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:

RCC Ltd

Environmental Chemistry & Pharmanalytics

CH.4452 Itlngen/Switzerland

Sponsor:

CIBA SPECiAL’rY CHEMICALS INC.

CH - 4002 Basel/Switzerland

RCC Study Number:

845423

Page 1 ~18

113
RCO 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

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 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

114
RCC 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

115
RCC Study Number 845423 REPORT Page 4

TKA 40278 (CG1827)

SIGNATURES

Study Director: ~dra Tognucci

Management: Dr. JSrn SchreitmOller

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RCC 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

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RCO 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:

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RCC 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

119
RCC Study Number 845423 REPORT Page 8

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.

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RCC 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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RCC Study Number 845423 REPORT Page 10

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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RCC Study Number 845423 REPORT Page 11

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 (0.45 pm).

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.

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RCC 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.

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RCC Study Number 845423 REPORT Page 13

TKA 40278 (CG1827)

2.6 DATA HANDLING

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 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 )

125
RCC 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:

’1’1.27 g/kg :1:0.5 glkg

The fat solubility was not corrected for the purity of the test item (as given under item 2.1).

126
RCC 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- tration of 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)

127
RCC 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-

j~ k

INo Timo (counts) Code

(rain)

128
RCC Study 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

129
RCC 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