Document 6E0OvwZ6QNGq3dG0XgEMakO4

3M M edical 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 Title 36> Extended Recovery Study Following a 26-W eek Capsule Toxicity Study (FACT-TO X-030) with Perfluorooctane Sulfonate Acid Potassium Salt (PFOS; T-6295.22) in Cynomolgus Monkeys IIO 3 A nalytical Laboratory Report Title Determination of the Presence and Concentration of PFOS in Serum and Liver Samples of Cynomolgus Monkeys Data Requirem ent Not Applicable A u th o r 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 TO X-160 3M LIMS No. E00-1668 Total Num ber o f Pages 113 CSS fz= O'~opm3 ro v> --r n S c~>m -X o O \0 rv> on 3M Environmental Laboratory 3M Environmental Laboratory 000055 -r. Page 1 Page 1 3M M edical 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 E00-1668 This page has been reserved for specific country requirements. 3M Environmental Laboratory 3M Environmental Laboratory 0000S6 Page 2 Page 2 3M M edical 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 TO X-160 LIMS E00-1668 Analytical Laboratory Report Title: Determination of the Presence and Concentration of PFO S in Serum and Liver Samples of Cynomolgus Monkeys Study Identification Numbers: T-6295.22, FACT TO X-160, LIM S-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 sam e test system. These studies include in-life studies Covance 6329-223 and Covance 6329-268 and analytical studies FACT-TO X-030 and FACT-TO X-160. The purity and stability of the reference standards are not included in this report, they are not known at this time. TYl. W Andrew Seacat, Ph.D., Study D irector ________ 3-- 2~&P~2--- D a te / John Butenhoff, Ph.D., Sponsor Representative f/U tjj, 2 t Date Z-- j(X * A .......... Lisa Clemen, Principal A nalytical Investigator OsllbZL Date W illiam Reagen, Ph.D., A nalytical Laboratory M anager O s -A / Date 3 M Environmental Lahnmtorv 3MEnvironmental Laboratory 000057 Paae 3 Page 3 3M M edical 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 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 TO X -160, LIM S-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 r/i/0 2 . Date 3M Environmental Laboratory 3MEnvironmental Laboratory 000058 Paae4 Page 4 3M M edical 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 EOO-1668 GLP Compliance Statement........................................................................................... 3 GLP Study - Quality Assurance Statem ent....................................................................................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 Sum m aries............................... 11 Preparatory Methods.............................................................................................................. 11 Analytical M ethods................................................................................................................ 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 R e fe re n c e s ................................... 17 Appendix A: Control Matrices.............................................................................................................18 Appendix B: Protocol, Amendments, and Deviation(s)................................................................ 19 Appendix C: Extraction and Analytical Methods............................................................................. 4 0 ETS-8-4.2, "Extraction of Fluorochemical Compounds from Serum for Analysis Using HPLC-Eiectrospray/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 0000S9 Page 5 Page 5 3M M edical 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 EOO-1668 Appendix F: Example Calculations................................................................ .................................. 107 Appendix G: Interim Certificate(s) of Analysis............................. .................................................108 Appendix H: Report Signature P a g e ................................................................................................113 List of Tables Table 1. Study Tim eline.....................................................................................................................8 Table 2. Cynomolgus Monkey Specimen Receipt for Study (# 6 32 9 -2 68 )...............................9 Table 3. Characterization of the Analytical Reference Substance in Study FACT-TO X-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 FA C T-TO X -160....................................... 18 Table 9. Data Summary for PFOS in Serum FACT-TO X-160 - pg/m L.................................... 91 Table 10. Data Summary for PFOS in Liver FACT-TO X-160 - pg/g.........................................91 3M Environmental Laboratory 3M Environmental Laboratory ooooeo ` Page 6 Page 6 3M M edical Department Study: T-6295.22 A nalytical Report: FACT-TOX-160 LIMS E00-1668 3M Medical Department Study: T-6295.22 Analytical Report: FACT TOX-160 _________________________ __________________ ___________________ L1MS 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 Liver Analyses 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 C FR 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 CFR Part 792 requirements. 3M Environmental Lahoratorv 3MEnvironmental Laboratory 000061 Pacte 7 Page 7 3M M edical 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: FACT TO X-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-W eek Capsule Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFO S; T 6295.22) in Cynomolgus Monkeys." The animals from study# 6329-268 were previously assigned to a completed in-life Covance study# 6329-223, "26-W eek Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295.7) in Cynomolgus Monkeys" and a completed analytical study# FACT-TO X-030, "Analytical Laboratory Report from the 26-W eek Capsule Toxicity Study with Perfluorooctanesulfonic Acid Potassium Salt (T-6295.7) in Cynomolgus Monkeys on the Determination of the Presence and Concentration of Perfluorooctanesulfonate (PFO S) 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 FACTTO X-160 (EOO-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 Medical Study: T-6295.7 52 Weeks Extended Recovery In-Life Study: Covance 6329-268 Analytical Study: FACT-TOX-160 3M Medical Study: T-6295.22 3M Environmental Laboratory 3M Environmental Laboratory 000062 Pane 8 Page 8 3M M edical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Analytical Report: FA C TTO X-160 LIMS EOO-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 Week 17 Week 25 Week 33 Week 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 Heart/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 I05552 Control matrices used in sera and liver analyses performed during FACT-TO X-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 Laboratory 3M Environmental Laboratory 000063 Paae9 Page 9 3M M edical 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 EOO-1668 Chemical Characterization of the Reference Substance Potassium perfluorooctanesulfonate (KPFOS) CAS Number: 2795-39-3 Chemical Formula: CeFtySOs'HC 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 Pate Storage Conditions Chemical Lot Number Physical Description 08/31/06 Frozen -20C +A10C 217 White Powder Purity 86.9%* N D -N o t determined NA - Not avaSable 'S e e Certificate o f Analysis from Centro Analytical Laboratories in AppendixG. SynQuest ND Frozen -20C W-10C Q-75-91 White Powder NA 3M Environmental Laboratory 3M Environmental Laboratory 00006^ Pane 10 Page 10 3M M edical Departm ent Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: FACT TO X-160 LIMS E00-1668 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-ferf-butyl ether (MtBE). Sample extracts were analyzed using high-performance liquid chromatography-electrospray/tandem mass spectrometry (HPLC-ESM SM S) 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-ferf-butyl ether (MtBE). Sample extracts were analyzed using high-performance liquid chromatography-electrospray/tandem mass spectrometry (HPLC-ESM SM S) in the multiple reaction mode versus an extracted rabbit liver curve. PFO S 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 Preparatory Methods E TS-8-4.2, "Extraction of Fiuorochemical Compounds from Serum for Analysis Using HPLCElectrospray/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 methylferf-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 Lahnratorv 3M Environmental Laboratory 0000&5 Pans 11 Page 11 3M M edical Departm ent 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 TH PFO S 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 E TS-8-5.2, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicais in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" E TS-8-7.0, "Analysis of Potassium Perfluorooctane-sulfonate or other Fluorochemicais 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/M S/M S. For example, molecular ion 499, selected as the primary ion for PFOS (CsF^SCV) analysis, was fragmented to produce ion 99 (F S 0 3-). 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 C hrom atograph: Hewlett-Packard Series 1100 Liquid Chromatograph system Analytical column: Keystone BetasilTM C i8 2x50 mm (5 pm) Column temperature: 30C Mobile phase components: Component A: 2mM ammonium acetate Component B: methanol Flow rate: 300 pL/min Injection volume: 1 0 |j L Solvent Gradient: 9.0 minutes tim e (minutes) %B 0.0 10% 1.0 10% 5.5 95% 7.5 95% 8.0 10% 3M Environmental Laboratorv 3M Environmental Laboratory 000066 Paae 12 Page 12 3M M edical 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 M ass S pectrom eterM icrom ass API/Mass Spectrometer Quattro IITM Triple Quadrupole system Software: Mass LynxTM 3.4 Cone Voltage: 3 0 -7 0 V Collision Gas Energy: 2 0 -5 0 eV Mode: Electrospray Negative Source Block Temperature: 150C 10C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (M RM ) Table 4. Target Ions M onitored 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 Deviations/Amendm 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 (DQ O s) 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 (LO Q ): The LOQ is 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% of the expected concentration. A cceptable Precision: Quality control samples are required to meet 25% precision for analyses, provided they are quantitated within the selected calibration range. A cceptable Spike R ecoveries: 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 of 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 Laboratoiv 3M Environmental Laboratory 000067 Paae 13 Page 13 3M M edical Departm ent Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 3M Medical Department Study: T-6295.22 Analytical Report: FACT TO X-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-TO X-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). Lim its 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-- Liver 4.92 ng/mL 12.6 ng/g B lanks: 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% . M atrix 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 000068 Paae 14 Page 14 3M M edical D epartm ent Study: T-6295.22 A nalytical 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 agreem ent 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. Ail 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 09/17/01 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 Redilution from original sample 11/07/01 11/15/01 Type Rabbit Monkey Monkey 2ug/g Monkey 10ug/g Monkey 2ug/g Monkey 10ug/g Monkey 2ug/g Monkey 10 ug/g Monkey 2 ug/g Monkey 10 ug/g % Recovery 102% 102% 298% 290% 55% 15% 60% Too Dilute 30% -11% 65% 43% 178% 61% 268% 216% 100% 90% 73% 61% Average 102% 294% 35% NA 9% 54% 119% 242% 95% 67% 3M Environmental Laboratory 3M Environmental Laboratory 000069 Paae 15 Page 15 3M M edical 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 (THPFO S) was added to all samples and standards. THPFO S 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 reextracts met surrogate criteria requirements. Statem ent o f 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 # 2 1 7 ) 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 fem ale livers. Detailed sample data tables are presented in Appendices D and E. 3M Environmental Laboratory 3M Environmental Laboratory 000070 Page 16 Page 16 3M M edical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: FACT TO X-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-TO X-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 (TO X-030). References None 3M Environmental Laboratory 3M Environmental Laboratory 000071 Paae 17 Page 17 3M M edical Departm ent Study: T-6295.22 3M Medical Department Study: T-6295.22 A nalytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: FACT TOX-160 LIMS E00-1668 Appendix A: Controi Matrices Table 8. Characterization o f 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 +/- 10QC 111022515 Monkey Serum Sigma-Aldrich 09/26/05 Frozen -20C +/- 10C 99H8400 Rabbit Serum Covance Laboratory NA Frozen -20C +/- 10C F04053 Rabbit Liver fiM Fnvtrnnmantal Lahnratmv 3M Environmental Laboratory 000072 Pans 1ft Page 18 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Analytical Report: FACTTOX-160 LIMS E00-1668 Appendix B: Protocol, Amendments, and Deviations 3M Environmental Laboratory 3M Environmental Laboratory 000073 Paae 19 Page 19 3M M edical Department Study: T-6295.22 3M Environmental Technology and Services PO Box 33331 St. Paul, MN 55133-3331 612 778 6442 A nalytical Report: FACT-TOX-160 LIMS EOO-1668 Protocol UFACT-TOX-160 3M Study Title E xtended R ecovery S tudy Follow ing a 2 6 -W e e k C apsule Toxicity S tudy (F A C T -T O X -0 3 0 ) w ith P erfluo ro octane Sulfonic Acid Potassium S a lt (P F O S ; T -6 2 9 5 ) in C ynom olgus M onkeys A N A LY TIC A L PH ASE PRO TO CO L A u th o r Lisa C lem en D a te : August 27, 2001 Perform ing Laboratories S era and L iver A n alyses 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue Sera and L iver E xtractions Pace Tier2 Facility 1700 Elm Street, Suite 200 Minneapolis, MN 55414 St. Paul, MM 55106 Laboratory Project Identification F A C T T O X -160 C ovance In-life S tudy Num ber: 6 3 2 9 -2 6 8 3M M edical D epartm ent Study: T -6 2 9 5 .2 2 E T& S S LIM S : E 0 0 -1 6 6 8 3M Environmental Laboratory 3M Environmental Laboratory 000074 Page 1 o f 9 Page 20 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Protocol #FACT-TOX-160 S tudy Id entification 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 Covance Laboratories, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Analytical Testing Laboratories (sera and liver analyses) 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 Experim ental S tart D ate Experim ental C om pletion D ate 27 August 2001 29 April 2002 3M Environmental Laboratory 3M Environmental Laboratory OOOOV5 Page 2 of 9 Page 21 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Protocol itFACT-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. Purpose This study is designed to continue the assessment o f perfluorooctanesulfonate (PFOS) levels in sera and liver during an extended recovery time o f 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. A t 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. Qu ality 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), C8F17S 0 3'K+, CAS 2795-39-3 3M Environmental Laboratory 3M Environmental Laboratory 000076 Page 3 o f 9 Page 22 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Protocol #FACT-TOX-160 The target analyte is perfluorooctanesulfonate (PFOS), CgF,7S 0 3'. 8. Te s t S ystem 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 Sam ple 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/fluld 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 of 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 I f 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 000077 Page 4 o f 9 Page 23 3M M edical Departm ent Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Protocol #FACT-TOX-160 11.A nalytical 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 o f Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry 11.3 If analytical methods other than those listed above are used, an amendment to this protocol will be written. Any deviations from these methods will be documented and included with the study data. 12. Data Qu ality 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 low est 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 Acceptabie Recoveries Matrix spikes and matrix spike duplicates required for analysis of 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 3 M Environmental Laboratory 000078 Page 5 of 9 Page 24 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Protocol #FACT-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 o f 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 of serum versus an unextracted calibration curve. After serum 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 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 tire 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 report. 13. Sub-C ontracted A nalysis 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. S tatistical 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 000079 Page 6 o f 9 Page 25 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Protocol itFACT-TOX-160 15. Report A report o f the results of 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 of 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 15.11Statistical 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 If 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 o f 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. L ocation 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 of 3M Environmental Laboratory. All corresponding 3M Environmental Laboratory 3M Environmental Laboratory 000080 Page? of 9 Page 26 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Protocol XFACT-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' 3MEnvironmental Laboratory 000031 Page 8 of 9 Page 27 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Protocol FACT-TOX-160 19. A ttachments 19.1 Attachm ent A: Material Safety Data Sheets (MSDS) for Reference Standard 20. Signatures A n d re v rS e a ca t, P h .D ., Study Director John B utenhoff, P h .D ., Sponsor Representative 27 ' D ate 20t pa* k _______________________________________________ QS-k i / p , Lisa Clemen, PrincipalAnalytical investigator D ate 3M Environmental Laboratory 3M Environmental Laboratory 000082 Page 9 o f 9 Page 28 3M M edical Departm ent 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 Am endm ent Date: April 4,2002 Perform ing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification FACTTOX-160 Covance In-life Study Number: 6329-268 3M Medical Department Study: T-6295.22 ET&SS LEMS: EOO-1668 3M Environmental Laboratory 3M Environmental Laboratory 000033 Page 29 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LEMS E00-1668 Protocol FACT-TOX-160 Amendment No. 1 T h is am endm ent m o d ifies th e fo llo w in g p o rtio n (s) o f th e p rotocol: 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 of 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 3MEnvironmental Laboratory 000084 Page 30 3M Medical Department Study: T-6295.22 Section 14.3.2 Method reads: NA Analytical Report: FACT-TOX-160 LIMS E00-1668 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 o f 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 000085 Page 31 3M M edical 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 Date -- f r . A r C W ^ ____________________________________________ ^ /o ? /D ? Lisa A. Clemen., Principal Analytical Investigator Date William K. Reagen, Ph.D., Performing Laboratory Management Date 3M Environmental Laboratory 3M Environmental Laboratory 000086 Page 32 3M M edical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LIMS EOO-1668 Record o f Deviation Study / Project No. FACT-TOX-160, EOO-1668 Deviation type . SOP (Check one) X Protocol Document number FACT-TOX-160, EOO-1668 1. identification Method Equipment Procedure Other: Date(s) o f occurrence 09/05/01 IL Description Required procedure/process: Protocol FACT-TOX-160, E00-1668 states: Matrix spikes and matrix spike duplicates required for analysis of iiver 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 fee calibration range of fee method and was diluted wife 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 fee 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 unextiacted calibration curve. If fee diluted liver matrix spikes do not show recoveries within 70-130% versus the extracted matrix calibration curve, then fee sample concentrations, at fee same dilution factor as the matrix spikes, will be corrected for fee spike recovery. . Actual procedure/process: The MKL090401 - 250 ppb - I05505M matrix spike/matrix spike duplicate (MS/MSD) average recovery was 294%. These samples were not spiked at fee appropriate level in relation to endogenous levels present in fee sample. III. Actions Taken (such as amendment issued, SOP revision, etc.) New matrix spikes were prepared at 2 ug/g & 10 ug/g then extracted on 10/02/01. Recorded by Authorized by A vJCt/twe/k Sfornar fleffejerA&W ' *Tc>\m u\tf%WCC OrtcAor". AimI-U/5 S tam i Date 15-1 d)oi Date s / / 1 % /1 3 /fil I Deviation No. (assigned by Study Director or Project Lead at the end of study or project) Attachment A 3M Environmental Laboratory ETS-4-8.2 Documentation of Deviations Page 1 of 1 oac*os7 Page 33 3M M edical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LIMS EOO-1668 Record of Deviation Study / Project No. FACT-TOX-160, EOO-1668 Deviation type SOP (Check one) X Protocol Document number FACT-TOX-160, EOO-1668 1. Identification Method Equipment Procedure Other: Date(s) of occurrence 10/04/01 II. Description \ I 1 \ Required procedure/process: Protocol FACT-TOX-160, EOO-1668 states: Matrix spikes and matrix spike duplicates required for analysis of liver samples should show spike recoveries within 70% -l 30%. After liver samples have been analyzed, sample concentrations wifi 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 wifi be prepared at the approximate measured concentrations o f the samples then subsequently diluted with the same volume of 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 sample concentrations, at the same dilution factor as the matrix spikes, will be corrected for the spike recovery. Actual procedure/process: The 2 ug/g matrix spike/matrix spike duplicate (MS/MSD) average recovery was 35% and the 10 ug/g MS/MSD was too dilute using a 1:500 dilution factor. III. Actions Taken (such as amendment issued, SOP revision, etc.) The 10 ug/g MS/MSD was re-diluted using a 1:50 dilution and analyzed on 10/16/01. The 1:50 dilution o f the 2 ug/g MS/MSDs was also reanalyzed on 10/16/01 to confirm the original low recovery. Recorded by A* Aorized-by ^ n ^ ^ Spoos&r 3bVu\ 'rtc-W'- Atvirtvo Sfcatc Date p - M 01 Date / 'z /r s / e f Deviation No. (assigned by Study Director or Project Lead at the end o f study or project) Attachment A 3M Environmental Laboratory ETS-4-8.2 Documentation of D*eviations Page 1 of 1 000088 Page 34 3M M edical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LIMS EOO-1668 Record of Deviation Study / Project No. FACT-TOX-160, E00-1668 Deviation type SOP (Check one) X Protocol Document number FACT-TOX-160, E00-1668 l. Identification Method Equipment Procedure Other: Date(s) o f occurrence 10/16/01 II. Description Required procedure/process: Protocol FACT-TOX-160, E00-1668 states: Matrix spikes and matrix spike duplicates required for analysis of 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 of 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 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 iiver 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. Actual procedure/process: The 2 ug/g and 10 ug/g matrix spike/matrix spike duplicate (MS/MSD) average recoveries were 9% and 52%. III. Actions Taken ________________________ (such as amendment issued, SOP revision, etc.)___________________ The MS/MSD and I05505M sample were all re-diluted from the original extracts at 1:50 then analyzed on 10/24/01.... ..................................................................................... ........... ' Recorded by fid A Authorized by Clji/iKu. . ' Date i3-licloi Sponsor fcefrew n laW . T (>Kn tiu W k v ? ' 1, s+ucL OirtcA-or' AftAreu) ^Acctr Deviation N o .------------3--------------- (assigned by Study Director or Project Lead at the end o f study or project) Attachment A 3M Environmental Laboratory ETS-4-8.2 Documentation of Deviations Page 1 o f 1. 000089 Page 35 3M M edical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LEMS EOO-1668 Record of Deviation Study / Project No. FACT-TOX-160, EOO-1668 Deviation type SOP (Check one) X Protocol Document number FACT-TOX-160, EOO-1668 1. Identification Method Equipment Procedure Other: Date(s) o f occurrence 10/24/01 11. Description Required procedure/process: Protocol FACT-TOX-160, EOO-1668 states: Matrix spilces and matrix spike duplicates required for analysis of 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 die method and was diluted with methanol into the validated calibration range, then additional matrix spikes will lie prepared at the approximate measured concentrations o f the samples then subsequently diluted with the same volume of 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 iiver 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. Actual procedure/process: The 2 ug/g and 10 ug/g matrix splke/matrix spike duplicate (MS/MSD) average recoveries were 119% and 242%. The sample recovery was not consistent with the previous analyses. III. Actions Taken _________________ ________ (such as amendment issued, SOP revision, etc.) Hie sample set was re-extracted on 11/07/01, then diluted 1:50 and analyzed. Recorded by ; A C li1--yK?^ A uthorized^ -Sponsor R^reseriW W .' *ToKr\ thrtcAvr'. A n rto Date )^l] I d | o i Date Deviation No. (assigned by Study Director or Project Lead at the end o f study or project) Attachment A 3M Environmental Laboratory ETS-4-8.2 Documentation of Deviations Page 1 of 1 000090 Page36 3M M edical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LIMS EOO-1668 Record of Deviation Study / Project No. FACT-TOX-160, EOO-1668 Deviation type SOP (Check one) X Protocol Document number FACT-TOX-160, EOO-1668 1. identification Method Equipment Procedure Other: Date(s) of occurrence 11/16/01 II. Description I Required procedure/process: _ Protocol FACT-TOX-160, EOO-1668 states: Matrix spikes and matrix spike duplicates required for analysis of liver samples should show spike recoveries within 7Q%~130%. After liver samples have been analyzed, sample concentrations will be evaluated. If a measured sample (s) concentration exceeded the calibration range of 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 die 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 dilutedliver 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. Actual procedure/process: The 10 ug/g matrix spike/matrix spike duplicate (MS/MSD) average recovery was 67%. ill. Actions Taken (such as amendment issued, SOP revision, etc.) The 10 ug/g MS/MSD was not reanalyzed to confirm recovery since the 2 ug/g MS/MSD average recovery, analyzed during the same run, was 95% and within die stated criteria. Also, all CCVs and other data quality, objectives were within 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 Authorized A* C >. 3 - ] h i lo{ Date, Sponsor R.pr*,jerAo<kvt,`. "3ohri 'Dire.cbr'. AncWu) SfcoccV Deviation No. (assigned by Study Director or Project Lead at the end of study or project) Attachment A 3M Environmental Laboratory ETS-4-8.2 Documentation of Deviations Page 1 of 1 000091 Page 37 3M M edical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LIMS E00-1668 Record of Deviation Study / Project No. Tox 160 Deviation type (Check one) Document number Ets 8-7.0 1. Identification ............ s o p ...... .. X Method Equipment Procedure Protocol Other: Date(s) o f occurrence 11-16-01 II. Description Required procedure/process: According to section 1i .i The average o f two standard curves will be plotted by linear regression. | Actual procedure/process: For the data set m O lll 16a, the unextracted curve was plotted using a quadratic fit. A linear curve that met the rA2 criteria and extended to 250 ppb (the level o f the CCVsj could not be generated. This deviation was written. HI. Actions Taken (such as amendment issued, SOP revision, etc.) Wo.AiW fc vmpoc .filiaWtltA, Vfersuti : Date Study / Project >rpqoy o/uuy director or Project Lead)________________________ P u kjttftJL i 'K i i . . f b j U , .. ' fftpv-Lr; V O ilr ig t t ip ^ it la o lf i, -fl?rnjpx UHrt, Authorized by Date 7, fe , j___________ '-- ^ .......................... ....................-- -- ^ D i i t c W A n d r ^ 'Secxtad W ^ / 2 ^ / ^ / D e w a f / o n N o . L r _ \ i _ j o i l PP (assigned by Study Director or Project Leacd at the end of study or project) J Pinter KeprWfcoTa+ive Jo no utenner r Attachment A 3M Environmental Laboratory ETS-4 -8.1 Documentation of Deviations Page 1 of 1 000092 Page38 3M M edical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LIMS E00-1668 R ecord of Deviation Study / Project No. FACT-TOX-160, EOO-1668 Deviation type SOP (Check one) X Protocol Document number FACT-TOX-160, EOO-1668 I, Identification Method Equipment Procedure Other: Date(s) of occurrence : 11/16/01 II. Description Required procedure/process:................................................ Protocol FACT-TOX-160, EOO-1668 states: Matrix spikes and matrix spike duplicates required for analysis of 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 of 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 the sample into the validated range of 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 sample concentrations, at the same dilution factor as the matrix spikes, will be corrected for the spike recovery. Actual procedure/process: 1) The 10 ug/g matrix spike/matrix spike duplicate (MS/MSD) average recovery was 67%. 2) No 1/500 liver dilution was reported during the course of this study. The 1/500 liver dilution was too dilute because the wrong iiver sample was used for preparing that MS/MSD level. __________________________ III. Actions Taken (such as amendment Issued. SOP revision, etc.) 1) No liver values were corrected for this low recovery since one set of MS/MSDs were within criteria and the average recovery for this set of MS/MSDs were within the extended 65-135% criteria as stated in an earlier deviation. 2) Dilution was not reextracted/rediluted using the correct sample since the 1/50 dilution was within criteria and this i/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 C ^ M ta Authorized by Date Sfcnscr Ricreiefrkki/t/ Direc-W "i Andrew) SeflCal Deviation No. Jl (assignedby Study Directoror Project Lead at theendof study or project) 2 . / ZsOOZ., 3M Environmental Laboratory 000093 Page 39 3M M edical 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 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-sulfonate or other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry," (14 pages) ETS-8-5.2, "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-sulfonate or other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry," (10 pages) 3M Environmental Laboratory 3M Environmental Laboratory 000094 Page 20 Page 40 3M M edical Department Study: T-6295.22 3M Environmental Laboratory Analytical Report: FACT-TOX-160 r LIMS E00-1668 Ekss Copy of Orpins! ! 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 po u n d s fr o m Ser u m f o r A n a ly sis U sin g H P L C -E lectro spr a y /M ass Spec tr o m etr y M ethod N um ber: ETS-8-4.2 Adoption Date: 03/01/99 Effective D ate: L/cL./0 ! Approved By: Laboratory Manager Group Leader Date OZ'IO ./O i Date 1.0 S c o pe a n d Ap p l ic a t io n _________________:_______________________________________ ' 1.1 Scope: This method is for the extraction o f fluorochemical compounds from serum. 1.2 Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, monkey, and human serum or other fluids as designated in the validation report. 2.0 S u m m a r y o f M e th o d _______________________ _________________________________________ 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-teri-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 o f methanol, then filtered through a 0.2 Jim nylon filter using a 3-mL plastic Word 6/95 3M Environmental Laboratory ETS-8-4.2 Fvhrortinn r*fP1nnrnrh<*mi/'nir from Sprtim Page 1 of 15 000095 Page 41 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 i 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 e fin itio n s}). These sample extracts are analyzed following method ETS-8-5.2 or other appropriate method. 3.0 D e fin itio n s______________________________________________ _____________________ 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) C8F i7S03` 3.2 PFOSA: perfluorooctane sulfonylamide C8F 17SO2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8Fi7S0 2N(CH2CH3)CH2C0 2 ' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonaimdo)-ethyl alcohol C8Fi7S02N(CH2CH3)CH2CH2 0 H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide CsFnS O2N(CHaCH^H 3.6 M556: C8Fi7S 02lSr(H)(CH2C00H) 3.7 Surrogate standard THPFOS: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 W a rn ing s and Ca utio ns _______________________ ;__________________________ ' 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 In te r fe r en c e s________________________________________________________________ 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 S u a n d M a ter ials ________ _________________________________________________ 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 ETS-8-4.2 T?..*__-- _ r m _________ x-------r . - i - r ____ o ---------- 000096 Page 2 of 15 Page 42 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 7.4 Volumetric flasks, glass, type A 7.5 40 mL glass vials (I-CHEM or equivalent) 7.6 4 7.7 Centrifuge tubes, polypropylene, 15 mL Labels 7.8 Bottle-top Dispenser - 3.0 to 10.0 mL or a 5-10 mL graduated pipette, glass 7.9 Syringes, capable of 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 e a g e n t s a n d S t a n d a r d s___________________________ ;__________________________ _____ 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 (Na2C 0 3), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Serum or blood, frozen from supplier 8.9 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 (1-H,1-H, 2-H, 2-H C8F13SO3.H, [THPFOS]), molecular weight = 428 8.9.8 Other fluorochemicals, as appropriate 3M Environmental Laboratory ETS-8-4.2 000097 Page 3 of 15 Page 43 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-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 m L o f 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 o f 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. 8.10.3.1 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 (Na2C0 3/NaHCC>3): Weigh approximately 26.5 g o f sodium carbonate (Na2C 0 3) and 21.0 g of sodium bicarbonate (N aH C03) 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 o f 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: molecular wt. PFOS ( 499 ) molecular wt. KPFOS ( 538 j 0.9275 ( Correction factor) 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. 1000 uglm Lx. 5 mL = 50 )ig lm L 100 mL 8.11.6 Dilute working standard 1 with methanol for a working standard 2 solution o f approx. 5.0 pg/mL. 3M Environmental Laboratory ETS-8-4.2 000098 Page 4 of 15 Page 44 3M M edical Departm ent Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 l'5 0 p g /m x l0 m > = 5.0 fig Im L 100 mL 8.11.7 Dilute working standard 1 with methanol for a working standard 3 solution of approx. 0.50 jig/mL. 8.12 Surrogate TH PFOS stock standard preparation 8.12.1 Weigh approximately 50-60 mg of surrogate standard 1-H.l-H, 2-H, 2-H, C8F13SO3H (THPFOS) into a 50 mL volumetric flask and record the actual weight. 8.12.2 Bring to volume with methanol for a surrogate stock of approximately 10001200 pg/mL. 8.123 Prepare a surrogate working standard. Transfer approximately 1 mL o f surrogate stock to a 10 mL volumetric flask and bring to volume with methanol for a working standard o f 100 pg/mL. Record the actual volume transferred. 9.0 S a m p l e H a n d l in g _____________________________________________________________________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw at room temperature or in lukewarm water prior to extraction. 10.0 Q u a l it y C o n t r o l __________ _____________________________________________________ 10.1 Solvent Blanks, M ethod Blanks and M atrix Blanks 10.1.1 Solvent Blanks: An aliquot o f 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 o f 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. 3MEnvironmental Laboratory ETS-8-4.2 000099 Page 5 of 15 Page 45 3M M edical 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 of matrix spikes (a set is a matrix spike/matrix spike duplicate at one level) using a commercially available matrix o f 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% o f the samples are <LOQ, two matrix spikes should be prepared at approximately 2 -5 times the expected LOQ. 10.2.7 If the majority o f 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 (CCVs) 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 - 00100 Page 6 of 15 Page 46 3M M edical Departm ent Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 10.3.3 The expected concentrations o f the CCVs will fall within the range o f the initial calibration curve; typically, the low to mid-range o f the curve. 10.3.4 Prepare, at a minimum, two continuing calibration verifications, each at a different concentration, per group o f 10 samples. For example, if a sample set = 3 4 , 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 C a l ib r a t io n a n d St a n d a r d iz a t io n ________________ ________ ' 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 of 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 o f 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 of 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 o f 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 OOOlOl Page 7 of 15 Page 47 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Table 1 Approximate spiking amounts for standards and spikes Using 1.0 m L of m atrix Working standard (approx, cone.) jjL 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 5. ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 1.00 ppm *ppm=iig/. 12.0 Pr o c ed u r e _________________________________________________________________________ 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 o r 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 o f 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 ETS-8-4.2 000102 Page 8 of 5 Page 48 3M M edical 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 D a t a A n a l y sis a n d Ca lc u l a t io n s___________________________________________ _ 13.1 Calculations 13.1.1 Calculate actual concentrations o f PFOS, or other applicable fluorochemical, in calibration standards using the following equation: ________mL o f std X concentra&on o f std( fJg/ mL)_______ mL o f std + mL o f surrogate std + initial matrix volume( mL) Final concentration ( fig / m L ) o f PFOS in matrix 14.0 M e t h o d P e r f o r m a n c e 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to M DL report for specific MDL and limit of quantitation (LOQ) values (see A ttachm ents B an d C). At the discretion o f 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 o f 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 o f the initial calibration curve. 14.3 Refer to section 14 of ETS-8-5.2 for method performance criteria. 3M Environmental Laboratory ETS-8-4.2 Af TlnAm/'boimir'olc' fVorv* Qflrtim 000103 Page 9 of 15 Page 49 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 15.0 Pollution Prevention and W aste Management ____________ ________ . 15.1 Dispose of sample waste, flammable solvent waste and used glass pipette waste using * 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 t ta c h m e n t s ________ :_____________ ;__________________________________ 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 R e fe r e n c e s____________________________________ ;__________ ;_________ ;_____________ 18.1 The validation report associated with this method is ETS-8-4.0 & 5.0-V -l. 18.2 FACT-M-3.1, "Analysis o f Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 18.3 ETS-8-5.2, "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" 19.0 A ffected Documents 19.1 ETS-8-5.2, "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" 20.0 Revisions_____________________________________________________ _______________ 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 Eliminate `potassium' from perfluorooctanesulfonate 8.9.1 Add K to PFOS 5.9.3 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 11.1 Added wording about control animal sera, using and not using commerciaUy available tissue In general, make less specific for equipment/suppiies. Substitute water for Milli-Q, Nalgene or equivalent bottle may be used. Revision Date 04/02/99 3M Environmental Laboratory ETS-8-4.2 000104 Page 10 of 15 Pae50 3M M edical Department Study: 1-6295.22 Analytical Report: FACT-TOX-160 Extraction Worksheet ETS-8-4.2 LIMS E00-1668 Studv # Matrix Box # Wk/Dav Date Spiked/ Analyst: Surrogate Std actual pg/mL # FC-Mix approx. 0.5 pg/mL # FC-Mix approx. 5 pg/mL # FC-Mix approx. 50 pg/mL # Comments Verified by: MS MSD -- - -- - -- - -* - - -- - -- - -- - - -- - - -~ - - - - Blank ID # - - - amount = - mL Serum Extraction Method Vortex 15 sec. _........... ..... ._J...................................................BatflJ&JmMals------- Pinette Matrix, snike with annronriate surroeate or FCMix Volume mL Pipette 1 mL of 0.5 M TBA. dH 10. pH = Std. # Pipette 2 mLof0.25Na2C03/0.25MNaHC03 buffer Std.# Dispense/pipette 5 mL of methvl-t-butvl ether TN-A- Dispenser ID #: Shake 20 min. Shaker ID #: Shaker speed: Centrifuge 20-25 min. Centrifuae ID#: Centrifuae speed: Remove a 4 mL aliouot of oreanic laver Put on Nitrnaen Evaoorator to drvness Add methanol Volume mL TN-A- Vortex 30 sec. F ilter using a 3m L B-D syringe w ith a 0.2um filter into a 1.5 mL autosam ole vial C ont Cal. Verifications used same matrix as for std curve. AoDendix A: Extraction Worksheet 3M Environmental Laboratory ETS-8-4.2 O O O IO S Page 11 of 15 Page 51 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 M DL/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/mL-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 000106 Page 12 of 15 Page 52 3M M edical Departm ent Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Compound: P]FOS Prepared range Rabbit Seram 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 4.92 - 247 High curve 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 RSD Range 4.67-11.0 5.34-12.0 4.84-9.80 4.60-10.5 % Recovery RSD Range . Range 88-103 87-105 93-102 94-103 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 % Recovery Range RSD Range 81-111 97-107 85-108 95-115 4.18-10.6 6.38-21.8 4.33-12.5 4.11-23.2 Appendix B: MDL/LOQ Values and Summary ETS-8-4.2 3M Environmental Laboratory (; Page 13 of 15 Page 53 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Compound: E tFOSE-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 1rx 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 Curve 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 000108 Page 14 of 15 Page 54 3M M edical Departm ent Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 E xam ple Ion Pair Standard Curves - Fluids A nalyses) Study number: ; Prep date(s): Analyte(s): Study Animal M atrix : Final solvent and TN: Blank flud/identifer: {Record matrix used to prepare curves, (c.g., ` Rabbil ' ID #XXX)} M ethod/revision: T arget 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 PFOSE - MSS6 Std cone Std cone Std conic Std cone Std cone Std cone pg/naL |ig/mL ug/mL ug/mL ug/mL ug/mL 0.500 0.507 0.532 0.501 0.521 0.501 0.500 0.507 0.532 0.501 0.521 0.501 5.00 5.07 5.32 5.01 5.21 5.01 5 .0 0 " ! r " f f 7 5.32 5.01 5.21 "1 5.01 5.00 1 1__ % W W T I 50. 1 so. i 1 k .. * * I 50.1 fl i 52.1 I j f s i n 50.0 J f L - s r f u f S r S t I Is.yM Jg 50.0 50.1 53.2 50.1 1 5 2 .1 50.1 50.0 50.1 53.2 50.1 . 52.1 50.1 All Am't spiked mL 0.010 0.020 0.005 0.010 0.020 0.005 0.010 0.015 0.020 All Final vol mL 1.015 1.025 1.010 1.015 1.025 1.010 1.015 1.020 1.025 Calculated concentrations of standards in the sera matrix: Rabbit Validated ranges - approximate concentrations Serum PFOS PFOSA PFOSAA Rabbit .5.00-1000 5.00-1000 5.00-1000 ng/mL ng/mL ng/tnL Bovine Estimates only. Use values for rabbit. Rat Estimates only. Use values for rabbit. Monkey & Plasma Estimates only. Use values for rabbit. Human Estimates only. Use values for rabbit. EtFOSE-OH 5.00-1000 ng/mL PFOSEA 5.00-1000 ng/mL M556 5.00-1000 ng/mL Attachment C: Ion Pair Standard Curves 3M Environmental Laboratory ETS-8-4.2 000109 Page 15 of 15 Page 55 3M M edical Departm ent Study: T-6295.22 3M Environmental Laboratory A nalytical Report: FACT-TOX-160 LIMS EOO-1668 m copycfO rfcind / /fe A i L tat&l _ M ethod E x t r a c t io n o f P o ta ssiu m P erflu o r o o cta n esu lfo n a te o r o t h er 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 f o r A n a ly sis u sin g H P L C - E l e ctrospray/M ass Spec tr o m etr y M ethod N um ber: ETS-8-6.0 Author: Lisa Clemen, Robert Wynne Approved By: t vr y \ -- Laboratory Manager u Adoption Date: Revision D ate: Wfr V^ h 7 Date Group Leader Technical Reviewer Date 07/ m h < l 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 ETS-8-6.0 . n._c T \ r* tm>.rK 000110 Page 1 of 14 Page 56 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 2.0 Summary 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-butyl 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 efinitio n s________ ;__________________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) C8FI7S 0 3 3.2 PFOSA: perfluorooctane sulfonylamide CgF17S0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate CgF,7S0 2N(CH2CH3)CH2C02 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F I7S 0 2N(CH2CH3)CH2CH20 H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8F17S 0 2N(CH2CH3)H 3.6 M556: C8F I7S 0 2N(H)(CH2C 0 0 H ) 3.7 Surrogate standard: lH-lH-2H-2H'perfluorooctane sulfonic acid 4,0. Warnings and Cautions_______________________________________________________ 4.1 H ealth and Safety'W arnings: 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 Eq u i p m e n t ____________________________________________ _ 6.1 The following equipment is used while performing this method. Equivalent equipmept is acceptable. 6.1.1 6.1.2 6.1.3 6.1.4 Ultra-Turrax T25 Grinder for grinding liver samples Vortex mixer, VWR, Vortex Genie 2 Centrifuge, Mistral 1000 or IEC Shaker, Eberbach or VWR 3M Environmental Laboratory ETS-S-6.0 n-PDCAC T Page 2 of 14 Page 57 3M M edical Department Study: T-6295,22. Analytical Report: FACT-TOX-160 LIMSEOO-1668 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivity to 0.100 g) < 7.0 S u ppl ie s and M a ter ia l s________________________________________ _____________________ 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable o f holding 250 mL and 1 L 7.5 Volumetric flasks, glass, type A 7.6 I-CHEM vials, 40 mL glass 7.7 Plastic sampule vials, Wheaton, 6 m L (or appropriate size) 7.8 Centrifuge tubes, polypropylene, 15 mL 7.9 Labels 7.10 Oxford D ispensor- 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 Reagents 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 b y 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 (NaaCOj), J.T. Baker or equivalent ^ 8.5 Sodium bicarbonate (N aH C 03), J.T. Baker or equivalent 8.6 Methyl-feri-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 D iy ice from supplier 8.10 Fluorochemical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 3M Environmental Laboratory ETS-8-6.0 . xv.__ r 000112 Page 3 o f 14 Page 58 3M M edical 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 C8F,3S 0 3H) molecular weight = 428 8.10.8 Other fluorochemicals, as appropriate .. 8.11 R eagent 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 m L beaker containing 500 mL Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.11.2 I N sodium hydroxide (NaOH): Dilute 1 0 N NaOH 1:10. Measure 10 m L o f 10 N NaOH solution into a 100 m L 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 caibonate/sodium bicarbonate buffer (N a^Q /N aH C O j): Weigh approximately 26.5 g o f sodium carbonate (N a^O j) and 21.0 g o f sodium bicarbonate (NaHCOa) into a l L volumetric flask and bring to volum e with M illi- QTM water. Store in a 1 L Nalgene bottle. 8.12 S tandards 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 .1 2 3 Weigh approximately 100 mg o f 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 (jig/mL). 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 3M Environmental Laboratory ETS-8-6.0 Page 4 o f 14 Page 59 3M M edical 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.l-H, 2-H, 2-H, CaFi3S 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 Handling________________ ;____________________________ ._________________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 10.0 Quality Control_________________________________________________ 10.1 M atrix blanks and m ethod 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 M atrix 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 w ith 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 C ontinuing 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 ETS-8-6.0 . -Timc -- t 000114 Page 5 o f 14 Page 60 3M M edical 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 o f the initial t . 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 -1 0 0 ppb, rather than 5 ppb -1 0 0 0 ppb). 11.0 Calibration and Standardization________ ______________________ _ 11.1 P rep are m atrix calibration standards 11.1.1 W eigh 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 b y shaking between aliquots while preparing a total o f eighteen 1 mL aliquots o f homogeneous solution in 15 m L 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 and ETS-8-7.0-V-1 or A ttachm ent B, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.9 Use Attachm ent 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-.8:6A 000115 Page 6 o f 14 Page 61 3M M edical Departm ent 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. '1 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, of 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. 1 2 3 Weigh the sample directly into a tared 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. v 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. 3M Environmental Laboratory ETS'8-6.0 000116 Page 7 o f 14 Page 62 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 12.11 Pipette 1.0 mL, or other appropriate volume, o f homogenate into a 15 mL polypropylene centrifuge tube. Label the centrifuge tube with the identical information as the sampule vial. Refer to attached worksheet for documenting the remaining steps. < 12.12 Pipette two 1 mL aliquots o f Milli-QTM water to centrifuge tubes. These will serve as method blanks. 12.13 Spike all samples, including blanks and standards ready for extraction with surrogate standard as described in section 11.2 . 12.14 Spike each matirx with the appropriate amount 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 m L 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 m L methyl-terf-butyl ether. 12.19 Cap each sample and put on the shaker at a setting o f300 ipm, for 20 minutes. 12.20 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 m L o f 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 ETr S-8-6r~.%0c.- . 000117 Page 8 of 14 Page 63 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 13.0 Data Analysis and Calculations________________________________ 13.1 C alculations: 13.1.1 Calculate the average density o f the liver homogenate by recording each mass o f ten separate 1.0 mL aliquots o f homogenate. Average density (mg/mL) - Average m ass (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 of homogenate suspension) using the following equation: g o f Liver x Average density* o f homogenate fmg/mLl (g o f 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 /m Ll = 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 Method Performance_________________________________________________________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to M DL report for specific MDL and limit o f quantitation (LOQ) values (refer to Attachm ents B an d C). 14.2 The following quality control samples are extracted with each batch o f samples to evaluate the quality o f the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision o f the extraction. 14.2.3 Continuing calibration 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 anagement___________:____________________ 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 000118 Page 9 o f 14 Page 64 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 16.0 R eco rds_________________________________ ________________________________ ;__________ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. *1* 17.0 T a b l e s. D ia g r am s. F l o w c h a rts, a n d V a lid a tio n D a ta ________________ __________ _ 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 efer en c es______________________________________________!____________________ 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 ofUltra-Turrax T-25" 18.3 FACT-M-1.1, "Extraction o f'PFOS or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 19.0 A ffec ted D ocum ents___________________ __________________ ________________________ _ 19.1 ETS-8-7.0, "Analysis o f Liver Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 R evisions Revision Tvhimfrpr ___________________ ____________________________________________________ Reason For Revision Revision Date 3M Environmental Laboratory ETS-8-6.0 000119 Pagel0of14 Page 65 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Study # Matrix Box?? Wk/Dav Date Spiked/Analyst ccv MS MSD Surrogate Std approx, ppm actual ppm # FC Mix Std approx. 0.5 ppm actual ppm # FC Mix Std approx. 5 ppm actual ppm #' FC Mix Std approx. 50 ppm actual ppm # i Comments - - - - -. - - - - - Blank Liver Homogenate: Std# Liver amount = Liver Extraction Method : Spike surrogate and Standard mix. Vortex 15 sec. PiDette-1 mL of Liver Solution Pipette 1 mL of t0.5 M TBA, pH 10. pH =* Std. # Pipette 2 mL of 0.25 Na2COy0.25M NaHCOi Buffer Std.# Dispense 5ml of Methyl-t-Butyl Ether TN-A- Shake 20 min. Shaker Speed Centrifuee 20-25 min. Centrifuge Speed Remove a 4 mL aliquot of organic layer Put on Nitrogen Evaoorator to dryness Evaporator Temperature Add 1.0 mL of Methanol TN-A- Vortex 30 sec. Filter using a 3cc B-D swinee with a 0.2um SRI filter into autosamolc vial Cont. Cal. Verifications used the same matrix as for the standard curve. - - - - - - - - - - - e Date & Initials -- Attachment B: MDL/LOQ Values 3M Environmental Laboratory ETS-8-6.0 000120 Page 11 o f 16 Page 66 3M M edical 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 -1 2 0 0 ppb EtFOSE-OH 108 345 60 ppb - 900 ppb* M556 82.3 262 60 ppb -1 2 0 0 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 o f these matrices were extracted and analyzed with the rabbit liver curves to determine equivalence. Responses in the rat, bovine, and monkey liver curves were equivalent to the rabbit responses, therefore, their MDL and LOQ will be assumed to be equivalent to those values as determined for the rabbit liver. - Refer to LOQ Summary and MDL study in ETS-8-6.0 & 7.0-V-l for further information * EtFOSE-OH estimates only for MDL and LOQ. Did not meet criteria for validation. Compound; PFOS __________________________________________ __ Liver matrix Prepared range of standards (ppb) (ng/mL) Range of average curve (ppb) (ng/mL) iLCRfipiir.. Vkv-'cve- (ppb) (ng/mL) Range of low Std curve (ppb) (ng/mL) :,;LC(UrdjtfJ Range of 'vLRj&fOTl' ; 10WJS!^ : high std .. curve f-|j curve ' (ppb) (ng/mL) |^ b ) % |/ m L ) 1; Rabbit 6*19 -1237 12 -1200 ^ 2 ^ 2 Q 1 " 6 - 300 - - / i f e s o O ^ 60-1200 Compound: PFOSA Prepared Liver matrix range of - standards (ppb) (ng/mL) Range o f LRifioih. , Range of average aye i u iy e low std curve curve (ppb) (ng/mL) . ,(ppb) (rig/mL) (ppb) (ng/mL) :/L^frpmf| Range of high, std curve .;(pp8)W^ni), (ppb) (ng/mL) Rabbit 6.19 -1237 12-1200 ' -1 2 ^ -2i*0 0 ^ 12 - 300 60-1200 Compound: PFOSAA Prepared Range of . L G tfip m . Liver . range of average ave curve matrix standards curve (ppb) (ng/mL) (ppb) (ng/mL) (ppb) (ng/mL) Rabbit 6.16 -1232 12 -1200 30 -1200 : Range of low std curve (PPb) (ng/mL) 30 - 900 LRfrpm:; Range of low std ; high std curve;'-: curve (ppb) (ng/mL) (ppb) (ng/mL) 60-900 N/A LCR fioul high std : curv : (ppb) (ng/mL) ;?.;N/A^--; Attachment B: MDL/LOQ Values SMEnvironmental Laboratory ETS-8-6.0 000121 Page 12 of 16 Page 67 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Compound: EtFOSE-OH Prepared Range of Liver range of average matrix standards curve (ppb) (ng/mL) (ppb) (ng/mL) Rabbit 6.17 -1235 31 - 900 LCR from ave curve (ppb) (ng/mL) 31-900 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 high std curve (ppb) (ng/mL) N/A Compound: PFOSEA Prepared Range o f Liver range of average matrix standards curve (ppb) (ng/mL) (ppb) (ng/mL) . LCRfrom . Range of av.curve.V low std curve (ppb)-(ng/mL)- (ppb) (ng/mL) Rabbit 6.17-1235 31 -1200 N/A L C R from . . low std curve (ppb):(ng/mL) Range of high std curve (ppb) (ng/mL) LCR from high std ;f ' c'curve-if'-r .(ppb) (ng/mL) N/A i-* Compound: M556 Prepared Liver range o f matrix standards (ppb) (ng/mL) Rabbit 6.17-1235 Range o f \;LOLfrQnpLfi- Range o f .;LCK:firom;--T Range of vrLCRfrni;';: average 'vavii^c^ low std `..Ilowisti! " ; high std curve curve curve (ppb) (ng/mL) . (Ppb);: (lig/mLL',. (ppb) (ng/mL) -:PPb)-r(ng/m: (PPb) (ng/mL) :?!(ppb)-(g/mL):!: :p.r'e :->. : L .rrpi. 31 - 1200 N/A ;>N/iA:ith. N/ Attachment C: Standard Calculations 3M Environmental Laboratory ETS-8-6.0 . . m r A O A . . 0 0 0 1 2 2 Page Page 68 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Ion Pair Standard Curves - Tissue '. 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 PFOS PFOSA PFOSAA EtFOSE PFOSEA Std cone Std cone Std cone Std cone Std cone ug/mL ug/mL ug/mL ug/mL ug/mL 0.500 0.500 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 M556 Std cone ug/mL 0.500 0.500 0.500 0.500 0.500 5.00 5.00 5.00 50.0 Std cone ug/mL All Ain't spiked mL 0.002 0.004 0.010 0.020 0.040 0.010 0.020 0.030 0.004 A ll Density S 0.167 0.167 0.167 0.167 0.167 0.167 0.167 0.167 0.167 Calculated concen bradons o f standards in the sam ple matrIX PFOS PFOSA PFOSAA EtFOSE PFOSEA M556 Final Final Final cone Final Final Final Std cone cnc cone ng/g cone cone cone ng/g ng/g ng/g ng/g 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 59.9 120 120 120 120 120 120 299 299 299 299 299 299 599 599 599 599 599 599 898 898 898 898 898 . 898 1198 1198 1198 1198 1198 1198 Surrogat Std cone ng/mL 100 Surrogate Final cone ng/mL ' 0.500 A ll Ain'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 Attachment C: Standard Calculations 3M Environmental Laboratory ETS-8-6.0 . . . .tvn < \r a . . 000123 Page 14 o f 14 Page 69 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 DKCcpyofOtalna 3M Environmental Laboratory *i M ethod A n a ly sis o f P o ta ssiu m P er flu o r o o c t a n e su lfo n a t e o r O t h e r F l u o r o c h e m ic a l s in Se r u m E x tr a c t s U sin g H P L C -E lectrospray/M ass Spectro m etry M ethod N um ber: ETS-8-5.2 Author: Lisa Clemen, Kris Hansen Adoption Date: 03/01/99 Revision Date: i) I 01 rr , ^ -------------------------------------Laboratory Manager K A * U r. Group Leader Technical Reviewer Date 0 ^ /o l/ Date > !_ - o f . o f Date 1.0 S c o pe and A ppl ic a tio n ______________________________________________________________ 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. Word 6/95 3 M Environm ental Laboratory ETS-8-5.2 A M nlvcc a C a*nm C v tr a A f T C C A /fC 0 0 0 1 2 4 Page 1 of 11. Page 70 3M M edical D epartm ent Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 2.0 S u m m a r y o f M e th 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 o f 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 A tm 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 (MS), Tandem Mass 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 electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e., Z-spray components are compatible with 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 a r n in g s a n d Ca u tio n s_______________________ ;_____________;_________________________ 4.1 H ealth and 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 3M Environmental Laboratory ETS-8-5.2 _ 0 0 0 1 , 2 5 Page 2 of II Page 71 3M M edical Departm ent Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 4.1.2 When handling samples o r 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 In t e r f e r e n c e s________________________________________ 5.1 To minimize interferences when analyzing samples, Teflon should not be used for sample storage or any part o f instrumentation that comes in contact with the sample or extract. 6.0 E quipm 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 or Ultima triple quadrupole Mass Spectrometer equipped with an electrospray ionization source H P1100 or Agilent low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 S u p p l ie s a nd M a t e r ia l s______________________________________________ ______________ 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 Re a g e n t s a n d S t a n d a r d s___________________________________________ _______________ 8.1 Reagents 8.1.1 M ethanol HPLC grade or equivalent 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. See ETS-8-4.2. 3M Environmental Laboratory ETS-8-5.2 ____ ,, 0 0 0 1 2 6 Page 3 of 11 Page 72 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 9.0 S a m pl e H a n d lin g __________________________________ _ _ __________________________ _ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. ' 9.2 I f analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C, or at room temperature, until analysis cm be performed. 10.0 Q u a l it y Co n t r o l __________________________________________________________ 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 Calibration Verifications (CCVs) 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy o f 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 of two per batch and always finishing an injection sequence with at least two calibration standards. 11.0 C a l ib r a t io n a n d S t a n d a r d iz a t io n _______ _________________________________________ 11.1 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 of 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 o f the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of 3 M Environm ental Laboratory ETS-8-5.2 a . . i ?_ _ j ? n vr eye*rs .ro __ 000127 Page 4 of 11 Page 73 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 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 r o c e d u r e s_________________ 12.1 Acquisition 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 o f 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 o f 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 of test (07) DD=day of test (12) ###=3-digit sequential file number starting with 1 through 999 (001) Also, as part o f 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. 3M Environmental Laboratory ETS-8-5.2 Page 5 of 11 A , f c T T n i ~ r> C C A / f C 000128 Page 74 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 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 jiL 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 Instrum ent 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 pL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 3M Environmental Laboratory ETS-8-5.2 c ___ T ? . T c e / i - t e 000129 Page 6 of 11 Pge "75 3M M edical 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 W ord 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 D a ta A n a l y sis and Ca lc u l a tio n s_____________________________ _____ ______________ 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: % Difference Expected Cone. --Calculated Cone. X 100 Expected Cone. 13.1.4 Calculate actual concentration o f PFOS, or other fluorochemical, in matrix (jXg/mL): .( Cone, o f PFOS Calc, from Std. Curve (ng/ mV) X Dilution Factor ) ( Initial Volume o f Matrix (mL) + mL o f Surrogate Standard) Final Volume ( mL ) 1m \0QOng 14.0 M e t h o d P erfo r m a n c e_________________________________________ ___________________ 14.1 Method Detection Limit (MDL) and Limit o f Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-4.2, 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 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 A n n lu c ic 1 ETS-8-5.2 Q ia r t im T 7 v tr ,rri T T c n r r T J /A /IT Page 7 of 11 000130 Page 76 3M M edical 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% o f 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 o f 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 V erifications (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. 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 P o l l u t io n Pr e v e n t io n a n d W a st e M a n a g e m e n t _______________ _________________ 15.1 Sample 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 ec o r d s____________________________________________________________________________ 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 A~ 000131 Page 8 of 11 Page 77 3M M/ edical Departm ent 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 o f 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 Attachm 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 a ta __________________________ 17.1 Attachment A: Data summary spreadsheet. 18.0 R e fe r e n c e s_______________;___________________________ ___________ 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 ffe 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 ETS-8-5.2 A c o_... IT, noA/fc Page 9 of 11 000132 Page 78 3M M edical Departm ent Study: T-6295.22 A nalytical Report: FACT-TOX-160 LIMSEOO-1668 20.0 Revisions_____________________ ;______________________________ _ _ _ __________ Revision i Number. Reason For Revision 1 Section 6.1.2 Clarification of H P1100 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 Clarified when blanks are run. 10.2 Added instructions for when, surrogate matrix is used. 10.3 Specify requirements for CCVs. 11.1 Requires only a calibration curve before the samples. 11.2 Clarify what to do if curve does not meet requirements. 11.3 Allow to truncate the curve. 12.1.1 Clarify sample list ID. 12.1.3 Clarify typical ran. 12.2 Changes to mobile phase gradient and specify flow rate. 14.3 Change acceptable limits. Add specifics for acceptance of calibration curve. 14.3.3 When to deactivate calibration standards based on blank response. 14.3.5 When to deactivate calibration standards within the linear range. 14.4 Modifies evaluation and use of matrix spikes. 14.5 Describes evaluation and use of CCVs. 14.5.1 Evaluation of CCVs. 16.0 Add requirements for records and documentation. Attachment A: Summary Spreadsheet i 3M Environmental Laboratory ETS-8-5.2 Page 10 of 11 r-' O m Page 79 000133 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Laboratory Study # * Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analvsis/Analyst: Group Dose Sam ple# Concentration ug/mL ' Initial Vol. ml Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration fug/mL): Taken from the MassLynx integration summary. Initial Volume fmL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet A n o lw c lo 3M Environmental Laboratory ETS-8-5.2 T Tcrrr ^ C /T V /IQ Page 11 of 11 000134 Page 80 3M M edical Departm ent Study: T-6295.22 3M Environmental Laboratory Analytical Report: FACT-TOX-160 LIMS EOO-1668 CxsotCopycf OryjpsO ki.v] BsOs M ethod A n a ly sis o f P o ta ssiu m P erflu 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 x tra cts Usin g H P L C -E lectro spra y /M ass Spectro m etry M ethod Num ber: ETS-8-7.0 Author: Lisa Clemen, Glenn Langenburg Approved By: 1M Laboratory Mfanager -- Group Leader AH'/)*- A Technical Reviewer Adoption Date: O jj Revision D ate: f\lft Date 9 -H H Ia Date 0 ? //v /? ? Date 1.0 Scope and A pplication 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 Com pounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 M atrices: Rabbit, rat, bovine, monkey liver, or other tissues as designated in the validation report. Word 6/95 3M Environmental Laboratory ETS-8-7.0 000135 Page 1 o f 10 Page 81 3M M edical Departm ent Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 2.0 S u m m a r y o f M ethod_____________________________________________ ____________________ 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 o f a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z = 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity o f a compound by detecting daughter ions o f the selected parent ion. 3.0 D efin itio n s_____________________ ;_______ ;______________________________________ 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 E lectrospray 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 Spectrom etry, 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 C onventional 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 w ith 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 U ser's Guide). 4.0 W a r n in g s a nd C autio ns_________________________ ____________:_________________ 4.1 H ealth and Safety W arnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. 3MEnvironmental Laboratory ETS-8-7.0 000136 Page 2 o f 10 Page 82 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIM S EOO-1668 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. 4.2 Cautions: 4.2.1 Operate the solvent pumps below a back pressure o f 400 bar (5800 psi). 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 te r fe r en c e s____________________________________________________________________ _ 5.1 To minimize interferences when analyzing samples, Teflon shall 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 ipm 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 .i 6.1.2 Micromass Quattro II triple quadrupole Mass Spectrometer equipped w ith an electrospray ionization source. H P1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 Su ppl ie s a nd M a ter ials________________________________________________________ 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 m l centrifuge tubes 8.0 R e a g en t s and St a n d a r d s____________________________________________________ ________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water (ASTM type I), all water used in this method should be A TSM 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 .3 0 0 g a m m o n i u m acetate. Pour into a 2 0 0 0 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 000137 Page 3 of 10 Page 83 3M M edical 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.Q. 9.0 Sam ple 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 u ality Control _________________________ _______________ _________________ __ 10.1 M ethod Blanks and M atrix Blanks 10.1.1 Solvent blanks, method blanks, and matrix blanks are prepared and analyzed w ith 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 sample?. With a minimum o f 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range o f the initial calibration curve. Additional spike concentrations may fall in the lowrange o f the initial calibration curve. 10.3 Continuing Calibration Checks 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy 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 Ca libratio 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. 3MEnvironmental Laboratory ETS*8-7 -0 -0 0 0 1 3 8 Page 4 o f 10 Page 84 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 11.3 For purposes o f 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 tire 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 P rocedures_________________________ ________________________________ ______________ 12.1 A cquisition 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 SERs. 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 12.2.2.2 Inject/sample = 1 12.2.23 Cycle time = 9 minutes 3M Environmental Laboratory ETS-8-7.0 000139 Page 5 o f 10 Page 85 3M M edical D epartm ent Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-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 Quattro n Triple Quadripole 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 O pai 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/m in 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 123.7.2 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 0Q0140 Page 6 o f 10 Page 86 3M M edical D epartm ent 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 U ser's Guide). Ensure start and end sample number includes all samples to be analyzed. 13.0 D a t a A n a l y sis and C a lc ula tio n s____________________________ _____________________ 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 (jig/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 e t h o d P erfo rm 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 r2 value 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% o f the spiked concentration. 14.6 I f criteria listed in the method performance section axe 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 a - . i . * : . ' - *t ETS-8-7.0 r : t t ~; -- t : c <n *c< 000141 Page 7 o f 10 Page 87 3M M edical 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 andl discussed in the text o f the report. 1 15,0 P ollutio n P reventio n a n d W a ste M anagem ent_________________ ________________ 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 eco rds___________________________________________________ _ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder..Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms from MassLynx and store in the study folder. 16.5 Summarize data using suitable software (Excel 5.0+) and store in the study folder, refer to A ttachm ent A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 T a b l e s. D iagram s. F lo w c h a rts, and V alidation D ata_________________________ 17.1 Attachment A: ETS-8-7.0 Data summary spreadsheet 18.0 R efer en c es__________ '______________________________ ;_________________ ______________ 18.1 FACT-M-2.1, "Extraction o f Potassium Perfluorooctanesiilfonate or Other Fluorochemical Compounds from Liver 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-8-6.0 & 7.0-V-l 19.0 A ffec ted 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" 3MEnvironmental Laboratory ETS-8-7.0 000142 Page 8 o f 10 Page 88 3M M edical 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 ETS-8-7.0 Page 9 o f 10 000143 Page 89 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept Date o f Extraction/Analyst: Date o f Analysis/Analyst: Croup Dose Sample# Concentration ng/g initial w t. g Dilution Factor fin al cone. ng/g - * - Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (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 ETS-8-7.0 3M Environmental Laboratory A alerete n f T fwir " P v tr a r t TTemer P S /M .Q Page 10 o f 10 000144 Pase9 3M M edical 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 Tlmepoint Week 9 Sex Male Female PFOS pgfrnL Average* SD 1 7 2 * .824 n=2 26.6 5.88 n=2 Week 17 Male Female 13.7 1.13 n=2 20.3*2.37 n=2 Week 25 Male Female 8.7*1.93 n=2 15.8*5.27 n=2 Week33 Male Female 8.10*0.768 n=2 12.4 4.2 n=2 Week41 Week53 ' Male Female Male Female 5.65 0.303 n=2 10.4*4.37 n=2 426 * 0.052 n=2 7.95*1.76 n=2 NOTE: It is not possible to verily true recovery o f endogenous analyte from tissues without radiolabeled reference material. T lie only measurement of aocuracy avaJable a t this Urn, matrix spike studies, indicates that the data are quantitative to 30% or greater. Table 10. Data Summary for PFOS in Liver FACT-TOX-160--pg/g Group Group 1 Sex Male PFOSpgfg Average* SD 3.56*0.436 n=2 Female 923*2.04 n=2 NOTE: It Is not possible to verity true recouBiy of endogenous analyte from tissues without radtolabeled reference materia). Tba only measurement of accuracy available at this lim e, matrix spike studies, Indicates that the data are quantitative to 35% or greater. 3M Environmental Laboratory 3M Environmental Laboratory Page 21 000145 Page 91 3M M edical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Appendix E: Data Spreadsheets Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: FACT TOX-160 LIMS E00-1668 3M Environmental Laboratory 3M E nvironm ental Laboratory 000146 Pge 22 Page 92 3M M edical Departm ent Study: T-6295.22 Study: FACT-TOX-160,E00-1668 ProductNutnber(TcstSubstance): Matrix: Mcthod/Rcvision: Analytical EquipmentSystemNumber: InstrumentSoftware/Version: Filename: R-SquaredValue: Slope: Y-Intercept: DatesofExtraction/Analyst: DatesofAnalysis/Analyst: DateofDataReduction/Analyst: Box: FACT-TQX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS E00-1668 ExtendedRecoveryStudyFollowinga26WeekCapsuleToxicityStdywithPerfluorooctaneSulfonicAcid PotassiumSalt(PFOS;T-6295)inCynomolgusMonkeys T-6295.22 MonkeySerum ETS-8-4.2&ETS-8-5.2versusanextractedrabbitseracurve Amelia062498 Masslynx3.4 SeeAttachments SeeAttachments SeeAttachments SeeAttachments 09/07/01 R.WW 09/13/01 MMH 09/14/01 MMH 01-042,01-043 S am p le D a ta MONKEY SERUM Group Dose Sample# MethodBUc MatrixBlk WB09060I-H20B!k-l WB090601-H20Blk-2 RBS090601-ScraBlk-l QC- 50ppb 250ppb RBS09001-SeraBlk-2 MKS090601-SeraBlk-1 MKS090601-SeraBlk-2 MKS090601-50ppb-MS MKS090601-50ppb-MSD MKS90601-230ppb-MS MKS090601-250ppb-MSD Group 1 Week9 I05505M 105523M I05539F I05332F Group1 Week17 I05S05M I05523M I0539F I55S2F Group1 Week25 Group1 Week33 Group1 Week41 I05305M 105523M I05539F I05552F 105505M 05523M 105339F I05552F 10505M 10S523M I05539F Group 1 I05552F I05J05M W eek 53 I0 5 5 2 3 M I05539F I05552F PPOS Perfluorooctanesu]fonate PFOS Cone. ng/mL 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 37.3 48.4 77.3 36.7 20.9 50.8 24.8 33.3 29.1 48.5 47.3 17.6 12.3 31.0 20.0 14.0 11.4 14.7 19.1 Concentration ofPFOS ug/mLor %Ree <LOQ(0.00492ug/mL) <LOQ(0.00492ua/mU <LOQ(0.00492ug/mL) <LOO(0.00492ue/mU 0.0102 0.00771 129% 113% 110% 101% 17.8 16.7 30.7 2Z4 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 13.5 7.28 4.30 4.22 9.19 6.70 Mean PFOS ug/mL <LOQ <LOO 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/MSDRPD NA NA 0.00177 13% 8% 0.824 5.88 1.13 X37 1.93 5.27 0.768 4.20 0.303 4.37 0.052 1.76 DateEnteied/By: 09/26/01 LAC . . i. DateVerified/By: 12/6/01mmh IAC >4 3M Environmental Laboratory 000147 Page 93 3M M edical Department Study: T-6295.22 FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Study: FACT-TOX-160,E00-1668 ProductNumber(TestSubstance): Matrix: Method/Revision: AnalyticalEquipmentSystemNumber: InstrumentSoftwatc/Versioir, Filename: R-SquaredValue: Slope: Y-Intercept: DatesofExtraction/Analyst: DatesofAnalysis/Anslyst DateofDataReduction/AnalysL* Box: ExtendedRecoveryStudyFollowinga26-WeekCapsuleToxicityStdywithPerfluoroocraneSulfonicAcid PotassiumSaJt(PFOS; T-6295)inCynomolgusMonkeys T-6295.22 MonkeySerum ETS-8-4.2&ETS-8-5.2versusanextractedrabbitseracurve Amelia062498 Mass>ynx3.4 SeeBelow SeeAttachments SeeAttachments SeeAttachments 09/07/01 RWW 09/13/01 MMH 09/14/01 MMH 01-042,01-043 Sample Data MONKEY SERUM Group Dose Sample# MethodBtk MatrixBlk QC-50ppb 2S0ppb WB090601-H20Blk-1 WB090601-H20Blk-2 RBS09060l-SeraBlk-1 RBS090601-SeraBlk-2 MKS090601-SeraBlk-I MKS090601-SeraBlk-2 MKS090601-50ppb-MS MKS090601-50ppb-MSD MKS090601-250ppb-MS MKS090601-250ppb-MSD Group1 Week9 Group1 Week17 Group1 Week25 Group1 Week33 Group1 Week41 Group1 Week53 I05505M I05523M 105539F I05552F I05505M I05523M 05539F 1055J2F I05505M 105S23M 105539F 105552F I05505M 105523M I0S539F I05552F I05505M I05523M I05539F 105552F I05505M 105523M I05539F 105552F PFOS 3 Pcrfluorootancsulfonate Extraction VoL mL 1 1 1 1 1 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 0.54 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 Factor 1 1 1 1 1 1 1 1 1 1 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 PFOS Cone. ng/mL 0.00 0.00 0.00 0.00 10.22 7.71 72.55 64.80 279.46 258,06 44.56 45.81 76,79 44.81 45.75 37.49 48.36 77.32 36.72 20.90 50.76 24.75 33.28 29.10 48.51 47.32 17.59 12.50 30.97 20.02 13.96 11.40 14.71 19.09 Filename 010913049 A010913050 A010913051 A01O913O52 A010913053 A01913054 A010913055 AO1O913056 010913057 AO10913O58 A010913088 A010913089 A010913090 A01091309! A010913081 A0109I3082 A010913083 A010913084 A010913077 A010913078 A010913079 A010913080 A010913070 A0109I3071 A010913075 A010913076 A010913066 A010913067 A010913068 A0109I3069 A010913062 AO10913063 A010913064 A0109I3065 Concentration o r pros ug/mLor% Ree <LOQ(0.00492ug/mL) <LOQ(0.00492ua/mL) <LOQ(0.00492Ug/mL) <LOO(0.00492ug/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 RSD Std. Dev. MS/MSDRPD NA NA 0.00177 13.0% 8.24% 0.824 5.83 1.13 2.37 1.93 5.27 0.768 4.20 0.303 4.37 0.052 1.76 DateEntered/By: 09/26/01 LAC DaleVerified/By: 12/6/01mmh 3MEnvironmental Laboratory O0O14S Page 94 3M M edical Department Study: T-6295.22 Study: FACT-TOX-160,E00-I668 Product Number(TestSubstance): Matrix: Method/Rcvision: Analytical EquipmentSystemNumber: InstrumentSoftware/Version: Filename: R-SquaredValue: Slope: Y-Intercept: DatesofExtraction/Analyst: DatesofAnalysis/Analyst DateofDataReduction/Analyst: Box: Sample Data FACT-TOX-160 Covance# 6329*268 Analytical Report: FACT-TOX-160 LIMS E00-1668 ExtendedRecoveryStudyFollowinga26-WeekCapsuleToxicityStdywithPerfluorooctaneSulfonicAcid PotassiumSa!t(PFOS;T-6295)inCynoraolgusMonkeys T-6295.22 MonkeySerum ETS-8-4.2&ETS-8-5,2versusanunextractedcurve Amelia062498 Masslynx3.4 SeeAttachments SeeAttachments SeeAttachments SeeAttachments 08/3I/OI RWW 09/05/01,09/06/01 MMH 09/06/01,09/07/01 MMH 01-042 MONKEY SERUM Group Sample# PFOS Concentration Dose Cone. ofPFOS ng/mL ug/mLor%Ree MethodBlank MKS083101-H20Blk-l 0.18 <LOQ(0.0012ug/mL) MKS08310l-H2OBlk-2 0.23 <LOO(0.0012ng/mL) MatrixBlank MKS083101-SetaBlk-l 9.69 0.00775 MKS083101-SeraBlk-2 9.05 0.00724 QC MKS083J01-MS-25ppb 32.6 118% MKS083101-MSD-25Bob 25.9 84% MK5083101-MS-500ppb 448 m% MKS0831O1-MSD-5O0ppb 449 112% PFOS*Pcrfluorooctanesulfcnatc LOQ=1.0ng/mLinstandardcalculatesto 1.2ng/mLinserum. LOQ* (1.0ng/mLunextstandard* 1.25extdii factor * 1dilutionfactoryiOOO**0.012ug/mLinserum Mean PFOS ug/mL <LOO 0.00750 101% 111% DateEntered/By: DateVerified/By: 09/12/01 LAC 16/6/0] mmh IkO RSD Std. Dev. MS/MSDRPD NA 0.000362 34% 0% 3M Environmental Laboratory 000149 Page 95 3M M edical Departm ent Study: T-6295.22 FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Study: FACT-TOX-160,EOO-1668 ProductNumbcr(TestSubstance): Matrix: Method/Revision: AnalyticalEquipmentSystemNumber InstrumentSoftware/Version; Filename: R-SquaredValue: Slope: Y-lntercepf. DatesofExtracdon/Analyst: DatesofAnalysis/Analysi: DateofDataReducdon/Analyst: Box; Sample Data ExtendedRecoveiyStudyFollowinga26-WeekCapsuleToxicityStdywithPerfluorooctaneSulfonicAcid PotassiumSalt(PFOS;T-6295)inCynomolgusMonkeys T-6295.22 MonkeySerum ETS-8-4.2& ETS-8-5.2versusanunextractedcurve Amelia062498 Masslynx 3.4 SeeBelow SeeAttachments SeeAttachments SeeAttachments 08/31/01 RWW 09/05/01,09/06/01 MMH 09/06/01,09/07/01 MMH 01-042 MONKEY SERUM Group Sample# Initial Extraction Surrogate Dose VoL Dilution Verified raL Factor MethodBlank MKS083101-H2OBlk-1 1 1.25 ConfirmedHigh MKS08310-H2OBlk-2 1 1.25 ConfirmedHigh MatrixBlank MKS083101-SeraBlk-1 1.25 ConfirmedHigh MKS083101-SeraBlk-2 1 1.25 ConfirmedHigh QC MKS083101-MS-2Sppb 1 1.25 ConfirmedHigh MKS083101-MSD-25ppb 1 1.25 ConfirmedHieh MKS083101-MS-500ppb 1 1.25 2ndanalysisOK MKS083101-MSD-500ppb 1 1.25 ConfirmedHigh PFOS=Peifluorooctanesulfonate LOQ* 1.0ng/mLinstandardcalculatesto1-2ng/mLinserum. LOQ=(1.0ng/mLuncxtstandard*1.25extdiifactor*l dilutionfactoryiOOO*0.0012ug/mLinserum PFOS Dilution Factor 1 1 1 1 1 l 1 1 DateEntered/By: 09/12/01 LAC DateVerified/By: 16/6/01mmh PFOS Filename Concentration Cone. ofPFOS ng/mL ug/mLor %Ree 0.18 A010906019 <LOQ(0.0012ug/mL) 0.23 AO10906020 <LOQ(0.0012ne/mLl 9.69 A010906021 0.00775 9.05 AOIO9O6022 0.00724 32.62 AOl0906023 118% 25.87 AO10906024 84% 447.77 AD10906025 111% 448.57 A010906026 112% Mean PFOS ug/mL <LOQ 0.00750 101% 111% RSD Std.Dev. MS/MSDRPD NA 0.000362 34% 0% 3M Environmental Laboratory Q O O IS O Page 96 3M M edical Department Study: T-6295.22 Study: FACT-TOX-160, E00-I668 ProductNumber(TestSubstance): Matrix: Method/Revision: AnalyticalEquipmentSystemNumber: InstrumentSoftware/Version: Filename: R-SquaredValue: Slope: Y-Inteieept: DatesofExtraction/Analyst: DatesofAnalysis/Analyst: DateofDataReducdon/Analyst: Box: Sample Data FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS E00-1668 ExtendedRecoveryStudyFollowinga26-WeekCapsuleToxicityStdywithPerfluorooctaneSulfonicAcid PotassiumSalt(PFOS;T-6295)inCynomolgusMonkeys T-6295.22 MonkeySerum ETS-8-4.2&ETS-8-5.2versusanunexCractedcurve Amelia062498 Masslynx3.4 SeeAttachments SeeAttachments SeeAttachments SeeAttachments 10/02/01 RWW 10/04/01 MMH 10/05/01 MMH 01-046 MONKEY SERUM Group Sample# PFOS Concentration Mean Dose Cone. ofPFOS PFOS ng/mL ug/mLor % Ree ug/mL MethodBlank WBI00201-H20Blk-l 0.00 <LOQ(0.00313ug/mL) MatrixBlank WB100201-H20Blk-2 RBS100201-SeraBlk-1 0.00 <LOQ(0.00313ug/mL) <LOO 0.09 <LOQ(0.00313ug/mL) RBS100201-ScraBlli-2 0.13 <LOQ(0.00313ug/mL) <LOQ MKS10020t-SeraBlk-3 7.23 0.00904 MKS100201-SeraBlk-4 7.14 0.00893 0.00898 QC MKS100201-MS-2ug/mL 8.69 113% MKS10020l-MSD-2ug/gmL 8.34 109% 111% MKS1002C1-MS-10ug/mL 45.6 115% MKS100201-MSD-10ug/mL 46.5 117% 116% PFOSPeriluorooctanesulfonate LOQ=2.50ng/mLinstandardcalculatesto3.13ng/mLinserum. LOQ=(2.50ng/mLunextstandard*1.25extddlfactor*1dilutionfactor)/1000*0.00313ug/mLinserum DateEntered/By: DateVerified/By: 10/11/01 LAC 12/10/01mmh \ \ r RSD Std. Dev. MS/MSDRPD NA NA 0.0000795 4% 2% 3M Environmental Laboratory 000151 Page 97 3M M edical 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 ProductNumber(TestSubstance): Matrix: Method/Revision: AnalyticalEquipmentSystemNumber InstrumentSoftware/Version: Filename: R-SquaredValue: Slope: Y-Intercept: DatesofExtraction/Analyst: DatesofAnalysis/Anaiyst: DateofDataReduction/Analyst: Box: ExtendedRecoveryStudyFollowinga26-WeekCapsuleToxicityStdywithPerfluorooctaneSulfonicAcid PotassiumSalt(PFOS;7*6295) inCynomolgusMonkeys T-6295.22 MonkeySerum ETS-8-4.2&ETS-8-5.2versusanunextractedcurve Amelia062498 Masslynx3.4 SeeBelow SeeAttachments SeeAttachments SeeAttachments 10/02/01 RWW 10/04/01 MMH 10/05/01 MMH 01-046 Sample Data MONKEY SERUM Group Sample# Initial Extraction Surrogate PFOS Dose Voi. Dilution Verified Dilution mL Factor Factor MethodBlank WB10020I-H20Blk-1 1 1.25 NA 1 WB100201-H20Blk-2 1 1.25 NA 1 MatrixBlank RBS100201-SeraBlk-1 1 1.25 NA 1 RBS100201-SeraBIk-2 1 1.25 NA 1 MKS100201-SeraBUc-3 1 1.25 NA 1 MKS100201-SeraBllt-4 1 1.25 NA 1 QC MKS100201-MS-2ug/mL 1 1.25 OutHigh 200 MKS10020l-MSD-2ug/gmL 1 1.25 NA 200 MKS100201-MS-10ug/mL 1 1.25 NA 200 MKS100201-MSD-10uft/mL 1 1.25 NA 200 PFOS=Perfluorooctanesulfonate LOQ932J0ng/mLinstandardcalculatesto3.13ng/mLinserum. LOQ=(2.50ng/mLunext standard* 1.25extdii factor *I dilutionfactor)/10000.00313ug/mLinserum DateEntered/By: 10/11/01 LAC DateVerified/By: 12/10/01nunh PFOS Filename Concentration Cone. ofPFOS ng/mL ug/mLor %Ree 0.00 A0U004082 <LOQ(0.00313ug/mL) 0.00 A011004083 <LOO(0.00313ug/mL) 0.09 A01004084 <LOQ(0.00313ug/mL) 0.13 A0I1004085 <LOO(0.00313ug/mL) 7.23 A011004086 0.00904 7.14 A011004087 0.00893 8.69 A011004088 113% 8.34 A011004089 109% 45.57 A011004090 115% 46.54 A011004091 117% Mean PFOS ug/mL <LOQ <LOQ 0.00898 111% 116% RSD Std.Dev. MS/MSDRPD NA NA 0.0000795 4% 2% 3M Environmental Laboratory 000152 Page 98 3M M edical Departm ent Study: T-6295.22 Study. FACT-TOX-160, EOO-166* Product Num bofTcst Substance): M atri x : Method/Revision: Analytical Equipment System Num ber Instrument Software/Versioo: Filename: R-Squared Value: Slope: Y-lntercept Dates o f Extraction/Analyst: Dates o f Anaiysis/Analyst: Date o f Data Reduction/Analyst Box: SampleData FACT-TOX-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 Scrum ETS-8-4,2 & ETS-8-5.2 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 10/05/0] MMH 01-046 MONKEYSERUM Group Dose Sample# Method Blank W B10020-H 20 Blk-1 W B 100201-H20 Blk-2 M atrix Blank RBS10020l-ScraBlk-l RBS10020I-Sera Blk-2 M KS100201-Sera Blk-3 M K S100201-Sera Blk-4 QC MKS I00201-MS-2 ug/mL MKS !00201-MSD-2 ua/ranL MKS1002OI-MS-I0 ug/mL MKS 10020 l-M SD -10 ua/mL PFOS Perfluorooctanesulfocute PFOS Cone. ne/raL 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 ue/mL) <LOQ (0.00492 ug/mL) <LOO fO.00492 ue/mL) 0.00802 0.00790 104% 99% 119% 121% Mean PFOS ug/mL <LOO <LOO 0.00796 102% 120% RSD Std. Dev. MS/MSD RPD NA NA 0.0000849 5% 2% Date Entercd/By. Date Verified/ By 10/11/0] LAC 12/10/01 mmh 3M Environmental Laboratory 000153 Page 99 3M M edical Department Study: T-6295.22 FACT-TOX-160 Covance# 6329-268 A nalytical Report: FACT-TOX-160 LIMS E00-1668 Study: FACT-TOX-160, EOO-1668 Produci Numbcr(Test Substance): Matrix: Mcthod/Rcvisico: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intcrccpt Dates o f Extraction/Analyst: Dates o f Analysis/Analyst: Date o f Data Reductioa/Amdyst: Box: SampleData Extended Recovery Study Following a 26-Wcck Capsule Toxicity Stdy with Peifluorooctane Sulfonic Acid Potassium Sa!t(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 Amelia 062498 Masslynx 3.4 See Below See Attachments See Attachments See Attachments 10/02/01 RWW 10/04/01 MMH 10/05/01 MMH 01-046 MONKEYSERUM C roup Dose Sample H Method Blank M atrix Blank W B10020I-H2O Blk-1 W B I00201-H20 Blk-2 RBS100201-Sera Blk-1 RBS100201-Sera Blk-2 M K S100201-Sera Blk-3 M K S10020]-Sera BUc-4 QC MKS 100201-MS-2 ug/mL MKS100201-MSD-2 uc/amL M KS10020i-MS-10 ug/mL M KS10020I-MSD-IO ue/tnL PFOS Pcrfluorooctanesulfonate Initial Voi. m l. 1 l 1 1 1 1 1 1 1 l Extraction Dilution Factor 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Date Entered/By: Date Verified/ By* 10/11/01 LAC 12/10/01 mmb IM ' Surrogate Verified NA NA NA NA NA NA NA NA NA NA PFOS Dilution Factor 1 1 l 1 1 I 200 200 200 200 PFOS Cone. ne/mL 0.00 0.00 0.00 0.00 8.02 7.90 9.96 9.49 58.97 60.27 Filename A 011004082 A0U004083 A 011004084 A 0 11004085 A 0 11004086 AOI1004087 A011004088 A 011004089 A011004090 A 011004091 Concentration of PFOS ue/xnL o r % Ree <LOQ (0.00492 ug/mL) <LOO (0.00492 uc/m Ll <LOQ (0.00492 ug/mL} <LOO (0.00492 ua/mLl 0.00802 0.00790 104% 99% 119% 121% Mean PFOS ne/m L <ux> <LOO 0 .0 0 7 9 6 102% 120% RSD S td . Dev. MS/MSD RPD NA NA 0 .0 0 0 0 8 4 9 5% 2% 3M Environmental Laboratory 0001S4 Page 100 3M M edical Department Study: T-6295.22 FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS E00-1668 Study. FACT-TOX-160, EO-16, Product Numbcr(Test Substance): Matrix: MethtxPRevisioo: Analytical Equipment System Number: Instrument Software/Versiotu Date o f Extraction/Analyst: Date of Anaiysia/Anaiyjt Date of Data Reduction/Analyst: SampleData Extended Recovery Study Following a 26-Week Capsule Toxicity Stdy with Periluorooctane Sulfonic Add Potassium SaJt(PFOS; T-6295) in Cyoomolgus Monkeys T-6295.22 Monkey Liver ETS-8-6.0 & ETS-S-7.0 versus an extracted rabbit liver curve Amelia 062498 Filename: See Attachments Masslynx 3.4 R-Squarcd Value: See Attachments 09/05/01 RWW 09/10/01,09/13/01,09/17/01 MMH Slope: Y-Intcrccpt: Sec Attachments See Attachments 09/11/01,09/14/01,09/18/01 MMH Box#: 01-042 MONKEYLIVER Group Dose Method Bik Matrix Bik QC Sample# RBL090501-H2O Blk-1 RBL090501-H2O Blk-2 KBL090501-LiverBlk*t RBL090501-Uver Blk-2 RBL090501-300 ppb-MS RBL090501-300 rob-MSD PFOS Calc. Coac. tltls 0.00 0.00 0.00 0.00 322 323 Concentration of PFOS ue/e or % Rec <LOQ (0.00629 ug/g) <LOO (0.00629 ue/el <LOQ (0.00629 ug/g) <LOO (0.00629 uc/fl 102% 102% Mean PFOS utAt <LOO <LOO 102% MKL090501-300 ppb-105505M-MS 879 298% MK1/09050I-300 m>b-IOS505M-MSD 854 290% 294% Group 1 I05505M 3251 3.25 I0S523M 3868 3.87 3.56 J05539F 10675 10.7 I05552F 7788 7.79 9.23 PFQS *Periluorooctanesulfonate * Matrix spikes weren't spiked at the appropriate level. Sample will be re-spiked, at the appropriate levels, at a later date. USD Std. Dev. MS/MSD r p d NA NA 0% 1% 0.436 2.04 Date Enlered/Aoalyst: 09/12*11, 09/26/01 LAC Dale Verified/Analyst: 12/10/01 mmh U l oMjasioa 3M Environmental Laboratory 000155 Page 101 3M M edical Departm ent Study: T-6295.22 FACT-TOX-160 Covance# 329-268 A nalytical Report: FACT-TOX-160 LIMS E00-1668 Study: FACT-T0X-I6, E00-I668 Product NumberfTesl Substance): Matrix: Method/Revision: Analytical Equipment SystemNumber: Instrument Software/Version: Date o f Extraoion/Analysi: Dateof Aaaiyui/Aiuiyst: Dateof Data Reduclion/Analyst: SampleData Extended Recovery Study Following a 26-Week Capsule Toxicity Sidy with Perfluorooctane Sulfonic Acid Potassium Salt(PFOS; 1-6295) in Cynomoigus Monkeys T-6295.22 Monkey Liver ETS-8-6,0 &ETS-8-7.0 versus an extracted rabbit liver curve Amelia 062498 Masslyax3.4 Filename: See Below R*Squared VSee Attachments 09/05*1 RWW Slope: See Attachments 09/10*1,09/13/01,09/17/01 MMH Y-Intcrcept: SecAttachments 09/11/C'l, 09/14/01,09/18/01 MMH Box#: 01-042 MONKEYLIVER Group Dose Method Blk Matrix BUc QC Group 1 Sample# RBL090501-H20 Blk-1 RBL090501-H20 Blk-2 RBLO90301-Liver Blk-1 RBLO90501-Liver Blk-2 RBL090501-300 ppb-MS RBL090501-300 oob-MSD MKL90501-3W) ppb-!05505M-MS MKLO90501-300 PO-I05505M-MSD 1O550SM I05523M I05539F 10S5S2F Surrogate Verified NA NA NA NA NA NA NA NA NA NA NA NA Initial Wt. 8 1.000 1.000 1.000 1.000 1.000 1.000 1.0160 1.0160 1.0160 1.0650 1.0155 1.0664 Total Mats of Liver a NA NA NA NA NA NA NA NA NA NA NA NA PFOS Perfluorooctanesulfonale * Matrix spikes weren't spiked at the appropriate level. Sample will be re-spiked, at the appropriate levels, at a later date. PFOS Cone, nt / t 0.00 0.00 0.00 0.00 321.51 322.78 83.93 83.43 66.07 82.38 21.68 L6.61 PFOS Dihuioa Factor 1 1 1 1 1 1 50 50 50 50 500 500 PFOS Calc. Cone. */ 0.00 0.00 0.00 000 322 323 879 854 3231 3868 1067S 7788 Filename Concentration of PFOS Mean PFOS AOI0910016 <LOQ(0.00629 ufi/g) AOI0910017 <LOO (0.00629 ue/rt <LOO AO10910015 <LOQ (0.00629 ug/g) A0109100I9 <LQO (0.Q629u/p> A010910022 102% <LOO A010910023 102% 102% A0I0917016 298% * A0I0917017 290% * 294% A010917U18 3.23 A01O917O19 3.87 3.56 A010913020 10.7 A010913021 7.79 9.23 RSD StiLDev. NA NA 0% 3% 0.436 2.04 Date Entered/Analyst: 09/12/01,09/26/01 LAC Date VerUkd/Analysl: 12/ 10/01 mmh 3M Environmental Laboratory 000156 Page 102 3M M edical Department Study: T-6295.22 FACT-TOX-160 Covance# 329-268 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Study: FACT-TOX-160. EOO-1665 Product Niunber(Test Substance): Matrix: Method/Rcvision: Analytical Equipment SystemNumber: Instrument Software/Version; Date of Extraction/Analysl: Date of Anslysis/Analyst: Date ofData Reduction/Analyst: Extended Recovery Study Following a 26-Week CapsuleToxicity Stdy with Periluorooctane Sulfonic Acid Potassium SaJt(PFOS; T-6295) in Cynomolgus Monkeys T-6295.22 Monkey Uver ETS-S-6.0 A ETS-8-7.0 versus an extracted rabbit liver curve Amelia 062498 Filename: See Attachments Masilynx 3.4 R-Squared Value: See Attachments 10/02/01 RWW Slope: See Attachments 10/04/01 MMH Y-lntercept: See Attachments 10/05/01 MMH Box#: 01-046 SampleData MONKEYLIVER Group Sample# PFOS Concentration Dose Method Blk RBL100201-H20 Blk-3 Cate. Cone ok/* 0.00 of PFOS ue/e or % Ree <LOQ(0.0126 ug/g) RBL10201-H20 Blk-4 0.00 <LOO 10.0126 ue/e) Matrix 81k QC RBL100201-Lrver Btk-1 RBL100201-Liver Blk-2 MKLI00201-2 ug/g-I05505M-MS MKL100201-2 ue/e-W5505M-MSD MKL100201-10 ug/g-I0555M-MS MKL100201-10 us/(t-I05503M-MSD 0.00 <LOQ (0.0126 ug/g) 0.00 <LOO(0.0126 ub/k) 4364 55% 3554 15% 9441 60% 5761 <LOO (0.0126 u*/K Group l 105503M 3237 3.24 PFOS * PeriUxmoctanesul fonate i 1:500 dilutionwas too dilute, theseextracts were re-diluted at 1:50 and analyzed 10/16/01. LAC 11/28/01 Mean PFOS ui/x <LOQ <LOO 35% 60% RSD Sti Dev. MS/MSD RPD NA NA 112% NA DateEntered/Analyst: 10/1MH LAC Date Verified/Analyst: 12/lQ/M mmh Study: FA CT-TO X -160. EOCM66X Product Nnmber(Tea Substance): Matrix; Mcthod/Revition: Analytical Equipment SystemNunr.ber. Instrument Software/Version: DateofExtraction/Anaiyst: Dateof Analysis/Analyst: Dateof Data Reduction/Analyst: SampleData Extended Recovery Study Followinga 26-Week Capsule Toxicity Stdy with Perfluorooctane Sulfonic Add Potassium Salt(PFOS; T-629S) in Cynomolgus Monkeys T-6295.22 Monkey Uver ETS-8-6.DA E T S -8-7.0 versus an extracted rabbit livercurve Amelia 062498 Filename: See Attachments Masilynx 3.4 R-Squared Value: See Attachments 10/02AH RWW Slope: See Attachments 10/16/01 MMH Y-lnterccpt: See Attachments 10/16/01 MMH Box#: 01-046 MONKEYLIVER Group Dose Method Blk Matrix BDt QC Sample# RBL100201-H2O BIk-3 RBL100201-H2O Blk-4 RfiLI0020l-Uver Blk-1 RBL100201-Uver Blk-2 MKL100201-2 ug/g-I05505M-MS MKL100201-2 ue/*-1055O5M-MSD MKL100201-10 ug/g-I05505M-MS MKL100201-10 UK/B-105503M-MSD p ro s Calc Cone C/K 0.00 " 0.00 1.33 0.25 3804 2995 9719 7466 Concentration of PFOS at/* or % Ree <LOQ (0.0126 ug/g) <LOO(0.0126 UK/8 <LOQ(0.0126 ug/g) <LOO(0.0126 un/nl 29% 11% 63% 41% Mean PFOS UR/C <LOO <LOO 9% 52% BSD StiLDev. MS/MSD RPD NA NA 438% 42% Groan! 05505M 3215 3.21 PFOS * Periluorooctaaesullbnate NOTE: Datawere not wtthin criteria, alt diluted samples were redUtUed 1:50 from original extract and analyzed on 10/24/01. LAC 11/28/01 Date Entered/Analyst: IQ/29/0I LAC Dale Verified/Analyst: 12/10/01 nunh / 3M Environmental Laboratory 000157 Page 103 3M M edical Departm ent Study: T-6295.22 FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS E00-1668 Study: FACT-TOX-160, EOO-665 Product NumberfTest Substance): Matrix: Method/Revisicn: Analytical Equipment System Number. Instrument Software/Version: Date of Extraction/Analyst; Date ofAnalysis/Analyst: Date ofData Reduction/Analyst: SampleData Extended Recovery Study Following a 26-Week CapsuleToxicity Stdy with Feifluoreocune Sulfonic Acid Potassium Salt(PF05; T-6295) in Cynomolgus Monkeys T-6295.22 Monkey Liver ET5-8-6.0 & ETS-8-7.0 versus an extractedrabbit Jivexcurve Amelia 062498 Filename: See Attachments Masslynx 3.4 R-Squared Value: See Attachments 10/02/01 RWW Slope: See Attachments 10/24/01 MMH Y-Intercept: See Auachtnems 10/26/01 MMH Box*: 01-046 MONKEYLIVER Group Dose Sample* Method Blk RBL10020I-H20 Blk-3 RBL100201H20 Blk-4 Matrix Blk RBL100201-LiverBUt-l RBL100201-Uver Blk-2 QC MKL100201-2 ug/g-I05505M-MS MKL100201-2 UR/K-I05505M-MSD MKL100201-10ug/g-I05505M*MS MKLI00201-10 UX/K-105505M-M5D Group 1 I05505M PFOS Perfluorooctanesulfonate PFOS Caie. Cene. f if 0.00 0.00 0.00 o.ou 14945 12530 38873 33522 11282 Concentration of PFOS t/e or % Ree <LOQ (0.0126 ug/g) <LOO (0.0126 ug/g) <LOQ (0.0126 ug/g) <LOO (0.0126 UK/E> 178% 61% 268% 216% 11.28 Mean PFOS Vi <LOO <LOO 119% 242% RSD Std. Dev. MS/MSDRPD NA NA 98% 21% DateEntered/Analyst: 11/06/01 LAC Date Verified/Anaiyst: 12/10/lH mmb Study: FACT-TOX-160, EOO-1668 Product NumberfTest Substance): Matrix: Mcthod/Revision: Analytical Equipment System Number Instrument Software/Venion: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reductkm/Analyst: SampleData Extended Recovery Study Followinga 26-WeekCapsule Toxicity Stdy with FerfluorooetaneSulfonic Acid Potassium SaltfPFOS; T-6295) in Cynomolgus Monkeys T-6295.22 Monkey Liver ETS-8-6.0A ETS-8-7.0 versus an extracted rabbitlivercurve Madeltne041098 Filename: See Attachments Masslynx 3.4 R-Squarcd Value: See Attachments 11/07/0] RWW Slope: See Attachments 11/15/01,11/16/01 MMH Y-lntercept See Attachments 11/16/0], 11/19/01 MMH Box*: 01-046 MONKEYLIVER Group Dose Method Blk Matrix Blk QC Group 1 PFOS- Sample# RBL11070I-H2O BLk-1 RBU1070I-H2O Blk-2 RBLl I0701-H2O Blk-3 RBL110701-H2O Blk-4 RBLl 10701-LiverBlk-I RBLl 10701-LiverBHt-2 RBLl 10701-Liver Blk-3 RBL11070]-Uver Blk-4 MKLII070I -2 ug/g-W5505M-MS MKL110701-2 UK/K-I05505M-MSD MKL110701-10 o*/g-10550JM-MS MKL110701-10 UC/8-I05S05M-MSD I05505M PFOS Calc. Cose, oefe 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 6328.44 6134.39 11836.78 10657.49 4241.25 Concentration of PFOS us/e or % Ree <LQQ (0.00629 ug/g) <LOQ (0.00629 ug/g) <LOQ (0.00629 ug/g) <LOO 0.00629 usJt) <LOQ (0.00629 ng/g) <LOQ (0.00629ug/g) <LOQ (0.00629ug/g) <LOO <0.00629 he/> 100% 90% 73% 61% 4.24 Mean PFOS ng/g <LOO <tOQ 95% 67% RSD Std. Dev. MS/MSD RPD 0 NA 0 NA 10% 17% Date Entered/Analyst: Date Verified/Anaiyst: I1/26/C1 LAC I2/I0/C1 nunh (AC. 0^jas|&2- 3MEnvironmental Laboratory 000158 Page 104 3M M edical Department Study: T-6295.22 FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS EOO-1668 Study: FACT-TOX-160, 00*1668 Product Nuraber(Tt Substance): Analytical Equipment System Number Instrument Software/Version: Dateof Extraction/Analyst Date of Analyiis'Analysl: Dateof Data Reduction/Analyst: Extended Recovery Study Following a 26-Week Capsule Toxicity Sidy with Periluorooctane Sulfonic Acid Potassium Salt<PFOS; T-629S) in Cynomolgus Monkeys T-6295 22 Monkey Liver ETS-8-5.0 A ETS-8-7.0 versus an extracted rabbit liver curve Amelia 062498 Filename: See Below Ma&slynx 2.4 R-Squared Value: See Attachments 10/02/01 RWW Slope: See Attachments 10/04/01 MMH Y-lfUercept See Attachments 10/05/01 MMH Box#: 01-046 SampleData MONKEYLIVER Group Dose Method Blk Matrix Blk QC Group 1 Sample# RBL10201-H20 Blk-3 RBL100201-H20 Blk-4 RBL100201-Liver Blk-1 RBL100201-Liver Blk-2 MKL10020I-2 ug/g-I05505M-MS MKL10020I-2 UH/0-IO35O5M-MSD MKL100201-10 ug/g-!03505M-MS MKL10020I-10 u*/r-I05J03M-MSD I05505M Surrogate Verified NA NA NA NA NA NA NA NA NA Initial W i g 1.0000 1.0000 1.0000 1.0000 1.0232 1.0232 1.0232 1.0232 1.0232 Total Mass of Liver K NA NA NA NA NA NA NA NA NA PFOS * Periloorooctanesulfonate * 1:500 dilution was toodilute, these extracts were re-diluted at 1:30 and analyzed J0/16/01. LAC 11/28/01 PFOS Cone n t ll 0.00 0.00 0.00 0.00 89.30 72.72 19.32 11.79 66.24 PFOS Dilution Factor 1 50 50 500 500 SO PFOS Calc. Cene. M /i 0.00 0.00 0.00 0.00 4364 3554 9441 5761 3237 Date Entered/Analyst: 10/11/01 LAC Date Verified/Analyit: 12/10/01 mmh FBcaame AO11004030 AOt1004031 A0UOO4O32 AOI1004033 A011004038 AOI1004039 AOI1004040 AOI1004041 AOI1004037 Concentration of PFOS uc/or% Ree <LOQ <0.0126 ug/g) <LOO (0.0126 ux/sl <LOQ <0.0126 ug/g) <LOO<0.0126 ug/z> 55% 19% 60% <LOO<0.0126 ug/*) 3.24 Mean PFOS U t/l <LOO <LOQ 35% 60% RSD Std. Dev. MS/MSD RPD NA NA 112% NA Study: FACT-TOX-160, EOO-1668 Product NuraberfTest Substance): Matrix: Metbod/Revisioo: Analytical Equipment SystemNumber. Instrument Software/VersiM: Date o f Extraction/Analyst: Dateof Anaiysis/AnaJyjt: Dateof Data Reduction/Analyst: SimpleDita ExtendedRecovery Study Following a 26-Week Capsule Toxidty Sidy with PeriluorooctaneSulfonic Acid Potassium Salt(PFOS; T-6295) in Cynomolgus Monitors T-6295.22 Monkey Liver ETS-8-6.OftETS-8-7.Ow isan extracted rabbit liver curve Amelia 062498 Filename: Sec Below Masslyxx 3.4 R-Squared Value: See Attachments 10/02/01 RWW Slope: See Attachments 10/16/01 MMH Y-Intercept: See Attachments 10/16/01 MMH Box#: 01-046 MONKEYLIVER Group Dote Method Blk Sample# RBL100201-H20 Blk-3 RBL100201-H20 Blk-4 Surrogate Verified NA NA Initial WL g 1.0000 J.0000 Total Mass of Liver c NA NA PFOS Coae. at/ 1 0.00 0.00 Matrix Blk QC RBLUXCOl-LiverBlk-1 RBL100201-Uver Blk-2 MKL100201-2 Ug/g-I0S505M-MS MKL100201-2 ue/e-I0550M-MSD NA NA NA NA 1.0000 1.0000 1.0232 1.0232 NA NA NA NA 1.33 0.25 77.84 61.29 MKL100201-10 vg/g-IOSSOSM-MS MKL100201-10 ue/g-105505M-MSO NA NA 1.0232 1.0232 NA NA 198.88 152.79 Group t I05505M NA 1.0232 NA 65.79 PFOS " PeriluoroocuuKsulfosale NOTE: Dau were not witkin criteria, all dilutod samples were rtdtimed 13 ) from original extract and analyzed on 10/24/0!. LAC 11/28/01 PFOS Dilution Factor 1 ] 1 1 50 SO 50 50 50 PFOS Calc. Cane na/* 0.00 0.00 1.33 0.25 3804 2995 9719 7466 3213 DateEntered/Analyse 10/29/01 LAC Date Verified/Analyst: 12/10/01 mmh Filename AOI1016Q30 A011016031 A011016032 A0110I6033 A011016038 AOI1016039 A011016040 A011016041 AOI1016037 Concentration of PFOS oe/*ar% Ree <LOQ (0.0126 ug/g) <LOO <0.0126 ui/*> <LOQ <0.0126 ug/g) <LOO <0.0126 us/e) 29% 11% 63% 41% 3.21 Mean PFOS " e'e <LOQ <LOO 9% 52% RSD Std. Dev. MS/MSD RPD NA NA 438% 42% 3M Environmental Laboratory 000159 Page 105 3M M edical 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(Tcjt Substance): Mauix: Method/Revision: Analytical Equipment SystemNumber: Instrument Scftware/Veraion: Date of Exinctiofl/An&iyst: Dale of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple D ata ExtendedRecovery StudyFollowing a 26-Week Capsule Toxicity Stdy with Perfluorooctane Sulfonic Add Potassium SaltfPTOS; T-6295) in Cyoonwlgus Monkeys T-6295.22 Monkey Liver ETS-8-6.0 ETS-8-7.Qversus an extracted rabbit liver curve Amelia 062498 Filename: See Below Masslyux 3.4 10/02/01 RWW R-5quarcdValue: See Attachments Slope: See Attachments 10/24/01 MMH Y-Inlercept: See Attachments 10/26/01 MMH Box#: 01-046 M O N K EY LIV ER Group Dose Sample# Method Blk Matrix Blk QC Group 1 RBL100201H20 Blk-3 RBLI0201-H20 Blk-4 RBL100201-Liver Blk-1 RBL100201-LiverBQc-2 MKL100201-2 ug/g-I05505M-MS MKL1002I-2 ue/z-t05S05M-MSD MKL100201-10 ug/g-105505M*MS MKL10020M0 ug/a-K)5505M-MSD IOJ505M Surrogate Verified Out High Out Hitch NA NA Out High OutHieh Otit High Out High Out High Initial Wt. B 1.0000 1.0000 1.0000 1.0000 1.0232 1.0232 1.0232 1.0232 1.0232 Total Mass of Liver a NA NA NA NA NA NA NA NA NA PFOS Cone. R/i 0.00 0.00 0.00 0.00 305.83 256.41 795.50 686.00 230.87 NOTE: Data were not consistent with previousanalyses. Samples were re-extracted, diluted 1:50, and analyzedon 11/16/01. LAC 11/28/01 PFOS Dilution Factor 1 1 1 1 50 50 50 50 50 PFOS Calc. Cone ne/e 0.00 0.00 0.00 0.00 14945 12530 38873 33522 11282 Date Entered/Analyst 11/06/01 LAC Date Vcrifcd/Anaiyst: 12/10/01 nunh Filename A011024030 A011024031 A011024032 A011024033 AO11024038 A011024039 AO11024040 A011024041 A011024037 Concentration of PFOS ue/e or % Ree <LOQ (0.0126 ug/g) <LOO (0.0126 uc/el <LOQ(0.0126 ug/g) <LOO 10.0126 uz/el 178% 61% 268% 216% 11.3 Mean PFOS ots/K -LOO <LOO 119% 242% RSD Std. Dev. MS/MSD RPD NA NA 98% 21% Study: FACT-TOX-160, EOO-1668 Product NamberfTest Substance): Matrix: Method/Reviaioo: Axuytical Equipment SystemNumber Instrument Software/Venon: Dateof Extraction/Analysf Dateof Analyris/Analyst DateofData Reduction/Analyst Sam ple D ata ExtendedRecovery StudyFollowinga 26-Week Capsule Toxicity Stdy with Perfluorooctane SulfonicAdd Potassium SaltfPFOS; T-6295) in Cynomdgus Monkeys T-6295.22 Monkey Liver ETS-8-6 0 St ETS-8-7.0versus an extracted rabbit liver curve Madeline041098 Filename: See Below Masslynx 3.4 R-Squand Value: SeeAttachments 11/07/01 RWW Slope: See Attachments 11/15/01.11/16/01 MMH Y-Intercept See Attachments 11/16/01.11/19/01 MMH Box#: 01-046 M O N K EY LIV ER Group Sample# Surrogate Initial W t Total Mass PFOS Doae Method Blk RBL110701-H20 Blk-1 Verified NS t 1.0000 of Liver 8 NA Cone. o*/* 0.00 RBL110701-H20 Blk-2 NS 1.0000 NA" 0.00 RBLI10701-H20 Blk-3 RBLI10701-HZO Blk-4 NA 1.0000 NA NA 1.0000 NA 0.00 0.00 Matrix Blk RBL110701-Liver Blk-1 RBLI 10701-LiverBlk-2 NS 0.9425 NA NS 0.9425 NA 0.00 0.00 RBLI 10701-Liver BBc-3 NA 0.942$ NA 0.00 RBLI 10701-Liver Blk-4 NA 0.9425 NA 0.00 QC MKL11070!-2 tfg/g-105505M-MS NA 1.0023 NA MKL110701-2 ue/e-105505M-MSD NA 1.0023 NA MKLI10701-10 ug/g-10550SM-MS MKLU0701-10 ug/x-lOSSOJM-MSD NA NA 1.0023 1.0023 NA NA 126.86 122.97 237.28 213.64 Group 1 I03503M NA 1.0023 NA 85.02 PFOS Periluofooctanesulfonaie NS Nor Spiked with Surrogate NOTE: Average recoyeiy ofthe 10ug/g matrix spikes was not within the */ 30%recovery as listed in the protocol. Adeviation will be written. DateEmered/Analyst: 11/26/01 LAC Dale Verified/Analyst: 12/lU/0t nunh PFOS Dilution Factor 1 1 1 1 1 1 1 50 50 30 30 50 PFOS Calc. Coac. m /t 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 6328 6134 11837 10637 4241 FBeaame M011I16031 M011U6032 M011116033 M011116034 M otil 16038 MO11116039 M011116040 MOI1116041 M011116046 M01UI6047 MOill 16043 MOH116049 M0111I6045 Concentration of PFOS uK/eor%Rec <LOQ(0.00629 ug/g) <LOQ (0.00629 ug/g) <LOQ (0.00629 ng/g) <LOO 0.00629 ue/e) <L0Q (0.00629 ug/g) <LOQ <0.00629 ug/g) <LOQ (0.00629 ug/g) <LOO (0.00629 ub/ ) 100% 90% 73% 61% 4.24 IH <LOO <LOO 95% 67% RSD Std. Dev. MS/MSD RPD NA NA 10% 17% 3MEnvironmental Laboratory 000160 Page 106 3M M edical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Appendix F: Example Calculations Analytical Report: FACT-TOX-160 LIMS EOO-1668 Analytical Report: FA C TTO X-160 LIMS EOO-1668 Formula Used for Sera Analyses in Study FACT TOX-160 AR (ng/mL) x D F x F V ( m L ) x 1.0 pg = R eported C oncentration (pg/m L) EV (m L) 1000 ng Calculation Used for Group 1, W eek 17, Animal ID 105505M 45.75 ng/mL x 200 x 1.0 mL x 1.0 pg = 14.5 pg/m L 0.63 mL 1000 ng AR-- Analytical result from M assLynx 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 3 curve 0) x DF x 1.0 pg = R eported C oncentration (pg/g) 3 sample 1000 ng (1) 3 curve is assumed to be: 1 g liver 5 m L H 20 Calculation Used fo r G roupl, Week 53, Animal ID 105505M 66.07 ng/g x 1 g / 5 m L x 50 x 1.0 pg = 3 .2 5 pg/g 1.0160 g/5mL 1000 ng AR-- Analytical result from MassLynx summary 3 curve--Density o f the liver standard curve, assumed to be lg liver/ 5 ml water 3 sample---Density of the liver sample (g sample/ 5 mL H20 ) DF-- Dilution factor 3M Environmental Laboratory 3M Environmental Laboratory 000161 Page 23 Page 107 3M M edical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: FACT TO X-160 LIMS E00-1668 Appendix G: Interim Certificate(s) of Analysis 3M Environmental Laboratory 3MEnvironmental Laboratory 000162 Paae 24 Page 108 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 ------------------------- LIMS 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 C entre Analytical Laboratories COA Reference #: 023-018A Bead Copy of Original 3M Product: PFO S,Lot217 "= T * R eference#: SD-018 __________________ P arity: 86.9% _____ __________________________ Test Name Specifications Result Purity1 86.9% Appearance Identification NMR M etals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium2 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (LC/MS) Total % Impurity (G C /M S) Related Compounds POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate4 Organic Acids *(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis0: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine White Crystalline Powder - 1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% Conforms Positive 1. 0.005 wt./wt.% 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 wt./wt.% 8.41 wt./wt.% None Detected 0.33 wt./wt.% None Detected N ot Applicable3 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 wt/wt.% 2. <0.1 wt/wt.% 3. 0.10 wt/wt.% 4. 0.28 wt/wt.% 1. 12.48 wt/wt.% 2. 0.244 wt/wt.% 3. 1.74 wt/wt.% 4. 8.84 wt/wt.% 5. 54.1 wt/wt.% COA023-018A 3M Environmental Laboratory 000163 Page 1 o f3 Page 109 3M M edical Departm ent 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 Date o f Last Analysis: 08/31/00 Expiration Date: 08/31/06 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/06 'Purity = 100% - (sum o f 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 endotherm 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. STFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonafluoropentanoic acid Pentafluoropropanoic acid th eo retical value calculations based on the empirical formula, CgFi7S03'K+(MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-018A 3M Environmental Laboratory 000164 Page 2 o f3 Page 110 3M M edical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 ------------------------- LIMS EOO-1668 Centre Analytical Laboratories, Inc. 3048 R esearch Drive S tate College, PA 16801 w w w .centrelab.com Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 C entre Analytical Laboratories COA Reference #: 023-018A LC/MS Purity Profile: Im purity C4 C5 C6 Cl T o tal w t/w t. % 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 /o//t /o! Date Reviewed By: n ,L m fLU % JPbhn Flaherty / Date Laboratory Manager, Centre Analytical Laboratories COA023-018A 3M Environmental Laboratory O O O ISS Page 3 o f 3 Page 111 3M M edical Department Study: T-6295.22 3M Environmental Laboratory Note to File Project o r Study Number: FACT-TCR-001 Associated Study Number: LIMS # E00-1682 Analytical Report: FACT-TOX-160 1 LIMS EQQ-4668 3MEnvironmental Laboratory Form ETS-4-15.0 000166 Exact Copy ot Original j al . jd ia lw Initial O a ts Page 112 3M M edical Departm ent Study: T-6295.22 3M Medical Department Study: T-6295.22 Appendix H: Report Signature Page Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: FACT TOX-160 LIMS E00-1668 Andrew Seacat, Ph.D., Study D irector M ur 3 H D ate' /-ytcf Jy John Butenhoff, Ph.D., Sponsor Representative Date C ^ 'fo . A . C ^ tvm Lisa Clemen, Principal A nalytical Investigator 05'lo i I DO. Date W illiam Reagen, Ph.D., Analytical Laboratory M anager Date 3M Environmental Laboratorv 3M Environmental Laboratory 0001S7 Pacte 25 Page 113