Page 1 o f 54 TITLE/COV ER PAGE

PHYSICOCHEMICAL PROPERTIES

Sponsor R esear ch Laboratory

Huntingdon Life Sciences, Ltd. Woolley Road Alconbury Huntingdon Cambridgeshire

PE28 4HS

ENGLAND

Final: 23 April 2007: 2 : TABLE OF CONTENT S

Page

T ITLE/COVER PAGE........................................................................................................ 1

TABL E OF CONTENTS.................................................................................................... 2

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS ................. 3

QUALITY ASSURANCE STATEMENT ......................................................................... 4

CONTRIBUTING SCIENTISTS........................................................................................ 5

SUMMARY ........................................................................................................................ 6

INTRODUCTION............................................................................................................... 7

TEST SUBSTANCE........................................................................................................... 8

PHYSICOCHEMICAL PROPERTIES

Vapour pressure ............................................................................................................... 9

Surface tension................................................................................................................. 15

Relative self-ignition temperature for solids.................................................................... 17

Particle size distribution................................................................................................... 19

Soil adsorption ................................................................................................................. 21

DEVIATIONS FROM PROTOCOL .................................................................................. 28

MAINTENANCE OF RECORDS...................................................................................... 28

REFERENCES.................................................................................................................... 29

APPENDIX

1. E y e Research Centre GLP Compliance Statement 2005.............................................. 30 2. Certificate of Analysis.................................................................................................. 32 3. Study Protocol .............................................................................................................. 34: 5 : CONTRIBUTING SCIENTIST S

Phy sicochemical Properties

The following staff member has reviewed this report.

A. L. Comb, B.Sc., Ph.D. (Scientific Manager, Product Chemistry)

The following staff were responsible for the conduct of the work and reporting of the results.

P. Sydney, B.Sc., M.Sc. (Study Director, Product Chemistry)

C. Pointer (Scientist, Product Chemistry)

C. Steil, B.Sc. (Scientist, Product Chemistry)

P. Woods, H.N.C. (Scientist, Product Chemistry): 6 : SUMMARY

s performed to determine certain specified physicochemical properties of The study was performed in accordance with the tests described in the EEC the Determination of Physicochemical Properties (Directive 92/69/EEC) 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 respective guideline methods followed and the result obtained for each test.

EEC OECD Test Result

Method

no. Guideline

no.

A.4 104 Vapour pressure 4 x 10 -9 Pa at 25 ° C

A.5 115 Surface tension 66.5 mN/m (90% saturated aqueous solution)

A.16 - Relative self-ignition

temperature for solids None below the melting temperature (approximately 150 ° C)

- - Particle size distribution 0% w/w is smaller than 10 μ m

C.19 121 Soil adsorption LogK
oc = 3.7 (immobile in soil): 7 : INTRODUCTION

was designed to determine certain specified physicochem ical properties of Information on physicochemical properties is important in the assessment of

effects of a substance both at work and in the environment.

The study was conducted in compliance with EEC Methods for the Determination of Physicochemical Properties (Directive 92/69/EEC) and the OECD Guidelines for the Testing of Chemicals. The physicochemical properties determined were vapour pressure, surface tension, relative self-ignition temperature for solids, particle size distribution and soil adsorption.

The protocol was approved by the Study Monitor for the Sponsor on 8 September 2006, and by the Sponsor, the Study Director and Huntingdon Life Sciences Management on 11 September 2006.

The experimental start and completion dates were 14 September 2006 and 11 October 2006, respectively.

Location of study : Huntingdon Life Sciences, Ltd. Eye Research Centre Eye

Suffolk

IP23 7PX: 8 : TEST SUBSTANCE

I dentity:

Chemical name:

Molecular fo rmula:

Molecular w eight:

CAS number:

L ot number:

Expiry date: 14 May 2007

Purity:

Appearance:

Storage conditions: Ambient temperature in a dark environmentally

controlled area

Date of sample receipt: 31 July 2006

Source:

Structure:: 9 : PHYSICOCHEMICAL PRO PERTIES

VAPOUR PRESSURE
(EEC Method A.4 and OECD Guideline 104)

METHOD

The vapour pressure was determined using a vapour pressure balance.

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).

INSTRUMENTATION

The vapour pressure balance, assembled by the Department of Facilities Management at Huntingdon Life Sciences, consisted of an evaporator furnace, which contained a small aperture of fixed size in its roof. Immediately above the aperture, the scale pan of a CI Electronics microbalance was suspended. The balance and furnace are contained within a bell jar, so that the whole of the apparatus can be evacuated.

CALIBRATION

The microbalance was calibrated with a UKAS calibrated 1 mg weight (certified weight = 1.0017 mg). During the calibration, the chart recorder was set to 5V full-scale (equivalent to 180 mm on the chart recorder trace). The mean deflection observed using the 1 mg weight in triplicate determinations was 99 mm. It was found that 1 μg was equivalent to 2.745 x 10 -3 V.

P ROCEDU RE

A quantity of test substance (0.15 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.

Two definitive runs were performed between temperatures of 89.5 and 125.0°C, the pressure being kept at less than 1 x 10 -5 Torr (1.3 x 10 -3 Pa) throughout. An initial run was

di sreg arded, the high and variable results being indicative of degassing and/or removal of volatile impurities. Mass differences, condensation rates and the sample temperature were monitored on a chart recorder on every occasion that the shutter was opened.: 10 :

CALCULATIONS

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

Vapour pressure = A g

m • ∆ 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 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 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 substituting equation 1 for vapour pressure in equation 2, and correcting for degree of condensation and enhancement, it is possible to calculate 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 ef fect 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 = Slope

T + I ntercept Equation 5

Consequently, a plot of log 10 Vp versus 1/T(K) should be linear, and by extrapolation the vapour pressure at 298.1 5K can be calculated.: 11 :

Glossary of ter ms 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)

RESULTS

The full results of the two definitive runs are presented in Tables 1 and 2, with a graphical representation of both data sets in Figure 1.

R un 1 Run 2

Corre lation (r): -0.9952 -0.9989 Slope: -7488 -8344 Intercept: 16.94 19.11 Vp (Pa) at 25°C: 6.6 x 10 -9 1.3 x 10 -9

The two runs give a mean vapour pressure value at 25°C of 4 x 10 -9 Pa.

CONCLUSION

The vapour pressure o f was found to be 4 x 10 -9 Pa at 25°C.: 12 :TABLE 1Determination of the Vapour Pressure of(Run 1)Temperature Mass Condensation Vapour pressureαCorrected vapour 1/Temperature Log(°C) difference rate(Pa)pressure (Pa)(1/K) vapour(μg) (μg/s) From FromFrom FrompressureMass Condensation Mass Condensationdifferencedifference90.5 0.11 NC 0.00018 NC NC 0.00018 NC 0.00275 -3.7495.0 0.20 0.010 0.00033 0.00041 0.765 0.00054 0.00054 0.00272 -3.27100.5 0.27 0.013 0.00045 0.00058 0.783 0.00074 0.00074 0.00268 -3.13105.0 0.48 0.023 0.00078 0.00100 0.777 0.00128 0.00128 0.00264 -2.89110.0 0.81 0.051 0.00133 0.00224 0.912 0.00245 0.00245 0.00261 -2.61115.0 1.59 0.086 0.00262 0.00377 0.837 0.00451 0.00451 0.00258 -2.35120.0 2.83 0.131 0.00467 0.00576 0.763 0.00755 0.00755 0.00254 -2.12125.0 5.48 0.211 0.00904 0.00937 0.682 0.01373 0.01373 0.00251 -1.86NC = no condensation observed: 13 :TABLE 2Determination of the Vapour Pressure of(Run 2)Temperature Mass Condensation Vapour pressureαCorrected vapour 1/Temperature Log(°C) difference rate(Pa)pressure (Pa)(1/K) vapour(μg) (μg/s) From FromFrom FrompressureMass Condensation Mass Condensationdifferencedifference89.5 0.07 NC 0.00012 NC NC 0.00012 NC 0.00276 -3.9393.5 0.13 NC 0.00022 NC NC 0.00022 NC 0.00273 -3.6699.5 0.24 0.009 0.00040 0.00040 0.669 0.00060 0.00060 0.00268 -3.22104.0 0.36 0.018 0.00060 0.00076 0.776 0.00098 0.00098 0.00265 -3.01109.5 0.75 0.046 0.00123 0.00199 0.893 0.00223 0.00223 0.00261 -2.65114.5 1.25 0.081 0.00207 0.00354 0.923 0.00384 0.00384 0.00258 -2.42119.5 2.57 0.141 0.00424 0.00621 0.846 0.00734 0.00734 0.00255 -2.13125.0 5.06 0.218 0.00834 0.00966 0.733 0.01317 0.01317 0.00251 -1.88NC = no condensation observed: 14 :FIGURE 1Plot of log10Vp versus 1/T-4.50-4.00-3.50-3.00-2.50-2.00-1.50-1.000.00245 0.00250 0.00255 0.00260 0.00265 0.00270 0.00275 0.002801/T

Log 10 V pRun 1Run 2: 15 : SURFA CE TENSION (EEC Method A.5 and OECD Guideline 115)

METHOD

The surface tension of a 90% saturated aqueous solution was determined at 20 ° C with a surface tension torsion balance using the OECD harmonised ring method.

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).

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,

w as determined in acco rdance with:

ø b = o

g 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

L iterature value for the surface tension of water at 20 ° C ( σ o ) = 72.7 mN/m.

Mean measur ed surf

ace tension of water at 20 ° C ( σ g ) = 72.5 mN/m.

Therefore, the calibration factor, ø b = 1.003

TEST SOLUTI ON PREPARATION

Test substance (approximately 70 mg) was combined with purified water (250 ml) and shaken at 30°C in a water bath for 3 days. After this period, the sample was equilibrated at 20°C for 6 hours and then duplicate portions (approximately 75 ml) were centrifuged (2000 rpm, 5 minutes, 20 ° C).

Aliquots (45 ml) of each clear supernatant were then diluted to 50 ml with purified water to produce 90% saturated aqueous solutions for measurement.: 16 :

PROCEDU RE

The test vessel was half filled with liquid, put on the tensiometer test platform, and raised so that the ring was 2-3 mm below the liquid surface. The platform was lowered to draw a lamella from the liquid surface at a rate of approximately 0.5 cm/min. The maximum force which arose just before the lamella was torn off (i.e. the uncorrected surface tension) was recorded. Measurements were made at 5-minute intervals until a constant value (to within 0.5 mN/m) was recorded.

RESULTS

Test temperature: 20°C

Solution concentration: 90% saturated

Solution age: Prepared immediately before measurement

Initial values for the surface tension were obtained as soon as possible after transfer of the aqueous solution to the measurement vessel.

Time Surface tension (mN/m) (minutes) Measu red Calibr ation corrected

A B A B

0 68.0 68.5 68.0 68.5

5 68.0 68.0 68.0 68.0

10 68.0 68.0 68.0 68.0

15 68.0 68.0 68.0 68.0

20 68.0 68.0 68.0 68.0

25 68.0 68.0 68.0 68.0

30 68.0 68.0 68.0 68.0

From the results there was no apparent time dependence of surface tension after transfer to the measuring vessel.

Overall mean calibration corrected surface tension = 68.0 mN/m (to the nearest 0.5 mN/m)

Harkins-Jordan* corrected value = 0.00181 x 2 + 0.862 x - 0.285 = 66.5 mN/m

where x = ove rall mean calibration corrected surface tension

CONCLUSION

The surface tension of a 90% saturated aqueous solut was found to be 66.5 mN/m. As the result is greater than 60 mN/m, considered to be

surface active.

*H ARKINS, W.D. and JO RDAN, H.F., J. Amer. Che m. Soc. 52 , 1751 (1930).: 17 : RELATIVE SE LF-I GNITION TEMPERATURE FOR SOLIDS

(EEC Method A.16)

METHOD

The presence or otherwise of a self-ignition temperature up to the melting temperature of the test substance was determined through the monitoring of the sample temperature while located in a steadily heated oven.

DEFINITION AND UNITS

The self-ignition temperature is defined as the minimum ambient te mper ature, expressed in degrees Celcius (°C), at which a certain quantity of a substance will ignite when mixed with air 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.

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. Metal base plate.

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.

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.

RESULTS

There was no exothermic reaction of below its melting point at approximately

150°C.

The temperature versus time graph is presented in Figure 2.

C ONCLUSION

did not self- ignite below its melting t emperature.: 18 :FIGURE 2Temperature Versus Time Curve0501001502002503003504000100 200 300 400 500 600 700 800Time (minutes)

Temperature (°C)SampleOven: 19 : PA RTICLE SIZE DISTRIBUTION

METHOD

The particle size distribution was initially examined using sieve analysis. As greater than 10% by weight of the test substance was found to pass a 75 micron sieve, it was further examined by image analysis.

PROCEDURE

Sieve analysis

A portion (10 g) of the test substance was weighed into the coarsest of a nest of sieves (100 mm diameter) of aperture sizes 400, 125, 75, 30 and 10 microns. The nest was shaken for 30 minutes with tapping (Pascall Inclyno sieve shaker), and the proportion of the test material passing through each sieve determined gravimetrically. The test was performed in duplicate, and the results are summarised thus:

Particle size % in range by weight (μm) Test 1 Test 2 Mean

>400

400 - 125

125 - 75

75 - 30

30 - 10

<10

Imag e analysis (based on BS3406, Part 4, 1993)

A suitable suspending medium was experimentally determined to fulfil the following properties.

i) low solubility ii) different refractive index iii) low volatility iv) good dispersion

Screening to remove particles greater than 125 μ m was not considered necessary, partly due to the relatively small proportion (less than 10% by mass) of the test substance found to be greater than this size in the sieve analysis test, but primarily due to the observations made on microscopic examination of an initial slide. This microscopic examination showed the particles to be predominantly long and thin, with none noticeably greater than 125 μ m in width. It was considered that the proportion of particles retained on the 125- μ m sieve in the sieve analysis test was influenced by the alignment of the particles on the sieve and that this proportion would have decreased on extended sieving.: 20 :

Si x replicate samples, each approximately 5-10 mg in weight, were suspended by agitation and mounted on a microscope slide for image analysis.

APPARATUS

The following conditions were employed:

Microscope: Leitz Laborlux S

Video monitoring: Ikegami charge coupling device (CCD) camera, model 1CD-42E, with monitor (NEC/Multisync 3D)

Illumination type: Kohler

Suspending medium: 1% v/v aqueous Tween 80

Size range: 0.5 - 600 μm

Magnification: X10, X40

Size parameters: Longest dimension, axial ratio

RESULTS

The reported results were prepared from combined data for both magnifications, with at least 50 fields counted at each magnification. The mean particle length and axial ratio are used to calculate particle volumes in each range which, when combined with the particle number distribution, give a volume or mass distribution.

The results can be summarised as follows:

Particle size % Mass (μm) Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 Mean

320 - 600

160 - 320

60.0 - 160

30.0 - 60.0

10.4 - 30.0

0.5 - 10.4

CONCLUSION

The results indicate that 0% w/w of i s smaller than 10 μm.: 21 : SOIL ADSORPTION (EEC Method C.19 and OECD Guideline 121)

METHOD

The sorption behaviour of the test substance on soil was examined using high performance liquid chromatography (HPLC).

HPLC is performed on commercially available analytical columns packed with a solid phasecontaining a moderately polar stationary phase with lipophilic and polar moieties (e.g. cyanopropyl bonded phase).

Chemicals injected onto such a column move along it by partitioning between the mobile phase and the stationary phase. The velocity of each component thereby depends on the degree of adsorption on the stationary phase. The dual nature of the stationary phase allows for interaction of polar and apolar parts of a molecule in a similar way as is the case for soil. This enables the relationship between the retention time on such a column and the adsorption coefficient on the organic parts of the soil to be established.

The adsorption coefficient is deduced from the capacity factor (k’) given by:

k’ =

0 0 r

t t

- t

where t r is the measured retention time of sample or reference; and

t 0 is the retention time of the internal de adtime standard.

DEFINITION AND UN ITS

The adsorption coefficient, K

oc , is defined as the distribution coefficient (K d ) normalised to

the or ganic carbon content of a sorbent. K d is defined as the ratio between the concentration

of the test substance in the soil and the concentration of the substance in the aqueous phase, both measured when adsorption equilibrium is reached.

The adsorption coefficient is usually given in the form of the logarithm to base ten (log 10 K
oc ).

PROCEDURE

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

oc , six

referenc e materials were selected from a list of recommended compounds. These reference compounds were selected to have log 10 K
oc values between 1.3 and 5.6. An internal deadtime standard ( I.D.S., formamide) with no retention on the HPLC column was used to determine the deadtime of the HPLC system.: 22 :

The following ref

erence mixture was prepared in HPLC mobile phase.

Reference substance Known* Concentration log 10 K

oc (mg /l )

Formamide (I.D.S.) Deadt ime only 1050

Acetanilide 1.3 54.0

Nitrobenzene 2.4 49.9 Naphthalene 2.8 50.6 2-Methylnaphthalene 3.9 62.2

Anthracene 4.4 47.3

DDT 5.6 107

*values f rom OECD Method 121, with the exception of nitrobenzene, 2-methylnaphthalene and anthracene, values for which came from ‘HODSON, J. and WILLIAMS, N.A., (1988). The Estimation of the Adsorption Coefficient (K

oc ) for Soils by High Performance Liquid Chromatog

raphy

, Chemosphere, Vol. 17, pp.67-77, Pergammon Journals Ltd’.

A solution of (52.0 mg/l in mobile phase, and containing I.D.S. at the same concentration a erence mixture) was prepared and chromatographed in duplicate, bracketed by samples of the reference mixture, using the HPLC conditions below.

HPLC CONDITIONS

Instrument:

Column:

Column temperature:

Mobile phase composition:

Flow rate:

Injection volume:

Detector:: 23 :

RESULTS

Table 3 pres ents the primar y data from which the calibration curve (Figure 3) was gen erated.

Figures 4 and 5 present examples of chromatograms generated during this test.

The mean calibration curve was used to interpolate the results.

The mean calibration curve produced was:

log 10 K
oc = 3.46 log 10 k’ + 3.86

w here k ’ = c apacity factor

=

0 0 r

0 r

t t

- t

= t t'

and t'

r = true retention time obtained by subtracting deadtime, or t 0 , of I.D.S. from measured

retention time of sample or ref erence standard.

A mean log 10 K
oc value for of 3.7 was thus obtained.

A r anking system of soil mobility based upon K

oc has been proposed by McCall et al (Measurement of Sorption Coefficients of Organic Chemicals and their use in Environmental Fate Analysis, Test Protocols for Environmental Fate & Movement of Toxicants, Proceedings of Association of Official Analytical Chemists, 1980):

K

oc Mobilit y clas s

0 - 50 ver y high

50 - 150 hig h

150 - 500 m ediu m 500 - 2000 lo w 2000 - 5000 slig ht

> 5000 i m mobile

T h e determined log 10 K
oc value of 3.7 (K

oc > 5000) suggests that the test substance will be

immobile in soil.

CONCLUSION

was found to have a lo g 10 K
oc value of 3.7.: 24 :TABLE 3HPLC Determination of log10KocforSampleComponenttr(minutes)k'log10k'log10KocStandard Run AAcetanilide3.7970.169-0.7731.3(t0= 3.249 minutes) Nitrobenzene4.4850.380-0.4202.4Naphthalene5.3920.660-0.1812.82-Methylnaphthalene 6.0000.847-0.0723.9Anthracene7.9851.4580.1644.4DDT13.1853.0580.4855.6Sample A6.1860.906-0.0433.7(t0= 3.246 minutes)Sample B6.1800.904-0.0443.7(t0= 3.246 minutes)Standard Run BAcetanilide3.7900.168-0.7741.3(t0= 3.244 minutes) Nitrobenzene4.4750.379-0.4212.4Naphthalene5.3740.657-0.1832.82-Methylnaphthalene 5.9770.842-0.0743.9Anthracene7.9461.4490.1614.4DDT13.1083.0410.4835.6Linear regression (based on mean of standard runs A and B): log10Koc= 3.46 log10k’ + 3.86: 25 :FIGURE 3log10k' Versus log10KocCalibration of Standard Runs A and Blog10Koc= 3.46 log10k' + 3.86r =0.98770.001.002.003.004.005.006.007.00-1.000 -0.800 -0.600 -0.400 -0.200 0.000 0.200 0.400 0.600 0.800 1.000log10k'

log 10 K oc: 26 :FIGURE 4Representative Chromatogram of Standard Mix with log10Koc between 1.3 and 5.6min02468101214mAU020040060080010001200VWD1 A, Wavelength=210 nm (0SA_00F\RSPSYS02.D)

3.249 3.797

4.485 5.392 6.000 7.985 13.185: 27 :FIGURE 5Representative Chromatogram of: 28 : DEVIATIONS FROM PROTOCOL

The final paragraph of Section 2.4.3 of the protocol states that samples w ill be ‘screened to remove particles of size greater than 125 μ m’ for image analysis. This was not done, the reasons for which are given on Page 19 of the report.

MAINTENANCE OF RECORDS

All raw data, samples and specimens (if appropriate) generated at Huntingdon Life Sciences during the course of this study, together with a copy of the final report, will be retained by Huntingdon Life Sciences in its archive for a period of five years from completion of the study, or until the Sponsor provides instructions for their delivery to the Sponsor’s designated archiving facility. After such time, the Sponsor will be contacted and advice sought on the return, disposal or further retention of the materials. Types of sample and specimen which are unsuitable, by reason of instability, for long term retention and archiving may be disposed of after the periods stated in Huntingdon Life Sciences Standard Operating Procedures and after contacting the Sponsor. Under no circumstances will any item be discarded without the Sponsor’s consent.

Huntingdon Life Sciences will retain the Quality Assurance records relevant to this study and a copy of the final report in its archive indefinitely.: 29 : REFERENCES

The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 No. 3106, as amended by Statutory Instrument 2004 No. 994).

OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17.

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

Directive 92/69/EEC, Official Journal No. L383A, 29.12.1992, Method A.4, Vapour

Pressure.

OECD Guideline 104 - Vapour Pressure, Adopted by the Council on 23 March 2006.

Directive 92/69/EEC, Official Journal No. L383A, 29.12.1992, Method A.5, Surface

Tension.

OECD Guideline 115 - Surface tension, Adopted by the Council on 27 July 1995.

Directive 92/69/EEC, Official Journal No. L383A, 29.12.1992, Method A.16, Relative Self-Ignition Temperature for Solids.

Directive 2001/59/EEC, Official Journal No. L225, 21.8.2001, Method C.19, Estimation of the Adsorption Coefficient (K

oc ) on Soil and on Sewage Sludge Using High Performance

L iquid Chromato graph y (HPLC).

OECD Guideline 121 - Estimation of the Adsorption Coefficient (K

oc ) on Soil and on Sewage Sludge Usin g High Performance Liquid Chromatography (HPLC), Adopted by the Council on 22 January 2001.

McCall et al , Measurement of Sorption Coefficients of Organic Chemicals and their use in Environmental Fate Analysis, Test Protocols for Environmental Fate & Movement of Toxicants, Proceedings Of Association of Official Analytical Chemists, 1980.: 30 :

AP PEN DIX 1

E YE RESEARCH CENTRE GLP CO MPLIANCE STATEMENT 2005: 31 :: 32 :

AP PEN DIX 2

CERTIFICATE OF ANALYSIS: 33 :: 34 :

AP PEN DIX 3

STUDY PROTOCOL: 35 :: 36 :: 37 :: 38 :: 39 :: 40 :: 41 :: 42 :: 43 :: 44 :: 45 :: 46 :: 47 :: 48 :: 49 :: 50 :: 51 :: 52 :: 53 :: 54 :