Document b56z9MdeL7gzDreZm0NQmoD6O

M u t . M i 20 i M U - ASM DuPont-13970 TRADE SECRET Study Title H-24616: Hydrolytic Stability of H-24616 as a Function of pH Test Guidelines: OECD Guideline 111 Author: Bogdan Szostek, Ph.D. Study Completed on: June 9,2004 Performing Laboratory: E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Laboratory Project ID: DuPont-13970 Work Request Number: 14644 Service Code Number: 392 Page 1 of 43 Company Sanitized. Does Not Contain T SCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with U.S. EPA TSCA (40 CFR part 792) Good Laboratory Practice Standards, which are consistent with the OECD Principles o f Good Laboratory Practice (as revised in 1997) published in ENV/MC/CHEM(98)17 and MAFF Japan Good Laboratory Practice Standards (59 NohSan Number 3850. The test substance was characterized by the sponsor prior to the initiation of this study. Although the characterization was not performed under Good Laboratory Practice Standards, the characterization was done by an ISO 9000 certified laboratory, and the accuracy of the data is considered sufficient for the purposes of this study. Applicant / Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Director: Bogdan Szostek, Ph.D. Senior Research Chemist O-JUjv) -3-ocH Date Applicant / Sponsor:_________________________________________ __________ DuPont Representative Date -2 Company Sanitized. Does Not Contain T SCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH QUALITY ASSURANCE STATEMENT Haskell Sample Number(s): 24616 Dates of Inspections: Conduct: 29-Jan-2004 Records, Reports: 29-30-Mar-2004; 2-Apr-2004 Dates Findings Reported to: Study Director: 29-Jan-2004; 7-Apr-2004 Management: 29-Jan-2004; 7-Apr-2004 DuPont-13970 Reported by: Quality Assurance Auditor Date -3 Company Sanitized. Does Not Contain TSCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Approved by: Jf* t-c. S. Mark Kennedy, Manager Issued by Study D irector: Bogdan Szostek, PhD . Senior Research Chemist q Q inJ" Z o Q t j Date Date -4 Company Sanitized. Does Not Contain T SC A CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 TABLE OF CONTENTS Page GOOD LABORATORY PR A C TIC E COM PLIANCE STA TEM EN T.....................................2 QUALITY ASSURANCE ST A T E M E N T ........................................................................................3 C ER TIFIC A TIO N ................................................................................................................................ 4 LIST OF TA B LES................................................................................................................................ 6 LIST OF FIG U R E S.............................................................................................................................. 7 STUDY IN FO R M A TIO N ....................................................................................................................8 STUDY PERSONNEL.......................................................................................................................... 9 SUM M ARY.......................................................................................................................................... 10 INTRODUCTION............................................................................................................................... 11 STUDY DESIGN................................................................................................................................. 11 M ATERIALS AND M E T H O D S............................................................. A. M aterials....................................................................................................................................11 1. Test Substance.............................................................................................................................................11 2. Residual 8-2 TBA in H-24616....................................................................................................................12 3. Preparation of buffer solutions...................................................................................................................12 a. pH 1.2:.................................................................................................................................................. 12 b. pH 4 : ......................................................................................................................................................12 c. pH 7 : ......................................................................................................................................................13 d. pH 9 : ......................................................................................................................................................13 4. Equipment.....................................................................................................................................................13 B. Methods......................................................................................................................................13 1. Preliminary test........................................................................................................................................... 13 2. pH measurement and temperature...............................................................................................................14 3. Analysis of test substance........................................................................................................................... 14 4. Analysis of 8-2 TBA....................................................................................................................................15 5. Calculations.................................................................................................................................................. 16 RESULTS AND D ISCU SSIO N ........................................................................................................18 CO NCLUSIO N S................................................................................................................................. 20 RECORDS AND SAM PLE ST O R A G E .........................................................................................21 REFER EN C ES.................................................................................................................................... 21 TABLES................................................................................................................................................ 22 FIG U R ES..............................................................................................................................................35 -5 Company Sanitized. Does Not Contain T SC A CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 LIST OF TABLES Page 1. Analytical results for aqueous stability of H-24616, pH 1.2,37C, Days 0 (12-Feb-2004) and 5 (17-Feb-2004)............................................................................................................................................................. 23 2. Analytical results for aqueous stability of H-24616, pH 4.0 phthalate buffer, 50C, Days 0 (5-Feb-2004) and 5 (10-Feb-2004).............................................................................................................................24 3. Analytical results for aqueous stability of H-24616, pH 4.0 citrate buffer, 50C, Days 0 (5-Feb-2004) and 5 (10-Feb-2004)....................................................................................................................................................25 4. Analytical results for aqueous stability of H-24616, pH 7.0, 50C, Days 0 (29-Jan-2004) and 5 (03-Feb-2004).............................................................................................................................................................. 26 5. Analytical results for aqueous stability of H-24616, pH 9.0, 50C, Days 0 (29-Jan-2004) and 5 (03-Feb-2004).............................................................................................................................................................. 27 6. Measured concentrations of 8-2 TBA for pH 1.2,37C,Days 0 (12-Feb-2004) and 5 (17-Feb-2004)..................28 7. Measured concentrations of 8-2 TBA for pH 4.0, 50C, pH 4.0 phthalate buffer, 50C, Days 0 (5-Feb-2004) and 5 (10-Feb-2004).............................................................................................................................29 8. Measured concentrations of 8-2 TBA for pH 4 .0 ,50C, pH 4.0 citrate buffer, 50C, Days 0 (5-Feb-2004) and 5 (10-Feb-2004).............................................................................................................................30 9. Measured concentrations of 8-2 TBA for pH 7.0,50C,Days0 (29-Jan-2004) and 5 (03-Feb-2004)..................31 10. Measured concentrations of 8-2 TBA for pH 9.0, 50C,Days0 (29-Jan-2004) and 5 (03-Feb-2003)..................32 11. Daily temperature readings of the shaking water bath.............................................................................................. 33 12. pH measurements for Day 0 and Day 5..................................................................................................................... 34 -6 Company Sanitized. Does Not Contain T SC A CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 LIST OF FIGURES Page 1. Example 19F NMR spectra of 1050 mg/L fluorinated active ingredient calibration standard (A), fluorinated active ingredient recovered in THF extract (B), and acetonitrile extract (C) for Day 5, pH 4.0 phthalate buffer...............................................................................................................................................36 2. Example 19F NMR spectra of 210 mg/L fluorinated active ingredient calibration standard (A), fluorinated active ingredient recovered in THF extract (B), and acetonitrile extract (C) for Day 5, pH 1.2 buffer...............................................................................................................................................................37 3. Example 19F NMR spectra of a blank control sample for Day 5, pH 1.2 buffer (A) and the fluorinated active ingredient recovered in THF extract (B) sample for Day 0, pH 1.2 buffer (B)........................................... 38 4. Example 19F NMR spectra of the fluorinated active ingredient recovered in THF extract for Day 0, pH 4.0 citrate buffer (A) and Day 5, pH 4.0 citrate buffer (B)................................................................................ 39 5. Example 19F NMR spectra of the fluorinated active ingredient recovered in THF extract for Day 0, pH 7.0 buffer (A) and Day 5, pH 7.0 buffer (B)....................................................................................................... 40 6. Example 19F NMR spectra of the fluorinated active ingredient recovered in THF extract for Day 0, pH 9.0 buffer (A) and Day 5, pH 9.0 buffer (B)....................................................................................................... 41 7. Representative calibration curve for 8-2 TBA.......................................................................................................... 42 8. Representative chromatograms of 102 pg/L 8-2 TBA calibration standard and 520 pg/L D-8-2 TBA internal standard.......................................................................................................................................................... 42 9. Representative chromatograms for Day 5, pH 4.0 citrate buffer hydrolytic stability sample and 520 pg/L D-8-2 TBA internal standard.............................................................................................................. 43 10. Representative chromatograms for Day 5, pH 4.0 citrate buffer blank sample and 520 pg/L D-8-2 TBA internal standard.......................................................................................................................................................... 43 -7 Company Sanitized. Does Not Contain TSCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 STUDY INFORMATION Substance Tested: [ ] Svnonvms/Codes: H-24616 [ ] Haskell Number: 24616 Composition: [ [ ] [ [ ] ] ] Known Impurities in Total Solids: 1010 ppm 46 ppm <10 ppm Total Elemental Iodine [I] Free Iodide Ion [I~] I2 (limit of detection = 10 ppm) Note: 964 ppm o f total iodine attributed to Telomer B Iodide. 0.44 Weight percent Telomer B Iodide is present in [ ] Physical Characteristics: Opaque tan liquid Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Completed: December 3, 2003/ (see report cover page) Experimental Start/Completion: December 22,2003 / March 2, 2004 -8 Company Sanitized. Does Not Contain TSCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH STUDY PERSONNEL Study Director: Bogdan Szostek, Ph.D. Analytical Associate: Keith B. Prickett, B.S. Management: S. Mark Kennedy, Ph.D. Report Preparation: Wanda F. Dinbokowitz Management: Kim D. Birkmeyer, M.S. DuPont-13970 -9-- Company Sanitized. Does Not Contain T SC A CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 SUMMARY Hydrolytic stability of H-24616 was tested in buffered aqueous solutions at pH 4.0, 7.0, 9.0 at 50C and pH 1.2 at 37C for five days. Concentrations of fluorinated active ingredient of H-24616 were measured by 19F NMR spectroscopy and percent recoveries were calculated for Day 0 and Day 5 replicate samples for pH 1.2, 4.0 phthalate, 4.0 citrate, 7.0, 9.0 buffers. The average percent recoveries for the Day 0 and Day 5, pH 1.2 replicate samples were 104 3.6% and 86 4.0%, respectively. The average percent recoveries for the Day 0 and Day 5, pH 4.0 phthalate buffer replicate samples were 93 2.9% and 82 6.9%, respectively. The average percent recoveries for the Day 0 and Day 5, pH 4.0 citrate buffer replicate samples were 92 3.2% and 89 3.1%, respectively. The average percent recoveries for the Day 0 and Day 5, pH 7.0 replicate samples were 97 2.0% and 91 1.8%, respectively. The average percent recoveries for the Day 0 and Day 5, pH 9.0 replicate samples were 98 4.2% and 92 0.81%, respectively. The fluorinated active ingredient of H-24616 was demonstrated to be hydrolytically stable at pH 9.0, pH 7.0, and pH 4.0 citrate buffer with ti/2 >one year using the criterion of Day 0 and Day 5 fluorinated active ingredient recoveries differing by no more than 10 percent. The data obtained for hydrolytic stability of fluorinated active ingredient of H-24616 at pH 1.2 and pH 4.0-phthalate buffers is not conclusive. The difference of recoveries for fluorinated active ingredient between Day 0 and Day 5 samples was larger than 10 percent. The data indicates that a significant portion of fluorinated active ingredient stays dissolved after ^ addition of acetonitrile to the samples that were exposed to test conditions in pH 1.2 and pH 4.0phthalate buffer. Accurate quantitation of the dissolved fraction of fluorinated active ingredient was not achievable for acetonitrile extract. Therefore, accurate mass balance of fluorinated active ingredient could not be achieved. The 19F NMR spectra o f the acetonitrile extracts for these pHs did not indicate any changes of the fluorinated active ingredient material. The measured concentration o f 8-2 TBA did not indicate any hydrolytic degradation of the fluorinated active ingredient material to 8-2 TBA for pH 4.0 and pH 7.0 buffers. The observed concentrations o f 8-2 TBA can be explained by the residual 8-2 TBA present in the test substance (H-24616). For pH 1.2 and 9.0 an increase of the 8-2 TBA concentration beyond that accounted for by residual 8-2 TBA present in the test substance was observed. It was estimated that this increase would represent hydrolysis of 0.06 and 0.03% the test substance for pH 1.2 and pH 9.0 buffers, respectively. - 10Company Sanitized. Does Not Contain TSC A CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 IN T R O D U C T IO N The purpose of the study was to investigate the hydrolytic stability o f H-24616 in pH 1.2, 4.0, 7.0, and 9.0 aqueous buffer solutions. The study was conducted according to the protocol: "Hydrolytic stability of H-24616 as a function of pH", which was patterned after the OECD Guideline 111.(1) Additionally, the concentration of the 8-2 Telomer B Alcohol (8-2 TBA) was monitored to investigate if 8-2 TBA is released from the test substance during the test. STUDY DESIGN A preliminary test was performed on the test substance at 50 0.1 C at pH 4.0, 7.0, 9.0 and at 37 0.1C for pH 1.2. If less than 10% test substance hydrolysis was observed after 5 days, the test substance was considered hydrolytically stable and no additional testing was performed. In addition to the test substance determination during the preliminary test, the samples were analyzed for 8-2 TBA. The study design complies with the following test guidelines: OECD Guideline 111(1) with the exception that the OECD Guideline 111 requires that the amount of the test substance used is the smaller o f 0.01 M or half the saturation concentration. The test substance fluorinated active ingredient [ ] of H-24616) is essentially not soluble in water. Therefore, the study was conducted with excess of undissolved fluorinated active ingredient and the amount of the test substance was sufficient to satisfy the sensitivity requirements of analytical method. The molar concentration of fluorinated active ingredient was estimated to be 0.002 M (in 5. Calculations). MATERIALS AND METHODS A. M aterials 1. Name: Composition: Haskell number: Test Substance H-24616 [ [ [ 24616 1 ] ] The test substance was characterized to determine % fluorine, % solids, pH, Mn, Mw, and polydispersity with the following results: - 11Company Sanitized. Does Not Contain TSCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH % Fluorine (w/w): % Solids (w/w) pH [] [] 6.3 Mn: Mw: polydispersity [] [] [] 2. Residual 8-2 TBA in H-24616 DuPont-13970 Residual 8-2 TBA content in the test substance (H-24616) was determined by LC/MS using the same analytical method as described in Analysis of 8-2 TBA in Methods. Five replicate samples of H-24616 (approximately 45 mg) were introduced to glass vials, weighed, 10 mL o f THF (tetrahydrofurane) was added, vials crimped, and vortexed for 10 minutes on a multitube vortexer. A 0.1 mL aliquot of dissolved sample was transferred to a glass LC vial and 0.9 mL acetonitrile and internal standard (D-8-2 TBA) was added. Calibration standards were made in 10/90% THF/acetonitrile in the range from 100 to 1020 ng/mL 8-2 TBA. 3. Preparation o f buffer solutions The following buffer solutions were prepared as described in the Annex to OECD guideline 111 .(1) a. pH 1.2: The 0.2 M potassium chloride (KC1) was prepared by dissolving 3.78 g of KC1 (purity 99.0%, Sigma) in 250 mL o f water. The 0.2 M hydrochloric acid (HC1) was prepared by diluting 4.93 g of hydrochloric acid (36.5-38%, Sigma) in 250 mL water. The pH 1.2 buffer solution was prepared by placing 125 mL of 0.2 M KC1 and 161.3 mL o f a 0.2 M HC1 solution into a 500-mL flask and bringing the flask to volume with water. The pH o f the buffer was adjusted to 1.2 with hydrochloric acid. b. pH 4: Phthalate buffer: 0.1 M solution o f potassium hydrogen phthalate was prepared by dissolving 10.21 g of potassium hydrogen phthalate (purity 99+%, Sigma) in 500 mL o f water. The pH 4.0 buffer solution was prepared by placing 2 mL of a 0.1 M sodium hydroxide solution and 250 mL of a 0.1 M potassium hydrogen phthalate solution into a 500-mL flask and bringing the flask to volume with water. The pH of the buffer was adjusted to 4.0 with hydrochloric acid. Citrate buffer: 0.1 M solution o f potassium dihydrogen citrate was prepared by dissolving 11.5 g of potassium dihydrogen citrate (purity 99+%, Fluka) in 500 mL of water. The pH 4 buffer solution was prepared by placing 45 mL o f a 0.1 M sodium hydroxide solution and 250 mL o f a 0.1 M potassium dihydrogen citrate solution into a 500-mL flask and bringing the flask to volume with water. The pH of the buffer was adjusted to 4.0 with 1 M sodium hydroxide. - 12Company Sanitized. Does Not Contain T SC A CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH c. pH 7: DuPont-13970 The 0.1 M solution o f potassium phosphate was prepared by dissolving 6.81 g of potassium phosphate, monobasic (KH2PO4, purity 99.5%, Sigma) in 500 mL of water. The pH 7 buffer solution was prepared by placing 148 mL of a 0.1 M sodium hydroxide solution and 250 mL of the 0.1 M potassium phosphate solution into a 500-mL flask and bringing the flask to volume with water. The pH of the buffer was adjusted to 7.0 with hydrochloric acid. d. pH 9: The 0.1 M boric acid (H3BO3) solution in 0.1 M potassium chloride (KC1) was prepared by dissolving 3.73 g o f KC1 (purity 99.0%, Sigma) and 3.09 g o f H3BO3 (purity 99.5%, Sigma) in 500 mL of water. The pH 9.0 buffer solution was prepared by placing 250 mL of a 0.1 M H3BO3 solution in 0.1 M KC1 and 106.5 mL of a 0.1 M sodium hydroxide solution into a 500-mL flask and bringing the flask to volume with water. The pH of the buffer was adjusted to 9.0 with sodium hydroxide. Water used for buffer preparation was obtained from a Bamstead NANOpure Diamond water purification system. The water resistivity is >18.2 megaohm*cm. All buffer solutions were sterilized prior to use by filtering through Coming Sterilization Filter systems with 0.22-pm cellulose acetate filter. The buffer solutions were stored at room temperature. 4. Equipment p H Meter: Water Bath: Autoclave: Automatic Pipettes: Balance: Centrifuge: Vortexer: Glass Vials: Orion Model 250 A Precision Reciprocal Shaking Water Bath, Model 50, Precision AMSCO Model 3023 Research Pro 100,1000, 5000; Eppendorff Mettler AE-100 Sorval Legend RT Multi-tube vortexer, VWR 10 mL borosilicate Serum Type Reaction vials (Supelco) with 20 mm Barrier Septa (Foil/Silicone), 0.1" thick (Supelco). The vials and septa were autoclaved at 121C for 30 min before use. B. Methods 1. Preliminary test The preliminary test was performed on H-24616 at 50C at each of the pH 4.0,7.0, 9.0 and at 37C for pH 1.2. The same sample preparation procedure and study design was followed for each of the pHs tested. Approximately 45 mg of the H-24616 was weighed into sixteen 10-mL autoclaved glass vials and 3 mL o f appropriate buffer solution was added. The vials were crimp caped with the aluminum foil covered part o f the septa facing inside the vial. Eight o f the vials were processed for analysis as the Day 0 samples. Remaining eight samples were wrapped with the aluminum foil, submerged in the water bath, and shaken at 100 rpm in the water bath for five days at the appropriate temperature. Eight vials containing only 3 mL of appropriate buffer were prepared to serve as the blanks. Four o f them were used as the Day 0 blanks and the other four - 13Company Sanitized. Does Not Contain T SCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 were wrapped in A1 foil and placed in the water bath to be the Day 5 blanks. Additionally, six vials containing 3 mL of appropriate buffer were spiked with 8-2 TBA stock solution (4.5 pL of 1020 mg/L 8-2 TBA). The vials were crimp capped with the aluminum foil covered part o f the septa facing inside the vial. Three of the vials were used as the QC samples for 8-2 TBA analysis on Day 0. The other three vials were wrapped in aluminum foil and placed in the water bath together with eight of the test substance vials. These served a role o f QC samples for 8-2 TBA analysis on Day 5. Four of the Day 0 or four of the Day 5 vials and two blank vials were processed for the fluorinated active ingredient of H-24616 determination. The other four of the Day 0 or Day 5 vials, two blanks vials, and 8-2 TBA QC vials were processed for the 8-2 TBA determination. 2. pH measurement and temperature The pH was measured on Day 5 in the vials containing the buffer and the test substance, designated for the H-24616 analysis. Inadvertently, only pH o f the buffers was measured on Day 0, not the pH o f samples containing the test substance. Day 5 sample vials were uncapped, the pH electrode was introduced to the vials and the pH reading was taken. These vials were further processed for H-24616 analysis. The temperature o f the water bath was monitored daily. The water bath has an automatic temperature control built in. The readings o f the temperature control were checked and recorded daily. 3. Analysis o f test substance Vials designated for H-24616 analysis were uncapped, 6 mL of acetonitrile was added, vials were recapped and vortexed for 10 minutes. Addition of acetonitrile to the vial containing test substance suspended in a buffer solution causes the fluorinated active ingredient to precipitate. The vials were centrifuged for fifteen minutes at 3200 rpm, uncapped, and the liquid phase was removed from the vial. Ten milliliters o f tetrahydrofiirane (THF) was added to the vial, the vial was capped, and then sonicated for 30 minutes. Approximately 1 mL of the THF solution was placed in a glass LC vial, 0.1 mL of methyl-d3-alcohol was added and subjected to 19F-NMR analysis. The blank samples were subjected to the same procedure, without pH measurement. The stock solution o f H-24616 was made by dissolving 0.45 g of H-24616 in 10 mL o f THF. Ten minutes of sonication was required to completely dissolve the test substance. Calibration standards for the H-24616 analysis were made by appropriate dilution of the stock. The calibration standards ranged from 200 to 2000 mg/L o f fluorinated active ingredient o f H-24616. Fresh stock solution and calibration standards were prepared for each instance o f analysis. Linear regression of the peak area vs. standard concentration was used to establish the calibration curves. - 14Company Sanitized. Does Not Contain T SC A CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 Instrumentation and conditions: NMR instrument: Bruker AMX360 with a l9F/'H dual probe Acquisition Parameters Pulse Program: zg Time Domain Size: 16 k Number of Scans: 128 D1 Delay: 10 sec Dummy Scan: 2 Pulse Length: 8 gsec Receiver Gain: automated by the instrument Instrument Frequency: 338.8314566 MHz Processing Parameters: Size of Real Spectrum: 16 k Window Function: EM Window Size: 50 Hz Peak Used for Quantitation -CF3resonance at 32.5 ppm 4. Analysis of 8-2 TBA Samples designated for 8-2 TBA analysis were processed by injecting 3 mL methyl-tert-butyl ether (MTBE) through the vial septum using a glass syringe. Neither Day 0 nor Day 5 samples were uncapped before MTBE injection to avoid any losses of 8-2 TBA. The vials containing injected MTBE were vortexed for 10 minutes on a multitube vortexer. Subsequently, the vials were uncapped and 0.1 mL of the MTBE extract was transferred to a glass LC vial, 0.9 mL acetonitrile added, spiked with internal standard, crimped capped, and subjected to LC/MS analysis for 8-2 TBA. The 1H, 1H, 2H, 2H-perfluorodecan-l-ol (97.6%, Oakwood Products, West Columbia, SC) was used as the analytical standard of 8-2 TBA. The ID, ID, 2D, 2D, 3-13C-heptadecafluoro decanol (M+5) (abbreviated as D-8-2 TBA) was used as the internal standard. Stock solutions (1000 mg/L) of the analytical standard and the internal standard were prepared in methanol and were stored refrigerated. The calibration standards were prepared fresh for each calibration in 10/90% MTBE/acetonitrile. The calibration standards were made in the range from 100 to 10000 pg/L 8-2 TBA. A constant level o f internal standard was used: 520 pg/L D-8-2 TBA. The calibration curves were contracted using the ratio of the peak area for ions m/z monitored for 8-2 TBA and peak area for ions monitored for D-8-2 TBA and the concentrations o f 8-2 TBA. - 15Company Sanitized. Does Not Contain T SC A CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 Instrumentation and conditions: HPLC Instrument: MS Instrument: Model 2795, Waters ZQ, Micromass LC Parameters: Column: Mobile Phase: Column Temperature: Injection Volume: Column Switch: Luna 3u C18 (2), 20 mm x 2mm A - water B- methanol Gradient: Time (min) %A 0 30 1 30 20 60 6.1 30 8 30 30C 20 pL 2 and 6 min MS Parameters: Ionization mode: Capillary voltage: Cone voltage: Source Temperature: Desolvation Temperature: Data Acquisition Function: Electrospray, negative ions 2.5 kV 10 V 120C 300C 8-2 TBA: SIR of 463, 523; 0-6 min D- 8-2 TBA: SIR of 467, 528 m/z; 0-6 min 5. Calculations %B 70 70 100 100 70 70 Flow (mL/min) 0.25 0.25 0.25 0.25 0.25 0.25 The following calculations were used to determine the nominal concentrations o f H-24616 fluorinated active ingredient (Tables 1-5). For example, if [ ] o f H-24616 containing [ ] fluorinated active ingredient was weighed into the vial and 3 mL of buffer was added to the vial the nominal concentration of fluorinated active ingredient is: [ ] 3390 mg/L o f fluorinated active ingredient. The % recovery is calculated by dividing the measured concentration by the nominal concentration, times 100%. The expected (nominal) concentration of 8-2 TBA in the spiked samples was calculated as follows: the 4.5 pL o f 1020 mg/L 8-2 TBA spike represents 4.5 pL x 1020 ng/pL= 4590 ng 8-2 TBA. This amount was extracted with 3 mL of MTBE. The expected nominal concentration of 8-2 TBA in the MTBE extract is: 4590 ng/3 mL=T530 ng/mL. The % recovery is calculated by dividing the measured concentration by the nominal concentration, times 100%. The molar concentration of fluorinated active ingredient can be estimated using average molecular weight (Mn) of fluorinated active ingredient: [ ] When [ ] of H-24616 is used, it represents [ ] 6.78 10"6 mole o f fluorinated active ingredient. Taking into account that 3 mL o f buffer is used this would represent the molar concentration: 6.78 10*6 mole/0.003 L = 0.0023 M. The expected concentration of 8-2 TBA in - 16Company Sanitized. Does Not Contain T SC A CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 the MTBE extract if 10% of the H-24616 hydrolyzed to form 8-2 TBA can be estimated in the following way. Ten percent of 45 mg of H-24616 represents 4.5 mg. Taking into account that the test substance contains [ ] fluorine and assuming that all fluorine is present as 8-2 TBA, the hydrolysis of the 4.5 mg of the test substance would release: [ ] 0.79 mg 8-2 TBA. This amount of 8-2 TBA would be contained in 3 mLs of MTBE extract. Therefore, expected concentration o f 8-2 TBA would be: 0.79 mg/0.003 L= 263 mg/L. The H-24616 contains residual 8-2 TBA. The expected, maximum concentration o f 8-2 TBA derived from residual 8-2 TBA in the MTBE extract (3 mL), assuming complete extraction for a 45 mg sample of H-24616 is: 45 mg of H-24616 x (0.019%/100)/0.003 L= 2.85 mg/L (2850 |rg/L). - 17Company Sanitized. Does Not Contain T SC A CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 RESULTS AND DISCUSSION Tables 1-5 present the results for measured concentrations of fluorinated active ingredient of H-24616 and percent recoveries obtained for Day 0 and Day 5 replicate samples for pH 1.2, 4.0, 7.0, 9.0, respectively. The average percent recoveries for the Day 0 and Day 5, pH 1.2 replicate samples are 104 3.6% and 86 4.0%, respectively. The average percent recoveries for the Day 0 and Day 5, pH 4.0 phthalate buffer replicate samples are 93 2.9% and 82 6.9%, respectively. The average percent recoveries for the Day 0 and Day 5, pH 4.0 citrate buffer replicate samples are 92 3.2% and 89 3.1%, respectively. The average percent recoveries for the Day 0 and Day 5, pH 7.0 replicate samples are 97 2.0% and 91 1.8%, respectively. The average percent recoveries for the Day 0 and Day 5, pH 9.0 replicate samples are 98 4.2% and 92 0.81%, respectively. The difference o f average recoveries between the Day 5 and Day 0 samples of the fluorinated active ingredient was less than 10% for pHs 4.0 citrate buffer, pH 7.0, and pH 9.0. This indicates that the fluorinated active ingredient of H-24616 is hydrolytically stable at these pHs {t\n >one year). The hydrolytic stability experiment for pH 4.0 was done with two different buffers: phthalate and citrate (Table 2 and Table 3). Two different buffers were used as initial experiments with phthalate buffer indicated that the buffer composition affects the solubility of fluorinated active ingredient in a buffer when subjected to prolonged exposure (5 days) at elevated temperature IiFni (50C). Simply, the addition of acetonitrile to the vial containing the test substance would not completely precipitate the fluorinated active ingredient. The amount of the active ingredient recovered in the THF extract would be 30-50% (Table 2) for the phthalate buffer. In order to determine if the unaccounted portion o f the fluorinated active ingredient was present in the acetonitrile extract, the NMR spectrum of the extracts was collected and a quantitation o f the fluorinated active ingredient in the acetonitrile extract was attempted. Accurate quantitation o f the fluorinated active ingredient in the acetonitrile extract (acetonitrile extract contains 6 mL acetonitrile and 3 mL of a buffer) was not possible because the calibration standards of fluorinated active ingredient in the acetonitrile extract could not be prepared using the THF stock of fluorinated active ingredient. The fluorinated active ingredient would precipitate if introduced to the acetonitrile extract using THF stock. In order to determine the sensitivity difference between the NMR measurements done for THF and acetonitrile extract matrix, a calibration standard of 8-2 TBA in both solvents was made and measured by the NMR methods. This allowed the derivation of the sensitivity-difference factor that was applied to correct for the measurement o f fluorinated active ingredient in the acetonitrile extract vs. the calibration standards made in THF. However, this procedure is considered to give only semi-quantitative results. Table 2 results indicate that significant portion of the fluorinated active ingredient is present in the acetonitrile extract for Day 5 samples. Comparison o f the 19F NMR spectra o f Day 5 THF extract, Day 5 acetonitrile extract, and a calibration standard indicates that the fluorinated active ingredient present in the acetonitrile extract did not undergo a chemical change that would be visible by 19F NMR (Figure 1). The average recovery of the fluorinated active ingredient for Day 5 samples is lower by 11% (Table 2) than the recovery for Day 0. However, taking into account the semi-quantitative nature of results for acetonitrile extracts, it is difficult to draw a conclusion about hydrolytic stability (tj/2 >one year) o f the fluorinated active """ ingredient as the difference may be coming from the inaccuracy o f the results obtained for the -- Company Sanitized. Does Not Contain TSCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 acetonitrile extracts. The observed solubility issues can be linked to buffer composition, not the buffer's pH for the pH 4.0 buffers. The solubility issue was not observed for citrate, pH 4.0 buffer (Table 3). The difference of average recoveries between the Day 5 and Day 0 samples for pH 1.2 is more than 10 percent. However, similarly to pH 4.0-phthalate buffer, the solubility issues were encountered for the pH 1.2 buffer. Table 1 indicates that a significant amount o f fluorinated active ingredient was detected in the acetonitrile extracts, but, for the reasons described above for the phthalate buffer, accurate quantitation o f the fluorinated active ingredient in the acetonitrile extract was not possible. Therefore, this data was not conclusive about the hydrolytic stability of the fluorinated active ingredient at pH 1.2. However, comparison of the NMR spectra of Day 5 THF extract, Day 5 acetonitrile extract for pH 1.2 buffer, and a calibration standard indicated that the fluorinated active ingredient present in the acetonitrile extract did not undergo a chemical change that would be visible by 19F NMR (Figure 2). Figure 3 presents the NMR spectra of a blank sample and the Day 0 THF extract for pH 1.2. Figures 4-6 present the comparison of NMR spectra obtained for Day 0 and Day 5 THF extracts for pH 4.0 citrate, pH 7.0, and pH 9.0 buffers, respectively. The content of residual 8-2 TBA in H-24616 was measured before the start of the hydrolytic study. Based on the results of five replicates of the H-24616, the content o f residual 8-2 TBA in H-24616 was calculated to be 0.0190.0003%. Assuming that the buffer extracts all the residual 8-2 TBA from the test substance during the time o f hydrolytic stability experiment, the expected concentration of 8-2 TBA in the test system would be 2.85 mg/L (see 5 Calculations). The hydrolysis of the fluorinated active ingredient of H-24616 would most likely result in the release of 8-2 TBA. If ten percent of H-24616 hydrolyzed releasing the 8-2 TBA, the expected concentration of 8-2 TBA in the test system would be 263 mg/L (see 5 Calculations). Tables 6-10 present the measured concentrations o f 8-2 TBA for Day 0 and Day 5 replicate samples for pH 1.2,4.0 phthalate, 4.0 citrate, 7.0, 9.0, respectively. The measured concentration of 8-2 TBA for pH 1.2 (Table 6) indicates the increase of 8-2 TBA concentration for Day 5 samples beyond the level that can be accounted for by the residual 8-2 TBA present in the test substance. The concentration of 8-2 TBA measured for the Day 0 samples is comparable with the expected concentration o f 8-2 TBA originating from residual 8-2 TBA. The Day 5 concentration of 8-2 TBA is higher by approximately 1.5 mg/L more than the expected concentration originating from the residual 8-2 TBA. The observed increase o f 8-2 TBA concentration indicates the hydrolysis of the test substance to form 8-2 TBA. This increase represents only estimated hydrolysis of approximately 0.06% (1.5 mg/L / 2630 mg/L * 100%) o f the test substance present in the test system. The acceptable recovery results for the quality control samples (QC) for Day 0 and Day 5 demonstrate the validity o f the 8-2 TBA measurement (Table 6). The measured concentrations of 8-2 TBA for pH 4.0 citrate, pH 4.0 phthalate, pH 7.0 test system do not exceed significantly the expected concentration of 8-2 TBA originating from the residual 8-2 TBA (Tables 7-9). This indicates that there is no contribution o f 8-2 TBA from the hydrolysis o f the test substance. The recovery results for QC samples are also in the acceptable range (Tables 7-9). The measured concentration of 8-2 TBA for pH 9.0 (Table 10) indicate the increase o f 8-2 TBA concentration for Day 5 samples beyond the level that can be accounted for by the residual 8-2 TBA present in the test substance. The increase is approximately 0.85 mg/L 8-2 TBA beyond the concentration explainable by the residual 8-2 TBA and is indicative of generation o f 8-2 TBA from hydrolysis o f fluorinated active - 19Company Sanitized. Does Not Contain TSCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 ingredient at pH 9.0. This increase represents an estimated hydrolysis of approximately 0.03% (0.85 mg/L / 2630 mg/L * 100 %) of the test substance present in the test system. Figure 7 shows a representative calibration curve obtained for 8-2 TBA. Figures 8-10 show chromatograms for the 8-2 TBA calibration standard, Day 5, pH 4.0 test sample, and Day 0, pH 4.0 blank sample, respectively. Table 11 summarizes the water bath temperature recording for pH 1.2,4.0, 7.0, and 9.0. In all cases, the water bath temperature was maintained at the specified level 0.1 C. Table 12 summarizes results of the pH measurements for pH 1.2,4.0, 7.0, and 9.0. Inadvertently, only pH of buffers was measured on Day 0, not the pH of samples containing the test substance. The measured pH of Day 0 buffers and Day 5 replicate samples for all of the investigated pHs was at the specified nominal level with the deviation not exceeding 0.1. CONCLUSIONS The hydrolytic stability of H-24616 was tested in buffered aqueous solutions at pH 4.0, 7.0, 9.0 at 50C and pH 1.2 at 37C for five days. The fluorinated active ingredient o f H-24616 was demonstrated to be hydrolytically stable at pH 9.0, pH 7.0, and pH 4.0 citrate buffer with t ]/2 >one year. The data obtained for hydrolytic stability of the fluorinated active ingredient of H-24616 at pH 1.2 and pH 4.0-phthalate buffers is not conclusive. The difference o f recoveries for fluorinated active ingredient between Day 0 and Day 5 samples was larger than 10 percent. The data indicates that a significant portion of fluorinated active ingredient stays dissolved after addition of acetonitrile to the samples that were exposed to test conditions in pH 1.2 and pH 4.0-phthalate buffer. Accurate quantitation of the dissolved fraction of fluorinated active ingredient was not achievable for acetonitrile extract. Therefore, accurate mass balance o f fluorinated active ingredient could not be achieved. The 19F NMR spectra o f the acetonitrile extracts for these pHs did not indicate any changes to the fluorinated active ingredient material. The measured concentration of 8-2 TBA did not indicate any hydrolytic degradation o f the fluorinated active ingredient material to 8-2 TBA for pH 4.0 and pH 7.0 buffers. The observed concentrations o f 8-2 TBA can be explained by the residual 8-2 TBA present in the test substance (H-24616). For pH 1.2 and 9.0 an increase of the 8-2 TBA concentration beyond that which can be accounted for by residual 8-2 TBA present in the test substance was observed. It was estimated that this increase would represent hydrolysis of 0.06 and 0.03% the test substance for pH 1.2 and pH 9.0 buffers, respectively. -20- Company Sanitized. Does Not Contain T SCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH mn. DuPont-13970 RECORDS AND SAMPLE STORAGE Specimens (if applicable), raw data, and the final report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. REFERENCES 1. Organisation for Economic Cooperation and Development (OECD). May 12, 1981. OECD Guidelines for Testing of Chemicals, Hydrolysis as a Function of pH, OECD Guideline No. 111. -21Company Sanitized. Does Not Contain T SC A CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 TABLES -22Company Sanitized. Does Not Contain T SC A CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 T ab le 1: A nalytical resu lts for aqueous stability o f H -24616, pH 1.2, 37C , D ays 0 (12-Feb-2004) and 5 (17-Feb-2004). F luorinated active F luorinated active ingredient of H-24616 ingredient of H-24616 N om inal C oncentration5 M easured C oncentration PH Day Rep (m g/L) (m g/L) 1.2 0 1 1.2 0 2 1.2 0 3 1.2 0 4 3320 3280 3330 3330 3570 3300 3520 3350 % Recovery 108 101 106 101 A verage Standard Deviation 104 3.6 1.2 5 1 1.2 5 2 1.2 5 3 1.2 5 4 3360 3320 3340 3260 2 6 6 0 `+ 2 3 6 2 2 7 103 2 7 9 0 '+ 2 3 4 2 2 4 4 0 '+ 2 5 5 2 864 82 914 834 A verage 86 Standard D eviation 4.0 1recovered in the THF extract 2recovered in the acetonitrile extract 3sample of acetonitrile extract not saved 4 represents total recovery from THF and acetonitrile extract 5represents nominal concentration of fluorinated active ingredient in the buffer -23Company Sanitized. Does Not Contain TSC A CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 Table 2: Analytical results for aqueous stability o f H -24616, pH 4.0 phthalate buffer, 50C, Days 0 (5-Feb-2004) and 5 (10-Feb-2004). F luorinated active F luorinated active ingredient o f H-24616 ingredient o f H-24616 pH Day R ep N om inal C oncentration4 M easured C oncentration (m g/L) (m g/L) 4.0 0 1 4.0 0 2 4.0 0 3 4.0 0 4 3380 3320 3340 3330 3100 2970 3180 3170 % Recovery 92 89 95 95 A verage Standard Deviation 93 2.9 4.0 5 1 4.0 5 2 4.0 5 3 4.0 5 4 3420 3330 3300 3410 1 5 1 0 '+ 9 4 0 2 13 2 0 '+ l 5402 1 0 9 0 '+ 1 6 7 0 2 1 3 4 0 '+ 1 5 9 0 2 723 863 843 863 A verage 82 Standard Deviation 6.9 recovered in the THF extract 2 recovered in the acetonitrile extract 3represents total recovery from THF and acetonitrile extract 4 represents nominal concentration of fluorinated active ingredient in the buffer - 24Company Sanitized. Does Not Contain TSCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 Table 3: A nalytical results for aqueous stability o f H -24616, pH 4.0 citrate buffer, 50C, Days 0 (5-Feb-2004) and 5 (10-Feb-2004). F luorinated active F luorinated active ingredient o f H-24616 ingredient of H-24616 PH Day Rep N om inal C oncentration5 M easured C oncentration (m g/L) (m g/L) 4.0 0 1 4.0 0 2 4.0 0 3 4.0 0 4 3380 3330 3320 3320 3080 3170 2920 3130 % Recovery 91 95 88 94 A verage Standard D eviation 92 3.2 4.0 5 1 4.0 5 2 4.0 5 3 4.0 5 4 3350 3320 3330 3360 2930 8 3 4 '+ 7 5 2 2940 3110 87 273 88 93 Average 89" Standard Deviation 3.14 1recovered in the THF extract 2recovered in the acetonitrile extract 3the vial most likely leaked during the experiment 4Day 5, replicate 2 excluded from calculation 5represents nominal concentration of fluorinated active ingredient in the buffer - 25Company Sanitized. Does Not Contain T SCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 Table 4: Analytical results for aqueous stability of H-24616, pH 7.0, 50C, Days 0 (29-Jan-2004) and 5 (03-Feb-2004). F luorinated active F luorinated active ingredient of H-24616 N om inal C oncentration ingredient of H-24616 pH Day Rep (m g/L)1 M easured Concentration (m g/L) 7.0 0 1 7.0 0 2 7.0 0 3 7.0 0 4 3420 3320 3360 3360 3290 3190 3360 3240 % Recovery 96 96 100 96 A verage Standard Deviation 97 2.0 7.0 5 1 7.0 5 2 7.0 5 3 7.0 5 4 3340 3340 3350 3330 3060 3100 2990 3000 92 93 89 90 A verage 91 Standard Deviation 1.8 1represents nominal concentration of fluorinated active ingredient in the buffer -26Company Sanitized. Does Not Contain TSCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 Table 5: A nalytical results for aqueous stability of H -24616, pH 9.0, 50C, Days 0 (29-Jan-2004) and 5 (03-Feb-2004). Fluorinated active F luorinated active ingredient of H-24616 ingredient of H-24616 P Day Rep N om inal C oncentration M easured C oncentration (m g/L)1 (m g/L) 9.0 0 1 9.0 0 2 9.0 0 3 9.0 0 4 3330 3340 3310 3300 3180 3180 3440 3240 % Recovery 95 95 104 98 Average Standard Deviation 98 4.2 9.0 5 1 9.0 5 2 9.0 5 3 9.0 5 4 3340 3330 3330 3330 3080 3080 3020 3110 92 92 91 93 A verage 92 Standard Deviation 0.81 1represents nominal concentration of fluorinated active ingredient in the buffer ,,Hi!*., - 27Company Sanitized. Does Not Contain T SC A CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 Table 6: M easured concentrations of 8-2 TBA for pH 1.2, 37C, Days 0 (12-Feb-2004) and 5 (17-Feb-2004). N om inai 8-2 TB A pH D ay Rep C oncentration (pg/L) 1.2 0 1 1.2 0 2 1.2 0 3 1.2 0 4 - A verage Standard Deviation 1.2 0 QC1 1.2 0 Q C 2 1.2 0 Q C 3 1530 1530 1530 1.2 5 1 1.2 5 2 1.2 5 3 1.2 5 4 - A verage Standard Deviation 1.2 5 QC1 1.2 5 QC2 1.2 5 QC3 1530 1530 1530 M easured 8-2 TBA C oncentration (pg/L) 2920 2950 2800 2950 2900 71 1580 1580 1580 A verage Standard Deviation 4010 4390 4060 4350 4200 200 1640 1660 1650 A verage Standard Deviation % Recovery - 103 103 103 103 0.0 - 107 108 108 108 0.6 - 28Company Sanitized. Does Not Contain T SC A CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 Table 7: M easured concentrations o f 8-2 TBA for pH 4.0, 50C, pH 4.0 phthalate buffer, 50C, Days 0 (5-Feb-2004) and 5 (10-Feb-2004). N om inai 8-2 TBA pH D ay R ep C o ncentration (|ig/L ) 4.0 0 1 4.0 0 2 4.0 0 3 4.0 0 4 - M easured 8-2 TBA C oncentration (pg/L) 2070 2210 2130 2070 % Recovery - A verage Standard Deviation 2120 66 4.0 0 QC1 4.0 0 QC2 4.0 0 QC3 1530 1530 1530 1570 1600 1580 103 105 103 A verage Standard Deviation 104 1.2 4.0 5 1 4.0 5 2 4.0 5 3 4.0 5 4 - 3400 3460 1710 1690 - A verage Standard Deviation 2570 1000 4.0 5 QC1 4.0 5 QC2 4.0 5 QC3 1530 1530 1530 1650 1430 1680 108 93 110 A verage Standard D eviation 104 9.3 -29Company Sanitized. Does Not Contain T SC A CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 T able 8: M easured concentrations o f 8-2 T B A for pH 4 .0 ,50C, pH 4.0 citrate buffer, 50C, Days 0 (5-Feb-2004) and 5 (10-Feb-2004). N om inal 8-2 TB A pH Day Rep C oncentration (pg/L) 4.0 0 1 4.0 0 2 4.0 0 3 4.0 0 4 - A verage Standard Deviation 4.0 0 QC1 4.0 0 QC2 4.0 0 QC3 1530 1530 1530 4.0 5 1 4.0 5 2 4.0 5 3 4.0 5 4 - A verage Standard Deviation 4.0 5 QC1 4.0 5 QC2 4.0 5 QC3 1530 1530 1530 M easu red 8-2 TBA C oncentration (pg/L) 2060 1990 2070 1600 1930 220 1740 1600 1560 A verage Standard Deviation 2720 3020 2960 2260 2740 350 1570 1620 1610 A verage Standard Deviation % Recovery - 114 105 102 107 6.2 - 103 106 105 105 1.5 - 30Company Sanitized. Does Not Contain T SCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 T able 9: M easu red concentrations o f 8-2 T B A for pH 7.0, 50C, D ays 0 (29-Jan-2004) and 5 (03-Feb-2004). pH Day 7.0 0 7.0 0 7.0 0 7.0 0 N om inai 8-2 T B A M easured 8-2 TBA Rep C oncentration C oncentration (pg/L) (Hg/L) 1234- 2240 2280 2240 1450' A verage Standard D eviation 7.0 0 7.0 0 7.0 0 QC1 QC2 QC3 15302 1530 1530 2250 23 1660 1660 1670 A verage Standard Deviation 7.0 5 7.0 5 7.0 5 7.0 5 1 2 3 4 - 3180 3190 3180 3180 A verage Standard D ev iatio n 7.0 5 7.0 5 7.0 5 QC1 QC2 QC3 1530 1530 1530 3180 5.0 1820 1760 1790 Average Standard Deviation 1Not used in the calculation of the average and standard deviation. % Recovery - 108 108 109 108 0.6 - 119 115 117 117 2.0 -31 Company Sanitized. Does Not Contain TSCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 Table 10: M easured concentrations o f 8-2 TBA for pH 9.0, 50C, Days 0 (29-Jan-2004) and 5 (03-Feb-2003). N om inai 8-2 TBA pH Day Rep C oncentration (0 g /L ) 9.0 0 9.0 0 9.0 0 9.0 0 1 2 3 4 - M easured 8-2 TBA C oncentration (pg/L) 2340 2340 2290 2360 A verage Standard D eviation 9.0 0 QC1 9.0 0 QC2 9.0 0 QC3 1530 1530 1530 2330 30 1660 1680 1520 Average Standard Deviation 9.0 5 9.0 5 9.0 5 9.0 5 1 2 3 4 - 3680 3720 3690 3710 A verage Standard D eviation 9.0 5 QC1 9.0 5 QC2 9.0 5 QC3 1530 1530 1530 3700 18 1670 1660 1690 A verage Standard Deviation % Recovery - 108 110 99 106 5.9 - 109 108 110 109 1.0 - 32Company Sanitized. Does Not Contain TSCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH Table 11: Daily tem perature readings o f the shaking water bath. DuPont-13970 Tim e (Day) 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 T em perature pH Date (C) 1.2 12-Feb-2004 37.0 1.2 13 - F e b - 2 0 0 4 37.0 1.2 14-Feb-2004 37.0 1.2 15-Feb-2004 37.0 1.2 16 - F e b - 2 0 0 4 37.0 1.2 17-Feb-2004 37.0 4.0 05-Feb-2004 50.0 4.0 06-Feb-2004 50.0 4.0 07-Feb-2004 50.0 4.0 08-Feb-2004 50.0 4.0 09-Feb-2004 50.0 4.0 10-Feb-2004 50.0 7.0 29-Jan-2004 50.0 7.0 30-Jan-2004 50.0 7.0 31-Jan-2004 50.0 7.0 01-Feb-2004 50.0 7.0 02-Feb-2004 50.0 7.0 03-Feb-2004 50.0 9.0 29-Jan-2004 50.0 9.0 30-Jan-2004 50.0 9.0 31-Jan-2004 50.0 9.0 01-Feb-2004 50.0 9.0 02-Feb-2004 50.0 9.0 03-Feb-2004 50.0 -33Company Sanitized. Does Not Contain TSCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH Table 12: pH m easurem ents for D ay 0 and Day 5. DuPont-13970 Day Rep N om inal pH M easured pH 0 1 ,2 , 3, 4 1.2 _1 5 1 1.2 1.30 5 2 1.2 1.29 5 3 1.2 1.28 5 4 1.2 1.30 0 1,2, 3, 4 4.02 _3 5 1 4.02 4.08 5 2 4.02 4.08 5 3 4.02 4.08 5 4 4.02 4.09 0 1,2, 3 ,4 4.04 5 5 1 4.04 4.06 5 2 4.04 4.05 5 3 4.04 4.06 5 4 4.04 4.06 0 1,2, 3 ,4 7.0 6 5 1 7.0 7.08 5 2 7.0 7.09 5 3 7.0 7.08 5 4 7.0 7.08 0 1,2, 3 ,4 9.0 7 5 1 9.0 9.05 5 2 9.0 9.05 5 3 9.0 9.05 5 4 9.0 9.06 1pH not measured for replicate samples; measured buffer pH was 1.20 2pH 4.0 phthalate buffer 3 pH not measured for replicate samples; measured buffer pH was 3.99 4pH 4.0 citrate buffer 5pH not measured replicate samples; measured buffer pH was 4.01 6pH not measured for replicate samples; measured buffer pH was 6.99 7pH not measured for replicate samples; measured buffer pH was 9.00 -34Company Sanitized. Does Not Contain T SC A CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 F IG U R E S - 35Company Sanitized. Does Not Contain T SC A CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 Figure 1: E xam ple I9F N M R spectra o f 1050 m g/L fluorinated active ingred ient calibration standard (A), fluorinated active ingredient recovered in THF extract (B), and acetonitrile extract (C) for Day 5, pH 4.0 phthalate buffer. So NRM M 4 . IB B ** IHW - I. N D F '.t. * S m * v4a V L Jo tVi 4 "-- AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 Figure 2: E xam ple 19F N M R spectra o f 210 m g/L fluorinated active ingred ient calibration standard (A), fluorinated active ingredient recovered in THF extract (B), and acetonitrile extract (C) for Day 5, pH 1.2 buffer. - 37Company Sanitized. Does Not Contain TSCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 Figure 3: E xam ple 19F N M R spectra o f a blank control sam ple for D ay 5, pH 1.2 buffer (A) and the fluorinated active ingredient recovered in TH F extract (B) sample for Day 0, pH 1.2 buffer (B). SC3SK* pH 1 , . p e p -- , (Mry O V a4> ~ao i -To" -38Company Sanitized. Does Not Contain TSCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 Figure 4: E xam ple 19F N M R spectra o f the fluorinated active ingred ient recovered in THF extract for Day 0, pH 4.0 citrate buffer (A) and Day 5, pH 4.0 citrate buffer (B). acasw? 4s oH i-c~`r.e O '~ 8 C H M i x m h o o ""& W " 30 "2ST ' io M K M f tM , SCarOQ! IO ~ O b r~ iH a 0 4 l> XM p***-K , day 9 S -It* AOP** Tlit -10 - 39Company Sanitized. Does Not Contain T SCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 Figure 5: Exam ple F N M R spectra o f the fluorinated active ingredient recovered in THF extract for Day 0, pH 7.0 buffer (A) and Day 5, pH 7.0 buffer (B). N R 1 4 B 4 4 , SC3HK? 9 - F M 1 - 2 D O X KM p*t 7 . <*av 0 lupm Jo "ST W t M i aC.3TC HF-a003, MM PK3P~*. 9 H 7 . d a y * T -VO l OP 2o W ' 3oT """ ~> IO " 2 ' " --Jo j00am. - 40Company Sanitized. Does Not Contain TSCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 Figure 6: E xam ple 19F N M R spectra o f the fluorinated active ingred ient recovered in THF extract for Day 0, pH 9.0 buffer (A) and Day 5, pH 9.0 buffer (B). acss met-*.S -T a rib -a o C K S i K N H . MW o T -41 Company Sanitized. Does Not Contain T SCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH Figure 7: Representative calibration curve for 8-2 TBA. DuPont-13970 Figure 8: Representative chrom atogram s of 102 pg/L 8-2 TBA calibration standard and 520 pg/L D-8-2 TBA internal standard. -42Company Sanitized. Does Not Contain TSCA CBI. AR-226 H-24616: Hydrolytic Stability of H-24616 as a Function of pH DuPont-13970 Figure 9: Representative chrom atogram s for Day 5, pH 4.0 citrate buffer hydrolytic stability sample and 520 pg/L D-8-2 TBA internal standard. Figure 10: Representative chromatograms for Day 5, pH 4.0 citrate buffer blank sample and 520 pg/L D-8-2 TBA internal standard. - 43- Company Sanitized. Does Not Contain T SCA CBI. AR-226