Document 2qnGjZLMyRYL3Jo1XM553oL35

AR226-2693 15 AR226-2693 DuPont EMSE Report No. 92-02 Study Title Aqueous Stability of 8-2 Telomer B Alcohol as a Function of pH Test Guideline OECD (1981). Hydrolysis as a Function o f pH. OECD Guideline for the Testing o f Chemicals Method 111, adopted 12-May-1981. Author William R. Berti, Ph.D. Study Completion Date 27-March-2003 Test Facilities E.I. du Pont de Nemours and Company Central Research & Development Corporate Center for Engineering Research Environmental and Microbiological Sciences & Engineering Glasgow Building 300, P.O. Box 6101 Newark, DE 19714-6101 USA and E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Newark, DE 19714 USA Sponsor Telomers Research Program The Rand Corporation 1200 South Hayes Street Arlington, VA 22202 USA EMSE Study /Project Number T0106 / 4842 Report Number EMSER 92-02 TO106 74842 Page 1 o f 31 DuPont EMSE Report No. 92-02 Page Reserved for Specific Country Requirements T 0 1 0 6 74842 Page 2 o f 31 DuPont EMSE Report No. 92-02 Certification of Authenticity Aqueous Stability of 8-2 Telomer B Alcohol as a Function of pH We, the undersigned, declare that the work described in this report was performed under our supervision, and that this report provides an accurate record o f the procedures and results. Report by: William R. Berti, Ph.D. Senior Research Biologist Study Initiation Date: 25-April-2002 Date Study Completed: 27-March-2003 Submitter: Telomers Research Program The Rand Corporation 1200 South Hayes Street Arlington, VA 22202 USA Date `nnw>- T0106 74842 Page 3 o f 31 DuPont EMSE Report No. 92-02 Table of Contents Page Reserved for Specific Country Requirements............................................................................... 2 Certification o f Authenticity..................................................................................................................... 3 Table o f Contents........................................................................................................................................ 4 1.0 Summary............................................................................................................................................ 6 2.0 General Study Information...............................................................................................................6 3.0 Materials and Methods............ .........................................................................................................8 3.1 Test Guidelines.............................................................................................................................. 8 3.2 Test System..................................................................................................................................... 8 3.2.1 Chemical System................................................................................................................. 8 3.2.2 Test System ........................................................................................................................ 10 3.3 Test Conduct.................................................................................................................................10 3.3.1 Buffer Solutions................................................................................................................. 10 3.3.2 Preliminary T est................................................................................................................ 11 3.3.3 Aqueous Stability o f an Unstable Substance................................................................. 11 3.4 Parameters Observed.................................................................................................................. 11 3.4.1 Analysis o f Test Systems................................................................................ 11 3.4.2 Analytical Methods for Test Substance and Products.................................................. 11 3.5 Statistical A nalysis...................................................................................................... 13 3.6 Protocol Deviations............................................................................................ 13 4.0 Results and Discussion..................................................................................................... 13 5.0 Conclusions...................................................................................................................................... 15 6.0 Retention o f Records......................................................................................................................15 7.0 Disposal o f Test Substance............................................................................................................15 8.0 References........................................................................................................................................ 15 Tables Table 1 Concentration o f 8-2 TBA at pH 1.2 at 37C and at pH 4, 7, and 9 at 50C after 0 and 5 Days........................................................................................................................................ 16 Table 2 pH o f Buffer Solutions Containing 8-2 TBA at Days 0 and 5.............................................17 Table 3 Daily Temperature Readings o f Shaking Water Bath for Aqueous Stability o f 8-2 TBA Conducted at pH 1 .2 ..................................... 18 Table 4 Daily Temperature Readings o f Shaking Water Bath for Aqueous Stability o f 8-2 TBA Conducted at pH 4 ......................................................................................................... 19 Table 5 Daily Temperature Readings o f Shaking Water Bath for Aqueous Stability o f 8-2 TBA Conducted at pH 7 and 9............. '.................................................................................20 Table 6 Sterility o f Test Solutions at Day 5 in Sterile Trypticase Soy Broth (TSB) M ed ium ............................................................................................................................................ 21 T0106 74842 Page 4 o f 31 DuPont EMSE Report No. 92-02 Appendix A Table A -l Analytical Results o f the Aqueous Stability o f 8-2 TBA, pH 1.2, 37C, Days 0 (21-Aug-2002) and 5 (26-Aug-2002)..........................................................................................22 Table A-2 Analytical Results o f the Aqueous Stability o f 8-2 TBA, pH 4.0, 50C, Days 0 (24-Jul-2002) and 5 (29-Jul-2002)................................................................................................23 Table A-3 Analytical Results o f the Aqueous Stability o f 8-2 TBA, pH 7.0, 50C, Days 0 (21-Aug-2002) and 5 (26-Aug-2002)........................................................................................... 24 Table A-4 Analytical Results o f the Aqueous Stability o f 8-2 TBA, pH 9.0, 50C, Days 0 (21-Aug-2002) and 5 (26-Aug-2002)........................................................................................... 25 Appendix B Table B -l Analytical Results o f the Aqueous Stability o f 8-2 TBA for Pilot Experiment Conducted at pH 4 and Room Temperature, Day 0 only (16-Jul-2002)f.... ........... ..............26 Table B-2 Analytical Results o f the Aqueous Stability o f 8-2 TBA, pH 1.2, 50?,C, Days 0 (31-Jul-2002) and 5 (5-Aug-2002)f............................................................................................. 28 Table B-3 Analytical Results o f the Aqueous Stability o f 8-2 TBA, pH 7.0, 50C, Days 0 (31-Jul-2002) and 5 (5-Aug-2002)f............................................................................................. 29 Table B-4 Analytical Results o f the Aqueous Stability o f 8-2 TBA, pH 9.0, 50C, Days 0 (31-Jul-2002) and 5 (5-Aug-2002)f............................................................................................. 30 Appendix C Appendix C Certificate o f Test Substance Analysis................................................ ii T0106 / 4842 Page 5 o f 31 DuPont EMSE Report No. 92-02 1.0 2.0 Aqueous Stability of 8-2 Telomer B Alcohol as a Function of pH Author William R. Berti, Ph.D. Summary Test System: The aqueous stability o f the test substance 8-2 Telomer B Alcohol (i.e., 8-2 TBA) in sterile aqueous solutions buffered at pH 1.2, 4.0, 7.0, and 9.0 was determined. Test systems consisted o f the test substance in aqueous buffered solutions in sterilized containers. The test system was incubated in the dark at 50C at pH 4, 7, and 9 and at 37C at pH 1.2. An unstable test substance is indicated by a 10% or greater loss o f test substance within 5 days. Findings: The test substance, 8-2 TBA, is stable under the conditions o f the test at pH 4 ,7 , and 9 at 50C and pH 1.2at37C . Conclusions: The time in which 50% o f the test substance will transform is estimated as greater than one year: >1 year. General Study Information .MATfOW Study Objectives This test generates essential environmental fate information relevant to the persistence o f the test substance. The aqueous stability o f a substance (e.g., hydrolytic stability) is one o f the most common reactions controlling abiotic degradation and is therefore one o f the main potential degradation paths o f substances in the environment. A procedure to determine aqueous stability rates also is important in indicating whether other testing should be performed on a parent substance or on its aqueous stability products. Aqueous stability behavior needs to be examined at pH values normally found in the environment (pH 4 to 9) at environmentally relevant temperatures (10 to 60C). It is also sometimes desirable to determine aqueous stability o f a test substance at physiologically relevant pH and temperatures, such as pH 1.2 and 37C, respectively. Specific objectives include: 1. To determine if a compound is stable in an aqueous environment, 2. To provide a material mass balance that accounts for greater then 90% o f the initially applied test substance. 3. For unstable compounds: a. To determine the aqueous stability rate o f the test substance in sterile buffer solutions at pH 4, 7, and 9 between 10 to 60C and at pH 1.2 and 37C, T0106 74842 Page 6 o f 31 DuPont EMSE Report No. 92-02 b. To identify and follow the formation and decline o f the aqueous stability produces) o f the test substance if formed, c. To determine a first-order aqueous stability rate constant and half-life o f the test substance (if rate constant and half-life o f major aqueous stability products cannot be determined, then a aqueous stability study with the aqueous stability produces) will be conducted exactly as the parent study). Test System Justification The test system is outlined by OECD Guideline 111 and was requested by the sponsor. Study Personnel E.I. du Pont de Nemours and Company Central Research and Development - Corporate Center for Engineering Research Environmental and Microbiological Sciences & Engineering Haskell Laboratory for Health and Environmental Sciences Management: John T. Gannon, Ph.D. E.I. du Pont de Nemours and Company Central Research and Development Corporate Center for Engineering Research Environmental and Microbiological Sciences & Engineering Glasgow Building 300, P.O. Box 6101 Newark, DE 19714-6101 USA and S. Mark Kennedy, Ph.D. E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Newark, DE 19714 USA Study Director: William R. Berti, Ph.D. E.I. du Pont de Nemours and Company Central Research and Development Corporate Center for Engineering Research Environmental and Microbiological Sciences & Engineering Glasgow Building 300, P.O. Box 6101 Newark, DE 19714-6101 USA Analytical Chemist: Bogdan Szostek, Ph.D. E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Newark, DE 19714 USA Technical Personnel: Lisa M. Sulecki, DuPont Central Research & Development Mark G. Starr, DuPont Central Research & Development Keith B. Prickett, DuPont Haskell Laboratory TO106 /4842 Page 7of31 DuPont EMSE Report No. 92-02 ) Study Execution Dates Study Initiation Date: 25-April-2002 Experimental Start Date: 15-July-2002 Experimental Completion Date: 9-September-2002 Study Completion Date: 27-March-2003 3.0 Materials and Methods 3.1 Test Guidelines The aqueous stability o f the test substance 8-2 TBA in sterile aqueous solutions buffered at pH 1.2,4 .0 ,7 .0 , and 9.0 was determined. Test systems consisted o f the test substance in aqueous buffered solutions in sterilized containers made o f non-adsorbing materials. The test systems were incubated in the dark at 50C at pH 4, 7, and 9 and 37C at pH 1.2. Day 0 and Day 5 samples were analyzed for the concentration o f the test substance. If less than 10% o f the test substance degrades in 5 days (t./2is greater than 1 year at 25C), the test substance is considered stable and no additional testing for aqueous stability is performed. If degradation o f the test substance had occurred at pH 4, 7, and/or 9 and 50C, aqueous stability would also be performed at two additional temperatures separated by at least 15C (e.g., 20C and 35C) at the pH values at which the test substance was shown to be unstable. To test for first-order behavior, each test vessel would have been analyzed in time intervals that provided a minimum o f six-spaced data points normally between 20% and 70% o f aqueous stability o f the test substance. Test solution would have been analyzed for the concentration o f the test substance and its transformation product(s). The test substance concentration would be plotted against the sampling intervals to determine its degradation rate constant and half-lives at each pH. 3.2 Test System 3.2.1 3.2.1.1 Chemical System Test Substance Name: Synonym: Active substance(s): Molecular Weight: CAS Name: CAS Number(s): 8-2 Telomer B Alcohol (Perfluorooctyl)ethanol, 8-2 TBA 8-2 Telomer B Alcohol, 99% 464.12 g mole"1 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro678-39-7 TO106 / 4842 . Page 8 o f 31 Structure: DuPont EMSE Report No. 92-02 F F 3.2.1.2 H Number: Lot Number: EMSE Sample Number: Concentration o f a.s., nominal: Concentration o f a.s., analyzed: Certificate o f Analysis Date: Date Received: Supplier: Solubility at 25C: Vapor pressure: Stability: Appearance/Color: Storage Conditions: Safety Precautions: H-24691 P.00/001 E93386-63 99% 99.2% 13-Sept-2001 26-Mar-2002 Clariant Co. -1 4 0 pg L'1 0.023 mm Hg Stable at ambient room temperature White solid Room temperature; keep tightly closed Wear lab coat, protective gloves, and safety glasses Reference Substance None 3.2.1.3 Test Vehicle Solutions buffered at pH 1 .2 ,4 ,7 , and 9 were added to glass test vessels containing 8-2 TBA L'1methanol to attain an initial concentration o f 150 pg 8-2 TBA L'1. For test solutions with a volume o f 4.5 mL, 33.8 pL o f 20 mg 8-2 TBA L"1methanol was used to attain a concentration o f 150 pg 8-2 TBA L'1. For test solutions with a volume o f 3.0 mL, 22.5 pL o f 20 mg 8-2 TBA L'1methanol was used to attain a concentration o f 150 pg 8-2 TBA L'1. T0106 74842 Page 9 o f 31 DuPont EMSE Report No. 92-02 3.2.1.4 Application Information The test substance in methanol was mixed with pH 1.2, 4, 7, or 9 buffer solutions to attain a concentration o f 150 pg 8-2 TBA L'1buffer solution. The amount o f methanol in the final test solution was less than 1% by volume. 3.2.2 3.2.2.1 3.2.2.2 Test System Test Units Different types o f test vessels were used to reduce or eliminate the amount o f test substance lost during the test period. Test vessels used to conduct this study were either 10 mL borosilicate glass vials with aluminum-lined crimp caps (pH 1.2, 7, and 9 samples) or 7 mL borosilicate glass vials with aluminum-lined screw caps (pH 4). All test vessels were gravity sterilized by autoclaving for 20 minutes at 121C. Test Conditions Test solutions buffered at pH 4 ,7 , and 9 were held at 50 1C for 5 days in the dark by covering the test vessels using the lid o f the shaking water bath. The test conditions were replicated 4 times at each pH and time. Test solutions buffered at pH 1.2 were held at 37 1C for 5 days in the dark by covering the test vessels using the lid o f the shaking water bath. This test condition was also replicated 4 times. 3.3 Test Conduct Sterile buffer concentrations o f 0.05 M were used. After preparation, each solution was filter ) sterilized by passing through a 0.2-mm filter. 3.3.1 3.3.1.1 Buffer Solutions pH 1.2 Buffer: 250 mL o f 0.200 M potassium chloride (KC1) and 475 mL o f 0.200 N HC1 were diluted to 1.000 L with water. Final pH adjustments were made with 1.0 N HC1 or NaOH as necessary. 3.3.1.2 3.3.1.3 3.3.1.4 pH 4 Buffer: 500 mL o f 0.100 M potassium hydrogen phthalate [KHlCgHjO^] and 4.00 mL o f 0.100 N sodium hydroxide (NaOH) were diluted to 1.000 L with water. No final pH adjustment was necessary. p H 7 Buffer: 500 mL o f 0.100 M potassium dihydrogen phosphate (KH2P 0 4) and 296 mL o f 0.100 M sodium hydroxide were diluted to 1.000 L with water. Final pH adjustments were made with 5N HC1. pH 9 Buffer: 500 mL 0.050 M boric acid (H2B 0 2) and 213 mL o f 0.100 M NaOH were diluted to 1.000 L with water. Final pH adjustments were made with 5N HC1. ) T0106 / 4842 Page 10 o f 31 DuPont EMSE Report No. 92-02 3.3.2 Preliminary Test A preliminary test was performed on the test substance at 50C at pH 4.0, 7.0, and 9.0 and at 37C and pH 1.2. 3.3.3 Aqueous Stabilitv o fan Unstable Substance The test substance is stable; therefore, testing for the aqueous stability o f an unstable substance was not performed. 3.3.4 3.3.4.1 3.3.4.2 Sample Collection and Storage Sampling Intervals Day 0 samples were frozen immediately after the addition o f the test substance until extracted and analyzed. Day 5 samples were shaken at 37C (buffer pH 1.2) or 50C buffer pH 4, 7, and 9) for 5 days, at which time they were frozen until extracted and analyzed. Test Solution Storage Test solutions were stored at approximately -20C. >..u iaiv ip b 3.4 Parameters Observed 3.4.1 Analvsis o f Test Svstems CJK 3.4.1.1 Sterility Measurements 1 Sterility was determined on Day 5 samples o f each o f the test systems. Sterile trypticase soy broth (TSB) medium was used to check the sterility o f each sample. At sampling, 0.5 mL o f the test solution was aseptically added to a culture tube containing 4.5 mL o f TSB medium. The TSB medium was incubated 3 to 4 days in darkness at 30C. The presence or absence o f cloudiness as compared to sterile TSB medium controls determined sterility. 3.4.1.2 pH Measurement Test solution pH was measured and documented at Day 0 and at the end o f the experiment. 3.4.1.3 Temperature Measurements The temperature o f the test system was monitored and recorded daily throughout the course o f the study. 3.4.2 3.4.2.1 Analytical Methods for Test Substance and Products Analysis o f Test Substance Sample preparation: The GC/MS analysis o f 8-2 TBA samples involved use o f an internal standard and surrogate spiked into each sample to trace the extraction recovery. The 1-Octanol, 3,3,4,4,5,5,6,6,7,8,8,8-dodecafluoro-7-(trifluoromethyl)- (CAS# 20015-46-7, 98%, Oakwood Products, West Columbia, SC), referred to as C9-iso, was used as the surrogate. The 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,10,10,10-hexadecafhioro-9-(trifhioromethy1)- (CAS# 31200-98-3, 98%, Oakwood Products, West Columbia, SC), referred to as Cl 1-iso, was used TO106 / 4842 Page 11 o f 31 DuPont EMSE Report No. 92-02 as the internal standard. The 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro(CAS# 678-39-7, 97.6%, Oakwood Products, West Columbia, SC), referred to as 8-2 TBA, was used as the analytical standard o f 8-2 TBA. Stock solutions at approximately 1000 pg mL'1o f internal standard and surrogate were prepared in methanol and stored refrigerated. The stocks were diluted to appropriate concentration with methanol before use for each set o f samples analyzed. The pH 1.2,7, and 9 samples (4.5-mL test solution volume) in crimp-capped 10-mL glass vials were spiked with 13.5 pL o f 50 pg C9-iso mL'1 methanol by injecting the solution into the closed vial through the vial septum using a syringe. Then, 2.25-mL propane, 2-methoxy2-methyl- [CAS# 1634-04-4 (methyl t-butyl ether, referred to as MTBE] was introduced into the vial using a syringe. The contents o f the vial were vortexed for 15 min using a multi-tube vortexer. The vial was opened and 1 mL o f the MTBE layer was sampled to a glass GC vial (1.7 mL), spiked with 6 pL o f a 50 pg C l 1-iso mL'1methanol, crimped, and subjected to GC/MS analysis. The calibration standards were prepared by spiking appropriate volumes o f 50 pg mL'1methanol o f C9-iso, C l 1-iso, 8-2 TBA to 1-mL MTBE. The calibration curves were constructed using peak area o f signal obtained for ion m/z: 95. The pH 4 samples (3-mL test solution volume) required a different preparation procedure because the presence o f phthalates in the sample buffer interfered with the analysis o f the test substance. The pH 4 samples in 7 mL glass vials with screw caps were opened and 9 pL o f 50-pg C9-iso mL'1methanol was added to the vial using a syringe, followed by 1.5 mL o f hexane (CAS# 110-54-3). The contents o f the vial were vortexed for 15 min using a multi-tube vortexer. The SPE column (Isolute Si, 100 mg /10mL, Jones Chromatography) was conditioned by cleaning it with 2 mL o f isopropanol (IPA) and conditioning with 2 mL hexane. The hexane extract was loaded onto the conditioned SPE column. After loading the hexane extract, the column was washed with 2 mL o f hexane and the analytes were eluted with 1 mL IPA. No drying o f the column was applied after the hexane wash. A 0.5 mL aliquot o f the IPA extract was sampled to a glass GC vial (1.7 mL), spiked with 3 pL o f 50 pg C l 1-iso mL'1methanol, crimped, and subjected to GC/MS analysis. The calibration standards for the pH 4 samples were prepared by spiking appropriate volumes o f 50 pg mL 1 methanol o f C9-iso, Cl 1-iso, 8-2 TBA to 1 mL o f matrix matched IPA. The matrix matched IPA was prepared by subjecting the blank pH 4 buffer to the SPE procedure, the same way as the samples. The IPA eluate was used to prepare the matrix matched calibration standards. The calibration curves were constructed using peak area o f signal obtained for ion m/z: 95. Instrumentation and conditions: GC/MS system: HP 6890 Plus GC (Agilent, Wilmington, DE USA), HP 5973 Mass Selective Detector (Agilent), MPS2-MultiPurposeSampler (Gerstal, Baltimore, MD USA) Column: DB-5MS, 30 m x 0.25 mm, 1 pm film (Agilent, Wilmington, DE USA) Temp, ramp: Initial: Flow rate: Split: Inlet temp.: Injection volume: MSD transfer line temp.: SIM ions monitored: 80C for 2 min 20C/minto 120C 50G/min to 300C and hold for 3 min 1.0 mL/min; He; constant flow mode 5:1 250C 2 pL 280C m/z: 31, 95, 131 T0106/4842 Page 12 o f 31 DuPont EMSE Report No. 92-02 Retention time: 8-2 TBA: C9-iso: Cl 1-iso: 4.93min 4.60min 5.38min 3.5 Statistical Analysis Paired t-test tests were used to determine if the mean test substance concentrations in paired Day 0 and Day 5 buffer solutions are the same (JMP, 1995). 3.6 Protocol Deviations None 4.0 Results and Discussion Under the conditions o f the test at pH 1.2 and 37C and at pH 4 ,7 , and 9 and 50C, 8-2 TBA is stable (Table 1). Tables in Appendix A (Tables A -l, A-2, A-3, and A-4) contain the analytical results o f the aqueous stability study o f the test substance that are summarized in Table 1. Stability o f the test substance, 8-2 TBA: At pH 1.2, 8-2 TBA concentration after five days at 37C was 195 8.00 pg L"1, compared to 183 10.9 pg L"`at the start o f the test (Day 0). The difference between the Day 5 and Day 0 concentrations o f the test substance was +6.7%, indicating that 8-2 TBA is stable at this pH. The difference between Day 5 and Day 0 was not statistically significant (p = 0.05). At pH 4, 8-2 TBA concentration after five days at 50C was 143 10.0 pg L'1, compared to 156 4.22 pg L*'at the start o f the test (Day 0). The difference between the Day 5 and Day 0 concentrations o f the test substance was -8.7%, indicating that 8-2 TBA is stable at this pH. The difference between Day 5 and Day 0 was not statistically significant (p = 0.05). At pH 7, 8-2 TBA concentration after five days at 50C was 181 12.6 pg L'1, compared to 199 + 9.12 pg L'1at the start o f the test (Day 0). The difference between the Day 5 and 0 concentrations o f the test substance was -9.6%, indicating that 8-2 TBA is stable at this pH. The difference between Day 5 and Day 0 was not statistically significant (p = 0.05). At pH 9, 8-2 TBA concentration after five days at 50C was 175 10.5 pg L'1, compared to 169 5.99 pg L"1at the start o f the test (Day 0). The difference between the Day 5 and Day 0 concentrations o f the test substance was +3.8%, indicating that 8-2 TBA is stable at this pH. The difference between Day 5 and Day 0 was not statistically significant (p = 0.05). Experimental conditions -p H : The pH 1.2 test solutions were pH 1.i 1 and 1.54 at days 0 and 5, respectively, a variation o f more 0.1 units (Table 2). This had no significant effect on the outcome o f the test because the compound was stable. The pH 4 test solutions were pH 4.02 and 4.12 at days 0 and 5, respectively; a variation o f more 0.1 units (Table 2). This had no significant effect on the outcome o f the test because the compound was stable. TO106 74842 Page 13 of31 DuPont EMSE Report No. 92-02 The pH 7 test solutions were pH 7.03 and 7.04 at days 0 and 5, respectively; and the pH 9 test solutions were pH 9.02 and 8.97 at days 0 and 5, respectively (Table 2). The pH o f the test solutions at these 2-pH levels did not change significantly (pH change less than 0.1 unit) during the test. Experimental conditions - temperature: The temperature o f the shaking water bath used to maintain the pH 1.2 test solution at a constant temperature did not fluctuate by a total o f more than 0.2C at 37C (Table 3). The average temperature o f the shaking water bath was 37.1C and the standard deviation was0.1C (n = 6). The temperature o f the shaking water bath used to maintain the pH 4 test solution at a constant temperature did not fluctuate by more than 0.1 C at 50C during the 5 days o f the test (Table 4). The average temperature o f the shaking water bath was 50.0C and the standard deviation was 0.05C (n = 6). The temperature o f the shaking water bath used to maintain the pH 7 and 9 test solutions at a constant temperature did not fluctuate by more than 0.1 C at 50C during the 5 days o f the test (Table 5). The average temperature o f the shaking water bath was 50.0C and the standard deviation was 0.1 C (n = 6). Experimental conditions - sterility: The sterility check solutions for the control, pH 1.2 at 37C, and pH 7 and 9 at 50C were sterile at the end o f the test, as indicated by the lack o f bacteria growth in the Trypticase Soy Broth (TSB) medium (Table 6). The sterility check solution for the pH 4 at 50C was not sterile as indicated by a slightly cloudiness o f the TSB medium after the incubation period. (Table 6). This did not affect the outcome o f the study because the test substance was stable at this pH and temperature (Table 1). Trial studies: Prior to the definitive studies, several trial studies were performed. The results o f these trial studies are found in Appendix B (Tables B -l, B-2, B-3, and B-4). The information below summarizes these results. Table B -l lists the results o f a pilot experiment that was conducted to demonstrate that the experimental design, implementation, and subsequent analysis o f the test substance was adequate to determine its aqueous stability. This study was conducted at pH 4 and room temperature for Day 0 only. Table B-2 lists the results o f the aqueous stability o f the test substance at pH 1.2 and 50C. The test substance was shown to be stable at this pH and temperature after 5 days. The study was repeated because it should have been conducted at a pH o f 1.2 and 37C. Both demonstrate that the test substance is stable (Tables 1 and A -l). Table B-3 lists the results o f the. aqueous stability o f the test substance at pH 7 and 50C on Day 0 and Day 5. This test was repeated using different test vessels because o f the large standard deviation o f the average test substance concentration at Day 5 (standard deviation o f 39.8 pg L'1for Day 5 compared to 6.25 pg L"1for Day 0), which appears to be due to the loss o f the test substance in two or more o f the Day 5 replications. The repeated study demonstrated that the test substance is stable at pH 7 (Tables 1 and A-3). T0106/ 4842 Page 14 o f 31 5.0 6.0 7.0 8.0 DuPont EMSE Report No. 92-02 Table B-4 lists the results o f the aqueous stability o f the test substance at pH 9 and 50C on Day 0 and Day 5. This test was repeated using different test vessels because o f the large standard deviation o f the average test substance concentration at Day 5 (standard deviation o f 42.9 pg L'1for Day 5 compared to 11.5 pg L 1for Day 0), which appears to be due to the loss o f the test substance in two o f the Day 5 replications. The repeated study demonstrated that the test substance is stable at pH 9 (Tables 1 and A-3). Conclusions The time in which 50% o f the test substance will transform in water at environmentally relevant pH ranges and temperatures is greater than one year (t >1 year), because >90% o f the test substance added at Day 0 was recovered after 5 days at pH 1.2 and 37C and pH 4.0, 7.0, and 9.0 and 50C. Retention of Records For the periods demanded by GLP guidelines and specific country requirements, study documents and materials will be stored in the archives o f the test facility, including but not limited to: study protocol; any protocol and/or report amendments or addenda or SOP deviations; all raw data; one original signed copy o f the final report; laboratory-specific or site-specific raw data such as personnel files, instrument, equipment, refrigerator, and/or freezer raw data. Documents and materials are archived according to the principles o f Good Laboratory Practice in the organization o f the testing facility. Disposal of Test Substance After issuance o f the final report, the remaining test substance will be stored at the DuPont EMSE laboratory until its expiration date and then destroyed by incineration, unless other arrangements are made between the submitter and the Test Facility. References 8.1 OECD Guidelines for the Testing o f Chemicals, Section 1, Method 111, Hydrolysis as a Function o f pH. 8.2 Environmental Protection Agency. 1989. Good Laboratory Practice Standards, 40 CFR, Part 160, Final Rule. EPA, Washington, DC. 8.3 JMP. 1995. JMP Statistical and Graphics Guide. Version 3 o f IMP. SAS Institute Inc. Cary, NC USA. T0106 / 4842 Page 15 o f 31 DuPont EMSE Report No. 92-02 Table 1 Concentration of 8-2 TBA at pH 1.2 at 37C and at pH 4, 7, and 9 at 50C AFTER0 AND 5 DAYS 8-2 TBA Average Difference between pH Time Date Concentration Day 5 and Day Of Day ugL-' % 1.2 0 21-Aug-2002 183 10.9:): 1.2 5 26-Aug-2002 195 8.00 +6.4 4 0 24-Jul-2002 156 4.22 4 5 29-JUI-2002 143 10.0 -8.7 7 0 21-Aug-2002 199 9.12 7 5 26-Aug-2002 181 12.6 -9.5 9 0 21-Aug-2002 169 5.99 9 5 26-Aug-2002 175 10.5 +3.5 f Difference between Day 5 and Day 0, % = (C5 - Co)/((C5 + C0)/2) x 100, where: Co = 8-2 TBA concentration at Day 0 C5 = 8-2 TBA concentration at Day 5 $ Mean standard deviation; n = 4. T0106 74842 Page 16 o f 31 DuPont EMSE Report No. 92-02 Table 2 pH of Buffer Solutions Containing 8-2 TBA at Days 0 and 5 Time Day 0 5 Date 21-Aug-2002 26-Aug-2002 Nominal dH s.u.t 1.2 1.2 Measured PH S.U. 1.11 1.54 Nominal temperature C Room temperature 37 Temperature of pH measurement C 22.5 37.1 0 24-Jul-2002 5 29-Jul-2002 4 4 4.02 Room temperature 4.12 50 23.0 50.0 0 21-Aug-2002 5 26-Aug-2002 7 7 7.03 Room temperature 7.04 50 22.5 50.0 0 21-Aug-2002 5 26-Aug-2002 9 9 f S.U. = Standard Units 9.02 Room temperature 8.97 50 22.5 50.0 TO106 74842 Page 17 o f 31 DuPont EMSE Report No. 92-02 Table 3 Daily Temperature Readings of Shaking Water Bath for Aqueous Stability of 8-2 TBA Conducted at pH 1.2 Time Day 0 1 2 3 4 5 Average, n = 6 Standard Deviation Date 21-Aug-2002 22-Aug-2002 23-Aug-2002 24-Aug-2002 25-Aug-20Q2 26-Aug-2002 Temperature C 37.1 37.0 37.2 37.0 37.1 37.1 37.1 0.1 T0106 / 4842 Page 18 o f 31 DuPont EMSE Report No. 92-02 Table 4 Daily Temperature Readings of Shaking Water Bath for Aqueous Stability of 8-2 TBA Conducted at pH 4 Time Day 0 1 2 3 4 5 Average, n = 6 Standard Deviation Date 24-Jul-2002 25-JU-2002 26-Jul-2002 27-JUI-2002 28-Jul-2002 29-Jul-2002 Temperature C 49.9 50.0 49.9 49.9 50.0 50.0 50.0 0.05 T0106 74842 Page 19 o f 31 DuPont EMSE Report No. 92-02 Table 5 Daily Temperature Readings of Shaking Water Bath for Aqueous Stability of 8-2 TBA Conducted at pH 7 and 9 Time Day 0 1 2 3 4 5 Average, n = 6 Standard Deviation Date 21-Aug-2002 22-Aug-2002 23-Aug-2002 24-Aug-2002 25-Aug-20Q2 26-Aug-2002 Temperature C 49.9 50.0 50.1 49.9 50.0 50.0 50.0 0.1 T0106 74842 Page 20 o f 31 DuPont EMSE Report No. 92-02 Table 6 Sterility of Test Solutions at Day 5 in Sterile Trypticase Soy Broth (TSB) Medium Treatment Result Conclusion Control Buffer 1.2 pH, 37C,Day5 Buffer 4 pH, 50C, Day 5 Buffer 7 pH, 50C, Day 5 Buffer 9 pH, 50C, Day 5 Clear Clear Cloudy Clear Clear Sterile Sterile Non-sterile Sterile Sterile t Sterile indicates that no bacteria were observed in 4.5 mL o f TSB containing 0.5 mL test solution after 3 to 4 days o f incubation in the dark at 30C. T0106 74842 Page 21 o f 31 DuPont EMSE Report No. 92-02 Table A-1 Analytical Results of the Aqueous Stability of 8-2 TBA, pH 1 .2 ,37C, Days 0 (21 -Aug-2002) and 5 (26-AUG-2002) No pH Time day 1 1.2 0 2 1.2 0 3 1.2 0 4 1.2 0 5 1.2 0 6 1.2 0 7 1.2 0 8 1.2 0 Average Standard Deviation Rep 1 1 2 2 3 3 4 4 Test Substance ugL-` 171 178 201 192 190 183 171 176 Test Substance Analytical Replication Average ugL-` 175 197 186 174 183 10.9 Difference Between Analytical Replicationsf % 4.0 4.6 4.0 2.9 9 1.2 5 1 200 200 0.0 10 1.2 5 1 11 1.2 5 2 200 186 189 4.0 12 1.2 5 2 13 1.2 5 3 193 204 204 0.2 14 1.2 5 3 15 1.2 5 4 205 203 188 15 16 1.2 5 4 174 Average 195 Standard Deviation 8.00 t Calculated by dividing the absolute difference o f the two analytical replications by their average value, times 100. TO106 / 4842 Page 22 o f 31 DuPont EMSE Report No. 92-02 I Table A-2 Analytical Results of the Aqueous Stability of 8-2 TBA, pH 4 .0 ,50C, Days 0 (24-JUL-2002) and 5 (29-Jul-2002) No pH 14 24 34 44 54 64 74 84 Average Standard Deviation Time day 0 0 0 0 0 0 0 0 Test Substance Test Analytical Replication Difference Between Rep Substance Average Analytical Replications! ugL-' ugL'1 % 1 162 1 162 2 156 2 157 3 150 3 156 4 155 162 157 153 153 0.2 0.4 3.7 3.1 4 151 156 4.22 9 4 51 128 130 2.6 10 4 51 132 11 4 52 155 154 1.3 12 4 52 153 13 4 53 143 143 0.2 14 4 53 143 15 4 54 145 145 0.0 16 4 Average Standard Deviation 54 145 143 10.0 t Calculated by dividing the absolute difference o f the two analytical replications by their average value, times 100. T0106 74842 Page 23 o f 31 DuPont EMSE Report No. 92-02 Table A-3 Analytical Results of the Aqueous Stability of 8-2 TBA, pH 7.0, 50C, Days 0 (21-Aug-2002) and 5 (26-AUG-2002) No pH 17 27 37 47 57 67 77 87 Average Standard Deviation Test Substance Test Analytical Replication Difference Between Time Rep Substance Average Analytical Replicationst day ugL-` ugC % 01 01 02 02 03 03 04 04 202 197 195 196 201 179 211 212 199 195 190 211 199 9.12 2.3 0.3 12 0.7 9 7 51 195 192 3.1 10 7 51 189 11 7 52 169 166 3.6 12 7 52 163 13 7 53 178 175 3.4 14 7 53 172 15 7 54 202 191 12 16 7 Average Standard Deviation 54 180 181 12.6 t Calculated by dividing the absolute difference o f the two analytical replications by their average value, times 100. TO106 74842 Page 24 o f 31 DuPont EMSE Report No. 92-02 Table A-4 Analytical Results of the Aqueous Stability of 8-2 TBA, pH 9 .0 ,50C, Days 0 (21-Aug-2002) and 5 (26-AUG-2002) Test Substance Test Analytical Replication Difference Between No PH Time Rep Substance Average Analytical Replicationsf day ugL-` u g v ' % 19 29 39 49 59 69 79 89 Average Standard Deviation 01 01 02 02 03 03 04 04 185 167 168 173 159 165 163 171 176 170 162 167 169 5.99 10 2.6 3.7 4.5 99 10 9 11 9 12 9 13 9 14 9 15 9 16 9 Average Standard Deviation 51 51 52 52 53 53 54 54 172 193 173 185 160 159 169 191 182 179 160 180 175 10.5 12 6.7 0.6 13 t Calculated by dividing the absolute difference o f the two analytical replications by their average value, times 100. T0106 / 4842 Page 25 o f 31 DuPont EMSE Report No. 92-02 Table B-1 Analytical Results of the Aqueous Stability of 8-2 TBA for Pilot Experiment Conducted at pH 4 and Room Temperature, Day 0 only (16-JuL-2002)f Test Substance Test Analytical Replication Difference Between No Rep Substance Average Analytical Replications^; --p ugL' % 1 1 106 112 10 2 1 117 3 2 104 o CO 8.2 4 2 113 5 3 109 110 2.4 6 3 111 7 4 102 113 19 8 4 123 9 5 114 110 7.3 10 5 106 11 6 125 117 13 12 6 109 13 7 103 106 6.9 14 7 110 15 8 118 115 4.9 16 8 112 17 9 114 115 3.2 18 9 117 19 10 100 94.1 12 20 10 88.4 t Experiment performed to assess the adequacy o f the test methods and analytical procedure.: It was performed using pH 4 buffer at room temperature, Day 0 samples only. Test vessels were 7 mL glass scintillation vials with aluminum-lined screw caps. 22.5 pL o f a 20 mg 8-2 TBA L'1 methanol solution was added to each test vessel with 3 mL o f the pH 4 buffer solution. After capping, the samples were vortex-mixed for 10 s. Samples were frozen until analyzed. $ Calculated by dividing the absolute difference o f the two analytical replications by their average value, times 100. T0106 74842 Page 26 o f 31 DuPont EMSE Report No. 92-02 Table B-1 (cont'd) Test Substance Test Analytical Replication Difference Between No Rep Substance Average Analytical Replications^ u g U ' % 21 22 23 24 25 26 27 28 29 30 31 32 Average Standard Deviation 11 11 12 12 13 13 14 14 15 15 16 16 108 104 120 84.9 117 122 101 115 112 104 106 121 106 103 119 108 108 114 110 6.09 3.6 35 4.4 12 7.4 13 t Experiment performed to assess the adequacy o f the test methods and analytical procedure. Ibwas performed using pH 4 buffer at room temperature, Day 0 samples only. Test vessels were 7*.mL > glass scintillation vials with aluminum-lined screw caps. 22.5 pL o f a 20 mg 8-2 TBAL'-h > methanol solution was added to each test vessel with 3 mL o f the pH 4 buffer solution. After capping, the samples were vortex-mixed for 10 s. Samples were frozen until analyzed. $ Calculated by dividing the absolute difference o f the two analytical replications by their average value, times 100. T0106 / 4842 Page 27 o f 31 DuPont EMSE Report No. 92-02 Table B-2 Analytical Results of the Aqueous Stability of 8-2 TBA, pH 1.2, 50C, Days 0 (31 -Jul-2002) and 5 (5-Auo-2002)t Test Substance Test Analytical Difference Between No pH Time Rep Substance Replication Average Analytical Replications^ day UgL-1 ugL"` % 1 1.2 0 1 136 137 2 1.2 0 1 138 3 1.2 0 2 117 119 4 1.2 0 2 120 5 1.2 0 3 157 150 6 1.2 0 3 144 7 1.2 0 3 152 8 1.2 0 3 149 1.2 4 114 110 1.2 4 105 Average Standard Deviation 129 18.4 1.2 2.0 8.7 1.6 8.8 9 1.2 5 1 143 141 3.8 10 1.2 5 1 138 11 1.2 5 2 140 134 9.0 12 1.2 5 2 128 13 1.2 5 3 102 102 14 1.2 5 4 165 164 15 1.2 5 4 163 Average 135 Standard Deviation 25.6 NA 0.8 t Test was performed at 50C instead o f 37C. Experiment was performed using pH 1.2 buffer at 50C. Test vessels were 7 mL glass scintillation vials with aluminum-lined screw caps. 22.5 pL o f a 20 mg 8-2 TBA L'1methanol solution was added to each test vessel with 3 mL o f the pH 1.2 buffer solution. After capping, the samples were vortex-mixed for 10 s. Samples were frozen until analyzed. { Day 0, replicate 3 measured four times; Day 5 replicate 3 measured once, f Calculated by dividing the absolute difference o f the two analytical replications by their average value, times 100. , NA = Not applicable; only one analytical measurement o f sample. TO106 / 4842 Page 28 o f 31 DuPont EMSE Report No. 92-02 Table B-3 Analytical Results of the Aqueous Stability of 8-2 TBA, pH 7 .0 ,50C, Days 0 (31-Jul-2002) and 5 (5-AuG-2002)t Test Substance Test Analytical Replication Difference Between No pH Time Rep Substance Average Analytical Replications^ day ugV u g L ' % 17 27 37 47 57 67 77 87 Average Standard Deviation 0 0 0 0 0 0 0 0 1 1 2 2 3 3 4 4 103 104 114 108 98.0 93.3 102 101 104 111 95.7 102 103 6.25 1.6 5.7 4.9 1.3 9 75 1 58.7 57.2 5.3 10 7 5 11 7 5 1 2 55.7 24.6 24.6 0.3 12 7 5 13 7 5 2 3 24.7 NDU 14 7 5 15 7 5 3 4 96.7 91.7 11 16 7 5 4 86.7 Average 43.4 Standard Deviation_____________________________________________ 39-8 ___________________________ f Test was repeated because o f the variation in replicate results o f test substance concentration at Day 5. Experiment was performed using pH 7 buffer at 50C. Test vessels were 7 mL glass scintillation vials with aluminum-lined screw caps. 22.5 pL o f a 20 mg 8-2 TBA L 1methanol solution was added to each test vessel with 3 mL o f the pH 1.2 buffer solution. After capping, the samples were vortex-mixed for 10 s. Samples were frozen until analyzed. Calculated by dividing the absolute difference o f the two analytical replications by their average value, times 100. f ND = Not determined; samples were lost prior to analysis T0106 / 4842 Page 29 o f 31 DuPont EMSE Report No. 92-02 Table B-4 Analytical Results of the Aqueous Stability of 8-2 TBA, pH 9.0, 50C, Days 0 (31-JUL-2002) and 5 (5-Auo-2002)t Test Substance Test Analytical Replication Difference Between No pH Time Rep Substance Average Analytical Replications! day ugL'1 ugL' % 19 29 39 49 59 69 79 89 Average Standard Deviation 0 0 0 0 0 0 0 0 1 1 2 2 3 3 4 4 162 163 149 135 153 147 132 139 162 142 150 136 147 11.5 0.6 9.6 4.4 5.2 9 95 1 150 143 9.8 10 9 5 11 9 5 1 2 136 71.3 80.8 24 12 9 5 13 9 5 2 3 90.3 59.7 59.0 2.3 14 9 5 15 9 5 3 4 58.3 131 142 15 16 9 5 4 152 Average 106 Standard Deviation 42.9 t Test was repeated because o f the variation in replicate results o f test substance concentration at Day 5. Experiment was performed using pH 9 buffer at 50C. Test vessels were 7 mL glass scintillation vials with aluminum-lined screw caps. 22.5 pL o f a 20 mg 8-2 TBA L"1methanol solution was added to each test vessel with 3 mL o f the pH 1.2 buffer solution. After capping, the samples were vortex-mixed for 10 s. Samples were frozen until analyzed. $ Calculated by dividing the absolute difference o f the two analytical replications by their average value, times 100. T0106 / 4842 Page 30 o f 31 DuPont EMSE Report No. 92-02 Appendix C Certificate of Test Substance Analysis CUriant GmbH, Werk Gendorf CQG / Quaumioigernent D-8*50* Burgkireiien Fw: -4-49 8679 / 2676 Clariant Inspection certificate according to E N 10204-3.13 Date: 13.11.2001 Page: 1 / 1 3-ur conaigr.m eni M aterial M aterial-r.o. s a c c a Mo. 1 - Decano 1, 3, 3 , 4 , 4 , 5 , 5, 6, 6 ,7 , 7 , 8 ,8 , 9, 9 / 1 0 ,1 0 , lQ -heptadecafluoro 104705 ?0C-/O01 bare.-:, of whirr. the nonaicr-fent is a part, the following values r.eu. They conform r.i agreed product specification. Inspection ctaracter ct i-;/ -methcdSpecif icatior. R esu lt ) >- 97,5 99,3% ia/a] Capii-ary uC AV U O VO CO o A O 0* Sun ?erfluoroaikyiethanol Capillary GC Sum reefluocalkylbutanoi Capillary GC Sura Ferfluorslkylethene Capillary GC <-1,0 99,9% ia/a) < 0 , 1 % [a/a] < 0,1%[a/a Water content Karl Fischer DIN 51777 M-Methylpyrrolidcne content Capillary GC o H V <-0,2 < 0, 1% The above particulars co not release the customer from the obligation carry out an inspection of goods received. to Ms. Eau.T.o-irtnar - Wcrksi.nspector T0106 / 4842 Page 31 o f 31