Document bB50pO1jXEgxXmOKd296NvNLo

3M IMedical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Study Title Oral (Gavage) Pharmacokinetic Study of PFOS in Rats Analytical Laboratory Report Title Determination of the Presence and Concentration of Perfluorooctanesulfonate (PFOS) in Serum, Liver, Urine, and Feces Samples Data Requirement' Not Applicable Author 3M Environmental Laboratory Study Completion Dipte at signing Performing Laboratories Urine Analyses Feces Analyses 3M Environmental Laboratory Building 2-3E-09,935 Bush Avenue St. Paul, MN 55106 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Liver Analyses Battelle Memorial Institute 505 King Avenue Columbus, OH 43201-2693 Serum Analyses Advanced BioanalyticalServices, Inc. 15 Catherwood Road Ithaca, NY 14850 Project Identification 3M Medical Department Study: T-6295.12 Argus In-Life Study: M18-013 Analytical Report: FACT TOX-110 3M Laboratory Request No. U2849 Total Number of Pages 354 3M EInvironrnental Laboratory Page 1 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 This page has been reserved for specific country requirements. 3M Environmental Laboratory Page 2 BACK TO MAIN 3M IWedical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 GLP Compliance Statement Analytical Laboratory Report Title: Determination of the Presence and Concentration of Perfluorooctanesulfonate (PFOS) in Serum, Liver, Urine and Feces Samples Study Identification Numbers:T-6295.12, FACT TOX-110, LRN-U2t349 This study was conducted in compliance with United States Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations 21 CFR Part 58, with the exceptions in the bulleted list below. Exceptions to GLP compliance: 0 Separate study directors were assigned to lead the in vivo and analytical portions of the study. 0 Some sample receipt data from specimens received on 29 January 1999 are missing the corresponding receipt acknowledgement forms 0 The study files do not contain the raw data documenting sample receipt for the Gestation Day 0 urine samples. 0 The original protocol called for study compliance with the FDA final rule 21 CFR Part 58; however, documentation from Advanced Bioanalytical Services, Inc. (the contract lab responsible for serum analyses) cites compliance with the EPA final rule 40 CFR Part 792. 0 Dose confirmation analyses were not performed in compliance with GLP regulations. 0 Sample WeightsNolumes worksheets for extraction, TOX-1I O , pages 2, 5, 9 and 11, on 9/13/99, are not completely attributable to the analyst performing the extraction. 0 Some of the "Argus Acknowledgement of Tissue Specimen Receipt" forms are missing the date and "reviewed by" signature. 0 Sample receipt documents for the 21Dec98 samples are not available. 0 The LC/MS instrument run information for the 20Jan99 samples, received on 1/22/99, are not available. 0 Not all reagent and solution bottles were labeled with an expiration date. 0 The analytical report from Advanced Bioanalytical Services, Inc. does not include the names of all of the participating scientists and professionals. 0 The stability of test and control articles under the conditions of administration were not determined. (Signatures on next page) 3M Einvironmental Laboratory Page 3 3M Medical DepartmentStudy: T-6295.12 Marvh T.' Case, D.M.V., Ph.D., Study Director Kris J. Hansen, Ph.D., Analytical investigator WilliGm Reagen, Ph.D.,-LaboratoryManager BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 w2.24 A d / I3aH 65/09 / 01 Date orA3A!/ Date 3N Environmental Laboratory Page 4 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 GLP Study-Quality Assurance Statement Analytical Laboratory Report Title: Determination of the Presence and Concentration of Perfluorooctanesulfonate(PFOS) in !Serum, Liver, Urine and Feces Samples Study Identification Numbers: T-6295.12, FACT TOX-110, LRN-U2849 This study has been inspected by the 3M Environmental Laboratory Quality Assurance Unit (QAU) as indicated in the following table. The findings were reported to the study I director and laboratory management. Inspection Dates Phase Date Reported to Management I Study Dlrector 5 Oct 1999 6 Oct 1999 Analytical Set-up I MS Data Reduction 13 Oct 1999 to 16 Dec 1999 First Draft Report 13 Oct 1999 1 13 Oct 1999 13 Oct 1999 13 Oct 1999 17 Jan 2000 17 Jan 2000 11 sept 2000 to 12 sept 2000, J I 18 Sept 2000, I 20 Sept 2000 to 22 Sept 2000, 25 Sept 2000 to 26 Sept 2000 Report 27 Sept 2000 27 Sept 2000 See contractor's analytical reports (Appendix G) for contract lab Quality Assurance statements. QAU Repredbntative Date 3M Environmental Laboratory Page 5 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Table of Contents GLP Compliance Statement.............................................................................................. 3 GLP Study-Quality Assurance Statement ....................................................................... 5 Study Personnel and Contributors..................................................................................... 10 Introduction and Purpose.................................................................................................. 11 Test System ................................................................................................................. 11 Specimen Collection..................................................................................................... 12 Specimen Receipt and Maintenance................................................................................. 12 Chemical Characterization................................................................................................ 13 Procurement................................................................................................................. 14 Stability Studies ............................................................................................................ 14 Dose Confirmation Analyses ........................................................................................ 14 Method Summaries ........................................................................................................... 14 3M Environmental Laboratory....................................................................................... 15 Deviations..................................................................................................................... 17 Data Quality Objectives and Data Integrity........................................................................ 17 Data Summary, Analyses, and Results ............................................................................. 17 Summary of Quality Control Analyses Results.............................................................. 17 Statement of Data Quality ............................................................................................ 18 Summary of Sample Results ........................................................................................ 19 Statistical Methods and Calculations ................................................................................. 19 Statement of Conclusion ................................................................................................... 19 Appendix A: Control Matrices and Dose Confirmation Analyses........................................ 20 Appendix 6: Protocol, Protocol Amendments and Deviation Summary ............................. 22 Appendix C: Extraction and Analytical Methods ................................................................ 55 ETS-8-96.0, "Extraction of Potassium Perfluorooctanesulfonateor Other FluorochemicalCompounds from Urine for Analysis Using HPLS-Electrospray/Mass Spectrometry/Mass Spectrometry," (14 pages) ............................................................. 56 ETS-8-97.0, "Analysis of Potassium Perfluorooctanesulfonateor Other FluorochemicalCompounds in Urine Extracts Using HPLC-Elect:rospray/Mass Spectrometry/MassSpectrometry," (10 pages) ............................................................. 70 Appendix D: Data Summary Tables .................................................................................. 80 Appendix E: Data Spreadsheets ........................................................................................ 83 Appendix F: Example Calculations .................................................................................... 97 Appendix G: Contract Lab Reports..................................................................................... 98 Battelle Memorial Institute, "Oral (Gavage) Pharmac:okinetic Study of PFOS in Rats," 73 pages ..................................................................................................................... 99 Advanced Bioanalytical Services, lnc., "Method Validation for the Quantitation of Perfluorooctanesulfonate(PFOS) in Rat Serum by Turbo Ion Spray LC/MS," 50 pages ....................................................................................................................... 172 3M E:nvironrnental Laboratory Page 6 BACK TO MAIN 3M IMedical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Advanced Bioanalytical Services, lnc., "Quantitative Determination of Perfiuorooctanesulfonate(PFOS) in Rat Serum from Study #FAC T-TOX-110 Using Turbo Ion Spray LC/MS," 24 pages .............................................................................. 214 Advanced Bioanalytical Services, Inc., "Addendum--Q uantitative Determination of Perfiuorooctanesulfonate (PFOS) in Rat Serum from Study #FACT-TOX-110 Using Turbo ion Spray LC/MS," 3 pages ................................................................................ 239 Centre Analytical Laboratories, Inc., "Oral (Gavage) Pharmacokinetic Study of PFOS in Rats," 103 pages ...................................................................................................... 243 Appendix H: interim Certificate of Analyses ...................................................................... 347 Appendix I: Report Signature Page ................................................................................... 354 3M Environmental Laboratory Page 7 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 List of Tables Table 1. Female Rat Population Demographics for Study #418-013 ................................. 11 Table 2. Custody Transfer of Specimens in the Study of the Determinationof the Presence and Concentration of PFOS in the Sera, Liver, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-6295.12 via Gavage ........................... 13 Table 3. Characterizationof Test Article ........................................................................... 14 Table 4. Negative Ions Monitored in 3M Laboratory Analyses .......................................... 16 Table 5. Description of the Control Matrices Used for Liver, Urine, Sera and Feces Analyses in Study FACT TOX-110....................................................................... 20 Table 6. Characterization of the Analytical Reference Substances used for Liver, Urine, Sera and Feces Analyses in Study FACT TOX-110 ............................................. 20 Table 7. Dose ConfirmationAnalyses for Perfluorooctanesulfonatefor Test Samples From Study In-life ##418-013-15 November 1998 .............................................. 21 Table 8. Deviation Summary for FACT TOX-110 ............................................................ .22 Table 9. Average Results for the Analyses of Serum Samples in the Study of the Determinationof the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-6295.12 via Gavage ................................................................................................................ 80 Table I O . Average Results for the Analyses of Liver Samples in the Study of the Determinationof the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-6295.12 via Gavage ............................................................................................................... .80 Table 11. Average Results for the Analysis of Urine Samples in the Study of the Determinationof the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-6295.12 via Gavage ................................................................................................................ 81 Table 12. Average Results for the Analysis of Feces Samples in the Study of the Determination of the Presence and Concentralion of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAFIPIUS@Rats Exposed to T-6295.12 via Gavage ................................................................................................................ 81 Table 13. LOQ Values Used in FACT TOX-110 Analyses................................................ .82 Table 14. Results of Analyses of Urine Samples in the Study of the Determinationof the Presence and Concentration of PFOS in the Sera, Livers, IJrine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-629!j.12 via Gavage .......................... .83 Table 14. Results of Analyses of Urine Samples in the Study of the Determinationof the Presence and Concentration of PFOS in the Sera, Livers, IJrine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-629!5.12 via Gavage (Continued)........84 Table 14. Results of Analyses of Urine Samples in the Study of the Determinationof the Presence and Concentration of PFOS in the Sera, Livers, IJrine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-629!5.12 via Gavage (Continued)........85 3M Environmental Laboratory Page 8 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Table 15. Results of Analyses of Serum Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-6295.12 via Gavage ....................... 86 Table 15. Results of Analyses of Serum Samples in the Study of the Determinationof the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-6295.12 via Gavage (Continued)....87 Table 15. Results of Analyses of Serum Samples in the Study of the Determination of the Presence and Concentration of PFOS in ttie Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposedto T-6295.12 via Gavage (Continued)....88 Table 15. Results of Analyses of Serum Samples in the Study of the Determinationof the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-6295.12 via Gavage (Continued)....89 Table 15. Results of Analyses of Serum Samples in the Study of the Determinationof the Presence and Concentration of PFOS in ttie Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-6295.12 via Gavage (Continued)....90 Table 16. Results of Analyses of Feces Samples in the Study of the Determinationof the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-629!5.12 via Gavage ........................... 91 Table 16. Results of Analyses of Feces Samples in the Study of the Determinationof the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-629!5.12 via Gavage (Continued)........92 Table 16. Results of Analyses of Feces Samples in the Study of the Determinationof the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-629!5.12 via Gavage (Continued)........93 Table 17. Results of Analyses of Liver Samples in the !Study of the Determinationof the Presence and Concentration of PFOS in the Sera, Livers, IJrine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-629!j.12 via Gavage .......................... .94 Table 17. Results of Analyses of Liver Samples in the !Study of the Determinationof the Presence and Concentration of PFOS in the Sera, Livers, IJrine, and Feces of Crl:CD@BRVAF/Plus@Rats Exposed to T-6295.12 via Gavage (Continued)........ 95 Table 17. Results of Analyses of Liver Samples in the !Study of the Determinationof the Presence and Concentration of PFOS in the S,era,Livers, IJrine, and Feces of Crl:CD@BRVAF/PIUS@Rats Exposed to T-6295.12 via Gavage (Continued)........96 3M Environmental Laboratory Page 9 ~~ 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Study Personnel and Contributors Study Director Sporisor Marvin T. Case, D.V.M., Ph.D. 3M Corporate Toxicology 3M Center, Building 220-2E-02 St. Paul, MN 55133-3220 (651)733-5180 3M Corporate Toxicology 3M Center, Building 220-2E-02 St. Paul, MN 55133-3220 John L. Butenhoff, Ph.D., Sponsor Representative Analytical Chemistry Laboratories Urine Analyses Liver Analyses 3M Environmental Laboratory Battalle Memorial Institute Kristen J. Hansen, Ph.D., Analytical lnvestigator Jon (2. Andre, Ph.D., Analytical lnvestigator Serum Analyses Advanced Bioanalytical Services, Inc. Dave Anderson, M.S., Analytical lnvestigator Feces Analyses Centre Analytical Laboratories Enaksha Wickremesinhe, Ph.D., Analytical Investigator 3M Environmental Laboratory Contributing Personnel David R. Barnidge" Lisa A. Clemen Kelly J. Dorweilei Mark E. Ellefson Sara E. Estes' Barb A. Gramenz' Sarah A. Heimdal' Cari S. Hewitt' .Marlene M. Heying' Contract lab professionalservice employees Harold C). Johnson Theresa King-Huntei Kelly J. Kuehlwein' Sally A. Linda* Joseph (2. Pilon* Scott R. Post* Ian A. Simith' Anh-Dao Vo' Bob W. Wynne' 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 from Battelle Memorial Institute, Centre Analytical Laboratories and Advanced Bioanalytical Services, Inc. (ABS) will be stored at the contract laboratories in such a mariner that the control substances will be preserved for use in ensuing studies. 3M EEnvironmental Laboratory Page 10 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 ~~ Introduction and Purpose The purpose of the study is to determine the presence and concentration of perfluorooctanesulfonate(PFOS) in serum, liver, urine and feces samples taken from the Gavage Study of T-6295.12(PerfluorooctanesulfonateCAS Number: 2795-39-3)in Crl:CD@BRVAF/PIUS@Rats. The study was initiated on 10 June 1999 and completed on 8 November 1999. Test System The test system species and strain selected was the Crl:CD@BRVAF/PIUS@(Sprague- J Dawle ) rat received from Charles River Laboratories, ilnc., permanently identified using Monel self-piercing ear tags. FO virgin female rats were at least 60 days of age and weighed approximately 200-225 g when received. Male rats of the same source and strain were used only as breeders and were not administered the test article or considered part of the test system. Five groups consisting of 16 female rats each (total of ;30rats) were used as the test system. All female rats were treated with the control or test article once daily beginning 42 days prior to cohabitation, and continued through day 14 or day 20 of presumed gestation. Table 1 presents the number of female rats in each group. Group I consisted of control FO female rats that were administered 0.0 mg/kg/day in 0.5% Tween 80 (vehicle). Group II through Group V FO female rats were administered 0.1,0.4, 1.6,or 3.2mg of PFOS per kg of body weightlday in 0.5% Twleen 80.At predetermined intervals during and at the end of the in-life phase of the study, sera, liver, urine and feces samples were taken from dams for analyses. Additionally, liver and serum samples were taken from the fetuses. Details of the in-life phase of the present study are presented in the Argus Laboratory final report, "Oral (Glavage) Pharmacokinetic Study of PFOS in Rats, M18-013." Table 1. Female Rat Population Demographics for Study #418-013 I Treatment Group (total) I 64 I EA I Group I I 16 1: 6 0.1 mg/kg/day Group I l l 16 16 0.4 mg/kg/day Group IV 16 16 1.6 mg/kg/day 3.2Gmrogu/kpgV/day 16 16 3M Environmental Laboratory Page 11 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Specimen Collection In the analytical study reported here, 822 serum, urine, liver and feces specimens were collected from adult female rats before mating and at gestation day (GD) 7, GD 15 and GD 21 and sent to the 3M Environmental Laboratory and the contract labs to be analyzed for PFOS. A total of 54 pooled serum and liver specimens were collected from fetuses. Specimens of pooled embryo/placenta, pooled liver/lung, pooled liver/placenta/liver-lungs,amniotic fluid, and carcasses from fetuses, as well as milk secreting glands and placentas from adult females, were collected by and received from Argus In-life Study W18-013, but were not a part of the scope of this study as determined by the study director. The number and type of specimens collected for analyses in the analytical phase of this study are presented below. Specimens Collected from Study Groups 1 through 5 (through 1/22/99): Serum Specimens-277 specimens Liver Specimens-I 00 specimens Urine Specimens-249 specimens Feces Specimens-250 specimens Blood specimens were separated by refrigerated centrifugation. Serum specimens were then harvested, immediately frozen on dry ice and maintained frozen at -70C or below until being shipped to the 3M Environmental Laboratory. Urine and feces samples were collected into centrifuge tubes, placed on dry ice and stored frozen at -70C or below until being shipped to the 3M Environmental Laboratory. Liver specimens collected from each animal were collected, frozen and then stored at -70C or below on dry ice until being shipped to the 3M Environmental Laboratory. All specimens were shipped to the 3M Environmental Laboratory frozen and on dry ice. Specimens were then shipped to the contract analytical laboratories for analysis. Urine samples were extracted beginning on 13 September 1999 using an ion-pairing reagent and methyl-tert-butyl ether (MtBE). Sample extracts were analyzed using highpressure liquid chromatography-electrospray/tandem mass spectrometry (HPLC-ES/MS/MS) in the multiple response monitoring mode. PFOS levels were quantitated by external calibration. Analytical details are included in this report. Specimen Receipt and Maintenance The 3M Environmental Laboratory received serum, liver, urine and feces specimens that were collected during premating, GD7, GD15 and GD21 of the in-life phase of this study W18-013 from Argus. All specimens were received frozen on dry ice and were immediately transferred to storage at -20C *I0C. Specimens that were analyzed at Battelle Memorial Institute (liver), at Advanced Bioanalytical Sciences-ABS (serum), or at Centre Analytical Laboratories (feces) were subsequently shipped frozen on dry ice. Table 2 lists specimen receipt information for this study. 3M Environmental Laboratory Page 12 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 ite Received February 1999 February 1999 I January 1999 I January 1999 February 1999 29 January 1999 February 1999 February 1999 February 1999 Date Shipped to Lab 19 August 1999 lSJune1999 - a 25 May 2000 19 August 1999 15 June 1999 Chemical Characterization Potassium Perfluorooctanesulfon*ate(KPFOS) CAS Number: 2795-39,-3 Chemical Formula: C8F&30iK+ Molecular Weight: 537.9 Chemical characterization information on potassium perfluorooctanesulfonate, CAS #2795-39-3, is presented in Table 3. 3M EInvironmental Laboratory Page 13 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Table 3. Characterization of Test Article Test Article" Chemical Name KPFOS Potassium Perfluorooctanesulfonate 3M Toximlogy Services, Medical Department I I I ExpirationDate I I I Storage Conditions ~~ I Chemical Lot # ~ 8/31101 Frozen 5 -10C 217 Physical Description Purity a The test article is defined as the substance gavaged to the test system. See Interim Certificate of Analysis in Appendix H for complete characterization information. Procurement Potassium 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 mg/mL to 0.64 mg/mL) collected on 15 November 1998 during the in-life phase of the study. The results are presented in Appendix A (see Table 8).The dose confirmation data were collected according to a method that was not fully validated. Dose confirmation was performed by diluting the dose samples with methanol into the linear range of the instrument. In all cases, samples were analyzed versus an unextracted calibration curve using HPLC-ES/MS/MS. Method Summaries Following is a brief description of the methods used during this analytical study by the 3M Environmental Laboratory and by contract labs. The methods used in this study are located in Appendix C and G. The methods and analytical equipment settings used by Battelle Mtsmorial Institute, Advanced BioanalyticalServices, Inc. and Centre Analytical Laboratories, Inc. are presented in the respective contract lab reports (see Appendix G). 3M Environmental Laboratory Page 14 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 3M Environmental Laboratory PREPARATORMY ETHOD 0 ETS-8-96.0, "Extraction of Potassium Perfluorooctanesulfonateor Other FluorochemicalCompounds from Urine for Analysis' Using HPL!S-Electrospray/Mass Spectrometry/Mass Spectrometry" This method is used to extract PFOS from urine by the use of an ion-pairing reagent and methyl-tert-butyl ether (MtBE). An ion-pairing reagent (tetrabutyl ammonium hydrogen sulfate) is added to 2 mL of the sample, and the analyte-ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 0.5 mL of methanol, then filtered through a 0.2 pm nylon filter attached to a 3-cc plastic syringe into glass autovials. Modification to ETS-8-96.0: Although the rat urine Lised to generate method validation data came from the same type of rat as the test animals, the validation rats and test animals were obtained from different suppliers. Upon analysis of urine extracts from the test animals, it was determined that an interferent, not present in the validation samples, was present in the urine extract of the test animals. The unidentified interferent proved problematic only in the analysis of the surrogate (THPFOS). As written, ETS-8-97.0 describes the quantitation of PFOS with respect to a surrogate. Due to the presence of the interferent in the surrogate analysis of the test animals, all quantitations for this study were conducted by external calibration (i.e. the surrogate was not used in any calculations). ANALYTICALMETHODS ETS-8-97.0, "Analysis of Potassium Perfluorooctanesulfonate or Other FluorochemicalCompounds in Urine Extracts Using HPLC-Electrospray/Mass Spectrometry/Mass Spectrometry" The analyses were performed by monitoring one or more product ions selected from a single primary ion characteristic of a particular fluorochemical using HPLCES/MS/MS. For example, molecular ion 499, selectced as the primary ion for PFOS (C8Fi7S03-)analysis, was fragmented further to produce ion 99 (FSO3-). The characteristic product ion 99 was monitored for quantitative analysis. 0 Battelle Preparatory and Analytical Method: Method for Analysis of Potassium Perfluorooctanesulfonate(PFOS) in Rat Liver by LC/MS/MS 0 ABS Preparatory and Analytical Method: Method Validation for the Quantitation of Perfluorooctanesulfonate(PFOS) in Rat Serum by `Turbo Ion Spray LC/MS 0 Centre Preparatory and Analytical Method: OOM-023-003(Revision 2), Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS 3M Environmental Laboratory Page 15 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 ANALYTICAELQUIPMENT The analytical equipment settings used in the analysis of urine samples varied slightly during actual data collection. The following is representative of the settings used during the analytical phase of this study by the 3M Environmental Laboratory. Liquid Chromatograph: Hewlett-Packard@Series 1100 Liquid Chromatograph system analytical column: Keystone@BetasilTMCIS 2x50 mm (5 pm) Column temperature: Ambient Mobile phase components: Component A: 2 mM ammonium acetate Component B: methanol Flow rate: 300 pL/min Injection volume: 10 pL Solvent Gradient: Time (min) 0.0 1.o 4.5 6.5 7.0 %B 40%B 40%B 95%B 95%B 40%B Mass Spectrometer: Micromass@API/Mass Spectrometer Quattro II" Triple Quadrupole system Software: Mass Lynx" 3.1 Cone Voltage: 50-70 V Collision Gas Energy: 30-50 eV Mode: Electrospray Negative Source Block Temperature: 125"C-150C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (MRM) Table 4. Negative Ions Monitored in 3M Laboratory Analyses Target Analyte I Primary Ion (MU) I Product Ion (MU) I I PFOS I 499.0 I 99.0 I THPFOS 427.0 80.0 3M Environmental Laboratory Page 16 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-I 10 LRN-U2849 Deviations It should be noted that as the analytical phase of this study progressed, method parameters were evaluated to improve analyses. Although the methods were validated using internal calibration, it was necessary to use exter'nalcalibration for quantitation in the study, due to an unknown contaminant that interfered with the surrogate response. Deviations from the original protocol and methods are documented in Appendix B. Data Quality Objectives and Data Integrity The following data quality objectives (DQOs) were indicated in the protocol for this study: 0 Linearity: The coefficient of determination (r2)equal to or greater than 0.98 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 0 Acceptable Spike Recoveries: 50-1 50% 0 Demonstration of Specificity: Specificity will be demonstrated by chromatographic retention time and mass spectral daughter ion characterization. Data Summary, Analyses, and Results Data quality objectives for the analytical phase of this study outlined in the 3M Environmental Laboratory protocol for FACT TOX-110 (see Appendix B) were met with the exceptions noted in this report. Summary of Quality Control Analyses Results 0 Linearity: The coefficient of determination (rz) of the standard curve was 20.980. 0 Calibration Standards: Quantitation of the target analytes wax 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 deactivated to provide a better linear fit over the curve range most appropriate to the data. Occasionally, EI single mid-range curve point that was an obvious outlier may have been deactivated. Quantitation of the analyte was based on the response of one specific product ion using the multiple response- monitoring mode of the instrument (see Appendix C,Analytical Methods). 3M Environmental Laboratory Page 17 BACK TO MAIN 3M Nledical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN42849 0 Limits of Quantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibration curve defined as a standard within *30% of the theoretical value. (See Appendix D, Table 12.) 0 Blanks: All blanks were below the lower limit of qualntitation for the compounds of interest. 0 Precision: Not determined for this study. Matrix Spike Recoveries: Matrix spikes and matrix spike duplicates were extracted with each set of samples and analyzed during analytical runs at the 3M Environmental Laboratory.Acceptable spike recoveries of 54-1 09% of expected values were achieved for 12 of 14 matrix spikes prepared in urine. The remaining two spikes were recovered at 46% and 39%. Low recoveries were confirmed, but were not re-extracted. Associated results, which may be underestimated by more than 50%, are specifically identified in the report tables as not meeting the data quality objectives of this study. 0 Surrogates: The surrogate (THPFOS) was added to all samples and standards. External calibration was used in quantitation of datal in this study due to the presence of an unidentified compound that interfered with the analysis of the surrogate. 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 quantitative to 50% or greater. Refer to contract lab reports in Appendix G for summary of quality control analysis results from contract labs. 3M Environmental Laboratory Page 18 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Summary of Sample Results Following is information regarding sample analyses at each of the analytical laboratories involved in the present study: Battelle Memorial InstitutRefer to the Battelle Final Report for information1 regarding liver analysis (Appendix G). For liver analysis data that has been corrected with the purity factor, see Appendix E. Advanced Bioanalytical Services, lnc.Refer to the ABS Bioanalytical Report and the A.BSAddendum to the Bioanalytical Report for information regarding swum analysis (Appendix G). For serum analysis data that has been corrected with the purity factor, see Appendix E. Centre Analytical LaboratoriesRefer to the Centre Analytical Report for information regarding feces analysis (Appendix G). 0 Samples from Control Animals: Low levels of PFOS were often detected in the sera, urine, feces and liver of the control animals. These levels were significantly lower than those found in the low dose test animals. 0 Samples from Dosed Animals: In general, PFOS levels found in the sera, urine, feces and liver of the test animals increased with each dose group within a particular time point. Detailed sample data tables are presentled in Appendices D and E. Statistical Methods and Calculations Statistical methods were limited to the calculation of means and stalndard deviations. See Appendix F for example calculations used to geneirate the urine sample data in FACT TOX-110. Statement of Conclusion Under the conditions of the present studies, PFOS wasi observed in the livers, urine, feces and sera of all female rats dosed with the T-6295.12 during the in-life phase of the study. Additionally, PFOS was observed in fetal liver and serum taken during gestation from the same group of female rats. 3M Ihvironmental Laboratory Page 19 3M Medical Department Study: T-6295.12 ~- BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Appendix A: Control Matrices and Dose ConfirmationAnalyses Table 5. Description of the Control Matrices Used for Liver, Urine, Sera and Feces Analvses in Studv FACT TOX-110 Lampire Biological Storage Conditions N / R - n o t recorded Table 6. Characterization of the Analytical Reference Substances used for Liver, Urine, Sera and Feces Analyses in Study FACT TOX-110 3M Environmental Battelle Memorial Advanced Bioanalytical Centre Analytical I I 1 Chemical , Name ~, Source Laboratow Institute KPFOS Potassium KPFOS Potassium Pefliiorooctanesulfonate* PerRuorooctanesulfonate* 3M ICPlPCP Division 3M ICPlPCP Division Services KPFOS Potassium PerfluorooctanesuIfiinate* 3M ICPlPCP Division Laboratory KPFOS Potassium PerRuorooctanesulfonate* 3MMTeodxiciaclolDogeypaSrebrnveicnet s, ~ Expiration Date 8/31/01 8131101 8/31101 8/31/01 Storage Conditions Frozen 5 -10C Frozen I-10C Frozen 5 -10C Frozen I - 10C Chemical Lot # Description I I 1 171 ~~ White crystalline powder 171 White crystalline powder 171 White crystalline powder 217 White crystalline powder Purity 86.4% 86.4% 86.4% 86.9% 3M IInvironmental Laboratory Page 20 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Repolt: FACT TOX-110 LRN-U2849 Table 7. Dose Confirmation Analyses for Perfluorooctanesulfonatefor Test Samples From Study In-life M18-013-15 November 1998 I GROUPDOSE TARGETCmc. PFOS (ng/mL) EXPECTEDCONC. PFOS (ng/mL)' MEASUREPDFOS CONC. (ng/rnLIb %OF EXPECTED GROUP1 Control 0.0 mglkglday Diluted 1/1 0.00 0.00 0.00 GROUP2 0.1 mg/kg/day 0.02 mg/mL Diluted 1/40 GROUP3 0.4 mg/kg/day 0.08 mglmL i i i Diluted 1/1000 GROUP4 1.6 mglkglday 0.32 mg/mL c 1 1 Diluted1/1000 GROUP5 20000 80000 320000 16100 64500 258000 14000 611130 246000 3.2 mg/kg/day 0.64 mglmL 640000 516000 I 417000 NA=not applicable "Calculationsused to obtain Expected ConcentrationPFOSare given below bMeasuredPFOSComntrafionvaluesare correctedusingthe PFOS correctionfactor andfor purity NA 87% 195% I96% 81Yo Formula used to calculate expected concentration From target concentration: Target Concentration x PFOS Purity x PFOS Correction Factor = Expected Concentration PFOS PFOS Purity = 86.9% PFOS Correction Factor = 0.9275 640000 ng/rnL x 0.869 x 0.9275 = 516000 ng/rnL Expected Concentration 3M EinvironmentalLaboratory Page 21 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-1IO LRN-U2849 Appendix B: Protocol, Protocol Amendments and Deviation Summary Table 8. Deviation Summary for FACT TOX-110 Deviation The original protocol states that the source of the control matrix used in the present study would be either Sigma Chemicals or Argus Research Laboratories.The physical description of the control substance was rat and/or rabbit liver andlor serum. Additional control substances used in this study were human urine and rat feces obtained from Lampire Biological Laboratories; Piperville, PA 18947. Date(s) of Occiurrence ~~ Entire study ETS-8-96.0 states 2.0 mL will be used as the initial volume. Samples 19516F, FO, G I , control-GDO premating had only 1.OmL available. 9114/99 Samples 19572F. FO, G5-3.2 MKD-G7,24 hour had no sample available. Samples 19543F, FO, G3-0.4 MKDGD21,24 hour did not have enough sample for extracting. Initial volumes were recorded in the Excel spreadsheet for calculating final concentrations. ETS-8-96.0 specifies to add 0.5 mL methanol final volume. For the following samples, 1.OmL of methanol final volume was added. 19540F, FO, G3-0.4MKD-GD15Terminal 19542F, FO, G3-0.4MKD-GD15-Terminal 19543F, FO, G3-0.4MKD-GD15-Predose 1 9 W F , FO, G3-OAMKD-GD1!%Terminal 19546F. FO. G3-0.4MKD-GD15-Predose 9114/99 9115/99 ETS-8-97.0, section 10.1. I states that the methanol (water and extraction) blanks will be analyzed with each analytical batch. The water and extraction blanks were not analyzed during the following runs: 092099A and 092199a. 9/20/93. 9/21/99 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. ETS-8-97.0 specifies the method of analysis requires the use of an internal calibration. The actual analyses used an external calibration method. 9/21199 9/20/9!3,9/21/99, 9/24/9!3, 9/27/99, 9/30/99, 1OI28199, 1 15/99 The control of bias document requires the calibration curve to have percent deviations GOYO.The actual percent deviation for the low calibration point is greater than 30%. 9/27/99 Impact on Study Successful validation studies usinc human urine and rat feces were performed; therefore, there was nc adverse impact on the quality of thf data. The LOQ was maintained for all samples; therefore, there was no adverse impact on the outcome of the study. Data quality will not be affected. No data were generated. The LOQ was maintained for all samples; therefore, there was no adverse impact on the outcome of the study. All blanks were analyzed at least once during the study. Methanol blanks are often analyzed more frequently to monitor possible contamination, but since the study data here were well above any analyte levels in the blanks, there was no adverse impact on the aualitv of the data. The analysis of Tween samples was not conducted under GLPs (as per discussions with study director). This deficiency is included in the final report. Unknown contaminant interfered with use of surrogate for internal calibration. Corrective action is an improvement. Data quality is satisfactory to *50% accuracy. The data points are at the midrange portion of the curve, so deviation at the low end curve points will not adversely affect the data. 3M Environmental Laboratory Page 22 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN42849 Table 8. Deviation Summary for FACT TOX-I10 Deviation I Protocol FACT-TOX-110and the method ETS-8-97.0 state the matrix spike/matrix spike duplicate analyses will be *30% of the spiked concentration. The actual acceptable result for the matrix spike/matrix spike duplicate analyses will be *50% of the spiked concentration. Dale(s) of Occiurrence I Impact on Study I Data quality will be expressed as having as accuracy of *50% rather than *30%. 3M Environmental Laboratory Page 23 3NI Medical Department Study: T-6295.12 3M Environmental Technology and Services I PO Box 33331 St. Paul, MN 55133-333 I 612 778 6442 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 PrOtOCOl #FACT-TOX-1I O Study Title Oral (Gavage) Pharmacokinetic Study of PFOS in Rats PROTOCOL Author Lisa Clemen Date: June 8,1999 Performing Laboratory 3M EnvironmentalTechnology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project IdenfiYkafion FACT-TOX-110 U2849 3M Environmental Laboratory 3NI Environmental Laboratory Page 1 of 70 Page 24 BACK TO MAIN 3M Medical Department Study: T-6295.12 I Analytical Report: FACT TOX-110 LRN-U2849 Prot0~01#FACT-TOX-7 10 Study ldentificatiori Oral (Gavage) Pharmacokinetic Study of PFOS in Rats Test Material Sponsor Perfluorooctane sulfonic acid potassium salt (T-6295) - 3M Toxic,ologyServices Medical Department 3M Cater, Building 220-213-02 St. Paul, IVIN 55144-1000 Sponsor Representative Marvin T. Case, D.V.M:.,Ph.D. 3M Toxic:ologyServices Telephone: 651-733-5180 Facsimile: 651-733-1773 Study Director Kristen J. Hansen, Ph.D. 3M Environmental Technology and Safety Services Building 2-3E-09 651-778-15018 Study Location(s) in vivo Testing Facility Analytical Testing Laboratory Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, PA 19044 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 3M Environmental Laboratory 3M EnvironmentalLaboratory Page 2 of IO Page 25 3M Medical Department Study: T-6295.12 . i Sub-Contract Laboratory Proposed Study Timetable Study Initiation Date Study Completion Date BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Advanced Bioanalytical Services, Inc. 15 Catherwood Road Ithaca,N11 14850 Battelle IvIemonal Institute 505 King Avenue Columbuci, Ohio 43201-2693 June 8, 1999 August 8,2000 1. STUDY Oral (gavage) pharmacokinetic study of potassium perfluorcioctanesulfonic acid (PFOS)inrats. 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 ##418-013. All serum samples will be extracted and analyzed at Advanced Bioanalytical Services, Inc. and all liver samples extracted and analyzed at Battelle Memorial Institute. Additional analyses may be performed at the: 3M Environmental Laboratory as methods are developed and validated. If additional analyses are performed an amendment to this protocol will be written. 3. REGULATORCYOMPLIANCE This study will be conducted in accordance with the United States Food and Drug Administration, Good Laboratory Practices Standards, Final Rule 21 CFR 58, With the exception that analysis of the test material mixture for concentration, solubility, homogeneity, and stability will not be conducted, and is the responsibility of the Sponsor. 4. QUAUTYASSURANCE The 3M Environmental Laboratory Quality Assurance Unit will review the protocol and audit study conduct, data, and final report to determine compliance with Good Laboratory Practice Standards and with 3M EnvironmentalLaboratory StandardOperating Procedures. The QA Unit at the sub-contract laboratory will audit their study conduct, data, and results report prior to submitting to the 3M EnvironmentalLaboratory. 3M Environmental Laboratory 3h4 Environmental Laboratory Page 3 of 10 Page 26 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Protocol #FACT-TOX-110 5. TESTMATERIAL 5.7 Refer to Argus Research Laboratory protocol for study H18-013. 6. CONTROMLATRICES 6.7 ldentificatfon Rat liver and serum andor rabbit liver and seruxn, traceability numbers will be recorded in the raw data and included in the final report 6.2 Source Argus Research and/or Sigma Chemical 6.3 Physical Description Rat liver and serum and/or rabbit liver and serum , 6.4 Purify and Stabi/ify Not applicable 6.5 Storage Conditions Frozen at -20 "Cf 10 OC! or -50 "C f 10"C 6.6 Reserve Mafrix A portion of the control matrix will be retained in the 3M archives for as long as the quality of the preparation affords evaluation, but not longer than ten years following the effective date of the final test rule (if applicable). 6.7 Disposition Matrices will be retained at the 3EvI 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 adequateprecautions for handling biological materials and preparing samples for analysis. 7. REFERENCMEATER~AL 7.1 ldentification Potassium perfluorooctanesulfonate (PFOS), lot #s 171,215, or 217 (equivalent lots) 7.2 Source 3M Specialty Chemicals 7.3 Physical Description White powder 7.4 Purify and Stability purity of PFOS is 99% or greater. Stability has not been determined. 7.5 Storage Conditions Room temperature 7.6 Reserve Material A reserve sample fiom each batch of PFOS used in this study will be retained as long as the quality of the preparation affords evaluation, but not longer than ten years following the effective date of the :ha1test rule (if applicable). 7,7 Disposition Unused reference material will be retained for use by the 3M Environmental Laboratory and will be discarded when the quality of preparation no longer affords evaluation. 3M Envimnmental Laboratory 3N1 Environmental Laboratory Page 4 of 10 Page 27 BACK TO MAIN 3M Medical Department Study: T-6295.12 I Analytical Report: FACT TOX-110 LRN-U2849 P r ~ t ~ C#oFlACT-TOX-llO 7.8 Safety Precautions Refer to MSDS for chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. 8. TESTSYSTEM Rats were used as thelest system and were maintained and closed as described in the Argus Research protocol #418-013. The female rats will be given the test material or control once daily beginning 42 days prior to cohabitation and continuethrougJ~day 14 or 20 of presumed gestation. See table 1for more dosage information. m ; g 7 1 Dosage Levels, Concentration, and Volumes k g / d a Conceyntratiori Dosage volume 1.6 0.32 16 3.2 0.64 9. SPECIMEN AND SAMPLE RECEIPT The 3M Environmental Laboratory will receive homogeneity samples for dose analysis and specimens of the followingbody tissues and fluids from the:indicated points in the study. See table 2 for specimen information. All specimens will be packed on dry ice for shipping. Body tissuehid Table 2 Specimen Information Collected Serum -. Dam and Fetus animals Urine and feces - Dam animals Liver - Dam and Fetus animals Milk Secreting Glands - Dam animals Amniotic Fluid - Dan1 animals Dam-Predose, Days 7,15, and 21 Fetus-At termination (ofthe study Predose, Days 7,15, ;md 21 Dam and Fetus-At termination of the study At the termination of the study Dav 15 and Dav 21 specimens 3M Environmental Laboratory 3M Environmental Laboratory Page 5 of 70 Page 28 BACK TO MAIN 3M Medical Department Study: T-6295.12 I Analytical Report: FACT TOX-110 LRN-U2849 ~ProtOCO#l FACT-TOX-110 Body tissudfluid Collected Embryo's with Placentae - Dam animals Carcass - Fetus animal Placentae - Fetus animal LiverAung - Fetus animal Day 15 and Day 2 1 At the termination of the study Day 15 and Day 2 1 At the termination of the study Total number of test animals: 64 Tbtal number of control animals: 16 Specimens sent to 3M Environmental Laboratories will be 1:eceived and backed according to applicable Standard Operating Procedures. IO. PREPARATORMYETHODS 10.1 FACT-M-I .l,Extraction of Potassium Perfluorooctanesulfonnesulfonateor Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPIX-Electrosprayhfass Spectrometry 10.2 ETS-84.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLCElectrospray/Mass Spectrometry 10.3 If preparatory methods other than those listed ahove are used, an amendment to this protocol will be written. Any deviations fiom tliese methods will be documented and included with the study data. 10.4 If analyses are sub-contractedto other laboratories, an amendment will be written to include their methods and copies of each method will be attached to this protocol. 1I.ANALYTICAMLETHODS 11.1 FACT-M-2.1, Analysis of Fluorochemicalsin Liver Extracts lJsing HPLCElectrosprayhfass Spectrometry 11.2 ETS-8-5.1, Analysis of Potassium Perfluorooctztnesulfonateor Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-13lectrospray/h.lassSpectrometry 11.3 If analytical methods other than those listed above are used, an amendment to this protocol will be written. Any deviations fiom tliese methods .willbe documented and included with the study data. 11.4 If analyses are sub-contractedto other laboratories, an amendment will be written to include their methods and copies of each method will be attached to this protocol. 3M Environmental Laboratory 3Nl Environmental Laboratory Page 6 of I O Page 29 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 PfOfOCOl #FACT-TOX-I10 12.DATAQUALITOYBJECWES The number of spikes/duplicates, use of surrogates, and informationon other data quality indicators are included in the analytical methods. In addition, the following criteria will be met: 12.1 Linearity 3 2 0.98 . 12.2 Limits ofdetection / guantitation 12.2.1 Method Detection Limit (MDL) for PI'OS a) Serum: 1.75 ppb 12.2.2 b) Liver: 15 ppb Limit of Quantitation (LOQ) - Equal to the lowest acxeptabie standard in the calibration curve 12.3 Duplicate acceptable precision c 30% for the method 12.4 Spike acceptable recoveries 70% 1 130% 12.5 Use of confirmatory methods 1ndetermina.tesampleswill be re-analyzed using a confirmatorymethod. H a contimatory method.is used, an amendment to this protocol will be Written. 12.6 Demonstrafion of specificity Chromatogrzphicretention time, mass spectral daughter ion characterization. 73.SUB-CONTRACTED ANALYSiS 73.1 All analyses as detailed in this protocol will be 11erformed at 3M Environmental Laboratories, Building 2-3E-09,935 Bush Avenue, St. Paul, MN 55106, at Advanced Bioanalytical Services, Inc., 15 Catherwood Road, Ithaca, Ny' 14850, or at Battelle Memorial Institute, 505 King Avenue, Columbus, Ohio 43201-2693. 73.2 An amendment to this protocol will be written if analyses are performed at laboratories other than 3M EnvironmentalLaboratories, Advanced Bioanalytical Services, Inc., or Battelle Memorial Institute. 14.STATlSTlCALANALYSIS Averages and standard deviations will be calculated. The si:atisticalmethods that will be used are described below: 14.7 Data transformations and analysis Data will be reported as the concentration (weighvweight or weighthol) of PFOS or metabolite per tissue or fluid. 3M Environmental Laboratory 3NI Environmental Laboratory Page 70f 70 Page 30 ~~ 3M Medical Department Study: T-6295.12 ,I BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 14.2 Statistical analysis Statisticsused may include regression analysis of concentrationsover time, and standard deviations calculated far the concentrations within each dose group. If necessary, simple statistical tests, such as Student's t test, may be applied to evaluate statistical difference. 75. 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 fcir inclusion in the 3M Environmental Laboratory report. Each report will include, but not be limited to, the following, when applicable: ' 75.1 Name and address of the facility perfoming the study . 75.2 Dates upon which the study was initiated and completed 75.3 A statementof complianceby the Study Director 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 substanceidentificationby name, chemical abstracts iiumber or code number, strength, purity, and compositionor other appropriate charactczistics,if provided by the Sponsor 15.6 Stability and the solubility of the test substances under the conditions of administration, if provided by the Sponsor 15.7 A description of the methods used to conduct the test(s) 75.8 A description of the test system 15.9 A description of any circumstancesthat may have affected the:quality or the integrity of the data 75.10 The name of the Study Director and the names of other scientists, professionals, and supervisorypersonnel involved in the study 75.71 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 75-14 The location where raw data and the final report are to be stored 3M Environmental Laboratory 3NI Environmental Laboratory Page 8 of I O Page 31 BACK TO MAIN 3Ni Medical Department Study: T-6295.12 I Analytical Report: FACT TOX-110 LRN-U2849 P r 0 t O ~ 0 l#FACT-TOX-T 70 75.75 A statement prepared by the Quality Assurance Unit listing the dates that study inspections and audits were made, and the dates of any findings reported to the Study Director and Management If it is necessary to make corrections or additions to a report after it has been accepted, the changes will be made in the form of an amendment issued by the Study Director. The amendment will clearly identify the part of the report that iis being amended, the reasons for the amendment, and will be signed by the Study Director. 16.LOCATION OFRAWDATA,RECORDSA,ND FINAL REPORT Original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress and before :acceptanceof the final report. When the final report is completed, all original paper data, includmg those items listed below, willbe retained in the archives of 3M EnvironmentalLaboratory for at least a period of time as specified by regulation, and as established by 3M EnvironmentalLahoratory Standard Operating Procedures. 76.7 The followingraw 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. 7 Approved protocol and amendments 16.1.2 Study correspondence 16.1.3 Shipping records 76.7.4 Raw data 76.7.5 Approved final report (original signed copy) 76.7.6 Electronic copies of data f6.2 The following supporting records will be retained separately h m the study folder in the archives according to 3M Environmental Laboratory Standard Operating Procedures: 76.2.1 Training records 76.2.2 Calibration records 76.2.3 Instrument maintenance logs 76.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 3M Environmental Laboratory 3M Environmental Laboratory Page 9 of 70 Page 32 3M Medical Department Study: T-6295.12 , I, BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 17. SPECIMENRETENTION Specimens will be maintained in the 3M EnvironmentalLalsoratory 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. 78,PROTOCOL AMENDMENTASND DMATlONS Planned changes to the protocol will be in the form of writtsn amendmen& signedby the Study Director and the Sponsor's Representative.Amendments will be considered as part of the protocol and will be attached to the final protocol. All changes to the protocol will be indicated in the final report. Any other changes will be in the form ofwritten deviations, signed by the Spdy Director and filed with the raw data. 19.ATTACHMENTS 79.1 Attachment A Preparatory and analytical methods 20. SIGNATURES A & KristeriJ. Hm'en, Ph.D., 3M Environmental Laboratory Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page I O of 70 Page 33 3NI Medical Department Study: T-6295.12 I I: BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Study Title Oral (Gavage) Pharmacokinetic Study of PFOS in Rats PROTOCOLAMENDMENT NO. 1 Amendment Date.: August 12,1999 Performing Laboratory 3M Environmental Technology & !Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project IdentNcation ETBSS FACT-TOX1LO LIRN U2849 3M Environmental Laboratoty 3M Environmental Laboratory Page 34 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 PrOtOcOl FACT-TOXI70 Amendment No. 1 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: Section 2.0 states this study is designed to determine potassium perfluorooctane sulfonic acid (PFOS) in specimens of liver and serurri of rats. AMENDTO READ: This study is designed to determinePFOS in spechens of rat Iiver, serum, and urine. All Day - and GestationDay 21 rat urine specimens will be extracted and analyzed by the 3M Environmental Laboratory. REASONT: he urine extraction and analytical methods were not validated and approved prior to protocol approval. -2. PROTOCOL READS: Section 6.0 lists rat or rabbit liver and serum. AMENDTOW D : Rat or rabbit urine from 3M Toxicologywith a physical descriptionof rat or rabbit urine. REASON: The rat urine matrix was added after the protocol was approved. 3. PROTOCOL READS: Section 10.0 and 11.0list the followingmethods to use for extraction and analysis: FACT-M-1.1 "Extraction of PotassiumPerfluorooctanesulfonateor Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" FACT-M-2.1 "Analysis of Fluorochemicalsin Liver Extracts Using HPIC-ElectrosprayMass Spectrometry" AMENDTOREAD: The extraction and analyticalmethods to follow at the 3M Environmental Laboratory are: ETS-8-6.0 "Extraction of PotassiumPerfluorooctanesulfonateor Other Fluorochemical Compounds from Liver for Analysis Using HPLC-ElectrosprayMass Spectrometry" ETS-8-96.0 "Extraction of Potassium Perfluorooctauesulfonateor Other Fluorochemical Compounds from Urine for Analysis Using HPLC-ElectrosprayMassSpectrometryMass Spectrometry" ETS-8-7.0 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/M:assSpectromc:try" ETS-8-97.0 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrospray/Mass Spectrom&y/h4ass Spectrometry" 3M Environmental Laboratory 3M EnvironmentalLaboratory Page 35 BACK TO MAIN 3h4 Medical Department Study: T-6295.12 I Ii Analytical Report: FACT TOX-110 LRN42849 ProtOCOl FACT-TOX?70 Amendment No. 7 REASON: The extraction and analytical methods FACT-M-1.1 and FACT-M-2.1, respectively, were updated on 07/22/99 to ETS-8-6.0 and ETS-8-7.0. Methods ETS-8-96.0 and ETS-8-97.0 were not validated and approved until after the protocol was approved. 4. PROTOCOLREADS: Section 10.4and 11.4 state that if the analyses are sub-contractedto other laboratories an amendment will be written to include these methods. AMENDTO READ: The extraction and analyticalmethods to follow at Advanced Bioanalytical Serviceswill be attachedto the protocol. The extraction and analyticalmethod to follow at BattelleIMemorial Institute is: "Method for Analysis of PerfluorooctaneSulfonate.(PFOS)in Rat sefeby LC/MS/MS, Version 4" 1.0 0 1;w 0 REASON:The analyticalmethods at the subcontract laboratorieswere not included in the original protocol. 5. PROTOCOL READS: Section 12.2.1b) Liver method detectionlimit is, 15ppb. AMENDTO READ: 12.2.1 b) Livermethod detection limit is 8.50 ppb (ng/g). 12.2.1 c) Urine method detectionlimit is 1.5 ppb (ng/g). RE~SONT:he validation supportingmethods ETS-8-6.0 and ETS-8-7.0 includeda lower method detection limit for PFOS in liver. A validation in urine was performed, after protocol approval, to support this method detection limit. 3M Environmental Laboratory 3M Environmental Laboratory Page 36 3bl Medical Department Study: T-6295.12 ., L II Amendment Approval BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 PrOtOCOl FACT-TOX170 Amendment No. I gc Marvin Case, D.V.&, PbD., Sponsor Representative / I &.vAJ- / 9 S $ * Date Kris J. Hanken, PbD., study Director 6*118&7 Date 3M Environmental Laboratory 3M Environmental Laboratory Page 37 3M Medical Department Study: T-6295.12 I, BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Study Title Oral (Gavage) Pharmacokinetic Study of PFOS in Rats PROTOCOL AMENDMENT NO. 2 Amendment Date: September 28,1999 Performing Laboratory 3M EnvironmentalTechnology& Safety Services 3M Environmental Labor<atory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOXl'lO LIRN U2849 3M Environmental Laboratory 3Nl Environmental Laboratory Page 38 BACK TO MAIN 3kl Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Protocol FACT-TOX710 Amendment No. 2 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: Section 2 states that all liver samples will be extracted and analyzed at Battelle Memorial Institute. AMENDTO READ: All dam liver specimens (female mother) and 27 fetus (pooled) liver specimens will be analyzed at Battelle Memorial Institute. REASON: , The fetal liver/lung is not a target matrix and will not be extracted or analyzed. 2. PROTOCOL READS: Section 9 states that 80 dam liver specimens (female mother) and 80 fetus (pooled) liver specimens will be sent to the Environmental Laboratory. AMENDTO READ: Seventy-three dam liver specimens (female mother), 25' fetus (pooled) liver specimens, and 81 fetal 1iverAungspecimens (27liverAung specimens isampled in triplicate) were sent to the Environmental Laboratory. REASON: The number of animalschanged during the c o m e of the study. 3. PROTOCOL READS: Section 12.2.1a) serurnmethod detection limit is 1.75 ppb b) Liver method detection limit is 15 ppb. AMENDTO READ: The method detectionlimitsfor all compounds and matrices will be tidcen from the methods used for extraction and analysis. REASON: The method detection limits listed are specific to the 3Ed Environmental Laboratory. Statementwas added to allow for sub-contractedanalyses andlor revised methods. 3M EnvironmentalLaboratory 3M Environmental Laborator), Page 39 BACK TO MAIN 3M Medical Department Study: T-6295.12 I, Analytical Report: FACT TOX-110 LRN-U2849 PrOtOCOl FACT-TOXI I O Amendment No. 2 4. PROTOCOL READS: Section 16 states that the original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance of the finalreport. When the final report is completed,all originalpaper data, including: approved protocol and amendments,study correspondence, shippingrecords, raw data, approved find report, electronic copies of data, triuningrecords, calibrationrecords, instrument maintenance logs and standard operatingprocedures, equipmentprocedures, and methods will be retained in the archives of the 3M EnvironmentalLaboratory. AMENDTO READ: Section 16states that the original data,or copiesthereof, will be availableat the 3M 1 Environmental Laboratory to facilitate audits of the study during its progress and before acceptanceof the final report. When the final report is oompleted, 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 archivesof the 3M Environmental Laboratory. All correspondinglrainingrecords, calibrationrecords, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained in the archives of the facility performing each analysis. REASON: Clarificationof the disposition of archived records if analysesare performed at a sub-contract laboratory. 5 . PROTOCOL READS: Section 17 states that specimens will be maintained in the 3M EnvironmentalLaboratory specimen archives. AMENDTO READ: Specimenswill be maintained in the 3M Environmental Laboratory specimenarchives. All . specimens sent to sub-contractlaboratorieswill be retuned to the 3M Environmental Laboratory upon completionof analysis and submissionof the sub-contractlaboratory(s) final report. The specimenswill be returned with the fclllowing documentation: the signed originalchain of custody and records of storageconditionswhile at the sub-contract facility. REASON: Clarificationof the dispositionof documentationwhen shipping specimens for analyses performed by a sub-contract laboratory. 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 40 3M Medical Department Study: T-6295.12 I Amendment Approval BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 ProtOCOl FACT-TOX110 Amendment No. 2 Marvin Case, D.V.M.-,PbD., Sponsor Representative WLf /P$w Dad Kriiten J. Hansen, Ph.D., StudyDirector ~ O /Pdw Date 3M Environmental Laboratory 3M Environmental Laboratory Page 41 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Study Title ' Analytical LaboratoryReport on the Determinationof Perfluorooctanesulfonate(PFOS) Presence and Concentration in Senun, Liver, and Urine @om the Gavage Study of T-6295.12 PROTOCOL AMENDMENT NO. 3 Amendment Datel: 20 January 2000 Urine Analyses 3M Environmental Technology and Safety Services Fluorine Analytical Chemistry Team Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Performing Laboratcries Liver Analyses Battelle Memorial Institute 505 King Avenue Columbus,OH 43201-2693 Serum Analyses Advanced Bioanalytical Services, Inc. 15 CatherwoodRoad Ithaca, NY 14850 Laboratory Project Identification ET&SS LRN-U2849 FACT TOX-110 Argus Study: 418-01.3 3M Medical Department Study: T-6295.12 3M Environmental Laboratory 3M Environmental Laboratory Page 42 BACK TO MAIN 3M Medical Department Study: T-6295.12 4 c Analytical Report: FACT TOX-110 LRN-U2849 PrOtOCOl LRN42849 Amendment Number 3 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: The study director for the present study was identified in the protocol as Kristen J. Hansen, Ph.D. AMENDTO READ; The role of study director for the present study WEIS reassignedto Marvin T. Case, D.V.M., Ph.D., as of 20 January 2000. The previous study director, Kristen J. Hansen, has been reassignedto the role of PrincipleAnalytical Investigator. REASON: The role of study directorwas reassignedin an effort to ensure compliancewith , 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 MarvinT. Case, D.V.M., Ph.D. AMENDTO READ: The role of sponsor for the presentstudy 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 EnvironmentalLaboratory 3M Environmental Laboratory Page 43 3M Medical Department Study: T-6295.12 c Amendment Approval John L. ButenhoB Ph.D., Sponsor Representative BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 PrOfOCOl LRN-U2849 Amendment Number 3 @' / z&!m Date & Kristen J; Hansen, Ph.D., Outgoing Study Director /l-F4b -25LW Date TL /sFA4-,- %%J Marvin T.Case, D.E M , Ph.D., Incoming Study Director &e 3M Environmental Laboratory 3M Environmental Laboratory Page 44 3M Medical Department Study: T-6295.12 I, BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Study Title pral (Gavage) PharmacokineticStudy of PFOS in Rats PROTOCOL AMENDMENT NO. 4 Amendment Date: 20 April ZOO0 Performing Laboratcries 3M Environmental Technolo_g_y and Safety Services Fluorine Analytical Chemistry Team Building 2-3E-09,935 Bush Avenue St. Paul, MN 55106 Battelle Memorial Institute 505 King Avenue Columbus, OH 43201-2693 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 =-Advanced BioanalyticalServices, Inc. 15 Catherwood Road Ithaca,NY 14850 Laboratory Project Identification ET&SS LRN-U284!) FACT TOX-110 Argus Study: 418-013 3M MedicaI Department Study T-6295.12 3M Environmental Laboratory 31\41Environmental Laboratory Page 45 BACK TO MAIN 3M Medical Department Study: T-6295.12 I .. ` Analytical Report: FACT TOX-110 LRN-U2849 PrOtOCOl LRN-U2849 Amendment Number 4 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: The amended section 2.0 text states that this study is designedto determinePFOS in specimens of rat ljver, serum, and urine. AMENDTO READ: This study is designed to determine PFOS in specimens of rat liver, serum, feces, and urine. REASON: The analysis of fecal tissue for the target chemical and/or its analytes was added to the scope of the study followingthe issuance of the protocol. Feces extractionand analyticalmethods , were not validated and approvedprior 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 descriptionof rz%otr rabbit feces. REASON: Analysis of fecal tissue for the target chemical andor irs analyteswas added to the scope of the study following the issuance of the original protocol. 3. PROTOCORLEADS: Section 13.1 lists all of the laboratories that will be conducting analyses for this study. AMENDTO READ: Add: Centre Analytical Laboratories, Inc., 3048 Research Drive, State College, PA 16801 REASON: Feces analyses were added to the scope of this study. The sub-contract laboratory performing analyses was not in the original protocol. 4. PROTOCORLEADS: Sections 10.4and 11.4 state that if the analyses are sub-contractedto other laboratories an amendment will be written to include these methods. AMENDTO READ: The feces extraction and analytical method used by Centre AnalyticaH Laboratories will be; 00hI-023-003 (Revision2), "Determinationof FluorochemicalResidues in Monkey/Rat Feces by LC/MS/MS." REASON: The sub-contract laboratory performing feces analyses was added to ihe scope of this study; this method was not validated and approved prior to protocol approval. 3M Environmental Laboratory 3M Environmental Laboratory Page 46 3M Medical Department Study: T-6295.12 c I Amendment Approval BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Protocol LRN-U2849 Amendment Number 4 John L.Butenhos Ph.D., Sponsor Representative Date Marvin T.Case, D.V.M., PhD., Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 47 3M Medical Department Study: T-6295.12 I BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Study Title Oral (Gavage) PharmacokineticStudy of PFOS in Rats REPORT AMENDMENT NO. 5 Amendment Date!: 26 July 2000 Performing Laboratory 3M EnvironmentalTechnology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 5510ti Laboratory Project ldenrtification ET&SS FACT Tox-110 ET&SS LRN-U2849 3M Environmental Laboratory 3Nl Environmental Laboratory Page 48 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 FACT TOX-110 Report Amendment No. 5 This amendment modifiesthe following portion@)of the protocol: 1. PROTOCORLEADS: The amended analyticalprotocol (AmendmentNo. 1,s,ection4.0) statesthe extraction and analytical method to follow at Battelle Memorial Institute is "Method for Analysis of Perfluorooctance Sulfonate(PFOS) in Rat Sera by LC/MS/MS,Version 1." AMENDTO READ: The extraction and analyticalmethod to follow at BattelleMemorial Institute is: "Method for Analysis of PerfluorooctaneSulfonate(PFOS) in Rat Liverby LC/MS/MS, Version 1.0" REASON: Liver analyses were sub-contractedto BattelleMemorial after the protocol was written; therefore, the method used to analyze liver samples also changed. The Battelle method listed in Amendment No. 1had a hand-written change, in the.t "sera" was crossed out and "liver" was written in. This hand-written change was dated after the study director had signed the amendment. 2. PROTOCORLEADS: In the Argus in-life protocol M18-013, the testing facilityis stated on page 1 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 directorresides. There cannot be two testing facilities. 3. PROTOCORLEADS: On the coverpage, the type of documentfor FACT-TOX-110 is listed as a "Protocol." On page 3, under Proposed Study Timetable, the "Study Initiation Date" and "Study Completion Date" are listed. AMENDTO READ: Ch,mge the document type for FACT-TOX-110 to "Analytical Phase: Protocol." Change "Study Initiation Date" to "Phase Initiation Date." Change "Study Completion Date" to "Phase CompletionDate." REASON: FACT-TOX-110 is the protocol for the analytical phase, while the Argus protocol #418-013 3M Environmental Laboratory 3Nl Environmental Laboratory Page 49 BACK TO MAIN 3Nl Medical Department Study: T-6295.12 I Analytical Report: FACT TOX-110 LRN-U2849 FACT TOX1-I O Report Amendment No. 5 is the protocol for the in-lifephase. 4. PROTOCORLEADS: The amended analyticalprotocol (AmendmentNo. 3, section 1) states that Marvin T. Case replaces Kristen J. Hansen as the study director. Kristen J. Hansen was reassigned to the role of Principle Analytical Investigator. The Argus Research Laboratories, Inc. protocol #418013 states that the study director for the in-life phase is Raymond York. AMENDTO READ: Marvin T. Case replacesboth Kristen J. Hansen and Raymond York ;Estudy 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. Thischange is retroactiveto February 10,2000. REASON: Per GLP regulations, only one study director is assigned to a study. Corrected spelling of Principal. 5. PROTOCORLEADS: The amended protocol (AmendmentNo. 4) states that rat feces sampleswill be analyzed for PFOS,as well as rat liver, urine and serum. Centre AnalyticalLaboratorieswill be conductingthe feces analyses. AMENDTO READ: Add that the Principal Analytical Investigator(PAI) at Centre Analytical Laboratoriesis Enaksha Wickremesinhe. REASON: The PAT for Centre was not listed in AmendmentNo. 4.. 3M Environmental Laboratory 3 M Environmental Laboratory Page 50 3NI Medical Department Study: T-6295.12 Amendment Approval hwdw- J O GL. Butenhog Ph.D., Sfinsor Representative e Madin f Case, D.KM., Ph.D., Study Director BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 FACT TOX-170 Repod Amendment No. 5 -7/5/@ Date Date 3M Environmental Laboratory - 3M Environmental Liiboratbw Page 51 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Study Title Oral (Gavage) Pharmacokinetic Study of PFOS in R.ats PROTOCOL AMENDMENT NO. 6 Amendment Date: October 3,2000 Performing Laboratlory 3 M EnvironmentalTechnology& !safety Services 3 M EnvironmentalLaboratory 935 Bush Avenue St. Paul, M N 551068 Laboratory Project ldentification ET&SS FACT TOX-110 3M LaboratoryRequest No,U2849 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 52 BACK TO MAIN 3hl Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Protocol FACT TOX-110 Amendment #6 This amendment modifies the following Iportion(s) 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 50%-150%. REASON: The analytical method and resulting QC data support a 50%-150% acceptable range for spike recoveries. 3M Environmental Laboratory 3M EnvironmentalLaboratory Page 53 BACK TO MAIN 3M Medical Department Study: T-6295.12 I. Analytical Report: FACT TOX-110 LRN-U2849 Protocol FACT TOX-7 70 Amendment #6 Amendment Approval $?AE?Xk.c.L* John L,.Butenhoff,Ph.D., Sponsor Representative oc.4854% ? , w e * Date 7-g& Marvin T. Case, D.V;M.,Ph.D., StudyDirector *- Date 3M Environmental Labofatow 3M Environmental Laboratory Page 54 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Appendix C: Extraction and Analytical Mlethods This appendix includes the following methods: ETS-8-96.0, "Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Urine for Analysis Using HPLS-Electrospray/Mass Spectrometry/Mass Spectrometry," (14 pages) ETS-8-97.0,"Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds in Urine Extracts Usirig HPLC-ElecArospray/Mass Spectrometry/MassSpectrometry," (10 pages) 3kl EnvironmentalLaboratory Page 55 BACK TO MAIN 3M Medical Department Study: T-6295.12 3M ENVIRONMENTLAALBORATORY Analytical Report: FACT TOX-1I O LRN-U2849 METHOD EXTRACTIOONF POTASSIUM PERnUOROOClTANESULFONATE OR OTHER FLUOROCHEMICALCOMPOUNDSFROM URINE FOR ANALYSIS USING HPLC- ELECTROSPRAYMSAPSESCTROMETRY/IMASSSPECTROMETRY Method Number: ETS-8-96.0 Adoption Date: 7-28-44 Author: Lisa Clemen, Glenn Langenburg Revision Date: r\l A Approved By: 1 Laborawv Mhager- Group Leader Technical Reviewer Date 3/zlr/g5 Data 71a--h97 Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method is for the extraction of potassiim perfluorooctanesulfonate (PFOS) or other fluorochemicalcompounds from urine. 1.2 Applicable compounds: Fluorochemicalsor other fluorinated compounds. 1.3 Matrices: Human, rat, and monkey urine or other fluids as designated in the validation report. Word 6/95 3 M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 1 of 14 Page 56 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 2.0 SUMMARY OF METHOD 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemicals fiom urine, or other fluids, using an ion pairing reagent and methyl-tert-butyl ether (MtBE). In this method, eight fluorochemicalsare extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, M556,M.570p,erfluorooctanoate (POAA),and surrogate standard (see 3.0 Definitions). An ion pairing reagent is added to two ml of sample and the analyte ion pair is partitioned into bAtE3E. The MtBE extract is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 0.5 ml of methanol, then filtered through a 0.2 pm nylon filter attached to 3 cc plastic syringe into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-97.0 or other appropriate method. 5.0 DEFINITIONS 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) C,F,,SO,' 3.2 PFOSA: perfluorooctane sulfonylamide C,F,,SO,NH, 3.3 PFOSAA:perfluorooctane sulfonylamido (ethy1)acetate C,F,,S0,N(CH2CH3)CH2CO~ 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamide)-ethyl alcohol C,F,,S 02N(CH2CH,)CH2CH,0H 3.5 M556:C8Fl,S0,N(H)(CH2COOH) 3.6 M570: C8F,,S02N(CH3)CH,COOH 3.7 POAA: perfluorooctanoate C,F,,COO- 3.8 Surrogate standard THPFOS: lH-lH-2H-2H perfluorooctane sulfonic acid, used as an internal standard in this method. 4.0 WARNINGS AND CAUTIONS 4.1 Health and safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 INTERFERENCES 5.1 At this time, it is unknown how the extraction method is affected by potential interferences that may be present such as conjugated fluorochemicds (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 57 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 6.0 EQUIPMENT 6.1 The following equipment is used while performing this method. Equivalent equipmentis acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge,Mistral 1000or IEC 6.1.3 Shaker, Eberbach or V W R 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance (*0.100 g) 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Eppendorf or disposablepipettes 7.3 Nalgene bottles, capable of holding 250 ml and 1 I, 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 ml glass 7.6 Centrifuge tubes, polypropylene, 15 ml 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0 ml 7.9 Syringes, capable of measuring 2.5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes,disposableplastic, 3 cc 7.12 Syringe filters, nylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers Note: Prior to using glasswareand 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 separatevials. 8.0 REAGENTS AND STANDARDS 8.1 Type I reagent gradewater, Milli-QTMor equivalent;all water used in this method should be Milli-Qm water and may be provided by a Milli-Q TOC PlusMsystem 8.2 Sodium hydroxide (NaOH), J.TBaker or equivalent 8.3 Tetrabutylammoniumhydrogen 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 EnvironmentalLaboratory ETS-8-96.0 Extraction ofPFOS from Urine Page 3 of 14 Page 58 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 8.6 Methyl-T-Butyl Ether,Omnisolv, glass distilled oir HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Urine frozen from supplier 8.9 Fluorochemical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecularweight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 8.9.3 PFOSAA (3M Specialty Chemical Division),moleculax 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 (I-H,l-H, 2-H, 2-H C,F,,SO,H) aiolecularweight =428 8.9.9 Other fluorochemicals, as appropriate 8.10 Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.10.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000ml beaker containing 500 ml Milli-Q""' water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.10.2 1N sodium hydroxide (NaOH): Dilute 1014NaOH 1:10. Measure 10ml of 10N NaOH solution into a 100 ml volumetric flask and dilute to volume using Milli- Q" water. Store in a 125ml 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-Q''Mwater. Adjust to pH 10 using approximately44 to 54 ml of 10N NaOH (While adding the last ml of NaOH, add slowly because the pH changes iibmptly). Dilute to volume with Milli-Qm water. Store in a 1 L Nalgene bottle. 8.10.3.1 TBA requires a check prior to each use to ensure pH = 10.0. Adjust as needed using 1N NaOH solution. 8.10.4 0.25 M sodium carbonate/sodium bicarb0nai.ebuffer (Na,CO,/NaHCO,): Weigh approximately 26.5 g of sodium carbonate QIJaJO,) and'Zl.O g of sodium bicarbonate (NaHCO,) into a 1 L volumetric flask and bring to volume with Milli- Q" water. Store in a 1 L Nalgene bottle, 8.11 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.OO ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA,and 1.10ppm EtFOSE-OH.) 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS fiom Urine Page 4 of 14 Page 59 BACK TO MAIN 3NI Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 8.11.3 Weigh approximately 100mg 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 ;standardof approximately1000ppm Wml). 8.11.5 Dilute the stock solutionwith methanol for a working standard 1solutionof approximately50 ppm. 8.11.6 Dilute working standard 1 with methanol fclr a working standard 2 solution of approx. 5.0 ppm. 8.11.7 Dilute working standard 1 with methanol folr a working standard 3 solution of approx. 0.50 ppm. 8.12 Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 rng of surrogatlestandard 1-H,l-H, 2-H, 2-H, C8F1,S03Hinto a 50 ml volumetric flask and record the i3Cctual weight. 8.12.2 8.12.3 Bring to volume with methanol for a surrogatestock of approximately1000-1200 PPm. Prepare a surrogateworking standard. Transfer approximately 1 ml of surrogate stock to a 10ml volumetricflask 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 fkozenand must be kept frozen until the extractionis performed. 9.2 Allow samplesto thaw to room temperatureprior to extraction. 10.0 QUALITY CONTROL 10.1 Solvent Blanks, Method blanks and matrix blanlu 10.1.1 An aliquot of 2.0 ml methanol is used as a solvent blank. 10.1.2 Extract two 2.0 ml aliquots of Milli-Qm water following this procedure and use as method blanks. 10.1.3 Extract two 2.0rnl aliquotsofthe urine follclwing this procedure and use as matrix blanks. See 11.1.4. 10.2 Matrix spikes 10.2.1 Prepare and analyzematrix 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 Expectedconcentrationsshould fall in the mid-range of the initial calibration curve. Additional spikes may be included arid may fall in the low-range of the initial calibration curve. 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Unine Page 5 of 14 Page 60 BACK TO MAIN 3h4 Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibrationverification !samplesto ensure the accuracyof the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuingcali'brationverification per group of 10 samples. For example, if a sample set = 34,four checks are prepared and extracted. 10.3.3 Prepare each continuing calibration verification fiom the same matrix used to prepare the initial curve. 10.3.4 The expected concentrationsWill fall within the mid-range of the initial calibrationcurve. Additionalspikesmay bt: includedthat fall in the low-rangeof the initial calibration curve. This is necessary if the analyst must quantitateusing 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 15 ml centrifuge tube. 11.1.2 Record each sample volume on the extracticln sheet. 11.1.3 While preparing a total of twenty-two aliquots in 15 ml centrifugetubes, mix or shake between aliquots. 11.1.4 Two 2.0 ml aliquots serve as matrix blanks. 11.1.5 Typically use the standardconcentrations 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 t w m~ethod blanks. 11.1.6 Refer to validation report FACT-TOX-131, W2067, which lists the working ranges and the Linear CalibrationRange (L<X)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 calculateactual concentrationsof PFOS in calibration standards. 11.2 To each standard,blank, or continuingcheck, add appropriateamount of surrogate working standardfor the concentrationto fall within the calibration curve range 10ppb - 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 EnvironmentalLaboratory ETS-8-96.0 Extraction of PFOS from Urine Page 6 of 14 Page 61 BACK TO MAIN 3R4 Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Table 1 Approximate spiking amounts for standards and spikes Using 2.0 ml of matrix Working standard PL (approx-. conc.) - 12.1 12.2 12.3 12.4 12.5 12.6 12.7 12.8 12.9 12.10 12.11 12.12 Obtain fiozen samples and allow to thaw at room temperature or in a lukewarm waterbath. Vortex mix for 15 seconds, then transfer 2.0 ml or other appropriatevolume to a 15 ml polypropylenecentrifuge tube. Return unused samples to freezer after extraction amounts have been removed. Record the initial volume on the sampleweightholiume worksheet. . See Attachment D. The original weightlvolume worksheet is included in the study binder. Label the tube with the studynumber, sampleID, date and analyst initials. See attached worksheet for documenting the remaining steps. Spike all samples,including blanks and standards, :ready for extractionwith surrogate standard as described in 11.2. Spike each matrix with the appropriateamount of standard as describedin 11.1, or Table 1 in that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibrationstandards. Vortex mix the standard curve samples, matrix spik:e samples, and continuing calibration samples for 15 seconds. Check to ensure the 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. To each sample, add 1 m10.5 M TBA and 2 ml of O.25Msodium carbonatdsodium bicarbonate buffer. Using an Oxford Dispenser, add 5 ml methyl-tert-butyl ether. Cap each sample and put on the shaker at a settingof 300 rpm,for 20minutes. 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 7 of 14 Page 62 BACK TO MAIN 3111 Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 12.13 Centrifugefor 20 to 25 minutes at a settingof 3500 rpm, or until layers are separated. 12.14 Label a &esh 15mi centrifugetube with the same informationas in 12.5. 12.15 Remove 4.0 ml of the organic layer to this clean 15 ml centrifbgetube. 12.16 12.11 Put each sample on the analyticalnitrogen evapor<ritoruntil dry,approximately30 to 60 minutes. Add 0.5 ml of methanol to each centrifuge tube using a graduated pipette. If excessive residue is present, add the methanol and allow the extract to sit for 30 minutes prior to vortexing. 12.17 Vortex mix for 30 seconds. 12.18 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extractiondate, fluorochemicalcomponents, extractiontype, vial file archive number, and analyst(s) performing the extraction. 12.19 Attach a 0.2 pm nylon mesh filter to a 3 cc syringa 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 refi-igerated at approximately4 "Cuntil 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 DATAANALYSIS AND CALCULATIONS 13.1 Calculations 13.1.1 Calculate actual concentrationsof PFOS, o:r other applicablefluorochemical,in calibrationstandardsusing the followingequation: ml of standard x concentrationof standard (UP /ml) - ml of standard + ml of surrogatestandard+ initial matrix volume (ml) Final Concentration (pg/ml) of PFOS in matrix 14.0 METHODPERFORMANCE 14.1 The method detection limit (h4DL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quantitation (LOQ) values (see Attachment B). 14.2 The followingqualitycontrol samples are extracted with each batch of samplesto 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-8-97.0for method perfonnancecriteria. 3M Environmental Laboratory ETS-8-96.0 ExtractionofPFOS from Urine Page 8 of 14 Page 63 BACK TO MAIN 3NI Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 15.0 POLLUTIOPNREVENTIOANND WASTMEANAGEMENT 15.1 Human and monkey sample waste is disposed in infectious biohazard waste containers, all other sample waste is disposed in noninfectious;biohazard waste containers. Flammable solvent waste is disposed in high BTU containers. Used glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 RECORDS 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 TABLESD. IAGRAMSF.LOWCHARTS, AND VALIDAT1ON DATE 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 17.4 Attachment D, Sample weightholume worksheet 18.0 REFERENCES 18.1 The validation report associated with this method is FACT-TOX-131, W2067. 19.0 AFFECTED OCUMENTS 19.1 ETS-8-97.0,"Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrospray Mass SpectrometryMass Spectrometry" 20.0 REVISIONS Revision Number Reason For Revisioil Revision Date 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 9 of 14 Page 64 3h4 Medical Department Study: T-6295.12 -nuuuuuLL Surrogate Std IV u l A J I l G G L 0 1 t>-O-YO.V DateSpikedAnalyst CCV BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 FC-Mix Comments approx. 50 ppm I I I I I I I I I - I Cont. Cal. Verifications used same matrix as for std curve. Attachment A 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 10 of 14 Page 65 BACK TO MAIN 3hrl Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Compound PFOS POAA PFOSA PFOSAA EtFOSE-OH M556 M570 MDL @Pb) 4.6 22.9 n/d n/d dd dd dd LOQ @Pb) 14.7 72.9 n/d nfd n/d dd nfd Linear Calibration Range (LCR) - 15 ppb 1500ppb (inh a 1 MeOH extract) 50 ppb - 1500 ppb (in h a lMeOH extract) - 25 ppb 1000 ppb (in final MeOH extract) 25 ppb - 1000 ppb (in find MeOH extract) 25 ppb - 1000 ppb (in final MeOH extract) 25 ppb - 1000ppb (in fiial MeOH extract) - 25 ppb 1000ppb (in final MeOH extract) NOTE:to calculate MDL, LOQ,and LCR values in ug/ml of cuine divide the above values by 4, MDLLOQ values in rat and monkey urine were not statistically determined. 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 of clean, control matrix is availabfe, samples will be evaluated versus a curve extracted from urine originating fiom the same species as the specimens. Please see LOQ Summaryand M D L study in FACT-TOX-131, W2067 for fitrther information. Attachment B:MDULOQ Summary 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 11 of 14 Page 66 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Human Urine Full Range Prepared range of standards (ppb) (n@ml) dd LCR from curve (PPW (ndml) dd Low Curve nfd dd High curve dd dd 1/X 2.5ppb- 1500 ppb 15 ppb- 1500 ppb % Recovery Range n/d dd idd 75-116 Compound: POAA Repared range of Human Urine standards (ppb) (ndml) Full Range dd Low Curve n/d LCR fkom curve (PPb) (ng/ml) dd n/d % n/d RSD Range dd dd n/d +I-30% n/d 1K quadratic 1 2.5ppb - 1500 ppb 1 50 ppb - 1500 ppb 1 86-105 I ~ ~~ +/-3O% Attachment B: MDULOQ Summary 3M EnvironmentalLaboratory ETS-8-96.0 Extractionof PFOS from Urine Page 12 of 14 Page 67 3M Medical Department Study: T-6295.12 Prep date(s): Analyte(s): Sample matrix: Ion Pair Standard Curves -Urine Standard number: Equipment number: Final solvent rrnd 'I": Blank fluidndentifier: Box Number: BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 PFOS Std conc Ug/d PFOSA Std conc ug/ml PFOSAA Std conc ug/d ---0.500 05.5.00 5- .- 00c -5.00 50.0 '0.501 0.501 , --II----- :x:.-- 5.01 -- .' 5.00 EtFOSE Std conc ug/d 5.01 POAA Std conc Ug/ml 49.9 M!i56 Std conc Ugid 5.00 -*-. M570 Std conc ug/ml All Am't spikedml All Find vol ml 0.010 0.025 0.005 0.010 0.025 0.005 0.0075 0.010 2.0125 2.0275 2.0075 2.0125 2.0275 2.0075%2.0100 2.0125 Calculated concentrations of standards in the sample matrix PFOS PFOSA PFOSAA EtFOSE POAA M5 56 M570 Surrogate All Final conc Final conc Find conc Final conc Final conc Final %:one Find conc Std wnc Am't -2.49 2.48 I 2.49 I 2.48 .-_I_-6--.17 i 6.18 6.17 ! ' - -----I 125 12.5 ! 12.5 __ ---24.8 ~24.9_ _ _24._8 _ _ 61.7 61.8 61.7 1 -. 11285T 112857 112857 fI 112857 186 248 249 372 372 j 372 371 2.48 2411 Final conc 125 PFOS PFOSA PFOSAA EtFOSE Final conc Final conc Final conc Final conc ng/ml ng/ml ng/ml ndml ng/rnl -- 5.00 -- -. 10.0 .---._I-- 25.0 5._.0_1_..-- 5.00 5.01 10.0 10.0 10.0 25.1 25.0 25.1 ---I_- 4.99 10.0 25.0 50.0 50.1 50.0 50.1 - - - - - _ _ _ I _ . - 100 ._. '00 100 100 100 - _ _ I 250 25 1 250 25 1 250 - --- - 500 501 500 501 499 I ---- 750 752 e--- 75 0 752 749 7fi0 .--- 1000 1002 IO00 1002 -- 998 I500 1503--- 1500 1503 1497 All Am't Surrogate Final conc ng/ml 500 Attachment C: Standard Calculation Sheet ETS-8-96.0 Extraction of PFOS from Urine 3M Environmental Laboratory Page 13 of 14 Page 68 31\11Medical DepartmentStudy: T-6295.12 Prep Date(s): Analyst(s): Sample Matrix: Methornevision: Target Analyte(s): BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Study Number: Equipment Number: Final Solvent & 'I"Number: Matrix BlanMIdentifier: Box: Summary of method: Notes: Attachment D: WeightNolume Sheet 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 14 of 14 Page 69 BACK TO MAIN 31W Medical Department Study: T-6295.12 3M ENVIRONMENTLAALBORATORY Analytical Report: FACT TOX-110 LRN-U2849 METHOD ANALYSIS OF POTASSIUM ~ l W L U O R O 0 C ' I ' ~ S U L F O N AOTREOTHER FLUOROCHEMICACLOMPOUNDSIN UIRINE EXTRACTS USING HPLC-ELECTROSPRAYMASS SPECTROM"RY/MASS SPECTROMETRY Method Number: ETS-8-97.0 Author: Lisa Clemen, Robert Wynne, Glenn Langenburg Adoption Date: ++28-44 Revision Date: )\I p Approved By: LaboratTv Manager L & t Group Leader Technical Reviewer Date ?-/w33 Date 42?h 9 Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method describes the analysis of urine extracts for fluorochemicals using HPLC-electrospray/mas spectrometry. 1.2 Applicable Compounds: Fluorochemicals or other ionizable compounds. 1.3 Matrices: Human,rat, and monkey urine, or other fluids as designated in the validation report. Word 6/95 3M Environmental Laboratory ETS-8-97.0 Analysis of Urine Extract Using ESIMS Page 1 of 10 Page 70 BACK TO MAIN 31W Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 2.0 SUMMARY OF METHOD 2.1 This method describes the analysis of fluorochemicals extracted from urine or other fluids, using HPLC-electrospray/mas spectrometry, or sinlilar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate(PFOS) anion, m/z= 499. Additionally, samples may be analyzed using a tand.em mass spectrometer to hrther veri@ the identity of a compound by detecting daughter ions of the parent ion. 3.0 DEFINITIONS 3.1 Atmospheric PressureIonization (API): The Microinass Quattro 11triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, et(:. The ionization process in these techniques occurs at atmospheric pressure (Le., not under a vacuum). 3.2 ElectrosprayIonization (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), Tantiem Mass Spectrometer (MSNS): The API Quattro 11triple quadrupole mass spectrometta is equipped with two quadrupole mass selective detectors and a collision cell. Ions are selectively discriminated by mass to charge ratio ( d z ) and subsequently detected. A single MS may be employed for ion detection or an ion may be selected in the first quadrupole, hgmented in the collision cell, and these fkagments may be analyzed inthe second quiidrupole. 3.4 Conventionalvs. Z-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. In the conventional.conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode, Though the configuration is different, the mfethodsof operation, cleaning, and maintenance are the same. However, Z-spray compone:nts and conventional components are not compatible with one another,but only with similar systems (i.e.*Z-spray components are compatible with some other Z-spray systems,etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these Quattro 11tripIe quadrupole systems. Currently MassLynx has WindowsNT 4.0 versions. For more details see the manual specific to the instrument (Micromass Quattro I1 triple quadrupole MassLym 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. 3M Environmental Laboratory ETS-8-97.0 Analysis of Urine Extract Using ESMS Page 2 of 10 Page 71 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. 4.2 Cautions: 4.2.1 Operate the solvent pumps below a back prc:ssure of 400 bar (5800 psi). If the back pressure exceeds 400 bar, the HP1100 Will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 INTERFERENCES 5.1 To minimize interferences when analyzing samples, teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 EQUIPMENT 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 Micromass Quattro I1 triple quadrupole Mass Spectrometer equipped with an electrospray ionization source 6.1.2 HPl 100 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 100psi. (House air system) 7.1.2 High purity grade argon regulated to approximately 6 psi. 7.1.3 HPLC analytical column, specifics to be deteirmined by the analyst and documented in the raw data. 7.1.4 Capped autovials or capped 15 mL centrifugt: tubes 8.0 REAGENTASND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-Qm water (ASTM type I), all water used in this method should be ASTM type I, or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.1.3.1 When preparing different amounts than those listed, adjust accordingly. 8.1.3.2 2.0 mM ammoniumacetate solution. Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 mL volumetric container containing 2000 mL Milli-Qm water, mix until all solids are dissolved. Store at room temperature. 3M Environmental Laboratory ETS-8-97.0 Analysis ofUrine Extract Using ESMS Page 3 of 10 Page 72 BACK TO MAIN 31W Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and twenty matrix standards are prepared during the extraction procedure. See ETS-8-96.0. 9.0 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. 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4' Cyor at room temperature, until analysis can be performed. 10.0 OUALITYCONTROL 10.1 Solvent Blanks, Method Blanks and Matrix Blaalks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a solvent blank, method blank, and matrix blank. prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to m e m e the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty samples, with a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lowrange of the initial calibration curve. 10.3 Continuing Calibration Verifications 10.3.1 Continuing calibration verifications are analyzed to veri@ 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. 11.0 CALIBRATION AND STANDARDIZATION 11.1 Analyze the extracted matrix standards prior to and following each set of extracts. The average of two standard curves will be plotted by regression (linear or otherwise, see 11.2), weighted 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 Extract Using ESMS Page 4 of 10 Page 73 BACK TO MAIN 31vl Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 11.2 Use the followingparameters for determining a calibration curve for each compound; Compound PFOS I Weighting I Il/X 1 I I Regression Fit i Linear Response type 12.0 PROCEDURES 12.1 Acquisition Set up 12.1.1 Set up the sample list. 12.1.1.1 Assign a sample list filename using MO-DAY-last two digits of yearincreasing letter of the alphabet starting with a 12.1.1.2 Assign a method (MSfile) for acquiring 12.1.1.3 Assign an HPLC program (Inlet file:) 12.1.1.4 Type in sample descriptions and vial position numbers 12.1.2 To create a method click on method in the Acquisition control panel then mass spectrometer headings and select SIR (Single Ion Recording) or MRM (Multiple Reaction Monitoring). Set Ionization Mode as appropriateand mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRS. Save acquisition method. If MSMS instruments are employed, additional product ion fkapentation informationmay be collected. Refer to Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM. 12.1.3 Typically the analyticalbatch run sequencebegins and ends with a set of extracted matrix standards. 12.1.4 Samplesare analyzed with a continuing calibrationverificationinjected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 3M Environmental Laboratory ETS-8-97.0 Analysis ofUrine Extract Using IWMS Page 5 of 10 s Page 74 BACK TO MAIN 3rd Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 12.2.2 Set-up the HP1lOO/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample sue = 10pL injection 12.2.2.2 Injectlsample = 1 12.2.2.3 Cycle time = 9.0 minutes 12.2.2.4 Solvent ramp = 0.00 min 1.Omin. 4.5 min. 6.5 min. Ammonium acetate 40% 60% 40% 60% 95% 95% 5% 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 Spectrometer Fitted with an Atmospheric Pressure Ionization Source," for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end of theprobe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Turn on the nitrogen. 12.3.5 Open the tune page. Click on operate to initiate source block and desolvation heaters. 12.3.6 Open the Inlet Editor. - 12.3.6.1 Set HPLC pump to "On" 12.3.6.2 Set the flow to 10 500 Urnin or as appropriate 12.3.6.3 Observe droplets coming out of the tip of the probe. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed 12.3.6.4 Allow to equilibrate for approximately 10 minutes. 12.3.7 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.7.1 Drying gas 250-400 literdhow 3M EnvironmentalLaboratory ETS-8-97.0 Analysis ofUrine Extract Using ESMS Page 6 of 10 Page 75 BACK TO MAIN 3rd Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 12.3.7.2 ESI nebulizing gas 10-15 litershorn 12.3.7.3 HPLC constant flow mode, flow rate 10- 500 pL/min 12.3.7.4 Pressure<400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7.5 Source block temperature 150 12.3.7.6 Desolvation temperature250 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, refer to appropriate MassLynx User's Guide). Ensure start and end sample number includes all samples to ble analyzed. 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations: 13.1.4 Calculatematrix spike percent recoveries using the followingequation: % Recovery = Observed Result - Backmound Result x 100 Expected Result 13.1.5 Calculatepercent differenceusing the following equation: % Difference = ExDected Conc. - Calculated Coilc, x 100 Expected Conc. 13.1.6 Calculate actual concentrationof PFOS, or olher fluorochemical,in matrix (IrgimL): LndmL of PFOS calc. b r n std. Curve x Dilution Factor) x Initial Volume of matrix (mL) Final Volume (mL) 1 ug; 1000 ng 14.0 METHODPERFORMANCE 14.1 Method Detection Limit (MDL) and Limit of Quantitation(LOQ)are method, d y t e , and matrix specific. Please see ETS-8-96.0, Attachment B, for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks, and Matrix Blanlcs 14.2.1 Solvent blanks, method blanks, and matrix blilnks values must be below the lowest standard in the calibrationcurve 14.3 Calibration Curves 14.3.1 The ? value for the calibration curve must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveriesmust be within f 30% of the spiked concentration. 3M Environmental Laboratory ETS-8-97.0 Analysis of Urine Extract Using E S M S Page 7 of 10 Page 76 BACK TO MAIN 3rd Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 14.5 Continuing Calibration Verifications 14.5.1 Continuing calibration verification percent recoveries must be within f 30% of the spiked concentration. 14.6 Ifcriteria 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 Ifdata are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 RECORDS 16.1 Each page generated for a study must have the followhg information included either in the header or hand written on the page: study or projeci number, acquisition method, integration method, sample name, extraction date, d:ilution 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) imd 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 n a m e and location of backup electronic data. 17.0 TABLESD,IAGRAMFSL.OWCHARTASN.D VALIDATIlON DATA 17.1 Attachment A: ETS-8-97.0 Data summary spreadshleet. 18.0 REFERENCES 18.1 ETS-9-24.0",Operation and Maintenance of the Micromass Atmospheric Pressure IonizatiodMass Spectrometer Quattro I1 triple quadrupole Systems" 18.2 The validation report associated with this method is IFACT-TOX-131,W2067 3Nl Environmental Laboratory ETS-8-97.0 Analysis of Urine Extract Using ESMS Page 8 of 10 Page 77 BACK TO MAIN 3Ml Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 19.0 AFFECTED DOCUMENTS 19.1 ETS-8-96.0,"Extractionof Potassium Perfluorooctanesulfonatear Other Fluorochemical Compoundsfrom Urine for Analysis Using HPLC-ElectrosprayhliissSpectrometry" 20.0 REvIsroNs Revision Number. Reason For Revisiay! Revision 3 M EnvironmentalLaboratory ET'S-8-97.0 Analysis of Urine Extract Using ESMS Page 9 of 10 Page 78 3CA Medical Department Study: T-6295.12 Laboratory Study # Study: Test Material: MatrixfFinal Solvent: MethodIRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept: Date of ExtractiodAnalyst: Date of Analysis/Analyst: Group Dose Sample# Concentration ug/d Initial Vol. mL Dilution Factor BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Final Cow. ug/mL Slope: Taken from linear regression equation. Attachment A Summary Spreadsheet ETS-8-97.0 Analysis of Urine Extract Using ESMS 3NI Environmental Laboratory Page 10 of 10 Page 79 BACK TO MAIN 3M Medical Department Study: T-6295.12 ~ Appendix D: Data Summary Tables Analytical Report: FACT TOX-110 LRN-U2849 Table 10. Average Results for the Analyses of Serum Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/Plus@Rats Exposed to 1r-6295.12 via Gavage D A Y ~FEI TAL 0.125 9.07 34.3 101 165 Table 11. Average Results for the Analyses of Liver Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/Plus@Rats Exposed to T-6295.12 via Gavage Dose Group Units I r 0 PFOS Concentration(pglg) Female Adult GO 0.288 PFOS Concentration (pglg) Fetal Liver I 0.169 0.1 29.2 7.93 0.4 107 30.6 1.6 347a 3.2 610 230 I I I I aResultfrom animal 19559 is an outlier and was not included in the average. NOTE: Liver concentrations are expressed as the average of samples within a dose group from all animals tested, not including samples that tested at below the limit of quantitation (LOQ=0.112 pg/g). Individual animal data are located in Attachment E, Data Spreadsheets. NOTE: Liver analysis were conductedat a contract laboratory but wssre corrected by 3M to reflectthe officialpurity values from the COA. Revisedreportsfrom the final contract laboratorieswill be addedas a report amendment at a later date. 3NI Environmental Laboratory Page 80 31vl Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Table 12. Average Results for the Analysis of Urine Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIus@Rats Exposedto T-62!35.12 via Gavage 0 mglkglday 0.1 mglkglday 0.4 --- mglkglday 1.6 mglkglday 3.2 mglkglday GESTATIONDAY0 GESTATIONDAY7 I GESTATIONDAY15 <LOQ~ <LOQ-l anomaly 0.00905 0.0497 0.0620 0.0685 0.302 0.959 1.53 I 1 0.308 1.10 1.60 I 1; 1 Or,.l 0.526 GESTATIONDAY21 0.0194 0.0574 0.555 "LOQ=Limit of quantitation (20.004 pg/mL) NOTE: Urine concentrations are expressedas pg/mLand are the average of samplesfrom all animals tested, not including samples that tested at or belowthe limit of quantitation(0.004 pglml-). Individualanirnaldata are located in the study binder archived for this report and also in Appendix E, Data Spreadsheets. Table 13. Average Results for the Analysis of Feces Samples in the Study of the Determination of the Presenceand Concentration of PFOS in the Sera, Livers, Urine, and Fecesof Crl:CD@BRVAF/Plus@Rats Exposedto T-6295.12 via Gavage GESTATIONDAY7 I 0.0155 I 0.490 I GESTATIONDAY15 0.0322 0.662 2.16 2.93 1 I 9.19 11.1 1 I 1:;33.0 1 I GESTATIONDAY21 0.0342 0.416 2.39 9.94 ~~ ~ ~ ~ ~ 'LOQ=Limit of quantitation (<0.010 pg/g) NOTE: Feces concentrations are expressed as pg/g and are the average of samples from all animals tested, not including samples that tested at or below the limit of quantitation (SO.010 pg/g:i, those reported as None Detected, or those reported as Not Quantifiable. Individual animal data are located in Appendix E, Data Spreadsheets and in Appendix G, Contract Lab Reports. 3M Environmental Laboratory Page 81 31\11Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Table 14. LOQ Values Used in FACT TOX-110 Analyses Urine Method I Effective Date I LOQ udmL Battelle I I Serum Fwes Advanced Bioanalytical I Centre I 8/23/99 8/26/99 4119/00 wI WmL I I 0.043 pgImL I 0.01 0 nala 3M Environmental Laboratory Page 82 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Appendix E: Data Spreadsheets Table 15. Results of Analyses of Urine Samples in the Study of the Determination of the Presence and Concentrationof PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/Plus@Rats Exposedto T-6295.12 via Gavage LOQ=Limitof quantitation (SO.004pg/mL) NS=Not sampled E=Not enough sample to complete the dilutiordanalysis L=Sample lost during extraction NOTE: PFOS concentrations in urineareexpressedas microgramsof PFOS/mLlof urinematrix (pghnL) 3M EnvironmentalLaboratory Page 83 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 GD15 GD21 ;td. external std. external std. 0.673 0.43'1 0.383 E 0.436 NS 0.600 E 0.29:3 NS 0.384 NS 0.502 NS 1.04 NS NS NS I I I 0:);: 1 1 1 I: 1 195456F 19547F 19548F <LOQ 0.0879 0.0N7S60 0.168 0.192 0.427 0.488 0.946 0.N5S34 0.248 0.359 NS L NS 0.593 0.688 19552F I 0.455 NS NS NS 19554F I 1.08 1.52 <LOQ Group 4 1.6 rngkglday 19557F 19560F I 1.13 1.11 <La1 0.718 I 19561F I 1.20 I 1.04 I 0.80!5 I NS I 19564F I I 0.336 I LOQ=Limitof Quantiition (<0.004p g h L ) NS=Not sampld E=Not enough sample to complete the dilutiodanalysis L=Sample lost during extraction NOTE: PFOS concentrations in urine are expressed as micrograms of PFOS/mL if urine matrix (pg/mL) 3kl EnvironmentalLaboratory Page 84 3rd Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Group/Dose Group 5 3.2 mglkglday Sample # (ail values in pg/mL) GDO external std. GD7 external std. GD15 external std. GD21 external std. 19565F 1.27 19566F 2.14 19567F 1.15 0.525 I9 5 6 8 ~ 0.961 1.82 I 19569F I 3.32 I 3.83 19571F 1.14 1.32 19572F 2.32 NS 0.381 I I I 0.647 1 0.391 0.973 1.39 1.51 I i::: ] I 19575F 1.44 I 0.865 0.551 I 19576F 2.98 NS NS NS 19577F 2.71 3.06 19578F 1.15 NS NS I 19579F 0.629 2.36 - 0.557 1.31 I 3kl Environmental Laboratory Page 85 3bl Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Table 16. Results of Analyses of Serum Samples in the Study of the Determination of the Presence and Concentrationof PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/Plus@Rats Exposedto T-6295.12 via Gavage Group/Dose Sample # (all values in pg/mL) GDO external std. GD7 external std. GDi5 external std. GD21 external std. 19501F 19502F 19503F 19504F 19505F 19506F 19507F 19508F 19509F 0.0968 0.0606 0.0524 0.0444 0.0475 0.267 0.0471 0.0473 cLOQ NS 0.189 0.051 <LOQ cLOQ 0.0560 cLOQ <LOQ 0.0708 <LOQ <LOQ Group 0.0 mg/kg/day 19512F I 19513F 19514F <LOQ 0.0455 cLOQ I 1 0.170 <LOQ 0.0677 <LOQ 0.0560 0.0720 I 19515F I <LOQ I <LOQ I 0.0502 I NS I 19516F I 0.0483 I cLOQ I cLOQ I <LOQ NS 0.0730 19505 N9 0.0760 I 19506 I NS I NS I NS I 0.231 19507 NS 19509 NS NS I I 19512 NS 19516 NS I NS NS NS NS 0.189 0.143 LOQ=Limitof quantiition (g0.043vg/mL) NS=Not sampled NOTE: PFOS concentrations in serumare expressedas microgramsof PFOSlml.of serum matrix (pg/rnL) NOTE: Serum analysis were conducted at a contract laboratory but were corrected by 3M to reflect the official purity values from the COA. Revisedreports from the final contract laboratorieswill be addedas a reportamendment at a later date. 3bl EnvironmentalLaboratory Page 86 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Table 16. Results of Analyses of Serum Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BR VAF/PIus@Rats Exposedto T-6295.12 via Gavage (Continued) Group/Dose Sample # (all values in pg/mL) external std. GD15 external std. GD21 external std. 19517F 19518F 9.94 NS 9.24 5.91 19519F 8.10 19520F 8.41 19521F 9.59 7.62 12.2 4.26 19522F 8.05 19523F 11.1 19524F 8.81 8.27 NS 9.42 4.62 19526F 8.19 19527F 10.4 Group 2 19528F 8.64 0.1 mglkglday 19529F 10.0 19530F 7.33 19531F 7.86 19532F 9.16 10.1 NS 1 16.95 5.35 6.32 2.63 6.61 10.2 6.54 8.99 8.73 8.35 19518 NS 19519 NS 19521 NS 9.68 19523 NS 8.81 19524 NS NS 8.90 19528 NS NS 9.33 19529 NS NS NS 8.99 NS=Not sampled ._ . NOTE: PFOSconcentrations inserum are expressedas microgramsof PFOSlmL of serum matrix (pg/mL) NOTE: Serum analysis were conducted at a contract laboratory but were corrected by 3M to reflect the official purity values from the COA. Revisedreports from the final contract laboratorieswill be added as a report amendment at a later date. 3NI Environmental Laboratory Page 87 3hl Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Table 16. Results of Analyses of Serum Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/Plus@Rats Exposed to T-6295.12 via Gavage (Continued) Group'Dose I Sample # (all values in pdmU1 GDO external std. I GD7 external std. I GD15 external std. I GD21 external std. 1 1 1 1 1 ii 19534F 1 40.5 43.2 48.5 I 19535F I 43.3 I 48.9 I 41.6 I 19536F 33.4 31.6 37.4 19537F 48.9 50.5 43.8 NS I 19538F 40.2 41.6 46.2 1 1 1 1 19541F 38.4 19542F 39.1 35.1 - 38.0 Group 3 19543F 40.0 39.8 - 37.0 19.9 0.4 mglkglday 19WF 39.6 I 19545F 41.4 I 19546F 37.6 19547F 37.0 - 42.4 I 40.2 36.5 ~ 3:: NS 19548F - 56.8 Fetal Serum 19533 NS 36.7 19536 NS 19543 NS NS 19546 NS NS 41.8 NS=Not samplkd .- , NOTE: PFOS concentrations inserum are expressedas microgramsof PFOSlmL.of serum matrix (pdrnL) NOTE: Serum analysis were conductedat a contract laboratory but were conectedby 3M to reflect the officialpurity values from the COA. Revised reports from the final contract laboratorieswill be added as a report amendment at a later date. 3M Environmental Laboratory Page 88 3bl Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Table 16. Results of Analyses of Serum Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/Plus@Rats Exposedto T-6295.12via Gavage (Continued) .OQ=Limit of qiiantitation (s0.043pg/mL) dS=Not sampled dOTE: PFOS concentrations inserclm areexpressedas microgramsof PFOS/mL of serum matrix (kig/mL) dOTE: Serum analysis were conducted at a contract laboratory but were corrected by 3M to reflect the official purity values from the COA. Revised reports from the final contract laboratorieswill be addedas a reportamendment at a later date. 3M Environmental Laboratory Page 89 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Table 16. Results of Analyses of Serum Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/PIus@Rats Exposedto T-6295.12 viia Gavage (Continued) GD15 GD21 td. external std. external std. 298 NS 269 NS 254 NS 290 208 271 123 232 NS 266 143 274 106 274 NS 258 NS 245 NS NS NS 312 191 NS NS 334 161 279 NS n I 19568 I NS I NS I NS 1 197 I I 19569 I NS I NS I NS I 152 I 19571 NS 19572 NS NS 19577 NS NS 195 NS=Not sampled ._ . NOTE PFOS concentrations in serum are expressed as micrograms of PFOS/mL of serum matrix (VglmL) NOTE: Serum analysis were conducted at a contract laboratory but were comscted by 3M to reflect the official purity values from the COA. Revised reportsfrom the final contract laboratorieswill be added as a report amendment at a later date. 3M Environmental Laboratory Page 90 3121 Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Table 17. Results of Analyses of Feces Samples in the Study of the Determination of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/Plus' Rats Exposedto T-6295.12 via Gavage Sample # (all values in pg/g) GDO external std. GD7 external std GD15 external std. GD21 external std. 19501F ND ND NC) NS 19502F ND NQ ND NS I 19503F I 4 O Q I NS I NS I NS I 19504F ND 19505F ND ND 19506F 0.0262 0.0155 0.0387 0.01 07 19507F ND Group 1 0.0 mglkglday 19508F 19509F <LOQ 0.0348 <LOQ ND 0.0210 0.0733 19511F I ND ND I 19512F I ND I ND I 0.0503 I 0.0187 I 19513F ND 19514F ND ND I I 19515F I ND I ND I NC) I NS I I 19516F 0.0529 NQ 0.01 88 <LOQ 1 19517F 1 0.609 0.356 0.659 NS 19519F 1 0.339 0.299 0.680 I 19520F I 0.587 I NS I NS I NS I 19522F I 0.331 0.676 Group 2 0.1 mglkglday 19525F 0.939 19526F 0.388 0.408 0.676 19527F 0.432 19528F 0.609 0.462 19529F 1 19530F I 19531F 0.693 0.638 0.647 0.542 1 19532F I 0.650 I 0.499 I 0.494 I NS I LOQ=Limitof quantiition (SO.010pug) NS=Not sampled ND=None detected NQ=Not quantifiable NOTE: PFOS concentrationsin feces are expressed as microgramsof PFOSlgof feces matrix (pg/g) 3M Environmental Laboratory Page 91 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Group/Dose Sample # (all values in pug) GDO external std. 19533F 2.26 19534F 3.26 I 19535F 2.43 19536F 2.76 GD7 external Std. 2.21 2.34 1.59 2.83 GD15 external std. 2.40 4.19 3.15 2.77 GD21 external std. 1.46 2.70 NS 1.61 19539F Group 3 1954OF 1.78 1.97 3.61 0.4 mg/kg/day I 19541F 1.83 19544F 2.98 2.93 19545F 2.39 19546F 2.03 1.32 I 1 I 19547F I 1.93 1 1.79 I 3.36 I NS 19548F 2.03 2.47 3.401 4.71 I 19549F I 12.3 NS NS NS 19551F I 9.03 NS I 19552F I 11.4 I NS I NS I NS 19554F 1 13.5 10.2 Group 4 1.6 rng/kg/day 19557F 7.79 1 19558F I 5.95 I 5.12 I 6.23 I NS 19561F I 10.5 7.71 19562F 8.33 19563F 6.49 91.34.20 19564F 14.7 11.9 3M EnvironmentalLaboratory Page 92 BACK TO MAIN 3rd Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Table 17. Results of Analyses of Feces Samples in the Study of the Determination 1 1 1 of the Presence and Concentration of PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/Plus@Rats Exposed to T-6295.12 via Gavage (Continued) ~~ GrouplDose Sample # (all values in pdg) GDO external std. external std. external std. external std. 19565F 20.4 G44D.97 :DG GiiI 19566F 25.7 23.0 13.2 I 19569F 19570F 32.5 27.0 19571F 26.0 I Group 5 19572F 22.1 3.2 rnglkglday 19573F 20.0 19574F 25.5 37.4 24.7 18.7 23.2 31.6 I 41.1 1 iz I 19575F 20.0 30.8 35.4 I 19576F I 31.6 I NS I NS I NS I 19577F 29.8 19578F 30.2 NS 19579F 22.0 31.2 29.1 24.6 19580F 21.6 26.1 28.4 NS 31\41 Environmental Laboratory Page 93 3NI Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Table 18. Resultsof Analyses of Liver Samples in the Study of the Determinationof the Presence and Concentrationof PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/Plus@Rats Exposedto T-6295.12 via Gavage Concentration of GroupDose I 1 Sample #/ 19517F 19518F 19519F Concentration of PFOS (all values in I i!m I I 42.6 I 21.8 I I 1 18.9 19515F 19516F 19504 19505 19506 19507 19509 19512 19516 0.286 0.138 0.153 <LOQ 0.149 <LOQ 0.204 <LOQ <LOQ 1 I Group 2 0.1 mg/kg/d,3y 1 19523F I 19525F 19526F 19527F z::17.7 1I 46.4 19530F 19531F 32.1 I 19532F 34.6 Fetal Liver 8.64 I 7.2-_3 I 7.68 3M Environmental Laborat.ory Page 94 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Table 18. Results of Analyses of Liver Samples in the Study of the Determination of the Presence and Concentrationof PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/Plus@Rats Exposed to T-6295.12 via Gavage (Continued) 19533F 19534F 19535F I 19536F I 88.1 I 19537F 19549F NS 342 19551F NS I I NS 19553F I 351 Group 3 0.4 mgkglday 19539F 1954OF 19541F 19542F 19543F 19544F 1 19556F 1 304 I 268 I 85.9 Group 4 1.6 mg/kg/day 19546F 19562F 215 19563F 19548F Fetal Liver 19533 27.5 I I I 19543 I 30.7 I I 19546 41.6 LOQ=Limitof quantitation(20.112 u. -dd-, NS=Not sampled NOTE: PFOS concentrations in liverare expressedas microgramsof PFOS/g of lwer mahix (pg/g) NOTE: Liver analysis were conducted at a contract laboratory but were corrected by 3M to reflect the official purity values from the COA. Revisedreports from the finalcontract laboratorieswill be addedas ,3 report amendment at a laterdate. 3M Environmental Laboratory Page 95 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Table 18. Results of Analyses of Liver Samples in the Study of the Determination of the Presence and Concentrationof PFOS in the Sera, Livers, Urine, and Feces of Crl:CD@BRVAF/Plus@Rats Exposed to T-6295.12 via Gavage (Continued) Group/Dose Concentration of I 19565F 19566F 594 I 19569F 468 Group 5 3.2 mg/kg/day 19573F 19574F 19578F 1 NS I Fetal Liver 19572 19577 339 19579 213 LOQ=Limitof quantiition ($0.112 pglg) NS=Not sampled NOTE: PFO8concentrations in liver are expressedas microgramsof PFOS/gof liver matrix (pglg) NOTE: Liveranalysis were conductedat a contract laboratorybut were corrected by 3M to reflectihe officialpulityvalues from the COA. Revisedreportsfrom the finalcontract laboratorieswill be addxias a reportamendment at a laterdate. 3M Environmental Laboratory Page 96 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Appendix F: Example Calculations Formula Used for Urine Analyses in Study FACT TOX-110 AR (ng/mL,) x DF x SC x 1.0 pg x PC = Reported Concentration(yglmL) 1000 ng Calculation Used for Group 2, GD15, Animal ID 19!522F 123.57ng/mL x 1 x 0.9275 x 1.0 pg x 0.864= 0.0990 pg PFOS/mL urine 1000 ng AR- Analytical result fiom MassLynx summary DF- Dilution factor SC-PFOS salt correction constant (0.9275) PC-PFOS purity correction factor (86.4%) 3M Environmental Laboratory Page 97 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 ~~ ~ ~ Appendix G: Contract Lab Reports This Appendix includes the following contract laboratory reports: Battelle Memorial Institute,"Oral (Gavage) PharmacokineticStudy of PFOS in Rats," 63 pages Advanced Bioanalytical Services, lnc., "Method Validation for the Quantitation of Perfluorooctanesulfonate(PFOS) in Rat Serum by Turbo Ion Spray LC/MS," 50 pages Advanced Bioanalytical Services, lnc., "Quantitative Determination of Perfluorooctanesulfonate(PFOS) in Rat Serum from Study #FAC T-TOX-110 Using Turbo Ion Spray LC/MS," 24 pages Advanced Bioanalytical Services,lnc., "AddendurrAuantitative Determination of Perfluorooctanesulfonate(PFOS) in Rat Serum from Study#FACT-TOX-110 Using Turbo Ion Spray LC/MS,"3 pages CentreAnalytical Laboratories,lnc., "Oral (Gavage) PharmacokineticStudy of PFOS in Rats," 103 pages 3bl EnvironmentalLaboratory Page 98 BACK TO MAIN 3NI Medical Department Study: T-6295.12 1L Analytical Report: FACT TOX-110 LRN-U2849 BIOLOGICAL.SAMPLE ANALYSIS Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I10 ##Baltelle . . . Pulting Technology To Work FINAL REPORT ORAL (GAVAGE) PHARMACOKINETIC STUDY OF PFOS IN RATS 3M Toxicology Services-MedicalDe,partment 3M Center, BuiIding 220-2E-02 St. Paul, MN 55144-1000 Testing Facility Battelle Memorial Institute 505 King Avenue Columbus, Ohio 43201-2693 Prepared By Patrick L. South,B.S. 3 M EnvironmentalLaboratory Page 99 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 Battelle Study Number: N003296-F 3M ToxicologyServicesProtocolNumber:FACT-TOX-I10 FINAL REPORT ORAL (GAVAGE) PHARMACOKINETIC STUDY OF PFOS IN RATS I Bhdlle Principal Investigator ii 3M EnvironmentalLaboratory Page 100 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Battclle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-110 ORAL (GAVAGE) PHARMACOKINETIC STUDY OF PFOS IN RATS EXECUTIVE SUMMARY Rat liver samples sent to Battelle by 3M Environmental Technology Serviceswere analyzed by the previously validated method "Method for Analysis of Potassium Perfluorooctanesulfonate(PFOS)in Rat Liver by LCIMSIMS". Samples were extracted and analyzed by High-Performance Liquid Chromatography Mass Spectroscopy(LC/MS/MS) for PFOS content only. Related fluorochemicals mentioned in the analytical method were not investigated. The results for the concentration determination of PFOS in the liver samples from this study are attached as appendicesto this report. Concentrations are reported as mass of PFOS (pg) per gram of liver tissue extracted. J iii 3M Environmental Laboratory Page 101 3M Medical DepartmentStudy: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-110 QUALITY ASSURANCE STATEMENT This study was inspected by the Quality Assurance Unit and reports were submitted to the task leader, study director, and associated management as follows: Phase Inspected Inspection Date Date Reported to Battelle Task Leader1Battelle Management Date Reported to Offsite Study Director1 Management Sample homogenization Sample weights Sample analysis Sample processing Audit final report Audft study file Audit final report 812711999 812711999 813011999 813011999 912011999 912011999 211 6l2OOl 912011999 912911999 912011999 912911999 9l2Ol1999 912911999 912011999 9/29/1999 912011999 912911999 912011999 912911999 211612001 2120l2001 I Qdality Assd&nce Udt Date Battelle Memorial Institute iv 3M Environmental Laboratory Page 102 3M Medical Department Study: T-6295.12 .. BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M ToxicologyServices Protocol Number: FACT-TOX-I10 GOOD LABORATORY PRACTICES COMPLIANCE STATEMENT Study Title: ORAL (GAVAGE) PHARMACOKINETICSTUDYOF 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 exceptionthat the mass spectrometry data for the liver samples was collected and processed with the MassLynx software system (version 3.1), which was not fully validated. The study was listed on Battelle'sMaster List of regulated studies. Jon C. U d r e , Ph.D. Date Battelle Principal Investigator TGV- Mkrvin T.Case, D.V.M., Ph.D. Study Director V 3M Environmental Laboratory Page 103 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology ServicesProtocol Number: FACT-TOX-1 10 Table of Contents Executive Summary .................................................................................................................................... ... 111 Quality Assurance Statement ................................................... ..................................................................... iv Compliance Statement .................................................................................................................................. v Tab 1.0 l e of I Contents ntroducti o..n.............................................................................................................................................................................................................................................................................v1i 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 4R.e2s.1ults.Q...u...a..l.i.t.y...C..o..n..t..r..o.l...._....._..............................................................................................................................................................................................22 4.2.2 Sample Results....................................................................................................... 3 5.0 Conclusions...................................................................................................................................... 3 6.0 Acknowledgements.......................................................................................................................... 3 7.0 Specimen Storage and Record Archives.......................................................................................... 3 Table 1. 1 List of Tables Example of Instrument ParametersUsed to Analyze Samples................................................ 2 Appendix A (Results) Summary Results for Rat liver Sample Analysis.. ...,......,.,,..,............................................:. ..A-I Appendix B (Daily Acceptance Criteria Summary) Daily Acceptance Criteria Summary .........................,...........,..........................................B-1 Appendix C (Method) Method for Analysis of Potassium Perfluorooctanesulfonate (PFOS)in Rat liver by LC/MS/MS.........C-1 Representative Chromatograms .....A...p..p..e.n..d..i.x..D...(.C...h..r.o..m...a.t.o..g..r.a..m...s.).......................................D-1 Appendix E (Protocol, Amendments, and Deviation) Protocol, Amendments, and Deviation .............,............................_.....................................E-1 PFOS Purity Report . . Appendix F (PFOS Purity Report) ._......................,.,...............,.....,..,,..,........ .. ..,.........................................F-1 vi 3M Environmental Laboratory Page 104 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M ToxicologyServices Protocol Number:FACT-TOX-I 10 1.O Introduction This report presents a description of the method used to analyze PFOS in rat liver samples from 3M Study Number FACT-TOX-110 and the results from this analysis. See Appendix E for a copy of the study protocol, amendments, and deviation reports. 2.0 Reference Substances The analytical reference substance for this study was potassium perfluorooctanesulfonate (PFOS) lot number 171, supplied by 3M. Note that based on information supplied to Battelle from 3M, PFOS has two equivalent names. The name appearing on the Material Safety Data Sheet and bottle label is potassium perfluoroalkyl sulfonate. The name more commonly used by 3M in analytical methods and correspondence is potassium perfluorooctanesulfonate. The latter name will be used in this report. See Appendix F for purity data supplied by 3M to Battelle. The surrogate standard was lH, 1H,2H,2H-Perfluorooctane sulfonic acid, lot number 59909, supplied by ICN. 3.0 Receipt of Samples Samples were received frozen and intact at Battelle, from 3M Environmental Technology and Services, in one batch on August 20,1999. Samples were generated by Argus Research under protocol number 418-013. The samples were stored at approximately-20C. 4.0 Analysis of Samples I? 4.1 Summary of Method Samples were analyzed by a previously validated method (Battelle study number N003604-A). The cument version of the method is attached to this report in Appendix C. Samples were analyzed by LC/MS/MS, and an example of the instrument parameters is listed in Table 1. Note that only PFOS itself (and the surrogate) was quantitated. The other related fluorochemicals,although present in the stock solutions, were not monitored. Quadratic regressions weighted l / x were used to construct the calibration curves. 1 3M Environmental Laboratory Page 105 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-110 'l'uhlr I . Esample of Instrument Parameters Used to Analyze Samples 4.2 R e s u l t s \ \ \ i L m witability acceptance cnteria were established during the method validation and .IIL I I I ~111,1~,1 111 \ppcndix C, Section IX Acceptaim Criteria. Relevant statistics from each sample .II ,111 1111)\ 1 1 1 ~ t1l11 Appendix B. Representative chromatogramsare given in Appendix D. L 3M Environmental Laboratory Page 106 3M Medical Department Study: T-6295.12 .- BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 4.2.2 Sample Results Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number:FACT-TOX-I IO The results of the sample analyses as well as a method detection limit determination are presented in Appendix A. The limit of quantitation is defined as the concentration of the lowest standard which meets acceptancecriteria for accuracy (25% RE). The notation BLOQ denotes "Below Limit of Quantitation" for samples that had concentrationslower than the theoretical concentration for the 0.13 pglg calibration standard. The notation ALOQ denotes "Above Limit of Quantitation" for samples that had concentrations higher than the theoretical concentration for the 13 pg/g calibration standard. Samples that were initially ALOQ were diluted with blank liver homogenate and re-extracted. Samples that were expected to be ALOQ were first diluted with blank liver homogenate before extraction. The "Corrected PFOS Conc" presented in the results tables is the concentration found for the diluted sample multiplied by its dilution factor (final volume ) sample homogenatevolume). The method detection limit (MDL) of PFOSwas calculated to be 0.0173 pg/g fiom the analysis of 7 replicate preparationsof 0.13 pg/g calibration standard. The h4DL was calculated by multiplying the standard deviationof the found concentrationsof the 7 reps by 3.143; the Signal-to-Noise ( S M ) ratio was calculated by dividing the mean found concentration of the 7 reps by their standard deviation. The method of MDL determinationwas provided by the Sponsor. 5.0 Conclusions ' All analyses met acceptance criteria unless otherwise noted. 6.0 Acknowledgements t Acknowledgement of principal contributors participating in the performance of this study at Battelle is presented in the following list. Participant Title Jon C. Andre, Ph.D. Richard W. Slauter, Ph.D., D.A.B.T. Patrick L. South, B.S. Battelle Principal Investigator Senior Program Director Mass Spectroscopist 7.0 Specimen Storage and Record Archives Refer to Appendix E, protocol amendment 2 for record archival information. All residual liver samples, extracts, and unused test article will be disposed of or returned to the Sponsor as directed by the Sponsor. 3 3M Environmental Laboratory Page 107 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FAST-TOX-110 LRN-U2849 Battelle Study Numba: N003296-F APPENDIX A -RESULTS 3M Environmental Laboratory Page 108 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Baaelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-1I O RAT LIVER SAMPLE RESULTS STUDY: N003296-F ANALYSIS DATE AND INSTRUMENTID: DATA ENTERED: SPREADSHEET SOFTWARE: Electronically Excel 97 25Aug99; 9053 FINAL LIVER SAMPLE RESULTS: Animal numbers ending in "P" indicate pooled fetal liver BLOQ = BELOW CONC OF LOWEST STANDARD IN CALIBRATION CURVE ALOQ = ABOVE CONC OF HIGHEST STANDARD IN CALIBRATION CURVE (1) = SAMPLE TO BE DILUTED BEFORE REPEAT EXTRACTION (2) = SAMPLE INITIALLY MARKEDAS 0.1 mg/kg/day; LABEL HAND-CORRECTEDTO INDICATEVEHICLE A- I 3M Environmental Laboratory Page 109 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Banelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I I O RAT LIVER SAMPLE RESULTS STUDY: N003296-F ANALYSIS DATE AND INSTRUMENT ID: DATA ENTERED: SPREADSHEET SOFTWARE: 28Aug99; 9053 Electronically Excel 97 FINAL LIVER SAMPLE RESULTS: Animal numbers ending in "P" indicate pooledfetal liver BLOQ = BELOW CONC OF LOWEST STANDARD IN CALIBRATION CURVE ALOQ =ABOVE CONC OF HIGHEST STANDARD IN CALIBRATION CURVE (1) = SAMPLE REPEATEDFROM25AUG99 ANALYSIS (2) = SAMPLE INITIALLY MARKEDAS 0.1 rng/kg/day; BAG LABEL HAND-CORRECTEDTO INDICATEVEHICLE A-7 3M Environmental Laboratory Page 110 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Banelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I IO RAT LIVER SAMPLE RESULTS STUDY: N003296-F ANALYSIS DATE AND INSTRUMENT ID: DATA ENTERED: SPREADSHEET SOFTWARE: 3OAug99; 9053 Electronically Excel 97 FINAL LIVER SAMPLE RESULTS: Animal numbers ending in "P" indicate pooled fetal liver Animal Number Dose Grp mglkglday Corrected PFOS Cone (P9/9) Note if Sample is a Rerun Note if Rerun is Needed BLOQ = BELOW CONC OF LOWEST STANDARD IN CALIBRATION CURVE ALOQ = ABOVE CONC OF HIGHEST STANDARD IN CALIBRATION CURVE A-3 3M Environmental Laboratory Page 111 BACK TO MAIN 3M Medical Department Study: T-6295.12 ___ Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I IO RAT LIVER SAMPLE RESULTS STUDY: N003296-F ANALYSIS DATE AND INSTRUMENT ID: DATA ENTERED: SPREADSHEET SOFTWARE: 0lSep99; 9053 Electronically Excel 97 FINAL LIVER SAMPLE RESULTS: Animal numbers ending in " P indicate pooled fetal liver Animal Number Dose Grp mglkglday Corrected PFOS Cone (IJg/g) Note if Sample is a Rerun Note if Rerun is Needed BLOQ = BELOW CONC OF LOWEST STANDARD IN CALIBRATION CURVE ALOQ = ABOVE CONC OF HIGHEST STANDARD IN CALIBRATION CURVE A-4 3M EnvironmentalLaboratory Page 112 3 M Medical Department Study: T-6295.12 BACK TO MAIN .. Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-1 IO METHOD DETECTION LIMIT (MDL) RESULTS STUDY: N003296-F ANALYSIS DATE AND INSTRUMENT ID: 28Aug99; 9053 DATA ENTERED: Electronically SPREADSHEET SOFTWARE: Excel 97 All concs in pg/g PFOS Calculated Concentration of Replicate 1 0.1184 Calculated Concentration of Replicate 2 0.1192 Calculated Concentration of Replicate 3 0.1251 Calculated Concentration of Replicate 4 0.1239 Calculated Concentration of Replicate 5 0.1351 I CalculatedConcentrationof Reulicate6 CalculatedConcentrationof Replicate 7 0.1248 0.1225 Mean Concentration Std. Dev. 0.1241 0.0055 Spike Level 0.1340 LOQ (det. from 10 x std.dev. "noise") LOQ (dct from cal curve low std) Curve Coeff of Determination Date analyzed K1c-vSpike Level too high Spike Level must be < lox MDL -2 Spike Level too low; SpikeLevel must be > MDL -3 SM too low; SM must be > 5 -4 Coeff of Det of calibration curve unacceptable A-5 3 M Environmental Laboratory Page 113 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Battclle Study Number: N003296-F 3M TosicologyServices Protocol Number: FACT-TOX- 1 IO 3M Environmental Laboratory Page 114 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number:FACT-TOX-I 10 DAILY ACCEPTANCE CRITERIA SUMMARY PFOS IN RAT LIVER STUDY NW3295-F SPREADSHEET SOFTWARE: Excel 97 DATA ENTERED MANUALLY NA=Not Appllable a ! CdCurve SyrSuR Homog.niz.r Analysis Date C o dof Det KRSD Fort Avo R.cov DllnStd A m R.cov Auputt 25,1999 0.9800 4.0 111.1 NA August 28.1999 0.9981 3.1 112.4 102.4 August 30.1999 0.9943 7.5 110.9 116.0 September 1.1999 0.9951 5.3 118.1 110.1 AveQCl %RE (KRSD) -7.1 (15.8) 2.7 (0.9) -1.8 (4.0) 1.9 (4.6) AveQC2 %RE (KRSD) O.O(l4.2) 2.3 (3.4) -1.9 (4.2) 1.6 (5.6) AveQCI AveQC4 %RE (%RSD) %RE (%RSD) -5.5(13.7) ' :5(16.8) -3.8 (4.1) -6.9 (0.9) -6.5 (6.4) 1.4 (1.1) -10.7 (4.6) 18.0 (7.4) B-I 3M Environmental Laboratory Page 115 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Baltclle Study Number: N003296-F 3M Toxicology ServicesProtocol Number: FACT-TOX- I IO APPENDIX C-METHOD 3M Environmental Laboratory Page 116 BACK TO MAIN 3h.1 Medical DeDartrnent Studv: T-6295.12 Analytical Report: FACT TOX-110 LRN42849 Battcllc Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I 10 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS)IN RAT LIVER BY LC/MS/MS Version 1.0 Study No.: AnaiystlDate: Revisions to the method r- I I .. G-eL L C V Approvedby: J&C. Andre. PBD. M&ager, Bionalytical Chemistry Date: U I3 1 /w 3M Environmental Laboratory Page I of 16 c- 1 Page 117 BACK TO MAIN 3M Medical DepartmentStudy: T-6295.12 Analytical Report: FACT TOX-110 LKN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I 10 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) CN RAT LWER BY LC/MS/MS Version 1.0 Study No.: AnalystlDatc: I. SUMMARY The extraction and analysisof potassium p c t t l u o r m m d f o n a t e and related fluorochemicalsin rat liver is performed Calibration styldvds are prcparcd by spiking blank Liver homogcmte with solvent standards from two independently-prcparcdstocks. The calibration n;mdardsarc fortifed with surrogatestandard, buEered, and cxuactcdwith ethyl acetate. The organic phases arc evaporated to dryness and reconstituted in methanol for analysis by Lc/Ms/MS. To extract and analyze potassium perfluorooctanesulfonateand rrlatedfluomhemid compounds found in Spgue-Dawley rat Liver. See Chain of Custody records ifapplicable. lV. GENERAL INS"RUCrr0NS d Calibrate all required balances according to the SOP on baiance usage. Make cquivalcntdilutions when thevolume necdcd variesfrom the volume stated in the mclhod I Labelall standardand magent solutionsas Spccificd in theappropriate SOP.If you intend to reuse a solutionfor fuhlre taslu,be nut the label includes the pnparatian date and study number for which the solution \MS initially pnpa& Sign on the final page of this method to signify that you have followed the method as written. all materials and reagents an? cumnt, and all equipmenthas been properly calibratedIf you deviatefrom the method, document the change, and obtainthe approval of the unit manager, study director, or taskleaderas soon as possible. e initial and datc all data entrieson the page on which they wen mnde. If only one puson enters all data on a single day. the documentdon may be made in a single locntioa on that page. If multiple staff make entries, the additional Cneicsmust k initialed and datedby the person meking the entry. Lincau or NA dmotcs"Not Applicable". e The method is h a e n in general chrowlo~caol rder, but the sequence of steps may be altered if the analyst deems it appropriate. unless the order for certain activities is specified e Stockswill be used for the d d o n of the study unless consumedor unless stability is considered suspect e No c o d o n wiU be made for purity or salt content of any test article but PFOSAA. e Use glassvolumelric. Eppendorf repeater, or positive-displaamentpipets for dispensing methanolic solutions. e Contact with Tdlon by the test article should be minimized. v. MATERIALS See Table I for all required chemids reagents. and solvents. UK Table 1 for documentation Check dl labels cycfully to e m tha~all mterials arc not expired i d that they are the proper purity or grade. Page 2 of 16 3M Environmental Laboratory Page 118 BACK TO MAIN 3-M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I 10 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS)IN EWT LIVER BY LC/MS/MS Version 1.0 Study No.: AndystlDate: Rat Liver Matrix Harlan spragueDawley -2ooc Ammonium Acetate. Mobile Phase RT a wclH3301 Sodium Hydmxide, Reagent Prep RT NaOH Tetrabutylammonim Extract Rep r RT Hydrogensult?te NkCO, SodiumBicahonate, ExaaRcp RT NaHC4 See Table 2 for dl required major pieces of equipment. Uw the table to document the aftu;ll p i m (e.g. make. model) of equipment Check calibration of all equipmentrequiringcalibration (e.g. balances) to ensure it is currcni. Page 3 of 16 3M Environmental Laboratory Page 119 BACK TO MAIN 3-M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number:N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I 10 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LLVER BY LCIMSIMS Version 1.0 Study No.: AnalydDatc: Refrigerator (1-9'C) Centrifuge Liver Store Buffer. Stocks Phase scparation d Tat Tubs Liver sample Stodwcll Polypropylene. swam homogenization Scientific 15 mL Centrifuge T u k s Extmt Samples Blue Falcon Polypropylene, 2096 r 15 mL Test T u b Evaporate Exbacts Blue Falcon Polypropylene, 2002 Tra~~sporttubeJ store Qcs Elkay 12x75m 3mL 127-TI60-56P polypropylene Magnetic stjxrcr Stir matrix Orbital Shaker Extract samples 3M Environmental Laboratory Page 4 of 16 C-4 Page 120 BACK TO MAIN 3-M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Banelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I10 METHOD FOR ANALYSIS OF POTASSrUM PERFLUOROOCTANESULFONATE (PFOS)IN RAT LNER BY LC/MS/MS Version 1.0 Study No.: AndystlDate: M. PROCEDURE A. Preparation of 2 mM Ammonium Acetate -+ Weigh 0.1500 0.0020g of ammonium acetate and tnnder to o 1000-mL volumetric flask. Dissolve the solid in water and dilute to volume with water. Solution may be used for one month stored at mm tcmpaanm. Aaual mass ofvnmOniumacetate: Actual final volume: Date of prepador Study NO: B. Preparation of -29y Sodium Hydroxide Solution Weigh 200 t 2 gof sodium hydroxide into abeaku. Add500 mL OfMilli-Qwaerand mix to dissolve. Cool andtransferto a polypropylene bottle for storage. Solutionmay be stored for 6 months at morn tempcrahrre. Aaual muss of sodium hydroxide: Actual volume Mifli-Q water: JI Date of prepamtion: Study No: C. Preparation O f -2.9% Sodium Hydroxide Solution Add 10 mL of -29% SodimHydroxide Solutionto a 1oO-t~Lvolumetriicfl& and dilute to volume with Milli-Qwater. Transferto a polypropylenebottle for storage. Solution may be stored for 6 months at mom tempcratun. Actual volume of -29% NaOH solution: Actual final volume: Date ofprcpera(io0: Study No: D. Preparation of TetrabutylammoniumHydrogensulfate(TBA) Solution, 0.5 M, @H 10) pH Meter Calibriuion pH bufer 7 pH butTer IO pH W i g : pH ruding: Add 169 t 1g ofTBA to -500 mL of h4illi-Q water in a beaker. Adjust the pH to 10.00 t 0.02using45-60mL of 29% Sodium Hyhxidc S~lutiond, ilute to lo00 mL with h4illi-Qwater, and mix. Adjust the pH to 10.00 t 0.02 using-2.9'0 NaOH and mkx. Transfer to a polypropylenebottle for storage. Solutionmay be used for one monh stored at rwrn tempcmturc. but the oH must k cheeked Dn o r to each u r Adjust to pH 10.0 x 0.02 with 2.9% SadiumHydroxide Solutionas mes~aty. Page 5 of 16 c-5 3M Environmental Laboratory Page 121 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M ToxicologyServices Protocol Number: FACT-TOX-I 10 METHOD FOR ANALYSIS OF POTASSIUM PERnUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LClMslMS Version 1.0 Study No.: AnnlydDatc: Actual mass of TEA: A d finalvolume: Actual finalp H Date of prepamion: ~ Study No: pH after rahccking and/or readjusting: E. Preparation of 0.25 M Carbonate Buffer Weigh 26.5 t 0.1 g of sodium cadmate and 21.0 x 0.1 g of d u m bicarbonate and transfer to Ihe sdmc 1OOO-mLvolumetricflatk. Dissolve the marcrialsin MilliQ WaDr. dilute to volume withW-Qwater. rnk and hansferto a polypropylenebottle for storage. Solution may be used for 1 month when stored d n g W Actual mass of sodium carbonate: Actual mass of sodium bicarbonate: Actual final volume: Date of prcwtion: Study No: F. Prepnration of Mobile Phase Component A: Mix togclha 600LULof 2 mMammoniumacetate and400mL of methanol. Solution may be used for 1 month when stored at mom tempcratun. .? Actual volume of 2 mM ammoniumacetate: mL Actual volume of methanol: mL Date ofp@on: Study No: ComponentB: Mix together 50 mL of2 mMammomumacetateand 950 mL of methanol $htion my be uwd for I month when storedat mom tempemtun?. Actual volumc of2 mMPmmDniUm acetate: mL Aaual volume of mcthanol: mL Date ofprepamtion: Study No: G. Preparation of Stock Surrogate Standard and Working Surrogate Standard (WSS) 1. Stock Surrogate Standard(250,OOO ng/mL): Weigh 25 1 2 mg of 1H. 1H.2H. ZH,-pc&~rooctane sulphonic acid and aansfer to a 1 0 0 4 volumetricflask Dissolve in methanol, dilute to volume with methanol. and mix. Storerrfrigcratd,pro& from W ti& Achul Wcigbt: Achul Dilution Volumc: Date of Prepmation: Study No: Page 6 of 16 C-6 3M Environmental Laboratory Page 122 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003196-F 3M Toxicology Services Protocol Number: FACT-TOX- 110 METHOD FOR ANALYSIS OF POTASSIURI PERFLUOROOCTANESULFONATE (PFOS)IN RAT LlVER BY LC/RlSIL\.IS Version 1.0 Study No.: Analystmate: 2. WSS (1000 ng/mL): Dilute 100WL of stock surrogate standad to 25 mL with methanol and mix. Actual Volume of Stock Internal SwndYd AchcPI Dilutioo volume: Date of heparation: H. Preparation of Calibration Solvent Stocks and Working Standards 1. Solvent Stocks: For eachd y t e weigh the specified a m k t of standard (independently weighed as A and B replicates) listed in Table 3 and &er into separate volumetricflasks. Dissolve in melhanol. dilute to volume with melhanol, and mix well. Ston refrigerated, protectedfrom W light. 2. Mixed Solvent Stocks: Pipet the spccificdamount of each analylicalstandard Replicate A as listed in Table 3 and lransfer into a single volumetricflask Dissolve in methanol, dilute to volume with methanol, and mix well. Ston rcfigentd. protectedfrom W li& Rcpcat the pnxrrs with Replicate B stocks. Theh d sobent stocks we .. used toprepare the working standards I Date of preparation: Study No: 3. working standards (WS): Dilute the mised stocks and working nandards with mctbanol as specified in Table 3 and mix wcll. Date of prepamtion: 3M Environmental Laboratory Page 7 of 16 C-7 Page 123 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I IO METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS Version 1.0 Study Na: Annlyst/DaL: Weigh all analytical standardsto at least the nearctt0.01 mg. ** Use volumetric or positive-displaccmartpipct(s). I. Preparation of Calibration Standards and Blanks 1. Liver homogenatc . Rrpsre blank liver homogenate io bulk by weighing appmximately 40 g of blank liver into a 500 mL Nalgenc bottle containing200 mL of MilliQ water. Grind to a homogeneous aspension Aliquot into approx 30 mL portions for frozen (approx -20C) storage. Actual Mass ofliver: Actual volume of water: Date of prep: Study: Determine dcnsity of dbrationlQC matrix: h4IX HOMOGENATE THOROUGHLYand determinethe mass in mUgrams of 10 mplicatewcighings of I mL p01ti0118ofthe THOROUGHLY homogenatt. MIXHOMOGENATEIMMEDIATELY PRIOR TO EACH ALIQUOT REMOVAL. 3M Environmental Laboratory 2. Liver Calibration Staodards Prepare uch Liverd b n t i o n slandard by adding0.45 mL of undilutedliver homogenate (STIR HOMOGENATE WHILE ALIQUOTXNG) into a 1s mL extraction tube and adding50 pL of WS or MeOK Prepare triplicated smndards and 6 blankr. See Table 5 for volumes. The diluted Liver density is assumed IO be approximately 150 mg/mL. Mix well. Page 8 of 16 C-8 Page 124 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX- I 10 METHOD FOR ANALYSIS OF POTASSlUM PERnUOROOCTANESULFONATE (PFOS) IN RAT L M ? R BY LC/MS/MS Version 1.0 Study No.: AnalydDate: Table 5. Calibmtion Standards and Blvlkc Date of preparation of cal stdrlblank: J. Preparation of Quality Control Liver Samples (QCs) I. Quality Conbnl Working Standards Dilute the following sourcevolumes methanol in volumetricflasks and mix well. Prcpam h hwhen used. Actual volumcsarc in parentheses. 2. Prepamtionof Quality ConmlLiver Samples Prepare eachQC in bulk by filling the volumetric flaskapproximatelyhalffull with undiluted liver homogenate(STIR HOMOGENATE WHILE ALIQUOTING). adding the appropriate QC WS, mixing and diluting to volume with undiluted livcr homogenate (STIR HOMOGENATEWHILE ALIQUOTING). MIX THOROUGHLYmddispense 2.5-mL aliqu~tsinto polypropylene tubes and store at approximately-2O'C. Date of QC prep: Shldy: K. Preparation of MS Check Standard for System Suitability Pipet 250 UL of WS 2 at -1O.OOO np/mL and 2.5 mL of WSS PI -1000 ng/mL in rnelhvlol into die sane SO-mL volumetric flask Dilute to volume with MeOH and mix c-9 3M Environmental Laboratory Page 125 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LKN-U2849 Banelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I 10 METHOD FOR ANALYSIS OF POTASSNM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LClhlSIMS Version 1.0 Study Ns: AnalydDate: L. Preparation of homogenizer recovery liver samples To determinethe recovery from the homogenizationprocess. unhomogenized blank liver will be fortified in duplicateat 3 concentrationlevels and homogenized as follows. This nccds to be done every day thathomogenizationof study samplesis performed. 1. Place approximately 0.5 g of unhomogenkd blank Liver into u c h of 6. 15 mL polypropylene centrifuge tubs. R m r d weights of liver. 2. Add 100 pL of WS 1.3. and 4 (one WS per duplicate tubes) to prepare fortifications at approximately 4, 0.8, and 0.4 3. Multiply the mass of liver in g by 2.5 andadd this m y mL of water. 4. Homogenize each liver sample. and rinse homogenizer probe with another volume ofwatcr used in step 3. addingrinsc'to homogenizedsample. 5. Clean homogenizerwithMeOH becwcensamples. 6. Cap and vortex homogenate for use in Uctraction. M. Preparation of Dilution Check Sample 1. Place 2.95 mL of undiluted liver homogenate (SZIRHOMOGENATE WWILE ALIQUOTING) into a IS mL e o n tube andadd 50 pL of Mixed Stock A 2. Dilute 50 pL of step 1 solution (VORTEX SOLUTION WHILE ALIQUOTING)with 0.45 mL of undilutedliver homogenate (STIR HOMOGENATE WHILE ALIQUOTING) in 3, 15 mL exbadion tukr. 3. Tbis sample shouldbe preparedfor d o nonly on days when study simples will bedilutedandextr;lct#t .r N. Homogenization of study samplu 1. Placeapproximately 0.5 g of unhomogcaizedstudy m p l e liver into a 15 mL polypropylene tube. Rccord weights of liver. 2. Multiplythe mass of liver in g by 2.5 and add thismany mL of water. 3. Homogenh each liver sample. andrinsc homogenizapmbc with another volumc o f waterused in stcp 2. addingrinse to homogenized sample 4. Clcao bomogeni2n with MeOH kcween samplu. 5. Cap a d w a x homogenatefor use in uctraction. 0. Analysis Standards, Blanks, QCs, and Samples 1. MIX LIVER HOMOGENA'ES THOROUGHLYBEFORE ALIQUOTNG and pipet 500 pL of each QC (4 replicates per I d ) . andother samples being unra*ed into 1 5 4 polypropylene ucuaction ~ubes.The cal stds and blanks - are already aliquotcd. 2. TOthe Blanks IS (3 reps). add 100 pL of MeOH and vortex. 3. To the Blanks +IS (3 mps) and to the remaining samples. add 100 pL WSS and VONX. 4. Add 0.5 mL of 0.5 M TBA (pH 10) to all Nbcs andvortex briefly. 5. Add I mL of 0.25 M carbonate M e r and vortex briefly. 6. Add 2.5 mL of ethyl accuIc. Place the tubcs sidewayson the orbid shaker at a settingof 300 for -20 minutes. 7. Ccnoi3uge tubes at a setting of 3500 `pm for -20 minutes IOsepam~elayers. 8. T d e r 2 mL of the top orgmic layer IOa clean polypropylene tube. Page IO of 16 3M Environmental Laboratory c-IO Page 126 BACK TO MAIN 3kl Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Banelk Study Number: N003296-F 3M Toxicology Services Protocol Number:FACT-TOX-I IO METHOD FOR ANALYSIS OF POTASSIUM PERFLL`OROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS Version 1.0 Study Na: AadyrtlDote: 9 Evaporate to dryness under nitrogen at a settingof 30'C for -60 minutes. IO. Rc~~nstjtuthee residues in 500 r L of methanal with vortexing. I I Syringe-filterexvdc1s into autosvnplcrvials for analysis. Store vials refrigerated (up to 1 month) if L C / M S M will not be performedthe same day. Since 3day room t e m p e m extiactstability was demonstrated during validationthe extracts of the calstds.blanks andQCs may be reusedfor up to 3 days after their initial prepamtion ifheld at room tempcnnve (recap the vials if reusing). Date of cal n d l b l d extractprep: Date of QC extractpep: WJ!&+? P. LC/NIS/MS Analysis QkW r@ I. use the system conditions spo~ifiedinT ~ MN C ~heconditionswhich m designatedmay be modified by the analyst to produceacceptablepeak shape. 3M Environmental Laboratory c-1 I Page 127 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I IO METHOD FOR ANALYSIS OF POTASSTUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LMCR BY LCfiISfiIS Version 1.0 Study No.: AnalydDatc 427281 MRM nansition for Surrogate Standard 499>99 MRM nansition Cor PFOS --556278MRM ansition forM-556 570> 169 MRM msition for M570 584>169 MRM hansition for PFOSAA 4 9 e 7 8 MRM transition for PFOSA 5262169 MRM transition for PFOSEA *11 minules( min) ss: 4 min L m i n ) PFOS: 4.3 min min) M-SW. 4.5 min L e ) M570:4.6 min min) PFOSAA: 4.7 min min) PFOSA: 5.1 min PFOSEA 5.7min ( min) e) Patameten that may be changed by the analyst. Actual values in ( ). 2. The above conditionsshould be suitable for the MicromassQunttroLC (SM 9053). Modiftcationsmay be ncceSSBly if anotherMicromass Quamn Series spectmmcter is used. Split the flow post-columnvia a Keystone BIO-tee or similar dcvia. 3. Calibrate the mass spearomctcrudnga suitable rcfcm~cceompound, or verify that the calibrationis suitable by visual inrpeCtion(on the tunc page) thata suioblemobile phase ion isstill ocnvarelydetermined.Ruolutionmay ncedto be higher than that used for analyzingsamples. , , 4. To check the proper pcrforina~~ofathe inseUmms inject tbe instrument check standard.The d t s should be comparable to a recent injection if available. 5. Usc an automatedchromatography integration SOAware system to collect the output from the analysis. 6. Loading Order: Sce the loading rcport from the automated duomatognphy integration roftwan system. 7. Make single injections ofeach cal rtandard QC. study sample, orblank Make at least 4 injcctionsof the inmuwnt check rtandnrd 8. Run set sizes should typically no(w r d 80 iqjcctionr due to inmumcnr nspansc roll&f considerations. Longer runs may be performed. but they pose a risk ofyieldingunaueptabk curve rrrultr. Vm. CALCULATIONS 1. Spreadsheet sofhviuc Version 2. MS Analysis Software: Version 3. Calculate the average density of the liver homogenate (10 reps) in mg/mL 4. Using the average density of the homogenate. calculate its liver density (mg of liver pcr mL of diluted homogenate): Undiluted Liver density (mg/mL) * (g of liver x avemge density of homogenate)@ of liver + g of water) where g of liver and g of water arc massc~used to prepare bulk homogenate: density of water is assumed to be I g/mL. Diluted liver density (mg/mL) = Undiluted density * Diln Factor Page I 2 of 16 c-12 3M Environmental Laboratory Page 128 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number:N003296-F 3M Toxicology Services Protocol Number:FACT-TOX- I 10 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE(PFOS) M RAT LIVER BY LC/MS/MS Vcnion 1.0 Study No.: AnalystlDnte: Where diln factor = Z1.8 = 1. I I 1 1 to account for 10% diln of liver homogenate in cal std and QC matrices. 5. Calculate the actual concentration( n g l d ) of PFOS and other fluomhemids in the suspensions of calibration stamhis and QCs by using the m s ofanalytes and dilution factors only (no liver density correction). Ua purity comction for PFOSAA only. 6. Calculatethe actual concentration of PFOS and other fluorochemicalsinliver for the calibrationswdardr and QCs DS follows: Conc(pglg) = C o x (nglmL) Diluted Liver density (mg/mL) x loo0mglg x 10' &ng 7. Assure that Iheintegrationsof the peak areas ofthe tcst d c l e and Jwogatc standard are correct Flag manualintegrationswhen pcrformcdCalculatethe exact concenhation of atch a liver s?andard 8. Calculatethe regression equation relating the peak responseratio (test articlc/SS) of each calibration standard(y-axis)to test article conantrationinliver (x-axis) for PFOS,M-556, h4570. and PFOSM. Calculate the regressionequationrelating the peak area of each caiibmionnandard to tejt aniclc concentrationin liverforPFOSA and PFOSEA. PFOS. M556. h4570.and PFOSAA arc quantitated by usingthe m g a t e standard as an intunal standard;PFOsA and PFOSEA are wwkated with0ut nfcrenceto the s w o r n standwd calibration m e ) .Use a quadratic regersion wagbtcd l/x ,origin ueluded, for 111 malyttl. 9. calculate a determined concentration for each injection of calibrationstandad, QC. and ' sample usingthe regression parameenand the peak response&os or arcas. 10. Calculatethe relative error, average relativeerror, standarddeviation,and relativestandard deviation for all QCs. Calculate the relative error for each injection of calibrationstandard. 11. Calculate the average recovery for the homogenizerrecoyrryfotti5cations. 12. Calculate the relative standaddeviation for the PFOS to SS peak area ratio of the replicate iqjcccions ofIhc c h a k surodard Ix ACCEPTANCECR~TERU~ A. MS Check Standard (System Suitability) At least 3 injectionsofthe MS Check Standiud must provide a %RSD of 10%or less for the PFOS to SS peak area ratio. B. Calibration Standards The p a n t relative C ~ O Kfor the concenation-levelavenges of the calibration amlards should meet the following Limits: 3M EnvironmentalLaboratory Page 13 of 16 c-13 Page 129 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analvtical ReDort: FACT TOX-110 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number:FACT-TOX- I IO METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE(PFOS) IN RAT LIVER BY LC/MS/klS Vcnion 1.0 Study No.: AnalysUDate: Up to 5 calibtatioastandard injections may be excludedfmm the cwc.providedthat one injection nmninsper Iml. Removal of an enlire Ievel may be done if approval is obtained. If an entire lcvcl is ranwedthe samplesb m k d by the remainingcalibration range *I1 be consideredacaptable. The calibrationcweshould have a mfficientof determinationof 0.91 or better. C. QCs The conanbation-levelamage pacmtrelative morsand perant relative standard deviations of the QCs should meet the following Limits: Table 10. QC Acceptance limits PFtX I 2n - I M-556 I 20 ic .M_'i7-1.) 2-1-) PFOSAA 20 PFOSA 20 PFOSEA 25 Removal of individual values fmm the QC calculatiolu may be done if pccompanied by a reasonableexplanation(e.&. iastmmentmalfunctionor Dixon's Q test d U ) . Ifthe w ~ g dceterminedconcentrationfor any QC lave1 excadr Iheacc+plsncslimit, &e task leadaorstudydimor should be notified.The NO maybe repeatedor aponim ofthe runmay be sousideredzsujmblc. For example, ifthc low QC faiIr the sated requircmcnu. samples maybe accepted thathave conceabatim bmckted by the highatcalibration nandvdand a mid-lcvel QC concenmtion D. Homogenizer Recovery and Dilutian Check Samples The average recovery across the 3 lmls of homogenizerrecovery samplesas well as that of the dilution chedc samplesshould fall within the rangeof 70-130% inclusive. Removal of individual outliers from ?heulcuhtions m a y be done ifaccompaniedby a reasonable ucplanaion. E. Sensitivity (LOQs) The validated limits of quantitationm noininally 0.13 pdg each for PFOS. M-556. M570. and PFOSAA. Page 14 of 16 3M Environmental Laboratory c-14 Page 130 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 Battellc Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I 10 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LrVER BY LC/MS/MS Venion 1.0 Study No.: AndydDate: For PFOSA and PFOSEA the validated LOQs arc nominally0.33 pug each. Due to the nature of the prepamtion of the calibration standards, lower concentrations of PFOSA and PFOSEA will be carried through the extraction. These lower concentdon values will be evaluated with each run set. and may be included in the regressions if they mect acceptance criteria Ifthey am included study samples which an quantitated to have concentrations below the validated level (nominally0.33 pug) will be appropriately flagged. F. Specificity 'Ibe merhod Men from endogcneous matrix interferences at levels sometimes exceeding 20% of LOQ. 'llu intuccpt of the c a l i o n c ~ w aeppears tooffer some wmftionfor any effect on quantitatiocu Acaptak purOrmana(error) ofthe lowest wd sadad, thacfore. will be considend sufficient evidencethatbradtetcdstudy samples arc quant5ed properly. G. General The above acceptancx criteriaindicate that thismethod is capableof producingaccasiod errors outside the normal acceptance Criteria of a validated method (15% normally). Where indicated,replicate arnlyscslessen the impactof these occasionaloutliers. See attachedhard copy of spreadsheet or seefile on network drive. 3M Environmental Laboratory Page 15 of 16 c-13 Page 131 BACK TO MAIN -3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Banelle Study Number:N003296-F 3M Toxicology Services Protocol Number:FACT-TOX-1 IO METEfOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS Version 1.0 Study No.: AnnlystlD;lte: XII. CONCLUSIONS Xm. SIGNATURES Analysts Technical Review QC Review Date: Date: Date: a ? DatC: Date: Date: Due: Datc: 3M Environmental Laboratory Page 16 of 16 C-16 Page 132 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Eattelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I 10 APPENDIX D-REPRESENTATIVECHROMATOGRAMS 3M Environmental Laboratory Page 133 3M Medical Department Study: T-6295.12 3attelle 2LC 9053 3- !8Aug06 Srn (Mn, 1x2) 1O( BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Battclle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-1 I O Calibration Blank 10 uL; N003296-F 35 20-~ug-i99! 13:33:0: 1.57e: PFOS Channel % 5.36 J fi . - + . 2: hRM of 2 ChanneldES 427.00 > 81.O 18 I' SurrogateChannel %- 4.46 0 1 . .. . . Time 3.50 3.f5 4 . b 4.15 4.50 4.95 5.b0 5.25 5.50 5.95 D-1 3M Environmental Laboratory Page 134 3M Medical Department Study: T-6295.12 lattelle 1LC 9053 :SI8Aug08 Sm (Mn, 1x2) BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Banelle Study Number: N003296-F 3M Toxicology Services ProtocolNumber: FACT-TOX-I IO 28-Au~-19s 1-3-:-5-5-:E_ - Operator: PL 2: MRM of 2 Channels E: 4.35 499.00 > 99.( 1.96~ PFOS Channel 00 0% Surrogate Channel 0- 3.bO 3 . b 4. 1 1.25 4.50 4.95 5.b0 5.55 5.80 5'15 . . Time D-2 3M Environmental Laboratory Page 135 3M Medical Department Study: T-6295.12 32LaCtte9ll0e53 ESIIBAug24 Sm (Mn, 1x2) 100 %. BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology ServicesProtocolNumber: FACT-TOX-I I O QC 4 10 UL; N003296-F 28-A~g-l9! 16~38~ :: ~ . . - Operator: PL 2: MRM of 2 Channels EZ 499.00 > 991 1.934 PFOS Channel I--, 1) 0- . . .-, . . BAul I'.","~ 2: LkM of 1 Channel& ES IOC 427.00 > a i.Q 1.26e Surrogate Channel ox I 1 . .,L. . . . 0 , ,, , , , , ,, , , , , , ., . . Time 3.bQ 3.f5 4.b0 4.15 4.50 4.f5 5.b0 5.55 5.bQ 5.f5 D-3 3M Environmental Laboratory Page 136 3M Medical Department Study: T-6295.12 3attelle 3LC 9053 ESI- !5Aug45 Sm (Mn, 1x2) 1O( BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I 10 Group 1 (Vehicle) DAM Li'ver 10 uL: N003296-F; 0 mgkglday 26-Aug-199 Operator: PLsl 2: MRM of 2 Channels ES-l % PFOS Channel . Surrogate Channel D-4 3M Environmental Laboratory Page 137 3M Medical Department Study: T-6295.12 3attelle L c 9053 31- BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number:N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I 10 Group II DAM Liver I O uL; N003296-F; 0.1 mgkglday 28Aug-I9! 16:12:1 0 erator: PL 2: MRM of !Channels E: 499.00 > 99.1 2.21a PFOS Channel 0 '. . . . . . . . . . . . , . . . !, ....:. 3Aug20 Sm (Mn. l x 2 j ".`""`.."`"`2~ Mor 427.00 > a i.o 7.lle Surrogate Channel 3.bO 3.75 41 L . . Time 2 5 4.bO 4.75 5.b0 5.55 5.b0 5.75 D-5 3M Environmental Laboratory Page 138 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Battellc Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I IO 1007 O&/ 4.2n 2 PFOS Channel 499.00 > 99.1 2.391 1Aug52Sm (Mn, lx2f 3 . h 3.f5 4.bO 4.25 4.b0 - 4 5 . 5 . b O 5.25 5.50 5.f5 . . Time D-6 3M Environmental Laboratory Page 139 3M Medical Department Study: T-6295.12 3attelle ISI- )lSep20 Srn (Mn, 1x2) ~~~ BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 BatteIlc Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I 10 Group IV DAM Liver I O uL; N003296-F;1.6 mglkglday 4.21 01-Sep-I 99 16:47:1$1 0 erator: PLS 2: MRMoffchannels Es-i 499.00 99.0 1.46e 100- 3.95 4 .A, 427.00> 81 4.04 i I %- Surrogate Channel 0 ........................................... 3.50 3.f5 4.bO 4.h5 4.bO 4.95 5.b0 5.h5 5.bO S.95 I I i Time 3M Environmental Laboratory Page 140 BACK TO MAIN 3M Medical Department Study: T-6295.12 3attelle JLC 9053 ISI- llSep63 Srn (Mn. 1x2) 1 oc Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX- I 10 Group V DAM Liver 10 uL; N003296-F; 3.2 mglkglday 4.20 02Sep-199 00:57:6 0 erator: PL 2: M R M of fChannels ES 499.00 > 99.C 1.85e % PFOS Channel 0 ... .... . . . . I . . . . ( lSep63 Sm (Mn. I x 2 i 100 . . . , . . . , I . .2: M Of channels' ES 3.84.3 Surrogate Channel % ) 0 , , . . , , , , , , ,. , ,- , , ,, ,, ,, ., , . Time s.bo 3.50 3.75 4 . b 4.ts 4.ho 4.f5 5.15 5.50 5.75 D-8 3M Environmental Laboratory Page 141 3M Medical Department Study: T-6295.12 ~~ BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Banelle Study Number: N003296-F 3M Toxicology ServicesProtocol Number: FACT-TOX-I IO 100, 4.25 499.00 > 99. 3.32 .I PFOS Channel 427.00 > 81.0 3.77s Surrogate Channel l o 1 ' ' 3.50 ' ' 3.f5 4,bo 4.15 4.50 4.75 s.bo 5.15 ' ' 5.50 ' ' 5.75 . ' ' Time D-9 3M Environmental Laboratory Page 142 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-UZ84Y Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I10 9 - atte e 2LC 9053 !SI18Aug50 Sm (Mn. 1x2) 1 oc 10 uL;N003296-F; 0.1 mglkg-lday. ug- 9 21:55:1: Operator:. PLi 2: MRM of 2 ChannelsE! 499.00> 9.9...l l.i8( PFOS Channel % a I 00, k,M. 0~ channels'ES 427.00 > 81.01 1.08e Surrogate Channel YO- 3M Environmental Laboratory D-IO Page 143 BACK TO MAIN 3M Medical DeDartment Studv: T-6295.12 Analvtical ReDort: FACT TOX-110 Jattelle x c 9053 31- IOAug20 Sm (Mn, 1x2) 1O( % Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number:FACT-TOX-I IO Group 111 Pooled ketal Liver 10 uL; N003296-F; 0.4 mglkglday 4. I 30-Aug-19 16:41:, Operator: PI 2: MRM of 2 Channels E: 499.00> 99. 1.11, PFOS Channel I I oc 97 Surrogate Channel % 0 3M Environmental Laboratory I D-1 I Page 144 BACK TO MAIN 3M Medical Department Study: T-6295.12 3attelle JLC 9053 31- IOAug26 Srn (Mn, 1x2) 1O[ Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number:FACT-TOX-I I O Group IV Pooled Fetal Liver I O uL; N003296-F; 1.6 mglkglday 4. 30Aug-199 17:49:3 0 erator: PL 2: MRM of !?4C9h9a.n0n0e>ls-gES.c 8.756 PFOS Channel % I a ... .... 3A~,,6 Sm (Mn, lx2$ I oc 3.96 427.00 > 81.o( 5.766 % Surrogate Channel 0 .' . . L. . 4.25 4.b0 4 . h , . 5..bO. . . 5.b5. . . 5, .50, , . 5..95. , . Time 3.50 3.f5 4 . h 3M Environmental Laboratory D-12 Page 145 3M Medical DepartmentStudy: T-6295.12 Sattelle 2LC 9053 SSIiOAug42 Sm (Mn, 1x2) BACK TO MAIN Analvti-ACT TOX- t l O LRN-U2849 Banelle Study Number: N003296-F 3M Toxicology Services ProtocolNumber: FACT-TOX-I10 Group V Pooled Fetal Liver 10 uL; N003296-F; 3.2 mglkglday 4.; 30-Aug-199 20:52:1 0 erator: PL! 2: MRM of !Channels ES 499.00 > 99.0 1.89e PFOS Channel 0 ... .... .".'""".'.""''".'".'''''2~ .Of Channell ES. 6.00a Surrogate Channel i 3M Environmental Laboratory D-13 lime Page 146 3M Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number: N003296-F 3M Toxjcology Services Protocol Number: FACT-TOX- I IO APPENDIX E-PROTOCOL, AMENDMENTS, A N D DEVIATION If 3M Environmental Laboratory Page 147 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LHN-U2849 AUG. 13. 1999 9:ZLAH c JM Ea+ammtal mdsunca Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-1 IO ENVIPONMENtkL LAB 2 3E 09 NO. 1106 P. 22 Tcckao*q PO Box 33331 S6I1. 1pnluil.6W4SuJ133-3331 P ~ O C O#FIACT-TOX-710 Study Title Oral (Gavage) Phrmacokinetic Study of PFOS hRQS PROTOCOL Author LisaClmen I' htc: ... . . - June 8,1999 .. Labomtory prolcd Idenlificrlion F A f f-'TQX-I10 u2849 3M Environmental Laboratory PqpldlO 651 778 6176 PFlGE.022 E-1 Page 148 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 AUC. 13. 1999 9:24AM Banelle Study Number: N003296-F 3M Toxicology Services ProtocolNumber: FACT-TOX-I 10 ENVIPONKENTAL LAB 2 3E 09 NO. 1406 P. 22 Study ldentificatlon Om1 (Gavage) Pharmrcoklnotic Study of PFOS In Rats Test Materiel Pduorooctaue sulfonic acid potassium salt (T-6295) Sponsor Sponsor Repmsentative Study Director .. . - .. Study Locrtfon(s) In vivo Testhg Fadity AnaWcal Testingbborafory Mpvin T.(he, D.V.M., P h 9 . 3 MToxicologySaviceS Telephone: 651-733-5180 F ~ c651:-733-1773 IbistenJ. Hanscn,Ph9. 3M EnvironmeatnlTdmology rlnrisafetysavices Building 2-33-09 651-7766018 ArguaResearchLabolaurrirs,Inc. 905 SheehyDxive, Building A HorshazaPA 19044 3MEn-d- B m 2-33-09 935 Bush Avenue SLPurl,MN 55106 3M Environmental Laboratory Page 2 of 10 ET1 7 7 R C17E DPGC a79 E-2 Page 149 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 AUC. 13. 1999 9:211iyI Banelte Study Number: N003296-F 3M Toxicology Services ProtocolNumber: FACT-TOX-I 10 ENVIRONMENTAL LAB 2 3E 09 NO. 1406 P. 24 pldoCd#FACT-tdx-7fO Sub-Contacl Labomtory Proposed Study Timetable study initiation Date Study Completior,Date Advmccd B i o d * c d S d c a , Inc. 15 CatherwoodRoad Ithaca,NY 14850 Battelle Mmonrl Institute 505 King Avmw C01Umbu0, Ohio 43201-2693 June 8,1999 August 8,2000 1. STUDY Oral (gavage)phanmcokjneticrtuayof potassium perfluomoctane sulfonic acid(PFOS) mnts. 3M Environmental Laboratory 651 778 6176 PAGE.024 E-3 Page 150 BACK TO MAIN -3bl Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 AUC. 13. 1999 9 : W Banelle Study Number: N003296-F 3M Toxicology Services ProtocolNumber: FACT-TOX- I 10 ENViRONMENTIL LAB 2 31 09 NO. 1106 P. 25 5. TESTMATERU 5.1 Refa to Argru R u w h Labontoyprotocol for study 11418-013. 6. C ~ N n z o r ~ T R l C E S 6.1 /dentitication Ra! liver and serum and/orrabbit livmmdsuum, traceabilitynumbm willbe recorded m the mu dataand includedinthe find rcport 6.2 SOUEOArgur Research rndlorS i p chemiul 6.3 Physkrl Description Rat livaand raumand/orrabbitliver and serum I 6.4 PufIW and Stabi/ity Not applicable 6.5 Storage Condlffons p 1 0 m a! -20 O C f 10'C or-SO 'c f 10 O C b l Resetve M a w A portion of the control mrtriX will be rarinSd inthe 3M rrdriver for u-1 m the quality of the prrpMtioa afkds evaluation, but not kmga thaa ten yepn followingthe effective date ofthe i i d testd e (ifrpplieable). 6.7 Disposltlon Ma!xiw will be retained a!the 3M EnvirOnmenbl IAxaOry per GLP =-.er mntriou (fees, Urine, md blood) may be dirpooed aft= QAU wifiwtion , ' 8.8 SafefyPmcautions R ~ f c r t a M s D S t o t ~ u a aWdaarpwroprirte lrbosstDIyattire,and foollow dequrttprctrutionsforhndling biologiaca.lnutarirlsand p~siInplcsi3rd* 7. RU?UIENCPMATERIAL 7.1 ldentttlcrtion P o ~ u m p ~ ~ t a n ~ l f o(PnFOaSt),elot #a 171,215, or217 (equivrlcnt lots) 7.2 SOUEO 3M SpecialtyChemic& 7.3 Physical DescripffonWhite pow& 7.4 PUrltyand StrbirHyPurity ofPFOS is 99% or gmbr. Stabilityh not been dctcrmincd 7.5 SZorap Condithnr Room tcmpnobrrr 7.6 R e 8 W M I k t f I / A nrnve sample from eachbatch of PFOS usedmthir study will be retained u long Y the quality of the preparationaffords evaluarion. but not longer thrpten yern followingthe effective date of the f i ~taI trule (ifapplicable). 7.7 DispodUon Unusednfinnrzm y d willbe ntaioedfor we by the 3M Envkomentrl Laboratory and will be discrrdcd whcn the q d t y ofprepadon no loonga afforb evllaatioa. 3M Environmental Laboratory Pa~o4of70 651 778 6176 PQGE.025 E-4 Page 151 BACK TO MAIN -3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 AUG. 13. 1999 9:251\y Banelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I10 ENVIBOWENTAL LAB 2 3E 09 NO. 1106 P. 26 P d o c d #FACT-TOX-110 7.8 Safety Precautions Refer toMSDSfor chemic& used Wear appropriate laboratorymire, md follow adequate pmlutionsforhandlingbiological materills and prepaIingumpl*lfordYsh. Body ttmdfldd Saum-Dan 8ndFetw mimrlr collected . Dam-Prrdoy Days 7,15, d 2 l Expseted # of rpedmem 320 D m and [Amniotic Fluid- Dun &ah I I I Day 15 and Day 21 3M EnvbonmcntY LIboratory RUG 13 ' 9 9 1 1 : 1 8 E-5 31bl Environmental Laboratory I II BO ' I1 PaQe5oIlO 6 5 1 7 7 8 6176 PAGE.026 Page 152 BACK TO MAIN -3M Medical Department Study: T-6295.12 Analvtical Reoort: FACT TOX-110 LRN-U2849 AUG. 13. 1999 9:25AM Banelle Study Number: N003296-F 3M Toxicology Services Prormol Number: FACT-TOX-IIO ENVIRONMENTAL LAB 2 3E 09 NO. 1406 P. 21 Table 2 Coat Specimen Infornution b Body tirsndflnid Collected - Embryo's With Plr~~nraCD a rnimalS -- CVCW Fthu pnimrl 'P1caent.e Fetw animal - - 'Livedlung FCW prirml Day 15 nnd Day 21 I I~t the tnminationof the study IDny 15 and Day 21 IAt the tamirUrtionofthe study i Expected # of spccimem 80 160 80 I 80 Total number of t#t mimrls. 64 ,Total numba of control anbalx 16 Spaimem rcmt to 3M Ewironmenht L*borrtolicswillbe reetivedand tracked to applicable standardopnatingw. lo,PREPAJUTORMYm o m 10,l FACT-M-1.1, ExtractionofPotouim P a f l u o w & or otherfionk FluomchemicalSurfactnnthon r Liver fbrAnalysisUsing H P L C - E I ~ Q ~ ~ ~ - SPectrOmetry 10.2 ETS-84.1, Exp;lction of Potassium Pdno-nate or Other .@.. o m w e a l compoandrfrom Serum or OtherFluid for Ad* UshgHPLC- I r Elatmpnym'sp~ 10.3 Ifprepantorymethods otherthan thoseliatd above ut used, mamendmentto &is protocol willbe writla Any deviations&om these methods willbe documcatcd and includedwiththestudydru 10.4 Ifaulysa urlub-contnctcdta other labontoria, aa vnmmnmtwillbe written to include thdrmethodc andcopicr of uch method will be rttrchedto thispmtDcoL 1f.f e FACT-M-2.1, ofPhrnrochcrnicritin L~VEWxtncb U% HPLC- E l t c t r o s p n y h SpeEtrOmetry 11.2 ETS-8-5,1,Anal* OfPoturimnPcrfluorooctaacsnlf~tocr OtherFluorochemicalr in Serum or OthetFluid ExmctaUsingHPLC-Ekctrospny/Maar Spectrometry 11.3 Ifanalytical methods omCr than thore listed above arc used, mamendment to this protocol will be written. Any deviations&om these methob will be documentedand included with the study dah 11.4 Ifanalyses arc sub-contnctedto other llbontaricqUI PnmQICnt will be writtento include their methods a d copiesofeachmcthodw i l l be rttrhedto thispxutocoL 3hA Environmental Laboratory Page6dlO 651 778 61?6 PACE.027 E-6 Page 153 BACK TO MAIN -3tvl Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 AUC. 13. 1999 9:25AM Banelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I IO EHVIRONHENTAL LIB 2 3E 09 NO. 1406 P. 28 Protocor #FACT-TOX-f f Q 12.DATA Q U A W OSJECnMS The number of spilcclldupllurct,use of s m g a t y and i n t o d o n on other data qllllity idcaton arc included inthe analyticalmethods. In addition,the folio* c r i t hwillbe met: 121 Linearity ? 20.98 * 12.2 Urnits of defocilon/ qurntitacion 12.2.1 Method DetectionLimi! (MDL)forPFOS a) Sarrm: 1.75ppb b) Liver. 1Sppb 1222 Limit of Qu8ntiratiaa~oQ)-Equrltothelawcst.ceeptablcstansMin the caliibrption m e 123 Dupllcrtoacceptoblcp h i o n <30% forthc method 124 Spike rcceprablemcoverler 7Ph - 13P? 125 Use of confirmatorymethods Indctmm* ate samples dlbe reanalyzedusing a mfimatorymethod Ifrconfirmntorymcthodisured,aumcndmeuttothis pmtocolwillbcwrittm. 126 bemonsmffonof SpeCMclty c h r o m r t o ~ h ircctcntiosltime,mus S p d d..au.ghter-ioncharo.tai.ption *I 14.S s A l l S n U r Averages md standard deviationrwillbe cplcut?tcd Ths Ptltioticilmethoda thatwill b t w d arc described below: 14.1 Data Innsfomations and rnalysh Data willbe reportedas tbc eoncentration (wei-aght or weighthol) of PFOS or metabolite pa tissue or fluid. 3Nl Environmental Laboratory Pwc 7ot 10 E-7 R S l 7 7 R R 1 7 R DEICE R?R Page 154 BACK TO MAIN -3Nl Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 AUC. 13. 1999 9:26AM Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number:FACT-TOX-IIO ENVlBONMENtlZl LAB 2 3E 09 NO. 1106 P. 29 P m C d #FACT-TOX-110 14.2 Statistical analysk Statistics used may includengrrzsioaanalysisof concentratioru o w time, and standnrd deviationscalculatedfor the concentrations within eachdose gruup. lfnecesruy, simple stltimcd tests, such uStudent's t ~ t , may be appliedto evrlpateStatistiddiffirsnce. 15.REPORT A mort eontnining dl the nsults of the study will bc p n p d by the 3M E!II*~ Laboratory. I f d y s c s are sub-cantnctd to otha labmtoriq erch lrboratorywinpnpsn a Rportmd submitit to the 3M psvironmerrtrlLabondoxy for inclusioninthe 3M Envkorunmtal Labonhy-oh EA& report willinclude,but not be limitedto, the followhg,when spplicable: 15.1 Nunc rad ddnu of the frcilitypcrformine the rtudy 15.2 Data upon which the study waa initiated md camplaed 15.3 A statanen! of complirnceby the Stpdy Dinctor dQeJsingmy a t c ~ t i o ntro Good Labontory Practice Senndydr 15.4 obj~ti~mdprocedurtJar~edintheapprovedprotowl,inchrdingpnyc~ intheori~protocol 155 - T h e test substanceidentificationby me,chemicalabstracts number or codenumber, strength purity, and compositionor otherappropxhkchawku&'cr,ifproYidedby e3ponror I 15.6 Stab.ili.tyiurdthe solubilityof thetsst substancesundmthe conditio& of rdmtntghrtioisifprovided by the Sponsor 15.7 A description of the metho& used to condud thetest(s) 15.8 Adeserigtionofthctestsy3tem 15.B A descriptionof my Eircrwrtpnctr that may haveaffected the quality or the htegdy ofttscdrt. 15.10 Thename of the StudyDinetor md the namuof other ccidsts, mfesdcm4md ~ p c n o n n c~ Il v c d i n T h Clmdy f5.11 A descriptionofthe transfonnationr.crrleutuiopr, 01 Opatiomip e h m e d on the data, 8sUmmIy md analysbof the d y t i w l c h & s k y d &m, d8strtancmt of the ~~mdram~thcdyaca 15.12 Statisrid methodswed to m h w the data, ifapplicable IS.13 Thcsigned mddated mpoits of eachof theindividd Midstsor otha profcsdonrk involved in the study,if applicable 15.14 The locationwhmiaw & t a d the 60111report uc to be rtored 3M EMmnmentd bboraloty AUG 13 ' 9 9 11:19 E-8 3M EnvironmentalLaboratory Page80flO 651 7 7 A K17K PClGF.R?q Page 155 BACK TO MAIN -3kl Medical DepartmentStudy: T-6295.12 Analvtical I?@@: FACT TOX-1 10 LRN-U2849 AUC. 13. 1999 9:26AM Battelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I I O iNVIRONMENTAL LIB 2 3E 09 NO. 1106 P. 30 R&W #FACT-7DX-110 15.15 A Ntanent prepared by the Quality Assurance Unit h h g thr dates thpt snrdy @#tiont md audits w a e made, and the dates of any reported to the Study D k t o r andMmrgQnent Ifit n e c to ~makc cotxection8or addition8b8 nport it ha6 been rcccpted, the changes will be madein the form of anammdmmtia8ued by the StudyDirector. Thc amendmentwill clearly identify theput of rhc report that is being mended,the rcwolufor the amendment, md will be signed by thc Study Dircctor. 16.1.1 Approved prototo1 md unandmalb 16.1.2 StudyCoPespoodmcc l e 1 3 Sliippingntordr ' I . .? 16.1.4 Rawdata 16.f.5 A p v e d finalrcport(original signed q y ) 18.1.6 Elactaunic copies of data f6.2 The fbbwing rupportinerocorbwillbe retainedseparately from.thertudy folder in the &VM accodagto 3M l h v k m a d Labors- S-dd Procodurrr: 16.21 Tzainingmonh 76.22 Cdi3raticmmrd~ $8.2.3 InrkumentmrHtmrncclogs 16.24 StmdprdOpcntinoProcedma,Equipment Pmcedum, and Mahods R U G 13 ' 9 9 1I:tB 3M Environmental Laboratory Page9dlO E-9 E51 7 7 R 6 1 7 G PCIGF.R.1R Page 156 BACK TO MAIN -3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LKN-W!Z~T .AUC. 13. 1999 9:26AM Banclle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-I IO ENVIPOMENTAL LAB 2 3E 09 NO. 1106 P. 31 P r o t o ~ a#l FACT-TOX-110 17. sPEunrEN&7ENnON Speciwu will bc Imbiucd inthe 3~ ~nviroDmmaLlab0ratrn-yspecimen archives for a period of time as apecifitd by regulationor IS long u the quality of thepreparation afords nrrhutiori,but not langerthan tenycan following the effective date of the h ltcst rule (if applicable), md as u t a b W by 3M Environmental Labontq StandaxdOpcrdng Procedurw. ~~.A~ACHMENTS f9.f AttachmentA Prrparato~a~nd analyticalmethods 3M Environmental Laboratory E-IO Page 157 BACK TO MAIN -3Ml Medical Department Study: T-6295.12 - Analytical Report: FACT TOX-110 LRN-U2849 SEP. 28. I999 ll:O!A1( Banclle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-1IO .. .. ENVIBONHENTAL LAB 2 3E 09 NO. 1936 P. 2 Study Title onl (Gavage)PhannacokindcStudy ofPFOS in Rab 1 PROTOCOL AMENDMENT NO. I If Amendment Date; Auguat 12,1999 PeHonning Laboratory 3M EnvironmmdTechnologyQ Safety Servicec 3M Environmental Laboratory 935 Burh Avenue SLPaul,MN 55106 f abontotyProlecfIdenthYcadl'on ETBSS FACT-TOXllO URN U2849 E-I I 3M Environmental Laboratory Page 158 BACK TO MAIN -3M Medical DepartmentStudy: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 SEP. 28. 1999 ll:O9AM Battclle Study Number: N003296-F 3~ Toxicology Sewices Protocol Number: FACT-fOX-I10 . .. ENVIRONMENTAL LAB 2 3E 09 NO. 1936 P. 3 mtocol FACT-TOX1 10 Amendment No. 1 This amendment modifirsthe followlng portfon(s)of the protocol: 1. PROTOCOL READS: Section 2.0 stater this study is designed to dctcnnine potassium perfluorooctanc sulfonic acid (PFOS)in specimensof liver #andsenm of rats. AMEND TO MAD: This study is designed to detenninc PFOS in,specimensof rat liver, smun, and urine. All Day -1 and GestationDay 21 rat urine apecimenil Will be extnctcd and analyzed by,the 3M Environmental Laboratory. REASONT: he urine attraction md anolyticd methodswere nolt vaIidatedand approvedprior to protocol approval. 2. PROTOCOL READS: Section 6.0 listsrat or rabbit liver andirerum. AMEND toREAD; Rat or rabbit urine from 3M ToxicologyWitla a physical description of rat or nbbit urine. RG~SONT;he rat urinemlrlrix w&) added after the protocolWII appmved 3. pRO7oc4LRULDS: Section 10.0 and 11.0 list the following:methoda to use for exfraction : # and analysis: FACT-M-1.1" E x u o n of PotassiumP s r f l w ~ ~ e s u l f o MOtreOtherAnionic Fhtorochemical Surfkctat hLiver forhalysisUsing HPLC-ElcctmsprayM~~ Specwm~~" FACT-M-2.1 "Analysis ofFluorochemicalsinLiverExtracbL r h HPLC4!lecmspray/Mbw Spemmctry" AMEND 70 READ: The cXtra&on mdd y t i c d methods to follow at tho 3M Envkonmmtal hbontory M: ETS-8-6.0 `'Exhdi~nof Pot+tSiUmP ~ U O K T J O C ~ C S U K ~ XOWtheCr Fl~~rochcmiCll Compounds b m Liver fir AnalysisUsing HPLC-Electrosp~aSyp~ectrornew ETS-8-96.0 "Extraction ofPotassium P ~ U O ~ O G U W S U E OorMO~thCer Flmchemical Compounds fromUrine for Anrlysis Using HPLC-ElcctroxprayMassSpech.ommy/Mpu SpscmmeW ETS-8-7.0 "halysir of PotassiumPerfluoroocturesulEouateor OthcrFluomchemical Compounds in Liver Extracts Using IIPLC-Elecuospny/Mus Spectrometry" ETS-8-97.0 "Analysir of PotassiumP a i l u o ~ o o c t a n e ~oor~Oether Fluorochcmicd Compoundr in Urine Extracts Using HPLC-ElcctmsprayMass!JpectrometryMass Spectromary" 3M EnvimnnmntalL a b w r t o ~ E-I2 3M Environmental Laboratory Page 159 3Wl Medical Department Study: T-6295.12 I - BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 SEP. 28. 1999 II:O9AM Battrlle Study Number: N003296-F 3M ToxicologyServices Protocol Number: FACT'-T'OX-I10 , .. ENVIRONMENTAL LAB 2 3E 09 ' NO. 1936 P. 1 P f d d FACT-TUXffO AmsndmmtNo. 1 RASON: The exaction and analyticalmethodsFACT-MA1 and FACT-M-2.1rc,spectjve]y, w e n updated on 07/22/99 to ETS-8-6.0 and En-8-7.0. Methods ETS-8.96.0 and ETS-8-97.0were not validrted and approveduntil rAa the protocol was approved. 4. PRorOCOr READS: Section10.4 and 11.4 statethat if the analysesarc rub-contnctedto o l h a labontoriaan amendment willbe writtento includethese methods. AMND IV RCID: The Cxttoctionand snalyticrl methods to follow11MvmcedBiornrlyticrl Savicer willbe attachedto the pmtocol. The d o n and analytical method to follow at Battclle M m d Institute is: v ~ 0 * 8 ' * ' ~ 0 "McthodforAnalysis ofPafluomoctane Sulfonate (PFOS) inElat Samby LC/MS/MS, 0 Liw REASONT: heanalytical methodsat the sub-contractlaboratorieswere not included in the original protocol. 2 JI 5. PROTCCOL RUDS: Seetion 12.2.1 b) Livamethod daectitm limit k 15 ppb. AMEND TOREAD: 122.1b) L i v amethod detectionlimit is 8.510 ppb (ng/g). 12.2.1 c) U M emethod detectionlimit is 1.5 ppb (nglg). R~SSONT:he validation supportingmethods BTS-8=6.0and ETS-8-7.0includeda lower method &taction for PFOS in liver. A validationin urine was performed. &uprotocol approval. to suppan rhir mahod detection limit 3M Environmental Laboratory E-13 Page 160 -3M Medical DepartmentStudy: T-6295.12 BACK TO MAIN - Analytical Report: FACT TOX-110 LKN-U~B~ Battclle Study Number: N003296-F - 3M Toxicology Services Protocol Number: FACT-TOX-I IO .- - . .- - - _. .. . NO. 1936 P. 5 FACT-TOX7 70 Amendment No. 1 Amendment Approval1 3M Environmental Laboratory E-14 Page 161 -3M Medical Department Study: T-6295.12 - BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Study Title Oral (Gavage) Pharmacokinetic Study of PFOS in Rats PROTOCOLAMENDMENT NO.2 Amendment Date: September 28,1999 Performing Laboratairy 3M EnvironmentalTechnology & Safcty Services 3M EnvironmentalLaboratory 935 -BushAvenue St. Paul, MN 55106 Laboratory Project Identii'ication ET&SS FACT-TOXl 10 LIRN U2849 3M Envlmnmental Laboratory 3Wl Environmental Laboratory E-15 Page 162 BACK TO MAIN -3M Medical Department Study: T-6295.12 - Analytical Report: FACT TOX-110 LRN-U2849 P ~ t o c oFlACT-TOX110 Amendment No. 2 4. PROTOCOL READS: Section 16 states that the original data, or copies thereof; will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance of the final report. When the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, electronic copies of data, training records, calibration records, instrument maintenance logs and standard operating procedures, equipment procedures, and methods will be retained in the archives of the 3M Environmental Laboratory. AMENDTO READ: Section 16 states that the originaldata, or copies thereof; will be available at the 3M EnvironmentalLaboratoryto facilitate audits of the study during its progress and before acceptanceof the final report.When the final report is completed, all originalpaper data, . including: @proved protocol and amendments, study correspondence, shipping records, raw data, approved finalreport, and electroniccopies of data will be retained in the archives of the 3M Environmental Laboratory. All correspondingt t d g records, calibration records, instrument maintenance logs,standard operating procediues, equipmentprocedures, and . methods will be retained in the archives of the facility ptxforming each analysis. REASON: Clarification of the dispositionof archived records if mllyses are performed at a sub-contract . laboratory. I 5. PROTOCOL READS: Section 17 states that specimens will be maintained in the 3M Environmental Laboratory specimen archives. AMENDTO READ: Specimens will be maintained in the 3M Envkonmental Laboratory specimen archives. All . specimenssent to sub-contract laboratorieswill be retunled to the 3M Environmental Laboratory upon completion of analysis and submission of the sub-contract laboratory(s) final report. The specimens will be retumed with the following documentation: the signed original chain of custody and records of storage conditions while at the sub-contract facility. REASON: Clarification of the disposition of documentation when shipping specimens for analyses performed by a sub-contract laboratory. 3M Environmental Laboratory 3hA Environmental Laboratory E-17 Page 163 3hl Medical Department Study: T-6295.12 I Amendment Approval BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 P ~ O ~ OFCACOT-~TOX7 70 Amendment No. 2 Marvin Case, D.V.M+Ph.D., SponsorRepresentative WLCf /P?U Datd I Kriiten J. Hamen, Ph.D., Study Director 5#rk /qw Date 3M Environmental Laboratory 3kl Environmental Laboratory E-18 Page 164 -3kl Medical Department Study: T-6295.12 - BACK TO MAIN Analytical ReDort: FACT TOX-110 LRN-U2849 Study Title Analytical Laboratory Report on the Determination of Perfluorooctanesulfonate(PFOS) Presence and Concentration in Serum, Liver, and Urine ikom the Gavage Study of T-6295.12 PROTOCOL AMENDMENT NO. 3 Amendment Date: 20 January 2000 Urine Analyst 3M Environmental Technologyand Safety Services Fluorine Analytical Chemistry Team Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Performing Laboratories Liver Analyses Battclle Memorial Institute 505 King Avenue Columbus, OH 43201-2693 Serum, ralyses Advanced Bioanalytical Services, Inc. I5 Catherwood Road Ithaca,NY 14850 Laboratory Project Identification ET&SS LRN-U2849 FACT TOX-1 10 Argus Study: 418-013 3M Medical Department Study: T-6295.12 3M Environmental Laboratory 3M Environmental Laboratory E-19 Page 165 BACK TO MAIN -3NI Medical Department Study: T-6295.12 nrt. F A f . T TOX-110 LRN-U2849 PrOtOCOl LRN-U2849 Amendment Number 3 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS.' The study director for the present study was identiified in the protocol as Kristen J. Hansen, Ph.D. AMENDTO READS The role of study director for the present study was reassigned to Marvin T. Case, D.V.M., Ph.D., as of 20 January 2000. The previous study director, Kristen J. Hansen, has been reassigned to the role of Principle Analytical Investigator. REASON: . The role of study director was reassigned in an effort to ensure compliance with Good Laboratory Practice Standards that outline study personnel requirements (refer to 21 CFR Part 58). 2. PROTOCOL READS: The sponsor for the present study was identified as Marvin T. Case, D.V.M., Ph.D. AMENDTO READ: The role of sponsor for the present study was reassigned to John L. Butenhoff, Ph.D., as of 20 January 2000. REASON: To ensure that the study director does not also cairry the duties of study sponsor, the sponsor role was reassigned. In this manner, personnel responsibilities and workload are more evenly balanced. .I 3M Environmental Laboratoty 3h4 Environmental Laboratory E-20 Page 166 -3kl Medical Department Study: T-6295.12 - Amendment Approval BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 PrOtocol LRN42849 Amendment Number 3 John L. Butenhog Ph.D., Sponsor Representative @' / Z A m Date Kristen J Hamen, Ph.D., Outgoing Study Director I / - F46 - 2 d U Date T&u Marvin T.Case, D.KM,Ph.D.,Incoming Study Director /dF&a.Lt&* & gate #I E-2 1 3M Environmental Laboratory 3M Environmental Laboratory Page 167 -3M Medical DepartmentStudy: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Baaelle Study Number: N003296-F 3M Toxicology Services ProtocolNumber: FACT-TOX-I 10 DEVIATION REPORll Battelle Study Number: N003296-F 3M Toxicology Services protocol Number. FACT-TOX-110 OKAL (GAVAGE) PHA.RMACOKINEI[ICSTUDY OF PFOS IN RATS TYPE OF DEVIATION: PROTOCOL DATE OF DEVIATION: August 19,1999 NATURE OF DEVIAlTON: The source ofcontrol rnabix will not be either Argus . Research or Sigma C h e q j d as specified in d o n 6.2 of the protocol. CAUSE OF DEVIATION: Harlan will be the supplier of control rat livers used to pnpare blanks, any standads, and QCs for the analyticallmtion of the study. IMPACT OF DEVIATION ON THE S T U D Y Harlan was used ils the control ma& supplierfor Battclle's valiciarion of the analytical method (Battelle study number NOO3604-A). This supplicrpmvidcd matrix that allowed :tchicvernent of the reported method acceptance criteria so that there is not impact on the study. I f CORRECTIVEA C n O N This protocol W o n report was prepared. APPROVED B Y p&/ . L Kristen J. Hansen, Ph.D. I Study Director 3M Environmental Laboratory E-22 Page 168 -3NI Medical DepartmentStudy: T-6295.12 - BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 Bottclle Study Number: N003296-F 3M Tosicology Services Protocol Number:FACT-TOX-I 10 APPENDIX F - PFOS PURITY IiEPORT 3NI Environmental Laboratory Page 169 3M Medical Department Study: T-6295.12 ETSS 2 3U BACK TO MAIN - Analytical Report: FACT TOX-110 LRN-U2849 Banelle Study Number: N003296-F 3M Toxicology Services Protocol Number: FACT-TOX-IIO f 651 na 4225 C4/26/59 G9:56 3 :02/03 ~ 0 : 3 4 1 -~ ~~ OW, 0 FC-95,lor 171 (PFOS).TN-A4834 Nomind produc: =CJn-SO,i:-) K(+) (white powder) This sample wus m b j e d d to 'H-NMR d :%NMZ d y s r t~o determine the purity of the nominal pmducl md to shu3ctuizc LI many impurity carnpancnts as possible. A podon of he sample was a c c w d y weighed. rpikad with P hrown amount of 1,bbis(uifluoromcrhyl)bencPe ( p h i .and then totally diuolved in D M S W for subsequent ;maly:iis by NMR A 400 M-YZ 'H-NMR ipesmnrm (# h57830.401) and r376 ,MHz "F-XMR spectrum (# f57830.4al) w e n squired using a Vyian UNrI"plus 400 FT-NMI( spxomcrcr Use of the p-HFX i n t e . d stimdud vas intended w permit the dcteminstion of the rbsolmeweight percent cancentnuow of the usigncd components withoot necessarily needingto identify or quantify all the components in the $amplemixnnz. .I The combined h=tlRr p c c a l dm were used to assign d l of h e major and most o f h e minor components in h i s sample as rccrivcd. The qualitative and quvltiuuvc compositional r e s u h hat were derived from the single vial W R internal r m d n r d i d o n analyses arc summarind in TABLE-1an l e following page. I have reponed both relative and absolute weight perctnt conctntrrtions. One possible muon that the absolute w.%valuer add up to marc than IC@% may be due to the fact that I assumed all of h e compcincnts conmined 8 carbons. If there w e n m y h o n e r chain homologs present (i.c.. 7,6,5, CIC. carbons). then rhe avenge compound moleculv weights would have been somewhat I u s thaD rhozc UJed in rbc calculsrionr. In pnu;rl. the %NMR technique is not p d c u l u l y well suited for identifying or quantifyingidnmounrs of v d o u r fluorochernicd homolog impurity compoacnuunlcrr tbe &aha arc very jhon. A more complete characttrization of m y otbcr fluorochem'cd homologs would require yldysh by elecmspny MS or isimilartechnique. Additional work would be requiredin M effon IOposiiively verify the tmatively assigned componenu listed in TASLE-I (dmotcd by possible). S d amountsofocher unidentifiedimpurities YC also detected in the n l R ipesua. but addiuonal work would be required in an elforr to identify cv quwrify these ulher rru:riols. Copier o f the %fR SFC:'JP will be provided for you at a later drte. If you have any questions sbaur b e ruultc in this initial repr: for FC-95.lac 171. please let me know. I aFologiL- fcr rhc delay in complc:ing this initid work, Tom Xcsmcr c: Rick Payfa - S A I C hnrly~kalW - 236-2a-1 I NIRBfClmC:: U i l l Q D C c 6 1 ?*.I. F-I 3NI Environmental Laboratory Page 170 -3M Medical DepartmentStudy: T-6295.12 ETSS 2 3U March 24 1999 BACK TO MAIN -- A.nalytical Report: FACT TOX-110 LRN-U2849 Battelle Study Number:N003296-F 3M Toxicology Services Protocol Number:FACT-TOX-1 IO 0 651 778 4226 04/26/99 09:56 6 :03/03 NO: 341 SAQC A m f y t i d Lab Rqum# S7830 u.1Rciort for FC-95. lot 171 3NI Environmental Laboratory h.2. F-2 Page 171 3M Medical Department Study: T-6295.12 i BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 METHOD VALIDATION REPORT TITLE: METHOD VALIDATION FOR THE QUANTITATION OF PERFLUOROOCTANESULFONATE(PFOS) IN RAT SERUM BY TlLJRBO ION SPRAY LC/MS DATE: 26 August 1999 REPORT: 99VDJAO1.MI.DOC AUTHORS: PREPARED FOR NUMBER OF PAGES: David J. Anderson, 1M.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 Ithaca. New York 14850 (607) 266-0665 Fax (607) 266-0749 3M Environmental Laboratory Page 172 BACK TO MAIN 3NI Medical Department Study: T-6295.12 AUTHORS: FOR DATE: TITLE : Analytical Report: FACT TOX-110 LRN-U2849 David J. Anderson, M.S. Amie J. Prince, B.S. Holly D. Ross, M.S. 3M Environmental Technology and Safety Services 26 August 1999 METHOD VALIDATION FOR THE QUANTITATION OF PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/MS ABSTRACT A sensitive, specific, accurate, and reproducibleanalyticalmethod was developedby Advanced BioAnalyticalServices,Inc., Ithaca, New York to quantitate perfluorooctanesulfonate(PFOS) in rat serum samples. Serum samples (50&)were extractedby a liquid-liquidextractionprocedure to isolatethe analyte from rat serum. Sample extractswere reduced to dryness, reconstituted, and analyzedby turbo ion spray liquid chromatography/massspectrometry(LCMS) in the negative ion mode. The assay demonstrated a lower limit ofquantitation (LLQ) of 0.05 p g / d using 50-& sample aliquots. The calibrationcurves were fit from 0.05 pg/mL to 20 pg/mL for PFOS by a weighted (l/f) quadraticequation. The coefficientsof determinationof the calibrationcurves ranged :!?om 0.9962to 0.9965. Precision and accuracy quality control (QC) samples were prepared at concentrations of 0.2,6,and 18 pg/mL PFOS. Quality control (QC)samples were prepared at a concentrationof 100pg/mL PFOS for partial volume analysis. The intra- and interassay precision (RSD) results calculated f h m all QC samplesranged from 1.92% to 4.87% for PFOS. The intra- and inter-assay accuracies(RE)calculated from QC samplesranged from -3.58% to 5.58% for PFOS. The mean extraction recoveries were from 87.6%to 100%for PFOS and 89.996for 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 EnvironmentalLaboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 2 of 50 99VDJA01.MLDOC Page 173 BACK TO MAIN 3NI Medical Department Study: T-6295.12 I Analytical Report: FACT TOX-110 LRN-U2849 after three freezehhaw cycles. Reliable results were obtained for sample extracts reinjected 27 hours after initial reconstitution. 3Nl Environmental Laboratory ADVANCED BIDANALYTICAL SERVICES, INC. Page 3 of 50 99VDJAO1.MLDOC Page 174 BACK TO MAIN 3NI Medical Department Study: T-6295.12 I Analytical Report: FACT TOX-110 LRN-U2849 QAU STATEhlENT Periodic inspections of the method validation for the quantitation of PFOS in rat serum were conducted by the Quality Assurance Unit of Advanced BioAnalytical Services(ABS)for compliancewith EPA GLP regulations (40 CFR Part 792). The study was inspected on the following dates: 13,21May 1999;7,8,14,15 June 1999; 1,2 July 1999; 3,4 August 1999. Results of the inspections were reported to ABS Management on: 13,21 May 1999; 7,8,14,15 Juxie 1999; 1,2 July 1999;3 , 4 August 1999. Results of the inspections were reported to the ,StudyDirector on 5 August 1999. Based on the inspections and the data reviewed, this report is a complete and accurate representation of the data. C L l IChthleen Com'ack, B.S. Quality Auditor 3NI Environmental Laboratory ADVANCED BIDANALYTICAL SERVICES, INC. Page 4 of 50 99VDJA01.MI.DOC Page 175 3NI Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 SIGNATURE PAGE TITLE: METHOD VALIDATIONFOR THE QUANTITATIONOF PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/MS Report Number: Reported by: Date Research Scientist Reviewed by: &7& kbthleen Cormack, B.S. Associate Auditor Authorized for Release by: John RIPerkins, Ph.D. Date Assistant ScientificDirector 3M EnvironmentalLaboratory 99VDJA01.MLDOC Page 176 BACK TO MAIN 3hl Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 TABLE OF CONTENTS ABSTRACT ........................................................................................................................ 2 QAU STATEMENT................................................................ 1........................................... 4 SIGNATUREPAGE........................................................................................................... 5 TABLE OF CONTENTS .................................................................................................6... LIST OF TABLES............................................................................................................... 8 LIST OF FIGURES ............................................................................................................. 9 1. INTRODUCTION............................................................................................ :.........10 2. EXPERIMENTAL.....................................................................................................10 2.1. CHEMICALSAND MATERIALS............................................................................... 10 2.2. Lc/Ms INSTRUMENTATION.................................................................................. 10 2.3. SAMPLEPREPARATIONAND EXTRACTIONPROCEDURE ......................................11 2.4. RAT SERUMVALIDATIONDATA...........................................................................11 2.5. ASSAY EVALUAnON.............................................................................................12 2.5.1. Intra- and Inter-AssayAccuracy.................................................................1..2. 2.5.2, Intra- and Inter-AssayPrecision ................................................ ...................12 2.5.3. PartialVolume Analysis................................................................................12 2.5.4. Lower Limit of Quantitation(LLQ) ...........................................................1..3. 2.5.5. Selectivity......................................................................................................13 2.5.6. Canyover Evaluation..................................................................................... 13 2.6. STABILITOYF PFOS IN QUALITCYONTROLSAMPLE.S....................................... 13 2.6.1. Ambient-TemperatureStabilityof PFOS in Rat Serum................................ 13 2.6.2. Freezer Stabilityof PFOS in Rat S ~ x u mat -20 'C ........................................14 2.6.3. Freezemaw Stabilityin Rat Serurn.............................................................. 14 2.7. REPRODUCIBILITYOF REINJECTINGEXTRACTESDAMPLES ................................ 14 2.8. EXTRACTIORNECOVERY ...................................................................................... 14 3. RESULTSAND DISCUSSION................................................................................ 15 3.1. ASSAY EVALUATION RESULTS ............................................................................. 16 3.1.1. Intra- and Inter-AssayAccuracy.................................................................... 16 3M Environmental Laboratory ADVANCED . BIOANALYTICAL SERVICES. INC Page 6 of 50 99VDJA01.MLDOC Page 177 BACK TO MAIN 3NI Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 3.1.2. Intra- and Inter-Assay Precision .................................................................... 16 3.1.3. Linearity......................................................................................................... 16 3.1.4. Partial Volume Analysis................................................................................ 16 3.1.5. Lower Limit of Qumtitation (LLQ:).............................................................. 17 3.1.6. Selectivity...................................................................................................... 17 3.1.7. Carryover Evaluation..................................................................................... 17 3.2. STABILITYOF PFOS IN QUALITYCOhTROLSAMPLES........................................17 3.2.1. Ambient Temperature Stability of IPFOS in Rat Serum ................................17 3.2.2. Freezer StabilityofPFOS in Rat Serum at -20 "C........................................ 18 3.2.3. FreezeA'haw Stabilityof PFOS in 'Rat Serum............................................... 18 3.3. REPRODUCIBILITYOF REMJECTTNG EXTRACTEDSAMPLES ................................ 18. 3.4. EXTRACTIONRECOVERY ...................................................................................... 18 4. CONCLUSIONS........................................................................................................19 5. DATA RETRIEVAL.................................................................................................. 19 6. REFERENCES..............................................................................'........................ 19 7. TABLES..................................................................................................................... 20 8. FIGURES................................................................................................................... 31 9. APPENDIX A QUANTITATIONOF PRFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/MS .................................. 37 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES. I N C. Page 7 of 50 ... I 99VDJA01.M[.DOC Page 178 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 LIST OF TABLES Table 1: Intra-AssayAccuracy and Precisionaf the PFOS Assay in Rat Serum for QC Samples ...................................................................................................... 20 Table 2: Inter-Assay Accuracy and Precision a dthe PFOS Assay inRat Serum for QC Samples fromThreeValidationItuns ........................................................ 21 Table 3: ASctacnudraacrydsafnrdomPrTechirseioenVoaflitdhaetiPoFnORSwAiss..s.a..y...i..n..R...a..t..S..e..r.u..m....f.o...r..C...a.l..i.b..r.a..t,i.o..n.........22 Table 4: CalibrationCurve Parameters for PFOS inRat Serum ..................................... 23 Table 5: PartialVolume Analysis of PFOS in :RatSerum............................................... 24 Table 6: Lower Limit of Quantitation of PFOS in Rat Serum......................................... 25 Table 7: Ambient-TemperatureStability of PFOS in Rat Serum After 24 Hours...........26 Table 8: Freezer Stabilityof PFOS in Rat Senun at -20 "C ............................................ 27 Table 9: StabilityofPFOS in Rat SerumAfter Three Freezemaw Cycles ..................2.8 Table 10:Reproducibilityof ReinjectingExtracted Samples ContainingPFOS after 27 Hours in ReconstitutionSolution................................................................. 29 Table 11:ExtractionRecovery of PFOS from Rat Serum......................................3.0.......... 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES,INC. Page 8 of 50 99VDJAO1 .MI.DOC Page 179 BACK TO MAIN 3NI Medical DepartmentStudy: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 LIST OF F'IGURES Figure 1: Full-Scan SingleMS Mass Spectrumof PFOS ................................................ .31 Figure 2: Full-Scan Single MS Mass Spectrum of Tetra-H-PFOS ...................................32 Figure 3: Mass Chromatograms of PFOS and its Internal Standard in Control Blank Rat Serum Extract............................................................................................. 33 Figure 4: Mass Chromatograms of PFOS in a F.at Serum Extract Sample Containing Internal Standard Only (Zero Sample).............................................................. 34 Figure 5: Mass ChromatogramsofCalibration Standard 1in Rat Serum Extract Containing PFOS (0.05 pg/mL) and the Internal Standard .............................. 35 Figure 6: Mass Chromatogramsof Calibration Standard 10in Rat Serum Extract Containing PFOS (20 pg/mL) and the Internal Standard ................................. 36 3 M Environmental Laboratory ADVANCED EIOANALYTICAL SERVICES, INC. Page 9 o f 50 99VDJA01.MI.DOC Page 180 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 1. INTRODUCTION The purpose of this report is to describe the method validation for the quantitative determination of perfluorooctanesulfonate(PFOS)in rat serum samples after modification of a previous method (1). The objectives of the method validation were to validate a simple extractionprocedure, detennine the lower limit of quantitation for routine analysis,determinethe extractionrecoveriesof the analyte and internal standard (IS), determinethe analyte stability in rat serum at ambient temperature, long-term freezer storage, and over three freezelthawcycles, and to provide specific, accurate, and reproducible quantitative results by turbo ion spray liquid chromatography/mass spectrometry (LCMS). 2. EXPERIMENTAL 2.1. CHEMICALSAND MATERIALS Perfluorooctanesulfonate(PFOS, Lot# 171)was obtained fkom 3M, Inc. The internal standard (IS) for PFOS was lH,IH,2H,2H-perfluorooctane sulfonic acid (Tetra-H- PFOS). The internal standard (Lot# 59909) was: obtained from 3M, Inc. A detailed list of chemicals and materials is found in Appendix A. \ Stock and working solutionswhich.were used to prepare the calibration curves for the analyteswere prepared as described in Appendix A. Stock solutions used in the preparationof quality control (QC) sampleswere prepared separately from those used in preparation of the calibrationcurves. Preparationof all solutionsused during extractionand analysis are described in Appendix A. 2.2. LCMS INSTRUMENTATION The liquid chromatography/massspectrometry system consisted of two LC-1OAJ3 pumps (Shimadzu, Columbia, MD 21046), a SCL-IOA pump controller (Shimadzy 3M Environmental Laboratory ADVAN CED BIOANALYTICAL SERVICES, INC. Page IO of SO 99VDJA01 .MI.DOC Page 181 BACK TO MAIN 3kl Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Columbia, MD 21046), a WISP 717plus autosampler (WatersAssociates, Millipore Corporation,Milford,MA 01757), a Betasil C,, (2 x 50 mm, 5 pm) column (KeystoneScientific,Inc., Bellefonte,PA 1682.3),and a PE SCIEX MI 365 mass spectrometer(PE SCIEX, Concord, Ontario). A detailed list of the instrumentation and instrument conditionsis found in Appendix.A. 2.3. SAMPLEPREPARATIONAND EXTRAC'CIONPROCEDURE For each analyhcal run (tray), duplicate5 0 - a idiquotsof the calibration curve samples were prepared as described in Appendix A. The nominal (theoretical) concentrationsof PFOS in the calibrationcurves were 0.05,0.1,0.25,0.5, 1,2,4, 8, 16, and 20 pg/mL. Rat serum quality control sampleswere prepared in advanceof the validation study at nominal (theoretical)concentrationsof 0.2,6, and 18 pg/mL for QCl, QC2, and QC3, respectively, as detailed in Appendix A. A dilution QC (QC4, 100 pg/mL) was preparedat a concentrationexceeding the upper limit of the calibration curve range (20 pg/mL), and was assayed using a 10-fold dilution for partial volume analysis. Calibrationstandardsand QC sampleswere exlmcted by the procedure detailed in i Appendix A. 2.4. RATSERUM VALIDATIODNATA The data were collected using selected ion mortitoring (SIM) turbo ion spray LCMS in the negative ion mode. Peak areas were integratedby the PE SCIEXpro* MacQuan, version 1.4, residing on a Macintosh computer. Following peak area integration,the results tables from MacQuan were saved as text files and uploaded to the Advanced BioAnalytical Services(ABS) file server where a weighted (1/y) quadratic regression was performed using the software package Watson v 5.3.1.01 ( P S S , Inc., Wayne, PA 19087).All data were rounded to no less than three significant figuresby ABS prior to reporting in Tables 1through 11. The data for the rat serum validation are stored in ABS Notebook 2304. 3M EnvironmentalLaboratory ABDI OVAANNACLEY DT I C A L SERVICES. I N C . Page 1 1 of 50 99VDJAOl .MI.DOC Page 182 3Wl Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 All calculationswere based on the peak area raiio of the analyte to the internal standard. Concentrationsof each analytein quality control samples were determined by inverse prediction from the calibrationcurve:. 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 concentrationfor each quality control level was divided by the theoretical concentration. One was subtracted fiom the result, converted to percent and expressed as RE. QCl through Q.C3 were assayed in replicates of five. In addition, the RE was reported for standardsat all levels over three runs. 2.5.2. Intra- and Inter-Assay Precision The intra- and inter-assay precision of the method was assessed by determining the Relative StandardDeviation (RSD)observed for quality control sample data. The mean concentrationand RSD were calculated for the first m and over three runs for intra-assay and inter-assay precision, respectivdy. QCl through QC3 were assayed in replicates of five. In addition, the RSD was reported for standards at all levels over three runs. 2.5.3. Partial Volume Analysis The effect of dilution on the analysis of PFOS in rat serum was determinedby partial volume analysis. QC4 (100 pg/mL) was prepared containing PFOS at approximately five times the upper limit of quantitation(ULQ).Five replicates of QC4 were diluted ten-fold and analyzed. The RSD and RE were reported. 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 12 of 50 99VDJA01 .MLDOC Page 183 BACK TO MAIN 3M Medical Department Study: T-6295.12 I Analytical Report: FACT TOX-110 LRN-U2849 2.5.4. Lower Limit of Quantitation (LLQ) Rat serum control samples from six separate individualswere spiked with PFOS at a concentrationof 0.05 pg/g/mL. The LLQ was determinedby obtaining the backcalculatedconcentrationsfiom these control samples. The overall mean, RSD, and RE were also calculated. 2.5.5. Selectivity The selectivity of the assay was determinedby LCMS. To monitor for interference from the biologicalmatrix, rat serum samples containingneither the analyte nor the internal standard (control blank) were assayed with all experiments. 2.5.6. Carryover Evaluation The carryover of analyte fiom one injection to ihe next was assessed by analyzing a control'blank injected immediately after a high calibration standard (STD10, 20 trg/ml). 2.6. STABILITYOF PFOS INQUALITCYoivmoL SAMPLES QCl through QC3 were used to determinethe 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 noininal timepoints of 0,2.5,6,and 24 hours after thawing. All QCs were assayed in replicates of five. 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 13 of 50 99VDJA01.MI.DOC Page 184 BACK TO MAIN 3M Medical Department Study: T-6295.12 I Analytical Report: FACT TOX-110 LRN-U2849 2.6.2. Freezer Stability of PFOS in Rat S'erumat -20 "C The freezer stability of PFOS was evaluated by storing QC samples at each concentrationlevel at -20 OC for 7, 13, and 33 days. Additional k e z e r stability timepoints will be reported in an addendum to ibis report. All QCs were assayed in replicates of four. 2.6.3. FreezdThaw Stability in Rat Serum The stability of PFOS was evaluated after three: fkeezehhaw cycles. The mean concentrationsof the QC samples after three freezehhawcycles @.e.,at least 2 hours frozen storage at the nominal temperature of -i!O "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. REPRODUCIBILITYQF REINJECTING EXTRACTED SAMPLES The reproducibilityof reinjectingreconstituted serum extracts was investigatedby reinjecting a set of previously-assayed standards and QC sampleswhich had been stored after injection at approximately 25 "C fcir 27 hours. The RE of the QC sampleswas used to assess processed sample stability in the reconstitution solution. All QCs were assayed in replicates of five. 2.8. EXTRACTION RJCCOVERY The extractionrecovery of PFOS f h m rat serumwas determinedby comparingthe peak area ratio (PAR) of samples (0.25,4, and 16 pg/mL) spiked after extraction (post-extract)with the PAR of samplesspiked before extraction (pre-extract). The internal standard was spiked post-extraction for all samples. The recovery of the internal standard (4 pg/mL) was assessed following a similar approach using PFOS as the reference. The extraction recovery (% Ekcovery)was determinedby dividing the pre-extract PAR by the post-extract P A R znd expressingthe result as a percentage. Five replicates were used at each concentration level. 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 14 of 50 99VDJAOI .MI.DOC Page 185 3NI Medical Department Study: T-6295.12 BACK TO MAIN Analytical Report: FACT TOX-110 LRN-U2849 3. RESULTS AND DISCUSSION A quantitativeanalyticalprocedure using turbo ion spray LCMS in the negative ion mode was developed to meet the high sensitivity, specificity, and reproducibility requirementsfor the determination of PFOS in rat serum. The fill-scan single MS mass spectrum of PFOS showed an abundant [Pld-KI- ion at m/z = 499 (Figure 1). The full-scan singleMS mass spectrumof Tetrij-H-PFOSshowed an abundant [M-HI- ion at d z = 427 (Figure 2). The following selected ion monitoring (SIM) was used to quantifythe analytesin rat serum: PFOS Tetra-H-PFOS (IS) m'z = 499.0 m'z = 427.0 The peak labelingof full-scan data does not accuratelyrepresent the performanceof 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 instrumentin the SIM mode. The mass chromatogramsof a representative control blank rat serum extract are shown in Figure 3. Mass chromatogramsfrom a representative control rat serum extract containing only the internal standard (zero sample), are shown in Figure 4. Figures 5 and 6 are mass chromatogramsof representativeextracts from calibration standards 1 and 10, respectively. 3M Environmental Laboratory ADVANCED EIOANALYTICAL SERVICES, INC. Page IS of 50 99VDJAOI .MI.DOC Page 186 BACK TO MAIN 31\11Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 3.1. ASSAY EVALUATIORNESULTS 3.1.1. Intra- and Inter-Assay Accuracy The intra-assay accuracy (RE) data from QC samplesranged from -2.35 to 5.58% for PFOS (Table 1). The inter-assay accuracyranged from -3.58 to 4.95% for PFOS (Table 2). These data indicate acceptable intra- and inter-assay accuracy for the determinationof PFOS in rat serum. 3.1.2. Intra- and Inter-Assay Precision The intra-assayprecision data (RSD) ranged h i m 1.99 to 3.28% for PFOS at all QC concentrationlevels (Table 1). The inter-assay precision results from QC samples ranged from 1.92 to 4.87% for PFOS (Table 2). The RSD ranged fkom 0.613 to 4.38% for calibrationstandardsat 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. Coefficientsof determination (r') were 20.9962 for PFOS in rat serum. The calibration curve statisticsare shom in Table 4. 3.1.4. Partial Volume Analysis The results of partial volume analysisof QC4 is shown in Table 5. The precision (RSD) of QC4 samples diluted 1in 10was 2.71%. The accuracywas 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 BOANALYTIC AL SERVICES, INC. Page 16 of SO 99VDJA01.MI.DOC Page 187 BACK TO MAIN 3 M Medical DepartmentStudy: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 3.1.5. Lower Limit of Quantitation (LLQ) Table 6 showsthe lower limit of quantitation(LLQ)data. The serum LLQ experiment demonstrated that 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 assaywas specificfor PFOS. No chromatographic interferenceswere observed in any of the control serum samples analyzed (Figure 3). The control blanks and zero samplesdid show some evidence of small chromatographicpeaks at the retention times of the analyte. Thesepeaks were not quantifiable as they were below the lower limit of quantitation (LLQ). 3.1.7. Carryover Evaluation Carryoverof PFOS and the IS was evaluatedb y injection of an extractedrat serum control blank following an injection of an extracted high standard (STD 10). The response for the small chromatographicpeak ai:the retention time for PFOS was comparableto the response of a rat serum conb-ol blank injected before the high standard. Thus, carryover is negligible for PFOlS. There was no evidence of carryover for the internal standard. . 3.2. STABILITYOF PFOS IN QUALITY CONTROL SAMPLES 3.2.1. Ambient Temperature Stability of'PFOS in Rat Semm The results of the ambient stability of PFOS in QC samples are shown in Table 7, The mean predicted concentrations deviated fiom -4.45 to 10.5% from the 0-hour values for all QC levels and time points. Based on these data, PFOS was stable in rat serum for up to 24 hours at ambient temperature. 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 17 of50 99VDJA01 .MLDOC Page 188 BACK TO MAIN 3M Medical Department Study: T-6295.12 I Analytical Report: FACT TOX-110 LRN-U2849 3.2.2. Freezer Stability of PFOS in Rat Serum at -20 "C The results of the long-term stability of PFOS in QC samples stored at -20 "C are presented in Table 8. The PFOS concentrationsdeviated from 0-hour values from -2.76 to 12.3%for all QC levels storedup to 33 days. Results of freezer storage after two, four, and eight months will be reported as an addendum to this report. 3.2.3. FreezePThaw Stability of PFOS in Rat Serum The results of the freezdthaw stability of PFOS in QC samples after three fieezdthaw cycles is shown in Table 9. PFOS was stable after three freezdthaw cycles with deviationsranging fkom -1.38% to 10.1%from the 0-cycle samples for all QC levels (Table 9). 3.3. REPRODUCIBILITYOF REINJEXXING]EXTRACTED SAMPLES The results of reinjectingreconstituted samplescontaining PFOS after storage for 27 hours at approximately 25 "Cin reconstitutionijolution are shown in Table 10. Reinjectingprocessed samples after 27 hours in1 reconstitution solutionwas appropriatewith RE values ranging'from -6.33 to 4.59% at 27 hours for all QC levels. 3.4. EXTRACTIONRECOVERY The mean recoveries of PFOS and Tetra-H-PFOS are shown in Table 11. The mean recoveriesranged from 87.6 to 100%for PFOS. The mean recovery for Tetra-HPFOS was 89.9%. 3M EnvironmentalLaboratory ADVANCED BIOANALYTICAL SERVICES. INC. Page 18 of 50 ~._ 99MJAO I .MLDOC Page 189 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 4. CONCLUSIONS The turbo ion spray LC/MS assay procedure for the determination of PFOS in rat serum has proven to be sensitive, specific, accurate, and reproducible. Its high sensitivity allows reliable and reproducible quentitationof PFOS down to a level of 0.05 pg/mL in rat serum based on 50-pL samples. 5. DATARETRIEVAL The data for the rat serum validation are stored in ABS Notebook 2304 and in the ABS Archives. 6. REFERENCES 1. Advanced BioAnalytical Services, Inc. Report 9 8 A G K P 0 2 . W . Analytical Report for the Determination.of Perfluorooctanoateand Perfluorooctanesulfonate in Human Serum by LC/MS. Grace K. Poon, Ph.D., David Hardwick, B.S., and Ellen Pace, M.A.T., 6 February 1998. ! 3M Environmental Laboratory ADVANCED BIDANALYTICAL SERVICES. INC. Page 19 of 50 99VDJAO1 .MI.DOC Page 190 BACK TO MAIN 3hl Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 7. TABLES Table 1: Intra-Assay Accuracy and Precision of the PFOS Assay in Rat Serum for QC Samples RSD = (SDMean) x 100 RE = [(Mean/Theoretical)-I] x 100 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 20 of 50 99VDJAO1.MI.DOC Page 191 BACK TO MAIN 3hl Medical Department Study: T-6295.12 I Analytical Report: FACT TOX-110 LRN-U2849 Table 2: Inter-Assay Accuracy and Preicision of the PFOS Assay in Rat Serum for QC Samples from Three Validation Runs Theoretical Conc. 0.2 QC2 QC3 6 18 Run 9 0.210 6.52 17.9 0.202 6.3 1 18.0 0.206 6.17 16.7 0.205 6.32 18.0 0.194 6.35 17.3 Run 10 0.187 6.44 16.9 0.186 6.44 17.3 0.182 6.32 17.5 0.189 6.19 18.0 RSD = (SDMean) x 100 RE = [$vfean/Theoretical)-l]x 100 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES, INC. Page 21 of 50 99VDJAO1 .MI.DM: Page 192 BACK TO MAIN 3hl Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Table 3: Accuracy and Precision of the PFOS Assay in Rat Serum for Calibration Standards from Three Validation Runs RSD = (SDh4ean) x 100 RE = [(Mean/Theoretical)-l]x 100 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES. INC. Page 22 of 50 99VDJA01.MI.DOC Page 193 BACK TO MAIN 3111 Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Table 4: Calibration Curve Parameters for PFOS in Rat Serum a: y = Ax2+Bx + C,weighted llf 3M Environmental Laboratory ADVAN CEO BIOANALY TICAL SERVICES. INC Page 23 of 50 99VDJA01.MI.DOC Page 194 BACK TO MAIN 3M Medical Department Study: T-6295.12 Analytical Report: FACT TOX-110 LRN-U2849 Table 5: Partial Volume Analysis of PFOS in Rat Serum 1 - TheoreticalConc. S Conc. (pgjmL) QC4 100 (1 in 10 dilution) 111 105 105 104 5.89 RSD = (SDMean) x 100 RE = [(Mean/Theoretical)-11x 100 3M Environmental Laboratory ADVANCED BIOANALYTICAL SERVICES. INC. Page 24 of 50 99VDJA01.MI.DOC Page 195 BACK TO MAIN 3M Medical Department Study: T-6295.12 ~ Analytical Report: FACT TOX-110 LRN-U2849 Table 6: Lower Limit of Quantitation of PFOS in Rat Serum - 4 PFOS nc.Found (pg/mL) 0.0548 0.0489 0.0499 0.0554 0.0527 0.0520 0.0523 4.95 RSD = (SDMcan) x 100 4.57 - RE = [(Mean/Theoretical)-1]1: 100 3M Environmental Laboratory ADVANCED BIOANALYTICAL S E R V I C E S , INC. Page 25 of 50 99VDJA01 .MI.DOC Page 196