REPORT

DETERMINATION OF THE WATER SOLUBILITY

OF TKA 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:

RCC Ltd

Environmental Chemistry & Pharmanalytics

CH- 4452 Itingen/Switzerland

ClBA SPECIALTY CHEMICALS INC,

CH - 4002 Basel/Switzeriand

RCC Study Number:

845422

Page 1 of 19

534
RCC 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

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 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.

Federal Office of Public Health

The Director

Bern, March 2003 Prof. Th. Zelther

535
RCC Study Number 845422 REPORT Page 3

TKA 40278 (CG1827)

GOOD LABORATORY PRACTICE

STATEMENT OF COMPLIANCE

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

536
RCC Study Number 845422 REPORT Page 4

TKA 40278 (CG1827)

SIGNATURES

Study Director: Mrs.

Management: Dr. JOrn SchreitmOller

537
RCC 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 to the 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

538
RCC 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:

539
RCC Study Number 845422 REPORT Page 7

TKA 40278 (CG1827)

CONTENTS

1 PURPOSE ...........................................................................................................

 ̄ 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 TKA4027817 (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

540
RCC Study Number 845422 REPORT Page 8

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.

541
RCC 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

542
RCC 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

543
RCC Study Number 845422 REPORT Page 11

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 times was

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.

544
RCC 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.

545
RCC 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

546
RCC Study Number 845422 REPORT Page 14

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 10-2 g/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 r2 fit 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).

547
RCC 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 Concentration Water 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

548
RCC 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

549
RCC 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 ~: s 2-72h 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

550
RCC 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

551
RCC 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