REPORT

Study Title:

ESTIMATION OF THE ADSORPTION COEFFICIENT OF TKA40278

(CGI827) ON SOIL USING HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY (HPLC)

Data Reoulrement I Test Guldellnes:

OECD Guideline No. 121

EC Directive 2001/59 0.19

Study Director:

Mrs. Alessandra Tognucci

Study Comeletlon Date:

April 25, 2003

Test Facility:

RCC Ltd

Environmental Chemistry & Pharmanalytlcs

CH-4452 Itingen/Switzerland

ClBA SPECIALTY CHEMICALS INC.

CH - 4002 Basel/Switzerland

RCC Study Number:

845431

Page 1 of 22

131 !
RCC Study Number 845431 REPORT Page 2

TKA 40278 (CGI 827)

GLP CERTIFICATE

The Swiss GLP Monitoring Authorities

,~ ~ ,~, sw medic

Statement of GLP Compliance

It is hereby confirmed that

duflng the pedod of

the following Test Facll~as 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 Land.~cape with respect to

the compliance with the Swiss legislation on Good Laboratory Prectlce.

Test Facilities 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 aforementionad test fadlltJes were operating In

compliance with the Swiss Ordinance relating to Good Laboratory Prac~ce [RS 813.016,5] at

the time they were Inspected,

Bem, March 2003

Federal Office of Publlo Health

The Dlre~,--tor

Prof. Th, Zeltner

132
RCC Study Number 84543"1 REPORT Page

TKA 40278 (CG1827)

GOOD LABORATORY PRACTICE

STATEMENT OF COMPLIANCE

RCC Study Number: 845431

Test Item: TKA 40278 (CGI 827)

Study Director: Mrs. Alessandra Tognucci

Title: Estimation of the Adsorption Coefficient of

TKA40278 (CGI827) on Soil using High

Performance Liquid Chromatography (HPLC)

This study was conducted in compliance with the Swiss Ordinance relating to Good

Laboratory Practice, adopted February 2’~, 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:

Date:

133
RCC Study Number 845431 REPORT Page 4

TKA 40278 (OG1827)

SIGNATURES

Study Director: i Tognucci

Management: Dr. JSrn Schreitmeller

134
RCC Study Number 845431 REPORT Page 5

TKA 40278 (CGI 827)

QUALITY ASSURANCE UNIT

RCC Ltd, Environmental Chemistry & Pharmanalytics, CH-4452 Itingen / Switzerland

STATEMENT

RCC Study Number:

Test Item:

Study Director:

Study Title:

845431

TKA 40278 (CGI 827)

Mrs. Alessandra Tognucci

Estimation of the Adsorption Coefficient of TKA 40278

(CGI 827) on Soil using High Performance Liquid

Chromatography (HPLC)

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 Report~ to t,he S,tudy

Director and to the Management

September 16, 2002 Study Plan September 16, 2002

December 09, 2002 Measurements December 09, 2002

April 17, 2003 Final report April 17, 2003

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

Quality Assurance: Ms. Jane Sanotra

135
RCC Study Number 845431 REPORT Page 6

TKA 40278 (CGI 827)

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:

136
RCC Study Number 84543’1 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 REAGENTS AND REFERENCE ITEMS ..................................................... 11

2.2.1 Reference Items ........................................................................... 11

2.2.2 Reagents and Apparatus .............................................................. 11

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

2.3.1 Principle .................................................................... , ...................12

2.3.2 Definitions ..................................................................................... 12

2.3.3 Test System .................................................................................. 13

2.3.4 Performance of the Test ............................................................... 13

2.3.5 HPLC-Conditions .......................................................................... 14

2.4 DATA HANDLING ...................................................................................... 14

3 RESULTS ............................................................................................................ 15

Table 1: Retention times in minutes of the reference items and the test item ........... 16

Table 2: Results of Linear Regression of Log k’ vs. Log Ko~ ..................................... 17

Table 3: Result of TKA 40278 (CGI 827) analyses ................................................... 18

Figure 1: Typical HPLC chromatogram of the reference item mixture ....................... 19

Figure 2: Typical HPLC chromatogram of a sample solution of TKA 40278

(CGI 827) ................................................................................................... 20

Figure 3: HPLC ¢hromatogram of the sodium nitrate solution ................................... 21

Figure 4: Adsorption coefficient calibration curve ...................................................... 22

137
RCC Study Number 845431 REPORT Page 8

TKA 40278 (CGI 827)

PREFACE

GENERAL

Study Title:

Sponsor:

Monitoring Scientist:

Test Facility:

Estimation of the Adsorption Coefficient of TKA 40278

(CGI 827) on Soil using High Performance Liquid

Chromatography (HPLC)

CIBA 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:

Reporting:

Head of RCC Quality Assurance:

Mrs. Alessandra Tognucci

Dr. Ute Schmiedel

Mrs. Josde Gustaw

Mrs. Miriam Weissenfeld

Mrs. Iris W(~thrich

SCHEDULE

Experimental starting date:

Experimental completion date:

Study completion date:

January21,2003

January 21,2003

April25,2003

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.

138
RCC Study Number 845431 REPORT Page 9

TKA 4O278 (CGI 827)

DATA REQUIREMENT / TEST GUIDELINES

This study followed the procedures indicated by the following internationally accepted

guidelines and recommendations:

OECD Guideline for the Testing of Chemicals, No. 121, Estimation of the Adsorption

Coefficient (Koc) on Soil and Sewage Sludge using High Performance Liquid

Chromatography (HPLC), Adopted Test Guideline (January 2001).

EC Directive 2001/59/EC, C. 19, EC publication L 225, August 2001.

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 provided by the sponsor

139
RCC Study Number 845431 REPORT Page 10

TKA 40278 (CGI 827)

1 PURPOSE

The adsorption coefficient is defined as the ratio between the concentration of the substance

in the soil/sludge and the concentration of the substance in the aqueous phase at adsorption

equilibrium. The adsorption coefficient normalised to the organic carbon content of the soil

Koc is a useful indicator of the binding capacity of a chemical on organic matter of soil and

sewage sludge and allows comparisons to be made between different chemicals.

The experimental method uses HPLC for the estimation of the adsorption coefficient Koc in

soil and sewage sludge,

2 MATERIAL AND METHODS

2.1 TEST ITEM

Data of test item as suppl{ed by the sponsor.

Identity: TKA 40278

Product Name: CGI 827

Batch: KL 01/02

Purity: 94.1% (see analytical report of Ciba Specialty

Chemicals Inc., Study No. 14026512-1)

Appearance: Solid, slightly yellow

Expiration date: August 30, 2004

Storage: At room temperature at about 20 °C.

The test item is light sensitive. Therefore, all test item handling was performed under red

light.

140
RCC Study Number 845431 REPORT Page 11

TKA 40278 (CGI 827)

2.2 REAGENTS AND REFERENCE ITEMS

2,2.1 Reference Items

Manufacturer. Lot&Filling Code

Identity Purity Expiry

Date Storage log K~

’ ArL-No. (~)

Acetanilide Fluka 415036/1 44400

00401 99.8 11/2005 RT 1.25

Isoproturon Dr. EhrenstQrfer 71009

C 144700 99.5 10~003 RT 1.86

Triadimenol Dr. Ehrenstorfer 00313

C 17620000 98.0 03~006 RT 2,40

Pymzophos Dr. Ehrenstorfer 91108

C 16610000 97.0 12~003 -18°C 3.65

Diclofop-methyl Dr. Ehrenstorfer 20308

C 12540000 99.0 03/2004 -18°C 4.20

2,4-DDT Dr, Ehrenstorfer 80717

120810 98.0 08/2004 RT 5.63

Sodium nitrate Fluka 39758911 34199

71757 100 10/2004 RT

RT = room temperature

to = dead time

2.2.2 Reagents ahd Apparatus

Water

Methanol

Tetrahydrofurane (THF)

pH meter

HPLC

RCC from Milli-Q water supply

Baker Art, No. 8402

Baker Art, No. 9441

Metrohm Titroprocessor 686 with electrode 6.0232.100

as specified below

141
RCC Study Number 845431 REPORT Page ’12

TKA40278 (CGI 827)

2.3 STUDY DESIGN

2.3.1 Principle

For the estimation of the adsorption coefficient, the HPLC-method was used. Therefore, an

analytical column packed with a commercially available, cyanopropyl solid phase containing

lipophilic and polar moieties was used. A moderately polar stationary phase based on a silica

matrix was used.

While passing through the column along with the mobile phase, the test item interacts with

the stationary phase. As a result of partitioning between mobile and stationary phases, the

test item is retarded. The dual composition of the stationary phase having polar and nonpolar sites allows for interaction of polar and non-polar groups of a molecule in a similar way

as is the case for organic matter in soil or sewage Sludge matrices. This enables the

relationship between the retention time on the column and the adsorption coefficient on

organic matter to be established.

2.3.2 Definitions

K~: Distribution coefficient defined as the ratio of equilibrium concentrations Ce of a dissolved

substance in a two-phase system consisting of a solid (soil or sewage sludge) and an

aqueous phase; it is a dimensionless value varying with solid properties.

C = Concentration of the test item at equilibrium (pg/g)

Koc: Distribution coefficient (1~) normalised to the organic carbon content (OC) of a solid;

particularly for non-ionised chemicals, it is an approximate indicator for the strength of

adsorption between a substance and the solid and allows comparisons to be made between

different chemicals.

Kd Koc = ~-~ (dim ensionless) (2)

The adsorption coefficient (Koc) is deduced from the capacity factor (k’) using a calibration

plot of log k’ versus log Koc of the selected reference items.

= tR -to- (3)

to

tR HPLC retention time of test or reference items (rain.)

HPLC dead time (min.)

(measurement of the retention time of sodium nitrate)

The adsorption coefficient (Koc) of the test item will be interpolated from the calibration curve

which will be established by correlation of the log Koc values of vadous reference items with

their log k’ values.

142
RCC Study Number 845431 REPORT Page 13

TKA 40278 (CGI 827)

2.3,3 Test System

This method utilises a high performance liquid chromatograph with UV-VIS detector,

reversed phase column and an aqueous mobile phase. Details are given below,

2.3.4 Performance of the Test

Test Item Solution

TKA 40278 (CGI 827) (13 rag) was dissolved in THF (10 ml) to prepare a stock solution of

1300 pg/ml. The stock solution (1 ml) was diluted with a solvent mixture of methanol and

water (55:45; v/v) to 10 ml to obtain a standard solution of 130 IJg/ml. The pH value of the

test item solution was determined to be 7.3. The test item solution was injected three times.

Reference Solutions

The reference items were chosen from the list given in the OECD Guideline No. 121 and EC

Directive C.19.

Stock solutions of the reference items in methanol were prepared as described:

24 mg of the reference items isoproturon and pyrazophos, 23 mg of the reference item

triadimenol and 25 mg of the reference items 2.4-DDT and diclofop-methyl were each

dissolved in 25 ml methanol. 10 mg of the reference item acetanilide were dissolved in 10 ml

methanol.

A combined reference solution was prepared by transferring 205 pl of the acetanilide stock

solution, 220 pl of the isoproturon. 1200 pl of the tdadimenol, 1800 pl of the pyrazophos,

330 pl of the diclofop-methyl and 1110 pl of the 2.4-DDT stock solution into one volumetric

flask aqd diluting to a volume of 10 ml with a solvent mixture of water and methanol (45:55;

v/v). This reference solution was injected six times.

Sodium nitrate (25,5 mg) was diluted to 100 ml with methanol to obtain a stock solution. The

stock solution (100 pl) was diluted to 1 ml with a solvent mixture of water and methanol

(45:55; v/v). This sodium nitrate solution was injected two times for the determination of the

dead time of the HPLC system.

143
RCC Study Number 845431 REPORT Page 14

TKA 40278 (CGI 827)

2.3.5 HPLC-Conditions

Apparatus:

Column:

Mobile Phase:

Detection:

Temperature:

Injection Volume:

Flow:

Vadan autosampler 9100

Varian pump 9010

Varian detector 9050

Jones column oven 7990

LiChrospher 100 CN, part. size 5 pro, 250 mm ̄ 4 mm

Water / methanol (45:55; vN); pH 6.8

210 nm for the reference items and for sodium nitrate

260 nm for the test item solution

25°C

10 pl

1.0 ml/min

2.4 DATA HANDLING

Based on the OECD test guideline No. 121 and the EC guideline C.19 the HPLC-method can

be used to accurately evaluate log Koc values in the range of 1.5 to 5.0. To this end, the

capacity factor (k’) is calculated from the retention time of the reference items, the test item

(tr) and an unretarded item (to):

k’ tr -to

to

where

Retention time of the test item or reference item

Time a non-sorbed chemical needs to pass the column

(dead-time, sodium nitrate solution)

Afterwards, a correlation of log k’ versus log Koc of the reference items is plotted using linear

regression according to the following equation.

Y = a+b.X X = (Y- a)/b

where

Y = log k’ values

X = log Koc values

a = Y-axis intercept of the regression curve

b = Slope

The log k’ value of the test item was then used to calculate its log Koc value.

144
RCC Study Number 845431 REPORT Page 15

TKA 40278 (CGI 827)

3 RESULTS.

The determination of the adsorption coefficient (Koc) of TKA 40278 (CGI 827) was performed

according to the OECD Guideline for the Testing of Chemicals, No. 121, Estimation of the

Adsorption Coefficient (Koc) on Soil and Sewage Sludge using High Performance Liquid

Chromatography (HPLC), January, 2001 and the EC Directive 2001159, C.19.

The test item solution was injected three times and the combined reference solution was

injected six times. The log Koc was calculated using a regression curve (log k’ vs. log Koc)

and was found to be 2.02 which is equal to a Koc value of 104. In the present study the

reference items covered the range of log Koc 1.25 to log Koc 5.63.

An example of an HPLC chromatogram of the reference standard mixture is shown in

Figure 1. An HPLC chromatogrem of the test item is shown in Figure 2. A typical

chromatogram of the determination of the dead-time is presented in Figure 3. The

chromatography of the test item resulted in one main peak with a well-defined and

reproducible retention time. The HPLC method was therefore considered to be suitable for

the purpose of the study. Based on the chromatographic data, TKA40278 (CGI 827) was

considered to be stable during the test procedure.

The retention times of the individual measurements of the reference items, test item and the

dead time are given in Table 1. The capacity factor (k’) was calculated for each reference

item using the retention times. Log Koc was plotted versus log k for each component to

obtain a regression curve (Table 2 and Figure 4).

The tabulated values may represent rounded or mean results, which were obtained by

calculation using the exact raw data.

In conclusion, the log of the adsorption coefficient (Koc) of TKA 40278 (CGI 827) was

estimated to be

log Koc = 2.02 which is equal to Koc value of t04

using the HPLC method. This value indicates that TKA 40278 (CGI 827) is moderately

mobile.

145
RCC Study Number 845431 REPORT Page 16

TKA 40278 (CGI 827)

Table 1: Retention times in minutes of the reference items and the test item

1. run 2. run 3. run 4, run 5. run 6. run

Sodium nitrate

(= dead time) 1.146 1.146

Reference Items:

Acetanilide 2.977 2,979 2,980 2.981 2.980 2.981

Isoproturon 3,536 3.540 3,542 3.544 3.542 3.544

Triadimenol 4.095 4.099 4.101 4.104 4.102 4.104

Pyrazophos 6.126 6.138 6.145 6.154 6.153 6.155

Diclofop-methyl 7.460 7.477 7.486 7,498 7.498 7.500

2.4-DDT 12.673 12.710 12.731 12.753 12.751 12.758

Test Item:

TKA 40278

(CGI 827): 3.681 3.681 3.681

146
RCC Study Number 845431 REPORT Page 17

TKA 40278 (CGI 827)

Table 2: Results of Linear Regression of Log k’ vs. Log Ko~

Number Mean log k’ log

of retention

analysis time [mini

Sodium nitrate

(= dead time) 2 1,15

Reference Items:

Acetanilide 6 2.98 1.60 0.20 1,25

Isoproturon 6 3.54 2.09 0.32 1,86

Triadimenol 6 4,10 2.58 0.41 2,40

Pyrazophos 6 6.15 4.36 0.64 3.65

Diclofop-methyl 6 7.49 5.53 0.74 4,20

2.4-DDT 6 12.73 10.11 1.00 5.63

slope = 0.t82

Linear Regression: y-axis intercept = -0.023

coefficient of regression = 1.000

147
RCC Study Number 845431 REPORT Page 18

TKA 40278 (CGI 827)

Table 3: Result of TKA 40278 (CGI 827) analyses

Number Retention log k’ log Koc Mean Koc Mean

of time log Koc Koc

analyses [min]

1 3.68 2.21 0.34479 2.02 104.4

2 3.68 2,21 0.34479 2.02 104.4

3 3.68 2.21 0.34479 2.02 2.02 104.4 104

The tabulated values represent rounded results, which were obtained by calculation using the

exact raw data.

148
RCC Study Number 845431 REPORT Page 19

TKA 40278 (CGI 827)

Figure 1: Typical HpLC chromatogram of the reference item mixture

Data Ftie: e:\vtLmodule 03~84543 l\7.nm Operator (Calc): Gwj

Channel: 1 = 210 nm RESULTS Calc Date: 23.01.2003 06:50:22

S ~’nple ID: Standard Mix Times Calc~ulated: 3

O~Jator (In.i): Gwj Calculation Method: c:\vl~module 03’~t 40278-std.mth

Injection Datz: 21.01.2003 19;08;49 Instrument (Ca]¢): V~tan St~r #1

Inje~tlon Method: ¢;\vl ~nodule 03~,a 40278*std.mth Run Mode: An~ysis

Run Time (rnin): |4.980 Peak Measurement: pe~tk Area

Workstation: WIN95 Calculation Type: Percent

]nstrament (Inj): V~dan Star #1 Calibration Level:

Verification Tolerance:

No

]

2

3

4

6

7

Pe~k Name Ret Peak Area Sep, Result (%)

Time ( ounta) Code

(rain)

Acetanilide 2.981 334972 W 7,08

Isoproturon 3,544 394052 W 8.33

3,956 307141 W 6.49

Tiadimenol 4.104 476631 VB 10,07

P’fazophos 6.155 650397 PV 13.74

Diclofopmethy[ 7.500 810262 VB 17,12

2.4-DDT 12,758 1759336 BB 37.17

Totah 4732791 100.00

149
RCC Study Number 84543"i REPORT Page 20

TKA 40278 (CGI 827)

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

Data File: c:\vlLmodule 03~84543 l~4.run Op~tor (Calc): Gwj Channel: 1 = 260 nm P~ESULTS Cato Date: 23.01.2003 06:44:54

Peak PeakNam= Ret Peak Are4t Sep. Result(e/.) No Time (counts) Code

(min)

I TKA 40278 3.681 2812049 VB 100.00

Totals 2812049 100.O0

150
RCC Study Number 845431

TKA 40278 (CG1827)

REPORT Page21

Figure 3: HPLC chromatogram of the sodium nitrate solution

Minute~

Peak Peak N=me Ret Peak Are~ Sep. Result (%)

No Time (count~) Code

(min)

] Na.nlt~at 1.146 979256 . BB 100.00

Totals 9792~6 100.00

151
RCC Study Number 845431 REPORT Page 22

TKA 40278 (CGI 827)

Figure 4: Adsorption coefficient calibration curve

1.5 n

Linear Regression

1.0 "~

0.5 -I

0.0 I ,

0,0 1.0 2.0 3.0 4.0 5.0 6.0

log Koc

Y = a+ b.x with Y = log k’ values

X = log Koc values

b = 0.182 (slope)

a = -0.023 (y-axis intercept)

r = 1.ooo

152