TEST SUBSTANCE:

DETERMINATION OF n -OCTANOL/WATER PARTITION COEFFICIENTGOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

SPONSOR:

~

### QUALITY ASSURANCE STATEMENT

This study was examined for compliance with OECD Principles of Good Laboratory Practice, (ENVIMCICHEM (98)17) and Title 40 of the Code of Federal Regulations, Parts 160 and 792, Good Laboratory Practice Standards, 1989. 

### The dates of all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows:

DATE REPORTED TO:

ACTIVITY: DATE CONDUCTED: STUDY DIRECTOR: MANAGEMENT:

Protocol August 9,2005 August 10,2005 August 22,2005

Sample Preparation August 18,2005 August 18,2005 September 29,2005

Raw Data and Draft Report September 27-28, 2005 September 28, 2005 September 29,2005

Final Report October 3,2005 October 3, 2005 October 4,2005



### All inspections were study-based unless otherwise noted.

Quality Assurance Representative

### REPORT APPROVAL

SPONSOR:

TITLE :

STUDY DIRECTOR:



# ,//~

/ 

# /& ,?J.('

~Koth? Z. Kendall, M.S. Laboratory Supervisor Wildlife International, Ltd.

MANAGEMENT:

Willard B. Nixon, P~.D. Director of Chemistry Wildlife International, Ltd. DATE



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DATE- 5 -

TABLE OF CONTENTS

Title/Cover Pa ge ............................................................................................................... ...................... 1

Good Laboratory Practice Compliance Statement .................................................................................. 2

Quality Assurance Statement

.................................................................................................... .............. 3

Report Approval ................................................................................................................ ..................... 4

Table of Contents.............................................................................................................. ...................... 5

Summary........................................................................................................................ ......................... 7

Introduction ................................................................................................................... ......................... 8

Purpose ........................................................................................................................ ........................... 8

Experimental Design ............................................................................................................ .................. 8

Materials and Methods.......................................................................................................... .................. 9
Test Substance................................................................................................................. ............... 9
Reference Standards ............................................................................................................ ........... 9 Solvents and Reagents.......................................................................................................... ........ 11 Mobile Phase Preparation....................................................................................................... ...... 11 Calibration Stocks and Standards Preparation .............................................................................. 11 Test Substance Stock and Solution Preparation............................................................................ 11

Test Procedure................................................................................................................. ............. 11 Data Analysis.................................................................................................................. .............. 12

Results ........................................................................................................................ .......................... 12

Conclusions .................................................................................................................... ...................... 13

References ..................................................................................................................... ....................... 14- 6 -

TABLE OF CONTENTS

- Continued -

TABLES

Table 1 - Preparation of Reference Stocks and Standard Solutions..................................................... 15

Table 2 - HPLC Operational Parameters ............................................................................................. 16

Table 3 - Reference Standards Retention Times, Capacity Factors, and Log K OW Values .................. 17

Table 4
- n -Octanol/Water Partition Coefficients (Log K OW ) for Test Substance................................ 18

FIGURES

Figure 1 - Reference standard calibration curve .................................................................................. 20

Figure 2 - A representative chromatogram of a thiourea reference standard ....................................... 21

Figure 3 - A representative chromatogram of an acet anilide reference standa rd ................................. 22

Figure 4 - A representative chromatogram of a 2-nitrophenol reference standard............................... 23

Figure 5 - A representative chromatogram of a toluene reference standard......................................... 24

Figure 6 - A representative chromatogram of a naphthalene reference standard................................. 25

Figure 7 - A representative chromatogram of a phenanthrene reference standard............................... 26

Figure 8 - A representative chromatogram of a ............................................ 27

Figure 9 - A representative chromatogram of the test substance ................................ 28

APPENDICES

Appendix 1 - Protocol..................................................................................................................... 29

Appendix 2 - Example Calculation................................................................................................. 39

Appendix 3 - Personnel Involved in the Study................................................................................ 40- 7 -

SUMMARY

Sponsor:

Sponsor’s Representative:

Location of study, raw data and a copy of the final report:

Wildlife International, Ltd. Project Number:

Test Substance:

Study:

Test Dates: Experimental Start (OECD): August 18, 2005 Experimental Start (EPA): September 6, 2005 Experimental Termination: September 7, 2005

Summary: Under the chromatographic conditions specified in guidelines, the test substance eluted as

15 resolved peaks. The corresponding partition coefficients (Log K OW ) for the test substance

were calculated as unretained, 1.06, 1.65, 2. 20, 2.62, 3.76, 4.19, 5. 33, 5.68, 5.79, 5.83,

(6.38), (6.47), (6.68) and (7.21). The values in brackets were extrapolated.- 8 -

INTRODUCTION

determined the n -octanol/water partition coefficient of the test substance

using the high performance liquid chromatography estimation method. The study was conducted at the

analytical chemistry facility in The study was performed

following procedures in the Product Properties Test Guidelines, OPPTS 830.7570, Partition Coefficient

(n-Octanol/Water), Estimation by Liquid Chromatography (1). This method is based on procedures

presented in the OECD Guideline for the Testing of Chemicals, 117: Partition Coefficient

(n-octanol/water), High Performance Liquid Chromatography (HPLC ) Method (2); Official Journal of the

European Communities No . L383 Method A.8: Partition Coefficient (3); and TSCA Title 40 of the

Federal Code of Regulations, Part 796, Section 1570: Partition Coefficient (n-Octanol/Water)
- Estimation by Liquid Chromatography (4).

The n -octanol/water partition coefficient of the test substance was determined using high

performance liquid chromatography (HPLC) with ultraviolet detection (UV). The study was identified as

Project Number All raw data generated by and a copy of the final

report are filed under Project Number in archives located on the

PURPOSE

The purpose of this study was to experimentally determine an estimate of the n -octanol/water

partition coefficient (K OW ) of the test substance, by a reverse-phase HPLC method.

EXPERIMENTAL DESIGN

The study was conducted according to procedures ou tlined in the corresponding protocol (Appendix

1). The test substance and a series of reference substances bracketing the applicable Log K OW range of the

HPLC estimation method were separately prepared in acetonitrile and diluted in 75% acetonitrile:25%

HPLC grade bottled water (v/v). The reference and test substance solutions were sequentially injected into

an HPLC system operating under standardized isocra tic, reverse-phase operating conditions employing a

C 18 -based stationary phase. Capacity factors were calculated for the reference standards and test substance

using thiourea to estimate column dead time ( i.e . the retention time of an unretained reference standard).

The logarithms of the capacity factors were then plotted against published Log K OW values to establish a- 9 -

linear regression calibration. The capacity factors of the test substance were used in conjunction with the

linear regression equation to calculate the Log K OW range for the test substance.

MATERIALS AND METHODS

Test Substance

The test substance was received from on April 19, 2005 a nd was assigned- 10 -

standard had a reported purity of 99.3%, was assigned an expiration date of March 19, 2006, and was

stored under ambient conditions.

Toluene was received from nd was assigned

The reference st andard, a liquid, was

identified as: toluene; lot number 06238 LQ; The reference standard had a

reported purity of 99.94%, was assigned an expiration date of March 19, 2006, and was stored under

ambient conditions.Solvents and Reagents

All solvents used in this study were HPLC grade or equivalent. All reagents were ACS reagent

grade or equivalent. A reagent blank prepared and analyzed concurrently with the reference and test

substances confirmed that the reagents were free of potentially interfering components.

Mobile Phase Preparation

The HPLC mobile phase was 75% acetonitrile : 25% water with 0.1% phosphoric acid (v/v). The

mobile phase was degassed prior to use.

Calibration Stocks and Standards Preparation

Six reference standards were used to generate a Log K Ow calibration curve. Calibration stock

solutions of the individual reference standards were prepared separately by dissolving each in acetonitrile.

Calibration standard solutions were prepared from these stock solutions of the reference standards by

diluting appropriately in 75% acetonitrile: 25% HPLC grade water (v/v). The concentrations of the

reference standards were adjusted, as necessary, to gi ve a detector response of at least 900 milli-absorbance

units for peak height. Details on the preparation of the calibration standards are provided in Table 1.

Test Substance Stock and Solution Preparation

A stock solution of the test subs tance was prepared by accurately weighing 0.500 g of the test

substance into a 20-mL glass scintillation vial. The test substance was then quantitatively transferred into

a 100-mL volumetri c flask. The stock solution was brought to volume using acetonitrile. This primary

stock solution contained 5.00 mg/mL. Subsequent dilution of the acetonitrile stock solution yielded a 50.0

μ g /mL solution that was prepared using mobile phase. Each of the triplicate injections (100 μ L) of this

solution gave a detector response of at least 100 m illi-absorbance units for the p eak height of the most

sensitive peak.

Test Procedure

Retention times of the test substance chromatographic components and reference standards were

determined using an Agilent Series 1100 High Performance Liquid Chromatograph (HPLC) equipped with

an Agilent Series 1100 variable wavelength UV detector. Chromatographic separations were achieved

using a YMC-Pack ODS-AM C 18 column (150 mm x 4.6 mm, 3- μ m particle size). The columntemperature was maintained at 40 ° C. The flow rate was 1.0 mL/minute. Prior to the test substance

analyses, the dead time was determined by injecting thiourea. The instrument parameters are summarized

in Table 2.

Reference standards (100 μ L injection volume) were injected individually prior to and after the test

substance to determine retention times. The test substance was injected, in triplicate, following the initial

reference standard injections and was analy zed under the same chromatographic conditions.

Data Analysis

The capacity factor (k ′ ), was calculated for the each of the test substance chromatographic

components and each reference standard using the following equation:

k ′ = (t R – t 0 )/t 0

where t R was the retention time of the test substance or reference standard and t 0 was the column dead time

established with thiourea. A correlation graph of Log k ′ versus Log K OW for the reference standards was

plotted and fitted to a regression equation in the form y = mx + b. The calibration curve is presented in

Figure 1. Log K OW for each component of the test substance was calculated by substituting the calculated

logarithm of the capacity factor for each component into the linear regression equation for the calibration

curve. An example calculation is presented in Appendix 2.

RESULTS

Along with analysis of reference standards, a standard of thiourea was injected in duplicate (once

near the beginning and once near the end of the HPLC sequence) to determine the analytical column dead

time (t 0 ) for use in calculating capacity factors (k ′ ) of the reference standards and the test substance. The

mean retention time of the duplicate thiourea standard injections was 1.619 minutes.

Six additional reference standards were analyzed with the test substance. The reference standard

series was injected at the beginning and at the end of the HPLC sequence. The capacity factors of each

standard were calculated based upon their retention times. Calculated Log k ′ values for acetanilide, 2-

nitrophenol, toluene, naphthalene, phenanthrene, and DDT reference standards are presented in Table 3.The reference standard calibration curve is presented in Figure 1. Typical reference standard

chromatograms are presented in Figures 2 through 8.

The test substance was sequentially injected in triplicate. The test substance eluted as 15 resolved

peaks. The peak retention times

The capacity factors (k ′ ) for the retained were then

calculated based on the above retention time range. The corresponding partition coefficients (Log K OW )

for the test substance were calculated to range from 1.06 to an extrapolated value of 7.21. The calculated

mean retention times, capacity factors, and mean partition coefficients for the test substance are presented

in Table 4. A representative chromatogram of the test substance is presented in Figure 9.

CONCLUSIONS

Under the chromatographic conditions specified in guidelines, the test substance eluted as

15 resolved peaks. The corresponding partition coefficients (Log K OW ) for the test substance were

calculated as unretained, 106, 1.65, 2.20, 2.62, 3.76, 4. 19, 5.33, 5.68, 5.79, 5.83, (6.38), (6.47), (6.68) and

(7.21). The values in brackets were extrapolated.REFERENCES

1. U.S. Environmental Protection Agency. 1996. Partition Coefficient (n-Octanol/Water), Estimation by the Liquid Chromatography . Series 830 – Product Properties Test Guidelines,

OPPTS Number 830.7570.

2. Organisation for Economic Cooperation and Development . 2001. Partition Coefficient (noctanol/water) High Performance Liquid Chromatography (HPLC) Met hod. OECD Guideline for Testing of Chemicals, Proposal for Updating Guideline 117. Revised draft document November

2001.

3. Annex to Commission Directive 92/69/EEC. 1992. Partition Coefficient . Official Journal of the European Communities No . L383. Method A.8.

4. TSCA Title 40 of the Code of Federal Regulations. 1994. Part 796, Section 1570; Partition Coefficient (n-Octanol/Water) – Es timation by Liqui d Chromatography.

5. Title 40 of the Code of Federal Regulations, Part 792 . 1989. Toxic Substances Control Act; (TSCA); Good Laboratory Practice Standards; Final Rule.

6. Organisation for Economic Cooperation and Development . 1998. OECD Principles of Good Laboratory Practice. ENV/MC/CHEM (98) 17, Environment Directorate, Paris.

7. Ministry of Agriculture, Forestry and Fisheries, Japan (MAFF). 1999. Good Laboratory Practice (GLP) for Agricultural Chemicals. 11 NohSan, Notification No. 6283, Agricultural Production Bureau, 1 October 1999.Table 1

Preparation of Reference Stocks and Standard Solutions

Reference

Substance Amount Weighed

(mg) Final

Volume

(mL) 1 Stock

Concentration (mg/mL) Initial

Volume

(mL) 2 Final

Volume

(mL) 3 Standard

Concentration ( μ g/mL)

1 Solutions were brought to final volume with acetonitrile in volumetric flasks.

2 Initial volumes measured with a gas-tight syringe.

3 Solutions were brought to final volume with 75% acetonitrile:25% HPLC grade water (v/v) in

graduated centrifuge tubes.Table 2

HPLC Operational Parameters

INSTRUMENT: Agilent Series 1100 High Performance Liquid Chromatograph (HPLC) with an Agilent Series 1100 Variable Wavelength (UV)

Detector

ANALYTICAL COLUMN: YMC-Pack ODS-AM C 18 (150 mm x 4.6 mm, 3- μ m)

STOP TIME: 40 minutes

FLOW RATE: 1.0 mL/minute

OVEN TEMPERATURE: 40 ° C

MOBILE PHASE: Acetonitrile:0.1% H 3 PO 4 ; 75:25, v/v

INJECTION VOLUME: 100 μ L

APPROXIMATE- 17 -

Table 3

Reference Standards Retention Times, Capacity Factors, and Log K OW Values

Reference
Standard Concentration ( μ g/mL) Retention
Time (minutes) Capacity

Factor (k ′ ) 1,2 Log k ′ 2 Log K OW 3

1 Capacity factor (k ′ ) is the standard retention time minus the mean column dead time (1.619 minutes for thiourea) divided by the mean column dead time.

2 Values obtained u tilizing Excel 2000 in full precision mode. Ma nual calculations may differ slightly.

3 Log K OW values are based on literature values (see reference 1).- 18 -

Table 4

n -Octanol/Water Partition Coefficients (Log K OW ) for Test Substance

Sample
Number- 19 -

Table 4 (Continued)

n -Octanol/Water Partition Coefficients (Log K OW ) for Test Substance

Sample
Number Retention
Time minutes) Mean (  ̄ x )
Standard Deviation (SD) 2 Capacity

Factor (k ′ ) 1,2 Log
k ′ 2 Log

K OW 2,3 Mean (  ̄ x ) 2,3

Standard Deviation (SD)

11.401
11.387
11.354  ̄ x = 11.381
SD = 0.02413 6.044
6.036
6.015 0.7813
0.7807
0.7792 5.83
5.83
5.82  ̄ x = 5.83
SD = 0.00

4 (Peak 12) 5 (Peak 12) 6 (Peak 12) 15.633
15.611
15.565  ̄ x = 15.603
SD = 0.03470 8.659
8.645
8.617 0.9375
0.9368
0.9354 (6.38) (6.38) (6.38)  ̄ x = (6.38)
SD = 0.00

4 (Peak 13) 5 (Peak 13) 6 (Peak 13) 16.493
16.465
16.416  ̄ x = 16.458
SD = 0.03897 9.190
9.173
9.143 0.9633
0.9625
0.9611 (6.47) (6.47) (6.47)  ̄ x = (6.47)
SD = 0.00

4 (Peak 14) 5 (Peak 14) 6 (Peak 14) 18.636
18.609
18.554  ̄ x = 18.600
SD = 0.04179 10.514
10.498
10.464 1.0218
1.0211
1.0197 (6.68) (6.68) (6.67)  ̄ x = (6.68)
SD = 0.00

4 (Peak 15) 5 (Peak 15) 6 (Peak 15) 25.665
25.624
25.548  ̄ x = 25.612
SD = 0.05937 14.857
14.832
14.785 1.1719
1.1712
1.1698 (7.21) (7.21) (7.20)  ̄ x = (7.21)
SD = 0.00

1 Capacity factor (k’) is the sample retention time minus the mean column dead time (1.619 minutes for thiourea) divided by the mean column dead time.

2 Values obtained utilizing Excel 2000 in full precision mode. Manual calculations may differ slightly.

3 Values in parenthesis are extrapolated values.- 20 -

- 1. 0 - 0. 5 0. 0 0. 5 1. 0

0. 0 1. 0 2. 0 3. 0 4. 0 5. 0 6. 0 7. 0

Log KowLog k

Figure 1. Reference standard calibration curve.min 0 5 10 15 20 25 30 35 mAU

0 500 1000 1500 2000 VWD1 A, Wavelength=210 nm (POW002\002-0201.D)

Figure 2. A representative chromatogram of a thiourea reference standard.- 22 -

min 0 5 10 15 20 25 30 35 mAU

0 500 1000 1500 2000 VWD1 A, Wavelength=210 nm (POW002\003-0301.D)

Figure 3. A representative chromatogram of an acetanilide reference standard.- 23 -

min 0 5 10 15 20 25 30 35 mAU

0 500 1000 1500 2000 VWD1 A, Wavelength=210 nm (POW002\004-0401.D)

Figure 4. A representative chromatogram of a- 24 -

min 0 5 10 15 20 25 30 35 mAU

0 500 1000 1500 2000 VWD1 A, Wavelength=210 nm (POW002\005-0501.D)

Figure 5. A representative chromatogram of a toluene reference standard.- 25 -

min 0 5 10 15 20 25 30 35 mAU

0 500 1000 1500 2000 VWD1 A, Wavelength=210 nm (POW002\006-0601.D)

Figure 6. A representative chromatogram of a naphthalene reference standard.- 26 -

min 0 5 10 15 20 25 30 35 mAU

0 500 1000 1500 2000 VWD1 A, Wavelength=210 nm (POW002\007-0701.D)

Figure 7. A representative chromatogram of a phenanthrene reference standard.min 0 5 10 15 20 25 30 35 mAU

0 200 400 600 800 1000 VWD1 A, Wavelength=210 nm (POW002\008-0801.D)12.168

Figure 8. A representative chromatogram of a DDT reference standard.min 0 5 10 15 20 25 30 35 mAU

0 50 100 150 200 VWD1 A, Wavelength=210 nm (POW002\009-0901.D)1.4491.8952.0582.2612.5382.8304.1684.9898.67910.49011.14211.40115.63316.49318.63625.665

Figure 9. A representative chromatogram of the test substance- 29 -

Appendix 1

Protocol- 30 -- 32 -- 35 -- 37 -- 38 -- 39 -

Appendix 2

Example Calculation

An example calculation is presented for the first injection of- 40 -

Appendix 3

Personnel Involved in the Study

The following key . personnel were involved in the conduct or

management of this study:

1. Willard B. Nixon, Ph.D., Director of Chemistry
2. Timothy Z. Kendall, Laboratory Supervisor

3. Frank J. Lezotte, B.S., Chemist