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Physicochemical Properties

HLS study number: CEF0001

Version ID: Draft 4 

Issue date: 03 October 2013

Page 1 of 53
Huntingdon Life Sciences

CEF0001

Sponsor and Test Facility 

Sponsor

Test facility Huntingdon Life Sciences

Eye Research Centre

Eye

Suffolk

IP23 7PX

UK

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Table of Contents

Sponsor and Test Facility...................................................................................................... 2

Compliance with Good Laboratory Practice .......................................................................... 6

Quality Assurance Statement................................................................................................ 7

Contributing Scientists ..........................................................................................................8

Summary ..............................................................................................................................9

1. Introduction ................................................................................................................ 10

1.1 Objective......................................................................................................... 10

1.2 Regulatory compliance ................................................................................... 10

1.3 Study schedule ............................................................................................... 10

1.4 Maintenance of records .................................................................................. 10

2. Test Substance .......................................................................................................... 11

3. Experimental Procedures ........................................................................................... 12

3.1 Melting temperature........................................................................................ 12

3.1.1 Method................................................................................................ 12

3.1.2 Definition and units.............................................................................. 12

3.1.3 Apparatus............................................................................................ 12

3.1.4 Procedure ........................................................................................... 12

3.1.5 Results................................................................................................ 12

3.1.6 Conclusion .......................................................................................... 13

3.2 Boiling temperature......................................................................................... 15

3.2.1 Method................................................................................................ 15

3.2.2 Definition and units.............................................................................. 15

3.2.3 Apparatus............................................................................................ 15

3.2.4 Procedure ........................................................................................... 15

3.2.5 Results................................................................................................ 15

3.2.6 Conclusion .......................................................................................... 15

3.3 Relative density .............................................................................................. 16

3.3.1 Method................................................................................................ 16

3.3.2 Definition and units.............................................................................. 16

3.3.3 Apparatus............................................................................................ 16

3.3.4 Reagents............................................................................................. 16

3.3.5 Suitability of displacement liquid.......................................................... 16

3.3.6 Procedure ........................................................................................... 16

3.3.7 Results................................................................................................ 18

3.3.8 Conclusion .......................................................................................... 18

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Table of Contents - continued

3.4 Vapour pressure ............................................................................................. 19

3.4.1 Method................................................................................................ 19

3.4.2 Definition and units.............................................................................. 19

3.4.3 Apparatus............................................................................................ 19

3.4.4 Calibration........................................................................................... 19

3.4.5 Procedure ........................................................................................... 19

3.4.6 Calculations......................................................................................... 20

3.4.7 Results................................................................................................ 21

3.4.8 Conclusion .......................................................................................... 21

3.5 Surface tension............................................................................................... 24

3.5.1 Method................................................................................................ 24

3.5.2 Definition and units.............................................................................. 24

3.5.3 Apparatus............................................................................................ 24

3.5.4 Test solution preparation..................................................................... 24

3.5.5 Procedure ........................................................................................... 25

3.5.6 Results................................................................................................ 25

3.5.7 Conclusion .......................................................................................... 25

3.6 Water solubility ............................................................................................... 26

3.6.1 Definition and units.............................................................................. 26

3.6.2 Preliminary test ................................................................................... 26

3.6.3 Procedure for the definitive test........................................................... 26

3.6.4 HPLC conditions ................................................................................. 26

3.6.5 Preparation of calibration..................................................................... 27

3.6.6 Calculations......................................................................................... 27

3.6.7 Results and discussion........................................................................ 27

3.6.8 Conclusion .......................................................................................... 27

3.7 Partition coefficient ......................................................................................... 34

3.7.1 Definition and units.............................................................................. 34

3.7.2 Preliminary estimation ......................................................................... 34

3.7.3 Method................................................................................................ 34

3.7.4 Procedure ........................................................................................... 35

3.7.5 HPLC conditions ................................................................................. 35

3.7.6 Results................................................................................................ 36

3.7.7 Conclusion .......................................................................................... 36

3.8 Flammability (solids) ....................................................................................... 43

3.8.1 Method................................................................................................ 43

3.8.2 Definition and units.............................................................................. 43

3.8.3 Procedure ........................................................................................... 43

3.8.4 Results................................................................................................ 43

3.8.5 Conclusion .......................................................................................... 43

3.9 Explosive properties........................................................................................ 44

3.9.1 Assessment of explosive properties .................................................... 44

3.9.2 Conclusion .......................................................................................... 44

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Table of Contents - continued

3.10 Relative self-ignition temperature for solids..................................................... 45

3.10.1 Definition and units.............................................................................. 45

3.10.2 Apparatus............................................................................................ 45

3.10.3 Procedure ........................................................................................... 45

3.10.4 Results................................................................................................ 45

3.10.5 Conclusion .......................................................................................... 45

3.11 Oxidising properties ........................................................................................ 47

3.11.1 Method................................................................................................ 47

3.11.2 Definition............................................................................................. 47

3.11.3 Procedure ........................................................................................... 47

3.11.4 Preliminary test ................................................................................... 47

3.11.5 Preliminary results............................................................................... 48

3.11.6 Conclusion .......................................................................................... 48

3.12 Particle size distribution .................................................................................. 49

3.12.1 Method................................................................................................ 49

3.12.2 Apparatus............................................................................................ 49

3.12.3 Procedure ........................................................................................... 49

3.12.4 Treatment of data................................................................................ 49

3.12.5 Results................................................................................................ 50

3.12.6 Conclusion .......................................................................................... 50

List of Annexes

Annex 1 Particle Size Distribution Data......................................................................51

Annex 2 Eye Research Centre GLP Compliance Statement 2012 .............................53

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Compliance with Good Laboratory Practice 

: Physicochemical properties 

The study described in this report was conducted in compliance with the following Good 

Laboratory Practice standards and I consider the data generated to be valid.

The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 No. 3106, 

as amended by Statutory Instrument 2004 No. 994). 

EC Commission Directive 2004/10/EC of 11 February 2004. 

OECD Principles of Good Laboratory Practice (as revised in 1997), 

ENV/MC/CHEM(98)17. 

These principles of Good Laboratory Practice are accepted by the regulatory authorities of 

the United States of America and Japan on the basis of intergovernmental agreements.

_________________________________ ___________________________ 

P Sydney BSc MSc Date

Study Director

Huntingdon Life Sciences

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Quality Assurance Statement

 Physicochemical properties 

The following inspections and audits have been carried out in relation to this study:

Study Phase Date(s) of Inspection Date of Reporting to Study 

Director and Management

Protocol Audit 14 Mar 2013 14 Mar 2013

Report Audit 17 Jul 2013 - 19 Jul 2013 19 Jul 2013

Process based inspections: At or about the time this study was in progress inspections of 

procedures employed on this type of study were carried out. These were conducted and 

reported to appropriate Company Management as indicated below: 

Process Based Inspections Date(s) of Inspection Date of Reporting to 

Management

Results processing 07 Feb 2013 07 Feb 2013

Standard preparation/Sample 

preparation

11 Feb 2013 11 Feb 2013

Measurements 12 Mar 2013 12 Mar 2013

Analytical procedures 10 Apr 2013 - 18 Apr 2013 18 Apr 2013

Study management and 

conduct 

06 June 2013 - 07 June 2013 07 Jun 2013

In addition, an inspection of the facility where this study was conducted was carried out on an 

annual basis. These inspections were promptly reported to Company Management. 

_________________________________ ___________________________ 

T Pullinger MRQA Date

Group Manager 

Department of Quality Assurance

Huntingdon Life Sciences

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Contributing Scientists

: Physicochemical properties 

Study management

P Sydney BSc MSc

Study Director 

Product Chemistry 

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Summary

A study was performed to determine some physicochemical properties of the test substance, 

. The methods followed are amongst those described in Commission Regulation 

(EC) No. 440/2008 and the OECD Guidelines for the Testing of Chemicals. The 

physicochemical properties which have been determined in this study are detailed below, 

together with the result obtained for each test. 

EC OECD Test Result

method no. method no.

A.1 102 Melting temperature 139°C (DSC)

A.2 103 Boiling temperature Not determinable; decomposes at 

temperatures above approximately 

230°C without boiling

A.3 109 Relative density )(D 20 

4 1.16 

A.4 104 Vapour pressure ≤ 3 x 10-8 Pa at 25°C

A.5 115 Surface tension 66.0 mN/m

A.6 105 Water solubility 27.5 mg/L at 20 ̊C

A.8 117 Partition coefficient Log10Pow < 0.3 at each of pH 4, 7 

and 9

A.10 - Flammability (solids) Not highly flammable

A.14 - Explosive properties

(theoretical assessment)

Not explosive

A.16 - Relative self-ignition temperature None below 400°C

A.17 - Oxidising properties Non-oxidising

- - Particle size distribution d(0.1) = 10 μm

d(0.5) = 37 μm

d(0.9) = 73 μm

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1. Introduction

1.1 Objective

This study was designed to determine some physicochemical properties of the test substance, 

. Information on physicochemical properties is important in the assessment of the 

potential effects of a substance both for human health and in the environment. 

1.2 Regulatory compliance

The study was conducted in accordance with accepted principles in order to meet the 

requirements of Commission Regulation (EC) No. 440/2008 and the OECD Guidelines for 

the Testing of Chemicals. 

1.3 Study schedule 

Study initiation date : 18 March 2013 

Experimental start date : 28 March 2013 

Experimental completion date : 08 August 2013 

Study completion date : 

1.4 Maintenance of records

Records and documentation relating to this study (including electronic records) will be 

maintained in the archives of Huntingdon Life Sciences for a period of one year from the date 

on which the Study Director signs the final report. 

Huntingdon Life Sciences will retain in its archive the Quality Assurance records relevant to 

this study for a period of 20 years and a copy of the final report indefinitely. 

1.5 Deviations from protocol

Although the definitive measured water solubility value was 27.5 mg/L, the surface tension 

test was run at the maximum concentration of 1 g/L rather than at 90% of this saturation 

concentration. The selected concentration was based on the fact that dissolved at 

between 1 and 2 g/L in water in a preliminary test (visual assessment). 

Definitive water solubility testing also showed the presence of a significant, soluble 

degradate component and it was thus apparent that the prepared surface tension solutions 

would contain both parent and degradate species. 

As it could be possible that either species may contribute to surface activity, it was 

considered valid to run the test at the higher concentration. As no significant surface activity 

was determined, there was deemed to be no impact on the outcome of the test. 

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2. Test Substance

Identity:

Structure:

Appearance: White crystalline powder

Storage conditions: Room temperature in the dark

Batch number: 12342

Purity: To be advised

Expiry date: To be advised

Date received: 04 March 2013

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3. Experimental Procedures

3.1 Melting temperature (EC Method A.1, OECD Method 102)

3.1.1 Method

The melting temperature was determined using the metal block method and confirmed by 

Differential Scanning Calorimetry (DSC).

3.1.2 Definition and units

The melting temperature of a substance is defined as the temperature (°C) at which the phase 

transition from solid to liquid state at normal atmospheric pressure takes place.

3.1.3 Apparatus

Melting/boiling point apparatus: Model B-545, Buchi. The apparatus is regularly calibrated 

by determination of the melting points of reference materials.

Mettler Toledo FP900 Thermosystem with FP85 DTA/DSC measuring cell.

3.1.4 Procedure

Dried test substance was tightly packed into a capillary tube to a height of 3 mm. Following 

an initial estimation, the melting point apparatus was set at approximately 10°C below the 

expected melting temperature. The capillary tube was placed in the apparatus and heated at 

1°C/minute. The procedure was performed in duplicate with a fresh sample on each occasion.

DSC was subsequently performed in a nitrogen atmosphere at a heating rate of 1°C/minute.

3.1.5 Results

The results of duplicate melting point determinations on the test substance were as follows:

Temperature (°C)

Melting stage* Sample I Sample II

A 123 123

B 133 133

C 138 138

D 139 139

E 140 140

*as referenced in OECD Guideline 102.

There was an unusually large gap between the first and second reported stages of melting and 

it was considered that true melting occurred over stages B to E.

The DSC trace (Figure 1) showed a sharp endotherm between approximately 135 and 140 ̊C

(peak maximum 139 ̊C), with no significant thermal event below this temperature range, 

confirming the earlier observation.

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3.1.6 Conclusion

was found to melt over the range 133 to 140°C (metal block method) and this 

result was confirmed by DSC (peak maximum temperature 139 ̊C). 

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Figure 1 DSC Trace of 

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3.2 Boiling temperature (EC Method A.2, OECD Method 103)

3.2.1 Method

The boiling temperature was examined by the Siwoloboff method. 

3.2.2 Definition and units

The standard boiling temperature is defined as the temperature (°C) at which the pressure of 

the saturated vapour of a liquid is equal to the standard pressure. 

3.2.3 Apparatus

Melting/boiling point apparatus: Model B-545, Buchi. 

Calibration: The apparatus is regularly calibrated by determination of the boiling points of 

reference materials. 

3.2.4 Procedure

A boiling tube (3.2 mm diameter) was filled to a height of approximately 10 mm with the 

sample. When the sample had melted, a boiling capillary was immersed, open end first.

The boiling tube containing the sample and the boiling capillary was inserted into the sample 

holder, and observations recorded as the temperature was raised. 

3.2.5 Results

The results of the definitive boiling point determinations on were as follows:

Barometric pressure: 1012 mbar.

As the tubes were heated at 1 ̊C/minute, samples were observed to darken from

approximately 230°C, indicating decomposition. No boiling was noted. 

3.2.6 Conclusion

The boiling temperature of was not determinable, as the test substance 

decomposed at temperatures above approximately 230°C without boiling. 

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3.3 Relative density (EC Method A.3, OECD Method 109)

3.3.1 Method

The relative density of was determined relative to purified water using a 

pyknometer at 20°C. Heavy distillate of petroleum was employed as the displacement liquid.

3.3.2 Definition and units

The relative density )(DT

4 of solids and liquids is defined as the ratio of the mass of a volume 

of substance to be examined, determined at T°C, and the mass of the same volume of water at 

4°C. 

3.3.3 Apparatus

Analytical balance: Model AT261, Mettler-Toledo

Pyknometer: Glass, nominal 25 cm3 capacity at 20°C, fitted with 

capillary stopper 

3.3.4 Reagents

Water: Purified by reverse osmosis and deionising; Elga 

Maxima

Displacement liquid: Heavy distillate of petroleum

3.3.5 Suitability of displacement liquid

The suitability of heavy distillate of petroleum as a displacement liquid was confirmed by the 

observation that approximately 10 mg of test substance did not dissolve in 10 mL of this 

solvent (solubility less than 0.1% w/v). 

3.3.6 Procedure

Test temperature: 20°C

A clean, dry pyknometer was accurately weighed (w1) then filled to the limits of its capacity 

with pure water. It was then carefully stoppered without trapping air, dried and re-weighed 

(w2). The procedure was repeated with the same pyknometer using displacement liquid (w3).

To the clean, dry and accurately weighed pyknometer, test substance (3 g) was added. The 

pyknometer was re-weighed (w4) and then the test substance covered with displacement 

liquid. After vigorous shaking of the stoppered pyknometer, to suspend the test substance, air 

bubbles were removed by ultrasonication. The pyknometer was filled with displacement 

liquid, stoppered and weighed as before (w5).

Two tests were performed concurrently using separate pyknometers.

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Parameters:

mass of pyknometer empty (g) = w1

mass of pyknometer + water (g) = w2

mass of pyknometer + displacement liquid (g) = w3

mass of pyknometer + test substance (g) = w4

mass of pyknometer + test substance + displacement liquid (g) = w5

Calculations:

mass of water to fill pyknometer (g) = w2 - w1 = W1

mass of displacement liquid to fill pyknometer (g) = w3 - w1 = W2

mass of test substance (g) = w4 - w1 = W3

mass of displacement liquid to fill pyknometer containing W3 g test substance (g) = 

w5 - w4 = W4

mass of displacement liquid equivalent to W3 g test substance (g) = W2 - W4 = W5

density of displacement liquid = (W2/W1) x T ρ w = 

T ρ l

volume of W3 g test substance (mL) = W5 / mean T ρ l value = Vs

relative density of test substance = W3/(Vs x 4 ρ w ) = T D4

where T ρ w is the density of water at the temperature of determination (= 0.998 g/mL)

4 ρ w is the density of water at 4°C (= 1.000 g/mL)

T ρ l is the density of the suspending displacement liquid at the temperature of 

determination (g/mL)

T D4 is the relative density of the test substance at the test temperature 

compared to water at 4°C

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3.3.7 Results

Parameter Determination 1 Determination 2

w1 19.28699 18.57529

w2 44.14280 43.61794

w3 39.39753 38.84603

w4 22.22016 21.55458

w5 40.29037 39.75273

W1 24.85581 25.04265

W2 20.11054 20.27074

W3 2.93317 2.97929

W4 18.07021 18.19815

W5 2.04033 2.07259

 T ρ l 0.80747 0.80783

mean

T ρ l 0.80756

Vs 2.52626 2.56620

20 D4 1.16 1.16

mean

20 D4 1.16

3.3.8 Conclusion

The relative density )(D20

4 of was found to be 1.16.

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3.4 Vapour pressure (EC Method A.4, OECD Method 104)

3.4.1 Method

The vapour pressure was determined using a vapour pressure balance.

3.4.2 Definition and units

The vapour pressure of a substance is defined as the saturation pressure above a solid or 

liquid substance. At the thermodynamic equilibrium, the vapour pressure of a pure substance 

is a function of temperature only. The SI unit of pressure is the Pascal (Pa).

3.4.3 Apparatus

The vapour pressure balance, assembled by the Department of Facilities Management at 

Huntingdon Life Sciences, consists of an evaporator furnace containing a small aperture of 

fixed size in its roof. Immediately above the aperture, the scale pan of a CI Electronics 

microbalance is suspended. The balance and furnace are contained within a bell jar, so that 

the whole of the apparatus can be evacuated.

3.4.4 Calibration

The microbalance was calibrated with a UKAS calibrated 1 mg weight. It was found that 

1 μg was equivalent to 2.875 x 10-3

V.

3.4.5 Procedure

A quantity of test substance (0.24 g) was added to the furnace, the apparatus assembled and 

evacuated to a pressure of less than 1 x 10-5 Torr (1.3 x 10-3 Pa).

After stabilisation at a given temperature, the shutter was opened to allow a stream of vapour 

to impact upon one balance pan. The temperature and pan deflection were recorded on a 

chart recorder. The trace obtained enabled the calculation of mass difference. The furnace 

temperature was then raised in steps of approximately 5oC and further measurements taken.

Six runs were performed at temperatures up to approximately 120°C, the pressure being kept 

at less than 1 x 10-5 Torr (1.3 x 10-3 Pa) throughout. Mass differences, condensation rates and 

the sample temperature were monitored on a chart recorder on every occasion that the shutter 

was opened.

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3.4.6 Calculations

Assuming no condensation, the vapour pressure is related to the observed mass difference by 

the relationship

Vapour pressure = A

∆m.g Equation 1

When condensation occurs the mass difference can, by reduced momentum transfer, be 

decreased by up to a factor of two from that appropriate to equation 1. The vapour pressure 

can also be obtained from the kinematic theory relationship for effusion or complete 

condensation

Vapour pressure = A

C

. M

2π.RT Equation 2

The efficiency of the condensation may be determined from the relative magnitudes of the 

measured mass difference and the condensation rate. For a degree of condensation, α, the 

measured condensation rate must be divided by this factor (α) to obtain the maximum 

condensation rate to be used in equation 2. Likewise, the experimental mass differences must 

be modified by a factor of 2/(2-α), which gives the appropriate factor of two enhancement in 

the vapour pressure calculation when the molecules do not bounce back.

By equating equations 1 and 2 at a given vapour pressure, it is possible to allow for the 

evaluation of α at each data point:

g

1

. M

2 .RT . 2

2 - = C

m π

α

∆ α Equation 3

Rearranging equation 3 gives equation 4 from which α can be calculated directly

1

+ 0.5

2 .RT

M . C

m.g =

−































 ∆

π

α Equation 4

The effect of α on the two methods for estimating vapour pressure yields identical results. 

Confidence in this system is demonstrated by the observation that the values of α approach, 

but never exceed, unity (when α=1 there is 100% condensation).

The vapour pressure-temperature relationship is as follows:

Log10Vp = T

Slope + Intercept Equation 5

Consequently, a plot of log10Vp versus 1/T(K) should be linear, and by extrapolation the 

vapour pressure at 298.15K can be calculated.

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Glossary of terms used in equations 1 to 5

A = Surface area of the aperture (5.952 x 10-6 m2) 

C = Maximum condensation rate (kg/s) 

g = Acceleration due to gravity (9.813 m/s2) 

M = Molar mass (kg/mol)

∆m = Mass difference (kg)

R = Universal gas constant (8.314 J/mol/K) 

T = Temperature (°C) + 273.15 (K) 

Vp = Vapour pressure (Pa) 

3.4.7 Results

The balance response was observed to decrease throughout the six runs, indicating continued 

degassing and/or removal of volatile impurities. 

The full results of the final run are presented in Table 1, with a graphical representation in 

Figure 2. The results can be summarised as follows:

Correlation (r): -0.9994

Slope: -6192

Intercept: 13.24

Vp (Pa) at 25°C: 3 x 10-8

The determined value should be regarded as a maximum possible vapour pressure given the 

decrease in response observed over the course of the test. 

3.4.8 Conclusion

The test substance, was found to have a vapour pressure of less than or equal to 

3 x 10-8 Pa at 25°C. 

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Table 1 Determination of the vapour pressure of 

Temperature Mass Condensation Vapour pressure α Corrected vapour 1/Temperature Log

(°C) difference rate (Pa) pressure (Pa) (1/K) vapour

(μg) (μg/s) From From From From pressure

Mass Condensation Mass Condensation

difference difference

102.0 0.25 0.008 0.00041 0.00027 0.489 0.00055 0.00055 0.00267 -3.26

108.0 0.51 0.007 0.00084 0.00026 0.266 0.00097 0.00097 0.00262 -3.01

112.0 0.80 0.010 0.00132 0.00034 0.230 0.00149 0.00149 0.00260 -2.83

117.0 1.27 0.010 0.00209 0.00036 0.158 0.00227 0.00227 0.00256 -2.64

121.0 1.92 0.017 0.00317 0.00060 0.172 0.00347 0.00347 0.00254 -2.46
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Figure 2 Plot of log10Vp versus 1/T for 

-4.0

-3.8

-3.6

-3.4

-3.2

-3.0

-2.8

-2.6

-2.4

-2.2

-2.0

0.00252 0.00254 0.00256 0.00258 0.00260 0.00262 0.00264 0.00266 0.00268

Log10

Vp

1/T
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3.5 Surface tension (EC Method A.5, OECD Method 115)

3.5.1 Method

The surface tension of a 1 g/L aqueous solution was determined with a surface tension torsion 

balance using the OECD harmonised ring method.

3.5.2 Definition and units

Surface tension (σ) is defined as the free surface enthalpy per unit of surface area and is 

reported in N/m (SI units) or mN/m (SI subunit).

3.5.3 Apparatus

Surface tension torsion balance: White Electrical Instrument Co., Malvern Link, 

Worcs.

Ring dimensions: Ring radius 6.37 mm

Wire radius 0.150 mm

Calibration: The calibration factor, øb, by which all instrument readings shall be 

multiplied, was determined in accordance with:

øb =

g

o 

σ

σ

where

σo = value cited in the literature for the surface tension of water (mN/m) at the test 

temperature

σg = measured value of the surface tension of water (mN/m) at that test temperature

Literature value for the surface tension of water at 20°C (σo) = 72.7 mN/m.

Measured surface tension of water at 20°C (σg) = 72.0 mN/m.

Therefore, the calibration factor, øb = 1.01

3.5.4 Test solution preparation

Test substance (approximately 50 mg) was dissolved in purified water (50 mL) to produce a 

1 g/l aqueous solution for measurement. Duplicate solutions were prepared in this manner.

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3.5.5 Procedure

The test vessel was half filled with liquid, put on the tensiometer test platform, and raised so 

that the ring was just below the liquid surface. The platform was lowered to draw a lamella 

from the liquid surface. The maximum force which arose just before the lamella was torn off 

is the surface tension and was recorded. Measurements were made at intervals until a 

constant value (to within 0.5 mN/m) was recorded. 

3.5.6 Results

Test temperature: 20°C

Solution concentration: 1 g/L (it was evident from the water solubility 

study (Section 3.6) that the prepared solution 

would contain both parent compound and 

degradate species). The test was run at this 

concentration as the preliminary water 

solubility test indicated a solubility in excess 

of 1 g/L.

Solution age: Initial measurements were made as soon as 

possible after dissolution of the test substance

Time Surface tension (mN/m)

(minutes) Measured Calibration corrected

A B A B

0 66.5 67.0 67.2 67.7

5 66.5 66.5 67.2 67.2

10 66.5 67.0 67.2 67.7

15 66.0 66.5 66.7 67.2

20 66.5 66.5 67.2 67.2

25 66.5 66.5 67.2 67.2

30 66.5 66.5 67.2 67.2

Overall mean calibration corrected surface tension = 67.0 mN/m

Harkins-Jordan* corrected value = 66.0 mN/m

3.5.7 Conclusion

The surface tension of a 1 g/L solution of was found to be 66.0 mN/m. As the 

result is greater than 60 mN/m, the test substance was not considered to be surface active.

*HARKINS, W.D. and JORDAN, H.F., J. Amer. Chem. Soc. 52, 1751 (1930).

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3.6 Water solubility (EC Method A.6, OECD Method 105)

3.6.1 Definition and units

The solubility in water is specified by the saturation mass concentration of the substance in 

water, and is a function of temperature. Solubility is specified in units of mass per volume of 

solution, and is reported as grams/litre (g/L). 

3.6.2 Preliminary test

A preliminary test was conducted by mixing a known mass (approximately 100 mg) of 

with increasing volumes of purified water and visually assessing dissolution. A 

solubility of approximately 1 to 2 g/L was estimated and a definitive test subsequently 

conducted using the flask method. 

3.6.3 Procedure for the definitive test

Glass-stoppered centrifuge tubes containing approximately 250 mg of test substance and 

25 mL of purified water were sealed and mixed at 20°C for 1, 2 and 3 days. On each 

occasion, the samples were filtered (0.45 μm, PTFE) and an aliquot (100 μL) of each clear 

filtrate was diluted to volume (10 mL) with mobile phase for analysis by high performance 

liquid chromatography (HPLC). The pH of each sample was measured. 

Blank and fortified control samples were processed and analysed as for the test samples.

3.6.4 HPLC conditions

Instrument: Agilent 1200 Series Liquid Chromatograph with 

Chemstation software

Column: Zorbax Eclipse XDB-C8 

(15 cm x 4.6 mm internal diameter)

Column temperature: 25 ̊C

Mobile phase composition: Methanol:pH 4 phosphate buffer (1:1 v/v)

Buffer composition: 0.2M aqueous potassium dihydrogen orthophosphate 

(110 mL) was mixed with 0.07M aqueous disodium 

hydrogen orthophosphate dodecahydrate (250 mL) and 

purified water (640 mL). The pH was adjusted to 

4.0 with orthophosphoric acid.

Flow rate: 1.0 mL/min

Injection volume: 10 μL

Detector: UV set at 210 nm

Retention time: Approximately 2.1 minutes

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3.6.5 Preparation of calibration

Stock calibration solutions of were prepared in acetonitrile. 

Calibration solutions in the ranges 0.1 to 5 mg/L, and 5 to 50 mg/L, were prepared by dilution 

of stock solutions with mobile phase. 

3.6.6 Calculations

The concentration of in the analysed solution (CA) was calculated from standards 

introduced before and after samples (bracketing standards) by the following equation: 

 CA (mg/L) = areapeak mean of bracketing standards

 sample areapeak x standard concentration (mg L)

The concentration of in the test solutions (CB) was then calculated thus:

 CB (mg/L) = CA (mg/L) x dilution factor

where the dilution factor was 100 for all samples.

3.6.7 Results and discussion

The detector calibration was found to be linear over the ranges investigated (Tables 2 and 3, 

Figure 3 and 4).

Table 4 presents a summary of the results of the test and shows that the water solubility of 

 was 27.5 ± 0.8 mg/L. Table 5 presents the primary data for this test, whilst 

Figures 5 and 6 present example standard and sample chromatograms from the analysis. 

It was evident from analysis of solutions from Days 1 to 3 of the definitive flask test that 

degradation of had occurred in aqueous solution. The analysed concentrations 

were significantly lower than that determined by visual assessment in the preliminary test and 

the chromatograms showed the presence of a much larger earlier-eluting peak, presumably a 

degradate. The magnitude of the two peaks was effectively unchanged over the three days of 

the test, indicating that an equilibrium had been achieved, within the first 24 hours of the test,

between these two dissolved species (in the presence of an excess of test substance).

A mean recovery of approximately 97% of from fortified control samples was 

achieved and deemed to be acceptable.

No significant interfering peaks were evident in blank control solutions. 

3.6.8 Conclusion

The water solubility of was found to be 27.5 mg/L at 20°C.

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Table 2 Standard calibration for by HPLC (0.1 to 5 mg/L) 

Standard concentration (mg/L) Peak area

5.985 446.86

2.993 220.30

1.496 110.32

0.7481 55.900

0.1496 10.958

Linear regression: y = 74.6x - 0.595 

(including x = 0, y = 0) r = 1.0000

x = concentration

y = peak area

Figure 3 Standard calibration for by HPLC (0.1 to 5 mg/L) 

0

50

100

150

200

250

300

350

400

450

500

0 1 2 3 4 5 6

Peak area

Concentration (mg/l)

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Table 3 Standard calibration for by HPLC (5 to 50 mg/L) 

Standard concentration (mg/L) Peak area

50.33 3909.2

40.26 3143.9

30.20 2366.1

20.13 1519.4

10.07 793.61

5.033 393.33

Linear regression: y = 77.9x - 2.97 

(including x = 0, y = 0) r = 0.9999

x = concentration

y = peak area

Figure 4 Standard calibration for by HPLC (5 to 50 mg/L) 

0

500

1000

1500

2000

2500

3000

3500

4000

4500

0 10 20 30 40 50 60

Peak area

Concentration (mg/l)

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Table 4 Measurements of water solubility at 20°C

Sample Time (days) Concentration Mean

number at 20°C (mg/L) concentration pH

(mg/L)

1A, 1B 1 28.1, 27.3 27.7 3.1, 3.1

2A, 2B 2 28.5, 27.0 27.7 3.1, 3.1

3A, 3B 3 27.7, 26.3 27.0 3.1, 3.1

Water solubility* = 27.5 ± 0.8 mg/L

(C. of V. = 3%)

*Determined as the mean of all samples

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Table 5 HPLC analysis of samples from the water solubility test for 

Sample Peak area CA (mg/L) Dilution factor CB (mg/L)

20.13 mg/L std 1510.4 - - -

Sample 1A 21.029 0.2808 100 28.08

Sample 1B 20.464 0.2732 100 27.32

Sample 2A 21.310 0.2845 100 28.45

Sample 2B 20.231 0.2701 100 27.01

20.13 mg/L std 1505.1 - - -

Sample 3A 20.587 0.2768 100 27.68

Sample 3B 19.542 0.2627 100 26.27

20.13 mg/L std 1489.3 - - -
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Figure 5 Chromatogram of standard (20.13 mg/L) 

0 1 2 3 4 min

mAU 

0

100

200

300

400

 VWD1 A, Wavelength=210 nm (E:\STUDIES\CEF0001\WATERS~1\0WS_00F\010-1001.D) 2.146

 
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Figure 6 Chromatogram of sample

0 1 2 3 4 min

mAU 

0

100

200

300

400

 VWD1 A, Wavelength=210 nm (E:\STUDIES\CEF0001\WATERS~1\0WS_00F\011-1101.D) 2.149

 

 
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3.7 Partition coefficient (EC Method A.8, OECD Method 117)

3.7.1 Definition and units

The partition coefficient (P) is defined as the ratio of the equilibrium concentrations of a 

dissolved substance in a two-phase system consisting of two largely immiscible solvents, in 

this case, n-octanol and water.

Pow =

water

octanol

 C

 C

The partition coefficient is usually given in the form of its logarithm to base ten (log10Pow).

3.7.2 Preliminary estimation

A value of log10Pow for was estimated from the ratio of the solubilities of the test 

substance in the pure solvents. The solubilities were estimated to be approximately 1 to 2 g/L

in each solvent by visual assessment. 

was thus estimated to have a log10Pow value of approximately 0 and it was 

considered that the high performance liquid chromatography (HPLC) method would be 

suitable for the measurement

3.7.3 Method

Partition coefficients in the range of log10Pow between 0 and 6 can be estimated using high 

performance liquid chromatography (HPLC). This is performed on columns containing 

hydrocarbon chains (e.g. C8, C18) chemically bound onto silica. The chemicals injected onto 

the column move along it by partitioning between the mobile solvent phase and the 

hydrocarbon stationary phase, their retention being proportional to their hydrocarbon-water 

partition coefficient. The partition coefficient is deduced from the capacity factor, k, given 

by

k = 

t - t

t

r o

o

where tr is the measured retention time of sample or reference

and to is the retention time of the internal deadtime standard.

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3.7.4 Procedure

As the test substance is ionisable, and it was not known how it would partition in the presence 

of its counter-ion, tributylammonium, the test was conducted at each of pH 4, 7 and 9, thus 

covering the pH range of environmental relevance. Each test was conducted as follows: 

In order to correlate the measured capacity factor (k) of the test material with its Pow, seven

reference materials were selected from a list of recommended compounds. These reference 

compounds were selected to have log10Pow values between 0.3 and 6.2. An internal standard 

(formamide) with no retention on the HPLC column was used to determine the deadtime of 

the HPLC system.

The following reference mixture was prepared in HPLC mobile phase.

Reference substance Known log10Pow* Concentration (mg/L)

Internal deadtime standard (I.D.S.) Deadtime only 200

2-Butanone 0.3 40000

Acetanilide 1.0 7.9

Nitrobenzene 1.9 8.2

Ethylbenzoate 2.6 20

Bromobenzene 3.0 10

Benzylbenzoate 4.0 10

DDT 6.2 20

*see EC Method A.8 for these values.

A solution of (approximately 5 mg/L in HPLC mobile phase, and also containing 

I.D.S. at the same concentration as in the reference mixture) was prepared and 

chromatographed in duplicate, bracketed by samples of the reference mixture, using the 

following conditions. 

3.7.5 HPLC conditions

Instrument: Agilent 1200 Series Liquid Chromatograph with Chemstation 

software

Column: Zorbax Eclipse XDB-C8 (15 cm x 4.6 mm internal diameter)

Column temperature: 25°C

Mobile phase composition: Acetonitrile:buffer solution (3:1 v/v) where phosphate 

buffers were used for the pH 4 and 7 tests and a borate buffer 

for the pH 9 test

Flow rate: 1.0 mL/minute

Injection volume: 10 μL

Detector: UV set at 210 nm

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3.7.6 Results

Example chromatograms are presented in Figures 7 to 12. It was evident that, at each pH 

value investigated, the retention time of was less than that of the lowest standard, 

2-butanone, which eluted after approximately 1.7 minutes under the test conditions. The peak 

at approximately 1.4 minutes in each chromatogram was that for the deadtime standard, 

formamide.

A value of log10Pow for of less than 0.3 was thus obtained over the range of pH 

values investigated. 

3.7.7 Conclusion

 was found to have a log10Pow value of less than 0.3 at each of pH 4, 7 and 9. 

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Figure 7 Chromatogram of standard materials with log10Pow between 0.3 and 6.2 at pH 4

0 2 4 6 8 10 12 14 min

mAU 

0

10

20

30

40

50

60

70

80

 VWD1 A, Wavelength=210 nm (0PC_0F\002-0201.D) 1.361 1.619 1.736 2.132 2.603 2.968 3.266

 8.780

I.D.S

Acetanilide

Nitrobenzene

Ethylbenzoate

Bromobenzene

Benzylbenzoate DDT

2-Butanone
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Figure 8 Chromatogram of at pH 4 0 2 4

68 10 12 14 min

mAU 0

20

40

60

80

100

120

 VWD1 A, Wavelength=210 nm (0PC_0F\003-0301.D) 1.363 1.477

I.D.S
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Figure 9 Chromatogram of standard materials with log10Pow between 0.3 and 6.2 at pH 7

024

68 10 12 14 min

mAU 0

10

20

30

40

50

60

70

80

 VWD1 A, Wavelength=210 nm (1PC_0F\005-0501.D) 1.376 1.631 1.743 2.156 2.650 3.025 3.377

 9.477

I.D.S

Acetanilide

Nitrobenzene

Bromobenzene

Ethylbenzoate

Benzylbenzoate

DDT

2-Butanone
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Figure 10 Chromatogram of at pH 7 0 2 4

68 10 12 14 min

mAU 0

20

40

60

80

100

120

 VWD1 A, Wavelength=210 nm (1PC_0F\003-0301.D) 1.247 1.372

I.D.S.
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Figure 11 Chromatogram of standard materials with log10Pow between 0.3 and 6.2 at pH 9

0 2 4 6 8 10 12 14 min

mAU 

0

10

20

30

40

50

60

70

80

 VWD1 A, Wavelength=210 nm (E:\STUDIES\CEF0001\POW\BUTANONE\2PC_0F\005-0501.D) 1.367 1.617 1.736 2.131 2.608 2.959 3.275 8.679

I.D.S

Acetanilide

Nitrobenzene

Ethylbenzoate

Bromobenzene

Benzylbenzoate DDT

2-Butanone
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Figure 12 Chromatogram of at pH 9 0 2 4

68 10 12 14 min

mAU 0

20

40

60

80

100

120

 VWD1 A, Wavelength=210 nm (2PC_0F\003-0301.D) 1.220 1.366

I.D.S.
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3.8 Flammability (solids) (EC Method A.10)

3.8.1 Method

The flammability (solids) was determined using a test mould and an ignition source.

3.8.2 Definition and units

A substance is considered to be highly flammable when the time taken for a pile of test 

substance to burn a distance of 100 mm, after having burned a distance of 80 mm, is less than 

45 seconds under the test conditions. 

3.8.3 Procedure

The metal mould of triangular cross-section of 20 mm base and 10 mm height and 250 mm in 

length, was loosely filled with test substance and dropped three times from a height of 2 cm 

onto a solid surface. The mould was removed from its holder and excess substance scraped 

off with a straight edge. A ceramic tile was placed on the mould, the apparatus inverted, and 

the mould removed. The tile was placed behind a screen, across the line of draught in a fume 

cupboard. 

A hot flame (minimum diameter 5 mm) was applied to one end of the pile for 2 minutes, and 

any propagation timed along 200 mm of its length. 

3.8.4 Results

The test substance melted and burned with a yellow/green flame which extinguished 

immediately on removal of the ignition source. There was no propagation of burning along 

the test pile. No further testing was necessary. 

3.8.5 Conclusion

was found not to be highly flammable.

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3.9 Explosive properties (EC Method A.14)

It was considered that the test substance was not explosive and therefore an assessment of 

this property was made using a theoretical consideration of its structure. 

3.9.1 Assessment of explosive properties

Chemical regulatory authorities require information on the explosive properties of substances 

for classification and labelling purposes. 

Test methods for the determination of explosive properties (EC Method A.14) involve 

measuring the effect of three stimuli; shock, heat and friction. The test substance is 

considered to possess explosive properties if a positive result is obtained in any one of these 

tests.

According to the United Nations Committee of Experts on the Transport of Dangerous Goods 

explosive properties are associated with the presence of certain chemical groups in a 

molecule which can react to produce a very rapid increase in temperature or pressure. Thus, 

it is possible to examine the chemical structure of a substance and ascertain whether or not 

there are any groups present that are associated with explosive properties. For this purpose a 

list of screening procedures has been published in Appendix 6 of the United Nations 

‘Recommendations on the Transport of Dangerous Goods, Manual of Tests and Criteria’.

The initial assessment of explosive properties is made by examining the chemical structures 

of the components with a list of functional groups that may indicate explosive properties in 

organic materials. This list is reproduced below:

Structural feature Examples

C-C unsaturation Acetylenes, acetylides, 1,2-dienes

C-Metal, N-Metal Grignard reagents, organo-lithium compounds

Contiguous nitrogen atoms Azides, aliphatic azo compounds, diazonium salts, hydrazines, 

sulphonylhydrazides

Contiguous oxygen atoms Peroxides, ozonides

N-O Hydroxylamines, nitrates, nitro compounds, nitroso compounds, 

N-oxides, 1,2-oxazoles

N-halogen Chloramines, fluoroamines

O-halogen Chlorates, perchlorates, iodosyl compounds

From an examination of the chemical structure of the test substance, it was concluded that it 

contained no functional groups which are characteristic of explosive properties. 

3.9.2 Conclusion

From the assessment of the chemical structure it was established that the test substance, 

, would not have explosive properties. 

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3.10 Relative self-ignition temperature for solids (EC Method A.16)

3.10.1 Definition and units

If the heat developed either by reaction of a substance with oxygen, or by exothermic 

decomposition, is not lost rapidly enough to the surroundings, self-heating, leading to 

self-ignition, occurs. Self-ignition therefore occurs when the rate of heat production exceeds 

the rate of heat loss.

The self-ignition temperature is defined as the minimum temperature, expressed in degrees 

Celsius (°C), at which a certain quantity of a substance will ignite under defined conditions. 

In accordance with the Directive, the self-ignition temperature in this test will be taken as the 

oven temperature at which the sample temperature reaches 400°C by self-heating.

3.10.2 Apparatus

Carbolite furnace with natural air circulation and explosion relief. 

Standard K-type thermocouples for temperature measurement. 

Open topped 20 mm cube made from stainless steel wire mesh with 0.045 mm openings. 

PICO ADC16 data logger with PICOLOG data logging software. 

Calibration: The furnace controller display is regularly calibrated against a standard 

thermometer. Instrument readings collected by the software are scaled from 

data points manually entered. 

3.10.3 Procedure

The mesh cube was filled with test substance by tapping gently and adding more until the 

cube was full. The cube was then suspended in the centre of the furnace at room temperature 

and a thermocouple placed in the centre of the cube. Another thermocouple was placed 

midway between the furnace wall and the cube in order to monitor the furnace temperature.

The temperature of the furnace and sample were then continuously recorded while the 

temperature of the furnace was increased at a rate of 0.5°C/min to 400°C. 

3.10.4 Results

There was no significant exothermic reaction of the test substance, indicating that it does not 

self-ignite below 400°C. 

The temperature versus time graph is presented in Figure 13. 

3.10.5 Conclusion

The test substance, , was found not to self-ignite below 400°C. 

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Figure 13 Temperature versus time curve 0

50

100

150

200

250

300

350

400

450

0 100 200 300 400 500 600 700 800

Temperature (°C)

Time (minutes)

Sample

Oven
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3.11 Oxidising properties (EC Method A.17)

3.11.1 Method

The substance to be tested and a defined combustible substance, cellulose, are mixed in 

various ratios. Each mixture is formed into a pile and ignited at one end. The maximum 

burning rate determined is compared with the maximum burning rate of the reference mixture 

of cellulose and barium nitrate.

3.11.2 Definition

A substance is considered as possessing oxidising properties when the maximum burning rate 

of the test mixtures is higher than, or equal to, the maximum burning rate of the reference 

mixture.

3.11.3 Procedure

The test substance, cellulose and barium nitrate (reference material) were dried and sieved 

(125 μm) prior to use. 

3.11.4 Preliminary test

The dried test substance (2 parts) was thoroughly mixed with dried cellulose (1 part) and 

formed into a cone shaped pile of dimensions 3.5 cm (diameter of base) x 2.5 cm (height). 

The cone, on a non-combustible, non-porous surface, was ignited with a gas burner. The 

vigour and duration of the combustion were observed, recorded and compared with that of a 

similarly prepared cone consisting of 3 parts of barium nitrate to 2 parts of cellulose. 

As the sample cone failed to burn to completion, cones consisting of 

(1 part) and cellulose (1 and 2 parts) were also prepared and tested. 

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3.11.5 Preliminary results

The test substance/cellulose mixtures did not burn to completion, whilst the barium nitrate 

reference burned vigorously to completion 23 seconds.

The following observations were noted for each of the weakly-burning test substance 

samples:

Test substance/cellulose ratio Duration of combustion

(seconds)

Observations

2:1 128 Did not burn to completion –

burned with a gentle, yellow, sooty 

flame, but some material remained 

unburned at the base of the pile

1:1 80 Did not burn to completion –

burned with a gentle, yellow, sooty 

flame, but some material remained 

unburned at the base of the pile

1:2 62 Did not burn to completion –

burned with a gentle, yellow, sooty 

flame, but some material remained 

unburned at the base of the pile

As none of the test substance/cellulose mixtures burned to completion, is 

considered to be non-oxidising. 

3.11.6 Conclusion

 was found to be non-oxidising. 

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3.12 Particle size distribution

3.12.1 Method

The size distribution of particles present in the test substance was assessed using particle size 

apparatus based on laser diffraction.

3.12.2 Apparatus

Particle sizer: Malvern Mastersizer 2000, with Mastersizer 2000 Control 

Software. The apparatus is regularly calibrated by determination 

of the particle size distribution of a reference material.

Sampling accessory: Hydro 2000S (A)

Dispersant: Heavy distillate of petroleum

Stirrer speed: 1800 rpm

Size range: 0.02 to 2000 μm

3.12.3 Procedure

A portion (30 mg) of test substance was transferred to a vial, dispersant (3 mL) was added 

and the sample shaken to suspend particles.

The entire contents of the vial were added directly to the sampling tank containing dispersant. 

Two measurements were taken 20 seconds apart and the mean data was calculated.

The test was performed in duplicate.

3.12.4 Treatment of data

The following parameters are calculated automatically by the instrument software:

d (0.1) is the diameter where 90% of the distribution is above and 10% is below.

d (0.5) is the diameter where 50% of the distribution is above and 50% is below. This value is 

also known as the Mass Median Diameter (MMD).

d (0.9) is the diameter where 10% of the distribution is above and 90% is below.

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3.12.5 Results

The overall mean values for the 10%, 50% and 90% quantile results are presented in the 

following table:

Parameter Size (μm)

d(0.1) 10.0

d(0.5) 36.5

d(0.9) 73.0

Copies of the original data from the particle size assessments are presented in Annex 1. These 

include all the calculated values, the tabulated data and graphical representations of the 

particle size distribution.

3.12.6 Conclusion

The test substance, , was determined to have d(0.1), d(0.5) and d(0.9) values of 10, 

37 and 73 μm respectively.

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Annex 1 Particle Size Distribution Data

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Annex 2 Eye Research Centre GLP Compliance Statement 2012

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