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TITLE/COVER PAGE 

WATER SOLUBILITY 

Sponsor Research Laboratory 

Huntingdon Life Sciences, Ltd. 

Woolley Road 

Alconbury 

Huntingdon 

Cambridgeshire 

PE28 4HS 

ENGLAND 

Final: 2 April 2008 
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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 


 

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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) 
 

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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. 
 

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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 
 

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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: 
 

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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. 
 

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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 (CA) was calculated from 

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

equation: 

 CA (mg/l) = 

sample peak area x standard concentration (mg l)

mean peak area of bracketing standards

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

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

where the dilution factor was 2 for all samples. 
 

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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. 
 

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TABLE 1 

Standard Calibration for by HPLC 

Standard concentration (mg/l) Peak area 

2.228 656.05 

1.782 518.93 

1.337 388.30 

0.8912 257.70 

0.4456 128.66 

0.2228 63.150 

0.08912 25.041 

Linear 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

100

200

300

400

500

600

700

0.00 0.50 1.00 1.50 2.00 2.50

Concentration (mg/l)

Peak area
 

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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 16.5 1.12 6.3 

2 17.5 1.09 6.4 

3 18.5 1.08 6.5 

4 19.0 1.08 6.4 

5 20.0 1.03 6.3 

6 21.0 1.05 6.4 

Mean 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 19.0 1.01 6.4 

10 21.0 1.01 6.4 

11 22.5 1.01 6.4 

12 24.0 0.99 6.4 

Mean solubility: 1.02 ± 0.03 mg/l 

Overall mean solubility: 1.05 ± 0.04 mg/l 

Coefficient of variation = 4% 
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TABLE 3 

HPLC Analysis of Samples from the Water Solubility Test for 

Sample Peak area CA (mg/l) Dilution factor 

0.8912 mg/l std 257.42 - - 

Sample 1 161.71 0.5600 2 Sample 2 156.88 0.5432 2 Sample 3 155.52 0.5385 2 Sample 4 156.10 0.5405 2 

0.8912 mg/l std 257.31 - - 

Sample 5 149.62 0.5170 2 Sample 6 152.42 0.5267 2 Sample 7 154.81 0.5349 2 Sample 8 153.91 0.5318 2 

0.8912 mg/l std 258.53 - - 

Sample 9 146.55 0.5053 2 

Sample 10 146.10 0.5038 2 Sample 11 145.70 0.5024 2 Sample 12 143.30 0.4941 2 

0.8912 mg/l std 258.39 - - 
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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. 
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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. 
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APPENDIX 1 

EYE RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2005
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APPENDIX 2 

EYE RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2008 
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APPENDIX 3

CERTIFICATE OF ANALYSIS
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APPENDIX 4 

STUDY PROTOCOL
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