Working for a better future



## P hysicochemical P roperties

HLS study number: CEF0001

Version ID: Draft 4

Issue date: 03 October 2013

Page 1 of 53Huntingdon Life Sciences

CEF0001



## Sponsor and Test Facility

Sponsor

Test facility 

### Huntingdon Life Sciences

Eye Research Centre

Eye

Suffolk

IP23 7PX

UK

2Huntingdon Life Sciences

CEF0001



## 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 Proc edure

................................ ................................ ........................... 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 den sity

................................ ................................ .............................. 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 Suitabili ty of displacement liquid

................................ .......................... 16

3.3.6 Procedure

................................ ................................ ........................... 16

3.3.7 Results

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

3.3.8 Conclusion

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

3Huntingdon Life Sciences

CEF0001



## Table of Contents

- continued

3.4 Vapour pre ssure

................................ ................................ ............................. 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 Proce dure

................................ ................................ ........................... 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 estima tion

................................ ................................ ......... 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 Flamma bility (solids)

................................ ................................ ....................... 43

3.8.1 Method

................................ ................................ ................................ 43

3.8.2 Definition and units

................................ ................................ .............. 43

3.8.3 Procedure

................................ ................................ ........................... 43

3.8.4 Result s

................................ ................................ ................................ 43

3.8.5 Conclusion

................................ ................................ .......................... 43

3.9 Explo sive properties

................................ ................................ ........................ 44

3.9.1 Assessment of explosive properties

................................ .................... 44

3.9.2 Conclusion

................................ ................................ .......................... 44

4Huntingdon Life Sciences

CEF0001



## Table of Contents

- continued

3.10 Relative self

- ignition tempera ture for solids

................................ ..................... 45

3.10.1 Definition and units

................................ ................................ .............. 45

3.10.2 Apparatus

................................ ................................ ............................ 45

3.10.3 Procedur e

................................ ................................ ........................... 45

3.10.4 Results

................................ ................................ ................................ 45

3.10.5 Conclusion

................................ ................................ .......................... 45

3.11 Oxidi sing properties

................................ ................................ ........................ 47

3.11.1 Method

................................ ................................ ................................ 47

3.11.2 Definiti on

................................ ................................ ............................. 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 Researc h Centre GLP Compliance Statement 2012

............................. 53

5Huntingdon Life Sciences

CEF0001



## Compliance with G ood L aboratory 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

6Huntingdon Life Sciences

CEF0001



## Quality Assurance Statement

Physicochemical properties

The following inspections and audits have been carried ou t 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 ab out 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

Stu dy manageme nt 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 Sc iences

7Huntingdon Life Sciences

CEF0001



## Contributing Scientists

: Physicochemical properties

Study management

P Sydney BSc MSc

Study Director

Product Chemistry

8Huntingdon Life Sciences

CEF0001



## 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 Testi ng of Chemicals. The

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

together with the result obtained for each test.

E C OECD Test Result

method no. method no.

A

. 1 102 Melting temperature 13 9 °C (DSC)

A

. 2 103 Boiling temperature Not determinable; decomposes at

te mperatures 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 m g/ L at 20 ̊C

A

. 8 1 1 7 Partition coefficient Log 10 P

ow < 0.3 at each of pH 4, 7

and 9

A

. 10

- Flammability (solids) Not h ighly flammable

A

. 14

- Explosive properties

(theoretical assessment) Not explosive

A

. 1 6

- 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

9Huntingdon Life Sciences

CEF0001



## 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 S tudy schedule

Study i nitiation d ate : 18 March 2013

Experimental start d ate : 28 March 2013

Experimental completion d ate : 08 August 2013

Study completion d ate :



## 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. T he 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, i t was

considered valid to run the t est at the higher concen tration. As no significant surface activity

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

10Huntingdon Life Sciences

CEF0001



## 2. Test S ubstance

Identity:

Structure:

Appearance: White crystalline powder

Storage conditions: Room temper ature in the dark

Batch number: 12342

Purity: To be advised

Expiry date: To be advised

Date received: 04 March 2013

11Huntingdon Life Sciences

CEF0001



## 3. Experimental Procedure s

3.1 Melting t emperature (E C Method A

. 1, OECD Method 102)

3.1.1 Method

The melting temperatur e 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

D ried test substance was tightly packed into a capi llary 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 capillar y t ube was placed in the apparatus and heated at

1°C/minute. The procedure was performed in duplica te 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 dupl icate 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 larg e gap between the first and second reported stages of melting and

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

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

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

confirming the earlier observation.

12Huntingdon Life Sciences

CEF0001



### 3.1.6 Conclusion

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

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

.

13Huntingdon Life Sciences

CEF0001



### Figure 1 DSC Trace of

14Huntingdon Life Sciences

CEF0001



## 3.2 Boiling temperature (E C 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.

Calibrat ion: 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 cap illary 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 determin ations on were as follows:

Barometric pressure : 10 12 mbar.

As th e tubes were heated at 1 ̊ C/minut e, 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 temperature s above approximately 230°C without boiling.

15Huntingdon Life Sciences

CEF0001



## 3.3 Relative density (EC Method A

. 3, OECD Method 109)

3.3.1 M ethod

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 )(D T

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 App aratus

Analytical balance: Model AT261

, Mettler

- Toledo

Pyknometer: Glass, nominal 25 cm 3 

### capacity at 20°C, fitted with

capillary stopper

3.3.4 Reagents

Water: Purified by reverse osmosis and deionising; Elga

Maxima

Displacement liquid: Heavy distillat e 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 (w 1 

### ) then filled to the limits of its capacity

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

- weighed

(w 2 

### ). The procedure was repeated with the same pyknometer using displacement liquid (w 3 

### ).

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

pyknometer was re

- weighed (w 4 

### ) and then the test substance covered with displacement

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

bubbles w ere removed by ultrasonication. The pyknometer was filled with displacement

liquid, stoppered and weighed as before (w 5 

### ).

Two tests were performed concurrently using separate pyknometers.

16Huntingdon Life Sciences

CEF0001



### Parameters:

mass of pyknometer empty (g) = w 1



### mass of pyknometer + water (g) = w 2



### mass of pyknometer + displacement liquid (g) = w 3



### mass of pyknometer + test substance (g) = w 4



### mass of pyknometer + test substance + disp lacement liquid (g) = w 5



### Calculations:

mass of water to fill pyknometer (g) = w 2



### - w 1 

### = W 1



### mass of displacement liquid to fill pyknometer (g) = w 3



### - w 1 

### = W 2



### mass of test substance (g) = w 4



### - w 1 

### = W 3



### mass of displacement liquid to fill pyknometer contai ning W 3 

### g test substance (g) =

w 5



### - w 4 

### = W 4



### mass of displacement liquid equivalent to W 3 

### g test substance (g) = W 2



### - W 4 

### = W 5



### density of displacement liquid = (W 2 

### /W 1 

### ) x T

w 

### ρ 

### = T

l 

### ρ

volume of W 3 

### g test substance (mL ) = W 5 

### / mean T

l 

### ρ value = V

s



### relative density of test substance = W 3 

### /(V

s 

### x 4

w 

### ρ 

### ) = T

4 

### D

where T

w 

### ρ 

### is the density of water at the temperatur e of determination (= 0.998 g/mL )

4

w 

### ρ 

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

T

l 

### ρ is the density of the suspending displacement liquid at the te mperature of

determination (g/mL )

T

4 

### D is the relative density of the test substance at the test temperature

compared to water at 4 ° C

17Huntingdon Life Sciences

CEF0001



### 3.3.7 Results

Parameter Determination 1 Determination 2

w 1 19.28699 18.57529

w 2 44.14280 43.61794

w 3 39.39753 38.84603

w 4 22.22016 21.55458

w 5 40.29037 39.75273

W 1 24.85581 25.0 4265

W 2 20.11054 20.27074

W 3 2.93317 2.97929

W 4 18.07021 18.19815

W 5 2.04033 2.07259

T

l 

### ρ 0.80747 0.80783

mean T

l 

### ρ 0.80756

V

s 2.52626 2.56620

20

4 

### D 1.16 1.16

mean 20

4 

### D 1.16



### 3.3.8 Conclusion

The relative density )(D 20

4 

### of was found to be 1.16.

18Huntingdon Life Sciences

CEF0001



## 3.4 Vapour pressure (E C 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 unit s

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 Pasc al (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 f ound 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 temper ature, 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 th en raised in steps of approximately 5 oC 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 a nd

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

was opened.

19Huntingdon Life Sciences

CEF0001



### 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 r elationship for effusion or complete

condensation

Vapour pressure =

A C

. M

RT

. 2 π 

### 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 degr ee 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 .RT 2

. 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 +

.RT 2 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:

Log 10 

### Vp =

T Slope 

### + Intercept Equation 5

Consequently, a plot of log 10 

### Vp versus 1/T(K) should be linear, and by extrapolation the

vapour pressure at 298.15K can be calculated.

20Huntingdon Life Sciences

CEF0001



### Glossary of terms used in equations 1 to 5

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

### m 2 

### )

C = Maximum condensation rate (kg/s)

g = Acceleration due to gravity (9.813 m/s 2 

### )

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 , wit h a graphical representation in

Figure 2. The results can be summarised as follows:

Correlation (r):

- 0.9 994

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.

2122Huntingdon Life SciencesCEF0001 

### Table 1 Determination of the vapour pressure of

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

(°C) diffe rence 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

- 2.4623Huntingdon Life SciencesCEF0001 

### Figure 2 Plot of log 10 

### Vp 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 0.00268Log10Vp

1/THuntingdon Life Sciences

CEF0001



## 3.5 Surface tension (E C 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 harmon ised 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, ø b 

### , by which all instrument readings shall be

multiplied, was determined in accordance with:

ø b 

### =

g o

σ σ



### where

σ o 

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

temperature

σ g 

### = 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 ( σ o 

### ) = 72.7 mN/m.

Measured surface tension of water at 20 ° C ( σ g 

### ) = 72 .0 mN/m.

Therefore, the calibration factor, ø b 

### = 1.01

3.5.4 Test solution preparation

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

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

24Huntingdon Life Sciences

CEF0001



### 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 l amella

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 temper ature: 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 wate r

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

Harkin s-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).

25Huntingdon Life Sciences

CEF0001



## 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/li tre (g/L ).

3.6.2 Preliminary test

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

with increasing volumes of purified water and visually ass essing 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 subs tance and

25 mL of purified water were sealed and mix ed 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

Chemstati on software

Column: Zorbax Eclipse XDB

- C8

( 1 5 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 di hydrogen 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

26Huntingdon Life Sciences

CEF0001



### 3.6.5 Preparation of calibration

Stock calibration solu tion s of were prepared in acetonitrile

.

Calibr ation 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 (C A 

### ) was calculated from standards

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

C A 

### (mg/L ) =

standards bracketing of areapeak mean L) (mg ion concentrat standard x areapeak sample



### The concentration of in the test solutions (C B 

### ) was then calculated thus:

C B 

### (mg/L ) = C A 

### (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 b e linear over the range s investigated (Table s 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 solution s from Days 1 to 3 of the definitive flask test th at

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 t o be 27.5 mg/L at 20 ° C.

27Huntingdon Life Sciences

CEF0001



### 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

1.496 110.3 2

0.7481 55.900

0.1496 10.958

Linear regression: y = 74.6 x - 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 6Peak area

Concentration (mg/l)

28Huntingdon Life Sciences

CEF0001



### Table 3 Standard ca libration 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 = 77.9 x - 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 60Peak area

Concentration (mg/l)

29Huntingdon Life Sciences

CEF0001



### Table 4 Measurements of water solubility at 20°C

Sample Time (days) Concentration Mean

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

(m g/ 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

3031Huntingdon Life SciencesCEF0001 

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

Sample Peak area C A (mg/L ) Dilution factor C B (mg/L )

20.13 mg/L std 1510.4

- - -

Sample 1 A 21.029 0.2808 100 28.08

Sample 1 B 20.464 0.2732 100 27.32

Sample 2A 21.310 0.2845 100 28.45

Sample 2B 20.231 0.27 01 100 27.01

20.13 mg/ L std 1505.1

- - -

Sample 3 A 20.587 0.2768 100 27.68

Sample 3 B 19.542 0.2627 100 26.27

20.13 mg/L std 1489.3

- - -32Huntingdon Life SciencesCEF0001 

### Figure 5 Chromatogram of standard (20.13 mg/L )

min 0 1 2 3 4 mAU

0 100 200 300 400 VWD1 A, Wavelength=210 nm (E:\STUDIES\CEF0001\WATERS~1\0WS_00F\010-1001.D)2.14633Huntingdon Life SciencesCEF0001 

### Figure 6 Chromatogram of sample

min 0 1 2 3 4 mAU

0 100 200 300 400 VWD1 A, Wavelength=210 nm (E:\STUDIES\CEF0001\WATERS~1\0WS_00F\011-1101.D)2.149Huntingdon Life Sciences

CEF0001



## 3.7 Partition coefficient (E C 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 sol vents, in

this case, n

- octanol and water.

P

ow 

### =

water octanol

C C



### The partition coefficient is usually given in the form of its logarithm to base ten (log 10 

### P

ow 

### ).

3.7.2 Preliminary estimation

A value of log 10 

### P

ow 

### 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 es t imated to have a log 10 

### P

ow 

### 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 log

10 

### P

ow 

### between 0 and 6 can be estimated using high

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

hydrocarbon chains (e.g. C 8 

### , C 18 

### ) 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 t

r 

### is the measured retention time of sample or reference

and t

o 

### is the retention time of the internal deadtime standard.

34Huntingdon Life Sciences

CEF0001



### 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 rel evance. Each test was conducted as follows:

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

ow 

### , seven

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

compounds were selected to have log 10 

### P

ow 

### 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 K nown log 10 P

ow * Concentration (mg/L )

Internal deadtime standard (I.D.S.) Deadtime only 20 0

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 Meth od 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 referen ce mixture, using the

following conditions.

3.7.5 HPLC conditions

Instrument: Agilent 1200 Series Liquid Chromatograph with Chemstation

software

Column: Zorbax Eclipse XDB

- C8 ( 1 5 cm x 4.6 mm internal diameter )

Column temperature: 25 ° C

Mobile phase c omposition: 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: 1 0 μ L

Detector: UV set at 210 nm

35Huntingdon Life Sciences

CEF0001



### 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, whic h 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 log 10 

### P

ow 

### 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 log 10 

### P

ow 

### value of less than 0.3 at each of pH 4, 7 and 9.

36Huntingdon Life SciencesCEF000137 

### Figure 7 Chromatogram of standard materials with log 10 

### P

ow 

### between 0.3 and 6.2 at pH 4

min 0 2 4 6 8 10 12 14 mAU

0 10 20 30 40 50 60 70 80 VWD1 A, Wavelength=210 nm (0PC_0F\002-0201.D)1.3611.6191.7362.1322.6032.9683.2668.780 I.D.S Acetanilide

Nitrobenzene

Ethylbenzoate

Bromobenzene

Benzylbenzoate DDT

2

- ButanoneHuntingdon Life SciencesCEF000138 

### Figure 8 Chromatogram of at pH 4

min 0 2 4 6 8 10 12 14 mAU

0 20 40 60 80 100 120 VWD1 A, Wavelength=210 nm (0PC_0F\003-0301.D)1.3631.477

I.D.SHuntingdon Life SciencesCEF000139 

### Figure 9 Chromatogram of standard ma terials with log 10 

### P

ow 

### between 0.3 and 6.2 at pH 7

min 0 2 4 6 8 10 12 14 mAU

0 10 20 30 40 50 60 70 80 VWD1 A, Wavelength=210 nm (1PC_0F\005-0501.D)1.3761.6311.7432.1562.6503.0253.3779.477 I.D.S Acetanilide

Nitrobenzene

Bromobenzene E thylbenzoate

Benzylbenzoate DDT

2

- ButanoneHuntingdon Life SciencesCEF000140 

### Figure 10 Chromatogram of at pH 7

min 0 2 4 6 8 10 12 14 mAU

0 20 40 60 80 100 120 VWD1 A, Wavelength=210 nm (1PC_0F\003-0301.D)1.2471.372

I.D.S.Huntingdon Life SciencesCEF000141 

### Figure 11 Chromatogram of standard materials with log 10 

### P

ow 

### between 0.3 and 6.2 at pH 9

min 0 2 4 6 8 10 12 14 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)1.3671.6171.7362.1312.6082.9593.2758.679 I.D.S Acetanilide

Nitrobenzene

Ethylbenzoate

Bromobenzene

Benzylbenzoate DDT

2

- ButanoneHuntingdon Life SciencesCEF000142 

### Figure 12 Chromatogr am of at pH 9

min 0 2 4 6 8 10 12 14 mAU

0 20 40 60 80 100 120 VWD1 A, Wavelength=210 nm (2PC_0F\003-0301.D)1.2201.366

I.D.S.Hunting don Life Sciences

PPM0011



## 3.8 Flammability (solids) (E C Method A

. 10)

3.8.1 M ethod

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 f ound not to be highly flammable.

43Huntingdon Life Sciences

CEF0001



## 3.9 Explosive properties (E C 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 ex plosive properties

Chemical regulatory authorities require information on the explosive properties of substances

for classification and labelling purposes.

Test methods for the determina tion of explosive properties (EC Method A

. 14) involve

measuring the ef fect 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 as certain 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 reprodu ced 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,

sulphonylh ydrazides

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 examina tion 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 subst ance,

, would not have explosive properties.

44Huntingdon Life Sciences

CEF0001



## 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 i n 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 rea ches 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 AD C16 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 thermocoupl e 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 40 0°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.

4546Huntingdon Life SciencesCEF0001 Figure 13 

### Temperature versus time curve

0 50 100 150 200 250 300 350 400 450

0 100 200 300 400 500 600 700 800Temperature (°C)

Time (minutes) Sample

OvenHuntingdon Life Sciences

CEF0001



## 3.11 Oxidising properties (E C 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 ra te

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

.

47Huntingdon Life Sciences

CEF0001



### 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 s ome 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 ge ntle, 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.

48Huntingdon Life Sciences

CEF0001



## 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

So ftware . 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: 18 00 rpm

Size range: 0.02 to 2000 μm

3.12.3 Procedure

A portion (3 0 mg) of test substance was tran sferred 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 t he 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.

49Huntingdon Life Sciences

CEF0001



### 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 determ ined to have d(0.1), d(0.5) and d(0.9) values of 10

, 37 and 73 μm respectively.

50Huntingdon Life Sciences

CEF0001



### Annex 1 Particle Size Distribution Data

51Huntingdon Life Sciences

CEF0001

52Huntingdon Life Sciences

CEF0001



### Annex 2 Eye Research Centre GLP Compliance Statement 20 1 2

53