Document mb9zZkyNeZEngBw8O1OyBN15k

tos b l ~ TRADE SECRET Study Title Hydrolytic Stability of H-25435 as a Function o f pH DuPont-14965 Test Guidelines: OECD Guideline 111 Author: Bogdan Szostek, Ph.D. Study Completed on: October 15,2004 Performing Laboratory: E.I. du Pont de Nemours and Company HaskellSMLaboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Laboratory Project ID: DuPont-14965 Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Work Request Number: 15028 Service Code Number: 392 Page 1 of 45 Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 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 compatible with the OECD Principles o f Good Laboratory Practice (as revised 1997), ENV/MC/CHEM(98)17, OECD, Paris, 1998, and MAFF Japan Good Laboratory Practice Standards (11 NohSan Number 6283), except for the item documented below. The item listed does not impact the validity of the study. 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 o f 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: k9 Bogdan Szostek, PhD . Senior Research Chemist 1^ - O C a Date Applicant / Sponsor:_________________________________________ DuPont Representative Date -2 - Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-2S435 as a Function of pH DuPont-14965 QUALITY ASSURANCE STATEMENT Work Request Number: 15028 Study Code Number: 392 The conduct o f this study has been subjected to periodic Quality Assurance inspections. The dates o f inspection are indicated below. Phase Audited Conduct: Report/Records: Audit Dates 06 May 2004 09-12 Aug 2004 Dates Reported to Study Director and Management 07 May 2004 24 Aug 2004 Reported by: DonnsfM. Johnston Quality Assurance Auditor iS 'Q c J ' 3 -& ' Date -3 - Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation o f data obtained from this study. Approved by: -jg- S. Mark Kennedy, Ph Issued by Study Director: i. 9 Bogdan Szostek, Ph.D. Senior Research Chemist t ST-OCX"' 2.0 Date 1\ Date -4 - Company Sanitized. Does Not Contain TSCA CBI. Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 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 PL IA N C E ST A T E M E N T ........................................ 2 Q U A L IT Y A SSU 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 LIST O F T A B L E S ........................................................................................................... 6 LIST O F F IG U R E S ..........................................................................................................................................7 ST U D Y IN F O R M A T IO N .............................................................................................................................. 8 S U M M A R Y .........................................................................................................................................................9 IN T R O D U C T IO N ........................................................................................................................................... 10 ST U D Y D E SIG N .......................................................................................... 10 M A T ER IA LS A N D M E T H O D S ............................................................ 10 A. M aterials.................................................................................... 10 1. Test Substance...........................................................................................................................................10 2. Residual 8-2 TBA in H-25435....................................................................................................................10 3. Preparation of buffer solutions....................................................................................................................11 4. Equipment....................................................................................................................................................12 B. Methods.....................................................................................................................................12 1. Preliminary test......................................................................................................................................... 12 2. pH measurement and temperature.............................................................................................................. 12 3. Analysis of test substance...........................................................................................................................12 4. Analysis of 8-2 TBA................................................................................................................................... 18 5. Calculations................................................................................................................................................ 20 R E SU L TS AN D D IS C U S S IO N .............................................................. 21 C O N C L U S IO N S ............................................................................................................................................. 22 R E C O R D S A N D SA M PLE S T O R A G E ................................................................................................. 23 R E F E R E N C E S............................................................................................................................. 23 T A B L E S............................................................................................................................................................. 24 F IG U R E S....................................................... 35 -5 - Company Sanitized. Does Not Contain TSCA CBI. AR-226 / --N Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 LIST OF TABLES Page 1. ANALYTICAL RESULTS FOR AQUEOUS STABILITY OF H-25435, pH 1.2, 37C, DAYS 0 (03-JUNE-2004) AND 5 (08-JUNE-2004)...............................................................................................................25 2. ANALYTICAL RESULTS FOR AQUEOUS STABILITY OF H-25435, pH 4.0, 50C, DAYS 0 (13-MAY-2004) AND 5 (18-MAY-2004)................................................................................................................26 3. ANALYTICAL RESULTS FOR AQUEOUS STABILITY OF H-25435, pH 7.0, 50C, DAYS 0 (06-MAY-2004) AND 5 (11-MAY-2004)................................................................................................................27 4. ANALYTICAL RESULTS FOR AQUEOUS STABILITY OF H-25435, pH 9.0, 50C, DAYS 0 (13-MAY-2004) AND 5 (18-MAY-2004)................................................................................................................28 5. MEASURED CONCENTRATIONS OF 8-2 TBA FOR pH 1.2, 37C, DAYS 0 (03-JUNE-2004) AND 5 (08-JUNE-2004).................................................................................................... 29 6. MEASURED CONCENTRATIONS OF 8-2 TBA FOR pH 4.0, 50C, DAYS 0 (13-MAY-2004) AND 5 (18-MAY-2004)......................................................................................................................................................... 30 7. MEASURED CONCENTRATIONS OF 8-2 TBA FOR pH 7.0, 50C, DAYS 0 (06-MAY-2004) AND 5 (ll-MAY-2004)......................................................................................................................................................... 31 8. MEASURED CONCENTRATIONS OF 8-2 TBA FOR pH 9.0, 50C, DAYS 0 (13-MAY-2004) AND 5 (18-MAY-2004)......................................................................................................................................................... 32 9. DAILY TEMPERATURE READINGS OF THE SHAKING WATER BATH....................................................33 10. pH MEASUREMENTS FOR DAY 0 AND DAY 5................................................................................................ 34 -6 - Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 LIST OF FIGURES Page 1. EXAMPLE CALIBRATION CURVES OBTAINED FOR MONITORED [ ]............ 36 2. EXAMPLE CHROMATOGRAMS OF MONITORED [ ] OBTAINED FOR A BLANK SAMPLE FOR pH 7.0 BUFFER ON DAY 0...........................................................................................37 3. EXAMPLE CHROMATOGRAMS OF MONITORED [ ] OBTAINED FOR A CALIBRATION STANDARD CONTAINING 5.3 mg/L H-25435......................................................................38 4. EXAMPLE CHROMATOGRAMS OF MONITORED [ ] OBTAINED FOR DAY 0, pH 7.0 HYDROLYTIC STABILITY SAMPLE....................................................................................... 39 5. EXAMPLE CHROMATOGRAMS OF MONITORED [ ] OBTAINED FOR DAY 5, pH 7.0 HYDROLYTIC STABILITY SAMPLE....................................................................................... 40 6. REPRESENTATIVE CALIBRATION CURVE FOR 8-2 TBA........................................................................... 41 7. REPRESENTATIVE CHROMATOGRAMS OF 104 ig/L 8-2 TBA CALIBRATION STANDARD AND 515 ftg/L D-8-2 TBA INTERNAL STANDARD......................................................................................... 42 8. REPRESENTATIVE CHROMATOGRAMS FOR DAY 5, pH 7.0 BUFFER BLANK SAMPLE....................43 9. REPRESENTATIVE CHROMATOGRAMS FOR DAY 5, pH 7.0 HYDROLYTIC STABILITY SAMPLE AND 515 jug/L D-8-2 TBA INTERNAL STANDARD........................................................................ 44 10. REPRESENTATIVE CHROMATOGRAMS FOR DAY 0, pH 7.0 HYDROLYTIC STABILITY SAMPLE AND 515 /g/L D-8-2 TBA INTERNAL STANDARD........................................................................ 45 -7 - Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 STUDY INFORMATION Substance Tested: [ ] Svnonvms/Codes: [ H-25435 [ ] ] Haskell Number: 25435 Composition: [ [ [ [ [ 1 ] ] ] ] [1 Known Impurities: None identified Physical Characteristics: Amber brown liquid Stability: The test substance appeared to be stable under the conditions o f the study; no evidence o f instability was observed. Study Initiated/Completed: May 4, 2004 / (see report cover page) In-Life Initiated/Completed: May 6,2004 / July 6,2004 if***^ Company Sanitized. Does Not Contain TSCA CBI. AR-226 suw**. Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 SUMMARY The hydrolytic stability of H-25435 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. [ ] representing major chemical species present in H-25435 were monitored during the hydrolytic stability study. Measured concentrations and recoveries o f individual [ ] reported as average concentrations and recoveries for Day 0 and Day 5 replicate samples, were used to evaluate the hydrolytic stability of H-25435. The average percent recoveries for the Day 0 and Day 5, pH 1.2 replicate samples were 76 5.9% and 63 28%, respectively. The average percent recoveries for the Day 0 and Day 5, pH 4.0 buffer replicate samples were 97 3.0% and 95 4.4%, respectively. The average percent recoveries for the Day 0 and Day 5, pH 7.0 replicate samples were 92 1.1% and 90 0.9%, respectively. The average percent recoveries for the Day 0 and Day 5, pH 9.0 replicate samples were 96 0.6% and 95 9.3%, respectively. The H-25435 was demonstrated to be hydrolytically stable at pH 9.0, pH 7.0, and pH 4.0. The data obtained for hydrolytic stability o f H-25435 at pH 1.2 show the difference o f average recoveries for monitored [ ] between Day 0 and Day 5 samples is larger than 10 percent. However, a precipitate was observed after addition o f the test substance to the pH 1.2 buffer. The precipitate was difficult io completely dissolve even after addition of acetonitrile and extended sonication, especially for the Day 5 samples. This lowered the observed recoveries, especially for the bis esters. Therefore, the observed change in the average / '"v recoveries between the Day 0 and Day 5 samples is most likely because o f observed solubility problems rather than hydrolysis. The measured concentration of 8-2 TBA did not indicate any hydrolytic degradation o f H-25435 to 8-2 TBA for any of the investigated pHs. The observed concentrations o f 8-2 TBA can be explained by the residual 8-2 TBA present in the test substance (H-25435). There was no significant increase o f 8-2 TBA concentration observed between Day 0 and Day 5 samples for any o f the investigated pHs. -9 -- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 IN T R O D U C T IO N The purpose of the study was to investigate the hydrolytic stability o f H-25435 in pH 1.2,4.0, 7.0, and 9.0 aqueous buffer solutions. The study was conducted according to the protocol: "Hydrolytic stability o f H-25435 as a function o f pH", which was patterned after the OECD Guideline l l l . (1) Additionally, the concentration o f 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 each o f the pH 4.0, 7.0, 9.0 and at 37 0.1 C for pH 1.2. If less than 10% o f the test substance hydrolysis is 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. MATERIALS AND METHODS A. M aterials 1. Name: Composition: Test Substance H-25435 [ [ [ [ [ Haskell number: 25435 2. Residual 8-2 TBA in H-25435 Residual 8-2 TBA content in the test substance (H-25435) was determined by LC/MS using the same analytical method as described in Analysis o f 8-2 TBA in Methods. Five replicate samples o f H-25435 (approximately 45 mg) were introduced to glass vials, weighed, 10 mL o f THF (tetrahydrofurane) was added, vials crimped, and sonicated for 1.5 hours. 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 10200 ng/mL 8-2 TBA. - 10- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 3. Preparation o f buffer solutions The following buffer solutions were prepared as described in the Annex to OECD guideline l l l . (1) a. pH 1.2: The 0.2 M potassium chloride (KC1) was prepared by dissolving 3.73 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 4.5 mL 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 sodium hydroxide. b. pH 4: The 0.1 M solution of potassium hydrogen phthalate was prepared by dissolving 5.1 g of potassium hydrogen phthalate (purity 99+%, Sigma) in 250 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 o f 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. c. pH 7: The 0.1 M solution of potassium phosphate was prepared by dissolving 3.4 g of potassium phosphate, monobasic (KH2PO4, purity 99.5%, Sigma) in 250 mL o f water. The pH 7 buffer solution was prepared by placing 148 mL o f 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 1.90 g o f KC1 (purity 99.0%, Sigma) and 1.55 g o f H3BO3(purity 99.5%, Sigma) in 250 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 hydrochloric acid. Water used for buffer preparation was obtained from a Bamstead NANOpure Diamond water purification system. The water resistivity was > 18.2 megaohm*cm. All buffer solutions were sterilized prior to use by filtering through Coming Sterilization Filter systems with 0.22-gm cellulose acetate filter. The buffer solutions were stored at room temperature. -11 - Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 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; Eppendorff Mettler AE-100 Sorval Legend RT Multi-tube vortexer, VWR 10 xnL 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-25435 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 of the pHs tested. Approximately 45 mg o f the H-25435 was weighed into sixteen 10-mL autoclaved glass vials and 3 mL o f appropriate buffer solution was added. The vials were crimp capped with the aluminum foil covering the part o f the septa facing the inside o f the vial. Eight o f the vials were processed for analysis as the Day 0 samples. The 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 were wrapped in aluminum 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 fiL o f 1040 mg/L 8-2 TBA). The vials were crimp capped with the aluminum foil covering the part of the septa facing the inside of the vial. Three o f 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 o f 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 o f the Day 5 vials and two blank vials were processed for the H-25435 determination. The other four o f 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 0 and Day 5 in the vials containing the buffer and the test substance, designated for the H-25435 analysis. The vials were uncapped, the pH electrode was introduced to the vials and the pH reading was taken. These vials were further processed for H-25435 analysis. The temperature of 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 H-25435 is a mixture of [ present in H-25435 [ - 12- Company Sanitized. Does Not Contain TSCA CBI. ] The [ ] ][ Hydrolytic Stability of H-25435 as a Function of pH '[ DuPont-14965 ] - 13- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH [ DuPont-14965 ] - 14- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH [ DuPont-14965 ] - 15- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH [ DuPont-14965 ] - 16- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 The test substance H-25435 has not been characterized to determine the actual contents o f the chemical species that were monitored during the study. The concentrations o f individual chemical species that were monitored during the study were represented as the total concentration of the H-25435. Vials designated for H-25435 analysis were uncapped, 6 mL of acetonitrile was added, vials were recapped and vortexed for 30 minutes. The obtained extract was diluted 1:1000 with 2:1 acetonitrile:water before analysis. The blank samples were subjected to the same procedure, except the pH measurement. The stock solution of H-25435 was made by dissolving 0.01 g o f H-25435 in 10 mL o f 2 mL isopropanol:8 mL water. Calibration standards for the H-25435 analysis were made by appropriate dilution o f the stock. The calibration standards ranged from 1 to 10 mg/L of H-25435. Fresh calibration standards were prepared for each instance o f analysis. Linear or quadratic regression of the peak area vs. standard concentration was used to establish the calibration curves. - 17- Company Sanitized. Does Not Contain TSCA CBI. Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 Instrumentation and conditions: HPLC Instrument: MS Instrument: Model Agilent 1100, Agilent Quattro Micro, Waters LC Parameters: Column: Mobile Phase: XterraMS C18,30 mm x 2.1 mm, 2.5 fim A- 0.15% (v/v) acetic acid/10 mM triethylamine in water B- 0.15% (v/v) acetic acid/10 mM triethylamine in methanol Gradient: Time (min) %B Flow (mL/min) 0.0 50 0.25 2.0 100 0.25 8.0 100 0.25 8.1 100 0.50 10.1 100 0.50 10.2 50 0.25 15.0 50 0.25 Column Temperature: Injection Volume: 30C 10 pL MS Parameters: Ionization mode: Capillary voltage: Cone voltage: Source Temperature: Desolvation Temperature: Data Acquisition Function: Electrospray, negative ions 3.2 kV 20 V 120C 450C C6-P mono ester ion: SIR of 443 m/z; 0-12 min C8-P mono ester ion: SIR of 543 m/z; 0-12 min C10-P mono ester ion: SIR of 643 m/z; 0-12 min C6-P-IPA mixed ester ion: SIR o f485 m/z; 0-12 min C8-P-IPA mixed ester ion: SIR of 585 m/z; 0-12 min C10-P-IPA mixed ester ion: SIR of 685 m/z; 0-12 min C6-P-C6 bis ester ion: SIR of 789 m/z; 0-12 min C6-P-C8 bis ester ion: SIR of 889 m/z; 0-12 min C8-P-C8/C6-P-C10 bis ester ion: SIR of 989 m/z; 0-12 min C10-P-C10 bis ester ion: SIR of 1089 m/z; 0-12 min 4. Analysis of 8-2 TBA Samples designated for 8-2 TBA analysis were processed by injecting 3 mL methyl-tertbutyl ether (MTBE) through the vial septum using a glass syringe. The pH 1.2 samples required neutralization before the extraction. Neutralization was done by injecting 0.27 mL of IN sodium hydroxide to the sample vial before the MTBE addition. Neither Day 0 nor Day 5 samples were uncapped before MTBE injection to avoid any losses o f 8-2 TBA. The vials containing injected MTBE were vortexed for 20 or 30 minutes on a multitube vortexer, centrifuged for 20 minutes at 4150 rpm at room temperature. Occasionally, the MTBE and aqueous layers would not separate even after centrifugation. In these cases, samples were be placed in a freezer at -20C for overnight storage, removed from the freezer, allowed to come to room temperature and centrifuged for 20 minutes. Subsequently, the sample vials were uncapped and 0.1 mL o f the -18- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability ofH-25435 as a Function of pH DuPont-14965 MTBE extract was transferred to a glass LC vial, 0.9 mL acetonitrile added, the extract was 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 o f 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) o f the analytical standard and the internal standard were prepared in methanol and were stored refrigerated. The calibration standards were prepared freshly for each calibration in 10/90% MTBE/acetonitrile. The calibration standards were made in the range from 100 to 10,000 1g/L 8-2 TBA. A constant level o f internal standard was used: 515 fig/L D-8-2 TBA. The calibration curves were constructed using the ratio o f 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 of 8-2 TBA. - 19- Company Sanitized. Does Not Contain TSCA CBI. Hydrolytic Stability of H-25435 as a Function of pH Instrumentation and conditions: DuPont-14965 HPLC Instrument: MS Instrument: Model 2795, Waters ZQ, Waters LC Parameters: Column: Mobile Phase: Column Temperature: Injection Volume: Column Switch: Xterra MS C18,30 mm x 2.1 mm, 2.5 /an A - water B- methanol Gradient: Time (min) %A 0 30 1 30 20 60 6.1 30 8 30 30C 20 (iL 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 o f 467, 528 m/z; 0-6 min %B 70 70 100 100 70 70 Flow (mL/min) 0.25 0.25 0.25 0.25 0.25 0.25 5. Calculations The following calculations were used to determine the recoveries o f individual chemical species monitored for H-25435 (Tables 1-4). For example, if 0.045 g o f H-25435 was weighed into the vial and 3 mL of buffer was added to the vial the nominal concentration that was assigned to each of the chemical species was: (0.045 g *1000 mg/g) / 0.003 L = 15,000 mg/L. The nominal concentration used for recovery calculation was an average o f four replicates. The % recovery was calculated by dividing the measured concentration by the nominal concentration, times 100% . The expected (nominal) concentration o f 8-2 TBA in the spiked samples was calculated as follows: the 4.5 /xL o f 1040 mg/L 8-2 TBA spike represents 4.5 iL x 1040 ng//xL= 4680 ng 8-2 TBA. This amount was extracted with 3 mL of MTBE. The expected nominal concentration o f 8-2 TBA in the MTBE extract is: 4680 ng/3 mL=1560 ng/mL. The % recovery is calculated by dividing the measured concentration by the nominal concentration, times 100%. The H-25435 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 o f H-25435 is: 45 mg of H-25435 x (0.129%/100)/0.003 L= 19.4 mg/L (19,400 /xg/L). -20- Company Sanitized. Does Not Contain TSCA CBI. Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 RESULTS AND DISCUSSION Tables 1-4 present the results for measured concentrations and recoveries o f individual [ ] measured for H-25435 and average concentrations and recoveries for Day 0 and Day 5 replicate samples for pH 1.2, 4.0, 7.0, 9.0, respectively. The average concentration and recoveries were calculated as an average of concentrations and recoveries of [ ] monitored for H-25435. The average percent recoveries for the Day 0 and Day 5, pH 1.2 replicate samples were 76 5.9% and 63 28%, respectively. The average percent recoveries for the Day 0 and Day 5, pH 4.0 buffer replicate samples were 97 3.0% and 95 4.4%, respectively. The average percent recoveries for the Day 0 and Day 5, pH 7.0 replicate samples were 92 1.1% and 90 0.9%, respectively. The average percent recoveries for the Day 0 and Day 5, pH 9.0 replicate samples were 96 0.6% and 95 9.3%, respectively. The difference of average recoveries between the Day 5 and Day 0 samples was less than 10% for pHs 4.0, pH 7.0, and pH 9.0 buffers. This indicates that the H-25435 is hydrolytically stable at these pHs (ti/2>one year). Similarly, the difference in recoveries [ ] for Day 0 and Day 5 samples at pH 4.0, pH 7.0, and pH 9.0 buffers were less than 10%, except the C10-P-C10 bis ester at pH 4.0 and pH 9.0 buffer. The average recoveries for Day 0 and Day 5 samples o f the pH 1.2 buffer differ by more than 10%. However, in case o f pH 1.2 buffer a precipitate was observed after addition o f the test substance to the buffer. The precipitate was difficult to completely dissolve even after addition o f acetonitrile and extended sonication, especially for the Day 5 samples. This lowered the observed recoveries, especially for the bis esters. Therefore, the observed change in the average recoveries between the Day 0 and Day 5 samples is most likely because of observed solubility problems rather than hydrolysis. Figure 1 shows example calibration curves for [ ] monitored during the hydrolytic stability study. Figures 2-5 show example chromatograms obtained for the monitored [ ] for a pH 7.0, Day 0 blank sample, calibration standard containing 5.3 mg/L H-25435, pH 7.0, Day 0 test sample, and pH 7.0, Day 5 test sample. The content o f residual 8-2 TBA in H-25435 was measured using the procedure described in Section A.2. Based on the results o f five replicates o f the H-25435, the content o f residual 8-2 TBA in H-25435 was calculated to be 0.1290.0048%. 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 19.4 mg/L (19,400 /xg/L). (see 5 Calculations). Tables 4-8 present the measured concentrations o f 8-2 TBA for Day 0 and Day 5 replicate samples for pH 1.2,4.0, 7.0, 9.0, respectively. The concentrations o f 8-2 TBA measured for the Day 0 samples are slightly higher than the expected concentration of 8-2 TBA originating from residual 8-2 TBA. This may be due to different extraction methods used for determination o f residual 8-2 TBA in the test substance and the method used for analysis of hydrolytic stability samples. The Day 5 concentrations o f 8-2 TBA are comparable with these observed for Day 0 samples. The observed differences in concentration o f 8-2 TBA between the Day 0 and Day 5 samples for pH 7.0, 9.0 buffers are in the range o f error for the analytical method used (15%). A decrease o f 8-2 TBA concentration between Day 0 and Day 5 may be explained by adsorption o f 8-2 TBA on the precipitate observed for this pH. The difference o f 8-2 TBA concentrations between Day 0 and Day 5 for pH 4.0 samples is larger than can be -21 - Company Sanitized. Does Not Contain TSCA CBI. Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 explained by the analytical method error (Table 6). However, the observed QC sample recoveries for Day 5 samples indicate higher than expected bias o f the analytical method for this set o f samples. Thus, the increase o f 8-2 TBA concentration for Day 5 samples is most likely due to analytical method performance rather than the test substance hydrolysis. Figure 6 shows a representative calibration curve obtained for 8-2 TBA. Figures 7-10 show chromatograms for 8-2 TBA calibration standard; Day 5, pH 7.0 blank sample; Day 5, pH 7.0 test sample; and Day 0, pH 7.0 test sample, respectively. Table 9 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 with deviation not exceeding 0.1 C. Table 10 summarizes results of the pH measurements for pH 1.2, 4.0, 7.0, and 9.0. The measured pH o f Day 0 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. CONCLUSIONS The hydrolytic stability o f H-25435 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 H-25435 was demonstrated to be hydrolytically stable at pH 9.0, pH 7.0, and pH 4.0 with X\n >one year. The data obtained for hydrolytic stability of H-25435 at pH 1.2 show the difference o f average recoveries for monitored [ ] between Day 0 and Day 5 samples is larger than 10 percent. However, in case o f pH 1.2 buffer a precipitate was observed after addition o f the test substance to the buffer. The precipitate was difficult to completely dissolve even after addition of acetonitrile and extended sonication, especially for the Day 5 samples. This lowered the observed recoveries, especially for the [ ] Therefore, the observed change in the average recoveries between the Day 0 and Day 5 samples is most likely because o f observed solubility problems rather than hydrolysis. The measured concentration o f 8-2 TBA did not indicate any hydrolytic degradation o f H-25435 to 8-2 TBA for any of the investigated pHs. The observed concentrations o f 8-2 TBA can be explained by the residual 8-2 TBA present in the test substance (H-25435). There was no significant increase o f 8-2 TBA concentration observed between Day 0 and Day 5 samples for any of the investigated pHs. - 22- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 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 o f Chemicals, Hydrolysis as a Function of pH, OECD Guideline No. 111. - 23- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 TABLES - 24- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 Table 1: A nalytical results for aqueous stability o f H -25435, pH 1 .2 ,37C, Days 0 (03-June-2004) and 5 (08-June-2004). PH Day Rep 11 (m g/L) I1 (m g/L) 1.2 0 1 1.2 0 2 1.2 0 3 1.2 0 4 17,800 16,400 17,100 18,300 17,200 16,400 17,100 18,200 [I (m g/L ) 14,200 13,400 13,900 14,500 11 (m g/L ) 11 (m g/L ) [1 (m g/L ) 11 (m g/L) 11 (m g/L ) I1 (m g/L ) I] (m g/L ) A verage (m g/L ) 16,900 15,600 16,800 16,700 16,500 15,000 15,700 16,500 14,200 12,800 13,500 14,400 15,700 12,800 14,700 15,100 8,670 5,810 7,420 7,740 2,970 1,630 2 ,5 7 0 2,620 1,270 648 1,130 1,300 12,500 11,000 12,000 12,500 A verage % RSD % Recovery 17,400 4.8 110 17,200 4.3 109 14,000 3.4 89 16,500 3.7 104 15,900 4.5 101 13,700 5.3 87 14,600 8.6 92 7,410 16.1 47 2 ,4 5 0 23.4 16 1,090 27.7 7.0 12,000 5.9 76 1.2 5 1 14,700 1.2 5 2 1 3,260 1.2 5 3 15,000 1.2 5 4 19,800 14,900 3,300 9 ,6 1 0 19,200 14,200 4,000 6,420 17,000 10,400 1,630 9 ,9 1 0 12,700 10,400 1,530 7,990 12,700 11,000 952 7,160 12,800 10,000 411 4 ,2 4 0 11,300 7,980 640 2,910 8,500 6,890 2 ,8 7 0 4 ,4 0 0 7,160 3,230 768 1,850 3,830 10,400 1,940 6,950 12,500 A verage % RSD % Recovery 16,500 17.3 109 14,600 33 97 12,500 44 83 11,000 13.5 73 10,400 22.7 69 10,300 27.9 68 8,510 44 56 6,460 47.8 43 6,150 24.7 41 2,970 34.2 20 9 ,9 5 0 28.2 66 1Replicate excluded from calculations of Average and %RSD. - 25- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 Table 2: Analytical results for aqueous stability o f H-25435, pH 4.0, 50C, Days 0 (13-M ay-2004) and 5 (18-M ay-2004). 11 (m g/L) pH Day Rep 1! (m g/L ) 4.0 0 1 15,300 4.0 0 2 16,100 4.0 0 3 16,200 4.0 0 4 16,600 14,600 15,300 16,700 16,500 11 (m g/L ) 11 (m g/L ) ii (m g/L ) 11 (m g/L) [C 6-P-C 6] (m g/L) II (m g/L ) [1 (m g/L ) 1I (m g/L ) A verage (m g/L ) 14,900 15,700 16,400 15,400 14,900 15,600 16,000 15,800 15,200 15,600 15,700 15,800 15,300 15,800 16,300 15,400 15,100 15,500 16,200 15,800 15,200 15,400 16,100 15,500 14,400 15,600 15,900 15,400 10,600 11,300 11,300 10,800 14,600 15,200 15,700 15,300 A verage % RSD % Recovery 16,100 3.4 103 4.0 5 1 4.0 5 2 4.0 5 3 4.0 5 4 15,500 15,400 14,400 16,200 15,800 6.3 101 15,300 16,600 15,500 16,700 15,600 4.0 100 14,300 15,300 14,400 15,600 15,600 3.1 100 15,100 14,900 13,800 15,300 15,600 1.7 100 14,700 14,600 13,400 15,100 15,700 2.9 100 15,000 15,100 13,600 15,200 15,700 3.0 100 15,000 15,000 14,100 15,400 15,600 2.5 100 15,000 15,100 14,400 15,900 15,300 4.2 98 14,300 14,800 13,800 15,300 11,000 3.2 70 8,760 8,800 8,220 9,890 15,200 3.0 97 14,300 14,600 13,600 15,100 A verage % RSD % Recovery 15,400 4.8 101 16,000 4.5 105 14,900 4.3 98 14,800 4.5 97 14,500 5.1 95 14,700 5.1 97 14,900 3.7 98 15,100 4.1 99 14,600 4.4 96 8,920 7.8 59 14,400 4.4 95 - 26Company Sanitized. Does Not Contain TSCA CBI. AR-226 ) Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 Table 3: Analytical results for aqueous stability of H-25435, pH 7 .0 ,50C, Days 0 (06-M ay-2004) and 5 (11-M ay-2004). 11 (m g/L ) pH Day Rep 11 (m g/L ) 11 (m g/L) 11 (m g/L ) I1 (m g/L ) 11 (m g/L) 11 (m g/L ) [1 (m g/L ) 1I (m g/L ) I1 (m g/L ) A verage (m g/L ) 7.0 0 1 7.0 0 2 7.0 0 3 7.0 0 4 15,800 15,700 15,300 15,300 15,700 15,900 16,500 16,200 15,500 15,300 15,300 14,800 15,400 15,000 15,400 15,000 15,400 15,200 15,300 15,100 14,800 15,100 15,300 15,200 15,600 15,500 15,400 15,100 15,800 14,900 14,800 14,300 15,000 14,700 14,300 14,000 9,890 9,720 9740 9570 14,900 14,700 14,700 14,500 A verage % RSD % Recovery 15,500 1.7 97 7.0 5 1 7.0 5 2 7.0 5 3 7.0 5 4 15,000 14,400 15,300 14,700 16,100 2.2 101 14,300 14,900 15,500 15,400 15,200 2.0 95 13,400 13,600 15,000 14,100 15,00 1.5 95 14,500 14,500 14,600 14,600 15,300 0.8 96 14,300 14,300 14,300 14,400 15,100 1.4 95 14,500 15,100 14,000 14,700 15,400 1.4 97 14,600 14,300 14,700 14,300 15,000 4.2 94 14,400 14,500 14,600 14,500 14,500 3.0 91 13,500 14,000 13,800 13,800 9,730 1.3 61 9,330 9,410 9 ,5 8 0 9,660 14,700 1.1 92 13,800 13,900 14,100 14,000 A verage % RSD % Recovery 14,900 2.6 96 15,000 3.7 96 14,000 5.1 90 14,600 0.4 94 14,300 0.3 92 14,600 3.1 94 14,500 1.4 93 14,500 0.6 93 13,800 1.5 89 9 ,5 0 0 1.6 61 1,4000 0.9 90 -27- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 Table 4: Analytical results for aqueous stability of H-25435, pH 9 .0 ,50C, Days 0 (13-M ay-2004) and 5 (18-M ay-2004). 11 pH D ay Rep (m g/L) Ii (m g/L ) 9.0 0 1 9.0 0 2 9.0 0 3 9.0 0 4 15,400 15,700 15,900 15,400 15,800 15,800 15,400 15,500 i1 (m g/L) 16,800 14,400 15,300 16,200 11 (m g/L) 1i (m g/L) 15,300 15,600 15,300 15,600 15,300 15,600 15,100 15,500 iI (m g/L) 15,300 15,500 15,300 15,400 11 (m g/L ) 15,400 15,500 15,200 15,300 I1 (m g/L ) II (m g/L ) [1 (m g/L) A verage (m g/L ) 15,200 15,400 15,100 15,200 15,000 15,200 14,600 15,200 12,700 12,500 12,500 12,700 15,200 15,100 15,000 15,200 A verage % RSD % Recovery 15,600 1.6 99 9.0 5 1 9.0 5 2 9.0 5 3 9.0 5 4 15,000 14,300 15,100 14,900 15,600 1.3 99 14,500 15,500 15,400 15,800 15,700 6.7 100 12,200 14,800 16,000 15,500 15,500 1.1 98 14,300 14,900 15,200 15,600 15,400 1.4 98 13,100 14,600 15,100 15,500 15,400 0.6 98 10,500 14,900 15,700 15,400 15,400 0.8 98 10,500 15,600 15,400 16,200 15,200 0.8 96 11,700 15,600 16,000 15,800 15,000 1.9 95 12,700 15,500 15,900 16,400 12,600 0.9 80 13,900 14,000 15,200 17,500 15,100 0.6 96 12,800 15,000 15,500 15,900 A verage % RSD % Recovery 14,800 2.4 95 15,300 3.7 98 14,600 11.6 94 15,000 3.7 96 14,600 7.2 94 14,100 17.3 91 14,400 18.3 93 14,800 13.9 95 15,100 11.0 97 15,200 11.0 98 14,800 9.3 95 -28- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 Table 5: M easured concentrations o f 8-2 TBA for pH 1 .2 ,37C, Days 0 (03-June-2004) and 5 (08-June-2004). N om inal 8-2 T B A pH Day Rep C on cen tration (/g/L) 1.2 0 1 1.2 0 2 1.2 0 3 1.2 0 4 - M easured 8-2 TBA C o n cen tration (/g/L) 25,300 25,700 25,600 2 4 ,8 0 0 % Recovery - A verage Standard D eviation 25,400 404 1.2 0 Q C1 1.2 0 Q C 2 1.2 0 Q C 3 1560 1560 1560 1680 1610 1670 108 103 107 A verage Standard D eviation 106 2.7 1.2 5 1 1.2 5 2 1.2 5 3 1.2 5 4 - 16,800 2 4 ,9 0 0 14,800 2 2 ,7 0 0 - A verage Standard D eviation 19,800 4 ,7 8 0 1.2 5 QC1 1.2 5 Q C 2 1.2 5 Q C 3 1560 1560 1560 1470 1500 1450 94 96 93 A verage Standard D eviation 94 1.5 - 29- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 Table 6: M easured concentrations of 8-2 TBA for pH 4 .0 ,50C, Days 0 (13-M ay-2004) and 5 (18-May-2004). N om inal 8-2 T B A PH Day Rep C oncentration (/tg/L ) 4.0 0 1 4.0 0 2 4.0 0 3 4.0 0 4 - M easured 8-2 TBA C oncentration Qtg/L) 25,400 24,300 21,900 23,200 % Recovery - A verage Standard D eviation 4.0 0 QC1 4.0 0 Q C 2 4.0 0 Q C3 1560 1560 1560 23,700 1,500 1680 1660 1620 108 106 104 A verage Standard D eviation 106 2.0 4.0 5 1 4.0 5 2 4.0 5 3 4.0 5 4 - A verage Standard D eviation 28,400 29,500 30,200 29,500 29,400 744 - 4.0 5 QC1 4.0 5 QC2 4.0 5 Q C3 1560 1560 1560 2010 2030 1630 129 130 104 A verage Standard D eviation 121 15 -30- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 Table 7: M easured concentrations of 8-2 TBA for pH 7.0, 50C, Days 0 (06-M ay-2004) and 5 (11-May-2004). pH Day 7.0 0 7.0 0 7.0 0 7.0 0 N om inal 8-2 T BA M easured 8-2 T BA R ep C o n c e n tr a tio n C o n c e n tr a tio n (igfL) (sTL) 1234- 2 2 ,3 0 0 2 2 ,3 0 0 22,800 23,900 % Recovery - A verage S ta n d a rd D eviation 7.0 0 7.0 0 7.0 0 QC1 QC2 QC3 1560 1560 1560 22,800 750 1640 1630 1600 105 104 103 A verage Standard D eviation 104 1.0 7.0 5 7.0 5 7.0 5 7.0 5 1 2 3 4 - 24,000 24,300 24,200 12,300 ' - A verage S ta n d a rd D eviation 7.0 5 7.0 5 7.0 5 QC1 QC2 QC3 1560 1560 1560 24,200 150 1590 1540 1580 102 99 101 A verage Standard D eviation 1Not used in the calculation of the average and standard deviation. 101 1.5 -31- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 Table 8: M easured concentrations o f 8-2 TBA for pH 9.0, 50C, Days 0 (13-M ay-2004) and 5 (18-May-2004). N om inal 8-2 T B A pH Day Rep C oncentration (pg/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 ) 25,000 2 5 ,3 0 0 25,300 25,700 % Recovery - A verage S ta n d a rd D eviation 9.0 0 QC1 9.0 0 Q C 2 9.0 0 Q C3 1560 1560 1560 25,300 287 1590 1640 1680 102 105 108 A verage Standard D eviation 105 3.0 9.0 5 9.0 5 9.0 5 9.0 5 1 2 3 4 - 26,300 26,000 25,700 25,900 - A verage Standard D eviation 9.0 5 QC1 9.0 5 Q C 2 9.0 5 Q C3 1560 1560 1560 26,000 250 1550 1620 1510 99 104 97 A verage Standard D eviation 100 3.6 - 32Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 Table 9: Daily tem perature readings of the shaking water bath. T im e (Day) pH 0 1.2 1 1.2 2 1.2 3 1.2 4 1.2 5 1.2 0 4.0 1 4.0 2 4.0 3 4.0 4 4.0 5 4.0 0 7.0 1 7.0 2 7.0 3 7.0 4 7.0 5 7.0 0 9.0 1 9.0 2 9.0 3 9.0 4 9.0 5 9.0 l T em perature was not recorded. D ate 03-June-2004 04-June-2004 05-June-2004 06-June-2004 0 7 -Ju ne-2004 08-June-2004 Tem perature (C ) 37.0 37.0 37.0 37.0 37.0 37.0 13-M ay-2004 14-M ay-2004 15-M ay-2004 16 -M a y -2 0 0 4 17-M ay-2004 18-M ay-2004 50.0 50.0 50.0 50.0 50.0 50.0 06-M ay-2004 07-M ay-2004 08-M ay-2004 09-M ay-2004 10-M ay-2004 11-M ay-2004 50.0 50.0 J J 50.0 50.0 13-M ay-2004 14-M ay-2004 15-M ay-2004 16 -M a y -2 0 0 4 17-M ay-2004 18-M ay-2004 50.0 50.0 50.0 50.0 50.0 50.0 - 33Company Sanitized. Does Not Contain TSCA CBI. AR-226 0 sa*N. Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 Table 10: pH m easurem ents for Day 0 and D ay 5. Day 0 0 0 0 5 5 5 5 0 0 0 0 5 5 5 5 0 0 0 0 5 5 5 5 0 0 0 0 5 5 5 5 R ep 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 N om inal pH 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 M easured pH 1.27 1.25 1.27 1.27 1.24 1.23 1.24 1.24 4.10 4.11 4.09 4.10 4.07 4.07 4.08 4.07 7.10 7.09 7.10 7.10 7.06 7.07 7.07 7.05 9.10 9.10 9.08 9.09 9.09 9.09 9.08 9.07 -34- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 FIGURES - 35Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH Figure 1: Exam ple calibration curves obtained for m onitored! DuPont-14965 ]. -36- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH Figure 2 Example chromatograms of monitored sam ple for pH 7.0 buffer on Day 0. DuPont-14965 1obtained for a blank 0p5H077,0B4bla0n6k 1, Day 0 _. 0.53 F3:SIR of 4 channels,ES4.678e+io4 %1 10 2.0 r 6.07 1 987,8 4.0 6.0 8.0 11.63 10.0 12.0 -37Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability o f H-25435 as a Function of pH DuPont-14965 Figure 3: Example chromatograms of monitored [ calibration standard containing 5.3 mg/L H-25435, obtained for a - 38Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH Figure 4: Example chromatograms of monitored [ hydrolytic stability sample. DuPont-14965 ]obtained for Day 0, pH 7.0 -39- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH Figure 5: Exam ple chromatograms of monitored { pH 7.0 hydrolytic stability sample. DuPont-14965 ]obtained for Day 5, 0p5H1710R4ebp25i, Day 5 r5l ' 236904.6 F2:SIR of 3 channels.ES2.676e+0D06 - 40Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH Figure 6: Representative calibration curve for 8-2 TEA. Compound name: 8-2TBA Correlation coefficient: r = 0.999939, rA2 = 0.999878 Calibration curve: 1.14978 * x+ -12.1779 Response type: Internal Std ( Ref 2 ), Area * ( IS Cone. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None 12045-j DuPont-14965 Response- -193 y lV ,T "l` "|"l l/lTT7"V"t 2000 4000 |`T rV i 6000 ( ,TT`V| i 8000 1, |..n .t1i0.0|..0i.0i ppb -41- Company Sanitized. Does Not Contain TSCA CB1. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 Figure 7: Representative chrom atogram s o f 104 /tg/L 8-2 TBA calibration standard and 515 ftg/L D-8-2 TB A internal standard. 051104b15 Smooth(Mn,1x3) std1 100-t 8-2TBA 3.50 16718.7 F1 :SIR of 2 channels,ES463,523 7.490e+004 %- 1.00 2.00 3.00 4.00 5.00 min F2:SIR of 2 channels,ES467,528 3.339e+005 m jn 5.00 - 42- Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 Figure 8: Representative chrom atogram s for Day 5, pH 7.0 buffer blank sample. -43Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 Figure 9: Representative chromatograms for Day 5, pH 7.0 hydrolytic stability sample and 515 pg/L D-8-2 TBA internal standard. 051104b38 Smooth(Mn,1x3) rep 1; pH 7; Day 5 F1 :SlR of 2 channels,ES463, 523 1.00 2.00 051104b38 Smooth(Mn,1x3) rep 1; pH 7; Day 5 100- 3.00 IS-8-2TBA 3.46 63591.5 4.00 5.00 F2:SIR of 2 channels,ES467, 528 2.392e+005 %- 1.00 2.00 5.00 min - 44Company Sanitized. Does Not Contain TSCA CBI. AR-226 Hydrolytic Stability of H-25435 as a Function of pH DuPont-14965 Figure 10: Representative chromatograms for Day 0, pH 7.0 hydrolytic stability sample and 515 ig/L D-8-2 TBA internal standard. 012904b37 Smooth(Mn,1x3) rep 1; pH 7; DayO 100-1 8-2TBA 3.51 353419.5 F1 :SIR of 2 channels,ES463,523 1.406e+006 %- 0 -i 1.00 2.00 012904b37 Smooth(Mn,1x3) rep 1; pH 7; Day 0 100-1 3.00 =4F.00r p f ^ f m57.00F f W p r r r min IS-8-2TBA 3.49 75001.1 F2:SIR of 2 channels,ES467, 528 2.833e+005 %- 1.00 2.00 3.00 4.00 5.00 min -45- Company Sanitized. Does Not Contain TSCA CBI. AR-226