TEST SUBSTANCE: ) 

DETERMI 
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT 

SPONSOR: 
QUALITY ASSURANCE STATEMENT 

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

the U.S. Environmental Protection Agency in 40 CFR Part 160 andlor 792, 17 August 1989 and OECD 

Principles of Good Laboratory Practice, (ENVIMCICHEM (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 7,2005 June 7,2005 September 14,2005 

Sample Collection and Processing August 29,2005 August 29,2005 September 7,2005 

Data and Draft Report September 12 and 13,2005 September 13,2005 September 15,2005 

Final Report September 20,2005 September 20,2005 September 20,2005 

All inspections were study-based unless otherwise noted. 

Marshall T. Hynson DATE ' L 

~ualig~ssurkce Program supe&isor 
-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 

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

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

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

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

Test Substance .....................................................................................................................................9 

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

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

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

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

Stock and Calibration Standards Preparation ......................................................................................9 

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

Calibration Curve...............................................................................................................................10 

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

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

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

- Continued - 

TABLES

Table 1 Typical HPLC Operational Parameters .....................................................................................13 

Table 2 Solubility Results of the Test Substance in HPLC-Grade Reagent Water ................................14 

FIGURES 

Figure 1 Analytical method flowchart for the analysis of the test substance in HPLC-grade 

reagent water ..........................................................................................................................15 

Figure 2 A representative calibration curve for the test substance .......................................................16 

Figure 3 A representative chromatogram of a low-level (5.00 mg/L) test substance 

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

Figure 4 A representative chromatogram of a high-level (50.0 mg/L) test substance 

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

Figure 5 A representative chromatogram of a Day 1 water solubility sample 

 19 

APPENDICES 

Appendix 1 Protocol, Protocol Amendments and Deviation................................................................20 

Appendix 2 Personnel Involved in the Study .......................................................................................34 

INTRODUCTION 

Tests were conducted to determine the solubility of the test substance in HPLC-grade reagent water. 

This study was conducted for 

 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 22 and 23, 2005. The definitive portion of this study was conducted between 

August 26 and August 29, 2005. The original raw data generated by and a copy 

of the final report are filed under Project Number 523C-104 in archives located on the 

OBJECTIVE 

The objective of this study was to determine the solubility of the test substance at ambient 

temperature. 

EXPERIMENTAL DESIGN 

The water solubility of the test substance was determined at ambient temperature (approximately 

22°C). The test consisted of equilibration of an excess amount of test substance with HPLC-grade reagent 

water at approximately 22°C. Quantitation of soluble test substance in the aqueous phase was performed on 

Days 1, 2 and 3. 

MATERIALS AND METHODS 

This study was conducted according to the procedures outlined in the corresponding protocol 

(Appendix 1). Due to physical properties of the test substance, the test procedure was modified and 

performed, based in part, on procedures in the U.S. EPA Product Properties Test Guidelines, OPPTS, 

Series: 830.7840, Water Solubility: Column Elution Method; Shake Flask Method (1) utilizing shake flask 

methodology. Additional guideline was provided in OECD Guideline for Testing of Chemicals, 105: 

Water Solubility (2), and TSCA Title 40 of the Federal Code of Regulations, Part 796, Section 1840: Water 

Solubility (3). 
 

 

 

 

 
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standards in 50% methanol : 50% H2O. 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 

500 

1500 

2500 

3500 

5000 

100 

100 

100 

100 

100 

5.00 

15.0 

25.0 

35.0 

50.0 

Analytical Method 

The method used for the analysis of the test substance in HPLC-grade reagent water was based upon 

methodology developed by The analytical method consisted of diluting the 

samples in 50% methanol : 50% HPLC-grade bottled water and submitting for analysis using high 

performance liquid chromatography. 

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

Model 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 

and a method flowchart is provided in Figure 1. 

Calibration Curve 

Five calibration standards of the test substance were prepared in 50% methanol : 50% H2O. The 

calibration standards, ranging in concentration from 5.00 to 50.0 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 2. Representative chromatograms of low- and 

high-level test substance calibration standards are presented in Figures 3 and 4, respectively. 

RESULTS AND DISCUSSION 

 The solubility of the test substance in water at 8500 mg/L was determined on Days 1, 2 and 3. Mean 

analytical results were 8098 mg/L ± 31.6 (RSD = 0.390%), 8225 mg/L ± 118 (RSD = 1.43%) and 

8261 mg/L ± 134 (RSD = 1.62%) mg/L, respectively. Results are summarized in Table 2. A representative 

chromatogram of a Day 1 water solubility sample is presented in Figure 5. 

CONCLUSIONS 

 The solubility of the test substance was determined in HPLC-grade reagent water at approximately 

22°C and analyzed by high performance liquid chromatography. Based on the results, the test substance is 

soluble at 8500 mg/L. 
REFERENCES 

 1. U.S. Environmental Protection Agency 1996. Water Solubility: Column Elution Method; Shake 

Flask Method. Product Property Test Guidelines. OPPTS 830.7840. 

 2. Organisation for Economic Cooperation and Development. 1995. Guideline for Testing of 

Chemicals, 105: Water Solubility.

 3. TSCA Title 40 of the Federal Code of Regulations. 1994. Part 796, Section 1840: Water 

Solubility.
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: 25.0 μL 

APPROXIMATE 

RETENTION TIMES: 

Component #1 – 4.4 minutes 

Component #2 – 5.5 minutes 

Component #3 – 7.8 minutes 

WAVELENGTH: 200 nm 
- 14 - 

Table 2 

Solubility Results of the Test Substance in HPLC-Grade Reagent Water 

Nominal 

Concentration 

(mg/L) 

Sample 

I.D. 

(523C-104-) 

Sampling 

Time 

(Day) 

Temp. at 

Sampling 

(°C) 

Measured 

Concentration 

(mg/L)1,2

Percent 

of 

Nominal1

Mean Measured 

Std. Dev. (SD) 

RSD.1

8500 

8500 

8500 

8500 

8500 

8500 

8500 

8500 

8500 

D1A 

D1B 

D1C 

D2A 

D2B 

D2C 

D3A 

D3B 

D3C 

1 

1 

1 

2 

2 

2 

3 

3 

3 

22.6 

22.5 

22.0 

8109 

8123 

8062 

8089 

8297 

8290 

8107 

8344 

8332 

95.4 

95.6 

94.9 

95.2 

97.6 

97.5 

95.4 

98.2 

98.0 

Mean = 8098 

SD = 31.6 

RSD = 0.390% 

Mean = 8225 

SD = 118 

RSD = 1.43% 

Mean = 8261 

SD = 134 

RSD = 1.62% 

 Mean = 

Std. Dev. = 

RSD = 

8195 

117 

1.43 

 

1

 Results were generated using Excel 2000 in the full precision mode. Manual calculations may differ slightly. 

2 The limit of quantitation (LOQ) was 1665 mg/L, calculated as the product of the concentration of the lowest 

calibration standard (5.00 mg/L) and the dilution factor of the samples (333). 
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Figure 1. Analytical method flowchart for the analysis of the test substance in HPLC-grade reagent 

water. 
0

200

400

600

800

1000

1200

0.00 10.00 20.00 30.00 40.00 50.00 60.00

Concentration (mg/L)

Peak Area 

Figure 2. A representative calibration curve for the test substance 

Slope = 20.63516; Y-Intercept = -9.28055; R2

 = 0.99991 
Figure 3. A representative chromatogram of a low-level (5.00 mg/L) test substance calibration standard. 
Figure 4. A representative chromatogram of a high-level (50.0 mg/L) test substance calibration standard. 
Figure 5. A representative chromatogram of a Day 1 water solubility sample (523C-104-D1B), nominal 

concentration 8500 mg/L. 
Appendix 1 

Protocol, Protocol Amendments and Deviation 



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Appendix 2 

Personnel Involved in the Study 

The following key