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 
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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 

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

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

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

Materials and Methods.................................................................................................................................8 

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

Fat Matrix ............................................................................................................................................9 

Solvents and Reagents .........................................................................................................................9 

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

 Preliminary Study ...........................................................................................................................9 

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

Stock and Calibration Standards Preparation ....................................................................................10 

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

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

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

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

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

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TABLES

Table 1 Typical HPLC Operational Parameters .....................................................................................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 curve 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 μg/g..........................................................19 

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

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

APPENDICES 

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

Appendix 2 Certificate of Analysis ......................................................................................................32 

Appendix 3 Personnel Involved in the Study .......................................................................................34 

INTRODUCTION 

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 high performance liquid chromatography with ultraviolet 

detection (HPLC/UV). The preliminary portion of this study was conducted on August 29 and 30, 2005. 

The definitive portion of this study was conducted 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 

 

 

 
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Fat Matrix 

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

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

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

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

conditions. A Certificate of Analysis is presented in Appendix 2. 

Solvents and Reagents 

All solvents used in the 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 centrifuge 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 aliquot 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, the sample was removed and centrifuged at 10,000 rpm for 

five minutes at 37°C. An initial dilution in tetrahydrafuron (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. Samples 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 three 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 performed 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 and 0.0100 mg/mL stock solutions. The 1.00 mg/mL stock 

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

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

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

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

Stock 

Concentration 

(mg/mL)

Aliquot 

(μL)

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 substance 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-μm particle size). Typical instrumental parameters are presented in Table 1. 
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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 analyzed concurrently with the solubility samples. 

The calibration set was injected at the beginning and end of each analytical sequence. 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 was 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. Representative 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 μg/g was determined on Days 0 and 1. Mean 

analytical results were 78668 μg/g ± 5179 (RSD = 6.58%) and 77302 μg/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 μg/g. 
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REFERENCES 

 1. Organisation for Economic Cooperation and Development. 1981. OECD Guideline for Testing 

of Chemicals, 116: Fat Solubility of Solid and Liquid Substances. Adopted 12 May 1981. 
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Table 1 

Typical HPLC Operational Parameters 

INSTRUMENT: Agilent Model 1100 High Performance Liquid Chromatograph 

(HPLC) equipped with an Agilent Series 1100 Variable 

Wavelength Detector 

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

STOP TIME: 

FLOW RATE: 

SOLVENT A: 

SOLVENT B: 

10 minutes 

1.000 mL/min 

10% (0.1% H3PO4) 

90% (CH3CN) 

OVEN TEMPERATURE: 40°C 

INJECTION VOLUME: 10.0 μL 

APPROXIMATE 

RETENTION TIMES: 

Component #1 – 4.4 minutes 

Component #2 – 5.5 minutes 

Component #3 – 7.8 minutes 

WAVELENGTH: 200 nm 
 

 
 

 

 

 
 


 
 



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Appendix 2 

Certificate of Analysis 

Appendix 3 

Personnel Involved in the Study 

The following key