TEST SUBSTANCE:

### GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

SPONSOR:

TITLE:



# A'*

### QUALITY ASSURANCE STATEMENT

This study was examined for compliance with Good Laboratory Practice Standards as published by

the US. Environmental Protection Agency in 

### 40 CFR Part 792, 17 August 1989 and OECD Principles of

Good Laboratory Practice (ENV/MC/CHEM (98) 

### 17). 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:

ACTIVITY: DATE REPORTED TO:

DATE CONDUCTED: STUDY DIRECTOR: MANAGEMENT:

Protocol June 14,2005 June 14,2005 September 30,2005

Sample Processing September 14,2005 September 14,2005 September 20,2005

Data and Draft Report September 27-29,2005 September 29,2005 September 30,2005

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

All inspections were study-based unless otherwise noted.

Susan L. Coleman, 

### B.A.

Senior Quality Assurance Representative DATE

### -4-



### REPORT APPROVAL

MANAGEMENT:TABLE OF CONTENTS

Title/Cover Page ............................................................................................................... ...........................1

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

Quality Assurance Statement.................................................................................................... ...................3

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

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

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

Introduc tion................................................................................................................... ...............................8

Objective...................................................................................................................... ................................8

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

Materials an d Methods.......................................................................................................... .......................8

Test Substance ................................................................................................................. ....................8

Fat Ma trix ..................................................................................................................... .......................9

Solvents a nd Reagents .......................................................................................................... ...............9

Test Procedures................................................................................................................ ....................9

Prelimin ary Study ............................................................................................................. ..............9

Definitive Study.............................................................................................................. ................9

Stock and Calibration St andards Preparation .................................................................................... 10

Analytical Method .............................................................................................................. ...............10

Calibration Curve.............................................................................................................. .................11

Results and Discussion ......................................................................................................... .....................11

Conclusions.................................................................................................................... ............................11

References..................................................................................................................... .............................12TABLE OF CONTENTS

- Continued -

TABLES

Table 1 Typical HPLC Operat ional Para meters .....................................................................................13

Table 2 Preliminary Solubility Results of the Test Substance in Fat .....................................................14

Table 3 Definitive Solubility Results of the Test Substance in Fat ........................................................15

FIGURES

Figure 1 A representative calibration curv e for the test substance .......................................................16

Figure 2 A representative chromatogram of a low-level (10.0 mg/L) test substance

calibration standard ......................................................................................................... .......17

Figure 3 A representative chromatogram of a high-level (100 mg/L) test substance

calibration standard ......................................................................................................... .......18

Figure 4 A representative chromatogram of a Day 0 fat solubility sample at 30°C

(523C-111-30A), nominal concentration 100000 μ g/g..........................................................19

Figure 5 A representative chromatogram of a Day 0 fat solubility sample at 50°C

(523C-111-50A), nominal concentration 100000 μ g/g..........................................................20

APPENDICES

Appendix 1 Protocol and Protocol Amendment...................................................................................21

Appendix 2 Certificate of Analysis ......................................................................................................3 2

Appendix 3 Personnel Involv ed in the Study .......................................................................................34INTRODUCTION

Tests were conducted to determine the fat solubility of the test substance. This study was conducted

for analytical chemistry facility in Easton, Maryland.

The analyses of the samples were performed using hi gh performance liquid chromatography with ultraviolet

detection (HPLC/UV). The pre liminary portion of this study wa s conducted on August 29 and 30, 2005.

The definitive portion of this study was conducte d between September 13 and September 14, 2005. The

original raw data generated by and a copy of the final report are filed under

Project Number 523C-111 in archives located on the- 9 -

Fat Matrix

The fat matrix was received from NATEC Institut on September 25, 2002 and was assigned

The fa t matrix, an opaque solid, was identified as: HB 307

Fat Simulant; Batch No.: 36. The fat simulant is a synt hetic mixture of saturated triglycerides with a fatty

acid and triglyceride distribution similar to that of co conut fat. The fat matrix was stored under ambient

conditions. A Certificat e of Analysis is presented in Appendix 2.

Solvents and Reagents

All solvents used in th e solubility analyses were HPLC grade and all reagents were ACS grade or

better.

Test Procedures

Preliminary Study

For the preliminary study, 10 grams of fat was added to a Teflon ® centrifuge tube. Approximately

one gram of the test substance was added to the cen trifuge tube. The sample was vortexed, sealed and

placed in an agitating waterbath at approximately 37° C. After approximately one hour of agitation, the

sample was removed and centrifuged at 10,000 rpm for five minutes at 37°C. An a liquot of the sample was

diluted in methanol and analyzed by HPLC/UV. The remaining fat sample was placed back into the

waterbath for agitation overnight. The next day, th e sample was removed and centrifuged at 10,000 rpm for

five minutes at 37°C. An initial dilution in tetrahyd rafuron (THF) and a secondary dilution in methanol was

performed and then the sample was analyzed. Results for the preliminary study are presented in Table 2.

Definitive Study

For the definitive study, eight aliquots of 10 grams of fat were weighed into Teflon ® centrifuge tubes.

One gram of test substance was added to each tube. Sa mples were vortexed and sealed. Four samples were

placed in an agitating waterbath at approximately 50°C and four were placed in an incubator on an agitating

table at approximately 30°C. After approximately one hour, all samples were placed into an agitating

waterbath at approximately 37°C. After a th ree hour equilibration, two samples from each initial

temperature were placed in a non-agitating 37°C waterbath for one hour. The samples were initially diluted

in THF and secondary dilutions were performed in methanol and analyzed by HPLC/UV. At hour 24,agitation was stopped and the above procedure was perfor med for the remaining four samples. Results for

the definitive study are presented in Table 3.

Stock and Calibration Standards Preparation

For the preliminary study, a primary stock solution of the test substance was prepared by accurately

weighing 0.100 g of the test substance and transferring into a 100-mL volumetric flask. The flask was then

brought to volume with methanol. The resulting stock concentration contained 1.00 mg/mL, and was

serially diluted in methanol to produce 0.100 a nd 0.0100 mg/mL stock solutions. The 1.00 mg/mL stock

was used to prepare a 0.0500 mg/mL calibration standa rd in 50% methanol : 50% HPLC reagent grade

water. The 0.0100, 0.0500, 0.100 and 1.00 mg/mL stock soluti ons were used as calibration standards.

The primary stock solution (1.00 mg/mL) was used to pr epare the set of standards in methanol used in

the definitive study. The following shows the dilu tion scheme for the set of calibration standards:

Stock

Concentration

(mg/mL) Aliquot

( μ L) Final

Volume

(mL) Standard

Concentration

(mg/L)

1.00

1.00

1.00

1.00

1.00 100

250

500

750

1000 10.0

10.0

10.0

10.0

10.0 10.0

25.0

50.0

75.0

100

Analytical Method

The method used for the analysis of the test s ubstance in fat was based upon methodology developed

by The analytical method consisted of diluting the samples in THF followed by

a secondary dilution in methanol and submitting for analysis using high performance liquid

chromatography.

Concentrations of the test substance in the samples were determined by HPLC using an Agilent Series

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

Wavelength Detector. Chromatographic separations were achieved using a YMC Pack ODS AM column

(150 mm x 4.6 mm, 3- μ m particle size). Typical instrumental parameters are presented in Table 1.- 11 -

Calibration Curve

Five calibration standards of the test substance were prepared in methanol. The calibration standards,

ranging in concentration from 10.0 to 100 mg/L, were anal yzed concurrently with the solubility samples.

The calibration set was injected at the beginning and end of each analytical seque nce. Linear regression

equations were generated using the sum of the peak area responses of the calibration standards. The

concentration of the test substance in the samples wa s determined by substituting the sum of the peak area

responses of the samples into the applicable linear regression equation. A representative calibration curve

for the test substance is presented in Figure 1. Repr esentative chromatograms of low- and high-level test

substance calibration standards are presented in Figures 2 and 3, respectively.

RESULTS AND DISCUSSION

The solubility of the test substance in fat at 100000 μ g/g was determined on Days 0 and 1. Mean

analytical results were 78668 μ g/g ± 5179 (RSD = 6.58%) and 77302 μ g/g ± 1549 (RSD = 2.00%),

respectively. Results are summarized in Table 3. Representative chromatograms of Day 0 fat solubility

samples are presented in Figures 4 and 5, respectively.

CONCLUSIONS

The solubility of the test substance was determined in fat at approximately 37°C and analyzed by high

performance liquid chromatography. Based on the results, the test substance is soluble at 77985 μ g/g.- 12 -

REFERENCES

1. Organisation for Economic C ooperation and Development. 1981. OECD Guideline for Testing

of Chemicals, 116: Fat Solubility of Solid and Liquid Substances. Adopted 12 May 1981.- 13 -

Table 1

Typical HPLC Operational Parameters

INSTRUMENT: Agilent Model 1100 High Perfor mance Liquid Chromatograph

(HPLC) equipped with an Ag ilent Series 1100 Variable

Wavelength Detector

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

STOP TIME:

FLOW RATE:

SOLVENT A:

SOLVENT B: 10 minutes

1.000 mL/min

10% (0.1% H 3 PO 4 )

90% (CH 3 CN)

OVEN TEMPERATURE: 40°C

INJECTION VOLUME: 10.0 μ L

APPROXIMATE

RETENTION TIMES: Component #1 – 4.4 minutes

Component #2 – 5.5 minutes

Component #3 – 7.8 minutes

WAVELENGTH: 200 nm- 24 -- 29 -Appendix 2

Certificate of AnalysisAppendix 3

Personnel Involved in the Study

The following key