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3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT T O X -110 LRN-U2849 Study Title Oral (Gavage) Pharmacokinetic Study of PFOS in Rats Analytical Laboratory Report Title Determination of the Presence and Concentration of Perfluorooctanesulfonate (PFOS) in Serum, Liver, Urine, and Feces Samples Data Requirement Not Applicable A u th o r 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 43201-2693 Serum Analyses Advanced Bioanalytical Services, Inc. 15 Catherwood Road Ithaca, NY 14850 Project Identification 3M Medical Department Study: T-6295.12 Argus In-Life Study: #418-013 Analytical Report: FACT TOX-110 3M Laboratory Request No. U2849 Total Number o f Pages 354 3M Environmental Laboratory Page 1 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTOX-110 LRN-U2849 This page has been reserved for specific country requirements. 3M [Environmental Laboratory Page 2 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTOX-110 LRN-U2849 GLP Compliance Statement Analytical Laboratory Report Title: Determination of the Presence and Concentration of Perfluorooctanesulfonate (PFOS) in Serum, Liver, Urine and Feces Samples Study Identification Numbers:T-6295.12, FACT TOX-110, LRN-U2849 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. Exceptions to GLP compliance: Separate study directors were assigned to lead the in vivo and analytical portions of the study. Some sample receipt data from specimens received on 29 January 1999 are missing the corresponding receipt acknowledgement forms The study files do not contain the raw data documenting sample receipt for the Gestation Day 0 urine samples. The original protocol called for study compliance with the FDA final rule 21 CFR Part 58; however, documentation from Advanced Bioanalytical Services, Inc. (the contract lab responsible for serum analyses) cites compliance with the EPA final rule 40 CFR Part 792. Dose confirmation analyses were not performed in compliance with GLP regulations. Sample WeightsA/olumes worksheets for extraction, TOX-110, pages 2, 5, 9 and 11, on 9/13/99, are not completely attributable to the analyst performing the extraction. Some of the "Argus Acknowledgement of Tissue Specimen Receipt" forms are missing the date and "reviewed by" signature. Sample receipt documents for the 21 Dec98 samples are not available. The LC/MS instrument run information for the 20Jan99 samples, received on 1/22/99, are not available. Not all reagent and solution bottles were labeled with an expiration date. The analytical report from Advanced Bioanalytical Services, Inc. does not include the names of all of the participating scientists and professionals. The stability of test and control articles under the conditions of administration were not determined. (Signatures on next page) 3M [Environmental Laboratory Page 3 3M Medical Department Study: T-6295.12 Marvin T.' Case, D.M.V., Ph.D., Study Director Kris J. Hansen, Ph.D., Analytical Investigator William Reagen, Ph.D., Laboratory Manager BACK TO MAIN Analytical Report: FACT TO X-110 LRN-U2849 V A */ Datif 6S-/cbjjo Date Date 3M Environmental Laboratory Page 4 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTOX-110 LRN-U2849 GLP Study--Quality Assurance Statement Analytical Laboratory Report Title: Determination of the Presence and Concentration of Perfluorooctanesulfonate (PFOS) in Serum, Liver, Urine and Feces Samples Study Identification Numbers: T-6295.12, FACTTOX-110, LRN-U2849 This study 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 14 Sept 1999 Extraction 4 Oct 1999 4 Oct 1999 5 Oct 1999 Analytical Set-up 13 Oct 1999 13 Oct 1999 6 Oct 1999 MS Data Reduction 13 Oct 1999 13 Oct 1999 13 Oct 1999 to 16 Dec 1999 First Draft Report 17 Jan 2000 17 Jan 2000 6 Sept 2000 to 8 Sept 2000, 11 Sept 2000 to 12 Sept 2000, ' ' 18 Sept 2000, 20 Sept 2000 to 22 Sept 2000, 25 Sept 2000 to 26 Sept 2000 Second Draft Report 27 Sept 2000 27 Sept 2000 See contractor's analytical reports (Appendix G) for contract lab Quality Assurance statements. Lj 5>C<^J ^ N /o t Date 3M Environmental Laboratory Page 5 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTOX-110 LRN-U2849 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........................................................................................................12 Specimen Receipt and Maintenance................................................................................... 12 Chemical Characterization...................................................................................................13 Procurement.................................................................................................................... 14 Stability Studies...............................................................................................................14 Dose Confirmation Analyses.......................................................................................... 14 Method Summaries.............................................................................................................. 14 3M Environmental Laboratory......................................................................................... 15 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.......................................................................................... 19 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................................................................. 55 ETS-8-96.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Urine for Analysis Using HPLS-Electrospray/Mass Spectrometry/Mass Spectrometry," (14 pages)............................................................. 56 ETS-8-97.0, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrospray/Mass Spectrometry/Mass Spectrometry," (10 pages)............................................................. 70 Appendix D: Data Summary Tables.....................................................................................80 Appendix E: Data Spreadsheets..........................................................................................83 Appendix F: Example Calculations.......................................................................................97 Appendix G: Contract Lab Reports.......................................................................................98 Battelle Memorial Institute, "Oral (Gavage) Pharmacokinetic Study of PFOS in Rats," 73 pages......................................................................................................................... 99 Advanced Bioanalytical Services, Inc., "Method Validation for the Quantitation of Perfluorooctanesulfonate (PFOS) in Rat Serum by Turbo Ion Spray LC/MS," 50 pages................................................................... ......................................................172 3M Environmental Laboratory Page 6 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTOX-110 LRN-U2849 Advanced Bioanalytical Services, Inc., "Quantitative Determination of Perfluorooctanesulfonate (PFOS) in Rat Serum from Study #FAC T-TOX-110 Using Turbo Ion Spray LC/MS," 24 pages................................................................................ 214 Advanced Bioanalytical Services, Inc., "Addendum--Quantitative Determination of Perfluorooctanesulfonate (PFOS) in Rat Serum from Study #FACT-TOX-110 Using Turbo Ion Spray LC/MS," 3 pages.................................................................................. 239 Centre Analytical Laboratories, Inc., "Oral (Gavage) Pharmacokinetic Study of PFOS in Rats," 103 pages.........................................................................................................243 Appendix H: Interim Certificate of Analyses....................................................................... 347 Appendix I: Report Signature Page..................................................................................... 354 3M (Environmental Laboratory Page 7 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTOX-110 LRN-U2849 List of Tables Table 1. Female Rat Population Demographics for Study #418-013................................. 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-6295.12 via Gavage........................... 13 Table 3. Characterization of Test Article............................................................................. 14 Table 4. Negative Ions Monitored in 3M Laboratory Analyses...........................................16 Table 5. Description of the Control Matrices Used for Liver, Urine, Sera and Feces Analyses in Study FACT TOX-110......................................................................... 20 Table 6. Characterization of the Analytical Reference Substances used for Liver, Urine, Sera and Feces Analyses in Study FACT TOX-110..............................................20 Table 7. Dose Confirmation Analyses for Perfluorooctanesulfonate for Test Samples From Study In-life #418-013-- 15 November 1998...............................................21 Table 8. Deviation Summary for FACT TOX-110...............................................................22 Table 9. Average Results for the Analyses of Serum 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-6295.12 via Gavage................................................................................................................... 80 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-6295.12 via Gavage.................................................................................................................... 80 Table 11. Average Results for the Analysis 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-6295.12 via Gavage.................................................................................................................... 81 Table 12. Average Results for the Analysis of Feces Samples in the Study of the Determination o f the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.12 via Gavage.................................................................................................................... 81 Table 13. LOQ Values Used inFACT TOX-110 Analyses.................................................. 82 Table 14. 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/PlusRats Exposed to T-6295.12 via Gavage.............................83 Table 14. 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-6295.12 via Gavage (Continued)....... 84 Table 14. 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-6295.12 via Gavage (Continued)....... 85 3M (Environmental Laboratory Page 8 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTOX-110 LRN-U2849 Table 15. Results of Analyses of Serum 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-6295.12 via Gavage.......................86 Table 15. Results of Analyses of Serum 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-6295.12 via Gavage (Continued)....87 Table 15. Results of Analyses of Serum 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-6295.12 via Gavage (Continued)....88 Table 15. Results of Analyses of Serum 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-6295.12 via Gavage (Continued)....89 Table 15. Results of Analyses of Serum 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-6295.12 via Gavage (Continued)....90 Table 16. 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-6295.12 via Gavage........................... 91 Table 16. 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-6295.12 via Gavage (Continued)....... 92 Table 16. 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-6295.12 via Gavage (Continued)....... 93 Table 17. Results of 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-6295.12 via Gavage........................... 94 Table 17. Results of 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-6295.12 via Gavage (Continued)....... 95 Table 17. Results of 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-6295.12 via Gavage (Continued)....... 96 3M [Environmental Laboratory Page 9 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTOX-110 LRN-U2849 Study Personnel and Contributors Study Director Sponsor Marvin T. Case, D.V.M., Ph.D. 3M Corporate Toxicology 3M Center, Building 220-2E-02 St. Paul, MN 55133-3220 (651)733-5180 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 Liver Analyses 3M Environmental Laboratory Battelle Memorial Institute Kristen J. Hansen, Ph.D., Analytical Investigator Jon C. Andre, Ph.D., Analytical Investigator Serum Analyses Advanced Bioanalytical Services, Inc. Dave Anderson, M.S., Analytical Investigator Feces Analyses Centre Analytical Laboratories Enaksha Wickremesinhe, Ph.D., Anal}/tical Investigator 3M Environmental Laboratory Contributing Personnel David R. Barnidge* Lisa A. Clemen Kelly J. Dorweiler* Mark E. Ellefspn Sara E. Estes* Barb A. Gramenz* Sarah A. Heimdal* Cari S. Hewitt* ^ Marlene M. Heying* Contract lab professional service employees Location of Archives Harold O. Johnson Theresa King-Hunter* Kelly J. Kuehlwein* Sally A. Linda* Joseph C. Pilon* Scott R. Post* Ian A. Smith* Anh-Dao Vo* Bob W. Wynne* 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 from Battelle Memorial Institute, Centre Analytical Laboratories and Advanced Bioanalytical Services, Inc. (ABS) will be stored at the contract laboratories in such a manner that the control substances will be preserved for use in ensuing studies. 3M [Environmental Laboratory Page 10 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTOX-110 LRN-U2849 Introduction and Purpose The purpose of the study is to determine the presence and concentration of perfluorooctanesulfonate (PFOS) in serum, liver, urine and feces samples taken from the Gavage Study of T-6295.12 (Perfluorooctanesulfonate CAS Number: 2795-39-3) in Crl:CDBR VAF/Plus Rats. The study was initiated on 10 June 1999 and completed on 8 November 1999. Test System The test system species and strain selected was the Crl:CDBR VAF/Plus (SpragueDawley) rat received from Charles River Laboratories, nc., permanently identified using Monel self-piercing ear tags. F0 virgin female rats were at least 60 days of age and weighed approximately 200-225 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. Five groups consisting of 16 female rats each (total of 80 rats) were used as the test system. All female rats were treated with the control or test article once daily beginning 42 days prior to cohabitation, and continued through day 14 or day 20 of presumed gestation. Table 1 presents the number of female rats in each group. Group I consisted of control F0 female rats that were administered 0.0 mg/kg/day in 0.5% Tween 80 (vehicle). Group II through Group V F0 female rats were administered 0.1, 0.4, 1.6, or 3.2 mg of PFOS per kg of body weight/day in 0.5% Tween 80. At predetermined intervals during and at the end of the in-life phase of the study, sera, liver, urine and feces samples were taken from dams for analyses. Additionally, liver and serum samples were taken from the fetuses. Details of the in-life phase of the present study are presented in the Argus Laboratory final report, "Oral (Gavage) Pharmacokinetic Study of PFOS in Rats, #418-013." Table 1. Female Rat Population Demographics for Study #418-013 Population Total Selected for Study Initial Quantity Acclimated (Total) Control Group I 0 mg/kg/day Treatment Group (total) Group II 0.1 mg/kg/day Group III 0.4 mg/kg/day Group IV 1.6 mg/kg/day Group V 3.2 mg/kg/day 120 16 64 16 16 16 16 80 16 64 16 16 16 16 3M [Environmental Laboratory Page 11 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FA C TTO X -110 LRN-U2849 Specimen Collection In the analytical study reported here, 822 serum, urine, liver and feces specimens were collected from adult female rats before mating and at gestation day (GD) 7, GD 15 and GD 21 and sent to the 3M Environmental Laboratory and the contract labs to be analyzed for PFOS. A total of 54 pooled serum and liver specimens were collected from fetuses. Specimens of pooled embryo/placenta, pooled liver/lung, pooled liver/placenta/liver-lungs, amniotic fluid, and carcasses from fetuses, as well as milk secreting glands and placentas from adult females, were collected by and received from Argus In-life Study #418-013, but were not a part of the scope of this study as determined by the study director. The number and type of specimens collected for analyses in the analytical phase of this study are presented below. Specimens Collected from Study Groups 1 through 5 (through 1/22/99): Serum Specimens--277 specimens Liver Specimens-- 100 specimens Urine Specimens--249 specimens Feces Specimens--250 specimens Blood specimens were separated by refrigerated centrifugation. Serum specimens were then harvested, immediately frozen on dry ice and maintained frozen at -70C or below until being shipped to the 3M Environmental Laboratory. Urine and feces samples were collected into centrifuge tubes, placed on dry ice and stored frozen at -70C or below until being shipped to the 3M Environmental Laboratory. Liver specimens collected from each animal were collected, frozen and then stored at -70C or below on dry ice until being shipped to the 3M Environmental Laboratory. All specimens were shipped to the 3M Environmental Laboratory frozen and on dry ice. Specimens were then shipped to the contract analytical laboratories for analysis. Urine samples were extracted beginning on 13 September 1999 using an ion-pairing reagent and methyl-terf-butyl ether (MtBE). Sample extracts were analyzed using highpressure 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. Specimen Receipt and Maintenance The 3M Environmental Laboratory received serum, liver, urine and feces specimens that were collected during premating, GD7, GD15 and GD21 of the in-life phase of this study #418-013 from Argus. All specimens were received frozen on dry ice and were immediately transferred to storage at -20C 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 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTOX-110 LRN-U2849 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-6295.12 via Gavage Tissue/Sample Generation Number of Samples Received Date Received Date Shipped to Lab Liver Serum Urine Feces Liver Serum Dam Dam Dam Dam Fetal (pooled) Fetal (pooled) 73 250 249 250 27 27 3 February 1999 2 February 1999 29 January 1999 30 January 1999 2 February 1999 29 January 1999 2 February 1999 3 February 1999 2 February 1999 19 August 1999 15 June 1999 a 25 May 2000 19 August 1999 15 June 1999 a 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 and are presented in Appendix A (see Table 6). Chemical Characterization Potassium Perfluorooctanesulfonate (KPFOS) CAS Number: 2795-39-3 Chemical Formula: C8F17S 0 3'K+ Molecular Weight: 537.9 Chemical characterization information on potassium perfluorooctanesulfonate, CAS #2795-39-3, is presented in Table 3. 3M [Environmental Laboratory Page 13 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTOX-110 LRN-U2849 Table 3. Characterization of Test Article Test Article Chemical Name KPFOS Potassium Perfluorooctanesulfbnate Source 3M Toxicology Services, Medical Department Expiration Date 8/31/01 Storage Conditions Frozen <-10C Chemical Lot # 217 Physical Description W hite crystalline powder Purity 86.9% 8 The test article is defined as the substance gavaged to the test system. See Interim Certificate of Analysis in Appendix H for complete characterization information. Procurement Potassium perfiuorooctanesulfonate was obtained in one lot (Lot Number #217) from 3M Toxicology Services. Stability Studies The stability of potassium perfiuorooctanesulfonate was not determined. Dose Confirmation Analyses Dose confirmation analyses were performed on test article samples (0 mg/mL to 0.64 mg/mL) collected on 15 November 1998 during the in-life phase of the study. The results are presented in Appendix A (see Table 8). The dose confirmation data were collected according to a method that was not fully validated. Dose confirmation was performed by diluting the dose samples with methanol into the linear range of the instrument. In all cases, samples were analyzed versus an unextracted calibration curve using HPLC-ES/MS/MS. Method Summaries Following is a brief description of the methods used during this analytical study by the 3M Environmental Laboratory and by contract labs. The methods used in this study are located in Appendix 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 lab reports (see Appendix G). 3M (Environmental Laboratory Page 14 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FA C TTO X -110 LRN-U2849 3M Environmental Laboratory Preparatory Method ETS-8-96.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Urine for Analysis Using HPLS-Electrospray/Mass Spectrometry/Mass Spectrometry" This method is used to extract PFOS from urine by the use of an ion-pairing reagent and methyl-tert-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-cc 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 HPLCES/MS/MS. For example, molecular ion 499, selected as the primary ion for PFOS (C8F17S03-) analysis, was fragmented further to produce ion 99 (F S 0 3-). The characteristic product ion 99 was monitored for quantitative analysis. 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 3M [Environmental Laboratory Page 15 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FAC TTO X-110 LRN-U2849 Analytical Equipment The 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 110(3 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: (min) 0.0 1.0 4.5 6.5 7.0 %B 40% B 40%B 95% B 95% B 40% B Mass Spectrometer: Micromass API/Mass Spectrometer Quattro IITMTriple Quadrupole system Software: Mass LynxTM 3.1 Cone Voltage: 50-70 V Collision Gas Energy: 30-50 eV Mode: Electrospray Negative Source Block Temperature: 125C-150C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (MRM) Table 4. Negative Ions Monitored in 3M Laboratory Analyses Target Analyte Primary Ion (a m u ) Product Ion (a m u ) PFOS 499.0 99.0 THPFOS 427.0 80.0 3M Environmental Laboratory Page 16 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FA C TTO X -110 LRN-U2849 Deviations 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 quantitation 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) were indicated in the protocol for this study: Linearity: The coefficient of determination (r2) equal to or greater than 0.98 Lim its o f 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 o f 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-110 (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 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, Analytical Methods). 3M Environmental Laboratory Page 17 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FA C TTO X -110 LRN-U2849 Lim its of Quantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibration curve defined as a standard within 30% of the theoretical value. (See Appendix D, Table 12.) Blanks: All blanks were below the lower limit of quantitation for the compounds of interest. Precision: 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 54-109% of expected values were achieved for 12 of 14 matrix spikes prepared in urine. The remaining two spikes were recovered at 46% and 39%. Low recoveries were confirmed, but were not re-extracted. Associated results, which may be underestimated by more than 50%, are specifically identified in the report tables as not meeting the data quality objectives of this study. Surrogates: The surrogate (THPFOS) was added to all samples and standards. External calibration was used in quantitation of data in this study due to the presence of an unidentified compound that interfered with the analysis of the surrogate. 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 contract lab reports in Appendix G for summary of quality control analysis results from contract labs. 3M Environmental Laboratory Page 18 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FA C TTO X -110 LRN-U2849 Summary of Sample 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 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 purity factor, see Appendix E. Centre Analytical Laboratories-- Refer to the Centre Analytical Report for information regarding feces analysis (Appendix G). Samples from Control Animals: Low levels of PFOS were often detected in the sera, urine, feces and liver of the control animals. These levels were significantly lower than those found in the low dose test animals. Samples from Dosed Animals: In general, PFOS levels found in the sera, urine, feces and liver of the test animals increased with each dose group within a particular time point. 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-110. Statement of Conclusion Under the conditions of the present studies, PFOS was observed iri the livers, urine, feces and sera of all female rats dosed with the T-629i.12 during the in-life phase of the study. Additionally, PFOS was observed in fetal liver and serum taken during gestation from the same group of female rats. 3M hnvironmental Laboratory Page 19 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTOX-110 LRN-U2849 Appendix A: Control Matrices and Dose Confirmation Analyses Table 5. Description of the Control Matrices Used for Liver, Urine, Sera and Feces Analyses in Study FACT TOX-110 Location of Analyses Control Matrix 3M Environmental Laboratory Human urine Battelle Memorial Institute Rat liver ABS Rat serum Centre Analytical Rat feces Source Expiration Date Storage Conditions Lot # Physical Description N/R--not recorded Lampire Biological 3 months Harlan Bioproducts for Science, Inc. 210 days Frozen at -20C10C Frozen at ~ -20C 66-11066 Human urine; no identity analyses performed Harlan Product #409 Rat liver; no identity analyses performed Harlan Bioproducts for Science, Inc. N/R Polypropylene vials frozen at -20C R80422 R80128 Rat serum; no identity analyses performed Lampire Biological N/R Frozen at<-10C N/R Rat feces; no identity analyses performed Table 6. Characterization of the Analytical Reference Substances used for Liver, Urine, Sera and Feces Analyses in Study FACT TOX-110 3M Environm ental Laboratory Battelle Mem orial Institute Advanced Bioanalytical Services Centre Analytical Laboratory Chem ical Name Source Expiration Date KPFOS Potassium Perfluorooctanesulfonate* 3M ICP/PCP Division KPFOS Potassium Perfluorooctanesulfonate* 3M ICP/PCP Division KPFOS Potassium Perfluorooctanesulfonate* 3M ICP/PCP Division KPFOS Potassium Perfluorooctanesulfonate* 3M Toxicology Services, Medical Department 8/31/01 8/31/01 8/31/01 8/31/01 S to ra g e C o n d itio n s Frozen <-10C Frozen <-10C Frozen < -10C Frozen <-10C Chem ical 171 171 171 217 L o t# Physical Description W hite crystalline powder W hite crystalline powder W hite crystalline powder Purity 86.4% 86.4% 86.4% *The reference standard used for analysis is KPFOS, but the target analyte is PFOS. W hite crystalline powder 86.9% 3M hnvironmental Laboratory Page 20 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTOX-110 LRN-U2849 Table 7. Dose Confirmation Analyses for Perfluorooctanesulfonate for Test Samples From Study In-life #418-013--15 November 1998 G roup Dose Target Conc. PFOS (ng/mL) Expected Conc. PFOS (ng/mL)a M e a s u r e d PFOS C o n c . (ng/mL)b % of Expected G roup 1 Control 0.0 mg/kg/day Diluted 1/1 0.00 0.00 0.00 G roup 2 0.1 mg/kg/day 0.02 mg/mL Diluted 1/40 20000 16100 14000 G roup 3 0.4 mg/kg/day 0.08 mg/mL Diluted 1/1000 80000 64500 61100 G roup4 1.6 mg/kg/day 0.32 mg/mL Diluted 1/1000 320000 258000 246000 Group5 3.2 mg/kg/day 0.64 mg/mL Diluted 1/1000 640000 516000 417000 NA=not applicable "Calculations used to obtain Expected Concentration PFOS are given below bMeasured PFOS Concentration values are corrected using the PFOS correction factor and for purity NA 87% 95% 96% 81% Formula used to calculate expected concentration From target concentration: Target Concentration x PFOS Purity x PFOS Correction Factor = Expected Concentration PFOS PFOS Purity = 86.9% PFOS Correction Factor = 0.9275 640000 ng/mL x 0.869 x 0.9275 = 516000 ng/mL Expected Concentration 3M [Environmental Laboratory Page 21 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Appendix B: Protocol, Protocol Amendments and Deviation Summary Table 8. Deviation Summary for FACT TOX-110 Deviation Date(s) of Occurrence The original protocol states that the source of the control matrix used in the present study would be either Sigma Chemicals or Argus Research Laboratories. The physical description of the control substance was rat and/or rabbit liver and/or serum. Additional control substances used in this study were human urine and rat feces obtained from Lampire Biological Laboratories; Piperville, PA 18947. Entiie study ETS-8-96.0 states 2.0 mL will be used as the initial volume. Samples 19516F, F0, G1, control-GDO premating had only 1.0 mL available. 9/14/99 Samples 19572F, F0, G5-3.2 MKD-G7, 24 hour had no sample available. Samples 19543F, F0, G3-0.4 MKDG D21,2 4 hour did not have enough sample for extracting. Initial volumes were recorded in the Excel spreadsheet for calculating final concentrations. ETS-8-96.0 specifies to add 0.5 mL methanol final volume. For the following samples, 1.0 mL of methanol final volume was added. 19540F, F0, G3-0.4MKD-GD15-Terminal 19542F, F0, G3-0.4MKD-GD15-Terminal 19543F, F0, G3-0.4MKD-GD15-Predose 19544F, F0, G3-0.4MKD-GD15-Terminal 19546F, F0, G3-0.4MKD-GD15-Predose 9/14/99 9/15/99 ETS-8-97.0, section 10.1.1 states that the methanol (water and extraction) blanks will be analyzed with each analytical batch. The water and extraction blanks were not analyzed I during the following runs: 092099A and 092199a. 9/20/99, 9/21/99 The analysis o f 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. 9/21/99 ETS-8-97.0 specifies the method of analysis requires the use o f an internal calibration. The actual analyses used an external calibration method. 9/20/99, 9/21/99, 9/24/99, 9/27/99, 9/30/9S, 10/28/99, 1-/5/99 The control of bias document requires the calibration curve to have percent deviations <30%. The actual percent deviation for the low calibration point is greater than 30%. 9/27/99 Impact on Study Successful validation studies using human urine and rat feces were performed; therefore, there was no adverse impact on the quality of the data. The LOQ was maintained for all samples; therefore, there was no adverse impact on the outcome of the study. Data quality will not be affected. No data were generated. The LOQ was maintained for all samples; therefore, there was no adverse impact on the outcome of the study. All blanks were analyzed at least once during the study. Methanol blanks are often analyzed more frequently to monitor possible contamination, but since the study data here were well above any analyte levels in the blanks, there was no adverse impact on the quality of the data. The analysis of Tween samples was not conducted under GLPs (as per discussions with study director). This deficiency is included in the final report. Unknown contaminant interfered with use of surrogate for internal calibration. Corrective action is an improvement. Data quality is satisfactory to 50% accuracy. The data points are at the mid range portion of the curve, so deviation at the low end curve points will not adversely affect the data. 3M [Environmental Laboratory Page 22 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTOX-110 LRN-U2849 Table 8. Deviation Summary for FACT TOX-110 Deviation Date(s) of Occurrence Protocol FACT-TOX-110 and the method ETS-8-97.0 state the matrix spike/matrix spike duplicate analyses will be 30% of the spiked concentration. The actual acceptable result for the matrix spike/matrix spike duplicate analyses will be 50% of the spiked concentration. 11/4/99 Impact on Study Data quality will be expressed as having as accuracy o f 50% rather than 30%. 3M [Environmental Laboratory Page 23 3M Medical Department Study: T-6295.12 3M Environmental Technology and Services PO Box 33331 St. Paul, MN 55133-3331 612 778 6442 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Protocol #FACT-TOX-110 3M Study Title Oral (Gavage) Pharmacokinetic Stu dy o f 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-110 U2849 3M Environmental Laboratory 3M Environmental Laboratory Page 1 o f 10 Page 24 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Protocol #FACT-TOX-110 Study Identification Oral (Gavage) Pharmacokinetic Study of PFOS in Rats 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 Sponsor Representative Marvin T. Case, D.V.M., Ph.D. 3M Toxicology Services Telephone: 651-733-5180 Facsimile : 651-733-1773 Study Director Kristen J. Hansen, Ph.D. 3M Environmental Technology and Safety Services Building 2-3E-09 651-778-6018 Study Location(s) In vivo Testing Facility Analytical Testing Laboratory 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 o f 10 Page 25 3M Medical Department Study: T-6295.12 Sub-Contract Laboratory Proposed Study Timetable Study Initiation Date Study Completion Date BACK TO MAIN I Analytical Report: FACTTOX-110 LRN-U2849 Protocol HFACT-TOX-110 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 study of potassium perfluorooctane sulfonic acid (PFOS) in rats. 2. Purpose This analytical study is designed to determine levels of potEissium perfluorooctanesulfonate (PFOS) in specimens of liver and serum of rats. The in-life portion of this study was conducted at Argus Research Laboratories, study #418-013. 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 Drug 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 tire 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 26 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Protocol #FACT-TOX-110 5. Test Material 5.1 Refer to Argus Research Laboratory protocol for study #418-013. 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 of the 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 of 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 3IVI Environmental Laboratory Page 4 o f 10 Page 27 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 _ Protocol #FACT-TOX-110 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 thelest system and were maintained and dosed as described in the Argus Research protocol #418-013. The female rats will be given the test material or control once daily beginning 42 days prior to cohabitation and continue through day 14 or 20 of presumed gestation. See table 1 for more dosage information. Table 1 - Dosage Levels, Concentration, and Volumes Dosage Group 1 2 3 4 5 Number of female rats 16 16 16 16 16 Dosage mg/kg/day 0 0.1 0.4 1.6 3.2 Concentration mg/kg/day 0 0.02 0.08 0.32 0.64 Dosage volume mL/kg 5 5 5 5 5 9. Specimen and Sample Receipt The 3M Environmental Laboratory will receive homogeneity samples for dose analysis and specimens of the following body tissues and fluids from the indicated points in the study. See table 2 for specimen information. All specimens will be packed on dry ice for shipping. Body tissue/fluid Table 2 Specimen Information Collected Serum - Dam and Fetus animals Urine and feces - Dam animals Liver - Dam and Fetus animals Milk Secreting Glands - Dam animals Amniotic Fluid Dam animals Dam-Predose, Days 7,15, and 21 Fetus-At termination of the study Predose, Days 7,15, ind 21 Dam and Fetus-At termination of the study At the termination of the study Day 15 and Day 21 Expected # of specimens 320 Dam and 320 Fetus (pooled) 320 urine and 320 feces 80 Dam and 80 Fetus (pooled) 80 80 3M Environmental Laboratory 3M Environmental Laboratory Page 5 o f 10 Page 28 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Protocol #FACT-TOX-110 Table 2 Cont Specimen Information Body tissue/fluid Collected Expected # of specimens Embryo's with Placentae - Dam Day 15 and Day 21 animals - Carcass - Fetus animal At the termination o f the study Placentae - Fetus animal Day 15 and Day 21 Liver/lung - Fetus animal At the termination of the study Total number o f expected specimens: 2000 Total number o f test animals: 64 Tbtal number of control animals: 16 80 160 80 80 Specimens sent to 3M Environmental Laboratories will be received and backed according to applicable Standard Operating Procedures. 10. Preparatory Methods 10.1 FACT-M-1.1, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 10.2 ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLCElectrospray/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 are 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. Analytical Methods 11.1 FACT-M-2.1, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry 11.2 ETS-8-5.1, Analysis of Potassium PerfluoroocUinesulfonate 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 of each method will be attached to this protocol. 3M Environmental Laboratory 3M Environmental Laboratory Page 6 o f 10 Page 29 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Protocol ttFACT-TOX-110 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 r2 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 12.2.2 Limit of 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 o f confirmatory methods Indeterminate samples will be re-analyzed using a confirmatory method. If a confirmatory method is used, an amendment to this protocol will be written. 12.6 Demonstration o f specificity Chromatographic retention time, mass spectral daughter ion characterization. 13. Sub-Contracted Analysis 13.1 All analyses as detailed in this protocol will be performed at 3M Environmental Laboratories, Building 2-3E-09,935 Bush Avenue, St. Paid, MN 55106, at Advanced Bioanalytical Services, Inc., 15 Catherwood Ro ad, 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 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) of PFOS or metabolite per tissue or fluid. 3M Environmental Laboratory 3M Environmental Laboratory Page 7 o f 10 Page 30 BACK TO MAIN 3M Medical Department Study: T-6295.12 ,, Analytical Report: FACT TOX-110 LRN-U2849 , Protocol #FACT-TOX-11Q 14.2 Statistical analysis Statistics used may include regression analysis of 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 15.3 A statement 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 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 of the test substances: under the conditions of administration, if provided by the Sponsor 15.7 A description of the methods used to conduct the test(s) 15.8 A description o f the test system - 15.9 A description of any circumstances that may have affected the quality or the integrity of the data 15.10 The name of the Study Director and the names of other scienti sts, professionals, and supervisory personnel involved in the study 15.11 A description of 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 of 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 3M Environmental Laboratory 3IVI Environmental Laboratory Page 8 o f 10 Page 31 BACK TO MAIN 3M Medical Department Study: T-6295.12 , Analytical Report: FACT TOX-110 LRN-U2849 , Protocol #FACT-TOX-110 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 of the study during its progress and before acceptance of 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 o f time as specified by regulation, and as established by 3M Environmental Laboratory Standard Operating Procedures. 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 of 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 3M Environmental Laboratory 3M Environmental Laboratory Page 9 o f 10 Page 32 BACK TO MAIN 3M Medical Department Study: T-6295.12 .( Analytical Report: FACT TO X-110 LRN-U2849 ,4 Protocol KFACT-TOX-110 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 of the preparation affords evaluation, but not longer than ten years following the effective date of the final test rule (if applicable), and as established by 3M Environmental Laboratory Standard Operating Procedures. 18. Protocol Amendments and deviations Planned changes to the protocol will be in the form of written amendments signed by the Study Director and die 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 of written deviations, signed by the Study Director and filed with the raw data. 19. A ttachments 19.1 Attachment A Preparatory and analytical methods 20. Signatures XC, Marvin T. Case, D.V.M., Ph.D., Sponsor Representative X Date ML Kristen J. Hansen, Ph.D., 3M Environmental Laboratory Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 10 o f 10 Page 33 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Study Title Oral (Gavage) Pharmacokinetic Stutty of 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-TOX110 LIRNU2849 3M Environmental Laboratory 3M Environmental Laboratory Page 34 BACK TO MAIN 3M Medical Department Study: T-6295.12 , ,, Analytical Report: FACT TOX-110 LRN-U2849 Protocol FACT-TOX110 Amendment No. 1 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL reads: Section 2.0 states this study is desiigned to determine potassium perfluorooctane sulfonic acid (PFOS) in specimens of liver and serum of rats. AMEND to read: This study is designed to determine PFOS in specimens of rat liver, serum, and urine. All Day - l and Gestation Day 21 rat urine specimens will be extracted and analyzed by the 3M Environmental Laboratory. REASON: The urine extraction and analytical methods were not validated and approved prior to protocol approval. 2. PROTOCOL reads: Section 6.0 lists rat or rabbit liver and serum. AMEND to read: Rat or rabbit urine from 3M Toxicology with a physical description o f rat or rabbit urine. REASON: The rat urine matrix was added after the protocol was approved. 3. 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 of Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" AMEND to read: The extraction and analytical methods to follow at the 3M Environmental Laboratory are: ETS-8-6.0 "Extraction of 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-8-7.0 "Analysis of 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" 3M Environmental Laboratory 3M Environmental Laboratory Page 35 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TO X-110 LRN-U2849 Protocol FACT-TOX110 Amendment No. 1 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. Methods ETS-8-96.0 and ETS-8-97.0 were not validated and approved until after the protocol was approved. 4. PROTOCOL reads: Section 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 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 of Perfluorooctane Sulfonate.(PFOS) in Rat Sera by LC/MS/MS, Version0i " 1to0 0Luer REASON: The analytical methods at the sub-contract laboratories were not included in the original protocol. 5. PROTOCOL reads: Section 12.2.1 b) Liver method detection limit is 15 ppb. AMEND TOread: 12.2.1 b) Liver method detection limit is 8.50 ppb (ng/g). 12.2.1 c) Urine method detection limit is 1.5 ppb (ng/g). REASON: The validation supporting methods ETS-8-6.0 and ETS-8-7.0 included a lower method detection lim it for PFOS in liver. A validation in urine was performed, after protocol approval, to support this method detection limit. l^k 3M Environmental Laboratory 3M Environmental Laboratory Page 36 3M Medical Department Study: T-6295.12 BACK TO MAIN Amendment Approval Analytical Report: FACT T O X-110 LRN-U2849 Protocol FACT-TOX110 Amendment No. 1 iC Marvin Case, D.V.Mr, Ph.D., Sponsor Representative /7 4 W - /W 'D ate Kris J. Hansen, Ph.D., Study Director ziizJzj Date 3M Environmental Laboratory 3M Environmental Laboratory Page 37 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Study Title Oral (Gavage) Pharmacokinetic Study of PFOS in Rats PROTOCOL AMENDMENT NO. 2 Amendment Date; September 28,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX110 LIRNU2849 3M Environmental Laboratory 3M Environmental Laboratory Page 38 BACK TO MAIN 3M Medical Department Study: T-6295.12 | Analytical Report: FACT TOX-110 LRN-U2849 Protocol FACT-TOX110 Amendment No. 2 This amendment modifies the following portion(s) of the protocol: l . Protocol reads: Section 2 states that all liver samples will be extracted and analyzed at Battelle Memorial Institute. Amend to read: All dam liver specimens (female mother) and 27 fetus (pooled) liver specimens will be analyzed at Battelle Memorial Institute. Reason: , The fetal liver/lung is not a target matrix and will not be extracted or analyzed. 2 . Protocol reads: Section 9 states that 80 dam liver specimens (female mother) and 80 fetus (pooled) liver specimens will be sent to the Environmental Laboratory. Amend to read: Seventy-three dam liver specimens (female mother), 27 fetus (pooled) liver specimens, and 81 fetal liver/lung specimens (27 liver/lung specimens sampled in triplicate) were sent to the Environmental Laboratory. Reason: The number of animals changed during the course of the study. 3. Protocol reads: Section 12.2.1 a) serum method detection lim it is 1.75 ppb b) Liver method detection lim it is 15 ppb. Amend to read: The method detection limits for all compounds and matrices w ill be taken from the methods used for extraction and analysis. Reason: The m ethod detection limits listed are specific to the 3M Environmental Laboratory. Statement w as added to allow for sub-contracted analyses and/or revised methods. 3M Environmental Laboratory 3M Environmental Laboratory Page 39 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Protocol FACT-TOX110 Amendment No. 2 4. Protocol reads: Section 16 states that the original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance of 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 of 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 of 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 of the facility performing each analysis. Reason: Clarification o f the disposition of archived records if analyses are performed at a sub-contract laboratory. 5. Protocol reads: Section 17 states that specimens will 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 will be returned to the 3M Environmental Laboratory upon completion of analysis and submission of the sub-contract laboratory(s) final report. The specimens will be returned with the following documentation: the signed original chain of custody and records of storage conditions while at the sub-contract facility. Reason: Clarification of the disposition of documentation when shipping specimens for analyses performed by a sub-contract laboratory. 3M Environmental Laboratory 3M Environmental Laboratory Page 40 3M Medical Department Study: T-6295.12 BACK TO MAIN Amendment Approval Analytical Report: FACT TO X-110 LRN-U2849 Protocol FACT-TOX110 Amendment No. 2 xs^ Marvin Case, D.V.M.-, Ph.D., Sponsor Representative Date Kristen J. Hansen, Ph.D., Study Director s'ot m i Date 3M Environmental Laboratory 3M Environmental Laboratory Page 41 31V Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTOX-110 LRN-U2849 Study Title Analytical Laboratory Report on the Determination of Perfluorooctanesulfonate (PFOS) Presence and Concentration in Serum, Liver, and Urine Gromthe Gavage Study of T-6295.12 PROTOCOL AMENDMENT NO. 3 Amendment Date: 20 January 2000 U rine 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 M emorial Institute 505 King Avenue Columbus, OH 43201-2693 Serum Analyses Advanced Bioanalytical Services, Inc. 15 Catherwood R oad Ithaca, NY 14850 Laboratory Project Identification ET&SS LRN-U2849 FACTTOX-110 Argus Study: 418-013 3M Medical Department Study: T-6295.12 3M Environmental Laboratory 3M Environmental Laboratory Page 42 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTOX-110 LRN-U2849 Protocol LRN-U2849 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. Amend 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 43 3M Medical Department Study: T-6295.12 Amendment Approval BACK TO MAIN Analytical Report: FACT TO X-110 LRN-U2849 Protocol LRN-U2849 Amendment Number 3 JohnL. Butenhoff, PhD., Sponsor Representative Date --------Kristen J. Hansen, PhD., Outgoing Study Director H 'F ^ b - -ZQ Date Marvin T. Case, D. V.M., PhD., Incoming Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 44 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Study Title Oral (Gavage) Pharmacokinetic Study of PFOS in Rats PROTOCOL AMENDMENT NO. 4 Amendment Date: 20 April 2000 Performing Laboratories U r in e A n a ly s e s 3M E nvironm ental Technology and Safety Services Fluorine Analytical Chemistry Team Building 2-3E-09, 935 Bush Avenue S t Paul, MN 55106 C entre A nalytical L aboratories, Inc. 3048 Research Drive State College, PA 16801 L iv e r A n a l y s e s Battelle M em orial Institute 505 King Avenue Columbus, OH 43201-2693 erum A nalyses Advanced Bioanalytical Services, Inc. 15 Catherwood R oad Ithaca, NY 14850 Laboratory Project Identification ET&SS LRN-U2849 FACT TOX-110 Argus Study: 418-013 3M Medical Department Study: T-6295.12 3M Environmental Laboratory 3M Environmental Laboratory Page 45 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Protocol LRN-U2849 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 o f fecal tissue for the target chemical and/or iis 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; 00M-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 the scope of this study; this method was not validated and approved prior to protocol approval. 3M Environmental Laboratory 3M Environmental Laboratory Page 46 3M Medical Department Study: T-6295.12 * Amendment Approval BACK TO MAIN Analytical Report: FACTTOX-110 LRN-U2849 Protocol LRN-U2849 Amendment Number 4 JohnL. Butenhoff, Ph.D., Sponsor Representative "fCw-C Marvin T. Case, D.V.M., Ph.D., Study Director Date A 7 / # # * * / O&tf- Date 3M Environmental Laboratory 3M Environmental Laboratory Page 47 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Study Title Oral (Gavage) Pharmacokinetic Study of PFOS in Rats REPORT AMENDMENT NO. 5 Amendment Date: 26 July 2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Labo ratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT Tox-110 ET&SS LRN-U2849 3M Environmental Laboratory 3M Environmental Laboratory Page 48 BACK TO MAIN 3M Medical Department Study: T-6295.12 (, Analytical Report: FACT TOX-110 LRN-U2849 - FACT Tox-110 Report Amendment No. 5 This amendment modifies the following portion(s) of the protocol: 1. Protocol Reads: The amended analytical protocol (Amendment No. 1, section 4.0) states the extraction and analytical method to follow at Battelle Memorial Institute is "Method for Analysis of Perfluorooctance 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 listed in Amendment No. 1 had a hand-written change, in tha.t "sera" was crossed out and "liver" was written in. This hand-written change was dated after the study director had signed the amendment. 2. Protocol Reads: In the Argus in-life protocol #418-013, the testing facil ity is stated on page 1 as Argus Research Laboratories, Inc. Amend to 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. 3. Protocol Reads: On the cover page, the type of document for FACT-TOX-110 is listed as a "Protocol." On page 3, under Proposed Study Timetable, the "Study Initiation Date" and "Study Completion Date" are listed. Amend to Read: Change the document type for FACT-TOX-110 to "Analytical Phase Protocol." Change "Study Initiation Date" to "Phase Initiation Dc.te." Change "Study Completion Date" to "Phase Completion Date." Reason: FACT-TOX-110 is the protocol for the analytical phase, while the Argus protocol #418-013 3M Environmental Laboratory 3IVI Environmental Laboratory Page 49 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 . FACT Tox-110 Report Amendment No. 5 is the protocol for the in-life phase. 4. Protocol Reads: The amended analytical protocol (Amendment No. 3, section 1) 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. protocol #418013 states that the study director for the in-life phase is Raymond York. Amend to Read: Marvin T. Case replaces both Kristen J. Hansen andRaymond 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 of Principal (corrected spelling) Analytical Investigator. This change is retroactive to February 10,2000. Reason: Per GLP regulations, only one study director is assigned to a study. Corrected spelling of Principal. 5. Protocol Reads: The amended protocol (Amendment No. 4) states that rat feces samples will be analyzed for PFOS, as well as rat liver, urine and serum. Centre Analytical Laboratories will be conducting the feces analyses. . Amend to Read: Add that the Principal Analytical Investigator (PAI) at Centre Analytical Laboratories is Enaksha Wickremesinhe. Reason: The PAI for Centre was not listed in Amendment No. 4. 3M Environmental Laboratory 3M Environmental Laboratory Page 50 3M Medical Department Study: T-6295.12 Amendment Approval (at Jnsor Representative BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 FACT Tox-110 Report Amendment No. 5 ijs h Date Case, D. V.M., Ph.D., Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 51 3M Medical Department Study. T-6295.12 BACK TO MAIN Analytical Report: FACTTOX-110 LRN-U2849 Study Title Oral (Gavage) Pharmacokinetic Stud]' of PFOS in Rats PROTOCOL AMENDMENT NO. 6 Amendment Date: October 3,2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT TOX-110 3M Laboratory Request No. U2849 3M Environmental Laboratory 3M Environmental Laboratory Page 52 BACK TO MAIN 3M Medical Department Study: T-6295.12 ., Analytical Report: FACT TOX-110 LRN-U2849 Protocol FACT TOX-110 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%. Amend 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 53 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TO X-110 LRN-U2849 Protocol FACT TOX-110 Amendment #6 Amendment Approval John L. Butenhoff, PhD., Sponsor Representative Date Marvin T. Case, D.V.M., PhD., Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 54 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTOX-110 LRN-U2849 Appendix C: Extraction and Analytical Methods This appendix includes the following methods: ETS-8-96.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Urine for Analysis Using HPLS-Electrospray/Mass Spectrometry/Mass Spectrometry," (14 pages) 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 Page 55 BACK TO MAIN 3M Medical Department Study: T-6295.12 3M Environmental Laboratory Analytical Report: FACT TOX-110 LRN-U2849 M ethod E x t r a c t io n o f P o ta ssiu m P e r fl u o r o o c t a n e su l fo n a t e o r o t h e r FLUOROCHEMICAL COMPOUNDS FROM URINE FOR ANALYSIS USING HPLC- E l e c t r o spr a y /M ass Spe c t r o m e t r y /M ass Spe c t r o m e t r y Method Number: ETS-8-96.0 Adoption Date: ^ Author: Lisa Clemen, Glenn Langenburg Revision Date: f \ j ( \ Approved By: 'dJL -- Laborafery M anager w Group Leader A d-Ln>J^v Technical Reviewer mn Date I'h -X h S ' Date Ir h lr i^ Date 1 .0 S c o p e a n d A p p l i c a t i o n _____________________________________________________________________ 1.1 Scope: This method is for the extraction o f potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from urine. 1.2 A pplicable com pounds: Fluorochemicals or other fluorinated compounds. 1.3 M atrices: 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 56 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 2 .0 S u m m a r y o f M e t h o d _________________________________________________________________ ___ 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-iert-butyl ether (MtBE). In this method, eight fluorochemicals are extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, M556, M.570, 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 D e f i n i t i o n s ________________________________________________________________ ;____________________ 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) c 8f I7s o 33.2 PFOSA: perfluorooctane sulfonylamide C8F17S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8F17S 0 2N(CH2CH3)CH2C 0 2` 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F17S 0 2N(CH2CH3)CH2CH20H 3.5 M556: C8F17S 0 2N(H)(CH2C 00H ) 3.6 M570: C8F17S 02N(CH3)CH2C 0 0 H 3.7 POAA: perfluorooctanoate C7F,5COO' 3.8 Surrogate standard THPFOS: 1H-1H-2H-2H perfluorooctane sulfonic acid, used as an internal standard in this method. 4.0 W a r n i n g s a n d C a u t i o n s _______________________________________________________________________ 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 I n t e r f e r e n c e s ___________________________________________ ;_____________________________________ 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 57 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 6.0 E q u i p m e n t ___________________________________________________________________________________ _ 6.1 The following equipment is used while performing ihis method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or EEC 6.1.3 Shaker, Eberbach or VWR 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) 7.0 S u p p l ie s a n d M a t e r ia l s 7.1 Gloves 7.2 Eppendorf or 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 o f measuring 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-QTM water. Rinse glass syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 R e a g e n t s a n d S t a n d a r d s ___________________________________________________________________ 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), Kodtik or equivalent 8.4 Sodium carbonate (NajCOj), 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 58 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, 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 M570 (3M Specialty Chemical Division), molecular weight = 571 8.9.7 POAA (3M Specialty Chemical Division), molecular weight = 452 8.9.8 THPFOS (1-H,1-H, 2-H, 2-H C8F13S 0 3H) 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 o f 10 N NaOH solution into a 100 ml volumetric flask and dilute to volume using MilliQTM water. Store in a 125 ml Nalgene bottle. 8.10.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g o f TBA into a 1 L volumetric containing 500 ml Milli-Q TMwater. 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 1 N NaOH solution. 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na.,C03/NaHC03): Weigh approximately 26.5 g of sodium carbonate (NajCOj) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with MilliQTM water. 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 59 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 8.11.3 Weigh approximately 100 mg of PFOS 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 (pg/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 o f 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 13S 0 3H 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 o f surrogate stock to a 10 ml volumetric flask and bring to volume with methanol for a working standard of 100-120 ppm. Record the actual volume transferred in the Standard Logbook. 9.0 S a m p l e H a n d l i n g _____________________________________________________________________________ 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 Q u a l i t y C o n t r o l ____________________________________________________________________________ 10.1 Solvent Blanks, Method blanks and matrix blanks 10.1.1 An aliquot o f 2.0 ml methanol is used as a solvent blank. 10.1.2 Extract two 2.0 ml aliquots o f 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 o f 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 o f the initial calibration curve. 3M Environmental Laboratory ETS-8-96.0 Extraction o f PFOS from Urine Page 5 o f 14 Page 60 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 10.2.4 Prepare one matrix spike and matrix 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 o f the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing calibration verification per group o f 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 o f 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). 1 1 .0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n _______________________________________________________ 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 a total 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 die end o f this section, to spike, in dupliccite, 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 the Linear Calibration Range (LCR) for calibration curves. 11.1.7 Use Attachm ent 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 o f 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 o f PFOS from Urine Page 6 o f 14 Page 61 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 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 O f 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 P r o c e d u r e ____________________________________________________________________________________ 12.1 Obtain frozen samples and allow to thaw at room temperature or in 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 A ttachm ent D. The original weight/volume worksheet is included in the study binder. 12.5 Label the tube with the study number, sample ID, elate 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 o f 0.25M sodium carbonate/sodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 ml methyl-teri-butyl ether. 12.12 Cap each sample and put on the shaker at a setting o f 300 rpm, for 20 minutes. 3M Environmental Laboratory ETS-8-96.0 Extraction o f PFOS from Urine Page 7 o f 14 Page 62 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 12.13 Centrifuge for 20 to 25 minutes at a setting o f 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 o f 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 analyst(s) 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. 1 3 .0 D a t a A n a l y s i s a n d C a l c u l a t i o n s _________________________________________________________ 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS, or other applicable fluorochemical, in calibration standards using the following equation: ml o f standard x concentration o f standard fug /m il_____________________= ml of standard + ml of surrogate standard + initial matrix volume (ml) Final Concentration (pg/ml) o f PFOS in matrix 1 4 .0 M e t h o d P e r f o r m a n c e ______________________________________________________________________ 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 o f 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 o f the initial calibration curve. 14.3 Refer to section 14 o f ETS-8-97.0 for method perfonnance criteria. 3M Environmental Laboratory ETS-8-96.0 Extraction o f PFOS from Urine Page 8 o f 14 Page 63 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 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 expropriate. 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-TO.X-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 Spectrometry" 20.0 Revisions_________________________________________________________________ Revision Number Reason For Revision Revision Date 3M Environmental Laboratory ETS-8-96.0 Extraction o f PFOS from Urine Page 9 o f 14 Page 64 BACK TO MAIN 3M Medical Department Study: T-6295.12 u A u u v u u i i tv v /A J v a iiC C l SZ-1 O - O - y O . U Studv # M atrix Box# Wk/Dav DateSpiked/Analyst CCV MS MSD Surrogate Std Approx, ppm Actual ppm # FC-Mix approx. 0.5 ppm actual ppm # FC-Mix approx. 5 ppm actual ppm # Analytical Report: FACT TOX-110 LRN-U2849 FC-Mix Comments approx. 50 ppm actual ppm # -------- -- ------- Blank Urine Extraction Method S td # : amount" Vortex 15 sec. Pipette M atrix . strike with appropriate surroeate or FC-Mix Volume Pipette 1 m l o f 0.5 M TBA , pH 10. pH= Std. # ml Pipette 2 ml o f 0.25 N a2CO y0.25M NaHCCh buffer Std. # Dispense 5 ml o f methyl-t-butyl ether TN-A- Shake 20 min. Shaker speed: Centrifuge 20-25 min. Centrifuge sneed: Remove a 4 ml aliauot of organic layer Put on Nitrogen Evaporator to dryness Temperature: Add methanol Volume ml TN-A- Vortex 30 sec. Filter using a 3cc B-D syringe with a 0.2um filter into an autosample vial Cont. Cal. Verifications used same 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 65 BACK TO MAIN 3M Medical Department Study. T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 MDL/LOQ valaes for human urine Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (PP*>) PFOS 4.6 14.7 15 ppb - 1500 ppb (in final MeOH extract) POAA 22.9 72.9 50 ppb - 1500 ppb (in final MeOH extract) PFOSA n/d n/d 25 ppb - 1000 ppb (in final MeOH extract) PFOSAA n/d n/d 25 ppb -1000 ppb (in final MeOH extract) EtFOSE-OH n/d n/d 25 ppb -1000 ppb (in final MeOH extract) M556 n/d n/d 25 ppb - 1000 ppb (in iinal MeOH extract) M570 n/d n/d 25 ppb - 1000 ppb (in final MeOH extract) n/d = not determined NOTE: to calculate MDL, LOQ, and LCR values in ug/ml of urine divide the above values by 4. MDL/LOQ values in rat and monkey urine were not statistically determined. Two curves in each o f these matrices were extracted and analyzed with the human urine curves to determine equivalence. Responses in the rat and monkey were similar to the human responses, therefore, their MDL and LOQ are assumed to be similar to the values determined for human urine. If a suitable amount o f clean, control matrix is available, samples will be evaluated versus a curve extracted from urine originating from the same species as the specimens. Please see LOQ Summary and MDL study in FACT-TOX-131, W2067 for farther information. Attachment B: MDL/LOQ Summary 3M Environmental Laboratory ETS-8-96.0 Extraction o f PFOS from Urine Page 11 o f 14 Page 66 BACK TO MAIN 3M Medical Department Study: T-6295.12 Compound: PFOS Prepared range of Human Urine 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 Prepared range of Human Urine 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 TOX-110 LRN-U2849 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 67 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Ion Pair Standard Curves - Urine Prep date(s): Analyte(s): Standard number: Equipment number: Sample matrix: Final solvent and TN: Method/revision: Blank fluid/identifier: 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 PFOSA PFOSAA EtFOSE POAA Mii56 M570 Std cone Std cone Std cone Std cone Std cone Std cone Std cone ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml 0.500 0.501 0.500 0.501 0.499 0.500 0.501 0.500 0.501 0.500 0.501 0.499 0.500 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 All Ain't spiked ml 0.005 0.010 0.025 0.005 0.010 0.025 0.005 0.0075 0.010 0.015 All Final vol ml 2.0075 2.0125 2.0275 2.0075 2.0125 2.0275 2.0075 2.0100 2.0125 2.0130 Calculated concentrations of standards in the sample matrix PFOS PFOSA PFOSAA EtFOSE POAA MS56 Final cone Final cone Final cone Final cone Final cone Final cone ng/ml 1.25 2.48 6.17 12.5 24.8 61.7 125 187 248 372 ng/ml 1.25 2.49 6.18 12.5 24.9 61.8 125 187 249 372 ng/ml 1.25 2.48 6.17 12.5 24.8 61.7 125 187 248 372 ng/ml 1.25 2.49 6.18 12.5 24.9 61.8 125 187 249 372 ng/m] 1.24 2.48 6.15 12.4 24.8 61.5 124 186 248 371 ng/ml 1.25 2.48 6.17 12.5 24.8 61.7 125 187 248 372 M570 Final cone ng/ml 1.25 2.49 6.18 12.5 24.9 61.8 125 187 249 372 Surrogate Std cone ng/ml 100 Surrogate Final cone ng/ml 125 All Ain't spiked ml 0.0025 Calculated concentrations of standards in methanol extract (0.5 ml final volume) PFOS PFOSA PFOSAA EtFOSE POAA M:556 M570 Final cone Final cone Final cone Final cone Final cone Final cone Final cone ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml 5.00 5.01 5.00 5.01 4.99 5.00 5.01 10.0 10.0 10.0 10.0 10.0 10.0 10.0 25.0 25.1 25.0 25.1 25.0 25.0 25.1 50.0 50.1 50.0 50.1 49.9 50.0 50.1 100 100 100 100 100 100 100 250 251 250 251 250 250 251 500 501 500 501 499 500 501 750 752 750 752 749 750 752 1000 1002 1000 1002 998 1000 1002 1500 1503 1500 1503 1497 1500 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 Extraction o f PFOS from Utine 3M Environmental Laboratory Page 13 o f 14 Page 68 3M Medical Department Study: T-6295.12 Prep Date(s): Analyst(s): Sample Matrix: Method/Revision : Target Analyte(s): Sample ID Initial Wt/Vol. g/mL/L BACK TO MAIN Analytical Report: FACT TO X-110 LRN-U2849 Study Number: Equipment Number: Final Solvent & TN Number: Matrix Blank/Identifier: Box: Comments ' Summary of method: Notes: Attachment D: Weight/Volume Sheet 3M Environmental Laboratory ETS-8-96.0 Extraction o f PFOS from Urine Page 14 o f 14 Page 69 BACK TO MAIN 3M Medical Department Study: T-6295.12 3M Environmental Laboratory Analytical Report: FACT TOX-110 LRN-U2849 M ethod A n a l y sis o f P o ta ssiu m P e r fl u o r o o c t a n e su l fo n a te o r O t h e r F l u o r o c h e m ic a l C o m p o u n d s in U irine E x t r a c t s U s in g H P L C -E lec tr o spr a y /M ass Spec tr o m etr y /M ass Spe c t r o m et r y M ethod N um ber: ETS-8-97.0 2 0 3Adoption Date: ^ -^ Author: Lisa Clemen, Robert Wynne, Glenn Langenburg Revision Date: Approved By: ------ :-- :__________________ Laboratory Manager & U 4 - ________ _ Group Leader A AlMm___________________________ Technical Reviewer 7/ u / ? 5 Date Date Date 1.0 S c o p e a n d A p p l i c a t i o n ________________________________ ;_________ _______________ 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 M atrices: Human, rat, and monkey urine, or other fluids as designated in the validation report. Word 6/95 3M Environmental Laboratory ETS-8-97.0 Analysis o f Urine Extract Using ES/MS Page 1 of 10 Page 70 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TO X-110 LRN-U2849 2.0 S u m m a r y o f M e t h o d _______________________ ;_________________________________________________ 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 tand em mass spectrometer to further verify the identity o f a compound by detecting daughter ions o f the parent ion. 3.0 D e f i n i t i o n s ____________________________________________________________________________________ 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods o f 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 ioni2:ation 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 toituous 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 w ith sim ilar 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 o f 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 a r n i n g s a n d C a u t i o n s _____________________________________________ ;________________ 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 o f 10 Page 71 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 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 f 400 bar (5800 psi). If the back pressure exceeds 400 bar, the HP 1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.Q I n t e r f e r e n c e s _________________________________________________________________________________ 5.1 To minimize interferences when analyzing samples, teflon should not be used for sample storage or any part o f instrumentation that comes in contact with the sample or extract. 6.0 E q u i p m e n t ______________________________________________________________________________________ 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 HP 1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 S u p p l i e s a n d M a t e r i a l s ______________________________________________________________________ 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 R e a o e n t s 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 titan those listed, adjust accordingly. 8.1.3.2 2.0 mM ammonium acetate solution:: Weigh approximately 0.300 g ammonium acetate. Pour into a 200C1mL 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 of 10 Page 72 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT T O X-110 LRN-U2849 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 S a m p l e H a n d l i n g _______________________________________________________________________ 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. 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 Q u a l i t y C o n t r o l ____________________________________________________________________________ 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix b lanks 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 o f 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 o f the initial calibration curve. 10.3 Continuing Calibration Verifications 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy o f 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 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n _______________________________________________________ 11.1 Analyze the extracted matrix standards prior to and foliowring each set of extracts. The average of two standard curves will be plotted by recession (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 73 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 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 Eitting 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 o f accuracy when quantitating low levels o f analyte, it may be necessary to use the low end of the calibration curve rather than the full range o f the standard curve. Example: when attempting to quantitate approximately 50 ppb of analyte, generate a calibration curve consisting of die 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 P r o c e d u r e s ___________________________________________________________________________________ 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 o f yearincreasing letter o f the alphabet starting with a 12.1.1.2 Assign a method (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 along with the SDRs. 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 o f 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 lis t 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 74 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 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 refil l 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 75 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 12.3.7.2 ESI nebulizing gas 10-15 liters/hour 12.3.7.3 HPLC constant flow mode, flow rate 10 - 500 pT./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. 1 3 .0 D a t a A n a l y s i s a n d C a l c u l a t i o n s _________________________________________________________ 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 oilier fluorochemical, in matrix (pg/mL): fng/m L o f PFOS calc, from std. Curve x Dilution Factor! x 1 pg______ Initial Volume of matrix CmLl 1000 ng Final Volume (mL) 14.0 M e th o d P er fo r m a n c e____________________________________ _______________________ 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 M atrix 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 M atrix 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 76 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT T O X -110 LRN-U2849 14.5 Continuing Calibration Verifications 14.5.1 Continuing calibration verification percent recoveries must be within 30% o f 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 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. 1 5 .0 P o l l u t i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t _______________________________________ 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. 1 6 .0 R e c o r d s _______________________________________________________________________________________ 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 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 o f backup electronic data. 1 7 .0 T a b l e s . D i a g r a m s . F l o w c h a r t s , a n d V a l i d a t i o n D a t a _______________________________ 17.1 Attachment A: ETS-8-97.0 Data summary spreadsheet. 1 8 .0 R e f e r e n c e s __________________________________________________________________________________ 18.1 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/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 77 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TO X-110 LRN-U2849 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 Number. 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 78 BACK TO MAIN 3M Medical Department Study: T-6295.12 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number Instrument Sofhvare/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: FACT TOX-110 LRN-U2849 Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from die study folder. Concentration (ug/mL): Taken from die 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 Analysis o f Urine Extract Using ES/MS 3M Environmental Laboratory Page 10 o f 10 Page 79 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTOX-110 LRN-U2849 Appendix D: Data Summary Tables Table 10. Average Results for the Analyses of Serum 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-6295.12 via Gavage 0 m g /k g /d a y 0.1 m g /k g /d a y 0 .4 m g /k g /d a y 1 .6 m g /k g /d a y 3 .2 m g /k g /d a y Da y O 0.0723 8.89 40.7 160 318 Day 7 0.126 7.82 40.9 154 105 Day 15 0.0926 8.80 41.4 156 275 D a y 21 0.0714 4.24 26.2 136 155 Day 21 Fetal 0.125 9.07 34.3 101 165 NOTE: Serum concentrations are expressed as pg/mL and 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 (0.043 pg/mL). Individual animal data are located in Attachment E, Data Spreadsheets. NOTE: Serum analysis were conducted at a contract laboratory but were corrected by 3M to reflect the official purity values from the COA. Revised reports from the final contract laboratories will be added as a report amendment at a later date. Table 11. 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-6295.12 via Gavage Dose G roup Units PFOS Concentration (pg/g) F e m a le A d u lt GO PFOS C oncentration (pg/g) Fetal Liver 0 0.288 0 .1 6 9 0.1 29.2 7.93 0.4 107 30.6 1.6 347a 86.7 3.2 610 aResult from animal 19559 is an outlier and was not included in the average. 230 NOTE: Liver concentrations are expressed as the average of samples within a dose group from all animals tested, not including samples that tested at below the limit of quantitation (LO Q =0.112 pg/g). Individual animal data are located in Attachment E, Data Spreadsheets. NOTE: Liver analysis were conducted at a contract laboratory but were corrected by 3M to reflect the official purity values from the COA. Revised reports from the final contract laboratories will be added as a report amendment at a later date. 3M Environmental Laboratory Page 80 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTO X-110 LRN-U2849 Table 12. Average Results for the Analysis 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-6295.12 via Gavage 0 m g /k g /d a y 0.1 m g /k g /d a y 0 .4 m g /k g /d a y 1 .6 m g /k g /d a y 3 .2 m g /k g /d a y G e s t a t io n D a y 0 <LOQa 0.0497 0.302 0.959 1.53 G e s t a t io n D a y 7 <LOQ-- 1 an om a ly 0.0620 0.308 1.10 1.60 G e s t a t io n D a y 1 5 G e s t a t io n D a y 21 0.00905 0.0194 0.0685 0.0574 0.526 0.555 0.622 2.71 0.563 1.61 aLOQ=Limit of quantitation (< 0.004 pg/mL) NOTE: Urine concentrations are expressed as pg/mL and are the average of samples from all animals tested, not including samples that tested at or below the limit of quantitation (0.004 pg/mL). Individual animal data are located in the study binder archived for this report and also in Appendix E, Data Spreadsheets. Table 13. Average Results for the Analysis 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-6295.12 via Gavage 0 m g /k g /d a y 0.1 m g /k g /d a y 0 .4 m g i/k g /d a y 1 .6 m g /k g /d a y 3 .2 m g /k g /d a y G e s t a t io n D a y 0 0.0380 0.499 2.42 10.3 23.9 G e s t a t io n D a y 7 0.0155 0.490 2.16 9.19 33.0 G e s t a t io n D a y 1 5 0.0322 0.662 2.93 11.1 29.5 G e s t a t io n D a y 21 0.0342 0.416 2.39 9.94 20.1 *LOQ=Limit of quantitation (<0.010 pg/g) NOTE: Feces concentrations are expressed as pg/g and are the average of samples from all animals tested, not including samples that tested at or below the limit of quantitation (<0.010 pg/g), those reported as None Detected, or those reported as Not Quantifiable. Individual animal data are located in Appendix E, Data Spreadsheets and in Appendix G, Contract Lab Reports. 3M Environmental Laboratory Page 81 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTOX-110 LRN-U2849 Table 14. LOQ Values Used in FACT TOX-110 Analyses Method Urine ETS-8-97.0 Liver Battelle Serum Advanced Bioanalytical Feces Centre I I | | | Effective Date 7/28/99 8/23/99 8/26/99 4/19/00 | LOQ p g /m L 0.004 pg/mL pg/g 0.112 pg/g p g /m L 0.043 pg/mL pg/g 0.010 ng/g 3M Environmental Laboratory Page 82 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Appendix E: Data Spreadsheets Table 15. 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-6295.12 via Ciavage Group/Dose Sample # (all values in pg/mL) GD0 external std. GD7 external std. GD15 external std. GD21 external std. 19501F 19502F 19503F 19504F 19505F 19506F 19507F <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 0.00891 <LOQ NS <LOQ <LOQ <LOQ <LOQ <LOQ 0.0101 NS 0.00406 0.0141 <LOQ <LOQ Group 1 0.0 mg/kg/day 19508F 19509F 1951OF <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 19511F 19512F <LOQ <LOQ <LOQ <LOQ <LOQ 0.00756 19513F <LOQ <LOQ <LOQ 19514F <LOQ <LOQ <LOQ 19515F <LOQ E <LOQ 19516F <LOQ <LOQ 0.00951 19517F 0.0396 0.0635 0.0456 19518F 0.0231 0.0648 0.00894 19519F 0.0487 0.0759 0.0324 19520F 0.0305 NS NS 19521F 0.0677 0.121 0.0419 19522F 19523F 0.0475 0.0562 0.0844 0.136 0.0990 0.180 Group 2 0.1 mg/kg/day 19524F 19525F 0.0374 0.0379 0.0806 0.0508 0.0981 0.0541 19526F 0.0268 0.0389 0.0493 19527F 0.0472 0.0319 0.0278 19528F 0.115 0.0357 0.0773 19529F 0.0444 0.0296 0.0641 19530F 0.0576 0.0266 0.132 19531F 0.0652 0.0541 0.0580 19532F 0.0504 0.0363 0.0601 LOQ=Limit of quantitation (<0.004 pg/mL) NS=Not sampled E=Not enough sample to complete the dilution/analysis L=Sample lost during extraction NOTE: PFOS concentrations in urine are expressed as micrograms of PFOS/mL of urine matrix (pg/mL) NS NS NS <LOQ <LOQ 0.0334 <LOQ NS 0.0161 NS NS 0.00887 NS NS NS <LOQ NS 0.0496 0.0772 NS 0.0420 NS 0.0726 0.0475 NS NS NS 0.0608 0.0519 NS NS NS 3M Environmental Laboratory Page 83 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTOX-110 LRN-U2849 Table 15. 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-6295.12 via Gavage (Continued) Group/Dose Sample # (all GD0 values in pg/mL) external std. GD7 external s;td. GD15 external std. Group 3 0.4 mg/kg/day 19533F 19534F 19535F 19536F 19537F 19538F 19539F 19540F 19541F 19542F 19543F 19544F 19545F 19546F 19547F 19548F 0.219 0.187 0.494 0.212 0.445 0.464 0.480 0.399 0.239 0.144 0.080 0.274 0.688 0.125 <LOQ 0.0879 0.343 0.400 0.191 0.533 0.485 0.536 0.712 0.207 NS 0.243 0.198 0.0334 NS 0.0769 0.168 0.192 0.673 0.431 0.436 0.600 0.293 0.384 0.502 1.04 NS 0.427 0.488 0.946 NS 0.534 0.248 0.359 Group 4 1.6 mg/kg/day 19549F 19550F 19551F 19552F 19553F 19554F 19555F 19556F 19557F 19558F 19559F 19560F 19561F 19562F 19563F 19564F 0.694 0.545 0.992 0.455 0.631 1.08 0.757 E 1.11 1.32 0.904 1.13 1.20 1.90 0.461 1.21 NS 0.771 NS NS 0.838 1.52 2.58 1.08 NS 1.05 0.297 1.11 1.04 1.11 0.546 1.28 NS 0.729 NS NS 0.378 <LOQ 0.765 <LOQ NS 0 .6 4 0 0.700 <LOQ 0.805 <LOQ <LOQ 0.336 LOQ=Limit of Quantitation (<0.004 pg/mL) NS=Not sampled E=Not enough sample to complete the dilution/analysis L=Sample lost during extraction NOTE: PFOS concentrations in urine are expressed as micrograms of PFOS/mL of urine matrix (pg/mL) GD21 external std. 0.383 E NS E NS NS NS NS NS NS L NS NS 0.593 NS 0.688 NS NS NS NS NS NS NS 3.33 NS NS 5.35 0.718 NS 1.45 NS NS 3M Environmental Laboratory Page 84 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTO X-110 LRN-U2849 Table 15. 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-6295.12 via Gavage (Continued) Group/Dose Sample # (all values In pg/mL) GDO external std. GD7 external std. GD15 external std. 19565F 1.27 0.908 0.585 19566F 2.14 1.63 0.188 19567F 1.15 0.525 <LOQ 19568F 0.961 1.82 0.381 19569F 3.32 3.83 0.647 19570F 1.04 1.03 0.150 19571F 1.14 1.32 0.391 Group 5 3.2 mg/kg/day 19572F 19573F 2.32 0.775 NS 0.702 0.973 0.970 19574F 0.685 1.29 0.693 19575F 1.44 0.865 0.551 19576F 2.98 NS NS 19577F 2.71 3.06 0.559 19578F 1.15 NS NS 19579F 0.629 2.36 0.557 19580F 0.844 1.49 0.677 LOQ=Limit of Quantitation (<0.004 pg/mL) NS=Not sampled E=Not enough sample to complete the dilution/analysls L=Sample lost during extraction NOTE: PFOS concentrations In urine are expressed as micrograms of PFOS/mL of urine matrix (pg'mL) GD21 external std. NS NS NS 1.39 1.51 NS 1.32 1.46 NS NS NS NS 2.69 NS 1.31 NS 3M Environmental Laboratory Page 85 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTOX-110 LRN-U2849 Table 16. Results of Analyses of Serum 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-6295.12 via Gavage Group/Dose Sample # (all values in pg/mL) GD0 external std. GD7 external std. GD15 external std. GD21 external std. 19501F 0.0968 0.343 0.0694 NS 19502F 0.0606 0.0462 0.0596 NS 19503F 19504F 0.0524 0.0444 NS 0.0800 NS <LOQ NS <LOQ 19505F 0.0475 0.0444 0.0526 <LOQ 19506F 0.267 0.189 0.172 0.0708 19507F 0.0471 0.051 0.0560 <LOQ 19508F 0.0473 <LOQ <LOQ NS 19509F <LOQ <LOQ <LOQ <LOQ 1951OF 19511F 0.0574 0.0535 0.0550 0.216 0.0530 0.152 NS NS Group 1 0.0 mg/kg/day 19512F 19513F <LOQ 0.0455 0.170 <LOQ 0.206 <LOQ 0.0720 NS 19514F <LOQ 0.0677 0.0560 NS 19515F 19516F <LOQ 0.0483 <LOQ <LOQ 0.0502 <LOQ NS <LOQ Fetal Serum 19504 NS NS NS 0.0730 19505 NS NS NS 0.0760 19506 NS NS NS 0.231 19507 NS NS NS 0.0727 19509 NS NS NS 0.0881 19512 NS NS NS 0.189 19516 NS NS NS 0.143 LOQ=Limit of quantitation (<0.043 pg/mL) NS=Not sampled NOTE: PFOS concentrations in serum are expressed as micrograms of PFOS/ml. of serum matrix (|jg/mL) NOTE: Serum analysis were conducted at a contract laboratory but were corrected by 3M to reflect the official purity values from the COA. Revised reports from the final contract laboratories will be added as a report amendment at a later date. 3M Environmental Laboratory Page 86 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Table 16. Results of Analyses of Serum 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-6295.12 via Gavage (Continued) Group/Dose Sample # (all values In pg/mL) GDO external std. GD7 external std. GD15 external std. GD21 external std. Group 2 0.1 mg/kg/day 19517F 19518F 19519F 19520F 19521F 19522F 19523F 19524F 19525F 19526F 19527F 19528F 19529F 19530F 19531F 19532F 7.84 8.53 8.10 8.41 9.59 8.05 11.1 8.81 10.4 8.19 10.4 8.64 10.0 7.33 7.86 9.16 7.46 8.90 5.90 NS 7.62 7.08 8.34 8.99 8.31 9.68 7.59 6.74 8.90 6.61 6.54 8.73 9.94 9.24 7.93 NS 12.2 8.27 9.42 7.95 8.64 10.1 7.59 6.95 6.32 10.2 8.99 8.35 NS 5.91 3.40 NS 4.26 NS 4.62 3.55 NS NS NS 5.35 2.63 NS NS NS Fetal Serum 19518 NS NS NS 10.5 19519 NS NS NS 7.36 19521 NS NS NS 9.68 19523 NS NS NS 8.81 19524 NS NS NS 8.90 19528 NS NS NS 9.33 19529 NS NS NS 8.99 LOQ=Umit of quantitation (<0.043 gg/mL) NS=Not sampled NOTE: PFOS concentrations In serum are expressed as micrograms of PFOS/mL of serum matrix (pg/mL) NOTE: Serum analysis were conducted at a contract laboratory but were conected by 3M to reflect the official purity values from the COA. Revised reports from the final contract laboratories will be added as a report amendment at a later date. 3M Environmental Laboratory Page 87 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTOX-110 LRN-U2849 Table 16. Results of Analyses of Serum 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-6295.12 via Gavage (Continued) Group/Dose Sample # (all GDO values in pg/mL) external std. GD7 external std. GD15 external std. GD21 external std. Group 3 0.4 mg/kg/day 19533F 19534F 19535F 19536F 19537F 19538F 19539F 19540F 19541F 19542F 19543F 19544F 19545F 19546F 19547F 19548F 46.6 40.5 43.3 33.4 48.9 40.2 39.1 37.1 38.4 39.1 40.0 39.6 41.4 37.6 36.5 49.8 38.3 43.2 48.9 31.6 50.5 41.6 38.5 39.6 NS 35.1 39.8 37.0 NS 40.2 36.5 51.8 Fetal Serum 40.9 48.5 41.6 37.4 43.8 46.2 32.1 42.0 NS 38.0 37.9 42.4 NS 40.9 37.4 50.1 17.6 17.7 NS 14.0 NS NS NS NS NS NS 19.9 NS NS 30.9 NS 56.8 19533 NS NS NS 36.7 19534 NS NS NS 35.8 19536 NS NS NS 27.8 19543 NS NS NS 29.5 19546 NS NS NS 41.8 LOQ=Limit of quantitation (<0.043 pg/mL) NS=Not sampled NOTE: PFOS concentrations in serum are expressed as micrograms of PFOS/mL. of serum matrix (pg/mL) NOTE: Serum analysis were conducted at a contract laboratory but were conected by 3M to reflect the official purity values from the COA. Revised reports from the final contract laboratories will be added as a report amendment at a later date. 3M Environmental Laboratory Page 88 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Table 16. Results of Analyses of Serum 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-6295.12 via Gavage (Continued) Gnoup/Dose Sample # (all GDO values in pg/mL) external std. GD7 external std. GD15 external std. GD21 external std. 19549F 143 NS NS NS 19550F 173 187 138 NS 19551F 159 NS NS NS 19552F 143 NS NS NS 19553F 157 138 117 NS 19554F 175 163 152 NS 19555F 143 168 194 NS 19556F 172 161 175 217 19557F 171 NS NS NS Group 4 1.6 mg/kg/day 19558F 169 148 142 NS 19559F 148 138 166 205 19560F 173 148 186 68.6 19561F 167 156 189 NS 19562F 160 155 123 55.0 19563F 141 145 158 NS 19564F 160 146 137 NS Fetal Serum 19560 NS NS NS 113 19562 NS NS NS 88.1 LOQ=Limit of quantitation (<0.043 pg/mL) NS=Not sampled NOTE: PFOS concentrations in serum are expressed as micrograms of PFOS/mL of serum matrix (pg/mL) NOTE: Serum analysis were conducted at a contract laboratory but were corrected by 3M to reflect the official purity values from the COA. Revised reports from the final contract laboratories will be added as a report amendment at a later date. 3M Environmental Laboratory Page 89 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTOX-110 LRN-U2849 Table 16. Results of Analyses of Serum 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-6295.12 via Gavage (Continued) Gnoup/Dose Sample # (all values in pg/mL) GDO external std. GD7 external std. GD15 external std. GD21 external std. 19565F 320 339 298 NS 19566F 291 242 269 NS 19567F 316 283 254 NS 19568F 293 294 290 208 19569F 289 279 271 123 19570F 303 291 232 NS 19571F 301 321 266 143 19572F 308 264 274 106 19573F 314 296 274 NS 19574F 315 340 258 NS 19575F 357 334 245 Group 5 19576F 331 NS NS 3.2 mg/kg/day 19577F 360 341 312 NS NS 191 19578F 322 NS NS NS 19579F 334 345 334 161 19580F 327 308 279 NS Fetal Serum 19568 NS NS NS 197 19569 NS NS NS 152 19571 NS NS NS 143 19572 NS NS NS 143 19577 NS NS NS 195 19579 NS NS NS 156 LOQ=Limit of quantitation (<0.043 pg/mL) NS=Not sampled NOTE: PFOS concentrations in serum are expressed as micrograms of PFOS/mL of serum matrix (pg/mL) N O TE: Serum analysis were conducted at a contract laboratory but were corrected by 3M to reflect the official purity values from the COA. Revised reports from the final contract laboratories will be added as a report amendment at a later date. 3M Environmental Laboratory Page 90 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 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-6295.12 via Gavage Group/Dose Sample # (all values in pg/g) GD0 external std. GD7 external std. GD15 external std. GD21 external std. 19501F ND ND ND 19502F ND NQ ND 19503F <LOQ NS NS 19504F ND NQ ND 19505F ND ND ND 19506F 0.0262 0.0155 0.0387 19507F ND ND ND Group 1 0.0 mg/kg/day 19508F 19509F <LOQ 0.0348 <LOQ ND ND 0.0210 1951OF ND ND <LOQ 19511F ND ND ND 19512F 19513F ND ND ND 0.0503 ND ND 19514F ND ND ND 19515F ND ND ND 19516F 0.0529 NQ 0.0188 19517F 0.609 0.356 0.659 19518F 0.396 0.488 0.584 19519F 0.339 0.299 0.680 19520F 0.587 NS NS 19521F 19522F 0.430 0.331 0.404 0.676 0.549 0.710 19523F 0.398 0.543 0.558 Group 2 0.1 mg/kg/day 19524F 19525F 19526F 0.724 0.411 0.388 0.500 0.517 0.408 0.725 0.939 0.676 19527F 0.432 0.645 0.694 19528F 0.609 0.462 0.734 19529F 0.693 0.647 0.629 19530F 0.342 0.369 0.630 19531F 0.638 0.542 0.675 19532F 0.650 0.499 0.494 LOQ=Limitof quantitation (<0.010 pg/g) NS=Not sampled ND=None detected NQ=Not quantifiable NOTE: PFOS concentrations in feces are expressed as micrograms of PFOS/g of feces matrix (pg/g) NS NS NS ND ND 0.0107 ND NS 0.0733 NS NS 0.0187 NS NS NS <LOQ NS 0.639 0.411 NS 0.351 NS 0.349 0.429 NS NS NS 0.396 0.340 NS NS NS 3M Environmental Laboratory Page 91 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTOX-110 LRN-U2849 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-6295.12 via Gavage (Continued) Group/Dose Sample # (all values in pg/g) GD0 external std. GD7 external std. GD15 external std. 19533F 2.26 2.21 2.40 19534F 3.26 2.34 4.19 19535F 2.43 1.59 3.15 19536F 2.76 2.83 2.77 19537F 2.41 2.27 2.95 19538F 2.68 2.55 2.73 19539F 3.20 1.82 2.69 Group 3 19540F 1.78 1.97 3.61 0.4 mg/kg/day 19541F 1.83 NS NS 19542F 2.86 2.61 1.64 19543F 1.84 2.43 2.97 19544F 2.98 1.98 2.93 19545F 2.39 NS NS 19546F 2.03 1.32 2.28 19547F 1.93 1.79 3.36 19548F 2.03 2.47 3.40 19549F 12.3 NS NS 19550F 14.7 13.0 12.1 19551F 9.03 NS NS 19552F 11.4 NS NS 19553F 11.9 10.7 12.9 19554F 13.5 10.2 12.3 19555F 8.98 8.35 14.1 Group 4 1.6 mg/kg/day 19556F 19557F 19558F 8.91 7.79 5.95 8.88 NS 5.12 7.88 NS 6.23 19559F 6.67 5.99 6.50 19560F 14.4 6.10 12.9 19561F 10.5 7.71 15.2 19562F 8.33 9.42 9.92 19563F 6.49 13.0 7.78 19564F 14.7 11.9 15.1 LOQ=Limit of quantitation (<0.010 pg/g) NS=Not sampled ND=None detected NQ=Not quantifiable NOTE: PFOS concentrations in feces are expressed as micrograms of PFOS/g of feces matrix (pg/g) GD21 external std. 1.46 2.70 NS 1.61 NS NS NS NS NS NS 1.94 NS NS 1.94 NS 4.71 NS NS NS NS NS NS NS 15.9 NS NS 9.19 9.72 NS 4.94 NS NS 3M Environmental Laboratory Page 92 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTOX-110 LRN-U2849 Table 17. Results of Analyses of Feces Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Seira, Livers, Urine, and Feces of Crl:CDBR VAF/Plus Rats Exposed to T-6295.12 via Gavage (Continued) Group/Dose Sample # (all values in pg/g) GDO external std. GD7 external s;td. GD15 external std. 19565F 20.4 44.9 30.6 19566F 25.7 23.0 13.2 19567F 26.3 31.2 17.7 19568F 21.0 20.0 40.8 19569F 23.5 37.3 31.6 19570F 17.1 32.5 27.0 19571F 26.0 37.4 24.7 Group 5 3.2 mg/kg/day 19572F 19573F 22.1 20.0 33.7 23.2 17.9 34.6 19574F 25.5 31.6 41.1 19575F 20.0 30.8 35.4 19576F 31.6 NS NS 19577F 29.8 59.4 41.1 19578F 30.2 NS NS 19579F 22.0 31.2 29.1 19580F 21.6 26.1 28.4 LOQ=Limit of quantitation (<0.010 -- pg/g) NS=Not sampled ND=None detected NQ=Not quantifiable NOTE: PFOS concentrations in feces are expressed as micrograms of PFOS/g ot feces matrix (pg/g) GD21 external std. NS NS NS 21.8 18.8 NS 18.7 13.0 NS NS NS NS 23.5 NS 24.6 NS 3M Environmental Laboratory Page 93 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTO X-110 LRN-U2849 Table 18. Results of 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-6295.12 via Gavage Group/Dose Sample # Concentration of PFOS (all values in ug/g) Group/Dose Sample # Concentration of PFOS (all values in pg/g) 19501F 0.346 19517F 42.6 19502F 0.305 19518F 21.8 19503F 19504F NS 0.125 19519F 19520F 18.9 15.4 19505F 0.174 19521F 20.1 19506F 0.403 19522F 32.0 19507F 19508F 0.168 0.230 19523F 19524F 17.7 19.6 19509F 1951OF 19511F 0.175 0.203 0.828 19525F 19526F 19527F 36.5 34.0 46.4 Group 1 0.0 mg/kg/day 19512F 19513F 19514F 19515F 0.353 0.258 0.325 0.286 Group 2 0.1 mg/kg/day 19528F 19529F 19530F 19531F 26.4 21.7 47.8 32.1 19516F 0.138 19532F 34.6 Fetal Liver Fetal Liver 19504 19505 0.153 <LOQ 19518 19519 7.32 8.64 19506 0.149 19521 7.23 19507 <LOQ 19523 7.68 19509 0.204 19524 8.54 19512 <LOQ 19528 9.33 19516 LOQ=Limit of quantitation (< 0.112 pg/g) NS=Not sampled <LOQ 19529 6.73 NOTE: PFOS concentrations in liver are expressed as micrograms of PFOS/g of liver matrix (pg/g) NOTE: Liver analysis were conducted at a contract laboratory but were corrected by 3M to reflect the official purity values from the COA. Revised reports from the final contract laboratories will be added as a report amendment at a later date. 3M Environmental Laboratory Page 94 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Table 18. Results of 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-6295.12 via Gavage (Continued) Group/Dose Sample # Concentration of PFOS (all values in pg/g) Group/Dose Sample # Concentration of PFOS (ail values in pg/g) 19533F 67.0 19549F NS 19534F 88.1 19550F 342 19535F 111 19551F NS 19536F 88.1 19552F NS 19537F 143 19553F 351 19538F 144 19554F 358 19539F 105 19555F 497 19540F 110 19556F 304 19541F 96.8 19557F 268 19542F 108 19558F 334 Group 3 0.4 mg/kg/day 19543F 19544F 85.9 113 Group 4 1.6 mg/kg/day 19559F 19560F 881 250 19545F NS 19561F 397 19546F 83.5 19562F 215 19547F 119 19563F 426 19548F 137 19564F 416 Fetal Liver Fetal Liver 19533 19534 19536 27.5 34.1 19.0 19560 19562 105 68.0 19543 19546 30.7 41.6 --------------------- LOQ=Limit of quantitation (< 0.112 pg/g) NS=Not sampled NOTE: PFOS concentrations in liver are expressed as micrograms of PFOS/g of I ver matrix (pg/g) NOTE: Liver analysis were conducted at a contract laboratory but were corrected by 3M to reflect the official purity values from the COA. Revised reports from the final contract laboratories will be added as a report amendment at a later date. 3M Environmental Laboratory Page 95 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTO X-110 LRN-U2849 Table 18. Results of 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-6295.12 via Gavage (Continued) Group/Dose Group 5 3.2 mg/kg/day Sample # Concentration of PFOS (all values in pg/g) 19565F 19566F 381 594 19567F 623 19568F 596 19569F 19570F 468 740 19571F 19572F 19573F 448 455 787 19574F 735 19575F 802 19576F NS 19577F 521 19578F NS 19579F 612 19580F 782 Fetal Liver 19568 260 19569 183 19571 199 19572 184 19577 339 19579 213 LOQ=Limit of quantitation (< 0.112 pg/g) NS=Not sampled NOTE: PFOS concentrations in liver are expressed as micrograms of PFOS/g of liver matrix (pg/g) NOTE: Liver analysis were conducted at a contract laboratory but were corrected by 3M to reflect trie official purity values from the COA. Revised reports from the final contract laboratories will be added as a report amendment at a later date. 3M Environmental Laboratory Page 96 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTOX-110 LRN-U2849 Appendix F: Example Calculations Formula Used for Urine Analyses in Study FACT TOX-110 A R (ng/mL) x DF x SC x 1.0 pg x PC = Reported (Concentration (pg/mL) 1000 ng Calculation Used for Group 2, GD15, Animal ID 19522F 123.57 ng/mL x 1 x 0.9275 x 1.0 p g x 0.864 = 0.0990 pg PFOS/mL urine 1000 ng AR-- Analytical result from MassLynx summary DF-- Dilution factor SC-- PFOS salt correction constant (0.9275) PC-- PFOS purity correction factor (86.4%) 3M Environmental Laboratory Page 97 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTOX-110 LRN-U2849 Appendix G: Contract Lab Reports This Appendix includes the following contract laboratory' reports: Battelle Memorial Institute, "Oral (Gavage) Pharmacokinetic Study of PFOS in Rats," 63 pages Advanced Bioanalytical Services, Inc., "Method Validation for the Quantitation of Perfluorooctanesulfonate (PFOS) in Rat Serum by Turbo Ion Spray LC/MS," 50 pages Advanced Bioanalytical Services, Inc., "Quantitative Determination of Perfluorooctanesulfonate (PFOS) in Rat Serum from Study #FAC T-TOX-110 Using Turbo Ion Spray LC/MS," 24 pages Advanced Bioanalytical Services, Inc., "Addendum--Quantitative Determination of Perfluorooctanesulfonate (PFOS) in Rat Serum from Study #FACT-TOX-110 Using Turbo Ion Spray LC/MS," 3 pages Centre Analytical Laboratories, Inc., "Oral (Gavage) Pharmacokinetic Study of PFOS in Rats," 103 pages 3M Environmental Laboratory Page 98 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTO X-110 LRN-U2849 BIOLOGICAL SAMPLE ANALYSIS Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-110 II Baifeiie Pu"ins Technology To Work FINAL REPORT ORAL (GAVAGE) PHARMACOKINETIC STUDY OF PFOS IN RATS SPONSOR 3M Toxicology Services-Medical Department 3M Center, Building 220-2E-C2 St. Paul, M N 55144-1000 Testing Facility Battelle Memorial Institute 505 K ing Avenue Columbus, Ohio 43201-2693 Prepared Bv Patrick L. South, B.S. 31V Environmental Laboratory Page 99 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 Battelli Study Number: N003296-F 3M Toxicology Services Protocol Number: FA C T-T O X -110 FINAL REPORT ORAL (GAVAGE) PHARMACOKINETIC STUDY OF PFOS IN RATS 3M Environmental Laboratory Page 100 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTOX-110 ' LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FA C T-T O X -110 ORAL (GAVAGE) PHARMACOKINETIC STUDY OF PFOS IN RATS 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 Perfluorooctanesulfonate (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 fluorochemicals mentioned in the analytical method were not investigated. 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 101 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: F A C T T Q X -1 10 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FA C T-T O X -110 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: Phase Inspected Inspection Date Date Reported to Battelle Task Leader/ Battelle Management Date Reported to Offsite Study Director/ Management______ Sample homogenization Sample weights Sample analysis Sample processing Audit final report Audit study file Audit final report 8/27/1999 8/27/1999 8/30/1999 8/30/1999 9/20/1999 9/20/1999 2/16/2001 9/20/1999 9/20/1999 9/20/1999 9/20/1999 9/20/1999 9/20/1999 2/16/2001 9/29/1999 9/29/1999 9/29/1999 9/29/1999 9/29/1999 9/29/1999 2/20/2001 Battelle Memorial Institute 3M Environmental Laboratory iv Page 102 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTQX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol N um ber: FA C T-T O X -110 GOOD LABORATORY PRACTICES COMPLIANCE STATEMENT Study Title: ORAL (GAVAGE) PHARMACOKINETIC 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 of regulated studies. Battelle Principal Investigator Study Director Date Date 3M Environmental Laboratory V Page 103 3M Medical Department Study: T-6295.12 --~ BACK TO MAIN ______________________________________ Analytical Report: FACT TOX-11Q LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Num ber: FA C T-T O X -110 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 of Tables Table 1. Example of Instrument Parameters Used to Analyze Samples.............................................2 A Appendix A (Results) Summary Results for Rat liver Sample Analysis............................................................................ '...A-l Appendix B (Daily Acceptance Criteria Summary) Daily Acceptance Criteria Summary..................................................................................................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 Deviation) Protocol, Amendments, and Deviation.................................................................................................... E-l Appendix F (PFOS Purity Report) PFOS Purity Report................................................................................................................................. F-l 3M Environmental Laboratory vi Page 104 3M Medical Department Study: T-6295.12 ' BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Num ber: FA C T-T O X -110 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-110 and the results from this analysis. See Appendix E for a copy of the study protocol, amendments, and deviation reports. 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 1H,1H,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 418-013. 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). 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, although present in the stock solutions, w ere n o t m onitored. Q uadratic regressions w eighted 1/x w ere used to construct the calibration curves. 3M Environmental Laboratory 1 Page 105 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT T O X -110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Num ber: FA C T-T O X -110 T ab le 1. E x a m p le o f I n s tr u m e n t P a r a m e te r s U sed to A n a ly z e S a m p le s LC/M S/M S System A utosam pler HPLC pumps Mass spectrom eter A nalytical column M obile phase co m ponents G radient profile Injection volume Flow f f Column temp HPLC pressure MS source Desolvation gas N ebulizer gas Source block temp Desolvation tem p Cone voltage Collision energy Collision gas Mu&pS* R e so lu tio n Ions m onitored Total ru n tim e Approxim ate retention times: Make: Gilson Model: 234 Make: Gilson Models: 305 and 306 Make: Micromass Model: Quattro LC with Z-spray source Keystone Betasil C18, 5pm, 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 11 0 0.3 10 pL LC column flow at start split to 25 uL/min into the MS Ambient Approximately 1000 psi at gradient start Electrospray, Negative Ion Nitrogen at -575 L/hr Nitrogen at -8 0 L/hr 140C 250C 70 V 40 eV Argon, gas cell, at ~2.5xlO J mb 650 V 12.0 for M SI; 10.0 for MS2 427>81 MRM transition for SS 499>99 MRM transition for PFOS 11 minutes SS: 4 min PFOS: 4.3 min 4.2 Results -4.2 I Q u ality C o n tro l S\sk-m suitability acceptance criteria were established during the method validation and .in iin.lulled m A ppendix C, Section IX Acceptance Criteria. Relevant statistics from each sample -i i a u pim u k d in Appendix B. Representative chromatograms are given in Appendix D. 2 3M Environmental Laboratory Page 106 3M Medical Department Study: T-6295.12 ' --' ' BACK TO MAIN ____________ Analytical Report: FACT TQX-110 ' LRN-U2849 4.2.2 Sample Results Battelle Study Number: N003296-F 3M Toxicology Services Protocol Num ber: FA CT-TO X -110 The results of the sample analyses as well as a method detection limit determination 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 notation ALOQ denotes "Above Limit of Quantitation" for samples that had concentrations higher than the theoretical concentration for the 13 pg/g calibration standard. Samples that were initially ALOQ were diluted with blank liver homogenate and re-extracted. Samples that were expected to be ALOQ were first diluted with blank liver homogenate before extraction. 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 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. The method of MDL determination was provided by the Sponsor. 5.0 Conclusions ' All analyses met acceptance criteria unless otherwise noted. 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. A ndre, Ph.D . Richard W. Slauter, Ph.D., D.A.B.T. Patrick L. South, B.S. Battelle Principal Investigator Senior Program Director Mass Spectroscopist 7.0 Specimen Storage and Record Archives Refer to Appendix E, protocol amendment 2 for record 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 Page 107 3M Medical Department Study: T-6295.12 ' BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FA CT-TO X -l 10 /I APPENDIX A -RESULTS 3M Environmental Laboratory Page 108 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol N um ber: F A C T -T O X -110 RAT LIVER SAMPLE RESULTS STUDY: N003296-F ANALYSIS DATE AND INSTRUMENT ID: DATA ENTERED: Electronically SPREADSHEET SOFTWARE: Excel 97 FINAL LIVER SAMPLE RESULTS: Animal numbers ending in "P " indicate pooled fetal liver 25Aug99; 9053 Corrected Note if Note if A nim al Dose Grp PFOS Sample is Rerun is Number__________ mg/kg/day________ Cone (pg/g)_________ a Rerun_________ Needed Animal 19501 Animal 19502 Animal 19504 Animal 19504P Animal 19505 Animal 19505P Animal 19506 Animal 19506P Animal 19507 Animal 19507P Animal 19508 Animal 19509 Animal 19509P Animal 19510 Animal 19511 Animal 19512 Animal 19512P Animal 19513 Animal 19514 Animal 19515 Animal 19516 Animal 19516P Animal 19520 Animal 19517 Animal 19518P Animal 19533 Animal 19533P Animal 19550 Animal 19560P Animal 19565 Animal 19568P 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 (SEE NOTE 2) 0.1 0.1 0.4 0.4 1.6 1.6 3.2 3.2 0.400 0.353 0.145 0.177 0.201 BLOQ 0.466 0.173 0.194 BLOQ 0.266 0.203 0.236 0.235 0.958 0.408 BLOQ 0.299 0.376 0.331 0.160 BLOQ ALOQ ALOQ 8.47 ALOQ ALOQ ALOQ ALOQ ALOQ ALOQ YES (1) YES (1) YES (1) YES (1) YES (1) YES (1) YES (1) YES (1) BLOQ = BELOW CONC OF LOWEST STANDARD IN CALIBRATION CURVE ALOQ = ABOVE CONC OF HIGHEST STANDARD IN CALIBRATION CURVE (1) = SAMPLE TO BE DILUTED BEFORE REPEAT EXTRACTION (2) = SAMPLE INITIALLY MARKED AS 0.1 mg/kg/day; LABEL HAND-CORRECTED TO INDICATE VEHICLE 3M Environmental Laboratory A -l Page 109 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TO X-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FA C T-TO X -II0 RAT LIVER SAMPLE RESULTS STUDY: N003296-F ANALYSIS DATE AND INSTRUMENT ID: 28Aug99; 9053 DATA ENTERED: Electronically SPREADSHEET SOFTWARE: Excel 97 FINAL LIVER SAMPLE RESULTS: Anim al numbers ending in "P " indicate pooled fetal liver Animal Number Dose Grp mg/kg/day Corrected PFOS Cone (pg/g) Note if Sample is a Rerun Note if Rerun is Needed Animal 19517 Animal 19518 Animal 19519 Animal 19519P Animal 19520 Animal 19521 Animal 19521P Animal 19522 Animal 19523 Animal 19523P Animal 19524 Animal 19524P Animal 19525 Animal 19526 Animal T 9527 Animal 19528 Animal 19528P Animal 19529 Animal 19529P Animal 19530 Animal 19531 Animal 19532 Animal 19533 Animal 19533P Animai 19550 Animal 19560P Animal 195 6 5 Animal 19568P 0.1 0.1 0.1 0.1 0 (SEE NOTE 2) 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 0.1 0.4 0.4 1 .6 1.6 3 .2 3.2 49.3 25.2 21.9 10.0 17.8 23.3 8.37 37.0 20.5 8.89 22.7 9.89 42.2 39.4 53.7 30.5 10.8 25.1 7.79 55.3 37.2 40.1 77.6 31.8 396 122 441 301 YES (1) YES (1) YES (1) YESm YES (1) YES (1) YES (1) YES (1) BLOQ = BELOW CONC OF LOWEST STANDARD IN CALIBRATION CURVE ALOQ = ABOVE CONC OF HIGHEST STANDARD IN CALIBRATION CURVE (1) = SAMPLE REPEATED FROM 25AUG99 ANALYSIS (2) = SAMPLE INITIALLY MARKED AS 0.1 mg/kg/day; BAG LABEL HAND-CORRECTED TO INDICATE VEHICLE 3M Environmental Laboratory A-2 Page 110 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TO X-110 LRKI-U249 Battelli Study Number: N003296-F 3M Toxicology Services Protocol Num ber: FA C T -T O X -110 RAT LIVER SA M P LE RESULTS STUDY: N003296-F A N A L Y S IS D A T E A N D IN S T R U M E N T ID: DATA ENTERED: SPREADSHEET SOFTWARE: 30Aug99; 9053 E le c tro n ic a lly Excel 97 FIN AL L IV E R SA M P LE RESULTS: A nim al n um bers en d in g in "P" indicate pooled fetal liver C o rre c te d Note if A nim al Dose Grp PFOS S am p le is ________N u m b e r____________ m g /k g /d a y __________C o n e (p g /g )___________ a R e ru n Note if Rerun is Needed Animal 19534 Animal 19534P Anim al 19535 Animal 19536 Animal 19536P Animal 19537 Animal 19538 Animal 19539 Animal 19540 Animal 19541 Animal 19542 Animal 19543 Animal 19543P Animal 19544 Animal 19546 Animal 19546P Animal 19547 Animal 19548 Animal 19562P Animal 19569P Animal 19571P Animal 19572P Animal 19577P Animal 19579P 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 1.6 3.2 3.2 3.2 3.2 3.2 102 39.5 128 102 22.0 165 167 121 127 112 125 99.4 35.5 131 96.6 48.2 138 159 78.7 212 230 213 392 246 BLOQ = B E L O W C O N C O F L O W E S T STAND ARD IN CALIBRATIO N CU R VE ALO Q = A B O V E C O N C O F H IG H E S T STAND ARD IN CALIBRATIO N CU RVE 3M Environmental Laboratory A-3 Page 111 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TQX-110 ' LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Num ber: FA C T-TO X -l 10 RAT LIVER S A M PLE RESULTS STUDY: N003296-F A N A LY S IS D A TE A N D IN S T R U M E N T ID: DATA ENTERED: SPREADSHEET SOFTWARE: 01Sep99; 9053 E le c tro n ic a lly Excel 97 FINAL LIVER S A M P LE RESULTS: A nim al n u m b e rs en d in g in "P" indicate pooled fetal liver C o rre c te d Note if Anim al Dose Grp PFOS S a m p le is ________N u m b e r____________ m g /k g /d a y __________ C o n e (p g /g )___________ a R e ru n Note if Rerun is Needed Animal 19553 Animal 19554 Animal 19555 Animal 19556 Animal 19557 Animal 19558 Animal 19559 Animal 19560 Animal 19561 Animal 19562 Animal 19563 Animal 19564 Animal 19566 Animal 19567 Animal 19568 Animal 19569 Animal 19570 Animal 19571 Animal 19572 Animal 19573 Animal 19574 Anim al 1957S Animal 19577 Animal 19579 Animal 19580 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 406 414 575 352 310 387 1.02E+03 289 460 249 493 482 687 721 690 542 856 518 527 911 851 928 603 708 905 f BLOQ = B E L O W C O N C O F L O W E S T STAN D A R D IN CALIBRATIO N CU R VE A LO Q = A B O V E C O N C O F H IG H E S T S TA N D A R D IN CA LIBR ATIO N C U R V E 3M Environmental Laboratory A-4 Page 112 BACK TO MAIN 3M Medical Department Study: T-6295.12____________________________________________ Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol N um ber: FA C T-T O X -l 10 M E TH O D DETECTION LIM IT (M DL) RESULTS STU D Y: N003296-F A N A L Y S IS DA TE AN D IN S T R U M E N T ID: 28Aug99; 9053 DATA ENTERED: E le c tro n ic a lly SPREADSHEET SOFTWARE: Excel 97 A ll cones in pg/g Calculated Concentration of Replicate 1 Calculated Concentration of Replicate 2 Calculated Concentration o f Replicate 3 Calculated Concentration of Replicate 4 Calculated Concentration of Replicate 5 Calculated Concentration of Replicate 6 Calculated Concentration o f Replicate 7 PFOS 0.1184 0 .1 1 9 2 0.1251 0.1239 0.1351 0.1248 0.1225 Mean Concentration Std. Dev. 0.1241 0.0055 Spike Level 0.1340 : : MDL determined ; S/N :001729 22.57 L O Q (det. from 10 x std.dev. "noise") LOQ (det from cal curve low std.) Curve Coeff of Determination Yes 0.05501 0.1340 0.9981 Date analyzed M ethod 08/28/99 L C /M S/M S Key 1 - Spike Level too high; Spike Level must be < lOx MDL 2 - Spike Level too low; Spike Level m ust be > MDL 3 - S/N too low; S/N must be > 5 4 - Coeff o f Det of calibration curve unacceptable 3M Environmental Laboratory A-5 Page 113 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FAC TTQ X-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-l I0 APPENDIX B-DAILY ACCEPTANCE CRITERIA SUMMARY .r 3M Environmental Laboratory Page 114 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTQX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Num ber: F A C T -T O X -110 DAILY ACCEPTANCE CRITERIA SUMMARY PFOS IN RAT UVER STUDY: N003296-F SPREADSHEET SOFTWARE: Excel 97 DATA ENTERED MANUALLY NA-Not Applicable ' ' Cal Curve Sys Suit Homogenizer Analysis Date C oelf o f Det %RSD Fort Ave Recov Diln Std Ave Recov Ave OC 1 %RE CtRSD) Ave QC 2 ARE (% RSD ) Ave QC 3 Ave QC 4 % R E CARSO) ARE (*ARSr August 25,1999 0.9699 4.0 111.1 NA -7.1 (15.8) 0.0 (14.2) -5.5(13.7) * 2^5 (16.8) August 28. 1999 August 30. 1999 0.9961 0.9943 3.1 7.5 112.4 110.9 102.4 116.0 2.7 (0.9) -1.8 (4.0) 2.3 (3.4) -1.9 (4.2) -3.8 (4.1) -6.5 (6.4) -6.9 (0.9) 1.4 (1.1) September 1, 1999 0.9951 5.3 118.1 110.1 1.9 (4.8) 1.8 (5.6) -10.7 (4.6) 18.0 (7.4) 3M Environmental Laboratory B-l Page 115 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT T O X -110 LRN-U2849 Battelie Study Number: N003296-F 3M Toxicology Services Protocol Number: FA C T-T O X -110 APPENDIX C-METHOD it 3M Environmental Laboratory Page 116 BACK TO MAIN 3M Medical Department Study: T-6295.12____________________________ Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol N um ber: F A C T -T O X -110 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS Version 1.0 Study No.: Analyst/Date: _ D ale of R e\ ision Revisions to the method R c\ iscd b\ A p p r o \ e d bv ^ fl Written by: Patrick L. South Date: 9*? Approved by:. V - C ' ____________ Date: JcfaJC. Andre, Ph.D. Manager, Bionalytical Chemistry v PI . > 3M Environmental Laboratory Page 1 of 16 C-l Page 117 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 --------------------------------------------------------------------------------------------------------------------------- LRN-U24 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FA C T-TO X -II0 METHOD FOR ANALYSIS OF POTASSIUM PER FLU O RO O CTA 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. S u m m a r y The extraction and analysis of potassium pertluorooctanesulfonate and related Quorochemicals in rat liver is performed. Calibration standards are prepared by spiking blank liver homogenate with solvent standards from two independently-prepared stocks. 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 pertluorooctanesulfonate and related fluorochemical compounds found in Sprague-Dawley rat liver. m. S a m p l e s See Chain of Custody records if applicable. IV. G en er a l Instr u c tio n s Calibrate all required balances according to the SOP on balance usage. Make equivalent dilutions when the volume needed varies from the volume stated in the method. Label all standard and reagent solutions as specified in the 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 the method document the change, and obtain the 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. the documentation may be made in a single location on that page. If multiple staff make entries, the additional entries must be initialed and dated by the person nnlrin; 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 the analyst deems it appropriate, unless the order for certain activities is specified % Stocks will be used for the 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. 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 documentation. Check all labels carefully to ensure that all materials are not expired and that they are the proper purity or grade. Page 2 of 16 3M Environmental Laboratory C-2 Page 118 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTO X-110 LRN-U2849 Bane lie Study Num ber: N 003296-F 3M Toxicology Services Protocol Num ber: F A C T -T O X -110 METHOD FOR ANALYSIS OF POTASSIUM PER FLU O RO O CTA N ESU LFO N A TE (PFO S) IN RAT LIV ER BY LC/M S/M S Version 1.0 Study No.:________________________ __ Analyjt/Date:_______________________ ___ Materials Use Potassium Perfiuorooctanesulfonate (PFOS) 1H.1H.2H.2HPerfluorooctane Sulphonic Acid M-556 M370 PFOSAA PFOSA PFOSEA Rat Liver Analytical Standard Surrogate Standard Analytical Standard Analytical Standard Analytical Standard Analytical Standard Analytical Standard Matrix Ammonium Acetate, N H ftO, Sodium Hydroxide, NaOH Tetrabutylammonium Hydrogensulfate (TBA), TOUCH,M.NfHSCM Sodium Carbonate, Na,CO, Sodium Bicarbonate, NaHCOi Ethyl Acetate Mobile Phase Reagent Prep Extract Prep Extract Prep Extract Prep Extract Prep Methanol Milli-Q Water pH 7 Buffer pH 10 Buffer M obile Phase, Stocks. WS Reagent Prep, Mobile Phase pH meter calibration pH meter calibration T ablet. Materials Supplier Grade or Puritv 3M ICN 3M 3M 3M 3M 3M Harlan SpragueDawley Storage Temp Room Temp Room Temp Room Temp Room Temp Room Temp Room Temp Room Temp --20C RT RT RT Lot o rli) Millipore ASTM Type I RT RT RT RT RT RT RT RT means Room Temperature. VI. E quipm ent 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 119 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTO X-110 LRN-U2849 Battelle Study Number: N 003296-F 3M Toxicology Services Protocol N um ber: FA C T -T O X -110 M ETHOD FO R ANALYSTS OF POTASSIUM PERFLU O RO O CTA NESU LFO NA TE (PFOS) IN RAT LIV ER BY LC/M S/M S Version 1.0 Study No.:_________________________ Analyst/Date:___________________________ E q u ip m en t Analytical Balance Weight Set Use Weigh Standards or Reagents Calibrate Balance Table 2. Equipment M anufacturer M m ld X nr SIN Pipettor Pipet Samples Pipettor Pipet Samples Pipettor Pipettor Pipet EtOAc extraction phase Pipet Reagents, WIS Eppendorf Repeater Vortexer Mix Samples Freezer (-20C) Refrigerator il-9 a 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 StockweU Scientific Blue Falcon Blue Falcon Elkay Polypropylene, 15 mL Polypropylene, 15 mL Polypropylene, 12 x 75 mm 5 mL polypropylene SW8599 2096 2002 127-T160-56P Evaporator Evaporate Extracts Zymaric Turbovap LV Syringe Filters Homogenizer pH meter Electrode Volumetric Flasks. Class A Volumetric Pipets, Class A Transfer Pipets, Plastic Filter Extract Grind liver Determine Buffer pH 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 120 BACK TO MAIN 3M Medical Department Study: T-6295.12 ----------------------------------------------- -- Analytical Report: FACT TOX-110 ' LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Num ber: FA CT-TO X -110 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS Version 1.0 Study No.: Analyst/Date: _ vn. P r o c e d u r e A. P reparation or 2 mM 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 No:______________ B. P re p a ra tio n o f--29% Sodium H ydroxide Solution Weigh 200 * 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: _________ t Actual volume Milli-Q water: ________ Date of preparation:___________________ Study No: C. P reparation 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 o f preparation: ______________________ Study No:______________ D. Preparation of Tetrabutylam m onium Hydrogensulfate (TBA) Solution, 0.5 M, (pH 10) pH Meter Calibration pH buffer 7 pH buffer 10 pH reading:_______ pH reading:_______ Add 1 69: 1 gofTBA to -500 mL of Milli-Q water in a beaker. Adjust the pH to 10.00 : 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 : 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 s 0.02 with 2.9% Sodium Hydroxide Solution as necessary. Page 5 of 16 3M Environmental Laboratory C-5 Page 121 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol N um ber: FA C T -T O X -110 METHOD FOR ANALYSIS OF POTASSIUM PERFLUO RO O CTA NESU LFO NA TE (PFOS) IN RAT LIV ER BY LC/M S/M S Version 1.0 Study No.: Analyst/Dale: _ 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 BufTer 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 idM 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 No:______________ Component B: Mix together SO 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 Diludon Volume:__________________ Date of Preparation: _________________ Study No: ________________ Page 6 of 16 3M Environmental Laboratory C-6 Page 122 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTO X-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Num ber: FA C T -T O X -110 METHOD FOR ANALYSIS OF POTASSIUM PER FLU O R O O CTA 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: _ 2. WSS (1000 ng/mL): Dilute 100 juL of stock surrogate standard to 25 mL with methanol and mix.. Actual Volume of Stock Internal Standard: ___________ Actual Dilution Volume:__________________ Date of Preparation: _________________ H . P reparation of Calibration Solvent Stocks and W orking Standards M andarti Stock 1A Stock IB Stock 2A Stock 2B Stock 3A Stock 3B Stock 4A Stock 4B Stock 5A Stock 5B 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 the 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 light Repeat the process with Replicate B stocks. The m ixed solvent stocks are used to prepare the w orking 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:______________________________ Siiiircc PFOS PFOS M-356 M-556 M570 M570 PFOSAA PFOSAA PFOSA PFOSA PFOSEA Table 3. Calibration Solvent Stocks and Working Standard T.if-ct Actual Amount Target Attuai Nominal Amount Anul\tii'.il Stil. Final Voi Final Voi. Cone Stock, or WS (mL) ImU (nji-ml.) 30 i 1 mg 10 5,000,000 25 i 0.5 mg - mg* 10 2,500,000 50 1 mg p" : ms* 10 5,000,000 25 z 0.5 mg mg* 10 2,500,000 50 t 1 mg . ' mg* 10 5,000.000 25 z 0.5 mg . mg* 10 2,500,000 93 z 1 mg - - ! mg* 10 5,000,000 46 z 0.5 mg mg* ;! 10 2,500.000 50 z 1 mg "IR* 25 z 0.5 mg ! mg* 10 10 5.000.000 2.500,000 50 z 1 mg mg* 10 5,000.000 Page 7 of 16 3M Environmental Laboratory C-7 Page 123 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTO X-110 ----------------------------- bR N-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FA C T-T O X -110 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/M S/M S Version 1.0 Study No.: Analyst/Date: _ Stock 6B Mixed Stock A Mixed Stock B WS l WS 2 WS 3 WS 4 WS 5 WS 6 WS 7 PFOSEA Stocks 1 thru 6 Rep A Stocks 1 thru 6 RepB Mixed Stock A Mixed Stock B WS 1 WS2 WS3 WS 4 WS 5 25 t 0.5 ma 5 mL each** 5 mL each** 1 mL** ma* ____mLeach mLeach ____ :mL - 1 mL** mLf, 2 mL** 2 mL** 2.5 mL** 2.5 mL** 2 mL** :; ! m l. 10 50 50 25 25 to 10 10 10 10 2.500,000 500,000 250,000 20,000 10,000 4000 2000 1000 500 200 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 -20"C) storage. Actual Mass ofLiver___________ Actual volume of water.__________ Date of prep :__________________ Study:_____________________ Replicate # Mass (ma) Determine density of calibration/QC matrix: MIX HOMOGENATE THOROUGHLY and determine the mass in milligrams o f 10 replicate weighings of l mL portions of the T H O RO U G H LY MDCHD homogenate. MDCHOMOGENATE IMMEDIATELY PRIOR TO EACH ALIQUOT REMOVAL. Table 4. Calibration Stda/QCi Matrix Density 2 3 4' ' 7 . , mtfvm 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 124 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FAC TTO X-110 LRN-U2849 Bauelle Study Number: N003296-F 3M Toxicology Services Protocol N um ber: FA C T -T O X -110 METHOD FOR ANALYSIS OF POTASSIUM PERJFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/M S/M S Version 1.0 Study No.: Analyst/Date: _ C.il Std/Blunk i 2 3 4 5 6 7 Blank Source WS 1 WS 2 WS 3 WS 4 WS 5 WS 6 WS 7 MeOH Table 5. Calibration Standards and Blanks Target Voi Actujl Voi Target Actual Nominili 0<L) (jwL) Final Voi Final Voi Conc (mL) (mL) (ni/mL) 50 0.5 2000 50 0.5 1000 50 0.5 400 50 0.5 200 50 0.5 100 50 0.5 50 50 0.5 20 50 0.5 0 Date of preparation of cal stds/blank: Nomin.il Conc OiU/ll) 13 6.6 2.6 1.3 0.66 0.33 0.13 0 J. P reparation of Q uality Control Liver Samples (QCs) 1. Quality Control Working Standards Dilute the following source volumes methanol in volumetric flasks and mix well. Prepare fresh when used. Actual volumes are in parentheses. Sotti ID OC WS 1 QC WS 2 QC WS 3 QC WS 4 Source Mixed Stock A ! Mixed Stock B QC WS 1 QC WS 2 Tabled. OC WS Preparation Voi Source. mL Final Voi, mL 1 3 f___ ir 100 ( :-A j. 50 ( _ : ) 100f 50 ( :... ) Conc. n"/mL 15.000 5000 1125 250 2. Preparation of Quality Control Liver Samples Prepare each QC in bulk by filling the volumetric flask approximately half full with undiluted liver homogenate (STIR HOMOGENATE WHILE ALIQUOTING), adding the appropriate QC WS, mixing, and diluting to volume with undiluted liver homogenate (STIR HOMOGENATE WHILE ALIQUOTING). MIX THOROUGHLY and dispense 2.5-mL aliquots into polypropylene tubes and store at approximately -20C. Table 7. QC Preparation QC Source Vol Source. mL Final Voi mL Conc. nj!/mL Cune, ni/*! 1 OC WS 1 2.5 ( - 25f;:: ; - ) -::. 1500 10 2 QC WS 2 2-5 ( 3 OC WS 3 23( ) : 25 ( ) ) M- n . r ) ; 500 112.5 3.3 0.7 4 QC WS 4 2.5 ( _ _ H 25 ( =: ) 25 0.16 Date of QC prep:__________________ Study:____________________ K. P reparation of MS Check Standard for System Suitability Pipet 250 u-L of WS 2 a t-10.000 ng/mL and 2.5 mL of WSS a t-1000 ng/mL in methanol into die same 50-mL volumetric flask. Dilute to volume with MeOH and mix. Page 9 of 16 3M Environmental Laboratory C-9 Page 125 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTO X-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Num ber: FA C T -T O X -110 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/M S/M S Version 1.0 Study No.:__________________________ Analyst/Date:____________________________ L. P reparation of homogenizer recovery liver samples To determine the recovery from the homogenization process, unhomogenized 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 0.3 g of unhomogenized blank liver into each of 6.13 mL polypropylene centrifuge tubes. Record weights of liver. 2. Add 100 pL of WS 1,3, and 4 (one WS per duplicate tubes) to prepare fortifications at approximately 4, 0.8, and 0.4 pg/g. 3. Multiply the mass of liver in g by 2.3 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. 3. Clean homogenizer with MeOH between samples. 6. Cap and vortex homogenate for use in extraction. M . Preparation of Dilution Check Sample 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 30 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. Homogenization of study samples 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. Cap and vortex homogenate for use in extraction. O. Analysis Standards, Blanks, QCs, and Samples 1. MIX LIVER HOMOGENATES THOROUGHLY BEFORE ALIQUOTING and pipet 500 n 't 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 >xL WSS and vortex. 4. Add 0.5 mL of 0.5 M TBA (pH 10) to all tubes and vortex briefly. 5. Add 1mL 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 dean polypropylene tube. Page 10 of 16 3M Environmental Laboratory C-10 Page 126 BACK TO MAIN 3M Medical Department Study: T-6295.12 ________________________________________________ Analytical Report: FACT T O X -110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FA C T-T O X -110 METHOD FOR ANALYSIS OF POTASSIUM PERFLU O RO O CTA NESU LFO NA TE (PFOS) IN RAT L IV E R BY LC/M S/M S Version 1.0 Study No.:__________________________ Analyst/Date:____________________ ________ 9. Evaporate to dryness under nitrogen at a setting of 30 C for -60 minutes. 10. Reconstitute the residues in 500 n L 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 preparadon if held at room temperature (recap the vials if reusing). Date of cal std/blank extract prep:_______________________ Date of QC extract prep:______________________________ P. LC/MS/MS Analysis iC P ffo fo S'. 1. Use the system conditions specified in Table yS. 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 Autosampler Make: ____ Model: ID: III'LC Pumps Make: ____ Model: ID: Mass Spectrometer Make: Model: ID: Anal\ tieal column Keystone Betasii 0 8 , 5 ^ 2 x 5 0 mm. Part No. 055-703-2, S/N: __________ L ot_______ Mobile Phase Component A: Ammonium acetate:methanol, 60:40, v:v Components Component B: Ammonium acetate:methanol. 5:95. v:v Gradient profile Time, min /oB Flow. mL/min 0 0 0.3 1 0 0.3 4.5 100 0.3 6 100 0.3 61 100 0.6 8.5 100 0.6 9 0 0.3 11 0 0.3 Injection soluine 10 uL ( _________________ Flow split 1LC column flow split to *30 uL/mia ( *iL/min) into the MS at run start Column Temp Ambient HPLC Pressure 1000 psi at gradient starti osi) MIS Source Electrospray. Negative Ion Desolsation g.ts Nitrogen at 575 L/hr ( L/hr) Nebulizer "as Nitrogen at 80 L/hr ( L/hr) Source Block Temp 140C( C) Desolsation Temp 250C( C) Cone voltage 70 V(____ V) PFOS,-f9"(D 20 V( V) PFOSA, PFOSAA. PFOSEA, M-556. M570 Collision energy 40 eV (___ eV) PFOS, I t PFOSA, PFOSAA. M-556, M570 30 eV ( eV) PFOSEA Collision gas Argon at *2.5 x 1O'3mb gas cell ( mb) Multiplier *650 V (___ V)_________________ Resolution *12.0 for MSI ( ); *10.0 for MS2( ) I I I 1 (bsnov& * ch>"sr"; *fcejfirM Page 11of 16 C-ll 3M Environmental Laboratory Page 127 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTQ X-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Num ber: F A C T -T O X -110 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS Version 1.0 Study No.: Analyst/Date: _ Inns monitored Total run time Approximate retention times: 427>81 MRM transition for Surrogate Standard (SS) 499>99 MRM transition for PFOS 556>78 MRM transition for M-556 570> 169 MRM transition for M70 384>169 MRM transition for PFOSAA 498>78 MRM transition for PFOSA 526>169 MRM transition for PFOSHA *11 minutes ( min) SS: 4 min ( min) PFOS: 4.3 min (___ __ min) M-556: 4.5 min (_____ min) M570:4.6 min (___ __min) PFOSAA: 4.7 min (______min) 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 maw spectrometer using a suitable reference compound, or verify that the calibration is suitable by visual inspection (on 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. 8. Run set sizes should typically not exceed 80 injections due to instrument responseroll-offconsiderations. Longertunsmaybeperformed,buttheyposea risk o f yielding unacceptable curve results. VUL 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 * Diln Factor Page 12 of 16 3M Environmental Laboratory C-12 Page 128 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTQ X-110 LRN-U2849 Battel le Study N um ber: N 003296-F 3M Toxicology Services Protocol Num ber: F A C T -T O X -110 METHOD FOR ANALYSIS OF POTASSIUM PERFLU OROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/M S/M S Version 1.0 Study No.:__________________________ Analyst/Date:____________________________ Where diln factor" 2/1.8 1.1111 to account for 10% diln of liver homogenate in cal std and QC matrices. 5. 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(pg/g) - Cone (ng/mL) Diluted Liver density (mg/mL) x 1000 mg/g x 10J pg/ng 7. Assure that the integrations of the peak areas of the test article and surrogate standard are correct. Flag manual integrations where performed. Calculate the exact concentration of each liver standard. 8. Calculate the regression equation relating the peak response ratio (test artide/SS) of each calibration standard (y-axis) to test article concentration in liver (x-axis) for PFOS, M-556, M570, 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, M356, M570, 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 1J 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 o f the check standard. DC A c c e p t a n c e C r it e r ia A. M S 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 129 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT T O X -110 ------------------------------- LR N-U 2B4y Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FA C T -T O X -110 METHOD FOR ANALYSIS OF POTASSIUM PER FLU O RO O CTA NESU LFO NA TE (PFOS) IN RAT LIVER BY LC/M S/M S Version 1.0 Study No.: Analyst/Date:_ Table 9. Calibration Standard Acceptance limits ANALYTE PFOS % Error 20 (25% at LOO) M-S56 M570 PFOSAA PFOSA 20 (25% at LOO) 20 (25% at LOO) 20 (25% at LOO) 20 (23% at LOO) PFOSEA 25 (30% at LOO) Up to 3 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 ANALYTE % 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). If the average determined concentration for any QC level exceeds the acceptance limit, the task leader or study director should be notified. The tun may be repeated or a portion of the run may be considered acceptable. For example, if the low QC tails the stated requirements, samples may be accepted that have concentrations bracketed by the 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 of 70-130% 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 pg/g each for PFOS, M-556, M570, and PFOSAA. Page 14 of 16 3M Environmental Laboratory C-14 Page 130 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 ------------------------------------ L K N -U 284 9 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Num ber: FA C T -T O X -110 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/M S/M S Version 1.0 Study No.:__________________________ Analyjt/Date:____________________________ For PFOSA and PFOSEA, the validated LOQs are nominally 0.33 ng/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. Acceptabe performance (error) of the lowest used standard, therefore, will be considered sufficient evidence that bracketed study samples are quantified properly. G. General X. 4 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. 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 131 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT T O X -110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol N um ber: FA C T -T O X -l 10 METHOD FO R ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/M S/M S Version 1.0 Study No.:_________________________ Analyst/Date:____________________________ xn. C o n c l u s io n s xm. Sig n a t u r e s Analysts ___________________________________________ ___________________________________________ ___________________________________________ ___________________________________________ Technical Review ___________________________________________ _____ '_____________________________________ Q C Review _________________________________________ ________ _ ___________________________________________ Date: Date: Date: Date: Date: Date: Date: Date: 3M Environmental Laboratory Page 16 of 16 C-16 Page 132 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT T O X -110 LRN-U2849 Battette Study Number. N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-110 " APPENDIX D-REPRESENTATTVE CHROMATOGRAMS 3M Environmental Laboratory Page 133 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTO X-110 LRN-U2849 Battelle Study Number: N 003296-F 3M Toxicology Services Protocol Num ber: F A C T -T O X -110 3M Environmental Laboratory D-l Page 134 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTQ X-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Num ber: FA C T-T O X -l 10 3M Environmental Laboratory D-2 Page 135 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTQ X-110 LRN-U2849 Battelle Study Number: N 003296-F 3M Toxicology Services Protocol N um ber: F A C T -T O X -110 3M Environmental Laboratory D-3 Page 136 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FAC TTQ X-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol N um ber: F A C T -T O X -110 3M Environmental Laboratory D-4 Page 137 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTQ X-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Num ber: FA C T -T O X -110 3M Environmental Laboratory D-5 Page 138 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTQ X-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol N um ber: FA C T-T O X -110 3M Environmental Laboratory D-6 Page 139 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTO X-110 LRN-U2849 Battette Study Number: N003296-F 3M Toxicology Services Protocol N um ber: FA CT-TOX -I IO 3M Environmental Laboratory D-7 Page 140 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT T O X -110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FA C T-TO X -II0 3M Environmental Laboratory D-8 Page 141 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTO X-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-l I0 3M Environmental Laboratory D-9 Page 142 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTO X-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol N um ber: F A C T -T O X -110 3M Environmental Laboratory D-10 Page 143 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTO X-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol N um ber: FA C T -T O X -110 3M Environmental Laboratory D-l I Page 144 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT T O X -110 LRN-U2849 Bane lie Study Number: N003296-F 3M Toxicology Services Protocol Num ber: F A C T -T O X -110 3M Environmental Laboratory D-12 Page 145 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol N um ber: FA C T -T O X -110 3M Environmental Laboratory D-13 Page 146 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FAC TTO X-11Q LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FA C T-T O X -110 APPENDIX E-PROTOCOL, AMENDMENTS, AND DEVIATION i/ 3M Environmental Laboratory Page 147 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTOX-110 ----------------------------- LRN-U2849 ADC. 13. 1999 9:24AM Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-110 EHVIEONMEHTAl LAB 2 3E 09 K0. 1406 ?. 22 JM Ea*troajDU] Techno)t7 nd Strricti POBox 33331 Sl P.ul. MN33133-3331 <12778 M2 Protocol FACT-TOX-110 S tu d y Title Oral (Gavage) Pharmacokinetic Study of PFOS in Rats PROTOCOL A u th o r Lisa Clema Date: June 8,1999 Performing Laboratory 3MEnvironmental TechnologySc SafetyServices 3MEnvironmental Laboratory 935 BushAvenue St Paul, MN 55106 Laboratory Project Identification FACT-TOX-110 U2849 3M Environmental Laboratory PUS 13 ' 99 1 I : 17 3M Environmental Laboratory E-l Page 1 of 10 651 779 6176 PAGE.022 Page 148 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 MIG, 13. 1999 9:24AM Battelle Study Number: N003296-F 3M Toxicology Services Protocol Num ber: FA C T -T O X -110 EHVliQNMEHTAL LAB 2 3E 09 HO. 1406 P. 23 Protocol HFACT-TQX-110 Study Identification Oral (Gavage) Pharmacokinetic Study of PFOS in Rats T est M aterial Perfluorooctane sulfonic acid potassium salt (T-6295) Sponsor 3MToxicology Services Medical Department 3M Center, Building220-2E-02 St. Paul, MN 55144-1000 . S pon sor R epresentative MarvinT. Case, D.VM., PhD. 3M Toxicology Services Telephone: 651-733-5180 Facsimile: 651-733-1773 ' S tu dy D irector >i KristenJ. Hansen, PhD. 3M Environmental Technology end Safety Services . Building 2-3E-09 651-778-6018 ' ' S tu dy Locationfs) . in vivo Testing Facility A n a ly tic a l T e s tin g L ab o ra to ry Aigus ResearchLaboratories, Inc. 905 SheehyDrive, Building A Horsham, PA 19044 3M Environmental Laboratory Building 2-3E-09 935 BushAvenue St Pad, MN 55106 ; 3M Environmental Laboratory AUG 13 '9 9 1 1 : 1 7 3M Environmental Laboratory . E-2 Page 2 of 10 77P R i7fi Pace avn Page 149 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT T O X -110 LRN-U2849 AUG. 13.1999 9:24AM Battel le Study Num ber: N 003296-F 3M Toxicology Services Protocol Num ber: F A C T -T O X -110 ENVIRONMENTAL LAB 2 3E 09 NO. 1406 P. 24 Protocol UFACT-TOX-110 Sub-Contract Laboratory Proposed S tu d y Timetable StudyInitiation Data Study Completion Date Advanced Bioanalytical Services, Inc. IS CatherwoodRoad Ithaca, NY 14850 BatteUeMemorial Institute SOSKing Avenue Columbus, Ohio 43201-2693 June 8,1999 August 8,2000 1. STUDY Oral (gavage) pharmacokineticstudyofpotassiumperfluorooctane sulfonic acid (PFOS) in rats. 2. P u r p o se This analytical studyis designedto determine levels ofpotassiumperfluorooctanesulfonate J (PFOS) in specimens of liverandserumofrats. Thein-Hfeportionofthis studywas conducted atArgusResearch Laboratories, study#418-013. All serumsamples will be extracted and analyzed at.AdvancedBioanalytical Services, Inc. and all liversamples extracted andanalyzedat ' Battelle Memorial Institute. Additional analyses maybeperformedatthe3MEnvironmental Laboratoryaamethoda are developedandvalidated. If additional analyses areperformed an . amendment to this protocol will bewritten. 3. R e g u la t o r y co m plian ce This studywill be conductedin accordancewiththe United States Food andDrug Administration, GoodLaboratory Practices Standards, Final Rule21 CFR 58, with die exception diet analysis ofthe testmaterialmixture forconcentration, solubility, homogeneity, and stability will notbe conducted, andis theresponsibility ofthe Sponsor. 4. Q u a lity A ssu r a n c e . The 3M Environmental LaboratoryQualityAssuranceUnitwill reviewtheprotocol and audit study conduct, data, and final report to determine compliancewith GoodLaboratoryPractice Standards andwith 3MEnvironmental LaboratoryStandardOperatingProcedures. The QAUnit at the sub-contract laboratorywill audittheirstudy conduct, data, andresults report pnorto submitting to the 3MEnvironmental Laboratory. 3M Environmental Laboratory BUG 13 ' 99 11:18 3M Environmental Laboratory E-3 page 3 of 10 651 778 6176 PAGE.024 Page 150 BACK TO MAIN 3M Medical Department Study: T-6295.12 -- ------------------------------------------------------------ - Analytical Report: FACT TOX-110 LRN-U2849 MIC. 13. 1999 9:24AM Battelle Study Number: N003296-F 3M Toxicology Services Protocol Num ber: FA C T -T O X -110 ENViRONMEMTAL LAB 2 3E 09 ' NO. 1406 P. 25 Protocol SFACT-TOX-110 5. Te s t Mater ial 5 ,1 R efer to Argus Research Laboratory protocol for study #418-013. 6. C o n t r o l M a t r ic e s 6.1 Identification Ratlive andserumand/orrabbitliver andserum, traceabilitynumbers will be recorded in therawdataandincluded inthe final report 6.2 S o u rc e Argus Research and/orSigma Chemical 6.3 P h y sic a l D escription Ratliver andserumand/orrabbitliver andserum . 6.4 P u rity a n d S ta b ility Not applicable 6.5 Storage C onditions Frozenat -20 6C 10 *Cor-SO78C 10 *C 6.6 R e s e r v e Matrix A portion of die control matrix will be retained in the 3M archives for as long as the quality of the preparation affords evaluation, but not longer than tea years following the effective date ofthe final testrule (ifapplicable). 6 .7 D isp o sitio n Matrices will be retained at the 3M Environmental Laboratoryper GLP regulation. Certain matrices (feces, urine, and blood) may be disposed after QAU verification. ' 6.6 S a fe ty P recau tion s RefertoMSDS forchemicalsused. Wearappropriate laboratoryattire, andfollow adequateprecautions forhandlingbiological materials and preparing samples foranalysis. ` f 7. R e f e r e n c e M a t e r ia l 7.1 Identification Potassiumperfluorooctsnesulfonate (PFOS), lot #s 171,213, or 217 (equivalent lots) 7.2 SOUrc* 3M SpecialtyChemicals 7.3 P h y s ic a l D escription Whitepowder 7.4 P u r ity a n d sta b ility PurityofPFOS is 99%orgreater. Stabilityhat not been determined. 7.5 S to ra g e C on ditions Roomtemperature ' 7.6 R e s e r v e M aterial A reserve sample fromeachbatchofPFOS used in thin studywill be retained as long as the qualityofthepreparationaffords evaluation, butnot longer thanten years following theeffective date ofthe final testrule (if applicable). 7.7 D isp o sitio n Unusedreferencematerial will be retainedforuse by the 3M Environmental Laboratoryandwill be discardedwhen the quality ofpreparation no longer affords evaluation. 3 U Environmental Laboratory AUG 13 '9 9 1 1 : 1 8 3M Environmental Laboratory E-4 Paga 4 of 10 SSl 778 6176 PAGE.025 Page 151 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 AG. 13. 1999 9:25A11 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Num ber: FA C T-T O X -110 EHVIEONMEHTAl UB 2 3E 09 HO. 1406 P.26 Protocol UFACT-TOX-110 7.8 S a fe ty P recau tion s RefertoMSDS for chemicals used. Wear appropriate laboratoryattire, andfollow adequateprecautions forhandlingbiological materials and preparing samples foranalysis. 8. Te s t S ystem Rats were used as thetest systemandweremaintainedanddosed as described is die Argus Research protocol #418-013. Thefemaleratswill be giventhetest material orcontrol once daily beginning 42 days prior to cohabitationandcontinuethroughday 14 or20 ofpresumed gestation. See table 1formore dosage information. ` Table 1 DosageLevels, Concentration, and Volumes Dosage Group 1 2 3 4 5 Numberof female rats 16 16 16 16 16 Dosage mg/kg/day 0 0.1 0.4 1.6 32 Concentration. mg/kg/day 0' 0.02 0.08 0.32 0.64 Dosage volume mL/kg 5 5 5 5 5 9. S p e c im e n a n d S a m p l e Re c e i p t . The 3M EnvironmentalLaboratorywill receive homogeneitysamples fordose analysis and specimens ofthe following bodytissues andfluids fromthe indicatedpoints in the study. See table 2 forspecimen information. All specimenswill bepackedon dryice forshipping. Body tissue/flnid Table 2 Specimen Information Collected . Serum-Dam andFetus animals Urine and feces -D im animals Liver--Dam andFetus animal Milk Secreting Glands- Dam animals Amniotic Fluid- Damanimals Dam-Predose, Days 7,13, and21 Fetus-Atterminationofthe study Predose, Days 7, IS, and21 DamandFetus-At terminationof the study At the termination of the study Day 15 andDay 21 Expected # of specimens 320 Dam and 320 Fetus (pooled) 320 urine and 320 feces 80 Dam and 80 Fetus (pooled) 80 80 3M Environmental Laboratory BUG I 3 *39 1 1 : IS 3M Environmental Laboratory E-5 Page 5 of 10 651 7?9 6176 PBGE.026 Page 152 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FA C T T O X -1 10 LR N -U 2849 AUC.l3.l999 9:25AM Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX -IIO ENVIRONMENTAL LAB 2 3E 09 NO. 1406 P.27 Protocol PFACT-TOX-110 Table 2 Cont Specimen Information Body tissue/iluid Collected Expected # of specimens Embryo's with Placentae- Dam Day IS andDay 21 animals Carcass - Fetus animal At the terminationofthe study Placentae- Fetus animal Day IS andDay 21 Livex/lung- Fetus animal Atdie terminationofthe study Total numberof expected specimens: 2000 Total numberofteat animals: 64 Total numberof control animals: 16 80 160 80 80 Specimens sent to 3M EnvironmentalLaboratorieswill bereceived andtracked according to applicable StandardOperatingProcedures. 10. P r e p a r a t o r y M e th o d s 10.1 FACT-M-1.1, ExtractionofPotassiumPerfluorooctanesulfonate or OtherAnionic Fluorochemical SurfactantfromLiver forAnalysisUsing HPLC-Electrospray/Mass Spectrometry 10.2 ETS-8-4.1, ExtractionofPotassiumPerfluorooctanesulfonate orOther Fluorochemical Compounds fromSerumorOtherFluid forAnalysis UsingHPLC- . r Electrospray/Mass Spectrometry 10.3 Ifpreparatorymethods otherthanthose listed above areused, m amendment to this protocol will bewritten. Any deviations fromthesemethodswill be documented and included with the studydata. 10.4 I f analyses are sub-contractedto other laboratories, anamendmentwill be written to include theirmethods andcopies ofeachmethodwill be attachedto this protocol H .A halytical Methods 11.1 FACT-M-2.1, Analysis ofFhrorochemieals in LiverExtracts Using HPLCElectrospray/Mass Spectrometry 11.2 ETS-8-S.1, Analysis ofPotassiumPerfluorooctanesulfonate or OtherFluorochemicals in Serumor OtherFluidExtractsUsing HPLC-Electrospray/Mass Spectrometry 11.3 Ifanalytical methods otherthanthose listed above areused, anamendmentto this protocol will be written. Any deviations fromthese methods ill be documented and includedwith the study data. 11.4 Ifanalyses are sub-contracted to otherlaboratories, anamendmentwill be written to include theirmethods andcopies ofeachmethodwill be attachedto thi protocol 3 U Environmental Laboratory AUG 13 ' 99 1 1 : 1 9 3M Environmental Laboratory E-6 Page 6 of 10 651 778 6176 PAGE.027 Page 153 BACK TO MAIN 3M Medical Department Study: T-6295.12 -------- ---------------------------------------------------------------------------- Analytical Report: FACT TOX-110 ' LRN-U2849 AUG, 11 1999 9:25AM Battelle Study Number: N003296-F 3M Toxicology Services Protocol Num ber: FA C T -T O X -110 ENVIRONMENTAL LAB 2 3E 09 NO. 1406 P. 28 Protocol #FACT-TQX-110 1 2 . D a t a Q u a l it y Ob je c tiv e s The number of spQces/duplicales, use ofsurrogates, andinformationon otherdataquality indicators are included in the analyticalmethods. In addition, the following criteriawill be met: 12.1 L in earity i2i 0.98 12.2 Lim its o f detection/quantitation 12.2.1 Method DetectionLimit (MDL) forFFOS a) Serum: 1.7Sppb b) Liver. ISppb 12.2.2 Limit of Quantitation(LOQ)-Equal to thelowest acceptable standard in the calibrationcurve 12.3 D u p lica te a c c e p ta b le p re cisio n <30% forthe method 12.4 S p ik e a c c e p ta b le r e c o v e rie s 70%-130% 1 Z S U se o f confirmatory m e th o d s Indeterminate samples will be re-analyzed using a confirmatory method. Ifmwifinwatniy method is used, an amendment to this protocol will be written. > 12.6 D em on stration o f sp e c ific ity Chromatographic retention time, mass spectral daughterion characterization. 13. Sub-C ontracted Analysis 13.1 All analyses as detailed in thisprotocolwill be performedat 3MEnvironmental Laboratories,Building 2-3E-09,935 Bush Avenue, St Faul, MN 55106, at Advanced Bioanalytieal Services, hoc., IS CatherwoodRoad, Ithaca, NY 14850, or at Battelle Memorial Institute, 505 King Avenue, Columbus, Ohio 43201-2693. 13.2 A n amendment to this protocol will be written i f analytes are performed at laboratories otherthan3MEnvironmental Laboratories, AdvancedBioanalytieal Services, Inc., orBattelle MemorialInsmute. 14. S ta tistica l A n alysis Averages and standarddeviations will be described below: The statisticalmethods that will be used are 14.1 D ata tran sform ations a n d a n a lysis Datawill be repottedas the concentration (weight/weight orweight/vol) ofPFOS ormetabolitepertissue or fluid. 3M Environmental Laboratory RUG 13 ' 9 9 11:19 3MI Environmental Laboratory E-7 Pace 7 of 10 RSI 7?n RI7R PORR Page 154 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TO X-110 LRN-U2849 AUG. 13. 1999 9:26AM Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FA C T -T O X -110 ENVIEONHENTAL LAB 2 3E 09 >0.1406 P.29 Protocol FACT-TOX-110 14.2 S ta tistic a l a n a lysis Statistics used may include regression analysis of concentrations overtime, andstandard deviations calculated for the concentrations within each dose group. Ifnecessary, simple statistical tests, such as Student's t test, maybe appliedto evaluatestatistical difference. IS .R s p o r T ' A report containing all theresults ofthe studywill be preparedbythe 3MEnvironmental Laboratory. Ifanalyses aresub-contractedto otherlaboratories, each laboratorywin prepare a report andsubmitit to the 3MEnvironmentalLaboratory forinclusioninthe 3MEnvironmental Laboratoryreport Eachreportwill include, butnot be limitedto, thefollowing, when applicable: 15.1 Name and address ofthe facilityperformingthe study 15.2 Dates upon which the studywas initiated andcompleted 15.3 A statement ofcompliancebythe StudyDirectoraddressing anyexceptions to Good Laboratory Practice Standards 15.4 Objectives andprocedures as statedin die approvedprotocol, includingany changes in the original protocol 15.5 The test substanceidentificationbyname, chemicalabstracts number orcode number, strength, purity, andcomposition orotherappropriatecharacteristics, if providedby the Sponsor ' ,. 15.8 Stability ind the solubilityofthetestsubstances underthe conditions of r administration, ifprovidedby the Sponsor 15.7 A description ofthemethods used to conduct thetest(s) 15.8 A description ofthe testsystem 15.9 A descriptionof anycircumstances thatmayhave affected the quality orthe integrity ofthe data 15.10 The name ofthe StudyDirector andthenames ofotherscientists, professionals, and supervisory personnel involved in die study 15.11 A description ofthe transformations, calculations, or operations performed on the data, a summaryandanalysisofdie analytical chemistrydata, anda statement ofthe conclusions drawnfromthe analyses 15.12 Statistical methodsused to evaluatethe data, if applicable 15.13 The signed anddatedreports ofeachof theindividual scientists or otherprofessionals involved in the study, if applicable 15.14 The location whererawdataandthe final report ire to be stored 3 U Environmental Laboratory AUG 13 ' 99 1 1 : 1 9 3M Environmental Laboratory E-8 Paga 8 of 10 651 77A R I76 POfiF.A?q Page 155 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT T O X -1 in LRN-U2849 . AUG, 13. 1999 9:26AM Battelle Study Number: N003296-F 3M Toxicology Services Protocol Num ber: F A C T -T O X -I10 ENVIRONMENTAL LAB 2 3E 09 NO, 1406 P.30 Protocol 9FACT-TOX-110 1 5 .1 5 A statementpreparedby the QualityAssurance Unit listing the dates that study inspections and weremade, andthe dates ofany findingsreported to the Study Director andManagement Ifit is necessary to make corrections oradditions to areport afterit has been accepted, the changes will be made in die formofanamendmentissued by the StudyDirector. The amendment will clearly identify thepartofthe reportthat is being amended, the reasons for the amendment, and will be signed by the StudyDirector. 1 $ . L o c a t io n o p R a w D a ta , R e c o r d s, a n d F in a l R e p o r t Original data, or copies thereof, will be available at the3MEnvironmentalLaboratoryto facilitate audits of the studyduring itsprogress andbefore acceptance ofthe final report When the final report is completed, all originalpaperdata, includingthose items listed below, will be retained in the archives of3MEnvironmental Laboratoryfor at least aperiod of time as specified by regulation, andas established by 3MEnvironmental LaboratoryStandardOperating Procedures. 16.1 The following rawdataandrecordswillbe retainedin the study folderin the study/project archivesaccordingto 3MEnvironmentalLaboratoryStandard Operating Procedures: 1 6 .1 .1 Approvedprotocol andamendments 1 6 .1 .2 Study correspondence 1 6 .1 .2 Shippingrecords ' 1 6 .1 .4 Raw data 1 6 .1 .5 Approved finalreport(original signedcopy) 1 6 .1 .6 Electronic copies ofdata - 1 6 .2 The following supportingrecordswill be retainedseparately fiom.the studyfolder in the archives according to 3M Environment] Laboratory Standard O perating Procedures: 1 6 .Z 1 Trainingrecords 1 6 .2 .2 Calibrationrecords 16.2.3 Instrument maintenance logs 16.2.4 Standard OperatingProcedures, EquipmentProcedures, and Methods 3W Environmental Laboratory f)U6 13 199 11:20 3M Environmental Laboratory E-9 Page S o l 10 S5i 77R fii7R poGr.a.na Page 156 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 ' " LKN-U2849 AUG. 13. 1999 9:26AM Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FA C T-T O X -110 ENVIRONMENTAL LAB 2 3E 09 NO. 1406 P. 31 Protocol FACT-TOX-110 17. S p e c im e n R e t e n t io n Specimens will be maintainedinthe 3MEnvironmental Laboratoryspecimen archives for a period of time as specified by regulationoras long as foe qualityoffoepreparation affords evaluation, but not longerthan tenyean following foe effective date ofthe final test rule (if applicable), and as establishedby 3MEnvironmental Laboratory StandardOperatingProcedures. 1 8 . P r o t o c o l A m e n d m e n t s a n d d e v ia t io n s Planned changes to theprotocol willbein foe form ofwrittenamendments signedby the Study Director andfoe Sponsor's Representative. Amendments will be considered as partof the protocol andwill be attachedto foe final protocol. All changes to the protocol will be indicated in foe final report Any otherchangeswillbe in foe formofwrittendeviations, signed by foe Study Director and filedwith foerawdata. 19.A tta c h m en ts 19.1 A tta c h m e n t A Preparatoryandanalyticalmethods 20. S ig n atu r es // ! J f J A / i ^ ^ _________________ Mama T. Cas, D.V.M., PhD., SponsorRepresentative 3M Environmental Laboratory fiUQ 13 '99 11:20 3M Environmental Laboratory E-10 Page 10 o f 10 pis i 77R fii7fi pace m i Page 157 BACK TO MAIN 3MI Medical Department Study: T-6295.12 ------ -' "' ___________ Analyticaj Report: FACT TOX-110 " * * LRN-U2849 Si?. 28.1999 11:09AM Battelle Study Number: N003296-F 3M Toxicology Services Protocol N um ber: F A C T -T O X -110 ENVIRONMENTAL LAB 2 3E 09 NO. 1936 P. 2 i Study Title Onl (Gavage)Phaimacoldnetic Study ofPFOS inRats PROTOCOL AMENDMENT NO. 1 Amendment Date; August 12,1999 Performing Laboratory 3M Environmental Technology &Safety Services 3MEnvironmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory P roject Identification ET&SS FACT-TOX110 URNU2849 It 3M Environmental Laboratory e c o 9 0 o o 3M Environmental Laboratory E-l 1 Page 158 BACK TO MAIN ilytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol N um ber: F A C T -T O X -110 SEP. 28. 1999 11:09AM ENVIRONMENTAL LAB 2 3E 09 NO. 1936 P. 3 Protocol FACT-TOX110 Amandment No. 1 This amendment modifies the following portlon(s) of the protocol: 1. P r o t o c o l r e a d s : Section 2.0 states this study is designed to determine potassium perfluorooctane sulfonic acid (PFOS) in specimens ofliver andserumofrats. AMEND TO read: This studyis designed to determine PFOS in specimens ofrat liver, serum, and urine. All Day -1 and GestationDay21 rat urine specimens will be extracted and analyzed by the 3M EnvironmentalLaboratory. REASON: The urine extraction andanalytical methods were not validated and approvedprior to protocol approval. 2. Protocol reads: Section 6.0 lists rat orrabbit liver andserum. AMEND TO read: Rat orrabbiturine from3MToxicology with aphysical description ofrat or rabbit urine. REASON: The rat urinematrix was addedaftertheprotocol was approved. 3. PROTOCOL reads: Section 10.0 and 11.0 list the following methods to use for extraction and analysis: FACT-M-1.1 "Extraction ofPotassiumPerfluorooctanesulfonate orOtherAnionic Fluorochemical Surfactant fromLiverforAnalysis Using HPLC-Electrospray/Mass Spectrometry" FACT-M-2.1 "Analysis ofFluorochemicals in LiverExtractsUsing HPLC-Electrospray/Mass Spectrometry" . A m e n d TO READ: T he extraction 4 analytical m ethods to follow at the 3M Environm ental Laboratory are; ETS-8-6.0 "ExtractionofPotassiumPerfhiorooctanesulfonateor OtherFluorochemical Compounds fromLiverforAnalysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-96.0 "Extraction ofPotassiumPerfluorooctanesulfouate or OtherFluorochemical Compounds fromUrine for Analysis Using HPLC-Electrospray/Msss Spectrometry/Mass Spectrometry" ETS-8-7.0 "Analysis ofPotassiumPerfluorooctanesulfonate orOtherFluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-97.0 "Analysis ofPotassiumPerfluorooctanesulfonate orOtherFluorochemical Compounds in Urine Extracts Using HPLC-Electrospray/Mass Spectrometry/Mass Spectrometry" 3M Environmental Laboratory P P PO *QO I T . . 3M Environmental Laboratory E-12 Page 159 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 SEP. 28. 1999 11:09AM Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FAC T-TO X-110 EHV1ROHMEKTAL LAB 2 3E 09 MO. 1936 P. 4 Protocol FACT-TOX110 AmendmentNo. 1 REASON: The extractionandanalytical methods FACT-M-1.1 andFACT-M-2.1, respectively, were updated on 07/22/99 to ETS-8-6.0 andETS-8-7.0. Methods ETS-8-96.0 and ETS-8-97.0 were not validated andapproveduntil aftertheprotocol was approved. 4. P r o t o c o l r e a d s : Section 10.4 and 11.4 state that if the analyses are sub-contracted to other laboratories an amendmentwill be writtento includethesemethods. AMEND TO r ead: The extractionand analytical methods to follow at AdvancedBioanalytical Services will be attachedto theprotocol. The extraction andanalytical methodto follow atBattelle Memorial Institute is: "Method forAnalysis ofPerfluorooctaneSulfonate (PFOS) inflat Set*by LC/MS/MS, Version^'"i,e O L 'w REASON: The analyticalmethods at thesub-contract laboratorieswere not included in the original protocol. 3. PROTOCOL r e a d s : Section 12.2.1 b) Livermethod detection limit is 15 ppb. A m e n d TO READ: 12.2.1 b) Livermethoddetection limit is 8.50 ppb (ng/g). 12.2.1 c) Urine method detection limit is 1.3ppb (ng/g). R e a s o n : The validation supportingmethods ETS-8-6.0 andETS-8-7.0 included a lower method detection limit forPFOS in liver. A validationin urine wasperformed, afterprotocol approval, to support thismethoddetection limit P ito /,. 3WEnvironmental Laboratory Q C D 7 0 >O O 3M Environmental Laboratory E-13 Page 160 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LK N -U 2tW SEP. 28. 1999 11:09AM Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FA C T-T O X -110 ENVIRONMENTAL LAB 2 3E 09 " . . . . . . . NO* 1936 ' Y 5 . . . . Amendment Approval Protocol FACT-TOX110 Amendment No. 1 Marvin Case, D.V,J o ., Ph.D., Sponsor Representative j 7 4a^ J - i m Date ____ fL _ Kris J. Hansen, PhJD., StudyDirector g //g /gy Date 3M EnvironmentalLaboratory WB no oo i o.nn 3M Environmental Laboratory E-14 Page 161 31V Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Study Title Oral (Gavage) Pharmacokinetic Study o f PFOS in Rats PROTOCOL AMENDMENT NO. 2 Amendment Date: September 28, 1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX110 LIRNU2849 / 3M Environmental Laboratory 3MI Environmental Laboratory E-15 Page 162 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Protocol FA C T-TO X110 Amendment No. 2 4. Protocol reads: Section 16 states that the original data, or copies thereof; will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance of 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 of the study during its progress and before acceptance of 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 ofdata 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 of the facility performing each analysis. Reason: Clarification of the disposition of archived records if analyses are performed at a sub-contract laboratory. < 5. PROTOCOL reads: Section 17 states that specimens will be maintained in the 3M Environmental Laboratory specimen archives. A mend to read: Specimens will be maintained in the 3M Environmental Laboratory specimen archives. All specimens sent to sub-contract laboratories will be returned to the 3M Environmental Laboratory upon completion of analysis and submission of the sub-contract laboratory(s) final report. The specimens will be returned with the following documentation: the signed original chain of custody and records of storage conditions while at the sub-contract facility. Reason: Clarification of the disposition of documentation when shipping specimens for analyses performed by a sub-contract laboratory. 3M Environmental Laboratory 3M Environmental Laboratory E-17 Page 163 3M Medical Department Study: T-6295.12 ' Amendment Approval BACK TO MAIN Analytical Report: FACT T O X -110 LRN-U2849 Protocol F A C T -T O X 1 10 Amendment No. 2 TQ ^ Marvin Case, D.V.M.-, Ph.D., Sponsor Representative Date ' fch * U2- ___________ Kristen J. Hansen, Ph.D., Study Director ' U Date 3M Environmental Laboratory 3M Environmental Laboratory E-18 Page 164 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TQX-110 LRN-U2849 Study Title Analytical Laboratory Report on the Determination o f Perfluorooctanesulfonate (PFOS) Presence and Concentration in Serum, Liver, and Urine from the Gavage Study of T-6295.12 PROTOCOL AMENDMENT NO. 3 Amendment Date: 20 January 2000 Urine Analyses 3M Environmental Technology and Safety Services Fluorine Analytical Chemistry Team Building2-3E-09 935 BushAvenue St. Paul, MN55106 Performing Laboratories Liver Analyses Battelle Memorial Institute 505 King Avenue Columbus, OH 43201-2693 Serum Analyses Advanced Bioanalytical Services, Inc. 15 Catherwood Road Ithaca, NY 14850 Laboratory Project Identification ET&SS LRN-U2849 FACTTOX-110 Argus Study: 418-013 3M Medical Department Study: T-6295.12 3M Environmental Laboratory 3M Environmental Laboratory E-19 Page 165 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Rpnnrt- F A C T T O X - U O LRN-U2849 Protocol LRN-U2849 Amendment Number 3 This amendment modifies the follow ing portion(s) o f the protocol: 1. Protocol reads: The study director for the present study was identified in the protocol as Kristen J. Hansen, Ph.D. Amend 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: T p 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 E-20 Page 166 3M Medical Department Study: T-6295.12 Amendment Approval BACK TO MAIN Analytical Report: FACT T O X -110 LRN-U2849 Protocol LRN-U2849 Amendment Number 3 JohnL. Butenhoff, Ph.D., Sponsor Representative Date /b z --------Kristen J Hansen, PhD., Outgoing Study Director / ! - F-tb -7C 0 Date &I Marvin T. Case, D. V.M., PhD., Incoming Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory E-21 Page 167 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTOX-110 LRN-U2849 Battelle StudyNumber: N003296-F 3MToxicologyServices Protocol Number: FACT-TOX-II0 DEVIATION REPORT Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number. FACT-TOX-110 ORAL (GAVAGE) PHARMACOKINETIC STUDY OF PFOS IN RATS TYPE OF DEVIATION: PROTOCOL . DATE OF DEVIATION: August 19, 1999 NATURE OF DEVIATION: The source of control matrix will not be either Argus - Research or Sigma Chemical as specified in section 6.2 of the protocol. CAUSE OF DEVIATION: Harlan will be the supplier of control rat livers used to prepare blanks, any standards, and QCs for the analytical portion of the study. IMPACT OF DEVIATION ON THE STUDY: Harlan was used as the control matrix supplier for Battelle's validation of the analytical method (Battelle study number N003604-A). This supplierprovided 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: C- Jon Cl Andre, Ph.D. Battelle Principal Investigator Kristen J. Hansen, Ph.D. Study Director io/1b y Date 3M Environmental Laboratory E-22 Page 168 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACTTOX-110 LRN-U2849 Battelle Study Number: N003296-F 3MToxicologyServicesProtocol Number: FACT-TOX-II0 APPENDIX F - PFOS PURITY REPORT t 3M Environmental Laboratory Page 169 BACK TO MAIN 3M Medical Department Study: T-6295.12 =TSS 2 3U Analytical Report: FACT TOX-110 LRN-U2849 Battelle StudyNumber: N003296-F 3MToxicology Services Protocol Number: FACT-TOX-110 ^ 651 778 4226 C 4 /2 6 / 9 9 C 9 :5 6 5 ;Q 2 /0 3 NO:341 121 From; Subject: Date: 3MSPECIALTYADHESIVES &CHEMICALS ANALYTICALLABORATORY LisaClemen(8-5568) - ET&SS- 2-03-09 TomKestner-(3-5633) -SA&CAnalyticalLab-236-23-11 Request # 57830 O u m ic a l CXum cUrizotion o f P O S F-B ostd Fluorochtm icak by 'H -N M R 4 HF-NM R Sptetroseopy March24.1999: PreliminaryreportforFC-95(PFOS), lot 171 SAMPLEPS5CKIPTTOf*i FC-95, lot 171(PFOS). TN-A-0834;Nominal product=C,FirS0j(-) K{+) (whitepowder) INTRPPVCTIQEi This samplewassubjectedto'H-NMRand!*F-NMRspectral analyses todeterminethepurityof thenominal product andtocharacterize as many impuritycomponents as possible. . EXPERIMENTAL; Aportionofthesamplewas accuratelyweighed, spikedwithaknownamountof 1,4-bis(trifluoromethyl)benzeoe (p-HFX), andthentotallydissolvedinDMSO-dforsubsequentanalysisbyNMR. A400MHz 'H-NMR spectrum(#h57830.40l) anda376MHz''F-NMRspectrum(#(57830.401) wereacquiredusingaYarian UNITYplus400FT-NMRspectrometer Useofthep-HFXinternal standardwas intendedtopermitthe determinationoftheabsoluteweightpercentconcentrationsoftheassignedcomponents without necessarily needingtoidentifyorquantifyall thecomponents inthesamplemixture. RESULTS; ThecombinedNMRspectral datawereusedtoassignall ofthemajorandmost of theminorcomponents inthis sample asreceived. The qualitativeandquantitativecompositional resultsthatwerederivedfromthesingle trial NMRinternal standardizationanalyses aresummarizedinTABLE-1 onthefollowingpage. Ihavereportedboth relative andabsoluteweightpercentconcentrations. Onepossiblereasonthattheabsolutewt.%values addupto morethan100%maybeduetothe factthatIassumedall ofthecomponentscontained8carbons. Iftherewere anyshorterchainhomologs present(i.e., 7, 6.5, etc. carbons), thentheaveragecompoundmolecularweights wouldhavebeensomewhat lessthanthoseusedinthecalculations. Ingeneral, the '*F-NMRtechniqueisnot p a rtic u la rly w e ll su ited fo r id e n tify in g o r q u an tify in g sm all am ounts o f various n u o ro c h e m ico l h o m o lo g im p u rity componentsunless thechains areveryshort. Amorecompletecharacterizationof anyotherfluorochemical homologs wouldrequireanalysis byelectrasprayMSorasimilartechnique. Additional workwouldberequiredinoneffort topositivelyverifythetentativelyassignedcomponents listedin TABLE-1 (denotedbypossible). Small amountsofotherunidentifiedimpurities arealsodetectedintheNMR spectra,but additional workwouldberequiredinaneffort toidentifyorquantifytheseother materials. Copies oftheNMRspectrawill beprovidedforyouatalaterdate. Ifyouhavemyquestions abouttheresults in this initial reportforFC-95. lot 171. pleaseletmeknow. Iapologizeforthedelay incompletingthis initial work. TomXestner e: R iei P ijrte - SAAC Analytical Lib - 236-23-11 Fil# Rafcmes: LCJTJ20.ECC'61 3M Environmental Laboratory riga - I F-l Page 170 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACTTOX-110 LRN-U2849 STSS 2 3W Mareb 24, 1999 Battdle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-110 "g* 651 77 8 4 2 2 6 0 4 /2 6 /9 9 0 9 :5 6 0 : 0 3 / 0 3 NO:341 SAdfcCAnalytical Lab Request # 37830 Initial Report for FC-93. lot 171 TABLE-1 Sample FC-95. lot 171 (PFOS), TN-A-0834 Overall Quantitative Compositional Results by 'H/'^F-NMRInternal Standardization Analyses Structural Assignment! ayC FjJ.-SC W -) K(+) (Normal chain: uium a x *7 for calculation purposes) NMR Absolute Weight% Coneentrsdons (liarle trial measuremenO 70.3% NMR Relative Weigbt% Concentrations (sintle trial measurement) 68.6% CFj(CFJ,-CF(CF1><CF0,-SO3(0 KM (Internal tnoaomethyl branch: assume x+y 3, a a 0, A y a 0, for calculation purposes) 17.7% 17.3% (CFj)jCF-(CFj),-SOj(-) K M (Isopropyl branch: assume x *3 for calculsdon purposes) 10.3% 10.2% CvF^-CFICFjl-SO A ) K M (Alpha branch; aasuma x * i for calculation purposes) Possible F-SFs-C Ja-SO jM K(+) (assume x 1 far calculitiOL purposes) 3.3% 0.37% 3.2% 0.36% C F jKCFjJx^C C FjJ H C F ^ -S O jC-) K M (Internal jem-dimethyl branch: assume x+y * and x a 0 for calculation purposes) 0.16% 0 .|6 ^ Possible CFrSFe-C.Fu.SOji-) K M (assume x * 7 for calculation purposes) Probable ClH to. r SOj(-) K(+) (Hydrocarbon sulfonate sale assume i a 8 for calculation purposes) (C F j) jC -(C F i ),-S O j{ - ) K M --(r-bucyl branch; tu u m e x 4 for caJcuiauon purpoMv) 0.11% 0.031% 0.027% 0.10% 0.030% 0.026% 3M Environmental Laboratory hn 2 F-2 Page 171 BACK TO MAIN 3M Medical Department Study: T-6295.12 ADVANCED BOANALYTICAL SERVICES, INC. Analytical Report: FACT TO X-110 LRN-U2849 METHOD VALIDATION 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 14 85 0 (6 0 7 ) 2 6 6 -0 6 6 5 Fax (6 0 7 ) 2 6 6 -0 7 4 9 3M Environmental Laboratory Page 172 BACK TO MAIN 3IVI Medical Department Study: T-6295.12 AUTHORS: FOR: DATE: TITLE: Analytical Report: FACT T O X-110 LRN-U2849 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) o f 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 of 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 173 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT T O X-110 LRN-U2849 after three freeze/thaw cycles. Reliable results were obtained for sample extracts reinjected 27 hours after initial reconstitution. 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S . INC. Page 3 of 50 99VDJA01-MI.DOC Page 174 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT T O X-110 LRN-U2849 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 3IVI Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S . INC. Page 4 of 50 99VDJA01.MI.DOC Page 175 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 TITLE: SIGNATURE PAGE METHOD VALIDATION FOR THE QUANTITATION OF PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/MS Report Number: Reported by: 2Musrm<( Date Reviewed by: Associate Auditor Authorized for Release by: O-- JohnR. Perkins, Ph.D. Assistant Scientific Director 7JouJ2$. Date o Date 3M Environmental Laboratory A D VA N CED BIO AN ALYTICAL S E R V I C E S . INC. Page 5 of 50 99VDJA01 .MI.DOC Page 176 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 TABLE OF CONTENTS ABSTRACT...................................................................... QAU STATEMENT............ .......................................................................... SIGNATURE PAGE................................................................. TABLE OF CONTENTS......................................................................................................6 LIST OF TABLES.................................................................................................. 8 LIST OF FIGURES...... .....................................................................................:................. 9 1. INTRODUCTION............................................................................................. :.........10 2. EXPERIMENTAL....................................................................................................... 10 2.1. Chemicals and Materials................................................................................. 10 2.2. LC/MS INSTRUMENTATION................................................... 2.3. Sample Preparation and Extraction Procedure..................................... 11 2.4. Rat Serum Validation Da ta ................................................................................. 11 2.5. Assay Evaluation........................................................... 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 of Quantitation (LLQ)............................................................... 13 2.5.5. Selectivity.......................................................................................... 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 -20 C....................................... 14 2.6.3. Freeze/Thaw Stability in Rat Serum............................................................... 14 2.7. Reproducibility of Reinjecting Extracted Sam ples.................................14 2.8. Extraction Recovery........................................................................................14 3. RESULTS AND DISCUSSION.................................................................... 3.1. Assay evaluation r esults................................................................................. 16 3.1.1. Intra- and Inter-Assay Accuracy................................................................... 16 4 10 1 13 15 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S . INC. Page 6 of 50 99VDJA01.MI.DOC Page 177 BACK TO MAIN 3M Medical Department Study: T-6295.12 , Analytical Report: FACT TOX-110 LRN-U2849 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 PFOS IN QUALITY CONTROL SAMPLES......... .............................. 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 of PFOS in Rat Serum................................................ 18 3 .3 . REPRO D U CIBILITY OF R EIN JEC TIN G E X TRA CTED S A M P L E S .................................... ....1 8 3 .4 . EXTRA CTIO N R E C O V E R Y ..................................................................................................................... 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 LC7MS...................................37 3M Environmental Laboratory ADVANCED BIDANALYTICAL S E R V IC E S . INC. Page 7 of 50 99VDJA01 .MI.DOC Page 178 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 LIST OF TABLES Table 1: Intra-Assay Accuracy and Precision of the PFOS Assay in Rat Seram for QC Samples .................................................................................................. 20 Table 2: Inter-Assay Accuracy and Precision of the PFOS Assay in Rat Serum for QC Samples from Tliree Validation Runs........................................................21 Table 3: Accuracy and Precision of 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 of 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 of 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 Etat Serum................................................. 30 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S , INC. Page 8 of 50 99VDJA01 .MI.DOC Page 179 BACK TO MAIN 3M Medical Department Study: T-6295.12 LIST OF FIGURES Analytical Report: FACT T O X-110 LRN-U2849 Figure 1: Full-Scan Single MS Mass Spectrum of 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 of 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 180 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT T O X-110 LRN-U2849 1. INTRODUCTION The purpose of this report is to describe the method validation for the quantitative determination of perfluorooctanesulfonate (PFOS) in rat serum samples after modification of 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. c h e m i c a l s a n d M a t e r i a l s 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. A detailed list of 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 of quality control (QC) samples were prepared separately from those used in preparation of the calibration curves. Preparation of all solutions used during extracti on and analysis are described in Appendix A. 2.2. LC/MS I n s t r u m e n t a t io n The liquid chromatography/mass spectrometry system consisted of two LC-10AD pumps (Shimadzu, Columbia, MD 21046), a SCL-10A pump controller (Shimadzu, AD VAN CED BIO AN ALYTICAL S E R V I C E S , INC. 3M Environmental Laboratory Page 10 of 50 99VDJA01.MI.DOC Page 181 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Columbia, MD 21046), a WISP 717plus autosampler (Waters Associates, Millipore Corporation, Milford, MA 01757), a Betasil C18(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 of the instrumentation and instrument conditions is found in Appendix A. 2.3. Sa m p l e P r e p a r a t io n a n d E x t r a c t io n P r o c e d u r e For each analytical run (tray), duplicate 50-pL aliquots of 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 o f 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 (QC4, 100 pg/mL) was prepared at a concentration exceeding the upper limit of 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 iri 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 50 99VDJA01.MI.DOC Page 182 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT T O X-110 LRN-U2849 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. A s s a y e v a l u a t i o n 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 of 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 |ig/mL) was prepared containing PFOS at approximately five times the upper limit of quantitation (ULQ). Five replicates of QC4 were diluted ten-fold and analyzed. The RSD and RE were reported. 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S . INC. Page 12 of 50 99VDJAOl.MI.DOC Page 183 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT T O X-110 LRN-U2849 2.5.4. Lower Lim it o f Quantitation (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 of 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 of analyte from one injection to Ihe next was assessed by analyzing a control'blank injected immediately after a high calibration standard (STD 10, 20 pg/mL). 2.6. STABILITY OF PFOS IN QUALITY CONTROL SAMPLES QC1 through QC3 were used to determine the stability of the analyte during sample storage, extraction, and analysis. 2.6.1. Ambient-Temperature Stability of PFOS in Rat Serum The stability of 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 S E R V IC E S , INC. Page 13 o f 50 99VDJA01.MI.DOC Page 184 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TO X-110 LRN-U2849 2.6.2. Freezer Stability of PFOS 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 Seram The stability of 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 of freshly thawed QC samples. All QCs were assayed in replicates of five. 2.7. repr o d u cibility of Reinjecting extracted Sam ples The reproducibility of 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. All QCs were assayed in replicates of five. 2.8. EXTRACTION RECOVERY 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 BIO ANALYTICAL S E R V IC E S , INC. Page 14 o f 50 99VDJA01 .MI.DOC Page 185 31V Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 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 fiill-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) mlz = 499.0 m/z = 427.0 The peak labeling o f full-scan data does not accurately represent the performance of 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 of 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 of representative extracts from calibration standards 1 and 10, respectively. BP A D V A N C E D BIOANALYTICAL S E R V IC E S . INC. 3M Environmental Laboratory Page 15 of 50 99VDJA01.MI.DOC Page 186 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 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 of 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 of 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. The calibration curve statistics are shown in Table 4. 3.1.4. Partial Volume Analysis The results of 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 of 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 187 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TO X-110 LRN-U2849 3.1.5. Lower Limit of Quantitation (LLQ) Table 6 shows the lower limit of quantitation (LLQ) data. The serum LLQ experiment demonstrated that 0.05 pg/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 of 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 of 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 PFO'S. There was no evidence of carryover for the internal standard.' 3.2. STABILITY OF PFOS IN QUALITY CONTROL SAMPLES 3.2.1. Ambient Temperature Stability of PFOS 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 AD VAN CED BIO AN ALYTICAL S E R V I C E S . INC. Page 17 of 50 99VDJA01.MI.DOC Page 188 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TO X-110 LRN-U2849 3.2.2. Freezer Stability of PFOS in Rat Serum at -20 C The results of the long-term stability of PFOS in QC samples stored at -20 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 of freezer storage after two, four, and eight months will be reported as an addendum to this report. 3.2.3. Freeze/Thaw Stability of PFOS in Rat Serum The results of the freeze/thaw stability of 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. R e p r o d u c ib il it y o f R e in j e c t in g E x t r a c t e d Sa m p l e s 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. E x t r a c t io n R e c o v e r y The mean recoveries of 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 f50 99VDJAOI.MI.DOC Page 189 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 4. CONCLUSIONS The turbo ion spray LC/MS assay procedure for the determination of PFOS in rat serum has proven to be sensitive, specific, accurate, and reproducible. Its high sensitivity allows reliable and reproducible quantitation of PFOS down to a level of 0.05 pg/mL in rat serum based on 50-pL samples. 5. DATA RETRIEVAL 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 S E R V IC E S , INC. 3M Environmental Laboratory Page 19 o f50 99VDJA01.MI.DOC Page 190 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 7. TABLES Table 1: Intra-Assay Accuracy and Precision of the PFOS Assay in Rat Serum for QC Samples Theoretical Cone. Run 9 PFOS Concentration (pg/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 2.93 1.77 6.34 1.99 5.58 17.6 3.28 -2.35 RSD = (SD/Mean) x 100 RE = [(Mean/Theoretical)-l] x 100 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S , INC. Page 20 of 50 99VDJA01.MI.DOC Page 191 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Table 2: Inter-Assay Accuracy and Precision of the PFOS Assay in Rat Serum for QC Samples from Three 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 0.187 0.186 6.44 6.44 16.9 17.3 0.182 6.32 17.5 0.189 0.182 6.19 6.31 18.0 17.4 Run 11 0.194 0.197 0.184 0.192 6.17 6.13 6.41 6.15 18.2 17.1 17.6 17.7 0.184 6.24 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)-l] x 100 3M Environmental Laboratory A D VAN CED BID AN ALVTICAL S E R V I C E S . INC. Page 21 of 50 99VDJA01 .MI.DOC Page 192 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Table 3: Accuracy and Precision of the PFOS Assay in Rat Serum for Calibration Standards from Three Validation Runs PFOS Calibration Standard Concentration (|ig/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 RSD (%) 4.38 2.65 2.07 1.15 0.613 RE (%) 2.20 -1.67 -0.207 -4.78 -5.53 2.14 1.90 7.05 4.50 7.78 1.62 2.13 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 A D VA N CED BID AN ALYTICAL S E R V I C E S . INC. Page 22 of 50 99VDJA01.MI.DOC Page 193 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Table 4: Calibration Curve Parameters for PFOS in Rat Serum Run# 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 BIDANALYTICAL S E R V IC E S . INC. Page 23 of 50 99VDJA01.MI.DOC Page 194 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Table 5: Partial Volume Analysis of PFOS in Rat Serum PFOS Cone. (ug/mL) QC4 Theoretical Cone. 100 (1 in 10 dilution) Run 9 111 105 105 104 104 Mean 106 RSD (%) RE (%) 2.71 . 5.89 RSD = (SD/Mean) x 100 RE = [(Mean/Theoretical)-l] x 100 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S , INC. Page 24 of 50 99VDJA01 .MI.DOC Page 195 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Table 6: Lower Limit of Quantitation of PFOS 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 100 RE = [(Mean/Theoretical)-1] x 100 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V IC E S , INC. Page 25 of 50 99VDJA01.MI.DOC Page 196