Document gbzX3mQrp0edBeGZOV6Gg8KoQ

3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 3M Medical Department Study: T-6295.22 Analytical Report: FACTTO X-160 M S E00-1668 Study Title Extended Recovery Study Following a 26-Week Capsule Toxicity Study (FACT-TOX-030) with Perfluorooctane Sulfonate Acid Potassium Salt (PFOS; T-6295.22) in Cynomolgus Monkeys Analytical Laboratory Report Title Determination of the Presence and Concentration of PFOS in Serum and Liver Samples of Cynomolgus Monkeys Data Requirement Not Applicable Author 3M Environmental Laboratory Study Completion Date May 3, 2002 Performing Laboratories Sera and Liver Analyses Sera and Liver Extractions 3M Environmental Laboratory Building 2-3E -09,935 Bush Avenue St. Paul, MN 55106 Pace Analytical Services, Inc.-- Tier2 Facility 1700 Elm Street, Suite 100 Minneapolis, MN 55414 Project Identification 3M Medical Department Study: T-6295.22 Covance In-Life Study: 6329-268 Analytical Report: FACT T O X -160 3M LIMS NO. E00-1668 Total Number of Pages 113 3M Environmental Laboratory 3M Environmental Laboratory Page 1 Page 1 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Analytical Report: FACT TO X-160 LIMS E00-1668 This page has been reserved for specific country requirements. 3M Environmental Laboratory 3M Environmental Laboratory Page 2 Page 2 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 GLP Compliance Statement Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: FACT TOX-160 LIMS E00-1668 Analytical Laboratory Report Title: Determination of the Presence and Concentration of PFOS in Serum and Liver Samples of Cynomolgus Monkeys Study Identification Numbers: T-6295.22, FACT TOX-160, LIMS-E00-1668 This study was conducted in compliance with United States Environmental Protection Agency (EPA) Good Laboratory Practice (GLP) Standards 40 CFR Part 792, 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 four separate studies. The in-life study phase was considered to end at the generation and shipment of specimens. The analytical study phase 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. There were two in-life studies and two analytical studies that utilized the same test system. These studies include in-life studies Covance 6329-223 and Covance 6329-268 and analytical studies FACT-TOX-030 and FACT-TOX-160. The purity and stability of the reference standards are not included in this report, they are not known at this time. Andrew Seacat, Ph.D., Study Director John Butenhoff, Ph.D., Sponsor Representative A CIwdA Lisa Clemen, Principal Analytical Investigator William Reagen, Ph.D., Analytical Laboratory Manager 'W rf&Al 3 -- Date / 3 t 2-00 Z Date OsitbX Date s - '/ / Date UM Environmental Lahnratorv 3M Environmental Laboratory Paae 3 Page 3 3M Medical Department Study: T-6295.22 3M Medicai Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Analytical Report: FACT TOX-160 LIMS E00-1668 GLP Study--Quality Assurance Statement Analytical Laboratory Report Title: Determination of the Presence and Concentration of PFOS in Serum and Liver Samples of Cynomolgus Monkeys Study Identification Numbers: T-6295.22, FACT TOX-160, LIMS-E00-1668 This study has been inspected by the 3M Environmental Laboratory Quality Assurance Unit (QAU) as indicated in the following table. The findings were reported to the study director and laboratory management. Inspection Dates Phase Date Reported to Management Study Director 06/19/01 Protocol 06/19/01 06/19/01 09/05/01 Extraction 09/05/01 09/05/01 09/11/01 02/01/02, 02/06/02-02/08/02, 02/18/02-02/22/02 02/21/02, 02/22/02 Analysis Data Draft report 09/12/01 02/26/02 02/26/02 09/12/01 02/26/02 02/26/02 QAU Representative S '/l/ Z Date 3M Environmental Laboratory 3M Environmental Laboratory Paae 4 Page 4 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Table of Contents Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: FACT TOX-160 LIMS E00-1668 GLP Compliance Statement.................................................................................................. 3 GLP Study - Quality Assurance Statement........................................................................... 4 List of Tables......................................................................................................................... 6 Study Personnel and Contributors........................................................................................ 7 Introduction and Purpose......................................................................................................8 Specimen Receipt and Maintenance.....................................................................................9 Chemical Characterization of the Reference Substance...................................................... 10 Sample Preparation and Analysis......................................................................................... 11 Sera Analyses................................................................................................................... 11 Liver Analyses.................................................................................................................. 11 Method Summaries...................................................................................................... ;...11 Preparatory Methods..................................................................................................11 Analytical Methods.................................................................................................... 12 Analytical Equipment..................................................................................................12 Deviations/Amendments.................................................................................................. 13 Data Quality Objectives and Data Integrity........................................................................... 13 Data Summary, Analyses, and Results................................................................................. 14 Summary of Quality Control Analyses Results................................................................. 14 Statement of Data Q uality................................................................................................ 16 Summary of Sample Results.............................................................................................16 Statistical Methods and Calculations.....................................................................................17 Statement of Conclusion....................................................................................................... 17 References.............................................................................................................................17 Appendix A: Control Matrices.................................................................................................18 Appendix B: Protocol, Amendments, and Deviation(s)......................................................... 19 Appendix C: Extraction and Analytical Methods.................................................................... 40 ETS-8-4.2, "Extraction of Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry," (15 pages)............................................................41 ETS-8-6.0, "Extraction of Potassium Perfluorooctane-sulfonate or other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry," (14 pages)............................................................. 56 ETS-8-5.2, "Analysis of Potassium Perfluorooctancesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry," (11 pages)........................70 ETS-8-7.0, "Analysis of Potassium Perfluorooctane-sulfonate or other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry," (10 pages)........................... 81 Appendix D: Data Summary Tables.......................................................................................91 Appendix E: Data Spreadsheets............................................................................................92 3M Environmental Laboratory 3M Environmental Laboratory Page 5 Page 5 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 3M Medicai Department Study: T-6295.22 Analytical Report: FACT TOX-160 LIMS E00-1668 Appendix F: Example Calculations....................................................................................... 107 Appendix G: Interim Certificate(s) of Analysis......................................................................108 Appendix H: Report Signature Page.....................................................................................113 List of Tables T a b let. Study Timeline........................................................................................................ 8 Table 2. Cynomolgus Monkey Specimen Receipt for Study (#6329-268)........................... 9 Table 3. Characterization of the Analytical Reference Substance in Study FACT-TOX-160.........................................................................................................10 Table 4. Target Ions Monitored in 3M Laboratory Analyses..................................................13 Table 5. Determinations of the LOQ For the Extracted Curve in the Analyses of Serum and Liver Extracts................................................................................................ ....14 Table 6. Liver Matrix Spike Recoveries.................................................................................15 Table 7. Sera Matrix Spike Recoveries................................................................................ 16 Table 8. Characterization of the Control Matrices Used for Serum and Liver Analyses in Study FACT-TOX-160..............................................................................................18 Table 9. Data Summary for PFOS in Serum FACT-TOX-160 - pg/mL................................ 91 Table 10. Data Summary for PFOS in Liver FACT-TOX-160 - pg/g.....................................91 3M Environmental Laboratory 3M En vironmental Laboratory Page 6 Page 6 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 3M Medical Department Study: T-6295.22 Analytical Report: FACT TOX-160 ______________________________________________________________________ LIMS E00-1668 Study Personnel and Contributors Study Director Andrew Seacat, Ph.D. 3M Corporate Toxicology Building 220-2E-02 St. Paul, MN 55144 Analytical Chemistry Laboratories Serum and LiverAnalyses 3M Environmental Laboratory (3M Lab) Lisa Clemen, Principal Analytical Investigator Sponsor 3M Corporate Toxicology 3M Medical Department Building 220-2E-02 St. Paul, MN 55144 John Butenhoff, Ph.D., Sponsor Representative Serum and Liver Extractions Pace Analytical Services, Inc.-- Tier2 Facility 3M Lab Contributing Personnel Rhonda S. Dick* Kelly Dorweiler* Kristen J. Hansen Marlene M. Heying* Harold O. Johnson `Contract lab professional service employees Ognjenka Krupljanin* Kelly J. Kuehlwein* Sally A. Linda* Bob W. Wynne* Location of Archives All original raw data, protocol, and analytical report have been archived at the 3M Environmental Laboratory and will be retained according to 40 CFR Part 792 requirements. The test substance 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 and will be retained according to 40 C FR Part 792 requirements. 3 M Pnvirnnmantal Lahoratorv 3M Environmental Laboratory Paae 7 Page 7 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Introduction and Purpose Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: FACTTOX-160 LIMS E00-1668 The purpose of the study is to determine the presence and concentration of PFOS in cynomolgus monkey sera and liver samples taken from Covance study# 6329-268, "Extended Recovery Study Following a 26-Week Capsule Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T6295.22) in Cynomolgus Monkeys." The animals from study# 6329-268 were previously assigned to a completed in-life Covance study# 6329-223, "26-Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295.7) in Cynomolgus Monkeys" and a completed analytical study# FACT-TOX-030, "Analytical Laboratory Report from the 26-Week Capsule Toxicity Study with Perfluorooctanesulfonic Acid Potassium Salt (T-6295.7) in Cynomolgus Monkeys on the Determination of the Presence and Concentration of Perfluorooctanesulfonate (PFOS) in Liver and Serum Samples". During study# 6329-223, the animals received 0.15 mg/kg/day of PFOS as a single daily capsule dose for at least 26 weeks followed by a 52-week recovery. At the end of the initial recovery the animals were transferred to a follow-up study (Covance 6329-268, T-6295-22) for evaluation of extended recovery. Animals were not treated in the follow-up study. The sera and liver samples for this study are the product of the in-life recovery study completed by Covance Analytical Research Laboratories under study# 6329-268 (T-6295.22). Analyses of sera and liver samples were completed by the 3M Environmental Laboratory under study number FACTTOX-160 (E00-1668), and the results of these analyses are presented in this report. The analytical portion of this study was initiated on 27 August, 2001. Table 1. Study Timeline 26 Weeks 52 Weeks Recovery In-Life Study: Covance 6329-223 Analytical Study: FACT-TOX-030 3M M edical Study: T-6 2 95 .7 52 Weeks Extended Recovery In-Life Study: Covance 6329-268 Analytical Study: FACT-TOX-160 3M M edical Study: T-6295.22 3M Environmental Laboratory 3M Environmental Laboratory Paae 8 Page 8 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: FACT TOX-160 LIMS E00-1668 Specimen Receipt and Maintenance The 3M Environmental Laboratory received cynomolgus monkey specimens collected at the end of the in-life study #6329-268 from 09 May, 2000 to 15 March, 2001. All specimens were received frozen in good condition on dry ice and were immediately transferred to storage at -50C 20C. Specimens that were extracted at Pace Tier2 were shipped frozen on dry ice. Table 2. Cynomolgus Monkey Specimen Receipt for Study (#6329-223) Receipt Date Timepolnt Specimen Number Received 05/09/00 07/06/00 08/30/00 10/24/00 12/21/00 03/13/01 03/15/01 Week 9 W eek 17 Week 25 Week 33 W eek 41 Week 53 Week 53 Serum Serum/Urine/Feces Serum Serum/Urine/Feces Serum Serum/Urine/Feces Liver/Lung Kidney/Spleen Thyroid/Brain Abdominal Fat Hearl/Bile Serum 4 4/4/4 4 4/4/4 4 4/4/4 4/4 4/4 4/4 4 4/4 3 additional vials from sample 105552 Control matrices used in sera and liver analyses performed during FACT-TOX-160 were obtained from commercial sources and are presented in Appendix A. Samples analyzed at the 3M Environmental Laboratory will be stored and maintained at the laboratory according to 40 CFR Part 792 requirements. 3M Environmental Laboratoiv 3M Environmental Laboratory Paae 9 Page 9 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Analytical Report: FACT TOX-160 LIMS EOO-1668 Chemical Characterization of the Reference Substance Potassium perfluorooctanesulfonate (KPFOS) CAS Number: 2795-39-3 Chemical Formula: C8F17S 03'K+ Molecular Weight: 537.9 Chemical characterization Information on the reference substances KPFOS used in this study is presented in tabular form below. Table 3. Characterization of the Analytical Reference Substances in Study FACT-TOX-160 Location 3M Lab Substance PFOS SD-018 THPFOS (Surrogate Standard) TCR-00017-055 Source 3M Expiration Date 08/31/06 Storage Conditions Chemical Lot Number Physical Description Purity Frozen -20C +/-10C 217 White Powder 86.9%* ND-Not determined NA - Not avalable *See Certificate of Analysis from Centre Analytical Laboratories inAppendixG. SynQuest ND Frozen -20C W-10C Q-75-91 White Powder NA 3M Environmental Laboratory 3M Environmental Laboratory Pane 10 Page 10 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: FACT TOX-160 LIMS EOO-1668 Sample Preparation and Analysis Sera Analyses As per the study protocol, all serum samples were analyzed in this phase study. Sera samples were extracted beginning on 31 August, 2001 using an ion pairing reagent and methyl-tert-butyI ether (MtBE). Sample extracts were analyzed using high-performance liquid chromatography-electrospray/tandem mass spectrometry (HPLC-ESMSMS) in the multiple reaction mode versus an extracted rabbit sera curve. PFOS levels were quantitated by external calibration. Liver Analyses As per the study protocol, all liver samples were analyzed in this phase study. Liver samples were extracted beginning on 05 September, 2001 using an ion pairing reagent and methyl-fert-butyl ether (MtBE). Sample extracts were analyzed using high-performance liquid chromatography-electrospray/tandem mass spectrometry (HPLC-ESMSMS) in the multiple reaction mode versus an extracted rabbit liver curve. PFOS levels were quantitated by external calibration. 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. 3M Environmental Laboratory Preparato ry Methods ETS-8-4.2, " Extraction of Fluorochemical Compounds from Serum for Analysis Using HPLC- Electrospray/Mass Spectrometry" Analytical samples were extracted using an ion-pairing extraction procedure: An ion-pairing reagent was added to a laboratory sample and the analyte ion pair was partitioned into methyltert-butyl-ether (MtBE). The MtBE extract was then removed and put into a nitrogen evaporator until dry. Each extracted laboratory sample was reconstituted in 1.0 mL of methanol and passed through a 0.2 pm nylon filter using a 3 mL disposable plastic syringe into glass autovials. 3M Environmental Lahnratmv 3M Environmental Laboratory Pane 11 Page 11 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 3M Medical Department Study: T-6295.22 Analytical Report: FACT TOX-160 LIMS EOO-1668 ETS-8-6.0, "Extraction of Potassium Perfluorooctane-sulfonate or other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry" Liver samples were homogenized in water. An aliquot of each homogenate was spiked with THPFOS and extracted using an ion-pairing extraction procedure. An ion-pairing reagent was added to the sample and the analyte ion pair was partitioned into MtBE. 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 passed through a 0.2 pm nylon filter, using a 3 mL disposable plastic syringe into glass autosampler vials. Analytical Methods ETS-8-5.2, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0, "Analysis of Potassium Perfluorooctane-sulfonate 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 a particular fluorochemical using HPLC/ES/MS/MS. For example, molecular ion 499, selected as the primary ion for PFOS (C8F17$ 0 3-) analysis, was fragmented to produce ion 99 (FS03-). The characteristic ion 99 was monitored for quantitative analysis. Analytical Equipment The following is representative of the settings used during the analytical phase of this study. Liquid Chromatograph: Hewlett-Packard Series 1100 Liquid Chromatograph system Analytical column: Keystone BetasilTM Ci82x50 mm (5 pm) Column temperature: 30C Mobile phase components: Component A: 2mM ammonium acetate Component B: methanol Flow rate: 300 pL/min Injection volume: 10 pL Solvent Gradient: 9.0 minutes Time (minutes) %B 0.0 10% 1.0 10% 5.5 95% 7.5 95% 8.0 10% 3M Environmental Laboratorv 3MEnvironmental Laboratory Paae 12 Page 12 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Analytical Report: FACT TOX-160 LIMS EOO-1668 Mass Spectrom eter: Micromass API/Mass Spectrometer Quattro IITMTriple Quadrupole system Software: Mass LynxTM 3.4 Cone Voltage: 30-70 V Collision Gas Energy: 20-50 eV Mode: Electrospray Negative Source Block Temperature: 150C 10C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (MRM) Table 4. Target Ions Monitored in 3M Laboratory Analyses Target Analyte Primary Ion (a m u ) Product Ion (a m u ) PFOS 499.0 99.0 THPFOS 427.0 80.0 Deviationa/Am endm erits There were one amendment and seven deviations from the original protocol. Amendments and deviations from the original protocol and methods are included in the Appendix B. Data Quality Objectives and Data integrity The following data quality objectives (DQOs) were indicated in the protocol for this study: Linearity: The coefficient of determination (r2) equal to or greater than 0.985 for liver analyses and equal to or greater than 0.990 for sera analyses using 1/x weighting. Lim its o f Q uantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibration curve, defined as the lowest standard that Is both 2 tim es the m atrix blank and is calculated within 30% of the expected concentration. Acceptable Precision: Quality control samples are required to meet 25% precision for analyses, provided they are quantitated within the selected calibration range. Acceptable Spike Recoveries: Matrix spikes and matrix spike duplicates required for analysis of liver and sera samples should show spike recoveries within 70-130%. C onfirm atory M ethods: If a confirmatory method is used, an amendment to this protocol will be written. Dem onstration o f S pecificity: PFOS identification will be substantiated by chromatographic retention time (approximately 8.00 minutes), by the characteristic primary ion (499) and characteristic product ion (99). 3M Environmental Laboratory 3M Environmental Laboratory Paae 13 Page 13 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 3M Medical Department Study: T-6295.22 Analytical Report: FACTTOX-160 LIMS E00-1668 Data Summary, Analyses, and Results Data quality objectives for the analytical phase of this study outlined in the 3M Environmental Laboratory protocol for FACT-TOX-160 (see Appendix B) were met with the exceptions noted in this report. Summary of Quality Control Analyses Results Linearity: The coefficient of determination (r2) of the standard curve was >0.985 for liver analyses and >0.990 for sera analyses. Calibration Standards: Quantitation of the target analytes was based on linear regression analysis, 1/x weighted of one or two extracted matrix curves bracketing each group of samples, except as noted in the deviation summary. High and/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 each analyte was based on the response of one specific product ion(s) 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 that is within 30% of the theoretical value, and is at least two times the analyte peak area detected in the extraction blanks. Table 5. Determinations of the LOQ For the Extracted Curve in the Analyses of Serum and Liver Extracts Analyte Method LOQ PFOS--Serum PFOS-- Uver 4.92 ng/mL 12.6 ng/g Blanks: All blanks were below the lower limit of quantitation for the compounds of interest. To simplify analyses that were complicated by endogenous levels of fluorochemicals in unexposed monkey sera which were above the lower limit of quantitation, rabbit sera was selected as a suitable surrogate matrix and all rabbit sera blanks were within criteria. Precision: precision was determined by analysis of CCVs in sera and was reproducible to within 25%, precision was determined by analysis of CCVs in liver and was reproducible to within 30%. Matrix Spikes: Matrix spikes and matrix spike duplicates were extracted with each set of liver and sera samples and analyzed at the 3M Environmental Laboratory (see tables 5 and 6). Two rabbit liver matrix spikes, extracted 09/05/01, were within criteria and two monkey liver matrix spikes were not within 30% of the theoretical concentration when analyzed 09/17/01. The monkey liver matrix spikes were not prepared at the appropriate concentration based on endogenous levels present in the samples. Additional monkey liver matrix spikes and one monkey liver sample were re-extracted on 10/02/01 at levels appropriate to the endogenous 3M Environmental Laboratory 3M Environmental Laboratory Paae 14 Page 14 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 3M Medical Department Study: T-6295.22 Analytical Report: FACT TOX-160 LIMS EOO-1668 levels. The re-extracted spikes did not meet criteria and the extraction was suspect due to an inconsistent sample concentration, inconsistent matrix spike recoveries, and high surrogate values - refer to deviations attached to this report for more information. Additional monkey liver matrix spikes and one monkey liver sample were re-extracted on 11/07/01. The extracted spikes and sample were in agreement with the initial extraction on 09/05/01. The rabbit liver matrix spike averages extracted with the samples on 09/05/01 and the monkey liver matrix spike averages extracted on 11/07/01 were within 35% of the theoretical concentration. All sera matrix spike recoveries, evaluated versus extracted and unextracted curves, were within 25% of the theoretical concentration. Extraction efficiency and absolute recovery were >100% in the sera matrix, based on average recoveries of 101%, 111%, 111 %, and 116% versus an unextracted (solvent) curve. Table 6. Liver Matrix Spike Recoveries Matrix Extraction Date Analysis Date Liver 09/05/01 09/10/01 Type Rabbit 09/17/01 Monkey 10/02/01 Suspect Extraction inconsistent sample concentration and high . surrogate deviation for 10/24/01 analysis 10/04/01 Initial Analysis 10/16/01 Reanalysis 10/24/01 Rediiution from original sample 11/07/01 11/15/01 _______1 Monkey 2 ug/g Monkey 10 ug/g Monkey 2 ug/g Monkey 10 ug/g Monkey 2 ug/g Monkey 10 ug/g Monkey 2 ug/g Monkey 10 ug/g % Recovery Average 102% 102% 298% 290% 55% 15% 60% Too Dilute 30% -11% 65% 43% 178% 61% 268% 216% 100% 90% 73% 61% 102% 294% 35% NA 9% 54% 119% 242% 95% 67% 3M Environmental Laboratorv 3M Environmental Laboratory Pane 15 Page 15 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: FACT TOX-160 LIMS E00-1668 Table 7. Sera Matrix Spike Recoveries Matrix Extraction Analysis Date Date Type % Average Recovery Sera 08/31/01 10/02/01 09/06/01 Evaluated Versus an Unextracted Curve = Absolute Recovery 10/04/01 Evaluated Versus an Extracted Curve Monkey 25 ng/mL Monkey 500 ng/mL Monkey 2.0 ug/mL Monkey 10 ug/mL 10/04/01 Evaluated Versus an Unextracted Curve = Absolute Recovery Monkey 2.0 ug/mL Monkey 10 ug/mL 118% 84% 111% 112% 104% 99% 119% 121% 113% 109% 115% 117% 101% 111% 102% 120% 111% 116% Surrogates: The surrogate (THPFOS) was added to all samples and standards. THPFOS was not used for quantitation, but was used to monitor for gross instrument failure. The surrogate response of each analytical run utilizing extracted matrix calibration curves was verified to determine that it did not vary more than 50% from the mean within each analytical run. All responses were within 50% except for analysis of liver dilutions on 10/24/01. These samples were >50% . These samples were considered suspect and re-extracted. Analysis of these re extracts met surrogate criteria requirements. S tatem 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, indicates that these data are quantitative to 25% or greater in sera and 35% or greater in liver. Summary of Sample Results PFOS results (those obtained using lot # 217) have been corrected for purity of the analytical reference material. Samples from Control Animals: No control animals were included in this study. Samples from Dosed Animals: In general, PFOS levels found in sera of the test animals decreased over time. PFOS levels in male livers were approximately half that determined in female livers. Detailed sample data tables are presented in Appendices D and E. 3M Environmental Laboratory 3M Environmental Laboratory Page 16 Page 16 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 3M Medical Department Study: T-6295.22 Analytical Report: FACT TOX-160 LIMS E00-1668 Statistical Methods and Calculations Statistical methods were limited to the calculation of means and standard deviations. See Appendix F for example calculations used to generate the liver and serum sample data in FACT-TOX-160. Statement of Conclusion Under the conditions of the present studies, the fluorochemical PFOS was observed in the serum and liver of all recovery study cynomolgus monkeys originally dosed with the test substance during the in-life phase of the study #6329-223 (TOX-030). References None 3M Environmental Laboratory 3M Environmental Laboratory Paae 17 Page 17 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: FACT TOX-160 LIMS E00-1668 Appendix A: Control Matrices Table 8. Characterization of the Control Matrices Used for Serum and Liver Analyses in Study FACT-TOX-160 Control Matrix Monkey Serum TCR-99131-022 Rabbit Serum TN-A-4511 Rabbit Liver TCR-99131-046 Source Expiration Date Storage Conditions Chemical Lot # Physical Description Lampire Biological 01/01/2010 Frozen-20C +/- 10C 111022515 Monkey Serum Sigma-Aldrich 09/26/05 Frozen -20C +/- 10C 99H8400 Rabbit Serum Covance Laboratory NA Frozen -20C +/- 10C F04053 Rabbit Liver Fnvirnnmontal Lahnratnrv 3M Environmental Laboratory Pane* 1ft Page 18 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: FACT TOX-160 LIMS E00-1668 Appendix B: Protocol, Amendments, and Deviations 3M Environmental Laboratory 3M Environmental Laboratory Paae 19 Page 19 3M Medical Department Study: T-6295.22 3M Environmental Technology and Services PO Box 33331 St. Paul, MN 55133-3331 6I2 778 6442 Analytical Report: FACT-TOX-160 L IM S E 00-1668 Protocol UFACT-TOX-160 rr,TM , io a Study Title Extended R ecovery Study Follow ing a 26-W eek Capsule T oxicity Study (FAC T-TO X-030) w ith Perfluorooctane Sulfonic Acid Potassium S alt (PFOS; T-6295) in Cynom olgus Monkeys ANALYTICAL PHASE PROTOCOL Author Lisa Clem en Date: A ugust 27, 2001 Performing Laboratories Sera and Liver Analyses 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MM 55106 Sera and Liver Extractions Pace Tier2 Facility 1700 Elm street, Suite 200 Minneapolis, MN 55414 Laboratory Project Identification FACTTO X-160 Covance In-life Study Number: 6329-268 3M M edical D epartm ent Study: T-6295.22 ET&SS LIMS: E00-1668 3M Environmental Laboratory 3M Environmental Laboratory Page 1 o f 9 Page 20 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Protocol UFACT-TOX-160 Study Identification Extended Recovery Study Following a 26-Week Capsule Toxicity Study (FACT-TOX-030) with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys Sponsor 3M Corporate Toxicology 3M Medical Department Building 220-2E-02 St. Paul, MN 55144 Sponsor Representative John Butenhoff, Ph.D. 3M Corporate Toxicology 3M Medical Department Building 220-2E-02 St. Paul, MN 55144 Telephone: 651-733-1962 Study Director Andrew Seacat, Ph.D. 3M Corporate Toxicology 3M Medical Department Building 220-2E-02 St. Paul, MN 55144 Telephone: 651-575-3161 Principal Analytical Investigator (PAI) Lisa Clemen Phase Locations In vivo Testing Facility Analytical Testing Laboratories (sera and liver analyses) Covance Laboratories, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 (sera and liver extractions) Pace Tier2 Facility 1700 Elm Street, Suite 200 Minneapolis, MN 55414 Proposed Study Timetable Experimental Start Date Experimental Completion Date 27 August 2001 29 April 2002 3M Environmental Laboratory 3M Environmental Laboratory Page 2 o f 9 Page 21 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Protocol UFACT-TOX-160 1. Study Extended Recovery Study Following a 26-Week Capsule Toxicity Study (FACT-TOX-030) with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys. 2. P urpose This study is designed to continue the assessment ofperfluorooctanesulfonate (PFOS) levels in sera and liver during an extended recovery time of approximately one year following the daily administration o f the test material by capsule to cynomolgus monkeys for at least 26 weeks and at least 52 weeks o f recovery. The animals used in this study were previously assigned to a recovery phase o f a completed study for at least 52 weeks. At the end o f this initial recovery phase study Covance #6329223, a partial hepatectomy was conducted on the animals, they were allowed to recover from the surgical procedure, then transferred to this follow-up study for evaluation o f recovery for an additional 52 weeks. The serum and liver o f cynomolgus monkeys will be analyzed for PFOS. Additional tissues or fluids may be analyzed at the discretion o f the PAI or study director. The in-life portion o f this extended recovery study was conducted at Covance Laboratories, study #6329-268. 3. Regulatory Compliance This study will be conducted in accordance with the United States Environmental Protection Agency Good Laboratory Practice Standards, 40 CFR 792. 4. Quality A ssurance The 3M Environmental Laboratory Quality Assurance Unit will review the protocol and audit study conduct, data, and the final report to determine compliance with Good Laboratory Practice Standards and with 3M Environmental Laboratory Standard Operating Procedures. 5. Test Material Refer to Covance Laboratory protocol for study #6329-268. Animals were not treated during this study. The FACT TOX-160 study is an extended recovery study o f cynomolgus monkeys from Covance study 6329-223 (analytical work performed under 3M Environmental Study # FACT-TOX-030), where animals received 0.15 mg/kg/day o f PFOS lot # 217 in a daily single capsule for at least 26 weeks, followed by a 52-week recovery period. 6. Control Matrices Types o f control matrices and their source, physical description, storage requirements, and traceability numbers will be recorded in the raw data and included in the final report. 7. Reference Material for FACT TOX-160 Potassium perfluorooctanesulfonate (KPFOS), C8F |7S 0 3`K+, CAS 2795-39-3 3M Environmental Laboratory 3M Environmental Laboratory Page 3 of 9 Page 22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Protocol UFACT-TOX-160 The target analyte is perfluorooctanesulfonate (PFOS), C8F17S 0 3'. 8. Test System Cynomolgus monkeys were used as the test system, and were maintained and dosed as described in the original Covance protocol #6329-223 (TOX-030). Two male and two female monkeys which were dosed during study #6329-223 (TOX-030) were included in this extended recovery study and all were allocated to Group 1. These animals received no further treatment during the recovery study. 9. Specimen and Sample Receipt The 3M Environmental Laboratory received specimens o f the following body tissues and fluids from the indicated points in the study from Covance Laboratories at the end o f the in life phase o f the study. All specimens were frozen and packed on dry ice for shipping. Body tissue/fluid Serum - all animals Liver - all animals Collected 2 ,4 , 6, 8,10, and 12 months following initiation o f study At scheduled sacrifices At scheduled sacrifices Expected # of specimens -24-28 serum samples 4 Total number o f test animals: 4 Modifications to the number o f samples analyzed may be implemented at the discretion o f the PAI and the study director. Specimens sent to 3M Environmental Laboratories were received and tracked according to the Sample Tracking System Standard Operating Procedure. Details o f specimen inspection for damage, receipt, storage, identification, chain o f custody, protocols and data will be presented in the phase report. 10. Preparatory Methods 10.1 ETS-8-6, Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 10.2 ETS-8-4, Extraction o f Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 10.3 If preparatory methods other than those listed above are used, an amendment to this protocol will be written. Any deviations from these methods will be documented and included with the study data. 3M Environmental Laboratory 3M Environmental Laboratory Page 4 o f 9 Page 23 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Protocol UFACT-TOX-160 11. Analytical Methods 11.1 ETS-8-7, Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry 11.2 ETS-8-5, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry 11.3 I f analytical methods other than those listed above are used, an amendment to this protocol will be written. Any deviations from these methods will be documented and included with the study data. 12. Data Quality Objectives The number o f spikes/duplicates, use o f surrogates, and information on other data quality indicators are included in the analytical methods. In addition, the following criteria will be met: 12.1 Linearity The coefficient o f determination (r2) o f the extracted liver standard curve must be equal to or greater that 0.985 using linear regression or quadratic fit. The coefficient o f determination (r2) o f the extracted serum standard curve must be equal to or greater that 0.990 using linear regression or quadratic fit. 12.2 Limits o f Quantitation (LOQ) The LOQ will be equal to the lowest acceptable standard in the calibration curve, defined as the lowest standard that is both 2 times the matrix blank and is calculated within 30% o f the expected concentration. 12.3 Acceptable Precision Quality control samples are required to meet 25% precision, provided they are quantitated within the selected calibration range. 12.4 Spike Acceptable Recoveries Matrix spikes and matrix spike duplicates required for analysis o f liver samples should show spike recoveries within 70%-130%. After liver samples have been analyzed, sample concentrations will be evaluated. If a measured sample (s) concentration exceeded the calibration range o f the method and was diluted with methanol into the validated calibration range, then additional matrix spikes will be prepared at the approximate measured concentrations o f the samples then subsequently diluted with the same volume o f methanol as the sample into the validated range o f the calibration curve. These liver matrix spikes will be evaluated versus an extracted matrix calibration curve and an unextracted calibration curve. If the diluted liver matrix spikes do not show recoveries within 70-130% versus the extracted matrix calibration curve, then the 3M Environmental Laboratory 3MEnvironmental Laboratory Page 5 of 9 Page 24 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Protocol UFACT-TOX-160 sample concentrations, at the same dilution factor as the matrix spikes, will be corrected for the spike recovery. For serum matrix spikes, a comparison of extracted (matrix) versus unextracted (solvent) calibration standards will be performed at two concentrations spanning the validated calibration range o f the method. This comparison will demonstrate the absolute recovery (recovery + serum matrix affect) o f analyte from the matrix for determining the extraction efficiency o f serum versus an unextracted calibration curve. After serum samples have been analyzed, sample concentrations will be evaluated. I f a measured sample (s) concentration exceeded the calibration range o f the method and was diluted with methanol into the validated calibration range, then additional matrix spikes will be prepared at the approximate measured concentrations of the samples then subsequently diluted with the same volume of methanol as sample into the validated range o f the calibration curve. These serum matrix spikes will be evaluated versus an extracted matrix calibration curve and an unextracted calibration curve. If the diluted serum matrix spikes do not show recoveries within 70-130% versus the extracted matrix calibration curve, then the sample concentrations, at the same dilution factor as the matrix spikes, will be corrected for the spike recovery. 12.5 Use o f Confirmatory Methods If a confirmatory method is used, an amendment to this protocol will be written. 12.6 Demonstration o f Specificity PFOS identification will be substantiated by chromatographic retention time (approximately 8 minutes), by the characteristic primary ion (499) and the characteristic product ion (99). Minor modifications to the Data Quality Objectives may be implemented at the discretion o f the PAL These will be documented in the raw data and the analytical re p o rt. 13. Sub-Contracted Analysis 13.1 All sera and liver extractions as detailed in this protocol will be performed at Pace T ier2 ,1700 Elm Street, Suite 200, Minneapolis, MN 55414. 13.2 All sera and liver analyses as detailed in this protocol will be performed at 3M Environmental Laboratory, Building 2-3E-09, 935 Bush Avenue, St. Paul, MN 55106. 13.3 An amendment to this protocol will be written if extractions and analyses are performed at laboratories other than the 3M Environmental Laboratory or Pace Tier2. 14. Statistical A nalysis Statistical methods will be limited to the calculation of means and standard deviations. Examples o f the calculations used in the analyses will be included in the analytical phase report. 3M Environmental Laboratory 3M Environmental Laboratory Page 6 o f 9 Page 25 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Protocot ttFACT-TOX-160 15. Report A report o f the results o f the study will be prepared by 3M Environmental Laboratory. The report will include, but not be limited to, the following, when applicable: 15.1 Name and address o f the facility performing the phase 15.2 Dates upon which the phase was initiated and completed 15.3 A statement o f compliance by the PAI addressing any exceptions to Good Laboratory Practice Standards 15.4 Objectives and procedures as stated in the approved phase protocol, including any amendments to the original phase protocol 15.5 Identity, purity, stability and the solubility o f the reference standard under conditions o f use 15.6 A description o f the methods used to conduct the test(s) 15.7 A description o f the specimens 15.8 A description o f any circumstances that may have affected the quality or the integrity o f the data 15.9 The name o f the PAI and the names o f other scientists, professionals, and supervisory personnel involved in the phase 15.10A description o f the transformations, calculations, or operations performed on the data, a summary and analysis o f the analytical chemistry data, and a statement o f the conclusions drawn from the analyses 15. `/'/Statistical methods used to evaluate the data, if applicable 15.12 The signed and dated reports o f each o f the individual scientists or other professionals involved in the phase, if applicable 15.13 The location where raw data and the final report are to be stored 15.14 A statement prepared by the Quality Assurance Unit listing the dates that study inspections and audits were made, and the dates o f any findings reported to the Study Director and Management I f it is necessary to make corrections or additions to a final report after it has been accepted, the changes will be made in the form of an amendment issued by the Study Director. The amendment will clearly identify the part o f the final report that is being amended, the reasons for the amendment, and will be signed by the Study Director. 16. Location of Ra w Data, Records, and Final Report Original data, or copies thereof, will be available at 3M Environmental Laboratory to facilitate audits o f the study during its progress and before acceptance of the phase report. When the phase report is completed, all original paper data, including those items listed below, will be retained in the archives o f 3M Environmental Laboratory. All corresponding 3M Environmental Laboratory 3M Environmental Laboratory Page 7 o f 9 Page 26 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Protocol UFACT-TOX-160 training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained at the 3M Environmental Laboratory. 16.1 The following raw data and records will be retained in the study folder in the study/project archives according to 3M Environmental Laboratory Standard Operating Procedures: 16.1.1 Approved protocol and amendments 16.1.2 Study correspondence 16.1.3 Shipping records 16. 1.4 Raw data 16.1.5 Approved final report (original signed copy) 16.1.6 Electronic copies o f data 16.2 The following supporting records will be retained separately from the study folder according to 3M Environmental Laboratory Standard Operating Procedures: 16.2.1 Training records 16.2.2 Calibration records 16.2.3 Instrument maintenance logs 16.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 17. Specimen Retention Specimens remaining after the analytical phase is completed will be sent to and maintained by: Lisa Clemen 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Telephone: (651) 778-6176 18. Protocol A mendments and deviations Planned changes to the protocol will be in the form o f written amendments signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part o f the protocol and will be attached to the final protocol. All changes to the protocol will be indicated in the final report. Any other changes will be in the form o f written deviations, signed by the Study Director and Sponsor Representative and filed with the raw data. 3M Environmental Laboratory' 3M Environmental Laboratory Page 8 o f 9 Page 27 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Protocol #FAGT-TOX-160 19. A ttachments 19.1 Attachment A: Material Safety Data Sheets (MSDS) for Reference Standard 20. Signatures A n d re 1 , Ph.D., S tudy D irector John Butenhoff, Ph.D., S ponsor R epresentative A___ . ------------------------------------------------------- Lisa Clem en, P rincipal A nalytical Investigator I Date 2 7 2 *2 / D a te 08/33lo , D a te 3M Environmental Laboratory 3M Environmental Laboratory Page 9 of 9 Page 28 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Study Title Extended Recovery Study Following a 26-Week Capsule Toxicity Study (FACT-TOX-030) with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 1 Amendment Date: April 4,2002 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project identification FACT TOX-160 Covance In-life Study Number: 6329-268 3M Medical Department Study: T-6295.22 ET&SS LIMS: E00-1668 Environmental Laboratorv 3M Environmental Laboratory Page 29 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Protocol FACT-TOX-160 Amendment No. 1 This am endm ent m odifies the follow ing portion(s) of th e protocol: 1. Protocol reads: Section 10 states that method ETS-8-6.0 will be used for extracting liver samples and section 12 of method ETS-8-6.0 references the use of equipment procedure AMDT-EP-22 for cleaning the tissue grinder. A mend to read: Section 12 of method ETS-8-6.0 changed to reference equipment procedure ETS-9-52.0 as of 11/07/01. Reason: Equipment procedure AMDT-EP-22 was replaced by equipment procedure ETS-9-52.0 on 11/07/01. Both cover the tissue grinder's cleaning procedure. 2. Protocol reads: Section 11 states that method ETS-8-7.0 will be used for analyzing liver extracts using HPLCElectrospray/Mass Spectrometry A mend to read: Method Modification Method: ETS-8-7.0 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" Section modified: 10.3.2,14.5.1, add sections 14.3.2-14.3.6 Effective date of modifications: July 22,1999 Section 10.3.2 Method reads: Analyze a mid-range calibration standard after every tenth sample, with a minimum o f one per batch. Modify method to read: Analyze a mid-range calibration standard at least after every ten samples, with a minimum of one per batch. Section 14.5.1 Method reads: Continuing calibration verification percent recoveries must be within 30% of the spiked concentration. Modify method to read: One continuing calibration verification per ten samples must show a percent recovery within +/-30% of the spiked concentration. 3M Environmental Laboratory 3M Environmental Laboratory Page 30 3M Medical Department Study: T-6295.22 Section 14.3.2 Method reads: NA Analytical Report: FACT-TOX-160 LEMS EOO-1668 Protocol FACT-TOX-160 Amendment No. 1 Modify method to read: 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. Section 14.3.3 Method reads: NA Modify method to read: 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 of the analyte. Section 14.3.4 Method reads: NA Modify method to read: 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: 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: A valid calibration curve must contain at least 5 active points. Reason: Method modifications are improvements/clarifications to the current method. 3M Environmental Laboratory 3M Environmental Laboratory Page 31 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Protocol FACT-TOX-160 Amendment No. 1 Amendment Approval Andrew Secat, Ph.D., Sponsor Representative/Study Director - C?`Vfc ClmjA_______________________________ c>H9l2. Lisa A. Clemen., Principal Analytical investigator Date O /c /o j- William K. Reagen, Ph.D., Performing Laboratory Management Date 3M Environmental Laboratory 3M Environmental Laboratory Page 32 3M Medical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LIMS E00-1668 Record of Deviation Study / Project No. FA C T -T O X -160, EOO-1668 D eviation type (Check one) . SOP X Protocol Document number FA C T -T O X -160, EOO-1668 /. Identification Method Equipment Procedure Other: Date(s) o f occurrence 09/05/01 II. Description Required procedure/process: Protocol FA C T-TO X -160, EOO-1668 states: M atrix spikes and m atrix spike duplicates required for analysis o f | liver sam ples should show spike recoveries w ithin 70% -130% . A fter liver sam ples have been analyzed, sam ple concentrations w ill be evaluated. I f a m easured sam ple (s) concentration exceeded die calibration range o f the m ethod and was diluted with m ethanol into the validated calibration range, then additional m atrix spikes w ill be prepared at the approxim ate m easured concentrations o f the sam ples then subsequently diluted w ith the sam e volum e o f m ethanol as the sample into the validated range o f the calibration curve. These liver m atrix spikes w ill lie evaluated versus an extracted m atrix calibration curve and an unextracted calibration curve. If thed ilu ted liver m atrix spikes do not show recoveries w ithin 70-130% versus the extracted m atrix calibration curve, then the sam ple concentrations, at the sam e dilution factor as the m atrix spikes, will be corrected for the spike recovery. Actual procedure/process: The M K L 09040i - 250 ppb - I05505M m atrix spike/m atrix spike duplicate (M S/M SD) average recovery was 294% . These sam ples w ere not spiked at the appropriate level in relation to endogenous levels present in the sam ple. III. Actions Taken j_________________________________ (such as amendment issued, SOP revision, etc.)____________________________ N ew m atrix spikes w ere prepared at 2 ug/g & 10 ug/g then extracted on 10/02/01. Recorded by Authorized by ... Sponsor fttpreaervU-ta/- 7o\m BuWtaXP Orte.W . Atstkvt-io StC-cA Date 13-1 io lo i Date 'z /r s fo / D eviation No. (assigned by Study Director or Project Lead at the end of study or project) A ttachm ent A 3M Environmental Laboratory ETS-4-8.2 Documentation of Deviations Page 1 of 1 Page 33 3M Medicai Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LIMS EOO-1668 Record of Deviation ___________________________ /. Identification___________________________ Study / Project No. FACT-TOX-160, E00-1668 D eviation type SOP Method Equipment Procedure (Check one) X Protocol Other: Document number Date(s) o f occurrence F A C T -T O X -1 6 0 , E 0 0 -1 6 6 8 ___________________________ 10/04/01______________________________________________ ____________________ ;_______ II. Description___________________________ Required procedure/process: Protocol FA C T-TO X -160, E0-1668 states: M atrix spikes and m atrix spike duplicates required for analysis o f liver sam ples should show spike recoveries w ithin 70% -130% . A fter liver sam ples have been analyzed, sam ple concentrations will be evaluated. If a m easured sam ple (s) concentration exceeded the calibration range o f the m ethod and was diluted w ith m ethanol into the validated calibration range, then additional m atrix spikes w ill be prepared at the approxim ate m easured concentrations o f the sam ples then subsequently diluted w ith the sam e volum e o f m ethanol as the sam ple into the validated range o f the calibration curve. These liver m atrix spikes w ill be evaluated versus an extracted m atrix calibration curve and an unextracted calibration curve. I f the diluted liver m atrix spikes do not show recoveries w ithin 70-130% versus the extracted m atrix calibration curve, then the sam ple concentrations, at the same dilution factor as the m atrix spikes, w ill be corrected for the spike recovery. Actual procedure/process: _ T he 2 ug/g m atrix spike/m atrix spike duplicate (M S/M SD ) average recovery was 35% and the 10 ug/g M S/M SD w as to o dilu te u sin g a 1:500 d ilu tio n facto r.*I2 III. Actions Taken _________________________________ (such as amendment issued, SOP revision, etc.)____________________________ I T he 10 ug/g M S/M SD w as re-diluted using a 1:50 dilution and analyzed on 10/16/01. The 1:50 dilution o f the 2 ug/g MS/M SDs was also reanalyzed on 10/16/01 to confirm the original low recovery. Recorded by Authorized by ^ ^ Spotu&r To\v\ bdkrAroYT rtcW- Atv^rtvo Statu Date l - i / i o l 01 , Date Deviation No. (assigned by Study Director or Project Lead at the end o f study or project) Attachm ent A 3M Environmental Laboratory ETS-4-8.2 Documentation of Deviations Page 1 of 1 Page 34 3M Medical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LIMS EOO-1668 Record of Deviation /. Identification Study / Project No. FACT-TOX-160, E00-1668 D eviation type SOP . Method Equipment Procedure (Check one) X Protocol Other: Document number Date(s) o f occurrence FA C T-TO X -160, EOO-1668 10/16/01 II. Description Required procedure/process: Protocol FACT-TO X -160, E00-1668 states: M atrix spikes and m atrix spike duplicates required for analysis o f liver sam ples should show spike recoveries w ithin 70% -130% . I A fter liver samples have been analyzed, sam ple concentrations will be evaluated. If a m easured sam ple (s) concentration exceeded the calibration range o f the m ethod and was diluted w ith m ethanol into the validated 1 calibration range, then additional m atrix spikes w ill be prepared at the approxim ate m easured concentrations o f the I sam ples then subsequently diluted w ith the sam e volum e o f m ethanol as the sam ple into the validated range o f the I calibration curve. These liver m atrix spikes w ill be evaluated versus an extracted m atrix calibration curve and an | unextracted calibration curve. If the diluted liver m atrix spikes do not show recoveries within 70-130% versus the extracted m atrix calibration curve, then the sam ple concentrations, at the sam e dilution factor as the m atrix spikes, w ill be corrected for the spike recovery. Actual procedure/process: T he 2 ug/g and 10 ug/g m atrix spike/m atrix spike duplicate (M S/M SD ) average recoveries w ere 9% and 52% . III. Actions Taken (such as amendment issued, SOP revision, etc.) T he M S/M SD and I05505M sam ple w ere all re-diluted from the original extracts at 1:50 then analyzed on 1 0 / 2 4 / 0 1. Recorded by Authorized by . / k u d u * * '/M . Sporuor ftc.prc,j{.r,\ciWc. -Aokrt tjuW'JnoV?' \ 1v _. s tw lj t W . W AnAreui W o t Date 13-j lo !oi /Z /tf/e l Deviation No. (assigned by Study Director or Project Lead at the end of study or project) Attachm ent A 3M Environmental Laboratory ETS-4-8.2 Documentation of Deviations Page 1 of 1 Page 35 3M Medical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LIMS EOO-1668 Record of Deviation Study / Project No. FA C T-TO X -160, EOO-1668 D eviation type SOP (Check one) X Protocol Document number FA C T-TO X -160, EOO-1668 1. Identification Method Other: Equipment Procedure Date(s) o f occurrence 10/24/01 II. Description Required procedure/process: Protocol FA C T -T O X -160, EOO-1668 states: M atrix spikes and m atrix spike duplicates required for analysis o f liver samples should show spike recoveries w ithin 70% -130% . A fter liver sam ples have been analyzed, sam ple concentrations will be evaluated. If a m easured sam ple (s) concentration exceeded the calibration range o f the m ethod and was diluted w ith m ethanol into the validated calibration range, then additional m atrix spikes will be prepared at the approxim ate m easured concentrations o f the sam ples then subsequently diluted w ith the sam e volum e o f m ethanol as the sam ple into the validated range o f the calibration curve. These liver m atrix spikes will be evaluated versus an extracted m atrix calibration curve and an unextracted calibration curve. I f the diluted liver m atrix spikes do not show recoveries within 70-130% versus the extracted m atrix calibration curve, then the sam ple concentrations, at the sam e dilution factor as the m atrix spikes, will be corrected for the spike recovery. Actual procedure/process: The 2 ug/g and 10 ug/g m atrix spike/m atrix spike duplicate (M S/M SD) average recoveries w ere 19% and 242% . The sam ple recovery w as n ot consistent w ith the previous analyses. III. Actions Taken (such as amendment issued, SOP revision, etc.) The sam ple set w as re-extracted on 11/07/01, then diluted 1:50 and analyzed. Recorded by Authorized Sponsor 'John . *. . U irtcW '. /Wlrtod ^>Cqt Date D a,e < * / * / '< f1 Deviation No. 1 . (assigned by Study Director or Project Lead at the end of study or project) A ttachm ent A 3M Environmental Laboratory ETS-4-8.2 D ocum entation o f D eviations Page 1 of 1 Page 36 3M Medical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LIMS EOO-1668 Record of Deviation Study / Project No. FA C T-TO X -160, EOO-1668 Deviation type (Check one) SOP X Protocol Document number F A C T -T O X -160, E00-1668 i. Identification Method Equipment Procedure Other: Date(s) o f occurrence 11/16/01 II. Description Required procedure/process: Protocol FA C T -T O X -160, EOO-1668 states: M atrix spikes and m atrix spike duplicates required for analysis o f liver sam ples should show spike recoveries w ithin 7Q%~130%. A fter liver samples have been analyzed, sam ple concentrations w ill be evaluated. If a m easured sam ple (s) concentration exceeded die calibration range o f die m ethod and was diluted w ith m ethanol into the validated calibration range, then additional m atrix spikes w ill be prepared at the approxim ate m easured concentrations o f the sam ples then subsequently diluted w ith the sam e volum e o f m ethanol as the sample into the validated range o f the calibration curve. These liver m atrix spikes will be evaluated versus an extracted m atrix calibration curve and an unextracted calibration curve. If die diluted liver m atrix spikes do not show recoveries w ithin 70-130% versus the | extracted m atrix calibration curve, then the sam ple concentrations, at the sam e dilution factor as the m atrix spikes, will be corrected for the spike recovery. Actual procedure/process: The 10 ug/g m atrix spike/m atrix spike duplicate (M S/M SD) average recovery was 67%. III. Actions Taken (such as amendment issued, SOP revision, etc.) The 10 ug/g M S/M SD was n ot reanalyzed to confirm recovery since the 2 ug/g M S/M SD average recovery, analyzed during the same run, was 95% and within the stated criteria. Also, all CCVs and other data quality, objectives w ere w ithin criteria for this analytical run. The accuracy for the liver data in this study will be changed from +/- 30% to +/- 35% in the final report. Recorded by Date Ct Authorized b y ^ ^ , ^ > )3-J loy Date Sponsor R.pri,ierAokvt/. lo h n CbrtcW. A<\(Wia) .SwxcolV '/ f t / * ) Deviation No. (assigned by Study Director or Project Lead at the end of study or project) A ttachm ent A 3M Environmental Laboratory ETS-4-8.2 Documentation of Deviations Page 1 of 1 Page 37 3M Medical Department Study: T-6295.22 3M Confidential Record of Deviation Analytical Report: FACT-TOX-160 LIMS EOO-1668 Study / Project No. Tox 160 D eviation type (Check one) Document number Ets 8-7.0 1. Identification SOP Protocol X Method Equipment Procedure Other: Date(s) o f occurrence 11-16-01 II. Description Required procedure/process: A ccording to section 11.1 T h e average o f tw o standard curves w ill be plotted by linear regression. I Actual procedure/process: F o r th e data set mO 11116a, the unextracted curve w as plo tted using a quadratic fit. A linear curve that m et th e r*2 criteria and extended to 250 ppb (the level o f the CCVs) could not be generated. This deviation was written. III. Actions Taken (such as amendment issued, SOP revision, etc.) Recorded b> Date fClm ppctjjn Study / Project ______________________________ fcomfletpciby s/idyDirector or Project Lead)________ ._____________________ tp..Av'tl r ! VcAuctlt, versus.. E*Wc.\eA- Cun*j. -Sititi^ fQrnpUs UHr lAt ^wjis/oi Authorized by Date --------- _ -- ------_ ----------- -- ----------------- OiietW'- Andrew 'Seated > *d ^ J te u r /Y & fDeviation No. L r- opc.ojor \ i . _ i f ) \ l P P (assigned by Study 'DDirierecctotorroorrPProroiejecct tLLeeaadd at the end of study or project) fttivt John Ijutenmlv A ttachm ent A 3M Environmental Laboratory ETS-4-8.1 Documentation of Deviations Page 1 of : Page 38 3M Medical Department Study: T-6295.22 3M Confidential Record of Deviation Analytical Report: FACT-TOX-160 LIMS EOO-1668 Study / Project No. FACT-TOX-160, EOO-1668 ......... Deviation type SOP (Checkone) X Protocol Document number FACT-TOX-160, EOO-1668__________ I. Identification Method Equipment Procedure Other: ! Date(s) o f occurrence 11/16/01 II. Description Required procedurc/process: ................................................................ Protocol FA C T-TO X -160, EOO-1668 states: M atrix spikes and m atrix spike duplicates required for analysis o f liver samples should show spike recoveries within 70% -130% . A fter Uver sam ples have been analyzed, sam ple concentrations will be evaluated. I f a m easured sam ple (s) concentration exceeded the calibration range o f the m ethod and was diluted with m ethanol into the validated calibration range, then additional m atrix spikes will be prepared at the approxim ate m easured concentrations o f the sam ples then subsequently diluted with the sam e volum e o f m ethanol as the sample into the validated range o f the calibration curve. These liver matrix spikes will be evaluated versus an extracted m atrix calibration curve and an unextracted calibration curve. If the diluted liver m atrix spikes d o not show recoveries within 70-130% versus the extracted matrix calibration curve, then the sample concentrations, at the sam e dilution factor as the m atrix spikes, will be corrected for the spike recovery. Actual procedure/process: ................................ ................................ 1) The 10 ug/g m atrix spike/m atrix spike duplicate (M S/M SD ) average recovery w as 67% . 2) N o 1/50 liver dilution w as reported dining the course o f this study. T he 1/500 Uver dilution w as to o dilute because the w rong liver sample w as used fo r preparing that M S/M SD level. III. Actions Taken {such as amendment Issued, SOP revision, etc.) 1) N o liver values were corrected for this low recovery since one set o f M S/M SD s w ere within criteria and the average recovery for this set o f M S/M SD s were within the extended 65-135% criteria as stated in an earlier deviation. 2) D ilution was not reextracted/rediluted using the correct sam ple since the 1/50 dilution w as within criteria and this 1/50 dilution was used to show that no effect was observed when liver dilutions were needed. Recorded by Lisa A. Clemen ^ / \ . Date 04/09/02 0 Authorized by Sponsor R t^ re ie d la K v t/ j Date / 2/ 6 T- D irecto r A n d itu ) S th C d l Deviation No. n <~> /} . j (assig n ed b y S tudy D irecto r o r P ro ject L ead a t the end o f stu d y o r p ro ject) 3M Environmental Laboratory Page 39 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 3M Medical Department Study: T-6295.22 Analytical Report: FACT TOX-160 UM S EOO-1668 Appendix C: Extraction and Analytical Methods This appendix includes the following methods: ETS-8-4.2, "Extraction of Fluorochemical Compounds from Serum for Analysis Using HPLCElectrospray/Mass Spectrometry," (16 pages) ETS-8-6.0, "Extraction of Potassium Perfluorooctane-suifonate or other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry," (14 pages) ETS-&-5.2, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry," (11 pages) ETS-8-7.0, "Analysis of Potassium Perfluorooctane-suifonate or other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry," (10 pages) 3M Environmental Laboratory 3MEnvironmental Laboratory Page 20 Page 40 3M Medical Department Study: T-6295.22 3M Environmental Laboratory Analytical Report: FACT-TOX-160 , LIMS EOO-1668 Des Cepy cf Orkin*I lz:: c3 .. M ethod E x t r a c t io n o f F l u o r o c h e m ic a l com pounds fr o m Se r u m f o r A n a ly sis Usin g H P L C -E lectro spra y /M ass Spec tr o m etr y M etbod Number: ETS-8-4.2 Adoption Date: 03/01/99 Effective Date: L /c it/( Approved By: Laboratory Manager K -k - H z -- Group Leader Date o^io-/c i Date 1.0 S cope an d Application_______________ :______________________________________' 1.1 Scope: This method is for the extraction o f fluorochemical compounds from serum. 1.2 AppGcable com pounds: 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. 2.0 S um m ary of M ethod_____________________________________________________________ 2.1 This performance-based method describes the procedure for extracting perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum, or other fluids, using an ion pairing reagent and methyl-/erf-butyl ether (MtBE). 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 0.2 |im nylon filter using a 3-mL plastic W ord 6/95 3M Environmental Laboratory Pvt-rorfinn E T S -8 -4 .2 PlurHmrhpmrils frnm fspnim Page 1 of 15 Page 41 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 1 2.2 syringe into glass autovials. (Application of this method to seven fluorochemicals was demonstrated: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556, and a surrogate standard {see 3 .0 D efinitions}). These sample extracts are analyzed following method ETS-8-5.2 or other appropriate method. 3.0 Definitions________________________________________________ _________ _____________ 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) CgF n S 0 3' 3.2 PFOSA: perfluorooctane sulfonylamide CgFi7S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate CgFi7S 0 2N(CH2CH3)CH2C 0 2' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol CbF 17S 0 2N(CH2CH3)CH2CH20 H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8F j7S 0 2N(CH2CH3)H . 3.6 M556: CgFn S 0 2N(H)(CH2C 0 0 H ) 3.7 Surrogate standard THPFOS: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 W arnings and Cautions___________________________;_________________________' 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 Interferences___________________________________________________________________ 5.1 There are no interferences known at this time. 6 .0 E q u ip 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 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) 7.0 Su and M aterials_________________________________________________________ 7.1 Gloves 7.2 Eppendorf or disposable pipettes, plastic or glass (or equivalent) 7.3 Polypropylene bottles, capable of holding 250 mL and 1 L (Nalgene or equivalent) 3M Environmental Laboratory E T S -8 -4 .2 t?...______ .rn .. ---.L_________:_. i c _______r*_________ P ag e 2 o f 15 Page 42 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 7.4 Volumetric flasks, glass, type A 7.5 40 mL glass vials (I-CHEM or equivalent) 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 Labels 7.8 Bottle-top Dispenser - 3.0 to 10.0 mL or a 5-10 mL graduated pipette, glass 7.9 Syringes, capable o f measuring 5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 mL (B&D or equivalent) 7.12 Syringe filters, nylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap glass autovials and caps 7.15 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with water. Rinse syringes a minimum o f 9 times with methanol, 3 rinses from 3 separate vials. 8.0 R eagents and Standards_________________________ :______________________________ 8.1 Reagent grade water, Milli-QTM, Nanopure II or equivalent 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent ' 8.4 Sodium carbonate (NaCCb), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHCCb), 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 Fluorochemical standards 8.9.1 KPFOS (PFOS) (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 8.9.3 PFOSAA+H (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 (l-H .l-H , 2-H, 2-H C8F 13SO3H, [THPFOS]), molecular weight = 428 8.9.8 Other fluorochemicals, as appropriate 3M Environmental Laboratory ETS-8-4.2 Page 3 of 15 Page 43 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 8.10 Reagent preparation NOTE: When preparing different 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 water, mix until all solids are dissolved. Store in a 1 L polypropylene (or equivalent) 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 bring to volume using water. Store in a 125 mL polypropylene (or equivalent) bottle. 8.10.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 mL water. Adjust to pH 10 using approximately 44 to 54 mL o f 10 N NaOH. (Add the last 1.0 mL of NaOH slowly, as the pH changes abruptly.) Dilute to volume with water. Store in a 1 L polypropylene (or equivalent) bottle. 8 .1 0 J.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na2C 0 3/N aH C03): Weigh approximately 26.5 g of sodium carbonate (Na2C 0 3) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with water. Store in a 1 L polypropylene (or equivalent) bottle. . 8.11 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing approximately 1.00 pg/mL of PFOS, PFOSA, PFOSAA, PFOSEA, M556 and EtFOSE-OH). . 8.11.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. For standards with K* or other salts, multiply by a correction factor. For example, the molecular weight of KPFOS = 538, and the molecular weight of PFOS = 499. Calculate the correction factor by using the following equation: molecularwt. PFOS( 499 ) = 0.9275 (Correction factor) molecularwt. KPFOS (5381 8.11.4 Bring to volume with methanol for a stock standard of approximately 1000 pg/mL. 8.11.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 pg/mL. I'lOOOW/mLxSjrf.'l = J 0 /mL 100 mL J 8.11.6 Dilute working standard 1 with methanol for a working standard 2 solution of approx. 5.0 pg/mL. 3M Environmental Laboratory ETS-8-4.2 Page 4 of 15 Page 44 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 ^50 p g / m L X 10 mL 100 mL = 5 .0 fig / mL 8.11.7 Dilute working standard 1 with methanol for a working standard 3 solution of approx. 0.50 pg/mL. 8.12 Surrogate THPFOS stock standard preparation 8.12.1 Weigh approximately 50-60 mg of surrogate standard l-H .l-H , 2-H, 2-H, CgFi3S0 3 H (THPFOS) into a 50 mL volumetric flask and record the actual weight. 8.12.2 Bring to volume with methanol for a surrogate stock o f approximately 1000 1200 pg/mL. 8.12.3 Prepare a surrogate working standard. Transfer approximately 1 mL o f surrogate stock to a 10 mL volumetric flask and bring to volume with methanol for a working standard of 100 pg/mL. Record the actual volume transferred. 9.0 Sam ple Handling_________________________________________________________________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw at room temperature or in lukewarm water prior to extraction. 10.0 Quality Control________________________________________________________________ 10.1 Solvent Blanks, M ethod Blanks and M atrix Blanks 10.1.1 Solvent Blanks: An aliquot of 1.0 mL methanol is used as a solvent blank. 10.1.2 Method Blanks: Following this procedure, extract two 1.0 mL aliquots o f water . and use as method blanks. 10.1.3 Matrix Blanks: Matrix blanks are prepared from one o f three sources: 1) a study control matrix from a study control animal received with each sample set; 2) a commercially obtained sample o f the same species as the study animals; or 3) a surrogate matrix, also obtained commercially, but of a different species than the study animal (e.g. if rabbit is used to generate standard curves and CCVs for a monkey or rat sera study). The matrix to use depends on what matrix is used for the curve. 10.1.3.1 Study control matrix curve--If the study control matrix is used for the curve, prepare two (2 ) matrix blanks using the study control matrix. 10.1.3.2 Commercially obtained (same species) matrix curve-- If the curve is prepared using commercially obtained matrix in the same species as the study animal, prepare two matrix blanks using this same commercially available matrix. 3M Environmental Laboratory ETS-8-4.2 Page 5 of!5 Page 45 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 10.1.3.3 Surrogate matrix curve-- If a surrogate matrix is used for the curve and continuing calibration verification samples: a) prepare two (2 ) matrix blanks using the surrogate matrix. b) Also, prepare a matrix blank with each set o f matrix spikes (a set is a matrix spike/matrix spike duplicate at one level) using a commercially available matrix of the same species as the study animals. 10.2 M atrix spikes 10.2.1 Study Control Matrix Curve No matrix spikes are prepared. 10.2.2 Commercially obtained matrix curve (same species) No matrix spikes are prepared. 10.2.3 Surrogate matrix curve Matrix spikes are necessary if matrix is not available in the same species as the study animal and a surrogate matrix is used for the curve and CCV samples (e.g., rabbit sera may be used to generate standard curves for a monkey sera study, due to measurable levels o f endogenous analyte in the monkey sera). Prepare and analyze matrix spike and matrix spike duplicate samples to verify the accuracy o f the extraction for target analytes. 10.2.4 Prepare each MS and MSD at two (2) levels (usually a low and mid-range concentration) using a sample chosen by the analyst, typically sera from a control group animal received with each sample set. 10.2.4.1 If there is not sufficient sera available from the control group, prepare matrix spikes using commercially available matrix from the same species as the study animal. 10.2.5 Prepare one matrix spike and matrix spike duplicate at each level listed in 10.2.4 per 20 samples, with a minimum o f 2 matrix spikes per level per batch. If a batch includes more than 20 samples, additional spikes may be prepared at the same, low, or high range levels. 10.2.6 If more than 25% of the samples are <LOQ, two matrix spikes should be prepared at approximately 2-5 times the expected LOQ. 10.2.7 If the majority of the samples are at or above the high range of the curve, additional matrix spikes should be prepared to approximate sample concentrations. 10.3 Continuing calibration verifications (CCYs) 10.3.1 Prepare continuing calibration verification samples for instrument stability verification during analysis. Prepare and analyze CCVs for every assay run, regardless of the matrix type used to prepare the standard curve. 10.3.2 Prepare each continuing calibration verification from the same matrix used to prepare the initial curve (i.e., either the study control matrix, commercial matrix of same species, or surrogate matrix). 3M Environmental Laboratory ETS-8-4.2 Page 6 of 15 Page 46 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1663 10.3.3 The expected concentrations of the CCVs will fall within the range of the initial calibration curve; typically, the low to mid-range of the curve. 10.3.4 Prepare, at a minimum, two continuing calibration verifications, each at a different concentration, per group of 10 samples. For example, if a sample set = 34, eight (8) checks are prepared; four (4) at a low range and four (4) at a mid-range concentration. 10.3.5 Additional continuing calibration verifications may be included at the same, low, mid or high-range concentration. 11.0 Calibratio n and Standardization____________________________ __________ 11.1 Prepare m atrix calibration standards 11.1.1 Transfer 1 mL o f appropriate serum to a 15 mL centrifuge tube. If commercially available sera is used, prepare two matrix blanks, using the volume o f sera available for most study animals. 11.1.1.1 Depending upon study goals or if analyte-free commercial sera is not available, a surrogate matrix may be used for generation of the calibration standard. For example, rabbit sera may be used as a surrogate matrix for rat studies if quantitation of extremely low levels o f the analyte is required. 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 o f thirteen aliquots in 15-mL centrifuge tubes, mix or shake sera between aliquots. 11.1.4 Two 1-mL aliquots, or other appropriate volume, serve as curve matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1 at the end of this section to spike one standard curve, for a total o f nine 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 See Section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each standard, blank, continuing check, and sample add an appropriate amount of surrogate working standard to achieve a constant concentration that falls within the calibration curve range o f 2.5 ng/mL-1000 ng/mL. Usually, samples are spiked for a constant concentration in all samples at approximately 500 ng/mL or other concentration as determined by the analyst. 11.3 Extract spiked matrix standards following 12.6-12.16 of this method. Use these standards to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory ETS-8-4.2 Page 7 of 15 Page 47 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Table 1 Approximate spiking amounts for standards and spikes Using 1.0 mL of matrix Working standard (approx, cone.) pL Approx, final cone, of 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 *p p m = |ig /m L 12.0 P rocedure_____________________________________________________________________ 12.1 Obtain frozen samples and allow to thaw at room temperature or in lukewarm water. 12.2 Label a 15 mL polypropylene centrifuge tube with the study number, sample ID, date and analyst initials. See attached worksheet (Attachment A or similar worksheet) for documenting the remaining steps. 12.3 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to the 15 mL polypropylene centrifuge tube. 12.4 Return unused samples to freezer after extraction amounts have been removed. 12.5 Record the initial volume on the extraction worksheet (Attachment A or similar worksheet). 12.6 Spike all samples, blanks and standards, that are ready for extraction with surrogate standard as described in 11.2. 12.7 Spike each calibration standard matrix with the appropriate amount of standard as described in 11.1, or Table 1 in that section, for the calibration curve standards. Also prepare matrix spikes if necessary and continuing calibration standards. 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.10 To each sample, add 1 mL 0.5 M TBA and 2 mL of 0.25M sodium carbonate/sodium bicarbonate buffer. 12.11 Using an bottle top dispenser or 5-10 mL graduated glass pipette, add 5 mL methyl-ferfbutyl ether. 12.12 Cap each sample and put on the shaker at a setting of 300 rpm for 20 minutes. 3M Environmental Laboratory .i* ETS-8-4.2 Page 8 of 15 Page 48 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 12.13 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm or until layers are well separated. 12.14 Label a fresh 15 mL centrifuge tube with the same information as in 12.5. 12.15 Remove 4.0 mL of the organic (top) 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, or longer if needed. 12.19 Label a 1.5 mL glass autovial (or low-volume autovial when necessary) with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.20 Attach a 25 mm, 0.2 pm nylon mesh filter to a 3 mL syringe and transfer the sample from step 12.18 to this syringe. Filter into the labeled autovial. 12.21 Cap and store extracts at room temperature or at approximately 4 C until analysis, 12.22 Complete the extraction worksheet (Attachment A or similar worksheet) and include in the study binder. 13.0 Data A nalysis and Calculations__________________________________________ ____ 13.1 Calculations 13.1.1 Calculate actual concentrations o f PFOS, or other applicable fluorochemical, in calibration standards using the following equation: _______ mLof stdXconcentrationof std( jig/mL)_______ mLof std+ mLof surrogatestd+ initial matrixvolume(mL) Final concentration( ftg/mL) of PFOS inmatrix 14.0 M e t h o d P e r fo rm 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 A ttachm ents B and C). At the discretion of the PAI, MDL may not be defined for some studies. 14.2 The following quality control samples are extracted with each batch o f samples to evaluate the quality of the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 If surrogate matrix is used for curve and QC, matrix spike and matrix spike duplicate samples to verify extraction efficiency. 14.2.3 Continuing calibration check samples to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-5.2 for method performance criteria. 3M Environmental Laboratory E T S -8 -4 .2 TJvt'O'trtrt a ClnnrrtAhmt/'alp #V irrt Qorn Page 9 of 15 Page 49 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 15.0 P ollution Prevention and W aste M a n a g e m e n t __________________ . 15.1 Dispose of sample waste, flammable solvent waste and used glass pipette waste using 1 proper methods and by placing each in their designated waste container. 16.0 R ecords________________________________ . ___________________ _____ ______ 16.1 Complete the extraction worksheet attached to this method (or a similar worksheet), and include in the study binder. 17.0 A ttachments____________ ;_______________________________ ________ ________ 17.1 Attachment A, Extraction worksheet (a similar worksheet may be substituted for Attachment A) 17.2 Attachment B, MDL/LOQ values and summary . 17.3 Attachment C, Ion Pair Standard Curves worksheet (a similar worksheet may be substituted for Attachment Q 18.0 References______________________________________________ ________ ____________ 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" 18.3 ETS-8-5.2, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" 19.0 A ffected D ocuments_______ ;_____________________________'____________ :_________ 19.1 ETS-8-5.2, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" 20.0 R evisions___________________________________________________________ Revision Number 1 2 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. Section 1.1,2.1 Elim inate `potassium' from perfluorooctanesulfonate 8.9.1 Add K to PFOS 8.9.3 Add H to PFOSAA 10.2-10.2.4, 14.2.2 Add information about using surrogate matrix, clarify control matrix and spiking the curve, change spikes to every 20 samples 10.3 Clarify purpose of continuing calibration checks 1l.l Added wording about control animal sera, using and not using commercially available tissue In general, make less specific forequipment/supplies. Substitute water for Milli-Q, Nalgene or equivalent bottle may be used. Revision Date 04/02/99 3M Environmental Laboratory ETS-8-4.2 Page 10 of 15 Page 50 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 Extraction Worksheet ETS-8-4.2 LIMS EOO-1668 Study # M atrix Box # W k/Dav D ate Spiked/ A n alyst: Surrogate Std actual pg/m L # FC-M ix approx. 0.5 pg/m L # FC-M ix a p p ro x .5 pg/m L # FC-M ix approx. 50 pg/m L # Com ments V erified by: MS M SD -- - -- - -- - --- -- - -- - -- - -- - --- -- - -- - - - - -- - -- - -- - Blank ID # -amount =. -------piL , -Serum Extraction M ethod .................. ..... !______ _ . .. . D ate & Initials . . Vortex 15 sec. _ Pinette Matrix, soike with appropriate surrogate orFCM ix Volume .. mL Pipette 1 mL o f 0.5 M TBA, pH 10. pH = ................ Std. # Pipette 2 mL of 0.25 Na2COj/0.25M NaHCOj buffer Std. # Dispense/pipette 5 mL of methvl-t-butyl ether TN-A- Dispenser ID #: Shake 20 min. Shaker ID #: Shaker speed: Centrifuge 20-25 min. Centrifuee ID#: Centrifuee speed: Remove a 4 mL aliauot of oreanic laver Put on Nitroeen Evaporator to drvness Add methanol Volume mL TN-A- Vortex 30 sec. .............. Filter using a 3mLB-D svrinee with a 0.2um filter into a 1,5 mLautosample vial Cont. Cal. V erifications used sam e m atrix as for std curve. ... . .... I .1 .1 ADDendix A: Extraction Worksheet 3M Environmental Laboratory ETS-8-4.2 Pase 11 of 15 Page 51 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 MDL/LOQ values for rabbit serum Compound MDL LOQ Linear Calibration Range (LCR) (ng/mL) (ng/mL) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 1.74 5.55 5 ng/m L-1000 ng/mL . PFOSA 1.51 4.79 5 ng/m L-1000 ng/mL PFOSAA 3.46 20.5 5 ng/mL-1000 ng/mL EtFOSE-OH 11.4 36.2 5 ng/mL-1000 ng/mL M556 6.03 19.2 5 ng/mL-1000 ng/mL PFOSEA 5.71 18.2 5 ng/mL-1000 ng/mL 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. Appendix B: MDL/LOQ Values and Summary 3M Environmental Laboratory ETS-8-4.2 Page 12 of 15 Page 52 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Compound: P FOS Prepared range Rabbit Serum of standards (ng/mL) Full Range 0.995 - 978 Low Curve 4.94 - 248 High curve 97.8 - 978 1/X 0.995 - 978 Compound: P FOSA Prepared range Rabbit Serum of standards (ng/mL) Full Range 0.993 - 976 Low Curve 4.93 - 97.6 High curve 24.8 - 976 1/X 0.993 - 976 Compound: P FOSAA Prepared range Rabbit Serum of standards (ng/mL) Full Range 0.991-974 Low Curve High curve 4.92 - 247 49.2 - 974 1/X 0.991-974 LCR from curve (ng/mL) 24.8 - 978 4.94 - 248 97.8 - 978 4.94 - 978 LCR from curve (ng/mL) 4.93-976 4.93 - 97.6 24.8-978 4.93 - 976 LCR from curve (ng/mL) 24.7 -974 9.74 - 247 97.4 - 974 9.74 - 974 % Recovery Range 83-108 85-104 85-106 94-111 % Recovery Range 88-103 87-105 93-102 94-103 %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 Appendix B: MDL/LOQ Values and Summary 3M Environmental Laboratory ETS-8-4.2 Page 13 of 15 Page 53 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Compound: EtFOSE-OH Prepared range Rabbit Serum of standards (ng/mL) Full Range 0.993 - 976 Low Curve 4.93 - 97.6 High curve 49.3 - 976 1/X 0.993 - 493 LCR from curve (ng/mL) 49.3 - 976 9.76 - 97.6 97.6 - 976 9.76 - 976 % Recovery Range 77-110 97-107 90-109 86-111 Compound: PIFOSEA Prepared range Rabbit Serum of standards (ng/mL) Full Range 0.993 - 976 Low Curve 4.93 - 248 High curve 49.3 - 976 1/X 0.993 - 976 LCR from curve (ng/mL) . 24.8 - 976 9.76 - 248 49.3 - 976 9.76 - 976 % Recovery Range 96-106 91-110 86-106 95-117 Compound: IV556 Prepared range Rabbit Serum of standards (ng/mL) Full Range 0.993 - 976 Low C urve 4.93 - 97.6 High curve 97.6 - 976 1/X 0.993 - 976 LCR from curve (ng/mL) 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 Appendix B: MDL/LOQ Values and Summary 3M Environmental Laboratory ETS-8-4.2 Page 14 of 15 Page 54 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 E xam ple Ion Pair Standard Curves - Fluids Analyses) Study number: Prep date(s): Analyte(s): Study Animal M atrix: Final solvent and TN: Blank fluid/identifier: Record matrix used lc>prepare curves, ic.g,, 'Rabbit,* ID #XXX)1 ~ 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: (Record the standards used to prepare the extracted curves.) Surrogate std approx. 100 ppm: Actual concentrations of standards in the FC mix in methanol PFOS PFOSA PFOSAA EtFOSE PFOSEA M556 Std cone Std cone Std cone Std cone Std cone Std cone pg/mL pg/mL pg/mL pg/mL pg/mL ng/mL All Am't spiked mL Calculated concentrations of standards in the sera matrix: Rabbit Validated ranges - approximate concentrations Serum PFOS PFOSA PFOSAA Rabbit 5.00-1000 ng/mL 5.00-1000 ng/mL 5.00-1000 ng/mL Bovine Estimates only. Use values for rabbit. Rat Estim ates only. Use values for rabbit. Monkey & Plasma Estimates only. Use values for rabbit. Human Estimates only. Use values for rabbit. E tF O S E -O H 5.00-1000 ng/mL PFOSEA 5.00-1000 ng/mL M556 5.00-1000 ng/mL A ttachm ent C: Ion Pair Standard Curves 3M Environmental Laboratory E T S -8 -4 .2 Page 15 of 15 Page 55 3M Medical Department Study: T-6295.22 3M Environmental Laboratory Analytical Report: FACT-TOX-160 LIMS EOO-1668 Exast Copy of Orlatoti / fa o ] . L Initial__ Data M ethod E x tr a c tio n o f P o ta ssiu m 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 C o m po u n d s f r o m L iv e r fo r A nalysis u sin g H P L C - E lectrospray/M ass Spectrom etry M ethod N um ber: ETS-8-6.0 Author: Lisa Clemen, Robert Wynne Approved By: y \A -- Laboratory Manager u Group Leader ----------- - Technical Reviewer Adoption Date: . Revision Date: hJft V Date 2A - f a WM Date . .... O i / w l v i Date _____ 1.0 Scope and Application 1.1 Scope: This method is for the extraction o f potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from liver. 1.2 A pplicable Com pounds: 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 3M Environmental Laboratory E T S -8 -6 .0 .. . c rt n.. Page 1 of 14 Page 56 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 2.0 S u m m ar y of M ethod___________________________________________ ______________ _ i 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-tert-hutyl 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. 3.0 D efinitions_____________________________ _______________________ _______________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) CgFI7S 0 3 3.2 PFOSA: perfluorooctane sulfonylamide C8F,7S 0 2NHj 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate CgF,7S0 2N(CH2CH3)CH2C0 2 3.4 EtFOSE-OH: 2 (N-ethylperfluorooctane sulfonamido)-ethyl alcohol CgF |7S 0 2N(CH2CH3)CH2CH20 H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8FJ7S 0 2N(CH2CH3)H 3.6 M556: C8F i7S 0 2N(H)(CH2C 0 0 H ) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid . 4.0 W arnings and Cautions_________________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 I n ter fer en ces ____________________________________________________________________ 5.1 There are no interferences known at this time. 6.0 E quipm ent ________________________________________________________________ 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 6.1.3 6.1.4 Vortex mixer, VWR, Vortex Genie 2 Centrifuge, Mistral 1000 or IEC Shaker, Eberbach or VWR ' 3M Environmental Laboratory E T S -8 -6 .0 in e r te f* Page 2 of 14 Page 57 3M Medical Department Study: T-6295.ZL Analytical Report: FACT-TOX-160 LIMS EOO-1668 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivity to 0.100 g) . 7.0 S upplies and M a ter ia ls__________________ ______________________________ ________ 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 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 D is p e n s o r-3.0 to 10.0 ml 7.11 Syringes, capable o f 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 MilliQTM water. Rinse syringes a minimum o f 9 times with methanol, 3 rinses from 3 separate vials. 8.0 R eagents and Standards________________________________________________________ 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and 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 (NajCOj), J.T. Baker or equivalent 8.5 Sodium bicarbonate (N aH C03), J.T. Baker or equivalent 8.6 Methyl-ter/-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 3M Environmental Laboratory E T S -8 -6 .0 -TTYPOkO r ___ T Page 3 o f 14 Page 58 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 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 CaF 13S 0 3H) molecular weight = 428 8.10.8 Other fluorochemicals, as appropriate . 8.11 Reagent preparation N O TE : 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 10 NNaOH 1:10. Measure 10 mL o f . 1 0 N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.11.3 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 o f NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-QTM water. Store 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 (NajCOj/NaHCOj): Weigh approximately 26.5 g o f sodium carbonate (NaaCOj) and 21.0 g o f sodium bicarbonate (NaHCOa) into a l L volumetric flask and bring to volum e with M illiQTM 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 o f approximately 1000 ppm (pg/mL). 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 3M Environmental Laboratory tr...__ ETS-8-6.0 Page 4 of 14 Page 59 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 8.12.6 Dilute the stock solution with methanol for a working standard 2 solution o f approx. 5.0 ppm. 8.12.7 Dilute the stock solution with methanol for a working standard 3 solution o f . . 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, CgF ,3S 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 o f approximately 1000-1200 ppm. 8.13.3 Prepare a surrogate working standard. Transfer approximately 1.0 m l o f 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 Sample H andling_________________ ;_______________________ ._________ .________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 10.0 Quality Control_____________________________________________ 10.1 Matrix blanks and method 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 liver homogenate following this procedure and use as matrix blanks. Refer to 11.1.6. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy o f the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually a control liver received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range o f 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 o f 2 matrix spikes per batch. 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibration verification samples to ensure the accuracy o f the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing calibration verification sample per group o f 10 samples. For example, if a sample set = 34, four verifications are prepared and extracted. 3M Environmental Laboratory E T S -8 -6 .0 f T i A C r ___ T Page 5 of 14 Page 60 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 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 o f 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 -1 0 0 0 ppb). 11.0 C alibration and Standardization____________________________ ______________ __ 11.1 P rep a re m atrix calibration standards 11.1.1 Weigh approximately 40 g o f liver into a 250 mL Nalgene bottle containing 200 mLs Milli-QTM water. Grind to a homogeneous solution. 11.1.2 I f 40 g is not available, use appropriate amounts o f 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 o f homogenate solution. 11.1.5 Add 1 mL o f homogenate to a 15 mL centrifuge tube. Re-suspend solution by shaking between aliquots while preparing a total o f eighteen 1 mL aliquots o f 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 o f this section, to spike, in duplicate, two standard curves, for a total o f eighteen samples, two matrix blanks, and two method blanks. 11.1.8 Refer to validation reports ETS-8-6.0 an d ETS-8-7.0-V-1 or A tta c h m e n ts , which lists the working ranges and the Linear Calibration Range (LCR) for ' calibration curves. 11.1.9 U se Attachment C as an aid in calculating the concentrations o f the working standards. Refer to 13.0 to calculate actual concentrations o f PFOS in calibration standards. 11.2 To each working standard, blank, or continuing verification, add appropriate amount o f surrogate working standard for the concentration to fall within the calibration curve range 5 ppb - lOOOppb. 3M Environmental Laboratory ETS-8-6.0 Page 6 of 14 Page 61 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 11.3 Extract spiked liver homogenates following 12.14-12.25 o f this method. Use these standards to establish each initial curve on the mass spectrometer. t 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 Approx, final cone, o f PFOS in liver - Blank 2 0.005 ppm 4 0.010 ppm 10 0.025 ppm 20 0.050 ppm ' 40 0.100 ppm 10 0.250 ppm 20 0.500 ppm 30 0.750 ppm 4 . 1.00 ppm 12.0 Procedure________________________________________________ ;_____________________ 12.1 Obtain frozen liver samples. 12.2 Cut approximately 1 g o f liver using a dissecting scalpel. This part o f the procedure is best performed quickly, not allowing the liver to thaw. 12.3 W eigh the sample directly into a fared plastic sampule vial. 12.4 Record the liver weight in the study notebook. 12.5 Return unused liver portions to freezer. . 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. Refer to AMDT-EP22. - 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. 3MEnvironmental Laboratory ETS-8-6.0 Page 7 of 14 Page 62 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 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 matiix with the appropriate amount o f standard as described in 11.1, or Table 1 o f 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. I f not, adjust accordingly. 12.17 To each sample, add 1 mL 0.5 M TBA and 2 mL o f the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.18 Using an Oxford Dispenser, add 5 mL methyl-ter/-butyl ether. 12.19 Cap each sample and put on the shaker at a setting o f 300 rpm, for 20 minutes. 1 2 .2 0 Centrifuge for 20 to 25 minutes at a setting o f 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 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. 12.27 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyses) 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. 3MEnvironmental Laboratory ETS-8-6.0 Page 8 of 14 Page 63 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 13.0 D a ta A n a l y sis and C alc ula tio n s______________________________ _________ ;_______ 13.1 Calculations: 13.1.1 Calculate the average density o f the liver homogenate by recording each mass o f ten separate 1.0 mL aliquots of homogenate. Average density (mg/mL) = Average mass (mg') o f the aliquots 1.0 mL aliquot 13.1.2 Calculate the amount o f liver (mg) per 1.0 mL homogenate (or concentration o f dispersed solid tissue per mL o f homogenate suspension) using the following equation: g o f Liver x Average density* o f homogenate Cmg/mLl (g of Liver + g o f 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: uL o f Standard x Concentration fug /mLl = Final Concentration (pg/g or mg/kg) mg Liver / 1 mL homogenate* o f PFOS in Liver *refer to 13.1.2 for details. 14.Q 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 M DL report for specific M DL and limit o f quantitation (LOQ) values (refer to Attachments B and C). 14.2 The follow ing 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 verification samples to determine the continued accuracy o f the initial calibration curve. . 14.3 Refer to section 14 o f ETS-8-7.0 for method performance criteria. 15.0 Pollution Prevention and W aste M anagem 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 broken glass containers located in the laboratory. 3M Environmental Laboratory ETS-8-6.0 Page 9 of 14 Page 64 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 16.0 R ecords_______________________________________________________________;_________ 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 T ables. D iagrams. Flowcharts, and Validation Data__________ ______________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard calculation and concentration worksheet 18.0 R eferences_______________________________________________ ____________________ 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 o f Ultra-Tuxrax T-25" 18.3 FACT-M-1.1, "Extraction of'PFOS or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 19.0 A ffected D ocuments___________________________________________________________ 19.1 ETS-8-7.0, "Analysis o f Liver Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 R evisions_______________________________________________________________________ Revision Number Reason For Revision Revision Date 3M Environmental Laboratory ETS-8-6.0 Page 10 of 14 Page 65 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 S tudy # M atrix Box W k/D ay D ate S p ik ed /A n aly st CCV MS M SD S u rro g ate S td approx, ppm actu al ppm # F C M ix S td ap p ro x . 0.5 p p m actu al ppm # F C M ix S td approx. 5 ppm actual p p m #. F C M ix S td approx. 50 p p m actu al ppm # i C om m ents -. - '- - - Blank Liver Homogenate: S td# Liver Extraction M ethod : Liver am ount Spike surrogate and Standard mix. Vortex IS sec. Pipette-1 mL o f Liver Solution Pipette 1 m L o ft0 .5 M T B A ,pH 10. p H S td .# Pipette 2 mL o f 0.25 Na^COV0.25M NaHCOi Buffer Std. # Dispense 5ml o f Methyl-t-Butyl Ether TN-A- ' Shake 20 min. Shaker Speed Centrifuge 20-25 min. Centrifuge Speed Remove a 4 mL aliauot o f oreanic layer Put on Nitrogen Evaporator to dryness Evaporator Temperature Add 1.0 mL o f Methanol TN-A- Vortex 30 sec. Filter usine a 3cc B-D syringe with a 0.2um SRI filter into autosamolc vial Cont. Cal. V erifications used the sam e m atrix as for the standard curve. - - - - - - '- - - e Date & Initials _ - ... .. - Attachment B: MDL/LOQ Values 3M Environmental Laboratory ETS-8-6.0 Page 11 o f 16 Page 66 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 MDL/LOQ values for rabbit liver Compound MDL LOQ Linear Calibration Range (LCR) (ppb) (Ppb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 8.45 26.9 30 ppb - 1200 ppb PFOSA 3.50 11.1 12 ppb - 1200 ppb . PFOSAA 24.6 78.3 30 ppb - 1200 ppb EtFOSE-OH 108 345 60 ppb - 900 ppb* M556 82.3 262 60 ppb - 1200 ppb . PFOSEA 33.9 108 30 ppb- 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 * EtFOS-OH estimates only for MDL and LOQ. Did not meet criteria for validation. Com pound: PFOS_____________________________________________________ Prepared R ange o f /L C R fro rrr. R ange o f : ;L C R fro m R a n g e o f T L C R f ro m L iv e r range o f average Vaveciive- lo w std .. ; lo w ,sfd `> h ig h std t, high s t d . m atrix standards (ppb) (ng/m L) curve (ppb) (ng/mL) - t--' (ppb) (ng/mL) curve . cu rv e ` i" curve -?jT c u rv e .1(p p b ) (n g /m L ) ( p p b ) ( n g /m L ) J (pp b ) (n g /m L ) "(p p b ) (n g /m L ) : R ab b it 6x19 - 1237 12 - 1200 - ^ 2 . 0 2 0 0 6-300 60-1200 r.'d L 'i o o J : Compound: PFOSA Prepared L iv e r m atrix range o f stan d ard s (ppb) (ng/m L) Range of average c u rv e (ppb) (ng/mL) LCR. from, aveburve- . (ppb) (ng/mL) Range of low std curve (ppb) (ng/m L) R abbit 6 .1 9 -1 2 3 7 12 - 1200 : : 12 412> : 12 - 3 0 0 L C R fro m 3 R a n g e o f :;LQR:f r o n i- low stdi^::' high std Kigli std curve (pp b ) (ng/m L ):1 (ppb) (n g /m L ) S ( p p b ) ( g /r f iL ) , 6 0 - 1200 i*'1 0 -T 2 0 f Compound: PFOSAA L iv e r m atrix Prepared . range o f standards (ppb) (ng/mL) Range of average curve (ppb) (ng/mL) R ab b it 6 .1 6 -1 2 3 2 12 -1 2 0 0 LCR from , ave curve (ppb) (ng/mL) 30-1200 Range of low std curve (ppb) (ng/mL) 30 - 900 LCR from lOW Std : curve ; (ppb) (ng/mL) Range of high std curve (ppb) (ng/mL) LCR front high std : curve . (ppb) (ng/mL) 6 0 -9 0 0 N /A .. N /A Attachment B: MDL/LOQ Values 3M Environmental Laboratory ETS-8-6.0 Page 12 of 16 Page 67 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Compound: EtFOSE-OH Prepared Range of L iv e r range o f average m atrix standards (ppb) (ng/mL) curve (ppb) (ng/mL) R a b b it 6 .1 7 - 1235 3 1 -9 0 0 LC R from ave curve (ppb) (ng/mL) 3 1 -9 0 0 Range of 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 from h igh std curve (ppb) (ng/mL) N /A Compound: PFOSEA Prepared Range of L iv e r m atrix range o f standards (ppb) (ng/mL) average curve (ppb) (ng/mL) . LCR from ave curve A (ppb)(ng/mL)' Range of low std curve (ppb) (ng/mL) R a b b it 6 .1 7 - 1 2 3 5 3 1 - 1 2 0 0 ! ;r3 l ) - : . p ^ N /A LCR from . low std : curve V ' (ppb) (ng/mL) Range of high std curve (ppb) (ng/mL) LC R from h ig h std c u rv e ) AA (ppb) (ng/mL) N /A V i } - N / A :^ P Compound: M556 Prepared Liver range of matrix standards (ppb) (ng/m L) Rabbit 6.17-1235 Range of V.LCRfrniA Range of average vavexuryiefp: low std curve A curve (ppb) (ng/m L ) v(ppb)>(ng/m L),'. (ppb) (ng/m L) 31 - 1200 A :60'A(2)f N/A LGR'from A Range of . LCR from Adowstd; ' ; high std '. high std ^ vV'ciyfcSft curve A^-ciyel' ''(p p b ) '( n g /ih L ) ': (p p b ) (n g /m L ) :* (p p b )-( g /rriL )'' 'N /A ;. N/A A ttachm ent C: Standard Calculations 3M Environmental Laboratory E T S -8 -6 .0 ___ . r n m o c Page 13 of 14 Page 68 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Ion Pair Standard Curves - Tissue i. Prep date(s): Analyte(s): Sample matrix: 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: Standard number: Equipment number: Final solvent and TN: Blank liver/identifier: Actual concentrations of standards in the FC mix PFO S . P F O S A PFO SA A E tFO SE PFO SE A Std cone S td cone Std cone Std cone Std cone ug/m L ug/m L ug/m L ug/m L ug/m L 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 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 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 50.0 50.0 50.0 50.0 50.0 3Y556 Std cone ug/m L 0.500 0.500 0.500 0.500 0.500 5.00 5.00 5.00 50.0 Std cone ' ug/m L A ll A m 't spiked mL 0.002 0.004 0.010 0.020 0.040 0.010 0.020 0.030 0.004 A ll D ensity S 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 F in a l PFOSA F in a l PFO SA A E tFO SE PFO SEA Final cone Final F in a l M 556 F in a l Std cone cone ng/g cone ng/g ng/g cone ng/g cone ng/g cone ng/g ng/g 5.99 5.99 5.99 5.99 5.99 5.99 12.0 12.0 . 12.0 12.0 12.0 12.0 29.9 29.9 29.9 29.9 29.9 29.9 59.9 59.9 59.9 59.9 59.9 59.9 120 120 120 120 120 120 299 299 299 299 299 299 599 599 599 599 599 599 898 1198 898 1198 898 1198 898 1198 898 . 1198 898 1198 S urrogate Std cone ng/m L 100 Surrogate Final cone ng/mL ' 0.500 A ll A m 't spiked mL . 0.005 Validated ranges - approximate concentrations Liver PFOS PFOSA PFOSAA Rabbit 5-1000 ppb 5-1000 ppb 5-1000 ppb Bovine Estimates only, use rabbit values. Rat Estimates only, use rabbit values. Monkey Estimates only, use rabbit values. EtFOSE-OH 5-1000 ppb POAA 5-1000 ppb PFOSEA 5-1000 ppb A ttachm ent C: Standard Calculations 3M Environmental Laboratory E T S -8 -6 .0 r*. . g____ . . r n r n n r ___________t Page 14 of 14 Page 69 3M Medical Department Study: T-6295.22 3M Environmental Laboratory Analytical Report: FACT-TOX-160 LIMS EOO-1668 DcciCcpyofGifgfeJ _ i 7 / } : cia .. M ethod A nalysis o f P o ta ssiu m P er flu o r o o c ta n esu lfo n a te o r O t h e r F lu o r o c h em ic a ls in Seru m E xtra cts U sin g H P L C -E lectrospray/M ass Spectro m etry M ethod Number: ETS-8-5.2 Author: Lisa Clemen, Kris Hansen Approved By: Laboratory Manager Group Leader o - A ^ -- . ___________ Technical Reviewer Adoption Date: 03/01/99 Revision Date: a&) i f 01 0 > /o /A /s Date O 7 ~ /0 l/0 l Date ~ o (.o ( Date 1.0 S cope and Application__________________________________________________________ 1.1 Scope: This method describes the analysis of serum extracts for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 M atrices: Rabbit, rat, bovine, monkey, and human serum, or other fluids as designated in the validation report. W ord 6/95 3M Environmental Laboratory E T S -8 -5 .2 A Q.a c i i m fc'tnfT C C A /f C Page 1 of 11. Page 70 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 2.0 S u m m a r y o f M eth o d ________________________________________ _ _ ______ ______________ 2.1 Although supported by a validation for most commonly used matrices, this is a performance-based method. Careful attention should be paid to method QC as there is great variability in sera. This method describes the analysis of fluorochemical surfactants extracted from serum or other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z= 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity of a compound by detecting daughter ions o f the parent ion. 3.0 D e f in it io n s ________ '________________________________________________________,_________ 3.1 Atm ospheric Pressure Ionization (API): The Micromass Quattro II and Ultima 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 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 M ass Spectrom etry, M ass Spectrom eter (M S), Tandem M ass Spectrom eter (M S/M S): The API Quattro II and Ultima 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 o f Micromass 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 e l e c t r o d e . T h o u g h t h e c o n f i g u r a t i o n i s different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e., Z-spray components are compatible with some other Z-spray systems, etc.) 3.5 M ass 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 or Ultima triple quadrupole MassLynx or MassLynx NT User's Guide). 4.0 W arnings and Cautions___________________________________________________ 4.1 H ealth an d Safety W arnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage o f approximately 5000 Volts. Word 6/95 3MEnvironmental Laboratory E T S -8 -5 .2 Page 2 of 11 Page 71 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. 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 HPLC will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences______________________________________________________ ____________ 5.1 To minimize interferences when analyzing samples, Teflon should not be used for sample storage or any part o f instrumentation that comes in contact with the sample or extract. 6.0 E q u ip m e n t __________________________________________ _________________________________________________________ _____________ 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 6.1.2 Micromass Quattro II or Ultima triple quadrupole Mass Spectrometer equipped with an electrospray ionization source HP 1100 or Agilent low pulse solvent pumping system, solvent degasser, column compartment, and autosampler ` 7.0 S upplies and M aterials_________________________________________________________ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (House air or nitrogen 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 and capped 15 mL centrifuge tubes 8.0 Reag ents and Standards_________________________________________ ;______________ 8.1 Reagents 8.1.1 Methanol* HPLC grade o r 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-8-4.2. 3M Environmental Laboratory ETS-8-5.2 Page 3 of 11 Page 72 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 9.0 S ample Handling________________________________________ ______________________ _ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped auto vials or capped 15 mL centrifuge tubes until analysis. * 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C, or at room temperature, until analysis can be performed. 10.0 Quality Control_______________________________________________________________ 10.1 Solvent Blanks, M ethod Blanks and M atrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed at least once during the course of the study to determine if contamination occurred during sample prep. 10.1.2 Analyze at least one solvent blank prior to each calibration curve. 10.1.3 Matrix blanks should be analyzed with each sample list that includes undiluted extracts. 10.2 M atrix Spikes 10.2.1 If curves and method QC are prepared in a surrogate matrix (e.g. curves in rabbit sera, samples are monkey sera), matrix spikes and matrix spike duplicates are prepared in blank sample matrix (e.g., monkey sera) and analyzed to verify extraction efficiency. 10.2.2 If curves and method QC are prepared in the same matrix as samples, no additional matrix spikes are required. 10.3 Continuing C alibration Verifications (CCVs) 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of . the calibration curve. 10.3.2 Analyze two calibration standards (one at each o f 2 levels) after every one to ten samples, with a minimum o f two per batch and always finishing an injection sequence with at least two calibration standards. 11.0 C alibration and S tandardization____________________________________________ 11.1 Analyze the extracted matrix calibration standards prior to each set of extracts. The curve will be plotted by linear regression, 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, reextract samples, or reanalyze the standard curve. 11.3 For purposes of accuracy when quantitating levels o f analyte at the limits of the curve range, it may be necessary to use either the low end or the high end of the calibration curve rather than the full range o f the standard curve. Example: when attempting to quantitate approximately 10 ppb of analyte, it may be beneficial to generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of 3M Environmental Laboratory ETS-8-5.2 -c r>... r r _ :___- n e rx .co Page 4 of 11 Page 73 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 high concentration standards. It is also acceptable to break the linear range into a low curve and a high curve. If this is done, no more than one point should be used in common between the curves. For example, the low curve may include the following points: 1, 5, 25, 100, 250 ppb and the high curve may include the points: 250, 500,750, 1000, 1250 ppb. 12.0 P rocedures____________________ ;___________ ;_______________ _________________ 12.1 A cquisition Set up 12.1.1 On the MassLynx main page, set up a sample list name. Save the list as instrument designator letter, last 2 digits of test year-mo-day, and a letter that will increase through the alphabet with each additional list for that day. Example Sample List: lYYMMDDa or D010712a I=instrument name (D for "Davey") YY=year o f test (01) MM=month of test (07) DD=day o f test (12) a=first sample list (run) o f the day (the next sample list will end with `b,' the next `c' and so on.) 12.1.2 Assign a filename using the instrument designator letter, the last 2 digits o f year-moday, and a 3-digit sequential file number that starts with 1 and increases by one for each filename. Example File Name: IYYMMDD### or D010712001 I=instrument name (D for "Davey") YY=year of test (01) MM=month o f test (07) DD=day o f test (12) ###=3-digit sequential file number starting with 1 through 999 (001) Also, as part of the sample list, assign a method (MS) for acquiring, an inlet file, a bottle number, an injection volume and sample descriptions. 12.1.3 To create a method, click on Method Editor button in the MS Status Pane and select SIR (Single Ion Recording) or MRM (Multiple Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. Also set the acquisition start and stop times. 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. 12.1.4 Typically the analytical batch run sequence begins with solvent blanks and a set o f extracted matrix standards. . 12.1.5 Sample extracts are analyzed with two CCVs injected every one to ten samples. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered sample extracts but may be included as such. 3MEnvironmental Laboratory ETS-8-5.2 e C C /M C Page 5 of 11 Page 74 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 12.2 Using the HPLC 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HPLC to 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 fiL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 10.0 minutes 12.2.2.4 Flow rate = 300 pL/min 12.2.2.5 Mobile Phase (program) Time 0.00 min. 1.00 min. 5.50 min. 7.50 min. 8.00 min. MeOH 10% 10% 95% 95% 10% 2.0 mM Ammonium acetate (in H20 ) 90% 90% 5% 5% 90% 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24 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 o f 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. The probe should be checked weekly. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 pL/min or as appropriate. Observe droplets coming out of the tip of the probe. 12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 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 (iL/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. 3M Environmental Laboratory E T S -8 -5 .2 a_ -- ec-- r?. . * i trc/A .rc* Page 6 of 11 Page 75 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 12.3.8 Print the tune page, MS file, HPLC parameters, sample list and the Microsoft Word summary page and store in the study binder with a copy taped into the instrument log. 12.3.9 Click on start button in the MassLynx main page (this may vary among MassLynx versions, see appropriate MassLynx USER'S GUIDE). Ensure start and end sample number includes all samples to be analyzed. 13.0 Data A nalysis and Calculations______________________________ ___________ _ 13.1 Calculations: 13.1.1 Calculate matrix spike percent recoveries using the following equation: % Recovery Observed Result - Matrix Blank Result X 100 Spiking Level 13.1.2 Calculate percent difference using the following equation: ____ Expected Cone. - Calculated Cone. % Difference = -- ------------------------- -------------------- x 100 Expected Cone. 13.1.4 Calculate actual concentration of PFOS, or other fluorochemical, in matrix (|Xg/mL): (C one, o f PFOS Calc, from Std. Curve (n g / m L ) x Dilution Factor) ( Initial Volume of Matrix (mL) + mL of Surrogate Standard)} Final Volume ( mL ) J 1pg iOOOng 14.0 M f.thot) Perform ance________________________________________________________ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-4.2, 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 values must be below the lowest active standard in the calibration curve. 14.3 Calibration Curves 14.3.1 The coefficient of determination value for the calibration curve must be 0.990 or better. 3M Environmental Laboratory E T S -8 -5 .2 A n o h / c i o n f i iT-vtr'w't Tici rr P/1U Page 7 of 11 Page 76 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 14.3.2 All active calibration curve points must be within 25% of the theoretical value with the exception o f the LOQ point, which may deviate up to 30%. 14.3.3 Calibration standards with peak areas less than two times the curve matrix blank must be deactivated to disqualify a data range that may be affected by background levels of the analyte. 14.3.4 Low or high curve points may be deactivated to optimize a linear range appropriate to the data. 14.3.5 Not including low or high points dropped to optimize the linear range, curve points may be deactivated if they deviate more than 25% from the theoretical value when the curve is evaluated over a linear range appropriate to the data. 14.3.6 One point below the LOQ may remain active even if it deviates more than 30% or has a peak area less than two times the matrix blank; however, the LOQ will be defined at the lowest point with acceptable deviation. 14.3.7 A valid calibration curve must contain at least 5 active points above and including the LOQ. 14.4 M atrix Spikes 14.4.1 The average matrix spike percent recoveries should be within 30% o f the spiked concentration. Recoveries outside o f this range should be discussed in the report. 14.5 Continuing Calibration Verifications (CCVs) . 14.5.1 Continuing calibration samples within the linear range o f the run must show a percent recovery within 25% o f the spiked concentration. If a CCV is outside o f this recovery, subsequent data should not be accepted. Acceptable data must be bracketed by the curve and passing CCVs. 14.6 If criteria listed in this method performance section isn't met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 1 4 .7 I f d a ta a re to b e re p o rte d w h e n p e rfo rm a n c e c rite ria h a v e n o t b e e n m e t, th e d a ta m u st b e footnoted on tables and discussed in the text of the report. 15.0 P ollution Prevention and W aste M anagement_______________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records ____________________________________________________________ 16.1 The first page of each data packet generated for a study must have the following information included either in the header, in the footer or hand written on the page: study or project number, instrument, sample matrix and time point, date, and analyst. 16.2 A data packet includes the following: data review summary form, MassLynx quantify compound summary report for each target analyte, quantify calibration report for each target analyte (curve), method report, Word document listing set-up parameters, tune 3M Environmental Laboratory ETS-8-5.2 Ac tl*: C C /N /C Page 8 of 11 Page 77 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 method report, MassLynx scanning method report, HPLC method report, sample list, and quantity sample report (chromatogram). 16.3 For each analysis, after printing the tune method report, Word document listing, set-up parameters, MassLynx scanning method report, and sample list, copy and tape into the instrument runlog. The original is maintained in the data packet. 16.4 On each page of the quantify compound report, quantify calibration report (curve), and quantify sample report (chromatogram), the following information must be included either in the header, the footer, or hand written on the page: study or project number, instrument, method, calibration (the method and calibration are usually assigned the same name), analyst, and date. . 16.5 The analyst must date and initial the first page in a packet as long as their initials and date are electronically included on each page. If initials and date are not electronically included, they must date and initial each page. 16.6 Summarize data using suitable software (e.g., Excel) for inclusion in the final report. See A ttachm ent A for an example of a summary spreadsheet. 16.7 Back up electronic data to appropriate medium. Record the file name and location o f backup electronic data in instrument log book. 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 ata __________________________ 17.1 Attachment A: Data summary spreadsheet. 18.0 R efe r en c es__________________________________________________________________ 18.1 FACT-M-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance o f 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 en ts___________________________________________ ;___________________ 19.1 ETS-8-4.2, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 3M Environmental Laboratory E T S -8 -5 .2 __ TTpU CC/X/IC P a g e 9 o f 11 Page 78 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 20.0 Revisions________________________________________________ ______ _________ _ Revision * Number. Reason For Revision 1 Section 6.1.2 Clarification of HP 1100 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 of solvent ramp. Section 17.1 Changed from attachment B to A. Revision Date 04/02/99 2 10.1 C larified w hen blanks are run. 10.2 A dded instructions for when, surrogate m atrix is used. 10.3 Specify requirem ents for C C V s. 11.1 R equires only a calibration curve before the sam ples. . 11.2 C larify w hat to do if curve does not m eet requirem ents. 11.3 A llow to truncate the curve. 12.1.1 C larify sam ple list ID . 12.1.3 C larify typical run. 12.2 C hanges to m obile phase gradient and specify flow rate. 14.3 C h a n g e acceptable Limits. A d d specifics for acceptance o f calib ratio n curve. 14.3.3 W hen to deactivate calibration standards based on blank response. 14.3.5 W hen to deactivate calibration standards w ithin the linear range. 14.4 M odifies evaluation and use o f m atrix spikes. 14.5 D escribes evaluation and use o f CCV s. 14.5.1 E valuation o f C C V s. 16.0 A dd requirem ents for records and docum entation. . A ttachm ent A: Sum m ary Spreadsheet E T S -8 -5 .2 a i r r* n . . t f .! . ., r n n r n 3M Environmental Laboratory Page 10 of 11 Page 79 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Laboratory Study # ` Study: - Test M aterial: M atrix/Final Solvent: M ethod/Revision: A nalytical Equipm ent System Num ber: Instrum ent Softw are/V ersion: Filenam e: R -Squared Value: Slope: Y Intercept: D ate o f Extracdon/A nalyst: D ate o f A nalysis/A nalyst: Group Dose S am p le# Concentration u e/m L Initial Vol. mL Dilution Factor Final Cone. u?/m l. S lope: Taken from linear regression equation. G ro u p /D o se: Taken from the study folder. S am p le# : Taken from the study folder. C o n c en tra tio n (ue/m L ): Taken from the M assLynx integration summary. In itia l V olum e (m L ): Taken from the study folder. D ilu tio n F a c to r: Taken from the study folder. F in al C one. (u g /m D : Calculated by dividing the initial volum e from the concentration A ttachm ent A: Sum m ary Spreadsheet A noK /etc 3M Environmental Laboratory E T S -8 -5 .2 C o m m R v t r o o t TTcintr P C /A /IC Page 11 of 11 Page 80 3M Medical Department Study: T-6295.22 3M Environmental Laboratory Analytical Report: FACT-TOX-160 , LIMS EOO-1668 Ex^CapyofCrigfci! d T J M ethod An a ly sis o f P o ta ssiu m P er flu o r o o c ta n esu lfo n a te o r O t h e r F lu o r o c h em ic a ls in L iv e r E xtracts Usin g H P L C -E lectro spra y /M ass Spec tr o m etr y M ethod Num ber: ETS-8-7.0 Author: Lisa Clemen, Glenn Langenburg Approved By: fY [ 4 Laboratory M*anager -- Adoption Date: Revision Date: -7 Date Group Leader /Ff)*- A Clt/fntiTechnical Reviewer Date o ? //y/W Date 1.0 Scope and Application 1.1 Scope: This method is for the analysis o f 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. W ord 6/95 3M Environmental Laboratory ETS-8-7.0 Page 1 of 10 Page 81 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 2.0 Summary of Method_______________________________________________________ _ 2.1 t This method describes the analysis o f 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 Definitions________________________________________________________ 3.1 A tm ospheric P ressure 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 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 o f a strong electrical field. 3 3 M ass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II triple quadrupole mass spectrometer is equipped with two quadrupole mass selective detectors and a collision cell. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or an ion may be selected in the first quadrupole, fragmented in the collision cell, and these fragments may be analyzed in the second quadrupole. 3.4 Conventional vs. Z-spray probe interface: The latest models o f 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 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 other Z-spray systems, etc.) 3.5 M ass 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 refer to the manual specific to the instrument (Micromass Quattro II triple quadrupole MassLynx or MassLynx N T User's Guide). 4.0 Warnings and Cautions________________________________ ;_______________ 4.1 H ealth an d Safety W arnings: 4.1.1 U se caution with the voltage cables for the probe. When engaged, the probe employs a voltage o f approximately 5000 Volts. 3M Environmental Laboratory ETS-8-7.0 Page 2 o f 10 Page 82 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 4.1.2 W hen 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). I f 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 In ter fer en c es_______________________________________________________________________ 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 ipm ent________________________________________________________ ;___________________ 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 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 eagents and Standards_________________________________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water (ASTM type I), all water used in this method should be 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. 3M Environmental Laboratory ETS-8-7.0 Page 3 of 10 Page 83 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 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 Q uality Co n t r o l _____________________________________________ ______________ _ 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 o r 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 samplep. W ith a minimum o f 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range o f the initial calibration curve. Additional spike concentrations may fall in the low- range o f the initial calibration curve. ' ' 10.3 Continuing Calibration Checks 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 every tenth sample, with a minimum o f one per batch. 11.0 C alibratio n and Standardization_________________________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set o f sample extracts. The average o f 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. 3M Environmental Laboratory ETS-8-7.0 Page 4 o f 10 Page 84 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 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 o f the standard curve. Example: when attempting to quantitate approximately 10 ppb o f analyte, generate a calibration curve consisting o f the standards from 5 ppb to 100 ppb rather than the full range o f the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting o f high concentration standards. 12.0 Procedures_____________________ __________________________________ ____________ 12.1 Acquisition Set up 12.1.1 Set up the sample list 12.1.1.1 Assign a sample list filename using MO-DAY-last 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 o f 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 12.2.2.2Tnject/sample = 1 12.2.2.3 Cycle time = 9 minutes ' 3M Environmental Laboratory ETS-8-7.0 Page 5 o f 10 Page 85 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 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 Instrum ent Set-up . 12.3.1 Refer to ETS-9-24.0, "Operation and Maintenance o f the Micromass Quatiro 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 o f the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. I f 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 o f the probe. A fine mist should be expelled with no nitrogen leaking around the tip o f 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 3MEnvironmental Laboratory ETS-8-7.0 Page 6 o f 10 Page 86 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 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 D a ta A n a l y sis and Calculatio ns________________________________________ _________ 13.1 C alculations: 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 (pg/g): fag o f PFOS calc, from std. Curve x Dilution Factor) (Initial Weight o f Liver (gl Final Volume (mL) x 1 tig 1000 ng 14.0 M eth o d P erform ance________________________________________________ _____________ 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 o f 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 r 1value 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% o f the spiked concentration. 14.5 C ontinuing Calibration Verification 14.5.1 Continuing calibration verification percent recoveries must be within 30% of the . spiked concentration. . 14.6 I f 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. 3MEnvironmental Laboratory ETS-8-7.0 A ,, n ;.* TT. tro r\ <rc* Page 7 of 10 Page 87 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 1 15.0 P ollutio n P revention and W aste 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 ecords____________________________________________________________________ _ 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 o f 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 bles. D iagram s. F low charts, and Validation D ata_________________________ 17.1 Attachment A: ETS-8-7.0 Data summary spreadsheet 18.Q R eferences_________ '_____________________________;_____________________________ 18.1 FACT-M-2.1, "Extraction o f 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 19.0 A ffected D ocum ents____________________________________________________ __________ 19.1 ETS-8-6.0, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver or Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" 3M Environmental Laboratory ETS-8-7.0 Page 8 o f 10 Page 88 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 20.0 R evisions___________________________________________________________________________ Revision Number . Reason For Revision Revision Date 3M Environmental Laboratory E T S -8 -7 .0 Page 9 o f 10 Page 89 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Laboratory Study # 1 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: G roup Dose Sam ple# C o n cen tratio n ng/g Initial w t. g D ilu tio n Factor Final Cone, ug/g - G roup/D ose: Taken from the study folder. Sam ple#: 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 Cone, (ug/g): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet A nalxrctc 3M Environmental Laboratory ETS-8-7.0 ix/*r " P v fra rt TTcmor "PS/K/f.Q Page 10 o f 10 Page 90 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Appendix D: Data Summary Tables Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: FACT TOX-160 LIMS E00-1668 Table 9. Data Summary for PFOS In Serum FACT-TOX-160--pg/mL Group Group 1 Tlmepoini Week 9 Sex Male Female PFOS pg/mL Average SD 17.2 .824 n=2 26.6 5.88 n=2 Week 17 Week 25 Male Female Male Female 13.7 1.13 n=2 20.3*2.37 n=2 8.7 1.93 n=2 15.8*5.27 n=2 Week 33 Male Female 8.10*0.768 n=2 12.4 4.2 n=2 Week 41 Male Female 5.65 * 0.303 n=2 10.4*4.37 n=2 Week 53 Male Female 426*0.052 n=2 7.95*1.76 (1=2 NOTE: It is notpossibletoverifytrue recouaryof endogenousanalyte fromtissueswffliout radto^abeled reference material. The onlymeasurementof accuracyavailableat thistime, matrixspite studies, incfcatesthat thedata are quantitativeto 30% orgreater. Table 10. Data Summary for PFOS In Liver FACT-TOX-160--|ig/g Group Group 1 Sex Male PFOS pg/g Average* SD 3.56 * 0.436 n=2 Female 9.23 2.04 n=2 NOTE: # isnot possibletoverilytrue reccwsiyof endogenousanalyte from tissueswithout rado-iabeled reference material. The onlymeasurement of accuracyavailableat thistime, matrixspikestucies, indicates thatthe data are quantitativeto 35% or greater. 3M Environmental Laboratory 3M Environmental Laboratory Page 21 Page 91 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Appendix E: Data Spreadsheets Analytical Report: FACT-TOX-160 LIMS EOO-1668 Analytical Report: FACT TOX-160 LIMS E00-1668 3M Environmental Laboratory 3M Environmental Laboratory Page 22 Page 92 3M Medical Department Study: T-6295.22 Study: FACT-TOX-160, EOO-1668 Product Numbcr\Test Substance): Matrix: Method/Revision: Analytical Equipment System Number Instrument Software/Versiort: Filename: R-Squared Value: Slope: Y-Intercept: Dates o f Extraction/Analyst: Dates of Analysis/Analyst: Date o f Data Reduction/Analyst: Box: Sample Data FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS E00-1668 Extended Recovety Study Following a 26-Week Capsule Toxicity Stdy with Perfluorooctane Sulfonic Acid Potassium Salt(PFOS; T-6295) in Cynomolgus Monkeys T-6295.22 Monkey Serum ETS-8-4.2 & ETS-8-5.2 versus an extracted rabbit sera curve Amelia062498 MasslynX 3.4 See Attachments See Attachments See Attachments See Attachments 09/07/01 RWW 09/13/01 MMH 09/14/01 MMH 01-042, 01-043 MONKEY SERUM Group Dose Sample U Method Blk WB090601-H20 BUt-l WB090601-H20 Blk-2 Matrix Blk RBS09060I-Scra Blk-I RBS090601-Sera Blk-2 MKS090601-Sera Blk-1 MKS090601-Sera Blk-2 QC - 50 ppb 250 ppb MKS090601-50 ppb-MS MKS090601-50 ppb-MSD MKS090601-250 ppb-MS MKS090601-250 ppb-MSD Group 1 Week 9 I05505M I05523M 105539F 1D5552F Group 1 Week 17 I05505M 105523M I05539F I05552F Group 1 Week 25 I05505M I05523M I05539F 105552F Group 1 Week 33 105505M I05523M I05539F 105552F Group 1 Week 4] 105505M I05523M 105539F I05552F Group t Week 33 I05505M I05539F I05552F ?FOS = Perfluorooctanesulfonate PFOS Cone 0.00 0.00 0.00 0.00 10.2 7.71 72.6 64.8 279 258 44.6 45.8 76.8 44.8 45.8 37.5 48.4 77.3 36.7 20.9 50.8 24.8 33.3 29.1 48.5 47.3 17.6 12.5 31.0 20.0 14.0 11.4 14.7 19.1 Concentration of PFOS ug/mL or % Ree <LOQ (0.00492 ug/mL) <LOO (0.00492 ug/mL) <LOQ (0.00492 ug/mL) <LOO (0.00492 ug/mL) 0.0102 0.00771 129% 113% 110% 101% 17.8 16.7 30.7 22.4 14.52 12.93 22.0 18.6 10.1 7.33 19.5 12.1 8.64 7.56 15.4 9.46 5.86 5.43 1 13.5 7.28 4.30 4.22 9.19 6.70 Mean PFOS ug/mL <LOQ <LOQ 0.00897 121% 105% 17.2 26.6 13.7 20.3 8.70 15.8 8.10 12.4 5.65 10.4 4.26 7.95 RSD Std. Dev. MS/MSD RPD NA NA 0.00177 13% 8% 0.824 5.88 1.13 2.37 1.93 5.27 0.768 4.20 0.303 4.37 0.052 1.76 Dam Entered/By: Date Verified/ By: 09/26/01 LAC 12/6/01 mmh IAL oh| a slo i 3M Environmental Laboratory Page 93 3M Medical Department Study: T-6295.22 FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS E00-1668 Study: FACT-TOX-160, EOO-1668 Product Number(Test Substance): Matrix: Mcthod/Revision'. Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-lntercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date o f Data Reduction/Analyst: Box: Extended Recovery Study Following a 26-Week Capsule Toxicity Stdy with Perfluorooctane Sulfonic Acid Potassium Salt(PFOS; T-6295) in Cynomolgus Monkeys T-6295.22 Monkey Serum ETS-8-4.2 & ETS-8-5.2 versus an extracted rabbit sera curve Amelia062498 Masslynx 3.4 See Below See Attachments See Attachments See Attachments 09/07/01 RWW 09/13/01 MMH 09/14/01 MMH 01-042, 01-043 Sample Data W U P i K b l S t K U Y1 Group Dose Sample # Method BLk Matrix Blk WB090601-H20 Blk-1 RBS09060l-Sera Blk-1 MKS090601-Scia Blk-1 QC - 50 ppb 250 ppb MKS090601-50 ppb-MS MKS090601-250 ppb-MS Group 1 Week 9 Group 1 Week 17 Group 1 Week 25 Group 1 Week 33 Group 1 Week 41 . Group 1 Week S3 PFOS * Perfluorooctanesulfonate I05505M I05539F I05552F I05505M I05539F I05552F I05505M 105539F I05552F 05505M I05539F I05552F I05505M I05539F I05552F 105505M I05539F 10S552F Extraction VoL mL 1 1 1 I 1 1 1 1 1 0.50 0.55 0.50 0.40 0.63 0.58 0.44 0.83 0.73 0.57 0.52 0.41 0.77 0.77 0.63 1.00 0.60 0.46 0.46 0.55 0.65 0.32 0.57 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 PFOS Dilution PFOS Cone. 1 0.00 1 0.00 1 0.00 1 0.00 1 10.22 7.71 1 72.55 1 64.80 I 279.46 1 258.06 200 44.56 200 45.81 200 76.79 200 44.81 200 45.75 200 37.49 200 48.36 200 77.32 200 36.72 200 20.90 200 50.76 200 24.75 200 33.28 200 29.10 200 48.51 200 47.32 200 17.59 200 12.50 200 30.97 200 20.02 200 13.96 200 11.40 200 14.71 200 19.09 Filename A 0 10913049 A01091305Q A010913051 A010913052 A010913053 A 010913054 AI0913055 A010913056 A010913057 A010913058 AOI0913088 A010913089 A010913090 A01091309I A010913081 A 010913082 A010913083 A 0 10913084 A010913077 AOI0913078 A010913079 A 010913080 A 010913070 A010913071 A 010913075 A 010913076 A010913066 A010913067 A010913068 A 010913069 A01091362 A010913063 A010913064 A 0 10913065 of PFOS ug/raL or % Ree <LOQ (0.00492 ug/mL) <LOO (0.00492 ue/mL) <LOQ (0.00492 ug/mL) <LOO (0.00492 ue/mL) 0.0102 0.00771 129% 113% 110% 101% 17.8 16.7 30.7 22.4 14.5 12.9 22.0 18.6 10.1 7.33 19.5 12.1 8.64 7.56 15.4 9 46 5.86 5.43 13.5 7.28 4.30 4.22 9.19 6.70 Mean PFOS ug/mL <LOQ <LOQ 0.00897 121% 105*/. 17.2 26.6 13.7 20.3 8.70 15.8 8.10 12.4 5.65 10.4 4.26 7.95 BSD Std. Dev. MS/MSD RPD NA NA 0.00177 13.0% 8.24% 0.824 5 88 1.13 2.37 1.93 5.27 0.768 4.20 0.303 4.37 0.052 1.76 Date Entered/By: 09/26/01 LAC Date Verified/ By: 12/6/01 nunh 3M Environmental Laboratory Page 94 3M Medical Department Study: T-6295.22 FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS E00-1668 Study: FACT-TOX-160, E00-1668 Product Number(Test Substance): Matrix: Method/Reviston: Analytical Equipment System Number: Instrument Softwaie/Version: Filename: R'Squared Value: Slope: Y-Intcrcept: Oates of Extraction/Analyst: Dates o f Analysis/Analyst: Date o f Data Reduction/Analyst: Box: Sample Data Extended Recovery Study Following a 26-Week Capsule Toxicity Stdy with Perfluorooctane Sulfonic Acid Potassium Salt(PFOS; T-6295) in Cynomolgus Monkeys T-6295.22 Monkey Serum ETS-8-4.2 & ETS-8-5.2 versus an unextracted curve Amelia 062498 Masslynx 3.4 See Attachments See Attachments See Attachments See Attachments 08/31/01 RWW 09/05/01,09/06/01 MMH 09/06/01,09/07/01 MMH 01-042 MONKEY SERUM Group Dose Sam ple# PFOS Cone. ng/mL Concentration of PFOS ug/mL or % Ree Method Blank Matrix Blank MKS083I0I-H2O BUc-1 MKS08310 1-H20 Blk-2 MKS083101-Sera Blk-t MKS083101-Sera Blk-2 0.18 <LOQ (0.0012 ug/mL) 0.23 <LOQ (0.0012 ng/mL) 9.69 0.00775 9.05 0.00724 QC MKS083101-MS-25 ppb 32.6 118% MKS083101-MSD-25 ppb 25.9 84% MK5O831O1-MS-S0O ppb 448 111% MKS08310 l-MSD-500 ppb 449 112% PFOS = Perfluorooctanesulfonate LOQ = 1.0 ng/mL in standard calculates to 1.2 ng/mL in sow n. LOQ (1.0 ng/mL unext standard * 1.25 ext dii factor * 1 dilution factor)/l000 0.0012 ug/mL in serum Mean PFOS ue/mL <LOQ 0.00750 101% 111% Date Entered/By: Date Verified/ By: 09/12/01 LAC 16/6/01 nunh U L oh\ ^ \ oL RSD Std. Dev. MS/MSD RPD NA 0.000362 34% 0% 3M Environmental Laboratory Page 95 3M Medical Department Study: T-6295.22 FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS E00-1668 Study: FACT-TOX-160, EOO-166S Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Softw&rc/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates o f Extraction/Analyst: Dates o f Analysis/Analyst: Date o f Data Reduction/Analyst: Box: Sample Data Extended Recovery Study Following a 26-Week Capsule Toxicity Stdy with Perfluorooclane Sulfonic Acid Potassium Salt(PFOS; T-6295) in Cynomolgus Monkeys T-6295.22 Monkey Serum ETS-8-4.2 & ETS-8-5.2 versus an unextracted curve Amelia 062498 Mas$>lynx 3.4 See Below See Attachments See Attachments See Attachments 08/3 L/01 RWW 09/05/01, 09/06/01 MMU 09/06/01,09/07/01 MMH 01-042 MONKEY SERUM Group Dose Sample # Inkial Voi. mL Extraction Dilution Factor Surrogate Verified Method Blank Matrix Blank MKS083101-H2O Blk-1 MKS083101-H 20 Blk-2 MKS08310!-Scra Blk-1 MKS083101-Sera Blk-2 1 1.25 Confirmed High 1 1.25 Confirmed High 1 1.25 Confirmed High 1 1.25 Confirmed High QC MKS083I01-MS-25 ppb 1 1.25 Confirmed High MKS083101-MSD-25 ppb 1 1.25 Confirmed High MKS083101-MS-500 ppb 1 1.25 2nd analysis OK MKS083101-MSD-500 ppb 1 1.25 Confirmed High PFOS - Perfluorooctanesulfonate LOQ - l .0 ng/mL in standard calculates to 1.2 ng/mL in serum. LOQ (1 .0 ng/mL unext standard * 1.25 ext dii factor * 1 dilution factor)/1000 0.0012 ug/mL in scrum Date Entered/By: 09/12/01 LAC Date Verified/ By: 16/6/01 mmh PFOS Dilution Factor 1 1 1 1 1 l 1 1 PFOS Cone. ng/mL 0.18 0.23 9.69 9.05 32.62 25.87 447.77 448.57 Filename A010906019 A010906020 A 010906021 A010906022 AO10906023 A 010906024 A 010906025 A 0 10906026 Concentration of PFOS ug/mL or % Ree <LOQ (0.0012 ug/mL) <LOO (0.0012 ng/mL) 0.00775 0.00724 118% 84% 111% 112% Mean PFOS ug/mL <LOO 0.00750 101% 111% RSD Std. Dev. MS/MSD RPD NA 0.000362 34% 0% 3M Environmental Laboratory Page 96 3M Medical Department Study: T-6295.22 Study: FACT-TOX-160, E00-1668 Product Nuinber(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-lntercept: Dates o f Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: Box: Sample Data FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Extended Recovery Study Following a 26-Week Capsule Toxicity Stdy with Perfluorooctane Sulfonic Acid Potassium Salt(PFOS; T-6295) in Cynomolgus Monkeys T-6295.22 Monkey Serum ETS-8-4.2 & ETS-8-5.2 versus an unextracted curve Amelia 062498 Masslynx 3.4 See Attachments See Attachments See Attachments See Attachments 10/02/01 RWW 10/04/01 MMH 10/05/01 MMH 01-046 MONKEY SERUM Group Dose Sample # PFOS Cone. ng/mL Concentration of PFOS ug/mL or % Ree Mean PFOS ug/mL Method Blank Matrix Blank WB100201-H20 Blk-1 W B100201-H20 Blk-2 RBS100201-Sera Blk-1 RBS100201-Sera Blk-2 MRS 100201-Sera Blk-3 MKS100201-Sera Blk-4 0.00 <LOQ (0.00313 ug/mL) 0.00 <LOQ (0.00313 ug/mL) <LOQ 0.09 <LOQ (0.00313 ug/mL) 0.13 <LOO (0.00313 ug/mL) <LOQ 7.23 0.00904 7.14 0.00893 0.00898 QC MKS100201-MS-2 ug/mL 8.69 113% MKS 10020 l-MSD-2 ug/gmL 8.34 109% MKS1002C1-MS-10 ug/mL 45.6 115% MKSI00201-M5D-10 ug/mL 46.5 117% 111% 116% PFOS = Perfluorooctanesulfonate LOQ 3 2.50 ng/mL in standard calculates to 3.13 ng/mL in serum. LOQ (2.50 ng/mL unext standard * 1.25 ext dii factor * I dilution factorVlOOO " 0.00313 ug/mL in serum Date Entered/By: Date Verified/ By: 10/11/01 LAC 12/10/01 mmh \ Kp RSD Std. Dev. MS/MSD RPD NA NA 0.0000795 4% 2% 3M Environmental Laboratory Page 97 3M Medical Department Study: T-6295.22 fa CT-TOX-i 60 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS 00-1668 Study: FACT-TOX-160, E00-1668 Product NumbcrfTcst Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst Date o f Data Reduction/Analyst: Box: Sample Data Extended Recovery Study Following a 26-Week Capsule Toxicity Stdy with Perfluorooctane Sulfonic Acid Potassium Salt(PFOS; T-6295) in Cynomolgus Monkeys T-6295.22 Monkey Serum ETS-S-4.2 & ETS-8-5.2 versus an unextracted curve Amelia 062498 Masslynx3.4 See Below See Attachments See Attachments See Attachments 10/02/01 RWW 10/04/01 MMH 10/05/01 MMH 01-046 MONKEY SERUM Group Dose Sample # Initial Voi. mL Extraction Dilution Factor Surrogate Verified PFOS Dilution Factor Method Blank W B10020I-H20 Blk-1 I 1.25 NA 1 WB100201-H20 Blk-2 1 1.25 NA 1 Matrix Blank RBS100201-Sera Blk-1 1 1.25 NA 1 RBS100201-Sera Blk-2 1 1.25 NA 1 MKS100201-Sera Blk-3 1 1.25 NA 1 MKSI00201-Sera Blk-4 1 1.25 NA 1 QC MKSI00201-MS-2 ug/mL I 1.25 Out High 200 MKS100201-MSD-2 ua/eroL I 1.25 NA 200 MKS100201-MS-10 ug/mL 1 1.25 NA 200 MKS100201-MSD-10 ug/mL 1 1.25 NA 200 PFOS = Perfluorooctanesulfonate LOQ 2.50 ng/mL in standard calculates to 3.13 ng/mL in serum. LOQ " (2.50 ng/mL unext standard * 1.25 ext dii factor 1 dilution factor)/1000 * 0.00313 ug/mL in serum PFOS Cone. ng/mL 0.00 0.00 0.09 0.13 7.23 7.14 8.69 8.34 45.57 46.54 Filename A 011004082 A 0 1 1004083 A 011004084 A 0 1 1004085 A 0 1 1004086 A 0 1 1004087 A 011004088 A 011004089 A 011004090 A 011004091 C o n cen tratio n of PFOS ue/mL or % Ree <LOQ (0.00313 ug/mL) <LOO (0.00313 ug/mL) <LOQ (0.00313 ug/mL) <LOO (0.00313 ug/mL) 0.00904 0.00893 113% 109% 115% 117% Mean PFOS ug/mL <LOQ <LOQ 0.00898 m% 116% Date Entered/By: Date Verified/ By: 10/11/01 LAC 12/10/01 mmh ^ RSD Std. Dev. MS/MSD RPD NA NA 0.0000795 4% 2% 3M Environmental Laboratory Page 98 3M Medical Department Study: T-6295.22 Study: FACT-TOX-160, EDO-1668 Product Number(Tcst Substance): Matrix; Method/Revision: Analytical Equipment System Num ber Instrument Softwarc/Version: Filename: R-Squarad Value: Slope: Y-lntercept: Dates of ExtractioiVAnalystDates of Analysis/Analyst: Date of Data Reduction/Analyst: Box: Sample Data F A C T -T O X -160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS E00-1668 Extended Recovery Study Following a 26-Week Capsule Toxicity Stdy with Perfluorooctanc Sulfonic Acid Potassium Saft(PFOS; T-6295) in Cynomolgus Monkeys T-6295.22 Monkey Serum ETS-8-4.2 A ETS-S-5.2 versus an extracted rabbit sera curve Amelia 062498 Masslynx 3.4 See Attachments See Attachments See Attachments See Attachments 10/02/01 RWW 10/04/01 MMH 10/05/01 MMH 01-046 MONKEY SERUM Group Dose Sample# Method Blank Matrix Blank W Bi0020I-H2OBlk-l WB10020I-H2O Blk-2 RBS10020I-Sem Dlk-I R BS100201 -Sera Blk-2 M RS10020I-Sera Blk-3 MKS 100201 -Sera Blk-4 QC MKS 100201-MS-2 ug/mL MKS 10020 l-MSD-2 iw/nmL MKSI00201-MS-10 ug/mL M K S100201-M SD -10ua/m L PFOS = Perfluorooctanesulfonate PFOS Cone. ne/mL 0.00 0.00 0.00 0.00 8.02 7.90 9.96 9.49 59.0 60.3 Concentration of PFOS ug/mL or % Ree <LOQ (0.00492 ug/mL) <LOO (0.00492 uis/mL) <LOQ (0.00492 ug/mL) <LOO (0.00492 ue/mL) 0.00802 0.00790 104% 99% 119% 121% Mean PFOS ug/mL <LOQ <LOO 0.00796 102% 120% RSD Std. Dev. MS/MSD RPD NA NA 0.0000849 5% 2% Date Enteted/By: Date Verified/ By: 10/11/01 LAC 12/10/01 mmh o h [ 0.5 it>a- 3MEnvironmental Laboratory Page 99 3M Medical Department Study: T-6295.22 F A C T -T O X -160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS E00-1668 Study: FACT-TOX-160, EOO-1668 Product Numbci^Tcst Substance): Matrix: Mcthod/Rcviston: Analytical Equipment System Number: Instrument Softwarc/Venuxi; Filename: R-Squarod Value: Slope: Y-Interccpt Dates o f Extraction/Analyjt: Dates ofAnalyiis/Analyst: Date of Data Reduction/Analyst: Box: Sample Data Fufirwfrft Recovery Study Following a 26-Week Capsule Toxicity Stdy with Perfluorooctane Sulfonic Acid Potassium Salt(PFOS; T-6295) in Cynomolgui Monkeys T-6295.22 Monkey Serum ETS-8-4.2 f t ETS-8-5.2 versus an extracted rabbit sera curve Amelia 062498 Masstynx 3.4 See Below Sec Attachments See Attachments See Attachments 10/02/01 RWW 10/04/01 MMH 10/05/01 MMH 01-046 MONKEY SERUM Croup Dose Sample a Method Blank W B100201-H20 Blk-1 W B 10020I-H20 Blk-2 Matrix Blank RBS10020I-Sera Blk-1 RBSI0020I-Sera Blk-2 M KS10020I-Sera Blk-3 M KS10020|-Sera Blk-4 QC MKS100201 -MS-2 ug/mL MKS 10020 l-MSD-2 ua/amL MKS 100201-M S-10 ug/mL MKS10020I-MSD-10 ug/mL PFOS * Periluorooctanesulfcnate Initial Val. 1 l 1 1 1 1 1 1 l 1 Extraction Dilution Factor 1.00 1.00 1.00 1.00 1.00 1.00 1.00 i.00 1.00 1.00 Date Entered/By. Date Verified! B y 1102//110m/01mLmAhC S u rro g a te Verified NA NA NA NA NA NA NA NA NA NA PFOS Dilution Factor 1 l l 1 1 1 200 200 200 200 PFOS Cone. ng/mL 0.00 0.00 0.00 0.00 8.02 7.90 9% 9.49 58.97 60.27 m enarne A 011004082 A 011004083 A 011004084 A 0 1 1004085 A 0 I1004086 A 0 I1004087 A 0 I1004088 A 011004089 A 01)004090 A 011004091 Concentration of PFOS ut/m L or % Ree <LOQ <0.00492 ug/mL) <LOO (0.00492 ue/m U <LOQ (0 00492 ug/mL) <LOQ <0.00492 un/mLl 0.00802 0.00790 104% 99% 119% 121% Mean PFOS uc/mL <LOO <LOO 0.00796 102% 120% RSD Std. Dev. MS/MSD RPD NA NA 0 0000849 5% 2% 3M Environmental Laboratory Page 100 3M Medical Department Study: T-6295.22 FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Study: FACT-TOX-160, EOO-166* Product NiunbcrfTest Substance): Matrix: Method/Revison: Analytical Equipment System Number: Instrument Soflware/Version: Date of Extract!on/Analyst: Dale of Analysis/Analyst: Date of Data Reduction/Analyst: Extended Recovery Study Following a 26-Week Capsule Toxicity Sidy with Periluorooctane Sulfonic Add Potassium SaltfPFOS; T-6295) in Cyoontolfus Monkeys T-6295.22 Monkey Liver ETS-S-60 & ETS-S-7.0 versus an extracted rabbit liver curve Amelia 062498 Filename: Sec Attachments Masslynx 3.4 R-Squared Value: See Attachments 09/03/01 RWW Slope: See Attachments 09/10/01,09/13/01.09/17/01 MMH Y-Interccpt: See Attachments 09/11/01,09/14/01,09/18/0] MMH Box: 01-042 Sample Data MONKEY LIVER Group Sample# PFOS Concentration Mean Dose Calc. Cone, of PFOS PFOS Method BU RBL09050I-H2O Blk-I ne/c neft or % Ree ut/x 0.00 <LOO 10.00629 u/e> RBL090501-H20 Blk-2 0.00 <LOO (0.00629 uo/cl <LOQ Matrix Btk RBL090501-Liver Blk-1 0.00 <LOQ (0.00629 ug/g) RBU m SO l-L iva Blk-2 0.00 <LOO (0.00629 ae/cl <LOO QC RBL090501-300 ppb-MS 322 102% RBL090501-300 ppb-MSD 323 102% 102% MXL090501-300 ppb-105505M-MS MKJ.090501-300 ppM0350SM-MSD 879 854 298% 290% 294% Group 1 105505M 3251 3.25 I05523M 3868 3.87 3.56 I05539F 10675 10.7 I05552F 7788 7.79 9.23 PFOS Periluorooctanesulfoute * Matrix spikes weren't spiked at the appropriate level. Sample will be re-spiked, at the appropriate levels, at a later date. RSD SUL Dev. MS/MSD RPD NA NA 0% 3% 0.436 2.04 Dale Entered/Analyst: 09/1^)1,09/26/01 LAC Dale Verified/Analy: 12/10/01 ninth Uc 3M Environmental Laboratory Page 101 3M Medical Department Study: T-6295.22 FACT-TOX-lO Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Study: FACT-TOX-160. EDO*1668 Product Number(Tetl Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Softwarc/Versioa: Date of Extraction/Analyst: Dale of Analysis/Analyst: Date of Data Reduction/Analyst: Extended Recovery Study Following a 26-Week Capsule Toxicity Stdy with Perfluorooctane Sulfonic Acid Potassium Sall(PFOS; T-6295) in Cynotnolgus Monkeys T-6293.22 Monkey Liver ETS-8-6.0 A ETS-8-7.0 versus an extracted rabbit liver curve Amelia 062498 Filename: See Below Masslynx 3,4 R-Squared V Sec Attachments 09/05/01 RWW Slope: See Attachments 09/10/11,09/13/01.09/17/01 MMH Y-Intercepl. See Attachments 09/11A 1,09/14/01,09/18/01 MMH Box#: 01-042 Sample Data MONKEY LIVER Creup Dose Sample# Surrogate Verified Initial Wt. X Total Masa of Liver Method Bik RBL09O5OI-H2O Olk-1 RBL09050I-H20 Blk-2 I NA 1.000 NA NA 1.000 NA Matrix Blk RBL090501-Liver Blk-I NA 1.000 NA RBL090501-Liver Blk-2 NA 1 000 NA QC RBL090501-300 ppb-MS NA 1.000 NA RBL090501-300 Dob-MSD NA 1.000 NA MKL090501-300 ppM0530SM-MS NA 1.0160 NA MKL090501-300 ppb-K)5305M-MSD NA 1.0160 NA Group 1 IO5505M NA 1.0160 NA IOSS23M NA 1.0650 NA I03539F NA 1.0155 NA I05552F NA 1.0664 NA PFOS = Periluorooctanesulfonalc * Matrix spikes weren't spiked at the appropriate level. Sample will be re-spiked, at the appropriate levels, at a later date. FFOS Com. ng/g 0.00 0.00 0.00 000 321.51 322.78 83.93 83.43 66.07 82.38 21.68 16.61 PFOS DUutioa Factor 1 1 1 1 1 1 50 30 50 50 500 500 PFOS Calc. Cone. nilc 0.00 0.00 0.00 0 00 322 323 879 854 3251 3868 10675 7788 Filename A0IO9I0OI6 A0I0910017 A01091001B A 0 10910019 A0I0910022 AO10910023 A010917016 A 0 10917017 A0I0917018 AOI0917019 A010913020 AO109I3O21 Conccntratiea of PFOS uf/e or 54 Ree <LOQ (0.00629 ug/g) <LOO (0.00629 ueJz) <LOQ (0.00629 ug/g) <LOO (0.00629 ua/jt) 102% 102% 298% 290% 3.25 3.87 10.7 7.79 Meaa PFOS s/ <LOO <LOO 102% 294% 3.56 9.23 USD Std. Dev. MS/MSD RPD NA NA 0% 3% 0.436 2.04 U K B R K IW A M iy K vn ia/ui, i/y/awvi lac Date Verified^Analyst: 12/10/01 mmh 3M Environmental Laboratory Page 102 3M Medical Department Study: T-6295.22 FACT-TOX-160 Covaace# 6329-268 Analytical Report: FACT-TOX-160 LIMS E00-1668 Study: FACT-TOX-160, E0Q-166I Product Numbcr(Test Substance): Matrix: Mcthod/Rcvision: Analytical Equipment System Number: Instrument Software/Versioa: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data RcductkWAnalyst: Sample Data Emended Recovery Study Following a 26-Week Capsule Toxicity Sidy with Perfluorooctane Sulfonic Acid Potassium SaltfPFOS; T-6295) in Cynomolgus Monkeys T-6295.22 Monkey Liver ETS-8-6 0 A ETS-8-7.0 versus an extracted rabbit liver curve Amelia 062498 Filename: See Attaduncnts MaasJynx 3.4 R-Squared Value: See Attachmenu 10/02/01 RWW Slope: See Attachmenu 10/04I MMH 10/05/01 MMH Y-Iniercept: Box: See A iuduncnu 01-046 MONKEY LIVER Group Sample PFOS Concentration Doee C tk . Cone, s f PFOS ae/c ae/e or K Ree Method Blk RBL100201-H2O BUc-3 0.00 <LOQ (0.0126 ug/g) RBL10020I-H20 Blk-4 0.00 <LOO (0.0126 ue/i) Matrix BUt RBLI00201-Liver BUt-1 RBLI00201Liver Blk-2 0.00 <LOQ (0.0126 ug/g) 0.00 <LOO (0.0126 ua/t) QC MKL100201-2 ug/g-105505M-MS 4364 55% MKL10020I-2 ue/e-I05305M-MSD 3554 15% MKL100201-10 ug/g-105505M-MS 9441 60% M KU0020M 0 oa/-I0S505M-MSD 5761 <LOO (0.0126 ue/e) Group 1 I05305M 3237 3.24 PFOS Perflucroocianesulfonate 1:500 dilution was u n dilute, these extracts were rc-diluledat 1JO tad analyzed 10/ 16/01. LAC 11/28/01 Mean PFOS <LOO <LOO 35% 60% RSD Std. Dev. MS/MSD RPD NA NA 112% NA Date Emeted/Analyst: 10/1 M1 LAC Date Verified/Analyst: 12/MMU mmh Study: FACT-TOX-160. EDO-166* Product NumberfTat Substance): Matrix: Mcthod/Rcvision: Analytical Equipment System Number Instrument Software/Verstai: Date of Exnaoion/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst Sample Data Extended Recovery Study Following a 26-Week Capsule Toxicity Sidy with Perfluomoctane Sulfonic Add Potassium SaltfPFOS; T-6295) in Cynomolgus Monkeys T-6295.22 Monkey Liver ETS-8-6.0A ETS-8-7.0 versus an extracted rabbit liver curve Amelia 062498 Filename: See Attachments Masdyax 3 4 A-Squared Value: See Attachments 10/02/01 RWW Slope: See Attachmenu 10/16/01 MMH Y-Interccpt: See Attachments 10/16/01 MMH Box: 01-046 MONKEY LIVER Group Doee Method Blk Matrix Blk Sample RBL100201-H20 Blk-3 RBL100201-H2O BHt-4 RfiLI00201-Liver Blk-I PFOS Cale. Coae. ae/8 0.00 0.00 1.33 Couceatratieu of PFOS u c /c e r % Ree <LOQ (0 0126 ug/g) <LOO (0 0126 u/l <LOQ (0.0126 ug/g) Mean PFOS tit <100 RSD Std. Der. MS/MSD RPD NA RBL100201-Liver Blk-2 02$ <LOO (0.0126 ua/e) <LOO NA QC MKL10020I-2 ug/g-I0550JM-MS 3804 29% MKL100201-2 UC/C-10550SM-MSD 2995 -11% 9% 438% MKI00201-10 ttg/g-1055O5M-MS MKL100201-10 UC/R-1O5505M-MSD 9719 7466 63% 41% 52% 42% Groan 1 I055U5M 3215 3.2) PFOS * Periluorooctaaesulienatc N O TE: D ata w ere BOt w ithin c rite ria , a ll d iluted sam ple* w ere rediluted 1:50 from o riainaJ extract and maaiyzed on 10/24/01. LAC 11/28/01 Dale Entered/Analyst: Date Verified/Analyst: KY29/C1 LAC I2/I0/C1 mmh 3M Environmental Laboratory Page 103 3M Medical Department Study: T-6295.22 J FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 r LIMS E00-1668 Study: FACT-TOX-160. EOO-1663 Product NumberfTest Substance): Matrix: Method/Rcvision: Analytical Equipment System Number: InstnuneM Software/Vcision; Date of Extraction/Analyst: Date of Analysis/Analyst: Date oTDau Reduction/Analyst: Sample Data Extended Recovery Study Following a 26-Week Capsule Toxicity Stdy with Periluoroocurte Sulfonic Acid Potassium Salt(PFOS; T-6295) in Cynomolgua Monkeys T-6295.22 Monkey Liver ETS-S-6.0 A ET5-8-7.0 versus an cxuacted rabbit liver curve Amelia 062498 Masslynx 3.4 I0/D2AH RWW Filename: R-Squared Value: Slope: See Attachments See Attachments See Attachments 10/24/01 MMH 10/26/01 MMH Y-Imercept: Box#: See Attachments 01*046 MONKEY LIVER Croup Dom Sample# Method Blk RBLI00201-H2O Blk-3 RBL10O20l-H2OBlk-4 Matrix Blk RBLI0020I-Liver BUt-l RBL100201-Liver 8lk-2 oc MKL10020I-2 ug/g-10S505M-MS MKL100201-2 UK/C-I0S505M-MSD MKLIOO201-10 ug/g-I05505M-MS MKLJOO201-10 UC/K-I05505M-MSD GreuD 1 I05505M PFOS a Perfluorooctanesulfonate pros Calc. Cone, mt/t 0.00 0.00 0.00 0.00 14943 12530 38873 33522 11282 Concentration of PFOS n/c or % Ree <LOQ (0.0126 ug/g) <LOQ(0 0126u*/e) <LOQ (0.0126 ug/g) <LOO (0.0126 us/tl 178% 61% 268% 216% 11.28 Mean pros art <LOO <LOO 119% 242% RSO Std-Dev. MS/MSD RPD NA NA 98% 21% Dale Entered/Analyst: Date Verified/Analyst: 11/06AH LAC 12/10/01 mmb Study: FACT-TOX-160, EOO-1668 Product NumberfTest Substance): Matrix: Mcthod/Revisioo: Analytical Equipment System Number Instrument Software/Vcnion: Date ot Extraction/Analyst Dale of Analysis/Analyst: Dale of Data Reductkxi/Analyst: Sample Data Extended Recovery Study Following a 26-Week Capsule Toxicity Stdy with Ferfluorooctaae Sulfonic Add Potassium Salt(PFOS; T-6295) in Cyaomolgus Monkeys T-6295.22 Monkey Liver ETS-8-6.0 A ETS4-7.0 versus an extracted rabbit liver curve MadelincO41096 Filename: See Auackmenta Masslynx 3.4 R-Squared Value: See Attachmenta 11/07/01 RWW Slope: See Attachments 11/15X11, 11/161)1 MMH Y-Intcrccpt: See Attachments 11/16/01, 11/19/01 MMH Box#: 01-046 MONKEY LIVER Group Dw Method Blk Matrix Blk QC Group 1 PFOS- Sample# RBL11070I-H2O Blk-1 RBL11070I-H2O Blk-2 RBL11070I-H20 Blk-3 RBL11070I-H2O Blk-4 RBL110701-Liver Blk-1 RBL110701-Liver Blk-2 RBL110701-Liver Blk-3 RBLI 10701-Liver Blk-4 MKL110701-2 ug/g-I0S505M-MS MKLI10701-2 ue/R-103S05M-MSD MKLI10701-10 u*/|-W5SOJM-MS MKL110701-10 UC/R-I05505M-MSD I05505M PFOS Calc. Cone. 00000000........800000000/000000001 6328.44 6134.39 11836.78 10657.49 4241.25 Concentration ef pros uc/f o r % Ree <LOQ (0.00629 ug/g) <LOQ (0.00629 ng/g) <LOQ (0.00629 ug/g) <LOO <0.00629 ue/e> <LOQ (0.00629 ug/g) <LOQ (0,00629 ug/g) <L0Q (0.00629 ug/g) 100<LOO (0.00629 uc/t) % 90% 73% 61% 4.24 Mean pros OE/f <LOO <LOO 95% 6756 RSD Std-Dev. MS/MSD RPD 0 NA 0 NA 10% 17% Date Entered/Analyst: Onte Verified/Analyst: 11/26/CI LAC I2/10A1 mmh 3MEnvironmental Laboratory Page 104 3M Medical Department Study: T-6295.22 FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS E00-1668 Study: FACT-TOX-160, E00-1668 Product Number(Test Subruoce): Matrix: Method/Revtsion: Analytical Equipment System Number: Instrument Software/Version: Date of Extraction/Analyst. Date of Analysis/Analyst: Date of Data Reduction/Analyst: Extended Recovery Study Following a 26*Week Capsule Toxicity Sidy with Perilucrooctane Sulfonic Acid Potassium Sait(PFOS; T-6293) in Cynotnolgus Monkeys T-6295 22 Monkey Liver ETS-8-5.0 A ETS-t-7.0 versus an extracted rabbit liver curve Amelia 062498 Filename: See Below Masslynx 3.4 R-Squarcd Value: See Attachments 10/02/01 RWW Slope: See Attachments 10/04/01 MMH 10/03/01 MMH Y-Intercept: Box#: See Attachments 01-046 Sample Data MONKEY LIVER Croup Dow Method Blk Sample# RBL100201-H2O Bflt-3 RBL100201-H20 Bflt-4 Surrogale Verified NA NA labial WL f 1.0000 1.0000 Total Mass of Liver 8 NA NA Matrix Blk RBL10020I-Uver Blk-I RBLI0020I-Uver BUc-2 QC MKL10020I-2 ag/g-I03S05M-MS MKL100201-2 uc/e-l05503M-MSD MKU0Q2Q1-1O ug/g-I05305M-MS MKL10020I -10 u*/*-l05505M-MSD Grouo 1 105305M PFOS PerilucrooclanesuDonate NA NA NA NA NA NA NA 1.0000 1.0000 1.0232 1.0232 1.0232 1.0232 1.0232 NA NA NA NA NA NA NA * 1:500 dilution was toodihue, these extracts were re-diluted at 1:30 and analyzed 10/16/01. LAC 11/28/01 PFOS Cone. "tit 0.00 0.00 0.00 0.00 89.30 72.72 19.32 11.79 66.24 PFOS Dilution Factor 1 1 30 50 500 500 50 PFOS C alc Cone. 8f t 0.00 0.00 0.00 0.00 4364 3554 441 5761 3237 Filename AO11004030 AO11004031 AO11004032 AO11004033 A011004038 A011004039 A011004040 A011004041 AOI1004037 Concentration of PFOS u c /e o rH Ree <LOQ 10.0126 uj/g) <LOO (0.0126 uc/tl <LOQ <0.0126 ug/g) <LOO (0.0126 ut/e) 55% 15% 60% < 1 0 0 (0 0126 ua/x) 3.24 Mean PFOS "tit <LOO <LOQ 33% 60% RSD Sid. Dev. MS/MSD RPD NA NA 112% NA Date EateteWAnalyst 10/11^31 LAC Date Verified/Analyst 12/10/0) mmh Study: FACT-TOX-160, E00-I66S Product Number(Tesi Substance): Matrix: Meihod/Rcviiion: Analytical Equipment System Number Instrurnem Software/Version: Date of Extraction/Analyst: Dale of Analysis/Analyst: Date of Dau Redaction/Analyst: Sample Data Extended Recovery Study Following a 26-Week Capsule Toxicity Sidy with Peifluorooctane Sulfonic Acid Potassium Salt(PFOS; T-6295) in Cynotnolgus Monkeys T-6295.22 Monkey Liver ETS-8-6.0 A ET5-8-7.0 versus aa extracted rabbil liver curve Amelia 062498 Filename: See Below Masslynx 3.4 1002/01 RWW 10/16A)I MMH 10/16/01 MMH R-Squared Value: Slope: Y-lniercept: Box#: See Attachments See Attachments See Attachments 01-046 MONKEY LIVER Group Sample# Surrogale Initial WL Total Maas PFOS Dam Method Blk Matrix Blk QC RBL100201-H20 Blk-3 RBM00201-H20 Blk-4 RBL10G201-Lrver Blk-1 RBL100201-Liver Blk-2 MKL100201-2 Ug/g-l03505M-MS MKLI0020I-2 IK/X-I05505M-MSD MKL100201-10 *g/g-IO5505M-MS MKL100201-10 ue/e-K)530$M-Mfin Vertfled NA NA NA NA NA NA NA NA 8 1.0000 1 0000 1.0000 1.0000 1.0232 1.0232 1.0232 10232 af Liver c NA NA NA NA NA NA NA NA Couc uc/t 0.00 000 1.33 025 77.84 61.29 191.88 132.79 Groua 1 I05505M NA 1.0232 NA 65.79 PFOS PeriluonKCUDCsuUbnate - NOTE: D au were am wia criteria, all diluted samples were redUuied 1 3 ) ftwn original extract and analyzed oo 10/24I. LAC 11/28/UI PFOS Dilution Factor 1 1 1 1 50 50 50 50 50 PFOS Cale Couc ng/g 0.00 0.00 1.33 0.25 3804 2993 9719 7466 3215 Date Entcred/Andyst: 10/29/01 LAC Date Verified/Analyst 12/10/01 math filename A11016030 A01101603I AOI1016032 AOI1016033 AOI1016031 AOI 1016039 AOI1016040 A0U016O41 A011016037 Concentration f PFOS nc/c or % Ree <LOQ (0.0126 ug/g) <LOO (00126 ue/e) <LOQ (0.0126 ug/g) <LOO (00126 ue/a) 29% -11% 63% 41% 3.21 Mean PFOS 8/1 <LOO <LOO 9% 52% RSD SULDcv. MS/MSD RPD NA NA 438% 42% 3M Environmental Laboratory Page 105 3M Medical Department Study: T-6295.22 FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Study: FACT-TOX-160. EOO-166* Product NumberiTcst SubiUncc): Matrix: Method/Rcvision: Analytical Equipment System Number: Instrument Softwaic/Veraion: Date of Extraflion/Analytt: Date of AnalysisAnalyst: Dale of Data Reduction/Analyst: Sample Data Extended Recovery Study Following a 26-Week Capsule Toxicity Sidy with Perfluorooctane Sulfonic Acid Potacsiuni Salt(PFOS; T-6295) in Cynomolgus Monkeys T-6295.22 Monkey Liver ETS-8-6.0 ft ETS-8-7.0 versus an extracted rabbit liver curve Amelia 062498 Masslynx 3.4 Filename: See Below R-Squared Value: See Attachments 10/02/01 RWW Slope: See Attachments 10/24/01 MMH Y-fnletcept; See Attachments 10/26/01 MMH Box: 01-046 MONKEY LIVER Croup Dose Sample# Surrogate Verified Initial W t S Tuta! Mass of Liver PFOS Cane Method Blk Matrix Bik QC RBLI002OI-H2O Blk-3 RBLI00201-H20 Blk-4 RBL 100201-Liver Blk-1 RBL 100201-Uver Blk-2 MKX1O0201-2 ug/g-!05505M-MS MKLI0020I-2 ux/e-I05505M-MSD MKL10020MO ug/g-105505M-MS MKL1002OM0 ue/*-I05505M-MSD Out High Out Rich NA NA Out High Out Hieh a n High Out High 1.0000 1.0000 1.0000 1.0000 1.0232 1.0232 1.0232 1.0232 _______ NA NA NA NA NA NA NA NA -, 0.00 000 0.00 0.00 305.83 256.41 795.50 686.00 Groua 1 I05505M Out Hink 1.0232 NA 230.87 PFOS - PeriluorooctanesuHonate NOTE: Dau Mere not consistent with previous analyses. Samples were re-extracted, diluted 1:50, and analyzed on 11/16/01. LAC 11/28/01 PFOS Dilution Factor I 1 1 1 50 50 50 50 50 PFOS Calc. Cone. -ft 0.00 0.00 0.00 0.00 14945 12530 38173 33522 11282 F ile n ame AO11024030 A0I1024O3I A011024032 AOl1024033 A011024038 AOl1024039 AOl 1024040 A011024041 A011024037 Concentration of PFOS ue/e or 96 Ree <LOQ (0.0126 ug/g) <LOO <0.0126 ue/e) <LOQ (0.0126 ug/g) <LOO (0.0126 ue/ft 178% 61% 268% 216% 11.3 Mean pros <LOO <LOO 119% 242% BSD Std. Dev. MS/MSD RPO NA NA 98% 21% Date Eatcred'Analytt: 11/06/01 LAC Date Verified/Analyst; 12/10/01 nunk Study: FACT-TOX-160, EOO-166 Product NumbetfTed Substance): Matrix: Method/Rcvision: Analytical Equipment System Number Instrument Softwan/Verexm: Date of Extraction'Analyst: Date of Analysis/Analyst: Dale o f Data Reduction/Analyst Sample Data Extended Recovery Smdy Following a 26-Week Capsule Toxjdty Stdy with Perilitorooclane Sulfonic Add Potassium SaltfPFOS; T-6295) in Cynomolgus Monkeys T-6295.32 Monkey Liver ETS-8-6 0 ft ETS-8-7.0 versus extracted rabbit liver curve MadeUne041098 Filename: See Below Masslynx 3.4 R-Squared Value: Sec Attackmenu 11/07/01 RWW 11/15/01, 11/16/DI MMH Slope: Y-lnlerccpt: See Attachments See Attachments 11/16/01. 11/19/01 MMH Box: 01-046 MONKEY LIVER Group Done Sample Method Blk Matrix Blk QC Group 1 RBL110701-H20 Blk-1 RBL110701-H20 Blk-2 RBLI10701-H2O Blk-3 RBL11U701-H2O Blk-4 RBU10701-W Blk-1 RBLI 10701-Liver Blk-2 RBL110701-Liver BBt-3 RBL1I070I-Uver Blk-4 MKLI10701-2 ng/g-I03505M-MS MKLI 10701-2 us/e-IOSSOSM-MSD MKLI 10701-10 up/-I05505M-MS MKLItOTOl-IO UK/K-I09903M-M5D 105505M Surrogate Verified NS NS NA NA NS NS NA NA NA NA NA NA NA Initial Wl B 1.0000 1.0000 1.0000 1.0000 0.9425 0.9425 09425 0.9425 1.0023 1.0023 1.0023 1.0023 1.0023 Total Mass of liver c NA NA NA NA NA NA NA NA NA NA NA NA NA PFOS Cm c. I/I 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 126.86 122.97 237.28 213.64 85.02 NS Not Spiked with Surrogate NOTE: Average recovery of the 10 ug/g matrix spikes was not within the / 30% recovery as listed in ihe protocol. A deviation will be written. Date Entered/AnaJyst: 11/26/01 LAC Date VeriAed/Anaiyst: 12/1UAM moth PFOS Dilation Factor 1 ! 1 1 I 1 1 1 SO 50 50 50 50 pros C ak.C oM . ug/g 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 6328 6134 11837 10657 4241 Filename M0] 1116031 MOI 1116032 MOI II 16033 MOU 116034 M0111I6038 MOI 1116039 MOI 1116040 MO11116041 MOI 1116046 MOI II 16047 MOI 1116048 MOI 1116049 MOI 1116045 Concentration ef PFOS u i/c or % Ree <LOQ (0.00629 ug/g) <LOQ (0.00629 ug/g) <LOQ (0.00629 ng/|) <LOO (0.00629 UR/s) <LOQ (0.00629 ug/g) <LOQ <0.00629 ug/g) <LOQ (0.00629 ug/g) <L00 (0.00629 UR/8) 100% 90% 73% 61% 4.24 Mean PFOS ut/c <LOO <LOO 95% 67% RSD Std. Dev. MS/MSD RPD NA NA 10% 17% 3M Environmental Laboratory Page 106 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: FACT TOX-160 LIMS E00-1668 Appendix F: Example Calculations Formula Used for Sera Analyses in Study FACT TOX-160 AR (ng/mL) x DF x FV (mL) x 1.0 pg = R eported Concentration (pg/mL) EV(m L) 1000 ng Calculation Used for Group 1, Week 17, Animal ID 105505M 45.75 ng/mL x 200 x 1.0 mL x 1.0 pg = 14.5 pg/mL 0.63 mL 1000 ng AR-- Analytical result from MassLynx summary DF-- Dilution factor FV--Final extract volume (1.0 mL unless otherwise noted) EV--Volume of sera extracted Formula Used for Liver Analyses In Study FACT TOX-160 AR (ng/g) x d curve(1) x DF x 1.0 pg = Reported Concentration (pg/g) d sample 1000 ng (1) d curve is assumed to be: 1 g liver 5 m L H 20 Calculation Used for Groupl, Week 53, Animal ID 105505M 66.07ng/g x 1 g /5 m L x 50 x 1 .0 pg = 3 .2 5 pg/g 1.0160 g/5m L 1000 ng AR-- Analytical result from MassLynx summary d curve--Density of the liver standard curve, assumed to be lg liver/ 5 ml water d sample--Density of the liver sample (g sample/ 5 mL H20 ) DF-- Dilution factor 3M Environmental Laboratory 3M Environmental Laboratory Page 23 Page 107 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Analytical Report: FACT TOX-160 LIMS EOO-1668 Appendix G: Interim Certificate(s) of Analysis 3M Environmental Laboratorv 3M Environmental Laboratory Paae 24 Page 108 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 ---------------------------LIMS E00-1668 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 www.centrelab.com Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A 3M Product: PFOS,Lot217 Reference#: SD-018 Purity: 86.9% Test Name Specifications Purity1 BtartCt^yofOriginal --l . Initial Oats Result 86.9% Appearance Identification NMR M etals (ICP/MS) 1. C alcium 2. M agnesium 3. Sodium 4. Potassium2 5. N ickel 6. fron 7. Manganese Total % Impurity (NM R) Total % Impurity (L C /M S) Total % Impurity (G C /M S) Related Compounds POAA Residual Solvents (TGA) Purity by D SC Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4 . Nitrate 5. Nitrite 6. Phosphate 7. Sulfate4 Organic A c id s5 (IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elem ental Analysis: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine White Crystalline Powder 1. Theoretical V alue = 17.8% 2. Theoretical V alue = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% Conforms P o sitiv e 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 wt/wt.% 6. 0.005 wt./wt.% 7. <0.001 wt/wt.% 1.91 w t/w t.% 8.41 wt/wt.% N one Detected 0.33 wt./wt.% N one Detected N ot Applicable1 1. < 0.015 wt./wt.% 2. 0.59 wt/wt.% 3. <0.040 wt/wt.% 4. <0.009 wt/wt.% 5. <0.006 wt/wt.% 6. <0.007 wt/wt.% 7. 8.76 wt/wt.% 1. <0.1 w t/w t.% 2. <0.1 w t/w t.% 3. 0.10 wt/wt.% 4. 0.28 wt/wt.% 1. 12.48 w t/w t.% 2. 0.244 wt/wt.% 3. 1.74 w t/wt.% 4. 8.84 w t/wt.% 5. 54.1 w t/w t.% C O A 023-018A 3M Environmental Laboratory Page t of 3 Page 109 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 ------------------------- fcIMS EOO-1668 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 www.centrelab.com Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A Date of Last Analysis: 08/31/00 Expiration Date: 08/31/06 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/06 'Purity = 100% - (sum of metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.905%+organic acid impurities, 0.38%+POAA, 0.33%) Total impurity from all tests = 13.07% Purity = 100% - 13.07% = 86.9% 2Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials of low purity. No endotheim was observed for this sample. 4Sulfur in the sample appears to be converted to SO4 and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity. 5TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonafluoropentanoic acid Pentafluoropropanoic acid `Theoretical value calculations based on the empirical formula, CsFnSCb'K* (MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). CO A 023-018A 3M Environmental Laboratory Page 2 o f3 Page 110 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 ------------------------- LIMS E00-1668 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 www.centrelab.com Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A LC/MS Purity Profile: Im parity C4 C5 C6 Cl Total wt/wt. % 1.22 1.33 4.72 1.14 8.41 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average result from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average result from the C6 and C8 standard curves. Prepared By: Scientist, Centre Analytical Laboratories iLReviewed By: m Shn Flaherty Date Laboratory Manager, Centre Analytical Laboratories CO A 023-018A 3M Environmental Laboratory Page 3 of 3 Page 111 3M Medical Department Study: T-6295.22 3M Environmental Laboratory Note to File Project or Study Number: FACT-TCR-001 Associated Study Number: LIMS # EOO-1682 Analytical Report: FACT-TOX-160 ------------------------- LIMS EOO 1668 3M Environmental Laboratory Form ETS-4-15.0 - Exact Copy of Original _ IP j Initial Data Page 112 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Appendix H: Report Signature Page Analytical Report: FACT-TOX-160 LIMS EOO-1668 Analytical Report: FACT TOX-160 LIMS EOO-1668 Andrew Seacat, Ph.D., Study Director 1% 3 ^-Oate/ 7. John Butenhoff, Ph.D., Sponsor Representative J , ZOOZDate A ________ Lisa Clemen, Principal Analytical Investigator O sloill Date William Reagen, Ph.D., Analytical Laboratory Manager / //O Date 3M Environmental Laboratorv 3M Environmental Laboratory Paae 25 Page 113