REPORT

DETERMINATION OF THE VAPOUR PRESSURE

OF TKA 40278 (CGI 827)

Data Reauirement / Test Guidelines:

OECD No. 104

Directive 92/69/EEC, A.4

Study Director:

Mrs. Alessandra Tognucci

Study Comoletlon Date:

June 04, 2003

Test Facility:

RCC Ltd

Environmental Chemistry & Pharmanalytics

CH-4452 Itingen/Switzedand

ClBA SPECIALTY CHEMICALS INC.

CH - 4002 Basel/Switzerland

RCC Study Number:

845420

Page 1 of 21

92
RCC Study Number 845420 REPORT Page 2 TKA 40278 (CG1827)

GLP CERTIFICATE

The Swlss GLP Monitoring Authorities

Statement of GLP Compliance

It is hereby confirmed that

dudng the pedod of

the following Test Facll~es 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 cornpllance with the Swiss legislation on Good Laboratory Practice.

Test Facllltlas Areas of expertise *

Toxicology

Environmental Chemistry and

Pharmanalytics

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 facJIIties were operating In

compliance with 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

93
RCC Study Number 845420 REPORT Page 3 TKA 40278 (CG1827)

GOOD LABORATORY PRACTICE

STATEMENT OF COMPLIANCE

RCC Study Number:

Test Item:

Study Director:

Title:

845420

TKA 40278 (CGI 827)

Mrs. Alessandra Tognucci

Determination of the Vapour Pressure of TKA 40278

(CGI 827)

This study, with exception of the model calculation was conducted in compliance with the

Swiss Ordinance relating to Good Laboratory Practice, adopted February 2

nd, 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

Date:

94
RCC Study Number 845420 REPORT Page 4

TI~ 40278 (CG1827)

SIGNATURES

Study Director: Mrs. Alessandra Tognucci

Date:

Management: Dr. JOrn SchreitmOller

95
RCC Study Number 845420 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:

845420

TKA 40278 (CGI 827)

Mrs. Alessandra Tognucci

Determination of the Vapour Pressure 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

January 28, 2003 Study Plan January28,2003

April 16, 2003 Process based Apd116,2003

(Analytical work)

June 02, 2003 Final report June 02, 2003

This statement also confirms that this final report reflects the raw data.

Quality Assurance: Mr. Henry Schneylin

Date:

96
RCC Study Number 845420 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:

845420

TKA 40278 (CGI 827)

Mrs. Alessandra Tognucci

Determination of the Vapour Pressure 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

January 28, 2003 Study Plan January 28,2003

April 16, 2003 Process based Apd116,2003

(Analytical work)

June 02, 2003 Final report June 02, 2003

This statement also confirms that this final report reflects the raw data.

Quality Assurance: Mr. Henry Schneylin

Date:

96
RCC Study Number 845420 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:

97
RCC Study Number 845420 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

SUMMARY ................................................................................................................... 10

1 PURPOSE ........................................................................................................... 11

2 MATERIAL AND METHODS ................................................................................ 11

2.1 TEST ITEM ................................................................................................. 11

2.2 REAGENTS AND APPARATUS ................................................................. 11

2.3 STUDY DESIGN ......................................................................................... 12

2.3.1 Model Calculation .......................................................................... 12

2,3.2 Principle ........................................................................................ 12

2.3.3 Trapping Efficiency Test ............................................................... 12

2,3.4 Performance of the Main Test ....................................................... 13

2.4 ANALYTICAL METHOD ............................................................................. 13

2.5 DATA HANDLING ...................................................................................... 14

3 RESULTS ............................................................................................................ 16

Table 1: Results of the gas saturation test ............................................................... 17

Figure t: Chromatogram of a calibration solution 0.0491 pg/ml ................................ 118

Figure 2: Chromatogram of sample 4 (at 60°C after 115.8 hours) ............................. 19

Figure 3: Chromatogram of a blank solution .............................................................. 20

Figure 3: An example of a calibration curve of TKA 40278 (CGI 827) ........................ 21

Table 2: An example of calibration data of TKA 40278 (CGI 827) ............................ 21

98
RCC Study Number 845420 REPORT Page 8

TKA 40278 (CG1827)

PREFACE

GENERAL

Title:

Sponsor:

Monitoring Scientist:

Test Facility:

Determination of the Vapour Pressure of TKA 40278

(CGI 827)

ClBA 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:

Head of RCC Quality Assurance:

Mrs. Alessandra Tognucci

Dr. Ute Schmiedel

Mrs. Christine Cadei, Mrs.

Patrick Schaniel

Mrs. Ids W(~thrich

Barbara Fl(~kiger, Mr.

SCHEDULE

Experimental starting date:

Experimental completion date:

Study completion date:

Apd109,2003

Apd124, 2003

June 04, 2003~nm

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.

99
RCC Study Number 845420 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. 104: "Vapour pressure", adopted July 27,1995

EEC Directive 92/69 A.4, Vapour pressure, Part: 1.4.6. Gas saturation method. EEC

Publication No. L383A, December 1992.

SUMMARY OF STUDY PLAN AMENDMENTS

First Amendment to Study Plan:

1) Concerning: Schedule

Alteration: New experimental starting date

Reason for Alteration: Delay in study

2) Concerning: 2.1 Test Item

Alteration: Purity

Reason for Alteration: New information given by the sponsor

100
RCC Study Number 845420 REPORT Page 10

TKA 40278 (CGI 827)

SUMMARY

The experimental results lead to the conclusion that the vapour pressure of the TKA 40278

(CGI 827) is:

1.7.10"= Pa at 50°C,

1.6.10"~ Pa at 60°C

and

1.6  ̄ 10"~ Pa at 70°C.

Based on the experimental results the vapour pressure of TKA 40278 (CGI 827) at 25°C is

expected to be

<< 1.6  ̄ t04 Pa at 25°C.

101
RCC Study Number 845420 REPORT Page 11

TKA 40278 (CG1827)

1 PURPOSE

The purpose of this study was to determine the vapour pressure of TKA 40278 (CGI 827) by

applying the gas saturation method.

2 MATERIAL AND METHODS

2.1 TEST ITEM

Data as provided by the sponsor.

Identity:

Product Name:

Batch:

Purity:

Molar mass:

Appearance:

Expiration date:

Storage:

TKA 40278

CGI 827

KL 01/02

94.1% (see analytical report of Ciba

Chemicals Inc., Study No. 14026512-1)

340.42 g/mol

Solid, slightly yellow

August 30, 2004

At room temperature at about 20 °C.

The test item is light sensitive.

Specialty

2.2 REAGENTS AND APPARATUS

Water RCC

Methanol Baker

Acetonitdle Baker

TENAX tubes

XAD-2 tubes

Glass Beads

Nitrogen

Balances

Digital flow meter

from Milli-Q water supply

Art. No. 8402

Art. No. 9017

SKC INC. Art. No. 226-35-03, Lot 2442

SKC INC. Art. No. 226-30-04, Lot 2455

Supelce Art. No. 59203

central supply, 99.99 %

Mettler PM 6100, AT 261

JRW Scientific ADM 3000

102
RCC Study Number 845420 REPORT Page 12

"rKA 40278 (CG1827)

2.3 STUDY DESIGN

2.3.1 Model Calculation

The model calculation was performed to decide which method should be used dudng the

main test. Several techniques are available to determine the vapour pressure of a test item

experimentally. These techniques are described in EEC guideline A.4 and in the OECD

guideline 104,

The value calculated, using the Modified Watson Correlation allows to choose the appropriate

method. In this case the gas saturation method was used. Details of the model calculation are

not given here but are part of the raw data.

2.3.2 Principle

The nitrogen flow from a central supply was split into three streams for each temperature,

The flow of each individual stream was regulated by needle valves to approx, 3 ml/min. Each

nitrogen stream flew through a 0.32 mm glass capillary, which lead into a thermostated oven

and through a septum to the bottom of a 20 ml glass vial, Two vials were filled with test item

adsorbed on glass beads. The third vial was empty and served as a control. The gas left the

vial through a capillary starting at the top of the vial. Outside the oven each gas stream

passed a separate TENAX adsorption tube. The adsorption tube of the control stream was

spiked with test item. Behind the adsorption tubes, the flow rate of the gas streams was

measured using a digital flow meter.

Depending on the expected vapour pressure the temperature of the oven and the duration of

the test are adjusted. This study was performed at 50, 60 and 70°C. After about 5 days the

adsorption tubes were eluted with a suitable solvent. The eluates were collected and

analysed using a suited quantification method.

By determining the amount of material transported by a known amount of carder gas the

vapour pressure at a given temperature can be calculated.

2.3.3 Trapping Efficiency Test

A trapping efficiency test was performed to select suitable adsorption tubes to collect the test

item and to select a suitable solvent for the elution of the test item from the tube.

The trapping efficiency was tested with TENAX and XAD-2 adsorption tubes. A spike solution

was prepared by dissolving 43.73 mg TKA 40278 (CGI 827) in 10 ml acetonitrile. 50 pl of this

solution were used as a spike solution and added by injection to the front part of the tube.

The tubes were dded and a stream of nitrogen flowed though the tubes for 30 minutes to test

the adsorption on each tube material. Afterwards the tubes were extracted with acetonitdle

and methanol, respectively. The tubes were eluted with 5 ml solvent and treated in the

ultrasonic bath for 15 minutes. Each extract was filled up to 10 ml with the respective solvent.

The solutions were analysed by HPLC to determine the trapping and elution efficiency.

Based on the results of the trapping efficiency test TENAX adsorption tubes and acetonitrile

as eluting solvent were used during the vapour pressure test.

103
RCC Study Number 845420 REPORT Page 13

TKA 40278 (CGI 827)

2.3.4 Performance of the Main Test

About 1.6 g of the test item were mixed on a Vortex mixer with about 14,1 g glass beads.

Two 20 ml headspace vials were filled with the coated glass breads. These two vials and one

empty control vial were placed in a temperature controlled oven. The two vials filled with the

test item mixture were each connected to a TENAX tube. The third empty vial was connected

to a TENAX tube, which was spiked with 219 pg test item using the stock solution. Afterwards

a stream of nitrogen passed through the apparatus. This test was performed at 50°C for

116.2 hours, at 60°C for 115.8 hours and at 70°C for 115.5 hours.

After completing the test the adsorption tubes were eluted with acetonitrile as described

under item 2.3.3. Each eluate was analysed using the analytical method as described later in

the report.

All test vessels were labelled with the following information: RCC study number and the

additional information necessary to assure unmistakable identification.

2.4 ANALYTICAL METHOD

Calibration Solution

A 32.71 mg sample of TKA 40278 (CGI 827) was dissolved in 100 ml acetonitdle / water (1:1;

v/v) to prepare a stock solution of 327.1 pg/ml TKA 40278 (CGI 827). Calculated volumes of

this stock solution were diluted with acetonitrile to obtain seven standard solutions in the

range from 0.0491 pg/ml to 32.71 pg/ml TKA 40278 (CGI 827). Solutions with concentrations

between 0.0491 pg/ml and 16.36 pg/ml were used as calibration solutions for samples and

blanks and solutions with concentrations between 2.617 pg/ml and 32.71 pg/ml were used as

calibration solutions for spiked solutions.

Using 33.64 mg, 31.30 mg and 30.59 mg respectively, stock solutions were prepared to

obtain test item solutions with a concentration between 0.626 pg/ml and 26.91 pg/ml TKA

40278 (CGI 827). These solutions were measured in respective to the calibration curve to

verify the HPLC-system dudng the test. The recovery of all samples was between 92 % and

100 %.

104
RCC Study Number 845420 REPORT Page 14

TKA 40278 (CG1827)

HPLC-Conditlons

Apparatus:

Column:

Mobile Phase:".,

Pump

Autosampler

Detector

Varian ProStar 230

Vadan ProStar 410

Varian ProStar 330

LiChrosphereRP 18e, part. size 5 pm, 250 mm, 4 mm

Eluent A: Milli-Q water

Eluent B: Acetonitrile

Eluent C: Tetrahydrofurane

0min 70%A 15%B 15%C

0- 3min 70%A 15%B 15%C

3-20 rain 0%A 85%B 15%C (gradient)

20-25min 0%A 85%B 15%C

25 - 26 min 70 % A 15 % B 15 % C (gradient)

26 - 35 min 70%A 15%B 15%C

Detection: 260 nm

Temperature: room temperature

Injection Volume: 50 pl

Flow: 1.0 ml/min

The retention time of TKA 40278 (CGI 827) was at about 12 minutes.

2.5 DATA HANDLING

The concentration of the test item in the eluted samples was interpolated from a calibration

curve, which was established by linear correlation of the peak area of the analytical standards

with their corresponding concentration. The correlation was performed using the linear

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

C=f.x

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 in the eluted samples (pg/ml)

The vapour pressure at a given temperature can be calculated by determining the amount of

material transported by a known amount of carrier gas.

105
RCC Study Number 845420 REPORT Page 15

TKA 40278 (CG1827)

Calculation of the vapour pressure

W’R’T

V.M

Pw = vapour pressure (Pa)

W = mass of evaporated test item (g)

R = gas constant

T = Temperature(K)

V = volume of nitrogen (m3)

M = molar mass of test item

calculated

determined by analysis

= 8.314 J/K mol

= measured

determined from flow rate

= 340.42 g/mol

Temperature dependence of vapour pressure:

In a limited temperature range, the logarithm of the vapour pressure of a pure item is a linear

function of the inverse of the thermodynamic temperature according to the simplified

Clapeyron-Clausius equation

log PvP = ~Hv/2.3  ̄ R. T + constant

PvP = vapour pressure (Pa)

,~Hv = heat of evaporisation (J  ̄ mol"~)

R = gas constant

T = temperature (K)

106
RCC Study Number 845420 REPORT Page 16

TKA 40278 (CG1827)

3 RESULTS

The vapour pressure of TKA 40278 (CGI 827) was determined according to the EEC

Directive 92/69, A.4 "Vapour Pressure" (1992) and the OECD guideline No. 104, "Vapour

Pressure" (1995) using the gas saturation method.

Based on the results of the vapour pressure model calculation the experiment was performed

at 50°C, 60°C and 70°C. These temperatures were chosen because the evaporation of the

test item was expected to be very low at lower temperatures. Details of the model calculation

are not given here but are part of the raw data.

The results and the amount of nitrogen used to calculate the vapour of the individual

measurements are summarised in Table 1. The tabulated values represent rounded results

which were obtained by calculation using the exact raw data. At none of the used

temperatures any significant amount of test item could be detected, thus the vapour pressure

was calculated using the least concentrated calibration solution. An extrapolation of the

results was not performed. Still it can be deduced that the vapour pressure at 25°C is

expected to be well below the calculated results.

A typical chromatogram of a standard calibration solution is given in Figure 1. An example of

a sample solution chromatogram equilibrated at 60°C is shown in Figure 2, A HPLC

chromatogram of a blank solution (acetonitrile and water 50:50; v/v) is shown in Figure 3. An

example of the calibration data for test item standards is given in Table 1. The R= fit was

1.0000 (optimum). This reflects the linearity of the HPLC-system within the calibration range

of 0.0491 - 16.355 pg/ml of the test item.

The experimental results lead to the conclusion that the vapour pressure of the TKA 40278

(CGI 827) is:

1.7.10"~ Pa at 50°C,

1,6.10~ Pa at 60°C

and

1.6.104 Pa at 70°C.

Based on the experimental results the vapour pressure of TKA 40278 (CGI 827) at 25°C is

expected to be

<< 1,6  ̄ 10~ Pa at 25°C,

107
RCC Study Number 845420 REPORT Page 17

TKA 40278 (CG1827)

Table 1: Results of the gas saturation test

Mean  ̄ Amount

Sample Temp, Flow Rate Time Volume extracted Dilution Recovery

110. [°¢] [ml/min] [hi [pg/ml] Factor [%]

1 5O 3.35 116.2 23,356 0.05 10

2 5O 3.04 116.2 21.177 0,05 10

3

Spike 50 * 5O 3.76 116.2 26,232 20.56 10

4 60 3.60 115.8 25.013 0.05 10

5 60 3.75 115.8 26.055 0.05 10

6

Spike 60 * 60 4.00 115.8 27.792 20.65 10

7 70 3.60 115.5 24.948 0.05 10

8 70 3.75 115.5 25.988 0.05 10

9

Spike 70 * 70 3.75 115.5 25,988 19.95 10

* TENAX tube spiked w~th 50 pl of a 4.37 mg/ml test item solution in acetonitdle

The recovery rate in the analysis of the fortified sample is high, showing that the trapping and

extraction methods were suited to detect the test item in the TENAX adsorber tubes.

An example of the calculation of the Vapour Pressure (test at 60°C)

The vapour pressure was calculated with the following equation:

W.R.T

PvP =

V.M

Values from the 60°C-test were used for the example-calculation

W = mass of evaporated substance

R = gas constant

T = Temperature (K)

V = volume of nitrogen

M = Molecularweight

SAMPLE I SAMPLE II

= 4.91 x 10.7 g

= 8.314 J/K mol

= 333 K

= 0.0250 m3

= 340.42 g/mol

Sample I: Pv~ = 0.00016 Jim3 = 1.6 x 104 N/m2 at 60°C

Sample It: Pw = 0.00015 J/m3 = 1.5 x 104 N/m2 at 60°C

Mean Vapour Pressure: 1.6 x 10"~ N/m2 = 1.6 x 10.4 Pa at 60°C

4.91 x 10.7 g

8.314 J/K mol

333 K

0.0261 m3

340.42 g/mol

108
RCC Study Number 845420 REPORT Page 18 TKA 40278 (CGI 827)

Figure 1: Chromatogram of a calibration solution 0.0491 pg/ml

Channel: Op~ra~r (Ca]0: SPA 1 = 260.00... nm RESULT~ Gale Dat~: 24.04,03 16:33:26

Peak P~k Name Result (%) Ret. Time

I TKA 40278 100.00~ _12.269 0.027

Area Sep. Width (cotmts) Co~e 1/2

(sec)

93569= _ BB 8-5

93569

109
RCC Study Number 845420 REPORT Page 19 TKA 40278 (CG1827)

Figure 2: Chromatogram of sample 4 (at 60°C after 115.8 hours)

Op~ator (CaIc): SPA Cha~ael: 1 = 260.00.. nm RESULTS Calc Date: 24.04.03 23:24:59

Injection Date: 24.04.03 22:49:54 InsOmment (Calc): Vari~m I£~ 31

Verification Tolerance: lqJA

Peak R~sult (%) ReL Time Area Sep. Width Time Offset (counts) Code 1,2

(rata) (rain)

100.¢~ 12.235 -0.007 40589 BB 100.00 -0.007 40589

11o
RCC Study Number 84..,’~20 REPORT Page 20 TKA 40278 (CG1827)

Figure 3: Chromatogram of a blank solution

Charm I: I = 260.00.,, nm RESULTS

Sample ID: 50_50 ACN~vl!LLI Q

Operator Cmj): SPA

Injection Dam: 24.04.03 20:20:15

Injection Metl~d: e:kle..3 lhnethods~ka 40278.ruth

Run l~tm¢ (mln): 35.013

Worksta~on:

lnstrarnent (Inj): VafianLC31

Ol~-ator (Calc): SPA

Ca~c IYat~: 24.04,03 20:55:t9

Peak Peak Name Result (%) Ret. Time Area Sep. Width So Time Offset (counts) Code 1/2 (m~) (m~) (~)

TKA 40278 i00.00 12,22~7 -0.015 32410 BB 0._~_0 Totals 100.00 -0.015 3241(I /

111
RCC Study Number 845420 REPORT Page 21 TKA 40278 (CGI 827)

Figure 3: An example of a calibration curve of TKA 40278 (CGI 827)

Linear regression

2,50EP07

~ 1,50B’07

~1,00~’07

2 4 6 8 10 12 14 16

Concentrat~o~

18

Y=b x+a with Y = peak area (counts)

x = concentration (IJg/ml)

b = 1165120 (slope)

a = 41182 (y-axis intercept)

r2 = 1.0000

Table 2: An example of calibration data of TKA 40278 (CGI 827)

Area Counts Concentration Deviation

[pg/ml] in %

(x)

93569 0.0491 -8.4%

793268 0.6542 -1.3%

3098359 2.6168 0.3%

9584018 8.1775 0.2%

19088284 16,355 0,0%

112