## Page 1 o f 34 TITLE/COVER PAGE

ABIOTIC DEGRADATION: HYDROLYSIS AS A FUNCTION OF pH ( P RELIMINARY TEST)

Sponsor R esear ch Laboratory

Huntingdon Life Sciences, Ltd. Woolley Road Alconbury Huntingdon Cambridgeshire PE28 4HS ENGLAND

Final: 2 April 2008

## : 2 : TABLE OF CONTENT S

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
ABIOTIC DEGRADATION: HYDROLYSIS AS A FUNCTION OF pH (PRELIMINARY TEST) .................................................................................................... 9

FIGURE

1. Standard Calibration for by HPLC.......................................................... 12

TABLES

1. Standard Calibration for by H 13 2. Preliminar y Test Results sis of 14 3. pH Change During Preliminary Hydrolysis 15

PROTOCOL DEVIATIONS............................................................................................... 16

MA INTENANCE OF RECORDS...................................................................................... 16

REFERENCES.................................................................................................................... 17

APPENDICES

1. Eye Research Centre GLP Compliance Statement 2005.............................................. 18 2. Eye Research Centre GLP Compliance Statement 2008.............................................. 20 3. Certificate of Analysis.................................................................................................. 22 4. Study Protocol .............................................................................................................. 24

### COMPLIANCE WITH GOOD LADORA TORY PRACTICE STANDARDS



### Abiotic Degradation: Hydrolysis as a Function of pH (Preliminary Test)

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 2004110/EC of 11 February 2004.

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

The OECD 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, B.Sc., M.Sc. Study Director Huntingdon Life Sciences Ltd. 

# ..**~** 

# ... 6-f..r:-.:'

...**~.~~** 

# ............ .

Date



### : 3:

## QUALITY ASSURANCE STATEMENT

Abiotic Degradation: Hydrolysis as a Function of pH (Preliminary Test)

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

Study Phase

Protocol Audit

Report Audit Date(s) oflnspection

17 May2007

14-16 October 2007 

### Date of Reporting to Study Director and Management

18 May2007

16 October 2007



## Process based inspections: At or about the time this study was in progress, inspections of procedures employed on this type of study were carried out. These were conducted and reported to appropriate Company Management as indicated below:

Process Based Inspections

Hydrolysis 

### Date(s) oflnspection

26 September 2007 

### Date of Reporting to Management

28 September 2007



### 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.

Vrt:ffiflKVIanager Department of Quality Assurance, Huntingdon Life Sciences Ltd.



### :4: 

# .. .**~.l:f.e.-:~~;-:-** 

# . .**~~'.(** 

# ........ .

Date

## : 5 : CONTRIBUTING SCIENTIST S

Abiotic De gradation: Hydrolysis as a Function of pH

(Preliminary Test)

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 : S UMMARY

A study was performed to determine the rate of hydrolysis of as a function of pH. The study was performed in accordance with the prelimi described in the Annex to EEC Directive 92/69/EEC, Method C.7, and the OECD Guidelines for the Testing of Chemicals, Method 111.

The preliminary study showed that at pH 4, 7 and 9 and 50 ± 0.5oC, less than 10% hydrolysis had occurred after 120 hours (5 days), equivalent to a half-life of greater than 1 year under environmental conditions (25°C); therefore, no further testing was necessary.

was determined to be hydrolytically stable under acidic (pH 4), neutral (pH 7) H 9) conditions

.

## : 7 : I NTRODUCTION

This study was designed to determine the rate of hydrolysis of as a function of pH. Determination of the rate of hydrolysis of a test subs in predicting its environmental fate.
The study was conducted in compliance with the requirements of the Annex to EEC Directive 92/69/EEC, Method C.7, and the OECD Guidelines for the Testing of Chemicals, Method 111.
The protocol was approved by the Study Director and Huntingdon Life Sciences Management on 15 May 2007, by the Study Monitor for the Sponsor on 17 May 2007 and by the Sponsor’s Representative on 31 May 2007.
The experimental start and completion dates were 15 August 2007 and 23 August 2007, respectively.
Location of study : Huntingdon Life Sciences, Ltd. Eye Research Centre Eye Suffolk IP23 7PX

## : 8 : T EST SUBSTANCE

Identity:

C hemical name:

Molecular fo rmula:

M olecular w eight:

CAS number:

Lot number:

Expiry date: 14 May 2008

Purity:

Appearance:

Storage conditions: Ambient temperature in a dark environmentally controlled area

Date of sample receipt: 31 July 2006

Source:

Structure:

## : 9 :

A BIOTIC DEGRADA TION: HYDROLYSIS AS A FUNCTION OF pH (PRELIMINARY TEST)

METHOD

The test was performed in accordance with the preliminary test as described in Method C.7 (92/69/EEC) and OECD Guideline 111.

DEFINITION AND UNITS

Hydrolysis refers to a reaction of a chemical RX with water. This reaction may be represented by the net exchange of the group X with OH:

RX + HOH → ROH + HX
The concentrations can be expressed in units such as milligrams per litre or moles per litre.

PROCEDURE

Preparation of buffer solutions

Buffer solutions were prepared as follows, using sterile, purified water:

pH 4.0 : Potassium dihydrogen orthophosphate (3.00 g) and disodium hydrogen

orthophosphate dodecahydrate (6.42 g) were dissolved in water (950 ml), and the pH was adjusted to 4.0 ± 0.05 with orthophosphoric acid. The volume was then adjusted to 1000 ml with water.

pH 7.0 : Potassium dihydrogen orthophosphate (6.82 g) was dissolved in water (950 ml),

1M sodium hydroxide (30 ml) was added and the pH was measured as 7.0 ± 0.05. The volume was then adjusted to 1000 ml with water.

pH 9.0 : Disodium tetraborate decahydrate (16.6 g) and potassium dihydrogen

orthophosphate (1.80 g) were dissolved in water (950 ml) and the pH was measured as 9.0 ± 0.05. The volume was then adjusted to 1000 ml with water.

Preliminary test

The following procedure was carried out in duplicate at each of pH 4, 7 and 9:

An aliquot (1 ml) of a stock solution of (approximately 50 mg/l in methanol) was added to a 100-ml volumetric flask co ffer solution (100 ml), which had been purged with nitrogen and pre-equilibrated at test temperature (50°C). The sample, of nominal concentration 0.5 mg/l, was placed in a 50°C water bath in the dark (the sample was foil-wrapped) until sampling was required (after a short incubation period of approximately 5 minutes, and then after 2.4 and 120 hours). At each sampling time, an aliquot (5 ml) was diluted to volume (10 ml) with methanol for analysis by high performance liquid chromatography (HPLC).

## : 10 :

S ample pH and incubation temperature were monitored over the period of the test using a Fisher Scientific Hydrus 400 pH meter and calibrated mercury-in-glass thermometer, respectively.

HPLC CONDITIONS

I nstrume nt:

Column:

Column temperature:

Mobile phase composition:

Flow rate:

Injection volume:

Detector:

Retention time:

PREPARATION OF CALIBRATION
A stock calibration solution of concentration 2.5 g/l was prepared by weighing test substance (approximately 120 mg) into a 50 ml volumetric flask and dissolving in and diluting to volume with methanol.
Calibration solutions in the range 0.05 to 0.5 mg/l were prepared by dilutions of the stock solution with mobile phase.

CALCULATIONS

The concentration of in the analysed solution (C A 

## ) was calculated from

s tandards introduced b fter samples (bracketing standards) by the following equation:

C A



## (mg/l) = standards bracketing of areapeak mean l) (mgion concentrat standard x areapeak sample



## The concentration of in each test solution (C B 

## ) was then determined thus:

C B 

## (mg/l) = C A



## (mg/l) x dilution factor

w here the dilution fa ctor was 2 for all samples tested.

## : 11 :

R ESULTS

Validation

Using the conditions described, the calibration of was found to be linear over the concentration range 0 to 0.5 mg/l, with sion coefficient of 1.0000 (Figure 1, Table 1).

The analytical method used was sufficiently precise and sensitive to detect a reduction of 10% of the initial test concentration.

Recoveries of from fortified control samples in each medium were found to be quantitative an orrection was necessary to the determined sample concentrations.

No significant interfering peaks were evident in blank control solutions.

Preliminary test

Results from th ry test showed that there was no significant change in the concentration of when incubated in pH 4, 7 and 9 buffer solutions at 50 ± 0.5oC (Table 2). Less hydrolysis had occurred after 120 hours (5 days) under these conditions, equivalent to a half-life of greater than 1 year under environmental conditions (25°C); therefore, no further testing was necessary.

pH of test solutions

The pH values of test solutions over the period of the test are presented in Table 3. The results show that there were no significant changes in pH with time.

CONCLUSION

was determined to be hydrolytically stable under acidic (pH 4), neutral (pH 7) H 9) conditions

.

### : 

## 12 :FIGURE 1Standard Calibrationforby HPLCy = 263.8x - 0.4848r = 1.000002550751001251500.00 0.100.200.300.400.50Concentration (mg/l)

Peak area

## : 13 : TABLE 1

Standard Calibration for by HPLC

Standard concentration (mg/l) Peak area

0.4918 129.7 2 0.3934 103.0 9 0.2951 77.22 5 0.1967 51.08 8 0.09835 25.23 3 0.04918 12.49 2

Li near regression: y = 263.8x - 0.4848 (including x = 0, y = 0) r = 1.0000

x = concentration y = peak area

## : 14 : TABLE 2

P reliminary Test Results for Hydrolysis of

C t 

### (mg/l)

pH t 0h 

### t 2.4h 

### t 120h 

### Measured Mean Measured Mean Measured Mean

4 0.47, 0.48 0.48 0.50, 0.48 0.49 0.48, 0.48 0.48

7 0.48, 0.48 0.48 0.48, 0.48 0.48 0.47, 0.47 0.47

9 0.48, 0.48 0.48 0.48, 0.48 0.48 0.47, 0.47 0.47

Where C t 

### is t he concentration of in solution at time t h 

### (in hours)

## : 15 : TABLE 3

pH Change During Prel iminary Hydrolysis Tests

Nominal pH Me an Initial pH (t 0h 

### ) Mean Final pH (t 120h 

### )

4 4.1 4.1

7 7.0 7.0

9 9.0 9.1

## : 16 : PROTOCOL DEVIATIONS

Blank control solutions were run at each pH during the course of method development work and no significant interfering peaks were evident. Such controls were not, however, run on each occasion of analysis as specified in the protocol.
This minor deviation was not considered to have any impact on the integrity of the study.

MAINTENANCE OF RECORDS

All raw data, samples and specimens (if appropriate) generate d 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.

## : 17 : 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 C.7, Abiotic Degradation, Hydrolysis as a Function of pH.
OECD Guideline 111 - Hydrolysis as a Function of pH, Adopted by the Council on 13 April 2004.

## : 18 :

AP PENDIX 1

EYE RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2005

## : 19 :

## : 20 :

A P PENDIX 2

EYE RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2008

## : 21 : 

## j 

# nli) Department**~** ofHealth

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

GOOD LABORATORY PRACTICE

STATEMENT OF COMPLIANCE IN ACCORDANCE WITH DIRECTIVE 2004/9/EC

TEST FACILITY

Huntingdon Life Sciences Eye Research Centre Oecoid Eye Suffolk IP23 7PX

DATE OF INSPECTION

28th January 2008 TEST TYPE

Analytical Chemistry Ecosystems Environmental Fate Environmental Toxicity Mutagenicity Phys/Chem Testing Toxicology

A general inspection for compliance with the Principles of Good Laboratory Practice

was carried out at the above test facility as part of the UK GLP Compliance Programme.

At the time of inspection no deviations were found of sufficient magnitude to affect the validity of non-clinical studies performed at these facilities

. 

# • Dr. Andrew 

### J. Gray Head, UK GLP Monitoring Authority

## : 22 :

A P PENDIX 3

CERTIFICATE OF ANALYSIS

## : 23 :

## : 24 :

A P PENDIX 4

STUDY PROTOCOL

## : 25 : Study Number: Enquiry Number:



### CONFIDENTIAL



## PROTOCOL 

# Huntingdon Life Sciences

ABIOTIC DEGRADATION: HYDROLYSIS AS A FUNCTION OF pH (PRELIMINARY TEST)

Sponsor

Total number of pages: 10



### Final Protocol Research Laboratory

Huntingdon Life Sciences Ltd Woolley Road Alconbury Huntingdon Cambridgeshire PE28 4HS ENGLAND

Page i

Huntingdon Life Sciences Ltd, registered in England No: 1815730

## : 26 : Study Number: Enquiry Number: 

# Huntingdon Life Sciences

PROTOCOL APPROVAL

ABIOTIC DEGRADATION: HYDROLYSIS AS A FUNCTION OF pH (PRELIMINARY TEST)

# p:·s;.fu~1:~·**·· ··············** **·············** Study Director Huntingdon Life Sciences Ltd



# ................

# ~.W- ..

# ~.L ... 

# Lt

...................... A. L. Comb,

# ~G~~. Ph.D.
, Management Huntingdon Life Sciences Ltd



# t? /J/

.

........ :1;

... 

# .,1............

. ..... A. I. Nikiforov, Ph.D. Study Monitor for the Sponsor Toxicology/Regulatory Services, Inc. 

## !.**f./~·7!·** 

## ?:E.!.

..... . Date



# }:.(. ;

.......**!'J.~** 

# ....**/.~v** 

# "{

Date



# /1.

...... 

# 1.J6Y..

... 2

- o OfDate 

# l--

Please sign both copies of this page, retain one for your records and return one to the Study Director at Huntingdon Life Sciences.

Final Protocol Page 

### ii

## : 27 : Study Number : Enquiry Number:

Study number:
Study title:

Test substance Identity:

Chemical name:

Molecular formula:

CAS number:

Lot number:
Expiry date:
Purity:
Appearance:
Storage conditions:

Date of sample receipt:

Source:
Sponsor:

Sponsor's Representative:

Study Monitor for the Sponsor:

Study Director:

Person acting in the temporary absence of the Study Director:

Location of study:

Proposed study dates Experimental start: Experimental completion: Audited draft report: 

# Huntingdon Life Sciences

STUDY DETAILS PAGE

Abiotic Degradation: Hydrolysis as a Function of pH (Preliminary Test)

To be included in the raw data when available

Ambient temperature in a dark envirorunentally controlled area

31 July 2006

Andrey I. Nikiforov, Ph.D., TRS, Inc., 2365 Hunters Way, Charlottesville, VA 22911, U.S.A.

P. Sydney

In the temporary absence of the Study Director, routine duties and scientific queries will be attended to by A. L. Comb, Scientific Manager, Product Chemistry. Formal GLP responsibilities will be assumed by Management. Decisions made during the temporary absence of the Study Director pertaining to this study will 

### be ratified by the Study Director upon return. Department of Product Chemistry, Eye Research Centre, Eye, Suffolk, IP23 7PX

September 2007 October 2007 October 2007 The actual dates will be documented in the fmal report.



### Final Protocol Page iii

## : 28 :

## : 29 :

## : 30 : Study Number: Enquiry Number:



### 2.**EXPE~ENTALPROCEDURES**


### 2.1 Method 

# Huntingdon 

# Life Sciences

The test will be performed in accordance with the preliminary test as described in Method C.7 (92/69/EEC) and OECD Guideline 111.

The test will be performed at 50± 0.5°C at three pH values: 4, 7 and 9. A sufficient number of
measurements will be made in order to be able to estimate whether the half-life time (t.,) is lower than 2.4 hours, or whether less than 10% hydrolysis is observed after 5 days. These values correspond respectively to half-life times lower than 1 day or higher than 1 year respectively, under conditions more representative of the environment {25°C).



### 2.2 Definition and units

Hydrolysis refers to a reaction of a chemical RX with water. This reaction may be represented by the net exchange of the group X with OH:

RX 

### + HOH**~** ROH 

### + HX

The concentrations can be expressed in units such as milligrams per litre or moles per litre.



### 2.3 Procedure

The test will be carried out in duplicate at pH 4, 7 and 9, using reagent grade chemicals and purified, sterile water. The test solutions will be contained in stoppered 100 

### m1 volumetric flasks and pH measurements will be made with a Corning Model 250 pH meter or a suitable alternative.

The test substance will be dissolved in the buffer solution at a concentration not exceeding 0.01 M

or half the saturation concentration, whichever is the lower. The composition of the buffer solutions employed may depend on the nature of the· test substance and the analytical procedure used. The concentration of the test substance in solution will be determined by a suitable analytical procedure at various time points which will include as a minimum 0 hours, 2.4 hours and 5 days. Full details of the analytical method will be documented in the study records.

The tests will be carried out using a water bath fitted with a thermostatic heater at 50 ± 0.5°C. The water bath temperature will be monitored with a calibrated mercury-in-glass thermometer.

Precautions will be taken to exclude oxygen by bubbling nitrogen through the buffer solution for a minimum of 5 minutes before preparation of the test solution. Light will be excluded from the test vessels by covering the flasks in aluminium foil.

Depending on the test concentrations used, the test solutions will be analysed in either of two ways:
1) Dilution of an accurately measured subsample with solvents compatible with the mobile phase being used; or
2) Extraction of an accurately measured subsample with a water immiscible solvent with a high solvation capacity for the test substance. Note

- if necessary; for reasons of low sensitivity or high test substance reactivity, the whole of the test solution may be used.



### Final Protocol Page 3

## : 31 :

## : 32 : Study Number: Enquiry Number:



### 3. REPORTING

3.1 Hydrolysis 

# Huntingdon 

# Life Sciences

The test report should, if possible, include but not necessarily be limited to the following information:

• test substance identity and impurities;

• detailed test procedure including the temperature, pH and buffer for each set of experiments;

• detailed analytical method used, including detailed method of extraction and recovery data if an extraction method is used to separate the chemical from the aqueous phase;

• all concentration-time data points for each test; and

• all information relevant for the interpretation of the results, especially with regard to impurities and physical state of the substance.



### 3.2 General reporting requirements

A summary of the test results will be sent to the Study Monitor for the Sponsor on completion of the experimental work.



### An audited draft report and a copy of the study raw data package will be provided to the Study Monitor for the Sponsor before the final report is issued. Unless otherwise requested, this draft report will be issued electronically (Microsoft Word format). Unless otherwise requested, one unbound and one electronic copy (PDF format) of the final report will be issued.



### In the absence of ongoing communications, Huntingdon Life Sciences reserves the right to finalise, sign and issue the final report from this study six months after issue of the draft. 

### In such

an event, all materials will be transferred to the archive. Any subsequent requests for modifications, corrections or additions to the final report will be the subject of a formal report amendment (or new study, as appropriate) and will be subject to additional charge.

The authorised final report will include the information and data required by Good Laboratory Practice standards and relevant guideline reporting requirements.

Corrections or additions to a final report will be in the form of an amendment by the Study Director. The amendment will clearly identify that part of the final report that is being added to or corrected, the reasons for the correction or addition and the impact the correction or addition will have on the study, and will be signed and dated by the Study Director and the Study Monitor for the Sponsor.



### Final Protocol Page 

### 5

## : 33 : Study Number: Enquiry Number: 

# Huntingdon Life Sciences

4. QUALITY ASSURANCE PROCEDURES AND RECORDS TO BE MAINTAINED

4.1 Quality assurance

The following will be inspected or audited in relation to this study.

Protocol Audit The protocol and any amendments specific to this study will be audited.

Process based inspections

Report Audit Routine and repetitive procedures will be inspected on representative studies, not necessarily on this study.

The draft report and study data will be audited before issue of the draft report to the Sponsor's Representative.

QA findings will be reported to the Study Director and Company Management promptly on completion of each action, except for process based inspections which will be reported to appropriate Company Management only.

4.2 Health & safety

In order for Huntingdon Life Sciences to comply with the Health and Safety at Work Act 1974, and the current Control of Substances Hazardous to Health Regulations, it is a condition of undertaking the study that the Sponsor shall provide Huntingdon Life Sciences with all information available to it regarding known or potential hazards associated with the handling and

use of any substance supplied by the Sponsor to Huntingdon Life Sciences. The Sponsor shall also comply with all current legislation and regulations concerning shipment of substances by road, rail, sea or air.

4.3 Archiving

All raw data, samples and specimens (if appropriate) generated at Huntingdon Life Sciences during the course of this study, together with the protocol and final report, will be retained by Huntingdon Life Sciences in its archive for a 'period of five years from completion of the study, unless the Sponsor provides instructions for their delivery to the Sponsor's designated archiving faciiity within this period. After such time, the Sponsor wiii 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.

Final Protocol Page6

## : 34 :