Study Title

Determining the Partition Coeffici ent (n-Octanol/Water) of EFKA 8530 by the Flask-Shaking Method Following OECD Guideline 107

Data Requirement

OECD Guideline 107

Author

Karen L. Proctor

Study Completed On

15 April 2008

Submitted To

Ciba Specialty Chemicals Company 540 White Plains Road Tarrytown, New York 10591

Performing Laboratory

Springborn Smithers Laboratories 790 Main Street Wareham, Massachusetts 02571-1037

Laboratory Project ID

Springborn Smithers Study No. 13658.6319

Page 1 of 43Springborn Smithers Study No. 13658.6319 Page 4



### KEY STUDY PERSONNEL

The following Springborn Smithers personnel were responsible for the conduct of the work and reporting of the study results.

Karen L. Proctor Study Director

Rebecca J. Smith Chemistry Technician II

James D. Hatch Chemist I

David O. Duval Chemistry Technician I

Andrea M. Soares Technical Report Writer

Ronald C. Biever Vice President, OperationsSpringborn Smithers Study No. 13658.6319 Page 5



### TABLE OF CONTENTS

Page

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT..........................................2 QUALIT Y ASSURANCE UNIT STATEMENT............................................................................ 3

KEY STUDY PERSONNEL

........................................................................................................... 4

TABLE OF CONTENTS

.............................................................................................................. ...5

SUMMARY

........................................................................................................................ .............7

1.0 INTRODUCTION ..................................................................................................................8

2.0 MATERIALS AND METHODS

........................................................................................... 8

2.1 Protocol ....................................................................................................................8 2.2 Test Substance and Standard Reagents

.................................................................... 8 2.2.1 Test Substance .............................................................................................8 2.2.2 Standard Reagents

........................................................................................ 9 2.3 n-Octanol-Saturated W ater and Wa ter-Saturated n-Octanol Solution Preparation ...............................................................................................................9 2.4 Stock Solution Preparation ....................................................................................10 2.5 Preparation of Quality Control Sam ples................................................................ 10 2.6 Preparation of Calibration Standards

..................................................................... 11 2.7 Test Apparatus .......................................................................................................11 2.8 Test Solution Preparation .......................................................................................11 2.8.1 Prelim inary Test......................................................................................... 11 2.8.2 Definitive T est ........................................................................................... 11 2.9 Test Procedure .......................................................................................................12 2.9.1 Prelim inary Test......................................................................................... 12 2.9.2 Definitive T est ........................................................................................... 12 2.10 Che mical Analysis ................................................................................................. 13 3.0 CALCULATIONS

............................................................................................................... 14

4.0 RESULTS .............................................................................................................................14

4.1 Prelim inary Results................................................................................................ 14 4.2 Definitive R esults................................................................................................... 14 PROTOCOL DEVIATI ON ........................................................................................................... 16

REFERENCES ..............................................................................................................................17 Table 1. Results of the prelim inary n-octano l/water partition coefficient test with EFKA 8530........................................................................................ 18Springborn Smithers Study No. 13658.6319 Page 6

Table 2. Results of the definitive n-octano l/water partition coefficient test with EFKA 8530........................................................................................ 19 Table 3. Results of the analysis of the EFKA 8530 n-octanol stock solution and quality control samples.

...................................................................... 20 Figure 1. HPLC chrom atogram of an EFKA 8530 water-saturated n-octanol phase test solution from the determination of the partition coefficient. ................................................................................................. 21 Figure 2. HPLC chrom atogram of a quality control sample (1032 mg a.i./L) from the determination of the partition coefficient for EFKA 8530.......... 22 Figure 3. HPLC chrom atogram of a calibratio n standard (25.0 mg a.i./L) from the determination of the part ition coefficient for EFKA 8530. ................. 23 APPENDIX 1 - STUDY PROTOCOL ..........................................................................................24

APPENDIX 2 - ANALYTICAL ME THODOLOGY.................................................................... 34 Table 1A. Analytic al results for the recove ry of EFKA 8530 from n-octanol during the method validation study............................................................39 Figure 1A. Representative chrom atogram of a 10.0 mg a.i./L calibration standard used for quantificati on during the method validation study with EFKA 8530........................................................................................ 40 Figure 2A. Representative chrom atogram of a 1032 mg a.i./L recovery sample fortified with EFKA 8530..........................................................................41 Figure 3A. Representative chrom atogram of a control sample during the method validation study with EFKA 8530. ............................................................ 42 Figure 4A. A typical regression analysis for the calibration standards used to quantitate E FKA 8530. ..............................................................................43Springborn Smithers Study No. 13658.6319 Page 7



### SUMMARY

Determining the Partition Coefficient (n-Octanol/Water) of EFKA 8530 by the Flask-Shaking Method Following OECD Guideline 107

STUDY SPONSOR: Ciba Specialty Chemicals Company

PROTOCOL TITLE: Determining the Partitioning Coefficient (n-Octanol/Water) of a Test Substance by the Flask-Shaking Method Following OECD Guideline 107, Springborn Smither s Laboratories Pr otocol No.: 040805/OECD/Kow

SPRINGBORN SMITHERS STUDY NUMBER: 13658.6319

TEST SUBSTANCE: EFKA 8530, Lot No. 15306, reported to have a purity of 93.56%, was received from Ciba Specialty Chemicals Company on 31 October 2007.

DEFINITIVE TEST DATE: 11 January to 4 February 2008

RESULTS: Analyses of the definitive test samples determined that the mean partition coefficient of the test substance was 24.9 (log P ow = 1.31).Springborn Smithers Study No. 13658.6319 Page 8



### 1.0 INTRODUCTION

The objective of this study wa s to determine the n-octanol/water partition coefficient (P ow ) for EFKA 8530 by the flask-shaking method. The n- octanol/water partition coefficient (P ow ) is the equilibrium ratio of the concentration of EFKA 8530 in n-octanol and water in a dilute solution. The test substance was analyzed by gel perm eation chromatography with evaporative light scattering detection (GPC/ELSD).

The study was initiated on 19 December 2007, the day the Study Director signed the protocol, and terminated the day the Study Director signed the final report. The definitive test was conducted from 11 January to 4 February 2008 at Springborn Smithers Laboratories (SSL) located in Wareham, Massachusetts. All raw da ta, the protocol and the final report produced during this study are stored in Springborn Smithers' archives at the above location.



### 2.0 MATERIALS AND METHODS

2.1 Protocol

The procedures followed during this study are detailed in the Springborn Smithers Laboratories protocol entitled "Determining the Partitioning Coefficient (n-Octanol/Water) of a Test Substance by the Flask-Shaking Method Followi ng OECD Guideline 107", Springborn Smithers

Laboratories Protocol No.: 040805/OECD/Kow ( Appendix 1 ).



### 2.2 Test Substance and Standard Reagents

2.2.1 Test Substance

The test substance, EFKA 8530, was received on 31 October 2007 from Ciba Specialty Chemicals Company, Allentown, Pennsylvania. The following information was provided:Springborn Smithers Study No. 13658.6319 Page 9

Name: EFKA 8530 Lot No.: 15306 CAS No.: Not Listed Purity: 93.56% Recertification Date: 31 January 2009

Upon receipt at Springborn Smithers, the test su bstance (SSL No. 126-09) was stored at room temperature in the original contai ner in a dark ventilated cabinet. Concentrations were adjusted for the purity of the test substance and ar e presented as active ingredient (a.i.).

Determination of stability and characterizati on, verification of the te st substance identity, maintenance of records on the test substance, and archival of a sample of the test substance are the responsibility of the Study Sponsor.

2.2.2 Standard Reagents

All solutions were prepared using reagent wate r (m eeting ASTM Type II requirements) purified with a Barnstead NANOpure ® system. The n-octanol used was obtained from Sigma-Aldrich Chemical Company, Inc. All chemicals were at least reagent grade a nd were obtained from

commercial sources.



### 2.3 n-Octanol-Saturated Water and Wate r-Saturated n-Octanol Solution Prep aration

n-Octanol-saturated water and water-saturated n-octanol were prepared simultaneously in Erlenmeyer flasks by combining 700 mL of n-oc tanol with 700 mL of purified reagent water (PRW). The flasks were tightly sealed and then placed on a shaker table (Lab-Line Orbital Model 3590). The flasks were shaken for 24 hour s at 150 rpm to saturate both phases. After being removed from the shaker table, the solutions were transferred into separatory funnels and allowed to settle until the two pha ses had completely separated. The two individual phases were transferred into separate amber bottles. This yielded n-octanol-saturated water and

water-saturated n-octanol.Springborn Smithers Study No. 13658.6319 Page 10



### 2.4 Stock Solution Preparation

A 2.02 mg a.i./mL stock solution was prepar ed by placing 0.4312 g of EFKA 8530 (0.4034 g as active ingredient) in a 200-mL volumetric flask a nd bringing it to volume with water-saturated

n-octanol. This stock solution was used to prepare test samples during preliminary testing.

A 2.05 mg a.i./mL stock solution was prepar ed by placing 0.4372 g of EFKA 8530 (0.4090 g as active ingredient) in a 200-mL volumetric flask a nd bringing it to volume with water-saturated

n-octanol. This stock solution was used to prepare test samples during definitive testing.

A 5.16 mg a.i./mL stock solution was prepar ed by placing 0.2760 g of EFKA 8530 (0.2582 g as active ingredient) in a 50.0-mL volumetric flask a nd bringing it to volume with n-octanol. This stock solution was used to prepare n-octanol quality control samples.

A 5.00 mg a.i./mL primary stock solution wa s prepared by placing 0.5344 g of EFKA 8530 (0.5000 g as active ingredient) in a 100-mL volumetric flask and bringing it to volume with tetrahydrofuran (THF). A 1000 mg a.i./L se condary stock solution was prepared by placing 10.0 mL of the 5.00 mg a.i./mL primary stock solution in a 50.0-mL volumetric flask and bringing it to volume with THF. A 100 mg a.i./L tertiary stock solution was prepared by placing 5.00 mL of the 1000 mg a.i./L secondary stoc k solution in a 50.0-mL volumetric flask and bringing it to volume with THF. The 1000 mg a.i./L secondary stock solution and the 100 mg a.i./L tertiary stock solution were used to prepare calibration standards.



### 2.5 Preparation of Quality Control Samples

n-Octanol quality control samples were pr epared at concentrations of 206, 1032 and 2064 mg a.i./L by fortification of water-saturated n-octanol with the 5.16 mg a.i./mL stock

solution.Springborn Smithers Study No. 13658.6319 Page 11



### 2.6 Preparation of Calibration Standards

Calibration standards were prepared at concentra tions of 2.00, 5.00, 10.0, 25.0, 35.0 and 50.0 mg a.i./L by fortifying THF with the 100 mg a. i./L (2.00 and 5.00 mg a.i./L standards) and 1000 mg a.i./L (10.0, 25.0, 35.0 and 50.0 mg a. i./L standards) stock solutions.



### 2.7 Test Apparatus

The test apparatus consisted of 50-mL glass centrifuge tubes with Teflon ® -lined screw caps. All testing was conducted in a temperat ure-controlled environmental ch amber designed to maintain a temperature of 20 ± 1 oC. The temperature extr emes were measured with a calibrated minimummaximum thermometer.



### 2.8 Test Solution Preparation

2.8.1 Preliminary Test

Test solutions were prepar ed in 50-mL glass centrifuge tubes using 20.0 mL of the

2.02 m g a.i./mL stock solution in water-sat urated n-octanol al ong with 20.0 mL of

n-octanol-saturated water.

2.8.2 Definitive Test

During the d efinitive experiment, three test solutions were prepared in duplicate using the volume ratios of 1:2, 1:1 and 2:1 n- octanol/water. In th e volume ratio of 1: 2 test, 10 mL of the 2.05 mg a.i./mL stock solution in water-saturat ed n-octanol along with 20 mL of corresponding n-octanol-saturated water was prep ared. In the volume ratio of 1:1 test, 15 mL of the stock

solution in water-saturated n- octanol along with 15 mL of co rresponding n-octanol-saturatedwater was prepared. In the volume ratio of 2:1 test, 20 mL of the stock solution in watersaturated n-octanol along with 10 mL of corresponding n-octanol-saturated water was prepared.Springborn Smithers Study No. 13658.6319 Page 12



### 2.9 Test Procedure

2.9.1 Preliminary Test

The centrifuge tubes con taining the test solutions were sealed with Teflon ® -lined screw caps and hand rotated through 180 degrees about their transv erse axes for approximately 100 rotations in five minutes to establish equilibrium. The samples were then centr ifuged at approximately 1000 rpm for 20 minutes in a Beckman Model GS-6R centrifuge. The temperature was maintained at 18.7 oC during this period.

A portion of the n-octanol phase was sampled by pi pet and transferred to a vial. The remainder of the n-octanol phase and inte rfacial layer was removed and di scarded and the water phase was sampled. The pH of the aqueous phase of each sample was determined using pH indicator strips after sampling.

After the n-octanol and water phases were sepa rated, the n-octanol po rtion of each sample (approximately 10 mL) was then analyzed by ge l permeation chromatography with evaporative light scattering detection (GPC/ELSD).

2.9.2 Definitive Test

The centrifuge tubes con taining the test solutions were sealed with Teflon ® -lined caps and hand rotated through 180 degrees about their transverse axes for ap proximately 100 rotations in five minutes to establish equilibrium. The samples were then centr ifuged for 20 minutes at approximately 1000 rpm in a Beckman Model GS-6R centrifuge. Test samples were allowed to re-equilibrate at the test temperature for approximately one hour. The temperature ranged from 25.8 to 26.6oC throughout the definitive test.

A portion of the n-octanol phase was removed by pi pet and transferred to a vial. The remainder of the n-octanol phase and inte rfacial layer was removed and di scarded. A portion of the water phase was sampled for pH measurements. Fina lly, the n-octanol samples were analyzed bySpringborn Smithers Study No. 13658.6319 Page 13

GPC/ELSD. Both water and n-oc tanol test samples were prepared for analysis in the same manner as described for the preliminary test.



### 2.10 Chemical Analysis

Blank water-saturated n-octanol samples and aliquots of the wate r-saturated n-octanol from the test samples were prepared and analyzed to determine EFKA 8530 concentration. For each test sample, one aliquot was removed from only the n-octanol phase. The octanol phase of the samples was diluted into the standard range with THF prior to analysis. In addition, a sample of the stock solution (2.02 mg a.i./mL preliminar y test, 2.05 mg a.i./mL definitive test) was analyzed.

Calibration standards were analyzed with the test samples, one set prior to analysis of the test samples and the other set after the test samples. Three QC samples were prepared and analyzed with each of the test samples. Analysis of th e QC samples was used to judge the precision and

instrumental control maintained during the analytical process.

All aqueous test and QC samples were analyz ed for EFKA 8530 concentration by gel permeation chromatography with evaporative light scatte ring detection (GPC/ ELSD) based on methodology validated at Springborn Smithers. The method validation study was conducted prior to the

initiation of the definitive test and establ ished an average reco very of EFKA 8530 of 91.8% ± 2.10% from n-octanol ( Appendix 2 ). A cceptance criteria for the recovery of QC samples were set at 80 to 120% of the prepared concentration. Conditions and procedures used throughout the analysis of the te st and QC samples during this study were the same as those described in the method validation study. All octa nol test and QC samples were analyzed using

the same standards and instrumental conditions as in the method validation following dilution

into the calibration stan dard range with THF.Springborn Smithers Study No. 13658.6319 Page 14



### 3.0 CALCULATIONS

The partition coefficient (P ow ) for EFKA 8530 was calculated us ing the following equation:



### P C C

OW n - octanol

water 

### =

Where:

C n-octanol = the concentration as moles/L of EFKA 8530 in the n-octanol phase at equilibrium C water = the calculated concentration as moles/L of EFKA 8530 in the water phase (based upon total measured mass minus the measured mass in the octanol fraction divided by the volume of PRW)



### 4.0 RESULTS

4.1 Preliminary Results

The temperature in the environmental chamber during the preliminary test was maintained at 18.7 oC. Results of the prelim inary test are presented in Table 1 . The mean partition coefficient of the test substance was determined to be 22.0 (log P ow = 1.30).



### 4.2 Definitive Results

The temperature in the environmental chamber during definitive tes ting ranged from 25.8 to 26.6 oC. The partition coefficient for defi nitive test samples is summarized in Table 2 . The mean partition coefficient of the test substance was 24.9 (log P ow = 1.31). A representative chromatogram for a water-saturated n-oc tanol phase sample is presented in Figure 1

.

The results of quality control sample analysis performed in n-octanol during preliminary and definitive testing and stock solution analysis are summarized in Table 3 . Water-saturated noctanol quality control sam ples resulted in m easurements ranging from 96.0% to 104% duringSpringborn Smithers Study No. 13658.6319 Page 15

preliminary and definitive testing. Based on these results, it was established that the appropriate quality control was maintained during the analyses . Results of the stock solution analysis during the preliminary test and definitive test resulted in a recovery of 106% and 99.5%, respectively, of the nominal concentration (2020 and 2050 mg a.i./L, respectively). Representative chromatograms from the analysis of a quality control sample and a calibration standard are presented in Figure 2 and Figure 3 , respectively.Springborn Smithers Study No. 13658.6319 Page 16



### PROTOCOL DEVIATION

The protocol states that prelimin ary testing will be conducted in an environmental chamber in the range of 20 to 25 oC, held constant at ± 1 oC. During the preliminary test, the thermometer on one side of the chamber was 20 oC. The minimum/maximum thermometer used for the study had a temperature of 18.7 oC throughout the entire preliminary test . Due to the short period of time required to invert samples to 180o for 100 in versions, this temperature deviation did not have a negative impact on the resu lts or interpretation of the study.Springborn Smithers Study No. 13658.6319 Page 17



### REFERENCES

Dean, J.A., Ed. 1985. Lange’s Handbook of Chemistry. pp. 5; 102-103, 13 th ed., McGraw-Hill Book Company, New York.

OECD, 1995. OECD Guideline for Testing of Chemicals. Partition Coefficient (n-octanol/water): Shake Flask Metho d. Guideline #107. Adopted 27 July 1995.

OECD, 1998. OECD Series on Principles of Good Laboratory Pr actice and Compliance Monitoring. Number 1. OECD Principles on Good Laboratory Practice (as revised in 1997). Environment Directorate Chemi cals Group and management Committee. ENV/MC/CHEM(98)17. OECD Paris. France. 41 pp.Springborn Smithers Study No. 13658.6319 Page 18



### Table 1. Results of the preliminary n- octanol/water partition coefficient test with EFKA 8530.

Measured Concentration

Ratio Replicate Number n-Octanol Phase (moles/L) Calculated Water Phase

(moles/L) P ow Log P ow

1:1 a 1 3.49E-03 2.82E-04 12.4 1.09

2 3.65E-03 1.15E-04 31.7 1.50

Mean: 22.0 1.30

a pH = 5Springborn Smithers Study No. 13658.6319 Page 19



### Table 2. Results of the definitive n-oc tanol/water partition coefficient test with EFKA 8530.

Measured Concentration

Ratio (n-octanol:water) Replicate Number n-Octanol Phase (moles/L) Calculated Water Phase

(moles/L) P ow Log P ow

1:1 a 1 3.47E-03 1.17E-04 29.7 1.47

2 3.51E-03 7.59E-05 46.3 1.67

2:1 a 1 3.41E-03 9.18E-05 37.1 1.57

2 3.25E-03 1.68E-04 19.3 1.29

1:2 a 1 3.39E-03 3.92E-04 8.65 0.937

2 3.38E-03 4.11E-04 8.24 0.916

Mean: 24.9 1.31 Std. Dev.: 15.5 0.321 % CV: 62.3 24.5

a pH = 4Springborn Smithers Study No. 13658.6319 Page 20



### Table 3. Results of the analysis of th e EFKA 8530 n-octanol stock solution and quality control samples.

Preliminary Test

Sample Prepared Concentration (mg a.i./L) Measured Concentration (mg a.i./L) Percent Recovery (%)

n-Octanol QC 1 206 207 100 QC 2 1032 991 96.0 QC 3 2064 2000 96.9

Stock 126-09E 2020 2140 106

Definitive Test

Sample Prepared Concentration (mg a.i./L) Measured Concentration (mg a.i./L) Percent Recovery (%)

n-Octanol QC 1 206 214 104 QC 2 1032 1019 98.8 QC 3 2064 2075 101

Stock 126-09F 2050 2039 99.5Springborn Smithers Study No. 13658.6319 Page 21



### Figure 1. HPLC chromatogram of an EFKA 8530 water-sa turated n-octanol phase test solution from the determination of the partition coefficient.Springborn Smithers Study No. 13658.6319 Page 22



### Figure 2. HPLC chromatogram of a qua lity control sample (1032 mg a.i./L) from the determination of the part ition coefficient for EFKA 8530.Springborn Smithers Study No. 13658.6319 Page 23



### Figure 3. HPLC chromatogram of a calibra tion standard (25.0 mg a.i./L) from the determination of the partit ion coefficient for EFKA 8530.Springborn Smithers Study No. 13658.6319 Page 24



### APPENDIX 1 - STUDY PROTOCOLSpringborn Smithers Study No. 13658.6319 Page 25Springborn Smithers Study No. 13658.6319 Page 26Springborn Smithers Study No. 13658.6319 Page 27Springborn Smithers Study No. 13658.6319 Page 28Springborn Smithers Study No. 13658.6319 Page 29Springborn Smithers Study No. 13658.6319 Page 30Springborn Smithers Study No. 13658.6319 Page 31Springborn Smithers Study No. 13658.6319 Page 32Springborn Smithers Study No. 13658.6319 Page 33Springborn Smithers Study No. 13658.6319 Page 34



### APPENDIX 2 - ANALYTICAL METHODOLOGYSpringborn Smithers Study No. 13658.6319 Page 35

SUMMARY Methodology was validated (15 January 2008) to quantify the amount of EFKA 8530 present in recovery samples prepared in n-octanol. Recove ry samples were diluted with tetrahydrofuran and analyzed by automated injection on a gel pe rmeation chromatography system equipped with evaporative light scatteri ng detection (GPC/ELSD).

This method was validated by fortification of n-octanol with EFKA 8530 at concentrations of 206, 1032 and 2064 mg a.i./L. Recoveries averag ed 91.8% ± 2.10% and a limit of quantitation (LOQ) of 29.3 mg a.i./L. Defined limits for accep tance of quality control sample performance in subsequent studies were set at 80 to 120%.

EXPERIMENTAL Equipment 1. Instrument: Hewlett-Packard 1050 isocratic pump equipped with a Hewlett-Packard 1050 autosampler, Polymer Laboratories PL-ELS1000 eva porative light scattering detector, Hewlett-Packard 35900E A/D interface and a Hewlett Packard ChemStation Version A.06.03 for data acquisition 2. Balance: Orion 0960, Mettler Toledo AB204 3. Laboratory equipment: syringes, volu metric flasks, volumetric pipets, Pasteur pipets, glass centrifuge tubes, autosampler vials and amber glass bottles with Teflon ® -lined caps

Reagents 1. n-Octanol: Sigma-Aldrich, reagent grade 2. Tetrahydrofuran (THF): Bu rdick & Jackson, reagent grade

Test Substance The test substance, EFKA 8530, was received on 31 October 2007 from Ciba Specialty Chemicals Company, Allentown, Pennsylvania. The following information was provided:Springborn Smithers Study No. 13658.6319 Page 36

Name: EFKA 8530 Lot No.: 15306 CAS No.: Not Listed Purity: 93.56% Recertification Date: 31 January 2009

Upon receipt at Springborn Smithers, the test su bstance (SSL No. 126-09) was stored at room temperature in the original contai ner in a dark ventilated cabinet. Concentrations were adjusted for the purity of the test substance and ar e presented as active ingredient (a.i.).

PROCEDURES Preparation of Stock Solutions A 5.16 mg a.i./mL primary stock solution was prepared by placing 0.2760 g (0.2582 g as active ingredient) in a 50.0 mL volumetric flask and bri nging it to volume with n-Octanol. This stock solution was used to fortify recovery samples. Th is stock was stored refrig erated (2 to 8 oC) in an amber glass bottle fitted with 20 mm septa.

A 5.00 mg a.i./mL primary stock solution was prepared by placing 0.5344 g (0.5000 g as active ingredient) of the test substance in a 100-mL volumetric fl ask and bringing it to volume with tetrahydrofuran (THF). A 1000 mg a.i./L se condary stock solution was prepared by placing 10.0 mL of the 5.00 mg a.i./mL stock solution in a 50.0-mL volumetric flask and bringing it to volume with THF. A 100 mg a.i./L tertiary st ock solution was prepared by placing 5.00 mL of the 1000 mg a.i./L stock solution in a 50.0-mL fl ask and bringing it to volume with THF. The 1000 mg a.i./L and 100 mg a.i./L stock solutions were used to fortify the calibration standards used during the study. All stock solutions were stored refrigerated (2 to 8 o C) in amber glass

bottles fitted with Teflon ® -lined caps.

Preparation of Calibration Standards Calibration standards were prep ared in THF by fortifying with the 1000 mg a.i./L and the 100 mg a.i./L stock solutions to yiel d concentrations of 2.00, 5.00, 10.0, 25.0, 35.0 and 50.0 mg a.i./L.Springborn Smithers Study No. 13658.6319 Page 37

Sample Fortification and Preparation A set of recovery samples was prepared by fo rtifying n-octanol at concentrations of 206, 1032 and 2064 mg a.i./L. Sample volumes were 5.00 mL. Each concentration was prepared in triplicate. Recovery samples were fortified usi ng the 5.16 mg a.i./mL stock solution. In addition three samples were left unfortified to serve as controls. All samples were diluted into the calibration standard range w ith THF prior to analysis.

ANALYSIS

Instrumental Conditions The GPC/ELSD analysis was conducted utiliz ing the following instru mental conditions:

Column: TOSOH Bioscience TSKGel G1000Hxl, 5 μm, 300 x 7.8 mm Mobile Phase: 100% THF Flow Rate: 1.0 mL/minute Injection Volume: 500 μL Retention Time: approximately 5.7 minutes

Detector Parameters Temperature: Evaporator: 100 oC Nebulizer: 50 oC Transfer Line Temperature: 50 oC Gas Flow Rate: 1.0 L/minute (Nitrogen)

Preparation of Standard Curve Two sets of calibration standards were analyzed with each recovery sample set; one set prior to analysis of the recovery samples, and the sec ond set immediately following the analysis of the recovery samples. Injection of recovery samples and calibration standards onto the chromatographic system was performe d by programmed automated injection.

CALCULATIONS A polynomial calibration curve wa s constructed by plotting the na tural logarithm (ln) of the analyte concentration (mg a.i./L) in the calibration standards ag ainst the natural logarithm (ln) of the peak area of the calibration standards. The equation of the line (equation 1) wasSpringborn Smithers Study No. 13658.6319 Page 38

algebraically manipulated to give equation 2. The concentration of the test substance within each recovery sample was determined using th e regression coefficients from the quadratic equation, the natural logarithm (ln) of the peak area of the recovery sample and the dilution factor. Equations 2, 3 and 4 were then used to calculate measured concentrations and analytical

results.



## ( ) DF x DCA 4 x)inverse(ln (x) DC(3) 2a 4aC 2 bb lnx (2) cb(lnx) 2 a(lnx)lny (1)

= = −+−

= ++=

where:

y = detector response (peak area) for analyte a, b, c = regression constants x = detected concentration (mg a.i./L) in the sample C = constant c minus the natural log of the peak area; C = (c - lny) DC (x) = detected concentration in the sample DF = dilution factor (the final sa mple volume divided by the original sample volume) A = concentration of the analyt e in the original sample

RESULTS AND DISCUSSION The mean recovery was 91.8% with a standard de viation of 2.10%. The limit of quantitation was set at 29.3 mg a.i./L. Defined limits for accepta nce of quality control sample performance in subsequent studies were set at 80 to 120%.

Analytical results for the recovery of EF KA 8530 from n-octanol are presented in Table 1A

. Representative chrom atograms from the analysis of a calibration standard, recovery sample and a control are presented in Figure 1A , Figure 2A and Figure 3A , respectively. A typical regression analysis for EFKA 8530 is presented in Figure 4A

.Springborn Smithers Study No. 13658.6319 Page 39



### Table 1A. Analytical results for the re covery of EFKA 85 30 from n-octanol during the method validation study.

Sample

ID Fortified Concentration (mg a.i./L) Dilution Factor Analytical Result (mg a.i./L) Percent of Fortified (%) Control A 0.00 20.0 < 29.3 a NA b Control B 0.00 20.0 < 29.3 NA Control C 0.00 20.0 < 29.3 NA

Low A 206 20.0 188 91.4 Low B 206 20.0 193 93.9 Low C 206 20.0 194 94.2

Mid A 1032 33.3 945 91.6 Mid B 1032 33.3 950 92.0 Mid C 1032 33.3 964 93.4

High A 2064 66.7 1800 87.3 High B 2064 66.7 1900 91.8 High C 2064 66.7 1870 90.4

Mean: 91.8

SD: 2.10 RSD: 2.29

N: 9

a Concentrations expressed as less than values were be low the limit of quantitati on (LOQ). The LOQ for each analysis is dependent upon the linear regression, the ar ea of the low standards and the dilution factor of the controls. The limit of quantitation for the method validation was 29.3 mg a.i./L. b NA = Not Applicable.

Note: Results were calculated using the actual analy tical (unrounded) results and not the rounded values presented in this table.Springborn Smithers Study No. 13658.6319 Page 40



### Figure 1A. Representative chromatogram of a 10.0 mg a.i./L calibration standard used for quan tification during the method validation study with EFKA 8530.Springborn Smithers Study No. 13658.6319 Page 41



### Figure 2A. Representative chromatogram of a 1032 mg a.i./L recovery sample fortified with EFKA 8530.Springborn Smithers Study No. 13658.6319 Page 42



### Figure 3A. Representative chromatogram of a control sample during the method validation study with EFKA 8530.

Note: No peak equivalent to retention time for EFKA 8530. EFKA 8530 is ordinarily detected at a retention time of approximately 5.7 minutes.Springborn Smithers Study No. 13658.6319 Page 43



### Figure 4A. A typical regression analysis for the calibration standards used to quantitate EFKA 8530.

Polynomial Regression Analysis

y = -6.1918E-02x 2 + 2.2171E+00x + 5.0862E+00 R

2 = 9.9961E-01

0 2 4 6 8 10 12 14

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

ConcentrationDetector Response

Polynomial Regression Analysis

y = -0.061918x 2 + 2.2171x + 5.0862

R 2 = 0.99961

ln Standard Standard ln standard Standard Concentration Concentration R esponse Response (b^2-4aC)

(mg a.i./L) (mg a.i./L) Area Area D 0.693 2.00 6.6574 778.54065 4.52638 1.609 5.00 8.4155 4516.70215 4.09095 2.303 10.0 9.8889 19710.8 3.72603 3.219 25.0 11.5905 108063 3.30461 3.555 35.0 12.1870 196212 3.15688 3.912 50.0 12.7738 352841 3.01154 0.693 2.00 6.5736 715.91473 4.54715 1.609 5.00 8.4353 4606.87646 4.08605 2.303 10.0 9.9361 20662.1 3.71436 3.219 25.0 11.5971 108780 3.30297 3.555 35.0 12.1759 194046 3.15962 3.912 50.0 12.8291 372920 2.99783