determination of the dissociation constant of h- 28308

Revision 1 Dupont-24198

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TRADE SECRET

Study Title

DETERMINATION OF THE DISSOCIATION CONSTANT OF H-28308

Test Guidelines U.S. EPA Product Properties Test Guidelines OPPTS 830.7370, Dissociation Constant (1996) OECD Guideline 112 (1981)

Authors Willard B. Nixon, Ph.D. Frank J. Lezotte, B.S.

Date Study Initiated February 21, 2008

Date Study Completed April 1, 2008

Date Revision Completed June 20, 2008

Performing Laboratory Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 U.S.A.

Sponsor E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A.

DuPont Project Identification Numbers DuPont Report No.: Dupont-24198 Work Request No.: 17474 Service Code: 1649

Wildlife International, Ltd. Study Number 112C-135

Revision 1 Determination of the Dissociation Constant of H-28308 Dupont-24198

2

PAGE RESERVED

STATEMENT OF CONFIDENTIALITY This report is the property of E.I. du Pont de Nemours and Company and contains confidential and trade secret information. Except as required by law, this report should not be partially or fully (i) photocopied or released in any form to an outside party without the prior written consent of E.I. du Pont de Nemours and Company or its affiliates, or (ii) used by a registration authority to support the registration of any other product without the prior written consent of E.I. du Pont de Nemours and Company or its affiliates.

Detennination of the Dissociation Constant ofH-28308 Revision l

Dupont-24198

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

This study was conducted in compliance with the following GLP principles:

• United States Environmental Protection Agency, (TSCA), Title 40 Code of Federal Regulations Part 792 (effective August 17, 1989)

Which are consistent with:

• The OECD Principles of Good Laboratory Practice (ENV/MC/CHEM(98)17) (Paris, 1998)

with the following exceptions:

The reference substance ,,vas not characterized in accordance with Good Laboratory Practice Standards.

The stability of the test substance and reference substance under the storage conditions at the test site was not determined in compliance with Good Laboratory Practice Standards.

Applicant/Sponsor

E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A.

DuPont Representative

Date

Date

3

Determination of the Dissociation Constant ofH-28308

QUALITY ASSURANCE STATEMENT

Study Number

112C-135 (Wildlife International, Ltd.)

Study Title

Detennination of the Dissociation Constant ofH-28308

Dates Findings Reported to Study

Activity Audited Audit Dates Director

Protocol February 15, 2008 February 15, 2008

Test Initiation February 21, 2008 February 21, 2008

Data and Draft Report March 29 - 31, 2008 March 31, 2008

Final Report April 1, 2008 April 1, 2008

Amended Report J1me 20, 2008 June 20, 2008

All inspections were study-based unless otherwise noted.

Linda R. Mitchell Wildlife International, Ltd.

Dates Findings Reported to Management

Revision 1 Dnpont-24198

February 28, 2008

February 25, 2008

April 1, 2008

April 1, 2008

Jnne 20, 2008

4

Determination of the Dissociation Constant ofH-28308 Revision I

Dupont-24198

CERTIFICATION

DETERMINATION OF THE DISSOCIATION CONSTANT OF H-28308 We, the undersigned, declare that the work described in this report was performed under our supervision, and that this report provides an accurate record of the procedures and results.

Senior Chemist

Approved by

Will!(n£,~ Director of Chemistry

Date Study Initiated

February 21, 2008

Date Study Completed

April I, 2008

Date Revision Completed

June 20, 2008

Sponsor

E.L du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A.

Date

5


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TABLE OF CONTENTS Page Reserved............................................................................................................... 2 Statement of Confidentiality ......................................................................................... 2 Good Laboratory Practice Compliance Statement........................................................ 3 Quality Assurance Statement........................................................................................ 4 Certification .................................................................................................................. 5 Table of Contents.......................................................................................................... 6 Basic Study Information ............................................................................................... 8 Reason for Revision 1................................................................................................... 9 1.0 Summary ............................................................................................................ 10 2.0 Introduction ........................................................................................................ 11 3.0 Objectives........................................................................................................... 11 4.0 Materials and Methods ....................................................................................... 11

4.1 Test Substance ..............................................................................................11 4.2 Experimental Design.....................................................................................11 4.3 Reference Substance .....................................................................................11 4.4 Reagents and Solvents ..................................................................................11 4.6 Determination of Dissociation Constant.......................................................12

5.0 Results and Discussion....................................................................................... 12 6.0 Conclusion.......................................................................................................... 12 7.0 References .......................................................................................................... 13


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TABLES Table 1 Typical Operating Parameters for Automatic Titrator.............14 Table 2 pKb and pKa Values of H-28308 in HPLC-Grade Bottled Reagent Water at 20°C ............................................................15 Table 3 Determination of pKb for H-28308 (Sample ID: 112C-135-1) .......................................................16 Table 4 Determination of pKa for Benzoic Acid (Sample ID: REF022108) .......................................................17

FIGURES Figure 1 Representative plot of titration curve for H-28308 (Sample ID: 112C-135-1) .......................................................18

APPENDICES Appendix I. Protocol ..............................................................................19 Appendix II. Certificate of Analysis .......................................................27


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BASIC STUDY INFORMATION

Study Title Determination of the Dissociation Constant of H-28308

Study Objectives The objective of this study was to determine the dissociation constant(s) of the test substance in water at 20°C.

Study Director Frank J. Lezotte, B.S. Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 U.S.A.

Sponsor’s Study Monitor Charles R. Powley, Ph.D. E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A.

Technical Staff, Wildlife International, Ltd. Will Schutt, B.S. John R. Murrell, B.S.

Test Item H-28308

Testing Facilities Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 U.S.A.


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REASON FOR REVISION 1

The report was revised as follows: 1. A calculated pKa value was added to section 1.0. 2. Section 4.6 was revised for the determination of dissociation constants before

and after the equivalence point. 3. The calculated pKa value for H-28308 was added to sections 5.0 and 6.0. 4. Table 2 was modified to correct the temperature for samples 112C-135-2 and

112C-135-3, and to include the individual and mean pKa values.


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DETERMINATION OF THE DISSOCIATION CONSTANT OF H-28308

1.0 SUMMARY STUDY: Determination of the Dissociation Constant of H-28308 SPONSOR: E.I. du Pont de Nemours and Company WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 112C-135

TEST DATES: Experimental Start (OECD) – February 21, 2008 Experimental Start (EPA) – February 21, 2008

Experimental Termination – February 22, 2008 RESULTS: The pKb value for H-28308 was 8.10 (SD = 0.0677, CV = 0.836%) at

20±1°C. The pKa value for H-28308 was 3.82 (SD = 0.0589, CV = 1.54%) at 20±1°C.


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2.0 INTRODUCTION This study was conducted by Wildlife International, Ltd. for E. I. du Pont de Nemours and Company and identified as Project Number 112C-135. Dissociation constant determinations were performed between February 21 and 22, 2008. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 112C-135 in archives located on the Wildlife International, Ltd. site.

3.0 OBJECTIVES The objective of this study was to determine the dissociation constant(s) of the test substance in water at 20°C.

4.0 MATERIALS AND METHODS The methods used in conducting this study were based upon procedures specified in the OECD Guideline 112, Dissociation Constants in Water (1) and U.S. Environmental Protection Agency Series 830-Product Properties Test Guidelines OPPTS Number 830.7370, Dissociation Constants in Water (2).

4.1 Test Substance The test substance was received from E. I. Du Pont de Nemours and Company on December 11, 2007 and was assigned Wildlife International, Ltd. identification number 8320 upon receipt. The test substance was a liquid, identified as: HFPO Dimer Salt (FRD 902). The test substance had a reported purity of 86% and reanalysis date of October, 2008. The certificate of analysis is presented in Appendix II. The test substance was stored at ambient temperature in locked storage at the Wildlife International, Ltd. facilities in Easton, Maryland.

4.2 Experimental Design The titration method was used to determine the dissociation constant K (expressed as its log value, pK) of the test substance in HPLC-grade bottled reagent water.

4.3 Reference Substance The benzoic acid reference substance was received from Sigma-Aldrich Chemical Company on June 10, 1998, and was assigned Wildlife International, Ltd. identification number 4505 upon receipt. The reference substance, a solid, was identified as: Benzoic acid; Lot Number 15422JR; CAS Number 65-85-0. The reference substance had a reported purity of 99.6% and was stored under ambient conditions.

4.4 Reagents and Solvents Water used in this study met or exceeded ASTM Type II standards (ASTM D1193-91). Other solvents and reagents were ACS reagent grade or equivalent.


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4.6 Determination of Dissociation Constant For the definitive study, three replicate samples, each at 1.00 mg/mL in HPLC-grade water, were prepared. For each preparation, 0.100 g of the test substance was brought to a final volume of 100 milliliters in a volumetric flask. The water was degassed with nitrogen to remove carbon dioxide prior to use. Each sample was equilibrated to 20 ± 1°C in a water bath then transferred into a beaker. The samples were then titrated against standardized 0.1N hydrochloric acid solution. At least ten incremental additions were made before the equivalence point and the titration was carried out past the equivalence point. Values of pK were calculated for a minimum of ten points prior to and after the equivalence point on the titration curve. The dissociation constants (Ka or Kb) and pKa or pKb were determined as follows:

pKa = pH – log([B]/[HB]) pKb = pH – log([HB/B]) Antilog (-pK) = K Where pH = pH of test solution at any point [B] = concentration of ionized species [HB] = concentration of unionized species

5.0 RESULTS AND DISCUSSION The calculated pKa value for the reference substance, benzoic acid, is summarized in Table 5. The calculated pKa value was 4.33 at 20.8°C. Although slightly elevated because of the temperature, the value is consistent with the value cited in OPPTS 830.7370 (4.19). The calculated pKb values for the test substance are summarized in Table 2, and a representative plot of a titration curve is presented in Figure 1. The mean pKb value was 8.10 (SD = 0.0677, CV = 0.836%) at 20±1°C. Representative titration data and pKb calculations for the test substance are summarized in Table 3. The corresponding calculated pKa values for the test substance are also summarized in Table 2. The mean pKa value was 3.82 (SD = 0.0589, CV = 1.54%) at 20±1°C.

6.0 CONCLUSION The pKb value for H-28308 was 8.10 (SD = 0.0677, CV = 0.836%) at 20±1°C. The pKa value for H-28308 was 3.82 (SD = 0.0589, CV = 1.54%) at 20±1°C.


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7.0 REFERENCES

1 Organization for Economic Cooperation and Development. 1981. Dissociation Constants in Water. OECD Guideline for Testing of Chemicals. Guideline 112.

2 U.S. Environmental Protection Agency. 1996. Series 830-Product Properties Test

Guidelines, OPPTS Number 830.7370: Dissociation Constants in Water.


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Table 1

Typical Operating Parameters for Automatic Titrator

Titrator: Metrohm Model 716 DMS Titrino

Titration parameters Definitive Trials

Mode MET

Volume Step 0.02 mL

Titration Rate Max. mL/min

Signal Drift 50 mV/min

Equilibration Time 26 seconds

Start Volume OFF

Pause 0 seconds

Measurement Input 1

Temperature 20 ± 1°C

Stop Volume 3.98 mL

Stop pH 3


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Table 2

pKb and pKa Values of H-28308 in HPLC-Grade Bottled Reagent Water at 20°C

Sample Number

Nominal Concentration

(mg/mL)

Temperature

(°C)

Equivalence Point (pH)

Titrant Volume

(mL) pKb 1

Mean pKb

Standard Deviation

112C-135-1

112C-135-2

112C-135-3

1.00

1.00

1.00

20.0

20.5

20.5

5.57

5.75

6.03

0.460

0.310

0.330

8.0815

8.1754

8.0439

8.10

0.0677 (CV = 0.836%)

Antilog (-pKb) = 7.94 x 10-9 1 Results were generated using Excel 2000 in the full precision mode. Manual calculations may differ slightly.

Sample Number

Nominal Concentration

(mg/mL)

Temperature

(°C)

Equivalence Point (pH)

Titrant Volume

(mL) pKa 1

Mean pKa

Standard Deviation

112C-135-1

112C-135-2

112C-135-3

1.00

1.00

1.00

20.0

20.5

20.5

5.57

5.75

6.03

0.460

0.310

0.330

3.7570

3.8622

3.8556

3.82

0.0589 (CV = 1.54%)

Antilog (-pKa) = 1.51 x 10-4 1 Results were generated using Excel 2000 in the full precision mode. Manual calculations may differ slightly.


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Table 3

Determination of pKb for H-28308 (Sample ID: 112C-135-1)

volume pH % ionized %unionized pKb0.000 8.385 0.0000 1.00000.020 8.413 0.0435 0.9565 7.07060.040 8.421 0.0870 0.9130 7.39980.060 8.411 0.1304 0.8696 7.58710.080 8.425 0.1739 0.8261 7.74830.100 8.402 0.2174 0.7826 7.84570.120 8.387 0.2609 0.7391 7.93470.140 8.395 0.3043 0.6957 8.03600.160 8.349 0.3478 0.6522 8.07600.180 8.341 0.3913 0.6087 8.14910.200 8.304 0.4348 0.5652 8.19010.220 8.252 0.4783 0.5217 8.21420.240 8.237 0.5217 0.4783 8.27480.260 8.147 0.5652 0.4348 8.26090.280 8.084 0.6087 0.3913 8.27590.300 8.093 0.6522 0.3478 8.36600.320 7.97 0.6957 0.3043 8.32900.340 7.907 0.7391 0.2609 8.35930.360 7.818 0.7826 0.2174 8.37430.380 7.818 0.8261 0.1739 8.49470.400 7.651 0.8696 0.1304 8.47490.420 7.356 0.9130 0.0870 8.37720.440 6.611 0.9565 0.0435 7.9534

Mean 8.0815Standard Deviation 0.3650

% RSD 4.5160 Equivalence Point 5.572% ionized = volume/equivalence point volume Volume (mL) 0.460% unionized= 1- % ionizedpKb = pH - log(% inionized / % ionized)Equivalence Point = change in pH/change in volume is greatest

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

0.000 0.500 1.000 1.500

Volume (mL)

pH


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Table 4

Determination of pKa for Benzoic Acid (Sample ID: REF022108)

volume pH % ionized %unionized pKa0.000 3.204 0.0000 1.00000.500 3.364 0.0621 0.9379 4.54301.000 3.506 0.1242 0.8758 4.35421.500 3.648 0.1863 0.8137 4.28822.000 3.786 0.2484 0.7516 4.26672.500 3.876 0.3106 0.6894 4.22243.000 3.979 0.3727 0.6273 4.20523.500 4.091 0.4348 0.5652 4.20494.000 4.213 0.4969 0.5031 4.21844.500 4.284 0.5590 0.4410 4.18105.000 4.426 0.6211 0.3789 4.21135.500 4.557 0.6832 0.3168 4.22326.000 4.658 0.7453 0.2547 4.19166.500 4.893 0.8075 0.1925 4.27047.000 5.164 0.8696 0.1304 4.34017.500 5.588 0.9317 0.0683 4.45338.000 7.276 0.9938 0.0062 5.0719

Mean 4.3279Standard Deviation 0.2226

% RSD 5.1424% ionized = volume/equivalence point volume% unionized= 1- % ionizedpKa = pH - log(% ionized / % unionized) Equivalence Point 7.94Equivalence Point = change in pH/change in volume is greatest Volume (mL) 8.050

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

0.000 5.000 10.000 15.000 20.000

Volume (mL)

pH


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Figure 1

Representative plot of titration curve for H-28308 (Sample ID: 112C-135-1)

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.40Volume (mL)

pH


19

Appendix I

Protocol


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21


22


23


24


25


26


27

Appendix II

Certificate of Analysis

determination of the dissociation constant of h- 28308

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