Document ZJJXN9bM4z5dB8Npo3oOJ7qap

M jsl _ 33/7 . j4 R . 2 . R 6 - 3 3 I 7 DuPont-13970 TRADE SECRET Study Title H-24616: Hydrolytic Stability of H-24616 as a Function o f pH T e s t G u id e l in e s : OECD Guideline 111 A u t h o r : Bogdan Szostek, Ph.D. S t u d y C o m p l e t e d o n : June 9, 2004 P e r f o r m in g L a b o r a t o r y : 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 L a b o r a t o r y P r o je c t ID: DuPont-13970 W ork Request N um ber Service C o de N um ber Page 1of43 Company Sanitized. Does no! contain TSCA CB H-24616: Hydrolytic Stability o f H-24616 as a Function o f pH DuPont-13970 GOOD LABORATORY PRACTICE COM PLIANCE 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 of 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 o f 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 o f 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 Date Applicant / Sponsor:__________________________________________ __________ DuPont Representative Date -2'Company Sanitized. Does not contain TSCA CBf H-24616: Hydrolytic Stability o f H-24616 as a Function o f pH QUALITY ASSURANCE STATEM ENT 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 CB1 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 o f data obtained from this study. Approved by: $. Mark Kennedy, Php Manager Issued by Study Director: ?W v Bogdan Szostek, Ph.D. Senior Research Chemist ( j J Z oo^j Date Date -4 - Company Sanitized. Does not contain TSCA CBi H-24616: Hydrolytic Stability o f H-24616 as a Function of pH___________________________________________ DuPont-13970 TABLE OF CONTENTS Page G O O D L A B O R A T O R Y PR A C T IC E C O M P L IA N C E S T A T E M E N T ............ ............................ 2 Q U A L IT Y A S S U R A N C E S T A T E M E N T ............................................ 3 C E R T IF IC A T IO N ...............................................................................................................................................4 L IS T O F T A B L E S ........................................................................................ 6 L IST O F F IG U R E S .............................................................................................................................................7 ST U D Y IN F O R M A T IO N .......................................................................... 8 ST U D Y P E R S O N N E L ..................................... 9 S U M M A R Y ..................................................................................................... 10 IN T R O D U C T IO N .............................................................................................................................................. 11 S T U D Y D E S IG N ........................... 11 M A T E R IA L S A N D M E T H O D S ..................................................................................................................11 A. M aterials......................................................................................................................................11 1. Test Sub^mce ...................................................................................................................................................11 2. R e s i d u a P B H H n H-24616........................................................................................................................... 12 3. Preparation o f buner 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 o ftest s u b t ree................................................................................................................................... 14 4. Analysis 15 5. Calculations...........................................................................................................................................................16 R E S U L T S A N D D IS C U S S IO N .................................................................................................................... 18 C O N C L U S IO N S .................................................................................................................................................20 R E C O R D S A N D S A M P L E S T O R A G E ....................................................................................................21 R E F E R E N C E S .................................................................................................................................................... 21 T A B L E S .......................................................................................................................................... 22 F IG U R E S ............................................................................................................................................................... 35 -5 - Company Sanitized. Does not contain TSCA CBI H-24616: Hydrolytic Stability o f H-24616 as a Function o f pH DuPont-13970 LIST OF TABLES Page 1. Analytical results for aqueous stability o f H-24616, pH 1.2, 37C, Days 0 (12-Feb-2004) and 5 (17-Feb-2004).......................................................................................................................................................................23 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)....................................................................................................................................24 3. Analytical results for aqueous stability o f 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 o f H-24616, pH 7.0, 50C, Days 0 (29-Jan-2004) and 5 (03-Feb-2004).......................................................................................................................................................................26 5. Analytical results for aqueous stability o f H-24616, pH 9 .0 ,50C, Days 0 (29-Jan-2004) and 5 (03-Feb-2004).......................................................................................................................................................................27 6. Measured concentrations o i j j p H y p b r pH 1.2,37C, Days 0 (12-Feb-2004) and 5 (17-Feb-2004)...................28 7. Measured concentrations o f ^ | | f o r pH 4.0, 50C, pH 4.0 phthalate buffer, 50C, Days 0 (5-Feb-2004) and 5 (10-Feb-2004)....................................................................................................................................29 8. Measured concentrations o f S j p H |& b r pH 4.0, 50C, pH 4.0 citrate buffer, 50C, Days 0 (5-Feb-2004) and 5 (10-Feb^)04).T*............................................................................................................................. 30 9. Measured concentrations o:^ B H M ^ for pH 7.0, 50C, Days 0 (29-Jan-2004) and 5 (03-Feb-2004).................. 31 10. Measured concentrations o f^ jB B B ^ o r pH 9.0, 50C, Days 0 (29-Jan-2004) and 5(03-Feb-2003)............ 32 11. Daily temperature readings o f the shaking water bath.................................................................................................. 33 12. pH measurements for Day 0 and Day 5............................................................................................................................ 34 -6Company Sanitized. Does not contain TSCA CM H-24616: .. ' Hydrolytic Stability o f H-24616 as a Function of pH DuPont-13970 LIST OF FIGURES 1. Example 19F NMR spectra o f 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........................................................................................................................................ 2. Example I9F NMR spectra o f 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......................................................................................................................................................... 3. Example 19F NMR spectra o f 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)............................... 4. Example 19F NMR spectra o f 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)...................................................................... 5. Example 19F NMR 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).............................................................................................. 6. Example I9F NMR spectra o f the fluorinated active ingredient recovered in THF extract for Day 0, pH 9.0 buffer (A) and Day 5, pH 9.0 buffer (B)........... ................................................................................... 7. Representative calibration curve fo: 8. Representative chromatograms of 102 p g internal standard........................... alteration standard and 520 ug/L 9. Representativg chromatograms for Day 5, pH 4.0 citrate buffer hydrolytic stability sample and 520 pg/L f B B B B ^ n t e m a l standard................................................................................ 10. Representative chromatograms for Day 5, pH 4.0 citrate buffer blank sample and 520 pg/ internal standard..... ......................................................................................................................... Page 36 37 38 39 40 41 42 42 43 43 -7Company Sanitized. Does not contain TSCA CB| H-24616: Hydrolytic Stability o f H-24616 as a Function o f pH STUDY INFORM ATION Substance Tested: Synonvms/Codes: H-24616 Haskell Number: 24616 DuPont-13970 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 Lad. Does not contain TSCA CBi H-24616: Hydrolytic Stability o f 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 - ^moany Sanitized. Does not contain TSCA CM Hydrolytic Stability o f 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 o f 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 fi/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 o f fluorinated active ingredient o f 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 o f 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 of the fluorinated active ingredient material. The measured concentration oijB B BB Jdidm otindicate any hydrolytic degradation of the fluorinated active ingredient material t( jp H H B fo r pH 4.0 a n d pH 7.0 buffers. The observed concentrations o f^ k B M ||c a n be explained by the residualg B B M y resent in the test substance (H-24616). ForjpH 1.2 and 9.0 an increase o f theJpH B B jconcentration beyond that accounted for by resid u al^ B H B ip resen t in the test substance was observed. It was estimated that this increase would represent hydrolysis o f 0.06 and 0.03% the test substance for pH 1.2 and pH 9.0 buffers, respectively. - 10Company Sanitized. Does not contain TSCA CBI H-24616: Hydrolytic Stability o f H-24616 as a Function of pH DuPont-13970 IN T R O D U C T IO N The purpose o f 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 QECj Guideline l l l . {1) Additionally, t h e ^ ncentration o f t h e ^ J P H H H H H M H I H H H ( p b y a s monitored to investigate i f S H H w s 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 0.1 C 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 forQpBHBQ^ 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 o f the test substance used is the smaller of 0.01 M or half the saturation concentration. The test substance fluorinated active ingredient is essentially not soluble in water. Therefore, the study was conducted with excess o f undissolved fluorinated active ingredient and the amount o f the test substance was sufficient to satisfy the sensitivity requirements of analytical method. The molar concentration o f fluorinated active ingredient was estimated to be 0.002 M (in 5. Calculations). MATERIALS AND M ETHODS A. M aterials 1. Test Substance Name: H-24616 Composition: Haskell number 24616 The test substance was characterized to determine % fluorine, % solids, pH, Mn, Mw, and polydispersity with the following results: - 11 ^ompany^anilze.^os hTcohtaln H-24616: - Hydrolytic Stability o f H-24616 as a Function o f pH % Fluorine (w/w): % Solids (w/w) pH Mn: Mw: polydispersity 12.3% 24.3% 6.3 1500 amu 1700 amu 1.13 2. R e s id u a llf lH B p n H-24616 DuPont-13970 ResidualjHpHBHpontent in the test substance (H-24616) was determined by LC/MS using the same analytical method as described in Analysis o f ^ p | | i n Methods. Five replicate samples o f 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 o f dissolved sample was transferred to a glass LC vial and 0.9 mL acetonitrile and internal s t a n d a r d f l ^ ^ H f l b w a s added. Calibration standards were made in 10/90% THF/acetonitrile in the rang^ronM TO to 1020 ng/mL 3. Preparation of buffer solutions The following buffer solutions were prepared as described in the Annex to OECD guideline 111 .cl-) a. pH 1.2: The 0.2 M potassium chloride (KC1) was prepared by dissolving 3.78 g o f 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 o f 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 o f 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 o f water. The pH 4 buffer solution was prepared by placing 45 mL of 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 o f the buffer was adjusted to 4.0 with 1 M sodium hydroxide. - 12- Company Sanitized. Does not contain TSCA CBl H-24616: Hydrolytic Stability o f H-24616 as a Function o f pH c. pH 7: DuPont-13970 The 0.1 M solution o f potassium phosphate was prepared by dissolving 6.81 g o f potassium phosphate, monobasic (KH2PO4, purity 99.5%, Sigma) in 500 mL o f 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 o f 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 o f a 0.1 M H3BO3 solution in 0.1 M KC1 and 106.5 mL o f a 0.1 M sodium hydroxide solution into a 500-mL flask and bringing the flask to volume with water. The pH o f 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 pH 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; Eppcndorff 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 o f 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 o f the pHs tested. Approximately 45 mg o f 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 of 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 o f appropriate buffer were prepared to serve as the blanks. Four o f them were used as the Day 0 blanks and the other four - 13 - Company Sanitized. Does not contain TSCA CB H-24616: Hydrolytic Stability o f H-24616 as a Function o f pH D u P o n t-13 970 were wrapped in A1 foil and placed in the water bath to be theD ay 5 blanks. Additionally, six vials containing 3 mL o f appropriate buffer were spiked w ith ttp H ^ p s to c k solution (4.5 pL of 1020 The vials were crimp capped with the aluminum foil covered p a rto f the septa facing inside the vial. Three o f the vials were used as the QC samples fo i^ H H M Q i analysis on Day 0. The other three vials were wrapped in aluminum foil and placed in the water bath together with eight o f the test substance vials. These served a role of QC samples for I H H f l p n a l y s i s on Day 5. Four o f the Day 0 or four o f the Day 5 vials and two blank vials were processed for the fluorinated active ingredient of ^j-24616 determination. The other four of die Day 0 or Day 5 vials, two blanks vials, a n d flH p p H O C vials were processed for the letermination. w^ 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 o f acetonitrile was added, vials were recapped and vortexed for 10 minutes. Addition o f 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 die liquid phase was removed from the vial. Ten milliliters o f tetrahydrofurane (THF) was added to the vial, the vial was capped, and then sonicated for 30 minutes. Approximately 1 mL o f the THF solution was placed in a glass LC vial, 0.1 mL o f 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 o f 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 of fluorinated active ingredient o f H-24616. Fresh stock solution and calibration standards were prepared for each instance of analysis. Linear regression o f the peak area vs. standard concentration was used to establish the calibration curves. - 14- Company Sanitized. Does not contain TSCA C8I H-24616: Hydrolytic Stability o f H-24616 as a Function o f pH DuPont-13970 Instrumentation and conditions: NMR instrument: Bruker AMX360 with a I9F/!H dual probe Acquisition Parameters Pulse Program: zg Time Domain Size: 16 k Number o f Scans: 128 D1 Delay: 10 sec Dummy Scan: 2 Pulse Length: 8 psec Receiver Gain: automated by the instrument Instrument Frequency: 338.8314566 MHz Processing Parameters: Size o f Real Spectrum: 16 k Window Function: EM Window Size: 50 Hz Peak Used for Quantitation -CF3 resonance at 32.5 ppm 4. Analysis of' Samples designated f o f l|0 jQ p n a ly s is were processed by injecting 3 mL methyl-tert-butyl ether (MTBE) through tKe vial septum using a glass syringe. ^Neither Day 0 nor Day 5 samples were uncapped before MTBE injection to avoid any losses o l||p fH H ^ $ T h e vials containing injected MTBE were vortexed for 10 minutes on a multitube vortexer. Subsequently, the vials were uncapped and 0.1 mL o f the MTBE extract was transferred to a glass LC vial, 0.9 m l, acetonitrile added, spiked with internal standard, crimped capped, and subjected to LC/MS analysis f o r ^ | m p ^ Oakwood Products, West Columbia, SC) was used as the analytical stan d ard o fjH H H K l as th ^ n te S S R tm a w d ^ to c k s o lu tio n ^ ^ ^ ^ ^ ^ * ^ I T 000 mg/L) o f 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 P g / L ^ m A constant level of internal standard was used: 520 The calibration curves were contracted using th e ra tio o fth e peak area for ions m7z monitored for | B B B e n d peak area for ions monitored f o f l H H H t p n d the concentrations of - 15Company Sanitized. Does not contain TSCA CBI H-24616: Hydrolytic Stability o f H-24616 as a Function of pH D uP ont-13 970 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 3D 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 _ IR o f 463, 523; 0-6 min ~ 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 0.045 g o f H-24616 containing 22.6% fluorinated active ingredient was weighed into the vial and 3 mL o f buffer was added to the vial the nominal concentration o f fluorinated active ingredient is: (0.045 g* 0.226*1000 mg/g) / 0.003 L = 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 j B B K n the spiked samples was calculated as follows: the 4.5 pL o f 1020 m g/L | (H B B b p ik e represents 4.5 pL x 1020 ng/pL= 4590 ng `i H & r h i s amount was extracted with 3 mL of MTBE. The expected nominal concentration MTBE extract is: 4590 ng/3 mL=1530 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) o f fluorinated active ingredient: 1500 g/mole. When 45 mg o f H-24616 is used, it represents (0.045 g * 0.226)/1500 g/mole = 6.78 10'6mole o f fluorinated active ingredient. Taking into account that 3 mL of buffer is used this would represent the molar concentration: 6.78 10'6mole/0.003 L = 0.0023 M. The expected concentration o lfiB B H B ln - 16- Company Sanitized. Does not contain TSCA CBf n-zwio: Hydrolytic Stability o f H-24616 as a Function of pH D uPont-13 970 the MTBE extract if 10% o f the H-24616 hydrolyzed to fo rm jp B M y c a n be estimated in the following way. Ten percent o f 45 mg of H-24616 represents 4.5 mg. Taking into account that_ the test substance contains 12.3% fluorine and assuming that all fluorine is present as| the hydrolysis o f flie 4.5 mg of the test substance_would release: 4.5 mg x 0J.23 /(324 mg F / m m o l e |m |H 4 6 4 . 1 mg/mmole) = 0.79 m g ^ H H B j This amount o f i p | | H w o u l d be contained in 3 mLs o f MTBE extract. Therefore, expected concentration o fM JP B M w o u ld be: 0.79 mg/0.003 L= 263 mg/L. The H-24616 contains r e s id u a J B B B The expected, maximum concentration o fj derived from residual'^ P J J J p n the MTBE extract (3 mL), assuming complete extraction for a 45 mg sample o f H-24616 is: 45 mg of H-24616 x (0.019%/100)/0.003 V= 2.85 mg/L (2850 pg/L). /**"N Company Sanitized. Does not contain TSC CBI H-24616: Hydrolytic Stability o f H-24616 as a Function o f pH DuPont-13970 RESULTS AND DISCUSSION Tables 1-5 present the results for measured concentrations o f fluorinated active ingredient o f 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 axe 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 of 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 o f H-24616 is hydrolytically stable at these pHs (ti/2>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 (50C). Simply, the addition o f acetonitrile to the vial containing the test substance would not completely precipitate the fluorinated active ingredient. The amount o f 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 NM R spectrum o f the extracts was collected and a quantitation o f the fluorinated active ingredient in the acetonitrile extract was attempted. Accurate quantitation of 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 theN M R measurements done for THF and acetonitrile extract matrix, a calibration standard o j^ B H M K n both solvents was made and measured by the NMR methods. This allowed the derivation o f 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 o f the fluorinated active ingredient is present in the acetonitrile extract for Day 5 samples. Comparison o f the 19F NMR spectra of 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 I9F NM R (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 o f results for acetonitrile extracts, it is difficult to draw a conclusion about hydrolytic stability (ti/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 CBl H-24616: Hydrolytic Stability o f H-:24616 as a Function of pH D u P o n t-13 970 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 o f 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 of fluorinated active ingredient was detected in the acetonitrile extracts, but, for the reasons described above for the phthalate buffer, accurate quantitation of 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 o f the NMR spectra o f 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 !9F NMR (Figure 2). Figure 3 presents the NMR spectra o f 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 j p j j j j n H-24616 was measured before the start of the hydrolytic study. Based on the results o f five replicates o f the H-24616, the content o f re sid u a lB B B ^ B in H-24616 was calculated to be 0.0190.0003%. Assuming that the buffer extracts all the residual M H ^ ^ M f o m the test substance during the time o f hydrolytic stability experiment, the expected concentration o f ^ I P P H ^ n the test system would be 2.85 mg/L (see 5 Calculations). The hydrolysis o f the fluorinated active ingredient o f H-24616 would jnost likgly result in the release If ten percent of H-24616 hydrolyzed releasing the H ^ H ^ |t h e expected concentration o f ^ m Q j n the test system would be 263 mg/L Calculations). Tables 6-10 present the measured concentrations o f f P P B Q f o r 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 o f f p B H ^ f o r pH 1.2 (Table 6) indicates the increase o f ^ ^ ^ K o n c e n f i a t i o n for Day 5 samples beyond the level that can b e accounted for by the^esIduaJ^PB B B jpres ent in the test substance. The concentration o f^ B M ^ Jm e a su re d for the Day Osamples is comparable with the expected concentration om pBBB^priginating from re s id u a l(J P B J 0 T h e Day 5 concentration o fjp B H B L p higher by approximately 1.5 mg/L more than the expected concentration originating from the residua!^ B S B M b se rv e d in c re a ^ o f^ p B B B j^ concentration indicates the hydrolysis o f the test substance to form V B B N jSi This increase represents only estimated hydrolysis of approximately 0.06% (1.5 mg/L7 2630 mg/L * 100%) of the test substance present in the test system. The acceptable recovery results for the quality control samples (QC) for Day 0 and Day 5 Remonstrate the validity o f th J& JJJJ^ n e a su re m e n t (Table 6). The measured concentrations o fV B B B Q b r pH 4.0 citratnplTH .0 phthalate, pH 7.0 test system dp not exceed significantly the expected concentration ofB B pj^Q brfrinatingfrom the r e s i d u a l m J f T a b l e s 7-9). This indicates that there is no contribution o fS p B B ^ p fo m the hydrolysis of the test substance. The recovery results for QC samples are also m the acceptable range (Tableg 1-9). The measured concentration o q H H H B F or pH 9.0 (Table 10) indicate the increase ofB pB B jftoncentration for Day 5 samples beyond the level that can be accounted for by the resi^alH jM jB Q p resen t in the test substance. The increase is ^approximately 0.85 m g /L ^ J j B p b e y o n R^he concentration explainable by the residual and is indicative o f generation oiV H B H K from hydrolysis o f fluorinated active 19- ,,oteoniainTSCACB! Company Sanit'ze<^' Doe ^#***>\ H-24616: Hydrolytic Stability o f H-24616 as a Function of pH D uP ont-13 970 ingredient at pH 9.0. This increase represents an estimated hydrolysis o f approximately 0.03% (0.85 mg/L / 2630 mg/L * 100 %) of the test substance present in the test system. Figure 7 shows a representativexalibration curve obtained f o n H B H f t Figures 8-10 show chromatograms for th q g p B ^ jjp a lib ra tio n 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 o f 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 o f samples containing the test substance. The measured pH of Day 0 buffers and Day 5 replicate samples for all o f the investigated pHs was at the specified nominal level with the deviation not exceeding 0.1. / ''"""'N CONCLUSIONS The hydrolytic stability o f 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 tj/2>one year. The data obtained for hydrolytic stability of the fluorinated active ingredient o f 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 o f fluorinated active ingredient stays dissolved after addition o f acetonitrile to the samples that were exposed to test conditions in pH 1.2 and pH 4.0-phthalate buffer. Accurate quantitation o f the dissolved fraction o f 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 to the fluorinated active ingredient material. The measured concentration ofM B B B M did n o t^ idicate any hydrolytic degradation o f the fluorinated activqjpgredig^t material t ^ H H M f o r pH 4.(Lpnd pH i O buffers. The observed concentrations o iJ p H H ^ p :a n be explamed by the resid u afip B B B B resen t in the test substance (H-24616). For pH 1.2 and 9X) an iny^ease o f th fip H H K o n c e n tra tio n beyond that which can be accounted for by residuajJpH H H p)resent 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. -20Company Sanitized. Does not eonWii troa H-24616: Hydrolytic Stability o f H-24616 as a Function o f pH 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. -21 - Company Sanitize Does not contain TSCA CB H-24t>16: Hydrolytic Stability o f H -24616 as a Function of pH D u P o n t-13 970 TABLES -22- Company Sanlftest!. Does yto!rta'mTSCA CB| H-24616: Hydrolytic Stability o f H-24616 as a Function o f pH DuPont-13970 T ab le 1: A nalytical results for aqueous stab ility o f H -24616, p H 1 .2 ,37C , D a y s 0 (12-Feb-2004) and 5 (17-Feb-2004). Fluorinated active Fluorinated active ingredient of H-24616 ingredient of H-24616 pH Day Rep Nominal Concentration5 Measured Concentration (mg/L) (mg/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 Average 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 2660]+2362 27103 2790`+2342 2440'+2552 864 82 914 834 Average 86 Standard Deviation 4.0 recovered in the THF extract 2 recovered in the acetonitrile extract 3 sample o f acetonitrile extract not saved 4 represents total recovery from THF and acetonitrile extract 5 represents nominal concentration o f fluorinated active ingredient in the buffer -23 - Omoany Sanitized, Poes not contain TSCA CBf H-24616: Hydrolytic Stability o f H-24616 as a Function o f pH DuPont-13970 Table 2: Analytical results for aqueous stability of H -24616, pH 4.0 phthalate buffer, 50C, Days 0 (5-Feb-2004) and 5 (lQ-Feb-2004). Fluorinated active Fluorinated active ingredient of H-24616 ingredient of H-24616 pH Day Rep Nominal Concentration'1 Measured Concentration (mg/L) (mg/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 Average 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 1510]+9402 1320`+15402 I090'+16702 1340`+15902 723 863 843 863 Average 82 Standard Deviation 6.9 recovered in the THF extract 2 recovered in the acetonitrile extract 3 represents total recovery from THF and acetonitrile extract 4 represents nominal concentration o f fluorinated active ingredient in the buffer - 24- Company Sanitized* not contain TSCA Cu, H-24616: Hydrolytic Stability o f H-24616 as a Function o f pH DuPont-13 970 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). Fluorinated active Fluorinated active ingredient of H-24616 ingredient of H-24616 pH Day Rep Nominal Concentration5 Measured Concentration (mg/L) (mg/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 Average Standard Deviation 92 3.2 4.0 5 1 4.0 5 2 4.0 5 3 4.0 5 4 3350 3320 3330 3360 2930 834`+752 2940 3110 87 273 88 93 Average Standard Deviation 8945 3.14 2 recovered in the acetonitrile extract 3 the vial most likely leaked during the experiment 4 Day 5, replicate 2 excluded from calculation 5 represents nominal concentration o f fluorinated active ingredient in the buffer Company S a n l . W T8CA C" H-24616: Hydrolytic Stability o f H-24616 as a Function o f pH DuPont-13970 Table 4: A nalytical results for aqueous stability o f H -24616, pH 7 .0 ,50C, D ays 0 (29-Jan-2004) and 5 (03-Feb-2004). pH Day Rep Fluorinated active ingredient of H-24616 Nominal Concentration (mg/L)1 Fluorinated active ingredient of H-24616 Measured Concentration (mg/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 Average 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 Average 91 Standard Deviation 1.8 1represents nominal concentration o f fluorinated active ingredient in the buffer - 26 0Mpany m l contain TSCA CBi H-24616: Hydrolytic Stability o f H-24616 as a Function o f pH DuPont-13970 Table 5: Analytical results for aqueous stability o f H -24616, pH 9 .0 ,50C, D ays 0 [ (29-Jan-2004) and 5 (03-Feb-2004). Fluorinated active Fluorinated active ingredient of H-24616 ingredient of H-24616 pH Day Rep Nominal Concentration Measured Concentration (mg/L)1 (mg/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 92 3080 92 3020 91 3110 93 Average 92 Standard Deviation 0.81 'represents nominal concentration o f fluorinated active ingredient in the buffer -27Company Sanitized. Does not contain TSCA CHI H-24616: Hydrolytic Stability o f H-24616 as a Function of pH T ab le 6: M easu red concentration s o: 5 (17-Feb-2004). DuPont-13 970 br pH 1 .2 ,37C, Days 0 (12-Feb-2004) and Nomina% B B j 3 i pH Day Rep C oncentranti (pg/l!) 12 0 1 12 0 2 12 0 3 12 0 4 - M easuredH H M l 1 Concentrano Ipg/L) % Recovery 2920 2950 2800 2950 - Average Standard Devitin 2900 71 12 0 QC1 12 0 QC2 12 0 QC3 1530 . 1530 1530 1580 103 1580 103 1580 103 Average Standard Deviation 103 0.0 12 5 1 12 5 2 12 5 3 12 5 4 - 4010 4390 4060 4350 - Average Standard Deviation 4200 200 12 5 QC1 12 5 QC2 12 5 QC3 1530 1530 1530 1640 107 1660 108 1650 108 Average Standard Deviation 108 0.6 -28- Company Sanitized. Does notcontam "IS&A.CD11 H-24616: Hydrolytic Stability o f H-24616 as a Function of pH DuPont-13970 Table 7: M easured concentrations o f J P H H J f o r pH 4.0, 50C, pH 4.0 phthalate buffer, 50C, Days 0 (5-Feb-2004) and 5 (10-Feb-2004). N o m in a fip |$ b pH Day Rep Concentratimi (pg/L) 4.0 0 1 4.0 0 2 4.0 0 3 4.0 0 4 - Average 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 - Average Standard Deviation 4.0 5 QC1 4.0 5 QC2 4.0 5 QC3 1530 1530 1530 M easured^jp^j^ Concentration (pg/L) % Recovery 2070 2210 2130 2070 - 2120 66 1570 103 1600 105 1580 103 Average Standard Deviation 104 1.2 3400 3460 1710 1690 - 2570 1000 1650 108 1430 93 1680 110 Average Standard Deviation 104 9.3 - 29Company Sanitized. Does not contain TSCA CBK H-24616: Hydrolytic Stability o f H-24616 as a Function of pH DuPont-13970 Table 8 M easured concentrations o f g B B ^ f o r pH 4.0, 50C, pH 4.0 citrate buffer, 50C, Days 0 (5-Feb-2004) and 5 (10-Feb-2004). N om inaM pH lR pH Day Rep Concentrauon (ug/L) 4.0 0 1 4.0 0 2 4.0 0 3 4.0 0 4 - - Average Standard Deviton 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 - Average Standard Deviton 4.0 5 QC1 4.0 5 QC2 4.0 5 QC3 1530 1530 1530 M easured^PH ^^ Concentration (pg/L) % Recovery 2060 1990 2070 1600 - 1930 220 1740 114 1600 105 1560 102 Average Standard Deviation 107 6.2 2720 3020 2960 2260 - 2740 350 1570 103 1620 106 1610 105 Average Standard Deviation 105 1.5 -30- Company Sanitized, Does not contain TSCA CB1 H-24616: -' Hydrolytic Stability o f H-24616 as a Function o f pH Table 9: M easured concentrations o 5 (03-Feb-2004). D uP ont-13 970 'or p H 7.0, 50C, D ays 0 (29-Jan -2004) and pH Day 7.0 0 7.0 0 7.0 0 7.0 0 Rep N om inaH p^H Concentratimi (Ug/L) M easured^j|pH B 9^ Concentraton (jig/L) % Recovery 1234- 2240 2280 2240 14501 - Average Standard Deviation 2250 23 7.0 0 QC1 15302 1660 108 7.0 0 QC2 1530 1660 108 7.0 0 QC3 1530 1670 109 Average Standard Deviation 108 0.6 7.0 5 7.0 5 7.0 5 7.0 5 1 2 3 4 - 3180 3190 3180 3180 - Average Standard Deviation 7.0 5 QC1 7.0 5 QC2 7.0 5 QC3 1530 1530 1530 3180 5.0 1820 119 1760 115 1790 117 Average Standard Deviation 1Not used in the calculation o f the average and standard deviation. 117 2.0 -31 - 'mpany San!Uzd. notcontain TSCA CI H-24616: Hydrolytic Stability o f H-24616 as a Function o f pH Table 10: M easured concentrations o 5 (03-Feb-2003). DuPont-13970 nr pH 9.0, 50C, D ays 0 (29-Jan-2004) and g. 11 pH Day Rep N onunanpH H Concentration U (Pg/L) M easure< fi|pM gl Concentration (pg/Lj* % Recovery ' 9.0 0 1 - 2340 - 9.0 0 2 - 2340 - 9.0 0 3 - 2290 - 9.0 0 4 - 2360 - Average Standard Deviation 9.0 0 QC1 9.0 0 QC2 9.0 0 QC3 1530 1530 1530 2330 30 1660 108 1680 110 1520 99 Average Standard Deviation 106 5.9 9.0 5 9.0 5 9.0 5 9.0 5 1 2 3 4 - 3680 3720 3690 3710 - Average Standard Deviation 9.0 5 QC1 9.0 5 QC2 9.0 5 QC3 1530 1530 1530 3700 18 1670 109 1660 108 1690 no Average Standard Deviation 109 1.0 -32Company Sanitized. Does not contain TSCA CBt H-24616: Hydrolytic Stability o f H-24616 as a Function of pH Table 11: D aily tem perature readings of the shaking w ater bath. DuPont-13970 Time (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 Temperature pH Date (C) 1.2 12-Feb-2004 37.0 1.2 13-Feb-2004 37.0 1.2 14-Feb-2004 37.0 1.2 15-Feb-2004 37.0 1.2 16-Feb-2004 37.0 12 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 Company Sanitized. Does not contain TSCA CB| H-24616: Hydrolytic Stability o f H-24616 as a Function of pH Table 12: pH m easurem ents for D ay 0 and D ay 5. DuPont-13970 Day Rep Nominal pH Measured pH 0 1, 2, 3, 4 1.2 J 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 J 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 J 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 J , 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 2 pH 4.0 phthalate buffer 3 pH not measured for replicate samples; measured buffer pH was 3.99 4 pH 4.0 citrate buffer 5 pH not measured replicate samples; measured buffer pH was 4.01 6 pH not measured for replicate samples; measured buffer pH was 6.99 7pH not measured for replicate samples; measured buffer pH was 9.00 -34- Company SanmzaO. Dona no. contain TSCA cm H-24616: Hydrolytic Stability o f H-24616 as a Function o f pH DuPont-13970 FIGURES -35- Company Sanitized, Does m l contain TSCA CBS H-24616: '' - ' Hydrolytic Stability ofH -24616 as a Function of pH___________________________________________ DuPont-13970 F igure 1: E xam ple 19F N M R spectra o f 1050 m g/L fluorinated active in gred ien t calibration standard (A), fluorinated active ingredient recovered in THF extract (B), and acetonitrile extract (C) for Day 5, pH 4.0 phthalate buffer. -36Company Sanitized, Does not contain TSCA CM H-24616: Hydrolytic Stability o f H-24616 as a Function o f pH DuPont-13970 Figure 2: E xam p le 19F N M R spectra o f 210 m g/L fluorinated active in g red ien t calibration standard (A), fluorinated active ingredient recovered in THF extract (B), and acetonitrile extract (C) for D ay 5, pH 1.2 buffer. -37Company Sanitized. Does not contain TSCA CBf H-24616: Hydrolytic Stability o f H-24616 as a Function of pH DuPont-13970 Figure 3: E xam ple 19F N M R spectra of a b lan k control sam ple for D a y 5, p H 1.2 buffer (A) and the fluorinated active ingredient recovered in THF extract (B) sample for Day 0, pH 1.2 buffer (B). ----------------------------------------------- r > - n || . 3 ------------------------------------------------- p w i .-it. ac-., .KH s A -38- c " y S a ,,ifeet,.0oM no*contain Tsca CBI H-24616: Hydrolytic Stability o f H-24616 as a Function of pH D uP ont-23 970 Figure 4: E xam p le I9F N M R spectra o f the flu orin ated active ingred ient reco v ered in T H F extract for Day 0, pH 4.0 citrate buffer (A) and D ay 5, pH 4.0 citrate buffer (B). -39 - Company SanilizadL. Qoes-nat contain- TSCACS# H-24616: Hydrolytic Stability o f H-24616 as a Function of pH DuPont-13970 F igure 5: E xam ple 19F N M R spectra o f th e fluorinated active in gred ien t recovered in THF extract for D ay 0, pH 7.0 buffer (A) and Day 5, pH 7.0 buffer (B). -40- Company Sanitized. Does not contain TSCA CB| H-24616: Hydrolytic Stability o f H-24616 as a Function of pH DuPont-13970 F igu re 6: E xam ple 19F N M R spectra o f the fiuorinated active in gred ien t recovered in THF extract for D ay 0, pH 9.0 buffer (A) and D ay 5, pH 9.0 buffer (B). -41 - Company Sanitized. Does not contain TSCA CBi H-246I6: Hydrolytic Stability o f H-24616 as a Function o f pH F igu re 7: R ep resentative calibration curve fo DuPont-13970 Figure 8: R eprsentatif chrom atograms o f 102 jig/Lj and 520 p g/L lB B B H B K n tern al standard. 021004b15 Smooth (Mn,1 x3) 1 3.61 40822.34 245953 %- 021004b 15 Smooth(Mn,1x3) 100- %- 3.59 221340.06 1293379 alibration standard SIR of 5 channels,ES463,523 2.496e+005 min SIR of 5 channels,ES- 467,528 1.301e+006 1.00 2.00 3.0 4.0 5.00 -42Company Sanitized. Does not contain TSCA CM H-24616: Hydrolytic Stability o f H -24616 as a Function of pH DuPont-13970 Figure 9: Representative chrom atogram ^for DayJL pH 4.0 citrate buffer hydrolytic stability sample and 520 p g /I^ H p B H H H n tern a l standard. Figure 10: R e p r s e n ta tif chromajjggrams for Day 5, pH 4.0 citrate buffer blank sam ple and 520 p g / H B lB I^ w n te r n a l standard. -43 - Company Sanitized. Does not contain TSCA CSt-