## European Union Risk Assessment Report

2-BUTOXYETHANOL

CAS No: 111-76-2 EINECS No: 203-905-0



### RISK ASSESSMENT

GENERAL NOTE

This document contains two different reports:

- Volume 68 Part I Environment (Publication: EUR 22501 EN) – pages 2-97

- Part II Human Health (Final approved version awaiti ng for publication) – pages 98-419

### OH H 3 

### C O



## 4 th 

## Priority List

Volume : 

# 68

Institute for Health and Consumer Protection



# European Chemicals Bureau

Existing SubstancesEuropean Chemicals Bureau 

# European Union Risk Assessment Report

CAS No: 111-76-2 EINECS No: 203-905-0



# 2-butoxyethanol (EGBE) Part I - environmentEuropeanUnion Risk Assessment Report2-butoxyethanol(EGBE)111-76-2203-905-0CAS:EC:

PL-4



# 68 EUR 22501 ENThe mission of the IHCP is to prov ide scientific support to the development and implementation of EU polices related to health and consumer protection. The IHCP carries out research to improve the understanding of potential health risks posed by chem ical, physical and biological agents from various sources to which consumers are exposed.

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EUR 22501 EN ISSN 1018-5593 Luxembourg: Office for Official Publ ications of the European Communities, 2006 © European Communities, 2006 Reproduction is authorised provided the source is acknowledged. Printed in Italy

## European Union Risk Assessment Report

2-BUTOXYETHANOL (EGBE)

Part I - Environment

CAS No: 111-76-2

EINECS No: 203-905-0


### RISK ASSESSMENTLEGAL NOTICE Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might be made of the following information

A great deal of additional info rmation on the European Union is available on the Internet. It can be accessed through the Europa Server (http://europa.eu.int).

Cataloguing data can be found at the end of this publication Luxembourg: Office for Official Public ations of the European Communities, 2006

© European Communities, 2006 Reproduction is authorised provided the source is acknowledged. Printed in Italy

### 2-BUTOXYETHANOL (EGBE)

Part I – Environment

CAS No: 111-76-2

EINECS No: 203-905-0


### RISK ASSESSMENT

Final Report, 2006

France

The environmental part of the risk assessment of 2-butoxyethanol (EGBE) has been prepared by Ministry of the Environment (MEDD) on behalf of the European Union.

The scientific work on this re port has been prepared by:

Institut National de l’Environnement Industriel et des Risques (INERIS) Direction des Risques Chroniques Unité Evaluation des Ris ques Ecotoxicologiques Parc Technologique ALATA BP n°2 60550 Verneuil-en-Halatte FranceDate of Last Literature Search: 2004 Review of report by MS Technical Experts finalised: 2005 Final report: 2006

### 0 OVERALL RESULTS OF TH E RISK ASSESSMENT

CAS No: 111-76-2 EINECS No: 203-905-0 IUPAC Name: 2-butoxytehanol Synonyms: EGBE (this synonym will be used in the present study to refer to the chemical Ethylene Glycol Buty l Ether). Other synonyms: 2-BE; butoxyethanol; 2-butoxy-1-ethano l; n-butoxyethanol; butyl ethoxol; 3-oxa-1-heptanol; o-butyl ethylen e glycol; butyl glycol; butyl monoether glycol; ethylene glycol butyl ether; EGBE; ethylene glycol n-butyl ether; ethylene glycol monobutyl ether; glycol butyl ether Commercial trade names: Dowanol EB; Butyl Cellosolve; Butyl Icinol; Butyl Oxitol; Eastman EB Solvent

Environment

Conclusions to the risk assessm ent for the aquatic compartment
Conclusion (ii) There is at present no need for further information and/or testing and for risk reduction measures beyond those wh ich are being applied already.
Conclusion (ii) is applied to al l levels of the lif e cycle of EGBE: production, formulation, processing and private use.

Conclusions to the risk assessment for the terrestrial compartment
Conclusion (ii) There is at present no need for further information and/or testing and for risk reduction measures beyond those wh ich are being applied already.
Conclusion (ii) is applied to al l levels of the lif e cycle of EGBE: production, formulation, processing and private use.

Conclusions to the risk assessmen t for the atmospheric compartment
Conclusion (ii) There is at present no need for further information and/or testing and for risk reduction measures beyond those wh ich are being applied already.
Conclusion (ii) is applied to al l levels of the lif e cycle of EGBE: production, formulation, processing and private use.

Conclusions to the risk asse ssment for secondary poisoning
Conclusion (ii) There is at present no need for further information and/or testing and for risk reduction measures beyond those wh ich are being applied already.
Conclusion (ii) is applied to al l levels of the lif e cycle of EGBE: production, formulation, processing and private use.

Human Health
(to be added later).

VII

### CONTENTS

1 GENERAL SUBSTANC E INFORMATION ................................................................................................ 5

1.1 IDENTIFICATION OF THE SUBSTANCE ........................................................................................ 5

1.2 PURITY/IMPURITIES, ADDITIVES .................................................................................................. 5

1.3 PHYSICO-CHEMICAL PROPERTIES .............................................................................................. 6 1.3.1 Physical state

............................................................................................................................... .. 6 1.3.2 Melting point

............................................................................................................................... .. 6 1.3.3 Boiling point

............................................................................................................................... ... 7 1.3.4 Relative density ............................................................................................................................. 7 1.3.5 Vapour pressure ............................................................................................................................. 7 1.3.6 Surface tension

.............................................................................................................................. 7 1.3.7 Water solubility ............................................................................................................................. 8 1.3.8 Partition coefficient n-octanol/water .............................................................................................. 8 1.3.9 Granulometry

............................................................................................................................... .. 9 1.3.10 Flash point

............................................................................................................................... ...... 9 1.3.11 Autoflammability ........................................................................................................................... 9 1.3.12 Flammability

............................................................................................................................... ... 9 1.3.13 Explosive properties ...................................................................................................................... 9 1.3.14 Oxidising properties ....................................................................................................................... 9 1.3.15 Viscosity

............................................................................................................................... ......... 10 1.3.16 Henry’s constant ............................................................................................................................ 10

1.4 CLASSIFICATION

............................................................................................................................... . 11 1.4.1 Current classification ..................................................................................................................... 11 1.4.2 Proposed classification (environmental part only) ......................................................................... 11

2 GENERAL INFORMATION ON EXPOSURE ........................................................................................... 12

2.1 PRODUCTION

............................................................................................................................... ........ 12 2.1.1 Production processes ..................................................................................................................... 12 2.1.2 Detailed production capacity ......................................................................................................... 12

2.2 USES

............................................................................................................................... ......................... 13 2.2.1 Paints and coatings ........................................................................................................................ 16 2.2.2 Detergents, cleaners ....................................................................................................................... 17 2.2.3 Chemicals used in synthesis: intermediate for EGBEA production .............................................. 18 2.2.4 Printing inks

............................................................................................................................... .... 18 2.2.5 Other uses

............................................................................................................................... ....... 18

3 ENVIRONMENT

............................................................................................................................... ............. 21

3.1 ENVIRONMENTAL EXPOSURE ........................................................................................................ 21 3.1.1 Environmental fate ......................................................................................................................... 21 3.1.1.1 Degradation in the environment ...................................................................................... 21 3.1.1.2 Distribution ...................................................................................................................... 24 3.1.1.3 Accumulation ................................................................................................................... 25 3.1.2 Environmental releases .................................................................................................................. 26 3.1.2.1 Release from production .................................................................................................. 26 3.1.2.2 Release from formulation, processing and private use .................................................... 29 3.1.2.2.1 Continental and regional releases ................................................................... 30 3.1.2.2.2 Local releases: EGBE used in leather finishing operations ............................ 31 3.1.2.2.3 Local releases: EGBE used for me tal cleaning operations (processing) ......... 31 3.1.2.2.4 Local releases: EGBE used in oilfield chemicals (processing) ....................... 32 3.1.2.2.5 Local releases: EGBE used in oil spill dispersants (marine compartment) ..... 36 3.1.2.2.6 Local releases for other uses ........................................................................... 37

13.1.3 Continental and regional predicted environmental concentrations ................................................ 42 3.1.4 Local predicted environmental concentrations (PEC local ) .............................................................. 43 3.1.4.1 Aquatic compartment ....................................................................................................... 43 3.1.4.1.1 PEC local for production .................................................................................... 43 3.1.4.1.2 Calculation of PEC local for formulation, processing and private use ............... 43 3.1.4.1.3 Measured levels .............................................................................................. 45 3.1.4.1.4 Comparison between predic ted and measured levels ...................................... 46 3.1.4.2 Terrestrial compartment ................................................................................................... 46 3.1.4.2.1 PEC local for production .................................................................................... 46 3.1.4.2.2 Calculation of PEC local for formulation, processing and private use ............... 46 3.1.4.3 Atmosphere ...................................................................................................................... 48 3.1.4.3.1 PEC local for production .................................................................................... 48 3.1.4.3.2 Calculation of PEC local for formulation, processing and private use ............... 48 3.1.4.3.3 Measured levels .............................................................................................. 49 3.1.4.3.4 Comparison between predic ted and measured levels ...................................... 49 3.1.4.4 Secondary poisoning ........................................................................................................ 50

3.2 EFFECTS ASSESSMENT: HAZARD IDENTIFICATION AND DOSE (CONCENTRATION) - RESPONSE (EFFECT ASSESSMENT) ............................................................................................... 51 3.2.1 Aquatic compartment (incl. sediment) ........................................................................................... 51 3.2.1.1 Fish

............................................................................................................................... ... 51 3.2.1.2 Aquatic invertebrates ....................................................................................................... 53 3.2.1.3 Algae

............................................................................................................................... . 58 3.2.1.4 Micro-organisms .............................................................................................................. 59 3.2.1.5 Endocrine disruption ........................................................................................................ 60 3.2.1.6 PNEC for the aquatic compartment ................................................................................. 61 3.2.1.6.1 Calculation of the intermittent PNEC for freshwater ...................................... 62 3.2.1.6.2 Calculation of the PNEC for the seawater compartment ................................ 62 3.2.1.6.3 Calculation of the intermittent PNEC for seawater ......................................... 62 3.2.1.7 Calculation of a PNEC for the sediment compartment .................................................... 62 3.2.1.7.1 Calculation of the PNEC for the marine sediment compartment .................... 63 3.2.1.8 PNEC for micro-organisms in STP ................................................................................. 63 3.2.2 Terrestrial compartment ................................................................................................................. 63 3.2.3 Atmosphere

............................................................................................................................... ..... 64 3.2.4 Secondary poisoning ...................................................................................................................... 64

3.3 RISK CHARACTERISATION ............................................................................................................. 65 3.3.1 Aquatic compartment (incl. sediment) ........................................................................................... 65 3.3.2 Terrestrial compartment ................................................................................................................. 67 3.3.3 Atmosphere

............................................................................................................................... ..... 68 3.3.4 Secondary poisoning ...................................................................................................................... 69

4 HUMAN HEALTH

............................................................................................................................... .......... 70

5 RESULTS

............................................................................................................................... .......................... 71

5.1 ENVIRONMENT

............................................................................................................................... ..... 71

5.2 HUMAN HEALTH

............................................................................................................................... .. 71

6 REFERENCES

............................................................................................................................... ................. 72

Euses Calculations can be viewed as part of the report at the website of the European Chemicals Bureau: http://ecb.jrc.it

2

### TABLES

Table 1.1 Physico-chemical properties ................................................................................................................ 6 Table 1.2 Peroxide levels in EGBE during storage under adverse (daylight) and recommended (dark) conditions. No antioxidants used. Results in mmol active oxygen/litre .............................................. 9 Table 1.3 Henry’s law constant values (at 25°C) in EPI suite (US EPA, 2001) .................................................. 10 Table 1.4 Henry’s law constant values as measured in Kim et al., 2000 ............................................................. 11 Table 2.1 Overview of EGBE production and sales in Europe for years 2001 to 2003 (data provided by CEFIC)

............................................................................................................................... .................. 12 Table 2.2 EGBE production sites in EU (larger than 1,000 tonnes/year) ............................................................ 13 Table 2.3 Breakdown of EGBE uses in Europe .................................................................................................. 14 Table 2.4 EGBE end use information (OECD, 1996) ......................................................................................... 16 Table 2.5 Detailed view of paint uses .................................................................................................................. 17 Table 2.6 EGBE concentrations in cleaning products ......................................................................................... 17 Table 3.1 Biodegradation test results for EGBE ................................................................................................. 22 Table 3.2 Estimated biodegradation rate constants fo r EGBE in WWTP, surface water, soil and sediment ...... 24 Table 3.3 Estimated solids / water partition coefficients ..................................................................................... 25 Table 3.4 Calculated distribution of EGBE in th e different compartments of the environment ......................... 25 Table 3.5 Estimated distribution in a STP (SIMPLETREAT) ............................................................................ 25 Table 3.6 Aquatic emission data from production sites of EGBE in EU ............................................................. 28

U Table 3.7 Atmospheric emissions of EGBE from European producers .............................................................. 29 Table 3.8 Emissions of EGBE to soil from European producers ........................................................................ 29 Table 3.9 Environmental exposure scenarios for formulation, processing and private uses of EGBE ................ 29 Table 3.10 Total continental and regional EGBE emissions ................................................................................. 30 Table 3.11 Default values to be used for hazard assessment of completion and workover chemicals (specified as ‘cleaning chemicals’, other chemical s’, ‘squeeze treatments’ and ‘hydrotest chemicals’) (Thatcher et al., 2004). ..................................................................................................... 33 Table 3.12 PEC intermittent for the use of oilfield chemicals containing EGBE on offshore platforms (marine compartment)

............................................................................................................................... ........ 34 Table 3.13 Examples of EGBE containing products registered in UK .................................................................. 35 Table 3.14 Dispersants used today, application methods and dosages (Bonn Agreement, 2001) ......................... 36 Table 3.15 Local releases of EGBE from formulation of paints ........................................................................... 38 Table 3.16 Local releases of EGBE from formulation of paints (continue) .......................................................... 38 Table 3.17 Local releases of EGBE from processing of paints ............................................................................. 39 Table 3.18 Local releases of EGBE from pr ocessing and private use of paints .................................................... 39 Table 3.19 Local releases of EGBE from formulati on, processing and private use of detergents ......................... 40 Table 3.20 Local releases of EGBE from formulation and processing of other uses ............................................ 40 Table 3.21 Local releases of EGBE from formulation and processing of other uses (continued) ......................... 41 Table 3.22 Local releases of EGBE from formulation and processing of other uses (continued) ......................... 41 Table 3.23 Local releases of EGBE from formulation and processing of other uses (continue) ........................... 42 Table 3.24 Regional PECs in air, water and soil (calculations made by EUSES – SIMPLEBOX model) ........... 43 Table 3.25 Local PEC in water at production ........................................................................................................ 43 Table 3.26 Local PEC STP and PEC aqua for EGBE .................................................................................................. 44 Table 3.27 PEClocal soil at production and in situ processing (according to EUSES) ............................................ 46 Table 3.28 Local PEC soil for EGBE ....................................................................................................................... 47 Table 3.29 Local PEC air for EGBE ........................................................................................................................ 48 Table 3.30 Short term fish toxicity data for EGBE ............................................................................................... 51 Table 3.31 Long term fish toxicity data for EGBE ............................................................................................... 53 Table 3.32 Short term invertebrate toxicity data for EGBE .................................................................................. 54 Table 3.33 Long term invertebrate toxicity data for EGBE .................................................................................. 56 Table 3.34 Pros and cons for the validation of the test performed with Brachionus calyciflorus ......................... 56 Table 3.35 Algae toxicity data for EGBE .............................................................................................................. 58 Table 3.36 Micro-organisms toxicity data for EGBE ............................................................................................ 60 Table 3.37 Toxicity tests retained for the derivation of PNEC aqua ......................................................................... 61 Table 3.38 QSAR ecotoxicity data for EGBE ....................................................................................................... 61 Table 3.39 Toxicity tests retained for the derivation of the intermittent PNEC aqua ............................................... 62 Table 3.40 Risk characterisation for micro-organisms in STP and aquatic organisms ......................................... 65 Table 3.41 Risk characterisation for terrestrial compartment ................................................................................ 67

3

### 1 GENERAL SUBSTANCE INFORMATION

1.1 IDENTIFICATION OF THE SUBSTANCE

CAS Number: 111-76-2 EINECS Number: 203-905-0 IUPAC Name: 2-butoxyethanol Molecular formula: C 6 H 14 O 2 Structural formula: CH 3 -CH 2 -CH 2 -CH 2 -O-CH 2 -CH 2 -OH

CH

3 O OH

Molecular weight: 118.17 g.mol -1 Synonyms: EGBE (this synonym will be used in the present study to refer to the chemical Ethylene Glycol Buty l Ether). Other synonyms: 2-BE; butoxyethanol; 2-butoxy-1-ethanol; n-butoxyethanol; butyl ethoxol; 3-oxa-1-heptanol; o-butyl ethylen e glycol; butyl glycol; butyl monoether glycol; ethylene glycol bu tyl ether; EGBE; ethylene glycol n-butyl ether; ethylene glycol monobutyl ether; glycol butyl ether Commercial trade names: Dowanol EB; Butyl Cellosolve; Butyl Icinol; Butyl Oxitol; Eastman EB Solvent Annex I entry: 603-014-00-0

1.2 PURITY/IMPURITIES, ADDITIVES
Purity: the purities quoted in IUCLID were all ≥ 99% w/w Impurity: as the synthesis reac tion produces other glycol ethers, small concentrations of them can be found. 2-butoxyethoxyethanol (CAS 112-34-5) ≤ 0.3% w/w 1,2-ethanediol (CAS 107-21-1) ≤ 0.3-0.5% w/w 1-butanol (CAS 71-36-3) ≤ 0.1-0.2% w/w water < 0.1-0.2% w/w
Additives: 0.008-0.012% w/w 2,6-bis(1,1- dimethylethyl)-4-methylphenol (CAS 128-37-0) added to prevent the formation of peroxides.

5EU RISK ASSESSMENT

– 2- BUTOXYETHANOL ( EGBE ) FINAL REPORT
, 2006

1.3 PHYSICO-CHEMICAL PROPERTIES
Note: When the reliability of values do not enab le a clear choice between one and another, a median value is chosen or calculated taking into consideration all figures supplied by the industry and only once the values found in handbooks or reports and which differ.
The physico-chemical properties are di scussed below and summarised in Table 1.1

.

Table 1.1 Physico-chemical properties

Property Value
Physical state Liquid Melting point -74.8°C Boiling point 171°C Relative density 0.9 at 20°C Vapour pressure 1.41 hPa, calculated at 25°C (initial value: 1 hPa at 20°C)

Surface tension 26.6 mN/m at 20°C Water solubility Highly miscible

Partition coefficient n-octanol/water (log value) 0.8

Granulometry Not applicable Flash point 67°C Autoflammability 244.5°C Flammability Upper limit: 12.7% (volume) Lower limit: 1.1% (volume)
Explosive properties Not explosive Oxidising properties No oxidising properties Viscosity 3.28 mPa.s at 20°C Henry’s constant 0.08 Pa.m 3 /mol at 25°C Conversion factors (101 kPa, 20°C) 1 ppm = 4.9 mg/m 3 1 mg/m 3 = 0.204 ppm

1.3.1 Physical state

EGBE is a neutral, colourle ss liquid with a very weak pl easant odour (Ullmann, 2000). An absolute perceptible limit in air of 0.1 ppm (50% recognition = 0.35 ppm and 100% recognition = 0.48 ppm) was referred to EGBE (Verschueren, 2001).

1.3.2 Melting point
Several values are reported in literature. The value of -77°C appears in several reports and studies (ECETOC, 1994; OECD, 1996; Dow Ch emical Co, 2001) and handbooks show values

6CHAPTER 1. GENERAL SUBSTANCE INFORMATION

ranging between -75 and -70°C (-74.8°C Lewis, 1999; < -70°C Ullmann, 2000; -70°C Howard, 1989; -75°C Eastman, 2001; -70.4°C BA SF, 2002; -70°C Verschueren, 2001).

The median of all different values f ound is retained for this study: -74.8°C.

1.3.3 Boiling point

Most of the values found in literature ar e in the following range: 168-172°C (BASF, 2002; Ullmann, 2000). A boiling point of 170.8°C for EG BE can be found (ECETOC, 1994) which is close to those appearing in handbooks (170° C in Kirk-Ohtmer, 1983 and Verschueren, 2001). Two other handbooks show values between 171 and 172°C (Lewis, 1999 ; Howard, 1989). Boiling point of 171°C was found in technical data sheets or product info rmation reports (BP, 2002; Dow Chemical Co, 2001).

The median of all the different values above is 171°C. This value will be used for the risk evaluation.

1.3.4 Relative density
Several relative density values are available too. Most of them are about 0.9 (Verschueren, 2001 - value measured at 20°C; Merck, 1996). Technical data sheets or product information reports give values of 0.9011 and 0.902, at 20°C (BP, 2002; Dow Chemical Co, 2001).
A rounded value of 0.9 will be chosen for this study.

1.3.5 Vapour pressure
Vapour pressures ranging from 0.79 to 1.3 hPa, at 20°C have b een reported. Data come from technical data sheets: 0.8 hP a (Shell Chemicals, 2001), 0.89 hPa (Dow Chemical Co, 2001), 1.3 hPa (BP, 2002). The same range appears in Sax, 1986 and other chemical handbooks give almost the same values: 0.89 hPa (Verschueren, 2001), 1 hPa (Ullmann, 2000).
The rounded value of the median give s a vapour pressure of 1 hPa, at 20°C. It will be used in the risk assessment (based on this value, a vapou r pressure of 1.41 hPa ha s been recalculated at 25°C, by EUSES, EC, 2004).

1.3.6 Surface tension
Several values of surface tension appear in technical data sheets for commercial EGBE: Dow Chemical Co, 2001 reports a value of 27.4 mN/m at 25°C, Eastman, 2001 gives a value of 26.6 mN/m at 20°C and Shell Chemicals, 2001 a nother of 28.9 mN/m. A value of 27.4 mN/m at 25°C is also reported in a handbook (Kirk-Ohtmer, 1983).
The value reported in Eastman, 2001 will be reta ined for the study: 26.6 mN/m, at 20°C, the temperature recommended in the OECD guideline No 115.
Surface active properties can be assumed for glycol ethers, and especially for ethylene glycol butyl ether, because of its quite long carbon chai n. The values reported in the literature for EGBE tend to indicate that this substance is a surface-active reagent ev en if no indication has been found about the concentratio n of the substance during the te sts listed above. Indeed, OECD

7EU RISK ASSESSMENT

– 2- BUTOXYETHANOL ( EGBE ) FINAL REPORT
, 2006

guideline n°115 suggests that surface tension m easurements should be performed using a concentration of 1 g/L for soluble substances.
The fact that EGBE shows surface-active proper ties could thus lead to the disturbance of analytical method employed to measure some physico-chemical characteristics.
However, there is a difference between the surface activity of traditional surfactants and substances that can reduce the su rface activity of solutions like EGBE. What is observed with the glycol ethers during the surface tension measurem ents is the typical non- ideal behaviour of a mixture of a water miscible solvent such as methanol and ethanol. The reason for the observed relationship between surface tens ion and concentration is the disruption of the hydrogen bonding of the water causing non-linear beha viour of the surface tension agai nst the concentr ation. In this case the substance is not migrating to the surface; it is not acting in the traditional surface-active manner. Therefore it would not affect the measurem ents of the physical chemical properties. One should also notice that glycol ethe rs do not form micelles. They ar e fully miscible with water and form clear solutions.
Furthermore, considering the other properties of this substance (EGBE is highly miscible in water, hydrosphere is the preferential targ et of EGBE in the environment: > 90%, see Section 3.1.1.2 ), surface active properties of EGBE will not be considered in this assessment.

1.3.7 Water solubility
EGBE is often reported as miscible with wate r in several data sheet s and product information reports (Hoechst, 1992; Hoechst, 1993; BP, 2002). A solubility of about 50,000 mg/L is reported by Eckel et al., 1996, 63,500 mg/L by ASTER, 1996. A value of 1.10 6 mg/L has been measured (Riddick et al., 1985 cited in HSDB, 2000).
EGBE can be considered as highly miscible in water and the upper value of the range advised by EUSES (EC, 2004), 1.10 5 mg/L, is retained for modelling purposes.

1.3.8 Partition coefficient n-octanol/water
Several QSAR calculations of partition coeffi cient n-octanol/water for EGBE have been performed. They are presented hereafter usin g an ascending order of logarithmic values: 0.57 (calculated with KOWWIN v. 1.66. - US EPA and Syracuse Research Corporation, 2001); 0.81 (Verschueren, 2001), 0.84 (ASTER, 1996).

Partition coefficient measurements have been performed (Tanii and Hashimoto, 1982): 0.77 is the mean value of three determinations. Measurements have been carried out with gas chromatography.
Other measured log K ow can be found in literature: 0.74 (Verschueren, 2001), 0.81 (Staples et al., 1998), 0.83 (Korenman and Dobromyslova, 1975; Ha nsch and Leo, 1985, cited in Prager, 1995; Staples et al., 1998).

The rounded value of the median obtained with all measured data gives a log K ow of 0.8 that is retained for this study.

8CHAPTER 1. GENERAL SUBSTANCE INFORMATION

1.3.9 Granulometry
Not applicable: the substance is a liquid.

1.3.10 Flash point
Values reported in literature range from 60 (INRS, 1996 and Merck, 1996) to 68°C (BP, 2002). Both values are measured with closed cup me thod. Two other values were obtained using this method: 67°C (Shell Chemicals, 2001; BASF, 2002) and 61°C (Sax, 1986), while methods leading to values of 65°Dow Chemical Co, 2001) and 67°C (Ullmann, 2000) are not mentioned.

The median of the values measured us ing a closed cup is retained: 67°C.

1.3.11 Autoflammability
Two temperatures of autoflammability have been found in technical data sheets: 244°Dow Chemical Co, 2001) and 245°C (BP, 2002). The median value of 244.5°C is retained.

1.3.12 Flammability
Limits of flammability are reported by BP, 2002 for EGBE. The upper limit is 12.7% (volume) and the lower one is 1.1% (volume).

1.3.13 Explosive properties
Not explosive.

1.3.14 Oxidising properties

There are some references which suggest that glycol ethers can be prone to the formation of peroxides on storage. However da ta from one of the producers, shown below, indicates that peroxide production levels for EGBE remain lo w, even during prolonged storage under adverse conditions, as shown in Table 1.2

.

Table 1.2 Peroxide levels in EGBE during storage under adverse

(daylight) and recommended (dark) conditions. No antioxidants used. Results in mmol active oxygen/litre
In daylight In the dark

Time 0 0.018 0.018

3 months 0.040 - 6 months 0.052 0.019

18 months 0.95 -

The National Fire Protection Association’s code for the reactivity of EGBE is 0 indicating minimal hazard. In consequence, there is no requirement for classification R19.

9EU RISK ASSESSMENT

– 2- BUTOXYETHANOL ( EGBE ) FINAL REPORT
, 2006

1.3.15 Viscosity
The range of viscosity values found in technical data sheets for EGBE goes from 2.9 mPa.s (at 25°C, Dow Chemical Co, 2001) to 3.3 mPa.s (Shell Chemicals, 2001). A value of 3.28 mPa.s is also reported by BP, 2002, at 20°C. BP Chemicals, 1998 also reports a range of viscosity values for several concentrations of EGBE in water (b etween 20 and 100% volume): 2-6 mPa.s at 20°C. Several values have been found in handbooks: 3.26 mPa.s, at 20°C (Ullmann, 2000), 2.83 mPa.s at 25°C (Prager, 1995) and a catalogue of commerc ial substances gives a viscosity of 5.83 mPa.s, at 20°C (Merck, 1996).
The most common values are rounded and a viscosity of 3.3 mPa.s (at 20°C) is retained for this study.

1.3.16 Henry’s constant
Different QSAR estimation methods (calcu lated with HENRYWIN v.3.10. - US EPA and Syracuse Research Corporation, 2001) give values of Henry’s law constant presented in Table 1.3

.

Table 1.3 Henry’s law constant values (at 25°C) in EPI suite (US EPA, 2001)

Method Value (Pa.m 3 /mole)

Bond method 0.009 9 Group method 0.002 1 Experimental value quoted in EPI suite database 0.162 1 Calculated with VP.M / WSol ratio using EPI suite estimated values (QSAR) 0.116 1
Henry’s law constant values have also been calculated using another program (MPBPVP), at 25°C. Depending on the method used, values of 0.01 and 0.002 Pa.m 3 /mol are reported (SRC, 1988, cited in Envirofate).
Another study shows a calculated Henry’s law constant of 0.551 Pa.m 3 /mol at 25°C ASTER, 1996).
Henry’s law constant can also be estimated from the ratio of the vapour pressure to the water solubility using selected values from th is study: 141 Pa for vapour pressure and 1.10 6 mg/L for water solubility. Calculation gives a Henry’s law constant of 0.017 Pa.m 3 /mol.
Experimental measurements of Henry’ s law constant have also been Kim et al., 2000). Two different methods were used to determine Henry’ s law constant for the equilibrium partitioning of EGBE contained in an aqueous solution and the air. Results of measurements are presented in Table 1.4

.

10CHAPTER 1. GENERAL SUBSTANCE INFORMATION

Table 1.4 Henry’s law constant values as measured in Kim et al., 2000
Bag method for equilibrium partitioning Batch stripping method
T = 20°C ; H = 0.041 Pa.m 3 /mol T = 22°C ; H = 0.063 Pa.m 3 /mol
T = 25°C ; H = 0.081 Pa.m 3 /mol T = 23°C ; H = 0.068 Pa.m 3 /mol
T = 25°C ; H = 0.082 Pa.m 3 /mol T = 30°C ; H = 0.101 Pa.m 3 /mol
T = 30°C ; H = 0.135 Pa.m 3 /mol
In the previous study, there is a good correlation between both method used, bag method for equilibrium partitioning and batch stripping method, and particularly at 25°C. Moreover, direct measurement of the Henry’s law constant is recommended for water miscible compounds (TGD - EC, 2003). So a Henry’s law constant of 0.08 Pa.m 3 /mol will be used.

1.4 CLASSIFICATION

1.4.1 Current classification

There is no classification for the environment.

1.4.2 Proposed classification (environmental part only)
According to the data presented and the criteria of Directive 67/ 548/EEC EGBE is not classified as dangerous for the environment.

11

### 2 GENERAL INFORMATION ON EXPOSURE

2.1 PRODUCTION
The chemical 2-butoxyethanol (here referred to as EGBE) belongs to the group of glycol ethers, which are mainly used as solvents. During 2003, the production of EGBE in the European Union was approximately 161,000 tonnes (personal co mmunication from CEFIC). According to information provided by industry and compiled by SICOS, 1999, the total EU production of all glycol ethers was 350,000 tonnes in 1998.

2.1.1 Production processes
EGBE is manufactured by addi ng n-butyl alcohol and ethylene oxide. The standard ethoxylation reaction for glycol ethers is as follows:

Equation 2.1 EGBE synthesis reaction

O 

# + H C 3 OH 

# → H C 3 O OH

ethylene oxide butanol EGBE

Ethylene oxide and butanol react together with a typical industrial catal yst. This catalysed reaction produces a range of butyl glycol ethe rs (monoglycol, diglycol and triglycol ethers) which are separated by fracti onation using, for example, vacuum distillation (personal communication from OSPA).

2.1.2 Detailed production capacity
The production and sales data for years 2001 to 2003 are given in Table 2.1

.

Table 2.1 Overview of EGBE production and sales in Eu rope for years 2001 to 2003 (data provided by CEFIC)
(In kilo tonnes) 2001 2002 2003 Figures retained

Production 135 158.5 161 155.1** Imports 9 6 6.5 6*** Exports 49 59 68 64***

Net into stock * 7.5 3 - Captive use (EGBEA production) 9 8 10 9

Table 2.1 continued overleaf

12CHAPTER 2. GENERAL INFORMATION ON EXPOSURE

Table 2.1 continued Overview of EGBE production and sales in Europe for years 2001 to 2003 (data provided by CEFIC)
(In kilo tonnes) 2001 2002 2003 Figures retained

Sales in EU 86 90 86.5 88.1

Total use in EU 95 97.5 96.5 97.1 ¤
* Included in exports figure ** The production volume has been obtained using the following calculat ion: (Exports + Captive use + Sales) – (imports) *** Mean of 2002 and 2003 figures. This figure has been retained in order to take into account the evolution of import and export volumes the last two years ¤ See Table 2.3 for a breakdown of total use in EU The figures presented above show that there is a trend for an increase in production year by year: 135, 158.5 and 161 kt for years 2001, 2002 and 2003 respectively. However this is almost entirely due to increased de mand for exports: 49, 59 and 68 kt each year between 2001 and 2003. The overall demand within the EU remains flat.
A production volume of 155,000 tonnes/ year will be retained for the risk assessment.
The production in the European Union is located at five different sites (see Table 2.2 ).

Table 2.2 EGBE production sites in EU (larger than 1,000 tonnes/year)

Company Location

BASF AG Ludwigshaven, Germany

BP* Lavera, S. France Dow Chemical Company** UCL, Wilton International, Middlesbrough, United Kingdom

Sasol Gmbh*** Marl, Germany

Shell Nederland BV Hoogvliet Rt, the Netherlands
* EGBE business of Hoescht renamed as Clariant in 1998 and then sold to BP (2001) ** EGBE business of ICI sold to Union Carbide in 1995 and then to Dow in 2001 / EGBE business of Enichem sold to Dow in 2001 *** Huels sold to Condea in 1998 and then EGBE business sold to Sasol in 2001

From the Table 2.2 , it appears that some production site s are located in the same area. Consequently the locations of bot h German sites and the Dutch one have been checked so as to establish whether they could pertain to the same region (T GD definition EC, 2003). Distances between Marl and Ludwigshaven on one hand an d Marl and Hoogvliet on the other hand are 259 and 188 km, respectively. So, in the regional assessment, none of th ese sites will be considered in a same region.
Ten EGBE importers are also repo rted. Most of them are paint or detergent manufacturers and some are distributors. They import less than 1,000 tonnes/year except one which has reported a higher tonnage (but detailed figures are not available).

2.2 USES
EGBE has a wide range of uses as a solvent in paints and surface coatings, detergents and surface cleaners, inks or dyes. A breakdown of th e uses of EGBE in Western Europe has been established based on the data collect ed for years 2001 to 2003 by CEFIC (see Table 2.3 ).

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Values presented in this table are based both on volumes shown in Table 2.1 and on the EGBE volumes declared by uses reported in 2001, 2002 and 2003. The analysis of this set of data has led to a choice which is meant to represent a r easonable worst case. The final data choice is based mainly on averages but some expert judgement has also been applied to adjust for market knowledge and the fact that supply via distributors adds some uncertainty to the numbers. This uncertainty explains the significan t fluctuation in the annual tonnage figures for the smaller uses. Typically, 25-40% of volume goes via distributors. To reflect these uncertai nties, the figures are quoted as rounded numbers. 2002 and 2003 data should be given more weight as some errors have possibly been made during assessment of the 2001 data in allocating users to the appropriate end use categories.

Table 2.3 Breakdown of EGBE uses in Europe
Retained proposal

End use Stage of the life cycle Industry category Use category 2001 ¤ 2002 2003 Quantity used (Tonnes) Percentage of total use

Paints and coatings (including estimation for indirect sales via distributors) Formulation Processing

Private use* 14: paints, lacquers and

varnishes industry 48: solvents 51,275 63,000 56,700 57,000 58.70

Detergents, cleaners Formulation
Private / public use 5: personal/ domestic 6: public domain 9: cleaning, washing agents 1,4921 7,000 10,000 11,000 11.33

Chemicals used in synthesis Processing 3: chemicals used in synthesis 33: organic intermediat

es 6,916 13,600 11,000 10,000 10.30

Captive use (EGBEA production) Processing 3: chemicals used in synthesis 33: organic intermediat

es 9,000 8,000 10,000 9,000 9.27

Printing inks Formulation Processing 12: pulp, paper and board industry 48: solvents 5,340 1,100 2,500 2,500 2.57

Oilfield chemicals Formulation Processing 6: public domain 48: solvents 0 3,000 2,000 2,500 2.57

Metal cleaning Formulation Processing 8 : metal extraction, refining and processing

industry 9: cleaning, washing agents 0 50 2,000 2,000 2.06

Electronic industry Processing 4: electrical / electronic industry 48: solvents 1,590 0 30 600 0.62

Table 2.3 continued overleaf

14CHAPTER 2. GENERAL INFORMATION ON EXPOSURE

Table 2.3 continued Breakdown of EGBE uses in Europe
Retained proposal

End use Stage of the life cycle Industry category Use category 2001 ¤ 2002 2003 Quantity used (Tonnes) Percentage of total use

Leather treatment operations Processing 7: leather processing

industry 48: solvents 1,349 0 160 500 0.51

Pharmaceuticals Processing 3: chemicals used in synthesis 48: solvents (extraction

agents, reaction media) 2,073 400 400 500 0.51

Adhesives Formulation Processing 6: public domain 48: solvents 223 500 500 500 0.51

Cosmetics / Personal care Formulation

Private use 5: personal / domestic 6: public domain 15: cosmetics 102 0 700 500 0.51

Fire foams Formulation Processing 6: public domain 25: foaming agents 65 200 330 300 0.31

Agricultural products Formulation Processing 1: agricultural industry 48: solvent 207 0 140 200 0.21

Paper industry 0 0 20 0 0.00

Textile manufacture 0 50 20 0 0.00 Miscellaneous (unknown) 917 0 0 0 0.00 Rubber, oil industry 674 0 0 0 0.00 Oil spill dispersants 191 0 0 0 0.00

Construction chemicals 153 0 0 0 0.00

Total 94,996 96,900 96,500 97,100 ~ 100
* For personal decorative paints only

¤ Original 2001 data provided by CEFIC have been modified so as to integrate sales via distributors and volume of EGBE used for EGBEA production on site (captive use) Four major uses can be identified from sale figures. The use of EGBE in paint and lacquer industry represents ~ 58% of the total volume used in EU. The two other main uses, intermediate for EGBEA synthesis (including ca ptive use) and cleaning agent, represent respectively ~ 20% and ~ 11% of the total quantity of EGBE used.

Information for other minor uses for EGBE is also available (light dimmed cells in Table 2.3 ). The sum of the other uses represents about 10 % of the total use of EGBE. Due to the low toxicity of the substance, these uses might be evaluated in a second tim e, if they show any concern. Moreover, some uses have been reported in the past that seem to no longer exist or errors could have occurred when allocating volumes to end-uses (dimmed lines in Table 2.3 ). For some of these uses, the percentage of tota l use has been set at 0 since no information has confirmed that EGBE was still used in this sector . For some other uses (e. g. oil spill dispersant),

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figures reported does not seem to indicate a real a nnual use of the substance since stockpiles could be made during several years w ithout using the produc t (see Section 3.1.2.2.5).
Another information source (OECD, 1996), presented in Table 2.4 , shows supplies of EGBE to EU market. It gives a more detailed view on the major uses for EGBE.

Table 2.4 EGBE end use information (OECD, 1996)

End use kt/year (% of total use)
Anticorrosion coatings 2.6 (2.9%)
Can coatings 9 (10.0%) Coil coatings 6.5 (7.2%)
Decorative trade coatings* 15.5 (17.2%) General industrial coatings* 16.8 (18.7%)
Automotive OEM coatings 1.3 (1.4%) Automotive OEM coatings* 6.5 (7.2%)
Wood coatings* 1.3 (1.4%) Detergents / cleaners 3 (3.3%) Inks (screen) 5 (5.6%)
Feedstock for EGBEA production 11 (12.2%)Industrial use

Subtotal 78.5 (87.3%)

Decorative retail coatings* 10.4 (11.6%)

Detergents / cleaners 1 (1.1%)Consumers uses

Subtotal 11.4 (12.7%)

Total 89.9 (100%)

* Water based In this study, the detailed percentages for paint and de tergent uses presented in Table 2.4 will be applied to the volumes retained in Table 2.3 . These figures will be presented with further explanations given in the following pa ragraphs concerning each reported use.

2.2.1 Paints and coatings
Glycol ethers and their acetates are effective in many surface coating technologies including, for example, high and low solid coatings, waterborne emulsions, water-reducible coatings. As ‘true solvents’ glycol ethers are able to dissolve many resins in all proportions, and are particularly useful in promoting flow properties. They also encourage good gloss and film properties by maintaining the resin(s) in solution throughout the film formation process (Shell Chemicals).
Detailed percentages for different paint t ypes were calculated from the figures in Table 2.3 and end use information for EGBE (OECD, 1996). They are presented in Table 2.5

.

16CHAPTER 2. GENERAL INFORMATION ON EXPOSURE

Table 2.5 Detailed view of paint uses

End use % of total use Typical maximum % of EGBE in formulated products
General industrial coatings* 14.11 3 Decorative trade coatings* 13.02 1.5 (3**)
Decorative retail coatings (consumers uses)* 8.73 1.5 (3**)

Can coatings 7.56 7 Coil coatings 5.46 7 Automotive OEM coatings* 5.46 8 Anticorrosion coatings 2.18 1 Automotive OEM coatings 1.09 2 Wood coatings* 1.09 2

Total 58.70

* Water based ** Personal communication, OSPA (value used for the assessment)

2.2.2 Detergents, cleaners
Glycol ethers can be used in al l types of cleaners. They reduce surface tension and are excellent coupling agents. Working in conjunction with a surf actant, they facilitate wetting of the surface, penetration of the soil, and help to suspend th e soil in water until it is wiped or rinsed away.
The analysis of uses of 434 cl eaning products identified during risk assessment of EGBE in cleaning products (NICNAS, 1996) revealed th e range of EGBE concentrations below.

Table 2.6 EGBE concentrations in cleaning products

EGBE conc. % of total products Examples of cleaning products

< 10% 68

10-30% 14

30-60% 1

10-60% 2

> 60% 1

unknown 14 Surface cleaner (multi-purpose cleaners) Floor stripper
Glass / window cleaner ; Oven cleaner Carpet cleaner Laundry detergent

Rust remover

In this study, most of the cleaning products ha ve an EGBE concentration < 10%. However, extreme values go from < 1% to 94%. The fraction of chemical in formulated detergent for both industrial and consumer uses (0.1) quoted in th e OECD, 1996 will be retained for this study. In this reference EGBE end use volumes show that 3⁄4 (~ 8.50% of total use) of EGBE used as a detergent takes place in the industrial domain and that only 1⁄4 (~ 2.83% of total use) remains for

consumers uses.

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2.2.3 Chemicals used in synthesis: in termediate for EGBEA production
EGBE is used in the synthesis of acetate esters as well as phthalate and stearate plasticisers. Although the major use of EGBE as an intermediate is in 2-butoxyeth anol acetate synthesis, it is also employed for the synthesis of di(-2butoxyethyl) phtha late (ATSDR, 1998).

2.2.4 Printing inks
Combination of glycol et hers and other solvents can be used to achieve the right evaporation rates, levelling properties, water miscibility and so lubility for ink formulations. They can also be used to adjust surface tension and viscosity, promoting deep and even penetration of inks in soft substrates (Shell Chemicals). The typical maximu m percentage of EGBE in formulated printing inks is 20% (OECD, 1996). Specifi c information shows a decline in this particular use due to the switch to radiation cure d screen printing inks.
Rastogi, 1991 analysed a series of printer’s inks in order to characterise their levels of organic solvents. EGBE was found in 2/29 samples and id entified, for example, in printer’s inks for screen printing on paper and paper boards. The EG BE concentrations range in such formulated products was 0.1-0.4%.
The value quoted in OECD, 1996, 20% , will be retained as the ty pical percentage of EGBE in printing inks.

2.2.5 Other uses
Oilfield chemicals (2.57%): oilfield chemicals include brines and brine components used as completion, workover 4 and drill-in base fluids. Other en d-use and intermediate applications include antifoams, drilling lubricants, emulsifi ers, corrosion inhibitors , surfactants, oilfield biocides and scale inhibitors. They can be used in offshore platforms. Based on a search of patents on such products (personal communication, OSPA), typical concen trations are in the range 5-40%. However, one product used for th e cleaning of oilfield equipment has been reported with concentrations of EGBE be tween 60 and 100%. This latter product is a concentrated one intended for dilution before use.
Metal cleaning (2.06%): metal cl eaning/degreasing is used in industries where the production process includes fabricating and/or assembling me tal parts: mainly the automotive, aviation, appliance and railroad industries. During the various steps of th e production process, metal parts must be cleaned of oils, fluxes and grease.
Electronic industry (0.62%): among the uses of glyc ol ethers in electronic industry, those in copper clad laminating, lamina tes or photoresistors are re ported (Shell Chemicals).
Pharmaceuticals (0.51%): glycol ethers are reported to be process chemicals in the synthesis of pharmaceuticals. Usually they provide the medium where reaction takes place but they are also used in separation steps in order to obtain the desired drug or to maximise its purity.
Leather treatment operations (0.51%): use as a so lvent in leather protect ion system confirmed by appropriate EU trade association (OSPA enquiries ). Nevertheless, EGBE is being substituted by other solvents (personal communication from the European Leather Industry).

4 Major maintenance or remedial treatments.

18CHAPTER 2. GENERAL INFORMATION ON EXPOSURE

Glycol ethers are used in aqueous degreasing systems for sheep and goatskin leather at concentrations of 1-5% (worst case). Only EGBE is reportedly us ed in this part of the process. Usage rates can therefore be calculated as up to 3 g/kg of dry finished leather (worst case based on 5% EGBE in formulation). Based on the to tal production information in the TGD of 314 Mm 2 leather per year, 25% of whic h is sheep/goatskin, and assumi ng a leather weight of 2 kg/m 2 would suggest total GE use in this application of around 500 tonnes/year.
Glycol ethers are also used in leather finishing. EG BE is quoted as being used in this application, which is effectively a “coating” operation wh ere a preparation is applied by air atomised spraying in a spray booth. Actual us age rates are quoted as up to 0.4 g/kg of dry finished leather, which, following the above assumptions, would l ead to total GE consumption of up to 250 tonnes/year. There will in addition be wastage from over spray and emissions to water from the over spray control systems. However, since th is is a coating operation, it is already covered by the existing painting scenarios. Consequently, ther e will not be a separate risk characterisation for this use.
Adhesives (0.51%): in the industry of adhesives and sealants, glycol ethers are employed as solvents and enter formulated products. They are employed both in domestic and industrial adhesives. EGBE is most likely us ed in water-based adhesives. A percentage of 10% of EGBE in formulation will be adopted for the assessment. This figure is similar to the highest figures used for water-based coatings (personal communication, OSPA).
Cosmetics/Personal care (0.51%): the solvent a nd coupling properties of glycol ethers are utilised in cosmetic formulations. They can be used to stabilise emulsions used in lotions, preventing the active ingredient particles fr om dropping out of suspension. This helps to maintain the aesthetic appearance of the product and can increase shelf-life. That usage is now believed to be superseded by DEGBE (OSPA e nquiries). Actually, EGBE is used in few cosmetic formulations and is only permitted in hair dyes up to a concentration of 10%.
Fire foams (0.31%): EGBE can be used in synthe tic foam formulations. No firm information was available but an estimation of the market size would be 1-3 kt/year. Some of this would be exported however (OSPA enquiries). Most synthetic foams use glycol ether solvents as a foam booster and freezing point depressant. Butyl glycol ether use is now more and more superseded by other glycol ether. The solvent composition of fire foams is typically 16% as the concentrate but 0.5% in use (personal communication, OSPA).
Agricultural products (0.21%): the use of EGBE as a solvent or a co-s olvent in pesticide formulations represents a minor application fo r this chemical. EGBE is used as a coupling solvent to help disperse the activ e component in water when the conc entrate is diluted for use. It may be used at a significant le vel in the concentrate but thes e are usually diluted manyfold before use. For a conservative worst case “in use” figure, 1% will be retained (this would correspond to 1 L diluted in 100 L which represents a low dilution f actor compared to typical use of pesticides for field application).

Construction chemicals: EGBE is an additive for construction materials. It is used as a water-reducing agent for example.
Cutting oils: declining use in soluble oils. Glycol ethers used ‘very occasionally’ at use levels of 5-7%. Total EU sales in 1996: 110 kt/y ear for such oils. As a worst-ca se, if 5% of oils use glycol ethers and all assumed to be EGBE, total market would be a maximum of 400 tonnes/year (~ 0.41% of total use) (OSPA enquiries).

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Oil spill dispersants: this use was reported to be declining. It is now only found in three of the 44 dispersants approved for use in UK waters. Total stocks of dispersants in EU are estimated at 5 kt and the estimated use in EU woul d be < 300 tonnes/year (OSPA enquiries).
Photographic solutions: EGBE seems to be no longer used in photographic solutions.
Rubber, oil industry: EGBE is reporte d as used in polymer manufacturing.

20

### 3 ENVIRONMENT

3.1 ENVIRONMENTAL EXPOSURE
The level of exposure of the environment to a chemical depends on the quantities and compartments of release and subsequent de gradation, distribution and accumulation in the environment. This section discusses the behaviour of EGBE and its releases into the environment.

3.1.1 Environmental fate

3.1.1.1 Degradation in the environment

Hydrolysis
No experimental data are available on hydrolysis . However, alcohols and ethers are generally resistant to hydrolysis (Lyman et al., 1990).

Photodegradation
If EGBE is present in ambient air it is expected to exist almost entirely in the vapour phase, based on a vapour pressure of 1 hPa at 20°C, where vapour phase reactions with photochemically produced hydroxyl radicals may be important. A QSAR method is applied for a first estimation of primary transformation rate s (SRC, 1988, cited in E nvironment Canada and Health Canada, 2000): the overall OH rate consta nt for EGBE has been calculated by the AOP program v1.90 and is estimated to be 2.3.10 -11 cm 3 /molecule.second at 25°C. The estimated value corresponds to an atmospheric half-l ife of about 16.7 hours at an atmospheric concentration of 5.10 5 hydroxyl radicals/cm 3 . Atkinson, 1987 has calculate d an OH rate constant for EGBE of 1.96.10 -11 cm 3 /molecule.second.
Estimated half-lives values for EGBE, based on photooxydation, are also mentioned in Howard et al., 1991, cited in Environment Canada and Health Canada, 2000. They range between 3.3 and 32.8 hours.
Values retained in this study to represent the ph otodegradation ability of EGBE were taken from a study performed by Aschmann and Atkinson, 1998. EGBE rate constants were measured using a relative method, i.e. measures were made in directly using a well-known compound, for three different sensitizers: OH, O 3 and NO 3 . Test conditions were as follow: light spectrum > 300 nm, EGBE concentration was 4.71.10 -3 mg/L. Measurements revealed that reactions between EGBE and O 3 or NO 3 are of minor or negligible importance as tropospheric loss processes. Indeed, rate constants with NO 3 and O 3 have been measured at 3.10 -15 cm 3 /molecule.second and < 1.1.10 -19 cm 3 /molecule.second respectively. The main loss process for EGBE in ambient air is the reaction with OH radicals. The corre sponding measured rate constant was 2.94.10 -11 cm 3 /molecule.second.
Using this last data, a pseudo firs t order rate constant for degrad ation in air is calculated using TGD (EC, 2003): K deg-air = 1.27 d -1 . This rate constant gives a half-life value of ~ 13 hours for EGBE in air.

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Degradation products of EGBE after its reaction with OH radicals have also been measured by Tuazon et al., 1998. An EGBE concentration of 4.7.10 -3 mg/L was used for the experiment performed at 25°C. Several products have b een identified: propiona ldehyde (CAS 123-38-6), butyraldehyde (CAS 123-72-8), butyl formate (CAS 592-84-7) and 2-hydroxyethyl formate (CAS 628-35-3), 3-hydroxybutyl formate, 2-hydr oxybutyl formate and an organic nitrate.

Since EGBE does not absorb ultraviolet radiation within the solar spectrum (> 290 nm), direct photolysis in the atmosphere is not expected to occur (Silverstein and Bassler, 1963, cited in HSDB, 2000).

Biodegradation
The available aerobic biodegradation test results for EGBE are summarised in Table 3.1 . A number of tests were carried ou t according to (inter national) standard te st guidelines. Although the current information on several technical aspect s is incomplete for some biodegradation tests the total data set available is regarded as being sufficient to draw conclusions on the biodegradation poten tial of EGBE.

Table 3.1 Biodegradation test results for EGBE

Test # Type of test Detection Result Day Method Conc. Of TS Conc. Of inoculum Reference Validity

1 BOD 5 - test O 2 uptake 73% (BOD/ThOD) 5 APHA n°219, 1971 Unknown 10 mL 2,c Bridié et al., 1979a Valid with restrictions

2 BOD 5 - test O 2 uptake 31% (BOD/ThOD) 5 APHA n°219, 1971 Unknown 10 mL 1,c Bridié et al., 1979a Valid with restrictions

3 BOD 5 - test O 2 uptake 8% (BOD/ThOD) 5 DEV H5 A2 Unknown River water taken below

a factory effluent Knappe and Popp, 1974 Valid with restrictions

4 BOD 20 -test O 2 uptake 75%

70%

64%

29% (BOD/ThOD) 20

15

10

5 APHA n°219, 1971 3, 7 and 10 mg/L** 3 mL 4 (filtered seawater

seed) Price et al., 1974 Valid with restrictions

5 BOD 20 -test O 2 uptake 88%

82%

74%

26% (BOD/ThOD) 20

15

10

5 APHA n°219, 1971 3, 7 and 10 mg/L** 3 mL 1,a Price et al., 1974 Valid with restrictions

6 Inherent biodeg Test DOC 95%

92%

8% 8

7

4 Zahn-Wellens test (OECD 302B) 750 mg/L 33 mg/L 1,b BASF AG, 1980 Valid with restrictions

Table 3.1 continued overleaf

22CHAPTER 3. ENVIRONMENT

Table 3.1 continued Biodegradation test results for EGBE

Test # Type of test Detection Result Day Method Conc. Of TS Conc. Of inoculum Reference Validity

7 Inherent biodeg Test DOC 100% 28 OECD 302B 500 mg/L Unknown 1,a Huels, 1982* Lack of data

8 Inherent biodeg Test DOC 100%

63%

22% 5

3

1 OECD 302B 450 mg/L Unknown 1,a Hoechst, 1976 Valid with restrictions

9 Ready biodeg Test O 2 uptake 88% 28 OECD 301C 100 mg/L 30 mg/L 3,b CEFIC, 1993* Lack of data

10 Ready biodeg Test DOC 95% 28 OECD 301E 10 mg/L

DOC Unknown 1,a Huels, 1982* Lack of data

11 Ready biodeg Test O 2 uptake 72%

57%

5% (BOD/ThOD) 20

10

5 Unknown Unknown Inoculum from a WWTP Dow cited in Gonsior, 1990 Valid with restrictions

12 Ready biodeg Test O 2 uptake 96% 14 OECD 301C 100 mg/L 30 mg/L 1,a CITI, 1992 Valid with restrictions

13 Ready biodeg Test O 2 uptake 75%

70%

47% 28

15

5 OECD 301D Unknown Unknown 1 Waggy et al., 1994 Valid with restrictions

a) Activated (domestic) sewage b) Activated sludge from (industrial) STP c) Filtered effluent from STP 1) Non-adapted inoculum 2) Adapted inoculum 3) No information on adaptation or non-adaptation of inoculum available 4) Seed source maintained by adding small amounts of settled raw wastewater * Original report not available ** Results of the biodegradation test were expressed taking into account all concentrations of EGBE used (“weighted average”) Several tests have been conducted according to APHA (American Public Health Association) method n°219. In tests 1 and 2, the Biological Oxygen Demand was measured after five days whereas in tests 4 and 5, the BOD was measured after 20 days. The four tests were performed with an inoculum constituted by filtered effluent from a STP excepted for test 4 in which the inoculum came from a filtered seawater seed. The domestic source of the inoculum was only specified for test 5. In tests 2, 4 and 5, the inoc ulum was reported as non-a dapted contrarily to that used in test 1. In test number 4, the inoculum source was maintained by adding small amounts of settled raw wastewater. In the f our tests, the biodegradation was quantified comparing the measured BOD to the Theoretical Oxygen Demand (ThOD). Concerning the three tests showing the biodegradati on of EGBE in the presence of non-adapted inoculum (#2,4,5), they all show similar values after five days even for the one performed with artificial saltwater (#4), respectively 31, 29 and 26%.
Tests number 6 to 8 carried according to the OE CD guideline 302B (Zahn- Wellens test) tend to demonstrate the inherent biodegradability of EGBE. Biodegradation percentages were > 70%

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, 2006

after 14 days in tests 6 (95% after 8 days) and 8 (100% degraded after 5 days) and reached 100% after 28 days in test 7. Each test was performe d using a non-adapted inoculum from domestic sewage (#7 and 8) or industrial STP (#6).
All ready biodegradation tests (#9-13) have shown a biodegradation > 60%. Excepted test number 9 where the inoculum were adapted and came from an industrial waste water treatment plant, all other tests have used a non-adapted inoc ulum from a domestic se wage (as specified in the OECD guidelines n°301). Tests 11, 12 a nd 13 where EGBE biodegradation reaches, respectively, 57% after 10 days, 96% after 14 days and 47% after 5 days (70% after 15 days), tend to confirm that the 10 days time window criterion is fulfilled.
To conclude, according to standard tests on read y biodegradation and further experimental data which confirmed high biodegradation rates, EGBE can be regarded as readily biodegradable.
As no result from biodegradation simulation test s in STP, surface freshwater, surface saltwater and soil is available, the degradation rates have to be estimated based on the “ready biodegradability” classification and the partition behaviour of EGBE according to the method described in the Technical Guidance Document for risk assessment of new and existing chemicals (TGD - EC, 2003). Results of this estimation are presented in Table 3.2

.

Table 3.2 Estimated biodegradation rate constants for EGBE in WWTP, surface water, soil and sediment
Compartment / medium Biodegradation rate Half-life Activated sludge (WWTP) K STP

= 1 h -1 0.7 hour

Surface water K freshwater = 4.7.10 -2 d

-1 15 days

Marine water K saltwater = 1.4.10 -2 d

-1 50 days

Soil* K soil = 2.3.10 -2 d

-1 30 days

Marine and freshwater sediments ** K sed

= 2.3.10 -3 d

-1 300 days * Biodegradation rates in sediment and soil take account of adsorption to solid matter (K oc

=27.3 L/kg, see below) ** Biodegradation rate in sediment takes account of the aerobic fraction of this compartment (0.1)

3.1.1.2 Distribution

Volatilisation
Based on the measured Henry’s law constant of 0.08 Pa.m 3 /mol at 25°C (Kim et al., 2000), the air-water partitioning coefficient (K air-water ) can be calculated. K air-water of 3.23.10 -5 indicates that volatilisation of EGBE from surface water and moist soil is expected to be very low.

Adsorption/desorption
Using the log K ow of 0.8 and according to the TGD (EC, 2003) (QSAR for soil and sediment sorption for nonhydrophobic chemicals) a K oc of 27.3 L/kg can be estimated. The solid/water partition coefficients in each co mpartment can be calculated as supplied in TGD (EC, 2003) (see Table 3.3 ).

24CHAPTER 3. ENVIRONMENT

Table 3.3 Estimated solids / water partition coefficients
Water / Compartment OC fraction in solid phase (F OCcomp ) Solids / water partition coefficients Total compartment water partition coefficient

Soil / water 0.02 K p_soil = 0.55 L/kg K soil-water = 1.03 m 3 /m

3

Sediment / water 0.05 K p_sed = 1.37 L/kg K sed-water = 1.49 m 3 /m

3 Suspended matter / water 0.1 K p_susp = 2.73 L/kg K susp-water = 1.58 m 3 /m

3

Distribution in the environment
The following theoretical distribution in the environment has been calculated using the multimedia fugacity model EQC (MacKay level I) and the physico-chemical properties given in Section 1.3.

Table 3.4 Calculated distribution of EGBE in the different compartments of the environment
Compartment % EGBE

Air 0.24

Water 99.20

Soil 0.55

Sediment 0.01
Regarding these results, the hydrosphere is the preferential target of the substance in the environment.

Distribution in STPs
Based on physical chemical properties discussed in this study (log H = -1.1 and K oc = 27.3 L/kg) as well as the biodegradation rate of 1 h -1 in STP, the elimination through biodegradation can be estimated with the model SIMPLETREAT in Table 3.5

.

Table 3.5 Estimated distribution in a STP (SIMPLETREAT)

Designation %

Air 9.54.10 -2

Water 12.6 Sludge 2.57.10 -1 % degraded 87.0

% removal 87.4

3.1.1.3 Accumulation
No experimental data on bioaccumulation is av ailable. Therefore, BCF-values for fish and earthworm are estimated using the log Kow of 0.8. The estimated BCF-values amount to 0.97 and 1.6 for, respectively, fish and worm. Anothe r calculated value is quoted: a BCF of 2.5 has been estimated by Syracuse program (US EPA and Syracuse Research Corporation, 2001).
In view of these BCF, EGBE is expected to have a low bioaccumulation potential.

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3.1.2 Environmental releases
The regional and continental releases include all relevant life cycle stages of EGBE. For production, it is assumed that there is only one production site in the region. The exposure assessment is based on the EU Technical Gu idance Documents (TGD - EC, 2003) applying the European Union System for the Evaluation of Substances, EUSES (EC, 2004).

3.1.2.1 Release from production
Ethylene oxide and anhydrous 5 alcohol (n-butanol for the production of EGBE) are added continuously to a reactor containing catalyst that operates at elevated temperature and pressure. To keep the proportion of lower molecular weight products as a high percentage of total production, a large excess of alcoho l is used. Unreacted excess al cohol is recycled using an alcohol recovery section (A and B parts in Figure 3.1 ). Some of the EGBE is also recycled as part of the production control process. The remain ing reactor stream is dr awn off for purification using a train of distillation columns (C, D and E parts in Figure 3.1 ). Relatively pure mono and diethylene glycol alkyl ether streams are drawn off along with a heavy stream (mainly tri but also containing penta and tetraet hylene glycol alkyl ether). Th e remaining low volume polymeric residue stream is either burnt or sold.
During normal operations, the process is entirely closed (due to physical properties of ethylene oxide: boiling point of 10.7°C) with little opportunity fo r environmental release. Such exposure is only likely during sampling, filling or loadin g operations along with campaign changes (most of producers revealed periodic campaigns of EGBE synthesis), when the plant is switched to a different glycol ether (differe nt alcohol or using propylene ra ther than ethylene oxide).

Figure 3.1 Basic schematic of t he EGBE productio n units (OSPA)

REACTO R A B C D E

Reaction section (Oxide-Alcohol-Catalyst) Alcohol removal Distillation section Mono-GE Di-GE Tri-GE... Alcohol + mono-GE

Polymeric
stream

5 Presence of water during the reaction is not expected as this reacts viol ently with ethylene oxide under the prevailing conditions.

26CHAPTER 3. ENVIRONMENT

Releases to water

Data of releases to water for EG BE production sites in Europe (see Table 2.2 ) are presented in Table 3.6 (Note: the site numbers in Table 3.6 do not directly corre spond to the order of companies in Table 2.2 ).
As there are only five EGBE production sites in Europe, the regional pro duction will not be set at 10% of total EGBE production (TGD default) but at the maximum vol ume produced at one site. The amount remaining corre sponds to the continental produc tion. Fraction releases and number of days of emission are taken fr om Table A1.1 and Table B1.5 (TGD - EC, 2003; Chapter 3 – Appendix I), with Main Category equa l to Ib (substances produced in a continuous production process) for EGBE is produced in a continuous production process.
All EGBE producers reported that releases to water enter a sewage treatment plant. During this step, 87.4% of EGBE is expected to be remove d by degradation processe s. When more precise data were available (EGBE concentration in STP effluents) they were also used.

Relevant data for the calculati on of Predicted Environmental Con centrations (PEC) in a STP and local concentrations in the aqua tic compartment are presented in Table 3.6 . When such data was not available the effluent concentration leaving the STP (PEC STP ) has been calculated according
to Equation 3.1 . This PEC STP is divided by a dilution fact or so as to obtain the local concentration in surface water - Clocal aqua (see Equation 3.2 ).

Equation 3.1 Calculation of PEC STP

STP STPwater water STP EFFLUENT F Elocal PEC 6 10 ××

=

Elocal water local release to waste wa ter during episode [kg/d] F

STPwater fraction of emission directed to water by STP 0.126 EFFLUENT STP effluent discharge rate of STP [L/day] PEC STP EGBE concentration in the STP effluent [mg/L]

Equation 3.2 Calculation of Clocal aqua

DILUTIO N PEC Clocal STP
aqua =

PEC STP concentration of the substance in the STP effluent [mg/L] Clocal aqua local concentration in surface wa ter during emission episode [mg/L] DILUTION (STP flow + river flow ) / STP flow [-]

The calculation of Clocal aqua is reduced to this equation due to the low adsorption of EGBE on suspended matter (Kp susp = 2.73 L/kg).

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Table 3.6 Aquatic emission data from production sites of EGBE in EU

Site # Emission in water

1 Reference years: 2001 and 2002 Release to wastewater treatment plant: 10* kg/day (300* days of production). Assuming 87.4% removal in STP: 1.3* kg/day in STP effluent. Before discharge to receiving waters, the effluent undergoes a dilution by a factor of 100 (mixing with seawater). Receiving water is the sea. The r eal dilution for marine environment is unknown. The releases of EGBE occur in a region where the tidal influences are really low. For those particular seas it is proposed in the TGD to use only a dilution factor of 10 instead of 100.
Flow of STP = 7,000* m 3 /day.

PEC STP

= 0.18* mg/L and Clocal aqua = 1.8.10 -4

* mg/L

2 Reference year: 1996 (production and processing volumes in 2000 were however comparable) Release to water: 1,680* kg/day in wastewater treatment plant influent and 8.7* kg/day in effluent (such values give 99.5*% removal of EGBE in STP). Emissions are calculated as a worst case using 90th percentile of daily measured concentrations in effluent, average STP flow and 365* days/year. Both production and processing releases are included.

Flow of STP = 5* m 3 /s; 10th percentile of receiving water flow = 734* m 3 /s; dilution in receiving water = 150*.

PEC STP

= 2.10 -2

* mg/L ; Clocal aqua = 1.3.10 -4

* mg/L Assuming 87.4% removal in STP: PEC STP = 0.49 mg/L ; Clocal aqua = 3.3.10 -3 mg/L

3 Reference year: 2000 Release to water: 21.9* kg/day in STP influent (365* d/y). Assuming 87.4% removal in STP: 2.8 kg/day in STP effluent.

Flow of STP = 0.83* m 3 /s; 10 th percentile of receiving water flow = 18.4* m 3 /s; dilution in receiving water = 23*.

PEC STP

= 3.9.10 -2 mg/L ; Clocal aqua = 1.7.10 -3 mg/L

4 Reference year: 2000 Release to water: 7.1* kg/d in STP influent (140* d/y). Assuming 87.4% removal in STP: 0.9 kg/d in STP effluent. Concentration in STP influent = 0.2* mg/L; concentration in STP effluent = 1.10 -3

* mg/L.

PEC STP

= 8.9.10 -2 mg/L ; Clocal aqua = 2.2.10 -3 mg/L (assuming a dilution factor of 40 and a STP flow of 10,000 m 3 /day – default value for chemical industry, Cf. emission sc enario documents, Chapter 7 of the TGD, E.C., 2003)

5 No data but total annual site production for EGBE < 1kt. No process water discharge is reported
* Original data provided by industry or calculated with orig inal data. Other data is calculated using default TGD values

Releases to air
Release data to air for EGBE produc tion sites in EU are presented in Table 3.7 . Local concentrations in air have been calculated based on information submitted by the producers, except for site 5.

28CHAPTER 3. ENVIRONMENT

Table 3.7 Atmospheric emissions of EGBE from European producers

Site # Release to air (kg/d) Clocal air,ann * (mg/m 3 ) Year

1 1.9 including emissions from plant and fugitive emissions (300 days/year) 4.34.10 -4 2002

2 0.6 at production and 5.4 at processing (365 days/year) Total (production + processing) 6 1.67.10 -4 mg/m 3 at production and 1.50.10 -3 at processing Total (production + processing) 1.67.10 -3 1996 (production and processing volumes in 2000 were however comparable)

3 ~0.1 (365 days/year) 2.78.10 -5 2000

4 ~0.4 (140 days/year) 4.27.10 -5 2000

5 No data but total annual site production for EGBE < 1kt. * EGBE concentration in air calculated at a 100 m distance from the source (annual average)

Releases to soil
Release data to soil for EGBE produc tion sites in EU are presented in Table 3.8 . Local concentrations in soil have been calculated based on information submitted by the producers, except for site 5.

Table 3.8 Emissions of EGBE to soil from European producers

Site # Total deposition flux during emission episode (μg.m -2

.j -1 ) Annual average deposition flux

(μg.m -2

.j -1 ) Local concentration in agricultural soil after 30 days (μg/kg ww) Local concentration in agricultural soil after 180 days (μg/kg ww) Local concentration in grassland after 180 days (μg/kg ww)

1 0.76 0.63 33.40 10.30 3.84

2* 2.40 2.40 0.27 0.27 0.50

3* 0.04 0.04 0 (5.10 -3

) 0 (5.10 -3

) 0 (8.10 -3

)

4* 0.16 0.06 0 (7.10 -3

) 0 (7.10 -3

) 0.01

5 No data but total annual site production for EGBE < 1kt.
* At this production site STP sludge is incinerated

3.1.2.2 Release from formulation, processing and private use
Generic exposure scenarios are used to estimate the releases from formulation, processing and private use of EGBE, as no actual data are available. The scenario s are based on the different use categories of EGBE (see Section 2.2 ). An overview of the various environmental exposure scenarios for formulation, processing a nd private use of EGBE is given in Table 3.9

.

Table 3.9 Environmental exposure scenarios for form ulation, processing and private uses of EGBE

Scenario names Designation Life cycle step

Paints I F / Paints I P ¤ General industrial coatings (water-based) Formulation/Processing

Paints II F / Paints II P ¤ Decorative trade coatings (water-based) Formulation/Processing

Paints III F / Paints III U Decorative retail coatings (water-based) Formulation/Private use

Paints IV F / Paints IV P ¤ Can coatings Formulation/Processing

Table 3.9 continued overleaf

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Table 3.9 continued Environmental exposure scenarios for formulat ion, processing and private uses of EGBE

Scenario names Designation Life cycle step

Paints V F / Paints V P ¤ Coil coatings Formulation/Processing

Paints VI F / Paints VI P ¤ Automotive OEM coatings (water-based) Formulation/Processing

Paints VII F / Paints VII P ¤ Anticorrosion coatings Formulation/Processing

Paints VIII F / Paints VIII P ¤ Automotive OEM coatings Formulation/Processing

Paints IX F / Paints IX P ¤ Wood coatings (water-based) Formulation/Processing
Detergents I F / Detergents I P Industrial detergents Formulation/Processing
Detergents II F / Detergents II U Domestic detergents Formulation/Private use

Intermediates I P Intermediate for chemicals synthesis Processing

Inks I F / Inks I P ¤ Printing inks Formulation/Processing

Oilfield I F / Oilfield I P ¤ Oilfield chemicals Formulation/Processing

Metal I F / Metal I P ¤ Metal cleaning Formulation/Processing

Elec I P Electronic industry Processing

Leather I P Leather treatment operations Processing

Pharm I P Pharmaceuticals Processing

Adhesives I F / Adhesives I P Adhesives Formulation/Processing

Cosmet I F / Cosmet I U Cosmetics / Personal care Formulation/Private use Agri I F / Agri I P Agricultural products Formulation/Processing

Fire I F / Fire I P Fire foams Formulation/Processing
P Processing F Formulation U Private use ¤ For these end uses there is a possibility that formul ation and processing steps take place at a same site. These cases will be treated dur ing risk characterisation.

3.1.2.2.1 Continental and regional releases
The total continental and regional EGBE emissi ons from formulation, processing and private uses are given in Table 3.10 . These results come from the calculation program EUSES (EC, 2004) and do not integrate the emi ssions due to the scenario “Agr i I” because of the scenario number limitations of the software. Nevertheless, the calculations have been made for this scenario in a separate file and it appears that the impact of emissions volumes for this scenario on total continental and regiona l emissions is negligible.

Table 3.10 Total continental and regional EGBE emissions

Air Water (total / waste water*) Soil

Continental 9.69.10 4 kg/day 4.90.10 4 kg/day/3.92.10 4 kg/day 6.81.10 3 kg/day

Regional 1.08.10 4 kg/day 5.56.10 3 kg/day/4.45.10 3 kg/day 7.59.10 2 kg/day

* It is assumed that 80% of the waste water is treated in a biological STP and the remaining 20% released directly into surface waters

30CHAPTER 3. ENVIRONMENT

3.1.2.2.2 Local releases: EGBE used in leather finishing operations

For the industrial use of EGBE in leather industry the dedicated scenario “leather processing industry’ has been used (for water releases). The emission of EGBE to waste water can be calculated using Equation 3.3 . Emissions to air and soil were estimated using A and B tables from Appendix 1 of the TGD (EC, 2003).

Equation 3.3 Calculation of waste water emission in leather processing industry 

## )1(

_ fix chemical product water local 

## F QQ E

− × × =

E local_water daily amount released to waste water [kg/d] Q

product mass of processed raw hide per day [15 t/d - default] Q chemical mass of substance used per mass of raw hide [3 kg/t 6 – personal communication from the European Leather Industry] F

fix

proportion of the substance chemically concerted or fixed to the raw hide during processing [0.01- Table A3.6]

3.1.2.2.3 Local releases: EGBE used for metal cleaning operations (processing)
During the various steps of the production process, metal parts must be cleaned of oils, fluxes and grease. Due to the properties of EGBE it is used in cleaning formulations as a wetting agent and dispersant. Typical cleaning fo rmulations contained 4% of th is kind of additives (cleaning formulations used for soak). In the calculation of EGBE daily releases to wastewater it will be assumed that the metal parts are cleaned by a static soak process. During this step of the process, losses of substance will be by dr ag out into the rinse bath and subsequent release of the rinse water. Here, as a worst case, th e following assumptions are made:

− it is considered that there is no return of the rinse water (F recycle = 0)

− the amount of solution removed from treatment bath due to drag out is taken as 0.3 L/m2 as a
worst case

− is a same way, the surface area of metal processed is taken at 40 m2/hr
If we consider a fraction of 0.04 for EGBE in the cleaning formulation, then, using a typical concentration of the formulation in the clea ning bath of 25-75 g/L, the concentration of substance in the treatment bath (C bath ) can be calculated as follow: 4%

. 75 g/L = 3 g/L.
Equation 3.4 is used to calculate the daily emission to wastewater:

6 This scenario only takes into account the use of EGBE in aqueous degreasing systems for leather. The other use of EGBE in the leather processing can be put in the category of “coating” operations and consequently is already covered by the dedicated scenarios for paints.

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Equation 3.4 Daily emission to wastewater during metal cleaning operations

process recycle bath metal process type dragout water process 

### TF C AREA Q Elocal

⋅−⋅⋅⋅ ⋅ = − 

### )1(10 3

, , , 

### dkg Elocal water process 

### /792.022)01(103403.0 3

, 

### =⋅−⋅⋅⋅⋅= −

Elocal process,water emission from process to water per day [kg/d] Q dragout,type amount of solution removed from treatment bath per unit area [0.3 L/m2] AREA process,metal surface area of metal proce ssed per hour [40 m2/hr] C

bath concentration of substance in treatment bath [3 g/L] 10 -3 conversion factor for g to kg [kg/g] F

recycle fraction of drag out returned to treatment bath [0] T

process number of hours worked per day [22, worst case]

In addition, it will be considered that the cleaning bath is dispos ed of every 4-8 weeks. Assuming a bath capacity of 1,000 L, the amount of substance released will be 3 g/L

. 1,000 L = 3 kg – source OECD, 2004. This release will be considered an intermittent emission.

Results are shown in Table 3.15 to Table 3.23

.

3.1.2.2.4 Local releases: EGBE used in o ilfield chemicals (processing)

a) Estimation of exposure concen trations using the CHARM model
EGBE has a wide range of uses as a solvent in paints and surface coatings, detergents and surface cleaners, inks or dyes. One of the report ed end use is as oilfield chemicals used on offshore platforms. Oilfield chemicals are in general brines and brin e components used as completion, workover and drill-in base fluids. A ge neric scenario has been applied in order to cover the use of EGBE in oilfield chemicals onshore (see Section 3.1.2.2.6).
The CHARM model (Thatcher et al., 2004) takes into account four different types of application, since they might be introduced into the envir onment in a different way. Application groups are:

− production chemicals (with injection chemi cals and surfactants as special cases)

− drilling chemicals (water based mud only)

− cementing chemicals (i.e., spacer and mixwater)

− completion and workover chemicals including we ll squeeze treatments and also pipeline hydrotest and preservation treating chemicals.
Due to the range of usual uses of EGBE, the application group, to which it belongs, seems to be “completion and workover chemicals”.
Two different scenarios are available dependi ng on the use of the chemicals during those operations. A distinction has been made betwee n surface/well cleaning and the other operations (other completion, workover, squeez e treatment and hydrotest chemical s). This is due to the fact that during cleani ng operations, as noted in the CHARM ma nual, discharge is considered to be

32CHAPTER 3. ENVIRONMENT

100% of the amount used, while for all other operati ons a fraction of the chem ical is retained in the formation by, for example, adsorption to the formation matrix during the operation.
The CHARM model considers that completion and workover chemicals are discharged in batches. Therefore a specific dilution factor ha s to be applied accounting for the discharge volumes. These releases should be considered as intermittent and compared to the appropriate PNEC for the risk characterisation.
The equations used for the calculation of PEC water for completion, workover squeeze treatment and hydrotest chemicals are presented below.
For surface and well cleaning chemical s, the PEC is calculated using:

PEC water = C i,cleaning × D batch, cleaning
With C i, cleaning = initial concentration of chemical in cleaning fluid (dosage, mg.L -1 )

D batch, cleaning = batchwise dilution fact or for cleaning fluids
For other completion, workover, squeeze treatm ent and hydrotest chemicals, the PEC is calculated using:

PEC water = f r × C i, completion × D batch, completion
With f r = fraction released - chemical

C i, completion = initial concentrat ion of chemical in completion and workover including squeeze treatments and hydrotest fluids (dosage, mg.L -1 )

D batch, completion = batchwise dilution factor for completion and workover including squeeze treatments and hydrotest fluids
Default values for the fraction released-chemical (f r ) and the batchwise dilution factor (D batch ) are reported in Table 3.11 as proposed in the CHARM model. Fo r cleaning chemicals, the fraction released should be set at 1 as the model a ssumes that the chemical is entirely released.

Table 3.11 Default values to be used for hazard assessment of completion and workover chemicals (specified as ‘cleaning chemicals’, other chem icals’, ‘squeeze treatments’ and ‘hydrotest chemicals’) (Thatcher et al., 2004).

Parameter Symbol Cleaning chemicals Other chemicals

Fraction released-chemical

Dilution factor at 500 m f r D batch n. r.
7.7 10 -5 (1:13,000) 0.1

7.1 10 -5 (1:14,000)

Parameter Symbol Squeeze treatments Hydrotest chemicals

Fraction released-chemical

Dilution factor at 500 m f r D batch 0.33

7.1 10 -5 (1:14,000) 1 0.001 (1:1,000)

EGBE has been reported in a manufactured pr oduct at concentrations between 60 and 100%. Different dilution ratios are suggested for this pr oduct, depending on the type of use. The lowest dilution ratio (1:10) applies to th e cleaning of drilling a nd construction equipmen t. This ratio will be used as a worst case and gives a concentratio n of EGBE in cleaning fl uids of 100 g/L. With this figure, the different scenarios ca n be used. Results are presented in Table 3.12

.

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Table 3.12 PEC intermittent for the use of oilfield chemicals containing EGBE on offshore platforms (marine compartment)

PEC seawater (mg/L)
Surface and well cleaning chemicals 7.7

Squeeze treatments 2.3

Hydrotest chemicals 100

b) Regulations on oilfield chemicals
In June 2000, OSPAR introduced Decision 2000/2 on a Harmonise d Mandatory Control System for the Use and Reduction of the Discharge of Offshore Chemicals. At the heart of OSPAR Decision 2000/2 are two R ecommendations: Recommendation 2000/4 on a Harmonised PreScreening Scheme for Offshore Chemicals, which facilitates the substitution of chemicals with certain characteristics by less hazardous alternatives and Recommendation 2000/5.
Decision 2000/2 and its supporti ng Recommendations entered into force on 16 January 2001. The Decision requires offshore chemicals to be ranked according to their calculated Hazard Quotients (HQ - ratio of Predicted Environmenta l Concentration (PEC) to Predicted No Effect Concentration (PNEC). It also obliges auth orities to use the CHARM “hazard assessment” module as the primary tool for ranking.
Details on the national regulations put in place in order to comply with OSPAR recommendations have been found for two count ries (United Kingdom and Norway). However other countries which have offshore oil and gas installations 7 may have similar regulations in place following harmonisation of such schemes in 1996.

− Norway
The Norwegian State Pollution Control Authority (SFT) regulates the use of drilling fluids/muds through discharge permits. Water based muds are tested under OSPAR formats for bioaccumulation potential and bio-degradability and given a discharge permit if judged to be environmentally friendly. Synthe tic muds are similarly evaluate d and can be given a discharge permit according to their properties. All oil-based muds are injected or taken to shore for treatment. The discharge of solids containing mo re than 1% oil, by weight, is forbidden - whether the drilling fluid is water- , oil- or synthetic-based. The re gulations also deal explicitly with well testing, workover and cementing.
Norwegian regulations:

1. Norwegian State Pollution Control Au thority (SFT). 1998. Requirements for Ecotoxicological Test ing and Environmental Assessmen t of Offshore Chemicals and Drilling Fluids. SFT, Oslo

2. Norwegian State Pollution Control Authority (SFT). 1999a. Environmental Monitoring of Petroleum Activities on the Norwegian Shelf; Guidelines 99:01. SFT, Oslo

3. Norwegian State Pollution Control Author ity (SFT). 1999b. Pollution Control Act, 1981. SFT, Oslo)

7 In some countries there are no offshore oil and gas inst allations in the OSPAR maritime area under the jurisdiction of the Contracting Parties (i.e. Belgium, Finland, France, Portugal, Sweden).

34CHAPTER 3. ENVIRONMENT

Norway uses the standard OSPA R “A” and “B” lists for offshore chemicals and requires that discharge of these “shall be reduced as much as possible, e.g., through recycling”. Operators are required to ensure the purity of the substances they use, with minimum contamination by other chemicals. Discharge of unused chemicals into the sea is expressly forbidden, even if they are on list A or B and their toxicity is therefore we ll known. All discharges mu st have a permit and chemicals not on the lists must be separately tested and notified.

− United Kingdom

The Offshore Chemical Notification Scheme (OCNS) was originally introduced in 1979. In 1993, the UK Government introduced a revised sche me, which classified chemicals using test protocols approved by the Oslo an d Paris Commissions (OSPAR). Th is was modified in detail, in early 1996, to meet the requirements of the OSPAR Harmonised Offshore Chemical Notification Format (HOCNF) whic h co-ordinates the testing requirements for oilfield chemicals throughout the NE Atlantic sector.
In June 2000, OSPAR introduced Decision 2000/2 on a Harmonise d Mandatory Control System for the Use and Reduction of the Discharge of Offshore Chemicals. In the UK this is to be administered under the Offshore Chemical Regu lations 2002 (OCR 2002) which came into force on 15th May 2002.
The UK operates the Offshore Chem ical Notification Scheme (OCNS 8 ). This is operated by the Centre for Environment, Fisheries and Aqu aculture Science (CEFAS ) on behalf of the Department for Trade and Industry (DTI). CE FAS is an Executive Agency of the UK Government's Department for Environment, Food and Rural Affairs (DEFRA). The OCNS requires the registration of all chemicals used in the actual exploration, exploitation and associated offshore processing of petroleum on th e UK Continental Shelf. Dossiers on individual products rather than substances have to be notified. The require d dossiers must include acute toxicity data on marine species from the three tropic levels as well as other data. Because individual products are notified, detailed in formation on usage rates and composition are available allowing more precise risk characterisations to be performed using the CHARM model and the products ranked according to the hazar d they present. However, for reasons of commercial confidentiality, the toxicity data , usage and composition information is not published.
EGBE has been registered by a number of supp liers to the industry under the name ‘EGMBE’ (ethylene glycol mono butyl ether), as shown below.

Table 3.13 Examples of EGBE containing products registered in UK
Product type or function Notifier Rating

Carrier solvent Baker Petrolite Silver

Drilling fluid Halliburton Gold

Well stimulant (30% EGBE) Baker Petrolite Gold

Gold is the highest possible rati ng out of 6 and is for products where the PEC:PNEC is less than one. Silver, the next highest, is for substances where the model PEC:PNEC is between 1 and 30. EGBE is also listed as a hydrotest chemical, but under a category of pr oducts where CHARM is

8 http://www.cefas.co.uk/ocns/index.htm

35EU RISK ASSESSMENT

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not regarded as suitable for assessing risk of use (not all uses are regarded as suitable for risk characterisation using CHARM). For this reason, it is only rated for hazard properties and falls into group E, the lowest hazard category available. It is also possible that EGBE and EGBE containing products may be registered under trade names (OSPA, personal communication).
Because they use detailed information supplied under confidentiality terms, specific assessments performed under such regulations should be regard ed as more precise than those carried out in this assessment. For instance, detailed inform ation is provided by the manufactures on actual usage and emission rates.

3.1.2.2.5 Local releases: EGBE used in oil spill dispersants (marine compartment)
The development of oil spill dispersants has started in the beginning of the 70’s with the increase in oil transportation and as a consequence the in crease in number of accidents. A summary of dispersants used t oday is provided in Table 3.14 and a list of agreed di spersants according to the Bonn Agreement is also available 9

.

Table 3.14 Dispersants used today, application methods and dosages (Bonn Agreement, 2001)

Standard name Generation Type Application method Solvent Dosage (dispersant / oil)

Conventional dispersants Second 1 Not diluted on ships No aromatic hydrocarbons 30 –100%

2 Diluted on ships Water-based (e.g., glycol ether) Concentrated dispersants Third
3 Not diluted on ships or airplanes Hydrocarbon-based 5 – 15% (concentrated

products)

It is difficult to know how often dispersants are used against oil sp ills. A survey of international uses of dispersants was performed by Lindgren et al., 2001. From this su rvey it seems clear that most of the time mechanical ac tions are recommended for combating oil spills and that the use of chemicals such as dispersants should be avoided as long as possible. This is particularly the case in the Baltic area, which falls under the HELC OM’s recommendations (Helsinki Commission; Denmark, Finland, Germany, Latvia, Lithuania, Poland, Sweden and the Russian Federation).
In specific situations dispersant s can nevertheless be used if the national authority approves it. This is also the case in the USA and Canada wh ere mechanical methods are used unless weather prevents the use of these methods.
In Belgium, France, United Kingdom and Germany, di spersants are used, part icularly in England where the total number of approved dispersants is high in comparison with other countries. However dispersants in these different count ries are applied under specific rules and recommendations and usually afte r approval by the government or the competent authority in this field. Apart from these theoretical considera tions, it seems that disper sants are nevertheless used in a lot of situations because it is often the easiest way to ma nage an oil spill (personal com. Cedre). Therefore small oil spills resulting from usual practice are most of the time treated with dispersants when of cour se this is possible.

9 This list of agreed dispersants will be updated in 2005.

36CHAPTER 3. ENVIRONMENT

Lessard and Demarco, 2000 report that the rate of usage of dispersants has gone up over the past 30 years and that dispersants have been successfu lly applied in half as many major spills during the 1990s as during the prior 25 years.
Among all reported dispersants some contain EGBE in non-negligible concentrations. This is the case for example of Wellchem Wella id 3315 (5% EGBE) and Corexit 9527 10 (38% EGBE). It was not possible to have the composition of all the agreed dispersants on the lists of the Bonn Agreement nevertheless from the two above examples it is clear that some of them contain high amounts of EGBE. Application rate s of dispersants depend on the amount of oil to be treated. Usual application rates seem to be around 5 to 15% from information compiled on product fact-sheets (Cedre, 2004).
EGBE sales for oil spill dispersant use have been reported in 2001 (191 tonnes). However, for the following years (2002 and 2003) no sale was repor ted. This is due to the mode of use of oil spill dispersant that required some stocks to be available. Indeed, oil spill dispersants are only used on occasions when an oil spill occurs in the environment and so may be used locally in relatively large amounts on very infrequent occasions . This should be conse quently treated as an intermittent release.
Figures on the number of oil spills detected ar e available for the Baltic Sea. Von Viebahn, 2002 reports that 250 to 650 oil spills are detected each year . It is also stated in the same report that the number of oil spills in the North Sea is higher, however exact figures are not available. More information on annual oil spills would be useful in order to propose a regional scenario. However due to a lack of time this is not possible at present.
In the same way, specific data (amount of oil re leased, dispersant used, etc.) are available on different oil spills, major as well as minor spills , in different locations. It is possible from the data available to construct worst case scenario s and assess local PEC, however this implies the development of specific and detailed emission scenario for major and minor spills. This could become difficult considering that a “typical oil spill” cannot be defined. Each presents unique circumstances, depending on location, quantity and t ype of oil, prevailing weather conditions etc.

It has also to be taken into consideration that the decision whether to use a dispersant is made with the regulatory authorities case-by-case and on a risk benefit basis. It is generally accepted that dispersants create adverse effects but th eir use is intended to mitigate the even worse potential effects of environmen tally hazardous oil products. For this reason, even if detailed composition information was available, it would s till not be possible to carry out such a risk benefit analysis within the framework of the Existing Substances risk evaluation process.

3.1.2.2.6 Local releases for other uses

Note: Tables used for local release estimates refer to TGD (EC, 2003), Tables A and B, Chapter 3, Appendix I; other Tables, Chapter 7.
The local release estimates for formulation, processing and use of EGBE are given in Table 3.15
to Table 3.20

.

10 This dispersant has been removed from the UK list of approved dispersant on 30/07/98 because it did not pass the Rocky Shore Test. However existing stock may have been used away from rocky shorelines in appropriate conditions.

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Table 3.15 Local releases of EGBE from formulation of paints

Scenario Paints I F Paints II F and Paints III F Paints IV F Paints V F
Main category Multi-purpose equipment Multi-purpose equipment Multi-purpose equipment Multi-purpose equipment

Total fraction and connected tonnage 0.1411/13,700 tonnes 0.2175 / 21,100 tonnes 0.0756/ 7,340 tonnes 0.0546 / 5,300 tonnes

Regional tonnage 1,370 tonnes 2,110 tonnes 734 tonnes 530 tonnes Typical max. % of EGBE 3 3 7 7 Number of days / Fraction of main source (Table B2.3)

300 / 0.6 (Table B2.3)

300 / 0.4 (Table B2.3)

300 / 0.7 (Table B2.3)

300 / 0.8

Release estimates (fraction)

- air

- waste water

- soil (Table A2.1)

0.005 0.003 0.0001 (Table A2.1)

0.005 0.003 0.0001 (Table 3.6)

0.03

0 0 (Table 3.5)

0.01

0 0 Amount released (kg/d)

- air

- waste water

- soil 13.7 8.2 0.3 14.1 8.4 0.3 51.4 0 0 14.1 0 0

Table 3.16 Local releases of EGBE from formulation of paints (continue)

Scenario Paints VI F Paints VII F Paints VIII F Paints IX F
Main category Multi-purpose equipment Multi-purpose equipment Multi-purpose equipment Multi-purpose equipment

Total fraction and connected tonnage 0.0546 / 5,300 tonnes 0.0218 / 2,120 tonnes 0.0109 / 1,060 tonnes 0.0109 / 1,060 tonnes Regional tonnage 530 tonnes 212 tonnes 106 tonnes 106 tonnes Typical max. % of EGBE 8 1 2 2 Number of days / Fraction of main source (Table B2.3)

300 / 0.8 (Table B2.3)

300 / 0.7 (Table B2.3)

300 / 0.8 (Table B2.3)

300 / 0.8

Release estimates (fraction)

- air

- waste water

- soil (Table A2.1)

0.005 0.02 0.0001 (Table A2.1)

0.005 0.02 0.0001 (Table 3.15)

0.03

0 0 (Table 3.19)

0.02 0.01

0 Amount released (kg/d)

- air

- waste water

- soil 7.1 28.3 0.1 2.5 9.9 0 8.5 0 0 5.7 2.8 0

38CHAPTER 3. ENVIRONMENT

Table 3.17 Local releases of EGBE from processing of paints

Scenario Paints I P Paints II P and Paints III U * Paints IV P Paints V P

Main category Non dispersive use Wide dispersive use Non dispersive use Non dispersive use

Total fraction and connected tonnage 0.1411 / 13,700 tonnes 0.2175 / 21,100 tonnes 0.0756 / 7,340 tonnes 0.0546 / 5,300 tonnes Regional tonnage 1,370 tonnes 2,110 tonnes 734 tonnes 530 tonnes Typical max. % of EGBE 3 3 7 7 Number of days / Fraction of main source (Table B3.13)

300 / 0.05 (Table B4.4)

300 / 0.002 (Table B3.13)

300 / 0.1 (Table B3.13)

300 / 0.1

Release estimates (fraction)

- air

- waste water

- soil (Table A3.15 water based)

0.8 0.1 0.001 (Table A4.5 water based)

0.8 0.15 0.01 (Table 3.6)

0.94

0 0.03 (Table 3.5)

0.01** 0.01

0 Amount released (kg/d)

- air

- waste water

- soil 182.7 22.8 0.2 11.3 2.1 0.1 230.0 0 7.3 1.8 1.8 0 * It has been confirmed that application conditions vary little between professional and retail decorative paints. The only difference is the classification of the persons applying the paints (professionals or private consumers). Consequently, these scenarios have been merged and the default emission factors for consumer products will be used (wide dispersive use: use outside of a factory). ** Assuming treatment of flue gases (98% if no treatment)

Table 3.18 Local releases of EGBE from processing and private use of paints

Scenario Paints VI P Paints VII P Paints VIII P Paints IX P
Main category Non dispersive use Non dispersive use Non dispersive use Non dispersive use

Total fraction and connected tonnage 0.0546 / 5,300 tonnes 0.0218 / 2,120 tonnes 0.0109 / 1,060 tonnes 0.0109 / 1,060 tonnes Regional tonnage 530 tonnes 212 tonnes 106 tonnes 106 tonnes Typical max. % of EGBE 8 1 2 2 Number of days / Fraction of main source (Table B3.13)

300 / 0.1 (Table B3.13)

300 / 0.1 (Table B3.13)

300 / 0.1 (Table B3.13)

300 / 0.1

Release estimates (fraction)

- air

- waste water

- soil (Table A3.15 water based)

0.01**

0.1 0.001 (Table A3.15 solvent based)

0.9 0.02 0.001 (Table 3.15)

0.01**

0 0 (Table 3.19)

0.96 0.01

0

Amount released (kg/d)

- air

- waste water

- soil 1.8 17.7 0.2 63.6 1.4 0.1 0.4 0 0 33.9 0.4 0 ** Assuming treatment of flue gases (98% if no treatmen t for solvent-based coatings and 80% for water-based coatings). Indeed, automotive producers operate VOC emission abatement systems to control air emissions

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Table 3.19 Local releases of EGBE from formulati on, processing and private use of detergents

Scenario Detergents I F Detergents I P Detergents II F Detergents II U
Main category Multi-purpose equipment Non dispersive use Non dispersive use Wide dispersive use

Total fraction and connected tonnage 0.0850 / 8,250 tonnes 0.0850 / 8,250 tonnes 0.0283 / 2,750 tonnes 0.0283 / 2,750 tonnes Regional tonnage 825 tonnes 825 tonnes 275 tonnes 275 tonnes Typical max. % of EGBE 10 10 10 10 Number of days / Fraction of main source (Table B2.3)

300 / 0.8 (Table B3.3 for waste water only)

200 / 0.002 (Table B2.3)

300 / 1 (Table B4.1 for waste water only)

365 / 0.002

Release estimates (fraction)

- air

- waste water

- soil (Table A2# - liquid)

0.00002 0.0009 0.0032 (Table A3.5)

0.0025

0.9 0.05 (Table A2# - liquid)

0.00002 0.0009 0.0032 (Table A4.1)

0 0.99 0.01 Amount released (kg/d)

- air

- waste water

- soil 0 2.0 7.0 - 7.4

- 0 0.8 2.9 - 1.5

-

Table 3.20 Local releases of EGBE from formulation and processing of other uses

Scenario Intermediates I P Inks I F Inks I P Pharm I P Elec I P
Main category Dedicated equipment Multi-purpose equipment Non dispersive

use Non dispersive

use Non dispersive

use
Total fraction and connected tonnage 0.103/10,000 t.
0.0927/9,000 t.

(captive use) 0.0257 / 2,500 t. 0.0257 / 2,500 t. 0.0051 / 500 t. 0.062 / 600 t.

Regional tonnage 10,000* t. 9,000* t. (captive use) 250 t. 250 t. 50 t. 60 t.

Typical max. % of EGBE 100 (default value) 20 20 100 (default value) 100 (default value) Number of days / Fraction of main source (Table B3.2)

300 / 0.25 (Table B2.3)

300 / 1 (Table B3.10 large companies)

300 / 0.333 (Table B3.2)

32 / 0.65 (Table B3.2)

12 / 0.5

Release estimates (fraction)

- air

- waste water

- soil (Table A3.3)

0.00001

0.007 0.0001 (Table A2.1)

0.005 0.02 0.0001 (Table A3.12 – printing and allied processes)
0.3 0.001 0.0001 (Table A3.3)

0.001 0.02 0.0001 (Table A3.4)

0.001 0.005 0.01

Amount released (kg/d)

- air

- waste water

- soil 0.1 / 0.1** 58.3 / 52.5**

0.8 / 0.8** 4.2 16.7 0.1 83.3 0.3 0 1.0 20.3

0 2.5 12.5 25.0 * The regional tonna ge is the total to nnage (3 EGBEA produ cers in the EU) ** Captive use: these amounts will be added to the releases at production

40CHAPTER 3. ENVIRONMENT

Table 3.21 Local releases of EGBE from formulat ion and processing of other uses (continued)

Scenario Leather I P Adhesives I F Adhesives I P Agri I F Agri I P Non dispersive use Multi-purpose equipment Non dispersive

use Multi-purpose equipment Wide dispersive use Main category

Total fraction and connected tonnage 0.0051 / 500 t. 0.0051 / 500 t. 0.0051 / 500 t. 0.0021 / 200 t. 0.0021 / 200 t.

Regional tonnage 50 t. 50 t. 50 t. 20 t. 20 t. Typical max. % of EGBE 5 10 10 100 (default value) 1

Number of days / Fraction of main

source (Table B3.4 except for waste water release)

200 / 0.5 (Table B2.3)

300 / 1 (Table B3.3 only for waste water)

50 / 0.002 (Table B2.2)

300 / 1 (Table B3.1)

2 / 0.001

Release estimates (fraction)

- air

- waste water

- soil (Table A3.6 except for waste water release)
0 specific scenario (see Section 3.1.2.2.2 ) 0.01 (Table A2.1)

0.005 0.02

0.0001 (Table A3.5)

0.05 0.45

0.45 (Table A2.1)

0.005 0.02

0.0001 (Table A3.1)

1 0

0

Amount released (kg/d)

- air

- waste water

- soil - 44.5 1.3 0.8 3.3 0 - 0.9

- 0.3 1.3 0 10 0 0

Table 3.22 Local releases of EGBE from formulat ion and processing of other uses (continued)

Scenario Oilfield I F Oilfield I P Metal I F Metal I P
Main category Multi-purpose equipment Wide dispersive

use Multi-purpose equipment Non-dispersive

use

Total fraction and connected tonnage 0.0257 / 2,500 t. 0.0257 / 2,500 t. 0.0206 / 2,000 t. 0.0206 / 2,000 t.

Regional tonnage 250 t. 250 t. 200 t. 200 t. Typical max. % of EGBE 100 100 4 4 Number of days / Fraction of main source (Table B2.3)

300 / 1 (Table B3.3 only for waste water)

50 / 0.002 (Table B2.34)

300 / 0.8 (Table B3.6)

300 / 0.7

Release estimates (fraction)

- air

- waste water

- soil (Table A2.1)

0.005 0.02

0.0001 (Table A3.5)

0.05 0.45

0.45 (Table A2.1)

0.01 0.02

0.0001 (Table A3.7)

0.25 Specific scenario (see point 3.1.2.2.3 ) 0.05

Table 3.22 continued overleaf

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Table 3.22 continued Local releases of EGBE from formulation and processing of other uses (continued)

Scenario Oilfield I F Oilfield I P Metal I F Metal I P
Main category Multi-purpose equipment Wide dispersive

use Multi-purpose equipment Non-dispersive

use Amount released (kg/d)

- air

- waste water

- soil 4.2 16.7

0.1 - 4.5 (specific scenario for the marine compartment – see

point 3.1.2.2.4 )

- 5.3 10.7

0.1 116.7 0. 8 (3 kg – intermittent

release)

23.3

Table 3.23 Local releases of EGBE from formulat ion and processing of other uses (continue)

Scenario Cosmet I F / Fire I F Cosmet I U Fire I P
Main category Multi-purpose equipment - Wide dispersive use

Total fraction and connected tonnage 0.0082 / 800 t. 0.0051 / 500 t. 0.0031 / 300 t.

Regional tonnage 80 t. 50 t. 30 t. Typical max. % of EGBE 10 (cosmetic products) / 16 (fire foam) 10 0.5

Number of days / Fraction of main source (Table B2.3)

300 / 1 (Table B4.1 only for waste water)

365 / 0.002 (Table B3.3 only for waste water)

50 / 0.002

Release estimates (fraction)

- air

- waste water

- soil (Table A2.1)

0.005 0.02 0.0001 (Table A4.1)

0 0.8 0.001 (Table A3.5)

0.05 0.45 0.45 Amount released (kg/d)

- air

- waste water

- soil 1.3 5.3 0 - 0.2

- - 0.5

-

3.1.3 Continental and regional predicted environmental concentrations
Continental and regional computations are done by means of multimedia fate models based on the fugacity concept. The standardised cont inental and regional environments of the TGD (EC, 2003) are used. Table 3.24 shows the calculated continental and regional PECs for air, water and soil using EUSES (EC, 2004). These results come from th e calculation program EUSES (EC, 2004) and do not integrat e the emissions due to the scenario “Agri I” because of the scenario number limitations of the software. Neve rtheless, the calculations have been made for this scenario in a separate file and it appears that the impact of emissions volumes for this scenario on total continental and regional emissions is negligible.

42CHAPTER 3. ENVIRONMENT

Table 3.24 Regional PECs in air, water and soil (calculations made by EUSES – SIMPLEBOX model)

Compartment PEC continental PEC regional

Air 1.10.10 -5 mg/m 3 1.15.10 -4 mg/m 3

Water 9.41.10 -4 mg/L 7.35.10 -3 mg/L Agricultural soil 8.44.10 -5 mg/kg (ww) 8.74.10 -4 mg/kg (ww) Pore water of agricultural soils 1.40.10 -4 mg/L 1.45.10 -3 mg/L

Natural soil 1.82.10 -4 mg/kg (ww) 1.89.10 -3 mg/kg (ww)

Industrial soil 4.39.10 -3 mg/kg (ww) 4.30.10 -2 mg/kg (ww)

Sediment 1.16.10 -3 mg/kg (ww) 9.08.10 -3 mg/kg (ww)

Seawater 6.09.10 -7 mg/L 6.12.10 -4 mg/L

Marine sediment 7.54.10 -7 mg/kg (dw) 7.57.10 -4 mg/kg (ww)

3.1.4 Local predicted environmental concentrations (PEC local )

3.1.4.1 Aquatic compartment

3.1.4.1.1 PEC local for production

PEC STP At production level, the local Pr edicted Environmental Concentr ation for micro-organisms in STP ranges from 0.02 to 0.49 mg/L (these values include emissions for EGBE processing, for on-site EGBEA production) (see Table 3.6 )

PEC aqua Emissions at production and pro cessing levels (EGBE captive use for EGBEA synthesis) result in a PEC for surface water ranging from 0.8 to 10.6 μg/L (including the PEC regional for the aquatic compartment). The PEC aqua for the different production sites are presented in Table 3.25

.

Table 3.25 Local PEC in water at production

Production sites #1 (PEClocal marine ) #2 #3 #4 #5

PEClocal aqua (μg/L) 0.8 10.6 9.0 9.6 -

3.1.4.1.2 Calculation of PEC local for formulation, processing and private use
Concentrations of EGBE in water for formulation, processing and private use in the EU are estimated with a generic scenario which was carried out based on default values (TGD – EC, 2003) because no site specific data were available.

It is assumed that the amounts released to wate r will enter a sewage treatment plant. During sewage treatment, 87.4% of EGBE is expected to be removed (see Table 3.5 ). The default flow rate of the treatment plant is 2,000 m 3 /day.

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The effluent concentration leaving the STP (Predi cted Environmental Concentration in a STP or PEC STP ) is calculated according to Equation 3.1 . This PEC STP is divided by a dilution factor (10: default value) to obtain the local PEC in surface water (see Equation 3.2 ). The daily amounts released for the generic s cenarios are the basis for th e calculation of the PECs. Table 3.26 gives the PECs for the aquatic compartment. PEC seawater have been calculated with EUSES 2.0 (EC, 2004).

Table 3.26 Local PEC ST P and PEC aqua for EGBE

Scenario Daily release to waste water (kg/d) PEC STP (μg/L) Local PEC aqua (μg/L) Total local PEC aqua * (μg/L) Total local PEC seawater ¤ (μg/L)

Paints I F / Paints I P 8.2 / 22.8 519 / 1,430 52 / 144 59 / 151 42 / 114

Paints II F & III F 8.4 533 54 61 43

Paints II P and Paints III U 2.1 132 13 21 11

Paints IV F / Paints IV P 0 / 0 - / - - / - 7 / 7 1 / 1

Paints V F / Paints V P 0 / 1.8 - / 110 - / 11 7 / 18 1 / 9

Paints VI F / Paints VI P 28.3 / 17.7 1,780/1,090 179 / 109 186 / 116 142 / 87

Paints VII F / Paints VII P 9.9 / 1.4 623 / 87 63 / 9 70 / 16 50 / 8

Paints VIII F / Paints VIII P 0 / 0 - / - - / - 7 / 7 1 / 1

Paints IX F / Paints IX P 2.8 / 0.4 178 / 22 18 / 3 25 / 10 15 / 2 Detergents I F / Detergents I P 2.0 / 7.4 125 / 464 13 / 47 20 / 54 11 / 37 Detergents II F / Detergents II U 0.8 / 1.5 52 / 93 6 / 10 13 / 17 5 / 8

Intermediates** I P *** 58.3 736 19 26 59

Inks I F / Inks I P 16.7 / 0.3 1,050 / 17 105 / 2 112 / 9 84 / 2

Pharm I P *** 20.3 254 7 14 21

Elec I P 12.5 791 80 87 63

Leather I P 44.5 2810 281 288 223

Adhesives I F / Adhesives I P 3.3 / 0.9 167 / 55 17 / 6 24 / 13 14 / 5 Agri I F / Agri I P 1.3 / 0 82 / - 8 / - 15 / 7 7 / 1

Oilfield I F / Oilfield I P Surface and well cleaning Squeeze treatments Hydrotest chemicals 16.7 / 4.5

-

-

- 1,050 / 276

-

-

- 105 / 28

-

-

- 112 / 35

-

-

- 84 / 23
7,700
2,300
100,000

Metal I F / Metal I P

Metal intermittent 10.7 / 0.8

3 673 / 51

1500 68 / 5

19 75 / 12

26 54 / 5

16

Table 3.26 continued overleaf

44CHAPTER 3. ENVIRONMENT

Table 3.26 continued Local PEC STP

and PEC aqua for EGBE

Scenario Daily release to waste water (kg/d) PEC STP (μg/L) Local PEC aqua (μg/L) Total local PEC aqua * (μg/L) Total local PEC seawater ¤ (μg/L)

Cosmet I F & Fire I F /Cosmet I U 5.3 / 0.2 335 / 14 34 / 2 41 / 9 27 / 3

Fire I P 0.5 32 4 11 2

* Total local PEC aqua = Local PEC aqua + regional PEC aqua
¤ Total local PEC seawater = Local PEC seawater + regional PEC seawater ** Captive use not included *** Dilution factor = 40 and EFFLUENT STP

= 10,000 m 3 /day (see scenario for IC3 chemicals used in synthesis) P Processing F Formulation U Private use

3.1.4.1.3 Measured levels

Contaminated sites
Ground water sample collections were performed in February 1974 near the “Valley of Drums”, Kentucky, USA, a contaminated site. EGBE was detected at a c oncentration of 2.3.10 -2 mg/L in one sample out of seven (Stonebreaker and Smith, 1980).
In April 1980, EGBE was detected by GC/MS at concentrations of 1.31 and 5.68 mg/L in the water of the Hayashida River, Hyogo prefecture, Japan. In this surface water, EGBE was considered as a contaminant from leather industr y effluents. The values represent respectively levels after steams and vacuum distillation (Yasuhara et al., 1981).
EGBE was measured in a municipal waste water treatment plant, in Göteborg, Sweden. Twentyfour hour flow proportional compos ite were taken in the influe nt (four samples during 1990 and eight samples in 1991) and the effluent (sev en samples during 1989-1991). EGBE was separated by liquid – liquid extraction with dichlorometh ane and analysed by GC/MS. The range of influent concentrations goes from 0 (non detected) to 2.5.10 -1 mg/L (average influent concentration in 1990 = 8.5.10 -2 mg/L and in 1991 = 2.3.10 -2 mg/L). The range of effluent concentrations was 0 (non detected) – 3.10 -3 mg/L (Paxeus et al., 1992).
In France, EGBE was detected in the influent of a domestic STP located in northern suburban Paris, in 1991 (November, 26). An alysis was performed with purge and trap – high resolution capillary GC/MS. EGBE was det ected at a concentration of 3.5.10 -2 mg/L Nguyen et al., 1994).

Concentrations of EGBE have b een measured with GC/MS in a queous samples from a municipal and an industrial landfill in the United States. They were ranging from < 0.4 (detection limit) to 84 mg/L. The municipal landfill cons isted mostly of municipal wast e (four wells were analysed). There, EGBE concentrations were detected in wells 3 (18 mg/L) and 4 (84 mg/L) which were located in the landfill. EGBE has not been detected in wells 1 and 2 which were located downgrade from the landfill. Indu strial landfill consisted mostly in industrial waste and EGBE concentrations were detected: 2.5 mg/L (well 8), 3 mg/L (wells 5 and 6). In well 7, EGBE has not been detected. Wells 5, 6 and 7 were located on the landfill whereas well 8 is a downgrade interceptor well located between the landfill and the treatment plant (Beihoffer and Ferguson, 1994).

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3.1.4.1.4 Comparison between predicted and measured levels

Exposure during formulation, processing and private use
Measured concentrations are only available for so me uses. In this section, they are compared to the concentrations predicte d by exposure scenarios.

EGBE concentrations measured by Yasuhara et al., 1981 can be put in parallel with values calculated with scenario “Leather I P ”. The measured values, ranging between 1.31 and 5.68 mg/L show factors 5 or 20 higher than the corresponding PEC calculated in surface water, 0.288 mg/L. But we must also co nsider that these data come from a 20 year-old study and measurements were performed in a place where EG BE contamination from the leather industry was expected. Therefore, these results shoul d be considered as a highly worst-case.
EGBE was also measured in municipal wast e water effluents. In the study by Paxeus et al., 1992 EGBE concentrations reach 3.10 -3 mg/L. In a first time this va lue can be compared to PEC STP presented in Table 3.26 . These concentrations range between 0.014 and 1.78 mg/L.

3.1.4.2 Terrestrial compartment

Different PECs can be determined to assess the exposure level in terrestrial compartment. The local PEC in soil is calculated according to the following equation:

Equation 3.5 Calculation of PEClocal soil

soil natural soil soil 

### l PECregiona Clocal PEClocal

_ 

### + =

3.1.4.2.1 PEC local for production

The different PEC local in soil at production and proces sing (EGBE captive use for on-site EGBEA production) levels are presented in Table 3.27

.

Table 3.27 PEClocal soil at production and in situ processing (according to EUSES)

Site # Local PEC in agricultural soil averaged over 30 days (μg/kg ww) Local PEC in agricultural soil averaged over 180 days (μg/kg ww) Local PEC in grassland averaged over 180 days (μg/kg ww)

1 34.25 11.15 4.72

2* 1.15 1.15 1.38

3* 0.88 0.88 0.89

4* 0.89 0.89 0.89

5 No data but total annual site production for EGBE < 1kt.
* At this production site STP sludge is incinerated

3.1.4.2.2 Calculation of PEC local for formulation, processing and private use
The EUSES models (EC, 2004) take into acc ount both the application of STP sludge on agricultural soil and deposition from air for the calculation of EGBE concentrations in the

46CHAPTER 3. ENVIRONMENT

terrestrial compartment. Table 3.29 8 gives the terrestrial PECs at local scale for the various generic scenarios.

Table 3.28 Local PEC soil for EGBE

Scenario PEC soil - average concentration in agricultural soil over 30 days (μg/kg ww) PEC soil - average concentration in agricultural soil over 180 days (μg/kg ww) PEC soil - average concentration in grassland over 180

days (μg/kg ww)

Paints I F / Paints I P 30 / 84 11 / 32 6 / 23

Paints II F & III F 31 11 6

Paints II P and Paints III U 9 4 3

Paints IV F / Paints IV P 4 / 10 4 / 10 5 / 17

Paints V F / Paints V P 2 / 8 2 / 4 3 / 3

Paints VI F / Paints VI P 96 / 59 31 / 20 13 / 8

Paints VII F / Paints VII P 5 / 9 12 / 6 6 / 7

Paints VIII F / Paints VIII P 2 / 2 2 / 2 2 / 2

Paints IX F / Paints IX P 12 / 4 5 / 3 3 / 4 Detergents I F / Detergents I P 9 / 26 4 / 9 3 / 5 Detergents II F / Detergents II U 5 / 7 3 / 3 2 / 2

Intermediates** I P 41 14 6

Inks I F / Inks I P 57 / 6 19 / 5 8 / 8

Pharm I P 15 6 3

Elec I P 44 15 7

Leather I P 150 47 19

Adhesives I F / Adhesives I P 11 / 5 5 / 3 3 / 2 Agri I F / Agri I P 7 / 3 3.8 / 2.5 3.0 / 2.5

Oilfield I F / Oilfield I P 57 / 17 19 / 6 8 / 4

Metal I F / Metal I P 38 / 9 13 / 7 6 / 10

Cosmet I F & Fire I F / Cosmet I U 20 / 3 7 / 2 4 / 2

Fire I P 4 2 2
* Captive use not included - PECregional natural_soil ~ 1.89 μg/kg (ww)Atmosphere P Processing F Formulation U Private use

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3.1.4.3 Atmosphere

3.1.4.3.1 PEC local for production
Emissions of EGBE in air, both at production an d processing (on-site EGBEA production), result in an average annual concentration in air (Clocal air,ann ) of 1.67 μg/m 3 in the worst case (site specific information for site #2). This results in a PEClocal air,ann 11 of 1.77 μg/m 3

.

3.1.4.3.2 Calculation of PEC local for formulation, processing and private use
The calculated annual average EGBE con centrations in air are presented in Table 3.29 for the different use patterns.

Table 3.29 Local PEC air

for EGBE

Scenario Concentration during emission (μg/m 3 ) C local_air,ann 100m from source

(μg/m 3 ) Annual deposition (μg/m2/d) PEC local_air,ann * (μg/m 3 )

Paints I F / Paints I P 3.81 / 50.40 3.13 / 41.40 4.51 / 59.60 3.25 / 41.50

Paints II F & III F 3.91 3.22 4.63 3.33

Paints II P and Paints III U 3.11 2.55 3.67 2.67

Paints IV F / Paints IV P 14.30 / 62.00 11.70 / 51.00 16.90 / 73.40 11.90 / 51.10

Paints V F / Paints V P 3.93 / 0.49 3.23 / 0.40 4.65 / 0.58 3.35 / 0.51

Paints VI F / Paints VI P 1.97 / 0.48 1.62 / 0.39 2.33 / 0.57 1.73 / 0.51

Paints VII F / Paints VII P 0.69 / 17.20 0.56 / 14.10 0.81 / 20.40 0.68 / 14.30

Paints VIII F / Paints VIII P 2.35 / 0.10 1.93 / 0.08 2.78 / 0.11 2.05 / 0.19

Paints IX F / Paints IX P 1.57 / 9.13 1.29 / 7.51 1.86 / 10.80 1.40 / 7.62 Detergents I F / Detergents I P 0.12 / 7.12.10 -4 0.10 / 3.90.10 -4 0.14 / 5.62.10 -4 0.22 / 0.12 Detergents II F / Detergents II U 5.09.10 -3 / 1.43.10 -4 4.19.10 -3 / 1.43.10 -4 6.12.10 -3 / 2.06.10 -4 0.16 / 0.12

Intermediates** I P 0.02 0.02 0.03 0.13

Inks I F / Inks I P 1.16 / 22.50 1.00 / 18.50 1.37 / 26.60 1.07 / 18.60

Pharm I P 0.30 0.02 0.04 0.14

Elec I P 0.70 0.02 0.03 0.14

Leather I P 4.29.10 -3 2.33.10 -3 3.35.10 -3 0.12

Adhesives I F / Adhesives I P 0.23 / 8.37.10 -5 0.19 / 1.15.10 -5 0.27 / 1.65.10 -5 0.27 / 0.12 Agri I F / Agri I P 0.09 / 27.6 0.08 / 0.15 0.10 / 0.22 0.23 / 0.30

Oilfield I F / Oilfield I P 1.16 / 4.22.10 -4 0.95 / 5.78.10 -5 1.37 / 8.31.10 -5 1.07 / 0.12

Table 3.29 continued overleaf

11 PEClocal air,ann = Clocal air,ann + PECregional air

48CHAPTER 3. ENVIRONMENT

Table 3.29 continued Local PEC air

for EGBE

Scenario Concentration during emission (μg/m 3 ) C local_air,ann 100m from source

(μg/m 3 ) Annual deposition (μg/m2/d) PEC local_air,ann * (μg/m 3 )

Metal I F / Metal I P 0.93 / 32.4 0.76 / 26.7 1.10 / 38.4 1.33 / 26.80

Cosmet I F & Fire I F / Cosmet I U 0.37 / 2.06.10 -5 0.30 / 2.06.10 -5 0.44 / 2.96.10 -5 0.42 / 0.12

Fire I P 4.82.10 -5 6.60.10 -6 9.49.10 -6 0.12

* PEC local_air,ann = C local_air,ann + regional PEC air ** Captive use not included P Processing F Formulation U Private use

3.1.4.3.3 Measured levels
EGBE was detected at 8 μg/m 3 in one out of six samples by GC/MS. Indoor air samples were collected from 14 homes and a small office in Italy (De Bortoli et al., 1986).
Concentrations of EGBE have been included in the Environmental Protection Agency’s volatile organic compounds national ambient database. This database compiles data on indoor air but not industrial space. The average EGBE co ncentration for 14 samples was 1.05 μg/m 3 (median = 0.37 μg/m 3 ; lower quartile = 0.15 μg/m 3 ; upper quartile = 1.77 μg/m 3 ) (Shah and Singh, 1988).

EGBE was also detected in the clean flue gas of a municipal wa ste incinerator in Germany, in 1995. It has been measured using the tec hnique of adsorption/thermodesorption and adsorption/elution on selected XAD resins and then detected with FID. EGBE concentration was 0.23 μg/m 3 (Jay and Stieglitz, 1995).
EGBE background levels have been measured by Ciccioli et al., 1993 and Ciccioli, 1996 and Ciccioli et al., 1996, in Northern and S outhern Europe, in Himalayas and in Antarctica. In the first study, concentrations ranged between 0.1 and 1.59 μg/m 3 and in the second between 1.26 and 14.85 μg/m 3 . Industrial or domestic emissions of EG BE were avoided in both studies. Major sources seemed to be vegetation in the first case and in the s econd, marine emission are proposed by the authors to explain the measurements.

3.1.4.3.4 Comparison between predicted and measured levels
The highest predicted EGBE concentrations in ai r take place during industrial uses of paints. For example, PEC local_air,ann reaches 51 μg/m 3 for the scenario “Paints IV P ”. No measured data is available in the neighbourhood of pl ants using paints at high scal e so comparison is not possible. EGBE emissions in air are often a ten order less at formulation th an at processing with most of the scenarios used for paints (“Paints I-II/VI-IX” ) and printing inks. For other uses, emissions to air are reduced: a maximum of ~ 41 μg/m 3 occurred for the processing of general industrial coatings. Some indoor measured concentrations of EGBE are only presented to get an order of magnitude.
EGBE background levels have been measured in a ppropriate places in or der to avoid industrial and domestic sources (by Ciccioli et al., 1993 and Ciccio li, 1996). These studi es show a natural

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production of EGBE by plants and the second st udy particularly underlined a possible marine source of EGBE.

3.1.4.4 Secondary poisoning

The bioconcentration factor for fish is very low, so it is not e xpected that there is a significant exposure for humans or predators via the local environment. Moreover, as EGBE is not classified as Very Toxic (T+), Toxic (T) or Harm ful (Xn and R48), it is assumed that there is a low potential for the substance to cause toxi c effects if accumulated in higher organisms.

50CHAPTER 3. ENVIRONMENT

3.2 EFFECTS ASSESSMENT: HAZ ARD IDENTIFICATION AND DOSE (CONCENTRATION) - RESPONSE (EFFECT ASSESSMENT)
Studies are classified as valid if they fully descri be the test material use d, the test organism, the test method and conditions and if the endpoint concentration is based upon measured levels. Where only some of these criteria are described th e tests may be used with care or considered not valid. Moreover for some studies or results, some da ta are lacking, i.e. the original paper is not available but only a citation.

3.2.1 Aquatic compartment (incl. sediment)

3.2.1.1 Fish

Acute toxicity
EGBE short term toxicity studi es for fish are summarised in Table 3.30

.

Table 3.30 Short term fish toxicity data for EGBE

Test # Species Duration Endpoint Result (mg/L) Method References Validity

1 Poecilia reticulata 7 days LC 50 983 Not specified Konemann, 1981 Valid with restrictions

2 Menidia beryllina * 96 hours LC 50 1,125 Not specified Dawson et al., 1975 Valid with restrictions

3 Lebistes reticulatus 96 hours LC 0 LC 50 LC 100 1,000 1,400 2,000 Not specified Knappe and Popp, 1974 Invalid

4 Oncorhynchus mykiss 96 hours LC 50 1,474 OECD guideline 203, 1999 INERIS, 1999b Valid

5 Lepomis macrochirus 96 hours LC 50 1,490 Not specified Dawson et al., 1975 Valid with restrictions

6 Leuciscus idus melanotus 48 hours LC 0 LC 50 LC 100 1,170-1,350 1,395-1,575 1,490-1,620 DIN 38412 part 15 Juhnke and Lüdemann, 1978 Valid with restrictions

7 Carassius auratus 24 hours LC 50 1,700 APHA n°231 Bridié et al., 1979b Valid with restrictions

8 Leuciscus idus 48 hours LC 50 1,880 DIN 38412 part 15 Huels, 1982 Invalid

9 Pimephales promelas 96 hours LC 50 2,137 US EPA Dow Chemical Co, 1979 Valid with restrictions

10 Lepomis macrochirus 96 hours LC 50 2,950 Not specified Dow Chemical Co, 1981, cited in Neely, 1984 Invalid

Table 3.30 continued overleaf

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Table 3.30 continued Short term fish toxicity data for EGBE

Test # Species Duration Endpoint Result (mg/L) Method References Validity

- Fundulus heteroclitus* 96 hours NOEC

LC 50 4

6.7 Stephan, 1974 Biospherics Inc., 1981b Invalid**

- Cyprinodon variegatus * 96 hours LC 50 116 US EPA, 1975 Welchem Inc, 1984 Invalid**

- Lepomis macrochirus 96 hours LC 50 127 Stephan, 1974 CIBA-GEIGY, 1976 Invalid**

* Marine and estuarine species ** Test performed with a preparation containing EGBE
Konemann, 1981 studied the effect of EGBE on a fresh water fish species: Poecilia reticulata (#1). This test lasted seven days in semi static conditions. The guideline followed was not recorded and the purity of EGBE was unknown. Groups of eight 2-3 month-old fish were exposed to each concentration tested in fo llowing conditions: 22 +/- 1°C, oxygen content > 5 mg/L, water hardness = 25 mg/L (CaCO 3 ). Test solutions were renewed daily and a solvent vehicle was also employed (aceto ne or 2-propanol). A log EC 50 was measured at 3.92 μmol/L (983 mg/L).
In test #2, performed by Dawson et al., 1975, a chemically pure gr ade EGBE was tested on the marine fish Menidia beryllina . Test was conducted in syntheti c seawater, at 20°C with mild aeration according to the oxygen demand, water hardness was 55 mg/L (CaCO 3 ). For other test conditions see test #5 description. A LC 50 of 1125 mg/L was obtained after 96 hours.
The toxicity of EGBE has been tested on the fish Oncorhynchus mykiss by INERIS, 1999b - test #4, according to the OECD guideline No 203. A LC 50 of 1,474 mg/L was obtained after 96 hours (95% confidence interval = 1,145-1,840 mg/L). Seve n fish were disposed per aquarium, in 15 L test medium with continuous aeratio n. Water hardness was 250 mg/L (CaCO 3 ), the range of pH during the test was 7.6-8.0 (measured before a nd after the test), DO between 8.6 and 10.1 and temperature was 16 +/- 1°C. Nominal concentrations tested in duplicates were as follow: 0, 100, 500, 1,000, 2,000 and 4,000 mg/L. The loss of test subs tance during the test was between 3 and 13%.
The test #5 was also performed by Dawson et al., 1975 on the fish species Lepomis macrochirus and a LC 50 of 1,490 mg/L was obtained after 96 hours. Th e test was conducted in well water, at 23°C with mild aeration supplied after 24 hours according to the oxygen demand measured daily. A range of pH has been measured between the beginning and the end of the test: 7.6-7.9. Fish have gone through an acclimation period of 14 days and have not been fed 48 hours prior to testing. A minimum of 1 L of test medium was us ed per gram of fish. No analytical monitoring was performed. Moreover, it is reported that some test substances in the study were diluted with distilled water or a solvent desc ribed as having relatively low t oxicity but most samples did not required dilution (it can be expected that EG BE did not undergo dilution for its high water solubility).
Test #6 is a static test performed on the fish Leuciscus idus melanotus in parallel in two different laboratories by Juhnke and Lüdemann, 1978. A continuous aeration is mentioned (mild bubbling) but no data on other test conditions is av ailable. Different Lethal Concentrations were measured after 48 hours. Results for both laboratories are presented in Table 3.30

.

52CHAPTER 3. ENVIRONMENT

Standard methods for the examination of wate r and wastewater, APHA n°231: static tank acute toxicity test, have been used by Bridié et al., 1979b so as to measure the toxicity of EGBE (#7). A LC 50 of 1,700 mg/L has been obtained after 24 hours. Ten fish ( Carassius auratus ) were used for each concentration, in 25 L test medium, at 20° C, pH 7 and solutions were aerated during the test period. Concentrations of EGBE in the test medium were determined using a total organic carbon analyser or by extraction and GC both at the beginning and at the end of the test but data are not available.
Test #9, conducted with a fresh water fish, Pimephales promelas , by Dow Chemical Co, 1979 gives a LC 50 of 2,137 mg/L after 96 hours (95% c onfidence interval = 2,022-2,263 mg/L – moving average method). The method followed came from the US EPA. A static open system was used with raw lake water dechlorinated with activated carbon used in test aquariums containing 10 fish. Other test conditions were as follow: temp erature = 12°C, pH between 7.6 and 7.8, water hardness 101 mg/L (CaCO 3 ), three test concentr ations (1,550, 2,100, 2,800 mg/L) plus control. A water temperature be tween 20 and 24°C is recommended for Pimephales promelas in the directive 92/CEE, met hod C1 “acute toxicity to fish”. The lo w water temperature employed in this test is thus a reason to restrict its validity.

Tests number 8 and 10 must be considered as inva lid for available data are reduced to minimum and, in some cases, reports are not available but only a quotation. Test number 3 is also classified as invalid for there were both insufficient data on test conditions and an insufficient number of fishes per test concentration (5 fish).

Long-term toxicity
A chronic toxicity test for EGBE is presented in Table 3.31

.

Table 3.31 Long term fish toxicity data for EGBE

Test # Species Duration Endpoint Result (mg/L) Method References Validity

1 Brachydanio rario 21 days NOEC > 100 Project of OECD guideline INERIS, 2001 Valid

Test conditions for this long term fish toxicity data are detailed in Section 3.2.1.5 of this study (endocrine disruption). Neither mortality nor endocrine disruption effect was observed at any test concentration. Thus a No Observed Effect Con centration can be set at > 100 mg/L, the highest tested concentration.

3.2.1.2 Aquatic invertebrates

Acute toxicity
Short term toxicity tests for EGBE are presented in Table 3.32

.

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Table 3.32 Short term invertebrate toxicity data for EGBE

Test # Species Duration Endpoint Result (mg/L) Method References Validity

1 Hydra attenuata

or H. vulgaris (cnideria) 72 hours EC 50 540

690 Regeneration potential test Morphological characteristics Bowden et al

. , 1995 Valid with restrictions

2 Crangon crangon (crustacea) 96 hours LC 50 698 Not specified Blackman, 1974 Lack of data

3 Daphnia magna 48 hours EC 50 835 Not specified Dow Chemical Co, 1979 Valid with restrictions

4 Artemia salina * (crustacea) 24 hours EC 50 1,000 Not specified Price et al

. , 1974 Valid with restrictions

5 Daphnia magna 24 hours EC 0 EC 50 EC 100 1,140 1,720 2,500 DIN 38412 part 11 Bringmann and Kühn, 1977 Valid with restrictions

6 Daphnia magna 24 hours EC 0 EC 50 EC 100 1,283 1,815 2,500 DIN 38412 part 11 Bringmann and Kuhn, 1982 Valid with restrictions

7 Daphnia magna 48 hours EC 50 1,550 OECD guideline n°202 INERIS, 1999a Valid

8 Daphnia magna 24 hours EC 50 5,000 DIN 38412 part 11 Hoechst, 1982 Lack of data

- Palaemonetes pugio* (crustacea) 96 hours NOEC
EC 50 2
5.4 US EPA, 1975 Biospherics Inc., 1981a Invalid**

- Crassostrea virginica (oyster) 96 hours LC 50 80.5 US EPA, 1975 Welchem Inc, 1984 Invalid**

- Panaeus setiferus (white shrimp) 96 hours LC 50 117 US EPA, 1975 Welchem Inc, 1984 Invalid**

* Marine species ** Test performed with a preparation containing EGBE

Bowden et al., 1995 have conducted thei r EGBE toxicity test on Hydra attenuata – test #1. The aim of this test was to measure the regeneration potential of this organi sm. Test conditions were as follow: 10 isolated digestive regions per test concentration, fi ve test concentrations (90, 180, 370, 740, 920 mg/L) plus one control, test soluti ons were renewed every 24 hours. At up to 370 mg/L, the digestive region had regenerated some tentacles and in some cases the basal disc. Only normal wound healing was observed at 740 mg/L while at 920 mg/L the wounds were healed but the region expanded. A 72-hour EC 50 was derived from these results: 540 mg/L. It should be noticed that there wa s no analytical monitoring and that the endpoint was unusual. Moreover no clear effect was shown in presence of the test substance. On the other side, another test performed with the same organism by the same team gives a 72-hour EC 50 of 690 mg/L. Here, the endpoint was based on morphological char acteristics. Test conditions were similar and 10 adult polyps were used per test concentratio ns. Varying degrees of shortened tentacles and body were seen at 740 mg/L and at 920 mg /L polyps were totally contracted.

54CHAPTER 3. ENVIRONMENT

Blackman, 1974 conducted a EG BE toxicity test on Crangon crangon and determined a LC 50 of 698 mg/L after 96 hours (test #2). 20 animals were us ed per test concentration and test solutions were renewed every 6, 12 or 24 hours. It should be noticed that the report is poorly documented.
Dow Chemical Co, 1979 has conducted a test on Daphnia magna . A 48-hour EC 50 was obtained: 835 mg/L (95% confidence interval = 640-1,051 mg/L ) – test #3. A static open system was used. Aquariums were filled with te n daphnids and raw lake water dechlorinated with activated carbon. Five test concentrations (370, 650, 1,000, 1,500, 2,800 mg/L) plus one control were tested in three replicates. Temperature was around 20°C, pH between 7.6 and 7.8 and water hardness was 101 mg/L (CaCO 3 ).
Test #4 has been performed with a crustacean, Artemia salina , by Price et al

. , 1974 and a 24-hour EC 50 of 1,000 mg/L has been determined. The te st medium was cons tituted by artificial seawater, at 24.5°C. 30 to 50 shrimp / mL were di sposed at the beginning of the test in 100 mL test solution. The following EGBE concentr ations were tested: 100, 1,000, 10,000 mg/L. At the end of the test, the number of alive shrimp was determined with a colony counter. The lack of analytical monitoring and the insufficient number of test concentrations have to be deplored.
In test #5, Bringmann and Kühn, 1977 have tested EGBE toxicity on Daphnia magna , according to the method DIN 38412 part 11: immobilisation in artificial fresh water. They noticed a 24-hour EC 50 of 1,720 mg/L. The test was conducted in an open system with 24 hour-old daphnids (wild species) in tap water. Temp erature ranged between 20 and 22°C and pH was about 7.6-7.7. Test solution was saturated with oxygen at the beginning of the essay but no mention of O 2 concentration at the end of the test wa s made. Three replicates were conducted for each concentration.
Test #6 has also been performed by Bringmann and Kuhn, 1982 on 24 hour-old Daphnia magna (IRCHA clone) with the same method as above. The EC 50 after 24 hours was 1,815 mg/L (95% confidence interval = 1,698-1,940 mg/L). Other te st conditions were as follow: open system, 20°C, pH = 8 +/- 0.2, water hardness was 250 mg/L (CaCO 3 ) and DO was measured at the end of the study to check that it was within the range tolerated by daphnids (2mg/L). Concentrations were tested in duplicate.
INERIS, 1999a has tested the toxicity of EGBE on Daphnia magna according to OECD guideline 202 (test #7). Five da phnids were used per containe rs and four replicates were performed for each test concentration: 0, 650, 1,000, 1,500, 2,200, 3,350, 5,000 mg/L. Other test conditions were as follow: wa ter hardness 25 0 mg/L (CaCO 3 ), pH = 7.8 +/- 0.2, DO = 5.3-6.8 mg/L. Less than 10% immobilisation was obs erved in the control after 48 hours and the analytical monitoring revealed a loss of substance during the test < 20%. A 48-hour EC 50 of 1,550 mg/L (95% confidence interv al = 1,390-1,720 mg/L) was observed.

We can notice another 24-hour EC 50 result of 5,000 mg/L with Daphnia Hoechst, 1982) – test #8. But as no further data is available, this test is clas sified as invalid.

Long-term toxicity
Chronic toxicity tests for EGBE are presented in Table 3.33

.

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Table 3.33 Long term invertebrate toxicity data for EGBE

Test # Species Duration Endpoint Result (mg/L) Method References Validity

1 Brachionus calyciflorus 48 hours EC 10 EC 20 EC 50 7.2 14.3 164 AFNOR NF T 90-377 DeVillers et al., 2002a Not useable for PNEC derivation

2 Daphnia magna 21 days NOEC EC 10 EC 20 EC 50 100 134 175 297 OECD guideline n°211 INERIS, 1999d Valid

3 Ceriodaphnia dubia 7 days EC 10 134.9 Draft AFNOR NF T 90 – 376 DeVillers et al., 2003 Valid with restrictions
The chronic toxicity of EGBE on the rotifer Brachionus calyciflorus has been determined by DeVillers et al., 2002a according to the French norm AFNOR NF T 90-377 – test #1. Cyst hatching was initiated in moderately hard water, about 20 hours before the beginning of the test (at 25°C under a light intensity of 3,000 lux.). pH was adjusted to 7.5. After 18 hours of incubation, cysts were regularly checked to ensu re the removal of test organisms within two hours of hatching. The assay was performed in a 48-well microplate (five concentrations plus one control with eight replicates). Test media c onsisted in synthetic fresh water solution with a suspension of the green alga Chlorella vulgaris as food source. One rotifer was disposed per well (newly hatched rotifer) and the in cubation occurred at 25°C in dar kness, in a covered microplate. After 48 hours, the tota l number of rotifers per well was counted and EC 10 , EC 20 and EC 50 were determined by non-linear regression using a log logistic model. The respective confidence intervals for these endpoints were as follow: EC 10 = 4.5-13.7 mg/L, EC 20 = 8.8-37.8 mg/L and EC 50 = 75-486 mg/L. No analytical mon itoring was performed during the test.
Several points can be highlighted so as to assess the validity of this test performed on Brachionus calyciflorus and showing the highest sensitivity. The pros and cons for the validation of the test are compiled in Table 3.34

.

Table 3.34 Pros and cons for the validation of the test performed with Brachionus calyciflorus

Issue Pros Cons
Analytical monitoring The test compound is considered stable in similar testing conditions, during 48 hours. Under static conditions, the monitoring of test concentrations during test #7 for invertebrates (INERIS, 1999a) has revealed a loss of the substance less than 20%, after 48 hours. In order to prevent evaporation of the solvent or the test substance during the test (EGBE evaporates more slowly than water), the microplate was covered. Indeed, the French norm recommends that the incubation is carried out in a water saturated atmosphere in order to avoid losses of test solution by evaporation; for example, the microplate can be arranged in a closed dish at the bottom of which a film of water has been previously deposited. Furthermore, an interlaboratory test has been successfully performed with copper sulphate and no evaporation problem has been encountered. No analytical monitoring has been performed during the test. Consequently, neither a loss of test substance nor a loss of solvent (water) during the test duration could not have been followed.

Table 3.34 continued overleaf

56CHAPTER 3. ENVIRONMENT

Table 3.34 continued Pros and cons for the validation of the test performed with Brachionus calyciflorus

Issue Pros Cons

Concentration / effect relationship Glycol ethers often show non-conventionalconcentration/effect relationships. Such nonconventional curves have already been reported for Xenopus exposed to 2-methoxyethanol (Daston et al., 1991) or for Hydra attenuata exposed to EGBE (Bowden et al., 1995).

DeVillers et al

. , 2002b has also reported the same difficulty in finding clear relationships between the tested concentrations and the endpoints studied for their tests with EGME/EGMA performed on algae, rotifers, molluscs, daphnids and fish (some of these tests where repeated two or more times so as to ensure that they reached their respective conditions of acceptance). The authors also report that similar conclusions can be drawn from an analysis of published papers in which the ranges of tested concentrations of EGME are given. These tests were performed with Pimephales promelas and Drosophila melanogaster (Daston et al

. , 1991), D. melanogaster again in Lynch and Toraason, 1996. The test results show a non-conventional concentration/effect relationship (a factor of 23 can be calculated between EC50 and EC10).
The studies from Daston et al., 1991 and Bowden et al., 1995 also show a conventional response for methoxyethanol with Hydra and, whilst the EGBE response is flat, it is still of conventional shape, which is not the case with the Brachionus

. Even if there are indications that the non conventional dose-response curves may be a general reaction to glycol ethers, no explanation has been found to this phenomenon.

Normalisation The test has been carried out following a French norm (NF T 90-377). An ISO norm is also in preparation. The methodology used for the test with rotifers is not as well normalised as the one for standard tests usually used for effect assessments of chemicals. Higher sensitivity For EGBE, the Brachionus test shows the highest sensitivity among all species tested. A ratio of 18.6 can be calculated between the ECs10 for Daphnia magna and Brachionus indicating a greater sensitivity of Brachionus compared to Daphnia magna . This is not in accordance with the trend generally observed when sensitivities of both species are compared, giving a ration of 2 between Brachionus and Daphnia indicating that Daphnia would be twice as sensitive as Brachionus (RIVM, 2004). Oxygenation of the test media Daphnia 48-hour test can be taken for comparison with the 48-hour Brachionus test. In an acute daphnids test (INERIS, 1999a), the measured dissolved oxygen concentration at the end of the test was between 5.3 and 6.8 mg/L. It can be expected that a similar DO level was maintained during the rotifer test. Although test solution volumes are lower in the rotifer test, both tests show similar conditions and the same duration Since no aeration took place and the test wells were covered during the test phase, it should have been reported how appropriate oxygen concentration was maintained during the whole test phase. This was not done. In fact, because EGBE is readily biodegradable, oxygen depletion is possible, which would mean that the low NOECs of the Brachionus calyciflorus studies were not caused by the toxic properties of EGBE. This may not occur to a significant degree over the timescale of the study but, with no oxygen data, it is not possible to be certain.
Considering all the elements from Table 3.34 no clear reason can be f ound to fully invalidate the test. However, considering all the elements highli ghted that have triggere d off some concerns for the validation of this test, this study will be ex cluded from the PNEC derivation. This decision is also supported by the availability of standard toxici ty test results such as the one performed with Daphnia magna (test #2).

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Test #2 performed by INERIS, 1999d on Daphnia magna according to OECD guideline No 211, revealed a 21-day NOEC of 100 mg/L. Five c oncentrations (0.1, 1, 10, 100 and 500 mg/L + control) were tested and solutions were renewed three times a week. Other test conditions were as follows: pH = 7.5-7.8, temperature = 20 +/- 2°C, DO ranged between 6.9 and 8.2 mg/L and water hardness was 220 mg/L (CaCO 3 ). A partial analytical monitoring has been performed, i.e. only for concentrations of 100 and 500 mg/L, a nd loss of substance over test duration was ranging between 3 and 14%. The c onfidence intervals for the endpoints measured were as follow: EC 10 = 110-140 mg/L, EC 20 = 150-180 mg/L and EC 50 = 282-300 mg/L.

DeVillers et al., 2003 – test #3 - have tested the toxicity of EGBE on Ceriodaphnia dubia according to the draft of the method AFNOR NF T90-376 (equivalent to OECD guideline No 211). An EC 10 of 134.9 mg/L has been determined after seven days. One daphnid was disposed per container, in ten replicates. The pa rental mortality and the number of offspring per living parent were used as endpoi nts. Other test conditions are s hown here: eight concentrations plus control were tested at a temperature of 23 +/- 1°C an d pH = 8-9, DO = 8.1-8.3 mg/L and water hardness was 200 +/- 40 mg/L (CaCO 3 ). There was no analytical monitoring because preliminary studies showed that EGBE was stab le at least during 24 hours, i.e. between two renewals. Furthermore, the dose – response curve was not clear and the 95% confidence interval for the EC 10 ranged between 25.3 and 147.4 mg /L. A NOEC (55.6 mg/L) and an EC 50 (138.2 mg/L) have also been calculated in anothe r report (INERIS, 1999c) but methods used for calculation tend to give a preference to the EC 10 value.

3.2.1.3 Algae
Toxicity tests for EGBE are summarised in Table 3.35

.

Table 3.35 Algae toxicity data for EGBE

Test # Species Duration Endpoint Result (mg/L) Method References Validity

1 Microcystis aeruginosa 8 days EC 3 (biomass) 35 Cell multiplication inhibition test Bringmann and Kühn, 1976 Not useable for PNEC derivation

2 Pseudokirchn eriella subcapitata 72 hours NOEC 286 OECD guideline n°201 INERIS, 1999e Valid

3 Selenastrum capricornutum 7 days NOEC LOEC EC 50 125 250 >1,000 US EPA 600/9-78- 018 Dill and Milazzo, 1988 Not useable for PNEC derivation

4 Scenedesmus quadricauda 8 days EC 3 (biomass) 900 Cell multiplication inhibition test Bringmann and Kühn, 1978 Not useable for PNEC derivation
Note: in some tests performed by Bringmann, T GK (Toxische GrenzKonzentration) values were established. As the percentage effect at the TG K is 3 or 5% (EC3 or EC5) these values are regarded as NOEC values.
Bringmann and Kühn, 1976 have performed a EGBE toxicity test on the algae Microcystis aeruginosa – test #1. The test occurred in a closed static system and 14 concentrations were tested in duplicates (dilution with a pace of tw o from a stock solution). Agitation took place once a day and the test was conducte d at 27°C in a neutral pH. The biomass was measured by spectrophotometry at 578 nm and an EC 3 of 35 mg/L was determined after eight days. This EC 3 can be considered as a NOEC. It should be noted that several drawbacks ar e associated with this

58CHAPTER 3. ENVIRONMENT

study. Indeed, the test should have been performed during the expone ntial growth phase of algal cells. However, algal cells could not have been ma intained in this phase within the duration of the study (7 days). There is also a deviation from more standard test results when converting the endpoint measured in the study (EC 3 ) to a NOEC or EC 10 . Finally, the result obtained in this study is based on biomass and is comparable with test #2 and #3 results for biomass measurements too. Nevertheless, growth rate m easurement is usually preferred when assessing the toxicity towards algae. Therefore, this study will not be used for the PNEC calculation.
A NOEC of 286 mg/L was determined in test #2 (INERIS, 1999e) base d on growth rate determination. This test was performed according to the OECD guideline No 201. Test conditions were as follow: 23 +/- 2°C, pH between 7.6 and 8.3, cell concentration = 10,000 cells/mL, illumination was set at 7,000 lux. and test medium was subject to agitation. Nominal concentrations are presented hereafter: 0, 88, 159, 286, 514, 926, 1,667 and 3,000 mg/L. There was an analytical monitoring a nd loss of substance over test duration ranged between 2 and 11%. Other endpoints have been measured during this test. A 72-hour NOEC of 88 mg/L, an EC 10 of 308 mg/L and an EC 50 of 911 mg/L were determined as far as biomass is concerned.
Test #3 has been performed by Dill and Milazzo, 1988 on the alga Selenastrum capricornutum

. A 7-day NOEC of 125 mg/L has been determined according to a method from the US EPA: the Selenastrum capricornutum Printz algal assay EPA-600/9- 78-018. Test conditions were as follow: pH was measured before and after the test and ranged between 7.4 and 7.8, temperature = 24 +/- 2°C, initial cell concentration = 10,000 cells/mL, illumination was set at 4,304 +/- 403 lux. Five concentrations ( 63, 125, 250, 500 and 1,000 mg/L) plus one control were tested in three replicates. Endpoints taken into account were the number of cells / mL and the total cell volume; two counts per replicate were performed with an electronic count er. It can be noticed that the highest concentration te sted did not inhibit 50% growt h. The comments made on test #1 for its validation apply also for this test.
Bringmann and Kühn, 1978 examined, in test #4, the toxicity of EGBE on Scenedesmus quadricauda . An EC 3 of 900 mg/L (equivalent to a NOE C) was determined according to biomass measurement by spectrometry at 578 nm. Test conditions are shown hereafter: closed static system, neutral pH, temp erature = 27°C, agitation once a day. 14 concentrations were tested in duplicate but no analy tical monitoring took place. The comments made on test #1 for its validation apply also for this test.

3.2.1.4 Micro-organisms
EGBE toxicity studies with micro-organisms are presented in Table 3.36

.

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Table 3.36 Micro-organisms toxicity data for EGBE

Test # Species Duration Endpoint Result (mg/L) Method References Validity

1 Entosiphon sulcatum * (protozoa) 72 hours EC 5 91 Cell multiplication inhibition test Bringmann, 1978 Valid with restrictions

2 Uronema parduzci (protozoa) 20 hours EC 5 463 Cell multiplication inhibition test Bringmann and Kühn, 1980 Valid with restrictions

3 Pseudomon as putida (bacteria) 16 hours EC 3 700 Cell multiplication inhibition test Bringmann and Kühn, 1976 Valid with restrictions

4 Chilomonas paramecium * (protozoa) 48 hours EC 5 911 Cell multiplication inhibition test Bringmann et al

. , 1980 Valid with restrictions

* Unciliated The toxicity of EGBE on protoz oa has been evaluated in three tests (#1,2,4). To assess the toxicity of a chemical on micro-organisms te sts conducted on ciliated pr otozoa are accepted but only one micro-organism is matching this characteristic: Uronema parduzci (#2), the others belong to unciliated protozoa. The cell multiplica tion inhibition was measured with an electronic cell counter. Tests were performed in a closed static system, at 25°C and neutral pH and 14 concentrations were tested in duplicate.
A test performed with the bacteria Pseudomonas putida (Bringmann and Kühn, 1976) is also quoted – test #3. The cell multiplication inhib ition was retained as an endpoint (biomass measurement via extinction at 436 nm) and the test was performed in a closed static system during 16 hours, at 25°C and neutra l pH. A range of 14 concentrati ons was tested in duplicate.

3.2.1.5 Endocrine disruption

A test has been conducted so as to identify a potential endocrine disrupting effect due to EGBE: INERIS, 2001. This study was performed accordi ng to an OECD guideline project based on the OECD guideline n°204. Fish ( Brachydanio rerio ) have been exposed during 21 days at three EGBE concentrations: 1, 10, 100 mg/L plus one c ontrol. No mortality was observed at any test concentration. Moreover the subs tance did not induce a higher vitellogenin synthesis (lower quantities than in the control were actually measured). Alth ough a lower gonado-somatic index 12 was observed at the lowest concen tration this was not the case fo r higher ones. After an exposure to the positive control substan ce (oestradiol, an oestrogenic chemical) the brachydanio female presented genito-urinary buds wh ich can normally only be found on the male. The percentage of apparition of genito-urinary buds was less than 10 % in all treatment groups compared to 40% in the group of fish in contact with oestradiol.
To conclude, EGBE has no potenti al for endocrine disruption.

12 Ratio of weight of gonads compared to weight of total organism.

60CHAPTER 3. ENVIRONMENT

3.2.1.6 PNEC for the aquatic compartment
Three long term test results from three species repres enting three trophic levels will be used to derive the PNEC aqua for EGBE. These tests are gathered in Table 3.37

.

Table 3.37 Toxicity tests retained for the derivation of PNEC aqu a Species Duration Endpoint Result (mg/L) Reference Lowest short term toxicity result for the same trophic level

Fish: Brachydanio rerio 21 days NOEC > 100 INERIS, 2001 Poecilia reticulata LC 50 after 7 days = 983 mg/L (Konemann, 1981).

Invertebrates: Daphnia magna 21 days NOEC 100 DeVillers et al

. , 2002a Hydra attenuata EC 50 after 72 hours =540 mg/L (Bowden et al., 1995) Daphnia magna EC 50 after 48 hours = 835 mg/L (Dow Chemical Co, 1979) Algae: Pseudokirchneriella subcapitata 72 hours NOEC 286 INERIS, 1999e Pseudokirchneriella subcapitata EC 50 after 72 hours = 911 mg/L (INERIS, 1999e)

An assessment factor of 10 is a pplied to the lowest test result in order to derive the PNEC aqua :

PNEC aqua = 10 mg/L
To support this assessment, we can add that EGBE is a non-polar narcotic (OECD, 1995) and acts with a non-specific mod of action in organisms. Although a smaller Assessment Factor can be used to derive a PNEC for non polar narcotics, a factor lower than 10 is not recommended by TGD (EC, 2003). Only additional studies, i.e. fiel d data or model ecosystems, could result, after a review on a case by case basis, in a lower assessment factor. Ne vertheless, the knowledge of a non-specific mode of action for EGBE seconds the PNEC aqua proposal.

Table 3.38 shows ecotoxicity results calculated from a log K ow of 0.8 and a molar weight of 118.17 g/mol. The equations for non-polar narcotics given in Table 1 of Chapter 4, Part III of TGD (EC, 2003) were used to estimate QSAR ecotoxicity data.

Table 3.38 QSAR ecotoxicity data for EGBE
Species Enpoint Result (mg/L)

Fish: Pimephales promelas 96-hour LC 50 1,006

Fish: Brachydanio rerio or Pimephales promelas 28, 32-day NOEC 113 Daphnia: Daphnia magna 48-hour EC50 983 Daphnia: Daphnia magna 16-day NOEC 242 Algae: Selenastrum capricornutum 72, 96-hour EC 50 1,103
The close relationship between predicted ecotoxicit y of EGBE and results from ecotoxicity tests confirms that EGBE does not ac t with a specific action manner.

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3.2.1.6.1 Calculation of the intermittent PNEC for freshwater
For substances subject to intermittent release, long-term effects are not likely to occur. Consequently, the effect assessment for substances with intermittent release is based on the acute toxicity data set. For EGBE, these tests are gathered in Table 3.39

.

Table 3.39 Toxicity tests retained for the derivation of the intermittent PNEC aqua Species Duration Endpoint Result (mg/L) Reference

Fish: Poecilia reticulata 7 days LC 50 983 Konemann, 1981

Invertebrates: Hydra attenuata 72 hours EC 50 540 Bowden et al., 1995

Algae: Pseudokirchneriella subcapitata 72 hours EC 50 911 INERIS, 1999e

Usually an assessment factor of 100 applies to the lowest result of toxicity tests performed on three trophic levels. It has been established that EGBE can be classified as non-polar narcotic. This could be a rationale for a lowering of the assessment factor. Consequently: PNEC aqua,inter = 540 / 10 = 54 mg/L.

3.2.1.6.2 Calculation of the PNEC for the seawater compartment
Chronic toxicity data on three freshwater species representing three trophic levels are available. Only acute toxicity data on marine organism s (fish and invertebra tes) are available. Nevertheless, the species tested do not show higher sensitivitie s than freshwater organisms. According to TGD (EC, 2003), both freshwater a nd seawater species are used to derive the PNEC for seawater. Thus the PNEC for marine organisms is determined from the lowest chronic test result (NOEC (21 days) = 100 mg/L on Daphnia magna ) to which an assessment factor of 100 is applied as proposed in the TGD. This gives a PNEC saltwater of 1 mg/L.

3.2.1.6.3 Calculation of the intermittent PNEC for seawater
An assessment factor of 1,000 should be applied on th e lowest acute toxicity test result in order to determine an intermittent PNEC for seawater. For the same reason explained above, this factor could be lowered. Consequently: PNEC saltwater,inter = 540/100 = 5.4 mg/L.

3.2.1.7 Calculation of a PNEC for the sediment compartment
As no specific data is available for this compartment, the PNEC sed will be calculated from the PNEC aqua using the equilibrium partitioning method.

62CHAPTER 3. ENVIRONMENT

Equation 3.6 Formula for the calculation of PNEC sed

using the equilibrium partitioning approach



### 1000 × × = − aqua
susp watersusp sed 

### PNEC RHO K PNEC

PNEC sed Predicted No Effect Concentrati on in sediment [mg/kg, wet weight] K susp-water partition coefficient susp ended matter / water [~1.58 m 3 /m 3 ] RHO susp bulk density of wet suspended matter [~1150 kg/m 3 ] PNEC aqua Predicted No Effect Concentration in water [10 mg/L]

This results in: PNEC sed = 13.7 mg/kg (ww)

3.2.1.7.1 Calculation of the PNEC for the marine sediment compartment
No test is available on sediment dwelling organisms exposed via se diment. The PNEC for organisms living in marine sediments may provi sionally be calculated using the equilibrium partitioning method from the PNEC for th e marine aquatic compartment (PNEC saltwater ).
Thus, the PNEC marine sed = 1.4 mg/kg wet weight of marine sediment.

3.2.1.8 PNEC for micro-organisms in STP
Four EGBE toxicity te sts on micro-organisms are quoted. Three tests were conducted with protozoa and one with an indivi dual bacteria species. Studies test ing ciliated protozoa can be used for the determination of a PNEC micro-organisms . That is why the test conducted on Uronema parduzci will be retained for the PNEC determination.

PNEC micro-organisms = 463 mg/L.

3.2.2 Terrestrial compartment
Since there are no EGBE toxicity data for terrestrial organisms, no PNEC soil can be derived directly. Therefore, this PNEC was estimated from the PNEC for aquatic organisms using the equilibrium partitioning approach.

Equation 3.7 Formula for the calculation of PNEC soil

using the equilibrium partitioning approach



### 1000 × × = − aqua soil watersoil soil 

### PNEC RHO K PNEC

PNEC soil Predicted No Effect Concentra tion in soil [mg/kg, wet weight] K soil-water partition coefficient soil water [~1.025 m 3 /m 3 ] RHO soil bulk density of wet soil [~1700 kg/m 3 ] PNEC aqua Predicted No Effect Concentration in water [10 mg/L]

This results in: PNEC soil = 6 mg/kg (ww)

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3.2.3 Atmosphere

No data are available in order to correctly assess the effect of EGBE for species living in the environment and exposed via the air compartment. In a first attempt to quan tify the risk for this compartment, inhalation toxicity da ta from the human risk assessment have been reported in this section.
In a repeat dose study with rats exposed by inhalation, a NOAEC value of 25 ppm (121 mg/m 3 ) has been identified from a sub-chronic study. Du ring these studies, haemol ysis was consistently observed and sometimes associated with hepati c effects. Effects on body weight gain, on the forestomach and on the WBC sub-populations (T limphocyte) were also observed. In a separate study a LOAEC of 31 ppm (150 mg/m 3 ) has been determined for mice and rats. Due to the closeness of the apparent LOAEC and NOAEC, it ha s been considered prudent to take the more conservative LOAEC of 31 ppm forward for the human health risk characterisation (with appropriate assessment factors). However, as the approach taken for the risk characterisation for the environmental section (atmospheric compartmen t) should be considered as a first tier, the NOAEC will be retained.

3.2.4 Secondary poisoning
No specific data available.

64CHAPTER 3. ENVIRONMENT

3.3 RISK CHARACTERISATION

3.3.1 Aquatic compartment (incl. sediment)

STP and surface water (including seawater)

Table 3.40 presents the calculated PEC / PNEC ratio s for the aquatic compartment. PECs for STP and surface water appear in Section 3.1.4.1 of this study whereas the corresponding PNECs are determined in Section 3.2.1 : 463 mg/L for the PNEC STP and 10 mg/L for PNEC aqua (1 mg/L for PNEC saltwater ).

Table 3.40 Risk characterisation for micr o-organisms in STP and aquatic organisms

Scenario RCR STP RCR aqua RCR seawater
Production site #1 0 (3.10 -5

) n. a. 0 (8.10 -4

)

Production site #2 0 (7.10 -5

) 0.001 n. a.
Production site #3 0 (6.10 -6

) 0.001 n. a.
Production site #4 0 (4.10 -6

) 0.001 n. a.
Paints I F / Paints I P 0 (7.10 -5

) / 0 (2.10 -4

) 0.006 / 0.015 0.042 / 0.114

Paints II F & III F 0 (7.10 -5

) 0.006 0.043

Paints II P and Paints III U 0 (3.10 -4

) 0.002 0.011

Paints IV F / Paints IV P - / - 0 (7.10 -4

) / 0 (7.10 -4

) 0.001 / 0.001

Paints V F / Paints V P - / 0 (1.4.10 -5

) 0 (7.10 -4

) / 0.002 0.001 / 0.009

Paints VI F / Paints VI P 0 (3.10 -4

) / 0 (1.4.10 -4

) 0.019 / 0.012 0.142 / 0.087

Paints VII F / Paints VII P 0 (7.10 -5

) / 0 (1.4.10 -5

) 0.007 / 0.002 0.050 / 0.008

Paints VIII F / Paints VIII P - / - 0 (7.10 -4

) / 0 (7.10 -4

) 0.001 / 0.001

Paints IX F / Paints IX P 0 (3.10 -5

) / 0 (4.10 -6

) 0.003 / 0.001 0.015 / 0.002 Detergents I F / Detergents I P 0 (2.10 -5

) / 0 (7.10 -5

) 0.002 / 0.005 0.011 / 0.037 Detergents II F / Detergents II U 0 (7.10 -6

) / 0 (1.10 -5

) 0.001 / 0.002 0.005 / 0.008

Intermediates** I P 0 (1.10 -4

) 0.003 0.059

Inks I F / Inks I P 0 (1.10 -4

) / 0 (3.10 -6

) 0.011 / 0 (9.10 -4

) 0.084 / 0.002

Pharm I P 0 (4.10 -5

) 0.001 0.021

Elec I P 0 (1.10 -4

) 0.009 0.063

Leather I P 0 (4.10 -4

) 0.029 0.223

Adhesives I F / Adhesives I P 0 (3.10 -5

) / 0 (7.10 -6

) 0.002 / 0.001 0.014 / 0.005 Agri I F / Agri I P 0 (1.10 -5

) / - 0.002 / 0 (7.10 -4

) 0.007 / 0.001

Table 3.40 continued overleaf

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Table 3.40 continued Risk characterisation for micro-organisms in STP and aquatic organisms

Scenario RCR STP RCR aqua RCR seawater
Oilfield I F / Oilfield I P 0 (1.10 -4

) / 0 (4.10 -5

) 0.011 / 0.004 0.084 / 0.023

Metal I F / Metal I P

Metal intermittent 0.001 / 0 (1.10 -4

)

0.003 0.007 / 0.001 0 (5.10 -4

) 0.054 / 0.005 0 (5.10 -4

)

Cosmet I F & Fire I F / Cosmet I U 0 (5.10 -5

) / 0 (2.10 -6

) 0.004 / 0 (9.10 -4

) 0.027 / 0.003

Fire I P 0 (5.10 -6

) 0.001 0.002
* Captive use not included P Processing F Formulation U Private use For the risk characterisation at production and processing taking place on-site, the worst case based on site-specific data gives a risk char acterisation ratio of 0.001 for freshwater and 8.10 -4 for seawater.
For some end uses, formulation a nd processing steps can be achieved at a same site. So, in order to characterise the total risk at such sites it is necessary to add the calculated risks for each step.
According to Table 3.40 no risk is identified for all end uses even when both formulation and processing can be considered at a same site (dimmed lines of Table 3.40 ).
No risk assessment could have been performe d concerning the use of EGBE in oil spill dispersants (see Section 3.1.2.2.5). Ne vertheless, no conclusion will be drawn concerning this use in the EU risk assessment since it has to be acknow ledged that the deci sion whether to use a dispersant is made with the regulatory authoritie s case-by-case and on a risk benefit basis. It is generally accepted that dispersants create adverse effects but their use is intended to mitigate the even worse potential effects of environmentally hazardous oil products. For this reason, even if detailed composition information was available, it would still not be possible to carry out such a risk benefit analysis within the framework of the Existing Substances risk evaluation process.

As far as the use of oilfield chemicals is concer ned, specific regulations ar e in place in different EU countries. Because they use detailed information supplied under confidentiality terms, specific assessments performed under these regulations should be regarded as more precise than those carried out in this assessment. For inst ance, detailed information is provided by the manufactures on actual usage and em ission rates. Consequently, in the knowledge that the use of such products is controlled by specific legislation, a Conclusion (ii) is appropriate.
EGBE is readily biodegradable and has a low pot ential for accumulation in biota. Consequently, this substance will not remain in the environment and secondary poisoning is not expected. Based on the risk assessment performed for fre shwater and on the lack of specific hazard identified for the marine environment, no risk is expected in the marine compartment.
Sediment (freshwater and marine sediments)
As neither monitoring da ta on levels of EGBE in sediment nor ecotoxicity data for benthic organisms are available, no risk characterisation is conducted for this compartment. In addition, the partition coefficient between sediment and wate r for EGBE is low. So it can be assumed that the risk assessment for the sediment is cove red by that for surface water (freshwater and seawater).

66CHAPTER 3. ENVIRONMENT

Conclusions to the risk assessm ent for the aquatic compartment
Conclusion (ii)

. Conclusion (ii) is applied to al l levels of the lif e cycle of EGBE: production, formulation, processing and private use.

3.3.2 Terrestrial compartment

Risk characterisation for the te rrestrial compartment has been performed calculating PEC/PNEC ratios. PECs for soil have been estimated in Section 3.1.4.2 and the corresponding PNEC has been determined in Section 3.2.2 (6 mg/kg, wet weight). Results are shown in Table 3.41

.

Table 3.41 Risk characterisation for terrestrial compartment

Scenario RCR agricultural_soil_over_30_days RCR grassland_over_180_days
Production site #1 0.006 0 (8.10 -4

)

Production site #2 0 (2.10 -4

) 0 (3.10 -4

)

Production site #3 0 (2.10 -4

) 0 (2.10 -4

)

Production site #4 0 (2.10 -4

) 0 (2.10 -4

)

Paints I F / Paints I P 0.005 / 0.014 0 (9.10 -4

) / 0.004

Paints II F and III F 0.005 / 0.013 0 (9.10 -4

) / 0.003

Paints II P and Paints III U 0.002 0 (6.10 -4

)

Paints IV F / Paints IV P 0 (5.10 -4

) / 0.002 0 (8.10 -4

) / 0.003

Paints V F / Paints V P 0 (3.10 -4

) / 0.001 0 (4.10 -4

) / 0 (3.10 -4

)

Paints VI F / Paints VI P 0.016 / 0.011 0.002 / 0.003

Paints VII F / Paints VII P 0.006 / 0.001 0 (9.10 -4

) / 0.001

Paints VIII F / Paints VIII P 0 (2.10 -4

) / 0 (4.10 -4

) 0 (3.10 -4

) / 0 (6.10 -4

)

Paints IX F / Paints IX P 0.002 / 0 (6.10 -4

) 0 (4.10 -4

) / 0 (6.10 -4

)
Detergents I F / Detergents I P 0.001 / 0.004 0 (3.10 -4

) / 0 (7.10 -4

)
Detergents II F / Detergents II U 0 (7.10 -4

) / 0.001 0 (2.10 -4

) / 0 (3.10 -4

)

Intermediates** I P 0.032 0.004

Inks I F / Inks I P 0.009 / 0 (9.10 -4

) 0.001 / 0.001

Pharm I P 0.011 0.001

Elec I P 0.007 0 (9.10 -4

)

Leather I P 0.025 0.003

Adhesives I F / Adhesives I P 0.002 / 0 (7.10 -4

) 0 (4.10 -4

) / 0 (2.10 -4

) Agri I F / Agri I P 0.001 / 0 (4.10 -4

) 0 (5.10 -4

) / 0 (4.10 -4

)

Oilfield I F / Oilfield I P 0.009 / 0.003 0.001 / 0 (4.10 -4

)

Table 3.41 continued overleaf

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Table 3.41 continued Risk characterisation for terrestrial compartment

Scenario RCR agricultural_soil_over_30_days RCR grassland_over_180_days
Metal I F / Metal I P 0.008 / 0.002 0.001 / 0 (4.10 -4

)

Cosmet I F and Fire I F / Cosmet I U 0.003 / 0 (3.10 -4

) 0 (5.10 -4

) / 0 (2.10 -4

)

Fire I P 0 (4.10 -4

) 0 (2.10 -4

)
* Captive use not included P Processing F Formulation U Private use

For the risk characterisation at production and processing taking pl ace on-site it can be noticed that no risk is expected. Accord ing to the figures presented in Table 3.41 , no risk is expected at formulation, processing or private use stages.
For some end uses, formulation and processing steps can be achieved at a same site (see Table 3.41 ). So, in order to characterise the total risk at such sites it is necessary to add the calculated risks for each step. According to Table 3.41 no risk is identified for all end uses where both formulation and processing are considered.

Conclusions to the risk assessment for the terrestrial compartment:
Conclusion (ii)

. Conclusion (ii) is applied to al l levels of the lif e cycle of EGBE: production, formulation, processing and private use.

3.3.3 Atmosphere
No specific effect data are avai lable in order to accurately asse ss the risk for the atmospheric compartment. However, due to the volatility of EGBE, direct emissions to air should not be overlooked. In a first attempt to quantify the risk for the air compartment, a NOAEC of 121 mg/m 3 will be compared to the PECs calculated for air. This NOAEC has been determined in a study where rats wher e exposed via inhalation.

The worst PEC local_air,ann of 51 μg/m 3 has been calculated for the scenario Paint IV P (use in can coatings).
Using the methodology described in the risk characterisation for c onsumers, a minimal margin of safety (MOS) can be calculated as follows:
Interspecies differences 0.1 (based on rationale described in the risk characterisation for workers part to account for relative species sensitivity to critical end point)
Intraspecies differences 10 (default for consumers)
Type of effect 1

Confidence of the database 1
Minimal MOS required 1

68CHAPTER 3. ENVIRONMENT

The ratio between the threshold reta ined in the effect assessment and this worst case exposure is about a factor of 2,400. This rough risk charac terisation for the air compartment leads to no concern by a sufficiently large margin that a more accurate assessment is not considered
necessary.

Conclusions to the risk assessment for atmosphere
Conclusion (ii)

. Conclusion (ii) is applied to al l levels of the lif e cycle of EGBE: production, formulation, processing and private use.

3.3.4 Secondary poisoning

Conclusions to the risk asse ssment for secondary poisoning
Conclusion (ii)

. Conclusion (ii) is applied to al l levels of the lif e cycle of EGBE: production, formulation, processing and private use.

69

### 4 HUMAN HEALTH

(to be added later).

70

### 5 RESULTS

5.1 ENVIRONMENT

Conclusions to the risk assessm ent for the aquatic compartment
Conclusion (ii) There is at present no need for furthe r information and/or testing and no need for risk reduction measures beyond t hose which are bei ng applied already.
Conclusion (ii) is applied to al l levels of the lif e cycle of EGBE: production, formulation, processing and private use.

Conclusions to the risk assessment for the terrestrial compartment
Conclusion (ii) There is at present no need for furthe r information and/or testing and no need for risk reduction measures beyond t hose which are bei ng applied already.
Conclusion (ii) is applied to al l levels of the lif e cycle of EGBE: production, formulation, processing and private use.

Conclusions to the risk assessmen t for the atmospheric compartment
Conclusion (ii) There is at present no need for furthe r information and/or testing and no need for risk reduction measures beyond t hose which are bei ng applied already.
Conclusion (ii) is applied to al l levels of the lif e cycle of EGBE: production, formulation, processing and private use.

Conclusions to the risk asse ssment for secondary poisoning
Conclusion (ii) There is at present no need for furthe r information and/or testing and no need for risk reduction measures beyond t hose which are bei ng applied already.
Conclusion (ii) is applied to al l levels of the lif e cycle of EGBE: production, formulation, processing and private use.

5.2 HUMAN HEALTH
(to be added later).

71

### 6 REFERENCES

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76

### ABBREVIATIONS

ADI Acceptable Daily Intake

AF Assessment Factor

ASTM American Society for Testing and Materials

ATP Adaptation to Technical Progress

AUC Area Under The Curve

B Bioaccumulation

BBA Biologische Bundesanstalt für Land- und Forstwirtschaft

BCF Bioconcentration Factor

BMC Benchmark Concentration

BMD Benchmark Dose

BMF Biomagnification Factor

BOD Biochemical Oxygen Demand

bw body weight / Bw
, bw

C Corrosive (Symbols and indications of danger for dangerous substances and preparations according to Annex II of Directive 67/548/EEC)

CA Chromosome Aberration

CA Competent Authority

CAS Chemical Abstract Services

CEC Commission of the European Communities

CEN European Standards Organisation / European Committee for Normalisation

CEPE European Committee for Paints and Inks

CMR Carcinogenic, Mutagenic and toxic to Reproduction

CNS Central Nervous System

COD Chemical Oxygen Demand

CSTEE Scientific Committee for Toxicity, Ec otoxicity and the Environment (DG SANCO)

CT 50 Clearance Time, elimination or de puration expressed as half-life

d.wt dry weight / dw

dfi daily food intake

DG Directorate General

DIN Deutsche Industrie Norm (German norm)

DNA DeoxyriboNucleic Acid

DOC Dissolved Organic Carbon

DT50 Degradation half-life or period required for 50 percent dissipation / degradation

DT90 Period required for 90 percent dissipation / degradation

E Explosive (Symbols and indications of danger for dangerous substances and preparations according to Annex II of Directive 67/548/EEC)

EASE Estimation and Assessment of Substance Exposure Physico-chemical properties [Model]

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EbC50 Effect Concentration measured as 50% reduction in biomass growth in algae tests

EC European Communities

EC10 Effect Concentration measured as 10% effect

EC50 median Effect Concentration

ECB European Chemicals Bureau

ECETOC European Centre for Ecotoxicology and Toxicology of Chemicals

ECVAM European Centre for the Validation of Alternative Methods

EDC Endocrine Disrupting Chemical

EEC European Economic Communities

EINECS European Inventory of Existin g Commercial Chemical Substances

ELINCS European List of New Chemical Substances

EN European Norm

EPA Environmental Protection Agency (USA)

ErC50 Effect Concentration measured as 50 % reduction in growth rate in algae tests

ESD Emission Scenario Document

EU European Union

EUSES European Union System for the Evaluation of Substances [software tool in support of the Technical Guidance Document on risk assessment]
F(+) (Highly) flammable (Symbols and indications of danger for dangerous substances and preparations according to Annex II of Directive 67/548/EEC)

FAO Food and Agriculture Organisation of the United Nations

FELS Fish Early Life Stage

foc Organic carbon factor (compartment depending)

GLP Good Laboratory Practice

HEDSET EC/OECD Harmonised Electronic Data Set (for data collection of existing substances)

HELCOM Helsinki Commission -Baltic Marine Environment Protection Commission

HPLC High Pressure Liquid Chromatography

HPVC High Production Volume Chemical (> 1000 tonnes/annum)

IARC International Agency for Research on Cancer

IC Industrial Category

IC50 median Immobilisation Concentration or median Inhibitory Concentration

ILO International Labour Organisation

IPCS International Programme on Chemical Safety

ISO International Organisation for Standardisation

IUCLID International Uniform Chemical In formation Database (existing substances)

IUPAC International Union for Pure and Applied Chemistry

JEFCA Joint FAO/WHO Expert Committee on Food Additives

JMPR Joint FAO/WHO Meeting on Pesticide Residues

78ABBREVIATIONS

Koc organic carbon normalised distribution coefficient

Kow octanol/water partition coefficient
Kp solids-water partition coefficient
L(E)C50 median Lethal (Effect) Concentration

LAEL Lowest Adverse Effect Level

LC50 median Lethal Concentration

LD50 median Lethal Dose

LEV Local Exhaust Ventilation

LLNA Local Lymph Node Assay

LOAEL Lowest Observed Adverse Effect Level

LOEC Lowest Observed Effect Concentration

LOED Lowest Observed Effect Dose

LOEL Lowest Observed Effect Level

MAC Maximum Allowable Concentration

MATC Maximum Acceptable Toxic Concentration

MC Main Category

MITI Ministry of International Trade and Industry, Japan

MOE Margin of Exposure

MOS Margin of Safety

MW Molecular Weight

N Dangerous for the environment (Symbols and indications of danger for dangerous substances and preparations according to Annex II of Directive 67/548/EEC

NAEL No Adverse Effect Level

NOAEL No Observed Adverse Effect Level

NOEL No Observed Effect Level

NOEC No Observed Effect Concentration

NTP National Toxicology Program (USA)

O Oxidising (Symbols and indications of danger for dangerous substances and preparations according to Annex II of Directive 67/548/EEC)

OC Organic Carbon content

OECD Organisation for Economic Cooperation and Development

OEL Occupational Exposure Limit

OJ Official Journal

OSPAR Oslo and Paris Convention for the protection of the marine environment of the Northeast Atlantic

P Persistent

PBT Persistent, Bioaccumulative and Toxic

PBPK Physiologically Based PharmacoKinetic modelling

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PBTK Physiologically Based ToxicoKinetic modelling

PEC Predicted Environmental Concentration
pH logarithm (to the base 10) (of the hydrogen ion concentration {H + }
pKa logarithm (to the base 10) of the acid dissociation constant
pKb logarithm (to the base 10) of the base dissociation constant

PNEC Predicted No Effect Concentration

POP Persistent Organic Pollutant

PPE Personal Protective Equipment
QSAR (Quantitative) Structure-Activity Relationship
R phrases Risk phrases according to Annex III of Directive 67/548/EEC

RAR Risk Assessment Report

RC Risk Characterisation

RfC Reference Concentration

RfD Reference Dose

RNA RiboNucleic Acid

RPE Respiratory Protective Equipment

RWC Reasonable Worst-Case
S phrases Safety phrases according to Annex IV of Directive 67/548/EEC

SAR Structure-Activity Relationships

SBR Standardised birth ratio

SCE Sister Chromatic Exchange

SCHER Scientific Committee on Health and Envionment Risks

SDS Safety Data Sheet

SETAC Society of Environmen tal Toxicology And Chemistry

SNIF Summary Notification Interc hange Format (new substances)

SSD Species Sensitivity Distribution

STP Sewage Treatment Plant
T(+) (Very) Toxic (Symbols and indications of danger for dangerous substances and preparations according to Annex II of Directive 67/548/EEC)

TDI Tolerable Daily Intake

TG Test Guideline

TGD Technical Guidance Document

TNsG Technical Notes for Guidance (for Biocides)

TNO The Netherlands Organisation for Applied Scientific Research

ThOD Theoritical Oxygen Demand

UC Use Category

UDS Unscheduled DNA Synthesis

UN United Nations

80ABBREVIATIONS

UNEP United Nations Environment Programme

US EPA Environmental Protection Agency, USA

UV Ultraviolet Region of Spectrum

UVCB Unknown or Variable composition, Complex reaction products of Biological material

vB very Bioaccumulative

VOC Volatile Organic Compound

vP very Persistent

vPvB very Persistent and very Bioaccumulative

v/v volume per volume ratio

w/w weight per weight ratio

WHO World Health Organisation

WWTP Waste Water Treatment Plant

Xn Harmful (Symbols and indications of danger for dangerous substances and preparations according to Annex II of Directive 67/548/EEC)

Xi Irritant (Symbols and indications of danger for dangerous substances and preparations according to Annex II of Directive 67/548/EEC)

81European Commission DG Joint Research Centre, Institute of Health and Consumer Protection European Chemicals Bureau EUR 22501 EN European Union Risk Assessment Report 2-butoxyethanol (EGBE) – Part I – Environment, Volume 68

Editors: S.J. Munn, K. Aschberger, O. Cosgrove, S. Pakalin, A. Paya-Perez, B. Schwarz-Schulz, S. Vegro

Luxembourg: Office for Official Publications of the European Communities

2006 – VIII pp., 82 pp. – 17.0 x 24.0 cm

EUR – Scientific and Technical Research series; ISSN 1018-5593

The report provides the comprehensive risk assessment of the substance 2-butoxyethanol (EGBE). It has been prepared by France in the frame of Council Regulation (EEC) No. 793/93 on the evaluation and control of the risks of existing substances, following the principles for assessment of the risks to humans and the environment, laid down in Commission Regulation (EC) No. 1488/94.

Part I – Environment

The evaluation considers the emissions and the resulting exposure to the environment in all life cycle steps. Following the exposure assessment, the environmental risk characterisation for each protection goal in the aquatic, terrestrial and atmospheric compartment has been determined.

The environmental risk assessment for 2-butoxyethanol (EGBE) concludes that there is at present no concern for the atmosphere, the aquatic ecosystem, the terrestrial ecosystem or for microorganisms in the sewage treatment plant. There is at present no need for further information and/or testing and no need for risk reduction measures beyond those which are being applied already.

Part II – Human Health

This part of the evaluation considers the em issions and the resulting exposure to human populations in all life cycle steps. The scenarios for occupational exposure, consumer exposure and humans exposed via the environment have been examined and the possible risks have been identified.

This part of the evaluation will be added later.The mission of the JRC is to provide custom er-driven scientific and technical support for the conception, development, implementati on and monitoring of EU policies. As a service of the European Commission, the JRC functions as a reference centre of science and technology for the Union. Close to the policy-making process, it serves the common interest of the Member States, wh ile being independent of special interests, private or national.

European Commission – Joint Research Centre Institute for Health and Consumer Protection European Chemicals Bureau (ECB)

European Union Risk Assessment Report

2-butoxyethanol (EGBE) Part I – environment

CAS No: 111-76-2 EINECS No: 203-905-0

Series: 4 th Priority List Volume: 68EU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 TITLE

RAPPORTEUR FRANCE R408_0808_ HH _C LEAN 

## European Union Risk Assessment Report

2-BUTOXYETHANOL

Part II – Human Health

CAS No: 111-76-2

EINECS No: 203-905-0



### RISK ASSESSMENT

FINAL APPROVED VERSIONEU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 TITLE

RAPPORTEUR FRANCE R408_0808_ HH _C LEAN LEGAL NOTICE

Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might be made of the following information

A great deal of additional in formation on the European Union is available on the Internet. It can be accessed through the Europa Server (http://europa.eu.int).

Cataloguing data can be found at the end of this publication

Luxembourg: Office for O fficial Publications of the European Communities

, [ECB: year] ISBN [ECB: insert number here]

© European Communities, [E CB: insert year here] Reproduction is authorised provided the source is acknowledged.

Printed in ItalyEU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 TITLE

RAPPORTEUR FRANCE R408_0808_ HH _C LEAN 

### 2-BUTOXYETHANOL

Part II – Human Health

CAS No: 111-76-2

EINECS No: 203-905-0



### RISK ASSESSMENT

FINAL APPROVED VERSION

August 2008

France

Rapporteur for the human health risk asse ssment of 2-BUTOXYETHANOL is BERPC.

Contact point:

BERPC

60-62 rue d’hauteville

75009 ParisEU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 TITLE

RAPPORTEUR FRANCE R408_0808_ HH _C LEAN Date of Last Literature Search : 2003 Review of report by MS Technical Experts finalised: 07-2007 Final report: 2008EU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 FOREWORD

RAPPORTEUR FRANCE R408_0808_ HH _C LEAN V Foreword
We are pleased to present this Risk Assessmen t Report which is the result of in-depth work carried out by experts in one Me mber State, working in co-opera tion with their counterparts in the other Member States, the Commission Se rvices, Industry and pub lic interest groups. The Risk Assessment was carried out in accordance with Council Regulation (EEC) 793/93 1 on the evaluation and control of the risks of “ex isting” substances. “Existing” substances are chemical substances in use within the European Community before September 1981 and listed in the European Inventory of Existing Commercial Chemical Substances. Regulation 793/93 provides a systematic framework for the evaluation of the risks to human health and the environment of these substances if they are produced or imported into the Community in volumes above 10 tonnes per year. There are four overall stages in the Regulation for reducing the risks: data collection, priority setting, risk assessment and risk reduction. Da ta provided by Industry are used by Member States and the Commission services to determine the priority of the substances which need to be assessed. For each substance on a priority list, a Member State volunteers to act as “Rapporteur”, undertaking the in-depth Risk Assessment a nd recommending a strategy to limit the risks of exposure to the substance, if necessary. The methods for carrying out an in-depth Risk Assessment at Community level are laid down in Commission Regulation (EC) 1488/94 2 , which is supported by a technical guidance document 3 . Normally, the “Rapporteur” and i ndividual companies producing, importing and/or using the chemicals work closely together to develop a draft Risk Assessment Report, which is then presented at a meeting of Memb er State technical experts for endorsement. The Risk Assessment Report is then peer-revie wed by the Scientific Committee on Health and Environmental Risks (SCHER) which gives its opinion to the Europ ean Commission on the quality of the risk assessment. If a Risk Assessment Report concludes that meas ures to reduce the risks of exposure to the substances are needed, beyond any measures which may already be in place, the next step in the process is for the “Rapporteur” to develop a proposal for a strategy to limit those risks. The Risk Assessment Report is also presented to the Organisation for Economic Co-operation and Development as a contribution to the Chapter 19, Agenda 21 goals for evaluating chemicals, agreed at the United Nations Conf erence on Environment and Development, held in Rio de Janeiro in 1992 and confirmed in the Johannesburg Declaration on Sustainable Development at the World Summ it on Sustainable Development, held in Johannesburg, South Africa in 2002. This Risk Assessment improve s our knowledge about the risk s to human health and the environment from exposure to chemicals. We h ope you will agree that the results of this indepth study and intensive co-operation will make a worthwhile contribution to the Community objective of reducing the overall risks from exposure to chemicals.

1 O.J. No L 084, 05/04/199 p.0001 – 0075 2 O.J. No L 161, 29/06/1994 p. 0003 – 0011 3 Technical Guidance Document, Part I – V, ISBN 92-827-801 [1234]EU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 FOREWORD

RAPPORTEUR FRANCE R408_0808_ HH _C LEAN VI Contact Details of the Rapporteur(s)
Rapporteur: France

Human Health: BERPC 60/62 rue d’Hauteville 75010 Paris France

Tel: + 33 1 55 07 89 89 Fax: + 33 1 47 70 63 13

The scientific work on the human health sections has been elaborated by:

Effects assessment, Institut National de Recherch e et de Sécurité (INRS) Exposure and Risk Expertise and Technica l Advice Division - RCB Characterisation for 30 rue Olivier Noyer Workers 75680 Paris Cedex 14 France And BERPC 60/62 rue d’Hauteville 75010 Paris France

Exposure and Risk Institut National de l'Environnement Industriel et Characterisation for des Risques (INERIS) Man via the environment Département TEC Parc Technologique ALATA BP n° 2 60550 Verneuil-en-Halatte France And BERPC 60/62 rue d’Hauteville 75010 Paris France

Exposure and Risk Centre Anti-Poison de Lille Characterisation for 5, avenue Oscar Lambret Consumers 59037 Lille cedex FranceEU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 CONTENTS

RAPPORTEUR FRANCE R408_0808_ HH _C LEAN 1 

### 0 OVERALL RESULTS OF THE RISK ASSESSMENT 4

CAS Number: 111-76-2 EINECS Number: 203-905-0 IUPAC Name: 2-butoxyethanol

Human health

Human health (toxicity)

Workers

Conclusion ( ii) There is at present no need for furt her information and/or testing and no need for risk reduction measures beyond those which are being applied already.

Consumers

Conclusion ( ii) There is at present no need for furt her information and/or testing and no need for risk reduction measures beyond those which are being applied already.

Conclusion (ii) applies to all scenar ios and all toxicological end-points

Humans exposed via the environment

Conclusion ( ii) There is at present no need for furt her information and/or testing and no need for risk reduction measures beyond those which are being applied already.

Human health (physico-chemical properties)

Conclusion ( ii) There is at present no need for furt her information and/or testing and no need for risk reduction measures beyond those which are being applied already.

4 Conclusion (i) There is a need for further information and/or testing. Conclusion (ii) There is at present no n eed for further information and/or testing and no need for risk reduction measures beyo nd those which are being applied already. Conclusion (iii) There is a need for limiting the risks; risk reduction measures which are already being applied shall be taken into
account.EU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 CONTENTS

RAPPORTEUR FRANCE R408_0808_ HH _C LEAN 2 

### CONTENTS

1 GENERAL SUBSTANCE INFORMATION ............................................................................................. 7

1.1 IDENTIFICATION OF THE SUBSTANCE ...................................................................................... 7

1.2 PURITY/IMPURITIES, ADDITIVES ............................................................................................... 7

1.3 PHYSICO-CHEMICAL PROPERTIES ............................................................................................. 7 1.3.1 Physical state ........................................................................................................................... 8 1.3.2 Melting point ........................................................................................................................... 8 1.3.3 Boiling point ............................................................................................................................ 9 1.3.4 Relative density ....................................................................................................................... 9 1.3.5 Vapour pressure ....................................................................................................................... 9 1.3.6 Surface tension ........................................................................................................................ 9 1.3.7 Water solubility ....................................................................................................................... 9 1.3.8 Partition coefficient n-octanol/water ........................................................................................ 10 1.3.9 Granulometry ........................................................................................................................... 10 1.3.10 Flash point

............................................................................................................................... 10 1.3.11 Autoflammability ..................................................................................................................... 10 1.3.12 Flammability ............................................................................................................................ 10 1.3.13 Explosive properties ................................................................................................................ 10 1.3.14 Oxidising properties ................................................................................................................. 10 1.3.15 Viscosity

............................................................................................................................... ... 11 1.3.16 Henry’s constant ...................................................................................................................... 11

1.4 CLASSIFICATION ............................................................................................................................ 12 1.4.1 Current classification (28 th TPA of directive 67/548/EEC) ..................................................... 12 1.4.2 Proposed classification ............................................................................................................ 12

2 GENERAL INFORMATION ON EXPOSURE ......................................................................................... 13

3 ENVIRONMENT

............................................................................................................................... ......... 14

4 HUMAN HEALTH

............................................................................................................................... ...... 15

4.1 HUMAN HEALTH (TOXICITY) ...................................................................................................... 15 4.1.1 Exposure assessment ............................................................................................................... 15 4.1.1.1 General discussion ..................................................................................................... 15 4.1.1.2 Occupational exposure ............................................................................................... 16 4.1.1.2.1 Scenario 1 : Manufacture and use as intermediate .................................... 20 4.1.1.2.2 Scenario 2 : Formulation of products containing EGBE ............. 24 4.1.1.2.3 Scenario 3 : Use of products containing EGBE ............................. 32 4.1.1.2.4 Summary of occupational exposure .......................................................... 54 4.1.1.3 Consumer exposure ................................................................................................... 55 4.1.1.3.1 Exposure from uses ................................................................................... 55 4.1.1.3.2 Summary of consumer exposure ............................................................... 63 4.1.1.4 Humans exposed via the environment ....................................................................... 64 4.1.2 Effects assessment: Hazard identification and do se (concentration)- response (effect) assessment 67 4.1.2.1 Toxicokinetics, metabolism and distribution ............................................................. 67 4.1.2.1.1 Studies in animals ..................................................................................... 67 4.1.2.1.2 Studies in humans ..................................................................................... 87 4.1.2.1.3 Other data ................................................................................................. 94 4.1.2.1.4 Summary of toxicokinetics, metabolism and distribution ......................... 101 4.1.2.2 Acute toxicity ............................................................................................................ 103 4.1.2.2.1 Studies in animals ..................................................................................... 103EU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 CONTENTS

RAPPORTEUR FRANCE R408_0808_ HH _C LEAN 3 4.1.2.2.2 Data from human exposure ....................................................................... 120 4.1.2.2.3 Specific toxicity: haematotoxicity ............................................................ 125 4.1.2.3 Irritation 153 4.1.2.3.1 Skin ........................................................................................................... 153 4.1.2.3.2 Eye ............................................................................................................ 155 4.1.2.3.3 Respiratory tract ........................................................................................ 160 4.1.2.4 Corrosivity ................................................................................................................. 161 4.1.2.5 Sensitisation ............................................................................................................... 161 4.1.2.5.1 Studies in animals ..................................................................................... 161 4.1.2.5.2 Studies in humans ..................................................................................... 162 4.1.2.5.3 Summary of sensitisation .......................................................................... 162 4.1.2.6 Repeated dose toxicity ............................................................................................... 163 4.1.2.6.1 Studies in animals ..................................................................................... 163 4.1.2.6.2 Studies in humans ..................................................................................... 189 4.1.2.6.3 Mechanistic studies of liver pathology ..................................................... 190 4.1.2.6.4 Mechanistic studies on forestomach pathology ........................................ 195 4.1.2.6.5 Summary of repeated dose toxicity ........................................................... 199 4.1.2.7 Mutagenicity .............................................................................................................. 200 4.1.2.7.1 Studies in vitro .......................................................................................... 200 4.1.2.7.2 Studies in vivo (table 4.81) ....................................................................... 208 4.1.2.7.3 Summary of mutagenicity ......................................................................... 210 4.1.2.8 Carcinogenicity .......................................................................................................... 212 4.1.2.8.1 Studies in animals ..................................................................................... 212 4.1.2.8.2 Studies in humans ..................................................................................... 217

PROPOSED MECHANISMS OF ACTION ASSESSED WITHIN THE IPCS FRAMEWORK .................... 229 4.1.2.9 Toxicity for reproduction ........................................................................................... 235 4.1.2.9.1 Effects on fertility ..................................................................................... 235 4.1.2.9.2 Developmental toxicity ............................................................................. 240 4.1.2.9.3 Summary of toxicity for reproduction ...................................................... 255 4.1.3 Risk characterisation ............................................................................................................... 256 4.1.3.1 General aspects .......................................................................................................... 256 4.1.3.2 Workers 259 4.1.3.2.1 Acute toxicity ............................................................................................ 260 4.1.3.2.2 Irritation and corrosivity ........................................................................... 263 4.1.3.2.3 Sensitisation .............................................................................................. 265 4.1.3.2.4 Repeated dose toxicity .............................................................................. 265 4.1.3.2.5 Mutagenicity ............................................................................................. 270 4.1.3.2.6 Carcinogenicity ......................................................................................... 270 4.1.3.2.7 Toxicity for reproduction .......................................................................... 270 4.1.3.2.8 Summary of risk characterisation for workers .......................................... 273 4.1.3.3 Consumers ................................................................................................................. 273 4.1.3.3.1 Acute toxicity ............................................................................................ 273 4.1.3.3.2 Irritation and corrosivity ........................................................................... 274 4.1.3.3.3 Sensitisation .............................................................................................. 275 4.1.3.3.4 Repeated dose toxicity .............................................................................. 275 4.1.3.3.5 Mutagenicity ............................................................................................. 278 4.1.3.3.6 Carcinogenicity ......................................................................................... 278 4.1.3.3.7 Toxicity for reproduction .......................................................................... 279 4.1.3.4 Humans exposed via the environment ....................................................................... 280 4.1.3.4.1 Summary of risk characterisation for exposure via the environment ........ 280

4.2 HUMAN HEALTH (PHYSICO-CHEMICAL PROPERTIES) ......................................................... 280

5 RESULTS

............................................................................................................................... .................... 281

5.1 INTRODUCTION .............................................................................................................................. 2 81

5.2 ENVIRONMENT

............................................................................................................................... 281EU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 CONTENTS

RAPPORTEUR FRANCE R408_0808_ HH _C LEAN 4 5.3 HUMAN HEALTH ............................................................................................................................ 281 5.3.1 Human health (toxicity) ........................................................................................................... 281 5.3.1.1 Workers 281 5.3.1.2 Consumers ................................................................................................................. 281 5.3.1.3 Humans exposed via the environment ....................................................................... 281 5.3.2 Human health (risks from p hysico-chemical properties) ......................................................... 282

6 REFERENCES

............................................................................................................................... ............. 283

ABBREVIATIONS

............................................................................................................................... ........... 301

Appendix A

............................................................................................................................... ........................ 308

Apppendix B

............................................................................................................................... ...................... 313

EUSES Calculations can be viewed as part of the repo rt at the website of the European Chemicals Bureau: http://ecb.jrc.it



### TABLES

Table 1.1.1: Summary of ph ysico-chemical properties............................................................................ ......... 8 Table 1.1.2: Henry’s law constant values (at 25°C) in EPI suite (US EPA and S RC, 2001)........................... 11 Table 1.1.3: Henry’s law constant values as measured in Kim et al. , 2000...................................................... 11 Table 4.1: Concentration of EGBE in the main use categories in the Danish pr oduct register (2001) ............. 16 Table 4.2: Concentration of EGBE in the main use categories in the Fr ench product register SEPIA (INRS, 2003) .......................................................................................................................... ....................................... 16 Table 4.3: OEL values (A riel Resear ch, 2003) ................................................................................... .............. 19 Table 4.4: Biological ex posure levels (BEL) .................................................................................... ................ 20 Table 4.5: Measured inhalation exposure for EGBE manufacture (SIDS, 1996, slightly modified with NICNAS, 1996) .......................................................................................................................... ....................................... 21 Table 4.6: Personal air measurements in Producer 1 enterprises (1995-2000) ................................................. 22 Table 4.7: Personal air measurements in Producer 2 plant (1995-2000)........................................................... 22 Table 4.8: Personal air measuremen ts in Producer 3 plant (2002)................................................................ .... 23 Table 4.9: Distribution of potential exposure duration (NICNAS, 1996) ......................................................... 2 5 Table 4.10: Exposure frequency and duration in the EU paints and inks manufacturing industry (CEPE, 2002) 26 Table 4.11: Atmospheric and biological monitoring during formulation of products (8 hr-TWA personal inhalation exposure and BA A urinary concentration)............................................................................. .......... 26 Table 4.12: Personal inhalation exposure in the MEGA databa se, 1996-2000 (BGA A, 2001) ........................ 27 Table 4.13: Results of measurements of potential hand exposure to DEGBE in loading mixers and filling containers with products containing DEGB E (after RISKOFDERM, 2002a and Gijsbers et al., 2004) .......... 28 Table 4.14: Contents of EGBE in paints (CEPE, 2002)............................................................................ ........ 33 Table 4.15: Relative frequencies of application tech niques in painting/surface coating (CEPE, 2002) ........... 33 Table 4.16: Atmospheric and biological monitoring during painting (8 hr-TWA inhalation exposure and BAA urinary concentration)......................................................................................................... .............................. 34 Table 4.17: Personal exposure in painting activities for measurements 60-480 minutes, years 1987-1998 (Vincent, 1999) ................................................................................................................ ................................. 36 Table 4.18: Personal exposure measurements in the MEGA database, 1996-2000 (BGAA , 2001) ................. 36 Table 4.19: Dermal exposure rates during brushing ( Gijsbers et al , 2004)...................................................... 38 Table 4.20: Atmospheric and biological monitoring during printing (8 hr-TWA inhalation exposure and BAA urinary concentration)......................................................................................................... .............................. 40 Table 4.21: Personal air measurements du ring screen printi ng (BP, 2002a)..................................................... 42 Table 4.22: Personal exposure in printing activities made for measurements 60-480 minutes, years 1987-1998 (Vincent, 1999). ............................................................................................................... ................................. 43 Table 4.23a: Potential dermal exposure measur ements during silkscreening (KRIOH, 2003) ......................... 44 Table 4.23b : Workers’internal exposures (Urany butoxyacetic acid (BAA) excretion, μmol/mol creatinine) 45 Table 4.24: Main uses of EGBE containing cleaners in Australia (NICNAS, 1996)........................................ 47 Table 4.25: Measured atmospheric ex posure (TWA) in cl eaning activ ities. .................................................... 48 Table 4.26: Exposure estimates for use of cleaning products (SIDS, 1996) ..................................................... 49 Table 4.27: Dermal exposure rates during sp raying and wiping (RISKOFDERM, 2003b).............................. 50EU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 CONTENTS

RAPPORTEUR FRANCE R408_0808_ HH _C LEAN 5 Table 4.28: Characterisation of ex posure by job category (Vincent et al. , 1993)............................................. 52 Table 4.29: Summary of proposed re asonable worst case exposures................................................................ 54 Table 4.30: summary of studies about expo sure by household cleaning pr oducts............................................ 57 Table 4.31: summary of studies about exposure by indoor air.................................................................... ...... 59 Table 4.32: Summary of studie s about exposure by paints........................................................................ ....... 61 Table 4.33: Summary of proposed “reasonable worst-case” exposu res in the main scenarios ......................... 62 Table 4.34: Summary of comb ined worst case scenarios.......................................................................... ....... 63 Table 4.1: exposure assessment for human via the environment (concentrations in foodstuff, air and drinking water and connected daily doses)............................................................................................... ....................... 64 Table 4.35: Average tissues concentrations of EGBE and BAA following 20 ppm exposure.......................... 66 Table 4.36: Excretion of [ 14 C]EGBE ................................................................................................................ 67 Table 4.37: Urinary metabolites of EGBE ........................................................................................ ................ 68 Table 4.38: Comparison of EGBE elimination between rats and mice at the same dose level tested............... 69 Table 4.39: Comparison of BAA urinary excretion between rats and mice...................................................... 70 Table 4.40: Effect of time and dose on th e excretion of BE me tabolites in urine............................................. 77 Table 4.41: Quantities of EGBE analysed in the di fferent system compartmen ts for the 2 tested concentrations (5 and 10 %)................................................................................................................... ................................... 88 Table 4.42: Kinetic parameters m easured in the Jakasa study .................................................................... ...... 92 Table 4.43: Intra and inter-individual variation of dermal fluxes after dermal exposure to 50 % EGBE in water.92 Figure 4.44: First PBPK developed for EGBE..................................................................................... ............. 95 Figure 4.45: Improvement of the PbPk model (from Dow, 1993) .................................................................... 97 Figure 4.46: Last PbPk model (Lee et al. , 1998) .............................................................................................. 99 Figure 4.47: Metabolism of EGBE (P atty, 2001).................................................................................. ............ 101 Table 4.48: Summary inhalation route........................................................................................... ................... 104 Table 4.49: Summary acute t oxicity via de rmal route. ........................................................................... .......... 110 Table 4.50: Summary of the LD50 reported in Carpenter et al ., 1956.............................................................. 113 Table 4.51: Summary of LD50 values after oral rout e administration............................................................. . 115 Table 4.52: Summary other routes ............................................................................................... ..................... 118 Table 4.53: Summary human acute toxi city data.................................................................................. ............ 121 Table 4.54: Summary of the erythrocyte toxicity in Carpenter et al. , 1956...................................................... 124 Table 4.55: Haemolytic activity of various concentrations of butoxyacetic acid incubated with erythrocytes from rat, human, do g and rabbit ..................................................................................................... ........................... 125 Table 4.56: Haemolytic activity of various concentrations of EGBE incubated with erythrocytes from rat, human, dog and rabbit. ................................................................................................................ .................................. 126 Table 4.57: Haemolysis of human and rat erythroc ytes by EGBE................................................................... . 126 Table 4.58: Haemolysis of human and rat erythroc ytes by BAA.................................................................... .. 127 Table 4.59: Effect s on rat blood............................................................................................... ......................... 131 Table 4.60: Effects on rabbit blood............................................................................................ ....................... 132 Table 4.61: Incidence of haemoglobinuria induced in rats of various ages by gavage administration of EGBE. 134 Table 4.62: In vivo and in vitro results.............................................................................................................. 140 Table 4.63: Effects of BAA on blood from diffe rent species..................................................................... ....... 144 Table 4.64: Effect of external su crose on BAA-indu ced cell sw elling ............................................................ . 146 Table 4.65: Effect of replacement of external sodium with potassium on haemolysis and MCV changes induced by 2.0 mM BAA. ................................................................................................................. ............................. 147 Table 4.66: Effects of divalent cathions and Ca chelation on BAA-induced h aemolysis ................................. 148 Table 4.67: Dose response rela tionship for BAA induced haemol ysis and cell swelling ................................. 148 Table 4.68: Effect of Ca on haemolysis ......................................................................................... ................... 150 Table 4.69: Irritation scores in th e Parent, 1992 Jacobs, 1992 study ........................................................... ..... 155 Table 4.70: Draize scores in the Kenn ah study.................................................................................. ............... 156 Table 4.70 bis: Individual score fo r Safepharm laborator ies study, 1994b)...................................................... 157 Table 4.70 ter: Individual sc ore for BASF study, 2000 .......................................................................... .......... 158 Table 4.71: Experimental pattern of exposure for Gage study, 1970.............................................................. .. 163 Table 4.71 bis: Incidence of non-neoplastic lesions in the urogenital system of male mice following 2-years exposure to EGBE............................................................................................................... .............................. 172 Table 4.72 : Summary of the studies on an imals performed by in halation route.............................................. 175 Table 4.73: Summary of the studies performed by dermal route ................................................................... ... 180 Table 4.74: Summary of studie s performed by oral route......................................................................... ........ 186 Table 4.75: Effects of EGBE on indicators of oxidative stress in B6C3F1 mice, mean & standard deviation & percentage of the control (100 %) (Kamendulis et al ., 1999) ........................................................................... 189EU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 CONTENTS

RAPPORTEUR FRANCE R408_0808_ HH _C LEAN 6 Table 4.76: Effects of EGBE, BAA and ferrous sulphate on indicators of oxidative stress in male B6C3F1 mouse and male F344 rat hepatocytes, mean & standard deviation & percentage of the control (100 %) (Kamendulis et al ., 1999; Park et al ., 2002a) ...................................................................................................................... ....... 193 Table 4.77: Effects of haemolysed erythrocytes (RBCs) on oxidative stress parameters in mouse hepatocytes (Park et al ., 2002a)...................................................................................................................... ...................... 194 Table 4.78: Halve lives and AUC in blood, saliva and tissues. (Poet et al. , 2003). ......................................... 197 Table 4.79: Test for gene muta tion induction in bacteria by EGBE and its me tabolites................................... 201 Table 4.80: Tests for genetic and related effects in cu ltured mammalian cells by EGBE and its metabolites.. 204 Table 4.80: (continued) Tests for genetic and related effects in cultured mammalian cells by 2-butoxyethanol and its meta bolites ................................................................................................................ ................................... 205 Table 4.80: (continued) Tests for genetic and related effects in cultured mammalian cells by 2-butoxyethanol and its meta bolites ................................................................................................................ ................................... 206 Table 4.81: In vivo tests in mammals for the genotoxicity of EGBE and its metabolites ................................. 210 Table 4.82: Neoplastic and non-neoplastic lesions of the adrenal medulla in female F344/N rats.................. 212 Table 4.83: Neoplastic and non-neoplastic lesions of the forestomach in male and female B6C3F 1 mice...... 214 Table 4.84: Neoplastic and non-neoplastic lesions of the liver in male and female B6C3F 1 mice .................. 215 Table 4.85: Summary of relationship between chemicals inducing haematoxicity and haemangiosarcomas in B6C3F1 mice and F344 rats Incidences are for control, low, middle and high dose groups, respectively. ..... 221 Table 4.85 bis: Data on estrous cy cling for rats exposed to EGBE fo r 13 weeks in drinking water................. 236 Table 4.85 ter: Vaginal cytology during the estrous cycle of the rat 1 ............................................................... 236 Table 4.86: effects of EGBE on the development of rat embryos explanted on day of gestation and cultured for 48h. ........................................................................................................................... ........................................ 246 Table 4.87: Effects of BAA on the development of rat embryos explanted on day 9 of gestation and cultured for 48 hr. ......................................................................................................................... ........................................ 247 Table 4.88: absorption coefficients taken into account for the calcula tions of intern al doses .......................... 254 Table 4.89: Summary of proposed reasonable worst case exposures (see also table 4.29 in & 4.1.1.2.4)........ 257 Table 4.90: Assessment factors applied for the calculation of minimal MOS for acute toxicity (for inhalation and dermal route).................................................................................................................. ................................... 258 Table 4.91: Occupational risk assessm ent of EGBE for acute toxicity............................................................ . 259 Table 4.91 bis: Occupational combined risk assessment for acute toxi city ...................................................... 2 61 Table 4.92: Occupational risk assessmen t of EGBE for eye and respiratory i rritation by vapour exposure..... 262 Table 4.92a: Equivalent human dose s for LOAEL(C) / NOAEL(C) ................................................................ 264 4.92b: Assessment factors fo r repeated do se toxicity ........................................................................... ............ 264 Table 4.92c: Risk assessment for inhalatio n and dermal exposure for RDT..................................................... 265 Table 4.92d: Risk characterisation fo r combined exposure and for RDT ......................................................... 26 7 Table 4.93: Assessment factors applied for the cal culation of minimal MOS fertility eff ects.......................... 268 Table 4.93bis: Risk charact erisation for reprotox icity ......................................................................... ............. 270 Table 4.94: Internal dose expo sure depending on scenarios ...................................................................... ....... 271 Table 4.95: Assessment factors applied for the calculation of minimal MOS for acute toxicity and for inhalation and dermal route. .............................................................................................................. ................................ 272 Table 4.96: MOSs and conclu sion for acut e toxicity ............................................................................. ........... 272 Table 4.97: Consumer risk assessment of EGBE for eye and respiratory irritation by vapour exposure.......... 273 Table 4.97 bis: Equivalent human doses for NOAEL(C) / LOAEL(C) ............................................................ 274 Table 4.97 ter: Assessment factors for oral exposure........................................................................... ............. 274 Table 4.98: Internal dose exposure depend ing on scenarios aver age over a year ............................................. 275 Table 4.99: MOSs and conclusion for repeated dose to xicity..................................................................... ...... 276 Table 4.99 bis: Risk charact erisation for car cinogenicity ...................................................................... ........... 277 Table 4.100: Assessment factors applied for the calculation of mi nimal MOS fertility effects........................ 277 Table 4.101: MOSs and conc lusion for re production .............................................................................. ......... 277EU RISK ASSESSMENT

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### 1 GENERAL SUBSTANCE INFORMATION

1.1 IDENTIFICATION OF THE SUBSTANCE

CAS Number: 111-76-2 EINECS Number: 203-905-0 IUPAC Name: 2-butoxyethanol Molecular formula: C 6 H 14 O 2 Structural formula: CH 3 -CH 2 -CH 2 -CH 2 -O-CH 2 -CH 2 -OH

Molecular weight: 118.17 g.mol -1 Synonyms: EGBE (this synonym will be used in the present study to refer to the chemical 2-butoxytehanol). Othe r synonyms: butoxyethanol ; 2-butoxy1-ethanol ; n-butoxyethanol ; butyl et hoxol ; 3-oxa-1-heptanol ; o-butyl ethylene glycol ; butyl glycol ; butyl monoether glycol ; ethylene glycol butyl ether ; 2-BE ; et hylene glycol n-butyl et her ; ethylene glycol monobutyl ether ; gl ycol butyl ether Commercial trade names : Dowanol EB ; Butyl Cellosolve ; Butyl Icinol ; Butyl Oxitol ; Eastman EB Solvent

1.2 PURITY/IMPURITIES, ADDITIVES
Purity : the purities quoted in IUCLID were all ≥ 99 % w/w
Impurities :

- as the synthesis reaction produces other gl ycol ethers, small concentrations of them can be found.

- 2-butoxyethoxyethanol (CAS 112-34-5) ≤ 0.3 % w/w

- 1,2-ethanediol (CAS 107-21-1) ≤ 0.3-0.5 % w/w

- 1-butanol (CAS 71-36-3) ≤ 0.1-0.2 % w/w

- water < 0.1-0.2 % w/w Additives : 0.008-0.012 % w/w 2,6-bis(1,1- dimethylethyl)-4-methylphenol (CAS 128-37-0) added to prevent th e formation of peroxides.

1.3 PHYSICO-CHEMICAL PROPERTIES
The physico-chemical properties are di scussed below and summarised in Table 1.1

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RAPPORTEUR F RANCE R408_0808_ HH _C LEAN 8 Table 1.1.1: Summary of physico-chemical properties

Property Value
Physical state Liquid Melting point -73.4°C Boiling point 170.5°C Relative density 0.9 at 20°C
Vapour pressure 1 hPa at 20°C

Surface tension 26.6 mN/m at 20°C Water solubility Miscible (~1.10 6 mg/L)

Partition coefficient n-octanol/water (log value) 0.8

Granulometry Not applicable Flash point 63.2°C Autoflammability 244.5°C Flammability Upper limit: 12.7 % (volume) Lower limit: 1.1 % (volume)
Explosive properties Not explosive Oxidising properties No oxidising properties Viscosity 3.28 mPa.s at 20°C Henry’s constant 0.08 Pa.m 3 /mol at 25°C Conversion factors (101 kPa, 20°C) 1 ppm = 4.9 mg/m 3 1 mg/m 3 = 0.204 ppm

1.3.1 Physical state
EGBE is a neutral, colourle ss liquid with a very weak pleasant odour (Ullmann, 2000). An absolute perceptible limit of 0.1 ppm (50 % recognition = 0.35 ppm and 100 % recognition = 0.48 ppm) was referred to EGBE (Verschueren, 2001).

1.3.2 Melting point
Several values are reported in literature. Th e value of -77°C appears in several studies (ECETOC, 1994 ; OECD SIAR, 1997 ; Dow, 2001) and handbooks show values ranging between -74.8 and -70°C (Lewis, 1999 ; Ullmann, 2000 ; Howard, 1989 ; Eastman, 2001 ; BASF, 2002 ; Verschueren, 2001).

The mean of all the values found is retained for this study : -73.4°C.EU RISK ASSESSMENT

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RAPPORTEUR F RANCE R408_0808_ HH _C LEAN 9 1.3.3 Boiling point

Most of the values f ound in literature are in the follo wing range: 168-172°C (Ullmann, 2000). A boiling point of 170.8°C for EGBE can be f ound (ECETOC, 1994) which is close to those appearing in handbooks (170°C in Kirk-Oth mer, 1983 and Verschueren, 2001). Two other handbooks show values between 171 and 172°C (Lewis, 1999 ; Howard, 1989). Boiling point of 171°C was found in technical data sheets or product information reports (BP, 2002b; Dow, 2001).

The mean of all the values above is 170.5°C. This value will be used for the risk evaluation.

1.3.4 Relative density
Several relative density values are available too. Most of them are about 0.9 (Verschueren, 2001 - value measured at 20°C ; Merck, 1996). T echnical data sheets or product information reports give values of 0.9011 and 0.902, at 20°C (BP, 2002b; Dow, 2001).
A rounded value of 0.9 will be chosen for this study.

1.3.5 Vapour pressure
Vapour pressures ranging from 0.79 to 1.3 hPa, at 20°C have been reported. Data come from technical data sheets: 0.8 hPa (Shell Chemicals, 2001), 0.89 hPa (Dow, 2001), 1.3 hPa (BP, 2002b). The same range appears in Sax (1986) an d other chemical handbooks give almost the same values: 0.89 hPa (Verschueren, 2001), 1 hPa (Ullmann, 2000).
The mean of these values gives a vapour pressure of 1 hPa, at 20°C. It will be used in the risk
assessment.

1.3.6 Surface tension
Several values of surface tension appear in technical data sheets for commercial EGBE: Dow Chemical Company (2001) reports a value of 27.4 mN/m at 25°C, Eastman (2001) gives a value of 26.6 mN/m at 20°C and Shell Chemi cals (2001) another of 28.9 mN/m. A value of 27.4 mN/m at 25°C is also reporte d in a handbook (Kirk-Othmer, 1983).
The value reported in Eastman (2001) will be re tained for the study: 26.6 mN/m, at 20°C, the temperature recommended in the OECD guideline No 115.

1.3.7 Water solubility
EGBE is often reported as miscible with water in several data sheets and product information reports (Hoechst, 1992 and 1993 ; BP, 2002b). A sol ubility of about 50000 mg/L is reported by Eckel et al. (1996), 63500 mg/L by ASTER (1996). A value of 1.10 6 mg/L has been measured (Riddick et al. , 1985 cited in HSDB).
EGBE can be considered as highly miscible in water and the maximum value permitted by EUSES (EC, 1996), 1.10 5 mg/L, is retained for modelling purposes.EU RISK ASSESSMENT

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RAPPORTEUR F RANCE R408_0808_ HH _C LEAN 10 1.3.8 Partition coefficient n-octanol/water
Several QSAR calculations of partition coefficient n-octanol/water for EGBE have been performed. They are presented he reafter using an ascending orde r of logarithmic values: 0.57 calculated with KOWWIN v.1.66. – (US EPA and SRC, 2001) ; 0.81 (Verschueren, 2001), 0.84 (ASTER, 1996).
Partition coefficient measurements have been performed (Tanii and Hashimoto, 1982): 0.77 is the mean value of three determinations. Meas urements have been carried out with gas chromatography.
Other measured log K ow can be found in lite rature: 0.74 (Verschuere n, 2001), 0.81 (Staples et al. , 1998), 0.83 (Korenman and Dobromyslova, 1975 ; Hansch and Leo, 1985 ; Staples et al.
, 1998).
Mean of all measured data gives a log K ow of 0.8 that is retained for this study.

1.3.9 Granulometry
Not applicable: the substance is a liquid.

1.3.10 Flash point
Values reported in literatu re range from 60 (INRS, 1996 a nd Merck, 1996) to 68°C (BP, 2002b). Both values are measured with closed cup method. Two other values were obtained using this method: 67°C (Shell Chemicals, 2001) and 61°C (Sax, 1986), while methods leading to values of 65°C (Dow Chem ical, 2001) and 67°C (Ullmann, 2000) are not mentioned.

The mean of the values measured us ing a closed cup is retained: 63.2°C.

1.3.11 Autoflammability
Two temperatures of autoflammability have been found in technical data sheets: 244°C (Dow Chemical Company, 2001) and 245°C (BP, 2002b) . The mean value of 244.5°C is retained.

1.3.12 Flammability
Limits of flammability are reported by BP (2002b) for EGBE. The upper limit is 12.7 % (volume) and the lower one is 1.1 % (volume).

1.3.13 Explosive properties
Not explosive.

1.3.14 Oxidising properties
No oxidising properties.EU RISK ASSESSMENT

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RAPPORTEUR F RANCE R408_0808_ HH _C LEAN 11 1.3.15 Viscosity
The range of viscosity values found in technical data sheets for EGBE goes from 2.9 (at 25°C, Dow, 2001) to 3.3 (Shell Chemicals, 2001) mPa.s. A value of 3.28 mPa.s is also reported by BP (2002b), at 20°C. BP (1998) also reports a range of vi scosity values for several concentrations of EGBE in water (between 20 and 100 % volume): 2-6 mPa.s at 20°C. Several values have been found in handbooks: 3.26 mPa.s, at 20°C (Ullmann, 2000), 2.83 mPa.s at 25°C (Prager, 1995) and a catalogue of commercial substances gives a viscosity of 5.83 mPa.s, at 20°C (Merck, 1996).

The most common values are rounded and a viscos ity of 3.3 mPa.s (at 20°C) is retained for this study.

1.3.16 Henry’s constant
Different QSAR estimation methods calculate d with HENRYWIN v.3.10. (US EPA and SRC, 2001) give values of Henry’s law constant presented in Table 1.1.2.

Table 1.1.2: Henry’s law constant values (at 25°C) in EPI suite (US EPA and SRC, 2001)
Method Value (Pa.m 3 / mole)

Bond method 0.009 9
Group method 0.002 1
Experimental value quoted in EPI suite database 0.162 1 Calculated with VP.M / WSol ratio using EPI suite estimated values (QSAR) 0.116 1

Henry’s law constant values have also been calculated using another program (MPBPVP), at 25°C. Depending on the method use d, values of 0.01 and 0.002 Pa.m 3 /mol are reported (SRC, 1988).
Another study shows a calculated Henry’s law constant of 0.551 Pa.m 3 /mol at 25°C (ASTER, 1996).
Henry’s law constant can also be estimated from the ratio of the vapour pressure to the water solubility using selected values from th is study: 100 Pa for vapour pressure and 1.10 6 mg/L for water solubility. Calculation give s a Henry’s law constant of 0.012 Pa.m 3 /mol.
Experimental measurements of Henry’s law constant have also been reported (Kim et al.
, 2000). Two different methods were used to determine Henry’s law constant for the equilibrium partitioning of EGBE contained in an aqueous solution and the air. Results of measurements are presented in Table 1.1.3.

Table 1.1.3: Henry’s law constant values as measured in Kim et al. , 2000
Bag method for equilibrium partitioning Batch stripping method
T = 20°C ; H = 0.041 Pa.m 3 /mol T = 22°C ; H = 0.063 Pa.m 3 /mol
T = 25°C ; H = 0.081 Pa.m 3 /mol T = 23°C ; H = 0.068 Pa.m 3 /mol
T = 25°C ; H = 0.082 Pa.m 3 /mol T = 30°C ; H = 0.101 Pa.m 3 /mol
T = 30°C ; H = 0.135 Pa.m 3 /molEU RISK ASSESSMENT

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RAPPORTEUR F RANCE R408_0808_ HH _C LEAN 12 In the previous study, there is a good correla tion between both method used, bag method for equilibrium partitioning and batch stripping method, and particularly at 25°C. Moreover, direct measurement of the Henry’s law cons tant is recommended in TGD (EC, 2003) for water miscible compounds. So a Henry’s law constant of 0.08 Pa.m 3 /mol will be used.

1.4 CLASSIFICATION

1.4.1 Current classification (28 th ATP of directive 67/548/EEC)
Classification: Xn; R 20/21/22
Xi; R 36/38

Labelling: Xn; R 20/21/22, 36/38

S 2 – 36/37 - 46

1.4.2 Proposed classification
Unchanged.EU RISK ASSESSMENT

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### 2 GENERAL INFORMATION ON EXPOSUREEU RISK ASSESSMENT

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### 3 ENVIRONMENTEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 15 

### 4 HUMAN HEALTH

4.1 HUMAN HEALTH (TOXICITY)

4.1.1 Exposure assessment

4.1.1.1 General discussion
Humans may be exposed to EGBE at workplace, via consumer products and indirectly via the environment. The highest potential exposure is likely to occur during occupational exposure.
Workers and consumers are primarily exposed via inhalation and dermal routes. EGBE is readily absorbed through the skin including ab sorption from direct contact with liquid or aerosol form or contact with vapours. Becau se this compound has a relatively low vapour pressure (0.1 kPa at 20°C), dermal absorption after direct contact with the liquid may be predominant or may contribute si gnificantly to overall exposure.
Environmental monitoring of brea thing zone or air c oncentrations in the work area has been found to be inadequate to asse ss overall exposures: the total exposure to EGBE should take into account the respiratory uptake of vapours and aerosols a nd the dermal absorption of EGBE in liquid, vapour and aerosol form. Biological monito ring of the common toxic urinary metabolite, 2-butoxyacetic acid (BAA), is consid ered necessary for a complete assessment (Angerer et al. , 1990 ; Johanson et al. , 1988 ; Sohnlein et al. , 1993 ; Vincent et al. , 1993). Limited data on urinary BAA concentrations ar e available for occupationally exposed groups of the population. Biomonitoring data in th e general population are not available.
Exposure may occur during manufacture and use as intermediate in the chemical and pharmaceutical industries, and during formulati on and use of products. EGBE is a solvent used in many industrial ac tivities or consumer applications. The main uses are in paints or surface coatings (solvent-based or water-bas ed), followed by cleaners and printing inks.
In the Swedish product regist er (KEMI, 2002), 882 products c ontaining EGBE have been identified, of which 58 % are paints or inks , 16 % cleaning agents, 8 % antirust and 8 % polishing agents.
In the Danish product register (Arbejdstilsyne t, 2001), 1 204 products containing EGBE have been identified, of which 76 were private household products. The most common uses were paints and varnishes (37 %), cleaning/washin g agents (20 %), surface treatment (8 %), corrosion inhibitors (6 %), surface active agen ts (3 %), adhesives/binding agents (3 %), solvents (2 %).
Other data extracted from the French product register SEPIA (INRS, 2003) showed that 368 products out of the 10 345 products register ed between 1997 and 2003 contained EGBE. The main use categories were: paints, varnishes an d inks (39 %), cleaning agents (37 %) and products for metallurgical and mechanical sectors, e.g. greas e cleaners (14 %).

The distribution of concentration intervals in the main type of products is presented in the tables 4.1 and 4.2.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 16 Table 4.1: Concentration of EGBE in the main use categories in the Danish product register (2001)

Content % Total Cleaning agents Solvents Paints Corrosion inhibitors Surface
treatment Surface active agents Adhesives

[0-1] 526 51 278 23 46 4 21 ]1-5] 276 74 72 36 14 4 ]5-10] 176 56 5 53 8 10 6 ]10-20] 147 40 8 31 27 4 6
]20-50] 51 13 6 4 8 ]50-80] 10 9 6 11
]80-100] 17 4 12 6

Table 4.2: Concentration of EGBE in the main use cat egories in the French product register SEPIA (INRS, 2003)

Concentration (%) Products for metallurgical and mechanical sectors Paints, varnishes and inks Cleaning agents

[0-1] 2 36 11 ]1-5] 36 41 53 ]5-10] 6 41 21 ]10-20] 2 9 13 ]20-50] 4 5 ]50-80] 2 ]80-100]

Other minor uses are reported: adhesives, metal cleaning, elec tronic industry, cutting oils, agricultural products, fire fighti ng foams, leather treatment, oil field chemicals and cosmetics (hair dyes) (SIDS, 1996 and OSPA, 2002).

4.1.1.2 Occupational exposure

Definitions and sources
In this document, unless otherw ise stated, the term exposure is used to denote external personal exposure as measured or otherwise assessed without taki ng into account the attenuating effect of any pers onal protective equipment (PPE) which might have been worn. This definition permits the effects of controls, other than PPE, to be assessed and avoids the considerable uncertainty associated with attempting to precisely quantify the attenuation of exposure brought about by the proper use of PPE . Furthermore, inappropriate use of gloves may even increase dermal uptake.
The worst-case estimates generated in this exposure assessment are considered to be reasonable worst-case estimates, as they descri be high-end or maximum exposures in feasible but not unrealistic situations. They are not intended to account for extreme or unusual use scenarios. The majority of exposures are expected to be well below these estimates.
Available recent high-quality doc umentation prepared by national or international bodies has been largely used in this report to avoid duplication of efforts. The following documents deserve here a specific mention: ATSDR, 1998 ; NICNAS, 1996 ; SIDS, 1996.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 17 Air sampling data are presented in this sec tion from a number of s ources and have been tabulated, where practicable. There is in general little or no information on the activities carried out while the sampling was running, the c oncentration of EGBE in the products, the control measures, and other important matters, such as sampling strategy and measurement methods, mean and 90 th or 95 th percentile of results; this is most often a serious difficulty for interpreting the data correctly.
Measured exposure data are compared with th at predicted from the EASE (Estimation and Assessment of Substance Exposure) model vers ion 2. EASE is a gene ral purpose predictive model for workplace exposure assessments. It is an electronic, knowledge based, expert system which is used where measured exposure da ta is limited or not available. The model is in widespread use across the European Union for the occupational exposure assessment of new and existing substances.
A few measured dermal exposure data are available for EGBE or analogous substances. They will be considered together with modelling to predict occupational dermal exposure to EGBE. Many of the references stress the importance of dermal exposure, particularly during use of products. All sections on dermal e xposure deal with liquid exposure.
All models are based upon assumptions. Their outputs are at best approximate and may be wrong. EASE is only intended to give genera lised exposure data; it predicts inhalation exposure as ranges for concentrations fo r continuous exposure at the process under consideration. Dermal exposure estimates are provided by EASE as the quantity of a product adhering to the skin due to a task, they do not take into account evaporation of the product.
In the present assessment all inhalation exposur es are expressed in parts per million (ppm), although the figures in the original pub lication are sometimes given as mg/m 3 . All mg/m 3 have been converted to ppm us ing the following approximation:
ppm = mg EGBE/m 3 x 24.05/118.2 = mg EGBE/m 3 x 0.2
mg EGBE/m 3 = ppm x 118.2/24.05 = ppm x 4.9
A number of biological monitoring data are available for potentially exposed workers. Although the dataset is not extensive, they give useful indica tions about the total exposure and are presented in this section.

Routes of exposure and relevant scenarios
The major occupational routes of exposure to EGBE are inhalation and skin contact. Assuming proper hygiene measures are applied, oral exposure would normally not occur in the workplace.
Extremely low exposures have been reported for non-industrial office settings. Data from the National ambient VOCs database indicate that for 14 samples from non- industrial offices, the daily mean and median indoor atmospheric concentrations of EGBE were 0.214 ppb (1.03 μg/m 3 ) and 0.075 ppb (0.36 μg/m 3 ), respectively (Shah and Heyderdahl, 1988 ; Shah and Singh, 1988 in ATSDR, 1998). Due to the very lo w atmospheric exposure levels (comparable to the general population), non-indu strial office settings will not be dealt with thereafter.
Workers may be significantly exposed during th e production of EGBE, its processing as an intermediate or during the formulation and use of EGBE containing products.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 18 Occupational exposure assessmen t will be carried out through three main categories of scenarios:
(a) the manufacture of EGBE a nd its use as an intermediate;
(b) the formulation of products containing EGBE;
(c) the use of products containing EGBE.
The third category will focus on particular sub- scenarios for exposure in the most frequent type of use, or particular pattern of use, when relevant.

Number of workers exposed
Due to the wide range of products containing EGBE, it is assumed that a large number of workers in many professional sectors ma y be exposed daily or occasionally.
Data from the National Occupational Exposur e Survey (NOES) conducted by NIOSH from 1980 to 1983 indicate that an estimated 2,139,292 workers in 2,259 industry/occupation categories were potentially expos ed to EGBE in the United States from 1981 to 1983. These numbers do not include workers potentially ex posed to trade-name compounds that contain EGBE (ATSDR, 1998). In Denmark, a survey of chemical exposure in the workplace estimated that there were a total of 55,000 exposures to EGBE in 1989 (Brandorff et al.
, 1995). Although these quantitative data are quite old, they confirm that EGBE is a widely used chemical with many workers potentially exposed.
Other data specific to some activities are reported further in this section.

Occupational exposure limits (OELs)
OELs apply to workplace air concentrations of chemicals. They are normally intended to protect workers against short-term adverse effect s (irritation, acute CNS effects) or long-term effects (e.g. on liver, lungs, ki dneys, or chronic CNS effects) after months or years of exposure. When applicable, a "short-term exposure limit" (STEL) may be proposed or imposed for the first ones, and/or a "time-weig hted average" (TWA) for the second. The first value ordinarily refers to a 15 minutes or so duration, the second to a shift (generally considered as an 8-hour shift).

Table 4.3 details the OELs recommended for EG BE in various countries. They are provided for information and are not an indication of the level of control of exposure achieved in practice in workplaces. All of them add a “skin” notation.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 19 Table 4.3: OEL values (Ariel Research, 2003) 8-hour TWA STEL, 15 min
Country mg/m 3 ppm mg/m 3 ppm

EU* 98 20 246 50

Austria 100 20 200 40
Belgium 98 20 246 50

Denmark 98 20 - -

Finland 98 20 250 50

France 9.8 2 147.6 30
Germany 98 20 392 80

Ireland 100 25
Italy 97 20

Netherlands 100 20 246 50
Norway 50 10 - -
Spain 98 20 - -

Sweden 50 10 100 20

Switzerland 100 20 200 40
United Kingdom 25 - 50
USA (ACGIH) - 20 - -
USA (NIOSH) 24 5
USA (OSHA) 120 25 - -

*Directive 2000/39/CE of 8 June 2000

Biological exposure levels (BELs)
Biological monitoring is recommended when app licable to measure both inhalation and skin exposure and can be carri ed out by determination of total urinary BAA (free and conjugated). Sensitive methods for the quantitative analysis of total urinary BAA were developed from the late 1980’s and adapted by successive improveme nts in analytical chemistry. Sampling at the end of the shift would be the most appropria te (Johanson and Johnss on, 1991 ; Laitinen, 1998 ; Sakai et al. , 1993).
BELs proposed to characterize occupational ex posure to EGBE are presented in table 4.4.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 20 Table 4.4: Biological exposure levels (BEL) Country Determinant Sampling time BEL Reference
Germany BAA in urine Post-shift at the end of the working week 100 mg/l DFG, 2002

USA BAA in urine Post-shift 60 mg/g creatinine NIOSH, 1990

France BAA in urine 80 mg/g creatinine INSERM, 1999

Finland BAA in urine 70 mmol/mol creatinine Laitenen, 1998
Nota :1 mmol/mol creatinine = 1.17 mg/g creatinine = 0.83 mg/l

The correlation between inhala tion exposure to EGBE and ur inary excretion of BAA was studied with persons mainly exposed through the respiratory system (Laitinen, 1998). The results indicate that an 8-hr inhalation exposure of 5 ppm in the absence of dermal exposure would correspond to a BAA excretion of about 75 mmol/mol creatinine ( 88 mg/g creatinine) in postshift urine samples.

4.1.1.2.1 Scenario 1 : Manufacture and use as intermediate

EGBE is manufactured continuous ly, either full-time during th e year or within periodic campaigns of several weeks or months. Worker s may be exposed approximately 8 hours for 5 days per week of campaign but typically th ere are no personnel working constantly on the plant, with only occasional visits from fitters , engineers and other technical staff. The process is enclosed as extensive precautions are taken to prevent and minimize exposure to workers in the production area, due to the toxicity of the ethylene oxide feedstock (SIDS, 1996).
There is the potential for exposure to the chemical in control rooms but this is minimal (2 to 4 people per shift per producti on facility) (ECETOC, 1994).
Exposure during transfer to tankers or drums is generally minimized by the use of automated filling, where the operator is segregated from the area during transfer, and the use of local exhaust ventilation. Accidental exposure may occur when the process is breached or when spills occur. Exposure may also occur during maintenance and cleaning activities; however, the purging of plant and equipment is genera lly standard practice (SIDS, 1996 and NICNAS, 1996).

Inhalation exposure

Measured data
Airborne measurements provided by manufacturer s of EGBE were collected for the OECD assessment in 1996. They are reported in table 4.5. No indication are available in the SIDS document whether they represent task measuremen ts or 8 hour TWA exposures. Clarifications were found and they are given for the Austra lian plant in the NICNAS report (NICNAS, 1996).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 21 Table 4.5: Measured inhalation exposur e for EGBE manufacture (SIDS, 1996, s lightly modified with NICNAS, 1996)

Manufacturer Monitoring area No of readings Mean (ppm) Maximum (ppm)
BASF (EU) Production 97 0.09 1.2
Filling 66 1.3 5.3*

Technical unit 9 0.25 1.2
Laboratory 14 1.3 11*

Various 8 0.5 2.7
BP (EU) All 311 <0.1 1.6
Eastman (US) Production 16 <0.04
Tanker loading 11 <0.25 1.8
Hüls (EU) Production 30 <0.14 <0.31
Filling 10 <0.14 0.22
Laboratory sampling 20 <0.38 1.1

ICI (Australia) All (STEL and TWA, personal) Maintenance (TWA, area) 0.1 1.8

*These values are reported as outliers by the department of Work Safety (Germany).
Older data are reported in open literature:

- measurements in a US plant showed that th e highest results were obtained during drum filling, with a TWA of 1.7 ppm in area monitoring. The highest personal monitoring reading was 0.1 ppm (Clapp et al. , 1984 ; SIDS, 1996).

- TWA personal exposures in the manufacture of EGBE in Europe were in the range of 0.01-0.4 ppm (production), 0.03-0.3 ppm (drum filling), 0.5-2.7 ppm (blending), <1.6 ppm (roadcar filling) (ECETOC, 1985).

- personal exposures at a numb er of production sites during 1988-1993 gave results in the range 0.1-1.6 ppm and a mean of typically 0.13 ppm (data received from CEFIC, 1993 in ECETOC, 1994).

More recent measurements were received in th e framework of this assessment from 4 EU producers of EGBE:
Producer 1: in 24 production and subsequent pr ocessing enterprises, analysis of 126 measurements (personal air sampling with e xposure duration >1hour) carried out during the period 1995-2000 leads to a 90 th percentile of 0.2 ppm. The highe st results are obtained in the laboratory with a 90 th percentile of 2.4 ppm. Deta ils are presented in table 4.6.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 22 Producer 2: 198 whole-shift personal measur ements carried out in the production plant between 1995 and 2000 result in 8-hr TWA expos ure <0.1 ppm in most operations. Details presented in table 4.7 show that personal exposure may be quite occasionally higher.
Producer 3: personal air measurements were ca rried out in 2002 in two departments of the production plant. Shift TWA exposures were all < 0.1 ppm, task levels were slightly higher in the despatch department (up to 0.6 ppm). Frequency and dura tion of exposure of the main tasks were given

. Details are presented in table 4.8.

Producer 4: 5 measurements made in the production plant in 2000 result in exposure <0.05 ppm (time span 420 to 690 minutes). No more details are available.

Table 4.6: Personal air measurements in Producer 1 enterprises (1995-2000) Type of
processing/workplace No of

results No of
enterprises 50 % value
(ppm) 90 % value
(ppm) 95 % value
(ppm) All 126 24 0.08 0.2 0.38

Production
( closed s y stem ) 21 1 0.02 0.06 0.07

Subsequent users
( closed s y stem ) 75 11 0.08 0.09 0.11

Laboratory
( with LEV ) 12 8 0.2 2.4 6.4

Filling/storage
( with LEV ) 18 4 0.08 0.6 0.68

Table 4.7: Personal air measurements in Producer 2 plant (1995-2000) Job function No of results Average TWA (ppm) Max TWA (ppm) TWA (ppm) 5 to 95 % confidence range
Lower Upper
Head of shift 106 0.2 9.0* <0.1 0.4

Chemist 10 <0.1 <0.1 <0.1 <0.1
Engineer 6 <0.1 <0.1

Fitter 2 <0.1 <0.1
Operator 57 0.3 9.7* <0.1 1.1
Physicist 4 <0.1 <0.1

Technician 13 <0.1 <0.1 <0.1 <0.1

* Statistical analysis of the raw data show s that these two high measurements should be considered as outliers.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 23 Table 4.8: Personal air measurements in Producer 3 plant (2002)
Job type No of samples 1 Sampling type Range 2 (ppm) Median (ppm) 90 % percentile (ppm)
Outside production operator 12 (3 <LOD) Shift TWA 0.002-0.03 0.01 0.024

Mechanical maintenance operator 2 (1 <LOD) Task 0.01-0.136

Mechanical maintenance o p erato r 1 Task 0.01

Road gantry operator 10 (9 <LOD) Shift TWA 0.002-0.008 0.002 0.004

Road gantry operator 8 (7 < LOD) Task 0.006-0.052 0.022 0.042
Road gantry operator 1 (< LOD) Task 0.003
Road gantry operator 1 (<LOD) Task 0.038
Ship/jetty outside operator 2 (1 <LOD) Shift TWA 0.02-0.366 3

Ship/jetty maintenance operator 1 (<LOD) Task 0.064

Ship/jetty maintenance operator 1 Task 0.628

Road/jetty outside operator 2(2 < LOD) Shift Both 0.002
Road/jetty outside operator 2(2 < LOD) Task Both 0.006

Note 1: LOD = lower limit of detection (5 μg which equates to 0.042 ppm for a 24 liter sample).
Note 2: for results <LOD, a value of 50 % of the LOD has been applied.

Note 3: one task ran over two shifts. Measurement was completed for the whole task and was therefore split between two operators, thus exposure approximates to 50 % of the level recorded.

Modelled data
The EASE model used to pred ict exposure during pr oduction in closed system with full containment provides an exposure estimation of 0 - 0.1 ppm. If the system is breached in some activities (like maintenance, sampling, clea ning, filling), concentrations could be in the range of 0.5 - 1 ppm (non dispersive use, lo w tendency to become airborne, presence of LEV).

Summary/statement of the exposure level
The results of monitoring data during producti on show very low workplace air levels. Due to automated processes for feeding reactors and fo r drum and tanker filling as well, typical inhalation exposure is < 0.5 ppm (using pers onal sampling) in most situations.
Higher exposure may occur during non-routine ma intenance activities or during rare incidents such as spills or leaks. Such incidents are presented as exceedingly rare by industry, addingEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 24 that workers would wear PPE in such circumstances. Some measurement results show concentrations up to 2.7 ppm (SIDS, 1996) or 2.4 ppm (90 % of laboratory results of producer 1).
SIDS (1996) retained a maximum atmospheric c oncentration of 3 ppm. Recent data confirm that exposure is lower in near ly all situations. It is proposed to adopt the value of 2.4 ppm (12 mg/m 3 ) as a reasonable worst-case TWA at mospheric concentr ation in production activities.

Dermal exposure
Due to the enclosure of the pro cess and control measur es taken to minimize skin contact, for example, during transfer to tankers, dermal expos ure at the plant is incidental and therefore likely to be low. The main s ource of potential exposure is during maintenance activities. Purging of plant and equipment is standard practice on site. Maintenance personnel are provided with butyl (or nitrile) rubber gloves and long-sleeved overalls, so exposure is not expected to be si gnificant (NICNAS, 1996).
Questioned on the possibility of real skin exposure, industry answered that: "Operating controls are such that accident al spills and leaks are extremely rare. Campaign changes would not normally result in breaking of confinement. Equipment for transfer to drums/trucks use engineering controls of exposure; equipment is designed to virtually eliminate the possibility of exposure during such operations. The only r eal possibility for exposure would be during maintenance, but even in such a situation, th e plant is normally purged to remove hazardous products before containment is broken and gloves would be recommended."
SIDS (1996) retained incidental contact tim e of 1 % of the working day with a 1000 cm 2 skin area exposed (a hand and a forearm). Incidental contact with the liquid seem appropriate for this scenario because exposure will be occasio nal (not daily) and intermittent contact would probably overestimate the exposure. The EASE model estimated a dermal exposure in the range of 0 - 0.1 mg/cm 2 /day (non dispersive use with direct handling and incidental contact). Assuming exposed skin surface area is 420 cm 2 (palms of hands for consistency with other EU occupational risk assessments), maximum ex ternal dermal exposure would be 42 mg/day. This exposure will be mitigated by the use of suitable gloves.

4.1.1.2.2 Scenario 2 : Formulation of products containing EGBE
During the formulation of products containi ng EGBE, workers may be exposed during preweighing before mixing, during transfer to the mixing tank, during mixing and during the filling of containers with pr oducts. The whole operation is ge nerally carried out at room temperature. Because of the similarity of scen arios, it will be assumed that exposure during formulation is the same whatever the final use of products is.
Quite a high number of workers are likely to be exposed during formula tion of products. In Australia, during the assessment of EGBE onl y in cleaning products, 82 companies were identified, some producing cleani ng products at more than one s ite, with at least 200 workers involved in formulation (NICNAS, 1996). An enquiry was recently conducted by CEPE (European council of the paint, printing and artis ts’ colours industry) on the industrial uses of 4 glycol ethers in paints or inks, one of which is EGBE: 109 an swers were received from all over Europe, 65 users and 44 non-users. They comprise both multinationals and small or medium size enterprises from most of the EU countries. The number of workers exposed wasEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 25 indicated by 43 user companies out of the 65, the answers were in the range of 1 to 250 and represent a total number of 1760 workers (CEPE, 2002).
Exposure strongly depends on the process, which may be enclosed or relatively open. When the transfer of EGBE to the mixing vessel is ca rried out in a sealed system, potential exposure will be minimal, but when the operator adds the raw materials directly by drum to the mixing tank, exposure may be greater due to possible sp lashing and vapour and/or aerosol generation. Information obtained from the national assessment of EGBE in Australia indicated that a number of formulators of cleaning products co ntaining EGBE use the latter procedure and that approximately 50 % of formulators carry out mixing in open top tanks, with greater potential for exposure (NICNAS, 1996). The situati on is likely to be sim ilar in the EU where many formulators are small or medium size enterprises.
Exposure will also strongly depend on the quantities handled, the concentration in the products and the duration and frequency of exposure.
While during preweighing and transfer to the mixing tank, wo rkers are potentially exposed to pure EGBE, they are exposed to a more dilute form during filling. However the frequency and duration of exposure may be greater. As operators may be involved in both mixing and filling, assessment of exposure is for the formulation process as a whole.
From responses to a questionnaire sent to formulators in Australia (NICNAS, 1996), workers are potentially exposed to EG BE for an average of 3 hours/ week (range 0.1-20). For most formulators, EGBE is an ingr edient in only some of their products, so exposure is not continuous on a daily or weekly basis. Fr om 74 responses to th e questionnaire, the distribution for potential expos ure duration was as follows:

Table 4.9: Distribution of potent ial exposure duration (NICNAS, 1996) Duration/week Number %

< 1 hour 30 41

1 hour 11 15

2 hours 11 14

3-4 hours 8 11

5-8 hours 6 8

Greater than 8 hours 8 11

This suggests that 89% of formulators use EGBE for 8 hours or less per week.
Information about exposure frequency and duration has also been recently collected in the EU by CEPE (table 4.10) which suggests more frequent and/or longer exposures.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 26 Table 4.10: Exposure frequency and duration in the EU paints and inks manufacturing industry (CEPE, 2002) Exposure in days/year (41 answers) Exposure in hours/day (42 answers) Arithmetic mean 173 5.1 Median 200 6.0 Range 2-250 0.5-8

Inhalation exposure

Measured data
There are a few measured data published fo r assessment of exposure during formulation. They are presented in table 4.11. C ontrol measures are generally unknown.

Table 4.11: Atmospheric and biological monitoring during fo rmulation of products (8 hr-TWA personal inhalation exposure and BAA urinar y concentration)

Products No
of samples Mean (range)
(ppm) No of postshift urinary samples Mean (range)
(mg/g creatinine) d Source

Printing inks 9 <1 Winchester, 1985

Varnishes (12) a 1.1 (0.1-8.1) (12) a 14.8 (0.8-42.7)
(post-shift) Angerer et al., 1990
(12) a 0.5 (<0.1- 1.4) b (12) a 0.30 (<0.03-1.83) b
(p ost-shift ) Varnishes (12) a 0.6 (<0.1- 1) b (12) a 23.1 (1.13-85.4) c
(p ost-shift ) Söhnlein et al., 1993

Paints 4 1.26 (0.41-3.13) Foo et al.
, 1994

Unknown 15 0.7 (max 1.5) SIDS, 1996 (industry)

Paints 328 0.4 (max 44.7) 300 3.9 (<2-59.6)
(post-shift) Vincent et al.
, 1996

Paints 179 0.1 (0.0-1.4) Wesolowski and Gromiec, 1997

a No of workers (No of samples not stated)

b Measurements on Monday

c Measurements on Tuesday

d BAA mg/l converted into BAA mg/g creatin ine using the relation mentioned in nota of table 4.4 In the inquiry of CEPE (2002), 20 facilities out of the 65 EGBE users who answered the questionnaire said they perf ormed workplace monitoring (11 replied negatively to theEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 27 question while the others did not answer at all). Twenty seven us ers indicated typical concentrations: 10 answers were <0.2 ppm, 5 in the range 0.2-2 ppm and 6 in the range 2- 5 ppm (10-25 mg/m 3 ). No details are available.

Information from database
In the German MEGA database, 165 exposure m easurements have been registered between 1996 and 2000, which were obtained in the pa int formulation industry. The results (measurement values with an exposure duration ≥ 1 hour and a sampling duration ≥ 1 hour converted to 8-hour weighted averages) are presented in table 4.12. When possible, a distinction is made on the basis of whether or not control measures (LEV) were taken. In this regard, the results present an apparent paradox that the workplaces with LEV frequently do not exhibit lower exposures than those without LEV and the exposures may even be higher.
Technical measures are mostly taken in plac e where the situation may result in a higher release of vapours, for instan ce when large quantities of s ubstance are handled or when process occurs at high temperat ure. By contrast, the release is comparatively low during use of small quantities or processing at ambient te mperature. In most cases, control measures create a situation where the exposure level of workplaces with large release approximately reaches the level of workplaces with only low release but without control measures.

Table 4.12: Personal inhalation exposure in the MEGA database, 1996-2000 (BGAA, 2001)
Paint production activity No of results No of companies 50% value (ppm) 75 % value (ppm) 90 % value (ppm) 95 % value (ppm) Raw EGBE handling, mixing and filling - without LEV - with LEV 165 71 92 39 23 28 a

a

a a

a

a 1.4. 1.9 1.4 2.4 2.5 2.4 Cleaning of containers - without LEV - with LEV 74 23 49 36 15 23 a

a

a a

a

a 2.7 3.2 2.2 7.6 6.8 6.6 a : measurement value < analytical determination limit

Modelled data
Using the EASE model (non dispersive use, lo w tendency to become airborne), the exposure estimate would be in the range of 0.5-1 ppm with LEV and 10-20 ppm in case of direct handling with dilution ventilation.

Summary/statement of the exposure level
SIDS (1996) and NICNAS (1996) estimated th at exposure during formulation of products would be 2 ppm for formulati ons including up to 10 % EGBE, and 10 ppm for those including 30 % or more. As very few measured data were available for formulation, this estimate was based on monitoring data of Vincent et al. (1993) and Salisbury a nd Bennett (1987) during cleaning activities.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 28 Although some of the published data are limited, results during formulation show that airborne concentrations are generally low. Recent data provided by industry indicate that exposure would not be higher than 5 ppm but very little information is av ailable to the context of this measurements. Based on the 90 th percentile of the MEGA database values, we propose in a first approach that wo rst case inhalation exposure dur ing formulation of products containing EGBE woul d be 3.2 ppm (15.7 mg/m 3 ). Typical exposure levels are probably much lower (<1 ppm).
Information presented in tables 4.9 and 4.10 s how that frequency and duration of exposure may considerably vary. A continuous exposur e for full shift (8 hour s per day) will be assumed, although the data available suggest that this is unlikely to be a daily exposure.

Dermal exposure

Measurements

As part of the “RISKOFDERM” project, a study was performed by the TNO (RISKOFDERM, 2002a ; Gijsbers et al ., 2004) to directly assess de rmal exposure to products containing 2-(2-butoxyethoxy)ethano l (DEGBE), a chemical of th e same family as EGBE, but much less volatile (vapour pressure 2.7 Pa at 20 °C compared to 100 Pa at 20 °C for EGBE). Hand exposure was measured during loading (typi cally at the beginnin g of the formulation process, handled product is the substance, short task dur ation ranged between 1 and 15 minutes) and during filling (typically at the end of the formulation process, handled product is the formulation, task duration ranged between 22 and 125 minutes). The measurements were made using cotton sampling gloves which were worn over new protective gloves, where present. Exposure was mainly due to exposur e on the hands. The most important source of variabilitywas due to between-company variability , rather than to either between-worker or within-worker variability. Results (given in DEGBE and product) are presented in table 4.13.

Table 4.13: Results of measurements of potential hand ex posure to DEGBE in loading mixers and filling containers with products containing DEGBE (aft er RISKOFDERM, 2002a and Gijsbers et al., 2004)

Exposure to DEGBE

or product N Range

(mg) AM

(mg) GM

(mg) GSD

(mg) AM

(μg/cm 2 /min) GM

(μg/cm 2 /min) GSD

(μg/cm 2 /min)

Loading (pure DEGBE)

Hands DEGBE 28 0.28-28300.0 3313.6 218.9 19.9 727.4 52.9 17.2

Hands Product* 28 0.31-27745.1 3215.0 217.0 19.3 708.8 52.4 16.7

Filling (all data)

Hands DEGBE 30 0.062 – 19000.0 1955.8 35.9 42.0 45.1 0.75 42.6

Hands Product* 30 4.1 – 18269.2 2726.6 555.4 9.4 58.5 11.5 9.6

Filling (only products < 10% DEGB E); data not published, but cal culated from original data

Hands Product* 21 4.1-11146 1216 249 8.5 15 4.1 7.6

N = number of measurements AM = arithmetic average GM = geometric average GSD = geometric standard deviation) * recalculated towards the full product by di viding the value measured for DEGBE by the fraction of DEGBE as analysed in the pr oductEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 29 The 90 th percentile from the measured data for loading was approximately 11,000 mg on 820

cm 2 (expressed as total product) , approximately 11,100 mg on 820 cm 2 for filling (all products, including (almost pur e) DEGBE) and approximat ely 3,300 mg for filling of products containing less than 10% of DEGBE (not published da ta, derived from original data). This value would lead to a level of 330 mg for DEGBE if the percentage of DEGBE would be 10%. It appears that the situations with handling (almost) pure products lead to higher exposure levels. That can be caused by the fact that products with small percentages of DEGBE, such as paints can be packaged in cans by highly automated equipment, while (almost) pure DEGBE is often packaged in la rger containers with more handling of the container by the workers.
Although assessed in a direct way and in actual working situations, these resu lts should not be given too much weight for the main following reasons:
- this is an isolated study, a nd it is known that there is an extreme variab ility in skin (and especially hand) exposure (Kromhout et al ., 2004), depending of a number of most often qualitative factors that have been divided in six categories (Marquart et al ., 2003);
- measurement durations are relatively shor t (range 1-139 min, arithmetic mean range 6- 74 min, all tasks counfounded; Gijsbers et al ., 2004) compared to a shift, so the results may represent rather task-associate d exposure measurements than shift exposure assessments.
- although from the same chemical fam ily as EGBE, DEGBE has very different physicochemical properties, among which volatilit y, viscosity and the octanol/water partition coefficient may play a significant role.
Biomonitoring studies
In this context, biomonitoring studies might be considered as an alternative means to assess skin exposure because: i) biom onitoring allows an assessment of the exposure on the whole shift, although it integrates dermal and inha lation routes; ii) several studies have been published on EGBE itself, so there is no need to extrapolate from other more or less related chemicals with the corresponding uncertain ties. Technically, only studies where total (i.e. free and conjugated) urinary butoxyacetic acid (BAA) has been evaluated are sufficiently reliable (Jones and Cocker, 2003; Franks et al ., 2006). In order to pr operly correct for EGBE absorption via the inhalation route, the latter must have been separately assessed on the same day and for the same worker and task as for de rmal exposure. Difficulti es may however arise, among which: i) too short sampling times when compared to a shift (e.g. more than 50% workers are exposed for 1 hour or less, accordin g to table 4.9); ii) sampling conditions often unspecified (e.g. was it area sampling or pers onal sampling? were there incidents when sampling was running? how was the mean inhala tion exposure concentration calculated?); iii) some skin absorption of vapours may occu r (up to 39% of the to tal uptake in extreme conditions, after Jones et al. , 2003); (note: these difficulties ma y lead to an overestimation of inhalation exposure with the asso ciated underestimation of skin exposure) iv) different values for the urinary excretion of total BAA per ppm EGBE i nhalation exposure have been proposed (e.g. 12 mmol/mol creat per ppm, Laitinen et al ., 1998; 15 mmol/mol creat per ppm, Laitinen, 1998; 10 mmol/mol creat per ppm, Franks et al ., 2006).
Keeping these difficulties in mind, evaluations are tentatively propos ed, based on biological monitoring with EGBE in real wo rking conditions. If A is the result of biomonitoring (total BAA, mg/g creatinine) and B the correspond ing inhalation exposure (ppm), a “dermal equivalent” (mg/day) may be evaluated using the formula:
[(A x 5/88) – B] x 88/5 /0.6 [which is equal to (5A – 88B) / 3 ]EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 30 where 5 and 88 are the correspond ence between inhalation expos ure (5 ppm during a shift) and the excreted BAA (88 mg/g creatinine, po st shift; after Laitin en, 1998), and 0.6 is the absorption efficiency by inhalation.
Among the studies from Angerer et al. (1990), Söhnlein et al. (1993) and Vincent et al. (1996), only peak measurements from Söhn lein show significant dermal absorption (85.4 mg/g creatinine with 1 ppm as a peak measured inhala tion exposure), leading to a dermal equivalent absorption of 113 mg/day, th at is to say a dermal load of 377 mg/day assuming 30% absorption. There is a high proportion of results entirely explained by the inhalation route or even leading to calculation of a negative dermal exposure, i.e. the biological monitoring results are (much) lower than would be expect ed due to the inhalation exposure data. This suggests a negative bias, especially for a ch emical being known to be eas ily absorbed through the skin; this may originate from the many unknowns in sampling conditions, as already mentioned, and also from remaining uncertainties in the biological disposition of 2-BE (Franks et al
., 2006).
If this bias is estimated as 30% (Franks et al ., 2006, estimate a 31% disc repancy solely due to sampling time differences), the dermal load would be re-assessed as ca. 500 mg/day.

Modelling
Considering the process and the tasks where exposure may occur, SIDS (1996) retained intermittent contact time of 20 % of the working day with a 1000 cm 2 skin area exposed (a hand and a forearm).
Taking into account the same a ssumption, the EASE model estim ates a dermal exposure in the range of 0.1-1 mg/cm 2 /day (non dispersive use with direct handling and intermittent contact). Assuming exposed skin surface area is 420 cm 2 (for consistency with other EU occupational risk assessments and default assu mptions recommended in table 3 of Appendix I of the Technical Guidance), maxi mum external exposure would be:

- 42-420 mg/day for loading (pure substance)

- 21-210 mg/day for filling (assuming 50% EGBE in the product as a worst case : in most situation, EGBE content will be much lower)

Assessment
Dermal exposure can occur duri ng part of the working day dur ing loading of mixers (short periods, up to approximately 20 minutes) and dur ing packaging of products containing EGBE (up to 120 minutes per day). Measur ed data for this scenario for EGBE are not available. Data from DEGBE for similar processes may be re levant. The measured values, using cotton gloves as samplers, for DEGBE are generally high compared to the estimates by EASE. Geometric mean exposure levels to products, for situations where formulations were made, so excluding the filling of (almost pure) DEGBE, were in the order of 200-250 mg and the 90 th percentile was in the order of 3,300 mg for filli ng of products with less than 10% DEGBE and 11,000 mg for loading of DEGBE into mixers, both on 820 cm 2 . The measured data for DEGBE may be an overestimate of potential dermal exposure for EGBE for two reasons. Firstly, the measurement method may have led to overestimation of dermal exposure, because cotton gloves are considered to retain more liquid than the skin would do. Secondly, EGBE is much more volatile than DEGBE and therefore, more of the substance may evaporate from the skin and not be available for uptake. The effe ct of both factors is di fficult to estimate. TheEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 31 estimate based on measured data is substantia lly higher than that based on EASE that is considered a weak model for dermal exposure. Although the measured data have a number of uncertainties, they should not be disregarded for risk characterisati on. Therefore, a rounded value of 10,000 mg EGBE/day will be used for the loading step of the process. In the evaluation of the Margin of Safety (MOS) it will be considered that this may be a (substantial) overestimation.
For the purpose of determining the evaporatio n time, the following equation can be used (TGD Appendix I.E):
T(s)= (mRT/M β pA)K
This equation leads for EGBE to an estimate of evaporation time of 3 hours, with the following input values: m = 10 000 mg, R = 8.134 J.K -1 .mol -1 , T = 305 Kelvin, K = 3.6 * 10 4

, M = 118.17, β = 8.7 m.h -1 (default), p = 100 Pa, A = 820 cm 2 . This indicates that in approximately 3 hours all EGBE would be eva porated from the skin.. For comparison, the same calculation with DEGBE (vapour pressure = 2.7 Pa) leads to an evaporation time of more than 100 hours.
The filling of packages with products leads to substantially lower exposure levels. Assuming a product with 50% EGBE and a 90 th percentile exposure level for the product of 3,300 mg leads to a reasonable worst case exposure leve l of approximately 1,600 for EGBE. The related evaporation time would be a pproximately half an hour.

Conservatively, it may be assumed that both ta sks are done by the same workers, leading to a total exposure of approximately 11 600 mg that will take approximately 3,5 hours to evaporate from the skin. Due to the sampling method for the measured data and the higher vapour pressure of EGBE compared to DEGBE, these are considered to be overestimates of the real exposure levels, which should be take n into account in the ev aluation of the MOS.

Biological monitoring data
On the one hand, exposure data have been obtai ned in real use conditions, which is a very strong argument to take them in consideration, but in an isolated work and with a chemical presenting physico-chemical prope rties very different from thos e of EGBE, which are strong arguments not to accept them at face value. On the other hand, limited biomonitoring data are available for EGBE itself, also in real conditions of use, with the advantage of not requiring any extrapolation of uncertain validity from another chemical substance. Its main weakness lies in the important pe netration of EGBE through the inhala tion route, which must then be assessed with sufficient reliability. Since neit her of these sources ca n be ignored, a final assessment must be based on both. The difficulty to do so lies here in assessments that differ by a factor of 23 (11,600 mg/day for Risk of Derm, 500 mg/day for biomonitoring). This factor is in fact relatively small considering the extreme variability evidenced in assessments made by RISKOFDERM in a variety of situati ons. Moreover, there ar e good reasons to think that data obtained with DEGBE may be overestimates when transposed to EGBE. On the other hand, based on extensive data from this RISKOFDERM project, Marquart et al . (2006, table 5) consider that 550 mg is more a typi cal exposure value than a reasonable worst-case. According to them, “individual m ean dermal exposure levels were on average within a 4-fold range”. In this context, it is proposed to re-evaluate the assessment of the limited data based on biomonitoring by a factor of 4. This eventua lly leads to propose a skin penetration of 2,000 mg/day for this scenario.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 32 4.1.1.2.3 Scenario 3 : Use of products containing EGBE
EGBE is used in a wide variety of products . The following scenarios are considered as representative:
- use of paints and coatings
- use of printing inks

- use of cleaners
Cleaning related to painting and printing acti vities are included in the first and second scenarios.

Measured exposure levels in general
Exposures to EGBE were f ound in a 1983 survey of 336 Belgian businesses (Veulemans et al., 1987a). In this study, EGBE was found in 25 of 94 air samples from sites using printing pastes; 10 of 81 samples where painting was done; 1 of 20 samples from automobile repair shops; and 17 of 67 samples from various othe r industrial sites where materials such as varnishes, sterilization agents and cleanin g agents were used. The geometric mean atmospheric concentrations and ranges of EGBE at various sites were as follows: printing shops, 0.8 ppm, range 0.3-3.7 ppm; painting areas, 3.9 ppm, range 0.7-19.4 ppm; automobile repair shops, 1.2 ppm (one sample); various other industries, 1.8 ppm, range <0.1-367 ppm (this data are considered further wh en relevant to a specific scenario).
In a survey on industria l solvents conducted in 1994 to 1996 by Yasugi et al. (1998) in 95 different plants (196 unit wo rk areas), EGBE was found 59 tim es (8 during printing and 51 during painting) with a medi an atmospheric concentratio n of 0.5 ppm and a maximum concentration of 1.3 ppm (10-l bag samp le, analysed the day of sampling).
From 1987 to 1998, the Fren ch COLCHIC database colle cted 10,593 personal sampling results of glycol ethers for 602 facilities (Vincent, 1999). EGBE was found 1,195 times; the arithmetic atmospheric mean value of the 60 to 480 minutes samplings (622 results) was 2.3 ppm (median 0.6 ppm; range 0.02-110 ppm; 95 th percentile 6.2 ppm). It should be noted that, globally, atmospheric concentrations have si gnificantly decreased fr om 1987-92 (arithmetic mean of 147 personal samplings 1.1 ppm, median 0.6 ppm, range 0.02-10.7 ppm) to 1993-98 (arithmetic mean of 178 pe rsonal samplings 0.28 ppm, medi an 0.1 ppm, range 0.02-3.8 ppm) (Vincent and Jeandel, 1999).
For the years 1999 to 2002, the COLCHIC data base collected 116 personal atmospheric sampling results of EGBE. The arithmetic mean value of 60 to 480 minutes samplings was found 0.52 ppm (median 0.14 ppm, range 0.02-14.84, 95 th percentile 1.22 ppm) (Vincent, 2003).

• Scenario 3-1 Painting/Surface coatings
EGBE is used as a solvent in paints and surface coatings, particularly in water-based type. It is the main application of EGBE and due to the high volume use, a large number of workers are potentially exposed.
Twenty seven water-based paints and formulations used in the automotive industry have been analysed (Jargot et al ., 1999). EGBE was found 18 times, with concentrations ranging from traces to 40.4 %; in thinners, its concentration ranged from 0 to 24.7 %.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 33 SIDS (1996) reports that coatings may t ypically contain 1 % up to 8 % EGBE.
Analysis of the answers collected in the pa int formulating industry by CEPE (2002) shows that the concentrations of EGBE range from traces to 39 % with an arithmetic mean up to 8 % (see table 4.14). Taking into account this data together with the information collected in European products registers (t ables 4.1 and 4.2), a worst case EGBE content of 20 % in industrial paints and surface coatings and 3 % in decorative paints will be assumed in this assessment. Therefore the conclusions in this section refer to solvent-based paints. Exposure from use of water-based pa ints (lower EGBE content) would be much lower.

Table 4.14: Contents of EG BE in paints (CEPE, 2002) Industrial paints Decorative paints

Water-based Solvent-based Water-based Solvent-based

Number of

answers 33 31 10 9

Arithmetic mean 7.8 % 7.1 % 1.4 % 1.6 %

Median 3.5 % 5 % 1.8 % 1 %
Range* 0.06-22.5 % 0.01-38.7 % 0.03-3.0 % 0.01-3.0 %
* minimum values probably correspond to incorporation of EGBE through additives.
Coatings and paints are applied by brushing, rolli ng, spraying or dipping in different industrial and skilled trade sectors, e.g. coating of metal and wood, vehicle production and repair, building trade. Application techniques inventorie d in the CEPE enquiry are presented in table 4.15 (CEPE, 2002).

Table 4.15: Relative frequencies of application te chniques in painting/surface coating (CEPE, 2002) Application technique No of mentions

Spray 40

Roll 22

Brush 21
Dipping 6

Roller coaters 3
Printing 3
Falling curtain 1EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 34 Inhalation exposure

Measured data
The data reported in the previous assessm ents (SIDS, 1996 ; NICNAS, 1996 ; ATSDR, 1998) completed by further references avai lable in open literature (Collinot et al. , 1996 ; Delest and Desjeux, 1995) are presented in table 4.16. Deta ils have been added when available.

Table 4.16: Atmospheric and biological monitoring during painting (8 hr-TWA inhalation exposure and BAA urinary concentration)

Activity No/type of sampling EGBE air concentration Mean (range)(ppm) No of post-shift

urinary samples Post-shift BAA urinary

excretion Mean (range) (mg/g creatinine) Reference

Furniture production/finishing 64/p 1.5
(0.07-9.9) Zaebst, 1984

Staining/varnishing 9 5

( ≤ 71) Denkhaus et al., 1986

Car repair 1/a 1.2 Veulemans et al., 1987a

General painting 10/a 3.9
(0.7-19.4) Veulemans et al., 1987a

House painting (water-based < 1.4 % EGBE) 15/p (0.4-12) Hansen et al.
, 1987

Cabinet finishing 6/p Nd 1 -0.4 Newman and Klein, 1990

Spray painting 11/p 0.4
(0.2-0.6) Winder and Turner, 1992

Painting (waterbased paints < 5 % EGBE) 54 0.1
(0.1-0.3) (<2-13.2) Delest and Desjeux, 1995

Indoors house painting
(water-based paints) 3 202/p 0.01
( < 0.01-0.15) Norbäck et al., 1996

Cataphoresis a ( ≤ 7.5) Collinot et al., 1996EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 35 Can coating

- external deco

- internal decor 20/p
11/p 0.73 (0.35-1.23) 0.44 (0.15—0.67) 12.2 (0.3-51.4) 9.2 (0.6-20.4) Haufroid et al.
, 1997

Painting of plastic hup caps 79/p < 0.1
( < 0.1-0.8) 19

n.d.

Cataphoresis 66/p 0.8
( < 0 .1-6.2) 51
17.9 (<2-210)

Can coating 168/p 0.2
( < 0 .1-2.4) 79
5 (<2-33.9)

New car painting 39/p < 0.1
( < 0.1-0.5) 40

n.d.

Coil coating 261/p 0.1
( < 0.1-1.0) 213
2.3 (<2-28.4)

Painting of metal frame 50/p < 0.1
( < 0.1-0.3) 46
9.4 (<2-63)

Painting of buildings 63/p < 0.1
( < 0.1-0.2) 63
<2 (<2-13.2)
Furniture varnishing n.d. 50 2.9 (<2-31.2)

Printed circuit boards varnishing 57/p < 0.2
( < 0.1-2.8) 56
4.6 (<2-30.4) Vincent et al., 1996

p: personal, a: area, Note 1: Limit of de tection not reported. Note 2 : No of workers (No of samples not stated) Note 3: EGBE found in 2 samples.

Information from database
In the HSE exposure database (Brown et al. , 1994), 12 results are av ailable for painting and 20 for lacquering; the ranges (a nd arithmetic means) were 0 – 1.4 ppm (0.15 ppm) and 0.2 – 7 ppm (1.69 ppm) respectively. Th e single high exposure was ta ken during the spraying of lacquer onto wooden furniture and local exhaust ventilation was provided; this high level was considered as probably due to a failure in the LEV system.
Exposure measurements (sampling period 60- 480 minutes) registered between 1987 and 1998 in the COLCHIC database were analysed by Vi ncent (1999). Results re lated to painting and coating are presented in table 4.17.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 36 Table 4.17: Personal exposure in painting activities fo r measurements 60-480 minutes, years 1987-1998 (Vincent, 1999)

Type of work No of results Mean (ppm) Range (ppm) Median (ppm) 95th percentile

(ppm)
Pneumatic spraying of paint or varnish 58 0.46 0.04-4.2 0.2 2.2

Varnishing (curtain) 19 0.14 0.02-0.6 0.1 0.6

Brush or roll coating of paint or varnish 21 0.18 0.04-0.6 0.12 0.42

Cataphoresis 3 0.2 0-0.6 - -

In the German MEGA database, 441 exposure m easurements have been registered between 1996 and 2000, which were obtained during applicati on of paints or coatings, mainly in the plastic or metal processing industries, in elec trical engineering or woodworking. The results (measurement values with an exposure duration ≥ 1 hour and a sampling duration ≥ 1 hour converted to 8-hour weighted averages) are presented in table 4.18. When possible, a distinction is made on the basis of whether or not control measures (LEV) were taken (see comments in scenario 2).

Table 4.18: Personal exposure measurements in the MEGA database, 1996-2000 (BGAA, 2001) Type of company/working

area No of results No of companies 50% value (ppm) 75 % value (ppm) 90 % value (ppm) 95 % value (ppm) Painting,brush and roller application, filling work - without LEV - with LEV 40

26 10 24

16 6 a

a

a a

a

a a

a

a 2.4

2.2

a Spraying (compressed air, airless, airmix) - without LEV - with LEV 224

23 189 112

14 98 a

a

a a

2.4

a a

11.6

a 3.8

24.6 2.6 Surface coating, mechanical - without LEV - with LEV 98 44 53 47 22 26 a

a

a a

a

a a

a

a a

a

a Surface coating, general - without LEV - with LEV 79 26 50 8 21 28 a

a

a a

a

a a

a

a 2.7 1.5 3.9 a : measurement value < analytical determination limitEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 37 Modelled data
Exposure to vapours during the use of paints or surface coatings is estimated by EASE to be in the range 100-140 ppm for wide dispersive, low tendency to become airborne, direct handling and dilution ventilation.
The model overestimates exposure levels, partic ularly because of non-consideration of the content of EGBE in the mixtures. The estimat es cannot be corrected for the partial vapour pressure because the composition of the formul ations is not known. A simple approach based on a reduction of the exposure by a factor equi valent to the EGBE concentration in the mixtures (up to 20 % for indus trial paints and 3 % for deco rative paints) would lead to exposure levels of 20-28 ppm fo r industrial paints and 3-4 ppm for decorative paints. However the validity of these extrap olations is rath er questionable.

Summary/statement of the exposure level
Exposure to EGBE during painting may be ex tremely variable, due to differences in frequency and duration of use, c oncentration of EGBE in the pa int, method of application and precautions taken during use. To some extent, th is variation is reflected in the atmospheric monitoring data available for EGBE during painting and surface treatment.
SIDS proposed a maximum atmospheric concentr ation of 10 ppm for use of a paint/surface coating containing 10 % EGBE, the justification fo r this estimation is not clear. On the basis of the available data, results of air monito ring are generally much lower except during spraying.
For spraying, the available measured data ar e mainly extracted from the COLCHIC and the MEGA databases. The 90 th percentile of the MEGA (11.6 ppm) database values seems to be the most representative wo rst case inhalation exposure.
For other application techniques, inhalation exposure is likely to be lower. The 95 th percentile of the COLCHIC and MEGA databa ses results are lower than 4 ppm. The highest result from the largest study (Vincent et al. , 1996) is 6.2 ppm. This value will be used for risk characterisation to ensure that highl y exposed workers are represented.
In conclusion, we propose in a first approach the following worst case inhalation exposures:

- 11.6 ppm (58.1 mg/m 3 ) for spray application of paint

- 6.2 ppm (30.4 mg/m 3 ) for other application techniques

Dermal exposure

Measurements

In a study performed by TNO (R ISKOFDERM, 2002a and Gijsbers et al., 2004), a part of the RISKOFDERM project, potential hand exposure to an analogous but less volatile glycol ether DEGBE (2-(2-butoxyethoxy)ethanol) was measur ed during indoors application of paint by brushing over of periods of 57-149 minutes. The sampling was e nded when the painters (who usually painted for most of the working day) took a break for coffee or lunch, resulting in the measurement durations mentioned. The measur ements were made using cotton sampling gloves which were worn over new protective gl oves, where present. The amount of used product was between 0.5 and 2.5 litre (AM 1.2 litre) and the paint contained between 0.4 andEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 38 3.2 % DEGBE (AM 2 %). The treated area dur ing measurements was between 2 and 15 m 2 (AM 6.4 m 2 ). Exposure was mainly due to exposure on the hands. Results (given in DEGBE and product) are presented in table 4.19.

Table 4.19: Dermal exposure rates during brushing ( Gijsbers et al , 2004)
Hands
exposure N W Range (mg) AM (mg) GM (mg) GSD (mg) AM c ( μ g/cm 2 /min) GM c ( μ g/cm 2 /min) GSD c ( μ g/cm 2 /min)

- DEGBE
- product 36

24 a 18

13 0.19-33.0

11.3-733.3 6.5

170.5 2.8

98.4 4

3 0.091

2.8 0.045

1.7 3.6

2.9

N, number of measurements; W, number of workers involved AM, arithmetic mean; GM, geometric mean ; GSD, geometric standard deviation Nota a : for 12 of the measurements, exposure to product could not be calculated due to contamination of samples by other sources of DEGBE.
Nota c : a surface area of 820 cm 2 was assumed for the hands exposure

The 90 th percentile of the measured exposure levels was approximately 400 mg product (unpublished data, derived from original data).
No measurements for dermal exposure in spra y painting of EGBE or other direct analogues (e.g. other glycol ethers) are available. A number of dermal exposure data sets is available for other spray applications. Exposur e to a non-volatile pigment wa s measured in car painting. Dermal exposure to the hands, expressed as tota l formulation, was between 2 and 211 mg (n = 30), with a median of 41 mg. Amount of produc t used was up to 1.5 kg in an average duration of 16 minutes (RISKOFDERM, 2002b ; Delgado et al., 2004). Exposure to another nonvolatile pigment was measured in marine anti -fouling painting. Dermal exposure to the hands, expressed as total formulation was between 286 and 27.000 mg (n = 24) for sampling periods of 1 to 3 hours. In this period 40 to 140 litres of paint were sprayed. Exposure was not only to workers actually doing spray painting, but also to auxiliary workers (so-called linesmen) assisting the spray painters. There was no di fference in exposure between the two jobs. (RISKOFDERM, 2002c ; Hughson and Aitken, 2004). Spray application of a cleaning agent containing DEGBE was measured 12 times in the food industry. Duration of measurements ranged between 6 and 18 minutes. The in-use concentration of DEGBE was between 0.007 and 1.1%. The total amount of diluted product used was between 16.5 and 97.2 L. Dermal exposure to the hands, expressed as tota l formulation, was between 37 and 1974 mg (RISKOFDERM, 2003a). The 90 th percentiles of these three data sets were respectively approximately 100, 13,600 and 1000 mg per meas urement period (unpublished data, derived from original data). Older data are reported in the TGD, where for spray painting on large surface areas a reasonable wo rst case value of 10 000 mg product per day (on 840 cm 2 ) is given (Appendix I.E).

Modelling
SIDS (1996) retained continuous skin contact over the work period (8 hours) and a 1000 cm 2 skin area exposed (a hand and a forearm). Intermittent contact seems more appropriate for tasks as brushing and rolling in this scenari o. For spray application, extensive contact is assumed., For wide dispersive us e, the EASE model es timates a dermal exposure in the range of 1-5 mg of product/cm 2 /day for intermittent contac t and 5-15 mg of product/cm 2 /day for extensive contact. The estimation is made from a formulation containi ng up to 20 % of EGBE (industrial paint) and a formulation containing up to 3 % of EGBE (decorative paint) and anEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 39 exposed skin surface area of 840 cm 2 (two hands for consistency with other EU occupational risk assessments). This leads to esti mated external dermal exposures of :

- 168-840 mg/day for industrial painting (exc luding spray applica tion)- 840-2520 mg/day for industrial spray painting

- 25-126 mg/day for decorative painting

Assessment
Skin contact due to manual transfer of liqui ds, spray application and brushing, rolling and cleaning is to be expected. In several of th e available references, the importance of skin exposure is stressed.
In the TNO study, cotton sample gloves can reta in more material than skin leads to overestimation of exposure. The size of this pos sible overestimation is difficult to estimate. It can be expected that this overestimation is sm aller when more viscous paints are used than when less viscous pure product is used. The fact that workers (except for breaks) may almost continuously do the same tasks suggests that the task based exposures s hould be extrapolated to a full work shift. Whereas extrapolation is clearly not va lid for high (product) exposures, due to saturation of the surface of the skin, this is not so much of a problem with lower task exposure levels. Given the fact th at painters spend part of their working pe riod in preparation, moving between rooms, cleaning up the material and other non-painting tasks, the exposure period for painting can be estimated to be up to 6 hours. Based on the 90 th percentile of the measured data (400 mg product in 2 hours), linear extrapolation to 6 hours of work and an assumed percentage of 20 % (industrial paint) or 3 % EGBE (decorative paint), the exposure value to be used in risk ch aracterisation is estimated as 400x 3x 0.2 = 240 mg or 400x 3x 0.03 = 36 mg EGBE per day for brushing and rol ling of industrial or decorative paints.
The exposure levels from industrial spray app lication apparently de pend on the scale of application, as well as on c ontrol measures in use. Without further information, it is assumed that large scale ap plication with limited exposure contro l can be done with paints containing up to 20% EGBE. The measured values over s hort periods cannot be extrapolated towards longer periods, because this woul d lead to oversatura tion of the skin. Therefore, a reasonable worst case exposure level of 10,000 mg product per day is assumed, based on the levels mentioned in the TGD and the measurements by Hughson and Aitken (2004). This leads to an estimated exposure to EGBE of 2000 mg on 840 cm 2 . Because EGBE is much more volatile than the measured substances, this may be an overestimation. Also, if less large scale tasks are done, the exposure levels may be substantially lo wer. These uncertainties should be taken into account in the evaluation of the MOS.
Dermal exposure may be lower if suitable gloves are worn.

Biomonitoring data
Three studies of biomonitoring have been reporte d for painting or coatin g activities. Results are presented in table 4.16.
In the study performed by INRS (Vincent et al., 1996), the workers most exposed to EGBE were found in the area of cataphoresis. The authors note that cutaneous exposure was prevalent particularly in workers who cleaned car bodies with a rag dipped in EGBE before installing the electrode. Working conditi ons may have greatly improved since 1993.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 40 In another French study (Delest and Desjeux, 1995), exposure of 54 hous e painters was also assessed by biological monitoring. Only 3 painte rs had BAA concentrations in urine higher than the detection limit (2 mg/g creatinine). The main reasons for such low figures were the application technique (brush or roller) a nd the low EGBE conten t in the products ( < 5 %).
An assessment of skin penetration may be ma de from these biomonitoring studies with the same reasons, techniques and limitations that have been presented in scenario 2. Highest results are also obtained with peak value measur ements, especially in this case from metal painting (63 mg/g creatinine and 0.3 ppm inha lation concentration), which gives a dermal equivalent of 96.2 mg/day, corresponding to a dermal load of 320 mg/day, assuming 30% absorption efficiency. As in scenario 2, a negative bias of 30% is also considered; the dermal load would then be re-assessed as ca. 430 mg/day.
In the case of building pain ting (“decorative”; 13.2 mg/g cr eatinine and 0.2 ppm inhalation concentration), the calculated dermal equivalent is 16 mg/day, corresponding to a dermal load of 54 mg/day, assuming 30% absorption efficienc y. If the same bias is also assumed, the dermal load would be assessed as ca. 70 mg/day.

• Scenario 3-2 Printing
EGBE is a solvent in a range of specialist i nks particularly silk-s creen inks used by professional trades. However there is a trend fro m solvent based inks to UV curing inks that contain no solvents.
SIDS (1996) reports that inks contain appr oximately up to 20 % EGBE. In the enquiry recently performed in the ink formulating indu stry by CEPE (2002), the information collected is very limited. Only 6 answers were obtained in relation with the EGBE content in printing inks. They indicated content from traces up to 31 %. Screen ink seem to contain the highest concentration. Other recent data provided by one of the main producer of screen printing show that typical percen tages range from 2 to 35 % (BP, 2002a ). Taking into account all this data, typical maximum contents of 35 % EGBE in silk-screen inks and 20 % in others will be assumed in this assessment.

Inhalation exposure

Measured data
Monitoring data are reported in previous reports (SIDS, 1996 ; NICNAS, 1996 ; ATSDR 1996), some of them are quite old and probabl y not representative of current working conditions. A compilation of these data is pres ented in table 4.20, completed with two further references (Auffarth et al. , 1998 and Laitinen, 1998). Details ha ve been added when available.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 41 Table 4.20: Atmospheric and biological monito ring during printing (8 hr-TWA inhalation exposure and BAA urinary concentration)
Activity No/type of sampling Mean (ppm) Range (ppm) Comments / Postshift
BAA urinary excretion (mg/g creatinine) Reference

Printing press operators 3/p <0.2 <0.04- 0.49 2 %, cleaning printing press rollers Lewis and Thoburn, 1981
Label production /plate makers and pressmen 7/p 2 1-2 Apol, 1981

Silk screening 6/p 3.4 1.1-5.3 Boiano, 1983
Silk screening
Spray printing p

p max 1.4

max 0.75 Clapp et al., 1984

Silk screening

Silk screeners
Spray printers

Controls 16/p
5/p
6/p 6.8

2.6

0.3 up to 45 % Baker et al., 1985

Silk screening 3/p 4 3-5 Apol, 1986

Silk screening 108/p 0.64 Max 1.14 Half-shift sampling Veulemans et al., 1987b

Printing (various) 25/a 0.8 0.3-3.5 Veulemans et al., 1987a

Silk screening 14/p

8/a 25

63 13-36

23-169 100 %, open spray troughs and wash table areas Kullman (NIOSH), 1988
Printing press operators 1/p 8.3 Lee, 1988

Silk-screening (cleanup and maintenance) 5/p 5.2 1.7-9.8 10-50 %; cleaning with ahydrocarbonbased solvent Salisbury and Bennett, 1987

Printing press operators 2/p <0.33 nd-0.53 2 %; cleaning printing press rollers Kaiser, 1990

General printing 9/p 0.64 0.4 - 0.8 9 urinary samples Sakai et al.,EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 42 3.9 (1.3-9.9) 1993

Tampography 84/p 0.2 < 0.1-0.7 48 urinary samples
2.2 (<2-7.1) Vincent et al.
, 1996

Silk screening 295/p 0.2 < 0.1-1.6 154 urinary samples

nd c Vincent et al.
, 1996

Offset printing 39 Nd b 11 urinary samples
2.2 (<2-3.8) Vincent et al.
, 1996

Printing press operation - semi automatic - 3/4 automatic process - fully automatic 5/p 14/p 3/p 0.5 0.5 0.5 0.3-0.9 0.3-0.9 0.3-0.9 Auffarth et al., 1998

Silk screening 5/p 0.5 0.2-0.7 Auffarth et al., 1998
p: personal sampling; a: area monitoring
b : any concentration upper than 0.1 ppm was detected
c: any concentration upper than 2 mg/l was detected
More recent exposure measurements made in 2001 and provided by one of the main producer of screen printing inks in the EU have been presented by industry (BP, 2002a). Printers are exposed 1-8 hours per day but 2-3 hours is the typical length time when workers are exposed to solvents. Nearly all operations use LEV as well as general ventilati on. EGBE was detected in 13 samples amongst 161. The results are pres ented in table 4.21. The figures represent TWA over 2-3 hours.

Table 4.21: Personal air measurements during screen printing (BP, 2002a)

Activity No of results where EGBE detected Mean (ppm) Maximum (ppm) 5-95 % percentile

(ppm)
Print shop 6 0.4 0.7 0.02-0.7

Reclaim 2 0.9 1.7

Other 5 <0.1 0.2

Information in database
In the HSE database (Brown et al ., 1994), 66 personal exposure (8-hour TWA) have been collected (1986-1991) for screen printing. The range was 0-9.2 ppm (arithmetic mean 1.54 ppm).
Referring to the already cited French COLCHI C database (Vincent, 1999), 45 atmospheric personal samplings have been made between 1987 and 1998 in the printing industry, resultingEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 43 in an arithmetic mean concentration of 0.6 ppm (range 0.02-5.2 ppm; median 0.04 ppm; 95 th percentile 2.6 ppm). Results collected during the same period for pr inting activities in different industrial sector s are presented in table 4.22.

Table 4.22: Personal exposure in printing activit ies made for measurements 60-480 minutes, years 1987-1998 (Vincent, 1999).
Activity No of results Mean (ppm) Range (ppm) Median (ppm) 95 th percentile (ppm)
Screen printing 92 1.1 0.02-6.5 0.4 4
Screen washing 9 0.4 0.04-1.6 - -
Offset printing 13 0.3 0.02-1.2 0.04 1.2
Cleaning in general 2 2.5 1.2-3.8 - -

Flexography 20 0.08 0.04-0.1 0.1 0.1
Rotogravure 7 0.04 0.02-0.1 -

In the printing industry, it should be noted that expos ure to EGBE has significantly decreased from 1987-92 to 1993-1998, with an arithmetic mean for personal samplings of 1.4 ppm (median 1.3 ppm; range 0.02-6.5 ppm; 68 samples) for the first period, and 0.3 ppm (median 0.1 ppm; range 0.02-2.5 ppm; 79 samples) for th e second (p<0.01) (Vincent and Jeandel, 1999).

Modelled data
SIDS calculated the exposure during silk scre ening and general printing tasks using inks containing 50 % and 20 % of EGBE. Exposu re estimates were 10 ppm and 2 ppm respectively. No information is available on the ca lculation method.
Exposure to vapours during printing is estimated by EASE to be in the range of 10-20 ppm for non dispersive use, direct handl ing with dilution ventilation.
The model overestimates exposure levels, partic ularly because of non-consideration of the content of EGBE in the products. The estimates cannot be corrected for the partial vapour pressure because the composition of the formul ations is not known. A simple approach based on a reduction of the exposure by a factor equi valent to the EGBE concentration in the mixture (35 % for screen inks and 20 % for others) would lead to exposure levels of:
- 3.5-7 ppm for silk screening
- 2-4 ppm for general printing.
However the validity of these extrap olations is rath er questionable.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 44 Summary/statement of the exposure level
Considering the above data, it seems reasonable to make distinctions between the different situations and uses. Mainly based on the data from COLCHIC, the reasonable worst-case exposures proposed would be the following:
- Silk screening (including washing): 4 ppm (20 mg/m 3 )
- General printing: 1 ppm (5 mg/m 3 )

Dermal exposure

Measurements
In a study performed in Finland by Kuopio Re gional Institute of Occupational Health (KRIOH)(RISKOFDERM, 2003b), a part of the EU RISKOFDERM project, potential hand exposure to EGBE was measured during silksc reening on 10 workers in three different enterprises. The measurements were made fo r 6 to 8 hours by giving protective gloves, which were collected after shift and analysed. Results (given in formulation) are presented in table 4.23a. In the same study, measurements of actual hand exposure indicate that the use of gloves does lower hand exposure significantly. Hands seemed to be the most dominant potential dermal route exposure because EGBE could be found only on a few parts of the body parts of printers.

Table 4.23a: Potential dermal exposure measurements during silkscreening (KRIOH, 2003)
1 st measurement day
( μ g/cm 2 /h) 2 nd measurement day
( μ g/cm 2 /h)
No of samples 6 10
Average 5.328 a 4.068 b

90 th % 15.684 12.937

a : 3 results were below the limit of detection

b : one result was below the limit of detection
The range of the measured exposure values for the formulation was <0.04-85 mg and the 90 th percentile was 65 mg/day (unpublished data, from the original data).

Modelling
Dermal exposure may occur during mixing, app lication and cleaning ac tivities. SIDS (1996) retained continuous skin contact over the work period (8 hours) and a 1000 cm 2 skin area exposed (a hand and a forearm). Intermittent cont act seems more appropriate for this scenario (as for painting). Assuming non dispersive use, direct handling and intermittent contact, the EASE model estimates a dermal exposure in the range of 0.1-1 mg of product/cm 2 /day. The estimation is made from a formul ation containing up to 35 % (scr een inks) or 20 % (others) of EGBE and an exposed skin surface area of 840 cm 2 (two hands for consistency with other EU occupational risk assessments). This leads to an estimated external dermal exposure of:EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 45 - 29-294 mg/day for silk screening

- 17-168 mg/day for general printing.

Assessment
For general printing, no relevant exposure data are available. The EASE estimate therefore has to be used for risk characterisation. For silk screen printing, a set of 16 potential full shift hand exposure data is available with a 90 th percentile of 65 mg/day of product. The “product” in this case consisted of pr inting ink with up to 10% of EGBE and retarder (up to 100% EGBE) that was used by just two of the printe rs (as part of the tota l ink-system). Assuming that the concentration of EGBE in screen prin ting inks can be up to 35 %, the reasonable worst case exposure level for EGBE in this process would be approximately 23 mg/day. Because the measured values are based on a more than 12 measurements and come from different workplaces, they can be considered sufficiently re presentative for use in risk characterisation. In conclusion, the following range are propos ed for dermal exposure during printing:
- 23 mg/day for silk screening
- 168 mg/day for general printing.
Dermal exposure may be much lower if suitable gloves are worn.

Biomonitoring data

The results of two studies of bi omonitoring performed in the printing industry are presented in table 4.20.
In the study performed by Vincent et al . (1996), EGBE was not the main solvent to which the workers were exposed to. BAA was not detected in the silk screening printing workshops while its concentration was up to 7.1 mg /g creatinine when pads were used.
In the study performed by Kuopio Regiona l Institute of O ccupational Health (KRIOH)(RISKOFDERM, 2003b) and also descri bed above in the paragraph “Dermal exposure – Measurements”, biological monito ring data were report ed. Workers internal exposure was evaluated by urinary excretion of 2-butoxyacetic acid (BAA) in the first measurements day and in the second measurements day; in the first measurements, the inhalation exposure was excluded by using a resp irator and in the second measurements, the dermal exposure was excluded by using prot ective overalls and glove s. The results are reported in the table 4.23b.

Table 4.23b : Workers’internal exposures (Urany buto xyacetic acid (BAA) excretion, μmol/mol creatinine) Urinary butoxyacetic acid (BAA) excretion, μmol/mol creatinine 1 st measurements day 2nd measurements day Before work shift After work shift Before work shift After work shift Average 3.2 3.8 3.5 18.4

90 th % 3.8 5.5 5.5 40.7

Because of many results below the limit of de tection, the correlations based on the results between the actual exposure and B AA excretion are highly inaccurate as it is mentioned in the report KRIOH ( RISKOFDERM, 2003b ).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 46 If a tentative assessment of skin penetration is made in the same conditions that have been presented in scenario 2, biomonitoring data may in this case be entirely explained by inhalation exposure alone. Supposing also a negativ e bias, it is proposed to come back to the low end of the EASE assessment.

• Scenario 3-3 - Cleaning
The NICNAS report (1996) focuses on the use of EGBE in cleaning products in Australia because of the specific concer n raised in this context :
« Due to the large number of cleaning products containing EGBE, a large number of workers may be exposed to the chemical. The main gr oups of workers who handle cleaning products containing EGBE include:
. carpet cleaners;
. contract cleaners;
. food process workers;
. hospital and nursi ng home workers;
. hospitality industry workers;
. householders;
. laundry cleaners and workers;
. mechanics;
. metal workers;
. school and office cleaners; and

. window cleaners
Exposure during cleaning is extremely variable, due to differences in frequency and duration of use, strength of solution used, method of application and precautions taken during use...
The strength of solution used in the cleaning proc ess is generally low as the product is usually diluted substantially before use; for example, most surface cleaners specify a dilution ratio in the range 1:3 to 1:100, depending on the applic ation and the soil load ing. A large proportion (68 %) of cleaning products contain less th an 10 % EGBE (see tabl e 4.24), so the final strength of solution is often less than 1%. A random survey of 20 general surface cleaning products containing <10 % EGBE i ndicated that the d ilution ratio ranged from 1:1 to 1:250, with most ratios in the 1:5 to 1:100 range...
Some products are sold as high level concentrates (>50 % EGBE) which must be diluted with large volumes of water before use. In some cas es, products are diluted with hot water (up to 80°C).

Exposure during dilution

Dilution is often carried out dail y at the beginning of the shift. While the dilution procedure is usually of short duration, the potential expos ure may be greater due to use of higher concentrations of EGBE and the possibility of splashing. Higher exposures may also occur ifEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 47 the product is diluted with hot water as vapour concentrations may be higher and skin absorption facilitated. If dilution is carried out in a confined space or poorly ventilated area, exposure may be increased.

Exposure during application of cleaning solution
A number of different methods are used to ap ply the cleaning solution, for example, washing, wiping, mopping and spraying. Approximately half of the cleaning produc ts were used in spray form, with a small number ma rketed in aerosol spray cans or trigger packs. This method of application will potentially increase bot h dermal and inhalation exposure as the atmospheric concentration of EGBE will be higher and dermal contact will be increased. The potential for exposure may also be increased where heat is appl ied during cleaning, for example, cleaning ovens and hot plates”.

Table 4.24: Main uses of EGBE containi ng cleaners in Australia (NICNAS, 1996) Use Number % of total EGBE %

Min. Max.

Surface cleaner 214 49 0.57 71
Floor stripper 49 11 <1 30.5

Glass/window cleaner 47 10 <1 40
Carpet cleaner 40 9 <1 10-30
Laundry detergent 15 4 <1.5 10-30

Rust remover 11 3 <10 30-60

Oven cleaner 11 3 <1 10-30

Ink/resin remover 9 2 1 10-93

Others 38 9 <10 94

AISE (BP, 2002b) sent recently a questionnaire to their 10 member companies most likely to be using EGBE. Six (belonging to the group of the biggest companies) responded to the request. According to the data collected, th e main uses of EGBE for cleaning are:

- industrial and institutional cleaners such as professional laundry products

- institutional kitchen/floor/window cleaners
- strippers

- other industrial cleaners for the food and beverage industry

- industrial car/truck cleaners.
The concentration of EGBE in these profe ssional products is always below 12 %, and typically not higher than 8 %.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 48 Taking into account the NICNAS da ta together with the information collected in European products registers (table 4.1 an d 4.2), a maximum content of 50 % EGBE in professional cleaners with dilution 1:1 and a task duration of 2 hours will be assumed in this assessment.

Inhalation exposure

Measured data
1) Workplace measurements
Monitoring data reported in NICNAS (1996) fo r cleaning activities ar e summarised in table 4.25.
Table 4.25: Measured atmospheric exposure (TWA) in cleaning activities.

Activity Nb/type of
sampling Mean (ppm) Range (ppm) EGBE % and comments Reference

Window cleaning in hospitals (4 w) <0.2 Cleaner applied in spray, gloves worn Apol and Cone, 1983

Cleaning in food plant (1 w) 1.6 0.3 %, mechanical floor scrubbing, 95 min sampling, gloves, overalls, boots worn Apol and Johnson, 1979

School cleaning (4 w) <0.7 0.25 %, cleaner applied in li q uid and s p ra y for m Rhyder, 1992 Window cleaning Cleaning cars: garage A

garage B

garage C

garage D

Office cleaners 1 : group A

group B 10/p(2 w)

10/p(6 w)

6/p (3 w)

4/p (2 w)

32/p(8 w)

8/p (2 w) 0.5

0.84

<0.1

4.9

0.32

<0.3 <0.1-1.2

<0.1-2.8

2.9-7.3

0.3-0.73 14.4 %, 0.8-5 h exposure, no gloves

worn 21.2 %, 0.3-4 h exposure, no gloves

worn 5.7 %, 0.7-2 h exposure, no gloves worn 21.2 %, 5.3 h exposure, short sleeves, gloves

worn 9.8 %, 15 min exposure, no gloves worn 0.9 %, 15 min exposure, no gloves worn Vincent et al. , 1993

p: personal sampling; a: area monitoring; w: workers.
Note 1: for office cleaners, results are for half-day shift air samples.
2) Experimental data
A series of experiments was conducted in a ro om-size, controlled environment chamber to evaluate a user's inhalation and dermal exposur e to constituents of a household spray cleaner dispensed from either a hand-trigger pump bottle or an aerosol can (Furtaw et al., 1997).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 49 Airborne concentrations and th e amounts transferred to gloves worn by the product user were measured for EGBE at 5 % weight product concentration. The measured air exposure concentrations of EGBE were on average 5.7 ppm; air exposure c oncentrations re sulting from a typical 20-minute usage were well below the OSHA OEL of 25 ppm.

NICNAS estimation
The inhalation exposure during cleaning activ ities was estimated by NICNAS (1996) for various EGBE concentration in products a nd taken up by SIDS (1996). The reasoning and the results are presented below.
In the monitoring data available, TWA air c oncentrations up to 9.7 ppm were obtained for print machine operators using a cleaning solv ent containing 10 – 50 % EGBE (Salisbury and Bennett, 1987) and up to 7.3 ppm for worker s cleaning car windows with a 21.2 % solution applied as a spray (Vincent et al. , 1993). Based on this data, th e EGBE air concentration selected for worst-case estimates for a 30 % cleaning solution was 10 ppm TWA.
For cleaning solutions containing <1% EGBE, th e atmospheric concentra tions were generally below the limit of detection; however, a persona l monitoring reading of 1.6 ppm was obtained during floor scrubbing with a 0.3 % solution (A pol and Johnson, 1979). Consequently, an air concentration of 2 ppm was selected for 0.1 and 1 % cleaning solutions.

Table 4.26: Exposure estimates for u se of cleaning products (SIDS, 1996) % EGBE Max. exposure (ppm)

0.1 2

1.0 2

10 4

30 10

Modelled data
Several parameters should be chosen to correspo nd to the real variability of conditions of use of cleaning products:

- wide dispersive use, low tendency to beco me airborne, direct handling would give 200- 300 ppm,

- wide dispersive use, low tendency to beco me airborne, direct handling and dilution ventilation, would give an estimation of 100-140 ppm.
Assuming the exposure would occur 2 hours a day, th is leads to exposure levels of 50-75 ppm or 25-35 ppm, averaged on a shift duration.
The model overestimates exposure levels, partic ularly because of non-consideration of the content of EGBE in the products. The estimates cannot be corrected for the partial vapour pressure because the composition of the formul ations is not known. A simple approach based on a reduction of the exposure by a factor equi valent to the EGBE concentration in the mixture (25 % if the estimation is made for a formulation containing up to 50 % of EGBEEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 50 with dilution 1:1) would lead to exposure levels of 12.5-18.7 ppm or 1-8.7 ppm. However the validity of these estimates is rather questionable.

Summary/statement of the exposure level
Only limited measured exposure data are availabl e and they are difficult to analyse because of the great variability of exposure conditions (in particular duration and content in the product).
The highest exposure of 7.3 ppm measured for workers cleaning cars with a 21.2 % solution may be considered as a worst case for this scenario. Therefore it seems cautious to keep the NICNAS estimation of 10 ppm (49 mg/m 3 ).

Dermal exposure

Measured data
A series of experiments was conducted in a ro om-size, controlled environment chamber to evaluate a user's inhalation and dermal exposur e to constituents of a household spray cleaner dispensed from either a hand-trigger pump bottle or an aerosol can (Furtaw et al., 1997). Approximately 10 % of the amount of product used was transferred to the user's gloves (simulating dermal exposure), with the great majority found on the palms rather than the backs of the hands (88 % according to data presented). Using results presented, skin contamination can be evaluated (for both hands) at 0.22 mg/cm2/day for a 5 % solution. Note also that the right hand is roughly 4 times more contaminated than the left one (right-handed worker).

In a study performed by TNO (RISKOFDERM, 2003a ), a part of the RISKOFDERM project, potential hand exposure to an analogous but less volatile glycol ether DEGBE (2-(2- butoxyethoxy)ethanol) was measur ed during spraying of a diluted cleaning agent (sampling duration between 6 and 18 min) and during wipi ng (car washing, sampling duration between 5 and 12 min and cumulative duration of wiping per day 24-90 min). For spraying, the measurements were made using butyl rubber protective (recommended by the employers), the in-use concentration was between 0.0007 and 1.1 % of DEGBE. For wiping, hand exposure was evaluated by the hand wash method, the in-use concentration was between 0.04 and 0.82 %. Exposure was mainly due to exposure on the hands . Results (given in product as well as recalculated to in-use produc t) are presented table 4.27. They show a substantially higher exposure rate during wiping activities.

Table 4.27: Dermal exposure rates during spraying and wiping (RISKOFDERM, 2003b) Hands exposure Range (mg) AM (mg) GM (mg) GSD (mg) AM ( μ g/cm 2 /min)* GM ( μ g/cm 2 /min)*
Spraying (n=12)

- DEGBE

- product 36-6302 a

37-1974 1000 a

719 438 a

522 3.73

2.72 0.15

68.4 0.04

48.1
Wiping (n=12)

- DEGBE

- product 1.8-29.8

1508-4861 10

3161 6.8

2985 2.50

1.45 1.8

535 1.1

499

Nota a : these values are in μ gEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 51 * Asurface area of 820 cm 2 was assumed for the hands

The 90 th percentiles (expressed in mg of total diluted product) fo r the sampling durations were 1000 mg for spray application and 4000 mg for car cleaning (unpublished data, derived from original data).

Modelled data
Dermal exposure is clearly predominant duri ng cleaning activities. SIDS (1996) retained continuous skin contact over the work period (8 hours) and a 1000 cm 2 skin surface area exposed (a hand and a forearm). Intermittent cont act seems more appropriate for this scenario because the duration will generally be a part of the shift and continuous contact would probably overestimate the exposure.
Assuming wide dispersive use, direct handl ing and intermittent contact, the EASE model estimates a dermal exposure in th e range of 1-5 mg of product/cm 2 /day. The worst case estimation is made for a formulation containing up to 50 % of EGBE with dilution 1:1 and a 420 cm 2 skin surface area exposed (one hand). This leads to an estimated external dermal exposure of 105 - 525 mg/day for cleaning.

Assessment
The results of the TNO study during spraying and wiping indicate relatively high potential for dermal exposure. However interpretation is difficult due to the relative small data set of measurements and the fact that during spraying th e use of sample gloves may lead to different exposure levels than direct cont act to the skin. Furthermore, EG BE is much more volatile than DEGBE and this may lead to higher evaporatio n from the skin and lower effective exposure levels. Also, for tasks with cumulative expos ure durations substant ially longer than the sampling durations, extrapolation is difficult due to the effects of evaporation as well as the possibility of saturation of the sk in with product, specifically for situations with direct contact and immersion, such as cleaning with a sponge or cloth. On the other hand, the kind of tasks performed with these produc ts will often not be done for many hours per day.
Although the measured data are difficult to inte rpret, they provide much more real exposure information than the EASE model, whose dermal exposure part is not based on real dermal exposure measurements. A rough estimator of reasonable worst case de rmal exposure (to the hands) is therefore assumed to be the 90 th percentile of the exposure levels during the measurement period. This appears to be reasonable, because the measurem ent periods were aimed at a covering one full “cycle” of the relevant activit ies and they were followed by pe riods without dermal exposure, when evaporation and other effects may lower the skin contamination levels before a new “cycle” will again contaminate the skin. Reas onable worst case exposure levels for hand exposure to diluted cleaning produc ts containing up to 25% of EGBE are therefore expected to be:

- spray application: 250 mg/day;

- manual application including immersion of the hands into the solution: 1000 mg/day.
The uncertainty in these values is rather large, because of the aspects mentioned earlier. This should be taken into account in the eval uation of the MOS. The estimate for manual application is substantially higher than that of EASE, while the estimate for spray applicationEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 52 is similar to that of EASE. However, the m easured data are preferred. Dermal exposure may be much lower if suitable gloves are worn.

Biological monitoring data
A study was conducted in France by INRS to eval uate the occupational exposure to EGBE of workers using window cleaning agents: 10 clea ning women working in a town hall and 13 automobile cleaners worki ng in 4 garages (Vincent et al., 1993). EGBE was de tected in all the window cleaning agents (0.9 to 21.2 %). The results of exposures by job category are presented in table 4.28.

Table 4.28: Characterisation of exposure by job category (Vincent et al. , 1993) Air concentration of EGBE (ppm) Pre-shift urinary concentration of BAA (mg/g creatinine) End-shift urinary concentration of BAA (mg/g creatinine) Job category

No of samples Mean Range No of samples Mean Range No of samples Mean Range

Cleaner of new

cars 15 2.33 <0.10- 7.33 14 17.9 <2-98.6 12 111.3 12.7- 371

Cleaner of used

cars 15 0.36 <0.10- 1.52 12 4.8 <2-33 11 6.3 <2-24.4

Office cleaner 32 0.32 <0.30- 0.73 32 2.1 <2-4.6 32 2.1 2-3.3

The authors noted that the high concentrations of urinary BAA relative to the EGBE TWA concentrations, indicated that dermal abso rption was the predominant route of worker exposure. They concluded that for workers in these occupations not wearing protective gloves, environmental sampling probably underestimates the exposure, and that biomonitoring of urinary BAA appears to be th e best method for estimating overall exposures.
To indirectly assess skin exposure using thes e data as already proposed in the preceding scenarios, with the same limitations, it is seen that highest results are also obtained with peak value measurements in new car cleaning activity (272.4 mg/g creatinine and 7.3 ppm inhalation concentration), which gives a dermal equivalent of 240 mg/day, corresponding to a dermal load of 800 mg/day, assuming 30% ab sorption efficiency. For the same reasons already discussed in scenario 2, a negative bias is also assumed as 30%, which gives a dermal load re-assessed as ca. 1040 mg/day.

Rettenmeier et al. (1993) reported biol ogical monitoring data on car cleaners. Total BAA (free BAA and BAA glutamine conjugate) in 6 post-shift samples aver aged 243 (range 67 to 935) mg/g creatinine (assuming an average urin ary creatinine concentration of 1.25g/l). No air monitoring was gathered in this study, which does not allow for anindirect evaluation of skin penetration.

• Data from other uses (miscellaneous)EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 53 Exposure of 13 workers using cutting oils containing EGBE (1 % to 5 %) in a mechanical workshop was measured by biological monitorin g. No atmospheric measurements were made in this case. Concentrat ions of BAA in post-shift urine varied from <2 to 8.3 mg /g creatinine. This result reflects a low exposure (Vincent et al. , 1996).
Exposure of 53 hairdressers working in 10 shops was evaluated. The composition of 43 products was analysed, EGBE was detected in 8 products (0.5 to 5 %). No solvent was detected in the atmosphere and BAA in the post-shift urine was <2 mg/g creatinine (detection limit). This result was probably related to the very low quantities of products handled and the use of gloves (Vincent et al. , 1996).
Seven personal exposure measurements made du ring removal of asbestos-containing mastic result in EGBE concentrations in the range fr om 2 to 22 ppm with a m ean of 12 ppm (Kelly, 1993 quoted in ATSDR, 1998).

4.1.1.2.4 Summary of occupational exposure

Differences in assessments of skin exposure are striking. One could a priori consider that the work undertaken within the “R ISKOFDERM” project should be given priority, since it is recent and has explored a diversity of real working conditions. In fact, this project has objectified the great difficulties of this exercise, leading Kromhout et al . (2004) to conclude: “For the time being, actual dermal exposure measurements and a better understanding of actual determinants of dermal exposure seem to be a necessity in orde r to evaluate dermal exposure hazards properly.” Impor tant characteristics that may be retained are the extreme variability of skin exposure (e.g. “geometric mean levels of dermal exposure range over up to 4 orders of magnitude between dermal exposur e operation units when exposure is expressed in milligrams.”) and the predominance of hand e xposure in very different tasks (e.g. Eriksson and Wiklund, 2004 ; Eriksson et al ., 2004 ; Fransman et al ., 2004 ; Gijsbers et al ., 2004 ; Roff et al ., 2004a and 2004b). In the present situation, skin exposure may also be indirectly assessed in real situations with EGBE itself (and not a parent compound with very different physico-chemi cal properties), so these data should also be considered as reliable although somewhat different. To take these 2 sources into ac count, it is proposed to take the available RISKOFDERM data as a basis and to adapt them to EGBE using bi omonitoring data when a pplicable. This is done in the following table 4.29 (last column) where a factor of 4 has b een applied to the biomonitoring data when already exposed reasons show that the RISKOFDERM results may not be directly used.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 54 Table 4.29: Summary of proposed reasonable worst case exposures.

Scenario 8-hour TWA inhalation (mg/m 3 ) Dermal worst case derived from biomonitoring data (mg/day) External dermal (mg/day) Dermal worst case, EASE & RISKOFDERM

& biomonit. Retained values for risk characterisation (mg/day) 1 - Manufacture 12 No data 42 b 42 2 - Formulation 15.7 500 11,600 c 2,000 e 3 - Use of products 3.1.1.1 - Coating/Painting – industrial: spraying 58.1 2,000 c 2,000 3.1.1.2 - Coating/Painting – industrial: other works 30.4 430 (metal painting) 240 c 430

3.1.2 - Coating/Paintingdecorative 30.4 70 36 c 70

3.2.1 – Printing - silk screening 20 Negligible 23 b 23 3.2.2 – Printing - general printing 5 Negligible d 168 b 168 3.3.1 – Cleaning - spraying 49 250 c 250 3.3.2 – Cleaning - wiping 49 1,040 1,000 c 1,040

Nota b : modelled data from EASE Nota c : data derived from measuremen ts with a less volat ile solvent (DEGBE)
Nota d: in this case, due to a probable negative bi as (see text), it is pr oposed to apply the low end of the EASE assessment.
Nota e: see explana tions in the § 4.1.1.2.2 – Assessment

4.1.1.3 Consumer exposure

4.1.1.3.1 Exposure from uses

EGBE is used as a solvent in many products available for consumers as, paints, paints thinners and cleaning products. EGBE is no more used in cosmetics.
Cleaning products which contain EGBE include general surface cleaners, floor strippers, window cleaners, carpet cleaners, spot cleaners, rust removers , oven and grease trap cleaners, laundry detergent, cars cleaners, bathrooms a nd toilets cleaners and disinfectants, ink and resin removers.

Paints that contain EGBE are both waterborne paints and solvent-borne paints (CEPE enquiry, 2002) including varnishes and products to preserve wood.
An investigation was made by the journal "Que choisir" in 2001 showing that two paints contained 1 % of EGBE and that in cleaners for cars, the concentrat ions of EGBE ranged from 0.3 to 4 %.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 55 Another source of exposure is linked to the stopp ers of bottles used for perfusion in medicine which are made with natural or artificial rubbers. Chemicals used in rubber formulations can leach from rubber stoppers during sterilisation and enter the bloodstream of patients when the perfusion is administered. The concentratio ns of leached stoppers components found in parenteral solutions concentrations were m easured by GC/MS after extraction by methylene chloride. Two parenteral solutions were tested containing 5 % dextrose and one containing 5 % sodium bicarbonate. Results indicate a mean release of 68 μg/150 ml EGBE in dextrose solutions (respectively 70 and 65 μg/150 ml for each sample) and a release of 13 μg/500 ml EGBE in the sodium bicarbonate solution. The si ze of the sampling is to o low to allow a good estimation of the real contamin ation of perfusion solutions by EGBE (Danielson, 1992). This scenario will not be used in the risk characteri sation as it is of concern for a few people and it is not a frequent event to th e single consumer. The consumer is exposed by this way only if two conditions are simultaneous: he needs a perfusion and the rubberstopper contains EGBE. By another way, the number of ev aluations of the release of EG BE from rubberstoppers is too weak to allow a good estimation of the exposure.

Scenario 1: Household surface cleaners
Two routes of exposures have to be consider ed during the use of household surface cleaners: inhalation and dermal exposure.
Cleaning frequency and duration we re assessed by a questionnaire applied to 342 subjects (75 % of women), 18 to 80 years old (Kovacs et al., 1997). Subjects report ed a longer duration for kitchen cleaning task that for the bathroom, with mean values of 37.7 and 30.7 mn respectively. (t = 3.67; p<0.0005). Depending on th e frequency of cleaning in a week, the duration of exposure to household surface cleaners can be estimated to mean values of 98 and 59 mn/week spent cleaning kitchen and ba throom respectively. When broken down by gender, the mean values for the kitchen are respectively 105 and 78 mn/week for women and men and, for the bathroom cleani ng, 64 and 44 mn/week respectively.
In the Technical Guidance Document, data fro m AISE (2002) are provided for the use of surface cleaners : the highest quantity of liquid surface cleaner used is 110 g/task when it is diluted in 5 l of wash water volume and the longest duration of exposure is 20 minutes. For sprays, the highest quantity of liqui d used is 30 g/task during 10 minutes.

Five tests were conducted in a 24.5 m 3 , controlled-environment chamber to evaluate a user's inhalation and dermal exposure to constituents of a household spray cleaner dispensed from either a hand-trigger pump bottle or an aerosol can (Furtaw et al., 1997). The temperature of the room was 23 °C, the humidity was monitored but not controlled, outdoo r air exchange rate was set at approximately 0.5 air changes per hour. The cleaning product contained 5 % EGBE. Subjects were asked to clean two "Formica" sheets (approximately 0.76 m x 1.83 m), one being placed horizontally in the room, the ot her being vertical on the wall. The cleaning process, which consisted on spraying the cl eaning product on the "Formica" surface then wiping it with a cotton cloth continued appr oximately for 20 minutes. About 28 g of the product was used during the cleaning process. Fi ve tests were performed. During the cleaning period, air from the breathing zone of the experi menter and air in a corner of the room were sampled on a charcoal tubes (type ORBO TM -32) and analysed on an automated gas chromatograph (Microsensor systems Inc. Model MSI-301). During the cleaning, the experimenter wore cotton gloves over PVC glove s. The purpose of the cotton gloves was to absorb cleaning solution th at would have been in contact with the hands of the person if he had not been wearing gloves. The amount of EG BE transferred to the gloves is about 6.3 % ±EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 56 3.8 % of the quantity used on the right hand and1.6% ± 3.0 % on the left one and a greater amount is found on the frontal area of the hand than on the back of the hand. The mass and size of particles emitted decay much faster than the air EGBE concentration in the room that suggests that EGBE is being reversibly abso rbed and re-emitted from chamber surfaces. Median diameters of partic les vary from 0.749 to 0.976 μm. The personal average ORBO TM

tube result was 5.70 ppm.

The interactions between EGBE and indoor ma terials were studied by placing the indoor material as a membrane between two air compartments. Both compartments are constantly flushed with air, the airflow containing EGBE leading to the primary cell, the other airflow with clean air entering the secondary cell. A permeation factor was defined as: PF(%) = C A x 100/C B where C A is the outlet concentration of the secondary cell and C B the outlet concentration of the primary cell. Averag e inlet concentration of EGBE was 483.7 μg/m 3 with a standard deviation of 2.2 μg/m 3 . PF values were 88.1 % a nd 9.4 % for wallpapers, 70.4 % for acrylic wall-covering, 61.6 % for carpets. Th e capacity of retention of each material was also calculated. It varies from 5.3 to 33.3 % (mean value = 19.2 %; SD = 9.8 %) according to the category of wall paper (n=6), 3.1 % for the textile wall-covering (n=1), 82.9 % for the carpet (n=1), 37.4 for the chipboa rd (n=1), 12.1 for the PVC flooring (n=1) and 80.4 % for the gypsum board (n=1). All those materials can re lease EGBE after the us e of cleaning products (Meininghaus and Knoppel

, 1999).
NICNAS (1996), using data av ailable from monitoring of workers using window cleaners (n = 23) or surface cleaners (n = 19), estim ates that a good worst-case estimate for concentrations in air during th e use of cleaning solutions is:
- 10 ppm (49 mg/m 3 ) for a 30 % cleaning solution
- 2 ppm (9.8 mg/m 3 ) for a 0.1 or 1 % cleaning solution
- 4 ppm (19.6 mg/m 3 ) for a 10 % cleaning solution
OECD (1997) estimated the exposure of c onsumers by cleaning products to 0.58 mg/kg/event assuming a respiratory rate of 0.8 m 3 /hour and a bodyweight of 60 kg for a 1 hour cleaning event. For dermal absorption, a sk in absorption rate of 0.2 mg/cm 2 /hour and a skin surface of 1,000 cm 2 were assumed.

Zhu et al. (2001) made the determination of EGBE emissions from selected consumer products and made assessment of the inhalation exposure associated with cleaning tasks. Two all-purposes cleaners were tested and two spray glass cleaners. Concentrations of the products ranged from 0.744 % to 1.28 % for general-purpos es cleaners, from 0.50 to 0.87 for glass cleaners. A standard room of volume 17.4 m 3 was used for the test. An average air exchange of 0.5 h -1 was applied to the standard room. It was assumed that fo r all-purpose spray-cleaners a mass of 76 g of product was applied on a surface of 4.5 m 2 for each task whereas a mass of 17 g of spray glass cleaners was applied to a surface of 2.3 m 2 for each task. The test included four various tasks for general-purposes cleaner s: clean outside of cab inets during 0.87 hours, clean counters during 0.42 hours, clean bathroom or other tiled or ceramic walls during 0.57 hours, clean outside of refriger ator and other appliances during 0.32 hours. Two tasks were assessed for glass cleaners: cl ean inside of windows during 2.12 hours, clean other glass surfaces like mirrors or tables during 0.40 hours. Samples of air in the chamber were collected every half hour or every hour or every 2 hour s depending on the stage of the emission. An inhalation rate of 1.3 m 3 /h and a mean bodyweight of 70.9 kg were used for exposure assessment. The daily exposure by task range d from 0.013 to 0.063 mg/kg bw/d if only one of the tasks were performed in one day (mean value = 0.0325mg/kg bw/d; standard deviation =EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 57 0.017 mg/kg bw/d). If they were all performed on the same day, the sum of daily intake by inhalation is 0.186 mg/kg bw/d with the first pr oduct and 0.075 mg/kg bw /d with the second. In the case of glass cleaners, the daily e xposure by task ranged from 0.001 to 0.004 mg/kg bw/d if only one of the task s were performed in one day (mean value = 0.003 mg/kg bw/d; standard deviation = 0.001 mg/kg bw/d). If they were all performed on the same day, the sum of daily intake by inhalation is 0.006 mg/kg bw/d with the first product and 0.004 mg/kg bw/d with the second.

Fortmann et al. (1999) performed tests to determine gas-phase and particulate emissions during application of a water-based cleaner wi th a hand-pump sprayer. Tests were performed in the bathroom of a test house and in an 18m 3 controlled-environment test-room. The waterbase cleaner which contained 50 mg/g of EGBE was applied to real istic surfaces (counter tops, glass, mirrors) with the manufacturer 's hand-pump spray. During application, air samples were collected on Tenax for an alysis of EGBE by gas chromatography. Concentrations of droplets were measured co ntinuously with aerodynamic particle analysers. The average concentration of EGBE measured by collection on Tenax for a 10-minute period in a closed bathroom during and following appli cation of 96 g of a 50/ 1 (v/v) dilution of the cleaner was 760 μg/m 3 . The mean particle droplet size during the application period was in the range of 1 to 2 μm. Particle concentrations increased during application with the sprayer but dropped quickly.

Table 4.30: summary of studies about exposure by household cleaning products Study Route of
exposure Concentration of EGBE in product Duration of
exposure number of
tests Concentration Daily exposure

Furtaw et al. , 1997 Inhalation 5 % 20 min n = 5 5.7 ppm (28 mg/m 3 ) in air

Furtaw et al. , 1997 Skin 5 % 20 min n = 5 6.3 ± 3.0 % (right hand)

1.6 ± 3.0 % (left hand)
Meininghaus and Knoppel, 1999 Inhalation 0.48 mg/m 3 in air

30 % 10 ppm (49 mg/m 3 ) in air
10 % 4 ppm (19.6 mg/m 3 ) in air NICNAS, 1996 Inhalation

0.1 to 1 % n = 42

2 ppm (9.8 mg/m 3 ) in air

OECD, 1997 Inhalation + skin 1 hour modelled data 0.58 mg/kg/event

Zhu, 2001 Inhalation 0.74 – 1.28 %

0.5 – 0.87 % 0.32-2.18 hr

0.4 – 2.52hr n = 2

n = 2 2.8 – 62 mg/m 3 0.013-0.063mg/kg/day
0.001-0.004mg/kg/day

Fortmann et al. , 1999 Inhalation 0.1 % 10 min n = 5 0.76 mg/m 3 in airEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 58 The risk assessment will be based on data from NICNAS (1996) and AISE (2002) for inhalation exposure, assuming a cleaning product containing 10 % EGBE leading to an air concentration of 19.6 mg/m 3 and a duration of exposure of 20 minutes (1/3 hour)for each event. The concentration of 10% will be chosen as a reasonable worst case as according to the Danish product register ( 2001) (cf table 4.1), products c ontaining 10 % EGBE or less represents 76 % of the whole market of produc ts containing EGBE (93 % contain 20 % or less EGBE). According to the french register SEPIA from INRS (2003), in 81 % of French cleaning products containing EGBE, its concentra tion is equal or less than 10 % (93% contain 20 % or less EGBE). These data are furnished without any distinction between products sold to general consumers and these sold in the o ccupational field and gene rally, cleaning products sold in the occupational field are more concentrat ed in cleaning substances than those sold to consumers are. Data indicates that the great majority of cleaning products available to consumers contain less than 10% EGBE. It ca nnot be excluded that some products may be available with higher levels. However, anecdotal evidence s uggests that such products are either used in small amounts (e .g. spot cleaners) or are diluted before use. Combined with other evidence to indicate a dec line in use in this application, a figure of 10% is set as a reasonable worse case for the composition of EG BE in consumer cleaning products for the scenarios modelled. The worst case consider ed will be the use of undiluted product.
For inhalation, based on a 20 m 3 respiratory volume a day for an adult weighing 60 kg, the exposure will be:

20 x 19.6 x 1

60 x 24 x 3 = 0.09 mg/kg/d

For skin contact, measured data from Furtaw et al. (1997) will be used to characterise each event : use of 28 g of undiluted product for a 20 minutes event (AISE, 2002) is considered as the worst case, transfer of 6.3 + 3.8 % of the quantity used on the right hand and of 1.6 + 3.0 % to the left hand. For a 10 % EGBE pr oduct, the dermal exposure will be:
0.1 x 28 x 1000 x (0.10 + 0.05)

60 = 7 mg/kg/d

Scenario 2: Measurements in indoor air
Measurements of EGBE concentrations were pe rformed in the indoor ai r of flats (Kirchner, 2002). Preliminary results indicate that the 90 th percentile is 3 μg/m 3 in the sleeping-room with a high value of 14 μg/m 3 in a flat. The 90 th percentile is 3.5 μg/m 3 in the kitchen with a high value of 24 μg/m 3 in a flat. Releases from buildi ng materials were also evaluated. Simulations were made by installing new carpets or new floor coverings or by applying paints on the walls of reference rooms. After 24 hours , the concentrations of EGBE in air ranged from below the detection limit to 3.8 μg/m 3 (mean value = 1.7 μg/m 3 ; SD = 1.5 μg/m 3 ) when release from carpets were tested. After 28 days , it ranged from non de tectable to 23.3 μg/m 3 (mean value = 6.1 μg/m 3 ; SD = 9.0 μg/m 3 ). With other categories of floor coverings, it ranged from non detectable to 5.6 μg/m 3 (mean value = 2.5 μg/m 3 ; SD = 2.7 μg/m 3 ) after 24 hours and from non detectable to 59.6 μg/m 3 (mean value = 27.5 μg/m 3 ; SD = 30.9 μg/m 3 ) after 28EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 59 days. Release by paints applied on the wall were at non detectable values after 24 hours and reached 24 and 298 μg/m 3 respectively for the two paints te sted 28 days after application.
The same type of measurements was performed in apartments in Italy: in five of them, EGBE was not detected, in a sixth, it was found at a concentration of 8 μg/m 3 (De Bortoli et al., 1986).
A study of the daily indoor concen trations for all indoor site type s (n = 14) gave the following results: average air pollution by EG BE = 0.214 ppb (1 μg/m 3 ); median concentration = 0.075 ppb (0.4 μg/m 3 ); 25 th percentile = 0.030 ppb (0.1 μg/m 3 ); 75 th percentile = 0.360 ppb (1.7 μg/m 3 ) (Shah and Singh, 1988).
According to data furnished by the FIPEC and SIGMAKALOM (Conseil supérieur d'hygiène publique de France (CSHPF), 2001), consumers shoul d inhale 1 g of EGBE during a period of 30 days.

Table 4.31: summary of studies about exposure by indoor air
Study Concentrations in air Number of
measurements Mean values Reasonable Worst
case exposure Bedroom : 3 μg/m 3* (highest value : 14 μg/m 3 in 1 flat) Kitchen : 3.5 μg/m 3 (highest value : 24 μg/m 3 in 1 flat) n = 57 24 μg/m 3 in 1 flat)

Release from carpets, 0- 28days : non detectable to 23.3 μg/m 3 n = 6 24 hours after : 1.7 μg/m 3 28 days after : 6.1 μg/m 3 23.3 μg/m 3

Release from new floor covering, 0-28days : non detectable to 59.6 μg/m 3 n = 5 24 hours after : 2.5 μg/m 3 28 days after : 27.5 μg/m 3 59.6 μg/m 3 Kirchner

(2002)

Release from paints, 0- 28days : non detectable to 298 μg/m 3 n = 2 298 μg/m 3

De Bortoli et

al. (1986) 8 μg/m 3 (Found in only 1 out of 6 flats) surveillance in 6 flats during

1983 et the beginning of 1984 n = ? 8 μg/m 3

Shah and Singh (1988) 1.7 μg/m 3 ** n = 14 1 μg/m 3 1.7 μg/m 3

CSHPF (2001) 1g/30 days modelled data by WPEM*** 1g/30 days

* 90 th percentile ** 75 th percentile *** WPEM = Wall Paint Exposure assessment developed by US EPAEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 60 As a worst case approach, the scenario chosen will be: rooms with paints releasing 298 μg/m 3 of EGBE and with a new covering floor releasing 59.6 μg/m 3 . For this scenario, the only exposure is by inhalation of indoor air. For inhalation; based on a 20 m 3 respiratory volume a day for an adult weighing 60 kg and a 20 hours duration of exposure, the exposure will be:
20 x (59 + 298) x 20

60 x 24 = 0.1 mg/kg/d

Scenario 3: Painting

Studies have been made to evaluate indoor concentrations of EGBE from paints.

Norbäck et al (1995 and 1996) measured painters exposur e to EGBE by sampling air in their breathing zone for one hour as they were appl ying various water-based paints: wall paints, ceiling paints, wood paints and wet room paints (N = 20 samples). All paints were applied by roller except one case of wall paint that was sp rayed. Most of the measurements (90 %) were performed during renovation of old buildings . The average room temperature was 19.3 °C (17.5-20.5 °C) and the relative air humidity ranged from 34 % to 83 % (arithmetic mean value = 58 %). EGBE was detected during painting of walls and wet room painting. The arithmetic mean for EGBE is a concentration of 59.6 μg/m 3 , the geometric mean is 8 μg/m 3 (geometric standard deviation = 4.2 μg/m 3 ) with a highest value of 730 μg/m 3

. Hansen et al. (1987) measured volatiles emitted during the use of waterborne paints in one room during 3 days. 13 samplings were perfor med by adsorbing the organic vapours present in the ambient air.. The organic vapours were adsorbed on polymer in cartridges of stainless steel (Tenax AT, 60-80 mesh). The samp ling time was 20 minutes corresponding to a sampling volume of 0.9 l. Samples were anal ysed by gas chromatography using a flameionisation detector. The content of EGBE in the paints ranged from 0 to 1.4 % w/w and the concentration in air of the wo rkplaces ranged from 2 to 60 mg/m 3

. OECD (1997) estimated the exposure of cons umers by paints and surface coatings to 1.05 mg/kg/event assuming a re spiratory rate of 0.8 m 3 /hour and a bodyweight of 60 kg for a 6 hour painting period. For dermal absorption, a skin absorption rate of 0.2 mg/cm 2 /hour and a skin surface of 1,000 m 2 were assumed.
EPA (2001) studied the emissions of EGBE from paints app lied on two types of surfaces: stainless steel plate and gypsum boa rd. The concentrations of EGBE in the paints ranged from 4.68 to 5.20 mg/g (mean value = 4.98, n = 4). Peak concentrations measured in the air of the environment chambers were higher when the surface is a stainless steel plate (11.6 mg/m 3 ) than on gypsum board (5.14 mg/m 3 ). 93 % of the EGBE is emitted during the 336-hour testing period when applied on stainless st eel plate, 14 % when painting on gypsum board. The study was prolonged with gyp sum board. After a rapid decrease in concentrations of EGBE in the environment chambers, levels of EGBE in the air remain at values near 0.01 mg/m 3 after 3 months.
CEPE (2002) considers that a 3% concentration in paints can be cons idered as a reasonable worst case for decorative paints based on data gathered from their member companies (see table 4.14).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 61 Table 4.32: Summary of studies about exposure by paints Study Average
exposure Reasonable Worst
case exposure number of
measurements Comment

Norback, 1995-1996 59.6 μg/m 3 730 μg/m 3 N = 20 Hansen et al. , 1987 2-60 mg/m 3 N = 13 EGBE content in paint 0-1.4 % OECD, 1997 1.05 mg/kg/event modelled data 6 hours painting period

5.14 mg/m 3 to 11.6 mg/m 3 11.6 mg/m 3 N = 4 2 weeks. Non adsorbent surface. Max EGBE content in paint 0.52 % EPA, 2001

0.01 mg/m 3 0.01 mg/m 3 value 3 months after painting CEPE, 2002 EGBE content in paint 3 %

The CONSEXPO model can also be used to es timate exposure. Another alternative model is the USEPA Wallpaint model. The parameters us ed in both models are shown in Appendix A. The calculated inhalation exposure results are :

CONSEXPO (worst case inhalation exposure) WPEM inhalation exposure

Exposure per event (over 133 minutes) 377 mg/m 3 144 mg/m 3 instantaneous (8 hr TWA = 27 mg/m 3 ) Calculated external dose per event (60 kg/person) 11.6 mg/kg/d Based on TWA : 3mg/kg/d Model prediction : 19.8 mg/kg/d

To verify if this value can be considered as realistic, the same calculation was performed with a 1.5 % concentration of EGBE in paints to co mpare with the value ob tained with measured data. A reasonable worst case inhalation exposure can be based on da ta from Hansen et al. (1987) for a 1.5% concentration of EGBE as the data were collected in real situ ations (renovation of old apartments in a building) and frequent sa mplings were performed during a 3 days work. Assuming a respiratory rate of 20 m 3 /day, a 6 hours event in one day and a mean bodyweight of 60 kg, the inhalation e xposure by paints is:

20 x 60 x 6

60 x 24 = 5 mg/kg/d

Modelling the exposure according to the T echnical Guidance Document, the inhalation exposure linked to the use of a 1.5% EGBE pa int should be: 64.2 mg/kg/d. This shows that in this case, the modelling strongly overestima tes the inhalation exposure (factor = 12.84).

For CONSEXPO and WPEM mode ls, calculations for a paint containing 1.5 % EGBE give the following results :EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 62 CONSEXPO (worst case inhalation exposure) WPEM inhalation exposure

Exposure per event (over 133 minutes) 188.5 mg/m 3 72 mg/m 3 instantaneous (8 hr TWA = 27 mg/m 3 ) Calculated external dose per event (60 kg/person) 5.8 mg/kg/d Based on TWA : 1.5 mg/kg/d Model prediction : 9.9 mg/kg/d Comparison with measured data 116 % of measured data 198 % of measured data

As all models give an overestimation of consumer exposures, we have chosen to consider that a 3 % EGBE concentration in paint leads to a co ncentration of EGBE in air of 120 mg/m 3 (60 mg/m 3 x 2 – 20 min TWA) and an external body burden exposure by i nhalation of : 10 mg/kg/d.

This value will be used as the worst case for inhalation exposure in this scenario.

For dermal exposure, the modelling proposed in the Technical Guidance document will be used. So the external exposure (E ed ) of skin to EGBE from paints will be:

C prod x TH der x AREA der x n E ed = D x BW

where : C prod = concentration of EGBE in paint (kg/m 3 ) TH der = thickness of product layer on skin AREA der = area of contact be tween paint and skin n = number of events (n = 1) D = dilution. There is no dilution so D = 1 BW = mean bodyweight for a consumer (60 kg)

Assuming the concentration of EGBE in the paint is 3 % i.e. 30 kg/m 3 (CEPE, 2002), the surface of the skin exposed is 840 cm 2 (840 x 10 –4 m 2 ), the thickness of paint layer on the skin is 10 -4 m, the external exposure of skin by paints will be:

30 x 10 -4 x 840 x 10 -4 x 1 E e = 1 x 60 = 4.2 mg/kg/d

4.1.1.3.2 Summary of consumer exposure

Table 4.33: Summary of proposed “reasonable worst-case” exposures in the main scenarios Scenario Inhalation (mg/kg/d) Skin (mg/kg/d)

1 – Household surface cleaners 0.09 7

2 – Indoor air 0.1
3 – Painting 10 4.2EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 63 For risk characterisation, inte rnal exposures will be estimated for each scenario.
As a consumer is also exposed to indoor air wh en he paints, or uses household cleaners, we will also include in the risk characterisation, the scenarios:

- household surface cleaners + indoor air
- paints + indoor air

Table 4.34: Summary of combined worst case scenarios Scenario Inhalation (mg/kg/d) Skin (mg/kg/d)

Household surface cleaners + indoor air 0.19 7
Paintings + indoor air 10.1 4.2

4.1.1.4 Humans exposed via the environment
The information relating to th e estimation of the indirect exposure of humans via the environment are presented in table 1. The conc entrations calculated in intake media (drinking water, fish, plant roots and leaves, milk, meat , air) and the subsequent estimation of human intakes via different routes ar e shown hereafter with the corre sponding total da ily intakes. Both local and regional levels are taken into consideration and the estimation of local environmental exposures has been performed for all scenarios listed in chapter 3.1.2.2. Concerning the production step, on ly the worst case has been re ported. Calculations have been performed using default parameters in EU SES except for the use of an absorption factor via inhalation of 60% and a body weight of 60kg.EURISK ASSESSMENT–2-BUTOXYETHANOLCAS111-76-2 CHAPTER4.HUMAN HEALTHRAPPORTEURFRANCER408_0808_HH_CLEAN64Table 4.1: exposure assessment for human via the environment (concentrations in foodstuff, airand drinking water and connecteddaily doses)Conc. in drinkingwater (mg.L-1) /Subsequent dailydose (mg.kg-1.d-1)Conc. in wet fish(mg.kg-1) /Subsequent dailydose (mg.kg-1.d-1)Conc. in plantroots (mg.kg-1) /Subsequent dailydose (mg.kg-1.d-1)Conc. in plantleaves (mg.kg-1) /Subsequent dailydose (mg.kg-1.d-1)Conc. in milk(mg.kg-1ww) /Subsequent dailydose (mg.kg-1.d-1)Conc. in meat(mg.kg-1ww) /Subsequent dailydose (mg.kg-1.d-1)Conc. in air(mg.m-3) /Subsequent dailydose (mg.kg-1.d-1)Total dailyintake(mg.kg-1.d-1)Production (site-specific,worst case)1.20.10-1/ 4.01.10-31.50.10-2/ 2.88.10-51.22.10-1/ 7.79.10-44.87.10-2/ 9.75.10-47.89.10-5/ 7.37.10-77.89.10-6/ 3.96.10-81.49.10-3/ 1.78.10-45.97.10-3Paints IFPaints IP5.00.10-2/ 1.67.10-31.25.10-1/ 4.16.10-37.06.10-2/ 1.35.10-41.76.10-1/ 3.38.10-41.81.10-2/ 1.16.10-45.34.10-2/ 3.41.10-49.75.10-2/ 1.95.10-31.24 / 2.48.10-27.72.10-5/ 7.22.10-77.61.10-4/ 7.11.10-67.72.10-6/ 3.87.10-87.61.10-5/ 3.82.10-73.25.10-3/ 3.89.10-44.15.10-2/ 4.98.10-34.26.10-33.46.10-2Paints IIFand IIIFPaints IIPand IIIU5.12.10-2/ 1.71.10-31.82.10-2/ 6.07.10-47.23.10-2/ 1.38.10-42.57.10-2/ 4.93.10-51.85.10-2/ 1.19.10-47.45.10-3/ 4.77.10-50.10 / 2.00.10-37.99.10-2/ 1.60.10-37.92.10-5/ 7.40.10-75.34.10-5/ 4.99.10-77.92.10-6/ 3.97.10-85.34.10-6/ 2.68.10-83.33.10-3/ 4.00.10-42.67.10-3/ 3.20.10-44.36.10-32.62.10-3Paints IVFPaints IVP7.35.10-3/ 2.45.10-41.70.10-2/ 5.67.10-41.04.10-2/ 1.99.10-51.04.10-2/ 1.99.10-56.40.10-3/ 4.09.10-51.72.10-2/ 1.10.10-43.54.10-1/ 7.08.10-31.53 /3.05.10-22.05.10-4/ 1.92.10-68.76.10-4/8.19.10-62.05.10-5/ 1.03.10-78.76.10-5/ 4.40.10-71.19.10-2/ 1.42.10-35.11.10-2/6.13.10-38.81.10-33.73.10-2Paints VFPaints VP7.35.10-3/ 2.45.10-41.64.10-2/ 5.47.10-41.04.10-2/ 1.99.10-52.32.10-2/ 4.45.10-54.05.10-3/ 2.59.10-56.27.10-3/ 4.01.10-50.10 / 2.00.10-31.56.10-2/ 3.12.10-46.01.10-5/ 5.62.10-71.60.10-5/ 1.50.10-76.01.10-6/ 3.02.10-81.60.10-6/ 8.03.10-93.35.10-3/ 4.01.10-45.14.10-4/ 6.17.10-52.69.10-31.01.10-3Paints VIFPaints VIP1.54.10-1/ 5.13.10-39.67.10-2/ 3.22.10-32.17.10-1/ 4.17.10-41.37.10-1/ 2.62.10-45.20.10-2/ 3.33.10-43.28.10-2/ 2.10.10-45.35.10-2/ 1.07.10-31.64.10-2/ 3.27.10-49.71.10-5/ 9.08.10-75.12.10-5/ 4.79.10-79.71.10-6/ 4.87.10-85.12.10-6/ 2.57.10-81.73.10-3/ 2.08.10-45.08.10-4/ 6.10.10-57.16.10-34.08.10-3Paints VIIFPaints VIIP5.86.10-2/ 1.95.10-31.45.10-2/ 4.83.10-48.27.10-2/ 1.59.10-42.05.10-2/ 3.92.10-52.02.10-2/ 1.30.10-49.42.10-3/ 6.03.10-52.10.10-2/ 4.20.10-44.26.10-1/ 8.52.10-33.73.10-5/ 3.49.10-72.49.10-4/ 2.33.10-63.73.10-6/ 1.87.10-82.49.10-5/ 1.25.10-76.79.10-4/ 8.15.10-51.43.10-2/ 1.71.10-32.74.10-31.08.10-2Paints VIIIFPaints VIIIP7.35.10-3/ 2.45.10-47.35.10-3/ 2.45.10-41.04.10-2/ 1.99.10-51.04.10-2/ 1.99.10-53.70.10-3/ 2.37.10-53.18.10-3/ 2.04.10-56.13.10-2/ 1.23.10-35.87.10-3/ 1.17.10-43.81.10-5/ 3.56.10-76.56.10-6/ 6.13.10-83.81.10-6/ 1.91.10-86.56.10-7/ 3.29.10-92.05.10-3/ 2.46.10-41.93.10-4/ 2.32.10-51.76.10-34.26.10-4Paints IXFPaints IXP2.20.10-2/ 7.33.10-49.12.10-3/ 3.04.10-43.11.10-2/ 5.95.10-51.29.10-2/ 2.47.10-58.35.10-3/ 5.35.10-55.82.10-3/ 3.72.10-54.22.10-2/ 8.44.10-42.28.10-1/ 4.55.10-33.36.10-5/ 3.14.10-71.34.10-4/ 1.25.10-63.36.10-6/ 1.68.10-81.34.10-5/ 6.70.10-81.40.10-3/ 1.69.10-47.62.10-3/ 9.15.10-41.86.10-35.83.10-3Detergents IFDetergents IP1.76.10-2/ 5.87.10-43.28.10-2/ 1.09.10-32.49.10-2/ 4.77.10-54.63.10-2/ 8.88.10-56.58.10-3/ 4.21.10-51.58.10-2/ 1.01.10-46.66.10-3/ 1.33.10-44.00.10-3/ 8.01.10-51.15.10-5/ 1.07.10-71.64.10-5/ 1.54.10-71.15.10-6/ 5.75.10-91.64.10-6/ 8.25.10-92.15.10-4/ 2.58.10-51.15.10-4/ 1.38.10-58.36.10-41.38.10-3Detergents IIF1.16.10-2/ 3.87.10-41.16.10-2/ 3.15.10-54.59.10-3/ 2.94.10-54.84.10-3/ 9.68.10-57.82.10-6/ 7.31.10-87.82.10-7/ 3.92.10-91.57.10-4/ 1.88.10-55.64.10-4EURISK ASSESSMENT–2-BUTOXYETHANOLCAS111-76-2 CHAPTER4.HUMAN HEALTHRAPPORTEURFRANCER408_0808_HH_CLEAN65Conc. in drinkingwater (mg.L-1) /Subsequent dailydose (mg.kg-1.d-1)Conc. in wet fish(mg.kg-1) /Subsequent dailydose (mg.kg-1.d-1)Conc. in plantroots (mg.kg-1) /Subsequent dailydose (mg.kg-1.d-1)Conc. in plantleaves (mg.kg-1) /Subsequent dailydose (mg.kg-1.d-1)Conc. in milk(mg.kg-1ww) /Subsequent dailydose (mg.kg-1.d-1)Conc. in meat(mg.kg-1ww) /Subsequent dailydose (mg.kg-1.d-1)Conc. in air(mg.m-3) /Subsequent dailydose (mg.kg-1.d-1)Total dailyintake(mg.kg-1.d-1)Detergents IIU1.67.10-2/ 5.56.10-42.35.10-2/ 4.51.10-55.69.10-3/ 3.64.10-53.63.10-3/ 7.27.10-59.32.10-6/ 8.72.10-89.32.10-7/ 4.68.10-91.15.10-4/ 1.38.10-57.24.10-4Intermediates IP2.29.10-2/ 7.63.10-43.17.10-2/ 6.08.10-52.31.10-2/ 1.48.10-44.83.10-3/ 9.66.10-51.25.10-5/ 1.17.10-71.25.10-6/ 6.27.10-91.34.10-4/ 1.61.10-51.09.10-3Inks IFInks IP9.36.10-2/ 3.12.10-38.75.10-3/ 2.92.10-41.32.10-1/ 2.53.10-41.24.10-2/ 2.37.10-53.19.10-2/ 2.04.10-48.73.10-3/ 5.58.10-53.29.10-2/ 6.59.10-45.56.10-1/ 1.11.10-25.93.10-5/ 5.54.10-73.21.10-4/ 3.00.10-65.93.10-6/ 2.97.10-83.21.10-5/ 1.61.10-71.07.10-3/ 1.28.10-41.86.10-2/ 2.24.10-34.37.10-31.37.10-2Metal IFMetal IP6.27.10-2/ 2.09.10-31.18.10-2/ 3.92.10-48.85.10-2/ 1.70.10-41.62.10-2/ 3.11.10-52.18.10-2/ 1.39.10-41.19.10-2/ 7.60.10-54.06.10-2/ 8.12.10-47.99.10-1/ 1.60.10-25.03.10-5/ 4.70.10-74.61.10-4/ 4.31.10-65.03.10-6/ 2.52.10-84.61.10-5/ 2.31.10-71.33.10-3/ 1.60.10-42.68.10-2/ 3.21.10-33.37.10-31.97.10-2Pharm IP9.95.10-3/ 3.32.10-41.12.10-2/ 2.14.10-51.01.10-2/ 6.44.10-54.52.10-3/ 9.04.10-56.84.10-6/ 6.39.10-86.84.10-7/ 3.43.10-91.39.10-4/ 1.67.10-55.25.10-4Elec IP2.44.10-2/ 8.13.10-41.41.10-2/ 2.69.10-52.46.10-2/ 1.58.10-45.00.10-3/ 1.00.10-41.32.10-5/ 1.24.10-71.32.10-6/ 6.64.10-91.38.10-4/ 1.65.10-51.11.10-3Leather IP1.60.10-1/ 5.33.10-32.26.10-1/ 4.33.10-47.95.10-2/ 5.09.10-46.36.10-3/ 1.27.10-47.25.10-5/ 6.78.10-77.25.10-6/ 3.64.10-81.17.10-4/ 1.40.10-56.41.10-3Adhesives IFAdhesives IP2.10.10-2/ 7.01.10-48.10.10-3/ 2.70.10-42.97.10-2/ 5.70.10-51.14.10-2/ 2.19.10-57.73.10-3/ 4.95.10-54.65.10-3/ 2.98.10-58.20.10-3/ 1.64.10-43.59.10-3/ 7.18.10-51.38.10-5/ 1.29.10-75.57.10-6/ 5.21.10-81.38.10-6/ 6.93.10-95.57.10-7/ 2.79.10-92.66.10-4/ 3.19.10-51.15.10-4/ 1.38.10-51.00.10-34.07.10-4Agri IFAgri IP1.46.10-2/ 4.87.10-47.54.10-3/ 2.51.10-42.06.10-2/ 3.95.10-51.07.10-2/ 2.04.10-56.48.10-3/ 4.14.10-54.13.10-3/ 2.64.10-57.02.10-3/ 1.40.10-45.07.10-3/ 1.01.10-41.03.10-5/ 9.67.10-86.19.10-6/ 5.78.10-81.03.10-6/ 5.19.10-96.19.10-7/ 3.10.10-92.27.10-4/ 2.73.10-51.65.10-4/ 1.98.10-57.35.10-44.19.10-4Oilfield IFOilfield IP9.36.10-2/ 3.12.10-31.11.10-2/ 3.71.10-41.32.10-1/ 2.53.10-41.57.10-2/ 3.01.10-53.19.10-2/ 2.04.10-41.07.10-2/ 6.82.10-53.29.10-2/ 6.59.10-43.81.10-3/ 7.62.10-55.93.10-5/ 5.54.10-76.94.10-6/ 6.49.10-85.93.10-6/ 2.97.10-86.94.10-7/ 3.48.10-91.07.10-3/ 1.28.10-41.15.10-4/ 1.38.10-54.37.10-35.60.10-4Cosmet IFand Fire IFFire IPCosmet IU3.49.10-2/ 1.16.10-37.78.10-3/ 2.59.10-48.70.10-3/ 2.90.10-44.93.10-2/ 9.44.10-51.10.10-2/ 2.11.10-51.23.10-2/ 2.35.10-51.23.10-2/ 7.90.10-54.02.10-3/ 2.57.10-53.53.10-3/ 2.26.10-51.29.10-2/ 2.58.10-43.57.10-3/ 7.14.10-53.55.10-3/ 7.10.10-52.25.10-5/ 2.10.10-75.42.10-6/ 5.07.10-85.28.10-6/ 5.44.10-82.25.10-6/ 1.13.10-85.42.10-7/ 2.72.10-95.28.10-7/ 2.92.10-94.18.10-4/ 5.02.10-51.15.10-4/ 1.38.10-51.15.10-4/ 1.38.10-51.64.10-33.91.10-44.21.10-4Regional 7.35.10-3/ 2.41.10-41.04.10-2/ 1.96.10-51.47.10-3/ 1.11.10-53.48.10-3/ 4.05.10-55.19.10-6/ 4.06.10-85.19.10-7/ 2.18.10-91.15.10-4/ 1.96.10-53.32.10-4EU RISK ASSESSMENT

– 2-BUTOXYETHANOL CAS 111-76-2 C HAPTER 4. HUMAN HEALTH

RAPPORTEUR F RANCE

R408_0808_ HH _C LEAN 66 The highest indirect exposure is estimated for the processing of can coating (Paints IV P ) : 3.73.10 -2 mg.kg -1 .day -1 . It can also be noted that the hi ghest exposures are to be expected through intake of drinking water and plants (leaves and roots). Moreover, based on the regional concentrations, the total daily intake for humans is 3.32.10 -4 mg.kg -1 .day -1 . These two figures will be taken forward into the risk characterisation.

4.1.2 Effects assessment: Hazard identifi cation and dose (concentration)- response (effect) assessment

4.1.2.1 Toxicokinetics, metabolism and distribution

4.1.2.1.1 Studies in animals

Inhalation

Rat
Sixteen male SD rats were exposed continuou sly (whole body) to EG BE (purity 99 %) by inhalation at doses of 20 pp m or 100 ppm for 1, 2, 3, 4, 6, 8, 10 or 12 days (Johanson, 1994). Urine was collected in 24-hr interval periods. After sacrifice, tissue samples of blood, muscle and liver were collected and analysed for EG BE and Butoxy Acetic Acid (BAA) content.
No clinical signs of toxicity were noted dur ing the duration of the study. In all the tissues examined, the EGBE concentration rapidly increas ed during the first three days and continue to increase slower during the remaining days of exposure. The table 4.35 shows the tissue concentrations reached in the 20 ppm exposure group.

Table 4.35: Average tissues concentrations of EGBE and BAA following 20 ppm exposure Tissue Blood Liver Muscle Testis

EGBE concentration 10 – 20 μ mol/l 10 μ mol/l 10 μ mol/l 5 μ mol/l

BAA concentration 30 – 40 μ mol/l 15 – 20 μ mol/l 10 μ mol/l 10 μ mol/l

Following a 100 ppm exposure the tissues concentr ations were approxim ately 5 times higher.
(5 times higher in blood, 3.5 and 3.6 in muscle and testis respectively and 7.5 in the liver). The blood clearance of EGBE was estimated to 2.6 ± 1.3 l/h/kg bw whichever the dose administered. The kinetic profile of BAA was approximately the same as EGBE. The 100ppm/20ppm concentration ratio were: 4.4 for blood, 4.7 for muscle, 2.3 for testis and 5.3 for liver. The urinary excretion of BAA aver aged 0.2 mmol/day in the 20 ppm group and 1.03 mmol/day in the 100 ppm group. This correspond to 64 % of the cal culated respiratory uptake. The renal clearance was 0.53 l/h/kg.
[ 14 C]EGBE (purity 99 %) was admi nistered to male F 344/N ra ts by inhalation (nose only – vapour form) for 6 hr at doses of 5, 50 or 450 ppm (Sabourin et al ., 1992a). One group of 5 rats was used to determine the fractional upt ake of inhaled EGBE and the body burden of theEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 67 compound at the end of the exposure. Respiratory measurements were performed on this group. After termination, the entire carcass was digested. The 14 C in these rats was used as a measure of the body burden. A second group of 4 rats was used to determine the paths of excretion of EGBE equivalents and the major metabolites excreted. Urine, faeces and exhaled breath were collected for 66 hr following the end of the 6 hr exposure. The rats were terminated and the carcass was digested and analysed for 14 C. A third group of 30 rats was used for analysis of blood 14 C and blood metabolite concentration at the 5 ppm exposure concentration only. At various ti me points and up to 24 hr fo llowing the start of exposure, 3 rats were euthanized and bled by cardiac puncture. Blood samples were analysed for haematocrit as well as 14 C associated with whole blood and plasma where

14 C associated with red blood cells = 14 C associated with whole blood - 14 C associated with plasma

Plasma metabolites were also analysed.
The respiratory rate and tidal volume were not significantly different form those of unexposed rats for any of the exposure. The amount of EGBE inhaled was proportioned to the exposure concentration in the 5 and 50 ppm exposures. A less than proportional amount of EGBE was inhaled at the high exposure concentr ation due to a lower minute volume.
At all exposure concentrations, the majority of 14 C was eliminated in the urine, less than 7 % of the parent compound was exhaled follo wing the exposure. 10-20 % of the ( 14 C)EGBE equivalent remained in the carcass up to 66 hr post exposure at all concentrations. 7 % of the excreted 14 C was in the form of 14 CO 2 (see table 4.36).

Table 4.36: Excretion of [ 14 C]EGBE [ 14 C]EGBE equivalents as percen tage of amount metabolised
Exposure concentration in ppm (values in μ mol)

Route of excretion 5 50 450

14 CO 2 during exposure 0.2 (0.008) 1.3 (0.6) 0.9 (3.5)

14 CO 2 post exposure 7.6 (0.25) 7.2 (3.3) 5.9 (22.7)
Urine 67.3 (2.3) 64 (31.4) 75.9 (296.8)
Faeces 1.6 (0.06) 2.3 (1.1) 1.2 (4.6)
Exhaled EGBE post exposure 3.3 (0.09) 5.1 (2.5) 2.3 (9)
Carcass 19.8 (.068) 19.1 (9.4) 12.9 (50.6)

Total 100 100 100

Urinary 14 C excreted during the first 41 hr represented more than 88 % of the total 14 C excreted in urine. BAA was the major metabolit e in urine at all exposure concentrations. EG and BEG were also found in lesser amounts (s ee table 4.37). As the exposure concentration increased, the proportion of 2 unidentified minor metabolites increased. At 5 ppm about 60 %EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 68 of the urinary 14 C was excreted during the 6 hr expos ure. Whereas at 450 ppm only 10 % was excreted during the exposure. The urinary me tabolites profile varied with time following exposure : metabolism to Glucuronide conjuga te of EGBE (BEG) wa s favoured during the exposure whereas metabolism to BAA and Ethyle ne Glycol (EG) was favoured at later time post exposure. This observation was more a pparent at the high exposure concentration. Metabolism of EGBE leading to EG and BAA appear to be easily saturable.

Table 4.37: Urinary metabolites of EGBE Percentage of retained dose excreted in urine by 41 hr
Exposure concentration in ppm (values in μ mol)

Metabolite 5 50 450
EG 16.1 (0.55) 13.4 (6.3) 7.9 (34)
BAA 43.2 (1.47) 41.9 (19.9) 36.6 (143)
BE glucuronide 3.4 (0.20) 6.7 (3.2) 10.4 (40)
4 min unknown ND 10.5 (0.5) 5.3 (22)
10 min unknown ND ND 1.5 (6)
Others 0.5 (0.03) 0.7 (0.4) 2.2 (10)

Analysis of whole blood as well as plasma indica ted that the majority of the EGBE equivalent were in the plasma.
During the first 2 hr of expos ure about 20 % of the blood 14 C was associated with the red blood cell fraction. However at the later time points the proportion of 14 C in the cellular fraction declined and was undete ctable following exposure. Bound 14 C metabolites was greater after the exposure than during the exposure.
In an inhalation study, F344 rats were expos ed whole body for 6h per day, 5 days per week for 104 weeks, to EGBE (purity > 99 %) at concentrations of 31.2, 62.5 and 125 ppm (Dill et al., 1998). Post exposure blood samp les were collected after 1 day, 2 weeks and 3, 6, 12 and 18 months for EGBE and BAA determination. The post exposure time points depends of the day of collection. Post exposure urine samples we re collected from anim als after 2 weeks and 3, 6, 12 and 18 months of exposure. Th is study was also performed on mice.
The EGBE blood concentrations showed a rapi d drop after the cessati on of exposure. The elimination t 1/2 for EGBE after 1 day of exposure was < 10 min, this t 1/2 does not depend on the dose level. Increases in th e initial blood concentration of EGBE and Area Under the Curve (AUC) were proportional to exposure concentrat ion, supporting that e limination of EGBE from the blood follows linear kinetics. At th e common concentration levels (62.5 and 125 ppm), mice eliminated EGBE fast er than rats, values of t 1/2 and AUC EGBE were significantly lower in mice than in rats (see table 4.38). The rate of EGBE elimination from blood slowed with increasing days of exposure, this change was more obvious in rats than mice as their t 1/2 and AUC dramatically increased after 3 months of exposure. Male rats generally exhibited larger AUC than females, indicating a lower blood clearance of EGBE. k was not significantly different between sexes, implying th at the volume of distri bution, rather than the rate of elimination might be responsible for the sex-related difference in EGBE clearance.EU RISK ASSESSMENT

– 2-BUTOXYETHANOL CAS 111-76-2 C HAPTER 4. HUMAN HEALTH

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R408_0808_ HH _C LEAN 69 Unlike EGBE, BAA was not rapidly cleared from the systemic circulation. The BAA concentrations in the blood did not start to decline until 20 to 80 min post exposure. It seems that BAA was eliminated from blood following sa turable, nonlinear kinetics. The k was not constant, decreasing as EGBE exposure concen trations increased. As the rate of BAA production reflects the EGBE elimination, it seem s that the nonlinear ch aracteristics of BAA kinetics likely result from BA A distribution or elimination pr ocesses. When the number of exposure days increased, the rate of 2BAA elimination tended to decrease. Females eliminated BAA more slowly than males (smaller k, longer t 1/2 and larger AUC). The maximum blood concentration was also higher in females at each exposure concentration. In mice, elimination of BAA was faster than rats when compared at common exposure concentrations. There was a clear difference in the urinary excretion of BAA between rats and mice (see table 4.39). Female rats excreted le ss BAA than males regardless of exposure concentration. Excretion of BAA te nded to increase with time.

Table 4.38: Comparison of EGBE elimination betw een rats and mice at the same dose level tested.

C 0 ( μ g/g) k T1/2 (min) AUC ( μ g/min/g)

62.5 ppm

Rats Mice Rats Mice Rats Mice Rats Mice

1d 1.86 1.59 1.29 1.35 0.0793 0.0809 0.229 0.185 8.74 8.56 3.03 3.75 23.5 19.6 5.6 7.3

2w 3.06 2.24 1.14 1.54 0.0828 0.0839 0.161 0.199 8.37 8.26 4.31 3.49 36.9 26.8 7.1 7.7

3m 0.54 0.47 1.30 1.3 0.0166 0.0248 0.126 0.199 41.66 27.9 5.51 3.49 32.3 18.9 10.3 6.5

6m 1.31 0.69 1.48 0.9 0.0308 0.0230 0.128 0.098 22.51 30.20 5.41 7.08 42.5 29.9 11.6 9.2

12m 1.08 0.97 1.17 1.54 0.0354 0.0439 0.091 0.129 19.6 15.79 7.63 5.38 30.5 22.0 12.9 12.0
125 ppm

1d 4.27 3.11 3.78 3.18 0.0735 0.0736 0.171 0.139 9.43 9.42 4.06 4.99 58.1 42.3 22.1 22.9

2w 5.36 4.10 2.13 2.31 0.082 0.0852 0.126 0.147 8.45 8.13 5.5 4.72 65.3 48.1 16.9 15.8

3m 3.17 2.75 4.36 6.53 0.0379 0.0411 0.153 0.188 18.30 16.87 4.53 3.69 83.6 67 28.5 34.7

6m 3.58 3.58 2.43 2.39 0.0514 0.0589 0.084 0.082 13.47 11.76 8.29 8.44 69.6 60.7 29 29.1

12m 2.23 1.33 2.19 2.31 0.0347 0.0342 0.094 0.094 19.96 20.28 7.41 7.41 64.3 38.8 23.4 24.7EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 70 Table 4.39: Comparison of BAA urinary excretion between rats and mice. k T1/2 (min) AUC ( μ g/min/g)

Rats Mice Rats Mice Rats Mice

62.5 ppm

1d 0.0174 0.0109 0.0221 0.0224 39.9 63.7 31.3 31 5541 8228 1330 2808

2w 0.0168 0.0088 0.0326 0.0304 41.2 78.4 21.3 22.8 4849 10605 797 1872

3m 0.0168 0.0094 0.0217 0.0178 41.3 73.7 31.9 39 - - 3059 3872

6m 0.0099 0.0090 0.0222 0.0225 70.3 76.8 31.2 30.8 - - 4592 7138

12m 0.0139 0.0087 0.0134 0.0108 49.8 79.6 51.6 64.1 7279 8445 9026 13941

18m 0.0067 0.0122 0.0144 0.0208 103.3 56.7 48.2 33.3 9351 8976 8158 7521
125 ppm

1d 0.011 0.0031 0.0241 0.0285 63.2 224.1 28.7 24.3 19010 33403 4475 8145

2w 0.0116 0.0053 0.0263 0.0237 59.5 130.2 26.4 29.3 13597 37943 4640 10912

3m 0.0059 0.0039 0.0248 0.0227 117.9 178.8 28 30.5 13554 29094 8295 11772

6m 0.0035 0.0018 0.0124 0.0167 195.9 395.8 55.8 41.6 14142 26485 30151 18022

12m 0.0037 0.0024 0.0124 0.0054 187 283.4 55.7 127.8 16649 25897 21094 40782

18m 0.0023 0.0058 0.0143 0.0166 297.5 119.3 48.5 41.7 30768 25745 34787 21126

Mouse
In an inhalation study, B6C3F1 mice were e xposed whole body for 6h per day, 5 days per week for 104 weeks, to EGBE (purity > 99 %) at concen trations of 62.5, 125 and 250 ppm (Dill et al., 1998). Post exposure blood samples were co llected after 1 day, 2 weeks and 3, 6, 12 and 18 months for EGBE and BAA determination. The post exposure time points depends of the day of collection. Post exposure urine samples were co llected from animals after 2 weeks and 3, 6, 12 and 18 months of exposure. Befo re the core study started, a separate set of mice was moved into the control chamber and designated as the “aged (naïve)” mice. At 18 months into the chronic study, these mice (about 19 months old) were moved to the 125 ppm exposure chamber and exposed for 3 weeks. Bl ood was collected after 1 day and 3 weeks of exposure at post exposure time points of 10, 20, 40, 80, 180, 360, 720 and 1440 min. Post exposure urine samples were collected from the aged mice for 16 h after 2 weeks of exposure. This study was also performed in rats.
The EGBE blood concentrations showed a rapi d drop after the cessati on of exposure. The elimination t 1/2 for EGBE after 1 day of exposure was < 5 min, this t 1/2 does not depend on the dose level. Increases in the initial blood con centration of EGBE and AUC were proportional to exposure concentration, supporti ng that elimination of EGBE from the blood follows linear kinetics. At the same concentration levels mice eliminated EGBE faster than rats. Values of
t 1/2 and AUC were significantly lower in mice at both exposure concentrations. The rate of EGBE elimination from blood slowed with incr easing days of exposure , this change was more obvious in rats than mice as their t 1/2 and AUC dramatically incr eased after 3 months of exposure. The kinetic parameters were not sign ificantly different between male and femaleEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 71 mice. Unlike EGBE, BAA was not rapidly cleare d from the systemic circulation. The BAA concentrations in the blood did not start to d ecline until about 40 min post exposure. It seems that BAA was eliminated from blood following sa turable, non-linear ki netics. The k was not constant, decreasing as EGBE exposure concen trations increased. As the rate of BAA production reflects the EGBE elim ination, it seems that the non- linear characteristics of BAA kinetics likely result from BA A distribution or elimination process. When the number of exposure days increased, the rate of 2BAA e limination tended to decrease. Elimination of BAA was faster than rats when compared at common exposure concentrations. No differences were seen between males and females. There was a clear difference in the urinary excretion of BAA between rats and mice. No sex differen ces in BAA excretion were seen regarding exposure concentration. Time-d ependent changes in urinary excretion of BAA were not comparable between males and females as the trend appeared to change over time in the opposite direction (decreasing in male s mice and increasing in females).
Elimination of EGBE and BAA in aged mice : EGBE was rapidly cleared from systemic circulation and kinetic parameters were not si gnificantly different fr om those of young mice. A slower terminal elimination phase was ob served in aged mice suggesting that timedependent reduction in the rate of EGBE elimin ation in chronically e xposed mice could be, in part, due to the age of animals. As withyoung mice, there was no sex difference in elimination kinetic. There was a great differe nce with young animals in blood concentration of BAA after 1 day of exposure. Aged mice el iminated BAA from blood more than 10 times slower than young mice. Consequently both t 1/2 and AUC were greater in old mice compared to young animals after 1 day of exposure. This ag e-related difference mostly disappeared after about 3 weeks of exposure.
In an inhalation study, female B6C3 F1 mice were exposed whole body to 2- butoxy[ 14 C]ethanol (purity 97.6 %) at dose of 250 ppm for 6 hours (Green et al. , 2000a). The mice (4 per time point) were terminated at 5 minutes, 24 and 48 hours post exposure. One animal for each time point was submitted to a whole body autoradiogr aphy, the remaining 3 animals were submitted to an analysis of the free and bound radioactivity of the stomach and
contents. The mice killed 5 min after the end of exposure contained high level of radioactivity without showing preferential labelling in any tissue or organ. The highest concentrations were found in liver, blood and nasal passages with lower concentrations in the gastro-intestinal. Similar concentration was observed in the glandular mucosa of the stomach but no radiolabelling were present in the forestomach. Very high leve ls of radiolabelling were present on the skin and fur near the hindquarters.
At 24 hours post exposure, the highest concentrat ions of radioactivity were observed in the liver and buccal cavity. Similar high amount of radi olabelling was seen in the mucosa of the caecum, in the forestomach mucosa, the lowe r gastro-intestinal tract mucosa and the oesophagus. The glandular stomach was conspic uously different from the forestomach in having low, background levels of labelling. High levels of radiol abel were seen in the skin and fur particularly on the back and near th e hind quarters. A lower level of labelling was observed in the salivary glands, thymus, ki dney medulla, adrenal and spleen. Background labelling was seen in the rest of the internal organs.
48 hours after exposure, high levels of radiolabelling was seen in the buccal cavity, the oesophagus, the forestomach and in the liver a nd the mucosa of the lower gastro-intestinal tract. The skin and fur, particularly near th e hind quarters, were also heavily radiolabeled.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 72 Low levels of labelling were observed in the duodenum, glandular stomach and the remainder of the internal organs.
The analysis of stomach and contents showed th at immediately after exposure, a greater level of radioactivity was present in the stomach and its contents than the two later time point and that most of this radioactivity was due to EGBE. At the 24 an d 48-hour time point, more of 80 % of the radioactivity present in the stomach tissues was covalently bound to protein. There was no significant difference between the glandular and forestomach.
High concentrations of radioactivit y were seen on the fur and skin of the animals in this study and according to the authors, this observation t ogether with the presence of radioactivity in the buccal cavity, oesophagus and stomach cont ents suggests that EGBE is ingested during grooming (during exposure and pos t exposure) and mucous removal (muco-ciliary clearance) through the nasopharynx (duri ng exposure). Although the whol e body autoradiograms very clearly demonstrated highly sele ctive labelling of the mucosa of the forestomach at the two later time points, the analysis of the stomachs failed to detect this, probably due to the high levels of background labelling in th e remainder of the stomach tissues.
In conclusion, the retention of radioactivity in the forestomach mucosa clearly indicates that the forestomach is a target organ followi ng an inhalation exposure to EGBE. The high concentrations of radi oactivity on the skin and fur and the presence of radioactivity in the buccal cavity and the oesophagus suggest that th e radioactivity enters the forestomach as a result muco-ciliary clearance from the na sopharynx and grooming and ingestion of the material present on the skin and fur.
A complementary study from Poet et al ., 2003 identified both BAA and EGBE in the saliva of mice administered EGBE by both oral gavage and intraperitoneal routes. The author concluded that EGBE can distribute to the fo restomach by multiple mechanisms: grooming of the fur, mucociliary clearance, saliva and from systemic blood circulation and that BAA can also distribute to forestomach tissues from saliva and blood circulation as well as being formed locally from EGBE.

In this series of studies, Poet et al ., 2003 also assessed the depos ition of EGBE on fur after inhalation exposure: female mi ce were exposed either by w hole-body or nose-only inhalation to a single 6-hour EGBE exposure of 250 ppm (va pour). At the end of each exposure, 5 mice were killed and immersed in hot water to co llect the EGBE deposited on the fur. Additional groups of 5 mice were killed immediately af ter inhalation exposure for blood analysis. Two other groups of 5 mice were subjected to an 18-hour urine collectio n after the inhalation
exposures. An average of 205 μ g of EGBE was detected on the fur of mice exposed whole-body. For mice exposed nose-only an average of 170 μ g was found. After corrections, the estimation of the authors was that whole body exposures averaged 25 % more EGBE on the fur than the corresponding nose-only exposure.
At the end of each exposure, the mean con centrations of EGBE in blood were 3.0 and 3.9 mg/l for whole-body exposure and nose-only expos ure respectively. As the same time, the concentration of BAA in blood were 235 a nd 390 mg/l for whole-body exposure and noseonly exposure respectively. Low levels of EG BE were found in the 18-hour post exposure urine collection (about 68 μ g) to either route. These concentration are supposed to come from the fur because EGBE is not expected to be excreted in the urine unconjugated. Concentrations of free BAA in the urine were about 2020 μ g or 1780 μ g for whole-body exposure and nose-only exposure respectively.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 73 Summary inhalation route :
Three studies performed with rats (two with F344 and one with SD rats) and 3 studies performed with B6C3F1 mice were reported. In one study, the distribution of EGBE and its metabolites was studied in relati on with time. This study clearly showed that the liver and the forestomach were the main target organs. The great amount of radiol abelling found in the gastro-intestinal tract demonstrated that EG BE was ingested duri ng grooming and by mucocilliary clearance from nasopharynx and not di rectly via inhalation route. However this mechanism cannot explain the great amounts found in the forestomach. EGBE metabolism leaded to the formation of a majority of BAA, EG and BEG and 2 metabolites unidentified in small quantities. BAA and EG formation followed a saturable mechanism and increased doses of EGBE leaded to a greater and greater form ation of BEG compared to BAA and EG. Like the other routes of administration, elimination via urinary route was ra pid and predominant. A small amount of the administrated dose was elim inated via inhalation ro ute in the form of CO 2 (less than 10 %).

The blood half-life of EGBE was about 10 minutes in rats and 5 minutes in mice whatever the level of exposure. Unlike BAA which seemed to be eliminated by a saturable mechanism, elimination of EGBE followed a linear kine tic. When repeated doses of EGBE were administered, the rate of BAA elimination tende d to decrease with increasing doses of EGBE.
In rats, elimination of BAA vari ed with the sex of animals: fe males tended to eliminate BAA slower. This difference between the sex could be due to a difference in the renal excretion between males and females. This sex differen ce was not found in mice. A species difference was also reported, mice eliminating 2 fold fast er than rats. An age difference was also described in mice, the older mice eliminating BAA 10 fold slower than the younger mice for an unique administration of EGBE. When exposu res were repeated , the difference between aged and young mice tended the di sappear. After 3 months of daily exposure, no difference was seen for the elimination of BAA when age is considered.

Dermal

In vivo studies

Rat
EGBE (99 % purity) was tested in a series of studies in ra ts for assessing its kinetic parameters via the dermal route (Bartnik et al ., 1987).
Cutaneous absorption in vivo : EGBE was applied to 6 males and 6 females Wistar rats (12

cm 2 on shaved skin) at a dose of 200 mg/kg. To a void oral uptake, the si te of application was kept covered with a perforated glass capsu le. Urine was collected for 48 hours. After termination, treated skin area was dissected for the determination of the radioactivity.
Percutaneous absorption was assessed by using the measurements of urinary excretion of 14 C from 0 to 48 hr following cutaneous and s ubcutaneous administration of EGBE and expressing the results as total radioactivity (urine) following topical application / total radioactivity (urine ) following subcutaneous application.
In a similar experiment, blood levels were de termined following cutaneous application of EGBE. EGBE was applied to twenty four fe male rats at a dose of 200 mg/kg (12 cm 2 on shaved skin). Animals were killed 0.5, 1, 2, 4, 6, 8, 16 and 24 hours after application of EGBE. EGBE and BAA were measured in blood.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 74 In vitro absorption of EGBE was also studied (see in vitro section)
Within 48 hr following topical application of EGBE, approximately 20 to 23 % of the applied radioactivity was found in the ur ine (95 % eliminated during the first 24 hr). A percutaneous absorption of approximately 25-29 % of the applied topical dose was determined.
The highest radioactivity in blood and plasma occurs at 2 hr following application. Measurement of BAA in the plasma presented the same qualitative course. Based on BAA levels in the plasma, it appears that the major part of the absorbed EGBE is metabolized to BAA.

Male F344/N rats were exposed dermally (non occlusively) to EGBE (99.9 % purity) (Sabourin et al ., 1992b) at dose levels of 122, 367 and 650 μ mol/rat ). Parent compound, CO 2 , urine and faeces were collected for 72 hr. After 72 hr, rats were killed and the skin around the site of dosing was removed from the carcass. Di gested tissues, urine and faeces were assayed for radioactivity.
Analysis of urine from [ 14 C]EGBE dosed animals indicated the presence of the parent compound. Some experiments demonstrated that 7 % of the volatilized [ 14 C]EGBE was trapped in the water in the urine receptacle. It demonstrate that urine dripping down the sides of the metabolic cage can absorb signif icant quantities of the volatilized EGBE.
At the mid dose, 3 rats per time point were ki lled at 0.5, 1, 2, 4, 8 a nd 16 hr after exposure. Blood was collected and haemat ocrit and radioactivity meas urement made. Urinary and plasma metabolites were analyzed.
The total recovery of the 14 C was 74 to 90 %. 43 to 64 % of the dermally applied dose was trapped as volatile 14 C. 20 to 25 % of the dermally applied dose were absorbed and metabolized. A small amount of the applied 14 C (0.3-2 %) was still present at the site of application 72 hr following dosing. The majority of radioactivity was excreted in urine. Small amounts of 14 C were found in faeces and exhaled CO 2 on air. There was no significant effect of the dose on the excretion of 14 C except for a slight increase in the proportion of 14 CO 2
. At the end of the collection period 7-16 % of the absorbed radiolabel remained in the carcass. Urinary metabolites were analyzed up to the time at which < 10 % of the 14 C excreted within 72 hr remained. For EGBE this included urine collected up to 23 hr after exposure. BAA was the major urinary metabolite. Amounts of gluc uronide conjugates were also detected. The metabolite profile was not significantly affected by the dose. Analysis of 14 C in whole blood indicated that over 80 % of ra dioactivity was associated with the plasma. The maximum concentration of total plasma metabolites wa s reached at about 1 hr following dermal application of the mid-dose of radiolabelled EGBE, after whic h the concentration decreased with a half-life of about 4 hr. Plasma BAA, the major metabolite, was measured at 1-2 and 4 hr post exposure and represented 53 to 75 % of plasma 14 C.

Guinea pig
The percutaneous uptake rate of EGBE was studied in Guinea Pigs (Johanson and Fernström, 1986d). Female Guinea-Pig (GP) were kept unde r anesthesia throughout the experiment. After Intra Venous (IV) bolus of 42 or 92 μ mol/kg EGBE, blood was sampled after 5, 10, 20, 30, 40, 50, 60, 75, 90, 105 and 120 min. At 150 min after th e IV dosing, animals were exposed toEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 75 1 ml of undiluted EGBE percutaneously (3.14 cm 2 of skin exposed) with occlusive conditions. Blood samples were collected at the same time intervals as the intravenous dose.

The concentration of EGBE in blood after IV administration declined rapidly. Estimates of the total clearance (Cl) was 128 ml/min/kg bw corresponding to 2.7 ml/min/g of liver (0.8 ml/min/g of liver in human and 2 ml/min/g of liver in perfused rat liver). After dermal administration, the blood concentration of EGBE rose rapidly and reach a plateau level during the later half of the e xposure period (average 21 μ mol/l). The average uptake rate was estimated to be 0.25 μ mol/min/cm 2

.

The influence of water on the percutaneous ab sorption of EGBE was studied in Guinea Pigs (Johanson and Fernström, 1988). Animals were kept under anesthesia for 6 hr and killed at the end of the experiment. GP were exposed for 2 hours to pure or aqueous EGBE concentrations of 80 %, 40 %, 20 %, 10 % and 5 %. After a recovery period of 2 hours a second 2-hour exposure to pure EGBE was performed. The skin uptake of diluted EGBE was compared with the skin uptake of undiluted EGBE, each animal serving as its own control. Arterial blood were collected at intervals of 10 to 30 min throughout the experiment and assayed for EGBE.

The concentration of EGBE in blood increase d with time after administration. After removal of the exposure source, concentration star ted to decreased immediately. During the 2 nd hour of exposure to pure EGBE, the blood level appeared to approach a plateau level. The average concentration during the last hour was 4.6 μ mol/l and the calculated skin uptake rate was 132 nmol/min/cm 2 . The absolute skin uptake rate and the average blood conc entration of EGBE during the first exposure period increased as the percentage of EGBE in the applied solution increased up to a maximum of about 40 % and then decrease again. The relative skin uptake rate showed a similar pattern. The average rela tive uptake rate of EG BE was approximately the same from the 5, 10 and 20 % solution as fr om the undiluted EGBE, while it was twice as high from the 40 and 80 % solutions. In contra st, the concentration of EGBE in blood declined approximately twice as rapidly after th e end of exposure to the 40 and 80 % solution (average t 1/2 : 28 min compared to 55 min for the other solutions).

In vitro studies : Cutaneous absorption of EGBE in vitro was assessed by a modifi ed method of Zesch and Schaefer (1973) (Bartnik et al ., 1987). These studies also incl uded an evaluation of cutaneous absorption of EGBE under semi occlusive and no n-occlusive conditions. Skin of rats, pigs and humans was used. Before and at the end of ea ch study, the skin was checked visually for integrity of the stratum corneum. For applic ation of test materi als, an area of 5 cm 2 (animal skin) or 3 cm 2 (human skin) was demarcated on the skin samples. 30 microliters of each of the following test solutions was a pplied : EGBE (100 %), EGBE ( 3.5 %) in water, EGBE (10 %) in water. All test solutions were evaluated fo r absorption in rat skin (1, 6 and 16 hr semiocclusive, 1hr exposure non-occlusive) and pi g skin (6 hr exposu re semi-occlusive). Absorption of EGBE (3.5 % in water) were also assessed on both rat and pig skin under nonocclusive conditions (10, 30 a nd 60 min exposure). A comparativ e study involving samples of rat, pig and human skin was performed with EGBE (10 % in water) using an exposure period of 1hr under both semi-occlusive and non-occlusiv e conditions. The studies with 3.5 and 10 % EGBE were repeated with 5 % 2-propanol (IPA) or 5 % alkyl sulfonate (LAS) added to the test solution. IPA nor LAS had any measurab le effect on the dermal uptake of EGBE.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 76 Under semi-occlusion in vitro , EGBE is readily absorbed and completely absorbed after 16 hr of exposure. Penetration depends on time as we ll as concentra tion. The penetration rate of pure EGBE is slower than from aqueous so lutions, but more complete after 16 hr. A comparison of EGBE penetration at 6 hr under semi-occlusive conditions showed that for all the tested formulations, the pene tration through the pig skin was 2 or 3 times le ss rapid than through rat skin. Application of EGBE on rat skin under non-o cclusive conditions results in apparently great reduction in the absorption due to the volatility of the compound. Within 10 min following application, a major proportion of the absorbed material had penetrated. In comparison with the pig skin, ra t skin showed an approximate 2 fold higher absorption. Under semi-occlusive condition, the penetration rate of EGBE through human skin was comparable with that through pig skin (much slower th an through rat skin). Under non-occlusive conditions, human skin exhibits the lowest percutaneous ab sorption (6.9 % of the applied dose).
Percutaneous absorption of EGEE and EGBE was studied in humans and rats (Lockley et al
., 1999 – Abstract). 2 butoxy [1- 14 C] ethanol (1.41 mg/cm 2 ) was applied to rat dorsal skin and human breast skin in vitro . The results were compared with an in vivo study in which EGBE (9.22 mg/cm 2 ) was applied to the skin of male Wist ar rats. Skin surface swab (unabsorbed material), tape strip (material in the stratum corneum), skin (material in the epidermis and remaining dermis) and absorbed dose (receptor fluids in vitro and mass balance cumulative dose in vivo ) were analyzed up to 24 hr after applic ation. Evaporated EGBE was trapped with carbon filters ( > 80% of the dose within 1 hr of application).
Absorption of EGBE in vitro through rat skin most clos ely reflected penetration in vivo

. Absorption through human skin in vitro was less than rat skin but reflected dermal absorption described in a previous study (Johanson et al. , 1988). This study demonstrates that for EGBE in vitro studies are able to reflect in vivo conditions.

Summary dermal route :
Five studies were reported on EG BE kinetic properties via derm al route. Two performed with Wistar rat, one performed with F344 rats and two performed with Guinea pigs. A series of in vitro studies was also available, studying percutaneous uptake of EGBE in various species, at different concentrations in different solvents and with various patterns of application (occlusive or not). Under semi-occlusive conditions, dermal uptake of pure EGBE was between 20 and 30 % of the administrated dose. De rmal uptake of aqueous dilutions of 5, 10 and 20 % EGBE was similar to that of the pur e substance. Uptake was increased and was maximal for the 40 and 80 % aqueous solutions of EGBE. The rate of penetration was less for pure substance than for concentrated aqueous solutions. Some in vitro studies demonstrated that dermal uptake for pig skin was 2 or 3 time s slower than rat ski n. The results found with human skin were more or less equivale nt to those seen with pig skin.

If EGBE was administrated under non-occlusiv e conditions, the dermal uptake decreased dramatically (uptake < 10 %), mainly because of the volatility of EGBE.
A plasma peak was obtained in about 2 hours after the beginning of the application and relatively constant until th e cessation of exposure.
The metabolism mainly lead to the formation of BAA and also to sma ller quantities of BEG (Sabourin et al ., 1992b). The half-life of metabolites in plasma was about 4 hours. The majority of metabolites was eliminated via ur inary excretion and only a very small amountEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 77 were found in faeces. One part of administ ered EGBE was metabolised in CO2 and eliminated via respiration, this amount increasing with th e increasing dose.

Oral

Rats
The metabolism and disposition of EGBE was studied in F344 rats (Ghanayem et al ., 1987a). Male rats were given a single doses of radiolabeled EGBE by gavage of either 125 or 500 mg/kg bw. Collection of urine, faeces and e xpired air was made during 48 hours after the administration. Biliary excretion of EGBE was also monitored. De termination of the radioactivity in each organ and in blood was performed at the end of the study.

The amount of 14 CO 2 exhaled in the first 48 hours afte r dosing was approximately 18 and 10 % of the administered doses of 125 and 500 mg/kg bw respectively. Exhaled Volatiles accounted for no more than 2 % of the administ ered doses. Faecal excretion of EGBE ranged between 2 and 3 % of th e administrated doses. Urine was th e major pathway of excretion and most of the radioactivity was excreted du ring the first 24 hours after dosing. A higher percentage (70 %) was found for the 125 mg/kg dose as compared to the 500 mg/kg dose (40 %). Two major metabolites were detected by chromatography in the urine (Butoxy Acetic Acid (BAA) and the Glucuronide conjugat e of EGBE (BEG). Three minor peaks corresponding to the Sulfate conjugate of EGBE (BES), EGBE and an unknown substance were also measured in urine. All 5 metabolites were observed in urine collected in the first 8 hours after treatment with 125 mg/kg bw; EGBE a nd BES were not seen in later time periods, and EGBE and BES were not seen at any time in the urine of rats tr eated with 500 mg/kg bw. Analysis of urine collected be tween 8 and 24 hr after dosing re vealed that approximately 90 % of the EGBE derived radi oactivity was BAA at both doses. While BAA was the only metabolite observed in the 24-48 hr urine of animals receiving 125 mg/kg bw, the ratio of BAA to BEG was approximately 3 to 1 in urin e of rats receiving 500 mg/kg bw (see table 4.40).

Table 4.40: Effect of time and dose on the excretion of BE metabolites in urine
Dose
125 mg/kg 500 mg/kg

8 hr 24 hr 48 hr 8 hr 24 hr 48 hr

Metabolite

X 3.0 <2 % ND <1 % <1 % <1 %

BAA 74.4 93.5 100 76.4 89.9 75.3

BEG 21.3 5.3 ND 23 9.2 23.8

BES 2.7 ND ND ND ND ND

BE <2 % ND ND ND ND ND

Significant increases in bile flow were seen as early as 0.5 hr after EGBE administration (for the 500 mg/kg dose) that returned to normal af ter 4 hr. Biliary excret ion of radioactivityEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 78 continued to increase in a manner parallel wi th the increase in bile flow. The cumulative excretion reached 8 % of the 500 mg/kg dose in 8 hr. BAA, BEG and EGBE were excreted in bile. EGBE was detected only during the firs t 2 hr after treatment. BEG was the major metabolite excreted in bile.
Organs which had the most radiolabelli ng were forestomach, liver and kidneys.
The modulation of EGBE metabolism after pyrazo le (alcohol dehydrogenase inhibitor) or cyanamide (aldehyde dehydrogenase inhibitor) administration was studied in F344 rats (Ghanayem et al ., 1987b). A single dose of 500 mg/kg of [ 14 C]EGBE was administered to rats by gavage and another group received the same dose after a pre-treat ment with 250 mg/kg Intra Peritoneal (IP) one hour before. Rats we re placed immediately in metabolism cages for urine and faeces collections (8, 24 and 48 hr after dosing for urine and 24 and 48 hr after dosing for faeces). Expired volatiles and 14 CO 2 were collected over the 48 hr housing period. At the end of the 48 hr rats were sacrificed and tissues were collected. Biliary excretion and radioactivity after treatment or rats with radiol abeled EGBE of radiolabeled EGBE + pyrazole was determined. Qualitative and qua ntitative determination of metabolites in urine and in bile was performed.
Approximately 11 % of the 500 mg/kg dose was exhaled as 14 CO 2 in 24 hr after dosing. However pre-treatment with pyrazole resulted in a significant decline in the percent of EGBE dose exhaled as 14 CO 2 . In contrast pyrazole significantly increased the urinary excretion of EGBE derived radioactivity. BAA was the major metabolite colle cted whatever the collection period (75 – 90 % of the total urine radioactivit y). Most of the remaining was the glucuronide conjugate of EGBE. In contrast the major meta bolite of pyrazole pre-treated rats was BEG (75 – 85 % of the total radioactivity). Furthermore, 8 – 19 % of the radioactivity was the sulfate conjugate which was not detected in rats treated with EGBE only. The increase of biliary excretion in general was more important for pyrazole pre-treated than EGBE only animals. Moreover, the biliary excretion of EGBE derived radioactivity wa s higher in pre-treated rats (16 % vs 8 % for animals treated with only EGBE. A similar metabolic profile was obtained in bile compared to urine (BAA constituted 10, 21 and 46 % of the total radioactivity excreted in the bile fractions at 0 to 1, 2 to 4 and 6 to 8 hr after treatment. In contrast, no BAA was detected in any of the bile fractions collecte d from pyrazole pre-treated rats. In these rats approximately 12 % of the radioactivity was excret ed in the first hr as unchanged EGBE with the remaining portion being BEG. In addition no metabolite other than BEG were detected in bile fractions collected at 2 to 4 and 6 to 8 hr after dosing with EGBE. With cyanamide pretreatment, the same results were obtained than with pyrazole. This st udy clearly demonstrates that metabolism of EGBE to BAA is mediated by alcohol and aldehyde dehydrogenases via the formation of BAL.
The metabolism and disposition of orally ad ministered EGBE (500 mg/kg) in young (4-5 weeks old) or adult F344 rats (9-13 weeks old) was compared (Ghanayem et al ., 1987c).
There was a significant hi gher percentage of th e EGBE exhaled as CO 2 , 48 hr after dosing, by young rats (approximately 10 and 22 % of the administered dose in adult and young rats respectively). Similarly, young rats excreted a significantly higher percentage of the administered dose in the urine than adult ra ts. BAA, BEG and BES were identified in the urine of EGBE treated rats. Young rats excr eted significantly less BAA than adult rats. Further, young rats excreted more BEG in urine than adults. No BES was detected in the urine of either age group. An unknown metabolite was detected in the urine of both age groups. It constituted less than 1 % of the total radioactivity excreted in the urine of adult rats comparedEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 79 to young rats. Lower levels of EGBE derived radi oactivity were seen in all tissues of young animals compared to tissues of old animals.
In a study performed by Medinsky et al ., in 1990, EGBE was admi nistrated via drinking water to male F344/N rats. In parallel 2 others ra t groups were dosed with Ethylene Glycol Methyl Ether (EGME) and Ethylene Glycol Et hyl Ether (EGEE) for comparison with EGBE. Rat were allowed access for 24 hours to 2-butoxy[U- 14 C]ethanol (50 mCi/mmol) (purity 99.9 %) at three doses: 290 ppm (237 μ mol/kg bw), 860 ppm (401 μ mol/kg bw) and 2590 ppm (1190 μ mol/kg bw). Exhaled CO 2 , urine and faeces were coll ected during 72 hours from the beginning of the exposure. At the end of the 72-hour collection period, cages were washed to determine the amount of radioac tivity remaining, the total amount of water consumed by each rat in the drinking wate r was measured and recorded. Rats were killed and carcasses analysed for total 14 C. Urine was analysed for pa rent compound and metabolites.
80 % of the radioactivity excreted appeared during the first 24-hour period. For metabolites, the major compound identified was BAA (50-60 % re latively constant for all doses), others compound identified were ethylene glycol, the glucuronide conjugate of EGBE and EGBE. No N-acetylglycine conjugate of BAA was identified in this study.
Effects of multiple dosing of male Fischer 344 rats with EGBE on its own disposition and metabolism were studied (Ghanayem et al ., 1992). This study was performed together with mechanistic studies on haematotoxicity (see § 4.1.2.2.3). Rats were treated with 125 mg/kg EGBE for 3 or 7 days by gavage follo wed by a single dose of 125 mg/kg dose of 14 C EGBE on day 4 and 8 respectively. Rats were placed in metabolism cages and EGBE metabolism analysis were performed.
No quantitative or qualitative alteration of EG BE metabolism and disposition were caused by repeat exposure compared to those observed in rats treated with a single dose. The ratio of BAA, the glucuronide and sulfate conjugates and parent EGBE excreted in the urine of rats treated for 4 or 8 days were relatively similar to those from rats treated with a single EGBE dose. Therefore, it is unlikely that toleran ce development to the hemolytic effects of EGBE (seen in many studies) is caused by increased de toxification of EGBE or inhibition of EGBE metabolism to BAA.
[ 14 C]EGBE (purity 96.3 %) was administered by gava ge to male F344 rats (3 rats/dose) at doses of 10 and 125 mg/kg bw as a solution in wa ter. An additional group of 3 rats was dosed with [ 14 C]EGBE at 10 mg/kg in a corn oil vehicle (Dow, 1993). Samples of blood were taken from the rats administered EGBE in wate r at 1, 3, 6, 12 and 24 hr after dosing for determination of total 14 C (plasma) and for analysis of EGBE and BAA (whole blood). Urine from each animal was collected at interval s of 0-12 and 12-24 hr after dosing and the metabolic profile assessed. Faeces were collected for 24 hr. Expired 14 CO 2 was collected throughout the study. Each animal wa s killed 24 hr after dosing.
Haematological effects were s een in 2 rats of th e 125 mg/kg bw dose group (the third rat was eliminated because of mis-dosing). 59 % of the do se was excreted in the urine during the first 24 hr following administration of 10 mg/kg EGBE in either corn oil or in water. 10 % was excreted as 14 CO 2 in 24 hr. At 125 mg/kg bw the 2 rats eliminated 37.7 and 70.3 % of the dose in urine during the first 24 hr and 7.6 and 8.5 % of the dose as 14 CO 2 . The maximum radioactivity plasma concentration was reached apparently just before the first blood sample was taken. Metabolite profiles in urine we re similar to than of the Medinsky et al. (1990) study and the Ghanayem et al ., 1987a study. Nearly 40 % of the 14 C in urine was present as BAA during the first 12 hr following 10 mg/k g bw and 65 % after 125 mg/kg bw EGBEEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 80 dosing. An additional 15 % of the 14 C in urine was present as the glucoronidase-labile conjugate of EGBE at 10 mg/kg bw and 10 % at 125 mg/kg bw. Ethylene Glycol (EG) was confirmed in this study as a metabolite of EGBE, but was present in small quantities.
The potential of EGBE to form fatty acids conjugates was studied in F344 rats and in vitro (Kaphalia et al. , 1996). [Ethyl-1, 2- 14 C] BE (purity 99 %) was administered as a solution of water via oral route to 3 rats at a dose of 500 mg/kg bw. Three other rats served as controls and were given the same amount of water. The animals were killed 2 hr after exposure. The liver was excised and analyzed for radiolabeled lipids.
Approximately 16 % of the total ra dioactivity detected in the liver was associated with lipids, mostly in the phospholipid fraction (about 85 % of the total lipids). 3 % radioactivity of the total lipids was detected in the ester fraction.

The effects of co-administrati on of EGBE (99 % purity) with alcohols (Ethanol (EtOH), nPropanol (n-PrOH) and n-Butanol (n-BuOH)) was studied (Morel et al. , 1996). Three sets of four groups of 10 male SD rats were used for the experiment in which all substances were administered orally by gavage (each set of groups differed fr om the others by the alcohol administered). In each set, the first group receive d distilled water and served as control. The second group was treated with onl y alcohol (10 or 30 mmol/kg) in distilled water, except for n-butanol (no vehicle), this group served as alcohol control group. Th e third group was dosed with EGBE at dose of 5 or 1 mmol/kg. The fourth group was given simultaneously alcohol and EGBE at the same doses than group 2 and 3 in distilled water (exc ept for n-butanol: no vehicle). The haemolytic effect of EGBE (5 mmol/kg bw) was evaluated 4 hr after treatment by Red Blood Cell (RBC)-counting. Urinary concen tration of BAA was determined on urine collected during 24 hr.
Administration of EGBE alone (5 mmol/kg) resulted in a signifi cant decrease on average RBC (26 %) accompanied by a large increase in the concentration of free plasma Haemoglobin (Hb). The co-administration of n-BuOH or n-PrOH (10 mmol/kg bw) partially reduced the haemolytic effect of EGBE. No ef fect was seen with co-administration of 10 mmol/kg bw EtOH. At the dose of 30 mmol/ kg bw, each of the three alcohols coadministrated with EGBE produced a complete protection against haem olytic effects. The alcohol control treatment did not have any effect on the haematological parameters.
Urinary excretion of BAA in rats treated only with 1 mm ol/kg bw EGBE was 0.083 mmol/24 hr, which represents about 30 % of the ingest ed dose. The simultaneous administration of each of the three alcohols at a dose of 10 mmol/kg with EGBE did not modify the 24 hr urinary excretion of BAA, whereas at dose of 30 mmol/kg, decreases of 43, 33 and 31 % were observed for EtOH, PrOH and BuOH respectively.

Mice
In a series of studies, the mechanism of EG BE (purity 99 %) accumu lation in forestomach was assessed (Poet et al ., 2003). Five experiment were conducted to examine various endpoints:
- target tissue histology / forestomach irritation
- tissue dosimetry and pharmacokinetics
- fur depositionEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 81 - salivary excretion

- retention in stomach content
For tissue dosimetry and pharmacokinetics, groups of 30 female mice were administered EGBE (in saline solution) at doses of 50 or 250 mg/kg by IP injecti on or 250 mg/kg by oral gavage. Blood samples were taken and then th e mice were killed 0.5, 1, 3, 6, 9, 12 and 24 hours after dosing. Kidney, liver and stomach tis sues were rapidly collected at each time point. The AUC and kinetic parameters for both EGBE and BAA were calculated.
EGBE concentration in tissues paralleled the le vels in blood regardle ss of dose or exposure route. For the 250 mg/kg dose to either rout e, concentration of EGBE were higher and persisted longer in forestomach than in blood or in other tissues. Regardless of the route, T1/2 and AUC were higher in forestomach than in other tissues (Blood T1/2 could not be calculated because it was shorter than the earliest time point collected. The T1/2 for BAA was 2.1 hours after gavage administration.
By 24h, about 50 % of the total dose was eliminat ed in the urine (48 % for oral route) as EGBE, BAA or a conjugate. Following gavage administration of EGBE, BAA was the major urinary metabolite (38 % of the dose); there was also a small quantity (less than 0.2 %) of free EGBE, a conjugate of EGBE (up to 3 % - pr obably glucuronide).a nd a conjugate of BAA (between 0 and 7 %).

Male and females B6C3F1 mice we re administered EGBE (in distilled water) at dose of 600 mg/kg (Deisinger and Boatman, 2004). Control anim als were treated with distilled water. At 5, 15, 45 and 90 min following the dose (5 min only for controls) animals were sacrificed and about 100 mg of blood, liver and forestomach were collected. Samples were analysed for EGBE, BAL and BAA by GC-MS.
Five minutes after the administration, forestom ach contained 50 fold more EGBE than blood or liver. Elimination from blood and liver sample s was rapid (but still measurable at 90 min post dosing) whereas EGBE concentration in the forestomach decreased gradually and represent still 62 % and 31 % of the dose measur ed 5 minutes after administration in females and males respectively. Low con centrations of BAL we re observed in all organs at maximum levels five minutes after administration. Forest omach levels were 10 fold higher than blood or liver concentrations. BAA concentrations incr eased till 45 and 90 min sample time. At all time points, BAA concentrations were lower in the forestomach than in blood or in the liver. No sex differences were detected for organ concentrations of EGBE and BAA but females seems to have higher BAL organ concentr ations than males (at all time points).

Summary oral route :
EGBE kinetic properties via oral route were re ported in up to 10 studies, 7 performed with F344 rats and one performed with SD rats. Two series of studies were also performed on mice These studies demonstrated the following results.
Absorption of EGBE orally administered was ra pid and essentially complete (assumed to be 100 %). Target organs in which the most radiolabeled compound were found were: the forestomach, the liver and the kidneys. The majo r metabolite of EGBE is BAA which formed through a mechanism involving alcohol and al dehyde dehydrogenases which seems to be saturable. Simultaneous administration of EGBE and a primary al cohol (ethanol, n-propanol or n-butanol) in sufficient quantity inhibited BAA fo rmation. The other metabolites were, inEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 82 order of magnitude: the glucuronide conjugate of EGBE (whose percentage increases relative to the dose at the expense of the BAA forma tion) and two minor metabolites, the sulphateconjugate of EGBE and ethylene glycol (which was not observed in all studies). Elimination is rapid and occurs mainly by urinary excre tion. A small amount of metabolised EGBE is eliminated as CO 2 in expired air (0 to 20 % for a hi gh and low dose respectively). A small amount of unchanged EGBE (approximately 1 %) is also eliminated in expired air. In two studies, BAA, BEG and EGBE were found in bile . EGBE did not accumulate in tissues. The metabolic profile was not changed after repeatedexposures compared to acute exposure. Agerelated difference was observed: young rats eliminating EGBE via CO2 and urine (with significantly less BAA and more BEG) to a greater extent than adult rats.

Other routes

Rat
Female SD rats (4 rats) were administered EGBE (purity unknown) via IP route at dose of 2.5 mmol/kg EGBE concurrently with 20 mmol/kg EtOH (Romer et al ., 1985).
The degradation of EGBE was almost completely inhibited in combination with simultaneous EtOH treatment.
Radiolabeled EGBE (purity 99 %) was administered subcutaneously to 3 male Wistar rats at the dose of 118 mg/kg. Faeces, urine and expired air were collected during the 72-hour period following EGBE administration. After terminati on the following organs and tissues were collected: liver, kidneys, spleen, fat, testes , thymus, sternum (including bone marrow) and blood (Bartnik et al ., 1987).
EGBE was metabolised and eliminated primarily in urine. Within 72 hr over 78 % of the radioactivity was found in the urine. A small amount (less than 1 %) was found in the faeces and 10 % was eliminated in the exhaled air as CO 2 . 1.6 % of the dose was found absorbed on activated charcoal indicating that the exhaled air contained only a small amount of the test compound or volatile metabolites. At the end of the experiment, 4.8 % of the radioactivity was found in the carcass. Spleen and thymus followed by the liver were the 3 major organs where the concentrations of radioactivity were highest.
In a series of studies, EGBE was administered via intravenous route in order to study the effect of dose, age and inhi bition of metabolism on the ki netic parameters of EGBE (Ghanayem et al ., 1990a). Male Fisher 344 rats (young: 3-4 mont hs old and old: 12-13 months old), three to four animals per treatmen t group were given IP doses of either pyrasole (250 mg/2.5 ml of 0.9 % saline/kg – alcoho l dehydrogenase inhibition), cyanamide (50 mg/2.5 ml of 0.9 % saline/kg - aldehyde dehydroge nase inhibition) or probenecid (50 mg/2.5 ml of 0.9 % saline alka linised with NaHCO 3 /kg – renal tubular transport inhibition). A second dose of probenecid was administered 4 hr later. Twenty to 30 minutes after initial treatment, all groups received a single bolus dose of ra diolabeled EGBE (31.25, 62.5 or 125 mg/kg) delivered IV. Blood samples were collected at 5, 15 and 30 min and at 1, 2, 4, 6 and 8 hours for analysis of radioactivity. EGBE and metabol ites were determined in whole blood and in plasma.

- Effect of dose and age: EGBE in blood is proportional to the administrated dose. At the same dose level, concentrations obtained in old animals were higher than in the young ones. The mean concentration of radioactivit y in plasma was higher than in whole blood.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 83 Only EGBE and BAA were found in the plasma . There was no significant effect of dose on T 1/2 or Vd (Volume of distribution) of EGBE. C max , AUC and Cl of EGBE were affected by the level of admi nistrated dose (increase of C max and AUC and decrease of Cl with increasing doses of EGBE). t 1/2 , Vd and Cl were not aff ected by the age of rats whereas C max and AUC were found to be age-dependent (increased in th e older rats). For BAA, there was a significant increase in t 1/2 in the older rats at the low and high dose but not in the middle. There is also a significant effect in AUC and C max , which tend to be higher in older animals than in young, this effect is more pronounced with high doses.

- Effect of alcohol dehydrogenase inhibition by pyrazole: the radioactivity was higher in plasma than in whole blood. The total radioact ivity was lower at all time point and at both dose levels than in rats treated with EG BE only. BE-glucuronide conjugate (BEG) was detected in the plasma (not in rats treated without pyrazo le). Pyrazole resulted in significant increases in t 1/2 , AUC and Cl of EGBE at both dose levels with no effect on Vd. For BAA, there was a significant decrease in the values of AUC, C max and t 1/2 in rats treated with pyrazole.

- Effect of aldehyde dehydrogenase inhibition by cyanamide: the mean concentration of radioactivity was higher in w hole blood than in plasma at all time points beyond the first 30 minutes after EGBE administration. Furthermore, the mean concentration of total plasma radioactivity time profiles was higher at all time points in EGBE treated rats compared with that in rats treated with cyanamide + EGBE. Increases of t 1/2 , AUC, Vd and Cl of EGBE were seen in the cyanam ide treatment group. No effect was seen on C max . The mean t 1/2 of BAA was more than doubled but the difference was not statistically significant. A significant decrease of the C max and AUC of BAA was seen.

- Effect of inhibition of renal tubular anion transport by prob enecid : a consistent increase in the mean whole blood and plasma concentrat ion of total radioactivity was seen. There were no significant changes in C max , AUC, t 1/2 , Vd or Cl of EGBE. In contrast, a significant increase in t 1/2 of BAA (the increase being greater at the lower dose). Probenecid also significantly increased the AUC of BAA (also greater at the lower dose). C max of BAA was not significantly affected.
In summary, this study has confirmed that BAA is the proximate hemolytic agent and that it is formed from EGBE by a metabolic pathway involving alcohol and aldehyde dehydrogenase. The renal organic acid transport may play a role in the clearance of BAA. The higher sensitivity of older rats compared to younger rats concerning haemotoxicity was due to a combination of factors: compromised renal cl earance of BAA by the renal anion transport system in older rats, increased EGBE metabo lism to BAA and diminished degradation of BAA to CO 2 and greater sensitivity of er ythrocytes in the older rats.

Mouse
Radiolabeled EGBE (purity 97.6 %) was injected intr avenously to female B6C3F1 mice at dose of 10 mg/kg (Green, 2000b). Animals were terminated at 4, 24 and 48 hours after dosing (4 animals per sacrifice time). One animal fo r each sacrifice time was submitted to whole body autoradiography. The remaining three animals were submitted to an analysis of stomach and contents, the determination of radioactivit y was made for forestomach, glandular stomach and stomach contents (free and bound radioactivity).
Four hours after dosing, the highest concentrat ions of radiolabel were found in the liver, Harderian glands, salivary gl ands, nasal passages, oesoph agus, buccal cavity and on theEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 84 surface of the feet. A lower concentration of radiolabel was seen in the stomach, Gastro Intestinal (GI) tract co ntents and mucosa, in the kidney cortex and associ ated with bones. Background levels of labelling were seen in the lung and the remaining internal organs.
After 24 hours, the highest concentrations of radiolabel were observed in the liver, bone, Harderian glands, surfaces of the feet and bu ccal cavity. Slightly lower amounts were seen in the mucosa of the stomach, GI tract and th e oesophagus. A lower level of labelling was observed in the salivary glands and kidney cort ex. A much lower level of labelling was seen in the spleen, GI content and the kidney medulla . Background labelling was seen in the lung and the rest of internal organs.
48 hours after dosing, radiolabel was observe d in the liver, bone, buccal cavity and oesophagus. The mucosa of the forestomach and glandular stomach were also radiolabeled although there was no significant difference between them. Lower levels were observed in the salivary glands and GI tract mu cosa. The lung and the remainder of the internal organs had background levels of labelling.
The total radioactivity in the forestomach and glandular stomachs was similar over the duration of the study with the bulk of the radio activity being present in the stomach walls: 80- 95 % of the radioactivity was bound to pr otein at the two later time points.
In this study, the radioactivity in the stomach tissue appears to be derived from the systemic circulation and by ingestion from the buccal cav ity. The origin of the latter is unknown, but according to the author, it may be derived from the salivary and Harderian glands which contained significant amounts of radioactivity.
In a series of studies, the mechanism of EG BE (purity 99 %) accumu lation in forestomach was assessed (Poet et al ., 2003). Five experiment were conducted to examine various endpoints:

- target tissue histology / forestomach irritation

- tissue dosimetry and pharmacokinetics

- fur deposition

- salivary excretion

- retention in stomach content

- For Tissue dosimetry and pharmacokinetics, gro ups of 30 female mice were administered EGBE (in saline solution) at doses of 50 or 250 mg/kg by IP injection or 250 mg/kg by oral gavage. Animals were sacrificed 0.5, 1, 3, 6, 9, 12 and 24 hours after dosing. The AUC and kinetic parameters for bo th EGBE and BAA were calculated.

EGBE concentration in tissu es paralleled the levels in blood regardless of dose or exposure route. For the 250 mg/kg dose to eith er route, concentration of EGBE were higher and persisted longer in forestomach than in blood or in other tissues. For BAA, during the first three hours after IP administ ration the maximum con centration were found in blood, kidneys and liver. After 3 hours, fore stomach concentration of BAA were higher than for other tissues. Unlike in the forestomach, BAA concentrations in the glandular stomach tissue were similar to other tissues.
Regarding kinetic parameters, for EGBE bot h T1/2 and AUC were higher for gavage dosing than IP dosing. Regardless of the r oute, T1/2 and AUC were also higher inEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 85 forestomach than in other tissues (Blood T1 /2 could not be calcu lated because it was shorter than the earliest time point collected. The T1/2 for BAA were 1h for IP injection.
By 24h, about 50 % of the total dose was elimin ated in the urine (54 % for IP) as EGBE, BAA or a conjugate for the 250 mg/kg administ rations. A majority of BAA was found (50 for the IP), a small quantity (l ess than 0.2 %) of free EGBE and of a conjugate of EGBE (up to 3 % - probably glucuron ide) was detected. The last substance detected was a conjugate of BAA (between 0 and 7 %).

- For saliva excretion assessment, groups of mi ce were administered EGBE at doses of 250 mg/kg by IP injection or oral gavage. Saliva tion was induced by injection of pilocarpine a few minutes before saliva collection. Saliva was collected under anes thesia at various times after administration (up to 2.5 hr) by peri ods of 15 to 30 min (t o obtain aliquots of saliva of 100-200 μ l). Blood was collected at the midpoint of each saliva collection interval and at the end of saliva collection. Kinetic parameter of EGBE and BAA for blood and saliva were calculated.
For EGBE, peak blood and sali va concentration were detect ed at 15 min and 7.5 min for blood and saliva respectively, regardless of th e route. Concentration in blood and saliva were nearly identical at all time points and were below the level of detection after 1.3h. AUC and T1/2 in blood and saliva were similar.
For BAA, the time curve was identical fo r blood and saliva, the values of saliva concentrations being 4 fold lower than the blood levels. The T1/2 was 2 fold higher in blood than in saliva (1.4 and 1.6 hr for IP and gavage resp ectively in blood).

- For retention in stomach contents, a group of animal was treated with 250 mg/kg EGBE by IP route. After sacrifice (3, 6 and 9 hours after exposure) EGBA and BAA were quantified in stomach tissue and stomach contents.

The concentration of EGBE was higher in the stomach content than that of the forestomach tissue. No EGBE was detected in either blood or glandular stomach tissue at any time point. The estimation of the T1/2 fo r EGBE in the content of the stomach was 4.8h following IP injections, l ongest of all the tissues.

Summary other routes :
Two studies were performed via IV route (one in rats, the other in mice), two studies were performed via IP route and one vi a SC route in rats. Target orga ns evidenced in the previous described studies were confirmed in these studies: spleen, liver, thymus and stomach. However, a slight difference was seen in th e distribution of the substance between the forestomach and the glandular stomach after an IV injection. The di stribution to stomach comes from systemic circulation and also from ingestion of EGBE (which could come from salivary glands).
In these studies, which are generally mechanis tic studies, it was demonstrated that the BAA formation was caused by Alcohol Dehydrogenase and Aldehyde Dehydrogenase in the liver. This finding is validated by competitive inhibi tion studies performed w ith Ethanol. Inhibition of renal tubular anion transport caused a decreas e in the renal excretion of BAA and therefore an increase of the EGBE toxicity.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 86 4.1.2.1.2 Studies in humans

Inhalation

Seven human male volunteers were exposed to 20 ppm EGBE (0.85 mmol/m 3 ) via inhalation route for a period of 2 hours during li ght physical exercise (50W) (Johanson et al ., 1986a). Blood samples were collected dur ing the exposure period and 3 hr afterwards and analysed for the determination of EGBE blood concentrat ion. Urine was collected for a period of 24 hours, the first sample was co llected immediately before th e volunteer entered the exposure chamber and thereafter sampling was made at 2- hour intervals for 6 hours. The samples were assayed for EGBE and BAA.
No signs of adverse effects c ould be related to the exposure of EGBE and no effects were seen on electrocardiograms. The concentration of EGBE in blood rose rapidly reaching an apparent plateau level within 1-2 hours. The decay after exposure was rapid, with a biphasic slope in a semi-logarithmic plot (data are too limited to permit the calculation of more than one slope constant). EGBE could no longer be de tected in the blood 2-4 hours after the end of exposure. The average elimination t 1/2 was 40 min, the average platea u level in blood was 7.4
μ mol/l, the average blood clearance was 1.2 l/min and the steady-state volume of distribution was 54l. The total amount of EGBE excreted in urine was less than 0.03 % of the total uptake and the t 1/2 of EGBE in urine was 1.36 hr. In urine, BAA reached a maximum concentration between 5 and 12 hours after the beginning of exposure. Elimination also reached a maximum between 2 and 10 hours after the beginning of th e exposure. Great vari ations are observed between subjects. T 1/2 for BAA in urine was calculated at 5.77 hours.
According to the authors and on the basis of the very high in vitro blood/air partition ratio (above 1000, Johanson and Dynesius, 1988), one w ould expect the respiratory retention of EGBE to be limited by the alveolar ventilati on and approximate 80 % at 50 W. However, the retention was about 57 % in the present study. The explanati on may be that EGBE is partly adsorbed on the surface of the respiratory airways during inhalation and desorbed during exhalation.
Five human volunteers were exposed to EGBE at 20 ppm for 2 hours during light physical exercise (50 W) (Johanson a nd Johnsson, 1991). Venous blood samples were collected and analysed for BAA before and immediately afte r exposure and also at 4 and 6hr after the beginning of exposure. Urine was collected every 2 hours and assessed for BAA content.
After 2 hours of exposure BAA was found in bl ood. An average maximum concentration of 45 μ M BAA was reached after 2-4 hr. The decrease in BAA blood levels from 4 to 6 hr corresponds to an average half-time of 4.3 hr. Th e time profile in blood seems to be similar to that for urinary excretion, wher e the maximum occurred at about 5 hr and the half-time was estimated to 4 hr. The average clearance of BAA was estimated to be 23-39 ml/min and is only about 1/3 of the glomerular filtration rate of about 125 ml/min. The low renal clearance may be due to binding of BAA to blood proteins and absence or low efficiency in tubular secretion. The low pKa of BAA ( 3.5) indicates that more of 99 % of BAA present in urine is present in the ionised form and is not availabl e for tubular re-absorption at normal urine pH. The average Vd of BAA was calculated to be 15 l. This value is approximately equal to the volume of extracellular water.
Respiratory uptake of EGBE was investigated (concurrently with 9 other solvents) (Kumagai et al ., 1999). The uptake of a chemical with high bl ood/air partition coeffici ent is predicted toEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 87 be high. But when a chemical is very water solubl e, it can be partially absorbed in the mucus layer during inhalation and rele ase during exhalation, leading to a low respiratory uptake.
Four human volunteers were submitted to 25 pp m of EGBE (whole body in an inhalation chamber) for 10 minutes. The same people were also submitted to inhalation of 9 other substances in the same test conditions. Exhale d air was collected from 1 min before exposure to the end of the exposure period.
The mean respiratory rate for each solvent was from 12.1 to 14 min -1 . The mean tidal volume was from 470 to 530 ml, no differences were found among the solvents. The results obtained with the different solvents suggests that the wash in/wash out behaviour cannot completely explain the actual respiratory behaviour of these solvents.

Summary human inhalation route:
Three studies were reported in which human volunteers were exposed to EGBE by inhalation route. Kinetic parameters found in these studies were equivalent to t hose described in human studies earlier in the report for others rout es of administration. Theoretical absorption (calculated) of EGBE via i nhalation route was found to be 80 %. However, measurements performed showed a real absorption of 55 to 60 %. This difference is explained by a “wash in / wash out” mechanism: due to its hydrophilic pr operties, EGBE is adsorbed to the surface of the respiratory tract during inspiration and it de sorbed during exhalation leading to a decrease in the real uptake of substance. In the risk characterisation section, a 60 % of absorption for EGBE inhalation is used .

Dermal

In vitro

In an in vitro skin penetration test, the mean absorption rate of undiluted liquid EGBE (purity > 99 %) through human epidermis assessed (IC I, 1982a and 1982b). In this test, the penetration rate of EGBE was compared with 3 other glycol ethers (EGEE, EGME and Propylene Glycol Methyl Ether (PGME)) and 1 acetate (EGEEA). Disks of human abdominal skin were placed in diffusion chambers, the membrane integrity was assessed before the test by measurement of their permeability to tritiated water. The glycol ether absorption rate was measured for a period of 8 hours. After the te st, the potential of the tested substance for impairing the epidermal diffusion barrier function was assessed by measuring the permeability to tritiated water again. A calculation of the damage ratio was made: Permeability constant after glycol ether contact / permeability constant before glycol ether
contact.
For EGBE the mean rate of penetrati on calculated in this study was 0.20 mg/cm 2 /hr ( ± 0.03 SEM, n=8). EGBE did not produce large alteration in permeability in this study: damage ratio = 2.07. Human skin in vitro will always deteriorate over time and a ratio greater than 1 would be expected. The measured ratio indicates a marginal effect w ith little damage to the skin following prolonged exposure.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 88 Another skin penetration study in vitro was performed with liquid EGBE (Eastman Kodak, 1991). Human abdominal skin (stratum corneu m) was exposed to EGBE in Franz-type diffusion cells. The integrity of each skin sample was determined before and after the test by measuring the permeability to tritiated water.
The skin absorption results for EGBE were measur ed twice due to high variability observed in the first study. The mean abso rption rates were: 0.857 mg/cm 2 /hour ± 0.282 in the first experiment and 1.52 mg/cm 2 /hour ± 0.37 in the second experiment. The damage ratio was 3.25 ± 3.33 in the first experiment and 5.14 ± 4.99 in the second experi ment. Due to the high variability in each experiment, mean absorpti on rates were calculated separately for the undamaged skin (n=8) and the damaged ones (n =4), the results from the damaged skin specimens were about 3 times higher than the undamaged ones (3.39 mg/cm 2 /hour vs 1.19 mg/cm 2 /hour). When the results from the four cells showing the high damage ratio are excluded from the calculation of the overall mean result, the mean damage ratio is 1.66 ± 1.31.
In a study performed by PMIC in 2001, in vitro percutaneous absorption of EGBE through human skin was assessed. Two different EGBE concentrations 10 and 5 % in an oxidative hair dye formulation was applied on human sk in during a period of 30 minutes. For each experiment 33 mg of the different fo rmulations (corres ponding to 20 mg/cm 2 ) were applied on the skin surface (1.76 cm 2 ). At the end of the 30 minutes of exposure, the surface of the skin was washed to eliminate the residual mixtur e. The diffusion of EGBE was monitored during the 24 hours following the applications. The rece ptor fluid was collected 2, 4, 6, 10, 21 and 24 hours after the beginning of e xposure. At the end of the 24 hr observation period, tissues (horny layer, epidermis and dermis) were analysed for EGBE remaining. This study was performed in compliance with GLP and fo llowing the recommendations of FDA, AAPS, COLIPA, SCCNFP and OECD for this kind of test.

Results are expressed in μ g equivalent of EGBE ( μ g/cm 2 ) and in % of the applied dose for all the compartment analysed (see table 41).

Table 4.41: Quantities of EGBE analysed in the di fferent system compartments for the 2 tested concentrations (5 and 10 %)
EGBE 5 % EGBE 10 %
μ g/cm 2 % of the applied dose μ g/cm 2 % of the applied dose Filter (F) 29 ± 3 3.13 ± 0.34 53 ± 8 2.82 ± 0.38 Washing (W) (30 min) 639 ± 66 68.6 ± 7.5 1223 ± 178 65.2 ± 9.5

Skin excess (F+W) 668 ± 68 71.7 ± 7.8 1275 ± 182 68.1 ± 9.8 Epidermis (E) Including stratum corneum (SC) 0.112 ± 0.065 0.012 ± 0.007 0.283 ± 0.128 0.015 ± 0.006

Dermis (D) 0.023 ± 0.01 0.002 ± 0.001 0.101 ± 0.051 0.005 ± 0.003

Receptor fluid (RF) 113 ± 54 12.1 ± 5.9 235 ± 138 12.5 ± 7.3 Total skin +RF (SC+E+D+RF) 113 ± 54 12.1 ± 5.9 235 ± 138 12.5 ± 7.3

Total recovery (%) 84 ± 4 81 ± 4EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 89 These results showed that no accumulation of EGBE in skin occured. For these two concentrations, the percentage of absorption we re similar (12.1 and 12.5 % for the 5 % and 10 % concentrations).

In vitro percutaneous absorption of liquid glycol ethers (including EGBE purity 98 %) was studied (Wilkinson and Williams, 2002). Human breast skin, full thickness or dermatomed (stratum corneum + upper dermis) was exposed to EGBE in aqueous solution at different concentrations (3 mg/ml or 6 mg/ml, 100 or 200 μ l in the skin) or undiluted (10.5 μ l). Percutaneous absorption was measured for 24 hours using flow through diffusion cells. Tissue culture medium was used as a receptor fluid wi th 2 % (w/v) Bovine Serum Albumin (BSA) or 2-6 % (w/v) PolyEthylene Glycol 20 (PEG 20) added for some studies.
In aqueous solution, a st eady state flux of 544 ± 64 nmol.cm -2 .h -1 (0.064 mg/cm 2 /hr) was found with dermatomed human skin. Reducing the dose to 100 μ l decreased the steady state flux of EGBE by about 55 %. Using full thickness skin increased the time to steady state (tau) and reduced the steady state flux. Absorption rates of undiluted EG BE in finite dose exceeded those measured with aqueous so lutions, though the apparent permeability coefficient was higher with the aqueous doses.

Modelling
An alternative for dermal permeation estimation is the model SKINPERM (version 3.0), which provides an estimation of the amount of EGBE that penetrates the skin either from the vapour phase, the neat liquid or an aqueous solution. SKINPERM describes dermal exposure and absorption using a quantitative structure activity relationship (QSAR) which is based solely on the biological structure of the skin and the physico-chemical properties of the substance under investigation. SKINPERM 3.0 was validated with an extensive database of experimentally measured permeation coefficients and is considered valid for substances with log Kow values in the range from 0 to 4. The model estimates the permeation rate for EGBE to be 0.63 mg/cm 2 /hr, which is consistent with the measured in vitro data. This model is used for EGBE only for comparative reasons as a lot of measured data is available.

In vivo
Five men, who have been involved in another study 2 years ago (Johanson et al. , 1986a), were exposed to neat EGBE for 2 hr by placing four finge rs of the left hand into a vessel filled with pure liquid solvent (Johanson et al. , 1988). At regular intervals, thickness of the skin and the finger volume were measured. Blood and urine analysis were performed during a 24-hour period after the begi nning of exposure.
The skin of the exposed fingers was not irritate d but appeared somewhat more rigid and less elastic after exposure and a wrinkled appearan ce. This effect reach es a maximum 2-4 hours after exposure and gradually disappeared. Conc urrently, the volume of the fingers and the thickness of the skin decreased a nd then return to the normal. EGBE was detected in the blood of all subjects after exposure. Estimates of th e skin uptake showed gr eat variations between individuals (more than ten-fold variation): 7 to 96 nmol/min/cm 2 (0.05 - 0.63mg/cm 2 /hr) with a geometric mean of 20 nmol/min/cm 2 (0.14mg/cm 2 /hr). The elimination t 1/2 for EGBE in blood ranged from 0.6 to 4.8 hr (mean 1.3 hr). The excretion rate of BAA in urine increased during the first hours of exposure with a maxi mum 5 hours after the beginning of exposure. The cumulative excretion of BAA ranged from 2.5 to 39 % of the uptake of EGBE (with a mean of 17 %).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 90 The percutaneous absorption of EGBE vap our was assessed in f our male volunteers (Johanson and Boman, 1991). Subj ects were exposed to EGBE by inhalation during 2 hours at concentration of 50 ppm (2 mmol/m 3 ). The subjects were breathi ng chamber air while sitting outside the exposure chamber, mouth breathing was assured by the use of a nose clip. After the two hours, one hour of pause was made then a second period of exposure was performed. For this second exposure, subjects were sit ting inside the exposure chamber (subjects were naked apart from a pair of shorts – the estimated surface area exposer being 16000 cm 2 ) but breathing air was supplied from a tank of clean ai r. Heart rate was monitored during the entire day. Each volunteer participated in two experiments, one at normal (23° C, 29 % relative humidity) and one at raised temperature and humidity (33° C, 71 %). The two experiments were separated by two weeks or more. Capill ary blood samples were collected during the experiments for the determination of EGBE . Based on earlier observations (Johanson et al.
, 1986a), the relative respiratory uptake of EGBE was assumed to be 60 %.
The EGBE blood concentration during the mouth exposure increased duri ng the first hour and seemed to reach a steady state at about 3 μ M (1.8-4 μ M) during the 2 nd hour. The mean respiratory uptake was 1.3 mmol or 11 μ mol/min and the apparent blood clearance 3.8 l/min. For the percutaneous exposure, the concentrat ion of EGBE in blood increased to about 9 μ M during the second hour of exposure. In se ven of the experiments the EGBE blood concentration was 2.4-5.5 times hi gher after skin expos ure than after mouth exposure, in one it was lower. This difference was also reflected in the high percutan eous absorption of 31 (8.6-48) μ mol/min, or 2.5-5.9 times higher than the re spiratory uptake. The half life of EGBE in blood after the skin exposure averaged 34 mi n (19-53 min). Only small and inconsistent differences in heart rate were found between the 2 routes of exposure, thus the observed differences in blood concentration and uptak e rates of EGBE cannot be explained by differences in pulmonary ventilation. The conc entration of EGBE in blood appeared to be slightly raised during mouth exposure to hot and humid, compared with normal chamber air. The increase may be partly explained by a raised pulmonary ventilation. The tendency towards higher blood concentrations in hot and humid conditions was more pronounced during the skin exposure. Howeve r, the differences between the two exposure conditions were not statistically significant.
Six human volunteers were exposed arm only to 50 ppm ( 13 C 2 )EGBE (purity 99.54 %) for 2 hr (Corley et al., 1997). Blood samples were collected into the antecubital vein of the unexposed arm for the analysis of both EG BE and its major metabolite BAA. Finger prickblood samples were taken from the exposed arm only at the end of the 2 hr exposure to compare with the blood obtained in the antecub ital vein. Blood samples were obtained before exposure and at 10, 20, 30, 40, 60 and 1.5, 2, 2.25, 2.5, 3, 3.5, 4, 8, 12, 16 and 24 hr after the initiation of the 2 hr exposure. Urine samples were collected before exposure and at 0.12 and 12-24 hr intervals following the initiati on of exposure for metabolite analysis.
EGBE was not detected in blood samples from the unexposed arm until 30 minutes. By 1.5 hr EGBE was detectable in all 6 subjects. An apparent steady state was reached for EGBE between 1.5 and 2 hr. EGBE was rapidly cleared from the blood with an apparent elimination
t 1/2 of 0.66 hr (in 2 of the 6 subjects).
As EGBE, BAA was not detected in blood samples from the unexposed arm until 30 minutes. BAA was detectable in all six subjects by 1 hr. Peak blood concentrations were reached 3-4 hr after the start of the 2 hr e xposure. BAA was less rapidly cl eared from the blood than EGBE, with an apparent elimination t 1/2 of 3.27 hr.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 91 The ratio of the finger prick blood sample ta ken at the end of the 2 hr exposure to the corresponding blood sample taken from the une xposed arm averaged nearly 1500/1 indicating a considerable influence by local absorption.
No free EGBE was detected in any urine sample s. Following hydrolysis to cleave acid-labile conjugates, EGBE was detectable in only 2 urine samples (0-12 hr). No EG nor glycolic acid were detected in urine.
BAA was eliminated in urine during the first 12hr collection interval .2/3 of the total amount of BAA excreted in the urine wa s in the form of an acid-lab ile conjugate. In this study the skin permeability coefficient was estimated to be 3 cm/hr.
Four volunteers (2 males and 2 females, aged 28-33) were exposed on 9 separate occasion, each occasion separated by at least 3 weeks (Jones et al. , 2003). All exposure were 50 ppm EGBE for 2h. Two exposure were performed w hole body and two skin only at 25° C, 40 % relative humidity and volunteers wearing shorts and T-shirts (baseline conditions). Other exposures were skin only and were performed with one parameter changed: high humidity (60 % and 65 %), low and high temperature (20 and 30° C), minimal clothing (shorts (and bra) only and overalls (all-in-one boiler suit) and an industrial scenario (overalls and high humidity and temperature (30° C and 60 % relative humidity). Total BAA in urine was measured to assess the absorbed EGBE. Urine samples were co llected before and after each exposure (0, 4, 6, 8, 10, 12, 22, 26, 30 and 34h). To record any phys iological changes in the volunteers under the different conditions, physiological monitori ng equipment was used. The results obtained for skin only exposure are expressed as a percentage of the whole body measurement.
The mean dermal absorption for baseline condi tions contributes for 11 % of the total body burden. Low temperature did not affect signi ficantly the dermal absorption whereas high temperature increased significantly the dermal absorption (14 %). Increasing humidity increased the dermal absorption but not significantly. Clothing has also a little effect on dermal absorption. In the industrial scenario skin absorption is significantly increased compared to baseline conditions. The maximum de rmal absorption contributes for 42 % of the total body burden (mean 39 %). No significant differences in any of the physiological parameters were seen. This study shows that temperature is an important factor to take into account for assessing the percutaneous absorption of EGBE and also th at the use of protectiv e equipement under high temperature and relative humid ity can lead to a high dermal absorption (higher than without protective equipment).
Six male volunteers were dermally exposed to EGBE at various concen tration: neat or 50%, 90 % in water for 4 hours on the forearm (about 40 cm 2 ) (Jakasa et al. , 2004). An inhalation exposure of EGBE vapour was also performed on each volunteer (93 ± 6.8 mg/m 3 for 30 minutes) and served as a reference dosage. De rmal absorption parameters were calculated during 24 hours after the beginning of exposures by measuri ng excretion of total BAA (free + conjugated) in urine and EGBE in blood. Blood samples were collected for 8 hours (16 samples per experiment). Urine samples were collected every 4 hours during the 24 hour period. Each volunteer was exposed twice to a 50 % EGBE concentration (on each arm), once to the 90 % concentration and once to the pure EGBE. The period between two dermal exposures of the same site was at least 4 weeks.

EGBE in blood was below the detection limits after exposure to pure EGBE. The average dermal flux and the apparent permeability coeffi cients were greater for the 50 % dilution than for the 90 % dilution (see table 4.42). The same re sults were obtained w ith urinary excretionEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 92 of BAA. The permeation rate reach a plateau between 1 and 2 hours after the beginning of exposure, indicating a steady state permeation. The maximum permeation rates are presented in the table 4.43 (apparent permeability coeffici ents). The uptake after dermal exposure was also compared with the uptake after inha lation exposure (see table 4.42) showing that significant amounts of EGBE are du e to dermal exposure. Half lif e of BAA was calculated to be 3.4 hr (range 1.3 to 3.8 hr for inhalation ex periment). From the inhalation study, it was calculated than 57 % of the inhaled EGBE was excreted as BAA in urine.

Table 4.42: Kinetic parameters measured in the Jakasa study
50 % EGBE 90 % EGBE Neat EGBE Average dermal flux (mg/cm 2 /h)

Based on blood data 0.92 +/- 0.34 0.74 +/- 0.25 ND
Based on urine data 1.34 +/- 0.49 0.92 +/- 0.60 0.26+/- 0.17 Apparent permeability coefficients (x10 - 3 cm/h) 1.75 +/- 0.53 0.88 +/- 0.42 ND

Estimated dermal uptake of EGBE(mg) 1,340 926 263 Pulmonary uptake 346

Intra and inter-individual varia tion of dermal fluxes were also studied with 50 % dilution. The results are reported in the table 4.43.

Table 4.43: Intra and inter-individual variation of dermal fluxes after dermal exposure to 50 % EGBE in water. Average dermal flux (mg/cm 2 /h) 90 % confidence interval Intra individual CV (%) Inter individual CV (%)

Blood data 0.92 +/- 0.34 0.63 – 1.19 16 35

Urine data 1.34 +/- 0.49 0.94 – 1.74 20 34

The results obtained in this study are in good agreement with animal data and in vitro data on human skin. It also demonstrate that demal ab soption of EGBE is important when compared with inhalation exposure.

Summary human dermal route:

Five in vivo studies on volunteers and four in vitro studies are available fo r this end point. The four in vitro studies, measuring the rate of absorp tion of liquid EGBE through human skin, gave results that varied by a factor of 25 (0.064 mg/cm 2 /hr for the lowest and 1.66 mg/cm 2 /hr for the highest). In vitro , rate of absorption is highly depe ndent on the con centration of the aqueous solution of EGBE used. In vivo , in one study, an estimation of the skin penetration gave results in the following range: 7 to 96 nmol/min/cm 2 (0.008 mg/cm 2 /hr to 0.0114 mg/cm 2 /hr) for pure liquid EGBE. A nother study performed with liquid EGBE has showed that greater absorption was f ound with EGBE 50 % in water th an neat EGBE (dermal flux 1.34 mg/cm 2 /h and 0.26 mg/cm 2 /h respectively). This is consistent with data available in vitro and in animals. This is also critical for risk characterization because percentage of absorptionEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 93 taken into account in each scenar io can be different if exposur e is exposure is to EGBE pure or EGBE in formulations.
EGBE half life was approximately 1 hour in al l studies whereas BAA half life was about 5 hours. If temperature and humidity conditions increased, the percutaneous uptake increased also (in contrast with the inhalati on uptake which remained constant).

In one in vivo study, percutaneous absorption of vapour EGBE was assessed. Depending on the external conditions during exposure, the internal dose of EGBE due to percutaneous absorption varies between 11 % and 39 %. The percentage of 11 % was found for “normal” conditions of use (temperature, humidity) and 39 % for the worst case of industrial use (high temperature, high humidity and overalls wearing). This worst case percentage is use in the risk assessment section to estimated the internal dose of EGBE due to dermal absorption of vapour EGBE.
Based on the PbPk modelling it is estimated th at for a worst-case exposure to EGBE vapour (100 % of the body exposed and no cloths), percutaneous absorption would account for 15-27 % of the internal dose of EGBE (Corley et al. , 1997).

Oral
In a suicide attempt, a 50-year woman inge sted 250-500 ml of a wi ndow cleaner containing 12 % of EGBE, corresponding to about 0.5 to 1 g/kg bw (Rambourg-Schepens et al. , 1988). On admission to the intensive care unit, the pa tient was comatose. Biologically she presented with metabolic acidosis, hypokala emia, a rise in serum crea tinine level and a markedly increased urinary excretion of oxalate crystals.
In another suicide attempt, quantitative analysis of blood BAA was performed (Gualtieri et al. , 2003). A high maximum blood concentra tion was found (4.86 mmol/L). Symptoms recorded were metabolic acidosis and hepatic biochemical disord ers. Si sign of haemolytic anaemia were described.

4.1.2.1.3 Other data

In vitro studies have been conducted to determine the relative degree to which some glycol ethers act as substrate for alcohol dehydroge nase (ADH) (Dow Chemicals, 1983). Four concentrations of EGBE (purity 98.55 %) were used to define the kinetic constants. Each concentration was run in duplic ate. Experiments were conduc ted at both pH 8.8 and 7.4 in order to determine if pH was a critical factor.
Assays conducted at pH 7.4 resulted in data sets which were not suitable for plotting. For EGBE, Vmax was 4.06 μ M/min, Km was 1.18x10 -3 M with a correlation coefficient of 0.98.

Kinetic of EGBE was studied in a perfused rat liver syst em with and without ethanol (Johanson et al. , 1986b). The liver of a Sprague-Dawley ra t was isolated and perfused with EGBE (99 % purity). Concentration ranging fr om 0.057 to 2.7 mM of EGBE were achieved and submitted to the perfusion system for period s of 10 min. Two samples from the perfusion medium and from the perfusate were collected du ring the last 4 minutes and were analysed for EGBE concentration determination. Preliminary studies indicated that the half life of the adaptation of the liver to new concentrations of EGBE was approximately 1 min. Hence, 6 min was regarded sufficient to reach a plateau leve l. At the end of 3 expe riments, the effect ofEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 94 EtOH was studied while maintaining the concen tration of EGBE at 0.45 mM. The liver was then perfused with a medium containing 17.1 mM EtOH during 20 min, followed by an equal period of time of perfusi on with EtOH free medium.

The elimination data fit well to the Michaelis-Menten equation. Th e estimates of the apparent maximum elimination rate of EGBE range between 0.59 and 1.3 μ mol/min/g. The estimate of the Km vary from 0.19 to 0.4 mM. Maximum cl earance ranged from 2.7 to 3.1 ml/min/g. In the presence of EtOH, the extraction ratio of EGBE decreased from 0.44 to 0.11. When EtOH is withdraw, the liver returned nearly to its pr evious elimination capacity in approximately 10 min. This finding supports the hypothesis that EGBE is mainly metabolised by ADH in the rat liver.
Testicular and hepatic capacities to metabo lise EGBE by alcohol dehydrogenase (ALD) was assessed (Moslen et al. , 1995). These comparisons were done w ith SD rats (highest testicular capacity to metabolise EGME) a nd with Syrian Golden Hamster (no detectable testicular capacity to metabolise EGME).
Testicular and hepatic activities of ALD for EGBE was greater in hamster than in rat. Comparing with other glycol et hers (GE), testicular and hepatic activities of ALD for EGBE were the less important in rats whereas testic ular ALD in hamster was greater for EGBE than for EGME or EGEE. In hamster liver, the AL D activity was less than those of EGME and EGEE.

In a series of in vitro studies the metabolites of some gl ycol ethers, EGME, EGEE and EGBE were determined in human and rat hepatocyte cultures (Green et al. , 1996). Adult male Fisher 344 hepatocytes and human hepatocytes were incubated with EGBE (purity unknown) at doses of 0.02 – 0.2 – 2 and 10 mM for 4 hr.
Over 90 % of the radioactivity was detected in 3 peaks: EGBE – BAA and EG. BAA was the major metabolite formed in both species. More than 90 % of the 0.2 mM EGBE was rapidly converted to BAA by rats hepatocytes (40 % for human hepatocytes). The percentage of EGBE that was converted to BAA after 4 hr of incubation with rat hepatocytes was similar at 0.02 – 0.2 and 2 mM. In human hepatocyte the per centage of EGBE that was metabolised to BAA decreased between 0.2 and 2 mM. The percentage of EG BE metabolised to EG was greatest at the lowest substrat e concentration for both human a nd rats suggesting that this pathway became saturated at relatively low glycol ether concentr ation. Another minor metabolite of EGBE was identified in th is study: glucuronide conjugate of EGBE.

V max values were 15–20 fold greater in rat he patocyte than in hum an hepatocytes (741 nmol/h/10 6 hepatocytes in rat and 113 in human). The Km value was similar for rats and humans (1 mM).
The potential of EGBE to form fatty acids conjugates was studied in F344 rats and in vitro (Kaphalia et al. , 1996). Fatty acid ethyl synthase (F AEES) was extracte d from rat liver microsomes. Different quantities of th is enzyme were incubated with 2 μ mol of [1- 14 C] oleic acid and 750 μ mol EGBE at 37 ° C for 2 hr. A contro l reaction mixture was also incubated without enzyme. After extracti on radioactivity was measured.
Incubation of [1- 14 C] oleic acid and EGBE in the presence of the FAEES results in the formation of 18:1 fatty acid 2-butoxyethyl ester ( not detected in the mixture incubated without enzyme).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 95 The role of liver alcohol dehydrogenase isoenzymes in the oxidation of glycol ethers in rats was explored (Aasmoe et al. , 1998). Wistar rats were kille d to prepare liver homogenate. ADH activity was assayed by gel electrophoresis after addition of EGBE at the following concentrations: 0.05-20 mM. Km and Vmax were calculated. Activities were expressed as specific activity (the rate of NADH forma tion/min/mg protein). This assay was also performed concurrently with Methanol (MeOH), Ethanol (EtOH),BuOH, EGME, EGEE, Ethylene Glycol Propyl Ether (EGPE) and Ethylene Glycol Hexyl Ether (EGHE).
After electrophoresis, the liver homogenate revealed one en zymatically active zone on staining with MeOH, EtOH, BuOH, EGME, EG EE, EGBE and EGPE (this pattern was identical for male and females). This enzymatic form is probably identical with the ADH-3. The intensity of the ADH-3 increas ed with increasing chain lengt h of the alcohol or glycol ether. The ADH-2 isoenzyme was only active w ith medium and long ch ain aliphatic alcohols (not with glycol ethers). The same ADH isoe nzyme was active in males and females, however the activity of ADH is sex-dependent, with higher enzyme activity in females than in males. This experiment also showed that this diffe rence was a result of hi gher amount of ADH in females rather than of sex-specific isoenzymes.
The substrate specificity (Vmax/Km) decrease in the following order: EGBE > EtOH > EGEE > EGME. The Km value for EGBE was approximate ly 10-fold lower than the other 2 glycol ethers, while the Vmax values were almost the same. Consequently, at low and identical glycol ether concentrations, the biotransformation of EGBE would be more efficient than that of the other two glycol ethers . At the same time, the low Km value for EGBE compared to EGME and EGEE indicates that metabolism of EGBE via ADH will be saturated at lower substrate concentrations than those of EGME and EGEE.
A PbPk model was developed with the results obtained from various studies (Johanson, 1986c). In his study, Johanson tried to extrapolate the in vitro rat data to man in vivo

. Concentration-time curves are generated by computer simulation. The outcome of the simulation is compared with th e results from the experimental exposure in male volunteers. The model used is designed for simulation of or ganic solvent kinetics in general. The organs are lumped into compartments according to blood flow and fat content. Muscles and skin form a separate compartment due to their wide va riation in blood flow at different work loads. It is assumed that solvent uptake occurs only in the lungs and eliminatio n only in the liver. It is further assumed that the solvent inst antaneously distribute homogenously in each compartment, and as a consequence the solv ent retained in the respiratory airways immediately reaches the arterial blood. The mode l structure is depicted in the figure 4.44:

Figure 4.44: First PBPK developed for EGBE All partition ratios between blood and other compartments are set to 1. Strict flow limitation, i.e. diffusion equilibrium between end-cap illary blood and tissue is assumed for all compartments. The relative pulmonary uptak e of EGBE was set to 0.6. Volumes and blood flows to the different organ groups were computed from literature data.
There is a good agreement between simulated and experimental blood concentration curves, indicating that the assumptions made have a certain degree of validity. Increased physical activity increased the bl ood concentration of EGBE due to increased pulmonary uptake rate. Similarly, the co-exposure to EtOH caused elevat ed blood concentration of EGBE due to a decrease in the elimination rate. The simulation also suggested that ve nous blood samples are useful, if not slightly more us eful, as arterial or capillary blood sample in the biologicalEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 96 monitoring of exposure to EGBE. In any case, blood samples should be collected during the exposure phase because of the rapid decay of EGBE. The risk of accumulation of unmetabolized solvent in the body appeared low. According to the model, non-linearities because of saturated elimination occur at concentrations well above 100 ppm, even in combination with physical exercise and etha nol, thus linear kine tics of EGBE may be expected at ordinary occ upational inhalation exposure.
The model was improved by Shyr et al. , 1993. Improvements consisted in the modelisation of the metabolite formation by three routes of exposure (oral, dermal, inhalation).
In order to improve the PbPk model desc ribed by Johanson in 1986, a series of Good Laboratory Practice (GLP) studies we re performed (Dow, 1993 – Corley et al. , 1994). The objectives were to :

- expand the PbPk model to include additional exposure routes (oral gavage, drinking water, IV infusion and dermal),

- determine the relative solubi lities of EGBE and BAA in rat blood, rat tissues and human blood for use in estimating partition coefficients

- incorporate the dispositio n of BAA into the model

- add allometric scaling factors for ra t and human physiological parameters

- add competing pathways for the metabolism of EGBE into the model.

- develop a method for analysing trace le vels of both EGBE and BAA in blood

- validate the new analytical method by administering 14 C-labeled BE to rats and analysing blood for BE and BAA in urine for BE and major metabolites.

- confirm the presence of the metabol ite, EG, in urine by mass spectroscopy

- validate the new PbPk model with data develope d as a part of these studies and relevant data from the literature.
The model used was significantly different from that of Johanson (see figure 4.45):EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 97 Figure 4.45: Improvement of th e PbPk model (from Dow, 1993)

Allometric-scalable physiological and biochemical parameters were used in place of standard values for a 70 kg man to normalise these parame ters to the actual body weights reported in several human studies. The rat was added to e xpand the database for model validation and to assist interspecies comparisons. Additional rout es of exposure were added for validation of the model simulations with data from published studies. A second model was linked to the EGBE model specifically to track the disposit ion of BAA. The kidney was added to the BAA model since it is an organ of elimination for BAA.
Nose only inhalation, IV infusion, oral gavage and drinking water simulations were modelled according to Andersen et al ., 1987. Dermal exposure to EGBE were modelled according to Mc Dougal et al (1986 and 1990) for neat and aqueous solutions and according to Johanson and Boman, 1991 for vapour absorptions.
Physiological constants were taken from th e literature (Anderson, 1987 ; Fiserova-Bergerova, 1983 ; Johanson, 1986c). The effect of exercise on the ventilatio n rates and blood flows were adapted from Astrand (1983) and Johanson (1986c).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 98 Partition coefficients were available only for EGBE in human blood/air and saliva/air (Johanson and Dynesius, 1988). Partition coefficien ts for EGBE and BAA in the blood, liver, kidney, skin, fat, muscle, stomach, small intest ine, caecum and lung tissues of male F344 rats and the blood of humans were determined experimentally.
After calculation of the partition coefficients the following assumptions were made for the PbPk model:

- the partition coefficients for EGBE and BAA for the rapidly perfused compartments were assumed to be equal to that of the liver

- the partition coefficients for the slowly perfused compartments were assumed to be equal to that of the muscle

- the average of the stomach, small intestine and caecum partition coefficients was used to represent GI tract

- the skin/air partition coeffici ent for dermal uptake of EGBE vapours was assumed to be equal to the blood/air partition coefficient

- the skin/liquid partition coefficient for derm al dosing of neat EGBE and solutions of EGBE was assumed to be equal to the m easured skin/saline partition coefficient.

Biochemical constants : the metabolism of EGBE and BAA was assumed to occur only in the liver through a single metabolic pathway (alcohol / aldehyde dehydrogenase) following Michaelis-Menten kinetics. Other minor metabolic pathways have been reported by various authors, since there was insufficient kinetic data available to differentiate the parameters associates with each of these other pathways, a second saturable pathway was included in the model to simulate the disappearance of EGBE from the liver by all other pathways combined. Parameters for this pathway were estimated (based on Medinsky et al., 1990 studies).

The model also assumed that BAA was bound to proteins in blood and was eliminated by a saturable process in the kidney. A model similar to that of Russel et al ., 1987 was used for describing protein binding and sa turable elimination of BAA.

C max and AUC of the concentratio n for BAA in blood were used as an estimates of the “delivered dose” or “internal dose surrogate” for high dose/low dose, dose/route and rat to human extrapolation.
A metabolism study was conducted to assist in va lidation of model parameters studied (see § 4.1.2.1.1.1). These results with othe r data sets were compared with model predictions.
Data are generally in good ag reement with the model except for dose levels which cause toxicity. The model has no systematic provisio n for correcting for potential diminished renal excretion and/or liver metabolis m that arise secondary to the haemolytic activity of BAA and will therefore tend to overpredict the amounts of BAA excreted by the kidneys in urine. Nevertheless the model satisfactorily predicts concentrations of BAA in blood and amount of BAA eliminated in the urine at dose le vels that do not cause toxicity.
The PbPk model of Corley et al .,1994 served as a background to develop a new model to take into account chronic inhalation and fe male rat and mice parameters. (Lee et al. , 1998). The 2- year toxicokinetic studies utilized in the mode l development were conducted as a part of the 2-year inhalation toxicity and carcinogenicity studies of EGBE (NTP, 1998) and are described previously in this report (Dill et al. , 1998).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 99 Compared to the previous model, the musc le compartment was included in the poorly perfused compartment, a kidney compartment wa s added for EGBE and spleen was added as a separate compartment. The same assumptions than before were made. It was also assumed that partition coefficients of EGBE and BAA in male rats were repres entatives of those for other species regardless of the sex. Some adjustments have been made due to chronic modifications induced by EGBE:

- cardiac output start to diminish after 6 month exposure (down to 2/3 of the estimate used for young animals by the end of the 18 months of exposure).

- volume of the fat compartment starts to increase after 6 month of exposure (up to 3 fold at the end of the 18 months of exposure).

- Total plasma protein binding sites in mice star t to increase after 6 months of exposure (up to 2 fold at the end of the study)

- The maximum rate of EGBE meta bolism to BAA for a male rat starts to increase after 12 months of exposure (2 fold at the end of exposure).

- The maximum rate of 2BAA renal elimination starts to decrease after 6 months of exposure (down to 3⁄4 of th e estimate for young animals at the end of exposure).

Figure 4.46: Last PbPk model (Lee et al. , 1998)

In human via inhalation and dermal route, the st udy of the PbPk model de monstrated that it is not possible to reach a BAA blood concentration that might be able to cause even a slight haemolysis in humans (Corley, 1996). This is based on the assumption that a concentration of 8mM of BAA in blood is needed in human to be able to develop sign of haemolysis (only 0.5 mM in rats) and was calculated for theoretical ex posure of 6 hr regardless of the concentration (even up to the saturated concentration of 1160 ppm EGBE) or inclusion of the dermal absorption of vapors.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 100 Recently, the model was improved to include key studies that independently determined sex, age and species differences in partition coeffi cients, metabolism and renal clearance (Corley et al ., 2005).

Summary other data :

In vitro studies have showed that EGBE transf ormation in BAA depended on ADH (isotype 3). This enzyme is more active in females than in males. Moreover, it has been demonstrated than metabolic rate in rat was 10 to 20 fold higher than in human.
A PbPk model has progressed with experimental data collected in humans and in animals. This model included at the present time various exposure route (inhalati on, oral, dermal, IV), the differences between humans and animals, th e kinetic parameters of the main metabolite BAA and allow also the modelling of repeated exposures. Recent studies have given results which are consistent with the current PbPk model.

4.1.2.1.4 Summary of toxicokinetics, metabolism and distribution
Oral administration of EGBE leads to a quite complete absorption of the substance. Via inhalation route, a “wash in / wash out” mechanism limits the absorption to 55 – 60 % of the administrated concentration. Via dermal r oute, uptake of liquid EGBE depends on the administration mode, the species and the concen tration of EGBE in the final product. An occlusive administration will be responsible ofa great percutaneous uptake whereas a nonocclusive administration will minimize abso rption due to the volatility of EGBE.
Rat skin seems to be readily permeable compared to pig or human skin (2 or 3 fold more). Percutaneous uptake also depends on the EGBE concentration in the te sted product : for 40 and 80 % aqueous solutions of EGBE, absorption was demonstrated to be maximum. In two rat studies, dermal absorption of liquid EGBE was estimated to be between 20 and 30 %.
In human, dermal studies with liquid EGBE give penetration uptakes wh ich vary by a factor of 10 between different subjects exposed to EG BE with the same experimental conditions. A percentage of absorptio n of about 12 % was calculated in one study using EGBE at 5 and 10 % in water.
Dermal penetration of vapour EGBE was studi ed in humans. Depending on the experimental conditions, the percentage of the absorbed subs tance due to dermal uptake varies between 11 and 39 % of the total body burden.

EGBE reaches a maximum blood concentration ra pidly after exposure whichever the route of exposure. EGBE is rapidly metabolised (with a plasmatic half lif e of about an hour).
Target organs are the liver, kidneys, thymus an d stomach, in particular forestomach in the rat whichever the route of administ ration (oral and inhalation route, no data for dermal route).
The main metabolism pathway leads to the fo rmation of BAA via Alcohol dehydrogenase and Aldehyde dehydrogenase in a saturable mechanis m. With increasing doses of EGBE, the formation of glucuronide conjugate of EGBE or BAA is enhanced. Minor metabolites of EGBE are also reported depending on the species used (see figure 4.47).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 101 Figure 4.47: Metabolism of EGBE (Patty, 2001)

Materials identified with an asterisk (*) have been identified in either rodents or humans.
**: BAA-Blu = N butoxyacetyl glutamine conjugate

Elimination is rapid and mainly via urinary route (80 to 90 % of the metabolites). The plasmatic half-life of metabol ites is about 4 hours. A small amount is eliminated as CO 2 by the respiration (10 to 20 %). Normal renal excr etion is conditioned by physiological state of the kidneys: females excreted less rapidly BAA th an males and aged animals have a trend to eliminate metabolites with more difficulties than young animals. Any renal injury will enhance BAA toxicity by incr easing its blood persistence.
However if renal integrity is respected, a re peated administration of EGBE lead to an adaptation of the metabolism. In this case el imination of BAA occurred more rapidly. This mechanism of extra hepatic adaptation is al so described for action of EGBE on red blood cells, especially on erythrocyte deformability (see § 4.1.2.2.3). EGBE BAA *

EGBE glucuronide *

BES (sulfate- conjugate) *
EG * BAA -– Glu ** Humans *

CO 2 *

URINE Mixed function oxydase Alcohol and aldehyde dehydrogenase

Butyraldehyde

Butyric acid CO 2 *

+ glycine Glycine conjugate oxalic acidEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 102 For risk characterisation, if absorption factors are needed, th e following values will be taken :

3⁄4 Oral route: 100 % absorption

3⁄4 Inhalation route: 60 % absorption

3⁄4 Demal route: distinction should be made between dermal absorption of EGBE vapour (in the atmosphere) and dermal absorption of liquid EGBE. For these two cases, the absorption factors should be discussed on a ca se by case basis because a lot of factors can modify these factors (temperature and humidity, clothing, con centration of EGBE if diluted, duration of the exposure...).
Overall, dermal absorption of EGBE vapour will contribute fo r 27 % of the total EGBE body burden in normal uses and 39 % if extreme conditions are expected. Dermal absorption of liquid EGBE will vary in function of the duration of exposure and the concentration of EGBE in the formulation, internal doses will be calculated on a case by case basis using the dermal fl ux calculated in the Jakasa study. Where required, a permeation rate of 0.63mg/cm 2 /hr will be used. This is the upper figure from the in vivo data and is consistent with the in vitro data.

It is considered that the PBPK model for EGBE is sufficiently well developed to justify its used to derive animal to man toxicokinetic ex trapolation factors for th e inhalation route. These factors are based on the toxokinetics of BAA since this is the meta bolite that causes the critical toxic effects. A detailed description of how the mode l is used is given in the General Aspects introduction to the risk ch aracterisation. Wher e required, the half life of BAA will be assumed to be 5 hours.

4.1.2.2 Acute toxicity

4.1.2.2.1 Studies in animals

Inhalation

Rats
Carpenter et al. 1956 and the report of the Mellon Institute of Industrial Research (1952) reported some experiments concerni ng acute toxicity of EGBE in various animals. For rats via inhalation route, a LT50 of 9 hours was reported for a con centration of 1300 ppm. At 800 ppm, mortality was 3/6 rats for a 8-hr exposur e period and 0/6 rats for a 4-hr exposure period. At 500 ppm no rats out of 6 were dead for a 8 hr exposure period and onl y one out of 6 for a 4 hr exposure period. When older rats were exposed to 375 ppm (age not reported) 11 out of 13 and 23 out of 23 died following a 7 hr exposure period.
Saturated vapour of EGBE caused no mortality am ong 6 adult female rats exposed for 8 hr and observed for a 14-day observation period. Clinical symptoms were limited to bloody urine and poor coordination. Concentration was measured to be 475 ppm.
Earlier results (1940 and 1943 cited in Carpenter et al., 1956) with saturated vapour give the following results: 4 ou t of 6 rats died following a 9 hr expos ure, 2 out of 6 rats died after a 4 hr exposure and no rats died after a 2 hr exposure.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 103 In a poorly reported study (Gage, 1970), rats (4 males and 4 females) were exposed whole body to EGBE (purity unknown) at an unique concentration of 2400 ppm (13 mg/l) during 5 hr. At this concentration test substance was in aerosol form.
All rats died within 2 days. Clinical sympto ms before death were a comatose state and haematuria. Blood Hb was measured 35 to 50 % of the normal.
Groups of Fisher 344 rats (6/sex/dose) were exposed whole body to EGBE at doses of 867, 523 or 202 ppm (purity 99.4 %) for 4 hr (Bus hy Run Research Center, 1980a). EC50 was calculated at the end of the 14-day observati on period. Clinical obs ervations and gross macroscopy was recorded during and at the end of the study.
In rats exposed to 867 ppm, loss of coordina tion was observed concu rrently with laboured breathing and red fluid discharge around the urogenital region. A ll animals were dead 2 days after exposure. In rats exposed to 523 ppm, coordination loss wa s also observed and red fluid discharge around the urogenital region. 2 male s and 3 females died during the 14-day observation period. At the end of the study, th e tail of 2 males and 2 females appeared necrotic. In rats exposed to 202 ppm, except a slight amount of dried red material observed on the tail the day following exposure, no other si gn of toxicity was obs erved. Body weight of the 523 and 202 ppm groups were depressed 1 da y after exposure but a normal body weight was recovered by 4 days. Animals that died had enlarged and discoloured kidneys with urinary bladder filled with red stained urine.
In this study, a LC50 of 486 ppm was calcu lated for males and 450 ppm for females.
In a validation study for the OECD method 403 (ring test), 8 chemicals including EGBE were tested (Shell Chemicals, 1982). Wistar rats (3/sex/group) were exposed whole body to saturated vapour concentration (t heoretically 617 ppm) for a variable period of time. The maximum exposure time was 7 hr, if deaths occurred during either the exposure period or the observation period, exposures were repeated for shorter interv als (eg. 3 hr, 1 hr, 30 min...).
For Butyl oxitol (EGBE – purity 99 %) durations of exposure were 7 hr, 3 hr and 1 hr. Measurements of the atmospheric concentra tions in the exposure chamber gave values between 750 and 910 ppm.
During exposure animals exhibited signs of le thargy. For rats exposed during 7 hr, mortality was 1/3 males and 3/3 females at the end of the observation period. Clinical signs were necrosis of the end of tails, blood in urine and paleness of eyes and feet. For the 3 hr exposure group mortality was 0/3 males and 1/3 females. Except for necrosis of the tail, the same symptoms than the 7 hr group were observed. For the 1 hr group, no mortality was observed, symptoms were limited to the presence of blood in urine and paleness eyes and feet. Recovery was full 2 days after exposures.
The results of other laboratories participat ing at the ring test were published (Klimisch et al.
, 1988). The nominal concentration at which animals were expose d varied from 3.3 mg/l to 4.2 mg/l (mean 3.7 mg/l). The LT 0 (time for which at least one d eath were found) was 3 hr for 5 laboratories and 1 hr for only one laboratory.

Mice
In a poorly reported inhalation study in mice,, th e effects of EGBE and other glycol ethers was studied (Werner et al. , 1943a). Swiss mice were exposed in groups of 16 for 7 hr periods.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 104 At 1210 ppm all animals died within 32 hr. At 920 ppm 11/16 animals died within 32 hr. At 770 ppm 14/16 animals died within 1 week afte r exposure. At 670 and 560 ppm, 2/16 animals died in 32 hr and 1 week respectively. No an imals died with exposur e to 390 ppm of EGBE.
Dyspnoea was the major toxic symptom describe d moreover when doses were near the lethal level, severe haemoglobinuria was seen. Moderate to marked follicular phagocytosis and congestion of the cavernous veins were frequent findings in spleens of exposed animals. Focal necrosis and lymphoid hyperplasia was also seen in 3 surviving animals. Interstitial nephritis and typical pictures of bronchopneumonia was seen in a few animals.

Guinea pigs
Carpenter et al

. , 1956 reported some experiments concer ning acute toxicity of EGBE in various animals. A LT50 of 7 hr was reported for guinea pigs for an exposure of 1300 ppm (Union Carbide Corp., 1943). Guinea pig morta lity was 1/6 in a 4 hr exposure (Mellon Institute of Industrial Research, 1952).
In a GLP study, EGBE (99.87 % purity) was admi nistrated via inhalation route (whole body) to Guinea Pigs for a single hr of exposure (Bushy Run Research Center, 1994). Target concentration for the exposure was the highest attainable EGBE con centration (saturated vapour concentration) at 20°C and 740 mm Hg: 791 ppm. Study design was identical to OECD guideline 403 except for th e use of GP and for the dur ing of the exposure (1hr).

Real concentration obtained were 633 a nd 691 ppm for the females and the males respectively.
No death nor clinical signs were seen duri ng the exposure or during the 14-day post-exposure period. No changes in body wei ght or bodyweight gain were observed. Gross findings at necropsy were observed in 4/5 males and 3/5 female s, all of these findings were considered to be incidental.
For this study, it can be concl uded that LC50 in GP in greater than 633 ppm in females and greater than 691 ppm in males.

Summary inhalation route:
Table 4.48: Summary inhalation route Lethal Concentration LC50 (mg/kg) Mortality and treatment related effects in function of exposure time Validity * Reference

Rats inhalation route
Exposure to 800 ppm during 8 hr caused 50 % mortality whereas an exposure time of 4 hr did not cause ant death (6 animals in each group)
Exposure to 500 ppm (2.45 mg/l) for 8 hr – did not cau se any mortality (0/6) wherease a tratment period of 4hr caused one death (out of 6)
If Older rats are exposed to 375 ppm: 2 Mellon Institute of Industrial Research, 1952
Carpenter et al. , 1956EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 105 11/13 and 23/23 died after 7 hr of
exposure LC50: 486 ppm (2.38 mg/l) for males and 450 ppm for females. 4hr exposure
Laboured breathing, loss of coordination, tail necrosis, renal toxicity, haemolysis.
Mortality from 523 ppm (2.56 mg/l) 2 Bushy Run Research Center, 1980a

Saturated vapours (617 ppm – 3 mg/l) and exposure time variable.
Mortality 4/6 for 7 hr exposure, 1/6 for 3 hr exposure and no mortality for 1 hr exposure.
Lethargy, necrosis of the tail and haemolysis. 2 Shell Chemicals, 1982

2400 ppm (11.76 mg/l) aerosol for 5 hr exposure. All rats died within 2 days. Comatose state and haemolysis. 3 Gage, 1970

Mice Inhalation route
Mortality from 670 ppm (3.28 mg/l) and 560 ppm (2.74 mg/l) for exposure of 32 hr and 1 week respectively. No dead for 390 ppm (1.91 mg/l).
Dyspnea and haemolysis. 3 Werner et al. , 1943a

Guinea Pigs Inhalation route
LT50: 7 hrs at 1300 ppm (6.37 mg/l) 2 Carpenter et al. , 1956
> 633 ppm (3.1 mg/l) (females)
> 691 ppm (3.39 mg/l) (males) 1 hr exposure.

No effects 1 Bushy Run Research Center, 1994.

* 1: valid (performed according to GL and GLP) – 2: valid with restriction (not performed according GL and/or not GLP but scientifically acceptable – 3: not valid or not assessment possible.

In a good quality study performed on rats, LC50 was calculated to be comprised between 450 and 486 ppm (for a 4 hour exposure (Bushy Run Research Center, 1980a). Other studies give results quite consistent with this one. In these studies, females and old animals were more sensitive than males or young animals.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 106 Clinical symptoms and pathology were: le thargy, ataxia, laboured breathing, loss of coordination, haemolysis and tail necrosis. Renal injuries were commonly seen during pathological examinations.
According to the data available, a classification R20 is needed.

Dermal

Rats
EGBE (purity unknown) was applied dermally (occlu sive) on rats for 4 hr (Mellon Institute of Industrial Research, 1961). Animals were obs erved during 14 days after application.
The calculated LD50 was 2275 mg/kg (1680 – 3079 mg/kg).
The dermal toxicity of EGBE (purity unkno wn) was assessed in rats in a GLP study (Safepharm laboratories, 1993a) according to OECD guideline 402. SD rats (5/sex) were given a single 24 hr, semi-occluded dermal app lication of undiluted test substance and then observed for 14 days.
No deaths were reported. No si gns of systemic toxicity or irritation were noted during the study. All rats gained body weight as expected during the study. No abnormalities were noted at necropsy. The LD50 in this study was great er than 2000 mg/kg for both males and females
rats. The dermal toxicity of EGBE (purity unkno wn) was assessed in rats in a GLP study (Safepharm laboratories, 1993b) according to OECD guideline 402. SD rats (5/sex) were given a single 24 hr, occluded dermal applicatio n of undiluted test substance. Animals were observed for 14 days following application.
One female was found dead 2 days after dosing. Comment signs of systemic toxicity noted were ataxia, pallor of the extremities, emac iation, lethargy, decreased respiratory rate, laboured respiration and tiptoe gait with incident s of ptosis and red/br own staining around the eyes. A full recovery was seen before the end of the study. No signs of irritation were noted during the study. Surviving animals showed ex pected gain in bodyweight during the study except for one female which showed bodyweight loss during the firs week. No abnormalities were noted at necropsy in surviving animals. Female that died during the study showed haemorrhagic lungs, dark liver, dark kidneys , sloughing of the non-glandular epithelium of the stomach and haemorrhage of the small and large intestines. The LD50 in this study was greater than 2000 mg/kg for both males and females rats.

Guinea pigs
Results of studies were repo rted briefly (Mellon Institute of Industrial Research, 1952). Dermal LD50 was found to be 6411 mg/kg (5400 to 7765 mg/kg).

LD50 in rabbits and in guinea pigs were compared (Roudabush et al. , 1965). Groups of Hartley guinea pigs and white rabbits (4 animals / dose) were dosed (at least 3 doses) with EGBE (purity unknown) in intact skin for GP and rabbits and also in abraded skin for GP only.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 107 The LD50 values were614 mg/kg in rabbits and 208 and 271 mg/kg for intact skin and abraded skin respectively in guinea pigs.
A comparative percutaneous toxicity study of 10 industrial solvents (including EGBE) was performed in guinea pig (Wahlberg and Boma n, 1979). A fixed volume of EGBE (purity > 99 %) was administrated occlusively to 20 guinea pigs for a period of time which allow the test substance to be totally absorbed (between 5 a nd 7 days). Animals were then observed for 35 days for clinical symptoms and were we ighted once daily except on week end. For comparison the same amount of EGBE was inject ed IP and the animals were followed in the same manner as the epicutaneous ly exposed guinea pigs. The a pplied volumes of EGBE were 450 and 1800 mg.
No deaths were seen for the 450 mg dose. For the high dose, 5 animals died on day 3 cumulative death reached 11 on day 4 and fina lly 13 on day 7 and after. No changes on bodyweight compared to controls were seen fo r the 450 mg dose. With IP injection of 1800 mg, 100 % mortality was reached in 6 hours, a nd with 450 mg dose, 70 % of the mortality was reached in 48 hr.
In a GLP study performed according to the OECD guideline 402, Hartley guinea pigs were exposed to 2000 mg/kg of EGBE (purity 99.8 %) via dermal route (Eastman Kodak, 1994b). The test substance undiluted was applied under occlusive conditions to the guinea pigs (5 /sex) for a test period of 24 hr.
No death was reported. All animals appeared clinically normal during the 14 day post exposure period. No effects were seen on body weight, bodyweight gain and no treatment related changes were noted at necropsy. Base d on this assay, the dermal LD50 of EGBE can be considered greater than 2000 mg/kg.

Rabbits

New Zealand White rabbits we re dosed with EGBE (purity unknown) via dermal route (Mellon Institute of Industri al Research, 1952 ; Carpenter et al. , 1956). Test material was applied occlusively during 24 hr at different doses. LD50 was calculated after a 14-day observation period.
Extreme congestion of the kidney s, bloody urine, pale liver and engorged spleen were noted in dead animals. The LD50 calculated was 560 mg/kg (480 to 640 mg/kg).
In the same report, previous studies were re ported: in 1947, 10 rabbits were doses with 505 mg/kg undiluted EGBE and all died within 48 hr. Bloody urine was a common finding. In 1948, 505 mg/kg was applied to 6 rabbits and 3 out of 6 survived but had bloody urine. Similarly in 1951, 4 out of 6 rabbits di ed when dosed with 505 mg/kg EGBE.
Ten New Zealand White rabbits were exposed dermally to Polysolv EB (EGBE – purity unknown) at dose of 2000 mg/kg during 24 hr (M B research laboratories, 1976). Epidermal abrasions were made every 2-3 cm over the expos ed area (sufficiently deep to penetrate the stratum corneum but not deep enough to prod uce bleeding). The wrapping was occlusive. After the 24 hr exposure period, anim als were observed for 14 days.
At 24 hr, all rabbits exhibited la crimation, bloody urine, flaccid muscle tone and anorexia. No spontaneous movement were noted. All animals died during the second day of observation. It can be concluded that LD50 is lesser than 2000 mg/kg.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 108 In order to estimate more precisely the valu e of the LD50, another st udy was performed with the same experimental conditions except for the dose level chosen and the number of animals. Four doses were applied to three groups of four NZ white rabbits: 250, 500 and 1000 mg/kg.
In this second study, mortality was seen at 500 mg/kg (1/4) and 1000 mg/kg (4/4). No toxic signs were reported for the 250 mg/kg gr oup, for 500 and 1000 mg/kg group blood was found in urine and for the 1000 mg/ kg group animals were flaccid. In died animals, necropsy findings were blood in urine and sometimes liv er and renal injuries. The calculated LD50 based on this study was 580 mg/kg (0.31 – 0.85).
The percutaneous toxicity of butyl cellosolv e was determined on rabbit skin (Duprat and Gradiski, 1978). Groups of 6 New Zealand White rabbits were exposed to undiluted EGBE (purity > 99.5 %) at doses of 72, 90, 108, 135, 180 and 225 mg/kg for a 8 hr period. Animals were observed daily over 15 days. Macroscopic and microscopic examination were performed on dead animals and those surviving on the 15 th day.
At the end of the exposure period, the to tal amounts of EGBE applied were absorbed. Mortality was 2/6 animals for 72 and 90 mg/ kg, 4/6 animals for 108 mg/kg, 5/6 animals for 135 and 180 mg/kg and 6/6 animals for 225 mg/kg. Death occurred between day 1 and day 8. Clinical signs were prostration, hypothermia and hemoglobinuria especially in the groups receiving the highest doses. Early death were consid ered to be caused by narcosis or failure of respiration and delayed death were probably due to renal impairment. Lungs appeared modified. Histopathological inve stigation in dead animals showed congestion of the liver, necrotic foci with mesenchymatous reactions a nd inconstant steatosis, passive congestion of the spleen, enlarged kidney w ith haemoglobinemic nephrosis, cutaneous lesions including necrosis. There were no evident dose-effect rela tionship since the findi ngs were exhibited at all dose levels. In surviving animals no effect s were seen for 90 and 72 mg/kg dose. Other animals showed signs of rena l toxicity and skin irritation.
In this study, the calcul ated LD50 was 100 mg/kg bw.
In a briefly reported study, groups of male New Zealand rabbits were exposed occlusively to EGBE during 24 hr at concentrations of 900 and 450 mg/kg (Bushy Run Research Center, 1980b). Mortality was 4/4 animals for the high dose and 1/4 for the low dose. Signs of erythema and necrosis were seen at the ad ministration point. In dead animals necropsy showed orange red lung and liver, dark spl een, dark red kidneys, orange peritonea and intestine, blood in urine. In this study LD50 was 569 mg/kg (348 – 931 mg/kg).
Nine glycol ethers including EGBE were test ed for determination of acute dermal LD50 (Eastman Kodak, 1981b). EGBE ( purity > 99.5 %) was administra ted dermally to groups of New Zealand White rabbits (5 animals/ group) at doses of 153, 307, 614 and 1239 mg/kg. After 24 hr of occluded exposure, the wrap was removed and animals were observed during 14 days for clinical and irrita tion signs. Body weights, gro ss pathology were also checked.
Clinical signs were anorexia, depression, cyanosis and ataxia for the lowest doses. Salivation, nasal discharge, iritis, depression, laboured breat hing and prostration were observed at higher doses. No gross pathological effects were s een at the 153 and 307 mg/kg doses. At the two highest doses, however, advers e and dose-related effects on ki dneys were recorded. Reddish coloured fluid was observed in the urinary bladder of animals. Some effects were also seen in liver and in thymus. Calculated LD50 was 435 mg/kg (range 330 to 578 mg/kg).
A GLP study was performed to assess the acute de rmal toxicity of EGBE (purity unknown) in the New Zealand White rabbit using a met hod described in the OECD guideline 402EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 109 (Safepharm laboratories, 1994a). 2 groups of 10 animals (5 males and 5 females) were given a single 24 hr semi-occluded dermal application to intact skin at dos e levels of 1000 and 2000 mg/kg. Animals were observed for 14 days after th e day of treatment and were killed for gross pathological examination.
There were no death for the 1000 mg/kg dose. Fo r the high dose, one female was found dead 2 days after dosing and 2 animals (1 male and 1 female) were killed in extremis one day after dosing. No signs of toxicity were seen for th e low dose, for the high dose, common signs of systemic toxicity related to EGBE administration were reporte d: lethargy, red stained urine, laboured breathing, hunched postu re and isolated incidents of loss of righting reflexes, hypothermia, ataxia and diarrhoea. Surviving animals recovered before the end of the observation period. Very slight to well-defined erythema with or w ithout very slight or slight oedema was noted at treatment sites of all an imals and at all doses, in high dose animals, some signs of necrosis were seen in 5 animals. All surviving animals except one female in the low dose group and one male in the high dose group showed expected gain in bodyweight during the study. For surviving animals, no a bnormalities were reported at necropsy (1000 and 2000 mg/kg), only the animal s dead in the high dose group s howed signs of hepatic and renal toxicity and haemorrhage of the gastric mucosa, of the non glandu lar epithelium of the stomach, of the small and large intestine and red fluid present in the bladder.
In this study, it can be conclude th at LD50 is greater than 2000 mg/kg.
A GLP study was performed to assess the acute de rmal toxicity of EGBE (purity unknown) in New Zealand White rabbit using a method desc ribed in the OECD guideline 402 (Safepharm laboratories, 1994b). 3 groups of 10 animals (5 ma les and 5 females) were given a single 24 hr occluded dermal application to intact skin at dose levels of 500, 707 and 1000 mg/kg. Animals were observed for 14 days after the da y of treatment and were killed for gross pathological examination.
One female treated with 500 mg/kg and 3 anim als treated with 1000 mg /kg were killed in extremis. Common signs of toxicity noted in all groups were ataxia, hunched posture, lethargy, laboured breathing, diures is, red-coloured urine and sk in and eyes pale yellow in appearance. Yellow coloured eyes was commonly noted in animals trea ted with 707 or 1000 mg/kg. Isolated incidents of toxicity noted in animals treated with 1000 mg/kg were diarrhoea, hypothermia and loss of righting reflexes. Common si gns of skin i rritation noted were very slight to well defined erythema, very slight to severe oedema, scattered areas of grey/green-coloured dermal necrosis, desquamati on, slight haemorrhage of dermal capillaries, small superficial scabs. Animals that died or we re killed in extremis during the study showed haemorrhagic lungs, dark or pale liver, dark ki dneys and red liquid presen t in the bl adder. In dead animals, slight haemorrhage of the gastri c mucosa and haemorrhage of the large and/or small intestines. No abnormalities were noted at necropsy of animals that were killed at the end of the study. The calculation of LD50 gi ves an overall value of 841 mg/kg, LD50 for males only was 1060 mg/kg and LD50 for females only was 667 mg/kg.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 110 Summary acute toxicity dermal route

Table 4.49: Summary acute toxicity via dermal route. LD50 (mg/kg) Experimental conditions Effects Validity * Reference

Rat dermal route
2275 mg/kg 4 hours occlusive 2 Mellon Institute of Industrial Research, 1961.
> 2000 mg/kg 24 hours, semi occlusive No irritation, no sign of toxicity 1 Safepharm laboratories, 1993a

> 2000 mg/kg 24 hours, occlusive Ataxia, pallor of extremities, lethargy, laboured breathing. No signs of irritation. 1 Safepharm laboratories, 1993b

Guinea-pigs Dermal route
6411 mg/kg 2 Mellon Institute of Industrial Research, 1952
208 mg/kg (intact skin)
271 mg/kg (abraded skin) 2 Roudabush et al.
, 1965

450 – 1800 mg/kg Mortality at 1800 mg/kg only (13/20 animals) 2 Wahlberg and Boman, 1979

> 2000 mg/kg 24 hours, occlusive no irritation, no signs of toxicity 1 Eastman Kodak, 1994b

Rabbits Dermal route
560 mg/kg

505 mg/kg 24 hours occlusive Haemolysis, renal – hepatic – splenic toxicity 2 Mellon Institute of Industrial Research, 1952
Carpenter et al.
, 1956
580 mg/kg 24 hours Anorexia, depression, 2 MB ResearchEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 111 occlusive, dermal abrasion cyanosis, ataxia, laboured breathing, renal – hepatic and thymic toxicity.
Mortality from 500 mg/kg. Laboratories, 1976

100 mg/kg 8 hours Haemolysis, narcosis, laboured breathing, skin irritation. Liver, splenic and kidney toxicity. 2 Duprat and Gradiski, 1978

569 mg/kg 24 hours occlusively Mortality from 450 mg/kg. 2 Bushy Run Research Center, 1980 b
435 mg/kg 24 hours occlusively Anorexia, depression, cyanosis, ataxia, laboured breathing, renal – hepatic and thymic toxicity. 2 Eastman Kodak, 1981b

> 2000 mg/kg 24 hours, semi occlusive Lethargy, haemolysis, ataxia, signs of irritation mild to severe at high doses (necrosis). Hepatic and renal toxicity. 1 Safepharm laboratories, 1994a

841 mg/kg
1060 mg/kg for males
667 mg/kg for females 24 hours, occlusively Mortality from 72 mg/kg. Signs of irritation mild to severe at high doses (necrosis). Very slight systemic effects. 1 Safepharm laboratories, 1994a

* 1: valid (performed according to GL and GLP) – 2: valid with restriction (not performed according GL and/or not GLP but scientifically acceptable – 3: not valid or not assessment possible.
In rats, three studies showed LD50 greater than 2000 mg/kg. In the most recent studies, performed according to the same experimental protocol except for occlusion (one occlusive and the other 1⁄2 occlusive) animals exhibited cl inical signs only when exposed to EGBE under complete occlusion. Clinical signs were the same than t hose seen with inhalation administration: mainly haemolysis, lethargy, atax ia, hepatic and renal toxicity. No signs of irritation were noted in any of the studies.
In guinea-pigs, great variations were seen depending on the studi es. LD50 ranged from 208 mg/kg to 6411 mg/kg. Only one recent study were performed according to guidelines. This study gave a LD50 of greater than 2000 mg/kg. For this study, no adverse effects were described (local or systemic). Very few deta ils are available about the local or systemic toxicity for the other studies.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 112 In rabbits, except for one study (Duprat and Grad iski, 1978), results we re quite consistent. When EGBE was applied occlusively, calculate d LD50 for a 24-hour application ranged from 435 to 841 mg/kg in 6 studies. When applied semi-occlusively, LD50 was greater than 2000 mg/kg, showing the importance of evaporation. Common systemic signs of toxicity usually seen with EGBE were described: ataxia, la boured breathing, depressi on, cyanosis, renal – hepatic, thymic and splenic toxic ity. Local signs of irritation were seen in some studies, mild irritation for lower doses and sometimes severe irritation , even necrosis for the higher doses.
Overall, for dermal toxicity, great differences were seen between the tested species and the mode of occlusion. When EGBE was administ ered non-occlusively or semi-occlusively the LD50 was much higher than when administered o cclusively. As for acute oral toxicity rabbit seems to be the most sensitive species with LD50 of about 500 mg/kg when administered occlusively whereas other species exhibite d LD50 greater than 2000 mg/kg in the same experimental conditions. Clinical signs were the same than those observed via oral route. Local signs of irritation were noted, mild for low doses and sometimes necrosis for the high doses. By dermal route, depending on the appl ication (occlusive or not) the LD50 was 500 mg/kg or > 2000 mg/kg respectively.
According to the data available, a classification R21 is needed.

Oral

Rat studies
Oral acute toxicity of 69 glycols and glycols deriva tives was assessed (Smyth et al. , 1941). The substance was administrated di luted in water via oral route. Ten Wistar male rats were used for each dosage and enough dosages were ad ministrated to include those at which all died. Observation period after dosing was 14 days. The maximum tested concentration was 2 %.
Most deaths occurred within the first 2 days after dosing Lethal Dose: LD50 was 1260 mg/kg (1120 to 1420).
In the publication of Carpenter et al., 1956 and Mellon Institute of Industrial Research, 1952, some LD50 results were repor ted. These tests were perfor med according to a standard procedure (exceptions were specified) on non fast en rats (5-6 weeks ol d) with dose levels differing by a factor of 1.26 or 2 in a geometric series. A 14-day post exposure observation period was made. Wistar rats were used until 1942, then Sherman rat from 1942 to 1952 and Carworth – Wistar from 1952.
Symptoms were sluggishness, ruff ling of coats, prostration, a nd narcosis. Autopsies on dead rats revealed congested of haemorrhaged l ungs, mottled livers, severely congested kidneys and haemoglobinuria.

Results are summarised in the table 4.50:EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 113 Table 4.50: Summary of the LD50 reported in Carpenter et al ., 1956 Sex Weight (g) Concentration EGBE in Water (%) LD50 and range (mg/kg) Year

M 90-120 10 1480 (1150 to 1910) 1938

M 90-120 10 2600 (2300 to 2900) 1951
F 90-120 10 2300 (1900 to 2800) 1951
M 90-120 5 2800 (2400 to 3300) 1952
F 90-120 5 1600 (1200 to 2100) 1952
M 90-120 5 1900 (1300 to 2600) 1953
F 90-120 5 1600 (1400 to 1900) 1954
M 30-60 5 3000 (2600 to 3400) 1954
F 30-60 5 2300 (2100 to 2600) 1954

M 335-460 5 560 1954
F 260-320 5 530 (380 to 750) 1954
M 90-120 5 2400 (2100 to 2700) 1954
F 90-120 Undiluted 2800 (2200 to 3700) 1954
M 90-120 5 2100 (1400 to 3100) 1955

In a range-finding test performed on rats (poorly described), EG BE (purity unknown) was administered orally in a water vehicle to gr oups of 5 animals at doses of 252, 500 and 1000 mg/kg (Dow, 1959).
Mortality was 3/5 animals at 500 mg/kg and 2/ 2 at 1000 mg/kg. From the lower dose, animals appeared sick and drowsy. Signs of haemat otoxicity appeared at the dose of 500 mg/kg (haematuria). From this study a LD50 of 470 mg/kg can be calculated.
EGBE (purity unknown) was administered by oral route to groups of 10 female rats at doses of 1000 – 1600 – 2000 – 2500 and 4000 mg/kg in a 10 % solution in corn oil. Rats were fasted 12 hours before administration. Post e xposure observation period was 7 days (Hoechst, 1966).
Mortality occurred from 1600 mg/kg bw dose a nd higher and between 1h30 and 24 hrs after treatment. In this study th e calculated LD50 was 1950 mg/kg.

Male Wistar rats were administrated Po lysolv EB (EGBE – purity unknown) orally by intubation ( MB research la boratories, 1976). Doses of 670, 1310, 2560 and 5000 mg/kg were used. Animals were observed fo r mortality, toxicity for 14 da ys and the LD50 calculated at the end of this period.
No mortality was found for the 670 mg/kg gr oup, 3/10 animals died in the 1310 mg/kg group, 9/10 animals died in the 2560 mg/kg group an d 10/10 animals died in the 5000 mg/kg group. Signs of toxicity were lethar gy in all groups and ataxia in the high dose group. At necropsy, signs of liver and kidney toxicity were s een in the 1310 mg/kg group. Blood in bladder wasEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 114 seen in all animals of the 2 highest dose gr oups. The LD50 calculated in this study was 1590 mg/kg.
Male Wistar rats (5/group) were dosed with EGBE at doses of 9030- 4515 - 2257 - and 1128 mg/kg (Bushy Run Research Center, 1980b). Anim als were observed during 14 days before calculating the LD50.
All animals died at doses of 9030 and 4515 mg/ kg during or the day after administration. At 2257 mg/kg two out of 5 animals died on day 5 af ter administration. No deaths were seen at 1128 mg/kg dose.
For the 2 high doses, clinical symptoms were difficult breat hing, sluggish and bloody salivation. For the 2257 mg/kg dos e, animals were sluggish and had an unsteady gait. Nothing remarkable was seen on animals of the lowest dose. On dead animals, necropsy revealed dark livers, distented stomach, filled with liquids an d gaz. Red kidneys, intestine containing blood, red adrenals were also reported. Nothing was seen in survivors. In this study, calculated LD50 was 2420 mg/kg (1670 - 3504 mg/kg).
EGBE undiluted (purity unknown) was administered orally to groups of females CDF rats (3/group) at doses of 130, 250, 500, 1000 and 2000 mg/kg (Dow, 1981).
Mortality was seen at 200 mg/kg (2 out of 3a nimals). Clinical signs were lethargy and respiratory difficulties at 2000 mg/kg and necrosis of the tails in all surviving rats of the 1000 and 2000 mg/kg dosed groups. For this study, the LD50 was between 1000 and 2000 mg/kg.

The acute oral LD50 was determined in fasted and fed rats (5/group) for 9 chemicals including EGBE (purity > 99.5 %) (Eastman Kodak, 1981a). Male CD/BR rats were given EGBE undiluted by gavage (five different doses progressing by a factor of 2).

The LD50 calculated for fasted or fed rats wa s 1746 mg/kg. For both groups (fed and fasted) clinical signs were: inactivity, laboured breath ing, anorexia, tremors and death. Haematuria was seen at high dose. In dead animals, blood was found in stomach and intestines.
Some LD50 studies were reported without deta ils concerning the method used (Tyler, 1984). A LD 50 of 620 mg/kg was reported in rats (Rowe and Wolf, 1982).

Mouse
Carpenter et al., 1956 reported some experiments concer ning acute toxicity of EGBE in various animals. A LD50 of 1230 mg/kg wa s reported for mice via oral route.

The acute oral LD50 was determined in fast ed and fed mice (5/group) for 9 chemicals including EGBE (purity > 99.5 %) (Eastman Kodak, 1981a). Male CD1 mice were given EGBE undiluted by gavage (five different doses progressing by a factor of 2).

The LD50 calculated for fasted mice was 1519 mg/kg, whereas LD50 for fed mice was 2005 mg/kg. For both groups (fed and fasted) clinic al signs were: inactiv ity, laboured breathing, anorexia, tremors and death. Haematuria was se en at high dose. In dead animals, blood was found in stomach and intestines.
Some LD50 studies were reported without deta ils concerning the method used (Tyler, 1984). Two LD 50 of 1170-1700 mg/kg and 1000-1600 we re reported (Rowe and Wolf, 1982 and Saparmamedov, 1974 respectively).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 115 Rabbit
Carpenter et al., 1956 reported some experiments concer ning acute toxicity of EGBE in various animals. A LD50 of 320 to 370 mg/kg was reported for rabbits via oral route.

Guinea-pig
In a GLP study (performed according to OECD GL 401), guinea pig were given orally EGBE (purity 99.8 %) in a distilled water vehicle at dose of 500, 1000 and 2000 mg/kg (Eastman Kodak, 1994a ).
Mortality was 3/5 in males and 5/5 in female s at the top dose. At 1000 mg/kg, the mortality was 1/5 for males and females. Clinical signs we re slight to severe weakness, sialorrhea and staining of the hair of the face and/or a bdomen for the 2000 mg/kg dose. At 1000 mg/kg animals evidenced weakness and prostration the day of administration. On ly a slight weakness was noted for the 500 mg/kg group. Sign of necros is and minor haemorrhage in the gastric mucosa was seen after termination in anim als doses with 1000 and 2000 mg/kg. For this study, the LD50 was calculated to be 1414 mg/kg.
Carpenter et al., 1956 reported some experiments concer ning acute toxicity of EGBE in various animals. A LD50 of 1200 mg/kg was re ported for guinea-pigs via oral route.

Summary oral route:
Table 4.51: Summary of LD50 values after oral route administration LD50 (mg/kg) Toxicological effects Validity * Reference

Rats Oral route
1260 mg/kg 3 Smith, 1941

560-2800 mg/kg for males
530 and 2300 mg/kg for females Narcosis - prostration 2 Mellon Institute of Industrial Research, 1952
Carpenter et al. , 1956

470 mg/kg Mortality from 500 mg/kg. Haematuria. 3 Dow, 1959

1950 mg/kg Mortality from 1600 mg/kg 2 Hoechst, 1966
1590 mg/kg Lethargy, laboured breathing, haemolysis, liver and kidney toxicity.
Mortality from 1310 mg/kg 2 MB Research Labs, 1976

2420 mg/kg Laboured breathing, sluggish and bloody salivation. Haemolysis, dark liver and red 2 Bushy Run Research Center, 1980 bEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 116 kidneys.
1000-2000 mg/kg Lethargy, laboured breathing, necrosis of the tail.
Mortality from 2000 mg/kg. 2 Dow, 1981

1746 mg/kg
(fasted or fed) Inactivity, laboured breathing, anorexia, tremors, haemolysis. 2 Eastman Kodak, 1981a

620 mg/kg 3 Rowe and Wolf, 1982

Mice Oral route
1230 mg/kg 2 Carpenter et al. , 1956
1170 – 1700 mg/kg
1000 – 1600 mg/kg 3 Rowe and Wolf, 1982
Saparmamedov, 1974
1519 mg/kg (fasted)
2005 mg/kg (fed) Laboured breathing, anorexia, tremors, haemolysis. 2 Eastman Kodak, 1981a

Rabbits Oral route
320 – 370 mg/kg 2 Carpenter et al. , 1956
Guinea pig oral route
1200 mg/kg 2 Carpenter et al. , 1956
1414 mg/kg Weakness, prostration, necrosis and haemorrhage of gastric

mucosa. Mortality from 1000 mg/kg 1 Eastman Kodak, 1994a

* 1: valid (performed according to GL and GLP) – 2: valid with restriction (not performed according GL and/or not GLP but scientifically acceptable – 3: not valid or not assessment possible.
In rats, numerous studies has been performed to assess the LD50 via oral route. Results varies a lot between 470 mg/kg and 2600 mg/kg. Recent studies (performed according to well defined experimental methods) have given results between 1000 and 2600 mg/kg. Clinical signs noted were: lethargy, laboured breathing, at axia. For pathology, haemolysis was seen in the majority of the studies, sometimes accompanie d with renal and hepatic lesions (certainly a consequence of haemolysis).
In mice, available studies exhibited LD50 ranging from about 1500 to 2000 mg/kg (these results are more consistent than those obtained in rat studies). Clinical symptoms similar than those of the rat studies were seen.
One study was performed in rabbits showing a LD50 ranging from 320 to 370 mg/kg. This value seems very low compared to other studies on other species via oral route. The rabbit can be considered to be the most sensitive species concerning acute oral toxicity of EGBE.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 117 Considering that this study is an old study, and th at only the result is available (no details on purity or experimental conditions), it is difficult to take this value in to account for the risk characterisation or cl assification purposes.
Two studies are available in Guinea-pigs. Th e LD50 calculated were 1414 and 1200 mg/kg. The same clinical signs and pathology than othe r species tested were seen in these studies. Necrosis and haemorrhage of the gastric mucosa was also seen.
According to the data available, a classification R22 is needed.

Other routes

Rats
Various results concerning IV administration of EGBE were reported (Mellon Institute of Industrial Researc h, 1952 ; Carpenter et al. , 1956): LD50 in female rats (170-230 g) when administrated as 3 % dilution was 380 mg/kg (2 90 – 500 mg/kg) – LD50 in female rats (90- 120 g) when administered undiluted was 340 mg/kg (300 to 380 mg/kg).
In a non-GLP study, unique IP injection of 2 different EGBE (n butyl oxitol ® 98-99 purity and Dowanol EB ® > 99 % purity) was performed on fema le SD rats (4/group). Doses of 200, 252, 316, 398 were tested for both substances with an additional dose of 500 mg/kg for Dowanol EB ® (Dow, 1972).
For both substances, mortality was seen from 252 mg/kg. In all dose groups and with both substances blood was seen in urine. A LD50 of 252 mg/kg was calculated for n butyl oxitol ® and a LD50 of 317 mg/kg was calculated for Dowanol EB ®

. Ten female SD rats were injected IV with 0.034 ml EGBE/kg (Freundt and Helm, 1986). Twenty four hour urine samples were collec ted during the next 4 days. The following parameters of kidney function were examined in the urine: volume, osmotic pressure (by means of freezing point depression), haematuria (using a semi-quantitative test), albumin, Lactate DeHydrogenase (LDH). Gel filtration of the urine was carried out before enzyme and albumin analyses.
After treatment, the following pa rameters were found to be cha nged significantly: the albumin level and the LDH activity was augmented only on the 2 nd day, and the GAL activity was decreased only in the 24-h urine samples from the 4 th day. The urinary LAP activity, the volume, and the osmotic pressure of the urine remained unchanged. EGBE caused a microhaematuria (approximately 50 erythroc ytes/μl) in 2 (of 10) rats on the 1 st dose day after its application.
SD rats were injected IV with 0.03 4 ml/kg EGBE (purity unknown) (Freundt et al. , 1993 – abstract). The activiti es of lactate dehydrogenase (LDH), leucine aminopeptidase (LAP) and beta-galactosidase (GAL), the concentrations of albumin and creatinine, the volume, the specific gravity and the pH, leucocytes, erythrocytes, nitrite, total protein, ketone, bilirubin and urobilinogen were analys ed in the 24 hr urine.
A significant increase of albumin and LDH activity was seen in the female rats at the 2 nd day after administration. All other parameters re mained normal. Because these changes were small and transient, the authors concluded th at EGBE have a slight nephrotoxic potential.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 118 Mice
SC injection of 0.5 cc/kg to white mice produced severe toxicity (Von Oettingen and Jirouch, 1931). For 0.25 cc/kg no mortality was seen and one out of two animals died with 0.5 cc/kg dose. The only animal treated with 0.75 cc/kg died. Due to the small number of animals treated no LD50 was calculated.
One study concerning IV administration of EGBE to mice was reported (Carpenter et al

. , 1956): LD50 in male and female mice when ad ministrated as 3 % dilution was 1130 mg/kg.
Acute toxicity was assessed in vitro and in vivo in mice (Tanii et al. , 1992). Male ddY mice (4 animals/dose – 4 doses) were given IP 2000 mg CCl 4 in olive oil or olive oil only 24 hr before dosing with EGBE. CCl 4 is known to inactivate the hepatic microsomal monooxygenase system and may influence the detoxi cation of EGBE. It also inhibits hepatic non-microsomal enzymes including ADH. An imals were observed 1 week and after termination the LD50 was calculated. Concurrent ly a cytotoxicity test was performed to determine ED50.

LD50 without CCl 4 was 6.77 mmol/kg whereas it was equal to 4.99 mmol/kg with CCl 4 . As CCl 4 at the dose used appears to inhibit all hepatic enzyme activities (microsomial and non microsomial), the authors concluded that it is unlikely that the metabolite contribute to the acute toxicity of EGBE.
There was no significant correlation between ED50 for the different cells used in the cytotoxicity studies and LD 50. A correlation was found betw een LD50 and logP for the majority of the tested substances. No quantitative relati onship was described.

Rabbits
The LD50 for male rabbits by IV route of undilu ted EGBE is 0.14 ml/kg (Mellon Institute of Industrial Research, 1952). Death occurred precipitally after 0.2 ml/kg while after 0.1 ml/kg the rabbits were prostrated and rapidly reco vered. At the 0.05 ml/kg dosage level, the only symptoms were increase d rate of respiration.
In the same report, various resu lts concerning IV administrati on of EGBE were reported: 3 % EGBE in 0.75 % NaCl solution was not haemolytic to rabbit red blood cells. LD50 of a 3 % EGBE solution was 500 mg/kg (380 – 650 mg/kg).

Table 4.52: Summary other routes LD50 (mg/kg) Experimental conditions Effects Validity * Reference

Rats
380 mg/kg IV route in 3 % water dilution 2 Mellon Institute of Industrial Research, 1952
317 mg/kg IP route. Mortality from 252 mg/kg. Haemolysis 2 Dow, 1972

IV route Increase of LDH activity and decrease of 3 Freundt and Helm, 1986EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 119 0.034 ml/kg GAL activity.

Microhaematuria

IV route
0.034 ml/kg Increase of albumin and LDH activity. 3 Freundt and Helm, 1986

Mice
About 0.5 cc/kg SC
0.25, 0.5 and 0.75 cc/kg 0.5 cc/kg produced severe toxicity (1 death out of 2 treated). 0.75 cc/kg: death of the only treated animal. 3 Von Oettingen and Jirouch, 1931

1130 mg/kg IV, 3 % dilution. 3 Carpenter et al

. , 1956
6.77 mmol/kg without CCl4
4.99 mmol/kg with CCl4 IP 3 Tanii et al., 1992

Rabbits
500 mg/kg IV route 2 Mellon Institute of Industrial Research, 1952
* 1: valid (performed according to GL and GLP) – 2: valid with restriction (not performed according GL and/or not GLP but scientifically acceptable – 3: not valid or not assessment possible.
Some studies performed via IV, IP and SC routes in various species gave different results. These studies are not suitable for RA because th ese routes of administ ration are not relevant with exposure scenarios. However, some of them give valuable data about the mode of action of EGBE for renal toxicity and valuable information about hepatic metabolism.

4.1.2.2.2 Data from human exposure

Inhalation
2 males and 1 female were exposed to 200 pp m two 4-hr periods separated by 30 minutes lunch (Mellon Institute of I ndustrial Research, 1955). Erythr ocyte fragility test, blood pressure and pulse-rate were determined the exposure day (3 measure before - during lunch and after exposure - for fragility test, one m easure was also made during exposure). Urine analysis for glucose and albumin were conduc ted the following morning, and the BAA levels were determined in 24-hour ur ine samples first collected at the end of the exposure day.

One male and the female excreted considerab le amount of BAA in the two 4 hrs periods following exposure but the other male excreted only trace amount of this metabolite for the same period. Clinical symptom (subjective) were : immediate irritation of the nose and throat,EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 120 followed by ocular irritation a nd disturbed taste was experienced by all three subjects. The female subject, who excreted the largest amount of BAA, reacted mo st adversely to the exposure, and acquired a headache which lasted about 24 hr. Unlik e rats, no effects were seen in the fragility test in human tested.
Another study was performed on human with lowe r doses: 2 females and 1 male and the male of the precedent study whom had only traces of BAA in urine were exposed to 100 ppm EGBE in the same conditions that the precedent study.
The only objective finding of si gnificance in the exposed subject s was the urinary excretion of BAA. Other measured parameters did not sh own significant differences. Even one subject who had not excreted significant quantities of the metabolite after the 200 ppm exposure, did eliminate 75.5 mg in 24 hours. The urinary BAA le vels of the other subj ects were similar to that found at the 200 ppm e xposure. Surprisingly, the subj ective response of the humans exposed to 100 ppm EGBE was at least as great, if not greater, than that elicited at the 200 ppm exposure level. The subject w hom participated at the former experiment reported that the EGBE odor was not as strong as at the 200 ppm leve l, although he did have a slight cold at the time of exposure. The two female subjects, appa rently experienced grea ter distress during and after exposure than did the males. One of them vomited after 7 hours of exposure and several times during the day after while the other contracted a headache the following day. The former subject stated that high air temperatur es often cause emesis, and was convinced that the present attack was caused by the relatively high chamber temperature (28.5° in the afternoon), rather than the solvent vapours. Th e other subject also though that the chamber temperature caused her headache.

Oral
In a suicide attempt, a 50-year woman inge sted 250-500 ml of a wi ndow cleaner containing 12 % of EGBE, corresponding to about 0.5 to 1 g/kg bw (Rambourg-Schepens et al. , 1988). On admission to the intensive care unit, the patient was comatose. Biochimically she presented with metabolic acidosis, hypokalaemia , a rise in serum creatinine level and a markedly increased urinary ex cretion of oxalate crystals.
In another case of attempted suicide (Gijsenbergh et al. , 1989), a 23-year-old woman had ingested about 500 ml of a mixture contai ning EGBE (12.7 %) and of EtOH (3.2 %) (about 57 g of EGBE corresponding to a dose of about 1g/kg bw).
Upon admission, she was in a comatose state, had breathing difficulties was in metabolic acidosis and had a concentration of EGBE in blood of 432 mg/l. 2 hr after admission, the blood concentration of EGBE was 304 mg/l . A fa ll in Hb from 11.9 g/dl on admission to 8.9 g/dl on the second day, together with the appearance of haematur ia was noticed. The half-life of EGBE in the blood was 210 min (greater than the commonly values described in the literature probably due to the pr esence of EtOH in the mixture). BAA concentration in the urine was maximum 4 hr after dialysis treatment was initiated.
A case of acute poisoning with EGBE was reported (Bauer et al. , 1992). A 53-year-old man ingested intentionally 500 ml of a mixture containing 9.1 % of EGBE (45.5 g – about 750 mg/kg bw) and 2.5 % EtOH.
Clinic signs were coma, tac hycardia, metabolic acidosis and hypoxemias, pulmonary oedema and Adult Respiratory Distress Syndrome (ARD S). By 36 hr after admission, the subjectEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 121 showed signs of non haemolytic hypochromic anaemia with thrombopenia. No signs of haemolytic anaemia were noticed.
Some cases of children poisoning which have occu rred in the Pittsburgh Poison Center were summarized in a publication (Dean and Kren zelok, 1992). The ingested products contained EGBE in concentrations ranging from 0.5 % to 9.9 %. The children poisoned ages ranged from 7 months to 9 years. The quantities range d from 5 to 300 mL - 24 children were studied - 2 of them were treated by gastric gavage followed by a 24 hr hospitalisation. The other children were treated at home w ith simple dilution. Common symptoms seen in acute animal dosing (haemolysis, CNS depression, metabolic acidosis and renal compromise) did not

occur. A 18-year-old male consumed between 360 a nd 480 ml of a glass cleaner containing 22 % EGBE (maximum 95 g of EGBE – about 1,25 g/kg bw) (Gualtieri et al. , 1995 –and Gualtieri et al. , 2003).
He was hospitalised with metabolic acidosis and hepatic biochemical abnormalities (Serum Glutamic Oxaloacetic Transaminase (SGOT), Se rum Glutamic Pyruvic Transaminase (SGPT) and hepatic bilirubin). After treatment with EtOH and haemodialysis, the patient returned home without sequelae. Nine days following his initial discharge, he was again admitted after ingestion of 480 ml of the same cleaner. Th e second hospitalisation was absent of any significant metabolic or hepatic abnormalities. Haematological and renal abnormalities were absent in both hospitalisations. Quantitati ve analysis for BAA has given a maximum concentration of 4.86 mmol/L approx imately 16 hours after ingestion.
A 19 year old man ingested about 20-30 oun ces of a product containing 25-35 % EGBE (maximum 336 g of EGBE – about 4.5 g/kg bw) (Burkhart and Donovan, 1998). The product had a pH of 13 and was therefore considered as an alkaline corrosive . The patient developed characteristic features of coma, acidosis a nd haematuria which were previously reported following EGBE ingestion. After treatment, the patient still exhibite d neurologic sequelae (difficulties with fine motor skills and stop speaking). However the patient medical history was significant for mental re tardation, depression, hyperactivit y and he was treated with various strong psychiatric medecine stopped af ter the ingestion. This weakens the direct relationship between EGBE and the sequelaes reported.
A case report of a massive ingestion of a mi xture containing 10-30 % of EGBE and 10-40 % of isopropanol was reported (McKinney et al. , 2000). A 51-year-old woman ingested 8 ounces of this mixture (corresponding to an estimate dose of 24-72g of EGBE corresponding to a range of 0.4 – 1.2 g/kg bw ) in a suicide attempt.
Clinical symptoms were metabolic acidosis a nd mental status depr ession. She was treated with EtOH therapy and recovere d without apparent sequelae. No signs of haematotoxicity were noticed.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 122 Table 4.53: Summary human acute toxicity data

Estimation of absorbed dose Patient history Patient pathology Reference

Between 0.5 and 1 mg/kg bw 50-year woman. Suicide attempt with glass cleaner. Coma, metabolic acidosis, hypokaliemia, increase in serum creatinine level and urinary excretion of oxalate crystals Rambourg-Schepens et al. , 1988.

About 1 g/kg bw 23-year woman. Suicide attempt with mixture containing EGBE. Coma, breathing difficulties and metabolic acidosis. Haematuria and decreased Hb for 2 days. Gijsenbergh et al.
, 1989.

About 750 mg/kg bw 53-year man. Suicide attempt with mixture containing EGBE. Coma, tachycardia, metabolic acidosis, hypoxemia, pulmonary oedema and ARDS. Non haemolytic anaemia with thrombopenia. Bauer et al ., 1992.

About 1.25 g/kg bw – 2 times separated by 9 days 18-year man. Ingestion of a glass cleaner. Metabolic acidosis and hepatic biochemical disorders.
Nothing after the second ingestion. Gualtieri et al. , 1995, Gualtieri et al. , 2003.

About 4.5 g/kg bw. 19-year man. Ingestion of a mixture containing EGBE. Coma, acidosis and haematuria. Burkhart and Donovan, 1998

Between 0.4 and 1.2 g/kg bw 51-year woman. Ingestion of a mixture containing EGBE. Metabolic acidosis and mental status depression. McKinney et al.
, 2000

According to this data, a LOAEL of 400 mg/ kg bw can be taken into account for acute toxicity by oral route in humans. It should be noted that this is a worst case estimation derived from the McKinney paper in which the possi ble range of exposure was between 0.4 and 1.2 g/kg bw.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 123 Summary human data:
Acute human toxicity data comes from children a ccidental ingestion or adult suicide attempts made with mixtures containing EGBE and from one study on human volunteers by inhalation. For oral route case reports, ingested doses are difficult to evaluate because of the lack of data concerning the body weight of all patients and the exact ingested dose, but a semi-quantitative estimation of the ingested doses was made for each case (see table 4.7). The range of doses which lead to clinical symptoms varies betw een 0.5 and 4.5 g/kg bw. In all cases, patients exhibited SNC depression and metabolic acidosis . Signs of haemolysis were seen in some cases but this finding was not syst ematic (this showed that human is much more resistant to haemolysis than rodents). After a first acute in gestion, a second administrator some days later did not exhibit the same symptoms, this finding was also seen with animals in some studies. In these cases, EGBE was ingested together with other substances (ethanol and/or unknown substances) that could have some influence on the symptoms seen. Between 0.5 and 1.5 g/kg bw the patients totally recovered after treatme nt. According to this data, a LOAEL of 400 mg/kg bw can be taken into account for acute t oxicity by oral route in humans in the risk characterisation section. If a risk characterisation by dermal and inhalation route is needed for acute effects, kinetic data is sufficient to extrapolate this oral LOAEL to inhalation and dermal LOAEL. Human data is preferred for risk characte risation, especially for EGBE because of its haematotoxicity more marked in animals than in humans. It should be noted that this is a worst case estimation derived from the McKinney paper in which the possible range of exposure was between 0.4 and 1.2 g/kg bw.
By inhalation route, symptoms reported by the th ree volunteers were signs of irritation (throat and ocular) and headache. These sympto ms did not seem to be dose-related.

Summary acute toxicity
For the inhalation route, the 4 hour LC 50 in rats, which are suscepti ble to haemolysis (see next section), was of the region of 450ppm (2214mg/m 3 ). Higher values are seen in other species.
For the dermal route, great differences were se en between the tested species and the mode of occlusion. The rabbit seems to be the most sensitive species with LD 50 of about 500 mg/kg when administered occlusively.
For the oral route, available studies considered as reliable show LD 50 values upwards from 1000mg/kg.
According to the data available, a classificatio n R20/21/22 is needed for all three routes of
exposure.
A number of human case studies are available fr om attempted suicides which suggest that the human LOEL is in the region of 400 mg/kg bw. Human data is preferred for risk characterisation, especially for EGBE, because it s haematotoxicity is more marked in animals than in humans. There is sufficient kinetic data available to enable route to route extrapolation to be carried out to risk assess th e more relevant dermal and inhalation routes. These extrapolations are carried out in the risk characterisation section. It should be noted that this is a worst case estimation of the LOEL derived from the McKinney paper in which the possible range of exposure was between 0.4 and 1.2 g/kg bw.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 124 4.1.2.2.3 Specific toxicity: haematotoxicity
Some studies were performed especially to assess the haematotoxic pot ential of EGBE in various conditions.

The studies described above are but a few that indica te that EGBE cause s acute haemolytic anaemia in rats and mice. This response is consistent and probabl y forms the basis for establishing an No Observable Adverse Effects Levels (NOAEL) in species used experimentally. It has also been suggested that it is an essential, early step in the development of haemangiosarcomas of the liver in male mice (described in 4.1.2.8.3). Clearly, if the haemangiosarcomas arising in male mice (see Section 4.1.2.8.3) are ultimately a consequence of anaemia induced by EGBE, the species and sex specificity of the neop lastic response must be due to some factor(s) subseque nt to the anaemia, since this also occurs in rats and female mice. Possible elements contributing to the sp ecificity of the response will be examined and the strength of the evidence evaluated in orde r to reach a conclusion on the human relevance of the haematological effects and the ne oplastic responses observed in rodents.

In vitro

In vitro and in vivo response of mammalian erythrocytes to EGBE and BAA was assessed (Carpenter et al. , 1956). The results are summ arized in the table 4.54:

Table 4.54: Summary of the erythrocyte toxicity in Carpenter et al. , 1956.

In vitro response In vivo response

2.5 % EGBE 0.1 % BAA

Animal Sex Max.time without haemolysis , Min Max. time to haemolysis, Min Max.time without haemolysis, Min Max. time to haemolysis, Min Osmotic fragility upon inhalation of EGBE Haemoglobinuria upon inhalation of EGBE

2 rats

3 rats

2 rats F

M

F 46 60-67

35-40

35-40 40-60

40-46 Significant ↑ at 62 ppm x 4h Seen at 203 ppm x 7h but not at 107 ppm x7h

2 mice

2 mice

1 mouse M

M

F 22-30 35-40

40-45

45 50-60

55 Significant ↑ at least as low as 100 ppm x 7h Seen at 200 ppm but not at 100 ppm x7h

2 rabbits

4 rabbits

2 rabbits M

M

F 110.113 123-125

60-90

80-90 80-100

85-100 Significant ↑ at 125 ppm x 7h Not seen as high as 197 ppm x 7h

1 monkey F 120 130 No Significant ↑ Not seen as high asEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 125 1 monkey M 103 120 at 200 ppm x 7h 200 ppm x 7h

2 dogs
2 dogs F

F 20 40-86

89-120 No Significant ↑ at 665 ppm x 7h x 2 days Not seen as high as 665 ppm x 7h

1 human

2 humans F

M 80

60-83 100

80-94 147-268 No Significant ↑ at 200 ppm x 8h Not seen at 200 ppm x 8h

2 guinea pigs
2 guinea pigs M

M 60-68 80

360 No Significant ↑ at 665 ppm x 8h Not seen at 665 ppm x 8h

Effects of EGBE and B AA on human, rat, dog and rabbit blood were assessed in vitro (ICI, 1985). EGBE and BAA solutions were prepared with veronal buffered saline. pH was adjusted to 7.2. One ml of erythr ocyte suspension was added to 3 ml of the test solution. The haemolytic activity was calculated using positive (totally lysed sample) and negative (no test substance) controls. Concentr ation range of 0.01 to 2 % BAA and 0.05 to 0.5 % EGBE were tested in the main study. With 2.5 and 1 % EG BE solutions, no analysis could be performed due to a discoloration of the test medi um. The problem was solved by employing 0.5 % EGBE as the maximum permissible concentration for incubation.
With BAA, in the preliminary study, no effect s were seen on human erythrocytes whereas marked haemolysis was noted in rat erythrocytes (38.4 %).
With BAA only rats erythrocytes readily lysed and a very mark ed haemolysis was seen from 0.05 %. With EGBE dog erythrocytes seemed to be very sensitive to haemolysis and various effects were seen from 0.4 % in other species (see tables 4.55 and 4.56).

Table 4.55: Haemolytic activity of various concentrations of butoxyacetic acid incubated wi th erythrocytes from rat, human, dog and rabbit Haemolysis (%)
Overall concentration (%) Species

0.01 0.02 0.03 0.04 0.05 0.1 0.2 0.5 2

Rat 0.8 4.4 8.4 11.1 53 91.9 97.9 98.8 24.9

Human 0.8 1.4 - - 0.2 0 1.2 0.8 0
Dog 0 13.6 - - 0 0 0 0 2.6

Rabbit 4.9 0 - - 3.2 1.2 0 1.1 1.7EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 126 Table 4.56: Haemolytic activity of various concentrations of EGBE incubated with erythrocytes from rat, human, dog and rabbit. Haemolysis (%)
Overall concentration (%) Species

0.05 0.1 0.25 0.4 0.5

Rat - 0 2.5 51.5 62

Human - 0 1.5 20.5 70.9
Dog 46.8 36.2 - 41.2 62.3

Rabbit - 0 2.8 83.7 72

A single dose of EGBE was applied dermally (semi- occlusively) to groups of 3 female Wistar rat at doses of 500, 375 and 260 mg/kg and to a gr oup of 6 female rats at 200 mg/kg (Bartnik et al. , 1987). Six hours after dosing, blood was sample d retro-orbitally. Animals were then killed and an additional blood sample was ta ken by heart puncture. Concurrently EGBE was injected intravenously to gr oups of 4 male rats at doses of 25, 37.5, 50, 62.5 and 75 mg/kg. At 3 and 6 hr following application blood was coll ected retro-orbitally. After termination, blood was also collected by heart puncture.
In this study, in vitro haemolysis was also studied, male Wistar rats and humans red blood cells were treated with various concentratio ns of EGBE and BAA. Positive and negative controls were performed, haem olytic activity was expressed as percentage of totally hemolyzed.

In vitro , BAA showed higher haemolytic potency th an EGBE. Hemolytic activity of EGBE and BAA was markedly lower on the human erythr ocytes compared with rat erythrocytes (see tables 4.57 and 4.58).

Table 4.57: Haemolysis of human and rat erythrocytes by EGBE Percentage haemolysis

15 min 30 min 45 min 60 min 120 min 180 min
EGBE (mmol/liter) R H R H R H R H R H R H

100 0 0 0 0 0 0 0

125 0 0 0 0 10 64 0

150 0 0 0 0 0 0 8 0 62 3 92 11

175 0 0 6 0 41 0 91 2 100 23 100 89

200 5 100 0 100 2 100 8 100 100 100 100

225 96 0 100 14 100 89 100 100 100 100 100 100

250 100 8 100 100 100 100 100 100 100 100 100 100EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 127 Table 4.58: Haemolysis of human and rat erythrocytes by BAA Percentage haemolysis

15 min 30 min 45 min 60 min 120 min 180 min
BAA (mmol/liter) R H R H R H R H R H R H

1.25 0 0 0 0 0 0 0 0 6 0 25 0

2.50 0 0 0 0 4 0 20 0 79 0 95 0

3.75 0 0 0 0 17 0 49 0 86 0 100 0

5.0 0 0 13 0 59 0 88 0 100 0 100 0

6.25 0 0 17 0 59 0 89 0 100 0 100 0

7.5 0 0 25 0 69 0 100 0 100 0 100 0

10 0 0 0 0 0 0

15 0 0 0 0 0 0

In vivo , a dermal dose of 200 mg/kg did not cause a ny haemolytic effect. Other tested doses caused haemolytic effects indicated by increase in mean cell volume, lower erythrocyte count and haemoglobin level and haemoglobinuria. No dose response relationship was found but according to the authors it might be due to i nherent biologic varia tion in percutaneous absorption and haemolytic susceptibility and al so to fewer animals per dose group. Following a IV injection of 62.5 mg/kg, no haemolysis was detectable whereas a dose of 75 mg/kg produced slight effects.

The effects of EGBE and its metabolites on hum an and rat erythrocytes were investigated in vitro (Ghanayem, 1989). Hepari nised blood was collected from a male and a female volunteer and cardiac blood was collected from male F344 rats, using EDTA as the anticoagulant. Blood-chemical mixtures were incubated in a shaking water bath at 37 ° for up to 4 hr. Incubation of whole blood from rats wi th 20 mM EGBE was associated with a slight, but significant decrease in haematocrit values and significant haemolysis, as determined by free plasma haemoglobin concentrations. No si gnificant changes were observed with 5 or 10 mM EGBE. Similar incubations, but with 2-(1-[ 14 C]butoxy)ethanol, followed by High Perfomance Liquid Chromatograpy (HPLC) analysis for metabolites failed to demonstrate the presence of either Butoxy Aldehyde (BAL) or BAA. Incubation of rat blood with BAL at 0.5, 1.0 or 2.0 mM resulted in both time- and concentr ation-dependent swelling of erythrocytes, as shown by an increase in haematocrit values, although significant haemolysis occurred only after incubation with 2 mM BAL for 4 hr. Sim ilar incubations of rat blood with BAA at 0.5, 1.0 or 2.0 mM also induced swelling of eryt hrocytes and haemolysis, but the highest concentration induced its maximum haematocri t response in 2 hr, which was followed by a rapid fall as haemolysis was essentially complete by 4 hr. Repetition of the incubations of rat blood with BAL, but supplemented with aldehyde dehydrogenase and NAD + , resulted in a significant potentiation of the effect of BAL, whereas addition of cyanamide (an inhibitor of aldehyde dehydrogenase) sign ificantly decreased the activity of BAL in both the presence and the absence of the exogenous enzy me. Therefore, it appeared that BAA was the haemolytic metabolite and that haemolytic activity of BAL was due to its metabolism to BAA byEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 128 dehydrogenases present in rat blood. It wa s also found that reduction of blood ATP concentrations was more effectively produced by the acid than by the aldehyde, while EGBE itself had no effect on ATP concentration, even at a 10-fold higher concentration. It can be concluded that erythrocyte swelling precedes haemolysis, strongly suggesting that the erythrocyte cell membrane is the target of the haemolytic species. It was not clear, however, whether cell swelling was mediated by loss of ATP, or whether this loss was a secondary effect.

In contrast to these data w ith rat blood, incubation of blood from volunteers with BAA at concentrations up to 2 mM had no effect on h aematocrit or plasma fr ee haemoglobin. Small, but significant increases in haematocrit were observed at 4 and 8 mM after 4 hr incubation and there was minimal, but significant haemolys is at 8 mM after 4 hr. A slight decrease in blood ATP was observed with 8mM BAA, but there was no time dependent relationship.
SD rats were administrated EGBE by gava ge at 50 and 500 mg/kg (Kurantsin-Mills et al
., 1992). Blood samples were taken at 0.5 – 2 and 4hr after dosage for determination of the physical characteristic of RBCs by scanning mi croscopy (SEM) and frequency distribution of their indices by flow cytometry.
SEM revealed discocyte-spherocyte transformati on in treated rats. Flow cytometric analysis of the frequency distribution of the RBCs sh owed that the MCV and Mean Cell Haemoglobin Concentration (MCHC) increased and decrease d, respectively compared to the controls. Whole blood viscosity was evalua ted by microviscometry at 37°C over a wide range of shear rates. The deformability of RBCs was estimated as the resistance of a cell flowing through microchannels that simulated mi crovessels. Whole blood viscosity increased at shear rates of 5.75 to 230/s for doses 50 and 100 mg/kg, and then decreased thereafter due to haemolysis. In vitro simulation of shear-dependent haemolysis us ing the microviscometer at shear forces of 0.62 to 13.20 dynes/cm 2 indicated that the RBCs of EGBE-treated rats hemolyzed significantly more than the contro ls. Flow studies in the microc hannels also showed that the deformability of RBCs of EGBE-treated rats wa s significantly less than the control cells due to the decreased surface area to volume ratio . These results demonstrate that membrane and cellular changes in the RBC due to EGBE a nd/or its metabolites alter its rheological properties, and therefore compro mise transit through the microve ssels of the microcirculation resulting in haemolysis.

In vitro haematopoietic toxicity of some glycol ethers and deri vatives (EGEE, EGME, EGBE, EGEEA, EGMEA, EGBEA, diEGBE, EGBE diA and PGME) was assessed using haematopoietic culture assays in mu rine, rat and human species (Ruchaud et al. , 1992).
Acute and subacute toxicities of the compounds were analysed: on established haemopoietic cell lines (either leukemic or growth-factor-dep endent cell lines), on haemopoietic progenitor growth and differentiation (CFU-c assay) and in bone marrow long- term cultures (Dexter type cultures) to determine the e ffects on both the hemopoietic and the stromal cell tissues.
Acute and subacute toxicity on haematopoietic cell line.

EGBE was tested for 48 hr at co ncentrations between 0.1 M and 0.1 μ M. Typical dose response curves were obtained after treatme nt of NB4 cells. For all compound tested doseresponses show the common feature of a very st eep slope on a semi-log plot, indicating that cell viability went from normal to nil by increasi ng concentrations by only a factor of 4 to 8. This steep slope and the sigmoid shape of th e curve suggested that the substance hindered some cellular mechanisms essential for cell survival. Combined dose-response/time-course analysis were carried out on th e human promyelocytic leukaemia celll line NB4 show that theEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 129 toxicity increased regularly with time over 96 hr treatment. Compared to other glycol ethers, EGBE caused a distinct toxicity feature for sh ort incubations (0 to 40 hr) indicating that injuries causing cell death had pr obably multiple origins. The first component of the toxic phase worked at doses > 1mM and was over by 48 hr incubation. The second component probably worked from the beginning of the incubation, when its effects accumulate with the previous ones, but persist for longer incubatio n times (> 91 hr) and at lower doses. No differences were found for the same cell line wh en growth was supported by one or the other of the two growth factors tested, IL3 and G-CSF.
Acute toxicity on Haemopoietic Progenitor Cell s (CFU-c): human umbilical cord blood cells were treated with EGBE for 15 hr before CF U-c assay plating, then incubated for 14 days with 5637 CM. Growth of haemopoietic colonies was dramatically reduced after treatment. Virtually no growth was observed at concentrat ions > 20 mM. Similar results were found with murine bone marrow cells.
Toxicity on haemopoietic cells and microenviro nment in Dexter Cultures: effects of EGBE were evaluated on Dexter type long-term cu ltures two weeks after their establishment. Haemopoiesis was followed for 2 months with a constant exposure to va rious concentrations of compound. An acute toxicity of EGBE became obvious from 4 days of exposure at 7.5 mM. Virtually no haemopoietic cells were f ound in the culture medium while only a few haematopoietic foci were still present within the layer of stromal cells. Increasing the concentrations definitely er adicated haemopoiesis, but stro mal cells remained unaffected. Large macrophages like cells in the adherent laye r where more sensitive than fibroblastic or preadipocytic stromal cells. After 2 weeks of exposure, aplastic cultures were obtained for doses between 7 and 15 mM. In these cultures, the stromal cell layer was apparently unaffected but no haemopoiesis persisted. Do ses exceeding 30 mM also destroyed the haemopoietic microenvironment. Haemopoietic cel l production in the liqu id phase of cultures was evaluated twice a week for 2 months. Results after 4 or 8 days of treatment showed that haemopoiesis was very rapidly destroyed with in a narrow dose range of the compound: for low doses low injury was apparent as judged by cell counts or cell morphology.
In summary, of these studies, EGBE was the most toxic compound. The IC50 after 96 hr of exposure was in the range 50-100 μ M. The pattern of toxicity suggested that cell death resulted from several mechanisms that had cumulative effects during the earliest period of exposure. This result is reminiscent of the haem olytic effect of EGBE reported by the others. By targeting the leucocyte membrane, EGBE could be responsible for both irreversible damage and cytolysis.

In vitro haemolytic effects of BAA were assess ed on human and rat erythrocytes (Udden, 2002). Human blood was sampled from healthy vol unteers, hospitalised patients and children and rat blood from Fisher 344 rats (9-11 weeks old). RBC were isolated, adjusted to a Packed Cell Volume (PCV) of 10 % and incubated in th e presence of BAA ( 99 % pure) for 1-4 hours at 37° C. Various parameters were assesse d: microhaematocrit, haemoglobin, RBC count, % haemolysis, erythrocyte deformability, erythroc yte osmotic fragility, density and Na and K + concentrations in erythrocytes. A microsc opic examination of er ythrocytes was also performed.
A time dependent increase of deformability was seen after exposure of human erythrocyte to 10 mM BAA compared to controls. No ha emolysis was observed. To compare subhaemolytic effects of BAA, human RBC were challenged with 0-10 mM BAA for 4 hours while rat erythrocytes were exposed to BAA at 100 fold lower concentrations. A comparable increase in cell rigidity with haemolysis was se en in both species. At th e highest concentrationEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 130 of BAA used, there was a 64 % in crease in Po of rat RBCs and an increase in Po of 40 % for human RBCs. A significant change in human erythrocyte deformability occurred at 7.5 and 10 mM BAA whereas decrease deformability in the rat erythrocyte was no ted first at 0.05 mM BAA. In Mean Cellular Volume (MCV), there was a small but significant increase in human red blood cell volume after exposure to the highe st concentration of BAA. For the size distribution, a main peak (very close to the MCV) was seen. A shoulder was also present. Both rats and human erythrocytes appeared to shift in size to a larger cell volume, as the number of smaller red blood cells diminished. For osmotic fragility, no significant changes in human erythrocytes were seen until they ha d been exposed to e ither 7.5 or 10 mM. Rat erythrocytes demonstrated a remarkable increas e in osmotic fragility after exposure to lower concentrations of BAA. For erythrocyte density, human red blood cells showed only a very small shift toward lower density, however, the rat erythrocytes showed a marked shift to lower cellular density. For erythrocyte cations contents, there was a significant increase in both human and rat erythrocyte Na concentration with exposure to high and low levels of BAA respectively. However, human red blood cell K + was slightly decreased and this occurred after incubation in the highest concen trations of BAA. Rat red blood cell K levels decreased after exposure to 0.025 mM BAA, th e lowest concentration used. Although there was a trend towards increased total cation cont ents in the human, only the rat erythrocytes showed a significant net change in total cati on content, a small net increase after their incubation with 0.1 mM BAA for 4 hours. The morphology of human erythrocytes was not changed after exposure to 10 mM BAA for 4 hours. However, rat erythrocytes exposed to 0.1 mM BAA showed increased numbers of spherocytes compared to the controls.
After corrections, there was no difference in h aemolysis of RBCs obtained from hospitalised adults and children compared to well adults when exposed to 10 mM BAA. MCV did increase as a result of BAA exposure, but no individual had a significant difference in the pattern of the frequency distri bution of cell size.

In vivo
A fragility test was performed by adding a drop of whole rat blood delivered with a needle into an agglutination tube cont aining 1 ml of saline (Mellon In stitute of Industrial Research, 1952) . The saline concentrati ons ranged from 0.36 % to 0.72 % in steps of 0.04 %. After addition of the blood the tubes were incubated at room temperature for 2 hr and read. The supernatant liquid is examined for any indication of haemolysis . Initial haemolysis was read as the highest concentration of saline in whic h haemolysis occurs and complete haemolysis the nearest in which there were no intact red cel ls visible. Cell detritus will not settle out in the 2 hr interval of the test. Initial haem olysis occurred at 0.44 % NaCl and complete haemolysis at 0.36 % NaCl in a remarkable consistent manner with rat blood. The exceptional normal reading was 0.48 % initial and 0.40 % complete.
Fifteen males and 15 females were exposed 7 hr /d to 500 ppm of EGBE. 13 out of 15 females died during or shortly after the first e xposure. Lung congestion and haemorrhage and generalised congestion of other organs was found at autopsy. Th e 2 other rats died after the second and the third exposures re spectively. Seven out of 15 males died during or after the first exposure, one each after 2 and 3 exposures, 3 after 4 exposures and 3 survived 30 exposures. Two of these survivors had mode rate cloudy swelling of convoluted and loop tubules of the kidney, one had moderate cloudy sw elling of the liver and one had completely normal kidneys, liver, lung, spleen and testis. Blood studies after 30 exposures revealed anaemia and increased fragility of the red cells.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 131 Regarding the high mortality at 500 ppm, another study was carried out with lesser concentrations. Animals were exposed to 314 pp m but lethality was found within 3 days of exposure in the 15 females exposed. Pathology was comparable to that described for the 500 ppm dose.
Another study using 200 ppm doses was performed to the fragility test. Fr agility of red cells on a group of rats was followed before and after exposure each day for a period of 9 days. Each day fragility of the red cells increased from initial haemolysis in 0.48 % saline before exposure to initial haemolysis at 0.52 % to 0.68 % after the e xposure (following an overnight or a 17 hr rest period red cell fragility had returned to the normal value.
Another group of 6 rats concurrently exposed at 200 ppm was used for enumeration of red blood cells and haemoglobin determinations. Th e protocols showed that the rats became anemic after 26 hr of exposure during 4 days . The red cell count was about half of preexposure level and haemoglobin had dropped from about 90 to 65 %. After 5 exposures of 6 or 7 hours duration followed by a 7-day although they were still below the pre-exposure levels in most cases. Thereupon the rats were again subjected to 3 more exposures. Again the red counts were depressed and the haemoglobin values lowered even more so than after the first 5 exposures. A subsequent re st period of 8 days resulted in a measurable increase in both values but the red cell counts were still subnormal. Fragility values were normal.
Further study on rats revealed that at a con centration as low as 25 ppm of EGBE a 7 hr exposure produced a rise in red cell fragility wh ereas after 10 ppm for 7 hr only 1 of 4 rats showed any increase.
Rats fed orally 1.48 g/kg as a 10 % aqueous diluti on showed a relatively enormous increase in red cell fragility. The results on these four rats are as follows:

Table 4.59: Effects on rat blood Time after dosing Initial and complete haemolysis (% Na Cl)

15 min 0.48 – 0.40

50 min 0.72 – 0.60

130 min 0.72 – 0.60

4 hr 1/2 0.72 – 0.60

2 Guinea pigs and 2 rabbits were followed for 3 days while being similarly exposed to 200 ppm EGBE. Guinea pigs were not affected whereas rabbits responded like rats.
One rabbit was exposed for 10 hours to 200 ppm of EGBE and the fragility or red cells was determined periodically until the blood had returned to normal.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 132 Table 4.60: Effects on rabbit blood Time after dosing Initial and complete haemolysis (% Na Cl)
Pre-exposure 0.48 – 0.40
3 – 180 min inclusive post-exposure 0.60 – 0.44
240 – 420 min inclusive post-exposure 0.52 – 0.40
420 min post exposure 0.48 – 0.40
25 hours post exposure 0.48 – 0.36

Two dogs were exposed to concentrations ranging form 200 to 665 ppm to determine the sensibility of their red blood cells to EGBE. No effects on RBC fragility were evidenced in these studies.
Two men were exposed to 100 ppm EGBE for a 4 hr period. Rats were concurrently exposed to serve as positive controls. RBC fragility tests were performed 4 times in men and twice in the rats. No response was seen in humans wh ereas rats elicited exp ected positive response showing that they are much more sensitive as humans to the haemolytic properties of EGBE.
Groups of 24 male F344 rats were dosed Per Os (PO) to EGBE (purity 99 %) at doses of 500 and 1000 mg/kg (Grant et al. , 1985). Six rats/group were bled and killed on day 1, 4, 8 and 22 after the last treatment. Analysis of RBC, White Blood Cell (WBC), Hb, MCV, Haematocrit (HCT), reticulocytes and differential white cell count were perfor med. After termination, spleen, thymus, liver, testes and kidneys were weighted and were examined by microscopy. Cervical and mesenteric lymph nodes were also examined by microscopy. Femoral bone marrow was also examined by electronic micros copy. This study was concurrently performed with EGME to compare haematological e ffects caused by the two glycol ethers.
Body weight gain was affected for high doses of EGBE. There was also an increase of the relative weights of spleen, liver and kidneys an d a decrease of thymus weight. These changes returned to normal by day 22 apart for liver and kidneys for which the weight was still slightly raised. Hyperplasia of marrow without evidence of haemorrhage was seen. Splenic extramedullary haemopoiesis was marked in both groups on day 1 but re turned to normal by day 8. A transient lymphocyte depletion of the t hymic cortex was also noted and returned to normal by day 4. Animals treated with the high dose had reduced RBC, HCT, HB and elevated MCV, reticulocytes counts and Mean Corpuscular H aemoglobin (MCH). Most of these changes disappeared ove r the recovery period, althoug h the MCV and MCH remained slightly elevated on day 22. The reticulocytosis was pa rticularly striking on day 1 and 4 but had returned to normal by day 8. In this group increased number of normoblastes, pronounced anisocytosis, polychromasia and presence of Ho well Jolly bodies were found but had resorbed by day 8. Leucocytes were also depressed on day 1 only due to a decrease number of circulating lymphocytes. These changes were also seen with the lower doses, but less in severity.
The role of Alcohol and Aldehyde dehydrogena ses in the haematotoxicity of EGBE was assessed (Ghanayem et al. , 1987b). Three groups of F344 rats (9-12 weeks old) were submitted to different treatments: rats of the first group were treated IP with pyrasole (250 mg/kg) one hour before gavage administration of water (control grou p) or EGBE. For theEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 133 second group, cyanamide (50 mg/kg) was admini strated IP to animals 0.5 hr before water (control) or EGBE administration by gavage. The third group received only a single dose of EGBE by gavage (500 mg/kg). Animals were pl aced immediately in metabolism cages after EGBE treatments to collect urine and faeces.
Concurrently, BAA or BAL was ad ministrated to rats by gavage at the molar equivalent of 125 mg/kg EGBE. Rats were also placed in metabolic cages.
After EGBE administration, blood samples were collected during termination of the study. Evaluated parameters were: RBC, Haemoglobin (Hb) concentration, PCV, HCT, MCV, MCH, MCHC and platelet. Free Hb was also quan tified in the plasma and urine of these rats. After collection of blood samples, liver, kidneys and spleen of treated animals were collected and weighted. Tissues were collected 24 hr and 48 hr after treatments.
Gavage administration of EGBE caused severe haemolysis accompanied by an increase in the relative spleen weight. The increase was signifi cant at all time points and was maximum 2 hr after EGBE administration. Recovery from trea tment was not complete even 48 hr after dosing. A significant decrease in the number of circulating RBCs and Hb concentration and HCT was seen in treated animals. These effect s were maximum within 8 to 24 hr after dosing and showed a rebound at 48 hr. Th ese finding were accompanied by increase of the free Hb in the plasma and haemoglobinuria. In the liver, histopathologic evaluati on made 24 hr after dosing showed coagulative necrosis and phagocyt osis of Hb by hepatocytes and Kupffer cells. In contrast, pre-treatment of rats with pyrasole resulted in complete prevention of the increase of the relative spleen weight and of the h aemolytic effects caused by EGBE alone. Liver effects were also completely prevented by pyras ole pre-treatment. With cyanamide treatment, significant increases of the relative spleen we ight, however the time to maximum effects was approximately doubled in rats treated with cyan amide and EGBE as compared to rats treated with EGBE alone, a significant decline in number of RBCs, HCT and Hb concentration in cyanamide treated rats was also seen but was significantly le ss than what was found after EGBE treatment only.
After BAA or BAL administration, the same effect s were seen on relative spleen weight than after EGBE administration. The maximum effects were observ ed at 4hr after dosing. No differences were seen on blood parameters be tween the three chemicals. Treatment with cyanamide before BAL administration resulte d in a significant protection against BAL induced toxicity.
Effect of age on the toxici ty was assessed (Ghanayem et al ., 1987c). EGBE (purity > 99 %) was administered orally to F344 rats (16 months old) at doses of 32, 63, 125, 250 and 500 mg/kg. Immediately after dosing ra ts were placed in metabolic cages and urine was collected at different times. Rats were killed at various time points af ter administration and blood was collected by cardiac punc ture. Spleen, liver, kidneys, testes and urinary bladder were collected and examined histologically. The following measurements were done on blood samples: RBC, WBC, Hb, HCT, MCV, MCH, MCHC and pl atelet count. Free con centrations of Hb were also determined in urine and in plasma.
Gavage administration of EGBE in young or adults rats resulted in a significant dose related increase of the spleen weight evidenced from 125 mg/kg. Young rats we re less sensitive than older ones. Maximum effects on re lative spleen weight were obser ved at 2 and 4 hr after the administration of 500 mg/kg to ra ts (young or old with a more marked effect in old animals). At 125 mg/kg dose, effects were maximum 4 hr after gavage in both age groups. After reaching the maximum, controls levels wher e obtained about 48 hr after treatment. OnEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 134 haematologic parameters, EGBE caused a signifi cant decrease in the number of circulating RBCs, Hb concentration and HCT. Other blood parameters showed minimal changes. These effects were dose dependent and older rats we re significantly more susceptible than younger rats. At 125 mg/kg, effects on RBCs, Hb and HCT were only seen in old animals 8 and 24 hr after administration. Young animals were not aff ected by treatment at this dose level. The decline in RBCs, Hb and HCT was accompanie d by a significant dose dependent increase in the concentration of free Hb in the plasma of treated rats. Th e peak level of free Hb in the plasma was lower in young animals compared to the older ones at all doses investigated. Maximum free plasma Hb levels were seen af ter the administration of 500 mg/kg EGBE and were approximately 5 times higher in the plasma of the older rats than those of measured in the plasma of the younger rats. For the 125 mg/kg dose, no effects were seen on free plasma Hb of young animals whereas a si gnificant increase was detected 8 hr after administration of the same dose to older rats. Haemoglobinuria was observed and was concomitant with the decline of free Hb in the plasma. A differen ce between old and young rats was also noted (see table 4.61).

Table 4.61: Incidence of haemoglobinuria induced in ra ts of various ages by gavage administration of EGBE. Dose (mg/kg)
Age 32 63 125 250 500

4-5 weeks 0/6 0/6 1/11 6/6 12/12

9-13 weeks 0/6 0/6 12/12 6/6 12/12

5-6 months 0/6 6/6 6/6 ND ND

16 months 6/6 6/6 6/6 ND ND

Histopathologic evaluation of tissu es of rats of various ages examined 24 hr after EGBE administration revealed that EG BE causes dose and age dependent liver changes. In 9 to 13 week old rats, EGBE caused focal disseminated coagulative necrosis of hepatocytes. In addition there was evidence of phagocytosis of dark pink prot ein compatible with Hb by Kupffer calls and hepatocytes. These effects we re observed at an incidence of approximately 16 and 83 % at 250 and 500 mg/kg doses, respectiv ely. Evidence of regression of these liver lesions was seen at 48 hr after EGBE dosing. No liver changes were observed at the same doses in 4 to 5 week old rats. In the kidneys, small and dark pink protein droplets, compatible with Hb, were seen in the proximal tubular epit helium of treated rats. In addition Hb casts were observed in the proximal tubules. The incide nce and severity of these effects were dose dependent and were observed in 9 to 13 week old rats but not in 4 to 5 week old rats. Similar to the liver, kidney lesions were more seve re at 24 hr after EGBE administration and exhibited signs of regression when examined at 48 hr after dosing.
The influence of the type of haematology analyser on measur ed parameters was assessed (Ghanayem et al. , 1990b). Groups of male F344 rats (9-1 1 weeks old, 3-5 animals per group) were treated by gavage with 125, 250 or 500 mg/ kg of EGBE (purity 99.9 %). Animals were held for 1, 2, 4, 8 or 24 hr after treatment. Contro l rats received the vehicl e (water). At the end of each holding period, blood samples were coll ected and analysed using both a laser-based and an impedance-based haematology analyser. The following parameters were measured : RBC and WBC counts, Hb, HCT, MCV, MCH, MC HC and platelet counts. In addition PCV were determined by centrifugation.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 135 Parameters obtained with the la ser-based analyser indicated dose and time-related decreases in RBC and Hb. Similar effects were observed with the impedance based analyser. Striking differences were seen in HCT MCVs MCH and MCHC of EGBE treated rats. While a dose and time dependant increase in HCTs was reco rded early after treatment (followed by a decline as haemolysis occurs) using the impedance based analyser a dose-dependant decrease was consistently recorded using the laser-based analyser.
By 24 hr HCT significantly decreased in all three dose groups and whichever the analyser used. The impedance based analyser also detect ed marked doses related early increases in MCVs followed by a decline to near normal by 24 hr after EGBE treatment. In contrast a small decrease in MCV was detected with the laser-based analyser. PCV confirmed the results obtained from the impedance-base d analyser showing an early in crease in PCVs followed by a decline as haemolysis of RBCs occurs.
In the 125 and 250 mg/kg dose groups, changes in MCHs recorded by both types of instruments were the same. However 4 hr afte r the high dose, a much greater increase was seen in MCHs from the impedance-based than from the laser-based analyser. Finally, while the laser-based analyser recorded a dose-dependent increase in MCHCs the impedance-based analyser recorded a dose-de pendent decrease in MCHCs in the same blood samples.
This study demonstrated the importance of the an alyser used to determine the early effects described for EGBE haematotoxicity: increas e of HCT and MCV. The impedance-based analyser is therefore more appropriate.
EGBE (99 % pure) was administrate d via oral route to groups of male SD rats at doses of 50 – 100 - 250 and 500 mg/kg (CMA, 1990). Blood sample s were collected at 0.5 – 2 and 4 hr after treatment for various analysis:

- Cell morphology was examined by microscopy a nd red cell indices we re recorded: RBCV and Hb concentration distribution. Plasma and urine Hb concentrations were also measured.

- The measure of the erythrocyte density distri bution was performed. This parameter reflect changes in the volume of the red cells and th e profile of the change s (entire population or a fraction of them).

- The ATP content of the red cells was determined to assess the ability of EGBE to perturb the energy state of the RBC and conse quently the function of ionic pumps.

- Effects on red cell lipid auto-oxydation was measured to see if haemolysis could be produced by a mechanism of peroxidation of uns aturated fatty acids of the cell membrane, MDA (malonyldialdehyde) served as a marker of lipid auto-oxidation.

- Viscosity and deformability of RBC was also measured after treatment.
The morphology of RBC showed a dose related tr end to the formation of spherocyte. For RBC indices, the MCV frequency distribution curve broadened significantly. The mean cell volume increased from 53.8 fL to 86.9 fL. The frequency distri bution of MCHC in contrast narrowed, and the mean value decreased from 35.2 gm/dl to 25.1 gm/dl. Similar data were obtained for all animals studied, and the MC V, MCHC, RDW and HDW values, as well as the other indices showed similar trends depe nding on the dose of EGBE administered and time of exposure prior to the sampling of blood. The red cell MC V increased steadily whereas the MCHC decreased as a functi on of the dose of ingested EG BE, suggesting an increase in cell water content. The mean value of MCV increased with increasing dose of ingestedEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 136 EGBE. The EGBE-treated animals had significa ntly higher MCV than the control group. The dose-response relationship noted for MCV was most apparent for 500 mg group which showed a significantly higher mean MCV values among the EGBE-treated rats as compared with the control group. Erythrocytes from the co ntrol animals had higher MCHC values than all EGBE-treated animals, with higher doses of the glycol ether re sulting in lower MCHC values. No clear dose response relationship was observed for other para meters. Using a 2-way ANOVA with time and dose as factors, a sign ificant difference betw een 50 and 500 mg/kg dose group was observed in the following parame ters: MCV, MCHC, HCT, RDW, HDW, Hb and RBC at 0.5 and 2 hr observation times. Haemoglobin in the plasma and urine was demonstrated 2 hours after EGBE administra tion. Dose-dependency was demonstrated 2 hours after the EGBE administration.
The median density of rat RBCs treated with EGBE decreased as a function of the dose of ingested compound. These changes in the profile indicated an increase in cell volume and a decrease in cell Hb concentration (perhaps as a result of an increased water cell content). There was no specific effect on any subpopulation of cells according to the density profiles.

No differences in the RBC ATP levels we re seen in EGBE treated animals.

The MDA concentration decreased to about 50 % regardless of the administered dose. The decline in MDA concentration is suggestive of the arrest of autooxidative processes in the cells by EGBE or its metabolites. There was no evidence for an increase in membrane lipid peroxidation.
An increase of 72 % of the pl asma viscosity was seen in tr eated animals (at dose of 500 mg/kg EGBE). At equal haematocrit of 40 %, the mean blood viscosity of the rats that received 50 and 100 mg/kg was 2 to 3-fold highe r than that of he co ntrol rats. The mean viscosity of the rat that rece ived 250 and 500 mg/kg was lowe r than that at the other two doses, but higher than th at of control rats.
The effects of a daily dose of EGBE on haemat ological parameters were assessed in rats (Ghanayem et al ., 1992). EGBE (purity 99 %) was admini strated by gavage to groups of male Fischer 344 rats (10-14 weeks old – 6 rats/group). Five groups of animals received 125 mg/kg EGBE during 1, 2, 3, 6 or 12 consecutive days. Tw enty four hours after the last dose animals were killed and 2 blood samples were collec ted, one with EDTA/dipotassiom salt as anticoagulant and the other with an acetate citrat e dextrose (ACD) solution (to be used for the determinaion of blood ATP concentration). Live rs and spleens were collected and weighted. Blood samples were analysed for the determin ation of RBC count, HB, HCT, MCV, MCH, MCHC and platelet counts. In addition PCV were determined on all blood samples. Other mechanistic studies were performed both in vitro and in vivo

. Some studies were performed in parallel to try to draw a mechanism of action of RBC toxicity:

- Experiment 1: Haematotoxicity of EGBE in pretreated/recovered rats in vivo was assessed: one group of rats received 125 mg /kg/d for 3 consecutive days and another group of untreated rats served as controls. Animals were then allowed to recover for a period of 7 days. Treated and untreated rats we re divided into 3 groups, the first receiving only water, the second and the third recei ving EGBE at doses of 125 and 250 mg/kg respectively. Blood samples were collected 2, 8 and 24 hr after dosing (the same analysis than the previous described study were pe rformed). Animals were killed after blood collection and the spleen was collected and weighed.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 137 - Experiment 2: The effects of BAA on blood fr om EGBE pretreated/r ecovered rats were also studied in vitro . Rats were treated with 125 mg /kg for 3 concecutive days and allowed a 7-day recovery period. Blood was inc ubated with BAA at concentration of 0 – 0.5 – 1 or 2 mM for 0.5 – 1 – 2 or 4 hr. At the end of the incubation period, PCV and blood ATP concentrations were measured.

- Experiment 3: The effects of EGBE in bled/recovered rats in vivo were also studied. Rats were bled and allowed a 7-day recovery period. After this period, treatments with 250 and 125 mg/kg via oral route were administered. Blood samples were collected 2, 8 and 24 hr after dosage and analysed as described below. After termination, the spleen was removed and weighted.

- Experiment 4: Effects of BAA in bled/recovered rats in vitro were also assessed. Rats were bled and allowed a 7-da y recovery period. Animals we re then killed and blood collected and pooled. The effects of BAA on PCV and ATP were investigated as described in the previous experiments.
Following repeated exposure (experiment 1), h aemolysis evidenced by a decrease RBC, HB and HCT was seen and become more pronoun ced following the third day of dosing but gradually recovery was observed thereafter. W ith continued repeated exposures to EGBE beyond day 3, rats exhibited a gradually incr ease in RBC and HB and approached pretreatment levels by day 12. HCT followed a similar pattern as RBCs and HB as dosing continued. On the other hand, MCV increased as the dosing regimen was extended to 6 days and declined to remain above control levels throughout the 12 day st udy. ATP concentrations, MCV and the number of reticulocytes increase d after 6 days and then declined by day 12. Reticulocytosis and increased ATP concentratio n were evident after the second day of dosing and persisted in EGBE treated rats. The relative weight of the spleen increased to a maximum after 6 days from which it declined between days 6 and 12. Relative liver weight was minimally affected by treatment and following a moderate decline on day 3 and 6, it was increased on day 12 compared to controls.
In the second experiment, EGBE pretreated/rec overed rats were found to be less sensitive to the haemolytic effects of EGBE than control rats. The RBC and HB declined in a dose and time dependent manner, moreover there was a mini mal decline of these parameters in rats pretreated with the low dose of EGBE. HCT increas ed at 2 hr and remained near or slightly below controls in EGBE pretreated/recovered rats . In contrast, rast which were not previously exposed to EGBE exhibited an increase in HCT early after treatment whic h later declined in a dose and time dependent manner. Treatment of pretreated animals with the high EGBE dose (250 mg/kg) caused more pronounced haematotoxi city than the low dose (125 mg/kg), this effect remained less than that observed in untreat ed rats of the same dose level. Pretreated animals were also less sensitive to the incr ease in MCV and ATP depletion caused by EGBE than non exposed rats. In addition the number of reticulocytes was significantly higher in rats pretreated with EGBE than in controls. Compar ison of the relative spleen weight showed that the increase caused by EGBE is less in pr etreated animals than in untreated.

In vitro (experiment 3), blood from pretreated rats was significantly less sensitive to BAA than untreated rats: increase in PCV was greater in untreated rats th an in treated rats. Similarly ATP concentrations in blood showed that pre-treatment decr ease the susceptibility of erythrocytes to the effects of BAA in vitro . Both effects of BAA became more dramatic as the concentration of BAA and the incubation time increased.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 138 Bled/recovered rats (experiment 4) were signifi cantly less sensitive to the haemolytic effects of EGBE than controls. The RBCs and HB dec lined in a dose and time dependent manner, however the decline in bled/recovered rats treated with the 125 mg/kg dose was minimal. HCT increased at 2 hr and remained near pre- treatment levels in bled/recovered rats. In contrast, EGBE administration to controls resulted in an early increase in HCT and then declined in a dose and time dependent manner. Although the number of reticulocytes in bled/recovered rats prior to the exposure to EGBE was significantly higher than in the corresponding controls. Treatment of bled/reco vered animals with the high dose of EGBE caused more pronounced haematotoxicity than th e low dose, however these effects continued to be less pronounced than those observed in control rats receiving the same EGBE dose. Comparison of the relative spleen weights demo nstrated that there was a significantly greater increase caused by EGBE in control ra ts compared to bled/recovered rats.

In vitro , blood obtained from bled/recovered rats was less sensitive to BAA–induced increases in PCV and ATP depletion. These effects were more pronounced with high BAA concentrations and with longer incubation times.

Some studies tried to demonstrate that a low dose of EGBE administra ted a few days before the administration of a lethal dose of EGBE can protect from haematological effects and then from death (Sivarao and Mehendale, 1995). In th ese studies, 3 groups of 12 female SD rats were treated orally with EG BE 500 mg/kg for groups 1 and 2 whereas group 3 served as controls and was administrate d with distilled water. On day 7 groups 1 and 3 were administrated LD50 dose of EGBE (1500 mg/kg) while group 2 received distilled water. Haematocrit of all rats was monitored on a daily basis as an index of toxicity. Rats were observed 14 days.
The effect of increasing the time gap between pre-treatment and lethal challenge on animal survival was studied by pretreat ing rats with 500 mg/kg EGBE on day 0. Rats were randomly divided into 3 groups (n=10, 8 and 6). On da y 7, 14 and 21 days, one group of these rats received a single administration of 1500 mg/kg. Rats were observed twice daily for 14 days.

In vivo effects of pyrasole (ADH inhi bitor) were tested with about the same experimental protocol (7 rats in each group): the treat ment group received pyr asole 250 mg/kg one hour before treatment with 500 mg EGBE. HCT were recorded before pyrasole administration, 24 hr following EGBE pre-treatment and on day 7 of the experiment.
In these studies, the in vitro haematotoxicity of EGBE was also assessed. Dose response curves were constructed by incubating 1 – 10 mM of BAA in salin e to rats blood. PCV (Packed Cell Volume – a marker of haematot oxicity found to be specific to BAA) were determined at various time points (1 - 4 hr). PCV are expressed as percent of controls.
Another experiment was carried out to i nvestigate if the loss of autoprotection in vivo could be related to a corresponding loss in resilience of RBC in vitro . Rats were administrated a single dose of 500 mg/kg EGBE following which they were randomly divided in 2 groups of 4 rats each. On 7 and 21 days one of the groups was killed and blood was incubated with BAA and PCV was determined in different concentrations of BAA.
To test the hypothesis that newly formed R BCs are primarily responsible for the EGBE induced autoprotection, bled/rec overed rats were used. Rats were bled 2.5 ml for 3 days. Blood letting was followed by a 7-day period of recovery. Recovery was ensured by the return of depleted haematocrit values to pr e-bled levels. Control rats underwent a sham treatment without blood letting. Both the bled/r ecovered rats as well as their sham treated controls were challenged with a lethal dose of EGBE (1500 mg/kg), and their survival wasEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 139 recorded, twice daily for 14 days. Incubation st udies with BAA, were also conducted using RBC from these rats, to corroborate the in vivo studies.
Moderately toxic symptoms were seen with the “protective” dose of EGBE: ataxia, haematuria, paleness of skin and morbidity. H aematocrit values fell to 18.6 twenty four hr after treatment. Recovery was rapid. HCT r eached control values level by 7 days. Rats received pre-treatment were fully protected agai nst the subsequent lethal challenge with 1500 mg/kg EGBE while “non protected” rats expe rimented high (> 90 %) mortality. Survivors showed an average HCT of 13.5 whereas the “pro tected” rats experienced a much lesser fall in their HCT with no mortality.
Regarding the effects of pyrasole pre-treatme nt on autoprotection, the initial haemolytic episode due to EGBE pre-trea tment was somehow attenuated (HCT recorded 24 hr after EGBE pre-treatment did not fall in the pyrasole treated rats) but following the administration, rats experienced profound ataxia that lasted seve ral hours more than observed in control rats. Following the lethal challenge, pyrasole pret reated rats suffered high mortality (87 %).
With increasing time between the pre-treatment and the lethal dose, the percentage of survival decreased indicating that the autoprot ection is gradually lost with time.
RBC isolated after EGBE pre-treatment showed a significantly higher susceptibility to BAA induced swelling than the cells isolated 7 days after EGBE treatment indicating that the resilience decrease with time.
When bled/recovered rats were challenged with lethal dose of EGBE, 87 % survived in sharp contrast to the 6 % survival of the corres ponding sham treated c ontrols. These findings suggested that newly formed RBCs were innately resistant to the haemolytic action of BAA.
Morphological changes of rat erythroc ytes after administration of EGBE in vivo or BAA in vitro were assessed (Udden, 2000). Fisher 344 rats were dosed orally with 125 and 250 mg/kg EGBE. Blood samples were collected before and 30 minutes, 1 hr, 2 hr and 4 hr after administration. Approximately 75 μ l of blood anticoagulated with EDTA was collected for microhaematocrit determination, fixation and for preparation of blood smears on glass slides.

For in vitro studies blood samples EDTA anti-coagulat ed were exposed to 1 or 2 mM BAA. Samples were taken at intervals for determinatio n of a spun haematocrit, preparation of blood smear and fixation. Washed red blood cells we re prepared by centrifugation followed by removal of the plasma and buffy coat and re-sus pension in buffer. Cells were washed 4 times and then re-suspended to 50 % haematocrit. B AA was added to achieve a final concentration of 1 or 2 mM.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 140 Table 4.62: In vivo and in vitro results
In vivo results In vitro results
30 min / 125 mg/kg
Erythrocytes with mouth shaped central pallor or stomatocytes. 2 hr / 125 mg/kg
Increase of the stomatocytes nb and increase of sphero-echinocytes.
4 hr / 125 mg/kg
Prevalence of stomatocytes and spherocytes

1 hr / 250 mg/kg
Impressive spherocytosis

HCT increased in a dose dependant manner. 30 min / both doses
Cupping and spherocytic changes more pronounced after 1 hr similar than EGBE administrated PO.

2 hr / 1 mM
Most of RBC were spherocytes. Wright stained: spherocytosis but only minimal stomatocytose. Sphero-echinocytosis was frequently observed after 1-2 hr of incubation.

Effects were more marked with 2 mM BAA.

Washed erythrocytes incubated with 1 or 2 mM BAA demonstrated haemolysis with ghost formation and agglutination.

Increase of HCT compar able to EGBE treated animals (1 hr / 125 mg/kg equivalent to 1hr / 1mM BAA).

No changes in human erythrocytes.

The difference between males and females Fischer 344 rats after exposure to an oral dose of EGBE was assessed (Ghanayem et al. , 2000). Fischer rats (12-16 weeks old) received an oral dose of 250 mg/kg EGBE (purity > 99 %). Control rats received 5 ml water/kg. Haematological parameters were analysed after termination (RBC – WBC – HB – HCT – MCV – MCH – MCHC – PCV). Blood smears were prepared from all samples. Morphological changes were ev aluated by light microscopy.

The effects of BAA were also evaluated in vitro . Blood samples were exposed to concentrations of 0.25 – 0.5 and 1 mM. Samp les of blood were also incubated with physiological saline to serve as controls. PCV was determined at 0.25 – 0.5 – 1 – 2 and 4 hr after incubation.
EGBE resulted in early swelling of erythrocyt es as evidenced by an increase in HCT and MCV. In comparison to males, female rats exhibited a greater increase in MCV at all time points. However HCT declined in both genders at 8 and 24 hr after dosing. At 4 and 8 hr after dosing, RBC, HCT and HB were significantly lower in female rats than in males, however theEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 141 difference in these parameters were not statistica lly significant at 24 hr. This reflects a faster onset of erythrocytes haemolysis in female af ter dosing compared to males. This is also confirmed by the greater increase of the relative spleen weight in females compared to males. Other haematological parameters were also consistent with a faster onset of haemolysis in female rats. In parallel reticulo cyte counts were greate r in females than in males at all time points, indicating a faster regenerative respons e. Morphological alterations were seen in blood smears prepared from rats at 4, 8 and 24 hr after EGBE administration. At 4 hr after administration blood smears exhibited evidence of erythrocyte destruction, schistocytes, stomatocytes, ghost cells and other deformed er ythrocytes. A decrease in the nb of RBC and absence of schistocytes with hypochromic RBC remaining at 8 and 24 hr was also observed. Aggregation of RBCs was evident by the formati on of rouleaux at 24 hr after administration. In males alteration in RBCs were quantitativel y similar than in females but these changes were observed more frequently and develope d at a faster onset in female rats.
EGBE (purity > 99 %) was administered by gavage to groups of Fisher 344 rats at doses of 250 mg/kg for 1, 2 or 3 cons ecutive days (Ghanayem et al. , 2001). Animals were sacrificed 24 or 48 hr after the last dose. Blood samples collected at termination were analysed for RBC, WBC, HB, HCT, MCV, MCH, MC HC and platelet count. In a ddition PCV were determined on all blood samples. Erythrocytes mor phological changes were evaluated by light microscopy. A complete necropsy examination was performed on all rats. The following tissues and organs were collected and examined: sternum, femur, vertebral column, tail, heart, lung, liver, spleen, kidneys, left eye and brain.
Severe acute regenerative anaemia in both sex in all treated animals were seen. However the progression of haemolytic anaemia as a function of the number of days of exposure varied and suggested a faster onset of haemolysis in fe males. After 24 hr following a single dose, the haematological parameters were relatively simila r in both males and females except for the reticulocyte count. Female rats exhibited approxi mately a 2 fold increase in reticulocytes with a minimal increase in males. In addition, haemolys is was more severe in females at 24 and 48 hr after the second daily dose, as evidenced by the continued decline of the haematological parameters at these time points. Reticulocyte counts in females doubled and quadrupled at 24 and 48 hr after treatment respectively. The progr ession of haemolytic anaemia was also more severe in females after the second daily exposure. At 24 hr after the third dose, the haematological parameters calculated as percenta ges of controls, were relatively similar in both sexes of rats and were indica ted severe regenerative anaemia.
Significant morphological changes in erythrocyte were also obs erved in treated animals. Slight macrocytosis with mode rate rouleaux formation and stom atocytosis were observed in female rats at 24 hr after a single dose. These morphologica l changes became progressively more severe as dosing continued and included the occurrence of occasional schistocytes and ghost cells and an increase d number of reticulocytes (micronuclei). Erythrocyte morphological changes were qualitatively similar in male rats after 1 or 2 doses and were most severe after 3 daily doses of EGBE.
Macroscopic abnormalities were noted in both males and females: watery blood, yellowish discoloured livers, dark brownish kidneys and multiple petechial haemorrhages on the sclera. In females, only bluish discolorat ion was noted on the tip of the ta il. In the 2 females that died within 24 hr after the first dose, thrombosis wa s noted only in submucosa of the most anterior section of the nasal cavity. Ot her lesions noted in these rats included focal hepatocellular necrosis and renal tubular necrosis associat ed with haemoglobin casts. Thrombosis was evident in females sacrificed 24 hr after a sing le dose. The vascular thrombi were occlusive and consisted of amorphous granular material or partly organized fibrin with varying numbersEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 142 of red blood cells. Widespread thrombi formation noted in the ocular ci liary processes and in the limbus associated with retinal haemorrhage and necrosis were sugge stive of an ischemicinfarctive process. Thrombosis was also obser ved in the submucosa of the most anterior section of the nasal cavity, mostly located in the nasal and maxillary turbinate as well as the septum. Evidence of EGBE-induced thro mbi was also observed in the pulmonary small caliber vessels and the right ventricle of the heart in association with foci of myocardial necrosis and inflammation. In ag reement with earlier findings, thrombosis also affected the dental pulp of the incisors in conjonction with focal stromal and odontoblastic coagulative necrosis. Lesions cons istent with acute infarction were noted in the coccygeal vertebrae and femur, where histopathological changes included intramedullary thrombosis, and necrosis of bone marrow cells, bone-lining cells, and the cortical and trabecular osteocytes. Dyi ng cells exhibited nuclear pyknosis and/or fragmentation to complete loss of nuclear staining, often appearing as ghost cells. Soi ne of the lacunae were empty. The changes in the femur were usually confined to the diaphysis (medullary cavity bone marrow and trabecular bone) and inner one-third of the cortex, while in the coccygeal vertebrae, the metaphyseal bony trabeculae and epiphysis were also necrotic. In rats sacrificed 48 hr after two or three daily EGBE doses, bone marrow regenerative erythroid hyperplasia was also observed in addition to the consistent presence of the fore menti oned lesions. Other not ed changes included multifocal hepatocellular necrosis in the liv er, extramedullary haematopoiesis in the spleen, and haemoglobinuric nephrosis.

In male rats treated with EGBE for one or two days, pathological changes were limited to those associated with haemolysis, consis ting of renal haemoglobinuric nephrosis and splenic extramedullary haematopoiesis. However, the 3-day EGBE dosing induced thrombosis and tissue infarctions similar to those seen in female rats. In particular, disseminated thrombosis was no ted in the longs, nasal subm ucosa, liver, heart, bones, tail and teeth. Degeneration, ne crosis and/or infarction were noted in the heart, eyes, teeth and bones.
Three groups of 3 female GP were treated with a unique subcutaneous dose of EGBE at 0.1 – 0.5 or 1 ml/kg (Unilever, 2001). Blood samples were taken before dosing and on day 1, 2 and 5 after dosing prior termination. On blood sampled, the following parameters were measured:
* Haematology: PCV – Hb – RBBC – WBCC – MCHC – MCV – MCH and reticulocytes
* Biochemistry: K + - Mg 2+ - Ca 2+ - free haemoglobin
At necropsy the following organs were examined macroscopically: liver, spleen and kidneys.
No effects were seen on any measured parameters.
In a recent publication (Koshkaryev et al. , 2003), the mechanism of EGBE-induced haemotoxicity was studied. Male and female Fi scher 344 rats were exposed to two, three or four daily doses of EGBE (purity > 99 %) at 250 mg/kg. Blood samples were taken from rats before sacrifice on days 2, 3 and 4 (groups 2, 3 and 4 respectively), each time 2h after administration and from group 5 before sacrifice 24 days after the last dosing. After isolation, RBC were subjected to determin ation of RBC flow properti es on the day of collection (aggregability, adherence and deformability).

RBC aggregability was not affected by the tr eatment. The effects on deformability were inconclusive. The main effect wa s an increase of the RBC adhere nce to Extra Cellular Matrix.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 143 There were no differences between male and female. The adheren ce was highest at day 2 (the first to be examined) after which it decreased sharply with time.
Further investigations were performed to elucidated the mechanism of thrombosis formation (Nyska et al. , 2003). Eyes from female Fischer 344 ra ts exposed to EG BE were studied histologically and immunohistoc hemically using specific monocl onal antibodies to determine the in situ protein expression of vascular cell adhe sion molecule-1 (VCAM-1), endothelial intercellular adhesion molecule-1 (ICAM-1), a nd P-selectin. Groups of female rats (4 per treated groups, 8 for control group) were treated with 250 mg/kg EGBE (100 % purity) in drinking water for 2, 3 or 4 consecutive daily treatment. Sacrificeof the animals was done 2 hours after the last scheduled treatment.
Histopathologically, lesions in the retina consisted of retina l thrombosis, haemorrhage, degeneration and necrosis. Changes were mainly located in proximity to capillaries and consited of multifocal haemorrhage within the retinal pigment epithelium (RPE) and choriocapillaries and between the RPE and photo receptors (leading to retinal detachement) and the photoreceptors and inner and outer nu clear layers. Haemorrhage in the RPE was associated with degeneration, exfoliation, loss of epithelial cells and presence of neutrophils. Haemorrhage whitnin the receptor layer cau sed organizational disruption and loss of photoreceptor cells. In the inner and outer nuclear layers, the haemorrhages and thrombosis were associated with degeneration and loss of cells resulting in thinning, disorganisation, and fusion of these layers. Thrombi were also noted within blood vessels of the ciliary processes, in the limbus and in the retina. Positive VCAM -1 expression was observed only in eyes of rats exposed to 3 and 4 doses of EGBE (mainly in the iris, ciliary processes and retina). Only weak effects were seen after one or two dos es. No effects were seen for ICAM-1 and Pselectin.

Studies with metabolites

The effects of BAA on human and rat blood cells were inve stigated (CMA, 1993). Human erythrocytes were obtained from healthy volunt eers and rats erythrocytes from Fischer 344 rats. Red cells were isolated and incubated in the presence of 0, 0.2 or 2mM BAA for 1-4 hr at 37 °C. Erythrocyte suspensions were samp led at intervals for determination of microhaematocrit, Hb, RBC count and percenta ge of haemolysis. Morphology of RBC was also observed. The deformability of erythrocytes was assessed.
A 30 % haemolysis was demonstrated in rat eryt hrocytes when incubated with 2 mM. For 0.2 mM only little haemolysis was seen (when incubate d for 6 hr or longer, mild haemolysis was detected). Less than 1 % haemolysis was seen in human erythrocyte when incubated for 4 hr with 2mM. For deformability, exposure of rats er ythrocytes to 2 mM for 1 hr resulted in a dramatic decrease in red blood cell deformabili ty (increase in cell rigidity). A longer incubation (4 hr) in the presence of 0.2 mM B AA also produced significant changes in the deformability curve in the absence of significant haemolysis. No deformation was seen in human erythrocytes expo sed to 2 mM for 4 hr.
Distinct changes in the morphology of rat R BCs exposed to BAA were observed. Rat RBCs incubated in buffer alone for 4 hr were found to be mostly normal appearing discocytes with occasional crenated cells identified. Incubati on in the presence of 0.2 mM BAA resulted in striking spherocytic and spheroechinocytic RB Cs. Also apparent was a tendency for BAA incubated RBCs to adhere to each other. Incu bation with 2 mM BAA re sulted in dramatic haemolysis with the appearance of RBC ghosts (which appear to be mutually adherent).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 144 Another finding was the pres ence of precipitate s of rat Hb. Human RBC morphology was unaffected by a 4 hr incubation in 2mM BAA.

Effects of BAA on RBCs from 10 mammalians were studied in vitro (Ward et al. , 1992 ; Ghanayem and Sullivan, 1993). To validate in vitro finding, the haematotoxic effects of EGBE was compared in vivo using one representative from each of the sensitive and less sensitive animals. Various animals were us ed to collect blood samples (see table 4.63)

Table 4.63: Effects of BAA on blood from different species Species Strain Average age

Mouse B6C3F1 9 wks

Rat F344 10 wks
Hamster Syrian (LVG:LAK) 27 wks

Rabbit New Zealand 37 wks
Guinea pig Hartley 28 wks

Canine Hound 18 wks
Feline Dom. Short Hair 7 yrs
Pig Poland China 2 yrs
Baboon P. cynocephalus 5 yrs

Human Adult

Erythrocytes were incubated with BAA at con centrations of 0, 1 or 2 mM. Complete Blood Counts (CBCs) and packed cell volume (spun haemat ocrit) were measured at 0, 1, 2 and 4hr after incubation. CBCs included white blood ce ll count, platelet co unt, RBC, HB, HCT, MCV, MCH and MCHC.
Male rats and male Guinea Pigs were admi nistrated EGBE at dose of 250 mg/kg. Blood samples were taken at 2, 8 or 24 hr after treatment for rats a nd 1, 2, 4, 8 hr for Guinea Pigs. CBC and spun HCT were performed at each time point.
All three rodent species were relatively susceptib le to the effects of BA A: It caused a time and concentration dependant increase in MCV and HCT. At high concentrations, swelling of RBCs was occasionally followed by a decline in the number of RBCs. Haemolysis was most significant in mouse blood incubated with the hi gh concentration of BAA. The 2 mM dose of BAA produced greater than 45 % increase above the correspondi ng control in MCV at 2 hr, with a 6 % decrease below control in the RBC count. At 4 hr after incubation, MCV declined from the 2 hr levels but remained significan tly higher than controls . The number of RBCs decreased but 30 % below the corresponding contro l levels. HB concentration declined in a manner parallel, but to a lesser extend, to th e number of RBCs. MCV increased by greater than 15 % and 35 % above contro ls after incubation with 1 or 2 mM BAA for 4 hr. In addition, HCT increased in para llel to MCV. Although significant swelling of RBC occurred, no significant haemolysis was observed in hamsters.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 145 For guinea pigs, there was no significant ch anges in MCV, HCT, RBC count or HB concentration in the blood of animals with vehicle, 1mM or 2mM BAA over the 4 hr study.

Blood from rabbits exhibited an increase of MCV and HCT in a concentration and time dependent manner. At the 2 mM BAA, a gr eater than 20 % increase in the MCV was observed above the corresponding control at 2 hr which further increased to 39 % above control levels at 4 hr. HC T increased in a manner parallel to MCV and was time and concentration dependent. Despite this extensive swelling, the RBC count and HB concentration remained essentially unaffected throughout the incubation.
Two species of carnivores were investigated fo r the haematological e ffects of BAA. Both canine and feline RBCs were found to be essentially insensitive to BAA.
Pig cells exhibited no significant change in size or number.
Two species of primates were investigat ed: baboon and human. In baboon blood incubated with BAA, MCV increased in a time and conc entration dependent manner approaching a 35 % above control increase after 4 hr incubation with 2 mM BAA. HCT increased in a fashion parallel to MCV and this increase was time and concentration dependent. The RBC counts declined slightly and this haemolysis was si gnificant in blood incubated with either BAA concentration for 4 hr. In contrast, incubation of human blood with the high concentration of BAA demonstrated minimal or no effect. The changes in red cells size in the 2mM dose varied slightly from one human to anothe r, with MCV increased of about 3.5 % above corresponding controls at 1, 2 a nd 4 hr. HCT also increased 2-4 % above controls in the 4 hr incubation period. The effects of BAA on MCV a nd HCT were not time dependent and were not statistically significant at any time poi nt during the incubation. These minimal effects were not statistically significant and were not associated with any significant changes in RBC count or HB concentration.

In vivo , gavage administration of EGBE caused an early increase in MCV and HCT in rat blood followed by a decline as a function of time. These e ffects wee associated with haemolysis and a decline in HB concentra tion and the number of RBCs. MCH and MCHC were affected in a manner reflective of the ch anges in RBC and HB. In contrast to the effects in rats, EGBE caused no significant changes in any parameters of guinea-pigs. These findings were in full agreement with the effects seen in the in vitro experiments.

The effects of BAA on rat and human erythrocytes were also studied by Udden and Patton, (1994) and Udden (1994). Heparinised human bl ood was obtained from healthy volunteers and rat blood was obtained by cardiac puncture of male F344 rats. Erythrocytes were washed free of plasma, re-suspended in buffer to a p acked cell volume of 10 % and incubated in buffer alone or buffer containi ng 0.2 or 2mM BAA (Shell Chem icals Development Co., purity 99 %) for up to 4 hr. BAA at 0.2 mM did not cau se haemolysis of rat erythrocytes, but the 2mM concentration had a rapid effect resulting in ca. 30 % haemolysis after 4 hr. Addition of glucose to the medium gave no protection. In co ntrast, incubation of human erythrocytes for 4 hr in the presence of 2mM BAA resulted in no haemolysis over that slight amount (< 1 %) owing to incubation of the cells in buffer alone. Rat erythrocytes also demonstrated decreased deformability (or increased cell rigidity), us ing a nucleopore filtration technique, and an increased mean cellular volume in the presence of 0.2 or 2mM BAA, whereas these effects were not observed with human erythrocytes.
Study of the effects in vitro of BAA on human blood samples was extended to include heparinised blood obtaine d from 9 healthy adults (mean ag e 41.6 years, range 31 – 56; 5 men, 4 women), 9 older people (mean age 71.9 years, range 64 – 79; 5 men, 4 women), 7 patientsEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 146 with sickle cell disease and 3 people with hereditary spherocy tosis (Udden and Patton, 1994). Erythrocytes were washed free of plasma, re-sus pended in buffer to a packed cell volume of 10 % and incubated in buffer alone or buffe r containing 2mM BAA (Shell Chemicals Development Co., purity 99 %) for up to 4 hr. Erythrocytes from the youn ger healthy subjects showed no significant increase in haemolysis in presence of BAA compared to that observed in the incubations in buffer alone. There was a greater tendency for er ythrocytes from the other volunteer groups to haemolyse more readil y in buffer alone than erythrocytes from the younger healthy group. However, none of the groups showed an increase in haemolysis due to the presence of 2 mM BAA. Erythrocyt e deformability was measured using a nuclepore filtration technique, but no cha nges due to BAA exposure was detected (Udden and Patton, 1994).
Mechanistic studies were performed by Udden and Patton, 2005 to elucidate the mechanism of BAA induced haemolysis. Red Blood Cells of Fi scher 344 rats (9-11 week old) were added to the following culture medium: 10 mM TR IS, pH 7.4; 140 mM KCl and 10 mM glucose with or without 2 mM CaCl 2 . The RBCs were incubated in the presence of BAA at 37° C. In some experiments the inhibito r charybdotoxin (inhib itor of the Ca activated potassium channel) was added 30 minutes be fore the addition of BAA.
To determine the effect of osmolarity, RBC were incubated with 1 or 2 mM BAA for 2 hours in the presence of 0 – 0.1 sucrose. Re sults are summarised in the table 4.64.

Table 4.64: Effect of external su crose on BAA-induced cell swelling

RBC was determined before (MCVt0) and after (MCV2hrs) a two hour incubation with 0 ( • ), 1 ( c ) or 2 mM (  ) BAA in the presence of 0-0.1 M sucrose. The ratio MCV 2hrs /MCV t0 is shown to demonstrate the relative decrease in cell swelling as a function of the increased external concentration of sucrose. The inse t figure shows impedance size distributions determined by a particle counter for rat red blood cel ls incubated in buffer, buffer with 0.1 MEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 147 sucrose, 2.0 mM BAA, or 2.0 mM BAA with 0.1 M sucrose after 2 hour incubation and resuspension in isotonic saline w ithout sucrose or additional BAA.

Haemolysis as a result of incubation with 2.0 mM BAA and 0-0.1 M sucrose.

Influence of K and Na content in the susp ending medium was also assessed. Rat red blood cells were incubated for 4 hours in calcium free media with ex ternal sodium concentration varying from 0-140 mM. Potassium was added to achieve a total external cation concentration of 140 mM.Results are summarised in the figure 4.65.

Rat red blood cells were incubated for 2 hour s in the presence of 0-2.0 mM BAA in low sodium (-o-) buffer containing 110 mM potassi um and 30 mM sodium or normal sodium (- • -) buffer containing 140 mM sodium Potassium was added to achieve a total external cation concentration of 140 mM.Results are summarised in the table 4.65.

Table 4.65: Effect of replacement of external sodium wi th potassium on haemolysis and MCV changes induced by 2.0 mM BAA.

RBC were incubated in the presence of 2 mM BAA for two hours in the presence of 0-4 mM CaCl 2 (-o-) or MgCl 2 (- • -). Results are summarised in the table 3a. In another experimentEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 148 EGTA (Ca chelating agent ) (-o-) was added to the medi um containing 2 mM CaCl 2 (- • -). Results are summarised in the table 4.66.

Table 4.66: Effects of divalent cathions a nd Ca chelation on BAA-induced haemolysis

When rat red blood cells were incubated for 2 hours with BAA at concentrations between 0 and 2 mM there was a dose related increase in haemolysis and MCV when calcium was present (- • -) (Figure 4a). The dose response for haem olysis showed a greater effect of BAA when calcium was absent (-o-). However, a dose response eff ect for BAA for increased MCV was not apparent in the cells treated in the absence of calcium (Table 4.67).

Table 4.67: Dose response relationship fo r BAA induced haemolysis and cell swellingEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 149 The morphology of rat red blood cells suspended in calcium free or calci um containing buffer was similar. Incubation with BAA in the pres ence of calcium produced spherocytes. In the absence of calcium, spherocytosis was more prominent as was ghost formation and fragmentation of the cells.

To confirm that the effect of Ca was to de lay the onset of haemolysis, RBC were incubated with 1 (figures 68a and 68c) or 2 mM (fi gures 68b and 68d) BAA in the presence (- • -) or absence (-o-) of 2 mM Ca for four hours.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 150 Table 4.68: Effect of Ca on haemolysis

These figures show that Ca delayed the onset of haemolysis caused by BAA. During the first hour of exposure, the initial incr ease in cell size did not depend on the presence of absence of Ca (fig c and d). At the onset of significant haemolysis more cells were destroyed when Ca was absent and the MCVof the remaining cells appeared to be much smaller.

An increased in RBC intracellular Ca was conf irmed by measurements after exposure to 0-2 mM BAA in the presence of Ca for 4 hours.

To verify that effects were due to an activati on of a specific Ca activ ated K channel (causing cells to lose K and prevent cell swelling – Gardos pathway), RBC we re incubated with a specific inhibitor of this channel (charybdotox in). Charybdotoxin in th e absence of BAA did not cause haemolysis, but haemolysis was greatly increased in presence of BAA. When Ca was present in the middle containing chary bdotoxin, the increase of haemolysis wasEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 151 equivalent to the haemolysis seen in the prev ious experiment when there were no Ca in the middle (figures 4.68a and 4.68b).

From these experiments, the authors suggested th at the mode of action of BAA begins with an influx of sodium that is not fully compensated by potassium loss and ends with osmotic lysis of the red cell because of an influx of water accompanying sodium. Our data also points to a role for calcium entry in the red blood cells.

Summary haematotoxicity
In studies performed in vivo , the same signs of toxicity seen in acute toxicity studies (LD50 studies) were recorded. Thrombosis were observed in various localisations sometimes leading to necrosis due to an infarction mechanism (tail, dental pulp, pulmonary, ocular).
Mechanistic studies have shown that EGBE causes haematotoxicity in vivo in rats and that BAA causes the same effects in vitro at very low concentration. When metabolic pathways leading to the formation of BAA were bloc ked, no effects were seen on RBC. It can be concluded that BAA is res ponsible of haematotoxicity in vivo

. Some species were very sensitive to EGBE- or BAA-induced haemolysis: rat, mouse, hamster baboon whereas other species were resistant to these effects: dog, guinea pig, pig, cat, rabbit and humans (30 x less sensitive than rats). In one study, dogs were very sensitive to EGBE but not to BAA.
In these studies, an increased sensitivity to h aemolysis was seen in old animals and in females in vitro and in vivo with BAA, showing that the differe nces of metabolism between male and female could not explain totally sex difference.
In one study, it was demonstrated that the type of analyser was important to detect early changes in blood parameters (i ncrease of HCT and MCV). An impedance based analyser was more sensitive than a laser based analyser.

In vivo or in vitro , haemolysis was due to a decrease of erythrocyte deformability due to erythrocyte swelling (this also explains the formation of thrombosis). Newly formed erythrocytes were more resistan t than old ones. It was also showed that EGBE pre-treatment gave a relative “protection” against higher doses administer ed later. More over, a study (Lomonova and Klimova, 1977) showed that repeated exposure to EGBE, 3 hours a day, 6 days a week for 4 months was more haematot oxic than the exposure to the same dose of EGBE, 6 hours a day, 3 consecutive days a week for 4 month. This study demonstrates an adaptive mechanism of “protection” when animals have a period ofrecovery time before a reexposure to EGBE.
The mechanism leading to erythrocyte swelling and loss of deformability is for the moment unknown. Apparently, there is no evidence of oxidative mechanism on erythrocyte membrane. Udden, 2002 showed that, at low do ses of BAA in rats, the increase of Na + intraerytrocytaire was not balanced by decrease in K + . It has been demonstrated that Ca could also play a role in the mechanism. This mechanism lead to osmotic regulation causing an increase in the size and the cell volu me of erythrocytes, a decreas e of the density and of the deformability and an increase of the osmotic fragility.
In humans, slight effects were seen with doses of 8 mM and 4 mM of BAA in vitro (Ghanayem, 1989).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 152 For the effects of EGBE in dog (and th e absence of effects when treated in vitro with BAA), another mechanism could be involved in EGBE induced haemolysis.

4.1.2.3 Irritation

4.1.2.3.1 Skin

Studies in animals
Guinea pigs
Primary skin irritation was determined by ap plication of the compound to the depilated abdomen of Guinea pigs at doses of 1, 5, 10 or 20 ml/kg (Eastman Kodak, 1981b).
Skin responses were compared with those se en in rabbits in the same study. A dose of 0.3 g/kg in rabbit lead to a moderate irritation wher eas a dose of 4.5 g/kg in guinea pigs lead to a strong irritation.

Rabbits
A skin irritation study was performed according to the method described in the US code of Federal Regulations (Huntingdon Life scien ce, 1979b). EGBE was administered on the abraded and intact skin of 6 rabbits duri ng 24 hours under semi-occlusive conditions. After exposure, scoring was performed (according to the Draize method) immediately and 48 hr after the first reading.
At the first reading time, 5 out of 6 animals exhi bited a slight to moderate erythema and 4 out of 6 exhibited a slight oedema. At the second re ading time, 4 out of 6 animals exhibited a very slight to moderate erythema and 3 ou t of 6 exhibited a slight oedema.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 153 24 hours 72 hours Rabbit

Intact skin Abraded skin Intact skin Abraded skin
Erythema 0 0 0 0 1

Oedema 0 0 0 0
Erythema 1 1 2 2 2

Oedema 0 0 0 0
Erythema 1 1 0 0 3

Oedema 1 1 0 0
Erythema 2 2 1 1 4

Oedema 1 1 1 1
Erythema 1 1 1 1 5

Oedema 1 1 1 1
Erythema 1 1 1 1 6

Oedema 1 1 1 1
According to Draize scoring.
In this study, EGBE can be considered to be slig htly irritant to rabbit skin (primary irritation index: 1.5).
EGBE was only mildly irritating to rabbit skin when applied and left uncovered for a 4 hr period on rabbit skin (Bushy Run Research Center, 1980b). More prolonged exposure to the undiluted chemical (24 hr) can lead to moderate to se vere irritation charac terised by erythema, oedema and necrosis (as seen in acute dermal toxicity test).
EGBE and others glycol ethers were tested to assess their cutaneous irritation properties. (Zissu, 1995). Test substance ( purity > 99 %) was administrated de rmally to rabbits according 2 test methods for assessing cutaneous irrita tion: the EEC testing method and the Draize protocol. Three New Zealand white rabbits were used in the EEC protoc ol and 6 in the Draize protocol. 0.5 ml of the test substance was applie d occlusively on the flank of each animal, 4 hr for the EEC protocol and 24 hr for the Draize prot ocol (for the later, EGBE was also applied on the second scarified flank). Animals were observed for er ythema and oedema. At 72 hr, histopathological control of the skin at the site of application completed the macroscopic observation.
According to the EEC method, EGBE is classifi ed irritant and acco rding to the Draize method, EGBE is classified as severely irrita nt. These results were confirmed by histological observations. No detailed observations were available in this publication.

Within the framework of the elaboration of a new EEC directive covering all preparations, different classes of substances were tested in different dilutions according to the experimental protocols and interpretation crit eria laid down in Annex V a nd VI, part ID of directive 79/831/EEC (Annex VI, 1983, Annex V, 1984) on da ngerous substances. In Jacobs andEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 154 Martens, 1985 and 1987, EGBE and other ethylene glycols (purity > 99 %) were administrated dermally to rabbits according the EEC testing method. Five New Zealand white rabbits were used. 0.5 ml of EGBE was appl ied under an exposure chamber on the flank of each animal, during 4 hrs. Maximal mean erythe ma was 1.8 but the results per animal were very divergent. (From not irritating to very ir ritating). When diluted in polyethylene glycol 400 (50%), no sign of any irritation was observed.

Summary on skin irritation:
All the studies performed on rabbits and Guinea- pigs have shown that EGBE have caused moderate irritation (erythema and oedema) when applied occlusively on the skin for a period of 4 hours (in studies corresponding to the EU crit eria for classification) . If the substance was applied on scarified skin or for a longer period of time, signs of severe irritation sometimes leading to necrosis were reported.
Overall, in animals, EGBE can be considered to be a skin irritant. Classification R38 is needed.

4.1.2.3.2 Eye

Animal studies
Marked irritation (Erythema and oedema) was s een when rabbits were instillated with one drop of pure EGBE (Von Oettingen and Jirouch, 1931). The corneal reflex was absent after
treatment. In an old study poorly reported, EGBE produced only moderate irritation when 0.02 mL was instilled into a rabbit eye (Carpenter and Smyth, 1946).
Eye irritation was assessed after administration of EGBE into a rabbit eye, pure or diluted (1/10 and 1/100) (Hoechst, 1966). Animals were examined 1, 3, 7 and 24 hours after application.
For pure substance, corneal and conjuctival injuries were note d (severe redness and chemosis and corneal opacification). For th e 1/10 dilution, only a slight conjuctival redness was seen. And for the 1/100 dilution, no effects were seen.
An eye irritation study was performed according to the method described in the US code of Federal Regulations (Huntingdon Life science, 1 979a). Six animals were instillated with 0.1 ml of pure EGBE and observed during 72 hours. A scoring of the effects at 24, 48 and 72 hours according to the method of Draize was performed.
Effects on cornea, conjunctivae and iritis were seen in 5, 2 and 6 out of 6 animals at observation time of 24, 48 and 72 hours respectivel y and were not revers ed after the 72 hr observation period. According to th e EU classification system, EGBE could be considered as a severe eye irritant due to the irreversible effects seen at the end of the observation period although this period was only 72 hr. This classifi cation rule is only rele vant when no other data are available from other eye irritation tests fo r the period after 72 hours. This study cannot be relevant to su pport R41 classification.

Instillation of 0.005 mL of undiluted EGBE into rabbit eyes produced seve re corneal injuries with iritis (Bushy Run Resear ch Center, 1980b). Moderate corneal injury was observed whenEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 155 0.5 mL of a 15 % aqueous dilution of EGBE was instilled into the eyes of rabbits and no injuries was noted for a 5 % aqueous dilution. No more details about this study are available, the value of 0.005mL reported in the study is qu ite surprising in rela tion with the effects observed and the other doses tested.
EGBE (99 % purity) was instille d in the right eye of 6 New Zealand white rabbits (Jacobs and Martens, 1987 “Evaluation of the enucleated eye test against the in vivo eye irritation test in the rabbit, Jacobs G. and Martens M.” IHE-re port 01/12/1987). - Pare nt, 1992). The test was performed in two phases. The test was perfor med according to OECD guideline 405 (version 1981). In the second phase, 3 other chemicals were tested and besides the clinical examination, corneal swelling was also measured.
Erythema, chemosis, iritis and corneal opacity we re scored according to the Draize scores at 4, 24, 48, 72, 96 and 168 hr after treatment. The m ean erythema, chemosis, iritis and corneal opacity values (average of the 24, 48 and 72 hour observation) are: 2. 59, 0. 78, 1.00 and 1. 33, respectively. The mean corneal upperlayer damage (loss of epithelium measured by fluorescein retention on the cornea using a hand- slit lamp) was 95% after 4 hrs. Pain response was measured and described as “a few blin ks only, normal within one or two minutes; animals didn’t squeal or rubbing the eyes”. The substance caused marked pannus in all 6 rabbits.
EGBE, according to this study, should be classifi ed as an irritant. (mean erythema score >2.5, iritis =1). Pain sensat ion after contact with th e product seems to be low, what can lead to underestimation of the irritating properties. Stud ies with fluorescein retention revealed a 90% loss of the upperlayer of the cornea after instillati on. Generally speaking, the pannus reported does not disappear in rabbits and remain through the lifetime . In contrast, pannus response when seen in humans is usually associated with chronic disease and is not an acute irritative effect. Pannus generally disapp ears in humans. For those reas ons pannus was not considered in the past as an irreversible effect for EC-classification howev er it must be noted that under the GHS it will be considered as leading to a classification of “severe irritating”.
The study was redone some years later under the same protocol (Jacobs, 1992). The authors came to nearly the same mean scores (avera ge of the 24, 48 and 72 hour observation of: 2.47, 0.83, 0.83 and 1.73 respectively.), but si nce the mean erythema score and iritis score is lower than 2.5 and 1 respectively, no classifi cation requirement is triggered.

Table 4.69: Irritation scores in the Parent, 1992 Jacobs, 1992 study Time after end of application 4 hr 24 hr 48 hr 72 hr 96 hr
Mean score of conjunctivitis (3 max) 1.4 2.4 2.6 2.4 1.6
Mean score of chemosis (4 max) 1.7 1.0 0.8 0.7 0.2
Mean score of iritis (2 max) 1.0 1.0 1.0 0.5 0.2
Mean score of corneal opacity (4 max) 0.7 1.5 1.5 2.2 1.2
Mean surface of corneal damage (100 % max) 95 94 81 60 23

EGBE (99 % purity) was instilled in the eye of 3 rabbits (ECETOC, 1998). The test was performed according to OECD guideline 405 (version 1981) and GLP. Erythema, chemosis, iritis and corneal opacity were scored according to the Draize scores at 1, 2,3,7,14, 21 daysEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 156 after treatment. The EC scores for erythema, ch emosis and corneal opacity values (average of the 24, 48 and 72 hour observation) are 2.33, 2.78 and 2.33 respectiv ely. Opacity resolved in all 3 animals althought very sli ght redness (score 1), resist in 2 of 3 animals and chemosis (score 1) in one animal. According to the sc ores a classification is needed as irritant.

Six New Zealand white rabbit were tested in a Draize irritancy test with about 100 chemicals including EGBE (purit y > 99 %) (Kennah et al ., 1989). The cornea, iris and conjunctivae were scored at 24, 48 and 72 hr and at 7, 10, 14 and 21 day. Ketamine was injected intramuscularly just prior to measurement at a do se of 40 mg/kg to sedate the rabbits. Corneal thickness was measured before instillation and thereafter at intervals coincident with the Draize scoring. Three measurements were made on each cornea and averaged. Results were expressed as swelling: corneal thickness on observed day / corneal thickness prior to instillation x 100. (a 100% swelling value represents no change).

Draize score obtained for EGBE are summarized in table 4.70

Table 4.70: Draize scores in the Kennah study concentration Draize score % swelling (%- value untreated cornea)
100 66, severely irrita ting, resolution of ocular irritation within 14 days. 181 (81)

70 49, moderately irritating 146 (46)
30 39, moderately irritating; relative high scores are due to conjunctival damages 114 (14)

20 2, minimal irritating 113 (13)
10 1, minimal irritating 91 (-9)

Based on all the substances studied, a significant linear correlation was established between Draize score and % swelling (r = 0.86). A great majority of chemicals showed agreement between the Draize score and the corneal swelli ng when compared with class irritancy rating (mild, slight, moderate, severe, extreme).
For EGBE, Draize scoring was more severe than % of swelling. These di fferences are due to conjonctival damage, more heavily weighted by Draize scoring. But, swelling seems to detect more persistent irritation than Draize score. Resolution of ocular irritation was within 14 days.
Draize score is a “weighted scoring system i nvolving multiplication factors” on a mix of all effects reported (e.g. discharge+ chemosis+redness+opacity,+area of opacity and iritis). It is not possible to convert this scori ng as such to the EC system.
In a similar study over 32 substances (Jacobs and Martens, 1989) using the EC method of calculation, a better linear corr elation was obtained when co mparing specific effects like corneal swelling and corneal opacity (r=0.94, n=32) as well as corneal swelling and erythema (r=0.93, n=32). When all eye lesions are taken into account, a threshold value of cornealEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 157 swelling of 60 (untreated cornea is set to 0%) can be accepted for classification purpose. For EGBE, the mean value of corneal swelling over 3 animals and 24,48,72 hrs was 70%. (In this study the observed pannus was not taken into consideration for th e threshold value).
Application of this threshold va lue for classification (60% of co rneal swelling) to the Kennah study results in:
EGBE (>99% pure) ; Ir ritating (Xi, R36)
EGBE (70%), EGBE (30%), EGBE( 20%), EGBE (10%) not classified
In a paper reporting only the resu lts of some studies, the ins tillation of a 5 % solution of EGBE according to Draize procedure did not ex hibited any irritant properties (Bagley, 1994)
A GLP study was performed to assess the eye irritation potential of EGBE according to OECD guideline 405 (Safepharm laboratories, 1994b). 6 New Zealand white rabbits were instilled 0.1 ml EGBE into the conjunctiva l sac of the right ey e. Amounts of ocular damage/irritation were noted 1, 24, 48 and 72 hr following treatment. Additional observations were made on days 7, 14 and 21 to assess th e reversibility of the ocular effects. No information on test substance purity was includ ed in the study report. The eyes were not rinsed 24 hours following treatment.
Areas of corneal opacity were noted in all tr eated eyes on 24 and 48 hr after treatment. Reversibility was not obtained in 21 days for 1 animal. Iridial inflammation was noted in all treated animals 1, 24 and 48 hr after instillation. Re versibility was obtained in 14 days. Moderate conjunctival irritation was observed at 1, 24 and 48 hr. Effects were present till D14. Petechial haemorrhage was se en in some animals at each observation time from 1 to 72 hr. Ectropion was noted from 72 hr observation time in some an imals and not reversible for 1 animal at the end of the study. One animal show ing signs of discomfort was therefore killed for humane reasons immediately after the 14 th day. The scores are shown in the table below.

Table 4.70 bis: Individual score fo r Safepharm laboratories study, 1994b) Animal Time (hrs) Opacity Iritis Redness Chemosis 24 1 1 2 2 48 1 1 2 2 72 1 1 2 2 1

7 day 0 0 0 0 24 1 1 2 2 48 1 1 2 2 72 1 1 2 2 2

14 days* 2* 1 2 2 24 1 1 2 2 48 1 1 2 2 72 1 1 2 2 3

7 days 0 0 0 0 24 1 1 2 2 48 1 1 2 2 72 2 1 2 2 4

21 days 2* 0 0 0 24 1 1 2 2 48 1 1 2 2 72 2 1 2 2 5

14 days 0 0 0 0 24 1 1 2 2 48 1 1 2 2 72 1 0 2 1 6

7 days 0 0 0 0 * vascularisation. Animal 2 sacrificed after observation at 14 daysEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 158 EGBE is considered to be a severe irritant to the eye in this study.

EGBE 5 % in water solution was tested in various in vitro assays: SIRC cytotoxicity assay, Neutral red uptake assay (48hr), cell protein assay, fibroblast cytotoxicity assay, corneal plasminogen activator assay (CEPA) (Harbell et al. , 1997).
Given that these assays are not yet (or will ne ver be) validated, the results obtained cannot be used in this risk assessment. (Howev er, the average Draize score obtained in vivo can be used: 2.7).
In a GLP study performed according to OECD guideline 405, 3 New Zealand white rabbits were instilled with EGBE (purity 99.6 %). Animals were observed 21 days (BASF, 2000). Eye washing was performed about 24 hours afte r treatment, before the 24 hour reading.
One animal exhibited a slight corneal opacity re versible in 21 days. Si gns of a slight iris injury was described, reversible in 7 days. A me dium to severe irritation of the conjunctivae was also observed and was reversible in 21 da ys. According to criteria for assessment of ocular lesions, EGBE can be considered as an eye irritant. The individual animal scores are shown below:

Table 4.70 ter: Individual score for BASF study, 2000 Animal Time (hrs) Opacity Iritis Redness Chemosis 24 1 0 2 2 48 1 0 2 2 72 1 1 3 2 1

14 days 0 0 0 0 24 0 1 3 2 48 1 1 3 2 72 1 1 3 2 2

21 days* 0 0 0 0 24 1 1 3 2 48 1 0 2 1 72 1 0 2 1 3

21 days 0 0 0 0

Human data:
After exposure to 200 ppm of EGBE, immediate ir ritation of the nose and throat, followed by ocular irritation and disturbed taste was experien ced by all three subjects (Mellon Institute of Industrial Research, 1955).

Summary of eye irritancy data
A lot of studies have been performed to assess the eye irritation proper ties of EGBE. Most of them were not performed according to guideline s, but overall, all studies have shown that EGBE is irritant or severely irritant to the eyes of rabbits with effects both on conjunctivae, iris and cornea. In one well performed study EGBE produced irreversible effects on the conjunctivae and on the cornea in at least one treated rabbit. Ac cording to the EU classification system, these effects would trigge r a classification R41, “severely irritant to eyes” because of the irreversibility of the e ffects at the end of the observation period. But these studies were performed according “old” guid elines. In the new guidelines, it is possible to rince the treated eye for an imal welfare purposes and to more closely simulate the human situation where lacrimation would clear the s ubstance from the eye. In a test, performedEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 159 according the recent guidelines, EGBE was onl y irritant (R36). This result was given precedence by the C&L group who conf irmed a classification of R36.
It was also demonstrated that dilution of EGBE in water decrease its irritant properties as well as rinsing of the eyes in case of exposure.

4.1.2.3.3 Respiratory tract

Animal data:
Sensory irritation was evaluated using an in vivo method with mice (Kane et al. , 1980). Male Swiss-Webster mice (4/group) were exposed whole body to a series of chemicals (including EGBE). The measured response was the maxi mum percent decrease in respiratory rate, averaged over 4 mice, simultaneously exposed for 10 minutes. The responses obtained for various concentrations of solvents were u tilized to develop a concentration-response relationship, for this, the concentration associ ated with a 50 % decrease in respiratory rate (RD50) was calculated.
Time response relationship: af ter an initial decr ease observed within a few seconds and characterized by a pause during the respirator y rate, a phase of re covery occurred but respiration rate was lesser than norm al until the end of the exposure period.
The response obtained within the concentration range tested was less than 50 % decrease in respiratory rate. The RD50 calcu lated by extrapolation was 2 825 ppm (confidence limit 1695 – 7278). These values are well in excess of th e saturated vapour pres sure. When compared with the criteria of evaluation, 0.01 RD50 (about 28 ppm) would cause minimal or no sensory irritation whereas 0.1 RD50 (about 280 ppm) woul d cause definite but tolerable sensory irritation.
An estimate of the RD50 of EGBE was repor ted using modelling based on physicochemical properties (Alarie et al. , 1995). Calculated log RD50 was 3.45 (2818ppm). This is in good agreement with the experimental data.

Human data:
Three volunteers were exposed to 100 and 200 pp m of EGBE for periods of 2 or 4 hours, separated by a 2 hour period of non-exposure (Mellon Institute of Industrial Research, 1955). Immediate irritation of the nose and throat, foll owed by ocular irritation and disturbed taste was reported by all three subjec ts Whether such ‘irritation’ was physiological or merely discomfort is not clear..
Some human studies by inhalation for toxokinetic studies (Johanson et al. , 1986a, Kumagai et al. , 1999) have shown that EGBE was not irritant to the eyes or respiratory tract at doses of 20 ppm during 2 hours or 25 ppm fo r exposure periods of 10 minutes. A study performed by Johanson and Bomanin 1991 in which human vol unteers were exposed to 50 ppm EGBE whole body (but breathing pure air) and mouth onl y did not describe ir ritant effects, but according to the description of the study, th is kind of effects were not recorded.
In a recent study (Jones et al. , 2003) no signs of irr itation were reported af ter exposure to 50 ppm EGBE (see § 4.1.2.1.2: Four volunteers (2 males and 2 females, aged 28-33) were exposed on 9 separate occasion, each occasion separated by at least 3 weeks. The exposure was 50 ppm EGBE for 2h. To record any physiol ogical changes in the voluntee rs under the different conditions, physiol ogical monitoring equipment wa s used.). The published studyEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 160 paper does not mention if these signs were ch ecked except the recordin g of the physiological changes. However, the author did indicate in a written communication that the volunteers were asked to report any adverse effects and none were reported (Jones, personnal communication).

Summary respirat ory irritation:
Animal studies available (including repeated dose toxicity studies performed by inhalation on rats and mice) did not show any si gns of significant respiratory i rritation. No classification is required for this end point.
From the human data it is apparent that the NOEC is >50ppm whilst the NOEC (based on effects of discomfort) is <100-200ppm. A NOEC of 50ppm is taken forward for risk characterisation.

4.1.2.4 Corrosivity
According to EU classification criteria EGBE ca nnot be considered as a corrosive substance. Some signs of necrosis were s een in some studies but only wh en animals were exposed intradermally or for a long period of time by occlusive wrapping.

4.1.2.5 Sensitisation

4.1.2.5.1 Studies in animals

Skin

In vivo studies
In a GLP study performed according to OECD gui deline 406, EGBE was tested in Guinea Pigs for sensitisation potential (Unilever, 1994). Induction was made with a dermal injection of 0.5 % EGBE in 0.9 % saline an d a topical application of a 25 % EGBE in 0.9 % saline. Challenge was made with a topical applicat ion of 10 % EGBE in saline. All topical applications were made occlusively. Concentr ations tested were ba sed on results found in preliminary studies (showing a Maximal Concen tration with No Irritation (CMNI) of 10 %). Two challenge applications were made at one week interval.

No evidence of sensitisation was seen in treat ed animals both at challenge and re-challenge.
EGBE was tested in a Maximised Magnusson & Kligman test (Zissu, 1995). After induction (test substance concentrations not reported) a 1 % concentration of EGBE was used for challenge.
No animals exhibited signs of sensitisation.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 161 4.1.2.5.2 Studies in humans

Skin
In a GCP study, the sensitising potential of 10 % (v/v) EGBE in aqueous solution was assessed in human on 201 volunt eers (TKL Research, 1992). I nduction phase consisted in 9 consecutive occlusive application of EGBE. Pa tches were removed 24 hr after application. Assessments of the sites of application were made at 48 hr interval s and after new patches were reapplied. Following the ninth evaluation, th e subjects were dismissed for a 14-day rest period. The challenge phase was in itiated during the sixth week of the study, identical patches were applied to sites previously unexposed to EGBE. They were removed after 24 hr of exposure and the sites graded after additiona l 24 hr and 48 hr periods. Re-challenge if required was to be performed whenever there wa s evidence of possible sensitisation. The rechallenge was conducted on naïve sites on the b ack under both occlusive and semi-occlusive conditions approximately one or two weeks afte r challenge had been completed. Patches were applied for 24 hr, the patches removed and the sites evaluated 48, 72 and 96 hr after patches applications.
Only 7 of the 48 hr evaluations and 12 of the 72 hr evaluations showed s light erythema. One subject at the 72 hr reading had definite eryt hema. More severe responses which would have been expected if delayed contact sensitisation had occurred were not observed. In conclusion, according to the authors, none of the 201 subj ects completing the study showed evidence of sensitisation. Re-challenge wa s not required for any subject.
Two cases of dermatitis herpetiformis (DH) temporally related to the use of a cleaning solution containing EGBE (Snider and Maize, 1993). Analysis of the mixture showed the presence of EGBE, trisodium phosphate, nonylphenol polyethylene glycol ether, anthraquinone, lemon fragrance and an antifoam agent. Patch testing with the individual chemicals at the same concentration than in the mixture (except for EGBE which was diluted to avoid irritation) and at the same time precipi tated a flare of DH in both patients. Causal agent remained unknown because each individual ingredient was not separately patch tested. These results are hardly reliable for EGBE risk assessment because of co-exposure.

Respiratory tract
Considering SAR in the glycol ether family, th e wide dispersive use of them and that no glycol ether has even been associated with cases of respiratory sensitisation, it can be considered that this toxicological property canno t be expected and is not relevant for risk
assessment.

4.1.2.5.3 Summary of sensitisation
No signs of skin sensitisation were seen in two animal studies or in a human patch test. Moreover, considering Structure Activity Relati onship (SAR) in the glycol ether family, the wide dispersive use of EGBE and that EGBE ha s never been associated with cases of skin sensitisation, it can be considered that skin sens itisation cannot be expect ed and is not relevant for risk assessment. No classification is needed for this end point.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 162 4.1.2.6 Repeated dose toxicity

4.1.2.6.1 Studies in animals

Inhalation

Rat
Three groups of 23 Wistar rats were expo sed to EGBE in an inhalation chamber at concentrations of 0, 135 and 320 ppm (0, 652 and 1546 mg/m 3 ), 7 h/day, 5 day/week for 5 weeks. Eight animals/group were used for path ological examination and were sacrificed by pair after 1, 3 and 5 weeks of exposure (Werner et al ., 1943b). The remaining animals were used for growth and haematological studies. Ha ematological examinations were made before – during and after the exposure (week 0-2-4-6-9) and consisted of red and white cell counts – differential counts, reticulocytes count and Hb estimations. Three weeks after the end of exposure and after a last blood analysis animals were sacrificed and organs examined (liver, kidney, heart, spleen and lung).
No effects on body weight gain were seen in either group of EGBE treated animals. No mortality or signs of systemic action were seen in the group of treated animals. The erythrocyte counts and haemoglobin concentration were significantly decreased at the end of 1 week of exposure to the high concentration of EGBE. Circulating reti culocytes were also increased over the same period.
For this study, transient effects on haematologica l parameters were seen at the high dose. The No Observable Adverse Effect Concentra tion (NOAEC) was 135 ppm based on transient haematological effects at 320 ppm.
Mortality of rats after two 8- hr exposures to 500 ppm (2415 mg/m 3 ) of EGBE was 3/6 animals (Mellon Institute of Industr ial Research, 1952). At 250 ppm (1208 mg/m 3 ), 0/6 animals died after or during six 8 hr exposure wh ereas 1/5 animals died af ter or during four 8- hour exposure. No deaths occured when 6 anim als were exposed to six 8-hour exposure of 125 ppm (604 mg/m 3 ) EGBE. No effects were seen in the control group. Due to the experimental conditions, this study cannot be taken into account for the Risk Assessment.
Groups of Alderly Park rats (4 males and 4 females/group) were exposed (whole body) to various concentrations of EGBE and for diffe rent periods of time (Gage, 1970). Rats were maintained in the exposure chamber for periods of up to 6 hours, and between repeated daily exposures they were returned to their cages wh ere food and water were freely available. In the initial experiments the concentrations were select ed to produce, if possible, acute effects after short exposures. Thereafter the exposure peri od was extended and the concentration lowered until the animals could survive 6-hour exposures, fi ve days a week, for up to four weeks. With liquids, the concentrations which could be tested in these acute and subacute experiments. The rats were weighted and examined each morn ing, and their conditions and behaviour were recorded throughout the exposure period. Urine was collected overnight after the last exposure day for biochemical te sts. Blood was collected during euthanasia for haematological tests. After a gross examination of the orga ns, the lung were collected and examined. The following organs were also taken for microscopi c examination: lungs, liver, kidneys, spleen, adrenals and occasionally heart, jejunum, ileum and thymus.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 163 Table 4.71: Experimental pattern of exposure for Gage study, 1970 Dose Number of exposures Symptoms
250 ppm
(1208 mg/m 3 ) 4 x 6-hr exposures initial haem aglobuniria and lethargy, low HB and MCHC, weight loss

100 ppm
(483 mg/m 3 ) 15 x 6-hr exposures no toxic signs, urine and blood tests normal apart from increased red cell osmotic fragility: autopsy, organs normal
50 ppm
(242 mg/m 3 ) 15 x 6-hr exposures as 100 ppm experiment

20 ppm
(97 mg/m 3 ) 15 x 6-hr exposures no toxic signs, blood normal: autopsy, organs normal

In this study, the Lowest Observable Adve rse Effect Concentration: LOAEC based on haematological signs was 50 ppm (15 exposures ) leading to a NOAEC of 20 ppm. Other effects than those seen on haematological pa rameters were seen only at 250 ppm (4 exposures)
Eighty five rats divided in thr ee groups were exposed to EGBE (purity unknown) at doses of 0 or 17 ppm (84 mg/m 3 ). The first group was exposed 3 hr day on 6 days per weeks and the second group was exposed to 6 hr/day on 3 consecutive days per week (Lomonova and Klimova, 1977). The third group served as contro l. It is noted that this study was not conducted according to GLP ans was poorly repo rted. Body weight of animals was measured regularly. A standardised battery of gene ral parameters and the level of blood cathecholamines were checked. For specific toxicity, blood parameters and erythrocyte fragility tests were performed. Catalase activ ity was also measured. Some functional tests were also performed: hexenal test, “cold te st” and water maze. After the 4 months of exposure, an unique dose of 800 mg/m 3 of EGBE was administered to assess the adaptative effects (if any) of regular admini stration of small doses of EGBE.
Overall, the results of all parameters assessed has shown that the toxici ty of EGBE was more severe in the first group (3 hr/d 6 days/week) than in the group 2. Moreover, the charge dose of 800 mg/kg administered at the end of the 4 month exposure period enha nced less effects in the second group than in the first one. Significan t toxic signs were limited to haemolysis and narcosis. Due to the experimental conditions and the specificity of the test, data obtained can only be qualitative and therefore is not suitable for risk characterisation. No NOAEC can be derived.
Groups of Fisher 344 rats (8 animals per sex ineach group with an addition of 7 females to the high exposure and control gr oups, 7 males to the control group and 8 males to the high exposure group - the additional animals were used to determine, if necessary, the reversibility of any effect - these animals were observed during 14 days after the last exposure) were exposed to 0, 20, 86 and 245 ppm (0, 97, 415 and 1183 mg/m 3 ) of EGBE 6 hr/day for 5 consecutive days –followed by 2 days without exposure and then 4 days of additional exposure (Bushy Run Research Center, 1981a). A ll animals were observed before, during and following exposure for any signs in dicative of toxic effects. The appearance and demeanor ofEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 164 all animals were recorded at these times. The modified Irwin Screen test was performed on five rats per sex from the 245 ppm exposure gr oup and five rats per sex from the control group for signs indicative of be havioural and/or neuromuscula r abnormalities. Animals were observed on exposure days 1, 2, 5, 6 and 7 prior to following exposure and preceding sacrifice. During the post-expos ure period, reversibility groups were screened on days 4, 7 and 14. Evaluations were made on the following: corneal response, pupil response, tail pinch, toe pinch, righting reflex, locomotor activity, imp aired gait, respiration, tremors, convulsions, salivation, piloerecti on, diarrhea, tail elev ation, lacrimation, ster eotypy. All rats had a ophthalmologic examination prior to group assignments. Rats were culled if abnormalities were observed. Except for reversibility groups, an eye examination was also done on all rats at sacrifice. The body weight for each rat was determined on the morning preceding the 1 st

, 2 nd , 5 th , 6 th and 7 th exposure days and again prior to sacr ifice. During the post-exposure period the body weights of the re versibility group were recorded on post-exposure days 4 and 7 and prior to sacrifice, day 14. Haematological tests were perf ormed on blood samples collected from all rats on the day prior to sacrifice. The analysis included: haemoglobin, haematocrit, erythrocyte count, white blood cell count, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration. Reticulocyte and white blood cell smears were made for each animal, but were only evaluated for high exposure and control animals. All rats were killed for necropsy on the day following the fina l exposure (except the rats of the reversibility groups which were sacr ificed on the 14 th day following the final exposure. Liver, lungs, kidneys and testes were weighed.
No deaths occurred during the study. After the first and second exposure, only red staining of urine was seen in animals of the high dose group. A slight increase over the control values for audible respiration and nasal discharge were observed in male rats of the 245 ppm group. Urogenital area wetness was observed in a few females of the 245 ppm exposure group. Male rats in the 245 ppm or contro l groups appeared normal duri ng the modified Irwin Screen testing sessions. One female rat of the 245 ppm exposure group exhibited an abnormal righting reflex following exposure. A decrease in body weight gain was observed for animals treated with 245 ppm and for fe males treated with 86 ppm. During post exposure observation, animals recovered and no differences were seen with controls at post exposure day 14. Erythrocyte count, haem oglobin and MCHC values were si gnificantly lower in males and females of the 245 ppm group compared to c ontrols. These animals also exhibited a significant increase in MCV, nucleated red bl ood cells, reticulocytes and in males only, lymphocytes. Haematological values in both male s and female rats exposed to 86 ppm were significant for haemoglobin (decrease) and MCV (increase). The female s of the 86 ppm group also had significant decreases in MCH and MCHC and an increase in the haematocrit values. All effects seems to be dose-re lated. No haematological eff ects were seen in the 20 ppm group. In the reversibility group, animals exhibited a substantia l recovery following 14 day post exposure period. However a significant decreas e in erythrocyte count (male only) and a significant increase in MCV (all animals) and in MCH (male only) were still present in treated animals. The leukocyte count returned to control value during this recovery period. The mean liver weight values (relative and absolute) for the female of the 245 ppm group were higher than the controls. In addition, the relative liver wei ght for the females of the 86 ppm group and for the males of the 245 ppm group were higher than the controls. The mean relative testes weight for the males of the 86 ppm group was lowe r than controls, this can be considered to be an isolated finding not relate d to the treatment. After 14 day recovery period, no significant differences from c ontrols in male or in female organ weight were observed. At necropsy, no finding could be specifically relate d to EGBE exposure. In this study, a NOAEC of 20 ppm can be indentified, according to the ef fects seen on haematological parameters at 86 ppm.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 165 EGBE (purity 99.4 %) vapour was administered by whole body exposure to Fischer 344 rats at concentrations of 0, 5 – 25 or 77 ppm 0, ( 24 – 121 – 372 mg/m 3 ), 6 hr/day, 5 day/week for approximately 13 weeks (16 males and 16 fe males/group) (Bushy Run Research Center, 1981b). Animals were observed for signs of toxi city before, during and after exposures. A modified Irwin screen test was performed on 5 rats/sex/group from the high concentration and controls (see 9 day study for details, ref: Bu shy Run Research Center, 1981a) and a sub-group was killed after 2 weeks (9 exposures) for in terim examination. Body weight determination were performed regularly. Food consumption was determined weekly. All rats had an ophthalmologic examination prior to group assi gnments, male rats of the 77, 25 ppm and controls were re-examined prior to terminal sacrifice. Only the females of the 77 ppm group were examined. Haematologic tests were perfor med on blood samples collected from all rats on the day prior to sacrifice. Clinical examinations includ ed urinalysis, haematology and serum clinical chemistry. Urine from rats of the high exposure group was analysed for haemoglobin and red blood cells. Urine from the sa me 5 rats per sex was analysed prior to exposure for the first 5 exposure days and prior to exposure each Wednesday thereafter. Urine was also collected and a more comprehensive analysis was performed one week prior to the terminal sacrifice from all rats. An interim sacrifice was performed on 6 rats per sex per exposure level. All remaining rats were necrop sied at the final sacrifice on the day following the last exposure. After sacrifice, a gross di ssection and an evaluation were performed on each animal. The following organs were weighed at terminal sacrifi ce: liver, kidneys, lungs, and testes. No organ weights were obtained for the rats at the interim sacrifice. At the time of the necropsy, histopathological examinations were performed on the usual tissues selected for such studies.

No death occurred related to treatment. No effects were seen concerning ophthalmologic examination and Irwin Screen test. For rats that were killed at the interim sacrifice time, after 9 exposures, the females of the 77 ppm group ha d lower body weight gain values comparing to the controls. No differences were found among the females of the lower exposure groups or males of any exposure group. For the main group, similar results were obtained for the same periods of time. However, following this ti me period the depression in body weight gain noted in the females was reversible and returned to values similar to the values of the control group. The decreases of food consumption are consistent with the changes noted in body weight gain. Slight signs of h aemolytic anaemia were seen in females exposed to high doses of EGBE. Subtle haematological effects noted in females were characterised by minimal depression of red blood cell c ounts, haemoglobin and haematocr it with an attendant slight increase in MCH. These effects were noted at the interim sacrifice time and persisted throughout the study without increasing in severi ty. There was no indication of red blood cells or haemoglobin in the urine collected daily during the first exposur e week or weekly thereafter from the male and female rats of the high exposure level. Moreover, there was no treatment related alteration in erythrocyte fragilit y at either the interim or terminal sacrifice times. No other haematological, urinary or c linical chemistry findings of toxicological significance were observed among the rats. Severa l incidental lesions we re present in various organs, however no treatment-related, gross or microscopic lesions were found in either the male or female rats sacrificed at the end of the study. On organ weights, the only significant finding was lower relative kidney weight among the males of a ll treated groups. This finding is not believe to be related to the treatment.
The NOAEC was 25 ppm, based on the non-progres sive haematological effects in females and a transient reduced body weight gain at tw o weeks seen in females of the 77 ppm dose
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R408_0808_ HH _C LEAN 166 EGBE was administered to groups of 10 ma le and 10 female F344/N rats by whole-body exposure to 0, 31, 62.5, 125, 250 or 500 ppm (0 – 150 – 302 – 604 – 1208 – 2416 mg/m 3 ) EGBE vapour (lot no. QP- 921215-26D2, Dow Chemical, Plaque mine, LA, USA, purity > 99 %) for 6 h per day, 5 days per week for 14 weeks (NTP, 2000 ; Nyska et al. , 1999a ; Long et al. , 2000).
Rats were held in the testing laboratory for 11 - 12 days and were 6 weeks old when the study began. They were housed indivi dually, water was available ad libitum and feed was available ad libitum except during the exposure periods. All rats were observed twice daily throughout the study. Body weights were recorded at the be ginning of the study and then weekly until the end of the experiment. Clinical observations also were recorded weekly throughout the experiment. Haematological examinations were performed at the end of the experiment, using an Ortho ELT-8/ds 9000 analyser. Differential leukocyte and nucleated erythrocyte counts were made by microscopic examination of blood films
Autopsies were made on all study rats. Organ weights measured were for the heart, right kidney, liver, lungs, right testis and thymus . A complete histological examination was performed on the chamber control and 500 ppm males and females and on the 250 ppm female rats.

Six female rats were found moribund and kille d during the study: five in the 500 ppm group (four in week 1, one in week 5) and one in th e 250 ppm group (in week 8). By the end of the study, body weight gains were significantly reduced in females of the 500 ppm group, but were unaffected in all other groups. Clinical findi ngs were most prevalent in rats of both sexes exposed to 125, 250 or 500 ppm and included a bnormal breathing, pallor, red urine stains, nasal and eye discharge, lethargy and either incr eased salivation or lacrimation. All females of the 500 ppm group, particularly du ring the first two weeks, deve loped alternating blue and white bands on their tails that caused them to se lf-mutilate and loose the distal portion of their tails.
Haematological examination showed that inhala tion of EGBE resulted in the development of a persistent and exposure-related macrocytic, normochromic, responsive anaemia, as indicated by decreased haematocrit values, haemoglobin concentrati ons and erythrocyte counts in the 125 ppm or greater group males and in all groups of exposed females. The effects were dose related. This evidence of a se x difference in the severity of the anaemia was also seen in the 500 ppm group, in which the indicators were slightly more severe in the females than in the males. Evidence of an er ythropoietic response was shown by increases in reticulocyte and nucleated erythrocyte counts in males of the 125 ppm or greater groups and females of the 62.5 ppm or greater groups. Othe r haematological change s were decreases in lymphocyte and monocyte counts in males of the 125 ppm or greater groups and increased platelet counts in females of the 125 or 500 ppm groups. Some organ weight changes were observed. These were: increases of the kidney of males in the 500 ppm group and females in the 125 ppm or greater groups; increases of th e liver of males in the 250 or 500 ppm and females in the 125 ppm or greater groups; and decr eases of the thymus of females in the 500 ppm group. Thrombosis was observed in the tail vertebr ae, femur, incisors, nasal cavity, lung, heart and liver of most females in the 500 ppm group, but not at a ll in the 250 ppm group. Additionally, in the 500 ppm group females, there was degeneration of the hepatic centrilobular areas and renal tubul es (4/5 in each case) and at rophy of the thymus (4/5) and spleen (1/5). Haematopoietic ce ll proliferation was observed in the spleen of 2/5 group same group. Bone marrow hyperplasia was recorded in males of the 250 and 500 ppm groups and females in all groups from 62.5 ppm. The severity of this response was dose related In the forestomach of females in the 500 ppm group, but not in lower dose groups, observationsEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 167 made were of inflammation (3/5), necrosis (2 /5), ulcers (2/5) and hype rplasia (1/5). Effects also observed in the 250 ppm group females were hepatic necrosis, pigmentation of Kupffer cells and renal tubule cells and bone ma rrow hyperplasia (NTP, 2000). No NOAEC was found for female rats. LOAEL was 31 ppm based on haematological effect s seen at all doses tested.A NOAEL of 62.5 ppm wa s found for male rats, based onhaematotoxic effects seen at 125 ppm..
EGBE was administered to groups of 50 ma le and 50 female F344/N rats by whole-body exposure to 0, 31.2, 62.5 or 125 ppm (0 – 150 – 302 – 604 mg/m 3 ) EGBE vapour (lot no. QP921215-26D2, Dow Chemical, Plaquemine, LA, USA, purity > 99 %)for 6 hr per day, 5 days per week for 104 weeks. For haematological an d bone marrow analyses, additional groups of 27 male and 27 female F344/N rats were exposed to 0, 62.5 or 125 ppm fo r evaluation at 3, 6 and 12 months and 9 male and 9 female rats were exposed to 31.2 ppm for 3 months (for haematological examination onl y) and 6 months (NTP, 2000).
Rats were held in the testing laboratory for 18 days and were 7 – 8 weeks old when the study began. They were housed indivi dually, water was available ad libitum and feed was available ad libitum except during the exposure periods. All rats were observed twice daily throughout the study. Body weights were recorded at the beginning of the study, after which they were recorded monthly from week 5 until week 89 and then every two weeks from week 92 until the end of the experiment. Clinical observatio ns also were recorded monthly from week 5 until week 89 and then every two weeks until the end of the experiment. Haematological examinations were performed on an Ortho ELT-8/ ds 9000 analyser at 3- and 6-months and on a Roche COBAS Helios analyser at 12-months. Differential leukocyte and nucleated erythrocyte counts were made by micros copic examination of blood films.
Autopsies and microscopic examinations were ma de on all of the main study animals that had not been used for blood and bone marrow samp ling. Organ weights were not measured. At autopsy, all organs and tissues we re examined for grossly visibl e lesions and all major tissues were fixed and processed for histological examination. A quality assessment pathologist evaluated the slides from all tumours and po tential target organs, which included bone marrow, forestomach, kidney, liver, lung, nose a nd spleen of males and females and the adrenal and clitoral glands of females. The slides and the preliminary quality assessment report were then submitted to a National Toxi cology Program Working Group of pathologists.
Survival of exposed male and female rats was similar to the chamber control groups, the mean numbers of survivors at the end of the expe riment in each of the 0, 31.2, 62.5 and 125 ppm groups were 19, 11, 21 and 24 males and 29, 27, 23 a nd 21 females, respectively. No clinical signs were attributed to EGBE exposure. Body weights of ma le and female rats in the 31.2 and 62.5 ppm groups and male rats of the 12 5 ppm group were generally similar to the controls throughout the experime nt. Body weights of female rats of the 125 ppm group were generally lower than those of the chamber control from week 17 until the end of the experiment.
Neoplastic effects observed in these e xperiments are described in section 4.1.2.8.1.3.
Haematological examination showed that inhala tion of EGBE resulted in the development of a persistent and exposure-related macrocytic, normochromic, responsive anaemia, as indicated by decreased haematocrit values, haemoglobin concentrati ons and erythrocyte counts. These changes occurred at 3, 6 and 12 months in 62.5 ppm group females and 125 ppm group males. Some anaemia also occurred at 3 and 6 months in the 31.2 ppm group females and at 12 months in the 62.5 ppm group males. Increases in circulating reticulocyteEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 168 and nucleated erythrocyte counts are consistent with an er ythropoietic response to the anaemia. In bone marrow there were approx imately 15 % - 35 % decreases in the myeloid/erythroid ratio in the 125 ppm rats of both sexes, bu t particularly in females. Bone marrow hyperplasia was recorded in males of the 250 and 500 ppm groups and females in all groups from 62.5 ppm. The severity of this response was dose related.
Non-neoplastic effects of exposure were observed in the nose, liver and spleen.
In the nose there were significant increases in the incidences of hyaline degeneration of the olfactory epithelium in all group s of males (chamber control, 13/48; 31.2 ppm, 21/49; 62.5 ppm, 23/49; 125 ppm, 40/50) and in females exposed to the two higher concentrations of EGBE (chamber control, 13/50; 31.2 ppm, 18/48; 62.5 ppm, 28/50; 125 ppm, 40/49). The severity of the lesion was mini mal and did not change with in creasing exposure concentration. It is the most common age-related change of th e nasal passages of rats (St. Clair and Morgan, 1992) and it has been proposed to have an adaptive or protective role (Buckley et al ., 1985).
Incidences of Kupffer cell pigmentation of th e liver increased significantly in all exposed groups of male rats (chamber control, 23/ 50; 31.2 ppm, 30/50; 62.5 ppm, 34/50; 125 ppm, 42/50) and in the two higher exposure groups of female rats (chamber control, 15/50; 31.2 ppm, 19/50; 62.5 ppm, 36/50; 125 ppm, 47/50). The se verity of the lesion increased in the 125 ppm group of both sexes.
In the spleen, incidences of fibrosis were significantly increased in the two higher exposure groups of male rats (chamber control, 11/ 50; 31.2 ppm, 14/50; 62.5 ppm, 19/50; 125 ppm, 20/50), but not in females (NTP, 2000). No NOAEC could be derived for non-neoplastic effects in this study. The LOAEC for both male and female rats was 31 ppm, based on an increased incidence of minimal Kupffer cell pigmentation and on significant haematological effects in females.

Mouse
EGBE (purity unknown) was administered by whole body exposure to dogs, monkeys, guinea pigs, mice and humans (Mellon Institute of Industrial Research, 1955). Groups of 70 male C3H mice were exposed to 0, 100, 200 or 400 ppm (0 – 483 – 966 – 1932 mg/m 3 ) EGBE 7 hr/day for up to 90 days. B ody weights were reco rded weekly. Serial sacrifices were performed on 15 mice from each group afte r 30 and 60 exposures, on 10 mice after 90 exposures, and on 11 to 18 mice after 42 days rest following 90 days of exposure. Ten mice from each group were used for serial determin ation of erythrocyte fragility after various exposure periods. The blood samples were obtaine d by section of the cervical cord. Liver and kidney weight were measured. Only the kidn eys were cut for histological examination (because repeated exposures to even fatal co ncentrations of EGBE produced undramatic micropathology in rats. No mortality or gros s pathology or renal tissue changes were observed. Statistically significant alterations of liver weights occurred in the mice exposed to 400 ppm for 30, 60 and 90 days. A similar occurren ce was noted in the mice exposed for 60 days to 200 ppm. The number of mice whic h had bloody urine immediately after exposure was proportional to the exposure concentration. No case of bloody urine was noted after the third exposure. Increased erythr ocyte fragility was found in the mice exposed to all three concentrations. The increase in erythrocyte fragili ty appeared to be as great after the first exposure as it was after the 89 th exposure. In all cases the eryt hrocyte fragility value fell to essentially normal levels when blood sample s were taken from mice after 17 hr rest.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 169 Sixty mice divided in three groups were exposed to EGBE (purity unknown) at doses of 0 and 17 ppm (84 mg/m 3 ). The first group was exposed 3 hr/day on 6 days per weeks and the second group was exposed to 6 hr/day on 3 consecutive days per week (Lomonova and Klimova, 1977). The third group served as control. It is noted that this study was not conducted according to GLP and was poorly reported. Body we ights of animals were recorded at regular intervals. A standardised battery of genera l parameters and the concentrations of blood cathecholamines were checked. For specific toxicity, blood parameters and erythrocyte fragility tests were performed. Catalase activ ity was also measured. Some functional tests were also performed: hexenal test, “cold te st” and water maze. After the 4 months of exposure, an unique dose of 800 mg/m3 of EGBE was administered to assess the adaptative effects (if any) of regular admini stration of small doses of EGBE.
Overall, the results of all parameters assessed ha ve shown that the toxicity of EGBE was more severe in the first group (3 hr/d 6 days/week) than in the group 2. Moreover, the charge dose of 800 mg/kg bw administered at the end of the 4 month exposure period enhanced less effects in the second group than in the first one. Significant toxic signs were limited to haemolysis and narcosis.
EGBE was administered to groups of 10 male and 10 female B6C3F 1 mice by whole-body exposure to 0, 31, 62.5, 125, 250 or 500 ppm (0 – 150 – 302 – 604 – 1208 – 2416 mg/m 3 ) EGBE vapour (lot no. QP- 921215-26D2, Dow Chemical, Plaque mine, LA, USA, purity > 99 %)for 6 hr per day, 5 days pe r week for 14 weeks (NTP, 2000).
Mice were held in the testing laboratory for 11 - 12 days and were 6 weeks old when the study began. They were housed indivi dually, water was available ad libitum and feed was available ad libitum except during the exposure periods. All mice were observed twice daily throughout the study. Body weights were recorded at the be ginning of the study and then weekly until the end of the experiment. Clinical observations also were recorded weekly throughout the experiment. Haematological examinations were performed at the end of the experiment, using an Ortho ELT-8/ds 9000 analyser. Differential leukocyte and nucleated erythrocyte counts were made by microscopic examination of blood films.
Autopsies were made on all study mice. Organ weights measured were for the heart, right kidney, liver, lungs, right testis and thymus . A complete histological examination was performed on the chamber control an d 500 ppm male and female mice.
In the 500 ppm group, in week 2 of the experime nt, two male and two female mice died and two male and two female mice were found mori bund and killed. All other mice survived. By the end of the study, body weight gains were significantly reduced in males of the 125, 250 and 500 ppm groups, but were unaff ected in all other groups. Clinical findings were found only in males and females of the 500 ppm gr oup that died or were killed and included abnormal breathing, red urin e stains and lethargy.
Haematological examination showed that inhala tion of EGBE resulted in the development of a persistent and exposure-rel ated normocytic (unlike rats ), normochromic, responsive anaemia, as indicated by decreased haematoc rit values, haemoglobin concentrations and erythrocyte counts in the 125 ppm or greater group males a nd in all groups of exposed females. Normocytic and normochromic erythr ocytes were demonstrated by the lack of change in the mean cell volumes and mean cell haemoglobin concentr ations, respectively. Other haematological changes were increased numbers of polychromatoph ilic erythrocytes in the 500 ppm group and increased platelet counts in males of the 500 ppm group and females of the 250 and 500 ppm groups. Increases were found in absolute and relative liver weightsEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 170 of males of the 500 ppm group an d in relative liver weights of males of the 250 ppm group and females of the 500 ppm group. Haemosiderin deposition in Kupffer cells was the only change observed in livers of the 500 ppm group males and the 250 and 500 ppm group females. Haemosiderin pigmentation was also increased in renal tubul e cells of both males and females of the 500 ppm group. Extrame dullary haematopoietic cell proliferation (primarily erythroid) and haemosiderin pigmen tation were present in males exposed to 125 ppm or greater and females exposed to 250 or 500 ppm. In the forestomach, there were significant increases in females only in the in cidence of inflammation in the 250 and 500 ppm groups and epithelial hyperplasi a in the 125 ppm and greater gr oups. Effects observed in the males and females of the 500 ppm group that were either killed or died included forestomach inflammation, necrosis and ulceration, suppura tive inflammation of the peritoneum and mediastinum, atrophy of the spleen, thymus a nd lymph nodes, renal tubule degeneration and, in males, testicular degeneration and epid idymidal necrosis (NTP, 2000). No NOAEC for female mice could be derived for non-neoplasti c effects in this study, the LOAEC for female mice being 31 ppm, the lowest dose tested, base d on significant haematological effects. The NOAEC for male mice was 62.5 ppm, based on haematological effects at 125 ppm.
EGBE was administered to groups of 50 male and 50 female B6C3F 1 mice by whole-body exposure to 0, 62.5, 125 or 250 ppm (0 – 150 – 302 – 604 mg/m 3 ) EGBE vapour (lot no. QP921215-26D2, Dow Chemical, Plaquemine, LA, USA, purity > 99 %) for 6 hr per day, 5 days per week for 104 weeks. For haematological an d bone marrow analyses, additional groups of 30 male and 30 female B6C3F 1 mice were exposed to 0, 62. 5, 125 or 250 ppm for evaluation at 3, 6 and 12 months (NTP, 2000).
Mice were held in the testing laboratory for 18 days and were 7 w eeks old when the study began. They were housed indivi dually, water was available ad libitum and feed was available ad libitum except during the exposure periods. All mice were observed twice daily throughout the study. Body weights were recorded at the beginning of the study, after which they were recorded monthly from week 5 until week 93 and then every two weeks from week 94 until the end of the experiment. Clinical observatio ns also were recorded monthly from week 5 until week 93 and then every two weeks from week 94 until the end of the experiment. Haematological examinations were performed on an Ortho ELT-8/ds 9000 analyser at 3- and 6-months and on a Roche COBAS Helios analyser at 12-months. Differential leukocyte and nucleated erythrocyte counts were made by mi croscopic examination of blood films. Bone marrow cellularity was measured using a Coulter Model Z H counter, while cytological evaluations of bone marrow cell morphology a nd myeloid/erythroid ratios were made microscopically.
Autopsies and microscopic examinations were made on all of the main study mice that had not been used for blood and bone marrow samp ling. Organ weights were not measured. At autopsy, all organs and tissues we re examined for grossly visibl e lesions and all major tissues were fixed and processed for histological examination. A quality assessment pathologist evaluated the slides from all tumours and po tential target organs, which included bone marrow, forestomach, kidney, liver, lung, nose a nd spleen of males and females and the preputial gland, prostate, skin (p repuce), testis and urinary bla dder of males. The slides and the preliminary quality assessment report were then submitted to a National Toxicology Program Working Group of pathologists.
Survival of male mice exposed to 125 or 250 ppm was significantly less than that of the chamber control group, whereas survival in th e lower dose group was similar to the chamber control group. The mean numbers of survivors at the end of the experime nt in each of the 0, 62.5, 125 and 250 ppm groups were 39, 39, 27 and 26 males and 29, 31, 33 and 36 females,EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 171 respectively. No clinical signs were attributed to exposur e to EGBE. Body weights of exposed male mice were generally less than those of th e chamber control during the last 25 weeks of the experiment. Body weights of female rats of the 250 ppm group were generally lower than those of the chamber control from week 30 until the end of the experiment, the difference being about 20 % for much of that time. Body weights of the 62.5 and 125 ppm group females were generally lower than the chamber controls from about w eek 60 until the end of the experiment.
Neoplastic effects observed in this e xperiments are described in section 4.1.2.8.1.3.
Haematological examination showed that inhala tion of EGBE resulted in the development of a persistent and exposure-related normocytic and normochromic, responsive anaemia, as indicated by decreased haematocrit values, haemoglobin concentrati ons and erythrocyte counts; it lacked, in general, changes in mean cell volumes and mean cell haemoglobin concentrations. These changes occurred at 3, 6 and 12 months in 125 and 250 ppm group male and female mice. Some anaemia also occurred at 6 months in the 62.5 ppm group females and there was a minimal increase in cell volume, suggesting a macrocytosis, in females of the 250 ppm group at 12 months. Increas es in circulating reticulocyte counts that were observed in 125 and 250 ppm male and fe male mice at 3 and 6 months and 250 ppm female mice at 12 months, are consistent with an erythropoietic response to the anaemia. In bone marrow there was no change in cell counts and no decrease in the myeloid/erythroid ratio, unlike the observations reported for rats (above). Additionally, thrombocytosis developed, as shown by increased platelet counts in the 250, 125 and 62.5 ppm exposed groups of males and females at 12 months, in the 250 ppm group males at 6 months and females at 6 and 3 months and in the 125 ppm group females at 6 months.
Incidences of haemosiderin pigmentation in Kupffer cells of the liver was significantly increased in males of the 125 and 250 ppm gr oups and females of all groups exposed to EGBE in a dose-dependent manner (see Table 33 in 4.1.2.8.1.3).
Haematopoietic cell prol iferation in the spleen was increased in male s exposed to 125 and 250 ppm and females exposed to 250 ppm, but it was not accompanied by any change in myeloid/erythroid cell ratio. In cidences of haemosiderin pigmentation in the spleen were significantly increased in all exposed groups of males and in the 125 and 250 ppm groups of females. These observations were attributed to the primary haemolytic effect of EGBE that was followed by regenerative hyperplasia of the haematopoietic tissue. Increases in the incidence of hyperplasia were also observed in the bone marrow of males exposed to 125 and 250 ppm.

In the nose there were increases in the incidences of hyaline degeneration in all groups of female mice of the olfactory epithelium (c hamber control, 6/50; 62.5 ppm, 14/50; 125 ppm, 11/49; 250 ppm, 12/50) and in the respiratory ep ithelium (chamber control, 17/50; 62.5 ppm, 35/50; 125 ppm, 26/49; 250 ppm, 23 /50). The severity of the lesion was minimal and did not change with increasing exposure concen tration. There was no clear dose response relationship. In males, the incidence was similar to the chamber controls.
Non-neoplastic lesions of the fo restomach consisted of ulcer (full thickness defect of the forestomach epithelium), particularly in fe males, epithelial hyperp lasia (an increased thickness of the stratified squamous epithe lium sometimes accompanied by an increased thickness of the keratinised layer) that was usually focal and, particularly in females, frequently associated with ul ceration ( see Table 5 in 4.1.2.8.1.3).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 172 There were a number of inflammatory changes in the urogenital system (NTP, 2000). These are shown in detail in the table below. Effects were only seen in male mice. In the kidney, incidences of glomeruloscler osis and hydronephrosis were si gnificantly increased at 125ppm (but not at 250ppm) whilst the incidence of chronic inflammati on was increased significantly at 250ppm. The incidences of inflammation of the preputial and prostate glands in males exposed to 250ppm were significan tly increased as were incide nces of inflammation of the bladder. The incidence of ulcer of the tran sitional epithelium of th e urinary bladder was significantly increased at 125ppm but not at 25 0ppm. The NTP authors concluded that these effects were indicative of an ascending urinary tract infecti on described as mouse urologic syndrome. The occurrence of this syndrome is pa rticularly common in i nhalation studies with mice, in which they are housed indivi dually in wire mesh cages (Everitt et al ., 1988). It is likely that the condition was exacerbated by the irritative effects of EG BE or its metabolites but is not primarily mediated by the substance.

Table 4.71 bis: Incidence of non-neoplastic lesions in the urogenital system of male mice following 2-years exposure to EGBE Control 62.5ppm 125ppm 250ppm Kidney a 50 50 47 50 Glomerulosclerosis 4 (1.3) 4 (1.3) 11* (1.3) 9 (1.4) Hydronephrosis 1 (2.0) 0 6* (1.8) 5 (2.2) Inflammation 0 1 (2.0) 2 (2.5) 4* (3.) Preputial gland a 49 49 49 49 Inflammation 2 (1.0) 7 (2.3) 6 (2.3) 8* (2.1) Prostate gland a 49 49 48 45 Inflammation 0 2 (2.0) 3 (2.3) 4* (1.8) Skin, prepuce a 50 50 49 50 Inflammation 2 (3.5) 3 (3.7) 13** (3.7) 8* (3.9) Ulcer 0 3 (2.7) 11** (2.8) 8* (2.5) Urinary bladder a 50 49 5046 45 Inflammation 0 2 (3.5) 5* (3.4) 4* (3.8) Transitional epithelium, ulcer 0 1 (3.0) 4* (2.8) 2 (3.0) Notes: Figures in brackets are severity of le sion. 1=minimal, 2=mild, 3=moderate, 4=marked

a =number of animals examined. *=significant p<0.05. **=significant p<0.01

No NOAEC could be derived for non-neoplastic effects in female mice in this study, the LOAEC being 62.5 ppm, the lowest dose test ed, based on marginal, but significant haematological effects. The NOAEC for ma le mice was 62.5 ppm, based on body weight gain decrement, marginal, significant haemat otoxic effects and inflammation of the kidney, urinary bladder and prepuce at 125 ppm.

Guinea-pig
EGBE (purity unknown) was administered by w hole body exposure to guinea pigs 7 hr/day (Mellon Institute of Industrial Research, 1955). Groups of 10 males were exposed to doses of 0,375 and 500 ppm for 30 days. In addition 6 male s and 6 females were added to the above groups for the study of sex differences in susc eptibility. Bw was recorded weekly. Liver and kidney weight were measured after euthan asia. Liver, lung, kidney were submitted to histological examination.
In the 500 ppm group, 2 male guinea pigs died within 12 exposure and one animal died after 7 exposures to 375 ppm. The mean body weight of both groups of animals fell slightly by the third exposure but only the weights of the gu inea pigs exposed to the lower level wereEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 173 significantly below the controls after 30 exposures. A signifi cant elevation in the kidney weights of both groups was also observed. No haematuria was observe d and only the 3 guinea pigs that which died during exposure showed any major tissue damage (lung haemorrhage and congestion).

Dog
Groups of 2 dogs were exposed to 415 ppm EGBE (42 % saturation) (purity unknown) 7 hours daily, five days a week for 12 weeks (Werner et al. , 1943c). Erythrocyte, reticulocyte, leucocyte and differential count s and haemoglobin estimations we re made routinely in the morning and in the afternoon. These determinati ons were made twice during the pre-exposure period, 3 or 4 times during the post-exposure period and 8 times during the 12 weeks of exposure. Sedimentation rates, haematocrit dete rminations and fragility tests were made only during the latter part of the experiment on af ternoon samples of blood. Urine was analysed for albumin, sugar and acetone. Urinary sediments were examined microscopically and average daily urine volumes were determined. Estimati on of urea were made on blood obtained in late afternoon immediately before feeding. After sacr ifice, histological examinations of the following organs were performed: lungs, liver , kidney, spleen, heart, urinary bladder, pancreas and large and small intestines. Additional examinations of the spleen were performed using a different trea tment (acidulated ferrocyanide) in order to demonstrate the presence or absence of iron-bearing pigment.
Dogs exposed to EGBE lost 6 and 9 % of thei r body weights during the study. Signs of central depression and slight increased of eyes and no se secretion were the only clinical effects detected. Signs of haemolytic anaemia (signifi cant decreases in haematocrit and haemoglobin) were seen in treated animals.
EGBE (purity unknown) was administered by w hole body exposure to dogs 7 hr/day (Mellon Institute of Industrial Res earch, 1955). Dogs were expos ed to 0, 100, 200, 385 or 617 ppm EGBE (0, 483, 966, 1860 or 2980 mg/m 3 ). For the 617 ppm dose, one black mongrel female was treated for 2 days. For the 385 ppm dose, one male and one female Basenji were exposed 7 hours a day for 28 and 8 days respectively (hae matological tests were performed daily but biochemistry only in male and after 26 a nd 27 exposure). For the 200 ppm group, one male and one female Basenji were exposed 7 hour s a day for 31 days. For the 100 ppm group, one male and one female wire-haired terrier we re exposed 7 hr/day for 91 days, usual blood chemistry were performed before the first expos ure and after the last exposure. Haematocrit values and icterus indices were observed in add to the regular haematological tests were performed weekly. Bw was record ed weekly. Erythrocyte count, leucocyte count, Hb level, erythrocyte fragility and haematocrit value we re measured immediatel y after exposure. For biochemistry, bromosulfalein retention time, sedimentation rate, icterus index, blood urea nitrogen and plasma fibrinogen levels were reco rded. Erythrocyte permeability test was made with the method of Bootman and Moses only for the 200 ppm group. Coloured photographs of the visceral organs as well as any stri king abnormality were performed. Histological examinations of adrenals appendix, gall bladder, gonads, heart, intestine, kidneys, liver, lung, pancreas, spleen, stomach, thyr oid, urinary bladder, uterus and tissue found abnormal at autopsy were realised.
For the 617 ppm group, the only dog exposed died after 2 days of exposure. Vomiting and weakness were seen during exposure. Autopsy re vealed congestion of the kidneys and lungs. For the 385 ppm group, male and female died after 28 d and 8 d respectively. Similar symptoms were seen in both male and female: loss of weight, increase in erythrocyte fragility,EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 174 weakness, apathy, anorexia and increase leucocyt e count. The erythrocyte fragility of male reached a maximum in 7 days and decrease pr ogressively through 27 days. Analysis of urine after the 25 th exposure indicated the presence of 55 mg of BAA. The only abnormality found in biochemistry was an elevated plasma fibr inogen concentration. At autopsy congestion of the lungs and of liver were obser ved in both animals. Congestion of kidneys were seen female only. For the 200 ppm group, slight evidence of toxi city were seen after 31 days of exposure. Erythrocyte fragility increased slightly and the erythrocyte fragility was higher than controls in both animals. Erythrocyte count and HB decrease slightly thr ough exposure. BAA was present in urine after the 14 th exposure: 100 and 42.5 mg in ma le and female respectively. For the 100 ppm group, only slight effects on haemat ocrit were seen. BAA in urine reached 100 and 94 mg in male and female respectively after 81 exposures.

Monkey
EGBE (purity unknown) was administered by whole body exposure to monkeys 7 hr/day (Mellon Institute of Industrial Research, 1955). Two Rhesus monkeys (1 male and 1 female) were treated with 100 ppm (483 mg/m 3 ) EGBE for 90 days (group 1). Another group of 2 Rhesus monkeys, were treated with 100 ppm, af ter 10 days of treatment no effects were seen and the dose was increased to 200 ppm (gr oup 2). No control group was used. Bw was recorded weekly. Erythrocyte count, leucocyt e count, HB level, erythrocyte fragility and haematocrit value were measured immediately af ter exposure, bromosulfalein retention time, sedimentation rate, icterus index, blood urea nitr ogen and plasma fibrinogen levels were also recorded. Coloured photographs of the visceral organs as well as any striking abnormality were noted. Histological examinations of adre nals appendix, gall bladder, gonads, heart, intestine, kidneys, liver , lung, pancreas, spleen, stomach, thyroi d, urinary bladder, uterus and tissue found abnormal at autopsy were performed.
In group 2, of 2 monkeys, 1 died of causes unrelat ed to treatment. The erythrocyte fragility values which were unchanged after 10 days exposure to 100 ppm rose after 4 days of treatment with 200 ppm. Erythrocyte fragility values were increased at the beginning of exposures. Values returned to normal until th e end of the study. A difference was seen between male and female: females were more sensitive than males. For group 1, erythrocyte fragility values rose to 0.56 – 0.48 (0.46 – 0.40 for pre-exposure values) several times after the 18 th exposure. A greater rise (0.62 – 0.54) was seen for the female after 7 exposures. These high values of the female returned to normal by the end of the 90-exposure period.

Summary inhalation route:
Many studies are available on rats , and mice. A few short studies using dogs, guinea-pigs and non human primates have also been conducted.
In rats and mice, common toxic ity signs together with effect s similar to those observed in acute administration. The main effect was haem olysis, which was consistently observed and sometimes associated with secondary hepatic e ffects (Kupffer cells pigmentation and absolute and relative liver weight increa ses). Other effects were decrea ses of body weight gain, hyaline degeneration of the olfactive epithelium, eff ects on the forestomach and effects on the WBC sub-populations (T lymphocyte). In these studies , a NOAEC of 25 ppm in rats and a LOAEC of 31 ppm in mice and rats can be establishe d (based on haemolysis, as the only significantEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 175 primary effect). The LOAEL of 31 ppm (coming from a six month satellite group in the NTP, 2000 104-week study) is taken into acc ount for the risk characterisation.

Table 4.72 : Summary of the studies on animals performed by inhalation route Study NOAEC (ppm) Effects Reference

Rats
5 weeks. Doses 0, 135, 320 ppm. 135 (for haematological effects) Poorly reported Werner et al.
, 1943b

Variable Number of exposures . Doses 0, 250, 500
ppm. - Poorly reported Mellon Institute of Industrial Research, 1952

15 exposures to 0, 20, 50 and 100
ppm. 4 exposures to 250
ppm. 20 ppm (haematological effects). Haematological effects Gage, 1970

4 months, 0, 17 ppm , 6 hours a day three days a week or 3 hours a day six days a week. - Poorly reported Lomonova and Klimova, 1977

9 days, 6 hours a day. Doses: 0, 20, 86, 245 ppm 20 ppm (haemotoxicity) Haematological effects and effects on liver and body weight gain. Bushy Run Research Center, 1981a

13 weeks. Doses: 0, 5, 25, 75 ppm 25 ppm (haematological effects only) Haematological effects only Bushy Run Research Center, 1981b

13 weeks. Doses: 0, 31, 62.5, 125, 250, 500 ppm LOAEC of 31 ppm Haematological effects.
Study performed to describe the vascular and bone lesion observed in moribund female. Nyska et al. , 1999a
Long et al. , 2000
NTP, 2000

104 weeks. Doses: 0, 31, 62.5, None identified for haematological effects; 31.2 ppm may be Haematological effects. Effects on liver (Kupffer cell NTP, 2000EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 176 125ppm considered as an LOAEC. pigmentation).

Mice
Doses: 0, 100, 200, 400 ppm. Duration: 30, 60 or 90 exposures < 100 ppm Haematological effects at all doses Mellon Institute of Industrial Research, 1955

4 months, 0, 17 ppm, 6 hours a day three days a week or 3 hours a day six days a week. Poorly reported Lomonova and Klimova, 1977

14 weeks. Doses: 0, 31, 62.5, 125, 250, 500 ppm None identified for haematological effects;
LOAEC of 31 ppm Haematological effects. Effect on body weight gain. Irritant effects on the forestomach. NTP, 2000

104 weeks. Doses 0, 62.5, 125, 250ppm none identified
LOAEC of 62.5 ppm Haematological effects. Effects on liver (Kupffer cell pigmentation). NTP, 2000

Guinea pigs
30 days
Dose: 0, 375 and 500 ppm none identified Mortality, effects on body weight and on kidneys. No effects on blood parameters. Mellon Institute of Industrial Research, 1955

Dogs

12 weeks
Dose: 415 ppm < 415 CNS depression, haemolytic anaemia. Werner et al.
, 1943c

Duration between 2 and 90 days.
Dose: 0, 100, 200, 385 and 617 - CNS depression, haemolytic anaemia. Effects on lungs, kidneys and liver. Mellon Institute of Industrial Research, 1955

Monkeys
90 days,
Dose: 0, 100, 200
ppm. - Haematological effects (haemolysis) Mellon Institute of Industrial Research, 1955EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 177 Dermal

Mice
EGBE was administered to BALB/c mice (5-6 w eeks old) to assess the effects on the immune system (Singh et al. , 2001). Doses of 0, 100, 500, 1000 and 1500 mg/kg/day (about 25 μ l in a 4:1 mixture of acetone and olive oil vehicle) were applied during 4 days to the backs of the mice. The numbers of animals per dose level is not indicated in the study report. Except for one group of mice tested for IgM plaque-forming cell (PFC) response, animals were killed 24 hours after the last dose. Body weights were r ecorded. Thymus and spleen were removed and weighed prior to preparation of cell suspension s. For evaluation of the PFC response, mice were immunised by Intraperitoneal injection w ith sheep erythrocytes on the third day of dosing and killed 72 hours after the final dose. Cell suspensions of lymphoid organ tissue were prepared for spleen and thymus, cellularity and viability were assessed. Lymphoproliferative assays (Non-specific m itogenic stimulation of either B or T lymphocytes), mixed lymphocyte response (MLR) a ssay, activity of natural killers (NK) cells, cytotoxic T lymphocyte (CTL) activity and plaq ue-forming cell response to sheep red blood cells were assessed using standard procedures.
No effects on body weight gain or thymus weight were seen at any dos e tested. At the 1500 mg/kg dose, an increase in the relative spleen we ight and an increase of splenic cellularity of 29 % were seen. There was no changes in th e lymphoproliferation assay in the B cells population whereas the T lymphocyte sub-population exhibited a decrease of the proliferative responses significant only at dos es of 500 and 1000 mg/k g (a decrease was also seen for the 1500 mg/kg dose but not statistically significant, this weaken the relevance of this finding). The same results were seen in the MLR assay (decreased responses at the high doses, significant only at 500 and 1000 mg/kg). NK, CTL activity and T-dependent PFC response was unchanged after treatment. The lack of a dose-related response in these observations throws doubt on their significance for EG BE toxicology. The NOAE L for immunological effects was 1000 mg/kg bw, based on spleen weight reduction at 1500 mg/kg bw per day.

Rabbits
EGBE was administered occlusively via dermal route to 4 groups of New Zealand (NZ) white rabbits (5 animals/sex) at concentrations of 100 - 50- 25 and 5 % (1 ml/kg of mixture corresponding to 900 – 450 – 225 and 45 mg/kg bw) during 6 hr/day for 9 days (dosed 5 days, no dosed 2 days and dosed 4 days) (Bushy R un Research Center, 1980c). Control group was treated with vehicle only. Dermal irritation scor e were determined afte r each test exposure. Body weight of animals was measured regularly : all males were submitted every day (and also after exposure period) to an ophthalmologic examination (d iscoloration, discharge, gross abnormality, reddening). Urine samples were collected prior to exposure on the 2 nd , 5 th and 9 th day of exposure and on the day of sacrifice. Th e following parameters were measured: colour, pH, protein, glucose, ketones, urobilinog en, bilirubin and blood. Blood samples were collected before dosing the last day of dosing and the day of sacrifice and assessed of RBC count, haematocrit, MCV, MCH, MCHC, WBC count. Reticulocytes and WBC differential smears were prepared for each animal but not ev aluated. Fourteen days after the last dosing, animals were euthanised. Tissues were coll ected and fixed, only kidneys were examinedEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 178 histopathologically. Liver and kidne ys of all animals were weighted, in males testes were also weighted.
In the preliminary studies: LD50 was calcula ted for males: 0.707 ml/kg and for females: 0.630 ml/kg. In the main study, no male rabbits died during the study. One female rabbit was found moribund on day 5, the condition was apparently unrelated to treatment. No body changes were seen in male whereas female rabbits treated with undiluted EGBE exhibited a decrease in bw gain throughout the direction of the study. A ll ophthalmologic examinations were negative for the male rabbits. The fema les were not examined. Severe necrosis and moderate oedema were observed in all an imals dosed with undiluted EGBE by the 6 th day of treatment. The 50 % dilution produced necrosis of the skin of 1 out of 5 males as compared to 4 out of 5 females. Erythema preceded necrosis in every instance. Slight oedema was also observed in those rabbits exhibiting necrosis. Dosage related erythema was observed at the lower dosage levels. Very slight reddening was seen in the male rabbits in the water control group. No statistical comparisons were performed on the skin irr itation readings. On day 2 of treatment there was haemoglobin in the urine of two of four male rabb its dosed with undiluted EGBE. There also was a slight increase in urin ary protein in four males dosed with undiluted material on day 2 of treatment. Both of these conditions returned to normal by treatment day 5. On day 2 of treatment there was blood in the urine of three of five female rabbits dosed with undiluted EGBE. There was a corresponding increase of urin ary protein in two of these animals. On day 5 of treatment, four of the five females dosed with undiluted material had blood in their urine, as well as one dosed with the 50 % diluti on. By day 9 of treatment only one dosed with undiluted sample s till had blood in the urine; how ever, at this time all five females dosed with the 50 % dilution had blood in their urine. There was no evidence of haemoglubinuria at any dosage level at the time of sacrifice (14 days post exposure). No abnormalities were seen in any of the other meas ures of urinary effect assessed and there was no occult found in the urine of rabbits dosed with the 25 % or the 5 % dilutions. Statistically significant depressions of m ean erythrocyte count, haemogl obin and mean corpuscular haemoglobin concentration were present on trea tment day 9 in females dosed with undiluted substance and the mean corpuscular haemogl obin was significantly increased. Although not statistically significant, the contemporaneous decreased haematocrit and increased mean corpuscular volume are consistent changes. The presence of larger red blood cells with more haemoglobin is indicative of response to red cell loss by early release of less mature cells into the circulation. These effects were no longer seen after the 14-day observation period. Females in the three lower dosage groups ha d no indication of biologically significant haematologic effects. The increased mean red bl ood cell counts on day 9 of treatment in the 5 % dilution group and on day 14 af ter exposure in the 25 % diluti on group are considered to be statistical artifacts. No changes were noted in any organ weight measured in all animals. Gross findings included some thickening of the skin in male rabbits given undiluted substance and possibly dose related patchy colour change of the kidneys of the three females.
For this study, the NOAEL of 450 mg/kg can be taken into account mainly based on gross findings observed in animals dosed with pure su bstance. Haematological effects seen during the study were transient and recovery was co mplete after the 14-day observation period. .
In a GLP study, 4 groups of 10 males and 10 female s NZ white rabbits were treated dermally (occlusively) with EGBE (high purity) at concentration of 0 % - 2.8 % - 14.3 % and 42.8 % in distilled water (corresponding to dose levels of 0 – 10 - 50 - 150 mg/kg) for a 6 hr period/day 5 d/w for 13 weeks (Wil Research laboratories in c., 1983). Animals were observed for clinical symptoms twice daily. Prior to treatment, the si te of application of each animal were graded using the Draize skin reaction scoring system . Scoring was performed daily for the first 3EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 179 weeks of the study, the once weekly thereafter. Bw and food consumption were measured weekly. Blood was collected before the study, duri ng week 4 and just before termination. The following parameters were determined:
Haematology: white blood cell count, mean corpuscular haemog lobin concentration, erythrocyte Count, mean corpuscular haemogl obin, haemoglobin, differential white blood cell count, haematocrit, erythrocyte fragility (t ermination only), mean corpuscular volume.
Clinical chemistry: glucose, di rect bilirubin, total bilirubin, albumin, blood urea nitrogen, total bilirubin, alkaline phosphatase.
All rabbits were subjected to a gross necr opsy examination. The following organs were weighted at sacrifice for at least 6 animals/ sex/level: heart, thymus, testes, spleen, liver, ovaries and kidneys. The following tissues were also examined briefly and conserved: adrenals, aorta, bone (ste rnebrae), brain (3 transverse sect ions), epididymis (both, tail), oesophagus, eyes, gall bladder, heart, intestines (caecum, duodenum, jejunum, ileum and colon), kidneys, liver (left & median lateral lobes), lung (lef t), lymph node (mesenteric & thoracic), mammary gland, ovaries , pancreas, parathyroids, pituit ary, prostate, sciatic nerve, seminal vesicles, skeletal muscle, skin (test site & adjacent normal skin), spleen, stomach (pylorus and fundus), submandibular salivary gl and, testes, thyroids, thymus, tongue, trachea, urinary bladder, uterus (both hor ns), vagina, All gross lesions.
Red coloured faeces and red liquid material on cage paper (probably blood) were seen in some treated animals (each group). Some irritant effects were sometimes seen in animals of both control and treated groups. There was no incr ease in the severity of these effects in treated animals versus controls. There were no tr eatment related deaths. No significant effects were seen on body weight gain or food consump tion in any group treated compared to control animals. Sporadic changes in haematology parameters and RBC fragility values were noted but values were within normal ranges for this laboratory and probably not related to the test material. Sporadic changes were also noted in WBC counts but were not related to test material administration. No changes in biochemistry were related to treatment. No test material related effects were noted on absolu te or relative orga n weights or final body weights.
The NOAEL for this study was equal to 150 mg/ kg bw, the highest tested dose. The actual NOEL may be of a higher value.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 180 Summary dermal dose:

Table 4.73: Summary of the studies performed by dermal route Study (rabbits) NOAEL (mg/kg bw/d) Effects Reference

Mice
4 days. Doses: 0, 100 – 500 – 1000 – 1500 mg/kg bw/day NOAEL=1000 Effects on splenic cellularity at 1500. Singh et al. , 2001.

Rabbits
9 days. Doses: 45 – 225 – 450 - 900 mg/kg bw/d NOAEL = 450 Transient haematological effects Bushy Run Research Center, 1980c

13 weeks. Doses: 10 – 50 – 150 mg/kg bw/d NOAEL > 150 No effects Wil Research laboratories inc., 1983

Two studies are available on rabb its to assess the toxicity of repeated doses of EGBE administered dermally. In only one study, signs of toxicity were recorded and were limited to transient signs of haemolysis. This study le d to a NOAEL of 450 mg/kg bw/d due to haematological effects seen at 900 mg/kg bw/d.
Given that this study was performed only dur ing 9 days, the NOAEL of the second study, which was performed during 13 weeks, could be mo re reliable for the risk characterisation. This NOAEL was 150 mg/kg bw/d.
The mouse study design for the assessment of EGBE effects on the immune system, give a NOAEL of 1000 mg/kg.

Oral

Rat
Five groups of Sherman rats (5/sex/group) were fed with diet containing 0, 0.03, 0.125, 0.5 and 2 % Butyl cellosolve (EGBE purity unknown) corresponding to doses of 0, 18, 76, 310 and 1540 mg/kg/d (Mellon Institute of I ndustrial Research, 1952) for 90 days.

No deaths were seen attributable to the dir ect action of EGBE. Appetit e was not affected and no pertinent micropathology was di scovered. Tests for blood in pooled urine sample from the 2 %, 0.5 % and controls groups after 3 and 6 days were negative. The mean weight gain was lower than controls for the 2 % group. Relative kidney and liver weight were increased at 2 % dose and relative liver weight only was increased at 0.5 % dose.

The NOAEL can be considered to be 0.125 % (76 mg/kg/d) but considering that the test conditions were poorly repor ted (no control of the administrate d substance, evaporation ?) this NOAEL will not be taken into accoun t for the risk characterisation.
Groups of young DW albino rats (10 males and 10 females/gr oup) were given food with EGBE (purity unknown) at doses of 1.25 – 0.25 – 0.05 or 0.01 % (cor responding to 919, 188, 38 and 7 mg/kg in males and 976, 222, 41 and 9 mg/kg in females respectively) during 3EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 181 months (Mellon Institute of Industrial Re search, 1963). A control group was given food without EGBE Animals were weighted 4 time s during the first week and once a week thereafter. After euthanasia liver and kidneys we re weighted. Organs were examined for signs of pathology, the urinary bladder was examined for concretions. Histology was performed on representative tissues.
One animal died at day 73 after the beginning of the study in the high dose group. Food consumption was decreased in both males and females at 1.25 %. In males a significant decrease of food consumption was also observed at 0.25 % dose. A significant decrease of body weight gain was seen in animals dosed with 1.25 % EGBE. It was also seen in males at doses of 0.25 %. A small decrease was seen on the 2 first days of dosing only with the 0.05 % dose. The mean liver and kidney we ight of males and females were definitely increased at the 1.25 % dose. At both 1.25 % and 0.25 % testes were atrophied. The NOAEL for this study can be considered to be 0.05 %. The purity of th e EGBE used in this study is not given, nor does there appear to be analytical verificati on of EGBE doses, homogeneity and stability. This makes this study difficult to interpret give n for example that testicular atrophy is not reported in other oral, repeated-dose studies ev en at higher doses of EGBE greater than 0.25% (e.g., 14-week drinking water study in rats a nd mice from NTP, 1993; continuous breeding study reported in Morrissey et al. , 1989). Given the availability of numerous other more recent and robust studies, this one cannot be considered reliable to derive the critical NOAEL.
Groups of 10 Albino rats (CR, COBS, CD-BR) were treated with various glycol ethers (including EGBE - purity > 99.5 %) via oral gava ge, 5 days per week for 6 weeks (Eastman Kodak, 1982). Doses administrated were: 0, 222, 443 and 885 mg/kg. Body weight were recorded twice a week during the first week of exposure and every week after until termination of the study. Animals were observe d daily for clinical signs. Blood samples were taken before euthanasia for the determina tion of haematology indices and biochemistry: SGOT, SGPT, alcaline phosphatase (ALP), LDH, urea nitrogen, cr eatinine and glucose. After termination, histological examinations were performed on the following organs: lung, heart, thymus, kidneys, liver, spleen, brain, salivary glands, stomach, intestinal tract, pancreas, oesophagus, adrenal glands, pituitary, thyroid, parathyroid, trachea, mesenteric lymph node (LN), testes and annexes, bone marrow, tongue, nasal cavities and eyes. Before fixation, liver, kidneys, heart, testes, brain and spleen were weighted.
2 out of 10 animals were found dead in the high dose. No mortality was seen in any other groups. A significant decrease in body weight gain was seen from D13 and after in animals treated with the high dose of EGBE. A signi ficant decrease of food consumption was also seen in this group but only from D3 to D20. In other groups there was a trend to a decrease in bw gain compabable to controls and a lowe r food consumption in the middle dose group (not significant). Clinical symptoms were limited to bloody urine in the mid and high dose groups. Only one of the ten rats given the low dose of EGBE had bloody urine. This persisted through the third week of treatment. Ot her clinical signs at the mi d and high dose were lethargy, unkempt hair coats, piloerecti on, rales, slight weakness and inactivity. All doses produced significant effects on RBC parame ters, showing a characteristic pattern of haemolytic anaemia. No effects were seen on WBC. For bi ochemistry: a significant increase of alkaline phosphatase (PAL) was seen from the middle dose. Moreover, in the high dose, a significant increase of SGPT and a decrease of glucose (G LU) were noted. These changes were slightly different compared to controls so their toxico logical significance is uncertain. Absolute and relative spleen weights were increased fo r the middle and high dose of EGBE. A small increase of the liver weight (only significant for relative weight) was seen for the high dose group. No effects were seen on testes weight (relative or absolute) at any dose group. ForEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 182 gross pathology, only enlarged, darkspleen were seen for high and intermediate dose. Histopathological lesions were hyperkeratosis and acanthosis in the stomach epithelium in all treated animals. Hepatomegaly was also seen in 4 out of 10 animals of the high dose group. Haemosiderin deposition was seen in some animals (7/10 and 6/10 in the high and mid dose group respectively). Thymus atrophy was se en in one animal of the high dose group. Congestion of the spleen was seen in all trea ted animals. Extramedulary haematopoiesis was reported for one animal in the high dose group and haemosiderin deposit in the majority of animals of the high and mid dose group. For this study, the NOAEL can be considered to be lower than 222 mg/kg (which is the Low Obse rvable Adverse Effect Level: LOAEL) based on effects seen on spleens and RBC parameters.
Toxic effects on the immune system of SD ra ts after 21 days of exposure to EGBE and EGME were assessed. Groups of 6 rats of each sex were exposed to EGBE (purity 97 %) in drinking water at concentra tion of0, 2 000 and 6 000 ppm corre sponding to about 0, 180 and 500 mg/kg respectively for males and 1600 – 4800 ppm for females corresponding to 200 and 444 mg/kg respectively (Exon et al ., 1991). All rats were injected SC at the base of the tail with 1 mg aqueous KLH in a 0.2-ml volume 7 days after initiation of gl ycol ether treatment. A second injection of aqueous KLH was given at th e base of the tail on Day 13 to initiate an IgG antibody response. All animals were in jected into the right footpad with 100 μ l of heataggregated KLH on Day 20. The left footpad was injected with 100 μ l of sterile saline. At the time of termination serum samples were collec ted for analysis of antibody levels to KLM. Spleens were used as the cell source to assess natural killer (NK) cell cytotoxicity and interleukin 2 (IL2) and interferon (IFN) producti on. All assays were performed on each rat on experiment.
The 24 hr Delayed-Type Hypersensitivity r eaction (DTH) response was assessed on Day 21 by measuring the thickness of th e heat-aggregated KLH-injected footpad and substracting the thickness of the saline-injected footpad. The reaction was expressed as millimetre difference in swelling between the two footpads.
Serum collected by cardiac puncture at time of termination (Day 21) was analysed for antiKLH IgG antibodies by an indirect enzyme -linked immunosorbent assay (ELISA).
Natural killer cell cytotoxicity: suspension of sp leen cells were prepared. Splenic natural killer cell cytotoxicity was assessed by the 4-hr 51 Cr release from YAC-1 tumour target cells as previously described (Exon et al ., 1991). Specific 51 Cr release was calculated by: cpm experimental release - cpm spontaneous rel ease x 100 / cpm maximum release by 2 % SDS - cpm spontaneous release. Spontaneous release of 51 Cr by target cells was less than 10 % in all
assays. Interleukin 2 production by rat spleen cells was measured by the capacity to support the growth of the IL2-dependent CTLL-2 cell line in vitro . This assay does not distinguish between IL2 and IL4 production. A sample of spl een cells was centrifuged and the cells were resuspended in RPMI (supplemented with peni cillin, streptomycin, 5 % heat-inactivated foetal bovine serum). Samples were incubated 24 hr with concanavalin A and the cell-free supernatants were harvested and te sted for IL2 activity. Briefly, 4 x 10 3 IL2-dependent murine CTLL-2 cells (in RPMI, 5 % foetal bovine serum, 5 x 10 -5 M 2-mercapto-ethanol) were cultured in replicate 200- μ l volumes in flatbottomed 96-cell cultu re plates in the presence of a log-2 dilution series of putative IL2- containing supernatants. After 18 hr, 1 μ Ci[ 3 H]TdR was introduced to each well for 4 hr, after which the cells harvested onto glass fiber filters, and the radioactivity measured by scintillation countering.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 183 Interferon concentrations in supernatants of spleen cells cultured for 48 hr with 1.0 μ g/ml concavalin A were assessed by the capacity to block the cytopathic effects of vesicular stomatitis virus on rat RT fibroblasts.
For pathological examination, the thymus, spl een, liver and right kidney and testis were collected and weighted at necropsy. The ti ssues were examined by light microscopy.
Body weights were decreased in male rats treated with 6000 ppm EGBE and in females treated with 1600 or 4800 ppm. No effects were seen in thymus or in testes (macroscopically or histologically). Relative liver weights were increased in male rats treated with the low dose of EGBE. Relative kidney weight was decreased in female rats treated with the low dose of BE. Microscopic changes were not observed in sections of liver and kidney taken from the BE-exposed rats. Histopathologic examination of the spleen was not done since all of this tissue was used as a source of cells for th e immune function assays. Immune Function assessment: Natural killer cytotoxic responses were enhanced in male and female rats treated with the low dose of BE. No effect on anti body production was observed in the BE-treated rats. No significant effects of glycol ether tr eatment were observed on DTH reactions of these rats. The cellular infiltrate in the KLH-injected area of the footpad consisted mainly of mononuclear phagocytes, typical of a Type IV hypersensitivity reaction. The authors concluded that the immune system is not a sensitive target for EGBE.
EGBE (lot no. BT00504LP, Aldrich Chemical Co., USA, purity ≈ 99 %) was administered in drinking water at concentrations of 0, 750, 1500, 3000, 4500 or 6000 pp m to groups of 10 male and 10 female F344/N rats for 13 weeks. These concentrations provided target dose levels of 0, 100, 150, 250, 400 or 650 mg/ kg bw per day. Estimates of compound consumption based on water consumption by rats were 69, 129, 281, 367 and 452 mg/kg/day for males and 82, 151, 304, 363 and 470 mg/kg/day fo r females. Supplemental groups of 10 rats/sex/group/time point were included for haematology and clinical chemistry observations at weeks 1 and 3 (NTP, 1993).
Rats were held in the testing laboratory for 11 - 12 days and were 6 weeks old when the study began. They were housed 5/cage, water and feed were available ad libitum . All rats were observed twice daily throughout the study. Body weights were recorded at the beginning of the study and then weekly until the end of the e xperiment. Clinical observations also were recorded weekly throughout the experiment. Haematological examinations were performed using a Series 7000 cell counter and a Series 810 whole blood platelet analyser (Baker Instruments). Clinical chemistry variables were measured with a Cobra Fara analyser (Roche Diagnostics). Evaluations of vaginal cytol ogy and sperm morphology were done for the three highest doses tested. Results c oncerning these examination are reported in the reprotoxicity section.
Complete autopsies were made on all study rats . Microscopic examinations were made on all control group and treated rats.

No rats treated with EGBE died or were killed before the scheduled end of the study and there were no clinical signs of toxicity. Body weight gains were reduced in both male and female rats of the 4500 and 6000 ppm treatment groups. In males and females there were reductions in drinking water consumption in the higher do se groups, this being clearly concentration related in females, from a mean of 18.8 ml/day in the contro l group to 10.7 ml/day in the 6000 ppm exposure group. A markedly macrocytic and mildly hypochromic anaemia was observed at each time point and reticulocyte counts were moderately increased in weeks 1 and 13. For males there was a decrease in erythrocyte co unts at all time point s in the 3000 ppm andEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 184 greater groups, while in fema les the decrease occurred in the 1500 ppm and greater groups. A consistent thrombocytoapenia was observed at all time points in males and females of the 4500 and 6000 ppm groups; it also occurred in fe males of the 3000 ppm group at week 13. The most consistent blood chemistry observati on was an increase in alkaline phosphatase, particularly in the high dose gr oups. This observation is consistent with mild cholestasis. Thymus weights were significantly reduced in males of the 4500 ppm group and males and females of the 6000 ppm group. Other organ wei ght changes were found, but these appeared to be secondary to body weight gain reduction (spleen was not weighed). Histopathological lesions occurred in the liver, spleen and bone marrow and male and female rats. Hepatic changes included primarily centrilobular he patocellular degeneration and primarily centrilobular Kupffer cell pigmentation. These cha nges were present in the majority of dosed rats, but they were more prevalent in the 3000 ppm and greater dose grou ps and were slightly more severe in females. In addition, the cytoplas m of hepatocytes of rats at all dose levels was more eosinophilic and lacked the ampholytic-to-ba sophilic granularity typi cal of the controls. In the spleen there was an increase in haemat opoiesis and deposition of haemosiderin. In bone marrow there was an hyperplasia characterised by an increase of haematopoietic cells and decrease in marrow fat cells. All of these lesion s were present in the majority of dosed rats, but they were more prominent in the 3000 pp m and greater groups (NTP, 1993). There was no treatment effect on testis weights but there was a reduction in the size of the uterus in females at 4500 and 6000 ppm. Changes in uterine weight were considered by the authors to be secondary to the reduction in body we ight gain rather than a dire ct effect of EGBE. The only spermatozoal measurement that showed significa nt change relative to the control group was sperm concentration which was slightly decrease d in all groups of treated males; however, this effect was not dose-related. There were no significant differences from the control group in oestrous cycle length for treated female s, although females treated at 4500 and 6000 ppm spent more time in dioestrous than the other gr oups. This correlates with the smaller uterine size, which was attributed to a secondary c onsequence of reduced body weight gain. These effects and their no relevance for risk asse ssment are more developed in the section 4.1.2.9.1 “other studies”. No NOAEL was identified in this study, based on cytoplasmic alterations in hepatocytes of both male and female rats at 750 ppm, equal to 69 mg/kg bw per day and 82 mg/kg bw per day in males and females respectively.
EGBE (purity > 99 %) was admi nistered to female Fischer 344 rat (10 – 12 w old) via oral route (Nyska et al. , 1999b). Two groups of 8 animals were treated with EGBE at dose of 250 mg/kg or vehicle during three days. Animals were sacrificed 24 hours after the last treatment. Complete necropsy examination were performed on all rats, the following tissues and organs were examined microscopically: sternum, femur, head, vertebral column, tail, heart, lungs, liver, spleen, kidneys, eyes and brain. Both eyes were bisected mid-sa gitally and 12 sections were prepared (6 stained with HE and six stained with PAS).
Gross abnormalities were noted only in EGBE treated animals and included bluish tail discoloration, watery blood, discoloured liv er, dark kidneys and multiple Petechial haemorrhages on the sclera. Some ocular change s were observed in 5/8 treated rats. Retinal changes were bilateral and occas ionally located in proximity to capillaries. Lesions included multifocal haemorrhage within the retinal pi gment epithelium (RPE), the choriocapillaris between the RPE and the photoreceptors leading to retinal detachment, and the photoreceptors and inner and outer nuclear layer. The haem orrhage in the RPE were associated with degeneration, exfoliation and loss of these cells . The haemorrhages within the photoreceptors layers, sometimes associated with minimal polymorphonuclear cell infiltration, caused organizational disruption of the r ods and cones and, at time, result ed in their loss. In the inner and outer nuclear layers, the haemorrhages were associated with degeneration and loss of cellEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 185 resulting in thinning, disorganization and fusion of these layers In a single rat, a patent retinal vessel was still surrounded by necr otic cells of the outer and i nner nuclear layers, suggesting an ischemic-infarctive process. In another case, an area of necr osis was present in the outer retinal layers, probably relate d to occlusion of the regiona l blood vessel of the ciliary processes and in the limbus.
In the organs other than the eyes, a range of histopathological changes were observed. Occlusive trombi were noted in the submucosa of the most anterior section of the nasal cavity and the small calibre pulmonary ve ssels. Thrombosis was also noted in the dental pulp of the incisors in association with focal stromal a nd odontoblast coagulative necrosis. The femur and coccygeal vertebrae at intermedullary locations also exhibited thrombosis in association with necrosis of marrow cells, bone-lin ing cells, and both cort ical and trabecular osteocytes. In the femur, the changes were usually confined to th e diaphysis (medullary cav ity and inner third of the cortex), while in the coccygeal vertebrae, the mete physis and epiphysis were alsoinvolved. Usual changes seen with EGBE were also noted in other organs: multifocal nonzonal hepatocellular necrosis, splenic extrame dullary haematopoiesis, bone marrow erythroid hyperplasia and haemoglobinuric nephrosis.
These effects, seen in female rats only, seem to be closely related to haemolytic toxicity. As the rats are one of the most sensitive species to haemolytic effects of EGBE and humans are resistant to these effects such symptoms could occur but at very high do ses (hardly obtainable in normal use conditions). The design and objecti ves of this study make it unsuitable to derive a NOAEL for Repeated Dose Toxicity.

Mouse
EGBE (lot no. BT00504LP, Aldrich Chemical Co., USA, purity ≈ 99 %) was administered in drinking water at concentrations of 0, 750, 1500, 3000, 4500 or 6000 pp m to groups of 10 male and 10 female B6C3F 1 mice for 14 weeks. These concentrations provided target dose levels of 0, 100, 150, 250, 400 or 650 mg/kg bw per day (NTP, 1993). Estimates of compound consumption based on water consump tion by mice were 118, 223, 553, 676 and 694 mg/kg/day for males and 185, 370, 676, 861 and 1306 mg/kg/day for females.
Mice were held in the testing laboratory for 11 - 12 days and were 6 weeks old when the study began. They were housed individually, and water and feed were available ad libitum . All mice were observed twice daily throughout the st udy. Body weights were recorded at the beginning of the study and then weekly until the end of the experiment. Clinical observations also were recorded weekly throughout the expe riment. Complete autopsies were made on all study mice. Microscopic examinations were made on all control group and treated mice.

No mice treated with EGBE died or were kill ed before the scheduled end of the study and there were no significant clinical signs of toxicity. Body weight gains were slight ly reduced in both male and female mice of the 3000 and 6000 ppm treatment groups. No particular pattern was evident in drinking water consumption. Organ weight changes that were found were secondary to body weight gain reduction. No tr eatment related gross or microscopic lesions in male or female mice were found (NTP, 1993). The NOAEL of 223/370 mg/kg is not very reliable because no haematologi cal analysis were performed on animals during or after the study.
In a series of studies, the mechanism of EG BE (purity 99 %) accumu lation in forestomach was assessed (Poet et al ., 2003). Five experiment were conducted to examine various endpoints:EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 186 - target tissue histology / forestomach irritation

- tissue dosimetry and pharmacokinetics

- fur deposition

- salivary excretion

- retention in stomach content
For target tissue histology assessment, B6C3F1 adult mice (16/sex/dose) were treated with EGBE orally at doses of 400, 800 or 1200 mg/kg for 4 days (i n fact due to an excessive unexpected mortality at these dose levels, surviving animals after 2 days of dosing were administered half of their corresponding original target dose). Half the animals were targeted for forestomach toxicity evaluation, the rema inder were examined for liver effects. To investigate if parenteral admi nistration could also produce fo restomach lesions, extra groups of mice were administered saline solutions of EGBE by either SC or IP injection of 400 mg/kg for 4 days (6 animal s/route of administration).
Following oral administration, neat EGBE cause d dose-related forestom ach lesions in all animals. This lesions were mainly epithelia l hyperplasia and inflam mation. Minimal lesions similar to those produced in the lower dose oral gavage groups were obser ved in 1/6 of the IP dosed mice and 2/6 of the SC dosed mice.
Summary oral route:

Table 4.74: Summary of studies performed by oral route Study NOAEL (mg/kg bw/d) Effects Reference

Rats 90 days in food 0, 18, 76, 310 and 1540 mg/kg/d NOAEL = 76 mg/kg/d,

LOAEL = 310 mg/kg/d Poorly reported Mellon Institute of Industrial Research, 1952

90 days in food 1.25 – 0.25 – 0.05 or 0.01 %
919, 188, 38 and 7 mg/kg in males and 976, 222, 41 and 9 mg/kg in females

NOAEL = 0.05 %
(38 and 41 mg/kg in male and females respectively) Decrease in food consumption and body weight gain in males. Testes atrophy. Mellon Institute of Industrial Research, 1963EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 187 6 weeks gavage 0, 222, 443 and 885 mg/kg
LOAEL = 222 mg/kg Haematological effects at all doses and irritant effects on the stomach Eastman Kodak, 1982

21 days in drinking
water 0, 180 and 500 mg/kg for males and 200 and 444 mg/kg for females.

Not suitable for RA No remarkable effects on the immune system Exon et al. , 1991

13 weeks in drinking
water 69, 129, 281, 367 and 452 mg/kg/day for males and 82, 151, 304, 363 and 470 mg/kg/day for females

LOAEL of 69 and 82 mg/kg bw/day for males and females respectively. Slight decrease in body weight gain and haematological effects. Liver effects. NTP, 1993

3 days 250 mg/kg

Not suitable for RA Haematological effects, trombi formation. Nyska et al. , 1999b

Mice
4 days Not suitable for RA Irritation of the forestomach Poet et al ., 2003

14 weeks in drinking
water 118, 223, 553, 676 and 694 mg/kg/day for males and 185, 370, 676, 861 and 1306 mg/kg/day for females.

NOAEL = 223 and 370 mg/kg bw/d for males and females respectively

LOAEL = 553 and 676 mg/kg bw/d for males and females respectively Slight decrease in body weight gain NTP, 1993

Six studies on rats and two on mice are availa ble. Effects seen by oral route were body weight reduction, haemolysis, hepatic effects a nd local irritation effect s. Irritation to theEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 188 forestomach was seen after gavage dosing and, to a far lesser extent, after subcutaneous and intraperitoneal injection. This difference is most likely due to the higher local concentration after gavage dosing. Overall, a LOAEL of 69 and 82 mg/kg bw/d (in males and females respectively) can be fixe d in a three-month study.

Other routes

EGBE was administered IP to female SD rats 5 times a week during 2 weeks at doses of 55 mg/kg (Bernard et al. , 1989). Urine collection was carried out before, every 2 days during the test and immediately after the last injecti on. The urinary concentration of albumin and β 2 - microglobulin ( β 2 -m) was determined and also the activity of N-acetyl- β -glucosaminidase.
No significant effects were seen on albumin, β 2 -m or N-acetyl- β -glucosaminidase urinary excretion. In conclusion, no glomerular da mages were induced by EGBE treatment.

4.1.2.6.2 Studies in humans

Inhalation
Twenty one male paint formulators with an av erage age of 41.3 years (27 to 53) exposed to various chemicals (IPA, acetone, EA, benzene, toluene, xylene, white spirit) including EGBE, at exposure level of 0.09 time Threshold Limit Value of index solvent mixture for 20.2 years were studied with a battry of neurobehavioural performance te sts including di git span, digit symbol, Benton visual retention, finger tapp ing, grooved peg board and aiming test (Foo et al ., 1994). A group of 21 male workers unexposed served as controls. Personal exposure levels to EGBE ranged from 0.41 ppm to 3.13 with a mean of 1.26 ppm (N=4). The performance of the paint formulators was worse than the controls in neurobehavioural tests. Statistically significant results were observed in aiming test error score, digit span and grooved peg board. These results ar e not relevant for risk assessment due to the co exposure to other solvents.
Nine floorers occupationally exposed for 8 to 35 years to mixtures of organic solvents were subjected to analysis and ch aracterisation of their lymphoc yte sub-population (Denkhaus et al ., 1986). They were matched with 9 healthy volunt eers to have control values. Ages ranged from 25 to 58 years old. As an indicator of solvent exposure, ai r and blood samples of exposed men were taken by personal monito ring during one shift and one week before analysis. In addition to cell typing, the followi ng parameters were obtained: haematological status, with differentiation, SGOT, SGPT, γ GT, AP, cholesterol and NF. Lymphocytes obtained were labelled with monoclonal an tibodies and counted for subpopulations: T4 helpers, T8 suppressor cells, total amount of T11 (all) cells, NK and B lymphocytes.
Some chemicals were identified by monito ring: butanol, iso-butanol, EGBE, EGEE, 2 methoxyethynol, toluene, m-xylene, 2-butanone, 2-hexanone. For EGBE, measurements gave the following results:
In air: mean concentration: 24.6 mg/m 3 (maximum concentration: 350 mg/m 3 )

In blood: mean concentration: 4.1 μ g/dl (maximum concentration: 108,2 μ g/dl)
Between the exposed and the unexposed group there were only negligible differences in Hb concentration and erythrocyte number (a very slight decrease). In the relative number ofEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 189 eosinophiles and stab cells (increase), segmented cells (decrease) and lymphocytes (increase), these differences were statistically significant. Others measured parameters where within the normal ranges. Significant differences in the di stribution of the lymphoc ytes sub-populations were seen between exposed and unexposed group. In the exposed workers, T11 cell and T4 helper were decreased, while NK and B lymphocy tes were increased. There were no changes in the T8 suppressor cell sub- population. In this study, the authors concluded that the observed decrease in helper/suppr essor ratio might be an indi cator of early haematological and/or immunological effect due to chemical exposure, which eventually may induce long term disorders of the haematopoetic and/or lymphopoetic system. But they also concluded than these results were only quantitative and did not reflect the f unctional state of each subpopulation. Since the workers were exposured to a variety of chemical s, it is not possible to draw conclusions for EGBE alone.

4.1.2.6.3 Mechanistic studies of liver pathology
Associated with haemolysis in rats and mice exposed to EGBE there were depositions of haemosiderin in Kupffer cells and hepatocytes. Haemosiderin is an in soluble product derived from iron-rich ferritin. In man, the major pathol ogical effects of chroni c hepatic iron overload are fibrosis and cirrhosis, porphyria cutanea tarda and hepatocellular carcinoma. Although precise pathogenic mechanisms remain unknow n, iron probably produces these and other toxic effects by increasing oxidative stress and lysoso mal lability (Bonkoavsky, 1991). Therefore, the development of the oxidativ e stress that is possible as a result of haemosiderosis was studied in male and fema le mice treated with EGBE by measuring the formation of 8-hydroxydeoxyguanosine (OH8dG), as an indicator of DNA damage, and malondialdehyde (MDA), as an indicator of lipid peroxidation, and the reduction in α

tocopherol concentration, as an indicator of a reduction in antioxida nt capacity (Kamendulis et al ., 1999). It is clear that α -tocopherol is not the only antioxida nt in the cells, so studies in which only α -tocopherol was measured are not assessing the full antioxidant condition of the cells. In a later study, these investigations were continued by comparing EGBE induced oxidative stress and altered cell growth in ma le B6C3F1 mice and male F344 rats (Siesky et al ., 2002). In addition, the relative sensitivities of male rat a nd male mouse hepatocytes to oxidative damage in vitro were examined (Kamendulis et al ., 1999; Park et al ., 2002a).
Groups of male a nd female B6C3F 1 mice were treated orally by gavage with EGBE at dose levels of 0, 300, 600 or 900 mg/kg bw for male s and 0, 200, 400 or 600 mg/kg bw for females then killed by cervical disloc ation and the livers removed, snap -frozen in liquid nitrogen and stored at -80 ° C until analysed for OH8dG, MDA and α -tocopherol. The results are presented in Table 4.75 as percentages of the control values.

Table 4.75: Effects of EGBE on indicators of oxidati ve stress in B6C3F1 mice, mean & standard deviation & percentage of the cont rol (100 %) (Kamendulis et al ., 1999)
Dose MDA α -Tocopherol OH8dG

Males mice

0 10.54 ± 2.74 5.59 ± 0.89 4.74 ± 1.17

300 122 58 97

600 143* 27* 162*EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 190 900 174* 22* 248*

Female mice

0 11.5 ± 2.72 7.05 ± 1.20 3.6 ± 1.09

200 182* 74 123

400 167* 62* 196*

600 229* 45* 303*

* P < 0.05 units: MDA, nmol/g liver;

α -tocopherol, nmol/g liver; OH8dG, pmol/ μ mol dG
The α -tocopherol concentration was higher in females and was spared to a greater extent than in male mice. This could be indicative of a grea ter protective effect in females than in males. The oxidative damage to DNA was, however, ve ry similar in male and female mice (high dose results being: males 11.76, females 10.91 pmol OH8dG / μ mol dG) (Kamendulis et al
., 1999).
In the more recent study, EGBE was administer ed daily by gavage to male B6C3F1 mice at doses of 0, 225, 450, or 900 mg/kg bw and to male F344 rats at doses of 0, 225 or 450mg/kg bw on 5 days per week for up to 90 days. Thes e doses correlated with those of the 2-year inhalation studies (NTP, 2000). After exposure for 7, 14, 28, or 90 days, 15 mice and 5 rats from each treatment group were killed by asphyxi ation. Seven days prior to sacrifice, an osmotic mini-pump filled with BrdU was implante d in each of five animals from each group. At sampling, livers were perfused with phosphate-buffered saline, removed, and weighed. A portion of liver was fixed in formalin, embe dded in paraffin, and sectioned. The remaining livers were snap frozen in liquid nitrogen and stored at -80oC. Blood was collected in microcapillary tubes for haematocrit measurements. Li ver sections were stained for ferric iron using the Perl method (Bugelski, 1985). Positive Perl staining was localized to Kupffer cells and approximately 2000 cells were counted for each an imal. A “Perl index” was determined (total number of labelled Kupffer cells/ total number of cells c ounted x 100). Immunohistochemical detection of BrdU in liver was performed as described (Eldridge et al., 1990). Cells in 15 random fields were counted (approximately 5000-6000 cells) in each animal. The labeling index was determined in both endothelial cell s and hepatocytes (total number of labeled cells/total number of cells x 100). Endothelial ce ll identity was confirme d by staining a serial section with anti-factor VIII (Sharifi et al ., 2000). Apoptosis and mitosis in the liver was defined and quantified as described (Bursch et al ., 1985). The identificati on of apoptotic cells was confirmed using TUNEL. Cells in 15 ra ndom fields were counted (approximately 5000- 6000 cells) in each animal. Apoptotic and mitotic indicies were determined (total number of labeled cells/total number of cells x 100). DNA damage expressed as OH8dG was measured in DNA isolated by sodium iodide extraction (Wang et al., 1994) and MDA was measured in liver as described by Bagchi et al., (1993).
EGBE decreased haematocrit and increased sple en weights in both rats (225 and 450 mg/kg bw dose groups) and mice (450 and 900 mg/kg bw dose groups). Kupffer cell Perl staining showed dose and time related increases in the Pe rl index that also oc curred in both rats and mice. These observations confirmed that haem olysis and hepatic deposition of iron after treatment with EGBE occurred in males of both rats and mice. There was also a reduction in antioxidant reserve as a result of this treatment, because hepatic α -tocopherol concentrationsEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 191 were decreased in both mice and rats at 7 a nd 90 days, although they were not significantly reduced at 14 or 28 days. An important differenc e between mice and rats is that control levels of α -tocopherol in this experiment were only a bout 1.5 nmol/g liver in mice, whereas they about 4 nmol/g liver in rats. Ev en after treatment, the reduced α -tocopherol levels in rats at both 7 and 90 days were higher than the cont rol levels in mice. Thus, rats remained adequately protected from oxida tive damage, whereas mice did not.

Treatment of mice with EGBE doses of 450 or 900 mg/kg bw resulted in an in crease in oxidative damage in both DNA (OH8dG) and lip id (MDA) at both the 7 and 90 day treatment points, but not at the intermed iate times of 14 and 28 days. In contrast, neither OH8dG nor MDA concentration was increased in rats at any dose level or at any sampling time.
DNA synthesis was increased in liver endothelial cells of mi ce at 7 and 14 days (all dose levels) while DNA synthesis in hepatocytes of mice was only increased at 90 days (all dose levels). No changes were seen at the other observations times in mice and no changes were seen in rats in either endothel ial cell or hepatocyte DNA synthe sis at any dose level or at any time point. No changes in either apoptosis or mitosis were observed in the liver of either species at any dose or time point.
In summary, EGBE caused an increase in oxida tive damage and DNA synthesis in mouse cell types that correspond to those from which the tumours were observed to arise, while no changes were seen in rats, which did not show an increase in either haemangiosarcomas or hepatocellular neoplasms (Siesky et al ., 2002).
Hepatocytes were isolated from male B6C3F 1 mice and male F344 rats by a two-step in situ collagenase perfusion, after which the cells were filtered and centrifuged (Kamendulis et al
., 1999; Park et al ., 2002a). Viability, determined by trypan blue exclusion, was routinely greater than 90 %. The cells were plated a nd cultured overnight. Th e medium was changed and replaced with EGBE or BAA at concentrati ons of 1, 5 or 25 mM, or ferrous sulphate at concentrations of 1, 5 or 25 μ M and incubations continued for 24 hr. Cytotoxicity was determined at that time by measurement of l actate dehydrogenase leakage into the medium and this compared with total lactate dehydrog enase in lysed cells. This experiment was performed on four independent occasions. For each of the substances, the lower two concentrations were not cytotoxic to either mouse or rat hepatocytes, whereas the highest concentration of each substance increased the concentration of lactate dehydrogenase in the medium in the range 1.6-fold to 2.6-fold.
Using the same exposure cond itions as in the previous in vitro study of cytotoxicity, measurements were made of OH8dG, MDA and α -tocopherol in three independent experiments. The summarised data are shown in Table 4.25. Neither EGBE nor BAA induced any increase in oxidative damage expressed as OH 8dG or MDA at any concentration in either rat or mouse hepatocytes. In contrast, ferrous sulphate induced significant increases in both OH8dG and MDA of mouse and rat hepatocytes. A clear species difference in sensitivity of the hepatocytes was seen, with mouse hepatocytes being much more sensitive than rat hepatocytes. In rat hepatocyte s, only the highest concentrati on of ferrous sulphate induced oxidative stress, while in mouse hepatocy tes OH8dG was elevated from the lowest concentration, 1 μ M, and cellular MDA was increased and α -tocopherol decreased at concentrations of 5 and 25 μ M ferrous sulphate. MDA in the culture medium reflects the amount of lipid oxidation products expelled from the cells during exposure, in contrast to cellular MDA that only indicates what remains at the end of exposure. Medium MDA was elevated at all ferrous sulphate concentratio ns in mouse hepatocytes cultures, whereas onlyEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 192 the highest concentration of fe rrous sulphate elevated medium MDA in the rat hepatocytes cultures.

Further studies in this series demonstrated that there was no inter action between ferrous sulphate and either EGBE or BAA in the produ ction of oxidative stress. Furthermore, when iron was presented as haemolys ed erythrocytes (Table 4.76) , there was a dose-dependent increase in oxidative stress in mouse hepatocy tes, demonstrated as increases in OH8dG and MDA and a decrease in α -tocopherol concentration (Park et al ., 2002b).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 193 Table 4.76: Effects of EGBE, BAA and fe rrous sulphate on indicators of oxidat ive stress in male B6C3F1 mouse and male F344 rat hepatocytes, mean & standard deviatio n & percentage of the co ntrol (100 %) (Kamendulis et al ., 1999; Park et al ., 2002a)
Dose OH8dG α -Tocopherol MDA (cells) MDA (medium)
Males mouse hepatocytes

0 2.78 ± 0.26 4.85 ± 0.42 0.70 ± 0.04 3.40 ± 0.39

EGBE (mM) 1.0 102 97 107 109

5.0 147 93 116 115

25.0 142 106 118 122

BAA ( mM ) 1.0 106 90 98 96

5.0 149 86 104 111

25.0 132 94 109 112

FeSO 4 ( μ M) 1.0 151* 96 112 177*

5.0 228* 67* 127* 335*

25.0 360* 57* 241* 458*
Male rat hepatocytes

0 3.63 ± 0.63 6.58 ± 0.59 0.80 ± 0.04 4.04 ± 0.19

EGBE (mM) 1.0 115 92 97 100

5.0 98 92 100 102

25.0 91 90 121 118

BAA ( mM ) 1.0 153 94 102 103

5.0 140 94 106 103

25.0 122 91 114 120

FeSO 4 ( μ M) 1.0 123 93 116 115

5.0 141 79 115 108

25.0 188* 75 165* 163*

* P < 0.05
units:MDA, nmol/10 6 cells or nmol/ml medium; α -tocopherol, ng/10 6 cells; OH8dG, pmol/ μ mol dG.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 194 Table 4.77: Effects of haemolysed erythrocytes (RBCs) on oxidative stress parameters in mouse hepatocytes (Park et al ., 2002a)
Treatment MDA α -Tocopherol OH8dG

Water 0.81 ± 0.21 4.35 ± 0.37 3.33 ± 0.52

0.1 x 10 6 RBCs 0.79 ± 0.15 3.25 ± 0.54 3.41 ± 0.62

1.0 x 10 6 RBCs 1.22 ± 0.34* 2.15 ± 0.63* 6.78 ± 0.43*

10 x 10 6 RBCs 2.36 ± 0.44* 2.25 ± 0.31* 13.40 ± 1.02*

units:MDA, nmol/10 6 cells or nmol/ml medium; α -tocopherol, ng/10 6 cells; OH8dG, pmol/ μ mol dG
* Significantly different from the control ( p < 0.05)
The effect of ferrous sulphate (to mimic the iron deposition in liver resulting from haemolysis) on morphological transformation of Syrian hamster embryo (SHE) cells was studied (Park et al ., 2002b). Significant increases in tr ansformation frequency were observed after exposure to concentrations of 2.5 and 5.0 μ g/ml for 7 days. The greatest effect was a transformation frequency of 0.71 %, compared w ith a medium control frequency of 0.06 %, at 2.5 μ g/ml. Co-incubation of this ferrous sulphate concentration with antioxidants [25 or 50
μ M (-)-epigallocatechin -3-gallate or 150 μ M α -tocopherol] significantly reduced the induced cell transformation frequency. Park et al. (2002b) also showed that 1.0 and 2.5 μ g/ml ferrous sulphate induced DNA strand breaks in SHE cells , as demonstrated with the single-cell gel electrophoresis (Comet) assay, as well as the formation of OH8dG and that co-incubation with either 25 μ M (-)-epigallocatechin-3-gallate or 200 μ M α -tocopherol completely abolished both of these expressions of ferrous sulphate-induced DNA damage.
In summary, these studies demonstrate that rat hepatocytes in culture are markedly less susceptible to oxidative stress than are mouse hepatocytes. In vivo there appears to be a greater antioxidant reserve in rat liver than in mouse liver, so that reduction in α -tocopherol levels observed in both species af ter EGBE treatment resulted in levels in rats that always remained higher than even the untreat ed control level in mice. Also in vivo , male mouse liver is marginally more susceptible than female mo use liver to oxidative da mage, as demonstrated by the higher background rate of hemangiosarcoma lesions. In addition, it has been shown that ferrous sulphate can induce DNA damage as well as morphological transformation in SHE cells and these effects can be re duced or abolished by antioxidants.

4.1.2.6.4 Mechanistic studies on forestomach pathology

Green et al ., 2000a, 2001 and 2002 have shown by whole body autoradiogr aphy that the administration of radiolabelled EGBE to mice by whole body exposure to its vapour results in a significant ingestion of the condensed s ubstance during grooming. The autoradiographs showed that the fur and skin contained high con centrations of radioactivity even 48 h after the end of exposure and there was evidence of se lective accumulation of radioactivity in the forestomach, the glandular stomach containing on ly low, background levels of radioactivity. Ingestion can, perhaps, also result from conde nsation of high vapour concentrations in the nasopharynx. In this way, EGBE can reach the st omach, where there is the potential to causeEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 195 the damage observed in the lifetime inhalati on exposure study (NTP, 2000). Subsequently, the capacity of the stomach to meta bolise EGBE was examined and a basis for the differential response of mice and rats investigated and comparison made between rodent and human stomachs. The study was not conducted to GLP guidelines.
Although this qualitative study suggests that signifi cant exposure of the forestomach to EGBE delivered as a vapour could occur as a result of ingestion of condensed material, this is not supported by the results of one quantitative study. In an inhalation experiment in which female mice were exposed (whole-body) to 229 ppm EGBE for 6 hr, an average of 205 ± 69
μ g EGBE was recovered from the fur of th e animals, i.e., < 10 mg/kg bw, while the concentration of EGBE in the blood at the end of the exposure period was 3.0 mg/L and the BAA concentration was 235 mg/L (Poet et al ., 2003). These blood concentrations were similar to those found in the inhalation exposure carcinogeni city assay (Dill et al ., 1998). Thus, it would appear that some, but not a great deal of EGBE could reach the forestomach as a result of grooming. The authors do acknowledg e, however, that some material could have been lost by evaporation and by the hot water washing procedure, which was adapted from a similar process that had been used for ethyle ne glycol, which is a less volatile material.
Female B6C3F1 mice were given a single intravenous dose of 2-butoxy[1- 14 C]ethanol and killed at 4, 24 and 48 h after dosing and the distri bution of radiolabel examined in the animals. High levels of radiolabel were present in the liver, oesophagus and buccal cavity throughout the time course of the study. Similar concentra tions of radioactivity were found in both the forestomach and glandular stomach. The radioa ctivity in the stomach tissues was derived from the systemic circulation and, in a ddition, there was evidence of ingestion of radiolabelled material from the buccal cavity. The origin of the la tter is unknown, but it may be derived from the salivary and Harderian glands, which contained significant amounts of radioactivity (B ennett, 2001).
Other experiments of Green et al ., 2001 and 2002 lend support to other aspects of the development of forestomach lesions. Groups of five female B6C3F 1 mice were administered orally by gavage either EGBE or BAA daily for 10 days at dose levels of 0, 50, 150 or 500 mg/kg bw per day (Green et al ., 2001 and 2002). Seventeen hours af ter the last dose and 1 hr before they were killed, the mice received a subcutaneous injection of bromodeoxyuridine, 15 mg/kg bw. Stomachs were removed for histology and quantification of cell proliferation, and blood samples were taken for haematology. In th e top dose group of mice receiving BAA, one died as a result of causes unrel ated to treatment. Haematologi cal changes typical for these compounds were observed in all dose groups ad ministered BAA, but of those receiving EGBE, they were largely confined to the 500 mg/kg bw per group. No abnormalities were seen in the glandular part of the stomach in any of the groups . No abnormalities were found in the forestomach of mice dosed with EGBE at doses of 50 or 150 mg/kg bw per day or with BAA at the 50 mg/kg bw dose level. The only abnormality observed in the forestomach was hyperkeratosis, which was minimal in two mice given EGBE at 500 mg/kg bw per day and in three mice given BAA at 150 mg/kg bw per day. In the 500 mg/kg bw per day group given BAA, all four mice that completed the study show ed hyperkeratosis, three to a slight degree and one to a moderate degree. The cell proliferat ion part of the study was invalid because of a high control group value. Within the groups tr eated with either compound, there were dose related increases in S-phase cells in the forestomach areas. Furthermore, the development of hyperkeratosis is, in itself, evidence of increased cellu lar proliferation.
As part of the same study, a group of 10 fe male mice were given oral doses of 2-(1- [ 14 C]butoxy)ethanol, 500 mg/kg bw pe r day for 5 days, then killed 17 hr later. Glandular stomach and forestomach areas were remove d, washed, separated and homogenised forEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 196 analysis. Homogenised washed forestomachs a nd glandular stomachs contained very little covalently bound radioac tivity and none was identified as asso ciated with particular protein bands following SDS-PAGE analysis. Therefore, it appears that covalent binding plays little, if any, role in the forestom ach toxicity of EGBE.

Untreated F344 rats and B6C3F 1 mice were killed, their stom achs removed and divided into forestomach and glandular stomach sections. Pooled samples of the same tissue were homogenised, stored at -70 ° until required and then alcohol and aldehyde dehydrogenase enzyme kinetics determined, using EGBE and BA L, respectively, as subs trates. Generally, the activities were similar in both regions of th e stomach for both enzymes and there were no major species differences in aldehyde de hydrogenase activity. In the case of alcohol dehydrogenase, however, there were major differences between rats and mice, both in affinity constants (K m ) and maximal rates (V max ). The K m

values were two orders of magnitude greater in both regions of the mouse stomach, compared to the rat, and V max was up to one order of magnitude higher in the stomach of th e mouse than in the rat. The absence of any marked regional difference in enzyme activity in the homogenates would appear to fail to explain the regional diffe rences in toxic and neoplasic resp onse. However, histocytochemical localisation of the enzymes showed that, in th e forestomach, the dehydr ogenase activities are strongly localised in the stom ach lining, whereas in the glandular stomach they are more homogeneously distributed thr oughout the stomach wall. Estima tion of the relative volumes of each stomach compartment containing enzyme activity shows an approximately 1:25 ratio between forestomach and glandular stomac h. Consequently, the enzyme activity is concentrated in a much smaller volume in the forestomach, so that, in a whole tissue homogenate, the activity is diluted (Green et al ., 2001 and 2002).
Independent experiments on the mode of action in the forestomach have been conducted elsewhere. Adult male and female B6C3F 1 mice were treated orally by gavage w ith undiluted EGBE at dose levels of 0, 400, 800 or 1200 mg/kg bw for 2 days, followed by 0, 200, 400 or 600 mg/kg bw for 2 days. The doses were redu ced because of excessive mortality caused, apparently, but unexpectedly, by the original doses. Survival did not improve at these lower doses, so treatment was discontinued. Two ot her groups of mice were treated by either subcutaneous or intraperitoneal injection with EGBE in saline at a dose level of 400 mg/kg bw for 5 days. Dose related incidences of forestomach lesions occurred in all male and female mice after oral administration of undiluted EGBE . These lesions included focal hyperplasia in the low dose group and more diffuse hyperplasi a at the higher doses associated with significant infiltration by neutrophils and mono nuclear inflammatory cells in the submucosa and muscularis. There were increases in the proliferating cell nuclear antigen labelling in regions surrounding the areas of hyperplasia. Minimal lesions similar to those found in the low dose (200 – 400 mg/kg bw) oral administrati on group were found in 1/ 6 intraperitoneally dosed mice and in 2/6 subcutaneously dosed mi ce, following 4 days administration of 400 mg/kg bw (Poet et al ., 2003).
As part of this same study, the tissue dose and pharmacokinetics of EGBE were also compared after an oral dose by gavage of 265 mg/kg bw (target dose 250 mg/kg bw) and an intraperitoneal inject ion of 53 mg/kg bw or 261 mg/kg bw (target doses 50 and 250 mg/kg bw). Regardless of the dose and the dose route, EGBE disappeared from the circulation within 1 hr, while BAA was detectable up to 12 hr after a target dose of 250 mg/kg bw by either route. EGBE concentrations in the forestomach and glandular stomach tissues were higher than in blood and other tissues and EGBE persis ted for a longer period in the stomach tissues, so that, regardless of the dose route, the t 1⁄2 and AUC for EGBE were higher in forestomachEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 197 tissues than in any other tissues. These data and the corresponding data for BAA are shown in Table 4.78.
In a separate experiment, the concentrations of EGBE and BAA in saliva were measured after oral and intraperitoneal dosing. The results are also shown in Table 4.78. Of particular significance is the relatively high concentrations of both compounds in the saliva after intraperitoneal dosing.

Table 4.78: Halve lives and AUC in blood, saliva and tissues. (Poet et al. , 2003).

EGBE BAA Route, dose in mg/kg bw Tissue

AUC ( μ g/g/h) t 1⁄2 (h) AUC ( μ g/g/h) t 1⁄2 (h)

Ip (261 ± 34) Blood NA NA 442.8 1.05

Liver 4.52 0.66 317.0 1.10
Kidney NA NA 507.4 1.44

Forestomach 274.2 2.60 468.4 4.62

Glandular stomach 34.7 1.86 289.6 1.31

Oral (265 ± 13) Blood NA NA 1740.7 2.11

Liver 16.7 0.57 604.1 2.09
Kidney NA NA 847.8 2.71

Forestomach 3063.9 3.40 539.3 3.96

Glandular stomach 746.5 4.91 186.6 1.57

Ip (258 ± 66) Blood 43.4 0.16 2194.0 1.42

Saliva 66.1 0.17 143.2 0.69
Oral ( 264 ± 42 ) Blood 27.1 0.35 900.6 1.55

Saliva 26.3 0.27 66.8 0.85

Probably as a result of the secretion of EGBE in saliva, significant quantities of the compound can reach the forestomach contents, even after intraperitoneal dosing.
Other, general factors contribut e to the preferential toxicity to the forestomach and are unrelated to the actual compound being investigated.
In rodents, including the mouse, rat, and Syrian hamster, the stomach is divided into two parts by the mucoepidermoid junction separating squamous from glandular epithelium. The proximal part, or forestomach, is non-glandular, continuous with the oesophagus, and lined by keratinised, stratified squamous epithelium. Th e distal part, or glandular stomach, empties into the duodenum and is lined by a specialized glandular epithelium. The forestomach is separated from the glandular stomach by a grossl y visible, elevated fold, the limiting ridge. This contrasts with the anatomy of the human stomach, the lining of which is entirelyEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 198 glandular, the human mucoepidermoid juncti on occurring where the oesophagus joins the stomach. There is thus no forestomach in human beings. There is a hi stological similarity between the rodent forestomach and the human oesophagus, but the physiological functions of these two organs are quite different. While the forestomach is a storage organ, where ingested material may reside for several hours before its transfer to the glandular stomach, the oesophagus is not, ingested material passing thro ugh in a matter of seconds. Exposure of the human (or rodent) oesophagus to ingested material is, therefore, brief and it is to be noted that the histology of the rodent oesophagus is not unlike that of the human organ, yet the oesophagus of the rats and mice exposed to EGBE in studies described here does not respond in the same way as the forestomach.

4.1.2.6.5 Summary of repeated dose toxicity
In rats and mice, haemolysis was consis tently observed (whichever the route of administration) and was sometimes associated with hepatic effects (Kupffer cell pigmentation and absolute and relative liver weight in creases), effects on body weight gain, hyaline degeneration of the olfactive epithelium (by inhalation), effects on the forestomach and effects on the WBC sub-populations (T lymphocyte ). In these studies and for the inhalation route, no NOAEC was identified for mice, wh ereas a NOAEC value of 25 ppm (121 mg/m 3 ) in rats was identified. In a separate study a LOAEC value of 31 ppm (150 mg/m 3 ) can be established in rats, based on haemolysis and Kupffer cell pigmentation. Due to the closeness of the apparent LOAEC and NOAEC, it is consider ed prudent to take th e more conservative LOAEC of 31 ppm forward for ri sk characterisation. However, the likelihood that this figure is close to the NOAEC will be ta ken into account in deriving a ppropriate assessment factors.

Slight effects on the immune system were s een in rats, mice and humans on NK cells or T lymphocyte sub-population. In the human study, co-exposure to a number of chemicals does not allow reliable conclusions to be drawn for EGBE alone. In the rodent studies, however a NOAEL of 1000 mg/kg bw in mice by dermal route ca n be established. Th e effects seen were small. A role of EGBE in the induction of i mmunotoxicity has not been developed to a point where it can be used in risk characterisation.
For the dermal route, a NOAEL of 150 mg/kg bw/d (the highe st dose tested) has been determined from a 13-week study in rabbits.
For the oral route, a LOAEL of 69 and 82 mg/kg/ day for male and female rats respectively, was found in a 13 -week drinking wa ter study(haemolytical effects).
As humans are far less sensitive than other sp ecies (except Guinea Pig) to the haemolytical properties of EGBE, we have tried to assess sepa rately haemolytical eff ects and related effects and other specific toxic effects which could be induced by EGBE. For all the studies, no specific relevant toxic effects, other th at haemotoxicity, can be identified.
For the risk characterisation, haemotoxicity will be the end point chosen keeping in mind the interspecies differences (human/rodents) to ca lculate margin of safety. No other lesion has been identified which can be specifically attributed to treatment with EGBE.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 199 4.1.2.7 Mutagenicity
The mutagenic properties of ethers of ethylene glycol and their metabolites in general and EGBE in particular have been reviewed (Elliott and Ashby, 1997 ; McGregor, 1984 and 1996; Tyler, 1982).

4.1.2.7.1 Studies in vitro

Bacterial studies
EGBE and its metabolites, BAL and BAA, have b een tested for their potential to induce gene mutations in bacteria on a number of occasions; these and the results are listed in Table 4.79.

Two studies in which five different strains of Salmonella typhimurium were used, null results were obtained with dose levels up to 20 mg per pl ate, in one case, and 10 mg per plate in the other. The latter was an Ames’ test conducte d by the US National Toxicology Program (NTP) in which supplementary metabolic activation co nditions were provided by rat liver S9 mixes at 10 % and 30 % and Syrian hamster liver S9 mixes at 10 % and 30 % (Zeiger et al ., 1992). Nevertheless, it was concluded in a later study that EGBE was mutagenic as demonstrated by its activity in S. typhimurium TA97a, at a minimal dose of 2.2 mg per plate in the absence of S9 mix and 9.0 mg per plate in th e presence of rat liver S9 mix at 2.5 %. Thus, the activity of EGBE was reduced by the presence of the s upplementary activation system. The mechanism of this reduction was not investigated. Th e authors imply, however, by referring to the absence of mutagenic activity in tests with BAL and BAA, that the reduction in mutagenic activity of EGBE was due to its metabolism to nonmutagens (Hoflack et al ., 1995). S. typhimurium TA97a is very similar to the strain TA97, the difference between the strains being in the length of the deletion through the uvrB locus. Consequently, important differences in the responsiveness of the strain s should not have been expected. Both strains carry the hisD6610 mutation that has a run of six cytosines at the mutation site and requires a frame shift mutation for its reversion to histidin e independence. Furtherm ore, EGBE is not the type of chemical structure that would be anticipated to produce a frameshift mutation.
Gollapudi et al . (1996) performed a carefully designed st udy that covered the conditions used by Hoflack et al ., 1995 and using S. typhimurium TA97a, TA100 and Escerichia coli WP2 uvrA . There was no indication of an increase, significant or not, at any EGBE dose level between 0.5 and 10.0 mg per plate. The po ssible contribution of peroxides to the mutagenicity observe d in the Hoflack et al ., 1995 study was discusse d, but dismissed. The lack of an effect in strain TA 97a in the Gollapudi et al ., 1996 study supports the absence of a mutagenic response from the closely re lated strain TA 97 in the Zeiger et al ., 1992 experiments. Nevertheless, the debate continues, because Hoflack et al. (1997) in a letter to the journal editor, reported that the activity in strain TA97a reported earlier was again observed upon re-testing in their laboratory.

Mammalian cell studies
EGBE was tested for gene mutagenic activity in Chinese hamster ovary (CHO) cells at the hprt locus (Slesinski and Weil, 1980). Null responses were obtaine d in this assay, in which test compound concentrations used were up to 1 % (v/v), approximately 9 mg/ml, in the absence of S9 mix and up to 0.5 % (v/v), approx imately 4.5 mg/ml, in the presence of S9 mix.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 200 Null responses were also obtained by Chiewchanwit and Au (1995), at the gptlocus in CHOAS52 cells exposed to either EG BE at concentrations up to 0 .9 mg/ml (relative survival 79 %) or BAL at concentrations up to 0.065 % (v/v) (relative survival 91 %), the next higher dose, 0.2 %, being too toxic to permit its evaluation for mutagenicity. These cells were derived from hprt -deficient CHO cells and contain a single autosomal copy of the xanthine guanine phosphoribosyl transferase ( gpt ) gene, which is the bacterial equivalent of the mammalian hprt gene. It had been demonstrated pr eviously in the same laboratory (Ma et al ., 1993) that methoxyacetaldehyde, a metabolite of EGME, is mu tagenic in CHO-AS52 cells, but not in the standard CHO cells. Methoxyacetaldehyde was used as a positive control in this study. However, Elias et al.
, . (1996) reported that both EGBE a nd BAL induced significant increases in 6-thioguanine-resista nt phenotypes (i.e., the hprt locus) in Chinese hamster lung V79 cells. Unfortunately, the data are inadequately describe d in this paper, there being no indication of variation or of survival. Readi ng from a graphical presentation (log dose scale), it appears that the reported significant response EGBE occurred at a concentration of about 9 mg/ml. This description is inadequate for evaluation purposes.EURISK ASSESSMENT–2-BUTOXYETHANOLCAS111-76-2 CHAPTER4.HUMAN HEALTHRAPPORTEURFRANCER408_0808_HH_CLEAN201Table 4.79: Test for gene mutation induction in bacteria by EGBE and its metabolitesResultaTest system Source & purity of chemicalWithoutexogenousmetabolicsystemWithexogenousmetabolicsystemDoseb(LED/HID)ReferenceEGBEBacteriophage T4D mutation Not stated- (severetoxicity)NT111μl/mlKvelland, 1988Salmonella typhimuriumTA100, TA1535,TA1537, TA1538, TA98, reverse mutationDupont de Nemours, USA (purity not stated)- -20,000μg/plateSippel and Krahn, 1977Salmonella typhimuriumTA100, TA1535,TA1537, TA97, TA98, reverse mutationFisher Scientific, USA > 99 %- -10,000μg/plateZeigeret al., 1992Salmonella typhimuriumTA100, TA98,TA102 reverse mutationMerck, Germany > 99 %- -14,000μg/plateHoflacket al., 1995Salmonella typhimuriumTA97a, reversemutationMerck, Germany > 99 %+ +2,200μg/plateHoflacket al., 1995Salmonella typhimuriumTA97a, TA100 &Eschichia coliWP2uvrAreverse mutationDow Chemical Co., USA 99.04 %- -10,000μg/plateGollapudiet al., 1996BALSalmonella typhimuriumTA100, TA98,TA97a, TA102 reverse mutationChemical synthesis > 91 %--7,000μg/plateHoflacket al., 1995BAASalmonella typhimuriumTA100, TA98,TA97a, TA102 reverse mutationJanssen Chimica, Belgium > 99 %- -1,000μg/plateHoflacket al., 1995a+, positive; –, negative; NT, not tested;bLED, lowest effective dose; HID, highest ineffective doseEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 202 In tests for the induction of sister-chroma tid exchanges (SCEs) in CHO cells, it was concluded in a US NTP-sponsored study (NTP, 2000) that there was no significant induction by EGBE concentrations up to 3.5 mg/ml in th e absence of S9 mix and 5.0 mg/ml in the presence of rat liver S9 mix, although a trend te st was significant (the reby judged to be an equivocal result) in the absenc e of S9 mix in one of two tr ials, the other being clearly negative. Slesinski and Weil (1980) also did not show an increase in SCE frequency at concentrations up to 0.25 %. On the other hand, Elias et al . (1996) reported a small induction of SCEs in V79 cells and Villalobos-Pietrini et al . (1989) found a significant increase in SCEs in cultured human lymphocytes in the absence of S9 mix of approximately 2.5-fold at a concentration of 0.5 mg/ml, the lowest tested, rising to approximately 4.3-fold at 3.0 mg/ml, the highest tested. Elias et al . (1996) reported a much larger induction of SCEs in V79 cells treated with BAL, but no effect of BAA. The data were presented only in a graphical form with no indication of variation or survival. It is noted that the reports of significant increases in the frequency of SCEs by Villalobos-Pietrini et al . (1989) and Elias et al . (1996) were observed at lower concentrations than used in the studies of Slesinski and Weil, 1980 and NTP (2000), neither of which reported a ny significant change in SCE frequency.
In three independent investigations, chromoso mal aberrations were not induced in cultured mammalian cells. These included an investigation with CHO cells in which EGBE concentrations up to 5.0 mg/ml did not induce aberrations in two of 3 trials in the absence of S9 mix or in a single trial in the presence of S9 mix (NTP, 2000); an experiment with V79 cells for which no experimental detail s or results were presented (Elias et al ., 1996); and two experiments with human lymphocytes with concentrations up to 3.0 mg/ml (VillalobosPietrini et al ., 1989) and 0.3 mM (Elias et al , 1996). It was reported that chromosomal aberrations (not including gaps) increased by BAL in both V79 cells and human lymphocytes by Elias et al . (1996). Once again, the data were presen ted only in a graphi cal form with no indication of variation or su rvival. A small increase in mi cronucleated cell frequency was observed, however, in V79 cells treated with EGBE at a con centration of least 1.0 mg/ml, where there were 1.50 % micronucleated cells compared with 0.89 % ± 0.24 % in the controls. A similar very small increase that was, nevertheless, statistically significant was observed with BAA (1.71 % at 0.66 mg/ml), while BAL treatment resulted in 1.91 % micronucleated cells at about 0.005 mg/ml and 48.13 % micronucleated cells at about 0.020 mg/ml. Many of these cells contained 3 – 4 nuclei per cell and cells with several micronuclei in place of the parental nuc leus. These latter observations are suggestive of aneuploidy. Investigation of the treated V 79 cells showed that the percen tage of hyperdiploid cells was increased by EGBE and by BAL in particular, whereas BAA had hardly any effect (Elias et al ., 1996). These in vitro mammalian cell studies are summarised in Table 4.80.

Other in vitro mammalian cell studies
Inhibition of gap-junctional intercellular comm unication was studied in a mixed culture of hprt + and hprt - V79 cells. EGBE was active in the assa y at non-cytotoxic concentrations, but neither BAL nor BAA showed any activity (Elias et al ., 1996).
In a test for morphological tran sformation of Syrian hamster embryo cells treated for 7 days, BAL up to 1.0 mg/ml was not active, but no details of the results were presented. It is not clear whether EGBE and BAA also were tested, but found to be inactive (Elias et al
., 1996).The text indicates that BAL was inactive, while a figure shows EGBE to be inactive. In a similar assay, but with a 24 hr exposure time, a significant result was recorded for two doses (1.0 and 1.25 mg/ml, with relative plating effici encies of 70 and 53 %, respectively), but not for a higher dose (1.5 mg/ml at which the rela tive plating efficiency was 42 %) (Kerckaert etEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 203 al ., 1996). This result was not repeated in anot her Syrian hamster embryo cell transformation study, in which both EGBE and BAA were tested at concentrations up to 20 mM for 7 days. No significant increase in transformation frequency was found with either compound, although at 8.0 mM EGBE the transformation fr equency was 0.52 when the relative plating efficiency was 73.1 %, compared with a contro l value for the transformation frequency of 0.27 (Park et al ., 2002b). Experiments were also conducted with ferrous sulphate in this system. Poly(ADP-ribosyl)ation is a re quirement for DNA base exci sion repair and chromosomal stability that is synthesised by the NAD + -dependent enzyme poly(ADP-ribose) polymerase (EC 2.4.2.30). The effect of EGBE on the activity of this enzyme wa s studied in Syrian hamster embryo cells treated with methyl meth anesulphonate. The glycol ether potentiates the clastogenic activity of this alky lating agent, perhaps by consuming NAD + when it is oxidised to the aldehyde, and the alkylating ag ent induces poly(ADP-ribose) metabolism. The study did not show that competition for NAD + was responsible for potentiation by EGBE (Hoflack et al ., 1997).
In Table 4.80 there is listed a study of uns cheduled DNA synthesis (UDS) by Slesinski and Weil, 1980. This is included for completeness sake. However, the sc intillation counting method used to assay for incorporation of tri tiated thymidine into DNA has been considered unacceptable for many years because of the possibi lity of a few cells escaping S-phase block and thereby distorting the results. Such complications of th e test, should they occur, can only be recognised and avoided by silver grai n counting with the us e of a microscope.EURISK ASSESSMENT–2-BUTOXYETHANOLCAS111-76-2 CHAPTER4.HUMAN HEALTHRAPPORTEURFRANCER408_0808_HH_CLEAN204Table 4.80: Tests for genetic and related effects incultured mammalian cells by EGBE and its metabolitesResultaTest system Source & purity of chemicalWithoutexogenousmetabolicsystemWithexogenousmetabolicsystemDoseb(LED/HID)ReferenceEGBEUnscheduled DNA synthesis, rat primaryhepatocytes (scintillation counting)*Union Carbide Corp., USA 99.4 %?0.0001 % Slesinski and Weil, 1980Gene mutation, Chinese hamster ovary CHOcells,hprtlocusUnion Carbide Corp., USA 99.4 %--1 % (v/v) Slesinski and Weil, 1980Gene mutation, Chinese hamster ovary CHOAS52 cells, grtlocusAldrich, USA (purity not stated)-7.6 mM Chiewchanwit and Au,1995Gene mutation, Chinese hamster lung V79cells,hprtlocusMerck, Germany 99 %+20 mM Eliaset al., 1996Sister chromatid exchange, Chinese hamsterovary CHO cellsin vitroUnion Carbide Corp., USA 99.4 %--0.25 % Slesinski and Weil, 1980Sister chromatid exchange, Chinese hamsterovary CHO cellsin vitroDow Chemical, USA > 99 %- -3,500μg/mlNTP, 2000Sister chromatid exchange, Chinese hamsterlung V79 cellsin vitroMerck, Germany 99 %±15 mM Eliaset al., 1996Sister chromatid exchange, humanlymphocytesin vitroNot stated+500 ppm Villalobos-Pietriniet al.,1989Chromosomal aberrations, Chinese hamsterovary CHO cellsin vitroDow Chemical, USA > 99 %- -5,000μg/mlNTP, 2000Chromosomal aberrations, Chinese hamsterlung V79 cellsin vitroMerck, Germany 99 %-0.3 mM Eliaset al., 1996EURISK ASSESSMENT–2-BUTOXYETHANOLCAS111-76-2 CHAPTER4.HUMAN HEALTHRAPPORTEURFRANCER408_0808_HH_CLEAN205Table 4.80: (continued) Tests for genetic and related effects incultured mammalian cells by 2-butoxyethanol and its metabolitesChromosomal aberrations, humanlymphocytesin vitroMerck, Germany 99 %-NT0.3 mM Eliaset al., 1996Chromosomal aberrations, humanlymphocytesin vitroNot stated-NT3,000 ppm Villalobos-Pietriniet al.,1989Micronuclei, Chinese hamster lung V79 cellsin vitroMerck, Germany 99 %±NT8 mM Eliaset al., 1996Aneuploidy, Chinese hamster lung V79 cellsin vitroMerck, Germany 99 %±NT16.8 mM Eliaset al., 1996Cell transformation, Syrian hamster embryocells, focus assayMerck, Germany 99 %-NT200 mM Eliaset al., 1996Cell transformation, Syrian hamster embryocells, focus assayNot stated+NT8 mM Kerckaertet al., 1996Cell transformation, Syrian hamster embryocells, focus assaySigma Chemical Co., USA >99 %-NT20 mM (7 days) Parket al., 2002bInhibition of gap-junctional intercellularcommunication, Chinese hamster lung V79cellsin vitroMerck, Germany 99 %+NT8 mM Eliaset al., 1996BALGene mutation, Chinese hamster ovary CHOAS52 cells, grtlocusAldrich, USA (purity not stated)-NT7.6 mM Chiewchanwit and Au,1995Gene mutation, Chinese hamster lung V79cells,hprtlocusChemical synthesis 91.4 %+NT20 mM Eliaset al., 1996Sister chromatid exchange, Chinese hamsterlung V79 cellsin vitroChemical synthesis 91.4 %±NT15 mM Eliaset al., 1996Chromosomal aberrations, Chinese hamsterlung V79 cellsin vitroChemical synthesis 91.4 %+NT0.2 mM Eliaset al., 1996Chromosomal aberrations, humanlymphocytesin vitroChemical synthesis 91.4 %+NT0.2 mM Eliaset al., 1996EURISK ASSESSMENT–2-BUTOXYETHANOLCAS111-76-2 CHAPTER4.HUMAN HEALTHRAPPORTEURFRANCER408_0808_HH_CLEAN206Micronuclei, Chinese hamster lung V79 cellsin vitroChemical synthesis 91.4 %±NT8 mM Eliaset al., 1996Table 4.80: (continued) Tests for genetic and related effects incultured mammalian cells by 2-butoxyethanol and its metabolitesAneuploidy, Chinese hamster lung V79 cellsin vitroChemical synthesis 91.4 %±NT0.09 mM Eliaset al., 1996Cell transformation, Syrian hamster embryocells, focus assayChemical synthesis 91.4 %-NTNG Eliaset al., 1996Inhibition of gap-junctional intercellularcommunication, Chinese hamster lung V79cellsin vitroChemical synthesis 91.4 %-NT8 mM Eliaset al., 1996BAASister chromatid exchange, Chinese hamsterlung V79 cellsin vitroJanssen Chimica, Belgium >99 %-NT0.9 mM Eliaset al., 1996Chromosomal aberrations, Chinese hamsterlung V79 cellsin vitroJanssen Chimica, Belgium >99 %-NTNG Eliaset al., 1996Micronuclei, Chinese hamster lung V79 cellsin vitroJanssen Chimica, Belgium >99 %+NT2.5 mM Eliaset al., 1996Aneuploidy, Chinese hamster lung V79 cellsin vitroJanssen Chimica, Belgium >99 %-NT0.36 mM Eliaset al., 1996Cell transformation, Syrian hamster embryocells, focus assaySpectrum Chemicals, USA >99%-NT20 mM (7 days) Parket al., 2002bInhibition of gap-junctional intercellularcommunication, Chinese hamster lung V79cellsin vitroJanssen Chimica, Belgium >99 %-NT8 mM Eliaset al., 1996a+, positive; ±, weak positive –, negative; ? questionable result; NT, not tested;bLED, lowest effective dose; HID, highest ineffective dose*Scintillation counting is nolonger considered a reliable method for measuring unscheduled DNA synthesis.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 207 4.1.2.7.2 Studies in vivo (table 4.81)
Sprague-Dawley rats (3/group, sex not specifie d) weighing approximate ly 260 g were given a single dose (route not stated) of EGBE, 0 or 120 mg/kg bw and killed 24 hr later. The brain, testes, liver, spleen and kidney were re moved and DNA extracted from each organ. The detection of 5-methyldeoxycyt osine and hydrophobic DNA adducts was performed using the
32 P-postlabelling technique. No significant differe nces were found between the groups in the levels of methylated deoxycytos ine, expressed as percentages of total cytidylic acid found in any organs. The percentages ranged from 3.4 % to 4.0 % in the controls and from 3.6 % to 4.0 % in the treated group. Furthermore, no obvious differences were found in the tlc hydrophobic adduct maps of 32 P-labelled nucleotides for any of the rat organs examined (Keith et al ., 1996).

A micronucleus induction test was performed with male F344/N ra ts (5/group) at dose levels selected on the basis of published toxicity info rmation and the results of a preliminary rangefinding study. The rats were inject ed intraperitoneally three times at 24 h intervals with EGBE dissolved in phosphate-buffered saline at dose levels of 0, 7.03, 14.06, 28.12, 56.25, 112.50, 225.00 or 450.00 mg/kg bw per day; the total do se volume was 0.4 mL. The positive control rats received injections of cyclophosphamide, 7.50 mg/kg bw per day. Rats were killed 24 hr after the final injection and smears prepared from femoral bone marrow, which were then fixed and stained. Two thousand polychromatic erythrocytes (PCEs) were scored for the frequency of micronucleated cells in each rat. Data were tabulated as the pooled mean of each exposure group and the standard error of the me an. They were analysed with a one-tailed Cochran-Armitage trend test and by pair-wise comparisons between each dosed group and the vehicle control group. Statistically si gnificant results were recorded for P ≤ 0.025 with the trend test and P ≤ 0.025 divided by the number of dose gr oups with the paired comparison test. A final decision on the biol ogical significance (the “final call”) was made on the basis of the statistical anal ysis, the reproducibility and the magnit ude of the effects observed. Two of five rats in the 450 mg/kg bw dose group died . The frequency of micronucleated PCEs per 1000 cells in the vehicle control group was 1.9 ± 0.2 and the range of means in the dose groups was from 1.2 ± 0.2 at 225.00 mg/kg bw to 2.2 ± 0.3 at 28.12 mg/kg bw and 2.2 ± 0.6 mg/kg bw at 450.00 mg/kg bw. The tr end test was not significant ( P = 0.570). The PCE/NCE ratios were not presented in the report (NTP, 2000).
Male and female F344 rats (tr eated, 5 – 8, controls 10 - 12/s ex/group) were administered EGBE by gavage at a dose level of 0 or 250 mg/kg bw per day for 1, 2 or 3 days and then killed 24 or 48h after the last dose. Haemoglobinuria was obs erved. Progressive timedependent macrocytic, hypochromic, regenerative haemolytic anaemia was was observed in both males and females treated with EGBE. A dditionally, EGBE treatment caused significant morphological changes in erythrocytes that were first observed just 24 h after a single dose, which included stomatocytosis, macrocytosis with moderate rouleaux formation and spherocytosis. These morphological changes be came progressively more severe with more EGBE doses and included the occasional occurren ce of schistocytes and ghost cells, rouleaux formation in rats of both sexes and, in fema le rats, an increased number of reticulocytes containing micronuclei. It appe ared that the progression of haemolytic anaemia was more rapid in female rats than in males. In response to the anaemia, extramedullary haematopoiesis was noted in the spleen. The general pathologi cal effects are described in more detail in section 4.1.2.2.3.2 (Ghanayem et al ., 2001).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 208 A micronucleus induction test was performed with CD-1 mice (4/sex/group/observation time) at dose levels selected on the basis of published LD 50 information. The mice were injected intraperitoneally once with EGBE dissolved in Hank’s balanced salts solution at dose levels of 0, 150, 300, 600, 800 or 1000 mg/ kg bw. The positive control mi ce received injections of cyclophosphamide, 25 mg/kg bw. Mice of the thr ee lower dose groups were killed 24, 48 and 72 hr after the injection, while mice of all othe r groups were killed at 24 hr after injection, and smears prepared from femoral bone marrow, wh ich were then fixed and stained. A minimum number of 1000 polychromatic erythrocytes (P CEs) were scored for the frequency of micronucleated cells in each mouse. In addition, the ratios of PCE to mature normochromatic erythrocytes (NCEs) were determined among 2000 erythrocytes per mouse. Data were tabulated as the pooled mean of each exposure group and the standard deviation. They were analysed according to the crite ria of Schmid (1976). Three ma le and three female mice of the1000 mg/g bw dose group died, the recorded resu lt at this dose level therefore being based on just one mouse of each sex. The frequency of micronucleated PCEs per 1000 cells in the vehicle control group was 1.41 ± 0.43 and the range of means in the dose groups was from 0.36 ± 0.19 at 300 mg/kg bw to 0.97 ± 0.39 at 800 mg/kg bw, both re sults being in the 24 hr samples. The mean result from the two surv ivors of the toxic, 1000 mg/kg bw dose was 2.46. There was no dose related change in the PCE/NCE ratio at either the 48 h or 72 h observation times from the common, 24 h control value of 1,53 ± 0. At 24 h, the ration was high at the lowest, 150 mg/kg bw dose level (2 .19 ± 0.32) and progressively fell to the contro l level with increasing dose (300 mg/kg bw, 2.11 ± 0.25; 600 mg/kg bw, 1.79 ± 0.23; 800 mg/kg bw, 1.54 ± 0.34; and 1000 mg/kg bw, 1.71, mean of two samp les). In comparison with other control group values in the same publi cation, none of the PCE/NCE rati os obtained with EGBE was outside of the control group range (Elias et al ., 1996).
In this study, mice were also treated with BAA at dose levels of 0, 50, 100 or 200 mg/kg bw, but sampling was restricted to 24 hr after the single in jection. The frequency of micronucleated PCEs per 1000 cells in the vehi cle control group was 0.00 for this particular experiment (but ranged upwards in ot her experiments in the series to 1.50 ± 0.46) and the range of means in the groups dosed with BAA was from 0.45 ± 0.03 at 50 mg/kg bw to 0.94 ± 0.5 at 200 mg/kg bw. BAA was toxic to bone marrow cells, there being statistically significant reductions in the PCE/NCE ratios from 1.79 ± 0.45 in the vehicle control group to 0.78 ± 0.19 and 0.79 ± 0.13 in the 100 and 200 mg/kg bw dose groups, respectively. The authors concluded that BAA did not increase the fre quency of micronucleated cells in mouse bone marrow (Elias et al ., 1996).

A micronucleus induction test wa s performed with male B6C3F 1 mice (5/group) at dose levels selected on the basis of published toxicity info rmation and the results of a preliminary rangefinding study. The mice were inject ed intraperitoneally three tim es at 24 hr intervals with EGBE dissolved in a total dose volume of 0.4 mL phosphate-buffered saline at dose levels of 0, 17.19, 34.38, 68.78, 137.50, 275.00, 550.00 or 1100.00 mg/kg bw per day. The positive control mice received injections of cyclophos phamide, 10.00 mg/kg bw per day. Mice were killed 24 hr after the final injection and sm ears prepared from femoral bone marrow, which were then fixed and stained. Two thousand polyc hromatic erythrocytes (PCEs) were scored for the frequency of micronucleated cells in ea ch mouse. Data were tabulated as the pooled mean of each exposure group and the standard erro r of the mean. They were analysed with a one-tailed Cochran-Armitage trend test and by pair-wise comparisons between each dosed group and the vehicle control gr oup. Statistically significant results were recorded for P ≤ 0.025 with the trend test and P ≤ 0.025 divided by the number of dose groups with the paired comparison test. A final decision on the biological significance (the “final call”) was made onEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 209 the basis of the statistical analysis, the repr oducibility and the magnitude of the effects observed. All mice in the 1100.00 mg/kg bw dose group died, whereas all mice in the 550 mg/kg bw dose group survived. The frequency of micronucleated PCEs per 1000 cells in the vehicle control group was 2.5 ± 0.2 and the range of means in the dose groups was from 2.3 ± 0.3 at 34.38 mg/kg bw to 3.8 ± 0.8 at 137.50 mg/kg bw. The trend test was not significant ( P = 0.236) and the paired comparison test gave P= 0.05, whereas P ≤ 0.008 was required for statistical significance (NTP, 2000).

4.1.2.7.3 Summary of mutagenicity
EGBE is not mutagenic in bacteria, not withst anding a significant response according to one report in S. typhimurium TA97a. This was not substantia ted by another study specifically designed to investigate this finding. Neither BAL nor BAA were mutagenic in bacteria. Two of three mammalian cell mutation assays did not indicate any mutagenic activity for EGBE and a significant result was obtai ned in an assay using a very high concentration (20 mM) that was poorly reported. The same publication report ed a significant result at 20 mM with BAL, whereas another study found no effect at concentr ations up to 7.6 mM. There have been no mammalian cell mutation studies with BAA.
There have been reports of significant activity of EGBE in tests for SCE induction and cell transformation, but, again, the results have been inconsistent. Furthermore, the significant SCE results could be artefacts due to cell cy cle delay. There is also some indication of inhibition of gap-junctional intercellular comm unication in a single study with EGBE and its two major metabolites. A single assay for UD S induction used a technique that is now considered to be invalid, if a significant response is obtained.

No evidence for chromosomal aberration induction has been found in a number of mammalian cell culture studies with EGBE, or in one with BAL or BAA, whereas weak aneugenic effects were obtaine d in the only available study with EGBE and BAL, but not with BAA. Micronuclei found in long exposure in vitro studies with BAL and, to a much lesser extent with EGBE itself, but not with B AA appear to be due to aneuploidy, rather than chromosomal breakage.

In vivo , there is no evid ence for micronucleus induction in bone marrow cells or interaction with DNA in several organs of rats. The possi bility of non-disjunct ion occurring and not being detected in these assays appears to be remote, because BAA produced no evidence of aneugenicity in vitro . BAA is rapidly formed in vivo and is by far the most prevalent blood metabolite of EGBE, so exposure of possible ta rget cells to either EGBE or BAL at high concentrations is brief. The balance of th e evidence suggests that EGBE do not pose a significant mutagenic potential in vivo

.EURISK ASSESSMENT–2-BUTOXYETHANOLCAS111-76-2 CHAPTER4.HUMAN HEALTHRAPPORTEURFRANCER408_0808_HH_CLEAN210Table 4.81:In vivotests in mammals for the genotoxicity of EGBE and its metabolitesResultaTest system Source & purity of chemicalDoseb(LED/HID)ReferenceEGBEDNA adducts, Sprague-Dawley rat brain,kidney, liver, spleen & testis,in vivo(32Ppost-labelling)Merck, Germany 99 %-(at 24 h)120 mg/kg bworally x 1Keithet al., 1996Methylation level ofDNA, Sprague-Dawleyrat brain, kidney, liver, spleen & testisin vivoMerck, Germany 99 %-Keithet al., 1996Methylation level of DNA, FVB/N transgenicmouse brain, kidney, liver, spleen & testisinvivoMerck, Germany 99 %-Keithet al., 1996Micronucleus test, CD-1 mouse bone-marrowcellsin vivoMerck, Germany 99 %-800 mg/kg bwi.p. x 1Eliaset al., 1996Micronucleus test, B6C3F1mouse bonemarrow cellsin vivoDow Chemical, USA >99 %-550 mg/kg bwi.p. x 3NTP, 2000Micronucleus test, male F344/N rat bonemarrow cellsin vivoDow Chemical, USA >99 %-450 mg/kg bwi.p. x 3NTP, 2000BALNo data availableBAAMicronucleus test, CD-1 mouse bone-marrowcellsin vivoJanssen Chimica, Belgium >99 %-200 mg/kg bwi.p. x 1Eliaset al., 1996a+, positive; –, negative; NT, not tested;bLED, lowest effective dose; HID, highest ineffective doseEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 211 4.1.2.8 Carcinogenicity
There appear to have been no epidemiological studies of a relationship between exposure to EGBE and human cancer, although there have case reports of te sticular cancer and leukaemia in occupational circumstances th at have involved co-exposure w ith other chemicals. There are only two experiments in rodents available, one in rats and one in mice, in which EGBE was administered by the inhalation route.

4.1.2.8.1 Studies in animals

Inhalation

Rat
The carcinogenic potential of EGBE was studied in an experiment in which it was administered by whole-body exposure of groups of 50 male and 50 female F344/N rats to 0, 31.2, 62.5 or 125 ppm (0, 151, 302 or 604 mg/m 3 ) EGBE vapour (lot no. QP-921215-26D2, Dow Chemical, Plaquemine, LA, USA, purity > 99 %) for 6 hr per day, 5 days per week for 104 weeks. For haematological and bone marrow analyses, additional groups of 27 male and 27 female F344/N rats were exposed to 0, 62.5 or 125 ppm for evaluation at 3, 6 and 12 months and 9 male and 9 female rats we re exposed to 31.2 ppm for 3 months (for haematological examination onl y) and 6 months (NTP, 2000).
Rats were held in the testing laboratory for 18 days and were 7 – 8 weeks old when the study began. They were housed indivi dually, water was available ad libitum and feed was available ad libitum except during the exposure periods. All rats were observed twice daily throughout the study. Body weights were recorded at the beginning of the study, after which they were recorded monthly from week 5 until week 89 and then every two weeks from week 92 until the end of the experiment. Clinical observatio ns also were recorded monthly from week 5 until week 89 and then every two weeks until the end of the experiment. Haematological examinations were performed on an Ortho ELT-8/ ds 9000 analyser at 3- and 6-months and on a Roche COBAS Helios analyser at 12-months. Differential leukocyte and nucleated erythrocyte counts were made by microscopi c examination of blood films. Bone marrow cellularity was made using a Coulter Model Z H counter, while cytologi cal evaluations of bone marrow cell morphology and myeloid/erythroi d ratios were made microscopically.
Autopsies and microscopic examinations were ma de on all of the main study animals that had not been used for blood and bone marrow samp ling. Organ weights were not measured. At autopsy, all organs and tissues we re examined for grossly visibl e lesions and all major tissues were fixed and processed for histological examination. A quality assessment pathologist evaluated the slides from all tumours and po tential target organs, which included bone marrow, forestomach, kidney, liver, lung, nose a nd spleen of males and females and the adrenal and clitoral glands of females. The slides and the preliminary quality assessment report were then submitted to a National Toxi cology Program Working Group of pathologists.
Survival of exposed male and female rats was similar to the chamber control groups, the mean numbers of survivors at the end of the expe riment in each of the 0, 31.2, 62.5 and 125 ppm groups were 19, 11, 21 and 24 males and 29, 27, 23 a nd 21 females, respectively. No clinicalEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 212 signs were attributed to EGBE exposure. Body weights of ma le and female rats in the 31.2 and 62.5 ppm groups and male rats of the 12 5 ppm group were generally similar to the controls throughout the experime nt. Body weights of female rats of the 125 ppm group were generally lower than those of the chamber control from week 17 until the end of the experiment.
Non-neoplastic findings are described in section 4.1.2.6.1.3.
The incidence of benign or malignant phaeochro mocytoma (combined) of the adrenal medulla in females exposed to 125 ppm EGBE was not significantly increased compared to the chamber controls, but it did exceed the historic al control range. There was only one malignant phaeochromocytoma, which occurred in the 125 ppm group. The data are summarised in Table 4.82.

Table 4.82: Neoplastic and non-neoplastic lesions of the adrenal medulla in female F344/N rats
Chamber

control 31.2 ppm 62.5 ppm 125 ppm

No. examined 50 50 49 49
Hyperplasia (severity) 11 (1.9) 11 (2.3) 8 (2.1) 17 (2.5)
Benign phaeochromocytoma 3/50 (6 %) 4/50 (8 %) 1/49 (2 %) 7/49 (14 %)
Benign or malignant phaeochromocytoma 3/50 (6 %) 4/50 (8 %) 1/49 (2 %) 8/49 (16 %)

It was on the basis of these data that the au thors of the report conc lude that there was no evidence for carcinogenicity in male rats and equivocal evidence for carcinogenicity in female rats. The primary criterion used to dis tinguish phaeochromocytomas from medullary hyperplasia was the presence of mild-to-moderate compression of the adja cent tissue. Most of the phaeochromocytomas were small and not substa ntially larger than th e more severe grades of adrenal medullary hyperplasia. Indeed, th ere was only one phaeochromocytoma that was graded as malignant. Therefore, it is considered that these data from female rats provide only the weakest of marginal evidence for carcinogenicity.

Two nasal tumours were discovered: a chondrom a in a 31.2 ppm male and an adenoma in a 62.5 ppm male. In the absence of any preneoplasti c changes, these are considered incidental findings. Although the incidence of hyaline degene ration was significantly increased in all exposed groups of male rats, th e severity of the lesion was mi nimal and was not affected by
exposure.

Mouse
The carcinogenic potential of EGBE was studied in an experiment in which it was administered by whole-body exposure of gr oups of 50 male and 50 female B6C3F 1 mice to 0, 62.5, 125 or 250 ppm (0, 302, 604 or 1208 mg/m 3 ) EGBE vapour (lot no. QP-921215-26D2, Dow Chemical, Plaquemine, LA, USA, purity > 99 %) for 6 hr per day, 5 days per week for 104 weeks. For haematological and bone marrow analyses, additional groups of 30 male and 30 female B6C3F 1 mice were exposed to 0, 62.5, 125 or 250 ppm for evaluation at 3, 6 and 12 months (NTP, 2000).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 213 Mice were held in the testing laboratory for 18 days and were 7 w eeks old when the study began. They were housed indivi dually, water was available ad libitum and feed was available ad libitum except during the exposure periods. All mice were observed twice daily throughout the study. Body weights were recorded at the beginning of the study, after which they were recorded monthly from week 5 until week 93 and then every two weeks from week 94 until the end of the experiment. Clin ical observations also were r ecorded monthly from week 5 until week 93 and then every two weeks from week 94 until the end of the experiment. Haematological examinations were performed on an Ortho ELT-8/ds 9000 analyser at 3- and 6-months and on a Roche COBAS Helios analyser at 12-months. Differential leukocyte and nucleated erythrocyte counts were made by mi croscopic examination of blood films. Bone marrow cellularity was made using a Coulter Model Z H counter, while cytological evaluations of bone marrow cell morphology an d myeloid/erythroid ratios were made microscopically.
Autopsies and microscopic examinations were ma de on all of the main study mice that had not been used for blood and bone marrow samp ling. Organ weights were not measured. At autopsy, all organs and tissues we re examined for grossly visibl e lesions and all major tissues were fixed and processed for histological examination. A quality assessment pathologist evaluated the slides from all tumours and po tential target organs, which included bone marrow, forestomach, kidney, liver, lung, nose a nd spleen of males and females and the preputial gland, prostate, skin (p repuce), testis and urinary bla dder of males. The slides and the preliminary quality assessment report were then submitted to a National Toxicology Program Working Group of pathologists.
Survival of male mice exposed to 125 or 250 ppm was significantly less than that of the chamber control group, whereas survival in all other treated groups was similar to the chamber control group. The mean numbers of surv ivors at the end of the experiment in each of the 0, 62.5, 125 and 250 ppm groups were 39, 39, 27 and 26 males and 29, 31, 33 and 36 females, respectively. No clinical signs were attributed to exposure to EGBE. Body weights of exposed male mice were generally less than those of the chamber control during the last 25 weeks of the experiment. Body weights of fema le rats of the 250 ppm group were generally lower than those of the chamber control from week 30 until the end of the experiment, the difference being about 20 % for much of th at time. Body weights of the 62.5 and 125 ppm group females were generally lower than the ch amber controls from about week 60 until the end of the experiment.
Non-neoplastic findings are described in section 4.1.2.6.1.
There was a positive trend in the incidences of forestomach squamous cell papilloma and squamous cell papilloma or carcinoma combined in female mice. The incidences were significantly increased in the 250 ppm group, in which the only squamous cell carcinoma occurred (Table 4.83). These incidences exceeded the historical control range for female mice (9/973 = 0.9 % ± 1.1 %; range 0 % - 3 %). There was no significant increase in the incidence of these neoplasms in male mice, but they di d exceed the historical control range for male mice (7/973 = 0.7 % ± 1.0 %; range, 0 % - 2 %). Agai n, there was one squamous cell carcinoma, this time in the 125 ppm group (Table 4.83).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 214 Table 4.83: Neoplastic and non-neoplastic lesions of the forestomach in male and female B6C3F 1 mice Chamber

control 62.5 ppm 125 ppm 250 ppm

Males

No. examined 50 50 50 50
Ulcer (severity) 1 (1.0) 2 (1.5) 9** (1.6) 3 (1.7)
Epithelial hyperplasia (severity) 1 (3.0) 7* (2.3) 16** (1.8) 21** (2.3)
Squamous cell papilloma or carcinoma (percentage) 1 (2 %) 1 (2 %) 2 (4 %) 2 (4 %)

Females

No. examined 50 50 50 50
Ulcer (severity) 1 (3.0) 7* (1.3) 13** (1.5) 22** (1.4)
Epithelial hyperplasia (severity) 6 (1.8) 27** (2.0) 43** (2.4) 44** (2.9)
Squamous cell papilloma or carcinoma (percentage) 0 (0 %) 1 (2 %) 2 (4 %) 6* (12 %)

* Significantly different fro m the chamber controls P ≤ 0.05; ** P ≤ 0.01
In the liver of male mice there was a positive trend in the incidence of haemangiosarcomas that led to a significant difference between the 250 ppm group and the chamber controls (Table 4.84). The incidence at 250 ppm also exceed ed the historical control range for this tumour in male mice (14/968 = 1.5 % ± 1.5 %; range, 0 % - 4 %). There was also a positive trend in the incidence of hepa tocellular carcinomas that led to a significant difference between males of the 250 ppm group and the chamber cont rols. There was, however, no change in the incidence of hepatocellular adenomas and carcinomas combined, because of a reduced incidence of hepatocellular ade nomas in the treated groups (Table 4.84). In female mice there was a single haemangiosarcoma, in the 62.5 pp m group, which was considered to be an incidental finding. Furthermore, the incidences of hepatocellular adenomas were reduced in the 125 and 250 ppm groups of female mice and there was no change in the incidence of hepatocellular carcinomas (Table 4.84).
One possible explanation for the liv er neoplasms is infection with Helicobacter hepaticus
, which had been previously implicated in significant liver diseas e of mice, including hepatocellular neoplasms and haemangiosarcomas (Hailey et al ., 1998). Investigation of liver samples from this experiment failed to demonstr ate the presence of this bacterium, therefore the experiment was not confounded by H. hepaticus infection.
The NOAEC for tumourigenicity in mice is 125 ppm, based on an increased incidence of haemangiosarcomas in males and squamous cell papillomas or carcinomas in females at 250
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R408_0808_ HH _C LEAN 215 Table 4.84: Neoplastic and non-neoplastic lesi ons of the liver in male and female B6C3F 1 mice Chamber

control 62.5 ppm 125 ppm 250 ppm

Males

No. examined 50 50 49 50
Kupffer cell, pigmentation, haemsiderin (severity) 0 0 8** (1.0) 30** (1.2)

Haemangiosarcoma 0/50 (0 %) 1/50 (2 %) 2/49 (4 %) 4/49* (8 %)
Hepatocellular adenoma 22 18 18 17
Hepatocellular carcinoma 10 11 16 21**
Hepatocellular adenoma or carcinoma 30 24 31 30

Females

No. examined 50 50 49 50
Kupffer cell, pigmentation, haemsiderin (severity) 0 5* (1.0) 25** (1.0) 44** (1.0)

Haemangiosarcoma 0 1 0 0
Hepatocellular adenoma 16 8* 7* 8*
Hepatocellular carcinoma 10 12 13 10
Hepatocellular adenoma or carcinoma 22 16 18 18

* Significantly different fro m the chamber controls P ≤ 0.05; ** P ≤ 0.01

Transgenic mouse models
A study for increased tumour incidence or decr eased tumour latency was conducted in FVB/N trangenic mice (Oncomice Neo 01 TM ), which carry the viral Harvey ras (v-Ha- ras ) oncogene controlled by the mouse mammary tumour vi rus (MMTV) promoter. These are mice that present a stable, heritable mutation in the Ha- ras gene. The endpoints of the assay can be achieved, in principle, by both genotoxic and no n-genotoxic agents, but the limitations of the assay have not been defined. Two groups of seven-week old mice (25/sex/group) were dorsally implanted with subc utaneous minipumps (2002 Alzet TM , Alza Corp.) delivering either saline or EGBE continuously at a rate of approximately 120 mg/k g bw perday for two weeks. Sub-groups of mice were killed for histological examination on days 5, 10, 15, 60 and 120 after implantation of the minipumps. Special attention was given to the development of mammary gland masses. There was no adverse ef fect upon either food or water consumption. At the 120-day sampling time, the mammary gla nds of six control female mice and five treated mice showed cellular hyp erplasia. In addition, a control female mouse had a papillary cystic mammary adenoma and a treated mouse ha d an anaplastic carc inoma of the bladder (Keith et al ., 1996). The authors conclude that EGBE does not promot e initiated cells carryingEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 216 the v-Ha- ras oncogene. There is some uncertainty a bout the numbers of mice used in the experiment and each sub-group.

Dermal

No data were available

Oral

No data were available

4.1.2.8.2 Studies in humans

No data.

Mechanism of haemangiosarcomas formation in male mice and its significance for human health
Haemangiosarcomas, which arise from the endothe lial cell component of the liver (Frith and Ward, 1979), have been observed to increase in incidence in male mice treated with EGBE, but not in female mice or rats of either sex at high exposur e concentrations of EGBE. The apparent species and sex specificity of th is response may impact upon the human risk assessment process so possible mechanisms for the induction of these tumours are addressed in this section. The compound-specific experimental data are described in preceding sections of this document.

While some in vitro studies for genotoxicity have repor ted significant responses to exposure to EGBE or its metabolites, others ha ve not and there is no evidence from in vivo studies for clastogenic activity or for covale nt interactions with DNA. It is considered, therefore, that there is a lack of evidence for a role for genotoxicity induced by EGBE or its metabolites in the neoplastic process.
A possible alternative mechanism of haeman giosarcomas induction can be based on the haematotoxicity of the major urinary metabolite, BAA. It has been established that BAA is the metabolite that induces haemolytic anaemia in mice of both sexes. However, it also induces anaemia in rats, this species being slightly more sensitive than mice. For the haemolysis hypothesis to be sustained, there should be, therefore, other species and sex differences amongst the rodents that are im portant components of this mechanism.
Following the haemolysis produced by exposure to EGBE, haemosiderin is deposited in several cell types in liver, including Kupffer cells and hepatocytes of mice and rats. In addition, it has been recognised that murine e ndothelial cells have a significant phagocytic activity (Steffan et al ., 1986), which may, therefore, serv e as a mechanism by which insoluble iron complexes or senescent erythrocytes can en ter these cells. Ferrous iron in haemosiderin can undergo redox cycling, the oxidative portion of which produces ferric iron and the highly reactive and damaging hydroxyl radical according to the Fenton reaction:
Fe(II) + H 2 O 2 → Fe(III) + • OH + OH -EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 217 The possible development of oxidative stress as a result of these iron deposits was studied in hepatocytes of male rats and mice (see Mechanistic studies of liver pathology, above), with the conclusion that rat hepatocytes in culture ar e markedly less susceptible to oxidative stress than are mouse hepatocytes. The antioxidant cap acity of the endothelial cells of the liver of rats is much less than that found in either th e hepatocytes or the Kupffer cells (DeLeve, 1998; Sporalics, 1999). Therefore, if there should be similar haemosiderin de position in endothelial cells, Kupffer cells and hepatocytes, the endot helial cells would be th e least well protected and thereby suffer the greatest oxidative damage. An in vivo study (Siesky et al ., 2002) has demonstrated that treatment of male mice with EGBE at dose levels (orally administered by gavage) equivalent to those that were associated with increased incidences of haemangiosarcomas in a 2-year inhalation expe riment leads to oxidative damage to hepatic DNA and lipid and increased DNA synthesis, par ticularly in endothelial cells, but also in hepatocytes. None of these effects occurred in rats. The in vivo and in vitro demonstrations of species differences in susceptibility to oxidativ e stress are, therefore, consistent with each other and the hypothesis that oxidative stress is an essential component of the process leading to haemangiosarcomas development in male mice treated with EGBE.
In addition, an in vivo study of possible differences between male and female mice does suggest that there is a marginally greater su sceptibility of male m ouse liver to oxidative damage than of female mouse liver. This sex di fference does not seem to be large in terms of measurements of either acute oxidative DNA damage or lipid peroxidation, but there did remain a larger reserve of antioxidant potential in the females than in the males. Accordingly, it would not be possible to predict with any a ssurance that haemangiosarcomas would develop in males, but not in females. Indeed, the neop lastic response was not large even in the male mice, so it could be that a pa rticular critical level of oxidativ e damage had been surpassed in the males, but had not been attained in the females. Deguchi et al . (1995) showed that the presence of female sex hormones imparts hi gher antioxidant properties than do male sex hormones. This same group (Okada, 1996) also f ound much lower levels of lipid peroxidation in female mouse kidney than in male mouse kidney. This result demonstrates that there is a higher level of protection in female than in male mouse kidney. It also demonstrates that sexrelated differences in susceptibility to oxidativ e damage are not restrict ed to the liver and, therefore, the consequences of such damage ma y show a sex-related inci dence pattern in other organs of the body.
In response to BAA, human erythrocytes in cu lture are clearly much less sensitive than the rodent cells by at least an or der of magnitude. Furthermore, haemotoxicity was not observed in the two available occupationally exposed people or in volunte ers under controlled conditions to EGBE (see 4.1.1.1). Exceptionally hi gh exposure to EGBE, as encountered in a single case of attempted suicide in an adult (e stimated dose 4500 mg/kg bw), did show that it is possible for human erythrocytes to be dama ged as a result of exposure to EGBE, but the effects require doses that are close to lethal (they were not seen in other attempted suicide cases where the estimated doses were at leas t 1000 mg/kg bw) and ar e not encountered in occupational circumstances or in normal consumer use (see 4.1.1.2). Consequently, they should not be considered in an assessment of risk. In case s of accidental poisonings of children, no evidence of haemolyt ic effects has been found. Theref ore, if haemolysis is an essential requirement for the induction of h aemangiosarcomas by EGBE then man is not a susceptible species.
The data available are consistent with the pr oposal that haemangiosarcomas observed in male mice could arise in mice of both sexes as a re sult of haemolysis leading to haemosiderin deposition. These deposits form nuclei for oxygen radical production that can damage manyEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 218 cellular components, including DNA, unless there is sufficient an tioxidant protection. When this deposition in the sinusoidal cells of the liv er reaches a certain level, the oxidative defence mechanisms available to the cells are overw helmed, creating the c onditions for neoplastic responses in the endothelial ce lls of the hepatic blood vessels. Since man is much less sensitive to the haemolytic effects of EGBE, damage to blood cells not having been observed except in cases of very high exposure found in attempted suicides, the low level of haemangiosarcomas induced in male mice, but not in either female mice or in rats of either sex might have no significance for human risk a ssessment. The weakness in this argument is that haemosiderin deposition was reported to oc cur in Kupffer cells, whereas its occurrence or otherwise in sinusoidal endot helial cells has not been reported. However, it has been demonstrated that active oxygen species can migrate from Kupf fer cells to the adjacent endothelial cells (Klaunig, 2004), and an endot helial cell response, in the form of proliferation, has been demonstrated in male mice, but not male rats (Siesky et al ., 2002), at dose levels that are associated with an incr eased incidence of haemangiosarcomas in male mouse liver.

Relevant information from studies not involving EGBE
The process by which haemangiosarcomas in cidence is increased in male B6C3F 1 mice treated with EGBE has been pr esented as a compound-specific m echanism. Elements of this mechanism may be applicable to other chemi cals that induce haemangiosarcomas, but it is extremely unlikely that generalisa tions can be made that include all of the steps described. This is because of:

• differences in metabolism and kinetics between species, sexes and routes of administration for specific compounds;

• the interplay of other toxico logical processes (e.g., genetic toxicity) that may modify fundamentally, or to a lesser degree, th e pathway leading to haemangiosarcomas development;

• chance differences in incidence between sexes (particularly when the incidence is low);

• missing information, such as the lack of h aematological data (particularly in older studies).
Results have been summarised (Table 4.85) on chemicals tested for carcinogenicity in B6C3F 1 mice and/or F344/N rats for which evidence of haemosiderin deposition is available in relatively complete reports that are in the public domain (spe cifically, NCI and NTP reports). In compiling this list, it was evident th at some chemicals did not cause haemosiderin deposition in the liver, but did so in other organs. Th ese have not been in cluded. Also, in the columns listing the haemangiosarcoma induction re sponse, it is stressed that the incidences refer only to the liver. Thus, the emphasis is on the relationships between haematotoxicity, haemosiderin deposition and haemangiosarcoma in th e liver. In this orga n, it was expected on the basis of the current hypothesis that, whil e the reasons for haematotoxicity might be different after exposure to different chemicals, the hepatic response to hepatic deposition of haemosiderin should be the same for any ch emical, including EGBE. In other organs, modifying factors may prevail. The spatial re lationships between the phagocytic cells and the cell populations in which the neoplastic response arises might be different from those found between Kupffer cells and hepati c sinusoidal endothelial cells; th ere may also be differencesEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 219 in protective capacity, e.g., sp lenic phagocytes have a higher antioxidant capacity than do sinusoidal endotheliam cells (DeLeve, 1998).
The data show that mice are more susceptibl e to haemangiosarcoma development than are rats, and male mice are more sensitive than female mice. None of the chemicals that are listed in section C (male mice) of table 4.85 induced haemangiosarcomas in section D (female mice) and only those chemicals in section C that de monstrably induced a lif e-long exposure of the liver to Kupffer cell pigmentation also induc ed increases in hepatic haemangiosarcoma incidence. At first glance, the main conclusion to be reached is that most of the data remain consistent with the suggested mechanism, in that significant haemosiderin deposition does not occur in cases where there is an absence of an increased incidence of haemangiosarcomas. However, some results require fu rther investigation. It is not suggested that haemosiderin deposition is the only mode of action that can result in haemangiosarcomas. The highest incidence of haemangiosarcomas occurred in male mice gavaged with pentachloroanisole. In this series of experiments, the Kupffer cell pigmentation observed at 13 weeks did not contain iron, bile or PAS-positive material. It app ears that special staini ng techniques were not applied to the pigment found in the 2-year study, therefore it is not known if it was haemosiderin or the non-iron pigment found in the 13 week st udy. It is al so noted that material was more prevalent in hepatocytes than in Kupffer cells. If it were not haemosiderin then pentachloroanisole would cease to be of use in looking for support for the mechanism.
Another case requiring discussion is C.I.Pigment Red 3, which did not induce haemangiosarcomas in either mice or rats, but haemosiderin deposition was observed in spleen and a “green-brown pigment” occurred in Kupffer cells. The latt er was not identified as haemosiderin in the report and, if it were not haemosiderin, then C.I.Pigment Red 3 would cease to be of use in looking for support for the proposed mechanism. Furthermore, if haemolytic anaemia was produced by C.I.Pigment Red 3 in mice, it was transient – unlike in rats – as suggested by the haematological chan ges observed at the end of the preliminary 2- week study, but not at the end of the 13-week study (p.62 of NTP TR 407). This contrasts with the haematotoxic activity of EGBE, which pe rsisted for at least 12 months (the longest period examined). This does lend some support to the hypothesis that life-long exposure of the liver to Kupffer cell pigmenta tion is required as a pre-requisite for an increase in liver haemangiosarcoma rates in male mice.
In the case of methyleugenol, haemosiderin depos its were found in the livers of female mice, but not of male mice or in rats of either sex. Haemangiosarcomas we re not induced, whereas there were increases in hepatocellular neop lasms. Although the mice in this study were infected with Helicobacter hepaticus , it was argued that this did not compromise the outcome. Even in the EGBE study, the increase in haem angiosarcomas incidence in male mice was modest and did not occur in female mice; theref ore, the absence of thes e neoplasms in female mice of the methyleugenol st udy does not conflict with th eir proposed mechanism of induction by EGBE.
The conclusion that can be reached is that, in addition to EGBE, only two compounds support the pattern of chronic haemosiderin deposition and increased incidence of haemangiosarcomas in male mice but not female mice, these being p -chloroaniline and p

nitroaniline. Another group has recently stud ied the possible association liver hepatic haemangiosarcomas and chemically induced haemosiderosis in mice in the US NTP database (Nyska et al ., 2004) and reached essentially the same conclusion. In addition to the qualitative association desc ribed here, however, Nyska et al . (2004) also showed that there was a very high statistically significant association between hepatic haemangiosarcomas andEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 220 Kupffer cell pigmentation.( p < 0.001). In all cases, the caus e of the haemosiderosis was haemolysis due to the chemical.
There are very few rodent studies with other compounds that are capable of providing support for the hypothesis proposed to explain the low level increased incidence of haemangiosarcomas in male mice exposed to EGBE. The available evidence is, however, consistent with the hypothesis. Currently, the most likely mechanism is that proposed, based on metabolism to BAA, followed by rodent eryt hrocyte-sensitive haemolysis, deposition of iron-protein complexes in the liver and the sust ained generation of toxi c (cytotoxic or DNA damaging) radicals from this source.
Human studies have demonstrated an enormously increased risk for hepatocellular cancer in hereditary haemochromatosis (e.g., Niederau et al ., 1985 found an increased risk of 219, based on 16 cases) that has been attributed to th e toxic effects of iron, but there also seems to be slightly increased risks for extra-hepati c cancers. Some studies have found genetic associations between several cancers and th e most common mutation, C282Y, of the HFE gene that causes hereditary haemochromatosis. Such associations only occur, however, in the presence of a particular allele of the transfer rin receptor gene. This s uggests that the increased cancer risk is due to the effects of iron (Dorak et al ., 2002). Additionally and independently of the genetic factors, dietary iron appears to be a risk factor fo r some cancers, e.g., colorectal cancer (Nelson, 2001), and dietary iron overloa d has been associated with about a 10- foldgreater risk of hepatocellular carc inoma among black Africans (Mandishona et al ., 1998). Focussing specifically upon liver angiosarcoma, however, the frequently quoted risk factors for this rare human cancer (0.5 – 2.5 cases per 10 7 people), which include hereditary haemochromatosis, contribute to explain no mo re than about 20 % of the published cases. Indeed, apart from occupational exposure to vinyl chloride, the ae tiology of this cancer remains largely unknown (Zocchetti, 2001). Therefor e, it is not possible to conclude whether iron plays any role in human angiosarcoma.EURISK ASSESSMENT–2-BUTOXYETHANOLCAS111-76-2 CHAPTER4.HUMAN HEALTHRAPPORTEURFRANCER408_0808_HH_CLEAN221Table 4.85: Summary of relationship between chemicals inducing haematoxicity and haemangiosarcomas in B6C3F1 mice and F344 ratsIncidences are for control, low, middleand high dose groups, respectively.A. Male RatsChemical Haematologyindicating toxicityto erythrocytesIncidence of Kupffer cell pigmentation(KCP) or hepatic haemosiderin (HS) at 2yearsKupffer cell pigmentation (KCP)or hepatic haemosiderin (HS)observed at 3 monthsIncidence of hepatichaemangiosarcomaReference**EGBE Yes, at 14 wks KCP: 23/50, 30/50, 34/50, 42/50 KCP 0/50, 0/50, 1/50, 0/50 TR484o-Nitroanisole Yes, at 2 & 13 wks KCP: 0/20, 1/20, 18/20 KCP None TR416p-Chloroaniline Yes, at 13 wks HS: 1/49, 0/50, 0/50, 26/50 KCP None TR351Pentachloroanisole Normal at 13 wks. KCP: 0/50, 1/50, 4/50 None (hypertrophy of Kupffer cells) None TR414Pyridine Yes, at 13 wks KCP: 4/50, 11/50, 20/50, 25/50 HS, but cell-type not identified None TR470Titanocene dichloride Yes, at 2 wks KCP: 1/60, 39/60, 41/60 None None TR399 (ratsonly)D&C Yellow No. 11 No haematology KCP: 7/50, 15/51, 23/51, 26/54 KCP None TR463 (ratsonly)Butyl benzyl phthalate KCP, 0/59, 1/58, 6/59, 6/58 None TR458Cupferron (Nhydroxy-N-nitrosobenzenamine)No haematology. HS: 0/49, 15/48, 28/43 No data None TR100No significant haemosiderin/pigmentation deposition was observed with: Methyleugenol, 2,4-diaminophenol.2HCl, CI Pigment Red 3.EURISK ASSESSMENT–2-BUTOXYETHANOLCAS111-76-2 CHAPTER4.HUMAN HEALTHRAPPORTEURFRANCER408_0808_HH_CLEAN222B. Female RatsChemical Haematologyindicating toxicityto erythrocytesIncidence of Kupffer cell pigmentation(KCP) or hepatic haemosiderin (HS) at 2yearsKupffer cell pigmentation (KCP)or hepatic haemosiderin (HS)observed at 3 monthsIncidence of hepatichaemangiosarcomaReferenceEGBE Yes, at 14 wks KCP: 15/50, 19/50, 36/50, 47/50 KCP None TR484o-Nitroanisole Yes, at 2 & 13 wks KCP: 8/20, 2/20, 20/20 KCP None TR416Pentachloroanisole Normal at 13 wks. Pigmentation in hepatocytes None (hypertrophy of Kupffer cells) None TR414Pyridine Yes, at 13 wks KCP: 6/50, 2/50, 6/50, 17/50 HS, but cell-type not identified None TR470Titanocene dichloride Yes, at 2 wks KCP : 3/60, 45/61, 50/60 None None TR399 (ratsonly)CI Pigment Red 3 Yes, at 2 & 13 wks. KCP : 0/50, 3/50, 14/50, 41/50* Pigment (type & cell-type notidentified)None TR407D&C Yellow No.11 No haematology KCP: 9/50, 11/51, 16/50, 32/51 KCP None TR463 (ratsonly)Butyl benzyl phthalate KCP: 4/60, 1/60, 6/60, 10/60 None TR458Cupferron (Nhydroxy-N-nitrosobenzenamine)No haematology HS: 0/48, 9/44, 33/44 No data None TR100No significant haemosiderin/pigmentation deposition was observed with: Methyleugenol (anaemia at 14 wks), 2,4-diaminophenol.2HCl, butylbenzyl phthalate,p-chloroanilineEURISK ASSESSMENT–2-BUTOXYETHANOLCAS111-76-2 CHAPTER4.HUMAN HEALTHRAPPORTEURFRANCER408_0808_HH_CLEAN223C. Male MiceChemical Haematologyindicating toxicityto erythrocytesIncidence of Kupffer cell pigmentation(KPC) or hepatic haemosiderin (HS) at 2yearsKupffer cell pigmentation (KPC)or hepatic haemosiderin (HS)observed at 3 monthsIncidence of hepatichaemangiosarcomaReferenceEGBE Yes, at 14 wks KCP: 0/50, 0/50, 8/50, 30/50 KCP 0/50, 1/50, 2/50, 4/50 TR484p-Chloroaniline Yes, at 14 wks. KCP/HS: 0/50, 0/49, 0/50, 50/50 KCP 2/50, 2/49, 1/50, 6/50 TR351Pentachloroanisole No haematology KCP: 1/50, 50/50, 50/50 KCP (but not containing iron) 2/50, 8/50, 10/50 TR414p-Nitroaniline Yes, at 2 & 13 wks KCP: 1/50, 1/50, 8/50, 50/50 KCP 0/50, 1/50, 2/50, 4/50 TR418o-Nitroanisole Yes, at 13 wks KCP: 0/50, 0/50, 3/50, 16/50 No 2/50, 2/50, 1/50, 0/50 TR416CI Pigment Red 3 Normal at 13 wks KCP : 0/50, 5/50, 30/50, 41/50* No 0/50, 1/50, 1/50, 0/50 TR4072,4-Diaminophenol.2HClNo haematology at13 wks. Yes, at 15monthsKCP: 0/50, 44/50, 47/50 No 1/50, 0/50, 1/50 TR401No significant haemosiderin/pigmentation deposition was observed with: Methyleugenol, Pyridine, CupferronEURISK ASSESSMENT–2-BUTOXYETHANOLCAS111-76-2 CHAPTER4.HUMAN HEALTHRAPPORTEURFRANCER408_0808_HH_CLEAN224D. Female MiceChemical Haematologyindicating toxicity toerythrocytesIncidence of Kupffer cell pigmentation(KPC) or hepatic haemosiderin (HS) at 2yearsKupffer cell pigmentation (KPC)or hepatic haemosiderin (HS)observed at 3 monthsIncidence of hepatichaemangiosarcomaReferenceEGBE Yes, at 14 wks KCP: 0/50, 5/50, 25/50, 44/50 KCP 0/50, 0/50, 1/50, 0/50 TR484p-Chloroaniline Yes, at 14 wks KCP: 0/50, 0/50, 1/50, 46/50 1/50, 0/50, 0/50, 1/50 TR351PentachloroanisoleNo haematology KCP: 0/50, 37/50, 48/50 0/50 0/50, 1/50 TR414p-Nitroaniline Yes, at 2 & 13 wks KPC: 1/50, 1/50, 4/50, 39/50 1/50, 1/50, 0/50, 0/50 TR418Methyleugenol No haematology HS: 0/50, 11/50, 24/50, 19/50* 0/50, 1/50, 0/50, 0/50 TR491CI Pigment Red 3 Normal at 13 wks KCP: 2/50, 1/50, 1/50, 29/50* 0/50, 0/50, 1/50, 0/50 TR4072,4-Diaminophenol.2HClNo haematology at13 wks. Yes, at 15monthsKCP, 0/50, 31/50, 50/50 None TR401No significant haemosiderin/pigmentation deposition was observed with: Pyridine, Cupferron,o-Nitroanisole*See discussion in the text.**NTP Technical Report NumbersEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 225 Mechanism of forestomach tumour formation in mice and its significance for human health

. Squamous cell papilloma and carcinoma of the forestomach have been induced in female mice, with some evidence (that does not reach a level of significance) for an increase in male mice. In contrast, there is no evidence for the induction of forestomach tumours or even preneoplastic effects, such as hyperplasia, in rats of either sex in the 2 year study, although hyperplasia was observed in female rats in a 14 week study in which higher exposure concentrations were used.
EGBE is a liquid with low vapour pressure that can therefore deposit on the fur of animals in whole body exposure experiments. The inha led vapour may also condense in the nasopharynx. By both this mechanism and as a result of grooming contaminated fur, EGBE can achieve a significant exposure by the oral route, even in inhala tion experiments. Although this is a plausible explanation for the toxi c and tumourigenic effects of EGBE on the forestomach, only small amounts of EGBE ( < 10 mg /kg) were found on the fur of mice at the end of 6 hr, whole body exposures to 250 ppm (t he highest concentration used in the NTP carcinogenicity experiment). In addition, dos age of mice by either intraperitoneal or subcutaneous injection also resulted in forestom ach lesions. It is clear, therefore, that EGBE accumulates in the forestomach as a result of a combination of processes. In addition to grooming of EGBE present on the fur of the mice and, possibly, mice licking the walls of the exposure chamber, it is likely that salivary ex cretion as well as mucociliary transport of material deposited in the bronchi, followed by ingestion, are other means by which exposure of the stomach could have occurred during the study.

The stomach of rats and mice consists of a fo restomach (not found in man) and a glandular portion. The forestomach acts as a storage orga n, where ingested material can remain for several hours, before it is transferred to the gl andular portion in which there is a rapid transit and the first stages of digestion occur. Again, this is not the only mechanism that results in prolonged exposure of this organ. EGBE and, to a lesser extent, BAA is al so eliminated more slowly from forestomach tissue than from eith er blood or other tissues following either oral gavage or intraperitoneal injection. Consequently , there is greater potenti al for damage to be induced by toxic substances in the fore stomach than in the glandular stomach.
Study of the stomachs of mice orally administer ed either EGBE or BAA shows that, at the doses used, there was no damage to the glan dular stomach by either compound and that damage in the form of hyperkeratosis occurred in the forestomach at lower doses of BAA than of EGBE. There is significant alcohol and alde hyde dehydrogenase activ ity in both parts of the stomach in rats and mice. While there wa s little difference in th e activity of aldehyde dehydrogenase in rats and mice, there were major species differences in alcohol dehydrogenase activity, the mouse enzyme havi ng very much higher affinity constants and maximal rates than the rat enzyme, when using EGBE as the substrate. Therefore, there is greater potential for EGBE to be metabolised to BAA in the forestomach of mice than in the forestomach of rats.
Hyperplasia and hyperkeratosis are histological responses to exposure to a wide range of chemicals that also produce papilloma and squamous cell carcinoma of the forestomach. Many of these chemicals are devoid of genotoxic ity, the neoplasms apparently developing as a result of persistent cellular damage and sustained hyperplasia (Kroes and Webster, 1986). While there is nothing unique to rodents about th is process, the fundamental differences in physiology and function between the rodent forestomach, on the one hand, and the humanEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 226 stomach and the rodent glandular stomach, on the other hand, point to the low probability that the latter would be targets for neoplasia by this mechanism. This proposal is substantiated by the lack of any neoplastic response in the gl andular stomach of mice exposed to EGBE under conditions that produce forestomach tumours.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 227 PROPOSED MECHANISMS OF ACTION ASSESSED WITHIN THE IPCS FRAMEWORK

Introduction
The experiments that form the bases for thes e evaluations of mechanism of carcinogenic action are summarised in the precedi ng sections of this document.

EGBE, when delivered as a va pour, induces tumours of the forestomach in female mice and haemangiosarcomas of the liver in male mice. There was also some evidence (which did not reach statistical significance) for an increase in forestomach tumours in male mice. Neither of these tumours do not occur in the other sex in mice and no significant elevation of tumour incidence is found in rats. These findings have not been verified in i ndependent experiments.

Postulated Mode of Action fo r the Induction of Tumours of the Forestomach in Female Mice

The proposed mechanism of action for production of tumours in the forestomach is the local generation, as well as accumulation of cyto xic metabolite(s) that induce a sustained, compensatory cell proliferation, neoplasia arising out of this proliferating cell population. The neoplasia was mainly papillomas, a singl e squamous cell carcinoma arising in the highest dose group of female mice.

Key Events

The following important steps are involved in the generation of forestomach tumours:

Enhanced exposure of the forestomach to EGBE and its metabol ites from multiple external and internal sources. EGBE delivered to female mice by inhalation was distributed throughout the internal organs and was present in the stom ach contents within 5 min after exposure. It was present in the mucosa of the forestomac h, the buccal cavity and the oesophagus 24 and 48 h after exposure, but the high leve ls observed in the forestomach was in contrast to the much lower levels found in the glandular stomach and the duodenum. EGBE was also found on fur and in the buccal cavity, oesophagus and stomach contents. EGBE delivered to female mice by intravenous injection was selectively concen trated in several tissu es, including liver, bone, Harderian glands and buccal cavity. The muco sa of the forestomach and glandular stomach were also labelled, but to similar extents. After EGBE administration by this route or subcutaneous injection, both EG BE and butoxyacetic acid were ex creted in saliva and found in the stomach periods for prolonged periods.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 228 Metabolism to cytotoxic metabolite(s) , most probably butoxyace tic acid, if not already presented to the forestomach in this form. EGBE is metabolised by preparations from the forestomach and glandular stomach of rats as well as mice. The enzymes principally responsible for EGBE metabolism are alco hol and aldehyde dehydrogenase (a minor oxidative pathway is mediated by cytochrome P450 enzymes). These enzymes are found in both the glandular stomach and forestomach of rats and mice, but whereas they are concentrated in the stratified squamous epith elium of the forestomach, their distribution is diffuse in the glandular stomach of both species . There are no major species differences in the tissue average activity of aldehyde dehydr ogenase, but the maximum rate of alcohol dehydrogenase activity is up to an order of ma gnitude higher in mice than in rats. The forestomach is a target tissue because EGBE en tering the stomach is held there and because the metabolising enzymes are concentrated in the superficial layers of the forestomach. BAA has shown to be a more potent in producing adve rse effects on the forestomach than EGBE. It may be that there is a species difference in part because the rate of metabolism of EGBE to 2-butoxyacetaldehyde is slower in rats than in mice, hence it is probable that either the generation of the more cytotoxi c butoxyacetic acid is slower, or its maximum concentration is lower in rats than in mice.

Cytotoxicity and cell proliferation , as indicated by epitheli al hyperplasia, was often accompanied by ulceration in a two- year inhalation experiment in mice, especially in females, but these responses were not repo rted in rats. Administration of undiluted EGBE to male and female mice by gavage induced dose-related irri tation of the forestomach; the compensatory cell proliferation was confirmed by immunoche mical staining. Similar forestomach lesions could be induced by EGBE admini stered by intraperitoneal or subcutaneous injection and by butoxyacetic acid given orally. This metabo lite was more potent than EGBE itself.

Squamous cell papillomas or carcinomas were significantly increased only at the highest concentration in female mice. It also app ears that mice may be more susceptible to the induction of forestomach tumours than rats, the background incidence being higher in mice.

3⁄4 Dose-Response Relationship . In female mice, the incidence and severity of epithelial hyperplasia of the forestomach in creased with rising dose in female mice, as did the incidence of ulceration of th e forestomach. Ulceration occurred in male mice also, but the incidences were lowe r and not clearly concentration related.

3⁄4 Temporal Relationship. In a 14 week inhalati on study in mice, epithelial hyperplasia of the forestomach was obs erved at 125 ppm and above and had progressed at 250 ppm or 500 ppm (the highe st concentration) to inflammation, necrosis and ulceration. Thus, preneoplas tic events preceded neoplasia, which was only observed in the two-year study.

3⁄4 Strength, Consistency and Specificity of Association of the Tumour Response with Key Events. There has not been any investiga tion of the association of key events with a neoplastic outcome at the le vel of individual female mice. In broad terms, however, it is clear from the dose-re sponse and the temporal relationships that there is consistency in thes e events. The induction of fo restomach tumours has been observed only in a single expe riment. The entire chain of events has not, therefore, been verified by independent study. Su ch verification shoul d be more readily achieved by oral administration of EGBE directly into the forestomach, by gavage;EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 229 however, such an experiment would have little applicability to occupational or
consumer exposures.

3⁄4 Biological Plausibility and Coherence. As generalisations, chemicals that do not induce carcinogenesis through mutagenesis do so by modes of action that include an increase in cell proliferation, either by mit ogenesis or by stimula ting reparative cell proliferation provoked by cytotoxicity. Base d on the evidence available, the latter mode of action is biologically plausible for the induction of forestomach tumours in female mice by EGBE.

3⁄4 Other Modes of Action. While no other mode of action has been proposed, genetic toxicity has been considered and rejected. Other possibilities could include aberrant control of gene expression because of alkyl ation of regulatory proteins (e.g., in chromatin), with ensuing loss of genetic stabilit y. No studies have been conducted to investigate such a mechanism.

3⁄4 Assessment of the Postul ated Mode of Action. The data available are fully consistent with the proposed mechanism for the induction of forestomach tumours in female mice. The apparent female sex sp ecificity is likely to be due to chance.

3⁄4 Uncertainties, Inconsistencies and Data Gaps. The experimental observations are consistent with the general hypothesis propo sed; however, there are two areas that require more specific reasoning. These are: the species difference in response and the progression from hyperplasia to neoplasia. The proposed reason for the difference in response between rats and mice is the differe nces in kinetic prope rties of th e rat and mouse alcohol dehydrogenase. Whether th is actually leads to a difference in concentration or quantity of butoxyacetic acid in the forestomach is unknown. As an alternative hypothesis, mice may be more su sceptible than rats to the induction of forestomach tumours. This suggestion is based on the observed higher incidence of forestomach tumours in untreated mice. It is not known how proliferation of apparently normal cells becomes transforme d into neoplasia. This criticism can be levelled at any non-genotoxic mechanism of carcinogenesis (but see 1.3.5). There is consistency within the database as current ly known. It would be expected, however, that exposure of male mice by inhalation to somewhat higher concentrations of EGBE should also lead to forestomach neoplasia. Similarly, exposure of female and male rats to higher concentrations could also lead to neoplasia if such concentrations could be tolerated. Since the lower concentrations used in the two-year inhalation experiment with rats were selected on th e basis of carefully executed shorter term studies to identify the maximum tolerated dose, this hypothetical experiment would be unlikely to be successful.

Postulated Mode of Action for the Induction of Hamangiosarcomas of the Liver in Male Mice
The proposed mechanism of action for production of haemangiosarcomas of the liver is the deposition of haemosiderin in relevant cell-t ypes, possibly including endothelial cells from which haemangiosarcomas arise, and the genera tion of cytotoxic reactive oxygen species that either induce genetic changes by this secondary mechanism or sustained cell proliferation within the endothelial ta rget tissue, neoplasia arising out of this pr oliferating cell population.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 230 Key Events
The following important steps are involved in the generation of haemangiosarcomas:

Metabolism to haemolytic metabolite(s) , most probably butoxyacetic acid. EGBE is principally metabolised by alcohol dehydr ogenase and aldehyde dehydrogenase to 2- butoxyacetaldehyde and butoxyacetic, respectivel y. Butoxyacetic acid is a more potent haemolytic agent than either of its precursors; furthermore, these precursors appear not to be haemolytic unless they are metabolised. Inhi bition of aldehyde dehydrogenase reduced the haemolytic activity of 2-but oxyacetaldehyde. At least in vitro , butoxyacetic acid causes haemolysis at much lower concentrations in rat erythrocytes than in human erythrocytes.

Haemolysis caused by butoxyacetic acid and followed by persistent, dose related anaemia results in haemosiderin deposition in the liver. This material has been observed in Kupffer cells and hepatocytes of both mice and rats , but there is currently no evidence for haemosiderin deposition in endothelial cells. Since haemolysis occurs in mice and rats of both sexes, it is necessary to provide a rationa le for the specificity of the neoplastic response to male mice; however, this could be difficu lt in view of the lo w (although statistically significant) tumourigenic response in male mice.

Cell toxicity resulting from the iron-mediated gene ration of reactive ox ygen species from haemosiderin. Compared to rats and female mice, male mice have a reduced antioxidant capacity in the liver, causing them to be more susceptible to oxidative damage; however, it is not clear that this difference is important, give n the greater inherent susceptibility of male mice to the neoplasm of interest. Oral administ ration of EGBE to mice at doses up to 600 mg/kg bw/day for up to 90 days produced increas ed DNA synthesis in en dothelial cells during the first 14 days of exposure and in hepatocy tes after 90 days. Incr eased oxidative damage was also observed mice in this experiment, while no change was observed in rats.

Haemangiosarcomas arise from the endothelial target ce lls and are significan tly increased in male mice to 8% in the 250 ppm group. It also appears that male B6C3F 1 mice may be more susceptible to haemangiosarcomas than are female mice (control incidence rates in NTP experiments being about 2.5% in males and 0.9% in females) and mice are more susceptible than F344 rats (none having been reported in co ntrol groups in NTP experiments). Thus, the apparent sex and species specificity could mere ly be a reflection of inherent sensitivity.

3⁄4 Dose-Response Relationship. Haemolysis and persistent anaemia have been shown to occur in a dose related manner in a number of experiments. In two-year inhalation experiments, Kupffer cell pigmentation was si gnificantly increased over that seen in control animals for male and female rats exposed at 62.5 or 125 ppm, male mice at 125 or 250 ppm and female mice at 62.5, 125 or 250 ppm. The increases observed in the male and female mice were dose rela ted, but the incidence of Kupffer cell pigmentation was greater in female mice than in male mice. A significant increase in the incidence of haemangiosarcomas occurred only in male mice and only at the highest exposure conc entration of EGBE.

3⁄4 Temporal Relationship. The experiments show that haemolysis is an acute response and anaemia is a sustained response to re peated exposure to EG BE. Haemosiderin deposition is observed in short-term expe riments and therefore it is present for aEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 231 considerable period before the emergence of the haemangiosarcomas, which occurred at increased incidence on ly in a two-year inhala tion exposure experiment.

3⁄4 Strength, Consistency and Specificity of Association of the Tumour Response with Key Events. Data from experiments with other chemicals show that mice are more susceptible to haemangiosarcoma development than are rats, and male mice are more sensitive than female mice, but the database in support of the hypothesis that life-long exposure of the liver to Kupffer ce ll pigmentation is a pre-requisite for an increase in liver haemangiosarcoma rates in male mice is small, after exclusion of doubtful examples.

3⁄4 Biological Plausibility and Coherence. It has long been proposed (e.g., for peroxisome proliferators) that increase d, persistent generation of reactive oxygen species can result in neoplasia by an indirect genotoxic mechanism. It has been shown that EGBE does increase oxidative da mage in cells, as demonstrated by the generation of malondialdehyde, a deposition product of lipids, and 8- hydroxydeoxyguanosine, an oxygen adduct of DNA, and that differences in reduced antioxidant concentrations within cells may be responsible for sex and species differences in response. Nevertheless, no substance has been shown unequivocally to induce tumours through this mechanism.

3⁄4 Other Modes of Action. While no other mode of action has been proposed, genetic toxicity has been considered and rejected. Other possibilities could include aberrant control of gene expression because of alkylation of regulatory proteins, as suggested for forestomach tumours, but there is no evid ence to support this suggestion either for the forestomach mucosa or endothelial cells of the hepatic sinusoids. Kinetic studies with liver slices suggest that concentr ations of 2-butoxyacetaldehyde that mightinduce genetic damage are unlikely to be reached under the conditions of the longterm inhalation experiments.

3⁄4 Assessment of the Postulated Mode of Action. Haemangiosarcomas are uncommon and their incidence in this experiment was low (4/49) although out with the historical control range, at the highest dose. Because this is a low increase in incidence, and because it has not been confirmed, it is di fficult to assign any mechanism. With the rejection of genotoxicity as a possible mechanism and the strong evidence for a potential source of reactive oxygen species within the liver, it is reasonable to presume that these may play a definitive role in neoplasia.

Uncertainties, Inconsistencies and Data Gaps. By their nature, r eactive oxygen species are difficult to localise in the target tissue. In addition, the genera tor of these cyto toxic species, haemosiderin, has not been demonstrated in endothelial cells, from which haemangiosarcomas arise. Haemosiderin was demonstrated in Kupffer cells, so reactive oxygen species could arise in thes e cells, and the recent demons tration in one study of the migration of reactive oxygen species from Kupffe r cells to endothelial cells does suggest a manner in which the latter might be damage . The involvement of cytokines secreted by Kupffer cells has been suggested in the case of peroxisome proliferators, but the target in those cases is the hepatocytes. A similar interplay of cytokines between Kupffer cells and endothelial cells may be involve d in the case of haemosiderin deposition, but this remains to be demonstrated and may not be necessary in view of the alternative suggestion. A fundamental major uncertainty is that it is unknown if the obse rvation in a single experimentEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 232 of a low, barely significant incidence of haem angiosarcomas in male mice is reproducible or whether it might also be obser vable in female mice, should the experiment be repeated. However, there is reason to expect that female mice would be less susceptible to this lesion.Summary of carcinogenicity
EGBE is carcinogenic in male mice, where it causes a low incidence of haemangiosarcomas, and female mice, where is causes an increased incidence of forestomach tumours. It is not carcinogenic in rats. Genotoxicity is not an impo rtant toxicological prope rty of this chemical and it is unlikely that the low, variable and unc ertainly defined genotoxic activity can be the cause of the carcinogenic responses. Hypothe ses have been proposed and supported by experiment data in an attempt to explain the carcinogenic responses. These have been described above. In the case of forestomach tumours, the argument that they arise in a tissue subject to sustained abuse and cons equent repair is clear. It is likely that this finding is in reality not sex specific but merely due to chance that the lo w level incidence in females rose above the level of statistical si gnificance but it did no t do so in males. In the case of the haemangiosarcomas, data from experiments with other chemicals show that mice are more susceptible to haemangiosarcoma developmen t than are rats, and male mice are more sensitive than female mice, but the database in support of the hyp othesis that life-long exposure of the liver to Kupffer cell pigmentation is a pre-requisite for an increase in liver haemangiosarcoma rates in male mice is small, after exclusion of doubtful examples. With the rejection of genotoxicity as a possible mech anism, strong evidence for a potential source of reactive oxygen species within the liver, and a mode of action where each step has at least some supporting data, it is reasonable to presume that these may play a role in the neoplasia.
With regard to human relevance, the mechanism proposed for the induction of haemangiosarcomas strongly suggests that EGBE is not likely to be a carcinogenic hazard under conditions of normal handling and use, because human erythrocytes are demonstrably more resistant to haemolysis than are rodent erythrocytes. The mechanism proposed for the induction of forestomach tumours would also point to a lack of human relevance under conditions of normal handling and use. As stated recently (IARC, 2003), while people do not possess forestomachs, they do have comparable squamous epithelial tissues in the oral cavity and the upper two-thirds of the oesophagus. T hus, in principle, carci nogens targeting the forestomach squamous epithelium in rodents are relevant for man. Howeve r, the relevance for man is probably low for agents that have no de monstrable genotoxicity and that are solely carcinogenic for the forestomach squamous epithe lium in rodents after oral administration. Consequently, for these agents, the mode of carcinogenic action could be specific to the experimental animals (IARC, 2003). EGBE satis fies only some of these conditions. On the other hand, there are proposed mechanisms that are supported by experimental evidence to show how this chemical, even when inhaled, can accumulate in the forestomach contents, where it can remain for many hours to cause damage directly or after its metabolism to BAA.
In conclusion, given the species and sex specificity of the ne oplastic responses and the current evidence supporting the hypothesis that the more likely mechanism of action is based on haematotoxicity, then EGBE is unlikely to be a human carcinogen.Therefore, an appropriate classification for “No classification” is proposed for carcinogenicity . This classification proposal was agreed by the C&L working grou p. Moreover, the last IARC evaluation (2004 and 2006) has classified EGBE in the list 3 of carcinogens : not classifiable as to their carcinogenicity to humans (Group 3) on the basis of limited evidence in experimental animals and inadequate evidence in humans.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 233 Since the only carcinogenic effects can be considered secondary to haemolysis, and haemolysis is the key end point for repeat dose toxicity, no separate ri sk characterisation is necessary for the cancer end point. If there are no concerns for re peat dose toxicity, it can be considered that there will be no concerns for cancer either.

4.1.2.9 Toxicity for reproduction

4.1.2.9.1 Effects on fertility

Oral route
Repeat Dose Toxicity Studies
Two of the repeated dose toxicity studies repo rted effects of EGBE on the testis in male rodents. The first of these studies was from the Mellon Institute of Industrial Research (1963), wherein rats were given diets c ontaining 0, 0.01, 0.05, 0.25 or 1.25% EGBE for 93 days. These levels of EGBE were approxi mately equivalent to 0, 7, 38, 188 and 919 mg/kg/day. At 1.25% EGBE, male rats had decreased appetite , decreased body weight gain (50% less than control animal s), increased liver and kidney we ights and testicular atrophy. Rats given 0.25% EGBE also exhibited decreased appetite and body weight gain, as well as testicular atrophy. There were no apparent effects at 0.05% EGBE . Note that the purity of the EGBE used in this study is not given, nor does there appear to be analytical verification of EGBE doses, homogeneity and stability. The resu lts of this study are difficult to interpret given that testicular at rophy is not reported in other oral, re peated-dose studies even at higher doses of EGBE greater than 0.25% (e.g., 14-week drinking water study in rats and mice from NTP, 1993; continuous breeding study reported in Morrissey et al. , 1989). The weight of evidence indicates that EGBE does not affect the testis at doses below those causing significant systemic toxicity.

In the 14-week inhalation study, B6C3F1 mice (10/sex/group) were exposed to 0, 31, 62.5, 125, 250 and 500 ppm EGBE vapor (6 hr/day, 5 days per week). During the first 2 weeks of study, 2 male and 2 female mice died and an additional 2 males and 2 females were killed moribund. These mice had numerous clinical signs of toxicity, including abnormal breathing, red urine stains, and lethargy. Hi stopathologically, male mice ha d ulcerations and necrosis of the forestomach and female mice had forestomach necrosis. Forestomach ulcerations and necrosis were accompanied by inflammation, which in some cases included suppurative inflammation. These animals also exhibited at rophy of the spleen, thymus, testis and lymph nodes, liver and epididymal necrosis, and rena l tubule degeneration. With 8 of 20 animals succumbing to toxicity at 500 ppm, it is appare nt that the maximum tolerated dose MTD) had been exceeded. Mice exposed to 500 ppm that survived to scheduled necropsy had effects on the spleen liver, forestomach and kidney, but did not exhibit testicular or epidiymal lesions. There were no effects on the testis or epididymis at lower concentrations, nor were there effects on the testis/epididymis in the 14-week rat study, the 2-year m ouse chronic study, or the 2-year rat chronic study. Thus, effects on the testis and epididymis at 500 ppm EGBE were not specific to the reproductive organs as numerous ti ssues were affected and systemic toxicity was lethal.

As mentioned previously, the most sensitive endpoint in the 14-week study was haematology changes that were seen in males at > 125 ppm and in females at al l concentrations of EGBE.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 234 Continuous breeding

Male and female Swiss CD-1 mice receiv ed 0 0.5, 1.0 or 2.0 % EGBE (purity > 99 %), equivalent to daily intakes of 0, 720, 1340 and 2050 mg/kg bw, in their drinking water during a continuous breeding phase (CBP) with a 7-da y pre-mating period and a 98-day cohabitation period (Morrissey et al ., 1988 and 1989 ; Heindel et al. , 1990). The study was performed according to GLP. During the cohabitation period, deaths occurred in the female mice: 13/20 in the 2 % group, 6/20 in th e 1 % dose group, 1/20 in the 0.5 % dose group and 1/40 in the control group. The average body we ights in the female 2 % dos e group were consistently lower than the controls. In the male mice, no deat hs occurred but weight loss (1-2 % of initial body weight) in the two highest doses and redu ced weight gain were noted. Reduced fluid consumption was observed at all dose levels in both sexes (22 %, 18 % and 36 % reduction relative to controls at 0.5 %, 1.0 % and 2.0 %, respectively after 14 weeks of dosing). The numbers of fertile pairs from the surviving pairs were 38/39, 19/19, 13/14 and 5/7 at 0, 0.5, 1.0 and 2.0 % dose levels, resp ectively. Significan t reduction in reproductive performance occurred at 1 and 2 % dose levels as indicated by dose-related decrease in number of litters per fertile pair, litter sizes, pup viability a nd live pup weight. A small but significant reduction (by 5 %) of live pup weight was also observed in the 0.5 % dose group without other significant reproductive effects.
At the completion of the con tinuous breeding phase, the F 0 breeding pairs were separated and housed individually and exposure to EGBE conti nued. When the last litter was weaned F0 males and females from the 1 % dose group were mated with male and female control animals in a one-week crossover mating study to determ ine any sex-related reproductive effects of EGBE. Exposure to EGBE was discontinued du ring the one-week mati ng period and then reintroduced at 1 % dose level (estimated daily intake 1830 mg/kg bw). Control males and females were also mated for comparative purpose s. The proportion of su ccessful copulations from the breeding pairs was similar in all groups . However, the number of fertile females was significantly reduced in the group where treated females we re mated with control males. When evaluated over the 7 day period prior to necropsy, proportionally more females (7/13) in this 1 % treated group than controls (9/38) had oestrus cycles lo nger than 7 days. At necropsy, male and female mice from this 1 % dose group had si gnificantly lower body weights and increased relative kidney weights,and female also had significant increases in relative liver weight. No signifi cant differences were observed be tween the control and treated animals for the weights of reproductive organs, sperm motility, morphology or averageoestrous cycle length and frequency. In the only histopathological examination carried out on the treated females, no treatment rela ted kidneys lesions were observed. The crossbreeding results suggest that the fertility effect s were only due to effects on the female mice. Furthermore, these effects may have been an indirect consequence of the severe general systemic toxicity rather than a direct effect of EGBE on the reproductive organs.
A final phase was conducted to assess the fertility and repr oductive effects of EGBE in second generation (F 1 ) pups. The pups select ed were those born afte r the CBP and when the maternal animals were individually housed. As th ere were insufficient pu ps in the 1 and 2 % dose groups, only the pups from the 0.5 % dose group were used. The F 1 generation pups were nursed, weaned and reared to sexual ma turity. After weaning, the mice received 0.5 % 2- butoxyethanol in their drinking wa ter (estimated daily intake 950 mg/kg bw). At 74 ± 10 days of age, the F 1 animals from different litters were ma ted. The animals were necropsied after delivery. No significant fertility and repr oductive effects were observed in the F 1 animals as indicated by the proportions of successful copulation and fertile females, litter size, pup viability and live pup weights. Similarly, no tr eatment-related change s in the weights ofEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 235 reproductive organs, sperm motility, morphology and the oestrous cycle length and frequency were noted. However, a significant increase in relative kidney weight in the females and a significant increase in relative liver weight in both the ma les and females were observed.
In summary, significant advers e reproductive effects were obser ved in the females at very high dose levels (1340 mg/kg and above) which al so caused severe toxicity, including death. Under the conditions of the study, the NOAEL fo r reproductive toxicity of EGBE (fertility) can be set as 720 mg/kg/day. For developm ental toxicity, no NOAEL can be derived. A conservative LOAEL of 720 mg/kg/day can be take n as only a very sli ght decrease in pup weight was observed at this dose. No NOAEL or LOAEL can be determined for systemic parental toxicity because this kind of study is not designed to assess systemic toxicity although it is of note that there we re effects (reduced fluid cons umption) even at the lowest dosage of 720 mg/kg/day.

Other studies:
Groups of five male JCL-ICR mice received doses of 500, 1000, or 20 00 mg/kg bw/d EGBE (purity and GLP status unknown) by gavage for 5 days/week for 5 weeks (Nagano et al.
, 1984). The mice were killed on day 6 of week 5 and the effects on the te stis and associated structures were observed. Controls received wate r. At the highest dose level all the mice died and dose-related haematotoxicity (reduced re d blood cell count) was observed at the other doses. There were no significant effects on the abso lute or relative weights of the testis or seminal vesicle and coagulating gland at any dose tested. An earlier publication (Nagano, 1979) mentioned testicular atr ophy in one of five mice dosed with EGBE (1000 mg/kg). This observation was not considered significant by Nagano and his co-authors in their later publication (Nagano et al. , 1984).
In 13 week duration toxicity studies in F344/N rats and B6C3F1 mice conducted by the National Toxicology Program (1993), EGBE ( purity 99 %) was administered in drinking water at concentrations of 0, 750, 1500, 3000, 4500 or 6000 ppm to groups of 10 male and 10 female rats and at 0, 750, 1500, 3000, 4500 or 6000 ppm to groups of 10 male and 10 female mice. These concentrations provided targ et dose levels of 0, 100, 150, 250, 400 or 650 mg/kg bw per day (NTP, 1993). In addition to co nventional toxicological endpoints, vaginal cytology and sperm morphology eval uations were performed. Vaginal cytology was assessed over the 7 days prior to sacrifice and sper m morphology and motility were estimated from epididymal sperm samples taken at necropsy. In the rat, haematotoxicity was evident in males at doses of and above 3000 ppm and in all female groups. Terminal body weight was decreased by 9 and 14% in males given 4500 a nd 6000 ppm EGBE. In females, body weights were decreased by 14 and 22% at these concentrations. There wa s no treatment effect on testis weights but there was a reduction in the size of the uterus in females at 4500 and 6000 ppm. Changes in uterine weight were considered by th e authors to be secondary to the reduction in body weight gain rather than a direct effect of EGBE. The only spermatozoal measurement that showed significant change relative to the control group was sperm concentration which was slightly decreased in all groups of treat ed males; however, this effect was not doserelated. There were no significant differences from the control group in oestrous cycle length for treated females, although females trea ted at 4500 and 6000 ppm spent more time in dioestrous than the other groups. This correlates with the smaller uterine size, which was attributed to a secondary consequence of reduc ed body weight gain. In mice there were no chemical-related gross or microscopic lesions an d no biological significa nt changes in any of the reproductive parameters evaluated. The small differences in the rat reproductiveEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 236 endpoints with EGBE were in marked contrast to the significant effects of EGME and EGEE in both species, where concurrent studies de monstrated treatment-related testicular degeneration/ atrophy and, for EGEE, extended oestrous cycle length.

Table 4.85 bis: Data on estrous cycling for rats exposed to EGBE for 13 weeks in drinking water. Study parameters 0 ppm 3000 ppm 4500 ppm 6000 ppm

n 7 6 7 6
Necropsy body weight (g) 1 186 + 4 172 + 2 160 + 2 145 + 2
Estrous cycle length (days) 6.50 + 0.70 6.83 + 0.95 7.57 + 0.53 5.83 + 0.70
Estrous stages (% of cycle)

Diestrus 28.9 45.6 52.8 67.5

Proestrus 8.8 11.4 13.9 7.0

Estrus 57.9 38.6 20.4 17.5

Metestrus 4.4 4.4 13.0 7.9

1 Based on n = 10.
With respect to oestrous cycle evaluations, no rmally cycling Fischer 344 females have 5-6 day oestrous cycles. While there were 10 females per dose group, 3-4 females per group had estrous cycles longer than 12 days or cycles th at were unclear, including the control group. These animals were excluded from the analys is, meaning that only 6-7 females per group were evaluated. Furthermore, given the approx imate times in the various stages of estrous (see table below from Zarrow et al ., 1964), it appears that the control animals spent an excessive amount of time in estrus. Typically, estrus would comprise approximately 30% of the estrous cycle, rath er than the 57.9% reported for the control females. Using Zarrow’s table, if one assumes 2 days for estrus and minimal durations for the other estrous stages, estrus would only comprise 44% of the cycle (2 da ys out of 4.5 days total estrous cycle). This value is well below the 57.9% reported in th e control animals in the NTP study. Vaginal smears were collected only once per day. With only 6-7 rats per group and stages of the cycle that are less than one day in duration, some phases could have gone undetected.

Table 4.85 ter: Vaginal cytology during the estrous cycle of the rat 1 Time (days) Stage Vaginal smear
0.5-1 Proestrus (P) Many rounded epithelial cells, few leukocytes
1-2 Estrus (E) Cornified epithelial cells, no leukocytes
0.5-1 Metestrus (M) Maximum number of leukocytes, often in clumps surrounding epithelial cells. For our purposes th is will be classified as part of Diestrus.
1.5-2 Diestrus (D) Few epithelial cells; few to many leukocytes. Sometimes divided into Diestrus I and II

1 Modified from Zarrow et al. (1964).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 237 These points, coupled with the lack of effect on overall estrous cycle length and absence of histopathologic lesions, suggest that the effects on estrous cycle length are unlikely to indicate a specific treatment-related reproductive effect.
Thus, the effects on male and female rat re productive endpoints are not indications of selective reproductive system toxicity. These effects were minor in nature, only present in one species and only occurred in the presence of other systemic toxicity (i.e., decreased body weights, haematotoxicity). There were no hi stopathological lesions in reproductive organ

Inhalation route
In an inhalation study (Doe, 1984) , male Alpk/Ap Wistar-derived rats were exposed whole body to 800 ppm (4 mg/l – saturated vapour) EGBE (purity unknown) for 3 hours (no GLP). This study was performed to compare the effects of EGBE and EGME in rats. The rats were then observed throughout the next 14 days. The animals were sacrificed on day 15 and subject to gross macroscopic post-mortem examinati on, which included the weighing of the testes. Clinical observation revealed haematuria in trea ted animals. EGBE had no effect on testicular weights.
In the 90 day toxicity study of EGBE (purity 99.4 %) administered by the inhalation route (whole body) to Fisher 344 rats at doses of 5, 25 or 75 ppm for 6 hours/day, 5 days/week for approximately 13 weeks (16 males and 16 fe males/group) (Bushy Run Research Center, 1981b), there were no adverse e ffects on testicular weights and no reported pathological changes in the reproductive organs.

Studies on metabolites :

In vitro studies
Primary testicular cell cultures were treated w ith a series of GE and Alkoxyacetic acids (Gray et al. , 1985). In contrast to the action of methoxy-acetic acid (MAA) and ethoxy-acetic acid (EAA), neither n-propoxy-acetic acid (PAA) nor n-butoxyacetic ac id (BAA) was overtly toxic at a concentration of 10 mM for 72 hours. Under these conditions BAA produced no discernible effects. At 20 mM, BAA was still without appreciable effects.
To explore the development of quantitative indices of toxicity in the cell cultures, measurements were made of Carnitine Acetyl Transferase (CAT) and lactate dehydrogenase-X (LDH-X), two enzymes whose activity is higher in the pachytene spermatocytes than in the other cell types present. When the 4 alkoxyacetic acids were compared, there was a parallel between their effects on CAT and LDH-X activ ity and the severity of the morphological changes. BAA produced a decrease in CAT ac tivity (10 mM) in the attached germ cell fraction less marked than those of others alkoxy acetic acids, although st atistically significant (88 nmol/mim/culture vs 121.7 nmol/mim/culture in controls). When expressed as specific activities, the re sult was still significant (54.8 nmol/m im/mg cell protein vs 68 nmol/mim/mg cell protein). No effect was seen with BAA treatment in LDH-X activity.
Mixed cultures of Sertoli and germ cells we re incubated during 24 hours in the presence of BAA at doses ranging from 0 to 10 mM (Foster et al. , 1987). Cultures were also made with MAA and EAA. BAA did not produ ce any changes when tested in Sertoli-germ cell cultures at 5mM in contrast with effects seen with MAA and EAA (to a lesser extent) for which anEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 238 enhancement of germ cell loss (pachytene spermato cytes) were seen at this dose level. Some non specific cell loss wa s observed at 10 mM..

In vivo studies
BAA was administered orally by gavage to young SD rats (4 weeks old) for 4 days concurrently with 3 other alkoxyacetic acids at equimolar doses to char acterise their relative testicular toxicity (Gray et al. , 1985). The rats were killed 24 hr after the last dose. With PAA (776 mg/kg) and BAA (868 mg/kg) no morphological changes in th e testis were observed at the dose studied. Only MAA produced testic ular toxicity at the dose of 592 mg/kg.
In male Wistar rats (Foster et al. , 1987), the administration by ga vage of a single dose of BAA did not result in any testicular damages at the highest dose tested of 868 mg/kg (the animals were terminated 14 days after dosag e). At the dose of 868 mg/kg, animals showed evidence of haematuria. No other systemic nor sp ecific effects on testes were seen at doses of 174 and 434 mg/kg. This study was concurren tly performed with MAA and EAA and confirmed the testicular toxic ity of these other alkoxyacids.
In all sub-acute, sub-chronic and chronic studies no direct effects were se en on male or female reproductive organs at all doses tested (even s ub-lethal doses). Metabol ism of EGBE leads to the formation of a majority of BAA, which is not a male reproductive toxic substance, unlike MAA or EAA (main metabolites of EGME and EGEE respectively).
In the continuous breeding study, effects were seen on fertility only at doses which were severly toxic to the animals (1340 and 2050 mg /kg bw/d). A NOAEL of 720 mg/kg bw/day can be fixed for fertility effects by oral route in mice.

4.1.2.9.2 Developmental toxicity

Studies in animals

Oral route

Studies in rats
The teratogenicity of EGBE (> 99.0 % purity) was evaluated us ing Fisher 344 rats (Sleet et al. , 1991; Schwetz and Harris, 1993 ; Research tria ngle institute, 1988 as part of the National Toxicology Programme (NTP)). The purpose of the st udy was to determine whether the exposure of pregnant rats to EGBE during critical periods of cardiovascular development adversely affected the structure of the foetal heart and great vessel s. Other developmental toxicity was also recorded. EGBE (in distilled water) was administered via gavage to a total of 298 rats; 104 rats served as controls. The di stribution of doses for the 184 animals dosed on gestation days (GD) 9 thr ough 11 was as follows: 46 rats (0 mg/kg/day), 46 rats (30 mg/kg/day), 47 rats (100 mg/kg/day), and 45 ra ts (200 mg/kg/day). A to tal of 93 rats (all dose groups includ ed) were killed on GD 12 of gestatio n, and the remaining 91 rats, on GD 20. For the 220 rats dose d on GD 11 through 13, the distributi on of doses was as follows: 58 rats (0 mg/kg/day), 52 rats (30 mg/kg/day) , 59 rats (100 mg/kg/ day), and 51 rats (300 mg/kg/day). A total of 104 rats (all dose groups included) we re killed on GD 14, and the remaining 116 rats, on GD 20. Al so, on GD 20, foetuses we re killed and examined.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 239 The maternal effects of EGBE given from GD 9 - 11 or from GD11 - 13 at doses of and greater than 100 mg /kg/day included marked reductions in body weight and/or weight gain, increases in organ weights (kidney and spleen) and severe haematotoxicity. In particular, dramatic reductions in circulat ing red blood cells, haematocri t and haemoglobin resulted by 24 hours after trea tment. By GD20 the haematotoxic effects were nearly reversed. The changes observed in haematologic al parameters and or gan weights in this study are typical of haemolytic anaemia and the compensatory haematopoietic response associated with recovery. The maternal NOAEL is 30 mg/kg/day based on th e effects desc ribes before.
Embryo/foetal effects were: incr eased resorptions, nonlive impl ants, and adversely affected implants per litter in the 200 mg/kg/day group dosed on GD 9 - 11, and decreased platelet count but no embryo lethality in the 300 mg/kg/day group dosed on GD 11 - 13.
EGBE exposure during gestation did not increase the incidence of foetal malformations; particularly, no cardiovascular malformations were observed. When 200 mg/kg were given from day 9 to 11, an in creased foetal lethalit y without malformation s was noted. When 300 mg/kg were given from day 11 to 13, a decrease plat elet count was seen in foetuses. For developmental toxicity the NOA EL is 100 mg/kg when EGBE is administered from GD 9 - 11 and the conservative NOAEL is 100 mg/kg when EGBE is administered GD11 – 13 (although the NTP co nclusion was for a NOAEL of equal to or greate r than 300 mg/kg/day).

Studies in mice
In a gavage probe study in pr egnant CD-mice (Exxon, 1985 ; Wier et al. , 1987), EGBE (purity 97 %)was administered in distille d water at 0 – 350 – 650 – 1000 – 1500 and 2000 mg/kg bw/day (6 animals per group) during gest ational days (GD) 8-14(Note GD0 = vaginal sperm plug and sacrificed GD18). Haemolytic effects in the dams were observed from 650 mg/kg/day. At 1500 mg/kg/day the maternal mort ality rate was 3/6 and at 2000 mg/kg/day 6/6. Increased resorption rates and numerically reduced number of viable foetuses were observed at 1000 and 1500 mg/kg/day. 4/43 foetus (i n one litter) at 1000 mg/kg/day and 1/25 at 1500 mg/kg/day had cleft palates. For this study, the NOAEL for matern al toxicity is 350 mg/kg/day and the NOAEL for developmen tal toxicity is 650 mg/kg/day.
In a subsequent GLP reproduction study (fo llowing a Chernoff modifi cation study design with treatment of the pregnant mice (20 pe r group) at 650 or 1000 mg/kg/day between GD 8- 14, the animals were allowed to give birth and the offspring were obser ved till Post natal Day (PND)22). No significant eff ects on pup growth or survival resulted. No adverse developmental effects were repor ted. Signs of haemolytic eff ects and bw gain reduction were seen at 1000 mg/kg in dams. The NOAEL for mate rnal toxicity is 65 0 mg/kg/day in this study, and as no effects were seen in pups, th e NOAEL for developmental toxicity can be considered to be greater than 1000 mg/kg/day.
In another Chernoff assay, fifty mated CD1 mi ce were orally administered EGBE (99 % purity) by gavage at 1180 mg/kg/day (calculated LD 10 based on a non-pregnant mouse pilot study) in corn oil from GD7-14 (GD1=vaginal sper m plug), then allowed to litter and to rear pups to PND3. 20 % of the dams died, maternal weight gain was reduced and, of 31 surviving pregnant females, there were only 24 viable lit ters (77 %) compared with 97 % control litter viability. No external malfor mations were seen, pup survival to PND was unaffected and no other indication of spec ific developmental toxicity was found (Schuler et al. , 1984). A maternal NOAEL could not be calculated and, although pup development was unaffected, theEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 240 observation of reduced numbers of live litters precludes a calc ulation of the developmental NOAEL.

Summary oral route :
One study is available in rats and 2 studies in mice. The lowest foetal NOAEL is 100 mg/kg/day in the Research triangle institute (1988) rat study. It is based on effects seen at 200 mg/kg/day: increase foetal le thality without malformations. Th ese effects were seen with maternal toxicity (haemolytic anaemia) and retarded body weight gain evidenced from 100 mg/kg/day. The maternal NOAEL is 30 mg/kg/day in this study.

Inhalation route

Studies in rats
In an initial GLP study, mated female Fische r 344 rats (30 per group) were exposed whole body to 0, 100, 200 or 300 ppm (0, 483, 966 or 1449mg/m 3 ) EGBE vapour for 6 hours/day on GD6-15 (GD0 = sperm plug positive) (Tyl et al. , 1984 ; Bushy Run Research Center, 1984). Maternal weight loss was observed at 300 ppm and 200 ppm and retarded weight gain at 100 ppm. During exposure, females were hypoactiv e, with a dose-related increase in the frequency of signs indicative of haematuria and with dose dependent reductions in food consumption at all levels and in water cons umption at 200 ppm a nd 300 ppm. At 300 ppm one animal died. In blood samples taken from females at necropsy (six days after the last exposure), red blood counts were reduced at 300 ppm and 200 ppm. At 300 ppm, 16 of 23 pregnant dams showed 100 % resorptions. There was no increase in resorptions at the lower doses, but low pregnancy incidences in all groups, including cont rols, resulted in only 7, 15, 21 and 21 litters being evaluated at 0, 100, 200 and 300 ppm, respectively. The authors considered this number of litters too few for definitive interpretation of results at higher doses. Interpretation of the results was complicated by maternal toxicity, which included decreased body weight gain at 100 ppm, and body weight losses at 200 and 300 ppm, a condition that causes marked maternal stress. Contributing to this stress was decrea sed food consumption at all concentrations, and decreased water cons umption at 200 and 300 ppm. Dams were hypoactive with signs of haemat uria, indicating maternal haemolysis. Red blood cell counts were reduced at necropsy in dams exposed to 2 00 and 300 ppm. Two foetuses in 2/7 litters at 300 ppm and 1/1 litter at 100 ppm had cardiovasc ular (ventricular se ptum) defects. The relevance of these findings as an indication of se lective toxicity to the foetus was considered unlikely by the authors, although it did trigger the conduct of a develo pmental phase staged oral study described above (Sleet et al. , 1991; Schwetz and Harris, 1993 ; Research Triangle Institute, 1988 as part of the National Toxicology Progra m (NTP)) which confirmed the lack of teratogenicity. In increased incidences of foetuses at 300 ppm in the inhalation study, the development of the inominate artery was aff ected (shortened or missing), the lateral brain ventricles were enlarged, ossification was reta rded and extra ribs were present. Foetal ossification was also retarded at 200 ppm and enlargement of the lateral brain ventricles was observed at increased incidences at 200 ppm and 100 ppm. Thes e variations were considered indicative of embryo/foetal toxi city at maternally toxic dose levels. To investigate these potential effects further, a sec ond inhalation study was performed in rats together with a study in rabbits. It should be noted that the effects seen on lateral ventricle size were not reproduced in the subsequent rat and rabbit studies, despite exposure over the same sensitive period with previously effective concentra tions of EGBE (100 and 200 ppm). In contrast, other effectsEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 241 (e.g., delayed ossification) were reproducible ac ross studies. Thus, the finding of increased incidence of enlarged lateral ventricl es cannot be considered significant.
In a GLP study, mated female Fischer 344 rats (36 per group) were exposed whole body to 0, 25, 50, 100 or 200 ppm (0, 121, 242, 483 or 966 mg/m 3 ) EGBE vapour (purity 99.6 %) for 6hr/day on GD 6-15 (Tyl et al. , 1984 ; Bushy Run Research Center, 1984)(GD0 = sperm plug positive). No adverse reproductive or developmental effects were observed in animals exposed to 25 ppm or 50 ppm. Maternal toxic ity was observed in a dose-related incidence during the exposure period, includ ed evidence of haematuria from 100 ppm and pale, cold extremities with necrosis of the tail tip at 200 ppm. Dose-dependent maternal weight loss was observed at 200 ppm and 100 ppm, associat ed with significant reductions of food consumption (for both groups) and wate r consumption (for the 200 ppm group). Haematological findings from blood samples ta ken at necropsy on GD21 (six days after the last exposure) evidenced signs of haemolytic anaemia for treatment with 100 ppm EGBE and higher.
At sacrifice, maternal gravide uterine weight was significantly reduced and the absolute and relative spleen and relative kidney weight were elevated compared to controls at 200 ppm.
Exposure to 200 ppm was associated with a statistically signifi cant increase in the number of totally resorbed litters and a reduction in the nu mber of viable implants and in percentage of live foetuses per litter. However, there were no statistical significant increases in incidences of external, visceral, skeletal, or total malfor mations associated w ith exposure to EGBE. Exposure to 200 ppm was also associated with a significant increase in the number of litters with one or more foetuses with unossified sk eletal elements and poor ly ossified skeletal elements (anterior arch of atlas, cervical cen tra 5 and 6 and forelimb proximal phalanges) and poorly ossified skeletal elements (cervical ar ches and sternebrae 1, 3, 4 and 6). Primarily because the skeletal elements were poorly ossifi ed there was a decreased incidence of bilobed cervical centra 5 and 6 and bilobed thoracic centra 9 and 13. There was also a decreased incidence of poorly ossified hindlimb proxima l phalanges. At 100 ppm, the number of litters with one or more foetuses with unossified cervical centrum 6 wa s significantly increased and, because fewer also showed ossification of cervical centrum 5, the number bilobed was significantly decreased. No signifi cant differences were observed for other aspects of skeletal ossification. The occurrence of unossified skelet al elements was considered by the authors to be an indication of delayed development in an imals exposed to EGBE under maternally toxic concentrations. For this study the NOAEL is 50 ppm for maternal toxicity based on haemotoxicity and bw and food consumption re tardation at 100 ppm. A LOAEL of 100 ppm for developmental toxicity is due to the small increase in skeletal ossi fication retardation, but without any other foetal effects, variant, observed at this dose le vel, this leads to a NOAEL of 50 ppm for developmental toxicity.
In another inhalation study, the te ratogenicity of EGBE (98 to 99.5 % purity) was evaluated using female SD rats (Nelson et al. , 1984). The animals were exposed whole body to EGBE vapour on GD 7 – 15 (GD0 = sperm positive vaginal smear) for 7hr/day (GLP status unknown). Sixteen and fifteen ra ts were exposed to 150 ppm and 200 ppm, respectively. The untreated control group consisted of 34 dams . Higher dose levels (250 - 500 ppm) in a preliminary study were associated with dose-related maternal deaths, haematuria and tail necrosis.
On the first day of exposure, haematuria was observed in the dams in 150 and 200 ppm groups. This was the only effect observed in da ms. There were no significant differences inEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 242 the number of resorptions foetal weights and th e incidence of malforma tions or variations between the group exposed to 200 ppm EGBE and the control group. A statistically significant decrease in foetal weights was observed in th e 150 ppm exposure group. Since differences in foetal weights between experime ntal and control groups were slight, and, also, that significant differences were not obs erved in the 200 ppm exposure group, these differences were not thought to have been of biological significance. Fo r this study a very conservative LOAEL of 150 ppm can be determined for dams for haematuria seen in the first day of treatment. For developmental toxicity, the NOAEL is 200 ppm.
In a briefly reported study (Barilyak, 1989 in Ru ssian), mated female rats were exposed to EGBE at concentrations of 77, 12 and 2 mg/m 3 , 4 hours a day during GD1 - GD19. At GD20, animals were killed and pups removed by lapa roscopy. Number of Corpora Lutea and, normal and abnormal pups were determined. A complete identification of teratogenic effects was performed in abnormal animals. In this study , EGME was also tested concurrently with Glycols.
For EGBE, at the two high dose tested, a signi ficant increase of total losses was seen compared to controls. These increases were due to an increase in pre-implantation loss for the high dose group (77 mg/m 3 ) and due to a post-implantation loss for the mid-dose group (12 mg/m 3 ). No abnormalities were seen in any trea ted group but 32.5 % of the pups of the high dose group exhibited a delayed development.
This publication is not detailed enough to consider it usefull for risk assessment.

Studies in rabbits
In a GLP study, mated New Zealand white rabbit s (24 per group) were exposed whole body to 0, 25, 50, 100 or 200 ppm (0, 121, 242, 483 or 966 mg/m 3 ) EGBE vapour (purity 99.6 %) for 6hr/day on GD 6-18 (GD0 = day of copulation) (Tyl et al. , 1984 ; Bushy Run Research Center, 1984). No adverse reproductive or deve lopmental effects were observed in animals exposed to 25 ppm or 50 ppm. At 200 ppm four fe males died or were sacrificed by the third day after the onset of dosing and four abor ted. The blood samples taken at necropsy on GD29 (eleven days after the last exposure) show ed increased haemoglobin concentration and haematocrit at 100 and 200 ppm, but statistical significance was only evident at 100 ppm.. Maternal body weight loss was obs erved in all groups including controls during exposure, but the difference was greatest at 200 ppm and, by GD15, the actual body weight at 200 ppm was significantly lower.. Exposure to 200 ppm produ ced a significant reduc tion in maternal body weight, gravide uterine weight a nd number of total implants and viable implants. There were no significant increases in the number of foetuses or litters with one or more affected foetuses with pooled external, visceral , skeletal (total) malformatio ns in any treatment group. For individual malformations, there wa s a significant increase relative to controls in the number of litters at 100 ppm with one or more foetuses exhibiting fusion of papillary muscles in the left ventricle. Five foetuses from 4/19 litters were affected. However, since this malformation was not observed at the higher or other concentra tions it was considered by the authors, the reviewers and the rapporteur as coincidental and not treatment-related. The incidences of skeletal variations did not indi cate an adverse treatment eff ect on ossification. There were significant decreases in the incidences of litte rs at 200 ppm with unossified sternebra 6 and with rudimentary ribs on lumbar vertebra 1. However the former observation was indicative of better ossification and the latter observati on was because more ribs at 200 ppm were full (extra) rather than rudimentary and there were actually no intergroup differences in supernumerary rib numbers. For this study, the NOAEL for maternal toxicity is 50 ppm andEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 243 the NOAEL for developmental toxicity is 100 ppm based on fewer viable implants seen at 200 ppm.

Summary inhalation route:
In both Tyl studies (1984), the developmental NOAEL of 50 ppm can be taken into account for the risk assessment (rats). This value is ba sed on variations seen at 100 ppm. These effects were seen in presence of mate rnal toxicity (haemolytic anae mia) which was seen at 100 ppm and higher. The maternal NOAEL for this effect was 50 ppm. In one study performed on rats, no effects were seen at the highest tested dose (200 ppm).

Dermal route:
EGBE was applied to the skin (unoccluded) of 9 pregnant SD rats to i nvestigate its potential for developmental toxicity (Hardin et al. , 1984). Four doses each 2.5 hours apart of 106 mg EGBE (total daily dose of 424 mg, 1760 mg/kg/day) were applied daily to shaved interscapular skin of rats on GD 7 – 16 (GD0 = sperm positive). A previous replicate of females dosed at a concentration three times hi gher had been terminated because of marked toxic effects (haematuria, tail necrosis, deat h after 3-7 days treatment). No maternal, embryotoxic, fetotoxic, or terat ogenic effects were detected with EGBE treatment at the lower concentration of approxi mately 1760 mg/kg/day.

No clear conclusion can be drawn from the findi ngs of this study since EGBE was applied to the skin without occlusion which would potentia lly enable evaporative loss from the site of application.

Other routes :

Studies in rats
Groups of rats were injected subcutaneously with aqueous solutions of EGBE to determine the teratogenicity of the chemi cal (Unilever, 1976). In the firs t of two preliminary studies to set the dose range, non-pregnant rats received a single injection with225-1350 mg/kg; 80% of the high dose animals died, haemoglobinuria was observed and general p oor condition of the rats resulted. In the second preliminary study, no effects were observed when a similar group of rats received repeated injections at 23-90 mg/kg/day. Experimental details for the preliminary studies were not available.
In the main study, conducted in accordance with OECD test guideline 414, groups of 20 pregnant CD rats received sub-cutaneous in jections of 0. 45, 90 or 180 mg/kg/day as an aqueous solution on GD6 - 15. No mortality re sulted but haemoglobinuria and body weight loss were observed in the medium and high dose dams after the first two injections only. No significant pathological findings were noted at necropsy. Pr e-implantation loss increased in the medium (13.8 %) and high dose (11.9 %) dams co mpared with the controls (6.9 %), but the values were within the laboratory's normal range. Other parameters were normal. In the foetuses, a slight increase in rib effects (wavy/thickened) and a dose-dependent increase in incomplete ossification of cranial bones were observed, but such changes are considered as variations and not malformations.
Under the conditions of the study, pre-implantati on loss occurred in dams at maternally toxic doses and there were no treatment related si gns of teratogenicity. In this study, the NOAELEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 244 for dams is 45 mg/kg/day and the NOAEL for de velopmental toxicity is 90 mg/kg/day based on the variations observed at 180 mg/kg/day.

In vitro studies
EGBE and other glycol ethers were tested in 2 series of Hydra Regeneration Assays (Bowden et al. , 1995 ; Johnson et al. , 1984 and 1988). The Hydra Regeneration Assay compares the toxicity of a test compound to an adult hydra polyp w ith its ability to in hibit the regeneration of an isolated digestive region (Bowden et al. , 1995) or the regenerati on of pellets prepared from non-specific dissociated adult cells (Johnson et al. , 1984 and 1988). In the Bowden study (1995), EGBE was administered at dos es ranging from 0.09 mg/ml to 0.92 mg/ml to groups of 10 polyps and 10 regenerates. The m ean morphological scores for polyps were 10, 8.6, 9.8, 9.0, 4.4 and 4.1 and for regenerates we re 8.1, 7.2, 6.6, 6.6, 3.0 and 1.9 for the control and concentrations of 0.09, 0.18, 0.37, 0.74 and 0.92mg /ml, respectively. Comparing the two polyp versus regenerate scores, the estimated T 50 (corresponding to the concentration 50 % toxic to the polyps) was 0.69 mg/ml and the I 50 (corresponding to the concentration which cause an inhibition of 50 % of the regeneration) was 0.54 mg/ml. However, if the treated concentrations versus their respective contro ls are compared, this gives equivalent T 50 /I 50 concentrations of approximately 0.6mg/ml . The T 50 /I 50 ratio is considered equivalent to the mammalian A/D ratio (adult to developmental toxicity) and is used as a predictor for developmental hazard. In this study the T 50 / I 50 ratio was 1.3 for the first calculation and approximately 1 for the second, indicating that the adult and develo pmental stages were similarly sensitive. If this ratio is compared to other glycol ethers, the developmental hazard was less than with EGEE and EGME and highe r or approximately equivalent to DGEE.

These results were not consiste nt with the Johnson study in which the “A/D” ratio was 4.4 for EGBE, 5 for EGEE and 1.3 for EGME, indicating that EGBE could be more toxic to the “developmental” stage than EGME. However, the developmental stages were different between the studies so a direct comparison is inappropriate. In any event, and assaid by the authors, these in vitro studies cannot be use for quantitative risk assessment.
EGBE and other glycol ethers were tested in the Rat Whole Embryo Culture (WEC) assay (Bowden et al. , 1995). The WEC system used mated Sprague-Dawley CD rats (GD1 = sperm positive). Animals were paired on a one-to-one basis. The morning following pairing was designated day 1 of gestation. On GD 10, dams were sacrificed and embryos dissected out. Embryos were cultured in a special medium wi th S9 metabolic activating system. EGBE in DMSO solution was diluted to 10 % (v/v) with sterile water and added to the culture medium. EGBE was tested at concentra tions of 0, 0.3, 0.5, 0.75 and 1 mg/m l. Culture were terminated after approximately 48 hours. To estimate grow th, the yolk sac diameter, crown-rump length and head length were measured and the num ber of somites counted. Each embryo was examined for dysmorphogeneses and the morphological score was assessed. The rate of dysmorphogeneses was calculated as: Malformati on index = n° of observed malformations / n° of embryos in the group. Re sults were compared with othe r glycol ethers: EGME – EGEE – DGEE – EGTA – EGS.
EGBE produced marginal reductions in growth and developmental parameters at 0.3 mg/ml; these effects being greatly increased at 0.5 mg/ml and above. Increased incidences of malformations were observed at 0.3 mg/ml a nd above, including poor yolk sac circulation, thin allantois, twisted flexions, incomplete fusion and/or irregular formation of the caudal neural tube and brains, irregul ar posterior neuropore and grow th-retarded forelimb buds. At 1 mg/ml, 5/10 embryos were dead. For this test , results obtained with glycol ethers are notEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 245 suitable for effect assessment because, in vivo , they have a very low half life in the circulating blood (< 10 min for EGBE) and the high doses at which glycol ethers produced effects in vitro are totally unrealistic in vivo . Although a S9 metabolic activat ion system was included in the embyo culture medium, the results might th erefore have been e xpected to show the toxicity profile of the metabolite. However, th e alcohol / aldehyde dehydrogenase system is not present in the S9, this may explain the discrepancy between embryo culture results and in vivo data. For these reasons in addition to the general difficulties in extrapolating in vitro data, the results obtained in this st udy cannot be taken into accoun t for the risk characterisation.
In another WEC assay (Jakobsen, 1995), rats em bryos were cultured in a medium containing Glycol ethers (EGME, EGEE, EGIPE and EG BE) and their alkoxy acids (MAA, EAA, IPAA and BAA). The doses tested were 2, 4, 8 a nd 16 mM for alkoxy acids and 10 20, 40 and 80 mM for the glycol ethers. After 48 hours of in cubation, effects on development were assessed.

The results obtained are summari zed in the following tables:EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 246 Table 4.86: effects of EGBE on the development of rat em bryos explanted on day of g estation and cultured for 48h.

*** p < 0.001 ** p < 0.01 * p < 0.05 Concentration Control EGBE 5 mM EGBE 10 mM EG BE 20 mM EGBE 40 mM

Nb of embryos evaluated 123 20 19 10 16

Development

Yolk sac ∅ (mm) 3.5 ± 0.4 3.5 ± 0.3 2.9 ± 0.3 *** 1.6 ± 0.5 *** 1.0 ± 0.3 ***

Crown rump lengh (mm) 2.8 ± 0.5 2.4 ± 0.3 *** 1.9 ± 0.2 *** 1.3 ± 0.2 *** -

Nb of somites 29.2 ± 2.2 29.6 ± 2.3 25.0 ± 4.4 - - Morphological

score 36.3 ± 2.1 35.5 ± 1.6 29.0 ± 5.2 *** 11.7 ± 10.1 *** 0.0 ***

% abnormalities
Grossly abnormal embryos 0 0 36.8 *** 100.0 *** 100.0 ***

Cranial
neuroporus, open 0 0 57.9 *** 100.0 *** 100.0 ***

Caudal
neuroporus, open 9.8 15.0 10.5 20.0 -

Brancial arches, abnormal 0 0 0 16.7 *** -

Vesicles 0 15.0 *** 5.3 * - -

Tail, abnormal 0.8 0 5.3 - -

Flexion, ventrally convex 2.4 5.0 10.5 60.0 *** -

Somites irregular 0 0 0 - -

Yolk sac, abnormal 0 0 0 0 0

Yolk sac, none circulation 30.9 100.0 *** 100.0 *** 100.0 *** 100.0 ***EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 247 Table 4.87: Effects of BAA on the development of rat embr yos explanted on day 9 of gestation and cultured for 48 hr.

*** p < 0.001 ** p < 0.01 * p < 0.05

With EGME and EGEE, specific embryotoxic e ffects are found following exposure to very low levels of their alkoxyacetic acids whereas no specific effects were found when embryos were exposed to comparable concentrations of EGBE and BAA. Th e reproductive effects observed were due to non specific toxic damage to the embryos at comparable concentration. Concentration Control BAA 2 mM BAA 4 mM BAA 8 mM BAA 16 mM

Nb of embryos evaluated 123 15 19 20 18

Development

Yolk sac ∅ (mm) 3.5 ± 0.4 3.6 ± 0.4 3.5 ± 0.3 3.4 ± 0.4 2.7 ± 0.2 ***

Crown rump lengh (mm) 2.8 ± 0.5 2.8 ± 0.5 2.7 ± 0.3 2.2 ± 0.3 *** 1.4 ± 0.4 ***

Nb of somites 29.2 ± 2.2 29.4 ± 2.1 30.5 ± 1.6 * 25.8 ± 4.3 *** 21.8 ± 3.0 ***
Morphological

score 36.3 ± 2.1 33.3 ± 9.5 35.3 ± 2.3 30.9 ± 3.1 *** 19.6 ± 5.3 ***

% abnormalities
Grossly abnormal embryos 0 6.7 ** 0 0 16.7 ***

Cranial
neuroporus, open 0 6.7 ** 0 25.0 *** 100.0 ***

Caudal
neuroporus, open 9.8 40.0 *** 68.4 *** 45.0 *** 0

Brancial arches, abnormal 0 0 0 10.0 *** 0

Vesicles 0 0 0 0 0

Tail, abnormal 0.8 0 0 15.0 *** 50.0 ***

Flexion, ventrally convex 2.4 0 0 0 22.2 ***

Somites irregular 0 0 0 0 0

Yolk sac, abnormal 0 0 0 0 0

Yolk sac, none circulation 30.9 46.7 57.9 * 90.0 *** 100.0 ***EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 248 This study demonstrates that embryotoxicity of glycol ethers increases with the increasing chain lengh of the alkyl group and also that the developmental toxicity of the alkoxyacetic acids decrease with th e increasing chain lengh of the alkyl group.
In another Rat Whole Embryo Culture assay (Giavini et al. , 1993) EGBE, Butoxy acetic acid (BAA) and 2 other glycol ethe rs and their corresponding alkoxyaci ds were tested. EGBE was tested at 3.12, 6.25, 12.5 and 25mM and BAA (adj usted to pH6 with Na OH solution) at 0.4, 0.8, 1.6 and 3.2mM. General embryotoxic effects, as shown by a reduction of the somite number and of total protein/embryo, were seen w ith EGBE concentrations of and greater than 6.25 mM. Embryonic development was blocked at the EGBE concentrations of 25 mM. Exposure of embryos to 12.5 mM resulted in severe dysmorphogenic effects (inhibition of rotation, severe reduction of telecephalic vesicles ). A dose dependant increasing percentage of abnormal development was seen with BAA at c oncentrations of 0.8, 1.6 or 3.2 mM. For this test, results obtained with glyc ol ethers are not suitable for effect assessment because, in vivo
, they have a very low half life in the circul ating blood (< 10 min for EGBE) and the high doses at which glycol ethers produced effects in vitro are totally unrealistic compared to the in vivo situation.
For BAA, effects were seen from 0.8 mM (40 % for 0.8 mM, 44.4 % for 1.6 mM and 60 % for 3.2 mM vs 22.2 % for controls and 20 % for 0.4 mM). Total proteins and somites numbers were also decreased for the 0.8 mM dose. This could suggest effects due to general toxicity rather than specific toxicity but as usual deve lopmental parameters were not assessed, this could not be fully confirmed (Saillenfait, pe rs. com.). The malformative effects affecting prosencephale in this study are comm on effects seen with such a test.
In Whole Embryo Cultures of rat BAA (in the form of sodium salt) and 6 other alkoxy acids were investigated (Rawlings et al. , 1985). At a concentration of 5 mM, BAA adversely affected the development of conceptuses both in growth and the incidence of abnormal embryos. The most frequent abnormality produced was an irregularity of the neural suture line. No effect was observed at 2mM. In co mparison with other al koxy acids (MAA, EAA, nPAA, 3MPA and 4MBA), it was apparent that the potency of the effects decreased with increasing length of the alkoxy chain. Dysmor phogenesis was least affected with BAA, nPAA, 3MPA and 4MBA. The authors state that the lack of teratogenicity of butoxyethanol in vivo is a reflection of the limited teratoge nic potential of its alkoxy acid metabolite.
EGBE was examined for its ability to block junction-mediated intercellular communication (Welsch and Stedman, 1984 ; Loch-Caruso et al. , 1984). The blockage of a specific type of intercellular communication, that is mediat ed by gap junctions, could be a possible mechanism of action of some teratogens. In terruption of intercellular communication was measured in vitro by an assay that depends on the transf er of metabolites via gap junctions, i.e., metabolic cooperation (V79 cell assay). EGBE was an effective blocker of intercellular communication at non cytotoxic doses in this assa y. Results obtained with other glycol ethers demonstrated that as the length of the alipha tic chain increased so did the cytotoxicity.

Summary in vitro studies:
In rat whole embryo culture assays EGBE and BAA seems to cause developmental abnormalities in conjuction with effects on growth. The NOAEL for these effects are 3.12 mM for EGBE and 0.4 mM for BAA (Giavini et al., 1993). These studies have demonstrated that the glycol ether embryotoxicity increased with increasing chain length (EGBE>EGEE>EGME). In contrast, the al koxyacid embryotoxicity decreased withEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 249 increasing alkoxy chain lengt h (MAA>EAA>BAA). The in vitro studies performed with EGBE (and glycol ethers in general) exibit effects which are not relevant in vivo (according to the kinetic of glycol ethers in vivo and the great doses needed to produce effects) and therefore are not directly usab le in the risk assessment.

Studies in humans
For developmental toxicity, no studies are ava ilable for assessing the risk of exposure to EGBE alone.
In an case-control study, the risk of congenital malformations re lated to glycol ether exposure during pregnancy have been evaluated (Ha et al. , 1996). Cases were any infant, born alive or stillborn, and foetuses of any gestationa l age aborted because of a malformation. Malformations of known origin were excluded. Control were normal neonates born within one week of the case in the same place (maternity or locality). This study involved 991 cases and 1144 controls. Mothers were submitted to a standard questionnaire including occupational history, socio-demographic char acteristics, pregnanc y history and hobbies. Father’s work was also determined. Occupational exposure to glyc ol ethers was assessed. Glycol ethers were classified in four group:

- group 1: EGME, EGEE and th eir acetates and some pol yethylenic compounds

- group 2: EGBE, EGPE, and their acetates

- group 3: PGME, Propylene Glycol Methyl Ether Acetate (PGMA) and some polyethylenic and propylenic compounds

- group 4: other glycol ethers
In this study, there was a significant excess of mothers occupationally exposed to glycol ethers among the cases (27 % versus 20 % of the controls). When types of malformations were studied, a significant excess of oral clefts and central nervous system malformations was found in glycol ether exposed mothers compared to controls (OR = 2, 95 % CI = 1.1 – 4.1 and OR = 1.8, 95 % CI = 1.1 – 3.3 respectively). An excess of musculoskeletal malformations in exposed mothers was also seen although it was on the borderline of significance.
In an case-control study (same data and same stu dy than the study described before ?), the risk of congenital malformations related to glycol ether exposure during pregnancy have been evaluated (Cordier et al. , 1997). The cases were women with any product of conception with a major congenital malformation diagnosed pren atally of during the perinatal period. The study period was between 1989 and 1992 and was conducted on 6 European registries (2 in France, one in UK, 2 in Italie and one in The Netherlands. Each case was match with a control chosen with the same birth date and born in the same town. Mothers was submitted a standardized questionnaire to assess their socio-economic status, medical and obstetric history, consumption of alcohol – tobacco – drugs, hobbies and occupation, both before and during each trimester of the pregnancy. Concer ning oral clefts, a distinction was made between cleft lip with or without cleft palate [C L(P)] and cleft palate only (CP) due to their etiological difference.
Between 1989 and 1992, a total of 984 cases of ma lformation were reported. The overall odds ratio (OD) of congenital malformation associat ed with glycol ether exposure was 1.44. after adjustment for several potentia l confounders. The associ ation with exposure to glycol ethersEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 250 appeared particularly strong in 3 malforma tion subgroups: neural t ube defects (OR=1.94, 95 % CI = 1.16 – 3.24), multiples anomalies (OR=2.00, 95 % CI = 1.24 – 3.23) and cleft lip (with or without cleft palate) (OR=2.03, 95 % CI = 1.11- - 3.73). In this last subgroup, risk, especially of an isolated defect, tende d to increase with level of exposure.
In the same study, the risk of oral cleft forma tion related to maternal occupation was studied in depth (Lorente et al ., 2000).
Upon the including 984 cases, 161 cases were oral clefts. Differences between working mothers concerned only the place of residence (mothers of CL(P) cases came from less urban background than their referent) and moth er’s age (older for CL(P) cases).
The analysis of occupational exposures wa s restricted to women who worked during pregnancy (100 cases and 75 refere nts). For occupational, the odds ratios were estimated for each type of cleft and were highest for serv ice occupation. Within this group the ORs were higher for CP than for CL(P) and significan t for CL(P) for only 2 occupations: housekeepers and hairdressers. For production workers, the el evated ORs concerned CL(P). For chemical exposure, Ors adjusted were estimated. For glycol ethers Ors significantly different for 1 were observed:
Exposure CL(P)

n=64 CP

n=36 Referents

n=751 OR
CL(P) 95 % CI
CL(P) OR

CP 95 % CI

CP
Glycol ethers 23 11 137 2.1 1.14-3.88 1.82 0.82-4.03

For CL(P) exposure to glycol ethers was associ ated with an excess ri sk. A more detailed analysis of the exposure variable s of the level, frequency, and reliability of exposure could be performed only when enough subjects were expo sed to each product. This analysis showed that, for glycol ethers among isolated CL(P) ca ses, the risk increased with the level and frequency.
In a case-control study, the risk of Neural Tube Defects related to maternal occupation al and hobby chemical exposure was assessed (Shaw et al. , 1999). 653 Neural Tube Defects cases (anencephaly, spina bifida cystica, craniorachis chisis or iniencephaly) were extracted from a cohort of 708129 births and foetal death betw een June 1989 and May 1991. An interview of 538 cases and 539 controls was performed. Inform ation about paternal occupation were also checked. Using information about occupation a nd hobbies, exposures to 74 chemical agents were classified as likely, maybe or not expos ed. A classification was also made regarding end-use of chemicals in 9 group.
Compared to control mothers, case mothers were more likely to be Hispanic, less than 25 years old and to have a househol d income under 10000 dollars. They also were less likely to have graduated from college, to have used mu ltivitamins in the 3 months before conception, to have smoked cigarettes in the first trimester or to have been employed in the periconceptional period.
Exposure to house keeping cleaner give an OD of 0.97. Overall there was no increase risk for solvent exposure based on combined occ upational and hobby/household exposure or for occupational exposure along among employed women.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 251 For glycol ethers and derivatives:

NTD cases Controls Odds ratio 95 % confidence interval

75 80 0.93 0.66-1.3

The risk of NTD was not increas ed for glycol ethers exposures.
A case control study similar than those already performed was made in Slovakia with data collection ranging from 1995 to 1996 (Cordier et al. , 2001). 196 case of live or stillborn babies with a major malformation or foetuses fr om therapeutic abortion were included in the study. Assessment of various chemical exposur e (including glycol et hers) was performed.
The distribution of congenital malformations in each broad group of anomalies was very similar to those observed in the previous study. Odds ratio for glycol ethers exposure during the first trimester of pregnanc y were estimated among working mothers for each subgroup of abnormalities, adjusted for matern al age, rural residence and mo ther’s socio-economic status. The overall risk of congenital abnormalities was elevated (OR = 2.3; 95 % CI = 0.7-7). Among 15 potentially exposed women, 10 (7 cases , 3 controls) were exposed to the highest exposure category; in this group, the OR for a ll malformations was slig htly higher (OR = 2.7; 95 % CI = 0.7-11). Malformation-specific Ors we re increased with high exposure for all except cardiac anomalies (unchanged) and urinar y anomalies (decreased ). Caution should be given to this study due to the very small size of the studied population.
In a case-control study, the risk of Neural Tube Defect rela ted to parental occupation was assessed among Mexican Americans (Brender et al , 2002). Case women were Mexican Americans whom had a product of conception (liv e or stillborn infant or spontaneous or induced abortion) with an NTD during the period of June 1995 to May 2000. NTD included were: anencephaly, spina bifida and encephalocele. A complete questionnaire was submitted to each case and control woman to determine th eir maternal health and reproductive history, family demographic data and medical histor y, and the use of medications, nutritional supplement, tobacco, alcohol, and drugs. Envi ronmental and occupational exposures were also assessed.
Of the 225 cases, 184 completed the interview ( 82 %). For the controls, only 225 of the 378 selected (60 %). Case women te nded to be slightly younger, less educated and significantly more case than controls where obese. For occupation, case women are more likely to work as cleaners and in health case. They are also more li kely to have work expos ures to glycol ethers (OR ∞ , 95 % CI 1.8 - ∞ ). Women who reported activities associated with solvent exposure outside the workplace also were li kely to have NTD-affected pr egnancies. For paternal work: risk estimates were close to the null for exposure to glycol ethers.
Based on these findings, maternal work exposure to solvents or glycol ether and maternal occupations in health care or cleaning may be risk factors for NTDs in the studied population.
Through a review of the epidemiol ogical studies available, Maldonado et al. (2003) demonstrated that the results available all suffers from methodological deficiencies which weaken the conclusions of these studies.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 252 Other relevant information
In a study by Wilson (1953), dams were bled to induce haemorrhagic anaemia at different stages of pregnancy. Dams were bled at 3 cr itical periods during de velopment: (1) on gd 7-9 to cause anaemia when the mesoderm is form ing and organogenesis is beginning on gd 9-10; (2) on gd 9-11 or gd 11-13 to produce anaemia during the most active period of organogenesis on gd 11-14; and (3) on gd 13-15 during late or ganogenesis and early histogenesis, which occurs on gd 15-16. Blood loss by dams wa s 5 cc or more per 100 g body weight, which caused a marked drop in haematocrit values. For dams bled on gd 7- 9, removal of 5 to 5.9 cc blood per 100 g body weight resulted in a 52 % decrease in haemat ocrit (relative to controls) and totally resorbed litters for 33 % of pregnancies (3 of 9). Haemorrhages in excess of 6 cc caused a 63 % decrease in haematocrit and earl y termination of 13 out of 15 pregnancies due to either maternal death (8 dams) and total re sorbed litters (5 dams). For dams bled on gd 9- 11, 25 % of dams losing 5-5.9 cc per 100 g body weight exhibited a 54 % decrease in haematocrit and totally resorbed litters with 1 of 8 term litters (gd 20) exhibiting retarded development. For dams with more severe anaemi a (in excess of 6 cc with a 59 % decrease in haematocrit), one third exhibite d total resorbed litters. Of the 13 pregnancies with fetuses surviving to term (gd 20), the mean rate of pr enatal survival was decreased (18 % of fetuses resorbed). Four litters exhibite d growth retardation (delay). In general, the authors concluded that “although an occasional abnormality may be produced at this time, maternal anaemia usually causes either early termination of pr egnancy or permits con tinuation of pregnancy without detriment to the young.” Bleeding the da ms on the 11-13 or 13-15 days of pregnancy caused a higher rate of maternal death (45 % of dams). In so me cases, death of the fetuses preceded death of the dam. Total litter loss due to resorption was not seen at these time points. Some delayed development was seen. Bleeding th at was initiated on gd 9 had the greatest likelihood of resulting in malforma tion, growth retardation (4 of 13 litters), or increased rate of resorption.
Similar results have been reported for pregnant ra bbits in the presence of maternal anaemia. In studies with diflunisal, an antiinflammatory drug, pregnant does given a single oral dose of diflunisal on gd 5 exhibited anaemia that pers isted through gd 15, desp ite clearance of the drug from maternal blood by gd 9 (Clark et al. , 1984). Signs of anaemia in pregnant does included haemoglobinuria, and decreased erythroc yte counts (a 74 to 46 % decrease relative to controls on gd 9 to 15) and decreased haemoglobin c oncentrations (a 63 to 21 % decrease relative to controls on gd 9 to 15). Haematotox icity was the most severe on gd 9, the first day of data collection, with some recovery by gd 15. These does showed an increase in preimplantation loss (10 of 25 inseminated doe s showed no evidence of implantations and there was an increase in preimplantation loss in other does), as well as an increase in totally resorbed litters (11 of 25 litters). The percen tage of resorptions and dead fetuses (mean of litter values) was 10.6 % in control does compar ed with 74.3 % in di flunisal-treated does, despite clearance of the test material from maternal blood by gd 9. These effects were attributed to maternal anaemia rather than a di rect effect of diflunisa l, because diflunisal is highly protein bound in maternal blood (greater than 98 %); thus, there are only low concentrations of diflunisal in rabbit embryos . Furthermore, rats, wh ich are resistant to diflunisal-induced maternal anaemia, do not demonstrate embryo/fetal effects, despite reaching approximately equivalent doses of diflunisal in rat embryos. Furthermore, there is little accumulation of diflunisal in embryos, which is likely a reflection of its low placental transfer. This study provides strong evidence th at the embryo/fetal effects (similar to those seen with EGBE) were sec ondary to maternal anaemia.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 253 4.1.2.9.3 Summary of toxicity for reproduction
Unlike EGME and EGEE, EGBE seems to have no specific effects on fertility (no effects were seen in the continuous breeding study an d neither macroscopic nor microscopic effects on reproductive organs in the repeated dose toxi city studies at doses which does not exhibit severe general toxicity.) A NOAEL of 720 mg/kg was derived from the continuous breeding study for fertility effects (it should be noted th at effects seen at the higher dose tested are certainly due to general toxicity)
For developmental toxicity, studies performed on animals via various administration routes did not demonstrate any teratoge nic potential, but foet otoxicity and embryot oxicity (lethality and resorptions) were often obs erved in relation with maternal toxicity (regenerative haemolytic anaemia). Other effects seen on fo etuses were an increase in the incidence of skeletal variations which are genera lly described as ossification delays. In vitro studies showed some adverse effects on development w ith EGBE and its metabolite BAA, but only in conjuction with growth effects. Effects seen in foetuses are certainly related to maternal toxicity. Some studies have previously shown a relationship between maternal haemotoxicity and effets seen with EGBE (resorption, growth retardation and variations).
Haemotoxicity described in § 4.1.2.2.3 generally o ccured at low doses of EGBE whatever the route of administration used. In theses studies, data on haemol ysis were often observed with acute dosing. Developmental toxic ity studies would require daily dosing with test material, which may produce more marked effects on haem atopoietic parameters. In addition, female mice and rats were more affected by EGBE haemolysis than males. Thus, these data demonstrate that the concentrations of EGBE used in these developmental toxicity studies were adequate to produce severe maternal anaemia of the magnitude sufficient to cause effects on embryo/fetal survival. These data gi ve plausible support to the hypothesis that the effects seen in developmental toxicity studi es with EGBE were due to haemolysis and subsequent maternal anaemia.
In human, all the epidemiologi cal studies, except one, studying glycol ethers, showed an increased risk of malformation (c left lip, neural tube defect). For EGBE, these studies did not allow to draw any conclusion about its potential effects on human because no studies are able to distinguish clearly an unique source of glyc ol ether, usually studi es described co-exposure to various glycol ethers, in cluding known developmental toxins such as EGME and other chemicals as well.
Overall, it is not possible to obtain a suitable NOAEL for developmental toxicity relevant for humans and based on animals studies. Regard ing kinetic properties and SAR with other glycol ethers, it can be assumed that devel opmental toxicity due to EGBE in humans could not be expected without maternal toxicity. C onsequently, there is no concern for this endpoint and no need for risk characterisation.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 254 4.1.3 Risk characterisation 5

4.1.3.1 General aspects
The human population may be exposed to EGBE at the workplace, both from use of consumer products and indirectly via th e environment (see 4.1.1.1, 4.1.1.2 and 4.1.1.3)
From the oral absorption studies it is concluded th at oral absorption is complete. For risk characterisation 100 % oral abso rption should be assumed.
From dermal absorption studies, a wide range of absorption values were observed depending on the species (rats having a greater dermal pene tration than humans), the dilution of EGBE (40 % or 80 % water solu tions of EGBE being absorbed at twice the rate compared to lower dilutions or undiluted EGBE), physical state of EGBE and occlusion stat us of administration. In general, dermal absorption of liquid EGBE varies between 20 to 30 % of applied dose in rats. Variations are seen in the reported valu es for individuals humans for the rate of absorption (ranging from 0.064 mg/cm 2 /hr to 1.66 mg/cm 2 /hr in vitro and from 0.826 to 11.3
μ g/cm 2 /hr in vivo ). For dermal absorption of vapour EG BE, studies on volunteers have shown a percentage of internal dose due to derm al absorption of 11 to 39 % (depending on the conditions of exposure). According to the PbPk model, for a worst case exposure (100 % of the body exposure with no cloths), internal dose of EGBE due to the percutaneous uptake when a subject is exposed to EGBE as vapour would be 15 to 27 % (this range being not negligible compared to the dose due to inhalation). For risk characterisation, dermal absorption of liquid EGBE can be assumed to be 30 % of applied dose. For dermal absorption of vapour a value of 39 % of the inte rnal dose due to dermal absorption can be taken into account but keeping in mind that this value has b een demonstrated only during extreme exposure conditions (high temperature, high humidity and overalls clothing) which can be considered to be the worst case of exposure.

From human volunteers inhalati on studies, an absorption of 55 % to 60 % has been measured. These values varies from the theoretical abso rption value of 80 %, cal culated from various studies, due to a wash in / wash out mechanism on the surface of the respiratory tract. For risk characterisation, 60 % inhalation exposure shoul d be assumed (highest measured value).

Table 4.88: absorption coefficients taken into account for the calculations of internal doses Oral Inhalation Dermal route
EGBE liquid EGBE vapour % of absorption 100 % 60 %

30 % 1 39 % of the internal dose 1

1 Maximum worst case percentage, can be reassessed for each scenario in a case by case basis if needed .

5 Conclusion (i) There is a need for further information and/or testing.

Conclusion (ii) There is at present no n eed for further information and/or testing and no need for risk reduction measures beyo nd those which are being applied already. Conclusion (iii) There is a need for limiting the risks; risk reduction measures which are already being applied shall be taken into
account.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 255 For interspecies extrapolati on, PBPK models exist for the rat, mouse and human. These enable the internal dose of EGBE to be es timated with some precision. Of the models available, that from Corley et al . 1994 and 1997 is considered the most complete and appropriate for potential use in the derivation of an interspecies extrapolation factor for oral exposure because it has been e xperimentally validated, covers relevant routes of exposure, and addresses both the di stribution and excretion of the metabolite BAA which is invariably the key substance of interest. For th e inhalation route, the model of Lee et al . (1998) was used to estimate BAA blood concen trations in female rats fo llowing inhalation exposure to EGBE because it is a recent extension of the Corley et al . (1994 and 1997) model for inhalation exposures and includes a dded parameters for female rats.

The PBPK model allows the concentration of th e proximate toxicant (BAA) to be predicted following either inhalation or oral exposure to EGBE. The following f our steps are carried out to obtain a human equivalent concentration (HEC), following a similar approach to that adopted by the US EPA (US EPA, 1999): (1 ) calculate the inte rnal dose metric (C max BAA in blood) corresponding to the female rat LOAEL, using the actual experimental exposure pattern (6hrs/day, 5 days/week) in the model simulation; (2) verify that steady state was achieved (e.g., no change in BAA C max as a result of extending th e exposure; (3) simulate the internal dose metric (C max BAA in blood) for humans inhali ng EGBE and 4) calculate the HEC (mg/m 3 ) that results in this internal dose C max BAA. The relationship between EGBE exposure and resultant BAA for humans and female rats as used in step 3 is shown below:

0 200 400 600 800 1000 1200 1400 1600 1800 2000

0 50 100 150 200 250 300 Concentration of EGBE in air (ppm)CmaxBAA in blood (μM)

Note: Data for rats is the upper line, data for humans the lower line

For acute toxicity, animal studies have give n a LD50 of 1000 mg/kg by or al route in reliable mice and rats studies. By derm al route, depending on the application (o cclusive or not) the LD50 was 500 mg/kg or > 2000 mg/kg respectivel y. By inhalation route, mortality was evidenced in reliable studies from about 500 pp m concentration (the LC50 being of about the same level). The effects seen in acute toxi city studies in animals were mainly acute haemolysis accompanied with non specific signs of toxicity (laboured breathing, lethargy, ataxia). Some hepatic and renal pathol ogy was also seen in these studies.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 256 In humans cases of massive ingestions the main toxic effect was a metabolic acidosis with sometimes haematotoxicity. For haematotoxicity, humans are much less sensitive than rodents and according to human studies, it appears that there is not a great intraspecies sensitivity (no influence of age or haematological status). Moreover, this end point was not the most sensitive end point for acute toxicity in hu man because in case of massive ingestions haemotoxicity was not observed in all cases. Bu t metabolic acidosis was observed in all case. The lowest dose leading to metabolic acidosis in human was 400 mg/kg (LOAEL).
As human data is preferred if exists, this LOAEL will be taken into account for the risk characterisation. As no data exists in human by dermal or inhalati on route, an extrapolation of this LOAEL will be done for risk characterisation for these routes using appropriate route to route extrapolation factors.
EGBE liquid is classified as irritating to the skin and to the eyes. For risk characterisation of EGBE vapour, a human NOEL of 50 ppm is used based on recent volunteer studies. No sensitisation properties liked directly to EGBE were seen from the available data on humans and animals.
For repeated dose toxicity, it has been shown that rodents ar e much more sensitive than human to the haemolytical eff ects of EGBE, therefore if a NOAEL based on these effects is used for risk characterisation, the MOS used shoul d reflect the sensitivit y of rodents. Kupffer cell pigmentation is secondary to the haemolytic effects. Effects on spleen (including spleen fibrosis) can also be relate d also to haemolysis. This effect usually occur following administration of substances which is able to cause iron accumulation in the splenic red pulp (Goodman et al. , 1984 ; Weinberger et al. , 1985). Effects on the forestomach of rodents do not appear to be relevant for humans. With regard to the increased incidence of hyaline degeneration of the olfactory ep ithelium observed in rodents, this appears to be an adaptive response, the severity of the lesion being una ffected by increasing exposure concentrations.
In other species than ro dents, old studies are av ailable. In dogs and monkeys, haemolysis was also described at relatively low doses. In guinea pigs, a species which is resistant to EGBE induced haemolysis, mortality was seen from 375 ppm and higher. Due to the limitations of these old studies these results cannot be taken into account for the risk assessment. The most reliable inhalation data is the LOAEL of 31 pp m derived from a a 6 month satellite group in a two-year study in rats. For oral route, a LOA EL of 69 mg/kg bw/d in male rats was derived from a 14 week oral study in rats. For dermal exposure, a NOAEL of 150mg/kg/day can be used.
Since all key effects are induced by haemolysis in rodents, a NOAEL based on haemotoxicity will be used in the risk characterisation. The sele ction of an appropriate interspecies chemical safety assessment factor (CSAF) must take into account the lower sensitivity of humans to BAA than rats (or mice). IPCS have pr oposed splitting the CSAF two components representing the toxokinetic and toxodynamic adjustment factors (AK AF and AD AF respectively). The toxicokinetic factor is taken account of by us e of the PBPK model described above. The AD AF factor needs to be set to an a ppropriate value to reflect the lowersensitivity of humans to haemolysis. The data available on the most sensitive measure (prehemolytic changes) suggests that a value of 0.01 would be realistic. However, a more cautious and conservative initial appro ach would be to propose a value of 0.1.
For mutagenicity, it has been demonstrated that EGBE is not a mutagenic substance in vivo.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 257 For reproductive toxicity, no specific effects were seen for fertil ity. In the continuous breeding study a NOAEL of 720 mg/kg was set ba sed on non specific effects observed at the higher doses tested. For developm ental studies, embryonic and foet al effects seen in animals were related to maternal toxicity (haemolysis).
For carcinogenicity, some effects were seen in mice in the 2-year studies. As the mechanism of haemangiosarcomas in male mice is related to haemotoxicity, the risk characterisation made for repeated dose toxicity is considered sufficient to also assess for carcinogenicity. The other tumours (mouse forestomach) are cons idered not relevant to humans; no risk characterisation is needed for them.

4.1.3.2 Workers
Assuming that oral exposure is prevented by personal hyg ienic measures, the risk characterisation for workers is limited to the de rmal and the inhalation routes of exposure.

Three sets of values have been utilised for dermal exposure: the EASE estimates, the data measured on DEGBE, and biomonitoring data from EGBE use in actual situations. As previously discussed in 4.1.1.1.2.2 and in 4.1.1.2.4, it is proposed to take the available RISKOFDERM data as a basis and to adapt th em using EGBE’s biom onitoring data when applicable. Summary of proposed reasonable wo rst case exposure is in dicated below. MOS are calculated and presented in the releva nt part of the risk characterisation.

Table 4.89: Summary of proposed reasonable worst case exposures (see also table 4.29 in & 4.1.1.2.4)

Scenario Inhalation (mg/m 3 ) External dermal exposure (mg/day)

1 - Manufacture 12 42

2 - Formulation 15.7 2,000

3 - Use of products 3.1 Coating/Painting a - industrial

- Spraying

- Other works - decorative 3.2 Printing

- silk screening

- general printing 3.3 Cleaning

- spraying

- wiping 58.1 30.4 30.4

20 5

49 49 2000 430 70

23 168

250 1,040EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 258 4.1.3.2.1 Acute toxicity
According acute toxicity data in human (suicide attempts ca ses) a conservative LOAEL of 400 mg/kg by oral route can be taken into account. As limited data exist in humans by dermal or inhalation route an extrapolation route to route of this LOAEL is done. The MOSs obtained are compared with minimal MOS calculated as follows:

Table 4.90: Assessment factors applied for the calculation of minimal MOS for acute toxicity (for inhalation and dermal route) Interspecies differences 1 (human data)
Intraspecies differences 3 (workers: homogen population)
Type of effect 1
Extrapolation LOAEL to NOAEL 5

Confidence of the database 1

Minimal MOS 15 for inhalation and dermal routes

Inhalation route:
Oral LOAEL of 400 mg/kg has to be extrapolate to a inha lation NOAEL to compare with inhalation exposure.
The dose of 400 mg/kg by oral route correspond to 400 mg/kg of in ternal dose (100 % absorption by oral route)
To obtain this internal dose by inhalation, it is n ecessary to add internal dose due to inhalation of vapours and internal dose due to percutaneo us penetration of vapours. The ratio between these two doses has been calculated 61% / 39 % respectively. Leading to:
400 x 0.61 = 244 mg/kg of internal do se due only to inhalation and
400 x 0.39 = 156 mg/kg of internal dose due onl y to percutaneous absortion of vapours.

- To obtain 244 mg/kg of internal dose in one day, a worker of 70 kg with a respiratory volume of 10 m 3 /worday, withan absorption factor of 60 % for inhalation uptake Should be exposed to
244 mg/kg * 70 kg / 10 m 3 / 0.60 = 2846 mg/m 3

. This value is the extrapolated LOAEC and should be compared with exposure levels.

The MOSs between the LOAEL and the inhalati on exposure levels are mentioned in table 4.91. The MOSs are evaluated by comparison with the minimal MOS (table 4.90). The conclusions are given in the ta ble 4.91. Based on the risk assessment for inhalation exposure, it is concluded that toxicity due to acute exposure are not expected.

Conclusion ii is reached for all occupational scenarios.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 259 Table 4.91: Occupational risk assessment of EGBE for acute toxicity
Risk assessment for inhalation
exposure Risk assessment for dermal exposure to liquid EGBE

Estimated Skin
exposure mg/day
(mg/kg bw/d Scenario

8-hour TWA inhalation (mg/m 3 ) MOS 1 Conclusion MOS Conclusion

1 - Manufacture 12 237 ii 42
(0.6) 2220 ii

2 - Formulation 15.7 181 ii 2,000
(28.57) 47 ii

3.1 Coating/Painting

3.11 –Industrial
-spraying 58.1 49 ii 2000
(28.57) 47 ii

-other works 30.4 94 ii 430
(6.14) 217 ii

3.12 - decorative 30.4 94 ii 70
(1) 1333 ii

3.2 Printing

3.21 Silk screening 20 142 ii 23
(0.33) 4039 ii

3.22 General printing 5 569 ii 168
(2.4) 555 ii

3.3 Cleaning
- spraying 49 58 ii 250
(3.57) 373 ii 3 - Use of end products

- wiping 49 58 ii 1040
(14.86) 90 ii

a: non dispersive use, b: wide dispersive use.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 260 1- calculation based on a respiratory volume of 10 m3/workday, a worker bw of 70 kg, an absorption factor of 60 %, an internal dose due to inhalation uptake (61 %) and dermal uptake of vapour (39 %) and an oral LOAEL of 400 mg/kg bw.

2- calculation based on a worker bw of 70 kg, an absorption factor of 30 % and an oral LOAEL of 400 mg/kg bw. Worst case.

Dermal route:
Oral LOAEL of 400 mg/kg has to be extrapolate to a dermal LOAEL to compare with dermal
exposure. Oral dose of 400 mg/kg would give an internal dose of 400 mg/ kg. To reach this dose with a dermal exposure to liquid EGBE, the external dose should be :
400 / 0.3 = 1333 mg/kg bw
because 30 is assumed to be the worst case pe rcentage of absorption of liquid EGBE by dermal route.
This LOAEL of 1333 mg/kg bw should be compar ed with estimated skin exposures. The MOSs between the LOAEL and the dermal expos ure levels are mentioned in table 4.91. The MOSs are evaluated by comparison with the mi nimal MOS (table 4.90). The conclusions are given in the table 4.91. Based on th e risk assessment for dermal exposure, it is concluded that toxicity due to acute exposure are not expected.

Conclusion ii is reached for all occupational scenarios.

Combined exposure : For the combined exposures the estimated internal doses are calculated from the biological exposure data. The oral LOAEL of 400 mg/mg would lead to an internal doses of 400 mg/kg because an absorption factor of 100 is taken into account for oral route. This LOAEL should be compared with internal doses calculated from exposures in each scenario (inhalation + dermal). The internal doses are calculated as follow :
Inhalation exposure will give internal dose of:
X ( value of the 8-hour TWA inhalation (mg/m 3 )) x 10 m 3 (inhaled air during a workday) x 0.6 (percentage of absorption by inhalation) / 70 (mean bw of a worker) = Y (inhalation internal dose). This value does not take into account the possi ble dermal absorption of vapour during the 8hr TWA. It has been demonstrated that dermal absorption of vapour EGBE could count for 39 % of the internal dose of EGBE. To take into account this value (which is not negligible) the value of internal dose due to dermal exposure to vapours (Z) should be added to the former value (Y). Z represent 39 % of the total in ternal dose and can be calculated as follow :

Z = 0.39/0.61 x Y = 0.64 Y
The total internal dose due to inhalation exposur e (inhalation output + derm al vapour penetration output) is Y + Z = 1.64 Y For dermal exposure internal dose is calculated for a 70 kg bw worker with a percentage of absorption of 30 % (liquid EGBE, worst case).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 261 Table 4.91 bis: Occupational combined risk assessment for acute toxicity

Scenario Internal dose after exposure to 8-hour TWA (mg/kg bw) Y+Z Internal dose after Dermal exposure to liquid EGBE

(mg/kg bw) worst case (based on maximal dose) Total internal dose (inhalation + dermal exposure) MOS Conclusion

1 - Manufacture 1.69 0.18 1.87 213 ii

2 - Formulation 2.2 8.58 10.78 37 ii

3 - Use of products 3.1 Coating/Painting - industrial

- Spraying

- Other works

- decorative 3.2 Printing

- silk screening

- general printing 3.3 Cleaning

- spraying

- wiping 8.17

4.28

4.28

2.81

0.70

6.89

6.89 8.58

1.84

0.30

0.10

0.72

1.07

4.46 16.75

6.12

4.58

2.91

1.42

7.96

11.35 24

65

87

137

281

50

35 ii

ii

ii

ii

ii

ii

ii

The calculated MOS should be compared with mi nimal MOS. To be in the worst case, the highest minimal MOS should be c hosen for the comparison: 15.
For acute toxicity a conclusion ii is reached for all scenarios for combined exposure.

4.1.3.2.2 Irritation and corrosivity

Skin
EGBE is classified as a skin irritant, and th e use of appropriate PPE is recommended. This endpoint is of concern, but th at this concern is managed by the information given by the classification and labelling of the substan ce so that no further concern remain. ( conclusion ii)

.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 262 Eye
Eye irritation (liquid) : The substance is classified as a eye irritant, and the use of appropria te PPE is recommended This endpoint is of concern, but that this c oncern is managed by the information given by the classification and labelling of the substan ce so that no further concern remain. ( conclusion ii ).

Eye and respiratory tract irritation (vapours):
Since the NOEL for respiratory tract irritati on is derived from humans, the only assessment factor needed is that to allow for possible intraspecies variation. This is particularly true since the effects are not physiological but more discomfort in nature . A factor of 3, and therefore minimal MOS of 3 is considered sufficient for this end point. The MOSs between the NOAEL and the inhalation exposure levels and the conclu sions of the risk assessment are mentioned in table 4.92.

Table 4.92: Occupational risk assessment of EGBE for eye and respiratory irritation by vapour exposure. Risk assessment for eye and respiratory irritation by vapour exposure Scenario

8-hour TWA inhalation (mg/m 3 ) MOS 1 Conclusion

1 - Manufacture 12 20 ii

2 - Formulation 15.7 16 ii
3.1 Coating/Painting

3.11 –Industrial
-spraying 58.1 4.2 ii

-other works 30.4 8 ii

3.12 - decorative 30.4 8 ii

3.2 Printing

3.21 Silk screening 20 12 ii

3.22 General printing 5 50 ii 3 - Use of end products

3.3 Cleaning 49 5 ii

For eye and respiratory tract irritation due to the exposure of EGBE in vapour form, conclusion ii is reached for all occupational scenarios.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 263 4.1.3.2.3 Sensitisation

Given the results from the dermal sensitisatio n studies it is conclude d that EGBE is of no concern for workers with rega rd to skin sensitisation (conclusion ii)

. There are neither data from human experi ence nor other indications for respiratory sensitisation but this toxicological end-point is not expected for EGBE (conclusion ii).

4.1.3.2.4 Repeated dose toxicity
It is clear from the available data that haemolys is is the primary and critical response elicited in the main animal test mode ls (rats and mice) following inha lation, oral or dermal exposure to EGBE. Blood from humans, pigs, dogs, cat s, and guinea pigs is less sensitive to haemolysis by BAA. In sensitive species, EGBE produces a characteristi c toxicity that is revealed clinically by the appearance of haemog lobinuria and pathologi cally by changes in a variety of blood parameters. A 100-fold greater concentration of BAA (10mM) is required for human erythrocytes to develop pre-hemolytic changes consistent with those seen in the rat (0.1mM). Such in vivo blood concentrations are unlikely to occur under normal conditions of human exposure to BE. Haemolysis did not oc cur with any blood from any individual, even sensitive sub-populations, when exposed in vitro to BAA(10 mM). Studies have also shown that potentially sensit ive human sub-populations, including th e children, the elderly and those with sickle cell anaemia are also equally resistant to the effect.
PBPK models exist for the rat, mouse and human.

These can be applied to deri ved the appropriate toxokinetic element of the interspecies assessment factor . The use of the model is de scribed in the General Aspects section and is applied here as follows:

Step 1: Calculate C max for BAA in blood corresponding to female rat LOAEL Female rat (critical species and sex) LOAEL = 31 ppm. Resultant C max BAA from such an exposure= 285 μM.

Step 2: Verify steady state. There were no changes in the C max of BAA in blood during any 24-hour simulation period using a 6 hours/day, 5 days/week exposure regime at the female rat LOAEL, indicating that stea dy state was achieved.

Steps 3 and 4: Calculate the C max for BAA in blood for humans continuously exposed to EGBE vapour

and calculate the LOAEL HEC for EGBE for human exposures producing the same C max of BAA in blood as that that produces effects in rats.

For a BAA concentration of 285 μM, the HEC = 98 ppm (474mg/m 3 ).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 264 Table 4.92a: Equivalent human do ses for LOAEL(C) / NOAEL(C) Oral Inhalation Dermal
End point LOAEL LOAEC NOAEL
Value 69mg/kg/day (male rat) 31ppm (152mg/m3) 150mg/kg/day

Equivalent BAA C max concentration 129 μM 285μM

Equivalent human dose 9.5 mg /kg/day 97ppm (474mg/m3)

A Risk Characterisation not using PbPk model is also proposed in Appendix B.
The risk characterisation for repeat dose then becomes as follows:

4.92b: Assessment factors for repeated dose toxicity Interspecies differences 0.1 (toxicodynamic factor)
Inhalation: toxicocinetic element taken into account by PBPK model

Dermal: Additional factor of 4 to account for allometric scaling of rats to humans
Intraspecies differences 5 (default for workers)
Duration of study No factor required. The critical study is 6 months in duration and the effects for this end-point (haemolysis) would not be expected to be more severe compared to lifetime exposure. (haemolysis due to EGBE is considered to be an acute or sub-acute effect on rodents, moreover in some studies animals tended to “recover” (red blood cells being less sensit ive ) with long times of
exposure. Type of effect 1
Extrapolation LOAEL to NOAEL 3 for oral and i nhalation. The effects were mild at this dose and there is evidence to show that the LOAEL is near the threshold level fo r effects of concern (eg NOAECs from other studies)
Factor of 1 for dermal exposure

Confidence of the database 1

Minimal MOS 1.5 for oral and inhalation
2.0 for dermal exposureEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 265 Note that the critical LOAEC used for the inhalation risk characterisation is derived from a whole body inhalation exposure study (as are all si milar studies). These animals will have been subjected to exposure by both the dermal and inhalation routes. The dermal absorption rates in rats and mice are also higher than in hu mans. Therefore it is no t necessary to make a correction for additional uptake fr om dermal absorption of vapours.

Risk characterisation
Oral exposure
No oral exposure has been identified for work ers, therefore there ar e no concerns for this route of exposure

Inhalation

Table 4.92c: Risk assessment for inhalation and dermal exposure for RDT
Risk assessment for inhalation
exposure Risk assessment for dermal exposure to liquid EGBE Scenario

8-hour TWA inhalation (mg/m 3 ) MOS 1 Conclu sion Estimated Skin exposure mg/day
(mg/kg bw/d MOS 2 Conclusion

1 - Manufacture 12 39 ii 42
(0.6) 250 ii

2 - Formulation 15.7 30 ii 2000
(28.57) 5 ii

3.1 Coating/Painting

3.11 –Industrial
-spraying 58.1 8 ii 2000
(28.57) 5 ii

-other works 30.4 16 ii 430
(6.14) 24 ii

3.12 - decorative 30.4 16 ii 70
(1) 150 ii

3.2 Printing 3 - Use of end products

3.21 Silk screening 20 24 ii 23
(0.33) 454 iiEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 266 3.22 General printing 5 95 ii 168
(2.4) 62 ii

3.3 Cleaning
spraying 49 10 ii 250
(3.57) 42

wiping 49 10 ii 1040
14.86) 10 ii

For exposure via the inhalation route, conc lusion (ii) is drawn for all scenarios.
Dermal exposure:
Exposure values are compared with the NOAEL of 150 mg/kg.
It is not possible to use the PBPK approa ch for the dermal assessment and a more conventional approach is therefore used.
See previous table and table showing derivation of MOSs
For exposure via the dermal route, conclu sion (ii) is drawn for all scenarios.

Combined exposure:
For the combined exposures the estimated internal doses are calculated from the biological exposure data. The inhalation LOAEC is chosen for comparison as this is lower than the equivalent for the dermal route. The LOAEC of 474mg/m3 would lead to an internal doses of 474 mg/m 3 x 10m3/day x 0.6/70kg = 40.6 mg/kg/day. This internal dose should be compared with internal dose calculated. This LOAEC should be compared with inte rnal doses calculated from exposures in each scenario (inhalation + de rmal). The internal doses are calculated as follow:

Inhalation exposure will give internal dose of:
X (value of the 8-hour TWA inhalation (mg/m 3 )) x 10 m 3 (inhaled air during a workday) x 0.6 (percentage of absorption by inhalation) / 70 (m ean bw of a worker) = Y (inhalation internal dose).
This value does not take into account the possi ble dermal absorption of vapour during the 8hr TWA. It has been demonstrated that dermal absorption of vapour EGBE could count for 39 % of the internal dose of EGBE. To take into account this value (which is not negligible) the value of internal dose due to dermal exposure to vapours (Z) should by added to the former value (Y). Z represent 39 % of the total in ternal dose and can be calculated as follow:

Z = 0.39/0.61 x Y = 0.64 Y

The total internal dose due to inhalation exposure (inhalation output + dermal vapour penetration output) is Y + Z = 1.64 YEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 267 For dermal exposure internal dose is calculated for a 70 kg bw worker with a percentage of absorption of 30 % (liquid EGBE, worst case)
The values are summarized in the following table:

Table 4.92d: Risk characterisation fo r combined exposure and for RDT

Scenario Internal dose after exposure to 8-hour TWA (mg/kg bw) Y+Z Internal dose after Dermal exposure to liquid EGBE

(mg/kg bw) worst case (based on maximal dose) Total internal dose (inhalation + dermal exposure) MOS Ccl

1 - Manufacture 1.69 0.18 1.87 22 ii

2 - Formulation 2.2 8.58 10.78 3.8 ii 3 - Use of products 3.1 Coating/Painting - industrial

- Spraying

- Other works - decorative 3.2 Printing

- silk screening

- general printing 3.3 Cleaning

- spraying

-

- wiping 8.17

4.28

4.28

2.81 0.70

6.89

6.89 8.58

1.84

0.30

0.10 0.72

1.07

4.46 16.75

6.12

4.58

2.91 1.42

7.96

11.35 2.4

6.6

8.8

14 29

5.1

3.6 ii

ii

ii

ii ii

ii

ii

1) internal dose for exposure to EGBE vapour is due to inhalation uptake (10 m 3 a workday, a worker of 70 kg bw and 60 % of absorption.

The calculated MOS should be compared with mi nimal MOS. To be in the worst case, the highest minimal MOS should be c hosen for the comparison: 2.

Conclusion ii is reached for all scenario for combined exposure. There are no concerns for repeat dose exposure for all scenarios. (conclusion ii ).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 268 4.1.3.2.5 Mutagenicity
Given the results from the mutagenicity studies it is concluded that EGBE is of no concern for workers with regard to mutagenicity (conclusion ii)

.

4.1.3.2.6 Carcinogenicity
Inhalation exposure:
As the mechanism of haemangiosarcomas in male mice is related to h aemotoxicity, the risk characterisation made for repeated dose toxicity (RDT) is also relevant for carcinogenicity. The other tumours seen in the animal studi es being not relevant to humans no risk characterisation is needed for them.

See RDT risk characterisation section

Results:
Exposure cannot be excluded for:

There are no concerns for carci nogenicity for all scenarios. (conclusion ii)

.

4.1.3.2.7 Toxicity for reproduction
It is possible to derive a no effect level of 720mg/kg/day for fertil ity effects based on a continuous breeding study in mice. The MOSs obtained are compared with minimal MOS calculated as follows:

Table 4.93: Assessment factors applied for the calculation of minimal MOS fertility effects. Interspecies differences 10
Intraspecies differences 5 (workers: homogen population)
Type of effect 1

Confidence of the database 1

Minimal MOS 50

Internal dose due to 720 mg/kg bw (NOAEL) would be 720 mg/kg bw (100 % oral absorption). This value should be compared w ith estimated internal doses due to exposure (see RDT section for calculation).
The following table summarise risk characterisa tion for inhalation exposure, dermal exposure and combined exposure.
It is necessary in this case to correct the inhalation value fo r the dermal absorption of vapour during the 8hr TWA as it is being compared to a NOAEL derived from an oral study. It has been demonstrated that dermal absorption of vapour EGBE could count for 39 % of the internal dose of EGBE. To take into account this value (which is not negligible) the value ofEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 269 internal dose due to dermal exposure to vapours (Z) should by added to the former value (Y). Z represent 39 % of the total internal dose and can be calculated as follow :

Z = 0.39/0.61 x Y = 0.64 Y

The total internal dose due to inhalation exposure (inhalation output + dermal vapour penetration output) is Y + Z = 1.64 Y. The values for Y are shown in the table used for the repeat dose combined route assessment.EURISK ASSESSMENT–2-BUTOXYETHANOL CAS111-76-2 CHAPTER4.HUMAN HEALTHRAPPORTEURFRANCER408_0808_HH_CLEAN270Table 4.93bis: Risk characterisation for reprotoxicityScenarioInternal doseafter exposureto 8-hourTWA (mg/kgbw)Y+Z1MOSInhalationonlyInternal dose afterDermal exposure toliquid EGBE(mg/kg bw)worst case (basedon maximal dose)MOSdermal onlyTotal internaldose(inhalation +dermalexposure)MOSCombinedCclinhalation,dermal andcombined1 - Manufacture 1.69 426 0.18 4000 1.87 385 ii2 - Formulation 2.2 327 8.58 84 10.78 67 ii3 - Use of products3.1 Coating/Painting- industrial-Spraying-Other works-decorative3.2 Printing-silk screening-general printing3.3 Cleaning-spraying-wiping8.174.284.282.810.706.896.898816816825610291041048.581.840.300.10.721.074.468438122866957100064015416.756.124.582.911.427.9611.35431181572475079063iiiiiiiiiiiiii1) internal dose for exposure to EGBE vapour is due to inhalation uptake (10 m3 a workday, a worker of 70 kg bw and 60 % of absorption) and topercutaneous absorption of vapour EGBE (39 % of the total internal dose).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 271 The calculated MOS should be compared with mi nimal MOS. To be in the worst case, the highest minimal MOS should be c hosen for the comparison: 50.

Conclusion ii for all scenar ios 3.1 industrial spraying.

For scenario 3.1. The calculated MOS is closed to the minimal MOS. Given the uncertainties of the dermal exposure assessment and the non specificity of the effects observed in the continuous breeding study, it can be considered th at there is no concern for this end-point.

Conclusion ii is reached for all scenarios.
The developmental toxicity studies for EGBE clearly indicate that the developmental effects observed are a consequence of and secondary to maternal toxicity. Any formal risk characterisation for humans using this data woul d not be meaningful. The available data does not suggest that EGBE shares the developmenta l toxicity properties of certain other glycol ethers. There is no other data which indicates potential developmental toxicity concerns. Therefore the conclusion is that there is no concern for this end point, conclusion (ii). (conclusion ii)

.

4.1.3.2.8 Summary of risk characterisation for workers
Conclusion (ii) applies for all scenarios concerning each endpoint.

4.1.3.3 Consumers
Assuming that oral exposure could only be acci dental by ingestion of a product, the risk characterisation for consumers is limited to the dermal and the inhalation routes of exposure.
For risk characterisation, a value of 30 % fo r dermal absorption and a value of 60 % for inhalation exposure can be taken into account.

Table 4.94: Internal dose exposure depending on scenarios Scenario Inhalation (mg/kg/d) Skin (mg/kg/d) Sum of exposures (mg/kg/d)

1 - Household surface cleaners 0.054 2.1 2.154

2 – Indoor air 0.06 0.06
3 – Painting 6 1.3 7.3

4 - Household surface cleaners + indoor air 0.114 2.1 2.214

5 - Painting + indoor air 6.06 1.3 7.4

4.1.3.3.1 Acute toxicity
According to acute toxicity da ta in human (suicide attempts cases), a LOAEL of 400 mg/kg can be taken into account. As limited data exis t in humans by dermal or inhalation route an extrapolation route to route of this LOAEL is done. The MOSs obtained are compared with minimal MOS calculated as follows:EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 272 Table 4.95: Assessment factors applied for the calculation of minimal MOS for acute toxicity and for inhalation and dermal route. Interspecies differences 1 (human data)
Intraspecies differences 10
Type of effect 1
Extrapolation LOAEL to NOAEL 5

Confidence of the database 1

Minimal MOS 50

The MOSs between the LOAEL and the inhalati on and skin exposures and for the sum of exposures are mentioned in table 4.96 :

Table 4.96: MOSs and conclusion for acute toxicity Scenario Inhalation Dermal Sum of exposures

MOS Conclusion MOS Conclusion MOS Conclusion

1 – Household surface cleaners 7407 ii 190 ii 185 ii

2 – Indoor air 6667 ii 6667 ii
3 – Painting 67 ii 308 ii 55 ii

4 – Household surface cleaners + indoor air 3508 ii 190 ii 180 ii

5 – Painting + indoor air 66 ii 308 ii 54 ii

Based on the risk assessment for the various scenar ios, it is concluded that the toxicity due to acute exposure is not expected. So conclusion ii is reached for all scenarios.

4.1.3.3.2 Irritation and corrosivity

Skin
EGBE is classified as a skin irritant, and th e use of gloves is recommended. This concern is managed by the information given by the classifi cation and labelling of the substance so that no further concern remains ( conclusion ii ).EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 273 Eye
Eye irritation (liquid):
The substance is classified as an eye irritant but in normal use, liquid should not be in contact with eyes. This concern is managed by the information given by th e classification and labelling of the substance so that no further concern remains ( conclusion ii ).

Eye and respiratory tract irritation (vapours):
Scenarios of concern are those for which an inhalation exposure by vapours of EGBE has been evaluated.
The NOAEL is 50 ppm that can be converted in mg/m 3 . So the NOAEL for this effect is :
50 x 4.9 = 245 mg/m 3

.
Table 4.97 : Consumer risk assessment of EGBE for eye and r espiratory irritation by vapour exposure. Scenario Inhalation
(mg/m3) MOS Conclusion

1 – Household surface cleaners 0.27 906 ii

2 – Indoor air 0.3 816 ii
3 – Painting 30 8 ii

4 – Household surface cleaners + indoor air 0.57 430 ii
5 - -Painting + indoor air 30.3 8 ii

For eye and respiratory tract irritation due to the exposure of EGBE in vapour form, conclusion ii is reached for all consumer scenarios.

4.1.3.3.3 Sensitisation

Skin

Given the results from the dermal sensitisatio n studies it is conclude d that EGBE is of no concern for consumers with re gard to skin sensitisation (conclusion ii)

.

Respiratory tract
There are neither data from human experi ence nor other indications for respiratory sensitisation.

4.1.3.3.4 Repeated dose toxicity
It is clear from the available data that haemolys is is the primary and critical response elicited in the main animal test mode ls (rats and mice) following inha lation, oral or dermal exposureEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 274 to EGBE. Blood from humans, pigs, dogs, cats, and guinea pigs is insensitive to haemolysis by BAA. In sensitive species, EGBE produces a characteristic toxicity that is revealed clinically by the appearance of haemoglobinuria and pathologically by changes in a variety of blood parameters. A 100-fold greater concentr ation of BAA (10mM) is required for human erythrocytes to de velop pre-hemolytic changes consistent with those seen in the rat (0.1mM). Such in vivo blood concentrations are unlikely to occur under normal conditions of human exposure to BE. Haemolysis did not occur with any blood from any indivi dual, even sensitive sub-populations, when exposed in vitro to BAA(10 mM). Studies have also shown that potentially sensitive human sub-populations, in cluding the children, the el derly and those with sickle cell anaemia are also e qually resistant to the effect.

The same models will be used for consumers than for workers to calculate the equivalent human EGBE concentration that would produce th is dose, and finally extrapolate this on the assumption. The NOAEL(C)/LOAEL(C) were calcul ated using the PBPK model in humans.

Table 4.97 bis: Equivalent human doses for NOAEL(C) / LOAEL(C) Oral Inhalation Dermal
End point LOAEL LOAEC NOAEL
Value 69 mg/kg/day (female rat)* 31 ppm (152mg/m3) 150mg/kg/day

Equivalent BAA C max concentration 129 μM 285 μM

Equivalent human dose 9.5 mg /kg/day** 97 ppm (474mg/m3)
* as quoted by US EPA. **assuming 70kg person drinking 2 litres water/day

To select an appropriate interspecies chemical safety assessment factor (CSAF), we use the same method than in the workers’ risk characte risation leading to an in terspecies factor of 0.1 (see Table 4.97 ter).
The risk characterisation for repeat dose then becomes as follows:

Table 4.97 ter: Assessment factors for oral exposure Interspecies differences 0.1 (toxicodynami c factor based on the difference in sensitivity between rodents and humans as described in the risk characterisation for workers part)
Inhalation: toxicokinetic element taken into account by PBPK model

Dermal: Additional factor of 4 to account for allometric scaling of rats to humans
Intraspecies differences 10 (default for consumers)EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 275 Duration of study No factor required. The critical study is 6 months in duration and the effects for this end-point (haemolysis) would not be expected to be more severe compared to lifetime exposure. (haemolysis due to EGBE is considered to be an acute or sub-acute effect on rodents, moreover in some studies animals tended to “recover” (red blood cells being more resistant) with long times of Type of effect 1
Extrapolation LOAEL to NOAEL 3 for oral and i nhalation The effects were mild at this dose and there is evidence to show that the LOAEL is near the threshold level fo r effects of concern (eg NOAECs from other studies)
Confidence of the database 1

Minimal MOS 3 for oral and inhalation
4 for dermal exposure

Risk characterisation
For repeated dose toxicity, daily exposure level ha s to be averaged over a year. So the internal exposure dose used for risk characterisation will be :
internal dose x number of events over a year

365

Table 4.98: Internal dose exposure depending on scenarios average over a year Scenario Number of events Inhalation (mg/kg/d) Skin (mg/kg/d) Sum of
exposures (mg/kg/d)

1 – Household surface cleaners 1.8-4.8 events/week (1) = 94-250 events/year 0.036 1.44 1.476

2 – Indoor air Each day 0.06 0.06
3 – Painting 10 events/year 0.16 0.036 0.196

4 – Household surface cleaners + indoor air 1.8-4.8 events/week (1) = 94-250 events/year 0.096 1.44 1.536

5 – Painting + indoor air 10 events/year 0.22 0.036 0.256
(1) Data from the Technical Guidance Document
The NOAEL is 474 mg/m 3 by inhalation and 150 mg/kg/day fo r dermal exposure. There is no oral exposure in normal use of products containing EGBE.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 276 Internal NOAELs have to be calculated and to be compared with the internal daily exposures by skin and by inhalation and to the sum of internal exposures.
For combined exposure, the NOAEL concerning the dermal exposure will be chosen as it is more protective for consumers.
The respiratory volume for an adult is 20 m 3 and the mean bodyweight is 60 kg. The absorption of EGBE by inhalation is 60 %. So the internal NOAEL by inhalation will be :

474 x 20 x 0.6

60 = 94.8 mg/kg/d

The dermal absorption factor is 30%. So th e internal NOAEL by derm al route will be :
150 x 0.3 = 45 mg/kg/d

Table 4.99: MOSs and conclusion for repeated dose toxicity Inhalation Dermal Sum of exposures Scenario MOS Conclusion MOS Conclusion MOS Conclusion

1 – Household surface cleaners 2633 ii 31 ii 30 ii

2 – Indoor air 1580 ii 750 ii
3 – Painting 592 ii 1250 ii 230 ii

4 – Household surface cleaners + indoor air 987 ii 31 ii 29 ii

5 – Painting + indoor air 431 ii 1250 ii 176 ii

Conclusion ii is reached for all scenarios

.

4.1.3.3.5 Mutagenicity
Given the results from the mutagenicity studies it is concluded that EGBE is of no concern for consumers with regard to mutagenicity (conclusion ii)

.

4.1.3.3.6 Carcinogenicity
As the mechanism of haemangiosarcomas in male mice is related to h aemotoxicity, the risk characterisation made for repeated dose toxici ty can also be used for carcinogenicity. The other tumours are not relevant to humans so no risk characterisation is needed for them.
For carcinogenicity, daily exposur e level has to be averaged ove r a year as for repeated dose toxicity. So the same values of internal e xposures will be compared to the NOAEL defined for carcinogenicity.

See RDT risk characterisation.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 277 The values are summarised in the following table:

Table 4.99 bis: Risk characterisation for carcinogenicity Inhalation Dermal Sum of exposures Scenario MOS Conclusion MOS Conclusion MOS Conclusion

1 – Household surface cleaners 2633 ii 31 ii 30 ii

2 – Indoor air 1580 ii 750 ii
3 – Painting 592 ii 1250 ii 230 ii

4 – Household surface cleaners + indoor air 987 ii 31 ii 29 ii

5 – Painting + indoor air 431 ii 1250 ii 176 ii

Conclusion ii is reached for all scenarios.

4.1.3.3.7 Toxicity for reproduction
It is possible to derive a no effect level of 720 mg/kg/day for fertility effects based on a continuous breeding study in mice. Daily exposu re level has been averaged over a year as reported in Table 4.98.
The MOSs obtained are compared with minimal MOS calculated as follows:

Table 4.100: Assessment factors applied for the calculation of minimal MOS fertility effects. Interspecies differences 10
Intraspecies differences 10
Type of effect 1

Confidence of the database 1

Minimal MOS 100

Risk characterisation

Table 4.101: MOSs and conclusion for reproduction Inhalation Dermal Sum of exposures Scenario MOS Conclusion MOS Conclusion MOS Conclusion

1 – Household surface cleaners 20000 ii 500 ii 488 ii

2 – Indoor air 12000 ii 12000 iiEU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 278 3 – Painting 4500 ii 20000 ii 3673 ii

4 – Household surface cleaners + indoor air 7500 ii 500 ii 469 ii

5 – Painting + indoor

3273 ii 20000 ii 2813 ii

Conclusion ii is reached for all scenarios:
The developmental toxicity studies for EGBE clearly indicate that the developmental effects observed are a consequence of and secondary to maternal toxicity. Any formal risk characterisation for humans using this data w ould not be meaningful. The available data does not suggest that EGBE shares the developmenta l toxicity properties of certain other glycol ethers. There is no other data which indicates potential developmental toxicity concerns. Therefore the conclusion is that there is no concern for this end point, conclusion (ii). (conclusion ii)

.

4.1.3.4 Humans exposed via the environment
The key health effect is repeated dose toxicity. Irritation (via de rmal or ocular routes)) is of low concern where exposure is dissipated throughout the environment. Comparison of the total internal dose of 94.8 mg.kg -1 (corresponding to the L OAEL of 31 ppm for RDT via inhalation route corrected with PbPk modelling to obain human internal dose see also calculation of internal NOAEL by inhalation in the consumer pa rt in chapter 4.1.3.3.4.) with the highest estimated exposure at regional (3.22.10 -4 mg.kg -1 .day -1 ) and local (3.73.10 -2 mg.kg -1 .day -1 ) levels leads to margins of safety of, respectively, 2.9.10 5 and 2.5.10 3 which do not lead to concern (compared to the mi nimal MOS of 3 calculated for consumers).

4.1.3.4.1 Summary of risk characterisation for exposure via the environment
(ii) There is at present no need for further information and/or testing and or risk reduction measures beyond those applied already. This conclusion applies for all endpoints in relation to local an d regional exposure.

4.2 HUMAN HEALTH (PHYSICO-CHEMICAL PROPERTIES)
EGBE has a low vapour pressure and is moderate ly flammable ( flash point is 60°C). It has no explosive or oxidising properties. However, it is noted that oxida tion by air may involve peroxidation of the substance, which may increas e explosive properties. A general warning to this effect is recommended. Use of antioxida nts reduces the potent ial to peroxidation.

It can be concluded that there is no co ncern for human health with regard physicochemical properties (conclusion ii).EU RISK ASSESSMENT

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### 5 RESULTS 6

5.1 INTRODUCTION

5.2 ENVIRONMENT

5.3 HUMAN HEALTH

5.3.1 Human health (toxicity)

5.3.1.1 Workers

Conclusion ( ii) There is at present no need for furt her information and/or testing and no need for risk reduction measures beyond those which are being applied already.

5.3.1.2 Consumers

Conclusion ( ii) There is at present no need for furt her information and/or testing and no need for risk reduction measures beyond those which are being applied already.
Conclusion (ii) applies to all end points and all scenarios:

5.3.1.3 Humans exposed via the environment

Conclusion ( ii) There is at present no need for furt her information and/or testing and no need for risk reduction measures beyond those which are being applied already.

6 Conclusion (i) There is a need for further information and/or testing.

Conclusion (ii) There is at present no n eed for further information and/or testing and no need for risk reduction measures beyo nd those which are being applied already. Conclusion (iii) There is a need for limiting the risks; risk reduction measures which are already being applied shall be taken into
account.EU RISK ASSESSMENT

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R408_0808_ HH _C LEAN 280 5.3.2 Human health (risks from physico-chemical properties)

Conclusion ( ii) There is at present no need for furt her information and/or testing and no need for risk reduction measures beyond those which are being applied already.EU RISK ASSESSMENT

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### 6 REFERENCES

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St. Clair M.B.G. and Morgan K.T., 1992. Changes in the upper respiratory tract. In Pathobiology of the Aging Rat (U.Mohr, D.L. Dungworth & C.C. Capen, Eds.) Vol.1, pp. 111-127. ILSI Press, Washington, DC.
Steffan A.-M., Gendrault J.-L., McCluskey R.S., McCluskey P.A. and Kirn, A., 1986. Phagocytosis, an unrecognised property of murine endothelial liver cells. Hepatology, 6, 830-836.
Tanii H. and Hashimoto K., 1982. Structure-toxicity relationship of acrylates and methacrylates. Toxicol. Letters 11, 125-129.
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Tyl R.W., Millicovsky G., Dodd D.E., Pritts I., France K. A. and Fisher L.C., 1984. Teratogenic evaluation of ethylene glycol monobutyl ether in Fischer 344 rats and new zealand white rabbits following inhalation exposure. Env. Health Perspect., 57, p47-68.
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– 2-BUTOXYETHANOL CAS 111-76-2 ABBREVIATIONS

RAPPORTEUR F RANCE 299 

### ABBREVIATIONS

ADH Alcohol Dehydrogenase
ADI Acceptable Daily Intake

AF Assessment Factor ALP Alcaline Phosphatase
ARDS Adult Respiratory Distress Syndrome
ASTM American Society for Testing and Materials
ATP Adaptation to Technical Progress
ATSDR Agency for toxic substances and disease registry (USA)

AUC Area Under The Curve

B Bioaccumulation
β 2-m β 2-microglobulin
BAA Butoxy Acetic Acid
BAL Buthoxy aldehyde
BBA Biologische Bundesanstalt für Land- und Forstwirtschaft

BCF Bioconcentration Factor
BEG Glucuronide conjugate of EGBE
BEI Biological Exposure Indice
BES Sulfo conjugate of EGBE

BMC Benchmark Concentration

BMD Benchmark Dose
BMF Biomagnification Factor

BSA Bovine Serum Albumin
bw body weight / Bw
, b.w. C Corrosive (Symbols and indications of danger for dangerous substances and preparations according to Annex III of Directive 67/548/EEC)

CA Chromosome Aberration
CA Competent Authority

CAS Chemical Abstract Services
CAT Carnitine Acetyl Transferase
CBC Complete Blood Count
CEC Commission of the European Communities
CEN European Standards Organisation / European Committee for Normalisation
CEPE European council of the paint, printing ink and artists’ colours industry

CI ClearanceEU RISK ASSESSMENT

– 2-BUTOXYETHANOL CAS 111-76-2 ABBREVIATIONS

RAPPORTEUR F RANCE 300 CMNI Maximal Concentration with No Irritation
CMR Carcinogenic, Mutagenic and toxic to Reproduction
CNS Central Nervous System
COD Chemical Oxygen Demand
CSTEE Scientific Committee for Toxicity, Ec otoxicity and the Environment (DG SANCO)

CT 50 Clearance Time, elimination or de puration expressed as half-life
d.wt dry weight / dw
dfi daily food intake

DG Directorate General
DIN Deutsche Industrie Norm (German norm)
DNA DeoxyriboNucleic Acid
DOC Dissolved Organic Carbon
DT50 Degradation half-life or period required for 50 percent dissipation / degradation
DT90 Period required for 50 percent dissipation / degradation
DTH Delayed Time Hypersensibility
E Explosive (Symbols and indications of danger for dangerous substances and preparations according to Annex III of Directive 67/548/EEC)
EASE Estimation and Assessment of Substance Exposure Physico-chemical properties [Model]

EbC50 Effect Concentration measured as 50 % reduction in biomass growth in algae tests
EC European Communities

EC10 Effect Concentration measured as 10 % effect

EC50 median Effect Concentration
ECB European Chemicals Bureau
ECETOC European Centre for Ecotoxicology and Toxicology of Chemicals
ECVAM European Centre for the Validation of Alternative Methods
EDC Endocrine Disrupting Chemical
EEC European Economic Communities
EG Ethylene Glycol
EGBE 2-Butoxyethanol
EGBEA 2-Butoxyethanol acetate
EINECS European Inventory of Existin g Commercial Chemical Substances
ELINCS European List of New Chemical Substances
EN European Norm EPA Environmental Protection Agency (USA)

ErC50 Effect Concentration measured as 50 % reduction in growth rate in algae tests

ESD Emission Scenario Document

EtOH Ethanol
EU European UnionEU RISK ASSESSMENT

– 2-BUTOXYETHANOL CAS 111-76-2 ABBREVIATIONS

RAPPORTEUR F RANCE 301 EUSES European Union System for the Evaluation of Substances [software tool in support of the Technical Guidance Document on risk assessment]
F(+) (Highly) flammable (Symbols and indications of danger for dangerous substances and preparations according to Anne x III of Directive 67/548/EEC)
FAO Food and Agriculture Organisation of the United Nations
FELS Fish Early Life Stage
G6P Glucose 6 Phosphate
G6PD Glucose 6 Phosphate Dehydrogenase
GE Glycol Ether
GGT Gamma Glutamyl Transpeptidase

GI Gastro Intestinal
GLP Good Laboratory Practice

GLU Glucose
GP Guinea Pig
Hb Haemoglobin

HCT Haematocrit
HEDSET EC/OECD Harmonised Electronic Data Set (for data collection of existing substances)

HELCOM Helsinki Commission -Baltic Marine Environment Protection Commission
HGB Haemoglobin
HPLC High Pressure Liquid Chromatography
HPVC High Production Volume Chemical (> 1000 t/a)
HSE Health and Safety Executive (UK)
IARC International Agency for Research on Cancer
IC Industrial Category
IC50 median Immobilisation Concentration or median Inhibitory Concentration

IL2 Inter Leukine 2
ILO International Labour Organisation

INF Interferon

INRS Institut National de Recherche et de Sécurité

IP Intra Peritoneal

IPA Intra Peritoneal
IPCS International Programme on Chemical Safety
ISO International Organisation for Standardisation

IUCLID International Uniform Chemical In formation Database (existing substances)
IUPAC International Union for Pure and Applied Chemistry

IV Intra Venous JEFCA Joint FAO/WHO Expert Committee on Food Additives
JMPR Joint FAO/WHO Meeting on Pesticide ResiduesEU RISK ASSESSMENT

– 2-BUTOXYETHANOL CAS 111-76-2 ABBREVIATIONS

RAPPORTEUR F RANCE 302 Koc organic carbon normalised distribution coefficient
Kow octanol/water partition coefficient
Kp solids-water partition coefficient
L(E)C50 median Lethal (Effect) Concentration

LAEL Lowest Adverse Effect Level
LAS Alkyl Sulfonate

LC50 median Lethal Concentration

LD50 median Lethal Dose
LDH Lactate DeHydrogenase

LEV Local Exhaust Ventilation
LLNA Local Lymph Node Assay
LN Lymph Node

LOAEC Low Observable Adve rse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level

LOEC Lowest Observed Effect Concentration

LOED Lowest Observed Effect Dose

LOEL Lowest Observed Effect Level

MAC Maximum Allowable Concentration
MATC Maximum Acceptable Toxic Concentration
MC Main Category
MCH Mean Corpuscular Haemoglobin
MCHC Mean Cell Haemoglobin Concentration
MDA Malonyl DiAldehyde

MeOH Methanol
MITI Ministry of International Trade and Industry, Japan
MOE Margin of Exposure
MOS Margin of Safety
MW Molecular Weight
N Dangerous for the environment (Symbols and indications of danger for dangerous substances and preparations according to Annex III of Directive 67/548/EEC

NAEL No Adverse Effect Level

nBuOH n-Butanol
NICNAS National Industrial Chemicals Notification and Assessment Scheme (Australia)
NIOSH National Institute for Occupational Safety and Health (USA)

NK Natural Killer

NOAEC No Observable Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NOEL No Observed Effect LevelEU RISK ASSESSMENT

– 2-BUTOXYETHANOL CAS 111-76-2 ABBREVIATIONS

RAPPORTEUR F RANCE 303 NOEC No Observed Effect Concentration
nPrOH n-Propanol
NTP National Toxicology Program (USA)

NZ Natural Killer
O Oxidizing (Symbols and indications of danger for dangerous substances and preparations according to Annex III of Directive 67/548/EEC)
OECD Organisation for Economic Cooperation and Development
OEL Occupational Exposure Limit

OJ Official Journal
OSPAR Oslo and Paris Convention for the protection of the marine environment of the Northeast Atlantic

P Persistent
PBT Persistent, Bioaccumulative and Toxic
PBPK Physiologically Based PharmacoKinetic modelling
PBTK Physiologically Based ToxicoKinetic modelling

PCV Packed cell volume

PEC Predicted Environmental Concentration
PEG PolyEthylene Glycol
PGME Propylene Glycol Methyl Ether
pH logarithm (to the base 10) (of the hydrogen ion concentration {H + }
pKa logarithm (to the base 10) of the acid dissociation constant
pKb logarithm (to the base 10) of the base dissociation constant

PNEC Predicted No Effect Concentration

PO Per os POP Persistent Organic Pollutant
PPE Personal Protective Equipment
QSAR (Quantitative) Structure-Activity Relationship
R phrases Risk phrases according to Annex III of Directive 67/548/EEC
RAR Risk Assessment Report

RBC Red Blood Cell

RC Risk Characterisation

RfC Reference Concentration

RfD Reference Dose

RNA RiboNucleic Acid
RPE Respiratory Protective Equipment

RWC Reasonable Worst Case
S phrases Safety phrases according to Annex III of Dir ective 67/548/EEC
SAR Structure-Activity RelationshipsEU RISK ASSESSMENT

– 2-BUTOXYETHANOL CAS 111-76-2 ABBREVIATIONS

RAPPORTEUR F RANCE 304 SBR Standardised birth ratio
SCE Sister Chromatic Exchange
SDS Safety Data Sheet
SEM Scanning electron microscopy
SETAC Society of Environmen tal Toxicology And Chemistry

SGOT Serum Glutamic Oxaloacetic Transaminase

SGPT Serum Glutamic Pyruvic Transaminase
SNIF Summary Notification Interc hange Format (new substances)
SSD Species Sensitivity Distribution
STEL Short-Term Exposure Limit
STP Sewage Treatment Plant
T(+) (Very) Toxic (Symbols and indications of danger for dangerous substances and preparations according to Anne x III of Directive 67/548/EEC)
TDI Tolerable Daily Intake

TG Test Guideline

TGD Technical Guidance Document

TLV Threshold Limit Value
TNsG Technical Notes for Guidance (for Biocides)
TNO The Netherlands Organisation for Applied Scientific Research
TWA Time-Weighted Average
UC Use Category UDS Unscheduled DNA Synthesis

UN United Nations
UNEP United Nations Environment Programme

US EPA Environmental Protection Agency, USA
UV Ultraviolet Region of Spectrum
UVCB Unknown or Variable composition, Complex reaction products of Biological material
vB very Bioaccumulative

Vd Volume of Distribution
vP very Persistent
vPvB very Persistent and very Bioaccumulative
v/v volume per volume ratio
w/w weight per weight ratio

WBC White Blood Cell
WEC Whole Embryo Culture
WHO World Health Organization

WWTP Waste Water Treatment Plant
Xn Harmful (Symbols and indications of danger for dangerous substances and preparationsEU RISK ASSESSMENT

– 2-BUTOXYETHANOL CAS 111-76-2 ABBREVIATIONS

RAPPORTEUR F RANCE 305 according to Annex III of Directive 67/548/EEC)
Xi Irritant (Symbols and indications of danger for dangerous substances and preparations according to Annex III of Directive 67/548/EEC)EU RISK ASSESSMENT

– 2-BUTOXYETHANOL CAS 111-76-2A PPENDIX A

RAPPORTEUR F RANCE 306 

### Appendix A

Methods of calculation of consumer exposures

Scenario 1 : Household surface cleaners

inhalation

V r x C air x t e E ei = W b x h d

E ei = external exposure by inhalation

V r = respiratory volume in one day

C air = concentration in air

t e = duration of exposure

W b = mean bodyweight for a consumer (60 kg)

h d = hours in one day

E ii = E ei x abs i

E ii = internal exposure by inhalation

abs i = percentage of absorption by inhalation

Dermal

C p x Q p x Q h E ed = W b

E ed = external dermal exposure

C p = concentration of EGBE in the cleaning product
Q p = Quantity of product usedEU RISK ASSESSMENT

– 2-BUTOXYETHANOL CAS 111-76-2A PPENDIX A

RAPPORTEUR F RANCE 307 Q h = Quantity of EGBE tran sferred to the hands

W b = mean bodyweight for a consumer (60 kg)

E id = E ei x abs i

E id = internal dermal exposure

abs i = percentage of absorption by skin

Scenario 2 : indoor air

inhalation

V r x C air x t e E ei = W b x h d

E ei = external exposure by inhalation

V r = respiratory volume in one day

C air = concentration in air

t e = duration of exposure

W b = mean bodyweight for a consumer (60 kg)

h d = hours in one day

E ii = E ei x abs i

E ii = internal exposure by inhalation

abs i = percentage of absorption by inhalation

Scenario 3 : painting

Inhalation

V r x C air x t e E ei = W b x h dEU RISK ASSESSMENT

– 2-BUTOXYETHANOL CAS 111-76-2A PPENDIX A

RAPPORTEUR F RANCE 308 E ei = external exposure by inhalation

V r = respiratory volume in one day

C air = concentration in air

t e = duration of exposure

W b = mean bodyweight for a consumer (60 kg)

h d = hours in one day

E ii = E ei x abs i

E ii = internal exposure by inhalation

abs i = percentage of absorption by inhalation

CONSEXPO model parameters : Contact scenario: evaporation due to Painting
Duration of contact per event: 133 min
Frequency of contact: 10/year
Person uses product (room volume, vent ilation and release area personal).

Personal volume=5.000000 m3.
Mean event concentration (a verage case): 3.772e+02 mg/m3
Year average (average case): 9.539e-01 mg/m3
Mean event concentration (cumul ative worst case): 3.772e+02 mg/m3
Year average (cumulative worst case): 9.539e-01 mg/m3

Exposure estimates based on the following parameters:

Release area: 28.000 m2

Room volume: 25.000 m3

Room Ventilation rate: 12.5 m3/hr
Product amount: 5.000 kg
product density: 1.300 mg/cm3
Weight fraction: 3%EU RISK ASSESSMENT

– 2-BUTOXYETHANOL CAS 111-76-2A PPENDIX A

RAPPORTEUR F RANCE 309 Fraction to upper layer: 1.000 fraction
Layer exchange rate: 1.000 1/min
Temperature: 293.000 Kelvin
Molecular weight matrix: 400.000 g/mol

Uptake

Model: fraction model

Cumulative worst case estimate: 7616 mg/year : 0.2979 mg/(kg.day)

Uptake estimates based on the following parameters:

Absorbed fraction: 60 %

Inhalation rate: 25300 cm3/min
Respirable fraction: 100%

WALLPAINT EXPOSURE MODEL (WPEM)

Resident DIY model

Room volume 25m 3

Painted area 28m 2
Air changes 0.5 per hour
Paint quantity:5kg, density 1.3, type: flat
Model type: empirical
Body mass: 60kg
Events per year: 10
Active/total lifetime: 40/70 years

No sinks

EGBE content: 3%
Painting time: 133 minutesEU RISK ASSESSMENT

– 2-BUTOXYETHANOL CAS 111-76-2A PPENDIX A

RAPPORTEUR F RANCE 310 Dermal

C prod x TH der x AREA der x n E ed = D x BW

where :

E ed = external exposure

C prod = concentration of EGBE in paint (kg/m 3 ) TH der = thickness of product layer on skin AREA der = area of contact be tween paint and skin n = number of events (n = 1) D = dilution. There is no dilution so D = 1 BW = mean bodyweight for a consumer (60 kg)

E id = E ei x abs i

E id = internal dermal exposure

abs i = percentage of absorption by skinEU RISK ASSESSMENT

– 2-BUTOXYETHANOL CAS 111-76-2 A PPENDIX B

RAPPORTEUR F RANCE 311 

### Appendix B

Risk characterisation without using PbPk extrapolation

In the kinetic studies, if the pbpk is not taken into account, some data can be used to derive MOS for interspecies differences. It is clearly demonstrated that rodent blood cells are much more sensitive than human ones (at least 100 times) , for this effect, a conservative factor of 0.1 will be used. For dermal pene tration is was also demonstrated than rat skin was 2-3 more permeable than human one, for this end-point a correcting factor of 0.5 will be taken into
account. The risk characterisation for repeat dose then becomes as follows:
Table: Assessment factors applied for the calcu lation of minimal MOS for occupational exposure
Interspecies differences 0.1 for greater sensitivit y of rodents blood cells
0.5 for dermal absorption

Dermal: Additional factor of 4 to account for allometric scaling of rats to humans
Intraspecies differences 5 (default for workers)
Duration of study No factor required
Uncertainties concerning dermal
exposure 0.2 for dermal and for scen arios using data derived from measurements with a less volatile solvent.*
Type of effect 1
Extrapolation LOAEL to NOAEL 3 for oral and i nhalation The effects were mild at this dose and there is evidence to show that the LOAEL is near the threshold level fo r effects of concern (eg NOAECs from other studies)
Factor of 1 for dermal exposure

Confidence of the database 1

Minimal MOS 1.5 for oral and inhalation
1 for dermal exposure

Note that the critical LOAEC used for the inhalation risk characterisation is derived from a whole body inhalation exposure st udy (as are all similar studies). These animals will have been subjected to exposure by both the dermal and inhalation routes.EU RISK ASSESSMENT

– 2-BUTOXYETHANOL CAS 111-76-2 A PPENDIX B

RAPPORTEUR F RANCE 312 Exposure will be compared with the LOAEC of 31 ppm (152 mg/m 3 ) derived from a 14-week study in rats.

Risk characterisation
Oral exposure
No oral exposure has been identified for work ers, therefore there are no concerns for this route of exposure.

Inhalation

Risk assessment for inhalation
exposure Risk assessment for dermal exposure to liquid EGBE Scenario

8-hour TWA inhalation (mg/m 3 ) MOS 1 Conclusion Estimated Skin
exposure mg/day
(mg/kg bw/d MOS 2 Conclusion

1 - Manufacture 12 12.7 ii 42
(0.6) 250 ii

2 - Formulation 15.7 9.7 ii 2000
(28.57) 5.3 ii

3.1 Coating/Painting

3.11 –Industrial
-spraying 58.1 2.6 ii 2000
(28.57) 5.3 ii

-other works 30.4 5 ii 430
(6.14) 24 ii

3.12 - decorative 30.4 5 ii 70
(1) 150 ii

3.2 Printing

3.21 Silk screening 20 7.6 ii 23
(0.33) 454 ii 3 - Use of end products

3.22 General printing 5 30.4 ii 168
(2.4) 63 iiEU RISK ASSESSMENT

– 2-BUTOXYETHANOL CAS 111-76-2 A PPENDIX B

RAPPORTEUR F RANCE 313 3.3 Cleaning - spraying 49 3.1 ii 250
(3.57) 42

3.3 Cleaning - wiping 49 3.1 ii 1040
(14.86) 10 ii

For exposure via the inhalation route, conc lusion (ii) is drawn for all scenarios.
Dermal exposure:
Exposure values are compared with the NOAEL of 150 mg/kg.

See previous table
For exposure via the dermal route, conclu sion (ii) is drawn for all scenarios.
Combined exposure:
For the combined exposures the estimated intern al doses are calculated from the biological exposure data. The inhalation LOAEC is chosen for comparison as this is lower than the equivalent for the dermal route. The LOAEC of 152 mg/m3 would lead to an internal doses of 152 mg/m 3 *10m3/day*0.6/70kg = 13 mg/kg/day This internal dose should be compared with internal dose calculated . This LOAEC should be compared with internal doses calculated from exposures in each scenar io (inhalation + dermal). The in ternal doses are calculated as follow :

Inhalation exposure will give internal dose of:
X (value of the 8-hour TWA inhalation (mg/m 3 )) x 10 m 3 (inhaled air during a workday) x 0.6 (percentage of absorption by inhalation) / 70 (mean bw of a worker) = Y (inhalation internal dose).
This value does not take into account the possible de rmal absorption of vapour during the 8hr TWA. It has been demonstrated that dermal absorption of vapour EGBE could count for 39 % of the internal dose of EGBE. To take into account this value (which is not negligible) the value of internal dose due to dermal exposure to vapours (Z) should by adde d to the former value (Y). Z represent 39 % of the total internal dose and can be calculated as follow :

Z = 0.39/0.61 x Y = 0.64 Y
The total internal dose due to inhalation exposur e (inhalation output + derm al vapour penetration output) is Y + Z = 1.64 Y

For dermal exposure internal dose is calculated for a 70 kg bw worker with a percentage of absorption of 30 % (liquid EGBE, worst case)EU RISK ASSESSMENT

– 2-BUTOXYETHANOL CAS 111-76-2 A PPENDIX B

RAPPORTEUR F RANCE 314 The values are summarized in the following table:

Scenario Internal dose after exposure to 8-hour TWA (mg/kg bw) Y+Z Internal dose after Dermal exposure to liquid EGBE

(mg/kg bw) worst case (based on maximal dose) Total internal dose (inhalation + dermal exposure) MOS Ccl

1 - Manufacture 1.69 0.18 1.87 7.0 ii

2 - Formulation 2.2 8.58 10.78 1.2 iii 3 - Use of products 3.1 Coating/Painting - industrial

- Spraying

- Other works - decorative 3.2 Printing

- silk screening

- general printing 3.3 Cleaning spraying 3.3 Cleaning wiping 8.17 4.28

4.28

2.81 0.70

6.89

6.89 8.58 1.84

0.30

0.10 0.72

1.07

4.46 16.75 6.12

4.58

2.91 1.42

7.96

11.35 0.77 2.1

2.8

4.5 9.2

1.63

1.14 iii ii

ii

ii ii

ii

iii

1) internal dose for exposure to EGBE vapour is due to inhalation uptake (10 m 3 a workday, a worker of 70 kg bw and 60 % of absorption.
The minimal MOS required for the inhalation route is 1.5 whilst for the dermal route it is 1.
According to the results obtained a conclusion iii is reached for scenario 2, scenario 3.1: industrial spraying and s cenario 3.3 cleaning wiping.
There are no concerns for repeat dos e exposure for the other scenarios. (conclusion ii)

.EU RISK ASSESSMENT

– 2-BUTOXYETHANOL CAS 111-76-2

RAPPORTEUR F RANCE 315 European Commission

EUR [ECB: click here to insert EUR No.] - European Union Risk Assessment Report [ECB: click here to insert SUBSTANCE NAME, and volume no.]

Editors: (keep this updated )

Luxembourg: Office for Official Publications of the European Communities

[ECB: insert year] – VIII pp., [ECB: insert number of pages] pp. – 17.0 x 24.0 cm

Environment and quality of life series

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Price (excluding VAT) in Luxembourg: EUR [ECB:insert price]

The report provides the comprehensive risk assessment of the substance 2-butoxyethanol. It has been prepared by France in the frame of Council Regulation (EEC) No. 793/93 on the evaluation and control of the risks of existing substances, following the principles for assessment of the risks to man and the env ironment, laid down in Commission Regulation (EC) No. 1488/94.

This part of the evaluation considers the em issions and the resulting exposure to human populations in all life cycle steps. The scenarios for occupational exposure, consumer exposure and humans exposed via the environ ment have been examined and the possible risks have been identified.

The human health risk assessment concludes that there is no concern for workers, consumers, for humans exposed via the environment and for human health (physicochemical properties).