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3M Medical Department Study: T-6395.14 AR226-1030A005 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Study Title Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats Analytical Laboratory Report Title Determination of the Concentration of Perfluorooctanesulfonate (PFOS) in the Serum, Liver, Urine, and Feces of Cri:CDBR VAF/Plus Rats Exposed to PFOS via Gavage Data Requirement Not Applicable Author 3M Environmental Laboratory Study Completion Date at signing Performing Laboratories Urine Analyses Feces Analyses 3M Environmental Laboratory Building 2-3E-09, 935 Bush Avenue St. Paul, MN 55106 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Liver Analyses Battelle Memorial Institute 505 King Avenue Columbus, OH 43201 Serum Analyses Advanced Bioanalytical Services, Inc. 15 Catherwood Road Ithaca, NY 14850 Project Identification 3M Medical Department Study: T-6295.14 Argus In-Life Study: 418-015 Analytical Report: FACT TOX-111 3M Environmental Laboratory Request No. U2994 Total Number o f Pages 341 3M Environmental Laboratory Page 1 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACTTOX-111 LRN-U2994 This page has been reserved for specific country requirements. 3M Environmental Laboratory Page 2 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 GLP Compliance Statement Analytical Laboratory Report Title: Determination of the Concentration of Perfluorooctanesulfonate (PFOS) in the Serum, Liver, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to PFOS via Gavage Study Identification Numbers: T-6295.14, FACT TOX-111, LRN-U2994 This study was conducted in compliance with United States Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations 21 CFR Part 58, with the exceptions in the bulleted list below. The analytical phase completed at the 3M Environmental Laboratory was performed in accordance with 3M Environmental Technology and Safety Services Standard Operating Procedures. Exceptions to GLP compliance: In the study binder, there are numerous Chain of Custody sheets that lack a signature, time, and date. In the study binder, there are packing lists sent by Argus that have evidence of having been whited-out with "White-out." Mass spectrometry data for liver and urine were collected and processed with the Mass Lynx software system that was not fully validated. Separate study directors were initially assigned to lead the in vivo and analytical portions of the study, respectively. This was eventually corrected. On several occasions, data entries or corrections were not documented exactly as required by the GLPs. Not all reagents and solutions used in this study included all the fields required by GLPs. Dose confirmation analyses were not performed in compliance with GLP regulations. (signatures on next page) 3M Environmental Laboratory Page 3 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 / ________________________ M.V., Ph.D., Study Director ^ 4 - -z- -- - ___________ Kris J. Hansen, Ph.D., Analytical Investigator 0 ^ } OHIO l Date -------- ______________________________ s ' j / q/ William Reagen, Ph.D., Laboratory Manager Date 4t 3M Environmental Laboratory Page 4 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -1 11 LRN-U2994 GLP Study--Quality Assurance Statement Analytical Laboratory Report Title: Determination of the Concentration of Perfluorooctanesulfonate (PFOS) in the Serum, Liver, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to PFOS via Gavage Study Identification Numbers: T-6295.14, FACT TOX-111, LRN-U2994 This analytical study performed at 3M Environmental Laboratory has been inspected by the 3M Environmental Laboratory Quality Assurance Unit (QAU) as indicated in the following table. The findings were reported to the study director and laboratory management. Inspection Dates Phase Date Reported to Management Study Director 10/7/99 Extraction 5/15/00 5/15/00 10/8/99 Analysis 5/15/00 5/15/00 8/29--9/1/00, 9/4-- 9/5/00 Data 9/6/00 9/6/00 i 10/12/00 10/26-- 10/27/00 Data Draft Report 10/12/00 10/30/00 10/12/00 10/30/00 11/13/00 Draft Report 11/13/00 11/13/00 See Appendix G for the individual contractor quality assurance statements. IWAfJ JLjuU _______________________ S '/j! ( QAU Representative Date 3M Environmental Laboratory Page 5 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 Table of Contents GLP Compliance Statement................................................................................................ 3 GLP Study--Quality Assurance Statement........................................................................ 5 Study Personnel and Contributors...................................................................................... 10 Introduction and Purpose....................................................................................................11 Test System................................................................................................................... 11 Specimen Collection and Analysis................................................................................. 11 Specimen Receipt and Maintenance.................................................................................. 12 Chemical Characterization of the Test Article..................................................................... 13 Procurement................................................................................................................... 14 Stability Studies.............................................................................................................. 14 Dose Confirmation Analyses..........................................................................................14 Method Summaries............................................................................................................. 15 3M Environmental Laboratory........................................................................................ 15 Preparatory Method............ ......................................................................................15 Analytical Methods.....................................................................................................15 Analytical Equipment.................................................................................................16 Deviations....................................................................................................................... 17 Data Quality Objectives and Data Integrity.........................................................................17 Data Summary, Analyses, and Results.............................................................................. 17 Summary of Quality Control Analyses Results...............................................................17 Statement of Data Quality..............................................................................................18 Summary of Sample Results......................................................................................... 18 Statistical Methods and Calculations.................................................................................. 19 Statement of Conclusion.....................................................................................................19 Appendix A: Control Matrices and Dose Confirmation Analyses........................................ 20 Appendix B: Protocol, Protocol Amendments and Deviation Summary.............................22 Appendix C: Extraction and Analytical Methods................................................................ 54 3M Environmental Laboratory, ETS-8-96.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Urine for Analysis using HPLC-Electrospray/Mass Spectrometry/Mass Spectrometry," (14 pages)...............55 3M Environmental Laboratory, ETS-8-97.0, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrospray/Mass Spectrometry/Mass Spectrometry," (10 pages)........................ 69 3M Environmental Laboratory, ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry," (9 pages)......................................................................81 Appendix D: Data Summary Tables...................................................................................91 Appendix E: Data Spreadsheets........................................................................................95 3M Environmental Laboratory Page 6 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Appendix F: Example Calculations..................................................................................... 101 Appendix G: Contract Laboratory Reports..........................................................................102 Advanced Bioanalytical Services, Inc., Quantitative Determination of Perfluorooctanesulfonate (PFOS) in Rat Serum from Study #FACT-TOX-111 Using Turbo Ion Spray LC/MS, ABS Report Number 99ADJA03.MI.DOC (19 pages).................103 Advanced Bioanalytical Services, Inc., "Method Validation for the Quantitation of Perfluorooctanesulfonate (PFOS) in Rat Serum by Turbo Ion Spray LC/MS," ABS Report Number 99VDJA01.MI.DOC (50 pages)................................................................. 122 Battelle Memorial Institute, Study Number N003296-G, Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats Final Report, (68 pages)................................................172 Centre Analytical Laboratories, Inc., Centre Study Number 023-017, Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats, (93 pages).........................................240 Appendix H: Certificate of Analysis.................................................................................... 334 Appendix I: Report Signature Page.................................................................................... 341 3M Environmental Laboratory Page 7 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -1 11 LRN-U2994 List of Tables Table 1. Female Rat Population Demographics for Study #418-015................................. 11 Table 2. Custody Transfer of Specimens in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Liver, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 via Gavage...........................13 Table 3. Characterization of Test Article in Study FACT TOX-111.................................... 13 Table 4. Negative Ions Monitored in 3M Laboratory Analyses for Study FACT TOX-111.. 17 Table 5. Characterization of the Control Matrices Used for Sera, Liver, Urine, and Feces Analyses in Study FACT TOX-111........................................................................ 20 Table 6. Characterization of the Analytical Reference Substances Used for Liver, Urine, Sera, and Feces Analyses in Study FACT TOX-111............................................ 20 Table 7. Dose Confirmation Analyses for Perfluorooctanesulfonate for Test Samples from In-Life Study #418-015...................................................................................21 Table 8. Deviation Summary for FACT TOX-11 1 ................................................................22 Table 9. Average Results for the Analyses of Sera Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 via Gavage................................................................................................................... 91 Table 10. Average Results for the Analyses of Liver Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 via Gavage................................................................................................................... 92 Table 11. Average Results for the Analyses of Urine Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 via Gavage................................................................................................................... 92 Table 12. Average Results for the Analyses of Feces Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 via Gavage................................................................................................................... 93 Table 13. LOQ Values Used in FACT TOX-111 Analysis by Method and Usage Dates ....94 Table 14. Results of Analyses of Sera Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 viaGavage............................95 Table 14. Results of Analyses of Sera Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Liver, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 viaGavage........................... 96 Table 15. Results of Analyses of Liver Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Liver, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 viaGavage........................... 97 3M Environmental Laboratory Page 8 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACTTOX-111 LRN-U2994 Table 15. Results of Analyses of Liver Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Liver, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 via Gavage...........................98 Table 16. Results of Analyses of Urine Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 via Gavage...........................99 Table 17. Results of Analyses of Feces Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 via Gavage.......................100 3M Environmental Laboratory Page 9 BACK TO MAIN 3M Medical Department Study: T-6395.14 Study Personnel and Contributors Analytical Report: FA C TTO X-111 LRN-U2994 Study Director Marvin T. Case, D.V.M., Ph.D. 3M Corporate Toxicology 3M Center, Building 220-2E-02 St. Paul, MN 55133-3220 Phone (651)733-5180 Sponsor 3M Corporate Toxicology 3M Center, Building 220-2E-02 St. Paul, MN 55133-3220 John L. Butenhoff, Ph.D., Sponsor Representative Analytical Chemistry Laboratories Urine Analyses 3M Environmental Laboratory Kristen J. Hansen, Ph.D., Analytical Investigator Serum Analyses Advanced Bioanalytical Services, Inc. (ABS) David J. Anderson, M.S., Analytical Investigator Liver Analyses Battelle Memorial Institute (Battelle) Jon C. Andre, Ph.D., Analytical Investigator Feces Analyses Centre Analytical Laboratories, Inc. (Centre) Enaksha Wickremesinhe, Ph. D. Analytical Investigator 3M Environmental Laboratory Contributing Personnel Lisa A. Clemen Kelly J. Dorweiler* Mark E. Ellefson Sara E. Estes* Barb A. Gramenz* Sarah A. Heimdal* Cari S. Hewitt* Marlene M. Heying* Megan C. Holloway* Harold O. Johnson Ognjenka Krupljanin* Kelly J. Kuehlwein* Sally A. Linda* Ian A. Smith* Bob W. Wynne* * Contract laboratory professional service employees Location of Archives All original raw data, the protocol, the analytical report, the test article and the analytical reference standard reserve samples, as well as the specimens pertaining to the analytical phase of this study, are archived at the 3M Environmental Laboratory for a minimum of ten years. Reserve samples of control matrices used at Battelle, Centre and ABS will be stored at the respective contract laboratories in such a manner that the control matrices will be preserved for use in ensuing studies. 3M Environmental Laboratory Page 10 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 Introduction and Purpose The purpose of the analytical study is to determine the concentration of perfluoro-octanesulfonate (PFOS) in sera, liver, urine, and feces specimens taken from the Gavage Recovery Study of T6295.14 (potassium perfluorooctanesulfonate (KPFOS), CAS Number: 2795-39-3) in Crl:CDBR VAF/Plus Rats. Rat dams were exposed to KPFOS via gavage prior to and during mating. Exposure to KPFOS was halted on the first day of presumed gestation. Pups were not directly exposed to KPFOS, but may have been exposed in utero and during lactation. The analytical phase of this study was initiated on 9 June 1999. Test System The test system species and strain selected was the Crl:CDBR VAF/Plus (Sprague-Dawley) rat, received from Charles River Laboratories, Inc., and permanently identified using Monel self piercing ear tags. Generation FO virgin female rats were approximately 60 days of age and weighed approximately 181 to 222 g when received. Male rats of the same source and strain were used only as breeders and were not administered the test article or considered part of the test system. Generation F1 rats were identified as part of Group I, II, or III litters, but were not individually tagged, and all parameters were evaluated in terms of the litter (Table 1). The test system comprised Generation FO rats and Generation F1 pups. In Generation FO, three groups of 8 female rats each (a total of 24) were used. Group I control FO female rats were administered Tween80 (carrier). All other FO female rats were administered KPFOS in 0.5% Tween80. At predetermined intervals during, and at the end of, the in-life phase of the study, sera, urine, feces, and liver specimens were collected from Generation FO rats, and sera and liver specimens were collected from Generation F1 pups. Table 1. Female Rat Population Demographics for Study #418-015 Population Selected for Study Total Litters Selected for Study Group I (Control) 0 mg/kg/day Group II 0.1 mg/kg/day Group III 1.6 mg/kg/day 8 8 8 T 7a 88 7b 7b a One Group I FO rat died from injury on DG 15 and did not give birth. 6 One Group III FO rat did not give birth. Specim en Collection and Analysis In the analytical study reported here, 440 serum, liver, urine, and feces specimens were collected from adult female rats (Generation FO) and their offspring (Generation FI). The specimens were sent to the 3M Environmental Laboratory and contract laboratories to be analyzed for PFOS. Sera specimens were collected from all Generation FO animals immediately 3M Environmental Laboratory Page 11 3M Medical Department Study: T -6395.14 BACK TO MAIN Analytical Report: FACT T O X -1 11 LRN-U2994 prior to cohabitation, on Gestation Days (DGs) 7,15, and 21 sand on Lactation Days (DLs) 14 and 22. Sera specimens were collected from pooled litter samples on DL 21. Urine and feces specimens were collected from all Generation FO animals beginning one day before cohabitation was initiated through the morning cohabitation was initiated, from DGs 6 through 7,14 through 15, and 20 through 21, and on DLs 21 through 22. On DL 22, all surviving Generation FO and Generation F1 animals assigned to the study were sacrificed, and a liver specimen was collected from each animal. The number and type of specimens collected for analyses in the analytical phase of this study are presented below. Specimens Collected from Study Groups I through III (through 20 February 99): Serum Specimens-- 161 specimens Liver Specimens--45 specimens Urine Specimens--117 specimens Feces Specimens-- 117 specimens Blood specimens were separated by refrigerated centrifuge. Resulting serum specimens were then transferred to labeled polypropylene tubes and immediately frozen on dry ice. Liver, urine, and feces specimens were collected and frozen on dry ice. All serum, liver, urine, and feces specimens were stored at -70 C or below until they were shipped, on dry ice, to the 3M Environmental Laboratory. Specimens were then shipped to the contract analytical laboratories for analysis. Urine samples were extracted beginning on 06 October 1999 using an ion-pairing reagent and methyl-ferf-butyl ether (MtBE). Sample extracts were analyzed using high-pressure liquid chromatography-electrospray/tandem mass spectrometry (HPLC-ES/MS/MS) in the multiple response monitoring mode. PFOS levels were quantitated by external calibration. Analytical details are included in this report. The methods and analytical equipment settings used by Battelle Memorial Institute, Advanced Bioanalytical Services, Inc. and Centre Analytical Laboratories, Inc. are presented in the respective contract laboratory reports (see Appendix G). Specimen Receipt and Maintenance The 3M Environmental Laboratory received serum, liver, urine and feces specimens that were collected during the in-life phase of this study, #418-015 from Argus. All specimens were received frozen on dry ice and were immediately transferred to storage at -20 10C. Specimens that were analyzed at Battelle Memorial Institute (liver), at Advanced Bioanalytical Sciences--ABS (serum), or at Centre Analytical Laboratories (feces), were subsequently shipped frozen on dry ice. Table 2 lists specimen receipt information for this study. 3M Environmental Laboratory Page 12 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Table 2. Custody Transfer of Specimens in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Liver, Urine, and Feces of Crl:CDBR VAF/PIus Rats Exposed to T-6295.14 via Gavage Tissue/Sample Liver Serum Urine Feces Liver Serum Generation Dam Dam Dam Dam Fetal (pooled) Fetal (pooled) Number of Samples Received 23 139 117 117 22 22 Date Received 09 March 1999 09 March 1999 09 March 1999 09 March 1999 09 March 1999 09 March 1999 Date Shipped to Contract Laboratory 19 August 1999 15 June 1999 a 27 June 2000 19 August 1999 15 June 1999 Shipment date not applicable: urine specimens were analyzed at the 3M Environmental Laboratory. Control matrices used in urine analyses were obtained from Lampire Biological Laboratories, Inc., and are presented in Appendix A. Chemical Characterization of the Test Article Potassium Perfluorooctanesulfonate (KPFOS) CAS Number: 2795-39-3 Chemical Formula: C8F17S 03'K+ Molecular Weight: 538.0 Chemical characterization information on potassium perfluorooctanesulfonate, CAS Number 2795-39-3, used in this study is presented in Table 3. Table 3. Characterization of Test Article in Study FACT TOX-111 Chemical Name Source Test Article KPFOS Potassium Perfluorooctanesulfonate 3M Toxicology Services Expiration Date Storage Conditions Chemical Lot # 31 August 2001 Ambient until 16 May 2000, then frozen -10 C 217 Physical Description White Crystalline Powder Purity 86.9% 3M Environmental Laboratory Page 13 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 See the Certificate of Analysis in Appendix H for complete characterization information. Preliminary characterization had been performed by NMR only, showing a purity of 99.28%, which was initially used for all calculations. A more complete characterization was performed later, which determined the purity of 86.9%. P rocurem ent Potassium perfluorooctanesulfonate was obtained in one lot (Lot number 217) from 3M Toxicology Services. Stability Studies The stability of potassium perfluorooctanesulfonate was not determined. Dose Confirm ation Analyses Dose confirmation analyses were performed on test article samples (0.00, 0.02, and 0.32 mg/mL) collected on 22 November 1998 and 07 January 1999, during the in-life phase of the study. The results are presented in Appendix A. The dose confirmation data were collected according to a method that was not fully validated. Dose confirmation was performed by diluting the KPFOS dose samples with methanol into the linear range of the instrument used for analysis. Samples were analyzed versus an unextracted 1/x-weighted calibration curve using HPLC-ES/MS/MS. 3M Environmental Laboratory Page 14 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 Method Summaries Following is a brief description of the methods used during this analytical study by the 3M Environmental Laboratory and by contract laboratories. The methods used in this study are located in Appendices C and G. The methods and analytical equipment settings used by Battelle Memorial Institute, Advanced Bioanalytical Services, Inc. and Centre Analytical Laboratories, Inc. are presented in the respective contract laboratory reports (see Appendix G). 3M Environmental Laboratory Preparatory Method ETS-8-96.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Urine for Analysis Using HPLC-Electrospray/Mass Spectrometry/Mass Spectrometry" This method is used to extract PFOS from urine by the use of an ion-pairing reagent and methyl-ferf-butyl ether (MtBE). An ion-pairing reagent (tetrabutyl ammonium hydrogen sulfate) is added to 2 mL of the sample, and the analyte-ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 0.5 mL of methanol, then filtered through a 0.2 pm nylon filter attached to a 3-mL plastic syringe, into glass autovials. Modification to ETS-8-96.0: Although the rat urine used to generate method validation data came from the same type of rat as the test animals, the validation rats and test animals were obtained from different suppliers. Upon analysis of urine extracts from the test animals, it was determined that an interferent, not present in the validation samples, was present in the urine extract of the test animals. The unidentified interferent proved problematic only in the analysis of the surrogate (THPFOS). As written, ETS-8-97.0 describes the quantitation of PFOS with respect to a surrogate. Due to the presence of the interferent in the surrogate analysis of the test animals, all quantitations for this study were conducted by external calibration (i.e., the surrogate was not used in any calculations). Analytical Methods ETS-8-97.0, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrospray/Mass Spectrometry/Mass Spectrometry" The analyses were performed by monitoring one or more product ions selected from a single primary ion characteristic of a particular fluorochemical, using HPLC-ES/MS/MS. For example, molecular ion 499 (C8F17S03-), selected as the primary ion for PFOS analysis, was fragmented further to produce ion 99 (FS03-). The characteristic product ion 99 was monitored for quantitative analysis. 3M Environmental Laboratory Page 15 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" The analyses were performed by monitoring the perfluorooctanesulfonate (PFOS) parent anion, m/z=499, and a daughter ion, m/z=99, to verify the presence of PFOS in the samples using a tandem mass spectrometer. Battelle Preparatory and Analytical Method: Method for Analysis of Potassium Perfluorooctanesulfonate (PFOS) in Rat Liver by LC/MS/MS ABS Preparatory and Analytical Method: Method Validation for the Quantitation of Perfluorooctanesulfonate (PFOS) In Rat Serum by Turbo Ion Spray LC/MS Centre Preparatory and Analytical Method: 00M-023-003 (Revision 2), Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS Analytical Equipment The actual analytical equipment settings used in the analysis of urine samples varied slightly during actual data collection. The following is representative of the settings used during the analytical phase of this study by the 3M Environmental Laboratory. Liquid Chromatograph: Hewlett-Packard Series 1100 Liquid Chromatograph system Analytical column: Keystone BetasilTM C182x50 mm (5 pm) Column temperature: Ambient Mobile phase components: Component A: 2 mM ammonium acetate Component B: methanol Flow rate: 300 pL/min Injection volume: 10 pL Solvent Gradient: Time (min) 0.00 1.00 5.50 7.50 8.00 %B 10.0 10.0 95.0 95.0 10.0 Mass Spectrometer: Micromass API/Mass Spectrometer Quattro IITMTriple Quadrupole system Software: Mass LynxTM 3.3 Cone Voltage: 60 V Collision Gas Energy: 40-45 eV Mode: Electrospray Negative Source Block Temperature: 150C 10C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (MRM) 3M Environmental Laboratory Page 16 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Table 4. Negative Ions Monitored in 3M Laboratory Analyses for Study FACT TOX-111 Target Analyte Primary Ion (a m u ) Product Ion (a m u ) PFOS THPFOS* 499.0 427.0 99.0 80.0 * THPFOS is a surrogate. D e v iatio n s It should be noted that as the analytical phase of this study progressed, method parameters were evaluated to improve analyses. Although the methods were validated using internal calibration, it was necessary to use external calibration for quantification in the study, due to an unknown contaminant that interfered with the surrogate response. Deviations from the original protocol and methods are documented in Appendix B. Data Quality Objectives and Data Integrity The following data quality objectives (DQOs) for 3M Environmental Laboratory assays were indicated in the protocol for this study: Linearity: The coefficient of determination (R2) > 0.980. Limits of Quantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibration curve. Acceptable Precision: Precision is within 30% for the method. Acceptable Spike Recoveries: 50-150% Demonstration of Specificity: Specificity will be demonstrated by chromatographic retention time and mass spectral daughter ion characterization. Data Summary, Analyses, and Results Data quality objectives for the analytical phase of this study outlined in the 3M Environmental Laboratory protocol for FACT TOX-111 (see Appendix B) were met with the exceptions noted in this report. Summary of Quality Control Analyses Results Linearity: The coefficient of determination (R2) of the standard curve was >0.980. Calibration Standards: Quantitation of the target analytes was based on linear regression analysis (1/x-weighted) of two extracted matrix curves bracketing each group of samples, except as noted in the deviation summary. High or low points on the curve may have been 3M Environmental Laboratory Page 17 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 deactivated to provide a better linear fit over the curve range most appropriate to the data. Occasionally, a single mid-range curve point that was an obvious outlier may have been deactivated. Quantitation of the analyte was based on the response of one specific product ion using the multiple response-monitoring mode of the instrument (see Appendix C). Limits of Quantitation (LOQ): For urine, the LOQ is 0.00295 pg/g. It is equal to the lowest acceptable standard in the calibration curve, defined as a standard within 30% of the theoretical value (see Appendix D). Blanks: All blanks were below the lower limit of quantitation for the compounds of interest. Precision: Precision was not determined for this study. Matrix Spike Recoveries: Matrix spikes and matrix spike duplicates were extracted with each set of samples and analyzed during analytical runs at the 3M Environmental Laboratory. Acceptable spike recoveries of 52 to 96% of expected values were achieved for all matrix spikes prepared in urine. Surrogates: The surrogate (THPFOS) was added to all urine samples and standards. External calibration was used to quantify urine values, because an unidentified compound interfered with the analysis of the surrogate in the samples. Statem ent of Data Quality It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data are quantitative to 50% or greater. Refer to Appendix G for contract laboratory summaries of quality control analysis results. Summary of Sam ple Results Following is information regarding sample analyses at each of the analytical laboratories involved In the present study: Battelle Memorial Institute: Refer to the Battelle Final Report for information regarding liver analysis (Appendix G). For liver analysis data that has been corrected with the Certificate of Analysis PFOS purity factor, see Appendix E. Advanced Bioanalytical Services, Inc.: Refer to the ABS Bioanalytical Report and the ABS Addendum to the Bioanalytical Report for information regarding serum analysis (Appendix G). For serum analysis data that has been corrected with the Certificate of Analysis PFOS purity factor, see Appendix E. Centre Analytical Laboratories, Inc.: Refer to the Centre Analytical Report for information regarding feces analysis (Appendix G). Feces analysis data is also presented in Appendix E. Samples from Control Animals: Low levels of PFOS were detected in the urine of some control animals. These levels were significantly lower than those found in the low dose test animals. 3M Environmental Laboratory Page 18 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 Samples from Dosed Animals: In general, PFOS levels found In the urine of the FO Generation animals increased with dose group. Detailed sample data tables are presented in Appendices D and E. Statistical Methods and Calculations Statistical methods were limited to the calculation of means and standard deviations. See Appendix F for example calculations used to generate the urine sample data in FACT TOX-111. Statement of Conclusion Under the conditions of the present study, perfluorooctanesulfonate was observed in all sample types of all Generation 0 test system animals dosed with the test substance during the in-life phase of the study, and in all sample types of their offspring (Generation F1). 3M Environmental Laboratory Page 19 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT T O X -111 LRN-U2994 Appendix A: Control Matrices and Dose Confirmation Analyses Table 5. Characterization of the Control Matrices Used for Sera, Liver, Urine, and Feces Analyses in Study FACT TOX-111 Location of Analyses 3M Environmental Laboratory Battelle Memorial Institute Advanced Bioanalytical Services, Inc Centre Analytical Laboratories, Inc. Control Matrix Human Urine Rat and/or Rabbit Livers Rat Serum Rat Feces Source Expiration Date Storage Conditions Chemical Lot # Physical Description Lampire Biological Laboratories, Inc. 01 August 2008 Frozen 66-11691 Urine Argus Research and/or Sigma N/A Frozen a t-20 10 C N/A Liver Harlan Bioproducts for Science, ine.. N/A Frozen R80128 Frozen Liquid Lampire Biological Laboratories, Inc. N/R Frozen at < -1 0 C N/R Rat feces N/A=Not Available N/R=Not Recorded Table 6. Characterization of the Analytical Reference Substances Used for Liver, Urine, Sera, and Feces Analyses in Study FACT TOX-111 Location Substance Source Expiration Date Storage Conditions Chemical Lot Number Physical Description Purity 3M Environmental Laboratory KPFOS Potassium Perfluorooctanesulfonate 3M Toxicology Services 31 August 2001 Ambient 171 White Crystalline Powder 86.4% Battelle Memorial Institute KPFOS Potassium Perfluorooctanesulfonate 3M Toxicology Services Advanced Bioanalytical Services, Inc KPFOS Potassium Perfluorooctanesulfonate 3M Toxicology Services Centre Analytical Laboratories, Inc. KPFOS Potassium Perfluorooctanesulfonate 3M Toxicology Services 31 August 2001 31 August 2001 31 August 2001 Ambient Ambient Ambient 171 171 217 White Crystalline Powder White Crystalline Powder White Crystalline Powder 86.4% 86.4% 86.9% 3M Environmental Laboratory Page 20 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 Table 7. Dose Confirmation Analyses for Perfluorooctanesulfonate for Test Samples from In-Life Study #418-015 Sample Number Group I Control 0.0 mg/kg/day Group II 0.1 mg/kg/day 0.02 mg/mL Group III 1.6 mg/kg/day 0.32 mg/mL B-418-015-A (11/22/98) Diluted 1/1 B-418-015-A (1/7/99) Diluted 1/1 B-418-015-B (11/22/98) Diluted 1/40 B-418-015-B (1/7/99) Diluted 1/40 B-418-015-C (11/22/98) Diluted 1/808 B-418-015-C(1/7/99) Diluted 1/808 Limit of Quantitation Limit (LOQ) = 0.030 pg/mL Target Cone. PFOS (pg/mL) Expected Cone. PFOS (pg/mL) Raw Measured Cone. PFOS Corrected Measured Cone. PFOS PFOS % Recovery (pg/mL) (pg/mL) Accuracy 0.00 0.00 0.00 0.00 <LOD 0.00 0.00 0.00 0.00 <LOD 20.0 16.1 0.543 17.4 108% 20.0 16.1 0.477 15.3 95% 320 258 0.441 285 111% 320 258 0.358 232 90% Formula used to calculate Expected Concentration from Target Concentration: Target Concentration x PFOS Purity (Test Article) x PFOS Correction Factor = Expected Concentration PFOS PFOS Purity (Test Article, Lot 217) = 86.9% PFOS Correction Factor = 0.9275 320 pg/mL x 0.869 x 0.9275 = 258 pg/mL Expected Concentration Formula used to calculate Corrected Measured Concentration of PFOS: Raw Measured Concentration x Dilution Factor x PFOS Purity (Reference Substance) x PFOS Correction Factor = Corrected Measured Concentration of PFOS PFOS Purity (Reference Substance, Lot 171) = 86.4% PFOS Correction Factor = 0.9275 0.358 pg/mL x 808 x 0.864 x 0.9275 = 232 pg/mL Corrected Measured Concentration of PFOS 3M Environmental Laboratory Page 21 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -1 11 LRN-U2994 Appendix B: Protocol, Protocol Amendments and Deviation Summary Table 8. Deviation Summary for FACT TOX-111 Deviation Date(s) of Occurrence Impact on Study Additional control matrices were used: human urine and rat feces. ETS-8-97.0 specifies the method of analysis requires the use of an internal calibration. The actual analyses used an external calibration method. The analysis of the dose samples was not conducted according to GLP regulations. ETS-8-5.1, a method validated for the analysis of sera extracts, was followed for the analysis of the Tween dose samples. Entire Study 06 October 1999 to 04 November 1999 21 September 1999 No negative impact on the study--A successful cross-validation was completed. Unknown contaminant interfered with use of surrogate for internal calibration. Corrective action is an improvement. Data quality is satisfactory to 50% accuracy. The analysis of Tween samples was not conducted under GLPs (as per discussions with study director). This deficiency is included in the final report. 3M Environmental Laboratory Page 22 3M Medical Department Study: T-6395.14 3M Environmental Technology and Services PO Box 33331 St. Paul, MN 55133-3331 612 778 6442 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Protocol UFAC T-TO X-111 3M Study Title Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats PROTOCOL Author Lisa Clemen Date: June 8,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification FACT-TOX-111 U2994 3M Environmental Laboratory 3M Environmental Laboratory Page 1 o f 10 Page 23 BACK TO MAIN 3M Medical Department Study: T-6395.14 ' , : Analytical Report: FACT T O X -111 LRN-U2994 Protocol #FACT-TOX-111 Study Identification Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats Test Material Sponsor Sponsor Representative Study Director Study Location(s) In vivo Testing Facility Analytical Testing Laboratory Perfluorooctane sulfonic acid potassium salt (T-6295) 3M Toxicology Services - Medical Department 3M Center, Building 22-2E-02 St. Paul, MN 55144-1000 Marvin T. Case, D.V.M.,Ph.D. 3M Toxicology Services Telephone: 651-733-5180 Facsimile: 651-733-1773 Kristen X. Hansen, Ph.D. 3M Environmental Technology and Safety Services Building 2-3E-09 651-778-6018 Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, PA 19044 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 3M Environmental Laboratory 3M Environmental Laboratory Page 2 of 10 Page 24 BACK TO MAIN 3M Medical Department Study: T-6395.14 : ,; '' Analytical Report: FACT T O X -111 ' LRN-U2994 Sub-Contract Laboratories Proposed Study Timetable Study Initiation Date Study Completion Date Protocol #FACT-TOX-111 Advanced Bioanalytical Services, Inc. 15 Catherwood Road Ithaca, NY 14850 Battelle Memorial Institute 505 King Avenue Columbus Ohio 43201-2693 June 8,1999 August 8,2000 1. Study Oral (gavage) pharmacokinetic recovery study of potassium perfluorooctane sulfonic acid (PFOS) in rats., 2. Purpose This analytical study is designed to determine levels of potassium perfluorooctanesulfonate (PFOS) in specimens of liver and serum of rats. The in-life portion o f this study was conducted at Argus Research Laboratories, study #418-015. All serum samples will be extracted and analyzed at Advanced Bioanalytical Services, Inc. and all liver samples extracted and analyzed at Battelle Memorial Institute. Additional analyses may be performed at the 3M Environmental Laboratory as methods are developed and validated.. If additional analyses are performed an amendment to this protocol will be written. 3. Regulatory Compliance This study will be conducted in accordance with the United States Food and Drag Administration, Good Laboratory Practices Standards, Final Rule 21 CFR 58, with the exception that analysis o f the test material mixture for concentration, solubility, homogeneity, and stability will not be conducted, and is the responsibility of the Sponsor. 4. Quality Assurance The 3M Environmental Laboratory Quality Assurance Unit will review the protocol and audit study conduct, data, and final report to determine compliance with Good Laboratory Practice Standards and with 3M Environmental Laboratory Standard Operating Procedures. The QA Unit at the sub-contract laboratory will audit their study conduct, data, and results report prior to submitting to the 3M Environmental Laboratory. 3M Environmental Laboratory 3M Environmental Laboratory Page 3 of 10 Page 25 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Protocol #FACT-TOX-111 5. Test Material 5.1 Refer to Argus Research Laboratory protocol for study #418-015. 6. Control Matrices 6.1 Identification Rat liver and serum, and/or rabbit liver and serum traceability numbers will be recorded in the raw data and included in the final report 6.2 Source Argus Research and/or Sigma Chemical 6.3 Physical Description Rat liver and serum, and/or rabbit liver and serum 6.4 Purity and Stability Not applicable 6.5 Storage Conditions Frozen at -20 C 10 C or -50 C 10 C 6.6 Reserve Matrix A portion of the control matrix will be retained in the 3M archives for as long as the quality of the preparation affords evaluation, but not longer than ten years following the effective date o f file final test rule (if applicable). 6.7 Disposition Matrices will be retained at the 3M Environmental Laboratory per GLP regulation. Certain matrices (feces, urine, and blood) may be disposed after QAU verification. 6.8 Safety Precautions Refer to MSDS for chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. 7. Reference Material 7.1 Identification Potassium perfluorooctanesulfonate (PFOS), lot #s 171,215, or 217 (equivalent lots) 7.2 Source 3M Specialty Chemicals 7.3 Physical Description White powder 7.4 Purity and Stability Purity of PFOS is 99% or greater. Stability has not been determined. 7.5 Storage Conditions Room temperature 7.6 Reserve Material A reserve sample from each batch o f PFOS used in this study will be retained as long as the quality of the preparation affords evaluation, but not longer than ten years following the effective date of the final test rule (if applicable). 7.7 Disposition Unused reference material will be retained for use by the 3M Environmental Laboratory and will be discarded when the quality of preparation no longer affords evaluation. 3M Environmental Laboratory 3M Environmental Laboratory Page 4 of 10 Page 26 BACK TO MAIN 3M Medical Department Study: T-6395.14 ; .; !' Analytical Report: FACT T O X -111 LRN-U2994 Protocol ttFACT-TOX-111 7.8 Safety Precautions Refer to MSDS for chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. . 8. Test System Rats were used as the test system and were maintained and dosed as described in Argus Research protocol #418-015. The female rats will be given the test material or control once daily beginning 42 days prior to cohabitation until day 0 of presumed gestation. See table 1 for more dosage information. . Dosage Group 1 2 3 Table 1 Dosage Levels, Concentration, and Volumes Number of female rats 8 8 8 Dosage mg/kg/day 0 0.1 --. Concentration mg/kg/day 0 0.02 0.32 . Dosage volume mL/kg 5 5 5 9. Specimen and Sample Receipt The 3M Environmental Laboratoiy will receive homogeneity samples and specimens o f the following body tissues and fluids from the indicated points in the study. All specimens will be packed on dry ice for shipping. See table 2 for more specimen information. Body tissue/fluid Table 2 Specimen Information Collected Serum - Dam and Pup animals Urine and Feces - Dam and Pup animals Liver - Dam and Pup animals Dam-Predose, Days 7,14,15,21, and 22 Pup-Day 21 Predose, Days 7,15,21, and 22 Dam-At the termination o f the study Pup-Day21 Expected# of specimens 144 Dam 24 Pup (pooled) 120 Urine and 120 Feces 24 Dam 24 Pup (pooled) Total number of expected specimens: 456 Total number of test animals: 16 Total number of control animals: 8 3M Environmental Laboratory 3M Environmental Laboratory Page 5 of 10 Page 27 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Protocol FACT-T0X-111 Specimens sent to 3M Environmental Laboratories will be received and tracked according to applicable Standard Operating Procedures. 10. Preparatory Methods 10.1 FACT-M -l.l, Extraction o f Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 10.2 ETS-8-4.1, Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC- , Electrospray/Mass Spectrometry 10.3 If preparatory methods other than those listed above are used, an amendment to this protocol will be written. Any deviations from these methods will be documented and included with the study data. 10.4 If analyses ar sub-contracted to other laboratories, an amendment will be written to include their methods and copies of each method will be attached to this protocol. 11. A nalytical Methods 11.1 FACT-M-2.1, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry 11.2 ETS-8-5.1, Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry 11.3 If analytical methods other than those listed above are used, an amendment to this . protocol will be written. Any deviations from these methods will be documented and included with the study data. 11.4 If analyses are sub-contracted to other laboratories, an amendment will be written to include their methods and copies o f each method will be attached to this protocol. 12. Data Quality Objectives The number of spikes/duplicates, use of surrogates, and information on other data quality indicators are included in the analytical methods. In addition, the following criteria will be met: 12.1 Linearity ri> 0.98 12.2 Limits of detection / quantitation 12.2.1 Method Detection Limit (MDL) for PFOS a) Serum: 1.75 ppb b) Liver: 15 ppb 3M Environmental Laboratory 3M Environmental Laboratory Page 6 of 10 Page 28 BACK TO MAIN 3M Medical Department Study: T-6395.14 ' :: ' : Analytical Report: FACT T O X -111 LRN-U2994 Protocol XFACT-TOX-111 12.2.2 Limit o f Quantitation (LOQ) - Equal to the lowest acceptable standard in the calibration curve 12.3 Duplicate acceptable precision < 30% for the method 12.4 Spike acceptable recoveries 70% -130% 12.5 Use of confirmatory methods Indeterminate samples will be re-analyzed using a confirmatory method. I f a confirmatory method is used, an amendment to this protocol will be written. 12.6 Demonstration of specificity Chromatographic retention time, mass spectral daughter ion characterization. 13. Sub-Contracted A nalysis 13.1 All analyses as detailed in this protocol will be performed at 3M Environmental Laboratories, Building 2-3E-09, 935 Bush Avenue, St. Paul, MN 55106, at Advanced Bioanalytical Services, Inc., 15 Catherwood Road, Ithaca, NY 14850, or at Battelle Memorial Institute, 505 King Avenue, Columbus, Ohio 43201-2693. 13.2 An amendment to this protocol will be written if analyses are performed at laboratories other than the 3M Environmental Laboratories, Advanced Bioanalytical Services, Inc., or Battelle Memorial Institute. . 14. Statistical Analysis Averages and standard deviations will be calculated. The statistical methods that will be used are described below: 14.1 Data transformations and analysis Data will be.reported as the concentration (weight/weight or weight/vol) o f PFOS or metabolite per tissue or fluid. 14.2 Statistical analysis Statistics used may include regression analysis o f concentrations over time, and standard deviations calculated for the concentrations within each dose group. If necessary, simple statistical tests, such as Student's t test, may be applied to evaluate statistical difference. 15. Report A report containing all the results of the study will be prepared by the 3M Environmental Laboratory. If analyses are sub-contracted to other laboratories, each laboratory will prepare a report and submit it to the 3M Environmental Laboratory for inclusion in the 3M Environmental Laboratory report. Each report will include, but not be limited to, the following, when applicable: 15.1 Name and address of the facility performing the study 15.2 Dates upon which the study was initiated and completed 3M Environmental Laboratory 3M Environmental Laboratory Page 7 o f 10 Page 29 BACK TO MAIN 3M Medical Department Study: T-6395.14 ., Analytical Report: FACT T O X -111 LRN-U2994 Protocol #FACT-TOX-111 15.3 A statement of compliance by the Study Director addressing any exceptions to Good Laboratory Practice Standards 15.4 Objectives and procedures as stated in the approved protocol, including any changes in the original protocol 15.5 The test substance identification by name, chemical abstracts number or code number, strength, purity, and composition or other appropriate characteristics, if provided by the Sponsor 15.6 Stability and the solubility o f the test substances under the conditions of administration, if provided by the Sponsor 15.7 A description o f the methods used to conduct the test(s) 15.8 A description o f the test system 15.9 A description o f any circumstances that may have affected the quality or the integrity o f the data 15.10 The name o f the Study Director and the names of other scientists, professionals, and supervisory personnel involved in the study 15.11 A description o f the transformations, calculations, or operations performed on the data, a summary and analysis of the analytical chemistry data, and a statement of the conclusions drawn from the analyses 15.12 Statistical methods used to evaluate the data, if applicable 15.13 The signed and dated reports of each o f the individual scientists or other professionals involved in the study, if applicable 15.14 The location where raw data and the final report are to be stored 15.15 A statement prepared by the Quality Assurance Unit listing the dates that study inspections and audits were made, and the dates of any findings reported to the Study Director and Management If it is necessary to make corrections or additions to a report after it has been accepted, the changes will be made in the form of an amendment issued by the Study Director. The amendment will clearly identify the part of the report that is being amended, the reasons for the amendment, and will be signed by the Study Director. 16. Location of Ra w Data, Records, and Final Report Original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits o f the study during its progress and before acceptance o f the final report. When the final report is completed, all original paper data, including those items listed below, will be retained in the archives of 3M Environmental Laboratory for at least a period of time as specified by regulation, and as established by 3M Environmental Laboratory Standard Operating Procedures. 3M Environmental Laboratory 3M Environmental Laboratory Page 8 o f 10 Page 30 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Protocol #FACT-TOX-111 16.1 The following raw data and records will be retained in the study folder in the study/project archives according to 3M Environmental Laboratory Standard Operating Procedures: 16.1.1 Approved protocol and amendments 16.1.2 Study correspondence 16.1.3 Shipping records 16.1.4 Raw data 16.1.5 Approved final report (original signed copy) 16.1.6 Electronic copies o f data 16.2 The following supporting records will be retained separately from the study folder in the archives according to 3M Environmental Laboratory Standard Operating Procedures: 16.2.1 Training records 16.2.2 Calibration records 16.2.3 Instrument maintenance logs 16.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 17. Specimen Retention Specimens will be maintained in the 3M Environmental Laboratory specimen archives for a period of time as specified by regulation or as long as the quality o f the preparation affords evaluation, but not longer than ten years following the effective date o f the final test rule (if applicable), and as established by 3M Environmental Laboratory Standard Operating Procedures. 18. Protocol A mendments and deviations Planned changes to the protocol will be in the form o f written amendments signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part of the protocol and will be attached to the final protocol. All changes to the protocol will be indicated in the final report. Any other changes will be in the form o f written deviations, signed by the Study Director and filed with the raw data. 19. A ttachments 19.1 Attachment A Preparatory and analytical methods 3M Environmental Laboratory 3M Environmental Laboratory Page 9 of 10 Page 31 BACK TO MAIN 3M Medical Department Study: T-6395.14 20. Signatures { j j y$AAtf~ir. J C.ylaJ--- Marvin T. Case, D.V.M., Ph.D., Sponsor Representative Analytical Report: FACT TOX-111 L R N -U 2 9 9 4 Protocol UFACT-TOX-111 *7 9/ ? .? Date ______________ _ Kristen J. Hansen, Ph.D., 3M Environmental Laboratory Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 10 o f 10 Page 32 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Study Title Oral (Gavage) Pharmacokinetic Recovery Study o f PFOS in Rats PROTOCOL AMENDMENT NO. 1 Amendment Date: August 12,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue . St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX111 LIRNU2994 3M Environmental Laboratory 3M Environmental Laboratory Page 33 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Protoco! FACT-TOX111 Amendment No. 1 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL reads: Section 10.0 and 11.0 list the following methods to use for extraction and analysis: FACT-M-1.1 "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic FluoroChemical Surfactant from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" FACT-M-2.1 "Analysis o f Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" AMEND TOread: The extraction and analytical methods FACT-M-1.1 and FACT-M-2.1, respectively, were updated on 07/22/99 to: ETS-8-6.0 "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" REASON: The extraction and analytical methods FACT-M-1.1 and FACT-M-2.1, respectively, were updated on 07/22/99 to ETS-8-6.0 and ETS-8-7.0. These methods were updated to replace the extraction solvent ethyl acetate with a different extraction solvent MTBE (methyl tert butyl ether), POAA and Monoester were removed from the standard mix, and M556 was added to the standard mix. The analytical method was updated to include linear regression with 1/x weighting and a few minor changes in the HPLC 1100 instrument parameters. 2. PROTOCOL reads: Section 10.4 and 11.4 state that if die analyses are sub-contracted to other laboratories an amendment will be written to include these methods. AMEND to read: The extraction and analytical methods to follow at Advanced Bioanalytical Services will be attached to the protocol. The extraction and analytical method to follow at Battelle Memorial Institute is: "Method for Analysis o f Perfluorooctane Sulfonate (PFOS) in Rat Sera-by LC/MS/MS, R e a s o n : The analytical methods at the sub-contract laboratories were not included in the original protocol. 3M Environmental Laboratory 3M Environmental Laboratory Page 34 BACK TO MAIN 3M Medical Department Study: T-6395.14 ,, Analytical Report: FACT T O X -111 LRN-U2994 Protocol FACT-TOX111 Amendment No. 1 3. PROTOCOL reads: Section 12.2.1 b) lists liver method detection limit as 15 ppb. AMEND TOread: The liver method detection limit is 8.50 ppb (ng/g). REASON: The validation supporting methods ETS-8-6.0 and ETS-8-7.0 included a lower method detection limit for PFOS. Amendment Approval t>-' arvin Case, D.V.M., Ph.D., Sponsor Representative ntA.u.i-j-!%9 ^ Date Kris J. Hansen, Ph.D., Study Director jlM is . Date ' 3M Environmental Laboratory 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Study Title Oral (Gavage) Pharmacokinetic Recovery Study o f PFOS in Rats PROTOCOL AMENDMENT NO. 2 Amendment Date: September 30, 1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX111 LIRNU2994 3M Environmental Laboratory 3MiEnvironmental Laboratory Page 36 BACK TO MAIN 3M Mediqal Department Study: T -6395.14 .; ' Analytical Report: FACT T O X -111 LRN-U2994 Protocol FACT-TOX111 Amendment No. 2 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: . Section 2 states the study is designed to determine levels of potassium perfluorooctanesulfonate (PFOS) in specimens of liver and sera in rats. Amend to read: The study is designed to determine levels o f potassium perfluorooctanesulfonate (PFOS) in specimens o f liver, sera, and urine in rats. . Reason: The urine analytical methods were validated and approved after approval o f the original protocol. 2. Protocol reads: Section 10.0 and 11.0 list the following methods to use for extraction and analysis: ETS-8-4.1 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-6.0 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-5.1 "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" Amend to read:: ETS-8-4.1 "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-6.0 "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-96.0 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Urine for Analysis Using HPLC-Electrospray/Mass Spectrometry/Mass Spectrometry" ETS-8-5.1 "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-97.0 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrospray/Mass Spectrometry/Mass Spectrometry" REASON: The extraction and analytical methods ETS-8-96.0 and ETS-8-97.0, were approved after approval o f the original protocol. . 3M Environmental Laboratory 3M nvironmental Laboratory Page 37 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Protocol FACT-TOX111 Amendment No. 2 3. Protocol reads: Section 6.1 lists control matrices as rat liver and serum or rabbit liver and serum. Source Argus and/or Sigma Chemical. Amend to read: Control matrices identification rat liver and serum, rabbit liver and serum, human urine and/or rat urine. Source Argus, Sigma Chemical, Lampire Biologicals, Biological Specialty Corp. and/or Golden West Biologicals. Reason: Addition o f control urine specifications. . 4. Protocol reads: Section 12.2.1 a) lists sera method detection limit as 1.75 ppb and b) lists liver method detection limit as 15 ppb. Amend to read: The method detection limits for all compounds and matrices will be taken from the methods used for extraction and analysis. Reason: The method detection limits listed are specific to the 3M Environmental Laboratory. Statement was added to allow for sub-contracted analyses and/or revised methods. 3M Environmental Laboratory Page 38 BACK TO MAIN 3M Medlcpl Department Study: T-6395.14 ' ;, Analytical Report: FACT T O X -111 LRN-U2994 Protoco/ FACT-TOX111 Amendment No. 2 5. Protocol reads: Section 16 states that the original data, or copies thereof, will be available at.the 3M Environmental Laboratory to facilitate audits o f the study during its progress and before acceptance o f the final report. When the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, electronic copies of data, training records, calibration records, instrument maintenance logs and standard operating procedures, equipment procedures, and methods will be retained in the archives of the 3M Environmental Laboratory. Amend to read: Section 16 states that the original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits o f the study during its progress and before acceptance o f the final report. When the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, and electronic copies o f data will be retained in the archives of the 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained in the archives o f the facility performing each analysis. Reason: Clarification o f the disposition of archived records if analyses are performed at a sub contract laboratory. 6. Protocol reads: Section 17 states that specimens will be maintained in the 3M Environmental Laboratory specimen archives. Amend to read: Specimens will be maintained in the 3M Environmental Laboratory specimen archives. All specimens sent to sub-contract laboratories will be returned to th 3M Environmental Laboratory upon completion o f analysis and submission of the sub-contract laboratory(s) final report. The specimens will be returned with the following documentation: the signed original chain o f custody and records o f storage conditions while at the sub-contract facility. Reason: Clarification o f the disposition o f specimens and documentation for analyses performed by a sub-contract laboratory. 3M Environmental Laboratory Page 39 BACK TO MAIN 3M Medical Department Study: T-6395.14 ., Amendment Approval Analytical Report: FACT T O X -111 ........... LRN-U2994 Protocol FACT-TOX111 Amendment No. 2 Marvin Case, D.V.M., Ph.D., Sponsor Representative Kristen J. Hansen, Ph.D., Study Director D ate' S ct D ate ? 3M Environmental Laboratory 3IVTEnvironmental Laboratory Page 40 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Study Tide Analytical Laboratory Report on the Determination of the Presence and Concentration of Potassium Perfluorooctanesulfonate (CAS Number 2759-39-3) in the Serum, Liver, and Urine of Crl:CDBR VAF/Plus Rats Exposed to PFOS Via Gavage PROTOCOL AMENDMENT NO. 3 Amendment Date: 20 January 2000 Performing Laboratories . Urine Analyses 3M Environmental Technology and Safety Services Fluorine Analytical Chemistry Team Building 2-3E-09 935 Bush Avenue St. Paul, M N 55106 Performing Laboratories Liver Analyses Battelle Memorial Institute 505 King Avenue Columbus, OH 43201-2693 Serum Analyses Advanced Bioanalytical Services, Inc. 15 Catherwood Road Ithaca, N Y 14850 Laboratory Project identification ET&SS LRN-U2994 FACT TOX-111 Argus Study: 418-015 3M Medical Department Study: T-6295.14 3 M Environmental Laboratory 3M Environmental Laboratory Page 41 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Protocol LRN-U2994 Amendment Number 3 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: The study director for the present study was identified in the protocol as Kristen J. Hansen, Ph.D. A mend to read: The role of study director for the present study was reassigned to Marvin T. Case, D.V.M., Ph.D., as of 20 January 2000. The previous study director, Kristen J. Hansen, has been reassigned to the role of Principle Analytical Investigator. . Reason: The role of study director was reassigned in an effort to ensure compliance with Good Laboratory Practice Standards that outline study personnel requirements (refer to 21 CFR Part 58). 2. Protocol reads: The sponsor for the present study was identified as Marvin T. Case, D.V.M., Ph.D. Amend to read: The role of sponsor for the present study was reassigned to John L. Butenhoff, Ph.D., as of 20 January 2000. Reason: To ensure that the study director does not also carry the duties of study sponsor, the sponsor role was reassigned, in this manner, personnel responsibilities and workload are more evenly balanced. 3M Environmental Laboratory 3M Environmental Laboratory Page 42 3M Medicpl Department Study: T-6395.14 Amendment Approval John L. Butenhoff, PhD., Sponsor Representative BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Protocol LRN-U2994 Amendment Number 3 z.><s>c> Date -i4 n ---------Kristen J. Hansen, PhD., Outgoing Study Director / / - <3uA ) - Z70 Date i-------------------------------------- /A Marvin T. Case, D. V.M., PhD,, Incoming Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 43 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C TT O X -111 L R N -U 2 9 9 4 Study Title Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats PROTOCOL AMENDMENT NO. 4 Amendment Date: 20 April 2000 Performing Laboratories U r in e A n a l y s e s 3M Environmental Technology and Safety Services Fluorine Analytical Chemistry Team Building 2-3E -09,935 Bush Avenue St. Paul, MN 55106 F ec e s A n aly s e s Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 L iv e r A n a l y s e s Battelle M emorial Institute 505 King Avenue Columbus, OH 43201-2693 S e r u m A n aly s e s Advanced Bioanalytical Services, Inc. 15 Catherwood Road Ithaca, N Y 14850 Laboratory Project identification ' ET&SS LRN-U2994 FACTTOX-111 Argus Study: 418-015 3M Medical Department Study: T-6295.14 3M Environmental Laboratory 3M Environmental Laboratory Page 44 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Protocol LRN-U2994 Amendment Number 4 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: The amended section 2.0 text states that this study is designed to determine PFOS in specimens of rat liver, serum, and urine. Amend to read: This study is designed to determine PFOS in specimens of rat liver, serum, feces, and urine. Reason: The analysis of fecal tissue for the target chemical and/or its analytes was added to the scope of the study following the issuance of the protocol. Feces extraction and analytical methods were not validated and approved prior to protocol approval. 2. Protocol reads: The amended section 6.0 lists rat or rabbit liver, serum, and urine. Amend to read: Add: rat or rabbit feces with a physical description of rat or rabbit feces. Reason: Analysis of fecal tissue for the target chemical and/or its analytes was added to the scope of the study following the issuance of the original protocol. 3. Protocol Reads: Section 13.1 lists all of the laboratories that will be conducting analyses for this study. Amend to read: Add: Centre Analytical Laboratories, Inc., 3048 Research Drive, State College, PA 16801 Reason: Feces analyses were added to the scope of this study. The sub-contract laboratory performing analyses was not in the original protocol. 4. Protocol Reads: Sections 10.4 and 11.4 state that if the analyses are sub-contracted to other laboratories an amendment will be written to include these methods. Amend to read: The feces extraction and analytical method used by Centre Analytical Laboratories will be; OOM-023-003 (Revision 2), "Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS." Reason: The sub-contract laboratory performing feces analyses was added to th scope of this study; this method was not validated and approved prior to protocol approval. 3M Environmental Laboratory 3M Environmental Laboratory Page 45 BACK TO MAIN 3M Medical Department Study: T-6395.14 Amendment Approval Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Protocol LRN-U2994 Amendment Number 4 John L. Butenhoff, Ph.D., Sponsor Representative ffy/*/"' / &> 5 Date fy X a u T Qsia- Marvin T. Case, D.V.M., Ph.D., Study Director J-L A/u*1_Qjyp- Date 3M Environmental Laboratory 3M Environmental Laboratory Page 46 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Study Title Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats PROTOCOL AMENDMENT NO. 5 Amendment Date: July 25,2000 Performing Laboratory 3M Environmental.Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX-111 U2994 3M Environmental Laboratory 3M Environmental Laboratory Page 47 BACK TO MAIN 3MMedicpl Department Study: T-6395.14 .. Analytical Report: FACT T O X -111 LRN-U2994 Protocol FACT TOX-111 Amendment No. 5 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: - In section 8, Table 1, the dosage level for dosage group 3 is listed as 0.6 mg/kg/day. Amend to read: Section 8, Table 1, the dosage level for dosage group 3 is 1.6 mg/kg/day. Reason: The dosage level for dosage group 3 was changed after the protocol was written. 2. Protocol reads: The amended analytical protocol (Amendment No. 1) states the extraction and analytical method to follow at Battelle Memorial Institute is: "Method for Analysis o f Perfluorooctane Sulfonate (PFOS) in Rat Sera by LC/MS/MS, Version 1" AMEND to read: The extraction and analytical method to follow at Battelle Memorial Institute is: "Method for Analysis of Perfluorooctane Sulfonate (PFOS) in Rat Liver by LC/MS/MS, Version 1.0" Reason: Liver analyses were sub-contracted to Battelle Memorial after the protocol was written; therefore the method used to analyze liver samples also changed. The Battelle method list in Amendment No. 1 contained a hand-written change, in that "sera" was crossed out and "liver" was written in. This hand-written change was dated after the study director had signed the amendment. 3. Protocol Reads: The Argus in-life protocol #418-015 lists the testing facility as Argus Research Laboratories, Inc. Amend t o Read: Change the testing facility to 3M Toxicology Services--Medical Department. Address: 3M Center, Building 220-2E-02, St. Paul, MN 55144-1000. This change is retroactive to February 10, 2000. Reason: Per GLP regulations, the testing facility must be where the study director resides. There cannot be two testing facilities. 3M Environmental Laboratory 3M Environmental Laboratory Page 48 BACK TO MAIN 3M Medical Department Study: T-6395.14 ' , Analytical Report: FACT T O X -111 LRN-U2994 Protocol FACT TOX-111 Amendment No. 5 4. Protocol Reads: The cover page of FACT-TOX-111 lists the document type as a "Protocol." On page three, under Proposed Study Timetable, the "Study Initiation Date" and the "Study Completion Date" are listed. Amend to Read: Change the document type for FACT-TOX-111 to "Analytical Phase Protocol." Change "Study Initiation Date" to "Phase Initiation Date." Change "Study Completion Date" to "Phase Completion Date." Reason: FACT-TOX-111 is the protocol for the analytical phase, while the Argus protocol #418-015 is the protocol for the in-life phase. 5. Protocol Reads: The amended analytical protocol (Amendment No. 3) states that Marvin T. Case replaces Kristen J. Hansen as the study director. Kristen J. Hansen was reassigned to the role of Principle Analytical Investigator. The Argus Research Laboratories, Inc. in-life protocol #418-015 states that the study director for the in-life phase is Raymond York. Amend to Read: Marvin T. Case replaces both Kristen J. Hansen and Raymond York as study director. . Raymond York has been reassigned to the role of Principal In-life Investigator. Kristen J. Hansen was reassigned to the role o f Principal (corrected spelling) Analytical Investigator. These changes are retroactive to February 10,2000. Reason: Per GLP regulations, only one study director is assigned to a study. Corrected spelling o f Principal. 6. Protocol Reads: The amended analytical protocol (Amendment No 4) states that rat feces will be analyzed for PFOS, as well as rat urine, liver and serum. Centre Analytical Laboratories will be conducting the feces analyses. Amend to Read: Add that the Principal Analytical Investigator (PAT) at Centre analytical Laboratories is Enaksha Wickremesirihe. Reason: The PAI for Centre was not listed in Amendment No. 4. 3M Environmental Laboratory 3M Environmental Laboratory Page 49 3M Medicpl Department Study: T-6395.14 '. BACK TO MAIN Amendment Approval Analytical Report: FACT T O X -111 LRN-U2994 Protocol FACT TOX-111 Amendment No. 5 John IButenhoff, Ph.D., Spoonnssoof-fR'k.eepprreesseentative 3 /j /t ._____ . Date Marvin T. Case, D.V.M., Ph.D., Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 50 3M Medicpl Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 j Study Title Oral (Gavage) Pharmacokinetic Study of PFOS in Rats PROTOCOL AMENDMENT NO. 6 Amendment Date: October 17,2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT TOX-111 3M Laboratory Request No. U2994 3 M Environmental Laboratory 3M Environmental Laboratory Page 51 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 ,. Analytical Report: FACT T O X -111 ..... LRN-U2994 Protocol FACT TOX-111 J Amendment #6 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: . Data Quality Objectives, Section 12.4, Spike Acceptable Recoveries are required to be 70% - 130%. . A m end to read: Spike Acceptable Recoveries are required to be 50-150%. - REASON: The analytical method and resulting QC data support a 50-150% acceptable range for spike recoveries. 3M Environmental Laboratory 3M Environmental Laboratory Page 52 BACK TO MAIN 3M Medical Department Study: T-6395.14 .; ' Analytical Report: FACT T O X -111 LRN-U2994 Protocol FACT TOX-111 J Amendment #6 Amendment Approval "T. John L. Butenhoff, PhD., Sponsor Representative Date in T. Case, D.V.M., PhD., Study Director Date 3 M Environmental Laboratory 3M Environmental Laboratory Page 53 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 Appendix C: Extraction and Analytical Methods This Appendix includes the following methods: 3M Environmental Laboratory, ETS-8-96.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Urine for Analysis using HPLC-Electrospray/Mass Spectrometry/Mass Spectrometry," (14 pages). 3M Environmental Laboratory, ETS-8-97.0, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrospray/Mass Spectrometry/Mass Spectrometry," (10 pages). 3M Environmental Laboratory, ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry," (9 pages). 3M Environmental Laboratory Page 54 BACK TO MAIN 3M Medical Department Study: T-6395.14 3M Environmental Laboratory Analytical Report: FACT T O X -111 LRN-U2994 M ethod E xtraction of P otassium Perfluorooctanesulfonate or other FLUOROCHEMICAL COMPOUNDS FROM URINE FORANALYSIS USING HPLC- Electrospray/M ass Spectrometry/M ass Spectrometry Method Number: ETS-8-96.0 Adoption Date: ^ Author: Lisa Clemen, Glenn Langeriburg Revision Date: (\Jf\ Approved By: 'OH Laborafory Manager''-' jjn-------- Group Leader A ClimrUK Technical Reviewer ifn h r Date Date -liai-ln Date 1.0 Scope and Application____________________________________________ _________ 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from urine. 1.2 Applicable compounds: Fluorochemicals or other fluorinated compounds. 1.3 Matrices: Human, rat, and monkey urine or other fluids as designated in the validation report. W ord 6/95 3M Environmental Laboratory ETS-8-96.0 Extraction o f PFOS from Urine Page 1 o f 14 Page 55 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 2.0 Summary of Method______________________________________ __________ 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemicals from urine, or other fluids, using an ion pairing reagent and methyl-ieri-butyl ether (MtBE). In this method, eight fluorochemicals are extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, M556, M570, perfluorooctanoate (POAA), and surrogate standard (see 3.0 Definitions). An ion pairing reagent is added to two ml of sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 0.5 ml of methanol, then filtered through a 0.2 pm nylon filter attached to 3 cc plastic syringe into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-97.0 or other appropriate method. 3.0 Definitions _____________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) C8F17S03' 3.2 PFOSA: perfluorooctane sulfonylamide C8F17S02NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8Fi7S02N(CH2CHj)CH2C02` 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8Fi7S 0 2N(CH2CH3)CH2CH20H 3.5 M556: C8F17S02N(H)(CH2C00H) 3.6 M570: C8F17S02N(CH3)CH2C00H 3.7 POAA: perfluorooctanoate C7FI5COO" 3.8 Surrogate standardTHPFOS: 1H-1H-2H-2H perfluorooctane sulfonic acid, used as an internal standard in this method. 4.0 Warnings and Cautions_______________ ;_____________ ;_________________________ 4.1 Health and safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 Interferences_____________ ;________________________;__________________________ 5.1 At this time, it is unknown how the extraction method is affected by potential interferences that may be present such as conjugated fluorochemicals (eg. Glucuronides). Conjugates may become deconjugated during extraction or analysis resulting in a high bias for reported results of target analytes. 3M Environmental Laboratory ETS-8-96.0 Extraction o f PFOS from Urine Page 2 o f 14 Page 56 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Rport: F A C T T O X -1 11 L R N -U 2 9 9 4 6.0 Equipment______________ :____________________________________________ _ 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR 6.1.4 Nitrogen evaporator, Organomation. 6.1.5 Balance ( 0.100 g) 7.0 Supplies and Materials______________________________________________ ~ 7.1 Gloves 7.2 Eppendorfor disposable pipettes 7.3 Nalgene bottles, capable of holding 250 ml and 1 L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 ml glass 7.6 Centrifuge tubes, polypropylene, 15 ml 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0 ml 7.9 Syringes, capable ofmeasuring 2.5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe filters, nylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli-QTMwater. Rinse glass syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards ________ ,_________________________________________ 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (Na2C03), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 3M Environmental Laboratory ETS-8-96.0 Extraction o f PFOS from Urine Page 3 o f 14 Page 57 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 L R N -U 2 9 9 4 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Onmisolv, glass distilled or HPLC grade 8.8 Urine frozen from supplier 8.9 Fluorochemical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 8.9.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.9.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.9.5 M556 (3M Specialty Chemical Division), molecular weight = 557 8.9.6 M 5 7 0 (3 M Specialty C hem ical D ivision), m olecular w eight = 571 8.9.7 POAA (3M Specialty Chemical Division), molecular weight = 452 8.9.8 THPFOS (1-H,1-H, 2-H, 2-H C8F13S03H) molecular weight = 428 8.9.9 Other fluorochemicals, as appropriate 8.10 Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.10.1 ION sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 ml beaker containing 500 ml Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.10.2 1 N sodium hydroxide (NaOH): Dilute 10N NaOH 1:10. Measure 10 ml of 10 N NaOH solution into a 100 ml volumetric flask and dilute to volume using MilliQTMwater. Store in a 125 ml Nalgene bottle. 8.10.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 ml Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 ml of 10 N NaOH (While adding the last ml of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-QTM water. Store in a 1 L Nalgene bottle. 8.10.3.1 TBA requires a check prior to each use to ensure pH = 10.0. Adjust as needed using 1N NaOH solution. 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na2C03/NaHC03): Weigh approximately 26.5 g of sodium carbonate (NajCO-j) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with MilliQTMwater. Store in a 1 L Nalgene bottle. 8.11 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 3M Environmental Laboratory ETS-8-96.0 Extraction o f PFOS from Urine Page 4 o f 14 Page 58 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 8.11.3 Weigh approximately 100 mg ofPFOS into a 100 ml volumetric flask and record the actual weight in the Standard Logbook. 8.11.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (jig/ml). 8.11.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.11.6 Dilute working standard 1 with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.11.7 Dilute working standard 1 with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.12 Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 mg of surrogate standard 1-H,1-H, 2-H, 2-H, C8F,3S03H into a 50 ml volumetric flask and record the actual weight. 8.12.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 PPm. 8.12.3 Prepare a surrogate working standard. Transfer approximately 1 ml of surrogate stock to a 10 ml volumetric flask andbring to volume with methanol for a working standard of 100-120 ppm. Record the actual volume transferred in the Standard Logbook. 9.0 Sample Handling________________________ '___________________________;_________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw to room temperature prior to extraction. 10.0 Quality Control________ _______________________________________ 10.1 Solvent Blanks, Method blanks and matrix blanks 10.1.1 An aliquot of 2.0 ml methanol is used as a solvent blank. 10.1.2 Extract two 2.0 ml aliquots of Milli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 2.0 ml aliquots of the urine following this procedure and use as matrix blanks. See 11.1.4. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually the control matrix received with each sample set. . 10.2.3 Expected concentrations should fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 3M Environmental Laboratory ETS-8-96.0 Extraction o f PFOS from Urine Page 5 o f 14 Page 59 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 10.2.4 Prepare one matrix spike andmatrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibration verification samples to ensure the accuracy of the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing calibration verification per group of 10 samples. For example, if a sample set = 34, four checks are prepared and extracted. 10.3.3 Prepare each continuing calibration verification from the same matrix used to prepare the initial curve. 10.3.4 The expected concentrations will fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb - 1000 ppb). 11.0 Calibration and Standardization____________________;_______________________ 11.1 Prepare matrix calibration standards 11.1.1 Transfer 2.0 ml of urine to a 15 ml centrifuge tube. 11.1.2 Record each sample volume on the extraction sheet. 11.1.3 While preparing atotal of twenty-two aliquots in 15 ml centrifuge tubes, mix or shake between aliquots. 11.1.4 Two 2.0 ml aliquots serve as matrix blanks. 11.1.5 Typically use the standard concentrations and spiking amounts listed in Table 1, at the end of this section, to spike, in duplicate, two standard curves, for a total of twenty standards, two matrix blanks, and two method blanks. 11.1.6 Refer to validation report FACT-TOX-131, W2067, which lists the working ranges and th Linear Calibration Range (LCR) for calibration curves. 11.1.7 Use Attachment D as an aid in calculating the concentrations of the working standards. See Section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each standard, blank, or continuing check, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 10 ppb 1500 ppb. 11.3 Extract spiked matrix standards following 12.6-12.16 of this method. Use these standards to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 6 o f 14 Page 60 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Table 1 Approximate spiking amounts for standards and spikes Using 2.0 ml of matrix Working standard pL Approx, final cone, Approx, final cone. (approx, cone.) of analyte in matrix Of analyte in solvent - - . Blank Blank 0.500 ppm 5 1.25 ppb 5.00 ppb 0.500 ppm 10 2.50 ppb 10.0 ppb 0.500 ppm 25 6.00 ppb 25.0 ppb 5.00 ppm 5 12.5 ppb 50.0 ppb 5.00 ppm 10 25.0 ppb 100.ppb 5.00 ppm 25 62.5 ppb 250 ppb 50.0 ppm 5 125 ppb 500 ppb 50.0 ppm 7.5 200 ppb 750 ppb 50.0 ppm 10 250 ppb 1000 ppb 50.0 ppm 15 375 ppb 1500 ppb 12.0 Procedure____________________________;______________________________ ^_______ 12.1 Obtain frozen samples and allow to thaw at room temperature orin a lukewarm waterbath. 12.2 Vortex mix for 15 seconds, then transfer 2.0 ml or other appropriate volume to a 15 ml polypropylene centrifuge tube. 12.3 Return unused samples to freezer after extraction amounts have been removed. 12.4 Record the initial volume on the sample weight/volume worksheet.. See Attachment D. The original weight/volume worksheet is included in the study binder. 12.5 Label the tube with the study number, sample ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike all samples, including blanks and standards, ready for extraction with surrogate standard as described in 11.2. 12.7 Spike each matrix with the appropriate amount of standard as described in 11.1, or Table 1 in that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. ` 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.10 To each sample, add 1 ml 0.5 M TBA and 2 ml of 0.25M sodium carbonate/sodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 ml methyl-ieri-butyl ether. 12.12 Cap each sample and put on the shaker at a setting o f300 rpm, for 20 minutes. 3M Environmental Laboratory ETS-8-96.0 Extraction o f PFOS from Urine Page 7 o f 14 Page 61 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 12.13 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm, or until layers are separated. 12.14 Label a fresh 15 ml centrifuge tube with the same information as in 12.5. 12.15 Remove 4.0 ml of the organic layer to this clean 15 ml centrifuge tube. 12.16 Put each sample on the analytical nitrogen evaporator until dry, approximately 30 to 60 minutes. 12.11 Add 0.5 ml of methanol to each centrifuge tube using a graduated pipette. If excessive residue is present, add the methanol and allow the extract to sit for 30 minutes prior to vortexing. 12.17 Vortex mix for 30 seconds. 12.18 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, fluorochemical components, extraction type, vial file archive number, and analyses) performing the extraction. 12.19 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 ml glass autovial or low-volume autovial when necessary. 12.20 Cap and store extracts at room temperature or refrigerated at approximately 4 C until analysis. ' 12.21 Complete the extraction worksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. 13.0 Data Analysis and Calculations_______________ ___________ ;_________________ 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS, or other applicable fluorochemical, in calibration standards using the following equation: ml of standard x concentration of standard (tie /mil _____________ _ = ml of standard + ml of surrogate standard + initial matrix volume (ml) Final Concentration (pg/ml) of PFOS in matrix 14.0 Method Performance_________________________;_____________ ;_______ ;_________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quantitation (LOQ) values (see Attachment B). 14.2 The following quality control samples are extracted with each batch of samples to evaluate the quality of the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision of the extraction. 14.2.3 Continuing calibration check samples to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-97.0 for method performance criteria. 3M Environmental Laboratory ETS-8-96.0 Extraction o f PFOS from Urine Page 8 o f 14 Page 62 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 15.0 Pollution Prevention and Waste Management________ ;________________ 15.1 Human and monkey sample waste is disposed in infectious biohazard waste containers, all other sample waste is disposed in noninfectious biohazard waste containers. Flammable solvent waste is disposed in high BTU containers. Used glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records ________________________________________________________________ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 Tables. Diagrams. Flowcharts, and Validation Date______ ' 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 17.4 Attachment D, Sample weight/volume worksheet -______ 18.0 References ____________________________________________________ 18.1 The validation report associated with this method is FACT-TOX-131, W2067. 19.0 Affected Documents_____________________ !__________ , '____________ _ 19.1 ETS-8-97.0, "Analysis of Potassium PerfluorooCtanesulfonate or Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrospray Mass Spectrometry/Mass S pectrom etry" 20.0 Revisions R ev isio n Number Reason For Revision R e v isio n D ate 3M Environmental Laboratory ETS-8-96.0 Extraction o f PFOS from Urine Page 9 o f 14 Page 63 BACK TO MAIN 3M Medical Department Study: T-6395.14 e x tra c tio n W o rksh ee t 1 S-S-yO .U Studv # M atrix B ox# W k/D av DateSpiked/A nalyst CCV MS M SD _______ Surrogate Std Approx, ppm Actual ppm #' FC-M ix approx. 0.5 ppm actual ppm # FC-M ix approx. 5 ppm actual ppm #. Analytical Report: FACT T O X -111 LRN-U2994 FC -M ix approx. 50 ppm actual ppm # C om m ents Blank Std # - -` -. - - '- - - - - - - amount = Urine Extraction M ethod : Vortex 15 sec. Pipette Matrix . spike with appropriate surrozate or FC-M ix Volum e ml Pipette 1 ml o f 0.5 M TBA, pH 10. pH = Std. # Pipette 2 ml o f 0.25 Na?COV0.25M NaHCO? buffer S td .# Dispense 5 ml o f methyl-t-butyl ether . TN-A- Shake 20 miri. Shaker speed: Centrifuee 20-25 min. Remove a 4 ml aliauot o f orzanic laver Centrifuse speed: 1 Put on N itrosen Evaporator to drvness Temperature: Add methanol Volume ml TN-A- Vortex 30 sec. Filter usine a 3cc B-D svrinee with a 0.2um filter into an autosample vial Cont. Cal. V erifications used sam e matrix as for std curve. - - - - - - - - ml . . Date & Initials Attachment A 3M Environmental Laboratory ETS-8-96.0 Extraction o f PFOS from Urine Page 10 o f 14 Page 64 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 MDL/LOQ values for human urine Compound MDL LOQ Linear Calibration Range (LCR) PFOS POAA PFOSA PFOSAA EtFOSE-OH (PPb) 4.6 22.9 n/d n/d n/d (PPb) 14.7 72.9 n/d n/d n/d 15 ppb -1500 ppb (in final MeOH extract) 50 p p b -1500 ppb (in final MeOH extract) 25 ppb - 1000 ppb (in final MeOH extract) 25 ppb - 1000 ppb (in final MeOH extract) 25 ppb - 1000 ppb (in final MeOH extract) M556 M570 n/d n/d 25 ppb -1000 ppb (in final MeOH extract) n/d n/d 25 ppb -1000 ppb (in final MeOH extract) n/d = not determined , NOTE: to calculate MDL, LOQ, andLCRvalues in ug/ml ofurine divide the above values by 4. MDL/LOQvalues in rat andmonkeyurine werenot statistically determined. Two curves in each of these matrices were extracted andanalyzedwith the humanurine curves to determine equivalence. Responses in the rat and monkey were similarto the humanresponses, therefore, theirMDL and LOQ are assumed to be similar to the values determined forhuman urine. If a suitable amount ofclean, control matrix is available, samples will be evaluatedversus a curve extracted fromurine originating fromthe same species as the specimens. Please see LOQ Summary andMDL study in FACT-TOX-131, W2067 for furtherinformation. Attachment B: MDL/LOQ Summary 3M Environmental Laboratory ETS-8-96.0 Extraction o f PFOS from Urine Page 11 o f 14 Page 65 BACK TO MAIN 3M Medical Department Study: T-6395.14 Compound: PFOS Preparedrange of HumanUrine standards (ppb) (ng/ml) Full Range n/d LCR from curve (PPb) (ng/ml) n/d . %Recovery Range n/d Low Curve n/d n/d n/d High curve n/d n/d n/d 1/X 2.5ppb -1500 ppb 15 ppb --1500 ppb 75-116 Compound: POAA Preparedrange of HumanUrine standards (ppb) (ng/ml) Full Range n/d LCR from curve (Ppb) (ng/ml) n/d %Recovery Range n/d Low Curve n/d n/d n/d High curve 1/X, quadratic n/d n/d 2.5ppb- 1500 ppb 50 ppb - 1500 ppb n/d . 86-105 Analytical Report: FACT TO X-111 LRN-U2994 RSD Range n/d n/d n/d +/- 30% RSD Range n/d ' n/d n/d +/- 30% Attachment B: MDL/LOQ Summary 3M Environmental Laboratory ETS-8-96.0 Extraction o f PFOS from Urine Page 12 o f 14 Page 66 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 LRN-U2994 Ion Pair Standard Curves - Urine Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Blank Guid/identiGer: Method/revision: Box Number: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Actual concentrations of standards in the FC mix PFOS Std cone ug/ml PFOSA Std cone ug/ml PFOSAA Std cone ug/ml EtFOSE Std cone ug/ml POAA Std cone ug/ml M556 M570 Std cone Std cone ug/ml ug/ml 0.500 0.500 0.501 0.501 0.500 0.500 0.501 0.501 0.499 0.499 0.500 0.500 0.501 0.501 0.500 0,501 0.500 0.501 0.499 0.500 0.501 5.00 5.01 5.00 5.01 4.99 5.00 5.01 5.00 5.01 5.00 5.01 4.99 5.00 5.01 5.00 5.01 5.00 5.01 4.99 5.00 5.01 50.0 50.1 50.0 50.1 49.9 50.0 50.1 50.0 50.1 50.0 50.1 49.9 50.0 50.1 50.0 50.1 50.0 50.1 49.9 50.0 50.1 50.0 50.1 50.0 50.1 49.9 50.0 50.1 Calculated concentrations of standards in the sample matrix PFOS PFOSA PFOSAA EtFOSE POAA M 556 Final cone Final cone Final cone Final cone Final cone Final cone ng/ml ng/ml ng/m l ng/ml ng/ml ng/ml 1.25 1.25 1.25 1.25 1.24 1.25 2.48 2.49 2.48 2.49 2.48 2.48 6.17 6.18 6.17 6.18 6.15 6.17 12.5 12.5 12.5 12.5 12.4 12.5 24.8 24.9 24.8 24.9 24.8 24.8 61.7 61.8 61.7 61,8 61.5 61.7 125 125 125 125 124 125 187 187 187 187 186 187 , 248 249 248 249 248 248 372 372 372 372 371 372 M 570 Final cone ng/ml 1.25 2.49 6.18 12.5 24.9 61.8 125 187 249 372 All All Ain't Final voi spiked ml ml 0.005 2.0075 0.010 2.0125 0.025 2.0275 0.005 2.0075 0.010 2.0125 0.025 2.0275 0.005 2.0075 0.0075 2.0100 0.010 . 2.0125 0.015 2.0130 Surrogate Std cone ng/ml 100 A ll Am't spiked ml 0.0025 Surrogate Final cone ng/ml 125 Calculated concentrations of standards in methanol extract (0.5 ml Gnal volume) PFOS PFOSA PFOSAA EtFOSE POAA M 556 M570 Final cone ng/ml 5.00 Final cone ng/ml 5.01 Final cone ng/ml 5.00 Final cone ng/ml 5.01 Final cone ng/ml 4.99 Final cone ng/ml 5.00 Final cone ng/ml 5.01 10.0 25.0 50.0 100 10.0 25.1 50.1 100 10.0 25.0 50.0 100 10.0 25.1 50.1 100 10.0 25.0 49.9 100 10.0 10.0 25.0 25.1 50.0 50.1 100 100 250 500 750. 1000 1500 251 501 752 1002 1503 250 500 750 1000 1500 251 501 752 1002 1503 250 499 749 998 1497 250 500 750 1000 1500 251 501 752 1002 1503 Surrogate Std cone ng/ml 100 Surrogate Final cone ng/ml 500 All Am't spiked ml 0.0025 Attachment C: Standard Calculation Sheet ETS-8-96.0 3M Environmental Laboratory Extraction o f PFO S from Urine Page 13 o f 14 Page 67 3M Medical Department Study: T-6395.14 PrepDate(s): Analyst(s): Sample Matrix: Method/Revision: Target Analyte(s): Sample ID Initial W t/V ol. g/mL/L BACK TO MAIN Analytical Report: FACT TO X -111 LRN-U2994 Study Number: Equipment Number: Final Solvent & TN Number: Matrix Blank/Identifier: Box: Comments Summary of method: Notes: Attachment D: W eight/Volume Sheet 3M Environmental Laboratory ETS-8-96.0 Extraction o f PFOS from Urine Page 14 o f 14 Page 68 BACK TO MAIN 3M Medical Department Study: T-6395.14 3M Environmental Laboratory Analytical Report: FACT T O X -111 LRN-U2994 Method Analysis of P otassium Perfluorooctanesulfonate or Oth er Fluorochem ical Com pounds in Urine Extracts Using H PL C -E lectrospray/M ass Spectrometry/M ass Spectrom etry Method Number: ETS-8-97.0 Author: Lisa Clemen, Robert Wynne, Glenn Langenburg Approved By: Laboratory Manager 7^ ------;------ - Group Leader Adoption Date: Revision Date: ]<4^ /? 5 Date Date Technical Reviewer Date 1.0 Scope and Application______________ :________ __________________ ,_________ 1.1 Scope: This method describes the analysis of urine extracts for fluorochemicals using HPLC-electrospray/mass spectrometry. . 1.2 Applicable Compounds: Fluorochemicals or other ionizable compounds. 1.3 Matrices: Human, rat, and monkey urine, or other fluids as designated in the validation report. W ord 6/95 3M Environmental Laboratory ETS-8-97.0 Analysis o f Urine Extract Using ES/MS Page 1 o f 10 Page 69 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 2.0 Summary of Method___________________________ .______ ;_____________ ___ 2.1 This method describes the analysis of fluorochemicals extracted from urine or other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z= 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity of a compound by detecting daughter ions of the parent ion. 3.0 Definitions ___________________________________________________ 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e., not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II triple quadrupole mass spectrometer is equipped with two quadrupole mass selective detectors and a collision cell. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or an ion may be selected in the first quadrupole, fragmented in the collision cell, and these fragments may be analyzed in the second quadrupole. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass Quattro II triple quadrupole systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e., Z-spray components are compatible with some other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these Quattro II triple quadrupole systems. Currently MassLynx has WindowsNT 4.0 versions. For more details see the manual specific to the instrument (Micromass Quattro II triple quadrupole MassLynx NT User's Guide). 4.0 W arnings and Cautions ______ :________________________________ _ 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. 3M Environmental Laboratory ETS-8-97.0 Analysis o f Urine Extract Using ES/MS Page 2 of 10 Page 70 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. 4.2 Cautions: 4.2.1 Operate the solvent pumps below a back pressure o f400 bar (5800 psi). If the back pressure exceeds 400 bar, the HP1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences__________________________________________________ . -______ 5.1 To minimize interferences when analyzing samples, teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 Equipment___________________;_____________ '___________________________ ' 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 6.1.2 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an electrospray ionization source HP1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 Supplies and Materials __________________ ;_______________________ ;_______ 7.1 Supplies 7.1.1 High purity grade nitrogen regulated to approximately 100 psi. (House air system) 7.1.2 High purity grade argon regulated to approximately 6 psi. 7.1.3 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7.1.4 Capped autovials or capped 15 mL centrifuge tubes 8.0 Reagents and Standards_____________________________ . ____________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water (ASTM type I), all.water used in this method should be ASTM type I, or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.1.3.1 When preparing different amounts than those listed, adjust accordingly. 8.1.3.2 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 mL volumetric container containing 2000 mL Milli-QTM water, mix until all solids are dissolved. Store at room temperature. 3M Environmental Laboratory ETS-8-97.0 Analysis o f Urine Extract Using ES/MS Page 3 o f 10 Page 71 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and twenty matrix standards are prepared during the extraction procedure. See ETS-8-96.0. 9.0 Sample Handling _________________________________ '_________________ 9.1 Fresh matrix standards are preparedwith each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C, or at room temperature, until analysis can be performed. 10.0 Quality Control __________________________________________ ;___________ 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. . 10.1.2 Analyze a solvent blank, method blank, and matrix blank prior to each calibration curve. 10.2 Matrix Spikes . 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty samples, with a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lowrange of the initial calibration curve. 10.3 Continuing Calibration Verifications 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum o f one per batch. 11.0 Calibration and Standardization _______________________________ . 11.1 Analyze the extracted matrix standards prior to and following each set of extracts. The average of two standard curves will be plotted by regression (linear or otherwise, see 11.2), weighted 1/x, not forced through zero, with IS reference (surrogate is used as an internal standard) using MassLynx or other suitable software. 3M Environmental Laboratory ETS-8-97.0 Analysis o f Urine Extract Using ES/MS Page 4 o f 10 Page 72 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report. FACT T O X -111 LRN-U2994 11.2 Use the following parameters for determining a calibration curve for each compound: Compound Weighting Regression Fit Response type PFOS 1/X Linear IS reference POAA 1/X Quadratic IS reference Suitability of curve String parameters for other fluorochemical compounds will addressed by the analyst. 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.4 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range ofthe standard curve. Example: when attempting to quantitate approximately 50 ppb of analyte, generate a calibration curve consisting of the standards from 25 ppb to 250 ppb rather than the full range of the curve (25 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 12.0 Procedures_______________________________ ;________ ;___________,_________ 12.1 Acquisition Set up 12.1.1 Set up the sample list. 12.1.1.1 Assign a sample list filename using MO-DAY-last two digits of yearincreasing letter of the alphabet starting with a 12.1.1.2 Assign amethod (MS file) for acquiring 12.1.1.3 Assign an HPLC program (Inlet file) 12.1.1.4 Type in sample descriptions and vial position numbers 12.1.2 To create a method click on method in the Acquisition control panel then mass spectrometer headings and select SIR (Single Ion Recording) or MRM (Multiple Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected long with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. Refer to Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM. 12.1.3 Typically the analytical batch run sequence begins and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration verification injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 3M Environmental Laboratory ETS-8-97.0 Analysis o f Urine Extract Using ES/MS Page 5 o f 10 Page 73 BACK TO MAIN 3M Medical Department Study: T-6395.14 . Analytical Report: FACT T O X -111 LRN-U2994 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample =1 12.2.2.3 Cycle time = 9.0 minutes 12.2.2.4 Solvent ramp = Time 0.00 min 1.0 min. 4.5 min. 6.5 min. 7.0 min. 9.0 min. MeOH 40% 40% 95% 95% 40% 40% 2.0 mM Ammonium acetate 60% 60% 5% 5% 60% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0, "Operation and Maintenance of the Micromass Quattro II Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source," for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Turn on the nitrogen. 12.3.5 Open the tune page. Click on operate to initiate source block and desolvation heaters. 12.3.6 Open the Inlet Editor. 12.3.6.1 Set HPLC pump to "On" 12.3.6.2 Set the flow to 10 - 500 uL/min or as appropriate 12.3.6.3 Observe droplets coming out of the tip of the probe. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed 12.3.6.4 Allow to equilibrate for approximately 10 minutes. 12.3.7 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.7.1 Drying gas 250-400 liters/hour 3M Environmental Laboratory ETS-8-97.0 Analysis o f Urine Extract Using ES/MS Page 6 o f 10 Page 74 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 12.3.7.2 ESI nebulizing gas 10-15 liters/hour . 12.3.7.3 HPLC constant flow mode, flow rate 10 - 500 pL/min 12.3.7.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7.5 Source block temperature 150 12.3.7.6 Desolvation temperature 250 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, refer to appropriate MassLynx User's Guide). Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations____________ '__________________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: %Recovery = Observed Result - Background Result x 100 Expected Result . . 13.1.5 Calculate percent difference using the following equation: %Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, in matrix (pg/mL): fag/mL of PFOS calc, from std. Curve x Dilution Factor! x 1 ue Initial Volume of matrix (mD 1000 ng Final Volume (mL) 14.0 Method Performance_______________________________________________________ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-96.0, Attachment B, for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix blanks values must be below the lowest standard in the calibration curve 14.3 Calibration Curves 14.3.1 The r2value for the calibration curve must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries must be within 30% of the spiked concentration. 3M Environmental Laboratory ETS-8-97.0 Analysis o f Urine Extract Using ES/MS Page 7 o f 10 Page 75 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 14.5 Continuing Calibration Verifications 14.5.1 Continuing calibration verification percent recoveries must be within 30% of the spiked concentration. 14.6 If criteria listed in this method performance section are not met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in file appropriate logbook. 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 Pollution Prevention and Waste Management______________________ __ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records___________________________________ ;_________________ |_______________ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study orproject number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by a linear or quadratic fit, referenced to the internal standard (surrogate), weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, from MassLynx, and store in the study folder. 16.5 Summarize data using suitable software (Excel 7.0) and store in the study folder, see Attachment A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location ofbackup electronic data. 17.0 TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA _______________________ 17.1 Attachment A: ETS-8-97.0 Data summary spreadsheet. 18.0 References____________ ________________________________________ :___________ 18.1 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure lonization/Mass Spectrometer Quattro II triple quadrupole Systems" 18.2 The validation report associated with this method is FACT-TOX-131, W2067 3M Environmental Laboratory ETS-8-97.0 Analysis o f Urine Extract Using ES/MS Page 8 o f 10 Page 76 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 19.0 Affected Documents____________________________________________________ _____ 19.1 ETS-8-96.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Urine for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 Revisions____________________________________________________________ _ Revision Nrimher Reason For Revision Revision Date 3M Environmental Laboratory ETS-8-97.0 Analysis o f Urine Extract Using ES/MS Page 9 o f 10 Page 77 BACK TO MAIN 3M Medical Department Study: T-6395.14 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squaxed Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Group Dose Sample# Concentration ug/mL Initial Vol. mL Dilution Factor Analytical Report: FA C T T O X -1 11 L R N -U 2 9 9 4 Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-97.0 3M Environmental Laboratory Analysis o f Urine Extract Using ES/MS Page 10 o f 10 Page 78 BACK TO MAIN 3M Medical Departm ent Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Method Modification M ethod: Sections m odified: ETS-8-97.0 10.3.2 and 14.5.1 Method reads: 10.3.2 14.5.1 Analyze a mid-range calibration standard after every tenth sample, with a minimum of one per batch. Continuing calibration verification percent recoveries must be within 30% of the spiked concentration. , *. . Modify method to read: 10.3.2 Analyze a mid-range calibration standard at least after every ten samples, with a minimum o f one per batch. 14.5.1 A t least one continuing calibration verification per ten samples must show a percent recovery within +/-30% oftire spiked concentration. Effective date o fmodification: tl*-- Signature ofPAI and date 7/28/99 z-', 3M Environmental Laboratory Bct Qq'O'j -- (Al ' Page 79 BACK TO MAIN 3M Medical Department Study: T-6395.14 . Analytical Report: F A C T T O X -1 11 .1 L R N -U 2994 Method Modification Methods: Section modified: ETS-8-7.0, ETS-8-5.1, ETS-8-97.0 add section 14.3.2 Method reads: NA Modify method to read: . 14.3.2 The second (bracketing) calibration curve may be deactivated if instrumental drift affects the data. The first curve and acceptable calibration checks shall bracket usable data. Effective date ofmodification ETS-8-7.0: 7/22/99 Effective date ofmodification ETS-8-5.1: 4/26/99 Effective date ofmodification ETS-8-97.0: 7/28/99 Signatureeof PAI and date i\ioHat? Exac Copy of Original 3M Environmental Laboratory Page 80 BACK TO MAIN 3M Medical Department Study: T-6395.14 3M Environmental Laboratory Analytical Report: FACT T O X -111 LRN-U2994 Method Analysis o f P otassium Perfluorooctanesulfonate o r Other Fluorochem icals in Serum Extracts Using H PLC -Electrospray/M ass Spectrometry Method Number: ETS-8-5.1 Author: Lisa Clemen, Robert Wynne Approved By: Adoption Date: 03/01/99 Revision Date: Laboratory Manager Date Group Leader fis*A Ctweu Technical Reviewer Date ' ohIuIw Date 1.0 Scope and Application_______________________________________________ _______ 1.1 Scope: This method describes the analysis of serum extracts for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey, and human serum, or other fluids as designated in the validation report. Word 6/95 3M Environmental Laboratory ETS-8-5.1 Analysis o f Serum Extract Using ES/MS Page 1 o f9 Page 81 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 2.0 Summary of Method ______;________________________________________ . 2.1 This method describes the analysis of fluorochemical surfactants extracted from serum or other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z= 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity of a compound by detecting daughter ions of the parent ion. 3.0 D efinitions___________________________ ;__________________________ ;_____________ 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e., not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II triple quadrupole systems are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass Quattro II triple quadrupole systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e., Z-spray components are compatible with some other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation ofthese Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details see the manual specific to the instrument (Micromass Quattro II triple quadrupole MassLynx or MassLynx NT User's Guide). 4.0 W arnings and Cautions____________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 2 o f9 Page 82 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 4.2 Cautions: 4.2.1 Do not operate solvent pumps above capacity o f400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HP1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences__________________________________________________ _________ 5.1 To minimize interferences when analyzing samples, teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 Equipment _______________________________________________________________ . 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an electrospray ionization source 6.1.2 HP1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 Supplies and Materials ___________________ ;__________ ;_____________ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (House air system) 7.1.2 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes 8.0 Reagents and Standards_______________________ 'v 8.1 Reagents : : '____________________ 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. SeeETS-8-4.1. 9.0 Sample Handling____________________ ;__________________________ ;_____________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 3M Environmental Laboratory ETS-8-5.1 Analysis o f Serum Extract Using ES/MS Page 3 o f9 Page 83 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C TT O X -111 L R N -U 2 9 9 4 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C, or at room temperature, until analysis can be performed. 10.0 Quality Control________;______________________________ 10.1 Solvent Blanks, Method Blanks and Matrix Blanks ____________ 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty samples, with a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lowrange of the initial calibration curve. 10.3 Continuing Calibration Verifications 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of the calibration curve. ' 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a m inim um of one per batch. 11.0 Calibration and Standardization______________ - '______________ ;________ 11.1 Analyze the extracted matrix standards prior to and following each set of extracts. The average of two standard curves will be plotted by linear regression (y = my + b), weighted 1/x, not forced through zero, using MassLynx or other suitable software. 11.2 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.3 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end o f the calibration curve rather than the full range of the standard curve. Example: when attempting to quantitate approximately 10 ppb of analyte, generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 3M Environmental Laboratory ETS-8-5.1 Analysis o f Serum Extract Using ES/MS Page 4 of 9 Page 84 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 L R N -U 2 9 9 4 12.0 Procedures _______________________;_____________________________________ __ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording) or MRM. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. IfMS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). 12.1.3 Typically the analytical batch run sequence begins with a set of extracted matrix standards and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample =1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 8.50 min. 11.0 min. 12.0 min. MeOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% . 10% 10% 60% 12.2.2.5 Press the "Start" button. 12,3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 3M Environmental Laboratory ETS-8-5.1 Analysis o f Serum Extract Using ES/MS Page 5 of 9 Page 85 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -111 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with nojagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fne mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing,gas 10-15 liters/hour 12.3.6.3 HPLC constant flow mode, flow rate 10- 500 pL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis ofbiological matrices. 12.3.10Click on start button in the Acquisition ControlPanel (this may vary among MassLynx versions, see appropriate MassLynx USER'S GUIDE). Press the start button. Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations______________- -________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: %Recovery - Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: %Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, in matrix (pg/mL): <hg of PFOS calc, from std. Curve x Dilution Factor) x 1 ue (Initial Volume of matrix ('mLl + mL of Surrogate Standard! lOOOng Final Volume (mL) 3M Environmental Laboratory ETS-8-5.1 Analysis o f Serum Extract Using ES/MS Page 6 of 9 Page 86 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 14.0 Method Performance _______________________________ ______________ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-4.1, Attachment B, for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix blanks values are must be below the lowest standard in the calibration curve 14.3 Calibration Curves 14.3.1 The r2value for the calibration curve must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries are must be within 30% of the spiked concentration. 14.5 Continuing Calibration Verifications 14.5.1 Continuing calibration verification percent recoveries must be 30% of the spiked concentration. 14.6 If criteria listed in this method performance section isn't met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 Pollution Prevention and Waste Management __________ , __________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records ______________________________ ^ ;______________ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, from MassLynx, and store in the study folder. 3M Environmental Laboratory ETS-8-5.1 Analysis o f Serum Extract Using ES/MS Page 7 o f9 Page 87 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 16.5 Summarize data using suitable software (Excel 5.0) and store in the study folder, see Attachment A for an example of a summaiy spreadsheet, 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 Tables. Diagrams. Flowcharts, and Validation Data 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet. ______ _______________ 18.0 References____________________________;_____________________________________ 18.1 FACT-M-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple quadrupole Systems" 18.3 The validation report associated with this method is ETS-8-4.0 & 5.0-V-l. . 19.0 Affected Documents _________________________________________ 19.1 ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" . 20.0 R e v isio n s_________________ ;______________ ______________________ ;____________________ Revision Number. 1 Reason For Revision Section 6.1.2 Clarification ofHPl 100 system components. Section 11.1 Average of two curves, not standard values, are used for plotting linear regression and added the 1/x weighting of the curve. Section 12.2.2.4 Clarification of solvent ramp. Section 17.1 Changed from attachment B to A. Revision Date 04/02/99 3M Environmental Laboratory ETS-8-5.1 Analysis o f Serum Extract Using ES/MS Page 8 o f9 Page 88 BACK TO MAIN 3M Medical Department Study: T-6395.14 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Group Dose Sample# Concentration ug/mL Initial Voi. mL Dilution Factor Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-5.1 3M Environmental Laboratory Analysis o f Serum Extract Using ES/MS Page 9 of 9 Page 89 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 Method Modification M ethod: Sections modified: ETS-8-5.1 10.3.2 and 14.5.1 Method reads: 10.3.2 14.5.1 Analyze a mid-range calibration standard after every tenth sample, with a minmnirn of one per batch. Continuing calibration verification percent recoveries must be within 30% ofthe spiked concentration. Modify method to read: 10.3.2 14.5.1 Analyze a mid-range calibration standard at least after every ten samples, with a rmriirmm Qf one per batch. At least one continuing calibration verification per ten samples must show a percent recovery within+/-30%ofthe spiked concentration. Effective date of modification: 4/26/99 M ?---------------- itjo %(&Q Signature of PAI and date 3M Environmental Laboratory Page 90 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -1 11 LRN-U2994 Appendix D: Data Summary Tables Table 9. Average Results for the Analyses of Sera Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 via Gavage DG0 PFOS Cone. (pg/mL) DG7 PFOS Cone. (pg/mL) DG15 PFOS Cone. (pg/mL) DG21 PFOS Cone. (pg/mL) DL14 PFOS Cone. (pg/mL) DL21 PFOS Cone. (pg/mL) DL22 PFOS Cone. (pg/mL) G roup I F0 0.0 mg/kg/day G roup I F1 0 .1 0 0 a'6 NS 0.0796af> 0.0742a'6 <LLQa'6 NS NS NS 0.0542a'6 NS 0.0492a'6 NS 0.0531a'6 NS G roup II F0 0.1 mg/kg/day G roup II F1 9.21a'6 NS 7.24a'6 NS 5.68a'6 NS 2.58a'6 NS 1.63a'6 NS NS 1.80a'6 0.979a'6 NS G roup III F0 1.6 mg/kg/day G roup III F1 161ab NS 129a'6 NS 90.6a'6 NS 39.5a'6 NS 2 o e a'6 NS NS 27.1a'6 1 4 1 a," NS 3 PFOS concentrations are the average of samples within a dose group from all animals tested, not Including samples that tested at or below the limit of quantitation (0.04 pg/mL). Individual animal data are located in Appendix E. b Sera analyses were conducted at a contract laboratory, but were corrected by 3M to reflect the official purity values from the Certificate of Analysis. A revised final report from the contract laboratory will be added as an amendment to this report. <LLQ=Less than the Limit of Quantitation NS=Not Sampled 3M Environmental Laboratory Page 91 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Table 10. Average Results for the Analyses of Liver Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 via Gavage DL22 PFOS Cone. (pg/g) Group IF 0 0.0 mg/kg/day 0 2 4 3 a'6 Group IF1 0.174a'6 Group II F0 0.1 mg/kg/day Group II F1 6.15a'6 5.00a'6 Group III F0 1.6 mg/kg/day Group III F1 59.7a'6 56.2a'6 a PFOS concentrations are the average of samples within a dose group from all animals tested, not including samples that tested at or below the limit of quantitation (0.11 pg/g). Individual animal data are located in Appendix E. b Liver analyses were conducted at a contract laboratory, but were corrected by 3M to reflect the official purity values from the Certificate of Analysis. A revised final report from the contract laboratory will be added as an amendment to this report. Table 11. Average Results for the Analyses of Urine Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 via Gavage Group 1F0 0.0 mg/kg/day Group II F0 0.1 mg/kg/day Group III F0 1.6 mg/kg/day DG0 PFOS Conc. (pg/mL) 0.00819a 0.0905a 2 .1 1 a DG6/7 PFOS Conc. (pg/mL) 0 .0 1 0 0 a 0.0307a 0 .8 8 8 a DG14/15 PFOS Conc. (pg/mL) 0.00685a 0.0327a 0.613a DG20/21 PFOS Conc. (pg/mL) 0.00614a 0.0231a 0.340a DL21/22 PFOS Conc. (pg/mL) <LOQa 0.00555a 0.0334a a PFOS concentrations are the average of samples within a dose group from all animals tested, not including samples that tested at or below the limit of quantitation (0.00295 pg/mL). Individual animal data are located in Appendix E. <LOQ=Less than Limit of Quantitation 3M Environmental Laboratory Page 92 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Table 12. Average Results for the Analyses of Feces Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 via Gavage Group IF0 0.0 mg/kg/day DG0 PFOS Cone. (pg/g) NDa D G 6 /7 PFOS Cone. (pg/g) NDa D G 1 4 /15 PFOS Cone. (pg/g) NDa DG20/21 PFOS Cone. (pg/g) NDa DL21/22 PFOS Cone. (pg/g) NDa Group II F0 0.1 mg/kg/day 0.601a'6 0.399a 0.294a 0.119a 0.0522a Group III F0 1.6 mg/kg/day 10.9a 8.39a 4.83a 2.06a 0.387a a PFOS concentrations are the average of samples within a dose group from all animals tested, not including samples that tested at or below the limit of quantitation (0.0100 pg/g). Individual animal data are located in Appendix E. and Appendix G. ^Animal 13745F result not included in average. ND=Not Detected 3M Environmental Laboratory Page 93 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Table 13. LOQ Values Used in FACT TOX-111 Analysis by Method and Usage Dates Sample LOQ Laboratory Method(s) Usage Date Urine Dosage Confirmation Liver Serum Feces 0.00295 |jg/mL 0.030 pg/mL 0 .1 1 pg/g 0.04 pg/mL 0 .0 1 0 0 pg/g 3M Environmental Laboratory 3M Environmental Laboratory Battelle Memorial Institute Advanced Bioanalytical Services, Inc. Centre Analytical Laboratories, Inc. ETS-8-96.0 ETS-8-97.0 ETS-8-5.1 N003604-A 6 October 1999 21 September 1999 29 October 1999 99VDJA01.MI.DOC 00M-023-003, revision 2 9 July 1999 6 July 1999 3M Environmental Laboratory Page 94 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 Appendix E: Data Spreadsheets Table 14. Results of Analyses of Sera Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 via Gavage. DG0 DG7 DG15 DG21 DL14 DL21 DL22 Sample # PFOS Cone. PFOS Cone. PFOS Cone. PFOS Conc. PFOS Conc. PFOS Conc. PFOS Conc. (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) 13726 13727 13728 13731 13734 13735 Group I 0.0 mg/kg/day 13736 13737 13726P 13727P 13728P 13734P 13735P 13736P 13737P 13739 13740 13741 13744 13745 13746 13748 Group II 13749 0.1 mg/kg/day 13739P 13740P 13741P 13744P 13745P 13746P 13748P 13749P 0.186a 0.105a 0.0804a 0.0727a 0 .1 0 2 a 0.0818a 0.0976a 0.0758a NS NS NS NS NS NS NS 8.45a 10.5a 1 0 .0 a 8.54a 8.54a 9.59a 8.81a 9.24a NS NS NS NS NS NS NS NS 0.152a 0.0721a 0.0614a 0.0754a 0.0556a 0.0605a 0.0933a 0.0663a NS NS NS NS NS NS NS 8 .2 1 a 6.78a 6.56a 7.93a 6.39a 7.34a NS 7.44a NS NS NS NS NS NS NS NS 0.145a 0.0805a 0.0523a 0.0527a 0.0575a 0.0496a 0.0599a 0.0959a NS NS NS NS NS NS NS 5.08a 6.77a 5.76a 5.22a 5.01a 5.75a 4.63a 7.26a NS NS NS NS NS NS NS NS 0.0734a 0.0507a <LLQa NS <LLQa <LLQa <LLQa 0.0459a NS NS NS NS NS NS NS 1.56a 3.46a 2.32a 2.63a 2 .0 2 a 2.45a 3.40a 2.83a NS NS NS NS NS NS NS NS 0.0710a 0.0551a 0.0458a NS 0.0513a <LLQa <LLQa 0.0478a NS NS NS NS NS NS NS 1.57a 1.72a 1.30a 1.69a 2 .2 0 a 1.75a 1.17a 1.62a NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS 0.0615a <LLQa <LLQa <LLQa <LLQa <LLQa 0.0448a NS NS NS NS NS NS NS NS 2 .0 2 a 1.83a 1.62a 1.98a 1.62a 2.05a 1.50a 1.79a 0.0516a 0.0447a <LLQa NS <LLQa <LLQa 0.0511a 0.0492a NS NS NS NS NS NS NS 1.15a 1.27a 1.03a 0.762a 0.942a 0.816a 0.814a 1.05a NS NS NS NS NS NS NS NS a Sera analyses were conducted at a contract laboratory, but were corrected by 3M to reflect the official purity values from the Certificate of Analysis. A revised final report f n o m the contract laboratory will be added as an amendment to this report. <LLQ=Less than the Limit of Quantitation (0.04 (pg/mL)) NS=No sample 3M Environmental Laboratory Page 95 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACTTOX-111 LRN-U2994 Table 14. Results of Analyses of Sera Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Liver, Urine, and Feces of Crl:CDBR VAF/PIus Rats Exposed to T-6295.14 via Gavage DGO DG7 DG15 DG21 DL14 DL21 DL22 Sam ple # PFOS Cone. PFOS Cone. PFOS Cone. PFOS Conc. PFOS Conc. PFOS Conc. PFOS Conc. (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) (Mg/mL) (pg/mL) 13751 13752 13753 13754 13755 13756 Group III 1.6 mg/kg/day 13758 13759 13751P 13752P 13753P 13754P 13756P 13758P 13759P 134a 142a 184a 171a 165a 162a 162a 171a NS NS NS NS NS NS NS 129a 146a 136a 131a 119a 137a 124a 115a NS NS NS NS NS NS NS 82.6a 94.2a 96.8a 89.0a 108a 93.3a 75.1a 85.9a NS NS NS NS NS NS NS 33.3a 30.1a 28.2a 32.1a 96.8a 34.9a 32.0a 28.5a NS NS NS NS NS NS NS 18.3a 2 0 .8 a 18.7a 18.3a NS 26.8a 23.9a 17.6a NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS 30.2a 29.3a 24.5a 2 0 .2 a 41.0a 2 2 .1a 22.7a 14.8a 16.8a 15.7a 8.19a NS 17.2a 12.4a 13.3a NS NS NS NS NS NS NS a Sera analyses were conducted at a contract laboratory, but were corrected by 3M to reflect the official purity values from the Certificate of Analysis. A revised final report from the contract laboratory will be added as an amendment to this report. LLQ= Limit of Quantitation (0.04 pg/mL) NS=No sample 3M Environmental Laboratory Page 96 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Table 15. Results of Analyses of Liver Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Liver, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 via Gavage Sample # DL22 PFOS Cone. (pg/g) 13726 0.295a Group 1 0.0 mg/kg/day 13727 13728 13731 13734 13735 13736 13737 13726P 13727P 13728P 13734P 13735P 0.371a 0.258a 0.257a 0.188a 0.217a 0.165a 0.189a 0.367a 0.137a 0.122a 0.135a 0.137a Group II 0.1 mg/kg/day 13736P 13737P 13739 13740 13741 13744 13745 13746 13748 13749 13739P 13740P 13741P 13744P 13745P 13746P 13748P 13749P BLOQa 0.148a 5.22a 5.84a 7.19a 5.41a 5.91a 5.97a 5.86a 7.77a 5.49a 5.11a 4.19a 5.47a 5.30a 5.24a 4.34a 4.90a a Liver analyses were conducted at a contract laboratory, but were corrected by 3M to reflect the official purity values from the Certificate of Analysis. A revised final report from the contract laboratory will be added as an amendment to this report. BLOQ=Below Concentration of Lowest Standard in Calibration Curve LOQ=0.11 pg/g 3M Environmental Laboratory Page 97 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -111 L R N -U 2 9 9 4 Table 15. Results of Analyses of Liver Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Liver, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 via Gavage ________________ Sample # DL22 PFOS Cone. (pg/g) Group III 1.6 mg/kg/day 13751 13752 13753 13754 13756 13758 13759 13751P 13752P 13753P 13754P 13756P 13758P 13759P 69.6a 88.1a 43.5a 42.9a 56.2a 49.0a 68.3a 50.7a 54.6a 49.8a 62.9a 72.0a 50.6a 52.6a a Liver analyses were conducted at a contract laboratory, but were corrected by 3M to reflect the official purity values from the Certificate of Analysis. A revised final report from the contract laboratory will be added as an amendment to this report. BLOQ=Below Concentration of Lowest Standard in Calibration Curve LOQ=0.11 pg/g 3M Environmental Laboratory Page 98 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Table 16. Results of Analyses of Urine Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 via Gavage DGO Sample # PFOS Cono. (pg/mL) DG6/7 PFOS Conc. (pg/mL) DG14/15 PFOS Conc. (pg/mL) DG20/21 PFOS Conc. (pg/mL) DL21/22 PFOS Conc. (pg/mL) 13726F 13727F 13728F G roup I 13731F 0.0 mg/kg/day 13734F 13735F 13736F 13737F 13739F 13740F 13741F G roup II 13744F 0.1 mg/kg/day 13745F 13746F 13748F 13749F 13751F 13752F 13753F G roup III 13754F 1.6mg/kg/day 13755F 13756F 13758F 13759F 0.00381 <LOQ 0.00464 0.00650 0.0108 0.0117 0.0117 <LOQ 0.0852 0.202 0.0587 0.0807 0.0520 0.0517 0.101 0.0929 1.91 1.49 3.33 1.48 0.859 2.55 2.74 2.50 <LOQ <LOQ 0.00312 <LOQ <LOQ <LOQ 0.0168 <LOQ 0.0454 0.0296 0.0256 0.0257 0.0394 0.0223 0.0248 0.0331 1.47 0.479 1.69 SL 0.403 0.597 0.438 1.14 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 0.00685 0.0135 0.0444 0.0335 0.0184 0.0244 0.0642 0.0253 0.0384 0.653 0.549 0.520 NE 0.953 0.798 0.273 0.548 <LOQ 0.00614 <LOQ NS <LOQ <LOQ <LOQ <LOQ 0.00531 0.0230 0.0368 0.0259 0.0298 0.0187 0.0266 0.0188 0.419 0.482 0.0898 0.183 0.363 0.416 0.558 0.208 <LOQ <LOQ <LOQ NS <LOQ <LOQ <LOQ <LOQ <LOQ 0.00981 0.00391 <LOQ <LOQ 0.00328 <LOQ 0.00520 0.0539 0.0472 0.0433 0.0118 NS 0.0279 0.0328 0.0169 <LOQ=Less than the Limit of Quantitation (0.00295(pg/mL) ND=Not Detected NS=No Sample SL=Sample lost during extraction NE=No sample extract remaining to perform dilution 3M Environmental Laboratory Page 99 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -111 L R N -U 2 9 9 4 Table 17. Results of Analyses of Feces Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.14 via Gavage Group 1 0.0 mg/kg/day G roup II 0.1 mg/kg/day G roup III 1.6 mg/kg/day Sample # 13726F 13727F 13728F 13731F 13734F 13735F 13736F 13737F 13739F 13740F 13741F 13744F 13745F 13746F 13748F 13749F 13751F 13752F 13753F 13754F 13755F 13756F 13758F 13759F Prior to DG6/7 Cohabitation PFOS Conc. PFOS Conc. (pg/g) (pg/g) ND ND ND ND ND ND ND ND ND 0.810 0.664 0.394 0.402 2.72a/0.9046 0.688 0.703 0.546 10.8 10.8 12.3 13.5 8.57 10.8 9.41 10.9 ND ND ND ND ND ND ND 0.514 0.537 0.318 0.309 0.382 0.389 0.322 0.420 8.51 9.61 9.03 8.86 12.2 6.70 7.65 4.47 DG14/15 PFOS Conc. (Mg/g) ND ND ND ND ND ND ND ND 0.298 0.311 0.308 0.416 0.294 0.345 0.204 0.176 5.00 6.31 4.02 3.80 5.65 5.13 5.30 3.41 DG20/21 PFOS Conc. (Mg/g) ND ND ND NS ND ND ND ND 0.133 0.0668 0.165 0.0883 0.0650 0.113 0.157 0.163 1.61 2.08 1.42 1.69 4.29 2.02 1.97 1.38 DL21/22 PFOS Conc. (Mg/g) 0.0204 ND ND NS ND ND ND ND 0.0674 0.0693 0.0389 0.0729 0.0339 0.0609 0.0508 0.0231 0.513 0.731 0.327 0.295 NS 0.336 0.211 0.296 Questionable residue found: re-extracted for confirmation. bRe-extracted value. Animal 13745F values were not included in the average values in Table 12. LOQ=0.0100 jjg/g ND=Not Detected NS=No Sample 3M Environmental Laboratory Page 100 BACK TO MAIN 3M Medical Department Study: T-6395.14 Appendix F: Example Calculations Formula Used for Urine Analyses in Study FACT TOX-111 1.0 u p AR (ng/mL) x DF x SC x ------x PC = Reported Concentration (pg/mL) lOOOwg Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Calculation Used for Group II DG 14-15, Animal Number 13739F 1.0u g 16.79 (ng/mL) x 1 x 0.9275 x ------------ x 0.864 =0.0135 (pg PFOS/mL urine) 1000n g AR--Analytical result from MassLynx summary DF-- Dilution factor SC-- PFOS salt correction constant (0.9275) PC-- PFOS purity correction factor (0.864) 3M Environmental Laboratory Page 101 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Appendix G: Contract Laboratory Reports This appendix includes the following contract laboratory reports: Advanced Bioanalytical Services, Inc., Quantitative Determination of Perfluorooctanesulfonate (PFOS) in Rat Serum from Study #FACT-TOX-111 Using Turbo Ion Spray LC/MS, ABS Report Number 99ADJA03.MI.DOC (19 pages). Advanced Bioanalytical Sen/ices, Inc., "Method Validation for the Quantitation of Perfluorooctanesulfonate (PFOS) In Rat Serum by Turbo Ion Spray LC/MS," ABS Report Number 99VDJA01 .Ml.DOC (50 pages). Battelle Memorial Institute, Study Number N003296-G, Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats Final Report, (58 pages). Centre Analytical Laboratories, Inc., Centre Study Number 023-017, Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats, (93 pages). 3M Environmental Laboratory Page 102 BACK TO MAIN 3M Mediqal Department Study: T-6395.14 |M lii I b | | ADVANCED f l l b io a n a l v t ic a l B H i SER V IC ES. INC. BIOANALYTICAL REPORT Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Title: Quantitative Determination o f Perfluorooctanesulfonate (PFOS) in Rat Serum from Study #FACT-T0X-111 Using Turbo Ion Spray LC/MS Date: II October 1999 Authors: David J. Anderson, M.S. Amie J. Prince, B.S. Holly D. Ross, M.S. Prepared For: 3M Environmental Technology and Safety Services St. Paul, MN 55133-3331 ABS Report No.: 99ADJA03.MI.DOC Study Protocol: FACT-TOX-111 Number o f Pages: 19 Summary and Conclusions The concentration o f perfluorooctanesulfonate (PFOS) was determined in rat serum samples collected during the study #FACT-TOX-l 11 using a sensitive, specific, accurate, and reproducible analytical method developed by Advanced BioAnalytical Services, Inc., Ithaca, NY. Rat serum samples (50 pL) were extracted by a liquid-liquid extraction procedure to isolate PFOS and the internal standard, lH,lH,2H,2H-perfluorooctane sulfonic acid (Tetra-H-PFOS). Following evaporation and reconstitution, sample extracts were analyzed by turbo ion spray liquid chromatography/mass spectrometry (LC/MS) in the negative ion mode. All samples were successfully analyzed within four runs. The lower limit o f quantitation was 0.05 pg/mL for PFOS. The precision o f this assay (RSD) as determined from the analysis o f quality control samples for PFOS was <3.61%. The precision o f this assay (RSD), as determined from the calibration standards for PFOS was <5.08%. The relative error (RE) of the assay, as determined from the analysis of the quality control samples ranged from 2.38 to 12.7%. The RE o f the assay, as determined from the analysis of calibration standards ranged from -5.31 to 10.5%. . 15 Catherwood Road Ithaca, New York 1 4 8 5 0 ( 6 0 7 ) 2 6 6 r 0 6 6 5 Fax ( 6 0 7 ) 2 6 6 - 0 7 4 9 3M Environmental Laboratory Page 103 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Title: ADVANCED BIOANALYTICAL SERVICES SIGNATURE PAGE Quantitative Determination of Perfluorooctanesulfonate (PFOS) in Rat Serum from Study #FACT-TOX-l 11 Using Turbo Ion Spray LC/MS ABS Report No.: Reported by: IK T J Date Reviewed by: Authorized for Release by: Melissa MM aanpfetss, AA .SS. f Associate Auditor ^(2 ^ -- John R | Perkins, Ph.D. Assistant Scientific Director Accepted by: Kris Hanson, Ph.D. 3M Study Director i/ T W Date fl. Date Date ADVANCED BIO ANA LYTICAL S E R V IC E S . IN C . 3M Environmental Laboratory Page 2 o f 19 99ADJA03.MI.DQC Page 104 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 QAU STATEMENT Periodic inspections o f the bioanalytical portion o f study #FACT-TOX-l 11 were conducted by the Quality Assurance Unit of Advanced BioAnalytical Services (ABS) for compliance with EPA GLP regulations (40 CFR Part 792). The study was inspected on the following dates: 25 June 1999; 7, 21 July 1999; 31 August 1999. Results o f the inspection were reported to ABS Management and Project Team Leader on: 25 June 1999; 7,21 July 1999; 31 August 1999. Results of the inspections ware reported to the Study Director on 2 September 1999. A Based on the inspections and the data reviewed, this report is a complete and accurate representation o f the data. Melissa Mapes, A.S; Associate Auditor lt> C T < Date ADVANCED BIO ANA LYTICAL S E R V IC E S , IN C . 3M Environmental Laboratory Page 3 of 19 99ADJA03.MI.DOC Page 105 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 -.1 . TABLE OF CONTENTS SIGNATURE PAGE............... 2 QAU STATEMENT................................................................................................................3 TABLE OF CONTENTS........................................................................................................ 4 LIST OF TABLES........................................................................ ........... ................ .'............ 5 1. INTRODUCTION.......................................................................................................... 6 1.1. Study Description and Objective................................................................................6 2. METHODS...'.................................................................. 6 2.1. Analytical Procedure(s).................................................................. ;...........................6 2.2. Assay Site......................................................................................................... 7 2.3. Data Processing........................................................................................................... 7 . 3. RESULTS......................... 7 3.1. Assay Performance..................................................................................................... 7 3.2. Analytical Results for Study #FACT-TOX-l 11..... 8 4. SUMMARY AND CONCLUSIONS.................................................... 8 5. DATA RETRIEVAL......................................................................... 8 6. REFERENCES............................................................................................................... 8 7. TABLES......................................................................................................................... 9 ADVANCED B IO A N A L Y T IC A L S E R V IC E S . INC. 3M Environmental Laboratory Page 4 of 19 99ADJA03.MI.DOC Page 106 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 LIST OF TABLES Table 1: Summary o f Sample and Assay Information for Protocol FACT-TOX-111..... 9 Table 2: Inter-Assay Precision and Accuracy for PFOS Quality Control Samples in Rat Serum for Study #FACT-TOX-l 11...................................................................10 Table 3: Inter-Assay Precision and Accuracy for PFOS Calibration Standards in Rat Serum for Study #FACT-TOX-l 11........................................................ .......... 11 Table 4: Calibration Curve Statistics for the Determination of PFOS in Rat Serum for Study #FACT-TOX- 111...................................................................................... 12 Table 5: Concentrations o f PFOS in Rat Serum Samples from Study #FACT-TOX-l 11 Following the 0 mg/kg Dose................................................................................13 Table 6: Concentrations o f PFOS in Rat Serum Samples from Study #FACT-TOX-l 11 Following the 0.1 mg/kg Dose..................... 15 Table 7: Concentrations of PFOS in Rat Serum Samples from Study #FACT-TOX-l 11 *' Following the 1.6 mg/kg Dose.................................................. ........................ 17 Table 8: Repeat Analysis of Rat Serum Samples from Study #FACT-TOX-l 1 1 ..........19 ADVANCED BIO ANA LYTICAL S E R V IC E S . IN C . 3M Environmental Laboratory Page 5 of 19 99ADJA03.MI.DOC Page 107 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FA C T T O X -1 11 L R N -U 2 9 9 4 1. INTRODUCTION 1.1. Study Description and Objective The objective o f this study was to determine the concentrations of perfluorooctanesulfonate (PFOS) in rat serum samples collected during the study #FACT- TOX-111, "Oral (Gavage) Pharmacokinetic Recovery Study o f PFOS in Rats." Rat serum samples were analyzed using a liquid-liquid extraction and turbo ion spray liquid chromatography/mass spectrometry (LC/MS) assay. . 2. METHODS 2.1. Analytical Procedure(s) - PFOS concentrations were determined in rat serum according to the validated LC/MS method (1). Serum samples (50 pL) were extracted by a liquid-liquid extraction procedure using ethyl acetate to isolate PFOS and the internal standard (IS), 1_H,lH,2H,2H-perfluorooctane sulfonic acid (Tetra-H-PFOS) from rat serum. Following evaporation to dryness and subsequent reconstitution, sample extracts were separated by reversed-phase chromatography on a 2 x 50 mm (5 pm) BetasilTM Clg column (Keystone Scientific, Inc., Bellefonte, PA) with an initial mobile phase o f 55% Eluent A (10:90 methanol:2 mM ammonium acetate) and 45% Eluent B (90:10 methanol:2 mM ammonium acetate). PFOS concentrations were determined by turbo ion spray liquid chromatography/mass spectrometry (LC/MS) in selected ion monitoring (SIM) mode. The negative ions monitored were: m/z = 499.0 for PFOS [M-K]m/z = 427.0 for Tetra-H-PFOS [M-H]' Study sample concentrations were determined from a weighted (1/y2), quadratic regression o f peak area ratios (peak area of PFOS/peak area o f Tetra-H-PFOS) versus nominal concentration often calibration standards. The nominal concentrations of the calibration standards were 0.0 5 ,0 .1 ,0 .2 5 ,0 .5 ,1 ,2 ,4 , 8,16, and 20 pg/mL PFOS. The lower limit o f quantitation (LLQ) for this assay was determined to be 0.05 pg/mL PFOS. Quality control (QC) serum samples at three different concentrations (0.2, 6, and 18 pg/mL PFOS) were analyzed with each assay batch in replicates o f four. Dilution QC samples (QC4, 100 pg/mL) were prepared at each dilution that was used during a particular assay and were analyzed with each assay batch in replicates of four as needed. The acceptance criteria for calibration standards stipulated that the back-calculated concentrations o f at least three-fourths o f the individual.calibration standard replicates must not deviate more than 15% from their nominal concentration (except at the LLQ). At least one duplicate at the LLQ must exhibit a deviation within 20% of the nominal ADVANCED BIOANALYTICAL S E R V IC E S . INC. Page 6 o f 19 99ADJA03.Ml.DOC Page 108 3M Medical Departm ent Study: T-6395.14 ., BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 concentration. The coefficient of determination (r2) must be >0.98. The acceptance criteria followed are in accordance with ABS Standard Operating Procedures. The acceptance criteria for the quality control samples stipulated that at least two-thirds of the individual QC sample replicates must not deviate more than 15% from their nominal concentrations. At least one replicate at each QC concentration must exhibit a deviation within 15%. A summary of the sample and assay information for study #FACT-TOX-l 11 is given in Table 1. 2.2. Assay Site All samples were analyzed at Advanced BioAnalytical Services, Inc., Ithaca, NY. 2.3. Data Processing The data were collected using turbo ion spray LC/MS selected ion monitoring (SIM) in the negative ion mode. Peak areas were integrated by the PE SCIEX program MacQuan, version 1.4, residing on a Macintosh computer. Following peak area integration, the results tables from MacQuan were saved as text files and uploaded to the ABS file server where a weighted (1/y2) quadratic regression was performed using the software package Watson (v 5.3.1.01 PSS, Inc., Wayne, PA 19087). Calculations were performed on unrounded numbers. All calibration standard and QC results were rounded to no less than three significant figures before reporting. 3. RESULTS 3.1. Assay Performance The performance o f the assay for PFOS as determined from the analysis o f daily quality control samples is documented in Table 2. The inter-assay precision (RSD) o f quality control samples ranged from 2.78 to 3.61% for PFOS. There was no marked inaccuracy in the results from these quality control samples; the relative error (RE) ranged from 2.38 to 12.7%. The performance of daily calibration curves is documented in Table 3. The inter-assay precision (RSD) o f the standards ranged from 1.52 to 5.08% for PFOS. There was no marked inaccuracy in the results from these standards; the relative error (RE) ranged between-5.31 to 10.5%. ADVANCED B IO A N A L Y T IC A L S E R V IC E S . INC. 3M Environmental Laboratory Page 7 of 19 99ADJA03-MI.DOC Page 109 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FA C T T O X -1 11 L R N -U 2 9 9 4 The slope, y-intercept, and coefficient of determination (r2) for each analytical run are presented in Table 4. The r2 values ranged from 0.9959 to 0.9977 for PFOS in rat serum. All ABS personnel assigned to analyze samples for this project were required to successfully extract quality control (QC) samples and calibration standards. Results of the extraction were reviewed by a trained analyst against the pre-defined acceptance criteria for this assay. Results are maintained in the study records of ABS. 3.2. Analytical Results for Study #FACT-TOX-l11 PFOS concentrations in rat serum from Study #FACT-TOX-l 11 are presented in Tables 5 through 7. All data were rounded to no less than three significant figures before reporting in Tables 5 through: 7. Study samples requiring repeat analysis for Study #FACT-TOX111 are presented in Table 8. 4. SUMMARYANDCONCLUSIONS The concentration of PFOS was determined in rat serum samples collected during the study #FACT-TOX-l 11. ' PFOS was isolated from serum by a liquid-liquid extraction procedure and determined by LC/MS. The quality of the determinations was satisfactory throughout. The LLQ was 0.05 pg/mL PFOS. The precision of the assay, as determined from the analysis o f quality control samples was 3.61% for PFOS. 5. DATARETRIEVAL The calculated concentration data from the analysis o f rat serum samples for Study #FACT-TOX-l 11 are maintained on file in the archives of the Advanced BioAnalytical Services, Inc., Ithaca, NY in ABS Notebook 2325. 6. REFERENCES 1. Advanced BioAnalytical Services Validation Report 99VDJA01 .MI.DOC. Method Validation for the Quantitation of Perfluorooctanesulfonate (PFOS) in Rat Serum by Turbo Ion Spray LC/MS. David J. Anderson, M.S. Amie J. Prince, B.S., and Holly D. Ross, M.S. 26 August 1999. ADVANCED BIOANALYTICAL S E R V IC E S , INC, Page 8 of 19 99ADJA03.MI.DOC Page 110 3M Medical Department Study: T -6 3 9 5 .14 ;, BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 ** . v 7. TABLES Table 1: Summary of Sample and Assay Information for Protocol FACT-TOX- 111 Species/Matrix: Rat/Serum Sample Collection and Storage Information: Anticoagulant/Stabilizer: Reported Sample Collection Dates: Dates Received at ABS: Storage Temperature at ABS: " None 12 January 1999 to 20 February 1999 16 June 1999 -2 0 C Assay Information: Assay Period: 9 July 1999 to 14 July 1999 Analyte: . Potassium perfluorooctanesulfonate (PFOS) Analytical Standard: Lot No; Source: PFOS 171 3M Internal Standard: Lot No: Source: 1H,1H,2H,2Hperfluorooctane sulfonic acid (Tetra-H-PFOS) 59909 3M Calibration Range: Regression Method: Weighting Factor: Lower Limit of Quantitation: Upper Limit of Quantitation: quadratic i/y2 0.05 pg/mL 20 pg/mL . ADVANCED I M f f l BIOANALYTICAL r if lB B H S E R V IC E S . INC. Page 9 of 19 99ADJA03.MI.DOC Page 111 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Table 2: Inter-Assay Precision and Accuracy for PFOS Quality Control Samples in Rat Serum for Study #FACT-TOX-lll PFOS Concentration (pg/mL) Run No. QCl 0.2 QC2 QC3 QC4 QC4 6 18 100 100 (1 in 10 Dilution) (1 in 20 Dilution) 1 0.232 6.17 18.2 0.212 6.28 18.4 0.210 6.11 18.2 0.216 5.97 18.2 NA ' NA NA NA NA NA NA NA 2 0.216 6.00 18.1 0.227 6.09 19.0 0.224 6.07 19.8 0.219 6.18 18.5 NA NA NA NA NA NA NA NA 3 0.211 5.97 18.8 0.210 5.84 18.2 116 114 NA NA 0.217 6.07 18.8 113 NA 0.214 5.99 17.9 106 NA 4 0.222 6.48 19.3 0.233 6.23 19.7 0.214 6.54 19.6 0.225 6.30 18.5 NA NA NA NA 112 109 117 113 Mean 0.219 6.14 18.7 RSD (%) 3.43 3.08 3.28 RE 9.39 2.38 3.86 112 3.61 12.3 113 2.78 12.7 RSD = (SD/Mean) x 100% RE = [(Mean-Nominal)/Nominal] x 100% NA: Not applicable. . ADVANCED BIO ANA LVTICAL S E R V IC E S . IN C . 31vr Envlrcnmtsnial Labuialury Page 10 o f 19 99ADJA03 .MI.DOC Page 112 3M Medical Department Study: T -6 3 9 5 .14 Table 3: Inter-Assay Precision and Accuracy for PFOS Calibration Standards in Rat Serum for Study #FACT-TOX-l 11 Run No. STD1 0.05 STD2 0.1 STD3 0.25 1 0.0496 0.0993 0.238 0.0513 0.101 0.243 2 0.0470 0.101 0.244 0.0538 0.101 0.244 3 0.0490 0.105 0.254 0.0511 0.0980 0.242 4 0.0522 0.0950 0.246 0.0491 0.104 0.246 Mean 0.0504 RSD (%) 4.24 RE 0.775 0.100 3.04 0.425 0.245 1.80 -2.16 RSD = (SD/Mean) x 100% RE = [(Mean-Nominal)/Nominal] x 100% PFOS Concentration (p.g/mL) STD4 STD5 STD6 STD7 0.5 1 2 4 0.493 0.957 2.13 4.39 0.491 0.963 2.12 4.38 0.542 0.899 2.03 4.30 0.539 0.928 2.03 4.43 0.482 0.980 2.15 4.49 0.487 0.937 2.01 4.39 0.484 0.953 2.11 4.47 0.481 . 0.958 2.13 4.50 0.500 0.947 2.09 4.42 5.08 2.66 2.67 1.52 -0.0325 -5.31 4.49 10.5 STD8 8 7.72 7.76 7.67 7.77 7.80 7.39 7.59 7.89 7.70 U98 -3.76 STD9 16 16.0 15.5 16.0 15.8 16.9 15.8 15.9 15.3 15.9 2.98 -0.651 STD 10 20 20.4 20.0 20.6 19.8 20.4 19.2 20.6 20.0 20.1 2.34 0.626 Analytical Report: F A C T T O X -1 11 LRN-U2994 Page 11 of 19 99ADJA03.M1.DOC > 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Table 4: Calibration Curve Statistics for the Determination of PFOS in Rat Serum for Study #FACT-TOX-lll Run No. Quadratic Linear Coefficient (A) Coefficient (B) 1 -0.0004154 0.1025 2 -0.0005419 0.1001 3 -0.0006070 0.1046 4 -0.0002665 0.1036 Mean -0.0004577 0.1027 y = Ax2+ Bx + C, weighted 1/y2 Intercept (C) -0.0006090 -0.0004172 -0.0001313 -0.0004533 -0.0004027 Coefficient of Determination ....... (i3) 0.9977 0.9959 0.9968 0.9966 0.9968 S ADVANCED BIO ANALYTICAL S E R V IC E S . IN C . 3M Environmental Laboialuiy Page 12 of 19 99ADJA03.MI.DOC Page 114 3M Medical Departm ent Study: T-6395.14 CUO lInD O> < > n ln 553 Table5: Concentrations ofPFOS in Rat SerumSamples fromStudy#FACT-TOX-l11 Followingthe 0 mg/kg Dose Sampling Time (day) 13726 13726P 13727 13727P PFOS Concentration (ug/mL) Rat Number* 13728 13728P 13731 13734 0 0.215 NS 0.121 NS 0.0930 NS 0.0842 0.118 7 0.176 NS 0.0835 NS 0.0711 NS 0.0873 0.0644 14 0.0822 NS 0.0638 NS 0.0530 NS NS 0.0594 15\ 21 22 0.168 NS 0.0932 NS 0.0605 NS 0.0610 0.0665 0.0849 0.0712 0.0587 <LLQ[0:04521<(0.05) <LLQ[0.0385]<(0.05) <LLQ[0.0343]<(0.05) NS <LLQ[0.0390]<(0.05) 0.0597 NS 0.0517 NS <LLQ[0.0431]<(0.05) NS NS <LLQ[0.0446]<(0.05) *Rat Number with a "P" indicates pup sample. <LLQ = Less than the Lower Limit of Quantitation (0.05 pg/mL). NS: No sample. , Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 "0 Page 13 of 19 99ADJA03.MLDOC (<QD > 3M Medical Departm ent Study: T-6395.14 3M ElWtlOlili inula! Laburalmy------------------- ami 0n3O> nm"'><chnoD3 7n Table5: (Continued) Concentrations ofPFOS in Rat SerumSamples fromStudy#FACT-TOX-l11 Followingthe Omg/kgDose Sampling Time (day) 13734P 13735 PFOS Concentration (pg/mL) Rat Number* 13735P 13736 13736P 13737 13737P 0 NS 0.0947 N S ' 0.113 NS 0.0877 NS 7 NS 0.0700 NS 0.108 NS 0.0767 NS 14 NS <LLQ[0.0492]<(0.05) NS <LLQ[0.0474]<(0.05) NS 0.0553 NS 15 NS 0.0574 NS 0.0693 NS 0.111 NS 21 <LLQ[0.0409]<(0.05) <LLQ[0.0418]<(0.05) <LLQ[0.0401]<(0.05) <LLQ[0.0491]<(0.05) <LLQ[0.0409]<(0.05) 0.0531 0.0518 22 NS <LLQ[0.0370]<(0.05) NS 0.0592 NS 0.0570 NS *Rat Number with a "P" indicates pup sample. . <LLQ = Less than the Lower Limit of Quantitation (0.05 pg/mL). NS: No sample. Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Page 116 Page 14 of 19 99ADJA03.MI.DOC 3M Medical Departm ent Study: T-6395.14 3M Environmental Labomluiy---------------- CD03 > 559 ni -tndS 7n Table 6: Concentrations ofPFOSin Rat SerumSamples fromStudy#FACT-TOX-l11 Followingthe'0.1 mg/kgDose Sampling Time (day) 13739 13739P PFOS Concentration (pg/mL) Rat Number* 13740 13740P 13741 13741P 13744 13744P 13745 13745P 13746 13746P 0 9.78 NS 12.1 NS 11.6 NS 9.88 NS 9.88 NS 11.1 NS 7 9.50 NS 7.85 NS 7.59 NS 9.18 NS 7.40 NS 8.49 NS 14 1.82 NS 1.99 NS 1.51 NS 1.96 NS 2.55 NS 2.03 NS 15 5.88 NS 7.83 NS 6.67 NS 6.04. NS 5.80 NS 6.65 NS 21 1.81 2.34 4.00 2.12 . 2.69 1.88 3.04 2.29 2.34 1.88 2.84 2.37 22 1.33 NS 1.47 NS 1.19 NS 0.882 NS 1.09 NS 0.945 NS *Rat Number with a "P" indicates pup sample. NS: No sample. Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Page 117 Page 15 of 19 99ADJA03.M1.DOC > 04 tnto> IIIB I R D n"m>cd<omn 7n U Table6: (Continued) Concentrations ofPFOS inRat SerumSamples fromStudy#FACT-TOX-lll Followingthe 0.1 mg/kgDose Sampling Time (day) PFOS Concentration (ug/mL) Rat Number* 13748 13748P 13749 13749P 0 10.2 NS 10.7 ' NS 7 NS NS 8.61 NS 14 1.35 NS 1.87 NS 15 5.36 NS 8.40 NS 21 3.94 1.74 3.27 2.07 22 0.942 NS 1.22 . NS *Rat Number with a "P" indicates pup sample. NS: No sample. CQoD'. 0 *(m30D- 3 (f)C31D *Ca--< 0cccoon5 >3CD o3.' 73 CO3oD- 73 O>--1 DO >o (10Q3] CD Page 16 of 19 99ADJA03.M1.DOC OH co X` H O 3M Medical Departm ent Study: T -6395.14 SivrtiWiranrnuMim L a b o r a t o r y ------------------------------- UcolmnU> S ll S <ims H 7n o Table 7: Sampling Time (day) 0 7 14 15 21 22 Concentrations ofPFOSin Rat SerumSamples fromStudy#FACT-TOX-l11 Followingthe 1.6mg/kgDose 13751 PFOS Concentration (pg/mL) Rat Number* 13751P 13752 13752P 13753 13753P 13754 13754P 13755 13756 13756P 13758 155 NS 164 NS 213 NS 198 NS 191 188 NS 188 149 NS 169 NS 157 NS 152 NS 138 158 NS 143 21.2 NS 24.1 NS 21.6 NS 21,2 NS NS 31.0 NS 27.7 95.6 NS 109 NS 112 NS 103 NS 125 108 NS 86.9 38.5 35.0 34.8 33.9 32.6 28.3 37.1 23.4 112 40.4 47.4 37.0 17.1 NS 19.5 NS 18.2 NS 9.48 NS NS 19.9 NS 14.3 *Rat Number with a "P" indicates pup sample. NS: No sample. Analytical Report: FA C T T O X -1 11 L R N -U 2 9 9 4 Page 119 Page 17 o f 19 99ADJA03.MI.DOC CO m3< O3*j CD s3 0) a o--t OCD 5 Table 7: (Continued) Concentrations ofPFOS in Rat SerumSamples fromStudy#FACT-TOX-lll Followingthe 1.6 mg/kg Dose Sampling Time (day) PFOS Concentration (ug/mL) Rat Number* 13758P 13759 13759P 0 NS 7 NS 14 NS 15 NS 21 25.6 22 NS 198 133 20.4 99.4 33.0 15.4 NS NS NS NS 26.3 NS . *Rat Number with a "P" indicates pup sample. NS:.No sample. CQD. o' D CD ~03o) 3 CD 3 *rca--< 7O00)1 CaDi > D rt1 oD' 73 *CO3oDr* 5 00 > o * (T0Q)3 CD Page 18 of 19 99ADJA03.MI.DOC sj 4Sk ' o hO o> Table8: Repeat AnalysisofRat SerumSamples fromStudy#FACT-TOX-lll Rat Treatment Time Number Original Cone. Pg/ml Original Curve Number 13753 3 Day 0 >ULQ-222.9138>(200) 3 Reason for Reassay 1 Reassay Cone. pg/ml t 213 Reassay Curve Number 4 Reported Cone. Uft/ml 213 Reason for Reported Cone. 1 >ULQ: Greater than the Upper Limit of Quantitation. REASONS FOR REASSAY: 1). Run 3 concentration exceeded calibration range. REASONS FOR REPORTED CONC: 1). Run 4 concentration within calibration range. -<CcO Cl 7! 05 CtoO at >3 *Q<J_ "(O3OD. 7Zf n>O c1 DO > o * "0 c0<n5D Page 19 of 19 99ADJA03.MLDOC C<tOD* o > BACK TO MAIN 3M Medical Department Study: T -6395.14 ADVANCED B ID A N A L Y T IC A L S E R V IC E S , IN C . ! ' Analytical Report: FACT T O X -111 LRN-U2994 M ETHO D V A L ID A T IO N REPORT TITLE: METHOD VALIDATION FOR THE QUANTITATION OF PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/MS DATE: 26 August 1999 REPORT: 99VDJA01.MI.DOC AUTHORS: David J. Anderson, M.S. Amie J. Prince, B.S. Holly D. Ross, M.S. PREPARED FOR: 3M Environmental Technology and Safety Services St. Paul, MN 55133-3331 NUMBER OF PAGES: 50 15 C a th e rw o o d R oad Ith a c a , N ew York 1 4 8 5 0 (6 0 7 ) 2 6 6 -0 6 6 5 F ax (6 0 7 ) 2 6 6 -0 7 4 9 3M Environmental Laboratory Page 122 BACK TO MAIN 3M Medical Department Study: T-6395.14 AUTHORS: FOR: DATE: TITLE: Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 David J. Anderson, M.S. Amie J. Prince, B.S. Holly D. Ross, M.S. 3M Environmental Technology and Safety Services 26 August 1999 METHOD VALIDATION FOR THE QUANTITATION OF PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/MS ABSTRACT A sensitive, specific, accurate, and reproducible analytical method was developed by Advanced BioAnalytical Services, Inc., Ithaca, New York to quantitate perfluorooctanesulfonate (PFOS) in rat serum samples. Serum samples (50 pL) were extracted by a liquid-liquid extraction procedure to isolate the analyte from rat serum. Sample extracts were reduced to dryness, reconstituted, and analyzed by turbo ion spray liquid chromatography/mass spectrometry (LC/MS) in the negative ion mode. The assay demonstrated a lower limit of quantitation (LLQ) of 0.05 pg/mL using 50-pL sample aliquots. The calibration curves were fit from 0.05 pg/mL to 20 pg/mL for PFOS by a weighted (1/y2) quadratic equation. The coefficients of determination of the calibration curves ranged from 0.9962 to 0.9965. Precision and accuracy quality control (QC) samples were prepared at concentrations of 0.2, 6, and 18 pg/mL PFOS. Quality control (QC) samples were prepared at a concentration o f 100 pg/mL PFOS for partial volume analysis. The intra- and inter assay precision (RSD) results calculated from all QC samples ranged from 1.92% to 4.87% for PFOS. The intra- and inter-assay accuracies (RE) calculated from QC samples ranged from -3.58% to 5.58% for PFOS. The mean extraction recoveries were from 87.6% to 100% for PFOS and 89.9% for the internal standard (IS). PFOS was measured as stable in rat serum for up to 24 hours at ambient temperature. PFOS was measured as stable in rat serum at -20 C, currently for up to 33 days, and 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S , INC. Page 2 of 50 99VDJA01 .MI.DOC Page 123 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 after three freeze/thaw cycles. Reliable results were obtained for sample extracts reinjected 27 hours after initial reconstitution. 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES. INC. Page 3 of 50 99VDJA01.MI.DOC Page 124 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 QAU STATEMENT Periodic inspections of the method validation for the quantitation of PFOS in rat serum were conducted by the Quality Assurance Unit o f Advanced BioAnalytical Services (ABS) for compliance with EPA GLP regulations (40 CFR Part 792). The study was inspected on the following dates: 13,21 May 1999; 7, 8,14,15 June 1999; 1,2 July 1999; 3 ,4 August 1999. Results of the inspections were reported to ABS Management on: 13,21 May 1999; 7, 8,14, 15 June 1999; 1,2 July 1999; 3 ,4 August 1999. Results of the inspections were reported to the Study Director on 5 August 1999. Based on the inspections and the data reviewed, this report is a complete and accurate representation of the data. Quality Auditor 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 4 of 50 99VDJA01.MI.DOC Page 125 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 SIGNATURE PAGE TITLE: METHOD VALIDATION FOR THE QUANTITATION OF PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/MS Report Number: Reported by: Zt,Mmsrm g Date Reviewed by: Associate Auditor Authorized for Release by: John R.fPerkins, Ph.D. Assistant Scientific Director TJcmlQEI Date vJ 2A A >AT.<]ct Date 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S . INC. Page 5 of 50 99VDJA01.MI.DOC Page 126 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 TABLE OF CONTENTS ABSTRACT....................................... .................................................................................. .2 QAU STATEMENT............ ..................................................... 4 SIGNATURE PAGE............................................:................................................................ 5 TABLE OF CONTENTS.................................................................. 6 LIST OF TABLES...................................................................................................i..............8 LIST OF FIGURES...... ........................................................................^............. :.................9 1. INTRODUCTION.............................................................................................. :......... 10 2. EXPERIMENTAL........................................................................... 2.1. CHEMICALS ANDMATERIALS.... ............................................................................. 10 2.2. LC/MS INSTRUMENTATION....................................................................................10 2.3. Sample Preparation and extraction procedu re................................. .....11 2.4. Rat Serum Validation Da ta ...,..........................................................................l l 2.5. Assay evaluation................................................................................................ 12 2.5.1. Intra- and Inter-Assay Accuracy................................................................. .....12 2.5.2. Intra- and Inter-Assay Precision......................................................................12 2.5.3. Partial Volume Analysis................................................................................... 12 2.5.4. Lower Limit o f Quantitation (LLQ)................................................ ............... 13 2.5.5. Selectivity.............. 13 2.5.6. Carryover Evaluation....................................................... 13 2.6. Stability of PFOS in quality Control Sa m ples.............................. ..........13 2.6.1 ' Ambient-Temperature Stability of PFOS in Rat Serum............................. 13 2.6.2. Freezer Stability of PFOS in Rat Serum at -2 0 C........................................ 14 2.6.3. Freeze/Thaw Stability in Rat Serum............................................................... 14 2.7. Reproducibility of reinjecting Extracted Sa m ples................................. 14 2.8. Extraction Recovery......................................................................................... 14 3. RESULTS AND DISCUSSION....................................................... .......:.................. 15 3.1. assay Evaluation r esu l ts......................................................... 16 3.1.1. Intra- and Inter-Assay Accuracy.................................................................... 16 ADVANCED BIOANALYTICAL S E R V IC E S . INC. 3M Environmental Laboratory Page 6 of 50 99VDJA01.MI.DOC Page 127 10 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 3.1.2. Intra- and Inter-Assay Precision.............. 16 3.1.3. Linearity.............................................. 16 3.1.4. Partial Volume Analysis................................................................................... 16 3.1.5. Lower Limit of Quantitation (LLQ)......... ......................................................17 3.1.6. Selectivity..................... 17 3.1.7. Carryover Evaluation....................................... 17 3.2. Stability of p f o s in Quality control Sa m ples.........:.............................. 17 3.2.1. Ambient Temperature Stability of PFOS in Rat Serum ................................ 17 3.2.2. Freezer Stability of PFOS in Rat Serum at -20 C......................................... 18 3.2.3. Freeze/Thaw Stability o f PFOS in Rat Serum................................................ 18 3.3. REPRODUCIBILITY OF REINJECTING EXTRACTED SAMPLES........................... ....18 3.4. EXTRACTION RECOVERY.................................... ................................................... 18 4. CONCLUSIONS............. ................................................................................. .....-.....19 5. DATA RETRIEVAL......................................................................................................19 6. REFERENCES......................................... 19 7. TABLES..................................................................................... 20 8. FIGURES.......................................... 31 9. APPENDIX A: QUANTITATION OF PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/M S................................... 37 3M Environmental Laboratory ADVANCED BIOANALYTIDAL SERVICES, INC. Page 7 of SO 99VDJAOt.MI.DOC Page 128 BACK TO MAIN 3M Medical Department Study: T-6395.14 LIST OF TABLES Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Table 1: Intra-Assay Accuracy and Precision of the PFOS Assay in Rat Serum for QC S a m p l e s ..................................................................................................... 20 Table 2: Inter-Assay Accuracy and Precision o f the PFOS Assay in Rat Serum for QC Samples from Three Validation R uns..........................................................21 Table 3: Accuracy and Precision o f the PFOS Assay in Rat Serum for Calibration Standards from Three Validation Runs............................................................... 22 Table 4: Calibration Curve Parameters for PFOS in Rat Serum ................................ .....23 Table 5: Partial Volume Analysis o f PFOS in Rat Serum............................................... 24 Table 6: Lower Limit of Quantitation of PFOS in Rat Serum.........................................25 Table 7: Ambient-Temperature Stability of PFOS in Rat Serum After 24 Hours..........26 Table 8: Freezer Stability o f PFOS in Rat Serum at -20 C .............................................27 Table 9: Stability of PFOS in Rat Serum After Three Freeze/Thaw Cycles...................28 Table 10: Reproducibility of Reinjecting Extracted Samples Containing PFOS after 27 Hours in Reconstitution Solution..................................................................29 Table 11: Extraction Recovery of PFOS from Rat Serum.................................. ...............30 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S , INC. Page 8 of 50 99VDJAOLMI.DOC Page 129 BACK TO MAIN 3M Medical Department Study: T-6395.14 LIST OF FIGURES Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Figure 1: Full-Scan Single MS Mass Spectrum o f PFOS................................................. 31 Figure 2: Full-Scan Single MS Mass Spectrum of Tetra-H-PFOS................................... 32 Figure 3: Mass Chromatograms of PFOS and its Internal Standard in Control Blank Rat Serum Extract............................................................................................... 33 Figure 4: Mass Chromatograms o f PFOS in a Rat Serum Extract Sample Containing Internal Standard Only (Zero Sample)......... ......................................................34 Figure 5: Mass Chromatograms of Calibration Standard 1 in Rat Serum Extract Containing PFOS (0.05 jig/mL) and the Internal Standard......................... .....35 Figure 6: Mass Chromatograms of Calibration Standard 10 in Rat Serum Extract Containing PFOS (20 pg/mL) and the Internal Standard................................. 36 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S , INC. Page 9 of 50 99VDJA01.MI.DOC Page 130 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 1. INTRODUCTION The purpose of this report is to describe the method validation for the quantitative determination o f perfluorooctanesulfonate (PFOS) in rat serum samples after modification o f a previous method (1). The objectives of the method validation were to validate a simple extraction procedure, determine the lower limit o f quantitation for routine analysis, determine the extraction recoveries of the analyte and internal standard (IS), determine the analyte stability in rat serum at ambient temperature, long-term freezer storage, and over three freeze/thaw cycles, and to provide specific, accurate, and reproducible quantitative results by turbo ion spray liquid . chromatography/mass spectrometry (LC/MS). . 2. EXPERIMENTAL 2.1. chemicals and Materials Perfluorooctanesulfonate (PFOS, Lot# 171) was obtained from 3M, Inc. The internal standard (IS) for PFOS was lH,lH,2H,2H-perfluorooctane sulfonic acid (Tetra-HPFS). The internal standard (Lot# 59909) was obtained from 3M, Inc. detailed list o f chemicals and materials is found in Appendix A. Stock and working solutions which were used to prepare the calibration curves for the analytes were prepared as described in Appendix A. Stock solutions used in the preparation o f quality control (QC) samples were prepared separately from those used in preparation of the calibration curves. Preparation of all solutions used during extraction and analysis are described in Appendix A. 2.2. LC/MS Instrumentation The liquid chromatography/mass spectrometry system consisted of two LC-10AD pumps (Shimadzu, Columbia, MD 21046), a SCL-10A pump controller (Shimadzu, ADVANCED BIOANALYTICAL S E R V IC E S , INC. 3M Environmental Laboratory Page 10 of 50 99VDJA01 .MI.DOC Page 131 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Columbia, MD 21046), a WISP 717plus autosampler (Waters Associates, Millipore Corporation, Milford, MA 01757), a Betasil C]g(2 x 50 mm, 5 pm) column (Keystone Scientific, Inc., Bellefonte, PA 16823), and a PE SCIEX API 365 mass spectrometer (PE SCIEX, Concord, Ontario). A detailed list o f the instrumentation and instrument conditions is found in Appendix A. 2.3. Sample Preparation and Extraction Procedure For each analytical run (tray), duplicate 50-pL aliquots o f the calibration curve samples were prepared as described in Appendix A. The nominal (theoretical) concentrations of PFOS in the calibration curves were 0.05,0.1,0.25,0.5, 1 ,2 ,4 , 8, 16, and 20 pg/mL. Rat serum quality control samples were prepared in advance of the validation study at nominal (theoretical) concentrations of 0.2, 6, and 18 pg/mL for QC1, QC2, and QC3, respectively, as detailed in Appendix A. A dilution QC (Q C 4,100 pg/mL) was prepared at a concentration exceeding the upper limit o f the calibration curve range (20 pg/mL), and was assayed using a 10-fold dilution for partial volume analysis. Calibration standards and QC samples were extracted by the procedure detailed in Appendix A. 2.4. Rat Serum validation data The data were collected using selected ion monitoring (SIM) turbo ion spray LC/MS in the negative ion mode. Peak areas were integrated by the PE SCIEX program MacQuan, version 1.4, residing on a Macintosh computer. Following peak area integration, the results tables from MacQuan were saved as text files and uploaded to the Advanced BioAnalytical Services (ABS) file server where a weighted (1/y2) quadratic regression was performed using the software package Watson v 5.3.1.01 (PSS, Inc., Wayne, PA 19087). All data were rounded to no less than three significant figures by ABS prior to reporting in Tables 1 through 11. The data for the rat serum validation are stored in ABS Notebook 2304. 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S . INC. Page 11 of SO 99VDJA01.MI.DOC Page 132 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 All calculations were based on the peak area ratio of the analyte to the internal standard. Concentrations of each analyte in quality control samples were determined by inverse prediction from the calibration curve. 2.5. Assay Evaluation 2.5.1. Intra-and Inter-Assay Accuracy The intra- and inter-assay accuracy of the method was assessed by determining the relative error observed in the analysis o f quality control samples. The mean concentration for each quality control level was divided by the theoretical concentration. One was subtracted from the result, converted to percent and expressed as RE. QC1 through QC3 were assayed in replicates o f five. In addition, the RE was reported for standards at all levels over three runs. 2.5.2. Intra- and Inter-Assay Precision The intra- and inter-assay precision o f the method was assessed by determining the Relative Standard Deviation (RSD) observed for quality control sample data. The mean concentration and RSD were calculated for the first run and over three runs for intra-assay and inter-assay precision, respectively. QC1 through QC3 were assayed in replicates o f five. In addition, the RSD was reported for standards at all levels over three runs. 2.5.3. Partial Volume Analysis The effect of dilution on the analysis of PFOS in rat serum was determined by partial volume analysis. QC4 (100 jig/mL) was prepared containing PFOS at approximately five times the upper limit o f quantitation (ULQ). Five replicates of QC4 were diluted ten-fold and analyzed. The RSD and RE were reported. 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES. INC. Page 12 o f 50 99VDJA01 .MI.DOC Page 133 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 2.5.4. Lower Limit ofQuantitation (LLQ) Rat serum control samples from six separate individuals were spiked with PFOS at a concentration of 0.05 pg/mL. The LLQ was determined by obtaining the backcalculated concentrations from these control samples. The overall mean, RSD, and RE were also calculated. 2.5.5. Selectivity The selectivity o f the assay was determined by LC/MS. To monitor for interference from the biological matrix, rat serum samples containing neither the analyte nor the internal standard (control blank) were assayed with all experiments. 2.5.6. Carryover Evaluation The carryover o f analyte from one injection to the next was assessed by analyzing a control blank injected immediately after ahigh calibration standard (STD10, 20 pg/mL).. 2.6. STABILITY OF PFOS IN QUALITY CONTROL SAMPLES QC1 through QC3 were used to determine the stability o f the analyte during sample storage, extraction, and analysis. 2.6.1. Ambient-Temperature Stability ofPFOS in Rat Serum The stability o f PFOS was evaluated by storing QC samples at each concentration level at ambient temperature (ca. 25 C) for nominal timepoints of 0 ,2 .5 ,6 , and 24 hours after thawing. All QCs were assayed in replicates of five. 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES. INC. Page 13 o f 50 99VDJA01 .MI.DOC Page 134 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 2.6.2. Freezer Stability ofPFOS in Rat Serum at -20 C The freezer stability of PFOS was evaluated by storing QC samples at each concentration level at -20 C for 7,13, and 33 days. Additional freezer stability timepoints will be reported in an addendum to this report. All QCs were assayed in replicates of four. 2.6.3. Freeze/Thaw Stability in Rat Serum The stability o f PFOS was evaluated after three freeze/thaw cycles. The mean concentrations of the QC samples after three freeze/thaw cycles (i.e., at least 2 hours frozen storage at the nominal temperature o f -20 C followed by thawing at ambient temperature for 30 minutes) were compared to the mean concentrations o f freshly thawed QC samples. All QCs were assayed in replicates of five. 2.7. reproducibility qf Reinjecting extracted Samples The reproducibility o f reinjecting reconstituted serum extracts was investigated by reinjecting a set of previously-assayed standards and QC samples which had been stored after injection at approximately 25 C for 27 hours. The RE of the QC samples was used to assess processed sample stability in the reconstitution solution. AH QCs were assayed in replicates of five. 2.8. EXTRACTIONRECOVERY The extraction recovery of PFOS from rat serum was determined by comparing the peak area ratio (PAR) of samples (0.25,4, and 16 pg/mL) spiked after extraction (post-extract) with the PAR of samples spiked before extraction (pre-extract). The internal standard was spiked post-extraction for all samples. The recovery of the internal standard (4 pg/mL) was assessed following a similar approach using PFOS as the reference. The extraction recovery (% Recovery) was determined by dividing the pre-extract PAR by the post-extract PAR and expressing the result as a percentage. Five replicates were used at each concentration level. 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 14 o f 50 99VDJA01.MI.DOC Page 135 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 . LRN-U2994 3. RESULTS AND DISCUSSION A quantitative analytical procedure using turbo ion spray LC/MS in the negative ion mode was developed to meet the high sensitivity, specificity, and reproducibility requirements for the determination of PFOS in rat serum. The firll-scan single MS mass spectrum of PFOS showed an abundant [M-K]' ion at m/z = 499 (Figure 1). The full-scan single MS mass spectrum of Tetra-H-PFOS showed an abundant [M-H]' ion at m/z = 427 (Figure 2). The following selected ion monitoring (SIM) was used to quantify the analytes in rat serum: PFOS / Tetra-H-PFOS (IS) m/z = 499.0 m/z = 427.0 The peak labeling of full-scan data does not accurately represent the performance o f the instrument in SIM mode. The SIM ions were derived from separate experiments using narrow-range scanning, which more accurately depicts the operation of the instrument in the SIM mode. The mass chromatograms o f a representative control blank rat serum extract are shown in Figure 3. Mass chromatograms from a representative control rat serum extract containing only the internal standard (zero sample), are shown in Figure 4. Figures 5 and 6 are mass chromatograms o f representative extracts from calibration standards 1 and 10, respectively. 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S . INC. Page 15 of 50 99VDJA01.MI.DOC Page 136 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 3.1. ASSAY EVALUATION RESULTS 3.1.1. Intra- and Inter-Assay Accuracy The intra-assay accuracy (RE) data from QC samples ranged from -2.35 to 5.58% for PFOS (Table 1). The inter-assay accuracy ranged from -3.58 to 4.95% for PFOS (Table 2). These data indicate acceptable intra- and inter-assay accuracy for the determination o f PFOS in rat serum. 3.1.2. Intra- and Inter-Assay Precision The intra-assay precision data (RSD) ranged from 1.99 to 3.28% for PFOS at all QC concentration levels (Table 1). The inter-assay precision results from QC samples ranged from 1.92 to 4.87% for PFOS (Table 2). The RSD ranged from 0.613 to 4.38% for calibration standards at all levels (Table 3). These data indicate acceptable intra-and inter-assay precision for the determination o f PFOS in rat serum. 3.1.3. Linearity The calibration curves were fit by a weighted (1/y2) quadratic regression. Coefficients of determination (r2) were >0.9962 for PFOS in rat serum. Die calibration curve statistics are shown in Table 4. 3.1.4. Partial VolumeAnalysis The results o f partial volume analysis of QC4 is shown in Table 5. The precision (RSD) of QC4 samples diluted 1 in 10 was 2.71%. The accuracy was 5.89%. These data indicate acceptable accuracy and precision for partial volume analysis for the determination o f PFOS in rat serum. 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S . INC. Page 16 of 50 99VDJA01 .MI.DOC Page 137 BACK TO MAIN 3M Medical Department Study: T-6395.14 ., Analytical Report: FACT T O X -111 LRN-U2994 3.1.5. Lower Limit ofQuantitation (LLQ) Table 6 shows the lower limit of quantitation (LLQ) data. The serum LLQ experiment demonstrated that 0.05 p.g/mL is an acceptable LLQ for PFOS. The precision (RSD) was 4.95% for PFOS. The RE was 4.57%. 3.1.6. Selectivity The assay was specific for PFOS. No chromatographic interferences were observed in any of the control serum samples analyzed (Figure 3). The control blanks and zero samples did show some evidence o f small chromatographic peaks at the retention times of the analyte. These peaks were not quantifiable as they were below the lower limit of quantitation (LLQ). 3.1.7. Carryover Evaluation Carryover of PFOS and the IS was evaluated by injection o f an extracted rat serum control blank following an injection of an extracted high standard (STD 10). The response for the small chromatographic peak at the retention time for PFOS was comparable to the response of a rat serum control blank injected before the high standard. Thus, carryover is negligible for PFOS. There was no evidence of carryover for the internal standard.' 3.2. STABILITYOFPFOS INQUALITYCONTROLSAMPLES 3.2.1. Ambient Temperature Stability ofPFOS in Rat Serum The results of the ambient stability of PFOS in QC samples are shown in Table 7. The mean predicted concentrations deviated from -4.45 to 10.5% from the 0-hour values for all QC levels and time points. Based on these data, PFOS was stable in rat serum for up to 24 hours at ambient temperature. 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S , INC. Page 17 ofSO 99VDJA01.MI.DOC Page 138 BACK TO MAIN 3M Medical Department Study: T-6395.14 . . Analytical Report: FACT T O X -111 LRN-U2994 3.2.2. Freezer Stability ofPFOS in Rat Serum at -20 C The results of the long-term stability of PFOS in QC samples stored at -2 0 C are presented in Table 8. The PFOS concentrations deviated from 0-hour values from -2.76 to 12.3% for all QC levels stored up to 33 days. Results o f freezer storage after two, four, and eight months will be reported as an addendum to this report. 3.2.3. Freeze/Thaw Stability ofPFOS in Rat Serum The results o f the freeze/thaw stability o f PFOS in QC samples after three freeze/thaw cycles is shown in Table 9. PFOS was stable after three freeze/thaw cycles with deviations ranging from-1.38% to 10.1% from the 0-cycle samples for all QC levels (Table 9). 3.3. Reproducibility of Reinjecting Extracted Samples The results of reinjecting reconstituted samples containing PFOS after storage for 27 hours at approximately 25 C in reconstitution solution are shown in Table 10. Reinjecting processed samples after 27 hours in reconstitution solution was appropriate with RE values ranging from -6.33 to 4.59% at 27 hours for all QC levels. 3.4. Extraction Recovery The mean recoveries o f PFOS and Tetra-H-PFOS are shown in Table 11. The mean recoveries ranged from 87.6 to 100% for PFOS. The mean recovery for Tetra-HPFOS was 89.9%. 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S . INC. Page 18 o f 50 99VDJA01.MI.DOC Page 139 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 4. CONCLUSIONS The turbo ion spray LC/MS assay procedure for the determination o f PFOS in rat serum has proven to be sensitive, specific, accurate, and reproducible. Its high sensitivity allows reliable and reproducible quantitation o f PEOS down to a level of 0.05 pg/mL in rat serum based on 50-jiL samples. 5. DATARETRIEVAL The data for the rat serum validation are stored in ABS Notebook 2304 and in the ABS Archives. 6. REFERENCES 1. Advanced BioAnalytical Services, Inc. Report 98AGKP02.MMM. Analytical Report for the Determination of Perfluorooctanoate and Perfluorooctanesulfonate in Human Serum by LC/MS. Grace K. Poon, Ph.D., David Hardwick, B.S., and Ellen Pace, M.A.T., 6 February 1998. ADVANCED BIOANALYTICAL SERVICES. INC. 3M Environmental Laboratory Page 19 o f50 99VDJA01 .MI.DOC Page 140 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 7. TABLES Table 1: Intra-Assay Accuracy and Precision ofthe PFOS Assay in Rat Serumfor QC Samples Theoretical Cone. Run 9 PFOS Concentration Qig/mL) QC1 QC2 QC3 0.2 6 18 0.210 6.52 17.9 0.202 6.31 18.0 0.206 6.17 16.7 0.205 6.32 18.0 0.194 6.35 17.3 Mean RSD (%) RE (%) 0.204 6.34 17.6 2.93 1.99 3.28 1.77 5.58 -2.35 RSD = (SD/Mean) x 100 RE = [(Mean/Theoretical)-l] x 100 . 3M Environmental Laboratory ADVANCED B IO A N A L Y T IC A L S E R V IC E S , INC. Page 20 of 50 99VDJA01.MI.DOC Page 141 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Table 2: Inter-Assay Accuracy and Precision ofthe PFS Assay in Rat Serumfor QC Samples fromThree Validation Runs PFOS Concentration (pg/mL) Theoretical Cone. Run 9 QC1 0.2 0.210 0.202 0.206 0.205 0.194 QC2 6 6.52. 6.31 6.17 6.32 6.35 QC3 18 17.9 18.0 16.7 18.0 17.3 Run 10 Run 11 0.187 0.186 0.182 0.189 0.182 0.194 0.197 0.184 0.192 0.184 6.44 6.44 6.32 6.19 6.31 6.17 6.13 6.41 6.15 6.24 16.9 17.3 17.5 18.0 17.4 18.2 17.1 17.6 17.7 17.0 Mean 0.193 6.30 17.5 RSD (%) 4.87 1.92 2.66 RE(%) -3.58 4.95 -2.73 RSD = (SD/Mean) x 100 RE = [(Mean/Theoretical)-1] x 100 3M Environmental Laboratory ADVANCED BIDANALYTICAL S E R V IC E S , INC. Page 21 of 50 99VDJA01.MI.DOC Page 142 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Table 3: Accuracy and Precision ofthe PFOS Assay in Rat Serum for Calibration Standards fromThree Validation Runs i iiiuhh-- itTirnr" M n im n aiiiim m n n " i ......... ' .... PFOS Calibration Standard Concentration (ng/mL) Theoretical STD1 STD2 STD3 STD4 STD5 STD6 STD7 STD8 Cone. 0.05 0.1 0.25 0.5 1 2 4 8 Run 9 0.0483 0.0972 0.251 0.480 0.942 2.10 4.48 7.61 0.0530 0.101 0.252 0.483 0.938 2.13 4.55 7.70 Run 10 0.0491 0.0951 0.239 0.469 0.954 2.16 4.58 8.05 0.0542 0.101 0.251 0.479 0.942 2.11 4.43 7.87 Run 11 0.0510 0.0960 0.251 0.469 0.945 2.21 4.40 7.64 0.0510 0.0995 0.252 0.477 0.948 2.13 4.53 7.78 Mean 0.0511 0.0983 0.249 0.476 0.945 2.14 4.50 7.78 RSD (%) 4.38 2.65 2.07 1.15 0.613 1.90 1.62 2.13 RE (%) 2.20 -1.67 -0.207 -4.78 -5.53 7.05 12.4 -2.80 RSD = (SD/Mean) x 100 RE = [(Mean/Theoretical)-l] x 100 STD9 STD10 16 20 15.8 20.0 16.2 19.9 15.9 19.6 15.7 20.0 15.8 20.3 15.6 20.0 15.8 20.0 1.22 1.08 -1.00 -0.0764 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S , INC. Page 22 of SO 99VDJA01.MI.DOC Page 143 BACK TO MAIN 3M Medical Department Study: T-6395.14 ,. Analytical Report: FACT T O X -111 LRN-U2994 Table 4: Calibration Curve Parameters for PFOS in Rat Serum Rim# Quadratic Coefficient Coefficient (A)" Linear (B) 9 -0.0010769 0.13730 10 -0.0010516 0.14535 11 -0.0012359 '0.15173 Mean -0.0011215 0.14479 a: y = Ax2+Bx + C, weighted 1/y2 Intercept Coefficient of (C) Determination (r2) 0.00042009 0.9965 0.0014910 0.9962 0.00088515 0.9964 0.00093206 0.9964 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES. INC. Page 23 of 50 99VDJA01 .MI.DOC Page 144 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Table 5: Partial VolumeAnalysis ofPFOS in Rat Serum Theoretical Cone. Run 9 PFOS Cone. (|ig/mL) QC4 100 Cl in 10 dilution) 111 105 105 104 104 Mean RSD (%) RE (%) 106 2.71 5.89 RSD = (SD/Mean) x 100 RE = [(Mean/Theoretical)-!] x 100 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 24 o f 50 99VDJA01 .MI.DOC Page 145 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Table 6: Lower Limit ofQuantitation ofPFOS in Rat Serum Run 11 PFOS Theoretical Concentration Cone. Found (pg/mL) 0.0548 LLQ (0.05 pg/mL) 0.0489 0.0499 0.0554 0.0527 0.0520 " Mean RSD (%) RE (%) 0.0523 4.95 4.57 RSD = (SD/Mean) x 1Q0, RE = [(Mean/Theoretical)-l] x 100 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S , INC. Page 25 o f 50 99VDJA01.MI.DOC Page 146 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Table 7: Ambient-Temperature Stability ofPFOS in Rat SerumAfter 24 Hours Run 11 QC (Theoretical Cone.) OHour PFOS (pg/mL) 2.5 Hour 6 Hour 24 Hour QC 1 (0.2 pg/mL) 0.194 0.197 0.184 0.192 0.184 0.187 0.181 0.184 0.180 0.178 0.177 0.179 0.180 0.183 0.189: 0.190 0.187 0.191 0.182 0.190 Mean RSD (%) %DEV from 0 Hour 0.190 3.03 NA 0.182 1.91 -4.20 0.182 2.52 -4.45 0.188 2.01 -1.16 6.17 6.17 6.36 6.55 QC 2 (6 pg/mL) 6.13 6.30 6.38 6.53 6.41 6.26 6.35 6.85 6.15 6.28 6.22 6.66 6.24 6.24 6.11 6.81 Mean RSD (%) %DEV from 0 Hour 6.22 6.25 6.28 1.82 0.797 1.86 NA 0.525 1.05 6.68 2.16 7.42 QC 3 (18 pg/mL) 18.2 18.0 18.1 19.1 17.1 18.0 18.0 19.4 17.6 17.7 17.6 19.2 Mean RSD (%) %DEV from 0 Hour .17.7 17.9 18.2 17.0 18.0 18.0 17.5 17.9 18.0 2.84 0.719 1.26 NA 2.33 2.39 19.4 19.7 19.4 1.19 10.5 NA: Not Applicable RSD = (SD/Mean) x 100 %DEV from 0 Hour = [(X Hour Cone. - 0 Hour Conc.)/0 Hour Cone.] x 100 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S , INC. Page 26 of SO 99VDJA01 .MI.DOC Page 147 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 L R N -U 2 9 9 4 Table 8: Freezer Stability ofPFOS in Rat Serum at -20 C QC Nominal Concentration QC 1 (0.2 ug/mL) Mean RSD (%) %DEV from 0 Hour OHour (Run 2)* 0.213 0.209 0.208 0.208 0.207 0.209 1.24 NA PFOS (ug/mL) 7 Days 13 Days (Run 6)* (Run 3)** 0.225 0.211 0.222 0.210 0.218 0.217 0.219 0.214 0.221 1.32 5.72 0.213 1.55 1.84 6.02 6.22 5.97 QC2 5.71 6.15 5.84 (6 ug/mL) 6.00 6.39 6.07 6.06 6.27 5.99 5.69 Mean 5.89 6.26 5.97 RSD (%) 3.08 1.62 1.58 %DEV from 0 Hour NA 6.19 1.20 QC3 (18 ug/mL)' Mean RSD (%) %DEV from 0 Hour 20.2 18.6 18.9 18.2 18.9 18.9 3.96 NA 19.1 18.8 19.5 18.2 19.8 18.8 19.3 17.9 19.4 18.4 1.37 2.42 2.52 -2.76 NA: Not Applicable RSD = (SD/Mean) x 100 %DEV from 0 Hour = [(Cone. - 0 Hour Conc.)/0 Hour Cone.] x 100 From Protocol FACT-TOX-110 From Protocol FACT-TOX-111 33 Days (Run 16) 0.245 0.234 0.228 0.232 0.235 3.17 12.3 6.45 6.13 6.58 6.37 6.38 2.97 8.28 18.6 18.8 18.7 18.7 18.7 0.395 -1.30 3M Environmental Laboratory ADVANCED B IO A N ALYTIC AL S E R V IC E S . INC. Page 27 of 50 . 99VDJA01.MI.DOC Page 148 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Table 9: Stability of PFOS in Rat Serum After Three Freeze/Thaw Cycles Run 10 QC (Theoretical Cone.) QC 1 (0.2 pg/mL) Mean RSD (%) %DEV from 0 Cycle PFOS (pg/mL) 0 Cycle 3 Cycles 0.187 0.177 0.186 0.177 0.182 0.185 0.189 0.185 0.182 0.189 0.185 0.182 1.70 3.12 NA. -1.38 QC2 (6 pg/mL) Mean RSD (%) %DEV from 0 Cycle 6.44 6.44 6.32 6.19 6.31 6.34 1.66 NA 6.59 6.61 6.44 6.39 6.40 6.49 1.63 2.35 QC3 (18 pg/mL) Mean RSD (%) %DEV from 0 Cycle 16.9 17.3 17.5 18.0 17.4 17.4 2.31 NA 19.2 19.1 19.2 19.6 18.7 19.2 1.80 10.1 NA: Not Applicable RSD = (SD/Mean) x 100 %DEV from 0 Cycle = [(3 Cycle - 0 CycIe)/0 Cycle] x 100 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S , INC. Page 28 of 50 99 VDJAO1.MI.DOC Page 149 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Table 10: Reproducibility of Reinjecting Extracted Samples Containing PFOS after 27 Hours in Reconstitution Solution . QC1 (0.2 ng/mL) Mean RSD (%) . RE (%) QC2 (6 pg/mL) Mean RSD (%) RE (%) QC3 (18 pg/mL) Mean RSD (%) RE (%) NA: N ot Applicable RSD = (SD/Mean) x 100 RE = [(M ean/Theoretical)-l] x 100 PFOS (ug/mL) t=0 Hours t=27 Hours (Run 10) (Run 12) 0.187 0.187 0.186 0.182 0.182 0.188 0.189 0.188 0.182 0.190 0.185 0.187 1.70 1.61 -7.50 -6.33 6.44 6.39 6.44 6.02 6.32 6.19 6.19 6.55 6.31 6.22 6.34 6.28 1.66 3.23 5.62 4.59 16.9 17.3 17.5 18.0 17.4 17.4 2.31 -3.29 17.3 18.2 18.1 17.3 17.6 17.7 2.40 -1.73 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S , INC. Page 29 of 50 99VDJA01 .MI.DOC Page 150 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Table 11: Extraction Recovery ofPFOSfrom Rat Serum Theoretical Cone. Pre-Extraction Post-Extraction in serum (pg/mL) Response (Area Ratio) Response (Area Ratio) % Recovery* PFOS 0.03160 0.02880 . no Spike 1 0.02850 0.03010 94.7 . (0.25 pg/mL) 0.02930 0.02950 99.3 Run 14 0.02790 0.02930 95.2 0.03050 0.02960 103 Mean 0.02956 0.02946 100 PFOS 0.4597 0.5270 87.2 Spike 2 (4 pg/mL) Run 14 0.4751 0.4776 0.4724 0.4772 . 0.5304 0.5335 0.5298 0.5392 89.6 89.5 89.2 88.5 Mean 0.4724 0.5320 88.8 PFOS 1.649 1.874 88.0 Spike 3 (16 pg/mL) Run 14 Mean 1.679 1.719 1.535 1.612 1.639 1.857 1.869 1.887 1.864 1.870 90.4 92.0 81.4 86.5 87.6 Tetra-H-PFOS Spike IS (4 pg/mL) Run 13 Mean 0.4068 0.3884 0.4043 0.3921 0.4078 0.3999 0.4460 0.4423 0.4445 0.4512 0.4412 0.4450 91.2 87.8 91.0 86.9 92.4 89.9 *(Individual Response for Pre-Ext./Individual Response for Post-Ext.) x 100 3M Environmental Laboratory ADVANCED B iO A N A L Y T IC A L S E R V IC E S , INC. Page 30 of 50 99VDJA01 .MI.DOC Page 151 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 8. FIGURES Figure 1: Full-Scan Single MS Mass Spectrum ofPFOS . Spectrum from PFOS Q1 ' 3.19e7 cps 3M Environmental Laboratory ADVANCED B IO A N A L Y T IC A L S E R V IC E S , INC. Page 31 o f 50 99VDJA01 .MI.DOC Page 152 BACK TO MAIN 3M Medical Department Study: T-6395.14 , ' Analytical Report: FACT T O X -111 LRN-U2994 Figure 2: Full-Scan SingleMS Mass Spectrum ofTetra-H-PFOS Spectrum from Tetra-H-PFOS Q1 ' .94e7 cps 3M Environmental Laboratory ADVANCED BIDANALYTICAL S E R V IC E S , INC. Page 32 o f 50 99VDJA01.MI.DOC Page 153 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Figure 3: Mass Chromatograms ofPFOS and its Internal Standard in Control Blank Rat SerumExtract m/z = 427.0 107e3 cps Time, min All quantitation results and figures were prepared from non-smoothed data. Analvte PFOS Tetra-H-PFOS (IS) Ion . m/z = 499.0 m /z = 427.0 Retention Time 3.1 2.9 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S , INC. Page 33 of 50 99VDJA01.MI.DOC Page 154 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Figure 4: Mass Chromatograms ofPFOS in a Rat SerumExtract Sample Containing Internal Standard Only (Zero Sample) m/z =427.0 2.34e5 cps Time, min A ll quantitation results and figures w ere prepared from non-smootbed data. A n a ly te PFOS Tetxa-H-PFOS (IS) Ism m/z = 499.0 m/z = 427.0 Retention Time 3.1 2.9 3M Environmental Laboratory ADVANCED B IO A N A L Y T IC A L S E R V IC E S . INC. Page 34 of 50 99VDJA01 .MI.DOC Page 155 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 LRN-U2994 Figure 5: Mass Chromatograms ofCalibration Standard 1in Rat Serum Extract Containing PFOS (0.05 pg/mL) and the Internal Standard m/z = 427.0 3.82e5 cps A ll quantitation results and figures were prepared from non-smoothed data. A n alv te PFOS Tetra-H-PFOS (IS) Ifin m/z = 499.0 m/z = 427.0 Retention Time 3.1 2.9 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S . INC. Page 35 of 50 99VDJA01 .MI.DOC Page 156 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Figure 6: Mass Chromatograms ofCalibration Standard 10in Rat Serum Extract Containing PFOS (20 pg/mL) and the Internal Standard m/z =427.0 3.52e5 cps Time, min All quantitation results and figures were prepared from non-smoothed data. A n alv te PFOS Tetra-H-PFOS (IS) I o n _______ m /z = 499.0 m/z = 427.0 Retention Time 3.1 - 2.9 3M Environmental Laboratory ADVANCED B iO A N A L V T IC A L S E R V IC E S . INC. Page 36 o f 50 99VDJAOI .Ml.DOC Page 157 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 9. APPENDIXA: QUANTITATION OF PERFLUOROOCTANESULFONATE (PFOS) INRAT SERUMBY TURBO ION SPRAYLC/MS AUTHORS: DavidJ. Anderson, M.S. AmieJ. Prince, B.S. HollyD. Ross, M.S. TABLE OF CONTENTS A.1.0 Chemical Structure(s)............................... v-38 A.2.0 Specimen(s)...................................................,.................. ...................................38 A.3.0 Assay Principle.................. 38 A.4.0 Compounds...... '.......... 38 A.5.0 Chemicals............ ........................................................ -38 A.6.0 Materials and Equipment.......................................................................................39 A.7.0 Preparation of Solutions.......... ................................... 42 A.8.0 Preparation of Quality Control Samples........ ...................................................... 45 A.9.0 Liquid-liquid Extraction Procedure...................................................................... 46 A.10.0 Instrument Conditions...........................................................................................47 A.11.0 Calculations............................... 49 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S . INC. Page 37 of 50 99VDJA01.MI.DOC Page 158 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 A.1.0 CHEMICAL STRUCTURE^) O II CgF'n----- S----- 0 "K + O Potassium Perfluorooctanesulfonate MW = 538 O 1H, 1H, 2H, 2H-Perfluorooctane Sulfonic Acid MW = 428, Internal Standard o A.2.0 SPECIMEN(S) This assay uses 50-pL aliquots of rat serum. Rat serum samples are stored at -20 C. A.3.0 ASSAY PRINCIPLE PFOS and its internal standard, Tetra-H-PFOS, are extracted from rat serum samples (50 pL) using a liquid-liquid extraction procedure. The organic layer is evaporated to dryness and then reconstituted. An aliquot is then analyzed by turbo ion spray liquid chromatography/mass spectrometry (LC/MS) in the negative ion mode. A.4.0 COMPOUNDS PFOS: Lot# 171,99.976% purity, 3M, Inc., Minneapolis, MN. Tetra-H-PFOS: Lot# 59909, 90% purity, 3M, Inc., Minneapolis, MN. A.5.0 CHEMICALS All chemicals may be substituted with that o f an equivalent manufacturer and grade of chemical. 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S , INC. Page 38 of 50 99VDJA01.MJ.DOC Page 159 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Acetonitrile Ammonium Acetate Ethyl Acetate Methanol Rhodapex mass calibration solution Sodium Bicarbonate Sodium Carbonate, Annhydrous Tetrabutylammonium Hydrogen Sulfate Water ' Rat Serum Rat Serum Cat# 015-4, Burdick & Jackson, Muskegon, MI 49442 Cat# 24,019-2, Aldrich, Milwaukee, WI 53201 . Cat# 100-4, Burdick & Jackson, Muskegon, MI 49442 Cat# 230-4, Burdick & Jackson, Muskegon, MI 49442 A mixture o f 0.11 mM trifluoroacetic acid (J.T. Baker, Phillipsburg, NJ 08865), 0.52 mM Rhodapex CO -436,1 mM ABEX EP110 and 1 mM ABEX EP-120 (all three supplied by Rhone-Poulenc, Inc., Cranbury, NJ 08512) in 1:1 methanokwater. Cat# 3509-01, J.T. Baker, Phillipsburg, NJ 08865 Cat# 3604-01, J.T. Baker, Phillipsburg, NJ 08865 HPLC Grade, Cat# J360-07, J.T. Baker, Phillipsburg, NJ 08865 High Purity (NANOpure), Bamstead Model D7331 Ultrapure Water System, Dubuque, LA 52001 . Lampire Biological Laboratories, Piperville, PA 18947 Harlan Bioproducts for Science, Inc., Indianapolis, IN 46229 A.6.0 MATERIALS ANDEQUIPMENT Mass Spectrometer PE SCIEX API 365 atmospheric pressure ionization tandem triple quadrupole mass spectrometer equipped with TurboIonSprayTM interface, PE SCIEX, Concord, Ontario L4K4V8 Data system API Standard Software, MacQuan v 1.4, LC2Tune v 1.3, MacDAD v 1.3, Bundler v 1.3, Multiview v 1.3, 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S . INC. Page 39 of 50 99VDJA01 .MI.DOC Page 160 BACK TO MAIN 3M Medical Department Study: T-6395.14 , Analytical Report: FACT T O X -111 LRN-U2994 HPLC Pump LC Pump Controller Autosampler HPLC Column Harvard syringe pump 11 Multi-tube vortexer Balance pH Meter Mechanical shaker NANOpure-UV water purification system Microbalance Micro weigh boats Weigh boats Sorvall RT-6000D refrigerated centrifuge Beckman GS6KR Refrigerated centrifuge and Sample Control v 1.3, on a Power Macintosh, PE SCIEX, Concord, Ontario L4K4V8 ShimadzuLC-lOAD pump, Shimadzu Co., Columbia, MD 21046 Shimadzu SCL-10A, Shimadzu. Co., Columbia, MD 21046 Waters 717plus, Waters Associates, Millipore Corporation, Milford, MA 01757 . Betasil C 18, 5 pm particle size, 2.1 x 50 mm, Cat #852055-701, Keystone Scientific, Inc., Bellefonte, PA 16823 Harvard Apparatus Inc., South Natick, MA 01760 Cat# 58816-115, VWR Scientific, West Chester, PA 19380 Model FX-300, AND Ltd., Tokyo, Japan Model 340, Coming Inc., Coming, NY 14830 Cat# 6000, Eberbach Corp., Ann Arbor, MI 48106 Bamstead, Dubuque, IA 52001 Model MT-5, Mettler-Toledo Inc., Hightstown, NJ 08520 Cat# 0219-0041, Perkin-Elmer Corp., Norwalk, CT 06859 Cat# 12577-025, VWR Scientific, West Chester, PA 19380 Cat# 83071, DuPont Co., Wilmington, DE 19898 Beckman, Palo Alto, CA 94304 H tW H lH dO B m m advanced b io a n a l y t ic a l S E R V IC E S . IN C . 3M Environmental Laboratory Page 40 o f50 9 9 V D J A O lM l.D O C Page 161 BACK TO MAIN 3M Medical Department Study: T-6395.14 , Analytical Report: FACT T O X -111 LRN-U2994 TurboVap LV Evaporator Cat# 43750, Zymark Instruments, Hopkinton, MA 01748 Pipettes Cat# P-5000, P-1000, P-200, P-100, Ramin Instrument Co., Woburn, MA 01888 Pipette tips RT-20, RT-200, C-5000, CP-25, CP-50, CP-250, Rainin Instrument Co., Woburn, MA 01888 Polypropylene tubes 13 mm x 100 mm, Cat# 15070-574, VWR Scientific, West Chester, PA 19380 Screw-capped Polypropylene vials 16.5 x 57 mm, Cat# 60.542, Sarstedt, Inc., Newton, SC 28658 Screw-capped Polypropylene vials 16.5 x 101 mm, Cat# 60.541, Sarstedt, Inc., Newton, SC 28658 Plug Tite Multipurpose Caps Cat# 78-127-0019-100, Elkay Products, Inc., Worcester, MA 01607 Pyrex volumetric flask Cat# 28014P-10, 28014P-25, 28014P-100,28014P1000, Kimble/Kontes, Vineland, NJ 08360 Solvent Filtration Apparatus Cat# 953781-0000, 953751-0000,953753-0000, 953826-0000, 953827-0000, Kimble/Kontes, Vineland, NJ 08360 Nylon Titan . 0.45 pm Scientific Resources Inc., Cat# 74547-NN, ,, Membrane Filters Eatontown,NJ 07724 PEEK tubing 0.005" i.d. x 1/16" o.d., Cat# 1535, Upchurch Scientific Inc., Oak Harbor, WA 98277 Liquid Nitrogen Supplied in-house from bulk tank, BOC Gasses, Buffalo, NY 14210-2005 Gastight Glass Syringes Cat# 1750, Hamilton Company, Reno, NV 89510 Other general laboratory glassware and supplies were used. H W K iH ADVANCED b io a n a l y t ic a l S E R V IC E S . IN C . 3M Environmental Laboratory Page 4) of 50 99VDJA01.MI.DOC Page 162 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 A.7.0 PREPARATION OF SOLUTIONS NANOpure water (18 megaohm-cm.) or equivalent should be used wherever water is called for. Mix all solutions well. A.7.1 Preparation of Analytical Standard Stock Solutions Analytical Standard Stock Solution, PFOS (1 mg/mL): Weigh approximately 5 mg of PFOS (after correction for purity) on a microbalance and transfer it to a polypropylene vial. Dilute with the appropriate volume Ofmethanol to yield a 1 mg/mL solution. Prepare a fresh solution every three months. Store the solution at 4 C and bring to ambient temperature before use. Standard Spiking Solutions Prepare Standard Working Solutions A through J in 5-mL class "A" volumetric flasks according to the following dilution scheme. Dilute to the 5-mL mark with water to yield the final concentration. Prepare fresh solutions every three months. Store the solutions at 4 C and bring to ambient temperature before use. Standard W orking A B C D E F G H I J Volume Spiked Solution Used (pL) 1000 Stock 800 Stock 400 Stock 200 Stock 100 Stock 250 200 ug/mL (A) 125 200 pg/mL (A) 62.5 200 pg/mL (A) 500 10'pg/mL (F) 250 10 jig/mL (F) PFOS Final Cone. (pg/mL) 200 160 80 40 20 10 5 2.5 1.0 0.5 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S . INC. Page 42 o f 50 99VDJAO1.MI.DOC Page 163 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 A.7.2 Preparation ofInternal Standard Stock Solutions Internal Standard Stock Solution, Tetra-H-PFOS (1 rag/mL): Weigh approximately 5 mg of Tetra-H-PFOS on a microbalance and transfer it to a polypropylene vial. Dilute with an appropriate volume o f methanol to yield a 1 mg/mL solution. Prepare a fresh solution every three months. Store the solution at 4 C and bring to ambient temperature before use. A.7.3 Preparation ofInternal Standard Working Solution Internal Standard Working Solution (4 pg/mL Tetra-H-PFOS): Add 400 pL of Internal Standard Stock Solution to a 100-mL class "A" volumetric flask. Dilute to the mark with water to give a 4 pg/mL Tetra-H-PFOS solution. Store the solution at 4 C and bring to ambient temperature before use. Prepare fresh solution as needed. A.7.4 Preparation ofStandard Curve and Control Blank Prepare fresh calibration standards for each analytical run by combining 360 pL of rat control serum and 40 pL of the analytical standard working solution (see table below) in labeled 1.7-mL microtubes. Prepare control blanks and zero samples by combining 360 pL of rat control serum with 40 pL of water. Vortex each for 60 seconds. 3M Environmental Laboratory ADVANCED B IO A N A L Y T IC A L S E R V IC E S , INC. Page 43 o f 50 99VDMOJ.MI.DOC Page 164 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Analytical Standard Dilution Table: Standard # 10 9 8 7 6 5 4 3 2 1 Volume Spiked (pL) 40 40 40 40 40 40 40 40 40 40 Solution to use A B C D E F G H I J pL control serum 360 360 360 360 360 360 360 360 360 360 Final Cone. (pg/mL) 20 16 8 4 2 1 0.5 0.25 0-1 0.05 A.7.5 Preparation of O ther Solutions 0.25 M Sodium Carbonate/0.25 M Sodium Bicarbonate: Weigh 26.5 g o f sodium carbonate and 21 g of sodium bicarbonate and dissolve in approximately 900 mL of HPLC-grade water. Transfer the solution to a 1000-mL volumetric flask and dilute to the mark with HPLC-grade water. Mix the solution thoroughly. Store at ambient temperature. Prepare a fresh solution every three months. 1 M Ammonium Acetate: Add 7.7 g of ammonium acetate to a 100-mL volumetric flask, dissolve in NANOpure water, and stir until completely dissolved. Dilute to the mark with NANOpure water. Store at ambient temperature. Prepare a fresh solution every three months. 2 mM Ammonium Acetate: Combine 2 mL o f 1M ammonium acetate and 900 mL NANOpure water in a 1000-mL volumetric flask; stir until completely mixed. Dilute to the mark with NANOpure water. Store at ambient temperature. Prepare a fresh solution every three months. 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S , INC. Page 44 of 50 99VDJA01 .MI.DOC Page 165 BACK TO MAIN 3M Medical Department Study: T-6395.14 . Analytical Report: FACT T O X -111 LRN-U2994 10 M Sodium Hydroxide: Add 40 g of sodium hydroxide to a 100-mL volumetric flask, dissolve in NANOpure water, and stir until completely dissolved. Dilute to the mark with NANOpure water. Store at ambient temperature. Prepare a fresh solution every three months. 1 M Sodium Hydroxide: Add 10 mL of 10 M sodium hydroxide to a 100-mL volumetric flask. Dilute to the mark with NANOpure water; stir until completely mixed. Store at ambient temperature. Prepare a fresh solution every three months. 0.5 M Tetrabutylamm onium Hydrogen Sulfate, pH 10: Weigh 16.98 g of tetrabutylammonium hydrogen sulfate and dissolve in 40 mL o f NANOpure water. Adjusted the pH to 10.0 with 10 M and 1 M sodium hydroxide. Transfer the solution to a 100-mL volumetric flask and dilute to the mark with NANOpure water. 10:90 M ethanol:2 mM Ammonium Acetate (Mobile Phase Eluent A): Add 100 mL o f methanol and 900 mL of 2 mM ammonium acetate to a 1000-mL Pyrex bottle. Mix the solution thoroughly and filter the solution through a 0.45 pM filter with a vacuum flask. Store at ambient temperature. Prepare a fresh solution every three months. 90:10 Methanol:2 mM Ammonium Acetate (Mobile Phase Eluent B): Add 900 mL o f methanol and 100 mL of 2 mM ammonium acetate to a 1000-mL Pyrex bottle. Mix the solution thoroughly and filter the solution through a 0.45 pM filter with a vacuum flask. Store at ambient temperature. Prepare a fresh solution every three months. A.8.0 PREPARATION OF QUALITY CONTROL SAMPLES A.8.1 Preparation of Quality Control Stock Solutions QC Stock Solution, PFOS (1 mg/mL): Weigh approximately 5 mg of PFOS (after correction for purity) on a microbalance and transfer it to polypropylene vial. Dilute 3M Environmental Laboratory ADVANCED BIOANALVTICAL SERVICES, INC. Page 45 o f 50 99VDJA01.MI.DOC Page 166 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 with the appropriate volume of methanol to yield a 1 mg/mL solution. Store the solution at 4 C and bring to ambient temperature before use. Prepare a fresh solution every three months. A.8.2 Preparation of Serum QC Samples QC4 (Dilution QC, 100 pg/mL PFOS): Add 1000 pL QC Stock Solution to a 10 mL class "A" volumetric flask to yield a 100-pg/mL solution. Dilute to the mark with control serum, cap and mix. Aliquots of approximately 0.2 mL are placed in polypropylene vials and stored frozen at -20 C. QC3 (18 pg/mL PFOS): Add 450 pL QC Stock Solution to a 25-mL class "A" volumetric flask to yield a 18-pg/mL solution. Dilute to the mark with control serum, cap and mix. Aliquots of approximately 0.5 mL are placed in polypropylene vials and stored frozen at -20 C. QC2 (6 pg/mL PFOS): Add 150 pL o f QC Stock Solution to a 25-mL class "A" volumetric flask to yield a 6-pg/mL solution. Dilute to the mark with control serum, cap and mix. Aliquots of approximately 0.5 mL are placed in polypropylene vials and stored frozen a t-20 C. QC1 (0.2 pg/mL PFOS): Add 111.1 p L o f QC3 to a 10-mL class "A" volumetric flask to yield a 0.2-pg/mL solution. Dilute to the mark with control serum, cap and mix. Aliquots of approximately 0.5 mL are placed in polypropylene vials and stored frozen at -20 C. A.9.0 LIQUID-LIQUID EXTRACTION PROCEDURE 1. Prepare fresh calibration standards for each run according to Section A.7.4. 2. Thaw QC samples. 3. Prepare QC4 samples (diluted 1 in 10) in two steps: a) Add 180 pL of control serum and 20 pL o f QC4 into 1.7-mL microtubes and 3M Environmental Laboratory ADVANCED BIOANALVTICAL SERVICES. INC. Page 46 of 50 99VDJAOt.MI.DOC Page 167 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 vortex. . b) Aliquot 50 pL of diluted QC4 into labeled 16 x 100 mm polypropylene tubes. 4. Aliquot 50 pL of each control blank, zero sample, calibration standard, and quality control samples 1-3 into labeled 16 x 100 mm polypropylene tubes. Standards and blanks are analyzed in duplicate. All QC samples are analyzed in replicates of five. 5. Add 1 mL of 0.5 M tetrabutylammonium hydrogen sulfate, pH 10 to each tube. 6. Add 2 mL of 0.25 M sodium carbonate/0.25 M sodium bicarbonate to each tube. 7. Add 500 pL of the Internal Standard Working Solution to each tube (except control blank, add 500 pL water). 8. Add 5 mL ethyl acetate to each tube. 9. Shake the tubes on a reciprocal shaker at medium speed for 20 minutes. 10. Centrifuge the tubes at 3000 rpm for 20 minutes at 20 C. 11. Freeze the aqueous phase in an acetone/dry ice bath and keep the tubes in the bath an additional 5 minutes. 12. Transfer the ethyl acetate layer to a fresh, labeled 13 x 100 mm polypropylene tube. . 13. Evaporate the ethyi acetate layer to dryness in a TurboVapTM at approximately 20 C under nitrogen. 14. Reconstitute the dried extracts in two steps: Add 500 pL acetonitrile and vortex for 60 seconds. Then add 500 pL water and vortex for 60 seconds. 15. Transfer 200 pL of the extract to a labeled polypropylene autosampler vial.. A.10.0 INSTRUMENTCONDITIONS HPLC Conditions: Eluent (gradient) Mobile Phase A: 10:90 methanol:2 mM ammonium acetate (v/v) . Mobile Phase B: 90:10 methanol:2 mM ammonium acetate (v/v) 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S . INC. Page 47 of 50 99VDJA01.MI.DOC P a g e t6 8 " BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Eluent Gradient Conditions: Time (min) 0 1.0 5 5.5 8 Flow Rate Autosampler Autosampler Temperature Injection Volume. HPLC Column HPLC Column Temperature Typical Initial Column Pressure Autosampler Run Time %B 45 ' 100 100 45% Stop 300 pL/min. Waters 717plus Ambient 3 to 5 pL Keystone Betasil C18, 5 pm particle size, 2.1 mm x 50 mm Ambient 90 bar 8.0 minutes A n alvte Representative Retention Time (minutes') PFOS Tetra-H-PFOS (IS) - 3.1 2.9 Mass Spectrometer Conditions: Curtain Gas Nebulizer Gas TurboIonSprayTM Temperature TurboIonSprayTM Auxiliary Gas UHP Nitrogen UHP Nitrogen 400 C UHP Nitrogen at 8 L/min 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S . INC. Page 48 o f 50 99VDJA01 .MI.DOC P a g e te - BACK TO MAIN 3M Medical Department Study: T-6395.14 . , ' Analytical Report: FACT T O X -111 LRN-U2994 Ions Monitored: Analvte PFOS Tetra-H-PFOS (IS) Ion Monitored m/z = 499.0 m/z = 427.0 Dwell Time 500 ms 500 ms Ionization Mode Ion Spray Voltage Declustering Potential MS Acquisition Time Pause Time . Negative Ion -3000 V -35 V 5 minutes 2 ms Calibrate th mass axis of the instrument by infusion of Rhodapex mass calibration solution (Section A.5.0) at a flow rate o f 10 pL/min. Optimize the sensitivity of the instrument using an infusion of a 10 pg/mL solution o f the analyte at 10 pL/min into a flow o f 190 pL/min of mobile phase using the gradient condition at which the analyte elutes from the LC column (100% eluent B). Mass spectral peak widths should be approximately 0.6 amu at half-height. One set of calibration standards was injected at the beginning and one set of calibration standards was injected at the end of each analytical run (tray). A.11.0 CALCULATIONS Calculated concentrations are based on peak area ratios o f PFOS to Tetra-H-PFOS. The peak area for the analyte ion is divided by the peak area for the corresponding internal standard ion. Data generated from samples in this study were acquired and integrated using PE SCIEX software applications Sample Control (v. 1.3) and MacQuan (v. 1.4). The validation data were formatted in the ABS laboratory information management system (Watson, v.5.3.1.01). All data were rounded to no less than three significant figures by ABS prior to reporting. ADVANCED BIOANALYTICAL SERVICES, INC. 3M Environmental Laboratory Page 49 o f 50 99VDJA01.M1.DOC Page 170 BACK TO MAIN 3M Medicai Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Acceptance Criteria for Sample Analysis Calibration standards: The coefficient of determination (r2) must be >0.98. At least three-fourths o f the individual calibration standard replicates will have deviations within 15% from their nominal concentrations. LLQ acceptance criteria: At least one replicate at the LLQ must exhibit a deviation within 20% from its nominal concentration. If this criterion is not met, both replicates are rejected and the standards at the next higher level are subjected to the same test. Quality control samples: At least two-thirds of the individual QC sample replicates will have deviations within 15% from their nominal concentrations, with at least one replicate at each QC concentration meeting this criterion. 3M Environmental Laboratory ADVANCED BIDANALVTICAL SERVICES. INC. Page 50 o f 50 99VDJAOl.MI.DOC Page 171 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 BIOLOGICAL SAMPLE ANALYSIS Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 . . . P uttin g T e c h n o lo g y To W ork FINAL REPORT ORAL (GAVAGE) PHARMACOKINETIC RECOVERYSTUDYOF PFOS INRATS SPONSOR 3M T oxicology Services-Medical Department 3M Center, Building 220-2E-02 \ St. Paul, MN 55144-1000 -- >* / Testing Facility Battelle Memorial Institute 505 King Avenue Columbus, Ohio 43201-2693 \ 3M Environmental Laboratory Prepared Bv Patrick L. South, B.S. Page 172 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Battelle Study Number: N 003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 FINAL REPORT ORAL (GAVAGE) PHARMACOKINETIC RECOVERYSTUDYOF PFOS INRATS 3M Environmental Laboratory Page 173 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TO X-111 ORAL (GAVAGE) PHARMACOKINETIC RECOVERY STUDYOF PFOS INRATS EXECUTIVE SUMMARY Rat liver samples sent to Battelle by 3M Environmental Technology Services were analyzed by the previously validated method "Method for Analysis of Potassium Perfluorooctanesiilfonate (PFOS) in Rat Liver by LC/MS/MS". Samples were extracted and analyzed by High-Performance Liquid Chromatography Mass Spectroscopy (LC/MS/MS) for PFOS content only. Related fluorochcmicals mentioned in the analytical method were not monitored. The results for the concentration determination of PFOS in the liver samples from this study are attached as appendices to this report. Concentrations are reported as mass of PFOS (pg) per gram of liver tissue extracted. 3M Environmental Laboratory iii Page 174 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Battelle Study Number: N 003296-C 3M Toxicology Services Protocol Number: FACT-TOX-111 QUALITY ASSURANCE STATEMENT This study was inspected by the Quality Assurance Unit and reports were submitted to the task leader, study director, and associated management as follows: Date Reported to Battelle Task Phase Inspected Inspection Date Leader/ Battelle _______________________________________;_______Management Date Reported to Offsite Study Director/ Management______ Standard Preparation Sample weights . Sample homogenization Extraction Sample analysis Audit study file Audit final report Audit final report 09/29/1999 09/30/1999 09/30/1999 10/01/1999 10/01/1999 10/11/1999 10/11/1999 02/19/2001 10/01/1999 10/01/1999 10/01/1999 10/11/1999 10/11/1999 10/11/1999 10/11/1999 02/19/2001 10/14/1999 10/14/1999 10/14/1999 10/14/1999 10/14/1999 10/14/1999 10/14/1999 03/14/2001 Quality Assurance UmK Battelle Memorial Institute Date 3M Environmental Laboratory IV Page 175 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT T O X -1 11 L R N -U 2 9 9 4 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 GOOD LABORATORYPRACTICES COMPLIANCE STATEMENT Study Title: ORAL (GAVAGE) PHARMACOKINETIC RECOVERY STUDY OF PFOS IN RATS This study was conducted in compliance with the Food and Drug Administration's Good Laboratory Practice Regulations (21 CFR 58), with the exception that the mass spectrometry data for the liver samples was collected and processed with the MassLynx software system (version 3.1), which was not fully validated. The study was listed on Battelle's Master List o f regulated studies. C CUrbJL_ _ _ _ _ _ Jon D. Andre, Ph.D. Battelle Principal Investigator Study Director f Date 3M Environmental Laboratory v Page 176 3M Medical Department Study: T-6395.14 ------------ :-------------------------- BACK TO MAIN _____________________________ Analytical Renort: FACT TOX-.1.11 '" LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-1! 1 Table of Contents Page Executive Summary.............................................................................................................................. iii Quality Assurance Statement................................................................................................................. iv Compliance Statement....................... v Table of Contents...................................................................................................................................vi 1.0 Introduction................................................................................................................................ I 2.0 Reference Substances..................................................................................................................1 3.0 Receipt of Samples..................................................................................................................... I 4.0 Analysis of Samples................................................................................................................... I 4.1 Summary of Method.................................... ,................................................................ 1 4.2 Results...........................................................................................................................2 4.2.1 Quality Control................................................................................................. 2 4.2.2 Sample Results.................................................................................................. 3 5.0 Conclusions................................................................................................................................3 6.0 Acknowledgements.................................................................................................................... 3 7.0 Specimen Storage and Record Archives......................................................................................3 List ofTables Table 1. Example of Instrument Parameters Used to Analyze Samples............................................. 2 Appendix A(Results) Summary Results for Rat liver Sample Analysis.................................................................................. A-l Appendix B(Acceptance Criteria Results, Calibration Curve) Acceptance Criteria Results, Calibration Curve........... ........................................................................ B-l Appendix C(Method) Method for Analysis of Potassium Perfluorooctanesulfonate (PFOS) in Rat liver by LC/MS/MS......... C-l Appendix D(Chromatograms) Representative Chromatograms.......................................................................................................... D-l Appendix E (Protocol, Amendments, and Deviations) Protocol, Amendments, and Deviations............................................................................................... E-l Appendix F (PFOS Purity Report) PFOS Purity Report......................................................................................... F-l 3M Environmental Laboratory vi Page 177 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT T O X -111 -- LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TO X-I1 1 Table of Contents Page Executive Summary.................................................................................................................................iii Quality Assurance Statement................................................................................................................... iv Compliance Statement............................................................................................................................. v Table of Contents....................................................................................................................................vi 1.0 Introduction................................................................................................................................. 1 2.0 Reference Substances...................................................................................................................1 3.0 Receipt of Samples.......................................................................................................................1 4.0 Analysis of Samples.................................................................................................................... 1 4.1 Summary of Method...................................................................................................... 1 4.2 Results................................................. :........................................................................2 4.2.1 Quality Control................................................................................................... 2 4.2.2 Sample Results................................................................................................... 3 5.0 Conclusions.................................................................................................................................3 6.0 Acknowledgements......................................................................................................................3 7.0 Specimen Storage and Record Archives......................................................................................... 3 List ofTables Table 1. Example of Instrument Parameters Used to Analyze Samples.......................................................2 Appendix A (Results) Summary Results for Rat liver Sample Analysis.........................................................................................A-l Appendix B(Acceptance Criteria Results, Calibration Curve) Acceptance Criteria Results, Calibration Curve......................................................................................... B-l Appendix C (Method) Method for Analysis of Potassium Perfluorooctanesulfonate (PFOS) in Rat liver by LC/MS/MS................. C-l AppendixD (Chromatograms) . Representative Chromatograms.......................................................................:....................................... D-l Appendix E (Protocol, Amendments, and Deviations) Protocol, Amendments, and Deviations...................................................................................................... E-l Appendix F (PFOS Purity Report) PFO S Purity R e p o r t..............................................................................................................................................................................F -l 3M Environmental Laboratory vi Page 178 3M Medical Department Study: T-6395.14 ........................................ ................. -- BACK TO MAIN Analytical Report: FACT T O X -111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 1.0 Introduction This report presents a description of the method used to analyze PFOS in rat liver samples from 3M Study Number FACT-TOX-111 and the results from this analysis. See Appendix E for a copy of the study protocol, amendments, and deviations. 2.0 Reference Substances The analytical reference substance for this study was potassium perfluorooctanesulfonate (PFOS) lot number 171, supplied by 3M. Note that based on information supplied to Battelle from 3M, PFOS has two equivalent names. The name appearing on the Material Safety Data Sheet and bottle label is potassium perfluoroalkyl sulfonate. The name more commonly used by 3M in analytical methods and correspondence is potassium perfluorooctanesulfonate. The latter name will be used in this report. See Appendix F for purity data supplied by 3M to Battelle. The surrogate standard was lH,lH,2H,2H-Perfluorooctane sulfonic acid, lot number 59909, supplied by ICN. 3.0 Receipt of Samples Samples were received frozen and intact at Battelle, from 3M Environmental Technology and Services, in one batch on August 20, 1999. Samples were generated by Argus Research under protocol number 418-015. The samples were stored at approximately -20C. 4.0 Analysis of Samples 4.1 Summary of Method Samples were analyzed by a previously validated method (Battelle study number N003604-A) in one batch. The current version of the method is attached to this report in Appendix C. Samples were analyzed by LC/MS/MS, and an example of the instrument parameters is listed in Table 1. Note that only PFOS itself (and the surrogate) was quantitated. The other related fluorochemicals were added to the stock solutions but not monitored. Quadratic regressions weighted 1/x were used to construct the calibration curves. The run order consisted of system suitabilities, 3 calibration curves analyzed at the start, near the middle, and at the end of the run and quadruplicate QCs interspersed throughout the run. The various recovery samples were also interspersed throughout the run. 3M Environmental Laboratory 1 Page 179 3M Medical Department Study: T-6395.14 ' '" BACK TO MAIN Analytical Report: FACT T O X -1 11 LRN-U2994 Baltelle Study Number: N 003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -1 1 1 Table 1. Example ofInstrument Parameters Usedto Analyze Samples LC/M S/MS System A u tosam pler HPLC pumps Mass spectrometer Analytical column Mobile phase components Gradient profile Injection volume Flow Column temp HPLC pressure MS source Desolvation gas Nebulizer gas Source block temp Desolv ation temp Cone voltage Collision energy Collision gas Multiplier Resolution Ions monitored Total run time A p proxim ate retention times: Make: Gilson Make: Gilson Make: Micromass Model: 234 Models: 305 and 306 Model: Quattro LC with Z-spray source Keystone Betasil C18, Sum, 2 x 50 mm. Part No. 055-701-2 Component A: Ammonium acetate(2mM):methanol, 60:40, v:v Component B: Ammonium acetate(2mM):methanol. 5:95, v:v Tim e, min %B Flow. mL/min 0 0 0.3 1 0 0.3 4.5 100 0.3 6 100 0.3 6.1 100 0.6 8.5 100 0.6 9 0 0.3 ___U ______________ 10 uL 0 0.3 LC column flow at start split to 20 pL/min into the MS Ambient Approximately 850 psi at gradient start Electrospray, Negative Ion Nitrogen a t-5 7 5 L/hr Nitrogen at ~80 L/hr 140C 250C 70 V 40 eV Argon, gas cell, at --2.5x10 mb 650 V 12.0 for M SI; 10.0 for MS2 427>8I MRM transition for SS 499>99 MRM transition for PFOS 11 minutes SS: 4 min PFOS: 4.2 min 4.2 Results 4.2.1 Quality Control System suitability acceptance criteria were established during the method validation and are included in A ppendix C, Section IX A cceptance Criteria. Relevant statistics from the sam ple set are provided in A ppendix B. Representative chromatograms are given in A ppendix D. 2 3M Environmental Laboratory Page 180 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Battelle Study Number: N 003296-G 3M Toxicology Services Protocol Number: FACT-TO X-111 4.2.2 Sample Results The results of the sample analyses are presented in Appendix A. The limit of quantitation is defined as the concentration of the lowest standard which meets acceptance criteria for accuracy (25% RE). The notation BLOQ denotes "Below Limit of Quantitation" for samples that had concentrations lower than the theoretical concentration for the 0.13 pg/g calibration standard. The "Corrected PFOS Cone" presented in the results tables is the concentration found for the diluted sample multiplied by its dilution factor (final volume ) sample homogenate volume). The method detection limit (MDL) of PFOS was calculated in Battelle study N003296-F to be 0.0173 pg/g from the analysis of 7 replicate preparations of 0.13 pg/g calibration standard. The MDL was calculated by multiplying the standard deviation of the found concentrations of the 7 reps by 3.143; the Signal-to-Noise (S/N) ratio was calculated by dividing the mean found concentration of the 7 reps by their standard deviation. 5.0 Conclusions The analysis met all acceptance criteria except for dilution recovery. The average dilution recovery was 133.3%. See Appendix E for deviation report. 6.0 Acknowledgements Acknowledgement of principal contributors participating in the performance of this study at Battelle is presented in the following list. Participant Title Jon C. Andre, Ph.D. Richard W. Slauter, Ph.D., D.A.B.T. Patrick L. South, B.S. Gerke H. van der Zwaag, M.S. Battelle Principal Investigator Senior Program Director Mass spectroscopist Chemist 7.0 Specimen Storage and Record Archives See Appendix E, protocol amendment 2 for records archival information. All residual liver samples, extracts, and unused test article will be disposed of or returned to the Sponsor as directed by the Sponsor. 3M Environmental Laboratory 3 Page 181 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 ~ ......... L R N -U 2 9 9 4 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 APPENDIX A-RESULTS 3M Environmental Laboratory Page 182 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TO X-111 RAT LIVER SAMPLE RESULTS STUDY: N003296-G ANALYSIS DATE AND INSTRUMENT ID: DATA ENTERED: SPREADSHEET SOFTWARE: 01Oct99; 9053 Electronically Excel 97 FINAL LIVER SAMPLE RESULTS: Animal numbers ending In "P" indicate pooled fetal liver Animal Number Corrected Dose Grp PFOS mq/kg/day_________Cone (pg/g) Note if Sample Is a Rerun Note if Rerun Is Needed Animal 13726 Animal 13726P Animal 13727 Animal 13727P Animal 13728 Animal 13728P Animal 13731 Animal 13734 Animal 13734P Animal 13735 Animal 13735P Animal 13736 Animal 13736P Animal 13737 Animal 13737P Animal 13739 Animal 13739P Animal 13740 Animal 13740P Animal 13741 A nim ali3741R Animal 13744 Animal 13744P Animal 13745 Animal 13745P Animal 13746 Anim al 137 46P Animal 13748 Animal 13748P Animal 13749 Animal 13749P Animal 13751 Animal 13751P Animal 13752 Animal 13752P Animal 13753 Animal 13753P Animal 13754 Animal 13754P Animal 13756 Animal 13756P Animal 13758 Animal 13758P Animal 13759 Animal 13759P 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 0.341 0.425 0.429 0.158 0.299 0.141 0.298 0.218 0.156 0.251 0.158 0.191 BLOQ 0.219 0.171 6.04 6.35 6.76 5.92 8.32 4.85 6.26 6.33 6.84 6.14 6.91 6.06 6.78 5.02 8.99 5.67 80.6 58.7 102 63.2 50.4 57.6 49.6 72.8 65.0 83.3 56.7 58.6 79.1 60.9 BLOQ = BELOW CONC OF LOWEST STANDARD IN CALIBRATION CURVE A -l 3M Environmental Laboratory Page 183 3M Medical Department Study: T-6395.14 " BACK TO MAIN Analytical Report: FACT T O X -111 LRN-2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number FACT-TOX-l 11 APPENDIX B- ACCEPTANCE CRITERIA RESULTS, CALIBRATION CURVE 3M Environmental Laboratory Page 184 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N 003296-G 3M Toxicology Services Protocol Number: FACT-TO X-l 11 P FO t N RAT UVEA STUDY: W N IH -O ANALYSIS DA HE AND INSTRUMENT: DATA ENTERED: ELECTAOWCALLY SPRE AD 8M U T SOFTWARE: EXCEL I? 01 Oct; I f S3 3 y * l m SuRabWty Ot-Oet-OI oi-oce-oz OI-Oot-03 01-Oct-04 0t-0et-05 F F O / 9 A rt a Ratte 7.778E+00 7.9076*00 7.822E*00 7.870E*00 7.3M E *00 7.893 0.252 3.3 QC 1 T h a o r Cane Roleata 1 2 3 4 QC 2 T h a o r Cone < |j0/g) Roleata 1 2 3 4 QC 3 Thaor Cone (M 'S ) R H ta tt 1 2 3 4 QC 4 T ha or C one (itala) R o in te 1 2 3 4 Dat'd. Cone, (w in I.292E+01 I.018E+01 9.22IE+00 1.0436*01 1.1156+01 Avg. Defd. Cone. (pg/Q) f.O M E +0 1 O e fd.C o M . M t 3.8ME+00 3.815E+00 3.8306*00 3.5386*00 3.321 E + M Aog. 0efd.CoM . (uofa) 3.883 E*00 0efd.CoM . 1.33260t 7.700601 7.702601 7.80IE-01 - ........ 7.S3BE-01 Avg. Oefd. Cone. 7.33 IE-01 Oefd. Cone. M 1.949601 1.871E-01 1.871601 1.72E-01 1.MOE-01 Avg. Defd. Cone. W 1.71 601 e ....- M i ) 1.5 to p 0.1 s ... (ta l 0.0 s iw ro) 0,0 % RSO -- O --------------- Avg. %RE .1 %RSD 3.9 %RE %RSO 3. %R %RSD 5.2 %R H om ogenizer Recovery T argai Conco Nominal Cone, ua/o Maw of Uvero S o m e Cone. ngftnL 4, Rep A 0.5207 4 , Rp f l 0.5833 0.8, Rap A 0.5141 0.8, Rep 0.5288 0.4, Rep A 0.4*67 0.4. Roo 8 0.5248 Targai tear cono (pgfg) - Mh o TA added (pg> / M a n o f tear (g) 2.0206*04 2.02BE+04 4.O56E+03 4.05E*03 f.978E*03 1.97BE+03 M a i* TA added, to 2.0206*00 2.0286*00 4.058E-O1 4.0566-01 1.970E-O1 1.0706-01 T a rm i Liver Cone, unfa 3.8956*00 3.0006*00 7.89064)1 7.05064)1 3.903601 3.789601 HoM ogantzar R ecovery ft acuiit Poutd Liver Coa unfa * 3.8956*00 4.107E+00 3.6006*00 7.890601 4.S22E*00 8.784 E*01 7.8ME-01 0.323601 3.983601 U17E+OQ 3.708E-O1 5.138E-01 % Recovery - f a n d Cone / Target Cong x 100% Rap A o f 0.4 ug/fl excluded by Q la d a t o u te r O - (355.9- 138JW 355.te105.4>- 1054 12$. 11W 1D0.7 358.1 138.3 0.877 DX.UTKJN TES T COHC U n d luta d ver danaity (ng te tL ), Prep J t l i p H , llu d y N0B32M-O Ave % R eco*: 117.4 t l d D ev: 13.1 %RSD: 11.1 OK to drop par SOP CHEM fV-015-03 at 95% conftdenea laval 187.8I 1 i 1 TA Source Cone. moOnl Fret M i Peetor firs t Din. ofalL 0.5070 60 M M E M J3 TO CONVERT CONCS PROM itgbnL TO ugfg: CONO (uofoi - COHC frw ftn ll / Am UwRutad U w r (tentit v (m o te l) x 1000 mora x 0.001 uatoa 5.0346*01 2nd M n Factor 10 Cone Exraewd. itelo 5.034 OButlon R ec o v ery R e tu ts Taraet Uor Cone. lkVd 5.034E*00 5.834E+00 5 .0 4 E - 0 C .16*00 8.4966*00 9796*00 % Recovery 132.3 129.0 138. Ava % Racav: 133.3 Std D av: 4.1 %RSD: 3.7 3M Environmental Laboratory B-l Page 185 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TO X-111 3M Medical Department Study: T-6395.14 Q uantify C a lib ra tio n R eport PFOS i n t a t L i v * r ; ND03ZS-G C a l i b r a t i o n : C:\KASSLTWXVHD032ft6G.PRO\CurvrilB\OIOct L aat w odifaad: S a t O ct 02 14:33:44 1999 " "* P rin t !: MS a t O c t 02 1 4 :S O :0 1 1999 S Compound 2 name: PFOS Coefficient of Determination: 0.992024 Calibration curve: -0.00950557 xA2 + 1.99272 x + -0.0790111 Response type: Internal Std ( Ref 1 ), Area * ( IS Cone. I IS Area ) Curve type: 2nd Order, Origin: Exclude, Weighting: 1/x, Axis trans: None BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Rag 2 03 3lw Environmental Laboratory Page 186 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N 003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 APPENDIX C-METHOD 3M Environmental Laboratory Page 187 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 Battelle Study Number: N 003296-G 3M Toxicology Services Protocol Number: FA C T-TO X -ll I METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS Version 1.0 Study No.:________________________ . Analyst/Date:.__________________________ O n to o ! K c \ j m o ii Revisions to the method K o \ i-sctl b \ I A p im n o J b \ ^sssss^sss Written by: .Date: 3^9 Approved by: Jcp/C. Andre, Ph.D. ger, Bionalytical Chemistry Date: 3M Environmental Laboratory Page 1 o fl6 C-l Page 188 BACK TO MAIN 3M Medical Department Study: T-6395.14 ------------------------------------------------------------------------ --- -------------------------------------------------------------- Analytical Report: FACT T O X -111 -- LRN-U2994 Batlelle Study Number: N003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -1 11 METHOD FOR ANALYSIS OF POTASSIUM PE R FLU O R O O C TA N ESU LFO N A TE (PFO S) IN RAT LIV ER BY LC /M S/M S Version 1.0 Study No.: Analyst/Date: _ L SUMMARY The extraction and analysis of potassium perfluorooctanesulfonate and related fluorochemicals in rat liver is performed. Calibration standards are prepared by spiking blank liver homogenate with solvent standards from two independently-prepared sucks. The calibration standards are fortified with surrogate standard, buffered, and extracted with ethyl acetate. The organic phases are evaporated to dryness and reconstituted in methanol for analysis by LC/MS/MS. n. Purpose To extract and analyze potassium perfluorooctanesulfonate and related fluorochemical compounds found in Sprague-Dawley rat liver. m . Samples See Chain of Custody records if applicable. IV. General Instructions Calibrate all required balances according to the SOP on balance usage. Make equivalent dilutions when the volume needed varies from the volume stated in foe method. Label all standard and reagent solutions as specified in foe appropriate SOP. If you intend to reuse a solution for future tasks, be sure the label includes the preparation date and study number for which the solution was initially prepared. Sign on the final page of this method to signify that you have followed the method as written, all materials and reagents are current, and all equipment has been properly calibrated. If you deviate from foe method, document the change, and obtain foe approval of the unit manager, study director, or task leader as soon as possible. Initial and date all data entries on the page on which they were made. If only one person enters all data on a single day, foe documentation may be made in a single location on that page. If multiple staff make entries, foe additional entries must be initialed and dated by the person snaking the entry. Line-outs or NA denotes "Not Applicable". The method is written in general chronological order, but the sequence of steps may be altered if foe analyst deems it appropriate, unless the order for certain activities is specified. Stocks will be used for foe duration of the study unless consumed or unless stability is considered suspect. No correction will be made for purity or salt content of any test article but PFOSAA Use glass volumetric, Eppendorf repeater, or positive-displacement pipets for dispensing methanolic solutions. a Contact with Teflon by the test article should be minimized. V. M aterials See Table 1 for all required chemicals, reagents, and solvents. Use Table 1 for documentarioa Check all labels careftiliy to ensure that all materials are not expired and that they are the proper purity or grade. Page 2 of 16 C-2 3M Environmental Laboratory Page 189 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Battel le Study Number: N 003296-G 3M Toxicology Services Protocol Number: F A C T -T O X -1 11 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFO S) IN RAT LIVER BY LC /M S/M S Version i.O Study No.:_________________________ ' Analyjt/Date: __________________________ [\L iic ri.iis l.\c Potassium Perfiuorooctanesulfonate (PFOS) 1H,1H,2H,2HPerfluorooctane Sulphonic Acid M-556 M570 PFOSAA PFOSA PFOSEA Rat Liver Analytical Standard Surrogate Standard Analytical Standard Analytical Standard Analytical Standard Analytical Standard Analytical Standard Matrix Ammonium Acetate. N IL C O , Sodium Hydroxide, NaOH Tetrabutylammonium Hydrogensulfate (TBA), fCH3(CH2)3]4N(HSO<) Sodium Carbonate, Na3C 0, Sodium Bicarbonate, NaHCOi Ethyl Acetate Mobile Phase Reagent Prep Extract Prep . Extract Prep Extract Prep Extract Prep Methanol Miili-Q Water pH 7 Buffer pH 10 Buifer Mobile Phase, Stocks. WS Reagent Prep, Mobile Phase pH meter calibration pH meter calibration Table 1. Materials S u p p lie r (drittle or P u rit\ 3M IN 3M 3M 3M 3M 3M Harlan SpragueDawley Storage Tem p Room Temp Room Temp Room Temo Room Temp Room Temp Room Temp Room Temp --20C RT RT RT Millipore RT ' RT RT RT ASTMTypel RT RT RT I nt n r ID RT means Room Temperature. VI. Equipment See Table 2 for all required major pieces of equipment Use the table to document the actual piece (e.g. make, model) of equipment Check calibration of all equipment requiring calibration (e.g. balances) to ensure it is current Page 3 of 16 3M Environmental Laboratory C-3 Page 190 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Batlelle Study Number: N003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -1 1 1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS Version 1.0 Study No.:_____________________ _ ' Analyst/Date:___________________________ F tiu rp n u 'n t Analytical Balance Weight Set t\c Weigh Standards or Reagents Calibrate Balance Table 2. Equipment M n m iliiftm v r Model \ or St\ 1 Pipettor Pipet Samples Pipettor Pipet Samples Pipettor Pipettor Pipet EtOAc extraction nhase Pipet Reagents, WIS Eppendorf Repeater Vortexer Mix Samples Freezer (-20C) Refrigerator Cl-^CI Centrifuge Store QCs, Blank Liver Store Buffer, Stocks Phase separation Test Tubes Centrifuge Tubes Test Tubes Transport tubes Magnetic stirrer Orbital Shaker Liver sample homogenization Extract Samples Evaporate Extracts Store QCs Stir matrix Extract Samples Stockwell Scientific Blue Falcon Blue Falcon Eikay Polypropylene, 15 mL Polypropylene, 15 mL Polypropylene, 12x75 mm 5 mL polypropylene SW8599 2096 2002 127-T160-56P Evaporator Syringe Filters Evaporate Extracts Filter Extract Zymark Turbovap LV Homogenizer dH meter Electrode Volumetric Flasks, Class A Volumetric Pipets, Class A Transfer Pipets, Plastic Grind liver Determine Buffer t)H Determine Buffer pH Make Volumetric Dilutions Make Volumetric Dilutions Transfer Extracts to Centrifuge Filters and LC Inserts NA NA Samco NA NA NA NA 3M Environmental Laboratory Page 4 of 16 C-4 Page 191 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -1 11 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFO S) IN RAT LIVER BY LC/M S/M S Version 1.0 Study No.: Analyst/Date: _ VII. Procedure A. Preparation of 2 raM Ammonium Acetate Weigh 0.1500 + 0.0020 g of ammonium acetate and transfer to a 1000-mL volumetric flask. Dissolve the solid in water and dilute to volume with water. Solution may be used for one month stored at room temperature. Actual mass of ammonium acetate:_________ Actual final volume:_________ Date of preparation:___________________ Study N o:_______________ B. Preparation o f ~29% Sodium Hydroxide Solution Weigh 2001 2 g of sodium hydroxide into a beaker. Add 500 mL ofMilli-Q water and mix to dissolve. Cool and transfer to a polypropylene bottle for storage. Solution may be stored for 6 months at room temperature. Actual mass of sodium hydroxide:_________ Actual volume Milli-Q w a te r ________ Date of preparation:___________________ Study N o:_______________ C. Preparation of ~2.9% Sodium Hydroxide Solution Add 10 mL of -29% Sodium Hydroxide Solution to a 100-mL volumetric flask and dilute to volume with Milli-Q'water. Transfer to a polypropylene bottle for storage. Solution may be stored for 6 months at room temperature. Actual volume of--29% NaOH solution:__________ Actual final volume: _________ Date of preparation:___________________ Study N o :_________________ D. Preparation of Tetrabutylammonium Hydrogensulfate (TBA) Solution, 0.5 M, (pH 10) pH Meter Calibration pH buffer: 7 pH buffer: 10 pH reading:_______ pH reading:_______ Add 1691 1g of TBA to -500 mL of Milli-Q water in a beaker. Adjust the pH to 10.00 t 0.02 using -55-60 mL of 29% Sodium Hydroxide Solution, dilute to 1000 mL with Milli-Q water, and mix. Adjust the pH to 10.00 s 0.02 using -2.9% NaOH and mix. Transfer to a polypropylene bottle for storage. Solution may be used for one month stored at room temperature, but the nH must be checked prior to each use. Adjust to pH 10.0 * 0.02 with 2.9% Sodium Hydroxide Solution as necessary. Page 5 of 16 C-5 3M Environmental Laboratory Page 192 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -1 1 1 L R N -U 2 9 9 4 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 M ETHOD FO R ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/M S/M S Version 1.0 Study No.: Analyst/Date: _ Actual mass of TBA: _________ Actual final volume:_________ Actual final pH: _________ ' Date of preparation:___________________ Study N o:______________ ' pH after rechecking and/or readjusting:________ E. Preparation of 0.25 M Carbonate Buffer Weigh 26.5 * 0.1 g of sodium carbonate and 21.01 0.1 g of sodium bicarbonate and transfer to the same 1000-mL volumetric flask. Dissolve the materials in Milli-Q water, dilute to volume with Milli-Q water, mix, and transfer to a polypropylene bottle for storage. Solution may be used for 1 month when stored refrigerated. Actual mass of sodium carbonate:_________ Actual mass of sodium bicarbonate:______ _ _ Actual final volume:_________ Date of preparation:___________________ Study N o:______________ F. Preparation of Mobile Phase Component A: Mix together 600 mL of 2 mM ammonium acetate and 400 mL of methanol. Solution may be used for 1 month when stored at room temperature. Actual volume of 2 mM ammonium acetate:_______ mL Actual volume of methanol: _______ mL Date of preparation:___________________ Study N o:______________ Components: Mix together 50 mL of 2 mM ammonium acetate and 950 mL of methanol. Solution may be used for 1 month when stored at room temperature. Actual volume of 2 mM ammonium acetate:_______ mL Actual volume of methanol: _______ mL Date of preparation:___________________ Study N o:______________ G. Preparation of Stock Surrogate Standard and Working Surrogate Standard (WSS) 1. Stock Surrogate Standard (250,000 ng/mL): Weigh 25 2 mg of 1H, 1H, 2H, 2H,-perfluorooctane sulphonic acid and transfer to a 100-mL volumetric flask. Dissolve in methanol, dilute to volume with methanol, and mix. Store refrigerated, protected from UV light Actual Weight:_______________ Actual Dilution Volume:__________________ Date of Preparation:_________________ Study No: ________________ . Page 6 of 16 3M Environmental Laboratory C-6 Page 193 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS Version t.O Study No.: Analyst/Date: _ 2. WSS (1000 ng/mL): Dilute 100 /uL of stock surrogate standard to 25 mL with methanol and mix.. Actual Volume of Stock Internal Standard: __________ Actual Dilution Volume:__________________ Date of Preparation:_________________ H. Preparation of Calibration Solvent Stocks and Working Standards Standard Stock 1A Stock IB Stock 2A Stock 2B Stock 3A Stock 3B Stock 4A Stock 4B Stock 5A Stock SB Stock 6A 1. Solvent Stocks: For each analyte weigh the specified amount of standard (independently weighed as A and B replicates) listed in Table 3 and transfer into separate volumetric flasks. Dissolve in methanol, dilute to volume with methanol, and mix well. Store refrigerated, protected from UV light 2. Mixed Solvent Stocks: Pipet die specified amount of each analytical standard Replicate A as listed in Table 3 and transfer into a single volumetric flask. Dissolve in methanol, dilute to volume with methanol, and mix well. Store refrigerated, protected from UV tight Repeat the process with Replicate B stocks. The m ixed solven t sto ck s a re u sed to prepare th e w orkin g standards. Date of preparation:___________________ Study N o:______________ 3. Working Standards (WS): . Dilute the mixed stocks and working standards with methanol as specified in Table 3 and mix well. Date of preparation:______________________________ Source PFOS PFOS M-S56 M-S56 M570 M570 PFOSAA PFOSAA PFOSA PFOSA PFOSEA T ab le 3 . C alibration Solven t S tocks and W orik--iung.-Svta/nad.arids Target Actual Amount Target Actual Nominai Amount Anahtieal Ntd. Final Voi iFinal V'ul. Stock, or WS (mL) (mL) Cone (m/rnL> 50 t 1 me mg 10 5,000,000 25 *0.5 mg 10 u;..:U-::-:, - 2,500,000 50 t 1 mg ' mg* 10 5,000,000 25 i 0.5 mg 10 2,500,000 50 - 1 mg mg* 10 5.000,000 25 e 0.5 mg 1 10 :: ::*. ' r--: .: 2.500,000 93 e 1 mg mg* 10 5.000,000 46 * 0.5 mg I: rng*i i' 10 r V ,'ii M r.. 2,500,000 50 1 mg mg* 10 --i:':: 'i' 5,000,000 25 : 0.5 mg mg* 10 ri-- :*=: ... 2.500,000 50 e 1mg 'm g * 10 1 5.000,000 Page 7 of 16 C-7 3M Environmental Laboratory Page 194 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -1 1 1 L R N -U 2 9 9 4 Battelle Study Number: N 003296-G 3M Toxicology Services Protocol Number: FACT-TO X-111 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS Version 1.0 ' Study No.: Analyst/Date: _ Stock 6B Mixed Stock A PFOSEA 25 i 0.5 mg __________ 2 S l_ Stocks 1 thru 5 mL mLeach 6 Reu A each** io 50 2.500.000 500.000 Mixed Stocks 1 thru Stock B 6RenB 5 mL each** _____mLeach 50 250,000 WS 1 Mixed Stock A 1 mL** _____ mL 25 20,000 WS 2 Mixed Stock B 1 mL** mL 25 10.000 WS 3 WS1 2 mL** -, mL . 10 4000 WS4 WS 2 2 mL** mL 10 2000 WS 5 WS 3 2.5 mL** m l,!.. 10 W M k ::i}X 1000 WS 6 WS 4 2.5 mL** mL 10 500 WS7 WS5 2 mL** 10 200 * Weigh all analytical standards to at least the nearest 0.01 mg. ** Use volumetric or positive-displacement pipet(s). L Preparation of Calibration Standards and Blanks 1. Liver homogenate Prepare blank liver homogenate in bulk by weighing approximately 40 g of blank liver into a 500 mL Nalgene bottle containing 200 mL of Milli-Q water. Grind to a homogeneous suspension. Aliquot into approx 30 mL portions for frozen (approx -20C) storage. Actual Mass of Liver____________ Actual volume of water:___________ Date of prep :__________________ Study:_____________________ Replicate # Mass (mg) Determine density of calibration/QC matrix: M IX HOMOGENATE THOROUGHLY and determine the mass in milligrams of 10 replicate weighings of 1 mL portions of the THOROUGHLY MIXED homogenate. MIX HOMOGENATE IMMEDIATELY PRIOR TO EACH ALIQUOT REMOVAL. Table 4. Calibration Stds/QCs Matrix Density i wm M . 3 i67 g io 2. Liver Calibration Standards Prepare each liver calibration standard by adding 0.45 mL of undiluted liver homogenate (STIR HOMOGENATE WHILE ALIQUOTING) into a 15 mL extraction tube and adding 50 pL of WS or MeOH. Prepare triplicate cal standards and 6 blanks. See Table 5 for volumes. The diluted liver density is assumed to be approximately 150 mg/mL. Mix well. 3M Environmental Laboratory Page 8 of 16 C-8 Page 195 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FA C T T O X -1 11 ------------------------ LRN-U994 Battelle Study Number: N 0 0 3 2 9 6 -0 3M Toxicology Services Protocol Number: FACT-TOX-t 11 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS Version 1.0 Study No.: Analyst/Date:_ fill Stil/Blank 1 2 3 4 5 6 7 Blank Sou ree W Sl WS 2 WS 3 WS4 WS 5 WS6 WS7 MeOH Table 5. Calibration Standards and Blanks Target Voi Actual Voi Target Attuili Nominal (L) 0'L) Final Vnl Final yol Conc (ml.) (ni Lj) (rii/mL) 50 0.5 2000 50 0.5 1000 50 v:' 0.5 "i;!&yN!NP 400 50 0.5 200 50 0.5 &j:" ;ITT K.j 100 50 0-3____ 50 50 0.5 20 50 0.5 1i ! > r 0 Nominal Conc (llit/n) 13 6.6 2.6 1.3 0.66 0.33 0.13 0 Date of preparation of cal stds/blank: J. Preparation of Quality Control Liver Samples (QCs) i 1. Quality Control Working Standards Dilute the following source volumes metbanot in volumetric flasks and mix well. Prepare fresh when used. Actual volumes are in parentheses. Soin 11) QC WS 1 QC WS2 QC WS 3 QCWS4 Source Mixed Stock A Mixed Stock B QC WS 1 QCWS2 Table 6, QC WS Preparation Vnl Source, mL Final Vol in L 5 f 1 100 ( t l( I 7.5 ( ) S0( ' > 100( y . ills fiiii 1 50 ( U r ' Came* nti/niL 15.000 5000 1125 250 2. Preparation of Quality Control Liver Samples Prepare each QC in bulk by filling the volumetric flask approximately halffilli with undiluted liver homogenate (STIR HOMOGENATE WHILE ALIQUOTING). adding the appropriate QC WS, mixing, and diluting to volume with undiluted liver homogenate (SUR HOMOGENATE WHILE ALIQUOTING). MIX THOROUGHLY and dispense 2.5-mL aliquots into polypropylene tubes and store at approximately -20C. Table 7. QC Preparation ____________________ Q C Source______ Voi Source, m l ______ Final Voi. m L Comi, ng/m L Cone m y; 1 QC WS 1 E 2S(- 2 QC WS 2 D 2-5 < :r, r ) - 25{ ... 3 QC WS 3 1 -:2 .J ( - r ;, - H 25 ( ) 4 QC WS 4 ' i s t - > 1500 500 112.5 25 I 10 1 3.3 0.7 0.16 Date of QC prep:__________________ Study:____________________ K. Preparation of MS Check Standard for System Suitability Pipet 250 juL of WS 2 at -10.000 ng/mL and 2.5 mL of WSS at -1000 ng/mL in methanol into the same 50-mL volumetric flask. Dilute to volume with MeOH and mix. Page 9 of 16 C-9 3M Environmental Laboratory Page 196 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 Battel le Study Number: N003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -1 1i M ETHOD FO R ANALYSIS OF POTASSIUM PERFLUO RO O C TANESULFO NATE (PFO S) IN RAT LIVER BY LC/M S/M S Version 1.0 Study No.: __________________________ ' Analyjt/Date: ____________________________ L. Preparation o f hom ogenizer recovery liver samples To determine the recovery from the homogenization process, anhomogenized blank liver will be fortified in duplicate at 3 concentration levels and homogenized as follows. This needs to be done every day that homogenization of study samples is performed. 1. Place approximately 0.5 g of unhomogenized blank liver into each of 6,15 mL polypropylene centrifuge tubes. Record weights of liver. 2. Add 100 |tL of WS 1,3, and 4 (one WS per duplicate tubes) to prepare fortifications at approximately 4, 0.8, and 0.4 (lg/g. 3. Multiply the mass of liver in g by 2.5 and add this many mL of water. 4. Homogenize each liver sample, and rinse homogenizer probe with another volume of water used in step 3, adding rinse to homogenized sample. 5. Clean homogenizer with MeOH between samples. 6. Cap and vortex homogenate for use in extraction. M . Preparation o f D ilution C heck Sam ple 1. Place 2.95 mL of undiluted liver homogenate (STIR HOMOGENATE WHILE ALIQUOTING) into a 15 mL extraction tube and add 50 pL of Mixed Stock A 2. Dilute 50 pL of step 1 solution (VORTEX SOLUTION WHILE ALIQUOTING) with 0.45 mL of undiluted liver homogenate (STIR HOMOGENATE WHILE ALIQUOTING) in 3,15 mL extraction tubes. 3. This sample should be prepared for extraction only on days when study samples will be diluted and extracted. N . H om ogenization o f study sam ples 1. Place approximately 0.5 g of unhomogenized study sample liver into a 15 mL polypropylene tube. Record weights of liver. 2. Multiply the mass of liver in g by 2.5 and add this many mL of water. 3. Homogenize each liver sample, and rinse homogenizer probe with another volume of water used in step 2, adding rinse to homogenized sample. 4. Clean homogenizer with MeOH between samples. 5. C ap and vortex homogenate for use in extraction. O. Analysis Standards, B lanks, QCs, and Samples 1. MIX LIVER HOMOGENATES THOROUGHLY BEFORE ALIQUOTING and pipet 500 juL of each QC (4 replicates per level), and other samples being extracted into 15-mL polypropylene extraction tubes. The cal stds and blanks are already aliquoted. 2. To the Blanks - IS (3 reps), add 100 ^L of MeOH and vortex. 3. To the Blanks + IS (3 reps) and to the remaining samples, add 100 /aL WSS and vortex. 4. Add 0.5 mL of 0.5 M TBA (pH 10) to all tubes and vortex briefly. 5. Add 1 mL of 0.25 M carbonate buffer and vortex briefly. 6. Add 2.5 mL of ethyl acetate. Place the tubes sideways on the orbital shaker at a setting of 300 for ~20 minutes. 7. Centrifuge tubes at a setting of 3500 rpm for ~20 minutes to separate layers. 8. Transfer 2 mL of the top organic layer to a clean polypropylene tube. Page 10 of 16 3M Environmental Laboratory C-10 Page 197 BACK TO MAIN 3M Medical Department Study: T-6395.14 ------------------------------------------------------------------------------------------------------------------------------------- - Analytical Report: FACT T O X -111 ~ LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: F A C T -T O X -1 1 1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/M S/M S Version 1.0 Study No.:_______________________ _ _ Analyst/Date:_____ __ 9. Evaporate to dryness under nitrogen at a setting of 30C for -60 minutes. 10. Reconstitute the residues in 500 /jL of methanol with vortexing. 11. Syringe-filter extracts into autosampler vials for analysis. Store vials refrigerated (up to 1 month) if LC/MS/MS will not be performed the same day. Since 3-day room temperature extract stability was demonstrated during validation, the extracts of the cal stds, blanks, and QCs may be reused for up to 3 days after their initial preparation if held at room temperature (recap the vials if reusing). Date of cal std/blank extract prep:_______________________ Date of QC extra prep:______________________________ P. LC/M S/M S Analysis 1. Use the system conditions specified in Table J0. The conditions which are designated may be modified by the analyst to produce acceptable peak shape. LC/MS/MS System Table 8 - LC/MS/MS Conditions Aitto.su in pier M ake:___ Model: ID: HPLC Pumps M ake:___ Model: ID: iYiass Spectrometer Make: Model: ID: Analytical column Keystone Betasil CI8, 5m , 2 x 50 mm, Part No. 055-701-2, S /N :____ :Lot: Mobile Phase Component A: Ammonium acetateanethanol, 60:40, v:v Components Comnonent B: Ammonium acetate:methanol. 5:95. v:v Gradient protile Time, min %B Flow, mL/min 0 0 0.3 1 0 0.3 4.5 100 0.3 6 100 0.3 6.1 100 0.6 8.5 100 0.6 9 0 0.3 11 0 0.3 Injection volume 10 uL ( ___ pL)___________ :_____ Flow split LC column flow split to *30 IJmm ( uL/min) into the MS at run start Column Temp Ambient HPLC Pressure 1000 psi at eradient start ( osi) MS Source Electrospritv. Negative Ion D erivation 'as Nitrogen at 575 L/hr ( L/hr) Nebulizer gas Nitrogen at 80 L/hr ( L/hr) Source Block Temp *140C ( C) Desolvation Temp *250C( a Cone voltage *70 V f V) PFOS.TS- *20 V f V) PFOSA, PFOSAA, PFOSEA. M-556. M570 Collision energy *40 eV ( _ eV) PFOS. ItPFO SA , PFOSAA, M-556, M570 *30 eV ( eV) PFOSEA Collision gas 1Arson at *2.5 x 10 mb eas cell ( mb) Multiplier *650 V ( V)_____________ _ Resolution 1 *12.0 for MSI f ):* 10.0 for MS2( ) OSHoui iSfh -Ltf I 1 I Page 11 of 16 C -ll 3M Environmental Laboratory Page 198 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FA C T-TO X -11 1 M ETHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC /M S/M S Version 1.0 Study No.: Analyst/Date: _ tons munitomi l'otal run time Approvili! ite retention times: 427>81 MRM transition for Surrogate Standard (SS) 499>99 MRM transition for PFOS 556>78 MRM transition for M-536 570>169 MRM transition for M570 584>169 MRM transition for PFOSAA 498>78 MRM transition for PFOSA 526>169 MRM transition for PFOSEA *11 minutest min) SS: 4 min ( min) PFOS: 4.3 min (_____ min) M-56: 4.5 min ( _ ___ min) M570:4.6 min (___ __min) PFOSAA: 4.7 m in e mini PFOSA: 5.1 min ( _ ___ min) PFOSEA: 5.7 min ( min) * Parameters that may be changed by the analyst Actual values in ( ). 2. The above conditions should be suitable for the Micromass Quattro LC (S/N 9053). Modifications may be necessary if another Micromass Quattro Series spectrometer is used. Split the flow post-column via a Keystone BlO-tee or similar device. 3. Calibrate the mass spectrometer using a suitable reference compound, or verify that the calibration is suitable by visual inspection (an the tune page) that a suitable mobile phase ion is still accurately determined. Resolution may need to be higher than that used for analyzing samples. 4. To check the proper performance of the instrument, inject the instrument check standard. The results should be comparable to a recent injection if available. 5. Use an automated chromatography integration software system to collect the output from the analysis. 6. Loading Order See the loading report from the automated chromatography integration software system. 7. Make single injections of each cal standard, QC, study sample, or blank. Make at least 4 injections of the instrument check standard. S. Run set sizes should typically not exceed 80 injections due to instrument response roll-offconsiderations. Longer runs may be performed, but they pose a risk of yielding unacceptable curve results. VIH. CALCULATIONS 1. Spreadsheet Software: __________________ Version_______ 2. MS Analysis Software:____________________ Version_______ 3. Calculate the average density of the liver homogenate (10 reps) in mg/mL. 4. Using the average density of the homogenate, calculate its liver density (mg of liver per mL of diluted homogenate): Undiluted liver density (mg/mL) = (g of liver x average density of homogenate)/(g of liver + g of water) where g of liver and g of water are masses used to prepare bulk homogenate; density of water is assumed to be 1 g/mL. - Diluted liver density (mg/mL) = Undiluted density * Diin Factor Page 12 of 16 C-12 3M Environmental Laboratory Page 199 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TO X-111 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS Version t.O Study No.:___________________________ Amdyst/Date: _ZIZZZZZZIZZZIZZZIIZZ Where diln factor = 2/1.8 = 1.1111 to account for 10% diln of liver homogenate in cal std and QC matrices. 3. Calculate the actual concentration (ng/mL) of PFOS and other fluorochemicals in the suspensions of calibration standards and QCs by using the mass of analytes and dilution factors only (no liver density correction). Use purity correction for PFOSAA only. 6. Calculate the actual concentration of PFOS and other fluorochemicals in liver for the calibration standards and QCs as follows: Conc(ug/g) = Cone (ng/mL) * Diluted Liver density (mg/mL) x 1000 mg/g x 10'3 pg/ng 7. Assure that the integrations of the peak areas of the test article and surrogate standard are correct Flag m anual integrations where performed. Calculate the exact concentration of each liver standard. 8. Calculate the regression equation relating the peak response ratio (test ardcle/SS) of each calibration standard (y-axis) to test article concentration in liver (x-axis) for PFOS, M-556, M370, and PFOSAA. Calculate the regression equation relating the peak area of each calibration standard to test article concentration in liver for PFOSA and PFOSEA. PFOS, M336, M370, and PFOSAA are quantitated by using the surrogate standard as an internal standard; PFOSA and PFOSEA are Quantitated without reference to the surrogate (external standard calibration curve). Use a quadratic regression weighted 1/x, origin excluded, for all analytes. 9. Calculate a determined concentration for each injection of calibration standard, QC, and sample using the regression parameters and the peak response ratios or areas. 10. Calculate the relative error, average relative error, standard deviation, and relative standard deviation for all QCs. Calculate the relative error for each injection of calibration standard. 11. Calculate the average recovery for the homogenizer recovery fortifications. 12. Calculate the relative standard deviation for the PFOS to SS peak area ratio of the replicate injections of the check standard. IX . accepta nce Cr iter ia A. MS Check Standard (System Suitability) At least 3 injections of the MS Check Standard must provide a %RSD of 10% or less for the PFOS to SS peak area ratio. B. Calibration Standards The percent relative errors for the concentration-level averages of the calibration standards should meet the following limits: 3M Environmental Laboratory Page 13 of 16 C-13 Page 200 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 1 1 L R N -U 2 9 9 4 Battelle Study Number: N 003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -1 1 1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS Version 1.0 Study No.: Analyst/Date: _ Table 9. Calibration Standard Acceptance limits ANALYTF PFOS M-556 M570 PFOSAA PFOSA PFOSEA " [li rot I 20 (23% at LOO) 20 (25% at LOO) 20 (25% at LOO) 20 (25% at LOO) 20 (25% at LOO) 25 (30% at LOO) Up to 5 calibration standard injections may be excluded from the curve, provided that one injection remains per level. Removal of an entire level may be done if approval is obtained. If an entire level is removed, the samples bracketed by the remaining calibration range will be considered acceptable. The calibration curve should have a coefficient of determination of 0.97 or better. C QCs The concentration-level average percent relative errors and percent relative standard deviations of the QCs should meet the following limits: Table 10. QC Acceptance limits ANALYTK % PFOS 20 M-556 20 M570 20 PFOSAA 20 PFOSA 20 PFOSEA 25 Removal of individual values from the QC calculations may be done if accompanied by a reasonable explanation (e.g., instrument malfunction or Dixon's Q test results). I f the average determined concentration for any QC level exceeds the acceptance limit, the task leader or study director should be notified. T he run m ay be repeated o r a portion o f the run may be considered acceptable. For example, if the low QC fails the stated requirements, samples may be accepted that have concentrations bracketed by tire highest calibration standard and a mid-level QC concentration. D. Homogenizer Recovery and Dilution Check Samples The average recovery across the 3 levels of homogenizer recovery samples as well as that of the dilution check samples should fall within the range o f70-130% inclusive. Removal of individual outliers from the calculations may be done if accompanied by a reasonable explanation. E. Sensitivity (LOQs) The validated limits of quantitation are nominally 0.13 pgfe each for PFOS, M-S56, M570, and PFOSAA. Page 14 of 16 3M Environmental Laboratory C-14 Page 201 BACK TO MAIN 3M Medical Department Study: T-6395.14 ' Analytical Report: FACT T O X -111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FA C T-TO X -111 M ETHOD FOR ANALYSIS OF POTASSIUM P E R F L U O R O O C T A N E S U L F O N A T E (P F O S ) IN RA T LITTER B Y L C /M S/M S Version 1.0 Study No.: __________________________ A n a ly st/D a te :____________________________ For PFOSA and PFOSEA, the validated LOQs are nominally 0.33 pg/g each. Due to the nature of the preparation of the calibration standards, lower concentrations of PFOSA and PFOSEA will be carried through the extraction. These lower concentration values will be evaluated with each run set, and may be included in the regressions if they meet acceptance criteria. If they are included, study samples which are quantitated to have concentrations below the validated level (nominally 0.33 pg/g) will be appropriately flagged. F. Specificity The method suffers from endogeneous matrix interferences at levels sometimes exceeding 20% of LOQ. The intercept of the calibration curve appears to offer some correction for any effect on quantitations. Aoceptabe performance (error) of the lowest used standard, therefore, will be considered sufficient evidence that bracketed study samples are quantified property. G. General . The above acceptance criteria indicate that this method is capable of producing occasional errors outside the normal acceptance criteria of a validated method (15% normally). Where indicated, replicate analyses lessen the impact of these occasional outliers. X . RESULTS See attached hard copy of spreadsheet or see file on network drive. XL Comments 3M Environmental Laboratory Page 15 of 16 C-15 Page 202 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Battelle Study Number: N 003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -1 1 1 M ETHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/M S/M S Version 1.0 Study No.: Analyst/Date: _ XXI. C o n c l u s io n s xm. sign a tu res Analysts ___________________________________________ ___________________________________________ __________________;________________________ _____________ _____________________________ Technical Renew ___________________________________________ ___________________________________________ QC Review Date: Date: Date: Date: Date: Date: Date: Date: 3M Environmental Laboratory Page 16 of 16 C-16 Page 203 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 APPENDIXD-REPRESENTATIVE CHROMATOGRAMS 3M Environmental Laboratory Page 204 3M Medical Department Study: T-6395.14 " BACK TO MAIN Analytical Report: FACT T O X -111 " LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 3M Environmental Laboratory D -l Page 205 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TO X-111 3M Environmental Laboratory D-2 Page 206 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 " LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TO X-111 3M Environmental Laboratory D-3 Page 207 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 3M Environmental Laboratory D-4 Page 208 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Battel le Study Number: N 003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 3M Environmental Laboratory D-5 Page 209 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRf'l-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 3M Environmental Laboratory D-6 Page 210 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT T O X-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TO X-111 3M Environmental Laboratory D-7 Page 211 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Battelle Study Number: N003296-G 3 M Toxicology Services Protocol Number: FACT-TO X-111 APPENDIX E-PROTOCOL, AMENDMENTS, AND DEVIATIONS 3M Environmental Laboratory Page 212 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 N -U 2994 Battelle Study Number: N003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -1 11 ABC. 13. 1999 9:28A M EHVIEONMEMTAL LAB 2 3E 09 * ' 3MSviroiBiilTfcJmolofy PO Box 33331 SU^iOrS5l33.3331 MO. 1406 P. 32 P rotO C O H tF A C T-TO X -111 3M Study Title Oral (Garage) Pharmacokinetic Recovery Study o f PFOS Is Rats PROTOCOL Author L in Clemen Date: June 8,1999 PerformingLaboratory 3M Environmental Technology & Safety Services. 3M Environmental Laboratory 935 Bush Avenue St. Paul. MN 55106 LaboratoryProjectidentification FACT-TOX-111 U2994 3MEnvironmentalLaboratory AUG 13 '99 11:29 3M Environmental Laboratory E-l Pageiof 10 651 778 6178 PAGE.032 Page 213 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 1 1 L R N -U 2 9 9 4 AliG. 1 3 .1 9 9 9 9:27A M . Battelle Study Number: N 003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -1 11 E N 7 M E N T A L LAB 2 3E 09 NO. 1408 P. 33 ProtocolSFACT-TQX-111 Study Identification Oral (Gavage) Pharmacokinetic Recovery Study o f PFOS in Rats TestMaterial Sponsor SponsorRepresentative StudyDirector StudyLoeatlonfs) In vivo Testing Facility Analytical Testing Laboratory Perfluorooctane sulfonic acid potassium salt . (T-6295) 3M Toxicology Services - M edical D epartm ent 3M Center, Building 220-2E-02 S t Paul, M N 55144-1000 M arvin T. Case, D .V.M ., P M ). 3M Toxicology Services Telephone: 651-733-5180 Facsim ile: 651-733-1773 K risten J. Hansen, P h D . 3M Environmental Technology and Safety Services Building 2-3E-09 651-778-6018 Argus Research Laboratories, Inc. 905 Sheehy Drive, B uilding A Horsham. PA 19044 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue S t Paul, M N 55106 3MEnvironmentalLaboratory fiUG 13 * 3 9 1 1 : 2 3 3M Environmental Laboratory E-2 Page2 of10 c o n s c i vc cccc Page 214 BACK TO MAIN 3M Medical Department Study: T-6395.14 AUG. 13. 1999 9:27A M . Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX -111 ENVIRONMENTAL LAB 2 3E 09 NO. 1406 P. 34 ProtocolFACT-TOX-111 Sub-Contract Laboratories Proposed Study Timetable Study tnitlatian Date StudyCompletionDate Advanced Bioanalytical Services, Inc. 15 CatherwoodRoad Ithaca, NY 14850 Battelle Memorial Institute 505 King Avenue Columbus Ohio 43201-2693 lune 8,1999 August 8,2000 1. Study O ral (gavage) pharm acokinetic recovery study o f potassium perfluorooctane sulfonic acid (PF O S )inrats. 2 . Purpose , T his analytical study is designed to determ ine levels o f potassium perfluorooctaaesulfonate (PFOS) in specim ens o f liver and serum o f rats. The in-life portion o f this study w as conducted at A rgus R esearch Laboratories, study #418-015. A ll sentm samples w ill be extracted and analyzed at Advanced B ioanalytieal Sendees, Inc. and all liver samples extracted and analyzed at B attelle M em orial Institute. A dditional analyses m ay be perform ed at the 3M Environm ental L aboratory as m ethods are developed and validated.. I f additional analyses are perform ed an am endm ent to this protocol w ill be w ritten. 3. RegulatoryCompliance T his Study w ill be conducted in accordance with the United States Food and Drug Adm inistration, G ood Laboratory Practices Standards, Final Rule 2 1 CFR 58, w ith the exception th at analysis o f the test m aterial m ixture to r concentration, solubility, homogeneity, and stability w ill not be conducted, and is th responsibility o f the Sponsor. 4. QUALITYASSURANCE The 3M Environm ental Laboratory Q uality Assurance U nit w ill review the protocol and audit study conduct, data, and final report to determ ine compliance w ith Good Laboratory Practice Standards and w ith 3M Environm ental Laboratory Standard Operating Procedures. The QA U nit a t the sub-contract laboratory w ill audit their study conduct, A*, and results report prior to subm itting to the 3M Environm ental Laboratory. 3MEnvironmentalLaboratory Oil fi Ifi ' fifi 1 1 :? ! E-3 Page3of10 7c c t * 7 - 7 0 e t C n-id a c e : a 3M Environmental Laboratory Page 215 BACK TO MAIN 3M Medical Department Study: T-6395.14 AUG. 13. 1999 9:27A M ^ jly t in a l Rennrt- F A C T T O X -1 11 L R N -U 2 9 9 4 Battelle Study Number: N003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -111 ENVIRONMENTAL LAB 2 3 09 HO. H 0 6 P. 35 P rotocol HFACT-TO X-111 5 . TestMaterial 5.1 R efer to Argus Research Laboratory protocol for study #418-015. 6 . ControlMatrices 8.1 IdentificationR at liver and serum, and/or rabbit liver and serum traceability num bers w ill be recorded in the raw data and included in the final report 6.2 Source ArgosResearch and/or Sigma Chemical 6.3 P hysical Description R at liver and serum, and/or rabbit liver and serum 6.4 PurityandStability Not applicable 8 .5 Storage Conditions Frozen at -2 0 C 1 0 aC o r -5 0 C 10 C 5 .8 Reserve Matrix A portion o f die control m atrix w ill be retained in the 3M archives . for as long as the quality o f the preparation affords evaluation, b ut n o t longer than ten years follow ing the effective date o f die final test rule O f applicable). 6.7 Disposition M atrices w ill be retained at the 3M Environm ental Laboratory p er GLP regulation. Certain m atrices (feces, urine, and blood) m ay be disposed after QAU verification. 8 .8 Safety Precautions Refer to MSDS for chemicals used. W ear appropriate laboratory attire, and follow adequate precautions for handling biological m aterials and preparing sam ples for analysis. 7 . R e f e r e n c e Ma t e r ia l 7.1 Identification Potassium perfluorooctanesulfonate (PFOS), lo t# s 171,215, or 217 (equivalent lots) 7 .2 Source 3M Specialty Chemicals 7.3 Physics! Description W hite p ow d er 7.4 PurityandStabilityPurity o f PFOS is 99% or greater. Stability has not been determ ined. 7 .5 Storage Conditions Room temperature 7 .8 Reserve Material A reserve sample fiom each batch ofPFO S used in this study w ill b e retained as long as die quality o f the preparation affords evaluation, b ut not longer than ten yean follow ing die effective date o f the final test rule (if applicable). 7 .7 Disposition Unused reference m aterial w ill be retained for use by die 3M Environm ental Laboratory and w ill be discarded when the quality o f preparation no longer affords evaluation. 3ME nvironm ental Laboratory fiUG 13 ' 9 9 1 1 : 2 1 3M Environmental Laboratory E-4 Page 4 o f 10 si 77B Rive Pace oo Page 216 BACK TO MAIN 3M Medical Department Study: T-6395.14 AUG. 13. 1999 9:28A M Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Battelle Study Number: N 003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -l 11 EHVIRQHMEHTAL LAB 2 3E 09 HO. 1406 P. 36 ProtocolHFACT-TOX-111 7.8 Safety Precautions Refer to MSDS for chemicals used. W ear appropriate laboratory attire, and follow adequate precautions for handling biological m aterials and preparing samples for analysis. 8. TestSystem R ats w ere used as foe test system and w ere m aintained and dosed as described in A rgus R esearch protocol #418-015. The fem ale rats w ill be given foe test material or control once daily 'beginning 42 days prior to cohabitation until day 0 o f presumed gestation. See table 1 fo r m ore dosage inform ation. D osage G roup 1 2 3 T ab lai D osage Levis C oncentration, and Volum es Number o f female rats 8 8 8 D osage m g/kg/day 0 0.1 0.6 Concentration m g/kg/day 0 0.02 . 0.32 D osage volum e m L/kg S 5. 9. Specimenand SampleReceipt The 3M Environm ental Laboratory w ill receive homogeneity sim ples and specim ens o f the follow ing body tissues and fluids from the indicated points in the study. A ll specim ens w ill be packed on dry ice for shipping. See table 2 for more specimen inform ation. B ody tissae/fluid T able 2 Specim en Inform ation C o llected Serum -D am and Pup animal U rine and Feces - Dam and Pup anim als L iv er--D am and Pup animal Dam-Predose, Days 7 ,1 4 ,1 5 ,2 1 , and 22 ' Pup-Day 21 Predose, Days 7 ,1 5 ,2 1 , and 22 Dam-At the termination o f foe study Pup-Day21 E xpected# o f specim ens 144 D am 24 Pup (pooled) 120 U rine and 120 Feces 24 Dam 24 Pup (pooled) Total num ber o f expected specim ens: 456 Total num ber o f test animal-16 Total num ber o f control anim als; 8 3MEnvironmentalLaboratory BUG 13 ' 3 9 I 1 : ?! 3M Environmental Laboratory E-5 Pane5 of10 Page 217 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 AUG. 13. 1999 9 : 28AK Battelle Study Number: N 003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -1 11 ENVIRONMENTAL LAB l 3E 09 KO. 1406 P. 33 ProtocoltFACT-TOX-W Specimens sent to 3M Environmental Laboratories w ill be received and tracked according to applicable Standard Operating Procedures. 10. PreparatoryMethods 10.1 FACT-M-1.1, Extraction o f Potassium Perfluorooctanesulfonate or O ther A nionic Fluorochem ical Surfactant from Liver for A nalysis Using HPLC-Electrospray/M ass Spectrom etry 10.2 ETS-8-4.1, Extraction o f Potassium Perfluorooctanesulfonate o r Other Fluorochem ical Compounds from Serum o r O ther Fluid for A nalysis U sing HPLCElectrospray/M ass Spectrometry 10.3 I f preparatory m ethods other than those listed above aroused, an am endment to this protocol w ill be w ritten. Any deviations from these methods w ill be docum ented and included w ith the study data. 10.4 I f analyses are sub-contracted to other laboratories, an amendment w ill be w ritten to include their methods and copies o f each method w ill be attached to this protocol. 11.Analytical Methods 11.1 FACT-M -2.1, A nalysis o f Fhiorochem icals in liv e r Extracts U sing HPLCElectrospray/M ass Spectrometry 11.2 ETS-8-5.1, Analysis o f Potassium Perfruorooctanesulfonate or Other Fluorochem icals in Serum or Other Fluid Extracts U sing HPLC-Electrospray/M ass Spectrom etry 11.3 I f analytical methods other Qian those listed above are used, an amendment to this protocol w ill be w ritten. Any deviations from these methods w ill be docum ented and included w ith the study data. 11.4 I f analyses are sub-contracted to other laboratories, an amendment w ill be w ritten to include their m ethods and copies o f each method w ill be attached to this protocol. iz d a t a Quality Objectives The num ber o f spikes/duplicales, use o f surrogates, and inform ation on other data qualify indicators axe included in foe analytical method*. In addition, foe following criteria w ill be met: 12.1 Linearity r*> 0.98 12.2 Limits ofdetection/ quantitation 12.2.1 M ethodD eteetionLim it (MDL) forPFO S a) Serum: 1.75 ppb b) Liver. 15 ppb 3MEnvironmentalLaboratory AUG 13 '99 11:22 E-6 Page8of10 651 778 6176 PAGE.037 3M Environmental Laboratory Page 218 BACK TO MAIN 3M Medical Department Study: T-6395.14 A nalytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 AUG. 13. 1999 9:28A M Battelle Study Number: N003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -1 11 ENVIRONMENTAL LAS 2 3E 09 NO. 1406 P. 38 Protocol#A A C 7-T 0X -f 11 12.2.2 Lim it o f Q uantitation (LOQ) - Equal to the lowest acceptable standard in the calibration curve 72.3 Duplicate acceptableprecision < 30% for the method 12.4 Spike acceptablencovartas 70% - 130% 12.5 Use ofconfirmatorymethods Indeterminate samples w ill be re-analyzed using a confirm atory m ethod. I f a confirm atory method is used, an am endment to th is protocol w ill be w ritten. 12.6 Demonstration ofspecificity Chromatographic retention time, m ass spectral daughter ion characterization. 13.Sub-ContractedAnalysis 13.1 A ll analyses as detailed in this protocol w ill be performed at 3M Environm ental Laboratories, B uilding 2-3E -09,935 Bush Avenue, S t Paul. M N 55106, at A dvanced Bioanalytical Services, hie., 15 Catherwood Road, Ithaca, NY 14850, or at B attelle M emorial Institute, 505 K ing Avenue, Columbus, Ohio 43201-2693. 13.2 A n am endment to this protocol w ill be w ritten if analyses are perform ed at laboratories other than the 3M Environmental Laboratories, Advanced Bioanalytical _ Services, Inc., or B attelle M emorial Institute. 14.StatisticalAnalysis Averages and standard deviations w ill be calculated. The statistical methods that w ill he used are described below: 14.1 Dahl transformations andanalysis D ata w ill be reported as die concentration (weight/weight or weight/VoI) o f PFOS or m etabolite par tissue or fluid. 14.2 Statisticalanalysis Statistics used may include regression analysis o f concentrations over tune, and standard deviation! calculated for the concentration* w ithin each dose' group. If necessary, sim ple statistical tests, such as Student's t test, m ay be applied to evaluate statistical difference. 75. REPORT A report containing all the results o f the study w ill be prepared by the 3M Environm ental Laboratory. I f analyses are subcontracted to other laboratories, each laboratory w ill prepare a report and subm it it to the 3M Environmental Laboratory for inclusion in the 3M Environm ental Laboratory rep o rt Each report w ill include, but not be lim ited to, the following, w hen applicable: 15.1 Name and address o f the facility performing the study 13.2 D ates upon which the study was initiated and completed 3MEnvironmentalLaboratory PageToUQ flUG 13 *99 11:22 E-7 651 778 6178 PAGE.838 3M Environmental Laboratory Page 219 BACK TO MAIN 3M Medical Department Study: T-6395.14 AUG. 1 1 1999 9:29A M Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Battel le Study Number: N 003296-G 3M Toxicology Services Protocol Number: FA C T-TO X -111 ENVIRONMENTAL LAB 2 3E 09 NO. U 0 6 P . 3 9 . ProtocolFACT-TOX-111 15.3 A statem ent o f com pliance by the Study D irector addressing any exceptions to Good Laboratory Practice Standards 15.4 O bjectives and procedures as stated in the approved protocol, including any changes in the original protocol 15.5 The test substance identification by nam e, chemical abstracts num ber o r code number, strength, purity, and com position or other appropriate characteristics, i f provided by the Sponsor . 15.6 Stability and the solubility o f the test substances under the conditions o f adm inistration, i f provided by the Sponsor 15.7 A description o f the m ethods used to conduct the test(s) 15.8 A description o f the test system 15.9 A description o f any circum stances that m ay have affected the quality o r the integrity o f the data 15.10 The name o f the Study D irector and the names o f other scientists, professionals, and supervisory personnel involved in the study 15.11 A description o f the transform ations, calculations, or operations perform ed cm the data, a summary and analysis o f foe analytical chem istry data, and a statem ent o f the conclusions drawn from foe analyses 15.12 Statistical method* used to evaluate the data, if applicable 15.13 T he signed and dated reports o f each o f foe individual scientists or other professionals involved in foe study, if applicable 15.14 The location where raw data and foe final report are to be stored 15.15 A statem ent prepared by the Quality Assurance U nit lilting the dates that study . inspections and audits w ere made, and foe dates o f any findings reported to foe Study D irector and M anagement I f it is necessary to make corrections or additions to a report after it has been accepted, the changes w ill be made in foe form o f an amendment issued by foe Study D irector. The am endm ent w ill clearly identify the part o f the report that is being amended, foe reasons for the am endm ent, and w ill be signed by foe Study Director. 16.Location ofRawData, R e c o w , anoFinalR e p o r t O riginal data, o r copies thereof, w ill be available at foe 3M Environmental Laboratory to facilitate audits o f the study during its progress and before acceptance o f the final rep o rt W hen foe fin al report is completed, all original paper data, including those item s listed below, w ill be retained in th e archives o f 3M Environmental Laboratory for at least a period o f tim e as specified by regulation, and as established by 3M Environmental Laboratory Standard Operating Procedures. 3MEnvironmentalLaboratory flUG 13 ' 9 3 1 t : 22 E-8 Paga8of 10 fiS 1 7 7 f l 8 1 7 6 p a c e m o 3M Environmental Laboratory Page 220 BACK TO MAIN 3M Medical Department Study: T-6395.14 AUG. 13. 1999 9:29A M - L R N -U 2 9 9 4 Battelle Study Number: N 003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -1 1 1 ENVIRONMENTAL LAB 2 3E 09 NO. 14 0 6 P. 40 Protocol#FACT-TOX-111 16.1 T he follow ing raw data and records w ill be retained in the study folder in die study/project archives according to 3M Environmental Laboratory Standard O perating Procedures: 16.1.1 Approved protocol and amendments 16.1.2 Study correspondence 16.1.3 Shipping records 16.1.4 Rawdata 16.1.5 Approved final report (original signed copy) 16.1.6 Electronic copies o f data 16.2 T he follow ing supporting records w ill be retained separately from the study folder in th e archives according to 3M Environm ental Laboratory Standard Operating Procedures: 16.2.1 Training records 16.2.2 Calibration records 16.2.3 Instrum ent m aintenance logs 16.2.4 Standard Operating Procedures, Equipm ent Procedures, and M ethods 4' 17. SpecimenRetention Specim ens w ill b e m aintained In the 3M Environm ental Laboratory specim en archives fo r a period o f tim e as specified by regulation or as long as the quality o fthe preparation affords evaluation, b ut n ot longer than ten years follow ing the effective date o f the final test rule (if applicable), and aa established by 3M Environm ental Laboratory Standard O perating Procedures. 18. ProtocolAmendmentsanddeviations Planned changes to the protocol w ill be in the form o f w ritten amendments signed by the Study D irector end die Sponsor's Representative. Amendments w ill be considered as p art o f the protocol and w ill be attached to the final protocoL A ll changes to the protocol w ill be indicated in foe fin al rep o rt A ny other changes w ill be in the form o f w ritten deviations, signed by the Study D irector and filed w ith the raw data. 19. Attachments 19.1 A ttachm ent A Preparatory and analytical methods 3MErtvlronmenMLaboratory flUG 13 '99 11:23 3M Environmental Laboratory E-9 Page9of10 SSI 778 617S PAGE.040 Page 221 BACK TO MAIN 3M Medical Department Study: T -6395.14 _______________________________Analytical Report: FACT T O X -111 ABC. 1 3 .1 9 9 9 9:29AM 20.Signatures Battel le Study Number: N 003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -1 11 ENVI M E N T A L LAB 2 3E 09 NO. 1 4 0 5 P. 41 Protocol FACT*TOX-111 7tiL T. Case, D.V.M ., P h D ., Sponsor Representative D ate M W l K ris;tten J ., Hansen, P hD ., 3M Envii:ronm ental Laboratory Study D irector Date 3MEnvironmentalLaboratory AUG 13 -99 11:23 3M Environmental Laboratory E-10 Page 10of10 <5il 77B R17S pose n ^i Page 222 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Battelle Study Number: N 003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -1 11 Study m e O ral (G avage) Pharm acokinetic R ecovery S tudy ofP F O S in R ats PROTOCOL AMENDMENT NO. 1 Amendment Date: August 12,1999 Performing Laboratory 3M E nvironm ental T echnology & S afety Services 3M E nvironm ental L aboratory 935 Bush Avenue St Paul, MN 55106 Laboratory Project Identification ET& SS F A C T -T O X lll L IR N U 2994 3M Environmental Laboratory 3M Environmental Laboratory E -ll Page 223 BACK TO MAIN 3M Medical Department Study: T-6395.14 AnalyticalReport^A^^O>^^1 L R N -U 2 9 9 4 Battelle Study Number: N003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -1 11 ProtocolFACT-TOX111 AmendmentNo. 1 This amendment modifies the following portions) o f the protocol: 1. Protocol reads: S ection 10.0 and 11.0 list the follow ing m ethods to use for extraction and analysis: FA C T-M -1.1 "E xtraction o f P otassium Perfluorooctanesulfonate or O ther A nionic F luorochem ical S urfactant from L iver for A nalysis U sing H PLC -Electrospray/M ass Spectrom etry" ^ FA C T-M -2.1 "A nalysis o f Fluorochem icals in L iver Extracts U sing H PLC -Electrospray/M ass S pectrom etry'' AM END t o r e a d : T he extraction and analytical m ethods FACT-M -1.1 and FA C T-M -2.1, respectively, w ere updated o n 07122199 to: E T S-8-6.0 "E xtraction o f P otassium Perfluorooctanesulfonate o r O ther Fluorochem ical *C om pounds from L iv er fo r A nalysis U sing H PLC -Electrospray/M ass Spectrom etry" E T S -8-7.0 "A nalysis o f P otassium Perfluorooctanesulfonate o r O ther F luorochem ical C om pounds in L iver E xtracts U sing H PLC -Electrospray/M ass Spectrom etry" Reason: T he extraction and analytical m ethods FACT-M -1.1 and FA C T-M -2.1, respectively, w ere updated on 07/22/99 to E T S-8-6.0 and ETS-8-7.0. T hese m ethods w ere updated to replace th e extraction solvent ethyl acetate w ith a different extraction solvent M TBE (m ethyl tert butyl eth er), P O A A and M onoester w ere rem oved ho rn the standard m ix, and M 556 w as added to die standard m ix. T h e analytical m ethod w as updated to include linear regression w ith 1/x w eighting and a few m inor changes in the H PLC 1100 instrum ent param eters. 2. PROTOCOL RE4D5.' S ection 10.4 and 11.4 state that i f the analyses are sub-contracted to o th er laboratories an am endm ent w ill b e w ritten to include these m ethods. A mend to read: T he extraction and analytical m ethods to follow at A dvanced B ioanalytical S ervices w ill b e attached to d ie protocol. T he extraction and analytical m ethod to follow at B attelle M em orial Institute is: "M ethod fo r A nalysis o f P erfluorooctane Sulfonate (PFOS) in R at S erab y LC/M S/M S, 1.0 REASON: T he analytical m ethods a t the sub-contract laboratories w ere not included in the original protocol. iA fc 3M EnvironmentalLaboratory E-12 3M Environmental Laboratory Page 224 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT T O X -1 11 L R N -U 2 9 9 4 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 ProtocolFACT-TOX111 AmendmentNo. 1 3. PROTOCOLreads: Section 12.2.1 b) lists liver method detection lim it as 15 ppb. AMENDTOread: The liver method detection lim it is 8.50 ppb (ng/g). R e a s o n : The validation supporting methods ETS-8-6,0 and ETS-8-7.0 included a low er method detection limit for PFOS. . Amendment Approval Nfarvin Case, D.V.M ., P hD ., Sponsor Representative . * Date K ris J. Hansen, P h D ., Study Director ( i p * ---------- D at 3 M Environm ental Laboratory 3M Environmental Laboratory E-13 Page 225 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 -- 1" Lkw-u-tflgr Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TO X-111 Study m e Oral (Gavage) Pharmacokinetic Recovery Study ofPFO S in Rats PROTOCOL AMENDMENT NO. 2 Amendment Date: September 30,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 93S.B ush A venue S t Paul, M N 55106 LaboratoryProject Identification ET&SS FACT-TOX111 L IR N U 2994 3M EnvironmentalLaboratory 3M Environmental Laboratory E-14 Page 226 BACK TO MAIN 3M Medical Departm ent Study: T -6395.14 ________________________ _________ Analytical Report: F A C T T Q X ^ U -1 L R N -U 2 9 9 4 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TO X-111 . ProtocolFACT-TOX111 AmendmentNo. 2 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: Section 2 states the study is designed to determine levels o f potassium perfluorooctanesulfonate (PFOS) in specimens o f liver and sera in rats. Amend to read: The study is designed to determine levels o f potassium perfluorooctanesulfonate (PFOS) in specimens o f liver, sera, and urine in rats. . Reason: .The urine analytical methods were validated and approved after approval o f the original protocol 1 Protocol reads: Section 10.0 and 11.0 list the following methods to use for extraction and analysis: ETS-8-4.1 "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-6.0 "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-5.1 "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochem ical Compounds in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" Amend to read:: ETS-8-4.1 "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis U sing HPLC-Electrospray/Mass Spectrometry" ETS-8-6.0 "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from L iver for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-96.0 "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochem ical Compounds from Urine for Analysis Using HPLC-Electrospray/Mass Spectrometry/Mass S pectrom etry" ETS-8-5.1 "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-97.0 "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrospray/Mass Spectrometry/Mass S pectrom etry" REASON: T he extraction and analytical m ethods ETS-8-96.0 and ETS-8-97.0, w ere ^ p ro v e d after approval o f the original protocol. 3 M Environm ental Laboratory E-15 3M Environmental Laboratory Page 227 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L K N -U 2 9 9 4 Battelle Study Number: N003296-G 3M T oxicology Services Protocol Number: FACT-TO X-111 ProtocolFACT-TOX111 AmendmentNo. 2 3. Protocol reads: Section 6.1 lists control matrices as rat liver and serum o r rabbit liver and serum. Source Argus and/or Sigm a Chemical. Amend to read: Control matrices identification rat liver and serum, rabbit liver and serum, human urine and/or rat urine. Source Argus, Sigma Chemical, Lampire Biologicals, Biological Specialty Corp. and/or Golden W est Biologicals. Reason: Addition o f control urine specifications. 4. Protocol reads: Section 12.2.1 a) lists sera m ethod detection lim it as 1.75 ppb and b) lists liver method detection lim it as 15 ppb. Amend toread: The m ethod detection lim its for all compounds and matrices 'will be taken from the methods used for extraction and analysis. Reason: The method detection lim its listed are specific to the 3M Environmental Laboratory. Statement w as added to allow for sub-contracted analyses and/or revised methods. 3 M Environm ental Laboratory 3M Environmental Laboratory E-16 Page 228 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Battelle Study Number: N 003296-G 3M Toxicology Services Protocol Number: FACT-TO X-111 Protocol FACT-TOX111 AmendmentNo. 2 5. Protocol reads: Section 16 states that th e original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits o f the study during its progress and before acceptance o f the final report. W hen die final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, electronic copies o f data, training records, calibration records, instrument maintenance logs and standard operating procedures, equipment procedures, and methods w ill be retained in the archives o f the 3M Environmental Laboratory. Amend to read: . Section 16 states that the original data, or copies thereof will be available at the 3M Environmental Laboratory to facilitate audits o f the study during its progress and before acceptance o f the final re p o rt W hen the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, and electronic copies o f data will be retained in the archives o f the 3M Environmental Laboratory. A ll corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods w ill be retained in the archives o f the facility performing each analysis. Reason: Clarification o f the disposition o f archived records i f analyses are performed at a sub contract laboratory. 6. Protocol reads: Section 17 states that specimens will be maintained in the 3M Environmental Laboratory specim en archives. Amend toread: Specimens w ill be maintained in the 3M Environmental Laboratory specimen archives. All specim ens sent to sub-contract laboratories will be returned to the 3M Environmental Laboratory upon completion o f analysis and submission o f the sub-contract laboratory(s) final rep o rt T he specimens w ill be returned w ith die following documentation: die signed original chain o f custody and records o f storage conditions while at die sub-contract facility. Reason: Clarification o f the disposition o f specimens and documentation for analyses performed by a sub-contract laboratory. 3 M Environm ental Laboratory 3M Environmental Laboratory E-17 Page 229 3M Medical Department Study: T-6395.14 ." BACK TO MAIN Analytical Report: FACT T O X -111 ' LRN-U2994 Battelle Study Number: N 003296-G 3 Mi Toxicology Services Protocol Number: FACT-TO X-111 Amendment Approval ProtocolFACT-TOX1 11 Amendment No. 2 M arvin Case, D .V Jii, PhJD., Sponsor Representative j j j ***** Kristen J. Hansen, P h D ., Study Director s Oct n w D ate 3M Environm entalLaboratory 3M Environmental Laboratory E-18 Page 230 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T TO X ^111 L R N -U 2 9 9 4 Study Title Analytical LaboratoryReport ontheDeterminationofthePresence andConcentration of PotassiumPerfluorooctanesulfonate (CASNumber2759-39-3) inthe Serum, Liver, andUrine of Crl:CDBRVAF/PlusRatsExposedtoPFOS Via Gavage PROTOCOL AMENDMENT NO. 3 Amendment Date: 20 January 2000 Performing Laboratories Urine Analyses 3M Environmental Technology and Safety Services Fluorine Analytical Chemistry Team Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Performing Laboratories Liver Analyses Battelle Memorial Institute 505 King Avenue Columbus, OH 43201-2693 Serum Analyses Advanced Bioanalytical Services, Inc. 15 Catherwood Road Ithaca, N Y 14850 Laboratory Project Identification ET&SS LRN-U2994 FACTTOX-111 Argus Study: 418-015 3M Medical Department Study: T-6295.14 3 M Environmental Laboratory E-19 3M Environmental Laboratory Page 231 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Protocol LRN-U2994 Amendment Number 3 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: The study director for the present study was identified in the protocol as Kristen J. Hansen, Ph.D. A mend to read: The role of study director for the present study was reassigned to Marvin T. Case; D.V.M., Ph.D., as of 20 January 2000. The previous study director, Kristen J. Hansen, has been reassigned to the role of Principle Analytical Investigator. R easo n: The role of study director was reassigned in an effort to ensure compliance with Good Laboratory Practice Standards that outline study personnel requirements (refer to 21 CFR Part 58). 2. Protocol reads: The sponsor for the present study was identified as Marvin T. Case, D.V.M., Ph.D. A mend to read: The role of sponsor for the present study was reassigned to John L. Butenhoff, Ph.D., as of 20 January 2000. R eason: To ensure that the study director does not also cany the duties of study sponsor, the sponsor role was reassigned. In this manner, personnel responsibilities and workload are more evenly balanced. 3M Environmental Laboratory 3M Environmental Laboratory E-20 Page 232 3M Medical Department Study: T-6395.14 Amendment Approval BACK TO MAIN Analytical R a n n rf FACT TDX -111 LRN-U2994 Protocol LRN-U2994 Amendment Number 3 John L. Butenhoff, PhD., Sponsor Representative Date -ib i-- -- Kristen J. Hansen, PhD., Outgoing Study Director f t - &xA> - 2<fltO Date S I a a m ^ r < s u , _____ . ...... .............. ........ / f b v u y %yt-r> Marvin T. Case, D. V.M., PhD., Incoming Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory E-21 Page 233 BACK TO MAIN 3M Medical Department Study: T-6395.14 ----------------------------------------- ----- " _______________ " Analytical Report- FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X - 111 DEVIATON REPORT Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 ORAL (GAVAGE) PHARMACOKINETIC RECOVERY STUDY OF PFOS IN RATS TYPE OF DEVIATION: PROTOCOL DATE OF DEVIATION: September 28,1999 NATURE OF DEVIATION: The source of control matrix will not be either Argus Research or Sigma Chemical as specified in section 6.2 o f the protocol. CAUSE OF DEVIATION: Harlan will be the supplier o f control rat livers used to prepare blanks, any standards, and QCs for the analytical portion o f the study. IMPACT OF DEVIATION ON STUDY: Harlan was used as the control matrix supplier for Battelle's validation of the analytical method (Battelle study number N003604-A). This supplier provided matrix that allowed achievement of the reported method acceptance criteria so that there is not impact on the study. CORRECTIVE ACTION: This protocol deviation report was prepared. APPROVED BY: Jon C. dre, Ph.D. Battelle rincipal Investigator Marvin T. Case, D.V.M., Ph.D. Study Director Date -7 f c / . Q +o/_ Date 3M Environmental Laboratory E-22 Page 234 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battelie Study Number: N003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -1 1 1 DEVIATON REPORT Battelie Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 ORAL (GAVAGE) PHARMACOKINETIC RECOVERY STUDY OF PFOS IN RATS TYPE OF DEVIATION: PROTOCOL DATE OF DEVIATION: October 4,1999 NATURE OF DEVIATION: The dilution recovery results for rat liver did not meet the method acceptance criteria o f 70-130%. This is required in section IX. D. of the method and section 12.4 o f the study protocol. CAUSE OF DEVIATION: The actual dilution recovery was 133%. IMPACT OF DEVIATION ON STUDY: The remaining method performance tests (calibration curve, QCs, and homogenization recoveries) met their respective acceptance criteria. Approximately 2/3 o f the study samples were diluted before extraction, and therefore potentially affected. The dilution recovery was not considered to be exceedingly high enough, at only approximately 3% above the normal acceptance level, to have significantly impacted the data. CORRECTIVE ACTION: The dilution recovery data will be presented in the final report. APPROVED BY: Battelie Principal Investigator Date Study Director 3M Environmental Laboratory E-23 Page 235 BACK TO MAIN 3M Medical Department Study: T-6395.14 Angly]jgg[ Renort: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -11 1 DEVIATON REPORT Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 ORAL (GAVAGE) PHARMACOKINETIC RECOVERY STUDY OF PFOS IN RATS TYPE OF DEVIATION: Protocol DATE OF DEVIATION: August 20, 1999 NATURE OF DEVIATION: Protocol states Battelle will analyze 24 dam and 24 pup (pooled). Chain o f Custody Record indicates only 45 samples were received. IMPACT OF DEVIATION ON STUDY: The impact of this is minimal to the interpretation of the study outcome. CORRECTIVE ACTION: This protocol deviation report was prepared. APPROVED BY: V W - C . CL-<u Jon C. ^ndre, Ph.D. Battelle Principal Investigator Date Study Director 3M Environmental Laboratory E-24 Page 236 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N 003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -1 11 i i APPENDIX F - PFOS PURITY REPORT 3M Environmental Laboratory Page 237 BACK TO MAIN 3M Medical Department Study: T-6395.14 ETSS 2 3W Analytical Report- F A C T T O X -1 11 L R N -U 2 9 9 4 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-t 11 ^ 651 778 4226 0 4 /2 6 /9 9 0 9 :5 6 0 :0 2 /0 3 NO:341 iE From: Subject: Date: 3M SPECIALTY ADHESIVES & CHEMICALS ANALYTICAL LABORATORY . Lisa Clem en. (8-3568) -ET & SS- 2-03-09 Request # 57830 Tom Kestner (3-3633) - SA&C Analytical Lob 236-2B-11 C h tm lc a l C h a r a c ttr tz a tio n o f P O S F - B a s td F lu o r e c h o m ie a ls b y 'J t - N M R ` ' F - N M R S p tc tro ic o p y March 24, 1999: Preliminary report for FC-95 (PFOS), lot 171 gAMELEPESCKKITOft FC-95, lot 171 (PFOS). TN-A-0834; Nominal product 'CiFirSOj(-) K(+) (white powder) INTRODUCTION This sample was subjected to 'H-NMR and "F-NMR spectral analyses to determine the purity o f the nominal product and to characterize as many impurity components as possible. EXPERIMENTAL A portion of the sample was accurately weighed, spiked with a known amount of l,4-bis(trifluoromethyl)benzene (p-HFX), and then totally dissolved in DMSO-d for subsequent analysis by NMR. A 400 M Hz 'H-NMR spectrum (# h57830.401) and e 376 MHz i'F-NMR spectrum (# (57830.401) were acquired using a Varian UNITYplui 400 FT-NMR spectrometer. Use of the p-HFX internal standard was intended to permit the determination of the absolute weight percent concentrations of the assigned components without necessarily needing to identify or quentify all the components in the sample mixture. KSSULISi The combined NMR spectral data were used to assign all of the major and most of the minor components in this sample as received. The qualitative and quantitative compositional results that were derived from the single trial NMR internal standardization analyses are summarized in TABLE-1 on the following page. I have reported both relative and absolute weight percent concentrations. One possible reason that the absolute wt.% values add up to more than 100% may be due to the fact that I assumed all of the components contained 8 carbons. If there were any shorter chain homologs present (i.e., 7 ,6 ,5 , etc. carbons), then the average compound molecular weights would have been somewhat less then those used in the calculations. In general, the "F-NMR technique is not ' particularly well suited for Identifying or quantifying small amounts of various fluorochemical homolog Impurity components unless the chains are veiy short. A more complete characterization of any other fluorochemical homologs would require analysis by electrospray MS or a similar technique. Additional work would be required in en effort to positively verify the tentatively assigned components listed in TABLE-1 (denoted by possible). Small amounts of other unidentified impurities are also detected in the NMR spectra, but additional work would be required in an effort to identify or quantify these other materials. Copies o f the NMR spectra will be provided for you at a later date. If you have any questions about the results in this initial report for FC-95, lot 171, please let me know. I apologize for the delay in completing this initial work. Tom Kestner e-. Rick Payfer - SA&C Analytical Lab - 23A-2B-11 Fite Ratone: LC37IM.DOC/St 3M Environmental Laboratory F ap-tF -l Page 238 BACK TO MAIN 3M Medical Department Study: T-6395.14 -- Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Battelle Study Number: N 003296-G 3 M Toxicology Services Protocol Number: F A C T -T O X -1 1 1 @651 778 4226 04/26/99 09:56 0 :03/03 NO:341 SA&C Analytical L ib Request #57830 Inlflri Report far P r.ofl |nt 171 TABLE-1 Sample: PC-95, lot 171 (PFOS), TN-A-0834 Overall Quantitative Compositional Results by ,H/'*F-NMR Internal Standardization Analyses Structural Assignments CFj(CFj),-SOj(-) K(+) . (Normal chain: atsuma a >7 for calculation purposes) NMR Absolut Weight* Concentrations (Inule trial measurement 70.3% NMR Relative Weight* Concentrations .,,(single trial measurement 68.6% CF*(CFa)t-CF(CFjHCFi},-$Oi(-) K(+) (Intomtl monomethyl branch; assume x+y 5 , a 0 0, A y # 0, for calctdsiloa purposes) 17.7% J7J% (CFj)}CF-{CFj)1-SO(-) K(+) (Isopropyl branch; assunw x 5 for calculation purposes) 10.5% 10.2% . C JW C F (C F ,K ra i(-) X(+) (Alpha branch; assume x * 6 for calculation purposes) 11 1 Possible F-SPi-CJ^-SCbW K(+) (assume x * 8 for calculation purposes) C FrC C FiV C iC FJH C FjV SO jO K(+) - (Internal gem-dimethyl btMch: assume x+y* 4 sad x # 0 for calculation purposes) 3.3% 0.37% 0.16% 3.2% 0.36% 0.16% Possible CF-SFs-CIFj,450)(-) K(+) (assume x * 7 for calculation purposes) Probable C,H,,.,-SO,(-) K(+) (Hydrocarbon sulfonate salt; assume x 8 for calculation purposes) (CFj)>C-{CFj)-$Oi (-) K(+) f brsnch: assume x 4 for calculation purposes) . 0.11% 0.031% 0.027% 0.10% . 0.030% 0.026% 3M Environmental Laboratory Pap-2F-2 Page 239 3M Medicpl Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 RE-ISSUED ANALYTIC AL REPORT STUDY TITLE Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats DATA REQUIREMENTS Analytical Method Requirements STUDY DIRECTOR Marvin T. Case, 3M PRINCIPAL INVESTIGATOR Enaksha Wickremesinhe, Centre RE-ISSUED ANALYTICAL REPORT COMPLETION DATE November 3,2000 PERFORMING LABORATORY / TESTING FACILITY Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive State College, PA 16801 Phone:814-231-8032 STUDY SPONSOR 3M Toxicology Services - Medical Department 3M Center, Building 220-2E-02 St. Paul, MN 55144-1000 PROJECT IDENTIFICATION Sponsor Protocol Number: FACT-TOX-111 Centre Study Number: 023-017 Total Pages: 92 3M Environmental Laboratory Page 240 BACK TO MAIN 3M Medical Department Study: T-6395.14 . Analytical Report: FACT T O X -111 . C entre S tudy N o.: 0@3rP4^994 S p o n s o r P ro to c o l N o : F A C T -T O X -1 11 GOOD LA B O R A TO R Y P R A C TIC E C O M P LIA N C E STATEM EN T Centre Study Number 023-017, entitled "Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats," conducted for 3M Toxicology Services - Medical Department, was performed in compliance with US FDA Good Laboratory Practice Standards (21 CFR 58) by Centre Analytical Laboratories, Inc. with the following exceptions: 1. The reference substances (analytical standards) used in this study (PFOS and THPFOS) have not been characterized under 21 CFR 58.105 2. The automated data collection systems used in this study are not fully compliant with 21 CFR 58.130(e). Enaksha Wicka'mfesinhe, Ph.D* Principal Investigator Centre Analytical Laboratories, Inc. Date *Enaksha Wickremesinhe is no longer employed at Centre, therefore Centre Management, Rick Grazzini will be signing in his stead. . Marvin T Case, D.V.M., Ph.D. Study Director 3M Toxicology Services Date 3M Environmental L a b o g ^ A n a lv tic a l L a b o ra to rie s , In c. Page 2 241 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Q UALITY ASSURANCE STATEMENT Centre Analytical Laboratories' Quality Assurance Unit reviewed Centre Study Number 023-017, entitled, "Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats". All phases were reviewed for conduct according to Centre Analytical Laboratories' Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Study Director and to management. Phase 1, Protocol Review 2. Extraction & Fortification 3. Raw Data & Summary Table Review 4. Draft Report Review 5. Final Report Review Date InsDected 5/17/00 Date Reported to Date Reported to Centre Study Director and Manasement SDOnsor Management 6/1/00 7/14/00 7/7/00 7/11/00 7/14/00 7/28,31/00 8/15/00 8/25/00 8/15/00 9/11/00 8/17/00 9/11/00 8/25/00 9/11/00 . ________ U J n Naomi Lovallo Quality Assurance Auditor ------ uh M Date Centre Analytical Laboratories, tic . 3M Environmental Laboratory Page 3 of 92 Page 242 BACK TO MAIN 3M Medical Department Study: T-6395.14 . Analytical Report: FACT T O X -111 LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 CERTIFICATION OF AUTHENTICITY This report, for Centre Study Number 023-017, is a true and complete representation of the raw data for the study. Submitted by: Centre Analytical Laboratories, Inc. 3048 Research Drive . State College, PA 16801 (814)231-8032 . Principal Investigator, Centre: . Date Centre Analytical Laboratories, Inc. *Enaksha Wickremesinhe is no longer employed at Centre, therefore Centre Management, Rick Grazzini will be signing in his stead. Centre Analytical Laboratories, Inc. Facility Management: Laboratory Manager Centre Analytical Laboratories, Inc. Study Director, 3M: J^ Marvin T. Case, D.V.M., Ph.D. 3M Toxicology Services %) AJq'W Date Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 4 of 92 Page 243- BACK TO MAIN 3M Medicpl Department Study: T-6395.14 , ' Analytical Report: FACT T O X -111 , LRN-U2994 . Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 STUDY IDENTIFICATION Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats SPONSOR PROTOCOL NUMBER: FACT-TOX- 111 TYPE OF STUDY: Oral (Gavage) Pharmacokinetic Recovery TEST SYSTEM: Rat Feces TEST MATERIAL: Perfluorooctane sulfonic acid potassium salt (T-6295) . SPONSOR: . 3M Toxicology Services - Medical Department 3M Center, Building 220-2E-02 St. Paul, MN 55144-1000 STUDY DIRECTOR: From 6/9/99 to 2/10/00 Kristen J. Hansen, Ph.D. 3M Environmental Technology and Safety Services Phone: (651) 778-6018 From 2/10/00 to Present Marvin T. Case, D.V.M., PhD . 3M Toxicology Services Phone:(651)733-5180 TESTING FACILITY: Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 PRINCIPAL INVESTIGATOR Enaksha Wickremesinhe, Ph.D. Centre Analytical Laboratories, Inc. Phone:(814)231-8032 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: Study Assignment Date:- Analytical Start Date: ' Analytical Termination Date: 06/09/99 04/19/00 07/06/00 07/20/00 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 5 of 92 Page 244 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 . ., '' Analytical Report: FACT T O X -111 LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 PROJECT PERSONNEL The Study Director for this project was Marvin T. Case at 3M Toxicology Services and the Principal Investigator for this project at Centre Analytical Laboratories, Inc. was Enaksha Wickremesinhe. The following personnel from Centre Analytical Laboratories, Inc., were associated with various analytical phases of the study: Name Enaksha Wickremesinhe Emily Stauffer Karen Smith Tiffany Proctor Rickey Keller Lawrence Ord Title Group/Team TparW Scientist Scientist Technician Sample Custodian " Sample Custodian Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 6 of 92 Page 245 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 Analytical Report: FACTTOX-111 LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 TABLE OF CONTENTS Page TITLE PAGE....................................................................................................................... 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT..............................2 QUALITY ASSURANCE STATEMENT...................................... ................................... 3 CERTIFICATION OF AUTHENTICITY.... .......................................................................4 STUDY IDENTIFICATION.................................................................................... 5 PROJECT PERSONNEL......................................................................................................6 TABLE OF CONTENTS..................................................................................................... 7 LIST OF TABLES..... ................................................................ 8 LIST OF FIGURES.... .'......................................................................................... ............9 LIST OF APPENDICES..................................................................................................... 10 1.0 SUMMARY.................................................. 11 2.0 OBJECTIVE........................................ 11 3.0 INTRODUCTION............................................................................................. .........11 4.0 TEST SYSTEM............................ 11 5.0 REFERENCE MATERIAL.............................................. 12 6.0 EXPERIMENTAL DESIGN...... .................... 13 7.0 DESCRIPTION OF ANALYTICAL METHOD........... 13 7.1 Extraction Procedure......... ................................................................................., 13 7.2 Preparation of Standards and Fortification Solutions......................................... '. 13 7.3 Chromatography................................................................................ .:............ 14 7.4 Instrument Sensitivity................................. 14 7.5 Description of Instrument and Operating Conditions........ ...................................14 7.6 Quantitation and Example Calculation.................... '........... 16 8.0 RESULTS AND DISCUSSION...................................................... 18 9.0 CIRCUMSTANCES THAT MAY HAVE AFFECTED THE DATA..................... 18 10.0 RETENTION OF DATA AND SAMPLES.... .............................................. ........., 18 11.0 TABLES..........................................................i.........................................................19 12.0 FIGURES....................................................................... .............30 13.0 APPENDICES........................................................................................... 38 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 7 of 92 Page 246 BACK TO MAIN 3M Medical Department Study: T-6395.14 r '` Analytical Report: FACT T O X -111 ' LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 LIST OF TABLES ' Page Table I. Summary of PFOS residues in Matrix and Matrix Zero Blanks......................... 20 Table IT. Summary of PFOS residues in G1 Prior to Cohabitation.................................. 21 Table IE. Summary of PFOS residues in G2 Prior to Cohabitation................................. 21 Table IV. Summary of PFOS residues in G3 Prior to Cohabitation.................................22 Table V. Summary of PFOS residues in G1 Day 6/7 Gestation....................................... 22 Table VI. Summary of PFOS residues in G2 Day 6/7 Gestation..................................... 23 Table VE. Summary of PFOS residues in G3 Day 6/7 Gestation.....................................23 Table VIE. Summary of PFOS residues in G1 Day 14/15 Gestation................................24 Table DC. Summary of PFOS residues in G2 Day 14/15 Gestation.......................... .......24 Table X. Summary of PFOS residues in G3 Day 14/15 Gestation.......................... .........25 Table XI. Summary of PFOS residues in G1 Day 20/21 Gestation ;.................................25 Table XE. Summary of PFOS residues in G2 Day 20/21 Gestation........................... . 26 Table XEI. Summary of PFOS residues in G3 Day 20/21 Gestation..............:................ 26 Table XTV. Summary of PFOS residues in G1 Day 21/22 Lactation............................... 27 Table XTV. Summary of PFOS residues in G2 Day 21/22 Lactation.................... ............ 27 Table XVI. Summary of PFOS residues in G3 Day 21/22 Lactation................................28 Table XVIL Summary of PFOS recoveries in Fortified Samples......... ...........................29 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 8 o f 92 Page 247 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Figure 1. LIST OF FIGURES Page Typical Calibration Curve for PFOS............. .................... ............................ 31 Figure 2. Typical Mean Response Factor for THPFOS................................................ 32 Figure 3. Chromatogram Representing a 5 ng/mL extracted standard for PFOS and 250 ng/mL fortification of THPFOS......... ............................................................ 33 Figure 4. Chromatogram Representing a 125 ng/mL extracted standard for PFOS and . 250 ng/mL fortification of THPFOS................ '........ .................................. . 34 Figure 5. Chromatogram Representing Control Rat Feces for PFOS and THPFOS , (Centre ID: 0005738 Blank B, Set: 070700A)........................ ....................... 35 Figure 6. Chromatogram Representing Control Rat Feces Fortified with 50 ng of PFOS and 500 ng of THPFOS (Centre ID: 0005803 Spk A, Set: 071000A)..........36. Figure 7. Chromatogram of Rat Feces Sample from G2 Day 6/7 Gestation (Centre ID: : 0005836, Set: 071000A)...................................................................... ..........37 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 9 of 92 Page 248 BACK TO MAIN 3M Medlcpl Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 ' LIST OF APPENDICES P age Appendix A Study Protocol FACT-TOX-111 (Centre Study No. 023-017) and Amendments and Deviations..................................................................... .. Appendix B Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS (Revision 2) Method #O0M-023-0O3, revision 2 and Deviations and Modifications............ ...................... ...........................66 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 10 o f 92 Page 249 BACK TO MAIN 3M Medical Department Study: T-6395.14 . Analytical Report: FACT T O X -111 , LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No.: FACT-TOX-l 11 1.0 SUMMARY The purpose of this study was to analyze perfluorooctanejsulfonate (PFOS) in rat feces as specified in 3M Protocol FACT-TOX-111. The analytical method used for this study was, "Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS, revision 2", Centre method number: 00M-023-003, revision 2, The limit of quantification for PFOS in rat feces established during the method validation was 10 ppb (0.0100 pg/g). The method validation did not determine a method detection limit. Residues (corrected for purity) ranging from non-detected levels to 13.5 pg/g were found in the rat feces samples. Fortification recoveries ranged from 73-136% with an average of 92% and relative standard deviation of 18%. 2.0 OBJECTIVE The objective of this study was to determine levels of periluorooctanesulfonate (PFOS) in specimens of feces of rats using the analytical method `Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS, revision 2", Centre method number 00M- 023-003, revision 2 .. , 3.0 INTRODUCTION The study was initiated on June 9, 1999, when the study director signed the protocol FACT-TOX-111. The study was assigned to Centre on April 14, 2000 when the study director signed Amendment #4. The complete protocol and amendments and deviations can be found in Appendix A. The analytical start date was July 6, 2000, -and the analytical termination date was July 20,2000. ;1 ' This report details the results of the residues of PFOS detected in rat feces, using the analytical method entitled, `Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS (revision 2)", Centre method number OOM-023-003, revision 2. Complete details of the analytical methodology can be found in Appendix B 4.0 TEST SYSTEM The 117 incurred rat feces samples analyzed in this study were received frozen on dry ice from 3M Environmental Laboratory St. Paul, MN on June 28, 2000. Samples were logged in and stored frozen (< -10C) on June 28, 2000 by Centre personnel. 3M Environmental Laboratory A m ended P age 11 o f 92 Page 250 BACK TO MAIN 3M Medical Department Study: T-6395.14 1 '' Analytical Report: FACT T O X -111 LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 The control rat feces used for standards and blanks were purchased from Lampire Biological Laboratories, Inc., Piperesville, PA and received frozen on dry ice at Centre on June 22,2000, logged in by Centre personnel and placed in frozen storage (<-10C). Sample login and chain of custody information can be found in the raw data package associated with this study. Storage records will be kept at Centre Analytical Laboratories, Inc. and a true copy of the storage records can be found in the raw data package associated with this study. 5.0 REFERENCE MATERIAL The analytical standard for PFOS determination (potassium perfluorooctane sulfonate) was received at Centre on November 12, 1999 and the surrogate standard THPFOS was received on December 3, 1999 from 3M Environmental Technology and Services. Characterization of the reference material PFOS was performed at Centre on August 31, 2000 and documentation can be found in the raw data package associated with this report. The available information for the reference materials is listed below. The reference materials were stored at room temperature. ' Compound PFOS THPFOS Centre Control No. 99-023-002 99-023-011 Batch No. 217 53406 - Purity (%) Expiration Date 86.9 08/31/01 NA 01/01/10 Molecular structures of PFOS and THPFOS are given below. ' PFOS Chemical Name: Perfluorooctane sulfonate Molecular weight: 499 (CSF17SO3") - O 0. N ote:.The neutral molecule and standard form from which PFOS (anion) is derived, is potassium perfluorooctane sulfonate [C8F17SO3K], molecular weight 538. . THPFOS Chemical Name: 4-H, perfluorooctane sulfonic acid Molecular weight: 428 1-H, 1-H, 2-H, 2-H, CaFuSOjH - Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 12 o f 92 Page 251 BACK TO MAIN 3M Mediqal Department Study: T-6395.14 ., . Analytical Report: FACT T O X -111 : LRN-U2994' Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 6.0 EXPERIMENTAL DESIGN Samples were extracted according to group number and sampling day. Each set of samples contained two matrix blanks, two matrix blanks spiked with the surrogate standard, two matrix spikes, and 7 - 1 6 samples. The extracts were analyzed by LC/MS/MS. Samples with residues outside the linear range of the calibration curve were diluted and re-analyzed. ' 7.0 DESCRIPTION OF ANALYTICAL METHOD Analytical method entitled "Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS (Revision 2)," Centre method number 00M-023-003, revision 2 (Appendix B) was used for this study. 7.1 Extraction Procedure One gram ( 0.05 g) of sample was weighed into 20 mL polyethylene scintillation vials . and fortified (if necessary) using disposable micropipettes. Ten mL of acetonitrile was added to the vial, capped tightly, and placed on a wrist-action shaker for - 30 min. The samples were filtered through a glass acrodisc filter. The filtered extract was passed through a conditioned carbon SPE column and collected. The columns were then eluted with -10 mL acetonitrile and -20 mL 90:10 acetonitrile:2% ascorbic acid in methanol. The combined extracts were evaporated down to near dryness with a rotary evaporator and then re-constituted with 2 mL methanol. The samples were analyzed using electrospray LC/MS/MS. 7.2 Preparation of Standards and Fortification Solutions ' . Standard solutions were prepared on May 3, 2000 as specified in Centre Analytical Laboratories' analytical method entitled, "Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS, (Revision 2)," Centre method number 00M-023-003, revision 2. An individual stock standard solution of PFOS was prepared at a concentration of 100 pg/mL by dissolving 10 mg of the standard (corrected for salt purity only) in methanol. From this solution, a 10 pg/mL fortification standard solution was prepared by taking 10 mL of the stock and bringing the volume up to 100 mL with methanol. Also, a stock standard of THPFOS was prepared at 100 pg/mL by dissolving 10 mg of the standard in methanol. An individual 10 pg/mL solution of THPFOS was prepared in the same fashion described above. A 2.5 pg/mL fortification standard of PFOS was prepared by taking 25 mL of the 10 pg/mL fortification standard of PFOS and bringing the volume up to 100 mL with methanol. An individual fortification solution of THPFOS was prepared in the same manner. A 6.5 pg/mL fortification standard of PFOS was prepared by taking 20 mL of Centre Analytical Laboratories, Inc. Page 13 o f 92 3M Environmental Laboratory Page 252 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 the 2.5 pg/mL standard of PFOS and bringing the volume up to 100 mL with methanol. To make the 0.1 pg/mL fortification standard, 20 mL of the 0.5 pg/mL was brought up to 100 mL with methanol. Calibration standards were processed through the extraction procedure, identically to the samples. The fortification of the standards before extraction was done according to the following table: . Cone, of PFOS Fortification Fortification Volume (pL) Solution (pg/ml) Weight of Control Sample (g) ( 0.05 g) .1 100 1.0 0.1 200 1.0 0.5 100 1.0 0.5 200 1.0 2.5 100 1.0 2.5 200 1.0 * 2.5 pg/ml THPFOS fortification solution. Fort. Level of Voi. of Extracted Surrogate Calibration Standard* Standard added (pL) (ppb) 10 200 20 200 50 200 100 200 250 200 500 200 The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation can be found in the raw data associated with this report. 7.3 Chromatography Quantification of PFOS and THPFOS was accomplished by electrospray LC/MS/MS analysis. The retention times of PFOS and THPFOS were ~ 5.2 min. and ~ 5.0 min., respectively, with no significant interfering peaks in the control matrices corresponding to either o f the analyte retention times. 7.4 Instrument Sensitivity The smallest standard amount injected during the chromatographic run was equivalent to 5 ng/mL of PFOS and 250 ng/mL of THPFOS in the rat feces matrix. 7.5 Description of Instrument and Operating Conditions A Micromass Quattro Ultima LC/MS/MS coupled to a Hewlet Packard HPLC system was used. Data acquisition and processing were performed using Masslynx 3.4 software. Detailed operating conditions are listed below: Centre Analytical Laboratories, Inc. 3M Environmental Laboratory " . Page 14 of 92 Page 253 BACK TO MAIN 3M Medicai Department Study: T-6395.14 Analytical Report: FACT TOX-111 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Instrument: Micromass Quattro Ultima ELECTROSPRAY ION SOURCE: Capillary: 3.0 kV Hexapole 2: 0.3 V Hexapole 1:0.1 V Source Block Temp.: 100C Aperture 1:0.2 V Desolvation Temp.: 350C ANALYZER: .. LM Res 1:10.5 V HMRes 1:10.5 V lEnergy 1:1.0 V Entrance: -2 V Exit: 2 V LM Res 2: 13.0 V HMRes 2 : 13.0 V lEnergy 2 :2.0 V Multiplier: 650 V GAS FLOWS AND PRESSURE: Desolvation N2Flow Rate: -650 L/hr Nebuliser N2Flow Rate: -150 L/hr . Gas Cell Pressure: -0.003 mbar Computer COMPAQ Professional Workstation AP200 . Software: Microsoft Windows NT: Version 4 Build 1381: Service Pack 5 Micromass Limited: Masslynx 3.4 Build 004 HPLC Equipment: Hewlett Packard (HP) Series 1100 HP Binary Pump HP Vacuum Degasser . HP Autosampler HP Column Oven HPLC Column: Genesis C-8, 5 cmx 2.1 mm i.d. x4 p Column Temperature: 35C Mobile Phase (A) : 2 mM AmmoniumAcetate in Type I Water Mobile Phase (B ): Methanol Gradient: Injected Volume: Ions monitored: Analvte PFOS THPFOS Time (min) 0.0 0.4 1.0 7.0 -, 7.5 10.5 11.0 14.5 15.0 10 pL %A 60.0 60.0 10.0 10.0 0.0 0.0 60.0 60.0 60.0 - %B 40.0 40.0 90.0 90.0 100.0 100.0 40.0 40.0 40.0 Flow Rate (mL/min) 0.3 0.3 0.3 0.3 0.3 0.4 0.4 0.4 ' 0.3 Transition Dwell (secs') Monitored . 499 --99 02 427 - 80 . 0.2 Coll Energy (eV) 43 35 Cone (VI 76 34 Centre Analytical Laboratories, Inc. . 3M Environmental Laboratory Page 15 o f 92 Page 254 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 . LRN-U2994-- 7.6 Quantitation and Example Calculation Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Ten microliters of sample or extracted calibration standard was injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using 1/x weighted linear regression) by Masslynx software using six concentrations of standards. The surrogate standard, THPFOS, was only used to monitor the efficiency of the extraction procedure and was not used for quantitation of PFOS. The residue concentration for rat feces was determined from the following equations: Use Equations 1 and 2 to calculate the amount of analyte found (in ppb, based on peak area) using the standard curve (linear regression parameters) generated by the Masslynx software program. _ Equation 1: Analyte found (ng/mL) = (Peak area - intercept') . slope Equation 2: Analyte found (ppb) = (analyte found (ng/mL) x final vol. (mlA x DFt sample wt. (g) where D F = dilution factor. For samples fortified with known amounts of analytes prior to extraction, use Equation 3 to calculate the percent recovery. quation 3: Recovery (%) = [analyte found (ng/mL)x final vol. (mL)xDF] analyte added (ng) ^ . . Equation 4: '. This calculation was only performed on actual samples and not QC recoveries. Corrected analyte found (ppb) = analyte found (ppb) x % purity % purity for PFOS lot 217 = 86.9% (0.869) An example of a calculation using an actual sample analyzed with extracted standards follows: Rat feces sample Centre ID 0005803 Spk A (Set: 071000A), fortified at 50 ng with PFOS. - Where: peak area intercept slope dilution factor = 19051 = 2021.14 = 673.327 =1 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 16 of 92 Page 255 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 ng added (fort level) = final voi. `= sample wt. = 50 2 mL 1.01 g Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 . From equation 1: Analyte found (ng/mL) ' = ri9 0 5 1 -2021.141 . 673.327 = 25.3 ng/mL Fromequation 2: Analyte found (ppb) = r25.3 x 2 mL x 11 1.01 g = 50.1 ppb Fromequation 3: % Recovery = f25.3 ng/mL x 2 mL x 1Y x 100 . 50 ng = 101% . Note: This example calculation was done using rounded numbers, and therefore may be slightly different from the values shown in the RAW DATA. The amount of surrogate standard THPFOS found was calculated by dividing the peak area for each sample by die mean response factor. ' Analyte found (ng/mL): peak area mean response factor . Other statistical methods used in analyzing this data were: . n Standard Deviation--' --------n-l. Mean = x =. n Relative StandardDeviation (RSD or Coefficient of Variation (CV)) - Standard Deviation x 100% . Mean Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 17 o f 92 Page 256 BACK TO MAIN 3M Medical Department Study: T-6395.14 . , Analytical Report: FACT T O X -111 L R N -U 2 9 9 4 - CentreStudyNo.: 023-017 SponsorProtocol No.: FACT-TOX-111 8.0 RESULTSANDDISCUSSION Although, the majority of the control samples did not contain interferences, a couple of samples didcontain residues. Howeverthese interferences were not significant enoughto adversely affect the data. A summary of residues found in all of the matrix blanks and matrix zero blanksis detailedinTable I. : Residues (correctedfor purity) ranging fromnon-detectedlevels to 13.5 pg/g were found in the rat feces samples. The residues found in all of the samples plus the averages and standarddeviations foreach groupateachinterval are detailedin Tables H-XVl For this report, the residues found in Tables I-XVI were corrected for the purity of the PFOS standard lot 217 based on the certificate of analysis finalized on September 7, 2000. . Fortification recoveries ranged from 73-136% with an average of 92% and relative standarddeviation of 18%. A summaryof all of the fortification recoveries can be found in Table XVII. : Typical calibration curves and chromatograms representing standards, controls, fortifications, andsamples aredepictedinFigures 1-7. 9.0 CIRCUMSTANCES THATMAYHAVEAFFECTEDTHEDATA Electronic records are not fully compliant with 21 CFR 11, "Electronic Records: Electronic Signature." However, fully approved SOP's were in place and all chromatograph instrumentation used in this study was fully validated (IQ, PQ, OQ), calibratedand operational. All original datawereprintedas hardcopies andfully audited ' by quality assurance. Verified exact copies and the electronic data have been stored in the archives at Centre Analytical Laboratories, Inc. Original rawdatahave been returned . to the sponsor. . v 10.0 RETENTIONOFDATAAND SAMPLES When the final report is complete, all original paper data generated by Centre Analytical Laboratories, Lie. will be shipped to the sponsor. This does not include facility-specific raw data such as instrument logs, however exact copies of temperature logs will be submitted. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific rawdata, will be retained in the Centre Analytical Laboratories, Inc. archives for the period of time specified in 21 CFR 58. Retained samples of reference substances arearchivedbythe sponsor. 3M Environmental Laboratory A m ended Page 18 o f 92 Page 257 3M Medicpl Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 C entre S tudy N o.: 023-017 S ponsor P rotocol N o: FA C T-TO X -111 11.0 TABLES Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 19 o f 92 Page 258 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 . Analytical Report: FACT T O X -111 LRN-U2994 ; Centre StudyNo.: 023-017 : SponsorProtocol No.: FACT-TOX-111 Table I. Summary ofPFOS residues in Matrix and Matrix Zero Blanks .i . S p o n so r ED C entre ID , Set Num Ur D ate E xtracted Corrected Residue R esidue Found Found (uste) (uste) na 0005738 B lank A 070600A R 7 /6 /0 0 ND ND na 0005738 B lank B 070600A R 7 /6 /0 0 ND ND na 0005738 Z ero B lank C 070600A R 7 /6 /0 0 ND ND . n a 0005738 Z ero B lank D 070600A R 7 /6 /0 0 ND ND : na 0005738 B lank A 070700A 7 /7 /0 0 NQ NQ na 0005738 B lank B 070700A 7 /7 /0 0 ND ND n a 0005738 Z ero B lank C 0707Q0A 7/7/00 ,, - ND ND n a 0005738 Z ero B lank D 070700A 7 /7 /0 0 ND ND na . 0005738 B lank A 071000A 7 /1 0 /0 0 ND1 ND na 0005738 B lank B 071000A 7 /1 0 /0 0 ND ND na 0005738 Z ero B lank C 071000A 7/10/00 ' ND ND na 0005738 Z ero B lank D 071000A 7 /1 0 /0 0 ND ND na .0 005738 B lank A 071100A 7 /1 1 /0 0 < 0 .0 1 0 0 < 0 .0 0 8 6 9 na 0005738 B lank B 071100A 7 /1 1 /0 0 NQ NQ na 0005738 Z ero B lank C 071100A 7 /1 1 /0 0 < 0 .0 1 0 0 < 0 .0 0 8 6 9 na 0005738 Z ero B lank D 071100A 7 /1 1 /0 0 NQ NQ na 0005738 B lank A 07120A 7 /1 2 /0 0 < 0 .0 1 0 0 < 0 .0 0 8 6 9 n 0005738 B lank B 071200A 7 /1 2 /0 0 0 .0 1 5 6 0 .0 1 3 6 na 0005738 Z ero B lank C 071200A 7 /1 2 /0 0 < 0 .0 1 0 0 < 0 .0 0 8 6 9 . na 0005738 Z ero B lank D 071200A 7 /1 2 /0 0 < 0 .0 1 0 0 < 0 .0 0 8 6 9 na 0005738 B lank A 071300A 7 /1 3 /0 0 NQ NQ na 0005738 B lank B 071300A 7 /1 3 /0 0 NQ: NQ na 0005738 Z ero B lank C 071300A 7 /1 3 /0 0 NQ NQ na 0005738 Zero B lank D 071300A 7 /1 3 /0 0 . NQ NQ . na 0005738 B lank A 071700A R 7 /1 7 /0 0 ND ND na 0005738 B lank B4 071700A R 7 /1 7 /0 0 ND ND na 0005738 Z ero B lank C 071700A R 7/17/00 ND ND na 0005738 Z ero B lank D 071700A R 7/17/00 ND ND na 0005738 B lank A 071800A 7 /1 8 /0 0 ND ND na 0005738 B lank B 071800A 7 /1 8 /0 0 ND' ND na 0005738 Z ero B lank C 071800A 7 /1 8 /0 0 ND ND N a 0005738 Zero B lank D 071800A 7 /1 8 /0 0 ND ND ND =sNot Detected andNQ= Not Quantifiable . Note: The average andstandarddeviation werecalculatedby using 0.0100 as the value forthose samples reportedas< 0.0100,0.00869 forthosereportedas< 0.00869, andzero forthose reportedas NDorNQ. .' 3M Environmental Laboratory Amended Page 20 of 92 Page 259 BACK TO MAIN 3M Medical Department Study: T-6395.14 r. / Analytical Report: FACT T O X -111 : LRN-U299T ' Centre StudyNo.: 023-017 SponsorProtocol No.: FACT-TOX-111 Table I (continued). Summary ofPFOS residues in Matrix and Matrix Zero Blanks Sponsor ID na na . na na C entre C orrected R esidue Set D ate R esidue F ound Found ID Num ber E xtracted (uste) (us/g) 000S738 B lank A 07200A 7 /2 0 /0 0 NQ . NQ 0005738 B lankB 072000A 7 /2 0 /0 0 NQ NQ 0005738 Z ero B lank C 072000A 0005738 Z ero B lank D 072000A 7 /2 0 /0 0 7 /2 0 /0 0 NQ . NQ n q NQ AVERAGE: < 0 .0 1 0 0 STANDARD DEVIATION: NQ < 0 .0 0 8 6 9 NQ ND = NotDetectedandNQ= Not Quantifiable Note: The average andstandarddeviationwerecalculatedbyusing0.0100 as th value forthose samplesreportedas < 0.0100,0.00869 forthosereportedas < 0.00869, andzero forthose reportedas NDorNQ. :i i 3M Environmental Laboratory Amended Page 20A of 92 Page 260 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 , Analytical Report: FACT T O X -111 LRN-U2994 Centre StudyNo.: 023-017 SponsorProtocol No.: FACT-TOX-111 Table II. Summary of PFOS residues in G1 Prior to Cohabitation S p o n so r ID 13726F G1 P rio r to C ohabitation 13727F G1 P rio r to C ohabitation 13728F G1 P rio r to C ohabitation 1373IF G1 P rio r to C ohabitation 13734F G1 P rio r to C ohabitation 1373SF G1 P rio r to C ohabitation 13736F G1 P rio r to C ohabitation 13737F G1 P rio r to C ohabitation C entre S et D ate ID N um ber E x tracted 0005799 070600A R 7 /6/00 0005800 070600A R 7 /6/00 0005801 070600A R 7 /6/00 0005802 070600A R 7 /6/00 0005803 070600A R 7 /6/00 0005804 070600A R 7 /6/00 0005805 070600A R 7/6/00 0005806 070600A R 7 /6/00 AVERAGE: STA N D A RD D E V IA T IO N : R esidue Found C orrected R esidue Found (u g /g ) ND ND .... (U g/g) ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND NQ NQ ND=Not DetectedandNQ=Not Quantifiable Table III. Summary ofPFOS residues in G2 Prior to Cohabitation S p o n so r ID 13739F G 2 P rio r to C ohabitation 13740F G 2 P rio r to C ohabitation 1374IF G 2 P rio r to C ohabitation 13744F G 2 P rio r to C ohabitation 13745F G 2 P rio r to C ohabitation 13745F G 2 P rio r to C ohabitation 13746F G 2 P rio r to C ohabitation*, 13748F G 2 P rio r to C ohabitation 13749F G 2 P rio r to C ohabitation C entre S et D ate ID N um ber E xtracted 0005807 0706-0700A D 7/6/00 0005808 0706-0700A D 7 /6/00 0005809 070600A R 7 /6/00 0005810 070600A R 7 /6/00 0005811 0706-0700A D 7/6 /0 0 . 0005811 0717-1800A D 7/1 7 /0 0 0005812 0706-0700A D 7 /6/00 0005813 0706-0700A D 7 /6/00 0005814 0706-0700AD 7/6/00 AVERAGE: STA N D A R D D E V IA T IO N : R esidue Found (u g /g ) 0 .9 3 3 0 .7 6 4 0 .4 5 3 0 .4 6 3 3 .1 3 1 .0 4 0 .7 9 1 0 .8 0 9 0 .6 2 8 1 .0 0 0 .820 C orrected R esidue Found (P g /g ) 0 .8 1 0 0 .6 6 4 0 .3 9 4 0 .4 0 2 2 .7 2 0 .9 0 4 0 .6 8 8 0 .7 0 3 0 .5 4 6 0 .8 7 0 0 .7 1 3 3M Environmental Laboratory A m ended Page 21 o f 92 Page 261 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 , ' Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 C e n tre S tu d y N o .: 0 2 3 -0 1 7 S p o n so r P ro to c o l N o .: F A C T -T O X -1 1 1 Table IV. Summary ofPFOS residues in G3 Prior to Cohabitation Sponsor ID 1375IF G 3 P rio r to C ohabitation 13752F G 3 P rio r to C ohabitation 13753F G 3 P rio r to C ohabitation 13754F G 3 P rio r to C ohabitation 13755F G 3 P rio r to C ohabitation 13756F G 3 P rio r to C ohabitation 13758F G 3 P rio r to C ohabitation 13759F G 3 P rio r to C ohabitation C entre S et D ate ID N um ber E xtracted 0005815 0706-0700A D 7/7/00 0005816 0706-0700A D 7 /7/00 0005817 0706-0700A D 7 /7/00 0005818 0706-0700AD 7 /7/00 0005819 0706-0700A D 7 /7/00 0005820 0706-0700A D 7/7/00 0005821 0706-0700A D 7 /7/00 0005822 0706-0700A D 7 /7/00 AVERAGE: STA N D ARD D E V IA T IO N : R esidue Found (P S fe ) 1 2 .4 1.2.5 14.1 1 5 .6 9 .8 6 12.4 10.8 12.5 1 2 .5 1 .7 7 C orrected R esidue Found (u g /g ) 10.8 10.8 12.3 13.5 8 .5 7 1 0 .8 9.41 1 0 .9 1 0 .9 1 .5 4 Table V. Summary ofPFOS residues in G1 Day 6/7 Gestation S p o n so r ID 13726F G1 D ay 6 /7 G estation 13727F G 1 D ay 6 /7 G estation 13728F G 1 D ay 6 /7 G estation 1373IF G l D ay 6 /7 G estation 13734F G1 D ay 6 /7 G estation 13735F G1 D ay 6 /7 G estation 13736F G 1 D ay 6 /7 G estation 13737F G 1 D ay 6 /7 G estation 1 C entre S et D ate ID N um ber E xtracted 0005823 070700A 7 /7 /0 0 0005824 070700A 7 /7 /0 0 0005825 070700A 7 /7 /0 0 0005826 070700A 7 /7/00 0005827 070700A 7 /7/00 0005828 070700A 7 /7 /0 0 0005829 070700A 7 /7/00 0005830 070700A 7 /7 /0 0 AVERAGE: STA N D A RD D E V IA T IO N : R esidue Found (U g/g) ND ND ND ND ND ND ND ND ND NQ C orrected R esidue Found (p g /g ) ND ND ND ND ND ND ND ND ND NQ N D = N o t D ete cted an d N Q = N o t Q u an tifiab le 3M Environmental Laboratory Amended Page 22 of 92 Page 262 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 . Analytical Report: FACT T O X -111 LRN-U2994 Centre StudyNo.: 023-017 SponsorProtocol No.: FACT-TOX-111 Table VI. Summary ofPFOS residues in G2 Day 6/7 Gestation S p o n so r ID 13739F G 2 D ay 6/7 G estation 13740F G 2 D ay 6 /7 G estation 13741F G 2 D ay 6 /7 G estation 13744F G 2 D ay 6 /7 G estation 13745F G 2 D ay 6/7 G estation 13746F G 2 D ay 6 /7 G estation 13748F G 2 D ay 6 /7 G estation 13749F G 2 D ay 6 /7 G estation C entre S et D ate ID N um ber E x tracted 0005831 071000A D 7/1 0 /0 0 0005832 071000A D 7/10/00 0005833 071000A 7 /1 0 /0 0 0005834 071000A 7 /1 0 /0 0 0005835 071000A 7/1 0 /0 0 0005836 071000A 7 /10/00 0005837 071000A 7 /1 0 /0 0 0005838 071000A 7 /1 0 /0 0 AVERAGE: STA N D ARD D E V IA T IO N : R esidue Found (tig /g ) 0 .5 9 1 0 .6 1 8 0 .3 6 6 0 .3 5 6 0.4 4 0 0 .4 4 8 0 .3 7 1 0 .4 8 3 0 .4 5 9 0 .1 0 1 C orrected R esidue Found (tig /g ) 0 .5 1 4 0 .5 3 7 0 ,3 1 8 0 .3 0 9 0 .3 8 2 0 .3 8 9 0 .3 2 2 0 .4 2 0 0399 0 .0 8 7 6 Table VTI. Summary ofPFOS residues in G3 Day 6/7 Gestation S p o n so r ID 13751F G 3 D ay 6/7 G estation 13752F G 3 D ay 6/7 G estation 13753F G 3 D ay 6 /7 G estation 13754F G 3 D ay 6 /7 G estation 13755F G 3 D ay 6 /7 G estation 13756F G 3 D ay 6 /7 G estation 13758F G 3 D ay 6 /7 G estation 13759F G 3 D ay 6/7 G estation C entre S et D ate ID N um ber E xtracted 0005839 071000A D 7/10/00 0005840 071000A D 7/10/00 0005841 071000A D 7/10/00 0005842 071000A D 7/10/00 0005843 071000A D 7/10/00 0005844 0005845 071000AD 071000AD 7 /1 0 /0 0 7 /1 0 /0 0 0005846 071000A D 7/10/00 AVERAGE: STA N D A RD D E V IA T IO N : R esidue Found (tig /g ) 9 .8 0 11.1 1 0 .4 1 0 .2 14.1 7 .7 1 8 .8 0 5 .1 5 9 .6 5 2 .6 0 C orrected R esidue Found, (tig /g ) 8 .5 1 9 .6 1 9 .0 3 8 .8 6 12.2 6 .7 0 7 .6 5 4 .4 7 839 2 .2 6 3M Environmental Laboratory Amended Page 23 of 92 Page 263 BACK TO MAIN 3M Medlcpl Department Study: T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 C e n tre S tu d y N o .: 0 2 3 -0 1 7 S p o n so r P ro to c o l N o .: F A C T -T O X -1 11 Table VIII. Summary of PFOS residues in G1 Day 14/15 Gestation S p o n so r ID 13726F G1 D ay 14/15 G estatio n 1 3 7 2 7 F G 1 D ay 14/15 G estatio n 13728F G 1 D ay 14/15 G estatio n 1373 IF G 1 D ay 14/15 G estatio n 1 3 7 3 4 F G 1 D ay 14/15 G estation 1 3 7 3 5 F G 1 D ay 14/15 G estation 1 3 7 3 6 F G 1 D ay 14/15 G estation 13737F G 1 D av 14/15 G estation C entre S et D ate ID N um ber E x tracted 0005847 071100A 7 /1 1 /0 0 0005848 071100A 7 /1 1 /0 0 0005849 071100A 7 /1 1 /0 0 0005850 071100A 7 /11/00 0005851 071100A 7/11/00 0005852 071100A 7 /11/00 0005853 071100A 7 /11/00 0005854 071100A 7 /11/00 AVERAGE: STA N D ARD D E V IA T IO N : R esidue Found (P g fe) ND ND ND ND ND ND ND ND ND NQ C orrected R esidue Found (p g /g ) ND ND ND ND ND ND ND ND ND NQ N D = N o t D e te c te d an d N Q = N o t Q u an tifiab le Table IX. Summary of PFOS residues in G2 Day 14/15 Gestation S p o n so r ID 13739F G 2 D ay 14/15 G estation 13740F G 2 D ay 14/15 G estation 13741F G 2 D ay 14/15 G estatio n 13744F G 2 D ay 14/15 G estatio n 13745F G 2 D ay 14/15 G estation 13746F G 2 D ay 14/15 G estation* 13748F G 2 D ay 14/15 G estation 13749F G 2 D ay 14/15 G estation C entre S et D ate ID N um ber E xtracted 0005855 071100A 7 /1 1 /0 0 0005856 071100A 7 /1 1 /0 0 0005857 071100A 7/1 1 /0 0 0005858 071100A 7/1 1 /0 0 0005859 071100A 7/1 1 /0 0 0005860 071100A 7 /1 1 /0 0 0005861 071100A 7 /1 1 /0 0 0005862 071100A 7 /1 1 /0 0 AVERAGE: STA N D A RD D E V IA T IO N : R esidue Found (tig fe ) 0 .3 4 3 0 .3 5 8 0 .3 5 4 0 .4 7 8 0 .3 3 8 0 .3 9 7 0 .2 3 5 0 .2 0 3 0338 0 .0 8 6 9 C orrected R esidue Found (P g/g) 0 .2 9 8 0 .3 1 1 0 .3 0 8 0 .4 1 6 0 .2 9 4 0 .3 4 5 0 .2 0 4 0 .1 7 6 0 .2 9 4 0 .0 7 5 5 3M Environmental Laboratory Amended Page 24 of 92 Page 264 BACK TO MAIN 3M Medical Department Study: T-6395.14 r ,. '' Analytical Report: FACT T O X -111 LRN-U2994 C e n tre S tu d y N o .: 0 2 3 -0 1 7 S p o n so r P ro to c o l N o .: F A C T -T O X -1 11 Table X. Summary ofPFOS residues in G3 Day 14/15 Gestation S p o n so r ID 1375 IF G 3 D ay 14/15 G estation 13752F G 3 D ay 14/15 G estation 13753F G 3 D ay 14/15 G estation 13754F G 3 D ay 14/15 G estation 13755F G 3 D ay 14/15 G estation 13756F G 3 D ay 14/15 G estation 13758F G 3 D ay 14/15 G estation 13759F G 3 D ay 14/15 G estation C entre S et D ate ID N um ber E xtracted 0005863 071200A 7/1 2 /0 0 0005864 071200A 7 /1 2 /0 0 0005865 071200A 7/1 2 /0 0 0005866 071200A 7 /1 2 /0 0 0005867 071200A 7 /12/00 0005868 071200A 7 /1 2 /0 0 0005869. 071200A 7 /12/00 0005870 071200A 7 /12/00 AVERAGE: STA N D A RD D E V IA T IO N : R esidue Found (P g fe) 5 .7 5 7 .2 6 4 .6 3 4 .3 7 6 .5 0 5 .9 0 6 .1 0 3 .9 2 5 .5 5 1 .1 5 C orrected R esidue Found (p g /g ) 5 .0 0 6 .3 1 4 .0 2 3 .8 0 5 .6 5 5 .1 3 5 .3 0 3 .4 1 4 .8 3 1 .0 0 TableXI. Summary ofPFOS residues in G1 Day 20/21 Gestation S p o n so r C entre ' S et D ate ID ID N um ber E xtracted 13726F G1 D ay 20/21 G estation 0005871 071200A 7 /1 2 /0 0 13727F G1 D ay 20/21 G estation 0005872 071200A 7 /1 2 /0 0 13728F G1 D ay 20/21 G estation 0005873 071200A 7 /1 2 /0 0 13734F G1 D ay 20/21 G estation 0005874 071200A 7/12/00 13735F G1 D ay 20/21 G estation 0005875 071200A 7/12/00 13736F G1 D ay 20/21 G estation 0005876 071200A 7/12/00 13737F G1 D ay 20/21 G estation . 0005877 071200A 7 /1 2 /0 0 AVERAGE: STA N D ARD D E V IA T IO N : R esidue Found (jig /g ) ND ND ND ND ND ND ND ND NQ C orrected R esidue Found (P g fe) ND ND ND ND ND ND ND ND NQ N D = N o t D e te c te d an d N Q = N o t Q u an tifiab le 3M Environmental Laboratory Amended Page 25 of 92 Page 265 BACK TO MAIN 3M Medical Department Study. T-6395.14 Analytical Report: FACT T O X -111 LRN-U2994 Centre StudyNo.: 023-017 SponsorProtocol No.: FACT-TOX-111 Table XII. Summary of PFOS residues in G2 Day 20/21 Gestation S p o n so r ID 13739F G 2 D ay 20/21 G estation 13740F G 2 D ay 20/21 G estation 13741F G 2 D ay 20(21 G estation 13744F G 2 D ay 20/21 G estation 13745F G 2 D ay 20/21 G estatio n 13746F G 2 D ay 20/21 G estation 13748F G 2 D ay 20/21 G estation 13749F G 2 D ay 20/21 G estation C entre C orrected R esidue R esidue Set D ate Found Found ID 0005878 N um ber E xtracted --We) -JHgfe? 072000A 7 /2 0 /0 0 0 .1 5 3 0 .1 3 3 0005879 072000A 7 /20/00 0.0769 0 .0 6 6 8 0005880 072000A 7 /2 0 /0 0 0.1 9 0 0 ,1 6 5 0005881 072000A 7 /2 0 /0 0 0.1 0 2 0 .0 8 8 3 0005882 072000A 7/20/00 0.0748 0 .0 6 5 0 0005883 072000A 7 /2 0 /0 0 0 .1 3 0 0 .1 1 3 0005884 072000A 7 /2 0 /0 0 0 .1 8 1 0 .1 5 7 0005885 072000A 7 /2 0 /0 0 0 .1 8 8 0 .1 6 3 AVERAGE: 0.1 3 7 0 .1 1 9 STA N D A R D D E V IA T IO N : 0.0483 04)420 Table XIII. Summary ofPFOS residues in G3 Day 20/21 Gestation Sponsor ID 13751F G 3 D ay 20/21 G estation 13752F G 3 D ay 20/21 G estation 13753F G 3 D ay 20/21 G estation 13754F G 3 D ay 20/21 G estation 13755F G 3 D ay 20/21 G estation 13756F G 3 D ay 20/21 G estation 13758F G 3 D ay 20/21 G estation t 13759F G 3 D ay 20/21 G estation C entre S et D ate ID N um ber E xtracted 0005886 0717-1800A D 7/17/00 0005887 0717-1800A D 7/17/00 0005888 0717-1800A D 7/17/00 0005889 0717-1800A D 7/17/00 0005890 0717-1800A D 7/17/00 0005891 0717-1800A D 7/17/00 0005892 0717-1800A D 7/17/00 0005893 0717-1800A D 7/17/00 AVERAGE: STA N D A RD D E V IA T IO N : R esidue Found (ti /g ) 1.86 2 .3 9 1.63 1 .9 4 4 .9 3 2 .3 2 2 .2 7 1 .5 9 237 1 .0 8 C orrected R esidue Found (tig /g ) 1.61 2 .0 8 1.42 1.69 4 .2 9 2 .0 2 1.97 1.38 2 .0 6 0 .9 4 0 3M Environmental Laboratory Amended Page 26 of 92 Page 266 BACK TO MAIN 3M Medical Department Study: T-6395.14 ' !. ' Analytical Report: FACT T O X -111 LRN-U2994 Centre StudyNo.: 023-017 SponsorProtocol No.: FACT-TOX-111 Table XIV. Summary ofPFOS residues in G1 Day 21/22 Lactation . S p o n so r ID 13726F G1 D ay 21/22 L actation 13727F G 1 D ay 21/22 L actation 13728F G1 D ay 21/22 L actation 13734F G1 D ay 21/22 L actation 13735F G1 D ay 21/22 L actation 13736F G1 D ay 21/22 L actation 13737F G1 D ay 21/2 2 L actation C entre R esidue Set D ate Found ID 0005894 N um ber 07130QA E xtracted 7 /1 3 /0 0 (n g fe ) 0 .0 2 3 5 0005895 071300A 7 /1 3 /0 0 ND 0005896 071300A 7 /1 3 /0 0 ND 0005897 071300A 7 /1 3 /0 0 ND 0005898 071300A 7 /1 3 /0 0 ND 0005899 071300A 7 /1 3 /0 0 ND 0005900 071300A 7 /1 3 /0 0 ND A V E R A G E : < 0 .0 1 0 0 STA N D ARD D E V IA T IO N : NQ C orrected R esidue Found (u ste) 0 .0 2 0 4 ND ND ND ND ND ND < 0 .0 0 8 6 9 NQ ND = Not Detected andNQ= Not Quantifiable Note: The average andstandarddeviationwerecalculatedbyusing 0.0100 asthe value forthose samples reportedas < 0.0100,0.00869 forthose reportedas < 0.00869, andzero forthose reportedas ND orNQ. Table XV. Summary ofPFOS residues in G2Day 21/22 Lactation Sponsor ID 13739F G 2 D ay 21/2 2 L actation 13740F G 2 D ay 21/22 L actation 13741F G 2 D ay 2 1/22 L actation 13744F G 2 D ay 2 1/22 L actation 13745F G 2 D ay 2 1/22 L actation ( 13746F G 2 D ay 21/22 L actation 13748F G 2 D ay 21/22 L actation 13749F G 2 D ay 21/22 L actation C entre S et D ate ID N um ber E xtracted 0005901 071800A 7 /1 8 /0 0 0005902 071800A 7 /1 8 /0 0 0005903 071800A 7 /18/00. 0005904 071800A 7 /1 8 /0 0 0005905 071800A 7 /1 8 /0 0 0005906 071800A 7 /1 8 /0 0 0005907 071800A 7 /1 8 /0 0 0005908 071800A 7 /1 8 /0 0 AVERAGE: STA N D ARD D E V IA T IO N : R esidue Found (u ste) 0 .0 7 7 6 0 .0 7 9 7 0 .0 4 4 8 0 .0 8 3 9 0 .0 3 9 0 0 .0 7 0 1 0 .0 5 8 5 0 .0 2 6 6 0 .0 6 0 0 0 .0 2 1 2 C orrected R esidue Found (U g/g) 0 .0 6 7 4 0 .0 6 9 3 0 .0 3 8 9 0 .0 7 2 9 0 .0 3 3 9 0 .0 6 0 9 0 .0 5 0 8 0 .0 2 3 1 0 .0 5 2 2 0 .0 1 8 5 3M Environmental Laboratory Amended Page 27 of 92 Page 267 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 , Analytical Report: FACT T O X -111 LRN-U2994 Centre StudyNo.: 023-017 SponsorProtocol No.: FACT-TOX-111 Table XVI. Summary ofPFOS residues in G3Day 21/22 Lactation Sponsor ID 13751F G 3 D ay 2 1 /22 L actation 13752F G 3 D ay 2 1 /22 L actation 13753F G 3 D ay 21/2 2 L actation 13754F G 3 D ay 2 1 /22 L actation 13756F G 3 D ay 2 1 /2 2 L actation 13758F G 3 D ay 21/2 2 L actation 13759F G 3 D ay 21/2 2 L actation C entre S et D ate ID N um ber E x tracted 0005909 0717-1800A D 7 /18/00 0005910 0717-1800A D 7 /18/00 0005911 071800A 7 /18/00 0005912 071800A 7 /18/00 0005913 0717-1800A D 7 /18/00 0005914 071800A 7 /1 8 /0 0 0005915 071800A 7/18/00 AVERAGE: STA N D A RD D E V IA T IO N : R esidue Found (U 8/g) 0 .5 9 0 0 .8 4 2 0 .3 7 6 0.3 4 0 0.3 8 6 0 .2 4 3 0 .3 4 1 0 .4 4 5 0 .204 C orrected R esidue Found G ig/g) 0 .5 1 3 0 .7 3 1 0327 0 .2 9 5 0.3 3 6 0 .2 1 1 0 .2 9 6 0387 0 .1 7 7 t 3M Environmental Laboratory Amended Page 28 of 92 Page 268 BACK TO MAIN 3M Medical Department Study: T-6395.14 ,. Analytical Report: FACT T O X -111 LRN-U2994 C entre S tudy N o.: 023-017 S p onso r P rotocol N o: F A C T -T O X -111 Table XVII. Summary of PFOS recoveries in Fortified Samples Sponsor C entre Set D ate . A m t. % ID ID N um ber E xtracted A dded Cna) R ecovery 13726F G1 P rio r to C ohabitation 0005799 S pk A 070600A R 7/6/00 50 118 13727F G1 P rio r to C ohabitation 0 0 0 5800 S p k B 070600A R 7/6/00 250 101 13728F G1 P rior to C ohabitation 0005801 S pk A 070700A 7/7/00 50 88 1373 IF G1 P rio r to C ohabitation 0005802 S p k B 070700A 7/7/00 250 86 13734FG 1 P rio r to C ohabitation 0005803 S pk A 071000A 7/10/00 50 101 13735F G1 P rior to C ohabitation 0005804 S p k B 071000A 7/10/00 2000 94 13736F G1 P rior to C ohabitation 0005805 Spk.A 071100A 7/11/00 50 85 13737F G1 P rior to C ohabitation 0005806 S p k B 071100A 7/11/00 20000 ' 84 13726F G1 P rior to C ohabitation 0005799 S pk A 071200A 7/12/00 50 136 13728F G1 P rior to C ohabitation 0005801 S p k B 071200A 7/12/00 20000 87 13734F G1 P rior to C ohabitation 0005803 Spk A 071300A 7/13/00 50 92 13735F G1 P rior to C ohabitation 0005804 S p k B 071300A 7/13/00 250 102 13726F G1 P rior to C ohabitation 0005799 Spk A 071700A R 7/17/00 50 79 13737F G1 P rio r to C o habitatio n 13735F G1 P rio r to C ohabitation 0005806 S p k B 071700A R 7/17/00 0005804 S pk A 071800A 7/18/00 20000 50 73 105 13 737F G1 P rio r to C o h ab itatio n 0005806 S p k B 071800A 7 /1 8 /0 0 20000 83 13727F G1 P rior to C ohabitation 0005800 Spk A 072000A 7/20/00 50 74 13734F G1 P rior to C ohabitation 0005803 S p k B 072000A 7/20/00 ' 250 AVERAGE: STANDARDDEVIATION: RELATIVESTANDARDDEVIATION: 74 92 16 18 C entre A nalytical L aboratories, Inc. 3M Environmental Laboratory .. Page 29 o f 92 Page 269 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 C entre S tudy N o.: 023-017 S ponsor P rotocol N o: FA C T -T O X -111 12.0 FIGURES Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 30 o f 92 Page 270 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Figure 1. Typical Calibration Curve for PFOS Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 31 of 92 Page 271 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 , LRN-U2994' Figure 2. Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Typical Mean Rsponse Factorfor THPFOS Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 32 of 92 Page 272 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Figure 3. C entre S tudy N o.: 023-017 S p o n so r P rotocol N o: F A C T -T O X -111 ChromatogramRepresenting a 5 ng/mL extracted standard ' for PFOS and 250 ng/mL fortification ofTHPFOS 1 : PFOS x c o ; r0600-1,5 ng/mL Standard : O S -Ju i-200010:37:59 0706 0700AD-3002 Sm (SG, 2x2) 1 100 ' ' 5.21 6692 i . . . L C /M S /M S # 6 M RM of 2 Channels ES499 > 99 6.77e4 ln Area %- V---- 'J ' ' ................ r ` * *` *' 1*` " ' 1 ' ` ` ' 1 1 " " 1.00 2.00 3.00 4.00 5.00 6.00 7.00 a.nn - Time 2: THPFOS ' x c o : ^0600-1, S ng/mL Standard 08-Jui-2000 1 0 :3 7 :5 9 " . LC/MS/MS #6 0706 0700AD-3002 Sm (SG, 2x2) .v 100 5.04 M RM of 2 Channels ES . 427 >80 . 9.57e5 Area %- * 1.00 * 2.00 ' 3.00 ' 4.00 * , t . . . . , 5.00 6.00 7.C0 T'-mi ! t *t t" Time 8.00 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 33 of 92 Page 273 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Figure 4. Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 ChromatogramRepresenting a 125 ng/mL extracted standard for PFOS and 250 ng/mL fortification of THPFOS Centre A nalytical Laboratories, Inc. 3M Environmental Laboratory Page 34 o f 92 Page 274 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Figure 6. ChromatogramRepresenting Control Rat Feces Fortified with 50 ng of PFOS and 500 ng of THPFOS (Centre ID: 0005803 Spk A, Set: 071000A) 1 : PFOS 0005803 Spk A 0710130A-1013 Sm (SG, 2x2) 100n %- 5.2S 19051 11-Jul-2000 01:17:25 LC/MS/MS #6i MRM of 2 Channels ES . 499 > 99 1.78e5 Area ' 1-bo ' 2.00 2 : THPFOS 803 Spk A 30A-1013 Sm (SG, 2x2) 3.0 4.00 ' 5.0 6.00 5.10 . ' ' ' 1" ' ' * l * 1] ' l ' *` ' ) ` ' ' ' ' - . - ' rT~ 1.00 2.00 . 3.00 1 4.00 L 5.00 6.00 ' ` 1` 1 7.00 8:00 1T'tne ' 11-Jul-2000 01:17:25 LC/MS/MS # 6 M RM ot 2 Channels ES427 > 80 ' . 9.47e5 ' Area ' 7.00 8.00 .o o ?...... $ ....... -? 9 ! Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 36 of 92 Page 276 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 . LRN-U2994 Figure 7. Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Chromatogram of Rat Feces Sample from G2 Day 6/7 Gestation (Centre ID: 0005836, Set: 071000A) 1 : PFOS 0005836 ___________ 071000A-1022 Sm (SG, 2x2) 100-1 %- X5.25 H -Jul-2000 03:48:34 ' LC/MS/MS #6 MRM of 2 Channels ES ' 499 > 99 . 1.15e6 Area 1.00 2.00 3.00 4.00 5.00 3.00 7.00 8.00 Time 2 : THPFOS 0005836 071000A-1022 Sm (SG, 2x2) 100-1 5.09 11-Ju!-2000 03:48:34 LC/MS/MS #6 MRM of 2 Channels ES427 >80 9.45e5 Area %-1 1 1.00 2.00 3.00 4.00 5.00 6.00 7,00 8.00 Time Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 37 of 92 Page 277 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 13.0 APPENDICES Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 38 of 92 Page 278 3M Medicpl Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 - Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 APPENDIX A Study Protocol FACT-TOX-111 (Centre Study No. 023-017) and Amendments and Deviations Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 3? of 92 Page 279 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 Analytical Report: FACT TOX-111 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 3M Environmeniai.Tedmolosy c ___ -___ PO Bo* 33331 S.l Pau*l, MN 35133-3331 612 773 6*W2 Protocol tfF A C T -T O X -1 11 Study Title Oral (Gavage) Pharmacokinetic Recovery Study ofPFOS in Rats PROTOCOL A u th o r Lisa Clemen Date: June 8,1999 Performing Laboratory 3M EnvironmentalTechnology & Safety Services 3M Environmental Laboratory 935 Bush Avenue ' ' St. Paul, MN 55106 Laboratory Project Identification FACT-TOX-111 U2994 Exact Copy of Original 3 M Environmental Laboratory Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 1 of to Page 40 of 92 Page 280 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Protocol X F A C T -T C X -1 11 S tu d y Id en tification Oral (G avage) P h arm acok in etic R e co v e r y . S tu d y o f PFO S in R ats Test Material . Sponsor. ' Sponsor Representative Study Director Perfluorooctane sulfonic acid potassium sait (T-6295) ' 3M Toxicology Services - M edical Department 3M Center, Building 220-2E-02 S t Paul, MN 55144-1000 Marvin T. Case, D .V .M ., Ph.D. 3M Toxicology Services Telephone: 651-733-5180 Facsimile: 651-733-1773 Kristen J. Hansen, PhJ3. ' 3M Environmental Technology and Safety Services Building 2-3E-09 ' 651-778-6018 ' . Study Location(s) . In vivo Testing FaciRty Analytical,Testing Laboratory Argus Research Laboratories, Inc. 905 Sheehy Drive, B uilding A Horsham, PA 19044 3M Environmental'Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 3 M Environmental Laboratory Centre Analytical Laboratories, Inc. 3M Environmental Laboratory ' Rage 2 of 10 Page 41 of 92 Page 281 3M Medicpl Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Sub-C ontract Laboratories Proposed Study Timetable S tu d y Initiation Date S tu d y Completion D ate Protocol ZF A C T-TQ X-111 Advanced B analytical Services, Inc. 15 Catherwood Road Ithaca, NY- 14850 Battello Memorial Institute :505 King Avenue Columbus Ohio 43201-2693 June 8,1999 August 8,2000 1. S tudy . Oral (gavage) phannacoldnetic recovery study o f potassium perfluorooctane sulfonie acid ' (PFOSJinrats. ' . 2. Purtpose This analytical Study is designed to determine levels o fpotassium perfluorooctanesulfonate . (PFOS) in specimens o f liver and serum o f rats. The in-life portion o f this study was conducted at Argus Research Laboratories, study #418-015. A ll serum samples w ill be extracted and analyzed at-Advanced Bioanalytical Services, Inc. and all liver samples extracted and analyzed at B attelle Memorial Institute. Additioftal analyses may be performed at the 3M Environmental Laboratory as methods are developed and validated.. I f additional analyses are performed an amendment to this protocol w ill b e written. ' 3. R g u la t o r y Co m p lia n c e This study w ill be conducted in accordance with the United States Food and Drug Administration, Good Laboratory Practices Standards; Final Rule 21 CFR58, w ith the exception that analysis o f the test material mixture for concentration, solubility, homogeneity, and stability w ill not be conducted, and is the responsibility o f the Sponsor. 4 . Qu ality A ssu r a n c e '. `. The 3M Environmental Laboratory Quality Assurance Unit w ill review the protocol and audit study conduct, data, and finjl report to determine compliance with Good Laboratory Practice S tandards and with 3M Environmental Laboratory Standard Operating Procedures.- The QA Unit at the sub-contract laboratory w ill audit their study conduct, data, and results report prior to submitting to the 3M Environmental Laboratory. 3 M Environmental Laboratory .. ' Pago 3 o f 10 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 42 of 92 Page 282 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Protocol 8FA C T-TO X-111 5. T e s t Ma t e r ia l ' 5.1 Refer to Argus Research Laboratory protocol for study #418-015. 6 . C o n t r o l Ma t r i c e s . . 6.1 IdentificationRat liver and serum, and/or rabbit liver and serum traceability numbers w ill be recorded in the raw data and included in the final report 6.2 Sourca Argus Research and/or Sigma Chemical . 6.3 PhysicalDescription Rat liver and serum, and/or rabbit liver and serum 6.4 PurityandStability Not applicable . 6.5 Storage Conditions Frozen at -20 8C 10 C or-5 0 C 10 C 6 .6 Reserve Matrix A portion o f the control matrix w ill be retained in the 3M archives for as long as the quality o f the preparation affords evaluation, but not longer than ten years following the effective date o f the final test rule (if applicable). 6 .7 Disposition Matrices w ill be retained at the 3M Environmental Laboratory per GLP regulation. Certain matrices (feces, urine, and blood) may be disposed after QAU verification. . . .' . 6 .3 SafetyPrecautions Refer to MSDS for chemicals used. Wear appropriate * ' laboratory attire, and follow adequate precautions for handling biological materials and. preparing samples for analysis. ' 7 . R e f e r e n c e Ma t e r ia l - 7 .1 Identiticatiori Potassium perfluorooctanesulfonate (PFOS), lot 8s171,215, or 217 (equivalent lots) ' . . 7 .2 S o u rce 3M Specialty Chemicals . 7.3 PhysicalDescription White powder . 7.4 Purityand StabilityPurity o fPFOS is 99% or greater. Stability has not been determined. 7.5 Storage Conditions Room temperature . 7 .6 ReserveMaterial A reserve sample from each batch o fPFOS used in this study w ill be retained as long as.the quality o f the preparation affords evaluation, but not longer than ten years following the effective date o f the final test rale (if applicable). 7.7 Disposition Unused reference material w ill be retained for use by the 3M - : . . Environmental Laboratory and w ill be discarded when the quality o f preparation no longer affords evaluation. - _; 3 M Environmental'Laboratory ' Centre Analytical Laboratories, Inc. 3M Environmental Laboratory P age 4 o f 10 Page 43 of 92 Page 283 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 ,; Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 - Centre Study No.: 0 2 3 -0 1 7 Sponsor Protocol No: FACT-T.OX-111 . Protocol 2FA C T.TO X -111 7.8 S a fe ty P reca u tio n s Refer to M SD Sfof chemicals used. Wear appropriars laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. 8. Te s t Sy s t e m Rats were used as the test system and were maintained and dosed as described in Argus Research protocol #418-015. The female rats w ill be given the test material or control once daily beginning 42 days prior to cohabitation until day 0 o fpresumed gestation. See table 1 for more dosage information. * .. Dosage Group i 2 3 Table 1 D osage Levels, Concentration, and Volumes Number o f Dosage - Concentration female rats mgfcg/day mg/kg/day 8 0. 0 8 0.1 0.02 8 &&-- . 032 <&) l . b *W f i Dosage volum e mLTcg 5 5 5. S. S pecim en a n d S am ple R e c e ip t ' The 3M Environmental Laboratory w ill receive homogeneity samples and specimens o f the . following body tissues and fluids from the indicated points in the study. A ll specimens w ill be packed on dry ice for shipping. See table 2 for more specimen information. . Table 2 . . Specimen Information Body tissue/Quid ' . Seram - Dam and Pup mrimal .Urine and Feces--Dam and Pup animals ' Liver--Dam and Pup animals C ollected Dam-Predose, Days 7 ,1 4 ,1 5 ,2 1 , and 22 ' Pup-Day 21 . Predose, Days 7 ,1 5 ,2 1 , and 22 Dam-At the termination o f the study Pup-Day21 Expected # o f specim ens 144 Dam 24 Pup (pooled) 120 Urine and 120 Feces 24 Dam - 24 Pup (pooled) Total number o f expected specimens: 456 Total number o f test animals: 16 Total number o f control animals: 8 3 M Enviwnmental Laboratory . ' Page 5 o f 10 Centre.Analytical Laboratories, Inc. 3IVI Environmental Laboratory ' . Page 44 of 92 Page 284 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 Analytical Report: F A C TT O X -111 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Protocol s p a c t - t o x - 11 1 Specim ens sent to 3M Environmental Laboratories w ill be received and tracked according to applicable Standard Operating Procedures. 10. P r e p a r a t o r y M e t h o d s . 10.1 FACT-M -l.l, Extraction o f Potassium Penluorooctanesulfonate or Other Anionic Fluorochenrical Surfactant fiom Liver for Analysis Using HPLC-EIectrospray/Mass Spectrometry " 1Q.2 ETS-8-4.1, Extraction o f Potassium Perfluorooctanesuifonate or Other Fluorochemical Compounds fiom Serum or OtherFluid for Analysis Using HPLC- Electrospray/Mass Spectrometry ,. 10.3 I f preparatory methods other than those listed above are used, an amendment to this protocol w ill be written. Any deviations from these methods w ill be documented and included with the study data. 10.4 I f analyses are sub-contracted to other laooratories, an amendment w ill be written to include their methods and copies o f each method w ill be attached to this protocol. 1 1 .A n a lyt ic a l Methods 11.1 FACT-M-Z1, Analysis ofFluorochemicals in Liver Extracts Using HPLC-. . Electrospray/Mass Spectrometry . 11.2 ETS-8-5.1, Analysis ofPotassium Perfluorooctanesuifonate or Other Fluorochetnicals in Serum or OtherFluid Extracts Using HPLC-EIectrospray/Mass Spectrometry 11.3 I f analytical methods'other than those listed above are used, an amendment to this protocol w ill he written. Any deviations from these methods w ill be documented and included with the study data. - 11.4 If analyses suesub-contracted to other laboratories, an amendment w ill be written to inrh^w theirmethods and copies o f each method w ill be attached to this protocol. 12. Da t a Qu au ty Objectives . .. The number o f spikes/duplicates, use o f surrogates, and information on other data quality . indicators are included in the analytical methods. In addition, the following criteriaw ill be met: 12.1 L in ea rity ri 2 0.98 12.2 L im its o f d etectio n /q u a n tita tio n 12.2.1 `Method Detection Limit (MDL) for PFOS : a) Serum: 1.75 ppb . b) Liver: 15 ppb . ' . 3 M E nvironm ental Laboratory. ' P a g e S o f 1C . Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 45 of 92 Page 285 3M Medicpl Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 - Centre Study No.: 023-017 ' Sponsor Protocol No: FACT-TOX-111 Protocol ZFACT-TO X-11 1 12.2.2 Limit o f Quantitation (LOQ)--Equal to the lowest acceptable standard in th calibration carve 122 Duplicate acceptable precision < 30% far the method 12.4 Spike acceptable recoveries 70% -130% ' 1 2 .5 Use of confirmatory methods Indeterminate samples w ill be re-analyzed using a confirmatory method. I f a confirmatory method is used, an amendment to this protocol w ill be written. 1 2 .8 Demonstration of specificity Chromatographic retention time, daughter ion characterization. spectrai 13. S ub-C o ntracted A n a ly sis 1 3 .1 A ll analyses as detailed in this protocol w ill be performed at 3M Environmental. Laboratories, Building 2-3E -09,935 Bush Avenue, S t Paul, M N 55106, at Advanced" Bioanalydcal Services, Inc., 15 Calhenvood Road, Ithaca, NY 14850, or at BatieHe Memorial Institute, 505 King Avenue, Columbus, Ohio 43201-2693. 1 3 .2 An amendment to this protocol w ill be written i f analyses are performed at laboratories other than the 3M Environmental Laboratories, Advanced Bioanalytical - Services, Inc., or Battelle Memorial Institute. 14. S t a v s v c a l A n a l y sis Averages and standard deviations w ill be calculated. The statistical methods that w ill be used are . described below: 1 4 .1 Data transformations and analysis Data w ill be reported as the concentration (weightAveight or weighl/voi) o f PFOS or metabolite per tissue or fluid. 1 4 .2 Statistical analysis Statistics used.may include regression analysis o f . concentrations over time, and standard deviations calculated for the concentrations within each dose group. I f necessary, simple statistical tests, such as Student's t test, " . . may be applied to evaluate statistical difference. 1 5 .R e p o p t .; A report containing all the results o f the study w ill be prepared by the 3M Environmental Laboratory. If analyses are sub-contracted to other laboratories, each laboratory w ill prepare a ' report and submit it to the 3M Environmental Laboratory for inclusion in the 3M Environmental. Laboratory,report Each report win include, but not be limited to, the following, when applicable: . ." ; 1 5 .1 Name and address o f the facility petfotming the study 1 5 .2 Daces upon which the study was initiated and completed 3MEnvironmental Laboratory . Page 7 o f 10 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 46 of 92 Page 286 3M Medical Department Study: T-6395.14 I BACK TO MAIN . Analytical Report: FACT T O X -111 LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Protocol " F A C T -T O X -1 11 15.3 A o f compliance by the Study Director addressing any exceptions to Good Laboratory Practice Standards 15.4 Objectives and procedures as stated in the approved protocol, including any changes in the original protocol 1 5 .5 The test substance identification by name, chemical abstracts number or code number, strength, purity, and composition or other appropriate characteristics, i f provided by the Sponsor _ 1 5 .8 Stability .and the solubility o f the test substances under the conditions o f administration, if provided by the Sponsor ` 1 5 .7 A'description ofthe methods used to conduct the testfs) 15.8 A description ofthe test system _ 15.9 A description o f any circumstances that may have afiected the quality or the integrity . ofthe data ' 15.10 The ofthe Study Director and the names o f other scientists, professionals, and supervisory personnel involved in the study 15.11 A description ofthe transformations, calculations, or operations performed on the a gmrnnflry and analysis o f the analytical chemisLy data, and a statement o f the conclusions drawn from the analyses .- 15.1 2 Statistical methods used to evaluate the data, i f applicable ' 1 5 .1 3 The signed and dated reports o f each ofthe individual scientists or other professionals ` involved in the study, i f applicable . 15.14 The location where raw data and the final report are to be stored 15.15 A statementprepared by the Quality Assurance Unit listing the dates that study - inspections and audits were made, and the dates o f any findings reported to the Study Director and Management ' I f it is necessary to make corrections or additions to a report after it has been accepted, the :changes will be mad* in the form o f an amendment issued by the Study Director. The . amendment vrill ciearly identify the part o f the report that is being amended, the reasons for the amendment, and will be signed by the Study Director. . 1 6 . L o c atio n o f Ra w Data, R e c o r d s, a n d Final R e p o r t Original data, or copies thereof will be.available at the 3M Environmental Laboratory to facilitate audits o fth e study during its progress and before acceptance o f the final report. When the final report is completed, all original paper data, including those items listed below, will he retained in the archives o f 3M Environmental Laboratory for at least a period*o f time as spedfied b y regulation, and as established by 3M Environmental Laboratory Standard Operating Procedures. . 3 M Environmental Laboratory . PagaSoHO Centre Analytical Laboratories, Inc. 3M Environmental Laboratory . Page' 4 7 of 92 . Page"2 8 f " 3M Medicpl Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 . LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Pretax! 0PACT-7CX-111 16.1 The follow ing raw data and records w ill be retained in the study folder in the study/project archives according to 3M Environmental Laboratory Standard Operating Procedures: 16.1.1 Approved protocol and amendments 16.1.2 Study correspondence 16.1.3 Shipping records 16.1.4 Raw data ' _ 16.1.5 Approved final report (original signed copy) 16.1.6 Electronic copies o f data 16.2 The follow ing supportingrecords w ill be retained separately fiom the study folder in the archives according to 3h i Environmental Laboratory Standard Operating Procedures: . 16.2.1 Training records ' 1 6 .Z 2 Calibration records 16.2.3 Instrument maintenance logs . 16.2.4 Standard Operating Procedures, Equipment Procedures, andMethods . 1 7 . S pecim en R etention ' Specim ens w ill be in the 3M Environmental Laboratory specimen archives for a period o f time as specified by regulation ctras long as the quality o f (he preparation affords evaluation, but not longer than ten years following the effective date o f the final test rule (if applicable), and as established by 3M Environmental Laboratory Standard Operating Procedures. 1 8 . P r o t o c o l A m endm ents a n d deviations . Planned changes to the protocol w ill he in the form o fwritten amendmentssigned by the Study . Director and the Sponsor's Representative. Amendments win be considered as part o f the protocol and w ill be attached to the final protocol. A ll changes to the protocol w ill be indicated in the final report. A ny other changes w ill be in the form o fwritten deviations, signed by the Study Director and filed with the raw data. .' 19.A ttachments . ' 19.1 A tta c h m e n t A Preparatory and analytical methods 3 M Environmental Laboratory Centre Analytical Laboratories, Inc. 3M Environmental Laboratory 9 of 10 Page-48 of 92 Page 288 3M Medicpl Department Study: T-6395.14 BACK TO MAIN A nalytical R eport: FACT T O X -111 LR N -U 2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 20. S ignatures Prstaccl XEACT-TOX-111 7 t& * - .______________ Marvin T. Case, D .V A 1, P h D , Sponsor Represeatarive" -7 / <?9rt Date -ijL /h- Knnsstteeaa JJ.. HHaannsseenn., PPhhJD ., 3M Environmental Laboratory Study D ir ec to r 1Date 3M Environmental Laboratory Centre Analytical Laboratories, Inc. 3M E nvironm ental Laboratory P age 10 o f to Page 49 of 92 Page 289 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Study Title Oral (Gavage) Pharmacokinetic Recovery Study o f PFOS in Rats PROTOCOL AMENDMENT NO. 1 AmendmentDate: August 12,1999 . PerformingLaboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory . 935 Bush Avenue SL Paul, MN 55106 LaboratoryProjectidentification ET&SS FACT-TOX111 ' L IR N U 2994 - act Copy of Original Initial [ata ~ 3M Environmental Laboratory Cntre A nalytical Laboratories, Lie. 3M Environmental Laboratory Pag 50 of 92 3M Medicpl Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 . LRN-U2994" . Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 P rotocol FA C T -T O X 1 11 Am endm ent N o. 1 This am endm ent modifies the following portion(s) of th e protocol: 1. P r o t o c o l r e a d s : Section 10.0 and 11.0 list the following methods to use for extraction and analysis: . FACT-M-l.I "ExtractionofPotassium Perfluorooctanesulfonate or OtherAnionic Fluorochemical Surfactant fiom Liver forAnalysis Using HPLC-Electrospray/Mass ' Spectrometry" . FACT-M-2.I "Analysis ofFIuorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" " A m e n d TO R EAD : The extraction and analytical methods FACT-M-1.1 andFACT-M -2.1, respectively, were updated on 07/22/99 to: ETS-8-6.0 "Extraction o f Potassium Perfluorooctanesulfonate or OtherFluorochemical Compounds fiom Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in liv e r Extracts Using HPLC-Electrospray/Mass Spectrometry" R e a s o n : The extraction and analytical methods FACT-M-1.1 and FACT-M-2.1, respectively, were updated on 07/22/99 to ETS-S-d.O and ETS-8-7.0. These methods were updated to replace the extraction solvent ethyl acetate with a different extraction solvent MTBE (methyl te n butyl ether), POAA and Monoester were removed fiom the standard m ix, andM 556 was added to the standard m ix. ; __ _ - The analytical method was updated to include linear regression w ith 1/x weighting and a few minor changes in the HPLC 1100 instrument parameters. 2. P rotocol READS: Section 10.4 and 11.4 state that if tire analyses are sub-contracted to other laboratories an amendment w ill be written to include these methods. . . A m e n d t o r e a d : The extraction and analytical methods to follow at Advanced Bioanalytical Services win be attached to the protocol. .' The extraction andanalyticalmethod to follow at BattelleMemonalInstitute is: "Method for Analysis Version^" ^ o f Ferfluorooctane . Sulfonate (PO S) in Rat Seta-by (PLivu- LC/MS/MS, . REASON.*The analytical methods at the sub-contract laboratories.were not included in the : : originalprotocol. . ' ` . I A C ^. 3M E nw cnm enial Laboratory ___Centre Analytical Laboratories; fiic. 3M Environmental Laboratory Pag51of92 Page 291~ BACK TO MAIN 3M Medical Departm ent Study: T -6395.14 _ : . . Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 ' : P ro to c o l F A C T -T O X 1 11 1. A m e n d m e n t M o . 3. P r o t o c o l r e a d s : Section 12.2.1 b) lists liver method detection lim it as 15 ppb. A m e n d t o r e a d : The liver method detection lim it is 8JO ppb (ng/g). . . R e a s o n : The validation supporting methods ETS-8-6.0 and ETS-8-7.0 included a lower method detection lim it for PFOS. . Amendment Approval ! _________ ;---- :-- i--------------- O 4 ^ 1 Marvin Case, D .V M ., PhD ., Sponsor Representative ? Date Kris JT.Hansen, Fh.D., Study Director ... ' 3M Environmental Laboratory ........................ C entre-A nalytical L aboratories, Inc. 3M Environmental Laboratory " . Page 52 of 92 -Page 292" 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 ' StudyTitle Oral (Gavage) Pharmacokinetic Recovery Study o f PFOS in Rats PROTOCOL AMENDMENT NO. 2 AmendmentDate: September 30,1999 . PerformingLaboratory 3M EnvironmentalTechnology & Safety Services 3M EnvironmentalLaboratory , 935 Bush Avenue . S t Paul, MN 55106 LaboratoryProjectldentlficatlon ET&SS FACT-TOX111 URNU2994 Copy of Origin/ 3M Environmental Laboratory Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Pag 53 of 92 Page 293 BACK TO MAIN Medicpl Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 - Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Aroteec/ FACT-TQX11 1 Amendment No. 2 This am endment m odifies the follow ing porfion(s) o f the protocol: 1. PROTOCOL READS: Section 2 states the study is designed to determine levels o f potassium perfluorooctanesolfonate (PFOS) in specimens o f liver and sera in rats. . A m end to read: The study is designedJo 'specimens o fliver, sera, and urine in rats. Reason: H ie urine analytical methods were validated and approved after approval'ofthe original protocol ' ' Z P rotocol reads: Section 10.0 and 11.0 list the following methods to use for extraction and analysis: ETS-8-4.1 "Extraction o fPotassium Perfluorooctanesulfbnate or OtherFluorochemical Compounds from Serum forAnalysis Using HPLC-EIectrospray/Mass Spectrometry'' . ETS-8-d.O "Extraction o fPotassium Perfluorooctanesulfbnate or Other Flnorochemical Compounds from Liver for Analysis Using HPLC-EIectrospray/Mass Spectrometry" - .EXS-8-5.1 "Analysis o fPotassium Perfluorooctanesulfonate or OtherFluotochemical . ' Compounds in Serum Extracts U sing HPLC-EIectrospray/Mass Spectrometry" ETS-8-7.0 "Analysis ofPotassium Perfhiorooctanesulfonate or Other Flnorochemical Compounds in Liver Extracts Using HPLC-EIectrospray/Mass Spectrometry" A m e n d t o r e a d ?. ' . ETS-8-4.1 "ExtractionofPotassium Perflnoroactanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-EIectrospray/Mass Spectrometry" ETS-8-i.O "Extraction ofPotassium Perfluorooctanesulfbnate or Other Fluorochemical ' Compounds from Liver for Analysis Using HPLC-EIectrospray/Mass Spectrometry" ETS-8-96.0 "Extraction ofPotassium Perfluorooctanesulfbnate or OtherFluorochemical Compounds from Urine for Analysis Using HPLC-EIectrospray/Mass Spectrometry/Mass Spectrometry" ' . ' ETS-3-S. 1 "Analysis ofPotassium Perfhiorooctanesulfbnate or Other Fluorochemical . Compounds in Serum Extracts Using HPLC-EIectrospray/Mass Spectrometry" ETS-8-7.0 "Analysis ofPotassium Perfhiorooctanesulfbnate or OtherFluorochemical Compounds in Liver Extracts Using HPLC-EIectrospray/Mass Spectrometry" ETS-8-97.0 "Analysis ofPotassium Perfluorooctanesulfbnate or Other Fluorochemical Compounds in Urine Extracts Using HPLC-EIectrospray/Mass Spectrometry/Mass- Spectrometry" . .' R e a s o n : The extraction and analytical methods ETS-3-96.0 and ETS-8-97.0, w ere approved after approval o f the original protocoL ' 3M E nvironm entalLaboratory Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 54 of 92 Page 294 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994- Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Protocol FACT-TOX111 AmendmentNo. 2 3. Protocol reads: Section 6.1 lists control matrices as rat liver and serum or rabbit liver and sen,TM c Argus and/or Sigma Chemical. . um . Source Amend to read: Control matrices identification rat liver and serum, rabbit liver and serum, human urin and/orraturine. Source Argus, Sigm a Chem ical, Lampire Biologicais, Biolorical Corp. and/or Golden W est B iologicais. Specialty Reason: Addition ofcontrol urine specifications. . . 4. P rotocol reads: Section 12.2. 1-a) lists sera method detection lim it as 1.75 ppb and b) lis ts livermetfmd detection lim it as 15 ppb. A mend to read: The method detection lim its for all compounds and matrices w ill be used forextraction and analysis. . . ^ me. . , ~ . R easo n : Tbemethod detection lim its listed are specific to the 3M Environmental Laboratory Statementwas added to allow for sub-contracted analyses and/orrevised methods ' 3M EnvxaumntalLaboratory ' Centre Analytical Laboratories, Inc. Page 55 o f 92 Page 295 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 . LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Protocol FACT-TOX11i Amendment No. 2 5. Pro tocol reads: Section 16 states that the original data, or copies thereof, w ill be available at the 3M : Environmental Laboratory to facilitate audits o fthe study during its progress ana before , acceptance o f die final report When the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records raw data, approved final report, electronic copies o f data, training records, calibration records, ' instrument maintenance logs and standard operating procedures, equipment procedures, and methods w ill be retained in the archives o f the 3M Environmental Laboratory. . A mend to read: Section 16 states that the original data, or copies thereoi w ill be available at the 3M ' EnvironmentalLaboratory to facilitate audits o f the study during its progress and before acceptance o fthe final report When the final reportis completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approYi final report, and electronic copies o f dataw ill be retained in the archives o f the 3M Environmental Laboratory. A ll corresponding training records, calibration records, instrument maintenance-logs, standard operating procedures, equipment procedures, and methods w ill be retained in the archives o f the facility performing each analysis. Re a so n : ' Clarification o f the disposition o f archived records if analyses are performed at a sub- - contract laboratory. - .. 6. P ro to col reads: ' . ' Section 17 states that specimens w ill be maintained in the 3M Environmental Laboratory specimen archives. .. ' Amend to read: ' . Specimens w ill be maintained in the 3M Environmental Laboratory specimen archives. A ll specimens sent to sub-contract laboratories w ill be returned to the 3M Environmental laboratory upon completion o f analysis and submission o f the sub-contract ]aboratory(s) - fined report The specimens w ill be returned with the following documentation: the signed original chain o f custody and records o f storage conditions while at the sub-contract facility. . R eason: Clarification-ofthe disposition o f specimens and documentation for analyses performed by a sub-contract laboratory. '- 3M Environmental Laboratory ,,. Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 56 o f 92 Page 296 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994..... Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Amendment Approval Protocol FAC r-T O X t 11 Amendment No. 2 -----T & * __________________________ Marvin Case, D.V.M ., PED., Sponsor Representative /-J- / ate ' K osten J. Hansen, PED., Study Director S__ct- 11f ? D is ;-- 3M Environmental Laboratory Centre A nalyticalLaboratories, Inc: 3M Environmental Laboratory Page 57 of 92 Page 297 3M Medlcpl Department Study: T-6395.14 BACK TO MAIN A nalytical R eport: FACT T O X -111 L R N -U 2994. . Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 _ v Study Title ' A nalytical Laboratory Report on the Determination o f the Presence and Conceitmtfn t Potassium Perfluorooctanesulfonate (CAS Number 2759-39-3) in the Serum. Live- 3 ? } * ; Crl:CD*BR VAF/Pius* Rats Exposed to PFOS V ia Gavage `,m d U n n e o f PROTOCOL AMENDMENT NO. 3 - AmendmentDate: 20 January 2000 Performing Laboratories Urine Analyses . 3M Environmental Technology and Safety Services Fluorine Analytical Chetnistiy Team Building 2-3E-09 - 93S Bush Avenue St Paul, MN S5106. . Performing Laboratories Liver Analyses Battelle Memorial Institute J05 King Avenue Columbus, OH 43201-2693 ' Serum Analyses Advanced Bioanalydcal Services, IS Catherwood Road Ithaca, NY 14350 Laboratory ProjectIdentification ET&SS LRN-U2994 . ' FACTTX-11I ' Argus Stud: 418-015 . 3M M edical Department Study: T36295.14 . ExactCopyof Original . Ltt- w ltfo , Inttaf Date . 3M Environmental Laboratory ___ _ Centre Analytical Laboratories, Lie. . 3M Environmental Laboratory . Page 58 of 92 Page 298 BACK TO MAIN Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Protocol LRN-U2SS4 . ' Amendment Number 3 T h is am endm ent m o d ifies th e follow ing p ortion (s) o f th e protocol: 1. Pro toco l h ead s: . The study director for the p resen t study w a s identified In th e protocol a s Kristen J H ansen, Ph.D. '' A mend to read: The role o f study director for th e presen t study w as reassign ed to Marvin T. C a se D.V.M., P h.D .f a s o f 2 0 January 2 0 0 0 . T he previous study director, Kristen J. ' H ansen, h as b een rea ssig n ed to the role o f Principle Analytical Investigator R easo n : ' The rafe o f study director w a s rea ssig n ed in an effort to ensure com pliance with G ood Laboratory Practice Standards that outline study personnel requirements (refer to 21 C F R P a r t5 8 ).. _ .' 2. P ro to co l r e a d s: T he sp on sor for th e p resen t study w a s identified a s Marvin T. C a se, D.V.M. Ph D A mend to read : *' ' ' The role o f sp onsor fo r the p resen t stu d y w as reassigned to John L. Butenhoff, Ph.D ., a s o f 2 0 January 2 0 0 0 . R easo n : ' ' To ensu re that th e study director d o e s not a lso cany fire duties o f study sp on sor th e ,, sp onsor role w a s reassig n ed , in this m anner, personnel responsibilities and '' workload are m ore even ly balan ced . ' 3M Environmental Laboratory Cntre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 59 of 92 Page 299 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994- Centre Study No.:.023-017 Sponsor Protocol No: FACT-TOX-111 Amendment Approval Prctoccl LF.N-U2994 Amendment Number 3 John L Butenhoff, l 'A?.,-Sponsor Representative -sUtatru* t>\ D ate . Kristen J. Hansen, P kD ., Outgoing Study Director // - Sjt)- Z7777) D ate . / u i _______ _ _ _______________________________ Marvin T Case, D.V.M., PkD ., Incoming Study Director A vjj, ^ f L ^ Data 3M Environmental Laboratory Centre Analytical Laboratories, Inc. ' 3M`Environmental Laboratory ' Page 60 of 92 Page 300 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994-. . Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Study Title Oral (Gavage) Pharmacokinetic Recovery'Study o f PFOS in Rats PROTOCOL AMENDMENT NO. 4 Amendment Date: 20 April 2000 Performing Laboratories | Ur in e A n a l y s e s | 3M Environmental Technology and Safety Services Fluorine Analytical Chemistry Team Building 2-3E-09,935 Bush Avenue St. Paul, MN5510 | 323 H Centre A naiytialL aborat0riej|h^ 3048 Research Drive State College, Pa 16801 ' Battette Memorial Institute . 505 KingAvenue Columbus, OH 43201-2693 1 Serum A n alyses 1 Advanced Bloanalytical Services, IneT 15Catherwood Road Ithaca, NY 14850 . Laboratory Project Identification ET&SS LRN-U2994 . FACTTOX-111 : Argus Study: 413-015 ' . 3M M edical Department Study: T-629S.14 3M Environmental Laboratory Exact Copy of Original _ Lac. oH12Llea Initial Date Centre Analytical Laboratories, Inc. Laboratory Page 61 of 92 Page 301 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 . . ' . Protocol LRN-U2354 ' AmendmentNumber4 This amendment modifies the following portion(s) of the protocol: 1. P r o t o c o l heads: . The amended section 2.0 text states that this study is designed to determine PFOS in specimens of rat liver, serum, and m ine. . : A mend to read: . This study is designed to determine PFOS in specimens o f rat liver, serum, feces, and urine . R eason: ' ` The analysis o f fecal tissue for the target chemical and/orits analytes was added to th scooe o f the study M ow ing the issuance o f the protocol. Feces extraction and analytical methods were not validated and approved prior to protocol approval. . 2. Protocol reads: . The amended section 6.0 lists rat or rabbit liver, serum, and urine. A mend to reao: ' Add: tat or rabbit feces with a physical description o f rat or rabbit feces. R eason: Analysis o f fecal tissue for the target chemical and/or its analytes was added to the scope o f the study foJlowmg tbe issuance of the original protocol. 3. Protocol Reads: Section 13.1 lists all of the laboratories that w ill be conducting'analyses for this study A m end to read: . ' Add: Centre Analytical Laboratories, Inc., 3048 Research Drive, State Colle PA 16801 R eason: ' T' 1 Feces analyses were 3dded to the scope o f this study. The sub-contract laboratory performin'? analyses was not in the original protocol. 4. Protocol Reads: . . Sections 10.4 and 11.4 state that if the analyses are sub-contracted to other laboratories an am endm entwillbewrittentoindudethesem ethods. ' '. A mend to read: The feces extraction and analytical method used by Centre Analytical laboratories w ill be- 00M -023-0C3 (Revision 2), "Detennmarion ofFloorocbemcal Residues in Monkey/Rat ' Feces by LC/MS/MS." . . R ea so n : - The sub-contract laboratory performing feces analyses was added to the scope o f this study; this method was not validated and approved prior to protocol approval.' . 3 M E nvironm ental Laboratory Centre Analytical Laboratories, Inc. 3M-nvironmental Laboratory Page 62 of 92 Page 302 BACK TO MAIN 3M Medical Department Study: T-6395.14 A nalytical Report: FACT T O X -111 L R N -U 2994. - Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Amendment Approval P ro to c o l L B N -U S 994 Amendment Num ber 4 ?- John L Buienhoff, PLD.,'Soonsor Representative /Q/S'7 / 9f Za 3^5 Qale 1^.7" ^, v_____ ________________ IS At u 'I <L__ , Marvin T. Case, M M .. PkJD., Study Director ------- 3M Environmental Laboratory Centre Analytical Laboratories, Inc. 3M"Environmental Laboratory Pag 63 of 92 Page 303 3M Medical Departm ent Study: T -6395.14 BACK TO MAIN ! ' . `` Analytical Report: FACT TOX-111 L R N -U 2 9 9 4 ' Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Centre Analytical Laboratories, In<r. 304S Research Drive, Slate Caileje, PA 16301. Phooe; (S14) 2314032, Facsimile (314)231-1233 PROTOCOL DEVIATION ' Deviation Number 1 - DateofOccurrence: {1)7/12-13/00 . Centre Study Number 023-017 Centre Protocol Number FACT-TOX-111 . DESCRIPTION OF.DEVIATION 1. 1Z4 Page 7: Accepted recovery of 136% for FFOS for 0005799 Spk A in Set 07I200A. . ACTIONS TAKEN 1*- amendment issued. SOP revisionctc1. Protocol deviation issued. Recorded By/Date:'. 4L i& .w fig g /' 8 h lz ) IMPACT ON THE STUDY . !. No negative impact on the study because 0005801 Spk B in Set 071200A was acceptable. Principal Investigator Signature o Date 7-- j/'t Sponsor Representative Signature ` Date p . r a t QAlTReview b e t . f f v / r e February 1Z 199S/2 .Centre Analytical Laboratories, Inc. 3M-Environrnental Laboratory Page 64 of 92 Page 304 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 ...... Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Centre Analytical Laboratories, Inc. 3043 Research D rive*Stale College, PA 26501. Phone: (314) 231-8032, Facsimile: (814)231-1253 . PROTOCOL DEVIATION Deviation Number 2 . Q / <?I ' . Date of Occurrence: (1 & 2) entire study . Ceaire Study Number: 023-017 \Centre ProtccolNumber: FACT-TOX-U1 DESCRIPTION OF DEVIATION . I. 123 Page 7: Did not perform duplicate extractions on any of the samples for this study. 1 Amendment 2 Item #4: A method detection limit was not established for rat feces during the method validation.' ACTIONS TAKEN _ _ . . i - amendment issued. SOP revision, etc. 1&2 Protocol deviation issued. RecordedPrTnrv ^ ' 7 ^ -'ftu a .f f t / ------- (\ IMPACT ON THE STUDY. 1 4 1 No negative impact. . .RATIONALE . 1. Although none of the study samples were duplicated, at least one of the matrix fortifications (QC's) in each set was prepared to match one of the extrataed calibration standards and a precision of< 30 was observed. 1 Any sample that contained'residue .with - 3:1 signafcnoise was integrated and any number that was greater than 0 was reported. The limit of quaniration was established at 10 ppb during the method validation. Principal Investigator Signature ' ' Date SponsorRepresentative Signature ' `f/r /z a e * Date CAL QAU Review M k - M k z ' h Jus/O aA^L,'/' $ 3 A*t.*.* A Fcaruary 12,1998/2 Centre Analytical Laboratories, Inc. Page 65 o f 92 Page 305 3M Medical Department Study: T -6395.14 , BACK TO MAIN , Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 APPENDIX B Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS (Revision 2) (Method #00M-023-003, revision 2), Deviations and Modifications Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 66 of 92 Page 306 3M Mediqal Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 LRN-U2994.. . Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 '. TITLE . Determination o f Fluorocnemical Residues in Monkey/Rat Feces bv LCAiS vr't (Revision 2) . ' *K. AUTHORS Enaksha Wickremesinhe, Shaozhi Zheng, andJohn Flaherty PATE ISSUED June 02,2000 SPONSOR 3M Environmental Technology and Safety Services - Building 2-3E-09 . FOBox3333I . StPauI,M N 5Jl33-333I . PERFORMING LABORATORY . '. Centre Analytical Laboratories, Inc. (Centre) ; 3048 Research Drive State College, PA 16801 . CENTRE STUDY NUMBER ' 023-003 . , . CENTRE METHOD NUMBER 00M-O23-O03, revision 2 ' ' ' .- .' ' . TOTAL-NUMBER OF PACTS . 20 ' ' . . " .. : Centre Analytical Laboratories, Inc. . Page 67 of 92 Page 307 BACK TO MAIN 3M Mediqal Department Study: T-6395.14 Analytical Report: FACT TOX-111 ........... ....... L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 : Cram Method No.: 00M-Q23-C03, revision 2 . MANAGEMENT APPROVAL Tne work cited here was performed in conibnnance with aoolicable procedures and general GLP's. " ncara operating Eaafcsha WickremSinhe, PhJ). Principal Investigator Centre Analytical Laboratories, file Date ' -c z ^ c o p i --J Shaozhi Zheng Principal Investigator .' Centre Analytical Laboratories, Inc. o --OZ --o-O Date . a L tL d d L jT ' /io h n Flaherty / Laboratory Manager Centre Analytical Laboratories, Ina Date i-JuArtr-ie _t. nuftj. gp <S> . A ft -JU N S -C O Date . 4 r ' _____ ' / b/o Kris Hansen, PhJO, Group Leader _ Date oM Environmental Laboratory CentreAnalytical Laboratories, Inc. Study " 023-003 - Page 2 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 68 of 92 Page 308 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -111 L R N -U 2 9 9 4 ' Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 C sam Method No. : OOM-023-003, revmoa 2 TABLE OF CONTENTS TITLE PAGE.. ..... l MANAGEMENT APPROVAL- ..... 2 TABLE OF CONTENTS______ 1. SUMMARY__________________ ____ _______ 2. FLUOROCHEMICAL STANDARDS-------------- ..... 3. .......4 :.-- 4 3. 'CHEMICALS AND SUPPLIES______________ ....... 7 3.1. Chemicals____________ _-- i--------------3.2. FLUOROCHEMICALSTANDARDS---------------- ----- 7 ....... 7 3.3.. Equipment and Supplies------------ --------- ____ 7 3.4. Solutions______________________ _____ .8 3-5. Preparation of Stock, Forttficatton, and Calibration Solutions........ 9 3.5.1. Stock solutions--------------------------- ---------------------- ------------ " 9 3.5.2. Fortification Solutions------------- ------- ------------ ----------- ----------- 9 38 8 . Calibration Standards -- ............... ........ ................... ------- 10 4. METHOD--------------------------------------------------------------------------- " ------- II 4.1. Flow Diagram____ .....----------------------------------------- --------- ------ II 4.2. Sample Processing .... -------- 11 4.3. Sample Preparation.. ------- 11 4 8 J . Matrix Blanks------------------------- ------- 12 4.3.2. Matrix-Zero Blanks----------------4 8 8 . Calibration Standards----------- ...... 12--------------- 12--------------- 4 8 .4. Continuing Calibration Verification Standards... ------- 12 4 8 8 . QC Recovery Samples.------------------- ------------ ------ 12 4.4. Extraction....________ _-- ------------------- ------------ -----12 4.4.1 SPE Column Preparation-------- ----- ------------ .... ----- 13 4.4.2 Silanization o f Pear-Shaped Flasks -------- -------- .......13 ' 4.48 Standardization o f SPE Columns--- ---------- ---- .....------13 4 8 . AnalysisbyLC/MS/MS--------------------- ----------------- --------- 14 4 8 .1 . .LC/MS/MS System and Operating Conditions (Electrospray)_____ 14 4.5.Z Example Tune H ie ParametersTM......................... ......... ........... ....... ..... 55 4 8 8 . Calibration Procedures-- --------------.-- '-------------------------------- 48.4. Sample Analysis---------------------------------------------- 17 4.6. Performance Criteria-------------------------------------- ----------------- ;______ j g 4.7. Time Required for Analysis----------- -----------..IS 5. CALCULATIONS....'.-------.........--------------- ----------- ,TM..IS 5.1. Analyte Found__________________ ---------- ;-- is 5.2. QC Recovery_____ -- ---------------- 19 5.3. Mean Response Factor... 6. SAFETY______________ 1 ..... ------- TM .:.....i9 19 ATTACHMENT 1________________________________________________________ 0 CentreAnalytical Laboratories, Inc. Study 023-003 Page 3 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 69 of 92 Page 39 BACK TO MAIN 3M Medical Department Study. T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-'l11 1. SUMMARY CentreMethod No,: 00M-023-003, revisin 2 . Tins document details a method o f analysis for residues o f perfluorooctane sulfonate (PFOS), perfluorooctane sulfonylamidi (PFQSA), perfluorooctane sulfonylamirioethvl- acetate (PFOSAA), perfluorooetanesulfonylethylamide (PFOSEA), perfluoroocmnoate (POAA), 2(N-ethyIperfIuorooctanesul&nanado)-ethyi alcohol (Et-FOSE-OH) MS70 and MS56 in monkey feces. The chemical formulas o f the analyres are given in Section v o f this method. . " This method is being re-issued as method OOM-023-003 revision 2 . It is being revised to add the rat feces as a matrix, to document the use o f20-m l. vials instead o f 15 m i, tubes for sample extraction, to add the option o f homogenizing feces sam ples before weighing to clarify the preparation o f QC recoyety samples and to correct Some typographical errors in method 00M-O23-003 revision T. Rat feces is being added to the method however, it was validated for the analysis ofPFO S only. Therefore, this method should be used for'the analysis ofPFO S only, in rat feces. Residues o f these fluorochemicals are extracted from feces with acetonitrile (ACM) The ACM extract is filtered and passed through a carbon solid-phase extraction (SPE) column Approximately 3-4 drops o f l-octanol are added to the extract and evaporated to near dryness using rotary evaporation. Each extract is then reconstituted with methanol Quantification ofPFOS, PFOSA, PFOSAA, PFOSEA Et-FOSE-OH, M 556, M 570, and POAA is accomplished by liquid chromatographyAandem m ass spectrometry. (LC/MS/MS) analysis using selected reaction monitoring (SRM). ' The proposed limit o f quantitation (LOQ) for this method is 10 ppb each for PFOS PFOSA PFOSAA PFOSEA, Et-FOSE-OH, M556, M570, and P O A A This is based on 2. FLTOROCHEMICAL STANDARDS . Molecular structures o f PFOS. PFOSA PFOSAA PFOSEA Et-FOSE-OH, M 556, MS70, and POAA are given below. ' PFOS . Molecular weight 499 (CjF^SO,- ) O C fuS ~ 0 Chnaical Name =Veriloorooctanesulfonate . ' Note: The neutral molecule and standard form that ihe.PFOS (anion) is derived from is potassium perfluorooctane sulfonate [CjFjjSOjK], m olecular weight 53S. Csatre AnalyticalLaboratories, tec. Study # 023-003 .p . Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 70 o f 92 ' Page 310 BACK TO MAIN 3M Medicpl Department Study: T -6395.14 Analytical Report: F A C T T O X -1 11 ; LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Ciane Method No. : OOM-O12-O03, .--vision 2 PFOSA Molecularweight: 499 o CsftyS--NHa 0 Chemical Name * Psnluorooctaae suifonylamide PFO SA A Molecularweight: 5S5 Cari7S--N/ CHjCOOH \ CHjCH3 Chemical Name perfhiorooeone sulfonylamidoethylaeetaic PFOSEA . Molecular weight: 527 '^ir\S--/N H . 0Il N hjCHj ChemicalName Perfluorooeamesulftmylethylanride POAA Molecular weight: 413 . .. C?r 15C00'. ChemicalName " Perxtuojooctanoate Note: The neutral molecule and standard form that the POAA (anion) is derived from is ammonium perfluoroctanoate [CjFjjCOONHJ, m olecular w eight 431. Centre Analytical Laboratories, Inc. Study # CCe-003 Centre Analytical Laboratories, Inc. 3M E nvironm ental Laboratory ' Page 5 Page 71 of 92 Page 311 BACK TO MAIN 3M Medical Department Study: T-6395.14 A nalytical Report: FACT T O X -111 LR N -U 2994. . Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-11 Et-FOSE-OH Molecular weight: 571 oysijCH CH7S--N C a m Method No. : OOM-O23-0O3, revision 2 Chemical Name - 2<NHsthylperauorooctanesulfonanrido)-hy! aleohot M 556 Molecularw eight 557 I yCHzCOCH Cj t it S-- H 0a - \ h Chemical Name " M556 M 570 Molecular w eight 571 o II / CHjCOOH N C,rio~ \ CHj Chemical Name " M370 THPFOS Molecular weight: 428 . I-H, 1-H, 2-H, 2-H, C gF^SO jH . ' ' ChemicalName * 4-H, perfluoroactane sulfonic acid Castre Analytical horatories, Inc. Study# 023-003 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory ' Page6 Page 72 of 92 ' Page 312 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 ; C tsas MehgdNo, : 00M-O23-CO3, revision 2 3. CHEMICALS AND SUPPLIES 3.1. Chemicals -. Chemical Acetonitrile (ACN) Carbon 120/400 mesh Methanof(MeOH) 1-O ctan ol. Ammonium Acetate L-Aseorhic add Toluene Dimetyldichlorosilane Water Grade HPLC -' EPLC Reagent Reagent Reagent Reagent Reagent Type I Source (VW R)J.T. Baker Suoelco (VW Rjj.T. Baker Aldrich Chemical Sigma-Aldrich Sigma-Aidrich ' (VW R)J.T. Baker Suseico in house Catalog No, . _ JT90I7-3~~ S72I0- JT9093-2 111-87-5 A -7330 L-5960 JT946Q-3 3-3009 (Type I w ater electrical resistivity, minimum o f 16.67 MO/cm at 25"C &-- Labcanco wateipro workstation) 'a 3.2. Fluorochemical Standards Standard Source PFOS PFOSA 3M 3M PFOSAA ' 3M PFOSEA . 3M. Et-FOSE-OH 3M M55 . ' 3M M570 : 3M . POAA 3M THPFOS 3M : 3.3. Equipmentai Supplies . __________________ Equipment Balance, analytical, (display at least O.OOOlg) Balance, top loading, (display at least 0.01 g) Rotary evaporator ' - Rotary evaporator trap Pear-shaped flasks (125 mL). 20-mL SPE tubes (cat no. N057177) with frits Vacuum pump _ ' ' Visiprep vacuum manifold Centre Analytical Laboratories, Inc. Study# 023-003 - : ' S ou rn -----M ettiw-----M ettle Buchi* Buchi Pyrex Suoelco ' Sunelco '-'Pa Centre A nalytical Laboratories, foe. 3M Environmental Laboratory Page 73 o f 92 Page 313 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Study No.: 023-017 S p o n so r P ro to co l N o: F A C T -T O X -1 11 Centre MethodNo. : 00M-O23-C03, rvision 2 Wrist-action shaker . 20-mL polyethylene scintillation vials with lids 20-mL syringe 25-mm diameter glass fibre acrodisc (cat "4523) Disposablepipets, test tubes etc.' Disposable micropipets, 100 - 200 pL. 125-mLLDPE narrow-mouth battles 2-mL clear HPLC vial kit (cat # J1S1-3400) Standard lab equipment (class A pipets and volumetric flask, graduated cylinders, etc,) LC/MS/MS and HPLC systems ' __________________ Burrell Wheaton (VWR) VWR Geiman . VWR Naigene gp various A s described in*123456 Section 4.5.1 Notes: ' .' 1. In order to avoid contamination, the use o f disposable containezs/tubes/'pipets etc. is highly recommended. 2. Teflon or teflon-lined containers or equipment, includin' teflon-lined HPLC vial caps should not be used. = 3. Pear-shaped flasks are silanized before use. ' ' 4. It may be necessary to check the solvents (methanol) for the presence o f contaminants (especially POAA) by LC/MS/MS before use. Certain lot numbers have been found to be " unsuitableforuse.' - 5. Diaposable micropipets or pipets should be used to aliquot standard solutions, when preparing standards and samples for extraction. . 6. Equivalent materials may be substituted for those specified in dais method. However, the use o f carbon from Supelco is strongly recommended. . *. 3.4. Solutions ' , . 1- 50 mM.Ammonium Acetate: D issolve 3.S5 g o f ammonium ^ in 1 L o f type I water. 2. 2 mM Ammonium Acetate: Dilute 40 mL o f the 100 tnM acetate solution in a liter o f type I water, for m obile phase A.'- 3. Saturated Ascorbic Acid in Methanol: D issolve - 10 g ascorbic acid in 100 mL methanol. '' ' 4. The 2 %-Ascorbic Acid in Methanol: D issolve 2 g ascorbic add in 100 mL methanol. - ' Note: The volumes shown are provided for guidance; alternative volum es . ' may be prepared. . Centre Analytical Laboratories, Inc.Study# 023-003 Centre Analytical Laboratories, Inc. . 3M Environmental Laboratory .. Page 74 of 92 Page 314 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 LRN-U29.94._~ Centre Study No.: 023-017 SponsorProtocol No: FACT-TOX-111 '- '. Csam Method No.: 00M-023-W3, visioa 2 3 .5 , Pr eparatio n o f S to c k , Fortification, a n d Calibr a tio n S o l u t io n s _1 Analytical standards are used for two pinposes: (1) to fortify control matrix samples designated to be extracted and processed as calibration standards that w ill be then used to calibrate the response o f the detector used in the anaivsis- and f '1 to fortify other control matrix samples to determine analytical rseovtsy. ' * The absolute volumes o f the standards may be varied by the analyst as Ion as the correct proportions o f solute to solvent are maintained. The solutions cited below are given as an example; alternative concentrations may be prepared-ifneeded ' 3,5.1. Stock solutions '. To prepare stock solutions o f 100 pg'mL each PFOS, PFOSA, PFOSAA. PFOSEA, Et-FOSE-OH, M556, M570, POAA, and the surrogate standard THPFOS, weigh out 10 mg o f analytical standard (corrected for purify and percent salt, if necessary) r.\d dilute to 100 mL with methanol in a 100-mL volumetric flask. Prepare a separate solution for each analyte. These stock solutions (in 125-mL LDPE bottles) are to be stored in a refiigeiator at 2C to 6C and are stable for a maximum period o f 6 months fiom the date o f . preparation. - . 3.5.2. Fortification Solutions . ' . a. 10 pg/mL Mixed Fortification Solution - Pipet 10.0 mL each o f PFOS PFOSA, PFOSAA, PFOSEA, Et-FOSE-OH, M556, M570, and POAA stock solution to a 100 mL volumetric flask. Bring up to volume with. methanoL ' . b .' 2.5 jigteL Mixed Fortification Solution-- Pip 25.0 mL o f the 10 uglmL mixed fortification solution to a 100-mL volumetric flask and bring un to volume with methanol. . ' c. 0.5 pg/mL Mixed Fortification Solution - P ip 20.0 mL o f the 2.5 psfajL mixed fortification solution to a 100-mL volumetric flask and bring up to . vo'lmne with methanoL . . d. 0.1 pg/mL Muted Fortification Solution-Pipet 20.0 mL o f the 0,5 jig/mL mixed fortification solution to a 100-mL volumetric flask and bring uo to volume with methanol. ;* Follow Steps a and b above to make a 25 pg/mL solution o f the . standard (THPFOS) from the stock solution.' Centre Analytical Laboratories, luc. Study# 023-003 * Page 9 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 75 of 92 Page 315 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 ... Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Caere MethodNo.: 00M-023-C03, revision 2 Store all fortification standard solutions (in 125-mL LDPE bottles) in a refrigerator at 2C to 6C for a maximum period o f 6 months from the date o f preparation. ' 3.5.3. Calibration Standards Prepare LC/MS/MS calibration standards by fordoing weighed out aliquots o f the same control feces sample (or a combined `bulk" control feces sample) beforeJhey are processed through the extraction procedure. . '1 The following is a typical example; additional concentrations may be prepared as.needed. It is recommended to prepare the fortification solutions so that the volume o f fortification is between 50 and 200 pL and to se discosable micropipets for aliquoting fortification volumes. ` .' 'Cone, o f Fortification Mixed ' Volume (pL) Fortification Weight o f ' Control Sample (g) Solution . (pgfod) NA 0.1 0.1 0.5 0.5 2.5 2-5 10 NA 1.0 100 1.0 200 1.0 100 1.0 200 1.0 100 1.0 200 1.0 100 1.0 * 2.5 pg/ml TBPFOS fortification solution. Cone, o f Voi. o f Extracted Surrogate Calibration Standard* Standard ' added (pL) (PPb) NA 200 10 200 20 200 -50 200 100 200 250 . 200 500 200 1000 200 Calibration standard preparations may be stored in a refrigerator at 2C to 6C, if needed, and used over a period o f time as long as its stability has been verified: '' Centre Analytical Laboratories, lac. Study tf 023-003 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 10 Page 76 of 92 Page 316 BACK TO MAIN 3M Mediqal Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 CentreMethodNo. : OOM-023-003, revision 2 4. . METHOD 4 .1 . Flow D iag ram The flow diagram o f the method is given heiow, followed bv ; descripdon o f each step. ' *ta:id Method Flow Diagram Weigh 1.0 g o f sample (fortify samples designated as calibration standards and QC recoveries) . 4* ` l Extract with ACN and filter 4 4. Carbon Solid-Phase Extraction 4 4 Concentrate to almost dryness ' '4 4. Adjust final volume (2.0 mL) 4' ' 4 j.OMS/MS . 4.2-, Sa m ple Processing All samples are received frozen and w ill bekept frozen (below -I0C ) until tim o f extraction. The rat feces do not need any processing, however, the monk * feces may need to be.homogenized in order to be able to weigh out'the suecifi*^ amounts. . ^ m ea 4 .3 . Sa m ple Preparation Prepare the following blank and calibration samples (Sections 4.3 .1 to 4.3 4} same control feces sample (or a combined "buik" control feces sample) ' m . Centre Analytical Laboratories, lac. Study# DZ3-003 . - .. . ' Page 11 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 77 o f 92 Page 317 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 LRN-U2994 - Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 ; Cram MethodNo.: 00M-023-C03, revision 2 4.3.1. M atrix B lanks : Prepare two Wank saxonies without any fortifications, per set o f saxnnl;- analyzed. These w ill be used as the manic blank. ' 4 3 .1 M a n x Zero Blanks Prepare two matrix blank samples fortified with the surrogate, per set o f samples analyzed. These will be used as the matrix zero blanks. 4.3.3. Calibration Standards Prepare calibration standards as described in Section 3-5.3. 4.3.4. Continuing Calibration Verification Standards ' Two of the extracted calibration standards, a low-mid and a mid-high level standard, w ill be used as continuing calibration verification (CCV) standards 4.3.5. OC Recovery Samples Prepare QC recovery samples to represent every control matrix included in the study. Prepare at least one QC recovery sample in the low-range, and one in the high-range, to approximately bracket the expected range o f analyte in the samples. For sets containing more than 20 samples, prepare an additional QC recovery sample for every group o f 10 additional samples (forexample, i f one set has 24 ssmples_then.3 levels of QC recovery samples wifi be prepared). Prepare these additional QC recovery samples to bracket the range o f analyte found in the sampies,.as appropriate. . Note: An example o f a sample extraction worksheet is Attachment 1. as 4 .4 ,'E x is a c h o n = - Note: A. PreparationfconditiomngofSPE columns is described in Section 4.4.1. B. Silanizattoa o f pear-shaped flasksis described in Section 4.41. * ' ' C Evaluation/standardizadon ofSPE columns (when a different supplier- is used etc.) is described in Section 4.4.3. I* a. Weigh approximately 1.0 g ( 0.05 g) o f sample in to 20 mL polyethylene scintillation vial (Note: designated samples need to be fortified with appropriate aliquots o f fortification standards). b. Add 10 mL o f ACN (note: break-up any dumps using a spatula or glass rod) . shake on a wrist-action shaker for 30 minutes. ' .' - -Centre Analyrica Laboratories, lac. Sftrdyi!'033*iX13 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory ' Pag 78 o f 92 Page 318 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 .... Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 ' . , Ccsac MeAod No.: OOM-OZJ.fi, revision 2 c. Let .he vials sir for - 5 minutes, carefully-decant, d irec tly on to a 20 mL disposable syringe barrel connected to a glass acroaisc (Gehnan) fiite- d. Transfer the filtrate to a conditioned S?E column (see Section 4.4. n Collect the elute in a I25-mL "siianized"(ses Section 4.4.2) pear-shaped flask ^ Note: The 20 mL SPE column fits well inside the mouth (sle*ve) o f th pear-shaped flask. Make sure that the pear-shaoed fiilc s w en . supported and will not topple. No vacuum is needed for this step e. Add 10 mL o f ACN and then 20 ml o f 90:10 ACN : 2% ascorbic acid in MeOH to the SPE column and collect the eluate in to the same Dear shi k flask, combining the eluates. f. Add 3-4 drop o f 1-octanol to the pear shaped flask and rotary evaoorate to near dtyness (a minute amount o f 1-octanol w ill remain) at a redaced oressur of-40C . *' 3 . Reconstitute by adding 2.0 mL methanol (final volum e= 2 mL) and swirl/m ix to dissolve. Transfer the extract to HPLC vials using disposable pipets 4.4.1 SPE Column Preparation . Pack 20-mL SPE tubes with 2 g carbon. Condition columns with 10 mL o f saturated ascorbic acid in MeOH followed by 10 mL ACM. Discard all washes. Do not allow the column to dry. Note: The SPE columns may be packed and conditioned at any point Use the vacuum manifold to condition the columns. * " Do not let the SPE columns to run dry at any time.' 4.4.2 SUanizadon o f Pear-Shaped F lasks ' Silanize the pear-shaped flasks before use, by rinsing with a 30% dintethvl- dichlbrosilane in toluene solution followed by a toluene rinse and finally a methanol rinse. ' 4.4.3 Standard& tion o fSPE Columns .' ' ' .. When using carbon from a different supplier, SPE columns shouid be : standardized in the following manner, prior.to analyzing samples: ' a. Using a mixed standard with a concentration between 100 and 500 ngr'mL (each o f all eight analytes) follow the elution schemes as outlined in ste-'s 2 and 3. Collect all eluting fractions. .' " Centre Analytical Laboratories, Inc. Study#023-003 .2 ,, Centre Analytical Laboratories, Inc. 3M Environmental Laboratory . Page 79 of 92 . Page 319 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 A nalytical R eport: FACT T O X -111 L R N -U 2994 - Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 - C am Mutbed No.: OOM-023-003, revision 2 b. Collect a post-elution fraction, after step 3, eluting with an additional ?n mL o f 90:10 (AC N: 2% ascorbic acid in MeOH). " c. A rtist the final volume o f all the fractions to 10 mL with methanol d. Analyze all the fractions fay LC/MS/MS. ' e. If the target fraction' contains a minimum o f 85% o f the resoe--' analytes, it may be considered acceptable. e If the "post-elation" fraction contains significant amount o f analvtes the target elution volume may be increased. ** ' 4.5. A nalysis b y LC/MS/MS 4.5.1. LC /M S/M S System an d Operating C onditions (Electrospray) Mass Spec: ' Micromass Quattro Ultima (Micromass) ' Interface: Electrospray (Micromass) ' Harvard infusion pump Computer: Software: COMPAQ Professional Workstation AP200 Windows NT, MassLynx 3.3 HPLC: ' Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP utosampler HP Column Oven Note: A 4 x 10 mm hypercarb drop-in guard cartridge (Keystone, part # ' 844017-400) is attached on-line after the purge valve and before the*samole injectorport to trap any residue contaminants that may be in the mobile p W and/or HPLC system. _ HPLC Column: Genesis C, (Jones Chromatography),2.1 mmx.50mm, 4p Column Temp.: 35 C ' Injection Voi.: 10 pL M obile Phase (A):2 mM ,tam onium Acetate in Type water M obile Phase (B): Methanol Time %A 0 60 - 0.4 60 . 1-0 7.0 10 10 7.3 0 9.0 o 9.5 6 I3 J 60 14.0 . 60 %B 40 - 40 90 90 100 100 40 40 40 Plow Rate tm /mini 0.3C0 0.3QQ 0300 0300 ' 0.300 : 0,400 ' 0.400 0.400 : 0300 Centre AiuEyricat Laboratories, Inc. Study # 023-003 . Page 14 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory . Page 80 o f 92 Page 320 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 ,. - Analytical Report: F A C T T O X -1 11 s LRN-U2994 ' . . Centre Study No.: 023-017 . . Sponsor Protocol No: FACT-TOX-111 . . . CentreMethcdNo.: OOM-023.003, revision! It may be necessary to adjust the HPLC. gradient in order to ootimia instrument performance. Columns with different dimensions (e. o f " 30 mm) and also columns from different manufacturers (Kevstone Be* etc.) canbe usedprovided equivalent chromatographyis obtained. " " Ions monitored: Analvte Mode PFOS negative PFOS ` negative PFOSAA n a tiv e PFOSEA negative Et-FOSE-OH negative `POAA negative M556 negative M570 negative THPFOS negative Parent Ion Product Ian 499 99 498 78 584 526 ' 526 169 630 59 413 369 556 498 570 419 427 80 Approximate 53 60 57 67 6.7 ** 5I 54 ' 56 5.1 " ' Note that the retention times may vary slightly, on a dav-to-dav basis depending on the batch o fmobile phase, etc. '* 4.5.2. Exam ple Time F ile Parameters . The M ow ing values are provided as an example. Actual values may v from instrument to instrument. A lso these values may be changed from tin f to time in order to optimize for greatest sensitivity. The mass spectrometer is tuned using a solution o f each analyte' at -0 5 - pgfmL, prepared via dilution o f the stock solution in methanol. The solution is infused (using a T cc::-..tor) at 10 pLtein into a 0.2 mL/min stream o f mobile phase consisting o f 40% methanol and 60% 2 mM The analytes are initially tuned for the parent ion and then hny-d for (he- production. The optimized parameters are saved as a "tune file" This tune file is then used duringroutine analysis. - ' Analvte PFOS PFSA PFOSAA PFOSEA Et-FOSE-OH POA M556 M570 THPFOS Dwell (s 02 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Centre Analytical Laboratories, Inc. Study* 023-003 Collision E nerw (VIA 43 23 . . 20 28 . 31 ' 11 28 . -20 35. ConefVT 76 34 37 49 30 25 50 60 ' 34 ' . Page 15 Centre A nalytical Laboratories, Inc. 3M Environmental Laboratory Page'81 o f 92 Page 321 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 LRN-U2994 - . Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Csatre Method Mo. : OOM-023-003, revision 2 ' ' Source Capillary Hexanole 1 Aperture I Hexapcle 2 SourceBlock Temp. DesoivanunTemp. ' Set 2.56 kV 0.5 V 0.2 V 0.8 V 100C 400C * Analyzer LM Resl HMR.es 1 . TFnergy 1 ' Entrance Exit LM Res2 HMRes2 IEnergy 2 Multiplier ' . Set 13.0 V 13.0 V . 0.7V -2 V ' IV 11.0 V 11.0 V 1.0 V 650 V Gas Flows Cone Gas Desolvathin Set - 150L/hr . ~700L /hr Pressures . Gas Cell Read back --3.0e-3 mbar Note: An alternative LC/MS/MS system may be used once >txm'T.^ nitrd to beequivalent. 4.5J . Calibration Procedures. ' t ' . a. Inject an aliquot (10 jtL) o f each calibration standard into the LC/M S/M S system. Include standards corresponding to six or more concentration levels (tanging from the LOQ to the highest concentration level to be . included in the analysis) in an analytical set. V b. U se a linear y = mx + b function for quantification. Linear standard '. curves are generated for each analyte by linear regression using i/x ' . weighting ofpeak area versus calibration standard concentration using the Windows NT, MassLynx 3 3 (or equivalent) software system . A ny * . extracted standards that fall outside the 70 to 130%, based on the' average response factor o f the surrogate standard, must be excluded from the calibration curve. _ Centre Analytical Laboratories. lac. Studyit 023-003- . Page 16 Centre A nalytical Laboratories, Inc. 3M Environmental Laboratory Page 82 o f 92 Page 322 BACK TO MAIN 3M Medicai Department Study. T-6395.14 Analytical Report: F A C T T O X -1 11 LRN-U2994 . Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Caare Metiad No. : 00M-023-O03, revision 2 c. T ie correlation eoem eieat (R) for calibration carves generated must be 0.98 (Rz0.96). I f calibration results fail outside these limits take appropriate steps to adjust instrument operation, and reanalyze the relevant .sets o fsamples. '' 4.5.4.- Sam pleA nalysis . a. Each set o f samples analyzed must include matrix blanks (without surrogate standard), matrise zero blanks (with the surrogate standard) a set o f extracted standards, and QC recovery samples. ' b. Inject an aliquot (10 jiL) o f each standard/sampI&'QC lecdvery/cantrol into the LCZMS/MS system. Begin a set o f samples with an entire set o f calibrations. Include two continuing calibration verification (CCV) standards (one from the low-mid and one ftom the mid-high range) after every 5 to 10 samples, atthe m ost ' "3 c. Determine the concentration o f each sample/QC recovety/control ftom the standard curve, based on the peak area o f each analyte. T ig standard responses must bracket responses o f the residue found in each sanrale set. Samples that fell outside the range o f the standard curve must be "diluted appropriately and re-analyzed. d. Evaluate the ongoing acceptability o f instrumental analysis M r i on the . CCV results. Samples analyzed w ill be acceptable as long as both CCV's fell within 30% o f their tree value. Calculate the CCV found using the linear calibration curve. If the CCV recovey iS outside 70% to 130%, re-analyze samples analyzed after th last acceptable rW fr e. Monitor the response' o f the surrogate standard (SS) for every samnle, extracted standard, blank, and QC recovery sample. The response is calculated using the "average response factor? function using MassLynx 3.3 (or .equivalent). Evaluate the acceptability o f the sample extraction process based bn the concentration o f the SS found in all extracted standards, QC samples, blanks, and samples. The average response factor for all .samples, blanks, extracted standards, and QC recovery samples must fall between 60% to 140% ( 40%). Those deviating by greater than 30% (outside 70% W130% range) must be checked for possible errors and may need to be re-extracted. Any responses deviating by greater than 40% (falling outside the 60% to 140% range) w ill require re-extraction. f. I f analysis is delayed, samples must be stored reftigerated at approximately 2C to 6'C until analysis, and analyzed preferably within i ' week. . Centre Analytical Laboratories, lac.-Study# 023-003 Page 17 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 83 o f 92 Page 323 BACK TO MAIN 3M Medicpl Department Study. T-6395.14 Analytical Report: FA C T T O X -1 11 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 tetre MethodNo.: 0OM-OZ3-O03, revision 2 4.6. Performance Criteria . The following two criteria Should be met before the initial analysis o f samles especially when using different instrumentation set-ups than those cited in'this* method. . First Criterion - Rim a standard solution on LC/MS/MS corresponding to the estimated LOQ (the 10 ppo extracted standard) and obtain a signal to noise ratio ',o f at least-9:l for all analytes. I f this criterion cannot be met, optimize and change instrument operating parameters. Second Criterion - Run a set o f standards o f six or more concentration levis ranging from the proposed LOQ up to the highest concentration, level to be included in the analysis. Generate a calibration curve for each analyte and obtain a linearregression with acorrelation coefficient o f at least 0.98 for each analyte 4.7. T im e R e q u ir e d f o r A n a l y s is A set o f 14 samples can b: taken through the extraction procedure in approximately six hours by one person. The LC/MS/MS analysis (12-14 standards and 14 samples) w ill take approximately seven hours. 5. CALCULATIONS . 5 .1 . Analyte Found Calculate the amount o f analyte found (in ppo) using the standard curve generated by the Mass Lynx software program using Equation.!. Correction for sample . weight (1 g) and final volume (2 mL) is not required since the samples and standards areextracted the same way. ' Equation!: (peak area -intercept) xDF Analyte found (ppb) - . slope Where DF= dilution factor, if samples were diluted. Centre Analytical Laboratories, Inc-StudyS023*003 ' Page 13 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 84 of 92 Page 324 BACK TO MAIN 3M Medicpl Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 C a m Method No. : OQM-023-003, revision 2 5.2. Q C R e c o v e r y Fcao.lrcus.laatme. pthleespeforcretinfiterdecowviethry kf.rnoomwE_nqaumat.oiounn_2ts. o f analJytes eD"r`iuerr to extraction, Equation 2: % Recovery = amount analyteadded (ppfa) 5.3. Mea n Response Facto r . The mean response factor (MRF) is calculated by the Mass Lynx softw arprogram by averaging the mean response for all calibration standards (response/amount added) and dividing by the amount added. The actual concentration from each injection is then calculated by dividing' the individualresponse by the MRF. The recovery for each injection is the percent o f the actual concentration to the amount added. . This calculation is performed by the' MassLynx 33 (or equivalent) software. 6. SAFETY A ll samples must be treated as potential biohazards and appropriate universal precautions must be taken (fellow standard operating procedures for the handling o f bioflnids). These include, but are not lim ited to, the use o f double layers o f latex or vinyl gloves, goggles, dust mask, and lab coat A ll disposable materials must be treated as biohazards and handled accordingly. - Acetonitrile, methanol, dimethyldichlorosilane and toluene are hih lv flammable. Open and use under fitme hood only. ' ~* A il organic waste and solvent waste must be segregated and disoosed o f : accordingly: '` Centre Analytical Laboratories, lee. Stray It023-003 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory /. Page 85 of 92 Page 325 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 . LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-U1 ATTACHMENT 1. C at Moilod Mo. : OOM.023^03, revision 2 je S5X C e n tr e A n a ly tic a l L a b o r a to r ie s. Inc 304* H o ra e *D riv e , S U *Cacjt. ? A 0 1 - P a e (IU )Z 3 M 0 J 2 HISPAItATtQNo r SAMPLES FOR EXTAACTIOXANOANALYSIS innoeolNa: NA , CoOTSMdrN S H S MirricFem Method Nc^ N A A n a ly te : P F 0 S . FF O S A - P O M . E<-FO S-O H.P FO S EA . P F O < u ..M . - Q u S p o rn Sam ple) C o u re Sam ple ID 1 ID S am ple D esm pm * 2ssftx~3m&ar TOKCST TTCSSZ W e ij& t (n/mL)I t ) " "tt F o rt. V o L (p U landa ID C ou. FoCaLV)oL Fon. Lve] (p p b ) uiI"Mam* acro Wan* 327" "SET" "StHT" aiflj " S E IT " -rar I ~rr irr ~33r "E" "W "rar "rar "Tiw "750" "rar "255""" "r a r Tirar F o rtifie d w tic _ _ C ap acity; Jm riaU /D m : /y OsS a p it u RM janqtta. n w r unie tn /w ? * m *jarnjicanot steaon. r e n i t arrows i* a oe tum x u ix it w ^ Sam ples rem oved fta m A te a r * __________ ' S a m p ic j o r w e jjh fd on B alance I ,, P a m p ig re tu rn e d to fa c e r * " Surrogate StdID: S a n p ta ttre te d w ich A O f and litte re d : C arbon SPS de an -op aad ra to v a p ; F in a l vo lu m e adjusted w 2 m L w nh jn e d o o o l: fc tn e i pla ced in re tn 'x n a tn ra ______ T n te In m a li/D a t T im e * la itia li/D tte T im e In ia k /P a lc In itia li/th x e _ In itia is /C o tc _ Iniisib/Dare^ tn m a ls /D a tt: l_ / / BUM A m m iri) 0> a A s4 b( C u to ftttO H M Aw atM cAcU M e * * t-O cnaal W is : TIh m U .I Ci2tre Analytical Lateralones, Inc. Study # 073*003 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 20. Page 86 o f 92 Page 326 3M Medicpl Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 3<WSResearch Drive,Stale Collese, PAISSO!. Phone: (814) 231-8032, Facsimile (814)231.] METHOD DEVIATION Deviation Number. 1 Date of Occurrence: (1)7/7/00 Page ! of l Centre Study Number 023-017 Sponsor Protocol Number FACT-TOX-111 DESCRIPTION OF DEVIATION Deviations to method tided "Determination of Fiuorochemical Residues in Monkey/Rat Fec-s bv LC/MS/MS (Revision 2)" (Centre Method 0OM-O23-0O3, revision 2) "' 1. Section 4.4.a: Only 0.78 g was weighed for Centre sample ID 0005822 because that was all of the sample available. ACITONSTAKEN i.e.. amendment issued. SOP revision, etc... 1. Method deviation issued. Recorded Bv/Date: ^ ^ : ! ^ ) ^ &lt((D _________ " ' A IMPACT ON THE STUDY I. No negative impact Pnaapai Investigator Signature ' t i n t *'DO Date Centre QAU Review ' _______ February 12. 1998/2 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 87 f 92 Page 327 3M Medicpl Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 ' Sponsor Protocol No: FACT-TOX-111 Centre Analytics! Laboratories, Inc. 3048 Research Drive,Stale CoOege, PA 168(11. Phone (814) 231-8032, Facsimile: (814)23M2S3 METHOD DEVIATION DeviationNumber 2 . Date ofOccurrence: (1)7/19/00 Page 1of ? I Centre Study Number: 023-017 SponsorProtocol Number FACT-T0X-U1 DESCRIPTION OF DEVIATION Deviations to method titled "Detennination of Fluorochemtcal Residues in Monkey/Rat Feces by LC/MS/MS (Revision 2)" (Centre Method OOM-023-003, revision 2) ' 1. Section 4.5.4.C : Quantitated Centre sample ID 0005890 residue outside the range of the standard curve in Set 077-1SOOAD.1 ACTIONS TAKEN i !. amendment issued. SOP revision, etc... 1. Method deviation issili Recorded By/Date, IMPACT ONTHE STUDY . ' 1. No negative impact because sample response only -1.1% higher than response of highest. standard. ' Principal Investigator Signature r/'S/eo Date Centre QAUReview A/I 5' I 'I J a n Frtnmyll. 1998/2 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 88 o f 92 Page 328 3M Medlcpl Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 LRISI-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 ' Centre Analytical Laboratories, Inc. 2043 Research Drive,SUIe College. PA 16301. Phone: (314) 231-3032, Facsimile: (S14)23M2S3 METHOD DEVIATION Deviation Humber: 3 lo fi " Dale o f Occurrence: (1)7/10-20/00 Centre StudyNumber: 023-017 SponsorProtocol Number: FACT-T0X-U1 DESCRIPTION OF DEVIATION Deviations to method titled "Determination ofHuorochetnicai Residues in Monkey/Rat Feces by LOMS/MS (Revision-2)" (Centre Method OOM-023-003. revision 2) 1. Section 4.3-5: Did not fortify any of the control samples from intervals Day 6/7 Gestation, Day 14/15 Gestation, Day 20/21 Gestation, or Day 21/22 Lactation. actio ns ta ken . ' I f - amendment issued. SOPrevision, eie-, 1. Method deviation issued- Recorded B N2 - * !\ w ' IMPACT ONTHESTUDY ' 1. No negative impact. ' '- Principal Investigator Signature P fW a o Date Centre QAUReview k/X ,L , f t ie t) Febnarv 12.1993/2 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 89 of 92 Page 329 3M Medicpl Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Centre Analytical Laboratories, Inc. 3048 Research Drive. State College. PA HS80I. Phom ; fS1412313032. Facsimile; <814^221-1153 METHOD M ODIFICATION FORM fest l o f I 4 Modification No.: 1 Study Director: Marvin T. Case Centre Study Nnmfyi- 023-017 Sponsor Protocol Number: FACT-TOX-111 Method Titig DeterminationofFltiorochenucal Residues in Monkey/Rat Feces by LC/MS/MS (Revision 2) Centre Method Number: Q0M-023-003, revision 2 MODlFTCATinNSection 3.4 Solutions X The 100 mM ammonium acetate solution should be SOmM ammounium acetate. JUSTIFICATION? . To correct for a typographical ecror. EFFECT ON STUDY:. No negative impact. Orignator/^cUA.Lj''^ 0 Principal Investigator: Sponsor Management: . rh jco rfifc o Date - Date ' February 12. I99S/3 Centre Analytical-Laboratories, Inc. 3M Environmental Laboratory Page 90 of 92 Page 330 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -111 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 3043 Research C ave,State Caliere, ?A 16801. Phones (814)231-8032, Facsimile: (814)231.1153 METHOD MODIFICATION FORM Modification No.: 2 . Study Director: Marvin T. Case ' Centre Study Nnrflfrrr 023-017 Sponsor Protocol Number: FACT-TOX-H1 Method Title; Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS (Revision 21) Centre Method Number: QQM-023-003. revision 2 MODIFICATION: !~ ~ ~~ ~ 1. Section 4.5.4. Sample Analysis (Item e.) Should read as follows: ' Monitor the response of the surrogate standard (SS) for every sample, extracted standard, blank, and QC recovery sample. The concentration is calculated using the "average response factor" function fromMassLynx 3 3 software (or equivalent). Evaluate the acceptability of the m pi. extraction process based on the concentration of the SS found in all extracted standards, QC samples, blanks, and samples. The percent recovery for all samples, blanks, extracted standards, ' andQC recovery samples must fall between 60% and 140%. Those deviating by greater than 30% must be checked for possible errors and may need extracted. Any recoveries deviating by greater than 40% (failing outside the 60% to 140% range) will require re-extraction, 2: Section 5.1 Analyte Found Remove second sentence. 3. Section 5.1 Equations Modify Equation I to: ' " Analyte found (ug/mL)'= (Peak area - intercept! slope - Change Equation 2 to: Analyte found (ppb) = fanalvte found fng/mU x final vol. tmD x DF) sample w t (g) ' Add Equation 3: Recovery (%)=> . - [analyte found (ng/mL)s< final voL(mL)xDF] ;cjqq analyte added (ng) 4. Section 53 Mean Response Factor ' Modify first sentence to read: . The mean response factor (MRF) is calculated by the MassLynx software program by averaging . the response for all calibration standards and dividing by the amount added. Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 91 o f 92 Page 331 3M Medlcpl Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 Centre Analytics! Laboratories, Inc. 304* Research Drive, Stale CoBese, PA KSO!. Phone: <S14)231.S032, Faaim ite: (841231-1I3 METHOD MODIFICATION FORM Pass 2 of 2 Modification No.: 2 Study Director: Marvin T. Case Centre Study Number 023-017 Sponsor Protocol Number: FACT-TOX-UI Method Titiff- Dtermination of Fluorochemical Residues in Monkey/Ra't Feces by LOMS/MS '_______ (Revision 21 Centr Method Number: QOM-023-003. revision 2 _________ IUST1HCATION: ' ' 1. To clarity SS acceptable criteria. , 2. Remove incorrect statement because calculations did include sample weight and final volume. 3. Modify calculations to correspond with those used in study. 4. Clarify calculation of mean resuonse factor _____________ 1 .____________ EFFECT ON STUDY: 1-4. No negative impact. x 4Originator: O \ .... 1y| Principal Investigator 0 . Sponsor-Management: / * ICo/gD Date Date Date .0 ^ , February 2, 1998/3 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 92 of 92 Page 332 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: F A C T T O X -1 11 ---------------------- CBKI-U2Sa4 3048 Research D rive, State College, PA 18801. in e. Phone: (814) 231-8032, Facsim ile: (814)231-1253 12 Amended pages + report amendment ANALYTICAL PHASE REPORT AMENDMENT RE-ISSUED Amendment Number: I Effective Date: 9/20/00 Centre Study Number: 023-017 Sponsor Protocol Number: FAGT-TOX-111 Report Title Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats Amended Section Section 1.0 Summary: 13548.2 ppb was changed to 13.5 pg/g in the third paragraph (See attached page 11). Section 7.6 Quantitation and Example Calculation: Equation 5: Residue Found (pg/g) = Residue Found (ng/g) * (1 pg / 1000 ng) Equation 6: Corrected Residue Found (pg/g) = Residue Found (pg/g) * 0.869 These equations were only applied to the tables in the report. Section 8.0 Results: 13548.2 was changed to 13.5 pg/g in the second paragraph (See attached page 18). Section 11.0: Tables I - XVI (See attached pages 20 - 28) Reason for Amendment ' T he units reported in the tables and text were changed from ng/g to pg/g, the am ount o f significant figures represented in the tables and text was changed to three, the zeros reported in Table I were changed to ND's, and any value less than 0.0100 pg/g (LOQ) was reported as < 0.0100. No negative impact Impact on the Study Fet'- PJ4fjri/i PrinciippaaQhnjystigator Signature* Study Director Signature JC - C -D i Date J 5 d ^ ..Q x)... Date * R ick G razzini signin g for Enaksha W ickrem esinhe who is no longer em ployed at Centre. CAL QAU Review USAs it,ju)vn November 1995/0 3M Environmental Laboratory Page 333 3M Medical Departm ent Study: T -6395.14 Appendix H: Certificate of Analysis BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 3M Environmental Laboratory Page 334 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 r paBk. Centre Analutical Laboratories. Inc. 3048 Research Drive Phone: (814) 231-8032 State College, PA 16801 Fax: (814) 231-1253 or (814) 231-1580 IN T E R IM C E R TIFIC A TE O F A N A LY S IS Revision 1(9/7/00) C entre A nalytical L aboratories COA Reference #: 023-018B 3M Product: P F O S ,L otl71 Reference #: SD-009 Purity: 86.4% Specifications _________ _________________ Result Purity1 V .M - 86.4% Appearance W hite Crystalline Powder Conforms Identification NMR M etals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium2 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (L C /M S) Total % Impurity (GC/MS) ' . .`t; ------------------ ?-'--------- T':'- ' ' ----------------- --------- ----- . .: " ' . /....... _ 77 "Tv.. . : ; : Positive 1. 0.017 w t./w t.% 2. 0.007 wt./wt.% 3. 1.355 wt./wt.% 4. 6.552 wt./wt.% 5. 0.003 w t/w t.% 6. 0.004 wt./wt.% 7. <0.001 w t/w t.% 1.00 w t/w t.% 10.60 w t/w t.% None Detected Related Compounds POAA Residual Solvents (TGA) Purity by DSC 0.30 w t/w t.% None Detected N ot Applicable^ Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. N itrite 6. Phosphate 7. Sulfate4 - ' ^ . .-.r'*:' ' m . y -:. 1. <0.015 w t/w t.% 2. 0.27 w t/w t.% 3. <0.040 w t/w t.% 4. <0.009 w t/w t.% 5. <0.006 w t/w t.% 6. <0.007 w t/w t.% 7. 8.82 w t/w t.% Organic A cids5(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elem ental Analysis": 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine ............ ' 1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% 1. <0.1 w t/w t.% 2. <0.1 w t./w t% 3. <0.1 w t/w t.% 4. <0.25 w t/w t.% 1. 12.08 w t/w t.% 2. 0.794 w t/w t.% 3. 1.61 w t/w t.% 4. 10.1 w t7w t.% 5. 50.4 w t/w t.% COA023-018B Page 1 o f3 3M Environmental Laboratory Page 335 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 r IN T E R IM C E R T IF IC A T E O F A N A LY S IS Centre Analytical Laboratories COAReference #: 023-018B Date o f Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 'Purity = 100% - (sum o f metal impurities, 1.39% +LC/MS impurities, 10.60%+Inorganic Fluoride, 0.27%+NMR impurities, 1.00%+POAA, 0.30%) Total impurity from all tests = 13.56% Purity = 100% - 13.56% = 86.4% 2Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials o f low purity. No endotherm was observed for this sample. 4Sulfur in the sample appears to be converted to SO4and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4is not considered an impurity.S6 STFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropentanoic acid Pentafluoropropanoic acid 6Theoretical value calculations based on the empirical formula, C8FnS03X + (MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 C F R 160). c. COA023-018B 3M Environmental Laboratory Page 2 o f 3 Page 336 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Analytical Laboratories, Inc. 3048 Research Drive Phone: (814) 231-8032 State College, PA 16801 Fax: (814) 231-1253 or (814) 231-1580 IN T E R IM C E R TIF IC A TE O F A N A LY S IS C entre A nalytical L aboratories COA Reference #: 023-018B LC/MS Purity Profile: Im purity C4 C5 C6 C7 T o tal w t./w t. % 1.03 1.56 6.38 1.63 10.60 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average response factors from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves. c: Prepared By: D a m S. Bell Date Scientist, Centre'Aniilytical Laboratories /f Reviewed By: CXk fll _____________ W/f& /John Flaherty ' D ate / Laboratory Manager, Centre Analytical Laboratories c. . . COA023-018B Page 3 o f 3 3M Environmental Laboratory Page 337 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Analytical Laboratories, Inc. 3048 Research Drive Phone: (814) 231-8032 State College, PA 16801 Fax: (814) 231-1253 or (814) 231-1580 ( IN T E R IM C E R T IF IC A T E O F A N A LY S IS Revision 1(9/7/00) C entre A nalytical L aboratories COA Reference #: 023-018A 3M Product: PFO S,L ot217 Reference #: SD-018 ____________________ P urity; 86.9%______ Test Name Specifications Result P u rity J 86.9% Appearance Identification NMR Metals (ICP/MS) White Crystalline Powder ; :;:v: ~'i . ;`' . . . . . . .. ... Conforms Positive 1. Calcium 2. Magnesium /. . 1. 0.005 w t./w t.% 2. 0.001 wt./wt.% 3. Sodium 4. Potassium2 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) 3. 1.439 wt./w t.% 4. 6.849 wt./wt.% 5. <0.001 wt./w t.% ' '' 6. 0.005 wt./wt.% . . i ... 7. <0.001 wt./wt.% 1.93 wt./wt.% Total % Impurity (L C /M S) 8.41 wt./wt.% Total % Impurity (GC/MS) None Detected .' Related Compounds - POAA Residual Solvents (TGA) ______________________ ' ' .v;.'. .. . 0.33 wt./wt.% None Detected Purity by DSC N ot Applicable4 Inorganic Anions (IC) . . . ; . ; '' 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate4 Organic A cid s5(IC) . ' T': - ;-v.. : :: : 1^ ' - . ' 1 1. <0.015 w t./w t.% 2. 0.59 wtVwt.% 3. <0.040 wt./wt.% 4. <0.009 wt./wt.% 5. <0.006 wt./wt.% 6. <0.007 wt./wt.% 7. 8.76 wt./wt.% 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis': 1. <0.1 w t./w t.% 2. <0.1 wt./wt.% 3. 0.10 wt./wt.% 4. 0.28 w t/w t.% 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur e 5. Fluorine 1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% 1. 12.48 w t./w t% 2. 0.244 w t./w t% 3. 1.74w t/w L% 4. 8.84 wt./wt.% 5. 54.1 wt./wt.% CO A 023-018A Page 1 o f3 3M"Environrrrerrtal Laboratory Page 338 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 r IN T E R IM C E R T IF IC A T E O F A N A LY S IS Centre Analytical Laboratories COA Reference #: 023-018A Date o f Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 'Purity = 100% - (sum o f metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.93%+organic acid impurities, 0.38%+POAA, 0.33% ) Total impurity from all tests = 13.09% Purity = 100% - 13.09% = 86.9% 2Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials of low purity. No endotherm was observed for this sample. 4Sulfur in the sample appears to be converted to SO4and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4is not considered an impurity.S6 STFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonofluoroperitanoic acid Pentafluoropropanoic acid 6Theoretical value calculations based on the empirical formula, CsFnSOs'K4' (MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). (. COA023-018A I Laboratory Page 2 o f 3 Page 339 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Centre Analytical Laboratories, Inc. 3048 Research Drive Phone: (814) 231-8032 State College, PA 16801 Fax: (814) 231-1253 or (814) 231-1580 IN T E R IM C E R TIFIC A TE O F A N A LY S IS Centre Analytical Laboratories COA Reference #: 023-018A LC/MS Purity Profile: Im purity C4 C5 C6 C7 T o ta l w t./wt. % 1.22 1.33 4.72 1.14 8.41 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average response factors from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves. / Prepared By: bavid S. Bell Date Scientist, Centre Analytical Laboratories // Reviewed By: f7 J *, fit /Jo h n Flaherty 9 Date Laboratory Manager, Centre Analytical Laboratories L COA023-018A Page 3 o f3 3M Environmental Laboratory Page 340 3M Medical Department Study: T-6395.14 Appendix I: Report Signature Page BACK TO MAIN Analytical Report: F A C T T O X -1 11 L R N -U 2 9 9 4 Marvin T. Case, D.M.V., Ph.D., Study Director / h j U f __ 0^~u / ?Date __________________________________ o .r ^ 6 3 y / William K. Reagen, Ph.D., Laboratory Manager Date J? John L. Butenhoff, Ph.D., Sponsor Representative --J - 4 * Kris J. Hansen, Ph.D., Analytical Investigator Date '/O V IO I Date 3M Environmental Laboratory Page 341
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