive 
rate for alcohols and the false negative rates for solids and surfactants (see 
paragraph 47) (4). When substances within these chemical and physical classes are excluded from the database, the accuracy of ICE across the EU, EPA, and GHS classification systems is substantially improved (4). 
Based on the purpose of this assay ( i.e., to identify ocular corrosives/severe 
irritants only), false negative rates are not critical since such substances would be subsequently tested in rabbits or with other adequately validated  
in vitro  tests, depending on regulatory requirements, using a sequential 
testing strategy in a weight of evidence approach. Furthermore, the current 
validation database did not allow for an adequate evaluation of some chemical or product classes ( e.g., mixtures). However, investigators could 
consider using this test method for testing all types of material (including 
mixtures), whereby a positive result could be accepted as indicative of an 
ocular corrosive or severe irritant response. However, positive results obtained with alcohols should be interpreted cautiously due to risk of over-prediction. 
7. All procedures with chicken eyes should follow the test facility’s applicable 
regulations and procedures for handling of human or animal-derived materials, which include, but are not limited to, tissues and tissue fluids. 
Universal laboratory precautions are recommended (15). 
8. A limitation of the test method is that, although it takes into account some of 
the ocular effects evaluated in the rabbit ocular irritancy test method and to 
some degree their severity, it does not consider conjunctival and iridal injuries. Also, although the reversibility of corneal lesions cannot be evaluated  per se  in the ICE test method, it has been proposed, based on 
rabbit eye studies, that an assessment of the initial depth of corneal injury 
can be used to distinguish between irreversible and reversible effects (16). Finally, the ICE test method does not allow for an assessment of the potential for systemic toxicity associated with ocular exposure. 
9. Efforts are ongoing to further characterize the usefulness and limitations of 
the ICE test method for identifying non-severe irritants and non-irritants (see also paragraph 48). Users are also encouraged to provide specimens and/or 
data to validation organizations for a formal evaluation of possible future 
uses of the ICE test method, including for the identification of non-severe ocular irritants and non-irritants. 
10. For any laboratory initially establishing this assay, the proficiency chemicals 
provided in Appendix 2 should be used. A laboratory can use these chemicals to demonstrate their technical competence in performing the ICE 
test method prior to submitting ICE data for regulatory hazard classification 
purposes.
 
PRINCIPLE OF THE TEST 
11. The ICE test method is an organotypic model that provides short-term main ­
tenance of the chicken eye  in vitro . In this test method, damage by the test 
substance is assessed by determination of corneal swelling, opacity, and fluorescein retention. While the latter two parameters involve a qualitative assessment, analysis of corneal swelling provides for a quantitative 
assessment. Each measurement is either converted into a quantitative score 
used to calculate an overall Irritation Index, or assigned a qualitative catego ­
rization that is used to assign an  in vitro  ocular corrosivity and severe 
irritancy classification. Either of these outcomes can then be used to 
predict the  in vivo  ocular corrosivity and severe irritation potential of a 
test substance (see Decision Criteria).
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 691
 
Source and Age of Chicken Eyes  
12. Historically, eyes collected from chickens obtained from a slaughterhouse 
where they are killed for human consumption have been used for this assay, 
eliminating the need for laboratory animals. Only the eyes of healthy animals considered suitable for entry into the human food chain are used. 
13. Although a controlled study to evaluate the optimum chicken age has not 
been conducted, the age and weight of the chickens used historically in this test method are that of spring chickens traditionally processed by a poultry 
slaughterhouse ( i.e., approximately 7 weeks old, 1,5-2,5 kg).
 
Collection and Transport of Eyes to the Laboratory  
14. Heads should be removed immediately after sedation of the chickens, usually 
by electric shock, and incision of the neck for bleeding. A local source of chickens close to the laboratory should be located so that their heads can be transferred from the slaughterhouse to the laboratory quickly enough to 
minimize deterioration and/or bacterial contamination. The time interval 
between collection of the chicken heads and use of eyes in the ICE test method should be minimized (typically within two hours) and should be demonstrated to not compromise the assay results. These results are based 
on the selection criteria for the eyes, as well as the positive and negative 
control responses. All eyes used in the assay should be from the same group of eyes collected on a specific day. 
15. Because eyes are dissected in the laboratory, the intact heads are transported 
from the slaughterhouse at ambient temperature in plastic boxes humidified 
with towels moistened with isotonic saline.
 
Selection Criteria for Eyes Used in the ICE  
16. Eyes that have high baseline fluorescein staining ( i.e., > 0,5) or corneal 
opacity score ( i.e., > 0,5) after they are enucleated are rejected. 
17. Each treatment group and concurrent positive control consists of at least 
three eyes. The negative control group or the solvent control (if using a solvent other than saline) consists of at least one eye.
 
PROCEDURE  
Preparation of the Eyes  
18. The eyelids are carefully excised, taking care not to damage the cornea. 
Corneal integrity is quickly assessed with a drop of 2 % (w/v) sodium 
fluorescein applied to the corneal surface for a few seconds, and then 
rinsed with isotonic saline. Fluorescein-treated eyes are then examined with a slit-lamp microscope to ensure that the cornea is undamaged ( i.e., 
fluorescein retention and corneal opacity scores ≤ 0,5). 
19. If undamaged, the eye is further dissected from the skull, taking care not to 
damage the cornea. The eyeball is pulled from the orbit by holding the 
nictitating membrane firmly with surgical forceps, and the eye muscles are cut with a bent, blunt-tipped scissor. It is important to avoid causing corneal damage due to excessive pressure ( i.e., compression artifacts). 
20. When the eye is removed from the orbit, a visible portion of the optic nerve 
should be left attached. Once removed from the orbit, the eye is placed on an 
absorbent pad and the nictitating membrane and other connective tissue are 
cut away.
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 692
 
21. The enucleated eye is mounted in a stainless steel clamp with the cornea 
positioned vertically. The clamp is then transferred to a chamber of the 
superfusion apparatus (16). The clamps should be positioned in the super ­
fusion apparatus such that the entire cornea is supplied with the isotonic saline drip. The chambers of the superfusion apparatus should be temperature 
controlled at 32 ± 1,5 °C. Appendix 3 provides a diagram of a typical 
superfusion apparatus and the eye clamps, which can be obtained commer ­
cially or constructed. The apparatus can be modified to meet the needs of an individual laboratory ( e.g., to accommodate a different number of eyes). 
22. After being placed in the superfusion apparatus, the eyes are again examined 
with a slit-lamp microscope to ensure that they have not been damaged 
during the dissection procedure. Corneal thickness should also be 
measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0,5; (ii) corneal opacity > 0,5; or, (iii), any additional signs of 
damage should be replaced. For eyes that are not rejected based on any of 
these criteria, individual eyes with a corneal thickness deviating more than 10 % from the mean value for all eyes are to be rejected. Users should be aware that slit-lamp microscopes could yield different corneal thickness 
measurements if the slit-width setting is different. The slit-width should be 
set at 0,095 mm. 
23. Once all eyes have been examined and approved, the eyes are incubated for 
approximately 45 to 60 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement is recorded for corneal thickness and opacity to serve as a baseline ( i.e., time = 
0). The fluorescein score determined at dissection is used as the baseline 
measurement for that endpoint.
 
Application of the Test Substance  
24. Immediately following the zero reference measurements, the eye (in its 
holder) is removed from the superfusion apparatus, placed in a horizontal position, and the test substance is applied to the cornea. 
25. Liquid test substances are typically tested undiluted, but may be diluted if 
deemed necessary ( e.g., as part of the study design). The preferred solvent 
for diluted substances is physiological saline. However, alternative solvents 
may also be used under controlled conditions, but the appropriateness of 
solvents other than physiological saline should be demonstrated. 
26. Liquid test substances are applied to the cornea such that the entire surface 
of the cornea is evenly covered with the test substance; the standard volume 
is 0,03 mL. 
27. If possible, solid substances should be ground as finely as possible in a 
mortar and pestle, or comparable grinding tool. The powder is applied to the cornea such that the surface is uniformly covered with the test substance; the standard amount is 0,03 g. 
28. The test substance (liquid or solid) is applied for 10 seconds and then rinsed 
from the eye with isotonic saline (approximately 20 mL) at ambient 
temperature. The eye (in its holder) is subsequently returned to the super ­
fusion apparatus in the original upright position.
 
Control Substances  
29. Concurrent negative or solvent/vehicle controls and positive controls should 
be included in each experiment.  ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 693
 
30. When testing liquids at 100 % or solids, physiological saline is used as the 
concurrent negative control in the ICE test method to detect non-specific 
changes in the test system, and to ensure that the assay conditions do not inappropriately result in an irritant response. 
31. When testing diluted liquids, a concurrent solvent/vehicle control group is 
included in the test method to detect non-specific changes in the test system, and to ensure that the assay conditions do not inappropriately result in an 
irritant response. As stated in paragraph 25, only a solvent/vehicle that has 
been demonstrated to have no adverse effects on the test system can be used. 
32. A known ocular irritant is included as a concurrent positive control in each 
experiment to verify that an appropriate response is induced. As the ICE assay is being used in this test method to identify corrosive or severe irri ­
tants, the positive control should be a reference substance that induces a 
severe response in this test method. However, to ensure that variability in the positive control response across time can be assessed, the magnitude of the severe response should not be excessive. Sufficient  in vitro  data for the 
positive control should be generated such that a statistically defined 
acceptable range for the positive control can be calculated. If adequate historical ICE test method data are not available for a particular positive control, studies may need to be conducted to provide this information. 
33. Examples of positive controls for liquid test substances are 10 % acetic acid 
or 5 % benzalkonium chloride, while examples of positive controls for solid 
test substances are sodium hydroxide or imidazole. 
34. Benchmark substances are useful for evaluating the ocular irritancy potential 
of unknown chemicals of a specific chemical or product class, or for evaluating the relative irritancy potential of an ocular irritant within a specific range of irritant responses.
 
Endpoints Measured  
35. Treated corneas are evaluated pretreatment and starting at 30, 75, 120, 180, 
and 240 minutes (± 5 minutes) after the post-treatment rinse. These time 
points provide an adequate number of measurements over the four-hour treatment period, while leaving sufficient time between measurements for 
the requisite observations to be made for all eyes. 
36. The endpoints evaluated are corneal opacity, swelling, fluorescein retention, 
and morphological effects ( e.g., pitting or loosening of the epithelium). All 
of the endpoints, with the exception of fluorescein retention (which is determined only at pretreatment and 30 minutes after test substance exposure) are determined at each of the above time points. 
37. Photographs are advisable to document corneal opacity, fluorescein retention, 
morphological effects and, if conducted, histopathology 
38. After the final examination at four hours, users are encouraged to preserve 
eyes in an appropriate fixative ( e.g., neutral buffered formalin) for possible 
histopathological examination.
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39. Corneal swelling is determined from corneal thickness measurements made 
with an optical pachymeter on a slit-lamp microscope. It is expressed as a 
percentage and is calculated from corneal thickness measurements according to the following formula:  
"
 
corneal thickness at time t  Ä corneal thickness at time  ¼ 0 
corneal thickness at time  ¼ 0 # 
Ü 100 
40. The mean percentage of corneal swelling for all test eyes is calculated for all 
observation time points. Based on the highest mean score for corneal 
swelling, as observed at any time point, an overall category score is then 
given for each test substance. 
41. Corneal opacity is calculated by using the area of the cornea that is most 
densely opacified for scoring. The mean corneal opacity value for all test 
eyes is calculated for all observation time points. Based on the highest mean 
score for corneal opacity, as observed at any time point, an overall category score is then given for each test substance (Table 1).
 
Table 1  
Corneal opacity scores  
Score Observation  
0 No opacity 
0,5 Very faint opacity 1 Scattered or diffuse areas; details of the iris are clearly visible 
2 Easily discernible translucent area; details of the iris are slightly obscured 
3 Severe corneal opacity; no specific details of the iris are visible; size of the pupil 
is barely discernible 
4 Complete corneal opacity; iris invisible 
42. The mean fluorescein retention value for all test eyes is calculated for the 30- 
minute observation time point only, which is used for the overall category 
score given for each test substance (Table 2).
 
Table 2  
Fluorescein retention scores  
Score Observation  
0 No fluorescein retention 
0,5 Very minor single cell staining 
1 Single cell staining scattered throughout the treated area of the cornea 
2 Focal or confluent dense single cell staining 3 Confluent large areas of the cornea retaining fluorescein
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 695
 
43. Morphological effects include ‘pitting’ of corneal epithelial cells, ‘loosening’ 
of epithelium, ‘roughening’ of the corneal surface and ‘sticking’ of the test 
substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.
 
DATA AND REPORTING  
Data Evaluation  
44. Results from corneal opacity, swelling, and fluorescein retention should be 
evaluated separately to generate an ICE class for each endpoint. The ICE 
classes for each endpoint are then combined to generate an Irritancy Clas ­
sification for each test substance.  
Decision Criteria  
45. Once each endpoint has been evaluated, ICE classes can be assigned based 
on a predetermined range. Interpretation of corneal thickness (Table 3), 
opacity (Table 4), and fluorescein retention (Table 5) using four ICE 
classes is done according to the following scales:  
Table 3  
ICE classification criteria for corneal thickness  
Mean Corneal Swelling (%) (*) ICE Class  
0 to 5 I 
> 5 to 12 II 
> 12 to 18 (> 75 min after treatment) II 
> 12 to 18 ( ≤ 75 min after treatment) III 
> 18 to 26 III > 26 to 32 (> 75 min after treatment) III > 26 to 32 ( ≤ 75 min after treatment) IV 
> 32 IV
 
(*) Corneal swelling scores only applicable if thickness is measured with a Haag-Streit BP900 slit-lamp 
microscope with depth-measuring device No I and slit-width setting at 9½, equaling 0,095 mm. Users 
should be aware that slit-lamp microscopes could yield different corneal thickness measurements if the 
slit-width setting is different.  
Table 4  
ICE classification criteria for opacity  
Mean Maximum Opacity Score (*) ICE Class  
0,0-0,5 I 
0,6-1,5 II 1,6-2,5 III 2,6-4,0 IV
 
(*) See Table 1.  ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 696
 
Table 5  
ICE classification criteria for mean fluorescein retention  
Mean Fluorescein Retention Score at 30 minutes 
post-treatment (*) ICE Class  
0,0-0,5 I 
0,6-1,5 II 
1,6-2,5 III 
2,6-3,0 IV  
(*) See Table 2.  
46. The overall  in vitro  irritancy classification for a test substance is assessed by 
reading the irritancy classification that corresponds to the combination of categories obtained for corneal swelling, corneal opacity, and fluorescein retention and applying the scheme presented in Table 6.
 
Table 6  
Overall  in vitro  irritancy classifications  
Classification Combinations of the 3 Endpoints  
Corrosive/Severe Irritant 3 × IV 
2 × IV, 1 × III 
2 × IV, 1 × II (*) 2 × IV, 1 × I (*) Corneal opacity ≥ 3 at 30 min (in at least 2 
eyes) 
Corneal opacity = 4 at any time point (in at least 2 eyes) Severe loosening of the epithelium (in at least 
1 eye)
 
(*) Combinations less likely to occur.  
47. As stated in paragraph 1, if the test substance is not identified as an ocular 
corrosive or severe irritant, additional testing should be conducted for 
classification and labelling purposes. The ICE test method has an overall 
accuracy of 83 % (120/144) to 87 % (134/154), a false positive rate of 6 % (7/122) to 8 % (9/116), and a false negative rate of 41 % (13/32) to 50 % (15/30) for the identification of ocular corrosives and severe irritants, when 
compared to  in vivo  rabbit eye test method data classified according to the 
EPA (1), EU (2), or GHS (3) classification systems. When substances within certain chemical ( i.e., alcohols and surfactants) and physical ( i.e., solids) classes 
are excluded from the database, the accuracy of ICE across the EU, EPA, and 
GHS classification systems ranges from 91 % (75/82) to 92 % (69/75), the false 
positive rates range from 5 % (4/73) to 6 % (4/70), and the false negative rates range from 29 % (2/7) to 33 % (3/9) (4). 
48. Even if an ocular corrosive or severe irritant classification is not obtained for 
a test substance, ICE data can be useful in conjunction with test data from the in vivo  rabbit eye test or from an adequately validated  in vitro  test to 
further evaluate the usefulness and limitations of the ICE test method for 
identifying non-severe irritants and non-irritants (a Guidance Document on 
the use of  in vitro  ocular toxicity test methods is under development).
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 697
 
Study Acceptance Criteria  
49. A test is considered acceptable if the concurrent negative or vehicle/solvent 
controls and the concurrent positive controls give an Irritancy Classification 
that falls within nonirritant and severe irritant/corrosive classes, respectively.  
Test Report  
50. The test report should include the following information, if relevant to the 
conduct of the study:  
Test and Control Substances  
Chemical name(s) such as the structural name used by the Chemical 
Abstracts Service (CAS), followed by other names, if known; 
The CAS Registry Number (RN), if known; Purity and composition of the substance or mixture (in percentage(s) by 
weight), to the extent this information is available; 
Physicochemical properties such as physical state, volatility, pH, stability, 
chemical class water solubility relevant to the conduct of the study; 
Treatment of the test/control substances prior to testing, if applicable ( e.g., 
warming, grinding); 
Stability, if known;
 
Information Concerning the Sponsor and the Test Facility  
Name and address of the sponsor, test facility and study director; 
Identification on the source of the eyes ( e.g., the facility from which they 
were collected); 
Storage and transport conditions of eyes ( e.g., date and time of eye 
collection, time interval prior to initiating testing); 
If available, specific characteristics of the animals from which the eyes were 
collected ( e.g., age, sex, weight of the donor animal);  
Justification of the Test Method and Protocol Used  
Test Method Integrity  
The procedure used to ensure the integrity ( i.e., accuracy and reliability) of 
the test method over time ( e.g., periodic testing of proficiency substances, 
use of historical negative and positive control data).  
Criteria for an Acceptable Test  
If applicable, acceptable concurrent benchmark control ranges based on 
historical data;  
Test Conditions  
Description of test system used; 
Slit-lamp microscope used ( e.g., model); 
Instrument settings for the slit-lamp microscope used; 
Information for the chicken eyes used, including statements regarding their 
quality;  ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 698
 
Details of test procedure used; 
Test substance concentration(s) used; Description of any modifications of the test procedure; 
Reference to historical data of the model ( e.g., negative and positive controls, 
proficiency substances, benchmark substances); 
Description of evaluation criteria used;
 
Results  
Description of other effects observed; 
If appropriate, photographs of the eye;  
Discussion of the Results  
Conclusion  
LITERATURE 
(1) U.S. EPA (1996). Label Review Manual: 2nd Edition. EPA737-B-96- 
001. Washington, DC: U.S. Environmental Protection Agency. 
(2) Regulation (EC) No 1272/2008 of the European Parliament and of the 
Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing 
Directive 67/548/EEC and 1999/45/EC, and amending 
Regulation (EC) No 1907/2006. OJ L 353, 31.12.2008, p. 1. 
(3) United nations (UN) (2007). Globally Harmonized System of Classifi ­
cation and Labelling of Chemicals (GHS), Second revised edition, UN 
New York and Geneva, 2007. Available at: 
[http://www.unece.org/trans/danger/publi/ghs/ghs_rev02/ 
02files_e.html] 
(4) ICCVAM (2007). Test Method Evaluation Report -  In Vitro Ocular 
Toxicity  Test Methods for Identifying Ocular Severe Irritants and 
Corrosives. Interagency Coordinating Committee on the Validation 
of Alternative Methods (ICCVAM) and the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). NIH Publication No: 07-4517. 
Available: 
[http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ocu_tmer.htm] 
(5) ESAC (2007). Statement on the conclusion of the ICCVAM retro ­
spective study on organotypic  in vitro  assays as screening tests to 
identify potential ocular corrosives and severe eye irritants. Available: 
[http://ecvam.jrc.it/index.htm]. 
(6) Regulation (EC) No 1907/2006 of the European Parliament and of the 
Council of 18 December 2006 concerning the Registration, Evalu ­
ation, Authorisation and Restriction of Chemicals (REACH), estab ­
lishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and 
Commission Regulation (EC) No 1488/94 as well as Council 
Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC. OJ L 396, 30.12.2006, p. 1. 
(7) OECD (2002). Test Guideline 405. OECD Guideline for Testing of 
Chemicals. Acute eye irritation/corrosion. Available: 
[http://www.oecd.org/document/40/0, 2340, en_2649_34377_37051 
368_1_1_1_1,00.html]
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 699
 
(8) ICCVAM (2007). ICCVAM Recommended ICE Test Method 
Protocol. In: ICCVAM Test Method Evaluation Report -  In Vitro  
Ocular Toxicity Test Methods for Identifying Ocular Severe Irritants and Corrosives. Interagency Coordinating Committee on the Vali ­
dation of Alternative Methods (ICCVAM) and the National Toxi ­
cology Program (NTP) Interagency Center for the Evaluation of Alter ­
native Toxicological Methods (NICEATM). NIH Publication No: 07- 4517. Available: 
[http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ocu_tmer.htm] 
(9) ICCVAM. (2006). Current Status of  In Vitro  Test Methods for Iden ­
tifying Ocular Corrosives and Severe Irritants: Isolated Chicken Eye Test Method. NIH Publication No: 06-4513. Research Triangle Park: 
National Toxicology Program. Available: 
[http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ 
ocu_brd_ice.htm] 
(10) Prinsen, M.K. and Koëter, B.W.M. (1993). Justification of the 
enucleated eye test with eyes of slaughterhouse animals as an alternative to the Draize eye irritation test with rabbits.  Fd. Chem. Toxicol.  31:69- 
76. 
(11) INVITTOX (1994). Protocol 80: Chicken enucleated eye test (CEET). 
Available: 
[http://ecvam.jrc.it/index.htm] 
(12) Balls, M., Botham, P.A., Bruner, L.H. and Spielmann H. (1995). The 
EC/HO international validation study on alternatives to the Draize eye irritation test.  Toxicol. In Vitro  9:871-929. 
(13) Prinsen, M.K. (1996). The chicken enucleated eye test (CEET): A 
practical (pre)screen for the assessment of eye irritation/corrosion 
potential of test materials.  Food Chem. Toxicol . 34:291-296. 
(14) Chamberlain, M., Gad, S.C., Gautheron, P. and Prinsen, M.K. (1997). 
IRAG Working Group I: Organotypic models for the assessment/pre ­
diction of ocular irritation.  Food Chem. Toxicol.  35:23-37. 
(15) Siegel, J.D., Rhinehart, E., Jackson, M., Chiarello, L., and the 
Healthcare Infection Control Practices Advisory Committee (2007). 
Guideline for Isolation Precautions: Preventing Transmission of Infectious Agents in Healthcare Settings. Available: 
[http://www.cdc.gov/ncidod/dhqp/pdf/isolation2007.pdf]. 
(16) Maurer, J.K., Parker, R.D. and Jester J.V. (2002). Extent of corneal 
injury as the mechanistic basis for ocular irritation: key findings and recommendations for the development of alternative assays.  Reg. Tox. 
Pharmacol.  36:106-117. 
(17) Burton, A.B.G., M. York and R.S. Lawrence (1981). The  in vitro  
assessment of severe irritants.  Fd. Cosmet.- Toxicol. - 19, 471-480.
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 700
 
(18) ICCVAM (2006). Background review document, Current Status of  In 
Vitro  Test Methods for Identifying Ocular Corrosives and Severe 
Irritants: Bovine Corneal Opacity and Permeability (BCOP) Test Method. Available: 
[http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ 
ocu_brd_bcop.htm] 
(19) ICCVAM (2006). Background review document, Current Status of  In 
Vitro  Test Methods for Identifying Ocular Corrosives and Severe 
IrritantsIsolated Chicken Eye (ICE) Test Method. Available: 
[http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ocu_brd_bcop.htm]
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 701
 
Appendix 1  
DEFINITIONS  
Accuracy:  The closeness of agreement between test method results and accepted 
reference values. It is a measure of test method performance and one aspect of 
‘relevance’. The term is often used interchangeably with ‘concordance’, to mean the proportion of correct outcomes of a test method.  
Benchmark substance:  A substance used as a standard for comparison to a test 
substance. A benchmark substance should have the following properties; (i), a consistent and reliable source(s); (ii), structural and functional similarity to the 
class of substances being tested; (iii), known physical/chemical characteristics; (iv), 
supporting data on known effects; and (v), known potency in the range of the desired response  
Cornea:  The transparent part of the front of the eyeball that covers the iris and 
pupil and admits light to the interior.  
Corneal opacity:  Measurement of the extent of opaqueness of the cornea 
following exposure to a test substance. Increased corneal opacity is indicative of damage to the cornea.  
Corneal swelling:  An objective measurement in the ICE test of the extent of 
distention of the cornea following exposure to a test substance. It is expressed as 
a percentage and is calculated from baseline (pre-dose) corneal thickness 
measurements and the thickness recorded at regular intervals after exposure to the test material in the ICE test. The degree of corneal swelling is indicative of damage to the cornea.  
EPA Category 1:  Corrosive (irreversible destruction of ocular tissue) or corneal 
involvement or irritation persisting for more than 21 days (1).  
EU Category R41:  Production of tissue damage in the eye, or serious physical 
decay of vision, following application of a test substance to the anterior surface 
of the eye, which is not fully reversible within 21 days of application (2).  
False negative rate:  The proportion of all positive substances falsely identified 
by a test method as negative. It is one indicator of test method performance.  
False positive rate:  The proportion of all negative substances that are falsely 
identified by a test method as positive. It is one indicator of test method 
performance.  
Fluorescein retention:  A subjective measurement in the ICE test of the extent of 
fluorescein sodium that is retained by epithelial cells in the cornea following exposure to a test substance. The degree of fluorescein retention is indicative of damage to the corneal epithelium.  
GHS (Globally Harmonized System of Classification and Labelling of 
Chemicals):  A system proposing the classification of chemicals (substances 
and mixtures) according to standardized types and levels of physical, health 
and environmental hazards, and addressing corresponding communication elements, such as pictograms, signal words, hazard statements, precautionary statements and safety data sheets, so that to convey information on their 
adverse effects with a view to protect people (including employers, workers, 
transporters, consumers and emergency responders) and the environment (3).
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 702
 
GHS Category 1:  Production of tissue damage in the eye, or serious physical 
decay of vision, following application of a test substance to the anterior surface 
of the eye, which is not fully reversible within 21 days of application (3).  
Hazard:  Inherent property of an agent or situation having the potential to cause 
adverse effects when an organism, system or (sub) population is exposed to that 
agent.  
Negative control:  An untreated replicate containing all components of a test 
system. This sample is processed with test substance-treated samples and other 
control samples to determine whether the solvent interacts with the test system.  
Non-irritant:  Substances that are not classified as EPA Category I, II, or III; EU 
Category R41 or R36; or GHS Category 1, 2A, or 2B ocular irritants (1)(2)(3).  
Ocular corrosive:  (a) A substance that causes irreversible tissue damage to the 
eye. (b) Substances that are classified as GHS Category 1, EPA Category I, or EU Category R41 ocular irritants (1)(2)(3).  
Ocular irritant:  (a) A substance that produces a reversible change in the eye 
following application to the anterior surface of the eye; (b) Substances that are 
classified as EPA Category II or III; EU Category R36; or GHS Category 2A, or 2B ocular irritants (1)(2)(3).  
Ocular severe irritant:  (a) A substance that causes tissue damage in the eye 
following application to the anterior surface of the eye that is not reversible within 21 days of application or causes serious physical decay of vision. (b) Substances that are classified as GHS Category 1, EPA Category I, or EU 
Category R41 ocular irritants (1)(2)(3).  
Positive control:  A replicate containing all components of a test system and 
treated with a substance known to induce a positive response. To ensure that 
variability in the positive control response across time can be assessed, the 
magnitude of the severe response should not be excessive.  
Reliability:  Measures of the extent that a test method can be performed repro ­
ducibly within and between laboratories over time, when performed using the 
same protocol. It is assessed by calculating intra- and inter-laboratory reproduci ­
bility and intra-laboratory repeatability.  
Slit-lamp microscope:  An instrument used to directly examine the eye under the 
magnification of a binocular microscope by creating a stereoscopic, erect image. 
In the ICE test method, this instrument is used to view the anterior structures of the chicken eye as well as to objectively measure corneal thickness with a depth-measuring device attachment.  
Solvent/vehicle control:  An untreated sample containing all components of a test 
system, including the solvent or vehicle that is processed with the test substance-treated and other control samples to establish the baseline response for the samples treated with the test substance dissolved in the same solvent 
or vehicle. When tested with a concurrent negative control, this sample also 
demonstrates whether the solvent or vehicle interacts with the test system.
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 703
 
Tiered testing:  A stepwise testing strategy where all existing information on a 
test substance is reviewed, in a specified order, using a weight of evidence 
process at each tier to determine if sufficient information is available for a hazard classification decision, prior to progression to the next tier. If the irritancy potential of a test substance can be assigned based on the existing 
information, no additional testing is required. If the irritancy potential of a test 
substance cannot be assigned based on the existing information, a step-wise sequential animal testing procedure is performed until an unequivocal classifi ­
cation can be made.  
Validated test method:  A test method for which validation studies have been 
completed to determine the relevance (including accuracy) and reliability for a 
specific purpose. It is important to note that a validated test method may not have sufficient performance in terms of accuracy and reliability to be found acceptable for the proposed purpose.  
Weight-of-evidence:  The process of considering the strengths and weaknesses of 
various pieces of information in reaching and supporting a conclusion concerning 
the hazard potential of a substance.
 ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 704
 
Appendix 2  
PROFICIENCY CHEMICALS FOR THE ICE TEST METHOD 
Prior to routine use of a test method that adheres to this test method, laboratories 
may wish to demonstrate technical proficiency by correctly identifying the ocular corrosivity classification of the 10 substances recommended in Table 1. These substances were selected to represent the range of responses for local eye irri ­
tation/corrosion, which is based on results in the  in vivo  rabbit eye test (TG 405) 
(i.e., Categories 1, 2A, 2B, or Not Classified or Labeled according to the UN 
GHS)(3)(7). However, considering the validated usefulness of these assays ( i.e., 
to identify ocular corrosives/severe irritants only), there are only two test 
outcomes for classification purposes (corrosive/severe irritant or 
non-corrosive/non-severe irritant) to demonstrate proficiency. Other selection criteria were that substances are commercially available, there are high quality  
in vivo  reference data available, and there are high quality data from the two  in 
vitro  methods for which Test Guidelines are being developed. For this reason, 
irritant substances were selected from the ICCVAM recommended list of 122 reference substances for the validation of  in vitro  ocular toxicity test methods 
(see Appendix H, ICCVAM Recommended Reference Substances List)(4). 
Reference data are available in the ICCVAM Background Review Documents 
for the Bovine Corneal Opacity and Permeability (BCOP) and the ICE test methods (18) (19).
 
Table 1  
Recommended substances for demonstrating technical proficiency with ICE  
Chemical CASRN Chemical Class (  1 ) Physical Form  In Vivo  
Classification (  2 ) In Vitro  lassification (  3 ) 
Benzalkonium 
chloride (5 %) 8001-54-5 Onium 
compound Liquid Category 1 Corrosive/Severe 
Irritant 
Chlorhexidine 55-56-1 Amine, 
Amidine Solid Category 1 Corrosive/Severe 
Irritant 
Dibenzoyl-L-tartaric 
acid 2743-38-6 Carboxylic 
acid, Ester Solid Category 1 Corrosive/Severe 
Irritant 
Imidazole 288-32-4 Heterocyclic Solid Category 1 Corrosive/Severe 
Irritant 
Trichloroacetic acid 
(30 %) 76-03-9 Carboxylic 
Acid Liquid Category 1 Corrosive/Severe 
Irritant 
2,6-Dichlorobenz-oyl 
chloride 4659-45-4 Acyl halide Liquid Category 2A Non-corrosive/Non- 
severe irritant 
Ethyl-2-methylaceto- 
acetate 609-14-3 Ketone, Ester Liquid Category 2B Non-corrosive/Non- 
severe irritant 
Ammonium nitrate 6484-52-2 Inorganic salt Solid Category 2A Non-corrosive/Non- 
severe irritant  ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 705
 
Chemical CASRN Chemical Class (  1 ) Physical Form  In Vivo  
Classification (  2 ) In Vitro  lassification (  3 ) 
Glycerol 56-81-5 Alcohol Liquid Not Labeled Non-corrosive/Non- 
severe irritant 
n-Hexane 110-54-3 Hydrocarbon 
(acyclic) Liquid Not Labeled Non-corrosive/Non- 
severe irritant  
Abbreviations: CASRN = Chemical Abstracts Service Registry Number 
( 1 ) Chemical classes were assigned to each test substance using a standard classification scheme, based on the National Library o f 
Medicine Medical Subject Headings (MeSH) classification system (available at http://www.nlm.nih.gov/mesh). 
( 2 ) Based on results from the  in vivo  rabbit eye test (OECD TG 405) and using the UN GHS (3)(7). 
( 3 ) Based on results in BCOP and ICE.  ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 706
 
Appendix 3  
Diagrams of the ICE superfusion apparatus and eye clamps  
(See Burton et al. (17) for additional generic descriptions of the superfusion apparatus and eye 
clamp)
  ▼M2 2008R0440 — EN — 22.03.2014 — 004.001 — 707
 
B.49.  IN VITRO  MAMMALIAN CELL MICRONUCLEUS TEST  
INTRODUCTION 
1. The  in vitro  micronucleus (MNvit) assay is a genotoxicity test for the 
detection of micronuclei (MN) in the cytoplasm of interphase cells. Micro ­
nuclei may originate from acentric chromosome fragments ( i.e. lacking a 
centromere), or whole chromosomes that are unable to migrate to the poles during the anaphase stage of cell division. The assay detects the activity of clastogenic and aneugenic chemicals (substances and mixtures) (1) (2) in 
cells that have undergone cell division during or after exposure to the test 
substance. This Test Method (TM) allows the use of protocols with and without the actin polymerisation inhibitor cytochalasin B (cytoB). The addition of cytoB prior to the targeted mitosis allows for the identification 
and selective analysis of micronucleus frequency in cells that have 
completed one mitosis because such cells are binucleate (3) (4). This TM also allows the use of protocols without cytokinesis block, provided there is 
evidence that the cell population analysed has undergone mitosis. 
2. In addition to using the MNvit assay to identify chemicals (substances and 
mixtures) that induce micronuclei, the use of a cytokinesis block, immuno ­
chemical labelling of kinetochores, or hybridisation with centromeric/te ­
lomeric probes (fluorescence  in situ  hybridisation (FISH)), also can 
provide information on the mechanisms of chromosome damage and micro ­
nucleus formation (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16). The labelling and hybridisation procedures can be used when there is an increase in micronucleus formation and the investigator wishes to determine if the 
increase was the result of clastogenic and/or aneugenic events. 
3. Micronuclei represent damage that has been transmitted to daughter cells, 
whereas chromosome aberrations scored in metaphase cells may not be transmitted. Because micronuclei in interphase cells can be assessed relatively objectively, laboratory personnel need only determine whether 
or not the cells have undergone division and how many cells contain a 
micronucleus. As a result, the preparations can be scored relatively quickly and analysis can be automated. This makes it practical to score thousands instead of hundreds of cells per treatment, increasing the power of the 
assay. Finally, as micronuclei may arise from lagging chromosomes, there 
is the potential to detect aneuploidy-inducing agents that are difficult to study in conventional chromosomal aberration tests,  e.g.  OECD Test 
Guideline 473 (Chapter B.10 of this Annex) (17). However, the MNvit 
assay does not allow for the differentiation of chemicals inducing poly ­
ploidy from those inducing clastogenicity without special techniques such as FISH described under paragraph 2. 
4. The MNvit assay is an  in vitro  method that typically uses cultured human 
or rodent cells. It provides a comprehensive basis for investigating chro ­
mosome damaging potential  in vitro  because both aneugens and clastogens 
can be detected. 
5. The MNvit assay is robust and effective in a variety of cell types, and in the 
presence or absence of cytoB. There are extensive data to support the validity of the MNvit assay using various rodent cell lines (CHO, V79, 
CHL/IU, and L5178Y) and human lymphocytes (18) (19) (20) (21) (22) 
(23) (24) (25) (26) (27) (28) (29) (30) (31). These include, in particular, the international validation studies coordinated by the Société Française de Toxicologie Génétique (SFTG) (18) (19) (20) (21) (22) and the reports of 
the International Workshop on Genotoxicity Testing (4) (16). The available 
data have also been re-evaluated in a weight-of-evidence retrospective
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 708
 
validation study by the European Centre for the Validation of Alternative 
Methods (ECVAM) of the European Commission, and the test method has 
been endorsed as scientifically valid by the ECVAM Scientific Advisory Committee (ESAC) (32) (33) (34). The use of the human TK6 lymphob ­
lastoid cell line (35), HepG2 cells (36) (37) and primary Syrian Hamster 
Embryo cells (38) has been described, although they have not been used in 
validation studies.
 
DEFINITIONS 
6. Definitions used are provided in Appendix 1.  
INITIAL CONSIDERATIONS 
7. Tests conducted  in vitro  generally require the use of an exogenous source of 
metabolic activation unless the cells are metabolically competent with respect to the substances being tested. The exogenous metabolic activation system does not entirely mimic  in vivo  conditions. Care should also be 
taken to avoid conditions that would lead to artefactual positive results 
which do not reflect intrinsic mutagenicity, and may arise from such factors as marked changes in pH or osmolality, or by high levels of cytot ­
oxicity (39) (40) (41). If the test chemical causes a change in the pH of the 
medium at the time of addition, the pH should be adjusted, preferably by 
buffering the stock solution so that all the volumes at all test concentrations, and for all controls, remain the same. 
8. To analyse the induction of micronuclei, it is essential that mitosis has 
occurred in both treated and untreated cultures. The most informative stage for scoring micronuclei is in cells that have completed one mitosis 
during or after treatment with the test substance.
 
PRINCIPLE OF THE TEST 
9. Cell cultures of human or mammalian origin are exposed to the test 
substance both with and without an exogenous source of metabolic 
activation unless cells with an adequate metabolising capability are used. Concurrent solvent/vehicle (VC) and positive control chemicals (PC) are included in all tests. 
10. During or after exposure to the test substance, the cells are grown for a 
period sufficient to allow chromosome or spindle damage to lead to the 
formation of micronuclei in interphase cells. For induction of aneuploidy, 
the test substance should ordinarily be present during mitosis. Harvested and stained interphase cells are analysed for the presence of micronuclei. Ideally, micronuclei should only be scored in those cells that have 
completed mitosis during exposure to the test substance or during the 
post-exposure period, if one is used. In cultures that have been treated with a cytokinesis blocker, this is achieved by scoring only binucleate cells. In the absence of a cytokinesis blocker, it is important to demonstrate 
that the cells analysed are likely to have undergone cell division during or 
after exposure to the test substance. For all protocols, it is important to demonstrate that cell proliferation has occurred in both the control and treated cultures, and the extent of test substance-induced cytotoxicity or 
cytostasis should be assessed in the cultures (or in parallel cultures) that 
are scored for micronuclei.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 709
 
DESCRIPTION OF THE ASSAY  
Preparations  
11. Cultured primary human peripheral blood lymphocytes (5) (19) (42) (43) 
and a number of rodent cell lines such as CHO, V79, CHL/IU, and L5178Y 
cells may be used (18) (19) (20) (21) (22) (25) (26) (27) (28) (30). The use of other cell lines and types should be justified based on their demonstrated performance in the assay, as described in the Acceptability Criteria section. 
Because the background frequency of micronuclei will influence the sensi ­
tivity of the assay, it is recommended that cell types with a low, stable background frequency of micronucleus formation be used. 
12. Human peripheral blood lymphocytes should be obtained from young 
(approximately 18-35 years of age), healthy, non-smoking individuals 
with no known recent exposures to genotoxic chemicals or radiation. If cells from more than one donor are pooled for use, the number of donors should be specified. The micronucleus frequency increases with age and this 
trend is more marked in females than in males (44) and this should be taken 
into account in the selection of donor cells for pooling.
 
Media and culture conditions 
13. Appropriate culture medium and incubation conditions (culture vessels, CO  2 
concentration, temperature, and humidity) should be used for maintaining 
cultures. Established cell lines and strains should be checked routinely for 
the stability of the modal chromosome number and the absence of myco ­
plasma contamination, and should not be used if contaminated or if the modal chromosome number has changed. The normal cell cycle time for the 
culture conditions used in the testing laboratory should be known. If the 
cytokinesis-block method is used then the concentration of the cytokinesis inhibitor should be optimised for the particular cell type and should be shown to produce a good yield of binucleate cells for scoring.
 
Preparation of cultures 
14. Established cell lines and strains: cells are propagated from stock cultures, 
seeded in culture medium at a density such that the cultures will not reach confluency in monolayers, and suspension cultures will not reach excessive density before the time of harvest, and incubated at 37 °C. 
15. Lymphocytes: whole blood treated with an anti-coagulant ( e.g. heparin), or 
separated lymphocytes, are cultured in the presence of a mitogen  e.g.  
phytohaemagglutinin (PHA) prior to exposure to the test substance and cytoB.
 
Metabolic activation 
16. Exogenous metabolising systems should be used when using cells with 
inadequate endogenous metabolic capacity. The most commonly used system is a co-factor-supplemented post-mitochondrial fraction (S9) prepared from the livers of rodents treated with enzyme-inducing agents 
such as Aroclor 1254 (45) (46) or a combination of phenobarbitone and β- 
naphthoflavone (46) (47) (48) (49). The latter combination does not conflict with the Stockholm Convention on Persistent Organic Pollutants (50) and Regulation (EC) No 850/2004 on Persistent Organic Pollutants (66) and has 
been shown to be as effective as Aroclor 1254 for inducing mixed-function 
oxidases (46) (47) (48) (49). The S9 fraction typically is used at concen ­
trations ranging from 1-10 % (v/v) in the final test medium. The condition of a metabolic activation system may depend upon the class of chemical 
being tested and in some cases it may be appropriate to utilise more than 
one S9 concentration.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 710
 
17. Genetically engineered cell lines expressing specific human or rodent 
activating enzymes may eliminate the need for an exogenous metabolic 
activation system, and may be used as the test cells. In such cases the choice of the cell lines used should be scientifically justified,  e.g.  by 
relevance of the mixed function oxidases for the metabolism of the test 
substance (51), and their responsiveness to known clastogens and aneugens 
(see separate section on Acceptability Criteria). It should be recognised that the substance being tested may not be metabolised by the expressed mixed function oxidase(s); in this case, the negative results would not indicate that 
the test substance cannot induce micronuclei.
 
Test substance preparation 
18. Solid chemicals should be dissolved in appropriate solvents or vehicles and 
diluted, if appropriate, prior to treatment of the cells. Liquid chemicals may 
be added directly to the test systems and/or diluted prior to treatment. Gases or volatile chemicals should be tested by appropriate modifications to the standard protocols, such as treatment in sealed vessels (52) (53). Fresh 
preparations of the test substance should be used unless stability data 
demonstrate the acceptability of storage.
 
Test Conditions  
Solvents/vehicles 
19. The solvent/vehicle should not react with the test substance, or be incom ­
patible with the survival of the cells or with the maintenance of S9 activity at the concentration used. If other than well established solvent/vehicles (e.g. water, cell culture medium, dimethyl sulfoxide) are used, their use 
should be supported by data indicating their compatibility with the test 
substance and their lack of genetic toxicity. It is recommended that, wherever possible, the use of an aqueous solvent/vehicle should be considered first.
 
Use of cytoB as a cytokinesis blocker 
20. One of the most important considerations in the performance of the MNvit 
assay is ensuring that the cells being scored have completed mitosis during 
the treatment or the post-treatment incubation period, if one is used. CytoB 
is the agent that has been most widely used to block cytokinesis because it inhibits actin assembly, and thus prevents separation of daughter cells after 
mitosis, leading to the formation of binucleated cells (5) (54) (55). Micro ­
nucleus scoring, therefore, can be limited to cells that have gone through mitosis during or after treatment. The effect of the test substance on cell proliferation kinetics can be measured simultaneously. CytoB should be 
used as a cytokinesis blocker when human lymphocytes are used because 
cell cycle times will be variable within cultures and among donors and because not all lymphocytes will respond to PHA. Other methods have been used when testing cell lines to determine if the cells being scored 
have divided; these are addressed below (see Paragraph 26). 
21. The appropriate concentration of cytoB should be determined by the 
laboratory for each cell type to achieve the optimal frequency of binucleated 
cells in the solvent/vehicle control cultures. The appropriate concentration 
of cytoB is usually between 3 and 6 μg/ml.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 711
 
Measuring cell proliferation and cytotoxicity and choosing exposure 
concentrations 
22. When determining the highest test substance concentration to be tested, 
concentrations that have the capability of producing artefactual positive responses, such as those producing excessive cytotoxicity, precipitation in the culture medium, and marked changes in pH or osmolality (39) (40) (41), 
should be avoided. 
23. Measurements of cell proliferation are made to ensure that the treated cells 
have undergone mitosis during the assay and that the treatments are 
conducted at appropriate levels of cytotoxicity (see Paragraph 29). Cytot ­
oxicity should be determined with and without metabolic activation in cells that require metabolic activation using the relative increase in cell counts (RICC) or relative population doubling (RPD) (see Appendix 2 
for formulas) unless cytoB is used. When cytoB is used, cytotoxicity can 
be determined using the replication index (RI) (see Appendix 2 for formula). 
24. Treatment of cultures with cytoB, and measurement of the relative 
frequencies of mononucleate, binucleate, and multi-nucleate cells in the culture, provides an accurate method of quantifying the effect on cell prolif ­
eration and the cytotoxic or cytostatic activity of a treatment (5), and 
ensures that only cells that divided during or after treatment are scored. 
25. In studies with cytoB, cytostasis/cytotoxicity can be quantified from the 
cytokinesis-block proliferation index (CBPI) (5) (26) (56) or may be 
derived from the RI from at least 500 cells per culture (see Appendix 2 
for formulas). When cytoB is used to assess cell proliferation, a CBPI or RI should be determined from at least 500 cells per culture. These measurements among others can be used to estimate cytotoxicity by 
comparing values in the treated and control cultures. Assessment of other 
markers of cytotoxicity ( e.g. confluency, cell number, apoptosis, necrosis, 
metaphase counting) can provide useful information. 
26. In studies without cytoB, it is necessary to demonstrate that the cells scored 
in the culture have undergone division during or following treatment with the test substance, otherwise false negative responses may be produced. Methods that have been used for ensuring that divided cells are being 
scored include incorporation and subsequent detection of bromodeo ­
xyuridine (BrdU) to identify cells that have replicated (57), the formation of clones when cells from permanent cell lines are treated and scored  in situ  
on a microscope slide (Proliferation Index (PI)) (25) (26) (27) (28), or the 
measurement of Relative Population Doubling (RPD) or Relative Increase 
in Cell Count (RICC) or other proven methods (16) (56) (58) (59) (see Appendix 2 for formulas). Assessment of other markers for cytotoxicity or cytostasis ( e.g. confluency, cell number, apoptosis, necrosis, metaphase 
counting) can provide useful information. 
27. At least three analysable test concentrations should be evaluated. In order to 
achieve this, it may be necessary to perform the experiment using a larger 
number of closely spaced concentrations and analyse micronucleus 
formation in those concentrations providing the appropriate range of cytot ­
oxicities. An alternative strategy is to perform a preliminary cytotoxicity test to narrow the range for the definitive test. 
28. The highest concentration should aim to produce 55 ± 5 % cytotoxicity. 
Higher levels may induce chromosome damage as a secondary effect of cytotoxicity (60). Where cytotoxicity occurs, the test concentrations 
selected should cover a range from that producing 55 ± 5 % cytotoxicity, 
to little or no cytotoxicity.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 712
 
29. If no cytotoxicity or precipitate is observed, the highest test concentration 
should correspond to 0,01 M, 5 mg/mL or 5 μl/mL, whichever is the 
lowest. The concentrations selected for analysis should, in general, be separated by a spacing of no more than 10. For test substances that exhibit a steep concentration-response curve, it may be necessary to more 
closely space the test substance concentrations so that cultures in the 
moderate and low toxicity ranges also will be scored. 
30. When solubility is a limiting factor, the maximum concentration, if not 
limited by cytotoxicity, should be the lowest concentration at which 
minimal precipitate is visible in cultures, provided there is no interference with scoring. Evaluation of precipitation should be done by methods such as light microscopy, noting precipitate that persists, or appears during 
culture (by the end of treatment).
 
Controls 
31. Concurrent positive and solvent/vehicle controls both with and without 
metabolic activation should be included in each experiment. 
32. PC are needed to demonstrate the ability of the cells used, and the test 
protocol, to identify clastogens and aneugens, and to affirm the metabolic 
capability of the S9 preparation. The PC should employ known inducers of 
micronucleus formation at concentrations expected to give small, but repro ­
ducible increases over background, and demonstrate the sensitivity of the test system. PC concentrations should be chosen so that the effects are clear 
but do not immediately reveal the identity of the coded slides to the reader. 
33. A clastogen that requires metabolic activation ( e.g. cyclophosphamide; 
benzo(a)pyrene) should be used to demonstrate both the metabolic 
competence and the ability of the test system to detect clastogens. Other 
PC may be used if justified. Because some PC that need metabolic activation may be active without exogenous metabolic activation under 
certain treatment conditions or in certain cell lines, the need for 
metabolic activation, and the activity of the S9 preparation, should be tested in the selected cell line and at the selected concentrations. 
34. At the present time, no aneugens are known that require metabolic 
activation for their genotoxic activity (16). Currently accepted PC for aneugenic activity are, for example, colchicine and vinblastine. Other chemicals may be used if they induce micronuclei solely, or primarily, 
through aneugenic activity. To avoid the need for two PC (for clastogenicity 
and aneugenicity) without metabolic activation, the aneugenicity control can serve as the PC without S9, and the clastogenicity control can be used to test the adequacy of the metabolic activation system used. PC for both 
clastogenicity and aneugenicity should be used in cells that do not 
require S9. Suggested PC are included in Appendix 3. 
35. The use of chemical class-related PC may be considered, when suitable 
chemicals are available. All PC used should be appropriate for the cell 
type and activation conditions.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 713
 
36. Solvent/vehicle controls should be included for every harvest time. In 
addition, untreated NC (lacking solvent/vehicle) should also be used 
unless there are published or laboratory historical control data demon ­
strating that no genotoxic or other deleterious effects are induced by the chosen solvent at the concentrations used.
 
TEST PROCEDURE  
Treatment Schedule  
37. In order to maximise the probability of detecting an aneugen or clastogen 
acting at a specific stage in the cell cycle, it is important that sufficient numbers of cells are treated with the test substance during all stages of their cell cycles. The treatment schedule for cell lines and primary cell cultures 
may, therefore, differ somewhat from that for lymphocytes which require 
mitogenic stimulation to begin their cell cycle and these are considered in Paragraphs 41-43 (16). 
38. Theoretical considerations, together with published data (18) indicate that 
most aneugens and clastogens will be detected by a short term treatment 
period of 3 to 6 hrs in the presence and absence of S9, followed by removal of the test substance and a growth period of 1,5-2,0 cell cycles (6). Cells are sampled at a time equivalent to about 1,5-2,0 times the normal ( i.e. 
untreated) cell cycle length either after the beginning or at the end of 
treatment (See Table 1). Sampling or recovery times may be extended if it is known or suspected that the test substance affects the cell cycling time (e.g. when testing nucleoside analogues). 
39. Because of the potential cytotoxicity of S9 preparations for cultured 
mammalian cells, an extended exposure treatment of 1,5-2,0 normal cell cycles is used only in the absence of S9. In the extended treatment, options are offered to allow treatment of the cells with the test chemical in the 
absence or presence of cytoB. These options address situations where there 
may be concern regarding possible interactions between the test substance and cytoB. 
40. The suggested cell treatment schedules are presented in Table 1. These 
general treatment schedules may be modified depending on the stability 
or reactivity of the test substance or the particular growth characteristics of the cells being used. All treatments should commence and end while the cells are growing exponentially. These schedules are presented in more 
details in paragraphs 41-47 following.
 
Table 1  
Cell treatment and harvest times for the MNvit assay  
Lymphocytes, primary cells and cell lines 
treated with cytoB + S9 Treat for 3-6 hrs in the presence of S9; 
remove the S9 and treatment medium; 
add fresh medium and cytoB; 
harvest 1,5-2,0 normal cell cycles later. 
– S9 
Short exposure Treat for 3-6 hrs; 
remove the treatment medium; 
add fresh medium and cytoB; harvest 1,5-2,0 normal cell cycles later.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 714
 
– S9 
Extended exposure  Option A:  Treat for 1,5-2 normal cell cycles in 
the presence of cytoB; 
harvest at the end of the exposure period.  
Option B:  Treat for 1,5-2,0 normal cell cycles; 
remove the test substance; 
add fresh medium and cytoB; 
harvest 1,5-2,0 normal cell cycles later. 
Cell lines treated without cytoB 
(Identical to the treatment schedules outlined above with the exception that no cytoB is added)  
Lymphocytes, primary cells, and cell lines with cytoB  
41. For lymphocytes, the most efficient approach is to start the exposure to the 
test substance at 44-48 hrs after PHA stimulation, when cycle synchroni ­
sation will have disappeared (5). In the initial assay, cells are treated for 3 to 6 hrs with the test substance in the absence and presence of S9. The 
treatment medium is removed and replaced with fresh medium containing 
cytoB, and the cells are harvested 1,5-2,0 normal cell cycles later. 
42. If both initial tests of the short (3-6 hrs) treatment are negative or equivocal, 
a subsequent, extended exposure treatment without S9 is used. Two treatment options are available and are equally acceptable. However, it might be more appropriate to follow Option A for stimulated lymphocytes 
where exponential growth may be declining at 96 hrs following stimulation. 
Also, cultures of cells should not have reached confluence by the final sampling time in Option B. 
— Option A: The cells are treated with the test substance for 1,5-2,0 
normal cell cycles, and harvested at the end of the treatment time. 
— Option B: The cells are treated with the test substance for 1,5-2,0 
normal cell cycles. The treatment medium is removed and replaced 
with fresh medium, and the cells are harvested after additional 1,5-2,0 
normal cell cycles. 
43. Primary cells and cell lines should be treated in a similar manner to lymp ­
hocytes except that it is not necessary to stimulate them with PHA for 44- 48 hrs. Cells other than lymphocytes should be exposed such that at the time of study termination, the cells are still in log-phase growth.
 
Cell lines without cytoB  
44. Cells should be treated for 3-6 hrs in the presence and absence of S9. The 
treatment medium is removed and replaced with fresh medium, and the cells 
are harvested 1,5-2,0 normal cell cycles later.  ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 715
 
45. If both initial tests of the short (3-6 hrs) treatment are negative or equivocal, 
a subsequent, extended exposure treatment (without S9) is used. Two 
treatment options are available, both of which are equally acceptable: 
— Option A: The cells are treated with the test substance for 1,5-2,0 
normal cell cycles, and harvested at the end of the treatment time. 
— Option B: The cells are treated with the test substance for 1,5-2,0 
normal cell cycles. The treatment medium is removed and replaced with fresh medium, and the cells are harvested after additional 1,5-2,0 normal cell cycles. 
46. In monolayers, mitotic cells (identifiable as being round and detaching from 
the surface) may be present at the end of the 3-6 hr treatment. Because 
these mitotic cells are easily detached, they can be lost when the medium 
containing the test substance is removed. Care should be taken to collect these when cultures are washed, and to return them to the cultures, to avoid losing cells that are in mitosis, and at risk for micronuclei, at the time of 
harvest.
 
Number of cultures  
47. Duplicate cultures should be used for each test substance concentration and 
for the vehicle/solvent and NC cultures. Where minimal variation between duplicate cultures can be demonstrated from historical laboratory data, it may be acceptable for single cultures to be used. If single cultures are used, 
it is recommended that an increased number of concentrations be analysed.
 
Cell harvest and slide preparation  
48. Each culture is harvested and processed separately. Cell preparation may 
involve hypotonic treatment, but this step is not necessary if adequate cell 
spreading is otherwise achieved. Different techniques can be used in slide preparation provided that high-quality cell preparations for scoring are obtained. Cell cytoplasm should be retained to allow the detection of micro ­
nuclei and (in the cytokinesis-block method) reliable identification of binu ­
cleate cells. 
49. The slides can be stained using various methods, such as Giemsa or fluor ­
escent DNA specific dyes (59). The use of a DNA specific stain ( e.g. 
acridine orange (61) or Hoechst 33258 plus pyronin-Y (62)) can 
eliminate some of the artefacts associated with using a non-DNA specific 
stain. Anti-kinetochore antibodies, FISH with pancentromeric DNA probes, or primed  in situ  labelling with pancentromere-specific primers, together 
with appropriate DNA counterstaining, can be used to identify the 
contents (chromosome/chromosomal fragment) of micronuclei if mech ­
anistic information of their formation is of interest (15) (16). Other methods for differentiation between clastogens and aneugens may be used if they have been shown to be effective.
 
Analysis  
50. All slides, including those of the solvent/vehicle and the controls, should be 
independently coded before the microscopic analysis. Alternatively, coded 
samples can be analysed using a validated, automated flow cytometric or 
image analysis system.  ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 716
 
51. In cytoB-treated cultures, micronucleus frequencies should be analysed in at 
least 2 000 binucleated cells per concentration (at least 1 000 binucleated 
cells per culture; two cultures per concentration). If single cultures are used, at least 2 000 binucleated cells per concentration should be scored from that culture. If substantially fewer than 1 000 binucleate cells per culture, or 
2 000 if a single culture is used, are available for scoring at each concen ­
tration, and if a significant increase in micronuclei is not detected, the test should be repeated using more cells, or at less toxic concentrations, whichever is appropriate. Care should be taken not to score binucleate 
cells with irregular shapes or where the two nuclei differ greatly in size; 
neither should binucleate cells be confused with poorly spread multi-nucleate cells. Cells containing more than two main nuclei should not be analysed for micronuclei, as the baseline micronucleus frequency 
may be higher in these cells (63) (64) Scoring of mononucleate cells is 
acceptable if the test substance is shown to interfere with cytoB activity. 
52. In cell lines assayed without cytoB treatment, micronuclei should be scored 
in at least 2 000 cells per concentration (at least 1 000 cells per culture; two 
cultures per concentration). Where only one culture per concentration is 
used, at least 2 000 cells should be scored from that culture. 
53. When cytoB is used, a CBPI or an RI should be determined to assess cell 
proliferation (see Appendix 2) using at least 500 cells per culture. When 
treatments are performed in the absence of cytoB, it is essential to provide evidence that the cells being scored have proliferated, as discussed in Para ­
graphs 24-27.
 
Acceptability criteria  
54. A laboratory proposing to use the MNvit assay described in this TM should 
demonstrate its ability to reliably and accurately detect chemicals of known aneugenic and clastogenic activity, with and without metabolic activation, 
as well as known negative chemicals, using the reference chemicals in 
Appendix 3. As evidence of its ability to perform this TM correctly, the laboratory should provide evidence that the cells being scored for micro ­
nucleus formation have completed one nuclear division if the test is 
performed without the use of cytoB. 
55. The chemicals in Appendix 3 are recommended for use as reference 
chemicals. Substitute or additional chemicals can be included if their 
activity is known and if they induce micronuclei by the same mechanisms 
of action, and if they are shown to be relevant to the chemicals that will be tested using the MNvit procedure. Justification could include a validation study employing a broad variety of substances or focused on a narrower 
spectrum based on the chemical class of the test substance or the 
mechanism of damage being studied. 
56. Solvent/vehicle control and untreated cultures should give reproducibly low 
and consistent micronuclei frequencies (typically 5-25 micronuclei/1 000 
cells for the cell types identified in paragraph 11). Other cell types may 
have different ranges of responses which should be determined when vali ­
dating them for use in the MNvit assay. Data from negative, solvent, and PC should be used to establish historical control ranges. These values 
should be used in deciding the adequacy of the concurrent NC/PC for an 
experiment.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 717
 
57. If minor changes to the protocol ( e.g. use of automated instead of manual 
scoring techniques; use of a new cell type) are proposed for the assay, then 
the effectiveness of the change should be demonstrated before the modified protocol can be considered acceptable for use. Demonstration of effec ­
tiveness includes demonstration that the major mechanisms of chromosome 
breakage and gain or loss can be detected, and that appropriate positive and 
negative results can be achieved for the class of the individual substance, or the broad range of substances, to be tested.
 
DATA AND REPORTING  
Treatment of results  
58. If the cytokinesis-block technique is used, only the frequencies of binucleate 
cells with micronuclei (independent of the number of micronuclei per cell) are used in the evaluation of micronucleus induction. Scoring of the 
numbers of cells with one, two, or more micronuclei could provide 
useful information, but is not mandatory. 
59. Concurrent measures of cytotoxicity and/or cytostasis for all treated and 
solvent/vehicle control cultures should be determined (58). The CBPI or 
the RI should be calculated for all treated and control cultures as measurements of cell cycle delay when the cytokinesis-block method is used. In the absence of cytoB, the RPD or the RICC or PI should be 
used (see Appendix 2). 
60. Individual culture data should be provided. Additionally, all data should be 
summarised in tabular form. 
61. Chemicals that induce micronuclei in the MNvit assay may do so because 
they induce chromosome breakage, chromosome loss, or a combination of 
the two. Further analysis using anti-kinetochore antibodies, centromere 
specific  in situ  probes, or other methods may be used to determine 
whether the mechanism of micronucleus induction is due to clastogenic and/or aneugenic activity.
 
Evaluation and interpretation of results  
62. There is no requirement for verification by additional testing of a clear 
positive or negative response. Equivocal results may be clarified by 
analysis of another 1 000 cells from all the cultures to avoid loss of 
blinding. If this approach does not resolve the result, further testing should be performed. Modification of study parameters over an extended or narrowed range of conditions, as appropriate, should be considered in 
follow-up experiments. Study parameters that might be modified include the 
test concentration spacing, the timing of treatment and cell harvest, and/or the metabolic activation conditions. 
63. There are several criteria for determining a positive result, such as 
a concentration-related increase or a statistically significant increase in the number of cells containing micronuclei. The biological relevance of the results should be considered first. Consideration of whether the observed 
values are within or outside of the historical control range can provide 
guidance when evaluating the biological significance of the response. Appropriate statistical methods may be used as an aid in evaluating the test results (65). However, the results of statistical testing should be 
assessed with respect to dose-response relationship. Reproducibility and 
historical data should also be taken into consideration.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 718
 
64. Although most experiments will give clearly positive or negative results, in 
some cases the data set will preclude making a definite judgement about the 
activity of the test substance. These equivocal or questionable responses may occur regardless of the number of times the experiment is repeated. 
65. Positive results from the MNvit assay indicate that the test substance 
induces chromosome breakage or chromosome loss, in cultured mammalian cells. Negative results indicate that, under the test conditions used, the test substance does not induce chromosome breaks and/or gain or 
loss in cultured mammalian cells.
 
Test Report  
66. The test report should include at least the following information, if relevant 
to the conduct of the study:  
Test chemical:  
— identification data and Chemical Abstract Services Registry Number and 
EC Number; 
— physical nature and purity; 
— physico-chemical properties relevant to the conduct of the study; — reactivity of the test chemical with the solvent/vehicle or cell culture 
media;  
Solvent/Vehicle:  
— justification for choice of solvent/vehicle; 
— solubility and stability of the test substance in solvent/vehicle;  
Cells:  
— type and source of cells used; 
— suitability of the cell type used; — absence of mycoplasma, if applicable; 
— information on cell cycle length, doubling time or proliferation index; 
— where lymphocytes are used, sex, age and number of blood donors, if 
applicable; 
— where lymphocytes are used, whether whole blood or separated lymp ­
hocytes are exposed; 
— number of passages, if applicable; 
— methods for maintenance of cell cultures, if applicable; 
— modal number of chromosomes; 
— normal (negative control) cell cycle time;  
Test Conditions:  
— identity of cytokinesis blocking substance (e.g. cytoB), if used, and its 
concentration and duration of cell exposure; 
— rationale for selection of concentrations and number of cultures, 
including cytotoxicity data and solubility limitations, if available; 
— composition of media, CO
 2 concentration, if applicable; 
— concentrations of test substance;  ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 719
 
— concentration (and/or volume) of vehicle and test substance added; 
— incubation temperature and time; 
— duration of treatment; 
— harvest time after treatment; 
— cell density at seeding, if applicable; 
— type and composition of metabolic activation system, including accepta ­
bility criteria; 
— positive control chemicals and negative controls; 
— methods of slide preparation and staining technique used; 
— criteria for micronucleus identification; 
— numbers of cells analysed; 
— methods for the measurements of cytotoxicity; 
— any supplementary information relevant to cytotoxicity; 
— criteria for considering studies as positive, negative, or equivocal; 
— method(s) of statistical analysis used; 
— methods, such as use of kinetochore antibody, to characterise whether 
micronuclei contain whole or fragmented chromosomes, if applicable;  
Results:  
— measurement of cytotoxicity used,  e.g.  CBPI or RI in the case of 
cytokinesis-block method; RICC, RPD or PI when cytokinesis-block 
methods are not used; other observations when applicable,  e.g.  cell 
confluency, apoptosis, necrosis, metaphase counting, frequency of binu ­
cleated cells; 
— signs of precipitation; 
— data on pH and osmolality of the treatment medium, if determined; 
— definition of acceptable cells for analysis; 
— distribution of mono-, bi-, and multi-nucleated cells if a cytokinesis 
block method is used; 
— number of cells with micronuclei given separately for each treated and 
control culture, and defining whether from binucleate or mononucleate cells, where appropriate; 
— concentration-response relationship, where possible; 
— concurrent negative (solvent/vehicle) and positive control chemical data 
(concentrations and solvents); 
— historical negative (solvent/vehicle) and positive control chemical data, 
with ranges, means and standard deviation and confidence interval ( e.g. 
95 %); 
— statistical analysis; p-values if any;  
Discussion of the results 
Conclusions
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 720
 
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Holland, N., Kirsch-Volders, M., Zeiger, E., Ban, S., Barale, R., Bigatti, M.P., Bolognesi, C., Jia, C., Di Giorgio, M., Ferguson, L.R., Fucic, A., Lima, O.G., Hrelia, P., Krishnaja, A.P., Lee, T.K., Migliore, L., 
Mikhalevich, L., Mirkova, E., Mosesso, P., Muller, W.U., Odagiri, Y., 
Scarffi, M.R., Szabova, E., Vorobtsova, I., Vral, A. and Zijno, A. (2001), HUman MicroNucleus Project: international database comparison for results with the cytokinesis-block micronucleus assay in human lymphocytes, I. 
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frequency of micronuclei,  Environ. Mol. Mutagen.  37, 31-45.
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(45) Maron, D.M. and Ames, B.N. (1983), Revised methods for the Salmonella 
mutagenicity test,  Mutation Res. , 113, 173-215. 
(46) Ong, T.-m., Mukhtar, M., Wolf, C.R. and Zeiger, E. (1980), Differential 
effects of cytochrome P450-inducers on promutagen activation capabilities and enzymatic activities of S-9 from rat liver,  J. Environ. Pathol. Toxicol. , 
4, 55-65. 
(47) Elliott, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., Gibson, 
G.G., Mackay, J.M. and Wolf, R.C. (1992), Alternatives to Aroclor 1254- induced S9 in  in-vitro  genotoxicity assays.  Mutagenesis , 7, 175-177. 
(48) Matsushima, T., Sawamura, M., Hara, K. and Sugimura, T. (1976), A safe 
substitute for Polychlorinated Biphenyls as an Inducer of Metabolic 
Activation Systems,  In: de Serres, F.J., Fouts, J. R., Bend, J.R. and 
Philpot, R.M. (eds),  In Vitro  Metabolic Activation in Mutagenesis 
Testing, Elsevier, North-Holland, pp. 85-88. 
(49) Johnson, T.E., Umbenhauer, D.R. and Galloway, S.M. (1996), Human liver 
S-9 metabolic activation: proficiency in cytogenetic assays and comparison 
with phenobarbital/beta-naphthoflavone or Aroclor 1254 induced rat S-9,  
Environ. Mol. Mutagen. , 28, 51-59. 
(50) UNEP (2001), Stockholm Convention on Persistent Organic Pollutants, 
United Nations Environment Programme (UNEP). Available at: [http:// 
www.pops.int/] 
(51) Doherty, A.T., Ellard, S., Parry, E.M. and Parry, J.M. (1996), An 
investigation into the activation and deactivation of chlorinated hydro ­
carbons to genotoxins in metabolically competent human cells,  Muta­
genesis , 11, 247-274. 
(52) Krahn, D.F., Barsky, F.C. and McCooey, K.T. (1982), CHO/HGPRT 
Mutation Assay: Evaluation of Gases and Volatile Liquids,  In: Tice, 
R.R., Costa, D.L. and Schaich, K.M. (eds), Genotoxic Effects of 
Airborne Agents. New York, Plenum, pp. 91-103. 
(53) Zamora, P.O., Benson, J.M., Li, A.P. and Brooks, A.L. (1983), Evaluation 
of an exposure system using cells grown on collagen gels for detecting highly volatile mutagens in the CHO/HGPRT mutation assay,  Environ. 
Mutagenesis  5, 795-801. 
(54) Fenech, M. (1993), The cytokinesis-block micronucleus technique: a 
detailed description of the method and its application to genotoxicity studies in human populations,  Mutation Res. , 285, 35-44. 
(55) Phelps, J.B., Garriott, M.L., and Hoffman, W.P. (2002), A protocol for the  
in vitro  micronucleus test. II. Contributions to the validation of a protocol 
suitable for regulatory submissions from an examination of 10 chemicals 
with different mechanisms of action and different levels of activity,  
Mutation Res. , 521, 103-112. 
(56) Kirsch-Volders, M., Sofuni, T., Aardema, M., Albertini, S., Eastmond, D., 
Fenech, M., Ishidate, M. Jr., Kirchner, S., Lorge, E., Morita, T., Norppa, H., 
Surralles, J., Vanhauwaert, A. and Wakata, A. (2004), Corrigendum to ‘Report from the  in vitro  micronucleus assay working group’,  Mutation 
Res., 564, 97-100. 
(57) Pincu, M., Bass, D. and Norman, A. (1984), An improved micronuclear 
assay in lymphocytes,  Mutation Res. , 139, 61-65. 
(58) Lorge, E., Hayashi, M., Albertini, S. and Kirkland, D. (2008), Comparison 
of different methods for an accurate assessment of cytotoxicity in the  in 
vitro  micronucleus test. I. Theoretical aspects,  Mutation Res. , 655, 1-3. 
(59) Surralles, J., Xamena, N., Creus, A., Catalan, J., Norppa, H. and Marcos, R. 
(1995), Induction of micronuclei by five pyrethroid insecticides in whole-blood and isolated human lymphocyte cultures,  Mutation Res. , 341, 
169-184. 
(60) Galloway, S. (2000), Cytotoxicity and chromosome aberrations  in vitro : 
Experience in industry and the case for an upper limit on toxicity in the 
aberration assay, Environ. Molec.  Mutagenesis  35, 191-201.
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(61) Hayashi, M., Sofuni, T., and Ishidate, M. Jr. (1983), An Application of 
Acridine Orange Fluorescent Staining to the Micronucleus Test,  Mutation 
Res., 120, 241-247. 
(62) MacGregor, J. T., Wehr, C. M., and Langlois, R. G. (1983), A Simple 
Fluorescent Staining Procedure for Micronuclei and RNA in Erythrocytes Using Hoechst 33258 and Pyronin Y,  Mutation Res. , 120, 269-275. 
(63) Hayashi, M., Sofuni, T. and Ishidate, M. Jr. (1983), An application of 
acridine orange fluorescent staining to the micronucleus test,  Mutation 
Res., 120, 241-247. 
(64) Fenech, M., Chang, W.P., Kirsch-Volders, M., Holland, N., Bonassi, S. and 
Zeiger, E. (2003), HUMN project: detailed description of the scoring criteria 
for the cytokinesis-block micronucleus assay using isolated human lymp ­
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(65) Hoffman, W.P., Garriott, M.L. and Lee, C. (2003),  In vitro  micronucleus 
test, In: Encyclopedia of Biopharmaceutical Statistics, Second edition. S. 
Chow (ed.), Marcel Dekker, Inc. New York, NY, pp. 463-467. 
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 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 725
 
Appendix 1  
Definitions  
Aneugen:  any substance or process that, by interacting with the components of 
the mitotic and meiotic cell division cycle, leads to aneuploidy in cells or organisms.  
Aneuploidy:  any deviation from the normal diploid (or haploid) number of chro ­
mosomes by a single chromosome or more than one, but not by entire set(s) of 
chromosomes (polyploidy).  
Apoptosis:  programmed cell death characterised by a series of steps leading to a 
disintegration of cells into membrane-bound particles that are then eliminated by 
phagocytosis or by shedding.  
Cell proliferation:  increase in cell number as a result of mitotic cell division.  
Centromere:  DNA region of a chromosome where both chromatids are held 
together and on which both kinetochores are attached side-to-side.  
Clastogen:  any substance or process which causes structural chromosomal aber ­
rations in populations of cells or organisms.  
Cytokinesis:  the process of cell division immediately following mitosis to form 
two daughter cells, each containing a single nucleus.  
Cytokinesis-Block Proliferation index (CBPI):  the proportion of second-division 
cells in the treated population relative to the untreated control (see Appendix 2 
for formula).  
Cytostasis:  inhibition of cell growth (see Appendix 2 for formula).  
Cytotoxicity:  harmful effects to cell structure or function ultimately causing cell 
death.  
Genotoxic:  a general term encompassing all types of DNA or chromosome 
damage, including breaks, adducts rearrangements, mutations, chromosome aber ­
rations, and aneuploidy. Not all types of genotoxic effects result in mutations or 
stable chromosome damage.  
Interphase cells:  cells not in the mitotic stage.  
Kinetochore:  a protein-containing structure that assembles at the centromere of a 
chromosome to which spindle fibres associate during cell division, allowing orderly movement of daughter chromosomes to the poles of the daughter cells.  
Micronuclei:  small nuclei, separate from and additional to the main nuclei of 
cells, produced during telophase of mitosis or meiosis by lagging chromosome 
fragments or whole chromosomes.  
Mitosis:  division of the cell nucleus usually divided into prophase, prometaphase, 
metaphase, anaphase and telophase.  
Mitotic index:  the ratio of cells in metaphase divided by the total number of cells 
observed in a population of cells; an indication of the degree of cell proliferation of that population.  
Mutagenic:  produces a heritable change of DNA base-pair sequences(s) in genes 
or of the structure of chromosomes (chromosome aberrations).
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 726
 
Non-disjunction:  failure of paired chromatids to disjoin and properly segregate to 
the developing daughter cells, resulting in daughter cells with abnormal numbers 
of chromosomes.  
Polyploidy:  numerical chromosome aberrations in cells or organisms involving 
entire set(s) of chromosomes, as opposed to an individual chromosome or chro ­
mosomes (aneuploidy).  
Proliferation Index (PI):  method for cytotoxicity measurement when cytoB is not 
used (see Appendix 2 for formula).  
Relative Increase in Cell Count (RICC):  method for cytotoxicity measurement 
when cytoB is not used (see Appendix 2 for formula).  
Relative Population Doubling (RPD):  method for cytotoxicity measurement when 
cytoB is not used (see Appendix 2 for formula).  
Replication Index (RI):  the proportion of cell division cycles completed in a 
treated culture, relative to the untreated control, during the exposure period 
and recovery (see Appendix 2 for formula).  
Test chemical (also referred to as test substance):  Any substance or mixture 
tested using this TM.  ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 727
 
Appendix 2  
Formulas for Cytotoxicity Assessment  
1. When cytoB is used , evaluation of cytotoxicity should be based on the 
Cytokinesis-Block Proliferation Index (CBPI) or Replicative Index (RI) (16) 
(58). The CBPI indicates the average number of cell cycles per cell during the 
period of exposure to cytoB, and may be used to calculate cell proliferation. The RI indicates the relative number of nuclei in treated cultures compared to control cultures and can be used to calculate the % cytostasis: 
% Cytostasis = 100 – 100{(CBPI
 T – 1) ÷ (CBPI  C – 1)} 
And: 
T = test chemical treatment culture C = vehicle control culture 
Where: 
CBPI ¼
 ððNo: mononucleate cells Þ þ ð2 Ü No : binucleate cellsÞ þ ð3 Ü No : multinucleate cells ÞÞ 
ðTotal number of cells Þ 
Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis. 
Cytostasis = 100 – RI 
RI ¼ ððNo: binucleated cellsÞ þ ð2 Ü No : multinucleate cells ÞÞ ö ðTotal number of cells Þ T 
ððNo: binucleated cellsÞ þ ð2 Ü No : multinucleate cells ÞÞ ö ðTotal number of cells Þ C Ü 100 
T = treated cultures 
C = control cultures 
2. Thus, an RI of 53 % means that, compared to the numbers of cells that 
have divided to form binucleate and multinucleate cells in the control culture, 
only 53 % of this number divided in the treated culture,  i.e. 47 % cytostasis. 
3. When cytoB is not used , evaluation of cytotoxicity based on Relative 
Increase in Cell Counts (RICC) or on Relative Population Doubling (RPD) is recommended (58), as both take into account the proportion of the cell popu ­
lation which has divided. 
RICC ¼
 ðIncrease in number of cells in treated cultures  ðfinal – startingÞÞ 
ðIncrease in number of cells in control cultures  ðfinal – startingÞÞ Ü 100 
RPD ¼ ðNo: of Population doublings in treated cultures Þ 
ðNo: of Population doublings in control cultures Þ Ü 100 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 728
 
where: 
Population Doubling = [log (Post-treatment cell number ÷ Initial cell number)] ÷ 
log 2 
4. Thus, a RICC, or a RPD of 53 % indicates 47 % cytotoxicity/cytostasis. 5. By using a Proliferation Index (PI), cytotoxicity may be assessed via 
counting the number of clones consisting of 1 cell (cl1), 2 cells (cl2), 3 to 4 cells (cl4) and 5 to 8 cells (cl8) 
PI ¼
 ðð1 Ü cl1Þ þ ð2 Ü cl2Þ þ ð3 Ü cl4Þ þ ð4 Ü cl8ÞÞ 
ðcl1 þ cl2 þ cl4 þ cl8Þ 
6. The PI has been used as a valuable and reliable cytotoxicity parameter also 
for cell lines cultured  in situ  in the absence of cytoB (25) (26) (27) (28).  ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 729
Appendix 3  
Reference chemicals recommended for assessing performance  ( 1 ) 
Category Chemical CAS No EC No  
1. Clastogens active without metabolic activation 
Cytosine arabinoside 147-94-4 205-705-9 Mitomycin C 50-07-7 200-008-6 
2. Clastogens requiring metabolic activation 
Benzo(a)pyrene 50-32-8 200-028-5 
Cyclophosphamide 50-18-0 200-015-4 
3. Aneugens 
Colchicine 64-86-8 200-598-5 Vinblastine 143-67-9 205-606-0 
4. Negative substances 
Di(2-ethylhexyl)phthalate 117-81-7 204-211-0 
Nalidixic acid 389-08-2 206-864-7 Pyrene 129-00-0 204-927-3 Sodium chloride 7647-14-5 231-598-3
 
( 1 ) The reference chemicals are the recommended chemicals for use. Substitution or adding of chemicals to the list of reference 
chemicals can be done if their activity is known and if they induce micronuclei by the same mechanisms of action, and if they 
are shown to be relevant to the chemicals that will be tested using the MNvit procedure. Depending on the purpose, justificatio n 
could also include a validation study employing a broad variety of substances or focused on a narrower spectrum based on the 
chemical class of the test substance or the mechanism of damage being studied.  ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 730
 
B.50.  SKIN SENSITISATION: LOCAL LYMPH NODE ASSAY: DA  
INTRODUCTION 
1. OECD Guidelines for the Testing of Chemicals and EU Test Methods are 
periodically reviewed in light of scientific progress, changing regulatory 
needs, and animal welfare considerations. The first Test Method (TM) (B.42) for the determination of skin sensitisation in the mouse, the Local 
Lymph Node Assay (LLNA; OECD Test Guideline 429) has been revised 
(1) The details of the validation of the LLNA and a review of the associated work have been published (2) (3) (4) (5) (6) (7) (8) (9). In the LLNA, radioisotopic thymidine or iodine is used to measure lymphocyte prolif ­
eration and therefore the assay has limited use where the acquisition, use, 
or disposal of radioactivity is problematic. The LLNA: DA (developed by Daicel Chemical Industries, Ltd) is a non-radioactive modification to the LLNA, which quantifies adenosine triphosphate (ATP) content via 
bio-luminescence as an indicator of lymphocyte proliferation. The LLNA: 
DA test method has been validated and reviewed and recommended by an international peer review panel as considered useful for identifying skin sensitising and non-sensitising chemicals, with certain limitations (10) (11) 
(12) (13). This TM is designed for assessing skin sensitisation potential of 
chemicals (substances and mixtures) in animals. Chapter B.6 of this Annex and OECD Test Guideline 406 utilise guinea pig tests, notably the guinea pig maximisation test and the Buehler test (14) The LLNA (Chapter 
B.42 of this Annex; OECD Test Guideline 429) and the two non-radioactive 
modifications, LLNA: DA (Chapter B.50 of this Annex; OECD Test Guideline 442 A) and LLNA: BrdU-ELISA (Chapter B.51 of this Annex; OECD Test Guideline 442 B), all provide an advantage over the guinea pig 
tests in B.6 and OECD Test Guideline 406 (14) in terms of reduction and 
refinement of animal use. 
2. Similar to the LLNA, the LLNA: DA studies the induction phase of skin 
sensitisation and provides quantitative data suitable for dose-response 
assessment. Furthermore, an ability to detect skin sensitisers without the 
necessity for using a radiolabel for DNA eliminates the potential for occu ­
pational exposure to radioactivity and waste disposal issues. This in turn may allow for the increased use of mice to detect skin sensitisers, which could 
further reduce the use of guinea pigs to test for skin sensitisation potential 
(i.e. B.6; OECD Test Guideline 406) (14).
 
DEFINITIONS 
3. Definitions used are provided in Appendix 1.  
INITIAL CONSIDERATIONS AND LIMITATIONS 
4. The LLNA: DA is a modified LLNA method for identifying potential skin 
sensitising chemicals, with specific limitations. This does not necessarily imply that in all instances the LLNA: DA should be used in place of the 
LLNA or guinea pig tests (i.e. B.6; OECD Test Guideline 406) (14), but 
rather that the assay is of equal merit and may be employed as an alternative in which positive and negative results generally no longer require further confirmation (10) (11). The testing laboratory should consider all available 
information on the test substance prior to conducting the study. Such 
information will include the identity and chemical structure of the test
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 731
 
substance; its physicochemical properties; the results of any other  in vitro  or 
in vivo  toxicity tests on the test substance; and toxicological data on struc ­
turally related chemicals. This information should be considered in order to determine whether the LLNA: DA is appropriate for the test substance (given the incompatibility of limited types of chemicals with the LLNA: DA (see 
paragraph 5) and to aid in dose selection. 
5. The LLNA: DA is an  in vivo  method and, as a consequence, will not 
eliminate the use of animals in the assessment of allergic contact sensitising 
activity. It has, however, the potential to reduce animal use for this purpose when compared to the guinea pig tests (B.6; OECD Test Guideline 406) (14). Moreover, the LLNA: DA offers a substantial refinement (less pain and 
distress) of the way in which animals are used for allergic contact sensiti ­
sation testing, since unlike the B.6 and OECD Test Guideline 406, the LLNA: DA does not require that challenge-induced dermal hypersensitivity reactions be elicited. Despite the advantages of the LLNA: DA over B.6 and 
OECD Test Guideline 406 (14), there are certain limitations that may 
necessitate the use of B.6 or OECD Test Guideline 406 (e.g. the testing of certain metals, false positive findings with certain skin irritants (such as some surfactant-type substances) (6) (1 and Chapter B.42 in this Annex), 
solubility of the test substance). In addition, chemical classes or substances 
containing functional groups shown to act as potential confounders (16) may necessitate the use of guinea pig tests (i.e. B.6; OECD Test Guideline 406 (14)). Limitations that have been identified for the LLNA (1 and Chapter 
B.42 in this Annex) have been recommended to apply also to the LLNA: DA 
(10). Additionally, the use of the LLNA: DA might not be appropriate for testing substances that affect ATP levels (e.g. substances that function as ATP inhibitors) or those that affect the accurate measurement of intracellular 
ATP (e.g. presence of ATP degrading enzymes, presence of extracellular 
ATP in the lymph node). Other than such identified limitations, the LLNA: DA should be applicable for testing any substances unless there are properties associated with these substances that may interfere with the 
accuracy of the LLNA: DA. In addition, consideration should be given to the 
possibility of borderline positive results when Stimulation Index (SI) values between 1,8 and 2,5 are obtained (see paragraphs 31-32). This is based on the validation database of 44 substances using an SI ≥ 1,8 (see paragraph 6) 
for which the LLNA: DA correctly identified all 32 LLNA sensitisers, but 
incorrectly identified three of 12 LLNA non-sensitisers with SI values between 1,8 and 2,5 (i.e. borderline positive) (10). However, as the same dataset was used for setting the SI-values and calculating the predictive 
properties of the test, the stated results may be an over-estimation of the 
real predictive properties.
 
PRINCIPLE OF THE TEST METHOD 
6. The basic principle underlying the LLNA: DA is that sensitisers induce 
proliferation of lymphocytes in the lymph nodes draining the site of test substance application. This proliferation is proportional to the dose and to 
the potency of the applied allergen and provides a simple means of obtaining 
a quantitative measurement of sensitisation. Proliferation is measured by comparing the mean proliferation in each test group to the mean prolif ­
eration in the vehicle treated control (VC) group. The ratio of the mean 
proliferation in each treated group to that in the concurrent VC group, 
termed the SI, is determined, and should be ≥ 1,8 before further
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 732
 
evaluation of the test substance as a potential skin sensitiser is warranted. 
The procedures described here are based on the use of measuring ATP 
content by bioluminescence (known to correlate with living cell number) (17) to indicate an increased number of proliferating cells in the draining auricular lymph nodes (18) (19). The bioluminescent method utilises the 
luciferase enzyme to catalyse the formation of light from ATP and 
luciferin according to the following reaction:  
ATP + Luciferin  + O
 2
 Oxyluciferin  + AMP  + PP  i + CO  2 + Light  
The emitted light intensity is linearly related to the ATP concentration and 
is measured using a luminometer. The luciferin-luciferase assay is a sensitive method for ATP quantitation used in a wide variety of appli ­
cations (20).
 
DESCRIPTION OF THE ASSAY  
Selection of animal species  
7. The mouse is the species of choice for this test. Validation studies for the 
LLNA: DA were conducted exclusively with the CBA/J strain, which is therefore considered the preferred strain (12) (13). Young adult female mice, which are nulliparous and non-pregnant, are used. At the start of the 
study, animals should be between 8-12 weeks old, and the weight variation 
of the animals should be minimal and not exceed 20 % of the mean weight. Alternatively, other strains and males may be used when sufficient data are generated to demonstrate that significant strain and/or gender-specific 
differences in the LLNA: DA response do not exist.
 
Housing and feeding conditions  
8. Mice should be group-housed (21), unless adequate scientific rationale for 
housing mice individually is provided. The temperature of the experimental animal room should be 22 ± 3 °C. Although the relative humidity should be at least 30 % and preferably not exceed 70 %, other than during room 
cleaning, the aim should be 50-60 %. Lighting should be artificial, the 
sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water.
 
Preparation of animals  
9. The animals are randomly selected, marked to permit individual identifi ­
cation (but not by any form of ear marking), and kept in their cages for 
at least five days prior to the start of dosing to allow for acclimatisation to 
the laboratory conditions. Prior to the start of treatment all animals are examined to ensure that they have no observable skin lesions.
 
Preparation of dosing solutions  
10. Solid chemicals should be dissolved or suspended in solvents/vehicles and 
diluted, if appropriate, prior to application to an ear of the mice. Liquid 
chemicals may be applied neat or diluted prior to dosing. Insoluble 
chemicals, such as those generally seen in medical devices, should be subjected to an exaggerated extraction in an appropriate solvent to reveal all extractable constituents for testing prior to application to an ear of the 
mice. Test substances should be prepared daily unless stability data demon ­
strate the acceptability of storage.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 733
 
Reliability check  
11. Positive control chemicals (PC) are used to demonstrate appropriate 
performance of the assay by responding with adequate and reproducible 
sensitivity to a sensitising test substance for which the magnitude of the response is well characterised. Inclusion of a concurrent PC is recommended because it demonstrates competency of the laboratory to successfully conduct 
each assay and allows for an assessment of intra- and inter-laboratory repro ­
ducibility and comparability. Some regulatory authorities also require a PC for each study and therefore users are encouraged to consult the relevant authorities prior to conducting the LLNA: DA. Accordingly, the routine use 
of a concurrent PC is encouraged to avoid the need for additional animal 
testing to meet such requirements that might arise from the use of a periodic PC (see paragraph 12). The PC should produce a positive LLNA: DA response at an exposure level expected to give an increase in the SI ≥ 1,8 
over the negative control (NC) group. The PC dose should be chosen such 
that it does not cause excessive skin irritation or systemic toxicity and the induction is reproducible but not excessive (e.g. SI > 10 would be considered excessive). Preferred PC are 25 % hexyl cinnamic aldehyde (Chemical 
Abstracts Service (CAS) number 101-86-0) and 25 % eugenol (CAS 
number 97-53-0) in acetone: olive oil (4:1, v/v). There may be circumstances in which, given adequate justification, other PC, meeting the above criteria, may be used. 
12. While inclusion of a concurrent PC group is recommended, there may be 
situations in which periodic testing (i.e. at intervals ≤ 6 months) of the PC 
may be adequate for laboratories that conduct the LLNA: DA regularly (i.e. 
conduct the LLNA: DA at a frequency of no less than once per month) and have an established historical PC database that demonstrates the laboratory’s ability to obtain reproducible and accurate results with PCs. Adequate profi ­
ciency with the LLNA: DA can be successfully demonstrated by generating 
consistent positive results with the PC in at least 10 independent tests conducted within a reasonable period of time (i.e. less than one year). 
13. A concurrent PC group should always be included when there is a 
procedural change to the LLNA: DA (e.g. change in trained personnel, change in test method materials and/or reagents, change in test method equipment, change in source of test animals), and such changes should be 
documented in laboratory reports. Consideration should be given to the 
impact of these changes on the adequacy of the previously established historical database in determining the necessity for establishing a new historical database to document consistency in the PC results. 
14. Investigators should be aware that the decision to conduct a PC study on a 
periodic basis instead of concurrently has ramifications on the adequacy and acceptability of negative study results generated without a concurrent PC 
during the interval between each periodic PC study. For example, if a 
false negative result is obtained in the periodic PC study, negative test substance results obtained in the interval between the last acceptable periodic PC study and the unacceptable periodic PC study may be ques ­
tioned. Implications of these outcomes should be carefully considered when 
determining whether to include concurrent PCs or to only conduct periodic PCs. Consideration should also be given to using fewer animals in the concurrent PC group when this is scientifically justified and if the laboratory 
demonstrates, based on laboratory-specific historical data, that fewer mice 
can be used (22).
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 734
 
15. Although the PC should be tested in the vehicle that is known to elicit a 
consistent response ( e.g. acetone: olive oil; 4:1, v/v), there may be certain 
regulatory situations in which testing in a non-standard vehicle (clinically/ chemically relevant formulation) will also be necessary (23). If the concurrent PC is tested in a different vehicle than the test substance, then 
a separate VC for the concurrent PC should be included. 
16. In instances where substances of a specific chemical class or range of 
responses are being evaluated, benchmark substances may also be useful to demonstrate that the test method is functioning properly for detecting the skin sensitisation potential of these types of substances. Appropriate 
benchmark substances should have the following properties: 
— structural and functional similarity to the class of the test substance being 
tested; 
— known physical chemical characteristics; — supporting data from the LLNA: DA; — supporting data from other animal models and/or from humans.
 
TEST PROCEDURE  
Number of animals and dose levels  
17. A minimum of four animals is used per dose group, with a minimum of 
three concentrations of the test substance, plus a concurrent NC group treated 
only with the vehicle for the test substance, and a PC (concurrent or recent, based on laboratory policy in considering paragraphs 11-15). Testing multiple doses of the PC should be considered, especially when testing the 
PC on an intermittent basis. Except for absence of treatment with the test 
substance, animals in the control groups should be handled and treated in a manner identical to that of animals in the treatment groups. 
18. Dose and vehicle selection should be based on the recommendations given in 
references (2) and (24). Consecutive doses are normally selected from an 
appropriate concentration series such as 100 %, 50 %, 25 %, 10 %, 5 %, 
2,5 %, 1 %, 0,5 %, etc. Adequate scientific rationale should accompany the selection of the concentration series used. All existing toxicological information (e.g. acute toxicity and dermal irritation) and structural and 
physicochemical information on the test substance of interest (and/or struc ­
turally related substances) should be considered, where available, in selecting the three consecutive concentrations so that the highest concentration maximises exposure while avoiding systemic toxicity and/or excessive 
local skin irritation (24) (25). In the absence of such information, an 
initial pre-screen test may be necessary (see paragraphs 21-24). 
19. The vehicle should not interfere with or bias the test result and should be 
selected on the basis of maximising the solubility in order to obtain the highest concentration achievable while producing a solution/suspension 
suitable for application of the test substance. Recommended vehicles are 
acetone: olive oil (4:1 v/v), N,N-dimethylformamide, methyl ethyl ketone, propylene glycol, and dimethyl sulphoxide (6) but others may be used if sufficient scientific rationale is provided. In certain situations it may be 
necessary to use a clinically relevant solvent or the commercial formulation 
in which the test substance is marketed as an additional control. Particular care should be taken to ensure that hydrophilic substances are incorporated into a vehicle system, which wets the skin and does not immediately run off, 
by incorporation of appropriate solubilisers (e.g. 1 % Pluronic® L92). Thus, 
wholly aqueous vehicles are to be avoided.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 735
 
20. The processing of lymph nodes from individual mice allows for the 
assessment of inter-animal variability and a statistical comparison of the 
difference between test substance and VC group measurements (see paragraph 33). In addition, evaluating the possibility of reducing the number of mice in the PC group is only feasible when individual animal 
data are collected (22). Further, some regulatory authorities require the 
collection of individual animal data. Regular collection of individual animal data provides an animal welfare advantage by avoiding duplicate testing that would be necessary if the test substance results originally 
collected in one manner (e.g. via pooled animal data) were to be considered 
later by regulatory authorities with other requirements (e.g. individual animal data).
 
Pre-screen test  
21. In the absence of information to determine the highest dose to be tested (see 
paragraph 18), a pre-screen test should be performed in order to define the appropriate dose level to test in the LLNA: DA. The purpose of the pre-screen test is to provide guidance for selecting the maximum dose 
level to use in the main LLNA: DA study, where information on the concen ­
tration that induces systemic toxicity (see paragraph 24) and/or excessive local skin irritation (see paragraph 23) is not available. The maximum dose level tested should be 100 % of the test substance for liquids or the 
maximum possible concentration for solids or suspensions. 
22. The pre-screen test is conducted under conditions identical to the main 
LLNA: DA study, except there is no assessment of lymph node proliferation 
and fewer animals per dose group can be used. One or two animals per dose 
group are suggested. All mice will be observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights are recorded pre-test and prior to termination (Day 8). Both ears of each mouse 
are observed for erythema and scored using Table 1 (25). Ear thickness 
measurements are taken using a thickness gauge ( e.g. digital micrometer 
or Peacock Dial thickness gauge) on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose), Day 7 (24 hours prior to termination) and Day 
8. Additionally on Day 8, ear thickness could be determined by ear punch 
weight determinations, which should be performed after the animals are humanely killed. Excessive local irritation is indicated by an erythema 
score ≥ 3 and/or ear thickness of ≥ 25 % on any day of measurement (26) 
(27). The highest dose selected for the main LLNA: DA study will be the next lower dose in the pre-screen concentration series (see paragraph 18) that does not induce systemic toxicity and/or excessive local skin irritation
 
Table 1  
Erythema Scores  
Observation Score  
No erythema 0 
Very slight erythema (barely perceptible) 1 Well-defined erythema 2
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 736
 
Observation Score  
Moderate to severe erythema 3 
Severe erythema (beet redness) to eschar formation preventing grading of 
erythema 4 
23. In addition to a 25 % increase in ear thickness (26) (27), a statistically significant 
increase in ear thickness in the treated mice compared to control mice has also been used to identify irritants in the LLNA (28) (29) (30) (31) (32) (33) (34). However, while statistically significant increases can occur when ear thickness is 
less than 25 % they have not been associated specifically with excessive irritation 
(30) (31) (32) (33) (34). 
24. The following clinical observations may indicate systemic toxicity (35) when 
used as part of an integrated assessment and therefore may indicate the 
maximum dose level to use in the main LLNA: DA: changes in nervous 
system function ( e.g. pilo-erection, ataxia, tremors, and convulsions); 
changes in behaviour ( e.g. aggressiveness, change in grooming activity, 
marked change in activity level); changes in respiratory patterns ( i.e. 
changes in frequency and intensity of breathing such as dyspnea, gasping, 
and rales), and changes in food and water consumption. In addition, signs of lethargy and/or unresponsiveness and any clinical signs of more than slight or momentary pain and distress, or a > 5 % reduction in body weight from 
Day 1 to Day 8 and mortality, should be considered in the evaluation. 
Moribund animals or animals showing signs of severe pain and distress should be humanely killed (36).
 
Main study experimental schedule  
25. The experimental schedule of the assay is as follows: 
— Day 1:  Individually identify and record the weight of each animal and 
any clinical observation. Apply 1 % sodium lauryl sulfate (SLS) aqueous solution to the dorsum of each ear by using a brush dipped in the SLS 
solution to cover the entire dorsum of each ear with four to five strokes. 
One hour after the SLS treatment, apply 25 μL of the appropriate 
dilution of the test substance, the vehicle alone, or the PC (concurrent or recent, based on laboratory policy in considering paragraphs 11-15), 
to the dorsum of each ear. 
— Days 2, 3 and 7:  Repeat the 1 % SLS aqueous solution pre-treatment 
and test substance application procedure carried out on Day 1. 
— Days 4, 5, and 6:  No treatment. 
— Day 8:  Record the weight of each animal and any clinical observation. 
Approximately 24 to 30 hours after the start of application on Day 7, humanely kill the animals. Excise the draining auricular lymph nodes from each mouse ear and process separately in phosphate buffered 
saline (PBS) for each animal. Details and diagrams of the lymph node 
identification and dissection can be found in reference (22). To further monitor the local skin response in the main study, additional parameters such as scoring of ear erythema or ear thickness measurements (obtained 
either by using a thickness gauge, or ear punch weight determinations at 
necropsy) may be included in the study protocol.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 737
 
Preparation of cell suspensions  
26. From each mouse, a single-cell suspension of lymph node cells (LNC) 
excised bilaterally is prepared by sandwiching the lymph nodes between 
two glass slides and applying light pressure to crush the nodes. After confirming that the tissue has spread out thinly pull the two slides apart. Suspend the tissue on both slides in PBS by holding each slide at an angle 
over the Petri dish and rinsing with PBS while concurrently scraping the 
tissue off of the slide with a cell scraper. Further, the lymph nodes in NC animals are small, so careful operation is important to avoid any artificial effects on SI values. A total volume of 1 mL PBS should be used for rinsing 
both slides. The LNC suspension in the Petri dish should be homogenised 
lightly with the cell scraper. A 20 μL aliquot of the LNC suspension is then 
collected with a micropipette, taking care not to take up the membrane that is visible to the eye, and subsequently mixed with 1,98 mL of PBS to yield a 2 
mL sample. A second 2 mL sample is then prepared using the same 
procedure so that two samples are prepared for each animal.
 
Determination of cellular proliferation (measurement of ATP content of 
lymphocytes)  
27. Increases in ATP content in the lymph nodes are measured by the luciferin/ 
luciferase method using an ATP measurement kit, which measures biolumi ­
nescence in Relative Luminescence Units (RLU). The assay time from time of animal sacrifice to measurement of ATP content for each individual 
animal should be kept uniform, within approximately 30 minutes, because 
the ATP content is considered to gradually decrease with time after animal sacrifice (12) Thus, the series of procedures from excision of auricular lymph nodes to ATP measurement should be completed within 20 minutes by the 
pre-determined time schedule that is the same for each animal. ATP lumi ­
nescence should be measured in each 2 mL sample so that a total of two ATP measurements are collected for each animal. The mean ATP lumi ­
nescence is then determined and used in subsequent calculations (see 
paragraph 30).
 
OBSERVATIONS  
Clinical observations  
28. Each mouse should be carefully observed at least once daily for any clinical 
signs, either of local irritation at the application site or of systemic toxicity. 
All observations are systematically recorded with records being maintained 
for each mouse. Monitoring plans should include criteria to promptly identify those mice exhibiting systemic toxicity, excessive local skin irritation, or corrosion of skin for euthanasia (36).
 
Body weights  
29. As stated in paragraph 25, individual animal body weights should be 
measured at the start of the test and at the scheduled humane kill.  
CALCULATION OF RESULTS 
30. Results for each treatment group are expressed as the mean SI. The SI is 
derived by dividing the mean RLU/mouse within each test substance group 
and the PC group by the mean RLU/mouse for the solvent/VC group. The 
average SI for the VCs is then one.  ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 738
 
31. The decision process regards a result as positive when SI ≥ 1,8 (10) 
However, the strength of the dose-response relationship, the statistical 
significance and the consistency of the solvent/vehicle and PC responses may also be used when determining whether a borderline result ( i.e. SI 
value between 1,8 and 2,5) is declared positive (2) (3) (37). 
32. For a borderline positive response between an SI of 1,8 and 2,5, users may 
want to consider additional information such as dose-response relationship, evidence of systemic toxicity or excessive irritation, and where appropriate, statistical significance together with SI values to confirm that such results are 
positives (10). Consideration should also be given to various properties of 
the test substance, including whether it has a structural relationship to known skin sensitisers, whether it causes excessive skin irritation in the mouse, and the nature of the dose-response relationship observed. These and other 
considerations are discussed in detail elsewhere (4) 
33. Collecting data at the level of the individual mouse will enable a statistical 
analysis for presence and degree of dose-response relationship in the data. Any statistical assessment could include an evaluation of the dose-response 
relationship as well as suitably adjusted comparisons of test groups ( e.g. 
pair-wise dosed group versus concurrent solvent/vehicle control compari ­
sons). Statistical analyses may include,  e.g.  linear regression or William’s 
test to assess dose-response trends, and Dunnett’s test for pair-wise 
comparisons. In choosing an appropriate method of statistical analysis, the 
investigator should maintain an awareness of possible inequalities of variances and other related problems that may necessitate a data trans ­
formation or a non-parametric statistical analysis. In any case, the inves ­
tigator may need to carry out SI calculations and statistical analyses with and without certain data points (sometimes called ‘outliers’).
 
DATA AND REPORTING  
Data  
34. Data should be summarised in tabular form showing the individual animal 
RLU values, the group mean RLU/animal, its associated error term ( e.g. SD, 
SEM), and the mean SI for each dose group compared against the concurrent solvent/vehicle control group.
 
Test report  
35. The test report should contain the following information: 
Test and control chemicals: 
— identification data ( e.g. CAS number and EC number, if available; 
source; purity; known impurities; lot number); 
— physical nature and physicochemical properties ( e.g. volatility, stability, 
solubility); 
— if mixture, composition and relative percentages of components; 
Solvent/vehicle: — identification data (purity; concentration, where appropriate; volume 
used); 
— justification for choice of vehicle;
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 739
 
Test animals: 
— source of CBA mice; — microbiological status of the animals, when known; — number and age of animals; — source of animals, housing conditions, diet, etc.; Test conditions: — the source, lot number and manufacturer’s quality assurance/quality 
control data for the ATP kit; 
— details of test substance preparation and application; — justification for dose selection (including results from pre-screen test, if 
conducted); 
— vehicle and test substance concentrations used, and total amount of test 
substance applied; 
— details of food and water quality (including diet type/source, water 
source); 
— details of treatment and sampling schedules; — methods for measurement of toxicity; 
— criteria for considering studies as positive or negative; 
— details of any protocol deviations and an explanation on how the 
deviation affects the study design and results; 
Reliability check: — a summary of results of latest reliability check, including information on 
test substance, concentration and vehicle used; 
— concurrent and/or historical PC and concurrent negative (solvent/vehicle) 
control data for testing laboratory; 
— if a concurrent PC was not included, the date and laboratory report for 
the most recent periodic PC and a report detailing the historical PC data for the laboratory justifying the basis for not conducting a concurrent PC; 
Results: — individual weights of mice at start of dosing and at scheduled kill; as 
well as mean and associated error term ( e.g. SD, SEM) for each 
treatment group; 
— time course of onset and signs of toxicity, including dermal irritation at 
site of administration, if any, for each animal; 
— time of animal termination and time of ATP measurement for each 
animal; 
— a table of individual mouse RLU values and SI values for each dose 
treatment group; 
— mean and associated error term ( e.g. SD, SEM) for RLU/mouse for each 
treatment group and the results of outlier analysis for each treatment 
group;
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 740
 
— calculated SI and an appropriate measure of variability that takes into 
account the inter-animal variability in both the test substance and control 
groups; 
— dose response relationship; — statistical analyses, where appropriate; Discussion of results: — a brief commentary on the results, the dose-response analysis, and stat ­
istical analyses, where appropriate, with a conclusion as to whether the test substance should be considered a skin sensitiser.
 
LITERATURE 
(1) OECD (2010),  Skin Sensitisation: Local Lymph Node Assay , Test Guideline 
No 429, Guidelines for the Testing of Chemicals, OECD, Paris. Available 
at: [http://www.oecd.org/env/testguidelines] 
(2) Chamberlain, M. and Basketter, D.A. (1996), The local lymph node assay: 
status of validation.  Food Chem, Toxicol. , 34, 999-1002. 
(3) Basketter, D.A., Gerberick, G.F., Kimber, I. and Loveless, S.E. (1996), The 
local lymph node assay: A viable alternative to currently accepted skin sensitisation tests.  Food Chem, Toxicol. , 34, 985-997. 
(4) Basketter, D.A., Gerberick, G.F. and Kimber, I. (1998), Strategies for iden ­
tifying false positive responses in predictive sensitisation tests.  Food Chem. 
Toxicol. , 36, 327-333. 
(5) Van Och, F.M.M., Slob, W., De Jong, W.H., Vandebriel, R.J. and Van 
Loveren, H. (2000), A quantitative method for assessing the sensitising potency of low molecular weight chemicals using a local lymph node 
assay: employment of a regression method that includes determination of 
uncertainty margins.  Toxicol. , 146, 49-59. 
(6) ICCVAM (1999), The murine local lymph node Assay: A test method for 
assessing the allergic contact dermatitis potential of chemicals/compounds: The results of an independent peer review evaluation coordinated by the 
Interagency Coordinating Committee on the Validation of Alternative 
Methods (ICCVAM) and the National Toxicology Program Center for the Evaluation of Alternative Toxicological Methods (NICETAM). NIH Publication No: 99-4494. Research Triangle Park, N.C. Available at: [http:// 
iccvam.niehs.nih.gov/docs/immunotox_docs/llna/llnarep.pdf] 
(7) Dean, J.H., Twerdok, L.E., Tice, R.R., Sailstad, D.M., Hattan, D.G., Stokes, 
W.S. (2001), ICCVAM evaluation of the murine local lymph node assay: II. Conclusions and recommendations of an independent scientific peer review panel.  Reg. Toxicol. Pharmacol.  34, 258-273. 
(8) Haneke, K.E., Tice, R.R., Carson, B.L., Margolin, B.H., Stokes, W.S. 
(2001), ICCVAM evaluation of the murine local lymph node assay: III. 
Data analyses completed by the national toxicology program interagency 
center for the evaluation of alternative toxicological methods.  Reg. Toxicol. 
Pharmacol. 34 , 274-286. 
(9) Sailstad, D.M., Hattan, D., Hill, R.N., Stokes, W.S. (2001), ICCVAM 
evaluation of the murine local lymph node assay: I. The ICCVAM 
review process.  Reg. Toxicol. Pharmacol.  34, 249-257. 
(10) ICCVAM (2010), ICCVAM Test Method Evaluation Report. Nonradio ­
active local lymph node assay: modified by Daicel Chemical Industries, Ltd, based on ATP content test method protocol (LLNA: DA). NIH Publication No 10-7551A/B. Research Triangle Park, NC: National 
Institute of Environmental Health Sciences. Available at: [http://icc ­
vam.niehs.nih.gov/methods/immunotox/llna-DA/TMER.htm]
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 741
 
(11) ICCVAM (2009), Independent Scientific Peer Review Panel Report: 
Updated validation status of new versions and applications of the murine 
local lymph node assay: a test method for assessing the allergic contact dermatitis potential of chemicals and products. Research Triangle Park, NC: National Institute of Environmental Health Sciences. Available at: [http:// 
iccvam.niehs.nih.gov/docs/immunotox_docs/LLNAPRPRept2009.pdf]. 
(12) Idehara, K., Yamagishi, G., Yamashita, K. and Ito, M. (2008), Character ­
ization and evaluation of a modified local lymph node assay using ATP content as a non-radio isotopic endpoint.  J. Pharmacol. Toxicol. Meth. , 58, 
1-10. 
(13) Omori, T., Idehara, K., Kojima, H., Sozu, T., Arima, K., Goto, H., Hanada, 
T., Ikarashi, Y., Inoda, T., Kanazawa, Y., Kosaka, T., Maki, E., Morimoto, 
T., Shinoda, S., Shinoda, N., Takeyoshi, M., Tanaka, M., Uratani, M., Usami, M., Yamanaka, A., Yoneda, T., Yoshimura, I. and Yuasa, A. (2008), Interlaboratory validation of the modified murine local lymph 
node assay based on adenosine triphosphate measurement.  J. Pharmacol. 
Toxicol. Meth. , 58, 11-26. 
(14) OECD (1992),  Skin Sensitisation , Test Guideline No 406, Guidelines for 
Testing of Chemicals, OECD, Paris. Available at: [http://www.oecd.org/env/ 
testguidelines] 
(15) Kreiling, R., Hollnagel, H.M., Hareng, L., Eigler, L., Lee, M.S., Griem, P., 
Dreessen, B., Kleber, M., Albrecht, A., Garcia, C. and Wendel, A. (2008), Comparison of the skin sensitising potential of unsaturated compounds as assessed by the murine local lymph node assay (LLNA) and the guinea pig 
maximization test (GPMT).  Food Chem. Toxicol. , 46, 1896-1904. 
(16) Basketter, D., Ball, N., Cagen, S., Carrillo, J.C., Certa, H., Eigler, D., 
Garcia, C., Esch, H., Graham, C., Haux, C., Kreiling, R. and Mehling, A. (2009), Application of a weight of evidence approach to assessing 
discordant sensitisation datasets: implications for REACH.  Reg. Toxicol. 
Pharmacol. , 55, 90-96. 
(17) Crouch, S.P., Kozlowski, R., Slater, K.J. and Fletcher J. (1993), The use of 
ATP bioluminescence as a measure of cell proliferation and cytotoxicity.  J. 
Immunol. Meth. , 160, 81-88. 
(18) Ishizaka, A., Tono-oka, T. and Matsumoto, S. (1984), Evaluation of the 
proliferative response of lymphocytes by measurement of intracellular ATP.  
J. Immunol. Meth. , 72, 127-132. 
(19) Dexter, S.J., Cámara, M., Davies, M. and Shakesheff, K.M. (2003), Devel ­
opment of a bioluminescent ATP assay to quantify mammalian and 
bacterial cell number from a mixed population.  Biomat. , 24, 27-34. 
(20) Lundin A. (2000), Use of firefly luciferase in ATP-related assays of 
biomass, enzymes, and metabolites.  Meth. Enzymol. , 305, 346-370. 
(21) ILAR (1996), Institute of Laboratory Animal Research (ILAR) Guide for 
the Care and Use of Laboratory Animals. 7th ed. Washington, DC: National 
Academies Press. 
(22) ICCVAM (2009), Recommended Performance Standards: Murine Local 
Lymph Node Assay, NIH Publication Number 09-7357, Research Triangle Park, NC: National Institute of Environmental Health Science. Available at: [http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna- 
ps/LLNAPerfStds.pdf] 
(23) McGarry, H.F. (2007), The murine local lymph node assay: regulatory and 
potency considerations under REACH.  Toxicol. , 238, 71-89. 
(24) Kimber, I., Dearman, R.J., Scholes E.W. and Basketter, D.A. (1994), The 
local lymph node assay: developments and applications.  Toxicol. , 93, 13-31. 
(25) OECD (2002),  Acute Dermal Irritation/Corrosion , Test Guideline No 404, 
Guidelines for Testing of Chemicals, OECD, Paris. Available at: [http:// 
www.oecd.org/env/testguidelines]
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 742
 
(26) Reeder, M.K., Broomhead, Y.L., DiDonato, L. and DeGeorge, G.L. (2007), 
Use of an enhanced local lymph node assay to correctly classify irritants 
and false positive substances.  Toxicologist , 96, 235. 
(27) ICCVAM (2009), Nonradioactive Murine Local Lymph Node Assay: Flow 
Cytometry Test Method Protocol (LLNA: BrdU-FC) Revised Draft Back ­
ground Review Document. Research Triangle Park, NC: National Institute 
of Environmental Health Sciences. Available at: [http://icc ­
vam.niehs.nih.gov/methods/immunotox/fcLLNA/BRDcomplete.pdf]. 
(28) Hayes, B.B., Gerber, P.C., Griffey, S.S. and Meade, B.J. (1998), Contact 
hypersensitivity to dicyclohexylcarbodiimide and diisopropylcarbodiimide in female B6C3F1 mice.  Drug. Chem. Toxicol. , 21, 195-206. 
(29) Homey, B., von Schilling, C., Blumel, J., Schuppe, H.C., Ruzicka, T., Ahr, 
H.J., Lehmann, P. and Vohr, V.W. (1998), An integrated model for the differentiation of chemical-induced allergic and irritant skin reactions.  
Toxicol. Appl. Pharmacol. , 153, 83-94. 
(30) Woolhiser, M.R., Hayes, B.B. and Meade, B.J. (1998), A combined murine 
local lymph node and irritancy assay to predict sensitisation and irritancy 
potential of chemicals.  Toxicol. Meth. , 8, 245-256. 
(31) Hayes, B.B. and Meade, B.J. (1999), Contact sensitivity to selected acrylate 
compounds in B6C3F1 mice: relative potency, cross reactivity, and 
comparison of test methods.  Drug Chem. Toxicol. , 22, 491-506. 
(32) Ehling, G., Hecht, M., Heusener, A., Huesler, J., Gamer, A.O., van 
Loveren, H., Maurer, T., Riecke, K., Ullmann, L., Ulrich, P., Vandebriel, R. and Vohr, H.W. (2005), A European inter-laboratory validation of alter ­
native endpoints of the murine local lymph node assay: first round.  Toxicol. , 
212, 60-68. 
(33) Vohr, H.W. and Ahr, H.J. (2005), The local lymph node assay being too 
sensitive?  Arch. Toxicol. , 79, 721-728. 
(34) Patterson, R.M., Noga, E. and Germolec, D. (2007), Lack of evidence for 
contact sensitisation by Pfiesteria extract.  Environ. Health Perspect. , 115, 
1023-1028. 
(35) ICCVAM (2009), Report on the ICCVAM-NICEATM/ECVAM/JaCVAM 
Scientific Workshop on Acute Chemical Safety Testing: Advancing  In Vitro  
Approaches and Humane Endpoints for Systemic Toxicity Evaluations. Research Triangle Park, NC: National Institute of Environmental Health 
Sciences. Available at: [http://iccvam.niehs.nih.gov/methods/acutetox/ 
Tox_workshop.htm] 
(36) OECD (2000),  Guidance Document on the Recognition, Assessment and 
Use of Clinical Signs as Humane Endpoints for Experimental Animals 
Used in Safety Evaluation , Environmental Health and Safety Monograph 
Series on Testing and Assessment No 19, ENV/JM/MONO(2000)7, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines] 
(37) Kimber, I., Hilton, J., Dearman, R.J., Gerberick, G.F., Ryan, C.A., 
Basketter, D.A., Lea, L., House, R.V., Ladies, G.S., Loveless, S.E. and 
Hastings, K.L. (1998), Assessment of the skin sensitisation potential of 
topical medicaments using the local lymph node assay: An interlaboratory exercise.  J. Toxicol. Environ. Health , 53 563-79. 
(38) OECD (2005),  Guidance Document on the Validation and International 
Acceptance of New or Updated Test Methods for Hazard Assessment , 
Environment, Health and Safety Monograph Series on Testing and Assessment No 34, ENV/JM/MONO (2005)14, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 743
 
Appendix 1  
DEFINITIONS  
Accuracy:  The closeness of agreement between test method results and accepted 
reference values. It is a measure of test method performance and one aspect of 
relevance. The term is often used interchangeably with ‘concordance’ to mean 
the proportion of correct outcomes of a test method (38).  
Benchmark substance:  A sensitising or non-sensitising substance used as a 
standard for comparison to a test substance. A benchmark substance should have the following properties; (i) a consistent and reliable source(s); (ii) structural 
and functional similarity to the class of substances being tested; (iii) known 
physicochemical characteristics; (iv) supporting data on known effects, and (v) known potency in the range of the desired response.  
False negative:  A substance incorrectly identified as negative or non-active by a 
test method, when in fact it is positive or active.  
False positive:  A substance incorrectly identified as positive or active by a test, 
when in fact it is negative or non-active.  
Hazard:  The potential for an adverse health or ecological effect. The adverse 
effect is manifested only if there is an exposure of sufficient level.  
Inter-laboratory reproducibility:  A measure of the extent to which different 
qualified laboratories, using the same protocol and testing the same test 
substances, can produce qualitatively and quantitatively similar results. Inter-laboratory reproducibility is determined during the pre-validation and vali ­
dation processes, and indicates the extent to which a test can be successfully 
transferred between laboratories, also referred to as between-laboratory reproduci ­
bility (38).  
Intra-laboratory reproducibility:  A determination of the extent that qualified 
people within the same laboratory can successfully replicate results using a specific protocol at different times. Also referred to as within-laboratory repro ­
ducibility (38).  
Outlier:  An outlier is an observation that is markedly different from other values 
in a random sample from a population.  
Quality assurance:  A management process by which adherence to laboratory 
testing standards, requirements, and record keeping procedures, and the 
accuracy of data transfer, are assessed by individuals who are independent 
from those performing the testing.  
Reliability:  Measures of the extent that a test method can be performed repro ­
ducibly within and between laboratories over time, when performed using the 
same protocol. It is assessed by calculating intra- and inter-laboratory reproduci ­
bility (38).  
Skin sensitisation:  An immunological process that results when a susceptible 
individual is exposed topically to an inducing chemical allergen, which provokes a cutaneous immune response that can lead to the development of 
contact sensitisation.  
Stimulation Index (SI):  A value calculated to assess the skin sensitisation 
potential of a test substance that is the ratio of the proliferation in treated groups to that in the concurrent vehicle control group.  
Test substance (also referred to as test chemical):  Any substance or mixture 
tested using this TM.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 744
 
B.51.  SKIN SENSITISATION: LOCAL LYMPH NODE ASSAY: 
BrdU-ELISA  
INTRODUCTION 
1. OECD Guidelines for the Testing of Chemicals and EU Test Methods are 
periodically reviewed in light of scientific progress, changing regulatory needs, and animal welfare considerations. The first Test Method (TM) 
(B.42) for the determination of skin sensitisation in the mouse, the Local 
Lymph Node Assay (LLNA; OECD Test Guideline 429) has been revised (1 and Chapter B.42 in this Annex). The details of the validation of the LLNA and a review of the associated work have been published (2) (3) (4) 
(5) (6) (7) (8) (9). In the LLNA, radioisotopic thymidine or iodine is used to 
measure lymphocyte proliferation and therefore the assay has limited use where the acquisition, use, or disposal of radioactivity is problematic. The LLNA: BrdU-ELISA (Enzyme-Linked Immunosorbent Assay) is a non- 
radioactive modification to the LLNA TM, which utilises 
non-radiolabelled 5-bromo-2-deoxyuridine (BrdU) (Chemical Abstracts Service (CAS) No 59-14-3) in an ELISA-based test system to measure lymphocyte proliferation. The LLNA: BrdU-ELISA has been validated 
and reviewed and recommended by an international independent scientific 
peer review panel as considered useful for identifying skin sensitising and non-sensitising chemicals with certain limitations (10) (11) (12). This TM is designed for assessing skin sensitisation potential of chemicals (substances 
and mixtures) in animals. Chapter B.6 of this Annex and OECD Test 
Guideline 406 utilise guinea pig tests, notably the guinea pig maximisation test and the Buehler test (13). The LLNA (Chapter B.42 of this Annex; OECD Test Guideline 429) and the two non-radioactive modifications, 
LLNA: BrdU-ELISA (Chapter B.51 of this Annex; OECD Test Guideline 
442 B) and LLNA: DA (Chapter B.50 of this Annex; OECD Test Guideline 442 A), all provide an advantage over the guinea pig tests in B.6 and OECD Test Guideline 406 (13) in terms of reduction and refinement of 
animal use. 
2. Similar to the LLNA, the LLNA: BrdU-ELISA studies the induction phase 
of skin sensitisation and provides quantitative data suitable for dose-response assessment. Furthermore, an ability to detect skin sensitisers without the necessity for using a radiolabel for DNA eliminates the 
potential for occupational exposure to radioactivity and waste disposal 
issues. This in turn may allow for the increased use of mice to detect skin sensitisers, which could further reduce the use of guinea pigs to test 
for skin sensitisation potential ( i.e. B.6; OECD Test Guideline 406) (13).
 
DEFINITIONS 
3. Definitions used are provided in Appendix 1.  
INITIAL CONSIDERATIONS AND LIMITATIONS 
4. The LLNA: BrdU-ELISA is a modified LLNA method for identifying 
potential skin sensitising chemicals, with specific limitations. This does not necessarily imply that in all instances the LLNA: BrdU-ELISA 
should be used in place of the LLNA or guinea pig tests ( i.e. B.6; 
OECD Test Guideline 406) (13), but rather that the assay is of equal merit and may be employed as an alternative in which positive and negative results generally no longer require further confirmation (10) (11). 
The testing laboratory should consider all available information on the test 
substance prior to conducting the study. Such information will include the
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 745
 
identity and chemical structure of the test substance; its physicochemical 
properties; the results of any other  in vitro  or in vivo  toxicity tests on the 
test substance; and toxicological data on structurally related chemicals. This information should be considered in order to determine whether the LLNA: BrdU-ELISA is appropriate for the test substance (given the incompatibility 
of limited types of chemicals with the LLNA: BrdU-ELISA (see paragraph 
5)) and to aid in dose selection. 
5. The LLNA: BrdU-ELISA is an  in vivo  method and, as a consequence, will 
not eliminate the use of animals in the assessment of allergic contact sensi ­
tising activity. It has, however, the potential to reduce the animal use for 
this purpose when compared to the guinea pig tests (B.6; OECD Test 
Guideline 406) (13). Moreover, the LLNA: BrdU-ELISA offers a substantial refinement of the way in which animals are used for allergic contact sensitisation testing, since unlike the B.6 and OECD Test Guideline 
406, the LLNA: BrdU-ELISA does not require that challenge-induced 
dermal hypersensitivity reactions be elicited. Furthermore, the LLNA: BrdU-ELISA does not require the use of an adjuvant, as is the case for 
the guinea pig maximisation test (Chapter B.6 of this Annex, 13). Thus, the 
LLNA: BrdU-ELISA reduces animal distress. Despite the advantages of the LLNA: BrdU-ELISA over B.6 and OECD Test Guideline 406 (13), there are certain limitations that may necessitate the use of B.6 or OECD Test 
Guideline 406 ( e.g. the testing of certain metals, false positive findings with 
certain skin irritants (such as some surfactant-type substances) (6) (1 and Chapter B.42 in this Annex), solubility of the test substance). In addition, chemical classes or substances containing functional groups shown to act as 
potential confounders (15) may necessitate the use of guinea pig tests ( i.e. 
B.6; OECD Test Guideline 406 (13)). Limitations that have been identified for the LLNA (1 and Chapter B.42 in this Annex) have been recommended to apply also to the LLNA: BrdU-ELISA (10). Other than such identified 
limitations, the LLNA: BrdU-ELISA should be applicable for testing any 
chemicals unless there are properties associated with these chemicals that may interfere with the accuracy of the LLNA: BrdU-ELISA. In addition, consideration should be given to the possibility of borderline positive results 
when Stimulation Index (SI) values between 1,6 and 1,9 are obtained (see 
paragraphs 31-32). This is based on the validation database of 43 substances using an SI ≥ 1,6 (see paragraph 6) for which the LLNA: BrdU-ELISA 
correctly identified all 32 LLNA sensitisers, but incorrectly identified two of 
11 LLNA non-sensitisers with SI values between 1,6 and 1,9 ( i.e. borderline 
positive) (10). However, as the same dataset was used for setting the SI-values and calculating the predictive properties of the test, the stated results may be an over-estimation of the real predictive properties.
 
PRINCIPLE OF THE TEST METHOD 
6. The basic principle underlying the LLNA: BrdU-ELISA is that sensitisers 
induce proliferation of lymphocytes in the lymph nodes draining the site of test substance application. This proliferation is proportional to the dose and to the potency of the applied allergen and provides a simple means of 
obtaining a quantitative measurement of sensitisation. Proliferation is 
measured by comparing the mean proliferation in each test group to the mean proliferation in the vehicle treated control group (VC). The ratio of the mean proliferation in each treated group to that in the concurrent VC 
group, termed the SI, is determined, and should be ≥ 1,6 before further 
evaluation of the test substance as a potential skin sensitiser is warranted.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 746
 
The procedures described here are based on the use of measuring BrdU 
content to indicate an increased number of proliferating cells in the draining 
auricular lymph nodes. BrdU is an analogue of thymidine and is similarly incorporated into the DNA of proliferating cells. The incorporation of BrdU is measured by ELISA, which utilises an antibody specific for BrdU that is 
also labelled with peroxidase. When the substrate is added, the peroxidase 
reacts with the substrate to produce a coloured product that is quantified at a specific absorbance using a microtitre plate reader.
 
DESCRIPTION OF THE ASSAY  
Selection of animal species  
7. The mouse is the species of choice for this test. Validation studies for the 
LLNA: BrdU-ELISA were conducted exclusively with the CBA/JN strain, 
which is therefore considered the preferred strain (10) (12). Young adult female mice, which are nulliparous and non-pregnant, are used. At the start of the study, animals should be between 8-12 weeks old, and the weight 
variation of the animals should be minimal and not exceed 20 % of the 
mean weight. Alternatively, other strains and males may be used when sufficient data are generated to demonstrate that significant strain and/or gender-specific differences in the LLNA: BrdU-ELISA response do not 
exist.
 
Housing and feeding conditions  
8. Mice should be group-housed (16), unless adequate scientific rationale for 
housing mice individually is provided. The temperature of the experimental animal room should be 22 ± 3 °C. Although the relative humidity should be at least 30 % and preferably not exceed 70 %, other than during room 
cleaning, the aim should be 50-60 %. Lighting should be artificial, the 
sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water.
 
Preparation of animals  
9. The animals are randomly selected, marked to permit individual identifi ­
cation (but not by any form of ear marking), and kept in their cages for at 
least five days prior to the start of dosing to allow for acclimatisation to the 
laboratory conditions. Prior to the start of treatment all animals are examined to ensure that they have no observable skin lesions.
 
Preparation of dosing solutions  
10. Solid chemicals should be dissolved or suspended in solvents/vehicles and 
diluted, if appropriate, prior to application to an ear of the mice. Liquid 
chemicals may be applied neat or diluted prior to dosing. Insoluble chemicals, such as those generally seen in medical devices, should be subjected to an exaggerated extraction in an appropriate solvent to reveal 
all extractable constituents for testing prior to application to an ear of the 
mice. Test substances should be prepared daily unless stability data demon ­
strate the acceptability of storage.
 
Reliability check  
11. Positive control chemicals (PC) are used to demonstrate appropriate 
performance of the assay by responding with adequate and reproducible 
sensitivity as a sensitising test substance for which the magnitude of the 
response is well characterised. Inclusion of a concurrent PC is recom ­
mended because it demonstrates competency of the laboratory to successfully conduct each assay and allows for an assessment of intra- 
and inter-laboratory reproducibility and comparability. Some regulatory 
authorities also require a PC for each study and therefore users are
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 747
 
encouraged to consult the relevant authorities prior to conducting the 
LLNA: BrdU-ELISA. Accordingly, the routine use of a concurrent PC is 
encouraged to avoid the need for additional animal testing to meet such requirements that might arise from the use of a periodic PC (see paragraph 12). The PC should produce a positive LLNA: BrdU-ELISA response at an 
exposure level expected to give an increase in the SI ≥ 1,6 over the negative 
control (NC) group. The PC dose should be chosen such that it does not cause excessive skin irritation or systemic toxicity and the induction is reproducible but not excessive ( e.g. SI > 14 would be considered 
excessive). Preferred PC are 25 % hexyl cinnamic aldehyde (CAS 
No 101-86-0) and 25 % eugenol (CAS No 97-53-0) in acetone: olive oil (4:1, v/v). There may be circumstances in which, given adequate justifi ­
cation, other PC, meeting the above criteria, may be used. 
12. While inclusion of a concurrent PC group is recommended, there may be 
situations in which periodic testing ( i.e. at intervals ≤ 6 months) of the PC 
may be adequate for laboratories that conduct the LLNA: BrdU-ELISA 
regularly ( i.e. conduct the LLNA: BrdU-ELISA at a frequency of no less 
than once per month) and have an established historical PC database that demonstrates the laboratory’s ability to obtain reproducible and accurate results with PCs. Adequate proficiency with the LLNA: BrdU-ELISA can 
be successfully demonstrated by generating consistent positive results with 
the PC in at least 10 independent tests conducted within a reasonable period of time ( i.e. less than one year). 
13. A concurrent PC group should always be included when there is a 
procedural change to the LLNA: BrdU-ELISA ( e.g. change in trained 
personnel, change in test method materials and/or reagents, change in test 
method equipment, change in source of test animals), and such changes 
should be documented in laboratory reports. Consideration should be given to the impact of these changes on the adequacy of the previously estab ­
lished historical database in determining the necessity for establishing a new 
historical database to document consistency in the PC results. 
14. Investigators should be aware that the decision to conduct a PC study on a 
periodic basis instead of concurrently has ramifications on the adequacy and 
acceptability of negative study results generated without a concurrent PC 
during the interval between each periodic PC study. For example, if a false negative result is obtained in the periodic PC study, negative test substance results obtained in the interval between the last acceptable periodic PC 
study and the unacceptable periodic PC study may be questioned. Impli ­
cations of these outcomes should be carefully considered when determining whether to include concurrent PCs or to only conduct periodic PCs. Consideration should also be given to using fewer animals in the concurrent 
PC group when this is scientifically justified and if the laboratory demon ­
strates, based on laboratory-specific historical data, that fewer mice can be used (17). 
15. Although the PC should be tested in the vehicle that is known to elicit a 
consistent response ( e.g. acetone: olive oil; 4:1, v/v), there may be certain 
regulatory situations in which testing in a non-standard vehicle (clinically/ chemically relevant formulation) will also be necessary (18). If the 
concurrent PC is tested in a different vehicle than the test substance, then 
a separate VC for the concurrent PC should be included.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 748
 
16. In instances where test substances of a specific chemical class or range of 
responses are being evaluated, benchmark substances may also be useful to 
demonstrate that the test method is functioning properly for detecting the skin sensitisation potential of these types of test substances. Appropriate benchmark substances should have the following properties: 
— structural and functional similarity to the class of the test substance 
being tested; 
— known physical chemical characteristics; 
— supporting data from the LLNA: BrdU-ELISA; 
— supporting data from other animal models and/or from humans.
 
TEST PROCEDURE  
Number of animals and dose levels  
17. A minimum of four animals is used per dose group, with a minimum of 
three concentrations of the test substance, plus a concurrent NC group treated only with the vehicle for the test substance, and a PC group (con ­
current or recent, based on laboratory policy in considering paragraphs 11- 
15). Testing multiple doses of the PC should be considered especially when testing the PC on an intermittent basis. Except for absence of treatment with the test substance, animals in the control groups should be handled and 
treated in a manner identical to that of animals in the treatment groups. 
18. Dose and vehicle selection should be based on the recommendations given 
in the references 2 and 19. Consecutive doses are normally selected from an 
appropriate concentration series such as 100 %, 50 %, 25 %, 10 %, 5 %, 2,5 %, 1 %, 0,5 %, etc. Adequate scientific rationale should accompany the selection of the concentration series used. All existing toxicological 
information ( e.g. acute toxicity and dermal irritation) and structural and 
physicochemical information on the test substance of interest (and/or struc ­
turally related substances) should be considered, where available, in selecting the three consecutive concentrations so that the highest concen ­
tration maximises exposure while avoiding systemic toxicity and/or 
excessive local skin irritation (19) (20 and Chapter B.4 of this Annex). In the absence of such information, an initial pre-screen test may be necessary (see paragraphs 21-24). 
19. The vehicle should not interfere with or bias the test result and should be 
selected on the basis of maximising the solubility in order to obtain the highest concentration achievable while producing a solution/suspension 
suitable for application of the test substance. Recommended vehicles are 
acetone: olive oil (4:1 v/v),  N,N -dimethylformamide, methyl ethyl ketone, 
propylene glycol, and dimethyl sulphoxide (6) but others may be used if sufficient scientific rationale is provided. In certain situations it may be 
necessary to use a clinically relevant solvent or the commercial formulation 
in which the test substance is marketed as an additional control. Particular care should be taken to ensure that hydrophilic test substances are incor ­
porated into a vehicle system, which wets the skin and does not 
immediately run off, by incorporation of appropriate solubilisers ( e.g. 1 % 
Pluronic® L92). Thus, wholly aqueous vehicles are to be avoided.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 749
 
20. The processing of lymph nodes from individual mice allows for the 
assessment of inter-animal variability and a statistical comparison of the 
difference between test substance and VC group measurements (see paragraph 33). In addition, evaluating the possibility of reducing the number of mice in the PC group is only feasible when individual animal 
data are collected (17). Further, some regulatory authorities require the 
collection of individual animal data. Regular collection of individual animal data provides an animal welfare advantage by avoiding duplicate testing that would be necessary if the test substance results originally 
collected in one manner ( e.g. via pooled animal data) were to be considered 
later by regulatory authorities with other requirements ( e.g. individual 
animal data).
 
Pre-screen test  
21. In the absence of information to determine the highest dose to be tested (see 
paragraph 18), a pre-screen test should be performed in order to define the appropriate dose level to test in the LLNA: BrdU-ELISA. The purpose of 
the pre-screen test is to provide guidance for selecting the maximum dose 
level to use in the main LLNA: BrdU-ELISA study, where information on the concentration that induces systemic toxicity (see paragraph 24) and/or excessive local skin irritation (see paragraph 23) is not available. The 
maximum dose level tested should be a concentration of 100 % of the 
test substance for liquids or the maximum possible concentration for solids or suspensions. 
22. The pre-screen test is conducted under conditions identical to the main 
LLNA: BrdU-ELISA study, except there is no assessment of lymph node proliferation and fewer animals per dose group can be used. One or two 
animals per dose group are suggested. All mice will be observed daily for 
any clinical signs of systemic toxicity or local irritation at the application site. Body weights are recorded pre-test and prior to termination (Day 6). Both ears of each mouse are observed for erythema and scored using Table 
1 (20 and Chapter B.4 of this Annex). Ear thickness measurements are 
taken using a thickness gauge ( e.g. digital micrometer or Peacock Dial 
thickness gauge) on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose), and Day 6. Additionally, on Day 6, ear thickness 
could be determined by ear punch weight determinations, which should 
be performed after the animals are humanely killed. Excessive local irri ­
tation is indicated by an erythema score ≥ 3 and/or ear thickness of ≥ 25 % 
on any day of measurement (21) (22). The highest dose selected for the 
main LLNA: BrdU-ELISA study will be the next lower dose in the 
pre-screen concentration series (see paragraph 18) that does not induce systemic toxicity and/or excessive local skin irritation.
 
Table 1  
Erythema Scores  
Observation Score  
No erythema 0 
Very slight erythema (barely perceptible) 1 Well-defined erythema 2
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 750
 
Observation Score  
Moderate to severe erythema 3 
Severe erythema (beet redness) to eschar formation preventing 
grading of erythema 4 
23. In addition to a 25 % increase in ear thickness (21) (22), a statistically significant 
increase in ear thickness in the treated mice compared to control mice has also been used to identify irritants in the LLNA (22) (23) (24) (25) (26) (27) (28). However, while statistically significant increases can occur when ear thickness 
is less than 25 % they have not been associated specifically with excessive 
irritation (25) (26) (27) (28) (29). 
24. The following clinical observations may indicate systemic toxicity (30) 
when used as part of an integrated assessment and therefore may indicate the maximum dose level to use in the main LLNA: BrdU-ELISA: changes 
in nervous system function ( e.g. pilo-erection, ataxia, tremors, and convul ­
sions); changes in behaviour ( e.g. aggressiveness, change in grooming 
activity, marked change in activity level); changes in respiratory patterns (i.e. changes in frequency and intensity of breathing such as dyspnea, 
gasping, and rales), and changes in food and water consumption. In 
addition, signs of lethargy and/or unresponsiveness and any clinical signs of more than slight or momentary pain and distress, or a > 5 % reduction in body weight from Day 1 to Day 6 and mortality should be considered in the 
evaluation. Moribund animals or animals showing signs of severe pain and 
distress should be humanely killed (31).
 
Main study experimental schedule  
25. The experimental schedule of the assay is as follows: 
— Day 1:  Individually identify and record the weight of each animal and 
any clinical observation. Apply 25 μL of the appropriate dilution of the 
test substance, the vehicle alone, or the PC (concurrent or recent, based 
on laboratory policy in considering paragraphs 11-15), to the dorsum of 
each ear. 
— Days 2 and 3:  Repeat the application procedure carried out on Day 1. 
— Day 4:  No treatment. 
— Day 5:  Inject 0,5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution 
intra-peritoneally. 
— Day 6:  Record the weight of each animal and any clinical observation. 
Approximately 24 hours (24 h) after BrdU injection, humanely kill the animals. Excise the draining auricular lymph nodes from each mouse ear and process separately in phosphate buffered saline (PBS) for each 
animal. Details and diagrams of the lymph node identification and 
dissection can be found in reference (17). To further monitor the local skin response in the main study, additional parameters such as scoring of ear erythema or ear thickness measurements (obtained either 
by using a thickness gauge, or ear punch weight determinations at 
necropsy) may be included into the study protocol.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 751
 
Preparation of cell suspensions  
26. From each mouse, a single-cell suspension of lymph node cells (LNC) 
excised bilaterally is prepared by gentle mechanical disaggregation 
through 200 micron-mesh stainless steel gauze or another acceptable technique for generating a single-cell suspension ( e.g. use of a disposable 
plastic pestle to crush the lymph nodes followed by passage through a #70 
nylon mesh). The procedure for preparing the LNC suspension is critical in 
this assay and therefore every operator should establish the skill in advance. Further, the lymph nodes in NC animals are small, so careful operation is important to avoid any artificial effects on SI values. In each case, the target 
volume of the LNC suspension should be adjusted to a determined 
optimised volume (approximately 15 mL). The optimised volume is based on achieving a mean absorbance of the NC group within 0,1-0,2.
 
Determination of cellular proliferation (measurement of BrdU content in 
DNA of lymphocytes)  
27. BrdU is measured by ELISA using a commercial kit ( e.g. Roche Applied 
Science, Mannheim, Germany, Catalogue Number 11 647 229 001). 
Briefly, 100 μL of the LNC suspension is added to the wells of a flat- 
bottom microplate in triplicate. After fixation and denaturation of the LNC, anti-BrdU antibody is added to each well and allowed to react. Subsequently the anti-BrdU antibody is removed by washing and the 
substrate solution is then added and allowed to produce chromogen. 
Absorbance at 370 nm with a reference wavelength of 492 nm is then measured. In all cases, assay test conditions should be optimised (see paragraph 26).
 
OBSERVATIONS  
Clinical observations  
28. Each mouse should be carefully observed at least once daily for any clinical 
signs, either of local irritation at the application site or of systemic toxicity. 
All observations are systematically recorded with records being maintained for each mouse. Monitoring plans should include criteria to promptly identify those mice exhibiting systemic toxicity, excessive local skin irri ­
tation, or corrosion of skin for euthanasia (31).
 
Body weights  
29. As stated in paragraph 25, individual animal body weights should be 
measured at the start of the test and at the scheduled humane kill.  
CALCULATION OF RESULTS 
30. Results for each treatment group are expressed as the mean SI. The SI is 
derived by dividing the mean BrdU labelling index/mouse within each test 
substance group and the PC group by the mean BrdU labelling index for the solvent/VC group. The average SI for the VCs is then one. 
The BrdU labelling index is defined as: 
BrdU labelling index = (ABS
 em – ABS blank  em ) – (ABS  ref – ABS blank  ref ) 
Where: em = emission wavelength; and ref = reference wavelength.  ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 752
 
31. The decision process regards a result as positive when SI ≥ 1,6 (10). 
However, the strength of the dose-response relationship, the statistical 
significance and the consistency of the solvent/vehicle and PC responses may also be used when determining whether a borderline result ( i.e. SI 
value between 1,6 and 1,9) is declared positive (3) (6) (32). 
32. For a borderline positive response between an SI of 1,6 and 1,9, users may 
want to consider additional information such as dose-response relationship, evidence of systemic toxicity or excessive irritation, and where appropriate, statistical significance together with SI values to confirm that such results 
are positives (10). Consideration should also be given to various properties 
of the test substance, including whether it has a structural relationship to known skin sensitisers, whether it causes excessive skin irritation in the mouse, and the nature of the dose-response observed. These and other 
considerations are discussed in detail elsewhere (4). 
33. Collecting data at the level of the individual mouse will enable a statistical 
analysis for presence and degree of dose-response relationship in the data. Any statistical assessment could include an evaluation of the dose-response 
relationship as well as suitably adjusted comparisons of test groups ( e.g. 
pair-wise dosed group versus concurrent solvent/vehicle control compari ­
sons). Statistical analyses may include,  e.g.  linear regression or William’s 
test to assess dose-response trends, and Dunnett’s test for pair-wise 
comparisons. In choosing an appropriate method of statistical analysis, 
the investigator should maintain an awareness of possible inequalities of variances and other related problems that may necessitate a data trans ­
formation or a non-parametric statistical analysis. In any case, the inves ­
tigator may need to carry out SI calculations and statistical analyses with and without certain data points (sometimes called ‘outliers’).
 
DATA AND REPORTING  
Data  
34. Data should be summarised in tabular form showing the individual animal 
BrdU labelling index values, the group mean BrdU labelling index/animal, its associated error term ( e.g. SD, SEM), and the mean SI for each dose 
group compared against the concurrent solvent/vehicle control group.
 
Test report  
35. The test report should contain the following information: 
Test and control chemicals: 
— identification data ( e.g. CAS number and EC number, if available; 
source; purity; known impurities; lot number); 
— physical nature and physicochemical properties ( e.g. volatility, stability, 
solubility); 
— if mixture, composition and relative percentages of components; 
Solvent/vehicle: — identification data (purity; concentration, where appropriate; volume 
used); 
— justification for choice of vehicle;
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 753
 
Test animals: 
— source of CBA mice; — microbiological status of the animals, when known; — number and age of animals; — source of animals, housing conditions, diet, etc.; Test conditions: — source, lot number, and manufacturer’s quality assurance/quality control 
data (antibody sensitivity and specificity and the limit of detection) for 
the ELISA kit; 
— details of test substance preparation and application; — justification for dose selection (including results from pre-screen test, if 
conducted); 
— vehicle and test substance concentrations used, and total amount of test 
substance applied; 
— details of food and water quality (including diet type/source, water 
source); 
— details of treatment and sampling schedules; — methods for measurement of toxicity; — criteria for considering studies as positive or negative; — details of any protocol deviations and an explanation on how the 
deviation affects the study design and results; 
Reliability check: — a summary of results of latest reliability check, including information on 
test substance, concentration and vehicle used; 
— concurrent and/or historical PC and concurrent negative (solvent/ve ­
hicle) control data for testing laboratory; 
— if a concurrent PC was not included, the date and laboratory report for 
the most recent periodic PC and a report detailing the historical PC data for the laboratory justifying the basis for not conducting a concurrent PC; 
Results: — individual weights of mice at start of dosing and at scheduled humane 
kill; as well as mean and associated error term ( e.g. SD, SEM) for each 
treatment group; 
— time course of onset and signs of toxicity, including dermal irritation at 
site of administration, if any, for each animal; 
— a table of individual mouse BrdU labelling indices and SI values for 
each treatment group; 
— mean and associated error term ( e.g. SD, SEM) for BrdU labelling 
index/mouse for each treatment group and the results of outlier 
analysis for each treatment group;
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 754
 
— calculated SI and an appropriate measure of variability that takes into 
account the inter-animal variability in both the test substance and 
control groups; 
— dose-response relationship; — statistical analyses, where appropriate; Discussion of results: — a brief commentary on the results, the dose-response analysis, and stat ­
istical analyses, where appropriate, with a conclusion as to whether the test substance should be considered a skin sensitiser.
 
LITERATURE 
(1) OECD (2010), Skin Sensitisation: Local Lymph Node Assay, Test 
Guideline No 429, Guidelines for the Testing of Chemicals, OECD, 
Paris. Available at: [http://www.oecd.org/env/testguidelines] 
(2) Chamberlain, M. and Basketter, D.A. (1996), The local lymph node assay: 
status of validation. Food Chem. Toxicol., 34, 999-1002. 
(3) Basketter, D.A., Gerberick, G.F., Kimber, I. and Loveless, S.E. (1996), The 
local lymph node assay: A viable alternative to currently accepted skin sensitisation tests. Food Chem. Toxicol., 34, 985-997. 
(4) Basketter, D.A., Gerberick, G.F. and Kimber, I. (1998), Strategies for iden ­
tifying false positive responses in predictive sensitisation tests. Food Chem. 
Toxicol., 36, 327-33. 
(5) Van Och, F.M.M., Slob, W., De Jong, W.H., Vandebriel, R.J. and Van 
Loveren, H. (2000), A quantitative method for assessing the sensitising potency of low molecular weight chemicals using a local lymph node 
assay: employment of a regression method that includes determination of 
uncertainty margins. Toxicol., 146, 49-59. 
(6) ICCVAM (1999), The murine local lymph node Assay: A test method for 
assessing the allergic contact dermatitis potential of chemicals/compounds: The results of an independent peer review evaluation coordinated by the 
Interagency Coordinating Committee on the Validation of Alternative 
Methods (ICCVAM) and the National Toxicology Program Center for the Evaluation of Alternative Toxicological Methods (NICETAM). NIH Publication No: 99-4494. Research Triangle Park, N.C. Available at: [http:// 
iccvam.niehs.nih.gov/docs/immunotox_docs/llna/llnarep.pdf] 
(7) Dean, J.H., Twerdok, L.E., Tice, R.R., Sailstad, D.M., Hattan, D.G., Stokes, 
W.S. (2001), ICCVAM evaluation of the murine local lymph node assay: II. Conclusions and recommendations of an independent scientific peer review panel. Reg. Toxicol. Pharmacol., 34(3), 258-273. 
(8) Haneke, K.E., Tice, R.R., Carson, B.L., Margolin, B.H., Stokes, W.S. 
(2001), ICCVAM evaluation of the murine local lymph node assay: III. 
Data analyses completed by the national toxicology program interagency 
center for the evaluation of alternative toxicological methods. Reg. Toxicol. Pharmacol., 34(3), 274-286. 
(9) Sailstad, D.M., Hattan, D., Hill, R.N., Stokes, W.S. (2001), ICCVAM 
evaluation of the murine local lymph node assay: I. The ICCVAM 
review process. Reg. Toxicol. Pharmacol., 34(3), 249-257. 
(10) ICCVAM (2010), ICCVAM Test Method Evaluation Report. Nonradio ­
active local lymph node assay: BrdU-ELISA Test Method Protocol (LLNA: BrdU-ELISA). NIH Publication No 10-7552A/B. Research Triangle Park, NC: National Institute of Environmental Health Sciences. 
Available at: [http://iccvam.niehs.nih.gov/methods/immunotox/llna-ELISA/ 
TMER.htm]
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 755
 
(11) ICCVAM (2009), Independent Scientific Peer Review Panel Report: 
Updated validation status of new versions and applications of the murine 
local lymph node assay: a test method for assessing the allergic contact dermatitis potential of chemicals and products. Research Triangle Park, NC: National Institute of Environmental Health Sciences. Available at: [http:// 
iccvam.niehs.nih.gov/docs/immunotox_docs/LLNAPRPRept2009.pdf] 
(12) Takeyoshi, M., Iida, K., Shiraishi, K. and Hoshuyama, S. (2005), Novel 
approach for classifying chemicals according to skin sensitising potency by non-radioisotopic modification of the local lymph node assay. J. Appl. Toxicol., 25, 129-134. 
(13) OECD (1992), Skin Sensitisation, Test Guideline No 406, Guidelines for 
Testing of Chemicals, OECD, Paris. Available at: [http://www.oecd.org/env/ 
testguidelines] 
(14) Kreiling, R., Hollnagel, H.M., Hareng, L., Eigler, L., Lee, M.S., Griem, P., 
Dreessen, B., Kleber, M., Albrecht, A., Garcia, C. and Wendel, A. (2008), Comparison of the skin sensitising potential of unsaturated compounds as 
assessed by the murine local lymph node assay (LLNA) and the guinea pig 
maximization test (GPMT). Food Chem. Toxicol., 46, 1896-1904. 
(15) Basketter, D., Ball, N., Cagen, S., Carrilo, J.C., Certa, H., Eigler, D., 
Garcia, C., Esch, H., Graham, C., Haux, C., Kreiling, R. and Mehling, A. (2009), Application of a weight of evidence approach to assessing 
discordant sensitisation datasets: implications for REACH. Reg. Toxicol. 
Pharmacol., 55, 90-96. 
(16) ILAR (1996), Institute of Laboratory Animal Research (ILAR) Guide for 
the Care and Use of Laboratory Animals. 7th ed. Washington, DC: National Academies Press. 
(17) ICCVAM (2009), Recommended Performance Standards: Murine Local 
Lymph Node Assay. NIH Publication Number 09-7357. Research 
Triangle Park, NC: National Institute of Environmental Health Sciences. Available at: [http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna- ps/LLNAPerfStds.pdf] 
(18) McGarry, H.F. (2007), The murine local lymph node assay: regulatory and 
potency considerations under REACH. Toxicol., 238, 71-89. 
(19) Kimber, I., Dearman, R.J., Scholes E.W. and Basketter, D.A. (1994), The 
local lymph node assay: developments and applications. Toxicol., 93, 13- 31. 
(20) OECD (2002), Acute Dermal Irritation/Corrosion, Test Guideline No 404, 
Guidelines for Testing of Chemicals, OECD, Paris. Available at: [http:// 
www.oecd.org/env/testguidelines] 
(21) Reeder, M.K., Broomhead, Y.L., DiDonato, L. and DeGeorge, G.L. (2007), 
Use of an enhanced local lymph node assay to correctly classify irritants and false positive substances. Toxicologist, 96, 235. 
(22) ICCVAM (2009), Nonradioactive Murine Local Lymph Node Assay: Flow 
Cytometry Test Method Protocol (LLNA: BrdU-FC) Revised Draft Back ­
ground Review Document. Research Triangle Park, NC: National Institute 
of Environmental Health Sciences. Available at: [http://icc ­
vam.niehs.nih.gov/methods/immunotox/fcLLNA/BRDcomplete.pdf]. 
(23) Hayes, B.B., Gerber, P.C., Griffey, S.S. and Meade, B.J. (1998), Contact 
hypersensitivity to dicyclohexylcarbodiimide and diisopropylcarbodiimide 
in female B6C3F1 mice. Drug Chem. Toxicol., 21, 195-206. 
(24) Homey, B., von Schilling, C., Blumel, J., Schuppe, H.C., Ruzicka, T., Ahr, 
H.J., Lehmann, P. and Vohr, V.W. (1998), An integrated model for the differentiation of chemical-induced allergic and irritant skin reactions. Toxicol. Appl. Pharmacol., 153, 83-94. 
(25) Woolhiser, M.R., Hayes, B.B. and Meade, B.J. (1998), A combined murine 
local lymph node and irritancy assay to predict sensitisation and irritancy 
potential of chemicals. Toxicol. Meth., 8, 245-256.
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(26) Hayes, B.B. and Meade, B.J. (1999), Contact sensitivity to selected acrylate 
compounds in B6C3F1 mice: relative potency, cross reactivity, and 
comparison of test methods. Drug. Chem. Toxicol., 22, 491-506. 
(27) Ehling, G., Hecht, M., Heusener, A., Huesler, J., Gamer, A.O., van 
Loveren, H., Maurer, T., Riecke, K., Ullmann, L., Ulrich, P., Vandebriel, R. and Vohr, H.W. (2005), A European inter- laboratory validation of 
alternative endpoints of the murine local lymph node assay: first round. 
Toxicol., 212, 60-68. 
(28) Vohr, H.W. and Ahr, H.J. (2005), The local lymph node assay being too 
sensitive? Arch. Toxicol., 79, 721-728. 
(29) Patterson, R.M., Noga, E. and Germolec D. (2007), Lack of evidence for 
contact sensitisation by Pfiesteria extract. Environ. Health Perspect., 115, 
1023-1028. 
(30) ICCVAM (2009), Report on the ICCVAM-NICEATM/ECVAM/JaCVAM 
Scientific Workshop on Acute Chemical Safety Testing: Advancing  In Vitro  
Approaches and Humane Endpoints for Systemic Toxicity Evaluations. 
Research Triangle Park, NC: National Institute of Environmental Health 
Sciences. Available at: [http://iccvam.niehs.nih.gov/methods/acutetox/ Tox_workshop.htm]. 
(31) OECD (2000), Guidance Document on the Recognition, Assessment and 
Use of Clinical Signs as Humane Endpoints for Experimental Animals Used 
in Safety Evaluation, Environmental Health and Safety Monograph Series on Testing and Assessment No 19, ENV/JM/MONO(2000)7, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines] 
(32) Kimber, I., Hilton, J., Dearman, R.J., Gerberick, G.F., Ryan, C.A., 
Basketter, D.A., Lea, L., House, R.V., Ladies, G.S., Loveless, S.E. and 
Hastings, K.L. (1998), Assessment of the skin sensitisation potential of topical medicaments using the local lymph node assay: An interlaboratory exercise. J. Toxicol. Environ.l Health, 53, 563-79. 
(33) OECD (2005), Guidance Document on the Validation and International 
Acceptance of New or Updated Test Methods for Hazard Assessment, 
Environment, Health and Safety Monograph Series on Testing and Assessment No 34, ENV/JM/MONO(2005)14, OECD, Paris. Available at: 
[http://www.oecd.org/env/testguidelines]
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 757
 
Appendix 1  
DEFINITIONS  
Accuracy:  The closeness of agreement between test method results and accepted 
reference values. It is a measure of test method performance and one aspect of relevance. The term is often used interchangeably with ‘concordance’ to mean 
the proportion of correct outcomes of a test method (33).  
Benchmark substance:  A sensitising or non-sensitising substance used as a 
standard for comparison to a test substance. A benchmark substance should have the following properties: (i) a consistent and reliable source(s); (ii) structural 
and functional similarity to the class of substances being tested; (iii) known 
physical/chemical characteristics; (iv) supporting data on known effects; and (v) known potency in the range of the desired response.  
False negative:  A test substance incorrectly identified as negative or non-active 
by a test method, when in fact it is positive or active (33).  
False positive:  A test substance incorrectly identified as positive or active by a 
test, when in fact it is negative or non-active (33).  
Hazard:  The potential for an adverse health or ecological effect. The adverse 
effect is manifested only if there is an exposure of sufficient level.  
Inter-laboratory reproducibility:  A measure of the extent to which different 
qualified laboratories, using the same protocol and testing the same test 
substance, can produce qualitatively and quantitatively similar results. 
Inter-laboratory reproducibility is determined during the pre-validation and vali ­
dation processes, and indicates the extent to which a test can be successfully transferred between laboratories, also referred to as between-laboratory reproduci ­
bility (33).  
Intra-laboratory reproducibility:  A determination of the extent that qualified 
people within the same laboratory can successfully replicate results using a specific protocol at different times. Also referred to as within-laboratory repro ­
ducibility (33).  
Outlier:  An outlier is an observation that is markedly different from other values 
in a random sample from a population.  
Quality assurance:  A management process by which adherence to laboratory 
testing standards, requirements, and record keeping procedures, and the 
accuracy of data transfer, are assessed by individuals who are independent 
from those performing the testing.  
Reliability:  Measures of the extent that a test method can be performed repro ­
ducibly within and between laboratories over time, when performed using the same protocol. It is assessed by calculating intra- and inter-laboratory reproduci ­
bility (33).  
Skin sensitisation:  An immunological process that results when a susceptible 
individual is exposed topically to an inducing chemical allergen, which provokes a cutaneous immune response that can lead to the development of contact sensitisation.  
Stimulation Index (SI):  A value calculated to assess the skin sensitisation 
potential of a test substance that is the ratio of the proliferation in treated groups to that in the concurrent vehicle control group.  
Test substance (also referred to as test chemical):  Any substance or mixture 
tested using this TM.
 ▼M3 2008R0440 — EN — 22.03.2014 — 004.001 — 758
 
B.52.  ACUTE INHALATION TOXICITY — ACUTE TOXIC CLASS 
METHOD  
INTRODUCTION 
1. This Test Method is equivalent to OECD Test Guideline (TG) 436 (2009). 
The first acute inhalation TG 403 was adopted in 1981, and has since been 
revised (see chapter B.2 of this Annex (1)). Development of an Inhalation 
Acute Toxic Class (ATC) method (2) (3) (4) was considered appropriate following the adoption of the revised oral ATC method (chapter B.1 tris of this Annex) (5). A retrospective performance assessment of the ATC test 
method for acute inhalation toxicity showed that the method is suitable for 
being used for Classification and Labelling purposes (6). The inhalation ATC Test Method will allow the use of serial steps of fixed target concen ­
trations to provide a ranking of test chemical toxicity. Lethality is used as 
key endpoint, however, animals in severe pain or distress, suffering or 
impending death should be humanely killed to minimise suffering. Guidance on humane endpoints is available in the OECD Guidance Document No 19 (7). 
2. Guidance on the conduct and interpretation of this Test Method can be 
found in the Guidance Document No 39 on Acute Inhalation Toxicity 
Testing (GD 39) (8). 
3. Definitions used in the context of this Test Method are provided in 
Appendix 1 and in GD 39 (8). 
4. The Test Method provides information on the hazardous properties and 
allows the test chemical to be ranked and classified according to the Regulation (EC) No 1272/2008 for the classification of chemicals that 
cause acute toxicity (9). In case point estimates of LC
 50 -values or 
concentration-response analyses are required, chapter B.2 of this Annex (1) is the appropriate Test Method to use. Further guidance on Test Method selection can be found in GD 39 (8). This Test Method is 
not specially intended for the testing of specialized materials, such as poorly 
soluble isometric or fibrous materials or manufactured nanomaterials.
 
INITIAL CONSIDERATIONS 
5. Before considering testing in accordance with this Test Method, all available 
information on the test chemical, including existing studies whose data 
would support not doing additional testing should be considered by the testing laboratory in order to minimize animal usage. Information that may assist in the selection of the most appropriate species, strain, sex, 
mode of exposure and appropriate test concentrations include the identity, 
chemical structure, and physico-chemical properties of the test chemical; results of any  in vitro  or in vivo  toxicity tests; anticipated use(s) and 
potential for human exposure; available (Q)SAR data and toxicological 
data on structurally related chemicals. Concentrations that are expected to 
cause severe pain and distress, due to corrosive (
 1 ) or severely irritant 
actions, should not be tested with this Test Method [see GD 39 (8)].  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 759  
( 1 ) The corrosivity evaluation could be based on expert judgment using such evidence as: 
human and animal experience, existing (in vitro) data, e.g. chapter B.40 (10), B.40 bis 
(11) of this annex or OECD TG 435 (12), pH values, information from similar chemicals 
or any other pertinent data.
 PRINCIPLE OF THE TEST 
6. It is the principle of the test that based on a stepwise procedure, sufficient 
information is obtained on the acute inhalation toxicity of the test chemical during an exposure period of 4 hours to enable its classification. Other 
durations of exposure may apply to serve specific regulatory purposes. At 
any of the defined concentration steps, 3 animals of each sex are tested. Depending on the mortality and/or the moribund status of the animals, 2 steps may be sufficient to allow judgement on the acute toxicity of the test 
chemical. If evidence is provided that one sex is more susceptible than the 
other, then the test may be continued with the more susceptible sex only. The outcome of the previous step will determine the following step such 
that: 
a) No further testing is needed, 
b) Testing of three animals per sex, or 
c) Testing with 6 animals of the more susceptible sex only  i.e. the lower 
boundary estimates of the toxic class should be based on 6 animals per 
test concentration group, regardless of sex. 
7. Moribund animals or animals obviously in pain or showing signs of severe 
and enduring distress should be humanely killed, and are considered in the 
interpretation of the test results in the same way as animals that died on test. 
Criteria for making the decision to kill moribund or severely suffering animals, and guidance on the recognition of predictable or impending death, are the subject of Guidance Document No 19 on Humane 
Endpoints (7).
 
DESCRIPTION OF THE METHOD  
Selection of animal species  
8. Healthy young adult animals of commonly used laboratory strains should be 
used. The preferred species is the rat and justifications should be provided if other species are used.
 
Preparation of animals  
9. Females should be nulliparous and non-pregnant. On the exposure day, 
animals should be young adults 8 to 12 weeks of age, and body weights 
should be within ± 20 % of the mean weight for each sex of any previously exposed animals at the same age. The animals are randomly selected, marked for individual identification. The animals are kept in their cages 
for at least 5 days prior to the start of the test to allow for acclimatisation 
to laboratory conditions. Animals should also be acclimatised to the test apparatus for a short period prior to testing, as this will lessen the stress caused by introduction to the new environment.
 
Animal husbandry  
10. The temperature of the experimental animal maintenance room should be 22 
± 3 °C. The relative humidity should ideally be maintained in the range of 30 to 70 %, though this may not be possible when using water as a vehicle. Before and after exposures, animals generally should be caged in groups by 
sex and concentration, but the number of animals per cage should not 
interfere with clear observation of each animal and should minimise losses due to cannibalism and fighting. When animals are to be exposed nose-only, it may be necessary for them to be acclimated to the restraining 
tubes. The restraining tubes should not impose undue physical, thermal, or 
immobilisation stress on the animals. Restraint may affect physiological
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 760
 
endpoints such as body temperature (hyperthermia) and/or respiratory 
minute volume. If generic data are available to show that no such 
changes occur to any appreciable extent, then pre-adaptation to the restraining tubes is not necessary. Animals exposed whole-body to an aerosol should be housed individually during exposure to prevent them 
from filtering the test aerosol through the fur of their cage mates. Conven ­
tional and certified laboratory diets may be used, except during exposure, accompanied with an unlimited supply of municipal drinking water. Lighting should be artificial, the sequence being 12 hours light/12 hours 
dark.
 
Inhalation chambers  
11. The nature of the test chemical and the objective of the test should be 
considered when selecting an inhalation chamber. The preferred mode of exposure is nose-only (which term includes head-only, nose-only or snout-only). Nose-only exposure is generally preferred for studies of liquid 
or solid aerosols and for vapours that may condense to form aerosols. Special 
objectives of the study may be better achieved by using a whole-body mode of exposure, but this should be justified in the study report. To ensure atmosphere stability when using a whole-body chamber, the total volume 
of the test animals should not exceed 5 % of the chamber volume. Principles 
of the nose-only and whole body exposure techniques and their particular advantages and disadvantages are described in GD 39 (8).
 
EXPOSURE CONDITIONS  
Administrations of concentrations  
12. A fixed duration of exposure for four hours, excluding equilibration time, is 
recommended. Other durations may be needed to meet specific require ­
ments, however, justification should be provided in the study report [see 
GD 39 (8)]. Animals exposed in whole-body chambers should be housed individually to prevent ingestion of test chemical due to grooming of cage mates. Feed should be withheld during the exposure period. Water may be 
provided throughout a whole-body exposure. 
13. Animals are exposed to the test chemical as a gas, vapour, aerosol, or a 
mixture thereof. The physical state to be tested depends on the 
physico-chemical properties of the test chemical, the selected concentration, 
and/or the physical form most likely present during the handling and use of the test chemical. Hygroscopic and chemically reactive test chemicals should be tested under dry air conditions. Care should be taken to avoid generating 
explosive concentrations.
 
Particle-size distribution  
14. Particle sizing should be performed for all aerosols and for vapours that may 
condense to form aerosols. To allow for exposure of all relevant regions of the respiratory tract, aerosols with mass median aerodynamic diameters (MMAD) ranging from 1 to 4 μm with a geometric standard 
deviation ( σ
 g ) in the range of 1,5 to 3,0 are recommended (8) (13) (14). 
Although a reasonable effort should be made to meet this standard, expert judgment should be provided if it cannot be achieved. For example, metal fumes may be smaller than this standard, and charged particles, fibres, and 
hygroscopic materials (which increase in size in the moist environment of 
the respiratory tract) may exceed this standard.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 761
 
Test chemical preparation in a vehicle  
15. A vehicle may be used to generate an appropriate concentration and particle 
size of the test chemical in the atmosphere. As a rule, water should be given preference. Particulate material may be subjected to mechanical processes to 
achieve the required particle size distribution, however, care should be taken 
not to decompose or alter the test chemical. In cases where mechanical processes are believed to have altered test chemical composition (e.g. extreme temperature from excessive milling due to friction), the composition 
of the test chemical should be verified analytically. Adequate care should be 
taken to not contaminate the test chemical. It is not necessary to test non-friable granular materials which are purposefully formulated to be 
un-inhalable. An attrition test should be used to demonstrate that respirable 
particles are not produced when the granular material is handled. If an attrition test produces respirable particles, an inhalation toxicity test should be performed.
 
Control animals  
16. A concurrent negative (air) control group is not necessary. When a vehicle 
other than water is used to assist in generating the test atmosphere, a vehicle control group should only be used when historical inhalation toxicity data 
are not available. If a toxicity study of a test chemical formulated in a 
vehicle reveals no toxicity, it follows that the vehicle is non-toxic at the concentration tested; thus, there is no need for a vehicle control.
 
MONITORING OF EXPOSURE CONDITIONS  
Chamber airflow  
17. The flow of air through the chamber should be carefully controlled, 
continuously monitored, and recorded at least hourly during each exposure. The monitoring of test atmosphere concentration (or stability) is an integral measurement of all dynamic parameters and provides an indirect 
means to control all relevant dynamic atmosphere generation parameters. 
Special consideration should be given to avoiding re-breathing in nose-only chambers in cases where airflow through the exposure system are inadequate to provide dynamic flow of test chemical atmosphere. 
There are prescribed methodologies that can be used to demonstrate that 
re-breathing does not occur under the selected operation conditions (8) (15). Oxygen concentration should be at least 19 % and carbon dioxide concen ­
tration should not exceed 1 %. If there is reason to believe that these 
standards cannot be met, oxygen and carbon dioxide concentrations 
should be measured.
 
Chamber temperature and relative humidity  
18. Chamber temperature should be maintained at 22 ± 3 °C. Relative humidity 
in the animals’ breathing zone, for both nose-only and whole-body 
exposures, should be monitored and recorded at least three times for durations up to 4 hrs, and hourly for shorter durations. The relative humidity should ideally be maintained in the range of 30 to 70 %, but 
this may either be unattainable (e.g. when testing water based mixtures) 
or not measurable due to test chemical interference with the test method.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 762
 
Test chemical: nominal concentration  
19. Whenever feasible, the nominal exposure chamber concentration should be 
calculated and recorded. The nominal concentration is the mass of generated test chemical divided by the total volume of air passed through the chamber 
system. The nominal concentration is not used to characterise the animals’ 
exposure, but a comparison of the nominal and the actual concentration gives an indication of the generation efficiency of the test system, and thus may be used to discover generation problems.
 
Test chemical: actual concentration  
20. The actual concentration is the test chemical concentration at the animals’ 
breathing zone in an inhalation chamber. Actual concentrations can be 
obtained either by specific methods ( e.g. direct sampling, adsorptive or 
chemical reactive methods, and subsequent analytical characterisation) or by non-specific methods such as gravimetric filter analysis. The use of gravimetric analysis is acceptable only for single component powder 
aerosols or aerosols of low volatility liquids and should be supported by 
appropriate pre-study test chemical-specific characterisations. Multi-component powder aerosol concentration may also be determined by gravimetric analysis. However, this requires analytical data which 
demonstrate that the composition of airborne material is similar to the 
starting material. If this information is not available, a reanalysis of the test chemical (ideally in its airborne state) at regular intervals during the course of the study may be necessary. For aerosolised agents that may 
evaporate or sublimate, it should be shown that all phases were collected 
by the method chosen. The target, nominal, and actual concentrations should be provided in the study report, but only actual concentrations are used in statistical analyses to calculate lethal concentration values. 
21. One lot of the test chemical should be used, if possible, and the test sample 
should be stored under conditions that maintain its purity, homogeneity, and 
stability. Prior to the start of the study, there should be a characterisation of the test chemical, including its purity and, if technically feasible, the identity, and quantities of identified contaminants and impurities. This can 
be demonstrated by, but is not limited to, the following data: retention time 
and relative peak area, molecular weight from mass spectroscopy or gas chromatography analyses, or other estimates. Although the test sample’s identity is not the responsibility of the test laboratory, it may be prudent 
for the test laboratory to confirm the sponsor’s characterisation at least in a 
limited way ( e.g. colour, physical nature, etc.). 
22. The exposure atmosphere shall be held as constant as practicable and 
monitored continuously and/or intermittently depending on the method of analysis. When intermittent sampling is used, chamber atmosphere samples should be taken at least twice in a four hour study. If not feasible due to 
limited air flow rates or low concentrations, one sample may be collected 
over the entire exposure period. If marked sample-to-sample fluctuations occur, the next concentrations tested should use four samples per exposure. Individual chamber concentration samples should not deviate 
from the mean chamber concentration by more than ± 10 % for gases 
and vapours, and by no more than ± 20 % for liquid or solid aerosols. Time to chamber equilibration (t
 95 ) should be calculated and recorded. 
The duration of an exposure spans the time that the test chemical is 
generated and this takes into account the times required to attain t  95 . 
Guidance for estimating t  95 can be found in GD 39 (8).  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 763
 
23. For very complex mixtures consisting of vapours/gases, and aerosols ( e.g. 
combustion atmospheres and test chemicals propelled from purpose-driven 
end-use products/devices), each phase may behave differently in an inha ­
lation chamber so at least one indicator substance (analyte), normally the principal active substance in the mixture, of each phase (vapour/gas and 
aerosol) should be selected. When the test chemical is a mixture, the 
analytical concentration should be reported for the total mixture and not just for the active ingredient or the component (analyte). Additional information regarding actual concentrations can be found in GD 39 (8).
 
Test chemical: particle size distribution  
24. The particle size distribution of aerosols should be determined at least twice 
during each 4 hour exposure by using a cascade impactor or an alternative instrument such as an aerodynamic particle sizer. If equivalence of the results obtained by a cascade impactor or an alternative instrument can be 
shown, then the alternative instrument may be used throughout the study. 
A second device, such as a gravimetric filter or an impinger/gas bubbler, should be used in parallel to the primary instrument to confirm the 
collection efficiency of the primary instrument. The mass concentration 
obtained by particle size analysis should be within reasonable limits of the mass concentration obtained by filter analysis [see GD 39 (8)]. If equiv ­
alence can be demonstrated in the early phase of the study, then further 
confirmatory measurements may be omitted. For animal welfare reasons, 
measures should be taken to minimize inconclusive data which may lead to a need to repeat an exposure. Particle sizing should be performed for vapours if there is any possibility that vapour condensation may result in the 
formation of an aerosol, or if particles are detected in a vapour atmosphere 
with potential for mixed phases (see paragraph 14).
 
PROCEDURE  
Main test  
25. Three animals per sex, or six animals of the more susceptible sex, are used 
for each step. If rodent species other than rats are exposed nose-only, maximum exposure durations may be adjusted to minimise 
species-specific distress. The concentration level to be used as the starting 
dose is selected from one of four fixed levels and the starting concentration level should be that which is most likely to produce toxicity in some of the dosed animals. The testing schemes for gases, vapours and aerosols 
(included in Appendixes2-4) represent the testing with the cut-off values 
of the CLP categories 1-4 (9) for gases (100, 500, 2 500, 20 000 ppm/4h) (Appendix 2), for vapours (0,5, 2, 10, 20 mg/l/4h) (Appendix 3) and for aerosols (0,05, 0,5, 1, 5 mg/l/4h) (Appendix 4). Category 5, which is not 
implemented in Regulation (EC) No 1272/2008 (9) relates to concentrations 
above the respective limit concentrations. For each starting concentration, the respective testing scheme applies. Depending on the number of humanely killed or dead animals, the test procedure follows the indicated 
arrows until a categorisation can be made. 
26. The time interval between exposure groups is determined by the onset, 
duration, and severity of toxic signs. Exposure of animals at the next 
concentration level should be delayed until there is reasonable confidence in the survival of the previously tested animals. A period of three or four days between the exposures at each concentration level is recommended to 
allow for the observation of delayed toxicity. The time interval may be 
adjusted as appropriate,  e.g. in case of inconclusive responses.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 764
 
Limit test  
27. The limit test is used when the test chemical is known or expected to be 
virtually non-toxic, i.e. eliciting a toxic response only above the regulatory limit concentration. Information about the toxicity of the test chemical can 
be gained from knowledge about similar tested substances or similar 
mixtures, taking into consideration the identity and percentage of components known to be of toxicological significance. In those situations where there is little or no information about its toxicity, or the test chemical 
is expected to be toxic, the main test should be performed [further guidance 
can be found in GD 39 (8)]. 
28. Using the normal procedure, three animals per sex, or six animals of the 
more susceptible sex, are exposed at concentrations of 20 000 ppm for gases, 20 mg/l for vapours and 5 mg/l for dusts/mists, respectively (if achievable), which serves as the limit test for this Test Method. When 
testing aerosols, the primary goal should be to achieve a respirable 
particle size ( i.e. an MMAD of 1-4 μm). This is possible with most test 
chemicals at a concentration of 2 mg/l Aerosol testing at greater than 2 mg/l should only be attempted if a respirable particle size can be achieved [see 
GD 39 (8)]. In accordance with GHS (16), testing in excess of a limit 
concentration is discouraged for animal welfare reasons. Testing in GHS Category 5 (16), which is not implemented in Regulation (EC) No 1272/2008 (9), should only be considered when there is a strong like ­
lihood that results of such a test would have direct relevance for protecting 
human health, and justification provided in the study report. In the case of potentially explosive test chemicals, care should be taken to avoid 
conditions favourable for an explosion. To avoid an unnecessary use of 
animals, a test run without animals should be conducted prior to the limit test to ensure that the chamber conditions for a limit test can be achieved.
 
OBSERVATIONS 
29. The animals should be clinically observed frequently during the exposure 
period. Following exposure, clinical observations should be made at least 
twice on the day of exposure, or more frequently when indicated by the response of the animals to treatment, and at least once daily thereafter for a total of 14 days. The length of the observation period is not fixed, but 
should be determined by the nature and time of onset of clinical signs 
and length of the recovery period. The times at which signs of toxicity appear and disappear are important, especially if there is a tendency for signs of toxicity to be delayed. All observations are systematically 
recorded with individual records being maintained for each animal. 
Animals found in a moribund condition and animals showing severe pain and/or enduring signs of severe distress should be humanely killed for animal welfare reasons. Care should be taken when conducting examinations 
for clinical signs of toxicity that initial poor appearance and transient 
respiratory changes, resulting from the exposure procedure, are not mistaken for treatment-related effects. The principles and criteria summarised in the Humane Endpoints Guidance Document should be 
taken into consideration (7). When animals are killed for humane reasons 
or found dead, the time of death should be recorded as precisely as possible.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 765
 
30. Cage-side observations should include changes in the skin and fur, eyes and 
mucous membranes, and also respiratory, circulatory, autonomic and central 
nervous systems, and somato-motor activity and behaviour patterns. When possible, any differentiation between local and systemic effects should be noted. Attention should be directed to observations of tremors, convulsions, 
salivation, diarrhoea, lethargy, sleep and coma. The measurement of rectal 
temperatures may provide supportive evidence of reflex bradypnea or hypo/ hyperthermia related to treatment or confinement.
 
Body weights  
31. Individual animal weights should be recorded once during the acclimati ­
sation period, on the day of exposure prior to exposure (day 0) and at 
least on days 1, 3 and 7 (and weekly thereafter), and at the time of death 
or euthanasia if exceeding day 1. Body weight is recognised as a critical indicator of toxicity and animals exhibiting a sustained decrement of ≥ 
20 %, compared to pre-study values, should be closely monitored. 
Surviving animals are weighed and humanely killed at the end of the 
post-exposure period.
 
Pathology  
32. All test animals, including those which die during the test or are euthanised 
and removed from the study for animal welfare reasons, should be subjected to gross necropsy. If necropsy cannot be performed immediately after a dead 
animal is discovered, the animal should be refrigerated (not frozen) at 
temperatures low enough to minimize autolysis. Necropsies should be performed as soon as possible, normally within a day or two. All gross pathological changes should be recorded for each animal with particular 
attention to any changes in the respiratory tract. 
33. Additional examinations included  a priori  by design may be considered to 
extend the interpretive value of the study, such as measuring lung weight of surviving rats and/or providing evidence of irritation by microscope exam ­
ination of the respiratory tract. Examined organs may include those showing 
evidence of gross pathology in animals surviving 24 or more hours, and organs known or expected to be affected. Microscopic examination of the entire respiratory tract may provide useful information for test chemicals that 
are reactive with water, such as acids and hygroscopic test chemicals.
 
DATA AND REPORTING  
Data  
34. Individual animal data on body weights and necropsy findings should be 
provided. Clinical observation data should be summarised in tabular form, 
showing for each test group the number of animals used, the number of animals displaying specific signs of toxicity, the number of animals found dead during the test or killed for humane reasons, time of death of indi ­
vidual animals, a description and time course of toxic effects and reversi ­
bility, and necropsy findings.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 766
 
Test report  
35. The test report should include the following information, as appropriate:  
Test animals and husbandry  
— Description of caging conditions, including: number (or change in 
number) of animals per cage, bedding material, ambient temperature 
and relative humidity, photoperiod, and identification of diet; 
— Species/strain used and justification for using a species other than the 
rat; 
— Number, age, and sex of animals; 
— Method of randomisation; 
— Details of food and water quality (including diet type/source, water 
source); 
— Description of any pre-test conditioning including diet, quarantine, and 
treatment for disease;  
Test chemical  
— Physical nature, purity, and, where relevant, physico-chemical properties 
(including isomerisation); 
— Identification data and Chemical Abstract Services (CAS) Registry 
Number, if known;  
Vehicle  
— Justification for use of vehicle and justification for choice of vehicle (if 
other than water); 
— Historical or concurrent data demonstrating that the vehicle does not 
interfere with the outcome of the study;  
Inhalation chamber  
— Description of the inhalation chamber including dimensions and volume; 
— Source and description of equipment used for the exposure of animals as 
well as generation of atmosphere; 
— Equipment for measuring temperature, humidity, particle-size, and actual 
concentration; 
— Source of air, treatment of air supplied/extracted and system used for 
conditioning; 
— Methods used for calibration of equipment to ensure a homogeneous test 
atmosphere; 
— Pressure difference (positive or negative); 
— Exposure ports per chamber (nose-only); location of animals in the 
system (whole-body); 
— Temporal homogeneity/stability of test atmosphere; 
— Location of temperature and humidity sensors and sampling of test 
atmosphere in the chamber; 
— Air flow rates, air flow rate/exposure port (nose-only), or animal load/ 
chamber (whole-body); 
— Information about the equipment used to measure oxygen and carbon 
dioxide, if applicable;  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 767
 
— Time required to reach inhalation chamber equilibrium (t  95 ); 
— Number of volume changes per hour; — Metering devices (if applicable);  
Exposure data  
— Rationale for target concentration selection in the main study; — Nominal concentrations (total mass of test chemical generated into the 
inhalation chamber divided by the volume of air passed through the chamber); 
— Actual test chemical concentrations collected from the animals’ 
breathing zone; for test mixtures that produce heterogeneous physical 
forms (gases, vapours, aerosols), each may be analysed separately; 
— All air concentrations should be reported in units of mass ( e.g. mg/l, 
mg/m
 3 , etc.), units of volume ( e.g. ppm, ppb) may also be reported 
parenthetically; 
— Particle size distribution, mass median aerodynamic diameter (MMAD), 
and geometric standard deviation ( σ g ), including their methods of calcu ­
lation. Individual particle size analyses should be reported;  
Test conditions  
— Details of test chemical preparation, including details of any procedures 
used to reduce the particle size of solid substances or to prepare 
solutions of the test chemical. In cases where mechanical processes may have altered test chemical composition, include the results of analyses to verify the composition of the test chemical; 
— A description (preferably including a diagram) of the equipment used to 
generate the test atmosphere and to expose the animals to the test atmos ­
phere; 
— Details of the chemical analytical method used and method validation 
(including efficiency of recovery of test chemical from the sampling medium); 
— The rationale for the selection of test concentrations;  
Results  
— Tabulation of chamber temperature, humidity, and airflow; — Tabulation of chamber nominal and actual concentration data; — Tabulation of particle size data including analytical sample collection 
data, particle size distribution, and calculations of the MMAD and σ
 g ; 
— Tabulation of response data and concentration level for each animal ( i.e. 
animals showing signs of toxicity including mortality, nature, severity, 
and duration of effects); 
— Individual body weights of animals collected on study days, date and 
time of death if prior to scheduled euthanasia; time course of onset of 
signs of toxicity, and whether these were reversible for each animal;  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 768
 
— Necropsy findings and histopathological findings for each animal, if 
available; 
— The CLP category classification and the LC  50 cut-off value;  
Discussion and interpretation of results  
— Particular emphasis should be made to the description of methods used 
to meet this Test Method’s criteria, e.g. the limit concentration or the 
particle size; 
— The respirability of particles in light of the overall findings should be 
addressed, especially if the particle-size criteria could not be met; 
— The consistency of methods used to determine nominal and actual 
concentrations, and the relation of actual concentration to nominal 
concentration should be included in the overall assessment of the study; 
— The likely cause of death and predominant mode of action (systemic 
versus local) should be addressed; 
— An explanation should be provided if there was a need to humanely 
sacrifice animals in pain or showing signs of severe and enduring 
distress, based on the criteria in the OECD Guidance Document on Humane Endpoints (7).
 
LITERATURE:  
(1) Chapter B.2 of this Annex, Acute Toxicity (Inhalation). 
(2) Holzhütter H-G, Genschow E, Diener W, and Schlede E (2003). Dermal 
and Inhalation Acute Toxicity Class Methods: Test Procedures and 
Biometric Evaluations for the Globally Harmonized Classification 
System.  Arch. Toxicol.  77: 243-254. 
(3) Diener W, Kayser D and Schlede E (1997). The Inhalation Acute-Toxic- 
Class Method; Test Procedures and Biometric Evaluations.  Arch. Toxicol.  
71: 537-549. 
(4) Diener W and Schlede E (1999). Acute Toxic Class Methods: Alternatives 
to LD/LC  50 Tests.  ALTEX  1: 129-134. 
(5) Chapter B.1 tris of this Annex, Acute Oral Toxicity — Acute Toxic Class 
Method. 
(6) OECD (2009). Report on Biostatistical Performance Assessment of the 
Draft TG 436 Acute Toxic Class Testing Method for Acute Inhalation Toxicity. Environmental Health and Safety Monograph Series on Testing and Assessment No 105, OECD, Paris. Available at: [http://www.oecd.org/ 
env/testguidelines] 
(7) OECD (2000). Guidance Document on the Recognition, Assessment and 
Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. Environmental Health and Safety Monograph 
Series on Testing and Assessment No 19. Available at: [http:// 
www.oecd.org/env/testguidelines] 
(8) OECD (2009). Guidance Document on Acute Inhalation Toxicity Testing. 
Environmental Health and Safety Monograph Series on Testing and 
Assessment No 39, OECD, Paris. Available at: [http://www.oecd.org/env/ 
testguidelines] 
(9) Regulation (EC) No 1272/2008 of the European Parliament and of the 
Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC 
and 1999/45/EC, and amending Regulation (EC) No 1907/2006 (OJ L 353, 
31.12.2008, p. 1).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 769
 
(10) Chapter B.40 of this Annex, In Vitro Skin Corrosion: Transcutaneous 
Electrical Resistance Test (TER). 
(11) Chapter B.40 bis of this Annex, In Vitro Skin Corrosion: Human Skin 
Model Test. 
(12) OECD (2005). In Vitro Membrane Barrier Test Method for Skin Corrosion. 
OECD Guideline for testing of chemicals No 435, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines] 
(13) Phalen RF (2009). Inhalation Studies: Foundations and Techniques. (2
 nd 
Edition) Informa Healthcare, New York. 
(14) SOT (1992). Technical Committee of the Inhalation Specialty Section, 
Society of Toxicology (SOT). Recommendations for the Conduct of Acute Inhalation Limit Tests.  Fund. Appl. Toxicol . 18: 321-327. 
(15) Pauluhn J and Thiel A (2007). A Simple Approach to Validation of 
Directed-Flow Nose-Only Inhalation Chambers. J. Appl. Toxicol. 27: 
160-167 
(16) UN (2007), United Nations Globally Harmonized System of Classification 
and Labelling of Chemicals (GHS), ST/SG/AC.10/30, UN New York and 
Geneva. Available: [http://www.unece.org/trans/danger/publi/ghs/ghs_wel ­
come_e.html]
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 770
 
Appendix 1  
DEFINITION  
Test chemical:  Any substance or mixture tested using this Test Method.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 771
 
Appendix 2  
Procedure to be followed by each of the starting concentrations for gases 
(ppm/4h)  
General remarks (  1 ) 
For each starting concentration, the respective testing schemes as included in this 
Appendix outline the procedure to be followed. 
Appendix 2a: Starting concentration is 100 ppm Appendix 2b: Starting concentration is 500 ppm 
Appendix 2c: Starting concentration is 2 500 ppm 
Appendix 2d: Starting concentration is 20 000 ppm Depending on the number of humanely killed or dead animals, the test procedure 
follows the indicated arrows.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 772  
( 1 ) In the following tables reference is made to GHS (Globally Harmonised System of 
Classification and Labelling of Chemicals (GHS). The EU equivalent is 
Regulation (EC) No 1272/2008. In the case of Acute Inhalation Toxicity, the 
Regulation (EC) No 1272/2008 (9) does not implement Category 5.
 Appendix 2a  
Acute Inhalation Toxicity:  
Test Procedure with a starting concentration of 100 ppm/4h for gases
  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 773
 
Appendix 2b  
Acute Inhalation Toxicity:  
Test Procedure with a starting concentration of 500 ppm/4h for gases
  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 774
 
Appendix 2c  
Acute Inhalation Toxicity:  
Test Procedure with a starting concentration of 2 500 ppm/4h for gases
  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 775
 
Appendix 2d  
Acute Inhalation Toxicity:  
Test Procedure with a starting concentration of 20 000 ppm/4h for gases
  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 776
 
Appendix 3  
Procedure to be followed by each of the starting concentrations for vapour 
(mg/l/4h)  
General remarks (  1 ) 
For each starting concentration, the respective testing schemes as included in this 
Appendix outline the procedure to be followed. 
Appendix 3a: Starting concentration is 0,5 mg/l Appendix 3b: Starting concentration is 2,0 mg/l 
Appendix 3c: Starting concentration is 10 mg/l 
Appendix 3d: Starting concentration is 20 mg/l Depending on the number of humanely killed or dead animals, the test procedure 
follows the indicated arrows.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 777  
( 1 ) In the following tables reference is made to GHS (Globally Harmonised System of 
Classification and Labelling of Chemicals (GHS). The EU equivalent is 
Regulation (EC) No 1272/2008. In the case of Acute Inhalation Toxicity, the 
Regulation (EC) No 1272/2008 (9) does not implement Category 5.
 Appendix 3a  
Acute Inhalation Toxicity:  
Test procedure with a starting concentration of 0,5 mg/L/4h for vapours
  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 778
 
Appendix 3b  
Acute Inhalation Toxicity:  
Test procedure with a starting concentration of 2 mg/L/4h for vapours
  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 779
 
Appendix 3c  
Acute Inhalation Toxicity:  
Test procedure with a starting concentration of 10 mg/L/4h for vapours
  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 780
 
Appendix 3d  
Acute Inhalation Toxicity:  
Test procedure with a starting concentration of 20 mg/L/4h for vapours
  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 781
 
Appendix 4  
Procedure to be followed by each of the starting concentrations for aerosols 
(mg/l/4h)  
General remarks (  1 ) 
For each starting concentration, the respective testing schemes as included in this 
Appendix outline the procedure to be followed. 
Appendix 4a: Starting concentration is 0,05 mg/l Appendix 4b: Starting concentration is 0,5 mg/l 
Appendix 4c: Starting concentration is 1 mg/l 
Appendix 4d: Starting concentration is 5 mg/l Depending on the number of humanely killed or dead animals, the test procedure 
follows the indicated arrows.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 782  
( 1 ) In the following tables reference is made to GHS (Globally Harmonised System of 
Classification and Labelling of Chemicals (GHS). The EU equivalent is 
Regulation (EC) No 1272/2008. In the case of Acute Inhalation Toxicity the 
Regulation (EC) No 1272/2008 (9) does not implement Category 5.
 Appendix 4a  
Acute Inhalation Toxicity:  
Test procedure with a starting concentration of 0,05 mg/L/4h for aerosols
  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 783
 
Appendix 4b  
Acute Inhalation Toxicity:  
Test procedure with a starting concentration of 0,5 mg/L/4h for aerosols
  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 784
 
Appendix 4c  
Acute Inhalation Toxicity:  
Test procedure with a starting concentration of 1 mg/L/4h for aerosols
  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 785
 
Appendix 4d  
Acute Inhalation Toxicity:  
Test procedure with a starting concentration of 5 mg/L/4h for_aerosols
  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 786
 
PART C: METHODS FOR THE DETERMINATION OF ECOTOXICITY  
TABLE OF CONTENTS 
C.1. ACUTE TOXICITY FOR FISH C.2. DAPHNIA  SP. ACUTE IMMOBILISATION TEST 
C.3. FRESHWATER ALGAE AND CYANOBACTERIA, GROWTH 
INHIBITION TEST 
C.4. DETERMINATION OF ‘READY’ BIODEGRADABILITY PART I. GENERAL CONSIDERATIONS PART II. DOC DIE-AWAY TEST (Method C.4-A) PART III. MODIFIED OECD SCREENING TEST (Method C.4-B) PART IV. CO
 2 EVOLUTION TEST (Method C.4-C) 
PART V. MANOMETRIC RESPIROMETRY TEST (Method C.4-D) PART VI. CLOSED BOTTLE TEST (Method C.4-E) PART VII. M.I.T.I. TEST (Method C.4-F) C.5. DEGRADATION — BIOCHEMICAL OXYGEN DEMAND C.6. DEGRADATION — CHEMICAL OXYGEN DEMAND C.7. DEGRADATION — ABIOTIC DEGRADATION: 
HYDROLYSIS AS A FUNCTION OF PH 
C.8. TOXICITY FOR EARTHWORMS C.9. BIODEGRADATION — ZAHN-WELLENS TEST C.10. SIMULATION TESTAEROBIC SEWAGE TREATMENT: C.10- 
A: ACTIVATED SLUDGE UNITS — C.10-B: BIOFILMS 
C.11. BIODEGRADATION — ACTIVATED SLUDGE 
RESPIRATION INHIBITION 
C.12. BIODEGRADATION — MODIFIED SCAS TEST C.13. BIOCONCENTRATION: FLOW-THROUGH FISH TEST C.14. FISH JUVENILE GROWTH TEST C.15. FISH, SHORT-TERM TOXICITY TEST ON  EMBRYO  AND 
SAC-FRY STAGES 
C.16. HONEYBEES — ACUTE ORAL TOXICITY TEST C.17. HONEYBEES — ACUTE CONTACT TOXICITY TEST C.18. ADSORPTION/DESORPTION USING A BATCH EQUI ­
LIBRIUM METHOD
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 787
 
C.19. ESTIMATION OF THE ADSORPTION COEFFICIENT ( K OC ) 
ON SOIL AND ON SEWAGE SLUDGE USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) 
C.20.  DAPHNIA MAGNA  REPRODUCTION TEST 
C.21. SOIL MICROORGANISMS: NITROGEN TRANSFORMATION 
TEST 
C.22. SOIL MICROORGANISMS: CARBON TRANSFORMATION 
TEST 
C.23. AEROBIC AND ANAEROBIC TRANSFORMATION IN SOIL 
C.24. AEROBIC AND ANAEROBIC TRANSFORMATION IN 
AQUATIC SEDIMENT SYSTEMS 
C.25. AEROBIC MINERALISATION IN SURFACE WATER — 
SIMULATION BIODEGRADATION TEST 
C.26.  LEMNA  SP. GROWTH INHIBITION TEST
 
▼M4 
C.27. SEDIMENT-WATER CHIRONOMID TOXICITY TEST USING 
SPIKED SEDIMENT 
C.28. SEDIMENT-WATER CHIRONOMID TOXICITY TEST USING 
SPIKED WATER 
C.29. READY BIODEGRADABILITY — CO  2 IN SEALED VESSELS 
(HEADSPACE TEST) 
C.30. BIOACCUMULATION IN TERRESTRIAL OLIGOCHAETES  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 788
 
C.1. ACUTE TOXICITY FOR FISH  
1. METHOD  
1.1. INTRODUCTION 
The purpose of this test is to determine the acute lethal toxicity of a 
substance to fish in fresh water. It is desirable to have, as far as 
possible, information on the water solubility, vapour pressure, chemical stability, dissociation constants and biodegradability of the substance to help in the selection of the most appropriate test 
method (static, semi-static or flow-through) for ensuring satisfactorily 
constant concentrations of the test substance over the period of the test. 
Additional information (for instance structural formula, degree of 
purity, nature and percentage of significant impurities, presence and amounts of additives, and n-octanol/water partition coefficient) 
should be taken into consideration in both the planning of the test 
and interpretation of the results.
 
1.2. DEFINITIONS AND UNITS 
Acute toxicity is the discernible adverse effect induced in an 
organism within a short time (days) of exposure to a substance. In 
the present test, acute toxicity is expressed as the median lethal 
concentration (LC  50 ) that is the concentration in water which kills 
50 % of a test batch of fish within a continuous period of exposure which must be stated. 
All concentrations of the test substance are given in weight by 
volume (milligrams per litre). They may also be expressed as weight by weight (mg/kg
 -1 ). 
1.3. REFERENCE SUBSTANCES 
A reference substance may be tested as a means of demonstrating 
that under the laboratory test conditions the response of tested species have not changed significantly. 
No reference substances are specified for this test.
 
1.4. PRINCIPLE OF THE TEST METHOD 
A limit test may be performed at 100 mg per litre in order to 
demonstrate that the LC  50 is greater than this concentration. 
The fish are exposed to the test substance added to water at a range 
of concentrations for a period of 96 hours. Mortalities are recorded at 
least at 24-hour intervals, and the concentrations killing 50 % of the 
fish (LCso) at each observation time are calculated where possible.  
1.5. QUALITY CRITERIA 
The quality criteria shall apply to the limit test as well as the full test 
method. 
The mortality in the controls must not exceed 10 % (or one fish if 
less than ten are used) by the end of the test. 
The dissolved oxygen concentration must have been more than 60 % 
of the air-saturation value throughout.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 789
 
The concentrations of the test substance shall be maintained to 
within 80 % of the initial concentrations throughout the duration 
of the test. 
For substances which dissolve easily in the test medium, yielding 
stable solutions i.e. those which will not to any significant extent 
volatilise, degrade, hydrolyze or adsorb, the initial concentration can 
be taken as being equivalent to the nominal concentration. Evidence shall be presented that the concentrations have been maintained throughout the test and that the quality criteria have been satisfied. 
For substances that are: 
(i) poorly soluble in the test medium, or 
(ii) capable of forming stable emulsions or dispersions, or 
(iii) not stable in aqueous solutions, 
the initial concentration shall be taken as the concentration measured 
in solution (or, if technically not possible, measured in the water column) at the start of the test. The concentration shall be determined after a period of equilibration but before the introduction 
of the test fish. 
In any of these cases, further measurements must be made during the 
test to confirm the actual exposure concentrations or that the quality 
criteria have been met. 
The pH should not vary by more than 1 unit.
 
1.6. DESCRIPTION OF THE TEST METHOD 
Three types of procedure can be used:  
Static test:  
Toxicity test in which no flow of test solution occurs. (Solutions 
remain unchanged throughout the duration of the test.)  
Semi-static test:  
Test without flow of test solution, but with regular batchwise 
renewal of test solutions after prolonged periods (e.g. 24 hours).  
Flow-through test:  
Toxicity test in which the water is renewed constantly in the test 
chambers, the chemical under test being transported with the water used to renew the test medium.
 
1.6.1.  Reagents  
1.6.1.1.  Solutions of test substances  
Stock solutions of the required strength are prepared by dissolving 
the substance in deionised water or water according to 1.6.1.2. 
The chosen test concentrations are prepared by dilution of the stock 
solution. If high concentrations are tested, the substance may be 
dissolved in the dilution water directly.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 790
 
The substances should normally only be tested up to the limit of 
solubility. For some substances (e.g. substances having low solu ­
bility in water, or high P  ow , or those forming stable dispersion 
rather than true solution in water), it is acceptable to run a test concentration above the solubility limit of the substance to ensure 
that the maximum soluble/stable concentration has been obtained. It 
is important, however, that this concentration will not otherwise disturb the test system (e.g. film of the substance on the water surface preventing the oxygenation of the water, etc.). 
Ultrasonic dispersion, organic solvents, emulsifiers or dispersants 
may be used as an aid to prepare stock solutions of substances with low aqueous solubility or to help to disperse these substances in the test medium. When such auxiliary substances are used, all test 
concentrations should contain the same amount of auxiliary 
substance, and additional control fish should be exposed to the same concentration of the auxiliary substance as that used in the test series. The concentration of such auxiliaries should be mini ­
mised, but in no case should exceed 100 mg per litre in the test 
medium. 
The test should be carried out without adjustment of the pH. If there 
is evidence of marked change in the pH, it is advised that the test 
should be repeated with pH adjustment and the results reported. In that case, the pH value of the stock solution should be adjusted to the pH value of the dilution water unless there are specific reasons 
not to do so. HCl and NaOH are preferred for this purpose. This pH 
adjustment should be made in such a way that the concentration of test substance in the stock solution is not changed to any significant extent. Should any chemical reaction or physical precipitation of the 
test compound be caused by the adjustment, this should be reported.
 
1.6.1.2.  Holding and dilution water  
Orinking-water supply (uncontaminated by potentially harmful 
concentrations of chlorine, heavy metals or other substances), good-quality natural water or reconstituted water (See Appendix 1) 
may be used. Waters with a total hardness of between 10 and 
250 mg per litre (as CaCO
 3 ) and with a pH from 6,0 to 8,5 are 
preferred.  
1.6.2.  Apparatus  
All apparatus must be made of chemically inert material: 
— automatic dilution system (for flow-through test), 
— oxygen meter, 
— equipment for determination of hardness of water, 
— adequate apparatus for temperature control, — pH meter.
 
1.6.3.  Test fish  
The fish should be in good health and free from any apparent 
malformation.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 791
 
The species used should be selected on the basis of practical criteria, 
such as their ready availability throughout the year, ease of main ­
tenance, convenience for testing, relative sensitivity to chemicals, and any economic, biological or ecological factors which have any bearing. The need for comparability of the data obtained and existing 
international harmonisation (reference 1) should also be borne in 
mind when selecting the fish species. 
A list of fish species which are recommended for the performance of 
this test is given in Appendix 2; Zebra fish and rainbow trout are the 
preferred species.
 
1.6.3.1.  Holding  
Test fish should preferably come from a single stock of similar 
length and age. The fish must be held for at least 12 days, in the following conditions:  
loading:  
appropriate to the system (recirculation or flow-through) and the fish 
species,  
water:  
see 1.6.1.2,  
light:  
12 to 16 hours illumination daily,  
dissolved oxygen concentration:  
at least 80 % of air-saturation value,  
feeding:  
three times per week or daily, ceasing 24 hours before the start of 
the test.
 
1.6.3.2.  Mortality  
Following a 48-hour settling-in period, mortalities are recorded and 
the following criteria applied: 
— greater than 10 % of population in seven days: 
rejection of entire batch, 
— between 5 and 10 % of population: 
holding period continued for seven additional days. 
If no further mortalities occur, the batch is acceptable, otherwise 
it must be rejected, 
— less than 5 % of population: 
acceptance of the batch.  
1.6.4.  Adaptation  
All fish must be exposed to water of the quality and the temperature 
to be used in the test for at least seven days before they are used.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 792
 
1.6.5.  Test procedure  
A range-finding test can precede a definitive test, in order to obtain 
information about the range of concentrations to be used in the main test. 
One control without the test substance is run and, if relevant, one 
control containing the auxiliary substance is also run, in addition to the test series. 
Depending on the physical and chemical properties of the test 
compound, a static, semi-static, or a flow-through test should be selected as appropriate, to fulfil the quality criteria. 
Fish are exposed to the substance as described below: 
— duration: 96 hours, — number of animals: at least seven per concentration, 
— tanks: of suitable capacity in relation to the recommended 
loading, 
— loading: maximum loading of 1 g per litre for static and semi- 
static tests is recommended; for flow-through systems, higher 
loading is acceptable, 
— test concentration: At least five concentrations differing by a 
constant factor not exceeding 2,2 and as far as possible 
spanning the range of 0 to 100 % mortality, 
— water: see 1.6.1.2, — light: 12 to 16 hours illumination daily, 
— temperature: appropriate to the species (Appendix 2) but within 
± 1
 o C within any particular test, 
— dissolved oxygen concentration: not less than 60 % of the air- 
saturation value at the selected temperature, 
— feeding: none. 
The fish are inspected after the first two to four hours and at least at 
24-hour intervals. Fish are considered dead if touching of the caudal 
peduncle produces no reaction, and no breathing movements are 
visible. Dead fish are removed when observed and mortalities are recorded. Records are kept of visible abnormalities (e.g. loss of equilibrium, changes in swimming behaviour, respiratory function, 
pigmentation, etc.). 
Measurements of pH, dissolved oxygen and temperature must be 
carried out daily.
 
Limit test  
Using the procedures described in this test method, a limit test may 
be performed at 100 mg per litre in order to demonstrate that the 
LC 50 is greater than this concentration. 
If the nature of the substance is such that a concentration of 100 mg 
per litre in the test water cannot be attained, the limit test should be performed at a concentration equal to the solubility of the substance 
(or the maximum concentration forming a stable dispersion) in the 
medium used (see also point 1.6.1.1).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 793
 
The limit test should be performed using seven to 10 fish, with the 
same number in the control(s). (Binomial theory dictates that when 
10 fish are used with zero mortality, there is a 99,9 % confidence that the LC
 50 is greater than the concentration used in the limit test. 
With 7, 8 or 9 fish, the absence of mortality provides at least 99 % 
confidence that the LC  50 is greater than the concentration used.) 
If mortalities occur, a full study must be carried out. If sublethal 
effects are observed, these should be recorded.  
2. DATA AND EVALUATION  
For each period where observations were recorded (24, 48, 72 and 
96 hours), plot percentage mortality for each recommended exposure 
period against concentration on logarithmic-probability paper. 
When possible and for each observation time, the LC  50 and the 
confidence limits (p = 0,05) should be estimated using standard 
procedures; these values should be rounded off to one, or at most two significant figures (examples of rounding off to two figures: 170 for 173,5; 0,13 for 0,127; 1,2 for 1,21). 
In those cases where the slope of the concentration/percentage 
response curve is too steep to permit calculation of the LC
 50 , a 
graphical estimate of this value is sufficient. 
When two consecutive concentrations, at a ratio of 2,2 give only 0 
and 100 % mortality, these two values are sufficient to indicate the 
range within which the LC  50 falls. 
If it is observed that the stability or homogeneity of the test 
substance cannot be maintained, this should be reported and care should be taken in the interpretation of the results.
 
3. REPORTING  
The test report shall, if possible, include the following information: 
— information about test fish (scientific name, strain, supplier, any 
pretreatment, size and number used in each test concentration), 
— dilution-water source and major chemical characteristics (pH, 
hardness, temperature), 
— in the case of a substance of low aqueous solubility, the method 
of preparation of stock and test solutions, 
— concentration of any auxiliary substances, 
— list of the concentrations used and any available information on 
the stability at the concentrations of the tested chemical in the 
test solution, 
— if chemical analyses are performed, methods used and results 
obtained, 
— results of the limit test if conducted, 
— reasons for the choice and details of the test procedure used (e.g. 
static, semi-static, dosing rate, flow-through rate, whether 
aerated, fish loading, etc.),  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 794
 
— description of test equipment, 
— lighting regime, — dissolved oxygen concentrations, pH values and temperatures of 
the test solutions every 24 hours, 
— evidence that the quality criteria have been fulfilled, 
— a table showing the cumulative mortality at each concentration 
and the control (and control with the auxiliary substance if 
required) at each of the recommended observation times, 
— graph of the concentration/percentage response curve at the end 
of the test, 
— if possible, the LC
 50 values at each of the recommended obser ­
vation times (with 95 % confidence limits), 
— statistical procedures used for determining the LC  50 values, 
— if a reference substance is used, the results obtained, 
— highest test concentration causing no mortality within the period 
of the test, 
— lowest test concentration causing 100 % mortality within the 
period of the test.  
4. REFERENCES  
(1) OECD, Paris, 1981, Test Guideline 203, Decision of the 
Council C(81) 30 final and updates. 
(2) AFNOR — Determination of the acute toxicity of a substance 
to Brachydanio rerio  — Static and Flow Through methods — 
NFT 90-303 June 1985. 
(3) AFNOR- Determination of the acute toxicity of a substance to  
Salmo gairdneri  — Static and Flow — Through methods — 
NFT 90-305 June 1985. 
(4) ISO 7346/1,/2 and/3 — Water Quality — Determination of the 
acute lethal toxicity of substances to a fresh water fish ( Bra­
chydanio rerio  Hamilton-Buchanan-Teleostei, Cyprinidae). Part 
1: Static method. Part 2: Semi-static method. Part 3: Flow- through method. 
(5) Eidgenössisches Department des Innern, Schweiz: Richtlinien 
fur Probenahme und Normung von Wasseruntersuchungsme ­
thoden -Part II 1974. 
6) DIN Testverfahren mit Wasserorganismen, 38 412 (11) und 1 
(15). 
(7) JIS K 0102, Acute toxicity test for fish. 
(8) NEN 6506- Water -Bepaling van de akute toxiciteit met behulp 
van Poecilia reticulata , 1980. 
9) Environmental Protection Agency, Methods for the acute 
toxicity tests with fish, macroinvertebrates and amphibians. The Committee on Methods for Toxicity Tests with Aquatic 
Organisms, Ecological Research Series EPA-660-75-009, 
1975. 
(10) Environmental Protection Agency, Environmental monitoring 
and support laboratory, Office of Research and Development, 
EPA-600/4-78-012, January 1978.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 795
 
11) Environmental Protection Agency, Toxic Substance Control, 
Part IV, 16 March 1979. 
(12) Standard methods for the examination of water and waste ­
water, fourteen edition, APHA-AWWA-WPCF,1975. 
13) Commission of the European Communities, Inter-laboratory 
test programme concerning the study of the ecotoxicityof a chemical substance with respect to the fish. EEC Study 
D.8368, 22 March 1979. 
(14) Verfahrensvorschlag des Umweltbundesamtes zum akuten 
Fisch-Test. Rudolph, P. und Boje, R. Okotoxikologie, Grundlagen fur die okotoxikologische Bewertung von Umwelt ­
chemikalien nach dem Chemikaliengesetz, ecomed 1986. 
(15) Litchfield, J. T.and Wilcoxon, F., A simplified method for 
evaluating dose effects experiments, J. Pharm, tExp. Therap., 1949, vol. 96,99. 
(16) Finney, D.J. Statistical Methods in Biological Assay. Griffin, 
Weycombe, U.K., 1978. 
17) Sprague, J.B. Measurement of pollutant toxicity to fish. 
Bioassay methods for acute toxicity. Water Res., 1969, vol. 3,793-821. 
(18) Sprague, J.B. Measurement of pollutant toxicity to fish. II 
Utilising and applying bioassay results. Water Res. 1970, 
vol. 4, 3-32. 
(19) Stephan, C.E. Methods for calculating an LC
 50 . In Aquatic 
Toxicology and Hazard Evaluation (edited by F.I. Mayer and J.L. Hamelink). American Society for Testing and Materials, 
ASTM STP 634,1977, 65-84. 
(20) Stephan, C.E., Busch, K.A., Smith, R., Burke, J. and Andrews, 
R.W. A computer program for calculating an LC
 50 . US EPA.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 796
 
Appendix 1  
Reconstituted water  
Example of a suitable dilution water  
All chemicals must be of analytical grade. 
The water should be good-quality distilled water, or deionised water with a 
conductivity of less than 5 μScm  -1 . 
Apparatus for distillation of water must not contain any parts made of copper.  
Stock solutions  
CaCI  2 . 2H  2 O (calcium chloride dihydrate): 
Dissolve in, and make up to 1 litre with water. 11,76 g 
MgSO  4 . 7H  2 O (magnesium sulphate heptahydrate): 
Dissolve in, and make up to 1litre with water. 4,93 g 
NaHCO  3 (sodium hydrogen carbonate): 
Dissolve in, and make up to 1 litre with water. 2,59 g 
KCI (potassium chloride): 
Dissolve in, and make up to 11itre with water. 0,23 g  
Reconstituted dilution water  
Mix 25 ml of each of the four stock solutions and make up to 1 litre with water. 
Aerate until the dissolved oxygen concentration equals the air-saturation value. The pH should be 7,8 ± 0,2. If necessary adjust the pH with NaOH (sodium hydroxide) or HCI (hydrochloric 
acid). 
The dilution water so prepared is set aside for about 12 hours and must not be 
further aerated. 
The sum of the Ca and Mg ions in this solution is 2,5 mmol per litre. The ratio 
of Ca:Mg ions is 4:1 and of Na:K ions is 10:1. The total alkalinity of this solution is 0,8 mmol per litre. 
Any deviation in the preparation of the dilution water must not change the 
composition or properties of the water.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 797
 
Appendix 2  
Fish species recommended for testing  
Recommended species Recommended 
range of test 
temperature (  o C) Recommended 
total length of test 
animal (cm)  
Brachydanio rerio (Teleostei, Cyprinidae)  
(Hamilton-Buchanan) Zebrab-fish 20 to 24 3,0 ± 0,5  
Pimephales promelas (Teleostei, Cyprinidae)  
(Rafinesque) Fathead minnow 20 to 24 5,0 ± 2,5  
Cyprinus carpio (Teleostei, Cyprinidae)  
(Linneaus 1758) Common carp 20 to 24 6,0 ± 2,0  
Oryzias latipes (Teleostei, Poeciliidae)  
Cyprinodontidae (Tomminck and Schlege 
1850) Red killifish 20 to 24 3,0 ± 1,0  
Poecilia reticulata (Teleostei, Poeciliidae)  
(Peters 1859) Guppy 20 to 24 3,0 ± 1,0  
Lepomis macrochirus (Teleostei, 
Centrarchidae)  (Rafinesque Linneaus 1758) 
Bluegill 20 to 24 5,0 ± 2,0  
Onchorhynchus mykiss (Teleostei, 
Salmonidae)  (Walbaum 1988) Rainbow trout 12 to 17 6,0 ± 2,0  
Leuciscus idus (Teleostei, Cyprinidae)  
(Linneaus 1758) Golden Orfe 20 to 24 6,0 ± 2,0  
Collection  
The fish listed above are easy to rear and/or are widely available throughout the 
year. They are capable of being bred and cultivated either in fish farms or in the 
laboratory, under disease — and parasite — controlled conditions, so that the test 
animal will be healthy and of known parentage. These fish are available in many parts of the world.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 798
 
Appendix 3  
Example of concentration: percentage mortality  
Example of determination of LC  50 using log-probit paper
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 799
 
C.2. DAPHNIA  SP. ACUTE IMMOBILISATION TEST  
1. METHOD  
This acute immobilisation testing method is equivalent to the OECD 
TG 202 (2004).  
1.1. INTRODUCTION 
This method describes an acute toxicity test to assess effects of 
chemicals towards daphnids. Existing test methods were used to 
the extent possible (1)(2)(3).  
1.2. DEFINITIONS 
In the context of this method, the following definitions are used:  
EC 50 : is the concentration estimated to immobilise 50 % of the 
daphnids within a stated exposure period. If another definition is 
used, this must be reported, together with its reference.  
Immobilisation:  those animals that are not able to swim within 
15 seconds, after gentle agitation of the test vessel are considered to be immobilised (even if they can still move their antennae).
 
1.3. PRINCIPLE OF THE TEST METHOD 
Young daphnids, aged less than 24 hours at the start of the test, are 
exposed to the test substance at a range of concentrations for a 
period of 48 hours. Immobilisation is recorded at 24 hours and 48 hours and compared with control values. The results are analysed in order to calculate the EC
 50 at 48h (see Section 1.2 for 
definitions). Determination of the EC  50 at 24h is optional.  
1.4. INFORMATION ON THE TEST SUBSTANCE 
The water solubility and the vapour pressure of the test substance 
should be known and a reliable analytical method for the quantifi ­
cation of the substance in the test solutions with reported recovery efficiency, and limit of determination should be available. Useful 
information includes the structural formula, purity of the substance, 
stability in water or light, P
 ow and results of a test for ready biode ­
gradability (see method C.4). 
Note: guidance for testing substances with physical chemical prop ­
erties that made them difficult to test is provided in (4).  
1.5. REFERENCE SUBSTANCES 
A reference substance may be tested for EC  50 as a means of assuring 
that the test conditions are reliable. Toxicants used in international ring-tests (1)(5) are recommended for this purpose (
 1 ). Test(s) with a 
reference substance should be done preferably every month and at 
least twice a year.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 800  
( 1 ) The results of these inter laboratory tests and a Technical Corrigendum to ISO 6341 give 
an EC  50 — 24 h of the potassium dichromate (K  2 Cr 2 O 7 ) within the range 0,6 mg/l to 
1,7 mg/l.
 1.6. QUALITY CRITERIA 
For a test to be valid, the following performance criteria apply: 
— in the controls, including the control containing the solubilising 
agent, not more that 10 % of the daphnids should have been 
immobilised; 
— the dissolved oxygen concentration at the end of the test should 
be ≥ 3 mg/l in control and test vessels. 
Note: For the first criterion, not more than 10 % of the control 
daphnids should show immobilisation or other signs of disease or 
stress, for example, discoloration, unusual behaviour such as trapping 
at surface of water.  
1.7. DESCRIPTION OF THE TEST METHOD  
1.7.1.  Apparatus  
Test vessels and other apparatus that will come into contact with the 
test solutions should be made entirely of glass or other chemically 
inert material. Test vessels will normally be glass test tubes or 
beakers; they should be cleaned before each use using standard laboratory procedures. Test vessels should be loosely covered to reduce the loss of water due to evaporation and to avoid the entry 
of dust into the solutions. Volatile substances should be tested in 
completely filled closed vessels, large enough to prevent oxygen becoming limiting or too low (see Section 1.6 and first paragraph of Section 1.8.3). 
In addition some or all of the following equipment will be used: 
oxygen-meter (with microelectrode or other suitable equipment for measuring dissolved oxygen in low volumes samples); pH-meter; adequate apparatus for temperature control; equipment for the deter ­
mination of total organic carbon concentration (TOC); equipment for 
the determination of chemical oxygen demand (COD); equipment for the determination of hardness, etc.
 
1.7.2.  Test organism  
Daphnia magna  Straus is the preferred test species although other 
suitable  Daphnia  species can be used in this test (e.g.  Daphnia 
pulex ). At the start of the test, the animals should be less than 
24 hours old and to reduce variability, it is strongly recommended they are not first brood progeny. They should be derived from a healthy stock (i.e. showing no signs of stress such as high mortality, 
presence of males and ephippia, delay in the production of the first 
brood, discoloured animals, etc.). All organisms used for a particular test should have originated from cultures established from the same stock of daphnids. The stock animals must be maintained in culture 
conditions (light, temperature, medium) similar to those to be used in 
the test. If the daphnids culture medium to be used in the test is different from that used for routine daphnids culture, it is good practice to include a pre-test acclimation period. For that, brood 
daphnids should be maintained in dilution water at the test 
temperature for at least 48 hours prior to the start of the test.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 801
 
1.7.3.  Holding and dilution water  
Natural water (surface or ground water), reconstituted water or dech ­
lorinated tap water are acceptable as holding and dilution water if daphnids will survive in it for the duration of the culturing, accli ­
mation and testing without showing signs of stress. Any water which 
conforms to the chemical characteristics of an acceptable dilution water as listed in Appendix 1 is suitable as a test water. It should be of constant quality during the period of the test. Reconstituted 
water can be made up by adding specific amounts of reagents of 
recognised analytical grade to deionised or distilled water. Examples of reconstituted water are given in (1) (6) and in Appendix 2. Note 
that media containing known chelating agents, such as M4 and 
M7 media in Appendix 2, should be avoided for testing substances containing metals. The pH should be in the range of 6 to 9. Hardness between 140 and 250 mg/l (as CaCO
 3 ) is recommended for  Daphnia 
magna , while lower hardness may be also appropriate for other  
Daphnia  species. The dilution water may be aerated prior to use 
for the test so that the dissolved oxygen concentration has reached saturation. 
If natural water is used, the quality parameters should be measured at 
least twice a year or whenever it is suspected that these character ­
istics may have changed significantly (see previous paragraph and Appendix 1). Measurements of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd, Ni) should also be made. If dechlorinated tap water is used, 
daily chlorine analysis is desirable. If the dilution water is from a 
surface or ground water source, conductivity and total organic carbon (TOC) or chemical oxygen demand (COD) should be measured.
 
1.7.4.  Test solutions  
Test solutions of the chosen concentrations are usually prepared by 
dilution of a stock solution. Stock solutions should preferably be 
prepared by dissolving the test substance in the dilution water. As 
far as possible, the use of solvents, emulsifiers or dispersants should be avoided. However, such compounds may be required in some cases in order to produce a suitably concentrated stock solution. 
Guidance for suitable solvents, emulsifiers and dispersants is given 
in (4). In any case, the test substance in the test solutions should not exceed the limit of solubility in the dilution water. 
The test should be carried out without the adjustment of pH. If the 
pH does not remain in the range 6-9, then a second test could be 
carried out, adjusting the pH of the stock solution to that of the 
diluition water before addition of the test substance. The pH adjustment should be made in such a way that the stock solution concentration is not changed to any significant extent and that no 
chemical reaction or precipitation of the test substance is caused. 
HCl and NaOH are preferred.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 802
 
1.8. PROCEDURE  
1.8.1.  Conditions of exposure  
1.8.1.1.  Test groups and controls  
Test vessels are filled with appropriate volumes of dilution water and 
solutions of test substance. Ratio of air/water volume in the vessel 
should be identical for test and control group. Daphnids are then placed into test vessels. At least 20 animals, preferably divided into four groups of five animals each, should be used at each test concen ­
tration and for the controls. At least 2 ml of test solution should be 
provided for each animal (i.e. a volume of 10 ml for five daphnids per test vessel). The test may be carried out using semi-static renewal 
or flow-through system when the concentration of the test substance 
is not stable. 
One dilution-water control series and also, if relevant, one control 
series containing the solubilising agent must be run in addition to the 
treatment series.
 
1.8.1.2.  Test concentrations  
A range-finding test may be conducted to determine the range of 
concentrations for the definitive test unless information on toxicity of the test substance is available. For this purpose, the daphnids are exposed to a series of widely spaced concentrations of the test 
substance. Five daphnids should be exposed to each test concen ­
tration for 48 hours or less, and no replicates are necessary. The exposure period may be shortened (e.g. 24 hours or less) if data suitable for the purpose of the range-finding test can be obtained 
in less time. 
At least five test concentrations should be used. They should be 
arranged in a geometric series with a separation factor preferably 
not exceeding 2,2. Justification should be provided if fewer than 
five concentrations are used. The highest concentration tested should preferably result in 100 % immobilisation, and the lowest concentration tested should preferably give no observable effect.
 
1.8.1.3.  Incubation conditions  
The temperature should be within the range of 18  o C and 22  o C, and 
for each single test it should be constant within ± 1  o C. A 16-hour 
light and eight-hour dark cycle is recommended. Complete darkness is also acceptable, especially for the test substances unstable in light. 
The test vessels must not be aerated during the test. The test is 
carried out without adjustment of pH. The daphnids should not be 
fed during the test.
 
1.8.1.4.  Duration  
The test duration is 48 hours.  
1.8.2.  Observations  
Each test vessel should be checked for immobilised daphnids at 24 
and 48 hours after the beginning of the test (see Section 1.2 for 
definitions). In addition to immobility, any abnormal behaviour or 
appearance should be reported.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 803
 
1.8.3.  Analytical measurements  
The dissolved oxygen and pH are measured at the beginning and end 
of the test in the control(s) and in the highest test substance concen ­
tration. The dissolved oxygen concentration in controls should be in 
compliance with the validity criterion (see Section 1.6). The pH 
should normally not vary by more than 1,5 units in any one test. The temperature is usually measured in control vessels or in ambient air and it should be recorded preferably continuously during the test 
or, as a minimum, at the beginning and end of the test. 
The concentration of the test substance should be measured, as a 
minimum, at the highest and lowest test concentration, at the 
beginning and end of the test (4). It is recommended that results be based on measured concentrations. However, if evidence is available to demonstrate that the concentration of the test 
substance has been satisfactorily maintained within ± 20 % of the 
nominal or measured initial concentration throughout the test, then the results can be based on nominal or measured initial values.
 
1.9. LIMIT TEST 
Using the procedures described in this Method, a limit test may be 
performed at 100 mg/l of test substance or up to its limit of solubility in the test medium (whichever is the lower) in order to demonstrate that the EC
 50 is greater than this concentration. The limit test should 
be performed using 20 daphnids (preferably divided into four groups 
of five), with the same number in the control(s). If any immobili ­
sation occurs, a full study should be conducted. Any observed abnormal behaviour should be recorded.
 
2. DATA  
Data should be summarised in tabular form, showing for each 
treatment group and control, the number of daphnids used, immo ­
bilisation at each observation. The percentages immobilised at 
24 hours and 48 hours are plotted against test concentrations. Data 
are analysed by appropriate statistical methods (e.g. probit analysis, etc.) to calculate the slopes of the curves and the EC
 50 with 95 % 
confidence limits (p = 0,05) (7) (8). 
Where the standard methods of calculating the EC  50 , are not 
applicable to the data obtained, the highest concentration causing 
no immobility and the lowest concentration producing 100 % immo ­
bility should be used as an approximation for the EC  50 (this being 
considered the geometric mean of these two concentrations).  
3. REPORTING  
3.1. TEST REPORT 
The test report must include the following: 
Test substance: 
— physical nature and relevant physical-chemical properties,  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 804
 
— chemical identification data, including purity. 
Test species: — source and species of  Daphnia , supplier of source (if known) and 
the culture conditions used (including source, kind and amount of 
food, feeding frequency). 
Test conditions: — description of test vessels: type of vessels, volume of solution, 
number of daphnids per test vessel, number of test vessels (rep ­
licates) per concentration, 
— methods of preparation of stock and test solutions including the 
use of any solvent or dispersants, concentrations used, 
— details of dilution water: source and water quality characteristics 
(pH, hardness, Ca/Mg ratio, Na/K ratio, alkalinity, conductivity, etc.); composition of reconstituted water if used, 
— incubation conditions: temperature, light intensity and period ­
icity, dissolved oxygen, pH, etc. 
Results: — the number and percentage of daphnids that were immobilised or 
showed any adverse effects (including abnormal behaviour) in the controls and in each treatment group, at each observation 
time and a description of the nature of the effects observed, 
— results and date of test performed with reference substance, if 
available, 
— the nominal test concentrations and the result of all analyses to 
determine the concentration of the test substance in the test 
vessels; the recovery efficiency of the method and the limit of 
determination should also be reported, 
— all physical-chemical measurements of temperature, pH and 
dissolved oxygen made during the test, 
— the EC
 50 at 48 h for immobilisation with confidence intervals and 
graphs of the fitted model used for their calculation, the slopes of 
the dose-response curves and their standard error; statistical procedures used for determination of EC
 50 ; (these data items 
for immobilisation at 24 h should also be reported when they 
were measured), 
— explanation for any deviation from the Testig Method and 
whether the deviation affected the test results.  
4. REFERENCES  
(1) ISO 6341. (1996). Water quality — Determination of the 
inhibition of the mobility of  Daphnia magna  Straus (Cladocera, 
Crustacea) — Acute toxicity test. Third edition, 1996. 
(2) EPA OPPTS 850.1010. (1996). Ecological Effects Test 
Guidelines — Aquatic Invertebrate Acute Toxicity Test, 
Freshwater Daphnids.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 805
 
(3) Environment Canada. (1996) Biological test method. Acute 
Lethality Test Using  Daphnia  spp. EPS 1/RM/11. Environment 
Canada, Ottawa, Ontario, Canada. 
(4) Guidance Document on Aquatic Toxicity Testing of Difficult 
Substances and Mixtures. OECD Environmental Health and Safety Publication. Series on Testing and Assessment. 
No 23. Paris 2000. 
(5) Commission of the European Communities. Study D8369. 
(1979). Inter-laboratory Test Programme concerning the study of the ecotoxicity of a chemical substance with respect to  
Daphnia.  
(6) OECD Guidelines for the Testing of Chemicals. Guideline 211:  
Daphnia magna  Reproduction Test, adopted September 1998. 
(7) Stephan C.E. (1977). Methods for calculating an LC
 50 . In 
Aquatic Toxicology and Hazard Evaluation (edited by F.I. Mayer and J.L. Hamelink). ASTM STP 634 — American 
Society for Testing and Materials. p. 65-84 
(8) Finney D.J. (1978). Statistical Methods in Biological Assay. 3
 rd 
ed. London. Griffin, Weycombe, UK.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 806
 
Appendix 1  
SOME CHEMICAL CHARACTERISTICS OF AN ACCEPTABLE 
DILUTION WATER  
Substance Concentration  
Particulate matter < 20 mg/l 
Total organic carbon < 2 mg/l 
Unionised ammonia < 1 μg/l 
Residual chlorine < 10 μg/l 
Total organophosphorus pesticides < 50 ng/l Total organochorine pesticides plus polychlorinated 
biphenyls < 50 ng/l 
Total organic chlorine < 25 ng/l
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 807
 
Appendix 2  
EXAMPLES OF SUITABLE RECONSTITUTED TEST WATER  
ISO Test water (1)  
Stock solutions (single substance) To prepare the reconstituted 
water, add the following 
volumes of stock solutions to 1 
litre water (*) Substance Amount added to 1 litre 
water (*)  
Calcium chloride 
CaCl  2 , 2H  2 O 11,76 g 25 ml 
Magnesium sulfate 
MgSO  4 , 7H  2 O 4,93 g 25 ml 
Sodium bicarbonate 
NaHCO  3 2,59 g 25 ml 
Potassium chloride 
KCl 0,23 g 25 ml  
(*) Water of suitable purity, e.g. deionised, distilled or reverse osmosis with conductivity 
preferably not exceeding 10 μS.cm  -1 . 
Elendt M7 and M4 medium  
Acclimation to Elendt M4 and M7 medium  
Some laboratories have experienced difficulty in directly transferring  Daphnia  to 
M4 and M7 media. However, some success has been achieved with gradual acclimation, i.e. moving from own medium to 30 % Elendt, then to 60 % Elendt and then to 100 % Elendt. The acclimation periods may need to be as 
long as one month.
 
Preparation  
Trace element  
Separate stock solutions (I) of individual trace elements are first prepared in 
water of suitable purity, e.g. deionised, distilled or reverse osmosis. From 
these different stock solutions (I) a second single stock solution (II) is 
prepared, which contains all trace elements (combined solution), i.e.:  
Stock solution(s) I (single 
substance) Amount added to water 
(mg/l) Concentration (related 
to medium M4) To prepare the combined stock solution II, add 
the following amount of stock solution I to 
water (ml/l) 
M4 M7  
H 3 BO 3 57 190 20 000-fold 1,0 0,25 
MnCl  2 .4H  2 O 7 210 20 000-fold 1,0 0,25 
LiCl 6 120 20 000-fold 1,0 0,25  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 808
 
Stock solution(s) I (single 
substance) Amount added to water 
(mg/l) Concentration (related 
to medium M4) To prepare the combined stock solution II, add 
the following amount of stock solution I to 
water (ml/l) 
M4 M7  
RbCl 1 420 20 000-fold 1,0 0,25 
SrCl  2 .6H  2 O 3 040 20 000-fold 1,0 0,25 
NaBr 320 20 000-fold 1,0 0,25 Na
 2 MoO  4 .2H  2 O 1 230 20 000-fold 1,0 0,25 
CuCl  2 .2H  2 O 335 20 000-fold 1,0 0,25 
ZnCl  2 260 20 000-fold 1,0 1,0 
CoCl  2 .6H  2 O 200 20 000-fold 1,0 1,0 
KI 65 20 000-fold 1,0 1,0 Na
 2 SeO  3 43,8 20 000-fold 1,0 1,0 
NH  4 VO  3 11,5 20 000-fold 1,0 1,0 
Na 2 EDTA.2H  2 O 5 000 2 000-fold — — 
FeSO  4 .7H  2 O 1 991 2 000-fold — — 
Both Na 2 EDTA and FeSO  4 solutions are prepared singly, poured together and autoclaved immediately. 
This gives: 
2 l Fe-EDTA solution 1 000-fold 20,0 5,0  
M4 and M7 media  
M4 and M7 media are prepared using stock solution II, the macro-nutrients and 
vitamin as follows:  
Amount added to water 
(mg/l) Concentration (related 
to medium M4) Amount of stock solution II added to prepare 
medium (ml/l) 
M4 M7  
Stock solution II (combined 
trace elements) 20-fold 50 50 
Macro nutrient stock 
solutions (single substance) 
CaCl  2 · 2H  2 0 293 800 1 000-fold 1,0 1,0 
MgSO  4 · 7H  2 O 246 600 2 000-fold 0,5 0,5 
KCl 58 000 10 000-fold 0,1 0,1 
NaHCO  3 64 800 1 000-fold 1,0 1,0 
Na 2 SiO  3 · 9H  2 O 50 000 5 000-fold 0,2 0,2 
NaNO  3 2 740 10 000-fold 0,1 0,1 
KH  2 PO 4 1 430 10 000-fold 0,1 0,1 
K 2 HPO  4 1 840 10 000-fold 0,1 0,1  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 809
 
Amount added to water 
(mg/l) Concentration (related 
to medium M4) Amount of stock solution II added to prepare 
medium (ml/l) 
M4 M7  
Combined Vitamin stock — 10 000-fold 0,1 0,1 
The combined vitamin stock solution is prepared by adding the 3 vitamin to 1 litre water, as shown below: 
Thiamine hydrochloride 750 10 000-fold 
Cyanocobalamine (B  12 ) 10 10 000-fold 
Biotine 7,5 10 000-fold 
The combined vitamin stock is stored frozen in small aliquots. Vitamins are 
added to the media shortly before use.  
N.B: to avoid precipitation of salts when preparing the complete media, add the 
aliquots of stock solutions to about 500-800 ml deionised water and then fill up to 1 litre.  
N.B: the first publication of the M4 medium can be found in Elendt, 
B. P. (1990). Selenium deficiency in crustacea; an ultrastructual approach to antennal damage in  Daphnia magna  Straus. Protoplasma, 
154, 25-33.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 810
 
C.3. FRESHWATER ALGAE AND CYANOBACTERIA, GROWTH 
INHIBITION TEST  
1. METHOD  
This method is equivalent to OECD TG 201 (2006) (1).  
1.1. INTRODUCTION 
Testing Methods are periodically reviewed and updated in the light 
of scientific progress. Testing Method C.3 needed to be revised to include additional species and to meet the requirements for hazard assessment and classification of chemicals. The revision has been 
completed on the basis of extensive practical experience, scientific 
progress in the field of algal toxicity studies, and extensive regu ­
latory use, which has occurred since the original adoption.
 
1.2. DEFINITIONS 
The following definitions and abbreviations are used for the purposes 
of this Testing Method:  
Biomass : is the dry weight of living matter present in a population 
expressed in terms of a given volume; e.g. mg algae/litre test 
solution. Usually ‘biomass’ is defined as a mass, but in this test this word is used to refer to mass per volume. Also in this test, surrogates for biomass, such as cell counts, fluorescence, etc. are 
typically measured and the use of the term ‘biomass’ thus refers to 
these surrogate measures as well.  
Coefficient of variation : is a dimensionless measure of the varia ­
bility of a parameter, defined as the ratio of the standard deviation to 
the mean. This can also be expressed as a percentage value. The mean coefficient of variation of average specific growth rate in replicate control cultures should be calculated as follows: 
1. Calculate % CV of average specific growth rate out of the daily/ ­
section-by-section growth rates for the respective replicate. 
2. Calculate the mean value of all the values calculated in point 1 to 
get the mean coefficient of variation of the daily/section-by- section specific growth rate in replicate control cultures.  
EC
 x : is the concentration of the test substance dissolved in the test 
medium that results in an x % (e.g. 50 %) reduction in growth of the test organism within a stated exposure period (to be mentioned 
explicitly if deviating from full or normal test duration). To 
unambiguously denote an EC value deriving from the growth rate or from the yield, the symbols ‘E
 r C’ and ‘E  y C’ are used respectively.  
Growth medium : is the complete synthetic culture medium in which 
test algae grow when exposed to the test substance. The test substance will normally be dissolved in the test medium.  
Growth rate (average specific growth rate) : is the logarithmic 
increase in biomass during the exposure period.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 811
 
Lowest Observed Effect Concentration (LOEC) : is the lowest 
tested concentration at which the substance is observed to have a 
statistically significant reducing effect on growth (at p < 0,05) when compared with the control, within a given exposure time. However, all test concentrations above the LOEC must have a harmful effect 
equal to or greater than those observed at the LOEC. When these 
two conditions cannot be satisfied, a full explanation must be given for how the LOEC (and hence the NOEC) has been selected.  
No Observed Effect Concentration (NOEC) : is the test concen ­
tration immediately below the LOEC.  
Response variable : is a variable for the estimation of toxicity 
derived from any measured parameters describing biomass by 
different methods of calculation. For this method, growth rates and yield are response variables derived from measuring biomass directly or any of the surrogates mentioned.  
Specific growth rate : is a response variable defined as the quotient 
of the difference of the natural logarithms of a parameter of obser ­
vation (in this Testing Method, biomass) and the respective time period.  
Yield : is the value of a measurement variable at the end of the 
exposure period minus the measurement variable’s value at the 
start of the exposure period to express biomass increase during the 
test.
 
1.3. APPLICABILITY OF THE TEST 
This Testing Method is most easily applied to water-soluble 
substances which, under the conditions of the test, are likely to remain in the water. For testing of substances that are volatile, 
strongly adsorbing, coloured, having a low solubility in water or 
substances that may affect the availability of nutrients or minerals in the test medium, certain modifications of the described procedure may be required (e.g. closed system, conditioning of the test vessels). 
Guidance on some appropriate modifications is given in (2), (3) 
and (4).
 
1.4. PRINCIPLE OF THE TEST 
The purpose of this test is to determine the effects of a substance on 
the growth of freshwater microalgae and/or cyanobacteria. Exponen ­
tially growing test organisms are exposed to the test substance in batch cultures over a period of normally 72 hours. In spite of the 
relatively brief test duration, effects over several generations can be 
assessed. 
The system response is the reduction of growth in a series of algal 
cultures (test units) exposed to various concentrations of a test substance. The response is evaluated as a function of the exposure 
concentration in comparison with the average growth of replicate, 
unexposed control cultures. For full expression of the system response to toxic effects (optimal sensitivity), the cultures are allowed unrestricted exponential growth under sufficient nutrient 
conditions and continuous light for a sufficient period of time to 
measure reduction of the specific growth rate.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 812
 
Growth and growth inhibition are quantified by measurements of the 
algal biomass as a function of time. Algal biomass is defined as the 
dry weight per volume, e.g. mg algae/litre test solution. However, dry weight is difficult to measure and therefore surrogate parameters are used. Of these surrogates, cell counts are most often used. Other 
surrogate parameters include cell volume, fluorescence, optical 
density, etc. A conversion factor between the measured surrogate parameter and biomass should be known. 
The test endpoint is inhibition of growth, expressed as the logar ­
ithmic increase in biomass (average specific growth rate) during the 
exposure period. From the average specific growth rates recorded in a series of test solutions, the concentration bringing about a specified x % inhibition of growth rate (e.g. 50 %) is determined and 
expressed as the E
 r C x (e.g. E  r C 50 ). 
For the application of this method within the EU regulatory 
framework, calculation of results should be based on an average specific growth rate for the reasons described in section 2.2 below. 
An additional response variable used in this Testing Method is yield, 
which may be needed to fulfil specific regulatory requirements in some countries. It is defined as biomass at the end of the exposure period minus the biomass at the start of the exposure period. From 
the yield recorded in a series of test solutions, the concentration 
bringing about a specified x % inhibition of yield (e.g. 50 %) is calculated and expressed as the E
 y C x (e.g. E  y C 50 ). 
In addition, the lowest observed effect concentration (LOEC) and the 
no observed effect concentration (NOEC) may be statistically deter ­
mined.  
1.5. INFORMATION ON THE TEST SUBSTANCE 
Information on the test substance, which may be useful in estab ­
lishing the test conditions, includes the structural formula, purity, 
stability in light, stability under the conditions of the test, light 
absorption properties, pKa, and results of studies of transformation including biodegradability in water. 
The water solubility, octanol water partition coefficient (P
 ow ) and the 
vapour pressure of the test substance should be known, and a 
validated method for the quantification of the substance in the test solutions with reported recovery efficiency and limit of detection should be available.
 
1.6. REFERENCE SUBSTANCE 
Reference substance(s), such as 3,5-dichlorophenol used in the inter ­
national ring test (4), may be tested as a means of checking the test procedure. Potassium dichromate can also be used as a reference substance for green algae. It is desirable to test a reference 
substance at least twice a year.
 
1.7. VALIDITY OF THE TEST 
For the test to be valid, the following performance criteria should be 
met: ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 813
 
— The biomass in the control cultures should have increased 
exponentially by a factor of at least 16 within the 72-hour test 
period. This corresponds to a specific growth rate of 0,92 day  –1 . 
For the most frequently used species, the growth rate is usually substantially higher (see Appendix 1). This criterion may not be 
met when species that grow slower than those listed in 
Appendix 1 are used. In this case, the test period should be extended to obtain at least a 16-fold growth in control cultures, while the growth has to be exponential throughout the test 
period. The test period may be shortened to at least 48 h to 
maintain unlimited exponential growth during the test, as long as the minimum multiplication factor of 16 is reached. 
— The mean coefficient of variation for section-by-section specific 
growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures (see section 1.2 under ‘coefficient of variation’) must not exceed 35 %. See the second paragraph in section 2.2.1 
for the calculation of section-by-section specific growth rate. This 
criterion applies to the mean value of coefficients of variation calculated for replicate control cultures. 
— The coefficient of variation of average specific growth rates 
during the whole test period in replicate control cultures must not exceed 7 % in tests with  Pseudokirchneriella subcapitata  and 
Desmodesmus subspicatus . For other less frequently tested 
species, the value should not exceed 10 %.
 
1.8. DESCRIPTION OF THE METHOD  
1.8.1.  Apparatus  
Test vessels and other apparatus, which will come into contact with 
the test solutions should be made entirely of glass or other 
chemically inert material. The items should be thoroughly washed to ensure that no organic or inorganic contaminants may interfere with the algal growth or composition of the test solutions. 
The test vessels will normally be glass flasks of dimensions that 
allow a sufficient volume of culture for measurements during the 
test and a sufficient mass transfer of CO
 2 from the atmosphere 
(see the second paragraph in section 1.8.9). Note that the liquid volume must be sufficient for analytical determinations (see the fifth paragraph in section 1.8.11). 
In addition, some or all of the following equipment will be required: 
— Culturing apparatus: a cabinet or chamber is recommended, in which 
the chosen incubation temperature can be maintained at ± 2 °C. 
— Light measurement instruments: it is important to note that the 
method of measurement of light intensity, and in particular the 
type of receptor (collector), will affect the measured value. 
Measurements should preferably be made using a spherical (4 π) receptor (which responds to direct and reflected light 
from all angles above and below the plane of measurement), or 
a 2 π receptor (which responds to light from all angles above the 
measurement plane).
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 814
 
— Apparatus to determine algal biomass. Cell count, which is the 
most frequently used surrogate parameter for algal biomass, may 
be made using an electronic particle counter, a microscope with counting chamber, or a flow cytometer. Other biomass surrogates can be measured using a flow cytometer, fluorimeter, spectrop ­
hotometer or colorimeter. A conversion factor relating cell count 
to dry weight is useful to calculate. In order to provide useful measurements at low biomass concentrations when using a spec ­
trophotometer, it may be necessary to use cuvettes with a light 
path of at least 4 cm.
 
1.8.2.  Test organisms  
Several species of non-attached microalgae and cyanobacteria may 
be used. The strains listed in Appendix 1 have been shown to be suitable using the test procedure specified in this Testing Method. 
If other species are used, the strain and/or origin should be reported. 
It has to be confirmed that exponential growth of the selected test alga can be maintained throughout the test period under the prevailing conditions.
 
1.8.3.  Growth medium  
Two alternative growth media, the OECD and the AAP medium, are 
recommended. The compositions of these media are shown in Appendix 2. Note that the initial pH value and the buffering capacity (regulating pH increase) of the two media are different. 
Therefore the results of the tests may be different depending on 
the medium used, particularly when testing ionising substances. 
Modification of the growth media may be necessary for certain 
purposes, e.g. when testing metals and chelating agents or testing 
at different pH values. Use of a modified medium must be described 
in detail and justified (3)(4).
 
1.8.4.  Initial biomass concentration  
The initial biomass in the test cultures must be the same in all test 
cultures and sufficiently low to allow exponential growth throughout 
the incubation period without risk of nutrient depletion. The initial 
biomass should not exceed 0,5 mg/l as dry weight. The following initial cell concentrations are recommended:  
Pseudokirchneriella subcapitata  5 × 10
 3 -10 4 cells/ml  
Desmodesmus subspicatus  2-5 × 10  3 cells/ml  
Navicula pelliculosa  10  4 cells/ml  
Anabaena flos-aquae  10  4 cells/ml  
Synechococcus leopoliensis  5 × 10  4 -10 5 cells/ml  
1.8.5.  Concentrations of test substance  
The concentration range in which effects are likely to occur may be 
determined on the basis of results from range-finding tests. For the 
final definitive test, at least five concentrations arranged in a 
geometric series with a factor not exceeding 3,2 should be selected. For test substances showing a flat concentration response curve, a higher factor may be justified. The concentration series 
should preferably cover the range causing 5-75 % inhibition of 
algal growth rate.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 815
 
1.8.6.  Replicates and controls  
The test design should include three replicates at each test concen ­
tration. If determination of the NOEC is not required, the test design may be altered to increase the number of concentrations and reduce 
the number of replicates per concentration. The number of control 
replicates must be at least three, and ideally should be twice the number of replicates used for each test concentration. 
A separate set of test solutions may be prepared for analytical deter ­
minations of test substance concentrations (see the fourth and sixth 
paragraphs in section 1.8.11). 
When a solvent is used to solubilise the test substance, additional 
controls containing the solvent at the same concentration as used in 
the test cultures must be included in the test design.
 
1.8.7.  Preparation of inoculum culture  
In order to adapt the test alga to the test conditions and ensure that 
the algae are in the exponential growth phase when used to inoculate the test solutions, an inoculum culture in the test medium is prepared 
2-4 days before start of the test. The algal biomass should be 
adjusted in order to allow exponential growth to prevail in the inoculum culture until the test starts. The inoculum culture shall be incubated under the same conditions as the test cultures. 
Measure the increase in biomass in the inoculum culture to ensure 
that growth is within the normal range for the test strain under the culturing conditions. An example of the procedure for algal culturing is described in Appendix 3. To avoid synchronous cell divisions 
during the test, a second propagation step of the inoculum culture 
may be required.
 
1.8.8.  Preparation of test solutions  
All test solutions must contain the same concentrations of growth 
medium and initial biomass of test algae. Test solutions of the 
chosen concentrations are usually prepared by mixing a stock 
solution of the test substance with growth medium and inoculum culture. Stock solutions are normally prepared by dissolving the substance in test medium. 
Solvents, e.g. acetone, t-butyl alcohol and dimethyl formamide, may 
be used as carriers to add substances of low water solubility to the test medium (2)(3). The solvent concentration should not exceed 100 μl/l, and the same concentration of solvent should be added to all 
cultures (including controls) in the test series.
 
1.8.9.  Incubation  
Cap the test vessels with air-permeable stoppers. The vessels are 
shaken and placed in the culturing apparatus. During the test it is necessary to keep the algae in suspension and to facilitate transfer of CO
 2 . To this end, constant shaking or stirring should be used. The 
cultures should be maintained at a temperature in the range of 21 
to 24 °C, controlled at ± 2 °C. For species other than those listed in Appendix 1, e.g. tropical species, higher temperatures may be appro ­
priate, providing that the validity criteria can be fulfilled. It is recom ­
mended to place the flasks randomly and to reposition them daily in 
the incubator.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 816
 
The pH of the control medium should not increase by more than 1,5 
units during the test. For metals and compounds that partly ionise at 
a pH around the test pH, it may be necessary to limit the pH drift to obtain reproducible and well defined results. A drift of < 0,5 pH units is technically feasible and can be achieved by ensuring an 
adequate CO
 2 mass transfer rate from the surrounding air to the 
test solution, e.g. by increasing the shaking rate. Another possibility is to reduce the demand for CO
 2 by reducing the initial biomass or 
the test duration. 
The surface where the cultures are incubated should receive 
continuous, uniform fluorescent illumination e.g. of ‘cool-white’ or 
‘daylight’ type. Strains of algae and cyanobacteria vary in their light requirements. The light intensity should be selected to suit the test organism used. For the recommended species of green algae, the 
light intensity at the level of the test solutions shall be selected 
from the range of 60-120 μE·m
 –2 ·s –1 when measured in the photo ­
synthetically effective wavelength range of 400-700 nm using an appropriate receptor. Some species, in particular  Anabaena flos- 
aquae , grow well at lower light intensities and may be damaged at 
high intensities. For such species an average light intensity in the range 40-60 μE·m
 –2 ·s –1 should be selected. (For light-measuring 
instruments calibrated in lux, an equivalent range of 4 440-8 880 
lux for cool white light corresponds approximately to the recom ­
mended light intensity 60-120 μE·m  –2 ·s –1 ). The light intensity 
shall not vary more than ± 15 % from the average light intensity over the incubation area.
 
1.8.10.  Test duration  
The test duration is normally 72 hours. However, shorter or longer 
test durations may be used, provided that all validity criteria in 
section 1.7 can be met.  
1.8.11.  Measurements and analytical determinations  
The algal biomass in each flask is determined at least daily during 
the test period. If measurements are made on small volumes removed from the test solution by pipette, these should not be replaced. 
Measurement of biomass is done by manual cell counting by 
microscope or an electronic particle counter (by cell counts and/or biovolume). Alternative techniques, e.g. flow cytometry, in vitro or in vivo chlorophyll fluorescence (6)(7), or optical density can be 
used providing a satisfactory correlation with biomass can be demon ­
strated over the range of biomass occurring in the test. 
The pH of the solutions shall be measured at the beginning and at 
the end of the test. 
Provided an analytical procedure for determination of the test 
substance in the concentration range used is available, the test 
solutions should be analysed to verify the initial concentrations 
and maintenance of the exposure concentrations during the test.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 817
 
Analysis of the concentration of the test substance at the start and 
end of the test of a low and high test concentration, and a concen ­
tration around the expected EC  50 may be sufficient where it is likely 
that exposure concentrations will vary less than 20 % from nominal values during the test. Analysis of all test concentrations at the start 
and end of the test is recommended where concentrations are 
unlikely to remain within 80-120 % of nominal. For volatile, unstable or strongly adsorbing test substances, additional sampling for analysis at 24 hour intervals during the exposure period is recom ­
mended in order to better define loss of the test substance. For these 
substances, extra replicates will be needed. In all cases, deter ­
mination of test substance concentrations need only be performed on one replicate vessel at each test concentration (or on the contents 
of the vessels pooled by replicate). 
Test media prepared specifically for analysis of exposure concen ­
trations during the test should be treated identically to those used 
for testing, i.e. they should be inoculated with algae and incubated 
under identical conditions. If analysis of the dissolved test substance concentration is required, it may be necessary to separate algae from the medium. Separation should preferably be made by centrifugation 
at a low g-force, sufficient to settle the algae. 
If there is evidence that the concentration of the substance being 
tested has been satisfactorily maintained within ± 20 % of the 
nominal or measured initial concentration throughout the test, analysis of the results can be based on nominal or measured initial values. If the deviation from the nominal or measured initial concen ­
tration is greater than ± 20 %, analysis of the results should be based 
on geometric mean concentration during exposure or on models describing the decline of the concentration of test substance (3)(8). 
The alga growth inhibition test is a more dynamic test system than 
most other short-term aquatic toxicity tests. As a consequence, the actual exposure concentrations may be difficult to define, especially for adsorbing substances tested at low concentrations. In such cases, 
disappearance of the substance from solution by adsorption to the 
increasing algal biomass does not mean that it is lost from the test system. When the result of the test is analysed, it should be checked whether a decrease in concentration of the test substance in the 
course of the test is accompanied by a decrease in growth inhibition. 
If this is the case, application of a suitable model describing the decline of the concentration of test substance (8) may be considered. 
If not, it may be appropriate to base the analysis of the results on the 
initial (nominal or measured) concentrations.
 
1.8.12.  Other observations  
Microscopic observation should be performed to verify a normal and 
healthy appearance of the inoculum culture and to observe any 
abnormal appearance of the algae (as may be caused by exposure 
to the test substance) at the end of the test.  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 818
 
1.8.13.  Limit test  
Under some circumstances, e.g. when a preliminary test indicates 
that the test substance has no toxic effects at concentrations up to 100 mg·l
 –1 or up to its limit of solubility in the test medium 
(whichever is the lower), a limit test involving a comparison 
of responses in a control group and one treatment group (100 mg·l
 –1 or a concentration equal to the limit of solubility), 
may be undertaken. It is strongly recommended that this be 
supported by analysis of the exposure concentration. All previously 
described test conditions and validity criteria apply to a limit test, with the exception that the number of treatment replicates should be 
at least six. The response variables in the control and treatment 
group may be analysed using a statistical test to compare means, e.g. a Student'’t's t-test. If variances of the two groups are unequal, a t-test adjusted for unequal variances should be performed.
 
1.8.14.  Modification for strongly coloured substances  
The irradiation (light intensity) should be in the highest end of the 
range prescribed in this Testing Method: 120 μE m-2 s-1 or higher. 
The light path should be shortened by reduction of the volume of the 
test solutions (in the range of 5-25 ml). 
Sufficient agitation (for example by moderate shaking) should be 
performed in order to obtain a high frequency of exposure of the 
algae to high irradiation at the surface of the culture.  
2. DATA  
2.1. PLOTTING GROWTH CURVES 
The biomass in the test vessels may be expressed in units of the 
surrogate parameter used for measurement (e.g. cell number, fluor ­
escence). 
Tabulate the estimated biomass concentration in test cultures and 
controls together with the concentrations of test material and the 
times of measurement, recorded with a resolution of at least 
whole hours, to produce plots of growth curves. Both logarithmic scales and linear scales can be useful at this first stage, but logar ­
ithmic scales are mandatory and generally give a better presentation 
of variations in growth pattern during the test period. Note that 
exponential growth produces a straight line when plotted on a logar ­
ithmic scale, and that the inclination of the line (slope) indicates the specific growth rate. 
Using the plots, examine whether control cultures grow exponen ­
tially at the expected rate throughout the test. Critically examine all data points and the appearance of the graphs, and check raw 
data and procedures for possible errors. Check in particular any 
data point that seems to deviate by a systematic error. If it is obvious that procedural mistakes can be identified and/or considered highly likely, the specific data point is marked as an outlier and not 
included in subsequent statistical analysis. (A zero algal concen ­
tration in one out of two or three replicate vessels may indicate the vessel was not inoculated correctly, or was improperly cleaned). Reasons for rejection of a data point as an outlier must 
be clearly stated in the test report. Accepted reasons are only (rare) 
procedural mistakes and not just bad precision. Statistical procedures for outlier identification are of limited use for this type of problem and cannot replace expert judgement. Outliers (marked as such) 
should preferably be retained among the data points shown in any 
subsequent graphical or tabular data presentation.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 819
 
2.2. RESPONSE VARIABLES 
The purpose of the test is to determine the effects of the test 
substance on the growth of algae. This Testing Method describes two response variables, as member countries have different pref ­
erences and regulatory needs. In order for the test results to be 
acceptable in all member countries, the effects should be evaluated using both response variables (a) and (b) described below. 
(a) Average specific growth rate: this response variable is calculated 
on the basis of the logarithmic increase of biomass during the test period, expressed per day. 
(b) Yield: this response variable is the biomass at the end of the test 
minus the starting biomass. 
For the application of this method within the EU regulatory 
framework, calculation of results should be based on an average specific growth rate for the reasons described below. It should be 
noted that toxicity values calculated by using these two response 
variables are not comparable and this difference must be recognised when using the results of the test. EC
 x values based upon average 
specific growth rate (E  r C x ) will generally be higher than results 
based upon yield (E  y C x ) if the test conditions of this Testing 
Method are adhered to, due to the mathematical basis of the respective approaches. This should not be interpreted as a difference in sensitivity between the two response variables, simply that the 
values are different mathematically. The concept of average 
specific growth rate is based on the general exponential growth pattern of algae in non-limited cultures, where toxicity is estimated on the basis of the effects on the growth rate, without being 
dependent on the absolute level of the specific growth rate of the 
control, on the slope of the concentration-response curve or on test duration. In contrast, results based upon the yield response variable are dependent upon all these other variables. E
 y C x is dependent on 
the specific growth rate of the algal species used in each test and on 
the maximum specific growth rate that can vary between species and even different algal strains. This response variable should not be used for comparing the sensitivity to toxicants among algal species 
or even different strains. While the use of average specific growth 
rate for estimating toxicity is scientifically preferred, toxicity estimates based on yield are also included in this Testing Method so as to satisfy current regulatory requirements in some countries.
 
2.2.1.  Average growth rate  
The average specific growth rate for a specific period is calculated as 
the logarithmic increase in biomass from the equation for each single vessel of controls and treatments:
 
μ i-j ¼ ln X j – ln X i 
t j – t i (day  –1 ) 
where: 
μ i-j : is the average specific growth rate from time  i to j; 
X i : is the biomass at time  i; 
X j : is the biomass at time  j. ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 820
 
For each treatment group and control group, calculate a mean value 
for growth rate along with variance estimates. 
Calculate average specific growth rate over the entire test duration 
(normally days 0-3), using the nominally inoculated biomass as the starting value rather than a measured starting value, because in this 
way greater precision is normally obtained. If the equipment used for 
biomass measurement allows sufficiently precise determination of the low inoculum biomass (e.g. flow cytometer) then the measured initial biomass concentration can be used. Also assess the section- 
by-section growth rate, calculated as the specific growth rates for 
each day during the course of the test (days 0-1, 1-2 and 2-3) and examine whether the control growth rate remains constant (See validity criteria, section 1.7). A significantly lower specific growth 
rate on day one than the total average specific growth rate may 
indicate a lag phase. While a lag phase can be minimised and prac ­
tically eliminated in control cultures by proper propagation of the pre-culture, a lag phase in exposed cultures may indicate recovery 
after initial toxic stress or reduced exposure due to loss of test 
substance (including sorption onto the algal biomass) after initial exposure. Hence the section-by-section growth rate may be assessed in order to evaluate effects of the test substance occurring 
during the exposure period. Substantial differences between the 
section-by-section growth rate and the average growth rate indicate deviation from constant exponential growth and that close exam ­
ination of the growth curves is warranted. 
Calculate the percent inhibition of growth rate for each treatment 
replicate from the equation:
 
%I r ¼ μ C – μ T 
μ C Ü 100  
where: 
%I r : percent inhibition in average specific growth rate; 
μ C : mean value for average specific growth rate ( μ) in the control 
group; 
μ T : average specific growth rate for the treatment replicate. 
When solvents are used to prepare the test solutions, the solvent 
controls rather than the controls without solvents should be used in calculation of percent inhibition.
 
2.2.2.  Yield  
The yield is calculated as the biomass at the end of the test minus 
the starting biomass for each single vessel of controls and treatments. For each test concentration and control, calculate a mean value for the yield along with variance estimates. The percentage inhibition in 
yield (%I
 y ) may be calculated for each treatment replicate as follows:  
%I y ¼ ðY C – Y T Þ 
Y C Ü 100  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 821
 
where: 
%I y : percentage inhibition of yield; 
Y C : mean value for yield in the control group; 
Y T : value for yield for the treatment replicate.  
2.3. PLOTTING CONCENTRATION RESPONSE CURVE 
Plot the percentage of inhibition against the logarithm of the test 
substance concentration and examine the plot closely, disregarding 
any such data point that was singled out as an outlier in the first 
phase. Fit a smooth line through the data points by eye or by computerised interpolation to get a first impression of the concen ­
tration response relationship, then proceed with a more detailed 
method, preferably a computerised statistical method. Depending 
on the intended usage of the data, the quality (precision) and amount of data, as well as the availability of data analysis tools, it may be decided (and sometimes well justified) to stop the data 
analysis at this stage and simply read the key figures EC
 50 and 
EC 10 (and/or EC  20 ) from the eye fitted curve (also see section 
below on stimulatory effects). Valid reasons for not using a statistical method may include: 
— data are not appropriate for computerised methods to produce 
any more reliable results than can be obtained by expert judgement — in such situations some computer programs may 
even fail to produce a reliable solution (iterations may not 
converge etc.), 
— stimulatory growth responses cannot be handled adequately using 
available computer programs (see below).
 
2.4. STATISTICAL PROCEDURES 
The aim is to obtain a quantitative concentration-response rela ­
tionship by regression analysis. It is possible to use a weighted 
linear regression after having performed a linearising transformation of the response data — for instance into probit or logit or Weibull units (9), but non-linear regression procedures are preferred tech ­
niques that better handle unavoidable data irregularities and devi ­
ations from smooth distributions. Approaching either zero or total inhibition, such irregularities may be magnified by the trans ­
formation, interfering with the analysis (9). It should be noted that 
standard methods of analysis using probit, logit, or Weibull 
transforms are intended for use on quantal (e.g. mortality or survival) data, and must be modified to accommodate growth or biomass data. Specific procedures for determination of EC
 x values 
from continuous data can be found in (10)(11) and (12). The use of 
non-linear regression analysis is further detailed in Appendix 4.  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 822
 
For each response variable to be analysed, use the concentration- 
response relationship to calculate point estimates of EC  x values. 
When possible, the 95 % confidence limits for each estimate should be determined. Goodness of fit of the response data to the regression model should be assessed either graphically or statis ­
tically. Regression analysis should be performed using individual 
replicate responses, not treatment group means. If, however, nonlinear curve fitting is difficult or fails because of too great a scatter in the data, the problem may be circumvented by performing 
the regression on group means as a practical way of reducing the 
influence of suspected outliers. Use of this option should be identified in the test report as a deviation from normal procedure because curve fits with individual replicates did not produce a good 
result. 
EC
 50 estimates and confidence limits may also be obtained using 
linear interpolation with bootstrapping (13), if available regression 
models/methods are unsuitable for the data. 
For estimation of the LOEC and hence the NOEC, and for effects of 
the test substance on growth rate, it is necessary to compare treatment means using analysis of variance (ANOVA) techniques. The mean for each concentration must then be compared with the 
control mean using an appropriate multiple comparison or trend test 
method. Dunnett’s or Williams’ test may be useful (14)(15)(16)(17) ­
(18). It is necessary to assess whether the ANOVA assumption of homogeneity of variance holds. This assessment may be performed 
graphically or by a formal test (18). Suitable tests are Levene’s or 
Bartlett’s. Failure to meet the assumption of homogeneity of variances can sometimes be corrected by logarithmic transformation of the data. If heterogeneity of variance is extreme and cannot be 
corrected by transformation, analysis by methods such as step-down 
Jonkheere trend tests should be considered. Additional guidance on determining the NOEC can be found in (12). 
Recent scientific developments have led to a recommendation of 
abandoning the concept of NOEC and replacing it with regression based point estimates EC
 x . An appropriate value for x has not been 
established for this algal test. A range of 10 to 20 % appears to be 
appropriate (depending on the response variable chosen), and preferably both the EC
 10 and EC  20 should be reported.  
2.5. GROWTH STIMULATION 
Growth stimulation (negative inhibition) at low concentrations is 
sometimes observed. This can result from either hormesis (toxic stimulation) or from addition of stimulating growth factors with the test material to the minimal medium used. Note that the 
addition of inorganic nutrients should not have any direct effect 
because the test medium should maintain a surplus of nutrients throughout the test. Low dose stimulation can usually be ignored in EC
 50 calculations unless it is extreme. However, if it is 
extreme, or an EC  x value for low x is to be calculated, special 
procedures may be needed. Deletion of stimulatory responses from the data analysis should be avoided if possible, and if available curve fitting software cannot accept minor stimulation, linear interpolation 
with bootstrapping can be used. If stimulation is extreme, use of a 
hormesis model may be considered (19).
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 823
 
2.6. NON-TOXIC GROWTH INHIBITION 
Light absorbing test materials may give rise to a growth rate 
reduction because shading reduces the amount of available light. Such physical types of effects should be separated from toxic 
effects by modifying the test conditions and the former should be 
reported separately. Guidance may be found in (2) and (3).
 
3. REPORTING  
3.1. TEST REPORT 
The test report must include the following: 
Test substance: — physical nature and relevant physiochemical properties, including 
water solubility limit, 
— chemical identification data, including purity. Test species: 
— the strain, supplier or source and the culture conditions used. 
Test conditions: 
— date of start of the test and its duration, 
— description of test design: test vessels, culture volumes, biomass 
density at the beginning of the test, 
— composition of the medium, 
— test concentrations and replicates (e.g. number of replicates, 
number of test concentrations and geometric progression used), 
— description of the preparation of test solutions, including use of 
solvents etc., 
— culturing apparatus, 
— light intensity and quality (source, homogeneity), 
— temperature, — concentrations tested: the nominal test concentrations and any 
results of analyses to determine the concentration of the test 
substance in the test vessels. The recovery efficiency of the method and the limit of quantification in the test matrix should be reported, 
— all deviations from this Testing Method, 
— method for determination of biomass and evidence of correlation 
between the measured parameter and dry weight. 
Results: — pH values at the start and end of the test at all treatments, 
— biomass for each flask at each measuring point and method for 
measuring biomass,
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 824
 
— growth curves (plot of biomass versus time), 
— calculated response variables for each treatment replicate, with 
mean values and coefficient of variation for replicates, 
— graphical presentation of the concentration/effect relationship, 
— estimates of toxicity for response variables e.g. EC  50 , EC  10 , EC  20 
and associated confidence intervals. If calculated, LOEC and 
NOEC and the statistical methods used for their determination, 
— if ANOVA has been used, the size of the effect which can be 
detected (e.g. the least significant difference), 
— any stimulation of growth found in any treatment, 
— any other observed effects, e.g. morphological changes of the 
algae, 
— discussion of the results, including any influence on the outcome 
of the test resulting from deviations from this Testing Method.  
4. LITERATURE  
(1) OECD TG 201 (2006). Freshwater Alga and Cyanobacteria, 
Growth Inhibition Test. 
(2) ISO 1998: Water quality — Guidance for algal growth 
inhibition tests with poorly soluble materials, volatile 
compounds, metals and waster water. ISO/DIS 14442. 
(3) OECD 2000: Guidance Document on Aquatic Toxicity Testing 
of Difficult Substances and mixtures. Environmental Health 
and Safety Publications. Series on Testing and Assessment, no. 23. 
(4) ISO 1998: Water quality — Sampling — Part 16: General 
Guidance for Biotesting. ISO 5667-16. 
(5) ISO 1993: Water quality — Algal growth inhibition test. ISO 
8692. 
(6) Mayer, P., Cuhel, R. and Nyholm, N. (1997). A simple in vitro 
fluorescence method for biomass measurements in algal growth inhibition tests. Water Research 31: 2525-2531. 
(7) Slovacey, R.E. and Hanna, P.J. In vivo fluorescence deter ­
minations of phytoplancton chlorophyll, Limnology & Ocean ­
ography 22,5 (1977), pp.919-925. 
(8) Simpson, S.L., Roland, M.G.E., Stauber, J.L. and Batley, G.E. 
(2003). Effect of declining toxicant concentrations on algal bioassay endpoints. Environ. Toxicol. Chem 22, 2073-2079. 
(9) Christensen, E.R., Nyholm, N. (1984): Ecotoxicological 
Assays with Algae: Weibull Dose-Response Curves. Env. 
Sci. Technol. 19, 713-718.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 825
 
(10) Nyholm, N. Sørensen, P.S., Kusk, K.O. and Christensen, E.R. 
(1992): Statistical treatment of data from microbial toxicity 
tests. Environ. Toxicol. Chem. 11, 157-167. 
(11) Bruce, R.D., and Versteeg, D.J. (1992). A statistical procedure 
for modelling continuous toxicity data. Env. Toxicol. Chem. 11:1485-1494. 
(12) OECD. (2004). Guidance Document on Statistical Analysis of 
Ecotoxicity Data. 
(13) Norberg-King T.J. (1988). An interpolation estimate for 
chronic toxicity: The ICp approach. National Effluent Toxicity Assessment Center Technical Report 05-88. USEPA, 
Duluth, MN. 
(14) Dunnett, C.W. (1955). A multiple comparisons procedure for 
comparing several treatments with a control. J. Amer. Statist. Assoc. 50: 1096-1121 
(15) Dunnett, C.W. (1964). New tables for multiple comparisons 
with a control. Biometrics 20: 482-491. 
(16) Williams, D.A. (1971). A test for differences between 
treatment means when several dose levels are compared with a zero dose control. Biometrics 27: 103-117. 
(17) Williams, D.A. (1972). The comparison of several dose levels 
with a zero dose control. Biometrics 28: 510-531. 
(18) Draper, N.R. and Smith, H. (1981). Applied Regression 
Analysis, second edition. Wiley, New York. 
(19) Brain P. and Cousens R. (1989). An equation to describe dose- 
responses where there is stimulation of growth at low doses. Weed Research, 29, 93-96.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 826
 
Appendix 1  
Strains shown to be suitable for the test  
Green algae  
— Pseudokirchneriella subcapitata , (formerly known as  Selenastrum capricor ­
nutum ), ATCC 22662, CCAP 278/4, 61.81 SAG 
— Desmodesmus subspicatus  (formerly known as  Scenedesmus subspicatus ) 
86.81 SAG  
Diatoms  
— Navicula pelliculosa , UTEX 664  
Cyanobacteria  
— Anabaena flos-aquae , UTEX 1444, ATCC 29413, CCAP 1403/13A 
— Synechococcus leopoliensis , UTEX 625, CCAP 1405/1  
Sources of Strains  
The strains recommended are available in unialgal cultures from the following 
collections (in alphabetical order): 
ATCC: American Type Culture Collection 
10801 University Boulevard Manassas, Virginia 20110-2209 
UNITED STATES 
CCAP, Culture Collection of Algae and Protozoa 
Institute of Freshwater Ecology, 
Windermere Laboratory 
Far Sawrey, Amblerside Cumbria LA22 0LP 
UNITED KINGDOM 
SAG: Collection of Algal Cultures 
Inst. Plant Physiology 
University of Göttingen 
Nicholausberger Weg 18 3400 Göttingen GERMANY 
UTEX Culture Collection of Algae 
Section of Molecular, Cellular and Developmental Biology School of Biological Sciences the University of Texas at Austin 
Austin, Texas 78712 
USA
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 827
 
Appearance and characteristics of recommended species  
P. subcapitata D. subspicatus N. pelliculosa A. flos-aquae S. leopoliensis  
Appearance Curved, 
twisted single 
cells Oval, mostly 
single cells Rods Chains of 
oval cells Rods 
Size (L × W) μm 8-14 × 2-3 7-15 × 3-12 7,1 × 3,7 4,5 × 3 6 × 1 
Cell volume ( μm 3 /cell) 40-60 (  1 ) 60-80 (  1 ) 40-50 (  1 ) 30-40 (  1 ) 2,5 (  2 ) 
Cell dry weight (mg/cell) 2-3 × 10  –8 3-4 × 10  –8 3-4 × 10  –8 1-2 × 10  –8 2-3 × 10  –9 
Growth rate (  3 ) day  –1 ) 1,5-1,7 1,2-1,5 1,4 1,1-1,4 2,0-2,4  
( 1 ) Measured with electronic particle counter 
( 2 ) Calculated from size 
( 3 ) Most frequently observed growth rate in OECD medium at light intensity approx. 70 μE·m  –2 ·s –1 and 21 °C  
Specific Recommendations on Culturing and Handling of Recommended 
Test Species  
Pseudokirchneriella subcapitata and Desmodesmus subspicatus  
These green algae are generally easy to maintain in various culture media. 
Information on suitable media is available from the culture collections. The cells are normally solitary, and cell density measurements can easily be performed using an electronic particle counter or microscope.
 
Anabaena flos-aquae  
Various growth media may be used for keeping a stock culture. It is particularly 
important to avoid allowing the batch culture to go past log phase growth when 
renewing, recovery is difficult at this point.  
Anabaena flos-aquae  develops aggregates of nested chains of cells. The size of 
these aggregates may vary with culturing conditions. It may be necessary to 
break up these aggregates when microscope counting or an electronic particle counter is used for determination of biomass. 
Sonication of sub-samples may be used to break up chains to reduce count 
variability. Longer sonication than required for breaking up chains into shorter lengths may destroy the cells. Sonication intensity and duration must be identical for each treatment. 
Count enough fields on the hemocytometer (at least 400 cells) to help 
compensate for variability. This will improve reliability of microscopic density determinations. 
An electronic particle counter can be used for determination of total cell volume 
of Anabaena after breaking up the cell chains by careful sonification. The sonifi ­
cation energy has to be adjusted to avoid disruption of the cells. 
Use a vortex mixer or similar appropriate method to make sure the algae 
suspension used to inoculate test vessels is well mixed and homogeneous.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 828
 
Test vessels should be placed on an orbital or reciprocate shaker table at about 
150 revolutions per minute. Alternatively, intermittent agitation may be used to 
reduce the tendency of Anabaena to form clumps. If clumping occurs, care must be taken to achieve representative samples for biomass measurements. Vigorous agitation before sampling may be necessary to disintegrate algal clumps.
 
Synechococcus leopoliensis.  
Various growth media may be used for keeping a stock culture. Information on 
suitable media is available from the culture collections.  
Synechococcus leopoliensis  grows as solitary rod-shaped cells. The cells are very 
small, which complicates the use of microscope counting for biomass measure ­
ments. Electronic particle counters equipped for counting particles down to a size of approximately 1 μm are useful. In vitro fluorometric measurements are also 
applicable.
 
Navicula pelliculosa  
Various growth media may be used for keeping a stock culture. Information on 
suitable media is available from the culture collections. Note that silicate is required in the medium.  
Navicula pelliculosa  may form aggregates under certain growth conditions. Due 
to production of lipids the algal cells sometimes tend to accumulate in the surface film. Under those circumstances special measures have to be taken when sub- samples are taken for biomass determination in order to obtain representative 
samples. Vigorous shaking, e.g. using a vortex mixer may be required.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 829
 
Appendix 2  
Growth media  
One of the following two growth media may be used: 
OECD medium: Original medium of OECD TG 201, also according to ISO 8692 US. EPA medium AAP also according to ASTM. When preparing these media, reagent or analytical-grade chemicals should be 
used and deionised water.
 
Composition of The AAP-medium (US. EPA) and the OECD TG 201 medium  
Component EPA OECD 
mg/l mM mg/l mM  
NaHCO  3 15,0 0,179 50,0 0,595 
NaNO  3 25,5 0,300 
NH  4 Cl 15,0 0,280 
MgCl  2 ·6(H  2 O) 12,16 0,0598 12,0 0,0590 
CaCl  2 ·2(H  2 O) 4,41 0,0300 18,0 0,122 
MgSO  4 ·7(H  2 O) 14,6 0,0592 15,0 0,0609 
K 2 HPO  4 1,044 0,00599 
KH  2 PO 4 1,60 0,00919 
FeCl  3 ·6(H  2 O) 0,160 0,000591 0,0640 0,000237 
Na 2 EDTA·2(H  2 O) 0,300 0,000806 0,100 0,000269 (*) 
H 3 BO 3 0,186 0,00300 0,185 0,00299 
MnCl  2 ·4(H  2 O) 0,415 0,00201 0,415 0,00210 
ZnCl  2 0,00327 0,000024 0,00300 0,0000220 
CoCl  2 ·6(H  2 O) 0,00143 0,000006 0,00150 0,00000630 
Na 2 MoO  4 ·2(H  2 O) 0,00726 0,000030 0,00700 0,0000289 
CuCl  2 .2(H  2 O) 0,000012 0,00000007 0,00001 0,00000006 
pH 7,5 8,1  
(*) The molar ratio of EDTA to iron slightly exceed unity. This prevents iron precipitation and, at the same 
time, chelation of heavy metal ions is minimised.  
In the test with the diatom Navicula pelliculosa, both media must be supple ­
mented with Na  2 SiO  3 ·9H  2 0 to obtain a concentration of 1,4 mg Si/l. 
The pH of the medium is obtained at equilibrium between the carbonate system 
of the medium and the partial pressure of CO  2 in atmospheric air. An 
approximate relationship between pH at 25 °C and the molar bicarbonate concen ­
tration is: 
PH eq = 11,30 + log [HCO  3 ] ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 830
 
With 15 mg NaHCO  3 , pH  eq = 7,5 (U.S. EPA medium) and with 50 mg 
NaHCO  3 /l, pH  eq = 8,1 (OECD medium).  
Element composition of test media  
Element EPA OECD 
mg/l mg/l  
C 2,144 7,148 
N 4,202 3,927 
P 0,186 0,285 
K 0,469 0,459 
Na 11,044 13,704 
Ca 1,202 4,905 
Mg 2,909 2,913 
Fe 0,033 0,017 
Mn 0,115 0,115  
Preparation of OECD medium  
Nutrient Concentration in stock solution 
Stock solution 1: macronutrients  
NH  4 Cl 
MgCl  2 ·6H  2 O 
CaCl  2 ·2H  2 O 
MgSO  4 ·7H  2 O 
KH  2 PO 4 1,5 g·l  –1 
1,2 g·l  –1 
1,8 g·l  –1 
1,5 g·l  –1 
0,16 g·l  –1 
Stock solution 2: iron  
FeCl  3 ·6H  2 O 
Na 2 EDTA·2H  2 O 64 mg·l  –1 
100 mg·l  –1 
Stock solution 3: trace elements  
H 3 BO 3 
MnCl  2 ·4H  2 O 
ZnCl  2 
CoCl  2 ·6H  2 O 
CuCl  2 ·2H  2 O 
Na 2 MoO  4 ·2H  2 O 185 mg·l  –1 
415 mg·l  –1 
3 mg·l  –1 
1,5 mg·l  –1 
0,01 mg·l  –1 
7 mg·l  –1 
Stock solution 4: bicarbonate  
NaHCO  3 50 g·l  –1 
Na 2 SiO  3 ·9H  2 0 
Sterilise the stock solutions by membrane filtration (mean pore diameter 0,2 μm) 
or by autoclaving (120 °C, 15 min). Store the solutions in the dark at 4 °C. 
Do not autoclave stock solutions 2 and 4, but sterilise them by membrane 
filtration.  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 831
 
Prepare a growth medium by adding an appropriate volume of the stock solutions 
1-4 to water: 
Add to 500 ml of sterilised water: 
— 10 ml of stock solution 1 — 1 ml of stock solution 2 — 1 ml of stock solution 3 — 1 ml of stock solution 4 
Make up to 1 000 ml with sterilised water 
Allow sufficient time for equilibrating the medium with the atmospheric CO
 2 , if 
necessary by bubbling with sterile filtered air for some hours.  
Preparation of AAP medium  
A1.1. Add 1 mL of each stock solution in A1.2.1-A1.2.7 to approximately 
900 mL of deionised or distilled water and then dilute to 1 L. 
A1.2. Macronutrient stock solutions are made by dissolving the following 
into 500 mL of deionised or distilled water. Reagents A1.2.1, A1.2.2, A1.2.3, and A1.2.4 can be combined into one stock solution. 
A1.2.1.  NaNO
 3 —12,750 g. 
A1.2.2.  MgCL  2 ·6H  2 O—6,082 g. 
A1.2.3.  CaCl  2 ·2H  2 O—2,205 g. 
A1.2.4.  Micronutrient Stock Solution —(see A1.3). 
A1.2.5.  MgSO  4 ·7H  2 O—7,350 g. 
A1.2.6.  K  2 HPO  4 —0,522 g. 
A1.2.7.  NaHCO  3 —7,500 g. 
A1.2.8.  Na  2 SiO  3 ·9H  2 O—See Note A1.1.  
Note A1.1  — Use for diatom test species only. May be added directly 
(202,4 mg) or by way of stock solution to give 20 mg/L Si final concentration in medium. 
A1.3. The micronutrient stock solution is made by dissolving the following 
into 500 mL of deionised or distilled water: 
A1.3.1.  H
 3 BO 3 —92,760 mg. 
A1.3.2.  MnCl  2 ·4H  2 O–207,690 mg. 
A1.3.3.  ZnCl  2 —1,635 mg. 
A1.3.4.  FeCl  3 ·6H  2 O—79,880 mg. 
A1.3.5.  CoCl  2 ·6H  2 O—0,714 mg. 
A1.3.6.  Na  2 MoO  4 ·2H  2 O—3,630 mg. 
A1.3.7.  CuCl  2 ·2H  2 O—0,006 mg. 
A1.3.8.  Na  2 EDTA·2H  2 O—150,000 mg. 
[Disodium (Ethylenedinitrilo) tetraacetate].  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 832
 
A1.3.9.  Na  2 SeO  4 ·5H  2 O—0,005 mg See Note A1.2. 
Note A1.2 — Use only in medium for stock cultures of diatom species. 
A1.4. Adjust pH to 7,5 ± 0,1 with 0,1  N or 1,0  N NaOH or HCl. 
A1.5. Filter the media into a sterile container through either a 0,22- μm 
membrane filter if a particle counter is to be used or a 0,45- μm filter 
if a particle counter is not to be used. 
A1.6. Store medium in the dark at approximately 4 °C until use.  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 833
 
Appendix 3  
Example of a procedure for the culturing of algae  
General observations  
The purpose of culturing on the basis of the following procedure is to obtain 
algal cultures for toxicity tests. 
Suitable methods must be used to ensure that the algal cultures are not infected 
with bacteria. Axenic cultures may be desirable but unialgal cultures must be 
established and used. 
All operations must be carried out under sterile conditions in order to avoid 
contamination with bacteria and other algae.  
Equipment and materials  
See under Testing Method: Apparatus.  
Procedures for obtaining algal cultures  
Preparation of nutrient solutions (media):  
All nutrient salts of the medium are prepared as concentrated stock solutions and 
stored dark and cold. These solutions are sterilised by filtration or by autoclaving. 
The medium is prepared by adding the correct amount of stock solution to sterile 
distilled water, taking care that no infections occur. For solid medium 0,8 % of agar is added.
 
Stock culture:  
The stock cultures are small algal cultures that are regularly transferred to fresh 
medium to act as initial test material. If the cultures are not used regularly, they are streaked out on sloped agar tubes. These are transferred to fresh medium at 
least once every two months. 
The stock cultures are grown in conical flasks containing the appropriate medium 
(volume about 100 ml). When the algae are incubated at 20 °C with continuous illumination, a weekly transfer is required. 
During transfer an amount of ‘old’ culture is transferred with sterile pipettes into 
a flask of fresh medium, so that with the fast-growing species the initial concen ­
tration is about 100 times smaller than in the old culture. 
The growth rate of a species can be determined from the growth curve. If this is 
known, it is possible to estimate the density at which the culture should be transferred to new medium. This must be done before the culture reaches the 
death phase.
 
Pre-culture:  
The pre-culture is intended to give an amount of algae suitable for the inocu ­
lation of test cultures. The pre-culture is incubated under the conditions of the 
test and used when still exponentially growing, normally after an incubation 
period of 2 to 4 days. When the algal cultures contain deformed or abnormal cells, they must be discarded.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 834
 
Appendix 4  
Data analysis by nonlinear regression  
General considerations  
The response in algal tests and other microbial growth tests — growth of 
biomass is by nature a continuous or metric variable — a process rate if 
growth rate is used, and its integral over time if biomass is selected. Both are 
referenced to the corresponding mean response of replicate non-exposed controls showing maximum response for the conditions imposed — with light and temperature as primary determining factors in the algal test. The system is 
distributed or homogenous and the biomass can be viewed as a continuum 
without consideration of individual cells. The variance distribution of the type of response for such a system relates solely to experimental factors (described typically by the log-normal or normal distributions of error). This is by contrast 
to typical bioassay responses with quantal data, for which the tolerance (typically 
binomially distributed) of individual organisms is often assumed to be the dominant variance component. Control responses here are zero or background level. 
In the uncomplicated situation, the normalised or relative response, r, decreases 
monotonically from 1 (zero inhibition) to 0 (100 per cent inhibition). Note that all responses have an associated error, and that apparent negative inhibitions can 
be calculated as a result of random error only.
 
Regression analysis  
Models  
A regression analysis aims at quantitatively describing the concentration-response 
curve in the form of a mathematical regression function Y = f (C) or more frequently F (Z) where Z = log C. Used inversely C = f
 –1 (Y) allows the 
calculation of EC  x figures including the EC  50 , EC  10 and EC  20 , and their 95 % 
confidence limits. Several simple mathematical functional forms have proved to successfully describe concentration-response relationships obtained in algal growth inhibition tests. Functions include, for instance, the logistic equation, 
the non-symmetrical Weibul equation and the log normal distribution function, 
which are all sigmoid curves asymptotically approaching one for C → 0, and 
zero for C → infinity. 
The use of continuous threshold function models (e.g. the Koyman model ‘for 
inhibition of population growth’ Kooijman et al. 1996) is a recently proposed or alternative to asymptotic models. This model assumes no effects at concen ­
trations below a certain threshold, EC0+, that is estimated by extrapolation of 
the response concentration relationship to intercept the concentration axis using a 
simple continuous function that is not differentiable in the starting point. 
Note that the analysis can be a simple minimisation of sums of residual squares 
(assuming constant variance) or weighted squares if variance heterogeneity is 
compensated.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 835
 
Procedure  
The procedure can be outlined as follows: select an appropriate functional 
equation, Y = f (C), and fit it to the data by non-linear regression. Preferably use the measurements from each individual flask rather than the mean values of the replicates, in order to extract as much information from the data as possible. 
If the variance is high, on the other hand, practical experience suggests that the 
mean values of the replicates may provide a more robust mathematical esti ­
mation, less influenced by systematic errors in the data, than with each individual data point retained. 
Plot the fitted curve and the measured data and examine whether the curve fit is 
appropriate. Analysis of residuals may be a particularly helpful tool for this 
purpose. If the chosen functional relationship to fit the concentration response does not describe the whole curve or some essential part of it, such as the response at low concentrations well, choose another curve fit option — e.g. a 
non-symmetrical curve like the Weibul function, instead of a symmetrical one. 
Negative inhibitions may be a problem with, for instance, the log-normal distribution function, likewise demanding an alternative regression function. It is not recommended to assign a zero or a small positive value to such negative 
values because this distorts the error distribution. It may be appropriate to make 
separate curve fits on parts of the curve such as the low inhibition part to estimate EC
 low x figures. Calculate from the fitted equation (by ‘inverse estima ­
tion’, C = f  –1 (Y), characteristic point estimates EC  x ’s, and report as a minimum 
the EC  50 and one or two EC  low x estimates. Experience from practical testing has 
shown that the precision of the algal test normally allows a reasonably accurate estimation at the 10 % inhibition level if data points are sufficient — unless stimulation occurs at low concentrations as a confounding factor. The precision 
of an EC
 20 estimate is often considerably better than that of an EC  10 , because the 
EC 20 is usually positioned on the approximately linear part of the central concen ­
tration response curve. Sometimes EC  10 can be difficult to interpret because of 
growth stimulation. So, while the EC  10 is normally obtainable with a sufficient 
accuracy, it is also recommended to report always the EC  20 . 
Weighting factors  
The experimental variance is not generally constant and typically includes a 
proportional component, a weighted regression is therefore advantageously 
carried out routinely. Weighting factors for such an analysis are normally assumed inversely proportional to the variance: 
W
 i = 1/Var(r  i ) 
Many regression programs allow the option of weighted regression analysis with 
weighting factors listed in a table. Conveniently, weighting factors should be normalised by multiplying them by n/ Σ w
 i (n is the number of data points) so 
that their sum equals one.  
Normalising responses  
Normalising by the mean control response gives some principle problems and 
gives rise to a rather complicated variance structure. Dividing the responses by the mean control response for obtaining the percentage of inhibition, one 
introduces an additional error caused by the error on the control mean. Unless 
this error is negligibly small, weighting factors in the regression and confidence limits must be corrected for the covariance with the control (17). Note that high precision on the estimated mean control response is important in order to 
minimise the overall variance for the relative response. This variance is as 
follows:
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 836
 
(subscript i refers to concentration level i and subscript 0 to the controls) 
Y i = Relative response = r  i /r 0 = 1 — I = f (C  i ) 
with a variance: 
Var (Y  i ) = Var (r  i /r 0 ) e¼ (∂Y i / ∂ r i ) 2 ·Var(r  i ) + (∂ Y i / ∂ r 0 ) 2 ·Var (r  0 ) 
and since 
(∂ Y i / ∂ r i ) = 1/r  0 and (∂ Y i / ∂ r 0 ) = r  i /r 0 2 
with normally distributed data and m  i and m  0 replicates: 
Var(r  i ) = σ 2 /m i 
the total variance of the relative response, Y  i thus becomes: 
Var(Y  i ) = σ 2 /(r 0 2 m i ) + r  i 2 · σ 2 /r 0 4 m 0 
The error on the control mean is inversely proportional to the square root of the 
number of control replicates averaged, and sometimes it can be justified to include historical data and in this way greatly reduce the error. An alternative 
procedure is not to normalise the data and fit the absolute responses, including 
the control response data, but introducing the control response value as an additional parameter to be fitted by non-linear regression. With a usual 2 parameter regression equation, this method necessitates the fitting of 3 
parameters, and therefore demands more data points than non-linear regression 
on data that are normalised using a pre-set control response.
 
Inverse confidence intervals  
The calculation of non-linear regression confidence intervals by inverse esti ­
mation is rather complex and not an available standard option in ordinary stat ­
istical computer program packages. Approximate confidence limits may be obtained with standard non-linear regression programs with re-parameterisation (Bruce and Versteeg, 1992), which involves rewriting the mathematical equation 
with the desired point estimates, e.g. the EC
 10 and the EC  50 as the parameters to 
be estimated. (Let the function be I = f ( α, β, concentration) and utilise the 
definition relationships f ( α, β, EC  10 ) = 0,1 and f ( α, β, EC  50 ) = 0,5 to substitute 
f (α, β, concentration) with an equivalent function g (EC  10 , EC  50 , concentration). 
A more direct calculation (Andersen et al, 1998) is performed by retaining the 
original equation and using a Taylor expansion around the means of r  i and r  0 . 
Recently ‘boot strap methods’ have become popular. Such methods use the 
measured data and a random number generator directed frequent re-sampling to estimate an empirical variance distribution.
 
Literature  
Kooijman, S.A.L.M.; Hanstveit, A.O.; Nyholm, N. (1996): No-effect concen ­
trations in algal growth inhibition tests. Water Research, 30, 1625-1632. 
Bruce, R.D. and Versteeg, D.J.(1992) A Statistical Procedure for Modelling 
Continuous Ecotoxicity Data. Env. Toxicol. Chem.11, 1485-1494. 
Andersen, J.S., Holst, H., Spliid, H., Andersen, H., Baun, A. & Nyholm, N. 
(1998): Continuous ecotoxicological data evaluated relative to a control response. Journal of Agricultural, Biological and Environmental Statistics, 3, 405-420.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 837
 
C.4. DETERMINATION OF ‘READY’ BIODEGRADABILITY  
PART I.  GENERAL CONSIDERATIONS  
I.1. INTRODUCTION 
Six test methods are described that permit the screening of chemicals 
for ready biodegradability in an aerobic aqueous medium: 
(a) Dissolved Organic Carbon (DOC) Die-Away (Method C.4-A) 
(b) Modified OECD Screening — DOC Die-Away (Method C.4-B) 
(c) Carbon dioxide (CO  2 ) Evolution (Modified Sturm Test) 
(Method C.4-C) 
(d) Manometric Respirometry (Method C.4-D) 
(e) Closed Bottle (Method C.4-E) 
(f) MITI (Ministry of International Trade and Industry — Japan) 
(Method C.4-F) 
General and common considerations to all six tests are given in 
Part I of the method. Items specific for individual methods are 
given in Parts II to VII. The appendices contain definitions, 
formulas and guidance material. 
An OECD inter-laboratory comparison exercise, done in 1988, has 
shown that the methods give consistent results. However, depending 
on the physical characteristics of the substance to be tested, one or 
other of the methods may be preferred.  
I.2. SELECTION OF THE APPROPRIATE METHOD 
In order to select the most appropriate method, information on the 
chemical's solubility, vapour pressure and adsorption characteristics 
is essential. The chemical structure or formula should be known in 
order to calculate theoretical values and/or check measured values of parameters, e.g. ThOD, ThCO
 2 , DOC, TOC, COD (see 
Appendices 1 and 2). 
Test chemicals which are soluble in water to at least 100 mg/l may 
be assessed by all methods, provided they are non-volatile and non- adsorbing. For those chemicals which are poorly soluble in water, volatile or adsorbing, suitable methods are indicated in 
Table 1. The manner in which poorly water-soluble chemicals and 
volatile chemicals can be dealt with is described in Appendix 3. Moderately volatile chemicals may be tested by the DOC Die-Away method if there is sufficient gas space in the test 
vessels (which should be suitably stoppered). In this case, an abiotic 
control must be set up to allow for any physical loss.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 838
 
Table 1:  
Applicability of test methods  
Test Analytical Method Suitability for substances which are: 
poorly soluble volatile adsorbing  
DOC Die-Away Dissolved organic carbon – – +/– 
Mod. OECD Die-Away Dissolved organic carbon – – +/– CO
 2 Evolution Respirometry: CO  2 
evolution + – + 
Manometric Respirometry Manometric respirometry: 
oxygen consumption + +/– + 
Closed Bottle Respirometry: dissolved 
oxygen +/– + + 
MITI Respirometry: oxygen 
consumption + +/– + 
Information on the purity or the relative proportions of major 
components of the test material is required to interpret the results 
obtained, especially when the results are low or marginal. 
Information on the toxicity of the test chemical to bacteria 
(Appendix 4) may be very useful for selecting appropriate test 
concentrations and may be essential for the correct interpretation 
of low biodegradation values.  
I.3. REFERENCE SUBSTANCES 
In order to check the procedure, reference chemicals which meet the 
criteria for ready biodegradability are tested by setting up an appro ­
priate flask in parallel to the normal test runs. 
Suitable chemicals are aniline (freshly distilled), sodium acetate and 
sodium benzoate. These reference chemicals all degrade in these methods even when no inoculum is deliberately added. 
It was suggested that a reference chemical should be sought which 
was readily biodegradable but required the addition of an inoculum. 
Potassium hydrogen phthalate has been proposed but more evidence 
needs to be obtained with this substance before it can be accepted as a reference substance. 
In the respirometric tests, nitrogen-containing compounds may affect 
the oxygen uptake because of nitrification (see Appendices 2 and 5).
 
I.4. PRINCIPLE OF THE TEST METHODS 
A solution, or suspension, of the test substance in a mineral medium 
is inoculated and incubated under aerobic conditions in the dark or in diffuse light. The amount of DOC in the test solution due to the inoculum should be kept as low as possible compared to the amount 
of DOC due to the test substance. Allowance is made for the endo ­
genous activity of the inoculum by running parallel blank tests with inoculum but without test substance, although the endogenous activity of cells in the presence of the substance will not exactly 
match that in the endogenous control. A reference substance is run in 
parallel to check the operation of the procedures.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 839
 
In general, degradation is followed by the determination of 
parameters, such as DOC, CO  2 production and oxygen uptake, and 
measurements are taken at sufficiently frequent intervals to allow the identification of the beginning and end of biodegradation. With automatic respirometers the measurement is continuous. DOC is 
sometimes measured in addition to another parameter but this is 
usually done only at the beginning and the end of the test. Specific chemical analysis can also be used to assess primary degradation of the test substance, and to determine the 
concentration of any intermediate substances formed (obligatory in 
the MITI test). 
Normally, the test lasts for 28 days. Tests however may be ended 
before 28 days, i.e. as soon as the biodegradation curve has reached 
a plateau for at least three determinations. Tests may also be prolonged beyond 28 days when the curve shows that biodegradation has started but that the plateau has not been reached day 28.
 
I.5. QUALITY CRITERIA  
I.5.1.  Reproducibility  
Because of the nature of biodegradation and of the mixed bacterial 
populations used as inocula, determinations should be carried out at 
least in duplicate. 
It is common experience that the larger the concentration of micro- 
organisms initially added to the test medium, the smaller will be the 
variation between replicates. Ring tests have also shown that there can be large variations between results obtained by different labora ­
tories, but good agreement is normally obtained with easily biode ­
gradable compounds.
 
I.5.2.  Validity of the test  
A test is considered valid if the difference of extremes of replicate 
values of the removal of test chemical at the plateau, at the end of 
the test or at the end of the 10-day window, as appropriate, is less 
than 20 % and if the percentage degradation of the reference substance has reached the level for ready biodegradability by 14 
days. If either of these conditions is not met, the test should be 
repeated. Because of the stringency of the methods, low values do not necessarily mean that the test substance is not biodegradable under environmental conditions, but indicates that more work will 
be necessary to establish biodegradability. 
If in a toxicity test, containing both the test substance and a reference 
chemical, less than 35 % degradation (based on DOC) or less than 
25 % (based on ThOD or ThCO
 2 ) occurred in 14 days, the test 
chemicals can be assumed to be inhibitory (see also Appendix 4). The test series should be repeated, if possible using a lower concen ­
tration of test chemical and/or a higher concentration of inoculum, 
but not greater than 30 mg solids/litre.
 
I.6. GENERAL PROCEDURES AND PREPARATIONS 
General conditions applying to the tests are summarised in 
Table 2. Apparatus and other experimental conditions pertaining 
specifically to an individual test are described later under the 
heading for that test.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 840
 
Table 2  
Test conditions  
Test DOC Die- 
Away CO 2 Evolution Manometric 
Respirometry Modified 
OECD Screeing Closed Bottle MITI (I)  
Concentration 
of Test Substance 
as mg/l 100 2-10 100 mg DOC/1 10-40 10-20 10-40 mg ThOD/1 50-100 5-10 
Concentration 
of Inoculum 
(in cells/l, approxi­
matively) ≤ 30 mg/l SS 
or ≤ 100 ml effluent/l 
(10
 7 -10 8 ) 0,5 
ml secondary 
effluent/1 
(10 5 ) ≤ 5 ml of 
effluent/l 
(10 4 -10 6 ) 30 mg/l SS 
(10 7 -10 8 ) 
Concentration 
of elements in mineral 
medium (in 
mg/l): 
P 116 11,6 29 N 1,3 0,13 1,3 Na 86 8,6 17,2 K 122 12,2 36,5 Mg 2,2 2,2 6,6 
Ca 9,9 9,9 29,7 
Fe 0,05 - 0,1 0,05 - 0,1 0,15 
pH 7,4 ± 0,2 preferably 7,0 
Temperature 22 ± 2
 o C 25 ± 1  o C 
DOC = Dissolved organic Carbon ThoD = Theoretical 
Oxygen Demand SS = Suspended Solids  
I.6.1.  Dilution water  
Deionised or distilled water, free from inhibitory concentrations of 
toxic substances (e.g. Cu  ++ ions) is used. It must contain no more 
than 10 % of the organic carbon content introduced by the test 
material. The high purity of the test water is necessary to 
eliminate high blank values. Contamination may result from inherent impurities and also from the ion-exchange resins and lysed material from bacterial and algae. For each series of tests 
use only one batch of water, checked beforehand by DOC 
analysis. Such a check is not necessary for the closed bottle test, but the oxygen consumption of the water must be low.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 841
 
I.6.2.  Stock solutions of mineral components  
To make up the test solutions, stock solutions of appropriate concen ­
trations of mineral components are made up. The following stock solutions may be used (with different dilution factors) for the 
methods DOC Die-Away, Modified OECD Screening, CO
 2 Evol­
ution, Manometric Respirometry, Closed Bottle test. 
The dilution factors and, for the MITI test, the specific preparation of 
the mineral medium are given under the headings of the specific 
tests.  
Stock solutions:  
Prepare the following stock solutions, using analytical grade 
reagents. 
(a) Monopotassium dihydrogen orthophosphate, 
KH  2 PO 4 8,50 g 
Dipotassium monohydrogen orthophosphate, 
K 2 HPO  4 21,75 g 
Disodium monohydrogen orthophosphate 
dihydrate Na  2 HPO  4 . 2 H  2 O 33,40 g 
Ammonium chloride, NH  4 Cl 0,50 g 
Dissolve in water and make up to 1 litre The pH 
of the solution should be 7,4. 
(b) Calcium chloride, anhydrous, CaCl  2 27,50 g 
or Calcium chloride dihydrate, CaCl  2 , 2 H  2 O 36,40 g 
Dissolve in water and make up to 1 litre 
(c) Magnesium sulphate heptahydrate, MgSO  4 . 7 H  2 O 22,50 g 
Dissolve in water and make up to 1 litre. 
(d) Iron (III) chloride hexahydrate, FeC1  3 . 6H  2 O 0,25 g 
Dissolve in water and make up to 1 litre.  
Note : in order to avoid having to prepare this solution immediately 
before use add one drop of conc. HCL or 0,4 g ethylenediaminetetra- 
acetic acid disodium salt (EDTA) par litre.  
I.6.3.  Stock solutions of chemicals  
For example, dissolve 1-10 g, as appropriate, of test or reference 
chemical in deionised water and make up to 1 litre when the solu ­
bility exceeds 1 g/l. Otherwise, prepare stock solutions in the mineral medium or add the chemical direct to the mineral medium. For the handling of less soluble chemicals, see Appendix 3, but in the MITI 
test (Method C.4-F), neither solvents nor emulsifying agents are to 
be used.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 842
 
I.6.4.  Inocula  
The inoculum may be derived from a variety of sources: activated 
sludge, sewage effluents (unchlorinated), surface waters and soils or from a mixture of these. For the DOC Die-Away, CO
 2 Evolution and 
Manometric Respirometry tests, if activated sludge is used, it should 
be taken from a treatment plant or laboratory-scale unit receiving predominantly domestic sewage. Inocula from other sources have been found to give higher scattering of results. For the Modified 
OECD Screening and the Closed Bottle tests a more dilute 
inoculum without sludge fiocs is needed and the preferred source is a secondary effluent from a domestic waste water treatment plant 
or laboratory-scale unit. For the MITI test the inoculum is derived 
from a mixture of sources and is described under the heading of this specific test.
 
I.6.4.1.  Inoculum from activated sludges  
Collect a sample of activated sludge freshly from the aeration tank of 
a sewage treatment plant or laboratory-scale unit treating predomi ­
nantly domestic sewage. Remove coarse particles if necessary by 
filtration through a fine sieve and keep the sludge aerobic thereafter. 
Alternatively, settle or centrifuge (e.g. at 100 g for 10 min) after 
removal of any coarse particles. Discard the supernatant. The sludge may be washed in the mineral medium. Suspend the concentrated sludge in mineral medium to yield a concentration of 3-5 g 
suspended solids/l and aerate until required. 
Sludge should be taken from a properly working conventional plant. 
If sludge has to be taken from a high rate treatment plant, or is thought to contain inhibitors, it should be washed. Settle or 
centrifuge the re-suspended sludge after thorough mixing, discard 
the supernatant and again re-suspend the washed sludge in a further volume of mineral medium. Repeat this procedure until the sludge is considered to be free from excess substrate or inhibitor. 
After complete re-suspension is achieved, or with untreated sludge, 
withdraw a sample just before use for the determination of the dry 
weight of the suspended solids. 
A further alternative is to homogenise activated sludge (3-5 g 
suspended solids/l). Treat the sludge in a mechanical blender for two min at medium speed. Settle the blended sludge for 30 min or 
longer if required and decant liquid for use as inoculum at the rate of 
10 mill of mineral medium.
 
I.6.4.2.  Other sources of inoculum  
It can be derived from the secondary effluent of a treatment plant or 
laboratory-scale unit receiving predominantly domestic sewage. Collect a fresh sample and keep it aerobic during transport. Allow to settle for 1 h. or filter through a coarse filter paper and keep the 
decanted effluent or filtrate aerobic until required. Up to 100 ml of 
this type of inoculum may be used per litre of medium.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 843
 
A further source for the inoculum is surface water. In this case, 
collect a sample of an appropriate surface water, e.g. river, lake, 
and keep aerobic until required. If necessary, concentrate the inoculum by filtration or centrifugation.
 
I.6.5.  Pre-conditioning of inocula  
Inocula may be pre-conditioned to the experimental conditions, but 
not pre-adapted to the test chemical. Pre-conditioning consists of aerating activated sludge in mineral medium or secondary effluent for five to seven days at the test temperature. Pre-conditioning 
sometimes improves the precision of the test methods by reducing 
blank values. It is considered unnecessary to pre-condition MITI inoculum.
 
I.6.6.  Abiotic controls  
When required, check for the possible abiotic degradation of the test 
substance by determining the removal of DOC, oxygen uptake or carbon dioxide evolution in sterile controls containing no inoculum. 
Sterilise by filtration through a membrane (0,2 - 0,45 micrometre) or 
by the addition of a suitable toxic substance at an appropriate concentration. If membrane filtration is used, take samples asep ­
tically to maintain sterility. Unless adsorption of the test chemical 
has been ruled out beforehand, tests which measure biodegradation 
as the removal of DOC, especially with activated sludge inocula, should include an abiotic control which is inoculated and poisoned.
 
I.6.7.  Number of flasks  
The number of flasks in a typical run is described under the headings 
of each tests. 
The following type of flask may be used: 
— test suspension: containing test substance and inoculum 
— inoculum blank: containing only inoculum — procedure control: containing reference substance and inoculum — abiotic sterile control: sterile, containing test substance (see 1.6.6) 
— adsorption control: containing test substance, inoculum and ster ­
ilising agent 
— toxicity control: containing test substance, reference substance 
and inoculum 
It is mandatory that determination in test suspension and inoculum 
blank is made in parallel. It is advisable to make the determinations 
in the other flasks in parallel as well. 
This may, however, not always be possible. Ensure that sufficient 
samples or readings are taken to allow the percentage removal in the 
10-day window to be assessed.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 844
 
I.7. DATA AND EVALUATION 
In the calculation of D  t , percentage degradation, the mean values of 
the duplicate measurement of the parameter in both test vessels and inoculum blank are used. The formulas are set out in the sections 
below on specific tests. The course of degradation is displayed 
graphically and the 10-day window is indicated. Calculate and report the percentage removal achieved at the end of the 10-day window and the value at the plateau or at the end of the test, 
whichever is appropriate. 
In respirometric tests nitrogen-containing compounds may affect the 
oxygen uptake because of nitrification (see Appendices 2 and 5).
 
I.7.1.  Degradation measured by means of DOC determination  
The percentage degradation D  t at each time a sample was taken 
should be calculated separately for the flasks containing test 
substance using mean values of duplicate DOC measurements in 
order that the validity of the test can be assessed (see 1.5.2). It is calculated using the following equation:
 
D t ¼ 1 Ä C t Ä C bt 
C o Ä C b0 Í Î  
Ü 100  
where: 
D t = % degradation at time t 
C o = mean starting concentration of DOC in the inoculated 
culture medium containing the test substance (mg DOC/l) 
C t = mean concentration of DOC in the inoculated culture 
medium containing test substance at time t (mg DOC/l) 
C bo = mean starting concentration of DOC in blank inoculated 
mineral medium (mg DOC/l) 
C bt = mean concentration of DOC blank inoculated mineral 
medium at time t (mg DOC/l). 
All concentrations are measured experimentally.  
I.7.2.  Degradation measured by means of specific analysis  
When specific analytical data are available, calculate primary biode ­
gradation from:  
D t ¼ S b Ä S a 
S b Ü 100  
where: 
D t = % degradation at time t, normally 28 days, 
S a = residual amount of test substance in inoculated medium at 
end of test (mg), 
S b = residual amount of test substance in the blank test with water/ 
medium to which only the test substance was added (mg).  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 845
 
1.7.3.  Abiotic degradation  
When an abiotic sterile control is used, calculate the percentage 
abiotic degradation using:  
% abiotic degradation  ¼ C sðoÞ Ä C sðtÞ 
C sðoÞ Ü 100  
Where: 
C s(o) = DOC Concentration in sterile control at day 0 
C s(t) = DOC Concentration in sterile control at day t.  
I.8. REPORTING 
The test report shall, if possible, contain the following: 
— test and reference chemicals, and their purity, — test conditions, — inoculum: nature and sampling site(s), concentration and any pre- 
conditioning treatment, 
— proportion and nature of industrial waste present in sewage if 
known, 
— test duration and temperature, — in the case of poorly soluble test chemicals, treatment given, — test method applied; scientific reasons and explanation should be 
given for any change of procedure, 
— data sheet, — any observed inhibition phenomena, — any observed abiotic degradation, — specific chemical analytical data, if available, — analytical data on intermediates, if available, — the graph of percentage degradation against time for the test and 
reference substances; the lag phase, degradation phase, 10-day window and slope should be clearly indicated (Appendix 1). If 
the test has complied with the validity criteria, the mean of 
the degradation percentages of the flasks containing test substance may be used for the graph, 
— percentage removal after 10-day window, and at plateau or at end 
of the test.
 
PART II.  DOC DIE-AWAY TEST  (Method C.4-A)  
II.1. PRINCIPLE OF THE METHOD 
A measured volume of inoculated mineral medium containing a 
known concentration of the test substance (10-40 mg DOC/l) as 
the nominal sole source of organic carbon is aerated in the dark or 
diffused light at 22 ± 2  o C. ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 846
 
Degradation is followed by DOC analysis at frequent intervals over a 
28-day period. The degree of biodegradation is calculated by 
expressing the concentration of DOC removed (corrected for that in the blank inoculum control) as a percentage of the concentration initially present. The degree of primary biodegradation may also be 
calculated from supplemental chemical analysis made at the 
beginning and end of incubation.
 
II.2. DESCRIPTION OF THE METHOD  
II.2.1.  Apparatus  
(a) Conical flasks, e. g. 250 ml to 2 l, depending on the volume 
needed for DOC analysis; 
(b) shaking machine to accommodate the conical flasks, either with 
automatic temperature control or used in a constant temperature room; and of sufficient power to maintain aerobic conditions in all flasks; 
(c) filtration apparatus, with suitable membranes; 
(d) DOC analyser; 
(e) apparatus for determining dissolved oxygen; 
(f) centrifuge.
 
II.2.2.  Preparation of mineral medium  
For the preparation of the stock solutions, see I.6.2. 
Mix 10 ml of solution (a) with 800 ml dilution water, add 1 ml of 
solutions (b) to (d) and make up to 11 with dilution water.  
II.2.3.  Preparation and pre-conditioning of inoculum  
The inoculum may be derived from a variety of sources: activated 
sludge; sewage effluents; surface waters; soils or from a mixture of these. 
See I.6.4., I.6.4.1., I.6.4.2. and I.6.5.
 
II.2.4.  Preparation of flasks  
As an example, introduce 800 ml portions of mineral medium into 2 
l conical flasks and add sufficient volumes of stock solutions of the 
test and reference substances to separate flasks to give a concen ­
tration of chemical equivalent to 10-40 mg DOC/l. Check the pH values and adjust, if necessary, to 7,4. Inoculate the flasks with 
activated sludge or other source of inocula (see I.6.4.), to give a 
final concentration not greater than 30 mg suspended solids/l. Also prepare inoculum controls in the mineral medium but without test or reference chemical. 
If needed, use one vessel to check the possible inhibitory effect of 
the test chemical by inoculating a solution containing, in the mineral medium, comparable concentrations of both the test and a reference chemical. 
Also, if required, set up a further, sterile flask to check whether the 
test chemical is degraded abiotically by using an uninoculated 
solution of the chemical (see I.6.6).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 847
 
Additionally, if the test chemical is suspected of being significantly 
adsorbed on to glass, sludge, etc., make a preliminary assessment to 
determine the likely extent of adsorption and thus the suitability of the test for the chemical (see Table 1). Set up a flask containing the test substance, inoculum and sterilising agent. 
Make up the volumes in all flasks to 11 with mineral medium and, 
after mixing, take a sample from each flask to de.termine the initial concentration of DOC (see Appendix 2.4). Cover the openings of the flasks, e.g. with aluminium foil, in such a way as to allow free 
exchange of air between the flask and the surrounding atmosphere. 
Then insert the vessels into the shaking machine for starting the test.
 
II.2.5.  Number of flasks in typical run  
Flasks 1 and 2: Test suspension 
Flasks 3 and 4: Inoculum blank 
Flask 5: Procedure control preferably and when necessary: 
Flask 6: Abiotic sterile control 
Flask 7: Adsorption control 
Flask 8: Toxicity control 
See also I.6.7.
 
II.2.6.  Performance of the test  
Throughout the test, determine the concentrations of DOC in each 
flask in duplicate at known time intervals, sufficiently frequently to 
be able to determine the start of the 10-day window and the 
percentage removal at the end of the 10-day window. Take only the minimal volume of test suspension necessary for each deter ­
mination. 
Before sampling make good evaporation losses from the flasks by 
adding dilution water (I.6.1) in the required amount if necessary. Mix the culture medium thoroughly before withdrawing a sample 
and ensure that material adhering to the walls of the vessels is 
dissolved or suspended before sampling. Membrane-filter or centrifuge (see Appendix 2..4) immediately after the sample has been taken. Analyse the filtered or centrifuged samples on the 
same day, otherwise store at 2-4
 o C for a maximum of 48 h, or 
below - 18  o C for a longer period.  
II.3. DATA AND REPORTING  
II.3.1.  Treatment of results  
Calculate the percentage degradation at time t as given under I.7.1 
(DOC determination) and, optionally, under I.7.2 (specific analysis). 
Record all results on the data sheets provided.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 848
 
II.3.2.  Validity of results  
See I.5.2.  
II.3.3.  Reporting  
See I.8.  
II.4. DATA SHEET 
An example of a data sheet is given hereafter.  
DOC DIE-AWAY TEST 
1. LABORATORY  
2. DATE AT START OF TEST  
3. TEST SUBSTANCE  
Name: 
Stock solution concentration: … mg/1 as chemical Initial concentration in medium, to: … mg/1 as chemical
 
4. INOCULUM  
Source: 
Treatment given: Pre-conditioning, if any: Concentration of suspended solids in reaction mixture: mg/1
 
5. CARBON DETERMINATIONS  
Carbon analyser:  
Flask nr DOC after n-days (mg/l) 
0 n  1 n 2 n 3 n x 
Test chemical plus 
inoculum 
1 a 1 
a 2 
a, mean 
C a(t) 
2 b1 
b2 
b, men 
C b(t) ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 849
 
Flask nr DOC after n-days (mg/l) 
0 n  1 n 2 n 3 n x 
Blank inoculum 
without test chemical 3 C
 1 
C 2 
C, mean C  c(t) 
4 d 1 
d 2 
d, mean 
C d(t) 
C blðtÞ ¼ C cðtÞ þ C dðtÞ 
2 
6. EVALUATION OF RAW DATA  
Flask nr % degradation afer n days 
0 n  1 n 2 n 3 n x 
1 D 1 ¼ Í 
1 Ä C aðtÞ Ä C blðtÞ 
C aðoÞ Ä C blðoÞ ! 
Ü 100 0 
2 D 2 ¼ Í 
1 Ä C bðtÞ Ä C blðtÞ 
C bðoÞ Ä C blðoÞ ! 
Ü 100 0 
Mean (*) D ¼ D 1 Ä D 2 
2 0 
(*) D1 and D  2 should not be averaged if there is a considerable difference.  
Note : similar formats may be used for the reference chemical and 
toxicity controls.  
7. ABIOTIC CONTROL  (optional)  
Time (days) 
0 t  
DOC conc. (mg/l) in sterile control C s(o) C s(t) 
% abiotic degradation  ¼ C sðoÞ Ä C sðtÞ 
C sðoÞ Ü 100  
8. SPECIFIC CHEMICAL ANALYSIS  (optional)  
residual amount of test chemical 
at end of test (mg/l) % primary degradation  
Sterile control S  b ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 850
 
residual amount of test chemical 
at end of test (mg/l) % primary degradation  
Inoculated test medium S  a S b Ä S a 
S b Ü 100  
PART III.  MODIFIED OECD SCREENING TEST  
(Method C.4-B)  
III.1. PRINCIPLE OF THE METHOD 
A measured volume of mineral medium containing a known concen ­
tration of the test substance (10-40 mg DOC/litre) as the nominal sole source of organic carbon is inoculated with 0,5 ml effluent per litre of medium. The mixture is aerated in the dark or diffused light 
at 22 ± 2
 o C. 
Degradation is followed by DOC analysis at frequent intervals over a 
28-day period. The degree of biodegradation is calculated by expressing the concentration of DOC removed (corrected for that in the blank inoculum control) as a percentage of the concentration 
initially present. The degree of primary biodegradation may also be 
calculated from supplemental chemical analysis made at the beginning and end of incubation.
 
III.2. DESCRIPTION OF THE METHOD  
III.2.1. Apparatus 
(a) Conical flasks, e.g. 250 ml to 2 litres, depending on the volume 
needed for DOC analysis; 
(b) shaking machine — to accommodate the conical flasks, either 
with automatic temperature control or used in a constant 
temperature room, and of sufficient power to maintain aerobic conditions in all flasks; 
(c) filtration apparatus, with suitable membranes; 
(d) DOC analyser; 
(e) apparatus for determining dissolved oxygen; 
(f) centrifuge.
 
III.2.2.  Preparation of mineral medium  
For the preparation of the stock solutions, see I.6.2. 
Mix 10 ml of solution (a) with 80 ml dilution water, add 1 ml of 
solutions (b) to (d) and make up to 1 litre with dilution water. 
This method uses only 0,5 ml effluent/litre as inoculum and therefore 
the medium may need to be fortified with trace elements and growth 
factors. This is done by adding 1 ml each of the following solutions 
per litre of final medium:  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 851
 
Trace element solution: 
Manganese sulfate tetrahydrate, MnSO  4 . 4H  2 O 39,9 mg 
Boric acid, H  3 BO 3 57,2 mg 
Zinc sulfate heptahydrate, ZnSO  4 . 7H  2 O 42,8 mg 
Ammonium heptamolybdate (NH  4 ) 6 Mo 7 O 24 34,7 mg 
Fe-chelate (FeCl  3 ethylenediamine-tetra-acetic acid) 100,0 mg 
Dissolve in, and make up to 1 000 ml with dilution 
water 
Vitamin solution: Yeast extract 15,0 mg 
Dissolve the yeast extract in 100 ml water. Sterilise by passage 
through a 0,2 micron membrane, or make up freshly.
 
III.2.3.  Preparation and pre-conditioning of inoculum  
The inoculum is derived from the secondary effluent of a treatment 
plant or laboratory scale unit receiving predominantly domestic 
sewage. See I.6.4.2. and I.6.5. 
0,5 ml per litre of mineral medium is used.  
III.2.4.  Preparation of flasks  
As an example, introduce 800 ml portions of mineral medium into 2- 
litre conical flasks and add sufficient volumes of stock solutions of 
the test and reference substances to separate flasks to give a concen ­
tration of chemical equivalent to 10-40 mg DOC/litre. Check the pH value and adjust, if necessary, to 7,4. Inoculate the flasks with sewage effluent at 0,5 ml/litre (see I.6.4.2). Also prepare inoculum 
controls in the mineral medium but without test or reference 
chemical. 
If needed, use one vessel to check the possible inhibitory effect of 
the test chemical by inoculating a solution containing, in the mineral 
medium, comparable concentrations of both the test and a reference chemical. 
Also, if required, set up a further, sterile flask to check whether the 
test chemical is degraded abiotically by using an uninoculated 
solution of the chemical (see I.6.6). 
Additionally, if the test chemical is suspected of being significantly 
adsorbed on to glass, sludge, etc., make a preliminary assessment to 
determine the likely extent of adsorption and thus the suitability of the test for the chemical (see Table 1). Set up a flask containing the test substance, inoculum and sterilising agent. 
Make up the volumes in all flasks to 1 litre with mineral medium 
and, after mixing, take a sample from each flask to determine the initial concentration of DOC (see Appendix 2.4). Cover the openings of the flasks, e.g. with aluminium foil, in such a way as to allow free 
exchange of air between the flask and the surrounding atmosphere. 
Then insert the vessels into the shaking machine for starting the test.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 852
 
III.2.5.  Number of flasks in typical run  
Flasks 1 and 2: test suspension 
Flasks 3 and 4: inoculum blank Flask 5: procedure control and preferably and when necessary: Flask 6: abiotic sterile control Flask 7: adsorption control Flask 8: toxicity control See also 1.6.7.
 
III.2.6.  Performance of the test  
Throughout the test, determine the concentrations of DOC in each 
flask in duplicate at known time intervals, sufficiently frequently to 
be able to determine the start of the 10-day window and the 
percentage removal at the end of the 10-day window. Take only the minimal volume of test suspension necessary for each deter ­
mination. 
Before sampling make good evaporation losses from the flasks by 
adding dilution water (I.6.1) in the required amount if necessary. 
Mix the culture medium thoroughly before withdrawing a sample and ensure that material adhering to the walls of the vessels is dissolved or suspended before sampling. Membrane-filter or 
centrifuge (see Appendix 2.4) immediately after the sample has 
been taken. Analyse the filtered or centrifuged samples on the same day, otherwise store at 2-4
 o C for a maximum of 48 h, or 
below 18  o C for a longer period.  
III.3. DATA AND REPORTING  
III.3.1.  Treatment of results  
Calculate the percentage degradation at time t as given under I.7.1 
(DOC determination) and, optionally, under I.7.2 (specific analysis). 
Record all results on the data sheets provided.  
III.3.2. Validity of results 
See I.5.2.  
III.3.3. Reporting 
See I.8.  
III.4. DATA SHEET 
An example of a data sheet is given hereafter  
MODIFIED OECD SCREENING TEST 
1. LABORATORY  
2. DATE AT START OF TEST  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 853
 
3. TEST SUBSTANCE  
Name: 
Stock solution concentration: … mg/litre as chemical Initial concentration in medium, to: … mg/litre as chemical
 
4. INOCULUM  
Source: 
Treatment given: Pre-conditioning, if any: Concentration of suspended solids in reaction mixture: mg/1
 
5. CARBON DETERMINATIONS 
Carbon analyser:  
Flask nr DOC after n-days (mg/l) 
0 n  1 n 2 n 3 n x 
Test chemical plus 
inoculum 
1 a 1 
a 2 
a, mean 
C a(t) 
2 b1 
b2 
b, men 
C b(t) 
Blank inoculum 
without test 
chemical 3 C 1 
C 2 
C, mean C  c(t) 
4 d 1 
d 2 
d, mean 
C d(t) 
C blðtÞ ¼ C cðtÞ þ C dðtÞ 
2 
6. EVALUATION OF RAW DATA  
Flask nr % degradation afer n days 
0 n  1 n 2 n 3 n x 
1 D 1 ¼ Í 
1 Ä C aðtÞ Ä C blðtÞ 
C aðoÞ Ä C blðoÞ ! 
Ü 100 0 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 854
 
Flask nr % degradation afer n days 
0 n  1 n 2 n 3 n x 
2 D 2 ¼ Í 
1 Ä C bðtÞ Ä C blðtÞ 
C bðoÞ Ä C blðoÞ ! 
Ü 100 0 
Mean (*) D ¼ D 1 Ä D 2 
2 0 
(*) D  1 and D  2 should not be averaged if there is a considerable difference.  
Note : similar formats may be used for the reference chemical and 
toxicity controls.  
7. ABIOTIC CONTROL  (optional)  
Time (days) 
0 t  
DOC conc. (mg/l) in sterile control C s(o) C s(t) 
% abiotic degradation  ¼ C sðoÞ Ä C sðtÞ 
C sðoÞ Ü 100  
8. SPECIFIC CHEMICAL ANALYSIS  (optional)  
residual amount of test chemical 
at end of test (mg/l) % primary degradation  
Sterile control S  b 
Inoculated test medium S  a S b Ä S a 
S b Ü 100 
PART IV. CO  2 EVOLUTION TEST (Method C.4-C)  
IV.1. PRINCIPLE OF THE METHOD 
A measured volume of inoculated mineral medium containing a 
known concentration of the test chemical (10-20 mg DOC or 
TOC/l) as the nominal sole source of organic carbon is aerated by 
the passage of carbon, dioxide-free air at a controlled rate in the dark or in diffuse light. Degradation is followed over 28 days by deter ­
mining the carbon dioxide produced, which is trapped in barium or 
sodium hydroxide and which is measured by titration of the residual 
hydroxide or as inorganic carbon. The amount of carbon dioxide produced from the test chemical (corrected for that derived from the blank inoculum) is expressed as a percentage of ThCO
 2 . 
The degree of biodegradation may also be calculated from supple ­
mental DOC analysis made at the beginning and end of incubation.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 855
 
IV.2. DESCRIPTION OF THE METHOD  
IV.2.1.  Apparatus  
(a) Flasks, 2-5 litres, each fitted with an aeration tube reaching 
nearly the bottom of the vessel and an outlet; 
(b) magnetic stirrers, when assessing poorly soluble chemicals; 
(c) gas-absorption bottles; 
(d) device for controlling and measuring airflow; 
(e) apparatus for carbon dioxide scrubbing, for preparation of air 
which is free from carbon dioxide; alternatively, a mixture of 
CO 2 -free oxygen and CO  2 -free nitrogen, from gas cylinders, in 
the correct proportions (20 % O  2 : 80 % N  2 ) may be used; 
(f) device for determination of carbon dioxide, either titrimetrically 
or by some form of inorganic carbon analyser; 
(g) membrane filtration device (optional); 
(h) DOC analyser (optional).  
IV.2.2.  Preparation of mineral medium  
For the preparation of the stock solutions, see I.6.2. 
Mix 10 ml of solution (a) with 800 ml dilution water, add 1 ml of 
solutions (b) to (d) and make up to 11 with dilution water.  
IV.2.3.  Preparation and pre-conditioning of inoculum  
The inoculum may be derived from a variety of sources: activated 
sludge; sewage effluents; surface waters; soils or from a mixture of 
these. 
See I.6.4., I.6.4.1., I.6.4.2. and I.6.5.  
IV.2.4.  Preparation of flasks  
As an example the following volumes and weights indicate the 
values for 5-litre flasks containing 3 l of suspension. If smaller 
volumes are used modify the values accordingly, but ensure that the carbon dioxide formed can be measured accurately. 
To each 5-litre flask add 2400 ml mineral medium. Add an appro ­
priate volume of the prepared activated sludge (see I.6.4.1 and I.6.5) to give a concentration of suspended solids of not more than 30 mg/l in the final 3 l of inoculated mixture. Alternatively first dilute the 
prepared sludge to give a suspension of 500-1000 mg/l in the 
mineral medium before adding an aliquot to the contents of the 5 litre flask to attain a concentration of 30 mg/l; this ensures greater precision. Other sources of inoculum may be used (see I.6.4.2.). 
Aerate these inoculated mixtures with CO
 2 -free air overnight to 
purge the system of carbon dioxide.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 856
 
Add the test material and reference substance, separately, as known 
volume of stock solutions, to replicate flasks to yield concentrations, 
contributed by the added chemicals, of 10 to 20 mg DOC or TOC/l; leave some flasks without addition of chemicals as inoculum controls. Add poorly soluble test substances directly to the flasks 
on a weight or volume basis or handle as described in Appendix 3. 
If required, use one flask to check the possible inhibitory effect of 
the test chemical by adding both the test and reference chemicals at the same concentrations as present in the other flasks. 
Also, if required, use a sterile flask to check whether the test 
chemical is degraded abiotically by using an uninoculated solution 
of the chemical (see I.6.6). Sterilise by the addition of a toxic substance at an appropriate concentration. 
Make up the volumes of suspensions in all flasks to 3 l by the 
addition of mineral medium previously aerated with CO
 2 -free air. 
Optionally, samples may be withdrawn for analysis of DOC (see Appendix 2.4.) and/or specific analysis. Connect the absorption bottles to the air outlets of the flasks. 
If barium hydroxide is used, connect three absorption bottles, each 
containing 100 ml of 0,0125 M barium hydroxide solution, in series to each 5-litre flask. The solution must be free of precipitated sulphate and carbonate and its strength must be determined 
immediately before use. If sodium hydroxide is used, connect two 
traps, the second acting as a control to demonstrate that all the carbon dioxide was absorbed in the first. Absorption bottles fitted with serum bottle closures are suitable. Add 200 ml 0,05 M sodium 
hydroxide to each bottle, which is sufficient to absorb the total 
quantity of carbon dioxide evolved when the test chemical is completely degraded. The sodium hydroxide solution, even when freshly prepared, will contain traces of carbonates; this is corrected 
by deduction of the carbonate in the blank.
 
IV.2.5.  Number of flasks in a typical run  
Flasks 1 and 2: Test suspension 
Flasks 3 and 4: Inoculum blank Flask 5: Procedure control and, preferably and when necessary: Flask 6: Abiotic sterile control 
Flask 7: Toxicity control 
See also I.6.7.
 
IV.2.6.  Performance of the test  
Start the test by bubbling CO  2 -free air through the suspensions at a 
rate of 30-100 ml/min. Take samples of the carbon dioxide absorbent periodically for analysis of the CO
 2 -content. During the first ten days 
it is recommended that analyses should be made every second or 
third day and then every fifth day until the 28th day so that the 10- 
day window period can be identified.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 857
 
On the 28th day, withdraw samples (optionally) for DOC and/or 
specific analysis, measure the pH of the suspensions and add 1 ml 
of concentrated hydrochloric acid to each flask; aerate the flasks overnight to drive off the carbon dioxide present in the test suspen ­
sions. On day 29 make the last analysis of evolved carbon dioxide. 
On the days of measurement of CO
 2 , disconnect the barium 
hydroxide absorber closest to the flask and titrate the hydroxide 
solution with HCl 0,05 M using phenolphthalein as the indicator. 
Move the remaining absorbers one place closer to the flask and place a new absorber containing 100 ml fresh 0,0125 M barium hydroxide at the far end of the series. Make titrations as needed, for example, 
when substantial precipitation is seen in the first trap and before any 
is evident in the second, or at least weekly. Alternatively, with NaOH as absorbent, withdraw with a syringe a small sample 
(depending on the characteristics of the carbon analyser used) of 
the sodium hydroxide solution in the absorber nearer to the flask. Inject the sample into the IC part of the carbon analyser for analysis of evolved carbon dioxide directly. 
Analyse the contents of the second trap only at the end of the test to 
correct for any carryover of carbon dioxide.
 
IV.3. DATA AND REPORTING  
IV.3.1.  Treatment of results  
The amount of CO  2 trapped in an absorber when titrated is given by: 
mgCO  2 = (100 × C  B - 0,5 × V × C  A ) × 44 
where: 
V = volume of HCl used for titration of the 100 ml in the 
absorber (ml) 
C B = concentration of the barium hydroxide solution (M) 
C A = concentration of the hydrochloric acid solution (M) 
if C  B is 0,0125 M and C  A is 0,05 M, the titration for 100 ml barium 
hydroxide is 50 ml and the weight of CO  2 is given by:  
0; 05 
2 Ü 44 Ü ml HCI titrated ¼ 1; 1 Ü ml HCI  
Thus, in this case, to convert volume of HCl titrated to mg CO  2 
produced the factor is 1,1. 
Calculate the weights of CO  2 produced from the inoculum alone and 
from the inoculum plus test chemical using the respective titration values and the difference is the weight of CO
 2 produced from the 
test chemical alone. 
For example, if the inoculum alone gives a titration of 48 ml and 
inoculum plus test chemical gives 45 ml, 
CO 2 from inoculum = 1,1 × (50-48) = 2,2 mg  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 858
 
CO 2 from inoculum plus test chemical = 1,1 × (50-45) = 5,5 mg 
and thus the weight of CO  2 produced from the test chemical is 
3,3 mg. 
The percentage biodegradation is calculated from:  
% degradation ¼ mg CO  2 produced Ü 100 
ThCO  2 Ü mg test chemical added; 
or, 
% degradation ¼ mg CO  2 produced Ü 100 
mg TO added in test  Ü 3; 67 
3,67 being the conversion factor (44/12) for carbon to carbon 
dioxide. 
Obtain the percentage degradation after any time interval by adding 
the percentage of ThCO  2 values calculated for each of the days, up 
to that time, on which it was measured. 
For sodium hydroxide absorbers, calculate the amount of carbon 
dioxide prodllced, expressed as IC (mg), by multiplying the concen ­
tration of IC in the absorbent by the volume of the absorbent. 
Calculate the percentage degradation from:  
% of ThCO  2 ¼ mg IC flask Ä mg IC blank 
MG TOC added as test chemical  Ü 100  
Calculate DOC removals (optional) as described under I.7. Record 
these and all other results on the data sheets provided.  
IV.3.2.  Validity of results  
The IC content of the test chemical suspension in the mineral 
medium at the beginning of the test must be less than 5 % of the TC, and the total CO
 2 evolution in the inoculum blank at the end of 
the test should not normally exceed 40 mg/1 medium. If values 
greater than 70 mg CO  2 /litre are obtained, the data and experimental 
technique should be examined critically. 
See also I.5.2.  
IV.3.3.  Reporting  
See I.8.  
IV.4. DATA SHEET 
An example of a data sheet is given hereafter.  
CARBON DIOXIDE EVOLUTION TEST 
1. LABORATORY  
2. DATE AT START OF TEST  
3. TEST SUBSTANCE  
Name: 
Stock solution concentration: … mg/litre as chemical  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 859
 
Initial conc. in medium: … mg/litre as chemical 
Total C added to flask: … mg C ThCO
 2 : mg CO  2 
4. INOCULUM  
Source: 
Treatment given: 
Pre-conditioning if any: 
Concentration of suspended solids in reaction mixture: mg/litre  
Time 
(day) CO 2 formed 
Test (mg) CO 2 formed 
blank (mg) CO 2 formed cumu ­
lative (mg) 
(test minus blank 
mean) ThCO  2 
cumulative  CO  2 
ThCO  2 Ü 100  
1 
2 mean 3 
4 mean 1 2 1 2 mean  
0 
n 1 
n 2 
n 3 
28 
Note : similar formats may be used for the reference chemical and 
toxicity controls.  
6. CARBON ANALYSIS  (optional) 
Carbon analyser:  
Time (day) Blank mg/l Test chemical mg/l  
0 C  b(o) C o 
28 (*) C  b(t) C t 
(*) Or at end of incubation  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 860
 
% DOC removed ¼ Í 
1 Ä C t Ä C bðtÞ 
C o Ä C bðoÞ Î 
Ü 100  
7. ABIOTIC DEGRADATION  (optional)  
% abiotic degradation  ¼ CO  2 formation in sterile in flask after 28 day  ðmgÞ 
ThCO  2 ðmgÞ Ü 100  
PART V.  MANOMETRIC RESPIROMETRY TEST  
(Method C.4-D)  
V.1. PRINCIPLE OF THE METHOD 
A measured volume of inoculated mineral medium, containing a 
known concentration of test chemical (100 mg/litre of the test substance, to give at least 50-100 mg ThOD/litre) as the nominal sole source of organic carbon, is stirred in a closed flask at a 
constant temperature (± 1
 o C or closer) for up to 28 days. The 
consumption of oxygen is determined either by measuring the quantity of oxygen (produced electrolytically) required to maintain 
constant gas volume in the respirometer flask, or from the change in 
volume or pressure (or a combination of the two) in the apparatus. Evolved carbon dioxide is absorbed in a solution of potassium hydroxide or another suitable absorbent. The amount of oxygen 
taken up by the test chemical (corrected for uptake by blank 
inoculum, run in parallel) is expressed as a percentage of ThOD or COD. Optionally, primary biodegradation may also be calculated from supplemental specific analysis made at the beginning and end 
of incubation, and ultimate biodegradation by DOC analysis.
 
V.2. DESCRIPTION OF THE METHOD  
V.2.1.  Apparatus  
(a) suitable respirometer; 
(b) temperature control, maintaining ± 1  o C or better; 
(c) membrane-filtration assembly (optional); 
(d) carbon analyser (optional).  
V.2.2.  Preparation of mineral medium  
For the preparation of the stock solutions, see I.6.2. 
Mix 10 ml of solution (a) with 800 ml dilution water, add 1 ml of 
solutions (b) to (d) and make up to 1 litre with dilution water.  
V.2.3.  Preparation and pre-conditioning of inoculum  
The inoculum may be derived from a variety of sources: activated 
sludge; sewage effluents; surface waters and soils or from a mixture 
of these. 
See I.6.4., I.6.4.1., I.6.4.2. and I.6.5.  
V.2.4.  Preparation of flasks  
Prepare solutions of the test and reference chemicals, in separate 
batches, in mineral medium equivalent to a concentration, 
normally, of 100 mg chemical/litre (giving at least 50-100 mg 
ThOD/litre), using stock solutions.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 861
 
Calculate the ThOD on the basis of formation of ammonium salts 
unless nitrification is anticipated, when the calculation should be 
based on nitrate formation (see Appendix 2.2) 
Determine the pH values and if necessary adjust to 7,4 ± 0,2. 
Poorly soluble substances should be added at a later stage (see 
below). 
If the toxicity of the test chemical is to be determined, prepare a 
further solution in mineral medium containing both test and reference 
chemicals at the same concentrations as in the individual solutions. 
If measurement of the physico-chemical uptake of oxygen is 
required, prepare a solution of the test chemical at, normally, 100 mg ThOD/litre which has been sterilised by the addition of a suitable toxic substance (see I.6.6). 
Introduce the requisite volume of solutions of test and reference 
chemicals, respectively, into at least duplicate flasks. Add to 
further flasks mineral medium only (for inoculum controls) and, if 
required, the mixed test/reference chemical solution and the sterile solution. 
If the test chemical is poorly soluble, add it directly at this stage on a 
weight or volume basis or handle it as described in Appendix 3. Add potassium hydroxide, soda lime pellets or other absorbent to the 
CO
 2 -absorber compartments.  
V.2.5.  Number of flasks in a typical run  
Flasks 1 and 2: test suspension 
Flasks 3 and 4: inoculum blank Flask 5: procedure control preferably, and when necessary: 
Flask 6: sterile control 
Flask 7: toxicity control See also I.6.7.
 
V.2.6.  Performance of the test  
Allow the vessels to reach the desired temperature and inoculate 
appropriate vessels with prepared activated sludge or other source 
of inoculum to give a concentration of suspended solids not greater than 30 mg/litre. Assemble the equipment, start the stirrer and check for air-tightness, and start the measurement of oxygen uptake. 
Usually no further attention is required other than taking the 
necessary readings and making daily checks to see that the correct temperature and adequate stirring are maintained. 
Calculate the oxygen uptake from the readings taken at regular and 
frequent intervals, using the methods given by the manufacturer of the equipment. At the end of incubation, normally 28 days, measure 
the pH of the contents of the flasks, especially if oxygen uptakes are 
low or greater than ThODNH
 4 (for nitrogen-containing compounds).  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 862
 
If required, withdraw samples from the respirometer flasks, initially 
and finally, for analysis of DOC or specific chemical (see 
Appendix 2.4). At the initial withdrawal, ensure that the volume of test suspension remaining in the flask is known. 'When oxygen is taken up by N-containing test substance, determine the increase in 
concentration of nitrite and nitrate over 28 days and calculate the 
correction for the oxygen consumed by nitrification (Appendix 5).
 
V.3. DATA AND REPORTING  
V.3.1.  Treatment of results  
Divide the oxygen uptake (mg) of the test chemical after a given 
time (corrected for that by the blank inoculum control after the same time) by the weight of the test chemical used. This yields the BOD 
expressed as mg oxygen/mg test chemical, that is:
 
BOD ¼ ðmg O  2 uptake by test chemical  Ä mg O  2 uptake by blankÞ 
ðmg test chemical in flask Þ 
= mg O  2 per mg test chemical 
calculate the percentage biodegradation either from:  
% biodegradation ¼ % ThOD ¼ BOD ðmg O  2=mg chemicalÞ 
ThOD ðmg O  2 chemicalÞ Ü 100  
or form  
% COD ¼ BOD ðmg O  2=mg chemicalÞ 
COD ðmg O  2 chemicalÞ Ü 100  
It should be noted that these two methods do not necessarily give the 
same value; it is preferable to use the former method. 
For test substances containing nitrogen, use the appropriate ThOD 
(NH  4 or NO  3 ) according to what is known or expected about the 
occurrence of nitrification (Appendix 2.2). If nitrification occurs but 
is not complete, calculate a correction for the oxygen consumed by nitrification from the changes in concentration of nitrite and nitrate (Appendix 5). 
When optional determinations of organic carbon and/or specific 
chemical are made, calculate the percentage degradation, as 
described under I.7. 
Record all results on the data sheets attached.
 
V.3.2.  Validity of results  
The oxygen uptake of the inoculum blank is normally 20-30 mg 
O 2 /litre and should not be greater than 60 mg/litre in 28 days. 
Values higher than 60 mg/litre require critical examination of the data and experimental techniques. If the pH value is outside the 
range 6-8,5 and the oxygen consumption by the test chemical is 
less than 60 %, the test should be repeated with a lower concen ­
tration of test chemical. 
See also I.5.2.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 863
 
V.3.3.  Reporting  
See I.8.  
V.4. DATA SHEET  
An example of a data sheet is given hereafter.  
MANOMETRIC RESPIROMETRY TEST 
1. LABORATORY  
2. DATE AT START OF TEST  
3. TEST SUBSTANCE  
Name: 
Stock solution concentration: … mg/litre Initial concentration in medium, C
 o : … mg/litre 
Volume in test flask (V): … ml ThOD or COD: … mg O
 2 /mg test substance (NH  4 or NO  3 ) 
4. INOCULUM  
Source: 
Treatment given: Pre-conditioning, if any: Concentration of suspended solids in reaction mixture: … mg/1
 
5. OXYGEN UPTAKE: BIODEGRADABILITY  
Time (Days) 
0 7 14 21 28  
O 2 upt. (mg) 
test chemical 1 
2 
a, mean 
O 2 upt. (mg) 
blank 3 
4 
b, mean 
Corrected BOD 
(mg) (a 1 - b m ) 
(a 2 - b m ) 
BOD per mg 
test chemical ða 1 Ä bÞ 
C o V 
ða 2 Ä bÞ 
C o V ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 864
 
Time (Days) 
0 7 14 21 28  
% degradation  
BOD 
ThOD Ü 100  D 1 (a 1 ) 
D 2 (a 2 ) 
Mean (*)  
V = volume of medium in test flask 
(*) D  1 and D  2 should not be averaged if there is a considerable difference.  
N.B.: Similar formats may be used for the reference chemical and the 
toxicity controls.  
6. CORRECTION FOR NITRIFICATION  (see Annex V)  
Day 0 28 Difference  
(i) Concentration of nitrate (mg N/litre) (N) 
(ii) Oxygen equivalent (4,57 × N × V) (mg) — — (iii) Concentration of nitrite (mg N/litre) (N) (iv) Oxygen equivalent (3,43 × N × V) (mg) — — (ii + iv) Total oxygen equivalent — —
 
7. CARBON ANALYSIS  (optional) 
Carbon analyser:  
Time (day) Blank mg/litre Test chemical mg/litre  
0 (C  blo ) (C  o ) 
28 (*) (C  blt ) (C  t ) 
(*) Or at end of incubation  
% DOC removed ¼ 1 Ä C t Ä C blt 
C o Ä C blo Í Î  
Ü 100  
8. SPECIFIC CHEMICAL  (optional) 
S b = concentration in physico-chemical (sterile) control at 
28 days 
S a = concentration in inoculated flask at 28 days,  
% biodegradation ¼ S b Ä S a 
S b Ü 100  
9. ABIOTIC DEGRADATION  (optional) 
a = oxygen consumption in sterile flasks after 28 days, (mg)  
oxygen consumption per mg test chemical  ¼ a Ü 100 
C o V ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 865
 
(see Sections 1 and 3)  
% abiotic degradation  ¼ a Ü 100 
C o V Ü ThOD  
PART VI.  CLOSED BOTTLE TEST  (Method C.4-E)  
VI.1 PRINCIPLE OF THE TEST METHOD 
The solution of the test chemical in mineral medium, usually at 2- 
5 mg/litre, is inoculated with a relatively small number of micro- organisms from a mixed population and kept in completely full, closed bottles in the dark at constant temperature. Degradation is 
followed by analysis of dissolved oxygen over a 28-day period. 
The amount of oxygen taken up by the test chemical, corrected for uptake by the blank inoculum run in parallel, is expressed as a percentage of ThOD or COD.
 
VI.2 DESCRIPTION OF THE METHOD  
VI.2.1.  Apparatus  
a) BOD bottles, with glass stoppers, e.g. 250-300 ml; 
b) water bath or incubator, for keeping bottles at constant 
temperature (± 1  o C or better) with the exclusion of light; 
c) large glass bottles (2-5 litres) for the preparation of media and for 
filling the BOD bottles; 
d) oxygen electrode and meter, or equipment and reagents for 
Winkler titration.  
VI.2.2.  Preparation of mineral medium  
For the preparation of the stock solutions, see I.6.2. 
Mix 1 (one) ml of solution (a) to (d) and make up to 1 litre with 
dilution water.  
VI.2.3.  Preparation of the inoculum  
The inoculum is normally derived from the secondary effluent of a 
treatment plant or laboratory-scale unit receiving predominantly domestic sewage. An alternative source for the inoculum is surface water. Normally use from one drop (0,05 ml) to 5 ml of filtrate per 
litre of medium; trials may be needed to discover the optimum 
volume for a given effluent (See I.6.4.2 and I.6.5).
 
VI.2.4.  Preparation of flasks  
Strongly aerate mineral medium for at least 20 min. Carry out each 
test series with mineral medium derived from the same batch. 
Generally, the medium is ready for use after standing for 20 h, at the test temperature. Determine the concentration of dissolved oxygen for control purposes; the value should be about 9 mg/litre 
at 20
 o C. Conduct all transfer and filling operations of the air- 
saturated medium bubble-free, for example, by the use of siphons.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 866
 
Prepare parallel groups of BOD bottles for the determination of the 
test and reference chemicals in simultaneous experimental series. 
Assemble a sufficient number of BOD bottles, including inoculum blanks, to allow at least duplicate measurements of oxygen consumption to be made at the desired test intervals, for example, 
after 0, 7, 14, 21 and 28 days. To ensure being able to identify the 
10-day window, more bottles may be required. 
Add fully aerated mineral medium to large bottles so that they are 
about one-third full. Then add sufficient of the stock solutions of the test chemical and reference chemical to separate large bottles so that the final concentration of the chemicals is normally not greater than 
10 mg/litre. Add no chemicals to the blank control medium 
contained in a further large bottle. 
In order to ensure that the inoculum activity is not limited, the 
concentration of dissolved oxygen must not fall below 0,5 mg/litre in the BOD bottles. This limits the concentration of test chemical to 
about 2 mg/litre. However, for poorly degradable compounds and 
those with a low ThOD, 5-10 mg/litre can be used. In some cases, it would be advisable to run parallel series of test chemical at two different concentrations, for example, 2 and 5 mg/litre. Normally, 
calculate the ThOD on the basis of formation of ammonium salts 
but, if nitrification is expected or known to occur, calculate on the basis of the formation of nitrate (ThOD
 NO3 : see Appendix 2.2). 
However, if nitrification is not complete but does occur, correct 
for the changes in concentration of nitrite and nitrate, determined 
by analysis, (see Appendix 5). 
If the toxicity of the test chemical is to be investigated (in the case, 
for example, of a previous low biodegradability value having been found), another series of bottles is necessary. 
Prepare another large bottle to contain aerated mineral medium (to 
about one-third of its volume) plus test chemical and reference 
chemical at final concentrations normally the same as those in the other large bottles. 
Inoculate the solutions in the large bottles with secondary effluent 
(one drop or about 0,05 ml, to 5 ml/litre) or with another source such as river water (see I.6.4.2.). Finally, make up the solutions to volume 
with aerated mineral medium using a hose which reaches down to 
the bottom of the bottle to achieve adequate mixing.
 
VI.2.5.  Number of flasks in a typical run  
In a typical run the following bottles are used: 
— at least 10 containing test chemical and inoculum (test suspen ­
sion), 
— at least 10 containing only inoculum (inoculum blank), — at least 10 containing reference chemical and inoculum (pro ­
cedure control),
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 867
 
— and, when necessary, six bottles containing test chemical, 
reference chemical and inoculum (toxicity control). However, 
to ensure being able to identify the 10-day window, about twice as many bottles would be necessary.
 
VI.2.6. Performance of the test 
Dispense each prepared solution immediately into the respective 
group of BOD bottles by hose from the lower quarter (not the bottom) of the appropriate large bottle, so that all the BOD bottles are completely filled. Tap gently to remove any air bubbles. Analyse 
the zero-time bottles immediately for dissolved oxygen by the 
Winkler or electrode methods. The contents of the bottles can be preserved for later analysis by the Winkler method by adding manganese (II) sulfate and sodium hydroxide (the first Winkler 
reagent). Store the carefully stoppered bottles, containing the 
oxygen fixed as brown manganese (III) hydrated oxide, in the dark at 10-20
 o C for no longer than 24 hours before proceeding with the 
remaining steps of the Winkler method. Stopper the remaining 
replicate bottles ensuring that no air bubbles are enclosed, and 
incubate at 20  o C in the dark. Each series must be accompanied 
by a complete parallel series for the determination of the inoculated blank medium. Withdraw at least duplicate bottles of all series for 
dissolved oxygen analysis at time intervals (at least weekly) over the 
28 days incubation. 
Weekly samples should allow the assessment of percentage removal 
in a 14-day window, whereas sampling every 3-4 days should allow the 10-day window to be identified, which would require about twice as many bottles. 
For N-containing test substances, corrections for uptake of oxygen 
by any nitrification occurring should be made. To do this, use the 
O
 2 -electrode method for determining the concentration of dissolved 
oxygen and then withdraw a sample from the BOD bottle for analysis for nitrite and nitrate. From the increase in concentration of nitrite and nitrate, calculate the oxygen used (see Annex V).
 
VI.3. DATA AND REPORTING  
VI.3.1.  Treatment of results  
First calculate the BOD exerted after each time period by subtracting 
the oxygen depletion (mg O  2 /litre) of the inoculum blank from that 
exhibited by the test chemical. Divide this corrected depletion by the concentration (mg/litre) of the test chemical, to obtain the specific BOD as mg oxygen per mg test chemical. Calculate the percentage 
biodegradability by dividing the specific BOD by the specific ThOD 
(calculated according to Appendix 2.2) or COD (determined by analysis, see Appendix 2.3), thus:
 
BOD ¼ ðmg O  2 uptake by test chemical  Ä mg O  2 uptake by blankÞ 
ðmg test chemical in flask Þ ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 868
 
= mg O  2 per mg test chemical  
% degradation ¼ BOD ðmg O  2=mg test chemicalÞ 
ThOD ðmg O  2=mg test chemicalÞ Ü 100  
Or 
% degradation ¼ BOD ðmg O  2=mg test chemicalÞ 
COD ðmg O  2=mg test chemicalÞ Ü 100  
It should be noted that these two methods do not necessarily give 
same value; it is preferable to use the former method. 
For test substances containing nitrogen, use the appropriate ThOD 
(NH  4 or NO  3 ) according to what is known or expected about the 
occurrence of nitrification (Appendix 2.2). If nitrification occurs but is not complete, calculate a correction for the oxygen consumed by nitrification from the changes in concentration of nitrite and nitrate 
(Appendix 5).
 
VI.3.2.  Validity of results  
Oxygen depletion in the inoculum blank should not exceed 1,5 mg 
dissolved oxygen/litre after 28 days. Values higher than this require investigation of the experimental techniques. The residual concen ­
tration of oxygen in the test bottles should not fall below 0,5 mg/litre 
at any time. Such low oxygen levels are valid only if the method of determining dissolved oxygen used is capable of measuring such levels accurately. 
See also I.5.2.
 
VI.3.3.  Reporting  
See I.8.  
VI.4. DATA SHEET 
An example of a data sheet is given hereafter.  
CLOSED BOTTLE TEST 
1. LABORATORY  
2. DATE AT START OF TEST  
3. TEST SUBSTANCE  
Name: 
Stock solution concentration: … mg/litre 
Initial concentration in bottle: … mg/litre ThOD or COD: … mg O
 2 /mg test substance  
4. INOCULUM  
Source: 
Treatment given:  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 869
 
Pre-conditioning if any: 
Concentration in the reaction mixture: … mg/litre  
5. DO DETERMINATION  
Method: Winkler/electrode  
Flask analyses  
Time of incubation (d) DO (mg/l) 
0 n  1 n 2 
Blank (without 
chemical) 1 C  1 
2 C  2 
Mean 
m b ¼ C 1 þ C 2 
2 
Test chemical 1 a  1 
2 a  2 
Mean 
m t ¼ a 1 þ a 2 
2 
Note : Similar format may be used for reference and toxicity control.  
6. CORRECTION FOR NITRIFICATION  (see Annex V)  
Time of incubation (d) 0 n  1 n 2 n 3 
(i) Concentration of nitrate (mg N/litre) 
(ii) Change in nitrate concentration (mg N/litre) — (iii) Oxygen equivalent (mg/litre) — (iv) Concentration of nitrite (mg N/litre) (v) Change in nitrite concentration (mg N/litre) — (vi) Oxygen equivalent (mg/litre) — (iii + vi) Total oxygen equivalent (mg/litre) —
 
7. DO DEPLETION: % DEGRADATION  
Depletion after n days (mg/litre) 
n 1 n 2 n 3 
FLASK 1: (m  to - m  tx ) - (m  bo - m  bx ) 
FLASK 2: (m  to - m  tx ) - (m  bo - m  bx ) ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 870
 
Depletion after n days (mg/litre) 
n 1 n 2 n 3 
FLASK 1: 
% D  1 ¼ ½ðm to Ä m tx Þ Ä ðm bo Ä m bx Þâ Ü 100 
conc : of test Ü ThOD chemical 
FLASK 2: 
% D  2 ¼ ½ðm to Ä m tx Þ Ä ðm bo Ä m bx Þâ Ü 100 
conc : of test Ü ThOD chemical 
% D mean (*) =  D 1 Ä D 2 
2 
(*) Do not take mean if there is considerable difference between duplicates.  
m to = value in the flask at time 0 
m tx = value in the flask at time x 
m bo = mean blank value at time 0 
m bx = mean blank value at time x 
Apply also correction for nitrification from iii + vi in section 6.  
8. BLANK DO DEPLETIONS  
Oxygen consumption by blank: (m  bo - m  b28 ) mg/litre. This 
consumption is important for the validity of the test. It should 
be less than 1,5 mg/litre.  
PART VII.  M.I.T.I. TEST  (Method C.4-F)  
VII.1. PRINCIPLE OF THE METHOD 
The oxygen uptake by a stirred solution, or suspension, of the test 
chemical in a mineral medium, inoculated with specially grown, unadapted micro-organisms, is measured automatically over a period of 28 days in a darkened, enclosed respirometer at 25 ± 
1
 o C. Evolved carbon dioxide is absorbed by soda lime. Biodegrad ­
ability is expressed as the percentage oxygen uptake (corrected for blank uptake) of the theoretical uptake (ThOD). The percentage of primary biodegradability is also calculated from supplemental 
specific chemical analysis made at the beginning and end of incu ­
bation and, optionally, by DOC analysis.
 
VII.2. DESCRIPTION OF THE METHOD  
VII.2.1.  Apparatus  
(a) Automatic electrolytic BOD meter or respirometer normally 
equipped with six bottles, 300 ml each and equipped with 
cups to contain CO  2 absorbent;  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 871
 
(b) constant temperature room and/or water-bath at 25  o C ± 1  o C or 
better; 
(c) membrane-filtration assembly (optional); 
(d) carbon analyser (optional).  
VII.2.2.  Preparation of mineral medium  
Prepare the following stock solutions, using analytical grade reagents 
and water (I.6.1.): 
(a) Monopotassium dihydrogen ortho phosphate, 
KH  2 PO 4 8,50 g 
Dipotassium monohydrogen ortho phosphate, 
K 2 HPO  4 21,75 g 
Disodium monohydrogen ortho phosphate 
dodecahydrate Na  2 HPO  4 12 H  2 O 44,60 g 
Ammonium chloride, NH  4 Cl 1,70 g 
Dissolve in water and make up to 1 litre 
The pH value of the solution should be 7,2 
(b) Magnesium sulphate heptahydrate, MgSO  4 7 H  2 O 22,50 g 
Dissolve in water and make up to 1 litre 
(c) Calcium chloride anhydrous, CaCl  2 27,50 g 
Dissolve in water and make up to 1 litre 
(d) Iron (III) chloride hexahydrate, FeCl  3 6 H  2 O 0,25 g 
Dissolve in water and make up to 1 litre 
Take 3 ml of each solution (a), (b), (c) and (d) and make up to 1 
litre. 
VII.2.3.  Preparation of inoculum  
Collect fresh samples from no fewer than ten sites, mainly in areas 
where a variety of chemicals are used and discharged. From sites 
such as sewage treatment works, industrial waste-water treatment, 
rivers, lakes, seas, collect 11 samples of sludge, surface soil, water, etc. and mix thoroughly together. After removing floating 
matter and allowing to stand, adjust the supernatant to pH 7 ± 1 
with sodium hydroxide or phosphoric acid. 
Use an appropriate volume of the filtered supernatant to fill a fill- 
and-draw activated sludge vessel and aerate the liquid for about 23 
1/2 h. 30 minutes after stopping aeration, discard about one third of 
the whole volume of supernatant and add an equal volume of a solution (pH 7) containing 0,1 % each of glucose, peptone and monopotassium ortho phosphate, to the settled material and 
recommence aeration. Repeat this procedure once per day. The 
sludge unit must be operated according to good practice: effluents should be clear, temperature should be kept at 25 ± 2
 o C, pH should 
be 7 ± 1, sludge should settle well, sufficient aeration to keep the 
mixture aerobic at all times, protozoa should be present and the 
activity of the sludge should be tested against a reference substance at least every three months. Do not use sludge as inoculum until after at least one month's operation, but not after 
more than four months. Thereafter, sample from at least 10 sites 
arregular intervals, once every three months.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 872
 
In order to maintain fresh and old sludge at the same activity, mix 
the filtered supernatant of an activated sludge in use with an equal 
volume of the filtered supernatant of a freshly collected ten-source mixture and culture the combined liquor as above. Take sludge for use as inoculum 18-24 h after the unit has been fed.
 
VII.2.4.  Preparation of flasks  
Prepare the following six flasks: 
No 1: test chemical in dilution water at 100 mg/1 
No 2, 3 and 4: test chemical in mineral medium at 100 mg/1 No 5: reference chemical (e.g. aniline) in mineral medium at 
100 mg/1 
No 6: mineral medium only 
Add poorly soluble test chemicals directly on a weight or volume 
basis or handle as described in Appendix 3, except that neither solvents nor emulsifying agents should be used. Add the CO
 2 
absorbent to all flasks in the special cups provided. Adjust the pH 
in flasks No 2, 3 and 4 to 7,0.  
VII.2.5.  Performance of the test  
Inoculate flasks No 2, 3 and 4 (test suspensions), No 5 (activity 
control) and No 6 (inoculum blank) with a small volume of the inoculum to give a concentration of 30 mg/1 suspended solids. 
No inoculum is added to flask No 1 which serves as an abiotic 
control. Assemble the equipment, check for air-tightness, start the stirrers, and start the measurement of oxygen uptake under conditions of darkness. Daily check the temperature, stirrer and 
coulometric oxygen uptake recorder, and note any changes in 
colour of the contents of the flasks. Read the oxygen uptakes for the six flasks directly by an appropriate method, for example, from the six-point chart recorder, which produces a BOD curve. At the 
end of incubation, normally 28 days, measure the pH of the contents 
of the flasks and determine the concentration of the residual test chemical and any intermediate and, in the case of water soluble substance, the concentration o f DOC (Appendix 2.4). Take special 
care in the case of volatile chemicals. If nitrification is anticipated, 
determine nitrate and nitrite concentration, if possible.
 
VII.3. DATA AND REPORTING  
VII.3.1.  Treatment of results  
Divide the oxygen uptake (mg) by the test chemical after a given 
time, corrected for that taken up by the blank inoculum conttol after 
the same time, by the weight of the test chemical used. This yields 
the BOD expressed as mg oxygen/mg test chemical, that is:  
BOD ¼ ðmg O  2 uptake by test chemical  Ä mg O  2 uptake by blankÞ 
ðmg test chemical in flask Þ 
= mg O  2 per mg test chemical  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 873
 
The percentage biodegradation is then obtained from:  
% biodegradation ¼ % ThOD ¼ BOD ðmg O  2=mg chemicalÞ 
ThOD ðmg O  2=mg chemicalÞ Ü 100  
For mixtures, calculate the ThOD from the elemental analysis, as for 
simple compound. Use the appropriate ThOD (ThOD  NH4 or 
ThOD  NO3 ) according to whether nitrification is absent or complete 
(Appendix 2.2). If however, nitrification occurs but is incomplete, 
make a correction for the oxygen consumed by nitrification 
calculated from the changes in concentrations of nitrite and nitrate (Appendix 5). 
Calculate the percentage primary biodegradation from loss of 
specific (parent) chemical (see 1.7.2).
 
D t ¼ S b Ä S a 
S b Ü 100 %  
If there has been a loss of test chemical in the flask No 1 measuring 
physico-chemical removal, report this and use the concentration of test chemical (S
 b ) after 28 days in this flask to calculate the 
percentage biodegradation. 
When determinations of DOC are made (optional), calculate the 
percentage ultimate biodegradation from:  
D t ¼ 1 Ä C t Ä C bt 
C o Ä C bo Í Î  
Ü 100 %  
as described under point I.7.1. If there has been a loss of DOC in the 
flask No 1, measuring physico-chemical removal, use the DOC 
concentration in this flask to calculate the percentage biodegradation. 
Record all results on the data sheets attached.  
VII.3.2.  Validity of results  
The oxygen uptake of the inoculum blank is normally 20-30 mg 
O 2 /1 and should not be greater than 60 mgl/l in 28 days. Values 
higher than 60 mgl/l require critical examination of the data and experimental techniques. If the pH value is outside the range 6-8,5 and the oxygen consumption by the test chemical is less than 
60 %, the test should be repeated with a lower concentration of test 
chemical. 
See also I.5.2. 
If the percentage degradation of aniline calculated from the oxygen 
consumption does not exceed 40 % after seven days and 65 % after 14 days, the test is regarded as invalid.
 
VII.3.3.  Reporting  
See I.8.  
VII.4. DATA SHEET 
An example of a data sheet is given below.  
MITI (I) TEST 
1. LABORATORY  
2. DATE AT START OF TEST  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 874
 
3. TEST SUBSTANCE  
Name: 
Stock solution concentration: mg/1 as chemical Initial concentration in medium, C
 o : … mg/1 as chemical 
Volume of reaction mixture, V: … ml ThOD: … mg O
 2 /1 
4. INOCULUM  
Sludge sampling sites: 
(1) … (6) … 
(2) … (7) … 
(3) … (8) … 
(4) … (9) … (5) … (10) … 
Concentration of suspended solids in activated sludge after 
acclimatisation with synthetic sewage = ... mg/1 
Volume of activated sludge per litre of final medium = ... ml Concentration of sludge in final medium = ... mg/1
 
5. OXYGEN UPTAKE: BIODEGRADABILITY  
Type of respirometer used:  
Time (Days) 
0 7 14 21 28  
O 2 upt. (mg) test 
chemical a 1 
a 2 
a 3 
O 2 upt. (mg) 
blank b 
Corrected BOD 
(mg) (a 1 - b 1 ) 
(a 1 - b 1 ) 
(a 1 - b 1 ) 
BOD per mg test 
chemical ða Ä bÞ 
C o V Flask 1 
Flask 2 
Flask 3 
% degradation  
BOD 
ThOD Ü 100  1 
2 
3 
Mean (*)  
(*) Do not take a mean if there are considerable differences between replicates.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 875
 
N.B.: Similar formats may be used for the reference compound.  
6. CARBON ANALYSIS  (optional) 
Carbon analyser:  
Flask DOC 
% DOC removed Mean 
Measured Corrected  
Water + test substance a — — 
Sludge + test substance b  1 b 1 - c 
Sludge + test substance b  2 b 2 - c 
Sludge + test substance b  3 b 3 - c 
Control blank c — — —  
% DOC removed :  a 1 Ä ðb Ä cÞ 
a Ü 100  
7. SPECIFIC CHEMICAL ANALYTICAL DATA  
Residual amount of test chemical at 
end of test % degradation  
blank test with water S  b 
inoculated medium S a1 
S a2 
S a3 
% degradation ¼ S b Ä S a 
S b Ü 100  
Calculate % degradation for flasks a  1 , and a  3 respectively  
8. REMARKS  
BOD curve against time, if available, should be attached.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 876
 
Appendix 1  
ABBREVIATIONS AND DEFINITIONS  
DO: Dissolved oxygen (mg/l) is the concentration of oxygen dissolved in an 
aqueous sample. 
BOD: Biochemical oxygen demand (g) is the amount of oxygen consumed by 
micro-organisms when metabolising a test compound; also expressed 
as g oxygen uptake per g test compound. (See method C.5). 
COD: Chemical oxygen demand (g) is the amount of oxygen consumed during 
oxidation of a test compound with hot, acidic dichromate; it provides 
a measure of the amount of oxidisable matter present; also expressed as 
9 oxygen consumed per g test compound. (See method C.6). 
DOC: Dissolved organic carbon is the organic carbon present in solution or 
that which passes through a 0,45 micrometre filter or remains in the 
supernatant after centrifuging at 40 000 m.s  -2 (± 4 000 g) for 15 min. 
ThOD: Theoretical oxygen demand (mg) is the total amount of oxygen required 
to oxidise a chemical completely; it is calculated from the molecular 
formula (see Appendix 2.2) and is also expressed as mg oxygen 
required per mg test compound. 
ThCO  2 : Theoretical carbon dioxide (mg) is the quantity of carbon dioxide 
calculated to be produced from the known or measured carbon 
content of the test compound when fully mineralised; also expressed 
as mg carbon dioxide evolved per mg test compound. 
TOC: Total organic carbon of a sample is the sum of the organic carbon in 
solution and in suspension. 
IC: Inorganic carbon 
TC: Total carbon, is the sum of the organic and inorganic carbon present in 
a sample.  
Primary biodegradation:  
is the alteration in the chemical structure of a substance, brought about by 
biological action, resulting in the loss of specific property of that substance.  
Ultimate biodegradation (aerobic):  
is the level of degradation achieved when the test compound is totally utilised by 
micro-organisms resulting in the production of carbon dioxide, water, mineral 
salts and new microbial cellular constituents (biomass).  
Readily biodegradable:  
an arbitrary classification of chemicals which have passed certain specified 
screening tests for ultimate biodegradability; these tests are so stringent that it 
is assumed that such compounds will rapidly and completely biodegrade in 
aquatic environments under aerobic conditions.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 877
 
Inherently biodegradable:  
a classification of chemicals for which there is unequivocal evidence of biode ­
gradation (primary or ultimate) in any recognized test of biodegradability.  
Treatability:  
is the amenability of compounds to removal during biological wastewater 
treatment without adversely affecting the normal operation of the treatment 
processes. Generally, readily biodegradable compounds are treatable but not all inherently biodegradable compounds are. Abiotic processes may also operate.
 
Lag time  
is the time from inoculation, in a die-away test, until the degradation percentage 
has increased to at least 10 %. The lag time is often highly variable and poorly reproducible.
 
Degradation time  
is the time from the end of the lag time till the time that 90 % of maximum level 
of degradation has been reached.  
10-day window  
is the 10 days immediately following the attainment of 10 % degradation.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 878
 
Appendix 2  
CALCULATION AND DETERMINATION OF SUITABLE SUMMARY 
PARAMETERS  
Depending on the method chosen, certain summary parameters will be required. 
The following section describes the derivation of these values. The use of these 
parameters is described in the individual methods.  
1. Carbon content  
The carbon content is calculated from the known elemental composition or 
determined by elemental analysis of the test substance.  
2. Theoretical oxygen demand (ThOD)  
The theoretical oxygen demand (ThOD) may be calculated if the elemental 
composition is known or derermined by elemental analysis. It is for the 
compound: 
C c H h Cl cl N n Na na O o P p S s 
without nitrification,  
ThOD  NH4 ¼ 16 ½2c þ 1=2 ðh Ä cl Ä 3nÞ þ 3s þ 5=2p þ 1=2na Ä oâ 
MW  mg/mg 
or with nitrification  
ThOD  NO3 ¼ 16 ½2c þ 1=2 ðh Ä clÞ þ 5=2n þ 3s þ 5=2p þ 1=2na Ä oâ 
MW  mg/mg  
3. Chemical Oxygen Demand (COD)  
The chemical oxygen demand (COD) is determined according to 
method C.6.  
4. Dissolved organic carbon (DOC)  
Dissolved organic carbon (DOC) is by definition the organic carbon of any 
chemical or mixture in water passing through a 0,45 micrometre filter. 
Samples from the test vessels are withdrawn and filtered immediately in the 
filtration apparatus using an appropriate membrane filter. The first 20 ml (amount can be reduced when using small filters) of the filtrate are 
discarded. Volumes of 10-20 ml or lower, if injected (volume depending 
on the amount required for carbon analyser) are retained for carbon analysis. The DOC concentration is determined by means of an organic carbon analyser which is capable of accurately measuring a carbon concen ­
tration equivalent or lower than 10 % of the initial DOC concentration used 
in the test. 
Filtered samples which cannot be analysed on the same working day can be 
preserved by storage in a refrigerator at 2-4
 o C for 48 h, or below - 18  o C 
for longer periods.  
Remarks:  
Membrane filters are often impregnated with surfactants for hydrophilis ­
ation. Thus the filter may contain up to several mg of soluble organic 
carbon which would interfere in the biodegradability determinations. 
Surfactants and other soluble organic compounds are removed from the filters by boiling them in deionised water for three periods each of one hour. The filters may then be stored in water for one week. If 
disposable filter cartridges are used each lot must be checked to confirm 
that it does not release soluble organic carbon.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 879
 
Depending on the type of membrane filter the test chemical may be 
retained by adsorption. It may therefore be advisable to ensure that the 
test chemical is not retained by the filter. 
Centrifugation at 40 000 m.sec  -2 (4 000 g) for 15 min may be used for 
differentiation of TOC versus DOC instead of filtration. The method is not reliable at initial concentration of < 10 mg DOC/l since either not all 
bacteria are removed or carbon as part of the bacterial plasma is redis ­
solved.
 
BIBLIOGRAPHY  
— Standard Methods for the Examination of Water and Wastewater, 12th 
ed, Am. Pub. Hlth. Ass., Am. Wat. Poll. Control Fed., Oxygen 
Demand, 1965, P 65. 
— Wagner, R., Von Wasser, 1976, vol. 46, 139. — DIN-Entwurf 38 409 Teil 41 Deutsche Einheitsverfahren zur Wasser-, 
Abwasser- und Schlammuntersuchung, Summarische Wirkungs- und 
Stoffkenngrößen (Gruppe H). Bestimmung des Chemischen Sauerstoff ­
bedarfs (CSB) (H 41), Normenausschuß Wasserwesen (NAW) in DIN Deutsches Institut für Normung e. V. 
— Gerike, P., The biodegradability testing of poorly water soluble 
compounds. Chemosphere, 1984, vol 13 (1), 169.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 880
 
Appendix 3  
EVALUATION OF THE BIODEGRADABILITY OF POORLY SOLUBLE 
SUBSTANCES  
In biodegradability tests with poorly soluble substances the following aspects 
should receive special attention. 
While homogeneous liquids will seldom present sampling problems, it is recom ­
mended that solid materials be homogenised by appropriate means to avoid errors 
due to non-homogeneity. Special care must be taken when representative samples of a few milligrams are required from mixtures of chemicals or substances with large amounts of impurities. 
Various forms of agitation during the tests may be used. Care should be taken to 
use only sufficient agitation to keep the chemical dispersed, and to avoid over ­
heating, excessive foaming and excessive shear forces. 
An emulsifier which gives a stable dispersion of the chemical may be used. It 
should not be toxic to bacteria and must not be biodegraded or cause foaming 
under test conditions. 
The same criteria apply to solvents as to the emulsifiers. It is not recommended that solid carriers be used for solid test substances but 
they may be suitable for only substances. 
When auxiliary substances such as emulsifiers, solvents and carriers are used, a 
blank run containing the auxiliary substance should be performed. 
Any of the three respirometric tests CO
 2 , BOD, MITI can be used to study the 
biodegradability of poorly soluble compounds.  
BIBLIOGRAPHY  
— de Morsier, A. et al., Biodegradation tests for poorly soluble compounds. 
Chemosphere, 1987, vol. 16, 833. 
— Gerike, P, The Biodegradability testing of poorly water soluble compounds. 
Chemosphere, 1984, vol. 13, 169.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 881
 
Appendix 4  
EVALUATION OF THE BIODEGRADABILITY OF CHEMICALS 
SUSPECTED TO BE TOXIC TO THE INOCULUM  
When a chemical is subjected to ready biodegradability testing and appears to be 
non-biodegradable, the following procedure is recommended if a distinction 
between inhibition and inertness is desired (Reynolds et al., 1987). 
Similar or identical inocula should be used for the toxicity and biodegradation 
tests. 
To assess the toxicity of chemicals studied in ready biodegradability tests, the 
application of one or a combination of the inhibition of Sludge Respiration rate (activated sludge respiration inhibition test — Directive 87/302/EEC), BOD 
and/or Growth Inhibition methods would seem appropriate. 
If inhibition due to toxicity is to be avoided, it is suggested that the test substance 
concentrations used in ready biodegradability testing should be less than 1/10 of the EC
 50 values (or less than EC  20 values) obtained in toxicity testing. 
Compounds with an EC  50 value of greater than 300 mg/1 are not likely to 
have toxic effects in ready biodegradability testing. 
EC 50 values of less than 20 mg/1 are likely to pose serious problems for the 
subsequent testing. Low test concentrations should be employed, necessitating the use of the stringent and sensitive Closed Bottle test or the use of
 14 C-labelled 
material. Alternatively, an acclimatised inoculum may permit higher test 
substance concentrations to be used. In the latter case, however, the specific criterion of the ready biodegradability test is lost.
 
BIBLIOGRAPHY  
Reynolds, L. et al., Evaluation of the toxicity of substances to be assessed for 
biodegradability. Chemosphere, 1987, vol. 16, 2259.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 882
 
Appendix 5  
CORRECTION FOR OXYGEN UPTAKE FOR INTERFERENCE BY 
NITRIFICATION  
Errors due to not considering nitrification in the assessment by oxygen uptake of 
the biodegradability of test substances not containing N are marginal (not greater 
than 5 %), even if oxidation of the ammonium-N in the medium occurs 
erratically as between test and blank vessels. However, for test substances containing N, serious errors can arise. 
If nitrification has occurred but is not complete the observed oxygen uptake by 
the reaction mixture may be corrected for the amount of oxygen used in 
oxidising ammonium to nitrite and nitrate, if the changes in concentration 
during incubation of nitrite and nitrate are determined by consideration of the following equations: 
2 NH
 4 Cl + 3 O  2 = 2 HNO  2 + 2 HCl + 2 H  2 O (1) 
2 HNO  2 + O  2 = 2 HNO  3 (2) 
Overall: 2 NH
 4 Cl + 4 O  2 = 2 HNO  3 + 2 HCl + 2 H  2 O (3) 
From equation (1), the oxygen uptake by 28 g of nitrogen contained in 
ammonium chloride (NH  4 Cl) in being oxidised to nitrite is 96 g, i.e. a factor 
of 3,43 (96/28). In the same way, from equation (3) the oxygen uptake by 28 g of nitrogen in being oxidised to nitrate is 128 g, i.e. a factor of 4,57 (128/28). 
Since the reactions are sequential, being carried out by distinct and different 
bacterial species, it is possible for the concentration of nitrite to increase or decrease; in the latter case an equivalent concentration of nitrate would be formed. Thus, the oxygen consumed in the formation of nitrate is 4,57 multiplied 
by the increase in concentration of nitrate, whereas the oxygen associated with 
the formation of nitrite is 3,43 multiplied by the increase in the concentration of nitrite or with the decrease in its concentration the oxygen loss is - 3,43 multiplied by the decrease in concentration. 
That is: 
O
 2 consumed in nitrate formation = 4,57 × increase in nitrate 
concentration (4) 
and 
O 2 consumed in nitrite formation = 3,43 × increase in nitrite 
concentration (5) 
and 
O 2 lost in nitrite disappearance = - 3,43 × decrease in nitrate 
concentration (6) 
So that 
O 2 uptake due to nitrification = ± 3,43 × change in nitrite 
conc. + 4,57 × increase in nitrate conc. (7) 
and thus 
O 2 uptake due to C oxidation = total observed uptake uptake 
due to nitrification (8). 
Alternatively, if only total oxidised N is determined, the oxygen uptake due to 
nitrification may be taken to be, as a first approximation, 4,57 × increase in oxidised N 
The corrected value for oxygen consumption due to C oxidation is then 
compared with ThOD NH
 3 , as calculated in Appendix 2.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 883
 
C.5 DEGRADATION — BIOCHEMICAL OXYGEN DEMAND  
1. METHOD  
1.1. INTRODUCTION 
The purpose of the method is the measurement of the biochemical 
oxygen demand (BOD) of solid or liquid organic substances. 
Data elaborated with this test pertain to water-soluble compounds; 
however, volatile compounds and those of low water solubility may also, at least in principle, be tested. 
The method is applicable only to those organic test materials which 
are not inhibitory to bacteria at the concentration used in the test. If 
the test material is not soluble at the test concentration, special measures, such as the use of ultrasonic dispersion, may have to be employed to achieve good dispersion of test material. 
Information on the toxicity of the chemical may be useful to the 
interpretation of low results and in the selection of appropriate test concentrations.
 
1.2. DEFINITION AND UNITS 
The BOD is defined as the mass of dissolved oxygen required by a 
specified volume of solution of the substance for the process of 
biochemical oxidation under prescribed conditions. 
The results are expressed as grams of BOD per gram of tested 
substance.  
1.3. REFERENCE SUBSTANCES 
The use of a suitable reference substance to check the activity of the 
inoculum is desirable.  
1.4. PRINCIPLE OF THE TEST METHOD 
A predetermined amount of the substance, dissolved or dispersed in 
a well-aerated suitable medium, is inoculated with micro-organisms 
and incubated at a constant defined ambient temperature in the dark. 
The BOD is determined by the difference in dissolved oxygen 
content at the beginning and at the end of the test. The duration of the test must be at least five days and not more than 28 days. 
A blank must be determined in a parallel assay containing no test 
substance.
 
1.5. QUALITY CRITERIA 
The BOD determination cannot be considered as a valid deter ­
mination of the biodegradability of a substance. This test may only be regarded as a screening test.
 
1.6. DESCRIPTION OF THE TEST METHOD 
A preliminary solution or dispersion of the substance is prepared to 
obtain a BOD concentration compatible with the method used. The 
BOD is then determined following any suitable national or inter ­
national standardised method.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 884
 
2. DATA AND EVALUATION  
The BOD contained in the preliminary solution is calculated 
according to the selected normalised method, and converted into grams of BOD per gram of tested substance.
 
3. REPORTING  
The method used shall be stated. 
The biochemical oxygen demand should be a mean of at least three 
valid measurements. 
All information and remarks relevant for the interpretation of results 
have to be reported, especially with regard to impurities, physical state, toxic effects and inherent composition of the substance which 
would affect the results. 
The use of an additive to inhibit biological nitrification must be 
reported.
 
4. REFERENCES 
List of standardised methods, for example: 
NF T 90-103: Determination of the biochemical oxygen demand. NBN 407: Biochemical oxygen demand. NEN 32355.4: Bepaling van het biochemish zuurstofverbruik 
(BZV). 
The determination of biochemical oxygen demand, Methods for 
the examination of water and associated materials, HMSO, London. 
ISO 5815: Determination of biochemical oxygen demand after n days.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 885
 
C.6. DEGRADATION — CHEMICAL OXYGEN DEMAND  
1. METHOD  
1.1. INTRODUCTION 
The purpose of the method is the measurement of the chemical 
oxygen demand (COD) of solid or liquid organic substances in a 
standard, arbitrary manner, under fixed laboratory conditions. 
Information on the formula of the substance will be useful to 
conduct this test and interpret the result obtained (e.g. halogen 
salts, ferrous salts of organic compounds, organochlorine compounds).
 
1.2. DEFINITIONS AND UNITS 
The chemical oxygen demand is a measure of the oxidisability of a 
substance, expressed as the equivalent amount in oxygen of an 
oxidising reagent consumed by the substance under fixed laboratory conditions. 
The result is expressed in grams of COD per gram of tested 
substance.
 
1.3. REFERENCE SUBSTANCES 
Reference substances do not need to be employed in all cases when 
investigating a new substance. They should serve primarily to calibrate the method from time to time and to allow comparison of 
results when another method is applied.
 
1.4. PRINCIPLE OF THE TEST METHOD 
A predetermined amount of the substance, dissolved or dispersed in 
water, is oxidised by potassium dichromate in a strong sulphuric acid medium with silver sulphate as a catalyst, under reflux for 
two hours. The residual dichromate is determined by titration with 
standardised ferrous ammonium sulphate. 
In case of chlorine-containing substances, mercuric sulphate (
 1 ) is 
added to reduce chloride interference.  
1.5. QUALITY CRITERIA 
Because of the arbitrary manner of determination, COD is an ‘oxi ­
disability indicator’ and as such is used as a practical method to measure organic matter. 
Chloride can interfere in this test; inorganic reducing or oxidising 
agents may also interfere with the COD determination. 
Some cyclic compounds and many volatile substances (e.g. lower 
fatty acids) are not fully oxidised by this test.
 
1.6. DESCRIPTION OF THE TEST METHOD 
A preliminary solution or dispersion of the substance is prepared to 
obtain a COD between 250 and 600 mg per litre.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 886  
( 1 ) After use, solutions containing mercury salts should be treated to avoid dissemination of 
mercury in the environment.
 Remarks:  
In the case of poorly soluble and non-dispersible substances, an 
amount of finely powdered substance or liquid substance 
corresponding to about 5 mg of COD can be weighed and put in the experimental apparatus with water. 
The chemical oxygen demand (COD) is often and especially in case 
of poorly soluble substances determined advantageously in a variant 
of the method, i.e., in a closed system with a pressure equaliser (H. 
Kelkenberg, 1975). In this modification compounds which are only with difficulty determined by the conventional method — e.g. acetic acid — may often be successfully quantified. The method also 
fails, however, in the case of pyridine. If the potassium dichromate 
concentration, as prescribed in ref.(1), is raised to 0,25 N (0,0416 M), the direct weighing-in of 5-10 mg of substance is facilitated which is essential for the COD determination of poorly 
water soluble substances (ref. (2)). 
Otherwise, the COD is then determined following any suitable 
national or international standardised method.
 
2. DATA AND EVALUATION  
The COD contained in the experimental flask is calculated following 
the selected normalised method, and converted to grams of COD per 
gram of tested substance.  
3. REPORTING  
The reference method used should be stated. 
The chemical oxygen demand should be a mean of at least three 
measurements. All information and remarks relevant to the interpre ­
tation of the results have to be reported, especially with regard to impurities, physical state and inherent properties of the substance (if 
known) which would affect the results. 
The use of mercuric sulphate to minimise the chloride interference 
must be reported.
 
4. REFERENCES  
(1) Kelkenberg, H.,Z. von Wasser und Abwasserforschung, 1975, 
vol. 8, 146. 
(2) Gerike, P. The biodegradability testing of poorly water soluble 
compounds. Chemosphere, 1984, vol. 13,169. 
List of standardised methods, for example: 
NBN T 91-201 Determination of the chemical oxygen 
demand. 
ISBN O 11 7512494 Chemical oxygen demand (dichromate 
value) of polluted and waste waters. 
NF T 90-101 Determination of the chemical oxygen demand. 
DS 217 = water analysis Determination of the chemical 
oxygen demand. 
DIN 38409-H-41 Determination of the chemical oxygen 
demand (COD) within the range above 15 mg per litre. 
NEN 3235 5.3 Bepaling van het chemisch zuurstofverbruik. ISO 6060 Water quality: chemical oxygen demand dich ­
romate methods.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 887
 
C.7. DEGRADATION — ABIOTIC DEGRADATION: HYDROLYSIS 
AS A FUNCTION OF PH  
1. METHOD  
This testing method is equivalent to the OECD TG 111 (2004).  
1.1. INTRODUCTION 
Chemicals can enter surface waters by such routes as direct appli ­
cation, spray drift, run-off, drainage, waste disposal, industrial, domestic or agricultural effluent and atmospheric deposition and may be transformed in those waters by chemical (e.g. hydrolysis, 
oxidation), photochemical and/or microbial processes. This Guideline 
describes a laboratory test method to assess abiotic hydrolytic trans ­
formations of chemicals in aquatic systems at pH values normally found in the environment (pH 4-9) and is based on existing 
Guidelines (1)(2)(3)(4)(5)(6)(7). 
The experiments are performed to determine (i) the rate of hydrolysis 
of the test substance as a function of pH and (ii) the identity or 
nature and rates of formation and decline of hydrolysis products to 
which organisms may be exposed. Such studies may be required for chemicals which are directly applied to water or that are likely to reach the environment by the other routes described above.
 
1.2. DEFINITIONS AND UNITS 
See Appendix 2  
1.3. APPLICABILITY OF THE METHOD 
The method is generally applicable to chemical substances (unla ­
belled or labelled) for which an analytical method with sufficient accuracy and sensitivity is available. It is applicable to slightly volatile and non-volatile compounds of sufficient solubility in 
water. The test should not be applied to chemicals that are highly 
volatile from water (e.g. fumigants, organic solvents) and thus cannot be kept in solution under the experimental conditions of this test. The test may be difficult to conduct with substances of minimal 
solubility in water (8).
 
1.4. PRINCIPLE OF THE TEST METHOD 
Sterile aqueous buffer solutions of different pH values (pH 4, 7 and 
9) are treated with the test substance and incubated in the dark under controlled laboratory conditions (at constant temperatures). After 
appropriate time intervals, buffer solutions are analysed for the test 
substance and for hydrolysis products. With labelled test substance (e.g.
 14 C), a mass balance can be more easily established. 
This testing method is designed as a tiered approach which is shown 
and explained in Appendix 1. Each tier is triggered by the results of 
the previous tier.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 888
 
1.5. INFORMATION ON THE TEST SUBSTANCE 
Non-labelled or labelled test substance can be used to measure the 
rate of hydrolysis. Labelled material is generally preferred for studying the pathway of hydrolysis and for establishing mass 
balance; however, in special cases, labelling may not be absolutely 
necessary.
 14 C-labelling is recommended but the use of other 
isotopes, such as  13 C, 15 N, 3 H, may also be useful. As far as 
possible, the label should be positioned in the most stable part(s) 
of the molecule. For example, if the test substance contains one ring, 
labelling on this ring is required; if the test substance contains two or more rings, separate studies may be needed to evaluate the fate of 
each labelled ring and to obtain suitable information on formation of 
hydrolysis products. The purity of the test substance should be at least 95 %. 
Before carrying out a hydrolysis test, the following information on 
the test substance should be available: 
(a) solubility in water [Testing Method A.6]; 
(b) solubility in organic solvents; 
(c) vapour pressure [Testing Method A.4] and/or Henry's Law 
constant; 
(d) n-octanol/water partition coefficient [Testing Method A.8]; 
(e) dissociation constant (pK
 a ) [OECD Guideline 112] (9); 
(f) direct and indirect phototransformation rate in water where 
appropriate. 
Analytical methods for quantification of the test substance and, if it 
is relevant, for identification and quantification of hydrolysis products in aqueous solutions should be available (see also Section 1.7.2).
 
1.6. REFERENCE SUBSTANCES 
Where possible, reference substances should be used for the identi ­
fication and quantification of hydrolysis products by spectroscopic 
and chromatographic methods or other suitably sensitive methods.  
1.7. QUALITY CRITERIA  
1.7.1.  Recovery  
Analysis of, at least, duplicate buffer solutions or of their extracts 
immediately after the addition of the test substance gives a first indication of the repeatability of the analytical method and of the uniformity of the application procedure for the test substance. 
Recoveries for later stages of the experiments are given by the 
respective mass balances (when labelled material is used). Recoveries should range from 90 % to 110 % for labelled and non labelled chemicals (7). In case it is technically difficult to 
reach this range, a recovery of 70 % for non labelled chemicals is 
acceptable, but justification should be given.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 889
 
1.7.2.  Repeatability and sensitivity of analytical method  
Repeatability of the analytical method(s) used to quantify the test 
substance and hydrolysis products at later times can be checked by duplicate analysis of the same buffer solutions (or of their extracts) 
after sufficient quantities of hydrolysis products have formed for 
quantification 
The analytical method should be sufficiently sensitive to quantify 
test substance concentrations down to 10 % or less of the initial concentration. If relevant, analytical methods should also be suffi ­
ciently sensitive to quantify any hydrolysis product representing 
10 % or more of applied (at any time during the study) down to 25 % or less of its peak concentration.
 
1.7.3.  Confidence intervals for hydrolysis kinetic data  
Confidence intervals should be computed and presented for all 
regression coefficients, rate constants, half-lives, and any other kinetic parameters (e.g. DT50).
 
1.8. DESCRIPTION OF THE TEST METHOD  
1.8.1.  Equipment and apparatus  
The study should be performed in glass containers (e.g. test tubes, 
small flasks) under dark and sterile conditions, if necessary, unless 
preliminary information (such as the n-octanol-water partition coef ­
ficient) indicates that the test substance may adhere to glass. In such cases, alternative materials (such as Teflon) may have to be 
considered. It may also be possible to alleviate the problem of 
adhere to glass by using one or more of the following methods: 
— determine the mass of test substance and hydrolysis products 
sorbed to the test vessel, 
— use of an ultrasonic bath, — ensure a solvent wash of all glassware at each sampling interval, 
— use of formulated products, 
— use an increased amount of co-solvent for addition of test 
substance to the system; if a co-solvent is used it should be a 
co-solvent that does not hydrolyse the test substance. 
Temperature-controlled water bath shakers or thermostatically 
controlled incubators for incubation of the various test solutions are normally required. 
Standard laboratory equipment is required, including, in particular, 
the following: 
— pH meter,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 890
 
— analytical instruments such as GC, HPLC, TLC equipment, 
including the appropriate detection systems for analysing radio ­
labelled and non-labelled substances or inverse isotopes dilution method, 
— instruments for identification purposes (e.g. MS, GC-MS, HPLC- 
MS, NMR, etc.), 
— liquid scintillation counter, — separating funnels for liquid-liquid extraction, — instrumentation for concentrating solutions and extracts (e.g. 
rotating evaporator), 
— temperature control devise (e.g. water bath). Chemical reagents include, for example: — organic solvents, analytical grade, such as hexane, dichlorome ­
thane, etc., 
— scintillation liquid, — buffer solutions (for details see Section 1.8.3). All glassware, reagent-grade water and buffer solutions used in the 
hydrolysis tests should be sterilised.
 
1.8.2.  Test substance application  
The test substance should be applied as aqueous solution into the 
different buffer solutions (see Appendix 3). If it is necessary for 
adequate dissolution, the use of low amounts of water miscible solvents (such as acetonitrile, acetone, ethanol) is permitted for appli ­
cation and distribution of the test substance but this should not 
normally exceed 1 % v/v. In case a higher concentration of 
solvents is considered (e.g. in the case of poorly soluble test substances), this could only be allowed when it can be shown that the solvent has no effect on the hydrolysis of the test substances. 
The use of formulated product is not routinely recommended, as it 
cannot be excluded that the formulation ingredients may influence 
the hydrolysis process. However, for poorly water-soluble test 
substances or for substances that adhere to glass (see Section 1.8.1), the use of formulated material may be an appropriate 
alternative. 
One concentration of the test substance should be used; it should not 
exceed 0,01 M or half of the saturation concentration (see 
Appendix 1).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 891
 
1.8.3.  Buffer solutions  
The hydrolysis test should be performed at pH values of 4, 7 and 
9. For this purpose, buffer solutions should be prepared using reagent grade chemicals and water. Some useful buffer systems are 
presented in Appendix 3. It should be noted that the buffer system 
used may influence the rate of hydrolysis and where this is observed an alternate buffer system should be employed (
 1 ). 
The pH of each buffer solution should be checked with a calibrated 
pH meter to a precision of at least 0,1 at the required temperature.  
1.8.4.  Test conditions  
1.8.4.1.  Test temperature  
The hydrolysis experiments should be carried out at constant 
temperatures. For extrapolation purposes, it is important to maintain the temperature to at least ± 0,5
 o C. 
A preliminary test (Tier 1) should be conducted at a temperature of 
50 o C if the hydrolytic behaviour of the test substance is unknown. 
Higher Tier kinetic tests should be carried out with a minimum of 
three temperatures (including the test at 50  o C) unless the test 
substance is stable to hydrolysis as determined by the Tier 1 testing. A suggested temperature range is 10-70
 o C (preferably 
with at least one temperature below 25  o C utilised), which will 
encompass the reporting temperature of 25  o C and most of the 
temperatures encountered in the field.  
1.8.4.2.  Light and oxygen  
All of the hydrolysis tests should be carried out using any suitable 
method to avoid photolytic effects. All suitable measures should be 
taken to avoid oxygen (e.g. by bubbling helium, nitrogen or argon for five minutes before preparation of the solution).
 
1.8.4.3.  Test duration  
The preliminary test should be carried out for 5 days whereas the 
higher Tier tests should be conducted until 90 % hydrolysis of the test substance or for 30 days whichever comes first.
 
1.8.5.  Performance of the test  
1.8.5.1.  Preliminary test (Tier 1)  
The preliminary test is performed at 50 ± 0,5  o C and pH 4,0, 7,0 and 
9,0. If less than 10 % of hydrolysis is observed after 5 days (t
 0,525  o C > 1 year), the test substance is considered hydrolytically 
stable and, normally, no additional testing is required. If the 
substance is known to be unstable at environmentally relevant temperatures (
 2 ), the preliminary test is not required. The analytical 
method must be sufficiently precise and sensitive to detect a 
reduction of 10 % in the initial concentration.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 892  
( 1 ) Mabey and Mill recommend the use of borate or acetate buffers instead of 
phosphate (11). 
( 2 ) Such information may come from other sources such as hydrolysis data of structurally 
similar compounds from the literature or from other preliminary, semi-quantitative 
hydrolysis tests with the test substance at an earlier development stage.
 1.8.5.2.  Hydrolysis of unstable substances (Tier 2)  
The higher Tier (advanced) test should be performed at the pH 
values at which the test substance was found unstable as defined by the preliminary test above. The buffered solutions of the test 
substance should be thermostated at the selected temperatures. To 
test for first-order behaviour, each reaction solution should be analysed in time intervals which provide a minimum of six spaced data points normally between 10 % and 90 % hydrolysis of the test 
substance. Individual replicate test samples (a minimum of duplicate 
samples contained in separate reaction vessels) should be removed and the contents analysed at each of at least six sampling times (for a 
minimum of twelve replicate data points). The use of a single bulk 
sample from which individual aliquots of the test solution are removed at each sampling interval is considered to be inadequate, as it does not allow for the analysis of data variability and it may 
lead to problems with contamination of the test solution. Sterility 
confirmation tests should be conducted at the end of the higher Tier test (i.e. at 90 % hydrolysis or 30 days). However, if no degradation (i.e. transformation) is observed, sterility tests are not considered 
necessary.
 
1.8.5.3.  Identification of hydrolysis products (Tier 3)  
Any major hydrolysis products at least those representing > 10 % of 
the applied dose should be identified by appropriate analytical methods.
 
1.8.5.4.  Optional tests  
Additional tests at pH values other than 4, 7 and 9 may be required 
for a hydrolytically unstable test substance. For example, for physio ­
logical purposes a test under more acidic conditions (e.g. pH 1,2) may be required employing a single physiologically relevant 
temperature (37
 o C). 
2. DATA  
The amounts of test substance and of hydrolysis products, if 
relevant, should be given as % of applied initial concentration and, 
where appropriate, as mg/L for each sampling interval and for each 
pH and test temperature. In addition, a mass balance should be given in percentage of the applied initial concentration when labelled test substance has been used. 
A graphical presentation of the log-transformed data of the test 
substance concentrations against time should be reported. Any major hydrolysis products at least those representing ≥ 10 % of 
the applied dose should be identified and their log-transformed 
concentrations should also be plotted in the same manner as the 
parent substance to show their rates of formation and decline.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 893
 
2.1. TREATMENT OF RESULTS 
More accurate determinations of half-lives or DT  50 values should be 
obtained by applying appropriate kinetic model calculations. The half-life and/or DT
 50 values (including confidence limits) should 
be reported for each pH and temperature together with a description 
of the model used the order of kinetics and the coefficient of determination (r
 2 ). If appropriate, the calculations should also be 
applied to the hydrolysis products. 
In the case of rate studies carried out at different temperatures, the 
pseudo first-order hydrolysis rate constants (k  obs ) should be 
described as a function of temperature. The calculation should be 
based on both the separation of k  obs into rate constants for acid 
catalysed, neutral, and base catalysed hydrolysis (k  H , k neutral  , and 
k OH respectively) and the Arrhenius equation:  
k obs ¼ k H ½H þ â þ k neutral þ k OH ½OH Ä â ¼ Σ 
i ¼ H;neutral ;OH A i e ÄB i=T 
where A  i and B  i are regression constants from the intercept and 
slope, respectively, of the best fit lines generated from linearly 
regressing ln k  i against the reciprocal of the absolute temperature 
in Kelvin (T). Through the use of the Arrhenius relationships for acid, neutral and base catalysed hydrolysis, pseudo first-order rate constants, and thus half-lives can be calculated for other 
temperatures for which the direct experimental determination of a 
rate constant is not practicable (10).
 
2.2. EVALUATION AND INTERPRETATION OF RESULTS 
Most hydrolysis reactions follow apparent first order reaction rates 
and, therefore, half-lives are independent of the concentration (see equation 4 in Appendix 2). This usually permits the application of laboratory results determined at 10
 -2 to 10  -3 M to environmental 
conditions ( ≤ 10  -6 M) (10). Several examples of good agreement 
between rates of hydrolysis measured in both pure and natural waters for a variety of chemicals were reported by Mabey and Mill (11), provided both pH and temperature had been measured.
 
3. REPORTING  
3.1. TEST REPORT 
The test report must include at least the following information: 
Test substance: — common name, chemical name, CAS number, structural formula 
(indicating position of label when radiolabelled material is used) and relevant physical-chemical properties (see Section 1.5); 
— purity (impurities) of test substance, — label purity of labelled chemical and molar activity (where appro ­
priate).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 894
 
— Buffer solutions: 
— dates and details of preparation, — buffers and waters used, 
— molarity and pH of buffer solutions. 
Test conditions: — dates of the performance of the studies, 
— amount of test substance applied, 
— method and solvents (type and amount) used for application of 
the test substance, 
— volume of buffered test substance solutions incubated, — description of the incubation system used, 
— pH and temperature during the study, 
— sampling times, — method(s) of extraction, 
— methods for quantification and identification of the test substance 
and its hydrolysis products in the buffer solutions, 
— number of replicates. Results: 
— repeatability and sensitivity of the analytical methods used, 
— recoveries (% values for a valid study are given in Section 1.7.1), — replicate data and means in a tabular forms, 
— mass balance during and at the end of the studies (when labelled 
test substance is used), 
— results of preliminary test, 
— discussion and interpretation of results, — all original data and figures. 
The following information is only required when hydrolysis rate is 
determined: 
— plots of concentrations versus time for the test substances and, 
where appropriate, for the hydrolysis products at each pH value 
and temperature; 
— tables of results of Arrhenius equation for the temperature 20
 o C/­
25 o C, with pH, rate constant [h  -1 or day  -1 ], half-life or DT  50 , 
temperatures [  o C] including confidence limits and the coefficients 
of correlation (r  2 ) or comparable information; 
— proposed pathway of hydrolysis.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 895
 
4. REFERENCES  
(1) OECD, (1981) Hydrolysis as a Function of pH. OECD 
Guideline for Testing of Chemicals No 111, adopted 12 May 1981. 
(2) US-Environmental Protection Agency, (1982) 40 CFR 
796.3500, Hydrolysis as a Function of pH at 25
 o C. 
Pesticide Assessment Guidelines, Subdivision N. Chemistry: Environmental Fate. 
(3) Agriculture Canada, (1987) Environmental Chemistry and Fate 
Guidelines for registration of pesticides in Canada. 
(4) European Union (EU), (1995) Commission Directive 95/36/EC 
amending Council Directive 91/414/EEC concerning the placing of plant protection products on the market. Annex V: 
Fate and Behaviour in the Environment. 
(5) Dutch Commission for Registration of Pesticides, (1991) 
Application for registration of a pesticide. Section G: Behaviour of the product and its metabolites in soil, water and air. 
(6) BBA, (1980) Merkblatt No 55, Teil I und II: Prüfung des 
Verhaltens von Pflanzenbehandlungsmitteln im Wasser 
(October 1980). 
(7) SETAC, (1995) Procedures for Assessing the Environmental 
Fate and Ecotoxicity of Pesticides. Mark R. Lynch, Ed. 
(8) OECD, (2000) Guidance document on aquatic toxicity testing 
of difficult substances and mixtures, OECD Environmental 
Health and Safety Publications Series on Testing and Assessment No 23. 
(9) OECD, (1993) Guidelines for the Testing of Chemicals. Paris. 
OECD (1994-2000): Addenda 6-11 to Guidelines for the 
Testing of Chemicals. 
(10) Nelson, H, Laskowski D, Thermes S, and Hendley P., (1997) 
Recommended changes in pesticide fate study guidelines for improving input to computer models. (Text version of oral 
presentation at the 14th Annual Meeting of the Society of 
Environmental Toxicology and Chemistry, Dallas TX, November 1993). 
(11) Mabey, W. and Mill, T., (1978) Critical review of hydrolysis 
of organic compounds in water under environmental 
conditions. J. Phys. Chem. Ref. Data 7, p. 383-415.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 896
 
Appendix 1  
Tiered hydrolysis test scheme
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 897
 
Appendix 2  
Definitions and units 
Standard International (SI) units  should be used in any case.  
Test substance:  any substance, whether the parent compound or relevant trans ­
formation products.  
Transformation products:  all substances resulting from biotic or abiotic trans ­
formation reactions of the test substance.  
Hydrolysis products:  all substances resulting from hydrolytic transformation 
reactions of the test substance.  
Hydrolysis  refers to a reaction of a test substance RX with water, with the net 
exchange of the group X with OH at the reaction centre: 
RX + HOH → ROH + HX [1] 
The rate at which the concentration of RX decreases in this simplified process is 
given by 
rate = k [H  2 O] [RX] second order reaction 
or 
rate = k [RX] first order reaction 
depending on the rate determining step. Because the water is present in great 
excess compared to the test substance, this type of reaction is usually described as a pseudo-first order reaction in which the observed rate constant is given by the relationship 
k
 obs = k [H  2 O] [2] 
and can be determined from the expression (*)  
k obs ¼ 1 
t ln C o 
C t [3] 
where: 
t = time 
and C  o , C t = concentrations of RX at times 0 and t. 
The units of this constant have the dimensions of (time)  -1 and the half-life of the 
reaction (time for 50 % of RX to react) is given by  
t 0;5 ¼ ln2 
k obs [4] 
Half-life:  (t 0,5 ) is the time taken for 50 % hydrolysis of a test substance when the 
reaction can be described by first order kinetics; it is independent of the concen ­
tration.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 898  
(*) If the plot of the log-transformed data  vs. time does not indicate a linear function 
(equated with a first-order reaction rate), then the use of equation [3] is not appropriate 
for determining the hydrolysis rate constant of the test compound.
 DT 50 (Disappearance Time 50):  is the time within which the concentration of 
the test substance is reduced by 50 %; it is different from the half-life t  0,5 when 
the reaction does not follow first order kinetics.  
Estimation of k at different temperature  
When the rate constants are known for two temperatures, the rate constants at 
other temperatures can be calculated using the Arrhenius equation:  
k ¼ A Ü e Ä E 
R Ü T or ln k ¼ ÄE 
R Ü T þ ln A 
A plot of ln k versus 1/T gives a straight line with a slope of - E/R 
where: k = rate constant, measured at different temperatures E = activation energy [kJ/mol] T = absolute temperature [K] R = gas constant [8,314 J/mol.K] The activation energy was calculated by regression analysis or the following 
equation:
 
E ¼ R Ü ln k 2 Ä ln k 1 
1 
T 1 Ä 1 
T 2 Í Î  
where: T  2 > T  1 . ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 899
 
Appendix 3  
Buffer Systems  
A. CLARK AND LUBS:  
Buffer mixtures of CLARK and LUBS  (*) 
Composition pH  
0,2 N HCl and 0,2 N KCl at 20  o C 
47,5 ml. HCl + 25 ml. KCl dil. to 100 ml 1,0 
32,25 ml. HCl + 25 ml. KCl dil. to 100 ml 1,2 20,75 ml. HCl + 25 ml. KCl dil. to 100 ml 1,4 13,15 ml. HCl + 25 ml. KCl dil. to 100 ml 1,6 
8,3 ml. HCl + 25 ml. KCl dil. to 100 ml 1,8 5,3 ml. HCl + 25 ml. KCl dil. to 100 ml 2,0 
3,35 ml. HCl + 25 ml. KCl dil. to 100 ml 2,2  
0,1 M potassium biphthalate + 0,1 N HCl at 20
 o C 
46,70 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml 2,2 39,60 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml 2,4 32,95 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml 2,6 26,42 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml 2,8 20,32 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml 3,0 14,70 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml 3,2 
9,90 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml 3,4 5,97 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml 3,6 2,63 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml 3,8  
0,1 M potassium biphthalate + 0,1 N NaOH at 20
 o C 
0,40 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml 4,0 3,70 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml 4,2 7,50 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml 4,4 
12,15 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml 4,6 17,70 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml 4,8
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 900  
(*) The pH values reported in these tables have been calculated from the potential measurements using Sörensen's 
standard equations (1909). The corresponding pH values are 0,04 units higher than the tabulated values.
 Composition pH  
23,85 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml 5,0 
29,95 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml 5,2 35,45 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml 5,4 39,85 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml 5,6 43,00 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml 5,8 45,45 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml 6,0
 
Buffer mixtures of CLARK and LUBS (Continued) 
0,1 M monopotassium phosphate + 0,1 N NaOH at 20  o C 
5,70 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml 6,0 8,60 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml 6,2 
12,60 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml 6,4 17,80 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml 6,6 23,45 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml 6,8 29,63 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml 7,0 35,00 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml 7,2 
39,50 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml 7,4 
42,80 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml 7,6 45,20 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml 7,8 46,80 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml 8,0  
0,1 M H
 3 B0 3 in 0,1 M KCl + 0,1 N NaOH at 20  o C 
2,61 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml 7,8 3,97 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml 8,0 5,90 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml 8,2 
8,50 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml 8,4 
12,00 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml 8,6 16,30 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml 8,8 21,30 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml 9,0 26,70 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml 9,2
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 901
 
32,00 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml 9,4 
36,85 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml 9,6 40,80 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml 9,8 43,90 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml 10,0
 
B. KOLTHOFF AND VLEESCHHOUWER:  
Citrate buffers of KOLTHOFF and VLEESCHHOUWER  (*)  
Composition pH  
0,1 M monopotassium citrate and 0,1 N HCl at 18  o C (*) 
49,7 ml. 0,1 N HCl + 50 ml. citrate to 100 ml 2,2 
43,4 ml. 0,1 N HCl + 50 ml. citrate to 100 ml 2,4 
36,8 ml. 0,1 N HCl + 50 ml. citrate to 100 ml 2,6 
30,2 ml. 0,1 N HCl + 50 ml. citrate to 100 ml 2,8 
23,6 ml. 0,1 N HCl + 50 ml. citrate to 100 ml 3,0 17,2 ml. 0,1 N HCl + 50 ml. citrate to 100 ml 3,2 
10,7 ml. 0,1 N HCl + 50 ml. citrate to 100 ml 3,4 
4,2 ml. 0,1 N HCl + 50 ml. citrate to 100 ml 3,6  
0,1 M monopotassium citrate and 0,1 N NaOH at 18
 o C (*) 
2,0 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml 3,8 9,0 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml 4,0 
16,3 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml 4,2 
23,7 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml 4,4 
31,5 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml 4,6 
39,2 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml 4,8 46,7 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml 5,0 
54,2 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml 5,2 
61,0 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml 5,4 
68,0 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml 5,6 
74,4 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml 5,8 81,2 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml 6,0
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 902  
(*) Add tiny crystal of thymol or a similar substance to prevent growth of molds.
 C. SÖRENSEN:  
Borate mixtures of SÖRENSEN  
Composition Sörensen 
18 o C Walbum, pH at 
ml. Borax ml. HCl/NaOH 10  o C 40  o C 70  o C 
0,05 M borax + 0,1 N HCl  
5,25 4,75 7,62 7,64 7,55 7,47 
5,50 4,50 7,94 7,98 7,86 7,76 
5,75 4,25 8,14 8,17 8,06 7,95 
6,00 4,00 8,29 8,32 8,19 8,08 6,50 3,50 8,51 8,54 8,40 8,28 7,00 3,00 8,08 8,72 8,56 8,40 7,50 2,50 8,80 8,84 8,67 8,50 8,00 2,00 8,91 8,96 8,77 8,59 8,50 1,50 9,01 9,06 8,86 8,67 9,00 1,00 9,09 9,14 8,94 8,74 
9,50 0,50 9,17 9,22 9,01 8,80 
10,00 0,00 9,24 9,30 9,08 8,86  
0,05 M borax + 0,1 N NaOH  
10,0 0,0 9,24 9,30 9,08 8,86 
9,0 1,0 9,36 9,42 9,18 8,94 8,0 2,0 9,50 9,57 9,30 9,02 7,0 3,0 9,68 9,76 9,44 9,12 6,0 4,0 9,97 10,06 9,67 9,28
 
Phosphate mixtures of SÖRENSEN  
Composition pH  
0,0667 M Monopotassium phosphate + 0,0667 M Disodium phosphate at 20  o C 
99,2 ml. KH  2 PO 4 + 0,8 ml Na  2 HPO  4 5,0 
98,4 ml. KH  2 PO 4 + 1,6 ml Na  2 HPO  4 5,2 
97,3 ml. KH  2 PO 4 + 2,7 ml Na  2 HPO  4 5,4 
95,5 ml. KH  2 PO 4 + 4,5 ml Na  2 HPO  4 5,6 
92,8 ml. KH  2 PO 4 + 7,2 ml Na  2 HPO  4 5,8 
88,9 ml. KH  2 PO 4 + 11,1 ml Na  2 HPO  4 6,0 
83,0 ml. KH  2 PO 4 + 17,0 ml Na  2 HPO  4 6,2 
75,4 ml. KH  2 PO 4 + 24,6 ml Na  2 HPO  4 6,4 
65,3 ml. KH  2 PO 4 + 34,7 ml Na  2 HPO  4 6,6 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 903
 
53,4 ml. KH  2 PO 4 + 46,6 ml Na  2 HPO  4 6,8 
41,3 ml. KH  2 PO 4 + 58,7 ml Na  2 HPO  4 7,0 
29,6 ml. KH  2 PO 4 + 70,4 ml Na  2 HPO  4 7,2 
19,7 ml. KH  2 PO 4 + 80,3 ml Na  2 HPO  4 7,4 
12,8 ml. KH  2 PO 4 + 87,2 ml Na  2 HPO  4 7,6 
7,4 ml. KH  2 PO 4 + 92,6 ml Na  2 HPO  4 7,8 
3,7 ml. KH  2 PO 4 + 96,3 ml Na  2 HPO  4 8,0 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 904
 
C.8. TOXICITY FOR EARTHWORMS  
ARTIFICIAL SOIL TEST  
1. METHOD  
1.1. INTRODUCTION 
In this laboratory test, the test substance is added to an artificial soil 
in which worms are placed for 14 days. After this period (and optionally after seven days) the lethal effect of the substance on the earthworms is examined. The test provides a method for 
relatively short-term screening of the effect of chemicals on earth ­
worms, by dermal and alimentary uptake.
 
1.2. DEFINITION AND UNIT 
LC 50 : the concentration of a substance estimated as killing 50 % of 
the test animals during the test period.  
1.3. REFERENCE SUBSTANCE 
A reference substance is used periodically as a means of demon ­
stration that the sensitivity of the test system has not changed signifi ­
cantly. 
Analytical grade chloroacetamide is recommended as the reference 
substance.  
1.4. PRINCIPLE OF THE TEST 
Soil is a variable medium, so for this test a carefully defined artificial 
loam soil is used. Adult earthworms of the species  Eisenia foetida  
(see note in Appendix) are kept in a defined artificial soil treated with different concentrations of the test substance. The content of the 
containers is spread on a tray 14 days (and optionally seven days) 
after the beginning of the test, and the earthworms surviving at each concentration counted.
 
1.5. QUALITY CRITERIA 
The test is designed to be as reproducible as possible with respect to 
the test substrate and organism. Mortality in the controls must not exceed 10 % at the end of the test, or the test is invalid.
 
1.6. DESCRIPTION OF THE TEST METHOD  
1.6.1.  Materials  
1.6.1.1. T e s t s u b s t r a t e 
A defined artificial soil is used as a basic test substrate. 
(a) Basic substrate (percentages are in terms of dry weight) 
— 10 % sphagnum peat (as close to pH 5,5 to 6,0 as possible 
with no visible plant remains and finely ground),  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 905
 
— 20 % kaolinite clay with preferably more than 50 % 
kaolinite, 
— about 69 % industrial quartz sand (dominant fine sand with 
more than 50 % of particle size 0,05 to 0,2 mm). If the 
substance is not sufficiently dispersible in water, 10 g per 
test container should be kept available for mixing with the test substance later on, 
— about 1 % calcium carbonate (CaCO
 3 ), pulverised, 
chemically pure, added to bring the pH to 6,0 ± 0,5. 
(b) Test substrate 
The test substrate contains the basic substrate, the test substance 
and deionised water. 
Water content is about 25 to 42 % of the dry weight of the basic 
substrate. The water content of the substrate is determined by 
drying a sample to constant weight at 105  o C. The key criterion 
is that the artificial soil must be wetted to a point where there is no standing water. Care should be taken in mixing to obtain an even distribution of the test substance and the substrate. The way 
of introducing the test substance to the substrate has to be 
reported. 
(c) Control substrate 
The control substrate contains the basic substrate and water. If an 
additive agent is used, an additional control should contain the 
same quantity of the additive agent.
 
1.6.1.2. T e s t c o n t a i n e r s 
Glass containers of about one litre capacity (adequately covered with 
plastic lids, dishes or plastic film with ventilation holes) filled with 
an amount of wet test or control substrate equivalent to 500 g dry 
weight of substrate.  
1.6.2.  Test conditions  
Containers should be kept in climatic chambers at a temperature of 
20 ± 2  o C with continuous light. Light intensity should be 400 to 
800 lux. 
The test period is 14 days, but mortality can be assessed optionally 
seven days after starting the test.  
1.6.3.  Test procedure  
T e s t c o n c e n t r a t i o n s 
Concentrations of the test substance are expressed as weight of 
substance per dry weight of basic substrate (mg/kg).  
R a n g e f i n d i n g t e s t 
The range of concentrations just causing mortalities of 0 to 100 % 
may be determined in a range-finding test to provide information on 
the range of concentrations to be used in the definitive test.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 906
 
The substance should be tested at the following concentrations: 
1 000; 100; 10; 1; 0,1 mg substance/kilogram test substrate (dry 
weight). 
If a full definitive test is to be carried out, one test batch per concen ­
tration and one for the untreated control, each with 10 worms, could be sufficient for the range-finding test.
 
D e f i n i t i v e t e s t 
The results of the range-finding test are used to choose at least five 
concentrations in a geometric series just spanning the range 0 to 100 % mortality and differing by a constant factor not exceeding 1,8. 
Tests using these series of concentration should allow the LC
 50 value 
and its confidence limits to be estimated as precisely as possible. 
In the definitive test at least four test batches per concentration and 
four untreated controls, each with 10 worms, are used. The results of these replicate batches are given as a mean and standard deviation. 
When two consecutive concentrations, at a ratio of 1,8, give only 
0 % and 100 % mortality, these two values are sufficient to indicate 
the range within which the LC
 50 falls.  
M i x t u r e o f t h e b a s i c t e s t s u b s t r a t e a n d t h e t e s t 
s u b s t a n c e 
The test substrate should, whenever possible, be made up without 
any additional agents other than water. Immediately before the start 
of the test, an emulsion or dispersion of the test substance in deionised water or other solvent is mixed with the basic test substrate, or sprayed evenly over it with a fine chromatographic or 
similar spray. 
If insoluble in water, the test substance can be dissolved in as small 
a volume as possible of suitable organic solvent (e.g. hexane, acetone or chloroform). 
Only agents which volatilise readily may be used to solubilise, 
disperse or emulsify the test substance. The test substrate must be 
ventilated before use. The amount of water evaporated must be 
replaced. The control should contain the same quantity of any additive agent. 
If the test substance is not soluble, dispersible or emulsifiable in 
organic solvents, 10 g of a mixture of fine ground quartz sand and 
a quantity of test substance necessary to treat 500 g dry weight of 
artificial soil are mixed with 490 g of dry weight of test substrate. 
For each test batch, an amount of wet test substrate equivalent to 
500 g dry weight is placed in each glass container and 10 earth ­
worms, which have been conditioned for 24 hours in a similar wet 
basic substrate and then washed quickly and surplus water absorbed 
on filter paper before use, are placed on the test substrate surface.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 907
 
The containers are covered with perforated plastic lids, dishes or film 
to prevent the substrate drying and they are kept under the test 
conditions for 14 days. 
The assessments should be made 14 days (and optionally seven 
days) after setting up the test. The substrate is spread on a plate made of glass or stainless steel. The earthworms are examined and 
the numbers of surviving earthworms determined. Earthworms are 
considered dead if they do not respond to a gentle mechanical stimulus to the front end. 
When the examination is made at seven days, the container is refilled 
with the substrate and the surviving earthworms are replaced on the 
same test substrate surface.
 
1.6.4.  Test organisms  
Test organisms should be adult  Eisenia foetida  (see note in 
Appendix) (at least two months old with clitellum) wet weight 300 to 600 mg. (For breeding method see Appendix.)
 
2. DATA  
2.1. TREATMENT AND EVALUATION OF RESULTS 
The concentrations of the substance tested are reported with 
reference to the corresponding percentages of dead earthworms. 
When the data are adequate the LC  50 value and the confidence limits 
(p = 0,05) should be determined using standard methods (Litchfield and Wilcoxon, 1949, for equivalent method). The LC
 50 should be 
given as mg of test substance per kilogram of the test substrate (dry 
weight). 
In those cases where the slope of the concentration curve is too steep 
to permit calculation of the LC  50 , a graphical estimate of this value 
is sufficient. 
When two consecutive concentrations at a ratio of 1,8 give only 0 % 
and 100 % mortality, the two values are sufficient to indicate the range within which the LC
 50 falls.  
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, contain the following: 
— statement that the test has been carried out in accordance with the 
abovementioned quality criteria, 
— test carried out (range finding test and/or definitive test), — exact description of the test conditions or statement that the test 
has been carried out in accordance with the method; any devi ­
ations have to be reported, 
— exact description of how the test substance has been mixed into 
the basic test substrate, 
— information about test organisms (species, age, mean and range 
in weight, keeping and breeding conditions, supplier),
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 908
 
— method used for determination of LC  50 , 
— test results including all data used, — description of observed symptoms or changes in behaviour of 
test organisms, 
— mortality in the controls, — LC
 50 or highest tested concentration without mortality and lowest 
tested concentration with a mortality of 100 %, 14 days (and optionally seven days) after setting up the test, 
— plotting of the concentration/response curve, 
— results obtained with the reference substance, whether in 
association with the present test or from previous quality control exercises.
 
4. REFERENCES  
1) OECD, Paris, 1981, Test Guideline 207, Decision of the Council 
C(81)30 final. 
2) Edwards, C. A. and Lofty, J. R., 1977, Biology of Earthworms, 
Chapman and Hall, London, p. 331. 
3) Bouche. M. B., 1972, Lombriciens de France, Ecologie et 
Systematique, Institut National de la Recherche Agronomique, p. 671. 
4) Litchfield, J. T. andWilcoxon, F., A simplified method of 
evaluation dose effect experiments. I. Pharm. Exp. Therap., 
vol. 96, 1949, p. 99. 
5) Commission of the European Communities, Development of a 
standardised laboratory method for assessing the toxicity of chemical substances to earthworms, Report EUR 8714 EN, 1983. 
6) Umweltbundesamt/Biologische Bundesanstalt für land- und 
Forstwirtschaft, Berlin, 1984, Verfahrensvorschlag ‘Toxizitätstest am Regenwurm  Eisenia foetida  in künstlichem Boden’, in: 
Rudolph/Boje, Ökotoxikologie, ecomed, Landsberg, 1986.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 909
 
Appendix  
Breeding and keeping of the worms before testing  
For breeding the animals, 30 to 50 adult worms, are put in a breeding box with 
fresh substrate and removed after 14 days. These animals maybe used for further breeding batches. The earthworms hatched from the cocoons are used for testing 
when mature (under the prescribed conditions after two to three months).
 
Keeping and breeding conditions  
Climatic chamber: temperature 20 ± 2  o C preferably with continuous light 
(intensity 400 to 800 lux). 
Breeding boxes: suitable shaI1ow containers of 10 to 20 l volume. Substrate:  Eisenia foetida  may be bred in various animal excrements. 
It is recommended to use as breeding medium a mixture of 
50 % by volume peat and 50 % cow or horse dung. The medium should have a pH value of about 6 to 7 (regulated with calcium carbonate) and a low ionic conductivity (less 
than 6 mmhos or 0,5 % salt concentration). 
The substrate should be moist but not too wet. 
Other successful procedures may be used besides the 
method given above.  
Note : Eisenia foetida  exists in two races which some taxonomists have separated 
into species (Bouche, 1972). These are morphologically similar but one,  Eisenia 
foetida foetida , has typically transverse striping or banding on the segments and 
the other,  Eisenia foetida andrei , lacks this and has a variegated reddish colour. 
Where possible  Eisenia foetida andrei  should be used. Other species may be 
used if the necessary methodology is available.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 910
 
C.9. BIODEGRADATION  
ZAHN-WELLENS TEST  
1. METHOD  
1.1. INTRODUCTION 
The purpose of the method is the evaluation of the potential ultimate 
biodegradability of water-soluble, non-volatile organic substances when exposed to relatively high concentrations of micro-organisms in a static test. 
Physico-chemical adsorption on the suspended solids may take place 
and this must be taken into account when interpreting results 
(see 3.2). 
The substances to be studied are used in concentrations 
corresponding to DOC-values in the range of 50 to 400 mg/litre or COD-values in the range of 100 to 1 000 mg/litre (DOC = dissolved organic carbon; COD = chemical oxygen demand). These relatively 
high concentrations have the advantage of analytical reliability. 
Compounds with toxic properties may delay or inhibit the degradation process. 
In this method, the measure of the concentration of dissolved organic 
carbon or the chemical oxygen demand is used to assess the ultimate 
biodegradability of the test substance. 
A simultaneous use of a specific analytical method may allow the 
assessment of the primary biodegradation of the substance (dis ­
appearance of the parent chemical structure). 
The method is applicable only to those organic test substances 
which, at the concentration used in the test: 
— are soluble in water under the test conditions, — have negligible vapour pressure under the test conditions, — are not inhibitory to bacteria, 
— are adsorbed within the test system only to a limited extent, 
— are not lost by foaming from the test solution. Information on the relative proportions of the major components of 
the test material will be useful in interpreting the results obtained, 
particularly in those cases where the results are low or marginal. 
Information on the toxicity of the substance to micro-organisms is 
desirable for the interpretation of low results and in the selection of 
appropriate test concentrations.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 911
 
1.2. DEFINITIONS AND UNITS 
The amount of degradation attained at the end of the test is reported 
as the ‘Biodegradability in the Zahn-Wellens test’:  
D T ð%Þ ¼ " 
1 Ä ðC T Ä C B Þ 
ðC A Ä C BA Þ # 
Ü 100  
where: 
DT = biodegradation (%) at time T, 
CA = DOC (or COD) values in the test mixture measured 
three hours after the beginning of the test (mg/l) (DOC = dissolved organic carbon, COD = chemical oxygen 
demand), 
CT = DOC or COD values in the test mixture at time of 
sampling (mg/l), 
CB = DOC or COD value of the blank at time of sampling 
(mg/l), 
CBA = DOC or COD value of the blank, measured three hours 
after the beginning of the test (mg/l). 
The extent of degradation is rounded to the nearest full percent. Percentage degradation is stated as the percentage DOC (or COD) 
removal of the tested substance. 
The difference between the measured value after three hours and the 
calculated or preferably measured initial value may provide useful information on the elimination of the substance (see 3.2, Interpre ­
tation of results).
 
1.3. REFERENCE SUBSTANCES 
In some cases when investigating new substances reference 
substances may be useful; however, specific reference substances 
cannot yet be recommended.  
1.4. PRINCIPLE OF THE TEST METHOD 
Activated sludge, mineral nutrients and the test material as the sole 
carbon source in an aqueous solution are placed together in a one to four litre glass vessel equipped with an agitator and an aerator. The mixture is agitated and aerated at 20 to 25
 o C under diffuse illumi ­
nation or in a dark room for up to 28 days. The degradation process 
is monitored by determination of the DOC (or COD) values in the filtered solution at daily or other appropriate regular time intervals. The ratio of eliminated DOC (or COD) after each interval to the 
value three hours after the start is expressed as percentage biodegra ­
dation and serves as the measure of the extent of degradation at this time. The result is plotted versus time to give the biodegradation curve. 
When a specific analytical method is used, changes in the concen ­
tration of the parent molecule due to biodegradation can be measured 
(primary biodegradability).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 912
 
1.5. QUALITY CRITERIA 
The reproducibility of this test has been proven to be satisfactory in 
a ring test. 
The sensitivity of the method is largely determined by the variability 
of the blank and, to a lesser extent, by the precision of the deter ­
mination of dissolved organic carbon and the level of test compound 
in the liquor.  
1.6. DESCRIPTION OF THE TEST PROCEDURE  
1.6.1.  Preparations  
1.6.1.1. R e a g e n t s 
Test water: drinking water with an organic carbon content < 5 mg/ ­
litre. The concentration of calcium and magnesium ions together 
must not exceed 2,7 mmole/litre; otherwise adequate dilution with deionised or distilled water is required. 
Sulphuric acid, analytical reagent (A.R.): 50 g/l 
Sodium hydroxide solution A.R.: 40 g/l 
Mineral nutrient solution: dissolve in one litre deionised 
water: 
ammonium chloride, NH4Cl, A.R.: 38,5 g 
sodium dihydrogenphosphate, NaH2PO4.2H2O, A.R.: 33,4 g 
potassium dihydrogenphosphate, KH2PO4, A.R.: 8,5 g di-potassium mono-hydrogenphosphate, K2HPO4, A.R.: 21,75 g 
The mixture serves both as a nutrient and as buffering system.
 
1.6.1.2. A p p a r a t u s 
Glass vessels with a volume of one to four litres (e.g. cylindrical 
vessels). 
Agitator with a glass or metal stirrer on a suitable shaft (the stirrer 
should rotate about 5 to 10 cm above the bottom of the vessel). A 
magnetic stirrer with a 7 to 10 cm long rod can be used instead. 
Glass tube of 2 to 4 mm inner diameter to introduce air. The opening 
of the tube should be about 1 cm above the bottom of the vessel. 
Centrifuge (about 3 550 g). 
pH-meter. 
Dissolved-oxygen meter. 
Paper filters.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 913
 
Membrane filtration apparatus. 
Membrane filters, pore size 0,45 μm. Membrane filters are suitable if 
it is assured that they neither release carbon nor absorb the substance in the filtration step. 
Analytical equipment for determining organic carbon content and 
chemical oxygen demand.
 
1.6.1.3. P r e p a r a t i o n o f t h e i n o c u l u m 
Activated sludge from a biological treatment plant is washed by 
(repeatedly) centrifuging or settling with test water (above). 
The activated sludge must be in an appropriate condition. Such 
sludge is available from a properly working waste-water treatment plant. To get as many different species or strains of bacteria as 
possible, it may be preferred to mix inocula from different sources 
(e.g. different treatment plants, soil extracts, river waters, etc.). The mixture is to be treated as described above. 
For checking the activity of the activated sludge see ‘Functional 
control’, below.
 
1.6.1.4. P r e p a r a t i o n o f t h e t e s t s o l u t i o n s 
To the test vessel add 500 ml of test water, 2,5 ml/litre mineral 
nutrient solution and activated sludge in an amount corresponding 
to 0,2 to 1,0 g/litre dry matter in the final mixture. Add sufficient 
stock solution of the substance to be tested so that a DOC concen ­
tration of 50 to 400 mg/litre results in the final mixture. The corresponding COD-values are 100 to 1 000 mg/litre. Make up 
with test water to a total volume of one to four litres. The total 
volume to be chosen is dependent on the number of samples to be taken for DOC or COD determinations and the volumes necessary for the analytical procedure. 
Normally a volume of two litres can be regarded as satisfactory. At 
least one control vessel (blank) is set up to run in parallel with each test series; it contains only activated sludge and mineral nutrient 
solution made up with test water to the same total volume as in 
the test vessels.
 
1.6.2.  Performance of the test  
The test vessels are agitated with magnetic stirrers or screw 
propellers under diffuse illumination or in a dark room at 20 to 
25 o C. Aeration is accomplished by compressed air cleaned by a 
cotton-wool strainer and a wash bottle if necessary. It must be ensured that the sludge does not settle and the oxygen concentration does not fall below 2 mg/litre. 
The pH-value must be checked at regular intervals (e.g. daily) and 
adjusted to pH 7 to 8, if necessary.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 914
 
Losses from evaporation are made up just before each sampling with 
deionised or distilled water in the required amounts. A good 
procedure is to mark the liquid level on the vessel before starting the test. New marks are made after each sampling (without aeration arid stirring). The first samples are always taken three hours after the 
start of the test in order to detect adsorption of test material by the 
activated sludge. 
The elimination of the test material is followed by DOC or COD 
determinations made daily or at some other regular interval. The 
samples from the test vessel and the blank are filtered through a carefully washed paper filter. The first 5 ml of test solution filtrate are discarded. Sludges difficult to filter may be removed previously 
by centrifugation for 10 minutes. DOC and COD determinations are 
made at least in duplicate. The test is run for up to 28 days.  
Note:  samples remaining turbid are filtered through membrane filters. 
The membrane filters must not release or adsorb any organic 
material.
 
F u n c t i o n a l c o n t r o l o f a c t i v a t e d s l u d g e 
A vessel containing a known substance should be run in parallel 
with each test series in order to check the functional capacity of the activated sludge. Diethyleneglycol has been found useful for 
this purpose.
 
A d a p t a t i o n 
If analyses are carried out at relatively short intervals (e.g. daily), 
adaptation can be clearly recognised from the degradation curve (see Figure 2). The test should therefore not be started immediately before the weekend. 
If the adaptation occurs in the end of the period, the test can be 
prolonged until the degradation is finished.  
Note:  if a broader knowledge of the behaviour of the adapted sludge 
is needed, the same activated sludge is exposed once again to the same test material in accordance with the following procedure: 
Switch of the agitator and the aerator and allow the activated sludge 
to settle. Draw off the supernatant liquid, fill up to two litres with test water, stir for 15 minutes and allow to settle again. After the supernatant liquid is drawn off again, use the remaining sludge to 
repeat the test with the same material in accordance with 1.6.1.4 and 
1.6.2, above. The activated sludge can also be isolated by centri ­
fuging instead of settling. 
The adapted sludge may be mixed with fresh sludge to a concen ­
tration of 0,2 to 1 g dry weight/litre.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 915
 
A n a l y t i c a l m e a n s 
Normally samples are filtered through a carefully washed paper filter 
(for washing use deionised water). 
Samples which remain turbid are filtered through membrane filters 
(0,45 μm). 
The DOC concentration is determined in duplicate in the sample 
filtrates (the first 5 ml are discarded) by means of the TOC 
instrument. If the filtrate cannot be analysed on the same day, it 
must be stored in the refrigerator until the next day. Longer storage cannot be recommended. 
The COD concentration: is determined in the sample filtrates with a 
COD analytical set-up by the procedure described in reference (2) below.
 
2. DATA AND EVALUATION  
DOC and/or COD concentrations are determined at least in duplicate 
in the samples according to 1.6.2 above. The degradation at time T is 
calculated according to the formula (with definitions) given unter 1.2 above. 
The extent of degradation is rounded to the nearest full percent. The 
amount of degradation attained at the end of the test is reported as the ‘Biodegradability in the Zahn-Wellens test’.  
Note:  if complete degradation is attained before the test time is over 
and this result is confirmed by a second analysis on the next day, the test can be concluded.
 
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, contain the following: 
— the initial concentration of the substance, 
— all other information and the experimental results concerning the 
tested substance, the reference substance if used, and the blank, 
— the concentration after three hours, 
— biodegradation: curve with description, 
— date and location where test organisms were sampled, status of 
adaptation, concentration used, etc., 
— scientific reasons for any changes of test procedure.  
3.2. INTERPRETATION OF RESULTS 
Removal of DOC (COD) which takes place gradually over days or 
weeks indicates that the test substance is being biodegraded.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 916
 
However, physico-chemical adsorption can, in some cases, play a 
role and this is indicated when there is complete or partial 
removal from the outset, within the first three hours, and the difference between control and test supernatant liquors remains at an unexpectedly low level. 
Further tests are necessary if a distinction is to be drawn between 
biodegradation (or partial biodegradation) and adsorption. 
This can be done in a number of ways, but the most convincing is to 
use the supernatant or sludge as inoculum in a base-set test (pre ­
ferably a respirometric test). 
Test substances giving high, non-adsorptive removal of DOC (COD) 
in this test should be regarded as potentially biodegradable. Partial, 
non-adsorptive removal indicates that the chemical is at least subject to some biodegradation. Low, or zero removals of DOC (COD) may be due to inhibition of microorganisms by the test substance and this 
may also be revealed by lysis and loss of sludge, giving turbid 
supernatants. The test should be repeated using a lower concentration of test substance. 
The use of a compound-specific analytical method or of
 14 C-labelled 
test substance may allow greater sensitivity. In the case of  14 C test 
compound, the recovery of the  14 CO 2 will confirm that biodegra ­
dation has occurred. 
When results are given in terms of primary biodegradation, an expla ­
nation should, if possible, be given on the chemical structure change 
that leads to the loss of response of the parent test substance. 
The validation of the analytical method must be given together with 
the response found on the blank test medium.  
4. REFERENCES  
(1) OECD, Paris, 1981, Test Guideline 302 B, Decision of the 
Council C(81) 30 final. 
(2) Annex V C.9 Degradation: Chemical Oxygen Demand, 
Commission Directive 84/449/EEC, (OJ L 251, 19.9.1984, p. 1).  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 917
 
Appendix  
EVALUATION EXAMPLE 
Organic compound: 4-Ethoxybenzoic acid 
Theoretical test concentration: 600 mg/1 Theoretical DOC: 390 mg/l 
Inoculum Sewage treatment plant of… 
Concentration: 1 gram dry material/litre Adaptation status: not adapted Analysis: DOC-determination 
Amount of sample: 3 ml 
Control substance: Diethyleneglycol Toxicity of compound: No toxic effects below 1 000 mg/l 
Test used: fermentation tubes test
 
Test time Control substance Test substance 
Blank 
DOC (  1 ) 
mg/l DOC (  1 ) 
mg/l DOC net 
mg/l Degradation 
% DOC (  1 ) 
mg/l DOC net 
mg/l Degradation 
% 
0 — — 300,0 — — 390,0 — 
3 hours 4,0 298,0 294,0 2 371,6 367,6 6 
1 day 6,1 288,3 282,2 6 373,3 367,2 6 
2 days 5,0 281,2 276,2 8 360,0 355,0 9 5 days 6,3 270,5 264,2 12 193,8 187,5 52 6 days 7,4 253,3 245,9 18 143,9 136,5 65 7 days 11,3 212,5 201,2 33 104,5 93,2 76 8 days 7,8 142,5 134,7 55 58,9 51,1 87 9 days 7,0 35,0 28,0 91 18,1 11,1 97 10 days 18,0 37,0 19,0 94 20,0 2,0 99
 
( 1 ) Mean values of triplicate determinations.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 918
 
Figure 1  
Examples of biodegradation curves
  
Figure 2  
Examples of sludge adaptation
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 919
 
C.10.  SIMULATION TESTAEROBIC SEWAGE TREATMENT: C.10-A: 
ACTIVATED SLUDGE UNITS — C.10-B: BIOFILMS  
C.10-A: Activated Sludge Units  
INTRODUCTION 
1. This Test Method is equivalent to OECD Test Guideline (TG) 303 (2001). 
In the 1950s it was realised that the newly introduced surfactants caused excessive foaming in waste water treatment plants and in rivers. They were not fully removed in the aerobic treatment and in some cases limited the 
removal of other organic matter. This instigated many investigations into 
how surfactants could be removed from waste waters and whether new chemicals produced by industry were amenable to waste water treatment. In order to do this, model units were used representing the two main types 
of aerobic biological waste water treatment (activated sludge and 
percolating, or trickling, filtration). It would have been impractical and very costly to distribute each new chemical and to monitor large-scale 
treatment plants, even on a local basis.
 
INITIAL CONSIDERATIONS  
Activated sludge units  
2. Model activated sludge units have been described ranging in size from 300 
ml up to about 2 000 ml. Some closely mimicked full-scale plants, having sludge settlement tanks with settled sludge being pumped back to the 
aeration tank, while others provided no settlement facilities e.g. Swisher 
(1). The size of the apparatus is a compromise; on the one hand, it must be large enough for successful mechanical operation and for the provision of sufficient volume of samples without affecting the operation, while on the 
other hand it should not be so large that it demands excessive space and 
materials. 
3. Two forms of apparatus which have been extensively and satisfactorily used 
are the Husmann units (2) and Porous Pot units (3)(4), first used in the study of surfactants; these are described in this Test Method. Others have also 
been used satisfactorily, e.g. Eckenfelder (5). Because of the relatively high 
cost and effort of applying this simulation test, simpler and cheaper screening tests, now embodied in chapter C.4 A-F of this Annex (6) were investigated in parallel. Experience with many surfactants and other 
chemicals has shown that those which passed the screening tests (readily 
biodegradable) also degraded in the simulation test. Some of those failing the screening tests passed the inherent biodegradability tests (chapters C.12 (7) and C.19 (8) of this Annex) but only some of this latter group were 
degraded in the simulation test, while those chemicals which failed tests for 
inherent biodegradability did not degrade in the simulation tests (9)(10)(11). 
4. For some purposes simulation tests carried out under a single set of 
operating conditions are sufficient; the results are expressed as a percentage removal of the test chemical or of dissolved organic carbon (DOC). A description of such a test is given in this test method. However, unlike 
the previous version of this chapter, which described only one type of 
apparatus treating synthetic sewage in the coupled mode using a relatively
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 920
 
crude method of sludge wastage, this text offers a number of variations. 
Alternatives to the type of apparatus, mode of operation, sewage and sludge 
wastage removal are described. This text closely follows that of ISO 11733 (12), which was carefully scrutinised during its preparation, though the method has not been subject to a ring test. 
5. For other purposes the concentration of the test chemical in the effluent is 
required to be known more accurately and for this a more extensive method 
is needed. For example, the sludge wastage rate must be more precisely controlled throughout each day and throughout the period of the test, and units have to be run at a number of wastage rates. For a fully comprehensive 
method, tests should also be run at two or three different temperatures: such 
a method is described by Birch (13)(14) and summarised in Appendix 6. However, present knowledge is insufficient to decide which of the kinetic models are applicable to the biodegradation of chemicals in waste water 
treatment and in the aquatic environment generally. The application of 
Monod kinetics, given in Appendic 6 as an example, is limited to chemicals present at 1 mg/l and above, but in the opinion of some even this remains to be substantiated. Tests at concentrations more truly reflecting 
those found in waste waters are indicated, in Appendix 7, but such tests, and 
those in Appendix 6, are included in Appendices instead of being issued as separate Test Methods.
 
Filters  
6. Much less attention has been given to model percolating filters, perhaps 
because they are more cumbersome and less compact than activated sludge plant models. Gerike et al developed trickling filter units and 
operated them in the coupled mode ((15). These filters were relatively 
large (height 2 m; volume 60 l) and each required as much as 2 l/h of sewage. Baumann et al (16), simulated trickling filters by inserting polyester ‘fleece’ strips into 1 m tubes (14 mm int. diameter) after the strips had been 
immersed in concentrated activated sludge for 30 min. The test chemical as 
sole C source in a mineral salts solution was fed down the vertical tube and biodegradation was assessed from measurements of DOC in the effluent and CO
 2 in the issuing gas. 
7. Biofilters have been simulated in another way (15); the inner surfaces of 
rotating tubes, inclined at a small angle to the horizontal, were fed with 
sewage (about 250 ml/h) with and without the test chemical, and the collected effluents analysed for DOC and/or the specific test chemical.
 
PRINCIPLE OF THE TEST 
8. This method is designed to determine the elimination and the primary and/or 
ultimate biodegradation of water-soluble organic chemicals by aerobic micro-organisms in a continuously operated test system simulating the activated sludge process. An easily biodegradable organic medium and the 
organic test chemical are the sources of carbon and energy for the 
micro-organisms. 
9. Two continuously operated test units (activated sludge plants or porous pots) 
are run in parallel under identical conditions which are chosen to suit the purpose of the test. Normally the mean hydraulic retention time is 6 h and 
the mean sludge age (sludge retention time) is 6 to 10 days. Sludge is 
wasted by one of two methods, the test chemical is normally added at a concentration of between 10 mg/l dissolved organic carbon (DOC) and 20 mg/l DOC, to the influent (organic medium) of only one of the units. The 
second unit is used as a control unit to determine the biodegradation of the 
organic medium.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 921
 
10. In frequently taken samples of the effluents, the DOC, preferably, or 
chemical oxygen demand (COD) is determined, together with the concen ­
tration of the test chemical (if required) by specific analysis, in the effluent from the unit receiving the test chemical. The difference between the effluent concentrations of DOC or COD in the test and control units is 
assumed to be due to the test chemical or its organic metabolites. This 
difference is compared with the influent concentration of DOC or COD due to the added test chemical in order to determine the elimination of the test chemical. 
11. Biodegradation may normally be distinguished from bioadsorption by 
careful examination of the elimination-time curve and may usually be confirmed by applying a test for ready biodegradation using an acclimatised 
inoculum from the unit receiving the test chemical.
 
INFORMATION ON THE TEST CHEMICAL 
12. The purity, water solubility, volatility and adsorption characteristics of the 
test chemical should be known to enable correct interpretation of results to be made. Normally volatile and insoluble chemicals cannot be tested unless 
special precautions are taken (see Appendix 5). The chemical structure, or at 
least the empirical formula, should also be known in order to calculate theoretical values and/or to check measured values of parameters, e.g. theor ­
etical oxygen demand (ThOD), dissolved organic carbon (DOC) and 
chemical oxygen demand (COD). 
13. Information on the toxicity of the test chemical to micro-organisms (see 
Appendix 4) may be useful for selecting appropriate test concentrations 
and may be essential for the correct interpretation of low biodegradation 
values.
 
PASS LEVELS 
14. In the original application of this simulation (confirmatory) test to the 
primary biodegradation of surfactants, a removal of more than 80 % of 
the specific chemical is required before the surfactant may be marketed. If 
the value of 80 % is not attained, this simulation (confirmatory) test may be applied and the surfactant may be marketed only if more than 90 % of the specific chemical is removed. With chemicals in general there is no question 
of pass/fail and the value of percentage removal obtained can be used in 
proximate calculations of the probable environmental concentration to be used in hazard assessments posed by chemicals. Results tend to follow an all or nothing pattern. In a number of studies of pure chemicals the 
percentage removal of DOC was found to be > 90 % in more than three 
quarters and > 80 % in over 90 % of chemicals which showed any significant degree of biodegradability. 
15. Relatively few chemicals (e.g. surfactants) are present in sewage at the 
concentrations (about 10 mg C/l) used in this test. Some chemicals may be inhibitory at these concentrations, while the kinetics of removal of others may be different at low concentrations. A more accurate assessment of 
the degradation could be made by using modified methods, using real ­
istically low concentrations of the test chemical, and the data collected could be used to calculate kinetic constants. However, the necessary experi ­
mental techniques have not yet been fully validated and neither have the 
kinetic models, which describe the biodegradation reactions, been estab ­
lished (see Appendix 7).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 922
 
REFERENCE CHEMICALS 
16. To ensure that the experimental procedure is being carried out correctly, it is 
useful occasionally to test chemicals whose behaviour is known simulta ­
neously when test chemicals are investigated. Such chemicals include adipic 
acid, 2-phenyl phenol, 1-naphthol, diphenic acid, 1-naphthoic acid, etc. 
(9)(10)(11).  
REPRODUCIBILITY OF TEST RESULTS 
17. There have been far fewer reports of studies of simulation tests than of tests 
for ready biodegradability. Reproducibility between (simultaneous) replicates is good (within 10-15 %) for test chemicals degraded by 80 % 
or more but for less well degraded chemicals variability is greater. Also, 
with some borderline chemicals widely disparate results (e.g. 10 %, 90 %) have been recorded on different occasions within the 9 weeks allowed in the test. 
18. Little difference has been found in results obtained with the two types of 
apparatus, but some chemicals have been more extensively and consistently degraded in the presence of domestic sewage than with OECD synthetic sewage.
 
DESCRIPTION OF THE TEST METHOD  
Apparatus  
Test system  
19. The test system for one test chemical consists of a test unit and a control 
unit; but when only specific analyses are performed (primary biodegra ­
dation) only a test unit is required. One control unit can be used for 
several test units receiving either the same or different test chemicals. In 
the case of coupling (Appendix 3) each test unit must have its own control unit. The test system may be either an activated sludge plant model, Husmann unit (Appendx 1, Figure 1) or a porous pot (Appendix 1, 
Figure 2). In both cases storage vessels of sufficient size for the influents 
and effluents are needed, as well as pumps to dose the influent, either mixed with solution of the test chemical or separately. 
20. Each activated sludge plant unit consists of an aeration vessel with a known 
capacity of about 3 litres of activated sludge and a separator (secondary 
clarifier) which holds about 1,5 litres; the volumes can, to some extent, be changed by adjusting the height of the separator. Vessels of different sizes are permissible if they are operated with comparable hydraulic loads. If it is 
not possible to keep the temperature in the test room in the desired range, 
the use of water-jacketed vessels with temperature controlled water is recommended. An airlift pump or a dosing pump is used to recycle the activated sludge from the separator to the aeration vessel, either 
continuously or intermittently at regular intervals. 
21. The porous pot system consists of an inner, porous cylinder with a conical 
bottom held in a slightly larger vessel of the same shape, but made of an impervious plastic material. A suitable material for the porous vessel is 
porous polyethylene of maximum pore size 90 μm and 2 mm thickness. 
Separation of the sludge from the treated organic medium is effected by differential passage through the porous wall. Effluents collect in the annular space from where it overflows into the collecting vessel. No settlement 
occurs and hence there is no sludge return. The whole system may be 
mounted in a thermostatically controlled water-bath. Porous pots become
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 923
 
blocked and could overflow in the initial stages. In such a case, replace the 
porous liner with a clean one by first siphoning the sludge from the pot into 
a clean bucket and removing the blocked liner. After wiping out the impervious outer cylinder insert a clean liner and return the sludge to the pot. Any sludge adhering to the sides of the blocked liner is also carefully 
scraped off and transferred. Clean blocked pots first by using a fine jet of 
water to remove remaining sludge and by soaking in dilute sodium hypo ­
chlorite solution, then in water, followed by thoroughly rinsing with water. 
22. For aeration of the sludge in the aeration vessels of both systems, suitable 
techniques are required, for example sintered cubes (diffuser stones) and compressed air. The air shall be cleaned, if necessary, by passing through 
a suitable filter and washed. Sufficient air must pass through the system to 
maintain aerobic conditions and to keep sludge flocs in suspension at all times during the test.
 
Filtration apparatus or centrifuge  
23. Device for filtration of samples with membrane filters of suitable porosity 
(nominal aperture diameter 0,45 μm) which adsorb soluble organic 
chemicals and release organic carbon to a minimum degree. If filters are used which release organic carbon, wash the filters carefully with hot water 
to remove leachable organic carbon. Alternatively, a centrifuge capable of 
producing 40 000 m/s
 2 may be used.  
Analytical equipment  
24. Apparatus required to determine: 
— DOC(dissolved organic carbon) and TOC (total organic carbon), or 
COD (chemical oxygen demand); 
— specific chemical, if required; — suspended solids, pH, oxygen concentration in water; — temperature, acidity and alkalinity; — ammonium, nitrite and nitrate, if the test is performed under nitrifying 
conditions.
 
Water  
25. Tap water, containing less than 3 mg/l DOC. Determine the alkalinity if not 
already known. 
26. Deionised water, containing less than 2 mg/l DOC.  
Organic medium  
27. Synthetic sewage, domestic sewage or a mixture of both is permissible as 
the organic medium. It has been shown (11)(14) that the use of domestic sewage alone often gives increased percentage DOC removal and even 
allows the removal and biodegradation of some chemicals which are not 
biodegraded when OECD synthetic sewage is used. Also, the constant or intermittent addition of domestic sewage often stabilises the activated sludge, including the crucial ability to settle well. Thus, the use of 
domestic sewage is recommended. Measure the DOC or COD concentration 
in each new batch of organic medium. The acidity or alkalinity of the organic medium should be known. The organic medium may require the addition of a suitable buffer (sodium hydrogen carbonate or potassium 
dihydrogen phosphate) if it is of low acidity or alkalinity, to maintain a 
pH of about 7,5 ± 0,5 in the aeration vessel during the test. The amount of buffer to be added, and when to add it, has to be decided in each individual case. When mixtures are used either continuously or intermittently, the DOC 
(or COD) of the mixture must be kept at an approximately constant value, 
e.g. by dilution with water.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 924
 
Synthetic sewage  
28. Dissolve in each litre of tap water: peptone, 160 mg; meat extract, 110 mg; 
urea, 30 mg; anhydrous dipotassium hydrogen phosphate (K  2 HPO  4 ), 28 mg; 
sodium chloride (NaCl), 7 mg; calcium chloride dihydrate (CaCl  2 .2H  2 O), 4 
mg; magnesium sulphate heptahydrate (Mg  2 SO 4 .7H  2 0), 2 mg. This OECD 
synthetic sewage is an example and gives a mean DOC concentration in the influent of about 100 mg/l. Alternatively, use other compositions, with about the same DOC concentration, which are closer to real sewage. If a less 
concentrated influent is required, dilute the synthetic sewage, for example 
1:1, with tap water to obtain a concentration of about 50 mg/l. Such a weaker influent will allow better growth of nitrifying organisms and this 
modification should be used if the simulation of nitrifying waste water 
plants is to be investigated. This synthetic sewage may be made up in distilled water in a concentrated form and stored at about 1 °C for up to one week. When needed, dilute with tap water. (This medium is unsatis ­
factory e.g. nitrogen concentration is very high, relatively low carbon 
content, but nothing better has been suggested, except to add more phosphate as buffer and extra peptone).
 
Domestic sewage  
29. Use fresh settled sewage collected daily from a treatment works receiving 
predominantly domestic sewage. It should be collected, prior to primary sedimentation, from the overflow channel of the primary sedimentation 
tank, or from the feed to the activated sludge plant, and be largely free 
from coarse particles. The sewage can be used after storage for several days (but generally should not exceed seven days) at about 4 °C, if it is proved 
that the DOC (or COD) has not significantly decreased (i.e. by less than 
20 %) during storage. In order to limit disturbances to the system, the DOC (or COD) of each new batch should be adjusted before use to an appropriate constant value, e.g. by dilution with tap water.
 
Activated sludge  
30. Collect activated sludge for inoculation from the aeration tank of a well 
operated waste water treatment plant or from a laboratory — scale activated 
sludge unit, treating predominantly domestic sewage.  
Stock solutions of test chemical  
31. For chemicals of adequate solubility, prepare stock solutions at appropriate 
concentrations (e.g. 1 to 5 g/l) in deionised water, or in the mineral portion of the synthetic sewage. (for insoluble and volatile chemicals, see Appendix 5). Determine the DOC and total organic carbon (TOC) of the stock solution 
and repeat the measurements for each new batch. If the difference between 
the DOC and TOC is greater than 20 %, check the water-solubility of the test chemical. Compare the DOC or the concentration of the test chemical measured by specific analysis of the stock solution with the nominal value, 
to ascertain whether recovery is good enough (normally > 90 % can be 
expected). Ascertain, especially for dispersions, whether or not DOC can be used as an analytical parameter or if only an analytical technique specific for the test chemical can be used. Centrifugation of the samples is required 
for dispersions. For each new batch, measure the DOC, COD or the test 
chemical with specific analysis.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 925
 
32. Determine the pH of the stock solution. Extreme values indicate that the 
addition of the chemical may have an influence on the pH of the activated 
sludge in the test system. In this case neutralise the stock solution to obtain a pH of 7 ± 0,5 with small amounts of inorganic acid or base, but avoid precipitation of the test chemical.
 
PROCEDURE 
33. The procedure is described for the activated sludge plant units; it has to be 
slightly adapted for the porous pot system.  
Preparation of the inoculum  
34. Inoculate the test system at the beginning of the test with either activated 
sludge or an inoculum containing a low concentration of micro-organisms. 
Keep the inoculum aerated at room temperature until it is used and use it within 24 h. In the first case, take a sample of activated sludge from the aeration tank of an efficiently operated biological waste water treatment 
plant, or a laboratory treatment plant, which receives predominantly 
domestic sewage. If nitrifying conditions are to be simulated, take sludge from a nitrifying waste water treatment plant. Determine the concentration of suspended solids and, if necessary, concentrate the sludge by settling so 
that the volume added to the test system is minimal. Ensure that the starting 
concentration of dry matter is about 2,5 g/l. 
35. In the second case, use 2 ml/l to 10 ml/l of an effluent from a domestic 
biological waste water treatment plant as an inoculum. To get as many different species of bacteria as possible, it may be helpful to add inocula from various other sources, for example surface water. In this case, the 
activated sludge will develop and grow in the test system.
 
Dosage of organic medium  
36. Ensure that influent and effluent containers and tubing from influent vessels 
and to effluent vessels are thoroughly cleaned to remove microbial growths 
initially and throughout the test. Assemble the test systems in a room where 
the temperature is controlled (normally in the range 20-25 °C) or use water-jacketed test units. Prepare a sufficient volume of the required organic medium (paragraphs 27-29). Initially fill the aeration vessel and 
the separator with the organic medium and add the inoculum (paragraphs 
34, 35). Start the aeration such that the sludge is kept in suspension and in an aerobic state and begin dosing the influent and recycling the settled sludge. Dose organic medium out of storage vessels into the aeration 
vessels (paragraphs 20, 21) of the test and control units and collect the 
respective effluents in similar storage vessels. To get the normal hydraulic retention time of 6 h, the organic medium is pumped at 0,5 l/h. To confirm this rate, measure the daily amount of organic medium dosed by noting the 
reduction in volumes of the medium in the storage vessels. Other modes of 
dosing would be necessary for determining the effects of intermittent release and ‘shock’ loading of chemicals. 
37. If the organic medium is prepared for use for a period longer than 1 day, 
cooling at about 4 °C, or other appropriate methods of conservation are necessary to prevent microbial growth and biodegradation outside the test 
units (paragraph 29). If synthetic sewage is used, it is possible to prepare, 
and store at about 4 °C, a concentrated stock solution (e.g. 10-fold the normal concentration, paragraph 28). This stock solution can be well mixed with the appropriate volume of tap water before use; alternatively, 
it can be pumped directly while the appropriate amount of tap water is 
pumped separately.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 926
 
Dosage of test chemical  
38. Add an appropriate volume of the stock solution of the test chemical 
(paragraph 31) to the storage vessel of the influent or dose it directly with a separate pump into the aeration vessel. The normal mean test concen ­
tration in the influent should be between 10 mg/l and 20 mg/l DOC, with an 
upper concentration of no more than 50 mg/l. If the water-solubility of the test chemical is low or if toxic effects are likely to occur, reduce the concentration to 5 mg/l DOC or even less, but only if a suitable specific 
analytical method is available and performed (dispersed test chemicals 
which are poorly soluble in water may be added using special dosing tech ­
niques, see Appendix 5). 
39. Start adding the test chemical after a period in which the system has 
stabilised and is removing DOC of the organic medium efficiently (about 80 %). It is important to check that all units are working equally efficiently before the addition of test chemical; if they are not, it usually helps to mix 
the individual sludges and to re-dispense equal volumes to individual units. 
When an inoculum of (about) 2,5 g/l (dry weight) activated sludge is used, the test chemical may be added from the start of the test since directly adding increasing amounts from the beginning has the advantage that the 
activated sludge may be better able to adapt to the test chemical. In 
whatever manner the test chemical is added, it is recommended that the relevant flow rate and/or the volumes in the storage vessel(s) are measured at regular intervals.
 
Handling of activated sludge  
40. The concentration of activated sludge solids normally stabilises between 
limits during the test, independent of the inoculum used, in the range 1 to 
3 g/l (dry weight) depending on the quality and concentration of the organic medium, operating conditions, the nature of the micro-organisms present and the influence of the test chemical. 
41. Either determine the suspended solids in the aeration vessels at least weekly 
and discard surplus sludge to maintain the concentration at 1 g/l to 3 g/l (dry weight), or control the mean sludge age at a constant value usually in the 
range 6 days to 10 days. If, for example, a sludge retention time of 8 days is 
chosen, remove daily 1/8 of the volume of the activated sludge in the aeration vessel and discard it. Carry this out on a daily basis or, preferably, by means of an automatic intermittently operating pump. Maintaining the 
concentration of suspended solids constant, or within narrow limits, does not 
maintain a constant sludge retention time (SRT), which is the operating variable that determines the value of the concentration of test chemical in the effluent. 
42. Throughout the test, remove, at least daily, any sludge adhering to the walls 
of the aeration vessel and the separator so that it is resuspended. Check and clean regularly all tubes and tubing to prevent growth of biofilm. Recycle 
the settled sludge from the separator to the aeration vessel, preferably by 
intermittent pumping. No recycling takes place in the porous pot system but ensure that clean inner pots are inserted before the volume in the vessel rises significantly (paragraph 21). 
43. Poor settlement and loss of sludge may occur in the Husmann plant units. 
These may be rectified by employing one or more of the actions, listed below, in parallel in test and control units: 
— fresh sludge or flocculant (for example 2 ml/vessel of 50 g/l FeCl
 3 ) 
could be added at regular intervals, e.g. weekly, but ascertain that no 
reaction or precipitation of the test chemical occurs with FeCl  3 ; ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 927
 
— the air-lift pump could be replaced by a peristaltic pump, thus enabling a 
sludge recirculation flow which about equals the influent flow to be used 
and allowing development of an anaerobic zone in the settled sludge (the geometry of the air-lift pump limits the minimum flow rate of returned sludge to be about 12-fold that of the influent); 
— sludge could be pumped intermittently from the separator to the 
aeration vessel (e.g. 5 min. every 2,5 h to recycle 1 l/h to 1,5 l/h; 
— a non-toxic, anti-foaming agent at minimal concentration could be 
used to prevent loss by foaming (e.g. silicone oil); 
— air could be passed through the sludge in the separator in short, 
shock bursts (e.g. 10 sec. every hour); 
— the organic medium may be dosed at intervals into the aeration 
vessel (e.g. 3 min. to 10 min. every hour).
 
Sampling and analysis  
44. At regular intervals measure the dissolved oxygen concentration, the 
temperature and the pH value of the activated sludge in the aeration vessels. Ensure that sufficient oxygen is always available (> 2 mg/l) and that the temperature is kept in the required range (normally 20 °C to 25 °C). 
Keep the pH at 7,5 ± 0,5 by dosing small amounts of inorganic base or acid 
into the aeration vessel or into the influent, or by increasing the buffering capacity of the organic medium (see paragraph 27). When nitrification occurs acid is produced, the oxidation of 1 mg N producing the equivalent 
of about 7 mg CO
 3 – . The frequency of measuring depends on the parameter 
to be measured and the stability of the system, and may vary between daily and weekly measurements. 
45. Measure the DOC or COD in the influents to the control and test vessels. 
Measure the test chemical concentration in the test influent by specific analysis or estimate it from the concentration in the stock solution 
(paragraph 31), the volume used and the amount of sewage dosed into 
the test unit. It is recommended that the concentration of the test chemical be calculated in order to reduce the variability of the concentration data. 
46. Take suitable samples from the collected effluent (e.g. 24 h composites) and 
filter through a membrane of pore size 0,45 μm or centrifuge them at about 
40,000 m/s
 2 for about 15 min. Centrifuging should be used if filtering is 
difficult. Determine DOC or COD at least in duplicate to measure ultimate 
biodegradation and, if required, primary biodegradation by an analysis specific for the test chemical. 
47. The use of COD may give rise to analytical problems at low concentrations 
and is therefore recommended only if a sufficiently high test concentration (about 30 mg/l) is used. Also, for strongly adsorbing chemicals, it is recom ­
mended that the amount of adsorbed chemical in the sludge be measured 
using an analytical technique specific for the test chemical. 
48. The frequency of sampling depends on the expected duration of the test. A 
recommended frequency is three times per week. Once the units are operating efficiently, allow from 1 week to a maximum of 6 weeks after the test chemical has been introduced, for adaptation to reach a steady state. 
Preferably obtain at least 15 valid values in the plateau phase (paragraph 
59), normally lasting 3 weeks, for the evaluation of the test result. The test may be completed if a sufficient degree of elimination is reached (e.g. > 90 %) and these 15 values, which represent analyses carried out each 
weekday over 3 weeks, are available. Normally, do not exceed a test 
duration of more than 12 weeks after addition of the test chemical.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 928
 
49. If the sludge nitrifies and if the effects of the test chemical on nitrification 
are to be studied, analyse samples from the effluent of the test and control 
units at least once per week for ammonium and/or nitrite plus nitrate. 
50. All analyses should be performed as soon as possible, especially the 
nitrogen determinations. If analyses have to be postponed, store the samples at about 4 °C in the dark in full, tightly stopped bottles. If samples have to be stored for more than 48 h, preserve them by 
deep-freezing, acidification (e.g. 10 ml/l of a 400 g/l solution of sulphuric 
acid) or by addition of a suitable toxic substance (e.g. 20 ml/l of a 10 g/l solution of mercury (II) chloride). Ensure that the preservation technique 
does not influence results of analysis.
 
Coupling of test units  
51. If coupling is to be used (Appendix 3), daily exchange the same amount of 
activated sludge (150 ml to 1 500 ml for aeration vessels containing 3 litres 
of liquor) between the aeration vessels of the test unit and its control unit. If 
the test chemical adsorbs strongly onto the sludge, change only the super ­
natant of the separators. In both cases use a correction factor to calculate the test results (paragraph 55).
 
DATA AND REPORTING  
Treatment of results  
52. Calculate the percentage of DOC or COD elimination of the test chemical 
for each timed assessment, using the equation: 
D t ¼ C s Ä ðE Ä E o Þ 
C s Ü 100 
where 
D t = % elimination of DOC or COD at time t 
C s = DOC or COD in the influent due to the test chemical, preferably 
estimated from the stock solution (mg/l) 
E = measured DOC or COD value in the test effluent at time t (mg/l) E
 o = measured DOC or COD value in the control effluent at time t (mg/l) 
53. The degree of DOC or COD elimination of the organic medium in the 
control unit is helpful information in assessing the biodegradative activity of the activated sludge during the test. Calculate the percentage elimination from the equation: 
D
 B ¼ C M Ä E o 
C M Ü 100 
where 
D B = % elimination of DOC or COD of the organic medium in the 
control unit at time t 
C M = DOC or COD of the organic medium in the control influent (mg/l)  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 929
 
Optionally, calculate the percentage elimination DOC or COD due to the 
organic medium plus test chemical in the test unit from the equation: 
D T ¼ C T Ä E 
C T Ü 100 
where 
D T = % elimination of total test influent DOC or COD 
C T = DOC or COD of total test influent or calculated from stock solutions 
(mg/l) 
54. Calculate the removal of the test chemical if measured with a specific 
analytical method at each time assessment from equation: 
D ST ¼ S i Ä S e 
S i Ü 100 
where 
D ST = % primary elimination of test chemical at time t 
S i = measured or estimated test chemical concentration in the test 
influent (mg/l) 
S e = measured test chemical concentration in test effluent at time t (mg/l) 
55. If the coupling mode has been used, compensate the dilution of the test 
chemical in the aeration vessel by the sludge exchange using a correction factor (see Appendix 3). If a mean hydraulic retention time of 6 h and an 
exchange of half of the volume of the activated sludge in the aeration vessel 
have been used, the determined daily elimination values (D
 t , paragraph 52) 
have to be corrected to obtain the true degree of elimination, D  tc , of the test 
chemical from the equation: 
D tc ¼ 4D t Ä 100 
3 
Expression of test results  
56. Plot the percentage elimination D  t (or D  tc ) and D  st , if available, versus time 
(see Appendic 2). From the shape of the elimination curve of the test chemical ( per se  or as DOC) some conclusions may be drawn about the 
removal process.
 
Adsorption  
57. If a high DOC elimination of the test chemical is observed from the 
beginning of the test, the test chemical is probably eliminated by adsorption onto the activated sludge solids. It is possible to prove this by determining the adsorbed test chemical by specific analysis. It is not usual for the 
elimination of DOC of adsorbable chemicals to remain high throughout 
the test; normally, there is a high degree removal initially which gradually falls to an equilibrium value. If, however, the adsorbable test chemical was able to cause acclimation of the microbial population in some way or other, 
the DOC elimination of the test chemical would subsequently increase and 
reach a high plateau value.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 930
 
Lag phase  
58. As in static, screening tests, many test chemicals require a lag phase before 
full biodegradation occurs. In the lag phase, acclimation or adaptation of the degrading bacteria takes place with almost no removal of the test 
chemical; then the initial growth of these bacteria occurs. This phase ends 
and the degradation phase is taken to begin when about 10 % of the initial amount of test chemical is removed (after allowing for adsorption, if it occurs). The lag phase is often highly variable and poorly reproducible.
 
Plateau phase  
59. The plateau phase of an elimination curve in a continuous test is defined as 
that phase in which the maximum degradation takes place. The plateau phase should be at least 3 weeks and have about 15 measured valid values.
 
Mean degree of elimination of test chemical  
60. Calculate the mean value from the elimination values (D  t ) of the test 
chemical at the plateau phase. Rounded to the nearest whole number 
(1 %), it is the degree of elimination of the test chemical. It is also recom ­
mended to calculate the 95 % confidence interval of the mean value.  
Elimination of organic medium  
61. Plot the percentage of elimination of the DOC or COD of the organic 
medium in the control unit (D  B ) versus time. Indicate the mean degree of 
elimination in the same way as for the test chemical (paragraph 60).  
Indication of biodegradation  
62. If the test chemical does not adsorb significantly on to activated sludge and 
the elimination curve has a typical shape of a biodegradation curve with lag, degradation and plateau phases (paragraphs 58, 59), the measured elim ­
ination can safely be attributed to biodegradation. If a high initial removal 
has taken place, the simulation test cannot differentiate between biological and abiotic elimination processes. In such cases, and in other cases where there is any doubt about biodegradation (e.g. if stripping takes place), 
analyse adsorbed test chemicals or perform additional static biodegradation 
tests based on parameters clearly indicating biological processes. Such tests are the oxygen uptake methods (chapter C.4 D, E and F of this Annex (6)) or a test with measurement of carbon dioxide production (chapter C.4 C of 
this Annex (6)) or the ISO Headspace method (18), using a pre-exposed 
inoculum from the simulation test. If both the DOC removal and specific chemical removal have been measured, significant differences (the former being lower than the latter) between the percentages removed indicate the 
presence in the effluents of intermediate organic products which may be 
more difficult to degrade than the parent chemical.
 
Validity of test results  
63. Information on the normal biodegradation behaviour of the inoculum is 
achieved if the degree of elimination of the organic medium (paragraph 53) in the control unit is determined. Consider the test to be valid if the 
degree of DOC or COD elimination in the control unit(s) is > 80 % after 
two weeks and no unusual observations have been made.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 931
 
64. If a readily biodegradable (reference) chemical has been used, the degree of 
biodegradation (D  t , paragraph 52) should be > 90 %. 
65. If the test is performed under nitrifying conditions, the mean concentration 
in the effluents should be < 1 mg/l ammonia-N and < 2 mg/l nitrite-N. 
66. If these criteria (paragraphs 63-65) are not met, repeat the test using an 
inoculum from a different source, test a reference chemical, and review all 
experimental procedures.  
Test Report  
67. The test report must include the following:  
Test chemical:  
— identification data; — physical nature and, where relevant, physical-chemical properties.  
Test conditions:  
— type of test system; any modifications for testing insoluble and volatile 
chemicals; 
— type of organic medium; 
— proportion and nature of industrial waste waters in sewage, if known; — inoculum, nature and sampling site(s), concentration and any 
pre-treatment; 
— test chemical stock solution: DOC and TOC content; how prepared, if 
suspension; test concentration used; reasons if outside range of 10-20 mg/l DOC; method of addition; date first added; any changes; 
— mean sludge age and mean hydraulic retention time; method of sludge 
wastage; methods of overcoming bulking, loss of sludge, etc.; 
— analytical techniques employed; 
— test temperature; 
— qualities of the sludge-bulking, sludge volume index (SVI), mixed liquor 
suspended solids (MLSS); 
— any deviations from standard procedures and any circumstances which 
may have affected results.  
Test results:  
— all measured data (DOC, COD, specific analyses, pH, temperature, 
oxygen concentration, suspended solids, N chemicals, if relevant; 
— all calculated values of D
 t (or D  tc ), D  B , D St obtained in tabular form and 
the elimination curves; 
— information on lag and plateau phases, test duration, the degree of elim ­
ination of the test chemical and that of the organic medium in the control unit, together with statistical information and statements of 
biodegradability and validity of the test; 
— discussion of results.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 932
 
LITERATURE:  
(1) Swisher RD (1987). ‘Surfactant Biodegradation’, 2nd Edn. Marcel Dekker 
Inc. New York, 1085 pp. 
(2) German Government (1962). Ordinance of the degradability of detergents 
in washing and cleaning agents. Bundesgesetzblatt, Pt.1 No 49: 698-706. 
(3) Painter HA and King EF (1978a). WRc porous-pot method for assessing 
biodegradability. Technical Report No 70, Water Research Centre, Medmenham, UK. 
(4) Painter HA and King EF (1978b). The effect of phosphate and temperature 
on growth of activated sludge and on biodegradation of surfactants. Wat. 
Res. 12: 909-915. 
(5) Eckenfelder, W.W (19) US EPA. (6) Chapter C.4 of this Annex, Determination of ‘Ready’ Biodegradability. (7) Chapter C.12 of this Annex, Biodegradation — Modified SCAS Test. (8) Chapter C.19 of this Annex, Estimation of the Adsorption Coefficient 
(K
 OC ) on Soil and on Sewage Sludge Using High Performance Liquid 
Chromatography (HPLC). 
(9) Gerike P and Fischer WK (1979). A correlation study of biodegradability 
determinations with various chemicals in various tests. Ecotox. Env. Saf. 3:157-173. 
(10) Gerike P and Fischer WK (1981), as (9), II Additional results and 
conclusions. Ecotox. Env. Saf. 5: 45-55. 
(11) Painter HA and Bealing D (1989). Experience and data from the OECD 
activated sludge simulation test. pp. 113-138, In: Laboratory tests for simu ­
lation of water treatment processes. CEC Water Pollution Report 18. Eds. Jacobsen BN, Muntau H, Angeletti G. 
(12) ISO 11733 (1995; revised 2004). Evaluation of the elimination and biode ­
gradability of organic substances in an aqueous medium — activated sludge simulation test. 
(13) Birch RR (1982). The biodegradability of alcohol ethoxylates. XIII Jornado 
Com. Espanol. Deterg.: 33-48. 
(14) Birch RR (1984). Biodegradation of noniomic surfactants. J.A.O.C.S. 61 
(2): 340-343. 
(15) Gerike P, Fischer WK and Holtmann W (1980). Biodegradability deter ­
minations in trickling filter units compared with the OECD confirmatory test. Wat.Res. 14: 753-758. 
(16) Baumann U, Kuhn G and Benz M. (1998). Einfache Versuchsanordnung 
zur Gewinnung gewässerökologisch relevanter Daten, UWSF — Z. 
Umweltchem. Ökotox. 10: 214-220. 
(17) Her Majesty’s Stationery Office (1982). Assessment of biodegradability. 
Methods for the examination of waters and associated materials. pp. 91- 
98 ISBN 0117516619. 
(18) ISO 14593 (1998). Water Quality — Evaluation in an aqueous medium of 
the ultimate biodegradability of organic compounds. Method by the analysis of inorganic carbon in sealed vessels.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 933
 
Appendix 1  
Figure 1  
Equipment used for assessment of biodegradability  
Husmann unit
  
A. Storage vessel 
B. Dosing pump C. Aeration chamber (3l capacity) D. Settling vessel E. Air lift pump 
F. Collection vessel G. Aerator H. Air flow meter
 
Figure 2  
Equipment used for assessment of biodegradability  
Porous pot
  
A. Storage vessel 
B. Dosing pump C. Porous aeration vessel D. Outer impermeable vessel E. Collection vessel 
F. Diffuser G. Air flow meter
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 934
 
Figure 3  
Details of 3 litre porous pot aeration vessel
  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 935
 
Appendix 2  
Example of an elimination curve
  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 936
 
Appendix 3  
[INFORMATIVE]  
COUPLING OF THE TEST UNITS 
In order to try to equalise the microbial populations in sludges in a test unit, 
receiving sewage plus a test chemical, and in a control unit, receiving only 
sewage, a daily interchange of sludge was introduced (1). The procedure was called coupling and the method is known as coupled units. Coupling was initially performed using Husmann activated sludge units but it has also been done with 
Porous Pot units (2)(3). No significant differences in results were found as 
between non-coupled and coupled units, whether Husmann or Porous Pot so there is no advantage in expending the time and energy needed in coupling the units. 
Sludge exchanges can give the appearance of quite a considerable removal, since 
some of the test chemical in transferred and the concentrations of test chemical in the test and control effluents become more nearly equal. Thus, correcting factors have to be used, which depend on the fraction exchanged and the mean hydraulic 
retention time. More details of the calculation have been published (1). 
Calculate the corrected DOC or COD elimination degree using the general 
formula: 
D
 tc ¼ ðD t Ä 100 · a · r=12Þ=ð1 Ä a · r=12Þ % 
where 
D tc = corrected % DOC or COD elimination 
D t = determined % DOC or COD elimination 
a = interchange fraction of the volume of the activated sludge units r = mean hydraulic retention time (h) If, for example, half of the volume of the aeration tank is exchanged (a = 0,5) 
and the mean hydraulic retention time is 6 h, the correction formula is: 
D
 tc ¼ 4D t Ä 100 
3 
LITERATURE:  
(1) Fischer W, Gerike P, Holtmann W (1975). Biodegradability Determinations 
via Unspecific Analyses (Chemical Oxygen Demand, DOC) in Coupled 
Units of the OECD Confirmatory Test. I The test. Wat. Res. 9: 1131-1135. 
(2) Painter HA, Bealing DJ (1989). Experience and Data from the OECD 
Activated Sludge Simulation Test. pp. 113-138. In: Laboratory Tests for Simulation of Water Treatment Processes CEC Water Pollution Report 18. 
Eds. Jacobsen BN, Muntau H, Angeletti G. 
(3) Painter HA, King EF (1978). Water Research Centre Porous Pot Method for 
Assessing Biodegradability. Technical Report TR70, Water Research Centre, Stevenage, UK.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 937
 
Appendix 4  
EVALUATION OF INHIBITION OF THE ACTIVATED SLUDGE  
Process by test chemicals  
1. A chemical (or a waste water) may not be degraded or removed in the 
simulation test and may even have an inhibitory effect on the sludge 
micro-organisms. Other chemicals are biodegraded at low concentrations but are inhibitory at higher concentration (hormesis). Inhibitory effects may have been revealed at an earlier stage or may be determined by applying a 
toxicity test, using an inoculum similar to or identical with that used in the 
simulation test (1). Such methods are inhibition of oxygen uptake (chapter C.11 of this Annex (2) and ISO 8192(3)) or inhibition of growth of sludge organisms (ISO 15522 (4)). 
2. In the simulation test any inhibition will be manifest by the difference in 
dissolved organic carbon (DOC) or chemical oxygen demand COD between 
the effluent from the test vessel and that from the control being greater than 
the DOC added as test chemical. Expressed in another way, the percentage removal of DOC (and biochemical oxygen demand BOD, chemical oxygen demand COD, and/or NH
 + 4 ) of the organic medium on treatment will be 
decreased by the presence of the test chemical. If this occurs, the test should 
be repeated reducing the concentration of the test chemical until a level is reached at which no inhibition occurs and perhaps further reducing the concentration until the test chemical is biodegraded. However, if the test 
chemical (or waste water) has adverse effects on the process at all concen ­
trations tested, the indications are that the chemical is difficult, if not impossible, to treat biologically, but it may be worth repeating the test with activated sludge from a different source and/or subjecting the sludge 
to a more gradual acclimation. 
3. Conversely, if the test chemical is bioeliminated at the first attempt in the 
simulation test, its concentration should be increased if it is required to be 
known whether the chemical could be inhibitory. 
4. It should be remembered in trying to determine degrees of inhibition that the 
activated sludge population can change, so that with time the micro-organisms may develop a tolerance towards an inhibitory chemical. 
5. Calculation of degree of inhibition: 
The overall percentage removals R
 o , of BOD, DOC, COD etc., for the test 
and control units can be calculated from: 
R o ¼ 100 ðI Ä EÞ=I % 
where: 
I = influent concentration of BOD, DOC, COD etc., for test or control 
vessels (mg/l) 
E = respective effluent concentrations (mg/l). I and E must be corrected for the DOC due to the test chemical in the test 
units, otherwise the calculations of percentage inhibition will be incorrect.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 938
 
The degree of inhibition caused by the presence of the test chemical can be 
calculated from: 
% inhibition ¼ 100 ðR c Ä R t Þ=R c 
where: 
R c = percentage removal in the control vessels 
R t = percentage removal in the test vessels  
LITERATURE:  
(1) Reynolds L et al. (1987). Evaluation of the toxicity of substances to be 
assessed for biodegradability. Chemosphere 16: 2259. 
(2) Chapter C.11 of this Annex, Biodegradation — Activated Sludge Respiration 
Inhibition Test. 
(3) ISO 8192 (2007) Water quality — Test for inhibition of oxygen consumption 
by activated sludge for carbonaceous and ammonium oxidation. 
(4) ISO 15522 (1999) Water Quality — Determination of the inhibitory effect of 
water constituents on activated sludge microorganisms.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 939
 
Appendix 5  
Poorly water-soluble test chemicals — volatile chemicals  
Poorly water-soluble chemicals  
Few reports seem to have been published on subjecting poorly water-soluble and 
insoluble chemicals to tests simulating waste water treatment (1)(2)(3). 
There is no single method of dispersal of the test chemical which is applicable to 
all insoluble chemicals. Two of the four types of method described in ISO 10634 (4) would seem to be suitable for attempting to disperse test chemicals for 
simulation testing; they are the use of emulsifying agents and/or of ultrasonic 
energy. The stability over at least 24h periods of the resulting dispersion should be established. Suitably stabilised dispersions, contained in a constantly stirred reservoir (paragraph 38), would then be dosed to the aeration tank separately 
from the domestic (or synthetic) sewage. 
If the dispersions are stable, investigate how the test chemical can be determined 
in the dispersed form. It is unlikely that DOC will be suitable, so that a specific analytical method for the test chemical would have to be established which could 
be applied to effluents, effluent solids and activated sludge. The fate of the test 
chemical in the simulation of the activated sludge process would then be determined in liquid and solid phases. Thus, a ‘mass balance’ would be estab ­
lished to decide whether the test chemical had been biodegraded. However, this 
would indicate only primary biodegradation. Demonstration of ultimate biodegra ­
dation should be attempted by applying a respirometric test for ready biodegrad ­
ability (chapter C.4 of this Annex (5) C, F or D) using as inoculum sludge exposed to the test chemical in the simulation test.
 
Volatile chemicals  
The application of waste water treatment simulations to volatile chemicals is both 
debatable and problematic. As with poorly water-soluble test chemicals, very few 
reports seem to have been published describing simulation tests using volatile chemicals. A conventional type of complete-mixing apparatus is adapted by sealing the aeration and settling tanks, measuring and controlling the air flow 
using flow-meters and passing the exit gas through traps to collect volatile 
organic matter. In some cases, a vacuum pump is used to draw the exit gas through a ‘cold’ trap or a purge-trap containing Tenax and silica gel for gas-chromatographic analyses. The test chemical present in the trap can be 
determined analytically. 
The test is carried out in two parts. The units are first operated without sludge 
but with the synthetic waste water plus test chemical being pumped into the aeration tank. Influent, effluent and exit gas samples are collected and 
analysed for the test chemical for a few days. From the data collected, the 
percentage (R
 vs ) of the test chemical stripped from the system may be calculated. 
Then the normal biological test (with sludge) is performed under operating 
conditions identical to those in the stripping study. DOC or COD measurements are also made to check that the units are performing efficiently. Occasional 
analyses are made to determine the test chemical in the influent, effluent and 
exit gas in the first part of the test; after acclimation more frequent analyses are made. Again, from the data in the steady state, the percentage of removal of the test chemical from the liquid phase by all processes (R
 T ) (physical and 
biological) may be calculated, as well as the proportion (R  V ) stripped from the 
system.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 940
 
Calculation: 
(a) In the non-biological test, the percentage (R  VP ) of the test material stripped 
from the system may be calculated from: 
R VP ¼ S VP 
S IP · 100 
where 
R VP = removal of test chemical by volatilisation (%), 
S VP = test chemical collected in trap expressed as equivalent concentration 
in liquid phase (mg/l), 
S IP = test chemical concentration in influent (mg/l). 
(b) In the biological test, the percentage (R  V ) of the test material stripped from 
the system may be calculated from: 
R V ¼ S V 
S I · 100 
where 
R V = removal of test chemical by volatilisation in biological test (%), 
S V = test chemical collected in trap in biological test, expressed as 
equivalent concentration in liquid influent (mg/l), 
S I = test chemical concentration in influent (mg/l). 
(c) In the biological test, the percentage (R  T ) of the test chemical removed by all 
processes is given by: 
R T ¼ 1 Ä S E 
S I · 100 
where 
S E = concentration of test chemical in the (liquid) effluent (mg/l). 
(d) Thus, the percentage (R  BA ) removed by biodegradation plus adsorption can 
be calculated from: 
R BA ¼ ðR T Ä R V Þ 
Separate tests should be carried out to determine whether the test chemical is 
adsorbed; if it is, then a further correction may be made. 
(e) A comparison between the proportion of test chemical stripped from the 
biological (R  v ) and non-biological test (R  vp ) systems indicates the overall 
effect that biological treatment has had on the emission of the test chemical 
into the atmosphere.  
Example:  Benzene 
Sludge retention time = 4 days 
A synthetic sewage; retention time = 8 h. 
S IP = S  I = 150 mg/l 
S VP = 150 mg/l (S  EP = 0) 
S V = 22,5 mg/l 
S E = 50 μg/l ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 941
 
Thus, 
R VP = 100, R  V = 15 
R T = 100 and R  BA = 85. 
Benzene was assumed not to be adsorbed onto sludge.  
LITERATURE:  
(1) Horn JA, Moyer JE, Hale JH (1970). Biological degradation of tertiary butyl 
alcohol. Proc. 25th Ind. Wastes Conference Purdue Univ.: 939-854. 
(2) Pitter P, Chudoba J (1990). Biodegradability of organic substances in the 
aquatic environment. CRC Press. Boston, USA. 
(3) Stover EL, Kincannon DF (1983). Biological treatability of specific organic 
compounds found in chemical industry waste waters. J. Wat. Pollut. Control 
Fed. 55: 97. 
(4) ISO 10634 (1995) Water Quality — Guidance for the preparation and 
treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium. 
(5) Chapter C.4 of this Annex, Determination of ‘Ready’ Biodegradability.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 942
 
Appendix 6  
Effects of sludge retention time (SRT) on treatability of chemicals  
INTRODUCTION 
1. The method described in the main text was designed to ascertain whether 
the chemicals tested (usually those known to be inherently, but not readily, 
biodegradable) can be biodegraded within the limits imposed in waste water treatment plants. The results are expressed in terms of percentage removal and percentage biodegradation. The conditions of operation of the activated 
sludge units and choice of influent allow rather wide variations in concen ­
tration of the test chemical in the effluent. Tests are carried out at only one nominal concentration of sludge solids or one nominal sludge retention time (SRT) and the sludge wastage regimes described can cause the value 
of SRT to vary considerably during the test, both from day to day and 
during a day. 
2. In this variant (1)(2) the SRT is controlled within much narrower limits 
throughout each 24h period (just as happens on the large-scale) which results in a more constant concentration in effluents. Domestic sewage is 
recommended since it gives more consistent and higher percentage 
removals. Also, the effects of a number of SRT values are investigated and in a more detailed study the effects of a range of temperatures on effluent concentration may be determined. 
3. There is no general agreement yet on which kinetic models operate when 
chemicals bio-degrade under conditions in waste water treatment. The Monod model of bacterial growth and substrate utilisation was chosen (1)(2) to be applied to the data collected, since the method was intended 
to be applied only to chemicals produced in high tonnages, resulting in 
concentrations in sewage of above 1 mg/l. The validity of the simplified model and the assumptions made was established using a series of alcohol ethoxylates having varying degrees of primary biodegradability (2)(3).  
Note:  This variant method follows closely much of the text of this test 
method C.10-A and only those details which differ are given hereafter.
 
PRINCIPLE OF THE TEST 
4. Activated sludge porous-pot units, designed to facilitate the (almost) 
continuous wastage of mixed liquor allowing very precise control of the 
sludge retention time (SRT, or θ s ), are operated in the non-coupled mode 
over a range of SRTs and, optionally, over a range of temperatures. The retention time is usually 2 to 10 days and the temperature between 5 and 
20 °C. Sewage, preferably domestic, and a solution of the test chemical are 
dosed separately to the units at rates to give the required sewage retention time (3 to 6 hours) and the required concentration of test chemical in the influent. Control units receiving no test chemical are operated in parallel for 
comparative purposes. 
5. Other types of apparatus can be used but great care should be exercised to 
ensure that good control of SRT is achieved. For example, when using plants, which incorporate a settler, allowance for loss of solids via the plant effluent may be necessary. Further, special precautions to avoid 
errors due to variation in the quantity of sludge in the settler should also 
be taken.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 943
 
6. The units are operated at each selected set of conditions and, after equi ­
librium has been reached, the average steady state concentrations in the 
effluents of test chemical and, optionally, DOC are obtained over a period of about three weeks. Besides assessing the percentage removal of test chemical and, optionally, DOC, the relationship between plant-operating 
conditions and the concentration in the effluent is expressed in graphical 
form. From this tentative kinetic constants may be calculated and the conditions under which the test chemical can be treated may be predicted.
 
INFORMATION ON THE TEST CHEMICAL 
7. Chapter C.10 A, paragraphs 12 and 13 apply.  
PASS LEVELS 
8. Chapter C.10 A, paragraphs 14 and 15 apply.  
REFERENCE TEST CHEMICAL 
9. Chapter C.10 A, paragraph 16 applies.  
REPRODUCIBILITY OF TEST RESULTS 
10. Chapter C.10 A, paragraphs 17 and 18 apply.  
DESCRIPTION OF THE METHOD  
Apparatus  
11. A suitable unit is the modified porous pot system (Appendix 6.1). It consists 
of an inner vessel (or liner) constructed from porous polypropylene of 3,2 mm thickness and pore size of approximately 90 μm, the joint being 
butt-welded. (This makes a more robust unit than that described in 
paragraph 21 of this chapter, C.10 A). The liner is fitted into an impervious polyethylene outer vessel, which consists of two parts: a circular base in which holes are bored to accommodate two air lines and a sludge-wastage 
line, and an upper cylinder which screws on to the base and which has an 
outlet placed so as to give a known volume (3 l) in the porous pot vessel. One of the air lines is fitted with a diffuser stone and the other is open-ended and set at right-angles to the stone in the pot. This system 
produces the necessary turbulence to ensure that the contents of the pot 
are completely mixed, as well as providing concentrations of dissolved oxygen greater than 2 mg/l. 
12. The appropriate number of units are maintained at controlled temperatures in 
the range of 5 to 20 °C (± 1 °C), either in water baths or in constant temperature rooms. Pumps are required to dose to the aeration vessels the solution of the test chemical and settled sewage at the required rates (0-1,0 
ml/min and 0-25 ml/min, respectively) and a third pump to remove waste 
sludge from the aeration vessels. The necessary very low flow-rate of waste sludge is achieved by using a pump set at a higher rate and operated inter ­
mittently by the use of a timer-switch, e.g. operating for 10 seconds per 
min, pump delivery rate of 3ml/min yielding a wastage rate of 0,5 ml/min.
 
Filtration apparatus or centrifuge  
13. Chapter C10 A, paragraph 23 applies.  
Analytical equipment  
14. Chapter C.10 A, paragraph 24 applies.  
Water  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 944
 
15. Chapter C.10 A, paragraphs 25 and 26 apply.  
Organic medium  
16. Chapter C.10 A, paragraph 27 applies.  
Synthetic sewage  
17. Chapter C.10 A, paragraph 28 applies.  
Domestic sewage  
18. Chapter C.10 A, paragraph 29 applies.  
Activated sludge  
19. Chapter C10 A, paragraph 30 applies.  
Stock solutions of test chemical  
20. Chapter C.10 A, paragraphs 31 and 32 apply.  
PROCEDURE  
Preparation of the inoculum  
21. Chapter C.10 A, paragraph 34 applies only — use activated sludge (about 
2,5 g/l).  
Number of test units  
22. For a simple test, ie. to measure percentage removal, only a single SRT is 
required, but in order to acquire data necessary to calculate tentative kinetic 
constants 4 or 5 SRT values are required. Values between 2 and 10 days are usually chosen. Practically, it is convenient to perform a test at 4 or 5 SRTs simultaneously at one temperature; in extended studies the same SRT 
values, or perhaps a different range of values, are used at other temperatures 
within the range 5 to 20 °C. For primary biodegradation (the main use), only one unit per set of conditions is normally required. However, for ultimate biodegradability a control unit is required, for each set of 
conditions, which receives sewage but not test chemical. If the test 
chemical is thought to be present in the sewage used, it would be necessary to use control units when assessing primary biodegradation, and making the necessary correction in the calculations.
 
Dosage of organic medium and test chemical  
23. Chapter C.10 A, paragraphs 36 to 39 apply, but note that the test chemical 
solution is dosed separately and that various sludge wastage rates are used. 
Also monitor and adjust, if necessary, to within ± 10 %, the flow-rates of 
influents, effluents and sludge wastage frequently, e.g. twice per day. If difficulties are encountered in the analytical methods when domestic sewage is used, carry out the test with synthetic sewage, but it must be 
assured that different media give comparable kinetic data.
 
Handling of activated sludge units  
24. Chapter C.10 A, paragraphs 40 to 43 apply, but control SRT only by ‘con ­
stant’ wastage of sludge.  
Sampling and analysis  
25. Chapter C.10 A, paragraphs 44 to 50 apply, except that the concentration of 
the test chemical is to be determined and DOC determined optionally; COD 
should not be used.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 945
 
DATA AND REPORTING  
Treatment of results  
26. Chapter C.10 A, paragraphs 52 to 54 apply.  
Expression of test results  
27. Chapter C.10 A, paragraphs 56 to 62 apply.  
Calculation of kinetic constants  
28. It is more realistic to quote the mean steady — state concentration of the 
test chemical in the effluent and to describe how this varies with plant-operating conditions than to quote percentage primary biodegradation. This can be done by consideration of equation (6) in Appendix 6.2, which 
can yield values for K
 S , μm and θ SC , the critical sludge retention time. 
(Alternatively, approximate values of K  S and μm may be obtained using a 
simple computer program to fit the theoretical curve calculated from equation 2 (Appendix 6.2) to the experimental values obtained. Although 
any given solution will not be unique, a reasonable approximation of K
 S and 
μ m can be obtained.)  
Variability of results  
29. It is common experience that variable values of kinetic parameters for 
individual chemicals are obtained. It is thought that the conditions under which the sludge has been grown, as well as the conditions prevailing in the 
test used (as in paragraph 5 and in other tests), have a large effect on the 
resulting values. One aspect of this variability has been discussed by Grady et al (4), who have suggested that the terms ‘extant’ and ‘intrinsic’ should be applied to two extreme conditions representing the limits of physiological 
state a culture may attain during a kinetic experiment. If the state is not 
allowed to change during the test, the kinetic parameter values reflect the conditions in the environment from which the micro-organisms were taken; these values are called ‘extant’ or currently existing. At the other extreme, if 
conditions in the test are such as to permit the full development of the 
protein-synthesizing system allowing maximum possible growth rate, the kinetic parameters obtained are said to be ‘intrinsic’, and are dependent only on the nature of the substrate and the types of bacteria in the 
culture. As a guide, extant values will be obtained by keeping the ratio of 
concentration of substrate to competent micro-organisms (S
 o /X o ) low, e.g. 
0,025, and intrinsic values arise when the ratio is high e.g. at least 20. In both cases, S
 o should equal or exceed the relevant value of Ks, the 
half-saturation constant. 
30. Variability and other facets of biodegradation kinetics were discussed at a 
recent SETAC workshop (5). From such studies, reported and projected, a clearer view of kinetics operating in waste water treatment plants should be 
forth-coming to enable a better interpretation of existing data to be made, as 
well as to suggest more relevant designs for future Test Methods.
 
LITERATURE:  
(1) Birch RR (1982). The biodegradability of alcohol ethoxylates. XIII Jornado 
Com. Espanol Deterg.: 33-48. 
(2) Birch RR (1984). Biodegradation of nonionic surfactants. J.A.O.C.S., 61(2): 
340-343. 
(3) Birch RR (1991). Prediction of the fate of detergent chemicals during sewage 
treatment. J. Chem. Tech. Biotechnol., 50: 411-422.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 946
 
(4) Grady CPL, Smets BF and Barbeau DS (1996). Variability in kinetic 
parameter estimates: A review of possible causes and a proposed 
terminology. Wat. Res., 30 (3): 742-748. 
(5) Biodegradation kinetics: Generation and use of data for regulatory decision 
making (1997). Workshop at Port Sunlight, UK. Eds. Hales SG, Feitjel T, King H, Fox K, Verstraete W. 4-6th Sept. 1996. SETAC- Europe, Brussels.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 947
 
Appendix 6.1  
Porous Pot with SRT Control
  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 948
 
Appendix 6.2  
Calculation of Kinetic Constants  
1. By assuming Monod kinetics apply and considering a mass balance of 
active solids and substrate across the activated sludge system (1), the following steady state expressions can be obtained:
 
1 
θ s ¼ μ m · S 1 
K s þ S 1 Ä K d [1] 
or 
S 1 ¼ K s · ð1 þ K d · θ s Þ 
θ s · ðμ m Ä K d Þ Ä 1 [2] 
where: 
S 1 = concentration of substrate in effluent, (mg/l) 
K S = half-saturation constant, the concentration at which μ = μ m /2 (mg/l) 
μ = specific growth rate (d  –1 ) 
μ m = maximum value of μ m (d –1 ) 
K d = specific decay rate of active solids (d  –1 ) 
θ S = sludge mean retention time, SRT (d) 
Examination of this equation leads to the following conclusions: 
(i) The effluent concentration is independent of that in the influent (S  0 ); 
hence, the percentage biodegradation varies with the influent concen ­
tration, S  0 . 
(ii) The only plant-control parameter affecting S  1 is the sludge retention 
time, θ S . 
(iii) For a given concentration in the influent, S  0 , there will be a critical 
sludge retention time, such that:  
1 
θ SC ¼ μ s · S 0 
K s þ S 0 Ä K d [3] 
where: 
θ SC = critical sludge retention time, below which the competent 
micro-organisms will be washed out of the plant. 
(iv) Since the other parameters in equation (2) are associated with growth 
kinetics, temperature is likely to affect the effluent substrate level and 
the critical sludge age, ie. the sludge retention time needed to obtain a 
certain degree of treatment would increase with decreasing temperature. 
2. From a mass balance of solids in the porous pot system, and assuming that 
the solids concentration in the plant effluent, X  2 is low compared with that 
in the aeration vessel, X  1 , the sludge retention time  
θ s ¼ V · X 1 
ðQ 0 Ä Q 1 Þ · X 2 þ Q 1 · X 1 [4] ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 949
 
and 
θ s ¼ V · X 1 
Q 1 · X 1 ¼ V 
Q 1 
where: 
V = volume of the aeration vessel (l) 
X 1 = concentration of solids in aeration vessel (mg/l) 
X 2 = concentration of solids in effluent (mg/l) 
Q 0 = flow rate of influent (l/d) 
Q 1 = flow rate of waste sludge (l/d) 
Thus, it is possible to control the sludge retention time at any pre-selected 
value by the control of the waste sludge flow rate, Q  1 . 
Conclusions: 
3. The main purpose of the test is thus to allow the effluent concentration, and 
hence the levels of test chemical in the receiving waters, to be predicted. 
4. By plotting S  1 , vs. θ S , the critical sludge retention time, θ SC , can sometimes 
be readily evaluated, eg. curve 3 in Figure 1. When this is not possible, θ SC 
may be calculated, together with approximate values of μ m and K  S , by 
plotting S  1 , vs. S  1 •θ S . 
Rearrangement of equation (1) gives  
S 1 · θ s 
1 þ θ s · K d ¼ K s 
μ m þ S 1 
μ m [5] 
If K  d is small, then 1 + θ s · Kd ~ 1 and [5] becomes:  
S 1 · θ s ¼ K s 
μ m þ S 1 
μ m [6] 
Thus, the plot should be a straight line (see Figure 2) of slope 1/ μ m and 
intercept K  S /μ m ; also θ S ~1/μ m . ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 950
 
Figure 1  
Three temperatures; five SRTs
  
Figure 2  
Regression Line SRT · S1 vs S1 at T = 5 °C
  
Glossary:  
Effluent concentration: Curve:
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 951
 
Appendix 7  
TEST AT LOW ( μg/l) CONCENTRATION RANGE 
1. Many chemicals are normally present in the aquatic environment, even in 
waste waters, at very low concentrations ( μg/l). At such concentrations, they 
probably do not serve as primary substrates resulting in growth, but are more likely to degrade as non-growth, secondary substrates, concurrent 
with a variety of naturally occurring carbon chemicals. Consequently the 
degradations of such chemicals will not fit the model described in Appendic 6. There are many models which could be applied and, under the conditions prevailing in waste water treatment systems, more than one may be simulta ­
neously operative. Far more research will be necessary to elucidate this 
problem. 
2. Meanwhile the procedure given in the main text (chapter C.10 A) can be 
followed, but only for primary biodegradability, using suitably low concen ­
trations (< 100 μg/l) and a validated analytical procedure. The percentage 
biodegradation may be calculated (see para. 54 of the Test Method) 
provided that abiotic processes (adsorption, volatility, etc.) are taken into account. An example is the study by Nyholm and his associates (1)(2) using a 4 h cycle in a fill and draw system. They reported pseudo first-order 
constants for 5 chemicals added in a synthetic sewage at 5 to 100 μg/l. 
(For ultimate biodegradability
 14 C-labelled test chemicals may be used. A 
description of this is beyond the scope of this Test Method since there are as yet no agreed procedures, though a proposed method for ISO 14592 (3) 
contains guidance on the use of
 14 C-labelled chemicals.  
SCAS test  
3. Later, a simpler two-stage test was proposed (4)(5)(6); the semi-continuous 
activated sludge (SCAS) method is followed by short-term kinetic tests on 
samples withdrawn from the SCAS units. The SCAS system is operated 
with known sludge wastage rates (unlike the original C.12 test method) and is fed a modified OECD synthetic sewage or domestic sewage. The synthetic sewage was modified (because of changing pH value and poor 
sludge settleability) by addition of phosphate as buffer, yeast extract, 
iron (III) chloride and trace element salts, and its COD was increased to about 750 mg/l by increasing the concentration of peptone and meat extract. The units were operated on a 24 h cycle: aeration for 23 h, wastage of 
sludge, settlement, withdrawal of supernatant (effluent) followed by addition 
of synthetic sewage plus test chemical, up to 100 μg/l, (i.e. at about the 
same concentration used in the short term test). Once per week 10 % of the total sludge was replaced by fresh sludge in order to maintain a balanced 
microbial population. 
4. The concentrations of test chemical initially and at the end of aeration are 
measured and the test is continued until a constant removal of test chemical 
is attained; this takes from one week to several months.
 
Short-term test  
5. A short test (e.g. 8 hours) is applied to determine the (pseudo) first order 
kinetic rate constant for the decay of the test chemical in activated sludge of 
known but different origins and histories. In particular, sludge samples are taken from the SCAS reactors — at the end of an aeration period when the concentration of organic substrate is low — during the course of an accli ­
matisation experiment (paragraphs 3, 4). Sludge may also be taken from a 
parallel SCAS unit not exposed to the test chemical, for comparison.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 952
 
Mixtures of sludge and the test chemical added at two or more concen ­
trations in the range 1-50 μg/l are aerated, without the addition of synthetic 
sewage or other organic substrate. The test chemical remaining in solution is determined at regular intervals e.g. hourly depending on the degradability of the chemical, for a period not longer than 24h. Samples are centrifuged 
before appropriate analysis.
 
Calculations  
6. Data from the SCAS units are used to calculate the percentage removal of 
test chemical (paragraph 54). Also, an average rate constant, K  1 , (normalised 
for concentration of suspended solids) can be calculated from: 
K 1 ¼ 1=t · ln C e 
C i · 1=SSð1=ghÞ 
where: 
t = aeration time (23h) C
 e = concentration at end of aeration period ( μg/l) 
C i = concentration at beginning of aeration ( μg/l) 
SS = concentration of activated sludge solids (g/l) 
7. In the short term test the log % concentration remaining is plotted against 
time and the slope of the initial part (10-50 % degradation) of the plot is equivalent to K
 1 , the (pseudo) first order constant. The constant is 
normalised with respect to the concentration of sludge solids by dividing 
the slope by the concentration of sludge solids. The reported result must also include details of initial concentrations of the test chemical and suspended solids, sludge retention time, sludge loading and source, and details of 
pre-exposure (if any) to the test chemical.
 
Variability of results  
8. Variability and other facets of biodegradation kinetics were discussed at a 
recent SETAC workshop (7). From such studies, reported and projected, a 
clearer view of kinetics operating in waste water treatment plants should be forth-coming to enable a better interpretation of existing data to be made, as 
well as to suggest more relevant designs for future Test Methods.
 
LITERATURE:  
(1) Nyholm N, Jacobsen BN, Pedersen BM, Poulsen O, Dambourg A and 
Schultz B (1992). Removal of micropollutants in laboratory activated 
sludge reactors. Biodegradability. Wat. Res. 26: 339-353. 
(2) Jacobsen BN, Nyholm N, Pedersen BM, Poulsen O, and Ostfeldt P (1993). 
Removal of organic micropollutants in laboratory activated sludge reactors under various operating conditions: Sorption. Wat. Res. 27: 1505-1510. 
(3) ISO 14592 (ISO/TC 147/SC5/WG4, N264) (1998). Water Quality — 
Evaluation of the aerobic biodegradability of organic compounds at low 
concentrations in water.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 953
 
(4) Nyholm N, Ingerslev F, Berg UT, Pedersen JP and Frimer-Larsen H (1996). 
Estimation of kinetic rate constants for biodegradation of chemicals in 
activated sludge waste water treatment plants using short-term batch experiments and μg/l range spiked concentrations Chemosphere 33 (5): 
851-864. 
(5) Berg UT and Nyholm N (1996). Biodegradability simulation Studies in 
semi-continuous activated sludge reactors with low ( μg/l range) and 
standard (ppm range) chemical concentrations. Chemosphere 33 (4): 711- 735. 
(6) Danish Environmental Protection Agency. (1996). Activated sludge biode ­
gradability simulation test. Environmental Project, No 337. Nyholm, N. Berg, 
UT. Ingerslev, F. Min. of Env. and Energy, Copenhagen. 
(7) Biodegradation kinetics: Generation and use of data for regulatory decision 
making (1997). Workshop at Port Sunlight, UK. Eds. Hales, SG. Feitjel, T. King, H. Fox, K. and Verstraete, W. 4-6th Sept. 1996. SETAC- Europe, 
Brussels.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 954
 
C.10-B: Biofilms  
INTRODUCTION 
1. Simulation tests are normally applied to chemicals which have failed a 
screening test for ready biodegradability (Chapter C.4 A to F of this Annex (9)), but have passed a test for inherent biodegradability. Excep ­
tionally simulation tests are also applied to any chemical about which 
more information is required, especially high-tonnage chemicals, and 
normally the activated sludge test is applied (C.10 A). In some circum ­
stances, however, specific information is required relating the behaviour of a chemical to methods of waste water treatment involving biofilms, 
namely, percolating or trickling filters, rotating biological contactors, 
fluidised beds. To meet this need various devices have been developed. 
2. Gerike  et al . (1) used large, pilot-scale trickling filters which they used in 
the coupled mode. These filters took up much space and required relatively large volumes of sewage or synthetic sewage. Truesdale  et al . (2) described 
smaller filters (6 ft × 6 in. diameter) which were fed surfactant-free natural 
sewage but still required rather large volumes. As many as 14 weeks were required for the development of a ‘mature’ biofilm and an additional 4-8 weeks were needed after first introduction of the test surfactant before 
acclimatisation took place. 
3. Baumann  et al.  (3) developed a much smaller filter which used polyester 
‘fleece’ previously steeped in activated sludge as the inert medium 
supporting the biofilm. The test chemical was used as the sole source of carbon and biodegradability was assessed from measurements of dissolved organic carbon (DOC) in the influent and effluent, and from the amount of 
CO
 2 in the exit gas. 
4. A quite different approach was made by Gloyna  et al.  (4) who invented the 
rotating tubular reactor. On the internal surface of the rotating tube a biofilm was grown on the known surface area by passage of influent introduced at the top end of the tube, inclined at a small angle to the horizontal. The 
reactor has been used to study the biodegradability of surfactants (5), as well 
as to investigate the optimal thickness of biofilm and diffusion through the film (6). These latter authors further developed the reactor, including modifying it to be able to determine CO
 2 in the exit gas. 
5. The rotating tubular reactor has been adopted by the Standing Committee of 
Analysts (UK) as a standard method for assessing both the biodegradability 
of chemicals (7) and the treatability and toxicity of waste waters (8). The 
method described here has the advantages of simplicity, compactness, repro ­
ducibility and the need for relatively small volumes of organic medium.  
PRINCIPLE OF THE TEST 
6. Synthetic or domestic sewage, and the test chemical, in admixture or alone, 
are applied to the internal surface of a slowly rotating inclined tube. A layer of microorganisms, similar to those present on bio-filter media, is built up on the internal surface. The conditions of operation of the reactor are chosen 
to give adequate elimination of organic matter and, if required, oxidation of 
ammonium.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 955
 
7. Effluent from the tube is collected and either settled and/or filtered before 
analysis for dissolved organic carbon (DOC) and/or the test chemical by a 
specific method. Control units receiving no test chemical are operated in parallel under the same conditions for comparative purposes. The difference between the concentrations of DOC in the effluent from the test and control 
units is assumed to be due to the test chemical and its organic metabolites. 
This difference is compared with the concentration of the added test chemical (as DOC) to calculate the elimination of the test chemical. 
8. Biodegradation may normally be distinguished from bio-adsorption by 
careful examination of the elimination-time curve. Confirmation may usually be obtained by applying a test for ready biodegradation (oxygen 
uptake or carbon dioxide production) using an acclimated inoculum taken at 
the end of the test from the reactors receiving the test chemical.
 
INFORMATION ON THE TEST CHEMICAL 
9. The purity, water solubility, volatile and adsorption characteristics of the test 
chemical should be known to enable correct interpretation of results to be 
made. 
10. Normally, volatile and poorly soluble chemicals cannot be tested unless 
special precautions are taken (see Appendix 5 to chapter C.10 A). The chemical structure, or at least the empirical formula, should also be known in order to calculate theoretical values and/or to check measured 
values of parameters, e.g. theoretical oxygen demand (ThOD), DOC. 
11. Information on the toxicity of the test chemical to micro-organisms (see 
Appendix 4 to chapter C.10 A) may be useful for selecting appropriate 
test concentrations and may be essential for the correct interpretation of low biodegradation values.
 
PASS LEVELS 
12. Originally, the primary biodegradation of surfactants was required to reach 
80 % or more before the chemical could be marketed. If the value of 80 % is not attained, this simulation (confirmatory) test may be applied and the surfactant may be marketed only if more than 90 % of the specific chemical 
is removed. With chemicals in general there is no question of a pass/fail 
level and the value of percentage removed can be used in proximate calcu ­
lations of the probable environmental concentration to be used in hazard assessments posed by chemicals. In a number of studies of pure chemicals 
the percentage removal of DOC was found to be > 90 % in more than 
three-quarters, and > 80 % in over 90 %, of chemicals which showed any significant degree of biodegradability.
 
REFERENCE CHEMICALS 
13. To ensure that the experimental procedure is being carried out correctly, it is 
useful occasionally to test reference chemicals whose behaviour is known. Such chemicals include adipic acid, 2-phenyl phenol, 1-naphthol, diphenic acid and 1-naphthoic acid.
 
REPRODUCIBILITY OF TEST RESULTS 
14. The relative standard deviation within tests was found by a laboratory in the 
UK to be 3,5 % and between tests to be 5 % (7).  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 956
 
DESCRIPTION OF THE METHOD  
Apparatus  
Rotating tubular reactors  
15. The apparatus (see figures 1 and 2 in the Appendix 8 consists of a bank of 
acrylic tubes each 30,5 cm long and 5 cm internal diameter, supported on 
rubber-rimmed wheels contained within a metal supporting frame. Each tube has an outside lip, approximately 0,5 cm deep, to retain it on the wheels, the internal surface is roughened with coarse wire wool and there is a 0,5 cm 
deep internal lip at the upper (feed) end to retain the liquid. The tubes are 
inclined at an angle of approximately one degree to the horizontal to achieve the required contact time when the test medium is applied to a clean tube. The rubber-tyred wheels are rotated using a slow, variable-speed motor. The 
temperature of the tubes is controlled by installation in a constant 
temperature room. 
16. By enclosing each tube reactor inside a slightly larger, capped tube and 
ensuring that connections were gas-tight, exit CO
 2 gas could be collected 
in an alkaline solution for subsequent measurement (6). 
17. A 24h supply, for each tube, of organic medium with added test chemical if 
applicable, is contained in a 20 l storage vessel (A)(see Figure 2). If required, the test chemical solution may be dosed separately. Near the 
bottom of each storage vessel there is an outlet which is connected by 
suitable tubing, e.g. silicone rubber, via a peristaltic pump (B) to a glass or acrylic delivery tube which enters 2-4 cm into the higher (feed) end of the inclined tube (C). Effluent is allowed to drip from the lower end of the 
inclined tube to be collected in another storage vessel (D). The effluent is 
settled or filtered before analysis.
 
Filtration apparatus-centrifuge  
18. Device for filtration of samples with membranes filter of suitable porosity 
(nominal aperture diameter 0,45 μm) which adsorb organic chemicals or 
release organic carbon to a minimum degree. If filters are used which release organic carbon, wash them carefully with hot water to remove 
leachable organic carbon. Alternatively a centrifuge capable of achieving 
40 000 m/sec
 2 may be used. 
19. Analytical equipment for determining: 
— DOC/total organic carbon (TOC), or chemical oxygen demand (COD); — specific chemical (HPLC, GC etc.) if required; 
— pH, temperature, acidity, alkalinity; 
— ammonium, nitrite, nitrate, if the tests are performed under nitrifying 
conditions.
 
Water  
20. Tap water, containing less than 3 mg/l DOC. 
21. Distilled or deionised water, containing less than 2 mg/l DOC.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 957
 
Organic medium  
22. Synthetic sewage, domestic sewage or a mixture of both may be used as the 
organic medium. It has been shown that the use of domestic sewage alone often gives increased percentage removed of DOC (in activated sludge 
units) and even allows the biodegradation of some chemicals, which are 
not biodegraded when OECD synthetic sewage is used. Thus, the use of domestic sewage is recommended. Measure the DOC (or COD) concen ­
tration in each new batch of organic medium. The acidity or alkalinity of 
the organic medium should be known. The medium may require the 
addition of a suitable buffer (sodium hydrogen carbonate or potassium hydrogen phosphate), if it is of low acidity or alkalinity, to maintain a 
pH of about 7,5 ± 0,5 in the reactor during the test. The amount of 
buffer, and when to add it, has to be decided in each individual case.
 
Synthetic sewage  
23. Dissolve in each 1 litre of tap water: peptone, 160 mg; meat extract, 110 
mg; urea, 30 mg; anhydrous dipotassium hydrogen phosphate, (K  2 HPO  4 ), 
28 mg; sodium chloride, (NaCl), 7 mg; calcium chloride dihydrate, 
(CaCl  2 .2H  2 O), 4 mg; magnesium sulphate heptahydrate, (MgSO  4 .7H  2 O), 2 
mg. This OECD synthetic sewage is an example and gives a mean DOC concentration in the influent of about 100 mg/l. Alternatively, use other 
compositions, with about the same DOC concentrations, which are closer 
to real sewage. This synthetic sewage may be made up in distilled water in a concentrated form and stored at about 1 °C for up to one week. When needed, dilute with tap water. (This medium is unsatisfactory e.g. nitrogen 
concentration is very high, relatively low carbon content, but nothing better 
has been suggested, except to add more phosphate, as buffer, and extra peptone).
 
Domestic sewage  
24. Use fresh settled sewage collected daily from a treatment works receiving 
predominantly domestic sewage. It should be collected from the overflow 
channel of the primary sedimentation tank, or from the feed to activated sludge plant, and be largely free from coarse particles. The sewage can be used after storage for several days at about 4 °C, if it is proved that the 
DOC (or COD) has not significantly decreased (i.e. by less than 20 %) 
during storage. In order to limit disturbances to the system, the DOC (or COD) of each new batch should be adjusted before use to an appropriate constant value, e.g. by dilution with tap water.
 
Lubricant  
25. Glycerol or olive oil may be used for lubricating the peristaltic pump rollers: 
both are suitable for use on silicone-rubber tubing.  
Stocks solutions of test chemical  
26. For chemicals of adequate solubility prepare stock solutions at appropriate 
concentrations (e.g. 1 to 5 g/l) in deionised water or in the mineral portion of the synthetic sewage. For insoluble chemicals, see Appendix 5 in chapter 
C.10-A. This method is not suitable for volatile chemicals without modifi ­
cations to the tubular reactors (paragraph 16). Determine the DOC and TOC of the stock solution and repeat the measurements for each new batch. If the difference between the DOC and TOC is greater than 20 %, check the 
water-solubility of the test chemical. Compare the DOC or the concentration 
of the test chemical measured by specific analysis of the stock solution with
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 958
 
the nominal value to ascertain whether recovery is good enough (normally > 
90 % can be expected). Ascertain, especially for dispersions, whether or not 
DOC can be used as an analytical parameter or if only an analytical technique specific for the test chemical can be used. Centrifugation of the samples is required for dispersions. For each new batch, measure the DOC, 
COD or the test chemical with specific analysis. 
27. Determine the pH of the stock solution. Extreme values indicate that the 
addition of the chemical may have an influence on the pH of the activated 
sludge in the test system. In this case neutralise the stock solution to obtain a pH of 7 ± 0,5 with small amounts of inorganic acid or base, but avoid precipitation of the test chemical.
 
PROCEDURE  
Preparation of organic medium for dosing  
28. Ensure that all influent and effluent containers and tubing from influent 
vessels and to effluent vessels are thoroughly cleaned to remove microbial growths, initially and throughout the test. 
29. Prepare the synthetic sewage (paragraph 23) freshly each day either from the 
solids or from the concentrated stock solution by appropriate dilution with tap water. Measure the required amount in a cylinder and add to a clean 
influent vessel. Also, where necessary, add the required amount of the stock 
solution of the test chemical or reference chemical to the synthetic sewage before dilution. If it is more convenient or necessary to avoid loss of the test chemical, prepare a separate diluted solution of the test chemical in a 
separate reservoir and deliver this to the inclined tubes via a different 
dosing pump. 
30. Alternatively (and preferably), use settled domestic sewage (paragraph 24) 
collected freshly each day if possible.
 
Operation of rotating tubular reactors  
31. Two identical tubular reactors are required for the assessment of one test 
chemical, and they are assembled in a constant temperature room normally 
at 22 ± 2 °C. 
32. Adjust the peristaltic pumps to deliver 250 ± 25 ml/h of the organic medium 
(without test chemical) into the inclined tubes, which are rotated at 18 ± 2 rpm. Apply the lubricant (paragraph 25) to the pump tubes initially and 
periodically through the test to ensure proper functioning and to prolong 
the life of the tubing. 
33. Adjust the angle of inclination of the tubes to the horizontal to produce a 
residence time of 125 ± 12,5 sec. for the feed in a clean tube. Estimate the retention time by adding a non-biological marker (e.g. NaCl, inert dye) to the feed: the time taken to reach peak concentration in the effluent is taken 
to be the mean retention time (when maximum film is present, the retention 
time can increase up to about 30 min.). 
34. These rates, speeds and times have been found to give adequate removals (> 
80 %) of DOC (or COD) and to produce nitrified effluents. The rate of flow should be changed if removal is insufficient or if the performance of a particular treatment plant is to be simulated. In the latter case, adjust the 
rate of dosing the organic medium until the performance of the reactor 
matches that of the treatment plant.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 959
 
Inoculation  
35. Airborne inoculation may be sufficient to start the growth of 
micro-organisms when synthetic sewage is used, but otherwise add 1 ml/l of settled sewage to the feed for 3 days.
 
Measurements  
36. At regular intervals check that the dose-rates and rotating speeds are within 
the required limits. Also, measure the pH of the effluent, especially if 
nitrification is expected.  
Sampling and analysis  
37. The method, pattern and frequency of sampling are chosen to suit the 
purpose of the test. For example, take snap (or grab) samples of influent 
and effluent, or collect samples over a longer period e.g. 3-6 h. In the first 
period, without test chemical, take samples twice per week. Filter the samples through membranes or centrifuge at about 40 000 m/sec
 2 for 
about 15 min (paragraph 18). It may be necessary to settle and/or 
coarse-filter the samples before membrane filtration. Determine DOC (or 
COD) at least in duplicate and if required BOD, ammonium and nitrite/ nitrate. 
38. All analyses should be performed as soon as possible after collection and 
preparation of the samples. If analyses have to be postponed, store the 
samples at about 4 °C in the dark in full, tightly stoppered bottles. If samples have to be stored for more than 48h, preserve them by deep-freezing, acidification or by addition of a suitable toxic chemical 
(e.g. 20 ml/l of a 10 g/l solution of mercury (II) chloride). Ensure that 
the preservation technique does not influence the results of analysis.
 
Running-in period  
39. In this period, the surface biofilm grows to reach an optimal thickness, 
usually taking about 2 weeks and should not exceed 6 weeks. The removal (paragraph 44) of DOC (or COD) increases and reaches a 
plateau value. When the plateau has been reached at a similar value in 
both tubes, one is selected to be a control in the remainder of the test, during which their performance should remain consistent.
 
Introduction of test chemical  
40. At this stage add the test chemical to the other reactor at the required 
concentration, usually 10-20 mg C/l. The control continues to receive the 
organic medium alone.  
Acclimation period  
41. Continue the twice weekly analyses for DOC (or COD) and, if primary 
biodegradability is to be assessed, also measure the concentration of the test chemical by specific analysis. Allow from one to six weeks (or longer under special conditions) after the test chemical has first been 
introduced for acclimation to occur. When the percentage removal (para ­
graphs 43-45) reaches a maximum value, obtain 12-15 valid values in the plateau phase over about 3 weeks for evaluation of the mean percentage removal. The test is considered completed if a sufficiently high degree of 
elimination is reached. Normally, do not exceed a test duration of more than 
12 weeks after the first addition of the test chemical.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 960
 
Sloughing of the film  
42. The sudden removal of large quantities of excess film from the tubes, or 
sloughing, takes place at relatively regular intervals. To ensure that the comparability of results is unaffected, allow tests to cover at least two 
full cycles of growing and sloughing.
 
DATA AND REPORTING  
Treatment of results  
43. Calculate the percentage DOC (or COD) elimination of the test chemical for 
each timed assessment using the equation: 
D t ¼ 100 ½C s Ä ðE Ä E o Þâ=C s % 
where: 
D t = percentage elimination of DOC (or COD) at time t; 
Cs = concentration of DOC (or COD) in the influent due to the test 
chemical, preferably estimated from the concentration in, and volume added, of the stock solution (mg/l); 
E = measured DOC (or COD) in the test effluent at time t (mg/l); 
Eo = measured DOC (or COD) in the control effluent at time t (mg/l). Repeat the calculation for the reference chemical, if tested.
 
Performance of the control reactor  
44. The degree of DOC (or COD) elimination (D  B ) of the organic medium in 
the control reactors is helpful information in assessing the biodegradative 
activity of the biofilm during the test. Calculate the percentage elimination 
from the equation: 
D B ¼ 100 ð1 Ä E o=C m Þ % 
where: 
Cm = DOC (or COD) of the organic medium in the control influent (mg/l). 
45. Calculate the removal (D  ST ) of the test chemical, if measured, by a specific 
analytical method at each time assessment from the equation: 
DST ¼ 100 ð1 Ä Se=SiÞ % 
where: 
Si = measured or, preferably, estimated concentration of test chemical in 
the test influent (mg/l) 
Se = measured test chemical concentration in the test effluent at time t 
(mg/l)  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 961
 
If the analytical method gives a positive value in unamended sewage 
equivalent to S  c mg/l, calculate the percentage removal (D  SC ) from: 
DSC ¼ 100 ðSi Ä Se þ ScÞ=ðSi þ ScÞ % 
Expression of test results  
46. Plot the percentage elimination D  t and D  ST (or D  SC ), if available, versus 
time (see Appendix 2 in chapter C.10- A). Take the mean (expressed to the 
nearest whole number) and standard deviation of the 12-15 values for D  T 
(and for D  ST , if available) obtained in the plateau phase as the percentage 
removal of the test chemical. From the shape of the elimination curve, some conclusions may be drawn about the removal processes.
 
Adsorption  
47. If a high DOC elimination of the test chemical is observed at the beginning 
of the test, the test chemical is probably eliminated by adsorption on to the 
biofilm. It may be possible to prove this by determining the adsorbed test chemical on solids sloughed from the film. It is not usual for the elimination of the DOC of adsorbable chemicals to remain high throughout the test; 
normally, there is an initial high degree of removal which gradually falls to 
an equilibrium value. If, however, the adsorbed test chemical was able to cause acclimation of the microbial population, the elimination of the test chemical DOC would subsequently increase and reach a high, plateau level.
 
Lag phase  
48. As in static, screening tests many test chemicals require a lag phase before 
full biodegradation occurs. In the lag phase, acclimation (or adaptation) of the competent bacteria takes place with almost no removal of the test chemical; then the initial growth of these bacteria occurs. This phase ends 
and the degradation phase is arbitrarily taken to begin when about 10 % of 
the initial amount of test chemical is removed (after allowing for adsorption, if it occurs). The lag phase is often highly variable and poorly reproducible.
 
Plateau phase  
49. The plateau phase of an elimination curve in a continuous test is defined as 
that phase in which the maximum degradation takes place. This phase 
should last at least 3 weeks and have about 12-15 measured valid values.  
Mean degree of elimination of the test chemical  
50. Calculate the mean value from the elimination values D  t (and D  st , if 
available) of the test chemical at the plateau phase. Rounded to the nearest whole number (1 %), it is the degree of elimination of the test chemical. It is also recommended to calculate the 95 % confidence 
interval of the mean value. In a similar way calculate the mean degree 
(D
 B ) of elimination of the organic medium in the control vessel.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 962
 
Indication of biodegradation  
51. If the test chemical does not adsorb significantly on to the biofilm and the 
elimination curve has a typical shape of a biodegradation curve with lag, degradation and plateau phases (paragraphs 48, 49), the measured elim ­
ination can safely be attributed to biodegradation. If a high initial removal 
has taken place, the simulation test cannot differentiate between biological and abiotic elimination processes. In such cases, and in other cases where there is any doubt about biodegradation (e.g. if stripping takes place), 
analyse adsorbed test chemical on samples of the film or perform additional 
static (screening) tests for biodegradability based on parameters clearly indicating biological processes. Such tests are the oxygen uptake methods 
(Chapter C.4 of this Annex D, E and F) (9) or a test which measures CO
 2 
production (Chapter C.4-C of this Annex or the Headspace method) (10); 
use as inoculum pre-exposed biofilm from the appropriate reactor. 
52. If both the DOC removal and specific chemical removal have been 
measured, significant differences (the former being lower than the latter) between the percentages removed indicate the presence in the effluents of intermediate organic products, which may be more difficult to degrade; 
these should be investigated.
 
Validity of test results  
53. Consider the test to be valid if the degree of DOC (or COD) elimination 
(D B ) in the control units is > 80 % after 2 weeks operation and no unusual 
observations have been made. 
54. If a readily biodegradable (reference) chemical has been tested, the degree 
of biodegradation should be > 90 % and the difference between duplicate 
values should not be greater than 5 %. If these two criteria are not met, review the experimental procedures and/or obtain domestic sewage from another source. 
55. Similarly, differences between biodegradation values from duplicate units (if 
used) treating a test chemical should not differ by more than 5 %. If this 
criterion is not met but the removals are high, continue analysis for a further 
three weeks. If removal is low, investigate the inhibitory effects of the test chemical if not known and repeat the test at a lower concentration of test chemical, if that is feasible.
 
Test Report  
56. The test report must include the following:  
Test chemical:  
— identification data; 
— physical nature and, where relevant, physico-chemical properties.  
Test conditions:  
— any modifications to test system, especially if insolubles or volatiles 
tested; 
— type of organic medium; 
— proportion and nature of industrial wastes in sewage, if used and if 
known; 
— method of inoculation;  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 963
 
— test chemical stock solution — DOC (dissolved organic carbon) and 
TOC (total organic carbon) content; how prepared, if suspension; test 
concentration(s) used, reasons if outside range 10-20 mg/l DOC; method of addition; date first added; any changes in concentration; 
— mean hydraulic retention time (with no growth); rotational speed of tube; 
approximate angle of inclination, if possible; 
— details of sloughing; time and intensity; 
— test temperature and range; 
— analytical techniques employed.  
Test results:  
— all measured data DOC, COD, specific analyses, pH, temperature, N 
chemicals, if relevant; 
— all calculated date of D
 t (or D  tc ), D  B , D s obtained in tabular form and 
elimination curves; 
— information on lag and plateau phases, test duration, the degree of elim ­
ination of the test chemical, of the reference chemical (if tested) and of 
the organic medium (in the control unit), together with statistical data 
and statements of biodegradability and validity of the test; 
— discussion of results.  
LITERATURE:  
(1) Gerike P, Fischer W, Holtmann W (1980). Biodegradability determinations 
in trickling filter units compared with the OECD Confirmatory Test. Wat. Res. 14: 753-758. 
(2) Truesdale GA, Jones K, Vandyke KG (1959). Removal of synthetic 
detergents in sewage treatment processes: Trials of a new biologically attackable material.Wat. Waste Tr. J. 7: 441-444. 
(3) Baumann U, Kuhn G and Benz M. (1998) Einfache Versuchsanordnung 
zur Gewinnung gewässerökologisch relevanter Daten, UWSF — Z. Umweltchem. Ökotox. 10: 214-220. 
(4) Gloyna EF, Comstock RF, Renn CE (1952). Rotary tubes as experimental 
trickling filters. Sewage ind. Waste 24: 1355-1357. 
(5) Kumke GW, Renn CE (1966). LAS removal across an institutional 
trickling filter. JAOCS 43: 92-94. 
(6) Tomlinson TG, Snaddon DHM, (1966). Biological oxidation of sewage by 
films of micro-organisms. Int.J. Air Wat. Pollut. 10: 865-881. 
(7) Her Majesty’s Stationery Office (1982). Methods for the examination of 
waters and associated materials. Assessment of biodegradability, 1981, 
London. 
(8) Her Majesty’s Stationery Office (1984). Methods for the examination of 
waters and associated materials. Methods for assessing the treatability of 
chemicals and industrial waste waters and their toxicity to sewage treatment processes, 1982, London. 
(9) Chapter C.4 of this Annex, Determination of ‘Ready’ Biodegradability 
A-F. 
(10) ISO 14593 (1998). Water Quality-Evaluation in an aqueous medium of the 
ultimate biodegradability of organic substances. Method by analysis of 
released inorganic carbon in sealed vessels.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 964
 
Appendix 8  
Figure 1  
Rotating tubes
  
Glossary:  
Plan view: 
View A/B: Driven wheels: Idling wheels: Drive motor: Reduction gear: Internal flange: Tilting mechanism: Bevel gear drive:
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 965
 
Figure 2  
Flow diagram
  
A: Feed Tank 
B: Peristaltic Pump C: Rotating Tube D: Effluent Collection Vessel
 
DEFINITIONS:  
Test chemical: Any substance or mixture tested using this Test Method. 
Chemicals: It should be noted that the term ‘chemical’ is used broadly in the 
UNCED agreements and subsequent documents to include substances, products, 
mixtures, preparations, or any other terms that may be used in existing systems to denote coverage.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 966
 
C.11.  BIODEGRADATION  
ACTIVATED SLUDGE RESPIRATION INHIBITION TEST  
1. METHOD  
1.1. INTRODUCTION 
The method described assesses the effect of a test substance on 
micro-organisms by measuring the respiration rate under defined conditions in the presence of different concentrations of the test substance. 
The purpose of this method is to provide a rapid screening method 
whereby substances which may adversely affect aerobic microbial 
treatment plants can be identified, and to indicate suitable non- inhibitory concentrations of test substances to be used in biodegrad ­
ability tests. 
A range-finding test may precede a definitive test. It provides 
information about the range of concentrations to be used in the 
main test. 
Two controls without test substance are included in the test design, 
one at the start and the other at the end of the test series. Each batch 
of activated sludge should also be checked using a reference substance. 
This method is most readily applied to substances which, due to their 
water solubility and low volatility, are likely to remain in water. 
For substances with limited solubility in the test media, it may not be 
possible to determine the EC
 50 . 
Results based on oxygen uptake may lead to erroneous conclusions 
when the test substance has the propensity to uncouple oxidative phosphorylation. 
It is useful to have the following information to perform the test: — water solubility, — vapour pressure, — structural formula, 
— purity of the test substance.
 
Recommendation  
Activated sludge may contain potentially pathogenic organisms and 
should be handled with care.  
1.2. DEFINITIONS AND UNITS 
The respiration rate is the oxygen consumption of waste-water 
micro-organisms in aerobic sludge, expressed generally as mg O  2 
per mg of sludge per hour.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 967
 
In order to calculate the inhibitory effect of a test substance at a 
particular concentration, the respiration rate is expressed as a 
percentage of the mean of the two control respiration rates:  
1 Ä 2R s 
Rc 1 þ Rc  2 Í Î  
Ü 100 ¼ % inhibition  
where: 
R s = oxygen-consumption rate at tested concentration of test 
substance 
R c1 = oxygen-consumption rate, control 1 
R c2 = oxygen-consumption rate, control 2. 
EC 50 in this method is the concentration of the test substance at 
which the respiration rate is 50 % of that shown by the control under conditions described in this method.
 
1.3. REFERENCE SUBSTANCES 
It is recommended that 3,5-dichlorophenol, as a known inhibitor of 
respiration, be used as a reference substance and tested for EC  50 on 
each batch of activated sludge as a means of checking that the sensitivity of the sludge is not abnormal.
 
1.4. PRINCIPLE OF THE TEST METHOD 
The respiration rate of an activated sludge fed with a standard 
amount of synthetic sewage feed is measured after a contact time 
of 30 minutes or three hours, or both. The respiration rate of the 
same activated sludge in the presence of various concentrations of the test substance under otherwise identical conditions is also measured. The inhibitory effect of the test substance at a particular 
concentration is expressed as a percentage of the mean respiration 
rates of two controls. An EC
 50 value is calculated from deter ­
minations at different concentrations.  
1.5. QUALITY CRITERIA 
The test results are valid if: 
— the two control respiration rates are within 15 % of each other, 
— the EC  50 (30 minutes and/or three hours) of 3,5-dichlorophenol is 
in the accepted range 5 to 30 mg/litre.  
1.6. DESCRIPTION OF THE TEST METHOD  
1.6.1.  Reagents  
1.6.1.1. S o l u t i o n s o f t h e t e s t s u b s t a n c e 
Solutions of the test substance are freshly prepared at the start of the 
study using a stock solution. A stock solution concentration of 0,5 g/ ­
litre is appropriate if the procedure recommended below is followed.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 968
 
1.6.1.2. S o l u t i o n o f c o n t r o l s u b s t a n c e 
A solution of 5,5-dichlorophenol can for example be prepared by 
dissolving 0,5 g 3,5-dichlorophenol in 10 ml of 1M NaOH, diluting to approximately 30 ml with distilled water, adding under stirring 
0,5M H
 2 SO 4 to the point of incipient precipitation — approximately 
8 ml of 0,5M H  2 SO 4 will be required — and finally diluting the 
mixture to one litre with distilled water. The pH should then be in the range 7 to 8.
 
1.6.1.3. S y n t h e t i c s e w a g e 
A synthetic sewage feed is made by dissolving the following 
amounts of substances in one litre of water: 
— 16 g peptone, 
— 11 g meat extract, 
— 3 g urea, — 0,7 g NaCl, 
— 0,4 g CaCl
 2 .2H  2 O, 
— 0,2 g MgSO  4 .7H  2 O, 
— 2,8 g K  2 HPO  4 . 
Note 1:  this synthetic sewage is a 100-fold concentrate of that 
described in the OECD Technical Report ‘Proposed method for the 
determination of the biodegradability of surfactants used in synthetic 
detergents’ (June 11, 1976), with the addition of dipotassium hydrogen phosphate.  
Note 2:  if the prepared medium is not used immediately, it shall be 
stored in the dark at 0 to 4
 o C, for no longer than one week, in 
conditions which do not produce any change in its composition. The 
medium may also be sterilised prior to storage, or the peptone and 
meat extract may be added shortly before carrying out the test. Before use, it shall be mixed thoroughly and the pH adjusted.
 
1.6.2.  Apparatus  
Measuring apparatus: the precise design is not critical. However, 
there should be head space and the probe should fit tightly in the neck of the measuring flask. 
Normal laboratory equipment and especially the following is 
necessary: 
— measuring apparatus, 
— aeration device, 
— pH-electrode and measuring equipment, — O
 2 -electrode.  
1.6.3.  Preparation of the inoculum  
Activated sludge from a sewage treatment plant treating predomi ­
nantly domestic sewage is used as the mircobial inoculum for the 
test. 
If necessary, on return to the larboratory, coarse particles may be 
removed by settling for a short period, e.g. 15 minutes, and 
decanting the upper layer of finer solids for use. Alternatively, the 
sludge may be mixed using a blender for a few seconds.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 969
 
In addition, if it is thought that inhibitory material is present, the 
sludge should be washed with tap water or an isotonic solution. 
After centrifuging, the supernatant is decanted (this procedure is repeated three times). 
A small amount of the sludge is weighed and dried. From this result, 
the amount of wet sludge can be calculated which must be 
suspended in water in order to obtain an activated sludge with a mixed liquor suspended solids range between 2 and 4 g/litre. This level gives a concentration between 0,8 and 1,6 g/litre in the test 
medium if the procedure recommended below is followed. 
If the sludge cannot be used on the day of collection, 50 ml of 
synthetic sewage is added to each litre of the activated sludge prepared as described above; this is then aerated overnight at 20 ± 
2
 o C. It is then kept aerated for use during the day. Before use the 
pH is checked and adjusted, if necessary, to pH 6 to 8. The mixed liquor suspended solids should be determined as described in the preceding paragraph. 
If the same batch of sludge is required to be used on subsequent 
days (maximum four days), a further 50 ml of synthetic sewage feed is added per litre of sludge at the end of each working day.
 
1.6.4.  Performance of the test  
Duration/contact time: 30 minutes and/or three hours, during 
which aeration takes place 
Water: Drinking water (dechlorinated if 
necessary) 
Air supply: Clean, oil-free air. Air flow 0,5 to 1 
litre/minute 
Measuring apparatus: Flat bottom flask such as a BOD-flask 
Oxygen meter: Suitable oxygen electrode, with a 
recorder 
Nutrient solution: Synthetic sewage (see above) Test substance: The test solution is freshly prepared at 
the start of the test 
Reference substance: e.g. 3,5-dichlorophenol (at least three 
concentrations) 
Controls: Inoculated sample without test 
substance 
Temperature: 20 ± 2
 o C 
A suggested experimental procedure which may be followed for both 
the test and reference substance for the three-hour contact period is given below: 
Several vessels (e.g. one-litre beakers) are used. 
At least five concentrations, spaced by a constant factor preferably 
not exceeding 3,2, should be used. 
At time ‘0’, 16 ml of the synthetic sewage feed are made up to 
300 ml with water. 200 ml of microbial inoculum are added and 
the total mixture (500 ml) poured into a first vessel (first control C
 1 ). ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 970
 
The test vessels should be aerated continuously such as to ensure 
that the dissolved O  2 does not fall below 2,5 mg/litre and that, 
immediately before the measurement of the respiration rate, the O  2 
concentration is about 6,5 mg/litre. 
At time ‘15 minutes’ (15 minutes is an arbitrary, but convenient, 
interval) the above is repeated, except that 100 ml of the test substance stock solution are added to the 16 ml of synthetic 
sewage before adding water to 300 ml and microbial inoculum to 
make a volume of 500 ml. This mixture is then poured into a second vessel and aerated as above. This process is repeated at 15-minute intervals with different volumes of the test substance stock solution 
to give a series of vessels containing different concentrations of the 
test substance. Finally, a second control is prepared (C
 2 ). 
After three hours the pH is recorded, and a well-mixed sample of the 
contents of the first vessel is poured into the measuring apparatus and the respiration rate is measured over a period of up to 
10 minutes. 
This determination is repeated on the contents of each vessel at 15- 
minute intervals, in such a way that the contact time in each vessel is 
three hours. 
The reference substance is tested on each batch of microbial 
inoculum in the same way. 
A different regime (e.g. more than one oxygen meter) will be 
necessary when measurements are to be made after 30 minutes of contact. 
If measurement of the chemical oxygen consumption is required, 
further vessels are prepared containing test substance, synthetic 
sewage feed and water, but no activated sludge. Oxygen 
consumption is measured and recorded after an aeration time of 30 minutes and/or three hours (contact time).
 
2. DATA AND EVALUATION  
The respiration rate is calculated from the recorder trace between 
approximately 6,5 mg O  2 /litre and 2,5 mg O  2 /litre, or over a 10- 
minute period when the respiration rate is low. The portion of the 
respiration curve over which the respiration rate is measured should 
be linear. 
If the respiration rates of the two controls are not within 15 % of 
each other, or the EC  50 (30 minutes and/or three hours) of the 
reference substance is not in the accepted range (5 to 30 mg/litre 
for 3,5-dichlorophenol), the test is invalid and must be repeated. 
The per - cent inhibition is calculated at each test concentration (see 
1.2). The per - cent inhibition is plotted against concentration on log- 
normal (or log-probability) paper, and an EC  50 value derived. 
95 % confidence limits for the EC  50 values can be determined using 
standard procedures.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 971
 
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, contain the following: 
— test substance: chemical identification data, — test system: source, concentration and any pre-treatment of the 
activated sludge, 
— test conditions: 
— pH of the reaction mixture before the respiration 
measurement, 
— test temperature, — test duration, — reference substance and its measured EC
 50 , 
— abiotic oxygen uptake (if any). 
— results: 
— all measured data, — inhibition curve and method for calculation of EC
 50 , 
— EC  50 and, if possible, 95 % confidence limits, EC  20 and 
EC 80 , 
— all observations and any deviations from this test method 
which could have influenced the result.  
3.2. INTERPRETATION OF DATA 
The EC  50 value should be regarded merely as a guide to the likely 
toxicity of the test substance either to activated sludge sewage 
treatment or to waste-water microorganisms, since the complex inter ­
actions occurring in the environment cannot be accurately simulated in a laboratory test. In addition, test substances which may have an 
inhibitory effect on ammonia oxidation may also produce atypical 
inhibition curves. Accordingly, such curves should be interpreted with caution.
 
4. REFERENCES  
(1) International Standard ISO 8192-1986. 
(2) Broecker, B., Zahn, R., Water Research 11,1977, p. 165. (3) Brown, D., Hitz, H. R., Schaefer, L., Chemosphere 10, 1981, 
p. 245. 
(4) ETAD (Ecological and Tox.icological Association of Dyestuffs 
Manufacturing Industries), Recommended Method No 103, also described by: 
(5) Robra, B., Wasserl Abwasser 117, 1976, p. 80. (6) Schefer, W., Textilveredlung 6,1977, p. 247. (7) OECD, Paris, 1981, Test Guideline 209, Decision of the 
Council C(84) 30 final.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 972
 
C.12.  BIODEGRADATION  
MODIFIED SCAS TEST  
1. METHOD  
1.1. INTRODUCTION 
The purpose of the method is the evaluation of the potential ultimate 
biodegradability of water-soluble, non-volatile organic substances when exposed to relatively high concentrations of micro-organisms over a long time period. The viability of the microorganisms is 
maintained over this period by daily addition of a settled sewage 
feed. (For weekend requirements, the sewage may be stored at 4
 o C. 
Alternatively, the synthetic sewage of the OECD confirmatory test 
may be used.) 
Physico-chemical adsorption on the suspended solids may take 
place and this must be taken into account when interpreting results 
(see 3.2). 
Because of the long detention period of the liquid phase (36 hours), 
and the intermittent addition of nutrients, the test does not simulate 
those conditions experienced in a sewage treatment plant. The results 
obtained with various test substances indicate that the test has a high biodegradation potential. 
The conditions provided by the test are highly favourable to the 
selection and/or adaptation of micro-organisms capable of degrading the test compound. (The procedure may also be used to produce acclimatised inocula for use in other tests.) 
In this method, the measure of the concentration of dissolved organic 
carbon is used to assess the ultimate biodegradability of the test substances. It is preferable to determine DOC after acidification 
and purging rather than as the difference of C
 total -C inorganic  . 
The simultaneous use of a specific analytical method may allow the 
assessment of the primary degradation of the substance (dis ­
appearance of the parent chemical structure). 
The method is applicable only to those organic test substances 
which, at the concentration used in the test: 
— are soluble in water (at least 20 mg dissolved organic carbon/ ­
litre), 
— have negligible vapour pressure, 
— are not inhibitory to bacteria, 
— do not significantly adsorb within the test system, 
— are not lost by foaming from the test solution. 
The organic carbon content of the test material must be established.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 973
 
Information on the relative proportions of the major components of 
the test material will be useful in interpreting the results obtained, 
particularly in those cases where the results are low or marginal. 
Information on the toxicity to microorganisms of the substance may 
be useful to the interpretation of low results and in the selection of 
an appropriate test concentration.  
1.2. DEFINITIONS AND UNITS 
C T = concentration of test compound as organic carbon as present 
in or added to the settled sewage at the start of the aeration 
period (mg/litre), 
C t = concentration of dissolved organic carbon found in the super ­
natant liquor of the test at the end of the aeration period 
(mg/litre), 
C c = concentration of dissolved organic carbon found in the super ­
natant liquor of the control at the end of the aeration period (mg/litre). 
The biodegradation is defined in this method as the disappearance of 
the organic carbon. The biodegradation can be expressed as: 
1. The percentage removal D
 da of the amount of substance added 
daily:  
D da ¼ C T Ä ðC T Ä C c Þ 
C T Ü 100  [1] 
where 
D da = degradation/daily addition. 
2. The percentage removal D  ssd of the amount of substance present 
at the start of each day:  
D ssd ¼ 2C T þ C ti Ä C ci Ä 3C  tðiþ1Þ þ 3C  cðiþ1Þ 
2C T þ C ti Ä C ci Ü 100  [2 (a)]  
≈ 2C T Ä 2ðC t Ä C c Þ 
2C T þ ðC t Ä C c Þ Ü 100  [2 (b)] 
where 
D ssd = degradation/substance start of day; 
the indices i and (i + 1) refer to the day of measurement. 
Equation 2(a) is recommended if effluent DOC varies from day to 
day, while equation 2(b) may be used when effluent DOC 
remains relatively constant from day to day.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 974
 
1.3. REFERENCE SUBSTANCES 
In some cases, when investigating a new substance, reference 
substances may be useful; however, no specific reference substance is recommended here. 
Data on several compounds evaluated in ring tests are provided (see 
Appendix 1) primarily so that calibration of the method may be performed from time to time and to permit comparison of results 
when another method is employed.
 
1.4. PRINCIPLE OF THE TEST METHOD 
Activated sludge from a sewage treatment plant is placed in a semi- 
continuous activated sludge (SCAS) unit. The test compound and 
settled domestic sewage are added, and the mixture is aerated for 
23 hours. The aeration is then stopped, the sludge allowed to settle and the supernatant liquor is removed. 
The sludge remaining in the aeration chamber is then mixed with a 
further aliquot of test compound and sewage and the cycle is repeated. 
Biodegradation is established by determination of the dissolved 
organic carbon content of the supernatant liquor. This value is compared with that found for the liquor obtained from a control tube dosed with settled sewage only. 
When a specific analytical method is used, changes in the concen ­
tration of the parent molecule due to biodegradation can be measured (primary biodegradability).
 
1.5. QUALITY CRITERIA 
The reproducibility of this method based on removal of dissolved 
organic carbon has not yet been established. (When primary biode ­
gradation is considered, very precise data are obtained for materials 
that are extensively degraded). 
The sensitivity of the method is largely determined by the variability 
of the blank and to a lesser extent by the precision of the deter ­
mination of dissolved organic carbon and the level of test compound 
in the liquor at the start of each cycle.  
1.6. DESCRIPTION OF THE TEST PROCEDURE  
1.6.1.  Preparations  
A sufficient number of clean aeration units, alternatively, the original 
1,5 litre SCAS test unit may be used, and air inlet tubes (Figure 1) 
for each test substance and controls are assembled. Compressed air supplied to the test units, cleaned by a cotton wool strainer, should be free of organic carbon and pre-saturated with water to reduce 
evaporation losses. 
A sample of mixed liquor, containing 1 to 4 g suspended solids/litre, 
is obtained from an activated sludge plant treating predominantly 
domestic sewage. Approximately 150 ml of the mixed liquor are 
required for each aeration unit.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 975
 
Stock solutions of the test substance are prepared in distilled water; 
the concentration normally required is 400 mg/litre as organic carbon 
which gives a test compound concentration of 20 mg/litre carbon at the start of each aeration cycle if no biodegradation is occurring. 
Higher concentrations are allowed if the toxicity to microorganisms 
permits it. 
The organic carbon content of the stock solutions is measured.
 
1.6.2.  Test conditions  
The test should be performed at 20 to 25  o C. 
A high concentration of aerobic microorganisms is used (from 1 to 
4 g/litre suspended solids), and the effective detention period is 
36 hours. The carbonaceous material in the sewage feed is 
oxidised extensively, normally within eight hours after the start of each aeration cycle. Thereafter, the sludge respires endogenously for the remainder of the aeration period, during which time the only 
available substrate is the test compound unless this is also readily 
metabolised. These features, combined with daily re-inoculation of the test when domestic sewage is used as the medium, provide highly favourable conditions for both acclimatisation and 
high degress of biodegradation.
 
1.6.3.  Performance of the test  
A sample of mixed liquor from a suitable predominantly domestic 
activated sludge plant or laboratory unit is obtained and kept aerobic until used in the laboratory. Each aeration unit as well as the control 
unit are filled with 150 ml of mixed liquor (if the original SCAS test 
unit is used, multiply the given volumes by 10) and the aeration is started. After 23 hours, aeration is stopped and the sludge is allowed to settle for 45 minutes. The tap of each vessel is opened in turn, and 
100 ml portions of the supernatant liquor are withdrawn. A sample 
of settled domestic sewage is obtained immediately before use, and 100 ml are added to the sludge remaining in each aeration unit. Aeration is started anew. At this stage no test materials are added, 
and the units are fed daily with domestic sewage only until a clear 
supernatant liquor is obtained on settling. This usually takes up to two weeks, by which time the dissolved organic carbon in the super ­
natant liquor at the end of each aeration cycle approaches a constant 
value. 
At the end of this period, the individual settled sludges are mixed, 
and 50 ml of the resulting composite sludge are added to each unit. 
95 ml of settled sewage and 5 ml of water are added to the control 
units, and 95 ml of the settled sewage plus 5 ml of the appropriate test compound stock solution (400 mg/litre) are added to the test 
units. Aeration is started again and continued for 23 hours. The 
sludge is then allowed to settle for 45 minutes and the supernatant drawn off and analysed for dissolved organic carbon content. 
The above fill-and-draw procedure is repeated daily throughout the 
test.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 976
 
Before settling, it may be necessary to clean the walls of the units to 
prevent the accumulation of solids above the level of the liquid. A 
separate scraper or brush is used for each unit to prevent cross contamination. 
Ideally, the dissolved organic carbon in the supernatant liquors is 
determined daily, although less frequent analyses are permissible. Before analysis the liquors are filtered through washed 0,45 μm 
membrane filters or centrifuged. Membrane filters are suitable if it 
is assured that they neither release carbon nor absorb the substance 
in the filtration step. The temperature of the sample must not exceed 40
 o C while it is in the centrifuge. 
The length of the test for compounds showing little or no biodegra ­
dation is indeterminate, but experience suggests that this should be at least 12 weeks in general, but not longer than 26 weeks.
 
2. DATA AND EVALUATION  
The dissolved organic carbon values in the supernatant liquors of the 
test units and the control units are plotted against time. 
As biodegradation is achieved, the level found in the test will 
approach that found in the control. Once the difference between the two levels is found to be constant over three consecutive 
measurements, such number of further measurements as are 
sufficient to allow statistical treatment of the data are made and the percentage biodegradation of the test compound is calculated (D
 da or D  ssd , see 1.2).  
3. REPORTING  
3.1. TEST REPORT 
The test report shall, if possible, contain the following: 
— all information on the kind of sewage, the type of unit used and 
the experimental results concerning the tested substance, the reference substance if used, and the blank, 
— the temperature, — removal curve with description, mode of calculation (see 1.2), 
— date and location where the activated sludge and the sewage 
were sampled, status of adaptation, concentration, etc., 
— scientific reasons for any changes of test procedure, 
— signature and date.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 977
 
3.2. INTERPRETATION OF RESULTS 
Since the substance to be tested by this method will not be readily 
biodegradable, any removal of DOC due solely to biodegradation will normally be gradual over days or weeks, except in such cases 
where acclimatisation is sudden as indicated by an abrupt 
disappearance occurring after some weeks. 
However, physico-chemical adsorption can sometimes play an 
important role; this is indicated when there is complete or partial 
removal of the added DOC at the outset. What happens subsequently 
depends on factors such as the degrees of adsorption and the concen ­
tration of suspended solids in the discarded effluent. Usually the difference between the concentration of DOC in the control and 
test supernatant liquors gradually increases from the initial low 
value and this difference then remains at the new value for the remainder of the experiment, unless acclimatisation takes place. 
If a distinction is to be drawn between biodegradation (or partial 
biodegradation) and adsorption, further tests are necessary. This 
can be done in a number of ways, but the most convincing is to 
use the supernatant liquor, or sludge, as inoculum in a base-set test (preferably a respirometric test). 
Test substances giving high, non-adsorptive removal of DOC in this 
test should be regarded as potentially biodegradable. Partial, non- 
adsorptive removal indicates that the chemical is at least subject to some biodegradation. 
Low, or zero removals of DOC may be due to inhibition of micro ­
organisms by the test substance and this may also be revealed by 
lysis and loss of sludge, giving turbid supernatants. The test should 
be repeated using a lower concentration of test substance. 
The use of a specific analytical method or of
 14 C-labelled test 
substance may allow greater sensitivity. In the case of  14 C test 
compound, the recovery of the  14 CO 2 will confirm that biodegra ­
dation has occurred. 
When results are also given in terms of primary biodegradation, an 
explanation should, if possible, be given on the chemical structure change that leads to the loss of response of the parent test substance. 
The validation of the analytical method must be given together with 
the response found on the blank test medium.
 
4. REFERENCES  
(1) OECD, Paris, 1981, Test Guideline 302 A, Decision of the 
Council C(81) 30 final.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 978
 
Appendix 1  
SCAS test: example of results  
Substance C T 
(mg/l) C t - C c 
(mg/l) Percentage 
biodegradition, 
D da Test duration 
(days)  
4-acetyl aminobenzene sulphonate 17,2 2,0 85 40 
Tetra propylene benzene sulphonate 17,3 8,4 51,4 40 4-nitrophenol 16,9 0,8 95,3 40 
Diethylene glycol 16,5 0,2 98,8 40 
Aniline 16,9 1,7 95,9 40 Cyclopentane tetra carboxylate 17,9 3,2 81,1 120
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 979
 
Appendix 2  
Example of test apparatus  
Figure 1
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 980
 
C.13  BIOCONCENTRATION: FLOW-THROUGH FISH TEST  
1. METHOD  
This bioconcentration method is a replicate of the OECD TG 305 
(1996).  
1.1. INTRODUCTION 
This method describes a procedure for characterising the bioconcen ­
tration potential of substances in fish under flow-through conditions. 
Although flow-through test regimes are much to be preferred, semi- static regimes are permissible, provided that the validity criteria are satisfied. 
The method gives sufficient details for performing the test while 
allowing adequate freedom for adapting the experimental design to 
the conditions in particular laboratories and for varying character ­
istics of test substances. It is most validly applied to stable organic chemicals with log P
 ow values between 1,5 and 6,0 (1) but may still 
be applied to superlipophilic substances (having log P  ow > 6,0). The 
pre-estimate of the bioconcentration factor (BCF), sometimes denoted as K
 B , for such superlipophilic substances will presumably 
be higher than the steady-state bioconcentration factor (BCF  SS ) value 
expected to be obtained from laboratory experiments. Preestimates of 
the bioconcentration factor for organic chemicals with log P  ow values 
up to about 9,0 can be obtained by using the equation of Bintein at al (2). The parameters which characterise the bioconcentration 
potential include the uptake rate constant (k
 1 ), the depuration rate 
constant (k  2 ) and the BCF  ss . 
Radio-labelled test substances can facilitate the analysis of water and 
fish samples and may be used to determine whether degradate identi ­
fication and quantification should be made. If total radioactive 
residues are measured (e.g. by combustion or tissue solubilisation), 
the BCF is based on the parent compound, any retained metabolites and also assimilated carbon. BCFs based on total radioactive residues may not, therefore, be directly comparable to a BCF derived by 
specific chemical analysis of the parent compound only. 
Clean-up procedures may be employed in radiolabelled studies in 
order to determine BCF based on the parent compound, and the 
major metabolites may be characterised if deemed necessary. It is also possible to combine a fish metabolism study with a bioconcen ­
tration study by analysis and identification of the residues in tissues.
 
1.2. DEFINITIONS AND UNITS 
Bioconcentration/Bioaccumulation is the increase in concentration of 
the test substance in or on an organism (specified tissues thereof) 
relative to the concentration of test substance in the surrounding medium. 
The bioconcentration factor (BCF or K
 B ) at any time during the 
uptake phase of this accumulation test is the concentration of test substance in/on the fish or specified tissues thereof (C
 f as μg/g 
(ppm)) divided by the concentration of the chemical in the 
surrounding medium (C  w as μg/ml (ppm)).  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 981
 
The steady-state bioconcentration factor (BCF  ss or K  B ) does not 
change significantly over a prolonged period of time, the concen ­
tration of the test substance in the surrounding medium being constant during this period of time. 
A plateau or steady-state is reached in the plot of test substance in 
fish (C
 f ) against time when the curve becomes parallel to the time 
axis and three successive analyses of C  f made on samples taken at 
intervals of at least two days are within ± 20 % of each other, and 
there are no significant differences among the three sampling periods. When pooled samples are analysed at least four successive 
analyses are required. For test substances which are taken up slowly 
the intervals would more appropriately be seven days. 
Bioconcentration factors calculated directly from kinetic rate 
constants (k
 1 /k 2 ) are termed kinetic concentration factor, BCF  K . 
The octanol-water partition coefficient (P  ow ) is the ratio of a 
chemical's solubility in n-octanol and water at equilibrium (Method A.8) also expressed as K
 ow . The logarithm of P  ow is used as an 
indication of a chemical's potential for bioconcentration by aquatic 
organisms. 
The exposure or uptake phase is the time during which the fish are 
exposed to the test chemical. 
The uptake rate constant (k  1 ) is the numerical value defining the rate 
of increase in the concentration of test substance in/on test fish (or 
specified tissues thereof) when the fish are exposed to that chemical 
(k 1 is expressed in day  -1 ). 
The post-exposure or depuration (loss) phase is the time, following 
the transfer of the test fish from a medium containing test substance to a medium free of that substance, during which the depuration (or 
the net loss) of the substance from the test fish (or specified tissue 
thereof) is studied. 
The depuration (loss) rate constant (k
 2 ) is the numerical value defining 
the rate of reduction in the concentration of the test substance in the test fish (or specified tissues thereof) following the transfer of the test fish 
from a medium containing the test substance to a medium free of that 
substance (k
 2 is expressed in day  -1 ). 
1.3. PRINCIPLE OF THE TEST METHOD 
The test consists of two phases: the exposure (uptake) and post- 
exposure (depuration) phases. During the uptake phase, separate groups of fish of one species are exposed to at least two concen ­
trations of the test substance. They are then transferred to a medium 
free of the test substance for the depuration phase. A depuration 
phase is always necessary unless uptake of the substance during the uptake phase has been insignificant (e.g. the BCF is less than 10). The concentration of the test substance in/on the fish (or 
specified tissue thereof) is followed through both phases of the test. 
In addition to the two test concentrations, a control group of fish is held under identical conditions except for the absence of the test substance, to relate possible adverse effects observed in the biocon ­
centration test to a matching control group and to obtain background 
concentrations of test substance.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 982
 
The uptake phase is run for 28 days unless it is demonstrated that 
equilibrium has been reached earlier. A prediction of the length of 
the uptake phase and the time to steady-state can be made from equation in Appendix 3. The depuration period is then begun by transferring the fish to the same medium but without the test 
substance in another clean vessel. Where possible the bioconcen ­
tration factor is calculated preferably both as the ratio (BCF
 ss ) of 
concentration of the fish (C  f ) and in the water (C  w ) at apparent 
steady-state and as a kinetic bioconcentration factor, BCF  K as the 
ratio of the rate constants of uptake (k  1 ) and depuration (k  2 ) 
assuming first-order kinetics. If first-order kinetics are obviously not obeyed, more complex models should be employed (Appendix 5). 
If a steady-state is not achieved within 28 days, the uptake phase 
should be extended until steady-state is reached, or 60 days, 
whichever comes first; the depuration phase is then begun. 
The uptake rate constant, the depuration (loss) rate constant (or 
constants, where more complex models are involved), the biocon ­
centration factor, and where possible, the confidence limits of each 
of these parameters are calculated from the model that best describes the measured concentrations of test substance in fish and water. 
The BCF is expressed as a function of the total wet weight of the 
fish. However, for special purposes, specified tissues or organs (e.g. 
muscle, liver), may be used if the fish are sufficiently large or the 
fish may be divided into edible (fillet) and non-edible (viscera) frac ­
tions. Since, for many organic substances, there is a clear rela ­
tionship between the potential for bioconcentration and lipophilicity, 
there is also a corresponding relationship between the lipid content 
of the test fish and the observed bioconcentration of such substances. Thus, to reduce this source of variability in test results for those substances with high lipophilicity (i.e. with log P
 ow > 3), bioconcen ­
tration should be expressed in relation to lipid content in addition to 
whole body weight. 
The lipid content should be determined on the same biological 
material as is used to determine the concentration of the test 
substance, when feasible.  
1.4. INFORMATION ON THE TEST SUBSTANCE 
Before carrying out the test for bioconcentration, the following 
information for the test substance should be known: 
— solubility in water — octanol-water partition coefficient P
 ow (denoted also as K  ow , 
determined by an HPLC method in A.8) 
— hydrolysis — phototransformation in water determined under solar or simulated 
solar irradiation and under the irridiation conditions of the test 
for bioconcentration (3)
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 983
 
— surface tension (i.e. for substances where the log P  ow cannot be 
determined) 
— vapour pressure — ready biodegradability (where appropriate) Other information required is the toxicity to the fish species to be 
used in the test, preferably the asymptotic LC
 50 (i.e. time-indepen ­
dent). An appropriate analytical method, of known accuracy, 
precision and sensitivity, for the quantification of the test substance in the test solutions and in biological material must be available, together with details of sample preparation and storage. 
Analytical detection limit of test substance in both water and fish 
tissues should also be known. When
 14 C labelled test substance is 
used, the percentage of radioactivity associated with impurities should be known.
 
1.5. VALIDITY OF THE TEST 
The following conditions should apply for a test to be valid: 
— the temperature variation is less than ± 2 °C; 
— the concentration of dissolved oxygen does not fall below 60 % 
saturation; 
— the concentration of the test substance in the chambers is main ­
tained within ± 20 % of the mean of the measured values during 
the uptake phase; 
— the mortality or other adverse effects/disease in both control and 
treated fish is less than 10 % at the end of the test; where the test is extended over several weeks or months, death or other adverse 
effects in both sets of fish should be less than 5 % per month and 
not exceed 30 % in all.
 
1.6. REFERENCE COMPOUNDS 
The use of reference compounds of known bioconcentration potential 
would be useful in checking the experimental procedure, when required. However, specific substances cannot yet be recommended.
 
1.7. DESCRIPTION OF THE TEST METHOD  
1.7.1.  Apparatus  
Care should be taken to avoid the use of materials, for all parts of 
the equipment, that can dissolve, sorb or leach and have an adverse effect on the fish. Standard rectangular or cylindrical tanks, made of chemically inert material and of a suitable capacity in compliance 
with loading rate can be used. The use of soft plastic tubing should 
be minimised. Teflon®, stainless steel and/or glass tubing are preferably used. Experience has shown that for substances with high adsorption coefficients, such as the synthetic pyrethroids, 
silanised glass may be required. In these situations the equipment 
will have to be discarded after use.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 984
 
1.7.2.  Water  
Natural water is generally used in the test and should be obtained 
from an uncontaminated and uniform quality source. The dilution water must be of a quality that will allow the survival of the 
chosen fish species for the duration of the acclimation and test 
periods without them showing any abnormal appearance or behaviour. Ideally, it should be demonstrated that the test species can survive, grow and reproduce in the dilution water (e.g. in 
laboratory culture or a life-cycle toxicity test). The water should 
be characterised at least by pH, hardness, total solids, total organic carbon and, preferably also ammonium, nitrite and alkalinity and, for 
marine species, salinity. The parameters which are important for 
optimal fish well-being are fully known, but Appendix 1 gives recommended maximum concentrations of a number of parameters for fresh and marine test waters. 
The water should be of constant quality during the period of a test. 
The pH value should be within the range 6,0 to 8,5, but during a 
given test it should be within a range of ± 0,5 pH units. In order to ensure that the dilution water will not unduly influence the test result (for example, by complexation of the test substance) or adversely 
affect the performance of the stock of fish, samples should be taken 
at intervals for analysis. Determination of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd, Ni), major anions and cations (e.g. Ca, Mg, Na, K, Cl, SO
 4 ), pesticides (e.g. total organophosphorous and total organoch ­
lorine pesticides), total organic carbon and suspended solids should 
be made, for example, every three months where a dilution water is known to be relatively constant in quality. If water quality has been demonstrated to be constant over at least one year, determinations 
can be less frequent and intervals extended (e.g. every six months). 
The natural particle content as well as the total organic carbon 
(TOC) of the dilution water should be as low as possible to avoid adsorption of the test substance to organic matter which may reduce its bioavailability (4). The maximum acceptable value is 5 mg/l for 
particulate matter (dry matter, not passing a 0,45 μm filter) and 
2 mg/l for total organic carbon (see Appendix 1). If necessary, the water should be filtered before use. The contribution to the organic carbon content in water from the test fish (excreta) and from the food 
residues should be as low as possible. Throughout the test, the 
concentration of organic carbon in the test vessel should not exceed the concentration of organic carbon originating from the 
test substance and, if used, the solubilising agent by more than 
10 mg/l (± 20 %).
 
1.7.3.  Test Solutions  
A stock solution of the test substance is prepared at a suitable 
concentration. The stock solution should preferably be prepared by simply mixing or agitating the test substance in the dilution water. The use of solvents or dispersants (solubilising agents) is not recom ­
mended; however this may occur in some cases in order to produce a 
suitably concentrated stock solution. Solvents which may be used are ethanol, methanol, ethylene glycol monomethyl ether, ethylene glycol dimethyl ether, dimethylformamide and triethylene glycol. 
Dispersants which may be used are Cremophor RH40, Tween 80, 
methylcellulose 0,01 % and HCO-40. Care should be taken when using readily biodegradable agents as these can cause problems with bacterial growth in flow-through tests. The test 
substance may be radio-labelled and should be of the highest 
purity (e.g. preferably > 98 %).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 985
 
For flow-through tests, a system which continuously dispenses and 
dilutes a stock solution of the test substance (e.g. metering pump, 
proportional diluter, saturator system) is required to deliver the test concentrations to the test chambers. At least five volume replacements through each test chamber per day are preferably 
allowed. The flow-through mode is to be preferred, but where this 
is not possible (e.g. when the test organisms are adversely affected) a semi-static technique may be used provided that the validity criteria are satisfied. The flow rates of stock solutions and dilution water 
should be checked both 48 h before and then at least daily during the 
test. In this check the determination of the flow-rate through each test chamber is included and ensured that it does not vary by more than 20 % either within or between chambers.
 
1.7.4.  Selection of species  
Important criteria in the selection of species are that they are readily 
available, can be obtained in convenient sizes and can be satisfac ­
torily maintained in the laboratory. Other criteria for selecting fish species include recreational, commercial, ecological importance as 
well as comparable sensitivity, past successful use etc. 
Recommended test species are given in Appendix 2. Other species 
may be used but the test procedure may have to be adapted to 
provide suitable test conditions. The rationale for the selection of 
the species and the experimental method should be reported in this case.
 
1.7.5.  Holding of fish  
Acclimate the stock population of fish for at least two weeks in 
water at the test temperature and feed throughout on a sufficient 
diet and of the same type to be used during the test. 
Following a 48-hour settling-in period, mortalities are recorded and 
the following criteria applied: 
— mortalities of greater than 10 % of population in seven days: 
reject the entire batch; 
— mortalities of between 5 and 10 % of population in seven days: 
acclimate for seven additional days; 
— mortalities of less than 5 % of population in seven days: accept 
the batch — if more than 5 % mortality during second seven 
days reject the entire batch. 
Ensure that fish used in tests are free from observable diseases and 
abnormalities. Discard any diseased fish. Fish should not receive 
treatment for disease in the two weeks preceding the test, or 
during the test.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 986
 
1.8. PERFORMANCE OF THE TEST  
1.8.1.  Preliminary test  
It may be useful to conduct a preliminary experiment in order to 
optimise the test conditions of the definitive test, e.g. selection of test 
substance concentration(s), duration of the uptake and depuration 
phases.  
1.8.2.  Conditions of exposure  
1.8.2.1. D u r a t i o n o f u p t a k e p h a s e 
A prediction of the duration of the uptake phase can be obtained 
from practical experience (e.g. from a previous study or an accumu ­
lation related chemical) or from certain empirical relationships 
utilising knowledge of either the aqueous solubility or the octanol/ ­
water partition coefficient of the test substance (see Appendix 3). 
The uptake phase should be run for 28 days unless it can be demon ­
strated that equilibrium has been reached earlier. If the steady-state has not been reached by 28 days, the uptake phase should be 
extended, taking further measurements, until steady-state is reached 
or 60 days, whichever is shorter.
 
1.8.2.2. D u r a t i o n o f t h e d e p u r a t i o n p h a s e 
A period of half the duration of the uptake phase is usually sufficient 
for an appropriate (e.g. 95 %) reduction in the body burden of the substance to occur (see Appendix 3 for explanation of the esti ­
mation). If the time required to reach 95 % loss is impractically 
long, exceeding for example twice the normal duration of the 
uptake phase (i.e. more than 56 days) a shorter period may be used (i.e. until the concentration of test substance is less than 10 % of steady-state concentration). However, for substances 
having more complex patterns of uptake and depuration than are 
represented by a one-compartment fish model, yielding first order kinetics, allow longer depuration phases for determination of loss rate constants. The period may, however, be governed by the 
period over which the concentration of test substance in the fish 
remains above the analytical detection limit.
 
1.8.2.3. N u m b e r s o f t e s t f i s h 
Select the numbers of fish per test concentration such that minimum 
of four fish per sample are available at each sampling. If greater statistical power is required, more fish per sample will be necessary. 
If adult fish are used, report whether male or female, or both are 
used in the experiment. If both sexes are used, differences in lipid content between sexes should be documented to be non-significant 
before the start of the exposure; pooling all male and all female fish 
may be necessary.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 987
 
In any one test, fish of similar weight are selected, such that the 
smallest are no smaller than two-thirds of the weight of the largest. 
All should be of the same year-class and come from the same source. Since weight and age of a fish appear sometimes to have a significant effect on BCF values (1) these details are recorded 
accurately. It is recommended that a sub-sample of the stock of 
fish is weighed before the test in order to estimate the mean weight.
 
1.8.2.4. L o a d i n g 
High water-to-fish ratios are used in order to minimise the reduction 
in C  W caused by the addition of the fish at the start of the test and 
also to avoid decreases in dissolved oxygen concentration. It is important that the loading rate is appropriate for the test species used. In any case, a loading rate of 0,1-1,0 g of fish (wet weight) 
per litre of water per day is normally recommended. High loading 
rates can be used if it is shown that the required concentration of test substance can be maintained within ± 20 % limits, and that the concentration of dissolved oxygen does not fall below 60 % satu ­
ration. 
In choosing appropriate loading regimes, account of the normal 
habitat of the fish species is taken. For example, bottom-living fish 
may demand a larger bottom area of the aquarium for the same 
volume of water than pelagic fish species.
 
1.8.2.5. F e e d i n g 
During the acclimation and test periods, fish are fed with an appro ­
priate diet of known lipid and total protein content, in an amount 
sufficient to keep them in a healthy condition and to maintain body weight. Fish are fed daily throughout the acclimation and test periods at a level of approximately 1 to 2 % of body weight per day; this 
keeps the lipid concentration in most species of fish at a relatively 
constant level during the test. The amount of feed should be re- calculated, for example, once per week, in order to maintain consistent body weight and lipid content. For this calculation, the 
weight of the fish in each test chamber can be estimated from the 
weight of the fish sampled most recently in that chamber. Do not weigh the fish remaining in the chamber. 
Uneaten food and faeces are siphoned daily from the test chambers 
shortly after feeding (30 minutes to 1 hour). Chambers are kept as clean as possible throughout the test so that the concentration of 
organic matter is kept as low as possible, since the presence of 
organic carbon may limit the bioavailability of the test substance (1). 
Since many feeds are derived from fishmeal, the feed should be 
analysed for the test substance. It is also desirable to analyse the 
feed for pesticides and heavy metals.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 988
 
1.8.2.6. L i g h t a n d t e m p e r a t u r e 
The photoperiod is usually 12 to 16 hours and the temperature (± 
2 °C) should be appropriate for the test species (see Appendix 2). The type and characteristics of illumination should be known. 
Caution should be given to the possible phototransformation of the 
test substance under the irradiation conditions of the study. Appro ­
priate illumination should be used avoiding exposure of the fish to unnatural photoproducts. In some cases it may be appropriate to use 
a filter to screen out UV irradiation below 290 nm.
 
1.8.2.7. T e s t c o n c e n t r a t i o n s 
Fish are exposed under flow-through conditions to at least two 
concentrations of the test substance in water. Normally, the higher 
(or highest) concentration of the test substance are selected to be about 1 % of its acute asymptotic LC
 50 , and to be at least ten-fold 
higher than its detection limit in water by the analytical method used. 
The highest test concentration can also be determined by dividing 
the acute 96 h LC  50 by an appropriate acute/chronic ratio (appro ­
priate ratios for some chemicals can be about 3 up to 100). If 
possible, choose the other concentration(s) such that it differs from the one above by a factor of ten. If this is not possible because of the 1 % of LC
 50 criterion and the analytical limit, a lower factor than ten 
can be used or the use of  14 C labelled test substance should be 
considered. No concentration used should be above the solubility of the test substance. 
Where a solubilising agent is used its concentration should not be 
greater than 0,1 ml/l and should be the same in all test vessels. Its contribution, together with the test substance, to the overall content 
of organic carbon in the test water should be known. However, every 
effort should be made to avoid the use of such materials.
 
1.8.2.8. C o n t r o l s 
One dilution water control or if relevant, one control containing the 
solubilising agent should be run in addition to the test series, provided that it has been established that the agent has no effects on the fish. If not, both controls should be set up.
 
1.8.3.  Frequency of water quality measurements  
During the test, dissolved oxygen, TOC, pH and temperature should 
be measured in all vessels. Total hardness and salinity, if relevant, should be measured in the controls and one vessel at the higher (or 
highest) concentration. As a minimum, dissolved oxygen and 
salinity, if relevant, should be measured three times — at the beginning, around the middle and end of the uptake period — and once a week in the depuration period. TOC should be measured at 
the beginning of the test (24 h and 48 h prior to test initiation of 
uptake phase) before addition of the fish and at least once a week, during both uptake and depuration phases. Temperature should be measured daily, pH at the beginning and end of each period and 
hardness once each test. Temperature should preferably be monitored 
continuously in at least one vessel.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 989
 
1.8.4.  Sampling and analysis of fish and water  
1.8.4.1. F i s h a n d w a t e r s a m p l i n g s c h e d u l e 
Water from the test chambers for the determination of test substance 
concentration is sampled before addition of the fish and during both 
uptake and depuration phases. As a minimum, the water is sampled 
at the same time as the fish and before feeding. During the uptake phase, the concentrations of test substance are determined in order to check compliance with the validity criteria. 
Fish is sampled on at least five occasions during the uptake phase 
and at least on four occasions during the depuration phase. Since on 
some occasions it will be difficult to calculate a reasonably precise estimate of the BCF value based on this number of samples, especially when other than simple first-order depuration kinetics 
are indicated, it may be advisable to take samples at a higher 
frequency in both periods (see Appendix 4). The extra samples are stored and analysed only if the results of the first round of analyses prove inadequate for the calculation of the BCF with the desired 
precision. 
An example of an acceptable sampling schedule is given in 
Appendix 4. Other schedules can readily be calculated using other assumed values of P
 ow to calculate the exposure time for 95 % 
uptake. 
Sampling is continued during the uptake phase until a steady-state 
has been established or for 28 days, whichever is the shorter. If the 
steady-state has not been reached within 28 days sampling continues 
until a steady-state has been attained or for 60 days, whichever is shorter. Before beginning the depuration phase the fish are transfered to clean tanks.
 
1.8.4.2. S a m p l i n g a n d S a m p l e P r e p a r a t i o n 
Water samples for analysis are obtained e.g. by siphoning through 
inert tubing from a central point in the test chamber. Since neither 
filtration nor centrifuging appear always to separate the non-bioa ­
vailable fraction of the test substance from that which is bioavailable (especially for super-lipophilic chemicals i.e. those chemicals with a 
log P
 ow > 5) (1) (5), samples may not be subjected to those treat ­
ments. 
Instead, measures should be taken to keep the tanks as clean as 
possible and the content of total organic carbon should be monitored during both the uptake and depuration phases. 
An appropriate number of fish (normally a minimum of four) is 
removed from the test chambers at each sampling time. The 
sampled fish are rinsed quickly with water, blot ‘dry’, killed 
instantly using the most appropriate and humane method, and then weighed. 
It is preferable to analyse fish and water immediately after sampling 
in order to prevent degradation or other losses and to calculate approximate uptake and depuration rates as the test proceeds. 
Immediate analysis also avoids delay in determining when a 
plateau has been reached.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 990
 
Failing immediate analysis, samples are stored by an appropriate 
method. Before the beginning of the study, information on the 
proper method of storage for the particular test substance — for example, deep-freezing, holding at 4
 0 C, duration of storage, 
extraction, etc. are obtained.  
1.8.4.3. Q u a l i t y o f A n a l y t i c a l m e t h o d 
Since the whole procedure is governed essentially by the accuracy, 
precision and sensitivity of the analytical method used for the test substance, check experimentally that the precision and reproduci ­
bility of the chemical analysis, as well as recovery of the test 
substance from both water and fish are satisfactory for the particular 
method. Also, check that the test substance is not detectable in the dilution water used. 
If necessary, the values of C
 w and C  f obtained from the test are 
corrected for the recoveries and background values of controls. 
Fish and water samples are handled throughout in such a manner as to minimise contamination and loss (e.g. resulting from adsorption by the sampling device).
 
1.8.4.4. A n a l y s i s o f F i s h S a m p l e 
If radiolabelled materials are used in the test, it is possible to analyse 
for total radio label (i.e. parent and metabolites) or, the samples may be cleaned up so that parent compound can be analysed separately. 
Also, the major metabolites may be characterised at steady-state or at 
the end of the uptake phase, whichever is the sooner. If the BCF in terms of total radiolebelled residues is ≥ 1 000 %, it may be 
advisable, and for certain categories of chemicals such as pesticides 
strongly recommended, to identify and quantify degradates repre ­
senting ≥ 10 % of total residues in fish tissues at steady state. 
If degradates representing ≥ 10 % of total radiolabelled residues in 
the fish tissue are identified and quantified, then it is also recom ­
mended to identify and quantify degradates in the test water. 
The concentration of the test substance should usually be determined 
for each weighed individual fish. If this is not possible, pooling of the samples on each sampling occasion may be done but pooling 
does restrict the statistical procedures which can be applied to the 
data. If a specific statistical procedure and power are important considerations, then an adequate number of fish to accommodate 
the desired pooling procedure and power should be included in the 
test (6) (7). 
BCF should be expressed both as a function of total wet weight and, 
for high lipophilic substances, as a function of the lipid content. 
Lipid content of the fish is determined on each sampling occasion 
if possible. Suitable methods should be used for determination of lipid content (ref. 8 and 2 of Appendix 3). Chloroform/methanol extraction technique may be recommended as standard method (9). 
The various methods do not give identical values (10), so it is 
important to give details of the method used. When possible, the analysis for lipid should be made on the same extract as that produced for analysis for the test substance, since the lipids often 
have to be removed from the extract before it can be analysed 
chromatographically. The lipid content of the fish (as mg/kg wet weight) at the end of the experiment should not differ from that at the start by more ± 25 %. The tissue percent solids should also be 
reported to allow conversion of lipid concentration from a wet to a 
dry basis.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 991
 
2. DATA  
2.1. TREATMENT OF RESULTS 
The uptake curve of the test substance is obtained by plotting its 
concentration in/on fish (or specified tissues) in the uptake phase 
against time on arithmetic scales. If the curve has reached a 
plateau, that is, become approximately asymptotic to the time axis, the steady-state BCF
 ss is calculated from:  
C f as steady Ä state ðmeanÞ 
C w as steady Ä state ðmeanÞ 
When no steady-state is reached, it may be possible to calculate a 
BCF  ss of sufficient precision for hazard assessment from a ‘steady- 
state’ at 80 % (1,6/k  2 ) or 95 % (3,0/k  2 ) of equilibrium. 
Also the concentration factor (BCF  K ) is determined, as the ratio 
k 1 /k 2 , the two first-order kinetic constants. The depuration rate 
constant (k  2 ) is usually determined from the depuration curve (i.e. 
a plot of the decrease in test substance concentration in the fish with time). The uptake rate constant (k
 1 ) is then calculated given k  2 and a 
value of C  f which is derived from the uptake curve (see also 
Appendix 5). The preferred method for obtaining BCF  K and the 
rate constants, k  1 and k  2 , is to use non-linear parameter estimation 
methods on a computer (11). Otherwise, graphical methods may be 
used to calculate k  1 and k  2 . If the depuration curve is obviously not 
first-order, then more complex models should be employed (see references in Appendix 3) and advice from a biostatistician sought.
 
2.2. INTERPRETATION OF RESULTS 
The results should be interpreted with caution where measured 
concentrations of test solutions occur at levels near the detection 
limit of the analytical method. 
Clearly defined uptake and loss curves are an indication of good 
quality bioconcentration data. The variation in uptake/depuration constants between the two test concentrations should be less than 
20 %. Observed significant differences in uptake/depuration rates 
between the two applied test concentrations should be recorded and possible explanations given. Generally the confidence limit of BCFs from well-designed studies approach ± 20 %.
 
3. REPORTING  
The test report must include the following information:  
3.1. TEST SUBSTANCE: 
— physical nature and, where relevant, physicochemical properties, 
— chemical identification data (including the organic carbon 
content, if appropriate), 
— if radio labelled, the precise position of the labelled atom(s) and 
the percentage of radioactivity associated with impurities.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 992
 
3.2. TEST SPECIES 
— scientific name, strain, source, any pre-treatment, acclimation, 
age, size-range, etc.  
3.3. TEST CONDITIONS: 
— test procedure used (e.g. flow-through or semi-static), 
— type and characteristics of illumination used and photoperiod(s), — test design (e.g. number and size of test chambers, water volume 
replacement rate, number of replicates, number of fish per 
replicate, number of test concentrations, length of uptake and depuration phases, sampling frequency for fish and water samples, 
— method of preparation of stock solutions and frequency of 
renewal (the solubilizing agent, its concentration and its contribution to the organic carbon content of test water must be given, when used), 
— the nominal test concentrations, the means of the measured 
values and their standard deviations in the test vessels and the 
method by which these were attained, 
— source of the dilution water, description of any pre-treatment, 
results of any demonstration of the ability of test fish to live 
in the water, and water characteristics: pH, hardness, temperature, 
dissolved oxygen concentration, residual chlorine levels (if measured), total organic carbon, suspended solids, salinity of the test medium (if appropriate) and any other measurements 
made, 
— water quality within test vessels, pH, hardness, TOC, temperature 
and dissolved oxygen concentration, 
— detailed information on feeding (e.g. type of food, source, 
composition — at least lipid and protein content if possible, 
amount given and frequency), 
— information on the treatment of fish and water samples, including 
details of preparation, storage, extraction and analytical procedures (and precision) for the test substance and lipid 
content (if measured).
 
3.4. RESULTS: 
— results from any preliminary study performed, 
— mortality of the control fish and the fish in each exposure 
chamber and any observed abnormal behaviour, 
— the lipid content of the fish (if determination on testing occa ­
sion), 
— curves (including all measured data) showing the uptake and 
depuration of the test chemical in the fish, the time to steady- 
state, ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 993
 
— Cf and Cw (with standard deviation and range, if appropriate) for 
all sampling times (Cf expressed in μg/g wet weight (ppm) of 
whole body or specified tissues thereof e.g. lipid, and Cw in μg/ml (ppm). Cw values for the control series (background 
should also be reported), 
— the steady-state bioconcentration factor (BCFss) and/or kinetic 
concentration factor (BCFK) and if applicable, 95 % confidence limits for the uptake and depuration (loss) rate constants (all 
expressed in relation to the whole body and the total lipid 
content, if measured, of the animal or specified tissues thereof), confidence limits and standard deviation (as available) and methods of computation/data analysis for each concentration of 
test substance used, 
— where radio-labelled substances are used, and if it is required, the 
accumulation of any detected metabolites may be presented, 
— anything unusual about the test, any deviation from these 
procedures, and any other relevant information, 
minimise results as ‘not detected at the limit of detection’ by pre-test 
method development and experimental design, since such results 
cannot be used for rate constant calculations.
 
4. REFERENCES  
(1) Connell D.W. (1988) Bioaccumulation behaviour of persistent 
chemicals with aquatic organisms. Rev. Environ. Contam. 
Toxicol. 102, p. 117-156. 
(2) Bintein S., Devillers J. and Karcher W. (1993) Nonlinear 
dependence of fish bioconcentration on n-octanol/water 
partition coefficient. SAR and QSAR in Environmental 
Research, 1, p. 29-390. 
(3) OECD, Paris (1996) Direct Phototransformation of chemicals 
in water. Environmental Health and Safety Guidance Document Series on Testing and Assessment of Chemicals. No 3. 
(4) Kristensen P. (1991) Bioconcentration in fish: Comparison of 
bioconcentration factors derived from OECD and ASTM testing methods; influence of particulate organic matter to the 
bioavailability of chemicals. Water Quality Institute, Denmark. 
(5) US EPA 822-R-94-002 (1994) Great Lake Water Quality 
Initiative Technical Support Document for the Procedure to 
Determine Bioaccumulation Factors. July 1994. 
(6) US FDA, (Food and Drug Administration) Revision. Pesticide 
analytical manual, 1, 5600 Fisher's Lane, Rockville, MD 
20852, July 1975. 
(7) US EPA (1974). Section 5, A(1) Analysis of Human or 
Animal Adipose Tissue, in Analysis of Pesticide Residues in Human and Environmental Samples, Thompson J.F. (ed.) Research Triangle Park, N.C. 27711. 
(8) Compaan H. (1980) in ‘The determination of the possible 
effects of chemicals and wastes on the aquatic environment: degradation, toxicity, bioaccumulation’ Ch. 2.3, 
Part II. Government Publishing Office, The Hague, The 
Netherlands.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 994
 
(9) Gardner et al, (1995) Limn. & Oceanogr. 30, p. 1099-1105. 
(10) Randall R.C., Lee H., Ozretich R.J., Lake J.L. and Pruell 
R.J. (1991). Evaluation of selected lipid methods for 
normalising pollutant bioaccumulation. Envir. Toxicol. Chem. 10, p. 1431-1436. 
(11) CEC, Bioconcentration of chemical substances in fish: the 
flow-through method-Ring Test Programme, 1984 to 
1985. Final report March 1987. Authors: P. Kristensen and 
N. Nyholm. 
(12) ASTM E-1022-84 (Reapproved 1988) Standard Practice for 
conducting Bioconcentration Tests with Fishes and Saltwater 
Bivalve Molluscs.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 995
 
Appendix 1  
Chemical characteristics of an acceptable dilution water  
Substance Limit concentration  
1 Particulate Matter 5 mg/l 
2 Total Organic Carbon 2 mg/l 3 Un-ionised ammonia 1 μg/l 
4 Residual chlorine 10 μg/l 
5 Total organophosphorous pesticides 50 ng/l 6 Total organochlorine pesticides plus polychlorinated 
biphenyls 50 ng/l 
7 Total organic chlorine 25 ng/l 
8 Aluminium 1 μg/l 
9 Arsenic 1 μg/l 
10 Chromium 1 μg/l 
11 Cobalt 1 μg/l 
12 Copper 1 μg/l 
13 Iron 1 μg/l 
14 Lead 1 μg/l 
15 Nickel 1 μg/l 
16 Zinc 1 μg/l 
17 Cadmium 100 ng/l 18 Mercury 100 ng/l 19 Silver 100 ng/l
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 996
 
Appendix 2  
Fish species recommended for testing  
Recommended species Recommended 
range of test 
temperature (  o C) Recommended 
total length of 
test animal (cm)  
1 Danio rerio  ( 1 ) (Teleostei, Cyprinidae) (Hamilton- 
Buchanan) Zebra — fish 20-25 3,0 ± 0,5 
2 Pimephales promelas  (Teleostei, Cyprinidae) (Rafi ­
nesque) Fathead minnow 20-25 5,0 ± 2,0 
3 Cyprinus carpio  (Teleostei, Cyprinidae) (Linnaeus) 
Common Carp 20-25 5,0 ± 3,0 
4 Oryzias latipes  (Teleostei, Poeciliidae) (Temminck 
and Schlegel) Ricefish 20-25 4,0 ± 1,0 
5 Poecilia reticulata  (Teleostei, Poeciliidae) (Peters) 
Guppy 20-25 3,0 ± 1,0 
6 Lepomis macrochirus  (Teleostei, Centrarchidae) 
(Rafinesque) Bluegill 20-25 5,0 ± 2,0 
7 Oncorhynchus mykiss  (Teleostei, Salmonidae) 
(Walbaum) Rainbow trout 13-17 8,0 ± 4,0 
8 Gasterosteus aculeatus  (Teleostei, Gasterosteidae) 
(Linnaeus) Three-spined stickleback 18-20 3,0 ± 1,0  
( 1 ) Meyer A., Orti G. (1993) Proc. Royal Society of London, Series B., Vol. 252, p. 231.  
Various estuarine and marine species have been used in different countries, for example: 
Spot Leiostomus xanthurus  
Sheepshead minnow  Cyprinodon variegatus  
Silverside  Menidia beryllina  
Shiner perch  Cymatogaster aggregata  
English sole  Parophrys vetulus  
Staghorn sculpin  Leptocottus armatus  
Three-spined stikleback  Gasterosteus aculeatus  
Sea bass  Dicentracus labrax  
Bleak  Alburnus alburnus  
Collection  
The fresh water fish listed in the table above are easy to rear and/or are widely available throughout 
the year, whereas the availability of marine and estuarine species is partially confined to the 
respective countries. They are capable of being bred and cultivated either in fish farms or in the laboratory, under disease-and parasite-controlled conditions, so that the test animal will be healthy and of known parentage. These fish are available in many parts of the world.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 997
 
Appendix 3  
Prediction of the duration of the uptake and depuration phases  
1. Prediction of the duration of the uptake phase  
Before performing the test, an estimate of k  2 and hence some percentage of 
the time needed to reach steady-state may be obtained from empirical 
relationships between k  2 and the n-octanol/water partition coefficient 
(P ow ) or k  2 and the aqueous solubility (s). 
An estimate of k  2 (day  -1 ) may be obtained, for example from the following 
empirical relationship (1): 
log 10 k 2 = 0,414 log  10 (P ow ) + 1,47 (r  2 = 0,95) [equation 1] 
For other relationships see Ref. (2). 
If the partition coefficient (P  ow ) is not known, an estimate can be made (3) 
from a knowledge of the aqueous solubility (s) of the substance using: 
log 10 (P ow ) = 0,862 log  10 (s) + 0,710 (r  2 = 0,994) [equation 2] 
where 
s = solubility (moles/l): (n=36) These relationships apply only to chemicals with log P
 ow values between 2 
and 6,5 (4). 
The time to reach some percentage of steady-state may be obtained, by 
applying the k  2 -estimate, from the general kinetic equation describing 
uptake and depuration (first-order kinetics):  
dC f 
dt ¼ k 1 · C w Ä k 2 · C f 
or if C  w is constant:  
C f ¼ k 1 
k 2 · C w ð1 Ä e Äk 2 t Þ [equation 3] 
When steady-state is approached (t → ∞), equation 3 may be 
reduced (5) (6) to:  
C f ¼ k 1 
k 2 · C w or C f /C w = k  1 /k 2 = BCF 
Then k  1 /k 2 · C w is an approach to the concentration in the fish at ‘steady- 
state’ (C  f,s ). 
Equation 3 may be transcribed to:  
C f ¼ C f;s ð1 Ä e Äk 2 t Þ or C f 
C fs ¼ 1 Ä e Äk 2 t [equation 4] 
Applying equation 4, the time to reach some percentage of steady-state may 
be predicted when k  2 is pre-estimated using equation 1 or 2.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 998
 
As a guideline, the statistically optimal duration of the uptake phase for the 
production of statistically acceptable data (BCF  K ) is that period which is 
required for the curve of the logarithm of the concentration of the test substance in fish plotted against linear time to reach its mid-point, or 1,6/k
 2 , or 80 % of steady-state but not more than 3,0/k  2 or 95 % of 
steady-state (7). 
The time to reach 80 % of steady-state is (equation 4):  
0; 80 ¼ 1 Ä e Äk 2 t 80 or t 80 ¼ 1; 6 
k 2 [equation 5] 
Similarly 95 percent of steady-state is:  
t 95 ¼ 3; 0 
k 2 [equation 6] 
For example, the duration of the uptake phase (up) for a test substance with 
log P  ow = 4 would be (using equations 1,5,6): 
log 10 k 2 = - 0,414.(4) + 1,47 k  2 = 0,652 days  -1 
up (80 %) = 1,6/0,652, i.e. 2,45 days (59 hours) 
or up (95 %) = 3,0/0,652, i.e. 4,60 days (110 hours) Similarly, for a test substance with s = 10
 -5 mol/l (log(s) = - 5,0), the 
duration of up would be (using equations 1,2,5,6): 
log 10 (P ow ) = 0,862 (- 5,0) + 0,710 = 5,02 
log 10 K 2 = 0,414 (5,02) + 1,47 
k 2 = 0,246 days  -1 
up (80 %) = 1,6/0,246, i.e. 6,5 days (156 hours) 
or up (95 %) = 3,0/0,246, i.e. 12,2 days (293 hours) Alternatively, the expression: t
 eq = 6,54 × 10  3 P ow + 55,31 (hours) 
may be used to calculate the time for effective steady-state to be 
reached (4).  
2. Prediction of the duration of the depuration phase  
A prediction of the time needed to reduce the body burden to some 
percentage of the initial concentration may also be obtained from the general equation describing uptake and depuration (first order 
kinetics) (1) (8). 
For the depuration phase, C
 w is assumed to be zero. The equation may be 
reduced to:  
dC f 
dt ¼ Ä k 2 C f or C f ¼ C f;o · e Äk 2 t ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 999
 
where  C f, o is the concentration at the start of the depuration period. 50 % 
depuration will then be reached at the time (t  50 ): 
C f 
C f;o ¼ 1 
2 ¼ e Äk 2 t 50 or t 50 ¼ 0; 693 
k 2 
Similarly 95 % depuration will be reached at:  
t 95 ¼ 3; 0 
k 2 
If 80 % uptake is used for the first period (1,6/ k 2 ) and 95 % loss in the 
depuration phase (3,0/ k 2 ), then the depuration phase is approximately twice 
the duration of the uptake phase. 
It is important to note, however, that the estimations are based on the 
assumption that uptake and depuration patterns will follow first order kinetics. If first order kinetics are obviously not obeyed, more complex 
models should be employed (e.g. ref (1)).
 
Literature  (of Appendix 3) 
(1) Spacie A. and Hamelink J.L. (1982) Alternative models for describing the 
bioconcentration of organics in fish. Environ. Toxicol. and Chem. 1, pp 
309-320. 
(2) Kristensen P. (1991) Bioconcentration in fish: comparison of BCF's 
derived from OECD and ASTM testing methods; influence of particulate matter to the bioavailability of chemicals. Danish Water 
Quality Institute. 
(3) Chiou C.T. and Schmedding D.W. (1982) Partitioning of organic 
compounds in octanol-water systems. Environ. Sci. Technol. 16 (1), pp 4-10. 
(4) Hawker D.W. and Connell D.W. (1988) Influence of partition coefficient 
of lipophilic compounds on bioconcentration kinetics with fish. Wat. 
Res. 22 (6), pp 701-707. 
(5) Branson D.R., Blau G.E., Alexander H.C. and Neely W.B. (1975) 
Transactions of the American Fisheries Society, 104 (4), pp 785-792. 
(6) Ernst W. (1985) Accumulation in Aquatic organisms. In: Appraisal of 
tests to predict the environmental behaviour of chemicals. Ed. by 
Sheehman P., Korte F., Klein W. and Bourdeau P.H. Part 4.4 pp 243-255. SCOPE, 1985, John Wiley & Sons Ltd. N.Y. 
(7) Reilly P.M., Bajramovic R., Blau G.E., Branson D.R. and Sauerhoff 
M.W. (1977) Guidelines for the optimal design of experiments to 
estimate parameters in first order kinetic models, Can. J. Chem. 
Eng. 55, pp 614-622. 
(8) Könemann H. and Van Leeuwen K. (1980) Toxicokinetics in fish: 
Accumulation and Elimination of six Chlorobenzenes by Guppies. Chemosphere, 9, pp 3-19.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1000
 
Appendix 4  
Theoretical example of sampling schedule for bioconcentration tests of substances with log P  ow = 4 
Fish sampling Sample time schedule 
No of water samples No of fish per sample Minimal required 
frequency (days) Additional sampling  
Uptake phase -1 
0 2 (*) 
2 add 45-80 fish 
1st 0,3 0,4 2 
(2) 4 
(4) 
2nd 0,6 0,9 2 
(2) 4 
(4) 
3rd 1,2 1,7 2 
(2) 4 
(4) 
4th 2,4 3,3 2 
(2) 4 
(4) 
5th 4,7 2 6 
Depuration phase Transfer fish to water 
free of test chemical 
6th 5,0 5,3 4 
(4) 
7th 5,9 7,0 4 
(4) 
8th 9,3 11,2 4 
(4) 
9th 14,0 17,5 6 
(4) 
(*) Sample water after minimum of 3 ‘chamber-volume’ have been delivered. 
Values in brackets are numbers of samples (water, fish) to be taken if additional sampling is carried out.  
Note : pre-test estimate of k  2 for log P  ow of 4,0 is 0,652 days  -1 . The total duration of the experiment is set to 3 × up = 3 × 4,6 days, i.e. 
14 days. For the estimation of ‘up’ refer to Appendix 3.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1001
 
Appendix 5  
Model discrimination  
Most bioconcentration data have been assumed to be ‘reasonably’ well described 
by a simple two-compartment/two-parameter model, as indicated by the rectilinear curve which approximates to the points for concentrations in fish, 
during the depuration phase, when these are plotted on semi-log paper. (Where 
these points cannot be described by a rectilinear curve then more complex models should be employed, see, for example, Spacie and Hamelink, Ref 1 in Appendix 3).
 
Graphical method for determination of depuration (loss) rate constant k  2 
Plot the concentration of the test substance found in each sample of fish against 
sampling time on semi-log paper. The slope of that line is k  2 . 
k 2 ¼ ln ðC f1=C f2 Þ 
t 2 Ä t 1
 
Note that deviations from straight line may indicate a more complex depuration 
pattern than first order kinetics. A graphical method may be applied for resolving 
types of depuration deviating from first order kinetics.  
Graphical method for determination of uptake rate constant k  1 
Given K  2 , calculate k  1 as follows:  
k 1 ¼ C f k 2 
C w Ü ð1 Ä e Äk 2 t Þ [equation 1] 
The value of C  f is read from the midpoint of the smooth uptake curve produced 
by the data when log concentration is plotted versus time (on an arithmetical 
scale).  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1002
 
Computer method for calculation of uptake and depuration (loss) rate 
constants  
The preferred means for obtaining the bioconcentration factor and k  1 and k  2 rate 
constants is to use non-linear parameter estimation methods on a computer. These programs find values for k
 1 and k  2 given a set of sequential time concentration 
data and the model:  
C f ¼ C w · k 1 
k 2 Ü 1 Ä e Äk 2 t Ê Ì  
0 < t < t  c [equation 2]  
C f ¼ C w · k 1 
k 2 Ü e Äk 2 ðtÄt c Þ Ä e Äk 2 t Ê Ì  
t > t  c [equation 3] 
where t  c = time at the end of the uptake phase. 
This approach provides standard deviation estimates of k  1 and k  2 . 
As k  2 in most cases can be estimated from the depuration curve with relatively 
high precision, and because a strong correlation exists between the two parameters k
 1 and k  2 if estimated simultaneously, it may be advisable first to 
calculate k  2 from the depuration data only, and subsequently calculate k  1 from 
the uptake data using non-linear regression.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1003
 
C.14.  FISH JUVENILE GROWTH TEST  
1. METHOD  
This growth toxicity test method is a replicate of the OECD TG 215 
(2000).  
1.1. INTRODUCTION 
This test is designed to assess the effects of prolonged exposure to 
chemicals on the growth of juvenile fish. It is based on a method, 
developed and ring-tested (1)(2) within the European Union, for assessing the effects of chemicals on the growth of juvenile rainbow trout  (Oncorynchus mykiss ) under flow-through conditions. Other well 
documented species may be used. For example, experience has been 
gained from growth tests with zebrafish ( Danio rerio ) (
 1 ) (3)(4) and 
ricefish (medaka,  Oryzias latipes ) (5)(6)(7). 
See also General introduction Part C.  
1.2. DEFINITIONS  
Lowest observed effect concentration (LOEC ): is the lowest tested 
concentration of a test substance at which the substance is observed 
to have a significant effect (at p < 0,05) when compared with the 
control. However, all test concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC.  
No observed effect concentration (NOEC) : is the test concentration 
immediately below the LOEC.  
EC
 x : in this test method is the concentration of the test substance 
which causes a x % variation in growth rate of the fish when 
compared with controls.  
Loading rate : is the wet weight of fish per volume of water.  
Stocking density : is the number of fish per volume of water.  
Individual fish specific growth rate : expresses the growth rate of 
one individual based on its initial weight.  
Tank-average specific growth rate : expresses the mean growth rate 
of a tank population at one concentration.  
Pseudo specific growth rate : expresses the individual growth rate 
compared to the mean initial weight of the tank population.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1004  
( 1 ) Meyer, A., Bierman, C.H. and Orti, G. (1993). The phylogenetic position of the zebrafish 
(Danio rerio ), a model system in developmental biology: an invitation to the comparative 
method. Proc. R. Soc. Lond. B. 252, 231-236.
 1.3. PRINCIPLE OF THE TEST METHOD 
Juvenile fish in exponential growth phase are placed, after being 
weighted, in test chambers and are exposed to a range of sublethal concentrations of the test substance dissolved in water preferably 
under flow-through, or, if not possible, under appropriate semi- 
static (static-renewal) conditions. The test duration is 28 days. Fish are fed daily. The food ration is based on initial fish weights and may be recalculated after 14 days. At the end of the test, the fish are 
weighed again. Effects on growth rates are analysed using a 
regression model in order to estimate the concentration that would cause a x % variation in growth rate, i.e. EC
 x (e.g. EC  10 , EC  20 , or 
EC 30 ). Alternatively, the data may be compared with control values 
in order to determine the lowest observed effect concentration (LOEC) and hence the no observed effect concentration (NOEC).
 
1.4. INFORMATION ON THE TEST SUBSTANCE 
Results of an acute toxicity test (see Test Method C. 1.) preferably 
performed with the species chosen for this test, should be available. 
This implies that the water solubility and the vapour pressure of the test substance are known and a reliable analytical method is available for the quantification of the substance in the test solutions with 
known and reported accuracy and limit of detection is available. 
Useful information includes the structural formula, purity of the 
substance, stability in water and light, pK
 a , P ow and results of a 
test for ready biodegradability (see Test Method C.4).  
1.5. VALIDITY OF THE TEST 
For the test to be valid the following conditions apply: 
— the mortality in the control(s) must not exceed 10 % at the end of 
the test; 
— the mean weight of fish in the control(s) must have increased 
enough to permit the detection of the minimum variation of 
growth rate considered as significant. A ring-test (2) has shown that for rainbow trout the mean weight of fish in the controls must have increased by at least the half (i.e. 50 %) of their mean 
initial weight over 28 days; e.g. initial weight: 1 g/fish 
(= 100 %), final weight after 28 days: ≥ 1,5 g/fish ( ≥ 150 %); 
— the dissolved oxygen concentration must have been at least 60 % 
of the air saturation value (ASV) throughout the test; 
— the water temperature must not differ by more than ± 1
 o C 
between test chambers at any one time during the test and 
should be maintained within a range of 2  o C within the 
temperature ranges specified for the test species (Appendix 1).  
1.6. DESCRIPTION OF THE TEST METHOD  
1.6.1.  Apparatus  
Normal laboratory equipment and especially the following: 
— oxygen and pH meters;  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1005
 
— equipment for determination of water hardness and alkalinity; 
— adequate apparatus for temperature control and preferably 
continuous monitoring; 
— tanks made of chemically inert material and of suitable capacity 
in relation to the recommended loading and stocking density (see Section 1.8.5 and Appendix 1); 
— suitably accurate balance (i.e. accurate to ± 0,5 %).
 
1.6.2.  Water  
Any water in which the test species shows suitable long-term 
survival and growth may be used as a test water. It should be of 
constant quality during the period of the test. The pH of the water 
should be within the range 6,5 to 8,5, but during a given test it should be within a range of ± 0,5 pH units. Hardness above 140 mg/l (as CaCO
 3 ) is recommended. In order to ensure that the 
dilution water will not unduly influence the test result (for example 
by complexion of test substance), samples should be taken at intervals for analysis. Measurements of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd and Ni), major anions and cations (e.g. Ca, Mg, Na, K, 
Cl and SO
 4 ), pesticides (e.g. total organophosphorus and total orga ­
nochlorine pesticides), total organic carbon and suspended solids should be made, for example, every three months where a dilution water is known to be relatively constant in quality. If water quality 
has been demonstrated to be constant over at least one year, deter ­
minations can be less frequent and intervals extended (e.g. every 6 months). Some chemical characteristics of an acceptable dilution water are listed in Appendix 2.
 
1.6.3.  Test solutions  
Test solutions of the chosen concentrations are prepared by dilution 
of a stock solution. 
The stock solution should preferably be prepared by simply mixing 
or agitating the test substance in the diluition water by using mech ­
anical means (e.g. stirring or ultrasonication). Saturation columns (solubility columns) can be used for achieving a suitable concen ­
trated stock solution. 
The use of solvents or dispersants (solubilising agents) may be 
required in some cases in order to produce a suitably concentrated 
stock solution. Examples of suitable solvents are acetone, ethanol, 
methanol, dimethylsulfoxide, dimethylformamide and triethyleneg ­
lycol. Examples of suitable dispersants are Cremophor RH40, Tween 80, Methylcellulose 0,01 % and HCO-40. Care should be 
taken when using readily biodegradable agents (e.g. acetone) 
and/or highly volatile compounds as these can cause problems with bacterial built-up in flow-through tests. When a solubilising agent is used it must have no significant effects on the fish 
growth nor visible adverse effects on the juvenile as revealed by a 
solvent-only control.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1006
 
For flow-through tests, a system which continually dispenses and 
dilutes a stock solution of the test substance (e.g. metering pump, 
proportional diluter, saturator system) is required to deliver a series of concentrations to the test chambers. The flow rates of stock solutions and dilution water should be checked at intervals, 
preferably daily, during the test and should not vary by more than 
10 % throughout the test. A ring-test (2) has shown that, for rainbow trout, a frequency of water removal during the test of six litres/g of fish/day is acceptable (see Section 1.8.2.2). 
For semi-static (renewal) tests, the frequency of medium renewal will 
depend on the stability of the test substance, but a daily water 
renewal is recommended. If, from preliminary stability tests (see 
Section 1.4), the test substance concentration is not stable (i.e. outside the range 80-120 % of nominal or falling below 80 % of 
the measured initial concentration) over the renewal period, 
consideration should be given to the use of a flow-through test.
 
1.6.4.  Selection of species  
Rainbow trout ( Oncorhynchus mykiss ) is the recommended species 
for this test since most experience has been gained from ring-test with this species (1)(2). However, other well documented species can be used but the test procedure may have to be adapted to provide 
suitable test conditions. For example, experience is also available 
with zebrafish ( Danio rerio ) (3)(4) and ricefish (medaka,  Oryzias 
latipes ) (5)(6)(7). The rationale for the selection of the species and 
the experimental method should be reported in this case.
 
1.6.5.  Holding of fish  
The test fish shall be selected from a population of a single stock, 
preferably from the same spawning, which has been held for at least 
two weeks prior to the test under conditions of water quality and 
illumination similar to those used in the test. They should be fed a minimum ration of 2 % body weight per day and preferably 4 % body weight per day throughout the holding period and during the 
test. 
Following a 48 h setting-in period, mortalities are recorded and the 
following criteria applied: 
— mortalities of greater than 10 % of population in seven days: 
reject the entire batch; 
— mortalities of between 5 % and 10 % of population: acclimation 
for seven additional days; if more than 5 % mortality 
during second seven days, reject the entire batch; 
— mortalities of less than 5 % of population in seven days: accept 
the batch. 
Fish should not receive treatment for disease in the two weeks 
preceding the test, or during the test.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1007
 
1.7. TEST DESIGN 
The ‘test design’ relates to the selection of the number and spacing 
of the test concentrations, the number of tanks at each concentration level and the number of fish per tank. Ideally, the test design should 
be chosen with regard to: 
— the objective of the study; 
— the method of statistical analysis that will be used; — the availability and cost of experimental resources. 
The statement of the objective should, if possible, specify the stat ­
istical power at which a given size of difference (e.g. in growth rate) 
is required to be detected or, alternatively, the precision with which the EC
 x (e.g. with x = 10, 20, or 30, and preferably not less than 10) 
is required to be estimated. Without this, a firm prescription of the 
size of the study cannot be given. 
It is important to recognise that a design which is optimal (makes 
best use of resources) for use with one method of statistical analysis 
is not necessarily optimal for another. The recommended design for the estimation of a LOEC/NOEC would not therefore be the same as that recommended for analysis by regression. 
In most of cases, regression analysis is preferable to the analysis of 
variance, for reasons discussed by Stephan and Rogers (8). However, when no suitable regression model is found (r
 2 < 0,9) NOEC/LOEC 
should be used.  
1.7.1.  Design for analysis by regression  
The important considerations in the design of a test to be analysed 
by regression are: 
— The effect concentration (e.g. EC  10,20,30  ) and the concentration 
range over which the effect of the test substance is of interest, 
should necessarily be spanned by the concentrations included in 
the test. The precision with which estimates of effect concen ­
trations can be made, will be best when the effect concentration is in the middle of the range of concentrations tested. A 
preliminary range-finding test may be helpful in selecting appro ­
priate test concentrations. 
— To enable satisfactory statistical modelling, the test should 
include at least one control tank and five additional tanks at different concentrations. Where appropriate, when a solubilising agent is used, one control containing the solubilising agent at the 
highest tested concentration should be run in addition to the test 
series (see Sections 1.8.3 and 1.8.4). 
— An appropriate geometric series or logarithmic series (9) (see 
Appendix 3) may be used. Logarithmic spacing of test concen ­
tration is to be preferred.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1008
 
— If more than six tanks are available, the additional tanks should 
either be used to provide replication or distributed across the 
range of concentrations in order to enable closer spacing of the levels. Either of these measures are equally desirable.
 
1.7.2.  Design for estimation of a NOEC/LOEC using Analysis of 
Variance (ANOVA)  
There should preferably be replicate tanks at each concentration, and 
statistical analysis should be at the tank level (10). Without replicate tanks, no allowance can be made for variability between tanks 
beyond that due to individual fish. However, experience has 
shown (11) that between-tank variability was very small compared with within-tank (i.e. between-fish) variability in the case examined. Therefore a relatively acceptable alternative is to perform statistical 
analysis at the level of individual fish. 
Conventionally, at least five test concentrations in a geometric series 
with a factor preferably not exceeding 3,2 are used. 
Generally, when tests are performed with replicate tanks, the number 
of replicate control tanks and therefore the number of fish should be 
the double of the number in each of the test concentrations, which 
should be of equal size (12)(13)(14). On the opposite, in absence of replicate tanks, the number of fish in the control group should be the same as the number in each test concentration. 
If the ANOVA is to be based on tanks rather than individual fish 
(which would entail either individual marking of the fish or the use of ‘pseudo’ specific growth rates (see Section 2.1.2)), there is a need 
for enough replication of tanks to enable the standard deviation of 
‘tanks-within-concentrations’ to be determined. This means that the degrees of freedom for error in the analysis of variance should be at least 5 (10). If only the controls are replicated, there is a danger 
that the error variability will be biased because it may increase with 
the mean value of the growth rate in question. Since growth rate is likely to decrease with increasing concentration, this will tend to lead to an overestimate of the variability.
 
1.8. PROCEDURE  
1.8.1.  Selection and weighing of test fish  
It is important to minimise variation in weight of the fish at the 
beginning of the test. Suitable size ranges for the different species 
recommended for use in this test are given in Appendix 1. For the 
whole batch of fish used in the test, the range in individual weights at the start of the test should ideally be kept to within ± 10 % of the arithmetic mean weight and, in any case, should not exceed 25 %. It 
is recommended to weight a subsample of fish before the test in 
order to estimate the mean weigh.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1009
 
Food should be withheld from the stock population for 24 h prior to 
the start of the test. Fish should then be chosen at random. Using a 
general anaesthetic (e.g. an aqueous solution of 100 mg/l tricaine methane sulphonate (MS 222) neutralised by the addition of two parts of sodium bicarbonate per part of MS 222), fish should be 
weighted individually as wet weights (blotted dry) to the precision 
given in Appendix 1. Those fish with weights within the intended range should be retained and then should be randomly distributed between the test vessels. The total wet weight of fish in each test 
vessel should be recorded. The use of anaesthetics likewise handling 
of fish (including blotting and weighing) may cause stress and injuries to the juvenile fish, in particular for those species of small size. Therefore handling of juvenile fish must be done with the 
utmost care to avoid stressing and injuring test animals. 
The fish are weighed again on day 28 of the test (see Section 1.8.6). 
However, if it is deemed necessary to recalculate the food ration, fish 
can be weighed again on day 14 of the test (see Section 1.8.2.3). Other method as photographic method could be used to determine 
changes in fish size from which food rations could be adjusted.
 
1.8.2.  Conditions of exposure  
1.8.2.1.  Duration  
The test duration is ≥ 28 days.  
1.8.2.2.  Loading rates and stocking densities  
It is important that the loading rate and stocking density is appro ­
priate for the test species used (see Appendix 1). If the stocking 
density is too high, then overcrowding stress will occur leading to reduced growth rates and possibly to disease. If it is too low, terri ­
torial behaviour may be induced which could also affect growth. In 
any case, the loading rate should be low enough in order that a 
dissolved oxygen concentration of at least 60 % ASV can be main ­
tained without aeration. A ring-test (2) has shown that, for rainbow trout, a loading rate of 16 trouts of 3-5 g in a 40-litre volume is 
acceptable. Recommended frequency of water removal during the 
test is 6 litres/g of fish/day.
 
1.8.2.3.  Feeding  
The fish should be fed with an appropriate food (Appendix 1) at a 
sufficient rate to induce acceptable growth rate. Care should be taken to avoid microbial growth and water turbidity. For rainbow trout, a rate of 4 % of their body weight per day is likely to satisfy these 
conditions (2)(15)(16)(17). The daily ration may be divided into two 
equal portions and given to the fish in two feeds per day, separated by at least 5 h. The ration is based on the initial total fish weight for each test vessel. If the fish are weighted again on day 14, the ration 
is then recalculated. Food should be withheld from the fish 24 h 
prior to weighing.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1010
 
Uneaten food and fecal material should be removed from the test 
vessels each day by carefully cleaning the bottom of each tank using 
a suction.  
1.8.2.4.  Light and temperature  
The photoperiod and water temperature should be appropriate for the 
test species (Appendix 1).  
1.8.3.  Test concentrations  
Normally five concentrations of the test substance are required, 
regardless of the test design (see Section 1.7.2). Prior knowledge of the toxicity of the test substance (e.g. from an acute test and/or 
from range-finding studies) should help in selecting appropriate test 
concentrations. Justification should be given if fewer than five concentrations are used. The highest tested concentration should not exceed the substance solubility limit in water. 
Where a solubilising agent is used to assist in stock solution prep ­
aration, its final concentration should not be greater than 0,1 ml/l and 
should preferably be the same in all test vessels (see Section 1.6.3). However, every effort should be made to avoid use of such materials.
 
1.8.4.  Controls  
The number of dilution-water controls depends on the test design 
(see Sections 1.7-1.7.2). If a solubilising agent is used, then the same 
number of solubilising-agent controls as dilution-water controls 
should also be included.  
1.8.5.  Frequency of analytical determinations and measurements  
During the test, the concentrations of test substance are determined at 
regular intervals (see below). 
In flow-through tests, the flow rates of diluent and toxicant stock 
solution should be checked at intervals, preferably daily, and should 
not vary by more than 10 % throughout the test. Where the test 
substance concentrations are expected to be within ± 20 % of the nominal values (i.e. within the range 80-120 %; see Sections 1.6.2 and 1.6.3), it is recommended that, as a minimum, the highest and 
lowest test concentrations be analysed at the start of the test and at 
weekly intervals thereafter. For the test where the concentration of the test substance is not expected to remain within ± 20 % of nominal (on the basis of stability data of the test substance), it is 
necessary to analyse all test concentrations, but following the same 
regime. 
In semi-static (renewal) tests where the concentration of the test 
substance is expected to remain within ± 20 % of the nominal values, it is recommended that, as a minimum, the highest and 
lowest test concentrations be analysed when freshly prepared and 
immediately prior to renewal at the start of the study and weekly thereafter. For tests where the concentration of the test substance is not expected to remain within ± 20 % of nominal, all test concen ­
trations must be analysed following the same regime as for more 
stable substances.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1011
 
It is recommended that results be based on measured concentrations. 
However, if evidence is available to demonstrate that the concen ­
tration of the test substance in solution has been satisfactorily main ­
tained within ± 20 % of the nominal or measured initial concen ­
tration throughout the test, then the results can be based on nominal 
or measured values. 
Samples may need to be filtered (e.g. using a 0,45 μm pore size) or 
centrifuged. Centrifugation is the recommended procedure. However, if the test material does not adsorb to filters, filtration may also be 
acceptable. 
During the test, dissolved oxygen, pH and temperature should be 
measured in all test vessels. Total hardness, alkalinity and salinity (if relevant) should be measured in the controls and one vessel at the highest concentration. As a minimum, dissolved oxygen and salinity 
(if relevant) should be measured three times (at the beginning, 
middle and end of the test). In semi-static tests, it is recommended that dissolved oxygen be measured more frequently, preferably 
before and after each water renewal or at least once a week. pH 
should be measured at the beginning and end of each water renewal in static renewal test and at least weekly in flow-through tests. Hardness and alkalinity should be measured once each test. 
Temperature should preferably be monitored continuously in at 
least one test vessel.
 
1.8.6.  Observations  
Weight: at the end of the test all surviving fish must be weighed as 
wet weights (blotted dry) either in groups by test vessel or individ ­
ually. Weighing of animals by test vessel is preferred to individual 
weights which require that fish be individually marked. In the case 
of the measurement of individual weights for determination of indi ­
vidual fish specific growth rate, the marking technique selected should avoid stressing the animals (alternatives to freeze marking 
may be appropriate, e.g. the use of coloured fine fishing line). 
The fish should be examined daily during the test period and any 
external abnormalities (such as hemorrhage, discoloration) and abnormal behaviour noted. Any mortalities should be recorded and 
the dead fish removed as soon as possible. Dead fish are not 
replaced, the loading rate and stocking density being sufficient to avoid effects on growth through changes in number of fish per tank. However, the feeding rate will need to be adjusted.
 
2. DATA AND REPORTING  
2.1. TREATMENT OF RESULTS 
It is recommended that a statistician be involved in both the design 
and analysis of the test since this test method allows for considerable variation in experimental design as for example, in the number of test chambers, number of test concentrations, number of fish, etc. In 
view of the options available in test design, specific guidance on 
statistical procedure is not given here.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1012
 
Growth rates should not be calculated for test vessels where the 
mortality exceeds 10 %. However, mortality rate should be 
indicated for all test concentrations. 
Whichever method is used to analyse the data, the central concept is 
the specific growth rate r between time t  1 and time t  2 . This can be 
defined in several ways depending on whether fish are individually 
marked or not or whether a tank average is required.  
r 1 ¼ log e w 2 Ä log  e w 1 
t 2 Ä t 1 Ü 100 
r 2 ¼ log e w 2 Ä log  e w 1 
t 2 Ä t 1 Ü 100 
r 3 ¼ log e w 2 Ä log  e w 1 
t 2 Ä t 1 Ü 100  
where: 
r 1 = individual fish specific growth rate 
r 2 = tank-average specific growth rate 
r 3 = ‘pseudo’ specific growth rate 
w 1 , w 2 = weights of a particular fish at times t  1 and t  2 , 
respectively 
log e w 1 = logarithm of the weight of an individual fish at the start 
of the study period 
log e w 2 = logarithm of the weight of an individual fish at the end 
of the study period 
log e w 1 = average of the logarithms of the values w  1 for the fish 
in the tank at the start of the study period 
log e w 2 = average of the logarithms of the values w  2 for the fish 
in the tank at the end of the study period 
t 1 , t 2 = time (days) at start and end of study period 
r 1 , r 2 , r 3 can be calculated for the 0-28 days period and, where 
appropriate (i.e. when measurement at day 14 has been done) for 
the 0-14 and 14-28 days periods.  
2.1.1.  Analysis of results by regression (concentration-response 
modelling)  
This method of analysis fits a suitable mathematical relationship 
between the specific growth rate and concentration, and hence 
enables the estimation of the ‘EC  x ’ i.e. any required EC value. 
Using this method the calculation of r for individual fish (r  1 ) is 
not necessary and instead, the analysis can be based on the tank- 
average value of r (r  2 ). This last method is preferred. It is also more 
appropriate in case of the use of smallest species. 
The tank-average specific growth rates (r  2 ) should be plotted 
graphically against concentration, in order to inspect the concen ­
tration response relationship.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1013
 
For expressing the relationship between r  2 and concentration, an 
appropriate model should be chosen and its choice must be 
supported by appropriate reasoning. 
If the numbers of fish surviving in each tank are unequal, then the 
process of model fitting, whether simple or non-linear, should be 
weighted to allow for unequal sizes of groups. 
The method of fitting the model must enable an estimate of, for 
example, the EC  20 and of its dispersion (either standard error or 
confidence interval) to be derived. The graph of the fitted model 
should be shown in relation to the data so that the adequacy of the fit of the model can be seen (8)(18)(19)(20).
 
2.1.2.  Analysis of results for the estimation of the LOEC  
If the test has included replication of tanks at all concentration levels, 
the estimation of the LOEC could be based on an analysis of variance (ANOVA) of the tank-average specific growth rate (see Section 2.1), followed by a suitable method (e.g. Dunnett's or 
Williams' test (12)(13)(14)(21)) of comparing the average r for 
each concentration with the average r for the controls to identify the lowest concentration for which this difference is significant at a 0,05 probability level. If the required assumptions for parametric 
methods are not met — non-normal distribution (e.g. Shapiro-Wilk's 
test) or heterogeneous variance (Bartlett's test), consideration should be given to transforming the data to homogenise variances prior to performing the ANOVA, or to carrying out a weighted ANOVA. 
If the test has not included replication of tanks at each concentration, 
an ANOVA based on tanks will be insensitive or impossible. In this situation, an acceptable compromise is to base the ANOVA on the 
‘pseudo’ specific growth rate r
 3 for individual fish. 
The average r  3 for each test concentration may then be compared 
with the average r  3 for the controls. The LOEC can then be 
identified as before. It must be recognised that this method 
provides no allowance for, nor protection against, variability between tanks, beyond that which is accounted for by the variability between individual fish. However, experience has shown (8) that 
between-tank variability was very small compared with within-tank 
(i.e. between fish) variability. If individual fish are not included in the analysis, the method of outlier identification and justification for its use must be provided.
 
2.2. INTERPRETATION OF RESULTS 
The results should be interpreted with caution where measured 
toxicant concentrations in test solutions occur at levels near the detection limit of the analytical method or, in semi static tests, when the concentration of the test substance decreases between 
freshly prepared solution and before renewal.
 
2.3. TEST REPORT 
The test report must include the following information:  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1014
 
2.3.1.  Test substance:  
— physical nature and relevant physical-chemical properties; 
— chemical identification data including purity and analytical 
method for quantification of the test substance where appropriate.  
2.3.2.  Test species:  
— scientific name, possibly 
— strain, size, supplier, any pre-treatment, etc.  
2.3.3.  Test conditions:  
— test procedure used (e.g. semi-static/renewal, flow-through, 
loading, stocking density, etc.), 
— test design (e.g. number of test vessels, test concentrations and 
replicates, number of fish per vessel), 
— method of preparation of stock solutions and frequency of 
renewal (the solubilising agent and its concentration must be 
given, when used), 
— the nominal test concentrations, the means of the measured 
values and their standard deviations in the test vessels and the method by which these were attained and evidence that the measurements refer to the concentrations of the test substance 
in true solution, 
— dilution water characteristics: pH, hardness, alkalinity, 
temperature, dissolved oxygen concentration, residual chlorine levels (if measured), total organic carbon, suspended solids, salinity of the test medium (if measured) and any other 
measurements made, 
— water quality within test vessels: pH, hardness, temperature and 
dissolved oxygen concentration, 
— detailed information on feeding, (e.g. type of food(s), source, 
amount given and frequency).
 
2.3.4.  Results:  
— evidence that controls met the validity criterion for survival, and 
data on mortalities occurring in any of the test concentrations, 
— statistical analytical techniques used, statistics based on replicates 
or fish, treatment of data and justification of techniques used, 
— tabulated data on individual and mean fish weights on days 0, 14 
(if measured) and 28 values of tank-average or pseudo specific 
growth rates (as appropriate) for the periods 0-28 days or possibly 0-14 and 14-28, 
— results of the statistical analysis (i.e. regression analysis or 
ANOVA) preferably in tabular and graphical form and the 
LOEC (p = 0,05) and the NOEC or ECx with, when possible, 
standard errors, as appropriate,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1015
 
— incidence of any unusual reactions by the fish and any visible 
effects produced by the test substance.  
3. REFERENCES 
(1) Solbe J.F. de LG (1987) Environmental Effects of Chemicals 
(CFM 9350 SLD). Report on a UK Ring Test of a Method for 
Studying the Effects of Chemicals on the Growth rate of Fish. 
WRc Report No PRD 1388-M/2. 
(2) Ashley S., Mallett M.J. and Grandy N.J., (1990) EEC Ring 
Test of a Method for Determining the Effects of Chemicals on 
the Growth Rate of Fish. Final Report to the Commission of the European Communities. WRc Report No EEC 2600-M. 
(3) Crossland N.O. (1985) A method to evaluate effects of toxic 
chemicals on fish growth. Chemosphere, 14, p. 1855-1870. 
(4) Nagel R., Bresh H., Caspers N., Hansen P.D., Market M., 
Munk R., Scholz N. and Höfte B.B., (1991) Effect of 3,4- dichloroaniline on the early life stages of the Zebrafish ( Bra­
chydanio rerio ): results of a comparative laboratory study. 
Ecotox. Environ. Safety, 21, p. 157-164. 
(5) Yamamoto, Tokio., (1975) Series of stock cultures in 
biological field. Medaka (killifish) biology and strains. 
Keigaku Publish. Tokio, Japan. 
(6) Holcombe, G.W., Benoit D.A., Hammermeister, D.E., 
Leonard, E.N. and Johnson, R.D., (1995) Acute and long-term effects of nine chemicals on the Japanese medaka ( Oryzias latipes ).Arch. Environ. Conta. Toxicol. 28, 
p. 287-297. 
(7) Benoit, D.A., Holcombe, G.W. and Spehar, R.L., (1991) 
Guidelines for conducting early life toxicity tests with 
Japanese medaka ( Oryzias latipes ). Ecological Research 
Series EPA-600/3-91-063. U.S. Environmental Protection Agency, Duluth, Minesota. 
(8) Stephan C.E. and Rogers J.W., (1985) Advantages of using 
regression analysis to calculate results of chronic toxicity tests. Aquatic Toxicology and Hazard Assessment: Eighth 
Symposium, ASTM STP 891, R C Bahner and D J Hansen, 
Eds., American Society for Testing and Materials, Philad ­
elphia, p. 328-338. 
(9) Environment Canada, (1992) Biological test method: toxicity 
tests using early life stages of salmonid fish (rainbow trout, coho salmon, or Atlantic salmon). Conservation and Protection, 
Ontario, Report EPS 1/RM/28, p. 81. 
(10) Cox D.R., (1958) Planning of experiments. Wiley Edt. 
(11) Pack S., (1991) Statistical issues concerning the design of tests 
for determining the effects of chemicals on the growth rate of fish. Room Document 4, OECD Ad Hoc Meeting of Experts 
on Aquatic Toxicology, WRc Medmenham, UK, 10- 
12 December 1991. 
(12) Dunnett C.W., (1955) A Multiple Comparisons Procedure for 
Comparing Several Treatments with a Control, J. Amer. Statist. Assoc., 50, p. 1096-1121. 
(13) Dunnett C.W., (1964) New tables for multiple comparisons 
with a control. Biometrics, 20, p. 482-491.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1016
 
(14) Williams D.A., (1971) A test for differences between treatment 
means when several dose levels are compared with a zero dose 
control. Biometrics 27, p. 103-117. 
(15) Johnston, W.L., Atkinson, J.L., Glanville N.T., (1994) A 
technique using sequential feedings of different coloured food to determine food intake by individual rainbow trout,  
Oncorhynchus mykiss : effect of feeding level. Aquaculture 
120, p. 123-133. 
(16) Quinton, J. C. and Blake, R.W., (1990) The effect of feed 
cycling and ration level on the compensatory growth response in rainbow trout,  Oncorhynchus mykiss . Journal of 
Fish Biology, 37, p. 33-41. 
(17) Post, G., (1987) Nutrition and Nutritional Diseases of Fish. 
Chapter IX in Testbook of Fish Health. T.F.H. Publications, Inc. Neptune City, New Jersey, USA. p. 288. 
(18) Bruce, R.D. and Versteeg D.J., (1992) A statistical procedure 
for modelling continuous toxicity data. Environ. Toxicol. 
Chem. 11, p. 1485-1494. 
(19) DeGraeve, G.M., Cooney, J.M., Pollock, T.L., Reichenbach, 
J.H., Dean, Marcus, M.D. and McIntyre, D.O., (1989) 
Precision of EPA seven-day fathead minnow larval survival 
and growth test; intra and interlaboratory study. report EA- 6189 (American Petroleum Institute Publication, No 4468). Electric Power Research Institute, Palo alto, CA. 
(20) Norbert-King T.J., (1988) An interpolation estimate for chronic 
toxicity: the ICp approach. US Environmental Protection 
Agency. Environmental Research Lab., Duluth, Minesota. Tech. Rep. No 05-88 of National Effluent Toxicity Assesment Center. Sept. 1988. p. 12. 
(21) Williams D.A., (1972) The comparison of several dose levels 
with a zero dose control. Biometrics 28, p. 510-531.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1017
 2008R0440 — EN — 22.03.2014 — 004.001 — 1018
 ▼B 
Appendix 1  
FISH SPECIES RECOMMENDED FOR TESTING AND SUITABLE TEST CONDITIONS  
Species Recommended test 
temperature range 
( o C) Photoperiod 
(hours) Recommended 
range for initial 
fish weight 
(g) Required 
measurement 
precision Loading rate 
(g/l) Stocking density 
(per litre) Food Test duration 
(days)  
Recommended species:  
Oncorhynchus mykiss  
rainbow trout 12,5-16,0 12-16 1-5 to nearest 
100 mg 1,2-2,0 4 Dry propietary salmonid fry 
food ≥ 28 
Other well documented 
species:  
Danio rerio  
zebrafish 21-25 12-16 0,050-0,100 to nearest 1 mg 0,2-1,0 5-10 Live food ( Brachionus Artemia ) ≥ 28 
Oryzias latipes  
ricefish (Medaka) 21-25 12-16 0,050-0,100 to nearest 1 mg 0,2-1,0 5-20 Live food ( Brachionus Artemia ) ≥ 28
Appendix 2  
SOME CHEMICAL CHARACTERISTICS OF AN ACCEPTABLE 
DILUTION WATER  
Substance Concentrations  
Particulate matter < 20 mg/l 
Total organic carbon < 2 mg/l 
Unionised ammonia < 1 μg/l 
Residual chlorine < 10 μg/l 
Total organophosphorus pesticides < 50 ng/l Total organochlorine pesticides plus polychlorinated 
biphenyls < 50 ng/l 
Total organic chlorine < 25 ng/l
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1019
 
Appendix 3  
Logarithmic series of concentrations suitable for toxicity test (9)  
Column (Number of concentrations between 100 and 10, or between 10 and 1) (*) 
1 2 3 4 5 6 7  
100 100 100 100 100 100 100 
32 46 56 63 68 72 75 10 22 32 40 46 52 56 
3,2 10 18 25 32 37 42 1,0 4,6 10 16 22 27 32 
2,2 5,6 10 15 19 24 
1,0 3,2 6,3 10 14 18 
1,8 4,0 6,8 10 13 1,0 2,5 4,6 7,2 10 
1,6 3,2 5,2 7,5 1,0 2,2 3,7 5,6 
1,5 2,7 4,2 
1,0 1,9 3,2 
1,4 2,4 1,0 1,8 
1,3 1,0
 
(*) A series of five (or more) successive concentrations may be chosen from a column. Mid-points between concentrations in colu mn 
(x) are found in column (2x + 1). The values listed can represent concentrations expressed as percentage per volume or weight 
(mg/l or μg/l). Values can be multiplied or divided by any power of 10 as appropriate. Column 1 might be used if there was 
considerable uncertainty on the toxicity level.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1020
 
C.15.  FISH, SHORT-TERM TOXICITY TEST ON  EMBRYO  AND 
SAC-FRY STAGES  
1. METHOD  
This short-term toxicity test method is a replicate of the OECD TG 
212 (1998).  
1.1. INTRODUCTION 
This short-term toxicity test on Fish Embryo and Sac-Fry stages is a 
short-term test in which the life stages from the newly fertilised egg to the end of the sac-fry stage are exposed. No feeding is provided in 
the embryo and sac-fry test, and the test should thus be terminated 
while the sac-fry are still nourished from the yolk-sac. 
The test is intended to define lethal, and to a limited extent, sub- 
lethal effects of chemicals on the specific stages and species tested. 
This test would provide useful information in that is could (a) form a bridge between lethal and sub-lethal tests, (b) be used as a screening test for either a Full Early Life Stage test or for chronic toxicity tests 
and (c) be used for testing species where husbandry techniques are 
not sufficiently advanced to cover the period of change from endo ­
genous to exogenous feeding. 
It should be borne in mind that only tests incorporating all stages of 
the life-cycle of fish are generally liable to give an accurate estimate 
of the chronic toxicity of chemicals to fish, and that any reduced exposure with respect to life stages may reduce the sensitivity and thus underestimate the chronic toxicity. It is therefore expected that 
the embryo and sac-fry test would be less sensitive than a Full Early 
Life Stage test, particularly with respect to chemicals with high lipo ­
philicity (log P
 ow > 4) and chemicals with a specific mode of toxic 
action. However smaller differences in sensitivity between the two 
tests would be expected for chemicals with a non-specific, narcotic 
mode of action (1). 
Prior to the publication of this test, most experience with this embryo 
and sac-fry test has been with the freshwater fish  Danio rerio  
Hamilton-Buchanan (Teleostei, Cyprinidae — common name zebrafish). More detailed guidance on test performance for this species is therefore given in Appendix 1. This does not preclude 
the use of other species for which experience is also available 
(Table 1).
 
1.2. DEFINITIONS  
Lowest Observed Effect Concentration (LOEC) : is the lowest 
tested concentration of a test substance at which the substance is 
observed to have a significant effect (at p < 0,05) when compared 
with the control. However, all test concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC.  
No Observed Effect Concentration (NOEC) : is the test concen ­
tration immediately below the LOEC.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1021
 
1.3. PRINCIPLE OF THE TEST 
The embryo and sac-fry stages of fish are exposed to a range of 
concentrations of the test substance dissolved in water. Within the protocol a choice is possible between a semi-static and a flow- 
through procedure. The choice depends on the nature of the test 
substance. The test is begun by placing fertilised eggs in the test chambers and is terminated just before the yolk-sac of any larvae in any of the test chambers has been completely absorbed or before 
mortalities by starvation start in controls. Lethal and sub-lethal 
effects are assessed and compared with control values to determine the lowest observed effect concentration and hence the no observed 
effect concentration. Alternatively, they may be analysed using a 
regression model in order to estimate the concentration that would cause a given percentage effect (i.e. LC/ECx, where x is a defined % effect).
 
1.4. INFORMATION ON THE TEST SUBSTANCE 
Results of an acute toxicity test (see Method C. 1) preferably 
performed with the species chosen for this test, should be available. 
The results may be useful in selecting an appropriate range of test concentrations in the early life stages test. Water solubility (including solubility in the test water) and the vapour pressure of the test 
substance should be known. A reliable analytical method for the 
quantification of the substance in the test solutions with known and reported accuracy and limit of detection should be available. 
Information on the test substance which is useful in establishing the 
test conditions includes the structural formula, purity of the 
substance, stability in light, stability under the conditions of the test, pKa, P
 ow and results of a test for ready biodegradability (see 
Method C. 4).  
1.5. VALIDITY OF THE TEST 
For a test to be valid, the following conditions apply: 
— overall survival of fertilised eggs in the controls and where 
relevant, in the solvent-only vessels must be greater than or equal to the limits defined in Appendices 2 and 3 
— the dissolved oxygen concentration must be between 60 and 
100 % of the air saturation value (ASV) throughout the test 
— the water temperature must not differ by more than ± 1,5
 o C 
between test chambers or between successive days at any time 
during the test and should be within the temperature ranges 
specified for the test species (Appendices 2 and 3).  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1022
 
1.6. DESCRIPTION OF THE TEST METHOD  
1.6.1.  Test chambers  
Any glass or other chemically inert vessels can be used. The 
dimensions of the vessels should be large enough to allow 
compliance with the loading rate (see Section 1.7.1.2). It is recom ­
mended that test chambers be randomly positioned in the test area. A randomised block design with each treatment being present in each block is preferable to a completely randomised design when there are 
systematic effects in the laboratory that can be controlled using 
blocking. Blocking, if used, should be taken account of in the subsequent data analysis. The test chambers should be shielded 
from unwanted disturbance.
 
1.6.2.  Selection of fish species  
Recommended fish species are given in Table 1A. This does not 
preclude the use of other species (examples are given in Table 1B), but the test procedure may have to be adapted to provide suitable test 
conditions. The rationale for the selection of the species and the 
experimental method should be reported in this case.
 
1.6.3.  Holding of the brood fish  
Details on holding the brood stock under satisfactory conditions may 
be found in OECD TG 210 (  1 ) and in references (2)(3)(4)(5)(6).  
1.6.4.  Handling of embryos and larvae  
Embryos and larvae may be exposed, within the main vessel, in 
smaller vessels fitted with mesh sides or ends to permit a flow of 
test solution through the vessel. Non-turbulent flow through these 
small vessels may be induced by suspending them from an arm arranged to move the vessel up and down but always keeping the organisms submerged; a siphon-flush system can also be used. 
Fertilised eggs of salmonid fishes can be supported on racks or 
meshes with apertures sufficiently large to allow larvae to drop through after hatching. The use of pasteur pipettes is appropriate to remove the embryos and larvaes in the semi-static tests with 
complete daily renewal (see paragraph 1.6.6) 
Where egg containers, grids or meshes have been used to hold eggs 
within the main test vessel, these restraints should be removed after 
the larvae hatch (
 1 ), except that meshes should be retained to prevent 
the escape of the fish. If there is a need to transfer the larvae, they should not be exposed to the air and nets should not be used to 
release fish from egg containers (such a caution may not be 
necessary for some less fragile species, e.g. the carp). The timing of this transfer varies with the species and transfer may not always be necessary. For the semi-static technique, beakers or shallow 
containers may be used, and, if necessary, equipped with a mesh 
screen slightly elevated above the bottom of the beaker. If the volume of these containers is sufficient to comply with loading requirements, (see 1.7.1.2) no transfer of embryo or larvae may be 
necessary.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1023  
( 1 ) OECD, Paris, 1992, Test Guideline 210, ‘Fish, Early-life Stage Toxicity Test’.
 1.6.5.  Water  
Any water which conforms to the chemical characteristics of an 
acceptable dilution water as listed in Appendix 4 and in which the test species shows control survival at least as good as that described 
in Appendices 2 and 3 is suitable as a test water. It should be of 
constant quality during the period of the test. The pH should remain within a range of ± 0,5 pH units. In order to ensure that the dilution water will not unduly influence the test result (for example by 
complexation of test substance), or adversely affect the performance 
of the brood stock, samples should be taken at intervals for analysis. Measurements of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd and Ni), 
major anions and cations (e.g. Ca, Mg, Na, K, Cl and SO
 4 ), 
pesticides (e.g. total organophosphorus and total organochlorine pesticides), total organic carbon and suspended solids should be made, for example, every three months, where a dilution water is 
known to be relatively constant in quality. If water quality has been 
demonstrated to be constant over at least one year, determinations can be less frequent and intervals extended (e.g. every six months).
 
1.6.6.  Test solutions  
Test solutions of the chosen concentrations are prepared by dilution 
of a stock solution. 
The stock solution should preferably be prepared by simply mixing 
or agitating the test substance in the dilution water by using mech ­
anical means (e.g. stirring and ultrasonication). Saturation columns (solubility columns) can be used for achieving a suitable concen ­
trated stock solution. As far as possible, the use of solvents or 
dispersants (solubilising agents) should be avoided; however, such 
compounds may be required in some cases in order to produce a suitably concentrated stock solution. Examples of suitable solvents are acetone, ethanol, methanol, dimethylformamide and triethyleneg ­
lycol. Examples of suitable dispersants are Cremophor RH40, Tween 
80, methylcellulose 0,01 % and HCO-40. Care should be taken when using readily biodegradable agents (e.g. acetone) and/or highly 
volatile as these can cause problems with bacterial built-up in 
flow-through tests. When a solubilising agent is used it must have no significant effect on survival nor visible adverse effect on the early-life stages as revealed by a solvent-only control. However, 
every effort should be made to avoid the use of such materials. 
For the semi-static technique, two different renewal procedures may 
be followed; either (i) new test solutions are prepared in clean vessels and surviving eggs and larvae gently transferred into the new vessels in a small volume of old solution, avoiding exposure 
to air, or (ii) the test organisms are retained in the vessels whilst a 
proportion (at least three-quarters) of the test water is changed. The frequency of medium renewal will depend on the stability of the test substance, but a daily water renewal is recommended. If, from 
preliminary stability tests (see Section 1.4), the test substance 
concentration is not stable (i.e. outside the range 80-120 % of nominal or falling below 80 % of the measured initial concentration) over the renewal period, consideration should be given to the use of 
a flow-through test. In any case, care should be taken to avoid 
stressing the larvae during the water renewal operation.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1024
 
For flow-through tests, a system which continually dispenses and 
dilutes a stock solution of the test substance (e.g. metering pump, 
proportional diluter, saturator system) is required to deliver a series of concentrations to the test chambers. The flow rates of stock solutions and dilution water should be checked at intervals, 
preferably daily, and should not vary by more than 10 % throughout 
the test. A flow rate equivalent to at least five test chamber volumes per 24 hours has been found suitable (2).
 
1.7. PROCEDURE 
Useful information on the performance of fish embryo and sac-fry 
toxicity tests is available in the literature, some examples of which are included in the literature section of this text (7)(8)(9).
 
1.7.1.  Conditions of exposure  
1.7.1.1.  Duration  
The test should start preferably within 30 minutes after the eggs have 
been fertilised. The embryos are immersed in the test solution before, or as soon as possible after, commencement of the blastodisc cleavage stage and in any case before the onset of the gastrula 
stage. For eggs obtained from commercial supplier, it may not be 
possible to start the test immediately after fertilisation. As the sensi ­
tivity of the test may be seriously influenced by delaying the start of the test, the test should be initiated within eight hours after fertili ­
sation. As larvae are not fed during the exposure period, the test 
should be terminated just before the yolk sac of any larvae in any of the test chambers has been completely absorbed or before mortalities by starvation start in controls. The duration will depend upon the 
species used. Some recommended durations are given in 
Appendices 2 and 3.
 
1.7.1.2.  Loading  
The number of fertilised eggs at the start of the test should be 
sufficient to meet statistical requirements. They should be randomly distributed among treatments, and at least 30 fertilised 
eggs, divided equally (or as equally as possible since it can be 
difficult to obtain equal batches when using some species) between at least three replicate test chambers, should be used per concen ­
tration. The loading rate (biomass per volume of test solution) should 
be low enough in order that a dissolved oxygen concentration of at 
least 60 % ASV can be maintained without aeration. For flow- through tests, a loading rate not exceeding 0,5 g/l per 24 hours and not exceeding 5 g/l of solution at any time has been recom ­
mended (2).
 
1.7.1.3.  Light and temperature  
The photoperiod and test water temperature should be appropriate for 
the test species (Appendix 2 and 3). For the purpose of temperature 
monitoring, it may be appropriate to use an additional test vessel.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1025
 
1.7.2.  Test concentrations  
Normally, five concentrations of the test substance spaced by a 
constant factor not exceeding 3,2 are required. The curve relating LC
 50 to period of exposure in the acute study should be considered 
when selecting the range of test concentrations. The use of fewer 
than five concentrations, for example in limit tests, and a narrower concentration interval may be appropriate in some circumstances. Justification should be provided if fewer than five concentrations 
are used. Concentrations of the substance higher than the 96 hour 
LC
 50 or 100 mg/l, whichever is the lower, need not be tested. 
Substances should not be tested above their solubility limit in the 
test water. 
When a solubilising agent is used to aid preparation of test solutions 
(see Section 1.6.6), its final concentration in the test vessels should 
not be greater than 0,1 ml/l and should be the same in all test vessels.
 
1.7.3.  Controls  
One dilution-water control (replicated as appropriate) and also, if 
relevant, one control containing the solubilising-agent (replicated as appropriate) should be run in addition to the test series.
 
1.7.4.  Frequency of analytical determinations and measurements  
During the test, the concentrations of the test substance are 
determined at regular intervals. 
In semi-static tests where the concentration of the test substance is 
expected to remain within ± 20 % of the nominal (i.e. within the range 80-120 %; see Section 1.4 and 1.6.6), it is recommended that, 
as a minimum, the highest and lowest test concentrations be analysed 
when freshly prepared and immediately prior to renewal on at least three occasions spaced evenly over the test (i.e. analyses should be made on a sample from the same solution — when freshly prepared 
and at renewal). 
For tests where the concentration of the test substance is not 
expected to remain within ± 20 % of nominal (on the basis of 
stability data of the substance), it is necessary to analyse all test concentrations, when freshly prepared and at renewal, but following the same regime (i.e. on at least three occasions spaced 
evenly over the test). Determination of test substance concentrations 
prior to renewal need only be performed on one replicate vessel at each test concentration. Determinations should be made no more than seven days apart. It is recommended that results be based on 
measured concentrations. However, if evidence is available to 
demonstrate that the concentration of the test substance in solution has been satisfactorily maintained within ± 20 % of the nominal or measured initial concentration throughout the test, then results can be 
based on nominal or measured initial values. 
For flow-through tests, a similar sampling regime to that described 
for semi-static tests is appropriate (but measurement of ‘old’ 
solutions is not applicable in this case). However, if the test duration is more than seven days, it may be advisable to increase the number of sampling occasions during the first week (e.g. three 
sets of measurements) to ensure that the test concentrations are 
remaining stable.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1026
 
Samples may need to be centrifuged or filtered (e.g. using a 0,45 μm 
pore size). However, since neither centrifuging nor filtration appears 
always to separate the non-bioavailable fraction of the test substance from that which is bioavailable, samples may not be subjected to those treatments. 
During the test, dissolved oxygen, pH and temperature should be 
measured in all test vessels. Total hardness and salinity (if relevant) 
should be measured in the controls and one vessel at the highest concentration. As a minimum, dissolved oxygen and salinity (if 
relevant) should be measured three times (at the beginning, middle 
and end of the test). In semi-static tests, it is recommended that dissolved oxygen be measured more frequently, preferably before and after each water renewal or at least once at week. pH should 
be measured at the beginning and end of each water renewal in semi- 
static test and at least weekly in flow-through tests. Hardness should be measured once each test. Temperature should be measured daily and it should preferably be monitored continuously in at least one 
test vessel.
 
1.7.5.  Observations  
1.7.5.1.  Stage of embryonic development  
The embryonic stage (i.e. gastrula stage) at the beginning of exposure 
to the test substance should be verified as precisely as possible. This can be done using a representative sample of eggs suitably preserved 
and cleared. The literature may also be consulted for the description 
and illustration of embryonic stages (2)(5)(10)(11).
 
1.7.5.2.  Hatching and survival  
Observations on hatching and survival should be made at least once 
daily and numbers recorded. It may be desirable to make more 
frequent observations at the beginning of the test (e.g. each 
30 minutes during the first three hours), since in some cases, survival times can be more relevant than only the number of deaths (e.g. when there are acute toxic effects). Dead embryos and 
larvae should be removed as soon as observed since they can 
decompose rapidly. Extreme care should be taken when removing dead individuals not to knock or physically damage adjacent eggs/ ­
larvae, these being extremely delicate and sensitive. Criteria for 
death vary according to life stage: 
— for eggs : particularly in the early stages, a marked loss of trans ­
lucency and change in colouration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance, 
— for embryos : absence of body movement and/or absence of heart 
beat and/or opaque discoloration in species whose embryos are 
normally translucent, 
— for larvae : immobility and/or absence of respiratory movement 
and/or absence of heart-beat and/or white opaque colouration of 
central nervous system and/or lack of reaction mechanical 
stimulus.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1027
 
1.7.5.3.  Abnormal appearance  
The number of larvae showing abnormality of body form and/or 
pigmentation, and the stage of yolk-sac absorption, should be recorded at adequate intervals depending on the duration of the 
test and the nature of the abnormality described. It should be 
noted that abnormal embryos and larvae occur naturally and can be of the order of several per cent in the control(s) in some species. Abnormal animals should only be removed from the test 
vessels on death.
 
1.7.5.4.  Abnormal behaviour  
Abnormalities, e.g. hyperventilation, uncoordinated swimming, and 
atypical quiescence should be recorded at adequate intervals depending on the duration of the test. These effects, although 
difficult to quantify, can, when observed, aid in the interpretation 
of mortality data, i.e. provide information on the mode of toxic action of the substance.
 
1.7.5.5.  Length  
At the end of the test, measurement of individual lengths is recom ­
mended; standard, fork or total length may be used. If however, 
caudal fin rot or fin erosion occurs, standard lengths should be 
used. Generally, in a well-run test, the coefficient of variation for length among replicates in the controls should be ≤ 20 %.
 
1.7.5.6.  Weight  
At the end of the test, individual weights can be measured; dry 
weights (24 hours at 60  o C) are preferable to wet weights (blotted 
dry). Generally, in a well-run test, the coefficient of variation for 
weight among replicates in the controls should be ≤ 20 %. 
These observations will result in some or all of the following data 
being available for statistical analysis: 
— cumulative mortality, 
— numbers of healthy larvae at end of test, — time to start of hatching and end of hatching (i.e. 90 % hatching 
in each replicate), 
— numbers of larvae hatching each day, 
— length (and weight) of surviving animals at end of the test, 
— numbers of larvae that are deformed or of abnormal appearance, — numbers of larvae exhibiting abnormal behaviour.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1028
 
2. DATA AND REPORTING  
2.1. TREATMENT OF RESULTS 
It is recommended that a statistician be involved in both the design 
and analysis of the test since the method allows for considerable 
variation in experimental design as, for example, in the number of 
test chambers, number of test concentrations, starting number of fertilised eggs and in the parameters measured. In view of the options available in test design, specific guidance on statistical 
procedures is not given here. 
If LOEC/NOECs are to be estimated, it will be necessary for vari ­
ations to be analysed within each set of replicates using analysis of 
variance (ANOVA) or contingency table procedures. In order to 
make a multiple comparison between the results at the individual concentrations and those for the controls, Dunnett's method may be found useful (12)(13). Other useful examples are also available 
(14)(15). The size of the effect detectable using ANOVA or other 
procedures (i.e. the power of the test) should be calculated and reported. It should be noted that not all the observations listed in Section 1.7.5.6 are suitable for statistical analysis using ANOVA. 
For example, cumulative mortality and numbers of healthy larvae at 
the end of the test could be analysed using probit methods. 
If LC/EC
 x s are to be estimated, (a) suitable curve(s), such as the 
logistic curve, should be fitted to the data of interest using a stat ­
istical method such as least squares or non-linear least squares. The curve(s) should be parameterised so that the LC/EC
 x of interest and 
its standard error can be estimated directly. This will greatly ease the 
calculation of the confidence limits around the LC/EC  x . Unless there 
are good reasons to prefer different confidence levels, two-sided 95 % confidence should be quoted. The fitting procedure should preferably provide a means for assessing the significance of the 
lack of fit. Graphical methods for fitting curves can be used. 
Regression analysis is suitable for all observations listed in Section 1.7.5.6.
 
2.2. INTERPRETATION OF RESULTS 
The results should be interpreted with caution where measured 
toxicant concentrations in test solutions occur at levels near the 
detection limit of the analytical method. The interpretation of results for concentrations above the water solubility of the substance should also be made with care.
 
2.3. THE TEST REPORT 
The test report must include the following information:  
2.3.1.  Test substance:  
— physical nature and relevant physical-chemical properties; 
— chemical identification data, including purity and analytical 
method for quantification of the tests substance where appro ­
priate.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1029
 
2.3.2.  Test species:  
— scientific name, strain, numbers of parental fish (i.e. how many 
females were used for providing the required numbers of eggs in the test), source and method of collection of the fertilised eggs 
and subsequent handling.
 
2.3.3.  Test conditions:  
— test procedure used (e.g. semi-static or flow-through, time period 
from fertilisation to start the test, loading, etc), 
— photoperiod(s), 
— test design (e.g. number of test chambers and replicates, number 
of embryos per replicate), 
— method of preparation of stock solutions and frequency of 
renewal (the solubilising agent and its concentration must be 
given, when used), 
— the nominal test concentrations, the measured values, their means 
and their standard deviations in the test vessels and the method 
by which these were attained and, if the test substance is soluble 
in water at concentrations below those tested, evidence should be provided that the measurements refer to the concentrations of the test substance in solution, 
— dilution water characteristics: pH, hardness, temperature, 
dissolved oxygen concentration, residual chlorine levels (if measured), total organic carbon, suspended solids, salinity of the test medium (if measured) and any other measurements made, 
— water quality within test vessels: pH, hardness, temperature and 
dissolved oxygen concentration.
 
2.3.4.  Results:  
— results from any preliminary studies on the stability of the test 
substance, 
— evidence that controls met the overall survival acceptability 
standard of the test species (Appendices 2 and 3), 
— data on mortality/survival at embryo and larval stages and overall 
mortality/survival, 
— days to hatch and numbers hatched, 
— data for length (and weight), — incidence and description of morphological abnormalities, if any, — incidence and description of behavioural effects, if any, 
— statistical analysis and treatment of data, 
— for tests analysed using ANOVA, the lowest observed effect 
concentration (LOEC) at p=0,05 and the no observed effect concentration (NOEC) for each response assessed, including a 
description of the statistical procedures used and an indication 
of what size of effect could be detected,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1030
 
— for tests analysed using regression techniques, the LC/ECx and 
confidence intervals and a graph of the fitted model used for its 
calculation, 
— explanation for any deviation from this testing method.  
3. REFERENCES  
(1) Kristensen P., (1990) Evaluation of the Sensitivity of Short 
Term Fish Early Life Stage Tests in Relation to other FELS 
Test Methods. Final report to the Commission of the European Communities, p. 60. June 1990. 
(2) ASTM (1988) Standard Guide for Conducting Early Life-Stage 
Toxicity Tests with Fishes. American Society for Testing and Materials. E 1241-88. p. 26 
(3) Brauhn J.L. and Schoettger R.A., (1975) Acquisition and 
Culture of Research Fish: Rainbow trout, Fathead minnows, 
Channel Catfish and Bluegills. p. 54, Ecological Research 
Series, EPA-660/3-75-011, Duluth, Minnesota. 
(4) Brungs W.A. and Jones B.R., (1977) Temperature Criteria for 
Freshwater Fish: Protocol and Procedures p. 128, Ecological Research Series EPA-600/3-77-061, Duluth, Minnesota. 
(5) Laale H.W., (1977) The Biology and Use of the Zebrafish 
(Brachydanio rerio ) in Fisheries Research. A Literature 
Review. J. Biol. 10, p. 121-173. 
(6) Legault R. (1958) A Technique for Controlling the Time of 
Daily Spawning and Collecting Eggs of the Zebrafish,  
Brachydanio rerio  (Hamilton-Buchanan) Copeia, 4, 
p. 328-330. 
(7) Dave G., Damgaard B., Grande M., Martelin J.E., Rosander B. 
and Viktor T., (1987) Ring Test of an Embryo-larval Toxicity Test with Zebrafish ( Brachydanio rerio ) Using Chromium and 
Zinc as Toxicants. Environmental Toxicology and Chemistry, 
6, p. 61-71. 
(8) Birge J.W., Black J.A. and Westerman A.G., (1985) Short-term 
Fish and Amphibian Embryo-larval Tests for Determining the Effects of Toxicant Stress on Early Life Stages and Estimating 
Chronic Values for Single Compounds and Complex Effluents. 
Environmental Toxicology and Chemistry 4, p. 807-821. 
(9) Van Leeuwen C.J., Espeldoorn A. and Mol F. (1986) Aquatic 
Toxicological Aspects of Dithiocarbamates and Related Compounds. III. Embryolarval Studies with Rainbow Trout (Salmo gairdneri ). J. Aquatic Toxicology, 9, p. 129-145. 
(10) Kirchen R.V. and W. R. West (1969) Teleostean Development. 
Carolina Tips 32(4): 1-4. Carolina Biological Supply 
Company. 
(11) Kirchen R.V. and W. R. West (1976) The Japanese Medaka. 
Its care and Development. Carolina Biological Supply 
Company, North Carolina. p. 36. 
(12) Dunnett C.W., (1955) A Multiple Comparisons Procedure for 
Comparing Several Treatments with Control. J. Amer. Statist. Assoc., 50, p. 1096-1121. 
(13) Dunnett C.W. (1964) New Tables for Multiple Comparisons 
with a Control. Biometrics, 20, p. 482-491. 
(14) Mc Clave J.T., Sullivan J.H. and Pearson J.G., (1980) Stat ­
istical Analysis of Fish Chronic Toxicity Test Data. 
Proceedings of 4th Aquatic Toxicology Symposium, ASTM, 
Philadelphia.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1031
 
(15) Van Leeuwen C.J., Adema D.M.M. and Hermes J., (1990) 
Quantitative Structure-Activity Relationships for Fish Early 
Life Stage Toxicity. Aquatic Toxicology, 16, p. 321-334. 
(16) Environment Canada., (1992) Toxicity Tests Using Early Life 
Stages of Salmonid Fish (Rainbow Trout, Coho Salmon or 
Atlantic Salmon). Biological Test Method Series. Report EPS 
1/RM/28, December 1992, p. 81. 
(17) Dave G. and Xiu R., (1991) Toxicity of Mercury, Nickel, Lead 
and Cobalt to Embryos and Larvae of Zebrafish,  Brachydanio 
rerio . Arch. of Environmental Contamination and Toxicology, 
21, p. 126-134. 
(18) Meyer A., Bierman C.H. and Orti G., (1993) The phylogenetic 
position of the Zebrafish ( Danio rerio ), a model system in 
developmental biology — an invitation to the comperative methods. Proc. Royal Society of London, Series B, 252: 231-236. 
(19) Ghillebaert F., Chaillou C., Deschamps F. and Roubaud P., 
(1995) Toxic Effects, at Three pH Levels, of Two Reference Molecules on Common Carp Embryo. Ecotoxicology and Environmental Safety 32, p. 19-28. 
(20) US EPA, (1991) Guidelines for Culturing the Japanese 
Medaka,  Oryzias latipes . EPA report EPA/600/3-91/064, 
Dec. 1991, EPA, Duluth. 
(21) US EPA, (1991) Guidelines for Conducting Early Life Stage 
Toxicity Tests with Japanese Medaka, ( Oryzias latipes ). EPA 
report EPA/600/3-91/063, Dec. 1991, EPA, Duluth. 
(22) De Graeve G.M., Cooney J.D., McIntyre D.O., Poccocic T.L., 
Reichenbach N.G., Dean J.H. and Marcus M.D., 
(1991) Validity in the performance of the seven-day Fathead minnow ( Pimephales promelas ) larval survival and growth 
test: an intra- and interlaboratory study. Environ. Tox. Chem. 
10, p. 1189-1203. 
(23) Calow P., (1993) Handbook of Ecotoxicology, Blackwells, 
Oxford. Vol. 1, chapter 10: Methods for spawning, culturing and conducting toxicity tests with Early Life stages of 
Estuarine and Marine fish. 
(24) Balon E.K., (1985) Early life history of fishes: New devel ­
opmental, ecological and evolutionary perspectives, Junk 
Publ., Dordrecht, p. 280. 
(25) Blaxter J.H.S., (1988) Pattern and variety in development, In: 
W.S. Hoar and D.J. Randall Eds., Fish Physiology, vol. XIA, Academic press, p. 1-58.
 
Table 1A  
Fish species recommended for testing  
FRESHWATER  
Oncorhynchus mykiss  
Rainbow trout (9)(16)  
Danio rerio  
Zebrafish (7)(17)(18)  
Cyprinus caprio  
Common carp (8)(19)  
Oryzias latipes  
Japanese ricefish/Medaka (20)(21)  
Pimephales promelas  
Fathead minnow (8)(22)  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1032
 
Table 1B  
Examples of other well-documented species which have also been used  
FRESHWATER SALTWATER  
Carassius auratus  
Goldfish (8)  Menidia peninsulae  
Tidewater silverside (23)(24)(25)  
Lepomis macrochirus  
Bluegill (8)  Clupea harengus  
Herring (24)(25)  
Gadus morhua  
Cod (24)(25)  
Cyprinodon variegatus  
Sheepshead minnow (23)(24)(25)  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1033
 
Appendix 1  
GUIDANCE ON PERFORMANCE OF A TOXICITY TEST ON 
EMBRYOS AND SAC-FRY OF ZEBRAFISH  (BRACHYDANIO RERIO)  
INTRODUCTION 
The zebrafish originates from the Coromandel coast of India where it inhabits 
fast-flowing streams. It is a common aquarium fish of the carp family, and 
information about procedures for its care and culture can be found in standard reference books on tropical fish. Its biology and use in fishery research have been reviewed by Laale (1). 
The fish rarely exceeds 45 mm in length. The body is cylindrical with 7-9 dark- 
blue horizontal silvery stripes. These stripes run into the caudal and anal fins. The back is olive-green. Males are slimmer than females. Females are more 
silvery and the abdomen is distended, particularly prior to spawning. 
Adult fishes are able to tolerate large fluctuations in temperature, pH and 
hardness. However, in order to get healthy fish which produce eggs of good 
quality, optimal conditions should be provided. 
During spawning the male pursues and butts the female, and as the eggs are 
expelled they are fertilised. The eggs, which are transparent and non-adhesive, 
fall to the bottom where they may be eaten by the parents. Spawning is influenced by light. If the morning light is adequate, the fish usually spawns in the early hours following daybreak. 
A female can produce batches of several hundreds of eggs at weekly intervals.
 
CONDITIONS OF PARENTAL FISH, REPRODUCTION AND EARLY-LIFE 
STAGES 
Select a suitable number of healthy fish and keep these in suitable water (e.g. 
Annex 4) for at least two weeks prior to the intended spawning. The group of 
fish should be allowed to breed at least once before producing the batch of eggs 
used in the test. The density of fish during this period should not exceed 1 gram of fish per litre. Regular changes of water or the use of purification systems will enable the density to be higher. The temperature in the holding tanks should be 
maintained at 25 ± 2
 o C. The fish should be provided with a varied diet, which 
may consist of, for example, appropriate commercial dry food, live newly hatched Arthemia, chironomids, Daphnia, white worms (Enchytraeids). 
Two procedures are outlined below, which in practice have led to a sufficient 
batch of healthy, fertilised eggs for a test to be run: 
i) Eight females and 16 males are placed in a tank containing 50 litres of 
dilution water, shielded from direct light and left as undisturbed as possible for at least 48 hours. A spawning tray is placed at the bottom of the aquarium in the afternoon the day before start of the test. The spawning tray consists of 
a frame (plexi-glass or other suitable material), 5-7 cm high with a 2-5 mm 
coarse net attached at the top and a 10-30 μm fine net at the bottom. A 
number of ‘spawning-trees’, consisting of untwisted nylon rope, are attached to the coarse net of the frame. After the fish have been left in 
dark for 12 hours, a faint light is turned on which will initiate the 
spawning. Two to four hours after spawning, the spawning tray is removed and the eggs collected. The spawning tray will prevent the fish from eating the eggs and at the same time permit an easy collection of the eggs. The 
group of fish should have spawned at least once before the spawning from 
which eggs are used for testing.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1034
 
ii) Five to 10 male and female fish are housed individually at least two weeks 
prior to the intended spawning. After 5-10 days, the abdomens of the females 
will be distended and their genital papillae visible. Male fish lack papillae. Spawning is performed in spawning tanks equipped with a false mesh bottom (as above). The tank is filled with dilution water, so that the depth of water 
above the mesh is 5-10 cm. One female and two males are placed in the tank 
the day before the intended spawning. The water temperature is gradually increased one degree higher than the acclimatisation temperature. The light is turned off and the tank is left as undisturbed as possible. In the morning a 
faint light is turned on which will initiate spawning. After two to four hours, 
the fish are removed and the eggs collected. If larger batches of eggs are needed than can be obtained from one female, a sufficient number of spawning tanks may be set-up in parallel. By recording the reproduction 
success of the individual females prior to the test (size of batch and 
quality), those females with highest reproduction success may be selected for breeding. 
The eggs should be transferred to the test vessels by means of glass tubes (inner 
diameter not less than 4 mm) provided with a flexible suction bulb. The amount of water accompanying the eggs on their transfer should be as small as possible. The eggs are heavier than water and sink out of the tube. Care should be taken to 
prevent eggs (and larvae) coming into contact with the air. Microscopic exam ­
ination of sample(s) of the batch(es) should be carried out to ensure that there are no irregularities in the first developmental stages. Disinfection of the eggs is not allowed. 
The mortality rate of the eggs is highest within the first 24 hours after fertili ­
sation. A mortality of 5-40 % is often seen during this period. Eggs degenerate as a result of unsuccessful fertilisation or development failures. The quality of the batch of eggs seems to depend on the female fish, as some females consistently 
produce good quality eggs, others never will. Also the development rate and the 
rate of hatching vary from one batch to another. The successfully fertilised eggs and the yolk sac larvae survive well, normally above 90 %. At 25
 o C the eggs 
will hatch three-five days after fertilisation and the yolk sac will be absorbed 
approximately 13 days after fertilization. 
The embryonic development has been well defined by Hisaoka and Battle (2). 
Due to the transparency of the eggs and post-hatch larvae, the development of 
the fish may be followed and the presence of malformations may be observed. 
Approximately four hours after spawning, the non-fertilized eggs may be distin ­
guished from the fertilized (3). For this examination, eggs and larvae are placed in test vessels of small volume and studied under a microscope. 
The test conditions, which apply to the early life stages, are listed in 
Appendix 2. Optimal values for pH values and hardness of the dilution water are 7,8 and 250 mg CaCO
 3 /l respectively.  
CALCULATIONS AND STATISTICS 
A two-stage approach is proposed. First, the data on mortality, abnormal devel ­
opment and hatching-time are analysed statistically. Then, for those concen ­
trations at which no adverse effects on any of these parameters have been 
detected, the body length is statistically evaluated. This approach is advisable since the toxicant may selectively kill smaller fish, delay hatching-time and induce gross malformations, thus leading to biased length measurements. 
Furthermore, there will be roughly the same number of fish to be measured 
per treatment, ensuring the validity of the test statistics.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1035
 
LC 50 AND EC  50 DETERMINATIONS 
The percentage of surviving eggs and larvae is calculated and corrected for 
mortality in the controls in accordance with Abbott’s formula (4):  
P ¼ 100 Ä C Ä P 0 
C Ü 100 Í Î  
where: 
P = corrected % survival P' = % survival observed in the test concentration C = % survival in the control 
If possible, the LC
 50 is determined by a suitable method at the end of the test. 
If the inclusion of morphological abnormalities in the EC  50 statistic is desired, 
guidance can be found in Stephan (5).  
ESTIMATION OF LOEC AND NOEC 
An objective of the egg and sac-fry test is to compare the non-zero concen ­
trations with the control, i.e. to determine the LOEC. Therefore multiple 
comparison procedures should be utilised (6)(7)(8)(9)(10).  
REFERENCES 
(1) Laale H.W., (1977) The Biology and Use of the Zebrafish ( Brachydanio 
rerio ) in Fisheries Research. A Literature Review. J. Fish Biol. 10, 
p. 121-173. 
(2) Hisaoka K.K. and Battle H.I., (1958). The Normal Development Stages of 
the Zebrafish  Brachydanio rerio  (Hamilton-Buchanan) J. Morph., 102, p. 
311. 
(3) Nagel R., (1986). Untersuchungen zur Eiproduktion beim Zebrabärbling 
(Brachydanio rerio  Hamilton-Buchanan). Journal of Applied Ichthyology, 
2, p. 173-181. 
(4) Finney D.J., (1971). Probit Analysis, 3rd ed., Cambridge University Press, 
Great Britain, p. 1-333. 
(5) Stephan C.E., (1982). Increasing the Usefulness of Acute Toxicity Tests. 
Aquatic Toxicology and Hazard Assessment: Fifth Conference, ASTM STP 766, J.G. Pearson, R.B. Foster and W.E. Bishop, Eds., American Society for Testing and Materials, p. 69-81. 
(6) Dunnett C.W. (1955). A Multiple Comparisons Procedure for Comparing 
Several Treatments with a Control. J. Amer. Statist. Assoc., 50, p. 1096 ­
-1121. 
(7) Dunnett C.W., (1964) New Tables for Multiple Comparisons with a 
Control. Biometrics, 20, p. 482-491. 
(8) Williams D.A., (1971). A Test for Differences Between Treatment Means 
when Several Dose Levels are Compared with a Zero Dose Control. 
Biometrics, 27, p. 103-117. 
(9) Williams D.A., (1972). The Comparison of Several Dose Levels with a 
Zero Dose Control. Biometrics 28, p. 519-531. 
(10) Sokal R.R. and Rohlf F.J., (1981). Biometry, the Principles and Practice of 
Statistics in Biological Research, W.H. Freeman and Co., San Francisco.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1036
 2008R0440 — EN — 22.03.2014 — 004.001 — 1037
 ▼B 
Appendix 2  
TEST CONDITIONS, DURATION AND SURVIVAL CRITERIA FOR RECOMMENDED SPECIES  
Species Temp 
( 0 C) Salinity 
(0/00) Photo-period 
(hrs) Duration of stages 
(days) Typical duration of test Survival of control, 
(minimum %) 
Embryo Sac-fry Hatching success Post-hatch  
FRESHWATER  
Brachydanio rerio  
Zebrafish 25 ± 1 — 12-16 3-5 8-10 As soon as possible after fertilisation 
(early gastrula stage) to 5 days post-hatch 
(8-10 days) 80 90  
Oncorhynchus mykiss  
Rainbow trout 10 ± 1 (  1 ) 
12 ± 1 (  2 ) — 0 (  a ) 30-35 25-30 As soon as possible after fertilisation 
(early gastrula stage) to 20 days post- hatch (50-55 days) 66 70  
Cyprinus carpio  
Common carp 21-25 — 12-16 5 > 4 As soon as possible after fertilisation 
(early gastrula stage) to 4 days post-hatch 
(8-9 days) 80 75  
Oryzias latipes  
Japanese ricefish/Medaka 24 ± 1 (
 1 ) 
23 ± 1 (  2 ) — 12-16 8-11 4-8 As soon as possible after fertilisation 
(early gastrula stage) to 5 days post-hatch (13-16 days) 80 80  
Pimephales promelas  
Fathead minnow 25 ± 2 — 16 4-5 5 As soon as possible after fertilisation 
(early gastrula stage) to 4 days post-hatch (8-9 days) 60 70
 
( 1 ) For embryos. 
( 2 ) For larvae. 
( a ) Darkness for embryo and larvae until one week after hatching except when they are being inspected. Then subdued lighting thro ughout the test.
 2008R0440 — EN — 22.03.2014 — 004.001 — 1038
 ▼B 
Appendix 3  
Test conditions, duration and survival criteria for other well documented species  
Species Temp (  o C) Salinity (0/00) Photo-period 
(hrs) Duration of stages (days) 
Typical duration of embryo and sac-fry test Survival of control (minimum %) 
Embryo Sac-fry test Hatching success Post-hatch  
FRESHWATER  
Carassius auratus  
Goldfish 24 ± 1 — — 3-4 > 4 As soon as possible after fertilisation 
(early gastrula stage) to 4 days post-hatch (7 days) — 80  
Leopomis macrochirus  
Blugill sunfish 21 ± 1 — 16 3 > 4 As soon as possible after fertilisation 
(early gastrula stage) to 4 days post-hatch 
(7 days) — 75 
SALTWATER  
Menidia peninsulae  
Tidewater silverside 22-25 15-22 12 1,5 10 As soon as possible after fertilisation 
(early gastrula stage) to 5 days post-hatch 
(6-7 days) 80 60  
Clupea harengus  
Herring 10 ± 1 8-15 12 20-25 3-5 As soon as possible after fertilisation 
(early gastrula stage) to 3 days post-hatch (23-27 days) 60 80  
Gadus morhua  
Cod 5 ± 1 5-30 12 14-16 3-5 As soon as possible after fertilisation 
(early gastrula stage) to 3 days post-hatch 
(18 days) 60 80  
Cyprinodon variegatus  
Sheepshead minnow 25 ± 1 15-30 12 — — As soon as possible after fertilisation 
(early gastrula stage) to 4/7 days post- hatch (28 days) > 75 80
Appendix 4  
SOME CHEMICAL CHARACTERISTICS OF AN ACCEPTABLE 
DILUTION WATER  
Substance Concentrations  
Particulate matter < 20 mg/l 
Total organic carbon < 2 mg/l 
Unionised ammonia < 1 μg/l 
Residual chlorine < 10 μg/l 
Total organophosphorus pesticides < 50 ng/l Total organochlorine pesticides plus polychlorinated 
biphenyls < 50 ng/l 
Total organic chlorine < 25 ng/l
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1039
 
C.16.  HONEYBEES — ACUTE ORAL TOXICITY TEST  
1. METHOD  
This acute toxicity test method is a replicate of the OECD TG 213 
(1998).  
1.1. INTRODUCTION 
This toxicity test is a laboratory method, designed to assess the oral 
acute toxicity of plant protection products and other chemicals, to adult worker honeybees. 
In the assessment and evaluation of toxic characteristics of 
substances, determination of acute oral toxicity in honeybees may 
be required, e.g. when exposure of bees to a given chemical is likely. The acute oral toxicity test is carried out to determine the inherent toxicity of pesticides and other chemicals to bees. The results of this 
test should be used to define the need for further evaluation. In 
particular, this method can be used in step-wise programmes for evaluating the hazards of pesticides to bees, based on sequential 
progression from laboratory toxicity tests to semi-field and field 
experiments (1). Pesticides can be tested as active substances (a.s.) or as formulated products. 
A toxic standard should be used to verify the sensitivity of the bees 
and the precision of the test procedure.
 
1.2. DEFINITIONS  
Acute oral toxicity : is the adverse effects occurring within a 
maximum period of 96h of an oral administration of a single dose of test substance.  
Dose : is the amount of test substance consumed. Dose is expressed 
as mass ( μg) of test substance per test animal ( μg/bee). The real dose 
for each bee can not be calculated as the bees are fed collectively, but an average dose can be estimated (totally consumed test 
substance/number of test bees in one cage).  
LD
 50 (Median Lethal Dose) oral : is a statistically derived single 
dose of a substance that can cause death in 50 % of animals when administered by the oral route. The LD
 50 value is expressed in μg of 
test substance per bee. For pesticides, the test substance may be 
either an active substance (a.s.) or a formulated product containing one or more than one active substance.  
Mortality : an animal is recorded as dead when it is completely 
immobile.
 
1.3. PRINCIPLE OF THE TEST METHOD 
Adult worker honeybees ( Apis mellifera ) are exposed to a range of 
doses of the test substance dispersed in sucrose solution. The bees are then fed the same diet, free of the test substance. Mortality is 
recorded daily during at least 48 h and compared with control 
values. If the mortality rate is increasing between 24 h and 48 h whilst control mortality remains at an accepted level, i.e. ≤ 10 %, it 
is appropriate to extend the duration of the test to a maximum of 96 
h. The results are analysed in order to calculate the LD
 50 at 24 h and 
48 h and, in case the study is prolonged, at 72 h and 96 h.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1040
 
1.4. VALIDITY OF THE TEST 
For a test to be valid, the following conditions apply: 
— the average mortality for the total number of controls must not 
exceed 10 % at the end of the test, 
— the LD  50 of the toxic standard meets the specified range.  
1.5. DESCRIPTION OF THE TEST METHOD  
1.5.1.  Collection of bees  
Young adult worker bees of the same race should be used, i.e. bees 
of the same age, feeding status, etc. Bees should be obtained from 
adequately fed, healthy, as far as possible disease-free and queen- 
right colonies with known history and physiological status. They could be collected in the morning of use or in the evening before test and kept under test conditions to the next day. Bees collected 
from frames without brood are suitable. Collection in early spring or 
late autumn should be avoided as the bees have a changed physiology during this time. If tests must be conduced in early spring or late autumn, bees can be emerged in an incubator and 
reared for one week with ‘bee bread’ (pollen collected from the 
comb) and sucrose solution. Bees treated with chemical substances, such as antibiotics, anti-varroa products, etc., should not be used for toxicity test for four weeks from the time of the end of the last 
treatment.
 
1.5.2.  Housing and feeding conditions  
Easy to clean and well-ventilated cages are used. Any appropriate 
material can be used, e.g. stainless steel, wire mesh, plastic or disposable wooden cages, etc. Groups of 10 bees per cage are 
preferred. The size of test cages should be appropriate to the 
number of bees, i.e. providing adequate space. 
The bees should be held in the dark in an experimental room at a 
temperature of 25 ± 2
 o C. The relative humidity, normally around 
50-70 %, should be recorded throughout the test. Handling 
procedures, including treatment and observations may be conducted under (day) light. Sucrose solution in water with a final concentration of 500 g/l (50 % w/v) is used as food. After given test 
doses, food should be provided  ad libitum . The feeding system 
should allow recording food intake for each cage (see Section 1.6.3.1). A glass tube (approximately 50 mm long and 10 mm wide with the open end narrowed to about 2 mm 
diameter) can be used.
 
1.5.3.  Preparation of bees  
The collected bees are randomly allocated to test cages, which are 
randomly placed in the experimental room.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1041
 
The bees may be starved for up to 2 h before the initiation of the 
test. It is recommended that the bees are deprived of food prior to 
treatment so that all bees are equal in terms of their gut contents at the start of the test. Moribund bees should be rejected and replaced by healthy bees before starting the test.
 
1.5.4.  Preparation of doses  
Where the test substance is a water miscible compound this may be 
dispersed directly in 50 % sucrose solution. For technical products and substances of low water solubility, vehicles such as organic solvent, emulsifiers or dispersants of low toxicity to bees may be 
used (e.g. acetone, dimethylformamide, dimethylsulfoxide). The 
concentration of the vehicle depends on the solubility of the test substance and it should be the same for all concentrations tested. However, a concentration of the vehicle of 1 % is generally appro ­
priate and should not be exceeded. 
Appropriate control solutions should be prepared, i.e. where a 
solvent or a dispersant is used to solubilise the test substance, two 
separate control groups should be used: a solution in water, and a sucrose solution with the solvent/carrier at the concentration used in dosing solutions.
 
1.6. PROCEDURE  
1.6.1.  Test and control groups  
The number of doses and replicates tested should meet the statistical 
requirements for determination of LD  50 with 95 % confidence limits. 
Normally, five doses in a geometric series, with a factor not 
exceeding 2,2, and covering the range for LD  50 , are required for 
the test. However, the dilution factor and the number of concen ­
trations for dosage have to be determined in relation to the slope of the toxicity curve (dose versus mortality) and with consideration 
taken to the statistical method which is chosen for analysis of the 
results. A range-finding test enables the choice of the appropriate concentrations for dosage. 
A minimum of three replicate test groups, each of 10 bees, should be 
dosed with each test concentration. A minimum of three control batches, each of 10 bees, should be run in addition to the test 
series. Control batches should also be included for the solvents/ ­
carriers used (see Section 1.5.4).
 
1.6.2.  Toxic standard  
A toxic standard should be included in the test series. At least three 
doses should be selected to cover the expected LD  50 value. A 
minimum of three replicate cages, each containing 10 bees, should 
be used with each test dose. The preferred toxic standard is dime ­
thoate, for which the reported oral LD  50 -24 h is in the range 0,10- 
0,35 μg a.s./bee (2). However, other toxic standards would be 
acceptable where sufficient data can be provided to verify the 
expected dose response (e.g. parathion).  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1042
 
1.6.3.  Exposure  
1.6.3.1.  Administration of doses  
Each test group of bees must be provided with 100-200 μl of 50 % 
sucrose solution in water, containing the test substance at the appro ­
priate concentration. A larger volume is required for products of low 
solubility, low toxicity or low concentration in the formulation, as higher proportions in the sucrose solution have to be used. The amount of treated diet consumed per group should be monitored. 
Once consumed (usually within 3-4 h), the feeder should be 
removed from the cage and replaced with one containing sucrose solution alone. The sucrose solutions are then provided  ad libitum . 
For some compounds, at higher concentrations rejection of test dose 
may result in little or no food being consumed. After a maximum of 6 h, unconsumed treated diet should be replaced with the sucrose solution alone. The amount of treated diet consumed should be 
assessed (e.g. measurement of volume/weight of treated diet 
remaining).
 
1.6.3.2.  Duration  
The duration of the test is preferably 48 h after the test solution has 
been replaced with sucrose solution alone. If mortality continues to rise by more than 10 % after the first 24 h, the test duration should 
be extended to a maximum of 96 h provided that control mortality 
does not exceed 10 %.
 
1.6.4.  Observations  
Mortality is recorded at 4 h after starting the test and thereafter at 24 
h and 48 h (i.e. after giving dose). If a prolonged observation period is required, further assessments should be made at 24 h intervals, up 
to a maximum of 96 h, provided that the control mortality does not 
exceed 10 %. 
The amount of diet consumed per group should be estimated. 
Comparison of the rates of consumption of treated and untreated 
diet within the given 6 h can provide information about palatability 
of the treated diet. 
All abnormal behavioural effects observed during the testing period 
should be recorded.
 
1.6.5.  Limit test  
In some cases (e.g. when a test substance is expected to be of low 
toxicity) a limit test may be performed, using 100 μg a.s./bee in 
order to demonstrate that the LD  50 is greater than this value. The 
same procedure should be used, including three replicate test groups for the test dose, the relevant controls, the assessment of the amount of treated diet consumed, and the use of the toxic standard. If 
mortalities occur, a full study should be conducted. If sublethal 
effects are observed (see Section 1.6.4), these should be recorded.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1043
 
2. DATA AND REPORTING  
2.1. DATA 
Data should be summarised in tabular form, showing for each 
treatment group, as well as control and toxic standard groups, the 
number of bees used, mortality at each observation time and number 
of bees with adverse behaviour. Analyse the mortality data by appro ­
priate statistical methods (e.g. probit analysis, moving-average, binomial probability) (3)(4). Plot dose-response curves at each 
recommended observation time and calculate the slopes of the 
curves and the median lethal doses (LD
 50 ) with 95 % confidence 
limits. Corrections for control mortality could be made using 
Abbott's correction (4)(5). Where treated diet is not completely 
consumed, the dose of test substance consumed per group should be determined. LD
 50 should be expressed in μg of test substance per 
bee. 
2.2. TEST REPORT 
The test report must include the following information:  
2.2.1.  Test substance:  
— physical nature and relevant physical-chemical properties (e.g. 
stability in water, vapour pressure), 
— chemical identification data, including structural formula, purity 
(i.e. for pesticides, the identity and concentration of active 
substance(s)).  
2.2.2.  Test species:  
— scientific name, race, approximate age (in weeks), collection 
method, date of collection, 
— information on colonies used for collection of test bees including 
health, any adult disease, any pre-treatment, etc.  
2.2.3.  Test conditions:  
— temperature and relative humidity of experimental room, 
— housing conditions including type, size and material of cages, 
— methods of preparation of stock and test solutions (the solvent 
and its concentration must be given, when used), 
— test design, e.g. number and test concentrations used, number of 
controls; for each test concentration and control, number of replicate cages and number of bees per cage, 
— date of test.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1044
 
2.2.4.  Results:  
— results of preliminary range-finding study if performed, 
— raw data: mortality at each dose tested at each observation time, — graph of the dose-response curves at the end of the test, — LD
 50 values with 95 % confidence limits, at each of the recom ­
mended observation times, for test substance and toxic standard; 
— statistical procedures used for determining the LD  50 , 
— mortality in controls, — other biological effects observed or measured e.g. abnormal 
behaviour of the bees (including rejection of the test dose), rate of consumption of diet in treated and untreated groups, 
— any deviation from the test procedures described here and any 
other relevant information.
 
3. REFERENCES  
(1) EPPO/Council of Europe (1993) Decision-Making Scheme for 
the Environmental Risk Assessment of Plant Protection Products — Honeybees. EPPO bulletin, vol. 23, N.1, 151-165. March 1993. 
(2) Gough, H. J., McIndoe, E.C., Lewis, G.B., (1994) The use of 
dimethoate as a reference compound in laboratory acute toxicity 
tests on honeybees ( Apis mellifera  L.) 1981-1992. Journal of 
Apicultural Research, 22, p. 119-125. 
(3) Litchfield, J.T. and Wilcoxon, F., (1949) A simplified method 
of evaluating dose-effect experiments. Jour. Pharmacol. and 
Exper. Ther., 96, p. 99-113. 
(4) Finney, D.J., (1971) Probit Analysis. 3rd ed., Cambridge, 
London and New-York. 
(5) Abbott, W.S., (1925) A method for computing the effectiveness 
of an insecticide. Jour. Econ. Entomol., 18, p. 265-267.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1045
 
C.17.  HONEYBEES — ACUTE CONTACT TOXICITY TEST  
1. METHOD  
This acute toxicity test method is a replicate of the OECD TG 214 
(1998).  
1.1. INTRODUCTION 
This toxicity test is a laboratory method, designed to assess the acute 
contact toxicity of plant protection products and other chemicals to 
adult worker honeybees. 
In the assessment and evaluation of toxic characteristics of 
substances, determination of acute contact toxicity in honeybees 
may be required, e.g. when exposure of bees to a given chemical is likely. The acute contact toxicity test is carried out to determine the inherent toxicity of pesticides and other chemicals to bees. The 
results of this test should be used to define the need for further 
evaluation. In particular, this method can be used in step-wise programmes for evaluating the hazards of pesticides to bees, based on sequential progression from laboratory toxicity tests to semi-field 
and field experiments (1). Pesticides can be tested as active 
substances (a.s.) or as formulated products. 
A toxic standard should be used to verify the sensitivity of the bees 
and the precision of the test procedure.
 
1.2. DEFINITIONS  
Acute contact toxicity : is the adverse effects occurring within a 
maximum period of 96 h of a topical application of a single dose of a substance.  
Dose : is the amount of test substance applied. Dose is expressed as 
mass (μg) of test substance per test animal ( μg/bee).  
LD
 50 (Median Lethal Dose) contact : is a statistically derived single 
dose of a substance that can cause death in 50 % of animals when administered by the contact. The LD
 50 value is given in μg of test 
substance per bee. For pesticides, the test substance may be either an 
active substance (a.s.) or a formulated product containing one or more than one active substance.  
Mortality : an animal is recorded as dead when it is completely 
immobile.
 
1.3. PRINCIPLE OF THE TEST METHOD 
Adult worker honeybees ( Apis mellifera ) are exposed to a range of 
doses of the test substance dissolved in appropriate carrier, by direct application to the thorax (droplets). The test duration is 48 h. If the 
mortality rate is increasing between 24 h and 48 h whilst control 
mortality remains at an accepted level, i.e. < 10 %, it is appropriate to extend the duration of the test to a maximum of 96 h. Mortality is recorded daily and compared with control values. The results are 
analysed in order to calculate the LD
 50 at 24 h and 48 h, and in 
case the study is prolonged at 72 h and 96 h.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1046
 
1.4. VALIDITY OF THE TEST 
For a test to be valid, the following conditions apply: 
— the average mortality for the total numbers of controls must not 
exceed 10 % at the end of the test, 
— the LD  50 of the toxic standard meets the specified range.  
1.5. DESCRIPTION OF THE TEST METHOD  
1.5.1.  Collection of bees  
Young adult worker bees should be used, i.e. bees of the same age, 
feeding status, race etc. Bees should be obtained from adequately fed, healthy, as far as possible disease-free and queen-right colonies with known history and physiological status. They could be collected 
in the morning of use or in the evening before test and kept under 
test conditions to the next day. Bees collected from frames without brood are suitable. Collection in early spring or late autumn should be avoided, as the bees have a changed physiology during the time. 
If tests have to be conduced in early spring or late autumn, bees can 
be emerged in an incubator and reared for one week with ‘bee bread’ (pollen collected from the comb) and sucrose solution. Bees treated with chemical substances, such as antibiotics, anti-varroa products, 
etc., should not be used for toxicity test for four weeks from the time 
of the end of the last treatment.
 
1.5.2.  Housing and feeding conditions  
Easy to clean and well-ventilated cages are used. Any appropriate 
material can be used, e.g. stainless steel, wire mesh, plastic, disposable wooden cages, etc. The size of test cages should be 
appropriate to the number of bees, i.e. providing adequate space. 
Groups of 10 bees per cage are preferred. 
The bees should be held in the dark in an experimental room at a 
temperature of 25 ± 2
 ο C. The relative humidity, normally around 
50-70 %, should be recorded throughout the test. Handling 
procedures, including treatment and observations may be 
conducted under (day) light. Sucrose solution in water with a final concentration of 500 g/l (50 % w/v) should be used as food and 
provided  ad libitum  during the test time, using a bee feeder. This can 
be a glass tube (approximately 50 mm long and 10 mm wide with the open end narrowed to about 2 mm diameter).
 
1.5.3.  Preparation of bees  
The collected bees may be anaesthetised with carbon dioxide or 
nitrogen for application of the test substance. The amount of anaes ­
thetic used and time of exposure should be minimised. Moribund 
bees should be rejected and replaced by healthy bees before starting 
the test.  
1.5.4.  Preparation of doses  
The test substance is to be applied as solution in a carrier, i.e. an 
organic solvent or a water solution with a wetting agent. As organic solvent, acetone is preferred but other organic solvents of low 
toxicity to bees may be used (e.g. dimethylformamide, dimethylsulf ­
oxide). For water dispersed formulated products and highly polar organic substances not soluble in organic carrier solvents, solutions may be easier to apply if prepared in a weak solution of a 
commercial wetting agent (e.g. Agral, Cittowett, Lubrol, Triton, 
Tween).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1047
 
Appropriate control solutions should be prepared, i.e. where a 
solvent or a dispersant is used to solubilise the test substance, two 
separate control groups should be used, one treated with water, and one treated with the solvent/dispersant.
 
1.6. PROCEDURE  
1.6.1.  Test and control groups  
The number of doses and replicates tested should meet the statistical 
requirements for determination LD  50 with 95 % confidence limits. 
Normally five doses in a geometric series, with a factor not exceeding 2,2, and covering the range for LD
 50 , are required for 
the test. However, the number of doses has to be determined in 
relation to the slope of the toxicity curve (dose versus mortality) 
and with consideration taken to the statistical method which is chosen for analysis of the results. A range-finding test enables the choice of the appropriate doses. 
A minimum of three replicate test groups, each of 10 bees, should be 
dosed with each test concentration. 
A minimum of three control batches, each of 10 bees, should be run 
in addition to the test series. If an organic solvent or a wetting agent 
is used three additional control batches of each 10 bees for the solvent or the wetting agent have to be included.
 
1.6.2.  Toxic standard  
A toxic standard must be included in the test series. At least three 
doses should be selected to cover the expected LD  50 value. A 
minimum of three replicate cages, each containing 10 bees, should be used with each test dose. The preferred toxic standard is dime ­
thoate, for which the reported contact LD
 50 -24 h is in the range 0,10- 
0,30 μg a.s./bee (2). However, other toxic standards would be 
acceptable where sufficient data can be provided to verify the expected dose response (e.g. parathion).
 
1.6.3.  Exposure  
1.6.3.1.  Administration of doses  
Anaesthetised bees are individually treated by topical application. 
The bees are randomly assigned to the different test doses and controls. A volume of 1 μl of solution containing the test 
substance at the suitable concentration should be applied with a 
microapplicator to the dorsal side of the thorax of each bee. Other 
volumes may be used, if justified. After application, the bees are allocated to test cages and supplied with sucrose solutions.
 
1.6.3.2.  Duration  
The duration of the test is preferably 48 hours. If mortality increases 
by more than 10 % between 24 h and 48 h, the test duration should 
be extended up to a maximum of 96 h provided that control 
mortality does not exceed 10 %.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1048
 
1.6.4.  Observations  
Mortality is recorded at 4 h after dosing and thereafter at 24 h and 
48 h. If a prolonged observation period is required, further assessments should be made, at 24 h intervals, to a maximum of 
96 h, provided that the control mortality does not exceeding 10 %. 
All abnormal behavioural effects observed during the testing period 
should be recorded.
 
1.6.5.  Limit test  
In some cases (e.g. when a test substance is expected to be of low 
toxicity) limit test may be performed, using 100 μg a.s./bee in order 
to demonstrate that the LD  50 is greater than this value. The same 
procedure should be used, including three replicate test groups for 
the test dose, the relevant controls, and the use of the toxic standard. If mortalities occur, a full study should be conducted. If sublethal effects are observed (see Section 1.6.4) these should be recorded.
 
2. DATA AND REPORTING  
2.1. DATA 
Data should be summarised in tabular form, showing for each 
treatment group, as well as, control and toxic standard groups, the 
number of bees used, mortality at each observation time and number 
of bees with adverse behaviour. Analyse the mortality data by appro ­
priate statistical methods (e.g. probit analysis, moving-average, binomial probability) (3)(4). Plot dose-response curves at each 
recommended observation time (i.e. 24 h, 48 h and, if relevant, 72 
h, 96 h) and calculate the slopes of the curves and the median lethal doses (LD
 50 ) with 95 % confidence limits. Corrections for control 
mortality could be made using Abbott's correction (4)(5). LD  50 
should be expressed in μg of test substance per bee.  
2.2. TEST REPORT 
The test report must include the following information:  
2.2.1.  Test substance:  
— physical nature and physical-chemical properties (e.g. stability in 
water, vapour pressure), 
— chemical identification data, including structural formula, purity 
(i.e. for pesticides, the identity and concentration of active 
substance(s)).  
2.2.2.  Test species:  
— scientific name, race, approximate age (in weeks), collection 
method, date of collection, 
— information on colonies used for collection of test bees including 
health, any adult disease, any pre-treatment, etc.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1049
 
2.2.3.  Test conditions:  
— temperature and relative humidity of experimental room, 
— housing conditions including type, size and material of cages, — methods of administration of test substance, e.g. carrier solvent 
used, volume of test solution applied anaesthetics used, 
— test design, e.g. number and test doses used, number of controls; 
for each test dose and control, number of replicate cages and 
number of bees per cage, 
— date of test.
 
2.2.4.  Results:  
— results of preliminary range-finding study if performed, 
— raw data: mortality at each concentration tested at each obser ­
vation time, 
— graph of the dose-response curves at the end of the test, 
— LD  50 values, with 95 % confidence limits, at each of the recom ­
mended observation times, for test substance and toxic standard, 
— statistical procedures used for determining the LD  50 , 
— mortality in controls, — other biological effects observed or measured and any abnormal 
responses of the bees, 
— any deviation from the test method procedures described here 
and any other relevant information.
 
3. REFERENCES  
(1) EPPO/Council of Europe (1993) Decision-Making Scheme for 
the Environmental Risk Assessment of Plant Protection Products — Honeybees. EPPO bulletin, vol. 23, N.1, p. 151-165. March, 1993. 
(2) Gough, H. J., McIndoe, E.C., Lewis, G.B., (1994) The use of 
dimethoate as a reference compound in laboratory acute toxicity tests on honeybees ( Apis mellifera  L.), 1981-1992. Journal of 
Apicultural Research 22, p. 119-125. 
(3) Litchfield, J.T. and Wilcoxon, F., (1949) A simplified method 
of evaluating dose-effect experiments. Jour. Pharmacol. and 
Exper. Ther., 96, p. 99-113. 
(4) Finney, D.J., (1971) Probit Analysis. 3rd ed., Cambridge, 
London and New-York. 
(5) Abbott, W.S., (1925) A method for computing the effectiveness 
of an insecticide. Jour. Econ. Entomol. 18, p. 265-267.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1050
 
C.18.  ADSORPTION/DESORPTION USING A BATCH EQUI ­
LIBRIUM METHOD  
1. METHOD  
This method is a replicate of the OECD TG 106, for the Deter ­
mination of Soil Adsorption/Desorption, using a Batch Equilibrium 
Method (2000).  
1.1. INTRODUCTION 
The method takes into account a ring test and a workshop for soil 
selection for the development of an adsorption test (1)(2)(3)(4) and also existing guidelines at national level (5)(6)(7)(8)(9)(10)(11). 
Adsorption/desorption studies are useful for generating essential 
information on the mobility of chemicals and their distribution in 
the soil, water and air compartments of the biosphere (12)(13)(14) 
(15)(16)(17)(18)(19)(20)(21). The information can be used in the prediction or estimation, for example, of the availability of a 
chemical for degradation (22)(23), transformation and uptake by 
organisms (24); leaching through the soil profile (16)(18)(19)(21) (25)(26)(27)(28); volatility from soil (21)(29)(30); run-off from land surfaces into natural waters (18)(31)(32). Adsorption data can 
be used for comparative and modelling purposes (19)(33)(34)(35). 
The distribution of a chemical between soil and aqueous phases is a 
complex process depending on a number of different factors: the 
chemical nature of the substance (12)(36)(37)(38)(39)(40), the character ­
istics of the soil (4)(12)(13)(14)(41)(42)(43)(44)(45)(46)(47)(48)(49), and climatic factors such as rainfall, temperature, sunlight and wind. 
Thus, the numerous phenomena and mechanisms involved in the 
process of adsorption of a chemical by soil cannot be completely defined by a simplified laboratory model such as the present method. 
However, even if this attempt cannot cover all the environmentally 
possible cases, it provides valuable information on the environmental relevance of the adsorption of a chemical. 
See also General Introduction.
 
1.2. SCOPE 
The method is aimed at estimating the adsorption/desorption 
behaviour of a substance on soils. The goal is to obtain a sorption value which can be used to predict partitioning under a variety of 
environmental conditions; to this end, equilibrium adsorption coef ­
ficients for a chemical on various soils are determined as a function of soil characteristics (e.g. organic carbon content, clay content and soil texture and pH). Different soil types have to be used in order to 
cover as widely as possible the interactions of a given substance with 
naturally occurring soils. 
In this method, adsorption represents the process of the binding of a 
chemical to surfaces of soils; it does not distinguish between different adsorption processes (physical and chemical adsorption) and such processes as surface catalysed degradation, bulk adsorption 
or chemical reaction. Adsorption that will occur on colloids particles 
(diameter < 0,2 μm) generated by the soils is not taken into account.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1051
 
The soil parameters that are believed most important for adsorption are: 
organic carbon content (3)(4)(12)(13)(14)(41)(43)(44)(45)(46)(47)(48); 
clay content and soil texture (3)(4)(41)(42)(43)(44)(45)(46) (47)(48) and pH for ionisable compounds (3)(4)(42). Other soil parameters which may have an impact on the adsorption/desorption of a particular 
substance are the effective cation exchange capacity (ECEC), the 
content of amorphous iron and aluminium oxides, particularly for volcanic and tropical soils (4), as well as the specific surface (49). 
The test is designed to evaluate the adsorption of a chemical on 
different soil types with a varying range of organic carbon content, 
clay content and soil texture, and pH. It comprises three tiers:  
Tier 1 : preliminary study in order to determine: 
— the soil/solution ratio, 
— the equilibrium time for adsorption and the amount of 
test substance adsorbed at equilibrium, 
— the adsorption of the test substance on the surfaces of 
the test vessels and the stability of the test substance during the test period.  
Tier 2 : screening test: the adsorption is studied in five different 
soil types by means of adsorption kinetics at a single concentration and determination of distribution coefficient 
K
 d and K  oc . 
Tier 3 : determination of Freundlich adsorption isotherms to 
determine the influence of concentration on the extent of 
adsorption on soils. 
Study of desorption by means of desorption kinetics/ 
Freundlich desorption isotherms (Appendix 1).  
1.3. DEFINITIONS AND UNITS  
Symbol Definition Units  
A t i adsorption percentage at the time t  i % 
A eq adsorption percentage at adsorption equilibrium %  
m s ads ðt i Þ mass of the test substance adsorbed on the soil at the 
time t  i μg 
m s ads ðΔt i Þ mass of the test substance adsorbed on the soil during the 
time interval Δt i μg 
m s ads ðeqÞ mass of the test substance adsorbed on the soil at 
adsorption equilibrium μg 
m 0 mass of the test substance in the test tube, at the beginning 
of the adsorption test μg 
m m ads ðt i Þ mass of the test substance measured in an aliquot ( v a A ) at 
the time point t  i μg 
m aq ads ðeqÞ mass of the substance in the solution at adsorption equi ­
librium μg 
m soil quantity of the soil phase, expressed in dry mass of soil g  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1052
 
Symbol Definition Units  
C st mass concentration of the stock solution of the substance μg cm  -3 
C 0 initial mass concentration of the test solution in contact 
with the soil μg cm  -3 
C aq ads ðt i Þ mass concentration of the substance in the aqueous phase 
at the time t  i that the analysis is performed μg cm  -3 
C s ads ðeqÞ content of the substance adsorbed on soil at adsorption 
equilibrium an equilibrium μg g  -1 
C aq ads ðeqÞ mass concentration of the substance in the aqueous phase 
at adsorption equilibrium μg cm  -3 
V 0 initial volume of the aqueous phase in contact with the soil 
during the adsorption test cm 3 
v a A volume of the aliquot in which the test substance is 
measured cm 3 
K d distribution coefficient for adsorption cm  3 g -1 
K oc organic carbon normalised adsorption coefficient cm  3 g -1 
K om organic matter normalised distribution coefficient cm  3 g -1 
K F ads Freundlich adsorption coefficient μg 1-1/n (cm  3 ) 1/n g -1 
1/n Freundlich exponent  
D t i desorption percentage at a point time t  i % 
D Δt i desorption percentage corresponding to a time interval Δt i % 
K des apparent desorption coefficient cm  3 g -1 
K F des Freundlich desorption coefficient μg 1-1/n (cm  3 ) 1/n g -1 
m aq des ðt i Þ mass of the test substance desorbed from soil at the time t  i μg 
m m des ðΔt i Þ mass of the test substance desorbed from soil during the 
time Δt i μg 
m m des ðeqÞ mass of the substance determined analytically in the 
aqueous phase at desorption equilibrium μg 
m aq des ðeqÞ total mass of the test substance desorbed at desorption 
equilibrium μg 
m s des ðΔt i Þ mass of the substance remaining adsorbed on the soil after 
the time interval Δt i μg 
m aq A mass of the substance left over from the adsorption equi ­
librium due to incomplete volume replacement μg 
C s des ðeqÞ content of the test substance remaining adsorbed on the 
soil at desorption equilibrium μg g -1 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1053
 
Symbol Definition Units  
C aq des ðeqÞ mass concentration of the test substance in the aqueous 
phase at desorption equilibrium μg cm  -3 
V T total volume of the aqueous phase in contact with the soil 
during the desorption kinetics experiment performed with the serial method cm
 3 
V R volume of the supernatant removed from the tube after the 
attainment of adsorption equilibrium and replaced by the same volume of a 0,01 M CaCl
 2 solution cm 3 
v a D volume of the aliquot sampled for analytical purpose from 
the time (i), during the desorption kinetics experiment performed with the serial method cm
 3 
V r r volume of the solution taken from the tube (i) for the 
measurement of the test substance, in desorption kinetics 
experiment (parallel method) cm 3 
V r F volume of the solution taken from the tube for the 
measurement of the test substance, at desorption equi ­
librium cm 3 
MB mass balance % 
m E total mass of the test substance extracted from soil and 
walls of the test vessel in two steps μg 
V rec volume of the supernatant recovered after the adsorption 
equilibrium cm 3 
P ow octanol/water partition coefficient 
pKa dissociation constant 
S w water solubility g l  -1 
1.4. PRINCIPLE OF THE TEST METHOD 
Known volumes of solutions of the test substance, non-labelled or 
radiolabelled, at known concentrations in 0,01 M CaCl  2 are added to 
soil samples of known dry weight which have been pre-equilibrated in 0,01 M CaCl
 2 . The mixture is agitated for an appropriate time. 
The soil suspensions are then separated by centrifugation and, if so 
wished, filtration and the aqueous phase is analysed. The amount of test substance adsorbed on the soil sample is calculated as the difference between the amount of test substance initially present in 
solution and the amount remaining at the end of the experiment 
(indirect method). 
As an option, the amount of the test substance adsorbed can also be 
directly determined by analysis of soil (direct method). This 
procedure which involves stepwise soil extraction with appropriate 
solvent, is recommended in cases where the difference in the solution concentration of the substance cannot be accurately deter ­
mined. Examples of such cases are: adsorption of the test substance 
on surface of the test vessels, instability of the test substance in the 
time scale of the experiment, weak adsorption giving only small concentration change in the solution; and strong adsorption yielding low concentration which cannot be accurately determined. 
If radiolabelled substance is used, the soil extraction may be avoided 
by analysis of the soil phase by combustion and liquid scintillation counting. However, liquid scintillation counting is an unspecific technique which cannot differentiate between parental and trans ­
formation products; therefore it should be used only if the test 
chemical is stable for the duration of the study.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1054
 
1.5. INFORMATION ON THE TEST SUBSTANCE 
Chemical reagents should be of analytical grade. The use of non- 
labelled test substances with known composition and preferably at least 95 % purity or of radiolabelled test substances with known 
composition and radio-purity, is recommended. In the case of short 
half-life tracers, decay corrections should be applied. 
Before carrying out a test for adsorption-desorption, the following 
information about the test substance should be available: 
(a) water solubility (A.6); 
(b) vapour pressure (A.4) and/or Henry's Law Constant; 
(c) abiotic degradation: hydrolysis as a function of pH (C.7); 
(d) partition coefficient (A.8); 
(e) ready biodegradability (C.4) or aerobic and anaerobic trans ­
formation in soil; 
(f) pKa of ionisable substances; 
(g) direct photolysis in water (i.e. UV-vis absorption spectrum in 
water, quantum yield) and photodegradation on soil.
 
1.6. APPLICABILITY OF THE TEST 
The test is applicable to chemical substances for which an analytical 
method with sufficient accuracy is available. An important parameter 
that can influence the reliability of the results, especially when the 
indirect method is followed, is the stability of the test substance in the time scale of the test. Thus, it is a prerequisite to check the stability in a preliminary study; if a transformation in the time 
scale of the test is observed, it is recommended that the main 
study be performed by analysing both soil and aqueous phases. 
Difficulties may arise in conducting this test for test substances with 
low water solubility (S
 w < 10  -4 g l -1 ), as well as for highly charged 
substances, due to the fact that the concentration in the aqueous 
phase cannot be measured analytically with sufficient accuracy. In these cases, additional steps have to be taken. Guidance on how to deal with these problems is given in the relevant sections of this 
method. 
When testing volatile substances, care should be taken to avoid 
losses during the study.
 
1.7. DESCRIPTION OF THE METHOD  
1.7.1.  Apparatus and chemical reagents  
Standard laboratory equipment, especially the following: 
(a) tubes or vessels to conduct the experiments. It is important that 
these tubes or vessels, 
— fit directly in the centrifuge apparatus in order to minimise 
handling and transfer errors, 
— be made of an inert material, which minimises adsorption of 
the test substance on its surface,  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1055
 
(b) agitation device: overhead shaker or equivalent equipment; the 
agitation device should keep the soil in suspension during 
shaking, 
(c) centrifuge: preferably high-speed, e.g. centrifugation forces > 
3 000 g, temperature controlled, capable of removing particles with a diameter greater than 0,2 μm from aqueous solution. The 
containers should be capped during agitation and centrifugation 
to avoid volatility and water losses; to minimise adsorption on 
them, deactivated caps such as teflon lined screw caps should be used, 
(d) optional: filtration device; filters of 0,2 μm porosity, sterile, 
single use. Special care should be taken in the choice of the 
filter material, to avoid any losses of the test substance on it; 
for poorly soluble test substances, organic filter material is not recommended, 
(e) analytical instrumentation, suitable for measuring the concen ­
tration of the test chemical, 
(f) laboratory oven, capable of maintaining a temperature of 103
 o C 
to 110  o C, 
1.7.2.  Characterisation and selection of soils  
The soils should be characterised by three parameters considered to 
be largely responsible for the adsorptive capacity: organic carbon, 
clay content and soil texture, and pH. As already mentioned (see Scope) other physico-chemical properties of the soil may have an impact on the adsorption/desorption of a particular substance and 
should be considered in such cases. 
The methods used for soil characterisation are very important and 
can have a significant influence on the results. Therefore, it is recom ­
mended that soil pH should be measured in a solution of 0,01 M CaCl
 2 (that is the solution used in adsorption/desorption testing) 
according to the corresponding ISO method (ISO-10390-1). It is 
also recommended that the other relevant soil properties be determined according to standard methods (for example ISO ‘Handbook of Soil Analysis’); this permits the analysis of sorption 
data to be based on globally standardised soil parameters. Some 
guidance for existing standard methods of soil analysis and char ­
acterisation is given in references (50-52). For calibration of soil 
test methods, the use of reference soils is recommended. 
Guidance for selection of soils for adsorption/desorption experiments 
is given in Table 1. The seven selected soils cover soil types 
encountered in temperate geographical zones. For ionisable test substances, the selected soils should cover a wide range of pH, in order to be able to evaluate the adsorption of the substance in its 
ionised and unionised forms. Guidance on how many different soils 
to use at the various stages of the test is given under ‘Performance of the test’ 1.9. 
If other soil types are preferred, they should be characterised by the 
same parameters and should have similar variation in properties to 
those described in Table 1, even if they do not match the criteria 
exactly.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1056
 
Table 1:  
Guidance for selection of soil samples for adsorption-desorption  
Soil Type pH range (in 0,01 M 
CaCl  2 ) Organic carbon 
content (%) Clay content (%) Soil texture (  1 ) 
1 4,5 - 5,5 1,0 - 2,0 65-80 clay 
2 > 7,5 3,5 - 5,0 20-40 clay loam 
3 5,5 - 7,0 1,5 - 3,0 15-25 silt loam 4 4,0 - 5,5 3,0 - 4,0 15-30 loam 5 < 4,0 - 6,0 (
 2 ) < 0,5 - 1,5 (  2 ) ( 3 ) < 10-15 (  2 ) loamy sand 
6 > 7,0 < 0,5 - 1,0 (  2 ) ( 3 ) 40-65 clay loam/clay 
7 < 4,5 > 10 < 10 sand/loamy sand  
( 1 ) According to FAO and the US system (85). 
( 2 ) The respective variables should preferably show values within the range given. If, however, difficulties in finding 
appropriate soil material occur, values below the indicated minimum are accepted. 
( 3 ) Soils with less than 0,3 % organic carbon may disturb correlation between organic content and adsorption. Thus, it is 
recommended the use of soils with a minimum organic carbon content of 0,3 %.  
1.7.3.  Collection and storage of soil samples  
1.7.3.1.  Collection  
No specific sampling techniques or tools are recommended; 
the sampling technique depends on the purpose of the study (53) ­
(54)(55)(56)(57)(58). 
The following should be considered: 
a) detailed information on the history of the field site is necessary; 
this includes location, vegetation cover, treatments with pesticides and/or fertilisers, biological additions or accidental contamination. Recommendations of the ISO standard on soil sampling 
(ISO 10381-6) should be followed with respect to the description 
of the sampling site; 
b) the sampling site has to be defined by UTM (Universal Trans ­
versal Mercator-Projection/European Horizontal Datum) or geographical co-ordinates; this could allow recollection of a 
particular soil in the future or could help in defining soil under 
various classification systems used in different countries. Also, only A horizon up to a maximum depth of 20 cm should be collected. Especially for the soil type No 7 if a O
 h horizon is 
present as part of the soil, it should be included in the sampling. 
The soil samples should be transported using containers and under 
temperature conditions which guarantee that the initial soil properties 
are not significantly altered.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1057
 
1.7.3.2.  Storage  
The use of soils freshly taken from the field is preferred. Only if this 
is not possible soil can be stored at ambient temperature and should be kept air-dried. No limit on the storage time is recommended, but 
soils stored for more than three years should be re-analysed prior to 
the use with respect to their organic carbon content, pH and CEC.
 
1.7.3.3.  Handling and preparation of soil samples for the test  
The soils are air-dried at ambient temperature (preferably between 
20-25  o C). Disaggregation should be performed with minimal force, 
so that the original texture of the soil will be changed as little as 
possible. The soils are sieved to a particle size ≤ 2 mm; recommen ­
dations of the ISO standard on soil sampling (ISO 10381-6) should be followed with respect to the sieving process. Careful homogeni ­
sation is recommended, as this enhances the reproducibility of the 
results. The moisture content of each soil is determined on three 
aliquots with heating at 105
 o C until there is no significant change 
in weight (approximately 12 h). For all calculations the mass of soil refers to oven dry mass, i.e. the weight of soil corrected for moisture 
content.
 
1.7.4.  Preparation of the test substance for application to soil  
The test substance is dissolved in a solution of 0,01 M CaCl  2 in 
distilled or de-ionised water; the CaCl  2 solution is used as the 
aqueous solvent phase to improve centrifugation and minimise cation exchange. The concentration of the stock solution should 
preferably be three orders of magnitude higher than the detection 
limit of the analytical method used. This threshold safeguards accurate measurements with respect to the methodology followed in this method; additionally, the stock solution concentration 
should be below water solubility of the test substance. 
The stock solution should preferably be prepared just before appli ­
cation to soil samples and should be kept closed in the dark at 4
 o C. 
The storage time depends on the stability of the test substance and its concentration in the solution. 
Only for poorly soluble substances (S
 w < 10  -4 g l -1 ), an appropriate 
solubilising agent may be needed when it is difficult to dissolve the 
test substance. This solubilising agent: (a) should be miscible with water such as methanol or acetonitrile; (b) its concentration should not exceed 1 % of the total volume of the stock solution and should 
constitute less than that in the solution of the test substance which 
will come in contact with the soil (preferably less than 0,1 %); and (c) should not be a surfactant or undergo solvolytic reactions with the test chemical. The use of a solubilising agent should be stipulated 
and justified in the reporting of the data. 
Another alternative for poorly soluble substances is to add the test 
substance to the test system by spiking: the test substance is dissolved in an organic solvent, an aliquot of which is added to the system of soil and 0,01 M solution of CaCl
 2 in distilled or de- 
ionised water. The content of organic solvent in the aqueous phase 
should be kept as low as possible, normally not exceeding 0,1 %. Spiking from an organic solution may suffer from volume unrepro ­
ducibility. Thus, an additional error may be introduced as the test 
substance and co-solvent concentration would not be the same in all 
tests.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1058
 
1.8. PREREQUISITES FOR PERFORMING THE ADSORPTION/ ­
DESORPTION TEST  
1.8.1.  The analytical method  
The key parameters that can influence the accuracy of sorption 
measurements include the accuracy of the analytical method in 
analysis of both the solution and adsorbed phases, the stability and purity of the test substance, the attainment of sorption equilibrium, the magnitude of the solution concentration change, the soil/solution 
ratio and changes in the soil structure during the equilibration 
process (35)(59-62). Some examples bearing upon the accuracy issues are given in Appendix 2. 
The reliability of the analytical method used must be checked at the 
concentration range which is likely to occur during the test. The experimenter should feel free to develop an appropriate method with appropriate accuracy, precision, reproducibility, detection 
limits and recovery. Guidance on how to perform such a test is 
given by the experiment below. 
An appropriate volume of 0,01 M CaCl
 2 , e.g. 100 cm  3 , is agitated 
during 4 h with a weight of soil, e.g. 20 g, of high adsorbability, i.e. 
with high organic carbon and clay content; these weights and volumes may vary depending on analytical needs, but a soil/solution ratio of 1:5 is a convenient starting point. The mixture is centrifuged 
and the aqueous phase may be filtrated. A certain volume of the test 
substance stock solution is added to the latter to reach a nominal concentration within the concentration range which is likely to occur 
during the test. This volume should not exceed 10 % of the final 
volume of the aqueous phase, in order to change as little as possible the nature of the pre-equilibration solution. The solution is analysed. 
One blank run consisting of the system soil + CaCl
 2 solution 
(without test substance) must be included, in order to check for 
artefacts in the analytical method and for matrix effects caused by the soil. 
The analytical methods which can be used for sorption 
measurements include gas-liquid chromatography (GLC), high- performance liquid chromatography (HPLC), spectrometry (e.g. GC/mass spectrometry, HPLC/mass spectrometry) and liquid scintil ­
lation counting (for radiolabelled substances). Independent of the 
analytical method used, it is considered suitable if the recoveries are between 90 % and 110 % of the nominal value. In order to allow for detection and evaluation after partitioning has taken 
place, the detection limits of the analytical method should be at 
least two orders of magnitude below the nominal concentration. 
The characteristics and detection limits of the analytical method 
available for carrying out adsorption studies play an important role 
in defining the test conditions and the whole experimental performance of the test. This method follows a general experimental path and provides recommendations and guidance for alternative 
solutions where the analytical method and laboratory facilities may 
impose limitations.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1059
 
1.8.2.  The selection of optimal soil/solution ratios  
Selection of appropriate soil to solution ratios for sorption studies 
depends on the distribution coefficient K  d and the relative degree of 
adsorption desired. The change of the substance concentration in the 
solution determines the statistical accuracy of the measurement based 
on the form of adsorption equation and the limit of the analytical methodology, in detecting the concentration of the chemical in solution. Therefore, in general practice it is useful to settle on a 
few fixed ratios, for which the percentage adsorbed is above 
20 %, and preferably >50 % (62), while care should be taken to keep the test substance concentration in the aqueous phase high 
enough to be measured accurately. This is particularly important in 
the case of high adsorption percentages. 
A convenient approach to selecting the appropriate soil/water ratios, 
is based on an estimate of the K
 d value either by preliminary studies 
or by established estimation techniques (Appendix 3). Selection of 
an appropriate ratio can then be made based on a plot of soil/solution 
ratio versus K  d for fixed percentages of adsorption (Fig.1). In this 
plot it is assumed that the adsorption equation is linear (  1 ). The 
applicable relationship is obtained by rearranging equation (4) of 
the K  d in the form of equation (1):  
V 0 
m soil ¼ Í 
m 0 
m s ads ðeqÞ Ä 1 ! 
K d (1) 
or in its logarithmic form assuming that R = m  soil=V 0 and A  eq %=100 =  m s ads ðeqÞ 
m 0 : 
log R ¼ Älog K  d þ log " 
ðA eq %=100Þ 
ð1 Ä A eq %=100Þ # (2)
 
Fig. 1  Relationship between soil to solution ratios and K  d at various percentages of 
adsorbed test substance  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1060  
( 1 ) C s ads ðeqÞ ¼ K  d · C aq ads ðeqÞ:
 Fig. 1 shows soil/solution ratios required as a function of K  d for 
different levels of adsorption. For example, with a soil/solution ratio 
of 1:5 and a K  d of 20, approximately 80 % adsorption would occur. 
To obtain 50 % adsorption for the same K  d , a 1:25 ratio must be 
used. This approach to selecting the appropriate soil/solution ratios 
gives the investigator the flexibility to meet experimental needs. 
Areas which are more difficult to deal with are those where the 
chemical is highly or very slightly adsorbed. Where low adsorption occurs, a 1:1 soil/solution ratio is recommended, although for some 
very organic soil types smaller ratios may be necessary to obtain a 
slurry. Care must be taken with the analytical methodology to measure small changes in solution concentration; otherwise the adsorption measurement will be inaccurate. On the other hand, at 
very high distribution coefficients K
 d , one can go up to a 1:100 
soil/solution ratio in order to leave a significant amount of chemical in solution. However, care must be taken to ensure good mixing, and adequate time must be allowed for the system to equili ­
brate. An alternative approach to deal with these extreme cases when 
adequate analytical methodology is missing, is to predict the K
 d 
value applying estimation techniques based, for example, on P  ow 
values (Appendix 3). This could be useful especially for low 
adsorbed/polar chemicals with P  ow < 20 and for lipophilic/highly 
sorptive chemicals with P  ow > 10  4 . 
1.9. PERFORMANCE OF THE TEST  
1.9.1.  Test conditions  
All experiments are done at ambient temperature and, if possible, at 
a constant temperature between 20  o C and 25  o C. 
Centrifugation conditions should allow the removal of particles 
larger than 0,2 μm from the solution. This value triggers the 
smallest sized particle that is considered as a solid particle, and is the limit between solid and colloid particles. Guidance on how to 
determine the centrifugation conditions is given in Appendix 4. 
If the centrifugation facilities cannot guarantee the removal of 
particles larger than 0,2 μm, a combination of centrifugation and 
filtration with 0,2 μm filters could be used. These filters should be 
made of a suitable inert material to avoid any losses of the test 
substance on them. In any case, it should be proven that no losses of the test substance occur during filtration.
 
1.9.2.  Tier 1 — Preliminary study  
The purpose of conducting a preliminary study has already been 
given in the Scope section. Guidance for setting up such a test is 
given with the experiment suggested below.  
1.9.2.1.  Selection of optimal soil/solution ratios  
Two soil types and three soil/solution ratios (six experiments) are 
used. One soil type has high organic carbon and low clay content, and the other low organic carbon and high clay content. The 
following soil to solution ratios are suggested: 
— 50 g soil and 50 cm
 3 aqueous solution of the test substance 
(ratio 1/1),  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1061
 
— 10 g soil and 50 cm  3 aqueous solution of the test substance 
(ratio 1/5), 
— 2 g soil and 50 cm  3 aqueous solution of the test substance 
(ratio 1/25). 
The minimum amount of soil on which the experiment can be 
carried out depends on the laboratory facilities and the performance of analytical methods used. However, it is recommended to use at 
least 1 g, and preferably 2 g, in order to obtain reliable results from 
the test. 
One control sample with only the test substance in 0,01 M CaCl
 2 
solution (no soil) is subjected to precisely the same steps as the test 
systems, in order to check the stability of the test substance in CaCl  2 
solution and its possible adsorption on the surfaces of the test 
vessels. 
A blank run per soil with the same amount of soil and total volume 
of 50 cm  3 0,01 M CaCl  2 solution (without test substance) is 
subjected to the same test procedure. This serves as a background control during the analysis to detect interfering substances or contaminated soils. 
All the experiments, included controls and blanks, should be 
performed at least in duplicate. The total number of the samples which should be prepared for the study can be calculated with 
respect to the methodology which will be followed. 
Methods for the preliminary study and the main study are generally 
the same, exceptions are mentioned where relevant. 
The air-dried soil samples are equilibrated by shaking with a 
minimum volume of 45 cm
 3 of 0,01 M CaCl  2 overnight (12 h) 
before the day of the experiment. Afterwards, a certain volume of 
the stock solution of the test substance is added in order to adjust the final volume to 50 cm
 3 . This volume of the stock solution added: (a) 
should not exceed 10 % of the final 50 cm  3 volume of the aqueous 
phase in order to change as little as possible the nature of the pre- 
equilibration solution; and (b) should preferably result in an initial concentration of the test substance being in contact with the soil (C
 0 ) 
at least two orders of magnitude higher than the detection limit of 
the analytical method; this threshold safeguards the ability to perform 
accurate measurements even when strong adsorption occurs (> 90 %) and to determine later the adsorption isotherms. It is also recom ­
mended, if possible, that the initial substance concentration (C
 0 ) 
not exceed half of its solubility limit. 
An example of how to calculate the concentration of the stock 
solution (C  st ) is given below. A detection limit of 0,01 μg cm  -3 
and 90 % adsorption are assumed; thus, the initial concentration 
of the test substance in contact with the soil should preferably be 1 μg cm
 -3 (two orders of magnitude higher than the detection limit). 
Supposing that the maximum recommended volume of the stock 
solution is added, i.e. 5 to 45 cm  3 0,01 M CaCl  2 equilibration 
solution (= 10 % of the stock solution to 50 cm  3 total volume 
of aqueous phase), the concentration of the stock solution should 
be 10 μg cm  -3 ; this is three orders of magnitude higher than the 
detection limit of the analytical method. 
The pH of the aqueous phase should be measured before and after 
contact with the soil since it plays an important role in the whole 
adsorption process, especially for ionisable substances.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1062
 
The mixture is shaken until adsorption equilibrium is reached. The 
equilibrium time in soils is highly variable, depending on the 
chemical and the soil; a period of 24 h is generally sufficient (77). In the preliminary study, samples may be collected sequentially over a 48 h period of mixing (for example at 4, 8, 24, 48 h). However, 
times of analysis should be considered with flexibility with respect to 
the work schedule of the laboratory. 
There are two options for the analysis of the test substance in the 
aqueous solution: (a) the parallel method and (b) the serial method. It should be stressed that, although the parallel method is experi ­
mentally more tedious, the mathematical treatment of the results is 
simpler (Appendix 5). However, the choice of the methodology to be 
followed, is left to the experimenter who will need to consider the available laboratory facilities and resources. 
(a) parallel method: samples with the same soil/solution ratio are 
prepared, as many as the time intervals at which it is desired to study the adsorption kinetics. After centrifugation and if so 
wished filtration, the aqueous phase of the first tube is recovered 
as completely as possible and is measured after, for example, 4 h, that of the second tube after 8 h, that of the third after 24, etc. 
(b) serial method: only a duplicate sample is prepared for each soil/ ­
solution ratio. At defined time intervals the mixture is centrifuged to separate the phases. A small aliquot of the 
aqueous phase is immediately analysed for the test substance; 
then the experiment continues with the original mixture. If filtration is applied after centrifugation, the laboratory should have facilities to handle filtration of small aqueous aliquots. It 
is recommended that the total volume of the aliquots taken not 
exceed 1 % of the total volume of the solution, in order not to change significantly the soil/solution ratio and to decrease the mass of solute available for adsorption during the test. 
The percentage adsorption
 A t i is calculated at each time point (t  i ) on 
the basis of the nominal initial concentration and the measured 
concentration at the sampling time (t  i ), corrected for the value of 
the blank. Plots of the  A t i versus time (Fig. 1 Appendix 5) are 
generated in order to estimate the achievement of equilibrium plateau (
 1 ). The K  d value at equilibrium is also calculated. Based 
on this K  d value, appropriate soil/solution ratios are selected from 
Fig.1, so that the percentage adsorption reaches above 20 % and preferably >50 % (61). All the applicable equations and principles of plotting are given in section on Data and Reporting and in 
Appendix 5.
 
1.9.2.2.  Determination of adsorption equilibration time and of the amount of 
test substance adsorbed at equilibrium  
As already mentioned, plots of  A t i or C aq ads versus time permit esti ­
mation of the achievement of the adsorption equilibrium and the amount of test substance adsorbed at equilibrium. Figs. 1 and 2 in the Appendix 5 show examples of such plots. Equilibration time is 
the system needs to reach a plateau.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1063  
( 1 ) Plots of the concentration of the test substance in the aqueous phase  C  aq ads Ê Ì  
versus time 
could also be used to estimate the achievement of the equilibrium plateau (see Fig. 2 in 
Appendix 5).
 If, with a particular soil, no plateau but a steady increase is found, 
this may be due to complicating factors such as biodegradation or 
slow diffusion. Biodegradation can be shown by repeating the experiment with a sterilised sample of the soil. If no plateau is achieved even in this case, the experimenter should search for 
other phenomena that could be involved in his specific studies; 
this could be done with appropriate modifications of the experiment conditions (temperature, shaking times, soil/solution ratios). It is left to the experimenter to decide whether to continue the test procedure 
in spite of a possible failure to achieve an equilibrium.
 
1.9.2.3.  Adsorption on the surface of the test vessel and stability of the test 
substance  
Some information on the adsorption of the test substance on the 
surface of test vessels, as well as its stability, can be derived by analysing the control samples. If a depletion more than the standard 
error of the analytical method is observed, abiotic degradation and/or 
adsorption on the surface of the test vessel could be involved. Distinction between these two phenomena could be achieved by 
thoroughly washing the walls of the vessel with a known volume 
of an appropriate solvent and subjecting the wash solution to analysis for the test substance. If no adsorption on the surface of the test vessels is observed, the depletion demonstrates abiotic unsta ­
bility of the test substance. If adsorption is found, changing the 
material of the test vessels is necessary. However, data on the adsorption on the surface of the test vessels gained from this experiment cannot be directly extrapolated to soil/solution 
experiment. The presence of soil will affect this adsorption. 
Additional information on the stability of the test substance can be 
derived by determination of the parental mass balance over time. This means that the aqueous phase, extracts of soil and test vessel 
walls are analysed for the test substance. The difference between the 
mass of the test chemical added and the sum of the test chemical masses in the aqueous phase, extracts of the soil and test vessel walls is equal to the mass degraded and/or volatilised and/or not extracted. 
In order to perform a mass balance determination, the adsorbion 
equilibrium should have been reached within the period of the experiment. 
The mass balance is performed on both soils and for one soil/ ­
solution ratio per soil that gives a depletion above 20 % and 
preferably > 50 % at equilibrium. When the ratio-finding experiment is completed with the analysis of the last sample of the aqueous phase after 48 h, the phases are separated by centrifugation and, if 
so wished, filtration. The aqueous phase is recovered as much as 
possible, and a suitable extraction solvent (extraction coefficient of at least 95 %) is added to the soil to extract the test substance. At least two successive extractions are recommended. The amount of test 
substance in the soil and test vessel extracts is determined and the 
mass balance is calculated (equation 10, Data and Reporting). If it is less than 90 %, the test substance is considered to be unstable in the time scale of the test. However, studies could still be continued, 
taking into account the unstability of the test substance; in this 
case it is recommended to analyse both phases in the main study.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1064
 
1.9.3.  Tier 2 — Adsorption kinetics at one concentration of the test 
substance  
Five soils are used, selected from Table 1. There is an advantage to 
including some or all of the soils used in the preliminary study, if 
appropriate, among these five soils. In this case, Tier 2 has not to be 
repeated for the soils used in preliminary study. 
The equilibration time, the soil/solution ratio, the weight of the soil 
sample, the volume of the aqueous phase in contact with the soil and concentration of the test substance in the solution are chosen based 
on the preliminary study results. Analysis should preferably be done 
approximately after 2, 4, 6, 8 (possibly also 10) and 24 h contact time; the agitation time may be extended to a maximum of 48 h in case a chemical requires longer equilibration time with respect to 
ratio-finding results. However, times of analysis could be considered 
with flexibility. 
Each experiment (one soil and one solution) is done at least in 
duplicate to allow estimation of the variance of the results. In every experiment one blank is run. It consists of the soil and 0,01 M CaCl
 2 solution, without test substance, and of weight and volume, 
respectively, identical to those of the experiment. A control sample 
with only the test substance in 0,01 M CaCl  2 solution (without soil) 
is subjected to the same test procedure, serving to safeguard against the unexpected. 
The percentage adsorption is calculated at each time point
 A t i and/or 
time interval  A Δt i (according to the need) and is plotted versus time. 
The distribution coefficient K  d at equilibrium, as well as the organic 
carbon normalised adsorption coefficient K  oc (for non-polar organic 
chemicals), are also calculated. 
Results of the adsorption kinetics test The linear K
 d value is generally accurate to describe sorptive 
behaviour in soil (35)(78) and represents an expression of inherent mobility of chemicals in soil. For example, in general chemicals with K
 d ≤ 1 cm  3 g -1 are considered to be qualitatively mobile. 
Similarly, a mobility classification scheme based on K  oc values has 
been developed by MacCall  et al . (16). Additionally, leaching clas ­
sification schemes exist based on a relationship between K  oc and 
DT-50 (  1 ) (32)(79). 
Also, according to error analysis studies (61), K  d values below 
0,3 cm  3 g -1 cannot be estimated accurately from a decrease in 
concentration in the aqueous phase, even when the most favourable (from point of view of accuracy) soil/solution ratio is applied, i.e. 1:1. In this case analysis of both phases, soil and solution, is recom ­
mended.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1065  
( 1 ) DT-50: degradation time for 50 % of the test substance.
 With respect to the above remarks, it is recommended that the study 
of the adsorptive behaviour of a chemical in soil and its potential 
mobility be continued by determining Freundlich adsorption isotherms for these systems, for which an accurate determination of K
 d is possible with the experimental protocol followed in this 
test method. Accurate determination is possible if the value which 
results by multiplying the K  d with the soil/solution ratio is > 0,3, 
when measurements are based on concentration decrease in the aqueous phase (indirect method), or > 0,1, when both phases are 
analysed (direct method) (61).
 
1.9.4.  Tier 3 — Adsorption isotherms and desorption kinetics/desorption 
isotherms  
1.9.4.1. A d s o r p t i o n i s o t h e r m s 
Five test substance concentrations are used, covering preferably two 
orders of magnitude; in the choice of these concentrations the water 
solubility and the resulting aqueous equilibrium concentrations should be taken into account. The same soil/solution ratio per soil should be kept along the study. The adsorption test is performed as 
described above, with the only difference that the aqueous phase is 
analysed only once at the time necessary to reach equilibrium as determined before in Tier 2. The equilibrium concentrations in the solution are determined and the amount adsorbed is calculated from 
the depletion of the test substance in the solution or with the direct 
method. The adsorbed mass per unit mass of soil is plotted as a function of the equilibrium concentration of the test substance (see 
Data and Reporting). 
Results from the adsorption isotherms experiment Among the mathematical adsorption models proposed so far, the 
Freundlich isotherm is the one most frequently used to describe adsorption processes. More detailed information on the interpretation 
and importance of adsorption models is provided in the references 
(41)(45)(80)(81)(82).  
Note : it should be mentioned that a comparison of K
 F (Freundlich 
adsorption coefficient) values for different substances is only possible if these K
 F values are expressed in the same units (83).  
1.9.4.2. D e s o r p t i o n k i n e t i c s 
The purpose of this experiment is to investigate whether a chemical 
is reversibly or irreversibly adsorbed on a soil. This information is important, since the desorption process also plays an important role 
in the behaviour of a chemical in field soil. Moreover, desorption 
data are useful inputs in the computer modelling of leaching and dissolved run-off simulation. If a desorption study is desired, it is recommended that the study described below be carried out on each 
system for which an accurate determination of K
 d in the preceding 
adsorption kinetics experiment was possible. 
Likewise with the adsorption kinetics study, there are two options to 
proceed with the desorption kinetics experiment: (a) the parallel method and (b) serial method. The choice of methodology to be 
followed, is left to the experimenter who will need to consider the 
available laboratory facilities and resources.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1066
 
(a) parallel method: for each soil which is chosen to proceed with 
the desorption study, samples with the same soil/solution ratio 
are prepared, as many as the time intervals at which it is desired to study the desorption kinetics. Preferably, the same time intervals as in the adsorption kinetics experiment should be 
used; however, the total time may be extended as appropriate 
in order the system to reach desorption equilibrium. In every experiment (one soil, one solution) one blank is run. It consists of the soil and 0,01 M CaCl
 2 solution, without test 
substance, and of weight and volume, respectively, identical to 
those of the experiment. As a control sample the test substance in 0,01 M CaCl
 2 solution (without soil) is subjected to the same 
test procedure. All the mixtures of the soil with the solution is 
agitating until to reach adsorption equilibrium (as determined 
before in Tier 2). Then, the phases are separated by centrifu ­
gation and the aqueous phases are removed as much as possible. The volume of solution removed is replaced by an equal volume 
of 0,01 M CaCl
 2 without test substance and the new mixtures 
are agitated again. The aqueous phase of the first tube is recovered as completely as possible and is measured after, for example, 2 h, that of the second tube after 4 h, that of the third 
after 6 h, etc. until the desorption equilibrium is reached. 
(b) serial method: after the adsorption kinetics experiment, the 
mixture is centrifuged and the aqueous phase is removed as much as possible. The volume of solution removed is replaced by an equal volume of 0,01 M CaCl
 2 without test substance. The 
new mixture is agitated until the desorption equilibrium is 
reached. During this time period, at defined time intervals, the mixture is centrifuged to separate the phases. A small aliquot of the aqueous phase is immediately analysed for the test substance; 
then, the experiment continues with the original mixture. The 
volume of each individual aliquot should be less than 1 % of the total volume. The same quantity of fresh 0,01 M CaCl
 2 
solution is added to the mixture to maintain the soil to 
solution ratio, and the agitation continues until the next time 
interval. 
The percentage desorption is calculated at each time point  ðD t i Þ 
and/or time interval  ðD Δt i Þ (according to the needs of the study) 
and is plotted versus time. The desorption coefficient of K  des at 
equilibrium is also calculated. All applicable equations are given in 
Data and Reporting and Appendix 5. 
Results from desorption kinetics experiment Common plots of the percentage desorption
 D t i and adsorption  A t i versus time, allow estimation of the reversibility of the adsorption 
process. If the desorption equilibrium is attained even within twice the time of the adsorption equilibrium, and the total desorption is 
more than 75 % of the amount adsorbed, the adsorption is 
considered to be reversible.
 
1.9.4.3. D e s o r p t i o n i s o t h e r m s 
Freundlich desorption isotherms are determined on the soils used in 
the adsorption isotherms experiment. The desorption test is performed as described in the section ‘Desorption kinetics’, with 
the only difference that the aqueous phase is analysed only once, 
at desorption equilibrium. The amount of the test substance desorbed is calculated. The content of test substance remaining adsorbed on soil at desorption equilibrium is plotted as a function of the equi ­
librium concentration of the test substance in solution (see Data and 
Reporting and Appendix 5).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1067
 
2. DATA AND REPORTING  
The analytical data are presented in tabular form (see Appendix 6). 
Individual measurements and averages calculated are given. Graphical representations of adsorption isotherms are provided. 
The calculations are made as described below. 
For the purpose of the test, it is considered that the weight of 1 cm
 3 
of aqueous solution is 1g. The soil/solution ratio may be expressed 
in units of w/w or w/vol with the same figure.  
2.1. ADSORPTION 
The adsorption  A  t i is defined as the percentage of substance 
adsorbed on the soil related to the quantity present at the beginning of the test, under the test conditions. If the test substance is stable and does not adsorb significantly to the 
container wall,
 A t i is calculated at each time point t  i , according to 
the equation:  
A t i ¼ m s ads ðt i Þ · 100 
m 0 ð%Þ (3) 
where:  
A t i = adsorption percentage at the time point t  i (%); 
m s ads ðt i Þ = mass of the test substance adsorbed on the soil at the 
time t  i (μg); 
m 0 = mass of the test substance in the test tube, at the 
beginning of the test ( μg). 
Detailed information on how to calculate the percentage of 
adsorption  A  t i for the parallel and serial methods is given in 
Appendix 5. 
The distribution coefficient K  d is the ratio between the content of the 
substance in the soil phase and the mass concentration of the 
substance in the aqueous solution, under the test conditions, when 
adsorption equilibrium is reached.  
K d ¼ C s ads ðeqÞ 
C aq ads ðeqÞ ¼ m s ads ðeqÞ 
m aq ads ðeqÞ · V 0 
m soil (cm  3 g -1 ) (4) 
where:  
C s ads ðeqÞ = content of the substance adsorbed on the soil at 
adsorption equilibrium ( μg g -1 ); 
C aq ads ðeqÞ = mass concentration of the substance in the aqueous 
phase at adsorption equilibrium ( μg cm  -3 ). This 
concentration is analytically determined taking into 
account the values given by the blanks;  
m s ads ðeqÞ = mass of the substance adsorbed on the soil at 
adsorption equilibrium ( μg); 
m aq ads ðeqÞ = mass of the substance in the solution at adsorption 
equilibrium ( μg); 
m soil = quantity of the soil phase, expressed in dry mass of 
soil (g); 
V 0 = initial volume of the aqueous phase in contact with 
the soil (cm  3 ). 
The relation between A  eq and K  d is given by:  
K d ¼ A eq 
100 Ä A eq · V 0 
m soil (cm  3 g -1 ) (5) ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1068
 
where: 
A eq = percentage of adsorption at adsorption equilibrium, %. 
The organic carbon normalised adsorption coefficient K  oc relates the 
distribution coefficient K  d to the content of organic carbon of the soil 
sample:  
K oc ¼ K  d · 100 
%OC  (cm  3 g -1 ) (6) 
where: 
%OC = percentage of organic carbon in the soil sample (g g  -1 ). 
K oc coefficient represents a single value which characterises the 
partitioning mainly of non-polar organic chemicals between 
organic carbon in the soil or sediment and water. The adsorption 
of these chemicals is correlated with the organic content of the sorbing solid (7); thus, K
 oc values depend on the specific character ­
istics of the humic fractions which differ considerably in sorption 
capacity, due to differences in origin, genesis, etc.  
2.1.1.  Adsorption isotherms  
The Freundlich adsorption isotherms equation relates the amount of 
the test substance adsorbed to the concentration of the test substance 
in solution at equilibrium (equation 8). 
The data are treated as under ‘Adsorption’ and, for each test tube, 
the content of the test substance adsorbed on the soil after the 
adsorption test ( C s ads ðeqÞ, elsewhere denoted as x/m) is calculated. 
It is assumed that equilibrium has been attained and that  C s ads ðeqÞ 
represents the equilibrium value:  
C s ads ðeqÞ ¼ m s ads ðeqÞ 
m soil ¼ C 0 Ä C aq ads ðeqÞ h i
: V 0 
m soil (μg g  -1 ) (7) 
The Freundlich adsorption equation is shown in (8):  
C s ads ðeqÞ ¼ K  F ads Ö C aq ads ðeqÞ 1=n (μg g  -1 ) (8) 
or in the linear form:  
log C  s ads ðeqÞ ¼ log K  F ads þ 1=n · log C  aq ads ðeqÞ (9) 
where:  
K F ads = Freundlich adsorption coefficient; its dimension is cm  3 g -1 
only if 1/n = 1; in all other cases, the slope 1/n is introduced 
in the dimension of  K F ads (μg 1-1/n (cm  3 ) 1/n g -1 ); 
n = regression constant; 1/n generally ranges between 0,7-1,0 
indicating that sorption data is frequently slightly non- 
linear. 
Equations (8) and (9) are plotted and the values of  K F ads and 1/n are 
calculated by regression analysis using the equation 9. The 
correlation coefficient r  2 of the log equation is also calculated. An 
example of such plots is given in Fig. 2.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1069
 
Fig. 2  Freundlich Adsorption Plot, normal and linearised  
2.1.2.  Mass balance  
The mass balance (MB) is defined as the percentage of substance 
which can be analytically recovered after an adsorption test versus the nominal amount of substance at the beginning of the test. 
The treatment of data will differ if the solvent is completely miscible 
with water. In the case of water-miscible solvent, the treatment of 
data described under ‘Desorption’ may be applied to determine the 
amount of substance recovered by solvent extraction. If the solvent is less miscible with water, the determination of the amount recovered has to be made. 
The mass balance MB for the adsorption is calculated as follows; it 
is assumed that the term (m
 E ) corresponds to the sum of the test 
chemical masses extracted from the soil and surface of the test vessel 
with an organic solvent:  
MB ¼ V rec · C aq ads ðeqÞ þ m  E Ê Ì  
· 100 
V 0 · C 0 ð%Þ (10) 
where: 
MB = mass balance (%); 
m E = total mass of test substance extracted from the soil and 
walls of the test vessel in two steps ( μg); 
C 0 = initial mass concentration of the test solution in contact 
with the soil ( μg cm  -3 ); 
V rec = volume of the supernatant recovered after the adsorption 
equilibrium (cm  -3 ). 
2.2. DESORPTION 
The desorption (D) is defined as the percentage of the test substance 
which is desorbed, related to the quantity of substance previously 
adsorbed, under the test conditions:  
D t i ¼ m aq des ðt i Þ 
m s ads ðeqÞ · 100ð%Þ (11) 
where:  
D t i = desorption percentage at a time point t  i (%); ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1070
 
m aq des ðt i Þ = mass of the test substance desorbed from soil at a time 
point t  i (μg); 
m s ads ðeqÞ = mass of the test substance adsorbed on soil at 
adsorption equilibrium ( μg). 
Detailed information on how to calculate the percentage of 
desorption  D  t i for the parallel and serial methods is given in 
Appendix 5. 
The apparent desorption coefficient (K  des ) is, under the test 
conditions, the ratio between the content of the substance 
remaining in the soil phase and the mass concentration of the 
desorbed substance in the aqueous solution, when desorption equi ­
librium is reached:  
K des ¼ m s ads ðeqÞ Ä m aq des ðeqÞ 
m aq des ðeqÞ V T 
m soil (cm  3 g -1 ) (12) 
where: 
K des = desorption coefficient (cm  3 g -1 ); 
m aq des ðeqÞ = total mass of the test substance desorbed from soil at 
desorption equilibrium ( μg); 
V T = total volume of the aqueous phase in contact with the 
soil during the desorption kinetics test (cm  3 ). 
Guidance for calculating the  m aq des ðeqÞ is given in Appendix 5 under 
the heading ‘Desorption’. 
Remark 
If the adsorption test which was preceded, was performed with the 
parallel method the volume V  T in the equation (12) is considered to 
be equal to V  0 . 
2.2.1.  Desorption isotherms  
The Freundlich desorption isotherms equation relates the content of 
the test substance remaining adsorbed on the soil to the concentration 
of the test substance in solution at desorption equilibrium (equation 16). 
For each test tube, the content of the substance remaining adsorbed 
on soil at desorption equilibrium is calculated as follows:
 
C s des ðeqÞ ¼ m s ads ðeqÞ Ä m aq des ðeqÞ 
m soil (μg g  -1 ) (13) 
m aq des ðeqÞ is defined as:  
m aq des ðeqÞ ¼ m  m des ðeqÞ · V 0 
V r F Ä m  aq A (μg) (14) 
where:  
C s des ðeqÞ = content of the test substance remaining adsorbed on 
the soil at desorption equilibrium ( μg g  -1 ); 
m m des ðeqÞ = mass of substance determined analytically in the 
aqueous phase at desorption equilibrium ( μg); ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1071
 
m aq A = mass of the test substance left over from the 
adsorption equilibrium due to incomplete volume 
replacement ( μg); 
m aq des ðeqÞ = mass of the substance in the solution at adsorption 
equilibrium ( μg); 
m aq A ¼ m  aq ads ðeqÞ · V 0 Ä V R 
V 0 Í Î  (15) 
V r F = volume of the solution taken from the tube for the 
measurement of the test substance, at desorption 
equilibrium (cm  3 ); 
V R = volume of the supernatant removed from the tube after the 
attainment of adsorption equilibrium and replaced by the 
same volume of a 0,01 M CaCl  2 solution (cm  3 ); 
The Freundlich desorption equation is shown in (16):  
C s des ðeqÞ ¼ K  F des · C aq des ðeqÞ 1=n (μg g -1 ) (16) 
or in the linear form:  
log C  s des ðeqÞ ¼ log K  F des þ 1=n · log C  aq des ðeqÞ (17) 
where:  
K F des = Freundlich desorption coefficient; 
n = regression constant;  
C aq des ðeqÞ = mass concentration of the substance in the aqueous 
phase at desorption equilibrium ( μg cm  -3 ). 
The equations (16) and (17) can be plotted and the value of  K F des and 
1/n are calculated by regression analysis using the equation 17. 
Remark: 
if the Freundlich adsorption or desorption exponent 1/n is equal to 1, 
the Freundlich adsorption or desorption binding constant ( K F ads and 
K F des ) will be equal to the adsorption or desorption equilibrium 
constants (K  d and K  des ) respectively, and plots of C  s vs C  aq will 
be linear. If the exponents are not equal to 1, plots of C  s vs C  aq will 
be non-linear and the adsorption and desorption constants will vary 
along the isotherms.  
2.2.2.  Test report  
The test report should include the following information: 
— complete identification of the soil samples used including, 
— geographical reference of the site (latitude, longitude), — date of sampling,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1072
 
— use pattern (e.g. agricultural soil, forest, etc.), 
— depth of sampling, — sand/silt/clay content; — pH values (in 0,01 M CaCl2), 
— organic carbon content, 
— organic matter content, — nitrogen content, — C/N ratio, — cation Exchange Capacity (mmol/kg), 
— all information relating to the collection and storage of soil 
samples, 
— where appropriate, all relevant information for the interpretation 
of the adsorption — desorption of the test substance, 
— reference of the methods used for the determination of each 
parameter, 
— information on the test substance as appropriate, 
— temperature of the experiments, 
— centrifugation conditions, — analytical procedure used to analyse the test substance, — justification for any use of solubilising agent for the preparation 
of the stock solution of the test substance, 
— explanations of corrections made in the calculations, if relevant, — data according to the form sheet (Appendix 6) and graphical 
presentations, 
— all information and observations helpful for the interpretation of 
the test results.
 
3. REFERENCES  
(1) Kukowski H. and Brümmer G., (1987) Investigations on the 
Adsorption and Desorption of Selected Chemicals in Soils. UBA Report 106 02 045, Part II. 
(2) Fränzle O., Kuhnt G. and Vetter L., (1987) Selection of Repre ­
sentative Soils in the EC-Territory. UBA Report 106 02 045, Part I. 
(3) Kuhnt G. and Muntau H. (Eds.) EURO-Soils: Identification, 
Collection, Treatment, Characterisation. Special Publication 
No 1.94.60, Joint Research Centre. European Commission, ISPRA, December 1994. 
(4) OECD Test Guidelines Programme, Final Report of the OECD 
Workshop on Selection of Soils/Sediments, Belgirate, Italy, 18- 20 January 1995 (June 1995). 
(5) US-Environment Protection Agency: Pesticide Assessment 
Guidelines, Subdivision N, Chemistry: Environmental Fate, 
Series 163-1, Leaching and Adsorption/Desorption Studies, 
Addendum 6 on Data Reporting, 540/09-88-096, Date: 1/1988.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1073
 
(6) US-Environment Protection Agency: Prevention, Pesticides and 
Toxic Substances, OPPTS Harmonized Test Guidelines, Series 
835-Fate, Transport and Transformation Test Guidelines, 0PPTS No: 835.1220 Sediment and Soil Adsorption/ ­
Desorption Isotherm. EPA No: 712-C-96-048, April 1996. 
(7) ASTM Standards, E 1195-85, Standard Test Method for Deter ­
mining a Sorption Constant (K
 oc ) for an Organic Chemical in 
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(8) Agriculture Canada: Environmental Chemistry and Fate. 
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 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1078
 
Appendix 1  
Testing scheme
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1079
 
Appendix 2  
INFLUENCE OF ACCURACY OF ANALYTICAL METHOD AND 
CONCENTRATION CHANGE ON ACCURACY OF ADSORPTION 
RESULTS  
From the following table (84) it becomes obvious that when the 
difference between the initial mass (m  0 = 110 μg) and equilibrium mass 
(m aq ads ðeqÞ = 100 μg) of the test substance in the solution is very small, an 
error of 5 % in the measurement of equilibrium concentration results in an error of 50 % in the calculation of the mass of the substance adsorbed in soil 
(
m s ads ðeqÞ) and of 52,4 % in the calculation of the K  d . 
Amount of soil m  soil = 10 g 
Volume of solution V  0 = 100 cm  3 
m aq ads ðeqÞ 
(μg) C aq ads ðeqÞ 
(μg cm  -3 ) R m s ads ðeqÞä 
(μg) C s ads ðeqÞä 
(μg g  -1 ) R‡ K  d * R‡  
FOR A = 9 %
 m 0 ¼ 110 μg or 
C 0 ¼ 1; 100 μg=cm3 
100 1,000 true value 10 1,00 true value 1 
101 1,010 1 % 9 0,90 10 % 0,891 10,9 % 
105 1,050 5 % 5 0,50 50 % 0,476 52,4 % 
109 1,090 9 % 1 0,10 90 % 0,092 90,8 %  
FOR A = 55 %
 m 0 ¼ 110 μg or 
C 0 ¼ 1; 100 μg=cm3 
50,0 0,500 true value 60,0 6,00 true value 12,00 
50,5 0,505 1 % 59,5 5,95 0,8 % 11,78 1,8 % 52,5 0,525 5 % 57,5 5,75 4,0 % 10,95 8,8 % 55,0 0,550 10 % 55,0 5,50 8,3 % 10,00 16,7 %  
FOR A = 99 %
 m 0 ¼ 110 μg or 
C 0 ¼ 1; 100 μg=cm3 
1,100 0,011 true value 108,9 10,89 true value 990 
1,111 0,01111 1 % 108,889 10,8889 0,01 % 980 1,0 % 
1,155 0,01155 5 % 108,845 10,8845 0,05 % 942 4,8 % 
1,21 0,0121 10 % 108,790 10,8790 0,10 % 899 9,2 %  
äm s ads ðeqÞ = m 0 Ä m aq ads ðeqÞ; C s ads ðeqÞ ¼ h 
C 0 Ä C aq ads ðeqÞ i 
V 0 
m soil: Kd ¼ m s ads ðeqÞ 
m aq ads ðeqÞ V 0 
m soil 
m s ads ðeqÞ = mass of the test substance in the soil phase at equilibrium, μg; 
m aq ads ðeqÞ = mass of the test substance in the aqueous phase at equilibrium, μg; 
C s ads ðeqÞ = content of the test substance in the soil phase at equilibrium, μg g -1 ; 
C aq ads ðeqÞ = mass concentration of the test substance in the aqueous phase at 
equilibrium, μg cm  -3 ; 
R = analytical error in the determination of the  m aq ads ðeqÞ; 
R‡ = calculated error due to the analytical error R.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1080
 
Appendix 3  
ESTIMATION TECHNIQUES FOR K  D 
1. Estimation techniques permit prediction of K  d based on correlations with, 
for example, P  ow values (12)(39)(63-68), water solubility data 
(12)(19)(21)(39)(68-73), or polarity data derived by application of HPLC 
on reversed phase (74-76). As shown in Tables 1 and 2, is the K  oc or K  om 
that are calculated from these equations and then, indirectly, the K  d from 
the equations:  
K oc ¼ K  d · 100 
%oc ðcm 3 g Ä1 Þ K  om ¼ K d 
1; 724 · 100 
%oc ðcm 3 g Ä1 Þ 
2. The concept of these correlations is based on two assumptions: (1) it is the 
organic matter of the soil that mainly influences the adsorption of a 
substance; and (2) the interactions involved are mainly non-polar. As a 
result, these correlations: (1) are not, or are only to some extent, applicable to polar substances, and (2) are not applicable in cases where the organic matter content of the soil is very small (12). In addition, although satis ­
factory correlations have been found between P
 ow and adsorption (19), the 
same cannot be said for the relationship between water solubility and extent of adsorption (19)(21); so far the studies are very contradictory. 
3. Some examples of correlations between the adsorption coefficient and the 
octanol-water partition coefficient, as well as water solubility are given in 
Tables 1 and 2, respectively.
 
Table 1  
Examples of correlations between the adsorption distribution coefficient and the octanol-water partition 
coefficient; for further examples (12) (68).  
Substances Correlations Authors  
Substituted ureas log K  om = 0,69 + 0,52 log P  ow Briggs (1981) (39) 
Aromatic chlorinated log K  oc = - 0,779 + 0,904 log P  ow Chiou  et al . (1983) (65) 
Various pesticides log K  om = 4,4 + 0,72 log P  ow Gerstl and Mingelgrin (1984) (66) 
Aromatic hydrocarbons log K  oc = - 2,53 + 1,15 log P  ow Vowles and Mantoura (1987) (67)  
Table 2  
Examples of correlations between the adsorption distribution coefficient and water solubility; for further 
examples see (68) (69).  
Compounds Correlations Authors  
Various pesticides log K  om = 3,8 - 0,561 log S  w Gerstl and Mingelgrin (1984) (66) 
Aliphatic, aromatic chlor ­
inated substances log K  om = (4,040 +/- 0,038) - (0,557 
+/- 0,012) log S  w Chiou  et al . (1979) (70) 
α-naphtol logK  oc = 4,273 - 0,686 log S  w Hasset et al. (1981) (71) 
Cyclic, aliphatic aromatic 
substances logK  oc = - 1,405 - 0,921 log S  w - 
0,00953 (mp-25) Karickhoff (1981) (72) 
Various compounds log K  om = 2,75 - 0,45 log S  w Moreale van Blade (1982) (73)  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1081
Appendix 4  
CALCULATIONS FOR DEFINING THE CENTRIFUGATION 
CONDITIONS  
1. The centrifugation time is given by the following formula, assuming 
spherical particles:  
t ¼ 9 
2 η 
ω 2 rp 2 ρ s Ä ρ aq Ä Ö " #  
ln ðR b=R t Þ (1) 
For simplification purposes, all parameters are described in non-SI units (g, cm). 
where: 
ω = rotational speed (=2 π rpm/60), rad s  -1 
rpm = revolutions per minute 
η = viscosity of solution, g s  -1 cm -1 
r p = particle radius, cm 
ρ s = soil density, g cm  -3 
ρ aq = solution density, g cm  -3 
R t = distance from the centre of centrifuge rotor to top of solution in 
centrifuge tube, cm 
R b = distance from the centre of centrifuge rotor to bottom in 
centrifuge tube, cm 
R b -R t =length of the soil/solution mixture in the centrifuge tube, cm. 
In general practice, double the calculated times is used to ensure complete 
separation. 
2. The equation (1) can be simplified further if we consider the viscosity ( η) 
and the density ( ρ aq ) of the solution as equal to the viscosity and density of 
water at 25  o C; thus, η = 8,95 × 10  -3 g s -1 cm -1 and ρ aq = 1,0 g. cm  -3 . 
Then, the centrifugation time is given by the equation (2):  
t ¼ 3:7 
rpmð Þ  2 · r p 2 ρ s Ä 1ð Þ  ln Rb 
Rt (2) 
3. From the equation (2) it becomes apparent that two parameters are 
important in defining the centrifugation condition, i.e. time (t) and speed (rpm), in order to achieve separation of particles with a specific size (in our 
case 0,1 μm radius): (1) the density of the soil and (2) the length of the 
mixture in the centrifuge tube (R
 b -R t ), i.e. the distance which a soil particle 
covers from the top of the solution to the bottom of the tube; obviously, for a fixed volume the length of the mixture in the tube will depend on the 
square of the radius of the tube. 
4. Fig. 1 presents variations in the centrifugation time (t) versus centrifugation 
speed (rpm) for different soil densities ( ρ
 s ) (Fig.1a) and different lengths of 
the mixture in the centrifuge tubes (Fig.2a). From Fig.1a the influence of 
the soil density appears obvious; for example, for a classical centrifugation of 3 000 rpm the centrifugation time is approximately 240 min for 1,2 g cm
 3 soil density, while it is only 50 min for 2,0 g cm  3 . Similarly, 
from Fig 1b, for a classical centrifugation of 3 000 rpm the centrifugation 
time is approximately 50 min for a length of the mixture of 10 cm and only seven min for a length of 1 cm. However, it is important to find an optimal relation between centrifugation which requires the less length possible and 
easy handling for the experimenter in separating the phases after centrifu ­
gation.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1082
 
5. Moreover, when defining the experimental conditions for the separation of 
soil/solution phases, it is important to consider the possible existence of a 
third ‘pseudo-phase’, the colloids. These particles, with a size less than 0,2 μm, can have an important impact on the whole adsorption 
mechanism of a substance in a soil suspension. When centrifugation is 
performed as described above, colloids remain in the aqueous phase and 
are subjected to analysis together with the aqueous phase. Thus, the information about their impact is lost. 
If the conducting laboratory has ultracentrifugation or ultrafiltration facil ­
ities, the adsorption/desorption of a substance in soil could be studied more 
in depth, including information on the adsorption of the substance on the 
colloids. In this case, an ultracentrifugation at 60 000 rpm/min or an ultra ­
filtration with filter porosity of 100 000 Daltons should be applied in order to separate the three phases soil, colloids, solution. The test protocol should 
also be modified accordingly, in order all three phases to be subjected to 
substance analysis.
 
Fig. 1a.
  
Variations of centrifugation time (t) versus centrifugation speed (rpm) for 
different soil densities ( ρ s ). R  t = 10 cm, R  b - R t = 10 cm, η = 8,95 × 10  -3 g 
s -1 cm -1 and ρ aq = 1,0 g. cm  -3 at 25  o C. 
Fig. 1b.
  
Variations of centrifugation time (t) versus centrifugation speed (rpm) 
for different lengths of the mixture in the centrifuge tube (R  b - R  t ) = L; 
R t = 10 cm, η = 8,95 × 10  -3 g s -1 cm -1 , ρ aq = 1,0 g. cm  -3 at 25  o C and 
ρ s = 2,0 g cm  -3 . ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1083
 
Appendix 5  
CALCULATION OF ADSORPTION A (%) AND DESORPTION D (%)  
The time scheme of the procedure is:
  
For all the calculations it is assumed that the test substance is stable and does not 
adsorb significantly to the container walls.  
ADSORPTION A (A%)  
a) Parallel method  
The percentage adsorption is calculated for each test tube (i) at each time 
point (t  i ), according to the equation:  
A t i ¼ m s ads ðt i Þ · 100 
m 0 (%) (1) 
The terms of this equation may be calculated as follows: 
m 0 = C  0 · V 0 (μg) (2)  
m s ads ðt i Þ ¼ m  0 Ä C aq ads ðt i Þ · V 0 (μg) (3) 
where:  
A t i = adsorption percentage (%) at the time point t  i 
m s ads ðt i Þ = mass of the test substance on soil at the time t  i that the 
analysis is performed ( μg) 
m 0 = mass of test substance in the test tube, at the beginning of the 
test (μg) 
C 0 = initial mass concentration of the test solution in contact with 
the soil ( μg cm  -3 ) ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1084
 
C aq ads ðt i Þ = mass concentration of the substance in the aqueous phase at 
the time t  i that the analysis is performed ( μg cm  -3 ); this 
concentration is analytically determined taking into account the values given by the blanks 
V
 0 = initial volume of the test solution in contact with the 
soil (cm  3 ). 
The values of the adsorption percentage  A t i or C aq ads ðt i Þ are plotted versus 
time and the time after which the sorption equilibrium is attained is deter ­
mined. Examples of such plots are given in Fig. 1 and Fig. 2 respectively.
  
Fig. 1.  
Adsorption equilibrium plot
  
Fig. 2.  
Mass concentration of the test substance in the aqueous phase (C  aq ) versus time  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1085
 
b) Serial method  
The following equations take into account that the adsorption procedure is carried out by measurements of the test 
substance in small aliquots of the aqueous phase at specific time intervals. 
During each time interval the amount of the substance adsorbed on the soil is calculated as follows: 
— for the first time interval Δt 1 = t 1 - t 0 
m s ads ðΔt 1 Þ ¼ m  0 Ä m m ads ðt 1 Þ · Í 
V 0 
v a A Î (4) 
— for the second time interval Δt 2 = t 2 - t 1 
m s ads ðΔt 2 Þ ¼ m  m ads ðt 1 Þ · Í 
V 0 
v a A Î 
Ä m  m ads ðt 2 Þ · Í 
V 0 Ä v a A 
v a A Î (5) 
— for the third time interval Δt 3 = t 3 - t 2 
m s ads ðΔt 3 Þ ¼ m m ads ðt 2 Þ · Í 
V 0 Ä v a A 
v a A Î 
Ä m  m ads ðt 3 Þ · Í 
V 0 Ä 2·v a A 
v a A Î (6) 
— for the nth time interval Δt n = t n - t n - 1 
m s ads ðΔt n Þ ¼ m  m ads ðt nÄ1 Þ · Í 
V 0 Ä ðn Ä 2Þ · v a A 
v a A Î 
Ä m m ads ðt n Þ · V 0 Ä ðn Ä 1Þ · v a A Ê Ì  
v a A 0 
@ 1 
A (7) 
The percentage of adsorption at each time interval,  A Δt i , is calculated 
using the following equation:  
A Δt i ¼ m s ads ðΔt i Þ 
m 0 · 100ð%Þ (8) 
while the percentage of adsorption  ðA t i Þ at a time point t  i is given by 
the equation:  
A t i ¼ Σ 
j ¼ Δt 1 Δt i 
m s ads ðjÞ 
m 0 · 100 ð%Þ (9) 
The values of the adsorption  A t i or A Δt i (with respect to the needs of 
the study) are plotted versus time and the time after which the sorption equilibrium is attained is determined. 
At the equilibration time t
 eq : 
— the mass of the test substance adsorbed on the soil is:  
m s ads ðeqÞ ¼ Σ n 
Δt i ¼ 1 m s ads ðΔt i Þ (10) ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1086
 
— the mass of the test substance in the solution is:  
m aq ads ðeqÞ ¼ m  0 Ä Σ n 
Δt i ¼ 1 m s ads ðΔt i Þ (11) 
— and the percentage of adsorption at equilibrium is:  
A eq ¼ m s ads ðeqÞ 
m 0 · 100ð%Þ (12) 
The parameters used above are defined as:  
m s ads ðΔt 1 Þ; m s ads ðΔt 2 Þ; :::; m s ads ðΔt n Þ = mass of the substance adsorbed 
on the soil during the time 
intervals Δ t1 , Δ t2 ,..., Δ tn 
respectively ( μg); 
m m ads ðt 1 Þ; m m ads ðt 2 Þ; :::; m n ads ðt n Þ = mass of the substance measured 
in an aliquot  v a A at the time 
points t  1 , t 2 ,t n respectively ( μg); 
m s ads ðeqÞ = mass of the substance adsorbed 
on the soil at adsorption equi ­
librium (μg); 
m aq ads ðeqÞ = mass of the substance in the 
solution at adsorption equi ­
librium (μg); 
v a A = volume of the aliquot in which 
the test substance is measured (cm
 3 ); 
A Δt i = percentage of adsorption 
corresponding at a time interval Δt
 i (%); 
A eq = percentage of adsorption at 
adsorption equilibrium (%).  
DESORPTION D (A %) 
The time t  0 that the desorption kinetics experiment begins, is considered as the 
moment that the maximal recovered volume of the test substance solution (after that the adsorption equilibrium is attained) is replaced by an equal volume of 0,01 M CaCl
 2 solution.  
(a) Parallel method  
At a time point t  i , the mass of the test substance is measured in the aqueous 
phase taken from the tube i  V  i 
r Ê Ì  
, and the mass desorbed is calculated 
according to the equation:  
m aq des ðt i Þ ¼ m  m des ðt i Þ · Í 
V 0 
v r i Î 
Ä m aq A (13) 
At desorption equilibrium t  i = t eq and therefore  m aq des ðt i Þ = m aq des ðeqÞ 
The mass of the test substance desorbed during a time interval ( Δt i ) is given 
by the equation:  
m aq des ðΔt i Þ ¼ m  aq des ðt i Þ Ä Σ 
j ¼ 1 i ¼ 1 
m aq des ðjÞ (14) 
The percentage of desorption is calculated: 
at a time point t  i from the equation:  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1087
 
D t i ¼ m aq des ðt i Þ 
m s ads ðeqÞ · 100 ð%Þ (15) 
and during a time interval ( Δt i ) from the equation:  
D Δt i ¼ m aq des ðΔt i Þ 
m s ads ðeqÞ · 100 ð%Þ (16) 
where:  
D t i = desorption percentage at a time point t  i (%) 
D Δt i = desorption percentage corresponding to a time interval 
Δt i (%) 
m aq des ðt 1 Þ = mass of the test substance desorbed at a time point t  i , (μg) 
m aq des ðΔt 1 Þ = mass of the test substance desorbed during a time interval 
Δt i (μg) 
m m des ðt i Þ = mass of the test substance analytically measured at a time t  i 
in a solution volume  V r i , which is taken for the analysis 
(μg) 
m aq A = mass of the test substance left over from the adsorption 
equilibrium due to incomplete volume replacement ( μg) 
m aq A ¼ m aq ads ðeqÞ · V 0 Ä V R 
V 0 Í Î  (17) 
m aq ads ðeqÞ = mass of the test substance in the solution at adsorption 
equilibrium ( μg) 
V R = volume of the supernatant removed from the tube after the 
attainment of adsorption equilibrium and replaced by the same volume of a 0,01 M CaCl
 2 solution (cm  3 ) 
V r i = volume of the solution taken from the tube (i) for the 
measurement of the test substance, in desorption kinetics 
experiment (cm  3 ). 
The values of desorption  D t i or D Δt i (according to the needs of the study) 
are plotted versus time and the time after which the desorption equilibrium is attained is determined.
 
(b) Serial method  
The following equations take into account that the adsorption procedure, 
which was preceded, was carried out by measurement of test substance in 
small aliquots  v  a A Ê Ì  
of the aqueous phase (serial method in ‘Performance 
of the test’ 1,9). It is assumed that: (a) the volume of the supernatant 
removed from the tube after the adsorption kinetics experiment was replaced by the same volume of 0,01 M CaCl
 2 solution (V  R ) and (b) 
and the total volume of the aqueous phase in contact with the soil (V  T ) 
during the desorption kinetics experiment remains constant and is given by the equation:
 
V T ¼ V  0 Ä Σ n 
i ¼ 1 v a A ðiÞ (18) ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1088
 
At a time point t  i : 
— The mass of the test substance is measured in a small aliquot  v  a D Ê Ì  
and 
the mass desorbed is calculated, according to the equation:  
m aq des ðt i Þ ¼ m  m des ðt i Þ · Í 
V T 
v a D Î 
Ä m  aq A · V T Ä ði Ä 1Þ · v a D Ê Ì  
V T 0 
@ 1 
A (19) 
— At desorption equilibrium t  i = t eq and therefore  m aq des ðt i Þ = m aq des ðeqÞ. 
— The percentage of desorption  D  t i is calculated, from the following 
equation:  
D t i ¼ m aq des ðt i Þ 
m s ads ðeqÞ · 100 ð%Þ (20) 
At a time interval ( Δt i ): 
During each time interval the amount of the substance desorbed is 
calculated as follows: 
— for the first time interval Δt 1 = t 1 -t 0 
m aq des ðΔt 1 Þ ¼ m  m des ðt 1 Þ · Í 
V T 
v a D Î 
Ä m  aq A and m  s des ðt 1 Þ ¼ m  s aq ðeqÞ Ä m aq des ðΔt 1 Þ (21) 
— for the second time interval Δt 2 = t 2 -t 1 
m aq des ðΔt 2 Þ ¼ m  m des ðt 2 Þ · Í 
V T 
v a D Î 
Ä m  aq des ðΔt 1 Þ · V T Ä v a D Ê Ì  
V T 0 
@ 1 
A Ä m  aq A · V T Ä v a D Ê Ì  
V T 0 
@ 1 
A 
and 
m s des ðt 2 Þ ¼ m  s ads ðeqÞ Ä m  aq des ðΔt 1 Þ þ m aq des ðΔt 2 Þ h i  
(22) 
— for the n  th interval Δt n = t n -t n-1 
m aq des ðΔt n Þ ¼ Ï 
m m des ðt n Þ · Í 
V T 
v a D Î 
Ä m  aq A · V T Ä n Ä 1ð Þ  · v a D Ê Ì  
V T 0 
@ 1 
A 
Ä Σ 
i ¼ 1; n ≠ 1 nÄ1 V T Ä n Ä ið Þ  · v a D Ê Ì  
V T · m aq des ðΔt i Þ 0 
@ 1 
A # 
and 
m s des ðt n Þ ¼ m  s ads ðeqÞ Ä Σ 
i ¼ 1; n ≠ 1 n 
m aq des ðΔt i Þ (23) 
Finally, the percentage of desorption at each time interval,  D  Δt i , is 
calculated using the following equation:  
D Δt i ¼ m aq des ðΔt i Þ 
m s ads ðeqÞ · 100 ð%Þ (24) 
while the percentage of desorption  D t i at a time point t  i is given by the 
equation:  
D t i ¼ Σ 
j ¼ Δt 1 Δt i 
m aq des ðjÞ 
m s ads ðeqÞ · 100 ¼ m aq des ðt i Þ 
m s ads ðeqÞ · 100 ð%Þ (25) ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1089
 
where the above used parameters are defined as:  
m s des ðΔt 1 Þ, m s des ðΔt 2 Þ, ..., m s des ðΔt n Þ = mass of the substance remaining 
adsorbed on the soil after the 
time intervals Δt 1 , Δt 2 , ..., Δt n 
respectively ( μg) 
m aq des ðΔt 1 Þ, m aq des ðΔt 2 Þ, ..., m aq des ðΔt n Þ = mass of the test substance 
desorbed during the time 
intervals Δt 1 , Δt 2 , ..., Δt n 
respectively ( μg) 
m m des ðt 1 Þ, m m des ðt 2 Þ, ..., m m des ðt n Þ = mass of the substance measured in 
an aliquot  v  D 
a Ê Ì  
at time points 
t 1 ,t 2 , ..., t  n , respectively ( μg) 
V T = total volume of the aqueous 
phase in contact with the soil during the desorption kinetics experiment performed with the 
serial method (cm
 3 ) 
m A 
aq = mass of the test substance left 
over from the adsorption equi ­
librium due to incomplete 
volume replacement ( μg) 
m A 
aq ¼ V 0 Ä Σ n 
i ¼ 1 v A 
a ið Þ Í Î 
Ä V R 
V 0 Ä Σ n 
i ¼ 1 v A 
a ið Þ Í Î 0 
B B B @ 1 
C C C A · m
 ads 
aq ðeqÞ (26) 
V R = volume of the supernatant 
removed from the tube after the 
attainment of adsorption equi ­
librium and replaced by the same volume of a 0,01 M CaCl
 2 
solution (cm  3 ) 
v D 
a = volume of the aliquot sampled for 
analytical purpose from the tube (i), during the desorption kinetics experiment performed with the 
serialmethod (cm
 3 ) 
v D 
a ≤ 0; 02 · V T (27) ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1090
 
Appendix 6  
ADSORPTION-DESORPTION IN SOILS: DATA REPORTING SHEETS  
Substance tested: 
Soil tested: 
Dry mass content of the soil (105  o C, 12h): . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . % 
Temperature: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  o C 
Suitability of the analytical method  
Weighed soil g 
Soil: dry mass g Volume CaCl
 2 sol. cm  3 
Nominal conc. final sol. μg cm  -3 
Analytical conc. final sol. μg cm  -3 
Principle of the analytical method used: 
Calibration of the analytical method: 
Substance tested: Soil tested 
Dry mass content of the soil (105
 o C, 12 h): . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . . . . . . . . . % 
Temperature: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  o C 
Analytical methodology followed: Indirect  □ Parallel  □ Serial  □ 
Direct □ 
Adsorption test: test samples  
Symbol Units Equilibration 
time Equilibration 
time Equilibration 
time Equilibration 
time 
Tube No 
Weighed soil — g Soil: dry mass m
 soil g 
Water volume in weighed soil 
(calculated) V WS cm 3 
Volume 0,01 M CaCl  2 sol. to 
equilibrate the soil cm 3 
Volume of stock solution cm  3 
Total volume of aq. phase in 
contact with soil V 0 cm 3 
Initial concentration Test solution C  0 μg cm  -3 
Mass test subst. at the beginning 
of the test m 0 μg ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1091
 
Symbol Units Equilibration 
time Equilibration 
time Equilibration 
time Equilibration 
time 
After agitation and centrifugation  
INDIRECT METHOD 
Parallel method 
Concentration test subst. aq. 
phase Blank correction included  C aq ads ðt i Þ μg cm  -3 
Serial method 
Measured mass test subst. in the 
aliquot V  a A m m ads ðt i Þ μg 
DIRECT METHOD 
Mass test substance adsorbed on 
soil m s ads ðt i Þ μg 
Calculation of adsorption 
Adsorption  A  t i % 
A Δt i % 
Means 
Adsorption coefficient K  d cm 3 g -1 
Means 
Adsorption coefficient K  oc cm 3 g -1 
Means 
Substance tested: 
Soil tested: Dry mass content of the soil (105
 o C, 12 h): . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . . . . . . . . . % 
Temperature: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  o C 
Adsorption test: blanks and control  
Symbol Units Blank Blank Control  
Tube No 
Weighed soils g 0 0 Water amount in weighed soil (calcu ­
lated) cm
 3 — — 
Volume of 0,01 M CaCl  2 solution 
added cm 3 
Volume of the stock solution of the 
test substance added cm 3 0 0 
Total volume of aq. phase (calculated) cm  3 — —  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1092
 
Symbol Units Blank Blank Control  
Initial concentration of the test 
substance in aqueous phase μg cm  -3 
After agitation and centrifugation  
Concentration in aqueous phase μg cm  -3 
Remark: add columns if necessary 
Substance tested: Soil tested: Dry mass content of the soil (105
 o C 12 h): . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . % 
Temperature: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  o C 
Mass balance  
Symbol Units  
Tube No 
Weighed soil — g 
Soil: dry mass ms  oil g 
Water volume in weighed soil (calcu ­
lated) V WS ml 
Volume 0,01 M CaCl  2 sol. to equilibrate 
the soil ml 
Volume of stock solution cm  3 
Total volume of aq. phase in contact 
with soil V 0 cm 3 
Initial concentration test solution C  0 μg cm  -3 
Equilibration time — h  
After agitation and centrifugation  
Concentr. test subst. aq. phase at 
adsorption equilibrium blank correction included
 C aq ads ðeqÞ μg cm  -3 
Equalibration time t  eq h 
1st dilution with solvent 
Removed volume aq. phase V  rec cm 3 
Added volume of solvent ΔV cm  3 
1st extraction with solvent 
Signal analysed in solvent S  E1 var. 
Conc. test subst. in solvent C  E1 μg cm  -3 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1093
 
Symbol Units  
Mass of substance extracted from soil 
and vessel walls m E1 μg 
2nd dilution with solvent 
Removed volume of solvent ΔV s cm 3 
Added volume of solvent ΔV' cm  3 
2nd extraction with solvent 
Signal analysed in solvent phase S  E2 var. 
Conc. test subst. in solvent C  E2 μg cm  -3 
Mass of substance extracted from soil 
and vessel walls m E2 μg 
Total mass test subst. extracted in two 
steps m E μg 
Mass balance MB % 
Substance tested: 
Soil tested: 
Dry mass content of the soil (105  o C, 12 h): . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . . . . . . . . . % 
Temperature: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  o C 
Adsorption isotherms  
Symbol Units  
Tube No 
Weighed soil — g 
Soil: dry mass E g Water volume in weighed soil 
(calculated) V
 WS cm 3 
Volume 0,01 M CaCl  2 sol. to 
equilibrate the soil cm 3 
Volume of stock solution added cm  3 
Total volume of aq. phase in 
contact with soil (calculated) V 0 cm 3 
Concentration solution C  0 μg cm  -3 
Equilibration time — h  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1094
 
Symbol Units  
After agitation and centrifugation  
Concentration subst. aq. phase, 
blank correction included  C aq ads ðeqÞ μg cm  -3 
Temperature  o C 
Adsorb. mass per unit soil  C s ads ðeqÞ μg g  -1 
Regression analysis: 
value of K  F ads : 
value of l/n: regression coefficient r
 2 : 
Substance tested: 
Soil tested: 
Dry mass content of the soil (105  o C, 12 h): . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . . . . . . . . . % 
Temperature: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  o C 
Analytical methodology followed: Indirect  □ Parallel  □ Serial  □ 
Desorption test  
Symbol Units Time 
interval Time 
interval Time 
interval Time 
interval  
Tube No coming from adsorption step 
Mass of substance adsorbed on soil at 
adsorption equilibrium  m s ads ðeqÞ μg 
Removed volume aq. phase, replaced by 
0,01 M CaCl  2 V R cm 3 
Total volume of aq. phase 
in contact with soil PM V  0 cm 3 
SM V  T cm 3 
Mass test subst. left over the adsorption 
equilibrium due to incomplete volume replacement
 m aq A μg 
Desorption kinetics  
Measured mass of substance desorbed 
from soil at time t  i m m des ðt i Þ μg 
Volume of the solution 
taken from the tube (i) for 
the measurement of the 
test substance PM V  r i cm 3 
SM v  a D cm 3 
Mass of substance desorbed from soil at 
time t  i (calculated)  m aq des ðt i Þ μg 
Mass of substance desorbed from soil 
during time interval Δt i (calculated)  m aq des ðΔt i Þ μg ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1095
 
Symbol Units Time 
interval Time 
interval Time 
interval Time 
interval  
Desorption percentage  
Desorption at time t  i D ti % 
Desorption at time interval Δt i D Δt i % 
Apparent desorption coefficient K  des 
PM: parallel method 
SM: serial method  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1096
 
C.19.  ESTIMATION OF THE ADSORPTION COEFFICIENT ( K OC ) 
ON SOIL AND ON SEWAGE SLUDGE USING HIGH 
PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)  
1. METHOD  
This method is a replicate of OECD TG121 (2001).  
1.1. INTRODUCTION 
The sorption behaviour of substances in soils or sewage sludges can 
be described through parameters experimentally determined by means of the test method C.18. An important parameter is the 
adsorption coefficient which is defined as the ratio between the 
concentration of the substance in the soil/sludge and the concen ­
tration of the substance in the aqueous phase at adsorption equi ­
librium. The adsorption coefficient normalised to the organic 
carbon content of the soil K
 oc is a useful indicator of the binding 
capacity of a chemical on organic matter of soil and sewage sludge and allows comparisons to be made between different chemicals. This parameter can be estimated through correlations with the 
water solubility and the n-octanol/water partition coefficient (1)(2) ­
(3)(4)(5)(6)(7). 
The experimental method described in this test uses HPLC for the 
estimation of the adsorption coefficient K
 oc in soil and in sewage 
sludge (8). The estimates are of higher reliability than those from 
QSAR calculations (9). As an estimation method it cannot fully replace batch equilibrium experiments used in the test method C18. However, the estimated K
 oc may be useful for choosing appro ­
priate test parameters for adsorption/desorption studies according to 
the test method C.18 by calculating K  d (distribution coefficient) or 
K f (Freundlich adsorption coefficient) according to the equation 3 
(see Section 1.2).  
1.2. DEFINITIONS  
K d : distribution coefficient is defined as the ratio of equilibrium 
concentrations C of a dissolved test substance in a two phase system consisting of a sorbent (soil or sewage sludge) and an aqueous phase; it is a dimensionless value when concentrations in 
both phases are expressed on a weight/weight base. In case the 
concentration in the aqueous phase is given on a weight/volume base then the units are ml · g
 -1 . K d can vary with sorbent properties 
and can be concentration dependent.  
K d ¼ C soil 
C aq or C sludge 
C aq (1) 
where: 
C soil = concentration of test substance in soil at equilibrium 
(μg· g -1 ) 
C sludge  = concentration of test substance in sludge at equilibrium 
(μg· g -1 ) 
C aq = concentration of test substance in aqueous phase at 
equilibrium ( μg· g -1 , μg· ml  -1 ). ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1097
 
K f : Freundlich adsorption coefficient is defined as the concentration 
of the test substance in soil or sewage sludge (x/m) when the equi ­
librium concentration C  aq in the aqueous phase is equal to one; units 
are μg.g -1 sorbent. The value can vary with sorbent properties.  
log x 
m ¼ log K  f þ 1 
n · log C  aq (2) 
where: 
x/m = amount of test substance x ( μg) adsorbed on amount of 
sorbent m (g) at equilibrium 
1/n = slope of Freundlich adsorption isotherm C
 aq = concentration of test substance in aqueous phase at equi ­
librium (μg· ml-  1 ) 
At C  aq ¼ 1; log K  f ¼ log x 
m 
K oc : distribution coefficient (K  d ) or Freundlich adsorption coefficient 
(K f ) normalised to the organic carbon content (f  oc ) of a sorbent; 
particularly for non-ionised chemicals, it is an approximate indicator for the extent of adsorption between a substance and the sorbent and allows comparisons to be made between different 
chemicals. Depending on the dimensions of K
 d and K  f , K oc can be 
dimensionless or have the units ml · g -1 or μg· g -1 organic matter.  
Koc ¼ Kd 
foc dimensionless or ml ·g Ä1 Ê Ì  
or Kf 
foc μg · g Ä1 Ê Ì  (3) 
The relationship between K  oc and K  d is not always linear and thus 
K oc values can vary from soil to soil but their variability is greatly 
reduced compared to K  d or K  f values. 
The adsorption coefficient (K  oc ) is deduced from the capacity factor (k') 
using a calibration plot of log k' versus log K  oc of the selected reference 
compounds.  
k 0 ¼ t R Ä t 0 
t 0 (4) 
where: 
t R = HPLC retention time of test and reference substance (min ­
utes); 
t 0 = HPLC dead time (minutes) (see Section 1.8.2).  
P OW : The octanol-water partition coefficient is defined as the ratio of 
the concentrations of dissolved substance in n-octanol and water; it is 
a dimensionless value.  
Pow ¼ Coctanol 
Caq ð¼ KowÞ (5) 
1.3. REFERENCE SUBSTANCES 
The structural formula, the purity and the dissociation constant (if 
appropriate) should be known before using the method. Information 
on solubility in water and organic solvents, on octanol-water 
partition coefficient and on hydrolysis characteristics is useful.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1098
 
To correlate the measured HPLC-retention data of a test substance 
with its adsorption coefficient K  oc , a calibration graph of log K  oc 
versus log k' has to be established. A minimum of six reference 
points, at least one above and one below the expected value of the test substance should be used. The accuracy of the method will be 
significantly improved if reference substances that are structurally 
related to the test substance are used. If such data are not available, it is up to the user to select the appropriate calibration substances. A more general set of structurally heterogeneous 
substances should be chosen in this case. Substances and 
K
 oc -values which may be used are listed in the Appendix in Table 
1 for sewage sludge and in Table 3 for soil. The selection of other calibration substances should be justified.
 
1.4. PRINCIPLE OF THE TEST METHOD 
HPLC is performed on analytical columns packed with a commer ­
cially available cyanopropyl solid phase containing lipophilic and polar moieties. A moderately polar stationary phase based on a 
silica matrix is used: 
— O — Si — CH
 2 — CH  2 — CH  2 — CN 
silica non-polar spacer polar moiety 
The principle of the test method is similar to testing method A.8 
(Partition coefficient, HPLC method). While passing through the column along with the mobile phase the test substance interacts 
with the stationary phase. As a result of partitioning between 
mobile and stationary phases the test substance is retarded. The dual composition of the stationary phase having polar and non- polar sites allows for interaction of polar and non-polar groups of 
a molecule in a similar way as is the case for organic matter in soil 
or sewage sludge matrices. This enables the relationship between the retention time on the column and the adsorption coefficient on organic matter to be established. 
pH has a significant influence on sorption behaviour in particular for 
polar substances. For agricultural soils or tanks of sewage treatment 
plants pH normally varies between pH 5,5 and 7,5. For ionisable 
substances, two tests should be performed with both ionised and non-ionised forms in appropriate buffer solutions but only in cases where at least 10 % of the test compound will be dissociated within 
pH 5,5 to 7,5. 
Since only the relationship between the retention on the HPLC 
column and the adsorption coefficient is employed for the evaluation, 
no quantitative analytical method is required and only the deter ­
mination of the retention time is necessary. If a suitable set of reference substances is available and standard experimental conditions can be used, the method provides a fast and efficient 
way to estimate the adsorption coefficient K
 oc . 
1.5. APPLICABILITY OF THE TEST 
The HPLC method is applicable to chemical substances (unlabelled 
or labelled) for which an appropriate detection system (e.g. spec ­
trophotometer, radioactivity detector) is available and which are 
sufficiently stable during the duration of the experiment. It may be particularly useful for chemicals which are difficult to study in other experimental systems (i.e. volatile substances; substances which are 
not soluble in water at a concentration which can be measured 
analytically; substances with a high affinity to the surface of incu ­
bation systems). The method can be used for mixtures which give unresolved elution bands. In such a case, upper and lower limits of 
the log K
 oc values of the compounds of the test mixture should be 
stated.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1099
 
Impurities may sometimes cause problems for interpretation of 
HPLC results, but they are of minor importance as long as the test 
substance can analytically be clearly identified and separated from the impurities. 
The method is validated for the substances listed in Table 1 in the 
Appendix and was also applied to a variety of other chemicals 
belonging to the following chemical classes: 
— aromatic amines (e.g. trifluralin, 4-chloroaniline, 3,5-dini ­
troaniline, 4-methylaniline, N-methylaniline, 1-naphthylamine), 
— aromatic carboxilic acid esters (e.g. benzoic acid methylester, 
3,5-dinitrobenzoic acid ethylester), 
— aromatic hydrocarbons (e.g. toluene, xylene, ethylbenzene, nitro ­
benzene), 
— aryloxyphenoxypropionic acid esters (e.g. diclofop-methyl, 
fenoxaprop-ethyl, fenoxaprop-P-ethyl), 
— benzimidazole and imidazole fungicides (e.g. carbendazim, 
fuberidazole, triazoxide), 
— carboxilic acid amides (e.g. 2-chlorobenzamide, N,N-dimethyl ­
benzamide, 3,5-dinitrobenzamide, N-methylbenzamide, 2-nitro ­
benzamide, 3-nitrobenzamide), 
— chlorinated hydrocarbons (e.g. endosulfan, DDT, hexachloro ­
benzene, quintozene, 1,2,3-trichlorobenzene), 
— organophosphorus insecticides (e.g. azinphos-methyl, disulfoton, 
fenamiphos, isofenphos, pyrazophos, sulprofos, triazophos), 
— phenols (e.g. phenol, 2-nitrophenol, 4-nitrophenol, pentachlorop ­
henol, 2,4,6-trichlorophenol, 1-naphthol), 
— phenylurea derivatives (e.g. isoproturon, monolinuron, pency ­
curon), 
— pigment dyestuffs (e.g. Acid Yellow 219, Basic Blue 41, Direct 
Red 81), 
— polyaromatic hydrocarbons (e.g. acenaphthene, naphthalene), 
— 1,3,5-triazine herbicides (e.g. prometryn, propazine, simazine, 
terbutryn), 
— triazole derivatives (e.g. tebuconazole, triadimefon, tradimenol, 
triapenthenol). 
The method is not applicable for substances which react either with 
the eluent or the stationary phase. It is also not applicable for substances that interact in a specific way with inorganic components (e.g. formation of cluster complexes with clay minerals). The method 
may not work for surface active substances, inorganic compounds 
and moderate or strong organic acids and bases. Log K
 oc values 
ranging from 1,5 to 5,0 can be determined. Ionisable substances must be measured using a buffered mobile phase, but care has to 
be taken to avoid precipitation of buffer components or test 
substance.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1100
 
1.6. QUALITY CRITERIA  
1.6.1.  Accuracy  
Normally, the adsorption coefficient of a test substance can be 
estimated to within +/- 0,5 log unit of the value determined by the 
batch equilibrium method (see Table 1 in the Appendix). Higher 
accuracy may be achieved if the reference substances used are struc ­
turally related to the test substance.  
1.6.2.  Repeatability  
Determinations should be run at least in duplicate. The values of log 
K oc derived from individual measurements should be within a range 
of 0,25 log unit.  
1.6.3.  Reproducibility  
Experience gained so far in the application of the method 
is supportive of its validity. An investigation of the HPLC 
method, using 48 substances (mostly pesticides) for which reliable 
data on K  oc on soils were available gave a correlation coefficient of 
R = 0,95 (10) (11). 
An inter-laboratory comparison test with 11 participating laboratories 
was performed to improve and validate the method (12). Results are given in Table 2 of the Appendix.
 
1.7. DESCRIPTION OF THE TEST METHOD  
1.7.1.  Preliminary Estimation of the Adsorption Coefficient  
The octanol-water partition coefficient P  ow (= K  ow ) and, to some 
extent, the water solubility can be used as indicators for the extent 
of adsorption, particularly for non-ionised substances, and thus may be used for preliminary range finding. A variety of useful correlations have been published for several groups of chemicals 
(1)(2)(3)(4)(5)(6)(7).
 
1.7.2.  Apparatus  
A liquid chromatograph, fitted with a pulse-free pump and a suitable 
detection device is required. The use of an injection valve with an 
injection loop is recommended. Commercial cyanopropyl chemically 
bound resins on a silica base shall be used (e.g. Hypersil and Zorbax CN). A guard column of the same material may be positioned between the injection system and the analytical column. Columns 
from different suppliers may vary considerably in their separation 
efficiency. As a guidance, the following capacity factors k' should be reached: log k'> 0,0 for log K
 oc = 3,0 and log k' > — 0,4 for log 
K oc = 2,0 when using methanol/water 55/45 % as mobile phase.  
1.7.3.  Mobile phases  
Several mobile phases have been tested and the following two are 
recommended: 
— methanol/water (55/45 % v/v) — methanol/0,01M citrate-buffer pH 6,0 (55/45 % v/v)
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1101
 
HPLC grade methanol and distilled water or citrate-buffer are used to 
prepare the eluting solvent. The mixture is degassed before use. 
Isocratic elution should be employed. If methanol/water mixtures are not appropriate, other organic solvent/water mixtures may be tried, e.g. ethanol/water or acetonitrile/water mixtures. For 
ionisable compounds the use of buffer solution is recommended to 
stabilise pH. Care must be taken to avoid salt precipitation and column deterioration, which may occur with some organic phase/ ­
buffer mixtures. 
No additives such as ion pair reagents may be used because they can 
affect the sorption properties of the stationary phase. Such changes 
of the stationary phase may be irreversible. For this reason, it is mandatory that experiments using additives are carried out on separate columns.
 
1.7.4.  Solutes  
Test and reference substances should be dissolved in the mobile 
phase.  
1.8. PERFORMANCE OF THE TEST  
1.8.1.  Test condition  
The temperature during the measurements should be recorded. The 
use of a temperature controlled column compartment is highly recommended to guarantee constant conditions during calibration 
and estimation runs and measurement of the test substance.
 
1.8.2.  Determination of dead time to  
For the determination of the dead time t  o two different methods may 
be used (see also Section 1.2).  
1.8.2.1.  Determination of the dead time to by means of a homologous series  
This procedure has proven to yield reliable and standardised t  o 
values. For details see Testing Method A.8: Partition coefficient 
(n-octanol/water), HPLC method.  
1.8.2.2.  Determination of the dead time to by inert substances which are not 
retained by the column  
This technique is based on the injection of solutions of formamide, 
urea or sodium nitrate. Measurements should be performed at least in duplicate.
 
1.8.3.  Determination of the retention times tR  
Reference substances should be selected as described in 
Section 1.3. They may be injected as a mixed standard to 
determine their retention times, provided it has been confirmed 
that the retention time of each reference standard is unaffected by the presence of the other reference standards. The calibration should be performed at regular intervals at least twice daily in order to 
account for unexpected changes in column performance. For best 
practice the calibration injections should be carried out before and after injections of the test substance to confirm retention times have not drifted. The test substances are injected separately in quantities 
as small as possible (to avoid column overload) and their retention 
times are determined.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1102
 
In order to increase the confidence in the measurement, at least 
duplicate determinations should be made. The values of log K  oc 
derived from individual measurements should fall within a range 
of 0,25 log unit.  
1.8.4.  Evaluation  
The capacity factors k' are calculated from the dead time t  o and 
retention times t  R of the selected reference substances according to 
equation 4 (see Section 1.2). The log k' data of the reference 
substances are then plotted against their log K  oc values from batch 
equilibrium experiments given in Tables 1 and 3 of the Appendix. Using this plot, the log k' value of a test substance is then used to 
determine its log K
 oc value. If the actual results show that the log 
K oc of the test substance is outside the calibration range the test 
should be repeated using different, more appropriate reference substances.
 
2. DATA AND REPORTING  
The report must include the following information: 
— identity of test and reference substances and their purity, and pK  a 
values if relevant, 
— description of equipment and operating conditions, e.g. type and 
dimension of analytical (and guard) column, means of detection, mobile phase (ratio of components and pH), temperature range 
during measurements, 
— dead time and the method used for its determination, — quantities of test and reference substances introduced in the 
column, 
— retention times of reference compounds used for calibration, 
— details of fitted regression line (log k' vs log K
 oc ) and a graph of 
the regression line, 
— average retention data and estimated d log K  oc value for the test 
compound, 
— chromatograms.  
3. REFERENCES  
(1) W.J. Lyman, W.F. Reehl, D.H. Rosenblatt (ed), (1990) 
Handbook of chemical property estimation methods, chapt. 4, McGraw-Hill, New York. 
(2) J. Hodson, N.A. Williams, (1988) The estimation of the 
adsorption coefficient (K
 oc ) for soils by HPLC. Chemosphere, 
17, p. 167. 
(3) G.G. Briggs, (1981) Theoretical and experimental relationships 
between soil adsorption, octanol-water partition coefficients, water solubilities, bioconcentration factors, and the parachor. 
J. Agric. Food Chem., 29, p. 1050-1059. 
(4) C.T. Chiou, P.E. Porter, D.W. Schmedding, (1983) Partition 
equilibria of nonionic organic compounds between soil organic matter and water. Environ. Sci. Technol., 17, p. 227-231. 
(5) Z. Gerstl, U. Mingelgrin (1984) Sorption of organic substances 
by soils and sediment. J. Environm. Sci. Health, B19, 
p. 297-312.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1103
 
(6) C.T. Chiou, L.J. Peters, V. H. Freed, (1979) A physical 
concept of soil water equilibria for nonionic organic 
compounds, Science, 106, p. 831-832. 
(7) S.W. Karickhoff, (1981) Semi-empirical estimation of sorption 
of hydrophobic pollutants on natural sediments and soils. Chemosphere, 10, p. 833-846. 
(8) W. Kördel, D. Hennecke, M. Herrmann, (1997) Application of 
the HPLC-screening method for the determination of the 
adsorption coefficient on sewage sludges. Chemosphere, 35(1/2), p. 121-128. 
(9) M. Mueller, W. Kördel (1996) Comparison of screening 
methods for the estimation of adsorption coefficients on soil. 
Chemosphere, 32(12), p. 2493-2504. 
(10) W. Kördel, J. Stutte, G. Kotthoff (1993) HPLC-screening 
method for the determination of the adsorption coefficient in soil-comparison of different stationary phases, Chemosphere, 
27(12), p. 2341-2352. 
(11) B. von Oepen, W. Kördel, W. Klein (1991) Sorption of 
nonpolar and polar compounds to soils: Processes, measurements and experience with the applicability of the 
modified OECD Guideline 106, Chemosphere, 22, p. 285-304. 
(12) W. Kördel, G. Kotthoff, J. Müller (1995) HPLC-screening 
method for the determination of the adsorption coefficient on soil-results of a ring test. Chemosphere, 30(7), p. 1373-1384.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1104
 
Appendix  
Table 1  
Comparison of K  oc values for soils and sewage sludges, and calculated values by the HPLC screening 
method  ( 1 ), ( 2 ) 
substance CAS-No log K  oc 
sewage 
sludges log K  oc 
HPLC Δ log K  oc 
soils log K  oc 
HPLC Δ 
Atrazine 1912-24-9 1,66 2,14 0,48 1,81 2,20 0,39 
Linuron 330-55-2 2,43 2,96 0,53 2,59 2,89 0,30 Fenthion 55-38-9 3,75 3,58 0,17 3,31 3,40 0,09 Monuron 150-68-5 1,46 2,21 0,75 1,99 2,26 0,27 Phenanthrene 85-01-8 4,35 3,72 0,63 4,09 3,52 0,57 Benzoic acid phenylester 93-99-2 3,26 3,03 0,23 2,87 2,94 0,07 Benzamide 55-21-0 1,60 1,00 0,60 1,26 1,25 0,01 4-Nitrobenzamide 619-80-7 1,52 1,49 0,03 1,93 1,66 0,27 Acetanilide 103-84-4 1,52 1,53 0,01 1,26 1,69 0,08 Aniline 62-53-3 1,74 1,47 0,27 2,07 1,64 0,43 2,5-Dichloroaniline 95-82-9 2,45 2,59 0,14 2,55 2,58 0,03
 
( 1 ) W. Kördel, D. Hennecke, M. Herrmann, (1997) Application of the HPLC-screening method for the determination of the adsorption 
coefficient on sewage sludges. Chemosphere, 35(1/2), p. 121-128. 
( 2 ) W. Kördel, D. Hennecke, C. Franke, (1997) Determination of the adsorption-coefficients of organic substances on sewage sludge s. 
Chemosphere, 35 (1/2), p. 107-119.  
Table 2  
Results of a laboratory inter-comparison test (11 participating laboratories) performed to improve and validate 
the HPLC-method  ( 1 ) 
substance CAS-No log K  oc K oc log K  oc 
[OECD 106] [HPLC-method]  
Atrazine 1912-24-9 1,81 78 ± 16 1,89 
Monuron 150-68-5 1,99 100 ± 8 2,00 Triapenthenol 77608-88-3 2,37 292 ± 58 2,47 Linuron 330-55-2 2,59 465 ± 62 2,67 Fenthion 55-38-9 3,31 2062 ± 648 3,31
 
( 1 ) W. Kördel, G. Kotthoff, J. Müller (1995). HPLC-screening method for the determination of the adsorption coefficient on soil- 
results of a ring test. Chemosphere, 30(7), 1373-1384.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1105
 
Table 3  
Recommended reference substances for the HPLC screening method based on soil adsorption data.  
Reference substance CAS-No log K  oc mean 
values from 
batch equilibrium number of K  oc 
data log S.D. source  
Acetanilide 103-84-4 1,25 4 0,48 (  a ) 
Phenol 108-95-2 1,32 4 0,70 (  a ) 
2-Nitrobenzamide 610-15-1 1,45 3 0,90 (  b ) 
N.N-dimethylbenzamide 611-74-5 1,52 2 0,45 (  a ) 
4-Methylbenzamide 619-55-6 1,78 3 1,76 (  a ) 
Methylbenzoate 93-58-3 1,80 4 1,08 (  a ) 
Atrazine 1912-24-9 1,81 3 1,08 (  c ) 
Isoproturon 34123-59-6 1,86 5 1,53 (  c ) 
3-Nitrobenzamide 645-09-0 1,95 3 1,31 (  b ) 
Aniline 62-53-3 2,07 4 1,73 (  a ) 
3,5-Dinitrobenzamide 121-81-3 2,31 3 1,27 (  b ) 
Carbendazim 10605-21-7 2,35 3 1,37 (  c ) 
Triadimenol 55219-65-3 2,40 3 1,85 (  c ) 
Triazoxide 72459-58-6 2,44 3 1,66 (  c ) 
Triazophos 24017-47-8 2,55 3 1,78 (  c ) 
Linuron 330-55-2 2,59 3 1,97 (  c ) 
Naphthalene 91-20-3 2,75 4 2,20 (  a ) 
Endosulfan-diol 2157-19-9 3,02 5 2,29 (  c ) 
Methiocarb 2032-65-7 3,10 4 2,39 (  c ) 
Acid Yellow 219 63405-85-6 3,16 4 2,83 (  a ) 
1,2,3-Trichlorobenzene 87-61-6 3,16 4 1,40 (  a ) 
γ-HCH 58-89-9 3,23 5 2,94 (  a ) 
Fenthion 55-38-9 3,31 3 2,49 (  c ) 
Direct Red 81 2610-11-9 3,43 4 2,68 (  a ) 
Pyrazophos 13457-18-6 3,65 3 2,70 (  c ) 
α-Endosulfan 959-98-8 4,09 5 3,74 (  c ) 
Diclofop-methyl 51338-27-3 4,20 3 3,77 (  c ) 
Phenanthrene 85-01-8 4,09 4 3,83 (  a ) 
Basic Blue 41 (mix) 26850-47-5 
12270-13-2 4,89 4 4,46 (  a ) 
DDT 50-29-3 5,63 1 — (  b ) 
( a ) W. Kördel, J. Müller (1994). Bestimmung des Adsorptionskoeffizienten organischer Chemikalien mit der HPLC. UBA R & D 
Report No. 106 01 044 (1994). 
( b ) B.V. Oepen, W. Kördel, W. Klein., (1991) Chemosphere, 22, p. 285-304. 
( c ) Data provided by industry.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1106
 
C.20  DAPHNIA MAGNA  REPRODUCTION TEST  
1. METHOD  
This Reproduction toxicity test method is a replicate of the OECD 
TG 211 (1998).  
1.1. INTRODUCTION 
The primary objective of the test is to assess the effect of chemicals 
on the reproductive output of  Daphnia magna . 
1.2. DEFINITIONS AND UNITS  
Parent Animals:  are those female  Daphnia  present at the start of the 
test and of which the reproductive output is the object of the study.  
Offspring:  are the young  Daphnia  produced by the parent animals 
in the course of the test.  
Lowest Observed Effect Concentration (LOEC):  is the lowest 
tested concentration at which the substance is observed to have a statistically significant effect on reproduction and parent mortality (at p < 0,05) when compared with the control, within a stated exposure 
period. However, all test concentrations above the LOEC must have 
a harmful effect equal to or greater than those observed at the LOEC. When these two conditions cannot be satisfied, a full explanation must be given for how the LOEC (and hence the NOEC) has been 
selected.  
No Observed Effect Concentration (NOEC):  is the test concen ­
tration immediately below the LOEC, which when compared with 
the control, has no statistically significant effect (p < 0,05), within a stated exposure period.  
EC
 x : is the concentration of the test substance dissolved in water that 
results in a x % reduction in reproduction of  Daphnia magna  within 
a stated exposure period.  
Intrinsic rate of increase:  is a measure of population growth which 
integrates reproductive output and age-specific mortality (20) (21) (22). In steady state populations it will be zero. For growing popu ­
lations it will be positive and for shrinking populations it will be 
negative. Clearly, the latter is not sustainable and ultimately will lead 
to extinction.  
Limit of Detection:  is the lowest concentration that can be detected 
but not quantified.  
Limit of Determination:  is the lowest concentration that can be 
measured quantitatively.  
Mortality:  an animal is recorded as dead when it is immobile, i.e. 
when it is not able to swim, or if there is no observed movement of appendages or postabdomen, within 15 seconds after gentle agitation 
of the test container. (If another definition is used, this must be 
reported together with its reference).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1107
 
1.3. PRINCIPLE OF THE TEST METHOD 
Young female  Daphnia  (the parent animals), aged less than 24 hours 
at the start of the test, are exposed to the test substance added to water at a range of concentrations. The test duration is 21 days. At 
the end of the test, the total number of living offspring produced per 
parent animal alive at the end of the test is assessed. This means that juveniles produced by adults that die during the test are excluded from the calculations. Reproductive output of parent animals can be 
expressed in other ways (e.g. number of living offspring produced 
per animal per day from the first day offspring were observed) but these should be reported in addition to the total number of juveniles 
produced per parent alive at the end of the test. The reproductive 
output of the animals exposed to the test substance is compared to that of the control(s) in order to determine the lowest observed effect concentration (LOEC) and hence the no observed effect concen ­
tration (NOEC). In addition, and as far as possible, the data are 
analysed using a regression model in order to estimate the concen ­
tration that would cause a x % reduction in reproductive output (i.e. the EC
 50 , EC  20 , or EC  10 ). 
The survival of the parent animals and time to production of first 
brood must also be reported. Other substance-related effects on 
parameters such as growth (e.g. length) and possibly intrinsic rate 
of increase, may also be examined.  
1.4. INFORMATION ON THE TEST SUBSTANCE 
Results of an acute toxicity test (see Method C.2, Part I) performed 
with Daphnia magna  should be available. The result may be useful 
in selecting an appropriate range of test concentrations in the repro ­
duction tests. The water solubility and the vapour pressure of the test 
substance should be known and a reliable analytical method for the 
quantification of the substance in the test solutions with reported recovery efficiency and limit of determination should be available. 
Information on the test substance which may be useful in estab ­
lishing the test conditions includes the structural formula, purity of 
the substance, stability in light, stability under the conditions of the 
test, pKa, P
 ow and results of the test for ready biodegradability (see 
Method C.4).  
1.5. VALIDITY OF THE TEST 
For a test to be valid, the following performance criteria should be 
met in the control(s): 
— the mortality of the parent animals (female  Daphnia ) does not 
exceed 20 % at the end of the test, 
— the mean number of live offspring produced per parent animal 
surviving at the end of the test is ≥ 60. 
1.6. DESCRIPTION OF THE TEST METHOD  
1.6.1.  Apparatus  
Test vessels and other apparatus which will come into contact with 
the test solutions should be made entirely of glass or other 
chemically inert material. The test vessels will normally be glass beakers. 
In addition, some or all of the following equipment will be required: — oxygen meter (with microelectrode or other suitable equipment 
for measuring dissolved oxygen in low volume samples),
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1108
 
— adequate apparatus for temperature control, 
— pH meter, 
— equipment for the determination of the hardness of water, — equipment for the determination of the total organic carbon 
concentration (TOC) of water or equipment for the determination 
of the chemical oxygen demand (COD), 
— adequate apparatus for the control of the lighting regime and the 
measurement of light intensity.
 
1.6.2.  Test organism  
The species to be used in the test is  Daphnia magna  Straus. Other  
Daphnia  species may be used providing they meet the validity 
criteria as appropriate (the validity criterion relating to the repro ­
ductive output in the controls should be relevant for the  Daphnia  
species). If other species of  Daphnia  are used they must be clearly 
identified and their use justified. 
Preferably, the clone should have been identified by genotyping. 
Research (1) has shown that the reproductive performance of 
Clone A (which originated from IRCHA in France) (3) consistently 
meets the validity criterion of a mean of ≥ 60 offspring per parent 
animal surviving when cultured under the conditions described in this method. However, other clones are acceptable provided that 
the Daphnia  culture is shown to meet the validity criteria for a test. 
At the start of the test, the animals should be less than 24 hours old 
and must not be first brood progeny. They should be derived from a healthy stock (i.e. showing no signs of stress such as high mortality, 
presence of males and ephippia, delay in the production of the first 
brood, discoloured animals etc.). The stock animals must be main ­
tained in culture conditions (light, temperature, medium, feeding and animals per unit volume) similar to those to be used in the test. If the  
Daphnia  culture medium to be used in the test is different from that 
used for routine  Daphnia  culture, it is good practice to include a pre- 
test acclimation period of normally about 3 weeks (i.e. one gener ­
ation) to avoid stressing the parent animals.
 
1.6.3.  Test medium  
It is recommended that a fully defined medium be used in this test. 
This can avoid the use of additives (e.g. seaweed, soil extract etc.), which are difficult to characterise, and therefore improves the oppor ­
tunities for standardisation between laboratories. Elendt M4 (4) and 
M7 media (see Appendix 1) have been found to be suitable for this 
purpose. However, other media (e.g. (5) (6)) are acceptable providing the performance of the  Daphnia  culture is shown to 
meet the validity criteria for the test. 
If media are used which include undefined additives, these additives 
should be specified clearly and information should be provided in the test report on composition, particularly with regard to carbon content as this may contribute to the diet provided. It is recom ­
mended that the total organic carbon (TOC) and/or chemical 
oxygen demand (COD) of the stock preparation of the organic additive is determined and an estimate of the resulting contribution to the TOC/COD in the test medium made. It is recommended that 
TOC levels in the medium (i.e. before addition of the algae) be 
below 2 mg/l (7).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1109
 
When testing substances containing metals, it is important to 
recognise that the properties of the test medium (e.g. hardness, 
chelating capacity) may have a bearing on the toxicity of the test substance. For this reason, a fully defined medium is desirable. However, at present, the only fully defined media which are 
known to be suitable for long-term culture of  Daphnia magna  are 
Elendt M4 and M7. Both media contain the chelating agent EDTA. Work has shown (2) that the ‘apparent toxicity’ of cadmium is generally lower when the reproduction test is performed in M4 
and M7 media than in media containing no EDTA. M4 and M7 
are not, therefore, recommended for testing substances containing metals, and other media containing known chelating agents should also be avoided. For metal-containing substances it may be advisable 
to use an alternative medium such as, for example, ASTM recon ­
stituted hard fresh water (7), which contains no EDTA, with added seaweed extract (8). This combination of ASTM reconstituted hard fresh water and seaweed extract is also suitable for long-term culture 
and testing of  Daphnia magna  (2), although it still exerts a mild 
chelating action due to the organic component in the added seaweed extract. 
At the beginning and during the test, the dissolved oxygen concen ­
tration should be above 3 mg/l. The pH should be within the range 6-9, and normally it should not vary by more than 1,5 units in any one test. Hardness above 140 mg/l (as CaCO
 3 ) is recommended. 
Tests at this level and above have demonstrated reproductive 
performance in compliance with the validity criteria (9) (10).  
1.6.4.  Test solutions  
Test solutions of the chosen concentrations are usually prepared by 
dilution of a stock solution. Stock solutions should preferably be 
prepared by dissolving the substance in test medium. 
The use of organic solvents or dispersants may be required in some 
cases in order to produce a suitably concentrated stock solution, but every effort should be made to avoid the use of such materials. Examples of suitable solvents are acetone, ethanol, methanol, 
dimethylformamide and triethylene glycol. Examples of suitable 
dispersants are Cremophor RH40, methylcellulose 0,01 % and HCO-40. In any case, the test substance in the test solutions should not exceed the limit of solubility in the test medium. 
Solvents are used to produce a stock solution which can be dosed 
accurately into water. At the recommended solvent concentration in the final test medium (i.e. ≤ 0,1 ml/l), the solvents listed above will 
not be toxic and will not increase the water solubility of a substance. 
Dispersants may assist in accurate dosing and dispersion. At the 
recommended concentration in the final test medium ( ≤ 0,1 ml/l), 
the dispersants listed above will not be toxic and will not increase 
the water solubility of a substance.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1110
 
1.7. TEST DESIGN 
Treatments should be allocated to the test vessels and all subsequent 
handling of the test vessels should be done in a random fashion. Failure to do this may result in bias that could be construed as being 
a concentration effect. In particular, if experimental units are handled 
in treatment or concentration order, then some time-related effect, such as operator fatigue or other error, could lead to greater effects at the higher concentrations. Furthermore, if the test results are likely to 
be affected by an initial or environmental condition of the test, such 
as position in the laboratory, then consideration should be given to blocking the test.
 
1.8. PROCEDURE  
1.8.1.  Conditions of exposure  
1.8.1.1.  Duration  
The test duration is 21 days.  
1.8.1.2.  Loading  
Parent animals are maintained individually, one per test vessel, with 
50-100 ml of medium in each vessel. 
Larger volumes may sometimes be necessary to meet requirements 
of the analytical procedure used for determination of the test substance concentration, although pooling of replicates for chemical analysis is also allowable. If volumes greater than 
100 ml are used, the ration given to the  Daphnia  may need to be 
increased to ensure adequate food availability and compliance with the validity criteria. For flow-through tests, alternative designs may, for technical reasons, be considered (e.g. four groups of 10 animals 
in a larger test volume), but any changes to the test design should be 
reported.
 
1.8.1.3.  Number of animals  
For semi-static tests, at least 10 animals individually held at each test 
concentration and at least 10 animals individually held in the control 
series. 
For flow-through tests, 40 animals divided into four groups of 10 
animals at each test concentration has been shown to be suitable (1). 
A smaller number of test organisms may be used and a minimum of 20 animals per concentration divided into two or more replicates with an equal number of animals (e.g. four replicates each with five daphnids) 
is recommended. Note that for tests where animals are held in groups, it 
will not be possible to express the reproductive output as the total number of living offspring produced per parent animal alive at the 
end of the test, if parent animals die. In these cases reproductive 
output should be expressed as ‘total number of living offspring produced per parent present at the beginning of the test’.
 
1.8.1.4.  Feeding  
For semi-static tests, feeding should preferably be done daily, but at 
least three times per week (i.e. corresponding to media changes). 
Deviations from this (e.g. for flow-through tests) should be reported.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1111
 
During the test the diet of the parent animals should preferably be living 
algal cells of one or more of the following:  Chlorella  sp, Selenastrum 
capricornutum  (now  Pseudokirchneriella subcapitata  (11)) and  
Scenedesmus subspicatus . The supplied diet should be based on the 
amount of organic carbon (C) provided to each parent animal. 
Research (12) has shown that, for  Daphnia magna , ration levels of 
between 0,1 and 0,2 mg C/ Daphnia /day are sufficient for achieving 
the required number of offspring to meet the test validity criteria. The ration can be supplied either at a consistent rate throughout the period 
of the test, or, if desired, a lower rate can be used at the beginning and 
then increased during the test to take account of growth of the parent animals. In this case, the ration should still remain within the recommended range of 0,1-0,2 mg C/ Daphnia /day at all times. 
If surrogate measures, such as algal cell number or light absorbance, 
are to be used to feed the required ration level (i.e. for convenience since measurement of carbon content is time consuming), each 
laboratory must produce its own nomograph relating the surrogate 
measure to carbon content of the algal culture (see Appendix 2 for advice on nomograph production). Nomographs should be checked at least annually and more frequently if algal culture conditions have 
changed. Light absorbance has been found to be a better surrogate 
for carbon content than cell number (13). 
A concentrated algal suspension should be fed to the  Daphnia  to 
minimise the volume of algal culture medium transferred to the test vessels. Concentration of the algae can be achieved by centrifugation followed by resuspension in distilled water, deionised water or  
Daphnia  culture medium.
 
1.8.1.5.  Light  
16 hours light at an intensity not exceeding 15-20 μE· m -2 ·s -1 . 
1.8.1.6.  Temperature  
The temperature of the test media should be within the range 18- 
22 o C. However, for any one test, the temperature should not, if 
possible, vary by more than 2  o C within these limits (e.g. 18-20, 
19-21 or 20-22  o C). It may be appropriate to use an additional test 
vessel for the purposes of temperature monitoring.  
1.8.1.7.  Aeration  
The test vessels must not be aerated during the test.  
1.8.2.  Test concentration  
Normally, there should be at least five test concentrations arranged in 
a geometric series with a separation factor preferably not exceeding 3.2, and the appropriate number of replicates for each test concen ­
tration should be used (see Section 1.8.1.3). Justification should be 
provided if fewer than five concentrations are used. Substances 
should not be tested above their solubility limit in the test medium.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1112
 
In setting the range of concentrations, the following should be borne 
in mind: 
If the aim is to obtain the LOEC/NOEC, the lowest test concen ­
tration must be low enough so that the fecundity at that concen ­
tration is not significantly lower than that in the control. If this is 
not the case, the test will have to be repeated with a reduced lowest concentration. 
If the aim is to obtain the LOEC/NOEC, the highest test concen ­
tration must be high enough so that the fecundity at that concen ­
tration is significantly lower than that in the control. If this is not the case, the test will have to be repeated with an increased 
highest concentration. 
If the EC
 X for effects on reproduction is estimated, it is 
advisable that sufficient concentrations are used to define the 
EC X with an appropriate level of confidence. If the EC  50 for 
effects on reproduction is estimated, it is advisable that the highest test concentration is greater than this EC
 50 . Otherwise, 
although it will still be possible to estimate the EC  50 , the 
confidence interval for the EC  50 will be very wide and it may 
not be possible to satisfactorily assess the adequacy of the fitted model. 
The range of test concentration should preferably not include any 
concentrations that have a statistically significant effect on adult survival since this would change the nature of the test from simply a reproduction test to a combined reproduction and 
mortality test requiring much more complex statistical analysis. 
Prior knowledge of the toxicity of the test substance (e.g. from an 
acute test and/or from range-finding studies) should help in selecting 
appropriate test concentrations. 
Where a solvent or dispersant is used to aid preparation of test 
solutions (see Section 1.6.4), its final concentration in the test 
vessels should not be greater than 0,1 ml/l and should be the same 
in all test vessels.
 
1.8.3.  Controls  
One test-medium control series and also, if relevant, one control 
series containing the solvent or dispersant should be run in 
addition to the test series. When used, the solvent or dispersant concentration should be the same as that used in the vessels containing the test substance. The appropriate number of replicates 
should be used (see Section 1.8.1.3). 
Generally, in a well-run test, the coefficient of variation around the 
mean number of living offspring produced per parent animal in the 
control(s) should be ≤ 25 %, and this should be reported for test 
designs using individually held animals.
 
1.8.4.  Test medium renewal  
The frequency of medium renewal will depend on the stability of the 
test substance, but should be at least three times per week. If, from 
preliminary stability tests (see Section 1.4) the test substance concen ­
tration is not stable (i.e. outside the range 80-120 % of nominal or falling below 80 % of the measured initial concentration) over the maximum renewal period (i.e. 3 days), consideration should be given 
to more frequent medium renewal, or to the use of a flow-through 
test.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1113
 
When the medium is renewed in semi-static tests, a second series of 
test vessels are prepared and the parent animals transferred to them 
by, for example, a glass pipette of suitable diameter. The volume of medium transferred with the  Daphnia  should be minimised.
 
1.8.5.  Observations  
The results of the observations made during the test should be 
recorded on data sheets (see examples in Appendices 3 and 4). If 
other measurements are required (see 1.3 and 1.8.8) additional obser ­
vations may be required.  
1.8.6.  Offspring  
The offspring produced by each parent animal should preferably be 
removed and counted daily from the appearance of the first brood, to prevent them consuming food intended for the adult. For the purpose of this method it is only the number of living offspring that needs to 
be counted, but the presence of aborted eggs or dead offspring 
should be recorded.
 
1.8.7.  Mortality  
Mortality among the parent animals should be recorded preferably 
daily, at least at the same times as offspring are counted.  
1.8.8.  Other parameters  
Although this method is designed principally to assess effects on 
reproduction, it is possible that other effects may also be sufficiently 
quantified to allow statistical analysis. Growth measurements are 
highly desirable since they provide information on possible sublethal effects, which may be more useful than reproduction measurement alone; the measurement of the length of the parent 
animals (i.e. body length excluding the anal spine) at the end of 
the test is recommended. Other parameters that can be measured or calculated include time to production of first brood (and subsequent broods), number and size of broods per animal, 
number of aborted broods, presence of males or ephippia and the 
intrinsic rate of population increase.
 
1.8.9.  Frequency of analytical determinations and measurements  
Oxygen concentration, temperature, hardness and pH values should 
be measured at least once a week, in fresh and old media, in the control(s) and in the highest test substance concentration. 
During the test, the concentrations of test substance are determined at 
regular intervals. 
In semi-static tests where the concentration of the test substance is 
expected to remain within ± 20 % of the nominal (i.e. within the range 80-120 % — see 1.4 and 1.8.4), it is recommended that, as a 
minimum, the highest and lowest test concentrations be analysed 
when freshly prepared and at the time of renewal on one occasion during the first week of the test (i.e. analyses should be made on a sample from the same solution — when freshly prepared and at 
renewal). These determinations should be repeated at least at 
weekly intervals thereafter.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1114
 
For tests where the concentration of the test substance is not 
expected to remain within ± 20 % of the nominal, it is necessary 
to analyse all test concentrations, when freshly prepared and at renewal. However, for those tests where the measured initial concen ­
tration of the test substance is not within ± 20 % of nominal but 
where sufficient evidence can be provided to show that the initial 
concentrations are repeatable and stable (i.e. within the range 80- 120 % of initial concentrations), chemical determinations could be reduced in weeks 2 and 3 of the test to the highest and lowest test 
concentrations. In all cases, determination of test substance concen ­
trations prior to renewal need only be performed on one replicate vessel at each test concentration. 
If a flow-through test is used, a similar sampling regime to that 
described for semi-static tests is appropriate (but measurement of ‘old’ solutions is not applicable in this case). However, it may be advisable to increase the number of sampling occasions during the 
first week (e.g. three sets of measurements) to ensure that the test 
concentrations are remaining stable. In these types of test, the flow- rate of diluent and test substance should be checked daily. 
If there is evidence that the concentration of the substance being 
tested has been satisfactorily maintained within ± 20 % of the nominal or measured initial concentration throughout the test, then results can be based on nominal or measured initial values. If the 
deviation from the nominal or measured initial concentration is 
greater than ± 20 %, results should be expressed in terms of the time-weighed mean (see Appendix 5).
 
2. DATA AND REPORTING  
2.1. TREATMENT OF RESULTS 
The purpose of this test is to determine the effect of the test 
substance on the total number of living offspring produced per parent animal alive at the end of the test. The total number of 
offspring per parent animal should be calculated for each test 
vessel (i.e. replicate). If, in any replicate the parent animal dies during the test or turns out to be male, then the replicate is excluded from the analysis. The analysis will then be based on a 
reduced number of replicates. 
For the estimation of the LOEC, and hence the NOEC, for effects of 
the chemical on reproductive output, it is necessary to calculate the 
mean reproductive output across replicates for each concentration 
and the pooled residual standard deviation, and this can be done using analysis of variance (ANOVA). The mean for each concen ­
tration must then be compared with the control mean using an 
appropriate multiple comparison method. Dunnett's or Williams' 
tests may be useful (14)(15)(16)(17). It is necessary to check whether the ANOVA assumption of homogeneity of variance holds. It is recommended that this be done graphically rather than 
via a formal significance test (18); a suitable alternative is to run a 
Bartlett's test. If this assumption does not hold, then consideration should be given to transforming the data to homogenise variances prior to performing the ANOVA, or to carrying out a weighted 
ANOVA. The size of the effect detectable using ANOVA (i.e. the 
least significant difference) should be calculated and reported.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1115
 
For the estimation of the concentration which would cause a 50 % 
reduction in reproductive output (i.e. the EC  50 ), a suitable curve, 
such as the logistic curve, should be fitted to the data using a statistical method such as least squares. The curve could be para ­
meterised so that the EC
 50 and its standard error can be estimated 
directly. This would greatly ease the calculation of the confidence 
limits about the EC  50 . Unless there are good reasons to prefer 
different confidence levels, two-sided 95 % confidence limits should be quoted. The fitting procedure should preferably provide 
a means for assessing the significance of the lack of fit. This can be 
done graphically or by dividing the residual sum of squares into ‘lack of fit’ and ‘pure error components’ and performing a significance test for lack of fit. Since treatments giving high 
fecundity are likely to have greater variance in the number of 
juveniles produced than treatments giving low fecundity, consideration to weighting the observed values to reflect the different variances in the different treatment groups should be 
given (see for background information ref. 18). 
In the analysis of the data from the final ring test (2), a logistic curve 
was fitted using the following model, although other suitable models can be used:
 
Y ¼ c 
1 þ x 
x 0 Í Î  b 
where: 
Y = the total number of juveniles per parent animal alive at the end 
of the test (calculated for each vessel) 
x = the substance concentration 
c = the expected number of juveniles when x = 0 x
 0 = the EC  50 in the population 
b = the slope parameter 
This model is likely to be adequate in a large number of situations, 
but there will be tests for which it is not appropriate. A check should be made on the validity of the model as suggested above. In some 
cases, a hormesis model in which low concentrations give enhanced 
effects may be appropriate (19). 
Other Effect Concentrations, such as the EC
 10 or EC  20 can also be 
estimated, although it may be preferable to use a different parame ­
terisation of the model from that used to estimate the EC  50 . 
2.2. TEST REPORT 
The test report must include the following:  
2.2.1.  Test substance:  
— physical nature and relevant physicochemical properties,  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1116
 
— chemical identification data, including purity.  
2.2.2.  Test species:  
— the clone (whether it has been genetically typed), supplier or 
source (if known) and the culture conditions used. If a 
different species to  Daphnia magna  is used, this should be 
reported and justified.  
2.2.3.  Test conditions:  
— test procedure used (e.g. semi-static or flow-through, volume, 
loading in number of  Daphnia  per litre), 
— photoperiod and light intensity, — test design (e.g. number of replicates, number of parents per 
replicate), 
— details of culture medium used, — if used, additions of organic material including the composition, 
source, method of preparation, TOC/COD of stock preparations, estimation of resulting TOC/COD in test medium, 
— detailed information on feeding, including amount (in mg C/ ­ 
Daphnia /day) and schedule (e.g. type of food(s), including for 
algae the specific name(species) and, if known, the strain, the culture conditions), 
— method of preparation of stock solutions and frequency of 
renewal (the solvent or dispersant and its concentration must 
be given, when used).
 
2.2.4.  Results:  
— results from any preliminary studies on the stability of the test 
substance, 
— the nominal test concentrations and the results of all analyses to 
determine the concentration of the test substance in the test vessels (see example data sheets in Appendix 4); the recovery 
efficiency of the method and the limit of determination should 
also be reported, 
— water quality within the test vessels (i.e. pH, temperature and 
dissolved oxygen concentration, and TOC and/or COD and 
hardness where applicable) (see example data sheet in 
Appendix 3), 
— the full record of living offspring by each parent animal (see 
example data sheet in Appendix 3), 
— the number of deaths among the parent animals and the day on 
which they occurred (see example data sheet in Appendix 3), 
— the coefficient of variation for control fecundity (based on total 
number of living offspring per parent animal alive at the end of the test), 
— plot of total number of living offspring per parent animal (for 
each replicate) alive at the end of the test versus concentration of 
the test substance, 
— the Lowest Observed Effect Concentration (LOEC) for repro ­
duction, including a description of the statistical procedures used and an indication of what size of effect could be detected and the No Observed Effect Concentration (NOEC) for repro ­
duction; where appropriate, the LOEC/NOEC for mortality of the 
parent animals should also be reported,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1117
 
— where appropriate, the EC  x for reproduction and confidence 
intervals and a graph of the fitted model used for its calculation, 
the slope of the dose-response curve and its standard error, 
— other observed biological effects or measurements: report any 
other biological effects which were observed or measured (e.g. growth of parent animals) including any appropriate justification, 
— an explanation for any deviation from the Test Method.
 
3. REFERENCES  
(1) OECD Test Guideline Programme, Report of the Workshop on 
the Daphnia magna  Pilot Ring Test, Sheffield University, UK, 
20-21 March 1993. 
(2) OECD Environmental Health and Safety Publications. Series 
on Testing and Assessment No6. Report of the Final Ring Test 
of the  Daphnia magna  Reproduction Test Paris. 1997. 
(3) Baird D.J., Barber J., Bradley M.C., Soares A.M.V.M. and 
Calow P. (1991) A comparative study of genotype sensitivity 
to acute toxic stress using clones of  Daphnia magna  Strauss. 
Ecotoxicology and Environmental Safety, 21, p. 257-265. 
(4) Elendt B.P., (1990) Selenium deficiency in Crustacea; An 
ultrastructural approach to antennal damage in  Daphnia 
magna  Straus. Protoplasma, 154, p. 25-33. 
(5) EPA (1993) Methods for Measuring the Acute Toxicity of 
Effluents and Receiving Waters to Freshwater and Marine Organisms. (Fourth ed.). EPA/600/4-90/027F. C. I. Weber (ed), USEPA, Cincinnati, Ohio. 
(6) Vigano L., (1991) Suitability of commercially available spring 
waters as standard medium for culturing  Daphnia magna . Bull. 
Environ. Contam. Toxicol., 47, p. 775-782. 
(7) ASTM (1988) Standard Guide for Conducting Acute Toxicity 
Tests with Fishes, Macroinvertebrates and Amphibians. E729- 
88a. American Society for Testing and Materials, Philadelphia P.A. p. 20. 
(8) Baird D.J., Soares A.M.V.M., Girling A., Barber J., Bradley 
M.C. and Calow P., (1989) The long term maintenance of  
Daphnia magna  Straus for use in ecotoxicological tests; 
problems and prospects. In: Proceedings of the 1st European Conference on Ecotoxicology. Copenhagen 1988 (H.Løkke, H. 
Tyle & F. Bro-Rasmussen. Eds.) p. 144-148. 
(9) Parkhurst B.R., Forte J.L. and Wright G.P., (1981) Reproduci ­
bility of a life-cycle toxicity test with  Daphnia magna . Bull. 
Environ. Contam. and Toxicol., 26, p. 1-8. 
(10) Cowgill U.M. and Milazzo D.P., (1990) The sensitivity of two 
cladocerans to water quality variables: salinity and hardness. 
Arch. Hydrobiol., 120(2), p. 185-196. 
(11) Korshikov (1990)  Pseudokirchneriella subcapitata  Hindak, 
F-1990. Biologice Prace, 36, p. 209. 
(12) Sims I.R., Watson S. and Holmes D., (1993) Toward a 
standard  Daphnia  juvenile production test. Environmental 
Toxicology and Chemistry, 12, p. 2053-2058.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1118
 
(13) Sims I., (1993) Measuring the growth of phytoplankton: the 
relationship between total organic carbon with three commonly 
used parameters of algal growth. Arch. Hydrobiol., 128, 459-466. 
(14) Dunnett C.W., (1955) A multiple comparisons procedure for 
comparing several treatments with a control. J. Amer. Statist. Assoc., 50, p. 1096-1121. 
(15) Dunnett C.W., (1964) New tables for multiple comparisons 
with a control. Biometrics, 20, p. 482-491. 
(16) Williams D.A., (1971) A test for differences between treatment 
means when several dose levels are compared with a zero dose control. Biometrics 27, p. 103-117. 
(17) Williams D.A., (1972) The comparison of several dose levels 
with a zero dose control. Biometrics, 28, p. 510-531. 
(18) Draper N.R. and Smith H., (1981) Applied Regression 
Analysis, second edition, Wiley, N.Y. 
(19) Brain P. and Cousens R., (1989) An equation to describe dose 
responses where there is stimulation of growth at low doses. 
Weed Research, 29, p. 93-96. 
(20) Wilson E.O. and Bossert, W.H., (1971) A Primer of Population 
Biology. Sinauer Associates Inc. Publishers. 
(21) Poole R.W., (1974) An Introduction to quantitative Ecology. 
Mc Graw Hill Series in Population Biology, New York, p. 532. 
(22) Meyer J.S., Ingersoll C.G., McDonald L.L. and Boyce M.S., 
(1986) Estimating uncertainty in population growth rates: 
Jackknife vs bootstrap techniques. Ecology, 67, p. 1156-1166.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1119
 
Appendix 1  
PREPARATION OF FULLY DEFINED ELENDT M7 AND M4 MEDIA  
Acclimation to Elendt M7 and M4 media  
Some laboratories have experienced difficulty in directly transferring  Daphnia  to 
M4 (1) and M7 media. However, some success has been achieved with gradual 
acclimation, i.e. moving from own medium to 30 % Elendt, then to 60 % Elendt 
and then to 100 % Elendt. The acclimation periods may need to be as long as one month.
 
PREPARATION  
Trace elements  
Separate stock solutions (I) of individual trace elements are first prepared in 
water of suitable purity, e.g. deionised, distilled or reverse osmosis. From 
these different stock solutions (I) a second single stock solution (II) is 
prepared, which contains all trace elements (combined solution), i.e.:  
Stock solutions I 
(single substance) Amount added to 
water 
mg/l Concentration (in 
relation to medium 
M4) 
fold To prepare the combined stock-solution 
II add the following amount of stock 
solution I to water 
ml/l 
M 4 M 7 
H 3 BO 3 57 190 20 000 1,0 0,25 
MnCl  2 * 4 H  2 O 7 210 20 000 1,0 0,25 
LiCl 6 120 20 000 1,0 0,25 RbCl 1 420 20 000 1,0 0,25 SrCl
 2 * 6 H  2 O 3 040 20 000 1,0 0,25 
NaBr 320 20 000 1,0 0,25 Na
 2 MoO  4 * 2 H  2 O 1 260 20 000 1,0 0,25 
CuCl  2 * 2 H  2 O 335 20 000 1,0 0,25 
ZnCl  2 260 20 000 1,0 1,0 
CoCl  2 * 6 H  2 O 200 20 000 1,0 1,0 
KI 65 20 000 1,0 1,0 
Na 2 SeO  3 43,8 20 000 1,0 1,0 
NH  4 VO  3 11,5 20 000 1,0 1,0 
Na 2 EDTA * 2 H  2 O 5 000 2 000 — — 
FeSO  4 * 7 H  2 O 1 991 2 000 — — 
Both Na  2 EDTA and FeSO  4 solutions are prepared singly, poured together and autoclaved immediately. This gives: 
21 Fe-EDTA solution 1 000-fold 20,0 5,0  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1120
 
M4 and M7 media  
M4 and M7 media are prepared using stock solution II, the macro-nutrients and 
vitamins as follows:  
Amount added to 
water 
mg/l Concentration 
(related to medium 
M4) 
fold Amount of stock solution added to 
prepare medium 
ml/l 
M 4 M 7 
Stock solution II combined trace elements 20 50 50 
Macro-nutrient stock solutions (single substance) CaCl
 2 * 2 H  2 O 293 800 1 000 1,0 1,0 
MgSO  4 * 7 H  2 O 246 600 2 000 0,5 0,5 
KCl 58 000 10 000 0,1 0,1 NaHCO
 3 64 800 1 000 1,0 1,0 
Na 2 SiO  3 * 9 H  2 O 50 000 5 000 0,2 0,2 
NaNO  3 2 740 10 000 0,1 0,1 
KH  2 PO 4 1 430 10 000 0,1 0,1 
K 2 HPO  4 1 840 10 000 0,1 0,1 
Combined Vitamin stock — 10 000 0,1 0,1 The combined vitamin stock solution is prepared by adding the 3 vitamins to 1 litre water as show below: 
Thiamine hydrochloride 750 10 000 — — 
Cyanocobalamine (B
 12 ) 10 10 000 — — 
Biotine 7,5 10 000 — —  
The combined vitamin stock is stored frozen in small aliquots. Vitamins are added to the media shortly before use.  
N.B. To avoid precipitation of salts when preparing the complete media, add the aliquots of stock solutions to about 500-800 ml 
deionised water and then fill up to 1 litre.  
N.N.B.  The first publication of the M4 medium can be found in Elendt, B.P. (1990). Selenium deficiency in crustacea; an ultrastructur al 
approach to antennal damage in  Daphnia magna  Straus. Protoplasma, 154, p. 25-33.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1121
 
Appendix 2  
TOTAL ORGANIC CARBON (TOC) ANALYSIS AND PRODUCTION OF 
A NOMOGRAPH FOR TOC CONTENT OF ALGAL FEED  
It is recognised that the carbon content of the algal feed will not normally be 
measured directly but from correlations (i.e. nomographs) with surrogate 
measures such as algal cell number or light absorbance). 
TOC should be measured by high temperature oxidation rather than by UV or 
persulphate methods. (See: The Instrumental Determination of Total Organic Carbon, Total Oxygen Demand and Related Determinands 1979, HMSO 1980; 49 High Holborn, London WC1V 6HB). 
For nomograph production, algae should be separated from the growth medium 
by centrifugation followed by resuspension in distilled water. Measure the 
surrogate parameter and TOC concentration in each sample in triplicate. Distilled water blanks should be analysed and the TOC concentration deducted from that of the algal sample TOC concentration. 
Nomograph should be linear over the required range of carbon concentrations. 
Examples are shown below.  
N.B.: These should not be used for conversions; it is essential that laboratories 
prepare their own nomographs.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1122
 
▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1123
 2008R0440 — EN — 22.03.2014 — 004.001 — 1124
 ▼B 
Appendix 3  
EXAMPLE DATA SHEET RECORDING MEDIUM RENEWAL, PHISICAL/CHEMICAL MONITORING DATA, FEEDING,  DAPHNIA  REPRODUCTION AND ADULT 
MORTALITY  
Experiment No: Data starded: Clone: Medium: Type of food: Test Substance: Nominal conc: 
Day 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21  
Medium renewal 
(tick) 
PH (  1 ) new 
old 
O 2 mg/l (  1 ) new 
old 
Temp ( °C) (  1 ) new 
old 
Food provided (tick) 
No live offspring (  2 ) Total 
Vessel 1 2 
3 
4 5
 2008R0440 — EN — 22.03.2014 — 004.001 — 1125
 ▼B 
Experiment No: Data starded: Clone: Medium: Type of food: Test Substance: Nominal conc: 
Day 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21  
6 
7 8 9 10 
Total 
Cumulative adult 
mortality (
 3 ) 
( 1 ) Indicate which vessel was used for the experiment. 
( 2 ) Record mortality of any adult animals as ‘M’ in relevant box. 
( 3 ) Record aborted broods as ‘AB’ in relevant box.
 Appendix 4  
EXAMPLE DATA SHEET FOR RECORDING RESULTS OF CHEMICAL ANALYSIS  
(a) Measured concentrations  
Nominal conc. Week 1 sample Week 2 sample Week 3 sample 
Fresh Old Fresh Old Fresh Old  
(b) Measured concentrations as a percentage of nominal  
Nominal conc. Week 1 sample Week 2 sample Week 3 sample 
Fresh Old Fresh Old Fresh Old  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1126
 
Appendix 5  
CALCULATION OF A TIME-WEIGHTED MEAN  
Time-weighted mean  
Given that the concentration of the test substance can decline over the period 
between medium renewals, it is necessary to consider what concentration should 
be chosen as representative of the range of concentrations experienced by the 
parent  Daphnia . The selection should be based on biological considerations as 
well as statistical ones. For example, if reproduction is thought to be affected mostly by the peak concentration experienced, then the maximum concentration 
should be used. However, if the accumulated or longer term effect of the toxic 
substance is considered to be more important, then an average concentration is more relevant. In this case, an appropriate average to use is the time-weighted mean concentration, since this takes account of the variation in instantaneous 
concentration over time.
 
Figure 1: Example of time-weighted mean
  
Figure 1 shows an example of a (simplified) test lasting seven days with medium 
renewal at Days 0, 2 and 4. 
— The thin zig-zag line represents the concentration at any point in time. The 
fall in concentration is assumed to follow an exponential decay process. 
— The six plotted points represent the observed concentrations measured at the 
start and end of each renewal period. 
— The thick solid line indicates the position of the time-weighted mean. The time-weighted mean is calculated so that the area under the time-weighted 
mean is equal to the area under the concentration curve. The calculation for the 
above example is illustrated in Table 1.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1127
 
Table 1:  
calculation of Time-weighted mean  
Renewal No Days Conc0 Conc1 Ln(Conc0) Ln(Conc1) Area  
1 2 10,000 4,493 2,303 1,503 13,767 
2 2 11,000 6,037 2,398 1,798 16,544 3 3 10,000 4,066 2,303 1,403 19,781 
Total Days: 7 Total Area 50,091 
TW Mean 7,156
 
Days  is the number of days in the renewal period  
Conc0  is the measured concentration at the start of each renewal period  
Conc1  is the measured concentration at the end of each renewal period  
Ln(Conc0)  is the natural logarithm of Conc0  
Ln(Conc1)  is the natural logarithm of Conc1  
Area  is the area under the exponential curve for each renewal period. It is calculated by:  
Area ¼ Conc 0 Ä Conc 1 
LnðConc 0Þ Ä LnðConc 1Þ Ü Days  
The time-weighted mean (TW  Mean ) is the  total area  divided by the  total days . 
Of course, for the  Daphnia  reproduction test the table would have to be extended 
to cover 21 days. 
It is clear that when observation are taken only at the start and end of each 
renewal period, it is not possible to confirm that the decay process is, in fact, exponential. A different curve would result in a different calculation for  Area . 
However, an exponential decay process is not implausible and is probably the 
best curve to use in the absence of other information. 
However, a work of caution is required if the chemical analysis fails to find any 
substance at the end of the renewal period. Unless it is possible to estimate how quickly the substance disappeared from the solution, it is impossible to obtain a realistic area under the curve, and hence it is impossible to obtain a reasonable 
time-weighted mean.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1128
 
C.21.  SOIL MICROORGANISMS: NITROGEN TRANSFORMATION 
TEST  
1. METHOD  
This test method is a replicate of OECD TG 216 (2000).  
1.1. INTRODUCTION 
This testing method describes a laboratory method designed to inves ­
tigate the long-term effects of chemicals, after a single exposure, on 
nitrogen transformation activity of soil microorganisms. The test is principally based on the recommendations of the European and 
Mediterranean Plant Protection Organization (1). However, other 
guideline, including those of the German Biologische Bundesanstalt (2), the US Environmental Protection Agency (3) SETAC (4) and the International Organization for Standardization (5), were also taken 
into account. An OECD workshop on soil/sediment selection held at 
Belgirate, Italy, in 1995 (6) agreed on the number and type of soils for use in this test. Recommendations for collection, handling and storage of soil sample are based on an ISO Guidance Document (7) 
and recommendations from the Belgirate workshop. In the 
assessment and evaluation of toxic characteristics of test substances, determination of effects on soil microbial activity may be required, e.g. when data on the potential side effects of crop protection 
products on soil microflora are required or when exposure of soil 
microorganisms to chemicals other than crop protection products is expected. The nitrogen transformation test is carried out to determine the effects of such chemicals on soil microflora. If agrochemicals 
(e.g. crop protection products, fertilisers, forestry chemicals) are 
tested, both nitrogen transformation and carbon transformation tests are conducted. If non-agrochemicals are tested, the nitrogen trans ­
formation test is sufficient. However, if EC
 50 values of the nitrogen 
transformation test for such chemicals fall within the range found for 
commercially available nitrification inhibitors (e.g. nitrapyrin), a carbon transformation test can be conducted to gain further information. 
Soils consist of living and non-living components which exist in 
complex and heterogeneous mixtures. Microorganisms play an important role in break-down and transformation of organic matter in fertile soils with many species contributing to different aspects of 
soil fertility. Any long-term interference with these biochemical 
processes could potentially interfere with nutrient cycling and this could alter soil fertility. Transformation of carbon and nitrogen occurs in all fertile soils. Although the microbial communities 
responsible for these processes differ from soil to soil, the 
pathways of transformation are essentially the same. 
This testing method described is designed to detect long-term 
adverse effects of a substance on the process of nitrogen trans ­
formation in aerobic surface soils. The test method also allows esti ­
mation of the effects of substances on carbon transformation by the 
soil microflora. Nitrate formation takes place subsequent to the degradation of carbon-nitrogen bonds. Therefore, if equal rates of nitrate production are found in treated and control soils, it is 
highly probable that the major carbon degradation pathways are 
intact and functional. The substrate chosen for the test (powdered lucerne meal) has a favourable carbon to nitrogen ratio (usually between 12/1 and 16/1). Because of this, carbon starvation is 
reduced during the test and if microbial communities are damaged 
by a chemical, they might recover within 100 days.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1129
 
The tests from which this testing method was developed were 
primarily designed for substances for which the amount reaching 
the soil can be anticipated. This is the case, for example, for crop protection products for which the application rate in the field is known. For agrochemicals, testing of two doses relevant to the 
anticipated or predicted application rate is sufficient. Agrochemicals 
can be tested as active ingredients (a.i.) or as formulated products. However, the test is not limited to agrochemicals. By changing both the amounts of test substance applied to the soil, and the way in 
which the data are evaluated, the test can also be used for chemicals 
for which the amount expected to reach the soil is not known. Thus, with chemicals other than agrochemicals, the effects of a series of concentrations on nitrogen transformation are determined. The data 
from these tests are used to prepare a dose-response curve and 
calculate EC
 x values, where x is defined % effect.  
1.2. DEFINITIONS  
Nitrogen transformation : is the ultimate degradation by micro ­
organisms of nitrogen-containing organic matter, via the process of 
ammonification and nitrification, to the respective inorganic end- product nitrate.  
EC
 x (effective concentration) : is the concentration of the test 
substance in soil that results in a x percent inhibition of nitrogen transformation to nitrate.  
EC
 50 (median effective concentration) : is the concentration of the 
test substance in soil that results in a 50 percent (50 %) inhibition of nitrogen transformation to nitrate.
 
1.3. REFERENCE SUBSTANCES 
None.  
1.4. PRINCIPLE OF THE TEST METHOD 
Sieved soil is amended with powdered plant meal and either treated 
with the test substance or left untreated (control). If agrochemicals are tested, a minimum of two test concentrations are recommended 
and these should be chosen in relation to the highest concentration 
anticipated in the field. After 0, 7, 14 days and 28 days of incu ­
bation, samples of treated and control soils are extracted with an 
appropriate solvent, and the quantities of nitrate in the extracts are 
determined. The rate of nitrate formation in treated samples is compared with the rate in the controls, and the percent deviation of the treated from the control is calculated. All tests run for at 
least 28 days. If, on the 28th day, differences between treated and 
untreated soils are equal to or greater than 25 %, measurements are continued to a maximum of 100 days. If non-agrochemicals are tested, a series of concentrations of the test substance are added to 
samples of the soil, and the quantities of nitrate formed in treated 
and control samples are measured after 28 days of incubation. Results from tests with multiple concentrations are analysed using a regression model, and the EC
 x values are calculated (i.e. EC  50 , 
EC 25 and/or EC  10 ). See definitions.  
1.5. VALIDITY OF THE TEST 
Evaluations of test results with agrochemicals are based on relatively 
small differences (i.e. average value ±25 %) between nitrate concen ­
trations in control and treated soil samples, so large variations in the 
controls can lead to false results. Therefore, the variation between 
replicate control samples should be less than ±15 %.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1130
 
1.6. DESCRIPTION OF THE TEST METHOD  
1.6.1.  Apparatus  
Test containers made of chemically inert material are used. They 
should be of a suitable capacity in compliance with the procedure 
used for incubation of soils, i.e. incubation in bulk or as a series of 
individual soil samples (see Section 1.7.1.2). Care should be taken both to minimise water loss and to allow gas exchange during the test (e.g. the test containers may be covered with perforated poly ­
ethylene foil). When volatile substances are tested, sealable and gas- 
tight containers should be used. These should be of a size such that approximately one quarter of their volume is filled with the soil 
sample. 
Standard laboratory equipment including the following is used: 
— agitation device: mechanical shaker or equivalent equipment; — centrifuge (3 000 g) or filtration device (using nitrate-free filter 
paper); 
— instrument of adequate sensitivity and reproducibility for nitrate 
analysis.
 
1.6.2.  Selection and number of soils  
One single soil is used. The recommended soil characteristics are as 
follows: 
— sand content: not less than 50 % and not greater than 75 %, 
— pH: 5,5-7,5, 
— organic carbon content: 0,5-1,5 %, — the microbial biomass should be measured (8)(9) and its carbon 
content should be at least 1 % of the total soil organic carbon. 
In most cases, a soil with these characteristics represents a worst case 
situation, since adsorption of the test chemical is minimum and its 
availability to the microflora is maximum. Consequently, tests with other soils are generally unnecessary. However, in certain circum ­
stances, e.g. where the anticipated major use of the test substance is 
in particular soils such as acidic forest soils, or for electrostatically 
charged chemicals, it may be necessary to use an additional soil.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1131
 
1.6.3.  Collection and storage of soil samples  
1.6.3.1.  Collection  
Detailed information on the history of the field site from where the 
test soil is collected should be available. Details include exact 
location, vegetation cover, dates of treatments with crop protection 
products, treatments with organic and inorganic fertilisers, additions of biological materials or accidental contaminations. The site chosen for soil collection should be one which allows long-term use. 
Permanent pastures, fields with annual cereal crops (except maize) 
or densely sown green manures are suitable. The selected sampling site should not have been treated with crop protection products for a 
minimum of one year before sampling. Also, no organic fertiliser 
should have been applied for at least six months. The use of mineral fertiliser is only acceptable when in accordance with the requirements of the crop and soil samples should not be taken 
until at least three months after fertiliser application. The use of 
soil treated with fertilisers with known biocidal effects (e.g. calcium cyanamide) should be avoided. 
Sampling should be avoided during or immediately following long 
periods (greater than 30 days) of drought or water logging. For ploughed soils, samples should be taken from a depth of 0 down 
to 20 cm. For grassland (pasture) or other soils where ploughing 
does not occur over longer periods (at least one growing season), the maximum depth of sampling may be slightly more than 20 cm (e.g. to 25 cm). 
Soil samples should be transported using containers and under 
temperature conditions which guarantee that the initial soil properties 
are not significantly altered.
 
1.6.3.2.  Storage  
The use of soils freshly collected from the field is preferred. If 
storage in the laboratory cannot be avoided, soils may be stored in 
the dark at 4 ± 2  o C for a maximum of three months. During the 
storage of soils, aerobic conditions must be ensured. If soils are collected from areas where they are frozen for at least three months per year, storage for six months at minus 18
 o C to minus 
22 o C can be considered. The microbial biomass of stored soils is 
measured prior to each experiment and the carbon in the biomass should be at least 1 % of the total soil organic carbon content (see Section 1.6.2).
 
1.6.4.  Handling and preparation of soil for the test  
1.6.4.1.  Pre-incubation  
If the soil was stored (see Section 1.6.3.2), pre-incubation is recom ­
mended for a period between two and 28 days. The temperature and 
moisture content of the soil during pre-incubation should be similar 
to that used in the test (see Sections 1.6.4.2 and 1.7.1.3).  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1132
 
1.6.4.2.  Physical-chemical characteristics  
The soil is manually cleared of large objects (e.g. stones, parts of 
plants, etc.) and then moist sieved without excess drying to a particle size less than or equal to 2 mm. The moisture content of the soil 
sample should be adjusted with distilled or deionised water to a 
value between 40 % and 60 % of the maximum water holding capacity.
 
1.6.4.3.  Amendment with organic substrate  
The soil should be amended with a suitable organic substrate, e.g. 
powdered lucerne-grass-green meal (main component:  Medicago 
sativa)  with a C/N ratio between 12/1 and 16/1. The recommended 
lucerne-soil ratio is 5 g of lucerne per kilogram of soil (dry weight).  
1.6.5.  Preparation of the test substance for the application to soil  
The test substance is normally applied using a carrier. The carrier 
can be water (for water soluble substances) or an inert solid such as 
fine quartz sand (particle size: 0,1-0,5mm). Liquid carriers other than water (e.g. organic solvents such as acetone, chloroform) should be 
avoided since they can damage the microflora. If sand is used as a 
carrier, it can be coated with the test substance dissolved or suspended in an appropriate solvent. In such cases, the solvent should be removed by evaporation before mixing with the soil. 
For an optimum distribution of the test substance in soil, a ratio of 
10 g of sand per kilogram of soil (dry weight) is recommended. Control samples are treated with an equivalent amount of water and/or quartz sand only. 
When testing volatile chemicals, losses during treatment should be 
avoided as far as possible and an attempt should be made to ensure 
homogeneous distribution in the soil (e.g. the test substance should 
be injected into the soil at several places).
 
1.6.6.  Test concentrations  
If agrochemicals are tested, at least two concentrations should be 
used. The lower concentration should reflect at least the maximum amount expected to reach the soil under practical conditions whereas the higher concentration should be a multiple of the lower concen ­
tration. The concentrations of test substance added to soil are 
calculated assuming uniform incorporation to a depth of 5 cm and a soil bulk density of 1,5. For agrochemicals that are applied directly to soil, or for chemicals for which the quantity reaching the soil can 
be predicted, the test concentrations recommended are the maximum 
Predicted Environmental Concentration (PEC) and five times that concentration. Substances that are expected to be applied to soils several times in one season should be tested at concentrations 
derived from multiplying the PEC by the maximum anticipated 
number of applications. The upper concentration tested, however, should not exceed 10 times the maximum single application rate. If non-agrochemicals are tested, a geometric series of at least five 
concentrations is used. The concentrations tested should cover the 
range needed to determine the EC
 x values.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1133
 
1.7. PERFORMANCE OF THE TEST  
1.7.1.  Conditions of exposure  
1.7.1.1.  Treatment and control  
If agrochemicals are tested, the soil is divided into three portions of 
equal weight. Two portions are mixed with the carrier containing the 
product, and the other is mixed with the carrier without the product (control). A minimum of three replicates for both treated and untreated soils is recommended. If non-agrochemicals are tested, 
the soil is divided into six portions of equal weight. Five of the 
samples are mixed with the carrier containing the test substance, and the sixth sample is mixed with the carrier without the 
chemical. Three replicates for both treatments and control are recom ­
mended. Care should be taken to ensure homogeneous distribution of the test substance in the treated soil samples. During mixing, compacting or balling of the soil should be avoided.
 
1.7.1.2.  Incubation of soil samples  
Incubation of soil samples can be performed in two ways: as bulk 
samples of each treated and untreated soil or as a series of individual and equally sized subsamples of each treated and untreated soil. 
However, when volatile substances are tested, the test should only 
be performed with a series of individual subsamples. When soils are incubated in bulk, large quantities of each treated and untreated soils are prepared and subsamples to be analysed are taken as needed 
during the test. The amount initially prepared for each treatment 
and control depends on the size of the subsamples, the number of replicates used for analysis and the anticipated maximum number of sampling times. Soils incubated in bulk should be thoroughly mixed 
before subsampling. When soils are incubated as a series of indi ­
vidual soil samples, each treated and untreated bulk soil is divided into the required number of subsamples, and these are utilised as needed. In the experiments where more than two sampling times can 
be anticipated, enough subsamples should be prepared to account for 
all replicates and all sampling times. At least three replicate samples of the test soil should be incubated under aerobic conditions (see Section 1.7.1.1). During all tests, appropriate containers with 
sufficient headspace should be used to avoid development of 
anaerobic conditions. When volatile substances are tested, the test should only be performed with a series of individual subsamples.
 
1.7.1.3.  Test conditions and duration  
The test is carried out in the dark at room temperature of 20 ± 2  o C. 
The moisture content of soil samples should be maintained during the test between 40 % and 60 % of the maximum water holding capacity of the soil (see Section 1.6.4.2) with a range of ±5 %. 
Distilled, deionised water can be added as needed. 
The minimum duration of tests is 28 days. If agrochemicals are 
tested, the rates of nitrate formation in treated and control samples 
are compared. If these differ by more than 25 % on day 28, the test 
is continued until a difference equal to or less than 25 % is obtained, or for a maximum of 100 days, whichever is shorter. For non-agro ­
chemicals, the test is terminated after 28 days. On day 28, the 
quantities of nitrate in treated and control soil samples are 
determined and the EC
 x values are calculated.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1134
 
1.7.2.  Sampling and analysis of soils  
1.7.2.1.  Soil sampling schedule  
If agrochemicals are tested, soil samples are analysed for nitrate on 
days 0, 7, 14 and 28. If a prolonged test is required, further 
measurements should be made at 14 days intervals after day 28. 
If non-agrochemicals are tested, at least five test concentrations are 
used and soil samples are analysed for nitrate at the beginning (day 0) and at the end of the exposure period (28 days). An intermediate measurement, e.g. at day 7, may be added if deemed necessary. The 
data obtained on day 28 are used to determine EC
 x value for the 
chemical. If desired, data from day 0 control samples can be used to report the initial quantity of nitrate in the soil.
 
1.7.2.2.  Analysis of soil samples  
The amount of nitrate formed in each treated and control replicate is 
determined at each sampling time. Nitrate is extracted from soil by shaking samples with a suitable extraction solvent, e.g. a 0,1 M 
potassium chloride solution. A ratio of 5 ml of KCl solution per 
gram dry weight equivalent of soil is recommended. To optimise extraction, containers holding soil and extraction solution should not be more than half full. The mixtures are shaken at 150 rpm 
for 60 minutes. The mixtures are centrifuged or filtered and the 
liquid phases are analysed for nitrate. Particle-free liquid extracts can be stored prior to analysis at minus 20 ± 5
 o C for up to six 
months.  
2. DATA  
2.1. TREATMENT OF RESULTS 
If tests are conducted with agrochemicals, the quantity of nitrate 
formed in each replicate soil sample should be recorded, and the 
mean values of all replicates should be provided in tabular form. Nitrogen transformation rates should be evaluated by appropriate and generally acceptable statistical methods (e.g. F-test, 5 % significance 
level). The quantities of nitrate formed are expressed in mg nitrate/kg 
dry weight soil/day. The nitrate formation rate in each treatment is compared with that in the control, and the percent deviation from the control is calculated. 
If tests are conducted with non-agrochemicals, the quantity of nitrate 
formed in each replicate is determined, and a dose-response curve is 
prepared for estimation of the EC
 x values. The quantities of nitrate 
(i.e. mg nitrate/kg dry weight soil) found in the treated samples after 28 days are compared to that found in the control. From these data, 
the % inhibition values for each test concentration are calculated. 
These percentages are plotted against concentration, and statistical procedures are then used to calculate the EC
 x values. Confidence 
limits (p = 0,95) for the calculated EC  x are also determined using 
standard procedures (10)(11)(12). 
Test substances that contain high quantities of nitrogen may 
contribute to the quantities of nitrate formed during the test. If 
these substances are tested at a high concentration (e.g. chemicals which are expected to be used in repeated applications) appropriate controls must be included in the test (i.e. soil plus test substance but 
without plant meal). Data from these controls must be accounted for 
in the EC
 x calculations.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1135
 
2.2. INTERPRETATION OF RESULTS 
When results from tests with agrochemicals are evaluated, and the 
difference in the rates of nitrate formation between the lower treatment (i.e. the maximum predicted concentration) and control is 
equal to or less than 25 % at any sampling time after day 28, the 
product can be evaluated as having no long-term influence on nitrogen transformation in soils. When results from tests with chemicals other than agrochemicals are evaluated, the EC
 50 , EC  25 
and/or EC  10 values are used.  
3. REPORTING  
The test report must include the following information: 
Complete identification of the soil used including: — geographical reference of the site (latitude, longitude), 
— information on the history of the site (i.e. vegetation cover, 
treatments with crop protection products, treatments with ferti ­
lisers, accidental contamination, etc.), 
— use pattern (e.g. agricultural soil, forest, etc.), 
— depth of sampling (cm), 
— sand/silt/clay content (% dry weight), — pH (in water), 
— organic carbon content (% dry weight), 
— nitrogen content (% dry weight), — initial nitrate concentration (mg nitrate/kg dry weight), 
— cation exchange capacity (mmol/kg), 
— microbial biomass in terms of percentage of the total organic 
carbon, 
— reference of the methods used for the determination of each 
parameter, 
— all information relating to the collection and storage of soil 
samples, 
— details of pre-incubation of soil if any. Test substance: 
— physical nature and, where relevant, physical-chemical properties, 
— chemical identification data, where relevant, including structural 
formula, purity (i.e. for crop protection products the percentage 
of active ingredient), nitrogen content. 
Substrate: 
— source of substrate, — composition (i.e. lucerne meal, lucerne-grass-green meal), 
— carbon, nitrogen content (% dry weight), 
— sieve size (mm).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1136
 
Test conditions: 
— details of the amendment of soil with organic substrate, 
— number of concentrations of test chemical used and, where 
appropriate, justification of the selected concentrations, 
— details of the application of test substance to soil, 
— incubation temperature, 
— soil moisture content at the beginning and during the test, 
— method of soil incubation used (i.e. as bulk or as a series of 
individual subsamples), 
— number of replicates, 
— sampling times, 
— method used for extraction of nitrate from soil, 
Results: 
— analytical procedure and equipment used to analyse nitrate, 
— tabulated data including individual and mean values for nitrate 
measurements, 
— variation between the replicates in treated and control samples, 
— explanations of corrections made in the calculations, if relevant, 
— the percent variation in nitrate formation rates at each sampling 
time or, if appropriate, the EC  50 value with 95 % confidence 
limit, other EC  x (i.e. EC  25 or EC  10 ) with confidence intervals, 
and a graph of the dose-response curve, 
— statistical treatment of results, 
— all information and observations helpful for the interpretation of 
the results.  
4. REFERENCES  
(1) EPPO, (1994) Decision-Making Scheme for the Environmental 
Risk Assessment of Plant Protection Chemicals. Chapter 7: Soil Microflora. EPPO Bulletin 24: 1-16, 1994. 
(2) BBA, (1990) Effects on the Activity of the Soil Microflora. 
BBA Guidelines for the Official Testing of Plant Protection Products, VI, 1-1 (2nd eds., 1990). 
(3) EPA (1987) Soil Microbial Community Toxicity Test. EPA 40 
CFR Part 797.3700. Toxic Substances Control Act Test Guide ­
lines; Proposed rule. September 28, 1987. 
(4) SETAC-Europe, (1995) Procedures for assessing the environ ­
mental fate and ecotoxicity of pesticides, Ed. M.R. Lynch, Pub. SETAC-Europe, Brussels. 
(5) ISO/DIS 14238 (1995) Soil Quality — Determination of 
Nitrogen Mineralisation and Nitrification in Soils and the Influence of Chemicals on these Processes. Technical 
Committee ISO/TC 190/SC 4:  Soil Quality — Biological 
Methods .
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1137
 
(6) OECD, (1995) Final Report of the OECD Workshop on 
Selection of Soils/Sediments, Belgirate, Italy, 18-20 January 
1995. 
(7) ISO 10381-6 (1993) Soil quality — Sampling. Guidance on 
the collection, handling and storage of soil for the assessment of aerobic microbial processes in the laboratory. 
(8) ISO 14240-1, (1997) Soil quality — Determination of soil 
microbial biomass — Part 1: Substrate-induced respiration 
method. 
(9) ISO 14240-2, (1997) Soil quality — Determination of soil 
microbial biomass — Part 2: Fumigation-extraction method. 
(10) Litchfield, J.T. and Wilcoxon F., (1949) A simplified method 
of evaluating dose-effect experiments. Jour. Pharmacol. and 
Exper. Ther., 96, p. 99-113. 
(11) Finney, D.J., (1971) Probit Analysis. 3rd ed., Cambridge, 
London and New-York. 
(12) Finney, D.J., (1978) Statistical Methods in biological Assay. 
Griffin, Weycombe, UK.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1138
 
C.22.  SOIL MICROORGANISMS: CARBON TRANSFORMATION 
TEST  
1. METHOD  
This method is a replicate of OECD TG 217 (2000).  
1.1. INTRODUCTION 
This testing method describes a laboratory method designed to inves ­
tigate long term potential effects of a single exposure of crop protection products and possibly other chemicals on carbon trans ­
formation activity of soil microorganisms. The test is principally 
based on the recommendations of the European and Mediterranean 
Plant Protection Organization (1). However, other guideline, including those of the German Biologische Bundesanstalt (2), the US Environmental Protection Agency (3) and SETAC (4), were 
also taken into account. An OECD Workshop on Soil/Sediment 
Selection held at Belgirate, Italy, in 1995 (5) agreed on the number and type of soils for use in this test. Recommendations for collection, handling and storage of soil sample are based on an ISO 
Guidance Document (6) and recommendations from the Belgirate 
Workshop. 
In the assessment and evaluation of toxic characteristics of test 
substances, determination of effects on soil microbial activity may be required, e.g. when data on the potential side effects of crop 
protection products on soil microflora are required or when 
exposure of soil microorganisms to chemicals other than crop protection products is expected. The carbon transformation test is 
carried out to determine the effects of such chemicals on soil micro ­
flora. If agrochemicals (e.g. crop protection products, fertilisers, forestry chemicals) are tested, both carbon transformation and nitrogen transformation tests are conducted. If non-agrochemicals 
are tested, the nitrogen transformation test is sufficient. However, 
if EC
 50 values of the nitrogen transformation test for such 
chemicals fall within the range found for commercially available nitrification inhibitors (e.g. nitrapyrin), a carbon transformation test 
can be conducted to gain further information. 
Soils consist of living and non-living components which exist in 
complex and heterogeneous mixtures. Microorganisms play an important role in breakdown and transformation of organic matter in fertile soils with many species contributing to different aspects of 
soil fertility. Any long-term interference with these biochemical 
processes could potentially interfere with nutrient cycling and this could alter the soil fertility. Transformation of carbon and nitrogen occurs in all fertile soils. Although the microbial communities 
responsible for these processes differ from soil to soil, the 
pathways of transformation are essentially the same.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1139
 
This testing method is designed to detect long-term adverse effects 
of a substance on the process of carbon transformation in aerobic 
surface soils. The test is sensitive to changes in size and activity of microbial communities responsible for carbon transformation since it subjects these communities to both chemical stress and carbon star ­
vation. A sandy soil low in organic matter is used. This soil is 
treated with the test substance and incubated under conditions that allow rapid microbial metabolism. Under these conditions, sources of readily available carbon in the soil are rapidly depleted. This causes 
carbon starvation which both kills microbial cells and induces 
dormancy and/or sporulation. If the test runs for more than 28 days, the sum of these reactions can be measured in (untreated soil) controls as a progressive loss of metabolically active 
microbial biomass (7). If the biomass in carbon-stressed soil, under 
the conditions of the test, is affected by the presence of a chemical, it may not return to the same level as the control. Hence, disturbances caused by the test substance at any time during the test will often last 
until the end of the test. 
The tests from which this testing method was developed were 
primarily designed for substances for which the amount reaching the soil can be anticipated. This is the case, for example, for crop protection products for which the application rate in the field is 
known. For agrochemicals, testing of two doses relevant to the 
anticipated or predicted application rate is sufficient. Agrochemicals can be tested as active ingredients (a.i.) or as formulated products. However, the test is not limited to chemicals with predictable envi ­
ronmental concentrations. By changing both the amounts of test 
substance applied to the soil, and the way in which the data are evaluated, the test can also be used for chemicals for which the amount expected to reach the soil is not known. Thus, with non- 
agrochemicals, the effects of a series of concentrations on carbon 
transformation are determined. The data from these tests are used to prepare a dose-response curve and calculate EC
 x values, where x is 
defined % effect.  
1.2. DEFINITIONS  
Carbon transformation : is the degradation by microorganisms of 
organic matter to form inorganic end-product carbon dioxide.  
EC x (Effective Concentration) : is the concentration of the test 
substance in soil that results in a x % inhibition of carbon trans ­
formation in carbon dioxide.  
EC 50 (Median Effective Concentration) : is the concentration of test 
substance in soil that results in a 50 % inhibition of carbon trans ­
formation in carbon dioxide.  
1.3. REFERENCE SUBSTANCES 
None.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1140
 
1.4. PRINCIPLE OF THE TEST METHOD 
Sieved soil is either treated with the test substance or left untreated 
(control). If agrochemicals are tested, a minimum of two test concen ­
trations are recommended and these should be chosen in relation to 
the highest concentration anticipated in the field. After 0, 7, 14 and 
28 days incubation, samples of treated and control soils are mixed with glucose, and glucose-induced respiration rates are measured for 12 consecutive hours. Respiration rates are expressed as carbon 
dioxide released (mg carbon dioxide/kg dry soil/h) or oxygen 
consumed (mg oxygen/kg soil/h). The mean respiration rate in the treated soil samples is compared with that in control and the percent 
deviation of the treated from the control is calculated. All tests run 
for at least 28 days. If, on the 28th day, differences between treated and untreated soils are equal to or greater than 25 % measurements are continued in 14 day intervals for a maximum of 100 days. If 
chemicals other than agrochemicals are tested, a series of concen ­
trations of the test substance are added to samples of the soil, and glucose induced respiration rates (i.e. the mean of the quantities of carbon dioxide formed or oxygen consumed) are measured after 28 
days. Results from tests with a series of concentrations are analysed 
using a regression model, and the EC
 x values are calculated (i.e. 
EC 50 , EC  25 and/or EC  10 ). See definitions.  
1.5. VALIDITY OF THE TEST 
Evaluations of test results with agrochemicals are based on relatively 
small differences (i.e. average value ±25 %) between the carbon dioxide released or the oxygen consumed in (or by) control and 
treated soil samples, so large variations in the controls can lead to 
false results. Therefore, the variation between replicate control samples should be less than ±15 %.
 
1.6. DESCRIPTION OF THE TEST METHOD  
1.6.1.  Apparatus  
Test containers made of chemically inert material are used. They 
should be of a suitable capacity in compliance with the procedure 
used for incubation of soils, i.e. incubation in bulk or as a series of 
individual soil samples (see Section 1.7.1.2). Care should be taken both to minimise water loss and to allow gas exchange during the test (e.g. the test containers may be covered with perforated poly ­
ethylene foil). When volatile substances are tested, sealable and gas- 
tight containers should be used. These should be of a size such that approximately one quarter of their volume is filled with the soil sample. 
For determination of glucose-induced respiration, incubation systems 
and instruments for measurement of carbon dioxide production or 
oxygen consumption are required. Examples of such systems and instruments are found in the literature (8) (9) (10) (11).
 
1.6.2.  Selection and number of soils  
One single soil is used. The recommended soil characteristics are as 
follows: 
— sand content: not less than 50 % and not greater than 75 %,  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1141
 
— pH: 5,5-7,5, 
— organic carbon content: 0,5-1,5 %, 
— the microbial biomass should be measured (12)(13) and its 
carbon content should be at least 1 % of the total soil organic 
carbon. 
In most cases, a soil with these characteristics represents a worst case 
situation, since adsorption of the test chemical is minimised and its availability to the microflora is maximum. Consequently, tests with 
other soils are generally unnecessary. However, in certain circum ­
stances, e.g. where the anticipated major use of the test substance is in particular soils such as acidic forest soils, or for electrostatically charged chemicals, it may be necessary to substitute an additional 
soil.
 
1.6.3.  Collection and storage of soil samples  
1.6.3.1.  Collection  
Detailed information on the history of the field site from where the 
test soil is collected should be available. Details include exact 
location, vegetation cover, dates of treatments with crop protection 
products, treatments with organic and inorganic fertilisers, additions of biological materials or accidental contaminations. The site chosen for soil collection should be one which allows long-term use. 
Permanent pastures, fields with annual cereal crops (except maize) 
or densely sown green manures are suitable. The selected sampling site should not have been treated with crop protection products for a minimum of one year before sampling. Also, no organic fertiliser 
should have been applied for at least six months. The use of mineral 
fertiliser is only acceptable when in accordance with the requirements of the crop and soil samples should not be taken 
until at least three months after fertiliser application. The use of 
soil treated with fertilisers with known biocidal effects (e.g. calcium cyanamide) should be avoided. 
Sampling should be avoided during or immediately following long 
periods (greater than 30 days) of drought or water logging. For 
ploughed soils, samples should be taken from a depth of 0 down to 20 cm. For grassland (pasture) or other soils where ploughing does not occur over longer periods (at least one growing season), 
the maximum depth of sampling may be slightly more than 20 cm 
(e.g. to 25 cm). Soil samples should be transported using containers and under temperature conditions which guarantee that the initial soil properties are not significantly altered.
 
1.6.3.2.  Storage  
The use of soils freshly collected from the field is preferred. If 
storage in the laboratory cannot be avoided, soils may be stored in the dark at 4 ± 2
 o C for a maximum of three months. During the 
storage of soils, aerobic conditions must be ensured. If soils are 
collected from areas where they are frozen for at least three 
months per year, storage for six months at minus 18  o C can be 
considered. The microbial biomass of stored soils is measured prior to each experiment and the carbon in the biomass should be 
at least 1 % of the total soil organic carbon content (see 
Section 1.6.2).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1142
 
1.6.4.  Handling and preparation of soil for the test  
1.6.4.1.  Pre-incubation  
If the soil was stored (see Sections 1.6.4.2 and 1.7.1.3), pre-incubation is 
recommended for a period between two and 28 days. The temperature 
and moisture content of the soil during pre-incubation should be similar 
to that used in the test (see Sections 1.6.4.2 and 1.7.1.3).  
1.6.4.2.  Physical-chemical characteristics  
The soil is manually cleared of large objects (e.g. stones, parts of 
plants, etc.) and then moist sieved without excess drying to a particle 
size less than or equal to 2 mm. The moisture content of the soil 
sample should be adjusted with distilled or deionised water to a value between 40 % and 60 % of the maximum water holding capacity.
 
1.6.5.  Preparation of the test substance for the application to soil  
The test substance is normally applied using a carrier. The carrier 
can be water (for water soluble substances) or an inert solid such as 
fine quartz sand (particle size: 0,1-0,5 mm). Liquid carriers other than water (e.g. organic solvents such as acetone, chloroform) should be avoided since they can damage the microflora. If sand 
is used as a carrier, it can be coated with the test substance 
dissolved or suspended in an appropriate solvent. In such cases, the solvent should be removed by evaporation before mixing with the soil. For an optimum distribution of the test substance in soil, a 
ratio of 10 g of sand per kilogram of soil (dry weight) is recom ­
mended. Control samples are treated with the equivalent amount of water and/or quartz sand only. 
When testing volatile chemicals, losses during treatment should be 
avoided and an attempt should be made to ensure homogeneous 
distribution in the soil (e.g. the test substance should be injected into the soil at several places).
 
1.6.6.  Test concentrations  
If crop protection products or other chemicals with predictable envi ­
ronmental concentrations are tested, at least two concentrations 
should be used. The lower concentration should reflect at least the 
maximum amount expected to reach the soil under practical conditions whereas the higher concentration should be a multiple of the lower concentration. The concentrations of test substance 
added to soil are calculated assuming uniform incorporation to a 
depth of 5 cm and a soil bulk density of 1,5. For agrochemicals that are applied directly to soil, or for chemicals for which the 
quantity reaching the soil can be predicted, the test concentrations 
recommended are the Predictable Environmental Concentration (PEC) and five times that concentration. Substances that are expected to be applied to soils several times in one season should 
be tested at concentrations derived from multiplying the PEC by the 
maximum anticipated number of applications. The upper concen ­
tration tested, however, should not exceed 10 times the maximum single application rate. 
If non-agrochemicals are tested, a geometric series of at least five 
concentrations is used. The concentrations tested should cover the 
range needed to determine the EC
 x values.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1143
 
1.7. PERFORMANCE OF THE TEST  
1.7.1.  Conditions of exposure  
1.7.1.1.  Treatment and control  
If agrochemicals are tested, the soil is divided into three portions of 
equal weight. Two portions are mixed with the carrier containing the 
product, and the other is mixed with the carrier without the product (control). A minimum of three replicates for both treated and untreated soils is recommended. If non-agrochemicals are tested, 
the soil is divided into six portions of equal weight. Five of the 
samples are mixed with the carrier containing the test substance, and the sixth sample is mixed with the carrier without the 
chemical. Three replicates for both treatments and control are recom ­
mended. Care should be taken to ensure homogeneous distribution of the test substance in the treated soil samples. During mixing, compacting or balling of the soil should be avoided.
 
1.7.1.2.  Incubation of soil samples  
Incubation of soil samples can be performed in two ways: as bulk 
samples of each treated and untreated soil or as a series of individual 
and equally sized subsamples of each treated and untreated soil. However, when volatile substances are tested, the test should only be performed with a series of individual subsamples. When soils are 
incubated in bulk, large quantities of each treated and untreated soils 
are prepared and subsamples to be analysed are taken as needed during the test. The amount initially prepared for each treatment and control depends on the size of the subsamples, the number of 
replicates used for analysis and the anticipated maximum number of 
sampling times. Soils incubated in bulk should be thoroughly mixed before subsampling. When soils are incubated as a series of indi ­
vidual soil samples, each treated and untreated bulk soil is divided 
into the required number of subsamples, and these are utilised as 
needed. In the experiments where more than two sampling times can be anticipated, enough subsamples should be prepared to account for all replicates and all sampling times. At least three replicate samples 
of the test soil should be incubated under aerobic conditions (see 
Section 1.7.1.1). During all tests, appropriate containers with sufficient headspace should be used to avoid development of anaerobic conditions. When volatile substances are tested, the test 
should only be performed with a series of individual subsamples.
 
1.7.1.3.  Test conditions and duration  
The test is carried out in the dark at room temperature of 20 ± 2  o C. 
The moisture content of soil samples should be maintained during the test between 40 % and 60 % of the maximum water holding 
capacity of the soil (see Section 1.6.4.2) with a range of ±5 %. 
Distilled, deionised water can be added as needed. 
The minimum duration of tests is 28 days. If agrochemicals are 
tested, the quantities of carbon dioxide released or oxygen consumed in treated and control samples are compared. If these 
differ by more than 25 % on day 28, the test is continued until a 
difference equal to or less than 25 % is obtained, or for a maximum of 100 days, whichever is shorter. If non-agrochemicals are tested, the test is terminated after 28 days. On day 28, the quantities of 
carbon dioxide released or oxygen consumed in treated and control 
soil samples are determined and the EC
 x values are calculated.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1144
 
1.7.2.  Sampling and analysis of soils  
1.7.2.1.  Soil sampling schedule  
If agrochemicals are tested, soil samples are analysed for glucose- 
induced respiration rates on days 0, 7, 14 and 28. If a prolonged test 
is required, further measurements should be made at 14 days 
intervals after day 28. 
If non-agrochemicals are tested, at least five test concentrations are 
used and soil samples are analysed for glucose-induced respiration at 
the beginning (day 0) and at the end of the exposure period (28 
days). An intermediate measurement, e.g. at day 7, may be added if deemed necessary. The data obtained on day 28 are used to determine EC
 x value for the chemical. If desired, data from day 0 
control samples can be used to estimate the initial quantities of 
metabolically active microbial biomass in the soil (12).  
1.7.2.2.  Measurement of glucose-induced respiration rates  
The glucose-induced respiration rate in each treated and control 
replicate is determined at each sampling time. The soil samples are mixed with a sufficient amount of glucose to elicit an immediate 
maximum respiratory response. The amount of glucose needed to 
elicit a maximum respiratory response from a given soil can be determined in a preliminary test using a series of concentrations of glucose (14). However, for sandy soils with 0,5-1,5 % organic 
carbon, 2 000 mg to 4 000 mg glucose per kg dry weight soil is 
usually sufficient. The glucose can be ground to a powder with clean quartz sand (10 g sand/kg dry weight soil) and homogeneously mixed with the soil. 
The glucose amended soil samples are incubated in a suitable 
apparatus for measurement of respiration rates either continuously, 
every hour, or every two hours (see Section 1.6.1) at 20 ± 2
 o C. The 
carbon dioxide released or the oxygen consumed is measured for 12 consecutive hours and measurements should start as soon as 
possible, i.e. within one to two hours after glucose supplement. 
The total quantities of carbon dioxide released or oxygen consumed during the 12 hours are measured and mean respiration rates are determined.
 
2. DATA  
2.1. TREATMENT OF RESULTS 
If agrochemicals are tested, the carbon dioxide released from, or 
oxygen consumed by each replicate soil sample should be recorded, and the mean values of all replicates should be provided in tabular form. Results should be evaluated by appropriate and 
generally acceptable statistical methods (e.g. F-test, 5 % significance 
level). Glucose-induced respiration rates are expressed in mg carbon dioxide/kg dry weight soil/h or mg oxygen/dry weight soil/h. The mean carbon dioxide formation rate or mean oxygen consumption 
rate in each treatment is compared with that in control, and the 
percent deviation from the control is calculated.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1145
 
If tests are conducted with non-agrochemicals, the quantities of 
carbon dioxide released or oxygen consumed by each replicate is 
determined, and a dose-response curve is prepared for estimation of the EC
 x values. The glucose-induced respiration rates (i.e. mg carbon 
dioxide/kg dry weight soil/h or mg oxygen/dry weight soil/h) found 
in the treated samples after 28 days are compared to that found in 
control. From these data, the % inhibition values for each test concentration are calculated. These percentages are plotted against concentration, and statistical procedures are used to calculate the EC
 x 
values. Confidence limits (p = 0,95) for the calculated EC  x are also 
determined using standard procedures (15)(16)(17).  
2.2. INTERPRETATION OF RESULTS 
When results from tests with agrochemicals are evaluated, and the 
difference in respiration rates between the lower treatment (i.e. the maximum predicted concentration) and control is equal to or less 
than 25 % at any sampling time after day 28, the product can be 
evaluated as having no long-term influence on carbon transformation in soils. When results from tests with chemicals other than agro ­
chemicals are evaluated, the EC
 50 , EC  25 and/or EC  10 values are 
used.  
3. REPORTING  
TEST REPORT 
The test report must include the following information: 
Complete identification of the soil used including: 
— geographical reference of the site (latitude, longitude), 
— information on the history of the site (i.e. vegetation cover, 
treatments with crop protection products, treatments with ferti ­
lisers, accidental contamination, etc.), 
— use pattern (e.g. agricultural soil, forest, etc.), — depth of sampling (cm), — sand/silt/clay content (% dry weight), 
— pH (in water), 
— organic carbon content (% dry weight), — nitrogen content (% dry weight); — cation exchange capacity (mmol/kg), — initial microbial biomass in terms of percentage of the total 
organic carbon, 
— reference of the methods used for the determination of each 
parameter, 
— all information relating to the collection and storage of soil 
samples, 
— details of pre-incubation of soil if any.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1146
 
Test substance: 
— physical nature and, where relevant, physical-chemical properties, 
— chemical identification data, where relevant, including structural 
formula, purity (i.e. for crop protection products the percentage of active ingredient), nitrogen content. 
Test conditions: 
— details of the amendment of soil with organic substrate, — number of concentrations of test chemical used and, where 
appropriate, justification of the selected concentrations, 
— details of the application of test substance to soil, — incubation temperature, 
— soil moisture content at the beginning and during the test, 
— method of soil incubation used (i.e. as bulk or as a series of 
individual subsamples), 
— number of replicates, 
— sampling times. 
Results: — method and equipment used for measurement of respiration rates, 
— tabulated data including individual and mean values for quan ­
tities of carbon dioxide or oxygen, 
— variation between the replicates in treated and control samples, 
— explanations of corrections made in the calculations, if relevant, 
— the percent variation of glucose-induced respiration rates at each 
sampling time or, if appropriate, the EC
 50 with 95 % confidence 
limit, other EC  x (i.e. EC  25 or EC  10 ) with confidence intervals, 
and a graph of the dose-response curve, 
— statistical treatment of results, where appropriate, — all information and observations helpful for the interpretation of 
the results.
 
4. REFERENCES  
(1) EPPO, (1994) Decision-Making Scheme for the Environmental 
Risk Assessment of Plant Protection Chemicals. Chapter 7: Soil Microflora. EPPO Bulletin 24: 1-16, 1994. 
(2) BBA, (1990) Effects on the Activity of the Soil Microflora. 
BBA Guidelines for the Official Testing of Plant Protection Products, VI, 1-1 (2nd eds., 1990). 
(3) EPA, (1987) Soil Microbial Community Toxicity Test. EPA 40 
CFR Part 797.3700. Toxic Substances Control Act Test Guide ­
lines; Proposed rule. September 28, 1987.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1147
 
(4) SETAC-Europe, (1995) Procedures for assessing the environ ­
mental fate and ecotoxicity of pesticides, Ed. M.R. Lynch, 
Pub. SETAC-Europe, Brussels. 
(5) OECD, (1995) Final Report of the OECD Workshop on 
Selection of Soils/Sediments, Belgirate, Italy, 18-20 January 1995. 
(6) ISO 10381-6, (1993) Soil quality — Sampling. Guidance on 
the collection, handling and storage of soil for the assessment 
of aerobic microbial processes in the laboratory. 
(7) Anderson, J.P.E., (1987) Handling and Storage of Soils for 
Pesticide Experiments, in ‘Pesticide Effects on Soil Micro ­
flora’. Eds. L. Somerville and M.P. Greaves, Chap. 3: 45-60. 
(8) Anderson, J.P.E., (1982) Soil Respiration, in ‘Methods of Soil 
Analysis — Part 2: Chemical and Microbiological Properties’. Agronomy Monograph No 9. Eds. A.L. Page, R.H. Miller and D.R. Keeney. 41: 831- 871. 
(9) ISO 11266-1, (1993) Soil Quality — Guidance on Laboratory 
Tests for Biodegradation in Soil: Part 1. Aerobic Conditions. 
(10) ISO 14239, (1997E) Soil Quality — Laboratory incubation 
systems for measuring the mineralisation of organic chemicals in soil under aerobic conditions. 
(11) Heinemeye r O., Insam, H., Kaiser, E.A, and Walenzik, G., 
(1989) Soil microbial biomass and respiration measurements; 
an automated technique based on infrared gas analyses. Plant and Soil, 116: 77-81. 
(12) ISO 14240-1, (1997) Soil quality — Determination of soil 
microbial biomass — Part 1: Substrate-induced respiration 
method. 
(13) ISO 14240-2, (1997) Soil quality — Determination of soil 
microbial biomass — Part 2: Fumigation-extraction method. 
(14) Malkomes, H.-P., (1986) Einfluß von Glukosemenge auf die 
Reaktion der Kurzzeit-Atmung im Boden Gegenüber Pflanzen ­
schutzmitteln, Dargestellt am Beispiel eines Herbizide. (Influence of the Amount of Glucose Added to the Soil on the Effect of Pesticides in Short-Term Respiration, using a 
Herbicide as an Example). Nachrichtenbl. Deut. Pflanzen ­
schutzd., Braunschweig, 38: 113-120. 
(15) Litchfield, J.T. and Wilcoxon, F., (1949) A simplified method 
of evaluating dose-effect experiments. Jour. Pharmacol. and Exper. Ther., 96, 99-113. 
(16) Finney, D.J., (1971) Probit Analysis. 3rd ed., Cambridge, 
London and New-York. 
(17) Finney D.J., (1978) Statistical Methods in biological Assay. 
Griffin, Weycombe, UK.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1148
 
C.23.  AEROBIC AND ANAEROBIC TRANSFORMATION IN SOIL  
1. METHOD  
This test method is a replicate of the OECD TG 307 (2002)  
1.1. INTRODUCTION 
This test method is based on existing guidelines 
(1)(2)(3)(4)(5)(6)(7)(8)(9). The method described in this test 
Method is designed for evaluating aerobic and anaerobic trans ­
formation of chemicals in soil. The experiments are performed to determine (i) the rate of transformation of the test substance, and (ii) the nature and rates of formation and decline of transformation 
products to which plants and soil organisms may be exposed. 
Such studies are required for chemicals which are directly applied to soil or which are likely to reach the soil environment. The results of such laboratory studies can also be used to develop sampling and 
analysis protocols for related field studies. 
Aerobic and anaerobic studies with one soil type are generally 
sufficient for the evaluation of transformation pathways (8)(10)(11). Rates of transformation should be determined in at least three additional soils (8)(10). 
An OECD Workshop on soil and sediment selection, held at 
Belgirate, Italy in 1995 (10) agreed, in particular, on the number 
and types of soils for use in this test. The types of soils tested should be representative of the environmental conditions where use or release will occur. For example, chemicals that may be released in 
subtropical to tropical climates should be tested with Ferrasols or 
Nitosols (FAO system). The Workshop also made recommendations relating to collection, handling and storage of soil samples, based on the ISO Guidance (15). The use of paddy (rice) soils is also 
considered in this method.
 
1.2. DEFINITIONS  
Test substance:  any substance, whether the parent compound or 
relevant transformation products.  
Transformation products:  all substances resulting from biotic or 
abiotic transformation reactions of the test substance including CO  2 
and products that are in bound residues.  
Bound residues: ‘Bound residues’ represent compounds in soil, plant 
or animal, which persist in the matrix in the form of the parent 
substance or its metabolite(s)/transformation products after 
extraction. The extraction method must not substantially change the compounds themselves or the structure of the matrix. The nature of the bond can be clarified in part by matrix-altering 
extraction methods and sophisticated analytical techniques. To 
date, for example, covalent ionic and sorptive bonds, as well as entrapments, have been identified in this way. In general, the 
formation of bound residues reduces the bioaccessibility and the 
bioavailability significantly (12) [modified from IUPAC 1984 (13)].  
Aerobic transformation:  reactions occurring in the presence of 
molecular oxygen (14).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1149
 
Anaerobic transformation:  reactions occurring under exclusion of 
molecular oxygen (14).  
Soil:  is a mixture of mineral and organic chemical constituents, the 
latter containing compounds of high carbon and nitrogen content and 
of high molecular weights, animated by small (mostly micro-) 
organisms. Soil may be handled in two states: 
(a) undisturbed, as it has developed with time, in characteristic 
layers of a variety of soil types; 
(b) disturbed, as it is usually found in arable fields or as occurs 
when samples are taken by digging and used in this test method (14).  
Mineralisation:  is the complete degradation of an organic 
compound to CO
 2 and H  2 O under aerobic conditions, and CH  4 , 
CO 2 and H  2 O under anaerobic conditions. In the context of this 
test method, when  14 C-labelled compound is used, mineralisation 
means extensive degradation during which a labelled carbon atom is oxidised with release of the appropriate amount of
 14 CO 2 (14).  
Half-life:  t 0,5, is the time taken for 50 % transformation of a test 
substance when the transformation can be described by first-order kinetics; it is independent of the concentration.  
DT
 50 (Disappearance Time 50):  is the time within which the 
concentration of the test substance is reduced by 50 %; it is 
different from the half-life t  0,5 when transformation does not 
follow first order kinetics.  
DT 75 (Disappearance Time 75):  is the time within which the 
concentration of the test substance is reduced by 75 %.  
DT 90 (Disappearance Time 90):  is the time within which the 
concentration of the test substance is reduced by 90 %.  
1.3. REFERENCE SUBSTANCES 
Reference substances should be used for the characterisation and/or 
identification of transformation products by spectroscopic and chro ­
matographic methods.  
1.4. APPLICABILITY OF THE TEST 
The method is applicable to all chemical substances (non-labelled or 
radiolabelled) for which an analytical method with sufficient 
accuracy and sensitivity is available. It is applicable to slightly volatile, non-volatile, water-soluble or water-insoluble compounds. The test should not be applied to chemicals which are highly 
volatile from soil (e.g. fumigants, organic solvents) and thus 
cannot be kept in soil under the experimental conditions of this test.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1150
 
1.5. INFORMATION ON THE SUBSTANCE 
Non-labelled or labelled test substance can be used to measure the 
rate of transformation. Labelled material is required for studying the pathway of transformation and for establishing a mass balance.
 14 C- 
labelling is recommended but the use of other isotopes, such as  13 C, 
15 N, 3 H, 32 P, may also be useful. As far as possible, the label should 
be positioned in the most stable part(s) of the molecule (  1 ). The 
purity of the test substance should be at least 95 %. 
Before carrying out a test on aerobic and anaerobic transformation in 
soil, the following information on the test substance should be available: 
(a) solubility in water (Method A.6) 
(b) solubility in organic solvents; 
(c) vapour pressure (Method A.4) and Henry's law constant; 
(d) n-octanol/water partition coefficient (Method A.8); 
(e) chemical stability in dark (hydrolysis) (Method C.7); 
(f) pK
 a if a molecule is liable to protonation or deprotonation 
[OECD Guideline 112] (16). 
Other useful information may include data on toxicity of the test 
substance to soil micro-organisms [testing methods C.21 and C.22] (16). 
Analytical methods (including extraction and clean-up methods) for 
quantification and identification of the test substance and its trans ­
formation products should be available.
 
1.6. PRINCIPLE OF THE TEST METHOD 
Soil samples are treated with the test substance and incubated in the 
dark in biometer-type flasks or in flow-through systems under controlled laboratory conditions (at constant temperature and soil 
moisture). After appropriate time intervals, soil samples are 
extracted and analysed for the parent substance and for trans ­
formation products. Volatile products are also collected for analysis using appropriate absorption devices. Using
 14 C-labelled 
material, the various mineralisation rates of the test substance can 
be measured by trapping evolved  14 CO 2 and a mass balance, 
including the formation of soil bound residues, can be established.  
1.7. QUALITY CRITERIA  
1.7.1.  Recovery  
Extraction and analysis of, at least, duplicate soil samples 
immediately after the addition of the test substance gives a first 
indication of the repeatability of the analytical method and of the uniformity of the application procedure for the test substance. Recoveries for later stages of the experiments are given by the 
respective mass balances. Recoveries should range from 90 % to 
110 % for labelled chemicals (8) and from 70 % to 110 % for non-labelled chemicals (3).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1151  
( 1 ) For example, if the test substance contains one ring, labelling on this ring is required; if 
the test substance contains two or more rings, separate studies may be needed to evaluate 
the fate of each labelled ring and to obtain suitable information on formation of trans ­
formation products.
 1.7.2.  Repeatability and sensitivity of analytical method  
Repeatability of the analytical method (excluding the initial 
extraction efficiency) to quantify test substance and transformation products can be checked by duplicate analysis of the same extract of 
the soil, incubated long enough for formation of transformation 
products. 
The limit of detection (LOD) of the analytical method for the test 
substance and for the transformation products should be at least 0,01 mg· kg
 -1 soil (as test substance) or 1 % of applied dose whichever is 
lower. The limit of quantification (LOQ) should also be specified.  
1.7.3.  Accuracy of transformation data  
Regression analysis of the concentrations of the test substance as a 
function of time gives the appropriate information on the reliability 
of the transformation curve and allows the calculation of the confidence limits for half-lives (in the case of pseudo first order kinetics) or DT
 50 values and, if appropriate, DT  75 and DT  90 values.  
1.8. DESCRIPTION OF THE METHOD  
1.8.1.  Equipment and chemical reagents  
Incubation systems consist of static closed systems or suitable flow- 
through systems (7)(17). Examples of suitable flow-through soil incubation apparatus and biometer-type flask are shown in Figures 1 and 2, respectively. Both types of incubation systems have 
advantages and limitations (7)(17). 
Standard laboratory equipment is required and especially the 
following: 
— analytical instruments such as GLC, HPLC, TLC-equipment, 
including the appropriate detection systems for analysing radio ­
labelled or non-labelled substances or inverse isotopes dilution method, 
— instruments for identification purposes (e.g. MS, GC-MS, 
HPLC-MS, NMR, etc.), 
— liquid scintillation counter, 
— oxidiser for combustion of radioactive material, — centrifuge, — extraction apparatus (for example, centrifuge tubes for cold 
extraction and Soxhlet apparatus for continuous extraction 
under reflux), 
— instrumentation for concentrating solutions and extracts (e.g. 
rotating evaporator), 
— water bath, — mechanical mixing device (e.g. kneading machine, rotating 
mixer).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1152
 
Chemical reagents used include, for example: 
— NaOH, analytical grade, 2 mol · dm  -3 , or other appropriate base 
(e.g. KOH, ethanolamine), 
— H  2 SO 4 , analytical grade, 0,05 mol · dm  -3 , 
— ethylene glycol, analytical grade, 
— solid absorption materials such as soda lime and polyurethane 
plugs, 
— organic solvents, analytical grade, such as acetone, methanol, 
etc., 
— scintillation liquid.  
1.8.2.  Test substance application  
For addition to and distribution in soil, the test substance can be 
dissolved in water (deionised or distilled) or, when necessary, in 
minimum amounts of acetone or other organic solvents (6) in which the test substance is sufficiently soluble and stable. However, the amount of solvent selected should not have a 
significant influence on soil microbial activity (see Sections 1.5 
and 1.9.2-1.9.3). The use of solvents which inhibit microbial activity, such as chloroform, dichloromethane and other halogenated 
solvents, should be avoided. 
The test substance can also be added as a solid, e.g. mixed in quartz 
sand (6) or in a small sub-sample of the test soil which has been air- 
dried and sterilised. If the test substance is added using a solvent the 
solvent should be allowed to evaporate before the spiked sub-sample is added to the original non-sterile soil sample. 
For general chemicals, whose major route of entry into soil is 
through sewage sludge/farming application, the test substance should be first added to sludge which is then introduced into the 
soil sample. (see Sections 1.9.2 and 1.9.3) 
The use of formulated products is not routinely recommended. 
However, e.g. for poorly soluble test substances, the use of 
formulated material may be an appropriate alternative.
 
1.8.3.  Soils  
1.8.3.1.  Soil selection  
To determine the transformation pathway, a representative soil can 
be used; a sandy loam or silty loam or loam or loamy sand (according to FAO and USDA classification (18)), with a pH of 5,5-8,0, an organic carbon content of 0,5-2,5 % and a microbial 
biomass of at least 1 % of total organic carbon is recommended (10). 
For transformation rate studies at least three additional soils should 
be used representing a range of relevant soils. The soils should vary 
in their organic carbon content, pH, clay content and microbial 
biomass (10).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1153
 
All soils should be characterised, at least, for texture (% sand, 
% silt, % clay) [according to FAO and USDA classification (18)], 
pH, cation exchange capacity, organic carbon, bulk density, water retention characteristic (
 1 ) and microbial biomass (for aerobic studies 
only). Additional information on soil properties may be useful in 
interpreting the results. For determination of the soil characteristics 
the methods recommended in references (19)(20)(21)(22)(23) can be used. Microbial biomass should be determined by using the substrate-induced respiration (SIR) method (25)(26) or alternative 
methods (20).
 
1.8.3.2.  Collection, handling, and storage of soils  
Detailed information on the history of the field site from where the test 
soil is collected should be available. Details include exact location, 
vegetation cover, treatments with chemicals, treatments with organic 
and inorganic fertilisers, additions of biological materials or other contamination. If soils have been treated with the test substance or its structural analogues within the previous four years, these should 
not be used for transformation studies (10)(15). 
The soil should be freshly collected from the field (from the A 
horizon or top 20 cm layer) with a soil water content which 
facilitates sieving. For soils other than those from paddy fields, sampling should be avoided during or immediately following long 
periods (> 30 days) of drought, freezing or flooding (14). Samples 
should be transported in a manner which minimises changes in soil water content and should be kept in the dark with free access of air, as much as possible. A loosely-tied polyethylene bag is generally 
adequate for this purpose. 
The soil should be processed as soon as possible after sampling. 
Vegetation, larger soil fauna and stones should be removed prior 
to passing the soil through a 2 mm sieve which removes small stones, fauna and plant debris. Extensive drying and crushing of the soil before sieving should be avoided (15). 
When sampling in the field is difficult in winter (soil frozen or 
covered by layers of snow), it may be taken from a batch of soil 
stored in the greenhouse under plant cover (e.g. grass or grass-clover 
mixtures). Studies with soils freshly collected from the field are strongly preferred, but if the collected and processed soil has to be stored prior to the start of the study storage conditions must be 
adequate and for a limited time only (4 ± 2
 o C for a maximum of 
three months) to maintain microbial activity (  2 ). Detailed instructions 
on collection, handling and storage of soils to be used for biotrans ­
formation experiments can be found in (8)(10)(15)(26)(27).  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1154  
( 1 ) Water retention characteristic of a soil can be measured as field capacity, as water 
holding capacity or as water suction tension (pF). For explanations see Appendix 1. It 
should be reported in the test report whether water retention characteristics and bulk 
density of soils were determined in undisturbed field samples or in disturbed (processed) 
samples. 
( 2 ) Recent research results indicate that soils from temperate zones can also be stored at – 
20 o C for more than three months (28)(29) without significant losses of microbial 
activity.
 Before the processed soil is used for this test, it should be pre- 
incubated to allow germination and removal of seeds, and to re- 
establish equilibrium of microbial metabolism following the change from sampling or storage conditions to incubation conditions. A pre- incubation period between two and 28 days approximating the 
temperature and moisture conditions of the actual test is generally 
adequate (15). Storage and pre-incubation time together should not exceed three months.
 
1.9. PERFORMANCE OF THE TEST  
1.9.1.  Test conditions  
1.9.1.1.  Test temperature  
During the whole test period, the soils should be incubated in the dark 
at a constant temperature representative of the climatic conditions 
where use or release will occur. A temperature of 20 ± 2  o C is 
recommended for all test substances which may reach the soil in temperate climates. The temperature should be monitored. 
For chemicals applied or released in colder climates (e.g. in northern 
countries, during autumn/winter periods), additional soil samples should be incubated but at a lower temperature (e.g. 10 ± 2
 o C). 
1.9.1.2.  Moisture content  
For transformation tests under aerobic conditions, the soil moisture 
content (  1 ) should be adjusted to and maintained at a pF between 2,0 
and 2,5 (3). The soil moisture content is expressed as mass of water 
per mass of dry soil and should be regularly controlled (e.g. in 2 
week intervals) by weighing of the incubation flasks and water losses compensated by adding water (preferably sterile-filtered tap water). 
Care should be given to prevent or minimise losses of test substance 
and/or transformation products by volatilisation and/or photodegra ­
dation (if any) during moisture addition. 
For transformation tests under anaerobic and paddy conditions, the 
soil is water-saturated by flooding.
 
1.9.1.3.  Aerobic incubation conditions  
In the flow-through systems, aerobic conditions will be maintained 
by intermittent flushing or by continuously ventilating with 
humidified air. In the biometer flasks, exchange of air is maintained by diffusion.
 
1.9.1.4.  Sterile aerobic conditions  
To obtain information on the relevance of abiotic transformation of a 
test substance, soil samples may be sterilised (for sterilisation methods see references 16 and 29), treated with sterile test 
substance (e.g. addition of solution through a sterile filter) and 
aerated with humidified sterile air as described in Section 1.9.1.3. For paddy soils, soil and water should be sterilised and the incubation should be carried out as described in 
Section 1.9.1.6.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1155  
( 1 ) The soil should neither be too wet nor too dry to maintain adequate aeration and 
nutrition of soil microflora. Moisture contents recommended for optimal microbial 
growth range from 40-60 % water holding capacity (WHC) and from 0,1-0,33 bar (6). 
The latter range is equivalent to a pF-range of 2,0-2,5. Typical moisture contents of 
various soil types are given in Appendix 2.
 1.9.1.5.  Anaerobic incubation conditions  
To establish and maintain anaerobic conditions, the soil treated with 
the test substance and incubated under aerobic conditions for 30 days or one half-life or DT
 50 (whichever is shorter) is then water-logged 
(1-3 cm water layer) and the incubation system flushed with an inert 
gas (e.g. nitrogen or argon) (  1 ). The test system must allow for 
measurements such as pH, oxygen concentration and redox potential and include trapping devices for volatile products. The 
biometer-type system must be closed to avoid entrance of air by 
diffusion.
 
1.9.1.6.  Paddy incubation conditions  
To study transformation in paddy rice soils, the soil is flooded with a 
water layer of about 1-5 cm and the test substance applied to the 
water phase (9). A soil depth of at least 5 cm is recommended. The system is ventilated with air as under aerobic conditions. pH, oxygen concentration and redox potential of the aqueous layer should be 
monitored and reported. A pre-incubation period of at least two 
weeks is necessary before commencing transformation studies (see Section 1.8.3.2).
 
1.9.1.7.  Test duration  
The rate and pathway studies should normally not exceed 120 
days (  2 ) (3)(6)(8), because thereafter a decrease of the soil 
microbial activity with time would be expected in an artificial laboratory system isolated from natural replenishment. Where 
necessary to characterise the decline of the test substance and the 
formation and decline of major transformation products, studies can be continued for longer periods (e.g. 6 or 12 months) (8). Longer 
incubation periods should be justified in the test report and accom ­
panied by biomass measurements during and at the end of these periods.
 
1.9.2.  Performance of the test  
About 50 to 200 g of soil (dry weight basis) are placed into each 
incubation flask (see Figures 1 and 2 in Appendix 3) and the soil 
treated with the test substance by one of the methods described in Section 1.8.2. When organic solvents are used for the application of 
the test substance, they should be removed from soil by evaporation. 
Then the soil is thoroughly mixed with a spatula and/or by shaking of the flask. If the study is conducted under paddy field conditions, soil and water should be thoroughly mixed after application of the 
test substance. Small aliquots (e.g. 1 g) of the treated soils should be 
analysed for the test substance to check for uniform distribution. For alternative method, see below.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1156  
( 1 ) Aerobic conditions are dominant in surface soils and even in sub-surface soils as shown 
in an EU sponsored research project [K. Takagi et al. (1992). Microbial diversity and activity in subsoils: Methods, field site, seasonal variation in subsoil temperatures and 
oxygen contents. Proc. Internat. Symp. Environm. Aspects Pesticides Microbiol., 
270-277, 17-21 August 1992, Sigtuna, Sweden]. Anaerobic conditions may only occur 
occasionally during flooding of soils after heavy rainfalls or when paddy conditions are 
established in rice fields. 
(
 2 ) Aerobic studies might be terminated much before 120 days provided that ultimate trans ­
formation pathway and ultimate mineralisation are clearly reached at that time. 
Termination of the test is possible after 120 days, or when at least 90 % of the test 
substance is transformed, but only if at least 5 % CO  2 is formed.
 The treatment rate should correspond to the highest application rate 
of a crop protection product recommended in the use instructions 
and uniform incorporation to an appropriate depth in the field (e.g. top 10 cm layer (
 1 ) of soil). For example, for chemicals foliarly or 
soil applied without incorporation, the appropriate depth for 
computing how much chemical should be added to each flask is 
2,5 cm. For soil incorporated chemicals, the appropriate depth is the incorporation depth specified in the use instructions. For general chemicals, the application rate should be estimated based 
on the most relevant route of entry; for example, when the major 
route of entry in soil is through sewage sludge, the chemical should be dosed into the sludge at a concentration that reflects the expected sludge concentration and the amount of sludge added to the soil 
should reflect normal sludge loading to agricultural soils. If this 
concentration is not high enough to identify major transformation products, incubation of separate soil samples containing higher rates may be helpful, but excessive rates influencing soil microbial 
functions should be avoided (see Sections 1.5 and 1.8.2). 
Alternatively, a larger batch (i.e. 1 to 2 kg) of soil can be treated with the test substance, carefully mixed in an appropriate mixing machine and then transferred in small portions of 50 to 200 g into 
the incubation flasks (for example with the use of sample splitters). 
Small aliquots (e.g. 1 g) of the treated soil batch should be analysed for the test substance to check for uniform distribution. Such a procedure is preferred since it allows for more uniform distribution 
of the test substance into the soil. 
Also untreated soil samples are incubated under the same conditions 
(aerobic) as the samples treated with the test substance. These 
samples are used for biomass measurements during and at the end 
of the studies. 
When the test substance is applied to the soil dissolved in organic 
solvent(s), soil samples treated with the same amount of solvent(s) are incubated under the same conditions (aerobic) as the samples treated with the test substance. These samples are used for 
biomass measurements initially, during and at the end of the 
studies to check for effects of the solvent(s) on microbial biomass. 
The flasks containing the treated soil are either attached to the flow- 
through system described in Figure 1 or closed with the absorption 
column shown in Figure 2 (see Appendix 3).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1157  
( 1 ) Calculation of the initial concentration on an area basis using the following equation:  
C soil mg =kg soil ½ â ¼ A½kg=haâ · 10 6 ½mg =kgâ 
l½mâ · 10 4 ½m 2=haâ · d kg  soil=m 3 h i  
C soil = Initial concentration in soil [mg ·kg -1 ] 
A = Application rate [kg · ha -1 ]; 1 = thickness of field soil layer [m]; d = dry bulk 
density of soil [kg · m -3 ]. 
As a rule of thumb, an application rate of 1 kg · ha -1 results in a soil concentration of 
approximately 1 mg · kg  -1 in a 10 cm layer (assuming a bulk density of 1 g · cm  -3 ).
 1.9.3.  Sampling and measurement  
Duplicate incubation flasks are removed at appropriate time intervals 
and the soil samples extracted with appropriate solvents of different polarity and analysed for the test substance and/or transformation 
products. A well-designed study includes sufficient flasks so that 
two flasks are sacrificed at each sampling event. Also, absorption solutions or solid absorption materials are removed at various time intervals (7-day intervals during the first month and after one month 
in 17-day intervals) during and at the end of incubation of each soil 
sample and analysed for volatile products. Besides a soil sample taken directly after application (0-day sample) at least five additional 
sampling points should be included. Time intervals should be chosen 
in such a way that pattern of decline of the test substance and patterns of formation and decline of transformation products can be established (e.g. 0, 1, 3, 7 days; 2, 3 weeks; 1, 2, 3 months, etc.). 
When using
 14 C-labelled test substance, non-extractable radioactivity 
will be quantified by combustion and a mass balance will be calculated for each sampling interval. 
In the case of anaerobic and paddy incubation, the soil and water 
phases are analysed together for test substance and transformation 
products or separated by filtration or centrifugation before extraction and analysis.
 
1.9.4.  Optional tests  
Aerobic, non-sterile studies at additional temperatures and soil 
moistures may be useful for the estimation of the influence of 
temperature and soil moisture on the rates of transformation of a test substance and/or its transformation products in soil. 
A further characterisation of non-extractable radioactivity can be 
attempted using, for example, supercritical fluid extraction.
 
2. DATA  
2.1. TREATMENT OF RESULTS 
The amounts of test substance, transformation products, volatile 
substances (in % only), and non-extractable should be given as % 
of applied initial concentration and, where appropriate, as mg · kg  -1 
soil (based on soil dry weight) for each sampling interval. A mass 
balance should be given in percentage of the applied initial concen ­
tration for each sampling interval. A graphical presentation of the 
test substance concentrations against time will allow an estimation of its transformation half-life or DT
 50. Major transformation products 
should be identified and their concentrations should also be plotted 
against time to show their rates of formation and decline. A major 
transformation product is any product representing ≥ 10 % of applied 
dose at any time during the study. 
The volatile products trapped give some indication of the volatility 
potential of a test substance and its transformation products from 
soil. ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1158
 
More accurate determinations of half-lives or DT  50 values and, if 
appropriate, DT  75 and DT  90 values should be obtained by applying 
appropriate kinetic model calculations. The half-life and DT  50 values 
should be reported together with the description of the model used, the order of kinetics and the determination coefficient (r
 2 ). First order 
kinetics is favoured unless r  2 < 0,7. If appropriate, the calculations 
should also be applied to the major transformation products. Examples of appropriate models are described in references 31 to 35. 
In the case of rate studies carried out at various temperatures, the 
transformation rates should be described as a function of temperature 
within the experimental temperature range using the Arrhenius rela ­
tionship of the form:
 
k ¼ A · e ÄB=T or lnk ¼ 1nA Ä B 
T , 
where ln A and B are regression constants from the intercept and 
slope, respectively, of a best fit line generated from linearly 
regressing ln k against 1/T, k is the rate constant at temperature T and T is the temperature in Kelvin. Care should be given to the 
limited temperature range in which the Arrehenius relationship will 
be valid in case transformation is governed by microbial action.
 
2.2. EVALUATION AND INTERPRETATION OF RESULTS 
Although the studies are carried out in an artificial laboratory system, 
the results will allow estimation of the rate of transformation of the test substance and also of rate of formation and decline of trans ­
formation products under field conditions (36)(37). 
A study of the transformation pathway of a test substance provides 
information on the way in which the applied substance is structurally changed in the soil by chemical and microbial reactions.
 
3. REPORTING  
TEST REPORT 
The test report must include: 
Test substance: — common name, chemical name, CAS number, structural formula 
(indicating position of label(s) when radiolabelled material is 
used) and relevant physical-chemical properties (see Section 1.5), 
— purity (impurities) of test substance, — radiochemical purity of labelled chemical and specific activity 
(where appropriate), 
Reference substances: — chemical name and structure of reference substances used for the 
characterisation and/or identification of transformation product, 
Test soils: — details of collection site, — date and procedure of soil sampling,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1159
 
— properties of soils, such as pH, organic carbon content, texture 
(% sand, % silt, % clay), cation exchange capacity, bulk density, 
water retention characteristic, and microbial biomass, 
— length of soil storage and storage conditions (if stored), Test conditions: — dates of the performance of the studies, 
— amount of test substance applied, 
— solvents used and method of application for the test substance, — weight of soil treated initially and sampled at each interval for 
analysis, 
— description of the incubation system used, 
— air flow rates (for flow-through systems only), — temperature of experimental set-up, — soil moisture content during incubation, — microbial biomass initially, during and at the end of the aerobic 
studies, 
— pH, oxygen concentration and redox potential initially, during 
and at the end of the anaerobic and paddy studies, 
— method(s) of extraction, 
— methods for quantification and identification of the test substance 
and major transformation products in soil and absorption materials, 
— number of replicates and number of controls. Results: 
— result of microbial activity determination, 
— repeatability and sensitivity of the analytical methods used, — rates of recovery (% values for a valid study are given in 
Section 1.7.1), 
— tables of results expressed as % of applied initial dose and, where 
appropriate, as mg · kg
 -1 soil (on a dry weight basis), 
— mass balance during and at the end of the studies, — characterisation of non-extractable (bound) radioactivity or 
residues in soil, 
— quantification of released CO
 2 and other volatile compounds, 
— plots of soil concentrations versus time for the test substance and, 
where appropriate, for major transformation products, 
— half-life or DT  50 , DT  75 and DT  90 for the test substance and, 
where appropriate, for major transformation products including 
confidence limits,  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1160
 
— estimation of abiotic degradation rate under sterile conditions, 
— an assessment of transformation kinetics for the test substance 
and, where appropriate, for major transformation products, 
— proposed pathways of transformation, where appropriate, — discussion and interpretation of results, — raw data (i.e. sample chromatograms, sample calculations of 
transformation rates and means used to identify transformation 
products).
 
4. REFERENCES  
(1) US — Environmental Protection Agency, (1982) Pesticide 
Assessment Guidelines, Subdivision N. Chemistry: Environ ­
mental Fate. 
(2) Agriculture Canada, (1987) Environmental Chemistry and Fate. 
Guidelines for registration of pesticides in Canada. 
(3) European Union (EU), (1995) Commission Directive 95/36/EC 
of 14 July 1995 amending Council Directive 91/414/EEC 
concerning the placing of plant protection products on the market. Annex II, Part A and Annex III, Part A: Fate and Behaviour in the Environment. 
(4) Dutch Commission for Registration of Pesticides, (1995) 
Application for registration of a pesticide. Section G: 
Behaviour of the product and its metabolites in soil, water and air. 
(5) BBA, (1986) Richtlinie für die amtliche Prüfung von Pflanzen ­
schutzmitteln, Teil IV, 4-1. Verbleib von Pflanzenschutzmitteln 
im Boden — Abbau, Umwandlung und Metabolismus. 
(6) ISO/DIS 11266-1, (1994) Soil Quality -Guidance on laboratory 
tests for biodegradation of organic chemicals in soil — Part 1: Aerobic conditions. 
(7) ISO 14239, (1997) Soil Quality — Laboratory incubation 
systems for measuring the mineralisation of organic 
chemicals in soil under aerobic conditions. 
(8) SETAC, (1995) Procedures for Assessing the Environmental 
Fate and Ecotoxicity of Pesticides. Mark R. Lynch, Ed. 
(9) MAFF — Japan 2000 — Draft Guidelines for transformation 
studies of pesticides in soil — Aerobic metabolism study in soil under paddy field conditions (flooded). 
(10) OECD, (1995) Final Report of the OECD Workshop on 
Selection of Soils/Sediments. Belgirate, Italy, 18-20 January 
1995. 
(11) Guth, J.A., (1980) The study of transformations. In Interactions 
between Herbicides and the Soil (R.J. Hance, Ed.), Academic Press, p. 123-157. 
(12) DFG: Pesticide Bound Residues in Soil. Wiley — VCH 
(1998). 
(13) T.R. Roberts: Non-extractable pesticide residue in soils and 
plants. Pure Appl. Chem. 56, 945-956 (IUPAC 1984). 
(14) OECD Test Guideline 304 A: Inherent Biodegradability in Soil 
(adopted 12 May 1981).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1161
 
(15) ISO 10381-6 (1993) Soil Quality — Sampling — Part 6: 
Guidance on the collection, handling and storage of soil for 
the assessment of aerobic microbial processes in the laboratory. 
(16) Appendix V to Directive 67/548/EEC. 
(17) Guth, J.A., (1981) Experimental approaches to studying the 
fate of pesticides in soil. In Progress in Pesticide Biochemistry. 
D.H. Hutson, T.R. Roberts, Eds. J. Wiley & Sons. Vol 1, 85-114. 
(18) Soil Texture Classification (US and FAO systems): Weed 
Science, 33, Suppl. 1 (1985) and Soil Sci. Soc. Amer. Proc. 
26:305 (1962). 
(19) Methods of Soil Analysis (1986) Part 1, Physical and Miner ­
alogical Methods. A. Klute, Ed.) Agronomy Series No 9, 2nd 
Edition. 
(20) Methods of Soil Analysis (1982) Part 2, Chemical and 
Microbiological Properties. A.L. Page, R.H. Miller and D.R. Kelney, Eds. Agronomy Series No 9, 2nd Edition. 
(21) ISO Standard Compendium Environment (1994) Soil Quality 
— General aspects; chemical and physical methods of analysis; 
biological methods of analysis. First Edition. 
(22) Mückenhausen, E., (1975) Die Bodenkunde und ihre geol ­
ogischen, geomorphologischen, mineralogischen und petrol ­
ogischen Grundlagen. DLG-Verlag, Frankfurt, Main. 
(23) Scheffer, F., Schachtschabel, P., (1975) Lehrbuch der Boden ­
kunde. F. Enke Verlag, Stuttgart. 
(24) Anderson, J.P.E., Domsch, K.H., (1978) A physiological 
method for the quantitative measurement of microbial biomass in soils. Soil Biol. Biochem. 10, p. 215-221. 
(25) ISO 14240-1 and 2 (1997) Soil Quality — Determination of 
soil microbial biomass — Part 1: Substrate-induced respiration 
method. Part 2: fumigation-extraction method. 
(26) Anderson, J.P.E., (1987) Handling and storage of soils for 
pesticide experiments. In Pesticide Effects on Soil Microflora. L. Somerville, M.P. Greaves, Eds. Taylor & Francis, 45-60. 
(27) Kato, Yasuhiro. (1998) Mechanism of pesticide transformation 
in the environment: Aerobic and bio-transformation of 
pesticides in aqueous environment. Proceedings of the 16
 th 
Symposium on Environmental Science of Pesticide, 
p. 105-120. 
(28) Keuken O., Anderson J.P.E., (1996) Influence of storage on 
biochemical processes in soil. In Pesticides, Soil Microbiology and Soil Quality, 59-63 (SETAC-Europe). 
(29) Stenberg B., Johansson M., Pell M., Sjödahl-Svensson K., 
Stenström J., Torstensson L. (1996) Effect of freeze and cold 
storage of soil on microbial activities and biomass. In 
Pesticides, Soil Microbiology and Soil Quality, 68-69 (SETAC-Europe). 
(30) Gennari, M., Negre, M., Ambrosoli, R., (1987) Effects of 
ethylene oxide on soil microbial content and some chemical 
characteristics. Plant and Soil 102, p. 197-200. 
(31) Anderson, J.P.E. (1975) Einfluss von Temperatur und Feuchte 
auf Verdampfung, Abbau und Festlegung von Diallat im 
Boden. Z. PflKrankh Pflschutz, Sonderheft VII, p. 141-146.
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(32) Hamaker, J.W., (1976) The application of mathematical 
modelling to the soil persistence and accumulation of 
pesticides. Proc. BCPC Symposium: Persistence of Insecticides and Herbicides, p. 181-199. 
(33) Goring, C.A.I., Laskowski, D.A., Hamaker, J.W., Meikle, 
R.W., (1975) Principles of pesticide degradation in soil. In 
‘Environmental Dynamics of Pesticides’. R. Haque and V.H. 
Freed, Eds., p. 135-172. 
(34) Timme, G., Frehse, H., Laska, V., (1986) Statistical interpre ­
tation and graphic representation of the degradational behaviour of pesticide residues. II. Pflanzenschutz — Nach ­
richten Bayer 39, p. 188-204. 
(35) Timme, G., Frehse, H., (1980) Statistical interpretation and 
graphic representation of the degradational behaviour of pesticide residues. I. Pflanzenschutz — Nachrichten Bayer 
33, p. 47-60. 
(36) Gustafson D.I., Holden L.R., (1990) Non-linear pesticide 
dissipation in soil; a new model based on spatial variability. Environm. Sci. Technol. 24, p. 1032-1041. 
(37) Hurle K., Walker A., (1980) Persistence and its prediction. In 
Interactions between Herbicides and the Soil (R.J. Hance, Ed.), 
Academic Press, p. 83-122.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1163
 
Appendix 1  
WATER TENSION, FIELD CAPACITY (FC) AND WATER HOLDING CAPACITY 
(WHC)  ( 1 ) 
Height of Water Column 
[cm] pF (  a ) bar (  b ) Remarks  
10 7 7 10  4 Dry Soil 
1,6 · 10 4 4,2 16 Wilting point 
10 4 4 10 
10 3 3 1 
6· 10 2 2,8 0,6 
3,3 · 10 2 2,5 0,33 (  c ) 
Range of 
Field capacity (  d ) 10 2 2 0,1 
60 1,8 0,06 33 1,5  9 
> > > > > > = 
> > > > > > ; 0,033 
10 1 0,01 WHC (approximation) 
1 0 0,001 Water saturated soil
 
( a ) pF = log of cm water column. 
( b ) 1 bar = 10  5 Pa. 
( c ) Corresponds to an approximate water content of 10 % in sand, 35 % in loam and 45 % in clay. 
( d ) Field capacity is not constant but varies with soil type between pF 1,5 and 2,5.  
Water tension  is measured in cm water column or in bar. Due to the large range of suction tension 
it is expressed simply as pF value which is equivalent to the logarithm of cm water column.  
Field capacity  is defined as the amount of water which can be stored against gravity by a natural 
soil two days after a longer raining period or after sufficient irrigation. It is determined in undis ­
turbed soil in situ in the field. The measurement is thus not applicable to disturbed laboratory soil samples. FC values determined in disturbed soils may show great systematic variances.  
Water holding capacity  (WHC) is determined in the laboratory with undisturbed and disturbed soil 
by saturating a soil column with water by capillary transport. It is particularly useful for disturbed soils and can be up to 30 % greater than field capacity (1). It is also experimentally easier to determine than reliable FC-values. 
Notes
 
( 1 ) Mückenhausen, E., (1975) Die Bodenkunde und ihre geologischen, geomorphologischen, mineralogischen und 
petrologischen Grundlagen. DLG-Verlag, Frankfurt, Main.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1164
 
Appendix 2  
SOIL MOISTURE CONTENTS (g water per 100 g dry soil) OF VARIOUS SOIL TYPES 
FROM VARIOUS COUNTRIES  
Soil moisture content at 
Soil type Country 
WHC (  1 ) pF = 1,8 pF = 2,5  
Sand Germany 28,7 8,8 3,9 
Loamy sand Germany 50,4 17,9 12,1 
Loamy sand Switzerland 44,0 35,3 9,2 
Silt loam Switzerland 72,8 56,6 28,4 Clay loam Brazil 69,7 38,4 27,3 Clay loam Japan 74,4 57,8 31,4 Sandy loam Japan 82,4 59,2 36,0 Silt loam USA 47,2 33,2 18,8 Sandy loam USA 40,4 25,2 13,3
 
( 1 ) Water holding capacity.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1165
 
Appendix 3  
Figure 1  
Example of a flow-through apparatus to study transformation of chemicals in soil  ( 1 ) ( 2 )
 
Figure 2  
Example of a biometer-type flask for studying the transformation of chemicals in soil  ( 3 )
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1166  
( 1 ) Guth, J.A., (1980) The study of transformations. In Interactions between Herbicides and the Soil (R.J. Hance, 
Ed.), Academic Press, p. 123-157. 
( 2 ) Guth, J.A., (1981) Experimental approaches to studying the fate of pesticides in soil. In Progress in Pesticide 
Biochemistry. D.H. Hutson, T.R. Roberts, Eds. J. Wiley & Sons. Vol 1, p. 85-114. 
( 3 ) Anderson, J.P.E., (1975) Einfluss von Temperatur und Feuchte auf Verdampfung, Abbau und Festlegung von 
Diallat im Boden. Z. PflKrankh Pflschutz, Sonderheft VII, p. 141-146.
 C.24.  AEROBIC AND ANAEROBIC TRANSFORMATION IN 
AQUATIC SEDIMENT SYSTEMS  
1. METHOD  
This test method is a replicate of the OECD TG 308 (2002).  
1.1. INTRODUCTION 
Chemicals can enter shallow or deep surface waters by such routes 
as direct application, spray drift, run-off, drainage, waste disposal, 
industrial, domestic or agricultural effluent and atmospheric 
deposition. This testing method describes a laboratory method to assess aerobic and anaerobic transformation of organic chemicals in aquatic sediment systems. It is based on existing Guidelines (1) ­
(2)(3)(4)(5)(6). An OECD Workshop on Soil/Sediment Selection, 
held in Belgirate, Italy in 1995 (7) agreed, in particular, on the number and type of sediments for use in this test. It also made recommendations relating to collection, handling and storage of 
sediment samples, based on the ISO Guidance (8). Such studies 
are required for chemicals which are directly applied to water or which are likely to reach the aqueous environment by the routes described above. 
The conditions in natural aquatic sediment systems are often aerobic 
in the upper water phase. The surface layer of sediment can be either 
aerobic or anaerobic, whereas the deeper sediment is usually 
anaerobic. To encompass all of these possibilities both aerobic and anaerobic tests are described in this document. The aerobic test simulates an aerobic water column over an aerobic sediment layer 
that is underlain with an anaerobic gradient. The anaerobic test 
simulates a completely anaerobic water-sediment system. If circum ­
stances indicate that it is necessary to deviate significantly from these recommendations, for example by using intact sediment cores or 
sediments that may have been exposed to the test substance, other 
methods are available for this purpose (9).
 
1.2. DEFINITIONS 
Standard International (SI) units should be used in any case.  
Test substance:  any substance, whether the parent or relevant trans ­
formation products.  
Transformation products:  all substances resulting from biotic and 
abiotic transformation reactions of the test substance including CO  2 
and bound residues.  
Bound residues: ‘bound residues’ represent compounds in soil, plant 
or animal that persist in the matrix in the form of the parent 
substance or its metabolite(s) after extractions. The extraction method must not substantially change the compounds themselves or the structure of the matrix. The nature of the bond can be 
clarified in part by matrix-altering extraction methods and sophis ­
ticated analytical techniques. To date, for example, covalent ionic and sorptive bonds, as well as entrapments, have been identified in this way. In general, the formation of bound residues reduces the 
bioaccessibility and the bioavailability significantly (10) (modified 
from IUPAC 1984 (11)).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1167
 
Aerobic transformation:  (oxidising): reactions occurring in the 
presence of molecular oxygen (12).  
Anaerobic transformation:  (reducing): reactions occurring under 
exclusion of molecular oxygen (12).  
Natural waters:  are surface waters obtained from ponds, rivers, 
streams, etc.  
Sediment:  is a mixture of mineral and organic chemical constituents, 
the latter containing compounds of high carbon and nitrogen content 
and of high molecular masses. It is deposited by natural water and 
forms an interface with that water.  
Mineralisation:  is the complete degradation of an organic 
compound to CO  2 , H  2 O under aerobic conditions, and CH  4 , CO  2 
and H  2 O under anaerobic conditions. In the context of this test 
method, when radiolabelled compound is used, mineralisation means extensive degradation of a molecule during which a labelled carbon atom is oxidised or reduced quantitatively with release of the 
appropriate amount of
 14 CO 2 or 14 CH 4 , respectively.  
Half-life , t 0,5 , is the time taken for 50 % transformation of a test 
substance when the transformation can be described by first-order kinetics; it is independent of the initial concentration.  
DT
 50 (Disappearance Time 50):  is the time within which the initial 
concentration of the test substance is reduced by 50 %.  
DT 75 (DISAPPEARANCE TIME 75):  IS THE TIME WITHIN 
WHICH THE INITIAL CONCENTRATION OF THE TEST 
SUBSTANCE IS REDUCED BY 75 %.  
DT 90 (Disappearance Time 90):  is the time within which the initial 
concentration of the test substance is reduced by 90 %.  
1.3. REFERENCE SUBSTANCES 
Reference substances should be used for the identification and quan ­
tification of transformation products by spectroscopic and chromato ­
graphic methods.  
1.4. INFORMATION ON THE TEST SUBSTANCE 
Non-labelled or isotope-labelled test substance can be used to 
measure the rate of transformation although labelled material is preferred. Labelled material is required for studying the pathway of transformation and for establishing a mass balance.
 14 C-labelling 
is recommended, but the use of other isotopes, such as  13 C, 15 N, 3 H, 
32 P, may also be useful. As far as possible, the label should be 
positioned in the most stable part(s) of the molecule (  1 ). The 
chemical and/or radiochemical purity of the test substance should 
be at least 95 %. 
Before carrying out a test, the following information about the test 
substance should be available: 
(a) solubility in water (Method A.6); 
(b) solubility in organic solvents; 
(c) vapour pressure (Method A.4) and Henry's Law constant;  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1168  
( 1 ) For example, if the substance contains one ring, labelling on this ring is required; if the 
test substance contains two or more rings, separate studies may be needed to evaluate the 
fate of each labelled ring and to obtain suitable information on formation of trans ­
formation products.
 (d) n-octanol/water partition coefficient (Method A.8); 
(e) adsorption coefficient (K  d , K  f or K  oc , where appropriate) 
(Method C.18); 
(f) hydrolysis (Method C.7); 
(g) dissociation constant (pK  a ) (OECD Guideline 112) (13); 
(h) chemical structure of the test substance and position of the 
isotope-label(s), if applicable.  
Note : the temperature at which these measurements were made 
should be reported. 
Other useful information may include data on toxicity of the test 
substance to microorganisms, data on ready and/or inherent biode ­
gradability, and data on aerobic and anaerobic transformation in soil. 
Analytical methods (including extraction and clean-up methods) for 
identification and quantification of the test substance and its trans ­
formation products in water and in sediment should be available (see Section 1.7.2).
 
1.5. PRINCIPLE OF THE TEST METHOD 
The method described in this test employs an aerobic and an 
anaerobic aquatic sediment (see Appendix 1) system which allows: 
the measurement of the transformation rate of the test substance 
in a water-sediment system, 
the measurement of the transformation rate of the test substance 
in the sediment, 
the measurement of the mineralisation rate of the test substance 
and/or its transformation products (when  14 C-labelled test 
substance is used), 
the identification and quantification of transformation products in 
water and sediment phases including mass balance (when 
labelled test substance is used), 
the measurement of the distribution of the test substance and its 
transformation products between the two phases during a period of incubation in the dark (to avoid, for example, algal blooms) at constant temperature. Half-lives, DT
 50 , DT  75 and DT  90 values 
are determined where the data warrant, but should not be extra ­
polated far past the experimental period (see Section 1.2). 
At least two sediments and their associated waters are required for 
both the aerobic and the anaerobic studies respectively (7). However, there may be cases where more than two aquatic sediments should 
be used, for example, for a chemical that may be present in 
freshwater and/or marine environments.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1169
 
1.6. APPLICABILITY OF THE TEST 
The method is generally applicable to chemical substances (unla ­
belled or labelled) for which an analytical method with sufficient accuracy and sensitivity is available. It is applicable to slightly 
volatile, non-volatile, water-soluble or poorly water-soluble 
compounds. The test should not be applied to chemicals which are highly volatile from water (e.g. fumigants, organic solvents) and thus cannot be kept in water and/or sediment under the experimental 
conditions of this test. 
The method has been applied so far to study the transformation of 
chemicals in fresh waters and sediments, but in principle can also be applied to estuarine/marine systems. It is not suitable to simulate 
conditions in flowing water (e.g. rivers) or the open sea.
 
1.7. QUALITY CRITERIA  
1.7.1.  Recovery  
Extraction and analysis of, at least, duplicate water and sediment 
samples immediately after the addition of the test substance gives 
a first indication of the repeatability of the analytical method and of 
the uniformity of the application procedure for the test substance. Recoveries for later stages of the experiments are given by the respective mass balances (when labelled material is used). 
Recoveries should range from 90 % to 110 % for labelled 
chemicals (6) and from 70 % to 110 % for non-labelled chemicals.
 
1.7.2.  Repeatability and sensitivity of analytical method  
Repeatability of the analytical method (excluding the initial 
extraction efficiency) to quantify test substance and transformation products can be checked by duplicate analysis of the same extract of 
the water or the sediment samples which were incubated sufficiently 
long enough for formation of transformation products. 
The limit of detection (LOD) of the analytical method for the test 
substance and for the transformation products should be at least 0,01 
mg· kg
 -1 in water or sediment (as test substance) or 1 % of the initial 
amount applied to a test system whichever is lower. The limit of quantification (LOQ) should also be specified.
 
1.7.3.  Accuracy of transformation data  
Regression analysis of the concentrations of the test substance as a 
function of time gives the appropriate information on the accuracy of 
the transformation curve and allows the calculation of the confidence 
limits for half-lives (if pseudo first-order kinetics apply) or DT  50 
values and, if appropriate, DT  75 and DT  90 values.  
1.8. DESCRIPTION OF THE METHOD  
1.8.1.  Test system and apparatus  
The study should be performed in glass containers (e.g. bottles, 
centrifuge tubes), unless preliminary information (such as n- 
octanol-water partition coefficient, sorption data, etc.) indicates that the test substance may adhere to glass, in which case an alternative material (such as Teflon) may have to be considered. Where the test 
substance is known to adhere to glass, it may be possible to alleviate 
this problem using one or more of the following methods:
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1170
 
— determine the mass of test substance and transformation products 
sorbed to glass, 
— ensure a solvent wash of all glassware at the end of the test, 
— use of formulated products (see also Section 1.9.2), — use an increased amount of co-solvent for addition of test 
substance to the system; if a co-solvent is used it should be a co-solvent that does not solvolyse the test substance. 
Examples of typical test apparatus, i.e. gas flow-through and 
biometer-type systems, are shown in Appendices 2 and 3, 
respectively (14). Other useful incubation systems are described in reference 15. The design of the experimental apparatus should permit the exchange of air or nitrogen and the trapping of volatile products. 
The dimensions of the apparatus must be such that the requirements 
of the test are complied with (see Section 1.9.1). Ventilation may be provided by either gentle bubbling or by passing air or nitrogen over the water surface. In the latter case gentle stirring of the water from 
above may be advisable for better distribution of the oxygen or 
nitrogen in the water. CO
 2 -free air should not be used as this can 
result in increases in the pH of the water. In either case, disturbance of the sediment is undesirable and should be avoided as far as 
possible. Slightly volatile chemicals should be tested in a 
biometer-type system with gentle stirring of the water surface. Closed vessels with a headspace of either atmospheric air or nitrogen and internal vials for the trapping of volatile products can 
also be used (16). Regular exchange of the headspace gas is required 
in the aerobic test in order to compensate for the oxygen consumption by the biomass. 
Suitable traps for collecting volatile transformation products include 
but are not restricted to 1 mol · dm
 -3 solutions of potassium 
hydroxide or sodium hydroxide for carbon dioxide (  1 ) and ethylene 
glycol, ethanolamine or 2 % paraffin in xylene for organic 
compounds. Volatiles formed under anaerobic conditions, such as methane, can be collected, for example, by molecular sieves. Such volatiles can be combusted, for example, to CO
 2 by passing the gas 
through a quartz tube filled with CuO at a temperature of 900  o C and 
trapping the CO  2 formed in an absorber with alkali (17). 
Laboratory instrumentation for chemical analysis of test substance 
and transformation products is required (e.g. gas liquid chroma ­
tography (GLC), high performance liquid chromatography (HPLC), 
thin-layer chromatography (TLC), mass spectroscopy (MS), gas 
chromatography-mass spectroscopy (GC-MS), liquid chroma ­
tography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), etc.), including detection systems for radiolabelled or non- 
labelled chemicals as appropriate. When radiolabelled material is 
used a liquid scintillation counter and combustion oxidiser (for the combustion of sediment samples prior to analysis of radioactivity) will also be required. 
Other standard laboratory equipment for physical-chemical and 
biological determinations (see Section Table 1, Section 1.8.2.2), 
glassware, chemicals and reagents are required as appropriate.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1171  
( 1 ) As these alkaline absorption solutions also absorb the carbon dioxide from the ventilation 
air and that formed by respiration in aerobic experiments, they have to be exchanged in 
regular intervals to avoid their saturation and thus loss of their absorption capacity.
 1.8.2.  Selection and number of aquatic sediments  
The sampling sites should be selected in accordance with the 
purpose of the test in any given situation. In selecting sampling sites, the history of possible agricultural, industrial or domestic 
inputs to the catchment and the waters upstream must be considered. 
Sediments should not be used if they have been contaminated with the test substance or its structural analogues within the previous four years.
 
1.8.2.1.  Sediment selection  
Two sediments are normally used for the aerobic studies (7). The 
two sediments selected should differ with respect to organic carbon 
content and texture. One sediment should have a high organic carbon content (2,5-7,5 %) and a fine texture, the other sediment should have a low organic carbon content (0,5-2,5 %) and a coarse texture. 
The difference between the organic carbon contents should normally 
be at least 2 %. ‘Fine texture’ is defined as a [clay + silt] (
 1 ) 
content of > 50 % and ‘coarse texture’ is defined as a [clay + silt] content of < 50 %. The difference in [clay + silt] content for the two 
sediments should normally be at least 20 %. In cases, where a 
chemical may also reach marine waters, at least one of the water-sediment systems should be of marine origin. 
For the strictly anaerobic study, two sediments (including their 
associated waters) should be sampled from the anaerobic zones of 
surface water bodies (7). Both the sediment and the water phases 
should be handled and transported carefully under exclusion of oxygen. 
Other parameters may be important in the selection of sediments and 
should be considered on a case-by-case basis. For example, the pH 
range of sediments would be important for testing chemicals for which transformation and/or sorption may be pH-dependent. pH- dependency of sorption might be reflected by the pK
 a of the test 
substance.  
1.8.2.2.  Characterisation of water-sediment samples  
Key parameters that must be measured and reported (with reference to 
the method used) for both water and sediment, and the stage of the test 
at which those parameters are to be determined are summarised in the Table hereafter. For information, methods for determination of these parameters are given in references (18)(19)(20)(21). 
In addition, other parameters may need to be measured and reported 
on a case by case basis (e.g. for freshwater: particles, alkalinity, hardness, conductivity, NO
 3 /PO  4 (ratio and individual values); for 
sediments: cation exchange capacity, water holding capacity, 
carbonate, total nitrogen and phosphorus; and for marine systems: 
salinity). Analysis of sediments and water for nitrate, sulfate, bioa ­
vailable iron, and possibly other electron acceptors may be also useful in assessing redox conditions, especially in relation to 
anaerobic transformation.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1172  
( 1 ) (Clay + silt) is the mineral fraction of the sediment with particle size of < 50 μm.
 Measurement of parameters for characterisation of water-sediment samples (7)(22)(23)  
Parameter Stage of test procedure 
field 
sampling post- 
handling start of 
acclimation start of test during test end of test  
Water  
Origin/source x Temperature x pH x x x x x 
TOC x x x 
O
 2 concentration* x x x x x 
Redox Potential* x x x x  
Sediment  
Origin/source x Depth of layer x pH x x x x x Particle size distribution x TOC x x x x Microbial biomass (*) x x x Redox potential (**) Obser ­
vation 
(colour/ 
smell) x x x x
 
(*) Microbial respiration rate method (26), fumigation method (27) or plate count measurements (e.g. bacteria, actinomy ­
cetes, fungi and total colonies) for aerobic studies; methanogenesis rate for anaerobic studies. 
(**) Recent research results have shown that measurements of water oxygen concentrations and of redox potentials have 
neither a mechanistic nor a predictive value as far as growth and development of microbial populations in surface 
waters are concerned (24)(25). Determination of the biochemical oxygen demand (BOD, at field sampling, start and 
end of test) and of concentrations of micro/macro nutrients Ca, Mg and Mn (at start and end of test) in water and the 
measurement of total N and total P in sediments (at field sampling and end of test) may be better tools to interpret 
and evaluate aerobic biotransformation rates and routes.  
1.8.3.  Collection, handling and storage  
1.8.3.1.  Collection  
The draft ISO guidance on sampling of bottom sediment (8) should 
be used for sampling of sediment. Sediment samples should be taken from the entire 5 to 10 cm upper layer of the sediment. Associated water should be collected from the same site or location and at the 
same time as the sediment. For the anaerobic study, sediment and 
associated water should be sampled and transported under exclusion of oxygen (28)(see Section 1.8.2.1). Some sampling devices are described in the literature (8)(23).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1173
 
1.8.3.2.  Handling  
The sediment is separated from the water by filtration and the 
sediment wet-sieved to a 2 mm-sieve using excess location water that is then discarded. Then known amounts of sediments and water 
are mixed at the desired ratio (see Section 1.9.1) in incubation flasks 
and prepared for the acclimation period (see Section 1.8.4). For the anaerobic study, all handling steps have to be done under exclusion of oxygen (29)(30)(31)(32)(33).
 
1.8.3.3.  Storage  
Use of freshly sampled sediment and water is strongly recom ­
mended, but if storage is necessary, sediment and water should be sieved as described above and stored together, water-logged (6- 
10 cm water layer), in the dark, at 4 ± 2°C
 4 for a maximum of 
four weeks (7)(8)(23). Samples to be used for aerobic studies should be stored with free access of air (e.g. in open containers), whereas those for anaerobic studies under exclusion of oxygen. Freezing of 
sediment and water and drying-out of the sediment must not occur 
during transportation and storage.
 
1.8.4.  Preparation of the sediment/water samples for the test  
A period of acclimation should take place prior to adding the test 
substance, with each sediment/water sample being placed in the 
incubation vessel to be used in the main test, and the acclimation to be carried out under exactly the same conditions as the test incubation (see Section 1.9.1). The acclimation period is the time 
needed to reach reasonable stability of the system, as reflected by 
pH, oxygen concentration in water, redox potential of the sediment and water, and macroscopic separation of phases. The period of 
acclimation should normally last between one week and two 
weeks and should not exceed four weeks. Results of determinations performed during this period should be reported.
 
1.9. PERFORMANCE OF THE TEST  
1.9.1.  Test conditions  
The test should be performed in the incubation apparatus (see 
Section 1.8.1) with a water sediment volume ratio between 3:1 and 4:1, and a sediment layer of 2,5 cm (± 0,5 cm). (
 1 ) A 
minimum amount of 50 g of sediment (dry weight basis) per incu ­
bation vessel is recommended. 
The test should be performed in the dark at a constant temperature in 
the range of 10 to 30 °C. A temperature of (20 ± 2)°C is appropriate. 
Where appropriate, an additional lower temperature (e.g. 10  o C) may 
be considered on a case-by-case basis, depending on the information required from the test. Incubation temperature should be monitored 
and reported.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1174  
( 1 ) Recent studies have shown that storage at 4  o C can lead to a decrease of the organic 
carbon content of the sediment which may possibly result in a decrease of microbial 
activity (34).
 1.9.2.  Treatment and application of test substance  
One test concentration of chemical is used (  1 ). For crop protection 
chemicals applied directly to water bodies, the maximum dosage on the label should be taken as, the maximum application rate 
calculated on the basis of the surface area of the water in the test 
vessel. In all other cases, the concentration to be used should be based on predictions from environmental emissions. Care must be taken to ensure that an adequate concentration of test substance is 
applied in order to characterise the route of transformation and the 
formation and decline of transformation products. It may be necessary to apply higher doses (e.g. 10 times) in situations where 
test substance concentrations are close to limits of detection at the 
start of the study and/or where major transformation products could not readily be detected when present at 10 % of the test substance application rate. However, if higher test concentrations are used they 
should not have a significant adverse effect on the microbial activity 
of the water-sediment system. In order to achieve a constant concen ­
tration of test substance in vessels of differing dimensions an adjustment to the quantity of the material applied may be considered 
appropriate, based on the depth of the water column in the vessel in 
relation to the depth of water in the field (which is assumed to be 100 cm, but other depths can be used). See Appendix 4 for an example calculation. 
Ideally the test substance should be applied as an aqueous solution 
into the water phase of the test system. If unavoidable, the use of 
low amounts of water miscible solvents (such as acetone, ethanol) is 
permitted for application and distribution of the test substance, but this should not exceed 1 % v/v and should not have adverse effects on microbial activity of the test system. Care should be exercised in 
generating the aqueous solution of the test substance — use of 
generator columns and pre-mixing may be appropriate to ensure complete homogeneity. Following addition of the aqueous solution to the test system, gentle mixing of the water phase is recommended, 
disturbing the sediment as little as possible. 
The use of formulated products is not routinely recommended as the 
formulation ingredients may affect the distribution of the test 
substance and/or transformation products between water and sediment phases. However, for poorly water-soluble test substances, 
the use of formulated material may be an appropriate alternative. 
The number of incubation vessels depends on the number of 
sampling times (see Section 1.9.3). A sufficient number of test 
systems should be included so that two systems may be sacrificed at each sampling time. Where control units of each aquatic sediment system are employed, they should not be treated with the test 
substance. The control units can be used to determine the 
microbial biomass of the sediment and the total organic carbon of the water and sediment at the termination of the study. Two of the control units (i.e. one control unit for each aquatic sediment) can be 
used to monitor the required parameters in the sediment and water 
during the acclimation period (see Table in Section 1.8.2.2). Two additional control units have to be included in case the test substance is applied by means of a solvent to measure adverse effects on the 
microbial activity of the test system.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1175  
( 1 ) Test with a second concentration can be useful for chemicals that reach surface waters by 
different entry routes resulting in significantly different concentrations, as long as the 
lower concentration can be analysed with sufficient accuracy.
 1.9.3.  Test duration and sampling  
The duration of the experiment should normally not exceed 100 days 
(6), and should continue until the degradation pathway and water/ ­
sediment distribution pattern are established or when 90 % of the test 
substance has dissipated by transformation and/or volatilisation. The 
number of sampling times should be at least six (including zero time), with an optional preliminary study (see Section 1.9.4) being used to establish an appropriate sampling regime and the duration of 
the test, unless sufficient data is available on the test substance from 
previous studies. For hydrophobic test substances, additional sampling points during the initial period of the study may be 
necessary in order to determine the rate of distribution between 
water and sediment phases. 
At appropriate sampling times, whole incubation vessels (in 
replicate) are removed for analysis. Sediment and overlying water 
are analysed separately (
 1 ). The surface water should be carefully 
removed with minimum disturbance of the sediment. The extraction and characterisation of the test substance and transformation products 
should follow appropriate analytical procedures. Care should be 
taken to remove material that may have adsorbed to the incubation vessel or to interconnecting tubing used to trap volatiles.
 
1.9.4.  Optional preliminary test  
If duration and sampling regime cannot be estimated from other 
relevant studies on the test substance, an optional preliminary test may be considered appropriate, which should be performed using the 
same test conditions proposed for the definitive study. Relevant 
experimental conditions and results from the preliminary test, if performed, should be briefly reported.
 
1.9.5.  Measurements and analysis  
Concentration of the test substance and the transformation products 
at every sampling time in water and sediment should be measured and reported (as a concentration and as percentage of applied). In general, transformation products detected at ≥ 10 % of the applied 
radioactivity in the total water-sediment system at any sampling time 
should be identified unless reasonably justified otherwise. Trans ­
formation products for which concentrations are continuously increasing during the study should also be considered for identifi ­
cation, even if their concentrations do not exceed the limits given 
above, as this may indicate persistence. The latter should be considered on a case by case basis, with justifications being provided in the report. 
Results from gases/volatiles trapping systems (CO
 2 and others, i.e. 
volatile organic compounds) should be reported at each sampling 
time. Mineralisation rates should be reported. Non-extractable 
(bound) residues in sediment are to be reported at each sampling point.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1176  
( 1 ) In cases where rapid re-oxidation of anaerobic transformation products may readily 
occur, anaerobic conditions should be maintained during sampling and analysis.
 2. DATA  
2.1. TREATMENT OF RESULTS 
Total mass balance or recovery (see Section 1.7.1) of added radio ­
activity is to be calculated at every sampling time. Results should be 
reported as a percentage of added radioactivity. Distribution of radio ­
activity between water and sediment should be reported as concen ­
trations and percentages, at every sampling time. 
Half-life, DT  50 and, if appropriate, DT  75 and DT  90 of the test 
substance should be calculated along with their confidence limits 
(see Section 1.7.3). Information on the rate of dissipation of the test substance in the water and sediment can be obtained through the use of appropriate evaluation tools. These can range from appli ­
cation of pseudo-first order kinetics, empirical curve-fitting tech ­
niques which apply graphical or numerical solutions and more complex assessments using, for example, single- or multi- compartment models. Further details can be obtained from the 
relevant published literature (35)(36)(37). 
All approaches have their strengths and weaknesses and vary 
considerably in complexity. An assumption of first-order kinetics may be an oversimplification of the degradation and distribution processes, but when possible gives a term (the rate constant or 
half-life) which is easily understood and of value in simulation 
modelling and calculations of predicted environmental concen ­
trations. Empirical approaches or linear transformations can result in better fits of curves to data and therefore allow better estimation 
of half-lives, DT
 50 and, if appropriate, DT  75 and DT  90 values., The 
use of the derived constants, however, is limited. Compartment models can generate a number of useful constants of value in risk assessment that describe the rate of degradation in different 
compartments and the distribution of the chemical. They should 
also be used for estimation of rate constants for the formation and degradation of major transformation products. In all cases, the method chosen must be justified and the experimenter should 
demonstrate graphically and/or statistically the goodness of fit.
 
3. REPORTING  
3.1. TEST REPORT 
The report must include the following information: 
Test substance: — common name, chemical name, CAS number, structural formula 
(indicating position of the label(s) when radiolabelled material is 
used) and relevant physical-chemical properties, 
— purity (impurities) of test substance, — radiochemical purity of labelled chemical and molar activity 
(where appropriate).
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1177
 
Reference substances: 
— chemical name and structure of reference substances used for the 
characterisation and/or identification of transformation products. 
Test sediments and waters: — location and description of aquatic sediment sampling site(s) 
including, if possible, contamination history, 
— all information relating to the collection, storage (if any) and 
acclimation of water-sediment systems, 
— characteristics of the water-sediment samples as listed in Table in 
section 1.8.2.2. 
Test conditions: — test system used (e.g. flow-through, biometer, way of ventilation, 
method of stirring, water volume, mass of sediment, thickness of 
both water and sediment layer, dimension of test vessels, etc.), 
— application of test substance to test system: test concentration 
used, number of replicates and controls mode of application of 
test substance (e.g. use of solvent if any), etc., 
— incubation temperature, — sampling times, — extraction methods and efficiencies as well as analytical methods 
and detection limits, 
— methods for characterisation/identification of transformation 
products, 
— deviations from the test protocol or test conditions during the 
study. 
Results: — raw data figures of representative analyses (all raw data have to 
be stored in the GLP-archive), 
— repeatability and sensitivity of the analytical methods used, — rates of recovery (% values for a valid study are given in 
section 1.7.1), 
— tables of results expressed as % of the applied dose and in 
mg· kg
 -1 in water, sediment and total system (% only) for the 
test substance and, if appropriate, for transformation products and non-extractable radioactivity, 
— mass balance during and at the end of the studies, 
— a graphical representation of the transformation in the water and 
sediment fractions and in total system (including mineralisation), 
— mineralisation rates,
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1178
 
— half-life, DT  50 and, if appropriate, DT  75 and DT  90 values for the 
test substance and, where appropriate, for major transformation 
products including confidence limits in water, sediment and in total system, 
— an assessment of the transformation kinetics of the test substance 
and, where appropriate, the major transformation products, 
— a proposed pathway of transformation, where appropriate, — discussion of results.
 
4. REFERENCES  
(1) BBA-Guidelines for the examination of plant protectors in the 
registration process., (1990) Part IV, Section 5-1: Degradability 
and fate of plant protectors in the water/sediment system. Germany. 
(2) Commission for registration of pesticides: Application for 
registration of a pesticide., (1991) Part G. Behaviour of the product and its metabolites in soil, water and air, Section 
G.2.1 (a). The Netherlands. 
(3) MAFF Pesticides Safety Directorate., (1992) Preliminary 
guideline for the conduct of biodegradability tests on pesticides 
in natural sediment/water systems. Ref No SC 9046. United- Kingdom. 
(4) Agriculture Canada: Environmental chemistry and fate., (1987) 
Guidelines for registration of pesticides in Canada. Aquatic (Laboratory) — Anaerobic and aerobic. Canada. p. 35-37. 
(5) US-EPA: Pesticide assessment guidelines, Subdivision N. 
Chemistry: Environmental fate (1982) Section 162-3, 
Anaerobic aquatic metabolism. 
(6) SETAC-Europe publication., (1995) Procedures for assessing 
the environmental fate and ecotoxicity of pesticides. Ed. Dr 
Mark R. Lynch. SETAC-Europe, Brussels. 
(7) OECD Test Guidelines Programme., (1995) Final Report of the 
OECD Workshop on Selection of Soils/sediments, Belgirate, Italy, 18-20 January 1995. 
(8) ISO/DIS 5667-12., (1994) Water quality — Sampling — Part 
12: Guidance on sampling of bottom sediments. 
(9) US-EPA (1998a) Sediment/water microcosm biodegradation 
test. Harmonised Test Guidelines (OPPTS 835.3180). EPA 
712-C-98-080. 
(10) DFG: Pesticide Bound Residues in Soil. Wiley-VCH (1998). 
(11) T.R. Roberts: Non-extractable pesticide residues in soils and 
plants. Pure Appl. Chem. 56, 945-956 (IUPAC 1984). 
(12) OECD Test Guideline 304A: Inherent Biodegradability in Soil 
(adopted 12 May 1981). 
(13) OECD (1993): Guidelines for Testing of Chemicals. Paris. 
OECD (1994-2000): Addenda 6-11 to Guidelines for the 
Testing of Chemicals. 
(14) Scholz, K., Fritz R., Anderson C. and Spiteller M. (1988) 
Degradation of pesticides in an aquatic model ecosystem. 
BCPC — Pests and Diseases, 3B-4, p. 149-158.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1179
 
(15) Guth, J.A., (1981) Experimental approaches to studying the 
fate of pesticides in soil. In Progress in Pesticide Biochemistry 
(D.H. Hutson, T.R. Roberts, Eds.), Vol. 1, 85-114. J. Wiley & Sons. 
(16) Madsen, T., Kristensen, P. (1997) Effects of bacterial inocu ­
lation and non-ionic surfactants on degradation of polycyclic 
aromatic hydrocarbons in soil. Environ. Toxicol. Chem. 16, 
p. 631-637. 
(17) Steber, J., Wierich, P. (1987) The anaerobic degradation 
of detergent range fatty alcohol ethoxylates. Studies with
 
14 C-labelled model surfactants. Water Research 21, p. 661-667. 
(18) Black, C.A. (1965). Methods of Soil Analysis. Agronomy 
Monograph No 9. American Society of Agronomy, Madison. 
(19) APHA (1989) Standard Methods for Examination of Water and 
Wastewater (17  th edition). American Public Health Associ ­
ation, American Water Works Association and Water 
Pollution Control Federation, Washington D.C. 
(20) Rowell, D.L. (1994) Soil Science Methods and Applications. 
Longman. 
(21) Light, T.S., (1972). Standard solution for redox potential 
measurements. Anal. Chemistry 44, p. 1038-1039. 
(22) SETAC-Europe publication (1991) Guidance document on 
testing procedures for pesticides in freshwater mesocosms. From the Workshop ‘A Meeting of Experts on Guidelines for Static Field Mesocosms Tests’, 3-4 July 1991. 
(23) SETAC-Europe publication. (1993) Guidance document on 
sediment toxicity tests and bioassays for freshwater and 
marine environments. From the Workshop on Sediment Toxicity Assessment (WOSTA), 8-10 November 1993. Eds.: I.R. Hill, P. Matthiessen and F. Heimbach. 
(24) Vink, J.P.M., van der Zee, S.E.A.T.M. (1997) Pesticide 
biotransformation in surface waters: multivariate analyses of environmental factors at field sites. Water Research 31, p. 2858-2868. 
(25) Vink, J.P.M., Schraa, G., van der Zee, S.E.A.T.M. (1999) 
Nutrient effects on microbial transformation of pesticides in 
nitrifying waters. Environ. Toxicol, p. 329-338. 
(26) Anderson, T.H., Domsch, K.H. (1985) Maintenance carbon 
requirements of actively-metabolising microbial populations 
under  in-situ  conditions. Soil Biol. Biochem. 17, p. 197-203. 
(27) ISO-14240-2., (1997) Soil quality — Determination of soil 
microbial biomass — Part 2: Fumigation-extraction method. 
(28) Beelen, P. Van and F. Van Keulen., (1990), The Kinetics of 
the Degradation of Chloroform and Benzene in Anaerobic 
Sediment from the River Rhine. Hydrobiol. Bull. 24 (1), 
p. 13-21. 
(29) Shelton, D.R. and Tiedje, J.M. (1984) General method for 
determining anaerobic biodegradation potential. App. Environ. Microbiol. 47, p. 850-857. 
(30) Birch, R.R., Biver, C., Campagna, R., Gledhill, W.E., Pagga, 
U., Steber, J., Reust, H. and Bontinck, W.J. (1989) Screening of chemicals for anaerobic biodegradation. Chemosphere 19, p. 1527-1550. 
(31) Pagga, U. and Beimborn, D.B. (1993) Anaerobic biodegra ­
dation tests for organic compounds. Chemoshpere 27, 
p. 1499-1509.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1180
 
(32) Nuck, B.A. and Federle, T.W., (1986) A batch test for 
assessing the mineralisation of  14 C-radiolabelled compounds 
under realistic anaerobic conditions. Environ. Sci. Technol. 30, p. 3597-3603. 
(33) US-EPA (1998b). Anaerobic biodegradability of organic 
chemicals. Harmonised Test Guidelines (OPPTS 835.3400). 
EPA 712-C-98-090. 
(34) Sijm, Haller and Schrap (1997) Influence of storage on 
sediment characteristics and drying sediment on sorption coef ­
ficients of organic contaminants. Bulletin Environ. Contam. Toxicol. 58, p. 961-968. 
(35) Timme, G., Frehse H. and Laska V. (1986) Statistical inter ­
pretation and graphic representation of the degradational behaviour of pesticide residues II. Pflanzenschutz — Nach ­
richten Bayer, 39, p. 187-203. 
(36) Timme, G., Frehse, H. (1980) Statistical interpretation and 
graphic representation of the degradational behaviour of 
pesticide residues I. Pflanzenschutz — Nachrichten Bayer, 33, p. 47-60. 
(37) Carlton, R.R., and Allen, R., (1994) The use of a compartment 
model for evaluating the fate of pesticides in sediment/water 
systems. Brighton Crop Protection Conference — Pest and Diseases, p. 1349-1354.
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1181
 
Appendix 1  
GUIDANCE ON THE AEROBIC AND THE ANAEROBIC TEST 
SYSTEMS  
Aerobic test system  
The aerobic test system described in this test method consists of an aerobic water 
layer (typical oxygen concentrations range from 7 to 10 mg ·l -1 ) and a sediment 
layer, aerobic at the surface and anaerobic below the surface (typical average redox potentials (E
 h ) in the anaerobic zone of the sediment range from — 80 to 
— 190 mV). Moistened air is passed over the surface of the water in each 
incubation unit to maintain sufficient oxygen in the head space.  
Anaerobic test system  
For the anaerobic test system, the test procedure is essentially the same as that 
outlined for the aerobic system with the exception that moistened nitrogen is 
passed above the surface of the water in each incubation unit to maintain a head 
space of nitrogen. The sediment and water are regarded as anaerobic once the redox potential (E
 h ) is lower than — 100 mV. 
In the anaerobic test, assessment of mineralisation includes measurement of 
evolved carbon dioxide and methane.  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1182
 
Appendix 2  
EXAMPLE OF A GAS FLOW-THROUGH APPARATUS
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1183
 
Appendix 3  
EXAMPLE OF A BIOMETER APPARATUS
  ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1184
 
Appendix 4  
EXAMPLE CALCULATION FOR APPLICATION DOSE TO TEST 
VESSELS  
Cylinder internal diameter: = 8 cm 
Water column depth not including sediment: = 12 cm Surface area: 3,142 × 4
 2 = 50,3 cm  2 
Application rate: 500 g test substance/ha 
corresponds to 5 μg/cm  2 
Total μg: 5 × 50,3 = 251,5 μg 
Adjust quantity in relation to a depth of 
100 cm: 
12 × 251,5 ÷ 100 = 30,18 μg 
Volume of water column: 50,3 × 12 = 603 ml Concentration in water: 30,18 ÷ 603 = 0,050 μg/ml or 50 μg/l
 ▼B 2008R0440 — EN — 22.03.2014 — 004.001 — 1185
 
C.25.  AEROBIC MINERALISATION IN SURFACE WATER — 
SIMULATION BIODEGRADATION TEST  
1. METHOD  
This method is equivalent to OECD TG 309 (2004) (1).  
1.1. INTRODUCTION 
The purpose of this test is to measure the time course of biodegra ­
dation of a test substance at low concentration in aerobic natural 
water and to quantify the observations in the form of kinetic rate expressions. This simulation test is a laboratory shake flask batch test to determine rates of aerobic biodegradation of organic substances in 
samples of natural surface water (fresh, brackish or marine). It is 
based on the ISO/DIS 14592-1 (2) and it also includes elements from the testing methods C.23 and C.24 (3)(4). Optionally, with long test times, semi-continuous operation replaces batch operation in order to 
prevent deterioration of the test microcosm. The principal objective 
of the simulation test is to determine the mineralisation of the test substance in surface water, and mineralisation constitutes the basis for expressing degradation kinetics. However, an optional secondary 
objective of the test is to obtain information on the 
primary degradation and the formation of major transformation products. Identification of transformation products, and if possible quantification of their concentrations, are especially important for 
substances that are very slowly mineralised (e.g. with half-lives for 
total residual
 14 C exceeding 60 days). Higher concentrations of the 
test substance (e.g. > 100 μg/l) should normally be used for identi ­
fication and quantification of major transformation products due to 
analytical limitations. 
A low concentration in this test means a concentration (e.g. less than 
1 μg/l to 100 μg/l) which is low enough to ensure that the biode ­
gradation kinetics obtained in the test reflect those expected in the 
environment. Compared to the total mass of biodegradable carbon 
substrates available in the natural water used for the test, the test substance present at low concentration will serve as a secondary substrate. This implies that the anticipated biodegradation kinetics 
is first order (‘non-growth’ kinetics) and that the test substance 
may be degraded by ‘cometabolism’. First order kinetics implies that the rate of degradation (mg/L/day) is proportional to the concen ­
tration of substrate which declines over time. With true first order 
kinetics the specific degradation rate constant, k, is independent of 
time and concentration. That is, k does not vary appreciably during the course of an experiment and does not change with the added concentration between experiments. By definition, the 
specific degradation rate constant is equal to the relative change in 
concentration per time: k = (1/C) · (dC/dt). Although first order kinetics are normally expected under the prescribed conditions, there may be certain circumstances where other kinetics are more 
appropriate. Deviations from first order kinetics may e.g. be 
observed if mass transfer phenomena such as the diffusion rate, rather than the biological reaction rate, is limiting the rate of biotransformation. However, the data can nearly always be 
described by pseudo first order kinetics accepting a concentration 
dependent rate constant.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1186
 
Information on biodegradability of the test substance at higher 
concentrations (e.g. from standard screening tests) as well as 
information on abiotic degradability, transformation products and relevant physico-chemical properties should be available prior to the test to help establish the experimental planning and interpret 
the results. The use of
 14 C labelled test substances and the deter ­
mination of the phase distribution of  14 C at the end of the test, 
enable ultimate biodegradability to be determined. When non- labelled test substance is used, ultimate biodegradation can only be 
estimated if a higher concentration is tested and all the major trans ­
formation products are known.
 
1.2. DEFINITIONS  
Primary biodegradation : The structural change (transformation) of 
a chemical substance by microorganisms resulting in the loss of 
chemical identity.  
Functional biodegradation : The structural change (transformation) 
of a chemical substance by microorganisms resulting in the loss of a specific property.  
Ultimate aerobic biodegradation : The breakdown of a chemical 
substance by microorganisms in the presence of oxygen to carbon dioxide, water and mineral salts of any other elements present (min ­
eralisation) and the production of new biomass and organic microbial 
biosynthesis products.  
Mineralisation : The breakdown of a chemical substance or organic 
matter by microorganisms in the presence of oxygen to carbon 
dioxide, water and mineral salts of any other elements present.  
Lag phase : The time from the start of a test until adaptation of 
the degrading micro organisms is achieved and the biodegradation degree of a chemical substance or organic matter has increased to a detectable level (e.g. 10 % of the maximum 
theoretical biodegradation, or lower, dependent on the accuracy of 
the measuring technique).  
Maximum level of biodegradation : The degree of biodegradation 
of a chemical substance or organic matter in a test, recorded in per cent, above which no further biodegradation takes place during the test. 
Primary substrate : A collection of natural carbon and energy 
sources that provide growth and maintenance of the microbial 
biomass.  
Secondary substrate : A substrate component present in such a low 
concentration, that by its degradation, only insignificant amounts of 
carbon and energy are supplied to the competent microorganisms, as compared to the carbon and energy supplied by the degradation of main substrate components (primary substrates).  
Degradation rate constant : A first order or pseudo first order 
kinetic rate constant, k (d
 –1 ), which indicates the rate 
of degradation processes. For a batch experiment k is estimated 
from the initial part of the degradation curve obtained after the 
end of the lag phase.  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1187
 
Half-life, t  1/2 (d): Term used to characterise the rate of a first order 
reaction. It is the time interval that corresponds to a concentration 
decrease by a factor 2. The half-life and the degradation rate constant are related by the equation t
 1/2 = ln2/k.  
Degradation half time, DT  50 (d): Term used to quantify the 
outcome of biodegradation tests. It is the time interval, including 
the lag phase, needed to reach a value of 50 % biodegradation.  
Limit of detection (LOD) and limit of quantification (LOQ) : The 
limit of detection (LOD) is the concentration of a substance below 
which the identity of the substance cannot be distinguished from 
analytical artefacts. The limit of quantification (LOQ) is the concen ­
tration of a substance below which the concentration cannot be determined with an acceptable accuracy.  
Dissolved organic carbon (DOC) : That part of the organic carbon 
in a sample of water which cannot be removed by specified phase separation, for example by centrifugation at 40 000 ms
 –2 for 
15 min. or by membrane filtration using membranes with pores of 
0,2 μm-0,45 μm diameter.  
Total organic  14 C activity (TOA) : The total  14 C activity associated 
with organic carbon.  
Dissolved organic  14 C activity (DOA) : The total  14 C activity 
associated with dissolved organic carbon.  
Particulate organic  14 C activity (POA) : The total  14 C activity 
associated with particulate organic carbon.  
1.3. APPLICABILITY OF THE TEST 
This simulation test is applicable to non-volatile or slightly volatile 
organic substances tested at low concentrations. Using flasks open to 
the atmosphere (e.g. cotton wool plugged), substances with Henry’s law constants less than about 1 Pa·m
 3 /mol (approx. 10  –5 atm·m  3 /­
mol) can be regarded as non-volatile in practice. Using closed flasks 
with a headspace, it is possible to test slightly volatile substances 
(with Henry’s law constants < 100 Pa·m  3 /mol or < 10  –3 atm·m  3 /mol) 
without losses from the test system. Loss of  14 C-labelled substances 
may occur, if the right precautions are not exercised, when the CO  2 
is stripped off. In such situations, it may be necessary to trap CO  2 in 
an internal absorber with alkali or to use an external CO  2 absorber 
system (direct  14 CO 2 determination; see Appendix 3). For the deter ­
mination of biodegradation kinetics, the concentrations of the test 
substance must be below its water solubility. It should be noted, 
however, that literature values of water solubility may be considerably higher than the solubility of the test substance in natural waters. Optionally, the solubility of especially poorly 
water-soluble test substances may be established by use of the 
natural waters being tested. 
The method can be used for simulating biodegradation in surface 
water free of coarse particles (pelagic test) or in turbid surface water 
which, e.g. might exist near a water/sediment interface (suspended 
sediment test).
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1188
 
1.4. PRINCIPLE OF THE TEST 
The test is performed in batch by incubating the test substance with 
either surface water only (pelagic test) or surface water amended with suspended solids/sediment of 0,01 to 1 g/L dry weight (sus ­
pended sediment test) to simulate a water body with suspended 
solids or re-suspended sediment. The suspended solids/sediment concentration in the lower range of this interval is typical for most surface waters. The test flasks are incubated in darkness at an envi ­
ronmental temperature under aerobic conditions and agitation. At 
least two different concentrations of test substance should be used in order to determine the degradation kinetics. The concentrations 
should differ from each other by a factor of 5 to 10 and should 
represent the expected range of concentrations in the environment. The maximum concentration of the test substance should not exceed 100 μg/L, but maximum test concentrations below 10 μg/L or less 
are preferred to ensure that the biodegradation follows first order 
kinetics. The lowest concentration should not exceed 10 μg/L, but 
lowest test concentrations of 1-2 μg/L or less than 1 μg/L are 
preferred. Normally an adequate analysis of such low concentration 
can be achieved by use of commercially available
 14 C-labelled 
substances. Because of analytical limitations, it is frequently impossible to measure the concentration of the test substance with the required accuracy, if the test substance is applied at a concen ­
tration ≤ 100 μg/L (see second paragraph in section 1.7.2). 
Higher concentrations of test substance (> 100 μg/L and 
sometimes > 1 mg/L) may be used for the identification and quan ­
tification of major transformation products or if a specific analysis 
method with a low detection limit is not available. If high concen ­
trations of test substance are tested, it may not be possible to use the results to estimate the first order degradation constant and half-life, as the degradation will probably not follow first order kinetics. 
Degradation is followed at appropriate time intervals, by measuring 
either the residual
 14 C or the residual concentration of test substance 
when specific chemical analysis is used.  14 C labelling of the most 
stable part of the molecule ensures the determination of the total mineralisation, while
 14 C labelling of a less stable part of the 
molecule, as well as the use of specific analysis, enable the 
assessment of only primary biodegradation. However, the most 
stable part does not necessarily include the relevant functional moiety of the molecule (that can be related to a specific property such as toxicity, bioaccumulation, etc.). If this is the case, it may be 
appropriate to use a test substance, which is
 14 C-labelled, in the 
functional part in order to follow the elimination of the specific property.
 
1.5. INFORMATION ON THE TEST SUBSTANCE 
Both radiolabelled and non-labelled test substances can be used in 
this test.  14 C-labelling technique is recommended and labelling 
should normally be in the most stable part(s) of the molecule (see 
also section 1.4). For substances containing more than one aromatic 
ring, one or more carbons in each ring should preferably be  14 C- 
labelled. In addition, one or more carbons on both sides of easily degradable linkages should preferably be
 14 C-labelled. The 
chemical and/or radiochemical purity of the test substance should 
be > 95 %. For radiolabelled substances, a specific activity of approx. 50 μCi/mg (1,85 MBq) or more is preferred in order to 
facilitate
 14 C measurements in tests conducted with low initial 
concentrations. The following information on the test substance 
should be available:  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1189
 
— solubility in water [Method A.6], 
— solubility in organic solvent(s) (substances applied with solvent 
or with low solubility in water), 
— dissociation constant (pKa) if the substance is liable to 
protonation or deprotonation [OECD TG 112] (5), 
— vapour pressure [Method A.4] and Henry’s law constant, — chemical stability in water and in the dark (hydrolysis) [Method C.7]. When poorly water-soluble substances are being tested in seawater, it 
may also be useful to know the salting out constant (or ‘Setschenow constant’) K
 s , which is defined by the expression: log (S/S’) = K  s 
C m , where S and S’ are the solubility of the substance in fresh water 
and seawater, respectively, and C  m is the molar salt concentration. 
If the test is carried out as a ‘suspended sediment test’ the following 
information should also be available: 
— n-octanol/water partition coefficient [Method A.8], 
— adsorption coefficient [Method C.18]. 
Other useful information may include: — environmental concentration, if known or estimated, — toxicity of the test substance to microorganisms [Method C.11], 
— ready and/or inherent biodegradability [Methods C.4 A-F, C.12, 
C.9, OECD TG 302 (5)], 
— aerobic or anaerobic biodegradability in soil and sediment/water 
transformation studies [Methods C.23, C.24].
 
1.6. REFERENCE SUBSTANCE 
A substance, which is normally easily degraded under aerobic 
conditions (e.g. aniline or sodium benzoate) should be used as 
reference substance. The expected time interval for degradation of aniline and sodium benzoate is usually less than 2 weeks. The purpose of the reference substances is to ensure that the microbial 
activity of the test water is within certain limits; i.e. that the water 
contains an active microbial population.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1190
 
1.7. QUALITY CRITERIA  
1.7.1.  Recovery  
Immediately after addition of the test substance, each initial test 
concentration should be verified by measurements of  14 C activity, 
or by chemical analyses in the case of non-labelled substances, in at 
least duplicate samples. This provides information on the applica ­
bility and repeatability of the analytical method and on the homo ­
geneity of the distribution of the test substance. Normally, the 
measured initial  14 C activity or test substance concentration is used 
in the subsequent analyses of data rather than the nominal concen ­
tration as losses due to sorption and dosing errors thereby are 
compensated. For  14 C-labelled test substance, the level of recovery 
at the end of the experiment is given by mass balance (see last paragraph in section 1.8.9.4). Ideally, the radiolabelled mass balance should range from 90 % to 110 %, whereas the analytical 
accuracy should lead to an initial recovery of between 70 % 
and 110 % for non-labelled test substances. These ranges should be interpreted as targets and should not be used as criteria for acceptance of the test. Optionally, the analytical accuracy may be 
determined for the test substance at a lower concentration than the 
initial concentration and for major transformation products.
 
1.7.2.  Repeatability and sensitivity of analytical method  
Repeatability of the analytical method (including the efficiency of 
the initial extraction) to quantify the test substance, and trans ­
formation products, if appropriate, should be checked by five 
replicate analyses of the individual extracts of the surface water. 
The limit of detection (LOD) of the analytical method for the test 
substance and for the transformation products should be at least 1 % 
of the initial amount applied to the test system if possible. The limit of quantification (LOQ) should be equal to or less than 10 % of the 
applied concentration. The chemical analyses of many organic 
substances and their transformation products frequently require that the test substance is applied at a relatively high concentration, i.e. > 100 μg/L.
 
1.8. DESCRIPTION OF THE TEST METHOD  
1.8.1.  Equipment  
The test may be conducted in conical or cylindrical flasks of appro ­
priate capacity (e.g. 0,5 or 1,0 litre) closed with silicone or rubber stoppers, or in serum flasks with CO
 2 -tight lids (e.g. with butyl 
rubber septa). Another option is to perform the test by use of 
multiple flasks and to harvest whole flasks, at least in duplicate, at each sample interval (see last paragraph in section 1.8.9.1). For non- volatile test substances that are not radiolabelled, gas-tight stoppers 
or lids are not required; loose cotton plugs that prevent contami ­
nation from air are suitable (see second paragraph in section 1.8.9.1). Slightly volatile substances should be tested in a biometer-type system with gentle stirring of the water surface. To be 
sure that no bacterial contamination occurs, optionally the vessels 
can be sterilised by heating or autoclaving prior to use. In addition, the following standard laboratory equipment is used: 
— shaking table or magnetic stirrers for continuous agitation of the 
test flasks,
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1191
 
— centrifuge, 
— pH meter, — turbidimeter for nephelometric turbidity measurements, 
— oven or microwave oven for dry weight determinations, 
— membrane filtration apparatus, — autoclave or oven for heat sterilisation of glassware, — facilities to handle
 14 C-labelled substances, 
— equipment to quantify  14 C-activity in samples from CO  2 -trapping 
solutions and, if required, from sediment samples, 
— analytical equipment for the determination of the test (and 
reference) substance if specific chemical analysis is used (e.g. 
gas chromatograph, high-pressure liquid chromatograph).  
1.8.2.  Stock solutions of test substance  
Deionised water is used to prepare stock solutions of the test and 
reference substances (see first paragraph in section 1.8.7). The 
deionised water should be free of substances that may be toxic to microorganisms, and dissolved organic carbon (DOC) should be no more than 1 mg/L (6).
 
1.8.3.  Collection and transport of surface water  
The sampling site for collection of the surface water should be 
selected in accordance with the purpose of the test in any given situation. In selecting sampling sites, the history of possible agricul ­
tural, industrial or domestic inputs must be considered. If it is known 
that an aquatic environment has been contaminated with the test substance or its structural analogues within the previous four years, it should not be used for the collection of test water, unless inves ­
tigation of degradation rates in previously exposed sites is the 
express purpose of the investigator. The pH and temperature of the water should be measured at the site of collection. Furthermore, the depth of sampling and the appearance of the water sample (e.g. 
colour and turbidity) should be noted (see section 3). Oxygen 
concentration and/or redox potential in water and in the sediment surface layer should be measured in order to demonstrate aerobic conditions unless this is obvious as judged from appearance and 
historic experience with the site. The surface water should be trans ­
ported in a thoroughly cleansed container. During transport, the temperature of the sample should not significantly exceed the temperature used in the test. Cooling to 4 °C is recommended if 
transport duration exceeds 2 to 3 hours. The water sample must not 
be frozen.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1192
 
1.8.4.  Storage and preparation of surface water  
The test should preferably be started within one day after sample 
collection. Storage of the water, if needed, should be minimised and must in any case not exceed a maximum of 4 weeks. The water 
sample should be kept at 4 °C with aeration until use. Prior to use, 
the coarse particles should be removed, e.g. by filtration through a nylon filter with about 100 μm mesh size or with a coarse paper 
filter, or by sedimentation.
 
1.8.5.  Preparation of water amended with sediment (optional)  
For the suspended sediment test, surface sediment is added to the 
flasks containing natural water (filtered to remove coarse particles as 
described in section 1.8.4) to obtain a suspension; the concentration 
of suspended solids should be between 0,01 and 1 g/L. The surface sediment should come from the same site as that from which the water sample was taken. Dependent on the particular aquatic 
environment, the surface sediment may either be characterised by a 
high organic carbon content (2,5-7,5 %) and a fine texture or by a low organic carbon content (0,5-2,5 %) and a coarse texture (3). The surface sediment can be prepared as follows: extract several sediment 
cores using a tube of transparent plastic, slice off the upper aerobic 
layers (from surface to a depth of max. 5 mm) immediately after sampling and pool them together. The resulting sediment sample should be transported in a container with a large air headspace to 
keep the sediment under aerobic conditions (cool to 4 °C if transport 
duration exceeds 2-3 hours). The sediment sample should be suspended in the test water at a ratio of 1:10 and kept at 4 °C 
with aeration until use. Storage of the sediment, if needed, should 
be minimised and must not in any case exceed a maximum of 4 weeks.
 
1.8.6.  Semi-continuous procedure (optional)  
Prolonged incubation (several months) may be necessary if a long 
lag time occurs before a significant degradation of the test substance can be measured. If this is known from previous testing of a substance, the test may be initiated by using a semi-continuous 
procedure, which allows periodical renewal of a part of the test 
water or suspension (see Appendix 2). Alternatively, the normal batch test may be changed into a semi-continuous test, if 
no degradation of the test substance has been achieved during 
approximately 60 days of testing using the batch procedure (see second paragraph in section 1.8.8.3).
 
1.8.7.  Addition of the test (or reference) substance  
For substances with high water solubility (> 1 mg/L) and low vola ­
tility (Henry’s law constants < 1 Pa·m  3 /mol or < 10  –5 atm·m  3 /mol), a 
stock solution can be prepared in deionised water (see section 1.8.2); the appropriate volume of the stock solution is added to the test 
vessels to achieve the desired concentration. The volume of any 
added stock solution should be held to the practical minimum (< 10 % of the final liquid volume, if possible). Another procedure is to dissolve the test substance in a larger volume of 
the test water, which may be seen as an alternative to the use of 
organic solvents.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1193
 
If unavoidable, stock solutions of non-volatile substances with poor 
water-solubility should be prepared by use of a volatile organic 
solvent, but the amount of solvent added to the test system should not exceed 1 % v/v and should not have adverse effects on the microbial activity. The solvent should not affect the stability of the 
test substance in water. The solvent should be stripped off to an 
extremely small quantity so that it does not significantly increase the DOC concentration of the test water or suspension. This should be checked by substance-specific analysis or, if possible, 
DOC analysis (6). Care must be taken to limit the amount of 
solvent transferred to what is absolutely necessary, and to ensure that the amount of test substance can dissolve in the final volume of test water. Other techniques to introduce the test substance into 
the test vessels may be used as described in (7) and (8). When an 
organic solvent is used for application of the test substance, solvent controls containing the test water (with no additions) and test water with added reference substance should be treated similarly to active 
test vessels amended with test substance in solvent carrier. The 
purpose of the solvent controls is to examine possible adverse effects caused by the solvent towards the microbial population as indicated by the degradation of the reference substance.
 
1.8.8.  Test conditions  
1.8.8.1.  Test temperature  
Incubation should take place in the dark (preferred) or in diffuse 
light at a controlled (± 2 °C) temperature, which may be the field temperature or a standard temperature of 20-25 °C. Field temperature may be either the actual temperature of the sample at the sampling 
time or an average field temperature at the sampling site.
 
1.8.8.2.  Agitation  
Agitation by means of continuous shaking or stirring must be 
provided to maintain particles and microorganisms in suspension. Agitation also facilitates oxygen transfer from the headspace to the 
liquid so that aerobic conditions can be adequately maintained. Place 
the flasks on a shaking table (approx. 100 rpm agitation) or use magnetic stirring. Agitation must be continuous. However, the shaking or stirring should be as gentle as possible, while still main ­
taining a homogeneous suspension.
 
1.8.8.3.  Test duration  
The duration of the test should normally not exceed 60 days unless 
the semi-continuous procedure with periodical renewal of the test suspension is applied (see section 1.8.6 and Appendix 2). However, the test period for the batch test may be extended to a 
maximum of 90 days, if the degradation of the test substance has 
started within the first 60 days. Degradation is monitored, at appro ­
priate time intervals, by the determination of the residual
 14 C activity 
or the evolved  14 CO 2 (see section 1.8.9.4) and/or by chemical 
analysis (section 1.8.9.5). The incubation time must be sufficiently 
long to evaluate the degradation process. The extent of degradation should preferably exceed 50 %; for slowly degradable substances, the extent of degradation must be sufficient (normally greater than 
20 % degradation) to ensure the estimation of a kinetic degradation 
rate constant.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1194
 
Periodic measurements of pH and oxygen concentration in the test 
system must be conducted unless previous experience from similar 
tests with water and sediment samples collected from the same site make such measurements unnecessary. Under some conditions, the metabolism of primary substrates at much higher concentrations 
within the water or sediment could possibly result in enough CO
 2 
evolution and oxygen depletion to significantly alter the experimental 
conditions during the test.  
1.8.9.  Procedure  
1.8.9.1.  Preparation of flasks for pelagic test  
Transfer a suitable volume of test water to the test flasks, up to about 
one third of the flask volume and not less than about 100 ml. 
If multiple flasks are used (to allow harvesting of whole flasks 
at each sampling time), the appropriate volume of test water is also about 100 ml, as small sample volumes may influence the length of the lag phase. The test substance is added from a stock 
solution as described in sections 1.8.2 and 1.8.7. At least two 
different concentrations of test substance differing by a factor of 5 to 10 should be used in order to determine degradation kinetics and calculate the kinetic degradation rate constant. Both of the 
selected concentrations should be less than 100 μg/L and preferably 
in the range of < 1-10 μg/L. 
Close the flasks with stoppers or lids impermeable to air and CO
 2 . 
For non-  14 C-labelled non-volatile test chemicals, loose cotton wool 
plugs that prevent contamination from air are suitable (see section 1.8.1) provided that any major degradation products are 
known to be non-volatile, and if indirect CO
 2 determination is 
used (see Appendix 3). 
Incubate the flasks at the selected temperature (see section 1.8.8.1). 
Withdraw samples for chemical analysis or  14 C measurements at the 
beginning of the test (i.e. before biodegradation starts; see 
section 1.7.1) and then at suitable time intervals during the course 
of the test. Sampling may be performed by withdrawal of sub- samples (e.g. 5 ml aliquots) from each replicate or by harvest of whole flasks at each sampling time. The mineralisation of the test 
substance may either be determined indirectly or directly (see 
Appendix 3). Usually, a minimum of five sampling points are required during the degradation phase (i.e. after ended lag phase) in order to estimate a reliable rate constant, unless it can be 
justified that three sampling points are sufficient for 
rapidly degradable substances. For substances that are not rapidly degraded more measurements during the degradation phase can easily be made and, therefore, more data points should be used 
for the estimation of k. No fixed time schedule for sampling can be 
stated, as the rate of biodegradation varies; however the recommen ­
dation is to sample once a week if degradation is slow. If the test substance is rapidly degradable, sampling should take place once a 
day during the first three days and then every second or third day. 
Under certain circumstances, such as with very rapidly hydrolysing substances, it may be necessary to sample at hourly intervals. It is recommended that a preliminary study is conducted prior to the test 
in order to determine the appropriate sampling intervals. If samples 
have to be available for further specific analysis, it is advisable to take more samples and then select those to be analysed at the end of the experiment following a backwards strategy, i.e. the last samples 
are analysed first (see second paragraph in section 1.8.9.5 for 
guidance on stability of samples during storage).
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1195
 
1.8.9.2.  Number of flasks and samples  
Set up a sufficient number of test flasks to have: 
— test flasks; at least duplicate flasks for each concentration of test 
substance (preferably a minimum of 3) or multiple test flasks for 
each concentration, if whole flasks are harvested at each sampling time (symbolised F
 T ), 
— test flasks for mass balance calculation; at least duplicate flasks 
for each test concentration (symbolised F  M ), 
— blank control, no test substance; at least one blank test flask 
containing only the test water (symbolised F  B ), 
— reference control; duplicate flasks with reference substance (e.g. 
aniline or sodium benzoate, at 10 μg/l) (symbolised F  C ). The 
purpose of the reference control is to confirm a minimum of microbial activity. If convenient, a radiolabelled reference substance may be used, also when the degradation of the test 
substance is monitored by chemical analyses, 
— sterile control; one or two flasks containing sterilised test water 
for examining possible abiotic degradation or other non- 
biological removal of the test substance (symbolised F
 S ). The 
biological activity can be stopped by autoclaving (121 °C; 20 min.) the test water or by adding a toxicant (e.g. sodium azide (NaN
 3 ) at 10-20 g/l, mercuric chloride (HgCl  2 ) at 
100 mg/l or formalin at 100 mg/l) or by gamma irradiation. If 
HgCl  2 is used, it should be disposed of as toxic waste. For water 
with sediment added in large amount, sterile conditions are not easy to obtain; in this case repeated autoclaving (e.g. three times) 
is recommended. It should be considered that the sorption char ­
acteristics of the sediment may be altered by autoclaving, 
— solvent controls, containing test water and test water with 
reference substance; duplicate flasks treated with the same 
amount of solvent and by use of the same procedure as that 
used for application of the test substance. The purpose is to examine possible adverse effects of the solvent by determining the degradation of the reference substance. 
In the design of the test, the investigator should consider the relative 
importance of increased experimental replication versus increased number of sampling times. The exact number of flasks required will depend on the method used for measuring the degradation 
(see third paragraph in section 1.8.9.1; section 1.8.9.4 and 
Appendix 3).
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1196
 
Two subsamples (e.g. 5 ml aliquots) should be withdrawn from each 
test flask at each sampling time. If multiple flasks are used to allow 
harvesting of whole flasks, a minimum of two flasks should be sacrificed at each sampling time (see first paragraph in section 1.8.9.1).
 
1.8.9.3.  Preparation of flasks for suspended sediment test [optional]  
Add the necessary volumes of test water and sediment, if required, to 
the test vessels (see section 1.8.5). The preparation of flasks for suspended sediment test is the same as for the pelagic test (see 
sections 1.8.9.1 and 1.8.9.2). Use preferably serum bottles or 
similar shaped flasks. Place the closed flasks horizontally on a shaker. Obviously, open flasks for non-
 14 C-labelled, non-volatile 
substances should be placed in upright position; in this case 
magnetic stirring and the use of magnetic bars coated with glass 
are recommended. If necessary, aerate the bottles to maintain proper aerobic conditions.
 
1.8.9.4.  Radiochemical determinations  
The evolved  14 CO 2 is measured indirectly and directly (see 
Appendix 3). The  14 CO 2 is determined indirectly by the difference 
between the initial  14 C activity in the test water or suspension and 
the total residual activity at the sampling time as measured after 
acidifying the sample to pH 2-3 and stripping off CO  2 . Inorganic 
carbon is thus removed and the residual activity measured derives from organic material. The indirect
 14 CO 2 determination should not 
be used, if major volatile transformation products are formed during 
the transformation of the test substance (see Appendix 3). If possible, the
 14 CO 2 evolution should be measured directly (see Appendix 3) at 
each sampling time in at least one test flask; this procedure enables 
both the mass balance and biodegradation process to be checked, but 
it is restricted to tests conducted with closed flasks. 
If the evolved  14 CO 2 is measured directly during the test, more 
flasks should be set up for this purpose at the start of the test. Direct
 14 CO 2 determination is recommended, if major volatile trans ­
formation products are formed during the transformation of the test 
substance. At each measuring point the additional test flasks are 
acidified to pH 2-3 and the  14 CO 2 is collected in an internal or 
external absorber (see Appendix 3). 
Optionally the concentrations of  14 C-labelled test substance and 
major transformation products may be determined by use of radio ­
chromatography (e.g. thin layer chromatography, RAD-TLC) or 
HPLC with radiochemical detection. 
Optionally the phase distribution of the remaining radioactivity (see 
Appendix 1) and residual test substance and transformation products 
may be determined.  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1197
 
At the end of the test the mass balance should be determined by 
direct  14 CO 2 measurement using separate test flasks from which no 
samples are taken in the course of the test (see Appendix 3).  
1.8.9.5.  Specific chemical analysis  
If a sensitive specific analytical method is available, primary biode ­
gradation can be assessed by measuring the total residual concen ­
tration of test substance instead of using radiolabelling techniques. If 
a radiolabelled test substance is used (to measure total minerali ­
sation), specific chemical analyses can be made in parallel to provide useful additional information and check the procedure. 
Specific chemical analyses may also be used to measure trans ­
formation products formed during the degradation of the test substance, and this is recommended for substances that are miner ­
alised with half-lives exceeding 60 days. The concentration of the 
test substance and the transformation products at every sampling 
time should be measured and reported (as a concentration and as percentage of applied). In general, transformation products detected at ≥ 10 % of the applied concentration at any sampling time should 
be identified unless reasonably justified otherwise. Transformation 
products for which concentrations are continuously increasing during the study should also be considered for identification, even if their concentrations do not exceed the limit given above, as this 
may indicate persistence. Analyses of transformation products in 
sterile controls should be considered, if rapid abiotic transformation of the test substance (e.g. hydrolysis) is thought possible. The need for quantification and identification of transformation products 
should be considered on a case by case basis, with justifications 
being provided in the report. Extraction techniques with organic solvent should be applied according to directions given in the respective analytical procedure. 
All samples should be stored at 2 to 4 °C and air-tight if analysis is 
carried out within 24 hours (preferred). For longer storage, the samples should be frozen below – 18 °C or chemically preserved. 
Acidification is not a recommended method to preserve the samples, 
because acidified samples may be unstable. If the samples are not analysed within 24 hours and are subject to longer storage, a storage stability study should be conducted to demonstrate the stability of 
chemicals of interest under – 18 °C storage or preserved conditions. 
If the analytical method involves either solvent extraction or solid phase extraction (SPE), the extraction should be performed immediately after sampling or after storing the sample refrigerated 
for a maximum of 24 hours. 
Depending on the sensitivity of the analytical method, larger sample 
volumes than those indicated in section 1.8.1 may be necessary. The test can easily be carried out with test volumes of one litre in flasks 
of 2-3 litre volume, which makes it possible to collect samples of 
approx. 100 ml.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1198
 
2. DATA AND REPORTING  
2.1. TREATMENT OF RESULTS  
2.1.1.  Plot of data  
Round off sampling times to a whole number of hours (unless the 
substance degrades substantially in a matter of minutes to hours) but 
not to a whole number of days. Plot the estimates of the residual activity of test substance (for
 14 C-labelled substances) or the residual 
concentration (for non-labelled substances), against time both in a 
linear and in a semi-logarithmic plot (see Figures 1a, 1b). 
If degradation has taken place, compare the results from flasks F  T 
with those from flasks F  S . If the means of the results from the flasks 
with test substance (F  T ) and the sterile flasks (F  S ) deviate by less 
than 10 %, it can be assumed that the degradation observed is predominantly abiotic. If the degradation in flasks F
 S is lower, the 
figures may be used to correct those obtained with flasks F  T (by 
subtraction) in order to estimate the extent of biodegradation. When 
optional analyses are performed for major transformation products, plots of their formation and decline should be provided in addition to a plot of the decline of the test substance. 
Estimate the lag phase duration t
 L from the degradation curve (semi- 
logarithmic plot) by extrapolating its linear part to zero degradation or alternatively by determining the time for approximately 10 % degradation (see Figures 1a and 1b). From the semi-logar ­
ithmic plot, estimate the first order rate constant, k, and its 
standard error by linear regression of ln (residual
 14 C activity or 
test substance concentration) versus time. With  14 C measurements 
in particular, use only data belonging to the initial linear part of 
the curve after the ended lag phase, and give preference to 
selecting few and representative data rather than selecting a greater number of more uncertain data. Uncertainty includes here errors inherent in the recommended direct use of measured residual
 14 C 
activities (see below). It may sometimes be relevant to calculate two 
different rate constants, if the degradation follows a biphasic pattern. For this purpose two different phases of the degradation curve are defined. Calculations of the rate constant, k, and the half-life t
 ½ = 
ln2/k, should be carried out for each of the individual replicate 
flasks, when sub-samples are withdrawn from the same flask, or by using the average values, when whole flasks are harvested at each sampling time (see last paragraph in section 1.8.9.2). When 
the first-mentioned procedure is used, the rate constant and half- 
life should be reported for each of the individual replicate flasks and as an average value with a standard error. If high concentrations of test substance have been used, the degradation curve may deviate 
considerably from a straight line (semi-logarithmic plot) and first 
order kinetics may not be valid. Defining a half-life has therefore no meaning. However, for a limited data range, pseudo first order kinetics can be applied and the degradation half-time DT
 50 (time to 
reach 50 % degradation) estimated. It must be borne in mind, 
however, that the time course of degradation beyond the selected data range cannot be predicted using the DT
 50 which is merely a 
descriptor of a given set of data. Analytical tools to facilitate stat ­
istical calculations and curve fitting are easily available and the use 
of this kind of software is recommended.  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1199
 
If specific chemical analyses are made, estimate rate constants and 
half-lives for primary degradation as above for total mineralisation. If 
the primary degradation is the limiting process data points from the entire course of degradation may sometimes be used. This is because measurements are direct by contrast to measurements of
 14 C activity. 
If 14 C-labelled substances are used, a mass balance should be 
expressed in percentage of the applied initial concentration, at least 
at the end of the test.  
2.1.2.  Residual activity  
When the  14 C-labelled part of an organic substance is biodegraded, 
the major part of the  14 C is converted to  14 CO 2 , while another part is 
used for growth of biomass and/or synthesis of extra-cellular meta ­
bolites. Therefore, complete ‘ultimate’ biodegradation of a substance does not result in a 100 % conversion of its carbon into
 14 CO 2 . The  
14 C built into products formed by biosynthesis is subsequently 
released slowly as  14 CO 2 due to ‘secondary mineralisation’. For 
these reasons plots of residual organic  14 C activity (measured after 
stripping off CO  2 ) or of  14 CO 2 produced versus time will show a 
‘tailing’ after degradation has been completed. This complicates a 
kinetic interpretation of the data and for this purpose, only the initial part of the curve (after the lag phase has ended and before approx. 50 % degradation is reached) should normally be used for the esti ­
mation of a degradation rate constant. If the test substance 
is degraded, the total residual organic
 14 C activity is always higher 
than the  14 C activity associated with the remaining intact test 
substance. If the test substance is degraded by a first order 
reaction and a constant fraction α is mineralised into CO  2 , the 
initial slope of the  14 C disappearance curve (total organic  14 C 
versus time) will be α times the slope of the corresponding curve 
for the concentration of test substance (or, to be precise, the part of 
the test substance labelled with  14 C). Using measurements of the 
total organic  14 C activity uncorrected, the calculated degradation 
rate constant will therefore be conservative. Procedures for esti ­
mating the concentrations of the test substance from the measured 
radiochemical activities based on various simplifying assumptions 
have been described in the literature (2)(9)(10)(11). Such procedures are most easily applied for rapidly degradable substances.
 
2.2. INTERPRETATION OF RESULTS 
If k is found to be independent of the added concentration (i.e. if the 
calculated k is approximately the same at the different concentrations of test substance), it can be assumed that the first order rate constant 
is representative of the testing conditions used, i.e. the test substance, 
the water sample and the test temperature. To what extent the results can be generalised or extrapolated to other systems must be evaluated by expert judgement. If a high concentration of test 
substance is used, and the degradation therefore does not follow 
first order kinetics, the data cannot be used for direct estimation of a first order rate constant or a corresponding half-life. However, data derived from a test using a high concentration of test substance may 
still be usable for estimating the degree of total mineralisation and/or 
detection and quantification of transformation products.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1200
 
If the rates of other loss processes than biodegradation are known 
(e.g. hydrolysis or volatilisation), they may be subtracted from the 
net loss rate observed during the test to give an approximated estimate of the biodegradation rate. Data for hydrolysis may, e.g. be obtained from the sterile control or from parallel testing using a 
higher concentration of the test substance. 
The indirect and direct determination of
 14 CO 2 (section 1.8.9.4 and 
Appendix 3) can only be used to measure the extent of minerali ­
sation of the test substance to CO  2 . Radiochromatography 
(RAD-TLC) or HPLC may be used to analyse the concentrations 
of 14 C-labelled test substance and the formation of major trans ­
formation products (third paragraph in section 1.8.9.4). To enable a direct estimation of the half-life, it is necessary that no major transformation products (defined as ≥ 10 % of the applied amount 
of test substance) be present. If major transformation products as 
defined here are present, a detailed evaluation of the data is required. This may include repeated testing and/or identification of the transformation products (see first paragraph in section 1.8.9.5) 
unless the fate of the transformation products can be reasonably 
assessed by use of experience (e.g. information on degradation pathway). As the proportion of test substance carbon converted to CO
 2 varies (depending largely on the concentration of test substance 
and other substrates available, the test conditions and the microbial 
community), this test does not allow a straightforward estimation of ultimate biodegradation as in a DOC die-away test; but the result is similar to that obtained with a respirometric test. The degree of 
mineralisation will thus be less than or equal to the minimum 
level of ultimate biodegradation. To obtain a more complete picture of the ultimate biodegradation (mineralisation and incorporation into biomass), the analysis of the phase distribution 
of
 14 C should be performed at the end of the test 
(see Appendix 1). The  14 C in the particulate pool will consist 
of 14 C incorporated into bacterial biomass and  14 C sorbed to 
organic particles.  
2.3. VALIDITY OF THE TEST 
If the reference substance is not degraded within the expected time 
interval (for aniline and sodium benzoate, usually less than two 
weeks), the validity of the test is suspected and must be further verified, or alternatively the test should be repeated with a new water sample. In an ISO ring-test of the method where seven labora ­
tories located around Europe participated, adapted degradation rate 
constants for aniline ranged from 0,3 to 1,7 day
 –1 with an average of 
0,8 d  –1 at 20  o C and a standard error of ± 0,4 d  –1 (t ½ = 0,9 days). 
Typical lag times were 1 to 7 days. The waters examined were 
reported to have a bacterial biomass corresponding to 10  3 -10 4 
colony forming units (CFU) per ml. Degradation rates in nutrient- 
rich Mid-European waters were greater than in Nordic oligotrophic waters, which may be due to the different trophic status or previous 
exposure to chemical substances. 
The total recovery (mass balance) at the end of the experiment 
should be between 90 % and 110 % for radiolabelled substances, whereas the initial recovery at the beginning of the experiment should be between 70 % and 110 % for non-labelled substances. 
However, the indicated ranges should only be interpreted as targets 
and should not be used as criteria for acceptance of the test.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1201
 
3. TEST REPORT  
The type of study, i.e. pelagic or suspended sediment test, must be 
clearly stated in the test report, which shall also contain at least the following information: 
Test substance and reference substance(s): — common names, chemical names (recommend IUPAC and/or 
CAS names), CAS numbers, structural formulas (indicating 
position of
 14 C if radiolabelled substance is used) and relevant 
physico-chemical properties of test and reference substance (see sections 1.5 and 1.6), 
— chemical names, CAS numbers, structural formulas (indicating 
position of
 14 C if radiolabelled substance is used) and relevant 
physico-chemical properties of substances used as standards for 
identification and quantification of transformation products, 
— purity (impurities) of test and reference substances, 
— radiochemical purity of labelled chemical and specific activity 
(where appropriate). 
Surface water: 
The following minimum information for the water sample taken 
must be provided: 
— location and description of sampling site including, if possible, 
contamination history, 
— date and time of sample collection, 
— nutrients (total N, ammonium, nitrite, nitrate, total P, dissolved 
orthophosphate), 
— depth of collection, — appearance of sample (e.g. colour and turbidity), — DOC and TOC, — BOD, — temperature and pH at the place and time of collection, 
— oxygen or redox potential (mandatory only if aerobic conditions 
are not obvious), 
— salinity or conductivity (in the case of sea water and brackish 
water), 
— suspended solids (in case of a turbid sample), 
— possibly other relevant information about the sampling location 
at the time of sampling (e.g. actual or historical data on flow rate 
of rivers or marine currents, nearby major discharges and type of discharges, weather conditions preceding the sampling time), 
and optionally: — microbial biomass (e.g. acridine orange direct count or colony 
forming units),
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1202
 
— inorganic carbon, 
— chlorophyll-a concentration as a specific estimate for algal 
biomass. 
In addition, the following information on the sediment should be 
provided if the suspended sediment test is conducted: 
— depth of sediment collection, — appearance of the sediment (such as coloured, muddy, silty, 
or sandy), 
— texture (e.g. % coarse sand, fine sand, silt and clay), 
— dry weight in g/l of the suspended solids, TOC concentration or 
weight loss on ignition as a measure of the content of organic 
matter, 
— pH, 
— oxygen or redox potential (mandatory only if aerobic conditions 
are not obvious). 
Test conditions: — delay between collection and use in the laboratory test, sample 
storage and pre-treatment of the sample, dates of performance of the studies, 
— amount of test substance applied, test concentration and reference 
substance, 
— method of application of the test substance including any use of 
solvents, 
— volume of surface water used and sediment (if used) and volume 
sampled at each interval for analysis, 
— description of the test system used. If dark conditions are not to be maintained, information on the 
‘diffuse light’ conditions: 
— information on the method(s) used for establishing sterile 
controls (e.g. temperature, time and number of autoclavings), 
— incubation temperature, 
— information on analytical techniques and the method(s) used for 
radiochemical measurements and for mass balance check and 
measurements of phase distribution (if conducted), 
— number of replicates. 
Results: — percentages of recovery (see section 1.7.1), 
— repeatability and sensitivity of the analytical methods used 
including the limit of detection (LOD) and the limit of quantifi ­
cation (LOQ) (see section 1.7.2),
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1203
 
— all measured data (including sampling time points) and calculated 
values in tabular form and the degradation curves; for each test 
concentration and for each replicate flask, report the linear correlation coefficient for the slope of the logarithmic plot, the estimated lag phase and a first-order or pseudo-first order rate 
constant (if possible), and the corresponding degradation half-life 
(or the half-life period, t
 50 ), 
— report relevant values as the averages of the results observed in 
individual replicates, e.g. length of lag phase, degradation rate 
constant and degradation half-life (or t  50 ), 
— categorise the system as either non-adapted or adapted as judged 
from the appearance of the degradation curve and from the possible influence of the test concentration, 
— the results of the final mass balance check and results on phase 
distribution measurements (if any), 
— the fraction of
 14 C mineralised and, if specific analyses are used, 
the final level of primary degradation, 
— the identification, molar concentration and percentage of applied 
and major transformation products (see first paragraph in section 1.8.9.5), where appropriate, 
— a proposed pathway of transformation, where appropriate, 
— discussion of results.
 
4. LITERATURE  
1. OECD TG 309 (2004) Aerobic Mineralisation in surface water 
— Simulation Biodegradation Test. 
2. ISO/DIS 14592-1 (1999) Water quality — Evaluation of the 
aerobic biodegradability of organic compounds at low concen ­
trations — Part 1: Shake flask batch test with surface water or 
surface water/sediment suspensions. 
3. Testing Method C.23. Aerobic and anaerobic transformation in 
soil. 
4. Testing Method C.24. Aerobic and anaerobic transformation in 
aquatic sediments. 
5. OECD (1993). Guidelines for the Testing of Chemicals. OECD, 
Paris. 
6. ISO 8245 (1999). Water quality — Guidelines on the deter ­
mination of total organic carbon (TOC) and dissolved organic carbon (DOC). 
7. ISO 10634 (1995). Water quality — Guidance for the prep ­
aration and treatment of poorly water-soluble organic 
compounds for the subsequent evaluation of their biodegrad ­
ability in an aqueous medium. 
8. OECD (2000). Guidance Document on aquatic toxicity testing of 
difficult substances and mixtures. Environmental Health and 
Safety Publications. Series on Testing and Assessment. No 22.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1204
 
9. Simkins, S. and Alexander, M. (1984). Models for minerali ­
sation kinetics with the variables of substrate concentration 
and population density. Appl. Environ. Microbiol.47, 394-401. 
10. Ingerslev, F. and N. Nyholm. (2000). Shake-flask test for deter ­
mination of biodegradation rates of  14 C-labeled chemicals at low 
concentrations in surface water systems. Ecotoxicol. Environ. 
Saf. 45, 274-283. 
11. ISO/CD 14592-1 (1999). Ring test report: Water Quality — 
Evaluation of the aerobic biodegradability of organic compounds at low concentrations part 1 — report of 1998/1999 ring-test. Shake flask batch test with surface water 
or surface water/sediment suspensions.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1205
 
Appendix 1  
Phase distribution of  14 C 
In order to check the procedure, the routine measurements of residual total 
organic  14 C activity (TOA) should be supplemented by mass balance 
measurements involving a direct determination of the evolved  14 CO 2 after 
trapping in an absorber (see Appendix 3). In itself, a positive  14 CO 2 formation 
is a direct evidence of biodegradation as opposed to abiotic degradation or other loss mechanisms, such as volatilisation and sorption. Additional useful information characterising the biodegradability behaviour can be obtained from 
measurements of the distribution of TOA between the dissolved state (dissolved 
organic
 14 C activity, DOA) and the particulate state (particulate organic  14 C 
activity, POA) after separation of particulate by membrane filtration or centrifu ­
gation. POA consists of test substance sorbed onto the microbial biomass and 
onto other particles in addition to the test substance carbon that has been used for 
synthesis of new cellular material and thereby incorporated into the particulate biomass fraction. The formation of dissolved
 14 C organic material can be 
estimated as the DOA at the end of biodegradation (plateau on the degradation 
versus time curve). 
Estimate the phase distribution of residual  14 C in selected samples by filtering 
samples on a 0,22 μm or 0,45 μm membrane filter of a material that does not 
adsorb significant amounts of the test substance (polycarbonate filters may be suitable). If sorption of test substance onto the filter is too large to be ignored (to 
be checked prior to the experiment) high-speed centrifugation (2 000 g; 10 min.) 
can be used instead of filtration. 
Proceed with the filtrate or centrifugate as described in Appendix 3 for unfiltered 
samples. Dissolve membrane filters in a suitable scintillation fluid and count as 
usually, normally using only the external standard ratio method to correct for 
quenching, or use a sample oxidiser. If centrifugation has been used, re-suspend the pellet formed of the particulate fraction in 1-2 ml of distilled water and transfer to a scintillation vial. Wash subsequently twice with 1 ml distilled 
water and transfer the washing water to the vial. If necessary, the suspension 
can be embedded in a gel for liquid scintillation counting.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1206
 
Appendix 2  
Semi-continuous procedure  
Prolonged incubation for up to several months may be required in order to 
achieve a sufficient degradation of recalcitrant substances. The duration of the test should normally not exceed 60 days unless the characteristics of the original 
water sample are maintained by renewal of the test suspension. However, the test 
period may be extended to a maximum of 90 days without renewal of the test suspension, if the degradation of the test substance has started within the first 60 days. 
During incubation for long periods, the diversity of the microbial community 
may be reduced due to various loss mechanisms and due to possible depletion of 
the water sample of essential nutrients and primary carbon substrates. It is therefore recommended that a semi-continuous test is used to adequately determine the degradation rate of slowly degrading substances. The test should 
be initiated by use of the semi-continuous procedure if, based on previous 
experience, an incubation period of three months is expected to be necessary to achieve 20 % degradation of the substance. Alternatively, the normal batch test may be changed into a semi-continuous test, if no degradation of the test 
substance has been achieved during approximately 60 days of testing using the 
batch procedure. The semi-continuous procedure may be stopped and the test continued as a batch experiment, when a substantial degradation has been recorded (e.g. > 20 %). 
In the semi-continuous test, every two weeks, about one third of the volume of 
the test suspension is replaced by freshly collected water with the test substance added to the initial concentration. Sediment is likewise added to the replacement water to the initial concentration (between 0,01 and 1 g/l), if the optional 
suspended sediment test is performed. Carrying out the test with suspended 
sediment solids, it is important that a fully suspended system is maintained also during water renewal, and that the residence time is identical for solids and water, as otherwise the intended similarity to a homogenous aqueous 
system with no fixed phases can be lost. For these reasons, an initial concen ­
tration of suspended sediments in the lower range of the specified interval is preferred, when the semi-continuous procedure is used. 
The prescribed addition of test substance implies that the initial concentration of 
test substance is not exceeded by the partial renewal of the test suspension and, 
hence, the adaptation, which is frequently seen with high concentrations of a test 
substance, is avoided. As the procedure comprises both a re-inoculation and a compensation of depleted nutrients and primary substrates, the original microbial diversity is restored, and the duration of the test can be extended to infinity in 
principle. When the semi-continuous procedure is used, it is important to note 
that the residual concentration of the test substance must be corrected for the amounts of test substance added and removed at each renewal procedure. The total and the dissolved test substance concentration can be used interchangeably 
for compounds that sorb little. Sorption is insignificant (< 5 %) under the 
specified conditions (0,1-1 g solids/l) for substances of log Kow < 3 (valid for neutral, lipophilic compounds). This is illustrated by the following calculation example. 0,1 g/l of solids roughly corresponds to 10 mg of carbon per litre 
(fraction of carbon, f
 C = 0,01). Assuming that: 
Log K  ow (of the test substance) = 3 
K oc = 0,42 × K  ow 
Partition coefficient, K  d = f C × K  oc 
then, the dissolved fraction of the total concentration (C-water (C  w )/C-total (C  t ) is: 
C w /C t = 1/(1 + K  d × SS) = 1(1 + K  oc × f C × SS) = 1/(1 + 0,42 × 10  3 × 0,01 × 
0,1 × 10  –3 ) = 0,999  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1207
 
Appendix 3  
Determination of  14 CO  2 
Indirect  14 CO  2 determination  
For routine measurements, the indirect method is normally the least time- 
consuming and most precise method if the test substance is non-volatile and is 
not transformed into volatile transformation products. Simply transfer unfiltered 
samples e.g. 5 ml size to scintillation vials. A suitable activity in samples is 5 000 dpm-10 000 dpm (80-170 Bq) initially, and a minimum initial activity is about 1 000 dpm. The CO
 2 should be stripped off after acidifying to pH 2-3 with 
1-2 drops of concentrated H  3 PO 4 or HCl. The CO  2 stripping can be performed 
by bubbling with air for about 1/2-1 hour. Alternatively, vials can be shaken vigorously for 1-2 hours (for instance on a microplate shaker) or with more gentle shaking be left overnight. The efficiency of the CO
 2 stripping procedure 
must be checked (by prolonging the aeration or shaking period). A scintillation 
liquid, suitable for counting aqueous samples should then be added, the sample homogenised on a whirling mixer and the radioactivity determined by liquid scintillation counting, subtracting the background activity found in the test 
blanks (F
 B ). Unless the test water is very coloured or contains a high concen ­
tration of particles, the samples will normally show uniform quenching and it will be sufficient to perform quench corrections using an external standard. If the test water is highly coloured, quench correction by means of internal standard 
addition may be necessary. If the concentration of particles is high it may not be 
possible to obtain a homogeneous solution or gel, or the quench variation between samples may be large. In that case the counting method described below for test slurries can be used. If the test is carried out as a suspended 
sediment test, the
 14 CO 2 measurement could be done indirectly by taking a 
homogeneous 10-ml sample of the test water/suspension and separating the phases by centrifugation at a suitable speed (e.g. at 40 000 m/s
 2 for 15 min.). 
The aqueous phase should then be then treated as described above. The  14 C 
activity in the particulate phase (POA) should be determined by re-suspending 
the sediment into a small volume of distilled water, transferring to scintillation vials, and adding scintillation liquid to form a gel (special scintillation liquids are available for that purpose). Depending on the nature of particles (e.g. their 
content of organic material), it may be feasible to digest the sample overnight 
with a tissue solubiliser and then homogenise on a whirling mixer prior to the addition of scintillation liquid. Alternatively, the POA can be determined by combustion in excess of oxygen by use of a sample oxidiser. When counting, 
internal standards should always be included, and it may be necessary to perform 
quench corrections using internal standard addition for each individual sample.
 
Direct  14 CO  2 determination  
If the evolved  14 CO 2 is measured directly, it should be done by setting up more 
flasks at the start of the test, harvesting the test flasks at each measuring point by 
acidifying the test flasks to pH 2-3 and collecting the  14 CO 2 in an internal 
(placed in each test flask at the start of the test) or external absorber. As absorbing medium either alkali (e.g. 1 N NaOH solution, or a NaOH pellet), 
ethanolamine or an ethanolamine-based, and commercially available absorbers 
can be used. For direct measurement of the
 14 CO 2 , the flasks should be closed 
with e.g. butyl rubber septa.  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1208
 
Figure 1a  
Example of arithmetic plot of data (residual activity versus time)
  
Figure 1b  
Example of semi-logarithmic plot of data (ln to residual activity versus time)
  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1209
 
C.26.  LEMNA  SP. GROWTH INHIBITION TEST  
1. METHOD  
This method is equivalent to OECD TG 221 (2006) (1). There has been 
broad agreement by EU authorities that the Lemna test is a suitable alternative to an algal test for strongly coloured substances (2)(3).
 
1.1. INTRODUCTION 
This Testing Method is designed to assess the toxicity of substances 
to freshwater aquatic plants of the genus  Lemna  (duckweed). It is 
based on existing guidelines (4)(5)(6)(7)(8)(9) but includes modifi ­
cations of those methods to reflect recent research and consultation 
on a number of key issues. The proposed method has been validated by an international ring-test (10). 
This Testing Method describes toxicity testing using  Lemna gibba  
and Lemna minor , both of which have been extensively studied and 
are the subject of the standards referred to above. The taxonomy of  
Lemna  spp. is difficult, being complicated by the existence of a wide 
range of phenotypes. Although genetic variability in the response to toxicants can occur with  Lemna , there are currently insufficient data 
on this source of variability to recommend a specific clone for use 
with this Testing Method. It should be noted that the test is not 
conducted axenically, but steps are taken at stages during the test procedure to keep contamination by other organisms to a minimum. 
Details on testing with renewal (semi-static and flow-through) and 
without renewal (static) of the test solution are described. Depending on the objectives of the test and on the regulatory requirements, it is recommended to consider the application of semi-static and flow 
through methods, e.g. for substances that are rapidly lost from 
solution as a result of volatilisation, photodegradation, precipitation or biodegradation. Further guidance is given in (11).
 
1.2. DEFINITIONS 
The following definitions and abbreviations are used for the purposes 
of this Testing Method:  
Biomass : is the dry weight of living matter present in a population. 
In this test, surrogates for biomass, such as frond counts or frond area, are typically measured and the use of the term ‘biomass’ thus refers to these surrogate measures as well.  
Chlorosis : is the yellowing of frond tissue.  
Clone : is an organism or cell arisen from a single individual by 
asexual reproduction. Individuals from the same clone are, therefore, genetically identical.  
Colony : means an aggregate of mother and daughter fronds (usually 
2 to 4) attached to each other. Sometimes referred to as a plant.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1210
 
EC x : is the concentration of the test substance dissolved in test 
medium that results in an x % (e.g. 50 %) reduction in growth of  
Lemna  within a stated exposure period (to be mentioned explicitly if 
deviating from full or normal test duration). To unambiguously denote an EC value deriving from growth rate or yield, the 
symbol ‘E
 r C’ is used for growth rate and ‘E  y C’ is used for yield, 
followed by the measurement variable used, e.g. E  r C (frond number).  
Flow-through : is a test in which the test solutions are replaced 
continuously.  
Frond : is an individual/single ‘leaf-like’ structure of a duckweed 
plant. It is the smallest unit, i.e. individual, capable of reproduction.  
Gibbosity : means fronds exhibiting a humped or swollen 
appearance.  
Growth : is an increase in the measurement variable, e.g. frond 
number, dry weight, wet weight or frond area, over the test period.  
Growth rate  (average specific growth rate ): is the logarithmic 
increase in biomass during the exposure period.  
Lowest Observed Effect Concentration (LOEC) : is the lowest 
tested concentration at which the substance is observed to have a 
statistically significant reducing effect on growth (at p < 0,05) when 
compared with the control, within a given exposure time. However, all test concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC. When these 
two conditions cannot be satisfied, a full explanation must be given 
for how the LOEC (and hence the NOEC) has been selected.  
Measurement variables : are any type of variables which are 
measured to express the test endpoint using one ore more different 
response variables. In this method frond number, frond area, fresh 
weight and dry weight are measurement variables.  
Monoculture : is a culture with one plant species.  
Necrosis : is dead (i.e. white or water-soaked) frond tissue.  
No Observed Effect Concentration (NOEC) : is the test concen ­
tration immediately below the LOEC.  
Phenotype : is the observable characteristics of an organism 
determined by the interaction of its genes with its environment.  
Response variables : are variables for the estimation of toxicity 
derived from any measured variables describing biomass by 
different methods of calculation. For this method, growth rates and 
yield are response variables derived from measurement variables like frond number, frond area, fresh weight or dry weight.  
Semi-static (renewal) test : is a test in which the test solution is 
periodically replaced at specific intervals during the test.  
Static test : is a test method without renewal of the test solution 
during the test.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1211
 
Test endpoint : describes the general factor that will be changed by 
the test chemical relative to the control as aim of the test. In this 
method the test endpoint is inhibition of growth, which may be expressed by different response variables which are based on one or more measurement variables.  
Test medium : is the complete synthetic growth medium on which 
test plants grow when exposed to the test substance. The test 
substance will normally be dissolved in the test medium.  
Yield : is the value of a measurement variable to express biomass at 
the end of the exposure period minus the measurement variable at 
the start of the exposure period.
 
1.3. PRINCIPLE OF THE TEST 
Exponentially growing plant cultures of the genus  Lemna  are 
allowed to grow as monocultures in different concentrations of the 
test substance over a period of seven days. The objective of the test is to quantify substance-related effects on vegetative growth over this period, based on assessments of selected measurement variables. 
Frond number is the primary measurement variable. At least one 
other measurement variable (total frond area, dry weight or fresh weight) is also measured, since some substances may affect other measurement variables much more than frond numbers. To quantify 
substance-related effects, growth in the test solutions is compared 
with that of the controls and the concentration bringing about a specified x % inhibition of growth (e.g. 50 %) is determined and expressed as the EC
 x (e.g. EC  50 ). 
The test endpoint is inhibition of growth, expressed as logarithmic 
increase in the measurement variable (average specific growth rate) 
during the exposure period. From the average specific growth rates 
recorded in a series of test solutions, the concentration bringing about a specified x % inhibition of growth rate (e.g. 50 %) is determined and expressed as the E
 r C x (e.g. E  r C 50 ). 
An additional response variable used in this Testing Method is yield, 
which may be needed to fulfil specific regulatory requirements in 
some countries. It is defined as the measurement variables at the end 
of the exposure period minus the measurement variables at the start of the exposure period. From the yield recorded in a series of test solutions, the concentration bringing about a specified x % inhibition 
of yield (e.g. 50 %) is calculated and expressed as the E
 y C x 
(e.g. E  y C 50 ). 
In addition, the lowest observed effect concentration (LOEC) and the 
no observed effect concentration (NOEC) may be statistically deter ­
mined.  
1.4. INFORMATION ON THE TEST SUBSTANCE 
An analytical method, with adequate sensitivity for quantification of 
the substance in the test medium, should be available.  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1212
 
Information on the test substance which may be useful in estab ­
lishing the test conditions includes the structural formula, purity, 
water solubility, stability in water and light, pK  a , K  ow , vapour 
pressure and biodegradability. Water solubility and vapour pressure can be used to calculate Henry’s Law constant, which will indicate if 
significant losses of the test substance during the test period are 
likely. This will help indicate whether particular steps to control such losses should be taken. Where information on the solubility and stability of the test substance is uncertain, it is recommended 
that these be assessed under the conditions of the test, i.e. growth 
medium, temperature, lighting regime to be used in the test. 
When pH control of the test medium is particularly important, e.g. 
when testing metals or substances which are hydrolytically unstable, 
the addition of a buffer to the growth medium is recommended (see first paragraph in section 1.7.4). Further guidance for testing substances with physicochemical properties that make them 
difficult to test is provided in (11).
 
1.5. REFERENCE SUBSTANCE 
Reference substance(s), such as 3,5-dichlorophenol used in the inter ­
national ring test (10), may be tested as a means of checking the test 
procedure. It is advisable to test a reference substance at least twice a year or, where testing is carried out at a lower frequency, in parallel to the determination of the toxicity of a test substance.
 
1.6. VALIDITY OF THE TEST 
For the test to be valid, the doubling time of frond number in the 
control must be less than 2,5 days (60 h), corresponding to approxi ­
mately a seven-fold increase in seven days and an average specific 
growth rate of 0,275 d  –1 . Using the media and test conditions 
described in this Testing Method, this criterion can be attained using a static test regime (8). It is also anticipated that this criterion will be achievable under semi-static and flow-through test 
conditions. Calculation of the doubling time is shown in section 2.1.
 
1.7. DESCRIPTION OF THE METHOD  
1.7.1.  Apparatus  
All equipment in contact with the test media should be made of glass 
or other chemically inert material. Glassware used for culturing and 
testing purposes should be cleaned of chemical contaminants that might leach into the test medium and should be sterile. The test vessels should be wide enough for the fronds of different colonies 
in the control vessels to grow without overlapping at the end of the 
test. It does not matter if the roots touch the bottoms of the test vessels, but a minimum depth of 20 mm and minimum volume of 100 ml in each test vessel is advised. The choice of test vessels is 
not critical as long as these requirements are met. Glass beakers, 
crystallising dishes or glass petri dishes of appropriate dimensions have all proved suitable. Test vessels must be covered to minimise evaporation and accidental contamination, while allowing necessary 
air exchange. Suitable test vessels, and particularly covers, must 
avoid shadowing or changes in the spectral characteristics of light.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1213
 
The cultures and test vessels should not be kept together. This is best 
achieved using separate environmental growth chambers, incubators, 
or rooms. Illumination and temperature must be controllable and maintained at a constant level (see section 1.7.8).
 
1.7.2.  Test organism  
The organism used for this test is either  Lemna gibba  or Lemna 
minor . Short descriptions of duckweed species that have been used 
for toxicity testing are given in Appendix 1. Plant material may be 
obtained from a culture collection, another laboratory or from the field. If collected from the field, plants should be maintained in culture in the same medium as used for testing for a minimum of 
eight weeks prior to use. Field sites used for collecting starting 
cultures must be free of obvious sources of contamination. If obtained from another laboratory or from a culture collection they should be similarly maintained for a minimum of three weeks. The 
source of plant material and the species and clone (if known) used 
for testing should always be reported. 
Monocultures, that are visibly free from contamination by other 
organisms such as algae and protozoa, should be used. Healthy plants of  L. minor  will consist of colonies comprising between two 
and five fronds, whilst healthy colonies of  L. gibba  may contain up 
to seven fronds. 
The quality and uniformity of the plants used for the test will have a 
significant influence on the outcome of the test and should therefore 
be selected with care. Young, rapidly growing plants without visible 
lesions or discoloration (chlorosis) should be used. Good quality cultures are indicated by a high incidence of colonies comprising at least two fronds. A large number of single fronds is indicative of 
environmental stress, e.g. nutrient limitation, and plant material from 
such cultures should not be used for testing.
 
1.7.3.  Cultivation  
To reduce the frequency of culture maintenance (e.g. when no  
Lemna  tests are planned for a period), cultures can be held under 
reduced illumination and temperature (4-10 °C). Details of culturing 
are given in Appendix 2. Obvious signs of contamination by algae or 
other organisms will require surface sterilisation of a sub-sample of  
Lemna  fronds, followed by transfer to fresh medium (see 
Appendix 2). In this eventuality, the remaining contaminated 
culture should be discarded. 
At least seven days before testing, sufficient colonies are transferred 
aseptically into fresh sterile medium and cultured for 7-10 days under the conditions of the test.
 
1.7.4.  Test medium  
Different media are recommended for  Lemna minor  and Lemna gibba , as 
described below. Careful consideration should be given to the inclusion 
of a pH buffer in the test medium (MOPS (4-morpholinepropane 
sulphonic acid, CAS No: 1132-61-2; EINECS No: 214-478-5) in  
L. minor  medium and NaHCO  3 in L. gibba  medium) when it is 
suspected that it might react with the test substance and influence the 
expression of its toxicity. Steinberg Medium (12) is also acceptable as 
long as the validity criteria are met.  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1214
 
A modification of the Swedish standard (SIS)  Lemna  growth 
medium is recommended for culturing and testing with  L. minor . 
The composition of this medium is given in Appendix 3. 
The growth medium, 20X — AAP, as described in Appendix 3, is 
recommended for culturing and testing with  L. gibba . 
Steinberg medium, as described in Appendix 3, is also suitable for  L. 
minor , but may also be used for  L. gibba  as long as the validity 
criteria are met.  
1.7.5.  Test solutions  
Test solutions are usually prepared by dilution of a stock solution. 
Stock solutions of the test substance are normally prepared by 
dissolving the substance in growth medium. 
The highest tested concentration of the test substance should not 
normally exceed the water solubility of the substance under the 
test conditions. It should be noted however that  Lemna  spp. float 
on the surface and may be exposed to substances that collect at the water-air interface (e.g. poorly water-soluble or hydrophobic 
substances or surface-active substances). Under such circumstances, 
exposure will result from material other than in solution and test concentrations may, depending on the characteristics of the test substance, exceed water solubility. For test substances of low 
water solubility it may be necessary to prepare a concentrated 
stock solution or dispersion of the substance using an organic solvent or dispersant in order to facilitate the addition of accurate 
quantities of the test substance to the test medium and aid in its 
dispersion and dissolution. Every effort should be made to avoid the use of such materials. There should be no phytotoxicity resulting from the use of auxiliary solvents or dispersants. For example, 
commonly used solvents which do not cause phytotoxicity at 
concentrations up to 100 μl·l
 –1 include acetone and dimethylform ­
amide. If a solvent or dispersant is used, its final concentration should be reported and kept to a minimum ( ≤ 100 μl·l
 –1 ), and all 
treatments and controls should contain the same concentration of 
solvent or dispersant. Further guidance on the use of dispersants is given in (11).
 
1.7.6.  Test and control groups  
Prior knowledge of the toxicity of the test substance to  Lemna , e.g. 
from a range-finding test, will help in selecting suitable test concen ­
trations. In the definitive toxicity test, there should normally be at 
least five test concentrations arranged in a geometric series. 
Preferably the separation factor between test concentrations should not exceed 3,2, but a larger value may be used where the concen ­
tration-response curve is flat. Justification should be provided if 
fewer than five concentrations are used. At least three replicates 
should be used at each test concentration. 
In setting the range of test concentrations (for range-finding and/or 
for the definitive toxicity test), the following should be considered:
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1215
 
— To determine an EC  x , test concentrations should bracket the EC  x 
value to ensure an appropriate level of confidence. For example, 
if estimating the EC  50 , the highest test concentration should be 
greater than the EC  50 value. If the EC  50 value lies outside the 
range of test concentrations, associated confidence intervals will 
be large and a proper assessment of the statistical fit of the model 
may not be possible. 
— If the aim is to estimate the LOEC/NOEC, the lowest test 
concentration should be low enough so that growth is not signifi ­
cantly less than that of the control. In addition, the highest test 
concentration should be high enough so that growth is signifi ­
cantly lower than that in the control. If this is not the case, the test will have to be repeated using a different concentration range 
(unless the highest concentration is at the limit of solubility or 
the maximum required limit concentration, e.g. 100 mg·l
 –1 ). 
Every test should include controls consisting of the same nutrient 
medium, number of fronds and colonies, environmental conditions 
and procedures as the test vessels but without the test substance. If 
an auxiliary solvent or dispersant is used, an additional control treatment with the solvent/dispersant present at the same concen ­
tration as that in the vessels with the test substance should be 
included. The number of replicate control vessels (and solvent 
vessels, if applicable) should be at least equal to, ideally twice, the number of vessels used for each test concentration. 
If determination of NOEC is not required, the test design may be 
altered to increase the number of concentrations and reduce the 
number of replicates per concentration. However, the number of control replicates must be at least three.
 
1.7.7.  Exposure  
Colonies consisting of 2 to 4 visible fronds are transferred from the 
inoculum culture and randomly assigned to the test vessels under 
aseptic conditions. Each test vessel should contain a total of 9 to 12 fronds. The number of fronds and colonies should be the same in each test vessel. Experience gained with this method and ring-test 
data have indicated that using three replicates per treatment, with 
each replicate containing 9 to 12 fronds initially, is sufficient to detect differences in growth of approximately 4 to 7 % of inhibition calculated by growth rate (10 to 15 % calculated by yield) between 
treatments (10). 
A randomised design for location of the test vessels in the incubator 
is required to minimise the influence of spatial differences in light intensity or temperature. A blocked design or random repositioning 
of the vessels when observations are made (or repositioning more 
frequently) is also required.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1216
 
If a preliminary stability test shows that the test substance concen ­
tration cannot be maintained (i.e. the measured concentration falls 
below 80 % of the measured initial concentration) over the test duration (7 days), a semi-static test regime is recommended. In this case, the colonies should be exposed to freshly prepared test 
and control solutions on at least two occasions during the test (e.g. 
days 3 and 5). The frequency of exposure to fresh medium will depend on the stability of the test substance; a higher frequency may be needed to maintain near-constant concentrations of highly 
unstable or volatile substances. In some circumstances, a flow- 
through procedure may be required (11)(13). 
The exposure scenario through a foliar application (spray) is not 
covered in this Testing Method, instead see (14).
 
1.7.8.  Incubation conditions  
Continuous warm or cool white fluorescent lighting should be used 
to provide a light intensity selected from the range of 85-135 
μE·m  –2 ·s –1 when measured in a photosynthetically active radiation 
(400-700 nm) at points the same distance from the light source as the  
Lemna  fronds (equivalent to 6 500-10 000 lux). Any differences 
from the selected light intensity over the test area should not 
exceed ± 15 %. The method of light detection and measurement, 
in particular the type of sensor, will affect the measured value. Spherical sensors (which respond to light from all angles above and below the plane of measurement) and ‘cosine’ sensors (which 
respond to light from all angles above the plane of measurement) are 
preferred to unidirectional sensors, and will give higher readings for a multi-point light source of the type described here. 
The temperature in the test vessels should be 24 ± 2 °C. The pH of 
the control medium should not increase by more than 1,5 units 
during the test. However, deviation of more than 1,5 units would not invalidate the test when it can be shown that validity criteria are met. Additional care is needed on pH drift in special cases such as 
when testing unstable substances or metals. See (11) for further 
guidance.
 
1.7.9.  Duration  
The test is terminated 7 days after the plants are transferred into the 
test vessels.  
1.7.10.  Measurements and analytical determinations  
At the start of the test, the frond number in the test vessels is 
counted and recorded, taking care to ensure that protruding, 
distinctly visible fronds are accounted for. Frond numbers 
appearing normal or abnormal, need to be determined at the beginning of the test, at least once every 3 days during the exposure period (i.e. on at least 2 occasions during the 7 day 
period), and at test termination. Changes in plant development, e.g. 
in frond size, appearance, indication of necrosis, chlorosis or gibbosity, colony break-up or loss of buoyancy, and in root length and appearance, should be noted. Significant features of the test 
medium (e.g. presence of undissolved material, growth of algae in 
the test vessel) should also be noted.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1217
 
In addition to determinations of frond number during the test, effects 
of the test substance on one (or more) of the following measurement 
variables are also assessed: 
(i) total frond area; 
(ii) dry weight; 
(iii) fresh weight. 
Total frond area has an advantage that it can be determined for each 
test and control vessel at the start, during, and at the end of the test. Dry or fresh weight should be determined at the start of the test from 
a sample of the inoculum culture representative of what is used to 
begin the test, and at the end of the test with the plant material from each test and control vessel. If frond area is not measured, dry weight is preferred over fresh weight. 
Total frond area, dry weight and fresh weight may be determined as 
follows: 
(i) Total frond area : The total frond area of all colonies may be 
determined by image analysis. A silhouette of the test vessel 
and plants can be captured using a video camera (i.e. by placing the vessel on a light box) and the resulting image digitised. By calibration with flat shapes of known area, the total frond area 
in a test vessel may then be determined. Care should be taken to 
exclude interference caused by the rim of the test vessel. An alternative but more laborious approach is to photocopy test vessels and plants, cut out the resulting silhouette of colonies 
and determine their area using a leaf area analyser or graph 
paper. Other techniques (e.g. paper weight ratio between silhouette area of colonies and unit area) may also be appro ­
priate. 
(ii) Dry weight : All colonies are collected from each of the test 
vessels and rinsed with distilled or deionised water. They are blotted to remove excess water and then dried at 60 °C to a constant weight. Any root fragments should be included. The 
dry weight should be expressed to an accuracy of at least 
0,1 mg. 
(iii) Fresh weight : All colonies are transferred to pre-weighed poly ­
styrene (or other inert material) tubes with small (1 mm) holes 
in the rounded bottoms. The tubes are then centrifuged at 3 000 rpm for 10 minutes at room temperature. Tubes, containing the now dried colonies, are re-weighed and the 
fresh weight is calculated by subtracting the weight of the 
empty tube.
 
1.7.10.1.  Frequency of measurements and analytical determinations  
If a static test design is used, the pH of each treatment should be 
measured at the beginning and end of the test. If a semi-static test design is used, the pH should be measured in each batch of ‘fresh’ 
test solution prior to each renewal and also in the corresponding 
‘spent’ solutions.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1218
 
Light intensity should be measured in the growth chamber, incubator 
or room at points the same distance from the light source as the  
Lemna  fronds. Measurements should be made at least once during 
the test. The temperature of the medium in a surrogate vessel held under the same conditions in the growth chamber, incubator or room 
should be recorded at least daily. 
During the test, the concentrations of the test substance are 
determined at appropriate intervals. In static tests, the minimum requirement is to determine the concentrations at the beginning 
and the end of the test. 
In semi-static tests where the concentration of the test substance is 
not expected to remain within ± 20 % of the nominal concentration, it is necessary to analyse all freshly prepared test solutions and the 
same solutions at each renewal (see third paragraph in section 1.7.7). 
However, for those tests where the measured initial concentration of the test substance is not within ± 20 % of nominal, but where sufficient evidence can be provided to show that the initial concen ­
trations are repeatable and stable (i.e. within the range 80-120 % of 
the initial concentration), chemical determinations may be carried out on only the highest and lowest test concentrations. In all cases, 
determination of test substance concentrations prior to renewal 
need only be performed on one replicate vessel at each test concen ­
tration (or the contents of the vessels pooled by replicate). 
If a flow-through test is used, a similar sampling regime to that 
described for semi-static tests, including analysis at the start, mid- 
way through and at the end of the test, is appropriate, but measurement of ‘spent’ solutions is not appropriate in this case. In this type of test, the flow-rate of diluent and test substance or test 
substance stock solution should be checked daily. 
If there is evidence that the concentration of the substance being 
tested has been satisfactorily maintained within ± 20 % of the nominal or measured initial concentration throughout the test, 
analysis of the results can be based on nominal or measured initial 
values. If the deviation from the nominal or measured initial concen ­
tration is greater than ± 20 %, analysis of the results should be based on the geometric mean concentration during exposure or models 
describing the decline of the concentration of the test substance (11).
 
1.7.11.  Limit test  
Under some circumstances, e.g. when a preliminary test indicates 
that the test substance has no toxic effects at concentrations up to 100 mg·l
 –1 , or up to its limit of solubility in the test medium 
(whichever is the lower), a limit test involving a comparison of 
responses in a control group and one treatment group (100 mg·l  –1 
or a concentration equal to the limit of solubility), may be under ­
taken. It is strongly recommended that this be supported by analysis 
of the exposure concentration. All previously described test 
conditions and validity criteria apply to a limit test, with the exception that the number of treatment replicates should be doubled. Growth in the control and treatment group may be 
analysed using a statistical test to compare means, e.g. a Student's 
t-test.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1219
 
2. DATA AND REPORTING  
2.1. DOUBLING TIME 
To determine the doubling time (T  d ) of frond number and adherence 
to this validity criterion by the study (section 1.6), the following 
formula is used with data obtained from the control vessels: 
T d = ln 2/μ 
where μ is the average specific growth rate determined as described 
in first and second paragraph in section 2.2.1.  
2.2. RESPONSE VARIABLES 
The purpose of the test is to determine the effects of the test 
substance on the vegetative growth of  Lemna . This Testing 
Method describes two response variables, as member countries have different preferences and regulatory needs. In order for the 
test results to be acceptable in all member countries, the effects 
should be evaluated using both response variables (a) and (b) described below. 
(a) Average specific growth rate: this response variable is calculated 
on the basis of changes in the logarithms of frond numbers, and in addition, on the basis of changes in the logarithms of another 
measurement parameter (total frond area, dry weight or fresh 
weight) over time (expressed per day) in the controls and each treatment group. It is sometimes referred to as relative growth rate (15). 
(b) Yield: this response variable is calculated on the basis of 
changes in frond number, and in addition, on the basis of 
changes in another measurement parameter (total frond area, 
dry weight or fresh weight) in the controls and in each treatment group until the end of the test. 
It should be noted that toxicity values calculated by using these two 
response variables are not comparable and this difference must be recognised when using the results of the test. EC
 x values based upon 
average specific growth rate (E  r C x ) will generally be higher than 
results based upon yield (E  y C x ) if the test conditions of this 
Testing Method are adhered to, due to the mathematical basis of the respective approaches. This should not be interpreted as a 
difference in sensitivity between the two response variables, simply 
that the values are different mathematically. The concept of average specific growth rate is based on the general exponential growth pattern of duckweed in non-limited cultures, where toxicity is 
estimated on the basis of the effects on the growth rate, without 
being dependent on the absolute level of the specific growth rate of the control, slope of the concentration-response curve or on test duration. In contrast, results based upon the yield response variable 
are dependent upon all these other variables. E
 y C x is dependent on 
the specific growth rate of the duckweed species used in each test and on the maximum specific growth rate that can vary between species and even different clones. This response variable should 
not be used for comparing the sensitivity to toxicants among 
duckweed species or even different clones. While the use of average specific growth rate for estimating toxicity is scientifically preferred, toxicity estimates based on yield are also included in this 
Testing Method to satisfy current regulatory requirements in some 
countries.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1220
 
Toxicity estimates should be based on frond number and on one 
additional measurement variable (total frond area, dry weight or 
fresh weight), because some substances may affect other measurement variables much more than the frond number. This effect would not be detected by calculating frond number only. 
The number of fronds as well as any other recorded measurement 
variable, i.e. total frond area, dry weight or fresh weight, are tabulated together with the concentrations of the test substance for 
each measurement occasion. Subsequent data analysis e.g. to 
estimate a LOEC, NOEC or EC
 x should be based on the values 
for the individual replicates and not calculated means for each treatment group.
 
2.2.1.  Average specific growth rate  
The average specific growth rate for a specific period is calculated as 
the logarithmic increase in the growth variables — rond numbers and one other measurement variable (total frond area, dry weight or fresh weight) — using the formula below for each replicate of control and 
treatments:
 
μ iÄj ¼ ln ðN j Þ – ln ðN i Þ 
t 
where: 
— μ i-j : average specific growth rate from time i to j 
— N  i : measurement variable in the test or control vessel at time i 
— N  j : measurement variable in the test or control vessel at time j 
— t: time period from i to j 
For each treatment group and control group, calculate a mean value 
for the growth rate along with variance estimates. 
The average specific growth rate should be calculated for the entire 
test period (time ‘i’ in the above formula is the beginning of the test and time ‘j’ is the end of the test). For each test concentration and control, calculate a mean value for average specific growth rate 
along with the variance estimates. In addition, the section-by- 
section growth rate should be assessed in order to evaluate effects of the test substance occurring during the exposure period (e.g. by inspecting log-transformed growth curves). Substantial differences 
between the section-by-section growth rate and the average growth 
rate indicate deviation from constant exponential growth and that close examination of the growth curves is warranted. In this case, a conservative approach would be to compare specific growth rates 
from treated cultures during the time period of maximum inhibition 
to those for controls during the same time period.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1221
 
Percentage inhibition of growth rate (I  r ) may then be calculated for 
each test concentration (treatment group) according to the following 
formula:  
% I r ¼ ðμ C – μ T Þ 
μ C Ü 100  
where: 
— % I  r : percentage inhibition in average specific growth rate 
— μ C : mean value for μ in the control 
— μ T : mean value for μ in the treatment group  
2.2.2.  Yield  
Effects on yield are determined on the basis of two measurement 
variables, frond number and one other measurement variable (total 
frond area, dry weight or fresh weight) present in each test vessel at the start and end of the test. For dry weight or fresh weight, the starting biomass is determined on the basis of a sample of fronds 
taken from the same batch used to inoculate the test vessels 
(see second paragraph in section 1.7.3). For each test concentration and control, calculate a mean value for yield along with variance estimates. The mean percentage inhibition in yield (% I
 y ) may be 
calculated for each treatment group as follows:  
% I y ¼ ðb C – b T Þ 
b C Ü 100  
where: 
— % I  y : percentage reduction in yield 
— b  C : final biomass minus starting biomass for the control 
group 
— b  T : final biomass minus starting biomass in the treatment 
group  
2.2.3.  Plotting concentration-response curves  
Concentration-response curves relating mean percentage inhibition of 
the response variable (I  r , or I  y calculated as shown in the last 
paragraph of section 2.2.1 or in section 2.2.2) and the log concen ­
tration of the test substance should be plotted.  
2.2.4.  EC  x estimation  
Estimates of the EC  x (e.g. EC  50 ) should be based upon both average 
specific growth rate (E  r C x ) and yield (E  y C x ), each of which should in 
turn be based upon frond number and one additional measurement variable (total frond area, dry weight, or fresh weight). This is 
because there are test substances that impact frond number and 
other measurement variables differently. The desired toxicity parameters are therefore four EC
 x values for each inhibition level 
x calculated: E  r C x (frond number); E  r C x (total frond area, dry weight, 
or fresh weight); E  y C x (frond number); and E  y C x (total frond area, 
dry weight, or fresh weight).  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1222
 
2.3. STATISTICAL PROCEDURES 
The aim is to obtain a quantitative concentration-response rela ­
tionship by regression analysis. It is possible to use a weighted linear regression after having performed a linearising transformation 
of the response data — for instance into probit or logit or Weibull 
units (16), but non-linear regression procedures are preferred tech ­
niques that better handle unavoidable data irregularities and devi ­
ations from smooth distributions. Approaching either zero or total 
inhibition, such irregularities may be magnified by the trans ­
formation, interfering with the analysis (16). It should be noted that standard methods of analysis using probit, logit, or Weibull 
transforms are intended for use on quantal (e.g. mortality 
or survival) data, and must be modified to accommodate growth rate or yield data. Specific procedures for determination of EC
 x 
values from continuous data can be found in (17), (18), and (19). 
For each response variable to be analysed, use the concentration- 
response relationship to calculate point estimates of EC  x values. 
When possible, the 95 % confidence limits for each estimate 
should be determined. Goodness of fit of the response data to the 
regression model should be assessed either graphically or statis ­
tically. Regression analysis should be performed using individual replicate responses, not treatment group means. 
EC
 50 estimates and confidence limits may also be obtained using 
linear interpolation with bootstrapping (20), if available regression models/methods are unsuitable for the data. 
For estimation of the LOEC and hence the NOEC, it is necessary to 
compare treatment means using analysis of variance (ANOVA) tech ­
niques. The mean for each concentration must then be compared 
with the control mean using an appropriate multiple comparison or 
trend test method. Dunnett’s or Williams’ test may be useful (21) ­
(22)(23)(24). It is necessary to assess whether the ANOVA assumption of homogeneity of variance holds. This assessment 
may be performed graphically or by a formal test (25). Suitable 
tests are Levene’s or Bartlett’s. Failure to meet the assumption of homogeneity of variances can sometimes be corrected by logarithmic transformation of the data. If heterogeneity of variance is extreme 
and cannot be corrected by transformation, analysis by methods such 
as step-down Jonkheere trend tests should be considered. Additional guidance on determining the NOEC can be found in (19). 
Recent scientific developments have led to a recommendation of 
abandoning the concept of NOEC and replacing it with regression based point estimates EC
 x . An appropriate value for x has not been 
established for this  Lemna  test. However, a range of 10 to 20 % 
appears to be appropriate (depending on the response variable chosen), and preferably both the EC
 10 and EC  20 should be reported.  
3. REPORTING  
3.1. TEST REPORT 
The test report must include the following: 
Test substance: 
— physical nature and physicochemical properties, including water 
solubility limit,  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1223
 
— chemical identification data (e.g. CAS Number), including purity. 
Test species: — scientific name, clone (if known) and source. Test conditions: — test procedure used (static, semi-static or flow-through), — date of start of the test and its duration, — test medium, — description of the experimental design: test vessels and covers, 
solution volumes, number of colonies and fronds per test vessel 
at the beginning of the test, 
— test concentrations (nominal and measured as appropriate) and 
number of replicates per concentration, 
— methods of preparation of stock and test solutions including the 
use of any solvents or dispersants, 
— temperature during the test, — light source, light intensity and homogeneity, — pH values of the test and control media, — test substance concentrations and the method of analysis with 
appropriate quality assessment data (validation studies, standard 
deviations or confidence limits of analyses), 
— methods for determination of frond number and other 
measurement variables, e.g. dry weight, fresh weight or frond 
area, 
— all deviations from this Testing Method. Results: — raw data: number of fronds and other measurement variables in 
each test and control vessel at each observation and occasion of analysis, 
— means and standard deviations for each measurement variable, — growth curves for each concentration (recommended with log 
transformed measurement variable, see second paragraph in 
section 2.2.1), 
— doubling time/growth rate in the control based on the frond 
number, 
— calculated response variables for each treatment replicate, with 
mean values and coefficient of variation for replicates, 
— graphical representation of the concentration/effect relationship, — estimates of toxic endpoints for response variables e.g. EC
 50 , 
EC 10 , EC  20 , and associated confidence intervals. If calculated, 
LOEC and/or NOEC and the statistical methods used for their 
determination,  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1224
 
— if ANOVA has been used, the size of the effect which can be 
detected (e.g. the least significant difference), 
— any stimulation of growth found in any treatment, 
— any visual signs of phytotoxicity as well as observations of test 
solutions, 
— discussion of the results, including any influence on the outcome 
of the test resulting from deviations from this Testing Method.  
4. LITERATURE  
(1) OECD TG 221 (2006) Lemna Sp. Growth Inhibition Test. 
(2) The use of Lemna studies for coloured substances is detailed in 
Section 13.5.3 of the EU Manual of Decisions dated July 2006, at http://ecb.jrc.ec.europa.eu/new-chemicals 
(3) Guidance on information requirements and chemical safety 
assessment — Chapter R.7b: Endpoint specific guidance; 
Table 7.8.3 Summary of difficult substance testing issues, 
available at: 
http://guidance.echa.europa.eu/docs/guidance_document/ ­
information_requirements_en.htm?time=1234958685#A 
(4) ASTM International. (2003). Standard Guide for Conducting 
Static Toxicity Test With  Lemna gibba  G3. E 1415-91 
(Reapproved 1998). pp. 733-742. In, Annual Book of ASTM Standards, Vol. 11.05 Biological Effects and Environmental Fate; Biotechnology; Pesticides, ASTM, West Conshohocken, PA. 
(5) USEPA — United States Environmental Protection Agency. 
(1996). OPPTS 850.4400 Aquatic Plant Toxicity Test Using Lemna spp., ‘Public draft’. EPA 712-C-96-156. 8pp. 
(6) AFNOR — Association Française de Normalisation. (1996). 
XP T 90-337: Détermination de l’inhibition de la croissance 
de Lemna minor . 10pp. 
(7) SSI — Swedish Standards Institute. (1995). Water quality — 
Determination of growth inhibition (7-d)  Lemna minor , 
duckweed. SS 02 82 13. 15pp. (in Swedish). 
(8) Environment Canada. (1999). Biological Test Method: Test for 
Measuring the Inhibition of Growth Using the Freshwater 
Macrophyte,  Lemna minor . EPS 1/RM/37-120 pp. 
(9) Environment Canada. (1993) Proposed Guidelines for Regis ­
tration of Chemical Pesticides: Non-Target Plant Testing and 
Evaluation. Canadian Wildlife Service, Technical Report Series No. 145. 
(10) Sims I., Whitehouse P., and Lacey R. (1999). The OECD  
Lemna  Growth Inhibition Test. Development and Ring-testing 
of draft OECD Test Guideline. R&D Technical Report EMA 003. WRc plc — Environment Agency. 
(11) OECD (2000). Guidance Document on Aquatic Toxicity 
Testing of Difficult Substances and Mixtures. OECD Environ ­
mental Health and Safety Publications, Series on Testing and 
Assessment No 23. 
(12) ISO DIS 20079. Water Quality — Determination of the Toxic 
Effect of Water Constituents and Waste Water to Duckweed 
(Lemna minor ) — Duckweed Growth Inhibition Test.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1225
 
(13) Walbridge C. T. (1977). A flow-through testing procedure with 
duckweed ( Lemna minor L .). Environmental Research 
Laboratory — Duluth, Minnesota 55804. US EPA Report No. EPA-600/3-77 108. September 1977. 
(14) Lockhart W. L., Billeck B. N. and Baron C. L. (1989). 
Bioassays with a floating plant ( Lemna minor ) for effects of 
sprayed and dissolved glyphosate. Hydrobiologia, 118/119, 
353-359. 
(15) Huebert, D.B. and Shay J.M. (1993). Considerations in the 
assessment of toxicity using duckweeds. Environmental Toxi ­
cology and Chemistry, 12, 481-483. 
(16) Christensen, E.R., Nyholm, N. (1984): Ecotoxicological Assays 
with Algae: Weibull Dose-Response Curves. Env. Sci. Technol. 19, 713-718. 
(17) Nyholm, N. Sørensen, P.S., Kusk, K.O. and Christensen, E.R. 
(1992): Statistical treatment of data from microbial toxicity 
tests. Environ. Toxicol. Chem. 11, 157-167. 
(18) Bruce R.D. and Versteeg D.J. (1992). A statistical procedure 
for modelling continuous toxicity data. Environmental Toxi ­
cology and Chemistry, 11, 1485-1494. 
(19) OECD. (2004). Guidance Document on Statistical Analysis of 
Ecotoxicity Data. 
(20) Norberg-King T.J. (1988). An interpolation estimate for chronic 
toxicity: The ICp approach. National Effluent Toxicity Assessment Center Technical Report 05-88. USEPA, Duluth, MN. 
(21) Dunnett, C.W. (1955). A multiple comparisons procedure for 
comparing several treatments with a control. J. Amer. Statist. Assoc., 50, 1096-1121. 
(22) Dunnett, C.W. (1964). New tables for multiple comparisons 
with a control. Biometrics, 20, 482-491. 
(23) Williams, D.A. (1971). A test for differences between treatment 
means when several dose levels are compared with a zero dose control. Biometrics, 27: 103-117. 
(24) Williams, D.A. (1972). The comparison of several dose levels 
with a zero dose control. Biometrics, 28: 510-531. 
(25) Brain P. and Cousens R. (1989). An equation to describe dose- 
responses where there is stimulation of growth at low doses. 
Weed Research, 29, 93-96.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1226
 
Appendix 1  
Description of  lemna  spp.  
The aquatic plant commonly referred to as duckweed,  Lemna  spp., belongs to the 
family  Lemnaceae  which has a number of worldwide species in four genera. 
Their different appearance and taxonomy have been exhaustively described 
(1)(2).  Lemna gibba  and  L. minor  are species representative of temperate areas 
and are commonly used for toxicity tests. Both species have a floating or submerged discoid stem (frond) and a very thin root emanates from the centre of the lower surface of each frond.  Lemna  spp. rarely produce flowers and the 
plants reproduce by vegetatively producing new fronds (3). In comparison with 
older plants, the younger ones tend to be paler, have shorter roots and consist of two to three fronds of different sizes. The small size of  Lemna , its simple 
structure, asexual reproduction and short generation time makes plants of this 
genus very suitable for laboratory testing (4)(5). 
Because of probable interspecies variation in sensitivity, only comparisons of 
sensitivity within a species are valid.
 
Examples of  Lemna  species which have been used for testing: Species 
Reference  
Lemna aequinoctialis : Eklund, B. (1996). The use of the red alga  Ceramium 
strictum  and the duckweed  Lemna aequinoctialis  in aquatic ecotoxicological 
bioassays. Licentiate in Philosophy Thesis 1996:2. Dep. of Systems Ecology, 
Stockholm University.  
Lemna major : Clark, N. A. (1925). The rate of reproduction of  Lemna major  as a 
function of intensity and duration of light. J. phys. Chem., 29: 935-941.  
Lemna minor : United States Environmental Protection Agency (USEPA). (1996). 
OPPTS 850.4400 Aquatic Plant Toxicity Test Using  Lemna  spp., ‘Public draft’. 
EPA 712-C-96-156. 8pp. 
Association Française de Normalisation (AFNOR). (1996). XP T 90-337: Déter ­
mination de l’inhibition de la croissance de  Lemna minor . 10pp. 
Swedish Standards Institute (SIS). (1995). Water quality — Determination of 
growth inhibition (7-d)  Lemna minor , duckweed. SS 02 82 13. 15pp. (in 
Swedish).  
Lemna gibba : ASTM International. (2003). Standard Guide for Conducting Static 
Toxicity Test With  Lemna gibba  G3. E 1415-91 (Reapproved 1998). pp. 733- 
742. 
United States Environmental Protection Agency (USEPA). (1996). OPPTS 
850.4400 Aquatic Plant Toxicity Test Using  Lemna  spp., ‘Public draft’. EPA 
712-C-96-156. 8pp.  
Lemna paucicostata : Nasu, Y., Kugimoto, M. (1981).  Lemna  (duckweed) as an 
indicator of water pollution. I. The sensitivity of  Lemna paucicostata  to heavy 
metals. Arch. Environ. Contam. Toxicol., 10:1959-1969.  
Lemna perpusilla : Clark, J. R. et al. (1981). Accumulation and depuration of 
metals by duckweed ( Lemna perpusilla ). Ecotoxicol. Environ. Saf., 5:87-96.  
Lemna trisulca : Huebert, D. B., Shay, J. M. (1993). Considerations in 
the assessment of toxicity using duckweeds. Environ. Toxicol. and Chem., 
12:481-483.  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1227
 
Lemna valdiviana : Hutchinson, T.C., Czyrska, H. (1975). Heavy metal toxicity 
and synergism to floating aquatic weeds. Verh.-Int. Ver. Limnol., 19:2102-2111.  
Sources of Lemna species  
University of Toronto Culture Collection of Algae and Cyanobacteria 
Department of Botany, University of Toronto 
Toronto, Ontario, Canada, M5S 3 B2 
Tel. +1-416-978-3641 
Fax +1-416-978-5878 
e-mail: jacreman@botany.utoronto.ca http://www.botany.utoronto.ca/utcc 
North Carolina State University 
Forestry Dept Duckweed Culture Collection Campus Box 8002 
Raleigh, NC 27695-8002 
UNITED STATES Tel. 001 (919) 515-7572 astomp@unity.ncsu.edu 
Institute of Applied Environmental Research (ITM) Stockholm University 
SE-106 91 Stockholm SWEDEN Tel. +46 86747240 
Fax +46 86747636 
Federal Environmental Agency (UBA) 
FG III 3.4 Schichauweg 58 
12307 Berlin 
GERMANY e-mail: lemna@uba.de http://www.umweltbundesamt.de/contact.htm
 
Literature  
(1) Hillman, W.S. (1961). The  Lemnaceae  or duckweeds: A review of the 
descriptive and experimental literature. The Botanical Review, 27:221-287. 
(2) Landolt, E. (1986). Biosystematic investigations in the family of duckweed 
(Lemnaceae ). Vol. 2. Geobotanischen Inst. ETH, Stiftung Rubel, Zürich, 
Switzerland. 
(3) Björndahl, G. (1982). Growth performance, nutrient uptake and human util ­
ization of duckweeds ( Lemnaceae  family). ISBN 82-991150-0-0. The Agri ­
cultural Research Council of Norway, University of Oslo. 
(4) Wang, W. (1986). Toxicity tests of aquatic pollutants by using common 
duckweed. Environmental Pollution, Ser B, 11:1-14. 
(5) Wang, W. (1990). Literature review on duckweed toxicity testing. Environ ­
mental Research, 52:7-22.  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1228
 
Appendix 2  
Maintenance of stock culture  
Stock cultures can be maintained under lower temperatures (4-10 °C) for longer 
times without needing to be re-established. The  Lemna  growth medium may be 
the same as that used for testing but other nutrient rich media can be used for 
stock cultures. 
Periodically, a number of young, light-green plants are removed to new culture 
vessels containing fresh medium using an aseptic technique. Under the cooler conditions suggested here, sub-culturing may be conducted at intervals of up to three months. 
Chemically clean (acid-washed) and sterile glass culture vessels should be used 
and aseptic handling techniques employed. In the event of contamination of the 
stock culture e.g. by algae or fungi, steps are necessary to eliminate the contami ­
nating organisms. In the case of algae and most other contaminating organisms, this can be achieved by surface sterilisation. A sample of the contaminated plant 
material is taken and the roots cut off. The material is then shaken vigorously in 
clean water, followed by immersion in a 0,5 % (v/v) sodium hypochlorite solution for between 30 seconds and 5 minutes. The plant material is then 
rinsed with sterile water and transferred, as a number of batches, into culture 
vessels containing fresh growth medium. Many fronds will die as a result of this treatment, especially if longer exposure periods are used, but some of those surviving will usually be free of contamination. These can then be used to re- 
inoculate new cultures.
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1229
 
Appendix 3  
Media  
Different growth media are recommended for  L. minor  and  L. gibba . For  L. 
minor , a modified Swedish Standard (SIS) medium is recommended whilst for  
L. gibba , 20X AAP medium is recommended. Compositions of both media are 
given below. When preparing these media, reagent or analytical-grade chemicals 
and deionised water should be used.  
Swedish Standard (SIS)  Lemna  growth medium  
— Stock solutions I-V are sterilised by autoclaving (120 °C, 15 minutes) or by 
membrane filtration (approximately 0,2 μm pore size). 
— Stock VI (and optionally VII) are sterilised by membrane filtration only; these 
should not be autoclaved. 
— Sterile stock solutions should be stored under cool and dark conditions. 
Stocks I-V should be discarded after six months whilst stocks VI (and 
optionally VII) have a shelf life of one month.  
Stock solution 
No. Substance Concentration in 
stock solution 
(g· l  –1 ) Concentration in 
prepared medium 
(mg·l  –1 ) Prepared medium 
Element Concentration 
(mg·l  –1 ) 
I NaNO  3 
KH  2 PO4 8,50 
1,34 85 
13,4 Na; N 
K; P 32; 14 
6,0; 2,4 
II MgSO  4 · 7H  2 O 15 75 Mg; S 7,4; 9,8 
III CaCl  2 · 2H  2 O 7,2 36 Ca; Cl 9,8; 17,5 
IV Na 2 CO 3 4,0 20 C 2,3 
V H 3 BO 3 
MnCl  2 · 4H  2 O 
Na 2 MoO  4 · 2H  2 O 
ZnSO  4 · 7H  2 O 
CuSO  4 · 5H  2 O 
Co(NO  3 ) 2 · 6H  2 O 1,0 
0,20 
0,010 
0,050 0,0050 0,010 1,00 
0,20 
0,010 
0,050 0,0050 0,010 B 
Mn 
Mo 
Zn Cu Co 0,17 
0,056 
0,0040 
0,011 0,0013 0,0020 
VI FeCl
 3 · 6H  2 O 
Na 2 -EDTA·2H  2 O 0,17 
0,28 0,84 
1,4 Fe 
— 0,17 
— 
VII MOPS (buffer) 490 490 — — 
— To prepare one litre of SIS medium, the following are added to 900 ml of 
deionised water: 
— 10 ml of stock solution I — 5 ml of stock solution II — 5 ml of stock solution III — 5 ml of stock solution IV
 ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1230
 
— 1 ml of stock solution V 
— 5 ml of stock solution VI — 1 ml of stock solution VII (optional)  
Note:  A further stock solution VII (MOPS buffer) may be needed for certain 
test substances (see last paragraph in section 1.4). 
— The pH is adjusted to 6,5 ± 0,2 with either 0,1 or 1 mol HCl or NaOH, and 
the volume adjusted to one litre with deionised water.
 
20X AAP growth medium  
Stock solutions are prepared in sterile distilled or deionised water. 
Sterile stock solutions should be stored under cool and dark conditions. Under 
these conditions the stock solutions will have a shelf life of at least 6-8 weeks. 
Five nutrient stock solutions (A1, A2, A3, B and C) are prepared for 20X — 
AAP medium, using reagent-grade chemicals. 20 ml of each nutrient stock 
solution are added to approximately 850 ml deionised water to produce the growth medium. The pH is adjusted to 7,5 ± 0,1 with either 0,1 or 1 mol HCl or NaOH, and the volume adjusted to one litre with deionised water. The 
medium is then filtered through a 0,2 μm (approximate) membrane filter into a 
sterile container. 
The growth medium intended for testing should be prepared 1-2 days before use 
to allow the pH to stabilise. The pH of the growth medium should be checked prior to use and readjusted if necessary by addition of 0,1 or 1 M NaOH or HCl 
as described above.
 
Stock solution 
No. Substance Concentration 
in stock solution 
(g·l –1 ) (*) Concentration in 
prepared medium 
(mg·l  –1 ) (*) Prepared medium 
Element Concentration 
(mg·l  –1 ) (*) 
A1 NaNO  3 
MgCl  2 ·6H  2 O 
CaCl  2 ·2H  2 O 26 
12 4,4 510 
240 90 Na; N 
Mg Ca 190; 84 
58,08 24,04 
A2 MgSO
 4 ·7H  2 O 15 290 S 38,22 
A3 K 2 HPO  4 ·3H  2 O 1,4 30 K; P 9,4;3,7 
B H 3 BO 3 
MnCl  2 ·4H  2 O 
FeCl  3 ·6H  2 O 
Na 2 EDTA·2H  2 O 
ZnCl  2 
CoCl  2 ·6H  2 O 
Na 2 MoO  4 ·2H  2 O 
CuCl  2 ·2H  2 O 0,19 
0,42 
0,16 
0,30 3,3 mg·l
 –1 
1,4 mg·l  –1 
7,3 mg·l  –1 
0,012 mg·l  –1 3,7 
8,3 
3,2 
6,0 66 μg·l
 –1 
29 μg·l –1 
145 μg·l –1 
0,24 μg·l –1 B 
Mn 
Fe 
— Zn Co 
Mo 
Cu 0,65 
2,3 
0,66 
— 31 μg·l
 –1 
7,1 μg·l –1 
58 μg·l –1 
0,080 μg·l –1 
C NaHCO  3 15 300 Na; C 220; 43  
(*) Unless noted  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1231
 
Footnote:  The theoretically appropriate final bicarbonate concentration (which will avoid 
appreciable pH adjustment) is 15 mg/l, not 300 mg/l. However, the historical use 
of 20X-AAP medium, including the ring test for this method, is based upon 300 mg/l. (I. Sims, P. Whitehouse and R. Lacey. (1999) The OECD  Lemna  
Growth Inhibition Test. Development and Ring-testing of draft OECD Test 
Guideline. R & D Technical Report EMA 003. WRc plc — Environment Agency.
 
STEINBERG medium (After ISO 20079)  
Concentrations and stock solutions  
— The modified Steinberg medium is used in ISO 20079 for  Lemna minor  alone 
(as only  Lemna minor  is allowed there) but tests showed good results could 
be reached with  Lemna gibba  too. 
— When preparing the medium, reagent- or analytical grade chemicals and 
deionised water should be used. 
— Prepare the nutrient medium from stock solutions or the 10-fold concentrated 
medium which allows maximum concentration of the medium without 
precipitation.  
Table 1  
pH-stabilised STEINBERG medium (modified acc. to Altenburger)  
Substance Nutrient medium  
Macroelements  mol weight mg/l mmol/l  
KNO  3 101,12 350,00 3,46 
Ca(NO  3 ) 2 · 4H  2 O 236,15 295,00 1,25 
KH  2 PO 4 136,09 90,00 0,66 
K 2 HPO  4 174,18 12,60 0,072 
MgSO  4 · 7H  2 O 246,37 100,00 0,41  
Microelements  mol weight μg/l μmol/l 
H 3 BO 3 61,83 120,00 1,94 
ZnSO  4 · 7H  2 O 287,43 180,00 0,63 
Na 2 MoO  4 · 2H  2 O 241,92 44,00 0,18 
MnCl  2 · 4H  2 O 197,84 180,00 0,91 
FeCl  3 · 6H  2 O 270,21 760,00 2,81 
EDTA Disodium-dihydrate 372,24 1 500,00 4,03  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1232
 
Table 2  
Stock solutions (Macroelements)  
1. Macroelements (50-fold concentrated)  g/l  
Stock solution 1:  
KNO  3 17,50 
KH  2 PO 4 4,5 
K 2 HPO  4 0,63 
Stock solution 2:  
MgSO  4 · 7H  2 O 5,00  
Stock solution 3:  
Ca(NO  3 ) 2 · 4H  2 O 14,75  
Table 3  
Stock solutions (Microelements)  
2. Microelements (1 000-fold concentrated)  mg/l  
Stock solution 4:  
H 3 BO 3 120,0  
Stock solution 5:  
ZnSO  4 · 7H  2 O 180,0  
Stock solution 6:  
Na 2 MoO  4 · 2H  2 O 44,0  
Stock solution 7:  
MnCl  2 · 4H  2 O 180,0  
Stock solution 8:  
FeCl  3 · 6H  2 O 760,00 
EDTA Disodium-dihydrate 1 500,00 
— Stock solutions 2 and 3 and separately 4 to 7 may be pooled (taking into 
account the required concentrations). 
— For longer shelf life, treat stock solutions in an autoclave at 121 °C for 
20 min. or alternatively carry out a sterile filtration (0,2 μm). For stock 
solution 8, sterile filtration (0,2 μm) is strongly recommended.  
Preparation of the final concentration of STEINBERG medium (modified)  
— Add 20 ml of stock solutions 1, 2 and 3 (see table 2) to about 900 ml 
deionised water to avoid precipitation. 
— Add 1,0 ml of stock solutions 4, 5, 6, 7 and 8 (see table 3). 
— The pH should be 5,5 ± 0,2 (adjust by addition of a minimised volume of 
NaOH solution or HCl).  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1233
 
— Adjust with water to 1 000 ml. 
— If stock solutions are sterilised and appropriate water is used no further 
sterilisation is necessary. If sterilisation is done with the final medium, 
stock solution 8 should be added after autoclaving (at 121 °C for 20 min).  
Preparation of 10-fold-concentrated STEINBERG medium (modified) for 
intermediate storage  
— Add to 20 ml of stock solutions 1, 2 and 3 (see table 2) to about 30 ml water 
to avoid precipitation. 
— Add 1,0 ml of stock solutions 4, 5, 6, 7 and 8 (see table 3). Adjust with water 
to 100 ml. 
— If stock solutions are sterilised and appropriate water is used no further 
sterilisation is necessary. If sterilisation is done with the final medium, 
stock solution 8 should be added after autoclaving (at 121 °C for 20 min). 
— The pH of the medium (final concentration) should be 5,5 ± 0,2.  ▼M1 2008R0440 — EN — 22.03.2014 — 004.001 — 1234
 
C.27  SEDIMENT-WATER CHIRONOMID TOXICITY TEST USING 
SPIKED SEDIMENT  
INTRODUCTION 
1. This Test Method is equivalent to OECD Test Guideline (TG) 218 (2004). 
This Test Method is designed to assess the effects of prolonged exposure of 
chemicals to the sediment-dwelling larvae of the freshwater dipteran  
Chironomus  sp. It is based on existing toxicity test protocols for  
Chironomus riparius  and  Chironomus tentans  which have been developed 
in Europe (1)(2)(3) and North America (4)(5)(6)(7)(8) and ring-tested 
(1)(6)(9). Other well documented chironomid species may also be used, 
e.g. Chironomus yoshimatsui  (10)(11). 
2. The exposure scenario used in this Test Method is spiking of sediment with 
the test substance. The selection of the appropriate exposure scenario 
depends on the intended application of the test. The scenario of spiking 
sediment is intended to simulate accumulated levels of chemicals persisting in the sediment. This exposure system involves spiking sediment of a sediment-water test system. 
3. Substances that need to be tested towards sediment-dwelling organisms 
usually persist in this compartment over long time periods. The sediment-dwelling organisms may be exposed via a number of routes. The relative importance of each exposure route, and the time taken for 
each to contribute to the overall toxic effects, is dependent on the 
physical-chemical properties of the chemical concerned. For strongly adsorbing substances (e.g. with log K
 ow > 5) or for substances covalently 
binding to sediment, ingestion of contaminated food may be a significant 
exposure route. In order not to underestimate the toxicity of highly lipo ­
philic substances, the use of food added to the sediment before application of the test substance may be considered. In order to take all potential routes of exposure into account the focus of this Test Method is on long-term 
exposure. The test duration is in the range of 20-28 days for  C. riparius  
and C. yoshimatsui , and 28-65 days for  C. tentans . If short-term data are 
required for a specific purpose, for example to investigate the effects of an unstable chemical, additional replicates may be removed after a 10-day 
period. 
4. The measured endpoints are the total number of adults emerged and the time 
to emergence. It is recommended that measurements of larval survival and growth should only be made after a 10-day period if additional short-term 
data are required, using additional replicates as appropriate. 
5. The use of formulated sediment is recommended. Formulated sediment has 
several advantages over natural sediments: 
— the experimental variability is reduced because it forms a reproducible 
‘standardised matrix’ and the need to find uncontaminated and clean 
sediment sources is eliminated; 
— the tests can be initiated at any time without encountering seasonal 
variability in the test sediment and there is no need to pre-treat the 
sediment to remove indigenous fauna; the use of formulated sediment 
also reduces the cost associated with the field collection of sufficient amounts of sediment for routine testing; 
— the use of formulated sediment allows for comparisons of toxicity and 
ranking substances accordingly. 
6. Definitions used are given in Appendix 1.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1235
 
PRINCIPLE OF THE TEST 
7. First instar chironomid larvae are exposed to a concentration range of the 
test chemical in sediment — water systems. The test substance is spiked into the sediment and first instar larvae are subsequently introduced into test 
beakers in which the sediment and water concentrations have been stabi ­
lised. Chironomid emergence and development rate is measured at the end of the test. Larval survival and weight may also be measured after 10 days if required (using additional replicates as appropriate). These data are analysed 
either by using a regression model in order to estimate the concentration that 
would cause × % reduction in emergence or larval survival or growth (e.g. EC
 15 , EC  50 etc.), or by using statistical hypothesis testing to determine a 
NOEC/LOEC. The latter requires comparison of effect values with control 
values using statistical tests.  
INFORMATION ON THE TEST SUBSTANCE 
8. The water solubility of the test substance, its vapour pressure, measured or 
calculated partitioning into sediment and stability in water and sediment 
should be known. A reliable analytical method for the quantification of 
the test substance in overlying water, pore water and sediment with known and reported accuracy and limit of detection should be available. Useful information includes the structural formula and purity of the test 
substance. Chemical fate of the test substance (e.g. dissipation, abiotic 
and biotic degradation, etc.) also is useful information. Further guidance for testing substances with physical-chemical properties that make them 
difficult to perform the test is provided in (12)
 
REFERENCE CHEMICALS 
9. Reference chemicals may be tested periodically as a means of assuring that 
the test protocol and test conditions are reliable. Examples of reference toxicants used successfully in ring-tests and validation studies are: 
lindane, trifluralin, pentachlorophenol, cadmium chloride and potassium 
chloride (1)(2)(5)(6)(13).
 
VALIDITY OF THE TEST 
10. For the test to be valid the following conditions apply: 
— the emergence in the controls must be at least 70 % at the end of the 
test. (1)(6); 
— C. riparius and C. yoshimatsui emergence to adults from control vessels 
should occur between 12 and 23 days after their insertion into the 
vessels; for C. tentans, a period of 20 to 65 days is necessary. 
— at the end of the test, pH and the dissolved oxygen concentration should 
be measured in each vessel. The oxygen concentration should be at least 
60 per cent of the air saturation value (ASV) at the temperature used, and the pH of overlying water should be in the 6-9 range in all test vessels; 
— the water temperature should not differ by more than ± 1,0 °C. The 
water temperature could be controlled by isothermal room and in that 
case the room temperature should be confirmed in an appropriate time 
interval.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1236
 
DESCRIPTION OF THE METHOD  
Test vessels  
11. The study is conducted in glass 600 ml beakers measuring 8 cm in diameter. 
Other vessels are suitable, but they should guarantee a suitable depth of overlying water and sediment. The sediment surface should be sufficient to provide 2 to 3 cm
 2 per larvae. The ratio of the depth of the sediment layer 
to the depth of the overlying water should be 1:4. Test vessels and other 
apparatus that will come into contact with the test system should be made entirely of glass or other chemically inert material (e.g. Teflon).
 
Selection of species  
12. The species to be used in the test is preferably  Chironomus riparius . 
Chironomus tentans  is also suitable but more difficult to handle and 
requires a longer test period.  Chironomus yohimatsui  may also be used. 
Details of culture methods are given in Appendix 2 for  Chironomus 
riparius . Information on culture conditions is also available for other 
species, i.e. Chironomus tentans (4) and  Chironomus yoshimatsui  (11). 
Identification of species must be confirmed before testing but is not required prior to every test if organisms come from an in-house culture.
 
Sediment  
13. Formulated sediment (also called reconstituted, artificial or synthetic 
sediment) should preferably be used. However, if natural sediment is 
used, it should be characterised (at least pH, organic carbon content, deter ­
mination of other parameters such as C/N ratio and granulometry are also recommended), and it should be free from any contamination and other organisms that might compete with, or consume the chironomids. It is 
also recommended that, before it is used in a chironomid toxicity test, the 
natural sediment be conditioned for seven days under the same conditions which prevail in the subsequent test. The following formulated sediment, based on the artificial soil used in Test Method C.8 (14), is recommended 
for use in this test (1)(15)(16): 
(a) 4-5 % (dry weight) peat: as close to pH 5,5 to 6,0 as possible; it is 
important to use peat in powder form, finely ground (particle size ≤ 
1 mm) and only air dried. 
(b) 20 % (dry weight) kaolin clay (kaolinite content preferably above 
30 %). 
(c) 75-76 % (dry weight) quartz sand (fine sand should predominate with 
more than 50 per cent of the particles between 50 and 200 μm). 
(d) Deionised water is added to obtain moisture content of the final mixture 
in a range of 30-50 %. 
(e) Calcium carbonate of chemically pure quality (CaCO
 3 ) is added to 
adjust the pH of the final mixture of the sediment to 7,0 ± 0,5. Organic carbon content of the final mixture should be 2 % (± 0,5 %) 
and is to be adjusted by the use of appropriate amounts of peat and 
sand, according to (a) and (c). 
14. The source of peat, kaolin clay and sand should be known. The sediment 
components should be checked for the absence of chemical contamination 
(e.g. heavy metals, organochlorine compounds, organophosphorous 
compounds, etc.). An example for the preparation of the formulated sediment is described in Appendix 3. Mixing of dry constituents is also acceptable if it is demonstrated that after addition of overlying water a 
separation of sediment constituents (e.g. floating of peat particles) does 
not occur, and that the peat or the sediment is sufficiently conditioned.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1237
 
Water  
15. Any water which conforms to the chemical characteristics of acceptable 
dilution water as listed in Appendices 2 and 4 is suitable as test water. Any suitable water, natural water (surface or ground water), reconstituted 
water (see Appendix 2) or dechlorinated tap water are acceptable as 
culturing water and test water if chironomids will survive in it for the duration of the culturing and testing without showing signs of stress. At the start of the test, the pH of the test water should be between 6 and 9 and 
the total hardness not higher than 400 mg/l as CaCO
 3 . However, if there is 
an interaction suspected between hardness ions and the test substance, lower hardness water should be used (and thus, Elendt Medium M4 must not be 
used in this situation). The same type of water should be used throughout 
the whole study. The water quality characteristics listed in Appendix 4 should be measured at least twice a year or when it is suspected that these characteristics may have changed significantly.
 
Stock solutions — Spiked sediments  
16. Spiked sediments of the chosen concentration are usually prepared by 
addition of a solution of the test substance directly to the sediment. A 
stock solution of the test substance dissolved in deionised water is mixed 
with the formulated sediment by rolling mill, feed mixer or hand mixing. If poorly soluble in water, the test substance can be dissolved in as small a volume as possible of a suitable organic solvent (e.g. hexane, acetone or 
chloroform). This solution is then mixed with 10 g of fine quartz sand for 
one test vessel. The solvent is allowed to evaporate and it has to be totally removed from sand; the sand is then mixed with the suitable amount of 
sediment per test beaker. Only agents which volatilise readily can be used to 
solubilise, disperse or emulsify the test substance. It should be born in mind that the sand provided by the test substance and sand mixture, has to be taken into account when preparing the sediment (i.e. the sediment should 
thus be prepared with less sand). Care should be taken to ensure that the test 
substance added to sediment is thoroughly and evenly distributed within the sediment. If necessary, subsamples can be analysed to determine degree of homogeneity.
 
TEST DESIGN 
17. The test design relates to the selection of the number and spacing of the test 
concentrations, the number of vessels at each concentration and the number 
of larvae per vessel. Designs for EC point estimation, for estimation of NOEC, and for conducting a limit test are described.
 
Design for analysis by regression  
18. The effect concentration (e.g. EC  15 , EC  50 ) and the concentration range, over 
which the effect of the test substance is of interest, should be spanned by the concentrations included in the test. Generally, the accuracy and especially 
validity, with which estimates of effect concentrations (EC
 x ) can be made, is 
improved when the effect concentration is within the range of concentrations tested. Extrapolating much below the lowest positive concentration or above the highest concentration should be avoided. A preliminary range-finding 
test is helpful for selecting the range of concentrations to be used (see 
paragraph 27).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1238
 
19. If the EC  x is to be estimated, at least five concentrations and three replicates 
for each concentration should be tested. In any case, it is advisable that 
sufficient test concentrations are used to allow good model estimation. The factor between concentrations should not be greater than two (an exception could be made in cases when the dose response curve has a shallow slope). 
The number of replicates at each treatment can be reduced if the number of 
test concentrations with different responses is increased. Increasing the number of replicates or reducing the size of the test concentration intervals tends to lead to narrower confidence intervals for the test. 
Additional replicates are required if 10-day larval survival and growth are 
to be estimated.
 
Design for estimation of a NOEC/LOEC  
20. If the LOEC or NOEC are to be estimated, five test concentrations with at 
least four replicates should be used and the factor between concentrations 
should not be greater than two. The number of replicates should be 
sufficient to ensure adequate statistical power to detect a 20 % difference from the control at the 5 % level of significance (p = 0,05). With the development rate, an Analysis of Variance (ANOVA) is usually appropriate, 
such as Dunnett-test and Williams-test (17)(18)(19)(20). In the emergence 
ratio the Cochran-Armitage, Fisher’s exact (with Bonferroni correction), or Mantel-Haenszel tests may be used.
 
Limit test  
21. A limit test may be performed (one test concentration and control) if no 
effects were seen in the preliminary range-finding test. The purpose of the 
limit test is to perform a test at a concentration sufficiently high to enable decision makers to exclude possible toxic effects of the test substance, and the limit is set at a concentration which is not expected to appear in any 
situation. 1 000 mg/kg (dry weight) is recommended. Usually, at least six 
replicates for both the treatment and control are necessary. Adequate stat ­
istical power to detect a 20 % difference from the control at the 5 % level of significance (p = 0,05) should be demonstrated. With metric response 
(development rate and weight), the t-test is a suitable statistical method if 
data meet the requirements of this test (normality, homogeneous variances). The unequal-variance t-test or a non parametric test, such as the Wilcoxon-Mann-Whithey test may be used, if these requirements are not 
fulfilled. With the emergence ratio, the Fisher exact test is appropriate.
 
PROCEDURE  
Conditions of exposure  
Preparation of spiked sediment — water system  
22. The spiking procedure described in Test Method C.8: Toxicity for 
Earthworms is recommended for application of the test substance (14). 
The spiked sediments are placed in the vessels and overlying water is added to produce a sediment-water volume ratio of 1:4 (see paragraphs 11 and 15). The depth of the sediment layer should be in the range of 
1,5-3 cm. To avoid separation of sediment ingredients and re-suspension 
of fine material during addition of test water in the water column, the sediment can be covered with a plastic disc while water is poured onto it, and the disc removed immediately afterwards. Other devices may also be 
appropriate. 
23. The test vessels should be covered (e.g. by glass plates). If necessary, during 
the study the water levels will be topped to the original volume in order to 
compensate for water evaporation. This should be performed using distilled 
or deionised water to prevent build-up of salts.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1239
 
Stabilisation  
24. Once the spiked sediment with overlying water has been prepared, it is 
desirable to allow partitioning of the test substance from the aqueous phase to the sediment (3)(4)(6)(13). This should preferably be done under 
the conditions of temperature and aeration used in the test. Appropriate 
equilibration time is sediment and chemical specific, and can be in the order of hours to days and in rare cases up to several weeks (4-5 weeks). As this would leave time for degradation of many chemicals, equilibrium is 
not awaited but an equilibration period of 48 hours is recommended. At the 
end of this further equilibration period, the concentration of the test substance should be measured in the overlying water, the pore water and 
the sediment, at least at the highest concentration and a lower one (see 
paragraph 38). These analytical determinations of the test substance allow for calculation of mass balance and expression of results based on measured concentrations.
 
Addition of test organisms  
25. Four to five days before adding the test organisms to the test vessels, egg 
masses should be taken from the cultures and placed in small vessels in culture medium. Aged medium from the stock culture or freshly prepared medium may be used. If the latter is used, a small amount of food e.g. green 
algae and/or a few droplets of filtrate from a finely ground suspension of 
flaked fish food should be added to the culture medium (see Appendix 2). Only freshly laid egg masses should be used. Normally, the larvae begin to hatch a couple of days after the eggs are laid (2 to 3 days for  Chironomus 
riparius  at 20 °C and 1 to 4 days for  Chironomus tentans  at 23 °C and  
Chironomus yoshimatui  at 25 °C) and larval growth occurs in four instars, 
each of 4-8 days duration. First instar larvae (2-3 or 1-4 days post hatching) should be used in the test. The instar of midges can possibly be checked 
using head capsule width (6). 
26. Twenty first instar larvae are allocated randomly to each test vessel 
containing the spiked sediment and water, using a blunt pipette. Aeration 
of the water has to be stopped while adding the larvae to test vessels and remain so for another 24 hours after addition of larvae (see paragraphs 25 and 32). According to the test design used (see paragraphs 19 and 20), the 
number of larvae used per concentration is at least 60 for the EC 
point estimation and 80 for determination of NOEC.
 
Test concentrations  
27. A range-finding test may be helpful to determine the range of concentrations 
for the definitive test. For this purpose a series of widely spaced concen ­
trations of the test substance are used. In order to provide the same density of surface per chironomids, which is to be used for the definitive test, chironomids are exposed to each concentration of the test substance for a 
period which allows estimation of appropriate test concentrations, and no 
replicates are required. 
28. The test concentrations for the definitive test are decided based on the result 
of the range-finding test. At least five concentrations should be used and 
selected as described in paragraphs 18 to 20.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1240
 
Controls  
29. Control vessels without any test substance but including sediment should be 
included in the test with the appropriate number of replicates (see para ­
graphs 19-20). If a solvent has been used for application of test substance 
(see paragraph 16), a sediment solvent control should be added.  
Test system  
30. Static systems are used. Semi-static or flow-through systems with inter ­
mittent or continuous renewal of overlying water might be used in excep ­
tional cases as for instance if water quality specifications become inappro ­
priate for the test organism or affect chemical equilibrium (e.g. dissolved oxygen levels fall too low, the concentration of excretory products rises too high or minerals leach from sediment and affect pH and/or water hardness). 
However, other methods for ameliorating the quality of overlying water, 
such as aeration, will normally suffice and be preferable.
 
Food  
31. It is necessary to feed the larvae, preferably daily or at least three times per 
week. Fish-food (a suspension in water or finely ground food, e.g. TetraMin 
or TetraPhyll; see details in Appendix 2) in the amount of 0,25-0,5 mg (0,35-0,5 mg for  C. yoshimatui ) per larvae per day seems adequate for 
young larvae for the first 10 days. Slightly more food may be necessary 
for older larvae: 0,5-1 mg per larvae per day should be sufficient for the rest 
of the test. The food ration should be reduced in all treatments and control if fungal growth is seen or if mortality is observed in controls. If fungal development cannot be stopped the test is to be repeated. When testing 
strongly adsorbing substances (e.g. with log K
 ow > 5), or substances 
covalently binding to sediment, the amount of food necessary to ensure survival and natural growth of the organisms may be added to the formulated sediment before the stabilisation period. For this, plant 
material must be used instead of fish food, e.g. addition of 0,5 % (dry 
weight) finely ground leaves of e.g. stinging nettle ( Urtica dioica ), 
mulberry ( Morus alba ), white clover ( Trifolium repens ), spinach ( Spinacia 
oleracea ) or of other plant material ( Cerophyl  or alpha-cellulose) may be 
used.
 
Incubation conditions  
32. Gentle aeration of the overlying water in test vessels is supplied preferably 
24 hours after addition of the larvae and is pursued throughout the test (care 
should be taken that dissolved oxygen concentration does not fall below 60 
per cent of ASV). Aeration is provided through a glass Pasteur pipette fixed 2-3 cm above the sediment layer (i.e. one or few bubbles/sec). When testing volatile chemicals, consideration may be given not to aerate the 
sediment-water system. 
33. The test is conducted at a constant temperature of 20 °C (± 2 °C). For  C. 
tentans  and C. yoshimatui  recommended temperatures are 23 °C and 25 °C 
(± 2 °C), respectively. A 16 hours photoperiod is used and the light 
intensity should be 500 to 1 000 lux.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1241
 
Exposure duration  
34. The exposure commences with the addition of larvae to the spiked and 
control vessels. The maximum exposure duration is 28 days for  C. 
riparius  and  C. yoshimatsui , and 65 days for  C. tentans . If midges 
emerge earlier, the test can be terminated after a minimum of five days 
after emergence of the last adult in the control.  
Observations  
Emergence  
35. The development time and the total number of fully emerged male and 
female midges are determined. Males are easily identified by their plumose antennae. 
36. The test vessels should be observed at least three times per week to make 
visual assessment of any abnormal behaviour (e.g. leaving sediment, 
unusual swimming), compared with the control. During the period of 
expected emergence a daily count of emerged midges is necessary. The sex and number of fully emerged midges are recorded daily. After identifi ­
cation the midges are removed from the vessels. Any egg masses deposited 
prior to the termination of the test should be recorded and then removed to prevent re-introduction of larvae into the sediment. The number of visible pupae that have failed to emerge is also recorded. Guidance on measurement 
of emergence is provided in Appendix 5.
 
Growth and survival  
37. If data on 10-day larval survival and growth are to be provided, additional 
test vessels should be included at the start, so that they may be used 
subsequently. The sediment from these additional vessels is sieved using a 250 μm sieve to retain the larvae. Criteria for death are immobility or lack 
of reaction to a mechanical stimulus. Larvae not recovered should also be 
counted as dead (larvae which have died at beginning of the test may have 
been degraded by microbes). The (ash free) dry weight of the surviving larvae per test vessel is determined and the mean individual dry weight per vessel calculated. It is useful to determine which instar the surviving 
larvae belong to; for that measurement of the width of the head capsule of 
each individual can be used.
 
Analytical measurements  
Concentration of the test substance  
38. Prior to test commencement (i.e. addition of larvae), samples of bulk 
sediment are removed from at least one vessel per treatment for the analytical determination of the test substance concentration in the sediment. It is recommended that, as a minimum, samples of the 
overlying water, the pore water and the sediment be analysed at the start 
(see paragraph 24) and at the end of the test, at the highest concentration and a lower one. These determinations of test substance concentration inform about the behaviour/partitioning of the test substance in the 
water-sediment system. 
39. When intermediate measurements are made (e.g. at day 7) and if the 
analysis needs large samples which cannot be taken from test vessels without influencing the test system, analytical determinations should be performed on samples from additional test vessels treated in the same 
way (including the presence of test organisms) but not used for biological 
observations.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1242
 
40. Centrifugation at e.g. 10 000 g and 4 °C for 30 min. is the recommended 
procedure to isolate interstitial water. However, if the test substance is 
demonstrated not to adsorb to filters, filtration may also be acceptable. In some cases it might not be possible to analyse concentrations in the pore water as the sample size is too small.
 
Physical-chemical parameters  
41. pH and temperature of the test vessels should be measured in an appropriate 
manner (see paragraph 10). Hardness and ammonia should be measured in the controls and one test vessel at the highest concentration at the start and the end of the test.
 
DATA AND REPORTING  
Treatment of results  
42. The purpose of this test is to determine the effect of the test substance on 
the development rate and the total number of fully emerged male and female 
midges, or in the case of the 10-day test effects on survival and weight of the larvae. If there are no indications of statistically different sensitivities of 
sexes, male and female results may be pooled for statistical analyses. The 
sensitivity differences between sexes can be statistically judged by e.g. a χ
 2 -r 
× 2 table test. Larval survival and mean individual dry weight per vessel must be determined after 10 days where required. 
43. Effect concentrations expressed and based on dry weight, are calculated 
preferably based on measured sediment concentrations at the beginning of the test (see paragraph 38). 
44. To compute a point estimate for the EC
 50 or any other EC  x , the per-vessel 
statistics may be used as true replicates. In calculating a confidence interval 
for any EC  x the variability among vessels should be taken into account, or it 
should be shown that this variability is so small that it can be ignored. When the model is fitted by Least Squares, a transformation should be applied to the per-vessel statistics in order to improve the homogeneity of 
variance. However, EC
 x values should be calculated after the response is 
transformed back to the original value. 
45. When the statistical analysis aims at determining the NOEC/LOEC by 
hypothesis testing, the variability among vessels needs to be taken into account, e.g. by a nested ANOVA. Alternatively, more robust tests (21) can be appropriate in situations where there are violations of the usual 
ANOVA assumptions.
 
Emergence ratio  
46. Emergence ratios are quantal data, and can be analyzed by the 
Cochran-Armitage test applied in step-down manner where a monotonic dose-response is expected and these data are consistent with this expec ­
tation. If not, a Fisher’s exact or Mantel-Haenszel test with 
Bonferroni-Holm adjusted p-values can be used. If there is evidence of greater variability between replicates within the same concentration than a binomial distribution would indicate (often referenced as ‘extra-binomial’ 
variation), then a robust Cochran-Armitage or Fisher exact test such as 
proposed in (21), should be used.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1243
 
The sum of midges emerged per vessel, ne, is determined and divided by 
the number of larvae introduced, n  a : 
ER ¼ n e 
n a 
where:  
ER = emergence ratio  
n e = number of midges emerged per vessel  
n a = number of larvae introduced per vessel 
47. An alternative that is most appropriate for large sample sizes, when there is 
extra binomial variance, is to treat the emergence ratio as a continuous response and use procedures such as William’s test when a monotonic 
dose-response is expected and is consistent with these ER data. Dunnett’s 
test would be appropriate where monotonicity does not hold. A large sample size is defined here as the number emerged and the number not emerging both exceeding five, on a per replicate (vessel) basis. 
48. To apply ANOVA methods values of ER should first be transformed by the 
arcsin-sqrt-transformation or Freeman-Tukey transformation to obtain an 
approximate normal distribution and to equalise variances. The Cochran-Armitage, Fisher’s exact (Bonferroni), or Mantel-Haenszel tests can be applied when using the absolute frequencies. The arcsin-sqrt trans ­
formation is applied by taking the inverse sine (sin
 -1 ) of the square root of 
ER. 
49. For emergence ratios, EC  x -values are calculated using regression analysis 
(or e.g. probit (22), logit, Weibull, appropriate commercial software etc.). If regression analysis fails (e.g. when there are less than two partial responses), 
other non-parametric methods such as moving average or simple inter ­
polation are used.
 
Development rate  
50. The mean development time represents the mean time span between the 
introduction of larvae (day 0 of the test) and the emergence of the experi ­
mental cohort of midges. (For the calculation of the true development time, the age of larvae at the time of introduction should be considered). The 
development rate is the reciprocal of the development time (unit: 1/day) and 
represents that portion of larval development which takes place per day. The development rate is preferred for the evaluation of these sediment toxicity studies as its variance is lower, and it is more homogeneous and closer to 
normal distribution as compared to development time. Hence, powerful 
parametric test procedures may be used with development rate rather than with development time. For development rate as a continuous response, EC
 x -values can be estimated by using regression analysis (e.g. (23), (24)). 
51. For the following statistical tests, the number of midges observed on 
inspection day × are assumed to be emerged at the mean of the time 
interval between day x and day x – l (l = length of the inspection interval, usually 1 day). The mean development rate per vessel ( x) is 
calculated according to:
 
x ¼ X  m 
i¼1 ƒ i x i 
n e ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1244
 
where:  
x: mean development rate per vessel 
i: index of inspection interval  
m: maximum number of inspection intervals  
ƒ i : number of midges emerged in the inspection interval i 
n e : total number of midges emerged at the end of experiment (=  P ƒ i ) 
x i : development rate of the midges emerged in interval i 
x i ¼ 1 
Ê 
day  i Ä 1 i 
2 Ì 
where: 
day  i : inspection day (days since application) 
l i : length of inspection interval i (days, usually 1 day)  
Test report  
52. The test report must at least provide the following information:  
Test substance:  
— physical nature and, where relevant, physical-chemical properties (water 
solubility, vapour pressure, partition coefficient in soil (or in sediment if 
available), stability in water, etc.); 
— chemical identification data (common name, chemical name, structural 
formula, CAS number, etc.) including purity and analytical method for 
quantification of test substance.  
Test species:  
— test animals used: species, scientific name, source of organisms and 
breeding conditions; 
— information on handling of egg masses and larvae; 
— age of test animals when inserted into test vessels.  
Test conditions:  
— sediment used, i.e. natural or formulated sediment; 
— for natural sediment, location and description of sediment sampling site, 
including, if possible, contamination history; characteristics: pH, organic 
carbon content, C/N ratio and granulometry (if appropriate). 
— preparation of the formulated sediment: ingredients and characteristics 
(organic carbon content, pH, moisture, etc. at the start of the test); 
— preparation of the test water (if reconstituted water is used) and char ­
acteristics (oxygen concentration, pH, conductivity, hardness, etc. at the start of the test); 
— depth of sediment and overlying water; — volume of overlying and pore water; weight of wet sediment with and 
without pore water;
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1245
 
— test vessels (material and size); 
— method of spiking sediment: test concentrations used, number of 
replicates and use of solvent if any; 
— stabilisation equilibrium phase of the spiked sediment-water system: 
duration and conditions; 
— incubation conditions: temperature, light cycle and intensity, aeration 
(frequency and intensity); 
— detailed information on feeding including type of food, preparation, 
amount and feeding regime.  
Results:  
— the nominal test concentrations, the measured test concentrations and the 
results of all analyses to determine the concentration of the test 
substance in the test vessel; 
— water quality within the test vessels, i.e. pH, temperature, dissolved 
oxygen, hardness and ammonia; 
— replacement of evaporated test water, if any; — number of emerged male and female midges per vessel and per day; — number of larvae which failed to emerge as midges per vessel; — mean individual dry weight of larvae per vessel, and per instar, if 
appropriate; 
— percent emergence per replicate and test concentration (male and female 
midges pooled); 
— mean development rate of fully emerged midges per replicate and 
treatment rate (male and female midges pooled); 
— estimates of toxic endpoints e.g. ECx (and associated confidence inter ­
vals), NOEC and/or LOEC,, and the statistical methods used for their determination; 
— discussion of the results, including any influence on the outcome of the 
test resulting from deviations from this Test Method.
 
LITERATURE:  
(1) BBA (1995). Long-term toxicity test with Chironomus riparius: Devel ­
opment and validation of a new test system. Edited by M. Streloke and H.Köpp. Berlin 1995. 
(2) Fleming R  et al.  (1994). Sediment Toxicity Tests for Poorly Water-Soluble 
Substances. Final Report to them European Commission. Report No: EC 
3738. August 1994. WRc, UK. 
(3) SETAC (1993). Guidance Document on Sediment toxicity Tests and 
Bioassays for Freshwater and Marine Environments. From the WOSTA Workshop held in the Netherlands. 
(4) ASTM International/E1706-00 (2002). Test Method for Measuring the 
Toxicity of Sediment-Associated Contaminants with Freshwater Invert ­
ebrates. pp 1125-1241. In ASTM International 2002 Annual Book of Stan ­
dards. Volume 11.05. Biological Effects and Environmental Fate;Biotech ­
nology; Pesticides. ASTM. International, West Conshohocken, PA. 
(5) Environment Canada (1997). Test for Growth and Survival in Sediment 
using Larvae of Freshwater Midges ( Chironomus tentans  or Chironomus 
riparius ). Biological Test Method. Report SPE 1/RM/32. December 1997.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1246
 
(6) US-EPA (2000). Methods for Measuring the Toxicity and Bioaccumulation 
of Sediment-associated Contaminants with Freshwater Invertebrates. 
Second edition. EPA 600/R-99/064. March 2000. Revision to the first edition dated June 1994. 
(7) US-EPA/OPPTS 850.1735. (1996): Whole Sediment Acute Toxicity Invert ­
ebrates. 
(8) US-EPA/OPPTS 850.1790. (1996): Chironomid Sediment toxicity Test. 
(9) Milani D, Day KE, McLeay DJ, and Kirby RS (1996). Recent intra- and 
inter-laboratory studies related to the development and standardisation of Environment Canada’s biological test methods for measuring sediment toxicity using freshwater amphipods  (Hyalella azteca)  and midge larvae  
(Chironomus riparius) . Technical Report. Environment Canada. National 
Water Research Institute. Burlington, Ontario, Canada. 
(10) Sugaya Y (1997). Intra-specific variations of the susceptibility of insec ­
ticides  in Chironomus yoshimatsui . Jp. J. Sanit. Zool. 48 (4): 345-350. 
(11) Kawai K (1986). Fundamental studies on Chironomid allergy. I. Culture 
methods of some Japanese Chironomids (Chironomidae, Diptera). Jp. J. 
Sanit. Zool. 37(1): 47-57. 
(12) OECD (2000). Guidance Document on Aquatic Toxicity Testing of 
Difficult Substances and Mixtures. OECD Environment, Health and Safety Publications, Series on Testing and Assessment No 23. 
(13) Environment Canada (1995). Guidance Document on Measurement of 
Toxicity Test Precision Using Control Sediments Spiked with a 
Reference Toxicant. Report EPS 1/RM/30. September 1995. 
(14) Test Method C.8 of this Annex, Toxicity for Earthworms. (15) Suedel BC and JH Rodgers (1994). Development of formulated reference 
sediments for freshwater and estuarine sediment testing. Environ. Toxicol. 
Chem. 13: 1163-1175. 
(16) Naylor C and C Rodrigues (1995). Development of a test method for 
Chironomus riparius using a formulated sediment. Chemosphere 31: 3291-3303. 
(17) Dunnett CW (1964). A multiple comparisons procedure for comparing 
several treatments with a control. J. Amer. Statis. Assoc., 50: 1096-1121. 
(18) Dunnett CW (1964). New tables for multiple comparisons with a control. 
Biometrics, 20: 482-491. 
(19) Williams DA (1971). A test for differences between treatment means when 
several dose levels are compared with a zero dose control. Biometrics, 27: 
103-117. 
(20) Williams DA (1972). The comparison of several dose levels with a zero 
dose control. Biometrics, 28: 510-531. 
(21) Rao JNK and Scott AJ (1992). A simple method for the analysis of 
clustered binary data. Biometrics 48: 577-585. 
(22) Christensen ER (1984). Dose-response functions in aquatic toxicity testing 
and the Weibull model. Water Research 18: 213-221. 
(23) Bruce and Versteeg (1992). A statistical procedure for modelling 
continuous toxicity data. Environmental Toxicology and Chemistry 11: 1485-1494. 
(24) Slob W (2002). Dose-response modelling of continuous endpoints. Toxicol. 
Sci. 66: 298-312.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1247
 
Appendix 1  
DEFINITIONS 
For the purpose of this Test Method the following definitions are used:  
Formulated sediment  or reconstituted, artificial or synthetic sediment, is a 
mixture of materials used to mimic the physical components of a natural sediment.  
Overlying water  is the water placed over sediment in the test vessel.  
Interstitial water  or pore water is the water occupying space between sediment 
and soil particles.  
Spiked sediment  is sediment to which test substance has been added.  
Test chemical:  Any substance or mixture tested using this Test Method.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1248
 
Appendix 2  
Recommendations for culture of  Chironomus riparius  
1. Chironomus  larvae may be reared in crystallising dishes or larger 
containers. Fine quartz sand is spread in a thin layer of about 5 to 10 mm deep over the bottom of the container. Kieselguhr (e.g. Merck, Art 8117) has also been shown to be a suitable substrate (a thinner layer 
of up to a very few mm is sufficient). Suitable water is then added to a 
depth of several cm. Water levels should be topped up as necessary to replace evaporative loss, and prevent desiccation. Water can be replaced if necessary. Gentle aeration should be provided. The larval rearing vessels 
should be held in a suitable cage which will prevent escape of the 
emerging adults. The cage should be sufficiently large to allow swarming of emerged adults, otherwise copulation may not occur (minimum is ca. 30 × 30 × 30 cm). 
2. Cages should be held at room temperature or in a constant environment 
room at 20 ± 2 °C with a photo period of 16 hour light (intensity ca. 1 000 
lux), 8 hours dark. It has been reported that air humidity of less than 60 % RH can impede reproduction.
 
Dilution water  
3. Any suitable natural or synthetic water may be used. Well water, dech ­
lorinated tap water and artificial media (e.g. Elendt ‘M4’ or ‘M7’ medium, see below) are commonly used. The water has to be aerated before use. If 
necessary, the culture water may be renewed by pouring or siphoning the 
used water from culture vessels carefully without destroying the tubes of larvae.
 
Feeding larvae  
4. Chironomus  larvae should be fed with a fish flake food (TetraMin  ® Tetra­
Phyll  ® or other similar brand of proprietary fish food), at approximately 
250 mg per vessel per day. This can be given as a dry ground powder or as 
a suspension in water: 1,0 g of flake food is added to 20 ml of dilution 
water and blended to give a homogenous mix. This preparation may be fed at a rate of about 5 ml per vessel per day (shake before use). Older larvae may receive more. 
5. Feeding is adjusted according to the water quality. If the culture medium 
becomes ‘cloudy’, the feeding should be reduced. Food additions must be 
carefully monitored. Too little food will cause emigration of the larvae towards the water column, and too much food will cause increased 
microbial activity and reduced oxygen concentrations. Both conditions 
can result in reduced growth rates. 
6. Some green algae (e.g.  Scenedesmus subspicatus , Chlorella vulgaris ) cells 
may also be added when new culture vessels are set up.
 
Feeding emerged adults  
7. Some experimenters have suggested that a cotton wool pad soaked in a 
saturated sucrose solution may serve as a food for emerged adults.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1249
 
Emergence  
8. At 20 ± 2 °C adults will begin to emerge from the larval rearing vessels 
after approximately 13-15 days. Males are easily distinguished by having plumose antennae.
 
Egg masses  
9. Once adults are present within the breeding cage, all larval rearing vessels 
should be checked three times weekly for deposition of the gelatinous egg masses. If present, the egg masses should be carefully removed. They should be transferred to a small dish containing a sample of the breeding 
water. Egg masses are used to start a new culture vessel (e.g. 2-4 egg 
masses/vessel) or are used for toxicity tests. 
10. First instar larvae should hatch after 2-3 days.
 
Set-up of new culture vessels  
11. Once cultures are established it should be possible to set up a fresh larval 
culture vessel weekly or less frequently depending on testing requirements, 
removing the older vessels after adult midges have emerged. Using this 
system a regular supply of adults will be produced with a minimum of management.
 
Preparation of test solutions ‘M4’ and ‘M7’  
12. Elendt (1990) has described the ‘M4’ medium. The ‘M7’ medium is 
prepared as the ‘M4’ medium except for the substances indicated in 
Table 1, for which concentrations are four times lower in ‘M7’ than in ‘M4’. A publication on the ‘M7’ medium is in preparation (Elendt, personal communication). The test solution should not be prepared 
according to Elendt and Bias (1990) for the concentrations of NaSiO
 3 5 
H 2 O, NaNO  3 , KH  2 PO 4 and K  2 HPO  4 given for the preparation of the stock 
solutions are not adequate.  
Preparation of the ‘M7’-medium  
13. Each stock solution (I) is prepared individually and a combined stock 
solution (II) is prepared from these stock solutions (I) (see Table 1). 
Fifty ml from the combined stock Solution (II) and the amounts of each 
macro nutrient stock solution which are given in Table 2 are made up to 1 litre of deionised water to prepare the ‘M7’ medium. A vitamin stock solution is prepared by adding three vitamins to deionised water as 
indicated in Table 3, and 0,1 ml of the combined vitamin stock solution 
are added to the final ‘M7’ medium shortly before use. (The vitamin stock solution is stored frozen in small aliquots). The medium is aerated and stabilised.
 
LITERATURE:  
BBA (1995). Long-term toxicity test with  Chironomus riparius : Development 
and validation of a new test system. Edited by M. Streloke and H. Köpp. Berlin 
1995.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1250
 
Table 1  
Stock solutions of trace elements for medium M4 and M7  
Stock solutions (I) Amount (mg) 
made up to 1 litre 
of deionised 
water To prepare the combined stock 
solution (II): mix the following 
amounts (ml) of stock solutions (I) 
and make up to 1 litre of deionised 
water Final concentrations in test solutions 
(mg/l) 
M4 M7 M4 M7  
H 3 BO 3 ( 1 ) 57 190 1,0 0,25 2,86 0,715 
MnCl  2 · 4 H  2 O (  1 ) 7 210 1,0 0,25 0,361 0,090 
LiCl (  1 ) 6 120 1,0 0,25 0,306 0,077 
RbCl (  1 ) 1 420 1,0 0,25 0,071 0,018 
SrCl  2 · 6 H  2 O (  1 ) 3 040 1,0 0,25 0,152 0,038 
NaBr (  1 ) 320 1,0 0,25 0,016 0,004 
Na 2 MoO  4 · 2 H  2 O (  1 ) 1 260 1,0 0,25 0,063 0,016 
CuCl  2 · 2 H  2 O (  1 ) 335 1,0 0,25 0,017 0,004 
ZnCl  2 260 1,0 1,0 0,013 0,013 
CaCl  2 · 6 H  2 O 200 1,0 1,0 0,010 0,010 
KI 65 1,0 1,0 0,0033 0,0033 
Na 2 SeO  3 43,8 1,0 1,0 0,0022 0,0022 
NH  4 VO  3 11,5 1,0 1,0 0,00058 0,00058 
Na 2 EDTA · 2 H  2 O (  1 ) ( 2 ) 5 000 20,0 5,0 2,5 0,625 
FeSO  4 · 7 H  2 O (  1 ) ( 2 ) 1 991 20,0 5,0 1,0 0,249  
( 1 ) These substances differ in M4 and M7, as indicated above. 
( 2 ) These solutions are prepared individually, then poured together and autoclaved immediately.  
Table 2  
Macro nutrient stock solutions for medium M4 and M7  
Amount made up to 1 
litre of deionised water 
(mg) Amount of macro nutrient 
stock solutions added to 
prepare medium M4 and 
M7 
(ml/l) Final concentrations in 
test solutions M4 and 
M7 
(mg/l)  
CaCl  2 · 2 H  2 O 293 800 1,0 293,8 
MgSO  4 · 7 H  2 O 246 600 0,5 123,3 
KCl 58 000 0,1 5,8 NaHCO
 3 64 800 1,0 64,8 
NaSiO  3 · 9 H  2 O 50 000 0,2 10,0 
NaNO  3 2 740 0,1 0,274 
KH  2 PO 4 1 430 0,1 0,143 
K 2 HPO  4 1 840 0,1 0,184  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1251
 
Table 3  
Vitamin stock solution for medium M4 and M7. All three vitamin solutions are combined to 
make a single vitamin stock solution  
Amount made up to 1 
litre of deionised water 
(mg) Amount of vitamin stock 
solution added to prepare 
medium M4 and M7 
(ml/l) Final concentrations in 
test solutions M4 and 
M7 
(mg/l)  
Thiamine hydrochloride 750 0,1 0,075 
Cyanocobalamin (B12) 10 0,1 0,0010 Biotine 7,5 0,1 0,00075
 
LITERATURE:  
Elendt, B.P. (1990). Selenium Deficiency in Crustacean.  Protoplasma  154: 25- 
33. 
Elendt, B.P. & W.-R. Bias (1990). Trace Nutrient Deficiency in  Daphnia magna  
Cultured in Standard Medium for Toxicity Testing. Effects on the Optimization of Culture Conditions on Life History Parameters of  D. magna . Water Research  
24 (9): 1157-1167.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1252
 
Appendix 3  
PREPARATION OF FORMULATED SEDIMENT  
Sediment composition  
The composition of the formulated sediment should be as follows:  
Constituent Characteristics % of sediment 
dry weight  
Peat Sphagnum moss peat, as 
close to pH 5,5-6,0 as 
possible, no visible plant 
remains, finely ground (particle size ≤ 1 mm) and 
air dried 4-5 
Quartz sand Grain size: > 50 % of the 
particles should be in the 
range of 50-200 μm 75-76 
Kaolinite clay Kaolinite content ≥ 30 % 20 
Organic carbon Adjusted by addition of peat 
and sand 2 (± 0,5) 
Calcium carbonate CaCO
 3 , pulverised, 
chemically pure 0,05-0,1 
Water Conductivity ≤ 10 μS/cm 30-50  
Preparation  
The peat is air dried and ground to a fine powder. A suspension of the required 
amount of peat powder in deionised water is prepared using a high-performance 
homogenising device. The pH of this suspension is adjusted to 5,5 ± 0,5 with CaCO
 3 . The suspension is conditioned for at least two days with gentle stirring at 
20 ± 2 °C, to stabilise pH and establish a stable microbial component. pH is 
measured again and should be 6,0 ± 0,5. Then the peat suspension is mixed with 
the other constituents (sand and kaolin clay) and deionised water to obtain a homogeneous sediment with a water content in a range of 30-50 per cent of dry weight of the sediment. The pH of the final mixture is measured once again and 
is adjusted to 6,5 to 7,5 with CaCO
 3 if necessary. Samples of the sediment are 
taken to determine the dry weight and the organic carbon content. Then, before it is used in the chironomid toxicity test, it is recommended that the formulated sediment be conditioned for seven days under the same conditions which prevail 
in the subsequent test.
 
Storage  
The dry constituents for preparation of the artificial sediment may be stored in a 
dry and cool place at room temperature. The formulated (wet) sediment should 
not be stored prior to its use in the test. It should be used immediately after the 7 days conditioning period that ends its preparation.
 
LITERATURE:  
Chapter C.8 of this Annex. Toxicity for Earthworms. 
Meller M, Egeler P, Rombke J, Schallnass H, Nagel R, Streit B (1998). 
Short-term Toxicity of Lindane, Hexachlorobenzene and Copper Sulfate on 
Tubificid Sludgeworms (Oligochaeta) in Artificial Media. Ecotox. and Environ. 
Safety 39: 10-20.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1253
Appendix 4  
Chemical Characteristics of an Acceptable Dilution Water  
Substance Concentrations  
Particulate matter < 20 mg/l 
Total organic carbon < 2 mg/l Unionised ammonia < 1 μg/l 
Hardness as CaCO
 3 < 400 mg/l (*) 
Residual chlorine < 10 μg/l 
Total organophosphorus pesticides < 50 ng/l Total organochlorine pesticides plus polychlorinated 
biphenyls < 50 ng/l 
Total organic chlorine < 25 ng/l
 
(*) However, it should be noted that if there is an interaction suspected between hardness 
ions and the test substance, lower hardness water should be used (and thus, Elendt 
Medium M4 must not be used in this situation).  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1254
 
Appendix 5  
Guidance for monitoring emergence of chironomid larvae  
Emergence traps are placed on the test beakers. These traps are needed from day 
20 to the end of the test. Example of trap used is drawn below:
  
A: the nylon screen 
B: the inverted plastic cups 
C: the lipless exposure beaker D: the water exchange screen ports 
E: water 
F: sediment  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1255
 
C. 28.  SEDIMENT-WATER CHIRONOMID TOXICITY TEST USING 
SPIKED WATER  
INTRODUCTION 
1. This Test Method is equivalent to OECD TG 219 (2004). This Test Method 
is designed to assess the effects of prolonged exposure of chemicals to the 
sediment-dwelling larvae of the freshwater dipteran  Chironomus  sp. It is 
mainly based on the BBA guideline using a sediment-water test system with artificial soil, and water column exposure scenario (1). It also takes 
into account existing toxicity test protocols for  Chironomus riparius  and  
Chironomus tentans  which have been developed in Europe and North 
America (2)(3)(4)(5)(6)(7)(8) and ring-tested (1)(6)(9). Other well docu ­
mented chironomid species may also be used, e.g.  Chironomus yoshimatsui  
(10)(11). 
2. The exposure scenario used in this Test Method is water spiking. The 
selection of the appropriate exposure scenario depends on the intended application of the test. The water exposure scenario, involving spiking of the water column, is intended to simulate a pesticide spray drift event and 
covers the initial peak of concentrations in pore water. It is also useful for 
other types of exposure (including chemical spills) except accumulation processes lasting longer than the test period. 
3. Substances that need to be tested towards sediment-dwelling organisms 
usually persist in this compartment over long time periods. The 
sediment-dwelling organisms may be exposed via a number of routes. 
The relative importance of each exposure route, and the time taken for each to contribute to the overall toxic effects, is dependent on the physical-chemical properties of the chemical concerned. For strongly 
adsorbing substances (e.g. with log K
 ow > 5) or for substances covalently 
binding to sediment, ingestion of contaminated food may be a significant exposure route. In order not to underestimate the toxicity of highly lipo ­
philic substances, the use of food added to the sediment before application 
of the test substance may be considered. In order to take all potential routes of exposure into account the focus of this Test Method is on long-term exposure. The test duration is in the range of 20-28 days for  C. riparius  
and C. yoshimatsui , and 28-65 days for  C. tentans . If short-term data are 
required for a specific purpose, for example to investigate the effects of unstable chemicals, additional replicates may be removed after a 10-day period. 
4. The measured endpoints are the total number of adults emerged and the time 
to emergence. It is recommended that measurements of larval survival and 
growth should only be made after a 10-day period if additional short-term data are required, using additional replicates as appropriate. 
5. The use of formulated sediment is recommended. Formulated sediment has 
several advantages over natural sediments: 
— the experimental variability is reduced because it forms a reproducible 
‘standardised matrix’ and the need to find uncontaminated and clean 
sediment sources is eliminated; 
— the tests can be initiated at any time without encountering seasonal 
variability in the test sediment and there is no need to pre-treat the sediment to remove indigenous fauna; the use of formulated sediment 
also reduces the cost associated with the field collection of sufficient 
amounts of sediment for routine testing;
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1256
 
— the use of formulated sediment allows for comparisons of toxicity and 
ranking substances accordingly: toxicity data from tests with natural and 
artificial sediments were comparable for several chemicals (2). 
6. Definitions used are given in Appendix 1.  
PRINCIPLE OF THE TEST 
7. First instar chironomid larvae are exposed to a concentration range of the 
test substance in sediment-water systems. The test starts by placing first 
instar larvae into the test beakers containing the sediment-water system and subsequently spiking the test substance into the water. Chironomid emergence and development rate is measured at the end of the test. 
Larval survival and weight may also be measured after 10 days if 
required (using additional replicates as appropriate). These data are analysed either by using a regression model in order to estimate the concen ­
tration that would cause x % reduction in emergence, larvae survival or 
growth (e.g. EC
 15 , EC  50 , etc.), or by using statistical hypothesis testing to 
determine a NOEC/LOEC. The latter requires comparison of effect values with control values using statistical tests.
 
INFORMATION ON THE TEST SUBSTANCE 
8. The water solubility of the test substance, its vapour pressure, measured or 
calculated partitioning into sediment and stability in water and sediment 
should be known. A reliable analytical method for the quantification of the test substance in overlying water, pore water and sediment with known and reported accuracy and limit of detection should be available. 
Useful information includes the structural formula and purity of the test 
substance. Chemical fate of the test substance (e.g. dissipation, abiotic and biotic degradation, etc.) also is useful information. Further guidance for testing substances with physical-chemical properties that make them 
difficult to perform the test is provided in (12).
 
REFERENCE CHEMICALS 
9. Reference chemicals may be tested periodically as a means of assuring that 
the test protocol and test conditions are reliable. Examples of reference toxicants used successfully in ring-tests and validation studies are: 
lindane, trifluralin, pentachlorophenol, cadmium chloride and potassium 
chloride. (1)(2)(5)(6)(13).
 
VALIDITY OF THE TEST 
10. For the test to be valid the following conditions apply: 
— the emergence in the controls must be at least 70 % at the end of the 
test. (1)(6); 
— C. riparius  and C. yoshimatsui  emergence to adults from control vessels 
should occur between 12 and 23 days after their insertion into the 
vessels; for  C. tentans , a period of 20 to 65 days is necessary. 
— at the end of the test, pH and the dissolved oxygen concentration should 
be measured in each vessel. The oxygen concentration should be at least 
60 % of the air saturation value (ASV) at the temperature used, and the 
pH of overlying water should be in the 6-9 range in all test vessels;  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1257
 
— the water temperature should not differ by more than ± 1,0 °C. The 
water temperature could be controlled by isothermal room and in that 
case the room temperature should be confirmed in an appropriate time intervals.
 
DESCRIPTION OF THE METHOD  
Test vessels  
11. The study is conducted in glass 600 ml beakers measuring 8 cm in diameter. 
Other vessels are suitable, but they should guarantee a suitable depth of overlying water and sediment. The sediment surface should be sufficient to 
provide 2 to 3 cm
 2 per larvae. The ratio of the depth of the sediment layer 
to the depth of the overlying water should be 1:4. Test vessels and other apparatus that will come into contact with the test system should be made entirely of glass or other chemically inert material (e.g. Teflon).
 
Selection of species  
12. The species to be used in the test is preferably  Chironomus riparius . 
Chironomus tentans  is also suitable but more difficult to handle and 
requires a longer test period.  Chironomus yohimatsui  may also be used. 
Details of culture methods are given in Appendix 2 for  Chironomus 
riparius . Information on culture conditions is also available for other 
species, i.e.  Chironomus tentans  (4) and  Chironomus yoshimatsui  (11). 
Identification of species must be confirmed before testing but is not required prior to every test if organisms come from an in-house culture.
 
Sediment  
13. Formulated sediment (also called reconstituted, artificial or synthetic 
sediment) should preferably be used. However, if natural sediment is 
used, it should be characterised (at least pH, organic carbon content, deter ­
mination of other parameters such as C/N ratio and granulometry are also recommended), and it should be free from any contamination and other 
organisms that might compete with, or consume the chironomids. It is 
also recommended that, before it is used in a chironomid toxicity test, the natural sediment be conditioned for seven days under the same conditions which prevail in the subsequent test. The following formulated sediment, 
based on the artificial soil used in Test Method C.8 (14), is recommended 
for use in this test (1)(15)(16): 
a) 4-5 % (dry weight) peat: as close to pH 5,5 to 6,0 as possible; it is 
important to use peat in powder form, finely ground (particle size ≤ 
1 mm) and only air dried. 
b) 20 % (dry weight) kaolin clay (kaolinite content preferably above 30 %). 
c) 75-76 % (dry weight) quartz sand (fine sand should predominate with 
more than 50 % of the particles between 50 and 200 μm). 
d) Deionised water is added to obtain moisture of the final mixture in a 
range of 30-50 %. 
e) Calcium carbonate of chemically pure quality (CaCO
 3 ) is added adjust 
the pH of the final mixture of the sediment to 7,0 ± 0,5. 
f) Organic carbon content of the final mixture should be 2 % (± 0,5 %) and 
is to be adjusted by the use of appropriate amounts of peat and sand, 
according to (a) and (c).  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1258
 
14. The source of peat, kaolin clay and sand should be known. The sediment 
components should be checked for the absence of chemical contamination 
(e.g. heavy metals, organochlorine compounds, organophosphorous compounds, etc.). An example for the preparation of the formulated sediment is described in Appendix 3. Mixing of dry constituents is also 
acceptable if it is demonstrated that after addition of overlying water a 
separation of sediment constituents (e.g. floating of peat particles) does not occur, and that the peat or the sediment is sufficiently conditioned.
 
Water  
15. Any water which conforms to the chemical characteristics of acceptable 
dilution water as listed in Appendices 2 and 4 is suitable as test water. 
Any suitable water, natural water (surface or ground water), reconstituted 
water (see Appendix 2) or dechlorinated tap water are acceptable as culturing water and test water if chironomids will survive in it for the 
duration of the culturing and testing without showing signs of stress. At 
the start of the test, the pH of the test water should be between 6 and 9 and the total hardness not higher than 400 mg/l as CaCO
 3 . However, if there is 
an interaction suspected between hardness ions and the test substance, lower 
hardness water should be used (and thus, Elendt Medium M4 must not be 
used in this situation). The same type of water should be used throughout the whole study. The water quality characteristics listed in Appendix 4 should be measured at least twice a year or when it is suspected that 
these characteristics may have changed significantly.
 
Stock solutions — Spiked water  
16. Test concentrations are calculated on the basis of water column concen ­
trations, i.e. the water overlying the sediment. Test solutions of the chosen concentrations are usually prepared by dilution of a stock solution. 
Stock solutions should preferably be prepared by dissolving the test 
substance in test medium. The use of solvents or dispersants may be required in some cases in order to produce a suitably concentrated stock solution. Examples of suitable solvents are acetone, ethanol, methanol, 
ethylene glycol monoethyl ether, ethylene glycol dimethyl ether, dimethyl ­
formamide and triethylene glycol. Dispersants which may be used are Cremophor RH40, Tween 80, methylcellulose 0,01 % and HCO-40. The solubilising agent concentration in the final test medium should be 
minimal (i.e. ≤ 0,1 ml/l) and should be the same in all treatments. When 
a solubilising agent is used, it must have no significant effects on survival or no visible adverse effect on the chironomid larvae as revealed by a solvent- only control. However, every effort should be made to avoid the use of such 
materials.
 
TEST DESIGN 
17. The test design relates to the selection of the number and spacing of the test 
concentrations, the number of vessels at each concentration and the number of larvae per vessel. Designs for EC point estimation, for estimation of 
NOEC, and for conducting a limit test are described. The analysis by 
regression is preferred to the hypothesis testing approach.
 
Design for analysis by regression  
18. The effect concentration (e.g. EC  15 , EC  50 ) and the concentration range, over 
which the effect of the test substance is of interest, should be spanned by the concentrations included in the test. Generally, the accuracy and especially 
validity, with which estimates of effect concentrations (EC
 x ) can be made, is 
improved when the effect concentration is within the range of concentrations  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1259
 
tested. Extrapolation much below the lowest positive concentration or above 
the highest concentration should be avoided. A preliminary range-finding 
test is helpful for selecting the range of concentrations to be used (see paragraph 27). 
19. If the EC
 x is to be estimated, at least five concentrations and three replicates 
for each concentration should be tested. In any case, it is advisable that 
sufficient test concentrations are used to allow a good model estimation. The factor between concentrations should not be greater than two (an exception could be made in cases when the dose response curve has a shallow slope). 
The number of replicates at each treatment can be reduced if the number of 
test concentrations with different responses is increased. Increasing the number of replicates or reducing the size of the test concentration intervals tends to lead to narrower confidence intervals for the test. 
Additional replicates are required if 10-day larval survival and growth are 
to be estimated.
 
Design for estimation of a NOEC/LOEC  
20. If the LOEC/NOEC are to be estimated, five test concentrations with at least 
four replicates should be used and the factor between concentrations should not be greater than two. The number of replicates should be sufficient to 
ensure adequate statistical power to detect a 20 % difference from the 
control at the 5 % level of significance (p = 0,05). With the development rate, an Analysis of Variance (ANOVA) is usually appropriate, such as Dunnett-test and Williams-test (17)(18)(19)(20). In the emergence ratio the 
Cochran-Armitage, Fisher’s exact (with Bonferroni correction), or 
Mantel-Haenszel tests may be used.
 
Limit test  
21. A limit test may be performed (one test concentration and control) if no 
effects were seen in the preliminary range-finding test. The purpose of the limit test is to indicate that the toxic value of the test substance is greater 
than the limit concentration tested. No suggestion for a recommended 
concentration can be made in this Test Method; this is left to the regulators’ judgement. Usually, at least six replicates for both the treatment and control are necessary. Adequate statistical power to detect a 20 % difference from 
the control at the 5 % level of significance (p = 0,05) should be demon ­
strated. With metric response (development rate and weight), the t-test is a suitable statistical method if data meet the requirements of this test (nor ­
mality, homogeneous variances). The unequal-variance t-test or a non para ­
metric test, such as the Wilcoxon-Mann-Whithey test may be used, if these 
requirements are not fulfilled. With the emergence ratio, the Fisher exact test is appropriate.
 
PROCEDURE  
Conditions of exposure  
Preparation of spiked water-sediment system  
22. Appropriate amounts of formulated sediment (see paragraphs 13-14 and 
Appendix 3) are added in the test vessels to form a layer of at least 
1,5 cm. Water is added to a depth of 6 cm (see paragraph 15). The ratio of the depth of the sediment layer and the depth of the water should not exceed 1:4 and the sediment layer should not be deeper than 3 cm. The 
sediment-water system should be left under gentle aeration for seven days 
prior to addition of test organisms (see paragraph 14 and Appendix 3). To avoid separation of sediment ingredients and re-suspension of fine material during addition of test water in the water column, the sediment can be 
covered with a plastic disc while water is poured onto it, and the disc is 
removed immediately afterwards. Other devices may also be appropriate.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1260
 
23. The test vessels should be covered (e.g. by glass plates). If necessary, during 
the study the water levels will be topped to the original volume in order to 
compensate for water evaporation. This should be performed using distilled or deionised water to prevent build-up of salts.
 
Addition of test organisms  
24. Four to five days before adding the test organisms to the test vessels, egg 
masses should be taken from the cultures and placed in small vessels in 
culture medium. Aged medium from the stock culture or freshly prepared medium may be used. If the latter is used, a small amount of food e.g. green algae and/or a few droplets of filtrate from a finely ground suspension of 
flaked fish food should be added to the culture medium (see Appendix 2). 
Only freshly laid egg masses should be used. Normally, the larvae begin to hatch a couple of days after the eggs are laid (2 to 3 days for  Chironomus 
riparius  at 20 °C and 1 to 4 days for  Chironomus tentans  at 23 °C and  
Chironomus yoshimatui  at 25 °C) and larval growth occurs in four instars, 
each of 4-8 days duration. First instar larvae (2-3 or 1-4 days post hatching) should be used in the test. The instar of midges can possibly be checked 
using head capsule width (6). 
25. Twenty first instar larvae are allocated randomly to each test vessel 
containing the spiked sediment and water, using a blunt pipette. Aeration 
of the water has to be stopped while adding the larvae to test vessels and remain so for another 24 hours after addition of larvae (see paragraphs 24 and 32). According to the test design used (see paragraphs 19 and 20), the 
number of larvae used per concentration is at least 60 for the EC 
point estimation and 80 for determination of NOEC. 
26. Twenty-four hours after adding the larvae, the test substance is spiked into 
the overlying water column, and slight aeration is again supplied. Small volumes of test substance solutions are applied below the surface of the water using a pipette. The overlying water should then be mixed with care 
not to disturb the sediment.
 
Test concentrations  
27. A range-finding test may be helpful to determine the range of concentrations 
for the definitive test. For this purpose a series of widely spaced concen ­
trations of the test substance are used. In order to provide the same density 
of surface per chironomids, which is to be used for the definitive test, chironomids are exposed to each concentration of the test substance for a period which allows estimation of appropriate test concentrations, and no 
replicates are required. 
28. The test concentrations for the definitive test are decided based on the result 
of the range-finding test. At least five concentrations should be used and 
selected as described in paragraphs 18 to 20.
 
Controls  
29. Control vessels without any test substance but including sediment should be 
included in the test with the appropriate number of replicates (see para ­
graphs 19-20). If a solvent has been used for application of test substance 
(see paragraph 16), a sediment solvent control should be added.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1261
 
Test system  
30. Static systems are used. Semi-static or flow-through systems with inter ­
mittent or continuous renewal of overlying water might be used in excep ­
tional cases as for instance if water quality specifications become inappro ­
priate for the test organism or affect chemical equilibrium (e.g. dissolved 
oxygen levels fall too low, the concentration of excretory products rises too high or minerals leach from sediment and affect pH and/or water hardness). However, other methods for ameliorating the quality of overlying water, 
such as aeration, will normally suffice and be preferable.
 
Food  
31. It is necessary to feed the larvae, preferably daily or at least three times per 
week. Fish-food (a suspension in water or finely ground food, e.g. TetraMin or TetraPhyll; see details in Appendix 2) in the amount of 0,25-0,5 mg 
(0,35-0,5 mg for  C. yoshimatui ) per larvae per day seems adequate for 
young larvae for the first 10 days. Slightly more food may be necessary for older larvae: 0,5-1 mg per larvae per day should be sufficient for the rest of the test. The food ration should be reduced in all treatments and control if 
fungal growth is seen or if mortality is observed in controls. If fungal 
development cannot be stopped the test is to be repeated. When testing strongly adsorbing substances (e.g. with log K
 ow > 5), or substances 
covalently binding to sediment, the amount of food necessary to ensure 
survival and natural growth of the organisms may be added to the 
formulated sediment before the stabilisation period. For this, plant material must be used instead of fish food, e.g. addition of 0,5 % (dry weight) finely ground leaves of e.g. stinging nettle ( Urtica dioica ), 
mulberry ( Morus alba ), white clover ( Trifolium repens ), spinach ( Spinacia 
oleracea ) or of other plant material ( Cerophyl  or alpha-cellulose) may be 
used.
 
Incubation conditions  
32. Gentle aeration of the overlying water in test vessels is supplied preferably 
24 hours after addition of the larvae and is pursued throughout the test (care 
should be taken that dissolved oxygen concentration does not fall below 
60 %of ASV). Aeration is provided through a glass Pasteur pipette fixed 2- 3 cm above the sediment layer (i.e. one or few bubbles/sec). When testing volatile chemicals, consideration may be given not to aerate the 
sediment-water system. 
33. The test is conducted at a constant temperature of 20 °C (± 2 °C). For  C. 
tentans  and  C. yoshimatui , recommended temperatures are of 23 °C and 
25 °C (± 2 °C), respectively. A 16 hours photoperiod is used and the 
light intensity should be 500 to 1 000 lux.
 
Exposure duration  
34. The exposure commences with the addition of larvae to the spiked and 
control vessels. The maximum exposure duration is 28 days for  C. 
riparius  and  C. yoshimatsui , and 65 days for  C. tentans . If midges 
emerge earlier, the test can be terminated after a minimum of five days 
after emergence of the last adult in the control.  
OBSERVATIONS  
Emergence  
35. The development time and the total number of fully emerged male and 
female midges are determined. Males are easily identified by their 
plumose antennae.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1262
 
36. The test vessels should be observed at least three times per week to make 
visual assessment of any abnormal behaviour (e.g. leaving sediment, 
unusual swimming), compared with the control. During the period of expected emergence a daily count of emerged midges is necessary. The sex and number of fully emerged midges are recorded daily. After identifi ­
cation the midges are removed from the vessels. Any egg masses deposited 
prior to the termination of the test should be recorded and then removed to prevent re-introduction of larvae into the sediment. The number of visible pupae that have failed to emerge is also recorded. Guidance on measurement 
of emergence is provided in Appendix 5.
 
Growth and survival  
37. If data on 10-day larval survival and growth are to be provided, additional 
test vessels should be included at the start, so that they may be used subsequently. The sediment from these additional vessels is sieved using a 250 μm sieve to retain the larvae. Criteria for death are immobility or lack 
of reaction to a mechanical stimulus. Larvae not recovered should also be 
counted as dead (larvae which have died at beginning of the test may have been degraded by microbes). The (ash free) dry weight of the surviving larvae per test vessel is determined and the mean individual dry weight 
per vessel calculated. It is useful to determine which instar the surviving 
larvae belong to; for that measurement of the width of the head capsule of each individual can be used.
 
Analytical measurements  
Concentration of the test substance  
38. As a minimum, samples of the overlying water, the pore water and the 
sediment must be analysed at the start (preferably one hour after application of test substance) and at the end of the test, at the highest concentration and 
a lower one. These determinations of test substance concentration inform on 
the behaviour/partitioning of the test substance in the water-sediment system. Sampling of sediment at the start of the test may influence the test system (e.g. removing test larvae), thus additional test vessels should 
be used to perform analytical determinations at the start and during the test 
if appropriate (see paragraph 39). Measurements in sediment might not be necessary if the partitioning of the test substance between water and sediment has been clearly determined in a water/sediment study under 
comparable conditions (e.g. sediment to water ratio, type of application, 
organic carbon content of sediment). 
39. When intermediate measurements are made (e.g. at day 7) and if the 
analysis needs large samples which cannot be taken from test vessels 
without influencing the test system, analytical determinations should be performed on samples from additional test vessels treated in the same way (including the presence of test organisms) but not used for biological 
observations. 
40. Centrifugation at e.g. 10 000 g and 4 °C for 30 min. is the recommended 
procedure to isolate interstitial water. However, if the test substance is demonstrated not to adsorb to filters, filtration may also be acceptable. In 
some cases it might not be possible to analyse concentrations in the pore 
water as the sample size is too small.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1263
 
Physical-chemical parameters  
41. The pH, dissolved oxygen in the test water and temperature of the test 
vessels should be measured in an appropriate manner (see paragraph 10). Hardness and ammonia should be measured in the controls and one test 
vessel at the highest concentration at the start and the end of the test.
 
DATA AND REPORTING  
Treatment of results  
42. The purpose of this test is to determine the effect of the test substance on 
the development rate and the total number of fully emerged male and female 
midges, or in the case of the 10-day test effects on survival and weight of the larvae. If there are no indications of statistically different sensitivities of sexes, male and female results may be pooled for statistical analyses. The 
sensitivity differences between sexes can be statistically judged by e.g. a χ
 2 -r 
× 2 table test. Larval survival and mean individual dry weight per vessel must be determined after 10 days where required. 
43. Effect concentrations expressed as concentrations in the overlaying water, 
are calculated preferably based on measured concentrations at the beginning 
of the test (see paragraph 38). 
44. To compute a point estimate for the EC
 50 or any other EC  x , the per-vessel 
statistics may be used as true replicates. In calculating a confidence interval 
for any EC  x the variability among vessels should be taken into account, or it 
should be shown that this variability is so small that it can be ignored. When the model is fitted by Least Squares, a transformation should be 
applied to the per-vessel statistics in order to improve the homogeneity of 
variance. However, EC
 x values should be calculated after the response is 
transformed back to the original value. 
45. When the statistical analysis aims at determining the NOEC/LOEC by 
hypothesis testing, the variability among vessels needs to be taken into 
account, e.g. by a nested ANOVA. Alternatively, more robust tests (21) can be appropriate in situations where there are violations of the usual ANOVA assumptions.
 
Emergence ratio  
46. Emergence ratios are quantal data, and can be analyzed by the 
Cochran-Armitage test applied in step-down manner where a monotonic 
dose-response is expected and these data are consistent with this expec ­
tation. If not, a Fisher’s exact or Mantel-Haenszal test with Bonferroni-Holm adjusted p-values can be used. If there is evidence of 
greater variability between replicates within the same concentration than a 
binomial distribution would indicate (often referenced as ‘extra-binomial’ variation), then a robust Cochran-Armitage or Fisher exact test such as proposed in (21), should be used. 
47. The sum of midges emerged per vessel, ne, is determined and divided by 
the number of larvae introduced, n
 a : 
ER ¼ n e 
n a ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1264
 
where:  
ER = emergence ratio  
n e = number of midges emerged per vessel  
n a = number of larvae introduced per vessel 
48. An alternative that is most appropriate for large sample sizes, when there is 
extra binomial variance, is to treat the emergence ratio as a continuous 
response and use procedures such as William’s test when a monotonic dose-response is expected and is consistent with these ER data. Dunnett’s test would be appropriate where monotonicity does not hold. A large sample 
size is defined here as the number emerged and the number not emerging 
both exceeding five, on a per replicate (vessel) basis. 
49. To apply ANOVA methods values of ER should first be transformed by the 
arcsin square roottransformation or Freeman-Tukey transformation to obtain 
an approximate normal distribution and to equalise variances. The 
Cochran-Armitage, Fisher’s exact (Bonferroni), or Mantel-Haenszel tests can be applied when using the absolute frequencies. The arcsin square root transformation is applied by taking the inverse sine (sine
 –1 ) of the 
square root of ER. 
50. For emergence ratios, EC  x -values are calculated using regression analysis 
(or e.g. probit (22), logit, Weibull, appropriate commercial software etc.). If regression analysis fails (e.g. when there are less than two partial responses), other non-parametric methods such as moving average or simple inter ­
polation are used.
 
Development rate  
51. The mean development time represents the mean time span between the 
introduction of larvae (day 0 of the test) and the emergence of the experi ­
mental cohort of midges. (For the calculation of the true development time, the age of larvae at the time of introduction should be considered). The development rate is the reciprocal of the development time (unit: 1/day) and 
represents that portion of larval development which takes place per day. The 
development rate is preferred for the evaluation of these sediment toxicity studies as its variance is lower, and it is more homogeneous and closer to normal distribution as compared to development time. Hence, powerful 
parametric test procedures may be used with development rate rather than 
with development time. For development rate as a continuous response, EC
 x -values can be estimated by using regression analysis (e.g. (23)(24)). 
52. For the following statistical tests, the number of midges observed on 
inspection day x are assumed to be emerged at the mean of the time 
interval between day x and day x – l (l = length of the inspection interval, usually 1 day). The mean development rate per vessel ( x) is 
calculated according to:
 
x ¼ X  m 
i¼1 ƒ i x i 
n e 
where:  
x: mean development rate per vessel 
i: index of inspection interval m: maximum number of inspection intervals
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1265
 
ƒ i : number of midges emerged in the inspection interval i  
n e : total number of midges emerged at the end of experiment (=  P ƒ i ) 
x i : development rate of the midges emerged in interval i 
x i ¼ 1= Í 
day  i Ä l i 
2 Î 
where: 
day  i : inspection day (days since application) 
l i : length of inspection interval i (days, usually 1 day)  
Test report  
53. The test report must at least provide the following information: 
Test substance:  
— physical nature and, where relevant, physical-chemical properties (water 
solubility, vapour pressure, partition coefficient in soil (or in sediment if available), stability in water, etc.); 
— chemical identification data (common name, chemical name, structural 
formula, CAS number, etc.) including purity and analytical method for 
quantification of test substance.  
Test species:  
— test animals used: species, scientific name, source of organisms and 
breeding conditions; 
— information on handling of egg masses and larvae; — age of test animals when inserted into test vessels.  
Test conditions:  
— sediment used, i.e. natural or formulated sediment; 
— for natural sediment, location and description of sediment sampling site, 
including, if possible, contamination history; characteristics: pH, organic carbon content, C/N ratio and granulometry (if appropriate). 
— preparation of the formulated sediment: ingredients and characteristics 
(organic carbon content, pH, moisture, etc. at the start of the test); 
— preparation of the test water (if reconstituted water is used) and char ­
acteristics (oxygen concentration, pH, conductivity, hardness, etc. at the 
start of the test); 
— depth of sediment and overlying water; — volume of overlying and pore water; weight of wet sediment with and 
without pore water; 
— test vessels (material and size); 
— method of preparation of stock solutions and test concentrations;
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1266
 
— application of test substance: test concentrations used, number of 
replicates and use of solvent if any; 
— incubation conditions: temperature, light cycle and intensity, aeration 
(frequency and intensity); 
— detailed information on feeding including type of food, preparation, 
amount and feeding regime.  
Results:  
— the nominal test concentrations, the measured test concentrations and the 
results of all analyses to determine the concentration of the test substance in the test vessel; 
— water quality within the test vessels, i.e. pH, temperature, dissolved 
oxygen, hardness and ammonia; 
— replacement of evaporated test water, if any; 
— number of emerged male and female midges per vessel and per day; 
— number of larvae which failed to emerge as midges per vessel; — mean individual dry weight of larvae per vessel, and per instar, if 
appropriate; 
— percent emergence per replicate and test concentration (male and female 
midges pooled); 
— mean development rate of fully emerged midges per replicate and 
treatment rate (male and female midges pooled); 
— estimates of toxic endpoints e.g. EC
 x (and associated confidence inter ­
vals), NOEC and/or LOEC, and the statistical methods used for their 
determination; 
— discussion of the results, including any influence on the outcome of the 
test resulting from deviations from this Test Method.  
LITERATURE:  
(1) BBA (1995). Long-term toxicity test with  Chironomus riparius : Devel­
opment and validation of a new test system. Edited by M. Streloke and H. Köpp. Berlin 1995. 
(2) Fleming R  et al.  (1994). Sediment Toxicity Tests for Poorly Water-Soluble 
Substances. Final Report to them European Commission. Report No: EC 
3738. August 1994. WRc, UK. 
(3) SETAC (1993). Guidance Document on Sediment toxicity Tests and 
Bioassays for Freshwater and Marine Environments. From the WOSTA 
Workshop held in the Netherlands. 
(4) ASTM International/E1706-00 (2002). Test Method for Measuring the 
Toxicity of Sediment-Associated Contaminants with Freshwater Invert ­
ebrates. pp 1125-1241. In ASTM International 2002 Annual Book of Stan ­
dards. Volume 11.05. Biological Effects and Environmental Fate; Biotech ­
nology; Pesticides. ASTM International, West Conshohocken, PA. 
(5) Environment Canada (1997). Test for Growth and Survival in Sediment 
using Larvae of Freshwater Midges ( Chironomus tentans  or Chironomus 
riparius ). Biological Test Method. Report SPE 1/RM/32. December 1997. 
(6) US-EPA (2000). Methods for Measuring the Toxicity and Bioaccumulation 
of Sediment-associated Contaminants with Freshwater Invertebrates. 
Second edition. EPA 600/R-99/064. March 2000. Revision to the first 
edition dated June 1994.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1267
 
(7) US-EPA/OPPTS 850.1735. (1996): Whole Sediment Acute Toxicity Invert ­
ebrates. 
(8) US-EPA/OPPTS 850.1790. (1996): Chironomid Sediment toxicity Test. (9) Milani D, Day KE, McLeay DJ, Kirby RS (1996). Recent intra- and 
inter-laboratory studies related to the development and standardisation of Environment Canada’s biological test methods for measuring sediment toxicity using freshwater amphipods  (Hyalella azteca)  and midge larvae  
(Chironomus riparius).  Technical Report. Environment Canada. National 
Water Research Institute. Burlington, Ontario, Canada. 
(10) Sugaya Y (1997). Intra-specific variations of the susceptibility of insec ­
ticides  in Chironomus yoshimatsui . Jp. J. Sanit. Zool. 48 (4): 345-350. 
(11) Kawai K (1986). Fundamental studies on Chironomid allergy. I. Culture 
methods of some Japanese Chironomids (Chironomidae, Diptera). Jp. J. 
Sanit. Zool. 37(1): 47-57. 
(12) OECD (2000). Guidance Document on Aquatic Toxicity Testing of 
Difficult Substances and Mixtures. OECD Environment, Health and 
Safety Publications, Series on Testing and Assessment No 23. 
(13) Environment Canada (1995). Guidance Document on Measurement of 
Toxicity Test Precision Using Control Sediments Spiked with a Reference Toxicant. Report EPS 1/RM/30. September 1995. 
(14) Chapter C.8 of this Annex, Toxicity for Earthworms, (15) Suedel BC and Rodgers JH (1994). Development of formulated reference 
sediments for freshwater and estuarine sediment testing. Environ. Toxicol. 
Chem. 13: 1163-1175. 
(16) Naylor C and Rodrigues C (1995). Development of a test method for 
Chironomus riparius using a formulated sediment. Chemosphere 31: 3291-3303. 
(17) Dunnett CW (1964). A multiple comparisons procedure for comparing 
several treatments with a control. J. Amer. Statis. Assoc. 50: 1096-1121. 
(18) Dunnett CW (1964). New tables for multiple comparisons with a control. 
Biometrics 20: 482-491. 
(19) Williams DA (1971). A test for differences between treatment means when 
several dose levels are compared with a zero dose control. Biometrics 27: 
103-117. 
(20) Williams DA (1972). The comparison of several dose levels with a zero 
dose control. Biometrics 28: 510-531. 
(21) Rao JNK and Scott AJ (1992). A simple method for the analysis of 
clustered binary data. Biometrics 48: 577-585. 
(22) Christensen ER (1984). Dose-response functions in aquatic toxicity testing 
and the Weibull model. Water Research 18: 213-221. 
(23) Bruce and Versteeg (1992). A statistical procedure for modelling 
continuous toxicity data. Environmental Toxicology and Chemistry 11:1485-1494. 
(24) Slob W (2002). Dose-response modelling of continuous endpoints. Toxicol. 
Sci. 66: 298-312.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1268
 
Appendix 1  
DEFINITIONS 
For the purpose of this method the following definitions are used:  
Formulated sediment  or reconstituted, artificial or synthetic sediment, is a 
mixture of materials used to mimic the physical components of a natural sediment.  
Overlying water  is the water placed over sediment in the test vessel.  
Interstitial water  or pore water is the water occupying space between sediment 
and soil particles.  
Spiked water  is the test water to which test substance has been added.  
Test chemical : Any substance or mixture tested using this Test Method.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1269
 
Appendix 2  
Recommendations for culture of  Chironomus riparius  
1. Chironomus  larvae may be reared in crystallising dishes or larger 
containers. Fine quartz sand is spread in a thin layer of about 5 to 10 mm deep over the bottom of the container. Kieselguhr (e.g. Merck, Art 8117) has also been shown to be a suitable substrate (a thinner layer 
of up to a very few mm is sufficient). Suitable water is then added to a 
depth of several cm. Water levels should be topped up as necessary to replace evaporative loss, and prevent desiccation. Water can be replaced if necessary. Gentle aeration should be provided. The larval rearing vessels 
should be held in a suitable cage which will prevent escape of the 
emerging adults. The cage should be sufficiently large to allow swarming of emerged adults, otherwise copulation may not occur (minimum is ca. 30 × 30 × 30 cm). 
2. Cages should be held at room temperature or in a constant environment 
room at 20 ± 2 °C with a photo period of 16 hour light (intensity ca. 1 000 
lux), 8 hours dark. It has been reported that air humidity of less than 60 % RH can impede reproduction.
 
Dilution water  
3. Any suitable natural or synthetic water may be used. Well water, dech ­
lorinated tap water and artificial media (e.g. Elendt ‘M4’ or ‘M7’ medium, see below) are commonly used. The water has to be aerated before use. If 
necessary, the culture water may be renewed by pouring or siphoning the 
used water from culture vessels carefully without destroying the tubes of larvae.
 
Feeding larvae  
4. Chironomus  larvae should be fed with a fish flake food (TetraMin  ® , Tetra­
Phyll  ® or other similar brand of proprietary fish food), at approximately 
250 mg per vessel per day. This can be given as a dry ground powder or as 
a suspension in water: 1,0 g of flake food is added to 20 ml of dilution 
water and blended to give a homogenous mix. This preparation may be fed at a rate of about 5 ml per vessel per day (shake before use.) Older larvae may receive more. 
5. Feeding is adjusted according to the water quality. If the culture medium 
becomes ‘cloudy’, the feeding should be reduced. Food additions must be 
carefully monitored. Too little food will cause emigration of the larvae towards the water column, and too much food will cause increased 
microbial activity and reduced oxygen concentrations. Both conditions 
can result in reduced growth rates. 
6. Some green algae (e.g.  Scenedesmus subspicatus , Chlorella vulgaris ) cells 
may also be added when new culture vessels are set up.
 
Feeding emerged adults  
7. Some experimenters have suggested that a cotton wool pad soaked in a 
saturated sucrose solution may serve as a food for emerged adults.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1270
 
Emergence  
8. At 20 ± 2 °C adults will begin to emerge from the larval rearing vessels 
after approximately 13-15 days. Males are easily distinguished by having plumose antennae.
 
Egg masses  
9. Once adults are present within the breeding cage, all larval rearing vessels 
should be checked three times weekly for deposition of the gelatinous egg masses. If present, the egg masses should be carefully removed. They 
should be transferred to a small dish containing a sample of the breeding 
water. Egg masses are used to start a new culture vessel (e.g. 2-4 egg masses/vessel) or are used for toxicity tests. 
10. First instar larvae should hatch after 2-3 days.
 
Set-up of new culture vessels  
11. Once cultures are established it should be possible to set up a fresh larval 
culture vessel weekly or less frequently depending on testing requirements, 
removing the older vessels after adult midges have emerged. Using this 
system a regular supply of adults will be produced with a minimum of management.
 
Preparation of test solutions ‘M4’ and ‘M7’  
12. Elendt (1990) has described the ‘M4’ medium. The ‘M7’ medium is 
prepared as the ‘M4’ medium except for the substances indicated in Table 1, for which concentrations are four times lower in ‘M7’ than in ‘M4’. A publication on the ‘M7’ medium is in preparation (Elendt, 
personal communication). The test solution should not be prepared 
according to Elendt and Bias (1990) for the concentrations of NaSiO
 3 5 
H 2 O, NaNO  3 , KH  2 PO 4 and K  2 HPO  4 given for the preparation of the stock 
solutions are not adequate.  
Preparation of the ‘M7’-medium  
13. Each stock solution (I) is prepared individually and a combined stock 
solution (II) is prepared from these stock solutions (I) (see Table 1). 50 ml from the combined stock Solution (II) and the amounts of each macro 
nutrient stock solution which are given in Table 2 are made up to 1 l of 
deionised water to prepare the ‘M7’ medium. A vitamin stock solution is prepared by adding three vitamins to deionised water as indicated in Table 3, and 0,1 ml of the combined vitamin stock solution are added to the final 
‘M7’ medium shortly before use. (The vitamin stock solution is stored 
frozen in small aliquots). The medium is aerated and stabilised.
 
Table 1  
Stock solutions of trace elements for medium M4 and M7  
Stock solutions (I) Amount (mg) 
made up to 1 litre 
of deionised 
water To prepare the combined stock 
solution (II): mix the following 
amounts (ml) of stock solutions (I) 
and make up to 1 litre of deionised 
water Final concentrations in test solutions 
(mg/l) 
M4 M7 M4 M7  
H 3 BO 3 ( 1 ) 57 190 1,0 0,25 2,86 0,715 
MnCl  2 · 4 H  2 O (  1 ) 7 210 1,0 0,25 0,361 0,090 
LiCl (  1 ) 6 120 1,0 0,25 0,306 0,077  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1271
 
Stock solutions (I) Amount (mg) 
made up to 1 litre 
of deionised 
water To prepare the combined stock 
solution (II): mix the following 
amounts (ml) of stock solutions (I) 
and make up to 1 litre of deionised 
water Final concentrations in test solutions 
(mg/l) 
M4 M7 M4 M7  
RbCl (  1 ) 1 420 1,0 0,25 0,071 0,018 
SrCl  2 · 6 H  2 O (  1 ) 3 040 1,0 0,25 0,152 0,038 
NaBr (  1 ) 320 1,0 0,25 0,016 0,004 
Na 2 MoO  4 · 2 H  2 O (  1 ) 1 260 1,0 0,25 0,063 0,016 
CuCl  2 · 2 H  2 O (  1 ) 335 1,0 0,25 0,017 0,004 
ZnCl  2 260 1,0 1,0 0,013 0,013 
CaCl  2 · 6 H  2 O 200 1,0 1,0 0,010 0,010 
KI 65 1,0 1,0 0,0033 0,0033 
Na 2 SeO  3 43,8 1,0 1,0 0,0022 0,0022 
NH  4 VO  3 11,5 1,0 1,0 0,00058 0,00058 
Na2EDTA · 2 H  2 O (  1 ) ( 2 ) 5 000 20,0 5,0 2,5 0,625 
FeSO4 · 7 H  2 O (  1 ) ( 2 ) 1 991 20,0 5,0 1,0 0,249  
( 1 ) These substances differ in M4 and M7, as indicated above. 
( 2 ) These solutions are prepared individually, then poured together and autoclaved immediately.  
Table 2  
Macro nutrient stock solutions for medium M4 and M7  
Amount made up to 1 
litre of deionised water 
(mg) Amount of macro nutrient 
stock solutions added to 
prepare medium M4 and 
M7 
(ml/l) Final concentrations in 
test solutions M4 and 
M7 
(mg/l)  
CaCl  2 · 2 H  2 O 293 800 1,0 293,8 
MgSO  4 · 7 H  2 O 246 600 0,5 123,3 
KCl 58 000 0,1 5,8 NaHCO
 3 64 800 1,0 64,8 
NaSiO  3 · 9 H  2 O 50 000 0,2 10,0 
NaNO  3 2 740 0,1 0,274 
KH  2 PO 4 1 430 0,1 0,143 
K 2 HPO  4 1 840 0,1 0,184  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1272
 
Table 3  
Vitamin stock solution for medium M4 and M7  
All three vitamin solutions are combined to make a single vitamin stock solution.  
Amount made up to 1 
litre of deionised water 
(mg) Amount of vitamin stock 
solution added to prepare 
medium M4 and M7 
(ml/l) Final concentrations in 
test solutions M4 and 
M7 
(mg/l)  
Thiamine hydrochloride 750 0,1 0,075 
Cyanocobalamin (B12) 10 0,1 0,0010 
Biotine 7,5 0,1 0,00075  
LITERATURE:  
BBA (1995). Long-term toxicity test with Chironomus riparius: Development and 
validation of a new test system. Edited by M. Streloke and H.Köpp. Berlin 1995. 
Elendt BP (1990). Selenium Deficiency in Crustacean.  Protoplasma  154: 25-33. 
Elendt BP and Bias W-R (1990). Trace Nutrient Deficiency in  Daphnia magna  
Cultured in Standard Medium for Toxicity Testing. Effects on the Optimization 
of Culture Conditions on Life History Parameters of  D. magna . Water Research  
24 (9): 1157-1167.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1273
 
Appendix 3  
PREPARATION OF FORMULATED SEDIMENT  
Sediment composition  
The composition of the formulated sediment should be as follows:  
Constituent Characteristics % of sediment 
dry weight  
Peat Sphagnum moss peat, as 
close to pH 5,5-6,0 as 
possible, no visible plant 
remains, finely ground (particle size ≤ 1 mm) and 
air dried 4-5 
Quartz sand Grain size: > 50 % of the 
particles should be in the 
range of 50-200 μm 75-76 
Kaolinite clay Kaolinite content ≥ 30 % 20 
Organic carbon Adjusted by addition of peat 
and sand 2 (± 0,5) 
Calcium carbonate CaCO
 3 , pulverised, 
chemically pure 0,05-0,1 
Water Conductivity ≤ 10 μS/cm 30-50  
Preparation  
The peat is air dried and ground to a fine powder. A suspension of the required 
amount of peat powder in deionised water is prepared using a high-performance 
homogenising device. The pH of this suspension is adjusted to 5,5 ± 0,5 with CaCO
 3 . The suspension is conditioned for at least two days with gentle stirring at 
20 ± 2 °C, to stabilise pH and establish a stable microbial component. pH is 
measured again and should be 6,0 ± 0,5. Then the peat suspension is mixed with 
the other constituents (sand and kaolin clay) and deionised water to obtain a homogeneous sediment with a water content in a range of 30-50 per cent of dry weight of the sediment. The pH of the final mixture is measured once again and 
is adjusted to 6,5 to 7,5 with CaCO
 3 if necessary. Samples of the sediment are 
taken to determine the dry weight and the organic carbon content. Then, before it is used in the chironomid toxicity test, it is recommended that the formulated sediment be conditioned for seven days under the same conditions which prevail 
in the subsequent test.
 
Storage  
The dry constituents for preparation of the artificial sediment may be stored in a 
dry and cool place at room temperature. The formulated (wet) sediment should 
not be stored prior to its use in the test. It should be used immediately after the 7 days conditioning period that ends its preparation.
 
LITERATURE:  
Chapter C.8 of this Annex, Toxicity for Earthworms 
Meller M, Egeler P, Rombke J, Schallnass H, Nagel R and Streit B (1998). 
Short-term Toxicity of Lindane, Hexachlorobenzene and Copper Sulfate on 
Tubificid Sludgeworms (Oligochaeta) in Artificial Media. Ecotox. and Environ. 
Safety 39: 10-20.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1274
Appendix 4  
Chemical Characteristics of Acceptable Dilution Water  
Substance Concentrations  
Particulate matter < 20 mg/l 
Total organic carbon < 2 mg/l Unionised ammonia < 1 μg/l 
Hardness as CaCO
 3 < 400 mg/l (*) 
Residual chlorine < 10 μg/l 
Total organophosphorus pesticides < 50 ng/l Total organochlorine pesticides plus polychlorinated 
biphenyls < 50 ng/l 
Total organic chlorine < 25 ng/l
 
(*) However, it should be noted that if there is an interaction suspected between hardness 
ions and the test substance, lower hardness water should be used (and thus, Elendt 
Medium M4 must not be used in this situation).  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1275
 
Appendix 5  
Guidance for monitoring emergence of chironomid larvae  
Emergence traps are placed on the test beakers. These traps are needed from day 
20 to the end of the test. Example of trap used is drawn below:
  
A: the nylon screen 
B: the inverted plastic cups 
C: the lipless exposure beaker D: the water exchange screen ports 
E: water 
F: sediment  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1276
 
C.29.  READY BIODEGRADABILITY — CO  2 IN SEALED VESSELS 
(HEADSPACE TEST)  
INTRODUCTION 
1. This Test Method is equivalent to OECD Test Guideline (TG) 310 (2006). 
This Test Method is a screening method for the evaluation of ready biode ­
gradability of chemicals and provides similar information to the six test methods described in chapter C.4 of this Annex A to F. Therefore, a chemical that shows positive results in this Test Method can be considered 
readily biodegradable and consequently rapidly degradable in the 
environment. 
2. The well established carbon dioxide (CO
 2 ) method (1), based on Sturm’s 
original test (2) for assessing biodegradability of organic chemicals, by the measurement of the carbon dioxide produced by microbial action, has 
normally been the first choice for testing poorly soluble chemicals and 
those which strongly adsorb. It is also chosen for soluble (but not volatile) chemicals, since the evolution of carbon dioxide is considered by many to be the only unequivocal proof of microbial activity. Removal of 
dissolved organic carbon can be effected by physico-chemical processes — 
adsorption, volatilisation, precipitation, hydrolysis — as well as by microbial action and many non-biological reactions consume oxygen; rarely is CO
 2 produced from organic chemicals abiotically. In the original 
and modified Sturm test (1)(2) CO  2 is removed from the liquid phase to the 
absorbing vessels by sparging (i.e. bubbling air treated to remove CO  2 
through the liquid medium), while in the version of Larson (3)(4) CO  2 is 
transferred from the reaction vessel to the absorbers by passing CO  2 -free air 
through the headspace and, additionally, by shaking the test vessel continu ­
ously. Only in the Larson modification is the reaction vessel shaken; stirring is specified only for insoluble substances in ISO 9439 (5) and in the original 
US version (6), both of which specify sparging rather than headspace 
replacement. In another official US EPA method (7) based on Gledhill’s method (8), the shaken reaction vessel is closed to the atmosphere and CO
 2 
produced is collected in an internal alkaline trap directly from the gaseous 
phase, as in classical Warburg/Barcroft respirometer flasks. 
3. However, inorganic carbon (IC) has been shown to accumulate in the 
medium during the application of the standard, modified Sturm test to a number of chemicals (9). A concentration of IC as high as 8 mg/l was found during the degradation of 20 mg C/l of aniline. Thus, the collection of CO
 2 
in the alkaline traps did not give a true reflection of the amount of CO  2 
produced microbiologically at intermediate times during the degradation. As 
a result, the specification that > 60 % theoretical maximum CO  2 production 
(ThCO  2 ) must be collected within a ‘10-d window’ (the 10 days 
immediately following the attainment of 10 % biodegradation) for a test 
chemical to be classified as readily biodegraded will not be met for some chemicals which would be so classified using dissolved organic carbon (DOC) removal. 
4. When the percentage degradation is a lower value than expected, IC is 
possibly accumulated in the test solution. Then, the degradability may be 
assessed with the other ready biodegradability tests.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1277
 
5. Other drawbacks of the Sturm methodology (cumbersome, time-consuming, 
more prone to experimental error and not applicable to volatile chemicals) 
had earlier prompted a search for a sealed vessel technique, other than Gledhill’s, rather than gas flow-through (10)(11). Boatman et al (12) reviewed the earlier methods and adopted an enclosed headspace system 
in which the CO
 2 was released into the headspace at the end of incubation 
by acidifying the medium. CO  2 was measured by gas 
chromatography (GC)/IC analysis in automatically taken samples of the headspace but dissolved inorganic carbon (DIC) in the liquid phase was 
not taken into account. Also, the vessels used were very small (20 ml) 
containing only 10 ml of medium, which caused problems e.g. when adding the necessarily very small amounts of insoluble test chemicals, and/or there may be insufficient or no microorganisms present in the 
inoculated medium that are competent to degrade the test chemicals. 
6. These difficulties have been overcome by the independent studies of Struijs 
and Stoltenkamp (13) and of Birch and Fletcher (14), the latter being inspired by their experience with apparatus used in the anaerobic biodegra ­
dation test (15). In the former method (13) CO
 2 is measured in the 
headspace after acidification and equilibration, while in the latter (14) 
DIC in both the gaseous and liquid phases was measured, without treatment; over 90 % of the IC formed was present in the liquid phase. Both methods had advantages over the Sturm test in that the test system was more compact 
and manageable, volatile chemicals can be tested and the possibility of delay 
in measuring CO
 2 produced is avoided. 
7. The two approaches were combined in the ISO Headspace CO  2 Standard 
(16), which was ring-tested (17) and it is this Standard which forms the basis of the present Test Method. Similarly, the two approaches have been 
used in the US EPA method (18). Two methods of measuring CO
 2 have 
been recommended, namely CO  2 in headspace after acidification (13) and 
IC in the liquid phase after the addition of excess alkali. The latter method was introduced by Peterson during the CONCAWE ring test (19) of this 
headspace method modified to measure inherent biodegradability. The 
changes made in the 1992 (20) revision of the methods in chapter C.4 of this Annex for Ready Biodegradability have been incorporated into this Test Method, so that the conditions (medium, duration etc.) are otherwise the 
same as those in the revised Sturm test (20). Birch and Fletcher (14) have 
shown that very similar results were obtained with this headspace test as were obtained with the same chemicals in the OECD Ring Test (21) of the revised Test Methods.
 
PRINCIPLE OF THE TEST 
8. The test chemical, normally at 20 mg C/l, as the sole source of carbon and 
energy, is incubated in a buffer-mineral salts medium which has been 
inoculated with a mixed population of micro-organisms. The test is 
performed in sealed bottles with a headspace of air, which provides a reservoir of oxygen for aerobic biodegradation. The CO
 2 evolution 
resulting from the ultimate aerobic biodegradation of the test chemical is 
determined by measuring the IC produced in the test bottles in excess of that 
produced in blank vessels containing inoculated medium only. The extent of biodegradation is expressed as a percentage of the theoretical maximum IC production (ThIC), based on the quantity of test chemical (as organic 
carbon) added initially. 
9. The DOC removal and/or the extent of primary biodegradation of the test 
chemical can also be measured (20).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1278
 
INFORMATION ON THE TEST CHEMICAL 
10. The organic carbon content (% w/w) of the test chemical needs to be 
known, either from its chemical structure or by measurement, so that the percentage degradation may be calculated. For volatile test chemicals, a 
measured or calculated Henry’s law constant is helpful for determining a 
suitable headspace to liquid volume ratio. Information on the toxicity of the test chemical to micro-organisms is useful in selecting an appropriate test concentration and for interpreting results showing poor biodegradability: it is 
recommended to include the inhibition control unless it is known that the 
test chemical is not inhibitory to microbial activities (see paragraph 24).
 
APPLICABILITY OF THE METHOD 
11. The test is applicable to water-soluble and insoluble test chemicals, though 
good dispersion of the test chemical should be ensured. Using the recom ­
mended headspace to liquid volume ratio of 1:2, volatile chemicals with a 
Henry’s law constant of up to 50 Pa.m  3 .mol  –1 can be tested as the 
proportion of test chemical in the headspace will not exceed 1 % (13). A smaller headspace volume may be used when testing chemicals, which are more volatile, but their bioavailability may be limiting especially if they are 
poorly soluble in water. However, users must ensure that the headspace to 
liquid volume ratio and the test chemical concentration are such that sufficient oxygen is available to allow complete aerobic biodegradation to occur (e.g. avoid using a high substrate concentration and a small headspace 
volume). Guidance on this matter can be found in (13)(23).
 
REFERENCE CHEMICALS 
12. In order to check the test procedure, a reference chemical of known biode ­
gradability should be tested in parallel. For this purpose, aniline, sodium 
benzoate or ethylene glycol may be used when testing water-soluble test chemicals and 1-octanol for poorly soluble test chemicals (13). Biodegra ­
dation of these chemicals must reach > 60 % ThIC within 14 days.
 
REPRODUCIBILITY 
13. In the ISO ring test of the method (17), the following results were obtained 
using the recommended conditions, including 20 mg C test chemical/l.  
Test Chemical Mean Percentage 
Biodegradation 
(28d) Coefficient of 
variation 
(%) Number of 
Laboratories  
Aniline 90 16 17 
1-Octanol 85 12 14 
Within-test variability (replicability), using aniline, was low with coef ­
ficients of variability not greater than 5 % in nearly all test runs. In the 
two cases in which the replicability was worse, the greater variability was probably due to high IC production in the blanks. Replicability was worse with 1-octanol but was still less than 10 % for 79 % of test runs. This 
greater within-test variability may have been due to dosing errors, as a 
small volume (3 to 4 μl) of 1-octanol had to be injected into sealed test 
bottles. Higher coefficients of variation would result when lower concen ­
trations of test chemical are used, especially at concentrations lower than 10 
mg C/l. This could be partially overcome by reducing the concentration of 
total inorganic carbon (TIC) in the inoculum.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1279
 
14. In an EU ring-test (24) of five surfactants added at 10 mg C/l, the following 
results were obtained:  
Test Chemical Mean Percentage 
biodegradation 
(28d) Coefficient of 
variation 
(%) Number of 
laboratories  
Tetrapropylene 
Benzene sulphonate 17 45 10 
Di-iso-octylsulpho- 
Succinate 
(anionic) 72 22 9 
Hexadecyl- 
trimethyl (*) 
Ammonium chloride 
(cationic) 75 13 10 
Iso-Nonylphenol - 
(ethoxylate)  9 
(non-ionic) 41 32 10 
Coco-amide-propyl 
Dimethylhydroxy 
Sulphobetaine (amphoteric) 60 23 11
 
(*) SiO  2 was added to neutralize toxicity.  
The results show that generally, the variability was higher for the less 
well-degraded surfactants. Within-test variability was less than 15 % for 
over 90 % of cases, the highest reaching 30-40 %. 
NOTE: Most surfactants are not single molecular species but are mixtures 
of isomers, homologues, etc. which degrade after different char ­
acteristic lag periods and at different kinetic rates resulting in 
‘blurred’, extenuated curves, so that the 60 % pass value may not be reached within ‘the 10-d window’, even though each indi ­
vidual molecular species would reach > 60 % within 10 days if 
tested alone. This may be observed with other complex mixtures as 
well.
 
DESCRIPTION OF THE METHOD  
Apparatus  
15. Normal laboratory apparatus and: 
(a) Glass serum bottles, sealed with butyl rubber stoppers and crimp-on 
aluminium seals. The recommended size is ‘125 ml’ which have a 
total volume of around 160 ml (in this case the volume of each bottle should be known to be 160 ± 1 ml). A smaller size of vessel may be used when the results fulfil the conditions described in paragraph 66 
and 67; 
(b) Carbon analyser or other instrument (e.g. gas chromatograph) for 
measuring inorganic carbon;
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1280
 
(c) Syringes of high precision for gaseous and liquid samples; 
(d) Orbital shaker in a temperature-controlled environment; 
(e) A supply of CO  2 free air — this can be prepared by passing air through 
soda lime granules or by using an 80 % N  2 /20 % 0  2 gas mixture 
(optional) (see paragraph 28); 
(f) Membrane-filtration device of 0,20–0,45 μm porosity (optional); 
(g) Organic carbon analyser (optional).  
Reagents  
16. Use analytical grade reagents throughout.  
Water  
17. Distilled or de-ionised water should be used containing ≤ 1 mg/l as total 
organic carbon. This represents ≤ 5 % of the initial organic carbon content 
introduced by the recommended dose of the test chemical.  
Stock solutions for the mineral salts medium  
18. The stock solutions and the mineral salts medium are similar to those in ISO 
14593 (16) and C.4 ‘ready biodegradability’ tests (20). The use of a higher 
concentration of ammonium chloride (2,0 g/l instead of 0,5 g/l) should only be necessary in very exceptional cases, e.g. when the test chemical concen ­
tration is > 40 mg C/l. Stock solutions should be stored under refrigeration 
and disposed of after six months, or earlier if there is evidence of precipi ­
tation or microbial growth. Prepare the following stock solutions: 
(a) Potassium dihydrogen phosphate (KH
 2 PO 4 ) 8,50 g 
Dipotassium hydrogen phosphate (K  2 HPO  4 ) 21,75 g 
Disodium hydrogen phosphate dihydrate (Na  2 HPO  4 .2H  2 O) 33,40 g 
Ammonium chloride (NH  4 Cl) 0,50 g 
Dissolve in water and make up to 1 litre. The pH of this solution should 
be 7,4 (± 0,2). If this is not the case, then prepare a new solution. 
(b) Calcium chloride dihydrate (CaCl  2 .2H  2 O) 36,40 g 
Dissolve in water and make up to 1 litre. 
(c) Magnesium sulphate heptahydrate (MgSO  4 .7H  2 O) 22,50 g 
Dissolve in water and make up to 1 litre. 
(d) Iron (III) chloride hexahydrate (FeCl  3 .6H  2 0) 0,25 g 
Dissolve in water and make up to 1 litre and add one drop of concen ­
trated HCl.  
Preparation of mineral medium  
19. Mix 10 ml of solution (a) with approximately 800 ml water (paragraph 17), 
then add 1 ml of solutions (b), (c) and (d) and make up to 1 litre with water (paragraph 17).
 
Other reagents  
20. Concentrated ortho-phosphoric acid (H  3 PO 4 ) (> 85 % mass per volume).  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1281
 
Sodium hydroxide solution 7M  
21. Dissolve 280 g of sodium hydroxide (NaOH) in 1 litre of water (paragraph 
17). Determine the concentration of DIC of this solution and consider this value when calculating the test result (see paragraphs 55 and 61), especially 
in the light of the validity criterion in paragraph 66 (b). Prepare a fresh 
solution if the concentration of DIC is too high.
 
Test chemical  
22. Prepare a stock solution of a sufficiently water-soluble test chemical in 
water (paragraph 17) or in the test medium (paragraph 19) at a concentration 
preferably 100-fold greater than the final concentration to be used in the test; it may be necessary to adjust the pH of the stock solution. The stock solution should be added to the mineral medium to give a final organic 
carbon concentration of between 2 and 40 mg C/l, preferably 20 mg C/l. If 
concentrations lower than these are used, the precision obtained may be impaired. Soluble and insoluble liquid chemicals may be added to the 
vessels directly using high precision syringes. Poorly soluble and 
insoluble test chemicals may require special treatment (25). The choices are: 
(a) direct addition of known weighed amounts; 
(b) ultrasonic dispersion before addition; 
(c) dispersion with the aid of emulsifying agents to be required to establish 
whether they have any inhibitory or stimulatory effects on microbial 
activity before addition; 
(d) adsorption of liquid test chemicals, or a solution in a suitable volatile 
solvent, on to an inert medium or support (e.g. glass fibre filter), 
followed by evaporation of the solvent, if used, and direct addition of known amounts; 
(e) addition of known volume of a solution of the test chemical in an easily 
volatile solvent to an empty test vessel, followed by evaporation of the solvent. 
Agents or solvents used in (c), (d) and (e) have to be tested for any 
stimulatory or inhibitory effect on microbial activity (see paragraph 42(b).)
 
Reference chemical  
23. Prepare a stock solution of the (soluble) reference chemical in water 
(paragraph 17) at a concentration preferably 100-fold greater than the final concentration to be used (20 mg C/l) in the test.
 
Inhibition check  
24. Test chemicals frequently show no significant degradation under the 
conditions used in ready biodegradation assessments. One possible cause is that the test chemical is inhibitory to the inoculum at the concentration 
at which it is applied in the test. An inhibition check may be included in the 
test design to facilitate identification (in retrospect) of inhibition as a possible cause or contributory factor. Alternatively, the inhibition check may rule out such interferences and show that zero or slight degradation 
is attributable solely to non-amenability to microbial attack under the 
conditions of the test. In order to obtain information on the toxicity of the test chemical to (aerobic) micro-organisms, prepare a solution in the test medium containing the test chemical and the reference chemical 
(paragraph 19), each at the same concentrations as added, respectively 
(see paragraph 22 and 23).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1282
 
Inoculum  
25. The inoculum may be derived from a variety of sources: activated sludge; 
sewage effluent (non-chlorinated); surface waters and soils; or from a mixture of these (20). The biodegradative activity of the source should be 
checked by using a reference chemical. Whatever the source, 
micro-organisms previously exposed to the test chemical should not be used if the procedure is to be used as a test for ready biodegradability.
 
Warning: Activated sludge, sewage and sewage effluent contain 
pathogenic organisms and must be handled with caution. 
26. Based on experience, the optimal volume for the inoculum is that which: 
— is sufficient to give adequate biodegradative activity; 
— degrades the reference chemical by the stipulated percentage (see 
paragraph 66); 
— gives 10  2 to 10  5 colony-forming units per millilitre in the final mixture; 
— normally gives a concentration of 4 mg/l suspended solids in the final 
mixture when activated sludge is used, concentrations up to 30 mg/l may be used but may significantly increase CO
 2 production of the blanks 
(26); 
— contributes less than 10 % of the initial concentration of organic carbon 
introduced by the test chemical; 
— is generally 1-10 ml of inoculum for 1 litre of test solution.  
Activated sludge  
27. Activated sludge is freshly collected from the aeration tank of a sewage 
treatment plant or laboratory-scale unit treating predominantly domestic sewage. If necessary, coarse particles should be removed by sieving (e.g. using a 1 mm
 2 mesh sieve) and the sludge should be kept aerobic until used. 
28. Alternatively, after removal of any coarse particles, settle or centrifuge (e.g. 
1 100 × g for 10 minutes). Discard the supernatant liquid. The sludge may be washed in the mineral solution. Suspend the concentrated sludge in mineral medium to yield a concentration of 3-5 g suspended solids/l. 
Thereafter aerate until required. 
29. Sludge should be taken from a properly working conventional treatment 
plant. If sludge has to be taken from a high rate treatment plant, or is 
thought to contain inhibitors, it should be washed. Settle or centrifuge the 
re-suspended sludge after thorough mixing, discard the supernatant liquid and again suspend the washed sludge in a further volume of mineral medium. Repeat this procedure until the sludge is considered to be free 
from excess substrate or inhibitor. 
30. After complete re-suspension is achieved, or with untreated sludge, 
withdraw a sample just before use for the determination of the dry weight 
of the suspended solids. 
31. A further alternative is to homogenise activated sludge (3-5 g suspended 
solids/l). Treat the sludge in a Waring blender for 2 minutes at medium speed. Settle the blended sludge for 30 minutes or longer if required and 
decant liquid for use as inoculum at the rate of about 10 mg/l of mineral 
medium.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1283
 
32. Still further reduction of the blank CO  2 evolution can be achieved by 
aerating the sludge overnight with CO  2 -free air. Use 4 mg/l activated 
sludge solids as the concentration of the inoculum in this test (13).  
Secondary sewage effluent  
33. Alternatively, the inoculum can be derived from the secondary effluent of a 
treatment plant or laboratory-scale unit receiving predominantly domestic sewage. Maintain the sample under aerobic conditions and use on the day of collection, or pre-condition if necessary. The effluent should be filtered 
through a coarse filter to remove gross particulate matter and the pH value is 
measured. 
34. To reduce its IC content, the filtrate is sparged with CO
 2 -free air (paragraph 
15-e) for 1 h while maintaining the pH at 6,5 using orthophosphoric acid (paragraph 20). The pH value is restored to its original value with sodium 
hydroxide (paragraph 21) and after settling for about 1 h a suitable volume 
of the supernatant is taken for inoculation. This sparging procedure reduces the IC content of the inoculum. For example, when the maximum recom ­
mended volume of filtered sparged effluent (100 ml) per litre was used as 
inoculum, the amount of IC present in blank control vessels was in the 
range 0,4 to 1,3 mg/l (14), representing 2-6,5 % of test chemical C at 20 mg C/l and 4-13 % at 10 mg C/l.
 
Surface waters  
35. A sample is taken of an appropriate surface water. It should be kept under 
aerobic conditions and used on the day of collection. The sample should be concentrated, if necessary, by filtration or centrifugation. The volume of 
inoculum to be used in each test vessel should meet the criteria given in 
paragraph 26.
 
Soils  
36. A sample is taken of an appropriate soil, collected to a depth of up to 20 cm 
below the soil surface. Stones, plant remains and invertebrates should be removed from the sample of soil before it is sieved through a 2 mm mesh (if the sample is too wet to sieve immediately, then partially air dry to facilitate 
sieving). It should be kept under aerobic conditions and used on the day of 
collection (If the sample is transported in a loosely-tied black polythene bag, it can be stored at 2 to 4 °C in the bag for up to one month).
 
Preconditioning of inoculum  
37. Inoculum may be pre-conditioned to the experimental conditions, but not 
pre-adapted to the test chemical. Pre-conditioning can reduce the blank CO  2 
evolution. Pre-conditioning consists of aerating activated sludge after 
diluting in test medium to 30 mg/l with moist CO  2 -free air for up to 5-7 
days at the test temperature.  
TEST PROCEDURE  
Number of bottles  
38. The number of bottles (paragraph 15-a) needed for a test will depend on the 
frequency of analysis and the test duration. 
39. It is recommended that triplicate bottles be analysed after a sufficient 
number of time intervals such that the 10-d window may be identified. Also at least five test bottles (paragraph 15-a) from sets (a), (b) and (c) (see paragraph 42) are analysed at the end of the test, to enable 95 % 
confidence intervals to be calculated for the mean percentage biodegradation 
value.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1284
 
Inoculated medium  
40. The inoculum is used at a concentration of 4 mg/l activated sludge dry 
solids. Prepare immediately before use sufficient inoculated medium by adding, for example, 2 ml suitably treated activated sludge (paragraphs 27 
to 32) at 2 000 mg/l to 1 litre of mineral salts medium (paragraph 19). 
When secondary sewage effluent is to be used add up to 100 ml effluent (paragraph 33) to 900 ml mineral salts medium (paragraph 19) and dilute to 1 litre with medium.
 
Preparation of bottles  
41. Aliquots of inoculated medium are dispensed into replicate bottles to give a 
headspace to liquid ratio of 1:2 (e.g. add 107 ml to 160 ml-capacity bottles). Other ratios may be used, but see the warning given in paragraph 11. When using either type of inoculum, care must be taken to ensure that the 
inoculated medium is adequately mixed to ensure that it is uniformly 
distributed to the test bottles. 
42. Sets of bottles (paragraph 15a) are prepared to contain the following: 
(a) Test vessels (denoted F
 T ) containing the test chemical; 
(b) Blank controls (denoted F  B ) containing only the test medium plus 
inoculum; any chemicals, solvents, agents or glass fibre filters used to 
introduce the test chemical into the test vessels must also be added; 
(c) Vessels (denoted F  C ) for checking the procedure containing the 
reference chemical; 
(d) If needed, vessels (denoted F  I ) for checking a possible inhibitory effect 
of the test chemical containing both the test chemical and reference 
chemical at the same concentrations (paragraph 24) as in bottles F  T 
and F  C , respectively; 
(e) Vessels (denoted F  S ) for checking a possible abiotic degradation as (a) 
plus 50 mg/l HgCl  2 or sterilised by some other means (e.g. by auto ­
claving). 
43. Water-soluble test chemicals and reference chemicals are added as aqueous 
stock solutions (paragraphs 22, 23 and 24) to give a concentration of 10 to 
20 mg C/l. 
44. Insoluble test chemicals and insoluble reference chemicals are added to 
bottles in a variety of ways (see paragraph 22a-e) according to the nature 
of the test chemical, either before or after addition of the inoculated medium, depending on the method of treatment of the test chemical. If one of the procedures given in paragraph 22a-e is used, then the blank 
bottles F
 B (paragraph 42b) should be treated in a similar fashion but 
excluding the test chemical or reference chemical. 
45. Volatile test chemicals should be injected into sealed bottles (paragraph 47) 
using a micro syringe. The dose is calculated from the volume injected and 
the density of the test chemical. 
46. Water should be added to vessels, where necessary, to give the same liquid 
volume in each vessel. It must be ensured that the headspace to liquid ratio 
(usually 1:2) and concentration of the test chemical are such that sufficient 
oxygen is available in the headspace to allow for complete biodegradation.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1285
 
47. All bottles are then sealed for example, with butyl rubber septa and 
aluminium caps. Volatile tests chemicals should be added at this stage 
(paragraph 45). If the decrease in DOC concentration of the test solution is to be monitored and for time zero analyses to be performed for initial IC concentration (sterile controls, paragraph 42e) or other determinands, 
remove an appropriate sample from the test vessel. The test vessel and its 
contents are then discarded. 
48. The sealed bottles are placed on a rotary shaker (paragraph 15d), with a 
shaking rate sufficient to keep the bottle contents well mixed and in 
suspension (e.g. 150 to 200 rpm), and incubated in the dark at 20 °C, to 
be kept within ± 1 °C.
 
Sampling  
49. The pattern of sampling will depend on the lag period and kinetic rate of 
biodegradation of the test chemical. Bottles are sacrificed for analysis on the 
day of sampling, which should be at least weekly or more frequently (e.g. 
twice per week) if a complete degradation curve is required. The requisite number of replicate bottles is taken from the shaker, representing F
 T , F B and 
F C and, if used F  I and F  S (see paragraph 42). The test normally runs for 
28d. If the biodegradation curve indicates that a plateau has been attained 
before 28d, the test may be concluded earlier than 28d. Take samples from the five bottles reserved for the 28th day of the test for analysis and use the results to calculate the confidence limits or coefficient of variation of 
percentage biodegradation. Bottles representing the checks for inhibition 
and for abiotic degradation need not be sampled as frequently as the other bottles; day 1 and day 28 would be sufficient.
 
Inorganic carbon (IC) analysis  
50. CO  2 production in the bottles is determined by measuring the increase in the 
concentration of inorganic carbon (IC) during incubation. There are two 
recommended methods available for measuring the amount of IC produced in the test, and these are described immediately below. Since the methods can give slightly different results only one should be used in 
a test run. 
51. Method (a) is recommended if the medium is likely to contain remnants of, 
for example, a glass-filter paper and/or insoluble test chemical. This analysis 
can be performed using a gas chromatograph if a carbon analyser is not 
available. It is important that the bottles should be at or close to the test temperature when the headspace gas is analysed. Method (b) can be easier for laboratories using carbon analysers to measure IC. It is important that the 
sodium hydroxide solution (paragraph 21) used to convert CO
 2 to carbonate 
is either freshly prepared or its IC content is known, so that this can be taken into account when calculating the test results (see paragraph 66-b.)
 
Method (a): acidification to pH < 3  
52. Before each batch of analyses, the IC analyser is calibrated using an appro ­
priate IC standard (e.g. 1 % w/w CO  2 in N  2 ). Concentrated orthophosphoric 
acid (paragraph 20) is injected through the septum of each bottle sampled to 
lower the pH of the medium to < 3 (e.g. add 1 ml to 107 ml test medium). The bottles are placed back on the shaker. After shaking for one hour at the test temperature the bottles are removed from the shaker, aliquots (e.g. 1 ml) 
of gas are withdrawn from the headspace of each bottle and injected into the 
IC analyser. The measured IC concentrations are recorded as mg C/l.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1286
 
53. The principle of this method is that after acidification to pH < 3 and equili ­
bration at 20 °C, the equilibrium constant for the distribution of CO  2 
between the liquid and gaseous phases in the test bottles is 1,0 when 
measured as a concentration (13). This should be demonstrated for the test system at least once as follows: 
Set up bottles containing 5 and 10 mg/l as IC using a solution of anhydrous 
sodium carbonate (Na
 2 CO 3 ) in CO  2 -free water prepared by acidifying water 
to pH 6,5 with concentrated ortho-phosphoric acid (paragraph 20), sparging 
overnight with CO  2 -free air and raising the pH to neutrality with alkali. 
Ensure that the ratio of the headspace volume to the liquid volume is the same as in the tests (e.g. 1:2). Acidify and equilibrate as described in paragraph 52, and measure the IC concentrations of both the headspace 
and liquid phases. Check that the two concentrations are the same within 
experimental error. If they are not, the operator should review the procedures. This check on the distribution of IC between liquid and gaseous phases need not be made every time the test is performed; it 
could presumably be made while performing the calibration. 
54. If DOC removal is to be measured (water-soluble test chemicals only), 
samples should be taken of the liquid phase from separate (non-acidified) 
bottles, membrane-filtered and injected into the DOC analyser. These bottles can be used for other analyses as necessary, to measure primary biodegra ­
dation.
 
Method (b): conversion of CO  2 to carbonate  
55. Before each batch of analyses, the IC analyser is calibrated using an appro ­
priate standard — for example, a solution of sodium bicarbonate (NaHCO  3 ) 
in CO  2 free water (see paragraph 53) in the range 0 to 20 mg/l as IC. 
Sodium hydroxide solution (7M, paragraph 21) (e.g. 1 ml to 107 ml 
medium) is injected through the septum of each bottle sampled and the 
bottles are shaken for 1 h at the test temperature. Use the same NaOH solution on all bottles sacrificed on a particular day, but not necessarily on all sampling occasions throughout a test. If absolute blank IC values 
are required at all sampling occasions, IC determinations of the NaOH 
solution will be required each time it is used. The bottles are removed from the shaker and allowed to settle. Suitable volumes (e.g. 50 to 1 000 μl) of the liquid phase in each vessel are withdrawn by syringe. The samples 
are injected into the IC analyser and the concentrations of IC are recorded. It 
should be ensured that the analyser used is equipped properly to deal with the alkaline samples produced in this method. 
56. The principle of this method is that after the addition of alkali and shaking, 
the concentration of IC in the headspace is negligible. This should be checked for the test system at least once by using IC standards, adding 
alkali and equilibrating, and measuring the concentration of IC in both 
the headspace and liquid phases (see paragraph 53). The concentration in the headspace should approach zero. This check on the virtually complete absorption of CO
 2 need not be made every time the test is performed. 
57. If DOC removal is to be measured (water-soluble test chemicals only), 
samples should be taken of the liquid phase from separate bottles (con ­
taining no added alkali), membrane filtered and injected into the DOC 
analyser. These bottles can be used for other analyses, as necessary, to 
measure primary biodegradability.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1287
 
DATA AND REPORTING  
Calculating of results  
58. Assuming 100 % mineralisation of the test chemical to CO  2 , the ThIC in 
excess of that produced in the blank controls equals the TOC added to each test bottle at the start of the test, that is: 
ThIC ¼ TOC 
The total mass (mg) of inorganic carbon (TIC) in each bottle is: 
TIC ¼ ðmg C in the liquid þ mg C in the headspace Þ 
¼ ðV
 L Ü C L Þ þ ðV H Ü C H Þ Equation [1] 
where: 
V L = volume of liquid in the bottle (litre); 
CL = concentration of IC in the liquid (mg/l as carbon); 
VH = volume of the headspace (litre); CH = concentration of IC in the headspace (mg/l as carbon). 
The calculations of TIC for the two analytical methods used for measuring 
IC in this test are described below in paragraphs 60 and 61. Percentage biodegradation ( % D ) in each case is given by: 
% D ¼
 ðTIC  t Ä TIC  b Þ 
TOC Ü 100 Equation [2] 
where: 
TIC  t = mg TIC in test bottle at time t; 
TIC  b = mean mg TIC in blank bottles at time t; 
TOC = mg TOC added initially to the test vessel. 
The percentage biodegradation % D is calculated for the test (F  T ), reference 
(F C ) and, if included inhibition monitoring control (F  I ) bottles from the 
respective amounts of TIC produced up to each sampling time. 
59. If there has been a significant increase in the TIC content of the sterile 
controls (F  S ) over the test period, then it may be concluded that abiotic 
degradation of the test chemical has occurred and this must be taken into account in the calculation of D in Equation [2].
 
Acidification to pH < 3  
60. Since acidification to pH < 3 and equilibration results in the equalisation of 
the concentration of TIC in the liquid and gaseous phases, only the concen ­
tration of IC in the gas phase needs to be measured. Thus, from Equation [1]
 TIC ¼ ðV L þ V H Þ Ü C H ¼ V B Ü C H , where V  B = volume of the serum 
bottle.  
Conversion of CO  2 to carbonate  
61. In this method calculations are performed as in Equation [1], but the 
negligible amount of IC in the gaseous phase is ignored, that is  
V H Ü C H ¼ 0, and  TIC ¼ V L Ü C L . ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1288
 
Expression of Results  
62. A biodegradation curve is obtained by plotting percentage biodegradation, 
D, against time of incubation and if possible, the lag phase, biodegradation phase, 10-d window and plateau phase, that is the phase in which the 
maximal degradation has been reached and the biodegradation curve has 
levelled out, are indicated. If comparable results are obtained for parallel test vessels F
 T (< 20 % difference), a mean curve is plotted (see Appendix 
2, Fig.1); if not, curves are plotted for each vessel. The mean value of the 
percentage biodegradation in the plateau phase is determined or the highest 
value is assessed (e.g. when the curve decreases in the plateau phase), but it is important to assess that in the latter case the value is not an outlier. 
Indicate this maximum level of biodegradation as ‘degree of biodegradation 
of the test chemical’ in the test report. If the number of test vessels was insufficient to indicate a plateau phase, the measured data of the last day of the test are used to calculate a mean value. This last value, the mean of five 
replicates, serves to indicate the precision with which the percentage biode ­
gradation was determined. Also report the value obtained at the end of the 10-d window. 
63. In the same way, a curve for the reference chemical, F
 C , is plotted and, if 
included, for the abiotic elimination check, F  S and the inhibition control, F  I . 
64. The amounts of TIC present in the blank controls (F  B ) are recorded as are 
those in flasks F  S (abiotic check), if these vessels were included in the test. 
65. Calculate D for the F  I vessels, based on the theoretical IC yield anticipated 
from only the reference component of the mixture. If, at day 28, [(D  FC ( 1 ) – 
D FI ( 2 )/D FC ] × 100 > 25 %, it may be assumed that the test chemical 
inhibited the activity of the inoculum, and this may account for low values of D
 FT obtained under the conditions of the test. In this case the 
test could be repeated using a lower test concentration and preferably 
reducing the DIC in the inoculum and TIC formed in the blank controls, since the lower concentration will otherwise reduce the precision of the method. Alternatively, another inoculum may be used. If in flask F
 S 
(abiotic) a significant increase (> 10 %) in the amount of TIC is 
observed, abiotic degradation processes may have occurred.  
Validity of results  
66. A test is considered valid if: 
(a) the mean percentage degradation in vessels F  C containing the reference 
chemical is > 60 % by the 14th day of incubation; and 
(b) the mean amount of TIC present in the blank controls F  B at the end of 
the test is > 3 mg C/l. 
If these limits are not met, the test should be repeated with an inoculum 
from another source and/or the procedures used should be reviewed. For 
example, if high blank IC production is a problem the procedure given in paragraphs 27 to 32 should be followed.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1289  
( 1 ) The percentage degradation in Vessels F  C containing the reference substance. 
( 2 ) The percentage degradation in Vessels F  I.
 67. If the test chemical does not reach 60 % ThIC and was shown not to be 
inhibitory (paragraph 65), the test could be repeated with increased concen ­
tration of inoculum (up to 30 mg/l activated sludge and 100 ml effluent/l) or inocula from other sources, especially if degradation had been in the range 20 to 60 %.
 
Interpretation of results  
68. Biodegradation > 60 % ThIC within the 10-d window in this test demon ­
strates that the test chemical is readily biodegradable under aerobic conditions. 
69. If the pass value of 60 % ThIC is not attained, determine the pH value in 
media in bottles which have not been made acid or alkaline; a value of less 
than 6,5 could indicate that nitrification had occurred. In such a case repeat the test with a buffer solution of higher concentration.
 
Test Report  
70. Compile a table of % D for each test (F  T ), reference (F  C ) and, if included, 
inhibition control bottle (F  I ) for each day sampled. If comparable results are 
obtained for replicate bottles, plot a curve of mean % D against time. 
Record the amount of TIC in the blanks (F  B ) and in the sterile controls 
(F S ) DOC and/or other determinands, and their percentage removal. 
71. Determine the mean value of % D in the plateau phase, or use the highest 
value if the biodegradation curve decreases in the plateau phase, and report this as the ‘degree of biodegradation of the test chemical’. It is important to 
ensure that in the latter case the highest value is not an outlier. 
72. The test report must include the following information:  
Test chemical:  
— common name, chemical name, CAS number, structural formula and 
relevant physical-chemical properties; 
— purity (impurities) of test chemical.  
Test conditions:  
— reference to this Test Method; — description of the test system used (e.g. volume of the vessel, head space 
to liquid ratio, method of stirring, etc.); 
— application of test chemical and reference chemical to test system: test 
concentration used and amount of carbon dosed into each test bottle, any 
use of solvents; 
— details of the inoculum used, any pre-treatment and pre-conditioning; — incubation temperature; — validation of the principle of IC analysis; — main characteristics of the IC analyser employed (and any other 
analytical methods used); 
— number of replicates.  
Results:  
— raw data and calculated values of biodegradability in tabular form;
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1290
 
— the graph of percentage degradation against time for the test and 
reference chemicals, the lag phase, degradation phase, 10-d window 
and slope; 
— percentage removal at plateau, at end of test, and after 10-d window; 
— reasons for any rejection of the test results; — any other facts that are relevant to the procedure followed; 
— discussion of results.
 
LITERATURE:  
(1) Chapter C.4 of this Annex Determination of ‘Ready’ Biodegradability — 
CO 2 Evolution Test (Method C.4-C). 
(2) Sturm RN (1973). Biodegradability of Nonionic surfactants: screening test 
for predicting rate and ultimate biodegradation. J.A,.Oil Chem Soc. 50: 159-167. 
(3) Larson RJ (1979). Estimation of biodegradation potential of xenobiotic 
organic chemicals. Appl Env. Microbiol. 38: 1153-1161. 
(4) Larson RJ, Hansmann MA and Bookland EA (1996). Carbon dioxide 
recovery in ready biodegradability tests: mass transfer and kinetic constants, Chemosphere 33: 1195-1210. 
(5) ISO 9439 (1990; revised 1999). Water Quality — Evaluation of ultimate 
aerobic biodegradability of organic compounds in aqueous medium — Carbon dioxide evolution Test (Sturm). 
(6) US EPA (1996). Fate, Transport and Transformation Test Guideline. 
835.3110 Carbon dioxide evolution test. Office, Prevention Pesticides and Toxic Substances Washington, DC. 
(7) US EPA (1996). Fate, Transport and Transformation Test Guideline. 835. 
3100. Aerobic aquatic biodegradation. Office, Prevention Pesticides and 
Toxic Substances Washington, DC. 
(8) Gledhill WE (1975). Screening test for assessment of biodegradability: 
Linear alkyl benzene sulfonate. Appl Microbiol. 30: 922-929. 
(9) Weytjens D, Van Ginneken I and Painter HA (1994). The recovery of 
carbon dioxide in the Sturm test for ready biodegradability. Chemosphere 
28: 801-812. 
(10) Ennis DM and Kramer A (1975). A rapid microtechnique for testing biode ­
gradability of nylons and polyamides. J. Food Sci. 40: 181-185. 
(11) Ennis DM, Kramer A, Jameson CW, Mazzoccki PH and Bailey PH (1978). 
Appl. Env. Microbiol. 35: 51-53. 
(12) Boatman RJ, Cunningham SL and Ziegler DA (1986). A method for 
measuring the biodegradation of organic chemicals, Env. Toxicol. Chem. 5: 233-243. 
(13) Struijs J and Stoltenkamp J (1990). Head space determination of evolved 
carbon dioxide in a biodegradability screening test. Ecotox. Env. Safety 19: 
204-211. 
(14) Birch RR and Fletcher RJ (1991). The application of dissolved inorganic 
carbon measurements to the study of aerobic biodegradability. Chemo ­
sphere 23: 507-524. 
(15) Birch RR, Biver C, Campagna R, Gledhill WE, Pagga U, Steber J, Reust 
H, and Bontinck WJ (1989). Screening of chemicals for anaerobic biode ­
gradation. Chemosphere 19: 1527-1550.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1291
 
(16) ISO 14593, (1999) Water Quality — Evaluation of ultimate aerobic biode ­
gradability of organic compounds in an aerobic medium-method by 
analysis of inorganic carbon in sealed vessels (CO  2 headspace test). 
(17) Battersby NS (1997). The ISO headspace CO  2 biodegradation test, Chemo ­
sphere 34: 1813-1822. 
(18) US EPA (1996). Fate, Transport and Transportation. 835.3120. Sealed 
vessel carbon dioxide production test. Office, Prevention Pesticides and 
Toxic Substance, Washington, DC. 
(19) Battersby NS, Ciccognani D, Evans MR, King D, Painter HA, Peterson DR 
and Starkey M (1999). An ‘inherent’ biodegradability test for oil products: description and results of an international ring test. Chemosphere 38: 3219- 
3235. 
(20) Chapter C.4 of this Annex, Determination of ‘Ready’ Biodegradability. (21) OECD (1988). OECD Ring-test of methods for determining ready biode ­
gradability: Chairman’s report (M. Hashimoto; MITI) and final report (M. Kitano and M. Takatsuki; CITI). Paris. 
(22) Chapter C.11 of this Annex, Activated sludge respiration inhibition test. (23) Struijs J, Stoltenkamp-Wouterse MJ and Dekkers ALM (1995). A rationale 
for the appropriate amount of inoculum in ready biodegradability tests. 
Biodegradation 6: 319-327. 
(24) EU (1999). Ring-test of the ISO Headspace CO
 2 method: application to 
surfactants: Surfactant Ring Test-1, Report EU4697, Water Research 
Centre, May 1999, Medmenham, SL7 2HD, UK. 
(25) ISO 10634 (1996) Water Quality — Guidance for the preparation and 
treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1292
 
Appendix 1  
ABBREVIATIONS AND DEFINITIONS  
IC: Inorganic carbon  
ThCO  2 : Theoretical carbon dioxide (mg) is the quantity of carbon dioxide 
calculated to be produced from the known or measured carbon content of the test chemical when fully mineralised; also expressed as mg carbon dioxide 
evolved per mg test chemical.  
DOC:  Dissolved organic carbon is the organic carbon present in solution or that 
which passes through a 0,45 micrometre filter or remains in the supernatant after centrifuging at approx. 4 000 g (about 40 000 m sec
 -2 ) for 15 min.  
DIC:  Dissolved inorganic carbon  
ThIC:  Theoretical inorganic carbon  
TIC:  Total inorganic carbon  
Readily biodegradable:  An arbitrary classification of chemicals which have 
passed certain specified screening tests for ultimate biodegradability; these tests 
are so stringent that it is assumed that such chemicals will rapidly and completely biodegrade in aquatic environments under aerobic conditions.  
10-d window:  The 10 days immediately following the attainment of 10 % 
biodegradation.  
Inherent biodegradability:  A classification of chemicals for which there is 
unequivocal evidence of biodegradation (primary or ultimate) in any test of biodegradability.  
Ultimate aerobic biodegradation:  The level of degradation achieved when the 
test chemical is totally utilised by micro-organisms resulting in the production of 
carbon dioxide, water, mineral salts and new microbial cellular constituents (biomass).  
Mineralisation:  Mineralisation is the complete degradation of an organic 
chemical to CO
 2 and H  2 O under aerobic conditions, and CH  4 , CO  2 and H  2 O 
under anaerobic conditions.  
Lag phase:  The time from the start of a test until acclimatization and/or adap ­
tation of the degrading microorganisms is achieved and the biodegradation degree of a test chemical or organic matter has increased to a detectable level 
(e.g. 10 % of the maximum theoretical biodegradation, or lower, dependent on 
the accuracy of the measuring technique).  
Degradation phase:  The time from the end of the lag period to the time when 
90 % of the maximum level of degradation has been reached.  
Plateau phase:  Plateau phase is the phase in which the maximal degradation has 
been reached and the biodegradation curve has levelled out.  
Test chemical:  Any substance or mixture tested using this Test Method.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1293
 
Appendix 2  
Example of a biodegradation curve  
Figure 1  
Biodegradation of 1-octanol in the CO  2 headspace test
  
Glossary 
Biodegradation: Degradation phase: Maximum level of biodegradation: Plateau phase: 
10-d(ay) window: 
Test time (days):
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1294
 
C. 30.  BIOACCUMULATION IN TERRESTRIAL OLIGOCHAETES  
INTRODUCTION 
1. This Test Method is equivalent to OECD Test Guideline (TG) 317 (2010). 
Among the Test Methods relating to environmental fate, the Bioconcen ­
tration: Flow-through Fish Test (chapter C.13 of this Annex (49)) and the 
Bioaccumulation in Sediment-dwelling Benthic Oligochaetes (53) were published in 1996 and 2008 respectively. The extrapolation of aquatic bioac ­
cumulation data to terrestrial organisms like earthworms is difficult, if 
possible at all. Model calculations based on a test chemical’s lipophilicity, 
e.g. (14) (37), are currently used for the assessment of bioaccumulation of chemicals in soil, as e.g. in the EU Technical Guidance Document (19). The need for a compartment-specific test method has already been addressed, 
e.g. (55). Such a method is especially important for the evaluation of 
secondary poisoning in terrestrial food chains (4). Several national test methods address the issue of bioaccumulation in organisms other than fish e.g. (2) and (72). A method on the measurement of bioaccumulation from 
contaminated soils in earthworms ( Eisenia fetida , Savigny) and potworms 
has been developed by the American Society for Testing and Materials (3). An internationally accepted method for the determination of bioaccumu ­
lation in spiked soil will improve the risk assessment of chemicals in 
terrestrial ecosystems e.g. (25) (29). 
2. Soil-ingesting invertebrates are exposed to soil bound chemicals. Among 
these animals, terrestrial oligochaetes play an important role in the 
structure and function of soils (15) (20). Terrestrial oligochaetes live in 
soil and partly at the soil surface (especially the litter layer); they frequently represent the most abundant species in terms of biomass (54). By biotur ­
bation of the soil and by serving as prey these animals can have a strong 
influence on the bioavailability of chemicals to other organisms like invert ­
ebrates (e.g. predatory mites and beetles; e.g. (64)) or vertebrate (e.g. foxes and gulls) predators (18) (62). Some species of terrestrial oligochaetes currently used in ecotoxicological testing are described in Appendix 5. 
3. The ASTM Standard Guide for Conducting Laboratory Soil Toxicity or 
Bioaccumulation Tests with the Lumbricid Earthworm  Eisenia fetida  and 
the Enchytraeid Potworm  Enchytraeus albidus  (3) provides many essential 
and useful details for the performance of the present soil bioaccumulation 
Test Method. Further documents that are referred to in this Test Method are chapter C.13 of this Annex, Bioconcentration: Flow-through Fish Test (49) and OECD TG 315: Bioaccumulation in Sediment-dwelling Benthic Oligo ­
chates (53). Practical experience with soil bioaccumulation studies and 
publications from LITERATURE e.g. (1) (5) (11) (12) (28) (40) (43) (45) (57) (59) (76) (78) (79) are also major sources of information for this Test Method. 
4. This Test Method is mostly applicable to stable, neutral organic chemicals, 
which tend to adsorb to soils. Testing for bioaccumulation of soil-associating, stable metallo-organic compounds may be possible with this Test Method. It is also applicable to metals and other trace elements.
 
PRE-REQUISITE 
5. Tests for measuring the bioaccumulation of a chemical in terrestrial oligo ­
chaetes have been performed with heavy metals (see e.g. (63)) and persistent, organic chemicals having log Kow values between 3,0 and 6,0, e.g. (40). Such tests also apply to: 
— Chemicals that show a log K
 ow of more than 6,0 (super-hydrophobic 
chemicals);  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1295
 
— Chemicals which belong to a class of organic chemicals known to have 
the potential to bioaccumulate in living organisms, e.g. surface active or 
highly adsorptive chemicals; 
— Chemicals that indicate the potential for bioaccumulation from structural 
features, e.g. analogues of chemicals with known bioaccumulation potential; and 
— Metals. 
6. Information on the test chemical such as common name, chemical name 
(preferably IUPAC name), structural formula, CAS registry number, purity, 
safety precautions, proper storage conditions and analytical methods should be obtained before beginning the study. In addition, the following information should be known: 
(a) solubility in water; 
(b) octanol-water partition coefficient, K
 ow ; 
(c) soil-water partition coefficient, expressed as K  oc ; 
(d) vapour pressure; 
(e) degradability (e.g. in soil, water); 
(f) known metabolites. 
7. Radiolabelled or non-radiolabelled test chemicals can be used. However, to 
facilitate analysis it is recommended to use a radiolabelled test chemical. The decision will be made based on the detection limits or a requirement to 
measure parent test chemical and metabolites. If a radiolabelled test 
chemical is used and total radioactive residues are measured, it is important that the radiolabelled residues in both the soil and the test organisms are characterised for percentages of parent test chemical and 
labelled non-parent, e.g. in samples taken at steady state or at the end of 
the uptake phase, to allow a bioaccumulation factor (BAF) calculation for the parent test chemical and for the soil metabolites of concern (see paragraph 50). The method described here may have to be modified, e.g. 
to provide sufficient biomass, for measuring non-radiolabelled organic test 
chemical or metals. When total radioactive residues are measured (by liquid scintillation counting following extraction, combustion or tissue solubili ­
sation), the bioaccumulation factor is based on the parent test chemical 
and metabolites. The BAF calculation should preferably be based on the 
concentration of the parent test chemical in the organisms and total radio ­
active residues. Subsequently, the biota-soil accumulation factor (BSAF), normalized to the lipid content of worm and organic carbon content (OC) 
of soil should be calculated from the BAF for reasons of comparability 
between results from different bioaccumulation tests. 
8. Toxicity of the test chemical to the species used in the test should be 
known, e.g. an effect concentration (ECx) or lethal concentration (LCx) for the time of the uptake phase (e.g. (19)). The selected concentration of 
the test chemical should preferably be about 1 % of its acute asymptotic 
LC
 50 , and at least 10-fold higher than its detection limit in soil by the 
analytical method used. If available, preference should be given to toxicity values derived from long-term studies on sublethal endpoints (51) 
(52). If such data are not available, an acute toxicity test will provide useful 
information (see e.g. (23)).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1296
 
9. An appropriate analytical method of known accuracy, precision, and sensi ­
tivity for the quantification of the chemical in the test solutions, in the soil, 
and in the biological material should be available, together with details of sample preparation and storage as well as material safety data sheets. Analytical detection limits of the test item in soil and worm tissue should 
also be known. If a 14C-labelled test chemical is used, the specific radio ­
activity (i.e. Bq mol-1) and the percentage of radioactivity associated with impurities should be known. The specific radioactivity of the test chemical should be high enough to facilitate analysis, and the test concentrations used 
should not elicit toxic effects. 
10. The test can be performed with an artificial soil or with natural soils. 
Information on characteristics of the natural soil used, e.g. origin of soil 
or its constituents, pH, organic carbon content, particle size distribution (percent sand, silt, and clay), and water holding capacity (WHC), should be known before the start of the test (3) (48).
 
PRINCIPLE OF THE TEST 
11. The parameters which characterise the bioaccumulation of a test chemical 
include the bioaccumulation factor (BAF), the uptake rate constant (k  s ) and 
the elimination rate constant (k  e ). Definitions are provided in Appendix 1. 
12. The test consists of two phases: the uptake (exposure) phase and the elim ­
ination (post-exposure) phase. During the uptake phase, replicated groups of 
worms are exposed to soil, which has been spiked with the test chemical. In 
addition to the test animals, groups of control worms are held under identical conditions without the test chemical. The dry weight and lipid content of the test organisms are measured. This can be done using 
worms of the control group. Analytical background values (blank) can be 
obtained by analysing samples of the control worms and soil. For the elimination phase, the worms are transferred to a soil free of the test chemical. An elimination phase is always required unless uptake of the 
test chemical during the exposure phase has been insignificant. An elim ­
ination phase provides information on the rate at which the test chemical is excreted by the test organisms (e.g. (27)). If a steady state has not been reached during the uptake phase, the determination of the kinetic parameters 
– kinetic bioaccumulation factor BAFk, uptake and elimination rate 
constant(s) – should preferably be based on simultaneous fitting of the results of the uptake and elimination phases. The concentration of the test chemical in/on the worms is monitored throughout both phases of the test. 
13. During the uptake phase, measurements are made at sampling times up to 
14 days (enchytraeids) or 21 days (earthworms) until the steady state is 
reached (11) (12) (67). The steady state occurs when a plot of the concen ­
tration in worms against time is parallel to the time axis, and three successive concentration analyses made on samples taken at intervals of at least two days do not vary more than ± 20 % of each other based on 
statistical comparisons (e.g. analysis of variance, regression analysis). 
14. The elimination phase consists of transferring the test organisms to vessels 
containing the same substrate without the test chemical. During the elim ­
ination phase, measurements are made at sampling times during 14 days (enchytraeids) or 21 days (earthworms) unless earlier analytical deter ­
mination showed 90 % reduction of the test chemical residues in worms. 
The concentration of the test chemical in the worms at the end of the 
elimination phase is reported as non-eliminated residues. The steady state
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1297
 
bioaccumulation factor (BAFss) is calculated preferably both as the ratio of 
the concentration in worms (Ca) and in the soil (Cs) at apparent steady state, 
and as a kinetic bioaccumulation factor, BAFK, as the ratio of the rate constant of uptake from soil (ks) and the elimination rate constant (ke) (see Appendix 1 for definitions) assuming first-order kinetics (see 
Appendix 2 for calculations). If first-order kinetics is obviously not appli ­
cable, other models should be employed. 
15. The uptake rate constant, the elimination rate constant (or constants, where 
other models are involved), the kinetic bioaccumulation factor (BAFK), and where possible, the confidence limits of each of these parameters are 
calculated from computerised model equations (see Appendix 2 for guid ­
ance). The goodness of fit of any model can be determined from e.g. the correlation coefficient or the coefficient of determination (coefficients close to one indicate a good fit) or chi-squared. Also the size of the standard error 
or confidence limit around the estimated parameters may be indicative of the 
goodness of fit of the model. 
16. To reduce variability in test results for test chemicals with high lipophilicity, 
bioaccumulation factors should be expressed in relation to lipid content and organic carbon content (kg soil organic carbon (OC) kg-1 worm lipid content). This approach is based on the fact that for some chemical 
classes, there is a clear relationship between the potential for bioaccumu ­
lation and lipophilicity; this has been well established for fish (47). There is a relationship between the lipid content of fish and the bioaccumulation of 
such chemicals. For benthic organisms, similar correlations have been found 
e.g. (30) (44). Likewise for terrestrial oligochaetes this correlation has been demonstrated e.g. (5) (6) (7) (14). If sufficient worm tissue is available, the lipid content of the test animals can be determined on the same biological 
material as the one used to determine the concentration of the test chemical. 
Alternatively, control animals can be used to measure the lipid content.
 
VALIDITY OF THE TEST 
17. For a test to be valid the following criteria should be fulfilled for both 
controls and treatments: 
— At the end of the test, the overall mortality during uptake and elim ­
ination phase should not exceed 10 % (earthworms) or 20 % (enchytraeids) of the total number of the introduced worms. 
— For  Eisenia fetida and Eisenia andrei , the mean mass loss as measured 
at the end of the uptake and at the end of the elimination phase should 
not exceed 20 % compared to the initial fresh weight (f.w.) at start of 
each phase.
 
DESCRIPTION OF THE METHOD  
Test species  
18. Several species of terrestrial oligochaetes are recommended for bioaccumu ­
lation testing. The most commonly used species Eisenia fetida or Eisenia 
andrei (Lumbricidae), or Enchytraeus albidus, Enchytraeus crypticus, or 
Enchytraeus luxuriosus (Enchytraeidae)) are described in Appendix 5.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1298
 
Apparatus  
19. Care should be taken to avoid the use of materials, for all parts of the 
equipment, which can dissolve, adsorb the test chemical or leach other chemicals, and have an adverse effect on the test animals. Standard 
rectangular or cylindrical vessels, made of chemically inert material and 
of suitable capacity can be used in compliance with the loading rate, i.e. the number of test worms. Stainless steel, plastic or glass may be used for any equipment having contact with the test media. The test vessels should 
be appropriately covered to prevent escaping of the worms, while allowing 
sufficient air supply. For chemicals with high adsorption coefficients, such as synthetic pyrethroids, silanised glass may be required. In these situations 
the equipment will have to be discarded after use (49). Radiolabelled test 
items and volatile chemicals should be prevented from escaping. Traps (e.g. glass gas washing bottles) should be employed containing suitable absorbents to retain any residues evaporating from the test vessels.
 
Soil 
20. The test soil should be of a quality that will allow the survival and 
preferably the reproduction of the test organisms for the duration of the 
acclimation and test periods without them showing any abnormal 
appearance or behaviour. The worms should burrow in the soil. 
21. The artificial soil described in the chapter C.8 of this Annex (48) is recom ­
mended for use as the substrate in the tests. Preparation of the artificial soil for use in the bioaccumulation tests and recommendations for the storage of 
artificial soil are given in Appendix 4. Air-dried artificial soil may be stored 
at room temperature until use. 
22. However, natural soils from unpolluted sites may serve as test and/or culture 
soil. Natural soils should be characterised at least by origin (collection site), pH, organic carbon content, particle size distribution (percent sand, silt, and 
clay), maximum water holding capacity (WHCmax), and percent water 
content (3). Analysis of the soil or its constituents for micro-pollutants prior to use should provide useful information. If field soil from agricultural land is used, it should not have been treated with crop protection products or 
with manure from treated animals as fertilizers for at least one year and with 
organic fertilizers for at least six months prior to sampling (50). Manipu ­
lation procedures for natural soils prior to use in ecotoxicological tests with oligochaetes in the laboratory are described in (3). For natural soils the 
storage time in the laboratory should be kept as short as possible.
 
Application of the test chemical  
23. The test chemical is incorporated into the soil. The physicochemical prop ­
erties of the test chemical should be taken into consideration. A 
water-soluble test chemical should be completely dissolved in water before it be mixed with the soil. The recommended spiking procedure for poorly water-soluble test chemical involves coating of one or more of the 
(artificial) soil constituents with the test chemical. For example, the quartz 
sand, or a portion thereof, can be soaked with a solution of the test chemical in a suitable organic solvent, which is then slowly evaporated to dryness. The coated fraction can then be mixed into the wet soil. The major 
advantage of this procedure is that no solvent is introduced into the soil. 
When a natural soil is used, the test chemical may be added by spiking an
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1299
 
air-dried portion of the soil as described above for the artificial soil, or by 
stirring the test chemical into the wet soil, with subsequent evaporating step 
if a solubilising agent is used. In general, the contact of wet soil with solvents should be avoided as far as possible. The following should be considered (3): 
— If a solvent other than water is used, it should be one that is 
water-miscible and/or can be driven off (for example, evaporated), 
leaving only the test chemical on the soil. 
— If a solvent control is used, there is no need for negative control. The 
solvent control should contain the highest concentration of solvent added to the soil and should use solvent from the same batch used to make the stock solution. Toxicity and volatility of the solvent, and solubility of 
the test chemical in the chosen solvent should be the main criteria used 
for the selection of a suitable solubilising agent. 
24. For chemicals that are poorly soluble in water and in organic solvents, 2,0- 
2,5 g of finely ground quartz sand per test vessel can be mixed with the quantity of test chemical, e.g. using mortar and pestle, to obtain the desired 
test concentration. This mixture of quartz sand and test chemical is added to 
the pre-moistened soil and thoroughly mixed with an appropriate amount of de-ionised water to obtain the required moisture content. The final mixture is distributed to the test vessels. The procedure is repeated for each test 
concentration, and an appropriate control with 2,0-2,5 g of finely ground 
quartz sand per test vessel is also prepared. 
25. The concentration of the test chemical in the soil should be determined after 
spiking. The homogenous distribution of the test chemical into the soil should be verified before introducing the test organisms. The method used 
for spiking, and the reasons for choosing a specific spiking procedure should 
be reported (24). 
26. Equilibrium between the soil and the pore-water phase should ideally be 
established before adding the organisms; a time period of four days at 20 °C is recommended. For many poorly water-soluble organic chemicals the time 
required to reach a true equilibrium between adsorbed and dissolved 
fractions can be counted in days or months. Depending on the purpose of the study, for example when the environmental conditions are to be mimicked, the spiked soil may be ‘aged’ for a longer period, e.g. for 
metals three weeks at 20 °C (22).
 
Culturing of the test organisms  
27. Worms should be preferably kept in permanent laboratory culture. Guidance 
on laboratory culture methods for  Eisenia fetida  and  Eisenia andrei , and 
Enchytraeid species, is provided in Appendix 5 (see also (48) (51) (52)). 
28. The worms used in the tests should be free from observable diseases, 
abnormalities and parasites.  
PERFORMANCE OF THE TEST 
29. The test organisms are exposed to the test chemical during the uptake phase. 
The uptake phase should be of 14 days (enchytraeids) or 21 days (earth ­
worms) unless it is demonstrated that steady state has been reached.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1300
 
30. For the elimination phase, the worms are transferred to a soil free of test 
chemical. The first sample should be taken at 4-24 h after the start of 
elimination phase. Examples of sampling schedules for a 21-day uptake phase and a 21-day elimination phase are given in Appendix 3.
 
Test organisms  
31. For many species of terrestrial enchytraeids the individual weight is very 
low (e.g. 5-10 mg wet weight per individual for  Enchytraeus albidus  and 
less for  Enchytraeus crypticus  or Enchytraeus luxuriosus ); in order to 
perform the weight measurements and chemical analysis, it may be 
necessary to pool the worms of the replicate test vessels (i.e. all the 
worms of a replicate vessel will be used for obtaining one analytical tissue result). 20 individual enchytraeids are added to each replicate, and at least three replicates should be used. If the analytical detection limit of the 
test chemical is high, more worms may be necessary. For test species with 
higher individual weight ( Eisenia fetida  and  Eisenia andrei ), replicate 
vessels containing one individual can be used. 
32. The earthworms used in a test should be of similar weight (e.g.  Eisenia 
fetida  and Eisenia andrei  should have an individual weight of 250-600 mg). 
Enchytraeids (e.g.  Enchytraeus albidus ) should have a length of approxi ­
mately 1 cm. All worms used in a particular test should come from the same 
source, and should be adult animals with clitellum (see Appendix 5). Since the weight and age of an animal might have an effect on the BAF-values (e.g. due to varying lipid content and/or presence of eggs), these parameters 
should be recorded accurately and taken into account in the interpretation of 
results. In addition, cocoons can be deposited during the exposure period, which will also have an impact on the BAF values. It is recommended that a sub-sample of the test worms be weighed before the test in order to 
estimate the mean wet and dry weights. 
33. A high soil-to-worm ratio should be used in order to minimise the decrease 
of the test chemical concentration in the soil during the uptake phase. For  
Eisenia fetida  and  Eisenia andrei  a minimum amount of 50 g dry weight 
(d.w.) of soil per worm, and for enchytraeids, a minimum of 10-20 g d.w. of soil per test vessel are recommended. The vessels should contain a soil layer 
of 2-3 cm (enchytraeids) or 4-5 cm (earthworms). 
34. The worms used in a test are removed from the culture (e.g. enchytraeids by 
using jeweller’s tweezers). Adult animals are transferred to non-treated test 
soil for acclimation, and fed (see paragraph 36). If the test conditions differ from the culture conditions, an acclimation phase of 24-72 h should be sufficient to adapt the worms to the test conditions. After acclimation, 
earthworms are rinsed by transfer to glass dishes (e.g. petri dishes) 
containing clean water, and subsequently weighed before they are added to the test soil. Prior to weighing, excess water should be removed from the worms by gently touching them against the edge of the dish or by 
blotting them cautiously dry by using a slightly moistened paper towel. 
35. Burrowing behaviour of the test organisms should be observed and 
recorded. In tests with earthworms, the animals (control and treatments) 
normally burrow in the soil within a period of a few hours; this should be checked no later than 24 h after addition of the worms to the test vessels. If the earthworms fail to burrow in the soil (e.g. more than 10 % 
over more than half of the uptake phase), this indicates that either the test 
conditions are not appropriate or the test organisms are not healthy. In such
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1301
 
a case the test should be stopped and repeated. Enchytraeids mainly live in 
the interstitial pores of the soil, and frequently their integument may be only 
partly in contact with the surrounding substrate; exposure of burrowing and non-burrowing enchytraeids is assumed to be equivalent and non-burrowing of the enchytraeids does not necessarily require the repetition of the test.
 
Feeding  
36. Feeding should be envisaged when a soil with low total organic carbon 
content is used. When an artificial soil is used, a weekly feeding rate (i.e. the worms should be fed once a week) of 7 mg of dried dung per g soil dry weight is recommended for earthworms, and a weekly rate of 2-2,5 mg of 
ground oat flakes per g soil dry weight is recommended for enchytraeids 
(11). The first food ration should be mixed with the soil immediately before the test organisms are added. Preferably the same type of food like in the cultures should be used (see Appendix 5).
 
Light and temperature  
37. The tests should be carried out under a controlled 16/8 hours light/dark 
cycle, preferably 400 to 800 lx in the area of the test vessels (3). The test temperature should be 20 ± 2 °C throughout the test.
 
Test concentrations  
38. A single concentration is used. Situations where additional concentration(s) 
is(are) required should be justified. If toxicity (ECx) of the test chemical is 
close to the analytical detection limit, the use of radiolabelled test chemical with high specific radioactivity is recommended. For metals, the concen ­
tration should be above the background level in tissue and soil.
 
Replicates  
39. For the kinetic measurements (uptake and elimination phase), the minimum 
number of treated replicate vessels should be three per sampling point. The total number of replicates prepared should be sufficient to cover all 
sampling times during the uptake and the elimination phase. 
40. For the biological observations and measurements (e.g. dry-to-wet weight 
ratio, lipid content) and for the analysis of background concentrations in 
worms and soil, at least 12 replicate vessels of a negative control (four 
sampled at start, four at end of uptake, and four at end of elimination) should be provided if no solvent other than water is used. If any solubilising agent is used for application of the test chemical, a solvent control (four 
replicate vessels should be sampled at start, four at the end of the uptake 
phase, and four at the end of the elimination phase) containing all consti ­
tuents except for test item should be run in addition to the treated replicates. In this case, four additional replicate vessels of a negative control (no 
solvent) may also be provided for optional sampling at the end of the 
uptake phase. These replicates can be compared biologically with the solvent control in order to gain information on a possible influence of the solvent on the test organisms. It is recommended establishing a sufficient 
number of additional reserve replicate vessels (e.g. eight) for treatment and 
control(s).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1302
 
Frequency of soil quality measurements  
41. Soil pH, soil moisture content and the temperature (continuously) in the test 
room should be measured at the start and end of the uptake and elimination phases. Once per week the soil moisture content should be controlled by 
weighing the test vessels and comparing actual weights with initial weights 
at test start. Water losses should be compensated by adding deionised water.
 
Sampling and analysis of worms and soil  
42. An example of schedule for the uptake and elimination phases in earthworm 
and enchytraeid bioaccumulation tests is given in Appendix 3. 
43. The soil is sampled from the test vessels for the determination of test 
chemical concentration before inserting the worms, and during the uptake 
and elimination phases. During the test the concentrations of test chemical 
are determined in the worms and the soil. In general, total soil concen ­
trations are measured. As an option, concentrations in pore water may be measured; in such case, rationale and appropriate methods should be 
provided prior to initiation of a study, and included in the report. 
44. The worms and soil are sampled at least at six occasions during the uptake 
and the elimination phases. If the stability of a test chemical is demon ­
strated, the number of soil analyses can be reduced. It is recommended 
analysing at least three replicates at the beginning and at the end of the uptake phase. If the concentration in soil measured at the end of the uptake phase deviates from the initial concentration by more than 30 %, the soil 
samples taken at other dates should also be analysed. 
45. Remove the worms of a given replicate from the soil at each sampling time 
(e.g. after spreading the soil of the replicate on a shallow tray and picking 
the worms using soft jewellers’ tweezers), rinse them quickly with water in 
a shallow glass or steel tray. Remove excess water (see paragraph 34). Transfer the worms carefully to a pre-weighed vessel, weigh them instantly, 
including gut content. 
46. The earthworms ( Eisenia  sp.) should then be allowed to purge their gut 
overnight e.g. on a moist filter paper in a covered petri dish (see 
paragraph 34). After purging, the weight of the worms should be determined 
in order to assess a possible decrease in biomass during the test (see validity criteria in paragraph 17). Weighing and tissue analysis of Enchytraeids is carried out without purging, as this is technically difficult due to the small 
size of these worms. After final weight determination, the worms should be 
killed immediately, using the most appropriate method (e.g. using liquid nitrogen, or freezing at temperatures below – 18 °C). 
47. During the elimination phase, the worms replace contaminated gut contents 
with clean soil. This means, measurements in un-purged worms (enchytraeids in this context) sampled immediately before the elimination phase include contaminated gut soil. For aquatic oligochaetes it is assumed 
that after the initial 4-24 h of the elimination phase, most of the 
contaminated gut content has been replaced by clean sediment e.g. (46). Similar findings have been reported for earthworms in studies on the accumulation of radiolabelled cadmium and zinc (78). In the non-purged 
enchytraeids, the concentration of this first sample of the elimination phase 
may be considered as the tissue concentration after gut purge. To account for dilution of the test item concentration by uncontaminated soil during the elimination phase, the weight of the gut content may be estimated from 
worm wet weight/worm ash weight or worm dry weight/worm ash weight 
ratios.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1303
 
48. The soil and worm samples should be preferably analysed immediately after 
removal (i.e. within 1-2 days) in order to prevent degradation or other losses, 
and it is recommended calculating the approximate uptake and elimination rates as the test proceeds. If the analysis is delayed, the samples should be stored by an appropriate method, e.g. by deep-freezing ( ≤ – 18 °C). 
49. It should be checked that the precision and reproducibility of the chemical 
analysis, as well as the recovery of the test chemical from soil and worm samples are satisfactory for the given method; the extraction efficiency, the 
limit of detection (LOD) and the limit of quantification (LOQ) should be 
reported. Likewise it should be checked that the test chemical is not detectable in the control vessels in concentrations higher than background. When the concentration of the test chemical in the test organism Ca is > 0 
in the control worms, this should be included in the calculation of the 
kinetic parameters (see Appendix 2). All samples should be handled throughout the test to minimise contamination and loss (e.g. resulting from adsorption of the test chemical on the sampling device). 
50. When working with radiolabelled test chemicals, it is possible to analyse 
parent and metabolites. Quantification of parent test chemical and meta ­
bolites at steady state or at the end of the uptake phase provides 
important information. The samples should then be ‘cleaned up’ so that 
the parent test chemical can be quantified separately. If single metabolites exceed 10 % of total radioactivity in the analysed sample(s), the identifi ­
cation of these metabolites is recommended. 
51. The overall recovery, and the recovery of test chemical in worms, soil, and 
if used, in traps containing absorbents to retain evaporated test chemical, 
should be recorded and reported. 
52. Pooling of the individuals sampled from a given test vessel is acceptable for 
enchytraeid worms which are smaller than earthworms. If pooling involves 
the reduction of the number of replicates, this limits the statistical 
procedures which can be applied to the data. If a specific statistical procedure and power are required, then an adequate number of replicate test vessels should be included in the test to accommodate the desired 
pooling, procedure and power. 
53. It is recommended that the BAF be expressed both as a function of total dry 
weight and, when required (i.e. for highly hydrophobic chemicals), as a 
function of the lipid content. Suitable methods should be used for deter ­
mination of lipid content (some existing methods – e.g. (31) (58) – should be adapted for this purpose). These methods use a chloroform/methanol extraction technique. However, to avoid the use of chlorinated solvents, a 
modification of the Bligh and Dyer method (9) as described in (17) should 
be used. Since the various methods may not give identical values, it is important to give details of the method used. When possible, i.e. if sufficient worm tissue is available, the lipid analysis should ideally be made on the 
same sample or extract as the one used for analysis of the test chemical, 
since the lipids often have to be removed from the extract before it can be analysed chromatographically (49). Alternatively, control animals may be used to measure the lipid content, which can then be used to normalise BAF 
values. This latter approach reduces the contamination of equipment with 
the test chemical.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1304
 
DATA AND REPORTING  
Treatment of results  
54. The uptake curve of the test chemical is obtained by plotting its concen ­
tration in/on the worms during the uptake phase against time on arithmetic scales. When the curve has reached a plateau, or steady state (see definitions in Appendix 1), the steady state bioaccumulation factor BAFss is calculated 
from:
 
C a at steady state or at end of uptake phase  ðmeanÞ 
C s at steady state or at end of uptake phase  ðmeanÞ 
C a is the concentration of test chemical in the test organism 
C s is the concentration of test chemical in the soil 
55. When no steady state is reached, the BAFK, based on the rate constants, 
should be determined instead of BAFss, as described below: 
— Determine the accumulation factor (BAF  K ) as the ratio k  s /k e . 
— Uptake and elimination rates are preferably calculated simultaneously 
(see Equation 11 in Appendix 2) 
— The elimination rate constant (k  e ) is usually determined from the elim ­
ination curve (i.e. a plot of the concentration of the test item in the worms during the elimination phase). The uptake rate constant k
 s is 
then calculated given k  e and a value of C  a which is derived from the 
uptake curve – See Appendix 2 for a description of these methods. The 
preferred method for obtaining BAF  K and the rate constants, k  s , and k  e , 
is to use non-linear parameter estimation methods on a computer. If the elimination is obviously not first-order, then more complex models 
should be employed.
 
Test report  
56. The test report should include the following information:  
Test chemical:  
— Any available information on acute or long term toxicity (e.g. EC  x , LC  x„ 
NOEC) of the test chemical towards soil-dwelling oligochaetes; 
— purity, physical nature and, physicochemical properties e.g. log K  ow , 
water solubility; 
— chemical identification data; source of the test item, identity and concen ­
tration of any solvent used; 
— if radiolabelled test chemical is used, the precise position of the labelled 
atoms, the specific radioactivity, and the radiochemical purity.  
Test species:  
— scientific name, strain, source, any pre-treatment, acclimation, age, 
size-range, etc..  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1305
 
Test conditions:  
— test procedure used; 
— type and characteristics of illumination used and photoperiod(s); — test design (e.g. number and size of test vessels, soil mass and height of 
soil layer, number of replicates, number of worms per replicate, number 
of test concentrations, duration of uptake and elimination phases, 
sampling frequency); 
— rationale for the choice of test vessel material; 
— method of test item preparation and application as well as reasons for 
choosing a specific method; 
— the nominal test concentrations, the means of the measured values and 
their standard deviations in the test vessels, and the method by which 
these values were obtained; 
— source of the constituents of the artificial soil or – if natural media are 
used – origin of the soil, description of any pre-treatment, results of the 
controls (survival, biomass development, reproduction), soil character ­
istics (pH, total organic carbon content, particle size distribution (percent sand, silt, and clay), WHC
 max , percent water content at start and at end 
of the test, and any other measurements made); 
— detailed information on the treatment of soil and worm samples, 
including details of preparation, storage, spiking procedures, extraction, and analytical procedures (and precision) for the test item in worms and soil, and lipid content (if measured), and recoveries of the test item.  
Results:  
— mortality of the control worms and the worms in each test vessel and 
any observed abnormal behaviour (e.g. soil avoidance, lack of repro ­
duction in a bioaccumulation test with enchytraeids); 
— the dry weight to wet weight ratio of the soil and the test organisms 
(useful for normalisation); 
— the wet weights of the worms at each sampling time; for earthworms, the 
wet weights at start of the test, and at each sampling time before and 
after gut purging; 
— the lipid content of the test organisms (if determined); 
— curves, showing the uptake and elimination kinetics of the test chemical 
in the worms, and the time to steady state; 
— C
 a and C  s (with standard deviation and range, if appropriate) for all 
sampling times (C  a expressed in g kg  –1 wet and dry weight of whole 
body, C  s expressed in g kg  –1 wet and dry weight of soil). If a biota-soil 
accumulation factor (BSAF) is required (e.g. for comparison of results from two or more tests performed with animals of differing lipid content), C
 a may additionally be expressed as g kg  –1 lipid content of 
the organism, and C  s may be expressed as g kg  –1 organic carbon (OC) 
of the soil; 
— BAF (expressed in kg soil ·kg –1 worm), soil uptake rate constant k  s 
(expressed in g soil kg  –1 of worm day  –1 ), and elimination rate 
constant k  e (expressed in day  –1 ); BSAF (expressed in kg soil OC kg  –1 
worm lipid content) may be reported additionally;  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1306
 
— if measured: percentages of parent chemical, metabolites, and bound 
residues (i.e. the percentage of test chemical that cannot be extracted 
with common extraction methods) detected in soil and test animals; 
— methods used for the statistical analyses of data.  
Evaluation of results:  
— compliance of the results with the validity criteria as listed in 
paragraph 17; 
— unexpected or unusual results, e.g. incomplete elimination of the test 
chemical from the test animals.  
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Chemikalien an Bodenraubmilben (Gamasina), Teil 2: Erste Ergebnisse mit Lindan und Kaliumdichromat in subletaler Dosierung. Zool. Beitr. NF 34: 413-433. 
(65) Schmelz R and Collado R (1999).  Enchytraeus luxuriosus  sp. nov., a new 
terrestrial oligochaete species (Enchytraeide, Clitellata, Annelida). 
Carolinea 57: 93–100.
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(66) Sims R W and Gerard BM (1985). Earthworms, In: Kermack, D. M. & 
Barnes, R. S. K. (Hrsg.): Synopses of the British Fauna (New Series) 
No 31.171 S. London: E. J. Brill/Dr W. Backhuys. 
(67) Sousa JP, Loureiro S, Pieper S, Frost M, Kratz W, Nogueira AJA, Soares 
AMVM (2000). Soil and plant diet exposure routes and toxicokinetics of lindane in a terrestrial isopod. Environ. Toxicol. Chem. 19: 2557–2563. 
(68) Spacie A and Hamelink JL (1982). Alternative models for describing the 
bioconcentration of organics in fish. Environ. Toxicol. Chem. 1, 309-320. 
(69) Stephenson GL, Kaushik A, Kaushik NK, Solomon KR, Steele T, 
Scroggins RP (1998). Use of an avoidance-response test to assess the toxicity of contaminated soils to earthworms. In: Advances in earthworm 
ecotoxicology. S. Sheppard, J. Bembridge, M. Holmstrup, L. Posthuma 
(eds.). Setac Press, Pensacola, 67-81. 
(70) Sterenborg I, Vork NA, Verkade SK, Van Gestel CAM, Van Straalen NM 
(2003). Dietary zinc reduces uptake but not metallothionein binding and elimination of cadmium in the springtail  Orchesella cincta.  Environ. 
Toxicol. Chemistry 22: 1167-1171. 
(71) UBA (Umweltbundesamt) (1991). Bioakkumulation — Bewertungskonzept 
und Strategien im Gesetzesvollzug. UBA-Texte 42/91. Berlin. 
(72) US EPA (2000). Methods for measuring the toxicity and bioaccumulation 
of sediment-associated contaminants with freshwater invertebrates. Second 
Edition, EPA 600/R-99/064, US, Environmental Protection Agency, 
Duluth, MN, March 2000. 
(73) Van Brummelen TC and Van Straalen NM (1996). Uptake and elimination 
of benzo(a)pyrene in the terrestrial isopod  Porcellio scaber.  Arch. Environ. 
Contam. Toxicol. 31: 277-285. 
(74) Van Gestel CAM. (1992). The influence of soil characteristics on the 
toxicity of chemicals for earthworms; a review, In: Ecotoxicology of Earthworms (Ed. Becker, H, Edwards, PJ, Greig-Smith, PW & Heimbach, F). Intercept Press, Andover (GB). 
(75) Van Gestel CA and Ma W-C (1990). An approach to quantitative 
structure-activity relationships (QSARs) in earthworm toxicity studies. 
Chemosphere 21: 1023-1033. 
(76) Van Straalen NM, Donker MH, Vijver MG, van Gestel CAM (2005). 
Bioavailability of contaminants estimated from uptake rates into soil invert ­
ebrates. Environmental Pollution 136: 409-417. 
(77) Venter JM and Reinecke AJ (1988). The life-cycle of the compost-worm  
Eisenia fetida  (Oligochaeta). South African J. Zool. 23: 161-165. 
(78) Vijver MG, Vink JPM, Jager T, Wolterbeek HT, van Straalen NM, van 
Gestel CAM (2005). Biphasic elimination and uptake kinetics of Zn and Cd in the earthworm  Lumbricus rubellus  exposed to contaminated 
floodplain soil. Soil Biol, Biochem. 37: 1843-1851. 
(79) Widianarko B and Van Straalen NM (1996). Toxicokinetics-based survival 
analysis in bioassays using nonpersistent chemicals, Environ. Toxicol. Chem. 15: 402–406.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1311
 
Appendix 1  
DEFINITIONS  
Bioaccumulation  is the increase in concentration of the test chemical in or on an 
organism relative to the concentration of the test chemical in the surrounding medium. Bioaccumulation results from both bioconcentration and biomagnifi ­
cation processes (see below).  
Bioconcentration  is the increase in concentration of the test chemical in or on an 
organism, resulting from the uptake of the chemical exclusively from the 
surrounding medium (i.e. via the body surface and ingested soil), relative to the concentration of the test chemical in the surrounding medium.  
Biomagnification  is the increase in concentration of the test chemical in or on an 
organism, resulting mainly from uptake from contaminated food or prey, relative to the concentration of the test chemical in the food or prey. Biomagnification 
can lead to a transfer or accumulation of the test item within food webs. 
The elimination  of a test chemical is the loss of this chemical from the test 
organism tissue by active or passive processes that occurs independently of 
presence or absence of the test item in the surrounding medium. 
The bioaccumulation factor  (BAF) at any time during the uptake phase of this 
bioaccumulation test is the concentration of test chemical in/on the test organism (C
 a in g·kg -1 dry weight of worm) divided by the concentration of the chemical 
in the surrounding medium (C  s as g·kg -1 of dry weight of soil); the BAF has the 
units of kg soil ·kg -1 worm. 
The steady state bioaccumulation factor  (BAF  ss ) is the BAF at steady state and 
does not change significantly over a prolonged period of time, the concentration of the test chemical in the surrounding medium (C
 s as g.kg  -1 of dry weight of 
soil) being constant during this period of time.  
Bioaccumulation factors  calculated directly from the ratio of the soil uptake rate 
constant and the elimination rate constant (k  s and k  e , see below) are termed 
kinetic bioaccumulation factor (BAF  K ). 
The biota-soil accumulation factor  (BSAF) is the lipid-normalised concentration 
of the test chemical in/on the test organism divided by the organic 
carbon-normalised concentration of the test chemical in the soil at steady state. C
 a is then expressed as g ·kg -1 lipid content of the organism, and C  s as g·kg -1 
organic content of the soil; the BSAF has the units of kg OC ·kg -1 lipid. 
A plateau  or steady state  is defined as the equilibrium between the uptake and 
elimination processes that occur simultaneously during the exposure phase. The 
steady state is reached in the plot of BAF against time when the curve becomes parallel to the time axis and three successive analyses of BAF made on samples taken at intervals of at least two days are within 20 % of each other, and there 
are no statistically significant differences among the three sampling periods. For 
test chemicals which are taken up slowly, more appropriate intervals would be seven days (49). 
The organic carbon-water partitioning coefficient  (K
 oc ) is the ratio of a 
chemical’s concentration in/on the organic carbon fraction of a soil and the chemical's concentration in water at equilibrium. 
The octanol-water partitioning coefficient  (K
 ow ) is the ratio of a chemical’s 
solubility in n-octanol and water at equilibrium, also sometimes expressed as 
P ow . The logarithm of K  ow (log K  ow ) is used as an indication of a chemical's 
potential for bioaccumulation by aquatic organisms.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1312
 
The uptake or exposure phase  is the time during which the test organisms are 
exposed to the test chemical. 
The soil uptake rate constant  (k s ) is the numerical value defining the rate of 
increase in the concentration of the test item in/on the test organism resulting from uptake from the soil phase. k
 s is expressed in g soil kg  -1 of worm d  -1 . 
The elimination phase  is the time, following the transfer of the test organisms 
from a contaminated medium to a medium free of the test item, during which the 
elimination (or the net loss) of the chemical from the test organisms is studied. 
The elimination rate constant  (k e ) is the numerical value defining the rate of 
reduction in the concentration of the test item in/on the test organism, following the transfer of the test organisms from a medium containing the test item to 
a chemical-free medium; k
 e is expressed in d  -1 . 
Test chemical:  Any substance or mixture tested using this Test Method.  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1313
 
Appendix 2  
Calculation of uptake and elimination parameters  
The main endpoint of a bioaccumulation test is the bioaccumulation factor, BAF. 
The measured BAF can be calculated by dividing the concentration in the test organism, C
 a , by the concentration in the soil, C  s , at steady state. If the steady 
state is not reached during the uptake phase, the BAF  K is calculated from the rate 
constants instead of BAFss. However, it should be noted if the BAF is based on 
steady state concentrations or not. 
The usual means for obtaining the kinetic bioaccumulation factor (BAF  K ), the 
soil uptake rate constant (k  s ) and the elimination rate constant (k  e ) is to use 
non-linear parameter estimation methods on a computer, e.g. based on the models described in (68). Given a set of sequential time concentration data 
and the model equations: 
C
 a ¼ k s 
k e Ü C s ð1 Ä e –k e t Þ 0 < t < t  c [equation 1] 
or 
C a ¼ k s 
k e Ü C s ðe –k e ðtÄtcÞ Ä e –k e t Þ t > t  c [equation 2] 
where: 
C a = concentration of chemical in worms [g kg-1 wet or dry weight] 
k s = uptake rate constant in tissue [g soil kg-1 of worm d-1] 
C s = concentration of chemical in soil [g kg-1 of wet or dry weight] 
k e = elimination rate constant [d-1] 
t c = time at the end of the uptake phase, 
these computer programs calculate values for BAF  K , k s and k  e . 
When the background concentration in the non-exposed worms e.g. on day 0 
differs significantly from zero (this may e.g. be the case for metals), this back ­
ground concentration (C  a,0 ) should be included in these equations, to make them 
read: 
C a ¼ C a;0 þ k s 
k e Ü C s ð1 Ä e –k e t Þ 0 < t < t  c [equation 3] 
and 
C a ¼ C a;0 þ k s 
k e Ü C s ðe –k e ðtÄtcÞ Ä e –k e t Þ t > t  c [equation 4] 
In cases where a significant decrease of the test chemical concentration in the soil 
is observed over time during the uptake phase, the following models can be used 
e.g. (67) (79): 
C s ¼ C 0 ðe –k 
0 t Þ [equation 5]  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1314
 
where: 
C s = concentration of chemical in the soil [g kg-1 wet or dry weight] 
k 0 = degradation rate constant in soil [d-1] 
C 0 = initial concentration of chemical in soil [g kg-1 of wet or dry weight] 
C a ¼ k s 
k e Ä k 0 Ü ðe –k 0 t Ä e –k e t Þ 0 < t < t  c [equation 6] 
C a ¼ k s 
k e Ä k 0 Ü e –k 0 tc Ä e –k e tc ä e ÄkðtÄtcÞ t > t  c [equation 7] 
where: 
C a = concentration of chemical in worms [g kg-1 wet or dry weight] 
k s = uptake rate constant in tissue [g soil kg-1 of worm d-1] 
k 0 = degradation rate constant in soil [d-1] 
k e = elimination rate constant [d-1] 
t c = time at the end of the uptake phase. 
When steady state is reached during the uptake phase (i.e. t = ∞), equation 1 
C a ¼ k s 
k e Ü C s ð1 Ä e –k e t Þ 0 < t < t  c [equation 1] 
may be reduced to: 
C a ¼ k s 
k e Ü C s 
or 
C a=C s ¼ k s=k e ¼ BAF  K [equation 8] 
Then k  s /k e x C  s is an approach to the concentration of the test item in the worm 
tissue at steady state (C  a,ss ). 
The biota-soil accumulation factor (BSAF) can be calculated as follows: 
BSAF ¼ BAF  K ä f oc 
f lip [equation 9] 
where f  oc is the fraction of soil organic carbon, and f  lip is the fraction of worm 
lipid, both preferably determined on samples taken from the test, and based either 
on dry weight or on wet weight, respectively. 
The elimination kinetics can be modelled using the data from the elimination 
phase and applying the following model equation and a computer-based 
non-linear parameter estimation method. If the data points plotted against time 
indicate a constant exponential decline of the test item concentration in the animals, a one-compartment model (equation 9) can be used to describe the 
time course of elimination. 
C
 a ðtÞ ¼ C a;ss Ü e –k e t [equation 10]  ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1315
 
Elimination processes sometimes appear to be biphasic, showing a rapid decline 
of C  a during the early phases, that changes to a slower loss of test items in the 
later phases of the elimination, e.g. (27) (68). The two phases can be interpreted by the assumption, that there are two different compartments in the organism, from which the test item is lost with different velocities. In these cases, specific 
LITERATURE should be studied e.g. (38) (39) (40) (78). 
Using the model equations above, the kinetic parameters (k
 s and k  e ) may also be 
calculated in one run by applying the first order kinetics model to all data from both the uptake and elimination phase simultaneously. For a description of a method that may allow for such a combined calculation of uptake and elim ­
ination rate constants, references (41), (73) and (70) may be consulted. 
C
 a ¼ " 
K s 
K e · C s ð1 Ä e –k e t Þ Ü ðm ¼ 1Þ # 
þ " 
K s 
k e Ü C s ðe ÄK e ðtÄt c Þ Ä e –K e t Þ Ü ðm ¼ 2Þ # 
[equation 11]  
Note:  When uptake and elimination parameters are estimated simultaneously 
from the combined uptake and the elimination data, ‘m’ as shown in equation 11 is a descriptor that allows the computer program to assign the equation’s sub-terms to the data sets of the respective phase and to 
perform the evaluation correctly (m = 1 for uptake phase; m = 2 for 
elimination phase). 
Nevertheless, these model equations should be used with caution, especially 
when changes in the test chemical's bioavailability, or (bio)degradation occur during the test (see e.g. (79)).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1316
 
Appendix 3  
EXAMPLES OF SCHEDULES FOR SOIL BIOACCUMULATION TESTS  
Earthworm test  
(a) Uptake phase with 8 sampling dates used for calculation of kinetics  
Day Activity  
– 6 Conditioning of the prepared soil for 48 h; 
– 4 Spiking of the soil fraction with the test chemical solution; evap ­
orating of any solvent; mixing of the soil constituents; distributing the soil to the test vessels; equilibration at test 
conditions for 4 days (3 weeks for metal-spiked soil); 
– 3 to – 1 Separation of the test organisms from the culture for acclimation; 
preparation and moisturising of the soil constituents; 
0 Measuring temperature, and soil pH; removing soil samples from 
treated vessels and solvent controls for determination of test 
chemical concentration; addition of food ration; weighing and 
randomised distribution of the worms to the test vessels; retaining of sufficient subsamples of worms for determination of analytical background values, wet and dry weight, and lipid 
content; weighing of all test vessels to control soil moisture; 
controlling air supply, if closed test system is used; 
1 Controlling air supply, recording worm behaviour and 
temperature; taking soil and worm samples for determination of test item concentration; 
2 Same as day 1; 
3 Controlling air supply, worm behaviour and temperature; 
4 Same as day 1; 
5-6 Same as day 3; 
7 Same as day 1; addition of food ration; control soil moisture by 
re-weighing the test vessels and compensate evaporated water; 
8-9 Same as day 3; 
10 Same as day 1; 
11-13 Same as day 3; 
14 Same as day 1; addition of food ration; control soil moisture by 
re-weighing the test vessels and compensate evaporated water; 
15-16 Same as day 3; 
17 Same as day 1; 
18-20 Same as day 3;
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1317
 
Day Activity  
21 Same as day 1; measuring temperature and soil pH; control soil 
moisture by re-weighing the test vessels; end of uptake phase; transfer worms from remaining exposed replicates to vessels 
containing clean soil for elimination phase (no gut-purging); 
sampling of soil and worms from solvent controls. 
Pre-exposure activities (equilibration phase) should be scheduled 
taking into account the properties of the test chemical. 
Activities described for day 3 should be performed daily (at least 
on workdays). 
(b) Elimination phase
 
Day Activity  
– 6 Preparation and moisturising of the soil constituents; conditioning 
of the prepared soil for 48 h; 
– 4 Mixing of the soil constituents; distributing the soil to the test 
vessels; incubation at test conditions for 4 days; 
0 (end of uptake 
phase) Measuring temperature and soil pH; weighing and randomised 
distribution of the worms to the test vessels; addition of food 
ration; transfer worms from remaining exposed replicates to 
vessels containing clean soil; taking soil and worm samples after 4-6 h for determination of test chemical concentration; 
1 Controlling air supply, recording worm behaviour and 
temperature; taking soil and worm samples for determination of 
test chemical concentration; 
2 Same as day 1; 
3 Controlling air supply, worm behaviour and temperature; 
4 Same as day 1; 
5-6 Same as day 3; 
7 Same as day 1; addition of food ration; control soil moisture by 
re-weighing the test vessels and compensate evaporated water; 
8-9 Same as day 3; 
10 Same as day 1; 
11-13 Same as day 3; 
14 Same as day 1; addition of food ration; control soil moisture by 
re-weighing the test vessels and compensate evaporated water; 
15-16 Same as day 3; 
17 Same as day 1;
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1318
 
Day Activity  
18-20 Same as day 3; 
21 Same as day 1; measuring temperature and soil pH; control soil 
moisture by re-weighing the test vessels; sampling of soil and 
worms from solvent controls. 
Preparation of the soil prior to start of elimination phase should 
be done in the same manner as before the uptake phase. 
Activities described for day 3 should be performed daily (at least 
on workdays).  
Enchytraeid test  
(a) Uptake phase with 8 sampling dates used for calculation of kinetics  
Day Activity  
– 6 Conditioning of the prepared soil for 48 h; 
– 4 Spiking of the soil fraction with the test chemical solution; evap ­
orating of any solvent; mixing of the soil constituents; 
distributing the soil to the test vessels; equilibration at test 
conditions for 4 days (3 weeks for metal-spiked soil); 
– 3 to – 1 Separation of the test organisms from the culture for acclimation; 
preparation and moisturising of the soil constituents; 
0 Measuring temperature, and soil pH; removing soil samples from 
treated vessels and solvent controls for determination of test chemical concentration; addition of food ration to soil; 
weighing and randomised distribution of the worms to the test 
vessels; retaining of sufficient subsamples of worms for deter ­
mination of analytical background values, wet and dry weight, and lipid content; weighing of all test vessels to control soil 
moisture; controlling air supply, if closed test system is used; 
1 Controlling air supply, recording worm behaviour and 
temperature; taking soil and worm samples for determination of 
test item concentration; 
2 Same as day 1; 3 Controlling air supply, worm behaviour and temperature; 4 Same as day 1; 
5-6 Same as day 3; 
7 Same as day 1; addition of food ration to soil; control soil 
moisture by re-weighing the test vessels and compensate evap ­
orated water; 
9 Same as day 1; 
10 Same as day 3;
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1319
 
Day Activity  
11 Same as day 1; 
12-13 Same as day 3; 
14 Same as day 1; addition of food ration to soil; measuring 
temperature and soil pH; control soil moisture by re-weighing the test vessels; end of uptake phase; transfer worms from remaining exposed replicates to vessels containing clean soil 
for elimination phase (no gut-purging); sampling of soil and 
worms from solvent controls. 
Pre-exposure activities (equilibration phase) should be scheduled 
taking into account the properties of the test chemical. 
Activities described for day 3 should be performed daily (at least 
on workdays).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1320
 
Appendix 4  
Artificial soil – preparation and storage recommendations  
Since natural soils from a particular source may not be available throughout the 
year, and indigenous organisms as well as the presence of micro-pollutants can 
influence the test, an artificial substrate, the artificial soil according to Chapter 
C.8 of this Annex, Toxicity for Earthworms (48), is recommended for use in this test. Several test species can survive, grow, and reproduce in this soil, and maximum standardisation as well as intra- and interlaboratory comparability of 
test and culture conditions are provided. 
Soil constituents: 
Peat: 10 % Sphagnum-peat, in accordance with the 
OECD Guideline 207 (48); 
Quartz sand: 70 % Industrial quartz sand (air dried); grain size: 
more than 50 % of the particles should be in the range of 50-200 μm, but all particles 
should be ≤ 2 mm; 
Kaolinite clay: 20 % Kaolinite content ≥ 30 %; 
Calcium carbonate: ≤ 1 % CaCO
 3 , pulverised, chemically pure. 
As an option, the organic carbon content of the artificial soil may be reduced, 
e.g. by lowering the peat content to 4-5 % of dry soil and increasing the sand 
content accordingly. By such a reduction in organic carbon content, the possi ­
bilities of adsorption of test chemical to the soil (organic carbon) may be decreased, and the availability of the test chemical to the worms may increase (74). It has been demonstrated that  Enchytraeus albidus  and  Eisenia fetida  can 
comply with the validity criteria on reproduction when tested in field soils with 
lower organic carbon content, e.g. 2,7 % (33), (61), and there is experience that this can also be achieved in artificial soil with 5 % peat.
 
Preparation  
The dry constituents of the soil are mixed thoroughly (e.g. in a large-scale 
laboratory mixer). This should be done about one week before starting the 
test. The mixed dry soil constituents should be moistened with deionised water 
at least 48 h before application of the test item in order to equilibrate/stabilise the acidity. For the determination of pH a mixture of soil and 1 M KCl solution in a 
1:5 ratio is used. If the pH value is not within the required range (6,0 ± 0,5), a 
sufficient amount of CaCO
 3 is added to the soil, or a new batch of soil is 
prepared. 
The maximum water holding capacity (WHC) of the artificial soil is determined 
according to ISO 11268-2 (35). At least two days before starting the test, the dry artificial soil is moistened by adding enough deionised or reconstituted water to 
obtain approximately half of the final water content. The final water content 
should be 40 % to 60 % of the maximum WHC. At the start of the test, the pre-moistened soil is divided into as many batches as the number of test concen ­
trations and controls used for the test, and the moisture content is adjusted to 40- 
60 % of WHC
 max by using the solution of the test item and/or by adding 
deionised or reconstituted water. The moisture content is determined at the beginning and at the end of the test (at 105 °C). It should be optimal for the species’ requirements (the moisture content can also be checked as follows: when 
the soil is gently squeezed in the hand, small drops of water should appear 
between the fingers).
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1321
 
Storage  
The dry constituents of the artificial soil may be stored at room temperature until 
use. The prepared, pre-moistened soil may be stored in a cool place for up to 
three days prior to spiking; care should be taken to minimise evaporation of water. Soil spiked with the test item should be used immediately unless there is information indicating that the particular soil can be stored without affecting 
the toxicity and bioavailability of the test item. Samples of spiked soil may then 
be stored under the conditions recommended for the particular test item until analysis.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1322
 
Appendix 5  
Species of terrestrial oligochaetes recommended for testing bioaccumulation 
from soil  
Earthworms  
The recommended test species is  Eisenia fetida  (Savigny 1826), belonging to the 
family Lumbricidae. Since 1972 it is divided into two subspecies ( Eisenia fetida  
and Eisenia andrei  (10)). According to Jaenike (36), they are true, separate 
species.  Eisenia fetida  is easily recognised by its bright intersegmental yellow 
stripes whereas  Eisenia andrei  has a uniform, dark red colour. Originating 
probably from the region of the Black Sea, they are distributed worldwide today, especially in anthropogenically modified habitats like compost heaps. 
Both can be used for ecotoxicological as well as bioaccumulation tests.  
Eisenia fetida  and Eisenia andrei  are commercially available, e.g. as fish bait. In 
comparison to other lumbricid earthworms, they have a short life-cycle, reaching maturity within ca. 2-3 months (at room temperature). Their optimum temperature is approximately at 20-24 °C. They prefer relatively moist substrates 
with a nearly neutral pH and a high content of organic material. Since these 
species have been widely used in standardised ecotoxicological tests for about 25 years, their culturing is well established (48) (77). 
Both species can be bred in a wide range of animal wastes. The breeding 
medium recommended by ISO (35) is a 50:50 mixture of horse or cattle 
manure and peat. The medium should have a pH value of about 6 to 7 (regulated with calcium carbonate), a low ionic conductivity (less than 6 mS/cm or less than 0,5 % salt concentration) and should not be contaminated excessively with 
ammonia or animal urine. Also, a commercial gardening soil free of additives, 
or artificial soil according to OECD (48), or a 50:50 mixture of both can be used. The substrate should be moist but not too wet. Breeding boxes of 10 litre to 50 litre volume are suitable. 
To obtain worms of standard age and mass, it is best to start the culture with 
cocoons. Therefore, adult worms are added to a breeding box containing fresh 
substrate to produce cocoons. Practical experience has shown that a population density of approximately 100 adult worms per kg substrate (wet weight) leads to good reproduction rates. After 28 days, the adult worms are removed. The 
earthworms hatched from the cocoons are used for testing when mature after 
at least 2 months but less than 12 months. 
Worms of the species described above can be considered healthy if they move 
through the substrate, do not try to leave the substrate, and reproduce continu ­
ously. Very slow motioning or a yellow posterior end (in the case of  Eisenia 
fetida ) indicates substrate exhaustion. In this case, fresh substrate and/or a lower 
number of animals per box is recommended.
 
Additional selected references  
Gerard BM (1964). Synopsis of the British fauna. No 6 Lumbricidae. Linnean 
Soc. London, 6: 1-58. 
Graff O (1953). Die Regenwürmer Deutschlands. Schr. Forsch. Anst. Land ­
wirtsch. 7: 1-81. 
Römbke J, Egeler P, Füll C (1997). Literaturstudie über Bioakkumulationstests 
mit Oligochaeten im terrestrischen Medium. Bericht für das UBA F + E 206 03 909, 86 S. 
Rundgren S (1977). Seasonality of emergence in lumbricids in southern Sweden. 
Oikos 28: 49-55. 
Satchell JE (1955). Some aspects of earthworm ecology. Soil Zoology (Kevan): 
180-201.
 ▼M4 2008R0440 — EN — 22.03.2014 — 004.001 — 1323
 
Sims RW and Gerard BM (1985). A synopsis of the earthworms. Linnean Soc. 
London 31: 1-171. 
Tomlin AD (1984). The earthworm bait market in North America. In: Earthworm 
Ecology — from Darwin to vermiculture. Satchell, J.E. (ed.), Chapman & Hall, London. 331-338 pp.
 
Enchytraeids  
The recommended test species is  Enchytraeus albidus  Henle 1837 (white 
potworm).  Enchytraeus albidus  is one of the biggest (up to 15 mm) species of 
the annelid oligochaete family Enchytraeidae and it is worldwide distributed e.g. 
(8). Enchytraeus albidus  is found in marine, limnic and terrestrial habitats, 
mainly in decaying organic matter (seaweed, compost) and rarely in meadows 
(42). This broad ecological tolerance and some morphological variations indicate 
that there might be different races for this species.  
Enchytraeus albidus  is commercially available, sold as food for fish. It should be 
checked whether the culture is contaminated by other, usually smaller species (60). If contamination occurs, all worms should be washed with water in a Petri 
dish. Large adult specimens of  Enchytraeus albidus  are then selected (by using a 
stereomicroscope) to start a new culture. All other worms are discarded. Its life cycle is short as maturity is reached between 33 days (at 18 °C) and 74 days (at 12 °C). Only cultures which have been kept in the laboratory for at least 5 weeks 
(one generation) without problems should be used for a test. 
Other species of the  Enchytraeus  genus are also suitable, especially  Enchytraeus 
luxuriosus.  This species is a true soil inhabitant, which has been newly described 
in (65). If other species of  Enchytraeus  are used, they should be clearly identified 
and the rationale for the selection of the species should be reported.  
Enchytraeus crypticus  (Westheide & Graefe 1992) is a species belonging to the 
same group as  Enchytraeus luxuriosus . It has not been found to exist with 
certainty in the field, having only been described from earthworm cultures and compost heaps (Römbke 2003). Its original ecological requirements are therefore 
not known. However, recent laboratory studies in various field soils have 
confirmed that this species has a broad tolerance towards soil properties like pH and texture (Jänsch et al. 2005). In recent years, this species has often been used in ecotoxicological studies because of the simplicity of its breeding 
and testing, e.g. Kuperman et al. 2003). However, it is small (3-12 mm; 7 mm on 
average (Westheide & Müller 1996), and this makes handling more difficult compared with  Enchytraeus albidus . When using this species instead of  
Enchytraeus albidus , the size of the test vessel can but needs not to be 
smaller. In addition, it should be considered that this species reproduces very 
rapidly having a generation time of less than 20 days at 20 ± 2 °C (Achazi et al. 1999) and even quicker at higher temperatures. 
Enchytraeids of the species  Enchytraeus albidus  (as well as other  Enchytraeus  
species) can be bred in large plastic boxes (e.g. 30 × 60 × 10 cm or 20 × 12 × 
8 cm which is suitable for culture of worms of small size) filled with a mixture 
of artificial soil and commercially available, uncontaminated garden soil free of additives. Compost material should be avoided since it could contain toxic chemicals like heavy metals. Fauna should be removed from the breeding soil 
before use by three times deep-freezing. Pure artificial soil can also be used but 
the reproduction rate could be slower compared to that obtained with mixed substrates. The substrate should have a pH of 6,0 ± 0,5. The culture is kept in an incubator at a temperature of 15 ± 2 °C without light. In any case, a 
temperature higher than 23 °C should be avoided. The artificial/natural soil 
moisture should be moist but not wet. When the soil is gently pressed by hand, only small drops of water should appear. In any case, anoxic conditions should be avoided (e.g. if a lid is used, the number of lid holes should be high 
enough to provide sufficient exchange of air). The breeding soil should be 
aerated by carefully mixing it once per week.
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The worms should be fed at least once per week ad libitum with rolled oats 
which are placed into a cavity on the soil surface and covered with soil. If food 
from the last feeding date remains in the container, the amount of food given should be adjusted accordingly. If fungi grow on the remaining food, it should be replaced by a new quantity of rolled oats. In order to stimulate reproduction, the 
rolled oats may be supplemented with commercially available, vitamin amended 
protein powder every two weeks. After three months, the animals are transferred to a freshly prepared culture or breeding substrate. The rolled oats, which have to be stored in sealed vessels, should be autoclaved or heated before use in order to 
avoid infections by flour mites (e.g.  Glyzyphagus  sp., Astigmata, Acarina) or 
predacious mites (e.g.  Hypoaspis (Cosmolaelaps) miles , Gamasida, Acarina). 
After disinfecting, the food is ground up so that it can easily be strewn on the soil surface. Another possible food source is baker’s yeast or the fish food 
TetraMin
 ® . 
In general, the culturing conditions are sufficient if worms do not try to leave the 
substrate, move quickly through the soil, exhibit a shiny outer surface without soil particles clinging to it, are more or less whitish coloured, and if worms of different ages are visible. Actually, worms can be considered healthy if they 
reproduce continuously.
 
Additional selected references  
Achazi RK, Fröhlich E, Henneken M, Pilz C (1999). The effect of soil from 
former irrigation fields and of sewage sludge on dispersal activity and colonizing success of the annelid  Enchytraeus crypticus  (Enchytraeidae, Oligochaeta). 
Newsletter on Enchytraeidae 6: 117-126. 
Jänsch S, Amorim MJB, Römbke J (2005). Identification of the ecological 
requirements of important terrestrial ecotoxicological test species. Environ. Reviews 13: 51-83. 
Kuperman RG, Checkai RT, Simini M, Phillips CT, Kolakowski JE, Kurnas CW, 
Sunahara GI (2003). Survival and reproduction of  Enchytraeus crypticus  (Oligo­
chaeta, Enchytraeidae) in a natural sandy loam soil amended with the nitro-heterocyclic explosives RDX and HMX. Pedobiologia 47: 651-656. 
Römbke J (2003). Ecotoxicological laboratory tests with enchytraeids: A review. 
Pedobiologia 47: 607-616. 
Westheide W and Graefe U (1992). Two new terrestrial  Enchytraeus  species 
(Oligochaeta, Annelida). J. Nat. Hist. 26: 479-488. 
Westheide W and Müller MC (1996). Cinematographic documentation of 
enchytraeid morphology and reproductive biology. Hydrobiologia 334: 263-267.
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