Document QgLYdKn4q1xa0KBbv4JVO602k

3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Study Title 104-Week Dietary Chronic Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats (Covance Study No.: 6329-183) Analytical Laboratory Report Title Determination of the Presence and Concentration of Perfluorooctanesulfonate (PFOS) in Liver and Serum Specimens of Crl:CD(SD) IGS BR Rats Exposed to Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) Data Requirement Not Applicable Author 3M Environmental Laboratory Study Completion Date At signing Performing Laboratories Liver and Serum Analyses 3M Environmental Laboratory Building 2-3E-09, 935 Bush Avenue St. Paul, MN 55106 Project Identification 3M Medical Department Study: T-6295.4 Covance In-Life Study: 6329-183 Analytical Report: FACT TOX-002 3M Laboratory Request No. U2121 Total Number of Pages 181 3M Environmental Laboratory Page 1 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 This page has been reserved for specific country requirements. 3M Environmental Laboratory Page 2 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 GLP Compliance Statement Analytical Laboratory Report Title: Determination of the Presence and Concentration of Perfluorooctanesulfonate (PFOS) in Liver and Serum Specimens of Crl:CD(SD) IGS BR Rats Exposed to Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) Study Identification Number: T-6295.4, FACT TOX-002, LRN-U2121 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: There were two study directors in this study. This study was designed as two separate studies. The in-life phase study was considered to end at the generation and shipment of specimens. The analytical study was considered to start at the receipt of these specimens for analysis. This resulted in having two separate study directors, one for each phase of the same study. However, since the technical performance of each phase was entirely separate, no effect is expected from this exception. Changes in the raw data entries were not all made in accordance with 21 CFR 58.130 (e). Specimen stability storage will not be determined. One of the control matrices (rabbit liver) was not identified in accordance with 21 CFR 58.120 (a)(2). The electronic data systems in use have not been validated and there is not an electronic audit trail of corrections currently available (21 CFR 58.130 (e)). Authenticated hard copies of chromatograms and associated documents will be considered as the original raw data. Characterization of the analytical standards is underway, but was not completed at the time this data was assembled (21 CFR 58.105 (a)). When Certificates of Analysis for all remaining lots of PFOS are available, an amendment to this report will be issued. Lot No. 59909 of THPFOS will not be characterized for purity since quantities of this standard are exhausted. Study Director Lvv. ^ Sponsor Represen Z* Date Or 0 4& - 3M Environmental Laboratory Page 3 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 GLP Study--Quality Assurance Statement Analytical Laboratory Report Title: Determination of the Presence and Concentration of Perfluorooctanesulfonate (PFOS) in Liver and Serum Specimens of Crl:CD(SD) IGS BR Rats Exposed to Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) Study Identification Number: T-6295.4, FACT TOX-002, LRN-U2121 This study has been inspected by the 3M Lab Quality Assurance Unit (QAU) as indicated in the following table. The findings were reported to the study director and laboratory management. Inspection Dates Phase Date Reported to Management Study Director 11/11/99 Extraction 11/30/99 11/30/99 08/28/00-09/18/00 10/05/00 - 10/06/00, 10/16/00 10/20/00,10/30/00-10/31/00 11/01/00-11/03/00 11/02/00-11/03/00, 11/06/00-11/07/00 01/08/01 -01/11/01 Data Data Data Data Draft Report 09/19/00 11/02/00 11/06/00 11/08/00 01/12/01 09/19/00 11/02/00 11/06/00 11/08/00 01/12/01 QAU Represe; live /~ h t 3 -0 O Date 3M Environmental Laboratory Page 4 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table of Contents GLP Compliance Statement............................................................................................... 3 GLP Study--Quality Assurance Statement....................................................................... 4 Study Personnel and Contributors..................................................................................... 9 Introduction and Purpose...................................................................................................10 Test System.................................................................................................................. 10 Specimen Collection and Analysis................................................................................ 10 Specimen Receipt and Maintenance................................................................................. 11 Chemical Characterization................................................................................................. 11 Dose Confirmation Analyses.........................................................................................12 Method Summaries............................................................................................................ 12 3M Environmental Laboratory....................................................................................... 12 Preparatory Methods................................................................................................12 Analytical Methods....................................................................................................13 Analytical Equipment................................................................................................ 14 Deviations...................................................................................................................... 14 Data Quality Objectives and Data Integrity........................................................................ 15 Data Summary, Analyses, and Results............................................................................. 15 Summary of Quality Control Analyses Results.............................................................. 15 Statement of Data Quality.............................................................................................16 Summary of Specimen Results.....................................................................................16 Statistical Methods and Calculations................................................................................. 16 Statement of Conclusion.................................................................................................... 17 Reference.......................................................................................................................... 17 Appendix A: Identification of Test Article and Control Matrices........................................ 18 Appendix B: Protocol, Amendments, Deviations...............................................................19 Appendix C: Extraction and Analytical Methods................................................................42 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry (8 pages)...................................................................................................... 43 FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry (8 pages)...................................................................................................... 51 ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages).......................................................................................................................... 59 ETS-8-6.0, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages).......................................................................................................................... 73 3M Environmental Laboratory Page 5 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry (8 pages)........................................................................87 FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry (8 pages)............................................................ 95 ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry, (9 pages)................... 103 ETS-8-7.0, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry, (10 pages).................... 114 Appendix D: Data Summary Tables....................................................................................126 Appendix E: Data Spreadsheets.........................................................................................156 Appendix F: Example Calculations......................................................................................172 Appendix G: Interim Certificates of Analysis....................................................................... 174 Appendix H: Report Signature Page.................................................................................. 181 3M Environmental Laboratory Page 6 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 List of Tables Table 1. Test System Population Demographics for Study (6329-183)........................... 10 Table 2. Characterization of the Analytical Reference Materials Used in Study FACT TOX-002...................................................................................................... 12 Table 3. Negative Ions Monitored in 3M Laboratory Analyses......................................... 14 Table 4. Characterization of the Control Matrices Used for Sera Analyses in Study FACT TOX-002...................................................................................................... 18 Table 5. Characterization of the Control Matrices Used for Liver Analyses in Study FACT TOX-002...................................................................................................... 18 Table 6. Identification of the Test Article in Study FACT TOX-002.................................. 18 Table 7. FACT TOX-002 Data Summary of PFOS Concentration--Serum(pg/mL)........127 Table 8. FACT TOX-002 Data Summary of PFOS Concentration--Liver (pg/g).............128 Table 9. Week 4 Reported PFOS Levels in Sera of Male Rats in Study FACT TOX-002...................................................................................................... 129 Table 10. Week 4 Reported PFOS Levels in Sera of Female Rats in Study FACT TOX-002...................................................................................................... 130 Table 11. Week 14 Reported PFOS Levels in Sera of Male Rats in Study FACT TOX-002...................................................................................................... 131 Table 12. Week 14 Reported PFOS Levels in Sera Female Rats in Study FACT TOX-002...................................................................................................... 132 Table 13. Week 53 Reported PFOS Levels in Sera of Male Rats in Study FACT TOX-002...................................................................................................... 133 Table 14. Week 53 Reported PFOS Levels in Sera of Female Rats in Study FACT TOX-002...................................................................................................... 133 Table 15. Day 719 Reported PFOS Levels in Sera of Female Rats in Study FACT TOX-002...................................................................................................... 134 Table 16. Week 105 Reported PFOS Levels in Sera of Male Rats in Study FACT TOX-002...................................................................................................... 135 Table 16. Week 105 Reported PFOS Levels in Sera of Male Rats in Study FACT TOX-002 (continued).................................................................................. 136 Table 16. Week 105 Reported PFOS Levels in Sera of Male Rats in Study FACT TOX-002 (continued).................................................................................. 137 Table 17. Week 105 Reported PFOS Levels in Sera of Female Rats in Study FACT TOX-002...................................................................................................... 138 Table 17. Week 105 Reported PFOS Levels in Sera of Female Rats in Study FACT TOX-002 (continued).................................................................................. 139 Table 17. Week 105 Reported PFOS Levels in Sera of Female Rats in Study FACT TOX-002 (continued).................................................................................. 140 3M Environmental Laboratory Page 7 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 18. Week 106 Reported PFOS Levels in Sera of Male Rats in Study FACT TOX-002...................................................................................................... 141 Table 19. Week 106 Reported PFOS Levels in Sera of Female Rats in Study FACT TOX-002...................................................................................................... 141 Table 20. Week 4 Reported PFOS Levels in Liver of Male Rats in Study FACT TOX-002...................................................................................................... 142 Table 21. Week 4 Reported PFOS Levels in Liver of Female Rats in Study FACT TOX-002...................................................................................................... 143 Table 22. Week 14 Reported PFOS Levels in Liver of Male Rats in Study FACT TOX-002...................................................................................................... 144 Table 23. Week 14 Reported PFOS Levels in Liver of Female Rats in Study FACT TOX-002...................................................................................................... 145 Table 24. Week 53 Reported PFOS Levels in Liver of Male Rats in Study FACT TOX-002...................................................................................................... 146 Table 25. Week 53 Reported PFOS Levels in Liver of Female Rats in Study FACT TOX-002...................................................................................................... 147 Table 26. Day 719 Reported PFOS Levels in Liver of Female Rats in Study FACT TOX-002...................................................................................................... 148 Table 27. Week 105 Reported PFOS Levels in Liver of Male Rats in Study FACT TOX-002...................................................................................................... 149 Table 27. Week 105 Reported PFOS Levels in Liver of Male Rats in Study FACT TOX-002 (continued).................................................................................. 150 Table 27. Week 105 Reported PFOS Levels in Liver of Male Rats in Study FACT TOX-002 (continued).................................................................................. 151 Table 28. Week 105 Reported PFOS Levels in Liver of Female Rats in Study FACT TOX-002...................................................................................................... 152 Table 28. Week 105 Reported PFOS Levels in Liver of Female Rats in Study FACT TOX-002 (continued).................................................................................. 153 Table 28. Week 105 Reported PFOS Levels in Liver of Female Rats in Study FACT TOX-002 (continued).................................................................................. 154 Table 29. Week 106 Reported PFOS Levels in Liver of Male Rats in Study FACT TOX-002...................................................................................................... 155 Table 30. Week 106 Reported PFOS Levels in Liver of Female Rats in Study FACT TOX-002...................................................................................................... 155 3M Environmental Laboratory Page 8 3M Medical Department Study: T-6295.4 Study Personnel and Contributors Study Director John L. Butenhoff, Ph.D., Study Director 3M Corporate Toxicology - Medical Department 3M Center, Building 220-2E-02 St. Paul, MN 55144-1000 651-733-1962 Sponsor Marvin T. Case, D.V.M., Ph.D., Sponsor Representative 3M Corporate Toxicology - Medical Department 3M Center Building, 220-2E-02 St. Paul, MN 55144-1000 Analytical Chemistry Laboratories Liver and Serum Analyses 3M Environmental Laboratory (3M Lab) Kristen J. Hansen, Ph.D., Principle Analytical Investigator Analytical Report: FACT TOX-002 LRN-U2121 3M Lab Contributing Personnel Lisa A. Clemen Kelly J. Dorweiler* Mark E. Ellefson Sara E. Estes* Barb A. Gramenz* Sarah A. Heimdal* Cari S. Hewitt* Marlene M. Heying* Megan C. Holloway* Joy D. Jenkins 'Contract lab professional service employees Harold O. Johnson Ognjenka Krupljanin* Kelly J. Kuehlwein* Glenn M. Langenburg* Sally A. Linda* Ian A. Smith* Kathleen M. Stock* Bob W. Wynne* Richard D. Youngblom* Location of Archives All original raw data, protocol, and analytical report have been archived at the 3M Environmental Laboratory. The test article and 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. 3M Environmental Laboratory Page 9 3M Medical Department Study: T-6295.4 Introduction and Purpose Analytical Report: FACT TOX-002 LRN-U2121 The purpose of the study is to determine the presence and concentration of PFOS in liver and serum specimens collected from Covance Study No.: 6329-183 titled: 104-Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS, T6295) in Rats, initiated on 22 May 1998. Test System 360 males and 360 females were used as the test system. Table 1 outlines the rat population demographics and dosage levels for study 6329-183. The test system species and strain selected was the Crl:CD(SD) IGS BR rat received from Charles River Laboratories, Inc., identified using an implanted microchip device. At the initiation of treatment the rats were approximately 6-8 weeks of age and weighed between 100-300 g. Table 1. Test System Population Demographics for Study (6329-183) Number of Animals Study Group Male Female Dietary Levels (ppm) Group 1 Control Group 2 Low Group 3 Mid Group 4 Mid-High Group 5 High Group 6 High Recovery 70 70 60 60 60 60 60 60 70 70 40 40 0 0.5 2.0 5.0 20.0 20.0 Specimen Collection and Analysis Sample specimens were collected by Covance (study 6329-183) and sent to the 3M Environmental Laboratory for analysis. On week 4 and week 14, 50 liver and 50 serum specimens were collected from Groups 1-5 males and females. On week 53, 39 liver and 19 serum specimens were collected from Groups 1 and 5 males and females. On day 719, 9 liver and 9 serum specimens were collected from Group 3 females. At the time of terminal sacrifice week 105), 164 liver and 164 serum specimens were collected from Groups 1-5 males and females. At the time of recovery sacrifice (week 106), 27 liver and 27 serum specimens were collected from Group 6 males and females. 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 4/21/00): Serum Specimens--292 specimens Liver Specimens--312 specimens Specimens Collected from Study Group 6 (Recovery) on 4/24/00 and 4/27/00: Serum Specimens--27 specimens Liver Specimens--27 specimens 3M Environmental Laboratory Page 10 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Blood specimens were allowed to clot at room temperature and separated by centrifugation. Serum was then harvested and stored in a freezer at -60C to -80C until shipped to the 3M Environmental Laboratory. Liver specimens collected from each animal were flash frozen in liquid nitrogen and then stored in a freezer at -60C to -80C until shipped to the 3M Environmental Laboratory. Liver and sera specimens were shipped to the 3M Environmental Laboratory frozen and on dry ice. Sera and liver specimens were extracted beginning on May 27, 1998 using an ion pairing reagent and either ethyl acetate or methyl-fe/f-butyl ether (MtBE). Liver specimens were homogenized prior to the extraction procedure. Specimen extracts were analyzed using high-pressure liquid chromatography-electrospray/tandem mass spectrometry (HPLC-ESMSMS) in the multiple reaction monitoring mode. PFOS levels were quantitated by external calibration of extracted curves. Analytical details are included in this report. Specimen Receipt and Maintenance The 3M Environmental Laboratory received liver and serum specimens collected at predetermined time points during and at the end of the in-life phase of Covance Study No. 6329-183 from May 1998 through May 2000 from Covance. All specimens were received frozen on dry ice and were immediately transferred to storage at -20C 10C. Control matrices used in liver and sera analyses performed during TOX-002 were obtained from commercial sources and are presented in Appendix A (see Tables 4 and 5). Specimens analyzed at the 3M Environmental Laboratory will be maintained for a period of 10 years and will be stored at the laboratory at -20C 10C. Chemical Characterization Chemical characterization information on the test article used in this study is presented in Appendix A (see Table 6). Chemical characterization information on the analytical reference materials used in this study is presented in tabular form (Table 2) below. 3M Environmental Laboratory Page 11 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 2. Characterization of the Analytical Reference Materials Used in Study FACT TOX-002 Reference Material / Formula Acronym Source Potassium Perfluorooctanesulfonate C8 F1 7 SO3 -K+ KPFOS 3M Specialty Chemicals 3M Specialty Chemicals 3M Specialty Chemicals 1H, 1H, 2H, 2HTetrahydroperfluorooctanesulfonic acid C8 H4 F13 SO3 H THPFOS ICN Biomedics, Inc. ICN aUsed for analysis of week 4 and week 14 sera samples. b Samples were not corrected for purity. TBD--to be determined N/A--not applicable. This lot is exhausted and cannot be characterized. Expiration Date 08/31/00 2010 2010 01/01/2010 01/01/2010 Storage Conditions Ambient temprature Ambient temprature Ambient temperature (dry) Ambient temperature Ambient temperature Chemical Lot Number 171 193a Physical Description White crystalline powder White powder 215a 53406 59909 White powder Brown waxy solid Brown powder Purity 86.4% 88.0%b TBD TBD N/A Dose Confirmation Analyses Dose preparation methods and analysis were performed by Covance, using a validated analytical method provided by the Sponsor (MP-M383-MA), and are reported separately (Reference Covance 6329-183). Method Summaries Following is a brief description of the methods used during this analytical study by the 3M Environmental Laboratory. Detailed descriptions of the methods used in this study are located in Appendix C. As the present study progressed, more advanced methods evolved and earlier methods were used with deviations until amendments to the protocol were written. Protocol and method deviations are located in Appendix B of this report. 3M Environmental Laboratory Preparatory Methods FACT-M-1.0. "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry". This method was used for week 4 specimens extracted on 6/11/98 and week 14 specimens extracted on 8/6/98. FACT-M-3.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Serum for Analysis using HPLC-Electrospray/Mass 3M Environmental Laboratory Page 12 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Spectrometry". This method was used for week 4 specimens extracted on 5/27/98 and week 14 specimens extracted on 7/27/98. ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry". This method was used for week 53 specimens extracted on 11/10/99, and day 719, week 105, and week 106 specimens extracted on 5/1/00 and 5/2/00. ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry". This method was used for week 53 specimens extracted on 11/10/99 and 1/25/00, day 719 specimens extracted on 5/16/00 and 5/18/00, week 105 specimens extracted on 5/16-18/00 and 5/22/00, and week 106 specimens extracted on 5/16/00 and 5/18/00. An ion-pairing reagent was added to the specimen and the analyte ion pair was partitioned into ethyl acetate (FACT-M-1.0 and FACT-M-3.0) or MtBE (ETS-8-4.1 and ETS-8-6.0). The extract was transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract was reconstituted in 1.0 mL of methanol, and then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. Analytical Methods FACT-M-4.0, "Analysis of Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry". With the exception of week 4 sera specimens, data was worked up according to ETS-8-5.1 or ETS-8-7.0 and following control of bias documentation included in study binder. ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry". Sera run 7/15/98 was accepted despite the failure of the continuing calibration checks. Since compliance with method ETS-8-5.1 was in the protocol, deviations from it, including this one are also protocol deviations. A method deviation has been written. ETS-8-7.0, "Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry". The analyses were performed by monitoring one or more product ions selected from a single primary ion characteristic of PFOS using HPLC-ES/MS/m S. For example, molecular ion 499, selected as the primary ion for PFOS (C8F17SO3-) analysis, was fragmented further to produce ion 99 (FSO3-). The characteristic product ion 99 was monitored for quantitative analysis. 3M Environmental Laboratory Page 13 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Analytical Equipment The actual analytical equipment settings used in the present analytical phase of this study varied slightly during actual data collection. The following is representative of the analytical equipment settings used during the analytical phase of this study. Liquid Chromatograph: Hewlett-Packard Series 1100 Liquid Chromatograph system Analytical column: Keystone BetasilTM Ci8 2x50 mm (5 pm) Column temperature: Ambient Mobile phase components: Component A: 2mM ammonium acetate Component B: methanol Flow rate: 300 pL/min Injection volume: 10 pL Solvent Gradient: 13.5 minutes Time (minutes) %B 0.0 40% 8.5 90% 11.0 90% 12.0 40% 13.5 40% Mass Spectrometer: Micromass API/Mass Spectrometer Quattro IITMTriple Quadrupole system Software: Mass LynxTM 3.1, 3.3, and 3.4 Cone Voltage: 30-60 V Collision Gas Energy: 25-45 eV Mode: Electrospray Negative Source Block Temperature: 150C 10C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (MRM) Table 3. Negative Ions Monitored in 3M Laboratory Analyses Target Analyte Primary Ion (amu) Product Ion (amu) PFOS 499.0 80.0, 99.0, 130.0 THPFOS 427.0 80.0 Deviations As the analytical phase of this study progressed, method parameters were evaluated to improve analyses. Earlier methods were used with deviations until amendments to the protocol were written. 3M Environmental Laboratory Page 14 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Data Quality Objectives and Data Integrity The following data quality objectives (DQOs) were indicated in the method performance section of ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry and ETS-8-7.0, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry: Linearity: The coefficient of determination (r2) equal to or greater than 0.980. Limits of Quantitation (LOQ): The LOQ for PFOS is 5.55 ng/mL for serum and 26.9 ng/g for liver in the respective method performance sections, however, the LOQ for a given run is based upon the concentration of the lowest acceptable standard in the run. Acceptable Matrix Spike Recoveries: 70-130% for sera / 50-150% for liver. Data Summary, Analyses, and Results Data quality objectives for the analytical phase of this study outlined in the 3M Lab methods ETS-8-5.1 and ETS-8-7.0 (see Appendix C) were met with the exceptions noted in this report. Summary of Quality Control Analyses Results Linearity: The coefficient of determination (r2) of the standard curve was >0.985. Calibration Standards: With the exception of the week 4 sera data, quantitation of the target analytes was based on linear regression analysis of two extracted matrix curves weighted 1/x bracketing each group of samples. Calibration standards were not diluted and were spiked to be within the linear range of the instrument. Week 4 sera data was evaluated using an unweighted curve. 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. Low curve points with peak areas less than two times that of the extraction blanks were deactivated to disqualify a data range that may have been significantly affected by background levels of the analyte. Occasionally, a single mid-range curve point that was an obvious outlier may have been deactivated. Quantitation of PFOS was based on the response of one or more specific product ions using the multiple response-monitoring mode of the instrument (see Appendix C, Analytical Methods). 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), and is at least two times the analyte peak area detected in the extraction blanks. For both liver and sera analysis, the LOQ decreased over the course of the study as methods improved. The highest LOQs in both analyses occurred in the week 4 analysis and were 23.2 ng/mL (sera) and 27.8 ng/g (liver). Blanks: All blanks were below the lower limit of quantitation for PFOS. To simplify analyses that were complicated by endogenous levels of fluorochemicals in unexposed rat sera and liver, rabbit sera and liver were selected as a suitable surrogate matrix for blanks, curves, and CCVs. Precision: Not determined for this study. 3M Environmental Laboratory Page 15 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Matrix Spike Recoveries: Rat sera from 18 control animals were spiked prior to extraction. PFOS was spiked at approximately 75 ng/g, 100 ng/g, and 250 ng/g levels. With two exceptions, all Matrix Spike/Matrix Spike Duplicate (MS/MSD) were recovered at 30% of expected values. One MS, analyzed with the week 4 samples had a recovery of 167%; one MS analyzed with the week 53 samples was recovered at 131%. These high recoveries were confirmed. Because the remaining 16 spikes were acceptable, the high recoveries do not indicate serious analytical deficiencies. Rat liver from 20 control animals were spiked prior to extraction. PFOS was spiked at approximately 100 ng/g and 250 ng/g levels. Thirteen of the 20 spikes were within 50% of the expected value; seven were outside of this range. Four of the deviant spikes, analyzed with the day 719 samples, were mistakenly prepared from G3 and G6 animals instead of from control animals (G1). The high levels of PFOS in the tissue of these test animals obscured the relatively low level of material spiked (spike was <3% of endogenous). This mistake in preparation invalidates these results as indicators of method QC. Two of the remaining deviant spikes were analyzed with the week 4 samples and one deviant MS was analyzed with the week 53 samples. Because 13 of 16 matrix spikes that were prepared correctly were acceptable, the deviant recoveries do not indicate serious analytical deficiencies. All matrix spikes were prepared at levels approximate to G1 samples. Surrogates: The surrogate (THPFOS) was added to all specimens and standards before extraction, as stated in the methods. THPFOS was spiked into samples at concentrations within the linear range when samples were analyzed without dilution. Prior to May 5, 2000, THPFOS was not used for quantitation, but was used to monitor for gross instrument failure (see Appendix C, Analytical Methods). In analyses performed after May 5, 2000, the surrogate response of each analytical run was verified to determine that it did not vary more than 50% from the mean within each analytical run. At no time was THPFOS used for quantitation as an internal standard. No problems were observed with these data. Statem ent of Data Quality It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. Summary of Specimen Results Specimens from Control Animals: Low levels of PFOS were often detected in the sera and liver of the control animals. Specimens from Dosed Animals: Detailed specimen data tables are presented in Appendices D and E. Statistical Methods and Calculations Statistical methods were limited to the calculation of means and standard deviations. All specimen results were used to calculate the mean values; the LOQ value was used for specimens with <LOQ results. See Appendix F for example calculations used to generate the liver and serum specimen data in FACT TOX-002. 3M Environmental Laboratory Page 16 3M Medical Department Study: T-6295.4 Statement of Conclusion Analytical Report: FACT TOX-002 LRN-U2121 Under the conditions of the present studies, PFOS was observed in the sera and liver of rats dosed with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) during the in-life phase of the study. Reference Covance Study No.: 6329-183, 104-Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Rats 3M Environmental Laboratory Page 17 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Appendix A: Identification of Test Article and Control Matrices Table 4. Characterization of the Control Matrices Used for Sera Analyses in Study FACT TOX-002 Control Matrix Rabbit Serum Rabbit Serum Rat Serum Rat Serum Source Expiration Date Storage Conditions Lot # Physical Description S ig m a 2010 Frozen 47H4641 Rabbit Serum S ig m a 2010 Frozen 118H8418 Rabbit Serum S ig m a 2010 Frozen 17H9306 Rat Serum S ig m a 2010 Frozen 19H89292 Rat Serum Table 5. Characterization of the Control Matrices Used for Liver Analyses in Study FACT TOX-002 Control Matrix Rabbit Liver Rabbit Liver Rabbit Liver Rabbit Liver Source Expiration Date Storage Conditions Lot # Physical Description N/R-- not recorded CHW 2010 Frozen F00014 Rabbit Liver CHW 2010 Frozen F00016 Rabbit Liver N/R 2010 Frozen N/R Rabbit Liver CHW 2010 Frozen F00007 Rabbit Liver Table 6. Identification of the Test Article in Study FACT TOX-002 Chemical Name Source Test Article KPFOS Perfluorooctane sulfonic acid potassium salt 3M ICP/PCP Division Expiration Date Storage Conditions Chemical Lot # 08/31/01 Ambient temperature 217 Physical Description W hite crystalline powder Purity 86.9% 3M Environmental Laboratory Page 18 3M Medical Department Study: T-6295.4 Appendix B: Protocol, Amendments, Deviations Analytical Report: FACT TOX-002 LRN-U2121 3M Environmental Laboratory Page 19 3M Medical Department Study: T-6295.4 .7 Analytical Report: FACT TOX-002 LRN-U2121 3M Environmental Laboratory Protocol - Analytical Study Phase 104-Week Dietary Chronic Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats In-Vivo Study Reference Number: Covance # 6329-183 Study Number: A M D T -112296.1 Test M aterial: T-6295 Name and Address of Sponsor: 3M Toxicology Services Building 220-2E-02,3M Center St. Paul, MN 55144-1000 Name and Address of Testing Facility: 3M Environmental Technology and Services 935 Bush Avenue St. Paul, MN 55106 Proposed Experimental Start Date: May 25,1998 Proposed Experimental Termination Date: December 31,2001 Method Numbers and Revisions FACT-M-1.0, Extraction of Perfluorooctanesulfonate from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry FACT-M-2.0, Analysis of Liver Extracts for Fluorochemicals using HPLC- Electrospray/Mass Spectrometry F A C T -M -3.0, Extraction o f Perfluorooctanesulfonate from Sera for A nalysis using HPLC-Electrospray/Mass Spectrometry FACT-M-4.0, Analysis of Sera Extracts for Fluorochemicals using HPLC- Electrospray/Mass Spectrometry Author: Lisa Clemen Kris J. Hansen, PhD Study Director PY/L- i / f i A i Date Dale Baccon Date Study Director Management Andrew M. Seacat, PhD Sponsor Representative 5 /2 $ , Date Page 1 of 5 3M Environmental Laboratory Page 20 3M Medical Department Study: T-6295.4 iAnajyticaleportiACT OX-002 LRN-U2121 1.0 PURPOSE___________________________________________________________________ 1.1 According to this analytical protocol, the 3M Environmental Laboratory will analyze the tissue and fluid samples from the Covance study number 6329-183, "104-Week Dietary Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats." The collected data will be provided to the sponsor for use in the assessment of toxicological effects of the test material when administered in the diets of rats for at least 104 weeks. 1.2 Data collected in the Environmental Laboratory will be considered non-quantitative screening data until future studies have been conducted to determine absolute recoveries o f specific or general fluorochemical compounds. 2.0 REGULATORY COMPLIANCE_______________________________________________ 2.1 This analytical phase of the study will be conducted in accordance with the FDA Good Laboratory Practices Regulations 21 CFR 58, with the following exceptions: 2.1.1 The analytical phase is being conducted as a separate study and therefore has a separate Study Director, protocol, and final report, from those listed in the Covance protocol 6329-183. 2.1.2 The characterization of the reference material, including purity, identity, and stability, are the responsibility of the sponsor. 2.1.3 Sample storage stability will not be determined. 3.0 TEST MATERIALS_________________________________________________________ 3.1 Control, and reference Materials and Matrices 3.1.1 Analytical Reference Material: T-6295, from 3MICP/PCP Division 3.1.2 Analytical Reference Matrix: Rat liver, from Covance and rat serum, from Sigma Chemical Company. 3.1.3 Analytical Control Material: None. 3.1.4 A nalytical Control M atrix: R at liver, from C ovance and rat serum , fro m S igm a Chemical Company. 3.2 Number of Test and Control Samples: Throughout the course of the study, liver and serum from 580 test animals and 140 control animals will be made available. Samples will be analyzed as requested by the sponsor or the study director. Other biological tissues (kidney, bile, dermal application site, and cellular fraction) will be available for analysis if deemed appropriate. 3.3 Identification of Test and Control Samples: The samples will be identified using the Covance animal identification number that consists of a letter and five-digit number, plus the tissue identity and day identity (serum). 3.4 Purity and Identity of Reference Material: To be determined by Sponsor. 3.5 Stability of Reference Material: To be determined by Sponsor. 3M Environmental Laboratory Page 2 of 5 Page 21 3M M edical Departm ent Study: T-6295.4 Analytical Report: FACTTOX-QQ2 LRN-U2121 3.6 Storage Conditions for Reference Materials: Reference materials will be stored at room temperature (3.1.1), and samples will be stored at -20 10C (3.1.2, 3.1.4). Test and Control samples will be received according to AMDT-S-10-0. 3.7 Disposition of Specimens: Biological tissues and fluids will be retained per GLP Regulation for the time period required for studies longer than 28 days. 3.8 Safety Precautions: Refer to appropriate MSDS. Wear appropriate laboratory attire. Use caution when handling knives for cutting tissue samples. 4.0 EXPERIMENTAL - Overview_________________________________________________ 4.1 The tissues from animals dosed as described (Covance# 6329-183), will be available for analysis for fluorine-containing compounds. At the discretion of the Study Director, a series of analytical tests can be performed. All high dose and control sera and livers will be analyzed initially using HPLC-electrospray mass spectrometry to identify fluorine-containing compounds of interest present in the sera and liver (if any). The screening for organic fluoride in liver via combustion may be performed to present definitive data for fluorine in the liver. Based on the findings from these analyses, additional samples, tissues, or fluids may be analyzed at the discretion of the Study Director to determine the presence of fluorochemicals in these matrices. 5.0 EXPERIMENTAL - Methods__________________________________________________ 5.1 Methods (attached): 5.1.1 FACT-M-1.0, "Extraction of Perfluorooctanesulfonate from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry" 5.1.2 FACT-M-2.0, "Analysis of Liver Extracts for Fluorochemicals using HPLCElectrospray/Mass Spectrometry" 5.1.3 FACT-M-3.0, "Extraction of Perfluorooctanesulfonate from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry" 5.1.4 FACT-M-4.0, "Analysis of Serum Extracts for Fluorochemicals using HPLCElectrospray/Mass Spectrometry" 6 .0 D A T A A N A L Y S I S __________________________________________________________________________ 6.1 Quality Control: Matrix spikes will be extracted and analyzed to determine accuracy of the method. Also, continuing calibration checks will be analyzed to determine response bias. 6.2 Transformations: Any transformations performed on data collected during the analytical phase of the study will be documented in the final report. 6.3 Statistics: At the discretion of the Study Director, statistics used may include regression analysis of serum concentrations with time, averages, and standard deviations of concentrations for the different dose groups. If necessary, simple tests such as the Student's t-test may be applied to determine statistical difference. Any statistical analysis performed will be documented in the final report. 6.4 Data Reporting: A final data package will be submitted to 3M Toxicology Services. The data package will include the following with additional data included as deemed appropriate. 3M Environmental Laboratory Page 3 of 5 Page 22 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 6.4.1 A summary of individual sample results, reported as a concentration (weight/weight, weight/volume) of fluoride per tissue or fluid, or as the mass of a specific fluorochemical (HPLC-electrospray mass spectrometry) per unit of tissue or fluid. 6.4.2 A summary of quality control results (continuing calibration checks, method blanks, instrument blanks, matrix spikes, and matrix spike duplicates). 6.4.3 Certified copies or originals of the written validated methods. 6.4.4 Certified copies or originals of sample identification sheets sent from Covance. 6.4.5 Certified copies or originals of study specific raw data. 6.4.6 A summary of key personnel involved with the analytical phase of the study. 6.4.7 A signed QAU statement listing the dates of inspections and reports of findings to management and Study Director. 7.0 MAINTENANCE OF RAW DATA AND RECORDS ____________________________ 7.1 The following raw data and records (or certified copies thereof) will be maintained in the study folder in the archives according to appropriate SOPs. 7.1.1 7.1.2 7.1.3 7.1.4 7.1.5 7.1.6 7.1.7 Approved protocol Approved methods Data summaries Study correspondence Shipping records Raw data Electronic copies of data 7.2 Supporting records to be retained separately from the study folder in the archives according to 3M ET & SS SOPs, will include, but not necessarily be limited to the following: 7.2.1 7.2.2 7.2.3 7.2.4 7.2.5 7.2.6 Approved validation reports T raining records Calibration records Instrument maintenance logs Standard operating procedures, equipment procedures, and methods Appropriate specimens 8.0 REFERENCES________________________ ______________________________________ 8.1 Approved AMDT standard operating procedures. 8.2 Approved ETS standard operating procedures. 9.0 ATTACHMENTS____________________________________________________________ 9.1 FACT-M-1.0, Extraction of Perfluorooctanesulfonate from Liver for Analysis using HPLCElectrospray/Mass Spectrometry Page 4 of 5 3M Environmental Laboratory Page 23 3M Medical Department Study: T-6295.4 Analytical Report: FACT_ J X-Q.2 LRN-U2121 9.2 FACT-M-2.0, Analysis of Liver Extracts for Fluorochemicals using HPLCElectrospray/Mass Spectrometry 9.3 FACT-M-3.0, Extraction of Perfluorooctanesulfonate from Serum for Analysis using HPLCElectrospray/Mass Spectrometry 9.4 FACT-M-4.0, Analysis of Serum Extracts for Fluorochemicals using HPLCElectrospray/Mass Spectrometry 3M Environmental Laboratory Page 5 of 5 Page 24 3M Medical Department Study: T-6295.4 3 \I Environmental Technology and Services PO Box 33331 St. Paul, MN 55133-3331 612 778 6442 3M Analytical Report: FACT TOX-002 Study Title 104-Week Dietary Chronic Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats PROTOCOL AMENDMENT NO. 1 Amendment Date: December 13,1999 Performing Laboratory 3M E n v iro n m e n ta l T e c h n o lo g y & S a fe ty S e rv ic e s 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX-002 LIRNU2121 3M Environmental Laboratory 3M Environmental Laboratory Page 25 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 BRW g BT Protocol FACT Tox-002 Amendment Number 1 This amendment modifies the following portion(s) of the protocol: 1. Protocol Reads: The following methods will be used: FACT-M-1.0, Extraction of Perfluorooctanesulfonate from Liver for Analysis usingHPLCElectrospray/Mass Spectrometry FACT-M-2.0, Analysis of Liver Extracts for Fluorochemicals using HPLCElectrospray/Mass Spectrometry FACT-M-3.0, Extraction of Perfluorooctanesulfonate from Sera for Analysis using HPLCElectrospray/Mass Spectrometry FACT-M-4.0, Analysis of Sera Extracts for Fluorochemicals using HPLC-Electrospray/Mass Spectrometry Amend to Read: The following methods will be used: ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry ETS-8-6.0, Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry ETS-8-7.0, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry Reason: The methods originally listed were superseded during the course of the study. Andrew M. Seacat, Ph.D., Sponsor Representative K >i - fk Kristen J. Hansen, Ph.D., Study Director 3M Environmental Laboratory 3M Environmental Laboratory ( 3 / 9 ? Date n -fz o H i Date Page 26 3M Medical Department Study: T-6295.4 AnalyticalReport:FA2iTOX202 LRN-U2121 Study Title 104-Week Dietary Chronic Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats PROTOCOL AMENDMENT NO. 2 Amendment Date: 20 January 2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS LRN-U2121 FACT TOX-002 Covance Study: 6329-183 3M Medical Department Study: T-6295.4 3M Environmental Laboratory 3M Environmental Laboratory Page 27 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Protocol LRN-U2121 Amendment Number 2 This amendment modifies the following portion(s) of the protocol: 1. Protocol Reads: There is no independent section of the protocol that addresses sample retention. Amend to Read: Specimens will be maintained in the 3M Environmental Laboratory specimen archives. Any specimens sent to sub-contract laboratories will be returned to the 3M Environmental Laboratory upon completion of analysis and submission of the sub contract laboratory(s) final report. Specimens analyzed at sub-contract laboratories will be returned with the following documentation: the signed original chain of custody and records of storage conditions while at the sub-contract facility. Reason: To define in detail the appropriate disposition of specimens analyzed at subcontract laboratories. 2. Protocol Reads: Section 7 states that the following raw data and records will be retained in the study folder in the archives according to AMDT-S-8: Approved protocol and amendments; approved methods; data summaries; study correspondence; shipping records; raw data; and electronic copies of data. Additionally, Section 7 states that supporting records to be retained separately from the study folder in the archives according to AMDT-S-8 will include at least the following: Approved validation reports; training records; calibration records; instrument maintenance logs; Standard Operating Procedures, Equipment Procedures, and Methods; and appropriate specimens. Amend to Read: Section 7 states: "The original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance of the final report. When the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, and electronic copies of data will be retained in the archives of the 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained in the archives of the facility performing each analysis." Reason: To direct subcontract laboratories in the disposition of the items listed above. 3M Environmental Laboratory 3M Environmental Laboratory Page 28 3M Medical Department Study: T-6295.4 A n a y ic il e p o rtiF C T O X -002 LRN-U2121 Protocol LRN-U2121 Amendment Number 2 3. Protocol reads: The study director for the present study was identified in the protocol as Kristen J. Hansen, Ph.D. Amend to read: The role of study director for the present study was reassigned to John L. Butenhoff, Ph.D., as of 20 January 2000. The previous study director, Kristen 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). 4. Protocol reads: The sponsor for the present study was identified as Andrew M. Seacat, Ph.D. Amend to read: The role of sponsor for the present study was reassigned to Marvin T. Case, D.V.M., Ph.D., as of 20 January 2000. Reason: The change was made at the request of the sponsor. 3M Environmental Laboratory 3M Environmental Laboratory Page 29 3M Medical Department Study: T-6295.4 Amendment Approval Analytical Report: FACT TOX-002 LRN-U2121 Protocol LRN-U2121 Amendment Number 2 /e /. An, Sponsor Representative Date K risten J. Hansen, P h D ., Outgoing Study Director // --F& b '-20O Date M arvin T. Case, D.V.M ., P h D ., Incoming Sponsor Representative John L. Butenhoff, P h D ., Incoming Study Director Date fO Date 3M Environmental Laboratory 3M Environmental Laboratory Page 30 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LR r a f f l Study Title 104-Week Dietary Chronic Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats PROTOCOL AMENDMENT NO. 3 Amendment Date: November 21,2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M E nvironm ental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS LRN-U2121 FACT TOX-002 Covance 6329-183 3M Medical Department Study: T-6295 3M Environmental Laboratory 3M Environmental Laboratory Page 31 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 Protocol FACT TOX-002 Amendment No. 3 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: 1.2 Data Collected in the Environmental Laboratory will be considered non-quantitative screening data until future studies have been conducted to determine absolute recoveries of specific or general fluorochemical compounds. Amend to read: 1.2 If matrix spike studies provide accurate representation of recovery of endogenous levels of PFOS, the 3M Environmental Laboratory will provide semi-quantitative data for sera and liver samples collected from test animals. Reason: Due to improved analytical methods, the decision was made to change the purpose of the study results from non-quantitative to semi-quantitative. 3M Environmental Laboratory 3M Environmental Laboratory Page 32 3M Medical Department Study: T-6295.4 Amendment Approval Analytical Report: FACT TOX-002 LRN-U2121 Protocol FACT TOX-002 Amendment No. 3 M arvin T. Case, D.V.M ., Ph.D., Sponsor Representative 2-1 Date %-+-<* John L. Butenhoff, Ph.D., Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 33 3M Medical Department Study: T-6295.4 Record of Deviation Analytical Report: FACT TOX-02 LRN-U2121 Study / Project No. TOXOQ2 (LIMS #U2121) Deviation type (Check one) Document number ETS-8-7.0 I. Identification SOP X Method Equipment Procedure Protocol Other: Date(s) o f occurrence 7/6/98, 5/25/00, 5/26/00, 6/12/00, 6/14/00, 6/13/00,5/19/00, 5/22/00 II. Description Required procedure/process: Section 10.1.2: Analyze a method blank and a matrix blank prior to each calibration curve. Actual procedure/process: On several occasions, the blanks were either a) analyzed after the calibration curves or b) not analyzed at all for a particular MS run III. Actions Taken (such as amendment issued, SOP revision, etc.) Deviation written. Recorded by fa IV. Impact on Study/ Project (completed by Study Director or Project Lead) Date ii m itro All blanks were analyzed at least once during the course of the study. When blanks were run after the calibration curve, all control-of-bias practices were followed. In the instances where blanks were not run at all, the samples being analyzed were well above the typical run LOQ. In addition, the vast majority of samples run without blanks were high level dilutions. The function of the blank is to determine if contamination was introduced during extraction; any low-level contamination would be inconsequential to extracts requiring dilution. This deviation has no adverse affect on the study data. b ...Il I H I t ^thorized byy ip o n s c r ' M ri/ Case- ^ Y ^ ^ Pate D irtc b rT o L n ultnltofC D e V 3 tO n N O . (assigned by Study Director or Project Lead at the end of study or project) Attachment A: Record of Deviation 3M Environmental Laboratory ETS-4-8.0 p.E-Hh HII4/00 -Page'4-of 4 Page 34 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 Record of Deviation I. Identification Study / Project No. f C T - T O *- 0 0 2 - Deviation Type (Check one) SOP 'Method S Equipment Procedure Protocol Other: Document Number Date(s) of occurrence C t l k d t o i l'CT-H- 2-g t M d u u J B T S-f- ?-0 77yW/*?y errici Ai d o 'z /0 'z / do yeuuosk- II. Description: So^fJU JU%\ Required Procedure/process: jLf/Aoh) s h-tsJt-d kt cH /mJ'/.A iK-b IT* /jj&fxCtA 'tUi. tXnd&K w -fu, 1*3 U F > A x d __CJLSJs__ l^W A j j ^ d g j t * V ..(/v b > h . .../ V . ______________________ __________________________________________________________________________ ___ Actual Procedure/process: O-g-SP'U __I^ g > _ #Scptm. GS/ wY-H hvev x/gjMJta \xha+_ liotvi W ky s \q i* U iu A __fWrvc ih J s -UJUDf w a s K a k tJtA , 1vc s a r e t e W hrA ..ck a.C'/sjd-fl.h/s, %a MS//M$ , .i .J X t/'i rim. /o fy lA2-n&ltQ Im0ts.J?rt1 A,,Jl&ydAu^iCM^vxrK^d^p^ Sctswi^aCf_______________ III. Actions Taken: (such as amendment issued, SOP revision, etc.) H ^ l u LIs \jsi JLi lag. (ver{jtcL l ( h Vg * fw. t~L* LVv tu^ot/v^p IV. Impact on Study/ Project ( q S f yJV- 4_I>Wy MaJMjto vJ\jUi hi Oi.'KSrtU.Md i o he ( vduVC. hj W [O/Q^ /tm Authorized By (Study D irector / P roject Lead) 10/ 0 3 / 0 0 Date ^pOMSor Rep - M&n/tn Gdy*L ludu Oirtcliof ' 3hn RultrToi'P 3M Environmental Laboratory Deviation No. (S. Form ETS-4-8.0 (assigned by Study Director or Project Lead at the end of study or project) 3M Environmental Laboratory Page 35 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 Record of Deviation 1. Identification Study / Project No. F A C T -T d x -O O Z Deviation Type SOP (Check one) Protocol CxxvEincf, L l 2 H ~ l 2 3 Method Equipment Procedure Other: Document Number F a c t - iyi- h , o ! Date(s) o f occurrence I II. Description: O lllsfo s (W k4 u**) Required Procedure/process: -- .vm.i..!__ .v..i JX,-P-ttr SeA.vl&erui--L11oft.--...._/n_p_ifc_,___W_l_L_Iv_. _______ _____________p_e_r____b___l_cL L d,-- .Con M u i/u j-- A a ltLaLiOo__Urii>' <i.-hbrv__ per.CAoi.__teC o i/rieJ:_.toujJL_..\>& .....L irntn JSf'" 3>7._0jL J ftc_Spiked.. ConcanWl ibo Actual Procedure/process: .._____ ^LldkiL_LPJuife^_CCilil?rc3'Cfe_Uric3a.ai_Lofera...l otlkK_V 3dv!..tttevt0. Ike^ rec-o^r'iP^i re.Aty>-f\ -Lrom ~3? Ln~38/______ ___ _________________ _______ Tive^dk..usua torilkn,_______ III. Actions Taken: (such as amendment issued, SOP revision, etc.) ....B*ceJ.Spreadskvd._Qad_dlitf--dviciliQo .uxw Recorded By Date D&Ja. IV. Impact on Study/ Project txv-d to r um - .tyiL..(UIJJOlO. M. Authorized By (Study D irecto r / P ro ject Lead) Date 'ifV tv'h + T 'i________26 x/lT /A&0-C Spooior f*tcif^ Cax- Direx-W I Totv\ Buk/tli?? 3M Environm ental Laboratory ^ Deviation No. 3 Form ETS-4-8.0 (assigned by Study Director or Project Lead at the end of study or project) 3M Environmental Laboratory Page 36 3M Medical Department Study: T-6295.4 AnalyticalReport: FACT TOX-002 LRN-U2121 Record of Deviation I. Identification ., Study / Project No. Covance Study Number: 6329-^03/ 3M Project Number Tox002 Deviation Type (Check one) SOP V Method Equipment Procedure Protocol Other: Document Number: FACT-M-4.0 i Date(s) of occurrence: 07/23/98 II. Description: Required Procedure/process: Method FACT-M.4.0, states 1 0 .3 .1 Analyze a mid-range calibration standard after every tenth sample. If a significant change ( 30%) in peak area occurs, relative to the initial standard curve, stop the run. Only those samples analyzed before the last acceptable calibration standard will be used. The remaining samples must be reanalyzed. Calibration checks in this data packet (collected 071598b, on amelia) are not with in the 30% criteria. Actual Procedure/process: Calibration checks within 40% criteria will be accepted for this data set. The 97 ppb standard points at the beginning and end of the run are excluded from the calibration curve due to the analyst adding the wrong amount o f standard. This error... appears in both curves and is therefore verified. Deletion of the 97 ppb curve point caused the calibration curve to have a greater r2value and an overall better fit to the points............. III. Actions Taken: .................. (such a s am endm ent issued, S O P revision, etc.) ................................ This method deviation, increasing the criteria range for this data set is the only action that will need to be taken................................................... This new data set will be used for final results. Recorded By: Harold Johnson c\ . S V fo i Date: I September 5, 2000 IV. Impact on Study/ Project .. ^ ...... Vo .. >.n .. .... y p i o f/ /o o Authorized By (Study D irecto r / P ro ject L ead) ^ i Date S'lud'j PhcW '1-Tohr 3M E nvironm ental L aboratory Form E T S-4-8.0 o r/o s / 'Sjfcxwcsr Ip flrv CW- Deviation No. (assigned by Study Director or Project Lead at the end of study or project) 3M Environmental Laboratory Page 37 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Record of Deviation /. Identification Study / Project No. Deviation Type (Check one) FACT- T x - 2_____Cooa/t L ___339- 183 SOP Method Equipment Procedure Protocol 1^ Other: Document Number Date(s) of occurrence 2\ CPU 5S.2 Led Cn) II. Description: Required Procedure/process: -SpDjfijr.-- CijtjiLMl oiLanl^ilCI pmtacoJ pim ~lt> j5 W v, JhrttJcpr__ CippiQiJi. ........................................................................................ Actual Procedure/process: Tvl... jud.^ __ P'ftcli!r...,,iiAnd:LG.j?p-Dveii__ ._nl.^n'Cl__pmii .x_R.prfrje ____________________________ III. Actions Taken: (such as am endm ent issued, SO P revision, etc.) 0.lo J k J u lu ri:* __QAcij^kaal_jiroinfeo-b__Uiill__bc.-ii nedi__ ...Rfepre-seolaiiVt-_ktfere* tivi. 31-u d ^ Dirf-ci ^r.____________________ Recorded By Date f' h A P IV. Impact on Study/ Project Uhi*...Ae.viEon__Lull__hei__CtduCri .. _______ ilva-_OjukotM__ .Q..IP... Authorized By (Study D irecto r y P ro ject Lead) 7 ^ ------- Date 3M E nvironm ental L aboratory Form E TS-4-8.0 Deviation No. (assigned by Study Director or Project Lead at the end of study or project) 3M Environmental Laboratory Page 38 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 L R T O ffH Record of Deviation I. Identification Study / Project No. f ^ l z J - 0 0 2 ________ C o v a n e L 3 2 9 - I S3 Deviation Type SOP Method Equipment Procedure (Check one) Protocol Other: Document Number P r to t FACT- Tt>y- OCX Date(s) of occurrence II. Description: Required Procedure/process: Piv^col -s-iciks medh&dl FACT- tn- 3.D u ;il W tu td . Actual Procedure/process: J ld io J .._.....................i4cu,,.lu eif. III. Actions Taken: (such as amendment issued, SOP revision, etcJ J l is__Dev/c-kon t/oj JL rdkn /-flt.d .e u t Recorded By /I'M_A CL/thM. IV. Impact on Study/ Project ,,M jE h j JA Jm pn xn M tdj o f/n fx e u tlsC su rf- L *.. Date la /o ih t ..... Authorized By (Study D irecto r / P ro ject Lead) 3M Environm ental Laboratory Form ETS-4-8.0 Deviation No. _k. (assigned by Study Director or Project Lead at the end of study or project) 3M Environmental Laboratory Page 39 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2WI Record of Deviation I. Identification Study / Project No. FACX-TM-0&3L Deviation Type (Check one) SOP Cova tici^__b>2>'Xci-lS 3 Method Equipment Procedure 0 Protocol Other: Document Number Pn>Wi f A C T ' T b x . n o 2. Date(s) of occurrence ulnhi II. Description: Required Procedure/process: Protocol Tale* rhL&od F A C T -ft- L b U ilL hi Um L Actual Procedure/process: MeiW __JS_z2_r_u.D_uo__iu e4 III. Actions Taken: (such as amendment issued, SO P revision, etc.) Thu V nj-Lo l & IU out. Recorded By A [AtfhK IV. Impact on Study/ Project Date u /w M Authorized By (Study D irecto r / P roject Lead) j Date tfh I 3M E nvironm ental Laboratory Form E TS-4-8.0 Deviation No. T (assigned by Study Director or Project Lead at the end of study or project) 3M Environmental Laboratory Page 40 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 Ml1 LRN-U2121 Record of Deviation I. Identification Study / Project No. J D X -Q 5- Deviation Type (Check one) SOP 0 Method Equipment Procedure Protocol Other: Document Number ST'S - o l- - o Date(s) of occurrence a/oo - slop II. Description: Required Procedure/process: /<y.4- / tfMSri-X Spi'k-t et (usncts*-AJZsfl urn ku. */- 3% Actual Procedure/process: USCAX. u p ^ t n __V - s v 7i Yx ^t- __ ___________________________________________________________ 3CVl/Zj6 III. Actions Taken: (such as am endment issued, SO P revision, etc.) US^ f Le. ______________ /jn Recorded By ^ to /o f/o o Date Vaa*f IV. Impact on Study/ Project ' rlv' PF&S ..aso ___________ ___ .(I'JX. UL Zke^<^-/A. kjh IO/0S/OO Authorized By (Study D ire c to r / P ro ject Lead) Date 9tL * T < ^ ^"poiuor Rep - Mo<V 3M E nvironm ental L aboratory Form E T S-4-8.0 J z z fc * # " f i/ * ^"Vuilw DirtcToe - Tokr\ GulttlkA-f Deviation No. A (assigned by Study Director or Project Lead at the end of study or project) 3M Environmental Laboratory Page 41 3M Medical Department Study: T-6295.4 Appendix C: Extraction and Analytical Methods Analytical Report: FACT TOX-002 LRN-U2121 This appendix includes the following methods: FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry (8 pages) FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry (8 pages) ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages) ETS-8-6.0, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages) FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry (8 pages) FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry (8 pages) ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry, (9 pages) ETS-8-7.0, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry, (10 pages) 3M Environmental Laboratory Page 42 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 3M Environmental Laboratory M ethod Extraction of P otassium perfluorooctanesulfonate or Other A nionic Fluorochem ical Surfactants from Liver for Analysis U sing H P L C -E lectrospray/M ass Spectrom etry M ethod Num ber: FACT-M-1.0 Author: Lisa Clemen Approved By: s Laboratory Manager lbl i W L v -------- -- Group Leader nlfr A- C W u . Technical Reviewer Adoption Date: s / j l t / f Revision Date: (jA 5 '/z c /? P Date 5 / z u /4 f Date sh?hf Date 1.0 S c o p e a n d A p p l ic a t io n 1.1 Scope: This method is for the extraction of Potassium Perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from liver. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, and monkey livers or other livers as designated in the validation report. Microsoft 7.0.1/95 FACT-M-1.0 Extraction of PFOS from Liver Page 1 of 8 3M Environmental Laboratory Page 43 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 2.0 S u m m a r y o f M e th o d __________________ ;__________ ____________________________________ 2.1 This method describes how to extract potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from liver using ion pairing reagent and 5.0 mLs o f ethyl acetate. An ion pairing reagent is added to each sample and partitioned into ethyl acetate. Four mLs of extract is removed to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL methanol then filtered through a 3 cc plastic syringe attached to a 0.2 pm filter into glass autovials. 3.0 D e fin it io n s_____________________________ ;________________ ___________________________ 3.1 None. 4.0 W a r n in g s a n d C a u t io n s____________ ____________________________________________ . 4.1 Health and Safety Warnings: 4.1.1 Use universal precautions when handling animal livers, they may contain pathogens. 5.0 In t e r f e r e n c e s____________________________ .____________________________________________ 5.1 There are no known interferences at this time. E q u ip m e n t ________________________________________________________________________ ~ 6.1 The following equipment is used while carrying out this method. Equivalent equipment is acceptable. 6.1.1 Ultra-Turrax T25 Grinder for grinding liver samples 6.1.2 Vortex mixer, VWR, Vortex Genie 2 6.1.3 Centrifuge, Mistral 1000 or IEC 6.1.4 Shaker, Eberbach or VWR 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance 7.0 S u p p l ie s a n d M a t e r ia l s _________ ______________________________________________ 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable o f holding 250 mL and 1 L 7.5 Glass, type A, volumetric flasks 7.6 40 mL glass I-CHEM vials 7.7 Plastic sampule vials, Wheaton, 6 mL 7.8 Polypropylene centrifuge tubes, 15 mL 7.9 Labels FACT-M-1.0 Extraction of PFOS from Liver Page 2 of 8 3M Environmental Laboratory Page 44 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 7.10 Syringes, capable of measuring 10 pL to 50 pL 7.11 Glass, type A, volumetric pipettes 7.12 Graduated pipettes 7.13 Electronic pipettor, Eppendorf or equivalent 7.14 Timer 7.15 Disposable plastic 3 cc syringes 7.16 Filters, nylon syringe filters, 0.2 pm, 25 mm 7.17 Crimp cap autovials Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli- QTM water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 R e a g e n t s a n d St a n d a r d s________________ ;______________;_____________________________ 8.1 Reagents 8.1.1 Sodium Hydroxide (J.T Baker or equivalent), (NaOH) 10N: weigh approximately 200 grams NaOH. Pour into a 1000 mL beaker containing 500 liters (L) Milli-QTM water, mix until all solids are dissolved. Store in a 1 L nalgene bottle. 8.1.2 Sodium Hydroxide (J.T Baker or equivalent), (NaOH) IN. Dilute 10N 1:10. Measure 10 mL of the 10N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL nalgene bottle. 8.1.3 Tetrabutylammonium hydrogen sulfate (Kodak or equivalent), (TBA) 0.5M: Weigh approximately 169 grams of TBA into a 1 L volumetric containing 500 L Milli-QTM water. Adjust to pH 10 using approximately 64 mL 10N NaOH and dilute to volume with Milli-QTM water. Add NaOH slowly while adding the last 1 mL of NaOH because the pH changes abruptly. Store in a 1 L nalgene bottle. 8.1.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using IN NaOH solution. 8.1.4 Sodium carboriate/Sodium Bicarbonate Buffer (J.T. Baker or equivalent), (Na2C 0 3/NaHC03) 0.25M: Weigh approximately 26.5 g of sodium carbonate (Na^CO^ and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and dilute to volume with Milli-QTM water. Store in a 1 L nalgene bottle. 8.1.5 PFOS (3M Specialty Chemical Division), molecular weight = 538. 8.1.6 Ethyl Acetate, OmnisolV, glass distilled or HPLC grade. 8.1.7 Methanol, Omnisolv, glass distilled or HPLC grade. 8.1.8 Liver and control liver, received frozen from testing laboratory. 8.1.9 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC Plus system. 8.2 Standards 8.2.1 Prepare PFOS standards for the standard curve. FACT-M-1.0 Extraction of PFOS from Liver Page 3 of 8 3M Environmental Laboratory Page 45 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 8.2.2 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.2.3 Bring to volume with methanol for a stock standard of approximately 1000 ppm (pg/mL). 8.2.4 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.2.5 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.2.6 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 9.0 S a m p l e H a n d l in g _____________ :____________________________________ ;__________________ 9.1 All livers are received frozen and must be kept frozen until the extraction is performed. 10.0 Q u a l it y C o n t r o l 10.1 M atrix Spikes ____________________________ .______________ ;_________________ 10.1.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.1.2 Prepare each spike using liver chosen by the analyst, usually a control liver. 10.1.3 Expected concentrations will fall in the mid-range of the initial calibration curve. 10.2 Continuing Calibration Checks 10.2.1 Prepare and analyze continuing calibration check samples to determine the continued linearity of the initial calibration curve. 10.2.2 One check is prepared per group of ten samples. For example, if a sample set = 34, four checks are prepared and extracted. 10.2.3 Prepare each continuing calibration check from the same liver homogenate used to prep the initial curve. 10.2.4 The expected concentration will fall within the mid-range of the initial calibration curve. 11.0 C a l ib r a t io n a n d S t a n d a r d iz a t io n _______________ _____________________________ 11.1 P rep are Liver Homogenate to Use for Standards 11.1.1 Weigh approximately 40 g of liver into a 250 mL Nalgene bottle containing 200 mLs Milli-QTM water. Grind to a homogeneous solution. 11.1.2 If 40 g is not available, use appropriate amounts of liver and water in keeping with a 1:5 ratio. 11.1.3 See section 13.0 to calculate the actual density of liver. FACT-M-1.0 Extraction of PFOS from Liver Page 4 of 8 3M Environmental Laboratory Page 46 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 11.1.4 Add 1 mL of homogeneous solution to a 15 mL centrifuge tube. Re-suspend homogeneous solution by shaking between aliquots while preparing a total of sixteen 1 mL aliquots of homogeneous solution in 15 mL centrifuge tubes. 11.1.5 Two 1 mL aliquots serve as matrix blanks. Use the standard concentrations and spiking amounts listed in table 1 to spike, in duplicate, two standard curves for a total of fourteen samples. Table 1 Approximate Spiking Amounts for Calibration Standards Working Standard (Approx. Cone.) - 0.50 ppm 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm 5.0 ppm 50 ppm pL Approx, final cone, of PFOS in liver - Blank 4 0.010 ppm 20 0.050 ppm 40 0.100 ppm 10 0.250 ppm 20 0.500 ppm 30 0.750 ppm 4 1.000 ppm 11.1.1 See section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 Extract spiked liver homogenates following 12.14-12.24 o f this method. Use these standards to establish each initial curve on the mass spectrometer. 12.0 P r o c e d u r e s _________________ _______________________ ____________________ ____________ 12.1 Obtain frozen liver samples. In spent tissue, note that the liver has not been packaged with other tissues. 12.2 Cut approximately 1 g o f liver using a dissecting scalpel. 12.3 Weigh the sample directly into a tared plastic sampule vial. 12.4 Record the liver weight in the study notebook. 12.5 Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 12.6 Add 2.5 mLs o f water to sampule vial. 12.7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the sample with 2.5 mLs water using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Follow AMDT-EP-22. 12.10 Cap the sample and vortex for 15 seconds. 3M Environmental Laboratory FACT-M-1.0 Extraction of PFOS from Liver Page 5 of 8 Page47 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 12.11 Pipette 1 mL homogenate into a 15 mL polypropylene centrifuge tube. Label the centrifuge tube with the identical information as the sampule vial. (See Worksheet for documenting the remaining steps.) 12.12 Spike liver homogenates with the appropriate amount of PFOS standard as described in section 11.1 or Table 1. 12.13 Pipette two 1 mL aliquots of Milli-QTM water to centrifuge tubes. These will serve as instrument blanks. 12.14 Add 1 mL 0.5 M TBA and 2 mL of the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.15 Using a volumetric pipette, add 5 mLs ethyl acetate. 12.16 Cap each sample and put on the shaker for 20 minutes. 12.17 Centrifuge for 20 to 25 minutes, until layers are well separated. Set power on the centrifuge to approximately 3500 rpm. 12.18 Remove 4 mLs of organic layer, using a 5 mL graduated glass pipette, to a clean 15 mL centrifuge tube. Label this fresh tube with the same information as in 12.5. 12.19 Put each sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 hours. 12.20 Add 1.0 mL o f methanol to each centrifuge tube using a graduated pipette. 12.21 Vortex mix for 30 seconds. 12.22 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial. 12.23 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyses) who performed the extraction. 12.24 Cap and hold for electrospray mass spectrometry analysis. 12.25 Complete the worksheet and tape to page of study notebook. 13.0 D a t a A n a l y s is a n d C a l c u l a t io n s____________________ __________ ;______________ _ 13.1 Calculations: 13.1.1 Calculate the density of liver (mg) in 1.0 mL homogenate using the following equation: g of Liver x Average weight of ten 1 mL aliquots (mg) (g of Liver + g of Water) 3M Environmental Laboratory FACT-M-1.0 Extraction of PFOS from Liver Page 6 of 8 Page-48 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 13.1.2 Calculate actual concentrations ofPFOS in calibration standards using the following equation: uL o f Standard x Concentration (ug /mL) = Final Concentration (pg/g or mg/kg) mg L iv e r'/1 mL homogenate of PFOS in Liver *Average weight of liver in solution as determined in 13.1.1, by weighing ten 1 mL homogenates of approximately 40 mg liver in 200 mL of Milli-Q water. 14.0 M e t h o d P e r fo r m a n c e ___________ __________________ ;______________________ ___ 14.1 The method detection limit is equal to half the lowest standard in the calibration curve. 15.0 P o l l u t io n P r e v e n t io n a n d W a st e M a n a g e m e n t _______ .___________________ ___ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in * high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R e c o r d s__________ __________________ ________________________________________ _________ 16.1 Complete the extraction worksheet and tape into the study notebook. 17.0 T a b l e s , D ia g r a m s, F l o w c h a r t s, a n d V a l id a t io n D a t a ______________________ 17.1 The validation report associated with this method is FACT-M-1.0 & 2.0-V -l. 18.0 R e fe r e n c e s_____________ ________ _________________________________ ____ ____ 18.1 AMDT-EP-22, "Routine Maintenance of Ultra-Turrax T-25" 19.0 A f f e c t e d D o c u m e n t s___________ ' - __________________________________ _________ 19.1 FACT-M-2, "Analysis of Liver Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 R e v is io n s _________ ___ ________________;____________ Revision Number. t. Reason For Revision Revision Date 3M Environmental Laboratory FACT-M-1.0 Extraction ofPFOS from Liver Page 7 of 8 3M Medical Department Study: T-6295.4 Extraction Worksheet for FACT-M-1 Analytical Report: FACT TOX-002 LRN-U2121 Study # _ Sam ple Num ber set# H70 Blank Liver Blank PFOS approx. 0.5 ppm actual ppm #W - PFOS approx. 5 ppm actual ppm #W - PFOS approx. 50 ppm actual ppm #W - O ate and In itia ls for Std. _ _ _ 1 - - r .- - '- -- -, .- .- .- -. - 1Study num her where the original woi-ksheet is located, __________________________ '----------------------- -- ------------ ---- Rlank Liver Homogenate: Std # Liver amount--------------------------g---------- ----------- liv e r Extraction Method : ... ...................................................Date & Imtmls-------- Vortex 15 sec. ------- -------- -- -------------------------------------------------- ----------------- ----------- Pinette 1 mL of Liver S o l u t i o n _______________________________________ ;_________________ _________ ______ _ Pipette 1 mL o ft0 .5 M TBA, pH 10. Std.# ................ . ... Pipette2 mL o f 0.25Na?COy0.25MNaHCO-? Buffer Std.# -- --------------- Pipette 5 mL o f Ethyl Acetate TN-A- ------------------------- -------- Shake 20 min. Centrifuse 20-25 min. _______:__________ -- --------------------------------------------------------------------- --- -- Centrifuge _________Speed___ _____________ ;---------------;---------------- ----------------- --------- Remove a 4 mL aliauot of organic layer .................................................... ;---------------------- -------------------------- Put on Nitrogen Evaporator to dryness Evaporator___________________ Temperature-------------------- ------------------------ -------- Add 1.0 mL of Methanol TN-A- ------------------------- ---------------------------- --- Vortex 30 sec. .................................................................................... ......................... ....... .......... Fr iillitcerr uussiuni ga aa J3UcVc/ UB--DU asyy ri nini ^gve wm iutht wa 0.2um SRI filter---i-n---to----a---1-.-5---m----L----a--u--t-o--s--a--m-- rp,.le..v.nial . --------------;------------------------------- .---------------- -W------- MS/MSD/___ Cont. Checks: Spiked______uL of a _____ ppm std (______________ ) for a final concentration ot ________ ppm. MS/MSD used sam ple_______ :_______ . Cont. Checks used same homogenate as for std curve. FACT-M-1.0 Extraction of PFOS from Liver Page 8 of 8 3M Environmental Laboratory 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 3M Environmental Laboratory M ethod Extraction of P otassium perfluorooctanesulfonate o r other Anionic Fluorochem ical surfactants from Serum for Analysis U sing H PL C -E lectrospray/M ass Spectrom etry M ethod Number: FACT-M-3.0 Author: Lisa Clemen Adoption Date: ^ j i z ci^ Revision Date: |df/| Group Leader & A ( L,'rvx.v- Technical Reviewer Date sink Date 1.0 Scope and A pplication 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, and bovine serum or other sera as designated in the validation report. Microsoft 7.0.1/95 3M Environmental Laboratory FACT-M-3.0 Extraction of PFOS from Serum Page 1 of 8 Page 51 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 2.0 Su m m a r y o f M eth o d _________________________________:_________ ;-------------------------------- 2.1 This method describes how to extract potassium perfluorooctanesulfonate (PFOS) or other anionic fluorochemical surfactants from serum using an ion pairing reagent and 5.0 mL of ethyl acetate. An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into ethyl acetate. Four mL of extract are removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL of methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 3.0 D e f in it io n s _______________________________________ __________________ :--------------------------3.1 None. 4.0 W a r n in g s a n d C a u tio n s________ :_______ ____________________________ ______________ 4.1 Health and Safety Warnings: 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal serum, it may contain pathogens. 5.0 In t e r f e r e n c e s _____________ ;_____________________ ;_________ _ _____ ____ .__________ 5.1 There are no known interferences at this time. 6.0 E q u i p m e n t ________ _________________ ___________________________________ _______________________ 6.1 The following equipment is used while carrying out this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 6.1.3 6.1.4 6.1.5 Centrifuge, Mistral 1000 or IEC Shaker, Eberbach or VWR Nitrogen evaporator, Organomation Balance, (0.100 gm) 7.0 S u p p l i e s a n d M a t e r ia l s _____________________^ 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Nalgene bottles, capable of holding 250 mL and 1 L 7.4 Glass, type A, volumetric flasks 7.5 40 mL glass I-CHEM vials 7.6 Polypropylene centrifuge tubes, 15 mL 7.7 Labels 7.8 Syringes, capable of measuring 10 pL to 50 pL 7.9 Glass, type A, volumetric pipettes 7.10 Graduated pipettes FACT-M-3.0 Extraction of PFOS from Serum Page 2 of 8 3M Environmental Laboratory Page 52 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 7.11 Electronic pipettor, Eppendorf or equivalent 7.12 Timer 7.13 Disposable plastic 3 cc syringes 7.14 Filters, nylon syringe filters, 0.2 pm, 25 mm 7.15 Crimp cap autovials Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli esTMwater. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 R e a g e n t s a n d S t a n d a r d s _________;--------------------------- ---- ----------------------- 8.1 Reagents 8.1.1 Sodium hydroxide (J.T Baker or equivalent), (NaOH) ION: weigh approximately 200 grams NaOH. Pour into a 1000 mL beaker containing 500 liters (L) Milli-Q water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.1.2 Sodium hydroxide (J.T Baker or equivalent), (NaOH) IN. Dilute 10N 1:10. Measure 10 mL of 10N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.1.3 Tetrabutylammonium hydrogen sulfate (Kodak or equivalent), (TBA) 0.5M:W eigh approximately 169 grams of TBA into a 1 L volumetric containing 500 L Milli-Q water. Adjust to pH 10 using approximately 64 mL of 10N NaOH and dilute to volume with Milli-QTM water. Add NaOH slowly while adding the last mL of NaOH because the pH changes abruptly. Store in a 1 L Nalgene bottle. 8.1.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using IN NaOH solution. 8.1.4 Sodium carbonate/sodium bicarbonate buffer (J.T. Baker or equivalent), (Na2C 0 3/NaHC03) 0.25M: Weigh approximately 26.5 g of sodium carbonate (N a^O j) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with Milli-QTM water. Store in a 1 L nalgene bottle. 8.1.5 PFOS (3M Specialty Chemical Division), molecular weight =538. 8.1.6 Other fluorochemicals, as appropriate. 8.1.7 Ethyl Acetate, Omnisolv, glass distilled or HPLC grade. 8.1.8 Methanol, Omnisolv, glass distilled or HPLC grade. 8.1.9 Serum, frozen liquid from Sigma. 8.1.10 Control serum received with each sample set. 8.1.11 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC Plus system. 3M Environmental Laboratory FACT-M-3.0 Extraction of PFOS from Serum Page 3 of 8 Page-53 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 8.2 Standards 8.2.1 Prepare PFOS standards for the standard curve. 8.2.2 Prepare other fluorochemical standards, as appropriate. 8.2.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.2.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (pg/mL). 8.2.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.2.6 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.2.7 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 9.0 Sa m pl e H a n d lin g ________________________ :----------------------- --- ------------------------9.1 All sera are received frozen and must be kept frozen until the extraction is performed. 10.0 Quality C o n t r o l _________________________________________ _-- --------------------------------10.1 Matrix Blanks and Method Blanks 10.1.1 Two 1.0 mL aliquots of the serum are extracted following this procedure and used as matrix blanks. See section 11.1.2. 10.1.2 Two 1.0 mL aliquots of Milli-QTM water are extracted following this procedure and used as method blanks. 10.2 Matrix Spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using serum chosen by the analyst, usually control serum received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.3 Continuing Calibration Checks 10.3.1 Prepare and analyze continuing calibration check samples to determine the continued linearity of the initial calibration curve. . 10.3.2 One check is prepared per group of ten samples. For example, if a sample set = 34, four checks are prepared and extracted. 3M Environmental Laboratory FACT-M-3.0 Extraction of PFOS from Serum Page 4 of 8 Page--54- 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 10.3.3 Prepare each continuing calibration check from the same serum used to prep the initial curve. 10.3.4 The expected concentration will fall within the mid-range of the initial calibration curve. 11.0 C a l i b r a t i o n a n d S t a n d a r d iz a t io n ______________________________________________________ 11.1 P repare Serum Standards 11.1.1 Transfer 1 mL o f serum to a 15 mL centrifuge tube. 11.1.2 If the majority of serum sample volumes are less than 1.0 mL, extract standards using serum volumes in the standards equal to the serum volumes in samples. Do not extract below 0.50 mL of serum. Record the serum volume on the extraction sheet. 11.1.3 Mix or shake between aliquots while preparing a total of sixteen aliquots of serum in 15 mL centrifuge tubes. 11.1.4 Two 1 mL or appropriate aliquots serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in table 1 to spike, in duplicate, two standard curves for a total of fourteen samples. 11.1.5 Refer to the validation report FACT-M-3.0-V-1 and FACT-M-4.0-V-1 which lists the working ranges for calibration curves. Table 1 Approximate Spiking Amounts for Standards and Spikes Using 1.0 mL of Serum Working Standard (Approx. Cone.) - 0.500 ppm 5.00 ppm 5.00 ppm 5.00 ppm 50.0 ppm 50.0 ppm 50.0 ppm fiL Approx, final cone, of PFOS in serum - Blank 20 0.010 ppm 5 0.025 ppm 10 0.050 ppm 20 0.100 ppm 5 0.250 ppm 10 0.500 ppm 15 0.750 ppm 11.1.4 See section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 Extract spiked serum standards following 12.6-12.16 of this method. Use these standards to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory FACT-M-3.0 Extraction of PFOS from Serum Page 5 of 8 Page 55^ 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 12.0 P r o c e d u r e s _______________________________ ;...................... .... ............................................ 12.1 Obtain frozen serum samples and allow to thaw. 12.2 Vortex mix for 15 seconds then remove 1.0 mL or appropriate volume to a 15 mL polypropylene centrifuge tube. 12.3 Return serum samples to freezer after extraction amount has been removed. 12.4 Record the serum volume on the extraction worksheet. The final methanol volume will equal the initial serum volume. 12.5 Label the tube with the study number, serum ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike serum with the appropriate amount of PFOS standard as described in section 11.1 or Table I for the calibration curve standards. Also spike matrix spikes and continuing calibration standards. 12.7 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.8 Add 1 mL 0.5 M TBA and 2 mL of the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.9 Using a volumetric pipette, add 5 mL ethyl acetate. 12.10 Cap each sample and put on the shaker for 20 minutes. 12.11 Centrifuge for 20 to 25 minutes, until layers are well separated. Set power on the centrifuge to approximately 3500 rpm. 12.12 Transfer 4 mL of organic layer, using a 5 mL graduated glass pipette, to a clean 15 mL centrifuge tube. Label this fresh tube with the same information as in 12.5. 12.13 Put each sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 hours. 12.14 Add 1.0 mL or appropriate volume of methanol to each centrifuge tube using a graduated pipette. (This volume equals the initial volume of serum used for the extraction.) 12.15 Vortex mix for 30 seconds. . 12.16 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial. 12.17 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) who performed the extraction. 12.18 Cap and hold for HPLC-electrospray/mass spectrometry analysis. Extracts may be stored at 4 C until analysis. 12.19 Complete the extraction worksheet, attached to this document, and tape to page of study notebook. 3M Environmental Laboratory FACT-M-3.0 Extraction of PFOS from Serum Page 6 of 8 Page- 56 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 13.0 D a t a A n a l y s is a n d C a l c u l a t io n s _______________________________ ;__________________ _____ 13.1 Calculations: 13.1.1 Calculate actual concentrations of PFOS, or other appropriate fluorochemical, in calibration standards using the following equation: mL o f Standard x Concentration Tug /mL) = Final Concentration (pg/mL) mL of Standard + Initial Serum Volume (mL) of PFOS in Serum 14.0 M e t h o d P e r f o r m a n c e _______________________________________________ ___________________ 14.1 The method detection limit is equal to half the lowest standard in the calibration curve. 15.0 P o llutio n P reventio n and W aste M anag em ent _______________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in brokn glass containers located in the laboratory. 16.0 R e c o r d s _________ __________________ !_______________________________________ _______________ 16.1 Complete the extraction worksheet attached to this method, and tape into the study notebook. 17.0 T a b l e s . D ia g r a m s . F l o w c h a r t s , a n d V a l id a t io n D a t a ________ __ ________________ 17.1 The validation report associated with this method is FACT-M-3.0 & 4.0-V -l. 18.0 R e f e r e n c e s 18.1 None ________________________________ :________;------- ---------------------------------- 19.0 A f f e c t e d D o c u m e n t s _______________________ :________________________ :------ .----------------------- 19.1 FACT-M-4, "Analysis o f Serum Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 R e v is io n s Revision Number. Reason For Revision Revision EM 3M Environmental Laboratory FACT-M-3.0 Extraction of PFOS from Serum Page 7 of 8 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Extraction Worksheet for FACT-M-3 Study # _ __ _ _ Sample Number set # H ,0 Blank Serum Blank PFOS approx. 0.5 ppm actual ppm #W - PFOS approx. 5 ppm actual ppm #W - PFOS approx. 50 ppm actual ppm #W - Date and Initials for Std. or Comments . 1 -- -- -- -- -' - --- -- -- -, - - .- -- -- -- -- -- -- - - -- 1Studv num her where the original wo rksheet is located. Blank Serum Std# --S-e-r-u--m---=E--xt:-r-a--c-t.i.on M. . et.h.--od;-- ---------- - Serum amount = " .... . -. - - - ..- - ` ...... ------- g------------- ---------------------------Date & Initials -- Vortex 15 sec._______________ Pioette Serum Pipette 1 mL o f 0.5 M TBA, pH 10. " ......... VplmiiS------------------------JSL_----------------- -------------- ----------------- - Std. #------------------------ ----------- ---------- ----------- --------------------- Pipette 2 mL o f 0.25 Na?COV0.25M NaHCO} buffer Std.# ... ------------------------ ---------------------- Pipette 5 mL o f ethyl a c e t a t e _________________ TN-A- -- Shake 20 min.______________ Centrifuge 20-25 min. , ' ...-- -------- ------------------------ ------- Centrifuge speed:---------------------------- ---------- -------------------------- .-- Remove a 4 mL aliauot of organic layer _______________________ ________________ ____________-- ------------- - r ~ Put on Nitrogen Evaporator to dryness Evaporator#:----------------------------- Temperature........................................................ Add methanol Volume mL TN A ------------ -- ------------ -- Vortex 30 sec. .. ................................................... ---------------------------------------------- ------ :-- - - Filter using a 3cc B-D svringe with a 0.2um SRI filter into a 1.5 mL autosample vial ---------------------------- -------------------- _ ppm. MS/MSD used sample . Cont. Checks used same serum as for std curve. FACT-M-3.0 Extraction of PFOS from Serum Page 8 of 8 3M Environmental Laboratory Page 58 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 3M Environmental Laboratory M ethod E x t r a c tio n o f P otassium P erflu o ro o cta n esu lfo n a te o r O t h e r F l u o r o c h e m ic a l co m po unds fr o m Ser u m f o r A nalysis U sin g HPLC- E lectrospray/M ass Spectro m etry M ethod N um ber: ETS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg Revision Date: w i m Approved By: 0 - f iS -- Laboratory Manager lx}?-----....--` Group Leader Technical Reviewer 4 /17/5^ Date v /a / ? Date ov/W ^ Date 1.0 S c o p e a n d A p p l i c a t i o n 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from serum. 1.2 Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, monkey, and human serum or other fluids as designated in the validation report. Word 6/95 3M Environmental Laboratory ETS-8-4.1 Extraction o f PFOS from Serum Page 1 o f 14 Page 59 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 2.0 S u m m a r y o f M e t h o d ____________________________ _________________________________ 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum, or other fluids, using an ion pairing reagent and methyl-tert-butyl ether (MtBE). In this method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556, and surrogate standard (see 3.0 D efin ition s). An ion pairing reagent is added to 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 1.0 mL of methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 jam nylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-5.1 or other appropriate methods. 3.0 D e f i n i t i o n s _________ __ ________________________________________________ ;___________________ 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) C8F I7S 0 3' 3.2 PFOSA: perfluorooctane sulfonylamide CgF I7S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate CgF17S 0 2N(CH2CH3)CH2C 0 2' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F 17S 0 2N(CH2CH3)CH2CH20 H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide CgF17S 0 2N(CH2CH3)H 3.6 M556: C8F 17S 0 2N(H)(CH2C 0 0 H ) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 W a r n in g s a n d C a u t io n s ____________________________________ ;________________________ 4.1 H ealth and safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 I n t e r f e r e n c e s ______________________________________________________________________'_______ 5.1 There are no interferences known at this time. 6.0 E q u i p m e n t ___________________________________________________________________________ 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 2 of 14 Page 60 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) 7 .0 S u p p l i e s a n d M a t e r ia l s _____________________;_______________________________________________ 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Nalgene bottles, capable o f holding 250 mL and 1 L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0 mL 7.9 Syringes, capable o f measuring 5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe filters, nylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli-QTM water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8 .0 R e a g e n t s a n d S t a n d a r d s _______________________________ __________________________________ _ 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate Nt^COj), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Serum or blood, frozen from supplier 8.9 Fluorochem ical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 ETS-8-4.1 Extraction of PFOS from Serum Page 3 of 14 3M Environmental Laboratory Page 61 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 8.9.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.9.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.9.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.9.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.9.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H,1-H, 2-H, 2-H C8F 13S 0 3H) molecular weight = 428 8.9.8 Other fluorochemicals, as appropriate 8.10 R eagent 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 . 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.10.2 1 N sodium hydroxide (NaOH): Dilute 10N NaOH 1:10. Measure 10 mL of 10 N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.10.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g o f TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL o f 10 N NaOH (While adding the last mL of ' NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-QTM water. Store in a 1 L Nalgene bottle. 8.10.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using I N NaOH solution. 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer (N a^O /N aH C O ^: Weigh approximately 26.5 g of sodium carbonate (NajCOj) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with MilliQTM water. Store in a 1 L Nalgene bottle. 8.11 S tandards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.11.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.11.4 Bring to volume with methanol for a stock standard o f approximately 1000 ppm (pg/mL). 8.11.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.11.6 Dilute working standard 1 with methanol for a working standard 2 solution of approx. 5.0 ppm. ETS-8-4.1 Extraction of PFOS from Serum Page 4 o f 14 3M Environmental Laboratory Page 62 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 8.11.7 Dilute working standard 1 with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.12 Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 mg of surrogate standard 1-H,1-H, 2-H, 2-H, C8F 13S 0 3H into a 50 mL volumetric flask and record the actual weight. 8.12.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 ppm. 8.12.3 Prepare a surrogate working standard. Transfer approximately 1 mL o f surrogate stock to a 10 mL volumetric flask and bring to volume with methanol for a working standard o f 100 ppm. Record the actual volume transferred. 9.0 S a m p l e H a n d l in g ___________ :_______________________________________________________________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw to room temperature prior to extraction. 10.0 Q u a l it y C o n t r o l __________________________ ;__________________ _____________________________ _ 10.1 Solvent Blanks, M ethod blanks and m atrix blanks 10.1.1 An aliquot o f 1.0 mL methanol is used as a solvent blank. 10.1.2 Extract two 1.0 mL aliquots o f Milli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots o f the serum following this procedure and use as matrix blanks. See 11.1.4. 10.2 M atrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually the control matrix received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range o f the initial calibration curve. 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 C ontinuing calibration checks 10.3.1 Prepare continuing calibration check samples to ensure the accuracy of the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing check 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 check from the same matrix used to prepare the initial curve. 3M Environmental Laboratory ETS-8-4.1 Extraction o f PFOS from Serum Page 5 o f 14 Page 63 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 10.3.4 The expected concentrations will fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end o f the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb - 1000 ppb). 11.0 C a l i b r a t i o n a n d S t a n d a r d iz a t io n ______________________ ;___________ ;___________________ 11.1 P repare m atrix calibration standards 11.1.1 Transfer 1 mL of serum to a 15 mL centrifuge tube. 11.1.2 If most sample volumes are less than 1.0 mL, extract standards with matrix volumes equal to the sample volumes. Do not extract less than 0.50 mL of matrix. Record each sample volume on the extraction sheet. 11.1.3 While preparing a total of twenty aliquots in 15 mL centrifuge tubes, mix or shake between aliquots. 11.1.4 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1, at the end o f this section, to spike, in duplicate, two standard curves, for a total of eighteen standards, two matrix blanks, and two method blanks. 11.1.5 Refer to validation report ETS-8-4.0 & ETS-8-5.0-V-1, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.6 Use Attachment D as an aid in calculating the concentrations of the working standards. See Section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each standard, blank, or continuing check, add appropriate amount o f surrogate working standard for the concentration to fall within the calibration curve range 5 ppb 1000 ppb. 11.3 Extract spiked matrix standards following 12.6-12.16 o f this method. Use these standards to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 6 of 14 Page ' 64 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 1 Approximate spiking amounts for standards and spikes Using 1.0 mL of m atrix Working standard pL Approx, final cone, of (approx, cone.) analyte in matrix - - Blank 0.500 ppm 10 0.005 ppm 0.500 ppm 20 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 0.050 ppm 5.00 ppm . 20 0.100 ppm 50.0 ppm 5 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 1.00 ppm 12.0 P r o c e d u r e __________________________ _______________________________________________________ 12.1 Obtain frozen samples and allow to thaw at room temperature or in a lukewarm waterbath. 12.2 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to a 15 mL polypropylene centrifuge tube. 12.3 Return unused samples to freezer after extraction, amounts have been removed. 12.4 Record the initial volume on the extraction worksheet. 12.5 Label the tube with the study number, sample ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike all samples, including blanks and standards, ready for extraction with surrogate standard as described in 11.2. 12.7 Spike each matrix with the appropriate amount o f standard as described in 11.1, or Table 1 in that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 1 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.10 To each sample, add 1 mL 0.5 M TBA and 2 mL of 0.25M sodium carbonate/sodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 mL methyl-teri-butyl ether. 12.12 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. 12.13 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm, or until layers are well separated. ETS-8-4.1 Extraction o f PFOS from Serum Page 7 of 14 3M Environmental Laboratory Page 65 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 12.14 Label a fresh 15 mL centrifuge tube with the same information as in 12.5. 12.15 Remove 4.0 mL of the organic layer to this clean 15 mL centrifuge tube. 12.16 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. 12.17 Add 1.0 mL of methanol to each centrifuge tube using a graduated pipette. 12.18 Vortex mix for 30 seconds. 12.19 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this . syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.20 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.21 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.22 Complete the extraction worksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. 13.0 D a t a A n a l y s is a n d C a l c u l a t io n s _____________________________________ ________________ 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS, or other applicable fluorochemical, in calibration standards using the following equation: mL of standard x concentration of standard fug /mLl___________ ;______ _ = . mL of standard + mL of surrogate standard + initial matrix volume (mL) Final Concentration (pg/mL) o f PFOS in matrix 14.0 M e t h o d P e r f o r m a n c e _________________________;___________________________________________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quantitation (LOQ) values (see Attachments B and C). 14.2 The following quality control samples are extracted with each batch o f samples to evaluate the quality o f 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 o f the extraction. 14.2.3 Continuing calibration check samples to determine the continued accuracy o f the initial calibration curve. 14.3 Refer to section 14 of ETS-8-5.1 for method performance criteria. 15.0 P o l l u t i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t ______________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. ETS-8-4.1 Extraction of PFOS from Semm Page 8 of 14 3M Environmental Laboratory Page 66 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 - LRN-U2121 1 6.0 R e c o r d s ________________ ;_________________________________________________ ________ 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 A t t a c h m e n t s ______________________________________________________________________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 1 8.0 R e f e r e n c e s ____________________________________ :____________________________________________ 18.1 The validation report associated with this method is ETS-8-4.0 & 5.0-V -l. 18.2 FACT-M-3.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 19.0 A f f e c t e d D o c u m e n t s _____________________________________________________________________ 19.1 ETS-8-5.1, "Analysis o f Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 R e v i s i o n s ____________________ :__________________________________________________________ Revision Number 1 Reason For Revision Section 12.21 Changed to include sample storage at room temperature. Section 12.13 Added the shaker speed. Section 12.17 Final volume is 1.0 mL; not adjusted for initial volumes less than 1.0 mL. Revision Date 04/02/99 3M Environmental Laboratory . ETS-8-4.1 Extraction of PFOS from Serum Page 9 o f 14 Page 67 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Extraction Worksheet ETS-8-4.1 Studv# Matrix Box# Wk/Dav DateSpiked/Analyst CCV MS MSD Surrogate Std approx, ppm actual ppm # F C -M ix approx. 0.5 pm actual ppm # FC-Mix approx. 5 ppm actual ppm # FC-Mix approx. 50 ppm actual ppm # Comments RiantSerum Extraction Method ---- ---------- -- '- - Std# amount = mL . .......................................________________________________ _______________Date & Initials---- Vortex 15 sec. Pinette Matrix .......................................j__________________________________-- ------------- ------------______Volume________________ mL______________ ;-------------------- ---------------- Pipette 1 mL o f 0.5 M TBA, pH 10. pH = Std. # ............ . ... Pipette 2 mL o f 0.25 Na2COV0.25M NaHCCri buffer Std. # . Dispense 5 mL o f methyl-t-butyl ether TN-A- _ Shake 20 min. Centrifuge 20-25 min. Shaker speed:......... ...... ...... ...........................Centrifuge speed:_______________________________ _______ ;---------- Remove a 4 mL aliauot o f organic l a y e r ________________________________________________ ______________________ Put on Nitrogen Evaporator to d r y n e s s _______________ Temperature:________________________ ____________ ________ Add methanol Volume mL TN-A- ----------------- Vortex 30 s e c . ______________________________ '---------------------------- -- ---------- -- --------------- Filter using a 3cc B-D syringe with a 0.2um filter into a 1.5 mL autosample vial Cont. Cal. Verifications used same matrix as for std curve. --------------------- Attachment A 3M-Environmental-Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 10 of 14 Page 68 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 . LRN-U2121 MDL/LOQ values for rabbit serum Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 1.74 5.55 5 p p b - 1000ppb PFOSA 1.51 4.79 5 ppb - 1000 ppb PFOSAA 3.46 20.5 5 ppb - 1000 ppb EtFOSE-OH 11.4 36.2 5 p p b - 1000 ppb M556 6.03 19.2 5 ppb -1000 ppb PFOSEA 5.71 18.2 5 p p b -1000 ppb MDL/LOQ values in rat, bovine, monkey, and human serum, and monkey plasma were not statistically determined. Two curves in each of these matrices were extracted and analyzed with the rabbit serum curves to determine equivalence. Responses in the rat, bovine, monkey, and human were equivalent to the rabbit responses, therefore, their MDL and LOQ will be the same values as determined in rabbit serum. Please see LOQ Summary and MDL study in ETS-8-4.0 & 5.0-V-l for further information. Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction o f PFOS from Seram 3M Environmental Laboratory Page 11 of 14 Page 69 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Compound: PFQS Prepared range R abbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve H igh curve 1/X 0.995 - 978 4.94 - 248 97.8 - 978 0.995 - 978 LCR from curve (pph) (ng/m L ) 24.8 - 978 4.94 - 248 97.8 - 978 4.94-978 % Recovery Range 83-108 85-104 85-106 94-111 Compound: PFOSA Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve H igh curve 1/X 0.993 - 976 4.93 - 97.6 24.8 - 976 0.993 - 976 LCR from curve (ppb) (ng/m L ) 4.93 - 976 % Recovery Range 88-103 4 .93-97.6 . 87-105 24.8-978 93-102 4.93-976 94-103 Compound: PFOSAA Prepared range R abbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve H igh curve 1/X 0.991 - 974 4.92 - 247 49.2-974 0.991 - 974 LCR from curve (PPb) (ng/m L ) 24.7 - 974 9.74 - 247 97.4 - 974 9.74-974 % Recovery Range 81-111 97-107 85-108 95-115 RSD Range 4.67-11.0 5.34-12.0 4.84-9.80 4.60-10.5 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 RSD Range 4.18-10.6 6.38-21.8 4.33-12.5 4.11-23.2 Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction o f PFOS from Serum 3M Environmental Laboratory Page 12 o f 14 Page 70 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 ' LRN-U2121 Compound: EtFOSE-OH Prepared range R abbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve H igh curve 1/X 0.993 - 976 4.93 - 97.6 49.3 - 976 0.993-493 LCR from curve (ppb) (ng/m L ) . 49.3 -976 9.76-97.6 97.6 - 976 9.76 - 976 % Recovery Range 77-110 97-107 90-109 86-111 Compound: PFOSEA Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve H igh curve 1/X 0.993-976 4.93 - 248 49.3 - 976 0.993-976 LCR from curve (Ppb) (ng/m L ) 24,8 - 976 9.76 - 248 49.3 - 976 9.76 - 976 % Recovery Range 96-106 91-110 86-106 95-117 Compound: M556 Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 97.6 97.6 - 976 0.993 - 976 LCR from curve (ppb) (ng/m L ) 24.8 - 976 9.76-97.6 97.6 - 976 9.76 - 976 % Recovery Range 88-106 100-105 81-111 97-110 RSD Range 11.2-25.5 14.1-21.3 11.5-19.6 11.1-21.2 RSD Range 10.1-16.2 11.8-19.5 10.2-18.2 10.1-19.1 RSD Range 4.82-17.9 5.95-18.2 5.11-9.74 4.77-19.5 Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction o f PFOS from Serum 3M Environmental Laboratory Page 13 o f 14 Page 71 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Ion Pair Standard Curves - Fluids Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Blank fluid/identifier: Method/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Actual concentrations of standards in the FC mix PFOS PFOSA PFOSAA EtFOSE PFOSEA Std cone Std cone Std cone Std cone Std cone ug/mL ug/mL ug/mL ug/mL ug/mL 0.500 0.507 0.532 0.501 0.521 0.500 0.507 0.532 0.501 0.521 5.00 5.07 5.32 5.01 5.21 5.00 5.07 5.32 5.01 5.21 5.00 5.07 5.32 5.01 5.21 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 M556 Std cone ug/mL 0.501 0.501 5.01 5.01 5.01 50.1 50.1 50.1 50.1 A ll Am't spiked mL 0.010 0.020 0.005 0.010 0.020 0.005 0.010 0.015 0.020 All Final vol mL 1.015 1.025 1.010 1.015 1.025 1.010 1.015 1.020 1.025 Calculated concentrations of standards in the sample matrix PFOS PFOSA PFOSAA EtFOSE PFOSEA M556 Surrogate Final cone Final cone Final cone Final cone Final cone Final cone Std cone ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL 4.93 5.00 5.24 4.94 5.01 5.13 100 9.76 9.89 10.4 9.78 9.93 10.2 24.8 25.1 26.3 24.8 25.2 25.8 Surrogate 49.3 50.0 52.4 . 49.4 50.1 51.3 Final cone 97.6 98.9 104 97.8 99.3 102 ng/mL 248 251 263 248 252 258 500 493 500 524 494 501 513 735 746 782 737 749 766 976 989 1038 978 993 1017 All Ain't spiked mL 0.005 Validated ranges - approximate concentrations Serum Rabbit PFOS PFOSA PFOSAA EtFOSE-OH PFOSEA M556 5.00-1000 I 5.00-1000 1 5.00-1000 \ 5.00-1000 \ 5.00-1000 | 5.00-1000 Bovine Estimates only. Use values for rabbit. . Rat Estimates only. Use values for rabbit. Monkey & Plasma Estimates only. Use values for rabbit. Human Estimates only. Use values for rabbit. Attachment C: Ion Pair Standard Curves ETS-8-4.1 Extraction o f PFOS from Serum 3M Environmental Laboratory Page 14 o f 14 Page 72 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 3I lMf 1 El'/nl vViUrAoV ynllmlTleJJnll tXravlu Laboratjlorya M ethod E xtraction of P otassium Perfluorooctanesulfonate or other HPLC-F l u o r o c h e m ic a l C o m p o u n d s f r o m L iv e r f o r A n a l y s is u s in g Electrospray/M ass Spectrom etry Method Number: ETS-8-6.0 Author: Lisa Clemen, Robert Wynne Adoption Date: Revision Date: tOlt Approved By: Laboratory ]N^anager /jLjh slltA , ------------ - -------- -------------------------- ------------------------- ------------------------------ Group Leader Date Date A Cfjpyimviu Technical Reviewer 07/nlw Date 6i 1.0 Sco pe and A pplication 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from liver. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, and monkey livers or other tissues as designated in the validation report. Word 6.0/95 ETS-8-6.0 Extraction of PFOS from Liver Page 1 o f 14 3M Environmental Laboratory Page 73 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 2.0 Su m m a r y o f M e th o d __________ _________________________________________________ __ -- 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate ^ (PFOS) or other fluorochemical surfactants from liver, or other tissues, using an ion pairing reagent and methyl-terributyl ether (MtBE). In this method, seven fluorochemicals can be extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH,.PFOSEA, M556, and surrogate standard. An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL methanol then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-7.0 or other appropriate methods. 3.n D e f in it io n s________________ __________________________________________-- --------- 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) C8F 17S 0 3 3.2 PFOSA: perfluorooctane sulfonylamide C8F 17S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate CgF I7S 0 2N(CH2CH3)CH2C 0 2 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F 17S 0 2N(CH2CH3)CH2CH20 H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8F n S 0 2N(CH2CH3)H 3.6 M556: C8F 17S 0 2N(H)(CH2COOH) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4 .0 W a r n in g s a n d C a u t io n s_____________ ___________________________ :----------------------- ---------- 4.1 Health and Safety Warnings: 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 INTERFERENCES________ _________________________________________________ ______________ 5.1 There are no interferences known at this time. 6.0 EQUIPMENT_____________ ____________________ _________________ __________ 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Ultra-Turrax T25 Grinder for grinding liver samples 6.1.2 Vortex mixer, VWR, Vortex Genie 2 6.1.3 Centrifuge, Mistral 1000 or IEC 6.1.4 Shaker, Eberbach or VWR ETS-8-6.0 Extraction o f PFOS from Liver Page 2 of 14 3M Environmental Laboratory Page 74 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivity to 0.100 g) 7 .0 S u p p l ie s a n d M a t e r i a l s ......................................................................................................................... .............. 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable of holding 250 mL and 1 L 7.5 Volumetric flasks, glass, type A . 7.6 I-CHEM vials, 40 mL glass 7.7 Plastic sampule vials, Wheaton, 6 mL (or appropriate size) 7.8 Centrifuge tubes, polypropylene, 15 mL 7.9 Labels 7.10 Oxford Dispensor - 3.0 to 10.0 ml 7.11 Syringes, capable of measuring 5 pL to 50 pL 7.12 Graduated pipettes 7.13 Syringes, disposable plastic, 3 cc 7.14 Syringe filters, nylon, 0.2 pm, 25 mm 7.15 Timer 7.16 Crimp cap autovials and caps 7.17 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli * QTM water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8 .0 R e a g e n t s a n d S t a n d a r d s ___________________ ________________________________ -- --------------- 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should * be Milli-QTM water and be provided by a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (Na2C 0 3), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8.6 Methyl-ieri-butyl ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Liver, frozen from supplier 8.9 Dry ice from supplier 8.10 Fluorochem ical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 ETS-8-6.0 Extraction of PFOS from Liver Page 3 of 14 3M Environmental Laboratory Page 75 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 8.10.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 8.10.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.10.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.10.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.10.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.10.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H,1-H, 2-H, 2-H C8F I3S 0 3H) molecular weight = 428 8.10.8 Other fluorochemicals, as appropriate 8.11 R eagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.11.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.11.2 1 N sodium hydroxide (NaOH): Dilute 10N NaOH 1:10. Measure 10 mL of 10N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.11.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL of 10 N NaOH (While adding the last mL o f NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-QTM water. S to re in a 1 L Nalgene bottle. 8.11.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.11.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na^Oj/NaHCOp: Weigh approximately 26.5 g o f sodium carbonate (Na2C 0 3) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with MilliQTM water. Store in a 1 L Nalgene bottle. 8.12 Standards preparation 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.12.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.12.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (qg/mL). 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 4 of 14 Page 76 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 8.12.6 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.12.7 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.13 Surrogate stock standard preparation 8.13.1 Weigh approximately 50-60 mg o f surrogate standard 1-H,1-H, 2-H, 2-H, C8F 13S 0 3H into a 50 ml volumetric flask and record the actual weight. 8.13.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 - PPm 8.13.3 Prepare a surrogate working standard. Transfer approximately 1.0 ml of surrogate stock to a 10 ml volumetric flask and bring to volume with methanol for a working standard o f 10-20 ppm. Record the actual volume transferred. 9.0 S a m p l e H a n d l in g _____________ _________________________________________________________ -- 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 10.0 Q u a l it y C o n t r o l _____________ _________________________________________ ______ 10.1 M atrix blanks and m ethod blanks 10.1.1 A n a liq u o t o f 1.0 m L m e th a n o l is u s e d a s a s o lv e n t b la n k . 10.1.2 Extract two 1.0 mL aliquots of Milli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots of liver homogenate following this procedure and use as matrix blanks. Refer to 11.1.6. 10.2 M atrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually a control liver received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibration verification samples to ensure the accuracy of the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing calibration verification sample per group o f 10 samples. For example, if a sample set - 34, four verifications are prepared and extracted. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 5 o f 14 Page-77 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 10.3.3 Prepare each continuing calibration verification from the same matrix used to prepare the initial curve. 10.3.4 The expected concentrations will fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb - 1000 ppb). 11.0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n ---------------------------------------------------------------------:----------- 11.1 Prepare m atrix calibration standards 11.1.1 Weigh approximately 40 g of liver into a 250 mL Nalgene bottle containing 200 mLs Milli-QTM water. Grind to a homogeneous solution. 11.1.2 If 40 g is not available, use appropriate amounts of liver and water to ensure a 1:5 ratio. 11.1.3 Refer to 13.0 to calculate the actual density o f liver homogenate and the concentration o f solid liver tissue dispersed in 1.0 mL of homogenate solution. 11.1.5 Add 1 mL of homogenate to a 15 mL centrifuge tube. Re-suspend solution by shaking between aliquots while preparing a total of eighteen 1 mL aliquots of homogeneous solution in 15 mL centrifuge tubes. 11.1.6 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. 11.1.7 Typically use the standard concentrations and spiking amounts listed in Table 1, at the end of this section, to spike, in duplicate, two standard curves, for a total of eighteen samples, two matrix blanks, and two method blanks. 11.1.8 Refer to validation reports ETS-8-6.0 and ETS-8-7.0-V-1 or Attachm ent B, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.9 Use A ttachm ent C as an aid in calculating the concentrations o f the working standards. Refer to 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each working standard, blank, or continuing verification, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 5 ppb-lOOOppb. 3M Environmental Laboratory ETS-8-6.0 Extraction o f PFOS from Liver Page 6 o f 14 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 11.3 Extract spiked liver homogenates following 12.14-12.25 of this method. Use these standards to establish each initial curve on the mass spectrometer. Table 1 Approximate Spiking Amounts for Calibration Standards Working Standard (Approx. Cone.) - 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm 5.0 ppm 50 ppm pi '2 4 10 20 40 10 20 30 4 Approx, final cone, of PFOS in liver Blank 0.005 ppm 0.010 ppm 0.025 ppm 0.050 ppm 0.100 ppm 0.250 ppm 0.500 ppm 0.750 ppm 1.00 ppm 1 2 .0 P r o c e d u r e ------------------------ --------------------------------------------------------------------------------------------------- 12.1 Obtain frozen liver samples. 12.2 Cut approximately 1 g of liver using a dissecting scalpel. This part of the procedure is best performed quickly, not allowing the liver to thaw. 12.3 Weigh the sample directly into a tared plastic sampule vial. 12.4 Record the liver weight in the study notebook. 12.5 R e tu r n u n u s e d liv e r p o rtio n s to fre ez er. 12.6 Add 2.5 mLs of water to sampule vial. 12.7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the sample with 2.5 mLs water using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Refer to AMDT-EP' 22. 12.10 Cap the sample and vortex for 15 seconds. Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 7 of 14 Page 79 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 12.11 Pipette 1.0 mL, or other appropriate volume, of homogenate into a 15 mL polypropylene centrifuge tube. Label the centrifuge tube with the identical information as the sampule vial. Refer to attached worksheet for documenting the remaining steps. 12.12 Pipette two 1 mL aliquots o f Milli-QTM water to centrifuge tubes. These will serve as method blanks. 12.13 Spike all samples, including blanks and standards ready for extraction with surrogate standard as described in section 11.2. 12.14 Spike each matirx with the appropriate amount of standard as described in 11.1, or Table 1 of that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. . 12.15 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.16 Check to ensure 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.17 To each sample, add 1 mL 0.5 M TBA and 2 mL of the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.18 Using an Oxford Dispenser, add 5 mL methyl-tert-butyl ether. 12.19 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. 12.20 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm, or until layers are well separated. 12.21 Label a fresh 15 mL centrifuge tube with the same information as in 12.10. 12.22 Remove 4.0 mL of the organic layer to the fresh 15 mL centrifuge tube. 12.23 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. 12.24 Add 1.0 mL to each centrifuge tube using a graduated pipette. 12.25 Vortex mix for 30 seconds. 12.26 Attach a 0.2 xm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.27 Label the autovial with the study number, animal number and gender, sample timepoint, * matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.28 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.29 Complete the extraction worksheet, attached to this document, and tape in study notebook or include in study binder, as appropriate. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 8 of 14 Page 80 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 1 3 .0 D a t a A n a l y s is a n d C a l c u l a t io n s _______________________________________________________ 13.1 Calculations: 13.1.1 Calculate the average density of the liver homogenate by recording each mass of ten separate 1.0 mL aliquots of homogenate. Average density (mg/mL) = Average mass (mg) of the aliquots 1.0 mL aliquot 13.1.2 Calculate the amount o f liver (mg) per 1.0 mL homogenate (or concentration of dispersed solid tissue per mL of homogenate suspension) using the following equation: g o f L iver x A verage density* o f hom ogenate (m g/m L) (g of Liver + g of Water) * refer to 13.1.1 for details. 13.1.3 Calculate actual concentrations o f PFOS and other fluorochemicals in calibration standards using the following equation: llL o f Standard x Concentration tug /mL') = Final Concentration (pg/g or mg/kg) mg Liver/ 1 mL homogenate* of PFOS in Liver *refer to 13.1.2 for details. 1 4 .0 Method Performance ______ ______________________________________________ ________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit o f quantitation (LOQ) values (refer to Attachments B and C). 14.2 The following quality control samples are extracted with each batch of samples to evaluate the quality o f 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 o f the extraction. 14.2.3 Continuing calibration verification samples to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 o f ETS-8-7.0 for method performance criteria. 1 5 .0 P o l l u t i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t ________________________ ____________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in * high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 3M Environmental Laboratory ETS-8-6.0 Extraction o f PFOS from Liver Page 9 of 14 Page 81 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 1 6 .0 R e c o r d s _______________ ___________________ ____________________________________ _-- :---------- 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. 1 7 -0 T a b l e s . D ia g r a m s . F l o w c h a r t s , a n d V a l i d a t io n D a t a ____________________ ________ 17.1 Attachment A, Extraction worksheet 17.2 A tta c h m e n t B, MDL/LOQ v a lu e s a n d s u m m a ry 17.3 Attachment C, Calibration standard calculation and concentration worksheet 18.0 REFERENCES_______________________________ _______________________________ ____ 18.1 The validation report associated with this method is ETS-8-6.0 & 7.0-V-l. 18.2 AMDT-EP-22, "Routine Maintenance of Ultra-Turrax T-25" 18.3 FACT-M-1.1, "Extraction o f PFOS or Other Anionic Fluorochemical Surfactants from * Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 19.0 A f f e c t e d D o c u m e n t s _________ --------------- -------------------------------------------------------------------19.1 ETS-8-7.0, "Analysis of Liver Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 2 0 .0 R e v i s i o n s _________________ ____________________________ R evision N um ber. ' .. R eason F or R evision R evision D ate 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 10 of 14 Page 82^ 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Attachment B: MDL/LOQ Values 3M Environmental Laboratory . ETS-8-6.0 Extraction of PFOS from Liver Page 11 o f 16 Page-83 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 MDL/LOQ values for rabbit liver Compound MDL (PPb) LOQ Linear Calibration Range (LCR) (Ppb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS PFOSA PFOSAA EtFOSE-OH 8.45 3.50 24.6 108 26.9 30 p p b - 1200 ppb 11.1 12 ppb - 1200 ppb 78.3 30 ppb - 1200 ppb 345 60 ppb - 900 ppb* M556 PFOSEA 82.3 262 60 ppb - 1200 ppb 33.9 108 30 p p b -1200 ppb MDL/LOQ values in rat, bovine, and monkey liver were not statistically determined. Two curves in each of these matrices were extracted and analyzed with the rabbit liver curves to determine equivalence. Responses in the rat, bovine, and monkey liver curves were equivalent to the rabbit responses, therefore, their MDL and LOQ will be assumed to be equivalent to those values as determined for the rabbit liver. Refer to LOQ Summary and MDL study in ETS-8-6.0 & 7.0-V-l for further information * EtFOSE-OH estimates only for MDL and LOQ. Did not meet criteria for validation. Compound: PFOS ______________ _______ Prepared 1 Range o f LCR from Liver range o f average ave curve matrix standards (ppb) (ng/mL) curve (ppb) (ng/mL) (ppb) (ng/mL) ________ :________________ Range o f LCR from Range o f low std low std high std curve curve curve (ppb) (ng/m L) (ppb) (ng/mL) (ppb) (ng/mL) Rabbit 6.19 - 1237 12 - 1200 12 - 1200 6-300 12 - 300 60 - 1200 LCR from high std curve (ppb) (ng/mL) 60 - 1200 Compouind: PFOSA Prepared Liver matrix range of standards (ppb) (ng/mL) Range o f average curve (P P b ) (ng/mL) Rabbit 6 .1 9 -1 2 3 7 12 - 1200 LCR from ave curve (ppb) (ng/mL) 12-1200 Range of low std curve (ppb) (ng/mL) 12-300 LCR from low std curve (ppb) (ng/mL) 12 - 300 Range of high std curve (ppb) (ng/mL) LCR from high std ...... curve (ppb) (ng/mL) 60 - 1200 60 1200 Compound: PFOSAA Prepared Range o f Liver range of average matrix standards curve (ppb) (ng/mL) (ppb) (ng/mL) Rabbit 6 .1 6 -1 2 3 2 12 - 1200 LCR from aye curve (ppb) (ng/mL) 30-1200 Range o f low std curve (ppb) (ng/mL) 30 - 900 LCR from low std curve . (ppb) (ng/mL) 60-900 Range of high std curve (ppb) (ng/mL) N /A _ LCR from high std curve (ppb) (ng/mL) ' N/A Attachment B: MDL/LOQ Values 3M Environmental Laboratory ETS-8-6.0 Extraction o f PFOS from Liver Page 12 o f 16 Page 84 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Compound: EtFOSE-OH Prepared Range o f Liver matrix range o f standards (ppb) (ng/mL) average curve (ppb) (ng/m L) Rabbit 6.17-1235 31 -9 0 0 LCR from ave curve (ppb) (ng/mL) 31 -9 0 0 Range o f low std curve (ppb) (ng/mL) N /A LCR from low std curve (ppb) (ng/mL) N /A Range o f high std curve (ppb) (ng/mL) N /A LCR from high std curve (ppb) (ng/mL) N /A Com pou nd: PFO SEA Prepared Range o f Liver range of average matrix standards (ppb) (ng/mL) curve (ppb) (ng/m L) Rabbit 6.17-1235 3 1 - 1200 LCR from ave curve (ppb) (ng/mL) 31-1200 Range o f low std curve (ppb) (ng/mL) N /A LCR from low std curve (ppb). (ng/mL) N /A Range of high std curve (ppb) (ng/mL) N /A LCR trom high std curve (ppb) (ng/mL) . N/A. ' C o m p o u n d : M556 Prepared Liver matrix range o f standards (ppb) (ng/mL) Rabbit 6 .1 7 -1 2 3 5 Range o f average curve (ppb) (ng/m L) 31 - 1200 .LCR from ave curve (ppb) (ng/mL) 60-1200 Range o f low s td curve (ppb) (ng/mL) N /A LCR from low std curve (ppb) (ng/mL) N /A 7 Range of high std curve (ppb) (ng/mL) LCR from high std. - curve (ppb) (ng/mL) N/A . N /A . Attachment C: Standard Calculations ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 13 o f 14 Page 85 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Ion Pair Standard Curves - Tissue Prep date(s): A nalyte(s): Sam ple m atrix: Method/revision: T arg et analyte(s): F C m ix std approx. 0.500 ppm : FC m ix std approx. 5.00 ppm : F C m ix std approx. 50.0 ppm : S u rro g ate std ap p ro x . 100 ppm : Standard num ber: E quipm ent num ber: F inal solvent and TN: B lank liver/identifier: A ctual concentrations of stan d ard s in the FC m ix PFOS Std cone ug/mL PFOSA Std cone ug/mL PFOSAA Std cone ug/mL EtFOSE Std cone ug/mL PFOSEA Std cone ug/mL 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 5.00 5.00 0.500 0.500 0.500 5.00 5.00 0.500 0.500 0.500 5.00 5.00 0.500 0.500 0.500 5.00 5.00 0.500 0.500 0.500 5.00 5.00 5.00 5.00 5.00 5.00 5.00 50.0 50.0 50.0 50.0 50.0 M556 Std cone ug/mL 0.500 0.500 0.500 0.500 0.500 5.00 5.00 5.00 50.0 Std cone ug/mL All Am't spiked mL 0.002 0.004 0.010 0.020 0.040 0.010 0.020 0.030 0.004 All Density g 0.167 0.167 0.167 0.167 0.167 0.167 0.167 0.167 0.167 Calculated concentrations of standards in the sample matrix PFOS Final cone PFOSA Final cone PFOSAA Final cone ng/g EtFOSE Final cone PFOSEA Final cone M556 Final cone Std cone ng/g ng/g 5.99 12.0 29.9 59.9 120 299 599 898 1198 ng/g 5.99 12.0 29.9 59.9 120 299 599 898 1198 5.99 12.0 29.9 59.9 120 299 599 898 1198 ng/g 5.99 12.0 29.9 59.9 120 299 599 898 1198 ng/g 5.99 12.0 29.9 59.9 120 299 599 898 1198 ng/g 5.99 12.0 29.9 59.9 120 299 599 898 1198 Surrogate Std cone ng/mL too Surrogate Final cone ng/mL 0.500 All Am't spiked mL 0;005 Liver Rabbit Bovine Rat Monkey PFOS PFOSA 5-1000ppb 5-1000ppb Estimates only, userabbitvalues. Estimates only, userabbitvalues. Estimatesonly, userabbit values. PFOSAA 5-1000ppb EtFOSE-OH 5-1000 ppb POAA PFOSEA 5-1000ppb 5-1000ppb _____ ___------- -------________ ________ ................. ............ .-- Attachment C: Standard Calculations ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 14 of 14 Page 86 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 3M E n v ir o n m e n t a l L a b o r a t o r y M ethod Analysis of Fluorochem icals in Liver Extracts U sing H PLC -Electrospray/M ass Spectrom etry Method Number: FACT-M-2.0 Author: Lisa Clemen Approved By: /H ^ Laborattonrryv MM aannaagaeerr /UsIa*. I b z --- -------" / ...... Group Leader Technical Reviewer Adoption Date: S/3( lc>s? Revision Date: fjft A DDaatete S Date r s h ih a Date 1.0 Scope and Application 1.1 Scope: This method is for the analysis of extracts of liver or other tissues for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Potassium perfluorooctanesulfonate, anionic fluorochemical surfactants, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, and monkey livers or other livers as designated in the validation report. Word 7.0.1/95 FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 1 of 8 3M Environmental Laboratory Page-87- 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 2.0 Summary of M ethod_______ ___________________________________________ 2.1 This method describes the analysis of fluoroehemical surfactants extracted from liver using HPLC-electrospray/mass spectrometry. The analysis is performed by monitoring a single ion characteristic of a particular fluoroehemical, such as the potassium perfluorooctanesulfonate (PFOS) anion, M/Z= 499. Samples may also be screened to verify compound identification. 3.0 Definitions________________ ___________________________________________ ______ 3.1 None. 4.0 W arnings and Cautions ____________________________ ____________ 4.1 H ealth and Safety W arnings: 4.1.1 Use caution with the voltage cable for the probe. When the voltage cable is plugged into the probe DO NOT TOUCH THE PROBE, there is risk of electrical shock. 4.2 Cautions: 4.2.1 Do not run solvent pumps above capacity of 400 bar (5800 psi). If pressure goes over 400 bar, the HP 1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences_____________________________________________________ ________ 5.1 Teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 E quipment__________ ___________ __________________________________ ________ 6.1 Equipment listed below may be changed in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HP 1100 low pulse solvent pumping system and autosampler. 7.0 Supplies and M aterials________ :________________ ;_______________ ___________ _ 7.1 Supplies 7.1.1 Nitrogen gas, refrigerated liquid, regulated to approximately 100 psi. 7.1.2 HPLC column, specifics to be determined by the analyst. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes. 8.0 Reagents and Standards _________________________________ _________ _-- 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent. Word 7.0.1/95 FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 2 of 8 3M Environmental Laboratory Page 88^ 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC Plus system. 8.1.3 Ammonium acetate, HPLC grade or equivalent. 8.2 Standards 8.2.1 Typically one H20 blank, one liver blank, and seven liver standards are prepared during the extraction procedure. See FACT-M-1. 9.0 Sample H andling ___________ _________________________________________ ______ 9.1 Fresh liver 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 may be refrigerated until analysis can be performed. 10.0 Quality Control ____________________________________________ ___ _______ 10.1 Matrix Blanks and Method Blanks 10.1.1 Analyze a method blank and matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Analyze a matrix spike and matrix spike duplicate with each analysis. 10.2.2 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the low-range of the initial calibration curve. 10.2.3 See section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 10.3.1 Analyze a mid-range calibration standard after every tenth sample. If a significant change ( 30%) in peak area occurs, relative to the initial standard curve, stop the run. Only those samples analyzed before the last acceptable calibration standard will be used. The remaining samples must be reanalyzed. 10.3.2 See section 13 to calculate percent difference. 10.4 System Suitability 10.4.1 System suitability (e.g. peak area, retention time and peak shape, etc.) will be assessed for each run. 11.0 C alibration and Standardization------------------------------------------ ------------------------ 11.1 Analyze the extracted liver standards prior to and following each set of extracts. The mean ' of two standard values, at each standard concentration, will be plotted by linear regression for the calibration curve using MassLynx or other suitable software. 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 3 of 8 Page"89 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 11.2 The r2value for the data should be 0.98 or greater. Lower values may be acceptable at the discretion of the analyst. 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 12.0 P rocedures_____________________________________________________________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using letter-MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A scan is usually collected along with the SIRs. Save method. 12.1.3 Typically the sample list begins with the first set o f liver standards and ends with the second set of standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection with a sample wash 12.2.2.2 Inject/sample =1 12.2.2.3 Cycle time =15 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 7.5 min. 11.0 min. 11.5 min. MeOH 45% 90% 90% 45% 2.0 mM Ammonium acetate 55% 10% 10% 55% Note: In this instrument configuration, the run must be set up on the electrospray software with a "Waiting for inlet start" message before the "Start" button is pressed on the HP Workstation. 12.2.2.5 Press the "Start" button. FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 4 of 8 3M Environmental Laboratory -Page~9fr 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 12.3 Instrument Sep-up 12.3.1 Refer to AMDT-EP-31 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eye piece 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 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 LC constant flow mode flow rate 1 0 -5 0 0 uL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the instrument is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Record tune parameters in the instrument log. 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis of biological matrices. 12.3.10 Click on start button in the Acquisition Control Panel. Press the start button at top of sample list. Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Ca lc u la tio n s____________________________ ______________ 13.1 Calculations: 13.1.1 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.2 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 5 of 8 Page-91 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 13.1.3 Calculate actual concentration o f PFOS anion in total liver (mg): ug PFOS anion calc, from std curve^ x Total mass of liver (g) 14.0 M ethod P erformance ______________________________________'___________ 14.1 The method detection limit is equal to at least three times the baseline noise in the matrix blank. 14.2 The practical quantitation limit is equal to the lowest standard in the calibration curve. 15.0 P ollution P revention and W aste M anagement______________________ ________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers. All containers are located in the laboratory. 16.0 R ecords__________________ ________________________________________ ________ 16.1 Store chromatograms in the study folder. Each chromatogram should have the following information included either in the header or hand written on the chromatogram: study number, sample name, extraction date, and dilution factor (if applicable). 16.2 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample list from MassLynx and tape into the instrument runlog. 16.4 Print data integration summary from MassLynx and tape into the instrument runlog. 16.5 Copy instrument runlog pages, including instrument parameters and sample results, and tape into appropriate study notebook. 16.6 Summarize data using suitable software and store in the study folder. 16.7 Back up electronic data to appropriate media. Record in study notebook the file name and location of backup electronic data. 17.0 T ables, Diagrams, F lowcharts, and Validation Data_____________________ _ 17.1 Attachment A: FACT-M-2 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT:M-1.0 & 2.0-V -l. 18.0 R eferences ____________ _____________________________________________ _ 18.1 AMDT-EP-31, "Operation of VG Platform Electrospray Mass Spectrometer" 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 6 of 8 Page 92 3M M edical Departm ent Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 19.0 Affected Documents _________________________________________ ' 19.1 FACT-M-1.0, "Extraction of Potassium Perfluorooctanesulfonate from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 Revisions Revision Miimher. Reason For Revision Revision Date 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 7 of 8 Page-93- 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date o f Extraction/Analyst: Date o f Analysis/Analyst: Group Dose S am p le# Concentration ug/m L Initial Vol. mL Dilution Factor Final Cone. u g/m L Slope: Taken from linear regression equation. G roup/D ose: Taken from the study folder. Sample#: Taken from the study folder. C oncentration (ug/m L): Taken from the MassLynx integration summary. Initial V olum e (m L): T a k e n from the study folder. D ilution Factor: Taken from the study folder. Final Cone. (ug/m L): Calculated by dividing the initial volume from the concentration 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 8 of 8 Page 94 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 3M Environment at.Labor a t o p v M ethod Analysis of Fluorochem icals in Serum Extracts U sing H PL C -E lectrospray/M ass Spectrom etry Method Number: FACT-M-4.0 Author: Lisa Clemen Approved By: Adoption Date: Revision Date: \)U L/ / W 9 f Date it_C Technical Reviewer Date h I ihIi s Date 1.0 Scope and Appu ca tio n ______ ____________________ 1.1 Scope: This method is for the analysis o f extracts o f serum or tissue for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Potassium perfluorooctanesulfonate, anionic fluorochemical surfactants, or other ionizable compounds. 1.3 M atrices: Rabbit, rat, and bovine serum or other sera as designated in the validation report. Word 7.0.1/95 SMEnWonmentartaboratoy FACT-M-4.0 Analysis o f Serum Extract Using ES/MS Page 1 o f 8 Page 95 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 2 .0 Su m m a r y o f M e t h o d _____________________________________________________________ ;________ 2.1 This method describes the analysis of fluorochemical surfactants extracted from serum using HPLC-electrospray/mass spectrometry. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the potassium perfluorooctanesulfonate (PFOS) anion, M/Z= 499. Samples may also be screened to verify compound identification. 3 .0 Definitions_________________ '____________________________________________________________ 3.1 None. 4 .0 W a r n in g s a n d C a u t io n s ___________________________________________________________________ 4.1 Health and Safety W arnings: 4.1.1 Use caution with the voltage cable for the probe. When the voltage cable is plugged into the probe DO NOT TOUCH THE PROBE, there is risk of electrical shock. 4.2 Cautions: 4.2.1 Do not run solvent pumps above capacity of 400 bar (5800 psi). If pressure goes over 400 bar, the HP 1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5 .0 I n t e r f e r e n c e s ____________________________________________________________________ __ 5.1 Teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6 .0 E q u i p m e n t __________________ ;________.________________________________________________________ 6.1 Equipment listed below may be changed in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HP 1100 low pulse solvent pumping system and autosampler. 7 .0 S u p p l i e s a n d M a t e r ia l s ___________________________________________________________________ 7.1 Supplies 7.1.1 Nitrogen gas, refrigerated liquid, regulated to approximately 100 psi. 7.1.2 HPLC column, specifics to be determined by the analyst. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes. 8 .0 R e a g e n t s a n d S t a n d a r d s _____________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent. 3M Environmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 2 of 8 Page 96 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water and may . be provided by a Milli-Q TOC Plus system. 8.1.3 Ammonium acetate, HPLC grade or equivalent. 8.2 Standards 8.2.1 Typically one H20 blank, one serum blank, and seven serum standards are prepared during the extraction procedure. SeeFACT-M-3. 9.0 Sample H andling________ ____________________________________________________ 9.1 Fresh serum 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 may be refrigerated at 4 C until analysis can be performed. 10.0 Quality Control________________________________________________ __________ 10.1 Matrix Blanks and Method Blanks 10.1.1 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Analyze a matrix spike and matrix spike duplicate with each analysis. . 10.2.2 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the low-range of the initial calibration curve. 10.2.3 See section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 10.3.1 Analyze a mid-range calibration standard after every tenth sample. If a significant change ( 30%) in peak area occurs, relative to the initial standard curve, stop the run. Only those samples analyzed before the last acceptable calibration standard will be used. The remaining samples must be reanalyzed. 10.3.2 See section 13 to calculate percent difference. 10.4 System Suitability 10.4.1 System suitability (e.g., peak area, retention time, peak shape, etc.) will be assessed for each run. 11.0 Calibration and Standardization___________________________________________ 11.1 Analyze the extracted serum standards prior to and following each set of extracts. The mean of two standard values, at each standard concentration, will be plotted by linear regression for the calibration curve using MassLynx or other suitable software. 3M Environmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 3 of 8 Page 97 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 11.2 The r2value for the data should be 0.98 or greater. Lower values may be acceptable at the discretion of the analyst. 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 12.0 P rocedures___________ ;_____________________________________________________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using letter-MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A scan is usually collected along with the SIRs. Save method. 12.1.3 Typically the sample list begins with the first set of serum standards and ends with the second set of standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection with a sample wash 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 15 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 7.5 min. 11.0 min. 11.5 min. MeOH 45% 90% 90% 45% 2.0 mM Ammonium acetate 55% 10% 10% 55% Note: In this instrument configuration, the run must be set up on the electrospray software with a "Waiting for inlet start" message before the "Start" button is pressed on the HP Workstation. 12.2.2.5 Press the "Start" button. FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 4 of 8 3M Environmental Laboratory Page 98 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 12.3 Instrument Set-up 12.3.1 Refer to AMDT-EP-31 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eye piece 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 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 HPLC constant flow mode flow rate 10 --500 pL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Record tune parameters in the instrument log. 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis of biological matrices. 12.3.10Click on start button in the Acquisition Control Panel. Press the start button at top of sample list. Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations_______________________________ :____________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 3M Environmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 5 of 8 Page 99 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, anion in serum (pg/mL): jag of PFO calc, from std. Curve x Dilution Factor x Final Volume (mL) Initial Volume of serum (mL) 1 4 .0 M e t h o d P e r f o r m a n c e ____________________________________________ ;__________________ 14.1 The method detection limit is equal to half the lowest standard in the calibration curve. 14.2 The practical quantitation limit is equal to the lowest standard in the calibration curve. 1 5 .0 P o l l u t i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t _____________________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 1 6 .0 R e c o r d s __________________________________________________________________________________ 16.1 Store chromatograms in the study folder. Each chromatogram must have the following information included either in the header or hand written on the chromatogram: study number, sample name, extraction date, and dilution factor (if applicable). 16.2 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample list from MassLynx and tape into the instrument runlog. 16.4 Print data integration summary from MassLynx and tape into the instrument runlog. 16.5 Copy instrument runlog pages, including instrument parameters and sample results, and tape into appropriate study notebook. 16.6 Summarize data using suitable software and store in the study folder. 16.7 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 1 7 .0 T a b l e s . D ia g r a m s . F l o w c h a r t s , a n d V a l i d a t io n D a t a _____________________________ 17.1 Attachment A: FACT-M-4 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT-M-3.0 & 4.0-V-l. 1 8 .0 R e f e r e n c e s ________________________________________________________________________ 18.1 AMDT-EP-31, "Operation of VG Platform Electrospray Mass Spectrometer" 1 9 .0 Affected Documents________________________________________ ;______________ 19.1 FACT-M-3.0, "Extraction of Fluorochemical Anions from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 3M Environmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 6 of 8 Page 100 3M Medical Department Study: T-6295.4 20.0 R e v isio n s Revision Number. Reason For Revision Analytical Report: FACT TOX-002 LRN-U2121 Revision Date 3M Environmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 7 of 8 Page 101 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date o f Extraction/Analyst: Date o f Analysis/Analyst: Group Dose Sam ple# Concentration ug/m L Initial Vol. mL Dilution Factor Final Cone. ug/m L Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. C oncentration (ug/m L): Taken from the MassLynx integration summary. Initial Volum e (mL): Taken from the study folder. D ilution Factor: Taken from the study folder. Final Cone. (ug/m L): Calculated by dividing the initial volume from the concentration 3M Environmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 8 of 8 Page 102 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 3M Environmental Laboratory M ethod Analysis of P otassium Perfluorooctanesulfonate o r O ther Fluorochem icals in Serum Extracts Using H PL C -E lectrospray/M ass Spectrometry Method Number: ETS-8-5.1 Author: Lisa Clemen, Robert Wynne Approved By: Adoption Date: 03/01/99 Revision Date: Laboratory Manager Group Leader __A CllAnt*-___________ _______________________ Technical Reviewer Date Date Date 1.0 Scope and Application____________ __________________________________________ 1.1 Scope: This method describes the analysis o f serum extracts for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey, and human serum, or other fluids as designated in the validation report. Word 6/95 ETS-8-5.1 Analysis o f Seram Extract Using ES/MS Page 1 o f 9 SM'Environmental Laboratory Page 103 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 2.0 Summary of M ethod______________________________________ __________________ 2.1 This method describes the analysis of fluorochemical surfactants extracted from serum or other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z= 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity o f a compound by detecting daughter ions of the parent ion. 3.0 Definitions________________________ ________________________ ________ _____ 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e., not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method o f ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II triple quadrupole systems are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass Quattro II triple quadrupole systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods o f operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e., Z-spray components are compatible with some other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation o f these Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details see the manual specific to the instrument (Micromass Quattro II triple quadrupole MassLynx or MassLynx NT User's Guide). 4.0 W arnings and Cautions_____________ ______________________________________ 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. E T S -8 -5 .1 Analysis o f Serum Extract Using ES/MS Page 2 o f 9 3M Environmental Laboratory------ Page 104 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 4.2 Cautions: 4.2.1 Do not operate solvent pumps above capacity of 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HP 1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 I nterferences__________________________________________________________ 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 E quipment__________________________ __________________ ______________________ 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 6.1.2 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an electrospray ionization source H P1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 Supplies and M aterials_______________ ________________ __________ ____________ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (House air system) 7.1.2 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes 8.0 Reagents and Standards__________ ______________________ ____________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. See ETS-8-4.1. 9.0 Sample Handling_______________________________________ _____________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 3M EnvironmentalLaboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 3 o f 9 P a g e 105 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C, or at room temperature, until analysis can be performed. 10.0 Q uality Control____________ ___________________________ 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty samples, with a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lowrange of the initial calibration curve. 10.3 Continuing Calibration Verifications 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy o f the calibration curve. ' 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a m in im u m of one per batch. 11.0 Calibration and Standardization _______________________________ ________ 11.1 Analyze the extracted matrix standards prior to and following each set o f extracts. The average o f two standard curves will be plotted by linear regression (y = my + b), weighted 1/x, not forced through zero, using MassLynx or other suitable software. 11.2 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.3 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end o f the calibration curve rather than the full range of the standard curve. Example: when attempting to quantitate approximately 10 ppb o f analyte, generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting o f high concentration standards. ^MEnvtronmentaftaboratery ETS-8-5.1 Analysis o f Serum Extract Using ES/MS Page 4 of 9 Page 106 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 12.0 P rocedures _________________ ____________________________________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using MO-DAY-last digit o f year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording) or MRM. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). 12.1.3 Typically the analytical batch run sequence begins with a set of extracted matrix standards and ends with a set o f extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosam pler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HP 1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 p,L injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 8.50 min. 11.0 min. 12.0 min. MeOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% 10% 10% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrum ent Set-up 12.3.1 Refer to ETS-9-24.0 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. ETS-8-5.1 Analysis o f Serum Extract Using ES/MS Page 5 of 9 Page 107 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to On . Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip o f the probe. Readjust the tip of the probe if no mist is observed. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 HPLC constant flow mode, flow rate 1 0 -5 0 0 pL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis of biological matrices. 12.3.10Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, see appropriate MassLynx USER'S GUIDE). Press the start button. Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and C alculations___________________________ ._________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration o f PFOS, or other fluorochemical, in matrix (pg/mL): (ng of PFOS calc, from std. Curve x Dilution Factor! x 1 qg (Initial Volume of matrix (mLl + mL of Surrogate Standard! 1000 ng Final Volume (mL) ETS-8-5.1 Analysis o f Serum Extract Using ES/MS Page 6 o f 9 3M'EnvironmentaFfcaboratory Page 108 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 14.0 M ethod P erformance 14.1 Method Detection Limit (MDL) and Limit o f Quantitation (LOQ) are method, analyte and matrix specific. Please see ETS-8-4.1, Attachment B, for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix blanks values are must be below the lowest standard in the calibration curve 14.3 Calibration Curves 14.3.1 The r2value for the calibration curve must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries are must be within 30% o f the spiked concentration. 14.5 Continuing Calibration Verifications 14.5.1 Continuing calibration verification percent recoveries must be 30% o f the spiked concentration. 14.6 I f criteria listed in this method performance section isn't met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 14.7 I f data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text o f the report. 15.0 P o l l u t i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t _____________ ________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R e c o r d s ______________________ ___________ ________ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable) and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, from MassLynx and store in the study folder. ' ETS-8-5.1 Analysis o f Serum Extract Using ES/MS Page 7 o f 9 Page 109 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 16.5 Summarize data using suitable software (Excel 5.0) and store in the study folder, see A ttachm ent A for an example of a summary spreadsheet. ' 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location o f backup electronic data. 17.0 T a b l e s , D ia g r a m s , F l o w c h a r t s , a n d V a l i d a t io n D a t a 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet. ______________________ 18.0 R e f e r e n c e s ______________________ ___________ _____________________________ 18.1 FACT-M-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple quadrupole Systems" 18.3 The validation report associated with this method is ETS-8-4.0 & 5.0-V -l. 19.0 A f f e c t e d D o c u m e n t s __________________________________ _______________ 19.1 ETS-8-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 R evisions________________________ ____________ ____ _________ Revision Number. 1 Reason For Revision Section 6.1.2 Clarification o f HP1100 system components. Section 11.1 Average of two curves, not standard values, are used for plotting linear regression and added the 1/x weighting o f the curve. Section 12.2.2.4 Clarification o f solvent ramp. Section 17.1 Changed from attachment B to A. Revision Date 04/02/99 SMEnvtronmentaftaboratory m i-s-ru Analysis o f Serum Extract Using ES/MS Page 8 o f 9 Page 110 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument Software/V ersion: Filename: R-Squared Value: Slope: Y Intercept: Date o f Extraction/Analyst: Date o f Analysis/Analyst: Group Dose S am p le# Concentration ug/m L Initial Vol. mL Dilution Factor Final Cone. ug/m L G roup/D ose: Taken from the study folder. Sample#: Taken from the study folder. C oncentration (ug/m L): Taken from the MassLynx integration summary. Initial V olum e (mL): Taken from the study folder. D ilution F actor: Taken from the study folder. Final C one. (ug/m L): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-5.1 Analysis o f Serum Extract Using ES/MS 31OTBnviTonmentat-b3boratery Page 9 o f 9 Page 111 3M Medical Department Study: T-6295.4 Study #: FACT-TOX-002 Analytical Report: FACT TOX-002 3M Environmental Lab --Method Modification M ethod: ETS-8-5.1 "A nalysis o f Potassium Perfluorooctanesulfonate or O ther Fluorochem icals in Sera Extracts U sing HPLC-Electrospray/M ass Spectrom etry" Section modified: Effective date of modifications: 10.3.2,14.5.1, add sections 14.3.2-14.3.6 April 26, 1999 Section 10.3.2______________________________________________________________ Method reads: 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum o f one per batch. Modify method to read: 10.3.2 Analyze a mid-range calibration standard at least after every ten samples, with a minimum o f one per batch. Section 14.5.1__________________________________________________________ Method reads: 14.5.1 Continuing calibration verification percent recoveries must be within 30% o f the spiked concentration. Modify method to read: 14.5.1 At least one continuing calibration verification per ten samples must show a percent recovery within +/-30% o f the spiked concentration. Section 14.3.2______________________________________________________________ Method reads: NA Modify method to read: 14.3.2 The second (bracketing) calibration curve may be deactivated if instrumental drift affects the data. The first curve and acceptable calibration checks shall bracket usable data. 3M Environmental Laboratory Page 112 3M Medical Department Study: T-6295.4 Study #: FACT-TOX-002 A fliltitiiiiA O ttiFA C U O X i0P 2 LRN-U2121 Section 14.3.3______________________________________________________________ Method reads: NA Modify method to read: 14.3.3 Calibration standards with peak areas less than 2 times the curve matrix blank should be deactivated to disqualify a data range that may be affected by background levels o f the analyte. Section 14.3.4______________________________________________________________ Method reads: NA Modify method to read: 14.3.4 Low or high curve points may be deactivated to optimize a linear range appropriate to the data. Section 14.3.5______________________________________________________________ Method reads: NA Modify method to read: 14.3.5 A curve point may be deactivated i f it deviates more than 30% from the theoretical value when the curve is evaluated over a linear range appropriate to the data. Section 14.3.6___________________ ;__________________________________________ Method reads: NA Modify method to read: X4.3.6 A valid calibration curve must contain at least 5 active points. . Signature of PAI and date \ i l Z 7 /o-u v 'J ' Signature of Sponsor and date 2. Signature of Study Director and date /J . 3M Environmental Laboratory Page 113 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 3M Environment at. Laboratory M ethod A nalysis o f P otassium P erflu o ro o cta n esu lfo n a te o r O t h e r F l u o r o c h e m ic a l s in L iv e r E x tra cts U sing H P L C -E lectrospra y/M ass Spectrom etry M ethod Number: ETS-8-7.0 Author: Lisa Clemen, Glenn Langenburg Approved By: '` Laboratory Manager - f b -------- Group Leader _A_______ _______________________________ Technical Reviewer Adoption Date: 0 2-X n A Revision Date: ?? / -a v Date W W /fa Date oilrtk^ Date 1.0 S c o p e a n d A p p l ic a t io n 1.1 Scope: This method is for the analysis of liver extracts for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 A pplicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 M atrices: Rabbit, rat, bovine, monkey liver, or other tissues as designated in the validation report. Word 6/95 ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 1 of 10 Page 114 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 2.0 S u m m a r y o f M e t h o d ________________________________________ ________________ __ ____________ 2.1 This method describes the analysis of fluorochemical surfactants extracted from liver using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z = 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity of a compound by detecting daughter ions o f the selected parent ion. 3.0 D e f in i t io n s _________________________________ '___________________ _____________________________ 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods o f ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e. not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of 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 o f a strong electrical field. 3.3 Mass Spectrom etry, Mass Spectrometer (MS), Tandem Mass Spectrom eter (MS/MS): The API Quattro II triple quadrupole mass spectrometer is equipped with two quadrupole mass selective detectors and a collision cell. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or an ion may be selected in the first quadrupole, fragmented in die collision cell, and these fragments may be analyzed in the second quadrupole. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass Quattro II triple quadrupole (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e. Z-spray components are compatible with other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details refer to the manual specific to the instrument (Micromass Quattro II triple quadrupole MassLynx or MassLynx NT User's Guide). 4.0 W a r n in g s a n d C a u t i o n s ____________________________ :_______ ._______________________ 4.1 Health and Safety W arnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. 3M-EnvifonmeutafLaboratory ETS-8-7.0 Analysis o f Liver Extract Using ES/MS Page 2 of 10 Page 115 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. 4.2 Cautions: 4.2.1 Operate the solvent pumps below a back pressure o f 400 bar (5800 psi). If the back pressure exceeds 400 bar, the HP 1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5 .0 I n t e r f e r e n c e s _________________________________________________ ______________________________ 5.1 To minimize interferences when analyzing samples, Teflon shall not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6 .0 E q u i p m e n t ________________________________________________________ __________________________ 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an electrospray ionization source. . 6.1.2 HP 1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7 .0 S u p p l i e s a n d M a t e r ia l s ________________________________________ .___________________________ 7.1 Supplies 7.1.1 High purity grade air regulated to approximately 100 psi (house air system) 7.1.2 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data 7.1.3 Capped autovials or capped 15 ml centrifuge tubes 8 .0 R e a g e n t s a n d S t a n d a r d s __________________________________________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent . 8.1.2 Milli-QTM water (ASTM type I), all water used in this method should be ATSM type I, or equivalent, and be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.1.3.1 When preparing different amounts than those listed, adjust accordingly. 8.1.3.2 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 mL volumetric container containing 2000 mL Milli-QTM water, mix until all solids are dissolved. Store at room temperature. ' ETS-8-7.0 Analysis o f Liver Extract Using ES/MS Page 3 o f 10 3M-Environmenfat-fcabratry------- ------------------------------- Page 116 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. Refer to ETS-8-6.0. 9.0 Sample H andling____________________________________________________________ 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 may be stored at room temperature, or refrigerated at approximately 4 C, until analysis can be performed. 10.0 Quality Control_________ '____________________________ _____________ ;________ 10.1 M ethod Blanks and M atrix Blanks 10.1.1 Solvent blanks, method blanks, and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 M atrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty sample^. 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 o f the initial calibration curve. 10.3 Continuing Calibration Checks 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard every tenth sample, with a minimum of one per batch. 11.0 C alibration and Standardization_____________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set o f sample extracts. The average of two standard curves will be plotted by linear regression (y = mx + b), weighted 1/x, not forced through the origin, using MassLynx or other suitable software. 11.2 If the curve does not meet requirements perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. ETS-8-7.0 Analysis o f Liver Extract Using ES/MS Page 4 o f 10 Page 117 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 11.3 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range of the standard curve. Example: when attempting to quantitate approximately 10 ppb of analyte, generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting o f high concentration standards. 12.0 P rocedures______________________ __________________________________________ 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 digit o f year-increasing letter o f the alphabet starting with a 12.1.1.2 Assign a method (MS file) for acquiring 12.1.1.3 Assign an HPLC program (Inlet file) 12.1.1.4 Type in sample descriptions and vial position numbers 12.1.2 To create a method click on method in the Acquisition control panel then mass spectrometer headings and select SIR (Single Ion Recording) or MRM (Multiple Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. Refer to Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM. 12.1.3 Typically the analytical batch run sequence begins and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration verification injected standard after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 9 minutes . 3MEnv ironmental-Laboratory ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 5 of 10 Page 118 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 12.2.2.4 Solvent ramp conditions Time MeOH 0.00 min. 1.0 min. 4.5 min. 6.5 min. 7.0 min. 9.0 mi. 40% 40% 95% 95% 40% 40% 2.0 mM Ammonium acetate 60% 60% 5% 5% 60% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0, "Operation and Maintenance of the Micromass Quattro II Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source," for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Turn on the nitrogen. 12.3.5 Open the tune page. Clicks on operate to initiate source block and desolvation heaters. 12.3.6 Open the Inlet Editor. 12.3.6.1 Set HPLC pump to "On" 12.3.6.2 Set the flow to 10 - 500 uL/min or as appropriate 12.3.6.3 Observe droplets coming out o f the tip of the probe. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip o f the probe if no mist is observed 12.3.6.4 Allow to equilibrate for approximately 10 minutes. 12.3.7 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.7.1 Drying gas 250-400 liters/hour 12.3.7.2 ESI nebulizing gas 10-15 liters/hour 12.3.7.3 HPLC constant flow mode flow rate 10 --500 pL/min 12.3.7.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7.5 Source block temperature 150 12.3.7.6 Desolvation temperature 250 ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 6 o f 10 3IMEnvifonmental Laboratory Page 119 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, refer to appropriate MassLynx User's Guide). Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations 13.1 Calculations: ______________________________ 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery - Observed Result - Background Result x 100 Expected Result ' 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentrations in matrix (jug/g): fag of PFQS calc, from std. Curve x Dilution Factor! x 1 fxg (Initial Weight of Liver fa) 1000 ng Final Volume (mL) 14.0 M ethod Performance_______________________________________________________ 14.1 Method Detection Limit (MDL) and Limit o f Quantitation (LOQ) are method, analyte, and . matrix specific. Refer to ETS-8-6.0, A ttachm ent B for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, M ethod Blanks and M atrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix blanks must be below the lowest standard in the calibration curve. . 14.3 C alibration Curves 14.3.1 The r2value for the calibration must be 0.980 or better. 14.4 M atrix Spikes 14.4.1 Matrix spike percent recoveries must be within 30% of the spiked concentration. 14.5 C ontinuing C alibration Verification 14.5.1 Continuing calibration verification percent recoveries must be within 30% o f the . spiked concentration. 14.6 If criteria listed in the 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. 3M- Environmental Laboratory ETS-8-7.0 Analysis o f Liver Extract Using ES/MS Page 7 o f 10 Page 120 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 P o l l u t io n P r e v e n t io n and W a st e M a n a g e m e n t _______________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 1 6 .0 R e c o r d s _______ :____________________________;__________ ;_________________ ^_______ ;____________ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms from MassLynx and store in the study folder. 16.5 Summarize data using suitable software (Excel 5.0+) and store in the study folder, refer to A ttachm ent A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location o f backup electronic data. 1 7 .0 T a b l e s . D ia g r a m s . F l o w c h a r t s , a n d V a l i d a t io n D a t a ______________________________ 17.1 Attachment A: ETS-8-7.0 Data summary spreadsheet 1 8 .0 R e f e r e n c e s _______________ ;_________________________________________________________________ 18.1 FACT-M-2.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple quadrupole Systems" 18.3 The validation report associated with this method is ETS-8-6.0 & 7.0-V-l 1 9 .0 A f f e c t e d D o c u m e n t s _____________________________________________________________________ 19.1 ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver or Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" 3M Environ-mental Laboratory ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 8 of 10 Page 121 3M Medical Department Study: T-6295.4 20.0 R e v isio n s Revision Number Analytical Report: FACT TOX-002 LRN-U2121 Reason For Revision Revision Date 3M 'ENviionmental'Laboratory ETS-8-7.0 Analysis o f Liver Extract Using ES/MS Page 9 o f 10 Page 122 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Num ber Instrument Software/V ersion: Filename: R-Squared Value: Slope: Y Intercept: Date o f Extraction/Analyst: Date o f Analysis/Analyst: Group Dose Sample# Concentration ng/g Initial Wt. g Dilution Factor ----- Final Cone.----ng/g Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. C oncentration (ng/g): Taken from the MassLynx integration summary. Initial W t. (g): Taken from the study folder. Dilution Factor: Taken from the study folder. Final C one, (ug/g): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-7.0 Analysis of Liver Extract Using ES/MS 3M'Environmental Laboratory Page 10 of 10 Page 123 3M Medical Department Study: T-6295.4 Study #: FACT-TO X-002 MA n a y j a l i o r t t C T i T &2 LRN-U2121 3M Environmental Lab -- Method Modification M ethod: ETS-8-7.0 "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochem icals in Liver Extracts Using HPLC-Electrospray/M ass Spectrom etry" Section modified: Effective date of modifications: 10.3.2,14.5.1, add sections 14.3.2-14.3.6 July 22, 1999 Section 10.3.2____________________________________ ^________________________ Method reads: 10.3.2 Analyze a mid-range calibration standard after every tenth sample, w ith a m inim um o f one per batch. Modify method to read: 10.3.2 Analyze a mid-range calibration standard at least after every ten samples, w ith a m inim um o f one per batch. Section 14.5.1_____________________________________________________________ Method reads: 14.5.1 Continuing calibration verification percent recoveries must be w ith in 30% o f the spiked concentration. Modify method to read: 14.5.1 A t least one continuing calibration verification per ten samples must show a percent recovery w ith in +/-30% o f the spiked concentration. Section 14.3.2_____________________________________________________________ Method reads: NA Modify method to read: 14.3.2 The second (bracketing) calibration curve may be deactivated i f instrumental d rift affects the data. The firs t curve and acceptable calibration checks shall bracket usable data. 3M Environmental Laboratory Page 124 3M Medical Department Study: T-6295.4 Study #: FACT-TOX-002 Analytical Report: FACT TOX-002 L & k -J ffl Section 1 4 .3.3_________________________________________________________ ___ Method reads: NA Modify method to read: 14.3.3 Calibration standards with peak areas less than 2 times the curve matrix blank should be deactivated to disqualify a data range that may be affected by background levels o f the analyte. Section 14.3.4_________________ _____________________________________________ Method reads: NA Modify method to read: 14.3.4 Low or high curve points may be deactivated to optimize a linear range appropriate to the data. Section 14.3.5_____________________________________________________________ Method reads: NA Modify method to read: 14.3.5 A curve point may be deactivated if it deviates more than 30% from the theoretical value when the curve is evaluated over a linear range appropriate to the data. Section 14.3.6_____________________________________________________________ Method reads: NA Modify method to read: 14.3.6 A valid calibration curve must contain at least 5 active points. iL Signature of PAI and date li/2 4 /O Signature of Sponsor and date Signature of Study Director and date SJ 3M Environmental Laboratory Page 125 3M Medical Department Study: T-6295.4 Appendix D: Data Summary Tables Analytical Report: FACT TOX-002 LRN-U2121 3M Environmental Laboratory Page 126 3M M edical Departm ent Study: T-6295.4 OM VS 3M Environm ental Laboratory Table 7. FACT TOX-002 Data Summary of PFOS Concentration--Serum (^g/mL) Tim e point Sex G roup 1 C ontrol Average SD G roup 2 Low Average SD G roup 3 Mid Average SD G roup 4 M id-High Average SD G roup 5 H ig h Average SD G roup 6 High Recovery Average SD Week 4a Week 14a Male Female Male Female 0.0259 0.00663 (n = 5) <LOQc (n = 5) 2.67 4.58 (n = 5) 0.907 0.0619 (n = 5) 1.61 0.207 (n = 5) 4.04 0.801 (n = 5) 6.96 0.993 (n = 4d) 4.33 1.16 (n = 5) 6.62 0.499 (n = 5) 17.1 1.22 (n = 5) 27.3 2.34 (n = 5) 7.57 2.17 (n = 5) 12.6 1.73 (n = 5) 43.9 4.90 (n = 5) 64.4 5.48 (n = 5) 41.8 7.92 (n = 5) 54.0 7.34 (n = 5) 148 13.8 (n = 5) 223 22.4 (n = 5) Week 53 Day 719 Week 105 Week 106 Male Female Male Female Male Female Male Female 0.0249 0.0182 (n = 5) 0.395 0.777 (n = 5) 0.0118 0.0104 (n = 11) 0.0836 0.134 (n = 24) 1.31 1.30 (n = 10) 4.35 2.78 (n = 15) 20.2 13.3 (n = 9) 7.60 8.60 (n = 17) 22.5 23.5 (n = 25) 75.0 45.7 (n = 15) 146 33.5 (n = 4) 220 44.0 (n = 5) 69.3 57.9 (n = 22) 233 124 (n = 25) 2.42 5.09 (n = 10) 9.51 8.70 (n = 17) Analytical Report: FACT TO X-002 LRN-U2121 a Not corrected for purity of the standard material. b LOQ-- Limit of Quantitation = 0.00910 pg/mL c LOQ-- Limit of Quantitation = 0.0457 pg/mL d C92987F sample spilled during extraction, no sample remaining for analysis. 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. Page 127 3M M edical Departm ent Study: T-6295.4 3M Environm ental Laboratory Table 8. FACT TOX-002 Data Summary of PFOS Concentration--Liver (Mg/g) Tim e point Sex G roup 1 C ontrol Average SD G roup 2 Low Average SD G roup 3 Mid Average SD G roup 4 M id-High Average SD G roup 5 H ig h Average SD G roup 6 High Recovery Average SD Week 4a Week 14a Week 53 Day 719 Week 105 Week 106 Male Female Male Female Male Female Male Female Male Female Male Female 0.104 0.0673 (n = 5) 0.107 0.0486 (n = 5) 0.459 0.0573 (n = 5) 12.0 22.4 (n = 5) 0.635 1.04 (n = 10) 0.923 1.77 (n = 10) 11.0 2.31 (n = 5) 8.71 0.552 (n = 5) 23.8 3.45 (n = 5) 19.2 3.77 (n = 5) 0.114 0.148 (n = 11) 0.185 0.184 (n = 24) 7.83 7.34 (n = 10) 12.9 6.81 (n = 15) 31.3 5.84 (n = 5) 25.0 6.11 (n = 5) 74.0 6.16 (n = 5) 69.2 3.46 (n = 5) 55.1 31.5 (n = 9) 26.4 20.4 (n = 17) 47.6 12.5 (n = 5) 83.0 14.1 (n = 5) 358 28.8 (n = 5) 370 22.3 (n = 5) 70.5 63.1 (n = 25) 131 61.4 (n = 15) 282 45.3b (n = 5) 373 44.1b (n = 5) 568 107 (n = 5) 635 49.0 (n = 5) 435 96.9 (n = 9) 560 180 (n = 10) 189 141 (n = 22) 381 176 (n = 25) 3.12 5.97 (n = 10) 12.9 10.4 (n = 17) Analytical Report: FACT TO X-002 LRN-U2121 a Not corrected for purity of the standard material. b The values in this mean were tentative 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. Page 128 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 9. Week 4 Reported PFOS Levels in Sera of Male Rats in Study FACT TOX-002 Dosage G roup Specimen ID PFOS pg/m L G roup 1 C o n tro l C92506M C92524M C92526M C92529M <LOQ <LOQ <LOQ <LOQ C92546M <LOQ C92574M 0.910* G roup 2 Low C92575M C92610M C92613M 0.989* 0.944* 0.852* C92618M 0.842* C92646M 3.97* G roup 3 Mid C92650M C92652M C92668M 2.85* 4.80* 5.98* C92678M 4.04* C92715M 8.46* G roup 4 M id-High C92718M C92731M C92734M 9.89* 4.03* 7.69* C92744M 7.81* C92752M 29.0* G roup 5 H ig h C92759M C92779M C92793M 40.7* 49.5* 42.7* C92798M 47.0* Note: Not corrected for purity of the standard material. LOQ-- Limit o f Quantitation = 0.00910 pg/mL * Continuing calibration verifications did not meet 30% criteria; data may be biased low. 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 129 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 10. Week 4 Reported PFOS Levels in Sera of Female Rats in Study FACT TOX-002 Dosage Group Specimen ID PFOS pg/m L G roup 1 C ontrol C92861F C92864F C92914F C92919F C92926F 0.0227 0.0309 0.0347 0.0193 0.0216 C92982F 1.69* G roup 2 Low C92941F C92945F C92950F 1.67* 1.89* 1.38* C92969F 1.44* C92996F 6.84* G roup 3 M id C93006F C93008F C93031F 5.75* 6.85* 6.65* C93045F 6.99* C93058F 15.3* G roup 4 M id-High C93067F C93070F C93075F 13.2* 11.6* 10.8* C93084F 12.1* C93114F 62.8* G roup 5 H ig h C93123F C93142F C93144F 52.4* 56.4* 42.7* C93159F 55.8* Note: Not corrected for purity of the standard material. * Continuing calibration verifications did not meet 30% criteria; data may be biased low. 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 130 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 11. Week 14 Reported PFOS Levels in Sera of Male Rats in Study FACT TOX-002 Dosage Group Specimen ID PFOS pg/m L C92509M <LOQ G roup 1 C ontrol C92511M C92521M C92528M <LOQ <LOQ <LOQ C92532M <LOQ C92593M 4.70 G roup 2 Low C92600M C92616M C92621M 4.58 4.09 4.13 C92627M 2.69 C92640M 15.9 G roup 3 M id C92645M C92662M C92676M 16.9 16.1 17.9 C92684M 18.8 C92713M 49.1 G roup 4 M id-High C92714M C92719M C92722M 37.2 41.6 43.3 C92728M 48.2 C92765M 139 G roup 5 H ig h C92777M C92789M C92799M 145 149 136 C92812M 171 Note: Not corrected for purity of the standard material. LOQ-- Limit of Quantitation = 0.0457 pg/mL 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 131 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 12. Week 14 Reported PFOS Levels in Sera Female Rats in Study FACT TOX-002 Dosage Group Specimen ID PFOS pg/m L C92880F 0.332 G roup 1 C ontrol C92887F C92898F C92903F 10.8 0.252 1.62 C92905F 0.333 C92944F 5.90 G roup 2 Low C92962F C92967F C92978F C92987F 8.21 7.23 6.50 * C92993F 28.7 G roup 3 M id C93000F C93018F C93023F 26.7 25.1 25.4 C93035F 30.6 C93051F 56.7 G roup 4 M id-High C93064F C93071F C93085F 64.5 71.3 62.3 C93109F 67.3 C93111F 229 G roup 5 H ig h C93127F C93143F C93155F 210 258 220 C93169F 199 Note: Not corrected for purity of the standard material. * Sample spilled during extraction, no sample remaining for analysis. 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 132 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 13. Week 53 Reported PFOS Levels in Sera of Male Rats in Study FACT TOX-002 D osage G roup Specimen ID PFOS pg /m L C92504M 0.0278 G roup 1 C o n tro l C92512M C92534M C92562M 0.0149 0.0251 0.0529 C92570M <LOQ C92778M 96.6 G roup 5 H igh C92795M C92796M 162 171 C92817M 154 LOQ-- Limit of Quantitation = 0.00395 pg/mL 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. Table 14. Week 53 Reported PFOS Levels in Sera of Female Rats in Study FACT TOX-002 D osage G roup Specimen ID PFOS pg /m L G roup 1 C o n tro l G roup 5 H igh C92876F C92889F C92899F C92923F C92930F C93112F C93120F C93154F C93163F C93171F 0.0310 1.78 0.0656 0.0891 <LOQ 240 144 223 245 250 LOQ-- Limit of Quantitation = 0.00395 pg/mL 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 133 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 15. Day 719 Reported PFOS Levels in Sera of Female Rats in Study FACT TOX-002 D osage G roup G roup 3 M id S p e cim e n ID C92991F G3 Day 719 C93003F G3 Day 719 C93007F G3 Day 719 C93011F G3 Day 719 C93022F G3 Day 719 C93039F G3 Day 719 C93040F G3 Day 719 C93047F G3 Day 719 C93048F G3 Day 719 PFOS pg /m L 15.1 13.0 30.9 3.57 2.02 27.6 43.8 21.9 24.3 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 134 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 16. Week 105 Reported PFOS Levels in Sera of Male Rats in Study FACT TOX-002 D osage G roup Specimen ID PFOS pg /m L C92503M 0.0148 C92505M 0.0161 C92510M 0.0285 C92513M 0.0127 G roup 1 C o n tro l C92514M C92518M C92541M 0.0325 <LOQ <LOQ C92542M <LOQ C92544M <LOQ C92551M <LOQ C92569M <LOQ C92577M 0.309 C92583M 0.212 C92590M 2.55 C92597M 1.95 G roup 2 Low C92598M C92601M 0.230 3.94 C92602M 1.39 C92619M 0.461 C92623M 2.05 C92626M 1.48 C92631M 8.98 C92633M 14.3 C92637M 1.81 C92641M 15.1 C92642M 6.05 C92643M 2.75 C92644M 1.21 G roup 3 M id C92649M C92653M C92657M 8.63 0.487 5.20 C92659M 14.4 C92660M 1.66 C92669M 3.13 C92674M 35.3 C92682M 4.77 C92683M 2.72 C92690M 2.83 LOQ-- Limit of Quantitation = 0.00428 pg/mL It is not possible to verify true recovery o f endogenous analyte from tissues w ithout radio labeled reference material. The only measurem ent of accuracy available at this time, matrix spike studies, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 135 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 16. Week 105 Reported PFOS Levels in Sera of Male Rats in Study FACT TOX-002 (continued) D osage G roup Specimen ID PFOS pg /m L C92691M 4.48 C92693M 7.71 C92694M 74.2 C92695M 75.6 C92696M 38.7 C92698M 12.1 C92699M 73.3 C92700M 28.8 C92702M 3.14 C92704M 10.9 C92705M 23.0 G roup 4 M id -H ig h C92708M C92716M C92717M 9.72 13.5 3.01 C92724M 12.8 C92726M 38.8 C92730M 6.72 C92735M 24.4 C92736M 4.10 C92739M 5.72 C92740M 35.7 C92742M 2.28 C92747M 45.7 C92748M 3.66 C92750M 3.40 LOQ-- Limit of Quantitation = 0.00428 pg/mL It is not possible to verify true recovery o f endogenous analyte from tissues w ithout radio labeled reference material. The only measurem ent of accuracy available at this time, matrix spike studies, indicated that the sera data are accurate to 30% ; liver data are accurate to 50%. 3M Environmental Laboratory Page 136 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 16. Week 105 Reported PFOS Levels in Sera of Male Rats in Study FACT TOX-002 (continued) D osage G roup Specimen ID PFOS pg /m L C92753M 35.8 C92757M 196 C92761M 27.3 C92762M 130 C92774M 10.4 C92775M 11.1 C92776M 108 C92780M 67.4 C92782M 16.7 C92786M 27.5 G roup 5 H igh C92788M C92791M 118 21.4 C92792M 18.0 C92800M 64.2 C92802M 169 C92803M 141 C92804M 127 C92808M 37.2 C92809M 6.02 C92814M 89.2 C92815M 11.9 C92818M 90.1 LOQ-- Limit of Quantitation = 0.00428 pg/mL It is not possible to verify true recovery o f endogenous analyte from tissues w ithout radio labeled reference material. The only measurem ent of accuracy available at this time, matrix spike studies, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 137 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 17. Week 105 Reported PFOS Levels in Sera of Female Rats in Study FACT TOX-002 D osage G roup Specimen ID PFOS pg /m L C92866F 0.624 C92867F 0.225 C92868F 0.0467 C92869F 0.306 C92871F 0.00650 C92873F 0.0602 C92874F 0.0427 C92877F 0.100 C92881F 0.0258 C92882F 0.0483 C92885F <LOQ (1) G roup 1 C o n tro l C92890F C92892F 0.0675 0.0140 C92895F 0.0233 C92896F 0.0407 C92897F 0.0443 C92906F 0.0795 C92907F 0.0176 C92909F 0.0402 C92911F 0.0687 C92912F 0.0311 C92913F 0.0575 C92925F <LOQ (2) C92927F 0.0229 C92936F 8.72 C92938F 0.349 C92946F 9.08 C92953F 6.64 C92957F 5.67 C92960F 1.70 G roup 2 Low C92964F C92970F C92973F 5.97 1.73 1.34 C92974F 4.06 C92975F 3.72 C92983F 6.81 C92984F 2.78 C92986F 5.24 C92988F 5.76 LOQ-- Limit o f Quantitation = (1) 0.00428 pg/mL, (2) 0.00848 pg/mL 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 138 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 17. Week 105 Reported PFOS Levels in Sera of Female Rats in Study FACT TOX-002 (continued) D osage G roup Specimen ID PFOS pg /m L C93059F 103 C93060F 89.5 C93062F 102 C93063F 171 C93072F 114 C93077F 38.0 G roup 4 M id -H ig h C93078F C93079F C93080F 25.9 72.8 73.6 C93081F 70.5 C93090F 99.1 C93098F 5.17 C93101F 117 C93102F 11.4 C93107F 33.2 LOQ-- Limit of Quantitation = (1) 0.00428 pg/mL, (2) 0.00848 pg/mL 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 139 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 17. Week 105 Reported PFOS Levels in Sera of Female Rats in Study FACT TOX-002 (continued) D osage G roup Specimen ID PFOS pg /m L C93113F 403 C93115F 286 C93124F 160 C93125F 353 C93133F 242 C93135F 274 C93137F 143 C93139F 375 C93140F 3.85 C93146F 51.5 C93149F 432 G roup 5 H igh C93151F C93153F C93157F 139 121 28.6 C93158F 287 C93161F 103 C93162F 446 C93164F 321 C93165F 165 C93166F 189 C93168F 339 C93174F 304 C93177F 248 C93178F 216 C93179F 190 LOQ-- Limit of Quantitation = (1) 0.00428 pg/mL, (2) 0.00848 pg/mL 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 140 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 18. Week 106 Reported PFOS Levels in Sera of Male Rats in Study FACT TOX-002 D osage G roup Specimen ID PFOS pg /m L C92821M 8.32 C92823M 0.533 C92827M 0.0303 C92829M 0.0243 G roup 6 H ig h R e co ve ry C92833M C92834M 0.0795 0.0138 C92848M 14.9 C92851M 0.0331 C92853M 0.210 C92856M <LOQ LOQ-- Limit of Quantitation = 0.00482 pg/mL 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. Table 19. Week 106 Reported PFOS Levels in Sera of Female Rats in Study FACT TOX-002 D osage G roup Specimen ID PFOS pg /m L G roup 6 H ig h R e co ve ry C93181F C93182F C93183F C93184F C93185F C93190F C93192F C93197F C93198F C93201F C93202F C93205F C93209F C93211F C93216F C93219F C93220F 5.76 1.69 5.68 1.64 11.8 31.6 16.8 9.35 8.63 27.0 10.1 0.0594 6.57 12.3 2.85 2.39 7.44 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 141 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 20. Week 4 Reported PFOS Levels in Liver of Male Rats in Study FACT TOX-002 D osage G roup Specimen ID PFOS pg/g G roup 1 C o n tro l C92506M C92524M C92526M C92529M <LOQ 0.169 0.186 <LOQ C92546M <LOQ C92574M 12.4 G roup 2 Low C92575M C92610M C92613M 9.67 8.54 14.2 C92618M 9.94 C92646M 40.7 G roup 3 M id C92650M C92652M C92668M 26.1 31.2 32.0 C92678M 26.7 C92715M 53.5 G roup 4 M id -H ig h C92718M C92731M C92734M 59.6 26.7 49.8 C92744M 48.3 C92752M 219* G roup 5 H igh C92759M C92779M C92793M 264* 342* 286* C92798M 298* Note: Not corrected for purity of the standard material. LOQ-- Limit of Quantitation = 0.0552pg/g * Results were outside the range of the curve and should be considered estimates. 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 142 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 21. Week 4 Reported PFOS Levels in Liver of Female Rats in Study FACT TOX-002 D osage G roup Specimen ID PFOS pg/g G roup 1 C o n tro l C92861F C92864F C92914F C92919F C92926F 0.0817 0.0738 0.184 0.0700 0.126 G roup 2 Low G roup 3 M id G roup 4 M id -H ig h G roup 5 H igh C92982F C92941F C92945F C92950F C92969F C92996F C93006F C93008F C93031F C93045F C93058F C93067F C93070F C93075F C93084F C93114F C93123F C93142F C93144F C93159F 9.09 7.99 9.40 8.43 8.67 28.9 14.6 29.7 27.1 24.8 107 83.9 77.0 70.7 76.7 435* 360* 395* 317* 359* Note: Not corrected for purity of the standard material. * Results were outside the range of the curve and should be considered estimates. It is not possible to verify true recovery o f endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 143 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 22. Week 14 Reported PFOS Levels in Liver of Male Rats in Study FACT TOX-002 D osage G roup Specimen ID PFOS pg/g C92509M 0.440 G roup 1 C o n tro l C92511M C92521M C92528M 0.542 0.492 0.403 C92532M 0.418 C92593M 24.6 G roup 2 Low C92600M C92616M C92621M 19.3 27.2 21.2 C92627M 26.6 C92640M 82.7 G roup 3 M id C92645M C92662M C92676M 73.5 68.3 68.3 C92684M 77.4 C92713M 375 G roup 4 M id -H ig h C92714M C92719M C92722M 372 377 308 C92728M 356 C92765M 722 G roup 5 H igh C92777M C92789M C92799M 618 559 456 C92812M 485 Note: Not corrected for purity of the standard material. 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 144 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 23. Week 14 Reported PFOS Levels in Liver of Female Rats in Study FACT TOX-002 D osage G roup Specimen ID PFOS pg/g C92880F 1.02 G roup 1 C o n tro l C92887F C92898F C92903F 52.0 0.697 5.26 C92905F 1.18 C92944F 18.9 G roup 2 Low C92962F C92967F C92978F 17.7 24.8 14.5 C92987F 19.8 C92993F 69.0 G roup 3 M id C93000F C93018F C93023F 71.1 67.4 64.9 C93035F 73.9 C93051F 355 G roup 4 M id -H ig h C93064F C93071F C93085F 361 355 373 C93109F 408 C93111F 692 G roup 5 H igh C93127F C93143F C93155F 619 621 675 C93169F 569 Note: Not corrected for purity of the standard material. 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 145 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 24. Week 53 Reported PFOS Levels in Liver of Male Rats in Study FACT TOX-002 D osage G roup Specimen ID PFOS pg/g C92501M 0.0326 C92504M 0.358 C92507M 0.301 C92512M 0.283 G roup 1 C o n tro l C92517M C92523M 0.761 0.529 C92534M 0.359 C92540M 0.106 C92562M 3.52 C92570M 0.103 C92751M 559 C92754M 486 C92755M 486 G roup 5 H igh C92778M C92790M C92795M 279 405 554 C92796M 427 C92817M 403 C92820M 316 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 146 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 25. Week 53 Reported PFOS Levels in Liver of Female Rats in Study FACT TOX-002 D osage G roup Specimen ID PFOS pg/g C92862F 5.66 C92876F 0.131 C92889F 2.08 C92894F 0.276 G roup 1 C o n tro l C92899F C92910F 0.217 0.198 C92917F 0.246 C92922F 0.0580 C92923F 0.154 C92930F 0.215 C93112F 296 C93116F 534 C93117F 514 C93120F 430 G roup 5 H igh C93130F C93138F 504 565 C93147F 619 C93154F 424 C93163F 883 C93171F 830 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 147 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 26. Day 719 Reported PFOS Levels in Liver of Female Rats in Study FACT TOX-002 D osage G roup G roup 3 M id S p e cim e n ID C92991F C93003F C93007F C93011F C93022F C93039F C93040F C93047F C93048F PFOS pg/g 43.6 51.7 97.2 13.2 9.02 79.7 94.1 53.3 53.8 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 148 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 27. Week 105 Reported PFOS Levels in Liver of Male Rats in Study FACT TOX-002 D osage G roup Specimen ID PFOS pg/g G roup 1 C o n tro l G roup 2 Low G roup 3 M id C92503M C92505M C92510M C92513M C92514M C92518M C92541M C92542M C92544M C92551M C92569M C92577M C92583M C92590M C92597M C92598M C92601M C92602M C92619M C92623M C92626M C92631M C92633M C92637M C92641M C92642M C92643M C92644M C92649M C92653M C92657M C92659M C92660M C92669M C92674M C92682M C92683M C92690M 0.152 0.106 0.511 0.0663 0.233 <LOQ 0.0283 0.0342 0.0291 <LOQ 0.0690 1.60 1.64 3.89 17.2 2.02 21.6 10.0 1.39 13.4 5.61 41.5 51.2 3.82 3.35 23.9 18.5 4.32 54.0 2.85 19.8 61.4 10.6 17.4 59.0 38.2 16.9 21.5 LOQ-- Limit of Quantitation = 0.0107 pg/g 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 149 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 27. Week 105 Reported PFOS Levels in Liver of Male Rats in Study FACT TOX-002 (continued) D osage G roup Specimen ID PFOS pg/g G roup 4 M id -H ig h C92691M C92693M C92694M C92695M C92696M C92698M C92699M C92700M C92702M C92704M C92705M C92708M C92716M C92717M C92724M C92726M C92730M C92735M C92736M C92739M C92740M C92742M C92747M C92748M C92750M 27.7 30.5 208 195 120 18.9 164 69.1 13.1 65.3 67.7 51.6 85.8 13.7 46.1 95.0 18.0 73.7 15.1 35.6 155 7.16 170 11.5 6.80 LOQ-- Limit of Quantitation = 0.0107 pg/g 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 150 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 27. Week 105 Reported PFOS Levels in Liver of Male Rats in Study FACT TOX-002 (continued) D osage G roup Specimen ID PFOS pg/g G roup 5 H igh C92753M C92757M C92761M C92762M C92774M C92775M C92776M C92780M C92782M C92786M C92788M C92791M C92792M C92800M C92802M C92803M C92804M C92808M C92809M C92814M C92815M C92818M 113 329 80.2 175 39.4 35.0 320 163 53.1 98.6 385 69.0 54.8 159 409 411 330 119 23.3 358 72.0 352 LOQ-- Limit of Quantitation = 0.0107 pg/g 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 151 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 28. Week 105 Reported PFOS Levels in Liver of Female Rats in Study FACT TOX-002 D osage G roup Specimen ID PFOS pg/g G roup 1 C o n tro l C92866F C92867F C92868F C92869F C92871F C92873F C92874F C92877F C92881F C92882F C92885F C92890F C92892F C92895F C92896F C92897F C92906F C92907F C92909F C92911F C92912F C92913F C92925F C92927F 0.907 0.367 0.108 0.250 <LOQ 0.254 0.151 0.271 0.0904 0.228 <LOQ 0.248 0.0490 0.113 0.116 0.158 0.328 0.0346 0.113 0.206 0.0667 0.183 <LOQ 0.161 LOQ-- Limit of Quantitation = 0.0107 pg/g 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 152 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 28. Week 105 Reported PFOS Levels in Liver of Female Rats in Study FACT TOX-002 (continued) D osage G roup Specimen ID PFOS pg/g G roup 2 Low G roup 4 M id -H ig h C92936F C92938F C92946F C92953F C92957F C92960F C92964F C92970F C92973F C92974F C92975F C92983F C92984F C92986F C92988F C93059F C93060F C93062F C93063F C93072F C93077F C93078F C93079F C93080F C93081F C93090F C93098F C93101F C93102F C93107F 10.5 1.72 15.3 20.1 14.5 4.94 20.1 6.69 5.08 15.8 13.5 27.0 8.58 13.2 17.2 183 133 242 133 167 152 61.7 113 149 118 186 19.2 193 40.0 79.8 LOQ-- Limit o f Quantitation = 0.0107 pg/g 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 153 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 28. Week 105 Reported PFOS Levels in Liver of Female Rats in Study FACT TOX-002 (continued) D osage G roup Specimen ID PFOS pg/g G roup 5 H igh C93113F C93115F C93124F C93125F C93133F C93135F C93137F C93139F C93140F C93146F C93149F C93151F C93153F C93157F C93158F C93161F C93162F C93164F C93165F C93166F C93168F C93174F C93177F C93178F C93179F 373 378 174 444 403 582 281 446 9.20 131 575 292 234 76.8 617 212 695 510 411 394 462 493 623 396 328 LOQ-- Limit of Quantitation = 0.0107 pg/g 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 154 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Table 29. Week 106 Reported PFOS Levels in Liver of Male Rats in Study FACT TOX-002 D osage G roup Specimen ID PFOS pg/g C92821M 9.68 C92823M 1.91 C92827M 0.144 C92829M 0.117 G roup 6 H ig h R e co ve ry C92833M C92834M 0.382 0.203 C92848M 17.9 C92851M 0.363 C92853M 0.431 C92856M 0.0332 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. Table 30. Week 106 Reported PFOS Levels in Liver of Female Rats in Study FACT TOX-002 D osage G roup Specimen ID PFOS pg/g C93181F 4.32 C93182F 2.10 C93183F 9.08 C93184F 1.69 C93185F 15.9 C93190F 22.8 C93192F 15.0 G roup 6 H ig h R e co ve ry C93197F C93198F C93201F 8.65 6.18 19.2 C93202F 17.0 C93205F 0.132 C93209F 14.9 C93211F 42.6 C93216F 14.7 C93219F 4.96 C93220F 19.5 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, indicated that the sera data are accurate to 30%; liver data are accurate to 50%. 3M Environmental Laboratory Page 155 3M Medical Department Study: T-6295.4 Appendix E: Data Spreadsheets Analytical Report: FACT TOX-002 LRN-U2121 3M Environmental Laboratory Page 156 3M Medical Department Study: T-6295.4 FA C T-M -4.0 Excel 97 3M Environmental Laboratory AMDT# 112296.1 Covance# 6329-183 3M Study Tille: Covance Study Title: Product N um ber(Test Substance): 104 W eek Dietary C hronic T oxicity and C arcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) it 104 W eek Dietary Chronic T oxicity Study w ith Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats T-6295 (PFOS) M a tr ix : M e lh o d /R e v is io n : Analytical Equipment System N um ber Instrument Software/Version: F ile n a m e : R-Squared Value: Slope: Y -I n te rc e p t : Date o f Ex traction/Analyst: Rat Serum - Curve linear regression, no weighting FACT-M-3.0 & FACT-M-4.0 - Reworked x dropping 97 ppb std Madeline 041098 M assLynx 3.1 See Attachments See Attachments See Attachments See Attachments 05/27/98 OK Blks G rp 1 G rp 2 G rp 3 G rp 4 G rp 5 M S, MSD Date o f Analysis/A nalyst: Date o f Data Reducdon/Analyst: 06/26/98,07/15/98 HOJ/MEE/KJH 07/02/98,07/23/98 H01 Sample Data WEEK 4 RAT SERA GZ T M ethod B Ik Sam ple# H 2 0 Blk-1 Initial Voi. tnL 1 PFO S Std C o rrectio n F a c to r 0 .9 2 7 5 PFO S Purity C o rrectio n Factor ND PFOS D ilution Factor 1 PFOS Cone. ng/m L 0.00 C onc entra tion of PFOS ug/mL o r % Ree. <LO Q (0.00453 ug/m L) PFOS ug/m L RSD Std. Dev. MS/M SD RPD M atrix Blk H 2 0 Blk-2 Rat Serum Blk-1 1 1 0.9275 0.9275 ND ND I 0.00 <LO O (0.00453 Ug/mL) * <L O Q (0.00453 ug/mL) 1 1.75 <LOQ (0.00453 Ug/mL) * NA QC-75 ppb G roup 1 Rat Serum Blk-2 MS MSD C92506M 1 1 1 1 0.9275 NA NA 0.9275 ND NA NA ND ! 1.73 <LO O (0.00453 ug/mL) < L O 0 (0.00453 ug/m L) 1 122 167% * 1 92.6 127% * 147% 1 1.57 <LO Q (0.00910 ug/mL) NA 27% Control C92524M 1 0.9275 ND l 0.00 <LOQ (0.00910 ug/m L) 0 .0 mg/kg C92526M 1 0.9275 ND 1 4.31 <LOQ (0.00910 ug/m L) C92529M C92546M C92861F 1 0.9275 1 0.9275 1 0.9275 ND ND ND 1 4.54 <LOQ (0.00910 ug/mL) 1 0.00 <LOO (0.00910 ug/m L) <L O Q (0.00910 ug/mL) 1 24.5 0.0227 NA NA C92864F 1 0.9275 ND 1 33.4 0 .0 3 0 9 C92914F 1 0.9275 ND 1 37.5 0.0347 C92919F C92926F 1 0.9275 0 .9 2 7 5 ND ND 1 20.8 23.3 0.0193 0.0216 0.0259 25.7 0.00663 G roup 2 C92574M 1 0.9275 ND 20 49.0 0.910 * L ow Dose C92575M 0.9275 ND 20 53.3 0.989 * 0.5 mg/kg C92610M 0.9275 ND 20 50.9 0.944 * C92613M C92618M C92982F 1 0.9275 1 0.9275 1 0.9275 ND ND ND 20 46.0 20 45.4 20 91.1 0.852 * 0.842 * 0.907 1.69 * 6.83 0 .0 6 1 9 C92941F 1 0.9275 ND 20 89.8 1.67 * C92945F 1 0.9275 ND 20 102 1.89 G roup 3 M id Dose C92950F C92969F C92646M C92650M 1 0,9275 1 0.9275 1 0.9275 l 0.9275 ND ND ND ND 20 74.6 20 77.5 50 85.5 50 61.4 1.38 1.44 3.97 2.85 * * * * 1.61 12.8 0 .2 0 7 2.0 mg/kg C92652M 0.9275 ND 50 103 4.80 * C92668M C92678M C92996F 1 0.9275 1 0.9275 0.9275 ND ND ND 50 129 50 87.2 50 147 5.98 * 4.04 * 4.33 6.84 * 26.7 1.16 C93006F 1 0.9275 ND 50 124 5.75 * C93008F 1 0.9275 ND 50 148 6.85 * C93031F C93045F 1 0.9275 1 0.9275 ND ND 50 143 50 151 6.65 * 6.99 * 6.62 7 .5 4 0 .4 9 9 G roup 4 C92715M 1 0.9275 ND 100 91.2 8.46 * Mid-High Dose C92718M 1 0.9275 ND 100 107 9.89 5.0 mg/kg C92731M 1 0.9275 ND 100 43.4 4.03 * C92734M C92744M C93058F 1 0.9275 ND 100 82.9 1 0.9275 ND 100 84.2 1 0.9275 ND 100 165 7.69 * 7.81 * 7.57 15.3 * 28.6 2 .1 7 C93067F 1 0.9275 ND 100 142 13.2 * G roup 5 C93070F C93075F C93084F C92752M 1 0.9275 ND 100 125 1 0.9275 ND 100 116 1 0.9275 ND 100 131 1 0.9275 ND 200 157 11,6 * 10.8 * 12.1 * 12.6 29.0 * 13.8 1.73 H igh Dose C92759M 1 0.9275 ND 200 219 40.7 * 20 mg/kg C92779M 1 0.9275 ND 200 267 49.5 * C92793M C92798M C93114F 1 0.9275 ND 200 230 1 0.9275 ND 200 253 1 0.9275 ND 200 338 42.7 47.0 62.8 * * 41.8 * 19.0 7 .9 2 C93123F 1 0.9275 ND 200 282 52.4 * C93I42F 1 0.9275 ND 200 304 56.4 C93144F C93159F 1 0.9275 ND 200 230 1 42.7 * 1 0.9275 ND 200 301 55.8 * 54.0 13.6 7 .3 4 inform ation on 10/04/00. LAC PFO S * Perfluorooctanesulfonate Correction factors n ot applicable for MS/M SD Q C data * Data entered, Continuing calibration verifications ND = Not Determined did not m eet +/ 30% criteria. LAC 09/05/00 NA= C om ponents were not integrated because they w ere not detected in the group 5 samples. Dale Entered/By. 07/30/98,09/05/00 LAC Date Verified/By: 08/24/00 KJH Purity Entered/Verificd: 09/12/00 LAC TOX-002-serai 83-3N Analytical Report: FACT TOX-002 LRN-U2121 Page 157 3M Medical Department Study: T-6295.4 AM DT# 112296.1 Covance# 6329-183 Analytical Report: FACT TOX-002 LRN-U2121 3M Study Title: Covance Study Title: Product Number(Test Substance): 104 Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats 104 Week Dietary Chronic Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats T-6295 (PFOS) Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squarcd Value: Slope: Y-Interccpt: Date of Extraction/Analyst: Date of Analysis/Analyst: Date o f Data Reduction/Analyst: Sample Data WEEK 14 RAT SERA REWORK Rat Serum - Curve linear regression, 1/X weighted FACT-M -3.0& ETS-8-5.1 Amelia 062498 MassLynx 3.3 See Attachments See Attachments See Attachments See Attachments 07/28/98 IAS 07/29/98,08/14/98,08/17/98 HOJ/MEE 02/01AX), 02/02/00 IAS Lot 193 Filenames Blks Grp 1 M Grp IF Grp 1 F Grp 2 Grp 3 Grp 4 Grp 5 M G rpSF MS, MSD PFOS A072998002-3 & 71-72 072998013-17 072998021,24, 25 81498012-13 81498016-26 81498029-40 81498043-54 81498064-68 08179825-29 D ilutions 1/1 1/1 1/1 1/25 1/25 1/50 1/100 1/250 1/500 81498071-72 1/1 Group Dose Method Blk Matrix Blk QC-100 ppb Rat Serum Group 1 Control 0.0 ppm in D iet Group 2 Low Dose 0.5 ppm in Diet Sample # H 20 Blk-1 H 2 0 Blk-2 Rabbit Serum Blk-1 Rabbit Serum Blk-2 TN-A-1902-MS TN-A-1902-MSD C92509M C9251IM C92521M C92528M C92532M C92880F C92887F C92898F C92903F C92905F C92593M C92600M C92616M C92621M C92627M C92944F C92962F Initial Voi. mL 1 1 1 1 1 1 1 1 1 l 1 1 1 1 1 1 1 1 1 1 PFOS Std Correction Factor 0.9275 0.9275 0.9275 0.9275 NA NA 0.9275 0.9275 0.9275 0 .9 2 7 5 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 PFOS Purity Correction Factor ND ND ND ND NA NA ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND PFOS Dilution Factor 1 ) 1 1 1 1 1 1 1 1 1 1 25 1 25 1 25 25 25 25 25 25 25 PFOS Cone. ng/mL ().(X) 7.82 0.00 6.79 105 105 18.0 17.4 17.2 18.9 17.5 360 468 273 70.0 361 204 198 177 179 117 256 356 Concentration of PFOS ug/m L o r % Ree. <LOQ (0.0457 ug/mL) <LOQ (0.0457 ug/mL) <LOQ (0.0457 ug/mL) <LOQ (0.0457 ug/mL) 107% 108% <LOQ (0.0457 ug/mL) <LOQ (0.0457 ug/mL) <LOQ (0.0457 ug/mL) <L O Q (0.0457 ug/m L ) <LOQ (0.0457 ug/mL) 0.332 10.8 0.252 1.62 0.333 4.70 4.58 4.09 4.13 2.69 5.90 8.21 M ean PFOS ug/mL <LOQ (0.0457 ug/mL) <LOO (0.0457 ug/mL) 108% <LOQ (0.0457 ug/mL) 2.67 4.04 RSD Std. Dev. MS/MSD RPD NA NA 1% NA NA 172 4.58 19.8 0.801 Group 3 Mid Dose 2.0 ppm in Diet Group 4 Mid-High Dose 5.0 ppm in Diet C92967F C92978F C92987F C92640M C92645M C92662M C92676M C92684M C92993F C93000F C93018F C93023F C93035F C92713M C92714M C92719M C92722M C92728M C93051F C93064F 1 0.9275 ND 25 314 1 0.9275 ND 25 282 1 0.9275 ND 25 ** 1 0.9275 ND 50 344 1 0.9275 ND 50 367 1 0.9275 ND 50 350 1 0.9275 ND 50 387 1 0.9275 ND 50 408 1 0.9275 ND 50 621 1 0.9275 ND 50 579 1 0.9275 ND 50 543 1 0.9275 ND 50 549 1 0.9275 ND 50 663 1 0.9275 ND 100 532 1 0.9275 ND 100 403 1 0.9275 ND 100 451 1 0.9275 ND 100 469 1 0.9275 ND 100 522 1 0.9275 ND 100 614 1 0.9275 ND 100 699 7.23 6.50 ** 15.9 16.9 16.1 17.9 18.8 28.7 26.7 25.1 25.4 30.6 49.1 37.2 41.6 43.3 48.2 56.7 64.5 14.3 6.96 0.993 7.13 17.1 1.22 8.57 27.3 2.34 11.2 43.9 4.90 Group 5 High Dose 20 ppm in Diet C93071F C93085F C93109F C92765M C92777M C92789M C92799M C92812M C93111F C93127F i 0.9275 ND 100 773 1 0.9275 ND I(X) 675 1 0.9275 ND 100 729 1 0.9275 ND 250 602 1 0.9275 ND 250 629 1 0.9275 ND 250 645 1 0.9275 ND 250 588 I 0.9275 ND 250 740 1 0.9275 ND 500 496 I 0.9275 ND 500 456 71.3 62.3 67.3 139 145 149 136 171 229 210 8.51 64.4 5.48 9.35 148 13.8 C93143F C93155F C93169F 1 0.9275 ND 500 559 1 0.9275 ND 500 477 1 0.9275 ND 500 432 258 220 199 10.0 223 22.4 Original PFOS LOQ (49.3 ng/mL) updated to reflect K+ information on 10/04/00. LAC 10/04/00 Correction factors not applicable for MS/MSD QC data ND = Not Determined PFO S = Perfluorooctanesulfonate ** Sample spilled during extraction, no sample remaining for analysis. Date Entered/By: 02/09/(K), 09/07/iK) LAC Date Verified/ By: 08/24/00 KJH Purity Entered/Verified: 09/12/00 LAC, 9112/00 MMH Original PFOS LOQ = 25 ng/mL updated to reflect purity information on 10/04/00. LAC 10/04/00 ETS-8-5.1 Excel 97 TiWEnvirOnmenfST aESrSTory TOX-002-sera 183-3N Page 158 3M Medical Department Study: T-6295.4 AM DT# 112296.1 Covance# 6329-183 Analytical Report: FACT TOX-002 LRN-U2121 3M Study Title: Covance Study Title: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sample Data 1()4 Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats 104 Week Dietary Chronic Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats T-6295 (PFOS) Rat Serum Filenames ETS-8-4.1 and ETS-8-5.1 Soup 020199 MassLynx 3.3 See Attachments See Attachments See Attachments See Attachments 11/10/99 SEE 11/15/99, 11/19/99, 11/22/99,11/24/99 IAS, 12/15/99 IAS Blanks Grp 1 M Grp I F Grp 1 F Grp 5 M Grp 5 F MS, MSD Box 99-220 PFOS S 121499043-45,63-65 111599019-25 111599026,28-32 121499057 112299017-20 112499016-19 121499058-59 Dilutions 1/1 1/1 1/1 1/2 1/200&2(KX> 1/1 1/1 11/16/99, 11/22/99, 11/23/99, 11/29/99 IAS, 12/13/99 MMH WEEK 53 RAT SERA Lot 171 G roup Dose Method Blk Matrix Blk Matrix Blk QC-250 ppb Group 1 Control 0.0 ppm in Diet Group 5 High Dose 20 ppm in Diet Sample# H 2 0 B lk-11 H 2 0 B lk-12 Rabbit Serum Blk-11 Rabbit Serum Blk-12 Rat Serum Blk-11 Rat Serum Blk-12 RTS 11109-250ppb-MS-l 1 RTS 11109-250ppb-MSD-l 1 C92504M C92512M C92534M C92562M C92570M C92876F C92889F C92899F C92923F C92930F C92778M C92795M C92796M C92817M C93112F C93120F C93154F C93163F C93171F Initial Voi. mL 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.600 0.700 0,700 0.700 0.700 0.800 0.700 0.800 0.700 0.700 0.8(X) 0.800 0.600 0.800 1.000 1.000 0.900 0.900 0.9IX) PFOS Std Correction Factor 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 NA NA 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 PFOS Purity Correction Factor 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 NA NA 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0,8640 0.8640 0.8640 0.8640 0.8640 0.8640 PFOS Dilution Factor 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 200 2(X) 200 200 2(XX) 200 2000 20(X) 2000 PFOS Cone. ng/mL 9.75 5.11 6.61 3.62 46.6 476 189 224 20.8 13.1 21.9 46.2 4.71 31.0 779 65.5 77.9 3.93 482 806 640 767 150 897 125 138 140 C oncentration M ean of PFOS PFOS ug/m L o r % Ree. ug/mL <LOQ (0.00782 ug/mL) <LOQ (0.00782 ug/mL) <LOQ (0.00782 ug/mL) <LOQ (0.00782 ug/mL) <LOQ (0.00782 ug/mL) <LOQ (0.00782 ug/mL) 0.0373 0.3814 * 0.209 76% 90% 83% 0.0278 0.0149 0.0251 0.0529 <LOQ (0.00395 ug/mL) 0.0249 0.0310 1.78 0.0656 0.0891 <LOQ (0.00395 ug/mL) 0.395 96.6 162 171 154 146 240 144 223 245 250 220 RSD Std. Dev. MS/MSD RPD NA NA 0.243 17% 73.2 0.0182 197 0.777 23.0 33.5 20.0 44.0 Original PFOS LOQ (4.93 ng/mL, 9.76 ng/mL) updated to reflect K+ and purity information on 10/04/00. LAC 10/04/00 NA Not Appicable PFOS = Perfluorooctanesulfonate * Second analysis confirmed high result. LAC 08/24/00 Date Entered/By: 11/22/99, 11/24/99 GML/LAC, 12/1/99 GML, 12/30/99 MMH Date Verified/ By: 08/31/00 KJH Purity Entered/Verified: 10/04/00 LAC, 10/04/00 KJH ETS-8-5.1 Excel 97 3WEnvrnmnTlTaboratory TOX-(X)2-seral83-3N Page 159 3M Medical Department Study: T-6295.4 AM DT# 112296.1 Covance# 6329-183 Analytical Report: FACT TOX-002 LRN-U2121 3M Study Title: Covance Study Title: Product Number(Test Substance): Matrix: Method/Revision: ANAlytical Equipment System Number: Instrument Software/Version: FileNAme: R-Squared Value: Slope: Y-Interccpt: Dates of Extraction/ANAlyst: Dates of ANAlysis/ANAlyst: Date of Data Reduction/ANAlyst: Sample Data 104 Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats 104 Week Dietary Chronic Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats T-6295 (PFOS) Rat Serum ETS-8-4.1 & ETS-8-5.1 Ruby 100699 MassLynx 3.3, 3.4 See listing to the right See Attachments See Attachments See Attachments 05/01/00, 05/02/CX) SAL/KJK/RWW 05/02/00 05/04/00,05/10/00 05/26/00, 05/3G/(X) MMH/IAS 05/04/00,05/05/00,05/11/00, 05/30/00, 06/01/00, 11/16/00IAS/MMH DAY 719 RAT SERA Group Dose Sample # Initial Voi. Surrogate Verified Original Purity Correction PFOS Purity Correction mL Factor Factor Group 3 mg/kg/day C92991FG3 Day719 C93(X)3FG3 Day719 1.00 1.00 NA NA 0.9949 0.9949 0.8640 0.8640 C93007F G3 Day719 0,80 NA 0.9949 0.8640 C93011FG3 Day719 0.75 NA 0.9949 0.8640 C93022FG3 Day719 0.85 NA 0.9949 0.8640 C93039F G3 Day719 1.00 NA 0.9949 0.8640 C93040FG 3 Day719 0.80 NA 0.9949 0.8640 C93047FG3 Day719 0.85 NA 0.9949 0.8640 C93048F G3 Day719 0.75 NA 0.9949 0.8640 Original PFOS LOQ (24.8 ng/mL, 4.93 ng/mL) updated to reflect purity information on 10/04/00. LAC 10/04/00 PFOS Dilution 50 50 50 50 50 50 2(X) 50 50 PFOS Cone. ng/mL 348 299 569 61.7 39.5 637 201 429 420 F ile N A m e (optional) D051(KXX)54 D 0 5 1000055 D 0 5 1000058 D 0 5 1000059 D 0 5 1000060 D 0 5 1000061 R0531(XX)30 D 0 5 1000065 D051(XXX)66 C o ncentration of PFOS 15.1 13.0 30.9 3.57 2.02 27.6 43.6 21.9 24.3 M ean PFOS 20.2 NA = Not Applicable D a teE ntered/B y: Date Verified/By: Purity Entered/Verified: 5/11/00,5/12/00,5/18/00,5/19/00,6/8/00, 6/19/00, 11/17/00 MMH/CSH/LAC 08/31/00 KJH, 11/21/00 MMH 10/04/1X) LAC, 10/04/00 KJH RSD S td. Dev. MS/MSD RPD 65.9 13.3 ETS-8-5.1 Excel 97 3MEnvrnmnTTbSrTSry TOX-002-seral 83-3N 2/13/01 10:06 AM Page 160 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 3M Study T itle: Covance Study Title: Product Number(Test Substance): Matrix: Method/RevLsion: ANAlytical Equipment System Number: Instrument Software/Version: FileNAme: R-Squared Value: Slope: Y-Intercept: Dates o f Extraction/Analyst: Dates o f Analysis/Analyst: Date o f Data Reduction/Analyst: Sample Data W E E K 105 R A T SERA mg/kg/day C92503M C92505M C92510M C92513M C92514M C92518M C92541M C92542M C92544M C92551M C92867F C92868F C92869F C92871F C92873F C92874F C92X77F C92881F C92882F C92885F C92890F C92892F C92895F C92896F C92897F C92906F C92907F C92909F C92911F C92912F C92913F C92925F C92577M mg/kg/day C92583M C92590M C92597M C92598M C92601M C92602M C92619M C92623M C92626M C92936F C92938F C92946F C92953F C92957F C92960F C92964F C92970F C92973F C92974F C92975F C92983F C92984F C92986F C92988F (roup 3 C92631M m g /k g /d ay C92633M C92637M C92641M C92642M C92643M C92644M C92649M C92653M C92657M C92659M C92660M C92669M C92674M C92682M C92683M C92690M O riginal PFOS LOQ (4,93 ng/mL, 9.76 ng/mL) i AMDT# 112296.1 Covance# 6329-183 104 W eek D ietary Chronic Toxicity and C arcinogenicity Study w ith Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T -6295) in Rats 104 W eek D ietary Chronic Toxicity Study w ith Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T -6295) in Rats T-6295 (PFOS) Rat Serum ETS-8-4.1 & ETS-8-5.1 Davey 070799, Ruby 100699, Amelia 062498 MassLynx 3,3, 3.4 See listing to the right See Attachments See Attachments See Attachments 05/01/00,05/02/00 SAL/KJK/RWW 05/02/00. 05/04/00. 0 5 /1 0/00.05/26/00.05/30/00 MMH/TAS 05/04/00,0 5 /0 5 /0 0 ,0 5 /1 1 /0 0 , 05/30/00.06411/00,11/16/00 IAS/M MH Initial Voi. mL 0.64 0.85 0.84 0.82 0.85 0.70 0.74 0.84 0.78 0.70 0.90 0.98 0.76 0.90 0.87 0.90 0.98 0.85 0.85 0.87 0.95 0.76 0.89 0.96 1.00 1.00 0.85 0.85 0.73 1.00 0.81 1.00 0.90 1.00 1.00 0.90 1.00 0.43 1.00 0.85 0.81 0.40 0.81 0.75 1.00 1.00 0.92 0.83 0.70 1.00 1.00 0.75 0.85 0.95 0.90 0.95 1.00 0.98 1.00 0.85 0.75 0.33 0.85 0.74 0.90 0.75 1.00 0.82 1.00 0.90 0.78 0.96 0.35 1.00 0.90 0.85 0.80 V erified NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA C o rrectio n F a c to r 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0.9949 0.9949 0.9949 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0.9949 0.9949 0.9949 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0.9949 0.9949 0.9949 0.9949 0.9949 0 .9 9 4 9 0.9949 0.9949 0.9949 0.9949 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0.9949 0.9949 0.9949 0.9949 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 0 .9 9 4 9 I PFOS Purity Correction 0.8640 0.8640 0.8640 0 .8 6 4 0 0 .8 6 4 0 0.8640 0.8640 0 .8 6 4 0 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0 .8 6 4 0 0 .8 6 4 0 0.8640 0 .8 6 4 0 0 .8 6 4 0 0 .8 6 4 0 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0 .8 6 4 0 0 .8 6 4 0 0.8640 0 .8 6 4 0 0 .8 6 4 0 0 .8 6 4 0 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0 .8 6 4 0 0 .8 6 4 0 0.8640 0.8640 0 .8 6 4 0 0 .8 6 4 0 0 .8 6 4 0 0.8640 0.8640 0 .8 6 4 0 0 .8 6 4 0 0 .8 6 4 0 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0 .8 6 4 0 0 .8 6 4 0 0 .8 6 4 0 0 .8 6 4 0 0.8640 0.8640 0.8640 0 .8 6 4 0 0 .8 6 4 0 0.8640 0.8640 0.8640 PFOS D ilu tio n 1 1 1 1 1 1 1 1 1 1 1 1 1 1 l 1 1 1 1 l 1 1 1 1 1 1 1 1 1 1 1 1 1 10 10 1 10 10 10 10 10 50 10 50 10 10 10 10 10 10 10 10 10 10 10 10 10 50 10 50 10 50 10 50 1 50 50 50 50 100 10 50 50 PFOS Cone. ng/m L 10.9 15.8 27.6 12.0 31.8 0.00 3 .2 8 3 .1 0 0.360 0.820 3.06 705 197 48.4 307 6 .7 4 68.0 41.8 97.9 25.9 52.8 0.00 69.2 15.5 26.9 46.8 43.3 77.9 14.8 46.3 64.1 35.8 59.6 3 .6 2 26.4 320 244 127 225 226 367 64.0 43.0 177 171 201 37.0 174 535 653 196 515 169 147 421 407 784 313 603 563 776 109 178 257 627 47.5 139 163 561 108 258 36.7 25.2 406 495 53.3 52.1 Filenam e (optional) A051000043 D050200028 A051000060 A051000061 A 0 5 1000046 D050200029 R053100019 R053100028 R 0 5 3 100020 D050200030 D050200033 A 051000048 A051000064 D050200034 A 051000067 D050200035 A051000049 D050200036 A 051000068 A 0 5 1000050 A051000053 R053100029 A051000055 A051000069 A051000070 D050200037 D050200040 D050200041 A 0 5 1000056 A051000071 A 051000074 A051000075 D050200042 A051000057 D050200043 R052600022 D050200044 D051000017 R050400017 D050200048 D 051000018 D051000019 D 051000020 R 051000023 R 0 5 1000024 R052500106 D 05I0000026 R052500107 R 0 5 1000030 R 051000031 R 051000032 R050400018 R050400019 R050400020 R051000033 R 0 5 1000034 R050400023 R051000037 R051000038 R050400024 R050400025 R 051000039 R050400026 R052500108 R050400027 R051000041 R050400030 R 0 5 1000044 R052600026 R 0 5 1000046 R 051000047 R 051000048 R 051000051 R050400031 R050400032 R 0 5 1000052 R 051000053 C onc entra tion of PFOS ug/m L o r % Ree 0 .0 1 4 8 0 .0 1 6 1 0 .0 2 8 5 0.0127 0 .0 3 2 5 < LOQ (0.00428 ug/mL) < LOQ (0.00428 ug/mL) < LOQ (0.00428 ug/mL) < LOQ (0.00428 ug/mL) < LO Q (0.00428 ug/mL) < LOO (0.00428 ue/mL) 0.624 0.225 0.0467 0.306 0.00650 0.0602 0 .0 4 2 7 0.100 0.0258 0 .0 4 8 3 < LOQ (0.00428 ug/mL) 0.0675 0.0140 0.0233 0.0407 0 .0 4 4 3 0 .0 7 9 5 0.0176 0 .0 4 0 2 0 .0 6 8 7 0 .0 3 1 1 0 .0 5 7 5 <LOQ (0.00848 ug/mL) 0 .0 2 2 9 0.309 0.212 2.55 1.95 0.230 3.94 1.39 0.461 2.05 1.48 8.72 0.349 9 .0 8 6.64 5 .6 7 1.70 5.97 1.73 1.34 4.06 6.81 2 .7 8 5 .2 4 5 .7 6 8 .9 8 14.3 1.8! 6.05 2.75 1.21 8.63 0.487 5.20 14.4 1.66 3.13 35.3 4.77 2 .7 2 2.83 PFOS 0.0118 0 .0 8 3 6 1.31 4 .3 5 7.60 LRN-U2121 RSD Std. Dev. 0.0104 161 1.30 2 .7 8 8 .6 0 NA Not Applicable Date Entered/By: D ate V erified/ By: Purity Enlered/Verified: 5/11/00, 5/12/00, 5/18/00, 5 /1 9/00,6/8/00, 6/19/00, 11/14/00, 11/17/00 M MH/CSH/LAC 08/31/00 KJH, 11/21/00 MMH 10/04/00 L A C / 10/04/00KJH E T S - 8 - 5 .1 Excel 97 rSWEnvrSnmenSr SBorTr^ T O X -002-seral83-3N 2/13/01 10:06 AM Page 161 3M Medical Department Study: T-6295.4 AMDT# 112296.1 Covance# 6329*183 Analytical Report: FACT TOX-002 LRN-U2121 3M Study Title: Covance Study Title: Product Number(Test Substance): 104 Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats 104 Week Dietary Chronic Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats T-6295 (PFOS) Rat Serum Method/Revision: ETS-8-4.1 & ETS-8-5.1 Analytical Equipment System Number: Ruby 100699. Davey 070799 Instrument Software/Version: MassLynx 3.3,3.4 Filename: R-Squared Value: See listing to the right See Attachments Slope: See Attachments V-Intercept: Dates of Extraction/Analyst: See Attachments 05/01/00. 05/02/00 SAL/KJK/RWW Dates of Analysis/Analyst: Date of Data Reduction/Analyst: 05/02/00. 05/04/00. 05/10/00,05/26/00. 05/30/00 MMH/IAS 05/04/00.05/05/00.05/11/00.05/30/00.06/01/00, 11/16/00 IAS/MMH Sample Data WEEK IQS RAT SERA _________ ___________ _________ _______________________ Lot 171 Dose S a m ple # Initial Voi. Surrogate Verified Original Purity Correction PFOS Purity Correction Factor PFOS Dilution Fa c to r PFOS Cone. ng/mL Filename (optional) Group 4 mg/kg/day C92691M C92693M C92694M C92695M 0.90 NA 0.9949 0.8640 0.85 NA 0.9949 0.8640 0.75 NA 0.9949 0.8640 0.85 NA 0.9949 0.8640 20 1(H) KM) KM) 232 R050400033 75.5 D051100016 640 R050400034 740 R050400037 C92696M C92698M C92699M 0.95 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 0.70 NA 0.9949 0.8640 100 100 100 423 R050400038 140 D0511OOOI7 591 R050400039 C92700M C92702M C92704M C92705M C92708M C92716M C927I7M C92724M C92726M C92730M C92735M C92736M C92739M C92740M C92742M C92747M C92748M C92750M 0.75 NA 0.9949 0.8640 0.90 NA 0.9949 0.8640 0.79 NA 0.9949 0.8640 0.73 NA 0.9949 0.8640 0.67 NA 0.9949 0.8640 0.75 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 0.85 NA 0.9949 0.8640 0.75 NA 0.9949 0.8640 0.70 NA 0.9949 0.8640 0.70 NA 0.9949 0.8640 0.75 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 0.85 NA 09949 0.8640 1.00 NA 0.9949 0.8640 0.57 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 0.85 NA 0.9949 0.8640 100 100 100 200 20 20 100 20 100 100 200 100 100 100 100 100 KM) 100 248 D051100018 32.6 D051100019 98.9 D051100020 96.8 R053100024 375 R053100025 584 R053100018 34.7 D051100024 626 R050400044 335 D051100025 54.1 D051100026 98.5 ROS0900029 35.5 D051100027 65.8 D051100030 349 D051100031 26,3 D051100032 300 D051100033 42.1 D051100034 33.3 D051100037 C93059F C93060F C93062F 0.80 NA 0.9949 0.8640 0.85 NA 0.9949 0.8640 1.00 NA * 0.9949 0.8640 500 5<M) 1000 189 R052500109 175 R0526OOO28 117 R050900030 C93063F C93072F C93077F C93078F C93079F C93080F C93081F 1.00 NA 0.9949 0.8640 0.85 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 0.94 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 0.90 NA 0.9949 0.8640 0.78 NA 0.9949 0.8640 1000 500 100 50 500 5(X> 500 197 R050900031 223 R052500109 438 D051100041 562 R050400048 168 R052500113 153 R052600029 127 R052500114 Group 5 mg/kg/day C93090F C93098F C93101F C93I02F C93107F C92753M C92757M C92761M C92762M C92774M 1.00 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 0.75 NA 0.9949 0.8640 0.80 NA 0.9949 0.8640 0.83 NA 0.9949 0.8640 0.80 NA 0.9949 0.8640 0.87 NA 0.9949 0.8640 0.65 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 200 KM) 500 100 50 2000 KMX) 2000 200 50 570 R050400051 59.5 D()51100047 202 R052500115 105 R051100051 635 R050400052 16.5 D051100052 196 R050900032 10.2 D051100053 751 R050400054 239 R052600033 C92775M C92776M C92780M C92782M C92786M C92788M C92791M 0.85 NA 0.9949 0.8640 0.96 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 0.57 NA 0.9949 0.8640 0.91 NA 0.9949 0.8640 0.80 NA 0.9949 0.8640 0.93 NA 0.9949 0.8640 50 2000 2000 50 50 2000 50 217 R052500117 59.9 D051100058 38.8 D051100059 220 R052500120 577 R050400055 54.4 D051100061 459 R050400058 C92792M C92800M C92802M 0.64 NA 0.9949 0.8640 0.91 NA 0.9949 0.8640 0.92 NA 0.9949 0.8640 2000 200 KXM) 6.62 D051100062 336 R050400059 179 R05090(K)33 C92803M C92804M C92808M C92809M C92814M C92815M 0.75 NA 0.9949 0.8640 0.88 NA 0.9949 0.8640 0.86 NA 0.9949 0.8640 0.65 NA 0.9949 0.8640 0.85 NA 0.9949 0.8640 0.70 NA 0.9949 0.8640 2000 200 50 50 2(XM) 2000 60.7 D051100065 645 R050400061 736 R050400062 90.1 R052600034 43.7 D051100067 4.79 D051100068 C92818M C93IL3F C93115F C93124F C93125F C93133F C93135F C93137F C93139F C93140F C93I46F C93149F C93151F C93153F C93157F C93158F C93161F C93162F C93164F C93165F C93166F 0.85 NA 0.9949 0.8640 0.94 NA 0.9949 0.8640 0.90 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 0.95 NA 0.9949 0.8640 0.75 NA 0.9949 0.8640 0.67 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 0.80 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 0.85 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 0.84 NA 0.9949 0.8640 1.00 NA 0.9949 (1.8640 1.00 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 0.70 NA 0.9949 0.8640 2000 2000 2000 2000 2000 2000 2000 2000 2000 50 2000 2000 2000 2000 2000 2000 200 2000 2000 2000 2000 44.1 D051100069 218 R050900036 148 D051100072 91.9 D051100073 203 R050900037 139 D051100074 158 R050900038 78.0 D051100075 162 D051100076 59.5 R052500123 29.6 DOS1100080 199 R050900039 80.1 D051100081 69.7 D051100082 14.0 D05U 00083 165 R050900040 499 R05040(K>72 257 R050900043 185 R050900044 94.7 D051100086 76.0 D051100087 C93168F C93174F C93177F C93178F C93179F 0.92 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 0.90 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 0.99 NA 0.9949 0.8640 2000 2000 2000 2000 2000 180 D051100088 175 R050900045 128 D051100089 124 D051100090 109 D051100093 Original PFOS LOQ updated to reflect purity information on 10/04/00. LAC 10/04/00 NA = Not Applicable Date Entered/By: Date Verified/ By: Purity Entered/Verified: 5/11/00. 5/12/00,5/18/00, 5/19/00, 6/8/00. 6/19/00. 11/14/00. 11/17/00 MMH/CSH/LAC 08/31/00 kjh. 11/21/00 MMH 10/04/00 LAC. 10/04/00 KJH Concentration of PFOS ug/mL o r % Ree 4.48 7.71 74.2 75.6 38.7 12.1 73.3 28.8 3.14 10.9 23.0 9.72 13.5 3.01 12.8 38.8 6.72 24.4 4.10 5.72 35.7 2.28 45.7 3.66 3.40 103 89.5 102 171 114 38.0 25.9 72.8 73.6 70.5 99.1 5.17 117 11.4 33.2 35.8 196 27.3 130 10.4 11.1 108 67.4 16.7 27.5 118 21.4 18.0 64.2 169 141 127 37.2 6.02 89.2 11.9 90.1 403 286 160 353 242 274 143 375 3.85 51.5 432 139 121 28.6 287 103 446 321 165 189 339 304 248 216 190 PFOS RSD Std. Dev. MS/MSD RPD 105 22.5 23.5 60.9 75.0 45.7 83.6 69.3 57.9 53.2 233 124 ETS-8-5.1 Excel 97 oM b llV iiuiim eiiia i Lauuiaiuiy TOX-002-seral83-3N 2/13/01 10:07 AM Page 162 3M Medical Department Study: T-6295.4 AM DT# 112296.1 Covance# 6329-183 Analytical Report: FACT TOX-002 LRN-U2121 3M Study Title: Covance Study Title: Product Number(Test Substance): Matrix: Method/Revision: A N A lytical E q uipm ent System N um ber: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: Sample Data 104 Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats 104 Week Dietary Chronic Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats T-6295 (PFOS) Rat Serum ETS-8-4.1 & ETS-8-5.1 Ruby 100699, Davey 070799 MassLynx 3.3, 3.4 See listing to the right See Attachments See Attachments See Attachments 05/01/00,05/02/00 SAL/KJK/RWW 05/02/00,05/04/00, 05/10/00, 05/26/00,05/30/00, 07/28/00 MMH/IAS 05/04/00,05/05/00, 05/11/00,05/30/00, 06/1AX), 07/31/00, 11/16/00 IAS/MMH WEEK 106 RAT SERA G roup Dose Sample # Initial Voi. Surrogate Original P uritj Verified Correction mL Factor Group 6 mg/kg/day C92821M C92823M 0.82 0.84 NA 0.9949 NA 0.9949 C92827M 0.96 NA 0.9949 C92829M 0.93 NA 0.9949 C92833M 0.66 NA 0.9949 C92834M 0.69 NA 0.9949 C92848M C92851M C92853M 0.80 0.84 0.84 NA 0.9949 NA 0.9949 NA 0.9949 C92856M 0.90 NA 0.9949 C93181F 0.86 NA 0.9949 C93182F 0.86 NA 0.9949 C93183F 1.00 NA 0.9949 C93184F 0.82 NA 0.9949 C93185F C93190F C93I92F 0.95 0.76 0.95 NA 0.9949 NA 0.9949 NA 0.9949 C93197F 1.00 NA 0.9949 C93198F 0.81 NA 0.9949 C93201F 0.79 NA 0.9949 C93202F 0.77 NA 0.9949 C93205F 0.78 NA 0.9949 C93209F 0.87 NA 0.9949 C93211F 0.88 NA 0.9949 C93216F UK) NA 0.9949 C93219F C93220F 0.85 1.00 NA 0.9949 NA 0.9949 Original PFOS LOQ (4.93 ng/mL) updated to reflect purity information on 10/04/00. LAC 10/04/00 PFOS Purity Correction Factor 0.8640 0.8640 0,8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 PFOS Dilution Factor 20 20 20 20 20 20 20 20 50 20 20 20 50 20 1 20 20 20 20 20 PFOS Cone. ng/mL 393 25.8 33,6 26.0 60.4 11.0 686 32.0 203 3.65 285 83.5 327 77.6 647 553 918 538 403 492 447 53.3 329 623 164 117 428 FileNAme (optional) R052600036 R052600037 R 0 5 3 100031 R 0 5 3 100032 R 0 5 3 100035 R0531(XX)36 R052600044 R 0 5 3 100037 D072800017 R 0 5 3 100038 R052600050 R052600051 R052600054 R052600055 R052600056 D0728(XX)18 R052600058 R052600061 R052600062 D072800019 R0526(XX)64 R0531(XX)39 R052600068 R052600069 R052600070 R052600071 R052600072 C oncentration of PFOS ug/mL o r % Ree 8.32 0.533 0.0303 0.0243 0.0795 0.0138 14.9 0.0331 0.210 < LOQ (0.00428 ug/mL) 5.76 1.69 5.68 1.64 11.8 31.6 16.8 9.35 8.63 27.0 10.1 0.0594 6.57 12.3 2.85 2.39 7.44 NA = Not Applicable Date Entered/By: Date Verified/ By: Purity Entered/Verified: 06/08/00, 06/19/00,08/14AX), 11/14/00, 11/17/1X) CSH/LAC 08/31/00 KJH, 11/21/00 MMH 10/04/00 LAC, 10/04/00 KJH Mean PFOS ug/m L 2.42 9.51 RSD Std. Dev. MS/MSD RPD 211 5.09 91.5 8.70 ETS-8-5.1 Excel 97 ^WEnVrnmflTbrTr TOX-(X)2-sera 183-3N 2/13/01 10:07 AM Page 163 3M Medical Department Study: T-6295.4 AMDT# 112296.1 Covance# 6329-183 Analytical Report: FACT TOX-002 LRN-U2121 3M Study Tide: Covance Study Tide: Product Number(Test Substance): M a tr ix : Method/Revision: AnalyticaJ E quipm ent System N um ber Instrument Software/Version : Date of Extracdon/Analyst: Date o f Analysis/Analyst: Date of Data Reduction/Analyst: Sample D ata 104 W eek Dietary Chronic T oxicity and Carcinogenicity Study w ith Perfluorooctane Sulfonic A cid Potassium Salt (PFOS T-6295) in Rats 104 W eek Dietary Chronic T oxicity Study w ith Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats T-6295 (PFOS) Rat Liver Filename: See L ist to Right FACT-M-1.0 & ETS-8-7.0 R-Squared Value: See Attachments Madeline 041098,Amelia 062498 Slope: See Attachments M assL y n x 3 .3 Y -Inte rc ept: See Attachments 6/11/98 IAS/RWW 07/06/98,07/14/98 KJH/M EE 02/02/00 IAS W E E K 4 R A T L IV E R R E W O R K ________________ ______________ L ot 193 G roup Sam ple# Initial WL Total M ass PFO S Std Dose Method Blk H 20 Blk-1 H 2 0 Blk-2 g 1.0000 1.0000 of Liver 8 NA NA Correction Factor 0 .9 2 7 5 0 .9 2 7 5 M atrix Blk Rabbit Liver Blk-1 1.0000 NA 0 .9 2 7 5 R abbit L iver Blk-2 1.0000 NA 0 .9 2 7 5 QC - 100 ppb C92506M-MS 1.0108 NA NA C92506M-MSD 1.0108 NA NA G roup 1 C92506M 1.0108 NA 0 .9 2 7 5 Control C92524M 0.9998 NA 0 .9 2 7 5 0.0 ppm C92526M 1.0287 NA 0 .9 2 7 5 in Diet C92529M 1.0117 NA 0 .9 2 7 5 C92546M 1.0034 NA 0 .9 2 7 5 C92861F 1.0140 NA 0 .9 2 7 5 C92864F 1.0172 NA 0 .9 2 7 5 C92914F 1.0186 NA 0 .9 2 7 5 C92919F 1.0145 NA 0 .9 2 7 5 C92926F 1.0092 NA 0 .9 2 7 5 G roup 2 C92574M 0 .9 9 2 8 NA 0 .9 2 7 5 Low Dose C92575M 1.0187 NA 0 .9 2 7 5 0.5 ppm C92610M 1.0060 NA 0 .9 2 7 5 in Diet C92613M 1.0075 NA 0 .9 2 7 5 C92618M 1.0133 NA 0 .9 2 7 5 C92982F 0 .9 9 7 1 NA 0 .9 2 7 5 C92941F 1.0104 NA 0 .9 2 7 5 C92945F 1.0161 NA 0 .9 2 7 5 C92950F 1.0132 NA 0 .9 2 7 5 C92969F 1.0038 NA 0 .9 2 7 5 G roup 3 C92646M 1.0116 NA 0 .9 2 7 5 M id Dose C92650M 1.0072 NA 0 .9 2 7 5 2.0 ppm C92652M 1.0030 NA 0 .9 2 7 5 in Diet C92668M 1.0036 NA 0 .9 2 7 5 C92678M 1.0010 NA 0 .9 2 7 5 C929%F 1.0047 NA 0 .9 2 7 5 C93006F 0.9943 NA 0 .9 2 7 5 C93008F 1.0158 NA 0 .9 2 7 5 C93031F 1.0166 NA 0 .9 2 7 5 C93045F 1.0076 NA 0 .9 2 7 5 G roup 4 C92715M 1.0003 NA 0 .9 2 7 5 Mid-High Dose C92718M 1.0026 NA 0 .9 2 7 5 5.0 ppm C92731M 1.0139 NA 0 .9 2 7 5 in Diet C92734M 0.9930 NA 0 .9 2 7 5 C92744M 1.0150 NA 0 .9 2 7 5 C93058F 1.0083 NA 0 .9 2 7 5 C93067F 1.0038 NA 0 .9 2 7 5 C93070F 0 .9 9 5 7 NA 0 .9 2 7 5 C93075F 1.0068 NA 0.9275 C93084F 1.0133 NA 0 .9 2 7 5 Group 5 C92752M 1 .0 1 0 0 NA 0 .9 2 7 5 High Dose C92759M 1.0122 NA 0 .9 2 7 5 20 ppm C92779M 1.0082 NA 0 .9 2 7 5 in Diet C92793M 0 .9 9 4 9 NA 0 .9 2 7 5 C92798M 1.0158 NA 0 .9 2 7 5 C93114F 1.0043 NA 0 .9 2 7 5 C93123F 1.0010 NA 0.9275 C93142F 1.0076 NA 0 .9 2 7 5 C93144F 1.0102 NA 0 .9 2 7 5 C93159F 1.0130 NA 0 .9 2 7 5 O riginal PFOS LO Q (59.5 ng/g) updated to reflect k+ information on 10/04/00, LA C 10/04/00 PFOS = Perfluorooctanesulfonate PFOS ng/R 0.00 0.00 0.00 0.00 231 311 21.8 182 207 0.00 0.00 89.4 80.9 202 76.6 137 398 319 278 465 326 293 261 309 277 282 444 283 337 346 289 313 157 325 297 270 577 645 292 533 528 1161 908 826 767 838 1190 1439 1858 1533 1630 2355 1943 2144 1728 1959 PFOS PFOS C o n cen tratio n D ilu tio n Calc. Cone. o f PFOS PFOS F a c to r _________ 5 1 _________ ug/g o r % R ee. 1 0.00 <LOQ (0.0552 ug/g) "g/g 1 0.00 <LOQ (0.0552 ug/g) <LO Q (0.0552 ug/g) 1 0.00 <LOQ (0.0552 ug/g) 0.00 <LOQ (0.0552 ug/g) <LO Q (0.0552 ug/g) 1 207 174% 1 286 241% 207% 1 20.0 <LOQ (0.0552 ug/g) 1 169 0.169 186 0.186 1 0.00 <L0Q (0.0552 ug/g) 1 0.00 <L0Q (0.0552 ug/g) 0.104 1 81.7 0 .0 8 1 7 1 73.8 0.0738 1 184 0.184 1 70.0 l 126 0.0700 0.126 0.107 33.3 12376 12.4 33.3 9670 9.67 33.3 8539 8.54 33.3 14248 14.2 33.3 9937 9.94 11.0 33.3 9089 9.09 33.3 7988 33.3 9398 7 .9 9 9.40 33.3 8429 8 .4 3 33.3 8669 8 .6 7 8.71 100 40692 40.7 100 26094 26.1 100 31167 31.2 100 31975 32.0 100 26736 26.7 31.3 100 28866 28.9 100 14601 14.6 100 29691 29.7 100 27099 27.1 100 24847 24.8 25.0 100 53503 53.5 100 59632 59.6 100 26740 26.7 100 49826 49.8 100 48285 48.3 47.6 100 106760 107 100 83858 83.9 100 76985 77.0 100 70660 70.7 100 76723 76.7 83.0 200 218558 219 * 200 263670 264 * 200 341904 342 * 200 285921 286 * 200 297613 298 * 282 200 434951 435 * 200 360089 360 * 200 394644 395 * 200 317269 317 * 200 358782 359 * 373 NAssNot Applicable RSD Std. Dev. M S/M SD RPD NA NA 3 2 .1 5 % 64.6 0 .0 6 7 3 45.4 0 .0 4 8 6 21.1 2.31 6.33 0 .5 5 2 18.7 5 .8 4 24.4 6.11 26.2 12.5 17.0 14.1 16.1 45.3 11.8 44.1 Correction Factors not applicable to M S/M SD Q C data * Results w ere outside the range o f the curve and should be considered to be estim ates. LA C 10/04/00 D ate Entered/Analyst: Date V erified/Analyst: Purity Entered/Verified : 02/08/00 L A C 10/03/00 K JH 10/04/00 L A C F A C T -M -2 .0 Excel 97 3M Environm ental Laboratory T O X -002-Iiverl83-4R 2/13/01 10:11 AM Page 164 3M Medical Department Study: T-6295.4 AMDT# 112296.1 Covanee# 6329-183 Analytical Report: FACT TOX-002 LRN-U2121 3M Study Title: Covance Study Title: Product N umber(Test Substance): M a tr ix : M e th o d /R e v is i o n : Analytical Equipm ent System Number: Instrument Software/Version: Date o f Extraction/Analyst: Date o f Analysis/Analyst: Date o f Data Reduction/Analyst: Sample Data 104 W eek Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic A cid Potassium S alt (PFOS T-6295) in Rats 104 W eek D ietary Chronic Toxicity Study with Perfluorooctane Sulfonic A cid Potassium Salt (PFOS T-6295) in Rats T-6295 R at Liver Filename: See L ist to Right FA C T-M -1.0& ETS-8-7.0 R-Squared Value: See Attachments Madeline 041098. Amelia 062498 Slope: See Attachments M assLynx 3.3 Y-Intercept: See Attachments 08/06/98 SAH 08/10/98.08/14/98,08/17/98 MEE/HOJ 02/03/00 IAS W E E K 14 R A T L IV E R R E W O R K t r Sample# Method Blk M atrix Blk Q C - lOOppb H 2 0 Blk-1 H 2 0 Blk-2 R abbit Liver Blk-1 Rabbit Liver Blk-2 C92509M-MS C92509M-MSD Initial W t g 1.0000 1.0000 1.0000 1.0000 0 .9 9 7 9 0 .9 9 7 9 Total Mass of Liver K NA NA NA NA NA NA L ot 193 PFO SStd C o rrectio n Factor 0 .9 2 7 5 0 .9 2 7 5 0 .9 2 7 5 0 .9 2 7 5 NA NA Group 1 C92509M 0.9979 NA 0 .9 2 7 5 Control C92511M 1.0044 NA 0.9275 0.0 ppm C92521M 0.9960 NA 0.9275 in Diet C92528M 1.0131 NA 0.9275 C92532M 1.0140 NA 0 .9 2 7 5 C92880F 1.0008 NA 0.9275 C92887F 1.0047 NA 0.9275 C92898F 1.0165 NA 0.9275 C92903F 1.0078 NA 0 .9 2 7 5 C92905F 0 .9 9 4 5 NA 0 .9 2 7 5 G roup 2 C92593M 0 .9 9 7 5 NA 0.9275 Low Dose C92600M 1.0058 NA 0 .9 2 7 5 0.5 ppm C92616M 1.0112 NA 0 .9 2 7 5 in Diet C92621M 1.0082 NA 0 .9 2 7 5 C92627M 0 .9 9 4 0 NA 0 .9 2 7 5 C92944F 1.0005 NA 0.9275 C92962F 0.9994 NA 0 .9 2 7 5 C92967F 1.0132 NA 0 .9 2 7 5 C92978F C92987F 1.0013 1.0095 NA 0.9275 NA 0.9275 Group 3 C92640M 1.0095 NA 0 .9 2 7 5 Mid Dose C92645M 0 .9 9 7 4 NA 0 .9 2 7 5 2.0 ppm C92662M 0 .9 9 7 7 NA 0.9275 in Diet C92676M 0.9926 NA 0.9275 C92684M 0 .9 9 8 7 NA 0.9275 C92993F 1.0006 NA 0 .9 2 7 5 C93000F 0.9994 NA 0.9275 C93018F 1.0081 NA 0 .9 2 7 5 C93023F 0.9965 NA 0 .9 2 7 5 C93035F 1.0019 NA 0 .9 2 7 5 G roup 4 C92713M 1.0048 NA 0.9275 M id-H igh Dose C92714M 1.0044 NA 0.9275 5.0 ppm C92719M 1.0050 NA 0 .9 2 7 5 in Diet C92722M 1.0095 NA 0 .9 2 7 5 C92728M 1.0011 NA 0.9275 C93051F 0.9944 NA 0.9275 C93064F 1.0103 NA 0 .9 2 7 5 C93071F 1.0001 NA 0.9275 C93085F 1.0038 NA 0.9275 C93109F 1.0022 NA 0.9275 G roup 5 C92765M 0.9983 NA 0.9275 High Dose C92777M 1.0067 NA 0.9275 20 ppm C92789M 0.9920 NA 0.9275 in Diet C92799M 1.0046 NA 0 .9 2 7 5 C92812M 1.0064 NA 0 .9 2 7 5 C93111F 0.9982 NA 0.9275 C93127F 1.0056 NA 0.9275 C93143F 1.0136 NA 0 .9 2 7 5 C93155F 1.0125 NA 0.9275 C93169F 0.9981 NA 0.9275 O riginal PFOS LOQ (61.3 ng/mL) updated to re f le a k+inform ation on 10/04/00. LAC 10/04/00 PFOS = Perfluorooctanesulfonale PFOSA = Perfluorooctanesulfonamide PFOSAA = Perfluorooctanesulfonamidoaceiate E tF O S E = N arrow Range N-Ethyl Perfluorooctanesulfonam ido ethyl alcohol Correction Factors not applicable to MS/MSD QC data PFOS ng/g 0 .0 0 60.9 0.00 12.1 533 658 473 587 529 440 457 110 563 76.4 572 126 265 209 296 230 285 204 191 271 156 216 180 158 147 146 167 149 153 146 139 160 406 403 409 336 384 381 393 383 404 441 777 670 598 494 526 745 672 679 737 612 PFOS D ilution 1 1 1 1 1 1 1 1 10 100 10 10 10 100 100 100 100 100 100 100 100 100 100 500 500 500 500 500 500 500 500 500 500 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 PFOS Calc. Cone. " 'S 0.00 56.5 0.00 11.2 59.6 184 440 542 492 403 418 1017 51996 697 5263 1175 24625 19274 27191 21166 26634 18933 17718 24839 14461 19842 82666 73468 68263 68329 77352 69020 71061 67380 64869 73916 374738 372357 377202 308422 356019 355218 360819 354845 373393 408111 722350 617695 559277 456225 485214 692038 619486 621011 674725 569017 C onc entra tion of PFOS ug/g o r % Ree. <LOQ (0.0569 ug/g) <LOO (0.0569 ug/g) <LOQ (0.0569 ug/g) <LO O (0.0569 ug/g) 50% 154% 0.440 0.542 0.492 0 .4 0 3 0.418 1.02 52.0 0.697 5 .2 6 1.18 24.6 19.3 27.2 21.2 26.6 18.9 17.7 24.8 14.5 19.8 82.7 73.5 68.3 68.3 77.4 69.0 71.1 67.4 64.9 73.9 375 372 377 308 356 355 361 355 373 408 722 618 559 456 485 692 619 621 675 569 NA = Not Applicable PFOS ug/R <LO Q (0.0569 ug/g) <LOQ (0.0569 ug/g) 102% 0.459 12.0 23.8 19.2 74.0 69.2 358 370 568 635 RSD Std. Dev. MS/M SD RPD NA NA 102% 12.5 0 .0 5 7 3 186 22.4 14.5 3 .4 5 19.7 3.77 8 .3 2 6.16 4.99 3 .4 6 8 .0 5 28.8 6 .0 3 22.3 18.8 107 7 .7 2 49.0 Dale Entered/Analyst: 02/09/00 LAC Date Verified/Analyst: 10/03/00 KJH Purity Entered/Verified: 10/04/00 LA C E T S -8 -7 .0 Excel 97 3M Environm ental Laboratory T O X -002-liverl83-4R 2/13/01 10:11 AM Page 165 3M Medical Department Study: T-6295.4 3M Study Title: Covance Study Title: Product N um ber(Test Substance): M atrix: M ethod/Revision: A nalytical Equipm ent System Num ber: Instrum ent Software/Version: Date o f Extraction/Analyst: D ate o f Analysis/Analyst: Date o f D ata R eduction/Analyst: Sample Data AMDT# 112296.1 Covance# 6329-183 104 W eek D ietary Chronic Toxicity and C arcinogenicity Study w ith Perfluorooctane Sulfonic A cid Potassium Salt (PFO S T-6295) in R ats 104 W eek D ietary C hronic T oxicity S tudy w ith Perfluorooctane Sulfonic A cid Potassium Salt (PF O S T-6295) in Rats T-6295 Rat L iver F ilenam e: See L ist to Right ET S-8-6.0 an d E TS-8-7.0 R-Squared V alue: See Attachm ents Soup 020199 M assL ynx 3.3 Slope: Y -Intercept: See A ttachm ents See A ttachm ents 11/11/99, 01/25/00 M CH /SA L 11/15/99, 11/19/99 11/22/99 IA S/M M H , 11/24/99 G M L , 12/16/99 IAS 11/16/99, 11/22/99, 11/23/99 IA S, 12/1/99 M M H , 12/17/99, 11/20/00 IA S/K JH F ilena m e s B lanks G rp 1 M G rp 1 F G rp 5 M G rp 5 F M S, MSD Box 99-222 Analytical Report: FACT TOX-002 LRN-U2121 PFOS 121699046-47,92-93 111999056-58, 121699067-76 111999059-, 121699063,77-87 112499088-99 112499100-115 121699060 PFO SA NA NA NA NA NA NA W EEK 53 RAT LIV ER ________ __________ ____ G roup Sam ple# Initial W L T otal M ass PFO S Std D ose M ethod Blk H 2 0 Blk-9 g 1.0000 o f Liver g NA C orrection F a c to r 0.9275 H 2 0 B lk-10 1.0000 NA 0.9275 M atrix Blk R abbit Liver Blk-9 1.0000 NA 0.9275 R abbit Liver Blk-10 1.0000 QC C 9 2 5 17 M -250 ppb-M S9 0.9979 NA NA 0.9275 NA C92876F-250 ppb-M SD10 0.9937 NA NA G roup 1 C92501M 1.0185 NA 0.9275 Control C92504M 1.0095 NA 0.9275 0.0 ppm in Diet C92507M C92512M 0.9916 0.9982 NA NA 0.9275 0.9275 M ales C92517M C92523M 1.0192 0.9909 NA NA 0.9275 0 .9 2 7 5 C92534M 1.0065 NA 0.9275 C92540M 1.0043 NA 0.9275 C92562M 1.0017 NA 0.9275 C92570M 1.0099 NA 0.9275 G roup 1 C92862F 1.0085 NA 0.9275 Control C92876F 0.9937 NA 0.9275 0.0 ppm C92889F 0.9965 NA 0.9275 in Diet C92894F 1.0128 NA 0.9275 Fem ales C92899F C92910F 0.9979 1.0171 NA NA 0.9275 0.9275 C92917F 1.0066 NA 0.9275 C92922F 1.0083 NA 0.9275 C92923F 1.0118 NA 0.9275 C92930F 1.0028 NA 0.9275 G roup5 C92751M 1.0034 NA 0.9275 High Dose C92754M 0 .9 9 0 7 NA 0.9275 20 ppm C92755M 1.0139 NA 0.9275 in Diet C92778M 0.9951 NA 0.9275 C92790M 1.0111 NA 0.9275 M ales C92795M 1.0179 NA 0.9275 C92796M 0.9930 NA 0.9275 C92817M 0.9950 NA 0.9275 C92820M 1.0019 NA 0.9275 G roup 5 C93112F 1.0040 NA 0.9275 High Dose C93116F 0.9931 NA 0.9275 20 ppm C93117F 0.9943 NA 0.9275 in Diet C93120F 1.0004 NA 0.9275 Fem aks C93130F C93138F 0.9912 0.9945 NA NA 0.9275 0.9275 C93147F 1.0040 NA 0.9275 C93154F 0 .9 9 6 7 NA 0.9275 C93163F 1 .0 1 4 1 NA 0.9275 C93171F 0.9000 NA 0.9275 O riginal PFO S LO Q (26.9 ng/g, 61.3 ng/g, 12.3 ng/g) u pd ated to reflect K+ an d purity inform ation on 10/04/00. L A C 10/04/00 C orrection Factors not applicable to M S/M SD Q C data PF O S - Perfluorooctanesulfonate N A = N ot Applicable Lot 171 PFO S Purity C orrection F a c to r 0.8640 0.8640 0.8640 0 .8 6 4 0 NA NA 0.8640 0 .8 6 4 0 0 .8 6 4 0 0.8640 0.8640 0.8640 0 .8 6 4 0 0.8640 0.8640 0.8640 0.8640 0 .8 6 4 0 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0 .8 6 4 0 0 .8 6 4 0 0.8640 0.8640 0 .8 6 4 0 0.8640 0.8640 PFOS Cone. HR/R 24.4 21.4 29.0 13.7 109 338 41.5 451 372 352 96.8 65.4 451 133 440 130 712 163 259 349 271 251 309 73.0 195 269 280 240 246 139 204 282 212 200 158 148 265 255 215 250 280 310 211 447 373 PFO S D ilu tio n F a c to r 1 10 10 10 10 I 10 10 1 1 1 1 1 1 1 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 PFO S Calc. C one, ng/g 19.6 17.1 23.2 11.0 119 176 32.6 358 301 283 761 529 359 106 3519 103 5657 131 2081 276 217 198 246 58.0 154 215 558532 485592 485843 279139 404861 554138 426888 403197 315897 296060 533843 513715 430218 504367 564538 619098 423936 883325 830454 C o n c en tra tio n of PFOS ug/g o r % Ree. <L O Q (0.0491 ug/g) < L O Q (0.0491 ug/g) < L O Q (0.0491 ug/g) <L O Q (0.0491 ug/g) 40% 58% 0 .0 3 2 6 0.358 0.301 0.283 0.761 0.529 0.359 0.106 3.52 0.103 5.66 0.131 2.08 0.276 0.217 0.198 0.246 0.0580 0.154 0.215 559 486 486 279 405 554 427 403 316 296 534 514 430 504 565 619 424 883 830 M ean PFOS u g /g <LO O (0.0491 ug/g) <LO Q (0.0491 ug/g) 49% 0.635 0.923 435 560 RSD S td. Dev. M S/M SD R P D NA NA 39% 163 1.04 191 1 .7 7 22.3 96.9 32.2 180 Date Entered/Analyst: D ate Verified/Analyst: Purity Entered/Verified: 11/24/99 L A C , 12/2/99 G M L , 01/24/00 M M H , 11/20/00 LAC 10/03/00 K JH , 11/21/00 M M H 10/04/00 L A C . 10/04/00 K JH E T S -8 -7 .0 Excel 97 3M Environm ental Laboratory T O X -002-Iiverl83-4R 2/13/01 10:11 AM Page 166 3M Medical Department Study: T-6295.4 AMDT# 112296.1 Covance# 6329-183 Analytical Report: FACT TOX-002 LRN-U2121 3M Study Title: Covance Study Title: Product NumbetfTest Substance): Matrix: M e tb o d /R e v is io n : Analytical Equipment System Number: Instrum ent Soflw are/V ersion: D ate o f Extraction/A nalyst: D ate o f Analysis/Analyst: D ate o f Data Reduction/Analyst: Sample Data 104 W eek D ietary Chronic Toxicity and Carcinogenicity Study w ith Perfluorooctane Sulfonic A cid Potassium Salt (PFOS T -6295) in Rats 104 W eek D ietary Chronic Toxicity Study with P erfluorooctane Sulfonic Acid Potassium Salt (PFO S T-6295) in Rats T-6295 Rat Liver Filename: See Below ETS-8-6.0 and ETS-8-7.0 R-Squared Value: See Attachments Amelia 062498 Slope: See Attachments M asslynx3.4 Y -Inte rc ept: See Attachments 05/16/0 0 ,0 5 /18/00 SAL/KJK 05/22/00,05/24/00,05/25/00. 06/12/00,06/14/00, 07/28/00,07/31/00 IAS/MMH 05/30/00, 06/15/00,06/16/00,07/31/00,08/01/00 IAS/M M H DAY 719 RAT L I V E R _____________________ _________________ ____________ ________ L ot 171 G roup Sam ple# Surrogate Initial WL Total M ass O riginal Purity PFOS Purity Dose M etbod Blk Matrix Blk QC 0 5 1 6 0 -H 2 0 B lk -5 05160-H 2 0 B lk -6 05 l8 0 -H 2 0 B lk -5 0518 0 -H 2 0 B lk -6 052200-H20Blk-3 052200-H 2 0 B lk -4 R B L05160-LiverB lk-5 RBL05160-LiverBlk-6 R B L05180-LiverBlk-5 R B L 05180-LiverB lk-6 RBL052200-LiverBlk-3 RBL052200-LiverBlk-4 C92503M G l-M S-250ppb-3 C92503M Gl-M SD-250ppb-3 C92505M G l-M S-250ppb-5 C92505M Gl-M SD-250ppb-5 C92866F-Gl-M S-250ppb-4 C 92866F-G 1-M SD-250ppb-4 C92868F Gl-M S-250ppb-6 C92868F Gl-M SD-250ppb-6 C92510M G 1-MS -250ppb-6 C92510M G 1-M SD-250ppb-6 C92991F G3-M S-250 ppb-5 C92991F G 3-M SD -250 ppb-5 C92821M G 6-M S-250 ppb-5 C92821M G6-M SD-250 ppb-5 Verified NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA g 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.0161 1.0161 0 .9 9 2 7 0.9927 0.9665 0.9665 1.0055 1.0055 0.9973 0.9973 1.0024 1.0024 0 .9 9 0 0 0.9900 of Liver g NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Correction Factor 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 NA NA NA NA NA NA NA NA NA NA NA NA NA NA Correction F a c to r 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 NA NA NA NA NA NA NA NA NA NA NA NA NA NA G roup 3 Mid Dose 2.0 ppm in D iet C92991F C93003F C93007F C93011F C93022F C93039F C93040F C93047F C93048F NA 1.0024 NA 0.9949 0.8640 NA 0.9967 NA 0 .9 9 4 9 0.8640 NA 1.0 0 1 0 NA 0 .9 9 4 9 0.8640 NA 1.0018 NA 0.9949 0.8640 NA 1.0008 NA 0.9949 0.8640 NA 1.0030 NA 0 .9 9 4 9 0.8640 NA 0.9967 NA 0 .9 9 4 9 0.8640 NA 1.0016 NA 0 .9 9 4 9 0.8640 NA 1.0020 NA 0 .9 9 4 9 0.8640 O riginal PFOS L 0 Q (1 2 .3 ng/g) updated r e f le c t purity information on 10/04/00. LA C 10/04/00 * R eanalyzed w ith the sam e results. LA C 08/14/00 a Endogenous level o f PFOS too high for MS/M SD analysis (diluted o ut o f accuracy range); do not include results in the body o f the final report. K JH 10/03/00 NA = Not applicable Date Entered/Analyst: Date Verified/Analyst. Purity Entered/V erifted: 06/01/00, 06/21/00, 0 6 /2 2/00,08/14/00 CSH/LAC 10/03/00 KJH 10/04/00 LA C, 10/04/00 KJH PFOS ng/g 0.740 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.570 0.00 0.00 357 411 496 478 125 135 379 433 803 966 492 517 319 309 504 593 1121 153 104 921 1080 615 620 PFOS D ilu tio n Factor 1 1 1 1 1 10 10 1 100 100 50 50 100 100 100 100 100 100 100 100 100 PFOS Calc. Cone. " s te 0.643 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.495 0.00 0.00 177 230 377 359 389 488 253 306 217 380 5430 7923 6423 5914 43639 51692 97210 13222 9018 79737 94108 53315 53775 Filenam e (optional) D061400077 D061400078 A052200060 A052200073 D 061400088 D 061400089 D061400079 D 061400080 A052200060 A052200074 D061400090 D061400091 A073100018 A073100019 A052200020 A052200021 A061200060 A061200061 D072800055 A052200025 D072800056 A052200076 D061400045 D061400046 D 0 6 1400047 D061400048 A052500015 A052500016 A052500017 A052500018 A052500019 A052500022 A052500023 A052500024 A052500025 C o n cen tratio n of PFOS ue/e o r % Ree < L O Q (0.0107 ug/g) < LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) < L O Q (0.0107 ug/g) < LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) < L O Q (0.0107 ug/g) 60% 78% 126% 120% 126% 158% 85% 102% 72% 126% 1816% 2650% 2134% 1965% 43.6 51.7 97.2 13.2 9.02 79.7 94.1 53.3 53.8 PFOS < LO Q (0.0107 ug/g) < L O Q (0.0107 ug/g) 69% 123% * 142% 94% 99% *a * a 2233% *a * a 2049% 55.1 RSD S td. Dev. M S/M SD RPD NA NA NA NA 26% 5% 22% 20% 55% 37% 8% 57.2 31.5 ETS-8-7.0 Excel 97 3M Environm ental Laboratory TOX-002-liverl 83-4R 2 /1 3 /0 1 10:12 AM Page 167 3M Medical Department Study: T-6295.4 AMDT# 112296.1 Covance# 6329-183 Analytical R e p o rL ^ A C T J O X flO 2 LRN-U2121 3M Study Title: Covance Study Title: Product NumheifText Substance): Matrix: Methnd/Revision: Analytical Equipment System Number Instrument Software/Version: Date of Extraction/Analyst Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple D ata 104 Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats 104 Week Dietary Chronic Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats T-6295 Rat Liver Filename: See Below ETS-8-fi.0and ETS-8-7.0 R-Squared Value: See Attachments Amelia 062498 Slope: See Attachments Masslynx3.4 Y-Intercept See Attachments 05/16/00-05/18/00,05/22/00 SAL/KJK 05/19/00, 05/22/00, 05/24/00, 06/12/00, 06/13/00,06/14/00, 07/28/00, 07/31/00 IAS/MMH 05/22/00, 05/23/00, 05/24/00, 05/25/00,06/13/00, 06/14/00, 06/15/00, 06/16/00, 07/31/00, 08/01/00, 11/20/00 IAS/MMH/KJH W E E K 105 R A T L IV E R ___________________________________________________________________________________________________ Lot 171 Do Method Blk Matrix Blk QC Group 1 Control 0.0 ppm in Diet Males Group 1 Control 0.0 ppm in Diet Females Group 2 Low Dose 0.5 ppm in Diet Males Group 2 Low Dose 0.5 ppm in Diet Females Sample # 05l60-H2OBlk-5 05160-H20Blk-6 05180-H20BUC-5 05180-H20Blk-6 052200-H20BC-3 052200-H20Blk-4 RBL05l60-LiverBlk-5 RBL05160-IiverBlk-6 RBL05180-LiverBlk-5 KBL05180-LiverBlk-6 RBL052200-UverBlk-3 RBL052200-LiverBlk-4 C92503M Gl-MS-250ppb-3 C92503M Gl-MSD-250ppb-3 C92505M Gl-MS-250ppb-5 C92505M Gl-MSD-250nnb-5 C92866F-G1 -MS-250ppb-4 C92866F-Gl-MSD-250nnb-4 C92868FGl-MS-250ppb-6 C92868F Gl-MSD-250ppb-6 C92510M Cl-MS-250ppb-6 C92510M C.l-MSD-250tJPb-6 C92991F G3-MS-250 ppb-5 C9299IF G3-MSD-250 nnb-5 C92821M G6-MS-250 ppb-5 C92821M G6-MSD-250 ppb-5 C92503M C92505M C92510M C92513M C925I4M C92518M C92541M C92542M C92544M C92551M C92569M C92866F C92867F C92868F C92869F C9287IF C92873F C92874F C92877F C9288IF C92882F C92885F C92890F C92892F C92895F C92896F C92897F C92906F C92907F C92909F C9291IF C92912F C92913F C92925F C92927F C92577M C92583M C92590M C92597M C92598M C92601M C92602M C92619M C92623M C92626M C92936F C92938F C92946F C92953F C92957F C92960F C92964F C92970F C92973F C92974F C92975F C92983F C92984F C92986F C92988F Surrogate Verified NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Confirmed Low NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Initial W t 8 l.(X) 1.00 1.00 1.00 1.0000 1.0000 1.00 1.00 1.00 1.00 1.0000 1.0000 1.0161 1.0161 0.9927 0.9927 0.9665 0.9665 1.0055 1.0055 0.9973 0.9973 1.0024 1.0024 0.9900 0.9900 1.0161 0.9927 0.9973 0.9947 0.9873 0.9939 1.0035 0.9861 0.9913 1.0077 1.0067 0.9665 0.9915 1.0055 1.0065 1.0005 1.0150 0.9957 0.9906 0.9994 1.0042 1.0344 0.9635 0.9984 1.0074 1.0100 0.9983 1.0148 0.9758 1.0083 1.0094 0.9960 1.0035 1.0722 0.9973 1.0121 0.9943 1.0076 1.0092 1.0050 0.9814 1.0729 1.0018 1.0866 1.0069 0.9944 1.0011 1.0030 1.0082 1.0272 1.0800 0.9955 1.0078 1.0016 0.9968 0.9967 0.9980 1.0023 0.9951 1.0009 Total Mass of Liver e NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Original Ihirity Correction Factor 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 PFOS Purity Correction Factor 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8fi40 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 Original PFOS LOQ (12.3 ng/g) updated to reflect purity information on 10/04/00. LAC 10/04/00 * Reanalyzed with the same results. LAC 08/14/00 a Endogenous level of PFOS too high for MS/MSD analysis (diluted out of accuracy range): do not include results in the body of the final report. KJH 10/03A)0 NA = Not Applicable Date Entered/Analyst: Date Verifted/AnaJyst Purity Entered/Verified: 5/24/00, 5/30/00, 5/31/00, 06/19/00.06/20/00, 06/21/00, 06/22/00, 08/14/00. 11/20/00 MMH/CSH/LAC 10/03/00 KJH, 11/21/00 MMH 10/04/00 LAC, 10/04/00 KJH PFOS 0.740 0.00 0.00 0.00 0.00 0.00 (MX) 0.00 0.00 1.14 0.00 0.00 357 411 496 478 125 135 379 433 803 965 492 517 319 309 177 122 587 75.9 265 8.00 32.7 38.9 33.2 0.00 80.0 101 419 125 290 25.0 297 173 309 104 264 0.00 275 56.3 131 135 182 384 38.9 131 239 76.5 211 6)6 185 186 188 90.2 200 233 244 124 32.1 167 130 120 20 176 234 172 614 230 77.7 58.6 181 155 310 99.0 151 198 PFOS Dilution Factor 1 1 1 1 1 1 1 1 10 10 1 1 1 100 100 50 50 1 1 1 1 1 1 1 10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 l 10 10 50 100 10 100 100 50 1(K) 50 100 100 1(H) 100 to o 10 100 100 100 100 100 100 100 100 100 PFOS Calc. Cone. 0.643 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.990 0.00 0.00 177 230 377 359 389 488 253 306 217 380 5430 7923 6423 5914 152 106 511 66.3 233 6.99 28.3 34.2 29.1 0.00 69.0 907 367 ]()8 250 21.7 254 151 271 90.4 228 0.00 248 49.0 113 116 158 328 34.6 113 206 66.7 183 4.99 161 1596 1641 3888 17190 2016 21574 10022 1390 13353 5606 10455 1719 15281 20115 14523 4938 2(H)50 6691 5084 15787 13476 26969 8576 13153 17189 Filename (optional) Concentration of PFOS PFOS D061400077 D061400078 A052200060 A052200073 D061400088 D061400089 D06I400079 D061400080 A052200060 A052200074 D061400090 D061400091 A073100018 A0731000I9 A052200020 A052200021 A061200060 A 0 6 1200061 D072800055 A052200025 D072800056 A052200076 D061400045 D061400046 D061400047 D061400048 A0524<HX)25 A052200026 A052200077 A052200080 A052400026 D061400081 A052200081 A052400027 A052200082 D061400084 A052200029 A061200062 A052200083 A052200032 A052200084 D061400085 A052400031 A052200034 A052200087 A052400032 A052400033 D061400092 A052400035 A052200088 A0522(XX)89 A052200035 A052200036 A052200039 A052400038 A052200090 A052200091 A052200092 A052200040 D061400095 A05220004I A 0 6 1200098 A051900015 D061400032 A061300015 A051900017 A061200089 A061200090 D061400033 A061200091 D061400034 D061400037 1)061400038 D061400039 A061200092 A06I200093 A061200099 A05I900018 A0519000I9 A051900023 D061400040 A061200096 A051900024 D061400041 A061200097 A051900025 < LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) < U > 0 (0.0107ue/e) < LOQ (0.0107 ug/g) < LOQ (0,0107 ug/g) < LOQ (0,0107 ug/g) < LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) < LOO (0.0107 ue/e) 60% 78% 126% 120% 126% 158%. 85% 102% 72% 126% 1816% 2650% 2134% 1965% 0.152 0.106 0.511 0.0663 0.233 < LOQ (0.0107 ug/g) 0.0283 0.0342 0.0291 < LOQ (0.0107 ug/g) 0.0690 0.907 0.367 0.108 0.250 < LOQ (0.0107 ug/g) 0.254 0.151 0.271 0.0904 0.228 < LOQ (0.0107 ug/g) 0.248 0.0490 0.113 0.116 0.158 0.328 0.0346 0.113 0.206 0.0667 0.183 < LOQ (0.0107 ug/g) 0.161 1.60 1.64 3.89 17.3 2.02 21.6 10.0 1.39 13.4 5.61 10.5 1.72 15.3 20.1 14.5 4.94 20.1 6.69 5.08 15.8 13.5 27.0 8.58 13.2 17.2 < LOQ (0.0107 ug/a) < LOQ (0.0107 ub/ b) * 69% 123% * 142% 94% 99%' *a * a 2233% *a a 2049% 0.114 0.185 7.83 12.9 RSI) Std. Dev. MS/MSD RPD NA NA NA NA 26% 5% 22% 20% 55% 37% 8% 130 0.148 99.4 0.184 93.8 7.34 52.7 6.81 ETS-8-7.1 Excel 97 3M Environmental Laboratory TOX-002-liverl 83-4R Page 168 3M Medical Department Study: T-6295.4 AMDT# 112296.1 Covance# 6329-183 3M Study Title: Covance Study Title: Product Number(Test Substance): Matrix: Metbod/Revision: Analytical Equipment System Number Instrument Snftware/Version: Date of Extraction/Analyst: Date of Analysis/AnaJyst: Date of Data Reduction/Analyst: Sample Data 104 Week Dietary Chronic Toxicity and Carcinogenicity Study with Periluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats 104 Week Dietary Chronic Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS T-6295) in Rats T-6295 Rat Liver Filename: See Below ETS-8-6.0 and ETS-8-7.0 R-Squared Value: See Attachments Amelia 062498, Davey 070799 Slope: See Attachments Masslynx 3.4 Y-lntercept See Attachments 5/16/00-5(18/00, 5/22/00 SAL/KJK 5/19AX), 5/22/00, 5/24/00, 5/26/00, 06/12/00,06/13/00, 06/14/00, 07/28/00,07/31/00 IAS/MMH 5/22/00, 5/23/00, 5/24/00. 5/25/00, 5/30/00, 06/13/00, 06/14/00, 06/15/00, 06/16/00, 07/31/00, 08/01/00 IAS/MMH WEEK 105 RAT LIVER_________________________________________________________________________________________________________ Lot 171 l)o*.P Method Blk Matrix Blk Sample # 0516O-H2OBIk-5 05160-H20BUC-6 05l80-H20Blk-5 05180-H20Blk-6 052200-H20Blk-3 052200-H20Blk-4 RBL05160-UverBlk-5 RBL05160-LiverBtk-6 Surrogate Verified NA NA NA NA NA NA NA NA Initial WL 8 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Total Mass of Liver 8 NA NA NA NA NA NA NA NA Original Purity Correction Factor 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0,9949 PFOS Purity Correction Factor 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 RBL05180-LiverBlk-5 RB L05180-LiverB Ik-6 NA NA 1.00 NA 0.9949 0.8640 1.00 NA 0.9949 0.8640 RBL052200-LiverBIk-3 NA 1.00 NA 0.9949 0.8640 RBL052200-LiverBlk-4 NA 1.00 NA 0.9949 0.8640 QC C92503M Gl-MS-250ppb-3 NA 1.0161 NA NA NA C92503M GI-MSD-250ppb-3 NA 1.0161 NA NA NA C92505M Gl-MS-250ppb-5 C92505M Gl-MSD-250pph-5 C92866F-Gl-MS-250ppb-4 C92866F-Gl-MSD-250ppb-4 C92868F Gl-MS-250ppb-6 C92868FG1-MSD-250ppb-6 NA NA NA NA NA NA 0.9927 0.9927 0.9665 0.9665 1.0055 1.0055 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA C92510M Gl-MS-250pph-6 C92510M G 1-MSD-250ppb-6 C92991F G3-MS-250 ppb-5 NA NA NA 0.9973 0.9973 1.0024 NA NA NA NA NA NA NA NA NA C92991FG3-MSD-250 ppb-5 C92821M G6-M5-250 ppb-5 C92821M G6-MSD-250 ppb-5 NA NA NA 1.0024 0.9900 0.9900 NA NA NA NA NA NA NA NA NA Group 3 Mid Dose 2.0 ppm C92631M C92633M C92637M NA 1.0036 NA 0.9949 0.8640 NA 0.9973 NA 0.9949 0.8640 NA 0.9931 NA 0.9949 0.8640 in Diet C92641M NA 1.0076 NA 0.9949 0.8640 Males C92642M C92643M NA 0.9928 NA 0.9949 0.8640 NA 0.9943 NA 0.9949 0.8640 C92644M NA 1.0045 NA 0.9949 0.8640 C92649M NA 1.0021 NA 0.9949 0.8640 C92653M NA 0.9991 NA 0.9949 0.8640 C92657M NA 1.0731 NA 0.9949 0.8640 C92659M NA 1.011 NA 0,9949 0,8640 C92660M NA 0.9929 NA 0.9949 0.8640 C92669M NA 1.0099 NA 0.9949 0.8640 C92674M NA 1.004 NA 0.9949 0,8640 C92682M NA 0.9935 NA 0.9949 0.8640 C92683M NA 1.0362 NA 0,9949 0.8640 C92690M NA 0.911 NA 0.9949 0.8640 Group 4 C92691M NA 0.9909 NA 0.9949 0.8640 Mid-High Dose 5.0 ppm C92693M C92694M NA 0.9722 NA 0.9949 0.8640 NA 1.0031 NA 0.9949 0.8640 in Diet C92695M NA 09918 NA 0.9949 0.8640 C92696M NA 1.0070 NA 0.9949 0.8640 Males C92698M NA 1.0065 NA 0.9949 0.8640 C92699M NA 1.0000 NA 0.9949 0.8640 C92700M NA 1.0539 NA 0.9949 0.8640 C92702M NA 0.9997 NA 0.9949 0.8640 C92704M NA 1.0042 NA 0.9949 0.8640 C92705M NA 0.9979 NA 0.9949 0.8640 C92708M NA 1,0022 NA 0.9949 0.8640 C92716M NA 0.9955 NA 0.9949 0.8640 C92717M NA 10129 NA 0.9949 0.8640 C92724M C92726M NA 1.0017 NA 0.9949 0.8640 NA 1 0698 NA 0.9949 0.8640 C92730M NA 1.0073 NA 0.9949 0.8640 C92735M C92736M NA 1.0086 NA 0.9949 0.8640 NA 09886 NA 0.9949 0.8640 C92739M NA 1 0888 NA 0.9949 0.8640 C92740M C92742M C92747M C92748M NA 09962 NA 0.9949 0.8640 NA 1 0035 NA 0.9949 0.8640 NA 0.9921 NA 0.9949 0.8640 NA 1.0012 NA 0.9949 0.8640 C92750M NA 1.0072 NA 0.9949 0.8640 Group 4 C93059F NA 0.9952 NA 0.9949 0.8640 Mid-High Dose C93060F NA 1.0789 NA 0.9949 0.8640 5.0 ppm C93062F NA 1,0068 NA 0.9949 0.8640 in Diet C93063F NA 0.9976 NA 0.9949 0.8640 C93072F NA 0.9991 NA 0.9949 0.8640 Females C93077F NA 1.0544 NA 0,9949 0.8640 C93078F NA 1.0089 NA 0.9949 0.8640 C93079F NA 0.9979 NA 0.9949 0.8640 C93080F NA 1.0316 NA 0.9949 0.8640 C93081F NA 09939 NA 0.9949 0.8640 C93090F NA 1 0008 NA 0,9949 0.8640 C93098F NA 09906 NA 0.9949 0.8640 C9310IF NA 0.9962 NA 0.9949 0.8640 C93102F C93107F NA 1.0888 NA 0.9949 0.8640 NA 1.0096 NA 0.9949 0.8640 Original PFOS LOQ (12-3 ng/g) updated to reflect purity information on 10/04/00. LAC 10/04/00 * Reanalyzed with the same results. LAC 08/14/00 a Endogenous level of PFOS too high for MS/MSD analysis (diluted out of accuracy range): do not include results in the body nf the final report. KJH 10/03/00 NA = Not Applicable PFOS Cone. 0.740 0,00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.570 0.00 0.00 357 411 496 478 125 135 379 433 803 966 492 517 319 309 480 588 43.7 38.9 273 212 50.0 623 131 245 715 121 202 682 437 202 226 317 136 240 223 139 87.6 189 335 60.1 756 311 595 393 638 531 468 83.7 856 68.8 179 711 33.1 776 52.9 31.5 841 660 112 611 771 740 287 519 709 539 858 87.6 888 201 371 PFOS Dilution Factor 1 1 1 10 10 1 to o 100 50 50 100 100 100 100 100 100 100 100 25 100 100 KK1 100 100 KM) 100 1(K) KM) 250 KMK) 1000 1000 150 1000 250 250 100 250 100 250 25 100 250 250 KM) 250 250 250 250 250 250 150 250 250 2500 250 160 250 250 250 250 250 250 25 250 150 250 PFOS Calc. Cone. 0.643 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.495 0.00 0.00 177 230 377 359 389 488 253 306 217 380 5430 7923 6423 5914 41.622 51167 3823 3352 23890 18505 43)8 54004 2855 19795 61389 tossi 17354 59020 38218 16893 21542 27748 30480 207597 194911 120002 18896 163768 69067 13052 65347 67702 51567 85794 13674 46056 95019 18049 73730 15111 35603 154965 7159 169815 11473 6796 183396 132866 241755 133009 167486 152272 61653 112894 149290 117702 186045 19199 193426 40012 79802 Dale Entered/Analyst: Date Verified/Analyst Purity ErrteresWerified: 5/24/00, 5/30/00. 5/31/00,6/2/00.06/20/00, 06/21/00, 06/22/00, 08/14/00 MMH/CSH/LAC 10/03/00 KJH 10/04/00 LAC, 10/04/00 KJH Analytical Report: FACT TOX-002 ^^TrN-U2T2^ Filename (optional) D061400077 D061400078 A0522OOO6O A052200073 D061400088 D061400089 DO614O0O79 D061400080 A05 2200060 A052200074 D061400090 D061400091 A073I00018 A0731000I9 A052200020 A052200021 A061200060 A061200061 D072800055 A052200025 D072800056 A0522(KM)76 D061400045 D061400046 D061400047 D061400048 A051900026 A05250W26 A051900027 A052500029 A051900031 A052600015 A051900032 A052500030 A061200100 D052600017 A052500031 A052500032 A052500033 A051900033 A051900034 D052600018 D052600019 A051900035 D052600022 A061300017 A061300018 A061300019 A052500036 A06I300022 D052600023 A052500037 A0519OOO43 D072800048 A051900047 A05 2500039 A061200I03 A051900048 D052600025 A052500040 A051900049 D052600026 D052600029 DOS2600030 AO5250OO43 A052500044 A052500045 A052500046 A05 2500047 DO526OO03I A061300016 A051900051 A052500050 D052600032 A051900055 A052500051 D052600033 A052500052 A051900056 A052500053 A0525(MH)54 D052600036 A0519OOO57 Concentration of PFOS ue/e or % Ree PFOS < LOQ (0.0107 ug/g) <LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) <LOQ (0.0107 ug/g) < LOO (0.0107 ub/e) <LOQ (0.0107 ug/g) < LOO (0.0107 ub/b) < LOQ (0.0107 ug/g) < I/>Q (0.0107 ug/g) <LOQ (0.0107 ug/g) <LOQ (0.0107 ug/g) < LOO (0-0107 ub/b) <LOO (0.0)07 ub/ b) 60% 78% 69%, 126% 120%, I23%- 126% 158% * 142% 85% 102% 94% 72%, 126% 1816% *a 99%. 2650% * a 2233%, 2134%, *a 1965%, * a 2049%- 41.5 51.2 3.82 3.35 23.9 18.5 4.32 54.0 2.85 19.8 61.4 10.6 17.4 59.0 38.2 16.9 21.5 26.4 27.7 30.5 208 195 120 18.9 164 69.1 13.1 65.3 67.7 51.6 85.8 13.7 46.1 95.0 18.0 73.7 15.1 35.6 155 7.16 170 11.5 6.80 70.5 183 133 242 133 167 152 61.7 113 149 118 186 19.2 193 40.0 79.8 131 RSI) Std. Dev. MS/MSD RPD NA NA NA NA 26% 5%, 22% 20% 55% 37% 8%, 77.5 20.4 89.4 63.1 46.7 61.4 ETS-8-7.1 Excel 97 3M Environmental Laboratory TOX-002-liverl 83-4R Page 169 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 AMDT# 112296.1 Covnace# 6329-183 LRN-U2121 3M Study Title: Covance Study Title: Product Numher(Test Substance): Matrix: Melhod/Revision: Analytical Equipment System Number: Instrument Soflware/Versdon: Date of Extraction/Analyst: Dale of Analysis/Analysi: Date of Data Reduction/Analysl: Sam ple Data 104 Week Dietary Chronic Toxidty and Carcinogenicity Study with Perfluorooctane Sulfonic Add Potassium Salt (PFOS T-6295) in Rats 104 Week Dietary Chronic Toxiaty Study with Perfluorooctane Sulfonic Add Potassium Salt (PFOS T-6295) in Rats T-6295 Rat Liver Filename: See Below ETS-8-6.0 and ETS-8-7.0 R-Squaied Value: See Attachments Amelia 062498. Davey 070799 Slope: SeeAttachment* Masslynx 3.4 5/16/00-5/1 SAX), 5/22/00 SAL/KJK Y-lntercept: SeeAttachments 5/19/00, 5/22/00, 5/24/00, 5/26/00.06/12/00,06/13/00.06/14/00,07/28/00,07/31/00 IAS/MMH 5/22/00. 5/23/00, 5/24/00. 5/25/00,5/30/00, 06/13/00,06/14/00,06/15/00,06/16/00,07/31/00, 08/01/00 IAS/MMH W EEK 105 RAT LIVER Group Duse Sample# G roups High Dose 20 ppm Males Groups High Dose 20 ppm in Diet Females RBL05160-LiverBlk-6 RBL05180-LiverBlk-6 C92S03M GI-MS-250ppb-3 C92503M Gl-MSD-250nt)b-3 C92505M Gl-MSD-250nnb-5 C92866F-GI-MSD-250nnb-4 CV2868FGI-MSD-250rnb-6 C92510M Gl-MSD-2S0ni*-6 C9299IFG3-MSD-250 nnb-5 C92821M G6-MSD-250 ppb-5 C92753M C92757M C9276IM C92762M C92774M C92776M C92780M C92786M C92788M C92791M C92792M C92800M C92802M C92803M C92804M C92808M C92809M C928I4M C92815M C93II3F C93115F C93I24F C93I25F C93I33F C93I35F C93I37F C93I39F C93140F C93151F C93I58F C93I6IF C93162F C93164F C93166F C93I68F C93I74F Surrogate Verified NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Initial Wt. LOO 1.00 1.00 1.00 1.0000 1.0000 1.00 1.00 1.00 1.00 1.0000 1.0161 1.0161 0.9927 0.9927 0.9665 0.9665 1.0055 1.0055 0.9973 0.9973 1.0024 1.0024 0.9900 0.9900 1,0015 1.0100 1.0065 0.9941 0.9768 1.0108 0.9953 0.9985 0.9923 1.0004 1.0336 1.0100 1.0046 1.0003 0.9946 1.0428 1.0029 0.9908 1.0161 1.0275 0.9995 1.0013 0.9987 1.0021 1.0015 1.0051 0.9705 1.0082 0.9941 0.9735 0.9965 1.0091 0.9962 1.0219 1.0057 0.9904 1.0034 0.9938 1.0044 1.0078 1.0317 0.9959 0.9930 0.9921 1.0740 0.9730 1.0472 Total Ma of Liver NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Correction Factor 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.944V 0.9949 Reanalyzed with the same results. LAC 08/I4AX) a Endogenous level of PFOS loo high for MS/MSD analysis (diluted out of accuracy range); do not indude results in the body of the final report. Kjh 10/03/00 NA a Not Applicable Dale Enlered/Analysl: Dale Verified/Analyst: Purity Entered/Verified: 05/24/00,05/30/00. 05/31A. 06/02/00.06/20/00,06/21/00.06/22AJ0.08/14/00 MMH/CSH/LAC 10/03/00 KJH 10/04/00 LAC, 10AI4AK) KJH PFOS Purity Correction Factor 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 PFOS 0.740 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.570 0.00 0,00 357 411 496 478 125 135 379 433 803 966 492 517 319 309 261 765 186 400 88.6 81.4 734 375 121 227 915 160 127 366 93.6 987 762 271 1091 847 166 812 85.8 872 401 103 900 135 643 999 21.1 304 132 687 541 175 143 486 161 118 976 903 1056 113 886 792 PFOS Dilution Factor 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I 1 10 to 100 100 50 50 500 500 500 500 500 500 500 500 500 500 500 500 500 500 5000 500 500 500 25 500 500 5(H) 5000 500 500 5000 500 5000 500 500 500 500 5000 500 500 500 5000 500 5000 5000 500 500 500 5000 50(10 500 500 PFOS Calc. Cone. Filename (optional) Concentration of PFOS 0.643 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.495 0.00 D061400077 D061400078 A052200060 A052200073 D061400088 D061400089 D061400079 D061400080 A052200060 A052200074 D06I400090 <LOQ (0.0107 ug/g) 177 230 377 359 389 488 253 306 217 380 5430 7923 6423 5914 113104 329066 80173 174603 39390 34980 320170 163214 53101 98636 384592 68954 54772 158784 408544 411159 330049 118664 23316 357873 71998 351938 373041 377647 173786 444108 402766 581680 280805 445629 9198 130983 575132 291713 233544 76847 617048 212218 694812 509959 410860 393880 461592 493038 62269* 395515 328294 A07310(H)18 A073IO0OI9 A052200020 A05220002I A061200061 A052200025 D072800056 A052200076 D061400046 D061400048 A05I900058 A052500057 A051900059 D052600038 A052500058 A052500059 D052600039 A052500060 A051900063 D052600040 A051900064 A05250006I A051900065 A06I300023 D052600043 A051900067 A051900071 A061200106 D052600045 A052500064 A052500065 A06I300024 A052500066 A052500067 A061300025 D052600046 A06I300026 A052500068 D052600047 A052500071 A052500072 A061300029 D052600050 A052500073 D052600051 A061300030 A051900080 A061300031 A061300032 D0S2600052 A052500074 A052500075 A061200105 D052600054 D052600057 102% 2650% 1965% *a *a 35.0 54.8 72.0 352 403 446 292 234 76.8 695 510 396 328 Mean PFOS 381 RSD Std. Dev. NA N\ NA NA '6% 5% 5551 176 3M Environmental Laboratory TDX-002-liverl83-4R Page 170 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 AM DT# 12296.1 Covance# 6329-183 LRN-U2121 3M Study Title: Covance Study Title: Product NumhertTeut Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Date of Extraction/Analyst: Date of Analysis/Analyst: Dale of Data Reduction/Analyst: Sample Data 104 Week Dietary Chronic Toxicity and Carcinogenicity Study with Periluorooctane Sulfonic Add Potassium Salt (PFOS T-6295) in Rats 104 Week Dietary Chronic Toxicity Study with Perfluoronctane Sulfonic Add Potassium Salt (PFOS T-6295) in Rats T-6295 Rat Liver Filename: See list to right ETS-S-6.0 and ETS-8-7.0 R-Squared Value: See Attachments Amelia 062498, Davey070799 Slope: See Attachments Masslynx 3,4 Y-Intercept: See Attachirents 5/16/00 5/18/00 SAL/KJK 5/25/00,06/13/00,06/14/00, 07/2MX). 07/31/00IAS/MMH 5/30/X). 06/14/00, 06/15/00, 07/31AX), 08/01/00 IAS/MMH W EEK 106 RA T L IVER Dose Method Blk Sample # 05160-H20Blk-5 Matrix Blk QC Group 6 RBL05160-LiverBlk-5 RBL05l80-LiverBlk-5 C92503M Gl-MSD-250onb-3 C92505M C.l-MS-250ppb-5 C92505M G1-MSD-250ppb-5 C92866F-Gl-MS-250ppb-4 C92866F-G l-MSD-250ppb-4 C92868F G 1-MSD-250pnb-6 C92510M GI-MSD-250ppb-6 C92991FG3-MSD-250 ppb-5 C92821M G6-MS-250 ppb-5 C92821M G6-MSD-250 ppb-5 C92821M C92823M C92827M C92829M C92833M C92834M C92848M C92851M C92853M C92856M C93181F C93182F C93183F C93184F C93185F C93190F C93192F C93197F C93198F C93201F C93202F C93205F C93209F C93211F C93216F C93219F C93220F VerlOed NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Initial WL 8 1.0000 1.0000 1.0000 1.0000 1.0000 1,0000 1.(XXX) 1.0000 1.0000 l.(XXX) 1.0000 1.0000 1.0161 1.0161 0.9927 0.9927 0.9665 0.9665 1.0055 1.0055 0.9973 0.9973 1.0024 1.0024 0.9900 0.99<X) 0.9900 1.0378 0.9974 1.0069 1.0462 1.0129 0.9561 1.0102 1.0350 1.0274 1.0108 1.0429 0.9968 0.9740 1.0186 1.0540 1.0134 1.0425 1.0660 1.0054 1.0631 1.0013 0.9948 1.0091 1.0096 1.0009 0.9938 of Uver NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Original Purity Correction Factor 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 PFOS Purity Correction Factor 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.8640 0.8640 0.8640 0.8640 0.8640 0.R640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 * Reanalyzed with the same results. LAC 08/14/00 a Endogenous level of ITOS too high for MS/MSD analysis (diluted out of accuracy range): do m lclude results in the body of the final report. Kjh 10/03AX) NA = Not Applicable Date Enlered/Analyst: Date Verified/Analysl: Purity Entered/Verified: 06AHAX), 06/21/00, 06/22/00, 08/14/00 CSH/LAC 10/03/00 KJH 10/04/00 LAC. 10/04AX) KJH PFOS 0.740 0.00 0.) ().(X) 0.00 0.00 0.00 0,(X) ().> 0.570 0.00 ().(X) 357 411 496 478 125 135 379 433 803 966 492 517 319 309 110 22.9 165 136 460 236 197 423 514 39.3 50.2 25.3 104 18.9 186 27.7 175 104 75.9 222 209 152 170 49.5 171 57.2 22.3 PFOS Dilution Factor 1 1 1 1 1 1 1 1 10 10 1 1 1 1 100 100 50 50 100 100 1 1 1 1 100 1 1 1 100 100 too 100 100 10) 100 l(K) 100 100 100 1 1) 1000 100 1) 1000 PFOS Calc. Coite. 0.643 0.00 0.) 0.00 0.(X) 0.CK) 0.00 0.00 ().) 0.495 0.) 0.00 177 230 377 359 389 488 253 306 217 380 5430 7923 6423 5914 9680 1915 144 117 382 203 17890 363 431 33.2 4316 2103 9079 1688 15896 22831 15018 8650 6184 19198 17050 132 14854 42582 14687 4962 19469 Filename (optional) Concentration of PFOS D061400077 D061400078 A052200060 A052200073 D061400088 D061400089 D0614(XX)79 D0614000R0 A052200060 A052200074 D0614X)90 D061400091 < LOQ (0.0107 ug/g) <LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) <LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) A073100019 78% A052200021 120% A061200061 D072800055 A052200025 D072X(XK)56 A052200076 DOfi1400045 D061400046 D061400047 D061400048 D061400020 D061400023 D061300032 D061300033 D0613X)34 D061300037 D061300016 D06I3X)38 D0613X)39 D0614000I6 D0614(XX)17 D061400018 D06I300019 D06I4X)19 D061300023 A052500085 D061300024 D061300025 D0613(XX)26 D061300027 D06I4K>24 D061300044 D061400025 A052500109 D061400027 D061400030 A0S2500114 158% 102% 126% 2650% 1965% 9.68 0.144 0.117 0.382 0.203 17.9 0.363 0.0332 2.10 9.08 1.69 15.9 22.8 15.0 8.65 6.18 17.0 0.132 14.9 42.6 14.7 4.96 19.5 * a *a ' Mean PFOS fr'fi , 12.9 RSD Sid. Dev. 10.4 3M Environmental Laboratory TOX-002-Bverl83-4R Page 171 3M Medical Department Study: T-6295.4 Analytical Report: FACT TO X-002 LRN-U2121 Appendix F: Example Calculations Formula Used for Sera Analyses in Study FACT TOX-002 A R (n g /m L ) x D F x SC x F V (m L ) x 1.0 pg x PC = R eported C oncentration (pg/m L) E V (m L ) 1000 ng OPC Calculation Used for Group 3, Day 719, Animal ID C92991F 348 n g /m L x 50 x 0.9275 x 1 m L x 1 .0 pg 1 mL 1000 ng x 0.864 = 14.0 pg/m L 0.9949 A R -- A n a ly tic a l resu lt fro m M a ssL yn x sum m ary D F -- D ilu tio n factor SC-- P FO S salt co rre ctio n constant (0 .9 2 7 5 ) F V -- F in a l e xtra ct v o lu m e (1.0 m L unless o the rw ise noted) E V -- V o lu m e o f sera extracted PC -- PFO S p u rity co rrectio n fa cto r (86.4% fo r lo t 171) O P C -- O rig in a l PFO S p u rity co rrectio n fa cto r (99 .49 % ) Formula Used for Liver Analyses in Study FACT TOX-002 A R (n g /g ) x d c u rv e (1) x SC x D F x 1 . 0 p g x P C = R e p o rte d C o n c e n tra tio n (pg/g) d specim en 1000 ng (1) d c u rv e is a ssum ed to b e: 1 g liv e r 5 m L H 2O Calculation Used for Group 3, Week 105, Animal ID C92631M 4 80 n g /g x 1 g / 5 m L x 100 x 1.0 p g x 0.864 1.0036 g / 5 m L 1000 ng 0.9949 = 41.5 pg/g A R -- A n a ly tic a l resu lt fro m M a ssL yn x sum m ary d curve-- D e n sity o f the liv e r standard curve, assumed to be 1g liv e r/ 5 m l w ater d specim en-- D e n s ity o f the liv e r specim en (g specim en/ 5 m L H 2O ) D F -- D ilu tio n factor PC -- PFO S p u rity co rrectio n fa cto r (86.4% fo r lo t 171) O P C -- O rig in a l PFO S p u rity co rrectio n fa cto r (99 .49 % ) 3M Environmental Laboratory Page 172 3M Medical Department Study: T-6295.4 S ta n d a rd d e via tio n s w e re ca lcu la te d using the equation: n ix 2 - (Ix )2 IISD = n(n - 1) Analytical Report: FACT TOX-002 LRN-U2121 3M Environmental Laboratory Page 173 3M Medical Department Study: T-6295.4 Appendix G: Interim Certificates of Analysis Analytical Report: FACT TOX-002 LRN-U2121 3M Environmental Laboratory Page 174 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023-018B 3M Product: PFO S,Lotl71 Reference #: SD-009 ____________________ Purity: 86.4% Test Name Specifications P u rity 1 Result 86.4% A ppearance Identification NMR M etals (ICP/M S) 1. C alcium 2. M agnesium 3. Sodium 4. Potassium 2 5. N ickel 6. Iron 7. M anganese Total % Impurity (NMR) Total % Impurity (L C /M S ) Total % Im purity (G C /M S) Related Compounds POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. C hloride 2. Fluoride 3. Brom ide 4. Nitrate 5. N itrite 6. Phosphate 7. S ulfate4 Organic Acids 5(IC) 1. T FA 2. PFPA 3. H FBA 4. NFPA Elem ental Analysis": 1. C arbon 2. Hydrogen 3. N itrogen 4. Sulfur 5. Fluorine W hite Crystalline Powder 1. Theoretical V alue = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical V alue = 60% C onform s Positive 1. 0.017 w t./w t.% 2. 0.007 wt.Avt.% 3. 1.355 wtVwt.% 4. 6.552 wt./wt.% 5. 0.003 wt./wt.% 6. 0.004 w t./w t% 7. <0.001 w t./wt.% 1.00 wt.Avt.% 10.60 wt./wt.% None Detected 0.30 wt./wt.% None Detected N ot Applicable"1 1. <0.015 wt.Avt.% 2. 0.27 wt.Avt.% 3. <0.040 wt.Avt.% 4. <0.009 wt.Avt.% 5. <0.006 wt.Avt.% 6. <0.007 wt.Avt.% 7. 8.82 wt.Avt.% 1. <0.1 wt.Avt.% 2. <0.1 wt.Avt.% 3. <0.1 wt.Avt.% 4. <0.25 wt.Avt.% 1. 12,08 wt.Avt.% 2. 0.794 wt.Avt.% 3. 1.61 wt.Avt.% 4. 10.1 wt.Avt.% 5. 50.4 wt.Avt.% COA023-018B Page 1 o f 3 Page 175 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B D ate o f Last A nalysis: 08/31/00 E xpiration D ate: 08/31/01 Storage C onditions: Frozen <-10C R e-assessm ent D ate: 08/31/01 P u rity = 100% - (sum o f m etal im purities, 1,39% +LC /M S im purities, 10,60% +Inorganic Fluoride, 0.27% +N M R im purities, 1.00% + PO A A , 0.30% ) T otal im purity from all tests = 13.56% Purity = 100% - 13.56% = 86.4% 2P o ta s s iu m is e x p e c te d in th is sa lt fo rm a n d is th e re fo re n o t c o n s id e re d a n im p u rity . 3P u rity b y D S C is g e n e ra lly n o t ap p lic a b le to m a te ria ls o f lo w p u rity . N o e n d o th e rm w a s observed for this sam ple. 4S u lfu r in th e sa m p le a p p e a rs to b e c o n v e rte d to S O 4 a n d h e n c e d e te c te d u s in g th e inorganic anion m ethod conditions. The anion result agrees w ell w ith the sulfur determ ination in the elem ental analysis, lending confidence to this interpretation. B ased on the results, the SO 4 is not considered an im purity. 5T F A HFBA NFPA PFPA Trifluoroacetic acid H eptafluorobutyric acid N onofluoropentanoic acid Pentafluoropropanoic acid ^Theoretical value calculations based on the em pirical form ula, C gFiySC blC (M W =538) T his w ork w as conducted under E PA G ood L aboratory P ractice Standards (40 C F R 160). /T ' COA023-018B 3M Environmental Laboratory Page 2 o f 3 Page 176 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Centre Analytical Laboratories, Inc. 3048 Research Drive State Coliege, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B LC /M S P urity Profile: Impurity C4 C5 C6 C7 Total wt./wt. % 1.03 1.56 6.38 1.63 10.60 Note: The C4 and C 6 values were calculated using the C4 and C 6 standard calibration curves, respectively. The C5 value was calculated using the average response factors fro m the C4 and C 6 standard curves. Likew ise, the C7 value was calculated using the average response factors from the C6 and C 8 standard curves. Prepared B y: f/so D a v id S. B e ll Date Sgietftist, C e^tr lytica l Laboratories R eview ed B y : LAK. ft) loh n Flaherty Date Laboratory Manager, Centre A n alytica l Laboratories COA023-018B 3M Environmental Laboratory Page 3 of 3 Page 177 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023-018A 3M Product: PFOS,Lot217 Reference#: SD-018 _______________ Purity: 86.9% Test Name Specifications P u rity 1 Result 86.9% A p pearance Identification NMR M etals (ICP/M S) 1. C alcium 2. M agnesium 3. Sodium 4. Potassium2 5. N ickel 6. Iron 7. M anganese Total % Im purity (NMR) Total % Im purity (L C /M S ) Total % Im purity (G C /M S) Related Compounds POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. C hloride 2. Fluoride 3. Brom ide 4. Nitrate 5. N itrite 6. Phosphate 7. Sulfate4 Organic A cid s3 (IC) 1. T FA 2. PFPA 3. HFBA 4. NFPA E lem ental A nalysis6: 1. C arbon 2. Hydrogen 3. N itrogen 4. Sulfur 5. Fluorine W hite Crystalline Powder C onform s Positive 1. 0.005 w t./w t.% 2. 0.001 wt./wt,% 3. 1.439 wt./wt.% 4. 6.849 wt./wt.% 5. <0.001 w t./w t% 6. 0.005 w t./w t.% 7. <0.001 w t./w t.% 1.93 w t./w t.% 8.41 wt./wt.% None Detected 0.33 wt./wt.% None Detected Not Applicable4 1. <0.015 w t./w t,% 2. 0.59 wt./wt.% 3. <0.040 wt./wt.% 4. <0.009 wt./wt.% 5. <0.006 wt./wt.% 6. <0.007 w t./w t% 7. 8.76 w t./wt.% 1. <0.1 w t/w t.% 2. <0.1 w t./wt.% 3. 0.10 w t/w t.% 4. 0.28 w t/w t.% 1. Theoretical V alue = 1 7 .8 % 1. 12.48 w t/w t.% 2. Theoretical V alue = 0% 2. 0.244 w t/w t.% 3. Theoretical Value = 0% 3. 1.74 w t/w t.% 4. Theoretical Value = 5.95% 4. 8.84 w t/w t.% 5. Theoretical V alue = 60% ' 5. 54.1 w t/w t.% | COAG23-G18A Pase 1 o f3 3M Environmental Laboratory Page 178 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A Date o f Last Analysis: 08/31/00 E xpiration Date: 08/31/01 Storage C onditions: Frozen <-10C Re-assessment Date: 08/31/01 p u r ity = 100% - (sum o f m etal im purities, 1.45% + L C /M S im purities, 8.41% +Inorganic Fluoride, 0 .59% +N M R im purities, 1.93%+organic acid im purities, 0.38% +P O AA , 0 .33% ) T otal im p u rity from a ll tests = 13.09% P urity = 100% - 13.09% = 86.9% 2Potassium is expected in th is salt fo rm and is therefore n o t considered an im p u rity . 3P u rity b y D S C is generally n o t applicable to m aterials o f lo w p u rity . N o endotherm was 1 observed fo r this sample. 4S u lfu r in the sample appears to be converted to S O 4 and hence detected u sin g the inorganic anion m ethod conditions. The anion result agrees w e ll w ith the su lfu r determ ination in the elemental analysis, lending confidence to this interpretation. Based on the results, the S O 4 is not considered an im p urity. 5T F A HFBA NFPA PFPA T rifluoroacetic acid H eptafluorobutyric acid N onofluoropentanoic acid Pentafluoropropanoic acid t h e o r e tic a l value calculations based on the e m p irical fo rm u la , CsF 17S(X K C (M W = 53 8) This w o rk was conducted under E P A G ood Laboratory Practice Standards (40 C F R 160). COA023-018A 3M Environmental Laboratory Page 2 of 3 Page 179 3M Medical Department Study: T-6295.4 Analytical Report: FACT TOX-002 LRN-U2121 Centre Analytical Laboratories, Inc. 3048 Research Drive state College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A LC /M S P urity Profile: Impurity C4 C5 C6 Cl Total wt./wt. % L22~ 1.33 4.72 1.14 8.41 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using die average response factors from the C4 and C6 standard curves. Likew ise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves. Prepared B y: cy//o fcso avid S. B e ll Scientist, Centn R eview ed B y : {J j* , /)} pI/L-, lytic a l Laboratories Date f/a / John Flaherty Date Laboratory Manager, Centre A n alytica l Laboratories COA023-018A 3M Environmental Laboratory Page 3 o f3 Page 180 3M Medical Department Study: T-6295.4 Appendix H: Report Signature Page Analytical Report: FACT TOX-002 LRN-U2121 John L. Butenhoff, Ph.D., Study Director Zt Q f Date J7^< TQk*. Marvin T. Case, D.V.M., Ph.D., Sponsor Representative $ -} fccJr Date Kristen J. Hansen, Ph.D., Principal Analytical Investigator / IH /# / Date William K. Reagen, Ph.D., Laboratory Manager Date 3M Environmental Laboratory Page 181