# REPORT

DETERMINATION OF THE WATER SOLUBILITY OFTKA 40278 (CGI 827) Data Requirement / Test Guidelines: OECD No. 105 EEC Directive 92/69, A.6 Study Director: Mrs. Alessandra Tognucci Study Completion Date: April 11, 2003 Test Facility: RCCLtd Environmental Chemistry & Pharmanalytics CH- 4452 Itingen/Switzerland

ClBA SPECIALTY CHEMICALS INC, CH - 4002 Basel/Switzeriand RCC Study Number: 845422 Page 1 of 19



### 534RCC Study Number 845422 REPORT Page 2

TKA 40278 (CGI 827) 

# GLP CERTIFICATE

The Swiss GLP Monitoring Authorities


# Statement of GLP Compliance

It is hereby confirmed that during the period of the following Test Facilities of November 18 - 22, 2002 RCC Ltd 4452Itingen Switzerland
were Inspected by the Federal Office of Public 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 Pharmanalytles Tax, 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 National GLP Inspections and Audits. It was found that the aforementioned test facilities were operating in compliance wlth the Swiss Ordinance relating to Good Laboratory Practice [RS 813.016.5] at the time they were Inspected. FederalOffice of Public Health The Director

Bern, March 2003 Prof. Th. Zelther



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TKA 40278 (CG1827) 

### GOOD LABORATORY PRACTICE 

# STATEMENTOFCOMPLIANCE
RCC Study Number: Test Item: Study Director: Title: 845422 TKA 40278 (CGI 827) Mrs. Alessandra Tognucci Determination of the Water Solubility of TKA 40278 (CGI 827) This study was cbnducted in compliance with the Swiss Ordinance relating to Good Laboratory Practice, adopted February 2n~,2000 [RS 813.016.5]. This Ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26~,1997 by decision of the OECD Council [C(97) 186/Final].

Study Director:

Date: Tognucci



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TKA 40278 (CG1827) 

# SIGNATURES

Study Director: Mrs.

Management: 

### Dr. JOrn SchreitmOller

537RCC Study Number 845422 REPORT Page 5

TKA 40278 (CGt 827) 

# QUALITY ASSURANCE UNIT RC(~’ Ltd, Environmental Chemistry & Pharmanalytics, CH-4452 Itingen / Switzerland
STATEMENT RCC Study Number: 845422 Test Item: TKA 40278 (CGI 827) 

### Study Director: Title: Mrs. Alessandra Tognucci Determination of the Water Solubility of TKA 40278 (CGI 827) 

### 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 tothe Management 

### June 19, 2002 Study Plan June 19, 2002 

### March 18, 2003 Process based, sample March 18, 2003



### preparation April 10, 2003 Final report April10,2003 This statement also confirms that this final report reflects the raw data. Quality Assurance: 

### Mr. Henry Schneylin

538RCC Study Number 845422 REPORT Page 6

TKA 40278 (CG182"7) 

# 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:



### 539RCC Study Number 845422 REPORT Page 7

TKA 40278 (CG1827) 

# CONTENTS

1PURPOSE ...........................................................................................................  ̄ 2 MATERIALAND METHODS ................................................................................ GLP CERTIFICATE ........................................................................................................ 2 STATEMENT OF COMPLIANCE..: ................................................................................. 3 SIGNATURES ................................................................................................................ 4 QUALI’I~y ASSURANCE UNIT ........................................................................................ 5 CERTIFICATION OF GLP AND VERIFICATION OF THEREPORT .............................. 6 CONTENTS ..............................................................................  ̄ .....................................

7 PREFACE

................................................... ! ................................................................ 8 DATA REQUIREMENT / TEST GUIDELINES ...................... . ......................................... 9 SUMMARY OF STUDY PLAN AMENDMENTS ..................................................... :: .......9 10 

### 10 2,1 TEST ITEM ................................................................................................. 10 2.2 REAGENTS AND APPARATUS ................................................................. 10 2.3 STUDY DESIGN ......................................................................................... 11 2.3.1 Principle ........................................................................................ 11 2.3.2 Preliminary Test ............................................................................ 11 2.3.3 Performance of the Main Test ....................................................... 11 2.4 ANALYTICAL METHOD ............................................................................. 12 2.5 DATA HANDLING ...... ................................................................................ 13 3 Table 1 : Table 2: Figure t: Figure 2: Figure 3: Figure 4: Table 3: 

### RESULTS

............................................................................................................. 14 Data of the performance of the water solubility test .................................... 15 Results of the water solubility of TKA 40278 (CGI 827) ................... ~ ..........15 Typical HPLC chromatogram of a standard solution of TKA40278 16 (CGI 827) (7.788 ~Jg/ml) ............................................................................. Typical HPLC chromatogram of a sample solution of TKA40278 17 (CGI 827) (72 hours) .....................................................................: ............ Typical HPLC chromatogram of a blank solution ........................................ 18 Calibration curve of TKA 40278 (CGI 827) .................................................19 The calibration data of TKA 40278 (CGI 827) ............................................. 19


### 540RCC Study Number 845422 REPORT Page8

3-KA 402"78 (CGI 827) 

# PREFACE
GENERAL Titlei Sponsor: Monitoring Scientist: Test Facility: Determination of the Water Solubility of TKA 40278 (CGI 827) 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 Coordinators: Reporting: Head of RCC Quality Assurance: Mrs. Alessandra Tognucci Dr. Ute Schmiedel Mr. Patrick Schaniel, Mr. Christoph Wild Mrs. Mirjam Weissenfeld Mrs. Iris W~thrich
SCHEDULE Experimental starting date: Experimental completion date: Study completion date: January 23, 2003 February 15, 2003 April 11, 2003/waj



### ARCHIVING RCC Ltd, CH-4452 Itingen/Switzeriand 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.



### 541RCC Study Number 845422 REPORT ,. Page 9

TKA 40278 (CG1827) 

# DATA REQUIREMENT / TEST GUIDELINES
This study followed the procedures indicated by the following internationally accepted guidelines and recommendations: OECD Guideline for Testing of Chemicals, No. 105, "Water solubility"; adopted July 27, 1995. EEC Directive 92/69, Part A, Methods for the determination of physico-chemical properties, A.6: "Water solubility", EEC Publication No. L383, December 1992.



# SUMMARY OF STUDY PLAN AMENDMENTS
First Amendment to Study Plan: 1) Concerning: Schedule Alteration: New experimental starting and completion date Reason for Alteration: Delay in study 

### 2) Concerning: 2.1 Test Item Alteration: Purity Reason for Alteration: New information given by the sponsor

542RCC Study Number 845422 REPORT Page 10

TKA 40278 (CG1827) 

# 1 PURPOSE The purpose of this study was to evaluate the solubility of TKA 40278 (CG1827) in water which is’ specified by the saturation mass concentration of the test item in water at a given teml~erature.


# 2 MATERIAL AND METHODS
2.1 TEST ITEM Data of test item as supplied by the sponsor. Identity: TKA 40278 Product Name: Batch: Purity: Appearance: Expiration date: Storage: CGI 827 KL 01/02 94.1% (see analytical report of Ciba Specialty Chemicals Inc., Study No. 14026512-1) Solid, slightly yellow August 30, 2004 At room temperature at about 20 °C. 

### The test item is light sensitive. Therefore, all test item handling was performed under red light. The solutions were stored in the dark and in amber coloured glass ware.



### 2.2 REAGENTS AND APPARATUS Water Acetonitrite Tetrahydrofurane (THF) Filter Nylon (0.2 pro) Milli-Q Supply RCC Baker Art. No, 9017 Baker Art. No. 9441 BGB Analytik AG Art. No. F2503-1 Centrifuge Water Bath Balances HPLC Heraeus HAAKE Mettler Toledo as specified below Megafuge

SWB 25 AT 261, PM 6100



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TKA 40278 (CGI 827) 

### 2.3 STUDY DESIGN ..... 2.3.1 Principle An excess amount o~the test item is~rought into oontact with water. When saturation is achieved, the mass concentration of the test item in the aqueous solution is determined by a suitable analytical method. A preliminary test is used to determine the appropriate method and the approximate amount of test item to be used in the main study, as well as the time necessary to achieve saturation. The test vessels were labelled with the following information: RCC study number and.the additional information necessary to assure unmistakable identification.


### 2.3.2 Preliminary Test A preliminary test using a simplified flask method with abbreviated equilibration timeswas performed to evaluate the magnitude of the water solubility of the test item. Details and results are not reported here, but are part of the raw data of the present study. As the water solubility was determined to be about 27 mg/I, the flask shaking method was usedffo~onduct the main test. 

### 2.3.3 Performance ofthe Main Test Test Solutions About 5 g of test item was weighed into each of 6 Edenmeyer flasks and 25 ml of water were added to each flask. The tightly closed flasks were shaken at about 30°C for 24, 48 and 72 hours, respectively. Afterwards, the flasks were equilibrated for another 24 hours at 20°C. The supernatant solutions were centrifuged (~2900 g) and filtrated (0.2 pm) prior to analysis. The quantification was performed using a HPLC method as described below. Details of sample treatment are given in Table 1.



### 544RCC Study Number 845422 REPORT Page 12

"rKA 40278 (CG1827) 

### 2.4 ANALYTICAL METHOD The concentration of the test item was analysed using HPLC. The test samples were quar~ified with reference to the calibration curves. Standard Calibration Solution A 38.94 mg sample of TKA 40278 (CGI 827) was dissolved in 50 ml acetonitrile and diluted to 100ml with water to prepare a stock solution of 389.4pg/ml TKA40278 (CGI827). Calculated volumes of this stock solution were diluted with a mixture of acetonitrile and water (1:1; v/v) to obtain seven standard solutions in the range from 0.7788 pg/ml to 116.82 pg/ml TKA 40278 (CGI 827). These solutions were used to calibrate the HPLC-system. A second stock solution of 381.9 IJg/ml TKA 40278 (CGI 827) was prepared by weighing 38.19 mg test item, dissolving in 50 ml acetonitrile and diluting to 100 ml with water. After respective dilution, a second set of standard solutions was obtained with concentrations in the range from 0.7638 IJg/ml to 114.57 IJg/ml TKA40278 (CGI 827). These solutions were measured in respective to the calibration curve to verify the HPLC-system durin~ the test. HPLC-Conditions Apparatus:
Column: Mobile Phase:

Detection: Temperature: Injection Volume: Flow: Varian autosampler 9095 Varian pump 9012 Varian detector ProStar 310 Lichrospher RP 18e, particle size 5 IJm, 250 mm  ̄ 4 mm Eluent A: Water Eluent B: Acetonitrile Eluent C: THF 0min 70%A 15%B 15%C 3min 70%A 15%B 15%C 20min 0%A 85%B 15%C 25 rain 0%A 85 % B 15 % C 26min 70%A 15%B 15%C 35rain 70%A 15%B 15%C 260 nm Room temperature 20 pl (gradient)



### 1.0 ml/min The retention time of TKA 40278 (CGI 827) was at about 17 minutes.



### 545RCC Study Number 845422 REPORT Page 13

TKA 40278 (CGI 827) 

### 2.5 DATA HANDLING Injected samples were quantified by peak area with refe, rence to the calibration curve. The lattet{was obtained by correlation of the peak area of the test item solutions with their corresponding concentration. The correlation was performed using the linear function given belo~v. From’this curve the concentration x of TKA 40278 (CGi 827) in the injected solutions was calculated by the following equation: Y=b.x+a ~> x=(Y-a)/b where Y = peak area of injected sample solutions

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

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TKA 40278 (CG1827) 

# 3 RESULTS
The determination of the water solubility of TKA 40278 (CGI 827) was performed according to the EEC Directive 92/69, A.6 "Water Solubility" (1992) and the OECD guideline No. 105, "Water Solubility" (1995) using the flask shake method. The saturation concentration of TKA 40278 (CGI 827) at room temperature was estimated by a simplified flask method in the preliminary test to be higher than 10-2g/I. Therefore, the flask shaking test was performed. The concentrations were determined after a shaking time of 24 hours, 48 hours and 72 hours, respectively at 30°C. Afterwards the samples were equilibrated for 24 hours at 20°C. The measured results differ by less than 15 %. Therefore the study is considered to be valid. The results of the individual measurements of the main test are summarised in Table 2. The tabulated values represent rounded results, which were obtained by calculation using the exact raw data. 

### A typical chromatogram of a standard solution is given in Figure 1. A typical chromatogram of a sample solution after 72 hours of incubation is given in Figure 2. A HPLC chromatogram of a blank solution is shown in Figure 3. The calibration data for test item-standards are given in Table 3. The r2fit was 0.9998 (optimum 1.0000). This reflects the tinearity of the HPLCsystem within the calibration range of 0.7788 t~g/ml- 116,82 pg/ml of the test item.
In conclusion, the water solubility of TKA 40278 (CGI 827) at 20°C was determined to be 9.9 mg/I (standard deviation+1.1 mg/l). The water solubility was not corrected for the purity of the test item (as given under item 2.1).



### 547RCC Study Number 845422 REPORT Page 15

TKA 40278 (CGI 827) 

### Table 1: Data of the performance of the water solubility test Sample solution no. 1 2 3 4 5 6 We~ghed sample of the 5.05. 5.31 5.56 5.97 

### test i~em (g) 5.07 5.21 Added volume of water 25 25 25 

### 25  ̄ 25 (ml) 25 Agitation time at 30°C 48 48 

### 24 24 (hours) 72 72 Equilibration time at 20°C 24 24 24 24 (hours) 24 24 

### pH value of test item 7.11 7.14 

### 7.14 ,7.12 

### solution 7.15 

### 7.14

, , 

### Dilution factor before 1 1 1 1 quantification 1

Table 2: Results of the water solubility of TKA 40278 (CGI 827)



### Sample No. Shaking ConcentrationWater solubility

time at analysed calculated 30°C [mg/I] [mg/I] 1 72 9.99 

### 2 10.39 

### 3 48 8.79 9.90 

### 4 10.87 SD =4-1.09 

### 5 24 8.38 

### 6 10.99 The saturation mass concentration is calculated from the bold values. 

### SD = standard deviation

548RCC Study Number 845422 REPORT Page 16

TKA 40278 (CGI 827) 

### Figuie 1: Typical HPLC chromatogram of a standard solution of TKA40278 (CGI 827)

(7.788 tJg/ml)

Data File: c:Wlhnodule 06~845422\140203\ 1020.ran Operator (Calc): Channel: 1 = 260 nm RESULTS Calc Date: 17.02.2003 14:44:32 Sample ID: e 2-5 7.788 Tim~s Calculated: 2 Operator (lnj): wic Calculation Method: c:\vl~nodul¢ 06~tka 40278.ruth Injection Date: 15.02,2003 02:56:36 Instrument (Cal¢): Varian Star #2 Injection Method: c:\vlkraodul¢ 06~tka 40278.ruth Run Mode: Analysis Run Time (rain): 34.983 Peak Measurement: Peak Area Workstation: WIN95 Calculation Type: Percent Instrument (Inj): Varian Star #2 Calibration Level: N/A Vedfication Tolerance: N!A

4c-I



### 11o 115 ~o ~ ~o Minula.~
Peak Peak Name Ret Peak Area Sep. Result (%) No Time (counts) Code (rain) i TKA 40278 17.233 591729 BB i00.00 Totals 591729 100.00



### 549RCC Study Number 845422 REPORT Page 17

TKA 40278 (CGI 827) 

### Figure 2: Typical HPLC chromatogram of a sample solution of TKA40278 (CGI 827)

(72 hours)

Data File: ¢:\vlhnodule 06k845422\140203\1014.run Operator(~Ic): w~c Channel: 1 = 260 nm RESULTS Calo Date: 18.02.2003 10:45:46 S~ple ~: s2-72h Tim~ Calculated: 4 O~mtor (hi): ~o Calculation M~od: c:~vl~odul¢ 0~tka 40278.ruth hj~tion Date: 14.02.2003 23:16:33 lnst~ment (Calo): V~ S~r #2 Inj~ion Method: cAvl~odul~ 06~tka 40278,ruth Run Mode: ~alysis Run Time (rain): 34.977 P~ M~urement: P~k ~ Wo~tion: ~95 ~Iculation T~ : Per~nt hs~ment (lnj): Vari~ S~ #2 ~libmtion ~v¢l: ~A Vefifi~tlon Toledo: ~A ~U

Peak Peak Name Ret Peak Area Sep. Result (%) No Time (counts) Code (rain) 1 TKA 40278 17.160 835861 BB 100.00 Totals 835861 100.00



### 550RCC Study Number 845422 REPORT Page 18

TKA 40278 (CGt 827) 

### Figu~:e 3: Typical HPLC chromatogram of a blank solution

Data File: c:\vl~module 06~845422\140203\1017.nan Channel: 1 = 260 nm RESULTS Sample ID: ACN-H20 1-1 Operator (Inj): Injection Date: 15.02.2003 01:06:3 Injection Method: e:\vl~aodule 06ktka 40278.ruth Run Time (rain): 34.978 Workstation: WIN95 lnslnament (Inj): Varlan Star #2 Operator (Cale): wie CaIe Date: 17.02.2003 14:40:01 Times Calculated: 2 Calculation Method: e:\v l~aodule 06ktka 40278.ruth Instrument (Cale): Varian Star #2 Run Mode: Analysis Peak Measurement: Peak Area Calculation Type: Percent Calibration Level: N/A Verification Tolerance: N/A rnAU

Peak Peak Name Ret Peak Area Sep. Result (%) No Time (counts) Code (rain) Totals 0 0.00



### 551RCC Study Number 845422 REPORT Page 19

TKA 40278 (CGI 827) 

### Figure 4: Calibration curve of TKA 40278 (CGI 827) Linear Regression

Y=b,x+a 

### with Y = peak area

x = concentration (tJg/ml) b = 80160.3459 (slope) a = 3373.4049 (y-axis intercept) r2 = 0.9998

Table 3: The calibration data of TKA 40278 (CGI 827)



### ,I Concentration Peak Area Deviation [IJg/ml] in % 

## (x) (Y) 0.7788 55871 -15.9 1.5576 125132 -2,5 7.788 591729 -5.8 15.576 1231948 -1.6 38.94 3190144 2.1 77.88 6306997 1.0 116.82 9310609 -0.6



### 552