P age 1 of 35 TITLE/COVER PAGE

WA TER SOLUBILITY

Sponsor Research Laboratory

Huntingdon Life Sciences, Ltd.

Woolley Road

Alconbury

Huntingdon

Cambridgeshire

PE28 4HS

ENGLAND

Final: 2 April 2008: 2 : TABLE OF CONTENTS

Page

TITLE/COVER PAGE ....................................................................................................... 1

TABLE OF CONTENTS.................................................................................................... 2

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

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

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

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

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

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

WATER SOLUBILITY...................................................................................................... 9

DEVIATIONS FROM PROTOCOL.................................................................................. 15

MAINTENANCE OF RECORDS...................................................................................... 15

REFERENCES ................................................................................................................... 16

APPENDICES

1. Eye Research Centre GLP Compliance Statement 2005 ............................................. 17

2. Eye Research Centre GLP Compliance Statement 2008 ............................................. 19

3. Certificate of Analysis.................................................................................................. 21

4. Study Protocol.............................................................................................................. 23: 5 : CONTRIBUTING SCIENTISTS

Water Solubility

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. Steil, B.Sc.

(Scientist, Product Chemistry): 6 : SUMMARY

A study was performed to determine the water solubility of The study was

performed in accordance with the test described in the EEC Methods for the Determination of

Physicochemical Properties (Directive 92/69/EEC, Method A.6) and the OECD Guidelines

for the Testing of Chemicals (Guideline 105).

was determined to have a water solubility of 1.0 mg/l at 20 ° C.: 7 : INTRODUCTION

A study was performed to determine the water solubility of The study was

performed in accordance with the test described in the EEC Methods for the Determination of

Physicochemical Properties (Directive 92/69/EEC, Method A.6) and the OECD Guidelines

for the Testing of Chemicals (Guideline 105).

The protocol was approved by the Study Director and Huntingdon Life Sciences

Management on 14 March 2007, by the Study Monitor for the Sponsor on 19 March 2007

and by the Sponsor’s Representative on 29 March 2007.

The experimental start and completion dates were 13 April 2007 and 26 April 2007,

respectively.

Location of study : Huntingdon Life Sciences, Ltd.

Eye Research Centre

Eye

Suffolk

IP23 7PX: 8 : TEST SUBSTANCE

Identity:

Chemical name:

Molecular formula:

Molecular weight:

CAS number:

Lot 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 : WATER SOLUBILITY

(EEC Method A.6, OECD Guideline 105)

METHOD

The water solubility was conducted by a flow through column elution method following a

preliminary examination.

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. The SI unit is grams/litre (g/l).

PROCEDURE

Preliminary test

A preliminary test was conducted by mixing a known mass (approximately 10 mg) of

with purified reverse-osmosis water (1000 ml) at room temperature and visually

assessing dissolution. The test substance was not completely dissolved after 96 hours,

indicating a solubility of less than 10 mg/l. The definitive test was thus performed by a flow

through column elution method.

Flow through column elution method

A stainless steel column (25 cm x 4.6 mm internal diameter, fitted with a 2 μm frit) was

packed with a sample (approximately 1 ml volume) of inert support material coated with the

test substance:

Inert support material: Sand, 50 - 150 mesh (BDH Chemicals Ltd.)

Loading of test substance onto support: Sand (4 g) was mixed with a solution of the test

substance in acetone (4 g/l, 50 ml). The solvent

was removed under vacuum at 40°C to give

approximately 50 mg test substance/g support.

A small glass wool plug was placed either side of the sample bed to keep the sample in place

and aid filtration. A control column containing sand only was also prepared.

The charged column was connected to a HPLC pump equipped with stainless steel

capillaries, and purified water was added to the solvent reservoir. The system was

equilibrated at the test temperature of 20°C.

The flow through the column was started at 0.4 ml/minute, and the eluent allowed to run to

waste for 16.5 hours before sampling commenced. At intervals separated from each other by

the passage of at least ten bed volumes, samples of the eluent (10 ml) were collected and a

portion (5 ml) diluted to volume (10 ml) with methanol for analysis by high performance

liquid chromatogaphy (HPLC).

After six samples had been taken, the flow rate was reduced to 0.2 ml/minute and the eluent

allowed to run to waste until collection of a further six samples. These were processed and

analysed as above.: 10 :

On each sampling occasion at both flow rates, an additional sample was collected for pH

measurement. All samples were also examined for light scattering and found to be haze-free.

HPLC CONDITIONS

Instrument:

Column:

Column temperature:

Mobile phase composition:

Flow rate:

Injection volume:

Detector:

Retention time:

VERIFICATION SAMPLES

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

PREPARATION OF CALIBRATION

A stock calibration solution of concentration 560 mg/l was prepared by weighing test

substance (approximately 28 mg) into a 50 ml volumetric flask and dissolving in and diluting

to volume with methanol.

Calibration solutions in the range of approximately 0.1 to 2 mg/l were prepared by dilutions

of the stock solution with methanol:water (1:1 v/v).

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) = sample peak area x standard concentration (mg l)

m ean peak area of bracketing standards

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 2 for all samples.: 11 :

RESULTS AND DISCUSSION

The detector calibration was found to be linear over the range 0 to 2 mg/l of standard

solutions in methanol:water (1:1 v/v) with a regression coefficient of 1.0000 (Table 1,

Figure 1).

A mean recovery of 103% of from fortified control samples was deemed to be

acceptable, and thus no correction was necessary to the determined sample concentrations.

No significant interfering peaks were evident in blank control solutions.

Table 2 presents a summary of the results of the test and shows that the water solubility of

is 1.05 ± 0.04 mg/l. Table 3 presents the primary data for this test.

CONCLUSION

was determined to have a water solubility of 1.0 mg/l at 20 ° C.: 12 : TABLE 1

Standard Calibration for by HPLC

Standard concentration (mg/l) P eak area

2.228 6 56.05

1.782 5 18.93

1.337 3 88.30

0.8912 2 57.70

0.4456 1 28.66

0.2228 6 3.150

0.08912 2 5.041

L inear regression: y = 293.6x - 2.009

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

x = concentration

y = peak area

FIGURE 1

Standard Calibration for by HPLC

0 1 00 200 300 400 500 600 700

0.00 0.50 1.00 1.50 2.00 2.50

Concentration (mg/l)Peak area: 13 : TABLE 2

Measurements of Water Solubility at 20°C

1. Column flow rate 0.4 ml/minute

Sample Sampling time Concentration pH

number ( hours) (mg/l)

1 1 6.5 1.12 6.3

2 1 7.5 1.09 6.4

3 1 8.5 1.08 6.5

4 1 9.0 1.08 6.4

5 2 0.0 1.03 6.3

6 2 1.0 1.05 6.4

M ean solubility: 1.08 ± 0.03 mg/l

2. Column flow rate 0.2 ml/minute

Sample Sampling time Concentration pH

number ( hours) (mg/l)

7 1 .0 1.07 6.3

8 2 .5 1.06 6.3

9 1 9.0 1.01 6.4

10 2 1.0 1.01 6.4

11 2 2.5 1.01 6.4

12 2 4.0 0.99 6.4

M ean solubility: 1.02 ± 0.03 mg/l

Overall mean solubility: 1.05 ± 0.04 mg/l

Coefficient of variation = 4%: 14 :TABLE 3HPLC Analysis of Samples from the Water Solubility Test forSamplePeak areaCA(mg/l)Dilution factorCB(mg/l)0.8912 mg/l std257.42---Sample 1161.710.560021.12Sample 2156.880.543221.09Sample 3155.520.538521.08Sample 4156.100.540521.080.8912 mg/l std257.31---Sample 5149.620.517021.03Sample 6152.420.526721.05Sample 7154.810.534921.07Sample 8153.910.531821.060.8912 mg/l std258.53---Sample 9146.550.505321.01Sample 10146.100.503821.01Sample 11145.700.502421.01Sample 12143.300.494120.990.8912 mg/l std258.39---: 15 : DEVIATIONS FROM PROTOCOL

There were no deviations from protocol.

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.: 16 : 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.6, Water

Solubility.

OECD Guideline 105 – Water Solubility, Adopted by the Council on 27 July 1995.: 17 :

APPENDIX 1

EYE RESEARCH CENTRE GLP COMPLIANCE STATEMENT 200518 :19 :

A PPENDIX 2

EYE RESEARCH CENTRE GLP COMPLIANCE STATEMENT 200820 :21 :

A PPENDIX 3

CERTIFICATE OF ANALYSIS22 :23 :

A PPENDIX 4

STUDY PROTOCOL24 :25 :26 :27 :28 :29 :30 :31 :32 :33 :34 :35 :