Document 2BaYJxJnEk2VwDZJvm6KYMdr

BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACTTOX-111 LRN-U2994 Study Title Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats Analytical Laboratory Report Title Determination of the Concentration of Perfluorooctanesulfonate(PFOS) in the Serum, Liver, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to PFOS via Gavage Data Requirement Not Applicable Author 3M Environmental Laboratory Study Completion Date at signing Performing Laboratories Urine Analyses Feces Analyses 3M Environmental Laboratory Building 2-3E-09,935 Bush Avenue St. Paul, MN 55106 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Liver Analyses Battelle Memorial Institute 505 King Avenue Columbus, OH 43201 Serum Analyses Advanced Bioanalytical Services, Inc. 15 Catherwood Road Ithaca, NY 14850 Project Identification 3M Medical Department Study: T-6295.14 Argus In-LifeStudy: 418-015 Analytical Report: FACT TOX-111 3M Environmental Laboratory Request No. U2994 Total Number of Pages 34 1 3M Environmental Laboratory Page 1 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-1II LRN-U2994 This page has been reserved for specific country requirements. 3M Environmental Laboratory Page 2 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-I 1I LRN-U2994 GLP Compliance Statement Analytical Laboratory Report Title: Determination of the Concentration of Perfluorooctanesulfonate(PFOS) in the Serum, Liver, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposedto PFOS via Gavage Study Identification Numbers: T-6295.14, FACT TOX-111, LRN-U2994 This study was conducted in compliance with United States Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations 21 CFR Part 58, with the exceptions in the bulleted list below. The analytical phase completed at the 3M Environmental Laboratory was performed in accordance with 3M Environmental Technology and Safety Services Standard Operating Procedures. Exceptions to GLP compliance: In the study binder, there are numerous Chain of Custody sheets that lack a signature, time, and date. In the study binder, there are packing lists sent by Argus that have evidence of having been whited-out with "White-out." Mass spectrometry data for liver and urine were collected and processed with the Mass Lynx software system that was not fully validated. Separate study directors were initially assigned to lead the in vivo and analytical portions of the study, respectively. This was eventually corrected. On several occasions, data entries or corrections were not documented exactly as required by the GLPs. Not all reagents and solutions used in this study included all the fields required by GLPs. Dose confirmation analyses were not performed in compliance with GLP regulations. (signatures on next page) 3M EnvironmentalLaboratory Page 3 3M Medical DepartmentStudy: T-6395.14 p L se, D.M.V., Ph.D., Study Director Kris J. Hansen, Ph.D., Analyfical lnvesfigator .--- William Reagen, Ph.D., Laboratory Manager BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 1 J W & 4 / 0/ Date UCh/u, Dat'e 3M EnvironmentalLaboratory Page 4 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 GLP Study-Quality Assurance Statement Analytical Laboratory Report Title: Determination of the Concentration of Perfluorooctanesulfonate (PFOS) in the Serum, Liver, Urine, and Feces of Cr1:CD"BR VAF/PIUS* Rats Exposed to PFOS via Gavage Study IdentificationNumbers: T-6295.14, FACT TOX-111, LRN42994 This analytical study performed at 3M Environmental Laboratory has been inspected by the 3M Environmental Laboratory Quality Assurance Unit (QAU) as indicated in the following table. The findings were reported to the study director and laboratory management. I 10/26-10/27/00 I 11/13/00 ~~ I I I I DraftReport I I I I DraftReport 10/30/00 11/13/00 10/30/00 11/13/00 See Appendix G for the individual contractor quality assurance statements. L W QAU Represdntative s-/g/uc Date 3M Environmental Laboratory Page 5 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Table of Contents GLP Compliance Statement.............................................................................................. 3 GLP Study-Quality Assurance Statement ....................................................................... 5 Study Personneland Contributors..................................................................................... 10 Introduction and Purpose .................................................................................................. 11 Test System ................................................................................................................. 11 Specimen Collection and Analysis ................................................................................ 11 Specimen Receipt and Maintenance................................................................................. 12 Chemical Characterizationof the Test Article.................................................................... 13 Procurement................................................................................................................. 14 Stability Studies ............................................................................................................ 14 Dose ConfirmationAnalyses ........................................................................................ 14 Method Summaries........................................................................................................... 15 3M EnvironmentalLaboratory....................................................................................... 15 Preparatory Method ................................................................................................. 15 Analytical Methods................................................................................................... 15 Analytical Equipment ............................................................................................... 16 Deviations..................................................................................................................... 17 Data Quality Objectives and Data Integrity........................................................................ 17 Data Summary. Analyses. and Results ............................................................................. 17 Summary of Quality Control Analyses Results.............................................................. 17 Statement of Data Quality ............................................................................................ 18 Summary of Sample Results ........................................................................................ 18 Statistical Methods and Calculations ................................................................................. 19 Statement of Conclusion ................................................................................................... 19 Appendix A: Control Matrices and Dose Confirmation Analyses........................................ 20 Appendix B: Protocol, Protocol Amendments and Deviation Summary ............................. 22 Appendix C: Extraction and Analytical Methods ................................................................ 54 3M Environmental Laboratory, ETS-8-96.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Urine for Analysis using HPLC-Electrospray/Mass Spectrometry/Mass Spectrometry," (14 pages)...............55 3M Environmental Laboratory, ETS-8-97.0, "Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrospray/Mass Spectrometry/Mass Spectrometry," (10 pages)........................ 69 3M Environmental Laboratoryl ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicals in Serum Extracts Using HPLC- Electrospray/MassSpectrometry," (9 pages).................................................................... 81 Appendix D: Data Summary Tables .................................................................................. 91 Appendix E: Data Spreadsheets ....................................................................................... 95 3M Environmental Laboratory Page 6 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Appendix F: Example Calculations .................................................................................... 101 Appendix G: Contract Laboratory Reports....................... ................................. .................I02 Advanced Bioanalytical Services, lnc., Quantitative Determination of Perfluorooctanesulfonate(PFOS) in Rat Serum from Study #FACT-TOX-111 Using Turbo Ion Spray LC/MS, ABS Report Number 99ADJA03.MI.DOC (19 pages).................103 Advanced Bioanalyfical Services, lnc., "Method Validation for the Quantitation of Perfluorooctanesulfonate(PFOS) in Rat Serum by Turbo Ion Spray LC/MS," ABS Report Number 99VDJAOI.MI.DOC (50 pages)................................................................ 122 Baffelle Memorial lnsfifute, Study Number N003296-G, Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats Final Report, (68 pages). .............................................. 172 Centre Analytical Laboratories, Inc., Centre Study Number 023-017, Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats, (93 pages). ....................................... 240 Appendix H: Certificate of Analysis ................................................................................... 334 Appendix I: Report Signature Page ................................................................................... 341 3M EnvironmentalLaboratory Page 7 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 List of Tables Table 1. Female Rat Population Demographicsfor Study #418-015 ................................. 11 Table 2. Custody Transfer of Specimens in the Study of the Determination of the Presenze and ConceEtration of PFOS in the Sera, Liver, Urine, and Feces of Crl:CD BR VAF/Plus Rats Exposed to T-6295.14 via Gavage .......................... . I 3 Table 3. Characterization of Test Article in Study FACT TOX-111 .................................... 13 Table 4. Negative Ions Monitored in 3M Laboratory Analyses for Study FACT TOX-111 ..17 Table 5. Characterization of the Control Matrices Used for Sera, Liver, Urine, and Feces Analyses in Study FACT TOX-111 ....................................................................... 20 Table 6. Characterization of the Analytical Reference Substances Used for Liver, Urine, Sera, and FecesAnalyses in Study FACT TOX-1I 1............................................ 20 Table 7. Dose Confirmation Analyses for Perfluorooctanesulfonate for Test Samples from In-Life Study #418-015 ................................................................................. 21 Table 8. Deviation Summary for FACT TOX-I 11 .............................................................. 22 Table 9. Average Results for the Analyses of Sera Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-6295.14 via Gavage ................................................................................................................ 91 Table I O . 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:CD@BRVAF/PIUS@Rats Exposed to T-6295.14 via Gavage ................................................................................................................ 92 Table 1I.Average Results for the Analyses of Urine Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-6295.14 via Gavage ................................................................................................................ 92 Table 12. Average Results for the Analyses of Feces Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-6295.14 via Gavage ................................................................................................................ 93 Table 13. LOQ Values Used in FACT TOX-111 Analysis by Method and Usage Dates ....94 Table 14. Results of Analyses of Sera Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-6295.14 via Gavage. .......................... 95 Table 14. Results of Analyses of Sera Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Liver, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-6295.14 via Gavage........................... 96 Table 15. Results of Analyses of Liver Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Liver, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-6295.14 via Gavage ........................... 97 3M EnvironmentalLaboratory Page 8 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Table 15. Results of Analyses of Liver Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Liver, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-6295.14 via Gavage........................... 98 Table 16. Results of Analyses of Urine Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-6295.14 via Gavage........................... 99 Table 17. Results of Analyses of Feces Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-6295.14 via Gavage...................... .IO0 3M EnvironmentalLaboratory Page 9 BACK TO MAIN 3M Medical Department Study: T-6395.14 Study Personnel and Contributors Analytical Report: FACT TOX-111 LRN-U2994 Study Director Marvin T. Case, D.V.M., Ph.D. 3M Corporate Toxicology 3M Center, Building 220-2E-02 St. Paul, MN 55133-3220 Phone (651) 733-5180 Analytical Chemistry Laboratories Urine Analyses 3M Environmental Laboratory Kristen J. Hansen, Ph.D., Analyfical lnvesfigafor Serum Analyses Advanced Bioanalytical Services, Inc. (ABS) David J. Anderson, M.S., Analyfical lnvesfigafor Sponsor 3M Corporate Toxicology 3M Center, Building 220-2E-02 St. Paul, MN 55133-3220 John L. Butenhoff, Ph.D., Sponsor Represenfafive Liver Analyses Battelle Memorial Institute (Battelle) Jon C. Andre, Ph.D., Analyfical lnvesfigafor Feces Analyses Centre Analytical Laboratories, Inc. (Centre) Enaksha Wickremesinhe, Ph. D. Analytical lnvesfigafor 3M EnvironmentalLaboratory Contributing Personnel Lisa A. Clemen Kelly J. Dorweiler* Mark E. Ellefson Sara E. Estes* Barb A. Gramenz* Sarah A. Heimdal* Cari S. Hewitt* Marlene M. Heying* Megan C. Holloway* Harold 0. Johnson Ognjenka Krupljanin* Kelly J. Kuehlwein* Sally A. Linda* Ian A. Smith* Bob W. Wynne" * Contract laboratory professional sewice employees Location of Archives All original raw data, the protocol, the analytical report, the test article and the analytical reference standard reserve samples, as well as the specimens pertaining to the analytical phase of this study, are archived at the 3M Environmental Laboratory for a minimum of ten years. Reserve samples of control matrices used at Battelle, Centre and ABS will be stored at the respective contract laboratories in such a manner that the control matrices will be preserved for use in ensuing studies. 3M Environmental Laboratory Page 10 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Introduction and Purpose The purpose of the analytical study is to determine the concentration of perfluoro-octanesulfonate (PFOS) in sera, liver, urine, and feces specimens taken from the Gavage Recovery Study of T- 6295.14 (potassium perfluorooctanesulfonate(KPFOS), CAS Number: 2795-39-3) in Crl:CD@BR VAF/PIUS@Rats. Rat dams were exposed to KPFOS via gavage prior to and during mating. Exposure to KPFOS was halted on the first day of presumed gestation. Pups were not directly exposed to KPFOS, but may have been exposed in utero and during lactation. The analytical phase of this study was initiated on 9 June 1999. Test System The test system species and strain selected was the Crl:CD@BRVAF/PIUS@(Sprague-Dawley) rat, received from Charles River Laboratories, Inc., and permanently identified using Monel@selfpiercing ear tags. Generation FO virgin female rats were approximately 60 days of age and weighed approximately 181 to 222 g when received. Male rats of the same source and strain were used only as breeders and were not administered the test article or considered part of the test system. Generation F1 rats were identified as part of Group I, II, or Ill litters, but were not individuallytagged, and all parameters were evaluated in terms of the litter (Table 1). The test system comprised Generation FO rats and Generation F1 pups. In Generation FO, three groups of 8 female rats each (a total of 24) were used. Group I control FO female rats were administered Tween@80(carrier). All other FO female rats were administered KPFOS in 0.5% Tween@80A. t predetermined intervals during, and at the end of, the in-life phase of the study, sera, urine, feces, and liver specimens were collected from Generation FO rats, and sera and liver specimens were collected from Generation F1 pups. Population Selected for Study Group I (Control) 8 0 mg/kg/day Group II a 0.1 mg/kg/day Group 111 8 1.6 mg/kg/day * OneGroupI FO rat died from injuryon DG 15 anddid notgive birth. One Group 111 FO rat did not give birth. Total Litters 7a 8 7b Selected for Study 7" 8 7b Specimen Collection and Analysis In the analytical study reported here, 440 serum, liver, urine, and feces specimens were collected from adult female rats (Generation FO) and their offspring (Generation FI). The specimens were sent to the 3M Environmental Laboratory and contract laboratories to be analyzed for PFOS. Sera specimens were collected from all Generation FO animals immediately 3M EnvironmentalLaboratory Page 11 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 prior to cohabitation, on Gestation Days (DGs) 7, 15, and 21 and on Lactation Days (DLs) 14 and 22. Sera specimens were collected from pooled litter samples on DL 21. Urine and feces specimens were collected from all Generation FO animals beginning one day before cohabitation was initiatedthrough the morning cohabitationwas initiated, from DGs 6 through 7, 14 through 15, and 20 through 21, and on DLs 21 through 22. On DL 22, all surviving Generation FO and Generation F1 animals assigned to the study were sacrificed, and a liver specimen was collected from each animal. The number and type of specimens collected for analyses in the analytical phase of this study are presented below. Specimens Collected from Study Groups I through 111 (through 20 February 99): Serum Specimens-161 specimens Liver Specimens45 specimens Urine Specimens-I 17 specimens Feces Specimens-I 17 specimens Blood specimens were separated by refrigerated centrifuge. Resulting serum specimens were then transferred to labeled polypropylene tubes and immediately frozen on dry ice. Liver, urine, and feces specimens were collected and frozen on dry ice. All serum, liver, urine, and feces specimens were stored at -70 "C or below until they were shipped, on dry ice, to the 3M Environmental Laboratory. Specimens were then shipped to the contract analytical laboratories for analysis. Urine samples were extracted beginning on 06 October 1999 using an ion-pairing reagent and methyl-fed-butyl ether (MtBE). Sample extracts were analyzed using high-pressure liquid chromatography-electrospray/tandemmass spectrometry (HPLC-ES/MS/MS) in the multiple response monitoring mode. PFOS levels were quantitated by external calibration. Analytical details are included in this report. The methods and analytical equipment settings used by Battelle Memorial Institute, Advanced Bioanalytical Services, Inc. and Centre Analytical Laboratories, Inc. are presented in the respective contract laboratory reports (see Appendix G). Specimen Receipt and Maintenance The 3M Environmental Laboratory received serum, liver, urine and feces specimens that were collected during the in-life phase of this study, M18-015 from Argus. All specimens were received frozen on dry ice and were immediately transferred to storage at -20 k 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 EnvironmentalLaboratory Page 12 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-I 11 LRN-U2994 Table 2. Custody Transfer of Specimens in the Study of the Determinationof the Presence and Concentrationof PFOS in the Sera, Liver, Urine,and Feces of Crl:CD@BRVAF/Plus@ Rats Exposed to T-6295.14 via Gavage TissudSample Date Received a Shipment date not applicable: urine specimenswere analyzedat the 3M EnvironmentalLaboratory. Control matrices used in urine analyses were obtained from Lampire Biological Laboratories, Inc., and are presented in Appendix A. Chemical Characterization of the Test Article Potassium Perfluorooctanesulfonate(KPFOS) CAS Number: 2795-39-3 Chemical Formula: C8FI7SO;K' Molecular Weight: 538.0 Chemical characterization information on potassium perfluorooctanesulfonate, CAS Number 2795-39-3, used in this study is presented in Table 3. Table 3. Characterization of Test Article in Study FACT TOX-111 Chemical Name I Test Article KPFOS Potassium Peffluorooctanesulfonate Source 3M Toxicology Services Expiration Date 31 Aug-ust 2001 Storage Conditions Ambient furonztiel 1n6SMIa0y"2C000, then I I I ChemicalLot# I I I Physical Description 217 White CrystallinePowder I Puritv I 86.9% I 3M Environmental Laboratory Page 13 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 See the Certificate of Analysis in Appendix H for complete characterization information. Preliminary characterization had been performed by NMR only, showing a purity of 99.28%, which was initially used for all calculations. A more complete characterizationwas performed later, which determined the purity of 86.9%. Procurement Potassium perfluorooctanesulfonatewas obtained in one lot (Lot number 217) from 3M Toxicology Services. Stability Studies The stability of potassium perfluorooctanesulfonatewas not determined. Dose Confirmation Analyses Dose confirmation analyses were performed on test article samples (0.00, 0.02, and 0.32 mg/mL) collected on 22 November 1998 and 07 January 1999, during the in-life phase of the study. The results are presented in Appendix A. The dose confirmation data were collected according to a method that was not fully validated. Dose confirmation was performed by diluting the KPFOS dose samples with methanol into the linear range of the instrument used for analysis. Samples were analyzed versus an unextracted l/x-weighted calibration curve using HPLC-ES/MS/MS. 3M EnvironmentalLaboratory Page 14 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Method Summaries Following is a brief description of the methods used during this analytical study by the 3M Environmental Laboratory and by contract laboratories. The methods used in this study are located in Appendices C and G. The methods and analytical equipment settings used by Battelle Memorial Institute, Advanced Bioanalytical Services, Inc. and Centre Analytical Laboratories, Inc. are presented in the respective contract laboratory reports (see Appendix G). 3M Environmental Laboratory PREPARATORMYETHOD ETS-8-96.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Urine for Analysis Using HPLC-Electrospray/Mass Spectrometry/Mass Spectrometry'' This method is used to extract PFOS from urine by the use of an ion-pairing reagent and methyl-fed-butyl ether (MtBE). An ion-pairing reagent (tetrabutyl ammonium hydrogen sulfate) is added to 2 mL of the sample, and the analyte-ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 0.5 mL of methanol, then filtered through a 0.2 pm nylon filter attached to a 3-mL plastic syringe, into glass autovials. Modificationto 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 (Le., 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/MassSpectrometry/Mass Spectrometry" The analyses were performed by monitoring one or more product ions selected from a single primary ion characteristic of a particular fluorochemical, using HPLC-ES/MS/MS. For example, molecular ion 499 (CsF&03-), selected as the primary ion for PFOS analysis, was fragmented further to produce ion 99 (FSO3-). The characteristic product ion 99 was monitored for quantitative analysis. 3M EnvironmentalLaboratory Page 15 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" The analyses were performed by monitoring the perfluorooctanesulfonate(PFOS) parent anion, m/z=499, and a daughter ion, m/z=99, to verify the presence of PFOS in the samples using a tandem mass spectrometer. e Battelle Preparatory and Analytical Method: Method for Analysis of Potassium Perfluorooctanesulfonate (PFOS) in Rat Liver by LC/MS/MS e 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 ANALYTICAELQUIPMENT The actual analytical equipment settings used in the analysis of urine samples varied slightly during actual data collection. The following is representative of the settings used during the analytical phase of this study by the 3M Environmental Laboratory. Liquid Chromatograph: Hewlett-Packard@Series 1100 Liquid Chromatograph system Analytical column: Keystone@BetasilTMCI8 2x50 mm (5 pm) Column temperature: Ambient Mobile phase components: Component A: 2 mM ammonium acetate Component B: methanol Flow rate: 300 pL/min Injectionvolume: 10 pL Solvent Gradient: Time (min) %B 0.00 10.0 1.oo 10.0 5.50 95.0 7.50 95.0 8.00 10.0 Mass Spectrometer: Micromass@API/Mass Spectrometer Quattro Ilm Triple Quadrupole system Software: Mass Lynxm3.3 Cone Voltage: 60 V Collision Gas Energy: 40-45 eV Mode: Electrospray Negative Source Block Temperature: 150C *I0C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (MRM) 3M EnvironmentalLaboratory Page 16 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-I I 1 LRN-U2994 Table 4. Negative Ions Monitored in 3M Laboratory Analyses for Study FACT TOX-11I I Targethalyte I Primarylon(wu) I Produdlon(mu) I PFOS 499.0 99.0 THPFOS* 427.0 80.0 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 quantification in the study, due to an unknown contaminant that interfered with the surrogate response. Deviations from the original protocol and methods are documented in Appendix B. Data Quality Objectives and Data Integrity The following data quality objectives (DQOs) for 3M Environmental Laboratory assays were indicated in the protocol for this study: 0 Linearity: The coefficient of determination (Rz) 10.980. 0 Limits of Quantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibration curve. 0 Acceptable Precision: Precision is within 30% for the method. Acceptable Spike Recoveries: 50-1 50% Demonstrationof Specificity: Specificitywill be demonstrated by chromatographic retention time and mass spectral daughter ion characterization. Data Summary, Analyses, and Results Data quality objectives for the analytical phase of this study outlined in the 3M Environmental Laboratory protocol for FACT TOX-111 (see Appendix B) were met with the exceptions noted in this report. Summary of Quality Control Analyses Results Linearity: The coefficient of determination (R2)of the standard curve was 20.980. Calibration Standards: Quantitation of the target analytes was based on linear regression analysis (l/x-weighted) of two extracted matrix curves bracketing each group of samples, except as noted in the deviation summary. High or low points on the curve may have been 3M Environmental Laboratory Page 17 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 deactivated to provide a better linear fit over the curve range most appropriate to the data. Occasionally, a single mid-range curve point that was an obvious outlier may have been deactivated. Quantitation of the analyte was based on the response of one specific product ion using the multiple response-monitoring mode of the instrument (see Appendix C). 0 Limits of Quantitation (LOQ): For urine, the LOQ is 0.00295 pg/g. It is equal to the lowest acceptable standard in the calibration curve, defined as a standard within +30% of the theoretical value (see Appendix D). 0 Blanks: All blanks were below the lower limit of quantitation for the compounds of interest. 0 Precision: Precision was not determined for this study. 0 Matrix Spike Recoveries: Matrix spikes and matrix spike duplicates were extracted with each set of samples and analyzed during analytical runs at the 3M Environmental Laboratory. Acceptable spike recoveries of 52 to 96% of expected values were achieved for all matrix spikes prepared in urine. Surrogates: The surrogate (THPFOS) was added to all urine samples and standards. External calibration was used to quantify urine values, because an unidentified compound interfered with the analysis of the surrogate in the samples. Statement 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 quantitativeto 50% or greater. Refer to Appendix G for contract laboratory summaries of quality control analysis results. 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 Certificate of Analysis PFOS purity factor, see Appendix E. Advanced Bioanalytical Services, Inc.: Refer to the ABS Bioanalytical Report and the ABS Addendum to the Bioanalytical Report for information regarding serum analysis (Appendix G). For serum analysis data that has been corrected with the Certificate of Analysis PFOS purity factor, see Appendix E. Centre Analytical Laboratories, Inc.: Refer to the Centre Analytical Report for information regarding feces analysis (Appendix G). Feces analysis data is also presented in Appendix E. 0 Samples from Control Animals: Low levels of PFOS were detected in the urine of some control animals. These levels were significantly lower than those found in the low dose test animals. 3M EnvironmentalLaboratory Page 18 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-1I 1 LRN-U2994 Samples from Dosed Animals: In general, PFOS levels found in the urine of the FO Generation animals increased with dose group. Detailed sample data tables are presented in Appendices D and E. ~ ~~ Statistical Methods and Calculations Statistical methods were limited to the calculation of means and standard deviations. See Appendix F for example calculations used to generate the urine sample data in FACT TOX-111. Statement of Conclusion Under the conditions of the present study, perfluorooctanesulfonatewas observed in all sample types of all Generation 0 test system animals dosed with the test substance during the in-life phase of the study, and in all sample types of their offspring (Generation FI). 3M Environmental Laboratory Page 19 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Appendix A: Control Matrices and Dose Confirmation Analyses Location of Analyses 3M Environmental Laboratory Control Matrix ~~~ Source Expiration Date Storage Conditions Chemical Lot # Physical Description Human Urine Lampire Biological Laboratories, Inc. 01 August 2008 Frozen 66-1 1691 Urine Battelle Memorial Institute Rat andor Rabbit Livers Argus Research and/or Sigma NIA Frozen at -20 f 10 "C NIA Liver Advanced Bioanalytical Services, Inc RatSerum Harlan Bioprcducts for Science, Inc.. N/A Frozen R80128 Frozen Liquid Centre Analytical Laboratories, Inc. Rat Feces Lampire Biological Laboratories, Inc. NIR Frozenat < -10 "C NIR Rat feces Table 6. Characterizationof the Analytical ReferenceSubstances Used for Liver, Urine, Sera, and Feces Analyses in Study FACT TOX-111 3M Environmental Laboratoty KPFOS Potassium Pefiuorwctanesulfonate Battelle Memorial KPFOS Potassium Petfluorooctanesulfonate Advanced Bioanalytical Services, Inc KPFOS Potassium Petfluorooctanesulfonate Centre Analytical Laboratories, Inc. KPFOS Potassium Peffluorooctanesulfonate 3M Toxicology Selvices 3M Toxicology Services 3M Toxicology Services 3M Toxicology Services 31 August 2001 Ambient 171 31 August 2001 31 August 2001 I Ambient I Ambient 171 171 31 August 2001 I Ambient I 217 Description Purity White Crystalline Powder 86.4% White Ctystalline Powder 86.4% White Crystalline Powder 86.4% White Ctystalline Powder 86.9% 3M EnvironmentalLaboratory Page 20 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Table 7. Dose ConfirmationAnalyses for Perfluorooctanesulfonatefor Test Samples from In-Life Study $418-015 Sample Number Group I Control 0.0 mglkglday B-' Group II B-4' 0.1 mglkglday 0.02mg1mL B-1 Target Conc. PFOS (IJglmL) Raw Corrected pFOs Expected 'One. pFos Measured Measured YORecovery Conc. PFOS Conc. PFOS ('glmL) (PglmL) (pg/mL) Accuracy Group 111 1.6 mglkgtday 0.32 mglmL I I B-418-015-- niliitnr Limit of Quantitation Limit (LOQ) = 0.030 pg/mL Formula used to calculate Expected Concentration from Target Concentration: Target Concentration x PFOS Purity (Test Article) x PFOS Correction Factor = Expected Concentration PFOS PFOS Purity (Test Article, Lot 217) = 86.9% PFOS Correction Factor = 0.9275 320 pg/mL x 0.869 x 0.9275 = 258 pg/mL Expected Concentration Formula used to calculate Corrected Measured Concentration of PFOS: Raw Measured Concentration x Dilution Factor x PFOS Purity (Reference Substance) x PFOS Correction Factor = Corrected Measured Concentration of PFOS PFOS Purity (Reference Substance, Lot 171) = 86.4% PFOS Correction Factor = 0.9275 0.358 VglmL x 808 x 0.864 x 0.9275 = 232 pg/mL Corrected Measured Concentration of PFOS 3M Environmental Laboratory Page 21 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Appendix 6: Protocol, Protocol Amendments and Deviation Summary Table 8. Deviation Summaryfor FACT TOX-11I Deviation IAdditional control matrices were used: human urine I and rat feces. Date(s) ofOccurrence Entire Study ETS-8-97.0 specifies the method of analysis requires the use of an internal 06 October 1999 to calibration. The actual 04 November 1999 analyses used an external calibration method. The analysis of the dose samples was not conducted according to GLP regulations. ETS-8-5.1, a method validated for the analysis of sera extracts, was followed for the analysis of the Tween dose samples. 21 September 1999 Impact on Study No negative impact on the study-A successful cross-validation was completed. Unknown contaminant interfered with use of surrogate for internal calibration. Corrective action is an improvement. Data quality is satisfactory to %50% accuracy. The analysis of Tween samples was not conducted under GLPs (as per discussions with study director). This deficiency is included in the final report. 3M Environmental Laboratory Page 22 3M Medical Department Study: T-6395.14 3M Environmental Technology and Services PO Box 33331 St. Paul, M N 55133-3331 612778 6442 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 PrOtOCOl #fACT-TOX-l I1 Study Tittle Oral (Gavage) PharmacokineticRecovery Study of PFOS in Rats PROTOCOL Aufhor Lisa Clemen Date: June 8,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M EnvironmentalLaboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification FACT-TOX-111 U2994 3M EnvironmentalLaboratory 3M Environmental Laboratory Page I of I O Page 23 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 PrOtOCOl #FACT-TOX-l7 I Study Identification Oral (Gavage) Pharmacokinetic Recovery Study of PFOS In Rats Test Material Sponsor Sponsor Representative SfudyDirector Study tocafion(s) In vivo Testing Facility Analytical Testing Laboratory Perfhorooctanesulfonic acid potassium salt ("-6295) 3M Toxicology Services - Medical Department 3M Center, Building 220-23-02 St. Paul, MN 55144-1000 Marvin T. Case, D.V.M., Ph.D. 3M Toxicology Services Telephone: 651-733-5180 Facsimile: 651-733-1773 Kristen J. Hansen, Ph.D. 3M Environmental Technology and Safety Services Building 2-333-09 651-778-6018 Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham,PA 19044 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 3M Environmental Laboraiory 3M Environmental Laboratory Page 2of I O Page 24 BACK TO MAIN 3M Medical Department Study: T-6395.14 Sub-Contract Laboraf odes Proposed Study Timetable Study inifiation Date Study Completion Date Analytical Report: FACT TOX-111 ! LRN-U2994 Profocol#FACT-TOX-?1f Advanced BiomalyticalServices, Inc. 15 Cathemood Road Ithaca,NY 14850 Battelle Memorial Institute 505 King Avenue Columbus Ohio 43201-2693 . June 8,1999 August 8,2000 Oral (gavage) pharmacokinetic recovery study of potassium perfluorooctane sulfonic acid (PFOS)in rats. 2. PURPOSE This analytical study is designed to determine levels of potassium perfluorooctanesulfonate (PFOS)in specimens of liver and serum of rats. The in-life portion of this study was conducted at Argus Research Laboratories, study #418-015. All serum sampleswill be extracted and analyzed at Advanced Bioanalytical Senrices, 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 thisprotocol willbe written. 3. REGffLATORY cOMiJLL4NCE This study will be conducted in accordance with the United States Food and Drug Administration, Good Laboratmy Practices Standards, Final Rule 21 CFR 58, withthe exception that analysis of the test material mixture for concentration, solubility, homogeneity, and stability will not be conducted, and is the responsibility of the Sponsor. 4. ' QUALITYASSURANCE The 3M Environmental Laboratory Quality Assurance Unit Will review the protocol and audit study conduct, data, and fmd 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 EnvimnrnenfalLaboratory 3M Environmental Laboratory Page 3 of 10 Page 25 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 PrOtOCOl #FACT-TOX-77 I 5. TESTMATERIAL 5.7 Refer to Argus Research Laboratory protocol for study #418-015. 6. CONTROL MATREES 6,f identification Rat liver and serum, and/or rabbit liver and serum traceabilitynumbers will be recorded in the raw data and included in the final report 6.2 Source Argus Research andor Sigma Chemical 6.3 Physical Description Rat liver and serum, and/orrabbit liver and serum 6.4 Purify and Stabiiify Not applicable 6.5 Storage Conditions Frozen at -20 "C f 10 "C or -50 "C f 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 frnal test rule (if applicable). 6.7 Dispositlon 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. REFERENCMEATERIAL 7.1 fdentificationPotassium perfluorooctanesulfonate (PFOS),lot #s 171,215, or 217 (equivalent lots) 7.2 Source 3M Specialty Chemicals 7.3 Physical Description White powder 7.4 Purify and StabilityPurity of PFOS is 99% or greater. Stabilityhas not been determined. 7.5 Storage Conditions Room temperature 7.6 Reserve Material A reserve sample fkom 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 followingthe effective date of the finaltest rule (if applicable). 7.7 Disposition Unused reference material will be retained for use by the 3M EnvironmentalLaboratory and will be discarded when the quality of preparationno longer affords evaluation. 3M Environmental Laboratory 3M Environmental Laboratory Page 4 of10 Page 26 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 P ~ O ~ O#CFAOC~T-TOX-f I f Dosage Group 1 2 3 Number of female rats 8 8 8 Dosage mg/kg/day 0 0.1 35-0 Concentration mg/kg/day 0 I 0.02 I 0.32 Dosage volume mwkg 5 15 15 Body tissue/fluid Collected Serum - Dam and Pup animals Urine and Feces - Dam and Pup - animals Liver Dam and Pup animals Dam-Predose, Days 7,14,15,21, and 22 Pup-Day 21 Predose, Days 7,15,21, and 22 Dam-At the termination of the study Pup-Day2 1 Total number of expected specimens: 456 Total number oftest animals: 16 Total number of control animals: 8 Expected # of specimens 144 Dam 24 Pup (pooled) 120 Urine and 120 Feces24 Dam 24 Pup (pooled) 3M Environmental Laboratory 3M Environmental Laboratory Page 5 of IO Page 27 BACK TO MAIN 3M Medical DepartmentStudy: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Protocol #FACT-TOX-I I I Specimens sent to 3M Environmental Laboratories will be received and tracked according to applicable Standard Operating Procedures. IO. PREPARATORMYETHODS 10,l FACT-M-1.1,Extraction ofPotassiumPerfluorooctanesulfonateor Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 10.2 ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC- , ElectrosprayMass 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 & sub-contracted to other laboratories, an amendment will be written to includetheir methods and copies of each method will be attached to thisprotocol. 1l.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 ofPotassium Perfluorooctanesulfonateor Other Flnorochemicals 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 wiIl be attached to this protocol. 12.DATAQUALITOYBJECTIVES The number of spikedduplicates, use of surrogates, and information on other data quality indicators are included inthe analytical methods. In addition, the followingcriteria will be met: I 2 I Linearity 12 2 0.98 12.2 Limits of detection / quantitafion 122.1 Method Detection Limit (MDL) for PFOS a) Serum: 1.75ppb b) Liver: 15 ppb 3M Environmental Laborafory 3M Environmental Laboratory Page 6of I O Page 28 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 PtVtOCOl #FACT-TOX-7 17 12.2.2 Limit of Quantitation (LOQ) - Equal to the lowest acceptable standard in the calibration curve 72.3 Duplicate acceptable precision e 30% for the method 72.4 Spike acceptable recoveries 70% - 130% 72.5 Use of confirmatory methods Indeterminate samples will be re-analyzed using a confirmatoq method. If a confirmaory method is used, an amendment to this protocol will be written. 72& Demonstration of specificity Chromatographic retention time, mass spectral daughter ion characterization. 73.S U B - C O N ~ ~ ; ~ACNTAELDYS~S 73.7 All analyses as detailed in this protocol will be performed at 3M Environmental Laboratories, Building 2-3B-09,935Bush Avenue, St. Paul, MN 55106, at Advanced Bioanalytical Services,Inc., 15 Catherwood Road, Ithaca, NY 14850,or at Battelle Memorial Institute, 505 King Avenue, Columbus, Ohio 43201-2693. 13.2 An amendment to thisprotocol will be written if analyses are perfomred at laboratories other than the 3M Environmental Laboratones, Advanced Bioanalytical Services, Inc., or BatteIle Memorial Institute. 14.STATISTEAL ANALYSIS Averages and standard deviations will be calculated. The statistical methods that will be used are described below: 14.7 Data transformationsand analysis Data will be reported as the concentration (weight/weight or weightlvol) of PFOS or metabolite per tissue or fluid. 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 3 M Environmental Laboratory report. Each report will include, but not be limited to, the following, when applicable: 75.7 Name and address of the facility performing the study 75.2 Dates upon which the study was initiated and completed 3M Environmental Laboratory 3M Environmental Laboratory Page 7 of 10 Page 29 BACK TO MAIN 3M Medicgl Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 PrOtOCOl #FACT-TOX-l I I 15.3 A statement of compliance by the StudyDirector addressing any exceptionsto 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-thetest 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 of the test system 15.9 A description of any circumstances that may have affected the quality or the integrity of the data 15.I O The name of the StudyDirector and the names of other scientists, professionals, and supervisory personnel involved in the study 15.I I 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 fiom the analyses 15.12 Statistical methods used to evaluate the data, if applicable 75.13 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 15.15 A statementpreparedby 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 wiIl be made in the form of an amendment issued by the Study Director. The amendment will clearly identifythe part of the report that is being amended, the reasons for the amendment, and will be signed by the Study Director. 16.LOCAT~OOFNRAWDATA,RECORDSA,ND FINALREPORT 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 originalpaper data, including those items listed below, will be retained in the archives of 3M Environmental Laboratory for at least a period of time as specified by regulation, and as established by 3M Environmental Laboratory Standard Operating Procedures. 3M Environmental Laboratory 3M Environmental Laboratory Page 8 of 10 Page 30 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Protocol #FACT-TOX-I 1I 7 6.7 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: 76.7.1 Approved protocol and amendments 76.1.2 Study correspondence 7 6.1.3 Shipping records 76.7.4 Raw data 76.7.5 Approved final report (original signed copy) 76.7.6 Electronic copies of data 16.2 The following supportingrecords will be retained separately from the study folder in the archives according to 3M Environmental Laboratory Standard Operating Procedures: 76.2.7 Training records 76.2.2 Calibration records 76.2.3 Instrument maintenance logs 76.2.4 Standard Operating Procedures, Ekpipment Procedures, and Methods 17.SPECIMERNETENTION Specimenswill be maintainedin 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.PROTOCOLAMENDMENATNSD DEVIATlONS Planned changes to the protocol will be in the form of written amendments signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part of the protocol and will be attached to the final protocol. All changes to the protocol will be indicated in the finalreport. Any other changes will be in the form of written deviations, signedby the Study Director and filed with the raw data 19.ATTACHMENTS 19.7 Atfachrnent A Preparatory and analytical methods 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 9 of I O Page 31 BACK TO MAIN 3M Medical Department Study: 1-6395.14 20. SIGNATURES A Ahwin T. Case, D.V.M.,PbD., SponsorRepresentative Analytical Report: FACT TOX-111 LRN-U2994 P t ~ t O c O#l FACT-TOX-I i1 Date # 6/4/99 Kriiten j,Hans&, Ph.D., 3M EnvironmentalLaboratory StudyDirectbr 'Date 3M EnvimnmenfalLaboratory 3M Environmental Laboratory Page 10 of I O Page 32 3M Medic@Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Study Title Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats PROTOCOL AMENDMENT NO. 1 Amendment Date: August 12,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M EnvironmentalLaboratory 935 Bush Avenue St. Paul, MIV 55106 Laboratory Project Identification ET&SS FACT-TOX111 LIRN U2994 3M Environmental Laboratory 3M Environmental Laboratory Page 33 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 PtUtOCOl FACT-TOXl I I Amendment No. I This amendment modifies the following portion(s)of the protocol: 1. PROTOCOLREADS: Section 10.0 and 11.0 list the following methods to use for extraction and analysis: FACT-M-1.1 "Extraction of Potassium Perfluorooctanesulfonateor Other Anionic Fluorochemicd Surfactant from Liver for Analysis Using HPLC-ElectsosprayMass Spectrometry" FACT-M-2.1 "Analysis of Fluomchemicals in Liver Extracts Using HPLC-ElectrosprayMass Spectrometry" AMENDTO READ; The extraction and analytical methods FACT-M-l .1and FACT-M-2.1, respectively, were updated on 07/22/99 to: ETS-8-6.0"Extraction ofPotassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Liver for Analysis Using HPLC-ElectrosprayMass Spectrometry' ETS-8-7.0 '"Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-ElectrosprayLMassSpectrometry" REASONT;he extraction and analytical methods FACT-M-1.1 and FACT-M-2.1 respectively, ' were updated on 07/22/99 to ETS-8-6.0 and ETS-8-7.0. These methods were updated to replace the extraction soIvent ethyl acetate with a different extraction solvent MTBE (methyl tert butyl ether), POAA and Monoester were removed flom the standard mix, and M556 was added to the standard mix. The analytical method was updated to include linear regression with l/x weighting and a few minor changes in the HPLC 1100 instrument parameters. 2. PROTOCOL READS: Section 10.4 and 11.4 state that if the analyses are sub-contracted to other laboratories an amendment will be written to include these methods. AMENDTO RAD: 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 *by LCIMSNS, Version$' Oi.;kLp \P REASON: The analytical methods at the sub-contract laboratories were not included in the original protocol. 3M Environmental Laboratory 3M Environmental Laboratory Page 34 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 PrOtOCOl FACT-TOXI I I Amendment No. 1 3. PROTOCOLREADS: Section 12.2.1 b) lists liver method detection limit as 15 ppb. AMENDToREAD: The liver method detection limit is 8.50 ppb (ng/g). REASON:The validation supportingmethods ETS-8-6.0and ETS-8-7.0included a lower method detection limit for PFOS. Amendment Approval -K W %*4 M'ant.in Case, D.V.M.,PhD., Sponsor Representative .. l7 & U L J l9W Date $LA &! Kris 5. Hansen, Ph.D., Study Director Datk 3M Environmental Laboratory 3 m r y - Page 35 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-I 11 LRN-U2994 Study Title Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats PROTOCOL AMENDMENT NO. 2 Amendment Date: September 30, 1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOXl 1 1 LIRN U2994 3M Envhnmentai Laboratory 3fvtnvironmental Laboratory Page 36 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Protocol FACT-TOXI71 Amendment No. 2 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: Section 2 states thestudy is designed to det&ne levels of potassium . perfluorooctanesulfonate(PFOS) in specimens of liver and sera in rats. AMENDTO READ: The study is designed to determine levels of potassium pduorooctmesulfonate (PFOS) in specimens of liver, sera, and urine in rats. REASON: The urine analytical methods were validated and approved after approval ofthe original protocol. PROrOCOL READS: Section 10.0 and 11.0 list the following methods to use for extraction and analysis: ETS-8-4.1 "Extraction of Potassium Perfluorooctanesulfonateor OtherFluorochemical Compounds from Serum for Analysis Using HPLC-ElectrosprayMass Spectrometry" ETS-8-6.0 "Extraction of Potassium Perfluorooctanesulfonateor OtherFluorochemical Compounds fkom Liver for Analysis Using HPLC-ElectrosprayjMasssSpectrometrf ' ETS-8-5.1 "Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds in Serum Extracts Using HPLC-ElectrosprayMass Spectrometry" ETS-8-7.0 "Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds in Liver Extracts Using HPLC-Electraspray/Mass Spectrometry" AMENDTO READ:: ETS-8-4.1 "Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Serum for Analysis Using HPLC-ElectrosprayMass Spectrometsy" ETS-86.0 "Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds h m Liver for Analysis Using HPLC-ElectrosprayMass Spectrometry" ETS-8-96.0 "Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds fiom Urine for Analysis Using HPLC-ElectrosprayMass SpectrometryMass Spectrometry" ETS-8-5.1 "Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds in Serum Extracts Using HPLC-ElectrosprayjMass Spectrometry" ETS-8-7.0 "Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds in Liver Extracts Using HPLC-ElectrosprayAdass Spectrometry" ETS-8-97.0 "Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds in Urine Extracts Using HPLC-ElectrosprayMass Spectrometryhfass Spectrometry'' REASONT: he extraction and analytical methods ETS-8-96.0 and ETS-8-97.0, were approved after approval of the originalprotocol. 3M Envlronmental Laboratory 3M-Emwironmental Laboratory Page 37 3M Medicgl Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Protocot FACT-TOXI I1 Amendment No. 2 3. PROTOCOLREADS: Section 6.1 lists control matrices as rat liver and serum or rabbit liver and senun. Source Argus andor Sigma Chemical. AMENDTO READ: Control matrices identificationrat liver and serum, rabbit liver and serum, human urine and/or rat urine. S o m e Argus, Sigma Chemical, Lampire Biologicals, Biological Specialty Corp. and/or Golden West Biologicals. REASON: Addition of control,urine specifications. 4. PROTOCOL READS: Section 12.2.1 a) lists sera method detection limit as 1.75 ppb and b) lists liver method detection limit as 15 ppb. AMENDTO READ: The method detection limits for all compounds and matrices will be taka &om the methods used for extraction and analysis. REASON: The method detection limits listed are specific to the 3M Environmental Laboratory. Statementwas added to allow for sub-contractedanalyses andor revised methods. -aboratory 3M Environmental Laboratory Page 38 BACK TO MAIN 3M Medicgl Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Protocol FACT-TOXI f I Amendment No. 2 5. PROTOCOL READS: Section 16 states that the original data, or copiesthereof, will be available at.the 3M Environmental Laboratory to hilitate auditsof the study during its progress and before acceptance of the final report. When the finalreport is completed, all originalpaper data, including: approved protocol and amendments, study correspondence, shipping records, raw data,approved final report, electronic copies of data, training records, calibrationrecords, instrumentmaintenance logs and standard operatingprocedures, equipment procedures, and methods will be retained in the archives of the 3M EnvironmentalLaboratory. AMENDTO READ: Section 16 states that the 0righ-ddata, or copies thereof, will be available at the 3M Environmental Laboratory to facilitateaudits of the study during its progress and before acceptanceof the finalreport. When the final report is completed, all originalpaper,data, including: approvedprotocol and amendments, study correspondence,shippingrecords, raw data, approved final report, and electronic copies of data will be retained in the archives of the 3M EnvironmentalLaboratory. All correspondingtrainingrecords,calibrationrecords, instrumentmaintenance logs, standardoperatingprocedures, equipmentprocedures, and methods will be retained in the archives of the facility performing each analysis. REASON: Clarificationof the dispositionof archived records if analyses are pe&omed at a subcontract laboratory. 6. f ROTOCOL READS: ' Section 17states that specimenswill be maintainedin the 3M EnvironmentalLaboratory specimen archives. AMENDTO READ: Specimenswill be maintained in the 3M Environm-entalLaboratory specimen archives. All specimens sent to sub-contract laboratories will be returned to the 3M Environmental Laboratory upon completion of aualysis and submission of the sub-contract laboratory(s) find report. The specimenswill be returnedwith the following documentation: the signed original chain of custody and records of storage conditions while at the sub-contract facility. REASON: Clarificationof the disposition of specimensand documentationfor analysesperformed by a sub-contract laboratory. 3M Environmental Laborafory boratory Page 39 3M Medical Department Study: T-6395.14 Amendment Approval BACK TO MAIN Analytical Report: FACT TOX-I 11 LRN-U2994 Protocol FACT-TOXI 1I Amendment No. 2 Marvin Case, D.V.M.,Ph.D., SponsorRepresentative - Kriskh J. Hansen, Ph.D., Study' U-'irector &O&f /PP* Date s 0 2 MYq Date 3M EnvironmentalLaboratory 3MEnvironrnental Laboratory Page 40 3M Medical DepartmentStudy: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-I 1 1 LRN-U2994 Study Titie Analytical Laboratory Report on the Determination of the Presence and Concentration of Potassium Perfluorooctanesulfonate (CAS Number 2759-39-3) in the Serum, Liver, and Urine of Crl:CD@BRVAFRluk Rats Exposed to PFOS Via Gavage PROTOCOL AMENDMENT NO. 3 Amendment Date: 20 January 2000 Performing Laboratories Urine Analyses 3M Environmental Technology and Safety Services Fluorine Analytical Chemistry Team Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Pedorming Laboratories Liver Analyses BatteIle Memorial Institute 505 King Avenue COlUmbW, OH 43201-2693 Serum Analyses Advanced Bioanalytical Services, Inc. 15 Cathenvood Road Ithaca,NY 14850 Laboratory Project Identification ET&SS LFW-U2994 FACT TOX-111 Argus Study: 418-015 3M Medical Department Study: T-6295.14 3M Environmental Laboratory 3M Environmental Laboratory Page 41 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Protocol LRN-U2994 Amendment Number 3 This amendmentmodifies the following portion@)of the protocol: f . PROTOCOL READS: The study director for the present study was identified in the protocol as Kristen J. Hansen, Ph.D. AMENDTO REAP: The role of study director for the present study was reassignedto MarvinT.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. AMENDTO READ: The role of sponsor for the present study was reassignedto John L. Butenhoff, Ph.D., as of 20 January 2000. REASON: To ensure that the study director does not also carry the duties of study sponsor, the sponsor role was reassigned. In this manner, personnel responsibilities and workload are more evenly balanced. 3M Environmental Laboratory 3M Environmental Laboratory Page 42 BACK TO MAIN 3M Medical Department Study: T-6395.14 Amendment Approval Analytical Report: FACT TOX-111 LRN-U2994 PrOfOCOl LRN42994 Amendment Number 3 John L. Butenhog Ph.D., Sponsor Representative F%&wwgt? I&/ Z@O& 1 Date Kristen J. Hansen, Ph.D., Outgoing Study Director /i-*&-W . Date - /d , f/ A L w 9d-&z? Marvin T. Case, D.V.M, Ph.D., Incoming Study Director . "Date 3M Environmental Laboratory 3M Environmental Laboratory Page 43 3M Medical DepartmentStudy: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Study Title Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats PROTOCOL AMENDMENT NO. 4 Amendment Date: 20 April 2000 Periorrning Laboratories 3M Environmental Technology and Safety Services Fluorine Analytical Chemistry Team Building 2-3E09,935 Bush Avenue St. Paul, MN 55106 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Battelle Memorial Institute 505 King Ayenue COlumbW, OH 43201-2693 Advanced Bioanalytical Services, Inc. 15 Catherwood Road Ithaca,NY 14850 Laboratory Project Identification ET&SS LRN-U2994 FACT TOX-111 A r e s Study: 418-015 3M Medical Department Study: T-6295.14 3M Environmental Laboratory 3M Environmental Laboratory Page 44 BACK TO MAIN 3M Medicjal Department Study: T-6395.14 Analytical Report: FACT TOX-111 .' LRN-U2994 Protocol LRN-U2994 Amendment Number 4 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: The amended section 2.0 text states that this study is designed to determine PFOS in specimens of rat liver, serum, and urine. AMENDTO READ: This study is designed to determine PFOS in specimens of rat liver, serum, feces, and wine. 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. AMENDTO READ: Add: rat or rabbit feces with a physical description of rat or rabbit feces. REASON: Analysis of fecal tissue for the target chemical and/or its analytes was added to the scope of the study following the issuance of the original protocol. 3. PROTOCOL READS: Section 13.1 Lists all of the laboratories that will be conductinganalyses for this study. AMENDTO READ: Add: Centre Analytical Laboratories, Inc., 3048 Research Drive, State College, PA 16801 RU~SON: Feces analyses were added to the scope of this study. The sub-contract laboratory performing analyses was not in the original protocol. 4. PRorocoL 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. AMENDTO READ: The feces extraction and analytical method used by Centre Analytical Laboratorieswill be; oOM-023-003 (Revision 2), "Determinationof FluorochemicalResidues in MonkeyRat Feces by LCIMSNS." 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 EnvironmentalLaboratory 3M Environmental Laboratory Page 45 BACK TO MAIN 3M Medical Department Study: T-6395.14 Amendment Approval Analytical Report: FACT TOX-111 LRN-U2994 Protocol LRN-U2994 Amendment Number 4 John L. Butenhog Ph.D., Sponsor Representative &w'/ 1 % Zd 08 Date D.V.M., Ph.D., Study Director /9 A , h J k Date . 3M Environmental Laboratory 3M Environmental Laboratory Page 46 3M Medical Department Study: 1-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Study Title Oral (Gavage)PharmacokineticRecovery Study of PFOS in Rats PROTOCOLAMENDMENT NO.5 Amendment Date: July 25,2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX-111 U2994 3M Environmental Laboratory 3M Environmental Laboratory Page 47 BACK TO MAIN 3M Medicjal Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 PrOtOCOl FACT TOX-1I I Amendment No. 5 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: In section 8, Table 1, the dosage level for dosage group 3 is listed as 0.6 mgikglday. AMENDTO READ: Section8 , Table 1,the dosage level for dosage group 3 is 1.6 mgkglday. REASON: The dosage level for dosage group 3 was changed after the protocol was written. 2. PROTOCOL READS: The amended analytical protocol (Amendment No. 1) states the extractian and analytical method to follow at Battelle Memorial Institute is: "Method for Analysis of Perfluorooctane Sulfonate (PFOS) in Rat Seraby LC/MS/MS, Version 1" AMENDTO READ: The extraction and analyticalmethod 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 BatteIle method list in Amendment No. 1contained a hand-written change, in that "sera" was crossed out and "liver" was written in. This hand-written change was dated afler the study director had signed the amendment. 3. PROTOCORLEADS: The Argus in-life protocol W18-015 lists the testing facility as Argus Research Laboratories, InC. AMENDTO READ: Change the testing facility to 3M Toxicology Services-Medical Department. Address: 3M Center, Building 220-2E-02, St. Paul, MN 55144-1000. This change is retroactive to February 10,2000. REASON: Per GLP regulations, the testing facility must be where the study director resides. There cannot be two testing facilities. 3M Environmental Laboratory 3M Environmental Laboratory Page 48 BACK TO MAIN 3M Medical Department Study: T-6395,14 Analytical Report: FACT TOX-111 LRN-U2994 PrOfOCOl FACT TOX-7 If Amendment No. 5 4. f ROTOCOL READS: The cover page of FACT-TOX-111 lists the document type as a "Protocol." On page three, under Proposed Study Timetable, the "Study Initiation Date" and the "Study Completion Date" are listed. AMENDTO READ: Change the document type for FACT-TOX-111 to "Analytical Phase Protocol." Change "Study Initiation Date" to "Phase Initiation Date." Change "Study Completion Date" to "Phase Completion Date." REASON: FACT-TOX-111 is the protocol for the analytical phase, while the Argus protocol #418-015 is the protocol for &e in-life phase. 5. PROTOCOL READS: The amended analytical protocol (Amendment No. 3) states that Marvin T. Case replaces Kristen J. Hansen as the study director. Kristen J. Hansen was reassigned to the role of Principle Analytical Investigator, The Argus Research Laboratories, Inc. in-life protocol #418-015 states that the study director for the in-life phase is Raymond York. AMENDTO READ: Marvin T. Case replaces both Kristen J. Hansen and Raymond York as study director. Raymond York has been reassigned to the role of Principal In-life Investigator. Kristen J. Hansen was reassigned to the role of Principal (corrected spelling) Analytical Investigator. These changes are retroactive to February 10,2000. REASON: Per GLP regulations, only one study director is assigned to a study. Corrected spelling of Principal. 6. PROTOCOLREADS: The amended analytical protocol (Amendment No 4) states that rat feces Will be analyzed for PFOS,as well as rat urine, liver and serum. CentreAnalytical Laboratories will be conducting the feces analyses. AMENDTO READ: Add that the Principal Analytical Investigator (`AI)at Centre analyticalLaboratories is Enaksha Wiclrremesinhe. REASON: The PAI for Centre was not listed in Amendment No. 4. 3M Environmental Laboratory 3M Environmental Laboratory Page 49 3M Medical Department Study: T-6395.14 BACK TO MAIN Amendment Approval Analytical Report: FACT TOX-111 LRN-U2994 PrOtOCOl FACT TOX-7 17 Amendment No. 5 Muhin T, Case,D.EM., Ph.D., Study Director Gd&k k Date 3M Environmental Laboratory 3M Environmental Laboratory .. Page 50 3M Medicpl DepartmentStudy: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Study Title Oral (Gavage) Pharmacokinetic Study of PFOS in Rats PROTOCOL AMENDMENT NO. 6 Amendment Date: October 17,2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55 106 Laboratory Project Identification ET&SS FACT TOX-111 3M Laboratory Request No. U2994 3M EnvitunmenfalLaboratory 3M Environmental Laboratory Page 51 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-I 11 LRN-U2994 PrOfOCOl FACT TOX-77 7 J Amendment #6 This amendment modifies the following portion($) of the protocol: 1. PROTOCOL READS: Data Quality Objectives, Section 12.4, Spike Acceptable Recoveries are required to be 70%130%. AMENDTO READ: Spike Acceptable Recoveries are required to be 50450%. REASONT: he analytical method and resulting QC data support a SO-150% acceptable range for spike recoveries. 3M Environmental Laboratory 3M Environmental Laboratory Page 52 BACK TO MAIN 3M ? Medic IDepartment Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 PrOtOCOl FACT TOX-7`lf J Amendment #6 Amendment Approval John L.Butenhofi PhD., Sponsor Representative L Q/+4@ Date M&n T.Case,D.Z M ,Ph.D., Study Director /bo& h Date 3M Environmental Laboratory 3M Environmental Laboratory rage 33 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Appendix C: Extraction and Analytical Methods This Appendix includes the following methods: 3M Environmental Laboratory, ETS-8-96.0, "Extractionof Potassium Perfluorooctanesulfonateor Other FluorochemicalCompoundsfrom Urinefor Analysis using HPLC-Electrospray/MassSpectrometry/MassSpectrometry," (14 pages). 3M Environmental Laboratory, ETS-8-97.0, "Analysis of Potassium Perfluorooctanesulfonate or Other FluorochemicalCompounds in Urine Extracts Using HPLC-Electrospray/Mass Spectrometry/MassSpectrometry," (I0 pages). 3M Environmental Laboratory, ETS-8-5.1,"Analysis of PotassiumPerfluorooctanesulfonate or Other Fluorochemicalsin Serum Extracts Using HPLC-Electrospray/MassSpectrometry," (9pages). 3M EnvironmentalLaboratory Page 54 BACK TO MAIN 3M Medical Department Study: T-6395.14 3M ENVIRONMENTLAALBORATORY Analytical Report: FACT TOX-111 LRN-U2994 METHOD EXTRACTION OF POTASSIUMPER.FI,UOROOCTANESvLFONATEOR OTHER FLUOROCEIE~CALCOMPOUNDSFROM URWE FOR ANALYSIS USING HPLCELECTROSPRAYMASPSESCTROmTRY/MASS SPECTROMETRY Method Number: ETS-8-96.0 Adoption Date: 3/ 28-4 Author: Lisa Clemen, Glenn Langenburg Revision Date: Approved By: Group Leader Technical Reviewer 7-lzgl3.5 Date TJ a d 9'1 Date 1.0 SCOPEAND APPLICATION 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate(PFOS) or other fluorochemical compounds fkom urine. 1.2 Applicable compounds: Fluorochemicals or other fluorinated compounds. 1.3 Matrices: Human,rat, and monkey urine or other fluids as designated in the validation report. Word 6/95 3M Environmental Laboratory ETS-8-96.0 Extraction ofPFOS from Urine Page 1 of14 Page 55 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 2.0 SUMMARY OF METHOD 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS)or other fluorochemicalsfrom urine, or other fluids, using an ion pairing reagent and methyl-tert-butyl ether (MtBE). In this method, eight fluorochemicals are extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, M556,M570,perfluorooctanoate (POAA), and surrogate standard (see 3.0DefuLifiorrs).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 prn nylon filter attached to 3 cc plastic syringe into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-97.0 or other appropriate method. 3.0 DEFINITIONS 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) C$,,SO, 3.2 PFOSA.perfluorooctme sulfonylamideC,F,,SO,NH, 3.3 PFOSAA:perfluorooctane sulfonylamido(ethy1)acetate C$,,S0,N(CH,CH3)CH&!O~ 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamide)-ethyl alcohol CgF17sO,N( CH2CH3)CHzC&OH 3.5 M556: C$ ,S02N(H)(CH2COOH) 3.6 M570: C,F,,SO,N(CH,)C~COOH 3.7 POAA:perfluorooctanoate C,F,5COO' 3.8 Surrogate standardTHPFOS:lH-lH-2H-ZH perfluorooctanesulfonic acid, used as an internal standard in this method. 4.0 WARNINGS AND CAUTIONS 4.1 Health and safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 INTERFERENCES 5.1 At this time, it is unknown how the extraction method is affected by potential interferences that may be present such as conjugated fluorochemicals (eg. Glucuronides). Conjugates may become deconjugated during extraction or analysis resulting in a high bias for reported results of target analytes. 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 2 of 14 Page 56 BACK TO MAIN 3M MedicalDepartmentStudy: T-6395.14 Analytical Report: FACT TOX-111 `LRN-U2994 6.0 EOUIPMENT 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge,Mistral 1000or IEC 6.1.3 Shaker, Eberbach or VWR 6.1.4 Nitrogen evaporator, Organomation. 6.1.5 Balance (k0.100 g) 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Nalgene bottles, capable of holding 250 ml and 1 L 1 7.4 Volumetric flasks,glass, type A 7.5 I-CHEM vials, glass, 40 ml glass 7.6 Centrifugetubes, polypropylene, 15ml 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0 ml 7.9 Syringes, capable of measuring 2.5 pL to 50 pL 7.1 0 Graduated pipettes 7.1 1 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: Priorto using glassware and bottles, rinse 3 times with methanol and 3 times with Milli-Qm water. Rinse glass syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 REAGENTASND STANDARDS 8.1 bTeypMeiIllrie-Qa%enwt gatreardeanwdamtear,yMbeillpir-oQvmideodr beqyuaivMalielnlit-;QalTl OwCatePrluussmedsiynsttheims method should 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutyfammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (Na$O,), J.T.Baker or equivalent 8.5 Sodium bicarbonate (NaHCO,), J.T.Baker or equivalent 3M Environmental Laboratory ETS-8-96.0 Extraction ofPFOS from Urine Page 3 of 14 Page 57 BACK TO MAIN 3M Medical DepartmentStudy: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 8.6 8.7 8.8 8.9 8.10 8.11 Methyl-T-Butyl Ether, Omnholv, glass distilled or HPLC grade Methanol, Omnisolv, glass distilled or HPLC grade Urine frozen from supplier 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 (3MSpecialty 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,l-H, 2-H, 2-H C8F,,S03H)molecular weight =428 8.9.9 Other fluorochernicals, as appropriate Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.10.1 10N sodium hydroxide (NaOH): Weigh a proximately 200 g NaOH. Pour into a 1000mlbeaker containing 500 ml Milli-Q%wlater, mix until all solids are dissolved. Store in a 1L Nalgene bottle. 8.10.2 1N sodium hydroxide (NaOH): Dilute 10N NaOH 1:lO. Measure 10mX of 10N NaOH solution into a 100ml volumetric flask and dilute to volume using MilliQm water. Store in a 125 ml Nalgene bottle. 8.10.3 0.5 M tetrabutylammoniumhydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1L volumetric containing 500 ml Milli-QTMwater. Adjust to pH 10 using approximately 44 to 54 ml of 10N NaOH (While adding the last ml of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-Qm water. Store in a 1L 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. 8.1 0.4 0.25 M sodium carbonate/sodium bicarbonate buffer (NqCO,/NaHCO,): Weigh approximately 26.5 g of sodium carbonate (NqCO,) and 21.Og of sodium bicarbonate (NaHCO,) into a 1 L volumetric flask and bring to volume with MilliQTMwater. Store in a 1L Nalgene bottle. Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.1 1.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.OO ppm PFOS,1.02 ppm PFOSA, 0.987 ppm PFOSAA,and 1.10 ppm EtFOSE-OH.) 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 4 of 14 Page 58 BACK TO MAIN 3M Medical DepartmentStudy: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 8.11.3 Weigh approximately 100 mg of PFOS into a 100ml 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 1000ppm (P&N* 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 1With methanol for a working standard 2 solution of approx. 5.0 ppm. 8.11.7 Dilute working standard 1with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.12 Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60mg of smogate standard 1-H,l-H, 2-H, 2-H, C8F,,S0,H into a 50 rnl volumetric flask and record the actual weight. 8.12.2 8.12.3 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 PPm. Prepare a surrogate working standard. Transfer approximately 1 ml of 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 SAMPLHEANDLING 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw to room temperature prior to extraction. 10.0 QUALITY CONTROL 10.1 Solvent Blanks, Method blanks and matrix blanks 10.1.1 An aliquot of2.0 ml methanol is used as a solvent blank. 10.1.2 Extract two 2.0 ml aliquots of Milli-Qm water following this procedure and use as method blanks. 10.1.3 Extract two 2.0ml aliquots of the urine following this procedure and use as matrix blanks. See 11.1.4. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually the control matrix received with each sample set. 10.2.3 Expected concentrations should fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 5 of 14 Page 59 BACK TO MAIN 3M Medical DepartmentStudy: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 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 of the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing calibration verification per group of 10 samples. For example, if a sample set =34, fourchecks are prepared and extracted. 10.3.3 Prepare each continuing calibration verification from the same matrix used to prepare the initial curve. 10.3.4 The expected concentrations will fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100ppb, rather than 5 ppb - 1000ppb). 11.o CALIBRATION AND STANDARDIZATION 11.1 Prepare matrix calibration standards 11.1.1 Transfer 2.0 ml of urine to a 15ml centrifbge tube. 11.1.2 Record each sample volume on the extraction sheet. 11.1.3 While preparing a total of twenty-twoaliquots in 15ml centrifuge tubes, mix or shake between aliquots. 11.1.4 Two 2.0 ml aliquots serve as matrix blanks. 11.1.5 Typically use the standard concentrations and spiking amounts listed in Table 1, at the end of this section, to spike, in duplicate, two standard curves, for a total of twenty standards, two matrix blanks, and two method blanks. 11.1.6 Refer to validation report FACT-TOX-131, W2067, which lists the working ranges and the Linear Calibration Range (LCR)for calibration curves. 11.1.7 Use Attachment D as an aid in calculating the concentrations of the working standards. See Section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each standard, blank, or continuing check, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 10 ppb - 1500 ppb. 11.3 Extract spiked matrix standards following 12.6-12.16 of this method. Use these standards to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 6 of 14 Page 60 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Working standard - (approx. conc.) clr, Approx. final conc. Approx. final conc. of analyte in matrix Of analyte in solvent - Blank Blank 12.0 PROCEDURE 12.1 Obtain fiozen 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 appropriatevolume to a 15 ml polypropylene centrihge tube. 12.3 Return unused samples to freezer after extraction amounts have been removed. 12.4 Record the initial volume on the sample weight'volume worksheet. . See Attachment D. The original weightlvolume worksheet is included in the study binder. 12.5 Label the tube with the study number, sample ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike all samples, including blanks and standards, ready for extraction with surrogate standard as described in 11.2. 12.7 Spike each matrix with the appropriate amount of standard as described in 11.1, or Table 1 in that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing Calibration samples for 15 seconds. 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.10 To each sample, add 1m10.5 M TBA and 2 ml of 0.25Msodium cwbonatehodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 ml methyl-tert-butyl ether. 12.12 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. 3M Environmental Laboratory . ETS-8-96.0 Extraction of PFOS from Urine - Pag-e 7 of 14 Page 61 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 12.13 Centrifbge for 20to 25 minutes at a setting of 3500rpm,or until layers are separated. 12.14 Label a fresh 15 ml centrifige tube with the same information as in 12.5. 12.15 Remove 4.0ml of the organic layer to this clean 15 ml centrifuge tube. 12.16 Put each sample on the analytical nitrogen evaporator until dry,approximately 30 to 60 minutes. 12.11 Add 0.5 ml of methanol to each centrihge 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, frnal 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. 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS, or other applicable fluorochemical, in calibration standards using the following equation: ml of standard x concentration of standard (ug /ml) - ml of standard + ml of surrogate standard + initial matrix volume (ml) Final Concentration (ydml) of PFOS in matrix 14.0 METHODPERFORMANCE 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quantitation (LOQ)values (see Attachment B). 14.2 The following quality control samples are extracted with each batch of samples to evaluate the quality of the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and . precision of the extraction. 14.2.3 Continuing calibration check samples to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-97.0 for method performance criteria. 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 8 of 14 Page 62 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 15.0 POLLUTION PREVENTIONAND WASTE MANAGEMENT 15.1 Human and monkey sample waste is disposed in infectious biohazard waste containers, all other sample waste is disposed in noninfmtiousbiohazard waste containers, Flasnmable solvent waste is disposed in high BTU containers. Used glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 RECORDS 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 TABLESD; IAGRAMSF.LOWCHARTASN.D VALIDATIONDATE 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDWLOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 17.4 Attachment D, Sample weightholume worksheet 18.0 REFERENCES 18.1 The validation report associated with thismethod is FACT-TOX-231, W2067. 19.0 AFFECTEDOCUMENTS 19.1 ETS-8-97.0, "Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrospray Mass SpectrometryMass Spectrometry" 20.0 REVISIONS Revision Number Reason For Revision Revision 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 9 of 14 Page 63 BACK TO MAIN 3M Medical Department Study: T-6395.14 bxtractton woruneet r;i S - ~ G Y ~ . O Study # Matrix Box # DateSpikedlAnalyst Surrogate Std Approx. pprn Actual pprn # FC-Mix approx. 0.5 ppm actual ppm # FC-Mix approx. 5 ppm actual pprn # MS MSD Analytical Report: FACT TOX-111 FC-Mix approx. 50 ppm actual ppm # Comments I I I I I- Blank Std # amount = ml .Urine Extraction Method Vortex 15 sec. Pipette Matrix ,spike with appropriate surrozate or FC-Mix Volume ml t Date & Initials Attachment A 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 10 of 14 Page 64 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-UZ994 Compound PFOS POAA PFOSA PFOSAA EtFOSE-OH M556 M570 MDL @Pb) 4.6 22.9 n/d n/d dd n/d n/d LOQ @Pb) 14.7 72.9 n/d n/d n/d n/d n/d Linear Calibration Range (LCR) 15 ppb - 1500ppb (infinalMeOH extract) 50 ppb - 1500ppb (infinalMeOH extract) - 25 ppb 1000ppb (in finalMeOH extract) 25 ppb - 1000ppb (in final MeOH extract) 25 ppb - 1000ppb (in linal MeOH extract) 1 25 ppb - 1000ppb (in finalMeOH extract) - I 25 ppb 1000ppb (infinalMeOH extract) NOTE:to calculate MDL, LOQ,and LCR values in ug/ml of urine divide the above values by 4. MDLnOQ values in rat and monkey Urine were not statisticallydetermined. Two curves in each of 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 ofclean, 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 further information. Attachment 13: MDL/LOQ Summary 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 11 of 14 Page 65 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Human Urine Full Range Prepared range of standards (ppb) (ng/ml) dd LCR from curve (ppb) (nglml) dd % Recovery Range n/d RSD Range dd Low Curve n/d dd . dd dd IHighcurve I dd I dd I dd I d d I 1/X 2.5ppb - 1500ppb 15 ppb- 1500ppb 75-116 +/- 30% Human Urine Full Range Repared range of standards (ppb) (ng/ml) dd LAW Curve dd High curve 1/x, quadratic dd 2.5ppb - 1500 ppb LCR from curve (PPb) (ndml) n/d dd dd 50 ppb- 1500ppb % Recovery Range dd dd dd 86-105 RSD Range dd n/d dd +I- 30% .. Attachment B:MDWLOQ Summary 3M Environmental Laboratory ETS-8-96.0 Extraction ofPFOS from Urine Page 12 of 14 Page 66 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 PFOS Std conc I u g / d 0.500 1 0.500 0.500 5.00 5.00 - - - _ I _ 5.00 50.0 50.0 50.0 50.0 PFOSA Std conc ug/ml 0.501 0,501 0.501 5.01 5.01 5.01 50.1 50.1 50.1 50.1 PFOSAA Std conc ug/d 1 0.500 1 0.500 1 0.500 5 .OO 5.00 5.00 50.0 1 50.0 1 50.0 50.0 EtFOSE Std couc ug/d 0.501 0.501 0.501 1~-5.01 5.01 5.01 50.1 50.1 50.1 50.1 POAA Std conc ug/ml 0.499 0.499 0.499 ____4.9-_9__ 4.99 4.99 49.9 49.9 49.9 49.9 M556 Std conc ug/d 0.500 0.500 0.500 - 5.00 5.00 5.00 50.0 50.0 50.0 50.0 M570 Std conc uglml I 0.501 I 0,501 1 0.501 5.01 5.01 5.01 50.1 50.1 I;-5o.1 50.1 Calculated concentrationsof standardsin the sample matrix PFOS , PFOSA PFOSAA EtFOSE POAA M556 Final conc Final conc Final conc Final conc Final conc Final conc M570 Final conc All All Am't Find vol spikedml ml 0.005 I 2.0075 0.010 2.0125 0.025 2.0275 0.005 2.0075 0.010 2.0125 0.025 0.005 1 2.0275 2.0075 0.0075 2.0100 0.010 2.0125 0.015 2.0130 Surrogate All Std conc Am't PFOS Final conc ng/ml 5.00 . 10.0 25.0 50.0 100 250 500 75 0 1000 1500 PFOSA Final conc nglml 5.01 10.0 25.1 50.1 100 25l-' 501 752 1002 1503- PFOSAA Final conc ng/d 5.00 10.0 25.0 50.0 100 250 500 750 1000 1500 EtFOSE Final conc ng/ml 5.01 10.0 25.1 50.1 IO0 25 1 501 752 1002 1503 POAA Final conc nglml 4.99 10.0 25.0 49.9 100 250 499 749 998 1497 M556 Final conc ngltnl 5.00 10.0 25.0 50.0 100 250 500 750 1000 1500 M570 Final conc ng/ml 5.01 10.0 25.1 50.1 IO0 25 1 --50-1 752 1002 1503 Surrogate Std conc nglml 100 Surrogate Final conc nglml 500 Attachment C Standard CaIculation Sheet ETS-8-96.0 3M Environmental Laboratory Extraction of PFOS from Urine Page 13 of 14 Page 67 3M Medical Department Study: T-6395.14 Prep Date(s): Analyst(s): Sample Matrix: Method/Revision: Target Analyte(s): BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Study Number: Equipment Number: Final Solvent & TN Number: Matrix Blanmdentifier : Box: Summary of method: Notes: Attachment D: WeighWolume Sheet 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 14 of 14 Page 68 BACK TO MAIN 3M Medical Department Study: T-6395.14 3M ENVIRONMENTLAALBORATORY Analytical Report: FACT TOX-111 LRN-U2994 METHOD &AI,YSIS OF POTASSruM ~ R 3 ? L U O R O O C T ~ S U L F O N A TOER OTHER FLUOROCHEMICCAOLMPOUNDINSURINEEXTRACTUSsmG HPLC-ELECTROSPMY/MSAPSESCTROMETRYIMIASSSPECTROMETRY Method Number: ETS-8-97.0 Author: Lisa Clemen, Robert Wynne, Glenn Langenburg Adoption Date: q-28-44 Revision Date: Approved By: V I Laboratory Manager Group Leader Technical Reviewer Date 't-lZ8149 Date 42d99 Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method describes the analysis of urine extractsfor fluorochemicalsusing HPLC-electrospraylmas spectrometry. 1.2 Applicable Compounds: Fluorochemicalsor other ionizablecompounds. 1.3 Matrices: Human,rat, and monkey Urine, or other fluids as designatedhthe validation report. Word 6/95 3M Environmental Laboratory ETS-8-97.0 Analysis ofUrine Extract Using ESMS Page 1 of IO Page 69 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 2.0 SUMMAROYF METHOD 2.1 This method describesthe analysisof fluorochemicals extracted fkom urine or other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysisis performedby monitoring a singleion characteristicof a particular fluorochemical,such as the perfluorooctanesulfonate(PFOS)anion, m/z= 499. Additionally, samplesmay be analyzed using a tandem mass spectrometerto furtherverify the identityof a compound by detecting daughterions of the parent ion. 3.0 DEFINITIONS 3.1 Atmospheric PressureIonization(API): The Micromass Q u a m II triple quadrupole systems allow for various methods of ionizationby utilizingvarious sources, probes, and interfaces. These includebut are not limitedto: Electrospray Ionization (ESI), Atmospheric Pressure chemicalIonization (APcI), Thermospray, etc. The ionizationprocess h these techniques occurs at atmosphericpressure (Le., not under a vacuum). 3.2 ElectrosprayIonization (E$, ESI):a method of ionizationperformed at atmospheric pressure,whereby ions in solution are transferredto the gas phase via tiny chargeddroplets. These charged droplets are produced by the applicationof a strong electrical field. 3.3 Mass Spectrometry,Mass Spectrometer (MS), TandemMass Spectrometer( M S M S ) : The MI Quattro II triple quadrupole mass spectrometeris equippedwith two quadrupole mass selectivedetectors and a collisioncell. Ions are selectivelydiscriminatedby mass to chargeratio ( d z )and subsequentlydetected. A singleMS may be employed for ion detection or an ion may be selected in the first quadrupole, fragmented in the collision cell, and these fi-agmentsmay be analyzedinthe second quadrupole. 3.4 Conventional vs. %spray probe interface: The latest models of Micromass Quattro I1 triple quadrupole systems (post 1998) utilize a "2-spray" conformation. The spray emitted from a probe is orthogonalto the cone aperture. In the conventionalconformationit is aimed directlyat the cone aperture,after passing through a tortuouspathway in the counter electrode. Though the configurationis different,the methods of operation,cleaning, and maintenanceare the same. However, 2-spray componentsand conventionalcomponents are not compatiblewith one another,but only with similar systems(i.e., Z-spray components are compatiblewith some other 2-spray systems, etc.) 3.5 Mass Lynx Software: System s o h a r e designed for the specific operationof these Quattro I1triple quadrupole systems. CurrentlyMassLynx has WindowsNT 4.0versions. For more details see the manual specificto the instrument(Micromass Quattro II triple quadrupole MassLynx NT User's Guide). 4.0 WARNINGS AND CAUTIONS 4.1 ,Healthand Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately5000 Volts. 3M Environmental Laboratory ETS-8-97.0 Analysis of Urine Extract Using ESMS Page 2 of 10 Page 70 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 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 beIow a back pressure of 400 bar (5800 psi). If the back pressure exceeds 400 bar, the HP 1100will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 INTERFERENCES 5.1 To minimize interferences when analyzing samples, teflon should not be used for sample storage or any part of instnunentationthat comes in contact with the sample or extract. 6.0 EQUIPMENT 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an electrospray ionization source 6.1.2 HP1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.1.1 High purity grade nitrogen regulated to approximately 100 psi. (House air system) 7.1.2 High purity grade argon regulated to approximately 6 psi. 7.1.3 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7.1.4 Capped autovials or capped 15 mL centrifuge tubes 8.0 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTMwater (ASTM type I), all water used in this method should be ASTM type I, or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.1.3.1 When preparing different amounts than those listed, adjust accordingly. 8.1.3.2 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000mL volumetric container containing 2000 mL Milli-QTMwater, mix until all solids are dissolved. Store at room temperature. 3M Environmental Laboratory ETS-8-97.0 Analysis of Urine Extract Using ES/MS Page 3 of IO Page 71 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 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 SAMPLEHANDLING 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 refiigerated at approximately A" C, or at mom temperature, until analysis can be performed. 10.0 OUALITYCONTROL 10.1 Solvent BIanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a solvent blank, method blank, and matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and andyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty samples, with a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrationswill fall in the mid-rage of the initial calibration curve. Additional spike concentrations may fall in the lowrange of the initial calibration curve. 10.3 Continuhg CalibrationVerifications 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum of one per batch. 11.o CALIBRATION AND STANDARDIZATION 11.1 Analyze the extracted matrix standards prior to and following each set of extracts. The average of two standard curves will be plotted by regression (linear or otherwise, see 11.21, weighted l/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 ofUrine ExtractUsing ESMS Page 4 of 10 Page 72 BACK TO MAIN Compound PFOS POM Weighting 1/X 1/X Regression Fit Linear Quadratic Response type I IS reference IS reference 12.0 PROCEDURES 12.1 Acquisition Set up 12.1.1 Set up the samplelist. 12.1.1.1 Assign a sample list filenameusing MO-DAY-last two digits of yearincreasing letter of the alphabet starting with a 12.1.1.2 Assign a method (MS file) for acquiring ' 12.1.1.3 Assign an HPLC program (Met fife) 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 spectrometerheadings and select SIR (SingleIon Recording) or MRM (Multiple Reaction Monitoring). Set IonizationMode as appropriateand mass to 499 or other appropriatemasses. A full scan is usually collected dong with the SIRS. Save acquisitionmethod. If MSMS instrumentsare employed, additionalproduct ion fragmentationinformationmay be collected. Refer to Micromass MassLynx GUIDE TO DATA ACQUISITIONfor additionalinformationand MRM. 12.13 Typically the analyticalbatch run sequencebegins and ends with a set ofextracted matrix standards. 12.1.4 Samplesare analyzedwith a continuingcalibrationverificationinjected after every tenth sample. Solventblanks shouldbe analyzedperiodicallyto monitorpossible analyte carryoverand are not consideredsamplesbut may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sampletray accordingto the sample list prepared in Section 12.1.1. 3M EnvironmentalLaboratory ETS-8-97.0 Analysis of Urine Extract Wsing EWMS Page 5 of 10 Page 73 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 12.2.2 Set-up the HP1lOO/autosamplerat the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook Time 0.00 min 1.0 min. 4.5 min. 6.5 min. 7.0 min. MeOH 40% 40% 95% 95% 40% 2.0 mM Ammonium acetate 60% 60% 5% 5% 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 I1 Triple Quadrupole Mass SpectrometerFitted with an Atmospheric Pressure Ionization Source," for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Turn on the nitrogen. 12.3.5 Open the tune page. Click on operate to initiate source block and desolvation heaters. 12.3.6 Open the Inlet Editor. 12.3.6.1 12.3.6.2 Set Set - HPLCpump to the flow to 10 "On" 500 u W m h 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 250-400litershow 3M Environmental Laboratory ETS-8-97.0 AnaIysis of Urine Extract Using ESMS Pa-ge 6 of IO Page 74 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-11? LRN-U2994 - 12.3.7.2 ESInebulizing gas 10-15literdhow 12.3.7.3 HPLC constant flow mode, flow rate 10 500 pL/min 12.3.7.4 Pressure~ 4 0 b0ar (Thisparameteris not set, it is a guide to enswe the HPLC is operatingcorrectly.) 12.3.7.5 Sourceblock 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 instrumentlog. 12.3.9 Click on start button in the AcquisitionControlPanel (this may vary among MassLynx versions, refer to appropriateMassLynx User's Guide). Ensure start and end sample number includes all samples to be analyzed. 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveriesusing the followingequation: % Recovery = ObservedResult - Backmound Result x 100 Expected Result 13.1.5 Calculatepercent difference using the following equation: % Difference = Exuected Conc. - CalculatedConc. x 100 Expected Conc. 13.1.6' Calculateactual concentrationof PFOS,or other fluorochemical,in matrix (P.g/mL): {ndmL of PFOS calc. fiom std. Curvex Dilution Factor) x Initial Volume of matrix hL> Final Volume (mL) 1 ug 1000 ng 14.0 METHOPDERFORMANCE 14.1 Method DetectionLimit (MDL) and Limit of Quantitation(LOQ)are method, amlyte, and matrix specific. Please see ETS-8-96.0,Attachment B, for a listing of currentvalidated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 Solventblanks,method blanks, and matrix blanks values must be below the lowest standardin the calibrationcurve 14.3 Calibration Curves 14.3.1 The 3 value for the calibrationcurvemust be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries must be within f 30% of the spiked concentration. 3M Environmental Laboratory ETS-8-97 ,O Analysis ofUrine Extract Using E S M S Page 7 of 10 Page 75 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 14.5 Continuing Calibration Verifications 14.5.1 Continuing calibration verification percent recoveries must be within f 30% of the spiked concentration. 14.6 If criteria listed in this method performance section are not met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by'the analyst. Document all actions in the appropriate logbook. 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 POLLUTIOPNREVENTIOANNI) WASTEMANAGEMENT 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 -CORDS 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 l/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.appropriatemedium. Record in study notebook the file name and location of backup electronic data. 17.0 TABLEDSI. AGRAMS.FLOWCHARATNSD,VALIDATION DATA 17.1 Attachment A: ETS-8-97.0 Data summary spreadsheet. 18.0 REFERENCES 18.1 ETS-9-24.0,"Operation and Maintenance of the Micromass Atmospheric Pressure IonizationMass Spectrometer Quattro XI 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 of Urine Extract Using ES/MS Page 8 of 10 Page 76 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 19.0 AFFECTEDOCUMENTS 19.1 ETS-8-96.0, "Extractionof Potassium Perfluorooctanesulfonateor 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 of Urine Extract Using ES/MS Page 9 of 10 Page 77 BACK TO MAIN 3M Medical Department Study: T-6395.14 Laboratory Study # Study: Test Material: Matrix/FinaI Solvent: MethodRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept: Date of Extractionfhalyst: Date of AnalysidAnalyst: Dilution Factor Analytical Report: FACT TOX-111 LRN-U2994 Final Conc. ug/mL Concentration (ug/mL): Taken fiom the MassLynx integrationsummary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken fiom the study folder. Final Conc. (uglmL): Calculatedby dividing the initid volume from the concentration AttachmentA Sununary Spreadsheet ETS-8-97.0 Analysis of Urine ExtractUsing ESlMS 3M Environmental Laboratory Page 10 of IO Page 78 BACK TO MAIN 3M Medical Department Study: T-6395.14 .1 Andlytical Report: FACT TOX-111 .1 LRN-U2994 Method Modification Method: Sections modified: ETS-8-97.0 10.3.2 and 14.5.1 Method reads: 10.3.2 14.5.1 AnaIyze a mid-range caliiration standardafter every tenthsample, with a minimumof one per batch ContinuiPgcalibration verification percent recoveriesmust be within i:30% of the spiked conccntcatio&, Modifjrmethodtored. 10.3.2 Analyzea mi&.raaga caliitim standard at least after e y c ~ yten samples, with a minimumofone per batch. 14.5.1 At least one continuingcalibrationverl6icatton per tensamplesmustshow a percent recovery ` within+/-30??ofthe spikedconcentration. E f f i v e date ofmodification: Signature ofPAI and date 7/28/99 $e#. ZI, zdtzo 3M Environmental Laboratory Page 79 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 .t LRN-U2994 Method Modification Methods: Section modified: ETS-8-7.0, ETS-8-5.I, ETS-8-97.0 add section 14.3.2 Method reads: NA Modify method to read: 14.32 The second(bracketing)calibrationcurve may be deactivatedifinsma1 drift affects the data. The first curve andacceptablecalibrationchecks shall bracket usable data. Effective date of modificationETS-8~7.0: 7/22/99 Effective date of modification ETS-8-5.1: 4/26/99 Effective date of modificationETS-8-97.0: 7/28/99 3M Environmental Laboratory Page 80 3M Medical Department Study: T-6395.14 3M ENVIRONMENTLAALBORATORY BACK TO MAIN Analytical Report: FACT TOX-1II LRN-U2994 A N A L Y S I S OF POTASSIUM PERFLUOROOCTANESULFONATEOR OTHER FLUOROCHEMICIANLSESRUM EXTRACTS USING HPLC-ELECTROSPRAYMSAPSESCTROMETRY Method Number: ETS-8-5.1 Author: Lisa Clernen, Robert Wynne Approved By: Adoption Date: 03/01/99 Revision Date: q l a f l Group Leader - c Technical Reviewer 4/w (29 Date Date 1.0 SCOPEAND APPLICATION 1.1 Scope: This method describes the analysis of s e m extracts for fluorochemical surfactants using HPLC-electrospray/mas spectrometry. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey, and human serum, or other fluids as designated in the validation report. Word 6/95 3M Environmental Laboratory ETS-8-5.1 Analysis ofSerum Extract Using ESMS Page I o f 9 Page 81 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-1I 1 LRN-U2994 2.0 SUMMARY O F METHOD 2.1 This method describes the analysis of fluorochemicalsurfactants extracted from serum or other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate(PFOS) anion, d z = 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity of a compound by detecting daughter ions of the parent ion. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro I1 tripXe quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization @SI), Atmospheric Pressure chemical Ionization (APcI); Thermospray, etc. The ionization process in these techniques occurs at atmosphericpressure (Le., not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS),Tandem Mass Spectrometer (MS/MS): The MI Quattro II triple quadrupole systems are equipped with quadrupole mass selective detectors. Ions are selectivelydiscriminatedby mass to charge ratio ( d z )and subsequently detected. A single M S may be employed for ion detection or a series ( M S M S ) for more specific fragmentation information. 3.4 Conventional vs. %spray probe interface: The latest models of Micromass Quattro I1 triple quadrupole systems (post 1998)utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. h the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e., 2-spray components are compatible with some other 2-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT4.0 versions. All versions are similar. For more details see the manual specific to the instrument (Micromass Q u a m I1 triple quadrupoleMassLynx or MassLynx NT User's Guide). 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum'ExtractUsing ES/MS Page 2 of 9 Page 82 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 4.2 Cautions: 4.2.1 Do not operate solvent pumps above capacity of 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HP1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 INTERFERENCES 5.1 To minimize interferenceswhen analyzing samples, teflon should not be used for sample storage or any part of instrumentationthat comes in contact with the sample or extract. 6.0 EQUIPMENT 6.1 Equipment listed below m a y be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 Micromass Quattro II triple quadrupoleMass Spectrometerequipped with an electrospray ionization source 6.1.2 WP1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 SUPPLIESAND MATERIALS 7.1 SuppIies 7.1.1 High purity grade nitrogen gas regulated to approximately 100psi (House air system) 7.1.2 HPLC analytical column, specificsto be determined by the analystand documented in the raw data. 7.i.3 Capped autovials or capped 15 mL centrifugetubes 8.0 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-Qm water, all water used in this method should be Milli-Qm water or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. See ETS-8-4.1. 9.0 SAMPLHEANDLING 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 3M Environmental Laboratory ETS-8-5.1 Analysis of Senun Extract Using ESMS Page 3 of9 Page 83 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 9.2 If analysis wil1 be delayed, extracted standards and samples can be refrigerated at approximately 4 O C, or at room temperature, until analysis can be performed. 10.0 OUALITCYONTROL 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the . recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty samples, with a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrationswill fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lowrange of the initial calibration curve. 10.3 ContinuingCalibrationVerifications 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of the calibrationcurve. 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum of one per batch. 11.0 CALIBRATIONAND STANDARDIZATION 11.1 Analyze the extracted matrix standardsprior to and following each set of extracts. The average of two standard curves will be plotted by linear regression (y =my +b), weighted l/x,not forced through zero, using MassLynx or other suitable software. 11.2 If the curve does not meet requirements, perform routine maintenanceor reexbct the standard curve (if necessary) and reanalyze. 11.3 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather thanthe full range of the standard curve. Example: when attemptingto quantitate approximateIy 10ppb of analyte, generate a calibration curve consistingof the standards from 5 ppb to 100ppb rather than the full range of the curve (5 ppb to 1000ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 4 of 9 Page 84 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report FACT TOX-1I 1 LRN-U2994 12.0 PROCEDURES 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording) or MRM. Set Ionization Mode as appropriate and mass to 499 or other appropriatemasses. A full scan is usually collected along with the SIICS. Save acquisitionmethod. If M S N S instruments are employed, additional product ion fkagmentation infomation may be collected. See Micromass MassLynx GULDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). 12.1.3' Typically the analyticalbatch run sequencebegins with a set of extracted matrix standards and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HP11OO/autosamplerat the following conditions or at conditions the analyst considers appropriatefor optimal response. Record actual conditions in the instrument logbook 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 8.50 min. 11.O min. 12.0 min. MeOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% , 10% 10% 60% 12.2.2.5 Press the "Start"button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 3M Environmental Laboratory ETS-8-5.1 Analysis ofSerum Extract Using ESMS Page 5 of 9 Page 85 BACK TO MAIN 3M Medical Depaftment Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 12.3.3 12.3.4 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. Set HPLC pump to "On".Set the flow to 10 - 500 uWmin or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A 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 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 12.3.6.2 12.3.6.3 12.3.6.4 Drying gas 250-400 litershour ESI nebulizing,gas 10-15 literskour HPLC constant flow mode, flow rate 10- 500 pL/min Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefilly guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Using the cross-flow counter electrode in the E S N S source is recommended for the analysis of biological matrices. 12.3.1OClick on start button in the Acquisition Control Pane1 (this may vary among MassLynx versions, see appropriateMassLynxUSER'SGUIDE). Press the start button. Ensure start and end sample number includes all samples to be analyzed. 13.0 DATAANALYSISAND CALCULATIONS 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: - % Difference = Exnected Conc. Calculated Conc. x 100 Expected Conc. . 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical,in matrix (tLdmL1: (ng of PFOS calc. from std. Curve x Dilution Factor1 x 1 UE (InitiaI Volume of matrix (mL)+ mL of Surrogate Standard) 1000 ng Final Volume (mL) 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ESMS Page 6 of 9 Page 86 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 14.0 METHODPERFORMANCE 14.1 Method Detection Limit (MDL)and Limit of Quantitation (LOQ)are method, analyte, and matrix specific. Please see ETS-841, AttachmentB, for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 SoIvent blanks, method blanks, and matrix blanks values are must be below the lowest standard in the calibration curve 14.3 Calibration Curves 14.3.1 The 3 value for the calibration curve must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries are must be within f 30% of the'spiked concentration. 14.5 Continuing Calibration Verifications 14.5.1 Continuing calibration verificationpercent recoveriesmust be f 30% of the spiked concentration. 14.6 If criterialisted in thismethod pmfomance sectionisn't met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 14.7 If data are to be reported when performance criteriahave not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 POLLUTION PREVENTIONAND WASTE MANAGEMENT 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken gIass containers located in the laboratory. 16.0 RECORDS 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and anaIyst. 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 calibrationcurve by linear regression, weighted l/x,then print these graphs and store in the study folder. 16.4 Print data integration summary, integration'method, and chromatograms, from MassLynx, and store in the study folder. 3M Environmental Laboratory ETS-8-5.1 Analysis ofSerum Extract Using ESMS Page 7 of 9 Page 87 BACK TO MAIN 3M Medical DepartmentStudy: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 16.5 Summarize data using suitable software(Excel 5.0) and store in the study folder, see Attachment A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 TABLESD.IAGRAMSF.LOWCHARTASN,D VALIDATION DATA 17.1 Attachment A: ETS-8-5.1 Data surnmary spreadsheet. 18.0 REFERENCES 18.1 FACT-M-4.1 "Extraction of Potassium Pduorooctanesulfomte or Other Fluorochemicai compounds fiom Serum for Analysis Using HPLC-ElectsosprayMass Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure IonizationMass SpectrometerQuattro I1 triple quadrupole Systems" 18.3 The validation report associated with this method is ETS-8-4.0 & 5.0-V-l . 19.0 AFFECTED DOCUMENTS 19.1 ETS-8-4.1,"Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 REXW3IONS Revision Number. 1 Reason For Revision Section 6.1.2 Clarification of HP1100 system components. Section 11.1 Average of two curves, not standard values, are used for plotting linear regression and added the l/x weighting of the curve. Section 12.2.2.4 Clarification of solvent ramp. Section 17.1 Changed fiom attachment B to A. Revision Date 04-99 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 8 of9 Page 88 3M Medical Department Study: T-6395.14 Laboratory Study # Study: Test Material: Matriflinal Solvent: MethodiRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept: Date of Extractiodhalyst: Date of AnalysisiAnaIysl BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Group/Dose: Taken from the study folder. Sample# Taken from the study folder. Concentration(ug/mL): Taken from the MassLynx integration summary. Initial Volume (a)Ta: ken from the study folder. Dilution Factor: Taken fiom the study folder. FinaI Cone. (ug/mL): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-5.1 3M Environmental Laboratory Analysis of Serum Extract Using ES/MS Page 9 of 9 Page 89 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 -1 LRN-U2994 Method Modification Method: Sections modified ETS-8-5.1 10.3.2and 14.5.1 Method reads: 10.3.2 14.5.1 Analyze a mid-range calibration standard after every tenthsampIe, with aminimumof one per batch Continujngcat'bratianverification percentrecoveries must be within f30% ofthe spiked concentration. Modify method to read: 10.3.2 Analyze a mid-range calibration standard at least afterevay ten samples, with a minim- of one per batch. 14.5.1 At Ieast one continuingcallbration verification per ten samples must show a percentrecovery within +1-30% of the spiked concentration. Effective date ofmodification: 4/26/99 3M Environmental Laboratory Page 90 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-11I LRN-U2994 Appendix D: Data Summary Tables Table 9. Average Results for the Analyses of Sera Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of DGO PFOS Conc. (IJglmL) DG7 PFOS Conc. (IJglmL) DGI 5 PFOS Conc. (lJdmL) DG21 PFOS Conc. (IJdmL) DL14 PFOS Conc. DL21 PFOS Conc. (W-W Group I Fo 0.0 mglkglday Group I F1 O.lOOa,b NS 0.0796arb 0.0742arb <LLQ"~ 0.0542arb NS 0.0492a3b NS NS NS NS 0.0531 a*b NS Group II FO 0.1 mglkglday Group II F1 9.2qa,b NS 7.24a*b 5.68a,b NS NS 2.Barb NS 1.63asb NS NS 1.80a'b O.97garb NS Group ill FO 1.6 mglkglday Group 111 F1 161a,b NS 129"jb NS 90.6a3b 39.5a*b 20.6a0b NS 14.1 NS NS NS 27.1asb NS a PFOS concentrationsare the average of samples within a dose groupfrom all animalstested, notincludingsamples that tested at or below the limitof quantitatiin (0.04 pg/mL). lndkidualanimal dataare located inAppendix E. Sera analyseswere conductedat a contract laboratory, butwere correctedby 3M to reflectthe official purityvaluesfrom the Certificate of Analysis. A revisedfinal reportfrom the contract laboratorywill be addedas an amendment to this report. cLLQ=Less than the Limit of Quantiiation NS=Not Sampled 3M Environmental Laboratory Page 91 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Table I O . Average Resultsfor the Analyses of Liver Samples in the Study of the Determinationofthe Presence and Concentrationof PFOS inthe Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/Plus@Rats Exposedto T-6295.14 via Gavage PFOS Conc. I GroupIF1 I 0.174"' I I Group II F1 I 5.0OaZb I I GroupIII F1 I 56.2a3b I a PFOS concentrations are the averageofsampleswithin a dose group from all animalstested, not including samplesthat tested at or below the limitof quantitation(0.11 Uglg). Individualanimaldata are locatedinAppendix E. Liveranalyseswere conductedat a contract laboratory, but were corected by 3M to reflect the officialpuntyvalues from the Certificateof Analysis. A revisedfinal reportfrom the contract laboratoryWill beaddedas an amendmentto this report. Table 11. Average Resultsfor the Analyses of Urine Samples inthe Study of the Determinationofthe Presence and Concentrationof PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/Plus@Rats Exposedto T-6295.14 via Gavage a PFOS concentrations are the averageof sampleswithin a dose group from all animalstested, not includingsamples that testedat or below the limit of quantitation (0.00295 pg/mL). Individualanimal data are located inAppendix E. <LOQ=Less than Limit of Quantitation 3M Environmental Laboratory Page 92 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-1I 1 LRN-U2994 i GroupIFO 0.0 mg/kg/day Group I1 FO 0.1 mgikglday Group 111 FO 1.6 mgkdday DGO PFOS Conc. (IJdg) NDa o.601a,b 10.9a DG6R PFOS Conc. (IJgkl) NDa 0.39ga 8.3ga DG14/15 PFOS Conc. Cdg) NDa 0.294a 4.83a DG20/21 PFOS Conc. (IJdg) N Da 0.119" 2.Na DL21122 PFOS Conc. (crdg) NDa 0.0522a 0.387' a PFOS concentrations are the average of samples within a dose group From all animals tested, not including samples that tested at or below the limit of quantitation (0.0100 Vg/g). Individualanimal data are locatedinAppendix E. and Appendix G. bAnimal13745Fresultnot includedin average. ND=Not Detected 3M Environmental Laboratory Page 93 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Table 13. LOQ Values Used in FACT TOX-111 Analysis by Method and Usage Dates Sample LOQ Laboratory Method@) Usage Date Urine Dosage Confirmation Liver Serum Feces 0.00295 pg/mL 0.030 pg/mL 0.11 pg/g 0.04 pg/mL 0.0100 pgrg 3M Environmental Laboratory 3M Environmental Laboratory Battelle Memorial Institute Advanced Bioanalytical Services, Inc. Centre Analytical Laboratories, Inc. ETS-8-96.0 ETS-8-97.0 ETS-8-5.1 N003604-A 99VDJA01 .MI.DOC 00 M-023-003, revision 2 6 October 1999 21 September 1999 29 October 1999 9 July 1999 6 July 1999 3M Environmental Laboratory Page 94 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Appendix E: Data Spreadsheets Table 14. Results of Analyses of Sera Samples in the Study of the Determinationof the Presenceand Concentrationof PFOS in the Sera, Livers, Urine,and Feces of Crl:CD@BRVM/Plus@Rats Exposedto T-6295.14 via Gavage. Group I 1.0mglkglda) ~__ 13734 0.102a 13735 0.0818' 13736 0.0976a 13737 0.0758a I 13726Pl NS 13727Pl NS I 0.0556' 0.0605' 0.0933a 0.0663' NS I NS I 0.0575a 0.0496a 0.059ga 0.0959' NS I NS I <LLQa <LLQa <LLQa O.045ga NS I NS I 0.0513a <LLQa <LLQa 0.0478' NS 1 NS I NS NS NS NS 0.0615' <LLQa <LLQ~ <LLQ~ 0.051 la 0.0492a I NS I NS 13741 I 10.Oa I 6.56' I 5.76a I 2.32a I 1.30' I NS I 1.03a I I I I I I I I I 13744 I 8.Sa I 7.93' I 5.22' I 2.63' I 1.69' 1 NS I 0.762a I 13745 8.Sa 6.3ga 5.0Ia 2.02a 2.20' NS 0.942a 13746 9.5ga 73Ia 5.7!ja 2.4!ja 1 .75' NS 0.81 6" 13748 8.81' NS 4.63' 3.40' 1.17a NS 0.814a Group II 13749 9.24' 7.44a 7.26' 2.83a 1 .62' NS 1 .OS' 1.1 mg/kg/da) 13739P NS NS NS NS NS 2.02a NS 13740P NS NS NS NS NS 1.83a NS 13741Pl NS I NS I NS I NS I NS I 1.62a I NS I 13744Pl NS I NS 1 NS I NS I NS 1 1.98' I NS I 13745Pl NS 1 NS I NS 1 NS I NS 1 1.62' I NS I 13746P1 NS I NS I NS I NS 1 NS I 2.05a I NS I I I I I 1 1 13748Pl NS I NS I NS 1 NS I NS I 1.50a I NS I 13749Pl NS NS NS NS NS 1.79' NS a Sera analyseswere conductedat a contractlaboratory, butwere corrected by 3M to reflect the officialpurity valuesfrom the Certifcateof Analysis. A revisedfinal report from thecontradlaboratorywill beaddedas anamendment to this report. <LLQ=Lessthan the Limitof Quantiition (0.04(pghL)) NS=No sample 3M Environmental Laboratory Page 95 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Table 14. Results of Analyses of Sera Samples in the Study ofthe Determinationof the Presence and Concentrationof PFOS in the Sera, Liver, Urine, and Feces of Crl:CD@'BR VAWPlus" Rats Exposed to T-6295.14 via Gavage I I 1 I 1 I I 3 DGO DG7 DG15 0621 DL14 DL21 DL22 Sample #/ PFOS Conc. PFOS Conc. PFOS Conc. PFOS Conc. PFOS Conc. PFOS ant. PFOS Conc. ( v d W W m L ) ( w W ( v d W (vdW (PdmL) (WmL) 13751 134a 12ga 82.6a 33.3a 18.3a NS 14.8a Group 111 I.6mglkglday 113751Pl NS I NS I NS I NS I NS I 30.2a I NS I I13752Pl NS I NS I NS I NS I NS I 29.3a I NS I 13753P NS NS NS NS NS 24.5a NS 13754P NS NS NS NS NS 20.2a NS 13756P NS NS NS NS NS 41 .Oa NS 13758P NS NS NS NS NS 22.1a NS 13759P NS NS NS NS NS 22.7 NS a Sera analyses were conducted at a contract laboratory, but were corrected by 3M to reflect the official punty values from the Certificate of Analysis. A revisedfinal reportfrom the contract laboratorywill beaddedas an amendmentto this report. LLQ= Limit of Quantitation (0.04 VglmL) NS=No sample 3M Environmental Laboratory Page 96 3M Medical Department Study: T-639514 BACK TO MAIN Analytical Report: FACT TOX-111 LRN42994 Table 15. Results of Analyses of Liver Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Liver, Urine, and Feces of Crl:CD@BRVAF/Plus@Rats Exposed to T-6295.14 via Gavage 13745P 13748P a Liveranalyseswere conductedat a contradlaboratow, butwere corrected by 3M to reflectthe officialpurityvalues from the Certiiite of Analysis. A revisedfinal report from the contract laboratorywill be addedas anamendmentto this report. BLOQ=Below Concentration of Lowest Standard in Calibration Curve LOQ=O.11 pg/g 3M Environmental Laboratory Page 97 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-1II LRN-U2994 Table 15. Results of Analyses of Liver Samples in the Study of the Determinationofthe Presenceand Concentrationof PFOS in the Sera, Liver, Urine, and Feces of Crl:CD@BRVAF/Plus@Rats Exposedto T-6295.14 via Gavage I 13756 I 56.2a I Group 111 1.6mglkglday 13756P 13758P 13759P 72 .Oa 50.6a 52.6a 3M Environmental Laboratory Page 98 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Sample # DGO PFOS Conc. bs/mL) DGW PFOS Conc. (PdW DG14/15 PFOS Conc. (PmL) DG20/21 PFOS Conc. bg/mL) DL21/22 PFOS Conc. (C19/mL) 13726F 0.00381 cLOQ cLOQ cLOQ cLOQ 13727F cLOQ <LOQ cLOQ 0.00614 cLOQ I13728Fl 0.00464 I 0.00312 I cLOQ I <LOQ I cLOQ I Group I 13731F 0.00650 cLOQ cLOQ NS NS O-Omg'kg'day 13734F 0.0108 <LOQ cLOQ cLOQ cLOQ 13735F 0.01 17 <LOQ cLOQ cLOQ cLOQ 13736F 0.0117 0.0168 cLOQ cLOQ cLOQ 13737F <LOQ cLOQ 0.00685 cLOQ cLOQ 13739F I 0.0852 I 0.0454 I 0.0135 I 0.00531 I <LOQ 13740F I 0.202 I 0.0296 I 0.0444 I 0.0230 I 0.00981 I 13741F I 0.0587 I 0.0256 I 0.0335 I 0.0368 I 0.00391 I Group II 13744F 0.0807 0.0257 0.0184 0.0259 cLOQ 0.1 mgkglday 13745F 0.0520 0.0394 0.0244 0.0298 cLOQ 13746F 0.0517 0.0223 0.0642 0.0187 0.00328 13758F 2.74 13759F 2.50 0.438 I.I4 cLOQ=Less than the Limit of Quantitation (0.00295(~glmL) ND=Not Detected NS=No Sample SL=Sample lostduringextraction NE=Nosample extract remainingto performdilution 0.273 0.548 0.558 0.208 0.0328 0.0169 3M Environmental Laboratory Page 99 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN42994 13756F 10.8 13758F 9.41 13759F 10.9 6.70 5.13 7.65 5.30 4.47 3.41 2.02 0.336 1.97 0.21 1 1.38 0.296 3M Environmental Laboratory Page 100 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Appendix F: Example Calculations Formula Usedfor UrineAnalyses in Study FACT TOX-11I AR (nglmL) x DF x SC x '*OPg x PC = Reported Concentration (pglmL) 1OOOng Calculation Used for Group II DG 14-15, Animal Number 13739F 16.79 (nglmL) x 1x 0.9275 x l*Oiug x 0.864 =0.0135(pg PFOSlmL urine) 1OOOng AR-Analytical result from MassLynx summary DF-Dilution factor SC-PFOS salt correction constant (0.9275) PC-PFOS purity correction factor (0.864) 3M Environmental Laboratory Page 101 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Appendix G: Contract Laboratory Reports This appendix includes the following contract laboratory reports: Advanced Bioanalyfical Services, Inc., Quantitative Determination of Perfluorooctanesulfonate(PFOS) in Rat Serum from Study #FACT-TOX-111 Using Turbo Ion Spray LC/MS, ABS Report Number 99ADJA03.MI.DOC (19 pages). Advanced BioanalyticalServices, Inc,, "MethodValidation for the Quantitation of Perfluorooctanesulfonate(PFOS) in Rat Serum by Turbo Ion Spray LC/MS," ABS Report Number 99VDJA01.MI.DOC (50 pages). BaffeIIeMemorial Institute, Study Number N003296-G, Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats Final Report, (58 pages). Centre Analytical Laboratories, Inc., Centre Study Number 023-017, Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats, (93 pages). 3M Environmental Laboratory Page 102 3M Medicpl Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-11I LRN-U2994 &.* I I i BIOANALYTICAL REPORT Title: Quantitative Determination of Perfluorooctanesulfonate (PFOS) in Rat Serum from Study #FACT-TOX-111Using Turbo Ion Spray LCMS Date: 11 October 1999 Authors: PreparedFor: . David J. Anderson, M.S. h i e J. Prince, B.S. Holly D. Ross, M.S. 3M Environmental Technology and Safety Services St. Paul, MN 55133-3331 A B S Report No.: 99ADJA03.MI.DOC Study Protocol: FACT-TOX-111 Number of Pages: 19 Summary and Conclusions The concentrationof perfluorooctanesulfonate(PFOS) was determined in rat serum samples collected during the study #FACT-TOX-111 using a sensitive, specific, accurate, and reproducible analytical method developed by Advanced BioAnalytical Services, Inc., Ithaca, NY. Rat serum samples (50 $) were extracted by a liquid-liquid extraction procedure to isolate PFOS and the internal standard, lH, lH,2H,2H-perfluorooctanesulfonicacid (Tetra-H-PFOS). Following evaporation and reconstitution, sample extracts were analyzed by turbo ion spray liquidchromatography/massspectrometry (LCMS) in the negative ion mode. AI1 samples were successfullyanalyzed within four runs. The lower limit of quantitation was 0.05 pg/mL for PFOS. The precision of this assay (RSD) as determined from the analysis of quality control samples for PFOS was 53.61%. The precision of this assay (RSD), as determined from the calibration standards for PFOS was 15.08%. The relative error (RE)of the assay, as determined from the analysis of the quality control samples ranged from 2.38 to 12.7%. The RE of the assay, as determined fkom the analysis of calibration standards ranged fiom -5.3 1 to 10.5%. 15 Catherwood Road I Ithaca, New York 14850 I (607)2664665 I Fax (607)266-0749 3M Environmental Laboratory Page 103 3M Medical DepartmentStudy: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-I 11 LRN-U2994 Title: ADVANCED BIOANALYTICAL SERVICES SIGNATUIW PAGE Quantitative Determination of Perfluorooctanesulfonate(PFOS) in Rat Serum fi-om Study #FACT-TOX-111 Using Turbo Ion Spray LCrMS Research ScieEtist CY Reviewed by: Melissa Mapes, AS. Associate Auditor Accepted by: Assistaht ScientificDirector Kris Hanson, Ph.D. 3M Study Director //OCT 99 Date Date 3M Environmental Laboratory ADVANCED BlOANALYTlCAL SERVICES. INC. Page 2 of 19 99ADJA03.MLDQC Page 104 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 QAU STATEMENT Periodic inspectionsof the biodytical portion of study #FACT-TOX-111were conducted by the Quality Assurance Unit of Advanced BioAnalytical Services (ABS) for compliance with EPA GLP regulations (40CFR Part 792). The study was inspected on the following dates: 25 June 1999; 7,21 July 1999;31 August 1999. Results of the inspectionwere reported to A B S Management and Project Team Leader on: 25 June 1999; 7,213uly 1999; 31 August 1999. Results of the inspections were reported to the Study Director on 2 September 1999. ' E k e d on the inspectionsand the data reviewed, this report is a complete and accurate representation of the data. w f l w Melissa Mapes, AS! Associate Auditor //de7 99 Date 3M Environmental Laboratory ADVANCED BlOANALYTlCAL SERVICES, INC. .. Page 3 of 19 99ADJA03.MLDOC Page 105 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 TABLEC)PCONTENTS SIGNATUREPAGE ............... ........................................................................................... 2 QAU STATEMENT............................................................................................................ 3 TABLEOF CONTENTS..................................................................................................... 4 LIST OF TABLES............................................................................................................... 5 . 1. INTRODUCTION....................................................................................................... 6 1.1. Study Description and Objective ............................................................................. 6 2. METHODS................................................................................................................... 6 2.1.Analytical Procedure(s)............................................................................................ 6 '.* 2.2. Assay Site................................................................................................................. 7 ... ..2.3. Data Processing........................................................................................................ 7 4 3 . RESULTS .................................................................................................................... 7 3.1. Assay Performance .................................................................................................. 7 3.2. Analytical Results for Study #FACT-TOX-111 ...................................................... 8 4. SUMMARY AND CONCLUSIONS.......................................................................... 8 5. DATA RETRIEVAL.................................................................................................... 8 6. REFERENCES ............................................................................................................ 8 7. TABLES ...................................................................................................................... 9 3M Environmental Laboratory ADVANCED . BlOANALYTlCAL SERVICES. INC Page 4 of 19 99ADJA03.MI.DOC Page 106 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 LIST OF TABLES Table 1: Summaryof Sample and Assay Information for Protocol FACT-TOX-1 11...... 9 Table 2: Inter-Assay Precision and Accuracy for PFOS Quality Control Samples in Rat Serum for Study #FACT-TOX-111............................................................,.... 10 Table 3: Inter-Assay Precision and Accuracy for PFOS Calibration Standardsin Rat Serum for Study #FACT-TOX-111....................................................... ~ ......... 11 . Table 4: CalibrationCurve Statisticsfor the Determination of PFOS in Rat Serum for Study #FACT-TOX-111 ...........,._...................................................................12 Table 5: CFoonllcoewnitnragtitohnes0omf PgFkOgSDinosRea..t..S...e..r.y...n...S.,a..m...p..l.e..s..f..r.o..m....S..t.u...d..y..#..F..A_C...T..-..T..O...X...-.1...1.113 Table 6: Concentrationsof PFOS in Rat Serum Samples from Study #FACT-TOX-1 11 Following the 0.1 m a g Dose......................................................................... 15 '.: Table 7: 2, Concentrationsof PFOS in Rat Serum Samples h Following the 1.6mgkg Dose..................................... m S.t..u...d..y....#..F...A....C...T....-.T....O...X-1 1 1 17 Table 8: Repeat Analysis of Rat Serum Samples from Study #FACT-TOX-111 .......... 19 ADVANCED BIOANALYTICAL SERVICES. INC. 3M Environmental Laboratory Page 5 of 19 99ADJA03.MI.DOC Page 107 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 1. INTRODUCTION 1.1. Study Description and Objective The objective of this study was to determine the concentrations of perfluorooctanesulfonate(PFOS) in rat serum samples collected during the study #FACTTOX-Ill, "Oral (Gavage) PharmacokineticRecovery Study of PFOS in Rats." Rat serum samples were analyzed using a liquid-liquid extraction and turbo ion spray liquid chromatography/massspectrometry (LC/MS) assay. 2. METHODS 2.1. Analytical Procedure(s) PFOS concentrationswere determined in rat serum according to the validated LC/MS method (1). Serum samples (50 $) were extracted by a liquid-liquid extraction procedure using ethyl acetate to isolate PFOS and the internal standard (IS), lH, IH,2H,2H-perfluorooctanesulfonic acid (Tetra-3-PFOS)from rat serum. Following evaporation to dryness and subsequentreconstitution, sample extracts were separatedby reversed-phasechromatographyon a 2 x 50 mm (5 p)B e t a s i P C,8column (Keystone Scientific, Inc., Bellefonte, FA) with an initial mobile phase of 55% Eluent A (10:90 methanol:2 mM ammonium acetate) and 45% Eluent B (90:10methanol:2 mM ammonium acetate). PFOS concentrationswere determinedby turbo ion spray liquid chromatography/mass spectrometry (LC/MS) in selected ion monitoring (SIM) mode. The negative ions monitored were: m/z = 499.0 for FFOS iM-K]m/z = 427.0 for Tetra-H-PFOS m-H]- Study sample concentrationswere determined fiom a weighted (l/f), quadratic regression of peak area ratios (peak area of PFOS/peak area of Tetra-H-PFOS)versus nominal concentrationof ten calibration standards. The nominal concentrationsof the calibration standards were 0.05,0.1,0.25,0.5,1,2,4,8, 16, and 20 pg/mL PFOS. The lower limit of quantitation (LLQ) for this assay was determined to be 0.05 pg/mL PFOS. Quality control (QC) serum samples at three different concentrations(0.2,6,and 18 pg/mL PFOS) were analyzed with each assay batch in replicates of four. Dilution QC samples (QC4, 100 pg/mL) were prepared at each dilution that was used during a particuIar assay and were analyzed with each assay batch in replicates of four as needed. The acceptance criteria for calibration standards stipulated that the back-calculated concentrationsof at least three-fourths of the individua1,calibrationstandardreplicates `mustnot deviatemore than k15% from their nominal concentration (except at the LLQ). At least one duplicate at the LLQ must exhibit a deviation within 120% of the nominal ADVANCED BIOANALYTICAL SERVICES, INC. 3ratory Page 6 of 19 99ADJA03.MI.DOC Page 108 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 concentration , The coefficient of determination (1')must be 20.98. The acceptance criteria followed are in accordance with ABS Standard Operating Procedures. The acceptancecriteria for the qualitycontrol samples stipulatedthat at least two-thirds of the individual QC sample replicates must not deviate more than 415% fiom their nominal concentrations. At least one replicate at each QC concentrationmust exhibit a deviation within f15%. A summary of the sample and assay information for study #FACT-TOX-1 1 1 is given in Table 1. 2.2. Assay Site All sampleswere analyzed at Advanced BioAnalytical Services,Inc., Ithaca, NY. 2.3. Data Processing The data were collected using turbo ion spray LCMS selected ion monitoring (SIM) in the negative ion mode. Peak areas were integrated by the PE SCIEX program MacQuan, version 1.4, residing on aMacintosh computer. Followingpeak area integration, the results tables &om MacQuan were saved as text files and uploaded to the ABS file server where a weighted (I/$) quadraticregressionwas performed using the software package Watson (v 5.3.1.01 PSS,Inc., Wayne, PA 19087).Calculationswere performed on unrounded numbers. AI1 calibrationstandard and QC results were rounded to no less than three significant figures before reporting. 3. RESULTS 3.1. Assay Performance The performance of the assay for PFOS as determined from the analysis of daily quality control samples is documented in Table 2, The inter-assay precision (RSD) of quality control samples ranged from 2.78 to 3.61% for PFOS. There was no marked inaccuracy in the results from these quality control samples; the relative error (RE) ranged from 2.38 to 12.7%. The performance of daily calibrationcurves is documentedin Table 3. The inter-assay precision (RSD)ofthe standardsranged from 1.52 to 5.08% for PFOS. There was no marked inaccuracy in the results from these standards; the relative error (RE) ranged between -5.31 to 10.5%. ADVANCED BI OANALYTICAL SERVICES. INC. 3fvEmtronmental Laboratory Page 7 of 19 99AD.lAO3.MI.DE Page 109 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 2 n . The slope, y-intercept, and coefficientof determination(Ifo)r each analytical run are presented in Table 4. The 3 values ranged firom 0.9959 to 0.9977 for PFOS in rat serum. All ABS personnel assigned to analyze samples for this project were required to successllly extract quality control (QC) samples and calibrationstandards. Results of the extraction were reviewed by a trained analyst against the pre-defined acceptance criteria for this assay. Results are maintainedin the study records of ABS. 3.2. Analytical Results for Study #FACT-TOX-111 PFOS concentrationsin rat serum from Study#FACT-TOX-111 are presented in Tables 5 through.7. All data were rounded to no less than three significant figures before reporting in Tables 5 through:7.Study samplesrequirhig repeat analysis for Study #FACT-TOX111are presented in Table 8, 4. SUMMARY AND CONCLUSIONS . The concentrationof PFOS was,determined in rat serum samples collected during the study #FACT-TOX-111 PFOS was isolated from serum by a liquid-liquid extractionprocedure and determinedby LCIMS. The quality of the determinationswas satisfactorythroughout. The LLQ was 0.05 pg/mL PFOS. The precision of the assay, as determined from the analysis of quality control samples was 53.61% for PFOS. 5. DATARETRIEVAL The calculated concentrationdata fiom the anaIysis of rat serum samples for Study #FACT-TOX-111 are maintained on file in the archives of the Advanced BioAnalytical Services, Inc., Ithaca, NY in ABS Notebook 2325. 6. REFERENCES 1. Advanced BioAnalyticalServicesValidationReport 99VDJAO1.MLDOC. Method Validation for the QuantitationofPertluorooctanesulfonate(F'FOS) in Rat Serumby Turbo Ion Spray LCMS. David J., Anderson, M.S. h i e J. Prince, . B.S.,and Holly D. Ross, M.S. 26 August 1999. -Caboratory ADVANCED BIDANALYTICAL SERVICES, INC. Page 8 of 19 99ADJA03.MI.DOC Page 110 3M Medical DepartmentStudy: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 7. TABLES Table 1: Summary of Sample and Assay Information for Protocol FACT-TOX111 SpeciesAUatrix: Rat/Serum Sample Collection and Storage Information: AnticoagulantLStabilizer: Reported Sample Collection Dates: Dates Received at ABS: Storage Temperature at ABS: .' None 12 January 1999to 20 February 1999 16 June 1999 -20 "C Assay Information: Assay Period .,.I ' Analyte: 9 July 1999 to 14 July 1999 Potassium perfluorooctanesulfonate (PFOS) Analytical Standard: Lot No: Source: PFOS 171 3M Internal Standard: Lot No: Source: 1Hy1H,2H,2Hperfluorooctane sulfonic acid (Tetra-H-PFOS) 59909 3M Calibration Range: Regression Method: Weighting Factor: Lower Limit of Quantitation: Upper Limit of Quantitation: quadratic lfy" 0.05 pg/mL 20 pg/mL ADVANC E O BIOANALYTICAL SERVICES. INC. Page 9 of 19 99ADJAO3.MI.DOC Jw Page 111 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-1 I 1 LRN-U2994 Table 2: Inter-Assay Precision and Accuracy for PFOS Quality Control Samples in Rat Serum for Study #FACT-TOX-111 &I 1 RE = [(Mean-Norninaf)/Nominal]x 100% NA: Not applicable. . ABDIDVAANNACLEYDTICAL SERVICES. INC. Page 10 of I9 P - 99ADJA03.MLDOC Page 112 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 ..t ADVANCED BlOANALYTICAL SERVICES, INC. D a Page 113 3 M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Table 4: Calibration Curve Statistics for the Determination of PFOS in Rat Serum for Study #FACT-TOX-lll s A. . y =Ax2+Bx f C,weighted 1/3 ADVANCED BIOANALYTICAL SERVICES. INC. Page 12 of 19 99ADJA03.MI.DOC Page 114 3M Medical Department Study: T-6395.14 ss n h VIm 99 \I \I n 8 h m vn , 00 n In 0 v3w 0 N BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 2 Ir 13 f a ADVANCED BIOANALYTICAL SERVICES, INC. Page 115 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Repod: FACT TOX-111 LRN-U2994 4 v 4 v 8 m v n 2 ADVANCED BIOANALYTICAL SERVICES, INC. 2 c f Page 116 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Page 117 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-I 11 LRN-U2994 ADVANCED BIOANALYTICAL SERVICES. INC. ry ... Page 118 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 AOVANCED BIOANALYTICAL SERVICES, INC. ~ Page 119 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 3M Envirdnmental Laaorarory Page 120 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-11I LRN-U2994 ADVANCED BIOANALYTICAL SERVICES. INC. 3M Envirdnmental Laboratory Page 121 3M Medical Department Study: T-6395.14 I BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 . . 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: 99VDJAO1.MI.DOC AUTHORS: PREPARED FOR. NUMBER OF PAGES: David J. Anderson, M.S. Amie J. Prince, B.S. Holly D. Ross, M.S. 3M Environmental Technology and Safety Services St. Paul, MN 55133-3331 50 15 Catherwood Road R Ithaca, New York 14850 I ( 6 0 7 ) 266-0665 rn Fax (607)266-0749 3M Environmental Laboratory Page 122 BACK TO MAIN 3M Medical Department Study: T-6395.14 AUTHORS: FOR DATE: TITLE: Analytical Report: FACT TOX-111 LRN-U2994 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 LCMS ABSTRACT A sensitive, specific, accurate, and reproducible analyhcal method was developed by Advanced BioAnalytical Services, Inc., Ithaca, New York to quantitate perfluorooctanesulfonate(PFOS) in rat serum samples. Serum samples (50 a)were extractedby a liquid-liquid extraction procedure to isolatethe d y t e fromrat serum. Sample extractswere reduced to dryness, reconstituted, and analyzed by turbo ion spray liquid chromatography/massspectrometry (LC/MS) in the negative ion mode. The assay demonstrated a lower limit of quantitation (LLQ) of 0.05 pg/nL using 50-JL sample aliquots. The calibration curves were fit from 0.05 pdmL to 20 pdmL for PFOS by a weighted (l/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) sampleswere prepared at a concentrationof 100 pglmL PFOS for partial volume analysis. The intra- and inter- assay precision (RSD) results calculated fiom 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 fiom 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 SERVICES, INC. Page 2 of 50 99VDJA01.MLDOC Page 123 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 after three fkeezekhaw cycles. Reliable resultswere obtained for sample extracts reinjected 27 hours after initial reconstitution. 3M Environmental Laboratory ADVANCED BIDANALYTICAL SERVICES. INC. Page 3 of 50 99VDJAO1.MLDOC Page 124 BACK TO MAIN 3M Medical Department Study: T-6395.14 I Analytical Report: FACT TOX-111 LRN-U2994 QAU STATEMENT Periodic inspectionsof the method validation for the quantitation of PFOS hrat sefum were conducted by the Quality Assurance Unit of Advanced Biohalytical 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 inspectionswere reported to ABS Management on: 13,21 May 1999; 7,8,14,15 Jurie 1999; 1,2 July 1999; 3 , 4 August 1999. Results of the inspections were reported to the Study Director on 5 August 1999. Based on the inspections and the data reviewed, this report is a complete and accurate representation of the data. Quality Auditor -- 3M EnvironmentalLaboratory ADVANCED 5lOANALYTICAL SERVICES, INC. Page 4 of 50 99VDJAO1.MI.DOC Page 125 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 SIGNATURE PAGE TITLE: METHOD VALIDATION FOR THE QUANTITATION OF PERFLUOROOCTANESULFONATE(PFOS)IN RAT SERUM BY TURBO ION SPRAY LCMS Report Number: 99VD Reported by: Research Scientist Reviewed by : 'Kgthleen Cormack, B.S. Associate Auditor Authorized for Release by: Assistant Scientific Director 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES. INC. , Page 5 of 50 99VDJAOI.MI.DOC Page 126 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 TABLE OF CONTENTS ABSTRACT ......................................................................................................................... 2 QAU STATEMENT............................................................................................................ 4 SIGNATUREPAGE ............................ ;...............;.............................................................. 5 TABLE OF CONTENTS .................................................................................................... 6 LIST OF TABLES............................................................................................................... 8 LIST OF FIGURES ............................................................................................................. 9 1. INTRODUCTION............................................................................................ :.........10 2. EXPERIMENTAL..................................................................................................... 10 2.1.CHEMICALSAND MATERIAL..S............................................................................. 10 2.2. LC/MS INSI-RUMENTA-II.O...N.............................................................................. 10 2.3. SAMPLEPREPARATIONAND EXTRACTIONPROCEDURE...................................... 11 2.4, U T SERUMVALIDATIONDATA........................................................................... 11 . 2.5. ASSAY EVALUATIO.N............................................................................................ 12 2.5.1. Intra- and Inter-Assay Accuracy.................................................................... 12 2.5.2. Intra- and Inter-Assay Precision ................................................ ................... 12 2.5.3. PartialVolume Analysis................................................................................ 12' 2.5.4. Lower Limit of Qumtitation (LLQ) .............................................................. 13 2.5.5. Selectivity........................................................................... :.......................... 13 2.5.6. CanyoverEvaluation..................................................................................... 13 2.6. STABILITYOF PFOS IN QUALITYCONTROLSAMPLES ........................................ 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. Freezemhaw Stabilityin Rat Serum............................................................... 14 2.7. REPRODUCIBILITYOFR E I " G EXTRACTED SAMPLES................................ 14 2.8. EXTRACTION RECOVERY ...................................................................................... 14 3. RESULTS AND DISCUSSION ............................................................. i.................. 15 3.1. ASSAY EVALUATIORNESULTS ............................................................................. 16 3.1.1. Intra- and Inter-Assay Accuracy.................................................................... 16 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES. I N C. Page 6 of 50 99VDJAOI.MI.DOC Page 127 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN.U2994 . 3.1.2. Intra- and Inter-Assay Precision .................................................................... 16 3.1.3. Line.ty. ....................................................................................................... 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 QUALITYCONTROL~AMPLES........................................ 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. Freezemaw StabilityofPFOS in Rat Serum............................................... 18 3.3. REPRoDUCIB~LITYOF REINJECITNGEXTRACTEDSAMPLES................................ 18' 3.4. EXTRACTION RECOVERY ...................................................................................... 18 4. CONCLUSIONS........................................................................................................ 19 5. DATA RETmEVAL.................................................................................................. 19 6. REFERENCES................................................................................... .'........................ 19 7. TABLES.................i................................................................................................... 20 8. FIGURES ................................................................................................................... 31 9. APPENDIX A: QUANTITATION OF PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUMBY TURBO ION SPWY LCMS .................................. 37 3M Environmental Laboratory ADVANCED BlOANALYTICAL SERVICES. INC. Page 7 of 50 .-. .................... 99VDJA01.MLDOC Page 128 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 LIST OF TABLES Table 1: Intra-Assay Accuracy and Precision of the PFOS Assay in Rat Serum for QC Samples ...................................................................................................... 20 Table 2: Inter-Assay Accuracy and Precision of the PFOS Assay in Rat Serum for QC Samples from Three Validation Runs ........................................................ 21 Table 3: Table 4: Table 5: Table 6: Accuracy and Precision of the PFOS Assay in Rat Serum for Calibration Standards from Three Validation Runs...........,....................................,............22 Calibration Curve Parameters for PFOS in Rat Serum ..................................... 23 Partial Volume Analysis of PFOS in Rat Serum ............................................... 24 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 Stabilityof PFOS in Rat Serum at -20 "C............................................ 27 Table 9: Stability of PFOS in Rat SerumAfter Three Freeze/Thaw Cycles .,.................28 Table 10:Reproducibility of Reinjecting Extracted Samples ContainingPFOS after 27 Hours inReconstitution Solution................................................................. 29 Table 11:Extraction Recovery ofPFOS from Rat Serum................................................. 30 -1 ADVANCED BIOANALYTICAL I SERVICES, INC. 3M Environmental Laboratory Page 8 of 50 99VDJAO1 .MI.DOC Page 129 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-I 11 LRN-U2994 LIST OF FIGURES Figure 1: Full-Scan SingleMS Mass Spectrumof PFOS...............,.................................31 Figure 2: Full-Scan SingleMS Mass Spectrumof Tetra-H-PFOS ................................... 32 Figure 3: Mass Chromatograms of PFOS and its Internal Standard in Control Blank Rat Senun Extract............................................................................................. 33 Figure 4: Mass Chromatograms of PFOS in a Rat Serum Extract Sample Containing Internal StandardOnly (Zero Sample).............................................................. 34 Figure 5: .. MCaossntaCinhirnogmPaFtoOgSra(m0.s0o5fpCg!amliLb)ratainodntShteanIndtaerrdn1alinStRanadt aSredru..m....E..x..tra..c..t.............35 Figure 6: Mass Chromatogramsof Calibration Standard 10in Rat S e m Extract Containing PFOS (20 pg/mL) and the Internal Standard ................................. 36 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 9 of 50 99VDJAOl.MLDOC Page 130 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 1. INTRODUCTION The purpose of this report is to describethe method validation for the quantitative determinationof perfluorooctanesulfonate(PFOS) in rat serum samplesafter modificationof aprevious method (1). The objectives of the method validation were to validate a simple extraction procedure, determine the lower limit of 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 fieezer storage, and over three freeze/thawcycles, and to provide specific, accurate, and reproducible quantitative results by turbo ion spray liquid chromatography/mass spectrometry(LCMS). 2. EXPERIMENTAL 2.1. CHEMICALASND MATERIALS Perfluorooctanesulfonate(PFOS, Lot# 171)was obtained from 3M, Inc. The internal standard (IS) for PFOS was lH,lH,2H,2H-perfluorooctanesulfonicacid (Tetra-HPFOS). The internal standard (Lot# 59909) was obtained fiom 3M,Inc. detailed list of chemicals and materials is found in Appendix A. Stock and working solutions whichwere used to prepare the calibration curves for the amlytes were prepared as describedin Appendix A. Stock solutionsused in the preparation of quality control (QC) sampleswere prepared separately from those used in preparation of the calibration curves. Preparation of all solutions used during extraction and analysis are described in Appendix A. 2.2. LC/MS INSTRUMENTATION The liquid chromatography/mass spectrometrysystem consisted of two LC-1O A D pumps (Shimadzu, Columbia, MD 21046), a SCL-1OA pump controller(Shimadzu, ADVANC ED BlOANALYTlCAL SERVICES, INC. 3M Environmental Laboratory Page 10 of 50 99VDJA01.MI.DOC Page 131 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Columbia, MD 21046), a WISP 717plus autosampler (Waters Associates, Millipore Corporation,Milford, MA 01757), a Betasil CI8(2 x SO 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. SAMPLEPREPARATIONAND EXTRACTIOPNROCEDURE For each analyticalrun(tray), duplicate 50-pL aliquotsof the calibration curve sampleswere prepared as describedin Appendix A. The nominal (theoretical) concentrationsof PFOS in the calibration curves were 0.05,0.1,0.25,0.5, 1,2,4,8, 16, and 20 &mL. Rat serum quality control sampleswere prepared in advance of the validation study at nomina1(theoretical)concentrationsof 0,2,6, and 18 p g / d for QCl, QC2, and QC3, respectively,as detailed in Appendix A. A dilution QC (QC4, 100 pghnL) was prepared at a concentrationexceedingthe upper limit of the calibration curve range (20 pg/mT.,), and was assayed using a 10-fold dilution for partial volume analysis. Calibration standardsand QC sampleswere extractedby the procedure detailed in i Appendix A. 2.4. RAT SERUM VALIDATION DATA The data were collectedusing selected ion monitoring ( S Wturbo ion spray LCMS in the negative ion mode. Peak areas were integrated by the PE SCIEX pro& MacQuan, version 1.4, residing on a Macintosh computer. Following peak area integration, the results tables fiom MacQuan were saved as text files and uploaded to the Advanced BioAnalyticalServices (ABS) file server where a weighted (llf) 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 figuresby A B S prior to reporting in Tables 1through 11. The data for the rat serum validation are stored in ABS Notebook 2304. --_ .I. -I - I I - _- _"_ ABDIOVAANNACLEYDTICAL SERVICES. INC. Page 1 1 ofSO 99VDJAOI .MI.DOC 3M Environmental Laboratory Page 132 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 All calculationswere based on the peak area ratio of the analyte to the internal standard. Concentrations of each analyte in quality control samples were determined by inverse prediction from the calibration curve. 2.5. ASSAY EVALUATION 2.5.1. Intra- and Inter-Assay Accuracy The intra- and inter-assay accuracy of the method was assessed by determining the relative error observed in the analysis of 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. QCl through QC3 were assayed in replicates of 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 determiningthe 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. QCl through QC3 were assayed in replicates of five. In addition, the RSD was reported for standards at all levels over three m s . 2.5.3. Partial Volume Analysis The effect of dilution on the analysis of PFOS in rat senun was determined by partial volume analysis. QC4 (100 pg/mL) was prepared containing PFOS at approximately five times the upper limit of quantitation WQ).Five replicates of QC4were diluted ten-fold and analyzed. The RSD and RE were reported. 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 12 of 50 99VDJA01.MI.DOC Page 133 BACK TO MAIN 3M Medical Department Study: T-6395.14 i Analytical Report: FACT TOX-111 LRN-U2994 2.5.4. Lower Limit of Quantitation (LLQ) Rat s e m control samples fkom six separateindividuals 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 m l y t e nor the internal standard (control blank) were assayed with all experiments. 2.5.6. Carryover Evaiuation The carryover of analyte from one injection to the next was assessed by analyzing a control'blank injected immediately after ahigh calibration standard (STD10, 20 PdmL). 2.6. STABILITY OF PFOS IN QUALITY CONTROL $AMPLES QCl 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 BIDANALYTICAL SERVICES, INC. Page 13 of50 99VDJA01.MI.DOC Page 134 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 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 fkeezer stability timepoints will be reported in an addendum to this report. All QCs were assayed in replicates of four. 2.6.3. keeze/Thaw Stability in Rat Serum The stability of PFOS was evaluated after three fieeze/thaw cycles. The mean concentrations of the QC samples after three freezehhaw cycles (i.e., at least 2 hours frozen storage at the nominal temperature of -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. REPRODUCIBlLITY QF REINJECTING EXTRACTED SAMPLES 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 mCOVERY The extractionrecovery of PFOS from rat serum was determinedby comparingthe peak area ratio (PAR)of samples (0.25,4, and 16 pdmL) spiked after extraction (post-extract)with the PAR of samples spiked before extraction @re-extract). The internal standard was spiked post-extraction for all samples. The recovery of the internal standard (4 pglmL) was assessed following a similar approach using PFOS as the reference. The extraction recovery (% Recovery) was determinedby 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 EIOANALYTICAL SERVICES, INC. Page 14 of50 99VDJAO1 .MLDOC Page 135 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 . LRN-U2994 3. RESULTS AND DISCUSSION A quantitative analytical procedure using turbo ion spray LC/MS in the negative ion mode was developed to meet the high sensitivity, specificity, and reproducibility requirements for the determination of PFOS in rat serum. The full-scan single MS mass spectrum of PFOS showed an abundant [M-KI-ion at d z =499 (Figure 1). The full-scan single MS mass spectrum of Tetra-H-PFOS showed an abundant [M-HI- ion at d z = 427 (Figure 2). The following selected ion monitoring (SIM) was used to quantify the analytes in rat serum: PFOS Tetra-H-PFOS (IS) m/z =499.0 d z =427.0 The peak labeling of Ml-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. 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 15 of 50 99VDJA01.MI.DOC Page 136 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 3.1. ASSAY EVALUATIONRF,SULTS 3.1.1. Intra- and Inter-Assay Accuracy The intra-assay accuracy (RE) data &omQC 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 &omQC samples ranged &om 1.92 to 4.87% for PFOS (Table 2). The RSD ranged fkom 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 (l/f) quadratic regression. Coefficients of determination {r2)were 20.9962 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 1in 10was 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 BIOANALYTIC AL SERVICES, INC. Page I6 of SO 99VDJA01 .MI.DOC Page 137 BACK TO MAIN 3M Medical Department Study: T-6395.14 I Analytical Report: FACT TOX-111 LRN-U2994 3.1.5. Lower Limit of Quantitation (LLQ) Table 6 shows the lower limit of quantitation (LLQ) data. The serum LLQ experimentdemonstratedthat 0.05 pg/mL is an acceptableLLQ 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 chromatographicinterferenceswere observed in any of the control serum samples analyzed (Figure 3). The control blanks and zero samples did show some evidenceof small chromatographicpeaks 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 Carryoverof 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 chromatographicpeak at the retention time for PFOS was comparableto the response of a rat serum control blank injected before the high standard. Thus, carryoveris negligible for PFOS. Therewas no evidence of carryover for the internal standard. 3.2. STASILITYOF PFOS IN QUALITY CONTROLSAMPLES 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 concentrationsdeviated from -4.45 to 10.5% from the 0-hour values for all QC levels &d time points. Based on these data, PFOS was stable in rat serum for up to 24 hours at ambient temperature. 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 17 of 50 99VDJAOl .MLDOC Page 138 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 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 fieezer storage after two,four, and eight months will be reported as an addendm to this report. 3.2.3. Freeze/Thaw Stability of PFOS in Rat Serum The results of the freezdthaw stability of PFOS in QC samples after three freezdthaw cycles is shown in Table 9. PFOS was stable after three freezekhaw cycles with deviations ranging fiom-1.38% to 10.1% from the 0-cycle samples for all QC levels (Table 9). 3.3. REPRODUCIBILITYOF REINJECTING EXTRACTESDAMPLES The results of reinjecting reconstituted samples containing PFOS after storage for 27 hours at approximately 25 "Cin reconstitution solution are shown in Table 10. Reinjectingprocessed samples after 27hours in reconstitution solution was appropriatewith RE values ranging-from-6.33 to 4.59% at 27hours for ali QC levels. 3.4. EXTRACTION RECOVERY The mean recoveries of PFOS and TetraH-PFOS are shownin Table 11. The mean recoveriesranged from 87.6 to 100%for PFOS.The mean recovery for Tetra-HPFOS was 89.9%. 3M Environmental Laboratory ADVANC ED 6I OANALYT IC AL SERVICES. INC. Page 18 of 50 99VDJAOI .MI.DOC Page 139 BACK TO MAIN 3M Medical Department Study: T-6395.14 ! Analytical Report: FACT TOX-111 LRN-U2994 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 an3 reproducible quantitation of PBOS down to a level of 0.05 p g / d 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 A B S Archives. 6. REFERENCES 1. Advanced BioAnalytical Services, Inc. Report 98AGKP02.MMM. Analytical Report for the Determination of Perfluorooctanoate and Perfluorooctanesulfonatein Human Serum by LCMS. Grace K. Poon, Ph.D., David Hardwick, B.S., and Ellen Pace, M.A.T., 6 February 1998. 3M Environmental Laboratory ADVANCED EIOANALYTICAL SERVICES. INC. Page 19 of 50 99VDJAO1.MLDOC Page 140 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-1I 1 LRN-U2994 7. TABLES Table 1: Intra-Assay Accuracy and Precision of the PFOS Assay in Rat Serum for QC Samples RSD = (SDMean) x 100 RE = [(Mean/Theoretical)-l] x 100 ADVANCED BIOANALYTICAL SERVICES, INC. 3M EnvironmentalLaboratory ' Page 20 of 50 99VDJAOl.MI.DOC Page 141 BACK TO MAIN 3M Medical Department Study: T-6395.14 I Analytical Report: FACT TOX-111 LRN-U2994 Table 2: Inter-Assay Accuracy and Precision of the PFOS Assay in Rat Serum for QC Samples from Three Validation Runs Theoretical Conc. Run 9 Run 10 Run 11 I 0.2 I 0.210 0.202 0.206 0.205 0.194. 0.187 0.186 0.182 0.189 0.182 0.194 6 652 6.3 1 6.17 6.32 6.35 6.44 . 6.44 6.32 6.19 6.31 6.17 RE (%) -3.58 I 4.95 18 17.9 18.0 16.7 18.0 17.3 16.9 17.3 17.5 18.0 17.4 18.2 17.1 17.6 - 17.7 17.0 17.5 2.66 -2.73 3M Environmental Laboratory AOVAN CED BIDANALYTICAL SERVICES.INC. Page 21 of 50 99VDJAO1 .MI.DOC Page 142 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Table 3: Accuracy and Precision of the PFOS Assay in Rat Serum for Calibration Standards from Three Validation Runs RSD = (SD/Mean) x 100 RE = [(Mean/Theoretical)-l]x 100 3M Environmental Laboratory ADVANCED BIDANALYTICAL SERVICES. INC. Page 22 of 50 99VDJA01.MI.DOC Page 143 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Table 4: Calibration Curve Parameters for PFOS in Rat Serum a: y =Axz+Bx + C,weighted l/g 3M Environmental Laboratory 99VDJAO1 .MI.DOC Page 144 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 , Table 5: Partial Volume Analysis of PFOS in Rat Serum I PFOS Conc. (pg/mL) Theoretical Conc. I I Run 9 QC4 100 (1in 10dilution) 111 RE (%) I 5.89 RSD = (SDMean) x 100 RE = [(Mean/Theoretical>-l]x 100 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 24 of 50 99VDJAOl .MI.DOC Page 145 BACK TO MAIN 3M Medical Department Study: T-6395.14 i Analytical Report: FACT TOX-111 LRN-U2994 Table 6 : Lower Limit of Quantitation of PFOS in Rat Serum Theoretical Concentration Conc. Found (pg/mL) 0.0548 LLQ 0.0489 RSD = (SDMean) x 100, RE = [(Mean/Theoretical)-l]x 100 3M EnvironmentalLaboratory ADVANCED BIOANALYTICAL SERVICES,INC. Page 25 of50 99VDJA01.MI.DOC Page 146 BACK TO MAIN 3 M Medical Department Study: T-6395.14 I Analytical Report: FACT TOX-111 LRN-U2994 Table 7: Ambient-Temperature Stability of PFOS in Rat Serum After 24 Hours QC (Theoretical Conc.) 0 Hour 0.194 2.5Hour I 6Hour I 24Hour N A Not Applicable RSD = %DEV (SD/Mean) x 100 from o HO-w= [(xHOW conc. - o HOW conc.)/o HOWconc.1 x 100 3M Environmental Laboratory ADVANCED BiOANALYTICAL SERVICES, INC. Page 26 of 50 99VDJA01.MI.DOC P a g e 147 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Table 8: Freezer Stability of PFOS in Rat Serum at -20 OC ; NA: Not Applicable RSD = (SDMean) x 100 %DEV fiom 0 Hour = [(Conc. - 0 Hour Conc.)/O Hour Conc.] x 100 *From Protocol FACT-TOX-110 **From ProtocolFACT-TOX-11I 3M Environmental Laboratory ABDI OVAANNACLEYDTICAL SERVICES, INC. Page 27 of 50 99VDJAOl.MI.DOC Page 148 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Table 9: Stability of PFOS in Rat Serum After Three FreezelThaw Cycles NA:Not Applicable RSD = (SDMean) x 100 %DEV &om 0 Cycle = [(3 Cycle - 0 Cyc1e)lO Cycle] x 100 3M EnvironmentalLaboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 28 of 50 99VDJA01.MLDOC Page 149 BACK TO MAIN 3M Medical Department Study: T-6395.14 i Analytical Report: FACT TOX-111 LRN-U2994 Table 10: Reproducibility of Reinjecting Extracted Samples Containing PFOS after 27 Hours in Reconstitution Solution Mean I PFOS (pg/mL) t==OHours I t=27Hours (Run 10) 0.187 (Run 12) 0.187 0.186 0.182 0.182 0.188 I 0.189 0.182 0.188 0.190 0.185 0.187 Mean 1 6.44 6.02 6.32 6.19 ' 6.19 6.55 6.3 1 6.22 6.34 6.28 17.3 17.5 18.0 17.4 Mean 17.4 N A Not Applicable RSD = (SDMean) x 100 RE = [(Mean/Theoretical)-l] x 100 18.2 18.1 17.3 . 17.6 17.7 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES. INC. Page 29 of50 99VDJAOl .MI.DOC Page 150 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Table 11: Extraction Recovery of PFOS from Rat Serum *(IndividualResponse for Pre-Ext./Individual Response for Post-Ext.)x 100 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 30 of 50 99VDJAO1 .MI.DOC Page 151 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 8. FIGURES Figure 1: Full-Scan Single MS Mass Spectrum of PFOS Spectrum from PFOS Q1 3.19c7 cps 499 [M-K]- 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 3 1 of 50 L 540 99VDJAO1.MI.DOC Page 152 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-I 11 LRN-U2994 Figure 2: Full-Scan Single MS Mass Spectrum of Tetra-H-PFOS Spectrum from Tetra-H-PPOS QI 1.94e7 cps I 350 400 450 m/Z 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 32 of 50 99VDJAO I.MI.DOC Page 153 BACK TO MAIN 3M Medical Department Study: T-6395.14 I Analytical Report: FACT TOX-111 LRN-U2994 Figure 3: Mass Chromatograms of PFOS and its internal Standard in Control Blank Rat Serum Extract mlz =427.0 1.07e3 cps J I 1 m/z = 499.0 1 1 1 2 3 Time, min I I 4 3.542 cps 1 Time, min All quantitation results and figures were prepared from non-smoothed data. Analvte PFOS Tetra-H-PFOS (IS) Iss m/z = 499.0 m/z = 427.0 Retention Time 3.1 2.9 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES,INC. Page 33 of SO 99VDJAOI .MI.DOC Page 154 BACK TO MAIN 3M Medical DepartmentStudy: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Figure 4: Mass Chromatograms of PFOS in a Rat Serum Extract Sample 1 1 d z =499.0 I 1 2 3 Time, min 3.78e2 cps 1 i 3 i Time, min All qoantitation results and figures were prepared from non-smoothed data. Anat* PFOS Tetra-H-PFOS (IS) Isn d z = 499.0 m/z = 427.0 Retention 3.1 2.9 3M EnvironmentalLaboratory ADVANCED E31OANALYTICAL SERVICES. INC. Page 34 of 50 99VDJAO1.MLDOC Page 155 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Figure 5: Mass Chromatograms of Calibration Standard 1 in Rat Serum Extract Containing PFOS (0.05 &mL) and the Internal Standard ndz = 421.0 3.82e5 cps 1. mlz =499.0 2 3 Time, min 4 2.11e3cps 1 2 3 4 Time, rnin All quantitation resuIts and figures were prepared from non-smoothed data. Analvte PFOS Tetra-H-PFOS (IS) h mlz =499.0 mlz = 427.0 Retention Time 3.1 2.9 3M Environmental Laboratory ADVANCED EIOANALYTICAL SERVICES, INC. Page 35 of 50 99VDJA01.MI.DOC Page 156 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Figure 6: Mass Chromatograms of Calibration Standard 10 in Rat Serum Extract Containing PFOS (20 pglmL) and the Internal Standard m/z =427.0 3.52e5 cps 30 20 10 1 d z =499.0 2 3 Time, min 4 5.14e5 cps 1 4 Time, min All quantitation results and figureswere prepared from non-smoothed data. Analvte PFOS Tetra-H-PFOS(IS) Ion m/z = 499.0 m/z = 427.0 Retention Time 3.1 2.9 3M Environmental Laboratory ADVANCED BiOANALYTlCAL SERVICES. INC. Page 36 of 50 99VDJAOI .MI.DOC Page 157 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-1I 1 LRN-U2994 9. APPENDIX A QUANTITATION OF PERFLUOROOCTANESULFONATE(PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/MS AUTHORS : David J. Anderson, M.S. Amie J. Prince, B.S. Holly D. Ross, M.S. TABLE OF CONTENTS A.l.O A.2.0 A.3.0 A.4.0 A.5.0 A.6.0 A.7.0 A.8.0 A.9.0 A.lO.O A.ll.O Chemical Structure(s) ........................................................................................ 38 Specimen(s) ......................................................................................................... h s a y Principle ........................................,...,..........,..........................................38 Compounds......;. .,,....;..................,................................,...................................38 Chemicals............................................................................. i......... ...................38 Materials andEquipment......................................................,................,......+....39 Preparation of Solutions ........................................................,...........................42 Preparation of Quality Control Samples............................................................ 45 Liquid-liquid Extraction Procedure...................................................................46 Instrument Conditions .......................................................................................47 Calculations .......................................,.,.......,......................................................49 3M Environmental Laboratory ADVANCED B IOANALYTICAL SERVICES, INC. Page 37 of 50 99MJAOl.MLDOC Page 158 3M Medical Department Study: T-6395.14 kl.O CHEMICAL STRUCTURE(S) BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 0 II C6F13-C-C-S-OH 11 H2 H2 0 IH, lH, 2H,2H-Perfiuorooctane SulfonicAcid MW =428, Internal Standard A.2.0 SPECIMEN(S) This assay uses 50-pL aliquots of rat s e w . Rat serum samples are stored at -20 "C. A.3.0 ASSAY PRINCIPLE PFOS and its internal standard, Tetra-H-PFOS, are extracted from rat serum samples (50 pL)using a liquid-liquid extraction procedure. The organic layer is evaporated to dryness and then reconstituted. An aliquot is then analyzed by turbo ion spray liquid chromatography/mass spectrometry (LC/MS) in the negative ion mode, A.4.0 COMPOUNDS PFOS: Lot##171,99.976% purity, 3M,Inc., Minneapolis, MN. Tetra-3-PFOS: Lot# 59909,90% purity, 3M, Inc., Minneapolis, MN. A.5.0 CHEMICALS All chemicals may be substituted with that of an equivalent manufacturer and grade of chemical. 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES. INC. Page 38of50 99VDJAOl.MI.DOC Page 159 BACK TO MAIN 3M Medical Department Study: T-6395.14 Acetonitrile Ammonium Acetate Ethyl Acetate Methanol Rhodapex mass calibration solution Sodium Bicarbonate Sodium Carbonate, Annhydrous TetrabutylammoniumHydrogen Sulfate Water Rat Serum Rat Serum Analytical Report: FACT TOX-111 LRN-U2994 Cat# 015-4, Burdick & Jackson, Muskegon, MI 494.42 Cat# 24,019-2, Aldrich, Milwaukee, WI 53201 Cat# 100-4,Burdick & Jackson, Muskegon, MI 49442 Cat# 230-4, Burdick & Jackson, Muskegon, MI 49442 A mixture of 0.1 1mM trifluoroacetic acid (J.T. Baker, Phillipsburg, NJ OSSSS), 0.52 mM Rhodapex CO-436,l mM ABEX EP- 110 and 1mM ABEX EP-120 (all three supplied by Rhone-Poulenc, Inc., Cranbury,NJO8512)in 1:l methano1:water. Cat# 3509-01, J.T. Baker, Phillipsburg, NJ 08865 Cat# 3604-01, J.T. Baker, Phillipsburg, NJ 08865 HPLC Grade, Cat# 5360-07, J.T. Baker, Phillipsburg, NJ 08865 High Purity (NANOpure), Barnstead Model D7331Ultrapure Water System, Dubuque, LA 52001 Lampire Biological Laboratories, Piperville, PA 18947 Harlan Bioproducts for Science, Inc., Indianapolis, IN 46229 A.6.0 MATERIALSAND EQUIPMENT Mass Spectrometer PE SCIEX API 365 atmospheric pressure ionization tandem triple quadrupole mass spectrometer equipped with TurboIonSprayTMinterface, PE SCIEX, Concord, Ontario L4K 4V8 Data system A P I Standard Software,MacQuan v 1.4, LC2Tunev 1.3, MacDAD v 1.3, Bundler v 1.3, Multiview v 1.3, ADVANCED BIDANALYTICAL SERVICES, INC. Page 39 of 50 99MJA01 .MLDOC 3M Environmental Laboratory Page 160 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 HPLC Pump LC Pump Controller Auto sampler HPLC Column Harvard syringe P U P 11 Multi-tube vortexer Balance pH Meter Mechanical shaker NANOpure-UV water purification system Microbalance Micro weigh boats Weigh boats S o r ~ a lRl T-6OOOD reiligerated centrihge Beckman GS6KR RefXgerated centrifuge and Sample Control v 1.3, on a Power Macintosh, PE SCIEX, Concord, Ontario L4K 4V8 Shimadzu LC-1OAD pump, Shimadzu Co., Columbia, MD 21046 Shimadzu SCL-lOA, Shim2 1046 Co., Columbia, MD Waters 717plus,Waters Associates, Millipore Corporation, Milford, MA 01757 Betasil C,*,5 pm particle size, 2.1 x 50 mm, Cat #852055-701,Keystone Scientific, Inc., Bellefonte, PA 16823 HarvardApparatusInc., South Natick, MA 01760 Cat# 58816-115,VWR Scientific,West Chester, PA 19380 Model FX-300, AND Ltd., Tokyo,Japan , Model 340, Coming Inc., Coming, NY 14830 Cat# 6000, Eberbach Corp., Ann Arbor, MI 48106 Bamstead, Dubuque, IA 52001 Model MT-5, Mettler-Toledo Inc., Hightstown, NJ 08520 Cat# 0219-0041, Perkin-Eher Corp., Norwalk, CT 06859 Cat# 12577-025, VWR Scientific, West Chester, PA 19380 Cat# 83071, W o n t Co., Wilmington, DE 19898 Beckman, Palo Alto, CA 94304 3M Environmental Laboratory ADVANCED BlOANALYTlCAL SERVICES. INC. Page40 of 50 99VDJAO1 .Ml.DOC Page 161 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 TurboVap LV Evaporator Pipettes Pipette tips ,Polypropylene tubes Screw-capped Polypropylene vials Screw-capped Polypropylene vials Plug Tite Multipurpose Caps Pyrex volumetric flask Solvent Filtration " Apparatus NylonTitan . Membrane Filters PEEK tubing Liquid Nitrogen , Gastight Glass Syringes Cat# 43750, ZymarkInstruments, Hopkinton, MA 01748 Cat# P-5000, P-1000, P-200, P-100, Rainin Instrument Co., Woburn, MA 01888 RT-20, RT-200, C-5000, CP-25, CP-50, CP-250, Rainin Instrument Co., Woburn, MA 01888 13 mfll x 100mm, Cat# 15070-574, VWR Scientific, West Chester, PA 19380 16.5 x 57 mm, Cat# 60.542, Sarstedt, Inc., Newton, SC 28658 16.5 x 101 mm, Catk 60.541, Sarstedt, Inc., Newton, SC 28658 Cat# 78-127-0019-100, Elkay Products, Inc., Worcester, MA 01607 Cat# 280141?-10,28014P-25,28014P-100,28014P1000,KimbleKontes,Vineland, NJ 08360 Cat##953781-0000,953751-0000,953753-0000, 953826-0000,953827-0000, KimbleKontes, Vineland, NJ 08360 0.45 pm Scientific Resources Inc., Cat# 74547-NNY Eatontoyn, NJ 07724 0.005" i.d. x 1/16" o.d., Cat# 1535, Upchurch Scientific Inc., Oak Harbor, WA 98277 Supplied in-house h m bulk tank, BOC Gasses, Buffalo, NY 14210-2005 Cat# 1750, Hamilton Company, Reno, NV 89510 Other general laboratory glassware and supplies were used. 3M Environmental Laboratory ADVANCED 8I OANALYTICAL SERVICES, INC. Page 41 of 50 99VDJA01.MI.DOC Page 162 BACK TO MAIN 3M Medical Department Study: T-6395.14 1 Analytical Report: FACT TOX-111 LRN-U2994 A.7.0 PREPARATION OF SOLUTIONS NANOpure water (1 8 megaohm-cm.) or equivalent should be used wherever water is called for. Mix all solutions well. k 7 . 1 Preparation of Analytical Standard Stock Solutions Analytical Standard Stock Solution, PFOS (1 mg/mL): Weigh approximately 5 mg of PFOS (after correction for purity) on a microbalance and transfer it to a polypropylene vial. Dilute with the appropriate volume of methanol to yield a 1 mg/mL solution. Prepare a fresh solution every three months. Store the solution at 4 "C and bring to ambient temperature before use. Standard Spiking Solutions Prepare Standard Working Solutions A through J in 5-mL. class "A"volumetric flasks according to the following dilution scheme. Dilute to the 5-mL, mark with water to yield the find concentration. Prepare fi-eshsolutions every three months. Store the solutions at 4 "Cand bring to ambient temperature before use. Standard Working A B C D E F G H I J Volume Spiked (PL 1 1000 800 400 200 100 250 I25 62.5 500 250 Solution Used Stock Stock Stock Stock Stock 200 &mL (A) 200 PdmL (4 200 P ! d d (4 10 P ! d d (F) 10 PdmL 02) PFOS Final Conc. (WmL) 200 160 80 40 20 10 5 2.5 1.0 0.5 3M Environmental Laboratory ADVANCED I3IOANALYT ICAL SERVICES, INC. Page 42 of 50 99MJA01.MLDOC Page 163 BACK TO MAIN 3M Medical Department Study: T-6395.14 ~ I Analytical Report: FACT TOX-111 LRN-U2994 A.7.2 Preparation of Internal Standard Stock Solutions Internal Standard Stock Solution, Tetra-H-PFOS (1 mg/mL): Weigh approximately 5 mg of Tetra-H-PFOS on a microbalance and transfer it to a polypropylene vial. Dilute with an appropriate volume of methanol to yield a 1m g / d solution. Prepare a fresh solution every three months. Store the solution at 4 "C and bring to ambient temperature before use. A.7.3 Preparation of Internal Standard Working Solution Internal Standard Working Solution (4 &mL Tetra-H-PFOS): Add 400 pL of Internal Standard Stock Solution to a 100-mI, class "A"volumetric flask. Dilute to the mark with water to give a 4 &mI, Tetra-H-PFOS solution. Store the solution at 4 "C and bring to ambient temperature before use. Prepare fresh solution as needed. A.7.4 Preparation of Standard Curve and Control Blank Prepare fresh calibrationstandards for each analyticalrunby combining 360 pL of rat control serum and 40 pL of the analytical standard working solution (see table below) in labeled I .7-mL microtubes. Prepare control blanks and zero samples by combining 360 pL,of rat control serum with 40 pL of water. Vortex each for 60 seconds. 3M Environmental Laboratory ABDIOVAANNACLEYDTICAL SERVICES, INC. Page 43 of50 99VDIAO1 .MJ.DOC Page 164 BACK TO MAIN 3M Medical Department Study: T-6395.14 I Analytical Report: FACT TOX-11I LRN-U2994 Analytical Standard Dilution Table: Standard # 10 9 8 7 6 5 4 3 2 1 Volume Spiked @L) 40 40 40 40 40 40 40 40 40 40 Sofution to use A B C D E F GH I J pL control serum 360 360 3 60 360 3 60 360 360 3 60 3 60 360 Final Conc. (PdmL) 20 16 8 4 2 1 0.5 0.25 0.1 0.05 A.7.5 Preparation of Other Solutions 0.25 M Sodium Carbonatd0.25 M Sodium Bicarbonate: Weigh 26.5 g of sodium carbonate and 21 g of sodium bicarbonate and dissolve in approximately 900 mL of HPLC-grade water. Transfer the solution to a 1000-mLvolumetric flask and dilute to the mark with HPLC-grade water. Mix the solution thoroughly. Store at ambient temperature. Prepare a fi-esh solution every three months. 1M Ammonium Acetate: Add 7.7 g of ammoniumacetateto a 100-mLvolumetric flask, dissolve in NANOpure water, and stir until completely dissolved. Dilute to the mark with NANOpure water. Store at ambient temperature. Prepare a fresh solution every three months. 2 mM Ammonium Acetate: Combine 2 rnL of 1M ammoniumacetate and 900 mL NANOpure water in a 1000-mL volumetric flask; stir until completelymixed. Dilute to the mark with NANOpurewater. Store at ambient temperature. Prepare a h s h solution every three months. 3M EnvironmentalLaboratory ADVANCED BIOANALYTICAL SERVICES. INC. Page 44of 50 99VDJA01.MI.DOC Page 165 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-I 11 LRN-U2994 10 M Sodium Hydroxide: Add 40 g of sodium hydroxide to a 100-mL volurnetnic flask, dissolve in NANOpure water, and stir until completely dissolved. Dilute to the mark with NANOpure water. Store at ambient temperature. Prepare a fresh solution every three months. 1 M Sodium Hydroxide: Add 10 mL of 10M sodium hydroxide to a 100-mL volumetric flask. Dilute to the mark with NANOpure water; stir until Completely mixed. Store at ambient temperature. Prepare a .freshsolution every three months. 0.5 M Tetrabutylammonium Hydrogen Sulfate, pH 10: Weigh 16.98 g of tetrabutylammoniumhydrogen sulfate and dissolve in 40mL of NANOpurewater. Adjusted the pH to 10.0with 10M and 1M sodium hydroxide. Transfer the solution to a 100-mLvolumetricflask and dilute to the mark with NANOpurewater. 10:90 Methanok2 mMAmmonium Acetate (Mobile Phase Eluent A): Add 100 mL of methanol and 900 mL of 2 mMammonium acetate to a 1000-mL Pyrex bottle. Mix the solution thoroughly and filter the solution through a 0.45 ph4 filter with a vacuum flask.Store at ambient temperature. Prepare a fresh solutim every three months. 9O:lO Methanok2 m M Ammonium Acetate (Mobile Phase Eluent B): Add 900 mL of methanol and 100niL of 2 mM ammonium acetateto a 1000-niLPyrex bottle. Mix the solution thoroughly and filter the solution through a 0.45 pM filter with a vacuum flask. Store at ambient temperature. Prepare a fresh solution every three months. A.8.0 PREPARATION OF QUALITY CONTROL SAMPLES A.8.1 Preparation of Quality Control Stock Solutions QC Stock Solution, PFOS (1 mglmL): Weigh approximately 5 mg of PFOS (after correction for purity) on a microbalance and transfer it to polypropylene vial. Dilute ADVANCED BIOANALYTICAL SERVICES, INC. Page 45 of 50 99VDJAO1 .MI.DOC 3M Environmental Laboratory Page 166 BACK TO MAIN 3M Medical Department Study: T-6395.14 I Analytical Report: FACT TOX-111 LRN-U2994 with the appropriatevolume of methanol to yield a 1mg/mL solution. Storethe solution at 4 "C and bring to ambient temperature before use. Prepare a fresh solution every three months. A.8.2 Preparation of Serym QC Samples QC4 (Dilution QC, 100 pg/mL PFOS): Add 1000 pL QC Stock Solution to a 10mL class "A" volumetric flask to yield a lOO-pg/mL solution. Dilute to the mark with control serum, cap and mix.Aliquots of approximately 0.2 mL are placed in polypropylene vials and stored frozen at -20 OC. QC3 (18 p g / d PFOS): Add 450 pL QC Stock Solution to a 25-mL class "A" volumetric flask to yield a 1S-pg/mL solution. Dilute to the mark with control serum, cap and mix. Aliquots of approximately 0.5 mL are placed in polypropylene vials and stored fiozen at -20 "C. QC2 (6 pg/mL PFOS): Add 150 1-11.of QC Stock Solution to a 25-mL class "A" volumetric flask to yield a 6-pg/mL solution. Dilute to the mark with control senun, cap and mix. Aliquots of approximately 0.5 mL are placed in polypropylene vials and stored frozen at -20 "C. QCl (0.2 p g / d PFOS): Add 111.1 pL of QC3 to a IO-mL class "A"vo1umetric flask to yield a 0.2-pg/mL solution. Dilute to the mark with control serum, cap and mix. Aliquots of approximately 0.5 mL are pIaced in polypropylene vials and stored frozen at -20 "C. A.9.0 LIQUID-LIQUID EXTRACTION PROCEDURE 1. Prepare fiesh calibration standards for each run according to Section A.7.4. 2. Thaw QC samples. 3. Prepare QC4 samples (diluted 1in 10)in two steps: a) Add 180 pL of control serum and 20 pL of QC4 into 1.7-mL microtubes and 3M Environmental Laboratory ABDIOVAANNACLEYDTICAL SERVICES. INC. Page 46 of 50 - 99VDJAOI .MI.DOC Page 167 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 vortex. b) Aliquot 50 pL of diluted QC4 into labeled 16 x 100mm polypropylene tubes. 4. Aliquot 50 pL of each control blank, zero sample, calibration standard, and quality control samples 1-3 into labeled 16 x 100 mm polypropylene tubes. Standards and blanks are analyzed in duplicate. All QC samples are analyzed in replicates of five. 5. Add 1mL of 0.5 M tetrabutylammoniumhydrogen sulfate, pH 10to each tube. 6. Add 2 mL of 0.25 M sodium carbonate/O.25M sodium bicarbonate to each tube. 7. Add 500 pL of the Internal Standard Working Solutionto each tube (except control blank, add 500 pL water). 8. Add 5 mL ethyl acetate to each tube. 9. Shake the tubes on a reciprocal shaker at medium speed for 20 minutes. 10. Centrifugethe tubes at 3000 rpm for 20 minutes at 20 "C. 11. Freeze the aqueousphase in an acetone/dry ice bath and keep the tubes in the bath an additional 5 minutes. 12. Transferthe ethyl acetatelayer to a fresh, labeled 13x 100mm polypropylene tube. 13. Evaporate-the ethyl acetate layer to dryness in a TurboVap'" at approximately 20 OC under nitrogen. 14. Reconstitute the dried extracts in two steps: Add 500 pL acetonitrile and vohex for 60 seconds. Then add 500 j& water and vortex for 60 seconds. 15. Transfer 200 pL of the extract to a labeled polypropylene autosampler Vial. A.10.0 INSTRUMENT CONDITIONS HPLC Condition@: Eluent (gradient) Mobile Phase A 10:90 methanol2 mM ammonium acetate (v/v) Mobile Phase B: 9O:lO methanok2 mM ammonium acetate (v/v) 3M Environmental Laboratory 'ADVANCED BIOANALYTICAL SERVICES. INC. Page 47 ofSO 99VDJAO 1 .MI.DOC Page+ 3M Medical Department Study: T-6395.14 i Eluent Gradient Conditions: Time (min) %B 0 45 1.o 100 5 100 5.5 45% 8 stop BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Analvte RmresentativeRetention Time (minutes) PFOS Tetra-H-PFOS (IS) 1 3.1 2.9 Mass SDectrometer Conditions: Curtain Gas Nebulizer Gas TurboIonSprayTMTemperature TurboIonSprayTMAuxiliary Gas UHP Nitrogen UHP Nitrogen 400 "C UHP Nitrogen at 8 L/min 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 48 of 50 99VDJA01.MI.DOC Page-l-69 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Ions Monitored Analvte PFOS Tetra-H-PFOS (IS) Ion Monitored m/z = 499.0 m/z = 427.0 - Dwell- Time 500 ms 500 ms Ionization Mode Ion Spray Voltage Declustering Potential MS Acquisition Time Pause Time Negative Ion -3000 V -35 v 5 minutes 2 ms Calibratethe mass axis of the instrumentby infusion of Rhodapexmass calibration solution (Section A.5.0)at a flow rate of 10 pL/min. Optimize the sensitivityof the instrument using an inhsion ofa 10 pg/mL solution of the d y t e at 10 pWmininto a flow of 190 pL/min of mobile phase using the gradient condition at which the analyte elutes from the LC column (100% eluent E). Mass spectralpeak widths should be approximately0.6 amu at half-height. One set of calibration standardswas injected at the beginning and one set of calibration standards was injected at the end of each analytical run (tray). A.ll.O CALCULATIONS Calculated concentrationsare based on peak area ratios of PFOS to Tetra-H-PFOS. The peak area for the analyte ion is divided by the peak area for the corresponding internal standard ion. Data generated from samples in this study were acquired and integrated using PE SCEX software applicationsSampleControl (v. 1.3) and MacQuan (v. 1.4). The validation data were formattedin the ABS laboratory informationmanagement system (Watson, v.5.3.1.01). All data were rounded to no less than three significant figures by A B S prior to reporting. ADVANCED BIOANALYTICAL SERVICES, INC. Page 49 of 50 ~ 99VDJA01.MI.DOC 3M Environmental Laboratory Page 170 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-11I LRN-U2994 Acceutance Criteria for Sample aalvsis Calibration standards: The coefficient of determination (3)must be 10.98. At least three-fourths of the individual calibration standard replicates will have deviations within i15% fkom their nominal concentrations. LLQ acceptance criteria: At least one replicate at the LLQ must exhibit a deviation within 120% h m its nominal concentration. If this criterion is not met, both replicates are rejected and the standards at the next higher level are subjected to the same test. Quality control samples: At least two-thirds of the individual QC sample replicates will have deviations *thin 515% fkom their nominal concentrations, with at least one replicate at each QC concentration meeting this criterion. 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES. INC. Page 50 of50 99VDJAO'I.MLDOC ' Page 171 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 BIOLOGICAL SAMPLE ANALYSIS Battelle Study Number: N003296-G 3M ToxicologyServices Protocol Number:FACT-TOX-111 RpBattelle @I& . . . Putting Technology Tu Work FINAL REPORT ORAL (GAVAGE)PHARMACOKINETIC RECOVERY STUDY OF PFOS IN RATS SPONSOR 3M Toxicology Services-MedicalDepartment 3M Center,Building220-2E-02 St. Paul, MN 55144-1000 \3M Environmental Laboratory Testinn Facilitv Battelle Memorial Institute 505 King Avenue COlUmbUS, Ohio 43201-2693 Prepared By Patrick L. South, B.S. Page 172 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX- II1 FINAL REPORT ORAL (GAVAGE) PHARMACOKINETIC RECOVERY STUDY OF PFOS IN RATS Date 3M Environmental Laboratory Page 173 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Battellt: Study Number: N003296-G 3M Toxicology Sewices Protocol Number: FACT-TOX-I I I O W L (GAVAGE) PaARMACOKINETIC RECOVERY 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 Perfluomoctanesdfonate(PFOS)inRat Liver by LCNSMS". Samples were extracted and andyzed by High-Performance Liquid Chromatography Mass Spectroscopy(LCMSMS) for PFOScontentonly. Relatedfluorochemicals mentioned in the analytical method were not monitored. The resdts for the concentrationdetermination of PFOS in the liver samplesfrom this study are attached as appendicesto thisreport. Concentrationsare reported as mass of PFOS (pg) per gram of liver tissue extracted. ... 111 3M Environmental Laboratory Page 174 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number:FACT-TOX-I1 1 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 Standard Preparation Sample weights , Sample homogenization Extraction Sample analysis Audit study file Audit final report Audit final report 09/29/1999 09/30/1999 09/30/1999 10/01/1999 10/01/1999 10111/1999 1011 111999 02/19/200 1 1o/o 1A999 10/01/1999 10/01/1999 10/11/1999 10/11/1999 10/11/1999 10111/1999 0211912001 1011411999 10/14/1999 10/14/1999 10/14/1999 1011411999 10/14/1999 10/14/1999 03/14/200 1 Battelle Memorial Institute iv 3M Environmental Laboratory Page 175 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Batrefle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-11 1 GOOD LABORATORY PRACTICES COMPLIANCE STATEMENT Study Title: ORAL (GAVAGE)PHARMACOKINETICRECOVERY STUDY OF PFOS IN RATS This study was conducted in compliance with the Food and Drug Administration's Good Laboratory Practice Regulations (21 CFR S8), with the exception that the mass spectrometry data for the liver sampleswas collected and processed with the MassLynx s o h a r e system (version 3.1), which was not fully validated. The study was listed on Battelle's Master List of regulated studies. Qv-ULrd4 Jon #. Andre, Ph.D. Battale Principal Investigator A Study Director Y-w-0 I Date V 3M Environmental Laboratory Page 176 3M Medical Department Study: T-6395.14 BACK TO MAIN Analvtical Reoort: FACT - 1 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-1 I 1 Table of Contents Executive Summary .................................................. ...................,.............................................................Piiai ge Quality Assurance Statement....................................I..................................................................................iv TCaobmleploifanCcoenStetanttesm...e.n..t..................................................,........................................................,...................................................................................................................................................... v vi 1.0 2.0 RInetfreordeunccteioSnu..b..s..t.a..n..c..e.s...............................................................................................................................................................................................................................................I1 3.0 4.0 RAencaelyipstisooffSSamampplelse.s.....................................................................................................................................................................................................................................I1.. 4.1 4.2 RSuemsumltsa.r.y...o..f.,M...e..t.h..o..d...............................................................................I.................................,...................................................................................................21 4.2.1 4.2.2 QSaumalpitlyeRCeosnutrltosl...................................................................................................................................................................................................23 5.0 ConcIusions...............,................................................................................................................... 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 Summary Results for Rat liver Sample AnAalpypsiesn..d..i.x..A....(.R..e..s.u..l.t.s.)............................,................A-1 Acceptance CriteriAa Rpepseunltdsi,xCBali(bAracticoenpCtaunrvcee..C..r.i.t.e..r.i.a...R..e..s.u..l.t.s..,.C...a.l.i.b..r.a..t.i.o..n.,C..u..r.v..e..)............,...B-1 Appendix C (Method) Method for Analysis of Potassium Perfluorooctanesulfonate(PFOS)in Rat liver by LCIMSNS.........C-1 RepresentativeChromatograms ......A..p..p..e.n..d.,i.x..D...(..C..h..r.o..m...a..t.o.g..r.a..m...s.)...................,............,......D-1 Protocol, Amendments,AanpdpDenedviiaxtiEon(sP.r,.o.t..o.c..o.l.,,.A..m...e..n.d..m...e.n..t.s..,.a..n...d..D...e.v.,i.a..t.i.o.n..s..)......................E-l Appendix F (PFOSPurity Report) PFOS Purity Report ................................................................................................................ F-1 vi 3M Environmental Laboratory Page 177 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Bnttelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-I 1I Table of Contents Executive Summary............................................................................................................................................. g..g. gg 111 QualityAssurance Statement.............................................................................................................................. iv ComplianceStatement................................................... ........................................................... ......................_..v._. Table of Contents................................................................................................................................................ v i 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 R4.e2s.u1lts..Q...u...a..l..i.t..y..C....o..n..t.r..o..I......................................................:..........................................,...................................................................................................................22 4.2.2 Sample Results ............................................................................................................ 3 5.0 ConcIusions............................................................................................................................................. 3 6.0 Acknowledgements................................................................................................................................. 3 7.0 Specimen Storageand RecordArchives................................................................................................. 3 List of Tables Table 1. Exampleof InstrumentParameters Used to Analyze Samples............................................................. 2 Appendix A (Results) Summary Results for Rat liver SampleAnalysis..........,......................................................._....A-1 Appendix B (Acceptance Criteria Results, Calibration Curve) AcceptanceCriteriaResults,Calibration Curve.. .........................................,..............................B-1 Appendix C (Method) Method forAnalysisof Potassium Perfluorooctanesulfonate(PFOS)in Rat liver by LC/MS/MS................C-1 Appendix D (Chromatograms) RepresentativeChromatograms ..........................................................:.................................D-1 Appendix E (Protocol, Amendments, and Deviations) Protocol, Amendments,and Deviations .....,..............,.......,........................................................E-I Appendix F (PFOS.Purity Report) PFOS Purity Report ......................................................................................................................F-l vi 3M Environmental Laboratory Page 178 3M Medical Department Study: T-6395.14 BACK TO MAIN - Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M ToxicologyServicesProtocol Number: FACT-TOX-1 I I 1.O Introduction This report presents a descriptionof the method used to analyze PFOS in rat liver samples from 3M Study Number FACT-TOX- I 1 1 and the results &om this analysis. See Appendix E for a copy of the study protocol, amendments, and deviations. 2.0 Reference Substances The analytical reference substancefor this studywas potassium perfluorooctanesulfonate(PFOS)lot number 171, suppliedby 3M. Note that based on informationsupplied to BattelIe&om 3M, PFOS has two equivalentnames. The name appearingon the Material SafetyData Sheet and bottle label is potassium perfluoroalkyl sulfonate. The name more commonly used by 3M in analyticalmethodsand correspondenceis potassium perfluorooctanesulfonate. The latter name will be used in this report. See Appendix F for purity data supplied by 3M to Battelle. The surrogate standard was lH,IH,2H,2H-Pertluorooctane sulfonic acid, lot number 59909, supplied by ICN. 3.0 Receipt of Samples SampIeswere receivedfrozen and intact at Battelle, fiom 3M EnvironmentalTechnologyand Services, in one batch on August20,1999. Sampleswere generatedby Argus Researchunder protocol numb.3418-015. The sampleswere stored at approximately-20C. 4.0 Analysis of Samples 4.1 Summary of Method Sampleswere analyzedby a previouslyvalidatedmethod (Battelle studynumber N003604-A) in one batch. The currentversion of the method is attachedto this report in Appendix C. Sampleswere analyzed by LC/MS/MS, and an exampleofthe instrumentparametersis listed in Table 1. Note that only PFOS itself (and the surrogate)was quantitated. The other related fluomchemicals were added to the stock solutionsbut not monitored. Quadraticregressionsweighted Ilx were used to constructthe calibration curves. The run order consistedof system suitabilities, 3 calibrationcurves analyzed at the start, near the midde, and at the end of the run and quadruplicate QCs interspersedthroughoutthe run. The various recovery sampleswere also interspersedthroughoutthe run. 1 3M Environmental Laboratory Page 179 3M Medical Department Study: T-6395.14 BACK TO MAIN -. A...n.-a..l.ytic_a..-l Report: FACT TOX-111 LRN-U2994 Battelle Study Number: NOO3296-G 3M Toficology ServicesProtocol Number: FACT-TOX-1 I I Table 1. Example of Instrument Parameters Used to Analyze Samples 4.2 Results 4.1.I Quality Control System suitability acceptancecriteria were establishedduring the method validation and are riicludcd in Appendix C,Section IXAcccprunce Criteria. Relevantstatistics from the sampleset are proi idcd in Appendix B. Representativechromatograms are given in Appendix.D. 2 3M Environmental Laboratory Page 180 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology ServicesProtocol Number: FACT-TOX-I11 4.2.2 Sample Results `Theresults of the sampleanalysesare presented in Appendix A. The limit of quantitation is definedas the concentrationof the lowest standard which meets acceptance criteria for accuracy (25% RE). The notation BLOQ denotes "BelowLimit of Quantitation"for samplesthat had concentrationslower than the theoretical concentrationfor the 0.13 pg/g calibrationstandard. The "Corrected PFOS Conc" presented in the results tables is the concentration found for the diluted sample multiplied by its dilution factor (final volume ) sample homogenate volume). The method detection limit (MDL) of PFOS was calculated in Battelle study N003296-F to be 0.0173 pg/g from the analysis of 7 replicate preparations of 0.13 pg/g calibration standard. The MDL was calculated by multiplyingthe standarddeviation ofthe found concentrationsof the 7 reps by 3.143; the Signal-to-Noise (S/N)ratio was calculated by dividing the mean found concentrationof the 7 reps by their standard deviation. 5.0 Conclusions The analysis met all acceptancecriteria except for dilution recovery. The average dilution recovery was 133.3%. See Appendix E for deviation report. 6.0 Acknowledgements Acknowledgement of principal contributorsparticipating in the perfomawe of thisstudy at Battelle is presented in the followinglist. Participant Title Jon C. Andre, Ph.D. Richard W. Slauter,Ph.D.,D.A.B.T. Patrick L. South, B.S. Gerke H.van der Zwaag, M.S. Battdle Principal Investigator Senior Program Director Mass spectroscopist Chemist 7.0 Specimen Storage and Record Archives See Appendix E, protocol amendment 2 for records archival information. All residual liver samples, extracts, and unused test article will be disposed of or returned to the Sponsor as directed by the Sponsor. 3 3M Environmental Laboratory Page 181 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical R- eport: FACT TOX-111 LRN-U2994 Battelle Study Number:N003296-G 3MToxicology ServicesProtocol Number: FACT-TOX-1I I APPENDIX A -RESULTS 3M Environmental Laboratory Page 182 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 BattelleStudy Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-I I 1 RAT LIVER SAMPLE RESULTS STUDY NO032964 ANALYSIS DATE AND INSTRUMENT ID: DATA ENTERED SPREADSHEET SOFTWARE: 010ct99; 9053 Electronlcally Excel 97 FINAL LIVER SAMPLE RESULTS Animal numbers endlng In "P" lndlcato poled fetal liver Animal Number Dose Grp mglkglday Corrected PFOS Cone W g ) Note if Sample Is a Rerun Note if Rerun lo Needed BLOQ = BELOW CONC OF LOWEST STANDARD IN CALIBRATION CURVE A- 1 3M Environmental Laboratory Page 183 3M Medical Department Study: T-6395.14 BACK TO MAIN - Analytical Report: FACT TOX-111 LRN-U2994 Bnttelle Study Number: N003296-G 3M Toxicology ServicesProtocol Number: FACT-TOX-I I 1 APPENDIX B-ACCEPTANCECIUTERIA RESULTS,CAIJBRATION CURVE 3M EnvironmentalLaboratory Page 184 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-t 11 oe 1moorConc (&I * l.IlIW1 3M Environmental Laboratory Page 185 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 . . . . . . . ....................... Compound 2 name: PFOS Coefficient of Determination: 0.992024 Calibration cuwe: -0.00950557 xA2+ 1.99272 x + -0.07901 I1 Response type: Internal Std ( Ref 1 ), Area ( IS Conc. I IS Area ) ,Curvetype: 2nd Order, Origin: Exclude, Weighting: l/x, Axis trans: None 27.7 i ................................ X Response -0.0790 ( 3 EnvironmentalLaboratory Page 186 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Bntteile Study Number: N003296-G 3M ToxicologyServices Protocol Number: FACT-TOX- I I I APPENDIX C-METHOD 3M Environmental Laboratory Page 187 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Baaelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-I 11 METHOD FOR ANALYSE OF POTASSIUM PERFLUOROOCTANESULFONATE(PFOS) IN RAT LIVER BY LC/MS/MS - Version 1.0 Study No.: AaalystlDate: Revisions to the method m* L C I Approvedby: J&E.Andre, Ph.D. Date: M&ger, BionaIytical Chemistry ? T , pv 3M Environmental Laboratory Page 1 of 16 c-1 Page 188 BACK TO MAIN 3M Medical Department Study: T-6395.14 - Analytical Report: FACT TOX-111 LRN-U2994 BafleNe Study Number:N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-I I 1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE(PFOS) IN RAT LIVER BY LClMSMS Venian 1.0 - Study No.: - AnalysUDate: L II. m. Iv. 2: V. SUMMARY The exbadon and analysisof potassium perfkumoctanesulfooatc andrelated ftuorwhemids in rat liver is perfomed. W i & o n standards are pnpared by spiking hlauk liverhomogenate with soIvent srandards from twoindependedy-preparedstocks. Thecalibrationstandards are fortified with sunogatestandard, buffered,and emacted with ethyl acetate. The organic p W are evaporatedtodryness and reconstitutedin methanol foranalysis by LCIMSIMS. PuWOSE To extractandanalyzepotassiumperflwmadanesulfonateand relatedfluomchemical compounds fomd in Spragw-DaWlty rat liver. SAMPLES See Chain of Custody words ifappliuble. GENERAL WSI'RUCTXONS Calibrateall rcquindbabcesaccording to the SOP on balancewage. Makeequivalentdilutionswhen thevolume neededvariesfrom thevolume stated in the method Label all standard and reagent solutionsas specifiedintheappropriateSOP.Ifyou intend to rmsea solutionfor futuretasks,be sure the label includesthe prepmion date and study number for which the solution was initiauy prepared. Sign on the finatpage of thismethod to si- that you havefollowed themethod as written, all materialsand reagentsare current,andall equipmentbas k e n properlycalibrated.If you deviatefrom the method, document the change,and obtain the approval of the unit manager, snuty director. or task leader as soon as possible. Initialanddareall data entrieson the page on which they were made. Ifonly one person entersall data on a single day, thedocumentation may be madc in asinglelocation on that page. If multiple staffmake enbies. the additionalentriesmust be hitided and dated by the pcrson making tho entry. LineoutsOTNA denotes"Not Applicable". The method is written in gene& chronologicalorder,but thescqwnce of steps may be altered if theanaiyst deems it apppdate. unlesstheorder for Cenainactivitiesis specified. Stockswill be used for thedurationof the study unlessconsumedor unlessstabiity is considered suspect. No correction will be made for purity or salt content of any test article but PFOSAA. Use glass volumetric. Eppendorfrepcater, or psitivedisplawnentpipets for dispensing methanolic solutions Contact with Teflon by the test article should be midmizcd. MATERIALS Set Table I for all reqired chemicals, reagents. and solvents. UseTabie 1for documentation Checkall labels c u e ~ l l yto ensure b tall materialsare not expired and that they are theproper purity or grade. Page 2 of 16 c-2 3M Environmental Laboratory Page 189 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-I11 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PPOS)LN RAT LIVER BY LC/MS/MS - Version 1.0 Study No.: AnmlystlDate: Tablel. Matcriah I I AmmoniumAcetate. NH&H,& II Mobilephase I Sodium Hydroxide. ReagentPnp NaOH Tetrabutylammonium Extract Prep Hydrogensulfate I I I RT I I RT RT NazCO, Sodium Bicarbonate. NaHCO, Ethyl Acetate Methanol Milli-QWatu pH 7 Bu&r pH 10 Buffer E m t Prep -Rep Mobile Phasc, stocks. ws RcagentRep. Mobile Phase pH meter calibration pH meter calibration , Millipon RT RT RT ASTMTypeI RT RT RT SaTable2 for all required major pieces of equipment.Usethe table to documentthe actual piece (e.g. make, model) of quipmeor Check calibrationof all quipmcnt requiringcalibmtion(eg. balances) to ensure it is cunrnt. Page 3 of 16 c-3 3M Environmental Laboratory Page 190 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M ToxicologyServices Protocol Number: FACT-TOX-I I 1 METHOD FOR ANALYSIS OF POTASSIUM PERnUOROOCTANESULFONATE (PFOS) LN RAT LIVER BY LC/MS/MS Version 1.0 Study No.: AnaIydDatc: Analytical Baiance Weight Set Pipettor Pipettor Pipettor Pipeaor Vortexcr Freezer (-2ODC) Refrigerator (L-QT) Centrifuge Weigh Strudardsor Reagents Calibrate Balance Pipet Samples Pipet samples Pipet EtOAc extraction phase Pipet Reagmts.WIS Mix samples I Eppendorf Store QCs Blank Liver Ston Buffer, Stocks Phase separation I Repeater 3M Environmental Laboratory Page 4 of 16 C-4 Page 191 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: NO032964 3M Toxicology Services Protocol Number: FACT-TOX-1 I 1 METHOD FOR ANALYSIS OF POTASSIUM PERFCUOROOCTANESULFONATE(PFOS) IN RAT LIVER BY LC/MS/MS Version 1.0 Study Na: AnralystlDnte: VIL PROCEDURE A. Preparation of 2 m M Ammonium Acetate -+ Weigh 0.1500 0.0020 g of ammonium acetateand transfer to a 1OOO-mL volumetric flask. Dissolve thc solidin water and dilutetovolume with water. Solutionmay be wd for one month stored at mom tempemlure. Actual mass of ammonium acetate: Actual final volume: Date of preparation: Study No: B. Preparation O f -29% Sodium Hydroxide Solution Weigh 200 t 2g of sodiumhydroxideinto akaker.Add 500 mL ofWQwater and mix to dissolve. Cool andtransfer toa polyprq)ylene boniefor storage. Solutionmay be stomi for 6 monthsat mom tempemtw. Actual mass of sodium hydmxide: Actual volumeMilli-Qwater: Date of prepamtiom Study No: C. Preparation of -2.9% Sodium Hydroxide Solution Add 10 mLof -29% Sodium Hyb'pxideSolutionto a 100-mLvolumetricflask and dilute to volumc with Milli-Qwater.Transferto a polypropylenebottle for storage. Solution may be stored for 6 months at room temperahue. Actual volume of-29% NaOH solution: Actual final volume: Date of pixpaxation: Study No: I). Preparation of Tetrabutylammonium Hydrogensulfate P A ) Solution, 0.5 M, @E 10) pH Meter Calibration pH buffer: 7 pH baer: IO pwnading:pH reading: Add 169 t 1 g of TBA to -500 mL 0fMilli-Qwater in a b k e r . Adjust the pH to 10.00 s 0.02 using4 5 - 6 0 mL of29% SodiumHydroxi& Solution, dilute to IO00 mL with MilliQ water, and mix. Adjust the pH to 10.00:0.02using -2.9% NaOH and mix. Transferto a polypropylcnc bottle for storage. Solution may be for one month stored at mom tcmpenturc,but the DEmust be checked Drior to eachuse. Adjust to pH 10.0 t 0.02wilh 2.9% Sodium Hydroxide Solutionas necessary. Page 5 of 16 C-5 3M Environmental Laboratory Page 192 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number:N003296-G 3M Toxicology ServicesProtocol Number: FACT-TOX-II 1 METHOD FOR ANALYSIS OF POTASSIUM ' PERFLUOROOCTANESULFONATE(PFOS) RAT W E R BY LC/ltIS/MS Venion 1.0 - Study No.: AnalystIDate Actual mass of TBA: Actu*ll flnai volume: Actual final pH: Date of pnparation: Study No: pH after rechecking and/or readjusting: E. Preparation of 0.25 M Carbonate Buffer Weigh 26.5 t 0.1 gof Soduimcarbonate and 21.0 t 0.1gof scdiumbicsrbonate and hausfcrto the same 1OOO-mtwlumcUicflask.Dissolvethematerials inMilli-Qwater, dilute to volume with MilliQ water,mix, and banskr to a polypropylenebottle for storage. Solutionmay be used for 1 monthwhen storedrcfrguatd Aaual~crfsodi~CarbOMte: Actual mass of sodium biaubonatc: Actual final volume: Date of preparation: Study No: F. Preparation of Mobile Phase i' ComponentA: Mix together600mt of 2 mM ammonium acetateand400 mL of methanol. Solutionmay be used for 1 month when stored 8t mom temperabun. Actual volume of 2 mM ammooium acetate: mL Actual volumeof methanol: mL Date of preparation: Study No: ComponentE: Mix together50 mL of2 mM ammoniumacetateand 950 mL of methanol.Solutionmaybe used for 1 monthw h storedat mom tempuature. Actual volumcof2 mMammoniumacelatc: mL Aclual volumc o? methanol: mt Date of preparation: Study No: G. Preparation of Stock Surrogate Standard and Working Surrogate Standard (WSS) 1. Stcck Surrogate Standard (250,ooO nglmL): weigh 25 t 2 mg of lH, 1% 2H, ZI,-perfluorooctanerulphonicacid and transfer to a 1OO-mL volumetric flask. Dissolve in m:thanol. dilute to volume with methanol, and mix. Store nfrigeratcd, protectedfromW UghL Actual Weight: Actual Dilution Volume: Dare of Preparation: Study No: Page 6 of 16 C-6 3M Environmental Laboratory Page 193 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battellr Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-II 1 iMETHOD FOR ANALYSIS OF POTASSTUM PERFLUOROOCTANESULFONATE(PFOS)KN RAT LIVER BY LCfiISMS Version 1.0 Study No.: AndyWDate: 2. WSS (1000 ng/mL): Dilute 100iuL of stock surrogatestandardto 25 aL with methanol and mix. Actual Volume of Stock Internal Standar& Actual Dilution Volume: Date of Prepamlion: H. Preparation of Calibration Solvent Stocks and Working Standards 1. Solvent stocks Foreachanal@ weigh the specified amountof stantlard( i n d c a t l y weighed as A and B replicates) listed in Table 3 and transfer into separate volumetric tlask. Dissolve in methanol. dilutetovolume with methanol. and mix well. Storenfiigigcnued, protectedh m W light 2. Mixed Solvent Stock Pipet specified amount of each analyticalstandardReplicate A as listed in Table 3 and transferintoa single volumetricflask Dissolve in methanol, dilute to volume withmethanol, and mix well. Ston refkigemted, protectedbornW light R e p a the pmcesswith %plicate B stocks. me mixedsohtontdochwe used toprcpmc!be working&wfmk Date of prepamtion: Study No: 3. workingstandards(WS): . Dilute the mixed stocks and working standmk with methanol as specified in Table 3 andmix well. Date of preparation: - 3M Environmental Laboratory Page 7 of 16 c-7 Page 194 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-II I METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT UVER BY LC/MS@S Version 1.0 - Study No.: AaalyNDak * Weighall analytical stadads to at leastthe nearest0.01 mg. ** Usevolumetricor pasitivcdisplaccmentpipet(s). L Preparation of Calibration Standardsand Blanks 1. Liver homogenate Prepare blank liverhomogenatein bulk by weighing approximately40g of blank liver into a 500 mL Nalgene bottlecontaining200 mL ofMilli-Q water. Grindto a homogeneoussuspension. Aliquot into approx30 mLportions for frozen (approx-2O'C) storage. Actual M a s of Liver: Actual volume of water: Date of prep: SW:. Determinedensity OrcalibratioOlQCmatrix: MIX HOMOGENATE TH0ROUGHI.Y anddeterminothe RL~SSin milligrams of 10 replicateweighings of 1OILportionsdthcTHOROUGHLY MIXED homogenate. M.IX HOMOGENATEIMMEDIATELYPRIORTO EACH ALIQUOT REMOVAL.. 3M EnvironmentalLaboratory 2. Liver Calibration Standards Prepare each liver calibration standardby adding0.45 mL of undiluted liver homogenate (STIR HOMOGENATE WHILE ALIQUOTING) into a IS mL cxcIxtion tube and adding 50 pL of WS or MeOH. Prepare hipticate cal stirndardsand6 blanks. SeeTable 5 for volumes. The dilutedliver density is assumed to be approximately 150 mglmL. Mi.. well. Page 8 of 16 C-8 Page 195 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LKN-U2994 Battelle Study Number: N003296-G ;bl Toxicology Services Protocol Number: FACT-TOX-1 I 1 METHOD FOR ANALYSIS OF POTASSCUI'+I PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER B Y LC/RIS/MS - Version 1.0 Study No.: Analyst/DB: Table 5. Calibration Standards and Blankr Date of preparationofcal stds/blank: J. Preparation of Quality Control Liver Samples (QCs) 1 1. Quality Control Working Staodards Dilute the following sourcevolumes methanol &volumetricflasks aodmix , well. Prepan h h when used. A c W volumes arc in parcnthcses 2. Prepamtion of Quality Control Liver Samples Prepare eachQC inbulk by fillingthevolumetricflaskapproximatelyWfult with undiluted liver homogenate (STIRHOMOGENATEWHILE ALIQUOTING), adding the appmpriate QC WS, mixing, and dilutingto VOlUme with undiluted liverhomogenate(STIRHOMOGENATE ALIQUOTING). MTX THOROUGHLYand dispc~m2.5& aIiquotsinto polypropylene tubes and storeat approximately-2OOC. Date of QC prep: Study: K. Prepamtionof MS Check Standard For System Suitability Pipet 250 crL of WS 2 at -10.000 nglmL and2.5 mL of WSS at -1000 ndmL in methanol into the same SO-mL volumetric flask Dilute to volume with MeOH and mix Page 9 of 16 c-9 3M Environmental Laboratory Page 196 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-I 1 I METHOD FOR ANALYSIS OF POTASSIUM PERFLJJOROOCTANESULFONATE(PFOS)IN RAT LIVER BY LC/MS/MS Version 1.0 Study No.: Anal yst/Date: L. Preparation of homogenizer recovery liver samples To determine the recwery from the homogenization process, rnbomopniztd blank liver will be fortified in duplicate at 3 concentlation levels and homogenized as follows. This needs to be done every day hat homogenization of study samplesis performed. 1. Place approximately0.5 gof unhomogeaizcdblank liver intoeach of 6.I5 mL polypropylenecen!rifi~getubes. Record weights of Liver. 2. Add 100 pL of WS 1.3. and 4 (one WS per duplicatetubes) to prepare fortificationsat approximately4. 0.8, and 0.4 pg/g 3. Multiply the mass of liver ing by 2.5 and addthis msny mL of water. 4. Homogenizeeach liver SampIe. and rirrSe homogenizerprobe with another volumeof water used in step 3. addingrinsetahomogenized sample. 5. Clean homogenimwithMdlH between samples. 6. Cap and vortex homogenate for use in extraction. M. Preparation of Dilution Check Sample I. Place 2.95 mLof undiluted liver homogenate (Sl'IR HOMOGENATEWHILE ALIQUOTING) into a 15 mL extractiontube andadd 50 pL of Mixed Stock A. 2. Dilute 50 pL of step 1 solution (VORTEX SOLUTIONWHILE ALIQUOTING)with 0.45 mL of undiluted liver hotnogenate(STIR HOMOGENATE WHlLE ALIQU-G) in 3.15 mL extraction tubes. 3. This sample should be prepiued for wctrilction only an days when study samples will be diluted and extracted N. Homogenization of study samples 1. Place approximately0.5 g ofunhomogenbred study sample liver into a 15 mL polypropylene tube. Xccord weights of liver. 2. Multiply the mass of liver in g by 2.5 and add this many mL ofwater. 3. Homogenizeeach liver sample, and rinse h o m o g e p~robewithanother volume of water used in step 2, adding rinse to homogenized sample. 4. Clean homogenizerwith MeOHbetween samples 5. Capind vortex homogenate for ILSC in cxbaction. 0. Analysis Standards, Blanks, QCs, and Samples 1. MIX LIVERHOMOGENATESTHOROUGMLYBEFORE ~IQUOTDJG and pipet 500pL of each QC (4 replicatesper Icvel), and othersamplesbeing extracted into 15-mL polypmpytenee m o ntubes. The cal stds and blanks me alreadydiquoted. 2. To the Blanks -IS (3 reps), add LOO of MeOH and vortex. 3. To the Blanks+ IS (3 reps) and to the remaining samples, add 100 pL WSS and vortex 4. Add 0.5 mL of 0.5 M T B A @H 10) to all tubsand vortex briefly. 5. Add I mL of 0.25 M carbonatebuffer and vortex briefly. 6. Add 2.5 mL of ethy1acetate. Placethe 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. Tnnsfer 2 mL of the top organic layer to a clean polypmpylcne tube. Page 10 of 16 c-IO 3M Environmental Laboratory Page 197 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-I II METHOD FOB ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/Ms Version 1.0 - Study No.: AnrlystlDate: 9. Evaporateto dryness undernitrogen at a settingof30C for 4 0minutes. 10. Reconslitutethe midues in 500 4 of metbaaol with vortexing. 11. Syringefifterextractsinto autosamplervialsfor anaiysis. Storevials rcfrgerated (up to 1month) if LCIMSIMSwill notbe performed thc same day. Since 3day mom temperaturecxbact stab~lyw d d~emonstratedduringvalidation, the exrmctsofthe cal stds,blanks, andQCs may be reusedforup to 3 days after theirinitial prepatationifheld at mom tempetature ( w pthe vials ifreusing). Date of cal stdlblank muact prep: Date of QC extract prep: QWW3MP P. LC/MS/MS Analysis 88 1. Use the systemconditionsSPeGified in Table#The conditionswhich are designated may be modified by tbe analyst bpmdutxacceptablepeak shape. c-I 1 3M Environmental Laboratory Page 198 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battelk SGdy Number: N003296-G 3 M Toxicology Services Prorocol Number: FACT-TOX- I1 I METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS Version 1.0 Study Na: AnalysUDatc 42-81 MRM m i t i o n for Sumgate Standard 499>99 MRM transitionfor PFOS 556>78 MRM transition for M-556 570s169 MRM transition for MS70 -- 584>169MRM transition forPFOSAA 49S-78 MRM transition for PFOSA 5 2 e I 6 9 MRM kansition for PFOSEA * l l r n i n ~ ( min) ss: 4 min L m i n ) PFoS4.3 m i n L m i n ) . M-556 4.5 min min) MS70: 4.6min min) PFOSAA.4.7 min min) PFOSA 5.1 min min) PFOSEA 5.fmin ( min) * Panmetersthat may be changed by the analyst. Actual values in ( ). 2. The aboveconditionsshouldbt suitablefor the Minomass QuattroLC (S/N 9053). Modificationsmay be necusary if anotherMicromess Quam0 Series spearometcr is used. Splittheflow postcolumnvia aKeystone BIO-tcc or similardevice. 3. Calibratethe massspectrometerusinga suitablerefercna compound, or verify thnt the calibrationis suitable by visual inspeaion (onthe tune page) thata suitable mobde phasc ion is stillaccllratelydetermined.Resolutionmay necdto be higher than that used for analyzing samples 4. To check the proper performawof theinsbumenSinject the instrumentcheck standard. 'Ihe resultsshouldkcomparableto a m n tirjedion if available. 5. Use an automatedcluamatoesaphy integrationsoftwaresystem to collect the output from rhe aualysis. 6. LoadingOrder Seethe loadingreportfromthe automatedchromatography integration wftware system. 7. Make single injections of each cal staadard QC. study sample, or blank Make at least4 injections of the inseument&e& staadard. 8. Run set sizes should typically not exceed 80 injectionsdue to instrument response mll-off considerations. Longer rims may be performed but they pose a riskofyielding uaacceptablecwe results. VKU CALCTOlLATIONS 1. SprcadshcctSoftwam Version 2. MS Adysis S o f l m : Version 3. CalcuIate the av- density of the liver homogenate (IO reps) in mdd. 4. Using the avenge density of the homogenate, calculate its liver density (mg of liver per mJ., of diluted homogenate): Undilutedliver density (mg/nL) = (g of liver x average density of homogenate)& of liver + g of water) where g of liver and gofwater an masses uscd to p n p bulk homogenate; density of water is assumedto be I g/mL. Diluted liver density (mg/mL) = Undiluted density + Diln Famor Page 12 of I6 42-12 3M Environmental Laboratory Page 199 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battclle Shidy Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-II t iMETHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) M RAT LIVER BY LC/&fs/Ms Version 1.0 - Study No.: AnaIysuDate: When diln hctor = 31.8 = I. I 111 to accOunt for 10% dila of liver homogenate incalstdandQCmatrias. 5. Calculate the 4concentration(ndmL) of PFOSand otherfluomchemicalsin the suspmslons of calibrationEtandardsand QCsby using the mass of analytcs and dilution facton only (no liver density comction). Use purity comctionfor PFOSAA only. 6. Calculate the actualconcentrationofPFOSand other fluomhemids in liver for the caliblationstandardsand Qcsasfollows: Conc(pg/g) = Conc (ndmLJ + Diluted Liver density (mg/mL) x loo0 mg/g x IO-' &ng 7. Assure that the integmions ofthe peak awasofthe test articlemi surmgatestandard are correct. Flag manualintegrationswhen performed.calculae the exactconcenhationof each liver standard 8. Calculatethe regrrssion equation relating the peak nsponse ratio (test articlelSS)of each calibrationstandard (y-axis) to test article conantration inLiver (x-axis) for PFOS, M-556, M570,and PFOSAA. calculatechc regression equationdating the peak area of each calibrationstandardto test article concentrationin liver for PFOSA andPFOSEk PFOS, M- 556. M570.and PFOSAA arc quantitated by using Ihesurrogatestandard as an inted standard;PFOSA and PFOSEAan amtit&&+'ithout nfcrcncc to the surrogate(md standardcalibrationcurve). Use a quadratic rcgr&n waghtcd Us ,origin excluded, for J all andytcs. 9. Calculatea determinedconcentrationfor each injectionof calibration-dad, Q C and sampleusingthe regression parameten andthe peak raponse ratiosor anas. 10. Calculate the relative error, averagerelative error. standarddeviationand relative standnrd deviation for aU QCs. Calculatethedative error for each injectionof calibrationstandard. 1I. Calculatethe avenge recovery for the homogenizMwvery fortifications. 12. Calculate the ~ h t i v setandard deviationfor the PFOS to SS peak a m ratio of the replicate injections of the check standard IX. ACCEPTANCECRITERIA A. MS Check Standard (System Suitability) At lcast 3 injectionsof the MS Check Stnndard 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-belaverages of the calibntion rtandanls should meet ?befollowing limits: 3M Environmental Laboratory Page 13 of 16 C-13 Page 200 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battclle Study Number: N003296-G 3M Toxicology Services ProtocolNumber: FACT-TOX-I I 1 METHOD FOR ANALYSIS OF POTASSTUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY L,C/MS/MS - - Version 1.0 Study No.: AnalystlDate: Up to 5 calibrationstandard injectionsmay be excludedfkom thecur~ep, rovidedtbatone injection remainsper level. Removal ofan entire level may be done ifapprovalis obtained. If an entire level is moved, the samplesbracketedby thexwnaiaiig calibration range willbe considendacceptable. The calibrationcurve should have a M c i e n t of determinationof 0.97 or beau. The concentmion-levelaverage percent relativeemrs and perceut relative standard deviationsofthe QCs should meet the foUowinglimits: 1: Removalof individualvalues from the QCcalculationsmaybe Line ifaccompanied by a reasonableexplanation(e.&. instrument malfunctionor Dixon's Q test results). If the average determined concentrationfor any QC l m l excccdsthe acceptance limit,the taslcleaderor study directorshouldk notitied The run may be repeated or a portion afrhe run may be consideredacceptable. For wcamplt, ifthe low QC fails thestatalrq&ment$ samples m y be acceptedthathave concenttationsbr;lcketcd by Uie highest calibration standard and a mid-level QC concentration D. Homogenizer Recovery and DiIution Check Samples The average recovery across the 3 levels of homogenizer m e r y samples aswell as that of the dilution check samples should fall within the range of 70-130'YO inclusive. Removal of individualoutliers h m thecalculationsm y be done if accompuedby a reasonable Pxplanation. E. Sensitivity (LOQs) The validated limits of quaatitationare nominally0.13 pglg each.for PFOS,M-556.M570. and PFOSAA. Page 14 of I6 3M Environmental Laboratory C-14 Page 201 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-II I METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LnlER BY LCMS/MS Version 1.0 - StudyNo.: AnaJystlDate: For PFOSAand PFOSEA, thc validated LOQs am nominally0.33 pg/g each. Due to &e n;lhueof the preparationof the calibrationstandards. lowerConcentrationsof PFOSAand ' PFOSEAwill be urried throughthe exmction. These lowerconcenaationvalues will be evaluated with each run K: and may be included in the relpessions if they meet acceptance criteria Ifthey arc indude& study samplewhich are qunnlitated to have comxntratior~~ below the validated level(nominally0.33 p@g)will be appmpriately flagged F. Specificity The method&ers horn cndogeneousmatrix interfermccsat levelssometimesexceeding 20% ofLOQ. The interaptoftha caIibrationcurve appearstooffer somc correctionfor any &aon quantitatiom. Acaptabepcrformanct(error)ofthe lowest used stadad, thenfore, will be considcrcdsufficientevidence that bracketed study samplesare quanti6cdproperly. G. General x. JI . The above acceptance crituiaindicatethat thismahodis capableofproducingoccasional errorsoutside the normal acceptance criteriaof a validated method(1% nonually). When indicated,replicate analyseslessen the impactof thcsc occasionaloutliers. RESULTS See attachedhard copyof spreadsheetor see file on network drive. XI. COlmaNTS 3M Environmental Laboratory Page IS of 16 c-15 Page 202 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protoco[Number: FACT-TOX-I I I METHODFOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE(PFOS) IN RAT L m R BY LC/Ms/MS Version 1.0 Study No.: Aaalystfllate: XIII. SIGNATURES Analysts Technical Review QC Review Date: Date: Date: Date: Date: Date: Date: Date: - - . 3M Environmental Laboratory Page 16 of 16 (2-16 Page 203 3M Medical Department Study: T-6395.14 .. BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Bottellr: Study Number: N003296-G 3M To~icologyServicesProtocol Number:FACT-TOX-I I I APPENDIX D-REPRESENTATIVECHROMATOGRAMS 3M Environmental Laboratory Page 204 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Battelie Study Number: N003296-G 3M Toxicoiogy Services Protocol Number: FACT-TOX-I I 1 100- 4.24 f 499.00 > 99.0 1.39e PFOS Channel %- a ,-0 too % ,,, ..,, Surrogate Channel 0 3.bO 3.f5 4.b0 4.h5 4.50 4.f5 5.b0 5.b5 5.bO 5.fS . . Erne D-1 3M Environmental Laboratory Page 205 3M Medical Department Study: T-6395.14 3attelle 2LC 9053 II1SOIc-to6 Sm (Mn, 1x2) 1 oc BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicotogy Services Protocol Number: FACT-TOX-I II 0.1310UuSL4;LNO0Q03S2t9a6n-Gdard 15 01a c t - I 99 1427:l 0 erator: PL: 2: MRM of !?ChanneIs Es 499.00 99.0 9.476 ?4 '! PFOS.Channel a ,... 100 ... , , , . , , ,-!. , , , ,&,-c,, , . , . . , . .- 4.52ed Surrogate Channel % 1 a 3M Environmental Laboratory Page 206 3M Medical Department Study: T-6395.14 lattelle 1LC 9053 SI101384Sm (Mn, 1x2) 100- BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Baaelle Study Number: NOO3296-G 3M Toxicology Services Protocol Number: FACT-TOX-II 1 oz-oct-I 99 05:01:3 Operator: PL! 2: MRM of 2 Channels ES 4.22 499.00 > 99.0 1.420 PFOS Channel %- i \ . . . . 0 , , . . ,..., I... I ~ ~ c t ~ ~ r n ( 1hx2n)'n , I'7 '9% Surrogate Channel i a n n e l s ES. 427.00 > 81.Ol 6.07e 3M Environmental Laboratory bo 4.55 4.bO 4.h 5.b0 5.hS 5.bO . . Time 5.f5 D-3 Page 207 3M Medical Department Study: T-6395.14 3attelle 2LC 9053 31ifoct25 Sm (Mn, 1x2) Ioc BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Banelle Study Number: N003296-G 3M Toxicology Services Protocot Number: FACT-TOX-II I 10 uL;ANn0im03a2l9163-7G3;50 MKD 01a c t - I99 17:42:5 0 erator: PL: 2: MRM of !Channels ES 499.00 > 99.0 2.30e % I I 0 101 100 PFOS Channel % Surrogate Channel 0 -. . . . . . .1 3.50 3 . h 4.b5 4.50 4.h 5.b0 5.125 5.50 5.fS . . Time D-4 3M Environmental Laboratory Page 208 3M Medical DepartmentStudy: T-6395.14 lattelle aLc 9053 !SI10~279Sm (Mn, 7x2) ioa BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology ServicesProtocolNumber:FACT-TOX-1 I 1 Animal 13756 10 uL; N003296-G; 1.6 MKD 02-0Ct499! 04:WO: Operator: PL! 2: MRM of 2 Channels ES- 499.00 > 99.Ol 5.21- PFOS Channel % a . . . . , , . . ai 10ct79Srn (kn, 1x2)' .o( . . . . I . . . . I . . . , I . , . , I . . . . I . , . , I . . , , ,,,, ,.,. 2: h R M of?! Channels ES- 3.97 427.00 > 6.33U Surrogate Channel 3M Environmental Laboratory . . . . . . I . . . . . . . . . . . . . . . . I . . . . Time .b5 4.50 4.75 5.bO 5.b5 5.50 5.45 D-5 Page 209 3M Medical Department Study: T.-_6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LR- BiittellcStudy Number:N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-II I 100- 4.22 499.00 > 99.0 1.45e PFOS Channel %- 1 oa 1 96: Surrogate Channel 3.34 . a I . Time 3.b0 3.45 4.b0 4.55 4.bO 4.95 5.bO 5.15 5.b0 5.f5 D-6 3M Environmental Laboratory Page 210 3M Medical Department Study: T-6395.14 3attelle 23SLIC- 9053 IqOcf89 Sm (Mn, 1x2) BACK TO MAIN Analytical Report: FAC. T_ T.O. X-111 LRN-U2994 Battelle study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-I1 I Animal 13756P 10 uL; N003296-G;1.6 MKD 02-oct-199 05:59:0 1.24e PFOS Channel 1 oc n Surrogate Channel % 1 3M Environmental Laboratory Page 211 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: NO032964 3M T o ~ i c o l oSe~rvices PrOtoCOl N u k FACT-TOX-I I I J APPENDIX E-PROTOCOL, AMENDMENTS, ANI)DEVIATIONS 3M EnvironmentalLaboratory Page 212 BACK TO MAIN 3M Medical Department Study: T-6395.14 I - Analytical Report: FACT TOX-111 LKN-U2994 AUG. 13. 1999 9:261uI 3MFaadsavlccr - Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-II 1 EHVIBONUENTIL LAB 2 3E 09 NO. 1406 P. 32 :&E:5,33*333l 612 778 6442 PIObW #FACT-TOX-lll SfUdL lide Oral (Gavage) Pharmacokinetic Recovery Study of PF'OS in Rats PROTOCOL ' Date: Yuno 8,1999 3M Environmental Laboratory P@310f10 651 778 6176 PFlGE.832 E-I Page 213 BACK TO MAIN 3M Medical Department Study: T-6395.14 A N . 13. 1999 9:21AM Analvtical qMplt' FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number:FACT-TOX-11I EH'IIPONENTIL LAB 2 3E 09 NO. 1406 P. 33 PIodocd WACT-TOX-jf f Study Identification Oral (Gavage) PhannacokindcRocevery Study ofPFOSin Rats Sponsor Representative , Stu@?Dlmtor study Locrilon(s) In Vivo Testing FWIiiy Ana&&al TestingLehratoty - 3M TOX~OIOSa~V(Yic~a Mcdicrrt DepaamCnt 3M C-:, Building2 2 @ 2 W StPaul,MN 55144-1000 MarvkrT,Case, D.V.M., Ph9. 3M To~cologySaViccs TelQhW 651-733-5180 PaCSimik 651-733-1773 KristenJ. Bansea, PbD. 3MEnvkonmmfa Tcchnotogy and Safety Services Building 2-3E-09 65 1-778-601 8 Argus Research Labaratoriiw. Inc. 905 Sheehy Drive, Building A Horsham,PA 19044 3MEnvironmentallry B ~ i l 2a-3399 935 Bush Avenue S t Paul, MN 55106 3M Environmental Laboratory Page 2 ofI O E-2 Page 214 BACK TO MAIN 3M Medical Department Study: T-6395.14 AUG. 13. 1999 9:21Afd Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-I II SNBIBONUHTAL LAB 2 3E 09 NO. 1106 P. 34 PdOCd #FACT-TOX-Ill Proposed S t u e Timetable Sttidy IniWon Data Study Completion Date Advanced Biouralytid Services, krc. is CztherwoodRotd IthacqNY 14850 B d c MrmorialInstitute 505 King Avenue C O ~ ~OUhioS43201-2693 Tunc 8,1999 August 8,2000 1. m D Y . Orat (gavage) phannacokinctk recovery study of potiusium pcrflnorooctane suKonic acid I (PFO3) in rats. 4 QUAUTYASSUR&WE The 3M E n h eLaboratoryQualityAssuranceUnitwill nview thc protocol and audit study tonduct, data, snd nporrtodettrminecomplisncewith Good Laboratory Practice Standazdsmd with 3M EnvimnmmtrlLaboratoryStandardOperatingProceduru. Tho QA Unit attho sub-eantnet labotafwywillaudit thdrstuayconducf data, andtesnlts report prior to submitting to the 3NEnvironmMalLabOXatOIy. E-3 3M Environmental Laboratory Page 215 BACK TO MAIN 3M Medical Department Study: T-6395.14 AUC. 13. 1999 9:2?A!d LRN-U2994 Battelle Siudy Number: NO032964 3M Toxicology Services Protocol Number: FACT-TOX-I 1 I ENVIRONMENTAL LAB 2 3E 09 NO. 1406 P. 35 6. C b m o L ~ T w C E s 8.1 IdentificationRat livroand sawn, d o r rabbit liver andac1w1traceabilitynumbers will be recordedin theraw data andinoludedinthe dinalreport 6.2 Source Argus Res& &or S@m Chemical 6.3 PhpiCar Destriptson Rat fiver and ;~~ll&m or, rabbitlivermdsaum 6.4 PudCy and S W M y Notrpplicabb 6.5 SWw COIIMWS Proz~aIt -20 "Cf 10 'C OT -50 'C f 10 'C 6.8 Resew Ma#x A portionof the oontsolmatrix will be retained in the 3M archives . for as long 1u the quality of thsprepsrafion afbdn duatiun, but not Soagcr thenten yean followingthe effcctivcdatc dtha finalttslrule (idOpplicablc). 6.7 Dlsposlflon Matriceswill be rrtained at the 3M EnvimnmgltalLabomtow per GLP repkatj~n Cabin matxiccs (fma, urine, and blood) may be disposed after QAU J vcrificstion, 8.8 Sa&@ Precautfons R e k toMSDS for Jlrmicalsused Wear appr~pxia!e laboratoryattire, andfbIlow adequateprecautions forhard@ biologicatmateridsand preparing spnrplcs for analysis. 7. REJVZRENCEMA~~~R~L 7.1 MentHlcPtron Po~mpafluomoctmcsuIfoaatG(PFOS), bt #a 171,215, or 217 (tquivaIent lots) 7.2 Source 3MS~altyChanicala 7.3 Physicel Destriptfon White powder 7.4 Purity and SiabuilyhaityofPFOS is99%or @eater. SWityhasnot been dctermioed. 7.5 &#3g@ CondfUons Robm t ~ p m r t u n 7.6 Resame Mafeda! A ressrve q 1 e born eachbatch ofPFaS uedin thisstudy will be retained 81 long as the quality of the Pnparation affords evaluation, but not longer than ten yan folIowing the effective date of the final test rule (ifapplicable). 7.7 DisposHlon Unused nfacnccmaterialwillbe mtained forwe by thc 3M Enviranmenta Labotatcty and will be discardedwhenthe quality ofpreparationno tonperpffordr avrlurtion. FlLlG 13 ' 9 9 1 1 : 2 1 E-4 3M Environmental Laboratory Page 216 BACK TO MAIN 3M Medical Department Study: T-6395.14 AUG. 13. 1999 9:2W Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3 M Toxicology Services ProtocolNumber: FACT-TOX-I 1 I EWIRONMENTLL LAB 2 3E 09 NO. 1406 P. 36 7.8 SrrOfyfncruilons RefktoMSDS fbrchemicalswed. Wear qpropriate laboratory attire, andfollcw adequatepredons forhzadling biologicalmateriala and preparing.samplesfor analysis. 8. T E S Z S V m Rats were used asthetut 3ystum md wexermincainrdanddosedu dcsaibed in ArgusRcsurrch protocol#415-015. The fitrmlerats will be giventhe tat mffteridor controlonce M y 'beginning42 dayspriorto oohabitatiom d dry 0 ofpresumedg d o n . Seetable 1formote Q 3 a g 'tn. ~ ' .. @.SPECIMENAND 8hMPl.E RE- The 3M EnvironmentalLobaratorywill receive homogeneitysunplea andspecimens of the followingbody t i m e s mdfluidsfromthe indicatedpoints inthe study. All specimam willb packedon dry ice f i r shipping. SeetabIe 2 formom spedmcn in for ma to^^ w Body thadfIuid r~erum-DamandPup Pnimpls UrinCandPeces-DamandPw lpnimalS Liver-DamandPup animals Speclmem Information Collected Erpested# of rpedmcns Dam-Predosc, Daya 7,14, IS,21, 144Dam I apld 22 (PUP-Day 21 Uhp(pa0led) IPn?do.se,D- a~.s7..15._ 21.ond~ I12OUrinSand 120 Pam sw Dam-At theterminationofthe Pup-Dafll 24 Dam 24Pup (pooled) Total numberofexpected specimens:456 Total number of teat mimalr, 16 TotpI M M of~cat101lo$rrls.8 E-5 3M Environmental Laboratory Page 217 BACK TO MAIN 3M Medical Department Study: T-6395.14 A R . 13. 1999 9:28AM Analytical Report: FACT TOX-111 LKN-UZ~W' Battelle Study Number: NO032964 3M Toxicology Services Protocol Number: FACT-TOX-I11 EBVIBOWEIERAL LA6 2 3E 09 NO. 1406 P. 31 fhfoed #FACT-ToX-il? SpccimerW seat to 3M EDviroMlentalLaboratorieswill be receivedand trarkeda c a r d i q to applicable StandadopaptinsProcedures. ~~~ATAQUAUTTOWEMMS The numberof SpiltddUpEoatu,u sof smogata,andinEDrmnti00onother dataquality indicatorsarehclndedin the pllplrtieplmethods. Inaddition,the following criteria will be met: 121 ffneoriiy Iz0.98 122 Limits of deloCtlon/quanfitaffon 12.2.f McdmiDdectionLimit(MDL)for PFOS a) Senrm: 1.75ppb b) L i v e lSppb RUG 13 '99 ll:B2 E-6 Pageeof10 651 778 6176 PAGE.837 3M EnvironmentalLaboratory Page 218 BACK TO MAIN 3M Medical Department Study: T-6395.14 A N . 13. 1999 9:2flAM Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-I I I ERVIlONM(ENTII LAB 2 3E 09 NO. 1406 .'E 38 72.3 724 125 7 26 14.STATISllCALh X L Y S l S Averages andstandarddeviatronr willbe calculatedThc statisticalmahodsthat will be uged.1~8 described below: 74.f M a frimstbmrtfons and anarvsiJ Datawillbereported asthe wwentration (weightkeightorwei&tkof) of PFOS ormetaboliteper tissueor fluid. 14.2 Statistical mna@sis Statisticsurd may includerepsdon aualysiso f co&aticnln o v a time. ands t a d d devialiona CntEIJOtcdfor &a UmcmPntiolU withinm h dose'gmup. Ifncceuuy, Simpleatadsticaltests, such ps Student's t test, maybe appIied to evaluate rtptiSticSr diikmcc 15.R~Po~t A nportcontaining all therrsurtSofthe study willbe p r e p d by the 3M Environmental Laboratory. Ifanalyses are subcootractedto other laboratonca, each hboratory willprepare a reportandsubmitIt to the3M EnvjronmcnMLabontoryforinclusioninthe 3M Emtimamental Laboratoryrcpozt, Each report will include, but notbe limited to,the hilowing, when applicable: 7!%f Name and ad&mof the facility perf& the study 15.2 Datesuponwhich the studywas initiated and wznpletd 3M EnvriOnmentd labontory RUG 13 ' 9 9 11:22 E-7 hge70flO 651 778 6176 PAGE.030 3M Environmental Laboratory Page 219 BACK TO MAIN 3M Medical DepartmentStudy: T-6395.14 AUG. 13. 1999 9:29AM Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M ToxicologyServices Protocol Number: FACT-TOX-I I1 EiiY[ROMENTAL LAB 2 3E 09 NO. 1106 P. 39 75.3 A statemmtof complianceby the StudyDk.ectatahfnssing q cxceptiOrUto Good Labaratory Practice stpndards 73.4 Objectivu aad proccdurm Idtakd hi the r p p r o v e d p t ~ ~in~clu, dingmyc a t s intlleori~protocot 765 The test substanceidendfiationby name, chemical abstractsnumberorcode number, sfrcngth, purity, and compositionOF other appropriatec W d s t i c a , ifprovidedby the Sponaor 15.8 Stabilityandthe solubilityof Ihstest sabstancw onder thscoditions of adminiamtion,ifprovidedby rha Sponsor 75.7 A desaIptionof themetbrdr usedto conducrthe terrt(rr) 75.8 A ducxiptionof the testsrstem 75.0 A d e s a i p t i o n o f a n y c ~ t h o t m o y h r v e ~ ~ t h e q u l loirttyheW@y ofthsctta 1 5 7 U Thc 1111111O f thestudyD b C t O I and the IlomesOfOther SCk&Sb, prOft&OIkdS, aad supmiaory psnonnel involved in the study 75.7 f A descriptionofthe trmbmhm,calrmlations,oropcrptionS~formcdonthc data,asrmrmpryand aulysia of the dyticd chcmiatry data, nndn s t a ~ motfthe conclusionsdrawnfromthe analyses 7S. f2 Statistical rncthodr ured to evaluatethe data, if applicable 15.73 The signed anddated reports of& ofthe individual Scientistsox other profksioaals involvedinthe study, ifapplicable 15.74 Tho locationwhere rawdata andthe finalreportara to be stored fZf5A statement prepared by the QualityAssmace Unit listingthe dates that SNdy inspectiom andauditswere madt, end the dahs of any findhgsreportedto the Study DirtctormdManapement Ifit ianeeeasarytomakecamctionsoradditiomto anport a&it b bmacccptaf,the changeswillbcmsdciathefbrmof~~rmendmartissuedbytheStudyDinctor.Thc amendmentwill clearIy id- tho patt of therep& that isbciug iunded. the reasma forthe pnmdmart,andwillbe signedby thcStudyDirector. fb.f.OCATiONOPRAWbATA, RECORDS, AUOknw. REPORT Originaldata, or ccpiss thereof, will be available at the 3M EnviroumcntalLaboratoryto facilitate audits ofthe study duringitspgrw andbefore acceptanceof the ha! mpart Whm the6nalreport is completed, all originalpapa data, including those items listedbelow, wiUbe retainedinthe archives of 3M EnvinmmtntalLabontgr foratleasta period of time 011 opacified by regulation,and as &abEshed by 3M EnviroMlmtalLabomtoxy StandardOperating Proctdurej. RUG 13 '99 11:2t E-8 3M EnvironmentalLaboratory Page 220 BACK TO MAIN 3M Medical Department Study: T-6395.14 AUC. 13. 1999 9:29itM Analvtical ReDort: FACT TOX-111 LRN-U2994 Battellr Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-1 I I EHVIBONMENTAL LAB 2 31 09 NO. 1406 P. 40 f6.f The followingraw dataand records will be retained inthe study folderinthe study/pmjectarchives spccardnigto 3MEnvironmental Laborawy StandardOpaating pmcedutco: f6.7,7 Approved protocol and ynanrhncnb 16.1.2 Study comspondence ~'6.1.3 Shipping rcconh 76.f.4 Rawdata 16.f.b Appm~tiarrlrepoa(original signedcopy) f6.f.6 Bleemniccopiesofdata 16.2ThefoUowing supporting rscordswill be retainedscpmtdy h m the study folderin the mbima c c d n g to3MEa-entalL.abo- StSaaaniopsatino ProCedUns: f6.21 Trainingrecord6 16.22 Caliitionrecon% 16.2.3 Inskummtm a h i mlop 76.2.4 StandardOpetatipSProdutw, EqmpmtRoceduru, and Methods f7. sPEW&W &7ENTlON Spacimcnswill be maintainedinthe 3 MEnvironmentalLabarato~sp~ecimenarchiveror8 M o d oftime aa spcoifiedby regulationorBI long Ithe qualityofthepreparationaffords evaluation,but not longerthantenyearsM o w i n gthecff#tive due ofthe ffnaltest rule (if applicable),and aa estabtishdby 3M BmrIronmentalLaborataryStandardOperating Procsduns. 78.PROTOCOL.AMENDMENTASND DEViA7YOM Planned &tinges totheprotocolwillbs in the fonn of wlittenpmsndmmtsSignedby the Study Directorand the Sponsor'. Rcpzesentath. Amuatbn~tswlllbe CoOdidCrCd Mpart Ofthe protocolandwill be attached to them pmtoeot All changesbthe pmtocolwill be iodicated inthe finalkport.Any other chrplgos will bc in the form of writtandaviations, signedby the StudyDinctor antifiIedwith tbe raw data. 3M Envhmenial Laboratoty R U G 13 ' 9 9 11:23 E-9 3M Environmental Laboratory Page 9 of to 651 778 6176 PRtE.040 Page 221 BACK TO MAIN 3M Medical Department Study: T-6395.14 AUG, 13. 1999 9:2911M Analytical Report: FACT TOX-111 - L Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX- 1 1 1 EHYIPONMENTII LIB 2 3E 09 IO. 1406 P. 41 .. PmbWWFACT-TOx-f 1.t n i .. 3htEn~montalL~ RUG 13 '99 11:23 E-IO 3M EnvironmentalLaboratory Pep3 1OOf 10 Page 222 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-Uz94 Battelle Study Number: N003296-G 3M Toxicology Services ProtocolNumber: FACT-TOX-I 1 I Study 77th Oral (Gavage) PhiumacokineticRecovery Study of PFOS inRats PROTOCOLAMENDMENT NO. 1 Amendment Date: August 12,1999 Performing Laboratory 3M EnviroamcntalTechnology& Safety Sexvices 3MEnvircmmmtalLaboratory 935 Bush Av-e St.Pad,MN 55106 Laboatory Prqject Identifhtion ET&SS FACT-TOX111 LDRN U2994 E-1 1 3M Environmental Laboratory Page 223 BACK TO MAIN 3M Medical DeDartment Studv: T-6395.14 Analvtical Reoort: FACT TOX-111 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-1I 1 PrOtOd FACT-TOX171 Amendmeni No, 1 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: Section 10.0 and 11.0 list the followingmethods to use for extraction and analysis: FACT-M-1.1 "Extractionof PotassiumPcrflumoctandonateor Other Anionic FluorochemicalSurtimnt from Liverfor AnalysisUsing HPLC-Electrospray~s Spectrometry" FACT-M-2.1 ``Analysis of Fluomchemicals&LiverExtractsUsing HPLC-EIectrospray~ Spectrometry" AMEND toREAD: The anddytkdmahodsFACT-M-1.1 andFACT-M-2.1, respectively, w m updated on07/22/99 to: - ETS-8-6.0 "ExtractionofPotassiumPcrfluorooctanesulfonateor Other Fluorochdcd Compounds&om LiverforAnalysisUsingHPLC-Electrospray/MassSpectromw ETS-8-7.0"Analysis of PotassiumPerfluotooctaneaulfonateor OtherFluomchemical Compoundsin LiverExtmcts Using HPLC-EIectrospray/MassSpectrometry" REASON: The extraction and analyticalmethodsFACT-M-1.1 andFACT-M-2.1, respectively, were updatedon 07/22/99to ETS8-6.0 and ETS-8-7.0. Thesemethodswereupdatedtoreplace ' the extraction solventethyl acetatewitha differentextractionsolventMTBE (methylrertbutyl ether), POAfi and Monoesterwere removed h nthe standard mix, andM556 was added to the standardmix. The analytical methodwas updatedto include linear regression with Ilx weighting and afew minor changes in the HPLC 1100instnmmtparameters. 2. PROTOCOL READS: Section 10.4 and 11.4 Statethat ifthe analyses arc sub-contractedto other laboratoriesan amendmentwill bewrittento includethese methods. AMEND TOREAD: The extraction and d y t i c a l methodsto follow at Advanced Bioanalytical SeMccs will be attached to the protoccI. The extractionand analyticalmethod to fallow at BattelleMemorialInstitute is: "Methodfos Analysisof Perfluomoctane Sulfonate(PFOS) in Rat *by 0 L;* LCIMSIMS, REASONT:he analyticalmethods at the sub-contractlaboratories were not includedin the original pmtocol. 3M Environmental Laborakxy 3M Environmental Laboratory E-12 Page 224 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX- 1 1 I P m W FAC%TOXlll AmendmentNa. 7 3, PROTOCOL READS: Section 12.2.1 b) IistslivermethoddetectionIimit as 15 ppb. AMENDTOREAD: The livermethoddetectionlimit is 8.50 ppb (nglg). RmsON: ThevalidationSupportingmethods ETS-84.0 andETS-8-7.0 includeda lower method detection limit for PFOS. Amendment Approval &vin Case, D.V.M..,PhD., SponsorRepresentative ./w9 Date E-I3 3M EnvironmentalLaboratory Page 225 3M Medical Department Study: T-6395.14 BACK TO MAIN - Analytical Report: FACT TOX-111 LKN-ULYY4 I Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX- 1 1 1 Stuq TIile oral (gavage) P l l a m a c o ~Rcecovery studyof PFOS inRats PROTOCOL AMENDMENT NO. 2 Amendment Date: September30,1999 Performing Labomhny 3M EnvironmentalTedmology & Safety S d c w 3MEsvkbnmmtaloratory 935.BuahAvmus StPaul,MN 55106 Laboratory Project ldenfiAcation ETBCSS FA(3I-TOXlll LIRN U2994 3M EnvironmentalLaboratory E-14 Page 226 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analvtical ReDort: FACT TOX-111 LRN-U2994 Battelle Study Number:N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-I1 I Protocol FACT-TWi 17 Amendment No. 2 This amendment modifles the following portion(s) of me protocol: 1. PRorocoLREADS: Section 2 states the study is designed to determine levels of potassium p ~ u o m o c t a n d o n a t(ePFOS) in spechens of liver and sera in rats. AMENDTO READ: The study is designedto determine levels ofpotassiumpduomctandonatc (PFOS) in s p t c i m ~o f liver, sera,and urine in rats. RCSSONN: .Theurine analyticalmethodsw m validatedand appmed after approvalof the original prowd. 2 PROTOCOL WEADS: Section 10.0 and 11.0 list the followingmethodsto use fir extractionand anslysis: ETS-8-4.1 ''Extradon ofPotassiumPcrfluomctanctanesulfonateor OtherFlaorochdcal Compoundsh m S m m for AnalysisUsing HPLC-EIcctrospray~Spectrometry" ETS-8-6.0 "Extraction ofPotassiumPeduorooctandonate or Other Fluorochdcal Compounds h m Liver fir AnalysisUsing D L C - E l c c h ~ h i a s Ss pectrometry" ETS-8-5.1 "Analysis o f Potassium Perfluorooctanesulfanateor Other Fluorochcmicd Compounds in Saum Extracts Using H P L C - E I c c t r o s p ~ y MS~pactrometry" ETS-8-7.0"Analysis ofPotasshmPcrfluorooctanesulfanatc:or OtherFhorochunical Compounds in Liver Extracts Using HPLC-EI~~spray/MasSapectrometry" AMENDTOREAD:: ETS-8-4.I '?ExtractionofPotassiumP ~ u o r o ~ d o or~OthecrFluomchemical * Compounds E m Saum for AnalysisUsing HPLC-EIectrospray/Mas4Spectromctrf' ETS-8-6.0 ''Extraction of PotassiumPafluorooctanesulfonateorOther Fluomchemical Compounds fimm Liver for Analysis Using HPLC-El-yW Spsctrolnstryn ETS-8-96.0 "Exbactionof PotassiumP e r f l u o m d a e or otherFluomhemid Compounds GpmUriOe for Analysis Using PLC-EIcctrospray/Mass Specttometry/Mas9 Spectrometry" ETS-8-5.1 ''Analysis OfPotassiumP ~ U O ~ O C ~ ~OTOOthIerMF l ~C~ r o c h a n i ~ a l Compoundsin Saum Extracts UsingHPLC-ElectrosprayhiassSpectrometry" ETS-8-7.0 "Analysis of PotassiumP c r f l u o ~ ~ o c t a n ~ noar tOctherFluomchemical Compounds in Liver Extracts Using HPLC-ElechsprayhiassSpcctrometIy" ETS-8-97.0 "Analysisof Potassium Pfzfluorocctanesulfonateor OtherHumchemical Compounds inUrine ExtractsUsingHPLC-ElectrosprayhiassSpectrometrylMass Spectrometry" REASON: The extraction and analytical methodsETS-8-96.0 and ETs-8-97.0,were approved after approval of the original protocol. .- 3M Envlmnmentel Laboratory E-15 3M Environmental Laboratory Page 227 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LKN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX- 1 1 1 .. protoed FACT-70x1 11 Amsndment No. 2 3. PROTOCOLREADS: Section6.1 lists control matrices 83rat fiverand s c ~ omrrabbit Ever and senam. Source Argus and/or S i pChemical. AMEND READ: Controlmatrices idcntitiCation ratliver audserum, rabbit liwrand serum, humanurine d o rraturine. SourceArgus,SigmaChemical,LampirsBiologicals, Biological Specialty Corp.and/orGoldenWest Biologicals. -SON: Additionof controlurine specXcatiom. 4 PRomCO&READs: Section 12.2.1 a) lists saamethoddetectionlimit88 1.75 ppb andb) listslivermethod detection limit as 15 ppb. AMEND~READ: The method detection limitsforall compoundsand d c e s willbe taken &omthemethods usedfor extractionand analysis. REASON: The methoddetectionlimitslistedam specificto the 3M EnviTanmental Laboratory. Statementwas addad to allow for sub-cantractedanalysesand/orrcvisadmdhods. .- 3M EnvimnmcMtal Laboratory 3M Environmental Laboratory E-16 Page 228 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: EA-CT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-I 1 I .. Protocd FACT-TOXl 1f Amendment Na 2 .. 5. PROTOCOL READS: Section 16states thatthe originaldata, or copiesthereof,willbe availableat the 3M Environmental Laboratory to facilitateauditsof the study duringitsprognss and before acceptanceof the finalreport.When the i%al reportis completed,all original paper data, including: approvedprotocoland Bmeflciments, study correspondence,shippingrecords,raw data,approved finalreport, electroniccopies of data, trainingmrds, calibrationmods, instrument maintenancelogs andStaDdard operatingp r o c c d ~e~qu~ip~m, entprocedures,and methodswill be retainedin the archives of the 3M EnvironmentalLaboratory. AMEND TO RCIID: Section 16 states that the on* data, or copiesthem&wilt be availableat the3M EnviromnentalLaboratoryto W t a t a auditsof the studydrpingitsprogress and before acceptanceof the WreporL When the finaI report is completqd, all originid paper data, iuclud@g: approved pFotOc01 and amendments,study correspondenc~s,hippingm&tg,w * data, approvedblreport, and electroniccopies of datawill be ntainedin the archivesof the 3MEnvirOnmental Laboratory. All comspondbgtrainingrecords, calibrationrecords, .. instrument maintenaoCcelogs, standard operatingpmcedrms, equipmentprocedures, and methoda will be retainedin the archi~eos f thefacilily performingeachanalysis. REASON: Clarificationof the dispositionof archivedmrdsif analysesareperformed at a sub- contract laboratory. 6. PROTOCORLEADS: Section 17 states that specimenSwillbe maintainedinthe 3M Environmental Laboratory specimen archives. AMWDTORE~D: Specimenswillbe maintained in the 3M EwiranmentaILaboratory specimen archives. AU SpeCimassent to subcontractlaboratories will be returnedtothe 3M Envhnmental Laboratoryupon compIetionofanalysisand submissionof the sub-contractlaboratory(s) finat report.The specimaW will be returnedwith the following documentation: the signed originalchain of custody and recordsof storageconditionswhile at the sub-contract Wty. REASON: Clarificationof the dispositionof specimensand documentationfor analysespcrfomedby a sub-contract laboratory, 3M Environmental LaboratOry 3M Environmental Laboratory E-17 Page 229 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Repzg-FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protoco1 Number: FACT-TOX-1 I I Amendment Approval PrOlbGdFACT-TOXI I1 Amendment No. 2 3M Environmental Laboratory E-I8 Page 230 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Study Title ' Analytical Laboratory Report on the Determination of the Presence and Concentration of . Potassium Perfluorooctanesulfonate(CAS Number2759-39-3) in the Serum, Liver, and Urine of Crl:CD@BR VAF/Plus* Rats Exposed to PFOS Via Gavage PROTOCOLAMENDMENT NO.3 a Amendment Date: 20 January 2000 Perfonning Laboratories Urine Ana1yse.s 3 M EnvironmentalTechnology and safety services Fluorine Analytical Chemistry Team Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Pe$ortning Laboratories Liver Analyses Battelle Memorial Institute 505 King Avenue Columbus, OH 43201-2693 Serum Analysu Advanced Bioanalytical Services, Inc. 15 Catherwood Road Ithaca, NY 14850 Laboratory Project Identification . ET&SS LRN-U2994 FACT TOX-I I 1 Argus Study: 418-015 ' 3M Medical Department Study: T-6295.14 3M EnvironmentalLaboratory E-19 3M EnvironmentalLaboratory Page 231 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 PrOtOCOl LRN-U2994 Amendment Number 3 This amendment modifies the following pottion(s) of the protocol: 1. PROTOCOL READS: The study director for the present study was identified in the protocol as Kristen J. Hansen,' Ph.0. AMENDTO READ: The rofe of study director for the present study was reassignedto MarvinT. 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 PrincipleAnalytical 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. PROTOCOLREADS: The sponsor for the present study was identified as Marvin T. Case, D.V.M., Ph.D. AMENDTO READ.' The role of sponsor for the present study was reassignedto 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 E-20 Page 232 3M Medical Department Study: T-6395.14 Amendment Approval 9&2- - F John L.ButenhoAf PhD., Sponsor Representative BACK TO MAIN W-A FACT TOX-111 LRN-U2994 Protocat LRN42994 Amendment Number 3 . we& Date 1, A 2 KristenJ Hamen, Ph.D., Outgoing Study Director /f-Gkb-~ Date rea, / b # 1 r L 5 k? M m i n 2: Case, D.KM,PhD., IncomingStudy Director "Date E-2 1 3M Environmental Laboratory 3M Environmental Laboratory Page 233 BACK TO MAIN 3M Medical Department Study: T-6395.14 Ana-l' FACT TOX-121 LRN-U2994 Batteile Study Number: N003296-G 3M ToxicologyServices ProtocoI Number: FACT-TOX-I 1i DEVIATON REPORT Battelle Study Number: N003296-G . 3M Toxicology Services Protocol Number: FACT-TOX-111 ORAL (GAVAGE) PHARMACOKINETIC RECOVERY STUDY OF PFOS IN RATS TYPE OF DEVIATION: PROTOCOL DATE OF DEVIATION: September 28,1999 NATURE OF DEVIATION The source of control matrix will not be either Argus Research or Sigma Chemical as specified in section 6.2 of the protocol. CAUSE OF DEVIATION. HarlanWiIl be the supplier of controlrat livers used to prepare blanks, any standards, and QCs for the analytical portion of the study. IMPACT OF DEVIATION ON STUDY Harlan was used as the control matrix supplier for Battelle's validation of the analytical method (Battelle study number N003604-A). This supplier prodled 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: Date Marvin T. Case, D.V.M.,Ph.D. Study Director 3M Environmental Laboratory E-22 Page 234 BACK TO MAIN 3M Medical DepartmentStudy: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Battelie Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 DEVIATON REPORT Battelle Study Number: N003296-G , 3M Toxicology Services Protocol Number: FACT-TOX-111 ORAL (GAVAGE)PHARMACOKINETICRECOVERY STUDY OF PFOS IN RATS TYPE OF DEVIATION: PROTOCOL DATE OF DEVIATION October 4, I999 NATURE OF DEVIATION: The dilution recovery results for rat liver did not meet the method acceptance criteria of 70430%. This is required in section E.D. of the method and section 12.4 of the study protocol. CAUSE OF DEVIATION: The actual dilution recovery was 133%. IMPACT OF DEVIATION ON STUDY: The remaining method performance tests (calibration curve, QCs, and homogenization recoveries) met their respective acceptance criteria. Apprbximately 2/3 of the study samples were diluted before extraction, and therefore potentially * affected. The,dilution recovery was not considered to be exceedingly high enough, at only approximately 3% above the normal acceptance level, to have significantly impacted the data. CORRECTIVEACTION The dilution recovery data will be presented in the final report. APPROVED BY: n Jon d hdre, Ph.D. Date BattelG Principal Investigator h r v i n T. Case, b.V.M., PbD. Study Director 37444 h i Date 3M EnvironmentalLaboratory E-2 3 Page 235 BACK TO MAIN 3M Medical Department Study: T-6395.14 AnalQ&&&port: FACT TOX-111 LRN-U2994 Batteile Study Number: N003296-G 3M Toxicology Services ProtocolNumber: FACT-TOX-11 1 DEVIATON REPORT Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 ORAL (GAVAGE) PHARMACOKINETICRECOVERY STUDY OF PFOS IN RATS TYPE OF DEVIATION Protocol DATE OF DEVIATION August 20,1999 NATURE OF DEVIATION: Protocol statesBattellewill analyze 24 dam and 24 pup (pooled). Chain ofCustodyRecord indicatesonly 45 sampleswere received. IMPACT OF DEVIATIONON STUDY: The impact of this is minimal to the interpretation of the study outcome. CORRECTIVE ACTION This protocol deviation report was prepared. APPROVED BY: L c *LL-dL Jon C. &e. Ph.D. Battelleydncipal Investigator k 7'6, M b i n T. Case, D.V.M., PbD. Study Director q-zy VI Date 27m 'Date 3M Environmental Laboratory E-24 Page 236 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN42994 Battelle Study Number: NOO3296-0 3M Toxicology Services Protocol Number: FACT-TOX- 111 I - APPENDM F PFOS PURITY REPORT dI 3M Environmental Laboratory Page 237 BACK TO MAIN 3M Medical Department Study: T-6395.14 ETSS 2 3U LRN-U2994 Battelle Study Number: N003296-G 3M Toxicology Services Protocol Number: FACT-TOX-111 P651 778 4226 04/26/99 09:56 fl :02/03 NO:341 3M SPECIM,TY APHESLYES 6r CHEMICALSUYALMlCAL LABORATORY - To; - - a m i Subiacr: RUG Lisa Clemco (8356B) ETbrSS- 2-03-09 Request Cy 57830 Tom Kcrtner (SS633) SAQC Analytkd tab 236-28.1 1 CItrmicalClbnrct.rirdlor of POSF-&und Fluortxhrmica& by 'H-NMR& "F-NMR Spr edrorcop) March 24,1999 Preliminary=port for FC-95(PFOS),lot 171 -FC-95, lot 171(PFOS). TN-A4834 Nomind product4 F i r S O s ( - )K(+) (white powder) I " I 'Ibistnrnple WDI subjected to 'H-NMR and %NMR spcctrat d y w s to duenninathe purity of the nominal product and to characterize 88 m p y impurhy components possible. EXPERlMENTAtr A portion of the sample w s accuratelyweighed, spiked with a b o r n amount of I,bbis(trifluoromcthyI)benzurc (p-HFX), andthen rotallydiwoIved inDMSO4 for subsequent analysisby NMR A 400 MHz 'H-NMR #patrum(# h57830.401) and 8 376 &!?-- spectrum (# f57830.401) wero rtcquired usinglr varian UNlTYplur 4W FT-NMR spcctromtter. Use of the pHFX Inred shndard was intended to permit the dclumination of the absoluteweight percent concentrations of theassigned components wirhout n e c e u d y needing to Identify or quantify d rhe components in the smple mixntre. The combined NMR spectral dnra werc used to assign dl of L c major andmost of the minorcomponentsin thir sampleu received. The qdIt#ive and qtranritatlve compositional rcSulrs tha?werc derived from the single vial N M R infernal standardimtioa M ~ Y S C Sarc summarized inTABLE-1on the following page. I have reponedboth relative andabsolute weight percent concentrations. One possible reasonthat the absolutewt.% values odd up to more than 100% may be due to the fact thai I rtsaumed all of the componentscontained8 carbons. If there wem any shorter chain homologs present (is..7.6. S, CIC. carbons), then the averagem T u r i d molecular weights would have been somewhatless than thore used in the ealculallonr. In general. the V-NMR technique i s na ' particularly well suited for identifying or quantifyings d a ~ ~ ~ oof~dl ot su s fluorochemicdhomologimpurity componenrrunless the chatu M vay shod. A morecompletechanrcudza?Ionof any o t h a fluorochemicd homologswould require anrtyrir by electresprayMS or 8 similartechnique. Additional work wohd berequiredin Meffort to positidy verify the tentativelyassignedcomponentr listed in TABLE-1 (denoted by possible). SmIl umOunts of other unidentified Impuritiesa ~aelso detected in the NMR spectra, but additional work would be required in an efforr to identi& or qunntify these other mnteriaIs. Copies of the NMR spectra will be provided for you at a later date. If you have any questions about the multr in thir initial repoftfor FC-95,h171. p l w e let me know. f apologize forthe delay in completingthis initial work. Tom ICcstncr ?roc.;. F-1 3M Environmental Laboratory Page 238 c 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Battelle Study Number: N003296-G 3M ToxicologyServices Protocol Number: FACT-TOX-1 I I 3M Environmental Laboratory M.2. F-2 Page 239 3M Medicpl Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-I 11 LRN-U2994 RE-ISSUED ANALYTICAL REPORT STUDY TITLE Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats DATA REOUIREMENTS Analytical Method Requirements STUDY DIRECTOR Marvin T. Case,.3M PRINCIPAL INVESTIGATOR Enaksha Wickremesinhe, Centre' RE-ISSUED ANALYTICAL REPORT COMF'LETION DATE November 3,2000 PERFORMING LABORATORY / TESTING FACILITY Centre Analytical Laboratories, hc. (Centre) 3048 Research Drive State College, PA 16801 Phone: 814-231-8032 STUDY SPONSOR 3M Toxicology Services - Medical Department 3M Center, Building 220-2E-02 St. Paul, MN 55144-1000 PROJECT IDENTIF'ICATION SponsorProtocoI Number: FACT-TOX-11I Centre Study Number: 023-017 Total Pages: 92 . 3M Environmental Laboratory Page 240 BACK TO MAIN 3M Medical DepartmentStudy: T-6395.14 Analytical Report: FACT TOX-111 . Centre Study No.:O&@&CJ~ SponsorProtocol No:FACT-TOX-111 GOOD LABORATORY PRACTICE CONPLIANCE STATEMENT Centre Study Number 023-017, entitled "Oral (Gavage) Pharmacokinetic Recovery Study .of PFOS in Rats," conducted for 3M Toxicology Services - Medical Department, was performed in compliance with US FDA Good Laboratory Practice Standards (21 CFR 58) by Centre Analytical Laboratories, Inc. with the following exceptions: 1. The reference substances (analytical standards) used in this study (PFOS and THPFOS) have not been characterized under 21 CFR 58.105 2: The automated data collection systems used in this study are not fully compliant with 21 CER 58.130(e). //EPrniankcsiphaalWInivc egatsoirnhe, Ph.D* Centre Analytical Laboratories, Inc. 3-W'bo Date *Enaltsha Wickremesinhe is no longer employed at Centre, therefore Centre Management, Rick Grazzini will be signing in his stead, $a&in T Case, D.V.M., Ph.D. Date Study Director 3M Toxicology Services 3M EnvironmentalL a b o @ % - eAnalytical Laboratories,Inc. . Page 2 di?E 241 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 SponsorProtocol No:FACT-TOX-111 OUALITYASSURANCE STATEMENT Centre Analytical Laboratories' Quality Assurance Unit reviewed Centre Study Number 023-017, entitled, "Oral (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats". All phases were reviewed for conduct according to Centre Analytical Laboratories' Standard Operating Procedures, the Study Protocol, and all appIicable Good Laboratory ' Practice Standards. All findings were reported to the Study Director and to management. Phase 1 I Protocol Review 2. Extraction & Fortification 3. RawData& Summary Table Review 4. Draft Report Review 5. Find Report Review Date Inspected 5/ 17/00 7/7/00 Date Reported to Centre Management 6/1/00 711 1/00 Date Reported to Study Director and ana ent 7/14/00 7/14/00 7/28,31/00 8/15/00 8/25/00 8/15/00 911 1/00 8/17/00 911 1/00 8/25/00 9111/00 \- u Naomi Lovallo Quality Assurance Auditor A Date Centre Analytical Laboratories, Inc. 3M EnvironmentalLaboratory Page 3 of 92 Page 242 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 CERTIFICATION OF AUTHENTICITY This report, for Centre Study Number 023-017, is a true and complete representation of the raw.data for the study. Submitted by: Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 (814) 231-8032 Principal Investigator, Centre: 3-rJw'dcr Date Centre Analytical Laboratories, Inc. *Enaksha Wiclcremesinhe is no longer employed at Centre, therefore Centre Management,Rick Grazzini will be signing in his stead. Centre Analytical Laboratories, Inc. Facility Management: &Vd& A o h n Flaherty Date J Laboratory Manager Centre Analytical Laboratories, Inc. Study Director, 3M: Ma&n T. Case, D.V.M., Ph.D. 3M Toxicology Services Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 4 of 92 Page 243- BACK TO MAIN 3M Medicpi Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.:023-017 SPOUSPOro~tocol NO:FACT-TOX-I 11 STUDY IDENTIFICATION Oral (Gavage) PharmacokineticRecovery Study of PFOS in Rats SPONSORPROTOCOLNUMBER: FACT-TOX-111 TY'E OF STUDY Oral (Gavage)PharmacokineticRecovery TEST SYSI"i' - TEST MATERML SPONSOR a . Rat Feces Perfluorooctanesulfonic acid potassium salt . (T-6295) . 3M Toxicology Services-Medical D e p m e n t 3M Center, Building 220-2E-02 St. Paul, MN 55144-1000 STUDY DIRECTOR From 6/9/99to 2/10/00 Kristen J. Hansen, Ph.D. 3MEnvironmentalTechnology and Safety Services Phone: (651)778-6018 From UlO/OOto Present Marvin T. Case, D.V.M.,Ph.D. 3 M ToxicologyServices Phone: (651)733-5180 TESTING FACILITY: Centre Analyticd hbomtories, Inc. 3048 Research Drive State College, PA 16801 PRINCIPAL INVESTIGATOR Enaksha Wickremesinhe,Ph.D. Centre Analyticd Laboratories, Inc. Phone: (814)231-8032' ANALYTICAL PHASE. TIMETABLE: Study Initiation Date: ' Study AssignmentDate: Analytical Start Date: ,AnalyticalTerminationDate: 06/09/99 04119/00 07/06/00 07/20/00 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 5 of92 Page 244 BACK TO MAIN 3M ? Medic I Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.:023-017 SponsorProtocol No:FACT-TOX-1 I1 PROJECT PERSONiWL The Study Director for this project was Mavin T. Case at 3M Toxicology Services and the Principal Investigator for this project at Centre Analytical Laboratories, Inc. was Enaksha Wickremesinhe. The following personnel from Centre Analytical Laboratories, Inc., were associatedwith various analytical phases of the study: - Nam-e Enaksha Wickremesinhe Emily Stauffer Karen Smith Tiffany Roctor Rickey Kefler Lawrence Ord Group/Team Leader Scientist Scientist Technician Sample Custodian Sample Custodian Centre Analytical Laboratories, Inc. 3M Environmental Laboraiory .. Page 6 of 92 .. Page 245 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 .. LRN-U2994 Centre Study No 023-017 Sponsor Protocol No:FACT-TOX-I 11 TABLE OF CONTE"S . Paw TITLE PAGE ...................................................................................................................... 1 GOODMORATORY PRACTICE C O M P W C E STATEMEhi .............................2 QUALITY ASSURANCE STATEMENT...................................... ................................... 3 CERTIFICATIONOFAUTHENTICITY........................................................................... 4 STUDY IDENTlFlCATION.............................................................................................5.. PROJECTPERSOPWEL.................................................................................................... 6 . TABLE OF CONTENTS.................................................................................................... 7 LLI3SSTTOOFFFTIAGBULREFSS.............:i................................-..................................................................................................................................................................89 LIST OF APPENDICES ...................................................................................................10 LO SmxMARY ............................................................................................................. 11 2.0 OBJECTlvE..............................................................................................................1..1 3.0 IN'IXODUCTION ...............................................................................................-....11 4.0 TEST SYSTEM ........................................................................................................1.1 5.0 REFERENCEMATERIAL.......................................................................................12 6.0 EXPERIMENTAL,DESIGN......................................................................................13 7.0 DESCRIFTIONOFANALYTICAL METHOD .......................................................13 . 7.1ExtractionProcedure............................................................................................ 13 7..2 Preparation of StandardsandFortificationSolutions.......................................... 13 7.3Chromatography...............................................................................:................. 14 7.4InstrumentSensitivity.......................................................................................... 14 7.5Description ofInstrumentand Operating Conditions.......................................... 14 7.6QumtitationandExample Calculation.......................:................:.............1.........16 8.0 RESULTS ANI> DISCUSSION..............................................................................1.8 9.0 . CLRCUMSTANCES"HATMAY HAVE AFFECTED THEDATA.................... 18 10.0 RETENTION OFDATA AND SAMJ?LEs............................................................L 18 11.0 TABLES............................................:.............i........................................................19 12.0 FIGURES................................................................................................................. 30 13.0 APPENDICES.......................................................................................................... 38 . CentreAnalytical Laboratories. Inc 3M Environmental Laboratory .. Page 7 of 92 Page 246 BACK TO MAIN 3M MedicjalDepartment Study: T-6395.14 Analytical Report: FACT TOX-111 .. LRN-U2994 Centre Study No 023-017 . SponsorProtocol No:FACT-TOX-11I LIST OF TABLES . Page Table I. Summary of PFOS residues in Matrix and Matrix Zero Blanks......................... 20 Table 1I. Summaryof PFOS residuesin GI Prior to Cohabitation..................................21 Table Ill. Summary of PFOS residues in G2 Prior to Cohabitation.................................21 Table N . Summary of PFOS residuesin G3 Prior to Cohabitation ................................22 Table V.Summaryof PFOS residues in G1 Day 617 Gestation.......................................22 Table VI. Summaryof PFOS residues in G2 Day 617 Gestation.....................................23 Table W. Summary of PFOSresiduesin G3 Day 6/7Gestation....................................23 Table VIII. Summaryof PFOSresidues in G1 Day 14/15 Gestation............................... 24 Table IX. SUmmary of PFOS residues in G2 Day 14/15 Gestation ................................. 24 Table X. Summary of PFOS residues in G3 Day 14/15 Gestation...................................25 TabIe XI. Summary of PFOSresidues in G1 Day 20121 Gestation................................. 25 Table XII. Summaryof PFOS residues in G2 Day 20/21 Gestation................................ 26 Table MII. Summary of PFOS residuesin G3 Day20/21 Gestation............................... 26 - Table XIV. Summary of PFOSresidues in GI Day 21122Lactation...............................27 Table XV . Summaryof PFOS residues in G2 Day 21/22 Lactation................................ 27 Table XVI. Summaryof PFOSresidues in G3 Day 21/22 Lactation............................... 28 Table X M , SummaryofPFOSrecoveriesin Fortified Samples.................................... 29 Centre Analytical Laboratories. Inc. 3M Environmental Laboratory Page 8 of 92 Page 247 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.:023417 . Sponsor Protocol No: FACT-TOX-111 Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. - LIST OF FIGURES Page Typical CalibrationCurve for PFOS.............................................................. 31 Typical Mean Response Factor for THPFOS .........................................,...,.. 32 Chromatogram Representing a 5 n@mLextracted standardfor PFOS and 250 nglmL fortificationof THPFOS........................,.... 33 C25h0ronmg/amtoLgrfaomrtiRfiecparteiosennotfinTgHaP1F2O5Sn..g.../...d..e..x...t.r.a.c..t.e..d..s..t.a..n..d..a..r.d...f.o..r..P...F..O...S...a..n..d.34 Chromatogram Representing Control Rat Feces for PFOS and THPFOS (CentreID: 0005738Blank B, Set: 070700A)...............................................35 .......... Chromatogram Representing Control Rat Feces Fortified with 50 ng of PFOS and 500 ng of THPFOS (Centre ID: 0005803 Spk A, Set: 071000A) 36 Chromatogramof Rat Feces SampIe from G2 Day 6/7Gestation(Centre ID: . 0005836, Set: 071000A)........................................................,.,.....,...........3..7. Centre AnalyticaI Lahntories, Inc. 3M Environmental Laboratory . -. Page9of92 Page 248 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-I 11 LRN-U2994 Centre Study No.: 023-017 . Sponsor Protocol No:FACT-TOX-I I1 ' LIST OFAPPEIYDICES Appendix A Study Protocol FACT-TOX-111(Centre Study NO.023-017) and Amendments and Deviations .................................................................... 39 Appendix B Determination of Fluorochemical Residues in MonkeyRat Feces by LC/MS/MS (Revision 2) Method #OOM-023-003, revision 2 and Deviations andModifications............................................................. 66 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 10 of 92 Page 249 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 i LRN-U2994' Centre Study No.: 023-017 SponsorProtocoI NO.: FACT-TOX-1I1 I 1.0 S W R Y The purpose of this study was to analyzepeffluorooctantkulfonate(PFOS) in rat feces 8s I specified in 3M Protocol FACT-TOX-111.The analytical method used for this study was, "Determination of Fluorochernical Residues in Monkeymat Feces by LC/MS/Ms, revision 2",Centremethodnumbex OOM-023-003r,evision 2. The limit of quantificationfor PFOS in rat feces establishedduring the method vatidation was 10ppb (0.0100'pg/g). The method validation did not determine a method detection limit. Residues (comted for purity) ranging from non-detectedlevels to 13.5pg/g were found in the rat feces samples. Fortification recoveries ranged from 73-136% with,an average of 92% and relative standard deviation of 18%. 2.0 OBJECTIVE The objectiveof this study was to determinelevels of peffluorooctanesdfonate(PFOS) in specimensof fecesof rats using the analytical method "Determinationof Fluomhemicat Residues in MonkeyRat Feces by LcIMs/MS, revision 2",Centre method number OOM023-003, revision 2, . 3.0 INTRODUCTION The study was initiated on June 9, 1999, when the study director signed the protocol FACT-TOX-111.The. study was assigned to Centre on April 14, ZOO0 when the study director signed Amendment #4. The complete protocol and amendments and deviations can be found in Appendix A. The analytical start date was July 6, 2000, -and the analytical termination date was July 20,2000. c This report details the results of the residues of PFOS detected in rat feces, using the analyticd method entitled, "Determination of Ruorochemical Residues in Monkeymat Feces by LC/MS/MS (revision 2)", Centre method number OOM-023-003, revision 2. Completedetailsof the analyticalmethodologycan be found in Appendix B 4.0 TEXTSYSTEM ' The 117 incurred rat feces samples analyzed in this study were received frozen on dry ice from 3M Environmental Laboratory St. Paul, MN on June 28, 2000. Samples were logged in and.stored frozen (< -1OOC) on June 28, 2000 by Centre personnel. 3M EnvironmentalLaboratory Amended Page 11 of 92 Page 250 BACK TO MAIN 3M Medical Department Study: T-6395.14 I Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 The control rat feces used for standards 'and blanks were purchased from Lampire * Biological Laboratories,Inc,, Piperesville,PA and receivedfrozen on dry ice at Centre on June 22,2000, logged in by Centre personnel and placedin frozen storage (<-lOC). Sample login and chain of custody infomation can be found in the raw data package associated with this study. Storage records will be kept at Centre Analytical Laboratories, Inc. and a true copy of the storage records can be found in the raw data package associatedwith this study. 5.0 REFERENCE MATERIAL The analytical standard for PFOS determination (potassium perfluorooctane sulfonate) was received at Centre on November 12,1999 and the surrogate standard THPFOS was -received on December 3, 1999 from 3M Environmental Technology and Services. Characterization of the reference material PFOS was performed at Centre on August 31, 2000 and documentationcan be found in the raw data package associated with this report. The available information for the reference materials is listed below. The reference materials were stored at morn temperature. Comaound PFOS THPFOS Centre Control No. 99-023-002 99-023-011 Batch NO. 217 53406 - Puritv 86.9 NA Molecular struc@res of PFOS and THPFOS are given beIow. Exoiration Date 08/31/01 0 ~/Ol/lO PFOS Chemical Name: Perfiuorooctanesulfonate Molecular weight: 499 (CgF17S033 . 0 Note: The neutral molecule and standard form from which PFOS (anion) is derived, is potassium perfiuorooctane sulfonate E C ~ I ~ S O ~mKo]Ie, cular weight 538. TKPFOS ChemicalNime: 4-H,perfluorooctanesulfonicacid Molecular weight: 428 1-H,1-H,2-El, 243, CSF&~O@ Centre Analyticai Laboratories. Inc. 3M Environmental Laboratory Page 12of 92 Page 251 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 . SponsorProtocol NO:FACT-TOX-111 6.0 EXPERIMENTAL DESIGN Samples were extracted according to group number 5nd sampling day. Each set of - samples contained two matix blanks, two matrix blanks spiked with the surrogate standard, two matrix spikes, and 7 16 samples. The extracts were analyzed by LC/MS/MS. Samples with residues outside the linear range of the calibration curve were diluted and re-analyzed. 7.0 DESCRIPTION OFANALYTICAL iMETHOD Analytical method entitled "Determination of Fluorochemical Residues in Monkeymat Feces by Lc/MS/MS (Revision ':)2 Centre method number OOM-023-003,revision 2 (AppendixB)was used for this study. 7.1 Extraction Procedure One gram (k 0.05 g) of sample was weighed into 20 mL polyethylene scintillation vids - and fortified (if necessary) using disposable micropipettes. Ten mL of acetonitrile was added to the vid, capped tightly, and placed on a wrist-action shaker for 30 min. The samples-were filtered through a glass acrodisc filter. The filtered extract was passed through a conditioned carbon SPE column and collected. The columns were then eluted with -10 mL acetonitrile and -20 mL 9O:lO acetonitrile:Z% ascoibic acid in methanol. The combined extracts were evaporated down to near dryness with a rotary evaporator and then re-constituted with 2 mL methand. The sampks were analyzed using electrospray LCYMSNS. 7.2 Prepamionof Standardsand FortificationSolutions ' Standard solutions were prepared on May 3, 2000 as specified in Centre Analytical Laboratories' analytical method entitled, "Lletermination of Fluorochemical Residues in Monkeymat Feces by LC/MS/MS, (Revision:)2 Centre method number OOM-023-003, revision 2. An individual stock standard solution of PFOS was prepared at a concentration of 100pg/mL by dissolving 10 mg of the standard (corrected for salt purity bnly) in methanol. From this solution, a 10 pglmL fortification standard solution was prepared by taking 10 mL of the stock and bringing the volume up 'to 100 mL with methanol. Also, a stock standard of THPFOS was prepared at 100 pg/mL, by dissolving 10 mg of the standard in methanol. An individual 10 p g / d solution of THPFOS was prepared in the same fashion described above. A 2.5 pg/& fortification standard of PFOS was prepared by taking 25 mL of the 10 pg/mt fortification standard of PFOS and bringing the volume up to 100 mL with methanol. An individual fortification solution of "HPFOS was prepared in the same manner. A 0.5 p&mL fortification standard of PFOS was prepared by taking 20 mL of Centre Analytical Laboratories, Inc. Page 13 of 92 3M Environmental Laboratory Page 252 BACK TO MAIN 3M Medical DepartmentStudy: T-6395.14 I Analytical Report: FACT TOX-111 LRN-U2994. Centre Study No.: 023-017 . Sponsor Protocol No: FACT-TOX-111 the 2.5 p@mLstandard of PFOS and bringing the volume up to 100 mL with methanol. * To make the 0.1 pg/mL fortification standard, 20 mL of the 0.5 p g / d was brought up to 100 mL with methanol. Calibration standards were processed through the extraction procedure, identically to the samples. The fortification of the standards before extraction was done according to the foIlowing tabIe: (a) Conc. of PFOS Fortification Weight of Fortification Volume Control - Solution (Pgm Sample (g) (20.05 g) 0.1 . 100 1.0 0.1 200 1.0 0.5 100 1 .o 0.5 200 1 .O 2.5 100 1 .o 2.5 200 1 .o * 2.5 p g / d TJPFOS fortificationsolution. Fort. Level of Extncted Calibration Standard (PPb) 10 20 50 100 250 500 Vol. of Surrogate Standard* added (&) 200 200 200 200 200 200 The stock standard solution and all fortification and cgibration standard solutions were stored in a refrigerator (4' f 2C) when not in use. Documentation' of standard preparation can be found in the raw data associatedwith this report. 7.3 Chromatography - Quantification of PFOS and THE'FOS was accomplished by elecuospray LC/MS/MS analysis, The retention times of PFOS and THPFOS were 5.2 min. and 4 5.0 min., respectively, with no significantigterfexingpeaks in the control matrices correspondingto either Df the analyte retention times. 7.4 Instrument Sensitivity The smallest standard amount injected during the chromatographic run was equivalent to 5 n g / d of PFOS and 250 ng/mL of 'f"F0S in the rat feces matrix, 7.5 Descriptionof Instrumentand Operathg Conditions A Micromass Quattro Ultima LC/MS/MS coupled to a Hewlet Packard HPLC system was used. Data acquisition and processingwere performed using Masslynx 3.4software. Detaiied opmting conditionsare listed below: Centre Andyticd Laboratories,Inc. 3M EnvironmentalLaboratory Page 14 of 92 Page 253 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRNyU2994 Centre Study No.: 023-017 . Sponsor Protocol No: FACT-TOX-111 Instrument: Micromass QuattroUltima ELEcTRoSpRAY ION SOURCE: Capillary: 3.0 kV Hexapole 1:0.1 V Aperture 1: 0.2 V Hexapole 2: 0.3 V SourceBlock Temp,: lW0C Desolvation Temp.: 350C ANALYZER: LM Res 1: 10.5 V FMRes 1: 10.5 V Energy 1: 1.0V Entrance: -2V Exit: 2 V LM Res 2: 13.0 V Hh4 Res 2 13.0 V Energy 2 2.0V . Multiplier: 650 V GAS FLOWS AM)PRESSURE. Desolvation N2Flow Rate: -650 L/hr NebuIiserNzFlow Rate: -150 L/hr . Gas Cell Pressure: -0.003 mbar Computer: COhpAQ ProfessionalWorkstbtionAP200 Software: Microsoft Windows NT:Version 4Build 1381: Service Pack 5 MicromassLimited: Masslynx 3.4 Build 004 - HPLC Equipment: Hewlett Packard (HP) Series 1100 HP Binary Pump HE' Vacuum Degasser . HP Autosampler HP Column Oven HPLC Column: Genesis C-8,5 cm x 2.1 mm i.d. x 4 p Column Temperature: 35OC MobilePhase (A) : 2 mM AmmoniumAcetatein Type I Water Mobile Phase (B)' : Methanol Gradient: .. Injected Volume: Ions monitored : Analvte PFOS THPFOS Time (min). 0.0 . 0.4 1 .o 7.0 * . 7.5 10.5 11.0 14.5 15.0 10 pL %A 60.0 60.0 10.0 10.0 0.0 0.0 60.0 60.0 60.0 Transition Dwell kecs) Monitored 499 +99 . 0.2 427+SO . 0.2 % B 40.0 40.0 90.0 90.0 100.0 100.0 40.0 40.0 40.0 Flow Rate (mvmin) 0.3 0.3 . 0.3 0.3 0.3 0.4 0.4 0.4 . 0.3 ColI Enerw (eV) Cone ("VI 43 . 76 35 34 Centre Analytical Laboratories, Inc. . 3M Environmental Laboratory Page 15 of 92 Page 254 BACK TO MAIN 3M Medicjal Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994- Centre Study No.: 023-017 . 'SponsorProtocol No:FACT-TOX-111 7.6 Quantitationand Example Calculation Ten microliters of sample or extracted calibration standard was injected into the LCMSIMS. The peak area was measured and the standard curve was generated (using l/x weighted linear regression) by MassIynx Software using six concentrations of standards. The surrogate standard, THPFOS,was only used to monitor the efficiency of the extraction procedure and was not used for quantitation of PFOS. The residue concentrationfor ratfeces was determined from the followingequations: Use Equations 1 and 2 to calculate the amount of analyte found (in ppb, based on peak area) using the standrvd curve (linear regression parameters) generated by the Masslynx software program. Eauation 1: - Analyte found (ng/mL) =.Peak area intercept1 . slope Eauation 2: h d y t e found (ppb) =janalvte found (ng/mL>x final vol.(mL>x DF) sample wt. (g) where DF = dilution factor. For samples fortified with known amounts of andytes prior to extraction, use Equation 3 to calculatethe percent recovery. Eauation 3: Recovery (%) = [analytefound (nglmL)x finalvol. (mL) x DF] anaIyteadded (ng) Eauation 4 This eaIcuIationwas only performedon acmd samples and not QC recoveries. Corrected analytefound (ppb) = analytefound @pb)x % purity % purity for PFOS lot 217 = 86.9% (0-869) An example of a calculation using an actual sample analyzed with extracted standards . follows: Rat feces sample Centre ID 0005803 Spk A (Set: 071000A), fortified at 50 ng with PFOS. Where: peak area = 19051 intercept = 2021.14 slope . = 673.327 dilution factor =1 CentreAnalytical Laboratories,Inc. 3M Environmental Laboratory Page 16of 92 Page 255 BACK TO MAIN 3M Medicjal Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 ' ng added (fort level) = final vol. sample wt. = - Centre Study No.: 023-017 ' Sponsor Protocol No: FACT-TOX-111 50 2mL 1.01 g From equation 1: Analyte found ( n g / d ) = 119051- 2021.141 673.327 = 25.3 ng/mL From equation2: Analyte found Gpb) = 125.3x 2 mL x 11 1.01 g = 50.1 ppb From equation 3: ?4 Recovery = (25.3 n d d x 2.mLx 1) x 100 50 ng = 10-1% Note: This example csllculation was done using rounded numbers, and therefore may be slightIydifferentfrom the vdues shown in the RAW DATA. The amount of surrogate standard TPFOS found was calculated by dividing the peak area for each sample by the mean response factor. Analyte found'(ng/mL): peak area mean response factor Other statisticalmethods used in analyzingthis data were: . Standard Deviation = x.,4 - Mean =x =,n Relative Standard Deviation (RSD or Coeflcient of Variation(CV))= StandardDeviation x 100% Mean Centre Analytical Laboratories,Inci 3M Environmental'Laboratory Page 17 of 92 ,. Page 256 BACK TO MAIN 3M B Medic IDepartmentStudy: T-6395.14 Analytical Report: FACT TOX-I 11 LRN-U2994 .- Centre Study No.: 023-017 Sponsor Protocol No.: FACT-TOX-111 8.0 RESULTSAND DISCUSSION Although, the majority of the control samples did not contain interferences, a couple of sampIes did contain residues. However these interferenceswere not significant enough to adversely affect the data. A sumrnary of residues found in all of the matrix blanks and matrix zero blanks is detailed in Table I. , Residues (corrected for purity) ranging from non-detected levels to 13.5 pg/g were found in the rat feces samples. Themidues found in all of the samples plus the averages and standard kviations for each group at each interval are detailed in Tables II-XVI For this report, the residues found in Tables I-XVI were corrected for the purity of the PFOS standard lot 217 bwed on the certificate of analysis finalized on September 7, 2900. Fortification recoveries ranged from 73-13696 with an average of 92% and relative standarddeviation of 18%. A summary of all of the fortificationrecoveries can be found in Table XVII. Typical calibration curves agd chromatograms representing standards, controls, fortifications,and samplesare depicted in Figures 1-7. 9.0 CIRCUMSTANCESTHATMAY HAVE AFFECTED THEDATA Electronic records are not fully compliant with 21 CFR 11, "E2ectronic Records: Electronic Signature." However, fully approved SOP'S were in place and all chromatograph instrumentation used in this study was fully validated (IQ,-PQ,OQ), calibrated and operational. All original data were printed as hard copies and fully audited . by quality assurance. Verified exact copies and the electronic data have been stored in the archives at Centre AnalyticalLaboratories,Inc. Original raw data have been returned to the sponsor. k 10.0 mTENTION OFDATAAND SAMPLES When the final report is complete, all original paper data generated by Centre.AnalyticaI Laboratories, Inc. will be shipped to the sponsor. This does not include facility-specific raw data such as instrument Jogs, however exact copies of temperature logs will be submitted. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specificraw data, will be retained in the Centre Analytical Laboratories, Inc. archives for the period of time specified in 21 CFR 58. Retained samples of reference substances are archived by the sponsor. 3M EnvironmentalLaboratory Amended Page 18 of 92 Page 257 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 Sponsor Protocol No: FACT-TOX-111 11.0 TABLES . Centre Analytical Laboratories,Inc. 3M Environmental Laboratory Page'19of 92 Page 258 BACK TO MAIN 3M Medical DepartmentStudy: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 . ! Centre Study No.: 023-017 SponsorProtocol No.:FACT-TOX-ill b Table 1. Summary of PFOS resi&es in Matrix and Matrix Zero Blanks I Sponsor ID na Centre ID 0005738 BIank A sei N&r 0706oOAR Corrected Residue Date . ResidueFound Found Extracted 7/6/00 (Pg/g) ND bdg) ND na 0005738 Blank B 070600AR 716iOO ND ND na 0005738 Zero Blank C 0706QOAR 7/6/00 ND ND na 0005738 Zero Blank D 070WAR 7/6/00 ND Nil na 0005738 Blank A 070700A 7/1/00 NQ NQ na 0005738 Blank B 070700A 7nm ND ND na 0005738ZeroBlank C 070700A 7/7/00 . .ND ND na 0005738 Zero Blank D 070700A 7/7/00 ND ND na . 0005738BlankA 071000A 7/1(xoo ND- ND na 0005738 Blank B MlWA 7/10/00 m ND lla 0005738Zero Blank C MlOOOA na 0005738 Zero Blank D 071WOA na .0005738BlankA 071l00A 7/1QMo 7/1W 711 1/00 ND ,ND c 0.0100 ND m e 0.00869 na 0005738 BlankB 071100A 7111/00 NQ NQ - nnaa 00000055773388ZZeerrooBBllaannkkDC 0077111lO0O0AA 77111111//0000 <0N.0Q100 e0N.00Q869 na 0005738 Blank A 071uwIA 7/12100 e 0.0100 e 0.00869 na 0005738 BlankB 07luxlA 7/12/00 0.0156 0.0136 na 0005738 Zero Blank C 071u)oA 7/121poo < 0.0100 < 0.00869 na 0005738ZeroBlankD 071uxIA 7/12/00 e 0.0100 e 0.00869 na 0005738 Blank A 071300A 7/13/00 NQ NQ ~ na 0005738 Blank B 0713OOA 7/13/00 na 0005738Zero Blank C 071uxIA 7/13/00 na 0005738 Zero Blank D 071300A 7/13/00 na 0005738Blank A 0717OOAR 7/17/00 NQ. NQ . NQ ND NQ NQ NQ . ND na 0005738 Blank B' 0717WAR 7/17/00 ND ND na 0005738 Zero Blank C 071700AR 7/17/00 ND ND na 0005738 Zero Blank D 071700AR 7/17/00 ND ND na 0005738 Blank A 071800A 7/18/00 ND ND na 0005738 Blank B 071800A 7/18/00' ND' ND na 0005738ZeroBlank C 071800A 7118/00. ND ND Na 0005738ZeroBlankD M1800A 7/18/00 ND ND ND =Not Detected andNQ =Not Quantifiable Note:The average and standard deviation were calculatedby using 0.0100 as the value for those samples reported as 0.0100,0.00869 for those reported as e 0.00869, and zero for those reported as ND or NQ. 3M EnvironmentalLaboratory Amended Page 20 of 92 Page 259 BACK TO MAIN 3M ? Medic I Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRNXJ299'4. . Centre StudyNo.:023-017 Sponsor Wtocol No.:FACT-TOX-111 Table I (continued). Summary of PFOS residues in Matrix and Matrix Zero Blanks Sponsor ID 118 na I M na Centre Corrected Residue Set Date Residue Found Found ID 0005738BlankA 0005738BlankB 0005738ZeroBlankC Number 072000A 072000A 072000A Extracted 7/20/00 7/20/00 7/20/00 (clg/g) NQ . NQ NQ (Wdd NQ NQ NQ 0005738ZeroBlankD 072000A 7/20/00 AVERAGE NQ 0.0100 NQ < 0.00869 STANDARDDEVIATION: NQ NQ =Not DetectedandNQ =Not Quantifiable Note:The average and standarddeiiationwere calculatedby using 0.0100as the value for those samples reported as e 0.0100,0.00869 for those reportedas c 0.00869,and zero .forthose reported as ND or NQ. .. 3M Environmental Laboratory Amended Page 20A of 92 Page 260 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 SponsorProtocol No.:FACT-TOX-111 Table 11. Summary of PFOS residues in Gl Prior to Cohabitation Sponsor ID 13726FG1Prior to Cohabitation 13727FG1prior to Cohabitation 13728FG1Priorto Cohabitation 13731FG1prior to Cohabitation 13734FGl Priorto Cohabitation 13735FG1Prior to Cohabitation 13736FG1 Priorto cohabitation 13737FG1 Priorto Cohabitation Centre Set Date ID Number Extracted 0005799 070600AR 7/6/00 0005800 070600AR 7/6/00 0005801 070600AR 7/6/00 0005802 070600AR 7/6/00 0005803 070600AR 7/6/00 0005804 0706oOAR 7/6/00 0005805 070600AR 7/6/00 0005806 070600AR 7/6/00 AVERAGE STANDARD DEVIATION Residue Found corrected Residue Found 0 4 i d L ND ND ND ND ND ND ND ND m ND m ND ND ND ND ND ND ND NQ NQ ND =Not Detected and NQ =Not Quantifiable Table III. Summary of PFOS residues in 6 2 Prior to Cohabitation Sponsor ID 13739FG2 Prior to Cohabitation 13740FG2 Prior to Cohabitation 13741FG2 Prior to Cohabitation 13744FG2 Priorto Cohabitation 1374% G2 Prior to Cohabitation 13745FG2 Prior to Cohabitation 13746FG2Prior toCohabitationc 13748FG2 Priorto Cohabitation 13749FG2 Prior to Cohabitation Centre Set Date ID Number Extracted 0005807 070647OOA.D 7/6/00 0005808 0706-0700AD 7/6/00 0005809 070600AR 7/6/00 0005810 070600AR 7/6/00 0005811 0706-0700AD 7/6/00 0005811 0717-18OOAD 7/17/00 0005812 0706-0700AD 7/6/00 0005813 0706M00AD 7/6/00 0005814 0706-07OOAD 7/6/00 AVERAGE STANDARDDEVIATION Residue Found (&g) 0.933 0.764 0.453 0.463 3.13 1.04 0.791 0.809 0.628 1.00 0.820 CornCted Residue Found 0.810 0.664 0.394 0.402 2.72 0.904 0.688 0.703 0.546 0.870 0.713 3M Environmental Laboratory AmendedPage 21 of 92 Page 261 BACK TO MAIN 3M ? Medic I DepartmentStudy: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre StudyNo.: 023-017 SponsorProtocol No.: FACT-TOX-111 Table IV. Sumxnary of PFOS residues in 6 3 Prior to Cohabitation Sponsor ID 13751FG3 Prior to Cohabitation 13752FG3 Prior to Cohabitation 13753FG3 Priorto Cohabitation 13754FG3 Priorto Cohabitation 13755F G3 Mor to Cohabitation 13756FG3 Prior toCohabitation 13758FG3 Prior toCohabitation 13759F0 3PriortoCohabitation Centre Set Date ID Number Extracted 0005815 0706-0700AD 7/7/00 0005816 0706-0700AD 7/7/00 00058 17 0706-0700AD 7/7/00 0005818 0706-0700AD 7/7/00 0005819 07064700AD 7/7/00 0005820 07oMnooAD 7/7/00 0005821 07064700AD 7/7/00 0005822 0706-0700AD 7/7/00 AVERAGE STANDARD DEVIATION: Residue Found corrected Residue Found 0 0 12.4 10.8 1.2.5 10.8 14.1 12.3 15.6 13.5 9.86 8.57 12.4 10.8 10.8 9.41 125 10.9 12.5 10.9 1.77 154 Table V. Snmmary of PFOSresidues in G l Day 6p7 Gestation sponsor ID 13726FG1 Day 6/7 Gestation 13727FG1Day 6/7 Gestation 13728FG1Day 6/7Gestation 13731FG1Day 6/7 Gestation 13734FG1Day 6/7 Gestation 1373sG1Day 6/7Gestation 13736FG1Day 6/7Gestation 13737FG1Day 6/7Gestation Centre Set Date ID Number Extracted 0005823 070700A 7/7/00 0005824 070700A 7/7/00 0005825 070700A 7/7/00 0005826 070700A 7/7/00 0005827 070700A 7/7/00 0005828 070700A 7/7/00 ' 0005829 070700A 7/7/00 0005830 070700A 7/7/00 AVERAGE STANDARD DEVIATION Residue Found (y&) ND ND ND ND ND ND ND ND ND NQ corrected Residue Found (wglg) ND ND ND ND ND ND ND ND ND NQ ND = Not Detected and NQ = Not Quantifiable 3M EnvironmentalLaboratory AmendedPage22 of 92 Page 262 BACK TO MAIN 3M Medici"I DepartmentStudy: T-6395.14 Analytical Report: FACT TOX-1I 1 LRN-U2994 Centre Study No.:023-017 Sponsor Protocol No.:FACT-TOX-111 Table VI. Summary of PFOS residues in 6 2 Day 6/7 Gestation Sponsor ID 13739FG2 Day 6/7 &station 13740FG2 Day 6/7 Gestation 13741F G2 Day 617 Gestation 13744FG2 Day 6/7 Gestation 13745FG2 Day 6/7Gestation 1374sG2 Day 6/7Gestation 13748FG2Day 6/7 Gestation 13749FG2 Day M Gestation Centre Set Date ID Number Extracted 0005831 071000AD 7/10/00 0005832 071OOOAD 7/10/00 0005833 071000A 7 / 1 W 0005834 071000A 7/10/00 0005835 071000A 7/10/00 0005836 MlOOOA 7/10/00 0005837 Wl000A 7/10/00 0005838 MlOOOA 7/10/00 AVERAGE STANDARD DEVIATION: Residue Found (&g) 0.591 0.618 0.366 0.356 0.440 0.448 0.371 0.483 0459 0.101 Corrected Residue Found (Cldg) 0.514 0.537 0,318 0.309 0.382 0.389 0.322 0.420 0399 0.0876 Table VII. Summary of PFOS residues in 6 3 Day 6/7 Gestation Sponsor Centre Set Date ID ID Number Extracted 13751FG3Day 6/7Gestation 0005839 071000AD 7/10/00 13752FG3 Day 6/7Gestation 0005840 071000AD 7/10/00 13753FG3 Day 6,#Gestation 0005841 071oooAD 7/10/00 13754FG3Day 6/7Gestation 0005842 071000AD 7/10/00 1375% G3Day 6/7Gestation 0005843 071OOOAD 7/10/00 13756FG3 Day 6/7Gestation 13758FG3 Day 6/7Gestation , 0005844 0005845 13759FG3 Day 6/7Gestation 0005846 071OOOAD 071OOOAD 071000AD 7/10/00 7/10/00 7/10/00 AVERAGE STANDARD DEVIATION. Residue Found (pdg) 9.80 11.1 10.4 10.2 14.1 7.71 8.80 5.15 9.65 2.60 corrected Residue Found (Pdfz) 8.51 9.61 9.03 8.86 12.2 6.70 7.65 4.47 8.39 2.26 3M EnvironmentalLaboratory Amended Page 23 of 92 Page 263 BACK TO MAIN 3M ? Medic I DepartmentStudy: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.:023-017 Sponsor Protocol No.: FACT-TOX-111 Table VIII. Summary of PFOS residues in G1 Day 14/15 Gestation Sponsor ID 13726FG1 Day 14/15Gestation 13727133G1Day 14/15Gestation 13728FG1Day 14/15Gestation 13731FG1 Day 14/15Gestation 13734FG1 Day 14/15Gestation 13735Fa 1 Day 14/15Gestation 13736FC3l Day 14-15Gestation 13737FGl Day 14/15Gestation Centre Set Date ID Number Extracted 0005847 071100A 7/11/00 0005848 071100A 7/11/00 0005849 071100A 7/11/00 0005850 071100A 7/11/00 0005851 071100A 7/11/00 0005852 071100A 7/11/00 0005853 071100A 7/11/00 0005854 07ll00A 7/11/00 AVERAGE STANDARD DEVIATKON Residue Found ND NQ N D =Not Detected and NQ =Not Quantifiable Corrected Residue Found ND NQ Table IX. Summary of PFOS residues in G2 Day 14/15 Gestation Sponsor ID 13739FG2Day 14/15Gestation 13740FG2Day 14/15Gestation 13741FG2 Day 14/15Gestation 13744FG2Day 14/15Gestation 13745FG2 Day 14/15Gestation 1374613G2Day 14/15Gestation' 13748FG2 Day 14/15Gestation 13749FG2 Day 14/15Gestation Centre Residue Set Date ' Found ID Number Extracted (piglp) 0005855 071100A 7/1yoO 0.343 0005856 0711OOA 7/11/00 0.358 0005857 071100A 7/11/00 0.354 0005858 071100A 7/11/00 0.478 0005859 071100A 7/11/00 0.338 0005860 071100A 7/11/00 0.397 0005861 071l00A 7/11/00 0.235 0005862 071100A 7/11/00 0.203 AVERAGE: 0.338 STANDARDDEVIATION 0.0869 Corrected Residue Found (Clglg) 0.298 0.311 0.308 0.416 0.294 0.345 0.204 0.176 0.294 0.0755 3M EnvironmentalLaboratory Amended Page 24 of 92 Page 264 BACK TO MAIN 3M Medici"I Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.:023-017 Sponsor Protocol No.:FACT-TOX-111 Table X. Summary of PFOS residues in 6 3 Day 14/15 Gestation Sponsor lD 13751F G3 Day 14/15 Gestation 13752F G3 Day 14/15 Gestation 13753F(33 Day 14/15 Gestation 13754FG3Day 14/15Gestation 13755F G3 Day 14/15 Gestation 13756F G3 Day 14/15 Gestation 13758F G3 Day 14/15 Gestation 13759F G3 Day 14/15 Gestation Centre Set Date ID Number Extracted 0005863 0712OOA 7/12/00 0005864 071200A 7/12/00 0005865 071200A 7/12/00 0005866 071200A 7/12/00 0005867 07lZWA 7/12/00 0005868 071200A 7/12/00 0005869. 071200A 7/12/00 oooJ870 071200A 7/12/00 AVERAGE: STANDARD DEVIATION. Residue Found (pg/g1 5.75 7.26 4.63 4.37 6.50 5.90 6.10 3.92 5.55 1.15 corrected Residue Found (Pdd 5.00 6.31 4.02 3.80 5.65 5.13 5.30 3.41 4.83 1.00 Table XI. Summary of PFQS residuesin GI Day 20/21Gestation Sponsor ID 13726FG1 Day 20/21Gestation 13727FG1 Day 2W1 Gestation 13728F G1 Day 20/21 Gestation 13734F G1 Day 20/21 Gestation 1373SFG1Day 20/21 Gestation 13736FG1Day 20/21 Gestation 13737FGl Day 20/21Gestation . \ Centre Set Date ID 0005871 Number Extracted 071200A 7/12/00 0005872 071200A 7/12/00 0005873 0712OOA 7/12/00 0005874 071200A 7/12/00 0005875 071200A 7/12/00 0005876 071200A 7/12/00 0005877 071200A 7/12/00 AVERAGE STANDARD DEVIATION Residue Found (pg/g) ND ND ND ND ND ND ND ND NQ ND = Not Detected and NQ = Not Quantifiable corrected Residue Found (P&) ND ND ND ND ND ND ND ND NQ 3M Environmental Laboratory Amended Page 25 of 92 Page 265 BACK TO MAIN 3M ? Medic IDepartment Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 CentreStudy No.:023-017 SponsorProtocol No.:FACT-TOX-111 Table XII. Summary of PFOS residues in 62Day 20/21Gestation Sponsor ID 13739FG2 Day 20/21Gestation 13740F G2 Day 20/21 Gestation 13741F G2 Day 2N21 Gestation 13744FG2 Day 20/21 Gestation 13745FG2 Day 20/21 Gestation 13746FG2 Day 20/21 Gestation 13748FG2 Day 20/21 Gestation 137493G2 Day 20/21 Gestation Centre Set Date ID Number Extracted 0005878 072000A 7/2Q100 0005879 072000A 7/20/00 0005880 072000A 7/2QHx) 0005881 072000A 7 / 2 0005882 072000A 7/2oMo 0005883 072000A 7/2oKx) 0005884 072000A 7/20/00 0005885 072000A 7/2WOO AVERAGE: STANDARDDEVIATION Residue Found bg/g) 0.153 0.0769 0.190 ~ 0.102 0.0748 0.130 0.181 0.188 0.137 0.0483 Corrected Residue Found (w&) 0.133 0.0668 0,165 0.0883 0.0650 0.113 0.157 0.163 0.119 0.0424 Table XIII. Summary of PFOS residues in 6 3 Day 20/21Gestation SPon~ ID 13751F G3 Day 20/21 Gestation 13752FG3Day 20/21 Gestation 13753FG3Day 20/21Gestation 13754FG3 Day 20/21Gestation 13755FG3 Day 20/21Gestation 13756FG3Day 20/21 Gestation 13758F G3 Day 20/21 Gestation , 13759BG3 Day 20/21 Gestation Centre Set Date ID Number Extracted 0005886 0717-18OOAD 7/17/00 0005887 0717-18OOAD 7/17/00 0005888 0717-18OOAD 7/17/00 0005889 0717-18OOAD 7/17/00 0005890 0717-18OOAD 7/17/00 0005891 0717-18OOAD 7/17/00 0005892 0717-18OOAD 7/17/00 0005893 0717-18OOAD 7/17/00 AVERAGE: STANDARD DEVIATION: Residue Found (pglg) 1.86 2.39 1.63 1.94 4.93 2.32 2.27 1.59 2.37 1.08 Correctwi Residue Found (PdL 1.61 2.08 1.42 1.69 4.29 2.02 1.97 1.38 2.06 0940 3M Environmental Laboratory AmendedPage 26 of 92 Page 266 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre Study NO.:023-017 SponsorProtocol No.:FACT-TOX-111 Table XIV. Summary of PFOS residues in G1 Day 2Y22 Lactation Sponsor ID 13726FG1 Day 21/22 Lactation . 13727FG1Day 21/22 Lactation 13728FG1 Day 21/22 Lactation 13734FG1 Day 21/22 Lactation 1373513G1 Day 21/22 Lactation 13736FGl Day 21/22 Lactation 13737FG1Day 21/22 Lactation Centre Set Date ID Number Extracted 0005894 0713OOA 7/13/00 0005895 071300A 7/13/00 0005896 071300A 7/13/00 0005897 071300A 7/13/00 0005898 071300A 7/13/00 0005899 071300A 7/13/00 0005900 071300A 7/13/00 AVERAGE: STANDARDDEVIATION Residue Found (&g) 0.0235 ND ND ND ND ND ND <0.0100 NQ ND =Not Detected and NQ =Not Quantifiable corrected Residue Found ~I.Ifzk) 0.0204 ND ND kD ND ND ND <0.00869 NQ Note: The average and standarddeviationwere calculatedby using 0.0100as the value for those samples reported as c 0.0100,0.00869 for those reported as 0.00869, and zero for those reported as ND or NQ. Table XV. Summary of PFOS residues in 6 2 Day 21/22 Lactation sponsor ID 13739FG2Day 21/22 Lactation 13740FG2Day 21/22 Lactation 13741FG2Day 21/22Lactation 13744FG2Day 21/22 Lactation 13745FG2 Day 21/22 Lactation 4 13746FG2Day 21/22 Lactation 13748FG2 Day 21/22Lactation 13749FG2 Day 21/22 Lactation Centre Set Date ID Number Exmcted 0005901 071800A 7/18/00 0005902 071800A 7/18/00 0005903 071800A 7/18/00. 0005904 071800A 7/18/00 0005905 071800A 7/18/00 0005906 071800A 7/18/00 0005907 071800A 7/18/00 0005908 071800A 7/18/00 AVERAGE: STANDARD DEVIATION Residue Found (pdg) 0.0776 0.0797 0.0448 0.0839 0.0390 0.0701 0.0585 0.0266 0.0600 0.0212 Corrected Residue Found bdg) 0.0674 0.0693 0.0389 0.0729 0.0339 0.0609 0.0508 0.0231 0.0522 0.0185 3M Environmental Laboratory Amended Page 27 of 92 Page 267 BACK TO MAIN 3M Medic?I Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 ' Centre Study No.:023-017 SponsorProtocol No.:FACT-TOX-111 Table XVI. Summaryof PFOS residues in 6 3Day 21/22 Lactation Sponsor ID 13751FG3Day 21/22 Lactation 13752F G3 Day 21/22 Lactation 13753F G3 Day 21/22 Lactation 13754F0 3 Day 21/22 Lactation 13756F0 3 Day 21122Lactation 13758F0 3 Day 21/22 Lactation 13759FG3 Day 21/22 Lactation Centre Set Date ID Number Extracted 0005909 0717-18oOAD 7/18/00 0005910 0717-1800AD 7/18/00 0005911 M1800A 7/18/00 0005912 071800A 7/18/00 0005913 0717-18oOAD 7/18/00 0005914 071800A 7/18/00 oooJ915 071800A 7/18/00 AVERAGE: STANDARD DEVIATION. Residue Found (pglg) 0.590 0.842 0.376 0.340 0.386 0.243 0.341 0.445 0.204 cotrected Residue Found Qgln) 0.513 0.731 0.327 0.295 0.336 . 0.21 1 0.296 0.387 0.177 3M Environmental Laboratory Amended Page 28 of 92 Page 268 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 . SponsorProtocol No: FACT-TOX-1I1 ' Table XVII. Summary of PFOS recoveries in Fortified Samples Sponsor Centre . Set Date a Amt. % ID ID Number Extracted Added (ng) Recovery 13726FG1 Prior to Cohabitation 0005799 Spk A 070600A.R 7/6/00 50 118 13727FG1 Prior to Cohabitation 0005800 Spk B 070600AR 7/6/00 250 101 13728FG1 Prior to Cohabitation 0005801Spk A 070700A 7/7/00 50 88 13731F Gl Prior to Cohabitation 0005802 Spk B 070700A 7/7/00 250 86 13734FGI Prior to Cohabitation 0005803 Spk A 071000A 7/10/00 50 101 13735FGI Prior to Cohabitation 0005804Spk B 071OOOA 7/10/00 2000 94 1373613:GI Prior to Cohabitation 0005805 Spk A 071l00A 7/11/00 50 85 13737FGI Prior to cohabitation 0005806 Spk B 071IOOA 7/11/00 20000 . 84 13726FGI Prior to Cohabitation 0005799 SpkA 071200A 7/12/00 50 136 1372823G1 Prior to Cohabitation 00058OI SpkB 071200A . 7/12/00 20000 87 13734FG1 Prior to Cohabitation 0005803Spk A 071300A 7/13/00 50 92 13735FG1 Prior to Cohabitation 0005804 Spk B 071300A 7/13/00 250 102 13726FG1Prior to Cohaktation 0005799 Spk A 071700AR 7/17/00 50 79 13737FG1prior to Cohabitation 0005806 Spk B 071700AR 7/17/00 20000 73 137352;G1prior to Cohabitation 0005804 Spk A 071SOOA 7/18/00 50 . 105 13737FG1Prior to Cohabitation 0005806 Spk B 071800A 7/18/00 20000 83 I3727F G1 Prior to Cohabitation 0005800 Spk A 072000A 7/20Mo 50 74 13734FGl Prior to Cohbitation 0005803 Spk B 072000A 7/20/00 . 250 74 AVERAGE ~ 92 STANDARD DEVIATION 16 RELATXVE STANDARD DEVIATION: 18 .. Centre An.aIytica1 Laboratories, Inc. 3M Environmental Laboratory Page 29 of 92 Page 269 3M Medicpl DepartmentStudy: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.:023-017 Sponsor Protocol No: FACT-TOX-111 12.0 FIGURES Centre Analytical Laboratories, Inc. 3M EnvironmentalLaboratory Page 30 of 92 Page 270 BACK TO MAIN 3M Medicpi DepartmentStudy: T-6395.14 Analytical Report: FACT TOX-111 . .. LRN-U-2994. Figure 1. Centre StudyNo.:023-017 Sponsor b t o c o l No:FACT-"OX-1 11 Typical Calibration Curve or PFOS Ompound 1 name: PFOS oefficientof Determination: 0.998459 alibration curve: 673.327 x + 2021.14 esponse type: External Std, Area U N e type: Linear, Origin: Exclude, Welghting: l/x, Axis trans:None . .. *... ,.. 1 56.0 ' , iib.0 . , ' , .. 150.0' , ' 200.0 I i '-25,0n.g0tmi-1I Centre Analytical Laboratories, Inc. * . 3M Environmental Laboratory Page 31,of 92 Page 271 BACK TO MAIN 3M Medical DepartmentStudy: T-6395.14 I Analytical Report: FACT TOX-111 .. CRN-U2994 Figure 2. Centre Study No.:023-017 . SponsorProtocoI No:FACT-TOX-111 Typical &leanResponse Factor for THPFOS impound 2 name: THPFOS sassppoonnssee tFyapcet:oEr:x4t4e8rn.1a1lS8td, Area . m e type: RF 1.21e! I I I X X qesponsr L'.. Sd.0 I , n@mL I 1ob.o' ' ' ' ' ' 15b.b 20b.0 ' ' . 250.0 Centre Analytic$ Laboratories, Inc. 3M EnvironmentalLaboratory Page 32 of 92 Page 272 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Figure 3. Centre Study No.: 023-017 . Sponsor Protocol No: FACT-TOX-1 I1 Chromatogm Representing a 5 ngh& extracted standard ' for PFOS and 250 ng/mL fortification of TflpFOS ' I: P?OS XC070600-1,5ng/rnL Standard 07064700AD-3002 Sm (SG,2x2) .. 08JuI-200010:37:59( LCIMSIMS 8q MRM of 2 Channels ES-I 499 > 99 6.77e4 Area 9.57e5 Area %- .. .. Centre ,4na1yticd Laboratories, Inc. 3M Environmental Laboratory Page 33 of 92 Page 273 BACK TO MAIN 3M Medical DepartmentStudy: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Figure 4. Centre StudyNo.:023-017 Sponsor Protwo1 No:FACT-TOX-1I1 Chromatogram Representing a 125 ng/mL extracted standard for PFOS and 250 ngmL fortificationof THPFOS 1: PPOS XCO70600-5,125ng/mL Standard 0706-0700AD-3006 Sm (SG, 2x2) 522 08-JUl-2000 11 344~563 1 LC/MS/MS t6i MF~Mof 2 Channels ES- . . 499 > 09, 1.01 e61 . Area i . o o " ' " ~ . ~ ' " " ' j . ~' ' 4.b0' " 5 . b o " " ' 6 . 0 0 !: THPSOS KC070600-5,125ng/mL Standard 0706-0700AD-3006 Srn (SG, 2x2) 5.03 " 7.00 ' 8.00 , . Time 08-JUi-200017 :44:561 LCIMSIMStQ . MRM of 2 Channels ES427 > 801 9.08e5 Area ' ' ' ' ' 2.bd ' ' ' ' ' 3.b; ' ' 4.00 ' ' ' ' ' 5.bO ' 6.00 ' ' 7.00 " ' 8.bO , . Time 1 Centre Analytic31hbmtories, hc. 3M Environmental Laboratory Page 34 of 92 Page 274 BACK TO MAIN 3M Medici"I Department Study: T-6395.14 I Analytical Report: FACT TOX-111 LRN-U2994 CentreStudy No.:023417 ' SponsorProtocol No:FAm-TOX-1I1 Figure 6. ChromatogramRepresenting Control Rat Feces Fortified with 50 ng of PFOS and 500 ng of "HE'FOS (Centre DD: 0005503 Spk A, Set': 071000A) 1: PFOS ooosao3 s p k A 071OOOA-1013 Sm (SG, 2k) 159.0256 1 .. . 11-JuI-200001:17:2$ LC/MS/MS t6i M R M of 2 Channels ES- 1 . , 499~99j 1.78e5 Area I"' ' I. . ,... , . . . I . . . . I . . , . 6.00 ' ' ' 7.00 * ' . 8 3 0 Time 2: THPFOS 0005803 Spk A 071OOOA-1013 Sm (SG, 2x2) 5.10 .' II-JUI-2000 01 :17:25/ LCIMSIMS $6 MAM of 2 Channels ES427 > ao 9.47e5 Area .. i.bi' ' 1 .. ,. '0 , . ( . ..., . . . ~ .. .. , . . , , . . . . Time .'I 3.bi'' 'i.bO' ",i.bO" " ~ . o O 7.00 I 8.6i ' Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 36 of 92 Page 276 BACK TO MAIN 3M ? Medic I Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 . SponsorProtocol No:FACT-TOX-I 1I Figure 7. Chromatogram of Rat Feces Sample from GZ Day 6/7 Gestation (Centre ID: 0005836, Set: 071000A) I ' ', '8' ' I' . ,. ,...' L ' . . . , . . . . Time Centre Analyticai Laboratories, hc. 3M Environmental Laboratory Page 37 of 92 Page 277 3M Medici"I Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023417 SponsorProtocol No:FACT-TOX-111 13.0 APPENDICES Centre Analytical Labontories;Inc. 3M Envlrorimental Laboratory Page 38 of 92 Page 278 3M Medical Department Study: T-6395.14 I BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994' Centre Study No.: 023-017 SponsorProtocol No:FACT-TOX-111 . APPENDIX A Study Protocol FACT-TOX-111 (Centre Study No. 023-017) and Amendments and Deviations .- Centre Analytical Laboratories, Tnc. 3M Environmental Laboratory Page 39 of92 Page 279 3M Medicpl Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 ,LRN-U2994. Centre Study No.:023-017 . SponsorProtocol No:FACT-TOX-111 Study Title . .. Om1 (Gavage) Pharmacokinetic Recovery Study of PFOS in Rats 1. PROTOCOL Author Lisa Clnncn Date: . June8,1999 -. Pertbmlng Lahorafory 3 MEnvironmaraLTcchnolog& Safety Sesvics 3M EnVimnmcntat Laboratory 935Bush Avenue St.Paul,MN 55106 Laharatmy Project ldenffficaficn FACT-TOX-111 u2994 - -. .. . Centre .: Analytical Laboratories, Inc. 3M Environmental Laboratory page I of io Page 40 Of 92 .. . . Page 280 3M Medici'I DepartmentStudy: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.:023-017 . Sponsor Protocol NO:FACT-TOX-I 1I Study tdenfffmtion Oral (Gavage) PharmacokineticRecovery. Study of PFOS in Rats Test Materfaf - Sponsor .. . Sponsor Representative Study Director Pdwmoctaned o n i c acid p c t a ~ s i ~smd: (T-6295) - 3 M ToxiCOlOgY s&CcS Medical D q m a t 3 M Center,Building 220-2E-02 St. Paul, MN 55144-1000 MarvinT.Case, D.V.M., PkD. 3M ToXicologySerfices Teiephone: 651-733-5180 Facsimife: 651-733-1773 - . KristtnJ.Hanscn,Ph.D. . - 3M Environment$ Tectplology andSafetySariq Buildiag2-3B-09. ' 651-778-6018 .- . . , - Study Location(s1 In Viva TestingFaciiXy. Anaiytlcd T d n q Laboratory .. Argus R e s m h LA~rat~riehsc,. : 905 Sh&y Drive, Building A Harsham,P.A 1.9044 3 M Emrirc~~~~exitdLaboratoatory Building 2-3E-09 935 Bush Avenue StPaul,M?i 55106 3M Slvhmental Laboratcry ... . . .Centre Analytical Laboratories, Inc. .. .. 3M EnvironmentalLaboratory Page 2 of 10 ' Page41 of92 Page 281 BACK TO MAIN 3M a Medic I DepartmentStudy: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 SponsorProtocol No:FACT-TOX-111 Sub-ConfracfLabantodes Proposed Study Tirn3tabIe Study Initiation Date Study ComplefionDah .; . Pmtucd SFACT-TOX-Ill ' Advmcd BioanalpicalS d c e s , h c . 15 Cathemood Road rrhayw 14850 BatteileMananalh h t e :505 King Avenue ColumbusOhio 43201-2693 June 8, I999 Aupust 8.2000 Centre Analytical LaboratoAes, hc. 3M EnvironmentalLaboratory .. Page 42 of 92 Page 282 3M Medicpl Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-I 11 LRN-U2994 ' Centre Study No,:023-017 Sponsor Protocol'No: FACT-TOX-111 Centre Analytical Laboratories,hc. I . ..... 3M Environmental Laboratory . . .. .-. -. Page 43 of 92 .- Page 283 3M Medical DepartmentStudy: T-6395.14 7.8 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 ' .. Centre Study NO.: 023-017 . Sponsor Protocol No: FACT-T.0X-111 TOMnumber ofexpected specimens:456 Total number of test animals: 16 Total numberofcolitralanimds: 8 .. .. CentreAnaIytical Laboratories, hc. ... 3M Environmental Laboratory . -. .. Page 5of :O Page .44of 92 . Page 284 3M Medici'I DepartmentStudy: T-6395.14 .. . ...-.._.. BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 CentreStudy No.:023-017 Sponsor Protocol No: FACT-TOX-111 .... . - 72.1 Lioearity f 2 0.98 12.2 Limits of defection/quantihtion 12.2. I 'Mebod Detection mt @DL) for PFos. a) . Serum: 1.75ppb b) Liver: l5ppb Centre Analytical Laboratories, Inc. 3M Environmental Laboratory %e 6 of ;C .. 'Page'45of 92 Page 285 3M ? Medic I Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-UZ999- Centre StudyNo.:023-017 ' Sponsor Protocol No: FACT-TOX-I 11 3 4 Envihmentd Lahoaiqry .....- ._... ' Centre Analytical Laboratories,hc. 3M Environmental Laboratory .. . .Page 7of i o .. Page 46 of 92 3M ? Medic I Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U299..4... . Centre Study No.:023-017 Sponsor FYotmOl No:FACT-TOX-11I .----....- .._..______C_e_n..treAnalytical Laboratories,T._nc...-. 3M Environmental Laboratory . . .- ._..-. .. .. .. .. ..._. ... Page'47of92 .. . Page* 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 I LRN-U2994, Centre Study No.:023417 Sponsor Protocol No:FACT-TOX-111 .. . . . . . ........-. '16.1.1 16.1.2 16.1.3 16.1.4 f6.1.5 16.7.6 Approved protocolmd amcndnlmts Study corropondenet - shippingraords Rawdata . ApprovedI i l d report (original signcd copy) Elcemnic copies of data ? ~ . A ~ A C H M E N T S. f9.f AttachmentA Prepqatoq and d f i c d methods .. 3M E3VkUnhen&r Laboratory ' .. . -.... ............ Centre AnalyticalLaboritones,lnc. ... .. 3M Environmental Laboratory ...... . PageScflO .. ,. .. .. Page48 of 92 Page 288 BACK TO MAIN 3M Medici"I DepartmentStudy: T-6395.14 20. SrGUAmREs Analytical Report: FACT TOX-111 LRN42994 Centre Study No.:023-017 ' Sponsor Protocol No:FACT-TOX-111 ------- . . ' ... . . . . _ _ ...- '.' ~ PfUfCCdPACT-TOX-117 MarvinT.Case, D.VM, p i a , SponsorR~~IWULU~YC Da1te5799 .. .. . . .- .. . .. Centre halyticaI Laboratories,Inc. 3M EnvironmentalLaboratory Page49of92 . --rageLas 3M i" Medic I Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-1I 1 LFtN-kJZB4..' 'CentreStudy No.:023417 ' Sponsor Protocpi No: FACT-TOX-111 .. Study Title oral(Gavage) PharmacokineticRecoveryStudy ofPFOS in PROTOCOL AMENDMEKT NO. I -Amendment Date: August 12,1999 . I . Performing Laboratory 3M EmriroMlcntal Technology8 Safety S d c e s 3M EnvhnxnuataI Laboratory 935 Bush Avmue St Pal4 MN 55106 . Laboratory Projecf identificatipn . . ET&sS FACT-TOXI 11 LlRN U2994 . - 3M Envipnmentai Laboraiory . .- ..-......-..."- _-"..-.. Centre Analytical.L..ab-o-r.a, tories,hc. -. 3M Environmental Laboratory .. .. , -. Page 50 of 92 - -Paye%e 3M ? Medic I DepartmentStudy: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U.2.-994 Centre Study No.: 023017 .Sponsor'ProtocolNo: FACT-~0x-111 PffiLocalFACf-TOXf7 7 Amendment NO. I .. -..Centre -_~cc_II___ ~ n a l y t i c..-.iL..i.a.~_b._o_r_a_tI.o. ries.;.I ~ C . ~ 3M Environmental Laboratory . . . - .. . . Page 51 of 92 P a g e f t x 3M Medici"I Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023417 . SponsorProtocol No:FACT-TOX-111 . ....-...-..-- Rctcc=lFACT-TOX: 1I Amendment No. 1 - Amendment Approval. . .. .. -... - ."__.__....._._ Centrekalyticd Laborator_ies._,&._IC... 3M Environmental Laboratory .. Page 52 of 92 3M ? Medic I Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 ' Centre Study No.:023-017 Sponsor Protocol No: FACT-TOX-111 . . __ PROTOCOL AMENDMENT NO. z Amendment Date: September 30,1999 .. ... . .- . . _ - . _ _ . Centre Analytical Laboratorie.s,.Inc. 3M Environmental Laboratory . .' .. Pag6.53of 92 BACK TO MAIN 3M Medical DepartmentStudy: T-6395.14 -- Analytical Report: FACT TOX-111 LRN-U2994 _ . _... __,. . . - .. Centre Study No.: 023-017 Sponsor pT0toc01 No: FACT-TOX-111 ProtoCcfFACT-TOXl1 I Amendment NO. 2 ibis amendment modifies the following portion(s) of the pmtoc& .....--.._..__ Centre Analytical Laboratories,hc. 3M EnvironmentalLaboratory Page 54 of 92 . .. I . Page 294 -.-3M Medical Department Study: T-6395.14 ....-......_. BACK TO MAIN Analytical Report: FACT TOX-111 LRN-UZ9.94.. . Centre Study No.:023-017 Sponsor Protocol No: FACT-TOX-111 ..... . . . . . .......-..._. I _ . .-.._.. -Laboratory centre Analytical Laboratories,Inc. -. .. .. .. .. .. . . Page 295 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.:023-017 'SponsorProtocol No:FACT-TOX-111 , FACT-TOXI 11' pmsndmenl No. 2 -.. --.- .... -. Centre AnalyticalLaboratories,Inc. * ' .. 3M EnvironmentalLaboratory Page 56 of92 ' Page 296 3M Mediwl DepartmentStudy: T-6395.14 .. ' BACK TO MAIN ! Analytical Report: FACT TOX-111 LRN-U2994...-. . . . . . ..-. ~ Centre Study No.:023-017 Sponsor Protocol No: FACT-TOX-I I1 Amendment Approval v Kristk J. Hartrep,Pm.S,tudy D b r 5 Oclc l4Sf Date -. .. .. .____-___ ~ Centre Analytical L.a-b_o_r_a-.t_-or-ie.s-,-_-I-n_.c.:._.-... 3M EnvironmentalLaboratory .. ..- ....- ..... _. . .. ... Page57'of92' Page 297 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994. . .. . .Centre Study No.: 023-017 . SponsorProtocol No:FACT-TOX-111 ... . . .-..__ .. P-ROTOCOLAMENDMENTNO.3 . . Amendment Date: 20 J a n e 2000 Perfomring Laboratories *_ Performing Laboaforks ' Li*a~lysl?s . Laboratory PmjeciIdentiticatton ET&SSLRN-U2994 , * FACTTOX-I 1I ' Study: 418-015 3M Medical Department Study: T4295.14 h d COPY Of OrigiMl IAC. W f % L Initial D& . .- ---.__.---._. Centre Analytica.l La,bora.-t.o...r_ies..,.I-_n.-c.._.. . 3M Environmental Laboratory . -.. - Page 58 of92 Page 298 3M Medical Department Study: T-6395.14 BACK TO MAIN AnaMical Report: FACT TOX-111 LRN-U2994 Centre Study No.:023-017 Sponsor Protocol No:FACT-TOX-111 ' .. .. . _-_ . -mental Centre Analytical Laboratories, I~C. Laboratory Page:59of 92 Page 299 3M Medical DepartmentStudy: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994... . Centre Study No.:.023-017 '. SponsorProtocolNo: FACT-TOX-I11 .. - .. . .. .-... ..____._, Amendment Approval L /h F,& &, . . PhD.,Incoming Study Director %ate .. .. .. .. .. .. .--.... .. . C. entreAnalytical Laborator.ie..s.,LI.nc. 3IW-tnvironmentalLaboratory Page 60of92 Page 300 3M Medical DepartmentStudy: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994.-. Centre Study No.: 023-017 . SponsorProtocol No:FACT-TOX-111 . .- Study Tiffe O d (Gavage) Pharmacokinetic RecoveryStudy ofPFOS in PROTOCOL AMENDMENT NO. 4 1- I . Amendment Date: 20 April 2000 Pertbrmhg Laboratories and Safety Services SO5 f i g Avenue .*C~t~mbus,O4H3201-2693 , IS M a w o o dRoad I -- IthasNY 14850 ' Laboratory'P&ct Identiiication ET&SS LRN3J2994 FACTTOX-1.11 AgusStudy: 415915 . 3 M Medical DepartmentStudy T-6295.14 3M *hnrnenta/ Laboratcry "C - . EXXX Copy of Original ' O.fl2Llrn . . Initial Date .. .. .. Centie.A. .n..,a..l..y.tica. lLaboht.o..r_ie_.~.J_,nc_....... . a1 - Laboratory Page.61of 92 Page 301 3M Medical Department Study: T-6395.24 BACK TO MAIN Analytical Report: FACT TOX-?Il LRN-U2994. , . . Centre Study No.:023-017 ' SponsorProtocol No:FA(=T-TOX-111 .. ....-..-..-. . PmtOCd UN42994 Amendment Number 4 .__._....___",_. ' centre Analytical Labo.rator.ies,hc. 3 m r o n m e n t a l Laboratow .. Page 62of 92 Page 302 3M Medical Department Study: T-6395.14 /* BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 - Centre Study No.: 023417 ' Sponsor Protocol No: FACT-TOX-111 Amendment Approval .....--... ~ ..-... Centre Analytical Laboratories, Inc. 3MEnvironmental Laboratory .. .. Page63of92 . Page 303 3M Medicgl Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 -. . Centre Study No.:023-017 SponsorProtocol No:FAa-TOX-I 1I ' I PROTOCOL DEVIATION fipI CF.1 .. .. .. .. .CentreAnalytical Labontories,'Inc. 3fvEnvirrrmnental Laboratory , -. Page,64of92 Page 304 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994.-. Centre Study No.:023-017 Sponsor ProtocolNo:FACT-TOX-I 11 Centre Analytical Laboratories, LUG a-- boratory .. .. Page 65 of 92 Page 305 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.:023-017 . Sponsor Protocol No: FACT-TOX-111 APPENDIX I3 Determination of Fluorochemical Residues in Monkey/Rat Feces by - LC/MS/&lS (Revision 2) (Method #OOM-O23-003, revision 2), Deviations and Modfieations Centre Analytical Laboratories, Inc. ' 3M Environmental Laboratory Page 66of 92 Page 306 3M Medical DepartmentStudy: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-I 11 LRN-U2994.. . Centre Study No.:023-017 S,ponsorPfotocol No:FACT-TOX-l lI . . .__.. June 02,2000 SPONSOR , . . ... - 3 Centre Analytical L&omtories, Inc. ratory .. . Page 67of92 Page 307 3M Medicpl Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 . .... ~ .. ..LRN-U2994--. Centre Study No.: 023-0 17 Sponsor Protocol No: FACT-TOX-I 11 ..-...-.. _...._-- Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 68of 92 Page 308 3M Medic$ Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.:023-017 . SponsorProtocol No:FACT-TOX-111 TABLE OF C&I"lTS ' - -X~a~WE PA.4GGEE.W......N..A...TP...P...R....O......V...".A......L.....................................-.I.-....-I......,.................................._.I................-......................-.-....-...............................21 TABU . 1. S OF C W YO.._m....S..." .~..."............-....*.."................................."...........""................................................"..............................................34 . 2. 3. .'FC3.L1H.UEOwCRcwO&ELCwsXcEuAMsN.C.D..A.S_LU_SPT.P.A.I.JN..ED..S.A_..R"...D.:. .. . .. S. .. . .. . .. . .. . .. . .. . _. . . . . . _. . . . . . ........_...-............................................"......".".........;..........47 3t . 3.3.. ~EuQrom~~% clrmp,mncSsnTutAPNPD.m~RD....S.............................."............................._..".._.........:.......................... 7 7 7 3.4. S O L ~ O,.,N...~..,..... .....__..I. ............... .... ..... .... ..... .... ..... .... .... ..... .._I .. .... ..... .-. -.~..-......8. 35-P . ~~ 33.1. 3.52. FStonorctkiOEOsmoFtlSu-oTtfinOoSCnoKsI.,.uF..t..i.oO...n..s..R........".~.......~........o........N........,...C.....A....L...l.sUn.o....n......s........o.....".~.e..~"....~.......o.........*.p............,.......99~ - 4. IMETE3O3D3.....c.a.lib..r.a..t...i.o....n.. ... S.. .t;. a... .n.....'d.i ...a....r...d....s.........-.._.............I .""....................................__..........-.......-.............................:............................................._-...........1190I . . 4.1. FLOWDUG&% 4.2 SMLEPR-G ................................. 4.3. ... SWUPREPARMION .............. ......."..._.................".-I:..".- .- ..I ........"".....".........111 4.33. 4.32 iMatrix W.Z B&lanks..B...lank.s...........-....--...............................................................-......... ..........."a."-........."...,_ 11 12 43.3. CJIibntionstanandards..-...........1................. ........".-"........1122* ...I. 4-4.E,44X.33T..45R..ACCQ~CoOnRtNine...ic.:ao.i.nv_ge.rc.y.a.Sl.i.ab.m-rp.a.lt"ei.os..n.-.V..-&......&"..t..i..o,...n.........-.S.t..d...n_...d.a...r.d...S............,..........."I.......1..........".....".,.......-..............I..:.!.........12 I 4.4.1 SPEColumnPr~~ti..d...n.......................................... -....... ........".......I123 "- 44..44.32 SStilaanndiaprtdichntoiofnPof~S-PSECbopludRmnissk..s............................................................................."................1133 . . . 4.5. AWYSBBYLWNS ............-.-....."....-................................................. 14 . 45.1. .LWS/&ISSptm luld Op-g Co~dition@~ I e c -..~-.-I~4 ~, ~ ~ 44..553.2. EWxaimmpileoAnTnauyPSnrieSo.kW..c."deuPr.ey..s.a.met.a..s..................-...."._.i .-.................._...._".."-.-...-...._............I.6-"....I5 . 45.4. Sample 4.6. PERFORXW~CRITERIA. ....__...... -..... ...... -.._...._. "" I..._ ..I_. I .... .*.*-*.-*-.. . .......... 17 18 ~ 5.. . .... 4.7- TIMEREpurrtEDFOR CALCULATIONS 1 ANAtYsrS.--.......-.....-....."........~................. ........................"-.-..."-....""......Is . 5.1. ANALYE FO.......................................................... ~ ................ -."...IS IS . . . . ~.:!:QCRECOVERY 5.3. Mun R E ~ W .N."S...E..F..LA. C...~... .L........ ...."...... ........... ....-..... ....._.............................................. "......_.1.9 *.-.; ... A6.l-TS.r4uc.fxl?b(.m" T..I....-....................L...................". .I "I .". .... .."........................".................................. 19 19 - .. .........I.." 20 .- .-_. . Centre Analytical Laboratories, Inc. 3M EnvironmentalLaboratory Pgge.69 of 92 Page 309 3M Medicgl Department Study: T-6395.24 BACK TO MAIN Analytical Report: FACT TOX-1.?..1 t LRN-U2994 CentreStudy NO.:023417 ' Sponsor Protocol No:FACT-TOX-'lll ............... - .. ........ Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 70of 92 Page 310 3M Medicgl DepartmentStudy: T-6395.14 BACK TO MAIN Analyticai Report: FACT TOX-111 L. .RN-UZ99.4.... ~ Centre Study No.: 023-017 . SponsorProtocol No: FACT-TOX-111 PFOSEA Molecularweight: 527 .. .. . . CentreAnaIyticd Laboratories,Inc. 3M Environmental Laboratory Page 71 of 92 Page 311 3M Medical Department Study: T-6395.14 . .. .. BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994. .. . Centre Study No.: 023-017 ' SponsorProtocol No:FACT-TOX-I11 . . ....._____ ." . . Centre Analytical Laboratories, Jnc. 3M 'EnvironmentalLaboratory . .. , .. ~age'72o i 92 Page 312 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-1I 1 LRN-U2994 . Centre Study No.:023417 SponsorProtocol No:FACT-TOX-1I I .. 3.2. FLUOROCI~EMSICTAAWLARDS Sbddd Source PFOS 3M PFOSA 3M PFOsAri PFOSW 3M . 3M. Et-FOSE-OH 3M MS56 M570 POAA W F O S .. 34 3M . 3M 3M 3.3. E Q ~ ~ ~ ~ ~ A N D S ~ ~ ~ L I E ~ Centre Analytical Laboratories, hc. 3M Environmental Laboratory Page 73 of92 Page 313 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-I 11 LRN-U2994 Centre Study No.:023-017 . Sponsor Protocol No:FACT-TOX-111 - As dcirniod in Section 4.5.1 .. Centre AnaIytic$ Laboratories, Inc. . .. 3M Environmental Laboratory Page74 of92 Page 314 3M Medicpl DepartmentStudy: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994.-... Centre Study No.: 023-017 . SponsorProtocol No:FACT-TOX-111 Page 315 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 ,. Centre Study No.: 023-017 . Sponsor Protocol No: FACT-TOX-111 -..-........ ....-__. .conc. of FoniEation Wei& of ` Mixcd ' VoInmt@) Control Fortit?cation Sam~lek) Solution . , (Pdd NA NA 1.0 0.1 100 ` 1.0 0.1 200 1.0 0.5 roo 1 .o 0.5 200 1.0 2.5 ' 100 1.0 2.5 200 I .o 10 100 1.0 * 2.5 p g h l TBFOS fortbinxion solution. Conc. of vel. of Extracted s-gak ~iration stam* . addcaw) (PPW NA 10 20 .50 . 100 200 200 . 200 ZW 200 250 200 500 2--fl-o 1000 200 ' -.. ..... Centre Analytical Laboratories, Inc. 3M Environmental Laboratory .... Page 76 of 92 Page 316 3M Medical Department Study: T-6395.14 4. . MEmOD 4.1. FLOWD R G W V BACK TO MAIN Analytical Report: FACT TOX-1I 1 LRN-U2994 Centre Study No.: 023-017 . SponsorProtocol No:FACX'-TOX-111 ... ..__..... -. .. . .. . c w t C u y t i c a l k b a m w Inc.Study#023-003 .. .. ___ ....-._.___ .._.. Centre Analyiid.L-&bratoriei,~ n c . .. 3M EnvironmentalLaboratory .. ., 'P& 11 ' ' . . .. ,. .. ' Page 77 of 92 ., Page 317 3M Medical DepartmentStudy: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-11'l LRN-U2994, . Centre Study No.:023-017 SponsorProtocol No:FACT-TOX-111 . . . _._.....__.. .. . . ......- _._, ... ' ,centreAnalytical Labor;ltories, IIIC. 3M EnvironmentalLaboratory .. , . Page-78 of92 Page 318 3M Medical DepartmentStudy: T-6395.14 c e. : .. f. s BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U299. 4I-.'.. . P CeatreStudy No.:023-017 SponsorProtocoI No:FACT-TOX-11I Page 319 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 . Centre Study No.: 023-017 - SponsorqOtOCOI NO:FACT-TOX-111 0 0.4 . 1.0 7.0 1.5 9.0 9.5 153 14.0 -% A . 60 . 60 10 IO .o .O 60 ' 60 60 -%B - `40' 40 90 90 100 100 40 40 40 maw Ratc {mUrnin) . 0.SGO 0300 0~00 0300 0.300 . 0.400 * 0.400 0.400 :. 0300 -. ~ . Centre Analytical Laboratories, Inc. 3M Environmental Laboratory .. Pais80.of9.2 Page 320 3M Medical Department Study: T-6395.14 .. I .. BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.:023017 . SponsorProtocol No:FACT-TOX-1I1 . ...._. PFOS PFOSA PFOSAA PFOSEA EbF6SE-OH 'POAA * hU56 M57Q TXPFOS negative . n&e n@ve negative n@ve negative negative negative negative PYent Ion ' 499 498 . 584 . 526 630 . 413 556 570 427 - Prodnu -Ian 99 78 526 169 . 59 569 498 419 80 -%roxknate RetentionT h e Iminl 5.3 56.70 6.7 . 6.1 * 5.1 5.4 5.6 >. 5.1 - . . AnJMe ' PFOS PPFF OO UW ' ,' PFOSEA Er-FOSE.-OH: POAA 1W56 M570 . mF0S . Wfl4 - Dwell (SI 02 0.2 ' 0.2 02 0.2 0.2 0.2 0.2 0.2 Sbdy". 023-003 Col$io. ! Encr&(eq 43 28 . 20 ' 25 . .;I . I1 , .25 .20 ' 35. . . Conem , 76 34 . 57 49 30 25 . 50 . 60 ' 54 . . Pasc.15 ' .. .' Centre AnalyticalLaboratories,Inc. 3M Environmental Laboratory ... Page81 of 92 Page 321 3M Medicpl Department Study: T-6395.14 .. BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 '- CentreStudy NO.:023-017 . Sponsor Protocol No:FACT-TOX-11I . _. .--. Ccntre?rl&od No.: m&otj.~~zd , o.n . Set 2.56kV 0.5 v 0.2 v 0.8 v 1oooc 400C, -Set 13.0 V 12.0 v . 0.7V -2v . 1v 11.0 v 11.0 v 1.0 v 650 V -Rmd back 3.Oe-3 mbar . . . - I .... Centre Analytical Laboratories. Inc. 3M Environmental Laboratory . PJSC16 .. .. Paggd'82of 92 Page 322 3M Medicjal Department Study: T-6395.14 . . _.--..- BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 . Centre Study No.: 023417 . SponsorProtocol No:FACT-TQX-111 , ... .-... , . . .. .. -.. ...-..___. .-. .. C e n e Analytical Laboratories, Inc. 3M EnvironmentalLaboratory Page 83 of 92 Page 323 3M Medicpl DepartmentStudy: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 SponsorProtocolNo:FACT-TOX-111 4.6. '.PERFORMA,,CEC " E U A 5.1. ANAL.Y~EFOUM~ Equation 1: &ytc found (ppb) = @e& iuu -intaCqt) XDF, . .. slope . Whm DF =dilutionfaor, if syaples were diluted .-. __..._.... . Centre Analytical LabOmtoI'kS,hc-' 3M EnvironmentalLaboratory Page 84 of 92 Page 324 3M Medicpi Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Centm Study No.:023-017 SponsorProtocol No:FACT-TOX-111 ... -. .-. . ... ,._..__... - anaiytehmdbb) aridytefoundinrnatrixblank(ppb) . arrioont dyteadded@pb) XI00 5.3. IMEANRESPONSEFACTOR ..._-.._...._. . Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Pnge19. . .. . a. Page 85 of 92 Page 325 3M Medical Department Study: T-6395.14 A'ITAWJXNT 1. BACK TO MAIN Analytical Report: FACT TOX-111 . .. LRN-U2994. . Cenae Study No.: 023-017 ' Sponsor Protocol No: FACT-TOX-Ill Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page26 of92 Page 326 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.:023-017 Sponsor Protocol No: FACT-TOX-111 .. ACTIONSTAKN Le. amendment isnred. SOP rcvidon. ctc.., . Method deviationissue. Centre Analytical Laboratories, Inc. 3M Environmental Laboratory ' P a s 87 of 92 Page 327 3M Medic9I 1DepartmentStudy: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.:023-017 . . ' . SponsorProtocolNo:FACT-TOX-111 Centre Analytical Laboratories,Inc. , 3M Environmental Laboratory . , . ' Page's8of92 Page 328 3M Medical DepartmentStudy: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRv-U2994 ' Centre Study No.: 023417 Sponsor Protocol No: FACT-TOX-111 . Centre Analytic+ Laboratories, hC. 3M Environmental Laboratory Page 89 of92 Page 329 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.:023-017 SponsorProtocol No:FACT-TOX-111 No negative impact. .. .. .. . Centre Andytical.Labontorks,Inc. 3M Environmental Laboratory Page '90 of 92 Page 330 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-1I 1 LRN-U2994 Centre Study No.:023-017 . Sponsor Protocol No:FACT-TOX-I11 -OD iLIODIFICATION FORM - h m StudvN u m b 023-017 Modification No.: 2 grudv Directpl: MYvinT. C3sc s- S Page 1 of 2 FACl'*TOX- i 11 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory ... Page 91 of 92 Page 331 3M Medical Department Study: T-6395.14 BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Centre Study No.: 023-017 SponsorProtocol No:FACT-TOX-111 .. ~ METXIOD MODIFICATION F 0 R ; i ' Page 3 of2 13. No ocg~tiveimpact Centre AnalyticalLaboratories,Inc. 3M EnvironmentalLaboratory Page 92 of 92 Page 332 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LHN-U2994 Cazntrcz Analytical Laboratories. Inr. 3048RcKprch Drive, State Cdlege,PA 16801. Phone: (814) 231-8032,Fadmile: (814)231-1253 12 Amended pages + report amendment ANALYTICAL PHASE REPORT AMENDMENT RE-ISSUED AmendmentNumber: 1 Effective Date: 9/2O/OQ Zentre Study Number: 023-017 SponsorProtocol Number: FACT-TOX-111 Report Title Oral (Gavage) PharmacokineticRecovery Study of PFOS in Rats Amended Section Section 1.0Summary: 13548.2ppb was changed to 13.5 pglg in the third paragraph (See attached Page 11). Section 7.6Quantitationand Example Calculation: Equation5: ResidueFound (pglg) = Residue Found (nglg) * (1pg/ loo0ng) Bquation6 Corrected ResidueFound (pglg) =ResidueFound (pglg) * 0.869 mese equationswere only appliedto the tables in the report. Section 8.0 Results: 13548.2 was changed to 13.5 pg/g in the second paragraph (See attached page 18). Section 11.0 - Tables I XVI (See - attachedpages 20 28) Reason for Amendment The units reported in the tables and text were changed Rom ngfg to pg/g. the amount of significant figuresrepresented in the tables and text was changed to three,the zeros reported in Table I were changed to ND's. and any value less than 0.0100 pglg (LOQ) was reported as c 0.0100. No negative impact Study Director S i g n a m Impact on the Study -Date * Rick Grazzini signing for Enaksha Wickremesinhe who is no longer employed at Centre. CAL QAU Review *uI z!LI!pd November 199510 3M Environmental Laboratory Page 333 3M Medical Department Study: T-6395.14 Appendix H: Certificate of Analysis BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 3M EnvironmentalLaboratory Page 334 BACK TO MAIN 3M Medical Department Study: T-6395.14 . - Analytical Report: FACT TOX-111 LRN-U2994 C m t w Analytical Laboratorie%Inc. 3048 Research Drive State College, PA 16801 / Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 ! INTERIM CERTIFICATE OF ANALYSIS Revision 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023-018B 3M Product: PFOS,Lot 171 Reference #: SD-009 1. Calcium 2. Magnesium 3. Sodium 4. PotassiumZ 5. Nickel 6. Iron -:GC/MS) 1. 0.017wt.lwt.% 2. 0.007 wt.lwt.% 3. 1.355 wt./wt.% I 4. NFPA I Elemental Analysis? 1. Carbon 2. Hydrogen 3. Nitrogen c 4. Sulfur 5. Fluorine COA023-018B 3M Environmental Laboratory 1. Theoretical Vah e = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% 4. <0.25 wt.fwt.% 1. 12.08 wt.fwt.% 2. 0.794 wtJwt.% 3. 1.61 wt.fwt.% 4. 10,lwtJwt.% 5. 50.4 wt./wt.% Page 1 of 3 Page 335 BACK TO MAIN 3M Medical DepartmentStudy: T-6395.14 Analytical Report: FACT TOX-111 a\ LRN-U2994 CEntrcE Analytical laboratories, Inc. 3048 Research Drive State College, PA 16801 f Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B Date of Last Analysis: 08131/00 ExpirationDate: 08/31/01 Storage Conditions: Frozen <-lOC Re-assessment Date: OW31/01 - 'Purity = 100% (sum of metal impurities, 1.39% +LC/MS impurities, 10.60%+InorganicFluoride, 0.27%+- impurities, 1.00%+ PO& 0.30%) Total impurity fi-om all tests = 13.56% Purity = 100% - 13.56% = 86.4% zPotassiumis expected in this salt form and is thereforenot consideredan impurity. 'Purity by DSC is generally not applicableto materialsof low purity. No endothermwas ( observed for this sample. 4 S u lin~the sample appears to be converted to SO4 and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determinationin the elemental analysis,lendingconfidenceto this interpretation. Based on the results, the SO4 is not considered an impurity. HFBA NFPA PFPA Trifluoroacetic acid Heptqfluorobutyrk acid Nonofluoropentanoic acid Pentafluoropropanoicacid 6Theoreticalvalue calculationsbased on the empiricalformula, C S F ~ ~ S O ~(MXW+=538) This work was conductedunder EPA Good LaboratoryPractice Standards(40 CFR 160). (. COAO23-018B 3M Environmental Laboratory Page 2 of 3 Page 336 BACK TO MAIN 3M Medical Department Study: T-6395.14 Analytical Report: FACT TOX-111 LRN-U2994 3048 Research Drive Phone: (814) 231-8032 State College, PA 16801 Fax: (814) 231-1253 or (814) 231-1580 INTEHM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-01SB LC/MS Purity Profile: 1 Imunritv wt./wt. Yo I c4 I 1.03 I 1- C6 I 6.38 I 1 Total 1 10.60 Note: The C4 and C6 valueswere calculated'usingthe C4and C6 standardcalibration curves, respectively. The C5 value was calculated using the averageresponsefactors from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves. PreparedBY: , K ~- H Davd S. Bell ?A/- Date ytical Laboratories f?4b Date anager, Centre Analytical Laboratories c COA023-018B Page 3 of 3 3M Environmental Laboratory Page 337 BACK TO MAIN 3M Medical Department Study: T-6395.14 1 Analytical Report: FACT TOX-11I LRN-U2994 am\ CEntrE Analytical Laboratorks, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERM CERTIFICATE OF ANALYSIS Revision 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023-018A 3M Product: PFOS,Lot 217 Reference #: SD-018 Purity: 86.9% 4. Potassium' (. 2. Fluoride c. COAO23-01SA 3kl-nvkonrr~ental Laboratory 1. 0.005 wt./wt.% 2. 0.001 wt./wt.% 3. 1.439 wt./wt.% 1. e0.015 wt./wt.% 2. 0.59wtJwt.% 3. <0.040 wt./wt.% 4. <0.009 wt./wt.% 5. c0.006 wt./wt.% 6. c0.007 wt./wt.% 2. co.1 wt./wt.% 2. 4 . 1 wt./wt.% 3. 0.10 wt./wt.% 1. 12.48 wt./wt% 2. 0.244 wt./wt.% 3. 1.74wt./~t.% 4. 8.84 wt./wt.% 5. 54.1 wt./wt.% Page 1 of 3 Page 338 BACK TO MAIN 3M Medical Department Study: T-6395.14 I Analytical Report: FACT TOX-111 LRN-U2994 am\ Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTER,IM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A Date of Last Analysis: 08131/00 Expiration Date: 08/31/01 StorageConditions: Frozen 510C Re-assessmentDate: 08/31/01 'Purity = 100% - (sum of metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.93%+0rganicacid impurities,0.38%+POAA, 0.33%) - Total impurity fkom all tests = 13.09% Purity = 100% 13.09% = 86.9% 'Potassium is expected in this salt form and is thereforenot considered an impurity. 3Purityby DSC is generally not applicableto materialsof low purity. No endothermwas observed for this sample. 4Sulfurin the sample appears to be converted to so4 and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity. 'TFA HFBA NFPA PFPA Trifluoroacetic acid Heptaffuorobutyric acid Nonofluoropentanoic acid Pentafluoropropanoic acid 6Theoreticalvalue calculationsbased on the empirical formula,C S F ~ ~ S O(~MXw+=538) This work,wasconducted under EPA Good LaboratoryPractice Standards (40 CFR 160). COAO23-018A - 3 'Laboratory Page 2 of3 Page 339 BACK TO MAIN 3M Medical DepartmentStudy: T-6395.14 I Analytical Report: FACT TOX-111 LRN-U2994 k Phone: (814) 231-8032 State College, PA 16801 Fax: (814) 231-1253 or (814) 231-1580 INTERZM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A LCMS PurityProfile: Impurity c4 c5 C6 c7 Total wt./wt. % 1.22 I .33 I 4.72 I 1.14 8.41 Note: The C4 and C6 values were calculatedusing the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average response factors fiom the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves. Prepared B y YjaIDate Reviewed By: &a Date Laboratory Manager, Centre Analytical Laboratories c COAO23-0 18A Page3 of3 3M Environmental Laboratory Page 340 3M Medical Department Study: T-6395.14 Appendix I: Report $ignature Page BACK TO MAIN Analytical Report: FACT TOX-111 LRN-U2994 Maw'n T. Case, D.M.V., Ph.D., Study Director William K. Reagen, Ph.D., Laboratory Manager 1 date ur%53/4/ Date - . John L. Butenhoff, Ph.D., Sponsor Representative - kris J. Hansen, Ph.D., Analytical lnvestigator Date o(-/QY JO / Date 3M Environmental Laboratory Page 341