## REPORT
Study Title: DETERMINATION OF THE FAT SOLUBILITY OF TKA 40278 (CGI 827)
Date Requirement I Test Guidelines: OECD No. 116 Directive 84/449 EEC, A.7 Study Director: Mrs. AIessandraTognuccl Study Completion Date: May19,2003 Test Facility: RCCLtd Environmental Chemistry & Pharmanalytics CH.4452 Itlngen/Switzerland Sponsor: CIBA SPECiAL’rY CHEMICALS INC. CH - 4002 Basel/Switzerland RCC Study Number: 845423 Page 1 ~18



### 113RCO Study Number 845423 REPORT Page 2

TKA 40278 (OG1827) 

## GLP CERTIFICATE

The Swiss GLP Monitoring Authorities



# Statement of GLP Compliance

It is hereby confinlled that dudng the pe,~cd of the following Test Facilities of 

### November18-22,

### 2002 RCC Ltd 4452 Itingen 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 w~th respect to the compliance with the Swiss legislation on Good Laboratory Practice. Test Facilities Areas of expertise *

-Toxicology

-Environmental Chemistry and Pharmanalytlcs 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 National GLP Inspections and Audits. It was found that the aforementioned test facilities were operating in compliance with the Swiss Ordinance ratatJng to Good Laboratory Practice [RS 813.016.5] at the time they were Inspected. Federal Office of Publlo Health The Director
Bsm, March 2003 Prof. Th. Zeltner



### 114RCC Study Number 845423 REPORT Page 8

TKA 40278 (CG1827) 

### GOOD LABORATORY PRACTICE 

## STATEMENT OF COMPLIANCE
RCC Study Number: Test Item: Study Director: Title: 845423 TKA 40278 (CGI 827) Mrs. Alessandra Tognucci Determination of the Fat Solubility of TKA 40278 (CGI 827) This study was conducted 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 26t~,1997 by decision of the OECD Council [C(97)186/Final].

Study Director: Mrs. Alessandra Tognucci



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TKA 40278 (CG1827) 

## SIGNATURES

Study Director: ~dra Tognucci

Management: Dr. JSrn SchreitmOller



### 116RCC Study Number 845423 REPORT Page 5

TKA 40278 (CG1827) 

## QUALITY ASSURANCE UNIT RCC Ltd, Environmental Chemistry & Pharmanalytics, CH-4452 Itingen / Switzerland
STATEMENT RCC Study Number: Test Item: Study Director: Title: 845423 TKA 40278 (CGI 827) Mrs. Alessandra Tognucci Determination of the Fat Solubility of TKA 40278 (CGI 827) The general facilities and activitiesare 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 June 19, 2002 Study Plan June 19,2002 March 18, 2003 Process based (Sample March 18, 2003

preparation / incubation) May 06 ÷ 07, 2003 Final report May 07,2003 This statement also confirms that this final report reflects the raw data. QualityAssumnce: ,~Mrs. Margot Richter-Auer



### 117RCO Study Number 845423 REPORT Page 6

TKA 40278 (CG1827) 

## 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:



### 118RCC Study Number 845423 REPORT Page 7

TKA 40278 (CG1827) 

## 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 1 PURPOSE

............................................................................................................ 10 2 MATERIAL AND METHODS

................................................................................ 10 2.1 TEST ITEM ................................................................................................. 10 2.2 TESTSYSTEM ........................................................................................... 10 2.3 REAGENTS ................ ~ ............................................................................... 10 2.4 STUDY DESIGN ......................................................................................... 11 2.4.1 Principle ................... : ..................................................................... 11 2.4.2 Performance of the Preliminary Test ............................................. 11 2.4.3 Performance of the Maln Test ....................................................... 11 2.5 ANALYTICAL METHOD .............................................................................. 12 2,6 DATA HANDLING ....................................................................................... 13 3 RESULTS ............................................................................................................. 14 Table 1: Results obtained for the solubility of TKA 40278 (CGI 827) in standard

fat

...................................................................................................... 15 Figure 1: Typical HPLC chromatogram of a standard solution of TKA 40278 (CGI

827) (93.66 pg/ml) ...................................................................................... 16 Figure 2: HPLC chromatogram of a sample solution of TKA 40278 (CGI 827) ........... 17 Figure 3: The calibration curve of TKA 40278 (CGI 827) ............................................ 18 Table 2: The calibration data of TKA 40278 (CGI 827) ........................... i .................

18



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TKA 40278 (CG1827) 

## PREFACE
GENERAL Title: Sponsor: Monitoring Scientist: Test Facility: Determination of the Fat Solubility of TKA 40278 (CGI 827) ClBA SPECIALTY CHEMICALS INC. CH-4002 BaseVSwitzerland Dr. Moung Sook Lee RCC Ltd Environmental Chemistry & Pharmanalytics Zelgliweg 1 CH-4452 Itingen/Switzerland

RESPONSIBILITIES Study Director: Deputy Study Director: Technical Coordinator: Head of RCC Quality Assurance: Mrs. Alessandra Tognucci Dr. Ute Schmiedel Mrs. Jos~e Gustaw, Mrs. Barbara Fl~ckiger Mrs. Iris W~thrich

SCHEDULE Experimental starting date: Experimental completion date: Study completion date: February 06,2003 April05,2003 May19,2003

ARCHIVING RCC Ltd, CH-4452 Itingen/Switzerland 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.



### 120RCC Study Number 845423 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. 116: "Fat solubility of solid and liquid substances"; adopted May 12, 1981. EEC Directive 84/449, A.7: "Fat solubility", L251, September 1984.



## 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



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TKA 40278 (CGI 827) 

## 1 PURPOSE The purpose of the study was to determine the fat solubility of the test item at 37°C, which is one of the relevant data for evaluating the possible accumulation of chemical substances in biological tissue, 

## 2 MATERIAL AND METHODS
2.1 TEST ITEM Data of test item as supplied by the sponsor. Identity: Product Name: Batch: Purity: Appearance: Expiration date: Storage: TKA 40278 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. 

### 2.2 TEST SYSTEM A commercially available standard fat was used Identity: HB 307 Supplier: Natec, Hamburg Batch: 36 Certificate of Analysis dated: 30.10.2000 Storage at RCC: Room temperature 

### 2.3 REAGENTS Water RCC Acetone Baker Acetonitdle Baker Tetrahydrofurane Baker Filters (0.45 pm, Nylon) BGB from Milli-Q water supply Art. No. 9254 Art. No. 9017 Art. No. 9441 Art. No. F2503-2



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TKA 40278 (CGI 827) 

### 2.4 STUDY DESIGN 2.4.1 Principle Usually, an excess amount of the test item is brought into contact with the liquefied standard fat, When saturation is achieved, the mass concentration of the test item in the standard fat is determined by a suitable analytical method. In case the.test item is miscible with standard fat in a 1:1 ratio, a visual test on solubility (miscibility) in 3 different ratios is required only. The test vessels were labelled with the following information: RCC study number and the additional information necessary to assure unmistakable identification.
2.4.2 Performance of the Preliminary Test The miscibility of the test item and the fat was tested at 3 different ratios with the following weights taken. The fat was liquefied in a water bath at 37°C.
Test Item Liquefied Standard Fat Ratio Visual judgement 

### [g] [g] 10,01 10.04 1:1 Not dissolved 19.01 1,03 

### 1:20 Not dissolved 1.01 19.01 20:1 Not dissolved The samples were incubated for 66 hours in a water bath at 37°C and agitated. Afterwards their appearance was evaluated visually. As the test item was not dissolved, the main test was started. 

### 2.4.3 Performance of the Main Test Since the test item was not miscible with the standard fat in a 1:1 ratio a main test was performed. In the main test, eight glass flasks were filled with about 1.5 g of test item and about 20 g standard fat. The flasks were tightly sealed. One half group (group 1) was shaken at 30°C and the other half group (group 2) was shaken at 50°C for about 24 hours each. Afterwards, two flasks of both groups were Incubated at 37.0°C for about 5.5 hours. The other flasks were incubated for 24 hours at 37,0°C. The supernatant solutions were centrifuged ~2900 g for 5 minutes and filtrated (0.45pm). 100 ,ul of the saturated solution was diluted to 10 ml with acetone. The quantification was performed using a HPLC method as described below. Details of sample treatment are given in Table 1.



### 123RCC Study Number 845423 REPORT Page 12

TKA 40278 (CGI 827) 

### 2,5 ANALYTICAL METHOD The concentration of the test item in the diluted fat samples of the main test were analysed by HPLC. The test samples were quantified with reference to a calibration curve.
Standard Calibration Solution For example 31.22 mg of TKA 40278 (CGI 827) were dissolved in 100 ml tetrahydrofurane to prepare a stock solution of 312.2 ,ug/ml TKA 40278 (CGI 827). Calculated volumes of this stock solution were diluted with the tetrahydrofurane to obtain six standard solutions in the range from 6,244 pg/ml to 124.88 pg/ml TKA 40278 (CGI 827). These solutions were used to calibrate the HPLC-system. HPLC-Condltlons Apparatus:

Column: Mobile Phase: Varian Pump 9010 Varlan Autosampler 9!00 Varian Detector 9050 Luna C18, particle size 5pm, 150 mm. 4,6 mm Eluent A: Water Eluent B: Acetonitrile Eluent C: Tetrahydrofurane 0min 70%A 15%B 15%C 0- 3 rain 70%A 15%B 15%C 3-20 rain 0%A 85%B 15%C 20- 25 min 0%A 85%B 15%C Detection: 260 nm Temperature: Room temperature Injection Volume: 10 pl Flow: 1 mVmin The retention time of TKA 40278 (CGI 827) was at about 12.2 minutes.



### 124RCC Study Number 845423 REPORT Page 13

TKA 40278 (CG1827) 

### 2.6 DATA HANDLING Injected samples were quantified bypeakarea 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 square fit of the function given below. From this curve the concentration x of TKA 40278 (CGI 827) in the injected samples were calculated by the following equation:

Y=b.x+a ~ x=(Y-a)/b

C=f.x where Y= peak area of injected sample solutions

x= concentration of injected sample solution (pg/ml) b = slope a = y-axis intercept f = factor of dilution C = concentration of the test item after saturation (pg/ml)
The fat solubility of a test item is specified by the saturation mass fraction of the test item in standard fat at 37.0+0.5°C and is expressed in g/kg. Therefore, the determined value measured by HPLC in g/I was converted into g/kg using the density (0.91 g/cm3). The following relation ship exists between the solubility in g per 1000 g of solution (A’) and the solubility in g per 1000 g of solvent (A):
Fat solubility(A) - where 

### A’ *1000

(lOO0 - A’ ) in g/kg ( test item / standard fat )
A’ = concentration analysed in g/kg (test item / saturated standard fat )



### 125RCC Study Number 845423 REPORT Page 14

TKA 40278 (CG1827) 

## 3 RESULTS
The determination of the fat solubility of TKA 40278 (CGI 827) was performed according to the Directive 84/449/EEC, A.7 "Fat Solubility" (1984) and the OECD guideline No. 116, "Fat Solubility of Liquids and Solid Substances" (1981) using a simplified flask method. In a preliminary test the miscibility of the test item and the fat was tested. Since the test item was not miscible with the standard fat in a 1:1 ratio the main test was performed. In the main test the solubility of the test item in standard fat at 37°C with a preliminary equilibration time at 30°C respectively 50°C was tested. The concentrations found ranged from 10.7 g/kg to 11.8 g/kg for the different temperatures and incubation times. The detailed results are presented in Table 1. The tabulated values represent rounded mean 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 incubation is given in Figure 2. The calibration data are presented on page 18. In conclusion, the solubility of TKA 40278 (CGI 827) in standard fat at 37°C was determined to be: ’1’1.27g/kg :1:0.5 glkg The fat solubility was not corrected for the purity of the test item (as given under item 2.1).



### 126RCC Study Number 845423 REPORT Page 15

TKA 40278 (CG1827) Table 1: Results obtained for the solubility of TKA 40278 (CGI 827) in standard fat
Sample Incubation Total Weight Dilution Measured Concen- Mean Mean* Solubility* No conditions Weight of Fat Factor Concen- trationof in fat

of tration the Test Mixture item 

### (g) (mg/I) 

### (g/~) (g/kg) (g/kg) 

### (Old) (g) 30°C/24.6h 1 and 1.48 20.03 100 94.655 9.466 37°C/5.6h 30°C/24.6h 2 and 1.57 20.03 100 100.013 10.001 9.733 10.696 37°C/5.6h 30°C/24.6h 3 and 1.52 20.02 100 100.021 t0.002 37°C/24h 30°C/24,6h 4 and 1.51 20.19 100 99.320 9.932 9.967 10.953 37°C/24h 50°C/24.6h 5 and 1.57 20.19 100 107.338 10.734 37°C/5.6h 50°C/24.6h 6 and 1.49 20.t7 100 107.442 10.744 t0.739 11.801 37°C/5.6h 50°C/24.6h 7 and 1.49 20.18 100 101.072 10.107 37°C/24h 50°C/24.6h 8 and 1,49 20.15 100 101.649 10.165 10.136 11.147 37°C/24h +0.47 11.138
Calculated with the density of 0.91 kg/t of the standard fat, SD = Standard Deviation The following relation ship exists between the solubility in g per 1000 g of solution (A’) and the solubility in g per 1000 g of solvent (A = concentration analysed in g/kg):

A’ * 1000 Fat solubility (A) - (1000 - A’) in g/kg ( test item / standard fat )

11.147 * 1000 Fat solubility (A) = = ’11.27 g/kg (test item / standard fat)

(1000 - 11.147)



### 127RCC Study Number 845423 REPORT Page 16

TKA 40278 (CG1827) Figure 1: Typical HPLC chromatogram of a standard solution of TKA 40278 (CGI 827)

(93.66 pg!ml)

Dala File: c:\vlkmodule 03\845423\1021.run Channeh SarnpIe ID: Operator Injection Date: Injection Method: Run Time (min): Workstation: Instrument (lnj): I = 260 nm RESULTS

E 93.66 04,04.2003 t4:02:49 c:\v I~module 03\tka 40278.mth 34.988 WIN95 Varian Star # Operator (Cale): Gwj Calc Date: 04.04.2003 14:38:05 Times Calculated: 1 Calculation Method: e:\vl~module 03\tka 40278.ruth Instrument (Cale): Varlan Star #I Run Mode: Analysis Peak Measurement: Peak Area Calculatlon Type: Percent Calibration Level: NIA VeriEeaEon Tolerance: N/A rnAU

300-

200-



### k

j~

INo Timo (counts) Code (rain)



### 128RCCStudy Number 845428 REPORT Page 17

TKA 40278 (CG1827) Figure 2: HPLC chromatogram of a sample solution of TKA 40278 (CGI 827)

Data File: c:\vlLmodule 03\845423\1030.run Operator (Calc): Gwj Channel: I = 260 nm RESULTS Cale Date: 04.04.2003 20:43:47 Sample ID: Probe 7 Times Calculated: I Operator (In j): Gwj Calculation Method: c:\vl~module 03\tka 40278.ruth Injection Date: 04.04.2003 20:08:30 Instrument (Calc): Va~an Star #1 Injection Method: e:\vl~module 03\tka 40278.ruth Run Mode: Analysis Run Time (min): 34.982 Peak Measurement: Peak Area Workstation: WIN95 Calculation Type: Percent Instrument (Inj): Varlan Star #I Calibration Level: N/A Verification Tolerance: N/A



### ~ ~o t~ 4o ~5 ~o

Peak Peak Name Ret Peak Area Sep. Result No Time (counts) Code (rain) l TKA 40278 12.201 2446936 BE= 100.00 Totals 2446936 100.00



### 129RCC Study Number 845423 REPORT Page 18

TKA 40278 (CGI 827) Figure 3: The calibration curve of TKA 40278 (CGI 827)
Linear Regression

0 20 40 60 80 100 120 140 Concentration

Y=b.x+a with Y= peak area

x = concentration ~ug/ml) b = 23985 (slope) a = 22744.8 (y-axis intercept) r2 = 0.9999 Table 2: The calibration data of TKA 40278 (CGI 827)

Area Counts Concentration Deviation

[,ug/ml] in %  ̄ (Y) 

### (x) 170967 6,244 -1.0 311475 12.488 -3.6 778006 31.22 0.9 1525618 62.44 0.4 2284010 93.66 0.7 3003767 124.88 -0.5



### 130