## REPORT

HYDROLYSIS DETERMINATION OF TKA 40278 (CGI 827) AT DIFFERENT pH VALUES Data Requirement / Test Guidelines: OECD No. 111 EEC Directive 92/69, C.7 StudyDirector: Mrs. Alessandra Tognucci Stud Cornletion Date: July 16, 2003 Test Fa~ RCCLtd Environmental Chemistry & Pharmanalytics CH-4452 ltingerdSwitzedand
Sponsor: CIBA SPECIALTY CHEMICALS INC. CH-4002 Basel/Switzerland RCC Study Number: 845449 Page 1 of 23



### 69RCC Study Number 845449 REPORT Page 2

TKA 40278 (CGI 827) 

## GLP CERTIFICATE

The Swiss GLP Monitoring Authorlties


# Statement of GLP Compliance

It is hereby confirmed that dudng the pedod of the following Test Facilities of November 18 - 22, 2002 RCC Ltd 4452 Itingen 

### Switzerland
were Inspected by the FederalO~ce of Publla Health, the Swiss Agency for Therapeutic Products and the Swiss Agency for the Environment, Forests and Landscape with respect to the compliance with the Swiss legislation on Good Laboratory Practice. Test Facilities Areas of expertise * - Toxicology

- Environmental Chemistry and Pharrnanalytlcs TOX, ACC. MUT, OTH (Safety Pharmacology) ACC, ECT, ENF, EMN, PCT, RES, OTH (Animal metabolism) The inspections were performed in agreement with the OECD Guidelines for Naf~onal GLP Inspections and Audits, It was found that the aforementioned test facilities were operating in compliance with the Swiss Ordinance relating to Good Laboratory Practice [RS 813.016.5] at the time they were Inspected. Federal Oft]ce of Public Health The Director

Bern, March 2003 Prof. Th. Ze]ther



### 70RCC Study Number 845449 REPORT Page 3

TKA 402"78(CG1827) 

### GOOD LABORATORY PRACTICE 

## STATEMENT OF COMPLIANCE
RCC Study Number: Test Item: Study Director: Title: 845449 TKA 40278 (CGI 827) Mrs. Alessandra Tognucci Hydrolysis Determination of TKA 40278 (CGI 827) at Different pH Values This study was conducted in compliance with the Swiss Ordinance relating to Good Laboratory Practice, adopted February 2nd,2000 [RS 813.016.5]. This Ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26t~, 1997 by decision of the OECD Council [C(97)186/Final].

Study Director: Ira Tognucci

Date: ~.~



### 71RCC Study Number 845449 REPORT Page 4

TKA 40278 (CGI 827) 

# SIGNATURES

Study Director:

Date: Tognucci

Management: Dr. JOrn SchreitmOller

Date:



### 72RCC Study Number 845449 REPORT Page 5

TKA 40278 (CGt 827) 

# QUALITY ASSURANCE UNIT RCC Ltd, Environmental Chemistry & Pharmanalytics, CH-4452 Itingen / Switzerland STATEMENT RCC Study Number: Test Item: Study Director: Title: 845449 TKA 40278 (CGI 827) Mrs. Alessandra Tognucci Hydrolysis Determination of TKA 40278 (CGI 827) at Different pH Values The general facilities and activities are inspected periodically and the results are reported to the responsible person and the management. Study procedures were periodically inspected. The study plan and this report were audited by the Quality Assurance Unit. The dates are given below: Dates and Types of QAU Inspections Dates of Reports to the Study Director and to the Management September 16, 2002 Study Plan September 16, 2002 May 12, 2003 Process based May 12, 2003

(Measurement + Sampling) July 08/09, 2003 Final report July 09, 2003 This statement also confirms that this final report reflects the raw data. Quality Assurance: Mrs. M. Richter-Auer



### 73RCO Study Number 845449 REPORT Page 6

TKA 40278 ((;(31827) 

## CERTIFICATION OF GLP AND VERIFICATION OF THE REPORT

The statement of Compliance with Good Laboratory Practice found in this report, and signed by the Study Director is truthful and accurate, and this report as provided by the testing facility is complete and unaltered.

Signature of the Sponsor:

Date:



### 74RCCStudy Number 845449 REPORT Page 7

TKA 40278 (CGI 827) 

## CONTENTS GLP CERTIFICATE

........................................................................................................ 2 STATEMENT OF COMPLIANCE .................................................................................... 3 SIGNATURES

................................................................................................................ 4 QUALITY ASSURANCE UNIT ........................................................................................ 5 CERTIFICATION OF GLP AND VERIFICATION OF THE REPORT .............................. 6 CONTENTS .................................................................................................................... 7 PREFACE

....................................................................................................................... 8 DATA REQUIREMENT / TEST GUIDELINES

................................................................ 9 SUMMARY OF STUDY PLAN AMENDMENTS .............................................................. 9 SUMMARY ....................... i .......................................................................................... 10 1 PURPOSE

........................................................................................................... 11 2 MATERIAL AND METHODS .................................................................. : ............. 11 2.1 TEST ITEM ................................................................................................. 11 2.2 REAGENTS ................................................................................................ 12 2.3 EQUIPMENT .............................................................................................. 12 2.4 STUDY DESIGN ............................................................................. ; ...........13 2.4.1 Principle of the Test ...................................................................... 13 2.4.2 Performance of the Test ............................................................... 13 2.4,3 Preparation of the Test Solutions (3r’~test) .................................... 13 2.5 ANALYTICAL METHOD ............................................................................. 14 2.5.1 Standard Solutions ........................................................................ 14 2.5.2 HPLC-Conditions .......................................................................... 15 2.6 EVALUATION OF RESULTS ...................................................................... 16 3 RESULTS ............................................................................................................ 17 Table 1 : Results of the Preliminary Test of TKA 40278 (CGI 827) .............................. 18 Table 2: The calibration data of TKA 40278 (CGI 827) ............................................... 23 Figure 1 : Typical HPLC chromatogram of a standard solution of TKA 40278 (CGI 827) (18.714pg/ml) .............................................................................. 19 Figure 2: Typical HPLC chromatogram of a sample solution at ~H 4.0 Conditions: 120 hours in aqueous buffered solution ~H 4.0 at 50°C ............. 20 Figure 3: Typical HPLC chromatogram of a sample solution at ~H 7.0 Conditions: 120 hours in aqueous buffered solution ~H 7.0 at 50°C ............. 21 Figure 4: Typical HPLC chromatogram of a sample solution at ~H 9.0 Conditions: 120 hours in aqueous buffered solution )H 9.0 at 50°C ............. 22 Figure 5: Example of a calibration curve of TKA 40278 (CGI 827) ............................... 23


### 75RCC Study Number845449 REPORT Page8

TKA 40278 (CGI 827) 

## PREFACE
GENERAL Title: Sponsor: Monitoring Scientist: Test Facility: Hydrolysis Determination of TKA 40278 (CGI 827) at Different pH Values CIBA SPECIALTY CHEMICALS INC. CH-4002 Basel/Switzerland Dr. Moung Sook Lee RCC Ltd Environmental Chemistry & Pharmanalytics Zelgliweg 1 CH-4452 Itingen/Switzerland
RESPONSIBILITIES Study Director: Deputy Study Director: Technical Coordinator: Reporting: Head of RCC Quality Assurance: Mrs. Alessandra Tognucci Dr. Ute Schmiedel Mrs. Manuela Heckel, Mr. Patrick Schanie[ Mrs. Doris Leiminer Mrs. Iris WLithrich
SCHEDULE Experimental starting date: Experimental completion date: Study completion date: January27,2003 June 03, 2003 July16,2003/sds
ARCHIVING RCC Ltd, CH-4452 Itingen/Swltzerland will retain the study plan, raw data, a sample of test item and the final report of the present study for at least ten years. No data will be discarded without the sponsor’s consent.



### 76RCC StudyNumber 845449 REPORT Page 9

TKA 40278 (CGI 827) 

## DATA REQUIREMENT / TEST GUIDELINES
This study followed the procedures indicated by the following internationally accepted guidelines and recommendations: OECD Guideline for Testing of ChemicalsNo. 111,"Hydrolysis as a Function of pH"; adopted May 12, 1981. (only at pH 4.0, pH 7.0 and pH 9.0) EEC Directive 92/69, Section C.7, "Abiotic Degradation: Hydrolysis as a Function of pH", L383 A, December 1992,



## SUMMARY OF STUDY PLAN AMENDMENTS
First amendment to study plan;
1)Concerning: Schedule Alteration: Reason: Change of experimental starting, completion date and date of draft

report - Delay in study - Technical reason 2)Concerning: Alteration: Reason: Test Item

Purity Information given by the sponsor



### 77RCC Study Number 845449 REPORT Page 10

TKA 40278 (CGI 827) 

# SUMMARY
The hydrolysis determination of TKA 40278 (CGI 827) at different pH values was based on the OECD Guideline No. 111, "Hydrolysis as a Function of pH"; adopted May 12, 1981 and on the EEC Directive 92/69, Section C.7, "Abiotic Degradation: Hydrolysis as a Function of pH", L383 A, December 1992. The results of pH 4.0, pH 7.0 and pH 9.0 showed no significant degradation of TKA 40278 (CGI 827) at 50°C. The degradation of TKA 40278 (CGI 827) was less than 10 % after 5 days. These results were summarised in the table below.



### pH Hydrolysis after 5 days, 50°C



### [%] 4.0 <1o 7.o <1o 9.0 <1o According to the EEC Directive 92/69, Section C.7 it can be concluded, that the estimated half-life time is higher than one year under representative environmental conditions (25°C). Therefore, TKA 40278 (CGI 827) was considered to be hydrolytically stable, and no further testing was necessary.



### 78RCC Study Number 845449 REPORT Page 11

TKA 40278 (OGI 827) 

# 1 PURPOSE The purpose of this study was to determine the hydrolysis behavlour of TKA 40278 (CGI 827) in aqueous buffered solutions adjusted to pH 4.0, pH 7.0 and pH 9.0. Hydrolysis is one of the most common reactions controlling abiotic degradation in the environment. This reaction is particularly relevant for substances with low biodegradability.


## 2 MATERIAL AND METHODS 2.1 TEST ITEM Data as provided by the sponsor. Identity: Product Name: Batch: Purity: Appearance: Expiration date: Storage: TKA 40278 CGI 827 KL 01/02 94.1% Solid, slightly yellow August 30, 2004 At room temperature at about 20 °C. The test item is light sensitive.



### 79RCO Study Number 845449 REPORT Page12

TKA 40278 (CG1827) 

### 2.2 REAGENTS Buffer pH 4, Biphthalate Baker Buffer pH 7, Phosphate Baker Buffer pH 9, Borate/Potassium chloride/NaOH Baker Art. No. 5657 Art. No. 5656 Art. No. 7145 Water RCC from Milli-Q supply Acetonitrile Baker Art. No. 9017 Tetrahydrofuran Baker Art. Nr. 9441 The buffer solutions were sterilized for 25 minutes in an autoclave prior to first use. Nitrogen was passed through the buffer solutions for 15 minutes except when freshly sterilized.


### 2.3 EQUIPMENT Glassware: All glassware, which must be inert in the pH range applied, was rinsed with sterile buffer. The hydrolysis was carried out in flasks, which were stoppered or sealed with an inert material (e. g. PTFE). The test vessels were labelled with the following information: RCC study number and the additional information necessary to assure unmistakable identification. Water Baths: Thermostatically controlled water bath Haake SWB 25



### 80RCC Study Number 845449 REPORT Page 13

TKA 40278 (CG1827) 

### 2.4 STUDY DESIGN 2.4.1 Principle of the Test The study was conducted as described in the guidelines and in accordance with the RCC Ltd Standard Operating Procedures. The test item was dissolved in the buffer solutions and incubated at 50°C with the water bath kept constant at ± 0.1°C, usually. The concentration of the test item was determined as a function of time at each pH. 

### 2.4.2 Performance of the Test Three preliminary tests were performed at 50°C ± 0.5°C at each of pH 4.0, pH 7.0 and pH 9.0. Aliquots of each test solution were analysed in time intervals using the analytical method as described later in this report. Analysis was performed before incubation, after 2.4 hours and after 120 hours. The first and the second test were performed under normal daylight. Probably due to the light sensitivity of the test item, the content of test item found in the sample solutions showed high differences. Therefore, a third preliminary test was performed under red-light. The first and the second preliminary test are not reported but part of the raw data. A main study was not performed, as the test item was found to be stable according to the criteria given in the guideline. 

### 2.4,3 Preparation of the Test Solutions (3rd

### test) pH 4.0 13.96 mg of TKA 40278 (CGI 827) were dissolved in 100 mt buffer solution (pH 4.0) containing 2 % acetonitrile as solubilizer. This mixture was ultrasonified for 10 minutes and submitted to a 0.2 pm filtration. To obtain a test solution of not more than half the water solubility, the solution was diluted 1:1 with the respective buffer. Two allquots of this test solution of approximately 50 ml each were transferred into 50 ml Erienmeyer flasks in order to perform a duplicate test. pH 7.0 13.93 mg of TKA 40278 (CGI 827) were dissolved in 100 ml buffer solution (pH 7.0) containing 2 % acetonitrile as solubilizer. This mixture was ultrasonified for 10 minutes and submitted to a 0.2 pm filtration. To obtain a test solution of not more than half the water solubility, the solution was diluted 1:1 with the respective buffer. Two aliquots of this test solution of approximately 50 ml each were transferred into 50 ml Erlenmeyer flasks in order to perform a duplicate test.



### 81RCC Study Number 845449 REPORT Page 14

TKA 40278 (CG1827) pH 9.0 13.45 mg of TKA 40278 (CGI 827) were dissolved in 100 ml buffer solution (pH 9.0) containing 2 % acetonitrile as solubilizer, This mixture was ultrasonified for 10 minutes and submitted to a 0.2 pm filtration. To obtain a test solution of not more than half the water solubility, the solution was diluted 1:1 with the respective buffer. Two aliquots of this test solution of approximately 50 ml each were transferred into 50 ml Erlenmeyer flasks in order to perform a duplicate test.


### 2.5 ANALYTICAL METHOD 2.5.1 Standard Solutions Analytical standards were prepared from stock solutions created by combining the test item with appropriate solvent. 31.19 mg of TKA 40278 (CGI 827) were dissolved in 100 ml of a mixture of acetonitdle/water (50:50, v:v) to prepare a stock solution of 311.9 pg/ml TKA 40278 (CGI 827). Calculated volumes of this stock solution were diluted with the same mixture to obtain six standard solutions in the range from 4.679 pg/ml to 109.165 ,ug/ml TKA 40278 (CGI 827). These solutions were used to calibrate the HPLC-system. A second stock solution was prepared: 31.31 mg test item were weighed and diluted to obtain three solutions with concentrations in the range from 9.393 pg/ml to 78.275 pg/ml TKA 40278 (CGI 827). These solutions were measured in respective to the calibration curve to verify the HPLC-system during the test. Sample Preparation After incubation 5 pl aliquots of the test solutions at each pH value were analysed without dilution by measuring the UV/VIS signal of TKA 40278 (CGI 827) after HPLC separation of the injected sample solution.



### 82RCC Study Number 845449 REPORT Page 15

TKA 40278 (CGI 827) 

### 2.5.2 HPLC-Conditions Apparatus: Merck Workstation D-7000 Merck-Hitachi Pump L-7100 Merck-Hitachi Autoaampler L-7200 Merck-Hitachi Detector DAD L-7450 Merck-Hitachi Column Oven L-7300 Column: LiChrosphere RP 18e, 5,um, 250 mm  ̄ 4 mm Mobile Phase: Eluent A: Water Eluent B: Acetonitrile Eluent C: Tetrahydrofuran Gradient: Time A B C 0 - 3 min 

### 7O % 15% 15% 3 - 20 

### o% 85 % 15% 20 - 25 min 0% 

### 85 % 15% 25 - 26 m]n 70 % 15% 15% 26 -35 min 70 % 15 % 15 % Detection: 260 nm Temperature: Room temperature ( 23 °C) Injection Volume: 5 Flow: 0.7 ml/min The retention time of TKA 40278 (CGI 827) was at about 14.5 minutes.



### 83RCC Study Number 845449 REPORT Page 16

TKA 40278 (CGI 827) 

### 2.6 EVALUATION OF RESULTS Injected samples were quantified by peak area with reference to the calibration curve. The latter was obtained by correlation of the peak area of the analytical standards with their corresponding concentration. The correlation was performed using a least squares fit of the function given below. From this curve the concentration x of TKA 40278 (CGI 827) was calculated by the following equation: Y=b.x+a ~ x=(Y-a)/b where Y = peak area of injected sample solutions (counts)

x = concentration of injected sample solution (,ug/ml) b = slope a = y-axis intercept



### 84RCC Study Number 845449 REPORT Page 17

TKA 40278 (CGI 827) 

## 3 RESULTS The hydrolysis determination of TKA 40278 (CGI 827) at different pH values was based on the OECD Guideline No. 111, "Hydrolysis as a Function of pH"; adopted May 12, 1981 and on the EEC Directive 92/69, Section C.7, "Abiotic Degradation: Hydrolysis as a Function of pH", L383 A, December 1992. The results of the individual measurements are summarised in the attached table. The tabulated values represent rounded results obtained by calculation used the exact raw data, The results of pH 4.0, pH 7.0 and pH 9.0 showed no significant degradation of TKA 40278 (CGI 827) at 50°C. The degradation of TKA 40278 (CGI 827) was less than 10 % after 5 days. According to the EEC Directive 92/69, Section C.7, it can be concluded, that the estimated half-life time is higher than one year under representative environmental conditions (25°C). Therefore, TKA 40278 (CGI 827) was considered to be hydrolytically stable, and no further testing was necessary. A typical chromatogram of a standard solution is given in Figure 1. Typical chromatograms of the test’solutions after an incubation time of 5 days are given in the Figures 2 to 4. The linearity of the analytical method was proved in the concentration range from 4.679 pg/ml to 109.165pg/mLAn example of a calibration curve of TKA 40278 (CGI 827) is shown in Figure 5. The calibration data are given in Table 2.



### 85RCC Study Number 845449 REPORT Page 18

TKA 40278 (CGI 827) Table 1: Results of the Preliminary Test of TKA 40278 (CGI 827)

Initial Concentration Concentration concentration measured after measured after measured 2.4 hours 5 days

incubation incubation [,ug/ml] [,ug/ml] 20.47 pH 4.0 19.38 20.99

19.67 17.92 21.38 10.79 11.18 pH 7.0 10.29

10.68 10.2.7 11.80 20.36 20.87 pH 9.0 19.51

19,36 20.59 19.73 Based on the measured concentrations it can be stated, that the test item is stable under the conditions of the test.



### 86RCC Study Number 845449 REPORT Page 19

TKA 40278 (CGI 827) Figure 1: Typical HPLC chromatogramof a standard solution of TKA 40278 (CGI 827)

(18,714 pg/ml)

D-7000 HSM: 845449 Series: 0005 Report: modified System: Sys 1



### D-7000 HPLC System Manager Report Analyzed: 03.06.03 13:32 Reported: 04.06.03 07:20 Data Path: C:\Win32App\HSM\845449\DATA\0005\ Processing Method: TKA 40278A System(acquisition): Sys 1 Application: 845449 S~le Name: E 4 18.714 Injection from this vial: 1 of 1 Sample Description: Processed: 04.06.03 07:19
Serles:0005 Vial Number: 3 Vial Type: UNK Volume: 5,0 ul 

### 845&45

Chrom Type: Fixed WL Chromatogram, 260 nm

Acquisition Method: TKA 40278A Colu~m Type: kelne Pump A Type: L-7100 Solvent A: Solvent C: Method Description: Discovery C 18 Developed by: FRJ Solvent B:
ChromType: FixedWL Chromatogram, 260 nm Peak Quantitation: AREA Calculation Method: AREA% Me. RT Name Area : Area % 1 14,49 859330 i00,000 859330 i00,000 Peak rejection level: i000



### 87RCC Study Number 845449 REPORT

TKA 40278 (¢GI 827) Page 20 Figure 2: Typical HPLC chromatogram of a sample solution at pH 4.0

Conditions: 120 hours in aqueous buffered solution pH 4.0 at 50°C

D.7000 HSM: 845449 Scdes: 0004 Report: modified Sys~m: SFs



### D-7000 IIPLC

### SystemManager Report Analyzed: 27.05.03 20:32 Reported: 02.06.03 14:21 Data Path: C:\Win32App\MSM\845449\DATA\0004\ Processing Method: T~A 40278A System(acquisition) : Sys 1 Application: 845449 Sample Name: p 19-4 120h Injection from this vlal: 1 ~f 1 Sample Description: Processed: 02.06.03 14:21
Series:0004

Chrom Type: Fixed WL Chromatogram, 260 nm

2,5 -

Acquisition Method: TKA 40278A Coloa%nType: kelne Pump A Type: L-7100 Solvent A: Solvent C: Method D~scription: Discovery C 18 Developed byt FRJ Solvent B: Chrom %~ype: Fixed WL Chromatogram, 260 nm Peak Quantitation: AREA Calculation Method: AREA% No. RT Name Area Area % 1 14,50 950150  ̄ 100,000 950150 100,000 Peak rejection level: i000



### 88RCC Study Number 845449 REPORT

TKA 40278 (CG1827) Page 21 Figure 3: Typical HPLC chromatogram of a sample solution at pH 7.0

Conditions: 120 hours in aqueous buffered solution pH 7.0 at 50°C

D-7000 HSM: g45449 Sed~s: 0004 RepoM: modified System: s~ 1



### D-7000 HPLC System Manager Report Analyzed: 27.05,03 22:22 Reported: 02.06.03 11:34 Processed: 02.06.03 ii:33 Data Path: C: \WIn32App\MSM\845449\DATA\0004\ Processing Method: TKA 40278A System(acquisitlon) : Sys 1 Series:0004 Application: 845449 Vial Number: 9 Sample Name: p 21-7 120h Vial Type: DNK Injection from this vlal: i ~f i Volume: 5,0 ul Sample Description:Chrom Type: Fixed WL Chromatogram, 260 run

Developed by: FRJ Acquisition Method: TKA 40278A Column Type: keine Pump A Type: L-7100 Solvent A: Solvent C: Method Description: Discovery C 18 Solvent B: Cb/om Type: Fixed WL Chromatogram, 260 nm Peak Quantitation: AREA Calculation Method: AREA% NO. RT Name Area Area % 1 14,49 455520 100,000 455520 i00,000 Peak rejection level : 1000



### 89RCC Study Number 845449 REPORT

TKA 40278 (CGI 827) Page 22 Figure 4: Typical HPLC chromatogram of a sample solution at pH 9.0

Conditions: 120 hours in aqueous buffered solution pH 9.0 at 50°C

D-7000 HSM: 845~49 Series: 0~04 Report: modified



### D-7000 HPLC System Manager Report Analyzed: 28.05.03 00:49 Reported: 02.06.03 ii:34 Data Path: C:\WIn32App\HSM\845449\DATA\0004\ Processed: 02.06.03 11:33 Processing Method: TKA 40278A System(acq~isitlon): Sys 1 Application: 845449 Sample Name: p 24-9 120h Injection from this vial: 1 of 1 Sample Description: Series:0004 Volume: 5,0 ul Chrom Type: Fixed WL Chromatogram, 260 nm

0,20

Acquisition Method: TKA 40278A Column Type: kelne Pump A T~rpe: L-7100 Solvent A: Solvent C: Method Descriptlon: Discovery C 18 Developed by: FRJ Solvent B:
Chrom Type: Fixed WL Chromatogram, 260 rum Peak Quantltatlon: AREA Calculation Method: AREA% No. RT Ma~e Area Area % 1 14,48 891730 i00,000 891730 i00,000 Peak rejection level: I000



### 9ORCC Study Number 845449 REPORT

TKA 40278 (CG1827) Page 23 Figure 5: Example of a calibration curve of TKA 40278 (CGI 827)
Unear Regression

0 10 20 30 40 50 60 70 80 Concentration

Y=b.x+a with y = Area Counts

x = concentration (.ug/ml) b = 46213.3 (slope) a = -20027.6 (y-axis intercept) r2 = 0.9999 Table 2: The calibration data of TKA 40278 (CGI 827)

Concentration Area Counts [,ug/ml] Deviation in% 

## (x) (¥) 4.6785 214680 8.6 46.785 2125650 -0.8 10.9165 470860 -2,7 18.714 845955 0.1 77.975 3593820 0.3



### 91