Document MG0QoBB8GLNava7ex42kwdJYj

ST-545 Final Report ToxDocs 15-039 TOXICOLOGY ASSESSMENT AND COMPLIANCE ASSURANCE GROUP 3M COMPANY 15-039: ORAL ADME STUDY OF MTDID 32159, 208, AND 1004 IN RATS FINAL REPORT ToxDocs Study Number 15-039 Test Material MTDID 32159, 208, and 1004 Lot Number(s) Strategic Tox Lab Study Number Other Identification Number(s) MTDID 32159: Lot 30019 MTDID 208: Lot 217 MTDID 1004: Lot 332 ST-545 MTDID 32159 3M Product Code 41-2603-3318-6; T-7252; F-33318 MTDID 208 FC-95; T-6295; perfluorooctane sulfonate potassium salt; PFOS 19F/1H-NMR GID Number MTDID 1004 Analytical Report MTDID 32159 FC-143; T-7507; perfluorooctanoate ammonium salt; PFOA LIMS M13-0520 & M13-0647 MTDID 208 LIMS Request #M121793 MTDID 1004 LIMS Request #M12-2198 Study Director Jill Hart, AAS, LATg Senior Laboratory Technologist Principle Investigator: (Study Monitor) Sue Chang, Ph.D. Senior Toxicology Specialist Testing Facility In-Life Start Date 3M Strategic Toxicology Laboratory 3M Center, Bldg 270, room SB324 Saint Paul, MN 55144 March 4, 2015 In-Life Termination Date March 5, 2015 1 of 12 ST-545 Final Report `ToxDocs 15-039 TOXICOLOGY ASSESSMENT3MANCDOCMOPMAPNLYIANCE ASSURANCE GROUP 15-039: ORAL ADME STUDY OF MTDID 32159, 208, AND 1004 IN RATS Compliance Statement and Signature Page Ttohirsegsutluadtyorwyagsuicdoenldinuecst.ed for research and development purposes and does not conform This final report has been reviewed and approved by: Cha hur TAIT, AAS, LATg Senior Laboratory Technologist Study Director . AF => < `Sue Chang, Ph.D. SSetnuidoyrMToonxiitcoorlogy Specialist Dapdrz0ite 27 Apr 2016 Date 20f12 ST-545 Final Report ToxDocs 15-039 1 Summary The objective of this study was to collect tissues from rats for analytical method development. Male Sprague Dawley rats received a single oral dose of either MTDID 32159 (FBSA), 208 (PFOS), or 1004 (PFOA). Urine and feces were collected overnight and gross necropsy was performed at 24 hours post-dose, with blood (processed to serum) and liver collection. The data demonstrated that recoveries of MTDID 32159 (FBSA) can be improved with enzymatic treatment or acid hydrolysis. When performing LC-MS/MS analysis of FBSA in rat specimens, pre-treatment with -glucuronidase or acid hydrolysis prior to SPE, improved the quantifiable levels of FBSA in the matrices evaluated. For acid treatment with 1 N formic acid, incubation at 60oC for 2 hours appeared to the critical step for optimal FBSA recovery among the matrices evaluated. For urine samples, treatment with -glucuronidase at 37oC for at least 48 hours had similar levels of recovery when compared to 1N formic acid treatment at 60oC for 2 hours. For serum, liver, and fecal samples, treatment with -glucuronidase at 37oC for at least 24 hours had similar levels of recovery when compared to 1N formic acid treatment at 60oC for 2 hours. Compared to enzymatic digestion, acid treatment with 1 N formic acid required a shorter incubation time but offered comparable FBSA recoveries in all four matrices. For MTDID 208 (PFOS) and 1004 (PFOA), the results from this study suggested that liver homogenization using an automatic homogenizer can lead to higher PFOS and PFOA recoveries when compared to the hand-held homogenizer. Additionally, the automatic homogenizer offers a time-saving advantage (~3X less). The recoveries between stainless steel bead and ceramic bead lysing kits were comparable; however, the stainless steel beads caused issues with the solid phase extraction (SPE) process as pieces of stainless steel flaked off into the liver samples due to the strong high speed vibrations (grayish color was observed in these samples), making it difficult to properly process the samples. Therefore, the stainless steel bead lysing kits are not recommended for use with liver samples in an automatic homogenizer. 2 Objective Objective (1): Previous studies have shown that MTDID 32159 can conjugate with glucuronide (see ToxDocs 13-165 and 14-171) hence it is important to account for the conjugated complex when analyzing samples containing MTDID 32159. Therefore, MTDID 32159 concentrations were determined for serum, liver, urine, and feces samples with various enzyme deconjugation methods. Objective (2): MTDID 208 and 1004 concentrations were determined for liver samples under with two different homogenization methods. 3 of 12 ST-545 Final Report ToxDocs 15-039 3 Test Material Information 3.1 Test Substance Test Article (MTDID 32159) Identity MTDID 32159 Product Code 41-2603-3318-6, lot 30019, dated 3-19-2013 Chemical Name Nonafluoro-1-butanesulfonamide Other Identifiers F-33318, T-7252 Chemical Structure C4F9SO2NH2 Appearance White solid at room temperature Purity 99.73% Density 1.68 g/mL at melting point Molecular Weight 299.0 Stability Stable Melting Point 158 F Expiration Date N/A Characterization See 3M MRD Analytical Lab LIMS Request #M13-0520 and M13-0647 (dated 4-10-2013) Test Article (MTDID 208) Identity MTDID 208 Product Code FC-95, lot 217 Chemical Name Perfluorooctanesulfonate, potassium salt Other Identifiers K+PFOS Chemical Structure K+C8F17SO3- Appearance White solid at room temperature Purity 88.9% Density N/A Molecular Weight 538 g/mol Stability Stable Melting Point N/A Expiration Date N/A Characterization See 3M MRD Analytical Lab LIMS Request #M121793 (dated 12-20-2012) 4 of 12 ST-545 Final Report ToxDocs 15-039 Test Article (MTDID 1004) Identity MTDID 1004 Product Code FC-143 Chemical Name Perfluorooctanoate, ammonium salt Other Identifiers NH4+PFOA Chemical Structure NH4+C7F15CO2- Appearance White solid at room temperature Purity 98.4% Density NA Molecular Weight 431 g/mol Stability Stable Melting Point N/A Expiration Date N/A Characterization See 3M MRD Analytical Lab LIMS Request #M12-2198, dated 12-23-2013 3.2 Dose Preparation Dose Preparation Physical State of administered material Route of Administration 4 Test System MTDID 32159 and MTDID 208 were each prepared in 0.5% Tween 20 (w/v) and solublized by heating (~ 50oC) and sonication for 15 - 30 minutes. MTDID 1004 was prepared in Milli-Q water. The final working concentrations were 0.6 mg/mL, 1 mg/mL, and 1 mg/mL for MTDID 32159, 208, and 1004, respectively. On the day of dosing, 5 mL of dosing solution per kg body weight was administered via oral gavage to nonfasted rats. Gentle mixing of the solution was performed before each sample was drawn-up in the syringe for dosing. Liquid Oral 5 of 12 ST-545 Final Report ToxDocs 15-039 4.1 Test System Information 4 Species Strain Source Age at Initiation of Treatment Weight at Initiation of Treatment Number and sex Identification IACUC Animal Usage Application Number Rat Sprague Dawley Charles River 6-8 weeks 178 - 199 g 9 males Unique tail mark in indelible ink 2014-0001 4.2 Animal Husbandry Housing Diet/Water Housing Environment All animals were single housed in wired bottom cages during the entire study until study termination. Harlan Teklad Rat/Mouse 2018 Diet (Harlan Teklad, Madison, WI) and tap water were available ad libitum throughout the study. Temperature Range 72 3F Humidity Range 30 - 70% Minimum of 10 exchanges of room air per hour 12 hour light/dark cycle 5 Study Design On Study Day 1, all animals received a single dose of test material via oral gavage. The dose group designation is presented below: Group MTDID Dose Sex N Euthanasia (mg/kg) 1 32159 (FBSA) 3 M 3 24 hours PD 2 208 (PFOS) 5 M 3 24 hours PD 3 1004 (PFOA) 5 M 3 24 hours PD 6 Parameters Evaluated 6.1 Clinical Observations Each animal was observed immediately following dosing and throughout the study for mortality and morbidity. 6.2 Body Weights Each animal was weighed prior to dose administration for dosing volume calculation purpose. 6 of 12 ST-545 Final Report ToxDocs 15-039 6.3 Gross Necropsy At the end of 24 hours, all rats were euthanized by CO2 and no gross necropsies were performed. 6.4 Analytical LC-MS/MS was used to determine the concentrations of the test materials. Because MTDID 32159 has been shown to conjugate with glucuronide (see ToxDocs 13-165 and 14-171), MTDID 32159 concentrations were determined for serum, liver, urine, and feces samples with various enzyme deconjugation methods. MTDID 208 and 1004 concentrations were determined for liver samples only but with two different homogenization methods. 7 Raw Data The raw data for the in-life study were recorded in 3M Notebook #166568, page 97. 8 Results 8.1 Clinical Observations Clinical observations were normal throughout the study period. All animals appeared normal during the study and all of them survived to the scheduled necropsy. 8.2 Analytical Results MTDID 32159: See Appendix 1 MTID 208 and 1004: See Appendix 2 9 Conclusion The data demonstrated that the recoveries of MTDID 32159 (FBSA) can be improved in rat specimens with enzymatic treatment or acid hydrolysis. When performing LCMS/MS analysis of FBSA in rat specimens, pre-treatment with -glucuronidase or acid hydrolysis prior to SPE improved the quantifiable levels of FBSA in the matrices evaluated. For acid treatment with 1 N formic acid, incubation at 60oC for 2 hours appeared to the critical step for optimal FBSA recovery among the matrices evaluated. For urine samples, treatment with -glucuronidase at 37oC for at least 48 hours had similar levels of recovery when compared to 1N formic acid treatment at 60oC for 2 hours. For serum, liver, and fecal samples, treatment with -glucuronidase at 37oC for at least 24 hours had similar levels of recovery when compared to 1N formic acid treatment at 60oC for 2 hours. Compared to enzymatic digestion, acid treatment with 1 N formic acid required a shorter incubation time but offered comparable FBSA recoveries in all four matrices. 7 of 12 ST-545 Final Report ToxDocs 15-039 For MTDID 208 (PFOS) and 1004 (PFOA), the results from this study suggested that liver homogenization using an automatic homogenizer can lead to higher PFOS and PFOA recoveries when compared to the hand-held homogenizer. Additionally, the automatic homogenizer offers a time-saving advantage (~3X less). The recovery between stainless steel bead and ceramic bead lysing kits were comparable; however, the stainless steel beads caused issues with the solid phase extraction (SPE) process as pieces of stainless steel flaked off into the liver samples due to the high speed vibrations (grayish color was observed in these samples), making it difficult to properly process the samples. Therefore, the stainless steel bead lysing kits are not recommended for use with liver samples in an automatic homogenizer. 8 of 12 ST-545 Final Report ToxDocs 15-039 Appendix 1: MTDID 32159 Analyses 9 of 12 ST-545 Final Report ToxDocs 15-039 10 of 12 ST-545 Final Report ToxDocs 15-039 Appendix 2: MTDID 208 and 1004 Analyses 11 of 12 ST-545 Final Report ToxDocs 15-039 12 of 12