Document EmYB08qYzJGm0VRn0Xy05Empb

3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 Study Title 104-Week Dietary Carcinogenicity Study with Narrow Range N-Ethyl Perfluorooctanesulfonamido Ethanol in Rats Analytical Laboratory Report Title Determination of the Presence and Concentration of PFOS, PFOSA, PFOSAA, EtFOSE-OH, M556, and PFOSEA in Serum and Liver Samples of Crl:CD(SD) IGS BR Rats Exposed to N-Ethyl Perfluorooctanesulfonamido Ethanol Data Requirement Not Applicable Author 3M Environmental Laboratory Study Completion Date June 06, 2001 Performing Laboratories Liver and Serum Analyses '1 3M Environmental Laboratory Building 2-3E-09, 935 Bush Avenue St. Paul, MN 55106 Project Identification 3M Medical Department Study: T-6316.1 Covance In-Life Study: 6329-228 Analytical Report: FACT TO X -003 3M Laboratory Request No. U2104 Total Number of Pages 211 ' 3M Environmental Laboratory 3MEnvironmental Laboratory Page 1 Page 1 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 This page has been reserved for specific country requirements. / 3M Environmental Laboratory 3MEnvironmental Laboratory Page 2 Page 2 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 GLP Compliance Statement Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 Analytical Laboratory Report Title: Determination of the Presence and Concentration of PFOS, PFOSA, PFOSAA, EtFOSE-OH, M556, and PFOSEA in Serum and Liver Samples of Crl:CD(SD) IGS BR Rats Exposed to N-Ethyl Perfluorooctanesulfonamido Ethanol Study Identification Numbers: T-6316.1, FACT TOX-003, LRN-U2104 This study was conducted in compliance with United States Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations 21 CFR Part 58, with the exceptions in the bulleted list below. The analytical phase completed at the 3M Environmental Laboratory was performed in accordance with 3M Environmental Laboratory Standard Operating Procedures. Exceptions to GLP compliance: There were two study directors in this study. This study was designed as two separate studies. The in-life phase study was considered to end at the generation and shipment of specimens. The analytical study was considered to start at the receipt of these specimens for analysis. This resulted in having two separate study directors, one for each phase of the same study. However, since ' the technical performance of each phase was entirely separate, no effect is expected from this exception. Sample storage stability will not be determined. . Characterization of the analytical standards is underway, but has not yet been completed (21 CFR 58.105 (a)). Lot No. 59905 of the surrogate, THPFOS, will not be characterized for purity since quantities of this standard are exhausted. Lot 936 of EtFOSE-OH, and Lot L2353 and TN-A-1886 of PFOSA will not be characterized since quantities of these standards are exhausted. T h e electronic d ata systems in use have not been validated and there is not an electronic audit trail of corrections currently available (21 CFR 58.130 (e)). Authenticated hard copies of chromatograms and associated documents will be considered as the original raw data. There were instances in the raw data where corrections were not made according to 21CFR58.130(e) GLP requirements. Data was crossed out with no reason given, or original entries were obscured or contained "write-overs" with no explanation, initial or date provided. Some reagents (00-022-2, 0.5M TBA solution and 00-003-28, 0.5M TBA solution) were not labeled with storage requirements. Persons responsible for equipment were not identified (021188-55 IEC Cetra GP*R and N-Evap.) (See next page for signatures) 3M Environmental Laboratory 3MEnvironmental Laboratory Page 3 Page 3 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 GLP Compliance Statement continued Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 John L. Butenhoff, Ph. D., Study director ^ Date Marvin T. Case, D.V.M., Ph.D., Sponsor Representative Kristen J. Hansen, Ph.D., Principal Analytical Investigator William K. Reagen, Ph.D., Laboratory Manager *i Date 2573/ 0 ! Date jy /o ; Date 3M Environmental Laboratory 3MEnvironmental Laboratory Page 4 Page 4 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 GLP Study--Quality Assurance Statement Analytical Laboratory Report Title: Determination of the Presence and Concentration of PFOS, PFOSA, PFOSAA, EtFOSE-OH, M556, and PFOSEA in Serum and Liver Samples of Crl:CD(SD) IGS BR Rats Exposed to N-Ethyl Perfluorooctanesulfonamido Ethanol Study Identification Numbers: T-6316.1, FACT TOX-003, LRN-U2104 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 Dater Reported to Management Study Director 4/20/00 Sample prep 8/8/00 8/8/00 6/30/00, 7/5/00 4/23/01--4/30/01 4/30/01--5/1/01 , , 5/22/01 Sample prep Data Report 7/21/00 5/2/01 5/23/01 7/21/00 5/2/01 5/23/01 QAU Representative i___3 - c)o I Date 73 3M Environmental Laboratory 3MEnvironmental Laboratory Page 5 Page 5 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Table of Contents Analytical Report: FACT TOX-003 LRN-U2104 GLP Compliance Statement...............................................................................................3 GLP Study--Quality Assurance Statement....................................................................... 5 Study Personnel and Contributors......................................................................................8 Introduction and Purpose.................................................................................................... 9 Test System....................................................................................................................9 Specimen Collection and Analysis.................................................................................9 Specimen Receipt and Maintenance.................................................................................. 10 Chemical Characterization of the Reference Standards.....................................................11 Dose Confirmation Analyses...........................................................................................11 Method Summaries.............................................................................................................. 12 3M Environmental Laboratory........................................................................................ 12 Preparatory Methods..................................................................................................12 Analytical Methods.....................................................................................................13 Analytical Equipment..................................................................................................13 Data Quality Objectives and Data Integrity......................................................................... 14 Data Summary, Analyses, and Results............................................................................... 15 Summary of Quality Control Analyses Results...............................................................15 Statement of Data Quality...............................................................................................17 Summary of Sample Results.......................................................................................... 17 Statistical Methods and Calculations.................................................................................. 17 Statement of Conclusion......................................................................................................17 References................................................................................................................. 17 Appendix A: Chemical Characterization and Control Matrices........................................... 18 Appendix B: Protocol, Amendments and Deviations.......................................... 19 Appendix C: Extraction and Analytical Methods.................................................. 77 Appendix D: Data Summary Tables................................................................................. 157 Appendix E: Data Spreadsheets........................................................................................ <|g<| Appendix F: Example Calculations................................................................................... -|g4 Appendix G: Interim Certificates of Analysis..................................................................... 186 Appendix H: Report Signature Page................................................................................. 211 3M Environmental Laboratory 3MEnvironmental Laboratory Page 6 Page 6 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 List of Tables Analytical Report: FACT TOX-003 LRN-U2104 Table 1. Test System Population Demographics for Study 6329-228.............................. 9 Table 2. Characterization of the Analytical Reference Standards in Study FACT TOX-003................................................................................................................ 11 Table 3. Target Ions Monitored in 3M Laboratory Analyses.............................................. 14 Table 4. Matrix Spikes--Sera........................................................................................... 16 Table 5. Matrix Spikes--Liver............................................................................................ 16 Table 6. Characterization of the Control Matrices Used for Sera Analyses in Study FACT TOX-003......................................................................................................18 Table 7. Characterization of the Control Matrices Used for Liver Analyses in Study FACT TOX-003......................................................................................................18 Table 8. Characterization of Test Article in StudyFACT TOX-003.................................... 18 Table 9. FACT TOX-003 Data Summary for PFOS, PFOSA, PFOSAA, EtFOSE, M556 and PFOSEA-- Rat Serum (pg/mL)..................................................................... 157 Table 9. (Continued) FACT TOX-003 Data Summary for PFOS, PFOSA, PFOSAA, EtFOSE, M556 and PFOSEA--Rat Serum (pg/mL)............................................. 158 T^tble 10. FACT TOX-003 Data Summary for PFOS, PFOSA, PFOSAA, EtFOSE, M556 and PFOSEA--Rat Liver (pg/g)............................................................................ 159 Table 10. (Continued) FACT TOX-003 Data Summary for PFOS, PFOSA, PFOSAA, EtFOSE, M556 and PFOSEA--Rat Liver (pg/g)................ ................ :............... 160 3M Environmental Laboratory 3MEnvironmental Laboratory Page 7 Page 7 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Study Personnel and Contributors Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 Study Director John L. Butenhoff, Ph.D. 3M Corporate Toxicology - Medical Department 3M Center Building 220-2E-02 St. Paul, MN 55144-1000 651-733-1962 Sponsor Marvin T. Case, D.V.M., Ph.D. 3M Corporate Toxicology - Medical Department 3M Center Building 220-2E-02 St. Paul, MN 55144-1000 Analytical Chemistry Laboratories Liver and Serum Analyses 3M Environmental Laboratory Kristen J. Hansen, Ph.D. Principal Analytical Investigator 3M Lab Contributing Personnel [List all personnel alphabetically] David R. Barnidge, Ph.D.* Lisa A. Clemen Rhonda Dick* Kelly J. Dorweiler* Mark E. Ellefson Barb A. Gramenz* Sarah A. Heimdal* Marlene M. Heying* Megan C. Holloway* 'C o n tra ct lab professional service em ployees Harold O. Johnson Kelly J. Kuehlwein* Ognjenka Krupljanin* Sally A. Linda* Ian A. Smith* Kathleen M. Stock* Anh-Dao Vo Bob W. Wynne* Richard Youngblom* Location of Archives All original raw data, protocol, and analytical report have been archived at the 3M Environmental Laboratory. The test substance and analytical reference standard resen/e samples, as well as the specimens pertaining to the analytical phase of this study are archived at the 3M Environmental Laboratory. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 8 Page 8 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Introduction and Purpose Analytical Report: FACT TOX-003 LRN-U2104 The purpose of the study is to determine the presence and concentration of PFOS, PFOSA, PFOSAA, EtFOSE-OH, M556, and PFOSEA in serum and liver specimens collected from Covance Study No.: 6329-228 titled: 104-Week Dietary Carcinogenicity Study with Narrow Range (98.1%) N-Ethyl Perfluorooctanesulfonamido Ethanol in Rats. The Covance in-life study was initiated on April 17, 1998. The analytical phase of the study was initiated on May 22, 1998. Test System A total of 140 males and 140 female rats were used as the test system. Table 1 outlines the rat population demographics and dosage levels for study 6329-228. The test system species and strain selected was the Crl:CD(SD) IGS BR rat received from Charles River Laboratories, Inc., identified using an implanted microchip device. At the initiation of treatment the rats were approximately 6-8 weeks old and weighed between approximately 100-300 g. Table 1. Test System Population Demographics for Study 6329-228 Number of Animals Study Group Approximate Dietary Levels of Male Female EtFOSE-OH (ppm) Group 8 Control Group 9 Treated 70 70 70 70 0. 1 Specim en Collection and Analysis Specimens were collected by C ovance (study 6 3 2 9 -2 2 8 ) and sent to the 3M Environm ental Laboratory for analysis. On week 4, 20 serum specimens and 20 liver specimens were collected from Groups 8 and 9 males and females. On week 14, 20 serum specimens and 20 liver specimens were collected from Groups 8 and 9 males and females. On week 53, 20 serum specimens and 20 liver specimens were collected from Groups 8 and 9 males and females. On week 105, 40 liver specimens were collected from Groups 8 and 9 males and females. On week 105, 40 serum specimens were collected from Groups 8 and 9 males and females. The number and type of specimens collected for analyses in the analytical phase of this study are presented. Specimens Collected from Study Groups 8 and 9 Serum Specimens-- 100 specimens Liver Specimens-- 100 specimens Liver and sera specimens were shipped to the 3M Environmental Laboratory frozen and on dry ice. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 9 Page 9 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Sera and liver samples were extracted beginning on May 27,1998 using an ion pairing reagent and either ethyl acetate or methyl-tert-butyl ether (MtBE). Liver samples were homogenized prior to the extraction procedure. Sample extracts were analyzed using high-pressure liquid chromatography-electrospray/tandem mass spectrometry (HPLC-ESMSMS) in the multiple reaction monitoring mode. PFOS, PFOSA, PFOSAA, EtFOSE-OH, M556, and PFOSEA levels were evaluated by external calibration using extracted curves. Analytical details are included in this report. Specimen Receipt and Maintenance The 3M Environmental Laboratory received liver and serum specimens collected at predetermined time points during and at the end of the in-life phase of Covance Study 6329-228 from Covance May 1998 through June 2000. All specimens were received frozen on dry ice and were immediately transferred to storage at -20C 10C. Control matrices used in liver and sera analyses performed during TOX-003 were obtained from commercial sources and are presented in Appendix A. Samples analyzed at the 3M Environmental Laboratory will be maintained for a period of 10 years and will be stored at the laboratory at -20C 10C. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 10 Page 10 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Chemical Characterization of the Reference Standards Chemical characterization information on the analytical reference materials used in this study is presented in tabular form below. Table 2. Characterization of the Analytical Reference Standards in Study FACT TOX-003 Reference Standard / Form ula Potassium Perfluorooctanesulfonate C 8 F17S O 3 -K + N-Ethyl Perfluorooctanesulfonamido ethyl alcohol C 8 F i 7S 0 2 N (C 2 H 5 )C H 2C H 20 H Sodium Perfluorooctanesulfonylamido(ethyl) acetate CsF^SO zNfCHaCHslCHaCOO -Na Perfluorooctanesulfonylamido(ethyl)acid C 8F i7S 0 2 N (C H 2 C H 3)C H 2 C O O - h Perfluorooctanesulfonylamide C8F17SO2NH2 Perfluorooctanesulfonylethylamide C8F17SO2NHCH2CH3 M 556 C 8F i7S 02N (H )CH 2C O O H 1H, 1H, 2H, 2H - T e tra h y d ro p e rflu o ro o c ta n e s u lfo n ic a c id C8H4F13SO3H T h is lot is exhausted and cannot be characterized. N R -- Not recorded NA-- Not applicable TB D -- To be determined Acronym KPFOS* EtFO SE-O H Source 3M 3M 3M 3M 3M Expiration Date 08/31/01 2010 2010 11/26/01 2010 Storage C o n d itio n s A m bient tem perature A m bient tem perature Ambient tem perature A m bient tem perature A m bient tem perature Chem ical Lot Num ber 171 193 215 936 Unknown (S D 013) P h y s ic a l D e s c rip tio n Light colored powder W hite crystals W hite powder Amber waxy solid Amber waxy solid P u rity 8 6 .4 % 8 8 .0 % TBDC NA* 8 8 .9 % 3M 0 1 /0 1 /2 0 1 0 Ambient tem perature PFOSAA" Yellow to am ber 617 TBDd liquid PFOSA PFOSEA M 556 3M 0 1 /0 1 /2 0 1 0 A m bient Unknown Light yellow TBDC tem perature (9 8 -0 2 0 7 -0 3 0 3 -3 ) powder A m bient 3M 01/01/2010 tem perature L2353 Am ber to brown waxy solid NA* 3M 0 1 /0 1 /2 0 1 0 A m bient tem perature L15709 Light yellow waxy solid TBDC 3M 0 1 /0 1 /2 0 1 0 A m bient tem perature 529 Amber waxy solid TBDC Ambient Unknown Amber waxy 3M 01/01/2010 NA* tem perature (TN -A -1885) solid A m bient 3M 01/01/2010 N B 113047-80 W hite powder TBDC tem perature THPFOS Ambient ICN 01/01/2010 tem perature 59909 Brown powder NA* T a rg e t analyte is P FO S , CaF i7S 03' T a rg et analyte is CaF17S02{(CH:,CH3)(CH2COO`)} "Unless otherwise indicated, at the tim e of quantitation, the purity for all analytes was assumed to be 100% . dFor reference lot 617, a purity of 53.82% was used for PFO SA A result quantitation. Dose Confirmation Analyses Dose preparation methods and analysis were performed by Covance, using a validated analytical method provided by Covance (MP-M312-MA), and are reported separately (Reference Covance 6329-228). 3M Environmental Laboratory 3MEnvironmental Laboratory Page 11 Page 11 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Method Summaries Analytical Report: FACT TOX-003 LRN-U2104 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. Data collected prior to November 1999 was reworked in 2000 to accommodate improvements in data reduction methods. Both the original and "reworked" data are archived; reworked data is presented in the final results. The improved methods are documented in the form of method modifications. As the present study progressed, more advanced methods evolved arid earlier methods were used with deviations until amendments to the protocol were written. Protocol and method deviations are located in Appendix B of this report. 3M Environm ental Laboratory Preparatory Methods FACT-M-1.0. "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry". This method was used for week 4 samples extracted on 6/9/98 and week 14 samples extracted on 7/17/98. FACT-M-3.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry". This method was used for week 4 samples extracted on 5/27/98 and week 14 samples extracted on 7/15/98. ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry". This method was used for week 53 samples extracted on 4/20/00, and week 105 samples extracted on 6/29/00. ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry". This method was used for week 53 and week 105 samples extracted on 4/14/00. An ion-pairing reagent was added to the sample and the analyte ion pair was partitioned into ethyl acetate (FACT-M-1.0 and FACT-M-3.0) or MtBE (ETS-8-4.1 and ETS-8-6.0). The extract was transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract was reconstituted in 1.0 mL of methanol, and then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 12 Page 12 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Analytical Methods FACT-M-2.0, "Analysis of Liver Extracts for Fluorochemicals Using HPLC-Electrospray/Mass Spectrometry". FACT-M-4.0, "Analysis of Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" . ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry". ETS-8-7.0, "Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry". The analyses were performed by monitoring one or more product ions selected from a single primary ion characteristic of a particular fluorochemical using HPLC-ES/MS/MS. For example, molecular ion 499, selected as the primary ion for PFOS (C8F17SO3) analysis, was fragmented further to produce ion 99 (FSO3 ). The characteristic product ion 99 was monitored for quantitative analysis. Analytical Equipment The actual analytical equipment settings used in the present analytical' phase of this study varied slightly during actual data collection. The following is representative of the settings used during the analytical phase of this study. Liquid Chromatograph: Hewlett-Packard Series 1100 Liquid Chromatograph system Analytical column: Keystone BetasilTM Ct8 2x50 mm (5 pm) Column temperature: Ambient Mobile phase components: Component A: 2mM ammonium acetate . Component B: methanol Flow rate: 300 pL/min Injection volume: 10 pL Solvent Gradient: 9.0 minutes Time (minutes) %B 0.0 40% 1.0 40% 4.5 95% 6.5 95% 7.0 40% 9.0 40% 3M Environmental Laboratory 3MEnvironmental Laboratory Page 13 Page 13 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Mass SpectrometerJMicromass API/Mass Spectrometer Quattro II TMTriple Quadrupole system Software: Mass Lynx TM3.1,3.2, 3.3, 3.4 Cone Voltage: 30-60 V Collision Gas Energy: 25-45 eV Mode: Electrospray Negative Source Block Temperature: 150C 10C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (MRM) Table 3. Target Ions Monitored in 3M Laboratory Analyses Target Analyte Primary Ion (a m u ) Product Ion (a m u ) PFOS 499 80, 99,130 PFOSA 498 78 PFOSAA 584 83, 169 EtFOSE-OH 630 59 PFOSEA 526 65 M556 556 65, 78, 83, 169 THPFOS 427 80 Data Quality Objectives and Data Integrity . The following data quality objectives were indicated in the method performance section of ETS8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry and ETS-8-7.0, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry: Linearity: The coefficient of determination (r2) equal to or greater than 0.980. Limits of Quantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibration curve. Acceptable Precision: Precision is better than 30% for the method. Acceptable Spike Recoveries: 70-130%. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 14 Page 14 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Data Summary, Analyses, and Results Analytical Report: FACT TOX-003 LRN-U2104 Data quality objectives for the analytical phase of this study outlined in the 3M Environmental Laboratory protocol for FACT TOX-003 (see Appendix B) were met with the exceptions noted in this report. Sum mary o f Q uality Control Analyses Results Linearity: The coefficient of determination (r2) of the standard curve was >0.980. Calibration Standards: Quantitation of the target analytes was based on linear regression analysis weighted 1/x of a single, opening or two bracketing extracted matrix curves for each group of samples. Rat and rabbit sera were used for matrix curves; in sera analyses, while rabbit liver was used for matrix curves in liver analyses. High or low points on the curve may have been deactivated to provide a better linear fit over the curve range most appropriate to the data. Low curve points with peak areas less than two times that of the extraction blanks were deactivated to disqualify a data range that may have been significantly affected by background levels of the analyte. Occasionally, a single mid-range curve point that was an obvious outlier may have been deactivated. Quantitation of each analyte was based on the response of one or more specific product ion(s) using the multiple reaction monitoring mode of the instrument (see Appendix C, Analytical Methods). Calibration standards were prepared to run, undiluted, approximately within the linear range of the instrument (approximately 5 1000 ng/g). Limits of Quantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibration curve (defined as a standard within 30% of the theoretical value), and is at least two times the analyte peak area detected in the surrogate matrix b'anks. (See Appendix D). Blanks: All blanks were below the lower limit of quantitation for the compounds of interest. To simplify analyses that were complicated by endogenous levels of fluorochemicals in unexposed rat sera and liver, rabbit sera and liver were selected as suitable surrogate matrices for method blanks. Precision: Instrumental precision was determined by replicate injections of a single serum extract from a related study (TOX-001). Instrumental precision was determined for PFOS, PFOSAA, and M556; variation was less than 8.5% for all targeted analytes. Matrix Spikes: Sera--Matrix spike samples were prepared from control rat sera along with each batch of sera samples. Samples were spiked between 75-250 ng/mL, levels that approximate the levels detected in Groups 8 and 9 samples, depending upon the analyte. All spikes were prepared to run, undiluted, within a ten-fold limit of the linear range of the calibration curve. For some analytes, samples for some high dose animals may be much higher than the range of these prepared spikes. Sera matrix spike recoveries are presented in tabular form below. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 15 Page 15 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 Table 4. Matrix Spikes--Sera # of Spikes Prepared Average Spike Recovery Range Standard Deviation PFOS 8 111% 73-144% 27% PFOSA 8 107% 79-126% 16% PFOSAA 8 106% 74-140% 26% M556* 4 139% 108-174% 35% EtFOSE-OH 4 39% 16-61% 25% PFOSEA 4 66% 36-90% 28% M 5 56 was identified as a metabolite of E tF O S E -O H after initiation of the analytical work. Standard material was unavailable and had to be synthesized. N o M 5 56 data is provided for the W e e k 4 through W e e k 14. Liver--Matrix spike samples were prepared from a control group rat liver specimen along with each batch of liver samples. Samples were spiked between 100-500 ng/g, levels that approximate the levels detected in Groups 8 and 9 samples, depending on the analyte. All spikes were prepared to run, undiluted, within a ten-fold limit of the linear range of the calibration curve. For some analytes, samples for some high dose animals may be much higher than the range of these prepared spikes. Liver matrix spike recoveries are presented in tabular form below. Table 5. Matrix Spikes--Liver PFOS PFOSA PFOSAA M556* EtFOSE-OH PFOSEA # of Spikes Prepared 12 12 12 6 4 6 Average Spike Recovery 94% 87% 96% 103% 118% 108% Range 65-137% 58-132% 53-200% 80-122% 77-154% 69-144% Standard Deviation 23% 23% 39% 18% 39% 27/> M 5 56 was identified as a metabolite of E tF O S E -O H after initiation of the analytical work. Standard material was unavailable and had to be synthesized. No M 556 data or Q C is provided for the W e e k 4 through W e e k 14 samples. Surrogates: The surrogate (THPFOS) was added to all samples and standards, with the exception of Week 4 liver and sera samples. THPFOS was not used for quantitation, but was used to monitor for gross (50%) instrument failure. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 16 Page 16 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Statem ent of Data Quality It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the sera and liver data can be considered to be accurate to within one standard deviation of the average fortified samples recovery. Refer to Tables 4 and 5 for matrix spikes from each analyte and matrix. The results of quality control analyses (curve fit, CCVs, and MS/MSDs) for EtFOSE-OH and PFOSEA were inconsistent and indicate that data presented for this analyte should be considered to be qualitative only. Values for these analytes are presented in data tables in the spirit of full disclosure, but should not be used in any quantitative assessment of the data. Sum mary o f Sam ple Results PFOS results (those obtained using lot # 171 and #193) and EtFOSE-OH results (those obtained with lot # SD013) have been corrected for purity of the analytical reference material. Uncorrected results are noted in the data tables. Samples from Control Animals: The target analytes were often detected in the sera and liver samples from the control animals. These levels were usually lower than those found in the low dose test animals. Samples from Dosed Animals: In general, target analytes were present in the sera and liver samples in the dose group. Detailed sample data tables are presented in Appendices D and E. Statistical Methods and Calculations Statistical methods were limited to the calculation of means and standard deviations. See Appendix F for example calculations used to generate the liver and serum sample data in FACT TOX-003. Statement of Conclusion Under the conditions of the present studies, the concentrations of PFOS, PFOSA, PFOSAA, EtFOSE-OH, M556, and PFOSEA were determined in the sera and liver of rats dosed with NEthyl Perfluoroctanesulfonamido Ethanol during the in-life phase of the study. References Covance Study No.: 6329-228 titled: 104-Week Dietary Carcinogenicity Study with Narrow Range (98.1%) N-Ethyl Perfluorooctanesulfonamido Ethanol in Rats 3M Environmental Laboratory 3MEnvironmental Laboratory Page 17 Page 17 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 Appendix A: Chemical Characterization and Control Matrices Table 6. Characterization of the Control Matrices Used for Sera Analyses in Study FACT TOX-003 Control Matrix Rat Serum Rat Serum Rabbit Serum Rabbit Serum Source Expiration D a te S to ra g e C o n d itio n s C hem ical Lot # P h y s ic a l D e s c rip tio n Sigm a 2010 -20C 10C 17H9306 Rat Serum S igm a 0 1 /0 1 /2 0 1 0 -20C 10C 19H89291 Rat Serum S igm a 0 1 /0 1 /2 0 1 0 -20C 10C 47H4641 Rabbit Serum S igm a 2010 -20C 10C 118H8418 R abbit Serum Table 7. Characterization of the Control Matrices Used for Liver Analyses in Study FACT TOX-003 Control Matrix Rabbit Liver Rabbit Liver Rabbit Liver S o u rce Expiration D ate S torag e Conditions C hem ical Lot # P h y s ic a l D e s c rip tio n Covance Laboratories, Inc. 2010 -20C 10C F00014 Covance Laboratories, Inc. 2010 -20C 10C F00007 Covance Laboratories, Inc. 2010 -20C 10C F00016 ' R abbit Liver Rabbit Liver R abbit Liver Table 8. Characterization of Test Article in Study FACT TOX-003 Chem ical Name Source Expiration Date Storage Conditions Chem ical Lot # Physical Description Purity T e st A rticle EtFOSE-OH N -E thyi Perfluorooctanesulfonam ido ethanol T -6 3 1 6 3M 1 1 /2 6 /0 1 A m bient tem perature (30035, 30037, 30039) Light yellow, w axy solid/flakes 9 7 .4 % 3M Environmental Laboratory 3MEnvironmental Laboratory Page 18 Page 18 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Appendix B: Protocol, Amendments and Deviations 3M Environmental Laboratory 3MEnvironmental Laboratory Page 19 Page 19 3M Medical Department Study: T-6316.1 C O V A tfC l> THE DEVELOPMENT SERVICES COMPANY Analytical Report: FACT-TOX-003 LRN-U2104 Sponsor: 3M St. Paul, Minnesota PROTOCOL Study Title: ii 104-W eek Dietary Carcinogenicity Study with Narrow Range (98.1%) N-Ethyl Perfluorooctanesulfonamido Ethanol in Rats Date: April 1, 1998 Performing Laboratory: Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 Laboratory Study Identification: Proposal No. 23124A Covance 6329-228 3M T-k3iUI 3MEnvironmental Laboratory Page 20 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6 3 29-2 28 ______________P a g e 2 Study 104-Week Dietary Carcinogenicity Study with Narrow Range (98.1%) N-Ethyl Perfluorooctanesulfonamido Ethanol in Rats Purpose To assess the carcinogenicity of the test material when administered in the diet to rats for at least 104 weeks Sponsor 3M Toxicology Services Building 220-2E-02, 3M Center St. Paul, Minnesota 55144-1000 Study Monitor Andrew M. Seacat, PhD 3M Telephone No.: 612.575.3161 Facsimile No.: 612.733.1773 Study Location Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 . Mailing Address: PO Box 7545 Madison, Wisconsin 53707-7545 Study Director Peter J. Thomford, PhD Covance Laboratories Inc. Telephone No.: 608.241.7207 Facsimile No.: 608.242.2736 3MEnvironmental Laboratory Page 21 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Toxicologist Thomas E. Ryan, BS Covance Laboratories Inc. Covance 63 2 9 -2 2 8 _______________ P a g e 3 Proposed Study Timetable In-Life Start Date: April 6, 1998 In-Life End Date: April 5, 2000 Regulatory Compliance This study will be conducted in compliance with the Food and Drug Administration G ood Laboratory Practice Regulations as set forth in Title 21 o f the US Code of Federal Regulations, Part 58, issued December 22, 1978 (effective June 20, 1979), and with any applicable amendments. Animal Care and Use Statement All procedures in this protocol are in compliance with the Animal Welfare Act Regulations, 9 CFR 1-4. In the opinion of the Sponsor and study director, the study does not unnecessarily duplicate any previous work. Quality Assurance The protocol, study conduct, and final report will be audited by the Covance Quality Assurance Unit (QAU). The proliferation cell nuclear antigen evaluation, data, and report will be audited by the QAU of Pathology Associates International Test Material Identification T-6316 (Narrow Range N-Ethyl Perfluorooctanesulfonamido Ethanol, NEtFOSE) Lot Numbers The lot numbers will be maintained in the raw data. Purity 98.1% NEtFOSE (w/w) 3MEnvironmental Laboratory Page 22 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 ________________________________________________________________________________ __________________________ ________P a g e 4 Stability Responsibility of the Sponsor Storage Conditions At room temperature C h aracteristics Information on synthesis methods, composition, or other characteristics that define the test material is on file with the Sponsor. Reserve (Archive) Samples A reserve sample (approximately 5 g) will be taken and stored at room temperature. This sample will be transferred to the Sponsor after completion of the in-life phase to be retained in accordance with 21 CFR 58.195. Disposition of Test M aterial After authorization from the Sponsor, any remaining test material will be returned to: Andrew M. Seacat, PhD 3M Toxicology Services Building 220-2E-02, 3M Center St. Paul, Minnesota 55144-1000 Telephone No.: 612.575.3161 Facsimile No.: 612.733.1733 Animals . Species Rat Strain Crl:CD(SD)IGS BR Source Charles River Laboratories, Inc., Raleigh, North Carolina 3MEnvironmental Laboratory Page 23 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 _____________________ ;____________________________________________________________________________ __ _________ P a g e 5 Age at Initiation of Treatment Preferably 6 weeks of age, but not more than 8 weeks of age Weight at Initiation of Treatment 100 to 300 g Number and Sex 140 males and 140 females Identification Implantable microchip identification device Husbandry Housing Individual (may be group-housed during acclimation) Diet Certified Rodent Diet #5002, meal, (PMI Nutrition International) ad libitum, unless otherwise specified. The diet is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Results of specified nutrient and contaminant analyses are on file at Covance-Madison. Water A d libitum. Samples o f the water are routinely analyzed for specified microorganisms and environmental contaminants. The results are on file at Covance-Madison. Contaminants There are no known contaminants in the diet or water at levels that might interfere with this study. Environment Environmental controls for the animal room will be set to maintain 18 to 26C, a relative humidity of 30 to 70%, a 12-hour light/12-hour dark cycle, and a minimum of 10 room air changes/hour. The light/dark cycle may be interrupted to accommodate study-related activities. 3MEnvironmental Laboratory Page 24 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Acclimation At least 1 week Covance 6329-228 ______________P a g e 6 Randomization Selection o f animals for the study will be based on body weights, clinical observations, and other data as appropriate. Animals will be assigned to treatment groups using a computerized blocking procedure designed to achieve body weight balance with respect to treatment groups. At the time of randomization, the weight variation o f the animals o f each sex used will not exceed 2 standard deviations o f the mean weight, and the mean body weight for each group of each sex will not be statistically different at the 5.0% probability level. Justification Rats historically have been used in safety evaluation studies and are recommended by appropriate regulatory agencies. Group Designations and Dietary Levels Group3 Number of Animals Male Female Dietary Levels (ppm NEtFOSE)b 8 (C ontrol)' 4'6 9 (T reated)46 70 70 70 70 0 1 a G roups will be designated as Groups 8 and 9. This study will be located in the same animal room as Covance 6329-212. b T-6316 is 98.1% n-ethyl perfluorooctanesulfonamido ethanol (NEtFOSE); dose levels are expressed as ppm of NEtFOSE. c The control animals will receive the basal diet only. d Five animals/sex/group will be sacrificed during Weeks 4 and 14 for hepatocellular proliferation rate measurements and biochemical analyses (palmitoyl-CoA oxidation), e Ten animals/sex/group will be designated as interim sacrifice animals and will be sacrificed after at least 78 weeks of treatment. Dosing Procedures Method of Administration Dietary. Animals will receive test diet for at least 104 weeks. 3MEnvironmental Laboratory Page 25 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 --------------------------------------------------------------------------------------------------------------- Page 7 Reason for Dosing Route The potential human exposure is by the oral route. Dose P reparation Dose preparations will be mixed at least once every 4 weeks according to the study-specific mixing procedure developed by Covance. Dose concentrations will be based on the NEtFOSE content as supplied; the Sponsor has stated the T-6316 is 98.1% NEtFOSE (w/w) as supplied. All dose preparations will be stored at room temperature. R etention Samples Samples (approximately 100 g) will be taken from each dose preparation and stored at room temperature. Unless used for analyses, these samples will be discarded at least 1 month after completion o f the in-life phase. Dose Analyses By Covance using a method supplied by the Sponsor and validated by Covance H om ogeneity Homogeneity will be determined for the 1-ppm diet preparation mixed for W eek 1. One sample (approximately 100 g) each from the top, middle, and bottom of the diet preparation will be collected, divided into three subsamples for extraction and analysis, and analyzed for test material content. AD samples will be stored at room temperature until analyzed within 7 days o f mixing. Homogeneity analysis will be repeated if the batch size changes by more than 30%. Stability The middle homogeneity sample will be analyzed on the day of mixing and serve as stability time 0. Two additional samples (approximately 100 g each) will be taken from the 1-ppm diet preparation mixed for Week 1 to establish stability. One sample will be stored at room temperature for at least 19 days, then analyzed. The other sample will be stored at room temperature after at least 32 days, then analyzed. 3MEnvironmental Laboratory Page 26 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-2 28 ______________ P a g e 8 Dose Confirm ation Samples (approximately 100 g each) will be collected from all dose preparations and analyzed in duplicate. Homogeneity samples collected from the middle o f the dose preparation for Week 1 will be used for dose confirmation results. All samples will be stored at room temperature until analyzed. Observation of Animals Clinical Observations Each animal win be observed twice daily (a.m. and p.m.) for mortality and moribundity, findings will be recorded as they are observed. Once prior to treatment and weekly thereafter, each animal will be removed from its cage and examined; abnormal findings or an indication of normal will be recorded. The following information on each grossly visible or palpable mass will be recorded. *i time of onset location size (small or large) appearance progression ' Body W eights Prior to treatment (at randomization), weekly for Weeks 1 through 17, once every 4 weeks thereafter, and at Week 105 Food Consumption Weekly for Weeks 1 through 16 and once every 4 weeks thereafter Clinical Pathology Frequency and Num ber of Animals Unscheduled Collection When possible, a blood film will be made and held for possible future examination from animals sacrificed at unscheduled intervals. 3MEnvironmental Laboratory Page 27 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 __ ___________ P a g e 9 Scheduled Collections Hematology, clinical chemistry, urinalysis, and urine chemistry will be done on 10 animals/sex/group during Weeks 14, 27, and 53. A blood film will be made and held for possible future examination from animals at scheduled sacrifices after at least 78 and 104 weeks of treatment. Method of Collection Hematology, Clinical Chemistry, Urinalyses, and Urine Chemistry Animals will be fasted overnight; blood will be collected from a jugular vein. The anticoagulant will be potassium EDTA for hematology tests. Samples for clinical chemistry will be collected without anticoagulant. Urine will be collected chilled overnight (approximately 16 hours). Blood Filins Blood films will be taken as part of the necropsy procedure. Tests Hematology red blood cell (erythrocyte) count hemoglobin hematocrit mean corpuscular volume mean corpuscular hemoglobin mean corpuscular hemoglobin concentration platelet count white blood cell (leukocyte) count differential blood cell count blood cell morphology reticulocyte smear (made, but not examined) glucose urea nitrogen creatinine total protein albumin globulin cholesterol total bilirubin Clinical Chemistry alanine aminotransferase gamma glutamyltransferase aspartate aminotransferase calcium inorganic phosphorus sodium potassium chloride 3MEnvironmental Laboratory Page 28 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 appearance volume specific gravity pH protein urobilinogen sodium potassium Covance 6329-228 ____________ P ag e 1 0 Urinalysis glucose ketones bilirubin blood microscopic examination of sediment Urine Chemistry 16 hour excretion of: sodium . potassium Serum Analyses Frequency and Number of Animals Five animals/sex/group during Weeks 4 and 14 (animals selected for hepatocellular proliferation and biochemical analyses), five animals/sex/group (from animals selected fo r interim sacrifice) after at least 78 weeks of treatment,.and five animals/sex/group at the terminal sacrifice Method o f Collection Animals will be fasted overnight; blood (approximately 2 mL) will be collected from a jugular vein. Samples will be collected without mticoagulant. Sample Handling Blood samples will be allowed to clot at room temperature and centrifuged. Serum samples will be harvested and stored in a freezer set to maintain -60 to -8 0 C. Samples will be packed on dry ice and shipped to: Kris J. Hansen, PhD 3M Environmental Technology and Safety Services 935 Bush Avenue Building 2-3E-09 St. Paul, Minnesota 55133-3331 Telephone No.: 612.778.6018 Facsimile No.: 612.778.6176 3MEnvironmental Laboratory Page 29 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 63 29-2 28 _________________________________________________________________________ ;______________________________ P a g e 11 Samples will be retained by the Sponsor for possible future analysis. Termination Unscheduled Sacrifices and Deaths Necropsies will be done. Animals to be sacrificed will be anesthetized with sodium pentobarbital, weighed, and exsanguinated. A blood film will be taken as part o f the necropsy procedure for sacrificed animals. Scheduled Sacrifices Interim Sacrifices During Week 4, five animals/sex/group will be fasted overnight, bled for serum samples, anesthetized with sodium pentobarbital, weighed, and exsanguinated. The abdominal cavity of each animal will be opened, the liver will be removed and weighed, and liver samples will be collected. Animals will be discarded after liver collection. During Week 14, five animals/sex/group will be fasted overnight, bled for serum samples, anesthetized with sodium pentobarbital, weighed, and exsanguinated. The abdominal cavity of each animal will be opened, the liver will be removed, weighed, and liver samples collected. Animals will be discarded after liver collection. After at least 78 weeks of treatment, 10 animals/sex/group will be fasted overnight, bled for serum samples (five animals/sex/group), anesthetized with sodium pentobarbital, weighed, exsanguinated, and necropsied. A blood film will be taken as part of the necropsy procedure. Terminal Sacrifice After at least 104 weeks of treatment, the remaining animals will be fasted overnight, bled for serum samples (five animals/sex/group), anesthetized with sodium pentobarbital, weighed, exsanguinated, and necropsied. A blood film will be taken as part of the necropsy procedure. 3MEnvironmental Laboratory Page 30 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 63 29-2 28 ......................................................... ............................................................................. Page 12 Postmortem Procedures Necropsy The necropsy will include an examination o f the external features o f the carcass; all external body orifices; the abdominal, thoracic, and cranial cavities; organs; and tissues. Cell Proliferation Tissue Collection and Inununohistochemical Evaluation A t the W eek 4 and 14 interim sacrifices, representative samples o f the left lateral lobe o f the liver and any macroscopic lesions of the liver will be collected and preserved in zinc formalin. A fter fixation, each sample of liver will be embedded in paraffin, and the paraffin blocks will be shipped to: Sandra R. Eldridge, PhD Pathology Associates International 15 Worman's Mill Court, Suite I Frederick, Maryland 21701 Telephone No.: 301.663.1644, ext. 2201 Facsimile No: 301.663.8994 ' Proliferation cell nuclear antigen (PCNA) evaluation will be done on the samples. In addition, liver sections will be stained with hematoxylin and eosin and examined microscopically. Results will be provided for inclusion in the final report. Paimitoyl-CoA Oxidase Tissue Collection and Analyses At the W eek 4 and 14 interim sacrifices, a sample (approximately 500 mg) o f the right lateral lobe of the liver will also be collected from each animal and flash-frozen in liquid nitrogen. The liver tissue will be stored in a freezer set to maintain -60 to -80C until analyzed by Covance for palmitoyl-CoA oxidase activity. O rgan W eights At the W eek 79 and terminal sacrifices, the following organs (when present) will be weighed; paired organs will be weighed separately: 3MEnvironmental Laboratory Page 31 3M M edical D epartm ent Study: T-6316.1 Analytical Report: FACT-TOX-003 L R N -U 2104 * - Covance 63 29-2 28 Pape 13 adrenal (2) brain kidney (2) liver lung ovary (2) spleen testis (2) thyroid (2) with parathyroid uterus with cervix Organ-to-body weight percentages and organ-to-brain weight ratios will be calculated. Bone M arrow Smear From the femur of each animal at the Week 79 and terminal sacrifices only; made but not examined A dditional Liver Sample Collection A portion o f the liver will be collected from five animals/sex/group at the interim and terminal sacrifices and stored in a freezer set to maintain -60 to -80C. Samples will be packed on dry ice and shipped to Kris J. Hansen, PhD, 3M Environmental Technology and Safety Services. Samples will be retained by the Sponsor for possible future analysis. Tissue Preservation The following tissues (when present) from each animal will be preserved in 10% neutral-buffered formalin: ad re n al (2 ) brain cecum cervix colon duodenum epididymis (2) esophagus eye (2) femur with bone marrow (articular surface of the distal end) Harderian gland heart ileum jejunum kidney (2) lesions liver lung with mainstem bronchi lymph node (mesenteric) mammary gland (females only) ovary (2) pancreas pituitary prostate rectum salivary gland (mandibular (2)] sciatic nerve seminal vesicle (2) skeletal muscle (thigh) 3MEnvironmental Laboratory Page 32 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Skin spinal cord (cervical, thoracic, and lumbar) spleen sternum with bone marrow stomach testis (2) Covance 6329-228 ____________ P a g e 1 4 thymus thyroid (2) with parathyroid trachea urinary bladder uterus vagina Histopathology Tissues will be held for possible future examination. Reports One copy o f each draft report will be sent to the Sponsor. The report for this study will be included as an appendix to the report for Covance 6329-212. The report will include the following information: Experimental Design and Methods /i . Results dose analyses mortality clinical observations body weights body weight changes food consumption test material consumption clinical pathology results palmitoyl CoA oxidase activities macroscopic observations cell proliferation assessments (provided by the Sponsor's designee) Statistical Evaluation body weights body weight changes food consumption survival rates clinical pathology values palmitoyl CoA oxidase activities Statistical methods will be those presented in Attachments Nos. 1 and 2. For each sex, Group 9 will be compared to G roup 8 (Control). 3MEnvironmental Laboratory Page 33 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 _______________________________________________________________________________________________________ P a g e 15 At the end o f 1 year after issuance of the audited draft report, if no requested revisions or instructions to finalize have been communicated by the Spons or, then the audited draft report will be considered 'final' and issued as the final report, signed by the study director, and submitted to the Sponsor. Any modifications or changes to the audited draft report requested 1 year after issuance will be performed at additional cost to the Sponsor. Record Retention All raw data, documentation, records, protocol, specimens, and final report generated as a result o f this study, including those items listed below, will be archived in the storage facilities of Covance-Madison for a period of 1 year following submission o f the final report to the Sponsor. All raw data stored on magnetic media, the protocol and protocol amendments, study correspondence, and the original report will be retained by Covance. One year after submission of the final report, all of the aforementioned materials will be sent to the Sponsor, and a return fee will be charged. The Spo nsor may elect to have the ' materials retained in the Covance archives for an additional period o f time, and Covance will charge a storage fee. If the Sponsor chooses to have Covance dispose o f the materials, a disposal fee will be charged. protocol and protocol amendments dose preparation records in-life records animal receipt acclimation animal room maintenance randomizations dose administration clinical observations body weights food consumption sample collection clinical pathology records anatomical pathology records statistical analyses study correspondence tissue specimens (wet) blood and bone marrow slides final report (original signed copy) 3MEnvironmental Laboratory Page 34 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 ________________ _ ____________________________________________________________ ______________________________ P a g e 1 6 The following supporting records will be retained at Covance-Madison but will not be archived with the study data. feed analysis records water analysis records animal room environment records refrigerator and freezer temperature records room temperature records for test material storage instrument calibration and maintenance records PCNA evaluation data and paraffin blocks and tissue slides will be retained by Pathology Associates International. . Serum and liver samples sent to the Sponsor will be retained by the Sponsor 3MEnvironmental Laboratory Page 35 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 63 29-2 28 ____________________________________________________________ ___________ P a g e 1 7 PROTOCOL APPROVAL /I mM ouj 7 . Q&uk' Andrew M. Seacat, PhD Study Monitor 3M `{ 1 7 / Date 3M Environmental Laboratory Page 36 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Attachment No. 1 Covance 6329-228 ____________ P a g e 1 8 Statistical Analyses Levene's test will be done to test for variance homogeneity. In the case of heterogeneity o f variance at p ^ 0.05, transformations will be used to stabilize the variance. Comparison tests will take variance heterogeneity into consideration. One-way analysis o f variance (ANOVA) will be used (if applicable) to analyze body weights, body weight changes, food consumption, continuous clinical pathology values, and organ weight data. If the ANOVA is significant, Student's t-test will be used for control versus treated group comparisons. If the ANOVA shows significance for body weights at Week 1, one-way analysis o f covariance (ANCOVA) will be used to analyze body weights, with initial body weights as the co variate. If the ANCOVA is significant, covariate-adjusted means will be used for control versus treated group comparisons. Group comparisons (Group 9 versus Group 8) will be evaluated at the 5.0% two-tailed probability level. Only data collected on or after the first day of treatment'will be analyzed statistically. 3MEnvironmental Laboratory Page 37 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Attachment No. 2 Covance 6 3 29-2 28 ____________ P a g e 1 9 Adjusted survival data are analyzed by the National Cancer Institute (NCI) lifetable package. The tests include: Graphical (Kaplan-Meier product-limit estimation curves), Cox-Tarone binary regression methods for trend and heterogeneity, and Gehan-Breslow nonparametric methods for trend and heterogeneity. 3MEnvironmental Laboratory Page 38 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 C O V /V fiC E > THE DEVELOPMENT SERVICES COMPANY PROTOCOL AMENDMENT NO 1 Covance 6329-228 104-W eek Dietary Carcinogenicity Study with Narrow Range (98.1%) N-Ethyl Perfluorooctanesulfonamido Ethanol in Rats Sponsor: Study Monitor: Testing Facility: Study Director: 3M, St. Paul, Minnesota Andrew M. Seacat, PhD Covance Laboratories Inc., Madison, Wisconsin Peter J. Thomford, PhD This amendment modifies the following portions of the protocol: y< Effective April 23,1998 1. Page 11, T erm ination. To reflect the decision to anesthetize animals to be sacrificed with carbon dioxide, delete the text in this section and replace with the following: Unscheduled Sacrifices and Deaths Necropsies will be done. Animals to be sacrificed will be anesthetized with carbon dioxide, weighed, and exsanguinated. A blood film will be taken as part o f the necropsy procedure for sacrificed animals. 3MEnvironmental Laboratory Page 39 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 63 29-2 28 Protocol A m e n d m en t N o . 1 ____________________________________________________________________________ Page 2 Scheduled Sacrifices Interim Sacrifices During W eek 4, five animals/sex/group will be fastied overnight, bled for serum samples, anesthetized with carbon dioxide, weighed, and exsanguinated. The abdominal cavity of each animal will be opened, the: liver will be removed and weighed, and liver samples will be collected. Animals will be discarded after liver collection. D uring W eek 14, five animals/sex/group will be fasted overnight, bled for serum samples, anesthetized with carbon dioxide, weighed, and exsanguinated. T he abdominal cavity o f each animal will be opened, the liver will be removed and weighed, and liver samples will be collected. Animals will be discarded after liver collection. A fter at least 78 weeks of treatment, 10 animals/sex/group will be fasted overnight, bled for serum samples (five animals/sex/group), anesthetized with carbon dioxide, weighed, exsanguinated, and necropsied. A blood film will be taken as part of the necropsy procedure. Term inal Sacrifice A fter at least 104 weeks o f treatment, the remaining animals will be fasted overnight, bled for serum samples (five animals/sex/group), anesthetized with carbon dioxide, weighed, exsanguinated, and necropsied. A blood film will be taken as part o f the necropsy procedure. 2 Page 12, Postm ortem Procedures, Cell Proliferation Tissue Collection and Im m unohistochem ical E valuation, P aragraph 1. To reflect the decision to collect liver samples for proliferation cell nuclear antigen evaluation at Week 14 only, delete the text in this paragraph and replace with the following: At the W eek 14 interim sacrifice, representative samples of the left lateral lobe of the liver and any macroscopic lesions of the liver will be collected and preserved in zinc formalin. 3MEnvironmental Laboratory Page 40 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 C ovance 6329-228 Protocol A m e n d m e n t N o . 1 ____________________________________________________________________________________________________ __ ___________ P a g e 3 Effective April 28,1998 3. Page 2, A lternate S tu d y M onitor. To include the alternate study m onitor in the protocol, add the following after "Study Monitor:" A lternate Study M onitor Marvin T. Case, DVM, PhD 3M Toxicology Services Telephone No.: 612.733.5180 Facsimile No.: 612.733.1773 4. Page 6, G roup D esignations an d D ietary Levels, Footnote e. To reflect the decision to change the W eek 79 interim sacrifice to Week 53, delete the text in this footnote and replace with the following: Ten animals/sex/group will be designated as interim sacrifice animals and will be / . sacrificed after at least 52 weeks of treatment. 5. Page 9, Clinical Pathology, F requency an d N um ber of A nim als, Scheduled Collections, P a ra g ra p h 2. The W eek 79 interim sacrifice will be moved to Week 53, therefore blood films will be prepared from animals sacrificed after 52 weeks of treatment. To reflect this decision, delete the text in this paragraph and replace with the following: A blood film will be made and held for possible future examination from animals at scheduled sacrifices after at least 52 and 104 weeks of treatment. 6. Page 10, Serum Analyses, Frequency an d N um ber of Animals. The W eek 79 interim sacrifice will be moved to Week 53, therefore serum samples will be collected from animals sacrificed after 52 weeks of treatment. To reflect this decision, delete the text in this section and replace with the following: 3MEnvironmental Laboratory Page 41 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 63 29-2 28 Protocol A m end m en t N o . 1 ______________________________________________________________________ Page 4 Five animals/sex/group during Weeks 4 and 14 (animals selected for hepatocellular proliferation and biochemical analyses), five animals/sex/group (from animals selected for interim sacrifice) after at least 52 weeks of treatment, and five animals/sex/group at the terminal sacrifice 7. Page 11, Term ination, Scheduled Sacrifices, Interim Sacrifices, P a ra g ra p h 3. To reflect the decision to change the Week 79 interim sacrifice to W eek 53, delete the text in this paragraph and replace with the following: After at least 52 weeks o f treatment, 10 animals/sex/group will be fasted overnight, bled for serum samples (five animals/sex/group), anesthetized with carbon dioxide, weighed, exsanguinated, and necropsied. A blood film will be taken as part of the necropsy procedure. 8. *i Page 12, Postm ortem Procedures, O rgan Weights. To reflect the decision to record organ weights at the W eek 53 interim sacrifice only, delete the text in this section and replace with the following: At the W eek 53 interim sacrifice, the following organs (when present) will be weighed; paired organs will be weighed separately: adrenal (2) brain kidney (2) liver lung ovary (2) spleen testis (2) thyroid (2) with parathyroid uterus with cervix Organ-to-body weight percentages and organ-to-brain weight ratios will be calculated. 9. Page 13, Postm ortem P rocedures, Bone M arrow Smear. The W eek 79 interim sacrifice will be moved to Week 53, therefore bone marrow smears will be collected from animals sacrificed after 52 weeks of treatment. To reflect this decision, delete the text in this section and replace with the following: 3MEnvironmental Laboratory Page 42 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 63 29-2 28 Protocol A m end m en t N o . 1 _________________________________________________________________ ____________ Page 5 From the femur of each animal at the Week 53 and terminal sacrifices only; made but not examined 10. Page 14, R eports, P a ra g ra p h 1. To reflect the decision to provide unaudited summary reports after the Week 14 and 53 interim sacrifices, delete the text in this paragraph and replace with the following: After the W eek 14 and 53 interim sacrifices, unaudited summary reports will be sent to the Sponsor in conjunction with the summary reports for Covance 6329-212. The summary reports will include a brief description of methods and results and summary tables o f in-life data, clinical pathology data, and anatomical pathology data. After completion of the study, one copy of each draft report will be sent to the Sponsor. The report for this study will be included as an appendix to the report for Covance 6329-212. The report will include the following information: Effective M ay 15,1998 11. P age 10, S erum A nalyses, M ethod o f Collection. To reflect the decision to increase the volume collected for serum analyses at the W eek 53 and 105 sacrifices, delete the text in this section and replace with following: Animals will be fasted overnight; blood (approximately 2 mL at the W eek 4 and 14 sacrifices, approximately 3 mL at the W eek 53 and 105 sacrifices) will be collected from a jugular vein. Samples will be collected without anticoagulant. 3MEnvironmental Laboratory Page 43 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 AMENDMENT APPROVAL Covance 6329-228 Protocol A m endm ent N o . 1 _______________________ P a g e 6 &L M. Seacat, PhD Study Monitor 3M Date *t 3MEnvironmental Laboratory Page 44 3M Medical Department Study: T-6316.1 V'' - Analytical Report: FACT-TOX-003 LRN-U2104 C O V A rliSlf^ TH C m O M ttH T SlUVtCCS COMPANY PROTOCOL AMENDMENT NO. 2 Covance 6329-228 104-W eek Dietary Carcinogenicity Study with Narrow Range (98.1%) N-Ethyl Perfluorooctanesulfonamido Ethanol in Rats Sponsor: Study Monitor: Testing Facility: Study Director: 3M , St. Paul, Minnesota Andrew ML Seacat, PhD Covance Laboratories Ine., Madison, Wisconsin Peter J. Thomford, PhD This amendment modifies the following portions o f the protocol: ` Effective A pril 1,1998 1. Page 4. To include the vehicle used to dissolve the test material before mixing with the diet, add the following section. Vehicle Identification Acetone Lot Numbers The lot numbers will be maintained in the raw data. Purity On file with the manufacturer Stability On file w ith the manufacturer 3MEnvironmental Laboratory Page 45 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Storage Conditions At room temperature C h aracteristics Information on synthesis methods, composition, or other characteristics that define the vehicle is on file with the manufac turer. Effective Ju n e 30,1998 2. P age 7, Dosing P rocedure, R etention Sam ples. To reflect the decision to send retention samples o f control diet to the Sponsor, delete die text in this section and replace with the following: Samples (approximately 100 g) will be taken from each dose preparation during the in-life phase and stored at room temperature. Unless used for analyses, retention samples from the treated groups will be discarded at least 1 month after completion of the in-life phase. Samples from control diels prepared for W eeks 14 and before will be shipped to: Kris J. Hansen, PhD 3M Environmental Technology and Safety Services 935 Bush Avenue Building 2-3E-09 St. Paul, Minnesota 55133-3331 Telephone No.: 651.778.6018 Facsimile No.: 651.778.6176 , . Effective July 20,1998 3. Page 2, Study M o n ito r an d A lternate Study M onitor. To indicate the change in the area code, delete the text in these sections and replace with the following: Study M onitor Andrew M. Seacat, PhD 3M Telephone No.: 651.575.3161 Facsimile No.: 651.733.1773 3MEnvironmental Laboratory Page 46 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 Protocol Amendment No. 2 ________________ Page3 A lternate Study M onitor M arvin T. Case, DVM, PhD 3M Toxicology Services Telephone No.: 651.733.5180 Facsimile No.: 651.733.1773 4. Page 4 , D isposition of Test M aterial. To indicate the change in the area code, delete th e text in these section and replace with the following: After authorization from the Sponsor, any remaining test material will be returned to: Andrew M. Seacat, PhD 3M Toxicology Services Building 220-2E-02,3M Center St. Paul, Minnesota 55144-1000 Telephone No.: 651.575.3161 Facsimile No.: 651.733.1733 : 5. Page 1 0 , S eru m Analyses, Sam ple H andling. T o indicate the change in the area code fo r the Sponsor, delete the text in these section and replace with the following: Blood sam ples will be allowed to clot at room temperature and centrifuged. Serum sam ples will be harvested and stored in a freezer set to maintain -60 to -8 0 C. Samples will be packed on dry ice and shipped to: Kris J. Hansen, PhD 3M Environmental Technology and Safety Services 935 B ush Avenue Building 2-3E-09 St. Paul, Minnesota 55133-3331 Telephone No.: 651.778.6018 Facsimile No.: 651.778.6176 Samples will be retained by the Sponsor for possible future analysis. 3MEnvironmental Laboratory Page 47 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Effective A pril 9,1999 Covance 6329-228 Protocol Amendment No. 2 ____________ Page4 6. Page 9, Clinical Pathology, Frequency a n d N um ber o f A nim als, S c h e d u led Collections, P a ra g ra p h 2. Hematology tests will be done for animals sacrificed at W eek 53 and blood films will not be necessary. Therefore, delete this paragraph and replace with the following. A blood film will be made and held for possible future examination from animals at scheduled sacrifice after at least 104 weeks o f treatment. 7. Page 11, Term ination, Scheduled Sacrifices, Interim Sacrifices, P a ra g ra p h 3. To reflect the decision to collect clinical pathology samples from animals at the W eek 53 interim sacrifice, delete the text in this paragraph and replace w ith the following: / After at least 52 weeks o f treatment, 10 animals/sex/group will be fasted overnight, bled for clinical pathology tests (all animals) and serum samples (five animals/sex/group), anesthetized with carbon dioxide, weighed, exsanguinated, and necropsied. 3MEnvironmental Laboratory Page 48 3M M edical D epartm ent Study: T-6316.1 Analytical Report: FACT-TOX-003 L R N -U 2104 AMENDMENT APPROVAL Covance 6329-228 Protocol Amendment No. 2 Pace 5 Andrew M. Seacat, PhD Study M onitor 3M ' PeteTJ. Thgrijf&d, PhD A ' Study Director Covance Laboratories Inc. 6/tifa Date 1 ' ax/Lut/Ke- D a t^ 3MEnvironmental Laboratory Page 49 3M Medical Department Study: T-6316.1 CO V/H C> THE DEVELOPMENT SERVICES COMPANY Analytical Report: FACT-TOX-003 LRN-U2104 PR O TO C O L AMENDMENT NO. 3 Covance 6329-228 104-W eek Dietary Carcinogenicity Study with Narrow Range (98.1%) N-Ethyl Perfluorooctanesulfonamido Ethanol in Rats Sponsor: Study Monitor: Testing Facility: Study Director: 3M, St. Paul, Minnesota Andrew M. Seacat, PhD Covance Laboratories Inc., Madison, Wisconsin*1 Peter J. Thomford, PhD______ .______ _______ This amendment modifies the following portion of the protocol. Effective January 26,2000 1. Page 14, Postmortem Procedures, Histopathology. The Sponsor has requested that all tissues from animals in Group 8 and selected tissues from animals in Group 9 be examined microscopically. To rellect this decision, delete the text in this section and replace with the following. Tissues (as appropriate) from each animal in Group 8 will be embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. Lesions, liver, lungs, kidneys, pancreas, thyroid, testes, and mammary glands (females) from each animal in Group 9 will be embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. Parathyroids will be processed with the thyroids, but will not be examined. 3MEnvironmental Laboratory Page 50 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covancc 63 2 9 -2 2 8 Protocol A m end m en t N o . 3 _________________ Page 2 Effective F e b ru a ry 18, 2000 2. Page 10, Serum Analyses, F requency an d N um ber o f A nim als. To reflect the decision to collect serum samples from 10 animals/sex/group at termination, delete the text in this section and replace with the following: Five animals/sex/group during Weeks 4 and 14 (animate selected for hepatocellular proliferation and biochemical analyses), five animals/sex/group (from an im al.; selected for interim sacrifice) after at least 52 weeks of treatment, and 10 animals/sex/group at the terminal sacrifice 3. Page 11, Term ination, Scheduled Sacrifices, Term inal Sacrifice. To reflect the decision to collect serum samples from 10 animals/sex/group at termination, delete the text in this section and replace with the following: After at least 104 weeks o f treatment, the remaining animals will be fasted overnight, bled for serum samples (10 animals/sex/group), anesthetized with sodium pentobarbital, weighed, exsanguinated, and necropsied. A blood film will be taken as part of the necropsy procedure. 4. Page 13, Postmortem Procedures, Additional Liver Sam ple Collection. decision to collect liver samples from 10 animals/sex/group at termination, delete the text in this section and replace with the following: A portion of the liver will be collected from five animals/sex/group at the interim sacrifice and 10 animals/sex/group at the terminal sacrifice and stored in a freezer set to maintain -60 to -80C. Samples will be packed on dry ice and shipped to Kris J. Hansen, PhD, 3M Environmental Technology and Safety Services. Samples will be retained by the Sponsor for possible future analysis. 3MEnvironmental Laboratory Page 51 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 AM ENDM ENT APPROVAL Covance 63 2 9 -2 2 8 P rotocol A m en d m en t N o . 3 __________ ____________ P a g e 3 3M PfetSr I Thgn*i<5rd, Study Director Covance Laboratories Inc. 2A Date * 3MEnvironmental Laboratory Page 52 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Environmental Laboratory Protocol - Analytical Study Phase 104-Week Dietary Carcinogenicity Study with Narrow Range N-Ethyl Perfluorooctanesulfonamido Ethanol in Rats In-Vivo Study Reference Number: Covance # 6329-228 ( g ) U < L T -h ifa Study Number: FACT-651498.L Test Material: T-6316 Name and Address of Sponsor: 3M Toxicology Services Building 220-2E-02,3M Center St. Paul, MN 55144-1000 Name and Address of Testing Facility: 3M Environmental Technology and Services 935 Bush Avenue St. Paul, MN 55106 Proposed Experimental Start Date: May 25, 1998 Proposed Experimental Termination Date: December 30,2001 Method Numbers and Revisions FACT-M-1.0, Extraction of Perfluorooctanesulfonate from Liver for ;\nalysis using HPLC-Electrospray/Mass Spectrometry FACT-M-2.0, Analysis o f Liver Extracts for Fluorochemicals using HPLC- Electrospray/Mass Spectrometry FACT-M-3.0, Extraction o f Perfluorooctanesulfonate from Sera for Analysis using HPLC-Electrospray/Mass Spectrometry FACT-M-4.0, Analysis o f Sera Extracts for Fluorochemicals using HPLC- Electrospray/Mass Spectrometry Author: Lisa Clemen Study Director Study Director Management Sponsor Representative 3MEnvironmental Laboratory Page 1 of 5 Page 53 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 1.0 PURPOSE_________________________________________________________________ 1.1 According to this analytical protocol, the 3M Environmental Laboratory will analyze the tissue and fluid samples from the Covance study number 6329-2.28, " 104-Week Dietary Carcinogenicity with Narrow Range N-Ethyl Perfluorooctanesulfonamido Ethanol in Rats." The collected data will be provided to the sponsor for use in the assessment o f toxicological effects o f the test material when administered in the diets o f rats for at least 104 weeks. 1.2 Data collected in the Environmental Laboratory will be considered non-quantitative screening data until future studies have been conducted to determine absolute recoveries o f specific or general fluorochemical compounds. 2.0 REGULATORY COMPLIANCE______________________________________________ 2.1 This analytical phase o f the study will be conducted in accordance with the FDA Good Laboratory Practices Regulations 21 CFR 58, with the following exceptions: 2.1.1 The analytical phase is being conducted as a separate study and therefore has a separate Study Director, protocol, and final report, from those listed in the Covance protocol 6329-228. 2.1.2 The characterization of the reference material, including purity, identity, and stability, are the responsibility of the sponsor. 2.1.3 Sample storage stability will not be determined. 3.0 TEST MATERIALS________________________________________________________ 3.1 Control, and reference Materials and Matrices 3.1.1 Analytical Reference Material: T-6316, from 3M ICP/PCP Division 3.1.2 Analytical Reference Matrix: Rat liver, from Covance and rat serum, from Sigma Chemical Company. 3.1.3 Analytical Control Material: None. 3.1.4 Analytical Control Matrix: Rat liver, from Covance and rat serum, from Sigma Chemical Company. 3.2 Number of Test and Control Samples: Throughout the course o f the study, liver and serum from 140 test animals and 140 control animals will be made available. Samples will be analyzed as requested by the sponsor or the study director. Other biological tissues (kidney, bile, dermal application site, and cellular fraction) will be available for analysis if deemed appropriate. 3.3 Identification of Test and Control Samples: The samples will be identified using the Covance animal identification number that consists of a letter and five-digit number, plus the tissue identity and day identity (serum). 3.4 Purity and Identity of Reference Material: To be determined by Sponsor. 3.5 Stability of Reference Material: To be determined by Sponsor. 3MEnvironmental Laboratory Page 2 of 5 Page 54 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3.6 Storage Conditions for Reference M aterials: Reference materials will be stored at room temperature (3.1.1), and samples will be stored at -20 lOooC (3.1.2, 3.1.4). Test and Control samples will be received according to AMDT-S-10-0. 3.7 Disposition of Specimens: Biological tissues and fluids will be retained per GLP Regulation for the time period required for studies longer than 28 days. 3.8 Safety Precautions: Refer to appropriate MSDS. Wear appropriate laboratory attire. Use caution when handling knives for cutting tissue samples. 4.0 EXPERIMENTAL - Overview__________________________________________ 4.1 The tissues from animals dosed as described (Covance# 6329-228), will be available for analysis for fluorine-containing compounds. At the discretion o f the Study Director, a series of analytical tests can be performed. All high dose and control sera and livers will be analyzed initially using HPLC-electrospray mass spectrometry to identify fluorine-containing compounds o f interest present in the sera and liver (if any). The screening for organic fluoride in liver via combustion may be performed to present definitive data for fluorine in the liver. Based on the findings from these analyses, additional samples, tissues, or fluids may be analyzed at the discretion o f the Study Director to determine the presence o f fluorochemicals in these matrices. 5.0 EXPERIMENTAL - Methods_________________________________________________ 5.1 Methods (attached): 5.1.1 FACT-M-1.0, "Extraction o f Perfluorooctanesulfonate from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry" 5.1.2 FACT-M-2.0, "Analysis o f Liver Extracts for Fluorochemicals using HPLCElectrospray/Mass Spectrometry" 5.1.3 FACT-M-3.0, "Extraction o f Perfluorooctanesulfonate from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry" 5.1.4 FACT-M-4.0, "Analysis o f Serum Extracts for Fluorochemicals using HPLCElectrospray/Mass Spectrometry" 6.0 DATA ANALYSIS___________________________________________________________ 6.1 Q uality C ontrol: Matrix spikes will be extracted and analyzed to determine accuracy of the method. Also, continuing calibration checks will be analyzed to determine response bias. 6.2 Transformations: Any transformations performed on data collected during the analytical phase o f the study will be documented in the final report. 6.3 Statistics: At the discretion o f the Study Director, statistics used may include regression analysis of serum concentrations with time, averages, and standard deviations of concentrations for the different dose groups. If necessary, simple tests such as the Student's t-test may be applied to determine statistical difference. Any statistical analysis performed will be documented in the final report. 6.4 Data Reporting: A final data package will be submitted to 3M Toxicology Services. The data package will include the following with additional data included as deemed appropriate. 3MEnvironmental Laboratory Page 3 of 5 Page 55 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 6.4.1 A summary o f individual sample results, reported as a concentration (weight/weight, weight/volume) o f fluoride per tissue or fluid, or as the mass o f a specific fluorochemical (HPLC-electrospray mass spectrometry) per unit o f tissue or fluid. 6.4.2 A summary of quality control results (continuing calibration checks, method blanks, instrument blanks, matrix spikes, and matrix spike duplicates). 6.4.3 Certified copies or originals of the written validated methods. 6.4.4 Certified copies or originals o f sample identification shee ts sent from Covance. 6.4.5 Certified copies or originals o f study specific raw data. 6.4.6 A summary o f key personnel involved with the analytical phase o f the study. 6.4.7 A signed QAU statement listing the dates o f inspections rind reports o f findings to management and Study Director. 7.0 MAINTENANCE OF RAW DATA AND RECORDS_____________________________ 7.1 The following raw data and records (or certified copies thereof) will be maintained in the study folder in the archives according to appropriate SOPs. 7.1.1 7.1.2 7.1.3 7.1.4 7.1.5 7.1.6 7.1.7 Approved protocol Approved methods Data summaries Study correspondence Shipping records Raw data Electronic copies of data ' 7.2 Supporting records to be retained separately from the study folder in the archives according to 3M ET & SS SOPs, will include, but not necessarily be limited to the following: 7.2.1 7.2.2 7.2.3 7.2.4 7.2.5 7.2.6 Approved validation reports Training records Calibration records Instrument maintenance logs Standard operating procedures, equipment procedures, and methods Appropriate specimens 8.0 REFERENCES_____________________________________________________________ 8.1 Approved AMDT standard operating procedures. 8.2 Approved ETS standard operating procedures. 9.0 ATTACHMENTS___________________________________________________________ 9.1 FACT-M-1.0, Extraction o f Perfluorooctanesulfonate from Liver for Analysis using HPLCElectrospray/Mass Spectrometry 3MEnvironmental Laboratory Page 4 of 5 Page 56 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 9.2 FACT-M -2.0, Analysis o f Liver Extracts for Fluorochemicals using HPLCElectrospray/Mass Spectrometry 9.3 FACT-M -3.0, Extraction o f Perfluorooctanesulfonate from Serum for Analysis using HPLCElectrospray/Mass Spectrometry 9.4 FACT-M -4.0, Analysis o f Serum Extracts for Fluorochemicals using HPLCElectrospray/Mass Spectrometry 4t 3MEnvironmental Laboratory Page 5 of 5 Page 57 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Study Title 104-Week Dietary Carcinogenicity Study with Narrow Range N-Ethyl Perfluorooctanesulfonamido Ethanol in Rats . PROTOCOL AMENDMENT NO. 1 Amendment Date: 20 January 2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS LRN-U2104 FACT TOX-003 Covance Study: 6329-228 3M Medical Department Study: T-6316.1 3M Environmental Laboratory 3MEnvironmental Laboratory Page 58 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 Protocol LRN-U2 ^ ^ N -T O IO 4 Amendment Number 1 This amendment modifies the following portion(s) of the protocol: 1. P r o t o c o l R e a d s : The following methods will be used: FACT-M-1.0, Extraction of Perfluorooctanesulfonate from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry FACT-M-2.0, Analysis of Liver Extracts for Fluorochemicals using HPLC- Electrospray/Mass Spectrometry . FACT-M-3.0, Extraction of Perfluorooctanesulfonate from Sera for Analysis using HPLC-Electrospray/Mass Spectrometry FACT-M-4.0, Analysis of Sera Extracts for Fluorochemicals using HPLC- Electrospray/Mass Spectrometry A mend to Read: The following methods will be used: ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical compounds from Serum for Analysis Using HPLC- Electrospray/Mass Spectrometry ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry ETS-8-6.0, Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry ETS-8-7.0, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Speptrometry Reason: The methods originally listed were superseded during the course of the study. 2. P r o to c o l R ea d s: There is no independent section of the protocol that addresses sample retention. A mend to Read: Specimens will be maintained in the 3M Environmental Laboratory specimen archives. Any specimens sent to sub-contract laboratories will be returned to the 3M Environmental Laboratory upon completion of analysis and submission of the sub contract laboratory(s) final report. Specimens analyzed at sub-contract laboratories will be returned with the following documentation: the signed original chain of custody and records of storage conditions while at the sub-contract facility. Reason: To define in detail the appropriate disposition of specimens analyzed at subcontract laboratories. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 59 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 Protocol L R N -U ^ W '^ 2104 Amendment Number 1 3. P r o to c o l R e a d s : Section 7 states that the following raw data and records will be retained in the study folder in the archives according to AMDT-S-8: Approved protocol and amendments; approved methods; data summaries; study correspondence; shipping records; raw data; and electronic copies of data. Additionally, Section 7 states that supporting records to be retained separately from the study folder in the archives according to AMDT-S-8 will include at least the following: Approved validation reports; training records; calibration records; instrument maintenance logs; Standard Operating Procedures, Equipment Procedures, and Methods; and appropriate specimens. A m end to R ea d : Section 7 states: "The original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance of the final report. When the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, and electronic copies of data will be retained in the archives of the 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained in the archives of the facility performing each analysis." Reason: To direct subcontract laboratories in the disposition of the items listed above. 4. Protocol reads: The study director for the present study was identified in the protocol as Kristen J. Hansen, Ph.D. ` A m en d to r ea d : The role of study director for the present study was reassigned to John L. Butenhoff, Ph.D., as of 20 January 2000. The previous study director, Kristen Hansen, has been reassigned to the role of Principle Analytical Investigator. Reason: ' The role of study director was reassigned in an effort to ensure compliance with Good Laboratory Practice Standards that outline study personnel requirements (refer to 21 CFR Part 58). 5. P r o t o c o l r e a d s : The sponsor for the present study was identified as Andrew M. Seacat, Ph.D. A m en d to r e a d : The role of sponsor for the present study was reassigned to Marvin T. Case, D.V.M., Ph.D., as of 20 January 2000. Reason: The change was made at the request of the sponsor. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 60 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 Protocol L R N -U ^ W '^ 2104 Amendment Number 1 Amendment Approval Q i& u 4 t'________________________ Andrew M. Seacat, Ph.D., Outgoing Sponsor Representative Date --------- - Kristen J Hansen, PhD ., Outgoing Study Director / t - F - c b "2COO Date / Fily+Jbu 2 - r f - iw ) Marvin T. Case, D.V.M., PhD., Incoming Sponsor Representative T)ate / John L. Butenhoff, PhD., Incoming Study Director Date 3M Environmental Laboratory 3MEnvironmental Laboratory Page 61 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Study Title 104-Week Dietary Carcinogenicity Study with Narrow Range N-Ethyl Perfluorooctanesulfonamido Ethanol in Rats PROTOCOL AMENDMENT NO. 3 Amendment Date: May 4, 2001 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Project Identification 3M Medical Department Study: T-6316.1 Covance In-Life Study: 6329-228 Analytical Report: FACT TOX-003 3M Laboratory Request No. U2104 3M Environmental Laboratory 3MEnvironmental Laboratory Exact Copy of Original Page 62 3M M edical D epartm ent Study: T-6316.1 Analytical Report: FACT-TOX-003 L R N -U 2104 Protocol FA C T TOX-003 Amendment No. 3 This amendment modifies the following portion(s) of the protocol: 1. P r o t o c o l r e a d s : PURPOSE: The FACT TOX-003 protocol does not clearly identify the intended analytes for the study. A m en d to r ea d : PURPOSE: Rat sera and liver samples will be analyzed for PFOS, PFOSA, PFOSAA, PFOSEA, EtFOSE-OH and M556. R easo n: The target analytes will be clearly specified. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 63 3M M edical D epartm ent Study: T-6316.1 Analytical Report: FACT-TOX-003 L R N -U 2104 Protocol FACT TOX-003 A m endm ent No. 3 Amendment Approval TM a rv in Case, D .M .V ., Ph. D ,, Sponsor Representative r Date John Butenhoff, P h .D ., Study Director C lu M A - ^ / 2o) Date i 3M Environmental Laboratory 3MEnvironmental Laboratory Page 64 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Study Title 104-Week Dietary Carcinogenicity Study with Narrow Range N-Ethyl Perfluorooctanesulfonamido Ethanol in Rat PROTOCOL AMENDMENT NO. 2 Amendment Date: January 8, 2001 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS LRN-U2104 FACT TOX-003 Covance 6329-228 3M Medical Department Study: T-6316.1 3M Environmental Laboratory 3MEnvironmental Laboratory Page 65 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 Protocol FA C T T O X - ^ -U 2104 Amendment No. 2 This amendment modifies the following portion(s) of the protocol: 1. P r o to c o l r e a d s : 1.2 Data collected in the Environmental Laboratory will be considered non-quantitative screening data until future studies have been conducted to determine absolute recoveries o f specific or general fluorochemical compounds. A m end to read: 1.2 If matrix spike studies provide accurate representation of recovery of endogenous levels of PFOS, PFOSA, PFOSAA, PFOSEA, M556, and EtFOSE-OH, the 3M Environmental Laboratory will provide semi-quantitative data for sera and liver samples collected from test animals. Reason: Due to improved analytical methods, the decision was made to change ihe purpose of the study results from non-quantitative to semi-quantitative. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 66 3M Medical Department Study: T-6316.1 Amendment Approval Analytical Report: FACT-TOX-003 Protocol FA C T T O X - ffi'^ 2104 Amendment No. 2 Marvin T. Case, D.V.M., Ph.D., Sponsor Representative John L. Butenhoff, PhD., Study Director Date s> Date P 3M Environmental Laboratory 3MEnvironmental Laboratory Page 67 3M Medical Department Study: T-6316.1 Record of Deviation I. Identification Analytical Report: FACT-TOX-003 LRN-U2104 Deviation Type (Check one) SOP ^ M e th o d Equipment Procedure Protocol Other: Docu: N u m b e r-, \ Date(s) o f occurrence II. Description: Required Procedure/process : A /V ss? * ' " ' A `" fy r y^zC a / V M - -rfCz^jo ) fk s s z * ' * Actual Procedure/process: ~ ^7 A - A A a A -- s 2 ^ - ja A Z j^ & C /0 '. S //Q / tA -toS :n Q sor> ^-J> c& ,___ / T 'p>ys~ y t ,, s ? / fifay ^ cA .J 7 2 t y / ? P /Q / 2 -^? & ?S? 'z>r? k. III. Actions Taken: (such as amendment issued, SOP revision, etc.) Recorded By IV. I vefy/Project K>o..cvdvi-rH- impaci-..on -fW....outcome...o .. itas t Date 'P ' ) ...... u ... .......k i n l *1 Authorized By (Study Director / Project Lead) ^ U ? /jy if# ' Date S t - i ^ O . r t t W - Tokn 3M Environmental`L' aboraatoorryy Form ETS-4-8.0 lp*r R i f r i t t e V i* -. M w O ui. `J C * * * ' w * = P * * ~ -.Y f lA t OuL Deviation No. *VM^u, .0^*1 + ^ (assigned by Study Director or Project Lead at the end of iludy or project) 3MEnvironmental Laboratory Page 68 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Record of Deviation I. Identification Study / Project No. Deviation Type (Check one) ..... SOP Protocol ....,,Cot/Mu. [>32cl - 2 ZS 0 Method Equipment Procedure Other: Document Number Date(s) o f occurrence II. Description: Required Procedure/process:________ ..^ ^ - z / f a S s Actual Procedure/process: ry-> P T Z py-. y x S & // JeP III. Actions Taken: (such as amendment issued, SOP revision, etc.) ^ /5 Z ^ .c P c /. 2^> 2&.-3L. ...L ^ya^ jp S , m P zP o 2 O^LJ. Recorded By Date Sp Irpct on Study / Project / S ' / pU sUefac. fl&C rrrvip- 7 V4 PCSfh*. j O^VX4tiSlJ ^<3 fyli Q)/o 5 / 0/ Authorized By (Study Director / Project Lead) Date 7 S L /> f L sponsor tprtsii\fahut/. Mc,,vi/i Cu, 3M Environmental Laboratory Form ETS-4-8.0 w ^ hirtclv Totin BuliniloCf Deviation No. (assigned by Study Director or Project Lead at the end of study or project) 3MEnvironmental Laboratory Page 69 3M Medical Department Study: T-6316.1 Record of Deviation I. Identification Analytical Report: FACT-TOX-003 LRN-U2104 Study / Project No. FACT-TOX-003 Covance 6329-228 Deviation Type (C heck one) Q SOP X Method Equipment Procedure Protocol Q Other: Document Number(s): FACT-M-4.0, Date(s) o f occurrence: 07/13/98, 04/21/00, ETS-8-5.1 04/24/00,08/15/00,08/28/00, 08/30/00 II. Description: Required Procedure/process: Section 10.1.1 and 10.1.2 states: "Analyze a method blank and a matrix blank: prior to each calibration curve."............ ................ ................. ....... ....... ... .................................. ............ .... ...... Actual Procedure/process: On several occasions, the blanks were either a) analyzed after the calibration curve or b) not analyzed at all for a particular MS analysis (08/30/00, Wk4, Serum). III. Actions Taken: (such as amendment issued, SOP revision, etc.) This deviation was written. Recorded By Date C h: IV..Imp act on Study / Project D s / o i / 101 All blanks were analyzed at least once during the course o f the study. When blanks were analyzed.......... after the calibration curve, all control-of-bias practices were followed. In one analysis, blanks were not analyzed since these blanks had been run previously and did not require reanalysis. In this particular analysis (08/30/00, Wk 4, Serum) instrument blanks were used to determine limits o f quantitation. This deviation has no adverse affect on these study data. ................................~ ......... ~ ............................................................'.h. O S '/o ^ /O ! Authorized By (S tu d y D ir e c to r 7 P r o je c t L ea d ) Date 5 h u ta O .rtcW Tohn BuknboVV' J/owor ReprwenUW Mv Cw, T 6. 3M Environmental Laboratory Form ETS-4-8.0 3MEnvironmental Laboratory Deviation No. 3_____ (assigned by Study Director or Project Lead at the end of study or project) Page 70 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Record of Deviation I. Identification Study / Project No. P A cT -r#*Deviation Type (Check one) A Cdvvcl. L3 2.$'2.2.$ SOP J3 Method Equipment Procedure Protocol Other: Document Number Datefs) o f occurrence z A r /f 2 __________ II. Description: Required Procedure/process: f - j y / V p os-s, A#*. / <?/ Ae> A ..__Sr*<)/ Actual Procedure/process: _______________ ____________________________________ _ 7/ ~*t tt /f t/ 3J Z/ . _ , 2 ++it ffi.l, f/<t JX co-ilA *y* > __ ^- / _S_-___1____J_"_>_S_1________________y__j_J__j_r________ .. 7 ^ J *___ / , / +_ / $ / / A ^ _A? S Jjfer / *t --_Se/(AJmSs'__Ar~L.____________ ?s~. Ac_/ ______A / 0t*-/ A _r~> _ f / s S _J. A A. >~./ J)___________________________________________ ___________ III. Actions Taken: ___________________ (such as amendment issued, SOP revision, etc.) A t* A *,,AArA... d g t/s j / S7Q.1 Recorded By Date ' A , IV. Impact on Study / Project c? > Jut*cA uutrrt /\Q'e*J-y<3 'fr'xA f {/o/orrxuo 1 - S~-^-U- ^_r2!3 /^KLlA/Q_________________________________________________________ Authorized B y (Study Director / Project Lead) kjh O ft/T -flo O j Date 9 /n V o v ______________ !I_cJ&t f&d. 2*-H OrtC-Tr. T>)v\ i.W iKc f t 3MVfEnvironmental Laboratory Form ETS-4-8.0 /j*arw o.* ^lj> Mftrt C c .it, Deviation No. H (assigned by Study Director or Project Lead at the end of study or project) 3MEnvironmental Laboratory Page 71 3M Medical Department Study: T-6316.1 Record of Deviation Analytical Report: FACT-TOX-003 LRN-U2104 /. Identification Study / Project No.tox003 Deviation Type (Check one) SOP V Method Equipment Procedure Protocol Other: Document Number:FACT-M-2.0 Date(s) of occurrence: 08/07/1998 II. Description: Required Procedure/process: .............. ..... Section 10.2 1 Analyze a matrix spike and matrix spike duplicate with each analysis With a minimum o f 2 spikes per batch. ___ Actual Procedure/process: The matrix spike and duplicates were analy2.ed in an earlier run and were not re-run with this analysis. III. Actions Taken: (such as amendment issued, SOP revision, etc.) This deviation from the stated method is noted. Recorded By: Harold Johnson C C 4 g i IV. Impact on Study / Project This will have no impact on the study. ^ Date May 4, 2001 /b<dloi Authorized By (Study Director / Project Lead) Date 5+udx^ Director Tahn BnWihoP Form ETS-4-8.0 Deviation No. (assigned by Study Director or Project Lead at the end of study or project) 3MEnvironmental Laboratory Page 72 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Record of Deviation I. Identification Study / Project No. Deviation Type (Check one) FflCT- TOx - 0 1 ..........Cm m &L___1*33*1- 3 3 3 SOP Method Equipment Procedure Protocol G! Other: Document Number 2 1 CFR 53. lap Date(s) o f occurrence II. Description: 05i z i l w Required Procedure/process: Snbc__ R p J m F filiv f__ Q pproui__ ojcL -C tn ftl^li C l_ p ro i glo.1___ p rio ji_1d__ Pire^L-W approval.________________________________________________________ Actual Procedure/process: Dirte la . Signed. / Ctpptr ite __h k k Spmvspr Kfcpr^tftl-Cii:bdL________ A a l^ k ^ __ p rp lo c o l III. Actions Taken: (such as amendment issued, SOP revision, etc.) _piciiJiLi__Lllt_Lt._________........ ~rW -S fte,prosfcn4o.k>v<- Jaezare._ikt-_______ HitLclnc.____________________ Recorded By A . CAjuman IV. Impact on Study / Project ~h> deualito_lo.il . nof. -lduerse^ AmcJ: Date 0 1 I n fo c& Fkis.itucl^. Authorized By STudy Director}/ Project Lead) Date /C. 2 ^ V __ C - l_________ _ D/ftctAWoril Totui tu/.S j t o t u o r f U p r t J c / i t a f t t e r v C .A C C 3MEnvironmental Laboratory v?Q&/vwi TCuo*. ^tha* Pi-o/ Deviation No. to_______ Form ETS-4-8.0 (assigned by Study Director or Project Lead at the end o f studjwr project) 3MEnvironmental Laboratory Page 73 3M Medical Department Study: T-6316.1 Record of Deviation I. Identification Analytical Report: FACT-TOX-003 LRN-U2104 Study / Project No. FACT-TOX-003 Covance 6329-228 Deviation Type (Check one) SOP X Method Equipment Procedure Protocol Other: Document Number(s): ETS-8-7.0 Date(s) o f occurrence: 04/14/00,04/17/00 II. Description: Required Procedure/process: Section 14.3.1 states, `T h e r 2 yalue for the calibration curve must be 0.980 or better; Section 14.3.6 states, "A valid curve must contain at least five active points." Actual Procedure/process: The EtFOSE-OH calibration curve for the W eek 104 liver generated on 04/14/00 had an r2 value o f 0.9687. A four point curve was used to plot the EtFOSE-OH calibration curve for the W eek 53 liver,/generated on 04/17/00. III. Actions Taken: (such as amendment issued, SOP revision, etc.) These data were flagged in the raw data and this deviation was written. -- Recorded By Date (y^"> A- 0 5 /d3/di IV. Impact on Study / Project EtFOSE-OH data is flagged as qualitative, so these data will not be adversely affected. ......................... je^ h O S/Q i-/O f Authorized By (Study Director / Project Lead) Date Stud-t Director ifornor i. "Johr\ uVtnhoif ^ ai- T ?<&/ 3M Environmental Laboratory Form ETS-4-8.0 Deviation No. If (assigned by Study Director or Project Lead at the end of study or project) 3MEnvironmental Laboratory Page 74 3M Medical Department Study: T-6316.1 Record of Deviation Analytical Report: FACT-TOX-003 LRN-U2104 I. Identification Study / Project No. I a. ,,^ ....fo ri- - ~TC>X-C 0 3 ../ .... COVOOCC...... - 3 3 2 ............. Deviation Type 0 SOP 0 Method ^ E q u ip m e n t Procedure (Check one) 0 Protocol 0 Other: Document Number . I Date(s) o f occurrence ETS-ci-a7.t> .Sgctioo I3.| ~TT,rv a ^ aooo A 'Siiiy 'p .Z m II. Description: Required Procedure/process: ensgr I5 used cn ck 6LP voider 1 it rYiusfc jpg. 'C&lJemdtd_befare dctuftoi a d v q I u x y v l _ .tfa e - - f o p., p i ^ i osg^__ur>i tl_ .-b e u i/sd? on _c" ' -------1 ___o p o r e e t o c __ .... u s l n ^ ^ u isp o o sg r... Actual Procedure/process: he CaU bnjchoo ChJLCX- loabpoio 'orci icached -fchaJ ( T i a r c J o - L ) ^ a o ___ l o q s ...d h e ^ J < f e t ... d i f n e ' f r t , B o tH - d - T a p j y ^ p c n ^ t r t o o o>___ C a J U b r o m a d , _________________________________ III. Actions Taken: (such as amendment issued, SOP revision, etc.) 'The Pispirfer yjit/ .be cMJWnzte< Tifare, U M .... d O J U L u _ . u j l d i i n . ,, M l ? p r o ^ d c i s ^ a j ^ ......d o o e L u - c t e d ^ O X J t ^ Mjl volum e used <sn ~tKuz. pm >.cT...pund t ______ |& t o c _ ..u t ^ ^ u i U J ^ ...ta d __ s tn C v ,, jf ( ( U L . Rcorted By Date K iL U j K u j ^ U j u o IV. Impact on Study / Project /8 /o o r.. Ct*vyj20.... .............................................................. ,................................................... ......SU)........................... cn^.... th<................... .....................-.........-...................-...... - , ............- ............ .......___________________________________ ____________ ? J z . f / ( r o .............. Authorized By (Study Director / Project Lead) /\ ^ ' Date jlvcD DirtC/W-. -lotav Bwh^ko(-p 3M Environmental Laboratory Form ETS-4-8.0 SjUiiu'cr . M a r/ Caw, Deviation No. _______z_____ (assigned by Study Director or Project Lead at the end o f study or project) 3MEnvironmental Laboratory Page 75 3M Medical Department Study: T-6316.1 Record of Deviation Analytical Report: FACT-TOX-003 LRN-U2104 I. Identification Study / Project No. FACT-TOX-003 Covance 6329-228 Deviation Type (Check one) D SOP X Method Q Equipment Procedure D Protocol Other: Document Number(s): ETS-8-5.1 Date(s) o f occurrence: 08/30/00 II. Description: Required Procedure/process: A five point curve or better is used for plotting the calibration curve. Actual Procedure/process: A four point curve was used for plotting the PFOSAA calibration curve for the Week 4 serum curve, generated on 08/30/00. III. Actions Taken: (such as amendment issued, SOP revision, etc.) These data were flagged in the raw data and this deviation was written. Recorded By O l'fc A C tW IV. Impact on Study / Project These data will not be adversely affected. tit QC. -OrviO................... Date sl^li yh 0 5 /OHIO! Authorized By (Study Director / Project Lead) Date OireC-tar ' Tohrv uk/\ho?l- Sponsor R.epresei'ilai^. Cwc Y h c u u ,^ 7<&_ 4*-f 3M Environmental Laboratory Form ETS-4-8.0 Deviation No. (assigned by Study Director or Project Lead at the end of study or project) 3MEnvironmental Laboratory Page 76 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Appendix C: Extraction and Analytical Methods This appendix includes the following methods: Preparatory Methods F A C T -M -1 .0. Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLG-Electrospray/Mass Spectrometry, (8 pages) F A C T -M -3 .0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry, (8 pages) E T S -8 -4 .1 , Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry, (14 pages) E T S -8 -6 .0 , Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry, (14 pages) Analytical Methods F A C T -M -2.0, Analysis of Liver Extracts for Fluorochemicals Using HPLC- Electrospray/Mass Spectrometry, (8 pages) , F A C T -M -4 .0, Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry, (8 pages) E T S -8 -5 .1 , Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry, (9 pages) E T S -8 -7 .0 , Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry, (10 pages) 3MEnvironmental Laboratory Page 77 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Environmental L aboratory Method Extraction of Potassium perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/M ass Spectrometry Method Number: FACT-M-1.0 Author: Lisa Clemen Approved By: Adoption Date: S'/zt/?/ Revision Date: fjy\ Laboratory Manager Date f(wH^ f i ------ -- Group Leader 5/z-u / 4 f Date qL*. i t CijLhUA. Technical Reviewer skih ? Date 2 t? m 7 a 1.0 S c o p e a n d A p p l i c a t i o n ~)> 1.1 Scope: This method is for the extraction of Potassium Perfluorooctanesulfonate (PFOS) or 9 other fluorochemical surfactants from liver. O D 1.2 A pplicable C om pounds: Fluorochemical surfactants or other fluorinated compounds. <D C* in 1.3 M atrices: Rabbit, rat, bovine, and monkey livers or other livers as designated in the validation report jO J* 2L Microsoft 7.0.1/95 FACT-M-1.0 Extraction of PFOS from Liver Page 1 of 8 3MEnvironmental Laboratory Page 78 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 2.0 S u m m a r y o f M e t h o d ________________;__________________________________________ 2.1 This method describes how to extract potassium perfluorooctaiiesulfonate (PFOS) or other fluorochemical surfactants from liver using ion pairing reagent and 5.0 mLs o f ethyl acetate. An ion pairing reagent is added to each sample and partitioned into ethyl acetate. Four mLs o f extract is removed to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL methanol then filtered through a 3 cc plastic syringe attached to a 0.2 pm filter into glass autovials. 3.0 D e f i n i t i o n s _____________________________ ________________________________________________________ 3.1 None. 4.0 W a r n i n g s a n d C a u t i o n s _____________ ' 4.1 Health and Safety Warnings: ________ ^___________'_______________________ . 4.1.1 Use universal precautions when handling animal livers, they may contain pathogens. . 5.0 I n t e r f e r e n c e s _______________________________ .________ .__________________________________________ 5.1 There are no known interferences at this time. i 6.0 E q u i p m e n t _______________________________________________ ;_____________ i___________ ' '" 6.1 The following equipment is used while carrying out this method. Equivalent equipm ent is acceptable. - 6.1.1 U ltra-Turrax T25 Grinder for grinding liver samples 6.1.2 Vortex mixer, VWR, Vortex Genie 2 6.1.3 Centrifuge, M istral 1000 or IEC 6.1.4 Shaker, Eberbach or VWR . 6.1.5 N itrogen Evaporator, Organomation 6.1.6 Balance . 7.0 S u p p l i e s a n d M a t e r i a l s __________ ;___________________________________________________ 7.1 Gloves 7.2 D issecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable o f holding 250 mL and 1 L 7.5 Glass, type A, volumetric flasks 7.6 40 mL glass I-CHEM vials 7.7 Plastic sampule vials, W heaton, 6 mL 7.8 Polypropylene centrifuge tubes, 15 mL 7.9 Labels FACT-M-1.0 Extraction of PFOS from Liver Page 2 of 8 3MEnvironmental Laboratory Page 79 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 7.10 Syringes, capable o f measuring 10 pL to 50 pL 7.11 Glass, type A, volumetric pipettes 7.12 Graduated pipettes 7.13 Electronic pipettor, Eppendorf or equivalent 7.14 Tim er 7.15 Disposable plastic 3 cc syringes 7.16 Filters, nylon syringe filters, 0.2 pm, 25 mm 7.17 Crimp cap autovials N ote: Prior to using glassware and bottles, rinse 3 times w ith methanol and 3 w ith M illi- QTM water. Rinse syringes a minimum o f 9 times w ith methanol, 3 rinses from 3 separate vials. 8.0 R e a g e n t s a n d S t a n d a r d s _________________ ._________________ _______________'___________________ 8.1 Reagents 8.1.1 Sodium Hydroxide (J.T Baker or equivalent), (NaOH) 10N: weigh approximately 200 grams NaOH. Pour into a 1000 mL beaker containing 500 liters (L) Milli-QTM w ater, m ix until all solids are dissolved. Store in a 1 L nalgene bottle. 8.1.2 Sodium Hydroxide (J.T Baker or equivalent), (NaOH) IN. Dilute 10N 1:10. M easure 10 mL o f the 10N NaOH solution into a 100 raL volumetric flask and ' dilute to volume using Milli-QTM water. Store in a 125 mL nalgene bottle. 8.1.3 Tetrabutylammonium hydrogen sulfate (Kodak or equivalent), (TBA) 0.5M: W eigh approximately 169 grams o f TBA into a 1 L volumetric containing 500 L Milli-QTM water. A djust to pH 10 using approximately 64 mL 10N NaOH and dilute to volum e w ith Milli-QTM water. Add NaOH slowly while adding the last 1 mL o f NaOH because the pH changes abruptly. Store in a 1 L nalgene hottle. 8.13.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using IN NaOH solution. 8.1.4 Sodium carbonate/Sodium Bicarbonate Buffer (J.T. Baker or equivalent), (N a^O j/N aH C O j) 0.25M: W eigh approximately 26.5 g o f sodium carbonate (Na2C03) and 21.0 g o f sodium bicarbonate (NaHCOj) into a 1 L volumetric flask and dilute to volume with Milli-QTM water. Store in a 1 L nalgene bottle. 8.1.5 PFOS (3M Specialty Chemical Division), molecular weight = 538. 8.1.6 Ethyl Acetate, Omnisolv, glass distilled or HPLC grade. 8.1.7 M ethanol, Omnisolv, glass distilled or HPLC grade. 8.1.8 Liver and control liver, received frozen from testing laboratory. 8.1.9 M illi-QTM water, all water used in this method should be Milli-QTM water and may be provided by a M illi-Q TOC Plus system. 8.2 Standards 8.2.1 Prepare PFOS standards for the standard curve. FACT-M-1.0 Extraction of PFOS from Liver Page 3 of 8 3MEnvironmental Laboratory Page 80 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 8.2.2 W eigh approximately 100 mg o f PFOS into a 100 mL volumetric flask and record the actual weight. 8.2.3 Bring to volume with methanol for a stock standard o f approximately 1000 ppm (gg/m L). 8.2.4 D ilute the stock solution with methanol for a working standard 1 solution o f approxim ately 50 ppm. 8.2.5 D ilute the stock solution with methanol for a working standard 2 solution o f approx. 5.0 ppm. 8.2.6 D ilute the stock solution with methanol for a working standard 3 solution o f approx. 0.50 ppm. 9.0 S a m p l e H a n d l i n g _______________;__________________ '_____________________ ;_______________________ 9.1 A ll livers are received frozen and m ust be kept frozen until the extraction is perform ed. 10.0 Q u a l i t y C o n t r o l 10.1 M atrix Spikes _____________________________________;________________ ;____________________ 10.1.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy o f the extraction. 10.1.2 Prepare each spike using liver chosen by the analyst, usually a control liver. 10.1.3 Expected concentrations will fall in the mid-range o f the initial calibration curve. 10.2 Continuing Calibration Checks 10.2.1 Prepare and analyze continuing calibration check samples to determ ine the continued linearity o f the initial calibration curve. 10.2.2 One check is prepared per group o f ten samples. For example, if a sample set = 34,1 fo u r c h e c k s are prepared and ex tra cted . 10.2.3 Prepare each continuing calibration check from the same liver homogenate used to prep the initial curve. 10.2.4 The expected concentration will fall within the mid-range o f the initial calibration curve. 11.0 C a l i b r a t i o n a n d S t a n d a r d iz a t io n _____________ ;______________________________ _ 11.1 P rep a re L iv er H om ogenate to Use fo r Standards 11.1.1 W eigh approximately 40 g o f liver into a 250 mL Nalgene bottle containing 200 m Ls Milli-QTM water. Grind to a homogeneous solution. 11.1.2 If 40 g is not available, use appropriate amounts o f liver and water in keeping with a 1:5 ratio. 11.1.3 See section 13.0 to calculate the actual density o f liver. FACT-M-1.0 Extraction of PFOS from Liver Page 4 of 8 3MEnvironmental Laboratory Page 81 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 11.1.4 Add 1 mL of homogeneous solution to a 15 mL centrifuge tube. Re-suspend homogeneous solution by shaking between aliquots while preparing a total o f sixteen 1 mL aliquots o f homogeneous solution in 15 mL centrifuge tubes. 11.1.5 Two 1 mL aliquots serve as m atrix blanks. Use the stan dard concentrations and spiking amounts listed in table 1 to spike, in duplicate, two standard curves for a total o f fourteen samples. T able 1 A pproxim ate Spiking A m ounts fo r C alibration Standards W orking Standard (Approx. Cone.) - 0.50 ppm 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm 5.0 ppm 50 ppm liL Approx, finiil cone, o f PFOS in liver - Blank 4 0.010 ppm 20 0.050 ppm 40 0.100 ppm 10 0.250 ppm 20 0.500 ppm 30 0.750 ppm 4 1.000 ppm 11.1.1 See section 13.0 to calculate actual concentrations o f PFOS in calibration standards. 11.2 Extract spiked liver homogenates following 12.14-12.24 o f this method. Use these standards to establish each initial curve on the mass spectrometer. 12.0 P r o c e d u r e s ________________________________ ^________________________________ :____________________ 12.1 Obtain frozen liver samples. In spent tissue, note that the liver lias not been packaged w ith other tissues. 12.2 C ut approxim ately 1 g o f liver using a dissecting scalpel. 1 2 3 W eigh the sample directly into a tared plastic sampule vial. 12.4 Record the liver weight in the study notebook. 12.5 Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 12.6 Add 2.5 m Ls o f water to sampule vial. 12.7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the sample with 2.5 mLs water using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Follow AMDT-EP-22. 12.10 Cap the sample and vortex for 15 seconds. 3MEnvironmental Laboratory FACT-M-1.0 Extraction of PFOS from Liver Page 5 of 8 Page 82 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 12.11 Pipette 1 mL homogenate into a 15 mL polypropylene centrifuge tube. Label the centrifuge tube w ith the identical information as the sampule vial. (See W orksheet for documenting the remaining steps.) 12.12 Spike liver homogenates w ith the appropriate amount o f PFOS standard as described in section 11.1 or Table 1. 12.13 Pipette two 1 mL aliquots o f Milli-QTM w ater to centrifuge tubes. These w ill serve as instrum ent blanks. 12.14 Add 1 mL 0.5 M TBA and 2 mL o f the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.15 Using a volum etric pipette, add 5 mLs ethyl acetate. 12.16 Cap each sample and put on the shaker for 20 minutes. 12.17 Centrifuge for 20 to 25 minutes, until layers are well separated, Set power on the centrifuge to approxim ately 3500 rpm. 12.18 Remove 4 m Ls o f organic layer, using a 5 mL graduated glass pipette, to a clean 15 mL centrifuge tube. Label this fresh tube with the same information as in 12.5. 12.19 Put each sample on the analytical nitrogen evaporator until dry , approximately 2 to 3 hours. 12.20 Add 1.0 mL o f methanol to each centrifuge tube using a graduated pipette. 12.21 Vortex m ix for 30 seconds. 12.22 A ttach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial. 12.23 Label the autovial w ith the study number, animal number and gender, sample tim epoint, m atrix, final solvent, extraction date, and analyses) who performed the extraction. 12.24 Cap and hold for electrospray mass spectrometry analysis. 12.25 Complete the worksheet and tape to page o f study notebook. 13.0 D a t a A n a l y s i s a n d C a l c u l a t i o n s 13.1 Calculations: _______________ ___________________________________ 13.1.1 Calculate the density o f liver (mg) in 1.0 mL homogenate using the following equation: g o f Liver x Average weight o f ten 1 mL aliquots (mg) (g o f Liver + g of Water) 3MEnvironmental Laboratory FACT-M-1.0 Extraction of PFOS from Liver Page 6 of 8 Page 83 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 13.1.2 Calculate actual concentrations o f PFOS in calibration standards using the following equation: mL o f Standard x Concentration (ug /mL) = Final Concentration (ng/g or mg/kg) mg Liver /1 mL homogenate o f PFOS in Liver *Average weight o f liver in solution as determined in 13.1.1, by weighing ten 1 mL homogenates o f approximately 40 mg liver in 200 mL o f M illi-Q water. 14.0 M e t h o d P e r f o r m a n c e ______________________________________ ;_______________________________ 14.1 The m ethod detection lim it is equal,to half the lowest standard in the calibration curve. 15.0. P o l l u t io n Pr e v e n t io n and W aste M a n a g em en t______ _______________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R e c o r d s _____________________________________________________________ i_________________________ ,16.1 Complete the extraction worksheet and tape into the study notebook. 17.0 T a b l e s , D i a g r a m s , F l o w c h a r t s , a n d V a l i d a t i o n D a t a _______________^_______________ 17.1 The validation report associated with this method is FACT-M-1.0 & 2.0-V -l. 18.0 R e f e r e n c e s ___________________________ ;___________________________________ , 18.1 AM DT-EP-22, "Routine M aintenance o f Ultra-Turrax T-25" _______________ 19.0 A f f e c t e d D o c u m e n t s ____________ _________________________________________________ ' ' 19.1 FACT-M -2, "Analysis o f Liver Extracts for Fluorochemicals using HPLC-Electrospray M ass Spectrometry" 20.0 R e v is io n s R ev isio n N um ber. Reason For Revision R evision D ate 3MEnvironmental Laboratory FACT-M-1.0 Extraction of PFOS from Liver Page 7 of 8 Page 84 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Extraction W orksheet for FACT-M-1 Study # S am ple Number set# - H ,0 Blank - Liver Blank - , --------------------- PFOS approx. 0.5 ppm actual ppm #W - PFOS approx. 5 ppm actual ppm #W - PFOS approx. 50 ppm actual ppm #W . '- Date and Initials for Std. r -'- - .- .- .- .- - . ' S tu d v n u m b e r w h e r e th e o rig in a l w o r k s h e e t is lo c a te d .___________________________ B lank L iver Homogenate: Std # Liver a m o u n t- e L iver E xtraction M ethod : D ate & Initials V ortex 15 sec. Pinette 1 mL o f Liver Solution Pinette 1 mL oftO .S M T B A ,p H 1 0 . S td .# '- Pipette2 mL o f 0 .2 5 N aiC O t/0.25M N aH C O t Buffer Std.# Pipette 5 m L o f Ethyl A cetate TN-A- Shake 20 min. Centrifuge 20-25 m in. C en trifu g e Remove a 4 mL aliauot o f organic laver Put on N itrogen Evaporator to drvness Evaoorator A dd 1.0 m L o f M eth an o l Speed TN-A- T em perature . --------------------. Vortex 30 sec. ' -------------------- Filter using a 3cc B -D sv rin e e w ith a 0 .2um SRI filter into a 1.5 m L autosam ole vial M S / M S D / ____ C o n t. C h e c k s : S p i k e d _______ u L o f a _______ p p m std ( __________________) f o r a f in a l c o n c e n tra tio n o f .. ________ __ p p m . M S / M S D u s e d s a m p le ________ :_________ . C o n t C h e c k s us ed s a m e h o m o g e n a te as f o r std c u rv e . FACT-M-1.0 Extraction of PFOS from Liver Page 8 of 8 3MEnvironmental Laboratory Page 85 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Environmental L aboratory Method Extraction of Potassium perfluorooctanesulfonate or other Anionic Fluorochemical surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry M ethod N um ber: FACT-M-3.0 Author: Lisa Clemen A doption D ate: t Revision D ate: Technical Reviewer o S5. 'K) Ol m X u' yluhs D ate oO l . Q S c o p e a n d A p p l i c a t i o n ~3 1.1 Scope: This method is for the extraction o f potassium perfluorooctanesulfonate (PFOS) or ^ other fluorochemical surfactants from serum. D 0 LA ~s 1-2 A pplicable C om pounds: Fluorochemical surfactants or other fluorinated compotmds. 0 ^ fP 1.3 M atrices: Rabbit, rat, and bovine serum or other sera as designated in the validation report. ix > M icrosoft 7.0.1/95 3MEnvironmental Laboratory FACT-M-3.0 Extraction of PFOS from Serum Page 1 of 8 Page 86 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 2.0 S u m m a r y o f M e t h o d ________________________________ ____________________ __ 2.1 This method describes how to extract potassium perfluorooctanesulfonate (PFOS) or other anionic fluorochemical surfactants from serum using an ion pairing reagent and 5.0 mL o f ethyl acetate. An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into ethyl acetate. Four mL o f extract are removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL o f methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 3.0 D e f i n i t i o n s ____________________________________________ __ ___________________;____________________ 3.1 None. 4.0 W a r n i n g s a n d C a u t i o n s 4.1 Health and Safety Warnings: _________________;________________ ' 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal serum, it may contain pathogens. 5.0 I n t e r f e r e n c e s _________________ ______________________________________________________________ __ 5.1 There axe no known interferences at this time. 6.0 E q u i p m e n t ____________________________ ______________________ ________________________;______________ 6.1 The follow ing equipment is used while carrying out this method. Equivalent equipm ent is acceptable. 6.1.1 6.1.2 6.1.3 6.1.4 6.1.5 V ortex mixer, VWR, Vortex Genie 2 Centrifuge, M istral 1000 or EEC Shaker, Eberbach or VWR N itrogen evaporator, Organomation Balance, (0.100 gm) . . 7.0 S u p p l i e s a n d M a t e r i a l s ____________________________________________ 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 N algene bottles, capable o f holding 250 mL and 1 L 7.4 Glass, type A, volumetric flasks 7.5 40 mL glass I-CHEM vials 7.6 Polypropylene centrifuge tubes, 15 mL 7.7 Labels 7.8 Syringes, capable o f measuring 10 pL to 50 pL 7.9 Glass, type A , volumetric pipettes 7.10 G raduated pipettes FACT-M-3.0 Extraction of PFOS from Serum Page 2 of 8 3MEnvironmental Laboratory Page 87 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 7.11 Electronic pipettor, Eppendorf or equivalent 7.12 Tim er . 7.13 Disposable plastic 3 cc syringes 7.14 Filters, nylon syringe filters, 0.2 pm, 25 mm 7.15 Crimp cap autovials N ote: Prior to using glassware and bottles, rinse 3 times with methanol and 3 tim es w ith M illi esTM 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 R eagents _________ ;________________________________________________ 8.1.1 Sodium hydroxide (J.T Baker or equivalent), (NaOH) 1ON: weigh approximately 200 grams NaOH. Pour into a 1000 mL beaker containing 500 liters (L) Milli-QTM w ater, m ix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.1.2 Sodium hydroxide (J.T Baker or equivalent), (NaOH) IN . D ilute 10N 1:10. M easure 10 mL o f 1ON NaOH solution into a 100 mL volum etric flask and dilute to volum e using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8 .1 3 Tetrabutylammonium hydrogen sulfate (Kodak or equivalent), (TBA) 0.5M : Weigj approxim ately 169 grams o f TBA into a 1 L volumetric containing 500 L M illi-Q^ ' w ater. Adjust to pH 10 using approximately 64 mL o f 1ON NaOH and dilute to volum e w ith Milli-QTM water. Add NaOH slowly while adding the last mL o f NaOH because the pH changes abruptly. Store in a 1 L Nalgene bottle. 8.1.3.1 TBA requires a check prior to each use to ensure pH = 10. A djust as needed using IN NaOH solution. 8.1.4 Sodium carbonate/sodium bicarbonate buffer (J.T. Baker or equivalent), (Na2C03/N aH C03) 0.25M: Weigh approximately 26.5 g o f sodium carbonate (N ajC O ^ and 21.0 g o f sodium bicarbonate (N aH C03) into a 1 L volum etric flask an d b rin g to v o lu m e w ith M illi-Q TM w ater. S to re in a 1 L n a lg en e b o td e. 8.1.5 PFOS (3M Specialty Chemical Division), molecular weight = 538. 8.1.6 O ther fluorochemicals, as appropriate. 8.1.7 Ethyl Acetate, Omnisolv, glass distilled or HPLC grade. 8.1.8 M ethanol, Omnisolv, glass distilled or HPLC grade. 8.1.9 Serum, frozen liquid from Sigma. 8.1.10 Control serum received with each sample set. 8.1.11 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC Plus system. 3MEnvironmental Laboratory FACT-M-3.0 Extraction of PFOS from Serum Page 3 of 8 Page 88 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 8.2 S tandards 8.2.1 Prepare PFOS standards for the standard curve. 8.2.2 Prepare other fluorochemical standards, as appropriate. 8.2.3 W eigh approximately 100 mg o f PFOS into a 100 mL volum etric flask and record the actual weight. . 8.2.4 Bring to volume with methanol for a stock standard o f approximately 1000 ppm (jig/m L ). 8.2.5 D ilute the stock solution w ith methanol for a working standard 1 solution o f approximately 50 ppm. 8.2.6 D ilute the stock solution w ith methanol for a working sitandard 2 solution o f approx. 5.0 ppm. 8.2.7 D ilute the stock solution w ith methanol for a working sitandard 3 solution o f approx. 0.50 ppm. 9.0 S a m p l e H a n d l i n g ___________________ ;_________ ____________________________________ ' 9.1 A ll sera are received frozen and m ust be kept frozen until the detraction is perform ed. 4 0 .0 Q u a l i t y C o n t r o l ___________________;________________________________________ ;__________ 10.1 M atrix B lanks an d M ethod B lanks 10.1.1 Two 1.0 mL aliquots o f the serum are extracted following this procedure and used as m atrix blanks. See section 11.1.2. 10.1.2 Two 1.0 mL aliquots o f Milli-QTM water are extracted fallowing this procedure and used as method blanks. 10.2 M atrix Spikes 10.2.1 Prepare and analyze m atrix spike and matrix spike duplicate samples to determ ine the accuracy o f the extraction. 10.2.2 Prepare each spike using serum chosen by the analyst, usually control serum received with each sample set. . 10.2.3 Expected concentrations will fall in the mid-range o f the initial calibration curve. Additional spikes may be included and may fall in the low-range o f the initial calibration curve. 103 C ontinuing C alibration Checks 10.3.1 Prepare and analyze continuing calibration check samples to determine the continued linearity of the initial calibration curve. 10.3.2 One check is prepared per group o f ten samples. For example, if a sample set = 34, four checks are prepared and extracted. 3MEnvironmental Laboratory FACT-M-3.0 Extraction of PFOS from Serum Page 4 of 8 Page 89 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 103.3 Prepare each continuing calibration check from the sam e serum used to prep the initial curve. 10.3.4 The expected concentration will fall within the mid-range o f the initial calibration curve. l l . n C a l i b r a t i o n a n d S t a n d a r d i z a t i o n _________ ;_____________________________ ___ ____________ _ 11.1 P rep are Serum S tandards 11.1.1 Transfer 1 mL o f serum to a 15 mL centrifuge tube. 11 .1 3 I f the m ajority o f serum sample volumes are less than 1.0 mL, extract standards using serum volumes in the standards equal to the serum volum es in samples. Do not extract below 0.50 mL o f serum. Record the serum volume on the extraction sheet. 11.1.3 M ix or shake between aliquots while preparing a total o f sixteen aliquots o f serum in 15 mL centrifuge tubes. 11.1.4 Two 1 mL or appropriate aliquots serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in table 1 to spike, in duplicate, two standard curves for a total o f fourteen samples. 11.1.5 R efer to the validation report FACT-M-3.0-V-1 and FACT-M-4.0-V-1 which lists . the working ranges for calibration curves. Table 1 A pproxim ate Spiking A m ounts fo r S tandards and Spikes Using 1.0 m L of Serum W orking Standard pL (Approx. Cone.) - .- 0.500 ppm 20 5.00 ppm 5 5.00 ppm 10 5.00 ppm 20 50.0 ppm 5 50.0 ppm 10 50.0 ppm 15 Approx, final cone, o f PFOS in serum B lan k 0.010 ppm 0.025 ppm 0.050 ppm 0.100 ppm 0.250 ppm 0.500 ppm 0.750 ppm 11.1.4 See section 13.0 to calculate actual concentrations o f PFOS in calibration standards. 11.2 Extract spiked serum standards following 12.6-12.16 o f this method. Use these standards to establish each initial curve on the mass spectrometer. 3MEnvironmental Laboratory FACT-M-3.0 Extraction of PFOS from Serum Page 5 of 8 Page 90 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 12.0 P r o c e d u r e s _____________________________________________________________________________ _ 12.1 Obtain frozen serum samples and allow to thaw. 12.2 Vortex mix for 15 seconds then remove 1.0 mL or appropriate volume to a 15 mL polypropylene centrifuge tube. 12.3 Return serum samples to freezer after extraction amount has been removed. 12.4 Record the serum volume on the extraction worksheet. The final m ethanol volum e w ill equal the initial serum volume. 12.5 Label the tube w ith the study number, serum ID, date and analyst initials. See attached worksheet for documenting the remaining steps. . 12.6 Spike serum w ith the appropriate amount o f PFOS standard as described in section 11.1 or Table I for the calibration curve standards. Also spike m atrix spikes and continuing calibration standards. . 12.7 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.8 Add 1 mL 0.5 M TBA and 2 mL o f the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.9 Using a volum etric pipette, add 5 mL ethyl acetate. 12.10 Cap each sample and put on the shaker for 20 minutes. 12.11 Centrifuge for 20 to 25 minutes, until layers are well separated. Set power on the centrifuge to approximately 3500 rpm. 12.12 Transfer 4 mL o f organic layer, using a 5 mL graduated glass pipette, to a clean 15 mL centrifuge tube: Label this fresh tube with the same information as in 12.5. 12.13 Put each sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 hours. 1 2 .1 4 A d d 1 .0 m L o r ap p rop riate v o lu m e o f m eth a n o l to ea c h c e n tr ifa g e tu b e u sin g a gra d u a ted pipette. (This volume equals the initial volume o f serum used for the extraction.) 12.15 Vortex mix for 30 seconds. 12.16 A ttach a 0.2 (im nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autoviaL 12.17 Label the autovial with the study number, animal number and gender, sample tim epoint, m atrix, final solvent, extraction date, and analyst(s) who performed the extraction. 12.18 Cap and hold for HPLC-electrospray/mass spectrometry analy sis. Extracts may be stored at 4 C until analysis. 12.19 Complete the extraction worksheet, attached to this document, and tape to page o f study notebook. 3MEnvironmental Laboratory FACT-M-3.0 Extraction of PFOS from Serum Page 6 of 8 Page 91 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 13.0 Data Analysis and Calculations_____________________________________ 13.1 Calculations: 13.1.1 Calculate actual concentrations of PFOS, or other appropriate fluorochemical, in calibration standards using the following equation: mL o f Standard x Concentration (ue /iriD = Final Concentration (pg/mL) mL o f Standard + Initial Serum Volume (mL) o f PFOS in Serum 14.0 M e t h o d P e r f o r m a n c e _______________________________ ______________________________________ 14.1 The method detection limit is equal to half the lowest standard in the calibration curve. 15.0 P o l l u t i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t ________;______________________ ;________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed iin broken glass containers located in the laboratory. 16.0 R e c o r d s _________________________ ;________________________________ - _______ 16.1 Complete the extraction worksheet attached to this method, and tape into the study notebook. 17.0 T a b l e s . D i a g r a m s . F l o w c h a r t s , a n d V a l i d a t i o n D a t a _______________________ 17.1 The validation report associated with this method is FACT-M-3.0 & 4.0-V -l. 18.0 R e f e r e n c e s _______________________________ '___________ ;________ ;_______________________________ 18.1 None . 19.0 A f f e c t e d D o c u m e n t s __________________ __________________ ._________ 19.1 FACT-M-4, "Analysis o f Serum Extracts for Fluorochemicals using HPLG-Electrospray Mass Spectrometry" 20.0 Revisions Revision Number. Reason For Revision Revision Date 3MEnvironmental Laboratory FACT-M-3.0 Extraction of PFOS from Serum Page 7 of 8 Page 92 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Extraction. Worksheet for FACT-M-3 S tu d y # i S a m p le Num ber #s e t H ,0 B lan k S erum B lan k PFO S a p p ro x . 0 .5 p p m ac tu a l #W - ppm PFO S approx. 5 p p m a c tu a l ppm #W - PFO S ap p ro x. 50 p p m a c tu a l ppm #W - ' D a te and In itia ls fo r S td . o r C o m m e n ts - -. .- - -- -- - - - - -- - - -- -- -- .- - -- -- - -. -. - - .- . -- - - - .- - - - . --- 1S tu d y n u m b e r w h e re th e o rig in a l w o t ksheet is located. B la n k Serum Std # - Serum amount - Serum E xtraction M eth o d ' : e D ate & In itials V o rte x 15 sec. PiDette Serum Pipette 1 m L o f 0.5 M T B A , p H 10. Volum e Std. # mL Pipette 2 m L o f 0.2 5 N a ? C O V 0 .2 5 M NaHCO- buffer Std. # Pipette 5 m L o f ethyl acetate TN -A - Shake 20 m in. Centrifuge 20 -25 m in. C entrifuge speed: Rem ove a 4 m L aliau o t o f organic layer Put on N itro g en E vaporator to drvness Evaporator#: A dd methanol Volum e mL Temperature: TN -A - .... . ... V o rtex 3 0 sec. F ilter using a 3cc B -D svringe w ith a 0.2um S R I filter into a 1.5 m L autosamole vial M S / M S D / ____ C o m . C h e c k s : S p ik e d _____ u L o f a _______ p p m std ( __________________ ) f o r a f in a l c o n c e n tr a tio n o f ___________ p p m . M S / M S D us ed s a m p le ___________________ . C o n t. C h e c k s us ed s a m e s e ru m as f o r std c u rv e . FACT-M-3.0 Extraction of PFOS from Serum Page 8 of 8 3MEnvironmental Laboratory Page 93 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Environmental L aboratory Method Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical compounds from Serum for Analysis Using HPLC- Electrospray/Mass Spectrometry M ethod N um ber: ETS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg . Revision Date: Approved By: Q -'f Laboratory Manager ---------- ------------------- y/s? Date , L ------Group Leader Ht u 3 tt Date A ________________________ Technical Reviewer m /ilIw Date Exact Copy of Original Initial 1.0 S c o p e a n d A p p l ic a t io n 1.1 Scope: This method is for the extraction o f potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from serum. 1.2 A pplicable 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. n,3io W o rd 6/9 5 3MEnvironmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 1 o f 14 Page 94 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 2.0 S u m m a r y o f M e t h o d _____________________________________________________ _____________________ 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum, or other fluids, using an ion pairing reagent and methyl-terf-butyl ether (MtBE). In this method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556, and surrogate standard (see 3.0 Definitions). 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 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-8-5.1 or other appropriate methods. 3.0 D e f i n i t i o n s _________________________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) C,F ,7S 0 3' 3.2 PFOSA: perfluorooctane sulfonylamide CgF I7S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8F17S 0 2N(CH2CH3)CH2C 0 2' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol ' CgF l7S 0 2N(CH2CH3)CH2CH20 H . 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide CgF,7S 0 2N(CH2CH3)H 3.6 M556: CgF I7S 0 2N(H)(CH2C 0 0 H ) . ' 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 W a r n i n g s a n d C a u t i o n s ______________________________________ ____________________________ 4.1 Health and safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 I n t e r f e r e n c e s _________________________________________________________________________ !________ 5.1 There are no interferences known at this time. 6.0 E q u i p m e n t __________________________________________________________________________________ _____ 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR ETS-8-4.1 Extraction of PFOS from Serum Page 2 of 14 3MEnvironmental Laboratory Page 95 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) 7.0 Supplies and Materials______________________________ _____________________ 7.1 Gloves 7.2 Eppendorfor disposable pipettes 7.3 Nalgene bottles, capable o f holding 250 mL and 1 L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0 mL 7.9 Syringes, capable of measuring 5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe filters, nylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers . Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli-QTM water. Rinse syringes a minimum 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 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC HusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (N a^O j), 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 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 ETS-8-4.1 Extraction of PFOS from Serum Page 3 of 14 3MEnvironmental Laboratory Page 96 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 8 .9 3 PFOS AA (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), molecuhir 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 CgF,jS03H) molecular weight = 428 8.9.8 Other fluorochemicals, as appropriate 8.10 Reagent preparation N O TE: W hen preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.10.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a . 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.10.2 1 N sodium hydroxide (NaOH): Dilute 10N NaOH 1:10. Measure 10 mL o f 10 N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.10.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g o f TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH , 10 using approximately 44 to 54 mL o f 10 N NaOH (While adding the last mL o f ' NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-QTM water. Store in a 1 L Nalgene bottle. 8.103.1 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 (Na^Oj/NaHCOj): Weigh approximately 26.5 g o f sodium carbonate (NajCOj) aad 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with MilliQTM water. Store in a 1 L Nalgene bottle. 8.11 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.11.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.11.4 Bring to volume with methanol for a stock standard of*approximately 1000 ppm (ug/mL). 8.11.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.11.6 Dilute working standard 1 with methanol for a working standard 2 solution of approx. 5.0 ppm. ETS-8-4.1 Extraction of PFOS from Serum Page 4 of 14 3MEnvironmental Laboratory Page 97 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 8.11.7 Dilute working standard 1 with methanol for a working standard 3 solution o f approx. 0.50 ppm. 8.12 Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 mg o f surrogate standard 1-H,1-H, 2-H, 2-H, CgF,3S 0 3H into a 50 mL volumetric flask and record the actual weight. 8.12.2 Bring to volume with methanol for a surrogate stock o f approximately 1000-1200 ppm. 8.12.3 Prepare a surrogate working standard. Transfer approximately 1 mL o f surrogate stock to a 10 mL volumetric flask and bring to volume with methanol for a working standard o f 100 ppm. Record the actual volume transferred. 9.0 Sample Handling _________:________ ;__________ ;__________ '_________ ;__________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw to room temperature prior to extraction. 10.0 Q u a l i t y C o n t r o l _____________________ ;_________________________________ 10.1 Solvent Blanks, Method blanks and matrix 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 ofMilli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots o f the serum following this procedure and use as matrix blanks. See 11.1.4. ` 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by th analyst, usually the control matrix received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range o f the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batcli. 10.3 Continuing calibration checks 10.3.1 Prepare continuing calibration check samples to ensure the accuracy o f the initial calibration curve. . 10.3.2 Prepare, at a minimum, one continuing check per group o f 10 samples. For example, if a sample set = 34, four checks are prepared and extracted. 10.3.3 Prepare each continuing calibration check from the san e matrix used to prepare the initial curve. 3MEnvironmental Laboratory ETS-8-4.1 Extraction of PFOS from Scrum Page 5 of 14 Page 98 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 10.3.4 The expected concentrations will fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range o f the initial calibration curve. This is necessary if the analyst must quantitate using only the low end o f the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 p p b - 1000 ppb). 11.0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n _______________________ ;____________ ;________ .___________ 11.1 Prepare matrix calibration standards 11.1.1 Transfer 1 mL o f serum to a 15 mL centrifuge tube. 11.122 I f 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 o f . matrix. Record each sample volume on the extraction sheet 11.1.3 W hile preparing a total o f twenty aliquots in 15 mL centrifuge tubes, mix or shake between aliquots. 11.1.4 Two 1 mL aliquots, or other appropriate volume, serves as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1, at the end o f this section, to spike, in duplicate, two sfctndard curves, for a total of eighteen standards, two matrix blanks, and two method blanks. 11.1.5 Refer to validation report ETS-8-4.0 & ETS-8-5.0-V-1, which lists the working , ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.6 Use Attachment D as an aid in calculating the concentrations o f the working standards. See Section 13.0 to calculate actual concentrations o f PFOS in calibration standards. 11.2 To each standard, blank, or continuing check, add appropriate amount o f surrogate working standard for the concentration to fall within the calibration curve range 5 ppb 1000 ppb. 113 Extract spiked matrix standards following 12.6-12.16 o f this method. Use these standards to establish each initial curve on the mass spectrometer. 3MEnvironmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 6 of 14 Page 99 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Table 1 Approximate spiking amounts for standards and spikes Using 1.0 mL of m atrix Working standard pL Approx, final cone, o f (approx, cone.) analyte in matrix - - Blank 0.500 ppm 10 0.005 ppm 0.500 ppm 20 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 . 0.050 ppm 5.00 ppm . 20 0.100 ppm 50.0 ppm 5 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 1.00 ppm 12.0 P r o c e d u r e ______________________________________ ______________________________ ________________ 12.1 Obtain frozen samples and allow to thaw at room temperature or in a lukewarm waterbath. 12.2 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to a 15 mL polypropylene centrifuge tube. . 12.3 Return unused samples to freezer after extraction, amounts have been removed. 12.4 Record the initial volume on the extraction worksheet. . 12.5 Label the tube with the study number, sample ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 1 2 .6 . Spike all sam ples, including blanks and standards, ready for extraction with surrogate standard as described in 11.2. 12.7 Spike each matrix with the appropriate amount of standard as described in 11.1, or Table 1 in that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. I f not, adjust accordingly. 12.10 To each sample, add 1 mL 0.5 M TBA and 2 mL of 0.25M so'iium carbonate/sodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 mL methyl-ferf-butyl ether, 12.12 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. 12.13 Centrifuge for 20 to 25 minutes at a setting o f 3500 rpm, or until layers are well separated. ETS-8-4.1 Extraction of PFOS from Serum Page 7 o f 14 3MEnvironmental Laboratory Page 100 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 12.14 Label a fresh 15 mL centrifuge tube with the same information as in 12.5. 12.15 Remove 4.0 mL of the organic layer to this clean 15 mL centrifuge tube. 12.16 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. . 12.17 Add 1.0 mL o f methanol to each centrifuge tube using a graduated pipette. 12.18 Vortex mix for 30 seconds. 12.19 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this . syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.20 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.21 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.22 Complete the extraction wprksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. 1 3 . 0 D a t a A n a l y s i s a n d C a l c u l a t i o 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 standard x concentration of standard (ue /m L1!___________ ;_______ = . mL o f standard + mL o f surrogate standard + initial matrix volume (mL) Final Concentration (pg/mL) o f PFOS in matrix 1 4 .0 M e t h o d P e r f o r m a n c e _________________________ ;__________________________ :____________________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit o f quantitation (LOQ) values (see Attachm ents B and C). 14.2 The following quality control samples are extracted with each batch o f samples to evaluate the quality o f the extraction and analysis. 14.2.1 Method blanks and matrix blanks. . 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision o f the extraction. 14.2.3 Continuing calibration check samples to determine the continued accuracy o f the initial calibration curve. 14.3 Refer to section 14 of ETS-8-5.1 for method performance criteria. 1 5 . 0 P o l l u t i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t ________________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. ETS-8-4.1 Extraction of PFOS from Scram Page 8 of 14 3MEnvironmental Laboratory Page 101 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 . LRN-U2104 16.0 Records_____________ ;__________________________ ____________ ___________ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 Attachm ents_____________________________ __________ _______________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 18.0 References_____________________ _________ :___________________________ 18.1 The validation report associated with this method is ETS-8-4.0 & 5.0-V -l. 18.2 FACT-M-3.1, "Analysis o f Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 19.0 Affected Documents________________________________________________________ 19.1 ETS-8-5.1, "Analysis o f Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 Revisions ________________ .___________________ ________________________ __ Revision Number 1 Reason For Revision Section 12.21 Changed to include sample storage at room temperature. Section 12.13 Added the shaker speed. Section 12.17 Final volume is 1.0 mL; not adjusted for initial volumes less than 1.0 mL. Revision Date 04/02/99 3MEnvironmental Laboratory . ETS-8-4.1 Extraction of PFOS from Serum Page 9 o f 14 Page 102 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Extraction Worksheet ETS-8-4.1 Study # M a trix Box# W k /D a v D ateS p iked/A nalyst CCV MS M SD Surrogate Std approx, ppm actual ppm # F C -M ix approx. 0.5 pm actual ppm # FC -M ix: approx. 5 ppm actual pp m # F C -M ix approx. 50 ppm actual ppm # C om m ents - .- - - -' - - - - - - .- - --- - - *r --- --- - - - - '- - Blank_________ '__________________ S td#__________________________ am ount- ___________mL S e ru m E x tra c tio n M e th o d ______________________________:____________________________________ _________________P a t e & In itia ls V ortex 15 sec._______________________________ |___________________' '________________________________________________ P ipette M atrix______________________ r _ ;__________ V olum e__________________ m L________________ .________________________ Pipette 1 m L o f 0.5 M TB A , pH 10. pH - S td .# Pipette 2 m L o f 0.25 N a2COVQ.2SM NaHCO^ buffer S td .# ~ D ispense 5 m L o f m cthyl-t-butyl ether_________________________________ T N -A - _________________________________ _ Shake 20 min. Shaker speed: ~ C entrifuge 20-25 m in.________ ;_____________________________ C entrifuge speed:____________________________________ R em ove a 4 m L aliq u o t o f organic l a y e r ________________________________________________________________________ ;_________ P u t on N itrogen E vaporator to dryness__________________________ T em perature:___________________________________ _____________ A dd m ethanol_______________ V olum e m L ______________ T N -A - _____________________ __ _________ V ortex 3 0 sec.____________________________________________ ______________________;____________________________________________ F ilter using a 3 cc B -D syringe w ith a 0 .2 u m filter into a 1.5 m L autosam ple vial_______________________________________ _________ C ont. C al. V erifications u sed sam e m atrix as for std curve. A ttachm ent A 3MEnvironmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 10 of 14 Page 103 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 MDL/LOQ values for rabbit serum Compound MDL LOQ Linear Calibration Range (LCR) (PP*>) (PPb) A pproxim ate concentrations to be used for p rep arin g the Standard Calibration Curve PFOS 1.74 5.55 5 ppb -1000 ppb PFOSA 1.51 4.79 5 ppb - 1000 ppb PFOSAA 3.46 20.5 5 ppb -1000 ppb EtFOSE-OH 11.4 36.2 5 ppb -1000 ppb M556 6.03 19.2 5 ppb -1000 ppb PFOSEA 5.71 18.2 5 p p b -1000 ppb MDL/LOQ values in rat, bovine, monkey, and human serum, and monkey plasma were not statistically determined. Two curves in each of these matrices were extracted and anal^rzed with the rabbit serum curves to determine equivalence. Responses in the rat, bovine, monkey, and human were equivalent to the rabbit responses, therefore, their MDL and LOQ will be the same values as determined in rabbit serum. Please see LOQ Summary and MDL study in ETS-8-4.0 & 5.0-V-l for further information. /' Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction of PFOS from Serum 3MEnvironmental Laboratory Page 11 of 14 Page 104 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Com pound: F FOS Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1IX 0.995-978 4.94 - 248 97.8-978 0.995 - 978 C om pound; PFOSA Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X . 0.993 - 976 4.93 - 97.6 24.8 - 976 0.993 - 976 C om pound: PFOSAA Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.991 - 974 4.92 - 247 49.2 - 974 0.991 - 974 LCR from curve (ppb) (n g/m L ) 24.8 - 978 4.94 - 248 9 7 .8 -9 7 8 4.94 - 978 % Recovery Range 83-108 85-104 85-106 94-111 LCR from curve (ppb) (n g/m L ) 4.93 - 976 % Recovery Range 88-103 4.93 - 97.6 . 87-105 24.8-978 93-102 4.93-976 94-103 LCR from curve (PPb) (ng/mL) 24.7 - 974 9.74 - 247 97.4 - 974 9.74-974 % Recovery Range 81-111 97-107 85-108 95-115 RSD Range 4.67-11.0 5.34-12.0 4.84-9.80 4.60-10.5 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 RSD Range 4.18-10.6 6.38-21.8 4.33-12.5 4.11-23.2 Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction of PFOS from Serum 3MEnvironmental Laboratory Page 12 o f 14 Page 105 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Compound: EtFOSE-OH Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range 0.993 - 976 LCR from curve (ppb) (ng/mL) . 49.3-976 Low Curve 4 .9 3 -9 7 .6 9 .7 6 -9 7 .6 H igh curve 49.3 - 976 97.6 - 976 1/X 0.993 - 493 9.76 - 976 Compound: PFOSEA Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 248 49.3 - 976 0.993-976 LCR from curve (Ppb) (ng/mL) 24,8 - 976 9.76 - 248 49.3 - 976 9.76 - 976 Compound: M556 Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93-97.6 97.6-976 0.993 - 976 LCR from curve (ppb) (ng/mL) 24.8-976 9.76 - 97.6 97.6 - 976 9.76-976 % Recovery Range 77-110 97-107 90-109 86-111 % Recovery Range 96-106 91-110 86-106 95-117 % Recovery Range 88-106 100-105 81-111 97-110 RSD Riinge ll.::-25.5 14.1-21.3 11.5-19.6 11.1-21.2 RSD Range 10.1-16.2 11.8-19.5 10.2-18.2 10.1-19.1 RSD Range 4.82-17.9 5.95-18.2 5.11-9.74 4.77-19.5 Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction of PFOS from Serum 3MEnvironmental Laboratory Page 13 of 14 Page 106 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Ion Pair Standard Curves - Fluids Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Method/revision: Blank fluid/identliler: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: . FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Actual concentrations of standards in the FC mix P FO S PFO SA P F O S A A E tF O S E P F O S E A Std cone Std cone S td cone S td cone Std cone u g /m L u g /m L u g /m L u g /m L u g /m L 0.5 00 0.5 07 0.5 32 0 .5 0 1 0 .5 2 1 0.5 00 0.5 07 0.5 32 0 .5 0 1 0 .5 2 1 5.0 0 5 .0 7 5.3 2 5 .0 1 5 .2 1 5.0 0 5 .0 7 5.3 2 5 .0 1 5 .2 1 5.0 0 5 .0 7 5 .3 2 5 .0 1 5 .2 1 5 0 .0 5 0 .1 5 3 .2 5 0 .1 5 2 .1 50 .0 5 0 .1 53 .2 5 0 .1 5 2 .1 50 .0 5 0 .1 5 3 .2 5 0 .1 5 2 .1 50 .0 5 0 .1 5 3 .2 5 0 .1 5 2 .1 M 556 Std cone u g /m L 0 .5 0 1 0 .5 0 1 5 .0 1 5 .0 1 5 .0 1 5 0 .1 5 0 .1 5 0 .1 5 0 .1 AU Am t spiked m L 0.0 10 0.0 20 0 .0 0 5 0 .0 1 0 0 .0 2 0 0 .0 0 5 0 .0 1 0 0 .0 1 5 0.0 20 AU F inal vo l mL 1 .0 1 5 1 .0 2 5 1 .0 1 0 1 .0 1 5 1 .0 2 5 1 .0 1 0 1 .0 1 5 1 .0 2 0 1 .0 2 5 Calculated concentrations of standards in the sample matrix PFOS Final cone ng/m L PFOSA Final cone ng/m L PFOSAA Final cone ng/m L E tFO S E Final cone ng/m L PFOSEA Final cone ng/m L M 556 Final cone ng/m L Surrogate Std cone n g /iriL 4.93 9.76 24.8 49.3 97.6 248 493 735 976 5 .0 0 9.89 25.1 50.0 9 8 .9 251 500 746 989 5.24 10.4 26.3 52.4 . 104 263 524 782 1038 4.94 9.78 24.8 49.4 97.8 248 494 737 978 5.01 9.93 25.2 50.1 99.3 252 501 749 993 5.13 10.2 25.8 51.3 102 258 513 766 1017 10(1 S u rro g a te Final cone ng/m L 500 ' AU A m t spiked mL 0.005 Validated ranges - approximate concentrations Serum PFOS PFOSA PFOSAA EtFO SE-O H PFOSEA M 556 R ab b it 5.00-1000 | 5.00-1000 | 5.00-1000 | 5.00-1000 | 5.00-1000 | 5.00-1000 B o v in e Estimates only. U se values for rab b it . Rat Estim ates only. U se values for rabbit M onkey & Plasm a Estim ates only. Use values for rabbit Human Estim ates only. Use values for rabbit Attachment C: Ion Pair Standard Curves ETS-8-4.1 Extraction of PFOS from Serum 3MEnvironmental Laboratory Page 14 of 14 Page 107 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Environmental L aboratory Method E x tractio n of P otassium Perfluorooctanesulfonate or other Fluorochem ical Compounds from Liver for An ^ ysis using H PLC - Electrospray/M ass Spectrometry Method Number: ETS-8-6.0 Adoption Date: Author: Lisa Clemen, Robert Wynne 4' Approved By: Laboratory Manager u ib * ---------- Group Leader Revision Date: ~ V 2. V h <7 Date 'W l*t-f& 3 Date fi/t% A d W t u Technical Reviewer Cf/whl Date o a>y2 . r*i* P (A Xm CJ o 1.0 S c o p e a n d A p p l ic a t io n 1.1 Scope: This method is for the extraction o f potassium perfluorooctanesulfonate PFOS) or other fluorochemical compounds from liver. D P 6"*^ O--* 1.2 A pplicable C om pounds: Fluorochemical surfactants or other fluorinated compounds. CD (A o 1.3 M atrices: Rabbit, rat, bovine, and monkey livers or other tissues as designated in the validation report. 5' oo W o r d 6 .0 /9 5 ETS-8-6.0 Extraction of PFOS from Liver Page 1 of 14 3MEnvironmental Laboratory Page 108 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 2.0 Summary of Method______________________________________ ______________ ' 2.1 This method describes the procedure for extracting potassium pcrfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from liver, or other lissues, using an ion pairing reagent and methyl-teri-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 smalyte ion pair is partitioned into MtBE. The MtBE extract is transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL methanol then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-7.0 or other appropriate methods. 3.0 D efinitions________________;_______________________________________ ;___________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) C,F.7!>03 3.2 PFOSA: perfluorooctane sulfonylamide C8F 17S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate CsF,7S 0 2N(CH2CH3)CH2C02 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol ' CsF 17S 0 2N(CH2CH3)CH2CH20 H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide CaF,7S0 2N(CH2CH3)H 3.6 M556: CgF I7S 0 2N(H)(CH2C 0 0 H ) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 Warnings and Cautions__________________________________ :______________ . ' 4.1 H ealth an d 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 E q u i p m e n t _______________________________________________________________________ :------------------ 6.1 The following equipment is used while performing this method . Equivalent equipment is acceptable. 6.1.1 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 ETS-8-6.0 Extraction of PFOS from Liver Page 2 of 14 3MEnvironmental Laboratory Page 109 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivity to 0.100 g) 7 .0 S u p p l i e s a n d M a t e r i a l s _______________________________________________________________________ 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable o f holding 250 mL and 1 L 7.5 Volumetric flasks, glass, type A . 7.6 I-CHEM vials, 40 mL glass 7.7 Plastic sampule vials, Wheaton, 6 mL (or appropriate size) 7.8 Centrifuge tubes, polypropylene, 15 mL 7.9 Labels 7.10 Oxford D 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 Milli- QTM 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 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and be provided by a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (NajCOj), I.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8.6 Methyl-ieri-butyl ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Liver, frozen from supplier 8.9 Dry ice from supplier 8.10 Fluorochem ical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 ETS-8-6.0 Extraction of PFOS from Liver Page 3 of 14 3MEnvironmental Laboratory Page 110 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 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 CjF13S 03H) molecular weight = 428 8.10.8 Other fluorochemicals, as appropriate 8.11 R eagent preparation N O TE : W hen preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.11.1 1 0N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are . dissolved. Store in a 1 L Nalgene bottle. 8.11.2 1 N sodium hydroxide (NaOH): Dilute 10N NaOH 1:10. Measure 10 mL o f 10 N NaOH solution into a 100 mL volumetric flask arid dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.11.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g ' o f TBA into a 1 L volumetric containing 500 mL Milli -QTM water. Adjust to pH 10 using approximately 44 to 54 mL o f 10 N NaOH (While adding the last mL o f NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-QTM water. Store in a 1 L Nalgene bottle. * 8.11.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.11.4 0.25 M sodium carbonate/sodium bicarbonate buffer (NajCOj/NaHCOj): Weigh approximately 26.5 g o f sodium carbonate (NajCOj) and 21.0 g o f sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volum e w ith M illiQTM water. Store in a 1 L Nalgene bottle. 8.12 Standards preparation 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) . 8.12.3 W eigh 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 of approximately 1000 ppm (pg/mL). 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution o f approximately 50 ppm. 3MEnvironmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 4 of 14 Page 111 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 8.12.6 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.12.7 Dilute the stock solution with methanol for a working standard 3 solution o f approx. 0.50 ppm. 8.13 Surrogate stock standard preparation 8.13.1 Weigh approximately 50-60 mg o f surrogate standard l-H .l-H , 2-H, 2-H, C8F 13S 0 3H into a 50 ml volumetric flask and record the actual weight. 8.13.2 Bring to volume with methanol for a surrogate stock o f approximately 1000-1200 . ppm. 8.13.3 Prepare a surrogate working standard. Transfer appro:rimately 1.0 ml o f surrogate stock to a 10 ml volumetric flask and bring to volum e with methanol for a working standard o f 10-20 ppm. Record the actual volume transferred. 9 .0 S a m p l e H a n d l i n g __________________________ :____________________________________________________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 10.0 Q u a l i t y C o n t r o l _______________________________________________________ 10.1 Matrix blanks and method blanks ' 10.1.1 An aliquot o f 1.0 mL methanol is used as a solvent blank. 10.1.2 Extract two 1.0 mL aliquots o f Milli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots o f liver homogenate following this procedure and use as matrix blanks. Refer to 11.1.6. - 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy o f the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually a control liver received with each sample set 10.23 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 of 2 matrix spikes per batch. 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibration verification samples to ensure the accuracy o f the initial calibration curve. 103.2 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. 3MEnvironmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 5 of 14 Page 112 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 10.3.3 Prepare each continuing calibration verification from the same matrix used to prepare the initial curve. 103.4 The expected concentrations will fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end o f the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb -1 0 0 0 ppb). 1 1 .0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n __________________________________________________ 11.1 P rep are m atrix calibration standards 11.1.1 Weigh approximately 40 g o f liver into a 250 mL Nalgene bottle containing 200 mLs Milli-QTM water. Grind to a homogeneous solution. 11.1.2 I f 40 g is not available, use appropriate amounts o f liver and water to ensure a 1:5 ratio. 11.13 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 of homogenate to a 15 mL centrifuge tube. Re-suspend solution by shaking between aliquots while preparing a total o f eighteen 1 mL aliquots o f homogeneous solution in 15 mL centrifuge tubes. 11.1.6 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. 11.1.7 Typically use the standard concentrations and spiking amounts listed in Table 1, at the end o f this section, to spike, in duplicate, two standard curves, for a total o f eighteen samples, two matrix blanks, and two method blanks. 11.1.8 Refer to validation reports ETS-8-6.0 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. 1 1 .1 .9 U s e A tta c h m e n t C as an aid in calculating the concentr ations o f the w orking 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 ihe calibration curve range 5 ppb-lOOOppb. 3MEnvironmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 6 of 14 Page 113 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 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. Table 1 Approximate Spiking Amounts for Calibration Standards Working Standard (Approx. Cone.) - 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm . 5.0 ppm 50 ppm pi '2 4 10 20 40 10 20 30 4 Approx, final cone, o f PFOS in liver Blank 0.005 ppm 0.010 ppm 0.025 ppm 0.050 ppm 0.100 ppm 0.250 ppm 0.500 ppm 0.750 ppm 1.00 ppm 12.0 Procedure___________________________________________________ .______________ 12.1 Obtain frozen liver samples. 12.2 Cut approximately 1 g o f liver using a dissecting scalpel. This part o f the procedure is best performed quickly, not allowing the liver to thaw. 12.3 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. 12.9 Take the grinder apart and clean it with methanol after each sample. Refer to AMDT-EP22. 12.10 Cap the sample and vortex for 15 seconds. Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 3MEnvironmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 7 of 14 Page 114 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 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 infoimation 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 matrix 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. If not, adjust accordingly. 12.17 To each sample, add 1 triL 0.5 M TBA and 2 mL o f the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.18 Using an Oxford Dispenser, add 5 mL methyl-tert-butyl ether. 12.19 Cap each sample and put on the shaker at a setting o f 300 tpm, 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 analyst(s) performing the extraction. 12.28 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.29 Complete the extraction worksheet, attached to this document, and tape in study notebook or include in study binder, as appropriate. 3MEnvironmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 8 of 14 Page 115 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 13.0 D a t a A n a l y s i s a n d C a l c u l a t i o n s ____________________________ ____________ _____________ 13.1 Calculations: 13.1.1 Calculate the average density o f the liver homogenate by recording each mass o f ten separate 1.0 mL aliquots o f homogenate. Average density (mg/mL) = Average mass Cme) o f the aliquots 1.0 mL aliquot 13.1.2 Calculate the amount o f liver (mg) per 1.0 mL homogen ate (or concentration o f dispersed solid tissue per mL o f homogenate suspension.) using the following equation: g o f Liver x Average density* o f homogenate faig/mO (g o f Liver + g of Water) * refer to 13.1.1 for details. 13.1.3 Calculate actual concentrations o f PFOS and other fhiorochemicals in calibration standards using the following equation: ' ' uL o f Standard x Concentration <ug /mL") = Final Concentration (pg/g or mg/kg) mg L iv er/1 mL homogenate* of PFOS in Liver refer to 13.1.2 for details. . 14.0 M e t h o d P e r f o r m a n c e ________________________________________________________________________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific M DL and limit o f quantitation (LOQ) values (refer to Attachm ents B and C). 14.2 The follow ing quality control samples are extracted with each batch o f samples to evaluate the quality o f the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and . precision o f the extraction. 14.2.3 Continuing calibration verification samples to determine the continued accuracy o f the initial calibration curve. 14.3 Refer to section 14 o f ETS-8-7.0 for method performance criteria. 1 5 .0 P o l l u t i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t ______________________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 3MEnvironmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 9 of 14 Page 116 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 1 6 .0 R e c o r d s _____________________________________________________________________ ;___________ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 1 7 . 0 T a b l e s . D i a g r a m s . F l o w c h a r t s , a n d V a l i d a t i o n D a t a _______________________________ 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 1 8 - 0 R E F E R E N C E S ____________________________________________________________________________________________________ 18.1 The validation report associated with this method is ETS-8-6.0 & 7.0-V-l. . 18.2 AMDT-EP-22, "Routine Maintenance o f Ultra-Turrax T-25" 18.3 FACT-M-1.1, "Extraction o f PFOS or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectromey" 1 9 .0 A f f e c t e d D o c u m e n t s ______________ ,__________________________________________________________ 19.1 ETS-8-7.0, "Analysis o f Liver Extracts for Fluorochemicals using HPLC-Electrospray ' Mass Spectrometry" 20.0 R e v is io n s Revision Number. Reason For Revision . Revision Date 3MEnvironmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 10 of 14 Page 117 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Studv # 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 # Comments - - .- *- ' -. - - - Liver Extraction M ethod Spike surrogate and S tandard m ix. V ortex 15 see." Pipette 1 m L o f Liver Solution Pipette 1 m L o f tO-5 M T B A , pH 10. pH ** _ Pipette 2 m L o f 0.25 N a2COVQ.25M N aH C O t Buffer Dispense 5ml o f M ethyl-t-Butyl E ther Shake 20 min. Centrifuge 20-25 min. Remove a 4 mL aliquot o f organic laycr_ Put on Nitrogen Evaporator to dryness Add 1.0 m L o f M ethanol Std. # S td .# TN-AShaker Speed Centrifuge Speed Evaporator Temperature TN-A- Vortcx 30 sec. Filter using a 3cc B -D syringe w ith a 0.2um SRI filter into autosam plevial ^ .a __ a * -- i . x L m*. 1 11 >1 ^i C ont. C a l. V e rific a tio n s used th e sam e m a trix as fo r the standard curve. Attachment B: MDL/LOQ Values 3MEnvironmental Laboratory . ETS-8-6.0 Extraction of PFOS from Liver - - - - '- - ... D ate & Initials Page 11 of 16 Page 118 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 MDL/LOQ values for rabbit liver Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 8.45 26.9 30 ppb - 1200 ppb PFOSA 3.50 11.1 12 ppb - 1200 ppb PFOSAA 24.6 78.3 30 ppb - 1200 ppb EtFOSE-OH 108 345 60 ppb - 900 ppb* M556 82.3 262 60 ppb -1200 ppb PFOSEA 33.9 108 30 ppb- 1200 ppb MDL/LOQ values in rat, bovine, and monkey liver were not statistically determined. Two curves in each 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________________________ _______ _____ L iver m atrix P re p a re d range o f standards (ppb) (ng/m L) R ange o f .\^IiQ R Sif& average ''.av elcn n ^ ^ J curve i-i - - `/ `i.)!W" (ppb) (ng/mL) : (ppb)" (ngfinL)'.. Range o f i lo w std .j, curve (ppb) (ng/m L) tpb)^ngfln) Range o f h ig h std curve (ppb) (ng/mL) .L C R fiO m r jrigfcstd j .f .Y t w r s f p 'X -<ppb) (ng?mL) ; R ab b it 6 .1 9 -1 2 3 7 12 - 1200 2 ,'- ^ f j f { ' 6 - 3 0 0 6 0 -1 2 0 0 i ^ r l2 0 0 ii Compound: PFOSA L iver m atrix P re p a re d range o f standards (ppb) (ng/mL) Range o f average curve (ppb) (ng/mL) L (3 t|rp m . ^ .a y p u w e 'i " ... . {ppb)';(n/ftiL); Range o f lo w std curve (ppb) (ng/mL) ! ; d b w ^ l i 'iiiV'i t. /(i#b > (ji /itt;) r Range o f h ig h std curve (ppb) (ng/mL) * L G ^ fifram ! n . h ig h stdr : :^ (ft3 b }.( n g f tM i) :: R ab b it 6 .1 9 -1 2 3 7 12 - 1200 1 2 4 1 2 0 0 ; ;, 1 2 - 3 0 0 : l'-JOO-' 6 0 - 1 2 0 0 . ' 6 0 - 1 2 0 0 ; . Compound: PFOSAA L iver m atrix P re p a re d range o f standards (ppb) (ng/mL) Range o f average curve (ppb) (ng/mL) R ab bit 6 .1 6 -1 2 3 2 12 - 1200 L C R from , ave curve (ppb) (ng/mL) 3 0 - 1200 Range o f lo w std curve (ppb) (ng/mL) 3 0 -9 0 0 L C R fro m - Range o f lo w stili : h ig h std curve (ppb) (ng/mL) curve (ppb) (ng/mL) 6 0 -9 C 0 N /A L C R from hig h std c u rv e (ppb) (ng/mL) ' N /A A tta ch m en t B : M D L /L O Q V alu es 3MEnvironmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 12 of 16 Page 119 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Compound: EtFOSE-OH L iver m atrix P re p a re d range o f standards (ppb) (ng/mL) Range o f average curve (ppb) (ng/mL) R ab bit 6 .1 7 -1 2 3 5 31 -9 0 0 L C R from ave curve (P Pb) (ng/mL) 3 1 -9 0 0 Range o f lo w std curve (ppb) (ng/mL) N /A L C R from lo w std curve (ppb) (ng/mL) N /A Range o f h ig h std curve (ppb) (ng/mL) N /A L C R from h ig h std curve (ppb) (ng/mL) N /A Compound: PFOSEA P re p a re d Range o f L iver m atrix range o f standards (ppb) (ng/mL) average curve (ppb) (ng/mL) L C R fro m ave curve- (ppbL (ng/mL) Range o f lo w std curve (ppb) (ng/mL) L C R from lo w stdi c in y (ppb). (ng/mL) Range o f h ig h std curve (ppb) (ng/mL) L C R from h ig h std c u rv e ." (ppb) (ng/mL) R ab bit 6 .1 7 -1 2 3 5 31 -1 2 0 0 3 1 - 1200- N /A N /A . ' N /A . ' N /A ' Compound: M556 P re p a re d L iver m atrix range o f standards (ppb) (ng/mL) R ab b it 6 .1 7 -1 2 3 5 Range o f average curve (ppb) (ng/mL) 3 1 - 1200 LC F& from ^ Range o f aye cu pe::';' lo w std curve (ppb)r(ng/ml)v. (ppb) (ng/mL) N /A L G R ^ o q f-: Range o f lOWStcti . h ig h std v V c u v e :^ s curve (ppb) ifng/in): (ppb) (ng/mL) L C R from h ig h s td T c u r v e 1;(ppb) (ng/iijL)" N /A . - 'r Attachment C: Standard Calculations ETS-8-6.0 Extraction of PFOS from Liver 3MEnvironmental Laboratory Page 13 of 14 Page 120 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 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 PFO S Std cone PFO SA Std cone PFO SA A S td cone E tF O S E Std cone PFO SEA S td cone u g /m L u g /m L u g /m L ug /m L u g /m L 0.5 00 0.5 00 0 .5 0 0 0 .5 0 0 0.5 00 0.5 00 0.5 00 0 .5 0 0 0 .5 0 0 0.5 00 0.5 00 0 .5 0 0 0 .5 0 0 0 .5 0 0 0.5 00 0.5 00 0 .5 0 0 0.5 00 0 .5 0 0 0.5 00 0.5 00 0 .5 0 0 0 .5 0 0 0 .5 0 0 0 .5 0 0 5 .0 0 5.0 0 5 .0 0 5 .0 0 5 .0 0 5 .0 0 5 .0 0 5 .0 0 5 .0 0 5 .0 0 5 .0 0 5.0 0 5 .0 0 5 .0 0 5 .0 0 5 0 .0 50 .0 5 0 .0 5 0 .0 5 0 .0 M 556 Std cone u g /m L 0 .5 0 0 0 .5 0 0 0.5 00 0 .5 0 0 0 .5 0 0 5 .0 0 5 .0 0 5 .0 0 5 0 .0 Std cone u g /m L A ll A in 't spiked mL 0.0 02 0.0 04 0.0 10 0.0 20 0.0 40 . 0.0 10 0.0 20 0.0 30 0.0 04 AU D en sity g 0.1 67 0.1 67 0.1 67 0 .1 6 7 0.1 67 0.1 67 0.1 67 0.1 67 0.1 67 Calculated concentrations of standards in the sample matrix PFO S F inal cone n g /g 5 .9 9 1 2 .0 2 9 .9 5 9 .9 120 299 599 898 1198 PFO SA F in al cone n g /g 5 .9 9 1 2 .0 2 9 .9 5 9 .9 120 299 599 898 1198 PFO SA A F in a l cone n g /g 5 .9 9 1 2 .0 2 9 .9 5 9 .9 120 299 599 898 1198 E tF O S E F in a l cone n g /g 5 .9 9 1 2 .0 2 9 .9 5 9 .9 120 299 599 898 1198 PFO SEA F in a l cone n g /g 5 .9 9 1 2 .0 2 9 .9 5 9 .9 120 299 599 898 1198 M 556 F in a l cone n g /g 5 .9 9 1 2 .0 2 9 .9 5 9 .9 120 299 599 898 1198 Std cone ng /g S urro gate S td cone n g /m L 100 Surrogate Final cone ng/m L 0.500 A ll A m 't s p ik e d mL 0.005 Validated ranges --approximate concentrations L iv e r PFOS PFOSA PFOSAA Rabbit 5-1000 ppb 5-1000 ppb 5-1000 ppb Bovine ' Estim ates only, use rabbit values. Rat Estimates only, use rabbit values. M onkey Estimates only, use rabbit values. EtFO SE-O H 5-1000 ppb POAA 5-1000 ppb PFOSEA 5-1000 ppb Attachment C: Standard Calculations ETS-8-6.0 Extraction of PFOS from Liver 3MEnvironmental Laboratory Page 14 of 14 Page 121 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Environmental L aboratory Method Analysis of Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry M ethod Num ber: FACT-M-2.0 Adoption Date: 5 / J l / ^ c? Author: Lisa Clemen Revision Date: A)A Approved By: ' I H >V -- Laboratory Manager f/p j^ Group Leader ------------- d*. A (W Technical Reviewer _________ r/ic fiy Date 5 7 z- Date s'h ih n Date ft Initial 1.0 Scope and Application ') 1.1 Scope: This method is for the analysis of extracts o f liver or other tissues for fluorochemical . surfactants using HPLC-electrospray/mass spectrometry. " 1.2 A pplicable Com pounds: Potassium perfluorooctanesulfonate, anionic fluorochemical , surfactants, or other ionizable compounds. ) 1.3 M atrices: Rabbit, rat, bovine, and monkey livers or other livers z.s designated in the validation report. Ul f j d) Date -- Word 7.0.1/95 FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 1 of 8 3MEnvironmental Laboratory Page 122 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 2.0 Summary of Method____________________________________________________ "' 2.1 This method describes the analysis o f fluorochemical surfactants extracted from liver using HPLC-electrospray/mass spectrometry. The analysis is performed by monitoring a single ion characteristic o f a particular fluorochemical, such as the pofcissium perfluorooctanesulfonate (PFOS) anion, M/Z= 499. Samples may also be screened to verify compound identification. 3.0 Definitions___________________________________________________________________ 3.1 None. 4.0 W arnings and Cautions_______________________________________________________ 4.1 H ealth an d Safety W arnings: 4.1.1 Use caution with the voltage cable for the probe. When the voltage cable is plugged into the probe DO NOT TOUCH THE PROBE, there is risk of electrical shock. 4.2 Cautions: 4.2.1 Do not run solvent pumps above capacity o f 400 bar (5800 psi). If pressure goes over 400 bar, the HP 1100 will initiate automatic shutdown. ' 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences_______________________________________________________ __________ 5.1 Teflon should not be used for sample storage or any part o f instiumentation that comes in contact with the sample or extract. 6.0 E quipment _______________________________________________________________ _ 6.1 Equipment listed below may be changed in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 H P1100 low pulse solvent pumping system and autosampler. 7.0 S u p p l i e s a n d M a t e r i a l s ________ :________________ ;____________________ ,_________________ 7.1 Supplies 7.1.1 Nitrogen gas, refrigerated liquid, regulated to approximately 100 psi. 7.1.2 HPLC column, specifics to be determined by the analyst. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes. 8.0 R e a g e n t s a n d S t a n d a r d s ________________________________________________________________ __ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent. Word 7.0.1/95 FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 2 of 8 3MEnvironmental Laboratory Page 123 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC Plus system. 8.1.3 Ammonium acetate, HPLC grade or equivalent. 822 S tandards 8.2.1 Typically one H20 blank, one liver blank, and seven liver standards are prepared during the extraction procedure. See FACT-M-1. 9.0 S a m p l e H a n d l i n g ________________________ '____________________________________________________ __ 9.1 Fresh liver standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. . 9.2 If analysis will be delayed, extracted standards and samples may be refrigerated until analysis can be performed. 10.0 Q u a l i t y C o n t r o l _______________________________________________________________ ^______________ 10.1 M atrix B lanks an d M ethod Blanks 10.1.1 Analyze a method blank and matrix blank prior to each calibration curve. 10.2 M atrix Spikes ' 10.2.1 Analyze a matrix spike and matrix spike duplicate with each analysis. 10.2.2 Expected concentrations will fall in the mid-range o f the initial calibration curve. Additional spike concentrations may fall in the low-range o f the initial calibration curve. " 10.2.3 See section 13 to calculate percent recovery. 10.3 C ontinuing C alibration Checks ' 10.3.1 Analyze a mid-range calibration standard after every tenth sample. If a significant ch an ge ( 30% ) in peak area occurs, relative to the initial standard curve, stop the run. Only those samples analyzed before the last acceptable calibration standard will be used. The remaining samples must be reanalyzed. 1023.2 See section 13 to calculate percent difference. 10.4 System Suitability 10.4.1 System suitability (e.g. peak area, retention time and peak shape, etc.) will be assessed for each run. 11.0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n _________________________________________________________ 11.1 Analyze the extracted liver standards prior to and following each set of extracts. The mean o f two standard values, at each standard concentration, will be plotted by linear regression for the calibration curve using MassLynx or other suitable software. 3MEnvironmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 3 of 8 Page 124 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 11.2 The r2 value for the data should be 0.98 or greater. Lower values may be acceptable at the discretion o f the analyst. 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 12.0 Procedures____________________________________^______________________________ 12.1 A cquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using letter-MO-DAY-last digit o f year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses.. A scan is usually collected along with the SIRs Save method. 12.13 Typically the sample list begins with the first set o f liver standards and ends with the second set of standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in section 12.1.1. 123.2 Set-up the HP 1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection with a sample wash . 12.2.2.2 Inject/sample =1 1 2 .2 .2 .3 C ycle tim e = 1 5 m inutes 123.2.4 Solvent ramp = Time 0.00 min. 7.5 min. 11.0 min. 11.5 min. MeOH 45% 90% 90% 45% 2.0 mM Ammonium acetate 55% 10% 10% 55% Note: In this instrument configuration, the run must be set up on the electrospray software with a "Waiting for inlet start" message before the "Start" button is pressed on the HP Workstation. 12.2.2.5 Press the "Start" button. FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 4 of 8 3MEnvironmental Laboratory Page 125 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 12.3 In stru m en t Sep-up 12.3.1 Refer to AMDT-EP-31 for more details. 123.2 Check the solvent level in reservoirs and refill if necessity. 12.33 Check the stainless steel capillary at the end o f the probe. Use an eye piece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 123.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out o f the tip o f the probe. Allow to equilibrate for approximately 10 minutes. 123.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. 12.3.6 The instrument uses these parameters at the following sittings. 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 1 2 3 .6 3 LC constant flow mode flow rate 10 - 5 0 0 uL/rain 123.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the instrument is operating correctly.) V . ' 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Record tune parameters in the instrument log. 123.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis of biological matrices. 123.10 Click on start button in the Acquisition Control Panel. Press the start button at top o f sample list. Ensure start and end sample number includes all samples to be a n a ly z e d . 13.0 D a t a A n a l y s i s a n d C a l c u l a t i o n s ____________________________________________ 13.1 Calculations: 13.1.1 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.2 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 3MEnvironmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 5 of 8 Page 126 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 13.1.3 Calculate actual concentration of PFOS anion in total liver (mg): f u g P F O S a n io n c alc, fro m s td curve^J v g o f liv er u sed fo r an aly sis ) . x T otal m ass o f liv er (g ) 14.0 M e t h o d P e r f o r m a n c e ________________________________________________________ ______________ 14.1 The method detection limit is equal to at least three times the biiseline noise in the matrix blank. 14.2 The practical quantitation limit is equal to the lowest standard in the calibration curve. 15.0 P o l l u t i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t _______________________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers. All containers are located in the laboratory. 16.0 R e c o r d s __________________________ .______________________________________________________________ 16.1 Store chromatograms in the study folder. Each chromatogram should have the following information included either in the header or hand written on the chromatogram: study ' number, sample name, extraction date, and dilution factor (if applicable). 16.2 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample list from MassLynx and tape into the instrument mnlog. 16.4 Print data integration summary from MassLynx and tape into the instrument runlog. 16.5 Copy instrument runlog pages, including instrument parameters and sample results, and tape into appropriate study notebook. 16.6 Summarize data using suitable software and store in the study folder. 16.7 Back up electronic data to appropriate media. Record in study notebook the file name and location o f backup electronic data. 17.0 T a b l e s , D i a g r a m s , F l o w c h a r t s , a n d V a l i d a t i o n D a t a ____________________________ __ 17.1 Attachment A: FACT-M-2 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT-M-1.0 & 2.0-V -l. 18.0 R e f e r e n c e s __________________________________________________________________ '_________________ 18.1 AMDT-EP-31, "Operation of VG Platform Electrospray Mass Slpectrometer" 3MEnvironmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 6 of 8 Page 127 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 19.0 A f f e c t e d D o c u m e n t s ________________________________________________ ________________ - 19.1 FACT-M-1.0, "Extraction o f Potassium Perfluorooctanesulfonate from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 Revisions_____________________ ;_____________________ ___________________ . Revision Number. Reason For Revision Revision Date 3MEnvironmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 7 of 8 Page 128 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Laboratory Study # S tudy: Test M aterial: M a trix /F in a l S olvent: M eth o d /R evisio n : A n a ly tic a l E q u ip m en t System N um ber. In strum ent S oftw are/V ersio n: F ilenam e: R -S quared V alu e: S lo p e : Y Intercep t: D ate o f E xtracd on /A nalyst: D ate o f A n a lysis /A n alyst G rou p Dose S am p le# C o n cen tratio n u g /m L In itia l V oL mL D ilu tio n F acto r F in a l Cone. u g /m L S lo p e : T a k e n fro m lin e a r regression equation. G ro u p /D o s e : T a k e n fro m the study folder. S a m p le # : T a k e n fro m th e study folder. C o n c e n tra tio n (u g /m L ): T aken fro m the M a ssL yn x integration sum m ary. In it ia l V o lu m e ( m L ) : T a k e n fro m the study fo lder. D ilu tio n F a c to r: T a k e n from the study folder. F in a l C o n e . (u g /m L ): C alculated by d ivid in g th e in itia l vo lu m e fro m the concentration 3MEnvironmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 8 of 8 Page 129 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 R N -U 2104 L 3M Environmental L aboratory Method Analysis of Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry M ethod N um ber: FACT-M-4.0 Author: Lisa Clemen Adoption Date: Revision Date: Approved By: \ , Laboratory Manager J4- * - --------------------- Group Leader Date ....................... Date ........ ... - . Technical Reviewer Ev m 'i I ihI i i_______________ Date 1.0 S c o p e a n d A p p l ic a t io n 1.1 Scope: This method is for the analysis o f extracts o f serum or tissue for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Com pounds: Potassium perfluorooctanesulfonate, arionic fluorochemical surfactants, or other ionizable compounds. 1 3 M atrices: Rabbit, rat, and bovine serum or other sera as designated in the validation report. Word 7.0.1/95 3MEnvironmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 1 of 8 Page 130 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 2.0 S u m m a r y o f M e t h o d ______________________________________________________________ ;_________ 2.1 This method describes the analysis o f fluorochemical surfactants extracted from serum using HPLC-electrospray/mass spectrometry. The analysis is performed by monitoring a single ion characteristic o f a particular fluorochemical, such as the potassium perfluorooctanesulfonate (PFOS) anion, M/Z= 499. Samples may also be screened to verify compound identification. 3.0 D e f i n i t i o n s _______________________ ^_________________________________ ______________________________ 3.1 None. 4.0 W a r n i n g s a n d C a u t i o n s _______________________________________________________________________ 4.1 H ealth an d Safety W arnings: 4.1.1 Use caution with the voltage cable for the probe. When the voltage cable is plugged into the probe DO NOT TOUCH THE PROBE, there is risk of electrical shock. 4.2 Cautions: 4.2.1 Do not run solvent pumps above capacity of 400 bar (5800 psi). I f pressure goes over 400 bar, the HP1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. i' 5.0 I n t e r f e r e n c e s _______________________________________________________________ _ 5.1 Teflon should not be used for sample storage or any part o f instrumentation that comes in contact with the sample or extract. * 6.0 E q u i p m e n t ____________________;________ ._____________________ ;___________________ ___________________ 6.1 Equipment listed below may be changed in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HP 1100 low pulse solvent pumping system and autosampler. 7.0 S u p p l i e s a n d M a t e r i a l s _______________________________________________________________________ 7.1 Supplies 7.1.1 Nitrogen gas, refrigerated liquid, regulated to approximately 100 psi. 7.1.2 HPLC column, specifics to be determined by the analyst. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes. 8.0 R e a g e n t s a n d S t a n d a r d s ______________________________________________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent. FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 2 of 8 3MEnvironmental Laboratory Page 131 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC Plus system. 8.1.3 Ammonium acetate, HPLC grade or equivalent. 8.2 Standards 8.2.1 Typically one H20 blank, one serum blank, and seven serum standards are prepared during the extraction procedure. SeeFACT-M-3. 9.0 S a m p l e H a n d l i n g _____________________________________ _____________________________ 9.1 Fresh serum standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 9.2 If analysis will be delayed, extracted standards and samples may be refrigerated at 4 C until analysis can be performed. 10.0 Q u a l i t y C o n t r o l __________ _______________________________________ ____________________________ 10.1 Matrix Blanks and Method Blanks 10.1.1 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes ' 10.2.1 Analyze a matrix spike and matrix spike duplicate with each analysis. . 10.2.2 Expected concentrations will fall in the mid-range o f the initial calibration curve. Additional spike concentrations may fall in the low-range o f the initial calibration curve. 10.2.3 See section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 10.3.1 Analyze a mid-range calibration standard after every tenth sample. If a significant change ( 30%) in peak area occurs, relative to the initial standard curve, stop the run. Only those samples analyzed before the last acceptable calibration standard will be used. The remaining samples must be reanalyzed. 103.2 See section 13 to calculate percent difference. 10.4 System Suitability 10.4.1 System suitability (e.g., peak area, retention time, peak shape, etc.) will be assessed for each run. 11.0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n _________________________________________________________ 11.1 Analyze the extracted serum standards prior to and following each set o f extracts. The mean o f two standard values, at each standard concentration, will be plotted by linear regression for the calibration curve using MassLynx or other suitable software. 3MEnvironmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 3 of 8 Page 132 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 11.2 The r2 value for the data should be 0.98 or greater. Lower values may be acceptable at the discretion o f the analyst. 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 12.0 P r o c e d u r e s _______________ ;_____________________ ,_____________________ .__________________________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using letter-MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A scan is usually collected along with the SIRs. Save method. 12.1.3 Typically the sample list begins with the first set of serum standards and ends with the second set of standards. 12.1.4 Samples are analyzed with a continuing calibration check: injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 11.2 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 conditio ns or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection with a sample wash 12.2.2.2 Inject/sample = 1 . 12.2.2.3 Cycle time = 15 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 7.5 min. 11.0 min. 11.5 min. MeOH 45% 90% 90% 45% 2.0 mM Ammonium acetate 55% 10% 10% 55% Note: In this instrument configuration, the run must be set up on the electrospray software with a "Waiting for inlet start" message before the "Start" button is pressed on the HP Workstation. 12.2.2.5 Press the "Start" button. FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 4 of 8 3MEnvironmental Laboratory Page 133 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 12.3 Instrument Set-up 12.3.1 Refer to AMDT-EP-31 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end o f the probe. Use an eye piece to check the tip. The tip should be flat with no jagged edge. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip o f the probe. Allow to equilibrate for approximately 10 minutes. 123.5 Turn on the nitrogen. A fine mist should be expelled wi1h no nitrogen leaking around the tip o f the probe. 12.3.6 The instrument uses these parameters at the following se ttings. 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 12.3.63 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.) * 123.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 123.8 Record tune parameters in the instrument log. 123.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis of biological matrices. 12.3.10Click on start button in the Acquisition Control Panel. Press the start button at top o f sample list. Ensure start and end sample number includes all samples to be analyzed. 13.0 D a t a A n a l y s i s a n d C a l c u l a t i o n s ____________________________________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the follovdng 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. 3MEnvironmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 5 of 8 Page 134 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, anion in serum (jag/mL): ug o f PFO calc, from std. Curve x Dilution Factor x Final Volume (mL) Initial Volume of serum (mL) 1 4 .0 M e t h o d P e r f o r m a n c e __________________________________________________________________ 14.1 The method detection limit is equal to half the lowest standard in the calibration curve. 14.2 The practical quantitation limit is equal to the lowest standard in the calibration curve. 1 5 .0 P o l l u t i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t ________________________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 1 6 .0 R e c o r d s _______________ '_____________________________________________________________ ;___________ 16.1 Store chromatograms in the study folder. Each chromatogram must have the following information included either in the header or hand written on the chromatogram: study number, sample name, extraction date, and dilution factor (if applicable). 16.2 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample list from MassLynx and tape into the instrument runlog. 16.4 Print data integration summary from MassLynx and tape into the; instrument runlog. 16.5 Copy instrument runlog pages, including instrument parameters and sample results, and tape into appropriate study notebook. 16.6 Summarize data using suitable software and store in the study folder. 16.7 Back up electronic data to appropriate medium. Record in study notebook the file name and location o f backup electronic data. 1 7 .0 T a b l e s . D i a g r a m s . F l o w c h a r t s , a n d V a l i d a t i o n D a t a ________________________________. 17.1 Attachment A: FACT-M-4 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT-M-3.0 & 4.0-V -l. 1 8 .0 R e f e r e n c e s _____________________________________________________________________________________ 18.1 AMDT-EP-31, "Operation of VG Platform Electrospray Mass Spectrometer" 1 9 .0 A f f e c t e d D o c u m e n t s _____________________________________________________ ;___________________ 19.1 FACT-M-3.0, "Extraction of Fluorochemical Anions from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 3MEnvironmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 6 of 8 Page 135 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 20.0 R e v i s i o n s Revision Number. ' Reaspn For Revision Revision Date i 3MEnvironmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 7 of 8 Page 136 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Laboratory Study # S tu d y: T e s t M a te ria l: M a trix /F in a l S o lv e n t: M e th o d /R e v is io n : A n a ly tic a l E q u ip m e n t S y s te m N u m b e r In s tru m e n t S o ftw a re /V e rs io n : F ile n a m e : R -S q u a re d V a lu e : S lo p e : Y In te rc e p t: D a te o f E x tra c tio n /A n a ly s t: D a te o f A n a ly s is /A n a ly s t: G ro u p D ose S a m p le # C o n c e n tra tio n u g /m L In itia l V o L mL D ilu tio n F a c to r F in a l C o n e. u g /m L S lo p e : T a k e n fro m lin e a r re g re s s io n e q u a tio n . G ro u p /D o s e : T a k e n fro m th e stu d y fo ld e r. S a m p le # : T a k e n fro m th e stu d y fo ld e r. C o n c e n tr a tio n (u g /m L ): T a k e n fro m th e M a s s L y n x in te g ra tio n s u m m ary. In it ia l V o lu m e (m L ) : T a k e n fro m th e s tu d y fo ld e r. D ilu tio n F a c to r: T a k e n fro m d ie stu d y fo ld e r. F in a l C o n e . (u g /m L ): C a lc u la te d b y d iv id in g th e in itia l v o lu m e fro m th e c o n c e n tra tio n 3MEnvironmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 8 of 8 Page 137 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Environmental L aboratory Method Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/M ass Spectrometry M ethod Num ber: ETS-8-5.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Robert Wynne Revision Date: Approved By: 9^1 iV--- Laboratory Manager Group Leader Date /S2 4 / $ ? Date 3 53' r *uJ\ Xi*- A ------------------------------------------------ m--.Technical Reviewer tXu o Date 3 1 .0 S c o p e a n d A p p l i c a t i o n 1.1 Scope: This method describes the analysis o f serum extracts for fluc rochemical surfactants O --s using HPLC-electrospray/mass spectrometry. 0 1.2 A pplicable C om pounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1 :5` 1.3 M atrices: Rabbit, rat, bovine, monkey, and human serum, or other fluids as designated in I W the validation report. W o rd 6 /9 5 ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 1 of 9 3MEnvironmental Laboratory Page 138 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 2.0 Summary of M ethod_________________________________________________ _________ 2.1 This method describes the analysis o f 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 o f a compound by detecting daughter ions o f the parent ion. 3.0 D efinitions___________________________ ;____________________________________ 3.1 A tm ospheric P ressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods o f ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e., not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method o f ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application o f a strong electrical field. 3.3 M ass Spectrom etry, M ass Spectrometer (MS), Tandem Mass Spectrom eter (MS/MS): The API Quattro II triple quadrupole systems are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently ' detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 C onventional vs. Z -spray probe interface: The latest models o f Micromass -Quattro II triple quadrupole systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods o f operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e., Z-spray components are compatible with some other Z-spray systems, etc.) 3.5 M ass L ynx Software: System software designed for the specific operation o f these Quattro n triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details see the manual specific to the instrument (Micromass Quattro II triple quadrupole MassLynx or MassLynx NT User's Guide). 4.0 W arnings and Cautions_____________________________________________________ 4.1 H ealth an d Safety W arnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 2 of 9 3MEnvironmental Laboratory Page 139 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 4.2 Cautions: 4.2.1 Do not operate solvent pumps above capacity o f 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HP1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences_________________________________________________________________ 5.1 To minimize 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 Equipment___________________________________________ ,_________________________ 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 6.1.2 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an electrospray ionization source H P1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 Supplies and Materials ____________________:__________ ;____________ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (House air system) 7.1.2 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes 8.0 Reagents and Standards_______________________ '______ ________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 S tandards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. See ETS-8-4.1. 9.0 Sample Handling______________________________________________________________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 3 of 9 3MEnvironmental Laboratory Page 140 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C, or at room temperature, until analysis can be performed. 10.0 Quality Control__________________________________________________________ _ 10.1 Solvent B lanks, M ethod Blanks and M atrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 M atrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the ' recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to meiisure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per foity samples, 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. 1(1.3 C ontinuing C alibration Verifications 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy o f the calibration curve. '` 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum of one per batch. . 11.0 Calibration and Standardization_____________ ;____________________ ;_________ 1 1 .1 A n a ly z e th e e x tr a c ted m atrix standards prior to and following each set o f extracts. The average o f two standard curves will be plotted by linear regression (y = my + b), weighted 1/x, not forced through zero, using MassLynx or other suitable software. 11.2 I f the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.3 For purposes o f accuracy when quantitating low levels o f 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 die 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. 3MEnvironmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 4 of 9 Page 141 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 12.0 Pr o c e d u r e s __________________________________________ _________________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using MO-DAY-last digit o f year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording) or MRM. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). 12.1.3 Typically the analytical batch run sequence begins with a set o f extracted matrix standards and ends with a set o f extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the A utosam pler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. t 12.2.2 Set-up the HP 1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 1 0 pL injection ' 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = Tim e 0.00 min. 8.50 min. 11.0 min. 12.0 min. M eOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% 10% 10% 60% 12.2.2.5 Press the "Start" button. 12.3 In stru m en t Set-up 12.3.1 Refer to ETS-9-24.0 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 3MEnvironmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 5 of 9 Page 142 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 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 shunless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out o f the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelled wi th 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 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 HPLC constant flow mode, flow rate 10 - 500 pL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. ' 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis o f biological matrices. 12.3.10Click on start button in the Acquisition Control Panel (this may vary-among M assLynx versions, see appropriate MassLynx USER'S GUIDE). Press the start button. Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations_______________ . ______ ;_________________ 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 concentration o f PFOS, or other fluorocheinical, in matrix (pg/mL): (he o f PFOS calc, from std. Curve x Dilution Factor! x 1 lie /Initial Volume o f matrix (riiLl + mL o f Surrogate Standard! 1000 ng Final Volume (mL) ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 6 of 9 3MEnvironmental Laboratory Page 143 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 14.0 M ethod Performance_______,___________________________________ _____________ 14.1 Method Detection Limit (MDL) and Limit o f Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-4.1, Attachm 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 are must be below the lowest standard in the calibration curve 14.3 C alibration C urves 14.3.1 The r2value for the calibration curve must be 0.980 or better. 14.4 M atrix Spikes 14.4.1 Matrix spike percent recoveries are must be within 30% o f the spiked concentration. 14.5 C ontinuing C alibration Verifications 14.5.1 Continuing calibration verification percent recoveries must be 30% o f the spiked concentration. 14.6 I f criteria listed in this method performance section isn't met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the , analyst. Document all actions in the appropriate logbook. 14.7 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 o f the report. 15.0 Pollution Prevention and Waste Ma n a g e m e n t _______________ ;___________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R E C O R D S ______________________________________________________________________________________ 16.1 Each page generated for a study must have the following informati on 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. 3MEnvironmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 7 of 9 Page 144 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 16.5 Summarize data using suitable software (Excel 5.0) and store in the study folder, see A ttachm ent A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location o f backup electronic data. 17.0 T ables. D iagrams. Flowcharts, and Validation Data 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet. ____________________ 18.0 References____________________________ :______________________________________ 18.1 FACT-M-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance o f the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro H triple quadrupole Systems" 18.3 The validation report associated with this method is ETS-8-4.0 S: 5.0-V -l. 19.0 A ffected Do c u m e n t s _____________________________________________________ 19.1 ETS-8-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 Revisions_____________ ________________ ______________________________________ _ Revision Number. 1 Reason For Revision . Section 6.1.2 Clarification o fH P l 100 system components. Section 11.1 Average o f two curves, not standard values, are used for plotting linear regression and added the 1/x weighting o f the curve. Section 12.2.2.4 Clarification o f solvent ramp. Section 17.1 Changed from attachment B to A. Revision Date 04/02/99 3MEnvironmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 8 of 9 Page 145 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Laboratory Study # S tu d y : Test M aterial: M a trix /F in a l S olvent: M eth o d /R evisio n : SystemA n a ly t ic a l E q u ip m e n t N um ber: Instrum ent S o fh vare/V ersio n : Filenam e: R -S qu ared V alu e: S lo p e : Y Intercept: D a te o f E xtraction /A nalysfc D ate o f A n a lysis /A n alyst G roup D ose Sam ple# C on cen tration ug/m L Initiai VoL mL D ilu tion Factor Final C one. ug/m L S lo p e : T a k e n fro m lin e a r regression equation. G ro u p /D o s e : T a k e n fro m th e study folder. S a m p le # : T a k e n fro m flic stu d y folder. C o n c e n tra tio n (u g /m L ): T a k e n fro m the M a s s L y n x integration sum m ary. In it ia l V o lu m e (m L ) : T a k e n fro m the study folder. D ilu tio n F a c to r: T a k e n fr o m the study folder. F in a l C o n e . (u g /m L ): C a lc u la te d by d ivid in g the in itia l vo lu m e fro m the concentration Attachment A: Summary Spreadsheet ETS-8-5.1 Analysis of Serum Extract Using ES/MS 3MEnvironmental Laboratory Page 9 of 9 Page 146 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Environmental L aboratory M ethod A n a ly sis o f Potassium Perfluorooctanesuleonate or Oth er F luorochem icals in Liver Extracts U sing H PL C -E lectrospray/M ass Spectrometry M ethod N um ber: ETS-8-7,0 Adoption Date: Author: Lisa Clemen, Glenn Langenburg Approved By: Revision Date: /\1/V y j Laboratory Manager Dat ' ufuA* { b -- -- Group Leader Date A__ _______________________________ Technical Reviewer 7V m- O l/l i / f t Date ! V 1.0 Scope and A pplication DO Co 77?CDI--h 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. 9 1.3 M atrices: Rabbit, rat, bovine, monkey liver, or other tissues as designated in the validation 5 ' report. Word 6/95 ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 1 of 10 3MEnvironmental Laboratory Page 147 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 2.0 Summary of M ethod__________________________________________________________ 2.1 This method describes the analysis o f fluorochemical surfactants extracted from liver using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z = 499. Additi onally, 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 Definitions___________________________ '_______________ ________________________ 3.1 A tm ospheric Pressure Ionization (API): The Micromass Quattro n triple quadrupole systems allow for various methods o f ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e. not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application o f a stron g electrical field. 3.3 M ass Spectrom etry, M ass Spectrom eter (MS), Tandem M ass Spectrom eter (MS/MS): The API Quattro II triple quadrupole mass spectrometer is equipped with two quadrupole y mass selective detectors and a collision cell. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion . detection or an ion may be selected in the first quadrupole, fragmented in the collision cell, and these fragments may be analyzed in the second quadrupole. 3.4 Conventional vs. Z-spray probe interface: The latest models c f Micromass Quattro II triple quadrupole (post 1998) utilize a "Z-spray" conformation, irhe 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 m a in ten a n ce are th e sam e. H o w ev er, Z -sp ray com p on en ts and co n v en tio n a l co m p o n en ts are not compatible w ith one another, but only with similar systems (i.e. Z-spray components are compatible with other Z-spray systems, etc.) 3.5 M ass Lynx Software: System software designed for the specific operation o f these Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details refer to the manual specific to the instrument (Micromass Quattro II triple quadrupole MassLynx or MassLynx NT User's Guide). 4.0 W arnings and Cautions_______________________ :__________________________ 4.1 H ealth and Safety W arnings: 4.1.1 U se caution with the voltage cables for the probe. When engaged, the probe employs a voltage o f approximately 5000 Volts. ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 2 of 10 3MEnvironmental Laboratory Page 148 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. '' 4.2 Cautions: 4.2.1 Operate the solvent pumps below a back pressure o f 400 bar (5800 psi). If the back pressure exceeds 400 bar, the HP1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 I n t e r f e r e n c e s ______________________________ ;__________________________ _________________________________________ 5.1 To minimize interferences when analyzing samples, Teflon shsill 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 i p m e n t ______________________________________________________________ ____ _____________ i_______________________ 6.1 Equipment listed below may be modified in order to optimize 1he system. Document any modifications in the raw data as method deviations. 6.1.1 6.1.2 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an electrospray ionization source. HP 1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 S u p p l i e s a n d M a t e r i a l s ____________________________________________ ________________________________________ 7.1 Supplies 7.1.1 High purity grade air regulated to approximately 100 psi (house air system) 7.1.2 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data 7.1.3 Capped autovials or capped 15 ml centrifuge tubes 8.0 R e a g e n t s a n d S t a n d a r d s ________________________________________________________________________ ____ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent . 8.1.2 Milli-QTM water (ASTM type I), all water used in this method should be ATSM type I, or equivalent, and be provided by a Milli-Q TOC!) Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.1.3.1 When preparing different amounts than those listed, adjust accordingly. 8.1.3.2 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 mL volumetric container containing 2000 mL Milli-QTM water, mix until all solids are dissolved. Store at room temperature. ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 3 of 10 3MEnvironmental Laboratory Page 149 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 8.2 S tandards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. Refer to ETS-8-6.0. 9.0 _ _S a m p l e H a n d l i n 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 may be stored at room temperature, or refrigerated at approximately 4 C, until analysis can be performed. 10.0 Q u a l i t y C o n t r o l ______________;_______________________________________ _________________________;_____________ 10.1 M ethod Blanks and M atrix Blanks 10.1.1 Solvent blanks, method blanks, and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 M atrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty sample^. With a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lowrange o f the initial calibration curve. 10.3 C ontinuing C alibration Checks 10.3.1 Continuing calibration verifications are analyzed to veri fy the continued accuracy o f the calibration curve. 10.3.2 Analyze a mid-range calibration standard every tenth sample, with a minimum of one per batch. 11.0 ;C a l i b r a t i o n a n d S t a n d a r d i z a t i o n _______________________________________________________ ___________ 11.1 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 Analysis of Liver Extract Using ES/MS Page 4 of 10 Page 150 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 11.3 For purposes o f accuracy when quantitating low levels o f analyte, it may be necessary to use the low end o f the calibration curve rather than the full range o f the standard curve. Example: when attempting to quantitate approximately 10 ppb o f analyte, generate a calibration curve consisting o f the standards from 5 ppb to 100 ppb rather than the full range o f the curve (5 ppb to 1000 ppb). This will reduce inacc uracy attributed to linear regression weighting o f high concentration standards. 12.0 P r o c e d u r e s ___________________ '_______________ 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 of the alphabet starting with a 12.1.1.2 Assign a method (MS file) for acquiring 12.1.1.3 Assign an HPLC program (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 was spectrometer headings and select SIR (Single Ion Recoiriing) or MRM (Multiple Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. IfMS/MS instruments are employed, additional product ion ' ' fragmentation information may be collected. Refer to flficromass 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 12.2.2.3 Cycle time = 9 minutes . 3MEnvironmental Laboratory ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 5 of 10 Page 151 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 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% 6(1% 5% 5% 60% 60% 12.2.2.5 Press the "Start" button. 12.3 In stru m en t Set-up 12.3.1 Refer to ETS-9-24.0, "Operation and Maintenance o f the Micromass Quattro II Triple Quadrupole Mass Spectrometer Fitted with an A tmospheric 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. If the tip is found to be ' unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Turn on the nitrogen. 12.3.5 Open the tune page. Clicks on operate to initiate source block and desolvation heaters. 12.3.6 Open the Inlet Editor. 12.3.6.1 Set HPLC pump to "On" 12.3.6.2 Set the flow to 10 - 500 uL/min or as appropriate 12.3.6.3 Observe droplets coming out o f the tip 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 m ist is observed 12.3.6.4 Allow to equilibrate for approximately 10 minutes. 12.3.7 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.7.1 Drying gas 250-400 liters/hour 12.3.7.2 ESI nebulizing gas 10-15 liters/hour 12.3.7.3 HPLC constant flow mode flow rate 10 --500 jj.L/min 12.3.7.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7.5 Source block temperature 150 12.3.7.6 Desolvation temperature 250 ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 6 o f 10 3MEnvironmental Laboratory Page 152 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, refer to appropriate MassLynx User's Guide). Ensure start and end sample number includes all samples to be analyzed. 13.0 D a t a A n a l y s i s a n d C a l c u l a t i o n s _____________________________ __________ _____________________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result ' 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentrations in matrix (pg/g): fne o f PFOS calc, from std. Curve x Dilution Factor! (Initial Weight o f Liver (gl Final Volume (mL) x 1 pg 1000 ng 14.0 M e t h o d P e r f o r m a n c e _________________________ ,__________________________ 14.1 Method Detection Limit (MDL) and Limit o f Quantitation (LOQ) are method, analyte, and . matrix specific. Refer to ETS-8-6.0, Attachm 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 stan d ard in th e ca lib ra tio n cu rve. . 14.3 C alibration C arves 14.3.1 The i2 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 C alibration Verification 14.5.1 Continuing calibration verification percent recoveries must be within 30% o f the . spiked concentration. 14.6 If criteria listed in the method performance section are not met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 3MEnvironmental Laboratory ETS-8-7.0 Analysis o f Liver Extract Using ES/MS Page 7 o f 10 Page 153 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 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 o f the report. 15.Q P o l l u t i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t _______________________________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. ' 16.0 ; ; ' R e c o r d s _________ ________________________________________________ ;______________________ _________ _______________ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, iicquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms from MassLynx and store in the study folder. ,16.5 Summarize data using suitable software (Excel 5.0+) and store in the study folder, refer to A ttachm ent A for an example o f a summary-spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file nams and location o f backup electronic data. ' 17.0 T a b l e s . D i a g r a m s . F l o w c h a r t s , a n d V a l i d a t i o n D a t a _________________________ 17.1 Attachment A: ETS-8-7.0 Data summary spreadsheet 1 8 .0 R e f e r e n c e s _____________________________________________ __________ _____________________________ 18.1 FACT-M-2.1, "Extraction o f Potassium Perfluorooctanesulfon&te or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple quadrupole Systems" 18.3 The validation report associated with this method is ETS-8-6.0 & 7.0-V -l 19.0 _ iA f f e c t e d D o c u m e n t s _____________________________________________ __________ _____________________________ 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 r Analysis of Liver Extract Using ES/MS Page 8 of 10 Page 154 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 2 Q .0 R e v is io n s Revision Number ________________________________________________ Reason For Revision Revision Date t4 3MEnvironmental Laboratory ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 9 of 10 Page 155 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Laboratory Study # Study: Test M aterial: M a trix /F in a l S olvent: M etho d/R evisio n: A n a ly tic a l E qu ipm ent System N u m b e r Instrum ent S o ftw are/V ersio n: Filenam e: R -S quared V alu e: S lo p e : Y Intercept: D a te o fE x tra c tio n /A n a ly s t D ate o f A n a lysis /A n alyst G roup Dose S am ple# C oncentration "TnffiaTVVE ng/g g D ilution Factor JKlnal C one, ng/g G ro u p /D o s e : T a k e n fro m the study folder. S a m p le # : T aken fro m th e study folder. C o n c e n tra tio n (n g /g ): T aken from the M assL yn x integration sum m ary. In it ia l W t . (g ): T a k e n fro m the study folder. D ilu tio n F a c to r: T a k e n fro m the study folder. F in a l C o n e , (u g /g ): C alcu lated b y d ivid in g the in itia l vo lu m e fro m the concentration Attachment A: Summary Spreadsheet ETS-8-7.0 Analysis of Liver Extract Using ES/MS 3MEnvironmental Laboratory Page 10 of 10 Page 156 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Appendix D: Data Summary Tables Analytical Report: FACT TOX-003 LRN-U2104 Analytical Report: FACT-TOX-003 LRN-U2104 Table 9. FACT TOX-003 Data Summary for PFOS, PFOSA, PFOSAA, EtFOSE, M556 and PFOSEA--Rat Serum (pg/mL) Tim ept G roup PFOS AvqSD PFOSA Avq SD PFOSAA Avq * SD E tF O S E ** A vq*S D M556 A vq tS D PFOSEA** Avq * SD Week 4 Group 8 0.0 ppm M F <LOQa (n=5). <LOQa (n=5) <LOQb (n=5) <LOQb (n=5> 0.00460 * 0.00158' (n=5) 0.0103 0.00375* __________ (n=5) M 0 .1 7 4 *0 .0 5 1 0 0.00500 * 0.000125 Group 9 <n=5) (n=5) 1.0 ppm F 0.270 * 0.0687 0.00778 * 0.00254 (n=5) (n=5) 0.247 * 0.195* (n=5) 0.294 * 0.111* <n=5) NA NA NA NA NA NA NA NA NA NA NA NA W eek 14 Group 8 0.0 ppm Group 9 1.0 ppm M 0.0393*0.0109 (n=5) <LOQc (n=5) <LOQd __________ (n=5) F 0.126*0.0 159 (n=5) <LOQc <n~5) <LOQd __________ (n=5) M 2 .1 9 *0 .7 3 3 0 .0 1 1 0 *0 .0 0 0 7 1 6 0.497 * 0.618 (n=5) (n=5) __________ (n=5) F 3.26 * 0.629 0.0 1 6 4 *0 .0 0 3 5 4 (n=6) (n=6) 0.578 * 0.425 (n=6) NA NA NA NA NA NA NA NA NA NA NA NA Week CyOu Group 8 0.0 ppm Group 9 M 0.0490 * 0.0352 <LOQe _________in =5)_________ ________ (n=5) F 0.0727 * 0.0565 (n=5) <LOQa (n=5) M 1.3 5*0.45 2 0.00672 * 0.00251 _________10=5}_________ ________ (n=5) <LOQ' (n=5) <LOQ' (n=5) 0.297 * 0.194 (n=5) <LOQ9 (n=5) <LOQ (n=5) <LOQs (n=5) <LOQh (n=5) <LOQh fn=5) 0.120 * 0.0289 (n=5) <LOQ' (n=5) <LOQ' /n-C \ <LOQ' (n=5) 1.0 ppm F NA-- Not applicable ` Qualitative data on 4 points in calibration curve 3 .0 2 * 1.54 0 .0 1 0 2 *0 .0 0 2 5 2 0.273 * 0.110 <LOQ3 0.202 * 0.0723 <LOQ' (n=5) ________ (n=S) . (n=5) ________ ^ _____ (n=5) (n=5 > LO Q -- Limit of Quantitation = 0 .0 2 3 0 pg/mL LO O -- Limit of Quantitation = 0 .0 282 pg/m L LO Q -- Limit of Quantitation = 0 .0 225 pg/mL bLO Q -- Limit of Quantitation = 0 .0 494 pg/m L eLO Q -- Limit of Quantitation = 0 .0 0 5 1 6 _p_g/mL hLO Q -- Limit of Quantitation = 0.0 049 4 pg/mL " E tF O S E and P F O S E A results are qualitative only. cLO Q -- Limit of Quantitation = 0 .0 0 9 8 9 pg/m L 'L O Q -- Limit of Quantitation = 0 .0 2 5 3 pg/mL 'LOQ-- Limit of Quantitation = 0.0 097 8 pg/mL N O T E : Results are expressed as group/gender averages * the standard deviation associated with that group/gender. N O T E : All P F O S A , P FO S A A , P FO S E A , M 556, and P F O S W e e k 4 results are not corrected for purity of the reference standard material. It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. T h e only m easurement of accuracy available at this time, matrix spike studies, indicate that, with the exception of P F O S E A and E tF O S E -O H , the sera and liver d ata can b e considered to b e accurate to within one standard deviation of the average fortified samples recovery. 3MEnvironmental Laboratory Page 157 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Analytical Report: FACT-TOX-003 LRN-U2104 Table 9. (Continued) FACT TOX-003 Data Summary for PFOS, PFOSA, PFOSAA, EtFOSE, M556 and PFOSEA--Rat Serum (pg/mL) Tim ept G roup PFOS Avg SD PFOSA A vgSD PFOSAA A vgSD E tF O S E ** A vg S D M556 A vgS D PFOSEA** Avg SD Week 105 G roup 8 0 .0 p p m G roup 9 M F M 0.0137 0.0181 (n=10) 0.0256 0 .014 5 (n=10) 1.12 1.10 (n=10) <LOQe (n = 1 0 )_______ <LOQ (n = 1 0 ) _______ 0.00720 0.00330 (n = 1 0 ) ________ LO Q 1 (n=10) LO O 1 (n=10) 0.252 0 .1 6 0 (n=10) <LO Q k (n=10) <LO Q k (n=10) <LO Q k (n=10) <LO Q ' (n=10) <LO Q ' (n=10) 0.0645 0.0445 (n=10) <LOQm (n=10) <LO Q m (n=10) <LO Q m (n=10) 1.0 p p m F 1.61 0 .6 3 1 0.00797 0.00198 0.259 0 .1 5 6 <LO Q k 0.0868 0.0294 <LOQm (n=10) (n=10) (n=10) ________ (n=10) ________ (n=10)________ _________ (n=1 ) " E tF O S E and P F O S E A results are qualitative LO Q-- Limit of Quantitation = 0 .0 282 pg/mL LO Q -- Limit of Quantitation = 0 .0 0 4 9 4 pg/mL only. eLO Q -- Limit of Quantitation = 0 .0 051 6 pg/m L 'LO Q -- Limit of Quantitation = 0 .0 0 9 7 8 pg/m L 'LOQ-- Limit of Quantitation = 0 .0 248 pg/m L aLO Q -- Limit of Quantitation = 0 .0 2 3 0 pg/mL 'LO Q -- Limit of Quantitation = 0 .0 2 5 3 pg/mL 'LOQ-- Limit of Quantitation = 0 .0 0 5 0 3 pg/mL "L O Q -- Limit of Quantitation = 0 .0 0 4 9 3 pg/m L bLO Q -- Limit of Quantitation = 0 .0 4 9 4 pg/m L 9LO Q -- Limit of Quantitation = 0 .0 2 2 5 pg/mL cLO Q -- Limit of Quantitation = 0 .0 0 9 8 9 pg/mL N O T E : Results are expressed as group/gender averages the standard deviation associated with that group/gender. N O T E : All P F O S A , PFO SA A , P F O S E A , M 556, and P F O S W e e k 4 results are not corrected for purity of the reference standard material. It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. T h e only m easurem ent of accuracy available at this time, matrix spike studies, indicate that, with the exception of P F O S E A and E tF O S E -O H , the sera and liver data can be considered to be accurate to within one standard deviation of the average fortified samples recovery. 3MEnvironmental Laboratory Page 158 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Analytical Report: FACT-TOX-003 LRN-U2104 Table 10. FACT TOX-003 Data Summary for PFOS, PFOSA, PFOSAA, EtFOSE, M556 and PFOSEA--Rat Liver (pg/g) Tim ept G roup PFOS A vgSD PFOSA Avg SD PFOSAA Avg SD E tF O S E * A vgSD M556 A vgSD PFOSEA* Avg SD Week 4 G roup 8 0 .0 p p m G roup 9 1 .0 p p m M 0.198 0.0822 (n=5) F 0.0891 0 .0 3 1 5 (n=5) M 6.53 0.628 (n=5) F 2.97 0.937 (n=5) <LOQa (n=5) <LOQa (n=5) 0.187 0.0401 (n=5) 0.222 0 .1 0 4 (n=5) <LOQb (n=5) <LOQb (n ~ 5 ) 0.683 0 .6 1 3 (n=5) 0.582 0 .1 9 3 (n=5) NR NR NR NR NA NA NA NA NA NA NA NA W eek 14 G roup 8 0 .0 p p m G roup 9 1 .0 p p m M 2.96 4.83 (n=5) F 0.411 0.0625 (n=5) M 12.4 6 .9 9 (n=5) F 9.19 1 .5 3 (n=5) 0.0822 0 .1 5 6 (n=5) 0.0130 0.000872 . (n=5) 0.366 0.221 (n=5) 0.580 0.239 (n=5) 0.330 0.656 (n=5) 0.0411 0.0121 (n=5) 2.89 1.88 (n=5) 2.61 2 .4 9 (n=5) NR NR NR NR NA NA NA NA NA NA NA NA M 1.44 1.18 <LOQ <LOQd <LOQe <LOQ' <LOQ9 G rou p 8 (n=5) (n=5) (n=5) (n=5) (n=5) (n=5) W eek 0 .0 ppm F 0 .1 7 8 0 .1 1 5 0.00647 0.0000786 (n=5) (n=5) <LOQd (n=5) <LOQe (n=5) <LOQ' (n=5) <LOQ9 (n=5) 53 M 37.1 10.9 G rou p 9 (n=5) 0.204 0.0769 fn=5) 0.906 0.605 rn-*a <LOQa /_C\ 0.336 0.0682 (n=5) <LOQ9 (n=5) 1 .0 p p m F 25.2 13.9 0.242 0.0999 0.527 0.131 <LOQa 0.267 0.0660 <LOQ9 ... te = 5 ) _ (n=5) E tF O S E and P F O S E A results are qualitative aLO Q -- Limit of Quantitation = 0.0121 pg/g (n=5) (n=5) ________ (p=5 ) (n= 5 > LO Q -- Limit of Quantitation = 0 .0 1 2 5 pg/g 9LO Q -- Limit of Quantitation = 0 .0 308 pg/g only. bLO Q -- Limit of Quantitation = 0.0341 pg/g 'L O O -- Limit of Quantitation = 0 .0 5 5 9 pg/g hL O Q -- Limit of Quantitation = 0.0 322 pg/g N R -- No result NA-- Not applicable cLO Q -- Limit of Quantitation = 0 .0 0 6 4 4 pg/g 'LO O -- Limit of Quantitation = 0 .0 3 0 7 pg/g l O Q -- Limit of Quantitation = 0.0314 pg/g N O T E : Results are expressed as group/gender averages the standard deviation associated with that group/gender. N O T E : All P F O S A , P FO S A A , P FO S E A , M 556 and E tF O S E -O H W eek 4 and W e e k 14 results are not corrected for purity of the reference standard material. It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. Th e only m easurement of accuracy available at this time, matrix spike studies, indicate that the sera and liver data can be considered to be accurate to within one standard deviation of the average fortified samples recovery. 3MEnvironmental Laboratory Page 159 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Analytical Report: FACT-TOX-003 LRN-U2104 Table 10. (Continued) FACT TOX-003 Data Summary for PFOS, PFOSA, PFOSAA, EtFOSE, M556 and PFOSEA--Rat Liver (pg/g) Tim ept W eek 105 G roup Group 8 0.0 ppm M F Group 9 1.0 ppm M F PFOS Avg SD 0.0653 0.0923 (n=10) 0.0872 0 .0 5 2 2 (n=10) 6.21 3.8 0 (n=10) 9.63 4 .3 5 (n=10) PFOSA A vgS D <LOQh (n=10) <LOQh (n=10) 0.371 0 .1 8 7 (n=10) 0.550 0 .1 1 2 (n=10) PFOSAA Avg SD <LOQ' ______________( Q z l O ) ______________ <LOQ' ___________ D=10)____________ . 0.736 0.551 (n=10) 0.771 0 .411 (n=10) E tF O S E * A vgSD <LOQ* (n=10) <LOQ* (n=10) <LOQ* (n=10) <LOQ* (n=10) M556 Avg SO <LOQ' (n=10) <LOQ' (n=10) 0.250 0.0865 (n=10) 0.336 0.0748 (n=10) PFOSEA* Avg SD <LOQ9 (h=10) <LOQ9 (n=10) <LOQ9 (n=10) <LOQ9 (n=10) E tF O S E and P F O S E A results are qualitative only. N R -- N o result NA-- Not applicable aLO Q -- Limit of Quantitation = 0.0121 pg/g bLO Q -- Limit of Quantitation = 0.0341 pg/g LO Q -- Limit of Quantitation = 0 .0 064 4 pg/g LO Q -- Limit of Quantitation = 0 .0 1 2 5 pg/g "LO Q -- Limit of Quantitation = 0 .0 5 5 9 pg/g fLO Q -- Limit of Quantitation = 0 .0 3 0 7 pg/g N O T E : Results a re expressed as group/gender averages th e standard deviation associated with that group/gender. 9LO Q -- Limit of Quantitation = 0 .0 308 pg/g hLO Q -- Limit of Quantitation = 0 .0 322 pg/g 'LOQ-- Limit of Quantitation = 0 .0 3 1 4 pg/g N O T E : All P F O S A , P FO S A A , P F O S E A , M 556 and E tF O S E -O H Week 4 and W e e k 14 results are not corrected for purity of the reference standard material. It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. T h e only m easurement of accuracy available at this time, matrix spike studies, indicate that the sera and liver data can be considered to be accurate to within one standard deviation of the average fortified samples recovery. 3MEnvironmental Laboratory Page 160 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Appendix E: Data Spreadsheets Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 /i 3MEnvironmental Laboratory Page 161 3M M edical D epartm ent Study: T-6316.1 ...... ....... C uvantr# 6329-228 TC3XU03 KMWm6 l>Ktry ('tooBvjen(y Sludy b San. k * r [ < .u . t ^ i m -- nn ia n-^TM ""*" t *--- ` - P-- Td'ItlEUOSEOH) Km Scab FACT M >0 * h A n tiH 0 EIS-t-5 I IM itaM IM MaoLyai 31 SccLwioRifti Y lW ty D w M Eim w A M lyA D * <4Aelyie/AeJy* Dm<ai DMH M u W M ih ' 5/27A4 OK 07/I3M.M/3OW Mti/MMH 2/14X10. VJIAUnio* Sa p k Data W EE K 4 I U T SERA Dm M A41K Mete BR MmU M auU O rayl CtlMgl 0 0H erra* La D ix D w E w iM y; D avV a^B f Pan* Eam Vm fe* Sarapta H O Bfcl 113) BR 2 Km Sdtaa B R I Km 5na b k 2 KTS-OttWM-MS RTS-032MS MSD RTS-052694-MS RTS452694 MSI) C92332F CV2352F C921J5tO IM C92373F C92192M C92196M C92207M O m itM CV2259M CV2435F CV2402F C92463F C92467F CV24XF CV2264M C9224IM C92244M C9230XM C93WM 03/2001^. VZ2A M 0*29*1 lA O EalrocOrai Vvl RMI 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 pros PPUS Partly Carrrdtoa ( 'x m llM Factor Factu 09275 Ctkaoaa 09275 09275 Latrai * Coluto-a 09275 l'otovaa NA SA NA SA NA NA NA NA 0 9275 lAAaowa 0 9275 UMava 0 9275 CMouwa 09275 Vokaoam 09275 l'alAuvn 0 9275 CalJMoa 09175 0 9275 Utlaoaa Datava* 0.9275 Vaiava* 0 9275 l 'MJtoa* 0 9275 Calao* 09275 Uataoaa 09275 Uataowa 09275 09275 Calaa Uata* 0.9275 Uatava* 0 9275 (Joaowa 09275 Catrama 09275 aaoaa 09275 Vaiava* NK aNst Rtpomd NA Na>Afffcrfcl LDQ>1m ii<Qm MMm 1.CCVitad Dm w M m i Km M 2 CCV* K M Dm r a n n i M a Kim M k>w ) CCV Ulted Dm wMMMl 4 Ecuad abm caldntaoa nofc 1M (M m M A QuiMcv <*3y, 4uktnuraipr acre <a4lopin te toktraaoaorra. LAC W3AM 07/1M * laUM LOQ agtej, CarracM LOQ ateL OUMD bOUdLOO^teL Crrctod LOQ tteL pros 241 00230 0.76 000905 pfo sa H 00 U H 4 94 00060* p fo sa a I5 001 J 20 000533 PFUS UKalivo Factor 1 NK NK 1 1 1 1 1 1 1 1 1 1 1 l 1 10 10 10 10 5 pros aatatL 000 NK NK 000 106 701 166 142 21.5 110 522 561 3 000 0.00 000 000 000 24.3 3*0 19.1 37 1 246 347 41.7 29.4 m 54.5 U niaai CaacaatratR A.rai* RSD PFOSA Partly m pros pros Sxd DT. Corradi / L or** Fra* M71MB011 <LOQ(00230 aftaL) Uakaoa* NK NR <1X30 NA Itakooa NK NR M7I39MU2 <1X30<0.02<0HttaLl <1230 NA Uakauwa Uakaoa M713MM2 M7I39S042 166 6 120% 40% NA NA A 3000016 2264 2a4 NA A30000I7 1934 2a4 210 16% NA M7I394QI3 <LOQ(00230 4*aL) 1 (Jatavo* M7I39BOI4 <LOQ(2JOi%taL) 1 Uakaoa M7IJ9R015 <LOQ(0.0230ajlaL) 1 Uakaoa M7I39B016 <LOQ(0.0230<%taLl 1 NA Uakaoa M7139BOI7 <10010.0230 u r ti) 1 <1X30 NA akaowa M7139W20 <LOQ(00230 utaLl 1 Uakaoo* M713WQ1 <LOQ|00230a|M.> \ IMoaoa M7IM022 <LOQ(00230 ^W .) 1 Uakaoa M7IMH23 <LOQ<OD230a*taL) 1 NA Uakaoa M7I39W24 <LOO(00230a rtU 1 <LOO NA Dottora* M7I39BQ27 0162 3 Uakaoa M7139B02* 0J34 3 Untai M71WR329 0143 3 LUooa M7V39B050 63M 3 234 Uakaoa M7139*331 0229 3 0270 00617 Uakaoa M71394034 0161 2 Uakaoa M7139U35 0193 2 Uakaoo* M71394036 0.136 2 Uakaoa M71394037 0.126 2 294 Uakaoa M7139403I 0233 2 0174 0X3310 Uakaoa FroSohrihi PFOSA- PtcOrarvoclinrailIfanV fFOSAA - PrtfciiMinr n ilff iil PFOSA PSUva Factor 1 1 1 1 1 1 NA NA 1 1 1 1 1 1 1 1 1 1 1 PFOSA ' FBcBUaa CoocMtraUaa f PFUSA a*L I h L r % Rc 1.99 M713940II <LOQ(0.0494 HpteL) NR NR NR NR NR NR 0.00 M71394002 <1230(00494 im/*L> 2.6 A 3000016 112% 1.7 A3000017 111% NA NA NA NA NA NA 044 AOXWKBI <LOQ(0.0494<*/L> 044 A3000022 <LOQ(0.0494H|AbL) 0 70 AQUQQ0023 <U3Q<<L<3494***.) 0.74 AM3000Q24 <LOQ(0.0494 u|/BaL) 043 AM3000023 <LOO(0.0494 (M/mLl 0.63 AM3000030 <L0Q(0.0494B|M.) 0.71 A043000031 <LDQ1004941lad.) 062 A043QQ0Q32 <LOQ<00494fkaL) 0.J7 A043000033 <LOQ(00494 H|teL) 066 A043000034 <LOO(00494 utkaL) 7.23 A04300009I 0X30723 443 AM3000039 0XU445 5.24 AW3000040 0X30524 116 A 3000041 0X3116 5.95 A043000042 0.00393 4.37 A043000047 < LOQ(0X3694tteL) 1.49 A043QQ0044 < LOQ(00494a3aiL) 5.22 A0430000* 000322 4.14 APPOPPO <LOQ(0.M94l'BlL) 449 A 3000051 < LOO(0 0494 aa/aiL) A n rifr PFUSA NM. <LO(> <IX)0 111% NA <LOO <LOO 0X10771 0.00500 RSD SI4. Dar. NA NA 1% NA NA NA NA NA 317 0X30234 2JI 0000125 Analytical Report: FACT-TOX-003 LRN-U2104 PTQSAA 044 Partly Factor 0J3O 0.5342 0.5342 0.5142 NA NA NA NA 0530 0530 0530 0530 0530 0530 0530 0530 0530 0530 0530 0530 0530 0530 0510 0530 0530 0530 0530 0530 PFOSAA Parity CvrracUua radar Uakaoa* Uakaoam Uakaoa* Uakaoa NA NA NA NA Uakaoaa Uakaoa Uakaoa Uakaoa Uakaoa* Uakaoa Uakaoa* Uakaoa Uakaoa Uataoa* Uakaoa Uakaoa Uaktoraa Uataoa* Uakaoa* Uakaoa* Uakaoaa Uakaoa LMaoa* Uakaoaa PFOSAA DRalk* Factor 1 1 1 1 1 1 NA NA 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 PFOSAA Crate. ta l NR NR NR NR 169 909 NA NA 117 19.1 117 161 305 564 11.9 545 IO 109 343 5 336 664 137 330 2*9 369 241 1100 n*M w NR NR NR NR AC4300U))* A3000017 \A SA A043000021 AC430OTI22 A30mO A043000024 A 3000025 AXR3000030 A 3000031 A 3000032 AW3WW033 A043000034 A43000Q3 A043000039 A3no AQ430UDM1 A .30000*2 A043000047 A043000044 A043000M9 Afl43000050 A043000QS1 CatTMrMtoa MPTUSAA mt/mL or % Rc NU NR SR NR 115% 133 SA NA 0X3101 0X3103 000629 0X10464 0X1164 0X10306 Q6640 000293 000475 000504 02 027 OUI 0551 0451 0.177 0154 0194 0134 0592 Arri|* PFOSAA bc/b L NE NR A 117% NA A * A A 00103 A A A A A 000460 A A A A 4A 029 A A A A 4A 0247 RSD Md. (tat. SR NR ?% SA 363 000375 343 0111 0195 3M Environmental Laboratory Page 162 3M Medical Department Study: T-6316.1 FACT M-iOBFAC-M-tO. ETS--3 I MmU mcMIOM MataLynx 3 I wR<s|e( 1 ) N-Efcyl taflua Covane# 6329-228 Analytical Report: FACT-TOX-003 LRN-U2104 Y -toriy Pat ti EaawrmtoAatlytt Oat ei Aaaiyta/Aaalya. Dak t Oat S a lip k Dala W EEK 14 RAT SERA C n^ Samptrt M a k lH M a il! oc loon* Cray 1 OaaU 0 0ffKD Gnar LaaDta H20BB 1 >127 BB 2 B aS tm B B I U S o w iA l KTS07I54MS RTS07I59MSU C922S5M C92250M CV2237M ey2iMM C922M3M C92396F CV2V7F C92MSF C9D99F C9240 C92US4 CV232aM CV2J27M C9232SM CV2330M CV2404FI/2 CV2464F2/2 C92443F C92466F CW4UF CV2409F I E a v l A m rttartmw na|c* S M i* ,n ib 4 * * * > Sat A n a t r a li 7/IW lAS/SAH 7/|7A*. (7A* MLUHO) / kkjom* Ellracfliw v6 n a b i i i i I I 1 i i i I i i i t pfos Factor 0.9275 0 9275 0 273 on SA NA 0.9273 0 9273 0 9273 09275 0 9275 0 9273 0 9275 09275 09275 0 9275 09275 0.9273 09273 0.9275 0 9Z73 09273 0.9273 0.9275 09275 09275 09275 FFOS Parti; I'w n n u Fa-tor lakDoam Uni iMiwn ttltv a Uatawwn NA NA Unkaowa Uakavwn Uakavwa Uakauwa Uotooaa Uakaowa Uakaowa Uataowa Uataowa Udaowa l'a U x i Uakaowa Udaowa Uakaowa Unknown Uataowa Uataowa Uataowa Uataowa Uakavwn Unknown pros UUalfco Fa-tor 1 1 1 1 l 1 1 1 1 1 1 1 l 1 1 1 1 25 23 23 23 30 1 30 30 30 30 pros nt/aL 121 000 4 60 2.04 107 114 31.0 31 0 403 390 132 130 120 15 121 1334 142 774 130 10* 793 33(4 599 79.6 301 (54 F illiat M7I790002 M7I79B043 (47179(003 M7179(00} (47179(040 347179(041 (47I79WI2 (471790013 M7I7H0I4 M7I79B0I5 NA M7I7M0K (47179(019 547179(020 M7179KU21 M71790022 (47179(025 AW79B0I4 A(07<0l5 A007VK016 Aaomoi? AJ0790020 (47179(033 Aa079K21 M Kfm at A4079W23 AJ079KB4 aTPFOS PFOS nitaL M ( ( nitaL <U3Q<<UI230 ttaL) <10010.0230 M .I <LOQ(Q4230 aftaL) ICO (Affilo lAaLt o-9 <LOO 109 117 113 0(346 0029 0(376 0(362 0.0393 0.141 0.120 o.m 0144 0112 0126 1.42 1 3.30 IJ0 1.92 2.51 2 19 3d 3.14 1 171 369 132 3.96 326 rrctfAahttaa rroAAaprin BSD SU. D ll. NA NA t% 270 0.0109 116 0(139 33.5 0733 193 0429 Lot L-2353 PFOSA Partly Carnclba Factor Unknown Uataowa Ukaun IN o m NA NA Uataowa Uataowa Uataowa Unknown Unknown Unknown Unknown Udaowa Udaowa Unknown Unknown Unknown Unknown Uataowa Uataowa Uataowa Uataowa Udaowa Uataowa Uataowa Uataowa PFOSA DUaltoa Factor 1 l 1 I 1 l 1 1 1 1 1 1 1 1 1 1 1 l 1 1 1 1 1 PFOSA 150 0.00 1.39 0.00 KD 109 193 16 1.39 1.63 1.69 164 1.33 1.34 1.61 11.2 11.6 116 7.9( 10.1 17.5 17. 14.7 11 13.1 212 ~ ------- C tM kU lM 4 PFOSA aita L or Roc (47179(0(12 <LOQ<0.009(9UftaL) (47179(043 <LOO<0409(9nitaL) (47179(003 <AjOQ( 000969ugtaL) (47279(044 LOO1Ottrd wctaL) (47179(040 (47179(041 103 110 (47179(012 <LOQ( 0.009(9u(taL) (47179(013 <LOQ10.00919ugtaL) (0179(014 <LOQ( 0.009(9uftaLl (47179(013 <LOQ< 0.009(9usteL) NA ** (0179(011 <LOQ( 0.009(9itaL> (47179(019 <LOQ( 0.009(9W|taL) (0179(020 <LOQ( 0.009(9u|taL) (0179(021 <LOQ<0.009(9ifta L) (0179(022 <LOO( 0.009(9 nataL) (0179(023 00112 (OI79HQ6 00116 (0179(027 00116 (47179(09 <LOQ( 0.009(9wftaL) (0179(029 O.OIOS (0179(032 0.0173 (01791033 0017 (0179(034 00147 (OI79W33 00129 (47179(036 00131 (0179(037 0.0222 Atra* PFOSA aataL <LOO <LOO 107 <LOO <LOO OOIIO 00164 D a h l w i l i y : 12/22*06 DalaVarita By: 0V2GI k^. V1/OI ana Puny baaat'VenikOVWAIl LAO BSD SU. Oar. NA NA 7 NA NA NA NA 644 0000716 214 000334 (617 PFOSAA OM Part Cornetto* Factor 033*2 033(2 033(2 033*2 NA NA 033(2 033(2 033(2 033(2 033(2 0330 0330 0330 0330 0330 0330 0330 0330 0330 0330 0330 0330 0330 0330 0330 330 PFOSAA Partly C w nato Factor Uataowa llwlnwa Uataowa llokmnww NA NA Uataowa Uataowa Uataowa Uataowa Uataowa Uataowa Uataowa Uataowa Uataowa Uataowa Uataowa Uataowa Uataowa Uataowa Uataowa Uataowa Uataowa Uataowa Uataowa PFOSAA DUaltoa Factor 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 23 30 1 1 1 1 30 PFOSAA Cm k. 000 000 000 000 766 til OI0 000 000 0.00 * 333 0.00 000 000 000 437 " ------- Caoctamfca -4 PFOSAA n ita L i r Bat (47179(003 <LOQi 0.02(2 |taL> (47179(043 <LOO I 0 02(2 wtaL I (47179(003 <LOQ 10.02(2 ugtaL1 74 (2 (47179(012 <LOQ<0.032 u*taL> (47179(013 <LOQ10 02(2 i^ /m Li <LOQ10.02(2 tal.) (47179(015 <LOQ10 0212 ta L j a 7179(01* <!OQ I 0.02*2 ta L l (47179(019 <LOQ<0 02(2 ta L i (47179(020 <LOQ<02192 ta L l M7I7MD2I <LOQ( 0092 nftaLj (47119(0221 <UJQ 10092 uataLi 333 0397 194 010 4W 0 ' 5.` 37* 1273 3(7 0316 317 413 AVAKQ24 1 12 Artrai PFUSAA M L BSD SU- Dm . LOO \A LOO 71 11 <LOO SA <LOO S6 SA 0 497 059 0425 07<13M laMMLOQ antaL CanadU LOQ wtaL 07/I7AN laMal LOQa^taL Carrada LOQ a ftaL 0M7/9S lallal LOQ a^taL Carrada U3Q W|teL pro s 24.1 0.0230 244 00230 141 00230 PFOSA 494 0.00494 9.(9 0009(9 9(9 00099 PFUSAA 10.5 000363 524 0.092 524 0.02(2 3MEnvironmental Laboratory Page 163 3M Medical Department Study: T-6316.1 Q ivmk* 6329-12* 1) N-Eatyl Perfluflr>nritiwiilf'->imlnEthanol BRatt PeutaaJFultnelTq SiiilTr Mani R-SquarU Vahe S*f Y-Utencpi. bau ul EmradmUAvalyiL ataau Aatym/Ajutffl bala u( bau Rcdta.tojUAaal)* fcTS-e i A h T S S I Rrty IUOWV Maa)) >4 Set to n i fe Ouu Se* Anacfcmoia Sk AVactineaU 04/20*0 SALKiK 0*21mi. 062*00. WtMW. tA&hOMHOi/SAli OSAtSAu.MC3A0.UkbU.UIUWn<>MSttUAS Sample Data W d E & S I RAT SLR-A C iu f baia Sample f MettudBft M20OU2UB*) Mura B* RfiS042WlSa*Bfc) RTS0t3S.BI. QC RTS042UF34S-230-3 RT3W2OMS-2.30-3 Eatmcttua VL 1 1 1 1 1 Uruuf OOpps ^lO kU LV JM l ,* 11 ----------- S u t ------------ ^ ii VM7 fi rm kftii V w tt lr t SA NA NA NA NA NA Ni" NA HA NA m>S OM Party Caenette o t9 0 9949 Q.9949 NA NA Dvm nw i *WI PTOShrtjf F ila r CJ640 0*6 016 0*6 NA NA PFQS Pttuttu Factor t 1 1 1 1 1 i 0*6 nos Cane. nc/aL 0 00 000 000 000 000 000 It2 107 NA NA ramarne R042IQQQ16 H042100017 RO42I000II RO42I0OW R042I00020 R042I00024 R42I00025 11042100026 NA NA Cascaterallaa uTPFOS t/aL ar Rac <LOQ (0.0042*u*teL) < LOO<0.00421w AbL i < LOQ(0.0042* anted.) < 1.00 (0.0042* U . I < LOQ(0.0042* lM .) LOO (0.0042* anted.) 73 79 NA NA 00271 701 RIMltOOQM 0.0617 147 R042I0033 < LOQ(0.0042* ut/mL) 25.) RO42I0QO36 00219 0090$ 1)6 R042100042 O li* Lw l * u LM r'.-.-aaa rrriT frti ---------- ---------------------nm nc 0*1*VIA NA NA NA HA ----- A----NA 1 NA n Ha BMI0 eaiuR nm a 0994 0*6 140 IBI 7 D0I1300029 A 417 D0I300032 4 D0lS0003J 432 DO300034 33* R0421000SI 1.21 0639 362 3.90 3.92 3.33 0294 lim a PFOS u/aL <LOO <LOO <LOO 76 NA 00490 00727 1.33 302 RAD SU. Dee. MS/MSDRPD NA NA NA 1 NA 71.7 0.0332 77.7 00363 334 0432 31.1 134 VerHM NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA PFOSA OMPutty CvrracUaa Factor 0.9310 0.9310 0.9310 09310 0.9310 0.9310 NA NA NA NA 0.9310 0.9310 0.9310 0.9310 09310 09310 0.9310 0.9310 09310 09310 09310 09310 0.9310 0.9310 0.9310 09310 09310 0.9310 0.9310 PFOSA P utty Carnetta radar Untava Untava Untava Untava Catana Untava NA NA NA Untava Untava Urtava Untava Untava Urtava Urtava Urtava Untava Untava Urtava Untava Urtava Urtava Uvtun Lutava Urtava Analytical Report: FACT-TOX-003 LRN-U2104 OSA M utua Factor 1 1 1 1 1 1 1 1 1 1 PFOSA Cooc. -tf-L fH tia i- CaacaUratton al PFOSA t/teL nettin e 020 R042100I7 < LOO<0.00316 unteL> PFOSA an/teL LOO RSD SU.be. MVMSD BPU SA ODO RO42I0U019 LOO(000316 untaLl LOQ NA 0.00 RO421OO0M <LOO(0 00316 Intel 1 LOO NA 222 R042100B26 90 *4 1? NA NA NA NA NA 0.00 RM2I0002I < LOQ(0*0316 upteL) 0.00 *042100031 <LOQ (000316 miteL) Q0Q LOQ(8.00516ufAaL) 0.00 RO42I0DOM < LOO(0.00316uated.1 NA 0.00 <LOQ1000316a|ted-t Q.OQ LOQ(0.00316te tetl 1 000 RD42100042 <LOO(0.00316latel 1 3.33 1 434 R0421OOO44 LOQ(0.00316nteU 1 1 104 R042I00049 1 4.09 0*109 1 1 1 1 1 NA 24.7 Due fcoMraVBr 0*12*0.06*1*0. OU22JOO. Ot/l00. OWOMOLAOMMHQVOMI Due V * * By 03/20*1 tgUOSIUWI LAC. 5/22/01 u d Piny EoKfcd/Vanfied: 03/29*1 LAO 04/21*0 04/24*0 (3*0 0t/2**0 tatted LOQ ^ /a L Cairelad LOQ ap/mL tatttal LOQ |/b L Careada* LOQ paL lattei LOQ apimL Carrete* LOQ a^teL lattei LOQa/aL Careaste* LOQ < |/aL rru s 4 9) 000421 NA NA 493 00042* 493 0.0042* PFUSA 491 0.00316 NA NA NA NA 4.91 000316 PPOfiAA 49.1 0*303 26.7 0.023) NA NA NA NA tifo si 24.7 0*223 NA NA 4.92 0*04 NA NA M3S6 49.3 0*494 97.6 0.097? 24* 0*24* 4.93 0*0494 fFOSCA 9.71 0*0971 NA NA 24* 0*24* NA NA PfOSKA P irta ro t^ ugooyl ntylaaMne 3M Environmental Laboratory Page 164 3M Medical Department Study: T-6316.1 Covane# 6329*228 SseJy Wu4nu NuBUwTca M m m u i Umu lo*-- Sotl*v/V<n TUXUjI 104Wa* Dwury Cariogca.dy StodywfcNansaRii^ir<W.I% N-ElfaylPertohroci T-6)J6ltlK)S.OHj Km Sciata ETS *-4 1* ETS-t-5 l R*y IOOWV M w lya J-4 SeelMUM> Ibcriffa! So Aaa*mrnu Dao o LurMat^AMJya D as o AaiJyaa/AtoJya ac uau Kcde.wa'Aeelya Sample D u C ru f D .h M cM Bt Mj b u BR QC Saatpkf 04209H2OM 3 KRS0t2D0ScnB4 3 RTS04209Stri BB 3 RTSO4209MS-25O-) 6T5O4209MS-2503 C ta pt Cmvul 0UH * C n a ft L n U it 1opp cnwvw o biv w m C9220VM C922XM C92MI* CVDlrf OilhTlAO d i o * C92267/M CV22J4-M c m 7/M CyZ2M<M C9M06*= CV24U cn tiw cnaU f 0924X4= See AM> hmrnl t U4/2U90 SALlKlK 0*21/00. 04/2-W0.09/1M, US/2W IAS/MMHHOJ/SAM 0VUVUO.IM/1VOO. 04VUajU.{WOOMMH/lAS Safrugai* Vrrifltd SA NA SA SA SA SA Sa SA NA NA SA NA NA NA PFOSAA OU Party Cornelina 09TM ovtoe 09to0 NA NA ootoo 0 9760 09760 09760 09760 0 97a0 09760 0 9760 09760 09760 09760 0.9760 09760 09760 09760 09760 09760 09760 rro&AA Partly Carracttaa lU asM Datato-- PFOSAA UOaiiua Farter 1 l t Dataos NA NA NA Uta-- Datas-- Uatea-- IU M W Uatao-- U ta n Datas-- Utaao-- IU h W Uta-- Dates-- Uatao-- Uta-- I 1 1 1 1 1 t l 1 1 1 1 l 1 U tos-- Uatao-- Wn n 1.latan-- PFOSAA Cuate. 0.00 000 000 000 0.00 207 244 NA NA 0.00 000 0.00 000 0.00 0.00 0.00 0.00 917 277 796 704 106 169 201 172 426 166 Fcoaai* ROI2400025 RM2400026 R042400027 6042400026 6042400029 R0424000)3 604210002$ 6042100026 NA NA 6D4240Q036 6042400017 6042400041 R042400042 6042400013 R042400044 R042400043 R042400049 R042400050 6042400031 6042100043 6041100044 R042I00044 R042I00049 R042I00050 R042100M R042I00032 6042100036 6042100057 R042100054 C--castrati-- o in tlS A A aefeiL ar % Rae < LOQ(0.0213 agtaL) < LOO 10.0233actaL) < LOQ (0.0233U|taL> LOO (0.9233--M .) LOQ(0.0253ugtaL) < LOO(0.0253 uaAaLI 13* 99* NA NA < LOQ (0.0233 ugtaU < LOQ(00233 agtaL) < LOQ(0.0233agtaL) < LOQ(0.023) agtaL) < LOO(0.0233attaL) < LOQ(0.0253agtaL) < LOQ(0.0233 itytaL) <LOQ(0.0233 agtaLI < LOQ(0.0253 agtaL) <tOf) 10.0253aataL) 0.0946 9.27 O.elO 0.311 0191 0.194 0206 0.340 0934 0.191 Maaa PFOSAA aa/aiL LOO LOO <LOQ 91* NA UTO <LOO 0297 0213 RSD SI4. Da*. MS/MSD 6PD NA NA NA 17 NA NA NA NA NA 63.3 0.194 40.3 0.1to Varillad NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Analytical Report: FACT-TOX-003 LRN-U2104 l EtFOSEQU Parity Cariarti-- Pactar 0.9770 09770 0.9770 9770 09770 99770 NA NA NA NA 0.9770 09770 09770 09770 09770 0.9770 0.9770 09770 09770 09770 09770 09770 09770 09770 09770 EtTOSEParity Carnett-- Factor w o a ElFOSE PMvttoa Case. Factor ptfmL C--aatarati-- ut ElFOSE aatotL ar Rae 04690 1 9.00 R042100D17 LOO (00223aataLl 04699 04490 044 NA NA NA NA l OOO *442100014 < LOO(0.0225axtaLV 1 000 R04210024 < LOO(00225 i t a L l 1 3.9 6442100026 16* * 1 90.2 1X41500022 M * tal* 04490 04690 04690 04690 04690 04690 04690 04690 04690 1 0.00 R442I00026 < LOQ(0.0225agtaLl 1 000 6042100032 < LOQ(0.0225agtaL) 1 0.00 6042100033 < LOQ(021225itytaLi 1 0.00 604210003 < LOO(0 0223arttoLi 1 040 6042100036 < LOQ(0 0223ftaLl 040 6042100040 < LOQ(0.0223agtaL) 040 6042)00041 <LOQ(40223 U . ) 0.00 6042100042 LOO(40223 aataLl 04690 04690 0J690 04690 1 eoo 604210006 LOQ(0.02239 M .I 0.00 6042100041 < LOQ(0.0223tataL) 040 6042100049 < LOQ(0.0223 agtoLl 900 6042100030 <1/70(0 0275-- J > 04690 04690 04690 04690 1 0.00 6042100032 LOQ(0.0223aftaL) 1 0.00 6042100016 < LOQ(40223 ttoL| 1 400 60(2100037 < LOQ(44223 itytaLl 1 400 6042100036 < LOO(0.0223wtaLl ElFOSE LOO LOO LOO 17 34 LOO LOO LOO LOO RSD N i D*r. MS/MSO RPD SA NA NA 13 13 SA NA NA SA SA r -- falrnifBji 0VI2O0.OMIAH. (4/Z2AI0. W/ Dde Yotfkd/Ey 01IM11 IjA to n W LAC. P irtj E a a td V a M oyiM Il LAC Ot^ino 04/24X0 OHM 06126X0 laHWLOUactaL Carracto LOQ agtaL Inaia LOQ ag/lsL C .n io a LOQ aaiL lama LOQatta*. C e n a li LOQ aglatL Inaia LOQ og/aeL Cerracted LOQ ag/aaL 3M Environmental Laboratory Page 165 3M Medical Department Study: T-6316.1 TOXUJ). 104W 1AVW.\EliA>Sfc\)W'> Am Senau ETS 1-4 l ETS-*-* I K*) I006W M**tya*J4 SacB u g lu feeRA * Rtiye<.!%)N EUtyJPi Cwviftcc 6329*228 Analytical Report: FACT-TOX-003 LRN-U2104 Dm M d k lM Mam B * QC 00 H Craap Low Du 1W * 04/2UM) SAL4UK 04/214JO.OV24). 01/13*0. Ofe2MO lAS/MMNHOJ/SAN ovvMn.otavw, o*/i 6a.oi/3oitommh/ias M20BK2OM-3 RBS42UOSaB A I RTSM20BSraBk 3 RT5043B.MS230-3 RTSO1200-MS-230-3 C4214VM (92I4*M C0220SM CV2231/M CO-HI/F (NI'**- O ililt# 012267*1 C92774/M CV2217/M CV221LM 00106 cyM ii* C924II<F C92422* 0(213 Sarrugalr VartfU* NA nA NA NA NA NA NA NA NA NA N'A NA NA NA NA NA NA NA MS34UM Par; i MSMParRj i M3J4 Cuenco* CutewSB* (MMta Futa 0.99 099 Uaknou* lU m ri 1 1 1 1 099 U mw r- 1 1 NA NA 1 1 NA NA 1 l 099 0.99 099 0.99 UBm M W m Utonow* Uaknou* t 1 1 1 l 099 U99 0.99 099 lU w n Itakaowu Uakaowa Uakaaneta 1 1 1 l 1 099 0.99 099 099 099 099 099 0.99 099 Utonovn Utoaoata OrtacM lU m i Uaknowa U nt UltBOMI (Mm Uakaowta 1 1 1 1 1 1 l 1 1 1 M54 u /a L 0.67 034 0 0.00 OOO OOO 261 27S NA NA 141 OOO OOO OOO OOO 163 OOO OOO 000 16 136 161 106 909 207 202 1*3 311 109 F IO M Cuacadrakfen 4M* i/M a r S Km RM2B0OOIJ <LOQ(000494 ag/nd.) RM2S00020 <LOQ1000194unfeL) Roarooou <LDQ(0.00494ug/aL) K0*2900021 LOOI000191 u/mLl A0C9000I7 LOQ(000191 M .) R082B00Q22 LOO(000191vataL) R0424OGO34 10 R042400033 11 NA NA NA Mcaooss <LQQ(.0M94|NaL) ROC2900ID6 LOQ(0.00!1u*/toL) R0S2B00Q27 LOQ<0.00194Uf/mL) RO*2B0002S LOQ(000194umL) 110*2*00029 LO 1000191uaftnLl R0C2KOO32 LOQ(000191 HptaL) RINOMILI) LOQ(0.00191 ifetoL) R0*2*00034 LOQ(0.00194 UtaL) R0B290O033 LOQ(000194ugteL) R0*2*0W36 LOO10.00194untai.) R042400032 00917 R042400033 0.136 R04240QQ51 0162 R042900031 0.106 R042400059 00990 R04240006 0301 R042400061 0301 R042400063 0113 R042400066 RM2400067 0.311 0.109 Mm MSS* LOO LOO LOO 10 NA LOO LOQ 0120 0202 USD St*. D. MS/MSD RPD NA NA NA 2* NA NA NA NA NA 2*2 002 t 331 00723 Sarrafato VrtA<4 NA NA NA NA NA NA NA na NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA rrosEA o u Pw*; C witcDN Fuete! 0.9930 0.9930 099 0.99 0.99 0.49 NA NA Na 099 0.99 0.99 0.99 0.99 099 099 0.9430 0.99 099 0.99 0.99 0.99 0.99 0.94 0.99 0.99 0.99 0.99 0.49 F r S A tu tj Camella* Factor IU m m Utoaaa* Utoaoan Utotaan* Utoavaa NA NA NA NA IW wM Uakaeen lirtiiim i IU m m U m m Uakaewa Uakaewa Uakaewa IM ** IU m h UtaMNM IU m m Uataeaa Utonoen Ite M Urtata W arn Ih tam PFDSCA DfeUlua Factor 1 1 1 1 1 1 1 1 1 t vtosea Cuoc. S*Sh 000 000 000 119 13) Firm an Canatorattaa utPFOSEA untaL ar % Km Moa r fosea RM2100017 LOO10.00971m AbL) LOO RM2IOOOI9 IOO t 0.0097* citai.) LOO R042100024 LOO( 000971 Mito! 1 LOO RM2100Q26 41* 42 DORI300022 34 2*T 41 1 (LOO R042100Q2* <LOQ ( 04097* tfetaL) 1 000 RM2100032 < LOQ ( 0.00971 1 OOO RO42100O33 < LOQ( 0.00971^ M . ) 0.00 RM2I00034 <LOO ( 0.00971uataL) U>0 Ii Ii i RO42KK036 < LOQ( 0 0 7ag/toU R042100040 LOQ( 0.00971gtaL> RM21O0O41 < LOQ ( 0 00971ifetaL) RO42I00M2 <LOO ( 0.00971a k l 1 LOO OOO R0421000M LOQ( O.OQ971BgtaL) 1 OOO R0421OOOU LOQ( 0 0M71 ag/ad-l 0.00 R042100049 LOQ( 0.00971 W .) 000 R042100Q30 LODI *0097* --Imi 1 LOQ iilil 1 RM2100Q32 <LOQ( 0.0097* igitad.) 1 R042HD036 LOQ<0.00971ptaOJ 1 RO42I00QS7 <LOQ( 0.00971a*/toL) 1 R042I000M LOO( 0.00971Mitol.) lOO R5D SU. Da. MS/MSD RPD NA NA Na 34 22 NA NA NA NA NA SA NA NA o viio j.ovovoo. rnmjoo. o u 01/2001 oviuoi lac. V 03/2M>l LAD filPOSE Nun Ai M3S6-CF17S02N((KCHC00> PfOSEAa (M IM I IMAM) OfelVOO 0U2KO laWai LUQufAaL Carretto* LOQ ag/aeC lattai LOQagML C atttcu* LOQ ag/L Initial LOQ^ftaL CmtmM LOQ USaL InWui LOQn |W l C a n t i t i LOQ D|Ml. 3M Environmental Laboratory Page 166 3M Medical Department Study: T-6316.1 Covane# 029-228 s**- Pruda! NuabafT^: U a w a So(lware/VeaMt F il m e R-Spwcd Vak. Y lM T .ff DateI EoaactoeWAtalyat Dates<Analyaa/Analy: Dateel Dai Brtaataa/Antly TUXIX13.104Week Dvuiy CuojpBkty Seto) To)|6<fclK>3fcOt|| BeaSena KTS-4-4 I a ETS I S | D o ty OTOTyy MataLyaa ! 4 Se* AkactootoMa S n A tu la c a tt See AIIu Bu b 06/29*0 SAL/KJK 07*M *. 07/19/00. 07/24*0. 06/24*0 MMII/5AH 07*7*0.07/25*0 07/27*0.06/24*0 MM1I/HOJ Range<96 l>NE*ayl Potatoi WEEK l>5 WAT SERA_____________________________________________ k . m Cnr Staapfcf E<rc*. Sw ni i r o s o u pmy Dot* Yto. Venn* 4'wrreclka OtVOlUOB&S Mem B RTSOtTVOSenBk' RTS0b2V0-Saa BR G ra fi OOppa RTSOe29l3MSD-2SOet>-5 C92I97/M 1 1 1 t 1 1 1 1 1 NA SA NA NA NA NA . NA NA NA NA NA NA NA 07W 09949 0*047 09949 0-AM7 0 7047 NA NA NA 07747 07747 07747 1 NA 07747 ____________________ __________________________________________________________________ ML-iSTW m e Portly (om cllui Fuetea pros Uta(tua Factor pros Cene. --1------- Ci t i niraton 4PFS itaL or Rae Mean BSD PFOS Sld. Dea. t/aL MS/MSP RPD `sw rT PFOGAOM Paito? Cornette Factor 04M0 04940 1 0 00 R07240000? <LCXJ(0.00421UfAnL) 1 0 33 R72400021 <LO0(0.00428untali LOO NA NA NA 0.9S10 0.9510 04640 04640 04640 04640 NA NA NA NA 1 0 39 B072400004 LOQ(0.00424ptaL) 1 047 R0724000Z2 <L00(000424lu/tal 1 LOO NA 1 3.23 R07M00009 LOQ(0.00424 ptaL, 1 395 R072400Q23 LOO(0.00424 ueAnl 1 LOO NA 1 340 0070600024 i 343 DQTO600023 137 * 136 1 1 343 ROT2400CO4 l 365 R072400025 146 2nd* 141 NA NA NA NA NA NA NA NA 0.9510 09510 0.9510 0.7310 NA NA NA NA 04640 04640 04640 1 4SI R072400CC7 <L0Q(0.00424ftaL) 1 301 R072400030 LOQ(000424ftaLl 1 3.3 R072400QJ1 <L0Q(000424 uptaL) NA 0.7510 NA 0.7310 NA 0.9510 Oloto NA 01640 1 27.4 11072400033 00242 NA 0.9510 04640 1 NA 04640 1 241 R07240003 <1j0Q(0.00424iif/nL) 07747 04640 NA 07747 04640 NA R072400041 412 00200 132 0.0111 NA NA NA NA 09510 0.9510 0.9510 0.9510 0.9510 Drap f 1Oppa C9226IM C9226WM C92314M 0 7747 04640 04640 1 LOQ(0.00428ugtaL) NA 04640 1 NA NA 04640 NA 00232 NA 00262 00241 0.0256 00145 NA l NA 09747 0.1640 644 D070600056 0594 NA 1 NA 07747 04640 10 110 R0724006I 0.933 NA NA NA 0 7747 04640 10 100 R072400063 006 NA 475 NA 07747 NA 07*47 04640 770 D070600064 00460 NA NA NA NA 1 NA 0 7747 04640 10 154 R071400065 1.33 7.4 NA NA 1 10 NA 0.9510 0.9510 0.9510 0.9510 0.9510 0.9510 0.9510 0.9510 0.9510 0.9310 07510 07510 NA NA NA NA NA NA NA et w tt 07/34) to0624*0 a ihc l a c o m ii M O W M O DEaemtoBy 07/13MO.OSa5W.('2lO.OM>]AN LAC DauVanfiatoBy: 0V2OOI k^. V22MI auto P m i LaeretoVwibek 0V29AI LAO 07747 0 7747 07747 07747 0 7747 04640 04640 10 01640 04640 10 10 04640 w d NA * Not AfplktaMe LOQ Laul 4QuNMWiga 137 R072400070 623 17 104 WTMoaan 124 R07240000 231 007 1.04 <01 124 1.76 215 NA NA NA NA NA NA NA 139.1 161 --------2J-------- NA NA PFOSAPa WTW|* - PTfttmTnmrtia*^Jfii--niiilni eia E U n s Nitro* Rafe N-Etayl PtotatorocnraidfoBanide etayl afccM M5.CJfl7S02N((H)CH2COO> ffO S A FirflHnockai luMooylkytamtoa 0.9510 0.9510 0.9510 0.9510 0.9510 0.9510 0.9510 0.7510 PFOSA PtoiRj PFOSA Cornetta DHtoltaa Fodor Unknown Uakno l 1 Unknown Unknown . Unknown Unknown 1 1 1 t NA 1 NA 1 NA NA NA NA Unknown Unknown 1 1 Unknown Unknown 1 1 lUnown 1 Unknown 1 Itakaown Unknown Unknown Unknown 1 Unknown Unknown Unknown Unknown 1 1 Unknown Unlaown Unknown Unknown 1 1 1 Unlaown Unknown Unknown Unknown Uttnowm Unknown Unknown 1 1 1 1 Unknown I Unknown 1 Unknown Unknown Unknown Unknown 1 Unknown 1 Unknown Unknown 1 1 Unknown 1 Unknown Unknown Unknown 1 1 1 Utaaown ----- U ----- PFOSA Cone oitaL 0.00 0.00 0.00 0.00 000 000 307 310 NA NA 0.00 0.00 000 0.00 0.00 0.00 0.00 0-00 0.00 0.00 0.00 ooo 0.00 0.00 0.00 000 0.00 0.00 0.00 0.00 5.51 342 144 9.7 3.5 606 205 05 3.52 141 473 11.3 13 5.3 7.13 5.49 7.26 7.2 9.23 5.37 Filettanti Ceeeraton f PFOSA attL or * Bee DoiMOoan <LDQ(0.0(2516ng/aL) D07060002I LOO(000516oe/tal 1 D070600004 <LOQ<<1<M516wgtaL) D070600022 <LOO<a<M5l6total 1 D0706000Q9 LOQ(000516 o*wl ) 0070600023 LOO(0.00516total.1 D070600024 IM D07060Q025 1264 NA NA NA NA D07060002B LOQ(0.0QSI6ptaL) 007060002$ LOQ<000516-iftal ) D070600030 LOQ(000316a^nL) DO70600031 LOQ(0.00516uptaL) D070600032 LOQ(000516^ W .) D070600D3 <LOQ(0.00514ftaL) D070600Q36 <LOQ<<LOQSI6ptaL) D070600037 <L0Q<0<M5I6 uptaL) D070600034 <LOQ<000516ptaL) D070600039 <100(000516 uwtal > D07Q600042 LOQ(O.OQ516uptaL) D070600043 LOQ(000516*pM .) D070600044 LOQ(0.00516uptaL) DO7W00CH5 LOQ(000516ptaL) D07060046 LOQ<0.00516gtaL) D070600049 LOQ(000516ptaL) 0070600050 <LOQ<<L<M5KptaL.) D07060QQS1 LOQ<000516tM .) 0070600052 LOQ(000316"g*-1 i 007060003 LOO<0.00516wwwl > 0070600056 000547 D070600Q57 LOQ(000516ptaL) 0070600054 00152 D07D6000S9 00103 D070600060 LOQ(000516iltaL| 0070600063 000637 D07060Q064 LOQ(000516p/taLl 0070600065 OOOP46 0070600066 LOQ(000516p/taLr 0070600067 LOO1000516toal.l D07060Q07D 000911 D07060007I 00119 0070600072 0.00042 D070600073 000541 D070600074 000750 D070600077 000577 0070600074 000763 0070600079 000767 0070600000 000971 DQ7060004I 000565 Mean PFOSA totaL LOO LOO LOO 125 NA LOO LOO 000720 000797 Analytical Report: FACT-TOX-003 L R N -U 2104 TOX003,104Watt Dietary Caauugcaa.iy Setto) wak.Voioa K a f >4 19 iN T-6314(ElFOS-OH) U S o ia ET5-4-4 1AETS-4-SI DaeyOTDM MeeaLim 1 4 | f--f - T ' -anal nm 06/2S900 SAL/KIK 07AWM07/19*0. 07/24). 06/24*0 MMH/SaH 07/07/00.07/25/00.07/27/00.0L2W MMHItOJ BSD Std.De*. MS/MSDBPD NA "3 T NA 1 NA NA NA NA NA NA NA NA NA NA NA NA " ' ' NA NA NA 434 0.00330 NA NA NA NA 000194 NA PFOSAOM Partly Cornetto 097N PFOSA P n rj Cmema PfOSAA PFOSAA FM--ta* Dtauto Cnae. In ln !*' 09760 09760 09760 09760 097e0 09760 0.9760 09760 09760 1eknnM --" j . i t 09760 .... J 0.9760 09760 i __ ^ 09760 Unknown --s * NA 1 1*7 1M 1'N -- I^ 3 ' M0 toi lata -.. U4 *75 4M in 161 " a^e il. wv. 1 231 DOTDeOOOil ITOMI) 07/1900 07/2400 04/2400 latte! LOQ apteL Carrtcted LOQ pAnL lattei LOQ ap/aL Cwrrected LOQ plaL lattai LOQ aptad. Cerratted LOQ ap/aL U ttta LOQ p/aL Ceerreoled LOQ f/aL PFOS 49 3 00421 4 93 0UD42I 493 000424 976 O.OU444 PFOSA 491 000316 NA NA NA NA NA NA PFOSAA 491 00503 4.91 O.OU.Vkl 4.91 000503 NA NA UFOSE 492 000441 NA NA NA NA NA NA MS54 493 000494 244 00241 241 00241 741 00244 PFOSSA 490 000493 NA NA NA NA NA NA 3M Environmental Laboratory Page 167 3M Medical Department Study: T-6316.1 ______ Covaner l 6329-228 Analytical Report: FACT-TOX-003 LRN-U2104 Saely PludULl NuobeflTca Saturata): " *>**>( Rail MctxxMtcvH. Itw in a l Sullwarc/VcntoB: Hknune HStfuaied Valu. Y'lgUUf*: Date*of EuracIMVAratyil: Date*ul Anaiytit/AraJya. Dale DateftcdiKUOb'AraiyU; Sampt DaU W EEK IBS RAT SERA__________________ Crtev Sdtopfc* Dm i ______________________________________________________taSP0l3 Ceacaalraltua Mean BSD Samcata EtfOSBOUFwlQ efPFOSAA rrosAA Sid. Da*. Correction attaL ar * Bac a*/*L MS/MSD BID Factor Matted BA 06290-H2DBB-5 <LOQ(0.00303up/uL) NA 09770 062VOH2OM-6 <LOO(000503aa/uL> <LOO NA NA 09770 Melm B* RBS06Su*Bfc-5 <LOQ(0.00303up/uL) NA 0.9770 RBS0629OS<n Bft-6 <LOO(0.00503ttattd.) NA NA 09770 Mam (ML RTS0629O-SaraBfc-3 <LOQ<0.00503apAnL) NA 09770 RTSU69SenBfc4 <LOQ(0.00303taa/nLl <LOO NA NA 09770 QC RTS0629OMS-2Mppb-5 131* NA NA RTS0629OMSD-2W6-5 140* 139* 2 * NA NA RTS6290-MS-250ppb-5 NA NA NA RTS06290-MSD-250 peb-5 NA NA NA NA NA Graapl Cc**u( C92I97M <LOQ(000503 upttaL) NA 0.9770 0/2203*4 <LOQ(0.00303 U fte U NA 0.9770 0 /2201*4 <LOQ(000503 aptaL) NA 09770 C92209M LOQ(000503 NptaL) NA 09770 c y n iiM L0Q (000503 aptaL) NA 09770 C92Z22M <LOQ(000503 aptaL) NA 09770 CV2231M <LOQ(000503 ap M .) NA 09770 0/2233*4 <LOQ<0.00503uptaL) NA 09770 CtfZ23WM L0Q(000503 aptoL) NA NA 0.9770 C92232M <LOO(000503wa/mLl <LOO NA NA 09770 C92533# <LOQ(000503aptari.) NA 0.9770 0/2334= <LOQ(000503 uptaL) NA 09770 C9Z34KF <LOQ(000503 aptaL) NA 09770 CV2354# <L0Q(000503 ap/toL) NA 09770 C92356T L0Q(000503 aptaL) NA 0.9770 0/23*04= <LOQ(000503 aptaL) NA 09770 003624= <LOQ(000503apAnL) NA 09770 003694= <LOQ<<10O5O3aptoL) NA 09770 0923744= <LOQ<<UP0503aptaL) NA NA 09710 003434= LOO<0.00503aiAnLl <LOO NA NA 0.9770 Greap 9 Law Date C9226I/M 0026644 0253 0124 NA 0.9770 NA 0.9770 C92270M C9227M4 09224144 0656 0371 0201 NA 0.9770 NA 0.9770 NA 0.9770 C9Z24344 02*9 NA 09770 C923MM 0.131 NA 09770 0031144 0231 NA 09770 CV23I444 C9232I44 C9MIQF 0924164= C924194= 0924234= 0.16 63.4 NA 0.135 0252 0160 NA 0165 NA 0.517 NA 0142 NA 0261 NA 09710 0.9770 0.9770 09770 0.9770 09770 0924304= 0117 NA 09770 0924314= 004394= 00443# 004314= 004324= 0.179 NA 0.123 NA 0200 NA 0569 602 NA 0236 0259 0156 NA 0.9770 0.9770 0.9770 0.9770 0.9770 * Hik revtftwici tiaa/rtnada>lb 07/24/00ad 0I/24AI0aai Dale tM m i/hy 07/13A0.0VI5O0.0l/2l/00,0a) Due VtnfoU&y 03/1001k^. S/22BIBah (yEe*etWmfied 03/2901 IMO EtFOSEFwtt; Cerraedee Factor 04090 04090 04090 04090 04090 orno NA NA NA NA 04090 04090 04490 04090 04490 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 KlFOS DtteUoa Factor 1 1 1 1 1 1 1 1 NA NA 1 1 1 1 1 1 1 1 1 ! 1 1 1 1 1 1 1 1 1 1 1 1 1 ElFOS Cone. mtfmL 0.40 M2 0.00 1.54 0.37 0.00 141 151 NA NA 1.21 0.00 0.95 0.56 0.15 017 041 0.3 012 0.97 0.07 077 0.34 036 005 057 006 0.27 0.25 0.24 0.92 054 0.79 257 015 1.42 219 1.37 OOi 006 072 0.63 0.61 0.56 0.96 1.03 1.00 026 OOO 020 fla u to Cetacea*ratto# aTEtFOS aaAaL *r * Bac 0070*00007 <L0Q ( 000441a^UL) D070600Q2I LOOI 0.00441aa/BiL) txnoooooM <LOQ( 000641ap/toL) D07D600022 LOCHO00441uc/ttLl D070600009 <LOQ( 000441* /u L | D070600023 <LOO< 0.00441aatotLl 0070*00024 0 D070600023 61* NA NA NA NA n r w i n a <LOQ( 0.00*41* U L ) 0070600029 <LDQ( 0.00441afUL) D07M00Q3Q <LOQ( 0.00*41* U L ) D07M0003I <LOQ( 0.00441 apAnL) DO7Q60GOJ2 LOQ( 0.00*41apUL) 0070600035 <LOQ( 0.00441qptaL) DV70600036 <LOQ( 0.00441apUL) D070600Q37 <LOQ( 0.00441ap/uL) D070600030 <LOQ( 00044apM.) D07060QQ39 <LOO100044u U L l D070400M2 <LOQ( 000*41 ^ftoL) D070600043 LOQ<040441 qpUL) D070600044 <LOQ<0.00441i|W .) 0070*00045 <LOQ( 0.00441* M - ) D070400046 <LOQ( 0.00441qprteL) DU70600049 <LOQ( 0.00441apM.) D070600050 <LOQ( 0.0044^ /ttL ) D07060005I <LOQ( 0.00*41apAaL) D070600B52 <LOQ( 000441apUL) 0070(00053 <LOO<000*4 tuiUL) D01D600Q56 <LOQ( 0.00*41^ M . | 0070600057 <LOQ< 0.00441ap/ui-) 0070*00051 <LOQ< 0.00*41ittL) D070600059 <LOQ( 0.00441 D070600060 <LOQ( 0.00441^ M L ) D070000063 <LOQ( 0.00*41t^ U L l D070600064 <LOQ( 0.00*4^ U L ) 1X170(00065 <LOQ( 0-00*4ataL) 0070*000*6 <LOQ( 000441 aptaL) D070600067 <LOO(OQ04U totoLP D070600070 <LOQ( 0.00*41^4nL) D07Q60007) <LOQ(000*41 ML) 0070*00072 LOQ( 000*4 dfUL) 0070*00073 <LOQ( 000*41 ^ /u L ) 0070600074 <LOQ<0.00441lyteL) D070600077 <LOQ<0.00*41i^toL) 0070*00071 <LOQ( 00044 ^ M .) 0070600079 <LOQ( 0.00*41a#AaL) 0070*00 <LOQ( 0.00*41V*> -) 0070*000(1 <LOOI 0.00*41ue/uLl Maaa EUDS ai/UL <LOO <LOO <200 60* NA LOO LOO LOO LOO BSD SU. Oca. MS/74SO BPt> NA NA NA 2* NA NA NA NA NA NA NA NA NA 070600 07/1900 07/2400 UV24O0 laMai LOQag/taL Corrected LOQ a^taL laMai LOQa^teL Carractad LOQ ( a L lottai LOQa^uL Carracted LOQ a|/a>L luttai LOQBgOtL Cerracted LOQ #f/uL 3MEnvironmental Laboratory Page 168 3M Medical Department Study: T-6316.1 TUX003. IM Wed l/tewy r a t | a t l ) Stody 16 Ni T-63l6|tlK)Sfc-UHi Km Sm b ETS-M I A KTS-S-5 I Davty OTOTyy MeaiLyu } Covane* 6329-22* Analytical Report: FACT-TOX-003 LRN-U2104 le* ( Atolyra/Aaalyn Dhc of Dau Radala**Analta 00/2X> SAL/KJK 07AMn.U7/|v0.U7/2Q0.MO4<UU MMli/SAH 07Ai7M/.07/2V.07/J7<l IM/2HAU (/(Minio) S*m |4( DU W E E K 1B$ R A T S E R A L*NBU0U7 SO Owt Meta B * M iU Stara B t QC Groa# 1 C ord OOfpa Sample* 0629OK3OBE 5 M.29*K2r>Bfc RBS0629*ScnBfc3 KBS0629* So* Bk RTS06290-S*B*-5 RrS0b29frSaiB* 6 RTSO62y0-MS-250fv6-3 KTVA29*MSD-25(iMto3 RTSO629&34S-2S0ppt-5 RTS0629 USD-250pet-5 CV2IV7/M 0/2203*4 0/22U6M SuitnfMe 3 rriflcM NA SA NA NA NA NA NA NA NA NA NA NA NA M M OM Partly Crrecito Factor ovsw om o w UWW Q*m OVAN NA NA NA NA ow n 0WM 09999 SI556 Partly ( uerrettoa 1Fatai 'a a n m 1LdiMiw 'liaiai 1 . 1'iaa..iir NA NA NA NA Untno l'a k a /n 0/22UWM NA oawN UaLau La* Owe '< > !* O/UIBM OI22I2/M C922MM cm > iM CVUiWU C92252M CV233S# CV2JM/F CV234I# C923S4/F e1 C92360F CYD62# C9236WF C92374# 0 /13*1* C9Z26I/M CV2266*i C9277UM CVZ27LM NA own Unkao NA *9999 D 4l NA o.*m Intatto NA OWN Intano NA r m Ontano NA own Unk'anwB NA ovm Untnown NA OWN Untano NA 0.9W Untano NA own Untano NA ovw UWama NA OMN Untano* NA OWN Untano NA OWN Untano NA 097*7 LlakaawB NA OWN U otn u NA 09SM UakBuwa NA 097*9 Untano NA 097*9 Untano NA oy7*y Untano C9231*l C9Z33M rviwwM C923IIM C92311/M C9232I/M CV241WF C924I6* C924IWF CV2423* CV243<3f cyzoi/F C9243Wf 072443# 0/2451* CY2452/F NA 097*9 Untat NA 097*9 Untano NA 091*9 lattano NA 0 97*9 UntanoS NA 0 97*7 UtWno.tt NA 0 97*9 1lllami NA 097*9 Untano NA 097*7 Untano NA 097*9 UMHn NA 097*9 Untano NA 0.97*9 Untano NA 0.97*9 Untano NA 097*9 Untat NA 097*9 Untat NA 097*9 Untano NA 097*9 l/nlavai n 97/2M ^ <m 600as. 07/13430.OWISOOOW2100.0** 03/2001 jb. 3/Z2/OI an* 0V2M>I LAC/ M4 DUaOua Factor 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11 SI556 na/mL 000 000 000 000 000 0.00 431 412 3*1 37* 000 0.00 000 000 000 000 000 0.00 000003.0000100000 0o00000..0000o000000o00 442 53.1 140 9216.41 129 13 424 776 257 124 97.4 914 106 U! 43 1 592 56.2 130 691 FHaaaaM A0C4000O5 A0*24000l9 A0V240Q6 A0C4UUO2O AOB2400007 AQC240002I A071900024 A<ni90002S AOC400022 A0*2400023 AOB2400006 A082400027 A0K24OO2I AOD4UU02V A0U4UO030 AOC24QOOJ3 AOC400034 40*2400033 A0C240QO36 A0C2400O37 AC2400040 AOK240004I AOC2400M2 A0S2400O43 A0S240QO44 A0*2400047 AQS240Q04* AOK2400049 AOK24O05O A0*240005t A07I9Q0076 A07190Q077 AOT190007I A071900079 A0S2400034 A<n17000*3 A0*2400055 A0717000*3 A0717000*6 A0719000*7 A071900070 A071700091 *071900092 A071900093 A071700094 A07I7QOOV7 A07170009* A07I9000W A071700100 A07I700IOI Cuecaaratta ai M ac/nL M 1 Bac <LOQ(0.0241|/L) LOO (0.0241uaOaLl LOQ (0.0241a|/aL) IOO (00241Ani.1 LOQ(0024 a*teU LOO (0.0241a^aL) 114* 16 134* 15 LOQ<0.0241ifte Ll LOQ(002417|AbL) LOQ(00241^ /a L ) LOQ(00241(*M .) LOQ(00241a|/BL) LOQ(00241a*M.) LOQ (00241aidO-l LOQ(00241|L) LOQ(002 M .) LOO<0.0241M/B>L) <LOQ(O02AL) LOQ<002414/mL) LOQ<00241^ te L ) LOQ(002 a*taL) LOQ(002 *taL) LOQ(002 d L I LOQ(002 pteLl LOQ(002 >|AaL) LOQ(002 h|/b L) lOO (0.02alBL> 0.0443 O532 0140 0*21103)903 0.129 0.0130 0.0425 0*777 0*237 0.123 0*975 000.*0.109013005 0*431 0.05)2 0.0303 0.130 0*092 2ar 2nd* Maa m/aiL LOO LOO LOO 170% 133% LOO LOO 0.0643 0.0060 BSD S( Dar. MS/M3D RPD NA NA NA 3% 1% NA NA NA NA 6*9 00443 33. 00294 SarrofaU VrrtAad NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA PFOSEA CM Patty CormUwi Pactar 09930 09930 09930 09930 09930 09930 NA NA NA NA 0.9930 0.9930 0.9930 0.9930 0.9930 0.9930 09930 0.9930 0.9930 0.9930 0.9930 0.9930 09930 0.9930 0.9930 0.9930 0.9930 0.9930 0.9930 0.9930 0.9930 0.9930 0.9930 0.9930 09930 0.9930 0.9930 0.9930 0.9930 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0 99 PPOSAA Parthaenurtanitol EiFOSE- Naa Rm* N-E MJ56 a C*F17S02N((H(CH2COO) PKJSEA- PF03A Party Camelia Factor Uakaotni IManwn Uakaow Untato Uaiao l lutatura NA NA NA NA Untato Untato Unta Unta Untno Unta Untato Untato Unacnw Unta Untato U nta i Un*o UMaora Untano Untee Untno Untaera Untno Udo UMaara Untato Usino Untno Untno Untato Untno Untno Unta Untno U n tn o Untno Untno Untno Untno Untno Untno Unta Unta Untato PFOSEA matto Factor 1 1 1 11 111 NA NA 1 1 1 1 1 1 1 1 11 111 1 l1 rrosEA Cotte. n*/*L 000 2.19 109 1*9 0*5 103 222 223 NA NA 0.00 0.00 0*2 12 07* ! 107 0.00 0.00 142 00..0970 0*4 01.0090 0001...*0092*0* 1.14 1.74 012*32 1.37 1.06 1.39 2.41 1.13 1.04 1 17 0.00 1.45 .7* 0.96 01.00. 1.23 1 17 110105 Fumante Canato rartan d rro sE A nnAL or Rae 0070600007 <LOQ(0.00493aM.) D070600Q2I LOQ(0.00493attaLl 007060000* <LOQ<0.00493HfteL) 0070600022 <LOO<<MXH93artO.) DQ70600009 <LOQ(0.00493a*M .) 0070600023 <LOQ(0*0491 aa/aLt D070600024 90% D070600Q25 90% NA NA NA NA D0706000a <LOQ<a00493a*M.) D070600Q29 <LOQ<000493a#W.) D0706000 <LOQ(00493upteL) 0070600031 LOQ(0.00493a6L) 007UKD032 LOQ(Q.004V3afttiL) 0070600035 <LOQ(0*0493 a^WiL) D070600036 <LOQ(*00493 BftaL) 0070600037 <LOQ(0*0493 aptnL) D0706000JI <LOQ(*00493 nftaL) D070600039 <LOO<0<XM93 atttoL) D07M00042 <LOQ(*00493 n*ML) 0070600043 <LOQ(*00493 a*ML) 0070600044 4 0 Q (&00493a^M.) D07060004S LOQ(*00493 a*M.) 0070600046 LOQ(*00493 a*ML) 0070600049 LOQ(*00493 B*taL) 00706000 <LOQ(*00493 a**L> D0706000SI <LOQ(*00493 a*tiL> D070600032 <LOQ(*00493 a*/toL> D07060D053 <LOO(000493anAnL) 0070600056 <LOQ(*00493 aptaL) D07060057 <LOQ(*00493 a*M-> D070605* LOQ(*00493 attML) D070600059 <LOQ(*00493 t^toL) 00706000 <LOQ(000493aftaL) 0070600063 <LOQ(000493 a*taL) 0070600064 <LOQ(*00493 aiWiL) o m tao o u <LOQ(*00493 a^ftoLy D070600066 LOQ(0*0493 0070601067 LOO(0*0493 taU D0706000 LOQ(0*049)a*ltoL) D07060ar71 <LOQ<*00493aftitaiL) OUAM4UI72 diJQ (0.00493i^taL) D070600071 <LOQ(0*0493 i^toL) D070600014 <LOQ(*00493 a/toL) 0070600077 <LOQ(*00493 a*ML) D07060007I <LOQ(*00493 af/toL) D070600079 <LOQ(*00493 tAM.) D0706000 <LOQ(*00493 a*/toL) 00706000*1 <LOO(*00493 M L l Mann PFOSEA ac/toL <LOO LOO LOO w% NA LOO LOO <LOO LOO BSD S i* De*. MS1MSD RPD NA NA NA 1% NA NA NA NA NA NA NA NA NA 07/1W00 07/14/00 (IOUO iMULOQnM. CafraM LOQ aftaL lto U M L O Q i* / L CarraOaO LOQ i ^ a l uuioQn^i- C x r a M LOQ ^ W L luBiM LOO to/tol. Canada LOQ aj/a L Date ofAaalyan/Aaaha. DwoftUatateow AM lya 06/3AX> Sa LIUK OlOttO* 07/1WOO0M*4i OfclAU) MM U7:A *?i54to (T.HAUl MM S aaifltD iii W EE K 198 RA T SERA Gnmf Srapttf pros Cmmrtoto Daw Coate. HPF6 aC/toL or % Bac MatauttBfe 06290H20 BR-5 OOO LQiG.00421 nf/toLi 06290H20 BEH 0 33 <LOO.GQU2iiuimL> Stara Bk RRS0629O S 8 k 5 039 LOQ >000421 nfaaLi RES06290S aB * < 0 47 LUO iUUIM* uajntLi MaraBR RTS0629O S m B *'5 >23 LO Q iaiw aafitoLi R750629O5aaBB -6 395 <LOQ iOUM3 a/toLi QC RT50629OM5-2 |f* 5 3 ! 35% RTS0629OS4SD-2 ta*-5 343 l>7% * RT50629OS1S-2 (96*5 343 1374 8TS0629OS4SD-230 nntt-5 365 14o % 2mT CM |i> C92I 97/M 451 <LOQ (00042S a* L i Coad Cy22DiM 301 LOQ 1*0043 a * L i OOppto CVZ20U 4 339 LOQ 1*0043 n^toLi c ia n i 3 37 diJQ ll/0043 !.) C 9Z21BM 27* 00242 CV2222M 775 0*0673 C92231A4 406 <U X/iO 0M9 ntoLi C92233M 241 LOQ (00043 at/toLt C9Z239M 701 OOttR C92252A4 CV2J 3LF 230 OOXjO 412 0*351 0/23M i Mia *tM39 CV2344# 53 9 U*4n* C92J54/F CyI3Staf 159 0* 13 4 43 <LOQ (00043 to L i C9236UF 236 0*3X3 C92J62 > 9 0*0711 C 9236WF C92374F CVIJ* 3# 03*3 0*333 3*1 *2n2 271 GO' II Gnayi Los Do* lOpfito CV226LM C9366M C92270M C92271M 1106*4 05tH 0 953 1001*3 3li 017 C9231A4 495 04W C 92281M C y l30WM 0174 301 V90 (*n) C 923ILM 3U3 0437 cyuiLM 154 13) C9I 32UM 3T7 0336 C924IOF CV2410F 2m " 7 137 CV241WF C92423# 101 0623 0541 * C92430F 199 173 1CV2431# Cy243WF 124 1 3 1C* CV244SF CVI451# 1141 1 3 113 * CV2452# 124 2 15 (6incavalcacakfinaeOw 07/3*00and003400raMym. LAC QW21AI0rad09433/00 07/13/00.0*/1SOD.002000.OMltaOO LAC 03/20101Ljfc. 5/3/01 i ^ n 03/7001 LAO 3MEnvironmental Laboratory Page 169 3M M edical D epartm ent Study: T-6316.1 _____ _ _ Covance# 6329-228 Study: Product NumbcrtTest Substance): Matrix: M clh tx i/Rcv is io n : Analytical Equipment System N um ber Instrument Software/Version: Date o Extraction/Analyst: Dale o f Analysis/Analyse Dale o f Data Reducuon/Analysl: Sample Data TOX003, 104 Week Dietary Carcinogenicity Study with Narrow Range (981% ) N-Ethyl Periluuroactanesulfanamido Ethanol in Rats T-6316 (EtFOSE-OH) R at Liver FA C T-M -1.0 FACT-M -2.0, ETS-8-7.0 M adeline 041098. Amelia 062498. Soup020199 M assLynx 3.1 6/09/98 RWW F lenm e: R-Squared Value: Slope: Y-lntcrcept See Below See Attachments Sec Attachments See Attachments 6/26/98, 6/3098, 10/1399 MEE/JJ/MMH 02/08/00.02/0900, 06/08/00. 05/04/01 MMH/1AS WEEK 4 RAT LIVER REWORK________________________________________ lot 193 Duse Sam ple# Initial W t T otal M aas of Liver PFOS Std C o rrec tio n PFOS Purity C o rrec tio n Method BUc Matrix Blk RBL06098-H20 BOt-1 RB L06098-H 20 BDt-2 RB1D6098Lvt Bflt-1 RBL06098-Lvr Bk-2 1.0000 1.0000 1.0080 1.0080 ______ ________ NA NA NA NA F a c to r 0.9275 0.9275 0.9275 0.9275 F a c to r Unknown Unknown Unknown Unknown QC - 100 ppb QC - 100 ppb QC - 500 ppb QC - 500 ppb Croup 8 Control 0 0 ppm G roup 9 Low Dose 1.0 ppm C92196M-MS C92196M-MSD C92I96M-MSD C92I96M-MSD C92192M-MSD C92192M-MSD C92196M C92207M C92216M C92259M C92264M C92280M C9224M C92300M C92329M 10106 1.0106 1.0106 10106 1.0052 10052 10106 1.0133 1.0029 1.0334 1.0512 10140 10217 1.0069 1.0390 10242 1.0230 1.0315 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0 9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 0.9275 NA NA NA NA NA NA NA NA NA NA Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown C92435F C92463F C92467F C92470F P P O S * Perfluurooctanesulfooate PK O SA a Pcrlluunxxtancsulfonam ide 1.0030 10376 10071 1.0170 0.9275 NA 0.9275 NA 0.9275 NA 0.9275 NA 0 9275 NR e Not Reported NA N ot Applicable Unknown Unknown Unknown Unknown Unknown PF O SA A = PeilluorooctMesulfonamidoacetalc LOQ Limit of Quantitation E tF O S E s Narrow Range N-Ethyl PerfluoruocLaoesuUboiotido ethyl alcohol PFOS Cone. ag/g 17.2 0 .7 3 14.5 5.95 334 368 413 456 NA NA 965 1067 NA NA 353 254 173 155 142 85.8 85.9 ISI 53.0 109 147 160 154 126 134 797 760 858 360 509 PFOS D ilution Factor NA NA NA NA 1 1 1 1 1 1 50 50 50 SO 50 5 5 5 5 5 PFOS Calc. Cone. a t/| 16.0 0.68 13.3 5.47 79.9 113 158 200 NA NA 608 710 NA NA 233 158 144 127 77.5 75.8 138 48.1 98.4 6788 7152 6978 5706 6034 3541 3513 3835 1658 2322 Filenam e C o n c en tra tio n of PFOS PFOS M062698002 A06309802 M062698003 A06309803 M062698040 M062698041 < LOO (0.0552 ug/g) 95% A06309841 NA NA S101399046 S101399047 NA NA 168% NA 119% NA 7nH 150% 3rd M062698013 M062698015 M06298016 M0626980I9 M062698020 M062698021 M062698022 M062698Q23 0 127 A0639814 A06309816 6 .03 A06309637 Analytical Report: FACT-TOX-003 LRN-U2104 RSD Std. Dev. Date Entered/By: 04/17/00, (W2Q/00 MMH/LAC, 058/01 mmh Dale Verified/ By: 03/22/Q l hoj.O 4 3/0l k jh .05/10/01 L A C 5122/01 m m h Purity Erucred/Venfied: 03/29/01 L A C /04/03/01 KJH1 FA C T-M -2.0 3MEnvironmental Laboratory 1 vr Week 4 rework Page 170 3M Medical Department Study: T-6316.1 A M U Iff U5U5V8.Z Covance# 6329-228 Analytical Report: FACT-TOX-003 LRN-U2104 Study: Product Nutnbcr(T< Substance): Matnx: M clb ud /K cv isi u n : Analytical Equipment System N um ber Instrument Software/Vertimi: Date of Extraction/A nalyte Dale o f Analysis/Altai) a Dale of D ata Reduction/Analyst: Sample Data TOXOU3. lO t W eek Dietary Carcinogenicity Study with Narrow Range (98.1) N-EihylPerfluorooetane*ulonamido Ethanol in Rats T-6316 (EtFOSE-OH) Rat liver FACT M I 0 A FA C T M 2 0. KTS B-7.0 Filename: k-Squared Value: See Below Sec Attachments Madeline 041098. Amelia 062498. Soup020l99 MassLynx 3.1 Slope: Y -Inle rc ept See Attachments See Attachments 6X19/98 RWW 6/26V8. 6/30-98.10/13/99 MEE/JJ/MMH 02/08/00, 02AJ9AJ0.06/0S/00.05/04/01 MMH/1AS WEEK 4 RAT LIVER REWORK Sam ple C PFOSA Purity PFOSA PFOSA PFOSA Duse C o rrectio n Cone. D ilution Calc. Coac. F a c to r Method Blk RBL06098 H 2 0 Blk-1 Unknown 0.00 1 0.00 RfiL06098-H20 Blk-2 Unknown 0 00 l 000 Matrix Blk R B li)6 0 9 8 L v r Blk 1 Unknown 000 l 0.00 RBL06098-Lvr B k-2 Unknown 1 27 1 1.26 Q C - lO Oppb C92I96M M S NA 104 1 103 C92I96M MSD NA 101 1 100 Q C - lOOppb C92196M-MS NA 107 1 106 CV2196M MSD NA 124 l 122 Q C lOOppb C92196M M S NA 84.4 l 84.4 C92196M M SD NA 68.7 1 68.0 Q C -SO Q ppb C92192M M S NA 382 . 1 380 C92192M MSD NA 352 1 350 Q C - 500 pph CV2192M-MS NA NA NA NA C92I92M M SD NA NA NA NA G roup t CN2192M Unknown 0.00 1 0.00 Cjtfilm l C92196M Unknown 0 00 l 0.00 0.0 ppm C92207M Unknown 000 1 0.00 CV22I6M CV2259M Unknown Unknown 000 000 1 1 0.00 0.00 C92332F Unknown 0.00 0.00 Cy2352F Unknown 5 06 l 4.81 C92355F C92366F Unknown Unknown 5.24 000 5.17 0.00 C92373F Unknown ooo 0.00 G roup 9 Low Dose CV2264M C92280M Unknown Unknown 194 244 192 234 1.0 ppm CV2284M C91300M Unknown Unknown 161 139 1 158 136 CV2329M Unknown 220 1 214 C92402F Unknown 227 217 C92435F Unknown 392 391 C92463F C92467F Unknown Unknown 200 203 193 201 C92470F Unknown 107 105 PROS * Pcrfluorooctaoesulfonaie NR = Not Reported P P 0S A * PaluorooctaA csulfooainidc NA = Not Applicable PROSAA c Perfluoruoctaoeculion am idoacctale LOQ = l.im il o f Quantitation EtFOSE Narrow Range N-Ethyi Pesiliaxooctanesulfonamido ethyl alcohol Filename M62698002 AO630WO2 M 62698003 A06309803 M6269804O M62698041 A0630S840 A 06309841 S1O1399054 S1OI399055 5101399046 S1O1399047 NA NA M62698012 M62698013 M 62698014 M62698015 M62698016 M 62698019 M 62698020 M6269S02I M62698022 M 62698023 M 62698026 M 62698027 M 62698028 M62698029 M 62698030 M62698Q33 M 62698034 M62698035 M 62698036 M62698037 C o n c en tra tio n of PFOSA u c/s o r % Ree- < LOQ (0.0121 ug/g) < LOQ (0.0121 u*/g) < LOQ (0.0121 ug/g) < LOO (0.0121 ug/R) 85% 83% 88% 2nd 101% 2nd 70% 3rd 56% 3rd 63% 58% NA 2nd NA 2nd < LO Q (0.0121 ug/g) < LO Q (0.0121 ug/g) < LO Q (0.0121 ug/g) < LOQ (0.0121 ug/g) < LOO (0.0121 us/ft) < LO Q (0.0121 ug/g) < LO Q (0.0121 ug/g) < L O Q (0.0121 ug/g) < LO Q(0.0121 ug/g) < LOO (0.0121 ui/jt) 0.192 0 .2 3 4 Q.1S8 0.136 0.214 0 .2 1 7 0.391 0.193 0201 0.105 PFOSA A LOO <LOQ 84% 94% 63% 60% NA <LOQ <LOQ 0.187 0.222 RSD Std. Dev. M S/M SD RPD NA NA 3% 14% 21% 8% NA NA NA NA NA 213 0.0401 47.2 0.104 PFOSAA O ld Purity PFOSAA Purity PFOSAA PFOSAA PFOSAA C o rrectio n C orrection Cone. D ilu tio n C alc. Cone. F a c to r 03382 F a c to r Unknown F a c to r 0.00 1 8/1 0.00 03382 Unknown 0.00 1 0.00 03382 Unknown 0.00 1 0.00 03382 Unknown 0.00 1 0 .0 0 NA NA 68.1 1 67.4 NA NA 158 1 156 NA NA 227 1 225 NA NA 254 1 251 NA NA NA NA NA NA NA NA NA NA NA NA 647 644 NA NA 763 759 NA NA NA NA NA NA NA NA NA NA 03382 Unknown 0.00 0.00 03382 Unknown 0.00 000 03382 U(known 0.00 0 .0 0 03382 Unknown 0.00 0 .0 0 05382 Unknown 0.00 0.00 05382 05382 Unknown Uaknown 0.00 0.00 0-00 0.00 05382 05382 05382 Unknown Unknown Unknown 0.00 0.00 0.00 1 0 .0 0 0 .0 0 0.00 05382 Unknown 160 428 05382 Unknown 132 342 05382 Unknown 225 590 05382 Unknown 111 5 293 05382 Unknown 675 1760 05382 Unknown 248 641 05382 Unknown 229 613 05382 Unknown m 446 05382 Unknown 133 355 05382 Unknown 324 856 PROS * Periluorooctancsulfonatc NR Not Reported PFOSA * Pcrfluorooctanesulfonamidc NA * Not A pplicate PFOSAA * Pcrfluorooctanesulfonamidoacetale LO Q * Limit o f Quantitation EtFO SE " Narrow Range N-Ethyl Parflnfmnriam.nf|frn t wiitn gthyi alcohol Filea a roe M062698002 A06309802 M062698003 A06309803 A06309640 A0630984I S 101399054 S10I3990SS NA NA S I0 I399046 S101399O47 NA NA M62698012 M62698013 M 62698014 M62698015 M62698016 M62698019 M62698Q20 M6269821 M 62698022 M62698023 M06309S26 M0309827 M06309828 M06309829 M06309830 M06309833 M063Q9834 M06309S3S M06309836 M06309837 C o n ccatratio B of PFOSAA / % Ree. < LO Q (0.0341 ug/g) < L O Q (0.0341 uc/s) < LO Q (0.0341 ug/g) < LO Q (0.0341 u*/e> 53% 123 177 198 2nd ?ruH NA 3rd NA 3rd 101 119 NA 2nd NA 7n/t < LOQ (00341 ug/g) < LO Q (0.0341 ug/g) < LO Q (0.0341 ug/g) < LO Q (0.0341 ug/g) < LO Q (0.0341 ue/c) < LO Q (0.0341 ug/g) < LO Q (0.0341 ug/g) < LO Q (0.0341 ug/g) < LO Q (0.0341 ug/g) < LO Q (0.0341 utit) 0.428 0342 0590 0393 1 76 0.641 0 .6 1 3 0 .4 4 6 0355 0356 M ean PFOSAA ug/g cLOO LOO 88 188 NA 110 NA <LOO <LOO 0683 0582 RSD Sid. l>cv. M S/M SD RPD NA NA 7W 11 NA 16 NA NA NA NA NA 89 8 0613 33 2 0193 Date Entered/By: 04/17AX), 06/20/00 MMH/LAC.05/08AJI mmh Dale V erified By: 03/22A)l boj. 04/03/01 kjb. 05/1001 LAC 5/22/01 mmh Purity Emered/Verified: 03/29/01 L A C /04/03/01 KJH Date EiMaed/By: D u e V aified/ By: Purity Enlered/Verified: 04/17/00.06/2QA MMH/LAC, 05/08/01 mm h 03/22/01 hoj. 04/03/01 kjh, 05/1001 LAC 5/22/01 mm h 03/29/01 L A C /04/03/0! KJH FA C T-M -2.0 3MEnvironmental Laboratory L vr W eek 4 (rework) Q5/2W2C1 Page 171 3M Medical Department Study: T-6316.1 .......... _ Covance# 6329*228 Study: Product Numbei^Teil Subslance): M am: Mctbod/Rcvision: Analytical Equipment System N ianbcr lntrumcnl Sofiware/Vcniatc Dale o f Extraction/Analyst; Dale o f Analysis/Analyst: Date o f Dots Reduction/Analyst: Sample Data TOX003, 104 Week Dictaiy Carcinogenicity Study with Narrow Range (98.1%) N-Ethyl Pcrfiuorooctaoesulfooamido T-6316 (EtFOSE-OH) R*4 Liv c r FA C T-M -1.0 & FACT-M-2.0, ETS-8-7.0 M adeline041098, Amelia 062498, Soup020l99 Filename; R-Squared Value: Slope- See Below See Attachments MassLynx 3.1 6 m m RWW Y -lntercept See Attachments 6 /2 6 98,6/3098.10/13/99 MEE/JJ/MMH 02/08/00,02/09AW, 06*08/00, QS/04/01 M MH/lAS WEEK 4 RAT LIVER REWORK__________ lot936 Dose Sam ple# E tF O S E Purity C o rrec tio n MamxBIk Q C - 100 ppb Q C - 100 ppb RBL06098-H20 B it- 1 RBL06098-H2 Blk-2 RBL06098-LvrBB:-2 C92196M-MSD F a c to r Unknown Unknown Unknown Unknown NA NA Q C - 100 ppb C92196M-MSD NA QC - >00 ppb QC - 500 ppb G roup 8 Control 0.0 ppm G roup 9 Low Dose 1.0 ppm C92196M-MSD C92192M-MSD C92I92M-MSD C92196M C92207M C92216M C922S9M C92352F C92355F C92366F C92373F C92280M C92284M C923M C92329M C9243SF C92463F C92467F C92470F NA NA NA NA NA Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown E tF O S E Cone. t/f NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR EtFO SE D ilution F a c to r l 1 1 l l 1 1 1 1 1 1 1 1 1 1 l 1 1 1 1 1 1 1 1 1 1 PFOSA = Pcrfluorooctonesul/ooamide NA N ot Applicable PFOSAA = PerfluorooctanesulfoaomidoaceUSe l.O Q B Limit o f Q um tjution EtFOSE = Narrow Range N-Ethyl Pcriluotooctanesulfoosmido ethyl alcohol EtFO SE Calc. Coac. NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR F ilenam e NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA C o n c en tra tio n o f EtFO SE M ean EtFO SE NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR RSD S td. Dev. Ml NR Dale Entcred/By: Dale Verified/ By: Purity Eniered/Verified: 04/17AW. 06/20AWMMH/LAC. 05/08/01 mmh 03/221 boj, 04/03/01 kjh, 05/1091 L A C 5/22/01 mmh 03/29/01 LAC / 04/03/01 KJH Analytical Report: FACT-TOX-003 LRN-U2104 F A C T -M -2 .0 3MEnvironmental Laboratory Page 172 3M Medical Department Study: T-6316.1 Iff U 9 V 3 7 0 . Covance# 6329-228 Study: Product NumbcrfTest Subunte): M in t Meibod/Kcvisiou: Analytical Equipment System N um ber Instrument Softw are/Vcniun: Dale o f Extract*'A nalyst: Dale of Analyst^Analyst: Dale al Dala Reductko/Analyst: Sample Data TUX03. 104 W eek Dietary Carcinogenicity Study with Narrow Range (98.1%) N Ethyl Pcrfluorooctanesulfonamido Elhanol in Rati T-6316 (EtFOSE-OH) Ral Liver Fiknane: Sec Bekjw FACT M -1 .0 * FACT M 2 0. ETS 8-7 0 R-Squared Value: See Attachments Madeline 041098. Amelia 062498. Soup 020199 Slope: See Attachments M atsLynx 3 1.3.2. 3.3 Y-Inlercept: Sec Attachments 0 7 /1 7 / 9 8 1AS/S AH 07/17/98. 7/29W , 08/07/98, 1O'12/99 HOJ/Sa H/M EE/M MH J0 /I3 W , 10/2&V9.02/09/00 MMH WEEK 14 RAT U VER REWORK_______________________________________ to!93___________ ______________________________________________________________ _______________ to L-23S3 G roup Duse Sam ple * Initia l \V L T o la l M aas 8 of Liver PFO S Sid C o rrectio n PFOS Purity C o rrectio n PFOS Cone. PFOS Dilution PFOS Calc. Cone. Filenam e C o n cen tratio n of PFOS M ean PFOS RSD Std. Dev. PFOSA Purity C o rrectio n Method Blk H 20 Blk-1 1.0000 t NA 09273 Factor Unknown *tl8 0 .0 0 Factor 1 8/8 0.00 A71798002 < LO Q (0.0114 ug/g) ug/g M S/M SD RPD F a c to r Unknown H 20B B -2 IOUOO NA 0 9275 Unknown 0 .0 0 1 0.00 A 7 1798044 < LO O <0.0114 uft/c) <LO O NA Uaknown M unB ik Rabbi! Liver Blk.- V 4013 NA 09275 Unknown 0 .0 0 1 0.00 A 7 1798003 < LO Q (0.0114 ug/g) Unknown Q C - lO Oppb Rabb Liver Blk-2 C92235M-MS 40 .1 3 1.0093 NA NA 0927$ NA Unknown NA 0 .0 0 1041 1 l 0.00 A71798045 < LO O (0.0114 ue/e) <LO O 76.9 A71798040 65% NA Unknown NA C92255M M SD 1.0093 NA NA NA 1100 l 136 A71798041 114% 89% 55% NA 1 0093 NA NA NA NA NA NA NA 2nd NA C922S5M-MSD 1.009) NA NA NA NA NA NA NA NA 2nd NA NA NA G roup 8 Conoui 0.0 ppm C92256M C92257M 10093 ions 0.9920 NA NA NA 0.9275 0.9275 09275 Unknown Unknown Unknown 963 2S3 1055 1 50 1 885 11589 987 A71798012 A80798080 A71798014 0.885 11.6 0.987 Unknown Unknown Unknown C92258M 1.0130 NA 09275 Unknown 674 1 616 A71798015 0.616 163 Unknown 09984 NA 0.9275 Unknown 772 1 717 A71798I6 0.717 2.96 4.83 Unknown C92397F 0 .9 9 4 9 NA 09275 Unknown Unknown 490 498 1 449 A71798019 464 A 7 1798020 0.449 0464 Unknown Unknown C92398F C92399F C92400F 10015 1.0009 09943 NA NA NA 0.9275 0.9275 0.9275 Unknown Unknown Unknown 491 362 375 1 I 1 455 A7179802! 335 A71798022 350 A 71798023 0.455 0.335 0.350 0.411 15.2 0.0625 Unknown Unknown Unknown G roup 9 Low Dote C92326M 0 9911 09983 NA 0.9275 0 9275 Unknown Unknown 531 254 1 80 497 18845 A71798026 A80798081 0.497 18.8 Unknown 1.0 ppm C92327M 09977 NA 0 9275 Unknown 177 80 13190 A80798062 13.2 Unknown C92328M CV233M 1009$ 1.0098 NA NA 09275 09275 Unknown Unknown 204 199 80 80 15021 14637 A80798083 A80798084 15.0 14.6 56.2 12.4 6.99 Ui&rown Unknown Unknown 11053 A 80798067 11.1 Unknown C9246SF C92466F 0.9997 1.0043 NA NA 0.9275 0.9275 Unknown Unknown 407 308 25 25 9443 7)10 A80798066 A80798089 9 .4 4 7.11 Unknown Unknown C92468F C924WF 1.01)4 1.0089 NA NA 0.9275 0.9275 Unknown Unknown 362 437 25 25 8299 10052 A80798090 A 80798091 6 .3 0 10.1 16.7 9.19 1.53 Unknown Unknown P F O S A fcifluorooctanesutfonainidc PF O SA A a Pertlucrooclancaulionamidoacelalc E tF O S E a Narrow Range N-Eihyl fcrfluoruociancsulfonam ido ethyl alcohol NA Not Applicable LOQ a Limit o f Quantitation PFOSA Cone. 4 .2 0 3 .8 2 4.99 3 .9 0 167 171 159 96.0 9.20 366 8.14 713 6.64 116 12.3 13.4 9 .4 5 14.3 7 .1 0 591 454 461 318 528 497 525 374 1001 Analytical Report: FACT-TOX-003 LRN-U2104 PFOSA D ilution F a c to r l l 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 PFOSA Calc. Cone. 4 .2 0 3.82 0 .1 2 4 0.0972 156 160 148 86 9 .1 2 8.21 7.12 8.65 114 113 13.4 9.44 14.4 7 .1 6 455 456 314 520 497 370 992 Filen am e C o n cen tratio n of PFOSA M ean PFOSA A 71798002 A71798044 A 71798003 A71798045 < LO Q (0.0124 ug/g) A71798041 132% S101299O47 7 1 2nd A 71798015 A71798016 < L O Q (0.0124 uk/ c) A71798023 A 7 1798026 0.0144 A71798030 A71798034 A 7 1798037 0.314 0 .9 9 2 0580 RSD S id . Dev. 0239 Date Eatcred/By: Dale Verified/By: Purity Entcred/Voificd: 04/17AXI m m h 12/1WOO bcj. 03/22/01 lx*. 04/03/01 kjh. 5/22AJ1 moth 03/294)1 L A C/ 04AI3/01 KJH F A C T-M -2.0 3MEnvironmental Laboratory L vr W eek 14 (rework') Page 173 3M M edical D epartm ent Study: T-6316.1 1J A l< IV lirV S V 3 7 0 > < Covane# 6329-228 Analytical Report: FACT-TOX-003 LRN-U2104 Study: Product Number<Tc*t Substance); M ate Mcihod/Rcvistoo: Analytical Equipment System Number bstw m c w Softwarc/Venicev. Date o f ExtrectioiVAnalyst Date erf Analysis/Analy st Date of Data Reduction/Analyst Sample Data TOX003, 104 Week Dietary Carcinogenicity Study with Narrow Range (98.1%) N-Ethyl Pcifluorooctancsulfonamido Ethanol in Rats T-63J6 (EiK JSE-OH ) Rat Liver FACT M -1 0 & FACT-M -20, ETS-8-7.0 Madeline 04)098. Amelia 062498. Soup 020199 MassLyn* 3.1. 3.2. 3.3 Filename: R-Squared Value: Slope: Y -l n te r c ep c See Below See Attachmeote See Attachments See Attachments 07/17/98 IAS/SAH 07/17/98. 7/29/99, 08/07/98. 10/12/99 HOi/SAH/MEE/MMH (0/13/99.10/26/99,02/09/00 MMH W E E K 14 R A T L IV E R R E W O R K U 6 1 7 ____________________________________________________ ____ ______________________________________________ _________________________________________lo t936 Dose Method Blk M anx Blk Q C lOOppb G roup 1 Control 0 0 ppm Sample # H20B9t 1 H20BIk 2 Rabbit Liver Blk-1 Rabbit Ijv er Blk-2 C92255M M S CV22S5MMSD C92255M M S C9225SMMSD C92255M C9225tM C92257M C92258M CV226UM C92396F C92397K C92398F C92399F C92400F PFOSAA O ld Purity C o rrec tio n Factor 05382 05382 05382 05382 NA NA NA NA 05382 05382 05382 05382 05382 05382 05382 05382 05382 05382 PFOSAA Purity C o rrectio n Factor Unknown Unknown Unknown Unknown NA NA NA NA Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown PFOSAA Cone. NR 0.00 NR 0.00 136 307 NA NA 51.1 283 21.7 31.0 72.5 118 10.1 40.6 53.8 690 PFOSAA Dilution Factor NR l NR 1 1 1 NA NA 1 10 1 1 1 1 l PFOSAA Calc. C one, c/f NR 0.00 NR 0 .0 0 83.7 254 NA NA 27.3 1504 11.8 16.4 39.1 62.6 5.45 21.8 28.9 37.4 F ilenam e NR S101299004 NR SI0129900S S 101299046 S 101299047 NA NA S10I299014 SI0I299O3O SI01299015 S101299016 S10I299017 S101299018 S 101299022 S101299023 S 101299024 SI0I299025 C o n c en tra tio n of PFOSAA M ean PFOSAA "V * K . NR <LOQ (0.0351 -- "rt <LOQ NR <LOQ <0.0351 u t/* ) 66% 200% <LOO 133% NA NA NA <l-OQ (0.0351 ug/g) 1.50 <LOQ (0.0351 ug/g) <LOQ (0.0351 ug/g) 0.0391 00626 <LOQ (0.0351 ug/g) 0.330 <LO Q (0.0351 ug/g) <LOQ (0.0351 ug/g) 0.0374 0.0411 RSD Std. Dee. M S/M SD RPD NA NA 101% NA 199 0.656 29.4 0.0121 EtFO SE Purity C o rrec tio n F a c to r Unknown Unknown Unknown Unknown NA NA NA NA Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown G roup 9 Low Dose 1.0 ppm C92325M C92326M C92327M 05382 05382 05382 Unknown 79.3 1 43.1 S101299026 0.0431 Unknown 469 20 5052 A102599062 5.05 Unknown 537 10 2899 S 101299042 2 .9 0 Unknown Unknown Unknown CV2328M C92330M C92464F C92465F 05382 05382 05382 05382 Unknown 461 10 2458 S101299032 2 .4 6 65.2 Unknown 747 10 3979 S10I299033 3.98 2 .8 9 1.88 Unknown 1149 10 6083 S 101299034 6.08 Unknown 421 10 2267 S101299038 2.27 Unknown Unknown Unknown Unknown C92466F C92468F C92469F 05382 05382 05382 Unknown 142 380 S 101299040 0.380 Unknown 103 5 275 S101299041 0275 95.1 Unknown 381 20 4063 A 102599063 4.06 2.61 2 .4 9 Unknown Unknown Unknown PPOS Peri)uoruucuncsulfonatc NR Not Reported PFOSA m FcrtluotoocUnesultunamidc NA " N ot Applicable PPOSAA fafluM ooclanesulfonam idoacetatc LO Q * Limit o f Quantitation EtFOSE * Narrow Range N-Etbyl Pafluotooctanesulfonam ido ethyl alcohol E tF O S E Cone. a rt NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR EtFO SE Dilution Factor 1 1 1 l 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 EtFO SE C ak. Cone. 8 /8 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR F ilenam e NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA C o n c en tra tio n of EtFO SE n t / t o r % Ree. NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR M ean EtFO SE 8/8 NR NK NK NR NR NK NR NR RSD Sid. Dev. M S/M SD RPD NR NR NR NR NR NR NR NR NR NR NR NR Dale Entocd/By: Date V aified/ By: Pinky EnienA/Vehfied: 04/17/00 mmh 12 /19 /00 Uj), 03/22/01 boj. 04/03I lyb. 5/22/01 nunh 03/29/01 L A O 04/03/01 KJH F A C T -M -2 .0 3MEnvironmental Laboratory L vr Week 14 (rework) 05/29/2001 Page 174 3M Medical Department Study: T-6316.1 Covance* 6329-228 Analytical Report: FACT-TOX-003 LRN-U2104 Study: Product NundwitTea SubMaete): M as: Mctbud/Revutoa: Amdyticnl Eifiifm-et Syucm Number IkMrumeei SoAware/Veaiua: Oale of ExtnciMWAulysi: Date U AaatyaiWAaatya: Date of Data Reduutoe/Aaaly: Sam ple Data TOX003. KM Week Dietary Carvinugeatciiy Study nib Narrow Raage (98.1%) N-Etby! PeriluotoocuaesuDoundo Ethaooi a T-6316 (ElFOSE-OHl Ral lavtf Flmame: See Below ETS 8 6 U 4 ETS-8-7.U R Squared Value: S e e A U th m ea u Amelia 06249*. Davey 070799 Maaal.yiut 3.3 A 3.4 Slope; Y-leicrcefX: See Attachments See Attachments 4/14/00 SAURWW 04/17/00. 05/03/00. 0/I5/0 lAS/MMK/lAS 04/18/00. 05/04/00. 05/16/00, 05/08/01 IAS/MMH W EEK 53 KAT LIVER G reup Du n Sampk Verified loiOnl Wl. 1 Tut*] Mam uf Liver Melbud Blk RBli/4 1400-i20Blk-1 RBL04I4001120BIL 2 M a tr aB lk RSLXMI40 LiverBlk-l Q C -300pi*. RBLW I400LttBlk 2 C92 19 7/M /G 8-M S-I C 9 2 197/M / 8-M S D I C92195/M/G 8MS-I C92I95/MA8-M S1 RBL04I400 MS I RBL041400-MSD-1 Greup I C92I95M 0.0 ppm C92I99M C92205M C9223IM C92246M C9234IF C92356F C92372F C92376F C92390F Greup f C92267M C92274M C92287M C9228SM C92320M C92406F CV24UF C9241IF CV2422F CV2438F Above bacar nage of Ibe tatuai a * Qualitative oaly NA NA NA NA NA NA High - Not Confirmed Utah - Not Cocfiirncd -- f S --------------NA High - Not Coafinncd NA NA llieh - Nut Confirmed NA NA NA NA NA High - Not Cueftrraed High - Nut Confirmed High - Noi Coufiimed High - Noi Confirmed llivh Not Confirmed High Noi Confirmed High - Not Confirmed 1Itgfa - Not Confirmed High - Not Confirmed 1 0000 NA 1.0000 NA 1.0000 NA 1.0000 NA 1 0453 NA 1 0453 NA 1.0243 NA 1 0243 NA 1.0000 NA 1.0000 NA 10243 NA 1 0416 NA 1 0515 NA 0 9947 NA 1 0013 NA 1 0282 NA 09926 NA 1.0245 NA 1.0418 NA 1.0329 NA 1.0137 NA 1.0853 NA 09901 NA 1.0555 NA 0.9914 NA 1.0459 NA l.QSIS NA 1.0528 NA 1 0250 NA 1.0810 NA NR = No Reponed NA &Noi Applicable LOQ a Limit of Quaauialioa Date Hatemd/Aaaly: Date Verifaed/AMlyu: P u n y Eatered/Veofied: 04/26/00 moth. 09/15/00 LAC. 05/09/01 mrab 03/22/01 boj. 04/03/01 kjb. 05/10/01 LAC 5/22/01 s 03/29/01 LAC/ 04/03/01 KJH PFOS OM Purity C erreclina Factor 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0 9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 PFOS Purity C trr ec U u n Factor 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 08640 0.1640 0.1640 0.1640 0.1640 08640 0.8640 0.1640 0.1640 0.8640 0.8640 0.8640 OA640 0.8640 0.1640 0.1640 0.1640 0.8640 0.1640 0.8640 0.1640 0.8640 0.8640 PFOS Cane. 0.00 0 .0 0 0 .0 0 0.00 289 287 U91 1112 232 261 920 171 741 433 298 0.00 m 223 349 334 497 478 572 410 239 362 460 372 323 1516 PFOS D ila ti eu Fecttir 1 1 1 1 1 l l 1 1 1 l 20 1 1 10 I 100 100 100 100 too 100 100 too too PFOS Calc. Cane. 0.00 0.00 0.00 4.77 226 224 403 324 232 261 780 2855 612 378 2581 0.00 100 189 291 281 2575 38241 50148 33720 20904 30075 36954 30647 27346 1211 F ilm arn e A041700004 D41400004 A041700005 D 04I400005 D 0 4 1400017 D 0 4 1400018 *041700017 A041700018 D 04I400076 D041400077 A0417000I9 A050300019 A04I700021 A041700024 A050300020 A 0 4 1700026 A041700027 A041700028 A04I700031 A 0 4 1700032 A050300028 A050300031 A050300032 A050300033 A050300034 A050300021 A05030024 A050300025 A050300026 A041700046 CoaccBUnUua ef PFOS a a /t e r % Ree < LOQ (0.0267 ug/g) < LOQ (0.0107 ug/g) < LOQ (0.0267 ug/g) < LOO (0.0107 ur/k) 78% 78% 137% 110% 76% 85% 0.710 2.86 0.6)2 0.378 2.58 < LOQ (0.0267 ug/g) 0.100 0.189 0.29) 0.281 42.6 38.2 50.1 33.7 20.9 30.1 37.0 30.6 27.3 1.22 Mean PFOS <LOO <LOO 78% A A 124% 80% A 1.44 0.178 37.1 A 25.2 PFOSA PeffluwooriaaeMlfoeamide PFOSAA * Perfluorooctaneaulfoaamidoneriate EtFOSE Narrow Raage N-Ethyl Pertluoroortairiulfbaa PPOSEA PerfhoroocUae aulfoayl Mhylanude M5S4 a C8FI7S02NH)CK2C00> RSD Sid. Dee. M S /M SD R PD NA NA NA NA 1% 22% 12% 81.7 1.18 64.6 0.115 29.4 10.9 55.0 13.9 S a rre gate Verified NA NA NA NA NA NA NA NA NA NA NA High - Not Confirmed NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA PFOSA Old Purity CerrecUou Feriar 0 9510 0.9510 0.9510 0.9510 NA NA NA NA NA NA 0.9510 0 9510 0.9510 0.9510 09510 0.9510 0.9510 0.9510 0.9510 0.9510 0.9510 0.95)0 0.9510 0.9S10 0.9510 0.9510 0.9510 0.9510 09510 0.9510 PFOSA Purity C a rre ctleu Feriar Unknown Unlrwnwa I'akaowa 1.'nknnwa NA NA NA NA NA NA Unknown Uakaowu Unknown Uakaowu Unknown Uakaowu Uaknowa I'akaowa Uakaowu Unknown Uakaowu Uukuowa Uakaowu Unknown Unknown Untaron Uakaowu Uakaowu Uakaowu Unknown PFOSA 2.39 0 00 209 000 239 262 291 196 283 260 408 272 2.67 406 3 01 3.07 3.66 1 91 6.55 306 132 147 227 321 170 289 197 179 316 160 PFOSA Dilutive Factor 1 1 1 1 1 1 i l 1 1 1 I 1 I 1 1 1 1 1 1 1 1 1 1 1 1 1 J PFOSA Calc. Cerne. Filename 2.51 AW 1700004 0 0 0 DWI40UOW 2.20 AW170000S 0.00 DW140U005 224 D W 1400017 246 D4I4O00I8 280 AWI7000I7 188 A041700018 282 D041400075 259 D041400076 4 19 A4I700019 275 AW 1700020 2 67 AW170002I 429 AW 1700024 3.23 AWI700025 3 14 AW 1700026 3.81 A041700027 1 96 AWI70028 a 61 AW I'uOOi 1 3 12 AW170U032 137 AW1700033 142 AW 1700034 241 AW1700035 320 AW170UU38 I II AW 1700039 290 A011700040 192 ACMI70U041 179 AW170U042 396 AW 1700045 156 AW 17000*6 3MEnvironmental Laboratory Page 175 3M Medical Department Study: T-6316.1 Cuvance# 6329-228 Analytical Report: FACT-TOX-003 LRN-U2104 CeoceM M iaa MPFOSA < LOQ (0.00644 ug/gl < LOQ (0 00644 ug/g) 78% 9*% 92% < LOQ (0.00644 ug/g) < LOQ (0 00644 ug/g) < LOQ 0 00644 ug/g) < LOQ (0.00644 ug/g) < LOQ (0.00644 ug/g) < LOQ (0.00644 ug/g) < LOQ (0.00644 ug/g) 0.00661 0.137 0.142 0.241 0320 0290 0.192 0.17 0.396 0 .1 5 6 PKiSA 0242 RSD S u l Do . SA SA NA 122 37.7 4)2 0 0999 Study PtoliKi Nutnbct(TcM Subawcc)-. Matrix: Mecbud/KrvuiuaAaalyiy-al bpupineai Syaera Number, luuuroeai Soiiware/Vmioa: Date ui xumuod/AMJyil: Dite o Aaalyiu/Aaalya. Dale o D au RaJucUoa/Aaalys. Sam ple Data TOX003. 104 Week Dwuiy Carciaogeaidty Study with Narrow R u g e (98.1%) N & byl T 6316 (ElFOSE-OH) Rai U v a ETS-8-6.0& ETS-8-7.0 Amelia 062498, Davey 010799 MattLya* 3.3 4 3.4 R-Sqnaied Value: Slope: Y-l 4/I 4/0 0S A L/R W W 04/17/00, 05/03AW, 05/15/00 IAS/MMH/IAS 04/18/00. 05/O4AX), 0S/16AX). 05/01/01 IAS/MMH SeeBelow See Aitartynea ti See A n a t n M i See AUadvnenta Dum Saapk* M etta) B)k Malnx 818 QC 300 ppb RB1.041400-1120B1L-1 RBLO4)40OLiverBlkl C92 197/M /G 8-M S 1 C92I9$(M (G I-M S-I RBL041400-MS-1 Gruup 1 OOppttt C92I95M C92I99M (.'9110SM C92238M G roup (.Oppia C9234IF C92J56F C92372F C92376F C92267M C92274M C92287M C92288M 1 A * Above barar raage of the ie 8 Qualitative oely C92406F C9241IF C924I8F C92422F C92438F V arlfM NA NA NA NA NA NA NA NA )Ugb - Noi CoaTumed NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA PFOSAA Old Purity Correctiva Factor 0.9760 0.9760 0.9760 0.9760 0.9760 0.9160 0.9760 09760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9160 0.9760 NA - Noi Applicable LOQ Unii o QuutHauoa D * eE aW ed /A eW y a: D a r VanfMd/Aaaly: Purtty EaUsced/VeofaML 04/26/00 mitih. 09/15/00 LAC.0S/09/1 nunh 03/22/01 ho). 04A)3A)I kjb. 05/I0A)! LAC 5/22/01 a 03/29/01 LA C/04/03/01 U H PFOSAA Purity Cerrectien Factor Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa U dana Uakaowa Unknown Uakaowa Uakaowa Uakaowa PFOSAA CUK. 0.00 0 .0 0 0 .0 0 0.00 224 260 251 254 232 266 0 .0 0 0.00 0.00 0.00 0.00 0.00 0.00 4.40 10.1 0.00 344 885 1872 678 729 464 406 583 72 530 PFOSAA D ila ti oa Factor 1 1 1 1 l 1 1 1 1 1 l 1 1 l 1 1 1 i 1 1 1 1 PFOSAA Caie. Cene, rt 0.00 0.00 0.00 0.00 215 249 240 243 222 266 0.00 0.00 4.40 9.96 0.00 348 836 1937 658 753 455 384 567 721 502 ilili F tteaam c C e w c te ir a O u e Mesa u i PFOSAA PFOSAA aa/c or % Ree F i A04)700004 < LOQ (0.0)25 ug/g) D041400004 < 1 .0 0 <0.0314 u t/a <LOQ A041700005 < LOQ (0.0125 ug/g) D041400005 < 1 .0 0 <0.0314ua/f) <LOO 004140001? 74% D04I4000I8 86% 80% A0417000)7 82% A04)700018 3 82% D 0 4 1400075 73% 004)400076 17% 10% A0417000) < LOQ (0.0125 ug/g) A041700020 < LOQ (0.0125 ug/g) A04I70Q021 < LOQ (0.0125 ug/g) A04)700024 < LOQ (0.0125 ug/g) A04)700025 < 1 .0 0 O.OJ25 a a /i) <LOO A041700026 < LOQ (P-OI25 ug/g) A041700027 < LOQ (0.0125 ug/g) A04)700028 < LOQ (0.0125 ug/g) A041700031 < LOQ (0.0)25 ug/g) A041700032 < LOO (0.0125 uc/a) <LOO A 0 4 1700033 A04)700034 0.348 0.136 A 0 4I7 00035 1.94 A A041700038 0.658 A04170Q039 0.753 0 .9 0 6 0.45 0.384 0.567 0.721 0.502 0.527 iilili 1. r r r r- 5 H ill] pfOSA Perttuorooctaa PPOSAA * E iF O S E N ano* Raagt N-Efcyl Pwffcionwaaet PFOSEA Perfucuooctaae adfoayl JjyUnyde M556 C8F17S02N((H)CH2C00) RSD $t d.D ev. MS/MSD RPD NA NA 15% 2% 18% NA NA NA NA 66.8 0.605 24.1 0.131 Su rra gai V triO td NA NA NA NA NA NA High - Net CouOraed NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA E4FOSE OM Purity C urrectaa Factor 0.9770 09770 SA SA SA 0.9770 0.9770 0.9770 0 9770 0.9770 0.9770 0 9770 0.9770 0 9770 0.9770 0.9770 0.9770 0.9770 09770 0.9770 0.9770 E lfQ SE Purity Cerrectiea F u to r 0.8890 l>8890 SA SA SA 0.8890 0.8890 0.8190 01890 0.8890 0.8890 0.8190 0 8890 01890 0.8190 08890 0 8890 Q.8890 0.8890 0.8190 08890 EtF O S E Ctac. 000 Oui) 211 49 SR OUU 000 0 00 000 0 0 00 000 0.00 0 .0 0 o.uo 0 .0 0 0.00 000 000 3M Environmental Laboratory Page 176 3M Medical Department Study: T-6316.1 Covane# 6329-228 Analytical Report: FACT-TOX-003 LRN-U2104 ElF O S E CaU- Coc. C v actalratlua uf ElFOSE # 1 2 ? Ree t LOQ <0.0559 ug/g) c 1.00 ( 00559 ug/g> c LOQ <0 0559 ug/g) c I.OQ ( 0 0559 utht) Mean E lF O S E 000 0.00 0.00 0.00 0 .0 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 000 NR NR A041700019 A041700020 A041700021 A041700024 A04I70002S A041700026 A 0 4I7 00027 A04170002 A04I700031 A041700032 A04I700033 A041700034 A041700035 A041700038 AP4I700039 A041700040 A04I100041 A041700042 A 04I7 00045 A04I700046 NR NR c LOQ (0.0559 ug/g) c LOQ ( 0.0559 ug/g) c LOQ ( 0.0559 ug/g) `'S> ?c LOQ ( 0.0559 ug/g) g 1 .0 0 1 0 0559 ut c LOQ <0 0559 ug/g) c LOQ ( 0.0559 ug/g) c LOQ ( 0.0559 ug/g) c LOQ ( 0.0SS9 ug/g) < LOQ <0 0559 u a /tl < LOQ ( 0.0559 ug/g) c LOQ <0.0559 ug/g) c LOQ ( 0.0559 ug/g) c LOQ ( 0 0559 ug/g) c LOO ( 0 0559 ut/) B < LOQ ( 0.0559 ug/g) t LOQ (00SS9 uglg) c LOQ ( 0 0559 ug/g) c LOQ ( 0.0559 ug/g) e l.O Q t 0.0559 u a /il <LOQ Study: Product NumberfTea Subrtaace): Matrix: Mctbod/Rcviiioa: Analytical Eqmp a ra i Syjtetn Number Inum ate Softwnia/Venioe: Date of EjtUKon/Analyit: Date of Aaalysu/AMlyM: Date of O va Reductioa/AMlya: S a n f k D ata TOX003,104 Week Dietary Caniaogeaiuty Study with Narrow Range (98.1%) N-Ethyl Prrfl't'TrTmtarailf**----^ T-6316 (BFQSE-OH) Rat Uvee Filesame: See Below ETS-8-6-0 A ETS-8-7.0 K.SiyafiOVg) ^ Amelia 062498. Davey 070799 Slope: See Auduaeetf M a u L y u 3.3 A 3.4 4J14/OOSAURWW Y'Intercept: S te A tlid u ie u i 04/17/00,05/03/00, 05/15/00IAS/MMH/1AS 04/18/00. 05/04/00, 05/16/00. 05/08/0) [AS/MMH E RSD Sid. Dev. MS/M SPRPD W EEK S3 R A T LIV ER RBL04)400-H20Blk-l RBL04l4Q0-H2OB|fc-2 RB LO 4l40 0-U verB lk l RBLO4l400-UverBlk-2 C92I97/M/GS-MS-I C92I97/M/G8-MSD-1 C92I95/M/GI-MS-1 C92195/MC8-MS-I RBL04I400-MS-I RBL04I400-MSD-I C92I95M C92I99M C92205M C92238M C92246M C92341F C92356F C92372F C92376F C92390F G raap 9 C92267M I.Oppnt C92274M C92287M C92288M C92320M C92406F C924I IF C924I8F C92422F C92438F Above linear nage of the initial curve,estimated vi Qualitative only Surrogate Verified lot NBI13047-80 M556 Old Parity CurrecUea Factor MSS Parity Correction High Not Confirmed NA 0.9989 0.9989 0.9989 0.9989 0.9989 0.9919 0.9989 0.9989 0.9989 0,9989 0.9989 0.9989 0.9989 0.9989 0,9989 0.9989 0.9989 0.9989 0.9919 0.9989 NR Not Repotted NA * Not Applicable LOQ Until of Quanwmioa NA NA Unknown Unknown Uakaowa Uakaowa Unknown Unknown Uakaowa Unknown Date Eateted/Aariy: Dale Verified/Analy*: Purity Ealcred/Venfied: 04/26/00 mob. 09/15/00 LAC, 05/09/01 mmh 03/22/01 boj,04/03/01 kjb, 0S/KV01 LAC 5/22/01 03/29/01 LAC/04/03/01 K3H MSS Coac. 0.00 0.00 0.00 0.00 0,00 0.00 0.00 0.00 MSS D tta U o a Factor MSS _8tftCalc. Cooc. 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 of MSS rtffS R K c LOQ ( 0.0307 ag/g) < LOQ (0.0307 ua/cl < LOQ ( 0.0307 ug/g) < LOQ ( 0.0307 tia/a) D 0 414000I7 0041400018 A041700017 A04I7000I8 D04I400075 D041400076 A 04I7 00019 < LOQ (0.0307 ug/g) A04)700020 < LOQ (0.0307 ug/g) A04)700021 < LOQ ( 0.0307 ug/g) A 0 4I7 00024 < LOQ ( 0.0307 ug/g) A041700025 < LOQ ( 0.0307 ug/ii A 0 4I7 00026 A041700027 < LOQ ( 0.0307 ug/g) < LOQ (0.0307 ug/g) A04I70002S A041700031 A041700032 A041700033 < LOQ (0.0307 ug/g) < LOQ (0.0307 ug/g) ( 0.0307Tuui g ig , 0.266 A041700034 0.331 A041700035 A04170003S A041700039 A041700040 A041700041 0.380 0.426 0.277 0 .2 2 6 0.300 A041700042 0.274 A041700045 A041700046 0.353 0.182 PFOS Pedluorooctaaetulfoaate PFOSA Perfluoreoctaaewlfoewi PFOSAA ElFOSE a Narrow Raage N-Elbyl PFOSEA a t>--r -------.-lr juKoay) cUryiataidc M556 - C8F17S02N((H)CK2C00) Sid. Dev. MVMSD RPD PFOSEa Old P a rti; Correct! _______ Factor_______ NA NA 0 9930 0 9930 0 9930 0 9930 0 9930 0 9930 0 9930 0 9930 MM 0 930 0 9930 0.9930 0 9930 0 9930 0 9930 0 9930 0.9930 0.9930 0.9930 0 9930 3MEnvironmental Laboratory Page 177 3M Medical Department Study: T-6316.1 PFOSEA Purity Carrectiun l akaotts PFOSEA CWK- ____s t t _ 000 I'aknuwi 0 00 NA NA I 'akaow* 208 254 442 35) 358 393 0 .0 0 LaUowa 0 00 tw k a o LUaowa Uaknowi U ataoM Uakaoaa lU aowa Cakaowa Uakaow* Uakaowa lakaowa lakaona 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0.00 0.00 PFOSEA DUaliwa Factor PFOSEA Calc. Cune. ___ F t 000 000 0.00 199 243 423 3)8 343 376 l 000 1 0 (XI 1 1)00 1 000 1 000 1 0.00 1 0.00 1 000 1 0.00 1 0.00 1 0 00 l 0.00 1 0.00 1 0.00 1 0.00 1 000 1 000 1 000 1 0.00 1 000 FUtuamr o PFOSEA A 0 4 1700004 D 0 4 1400004 A 041700005 D 0 4 1400005 D 0 4 1400017 D 0 4 1400018 A04I7000I7 A04I70U0IS D 0 4 1400075 D 0 4 1400076 A04I700019 A04I70U020 A041700021 A041700024 A04170002$ A 0 4 1700026 A041700027 A041700028 A04I70003I A041700032 A041700033 A041700034 A041700035 A0417000)8 A0417000)9 A041700040 A04I70U041 A04I70042 A041700045 A 0 4I7 00046 < 1.0y (0.0308 ug/g) < LOQ (0.0308 ug/g 69% 144% 115% 112% 123% < LOQ (0 0308 ug/g) < lt> Q (0 0.108 ug/g) *. LOO (0 0308 ue/v) < LOQ (0.0308 ug/g) < 1.00 (0.0308 ue/f) < LOQ (0 0308 ug/g) < LOO <0.0308 ua/al < LOO (0.0308 u a/i l < LOQ (0 0308 ug/g) LO O (0.0308 uc/i) < LOQ (0.0308 ug/g) < LOQ (0.0308 ug/g) < LOO 10.0301 ua/a) < LOQ (0 0308 ug/g) < 1.00 <0.0308 ua/a) < 1 .0 0 <0.0308 ua/e) < LOO <0.0308 ue/v) < LOQ (0 0308 ug/g) < LOQ (0.0308 ug/g) < 1 .0 0 (0 0308 ua/e) PFOSEA LOO NA NA Covance# 6329-228 Analytical Report: FACT-TOX-003 LRN-U2104 Study; Product NumbeffTesl Subsuace): Matrix: Me thod/Revuion : Aaalyikal Equipment System Number: Ituirumeai Softwaie/Venioa: Date of Extractiou/Aaalysi: Dale of Aoalyau/Aaalysl: Dale of Data Reductioii/Aulyst: Sam ple D ata TOX003.104 Week Dietary Caruaogeucuy Study with Narrow Raage (98.1%) NEibyl PerikoroocUBeauloaamido T-6316 (EtFOSE-OH) ETS-S-6.0A t i - l - 7 .0 Amelia 06249, Davey 070799 M assLyu 3J * 3.4 4/14/00 SAL/RWW 04/17/00, 05/03/00, 05/15/00IAS/MMH/1AS 04/18/00.05/04/00, 05/16/00. 05AAI1 1AS/MMH Fiteaatne: R-Squared Value: s lo p e : See Aar m a i See AAudunealt See A nadum att U, JUUd>U B . , W EE K S3 R A T LIVER Grwnp D u tc S am p le R B L 04l4 00-H 20B lk -2 RBL04l400-LiverBlk-2 C92197/M/G8-MSD-I C92195/M/G8-MSD-I RBL04I400-MSD-1 C92I99M C92205M C92238M C92246M C92356F C92372F C92376F C92390F C92274M C92287M C9228IM C92320M C92411F C924I8F C92422F C92438F PFOS Cale. Cune, ua/l 0.00 4.77 224 324 261 2855 612 378 2581 100 189 291 281 38248 50148 33720 20904 36954 30647 27346 1218 CencaotraUeo f PFOS a*/ e r % Rae < LOQ (0.0267 ug/g) <LO O (0.0107 ua/at < LOQ (0.0267 ug/g) < LOO (0.0107 u e/t) 78% 110% 85% 0.780 2.86 0.612 0.378 2.31 A 0.100 0.189 0.291 0.28) 42.6 38.2 50.1 33.7 20.9 37.0 30.6 27.3 1.22 A Marni PFOS <LOO <LOO 78% 124% 80% 1.44 0.178 37.1 23.2 Date Eaterod/Aaaly: Date Verified/Aaaly: Purity Emeted/Vecified: LOQ LimA o f Q u m h m 04/26/00 mrah. 09/15/00 LAC, QS/D9/01 mmb 03/22/01 boj, 04A)3A)t kjh, 05/t0A)l LAC 5/22/01 mmb 03/29/01 LAC/ 04/03/01 KJH RSD S U De*. M S /M SD R PD NA NA 1% 22% 12% 81.7 1.18 64.6 0.115 29.4 10.9 55.0 13.9 PFOSA Cale. Cede. 2.51 000 2.20 0.00 224 246 280 ISS 282 259 4.19 2.75 2.67 4.29 3.23 3.14 3.18 1.96 6.61 3 12 137 142 241 320 ISI 290 192 179 196 156 Cancan!ratina rfPFOSA u c /i u r % Rae < LOQ (0.00644 ug/g) Mean PFOSA < LOQ (0.00644 ug/g) 78% 86% 94% 63% 92% 15% < LOQ (0.00644 ug/g) < LOQ (0.00644 ug/g) < LOQ (0.00644 ug/g) < LOQ (0.00644 ug/g) < LOO (0.00644 ue/a) < LOQ (0.00644 ug/g) < LOQ (0.00644 ug/g) < LOQ (0.00644 ug/g) 0.00661 0.137 0.142 0.241 0.320 0.181 0.290 0.192 0.179 0 .3 9 6 0 156 oaate en SU. Da*. NA 1 2' 37.7 41.2 PKJSAA . EtFOSE Narrow R u g a N-Etbyl p*A "T ~ m a f iii|t"" -<- tbyl PFOSEA m Pcrfuoroocuee --*f~iyl etbyUmde M556 * C8FI 7S02N<(HJCH2C00) fFOS.AA Cale Cene 000 0.00 215 240 222 000 000 000 000 0.00 0.00 4 40 348 836 1937 658 455 384 567 728 r --caniratien m o& A A <LOQ (0 0125 ug/gj < LOQ (00125 ugg) 74% 12% 73% < LOQ (00125 ug/g) < LOQ (0 0125 ug/g) < LOQ (0.0125 ug/g) < LOQ (0.0125 ug/g) < LOQ (0 0125 ug/g) < LOQ (0 0125 ug/g) < tO Q 0 0125 u gg < LOQ )O.OI25 ug g) 0 348 0 836 1 94 0 658 0455 0 384 0.567 0.728 3MEnvironmental Laboratory Page 178 3M Medical Department Study: T-6316.1 Covane# 6329-228 Analytical Report: FACT-TOX-003 LRN-U2104 Study: Produci Nuinber(Tcd Subxuacc): Mau: Metbod/Rcvuioa: Aariyucri Frpapaieal Syttum N u o te r Imammeat Sofwant/Veoioa: Date of EiUKUQriAariyti: Due ri Aariytu/Aariys: Dale of Data RerfuOjoa/AaaJyH: S a u f k D ata TOX003. 104 Week Dietary Cuviaugrouity Study with Narrow Range (9I.I*)N -E thyl Perflumooctaneautfoaamido Ethanol is Rau T-63I6 (txFOSE-OH) R a Liver ETS * 6 0 A ETS I 7.0 Ftleaame; R-Squared Value: See Below SeeAttachment! Amelia 062491. Davey 070799 Slope; SeeAttachment M aisL yu 3.3 A 3.4 Y-lMenefd: SeeAttachment 4/14/00 SALVRWW 4/17/00. 5/15/00IAS/MMIMAS 4/1 8/00, 5/1 WOO, 05/08/01 1AS/MMH W E E K IOS R A T L 1 V E K _______________________________________________________________________________________ 1 171_______________________________________________________________________________________________________________________________________________________lot L-1S709 Gnmp Dune Method Blk M alm Blk Q C -300J1* S am p le* R B L 0 41 40 0-K 20 Blk -l KBIJ34140 lUOBIk 2 RBL04I400 UvetBUt l RB13NI400 IjverBIk 2 C92197/M/G8-MS l C92I97/M A 8M SD -1 C92I95/M/G8-MS-1 C 9 2 I95/M /U 8M S D -1 RBL04I400-MS-1 RBL041400- MSD-1 Su rra gate Verified NA NA NA NA NA NA High Not Confirmed llieb Not Coo/imel NA NA Initial Wl. 8 i oooo 1 0000 1.0000 1 IXXHJ 1.045? 1.0453 1 0243 1 0243 1 0000 1.0000 Total Mam of Liver NA NA NA NA NA NA NA NA NA NA PFO S OM Partly C arrectW n Factor 0.9949 0.9949 09949 0.9949 0.9949 0.9949 09949 0-9949 0.9949 0.9949 PFOS Purity C a rre cU on Factor 0 .8 6 4 0 0 864Q 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0 .8 6 4 0 0 .8 6 4 0 PFOS Cane. 0.00 0.00 0.00 0.00 289 287 1192 1112 232 261 PFOS D ilatiti* Factor 1 l 1 1 `1 1 1 1 1 l PFOS C ric Cane rt 0.00 0 .0 0 0.00 4.77 226 224 403 324 232 261 FUaname A04170Q004 0041*00004 A041700005 D 0 4 1400005 D 0 4 1400017 D04I400018 A041700017 A041700018 D041400076 D 0 4 1400077 CeDccntration of PFOS n ria or % Ree < LOQ (0.0267 ug/g) < LOO (0.0107 ut/e l < LOO (0.0267 uric) < LOO (0.0107 urie) 78% 78% 137% 110% 76% 85% Mean PFOS <LOO <LOO 78% 124% 80% RSD Std. Dev. MS/MSD RPD NA NA NA NA 1% 22% 12% Vcrid* I- NA NA NA NA NA NA NA NA NA NA PFOSA Old Parity Correction Factor 0.9510 0 9510 0.9510 0.9510 0.9510 0.9510 0.9510 0.9510 0.9510 0 .9 5 1 0 Grwap 8 0.0 ppm C92I97M C92203M C92208M C92209M C92218M NA 1.0453 NA 09949 0.8640 53.1 l 44.1 D041400019 0.0441 NA 1 0172 NA 0-9949 0.8640 3.44 l 2.94 D041400020 < LOQ (0.0107 ug/g) NA 1.0160 NA 0.9949 0.8640 9.43 l 8.06 D04140002) < LOQ (0.0107 ug/g) NA 1.0160 NA 0.9949 0.8640 0.00 1 0.00 D041400024 < LOQ (0.0107 ug/g) NA 0.9845 NA 09949 0.8640 83.0 l 73.2 D041400025 0.0732 NA 0.9510 NA 0.9510 NA 0.9510 NA 0.9510 NA 0.9510 CV2222M C9223IM C92233M C92239M C92252M C92333F C92334F C92348F C92354F C9235IF NA 1 0021 NA 0.9949 0.8640 3.96 1 3.43 D04I400026 < LOQ (0.0107 ug/g) NA 0.9510 NA 1.0036 NA 0.9949 0.8640 2.78 l 2.41 D041400027 <L O Q (0.0107 ug/g) NA 0.9510 NA 0.9986 NA 09949 0.8640 0.00 1 0.00 D041400028 < LOQ (0.0107 ug/g) NA 0.9510 NA 1.0052 NA 0.9949 0.8640 261 1 225 D041400031 0.225 141 NA 0.9510 NA 09930 NA 0.9949 0.8640 281 1 246 D041400032 0.246 0.0653 0.0923 NA 0 9510 KA 0.9977 NA 0.9949 0.8640 125 109 D041400033 0.109 NA 0.9510 NA 1.0419 NA 0.9949 0 .8 6 4 0 112 93.0 D041400034 0.0930 NA 0.9510 NA 1.0545 NA 0.9949 0 .8 6 4 0 186 l 153 D04I400035 0.153 NA 0.9510 NA 1.0607 NA 0-9949 0.8640 16.9 l 13.8 D041400039 0.0131 NA 0.9510 NA 0.9868 NA 0-9949 0.8640 7.29 l 6.42 D041400039 < LOQ (0.0107 ug/g) NA 0.9510 C92360F CV2362F C92369F CV2374F C92383F NA 1.0691 NA 0-9949 0.8640 109 l 81.4 D041400040 0.0884 NA 0.9510 NA 1.0245 NA 09949 0.8640 35.3 29.9 D041400Q41 0.0299 NA 0.9510 NA 1 0813 NA 0.9949 0 .8 6 4 0 140 112 D041400042 0.112 NA 0.9510 NA 1.0801 NA 0.9949 0 .8 6 4 0 138 M l D04I400045 0.111 59.9 NA 0 .9 5 1 0 NA 1.0817 NA 0.9949 0 .8 6 4 0 187 150 D041400046 0.150 0.0872 0.0522 NA 0.9510 G nap 9 1.0 ppm C9226IM C92266M C92270M C91278M C92281M C92283M C92309M C9231IM C923l*M C92321M C92410F C92416F C92419F C92423F C92430F C9243IF C'92439F C92443F C9245IF C92452F NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA High - Not Confirmed NA NA 0.9963 0.9909 1.0076 1.0037 ] .1X152 1 0182 0.9823 0.9877 0 9963 0 9981 1.0894 0.9916 1.0143 0.9980 0 9956 1.0113 1.0007 0.9890 0.9974 0.9927 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0.9949 0 .8 6 4 0 0 .8 6 4 0 0 .8 6 4 0 0.8640 0 .8 6 4 0 0 .8 6 4 0 0 .8 6 4 0 0 .8 6 4 0 0 .8 6 4 0 0 .8 6 4 0 0.8640 0 .8 6 4 0 0.8640 0 .8 6 4 0 0 .8 6 4 0 0 .8 6 4 0 0 .8 6 4 0 0 .8 6 4 0 0 .8 6 4 0 0 .8 6 4 0 289 20 429 20 737 20 218 20 270 20 636 20 643 MS 20 498 20 359 10 672 20 623 20 127 20 294 20 479 20 360 20 570 20 934 20 667 20 856 20 5037 7515 12702 3781 4660 10141 568 2084 8678 6249 10717 10911 2177 5114 8351 6178 9900 16395 11614 14968 D051500017 D051500018 DOS 1500019 DOS1500020 DOS1500021 D 0 5 1500025 D041400055 D051500027 D051500028 D051300029 DOS1500033 D051500034 D051500035 DOS1500036 D 05I5 00045 D 0 5 1500037 D 0 5 1500041 D 0 5 1500042 D051500043 DOS1500044 5.04 7.51 12.7 3.78 4 .6 6 10.8 0.57 2.08 8.68 6.25 10.7 10.9 2.18 5.11 1.35 6.18 9.90 16.4 11.6 15.0 61.2 6.21 3.80 45.2 9.63 4.35 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.9510 0.9510 0.9510 0.9510 0.9510 09510 0.9510 0.9510 0.9510 0.9510 0.9510 09510 0.9510 09510 0.9510 0 .9 5 1 0 0.9510 0.9510 0.9510 09510 A - QualitUive oety NR a Not Reponed PFOS a Perfluoiooaaaeaulfoaate NA a Nut Applicable PFOSA Perfluofooclaaeaulfonamide LOQ a UtttofQuaBtMioa PF5>ft*i4 ~ P-tAuait--(I*fttliif',ainidnarflfl1f EIFOSE Narrow Range N-Ethyl Pmrhmmnnaiuiuifnnamidn ethyl alcohol Date Eatcred/AariyM: 05/01/00. 05/26/00 MMH, CSH PFOSEA Perfuorooctaite aulfooyl etbylamide Date Vcoficd/AariyM: 03/22J boj. 04/03/01 kyh. 5/22/01 romh M554 C8F17S02N((H)CH2C00) Purity EatereriVeofied: 03/29/01 LAC / 04/03/01 1CIH PFOSA Parity C o rrec tiv a Factor Uakaowa Uakaowa Uakaowa Itafcanwa Urimom* llekacua* Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Unkaowa Uakaowa Unkaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa 11ntana* Uakaowa 1Inlrvnn 1Inlranm Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa Uakaowa PFOSA Cune 2.39 00 209 0.00 239 262 291 196 283 260 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .2 3 0.000 0.000 0.000 0.000 0 .0 0 0 0000 0.000 0.37 009 0 .0 0 0 0.08 0 .2 6 0.53 0.000 0.47 0.33 400 264 795 482 235 341 169 355 251 245 566 729 494 592 380 465 524 631 464 421 PFOSA Diteli** Factur l l 1 1 1 1 1 1 1 1 1 1 1 l l I l 1 1 1 1 1 1 l 1 l 1 PFOSA C r ic Cune. 2.51 0.00 2.20 0 00 224 246 280 188 282 259 000 0.00 0.00 0.24 0.00 0.00 0.00 0.00 0.00 000 000 0.37 0.090 0.00 0.09 0.26 0.54 0.00 0.46 0.32 422 280 829 505 246 352 180 378 265 258 546 773 512 624 402 484 551 671 490 446 Filename A04I7000O4 DOXHUOOCU A041700005 DMI400MA DM1400017 DM 14000 I t AW1700017 ACW17000 l i DOMI400075 DM1400076 D041400019 D M I400020 D04140002I DM1400024 D ot 1*00025 DM1400026 DM1400027 DMI 400028 DM1400031 DM 14000)2 DM1400033 D M 1 400034 DM1400035 DM1400038 DM1400039 DM1400040 DMI400M1 DMI40UM2 DM 1400045 DM 1400046 DM1400047 DM140M9 DM 1400052 DM1400053 DM1400054 DM1400055 DM 1400059 DM 1400060 D M 1400061 DM1400062 DMI40U63 DM1400066 DM1400074 DM1400068 DM1400069 DM1400070 DM140007) 3MEnvironmental Laboratory Page 179 3M Medical Department Study: T-6316.1 A M U l f VdUdVS.2 Covaoce# 6329-228 Analytical Report: FACT-TOX-003 LRN-U2104 C eew tm lee dPFOSA un/n e r * Ree < LOQ (0.00644 ug/g) < LOO (0.0322 ue/el < LOQ (0 00644 ug/g) L O O <00322 u r 'il 71% 86% 94% 63% 92% 13% 1 < LOQ (0.0322 ug/g> < LOQ (0.0322 ug/g) < LOQ (0.0322 ug/g) < LOQ (0.0322 ug/g) < LOQ (0.0322 ug/g) < LOQ (a0322 u*/g> < LOQ (0.0322 ug/g) LOQ (0.0322 ug/g) < LOQ (0.0322 ug/g) < LOO (0.0322 ue/f < LOQ (0.0322 ug/g) < LOQ (0.0322 ug/g) LO Q (0.0322 ug/g) LO Q (0.0322 ug/g) < LOQ (0 0322 ug/g) < LOQ (0 0322 ug/g) L O Q (0 0322 ug/g) LO Q (0 0322ug/g) LOQ (0.0322 ug/g) < LOO <0 0322 ua/) 0422 0.210 0.129 0.505 0.246 0.332 0.110 0.371 0.265 0.546 0.773 0.312 0.624 0.402 0.414 0.551 0.671 0.490 0.446 Meaa PFOSA b l LOO <1.00 82% 79% 88% LOO LOO 0 37) 0.350 Study: Pigduct NumberiTc Subsuota): Mauu: Mctbod/Kevieioa: Analytic^ EtpufcncM Syncro Number: lu m iroani Soilwwe/Vcfitoo Dale o ExUacUoa/Aaalytf: Date o Analyitt/Analytt. Date ui Data Reduction/Aadyal: Sam ple D ata TOX003,104 Week Dietary Cairinogenirily Study with Narrow Range (98.1%) N-Ethyl Perfluorooctaartulfnaamido Blume! in Rau T-6316 (EtFOSE-OH) R a Liver ETSS-6.0dt ETS-l-7.0 Amalia 062491. Davey 070799 M a u L y u 3.3 3.4 Filename: See Bdow R-Squamd Valu See Auaduuenu Slope: S eeA tu d u n e n u Y-Ialercept: See Aaaduneou 4/t4/QQ SAL/RWW 4/17/00.3/13/00 lAS/MMH/TAS 4/18/00, 5/1WO, 05/08/01IAS/MMH RSD SM. Dt. MS/MSD KPD NA NA NA NA 9% 39% 9% NA NA NA NA 504 0.187 20.3 0 112 W KEK 10S Ra T L IV L K G nap D e M e tt u d B I k M uruBlk QC Gruep g DOppm Greap 9 1.0 ppm Sample R B i0 4 ]4 0 0 -H 2 0 B l k -l RBi CMMO lO BIk 2 RBI ,W 14t-lj verBlk 1 KBI.IHI40U l.iveiBlk 2 l'V 2197/M /(;8 -M S-l C92I97/MA18 MSO ) C92I95/M/G8 MS-I C92195/MA.8-MSD 1 RB1.04I400 MS 1 RB1.04U0OMSDI C92I97M C92203M C9 2W 8M C92209M C92218M C9I222M C9223IM C92233M C92239M C92252M C92333F C92334F C924SV C92354F C92358F C92360F C92362F C92369F C92374F C92383F C9226IM C92266M C92270M C92278M C9221IM C92283M C92309M C923IIM C923I8M C9232IM C924I0F C924V6F C92419F C92423F CV2430F C92431F C92439F C92443F C9241F C92452F Surrogate VeriHed NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA lot 98-0207-0303 3 PFOSAA OM Parity Cerrectioa F a c ter 0.9760 0.9760 0.9760 09760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0 .9 7 6 0 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0 .9 7 6 0 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.97(A) 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 0.9760 NR * Not Reponed NA Not Applicable LOQ Limit o Quaauimoo Data HatemVAaalyil: Date VenTied/Aaaly: Punty t a l a ed/V oitad. 03/01/00.05/26AW MMH, CSH 03/22/0) boj. 04/03/01 kjh. 3/22/01 n 03/29/0) LAC 104/03/01 KJH PFOSAA Purity C o rrectio n F a c ter Unknown Ualmowi Uoknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Udmowa Unknown Unknown Unknown Unknown Unknown Unknown Uolcnnwn Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown PFOSAA Cone. 0 .0 0 0 .0 0 0 .0 0 0.00 224 260 251 254 232 266 0.00 0 .0 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0 .0 0 0.00 0.00 0.00 0.00 0.00 0 .0 0 574 383 112 762 600 527 497 545 482 578 580 88.6 420 813 482 698 404 753 162 771 PFOSAA DUnUen Factor 1 ! 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 20 1 l 20 t l 1 1 1 l 1 \ PFOSAA Calc. Cone. ____ t f t 0.00 0.00 0.00 0.00 215 249 240 243 222 266 0.00 0.00 0.00 0.00 0.00 0 .0 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0 .0 0 0.00 0.00 0.00 591 396 2279 771 612 531 511 565 496 593 545 1831 424 135 496 707 414 710 115 796 Filenaan A 0 4 1700004 D 0 4 1400004 A041700005 DIM1400005 D 0 4I4 00017 D 0 4 1400018 A041700017 A 0 4I7 00018 D04140007S D 0 4 1400076 D041400019 D 0 4 1400020 D41400021 D04I40Q024 D041400025 DQ41400026 D 0 4 1400027 D 0 4 1400028 D04I400031 D041400037 D041400033 D 0 4I4 00034 D041400035 D 04I4 0003S D041400039 D041400040 D 0 4 1400041 D 0 4 1400042 D041400045 D 0 4 1400046 D 0 4 1400047 D041400041 D0515000I9 D041400052 D041400053 D 0 4 1400054 D041400055 D 0 4 1400056 D 0 4 1400059 D041400060 D 0 4 1400061 D 0 5 1500034 DQ41400063 D 0 4I4 00066 D 0 4 1400074 D 0 4 1400067 D041400068 D 0 4 1400069 D 0 4 1400070 D041400077 C tM utnliM e i PFOSAA /nor % Re < LOQ (0.0125 ug/g} < LOO (0.0314 ue/r) < LOQ (0.0125 ug/g) 1 .0 0 (0.0314 u i/i) 74% 86% 12% 13% 73% 87% < LOQ (0.0314 ug/g) < LOQ (0.0314 ug/g) <L O Q (0 .0 3 M u g /g > < LOQ (0.0314 ug/g) LOQ (0.0314 ug/g) < LOQ (0.0314 ng/g) < LOQ (0.0314 ug/g) < LOQ (0.0314 tlg/g) < LOQ (0.0314 ug/g) < LOO <0.0314 u*/> < LOQ (0.0314 ug/g) < LOQ (0.0314 ug/g) < LOQ (0.0314 ug/g) LOQ (0.0314 ug/g) < LOQ (0.0314 ug/g) < LOQ (0.0314 ug/g) < LOQ (0.0314 ug/g) < LOQ (0.0314 ug/g) < LOQ (0.0314 ug/g) L O O (0.03l4ua/>) 0.591 0.396 2.21 0.771 0.612 Q.S31 0.518 0565 0.496 0.593 0.S4S 1.13 0.424 0.835 0.496 0.707 0.414 0.710 0.815 0.796 Mean PFOSAA Ft LOO LOO 10% 12% 10% LOO LOO 0 .7 3 6 0.771 nqt StA Dev. MS/MSD KPD NA NA NA NA 15% 2% 11% NA NA NA NA 74.9 0.551 53.2 0.411 V erttd NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Na NA NA NA NA NA NA NA NA NA NA NA NA EtFOSE Old Parity C e rrec tien P a c tar 0.9770 0.9770 0.9770 09771) 0.9770 09770 0.9770 0.9770 0.9770 0.9770 0.9770 0.9770 09770 0.9770 0.9770 0.9770 0.9770 0.9770 0.9770 0.9770 0 9770 0.977Q 0.9770 0.9770 0.9770 0.9770 0.9770 0.9770 0.9770 0.9770 0.9770 0.9770 0.9770 0.9770 0.9770 0.9770 0.9770 0.9770 0.9770 0.9770 0.9770 09770 0.9770 0.9770 0.9770 0 9770 09770 0.9770 0.9770 0.9770 EtFOSE Purity CerrecUoB Pactar 0.1190 0 8190 0 1190 0 8890 0 1890 0 8190 01890 0 8890 0.8190 0 8890 0.1890 0.1890 0.1190 0.1190 01190 0.1190 01890 0.1190 0 1190 0 >190 08890 Q.%894 0.8890 0.8890 0 8890 0.8190 0 8890 0.1190 01190 0.1890 0.8190 0 1890 0.1190 0.8190 0.1190 01190 0 1190 0.1890 0.8190 0 8890 0.1190 01190 0.8890 0.1190 0 1190 0.8190 01190 0.1190 0.1190 Q.1190 PFOSA PuduonioctM ee PFOSAA - Perfluocoortiim ul EtFOSE Narrow Range N-Etbyl Petflyoroocuaratilioeanudo etbyi al PFOSEA Perfuoroocta ae ntlionyl etbyiaeude M356 a C8FI7S02N((H)CH2COO) EtFOSE Cue. *t 0.00 0 .0 0 0.00 000 277 231 472 459 NR NR 000 0.00 0.00 0.00 0.00 0.00 0.00 000 0 00 000 0 00 .O 0 00 0.00 0.00 000 0.00 000 0.00 0.00 0.00 0.00 0.00 0.00 0.00 000 0.00 0.00 ooo 0.00 0.00 000 0.00 60.4 000 0.00 0.00 000 0.00 000 EtFOSE D ila tie n Facter 1 1 1 1 1 1 NR NR 1 1 1 l 1 EtF O S E Cale. Cene. 0 00 0 uO 0 00 ooo 265 221 452 439 NR NR 000 0 00 0 .0 0 0 .0 0 0 00 000 000 0 uO 0 00 0 00 0 00 0.00 0 O 000 000 000 0 .0 0 000 0.00 000 000 0O 0 00 0.00 000 0.00 000 0.00 0.00 000 0.00 000 0 .0 0 55 1 000 ooo .0 ooo 0 .0 0 0.00 3MEnvironmental Laboratory Page 180 3M Medical Department Study: T-6316.1 Covaoce# 6329-228 F ttcn a m MM1100004 0041400004 A041700005 D 0 4 1400005 D041400017 D04I4000U A041700017 A 0 4I7 0001 NR NR D04I4000I9 D041400020 D 0 4 1400021 D 0 4 1400024 D041400Q2S D 0 4 1400026 D 0 4 1400027 D 0 4 1400021 D041400031 D 0 4I4 00032 D041400033 D 0 4 1400034 D 0 4 1400035 D 0 4 1400031 0041400039 D041400040 D041400041 D041400042 004140004$ D 0 4 1400046 D 0 4 1400047 D04140004? DG4140Q049 D041400052 D 0 4 1400053 D 0 4 1400054 D0414000S5 D 04I400056 D041400059 D 0 4 1400060 D041400061 D 0 4 1400062 D 0 4 1400063 D 0 4 1400066 D041400074 D041400067 D04140006 D041400069 D 0 4 1400070 0041400073 C*accnUaUoa Mum f E IF O S E EtFOSE u /ftf R m til < LOQ (0.0559 ug/g) A < LOO (0.0559 us/ul A <LOO < LOQ (0.0559 ug/g) A < LOO (0.0559 ut/Bl A <LOO 92% A 77* A 4* 154* 149* A A 151* NR A NR A NR <L O Q ( 0.0559 ug/g) A <L O Q ( 0.0559 ug/g) A < LOQ (0.0559 ug/g} A <L O Q ( 0.0559 ug/g) A < LOQ {0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOO i 0.0559 uu/ak A <LOO < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0S59 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOO (0.0559 u* /tl A <LOO < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOO (0.0359 ua/a> A <LOQ < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (0.0559 ug/g) A < LOQ (Q.05S9 ug/g) A < LOQ (0.0559 ug/g) A < LOO (0.0559 u r /il A 0.00 3MEnvironmental Laboratory SU.D**. MS/MSD KPD Analytical Report: FACT-TOX-003 LRN-U2104 Page 181 3M Medical Department Study: T-6316.1 Covance# 6329-223 Product NumberCTea Subitanee): Matrix: Metbod/Revutoa: Aanlyucal E ifftB aii Syaem Number. ItstnuMM Soft* Mt/Vcntoa : Dam of Exincuoa/Aaalysi: Date u Aaalytu/Aialya: Dale of D au Reductjoa/Andy. S a n flt Data 70X003, )04 Week Dietary CarunugeQicHy Study with Narrow Raage (98.1%) N-Etbyl Petfluora.-iwesuUoBuauSo Ethn&al is Rat* T -6316 (EiFOSE-OH) Rjt Liver filename: See Below ETS I 6.0 A ETS-S7 0 Amelia 062498. Davey 070799 MauLyax J.3 A 3.4 R-Squared Value; Slope; Y Intercept; See Auadunenu See Attadunenu See Attachments 4/I4/00SAL/RWW 4/17W . 5/15/00 lAS/MSllWAS 4/18/00, 5/16/00, 05/08/01 IAS/MMII R a t L iv e r w eek 104 G roa p Ova* M a ted Blk Maini Blk QC Group 1 0.0 ppm G ro u p * l.Opptu A s Q ualm ive only Date Entered/Andya: Date Veaiied/Aadya; P u n y Eatered/Vcofied: Sam plet RBI.04l400-H20Blk 1 RBU4l40tt-lttOBIk-2 RBLOmOO-verBIkl KBL04I400 IjverBlk-2 C92I97/M /G 8-M S -I O II97/M /G 8-M S D -1 C9219SIMJG8-MS-1 C 9 2 195/M /G 8M S D -I RB1.041400 MS I RB1.04I4(-MS1>1 C92I97M C92203M C922U8M C922Q9M C922I8M C92222M C9223IM C92233M C92239M C92252M C92333F C92334F C92348F C92354F C92358F C92360F C92362F C92369F C92374F C92383F C9226IM C92266M C92270M C92178M C92281M C92283M C92309M C923IIM C923IIM C92321M C92410F C92416F C92419F C92423F C92430F C9243IF C92439F C92443F C92451F C92452F Verified NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 05/01/00. 05/26/00 MMH. CSH 03/22/01 boj. 04/03/01 kjh. 5/22/01 mmb 03/29X11 LAC / 04/03/01 KJH A NB 11 >047.80 MSS* O ld Purity Corredivi! MSS* Purity Correction Factor 0 9989 0.9989 09989 0 9989 0 9989 0.9989 0 9989 0.9989 0.9989 C a la o * 1lakauw Uakaow Colmowa Uakaow Cnkaowi tlakwnww Ca kao wa Cakao* 0 9989 Cakao 0.9989 0 9989 0.9989 Unkao Cakao Cokao 0.9989 0.9989 0.9989 9989 Unkaoa Cakao Cakao Cakao* 09989 0.9989 0.9989 09989 0 9989 Cakao Uakaow Unknown Uakaow Uakaow 0.9989 0.9989 0 9989 0 9989 0.9989 0 9989 Uakaow Uakaow Unknown Uakaow Unknown Unkao* 09989 0.9989 Uakaow Uakaow 0.9989 0.9989 Unkao* Uakaow 0 9989 0.9989 Uakaow Cakao* i 0.9989 0.9989 Unknown Uakaow 0.9989 09919 0.9989 .9989 09989 0.9989 0.9989 0.9989 Unknown Unknown Uakaow llot-nniM Unkao C a la o wn Unkao Cakao 0.9919 0.9989 0.9989 0.9989 0.9989 0.9919 Uakaow Unkao Unkao Unkao Unknown Ubkaow Not Reported NA = Not Applicable LOQ * Limit o( Quantitation MSS* CMC. 0.00 0.00 0.00 0.00 361 370 316 331 256 257 0.00 0 00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 230 314 402 313 194 306 84 212 240 201 454 486 318 385 245 267 339 302 310 268 MSS* DUutleu Fader 1 1 1 1 1 1 1 l 1 1 l l 1 1 l 1 1 1 I 1 1 1 1 1 1 l 1 1 1 1 l 1 1 l l 1 1 1 1 1 1 1 1 1 1 l 1 1 1 l MSS* Calc. Cone. 0.00 0 .0 0 0 .0 0 0.00 345 354 303 317 245 257 0 .0 0 0.00 0 .0 0 0 .0 0 0.00 0.00 o .o o 0 .0 0 0.00 0 .0 0 0.00 0.00 o .o o 0.00 0.00 0.00 0.00 0 .0 0 0.00 0.00 231 317 399 312 193 300 85.9 214 241 202 417 491 313 386 246 265 339 306 311 291 FUeauue A041700004 D 0 4 1400004 A041700005 13041400005 D041400017 D04I4000I8 A04I700017 A041700018 D 0 4 1400075 D 0 4 1400076 D 0 4 1400019 D041400020 D 0 4 1400021 D 041400024 D 041400025 D041400026 D 0 4 1400027 D 041400028 D 041400031 D 0 4 1400032 D 0 4 1400033 D041400034 D 0 4 1400035 D 0 4 1400038 D 041400039 D041400040 D 041400041 D 041400042 D 041400045 D 0 4 1400046 D 041400047 D 041400048 D041400049 D041400052 D0414000S3 D 0 4 1400054 D041400QS5 D 041400056 D 041400059 D 041400060 D041400061 D 0 4 1400062 D 041400063 D 041400066 D 0 4 1400074 D 041400067 D 041400068 D 0 4 1400069 D 0 4 1400070 D 041400073 Concentration f MSS6 tte /t nr 1* Ree < LOQ (0.0307 Uf/g) < LOO ( 0.0307 ut/e) < LOQ ( 0.0307 ug/g) < LOO ( 0.0307 ui/el 119% 122% 103% 108% 80% 84% < LOQ ( 0.0307 ug/g) < LOQ (0.0307 ug/g) <LQQ(Q.03Q7ut/g) < LOQ ( 0.0307 ug/g) < LOQ ( 0.0307 ug/f) < LOQ ( 0.0307 ug/g) < LOQ (0.0307 ug/g) < LOQ ( 0.0307 ug/g) < LOQ (0.0307 ug/g) < LOO <0.0307 ue/fl < LOQ (0.0307 ug/g) < LOQ (0.0307 ug/g) < LOQ (0.0307 ug/g) < LOQ (0.0307 ug/g) < 1 0 0 ( 0 .0 3 0 7 ue/el < 1.00 ( 0.0307 ua/f 1 < LOQ (0.0307 ug/g) < LOQ (0.0307 ug/g) < LOQ (0.0307 ug/g) < LOO ( 0.0307 us/ffl 0.231 0.317 0.399 0.312 0.193 0.300 0.0859 0.214 0.241 0.202 0.417 0.491 0.313 0.386 0.246 0.265 0.339 0.306 0.311 0.291 I <LOO <LOQ 121% 105% 82% <LOO <LOO 0.250 0.336 RSD Surrogate PFOSEA O U Purity Sid. Dev. Verified Correction MS/MSD RPD Factor NA NA Q.990 NA NA 0.9930 NA NA 0.9930 NA NA 09930 NA 0.9930 2.42% NA 0.9930 NA 0.9930 4.58% NA 0.9930 NA 0.9950 5% NA 0.9930 NA 0.9930 NA 0.9930 NA Q.990 NA 0.9930 NA 0.9930 NA 0.9930 NA 0.9930 NA 0.9930 NA NA 0.9930 NA NA 0.9930 NA 0.9930 NA 0.9930 NA 09930 NA 0.9930 NA 0.9930 NA 0.9930 NA 0.9950 NA 0.9930 NA NA 0.9930 NA NA 0.9930 NA 0.9930 NA 0.9930 NA 0.9930 NA 0.9930 NA 0.9930 NA 0.9930 NA 0.9930 NA 0.9930 34.7 0.0865 NA NA 0.9930 0.9930 NA 0.9930 NA 0.9930 NA 0.9930 NA 0.9930 NA 0.9930 NA 0.9930 NA 0.9930 NA 0.9930 22.2 NA 0.9930 0.0748 NA 0.9930 P f OS * PFOSA Petfluomoriaaeiiilfnaamide PFOSAA Peafluctnon narwiUnaanudoiTtaci EtFOSE Narrow Raage N-Elbyl PMfhwrooaaMHitfotMido etbyl al PFOSEA Petiuocoooaae sul/onyi etbylaoude M556 C8FI 7S02N((H)CH2C00) Analytical Report: FACT-TOX-003 LRN-U2104 PFOSEA Parity C o rrectio n Factor Unknown Uakaow Unkao Unknown Uakaow Uakaow Unkao Unknown Uakaow Unknown Uakaow Uakaow Unknown Unknown Unknown Uakaow Uakaow Unknown Udmowa Unknown Cakao Uakaow Uakaow Uaknowi U^aow Uakaow Unknown Uakaow Uakaow Unknow Unknown Uakaow Unknown Unknown Unknown Unkao Unknown Uakaow Unkao Uakaow Unknown Uakaow Unkaowa Unknown Unknown Uakaow Uakaow Uakaow Ualeno Unknown PFOSEA Cm c. -* 0 .0 0 0.00 000 000 208 254 442 353 351 393 000 0 .0 0 0 .0 0 0.00 0 .0 0 0 .0 0 0.00 0.00 0 .0 0 0.00 0 .0 0 0.00 0 00 0 .0 0 0.00 0.00 0.00 0 .0 0 0.00 0.00 0 .0 0 0.00 000 0 .0 0 0.00 0.00 000 000 0 .0 0 0.00 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0.00 0 .0 0 0 .0 0 0.00 0 .0 0 PFOSEA Dilativa Factor 1 1 1 1 l 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 l 1 1 1 1 1 1 l 1 1 1 1 1 1 1 1 1 1 1 PFOSEA Calc. Coite. 0.00 000 0.00 000 199 243 423 338 343 376 000 o .o o 0.00 0.00 0.00 000 0.00 000 000 0.00 000 0.00 0.00 0.00 0.00 0.00 0.00 0.00 000 0.00 0.00 0 00 0.00 0.00 0.00 0 00 0.00 C.0 0.00 0 .0 0 0 .0 0 0.00 0.00 0.00 0.00 0.00 0.00 000 000 000 Fumarne A 0 4 1700004 D 0 4 1400004 A041700005 D04I400005 0041*00017 (XM14000 It A041700017 A04I7UUG1I D041400075 0041400076 D041400019 D04I40020 D04140U021 D041400024 D041400025 D041400026 D 0 4 1400027 D041400021 0041400031 DO4I4032 D 0 4 1400033 D04I400034 D04I4035 D04I400038 D 0 4 1400039 D041400040 D 0 4 1400041 D 0 4 1400042 DOtl 400045 DO* 1400046 D041400047 D04I4U004I D 0 4 1400049 D041400032 D041400053 D041400054 DO4I4053 DC440056 D04I40059 D 0 4 1400060 D 041400061 DO*1400062 D 0 4 1400063 D041400066 DOt 1400074 DOt1400067 D041400061 D O t1400069 D O t1400070 D 0 4 1400073 Concentrati ut PFOSEA un/e or % Ree < LOO IO 0308 us/ei < LOO 10 0308 us t i < LOQ ,0.0308 ug'g) < L(X> 10 030* v a 'ti 69 2% 84 4% 1444 1154 1124 1234 < LOQ (0.0308 ug/g < LOQ (0 030 ug'g < LOQ (0 0308 ug/gj < LOQ (0.0306 ug/g < LOQ (0.0308 Ug/g) < LOQ (0 0306 ug/g < LOQ (0.0308 ug/g) < LOQ ,0.0308 ug/g < LOQ ,0.0308 ug.g) < LOO 10.0306 un e i < L(X) (0.0306 ug/g) < LOQ ,0 0308 ug/g) < LOQ ,0.0308 ug/g) < LOQ (0.0308 ug/g) < LOQ (0.0308 ug/g < LOQ ,0.0301 ug/g) < LOQ (0.0308 ug/g) < LOQ (0.0301 ug/g) < LOQ (0.0308 ug/g) LOG (0.0308 U d ii < LOQ ,0.0308 ug/g) < LOQ (0.0306 ug/g) < LOQ (0.0301 ug/g) < LOQ (0 0301 ug/g < LOQ (0.0306 ug/g; < LOQ (0.0308 upg) < LOQ (0 0306 ug/gj < LOQ <0.0308 ug/g) < LOQ ,0.0306 ug/g) < LOO iOllO u i / u < LOQ (0.0306 ug/g) < LOQ (0.0306 ug/g) < LOQ (0.0306 ug/g) < LOQ <0.0306 ug/g) < LOQ (0.0301 ug/g) < LOQ (0.030g ug/g < LOQ (0.0308 Ug/f) < LOQ ,0.0306 Ug/g) < LOQ <00306 ug/g) < LOQ ,0 0306 Uk'kJ 3MEnvironmental Laboratory 05/29/2001 Page 182 3M Medical Department Study: T-6316.1 Cuvance# 6329-228 Mean PFOSEA 'I <u>o <UK> 77% 130% 117% 1 <LOO <LOO 1 <LOO 1___ m i RSD SO. Dev. MS/MSD RPD NA NA NA NA 20% 22% 9% NA NA NA NA NA NA NA NA Study: Piu Iuli NurabefjTe SubMaBte): M am MelhWtUvuioa: Analytical Equipoen Syeo) Number: ItuUutueal Suflwue/Ventoa: Oule u EiUamon/AjuJyM: Dale jf Aoulyua'AsaJy: Dele o DuU Reduction/AaiJyB: S am ple Orna TOXUU3, 104 Week Dietary Carcinogenicity Study wilh Narrow Range (98.1%) N-Eibyl PerilooroocUnesulfoflnmido T-6316 (EtFOSE-OH) Ral Liver Filename: SeeAttachment ETS-8-6.0 & ETS-8-7.0 R-SquaredValue: See Attachment* Amelia 062498. Davey 070799 Slope: SeeAttachment* Ma&sl.yu 3.3 A 3 4 Y-Intercept: See Altatmenle 4/14/IX) SAL/RWW 4/17/00, S/15/001AS/MMH/IAS 4/18/00, 5/16/00,05/08/01 1AS/MMH in Rata W E E K 105 HAT LIV E R Group Dole Sample 1 Method Blk M u m Blk QG 3UUpfh Group 1 0.0 ppm RB 1.041400-H 2 0 B lk -l RBlJ)41400-H2OBlk 2 RBI4I400 IjvtuBlk-l KBI.04i40UljverBlk -2 C92I97/M/G8MS 1 092I97/M /G8M SIM C9 21 95/M /G 8-M S -1 (V2I95/M A8M SDI RBL04I400-MS 1 R B l-041400-M S D l C92I97N1 C92203M C92208M C92209M C922I8M C92222M C92231M C92233M C92239M C92252M C92333F C92348F C92354F C92358F C92360F p ro s Calc. Cuoc. "tflt 0.00 0 00 000 4 77 226 224 403 324 232 261 44.1 2.94 1.06 73 2 3.43 225 109 13 8 6.42 88.4 Group 9 l.Oppm C92383F C9226IM C92266M C92281M C92283M C923IIM C923I8M C92410F 2084 L-- -------;--------------1 C92452F Date EaMed/A ady: D ue Veofwd/Aaalya. Punty Eatered/Vcrified: 05/01/00.05/26/00 MMH. CSH 03/22/0) bo), 04/03/01 kjfa, 5/22/01 mmh 03/29/01 LAC / 04/03/01 KJH C o a c e n lr u U e a aTPFOS u u / e a r * Bee < LOQ (00267 ug/g) < 1.00 (00107 u t/t) < LOQ (0.0267 ug/g) < 1 .0 0 (0.0107 ut/t) 78% 71% 137% 110% 76% 85% 0.0441 < LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) <LO Q <0.0107 ug/g) 0.0732 < LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) < LOQ (0.0107 ug/g) 0.225 0.246 0.109 0.0930 0.153 0.0131 < LOQ (0.0107 ug/g) 0.0g|4 0.0299 Mean FF0S ii <L00 71% 124% 80% 0.150 0.087)7 3.78 4.66 10.8 2.08 8.68 10.7 10.9 11.6 9.63 NA Not Applicable LOQ n Limit of Quamitalios BSD Sid. Dev. MS/MSD RPD NA 1% 22% 12% 141.4 0.0522 61.2 PFOSA Calc. Cone. -- "Bfc 2.51 0.00 2.20 0 .0 0 224 246 280 188 282 259 0 .0 0 0.00 0.00 0.24 0 .0 0 0 .0 0 0 .0 0 0.00 0 .0 0 0 .0 0 0.00 0.37 0.09 0 .0 0 0.09 0 .2 6 0.54 0.00 0.46 0.32 422 280 829 505 246 352 180 378 265 258 546 773 512 624 402 484 551 490 446 C u n c ea lr u tl M i PFOSA ua/a *r % Bee < LOQ (0.00644 ug/g) < LOO (0.0322 ue/t) < LOQ (0.00644 ug/g) < LOO (0.0322 ua/c> 78% 86% 94% 63% 92% 85% < LOQ (0.0322 u*/g) < LOQ (0.0322 ug/g) < LOQ (0.0322 ug/g) < LOQ (0.0322 ug/g) < LOQ (0.0322 Ug/g) < LOQ (0.0322 ug/g) < LOQ (0.0322 Ug/g) < LOQ (0.0322 Ug/g) < LOQ (0.0321 ug/g) < LOO (0.0322 ut/e l < LOQ (0.0322 ug/g) < LOQ (0.0322 ug/g) < LOQ (0.0322 ug/g) < LOQ (0.0322 ug/g) < LOQ (Q,Q3U ugfg) < LOQ (0.0322 ug/g) < LOQ (0.0322 ug/g) < LOQ (0.0322 ug/g) < LOQ (0.0322 ug/g) < LOO (0.0322 uo/ii 0.422 0.280 0.829 0.505 0.246 0.352 0.180 0.378 0.265 0.258 0.546 0.773 0.512 0.624 0.402 0.484 0.551 0.671 0.490 0.446 Mean PFOSA .. r t <LOO <LOO 2% 79% 88% <LOO <LOO 0.371 0.550 BSD Sid. Dev. MS/MSD RPD NA NA 9% 39% 9% NA NA NA NA 50.4 0.187 20.3 0.112 PFOSAA Calc. Cauc. ua/g 0.00 000 0.00 0.00 215 249 240 243 222 266 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0.00 0.00 0 .0 0 0 .0 0 0.00 0 .0 0 0 .0 0 0 .0 0 0.00 0 .0 0 0 .0 0 0.00 0.00 59) 396 2279 778 612 531 5)8 565 496 591 545 1831 424 835 496 707 414 780 885 796 PFOSA * PerfbioroontnetulfonMnidc PFOSAA a P#fliinmrtiiiirftaifAwl. EiFOSE a Narrow Rang* N -Eabyl Parf)ummnw ulf<*nf|,t`dn ethyl alcohol PFOSEA s Petfuotooclane aulfoayl cthylamide M556 a C8F17S02N((H JCH2COO) CMaccalruUeu ufPFOSAA e/e e r % Rac < LOQ (0.0125 ug/g) < LOO (0.0314 ue/et < LOQ (0.0125 ug/g) < 1 .0 0 (0.0314 uf/e) 74% 86% 82% 13% 73% 17% < LOQ (0.0314 ug/g) < LOQ (0.0314 ug/g) < LOQ (0.0314 ug/g) < LOQ (0.0314 ug/g) < LOQ (0.0314 ug/g) <LO Q (0.0314 ug/g) < LOQ (0.0314 ug/g) < LOQ (0.0314 ug/g) < LOQ (0.0314 ug/g) < LOO (0.0314 ue/l < LOQ (0.0314 ug/g) <LOQ (0.0314ug/g) <LO Q (0.0314 ug/g) < LOQ (0.0314 ug/g) < LOQ (0.03)4 ug/g) < LOQ (0.0314 ug/g) < LOQ (0.0314 ug/g) < LOQ (0.0314 ug/g) < LOQ (0.03)4 ug/g) < LOO (0.0314 u i/f) 0.591 0.396 2.279 0.778 0.612 0.531 0.5)8 0.565 0.496 0.593 0.545 I.S3I 0 .4 2 4 0.835 0 .4 9 6 0.707 0.414 0.780 0.885 0.796 Mean PFOSAA F i <LOO <LOO 80% 82% 80% <LOO <LOO 0.736 0.771 Analytical Report: FACT-TOX-003 LRN-U2104 BSD Sid. Dev. MS/MSD RPD NA NA 15% 2% 11% NA NA NA NA 74.9 0.551 53.2 0.411 ElFOSE Cate. Ceoc. 0.00 000 0.00 221 439 NR NR 0.00 0.00 0.00 0.00 000 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 55.1 0.00 0.00 0.00 0.00 0.00 0.00 Cancera rH ou uf ElFOSE Mean ElFOSE < LOQ ( 0 0559 ug/g> < LOQ ( 0 0559 ug/g) , 77% 14% NR < LOO (0.0559 u / n < LOQ ( O.OS59 ug/g) < LOO ( 0.0559 ue/r i < LOQ (0.0559 ug/g) < LOQ (0.0559 ug(g) < LOQ (0.0559 ug/g) < LOQ (0.0559 ug/g) < LOQ (0.0559 ug/g) < LOO ( 0.0559 ua/e) NR < LOO (0.0559 ut/e l < LOO (0.0559 ue/t > < LOO (0.0559 u t/ti < LOO (0.0559 ue/t) <LOO < LOO ( 0 0559 u t/t i < LOO (0.0559 u t/t i < LOQ (0.0559 ug/g) < LOO (0.0559 u t/ti A <LOO BSD Sid. Dec. NA ETS-8-7.0 3MEnvironmental Laboratory Lvc Weak 105 Page 183 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Appendix F: Example Calculations Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 Formula Used for Sera Analyses in Study FACT TOX-003 AR (ng/mL) x DF x PC2 x FV (mL) x 1.0 pg = R eported C oncentration (pg/m L) PC I EV (mL) 1000 ng Calculation Used for Group 9, Week 105, Animal ID C92430/F 199 ng/mL x 10 x 0.8640 x 1 mL x 1.0 pg = 1.73 pg/m L 0.9949 1 mL 1000 ng AR-- Analytical result from MassLynx summary DF-- Dilution factor PC I-- Purity Correction-1 PC2-- Purity Correction-2 FV-- Final extract volume (1.0 mL unless otherwise noted) EV-- Volume of sera extracted Formula Used for Liver Analyses in Study FACT TOX-003 AR (ng/g) x d curve (1) x SC x DF x 1.0 p g = R eported C oncentration (pg/g) d sample 1 0 0 0 ng (1> d curve is assumed to be: 1 g liver 5 mL H20 Calculation Used for Group 9, Week 53, Animal ID C92267M 497 ng/g x 1 g/ 5 mL x 0.8640 x 100 x 1.0 pg = 42.6 pg/g 1.0137 g/5m L 0.9949 1000 ng AR-- Analytical result from MassLynx summary 3 curve-- Density of the liver standard curve, assumed to be lg liver/ 5 ml water d sample-- Density o f the liver sample (g sample/ 5 mL H2 0 ) PC I-- Purity Correction-1 PC2-- Purity Correction-2 DF-- Dilution factor 3MEnvironmental Laboratory Page 184 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Appendix F: Example Calculations Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 Formula Used for Sera Analyses in Study FACT TOX-003 AR (ng/mL) x DF x PC2 x FV (mL) x 1.0 pg = R eported C oncentration (pg/m L) PCI EV (mL) 1000 ng Calculation Used for Group 9, Week 105, Animal ID C92430/F 199 ng/mL x 10 x 0.8640 x 1 mL x 1.0 pg = 1.73 pg/m L 0.9949 1 mL 1000 ng AR-- Analytical result from MassLynx summary DF-- Dilution factor PC I-- Purity Correction-1 PC2--Purity Correction-2 FV-j--Final extract volume (1.0 mL unless otherwise noted) EV-- Volume of sera extracted Formula Used for Liver Analyses in Study FACT TOX-003 AR (ng/g) x 9 curve (1) x SC x DF x 1.0 p g = R eported C oncentration (pg/g) 3 sample 1 0 0 0 ng (l) 3 curve is assumed to be: 1 g liver 5 m L H 20 Calculation Used for Group 9, Week 53, Animal ID C92267M 497 ng/g x 1 g/ 5 mL x 0.8640 x 100 x 1.0 pg = 42.6 pg/g 1.0137 g/5m L 0.9949 1000 ng AR-- Analytical result from MassLynx summary 3 curve-- Density of the liver standard curve, assumed to be lg liver/ 5 ml water 3 sample-- Density o f the liver sample (g sample/ 5 mL H20 ) PC 1-- Purity C orrection-1 PC2-- Purity Correction-2 DF-- Dilution factor 3MEnvironmental Laboratory Page 185 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Appendix G: Interim Certificates of Analysis 3MEnvironmental Laboratory i Page 186 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 L R N -U 2104 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 1(9/7/00) C entre Analytical Laboratories COA Reference #: 023-018B 3M P ro d u ct: PFO S, L ot 171 R eference#: SD-009 _____________________ P u rity : 86.4% _____________________ Test Nam e S p ecificatio n s Purity1 R e s u lt 86.4% Appearance Identification NMR Metals (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 (GC/MS) Related Compounds POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. Chloride 2 . F lu o rid e 3. Bromide 4. Nitrate 5. Nitrite 6 . Phosphate 7. Sulfate4 Organic Acids 5 (IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis6: 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. 0.017 wt./wt.% 2. 0.007 wt7wt.% 3. 1.355 wt./wt.% 4. 6.552 wt./wt.% 5. 0.003 wt./wt.% 6 . 0.004 wt./wt.% 7. <0.001 wt./wt.% 1 . 0 0 wt./wt.% 10.60 wt./wt.% None Detected 0.30 wt7wt.% None Detected Not Applicable-1 1 . <0.015 wt./wt.% 2. 0.27 wt./wt.% 3. <0.040 wt./wt.% 4. <0.009 wt./wt.% 5. <0.006 wt./wt.% 6 . <0.007 wt./wt.% 7. 8.82 wt./wt.% 1 . <0 . 1 wt./wt.% 2 . <0 . 1 wt./wt.% 3. <0.1 wt./wt.% 4. <0.25 wt./wt.% 1 . 12.08 wt./wt.% 2. 0.794 wt./wt.% 3. 1.61wt./wt.% 4. 10.1 wt./wt.% 5. 50.4 wt./wt.% C O A 0 2 3 -0 1 8 B 3MEnvironmental Laboratory Page 1 o f 3 Page 187 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Analytical Laboratories. Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 IN TER IM CERTIFICATE OFANALYSIS C e n tre A nalytical L ab o rato ries C O A R eference #: 023-018B Date o f Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 'Purity = 100% - (sum o f metal impurities, 1.39% +LC/MS impurities, 10.60%+Inorganic Fluoride, 0.27%+NMR impurities, 1.00%+ PO.AA, 0.30%) Total impurity from all tests = 13.56% Purity = 100% -13.56% = 86.4% 2Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials o f low purily. 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. . 5T F A HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropentanoic acid Pentafluoropropanoic acid 6Theoretical value calculations based on the empirical formula, CgFi7 S0 3 TC+ (MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). C O A 0 2 3 -0 1 8 B 3MEnvironmental Laboratory Page 2 o f 3 Page 188 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 IN TER IM CERTIFICATE OFANALYSIS C entre Analytical Laboratories COA Reference #: 023-018B LC/MS Purity Profile: Im purity C4 C5 C6 Cl Total wt./wt. % 1,03 1,56 6,38 1.63 10.60 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 response factors from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves. ( Prepared By: s2 S //o/* David S. Bell Date Sdentist, C entr^g& lytical Laboratories Reviewed By: CJaA. ft) y o h n Flaherty Date / LT aa bh on rraa tton rr yv MV anager, Centre Analytical Laboratories C O A 0 2 3 -0 1 8 B 3MEnvironmental Laboratory Page 3 o f 3 Page 189 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OFANALYSIS C e n tre A nalytical L ab o rato ries C O A R eference #: 023-022-2 3M P ro d u ct: E tF O S E -O H T est C ontrol R eference #: TC R -00017-52 P u rity : 97.4% Test Name Purity1 Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium 5. Nickel 6. Iron 7. Manganese Total Total %% Impurity Impurity (NMR) (LC/MS) Total % Impurity (GC/MS) Related Compounds - POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. N itrate 5. Nitrite 6. Phosphate 7. Sulfate Organic Acids'4(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis3: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine Specifications Yellow-white, waxy solid Result 97.4% Conforms ;: ; . ,v j..:;'1. '- ' :. .; v ; . : : ' ; - ' o - " <:./" ' r.' Positive 1. <0.001 wt./wt.% 2. <0.001 wt./wt.% 3. <0.001 wt./wt.% 4. <0.001 wt./wt.% 5. <0.001 wt./wt.% 6. <0.001 wt./wt.% 7. <0.001 wt./wt.% 1.26 wt./wt.% None Quantified 1.29 wt./wt.% 0.10 wt./wt.% Nne Detected 90.3 wt./wt.%. . ; ' '' ;<: :.;4\/: 1. <0.015 wt./wt.% 2. <0.005 wt./wt.% 3. <0.040 wt./wt.% ;. 4 . < 0 . 0 0 9 w t . / w t . % 5. <0.006 wt./wt.% 6. <0.007 wt./wt.% 7. <0.154 wt./wt.% . .......... ' ' 1. <0.1 wt./wt.% 2. <0.1 wt./wt.% 3. <0.1wt./wt.% 4. <0.25 wt./wt.% 1. Theoretical Value = 25.2% 2. Theoretical Value = 1.75% 3. Theoretical Value = 2.45% 4. Theoretical Value = 5.60% 5. Theoretical Value = 56.6% 1. 25.04 wt./wt.% 2. 1.69wt./wt.% 3. 2.61 wt./wt.% 4. 8.88 wt./wt.% 5. 56.8 wt./wt.% CO A 023-022-2 3MEnvironmental Laboratory Page 1 of 3 Page 190 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Analytical Laboratories, Inc 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OFANALYSIS C e n tre A nalytical L ab o rato ries C O A R eference #: 023-022-2 3M P roduct: E tF O S E -O H T est C ontrol R eference #: TC R -00017-52 Date o f Last Analysis: 11/26/00 Expiration Date: 11/26/01 Storage Conditions: <-10 C Re-assessment Date: 11/26/01 P u rity = 100% - (total NMR impurities, 1.26% + GC/MS impurities, 1.29 + POAA, 0. 10% ) Total impurity from all tests = 2.65% Purity = 100% - 2.65% = 97.4% ' 2TFA Trifluoroacetic acid r' HFBA Heptafluorobutyric acid NFPA Nonafluoropentanoic acid PFPA Pentafluoropropanoic acid _ 3Theoretical value calculations based on the empirical formula, C12H 10F 17NO3 S (MW=571) CO A 023-022-2 3MEnvironmental Laboratory Page 2 o f3 Page 191 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 r IN TE R IM CERTIFICATE OF ANAL YSISPhone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 C e n tre A nalytical L ab o rato ries C O A R eference #: 023-022-2 3M P roduct: E tF O S E -O H T est C ontrol R eference #: TC R -00017-52 GC/MS Purity Profile Peak# 1 2 Total Retention Time (min) 13.934 17.307 - Identity PFOSDEA C7 - % Impurity 0.36 0.93 1.29 This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). Prepared By: id S. Bell Scientist Ceptre Anal ell Laboratories Reviewed By: 0 John Flaherty Laboratory Manager Centre Analytical Laboratories COA023-022-2 3MEnvironmental Laboratory */<>?/> Date Date Page 3 o f 3 Page 192 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OFANALYSIS C e n tre A nalytical L ab o rato ries C O A R eference #: 3M P roduct: E tF O S E -O H T est C o n tro l R eference #: SD -013 P u rity : 88.9% 023-022-1 Test Name Purity1 Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (LC/MS) Total % Impurity (GC/MS) 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. Sulfate Organic Acids'1(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis'1: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine Specifications ; ^ HSP|pit { Yellow-w'h'il>tet, wa<x1y so-`liid . f ! i 1^ 1i^r *f -.;r: ;;; .... r;;:... ^ . ` , , . ; " Result 88.9% Conforms Positive 1. <0.001 wt./wt.% 2. <0.001 wt./wt.% 3. <0.001 wt./wt.% 4. 0.002 wt./wt% 5. <0.001 wt./wt.% 6. <0.001 wt./wt.% 7. <0.001 wt./wt.% 0.90 wt./wt.% None Quantified 10.21 wt./wt.% 0.03 wt./wt.% None Detected 87.6 wt./wt.%. 1. .<0.015 wt./wt.% 2. <0.005 wt./wt.% 3. <0.040 wt./wt.% 4. <0.009 wt./wt.% . ". . 5. <0.006 wt./wt.% 6. <0.007 wt./wt.% 7. <0.040 wt./wt.% 1. <0.1 wt./wt.% 2. <0.1 wt./wt.% 3. <0.1 wt./wt.% 4. <0.25 wt./wt.% 1. Theoretical Value = 25.2% 2. Theoretical Value =1.75% 3. Theoretical Value = 2.45% 4. Theoretical Value = 5.60% 5. Theoretical Value = 56.6% 1. 24.42 wt./wt.% 2. 1.78wt./wt.% 3. 2.72 wt./wt.% 4. 9.34 wt./wt.% 5. 58.4 wt./wt.% COA023-022-1 3MEnvironmental Laboratory Page 1 of 3 Page 193 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Analytical Laboratories, Ine. 3048 Research Drive State College, PA 16801 n IN TER IM CERTIFICATE OF ANALYSISPhone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 C e n tre A nalytical L ab o rato ries C O A R eference #: 023-022-1 3M P roduct: E tFO S E -O H T est C ontrol R eference #: SD-013 Date o f Last Analysis: 11/26/00 Expiration Date: 11/26/01 Storage Conditions: <-10 C Re-assessment Date: 11/26/01 1Purity = 100% - (total metal impurities, 0.002% + total NMR impurities, 0.90% + GC/MS impurities, 10.21 + POAA, 0.03%) Total impurity from all tests = 11.14% Purity = 100% -11.14% = 88.9% 2TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonafluoropentanoic acid Pentafluoropropanoic acid . th e o re tic al value calculations based on the empirical formula, C12H10F 17NO3 S (MW=571) COA023-022-1 3MEnvironmental Laboratory Page 2 o f 3 Page 194 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone:(814)231-8032 Fax: (814) 231-1253 or (814) 231-1580 O INTERIM CERTIFICATE OFANALYSIS Centre Analytical Laboratories COA Reference #: 023-022-1 3M Product: EtFOSE-OH Test Control Reference #: SD-013 r' GC/MS Purity Profile P eak# 1 2 3 4 5 6 7 8 9 10 11 12 13 T o ta l R eten tio n T im e (m in ) 6 .1 6 3 8 .0 1 1 8 .2 0 6 9 .0 6 5 9 .8 4 4 13.93 1 4 .2 3 8 1 5 .1 3 0 15.52 15.941 1 6 .3 7 9 16.801 1 7 .2 2 2 - Id e n tity Unknown Unknown Unknown Unknown Unknown Unknown Unknown C2 C3 C4 C5 C6 Cl - ' % Im p u rity 0 .1 2 0 .2 3 0 .5 1 0 .2 1 0 .3 4 0 .6 2 0 .1 1 0 .1 1 1.11 1 .5 5 1 .0 7 3 .3 0 0 .9 3 1 0 .2 1 This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). Prepared By: atn d S .B eli Scientist Centre Anal Reviewed By: CjJL. PO ratones John Flaherty Laboratory Manager Centre Analytical Laboratories C O A 0 2 3 -022-1 3MEnvironmental Laboratory Date Date Page 3 o f3 Page 195 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 ANALYTICAL REPORT STUDY TITLE PURITY DETERMINATION OF SAMPLE LOTS OF PFOS Sample # TCR-00065-022 DATA REQUIREMENTS Test Article Characterization STUDY DIRECTOR Kevin Lloyd -T H IS IS AN EXACT COPY OF THE ORIG INAL DOCUM ENT" 8 Y. lL . .DATE isj/f ANALYTICAL REPORT COMPLETION DATE October 25,2000 PERFORMING LABORATORIE / TESTING FACILITIES Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive State College, PA 16801 Phone: 814-231-8032 STUDY SPONSOR 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 PROTECT IDENTIFICATION Centre Study Number: 023-045 Total Pages: 15 3MEnvironmental Laboratory Page 196 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 . LRN-U2104 Centre Study No.: 023-045 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Centre Study Number 023-045, entitled "Purity Determination of Sample Lots of PFOS, Sample # TCR-00065-022" conducted for 3M Environmental Laboratory, was performed in compliance with US EPA Good Laboratory Practice Standards (40 CFR Part 160) by Centre Analytical Laboratories, Inc. with the following exceptions: The automated data collection systems used in this study were not fully compliant with 21 CFR 58.130 (e). Study Director ' ' Centre Analytical Laboratories, Inc. U> lS>(o Date 3M Environmental Technology and Safety Services Date *3Tt Centre Analytical Laboratories, Inc. 3MEnvironmental Laboratory Page 2 of 15 Page 197 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 (Centre Study No.: 023-045 QUALITY ASSURANCE STATEMENT Centre Study Number 023-045, entitled "Purity Determination of Sample Lots of PFOS, Sample # TCR-00065-022" was reviewed by Centre Analytical Laboratories' Quality Assurance Unit. All reviewed phases were reviewed for conduct according to Centre Analytical Laboratories' Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Study Director and to management. Phase 1. Protocol Review Date Instiected 10/19/00 Date Reported to Study Director and Centre Management Date Reported to Sponsor Management 10/25/00 10/25/00 2. Raw Data Review 10/19/00 10/25/00 10/25/00 3. Report Review 10/24/00 10/25/00 10/25/00 Wilfiam Spare ' Quality Assurance Officer /o /z s '/o o Date THE ORIGINAL DOCUMENT BY------2 I L -------- DATE Centre Analytical Laboratories, Inc. 3MEnvironmental Laboratory Page 3 o f 15 Page 198 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 CERTIFICATION OF AUTHENTICITY This report, for Centre Study Number 023-045, is a true and complete representation of the raw data for the study. Submitted by: Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 (814) 231-8032 Study Director, Centre: Kevin Lloyd Study Director Centre Analytical Laboratories, Inc. 4 Centre Analytical Laboratories, Inc. Facility Management: Richard A President Centre Analytical Laboratories, Inc. z s ~ OCT-oo Date DOCUMENT" ----------- D A T E Centre Analytical Laboratories, Inc. 3MEnvironmental Laboratory Page 4 of 15 Page 199 3M M edical D epartm ent Study: T-6316.1 Analytical Report: FACT-TOX-003 L R N -U 2104 Centre Study No.: 023-045 STUDY IDENTIFICATION PURTTY DETERMINATION OF SAMPLE LOTS OF PFOS Sample # TCR-00065-022 TYPE OF STUDY: TEST SYSTEM: TEST ARTICLE: SPONSOR: STUDY DIRECTOR: TESTING FAdLFTIES: ANALYTICAL PHASE TIMETABLE: Characterization Not Applicable PFOS, Test Control Reference # TCR-00065-022 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 Kevin Lloyd Cent- Analytical Laboratories, Inc. Phone: (814) 231-8032 Centre Analytical Laboratories, Inc. (Centre) 3048 Research D riv e State College, PA 16801 Phone:814-231-8032 Study Initiation Date: Analytical Start Date: Analytical Termination Date: 09/28/00 10/05/00 10/24/00 THIS IS AN EXACT COPY OF THE ORIGINAL DOCUMENT" RY... RATF1Cjj&S'/cO - Centre Analytical Laboratories, Inc. 3MEnvironmental Laboratory Page 5 of 15 Page 200 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 PROJECT PERSONNEL The Study Director for this project was Kevin Lloyd at Centre Analytical Laboratories, Inc. The following personnel from Centre Analytical Laboratories, Inc., were associated with various phases of the study: Name Gerry Shero David S. Bell Emily R. Stauffer Mark Ammerman Title Scientist Scientist Scientist Sample Custodian i n e K j w K allMAL DOCUMENT' BY----- 22L _____date Centre Analytical Laboratories, Inc. 3MEnvironmental Laboratory Page 6 of 15 Page 201 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 TABLE OF CONTENTS Page TITLE PA G E ............................................................................................................................ 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT............................... 2 QUALITY ASSURANCE STATEMENT...............................................................................3 CERTIFICATION OF AUTHENTICITY................................................................................4 STUDY IDENTIFICATION.................................................................................................... 5 PROJECT PERSONNEL......................................................................................................... 6 TABLE OF CONTENTS......................................................................................................... 7 1.0 SUMMARY.....................................................................................................................8 2.0 INTRODUCTION...............................................................................................................8 3.0 TEST SYSTEM ..................................................................................................................8 4.0 TEST ARTICLE..................................................................................................................8 5.0 EXPERIMENTAL PROCEDURES..................................................................................9 6.0 RESULTS AND DISCUSSION....................................................................................... 10 8.0 CIRCUMSTANCES THAT MAY HAVE AFFECTED THE DATA...................... 10 9.0 RETENTION OF DATA AND SAMPLES................................................................ 10 Table I. Results From LC/MS for TCR-00065-022............................................................ 1 1 Figure 1. PFOS Calibration Standard (C100500-4) at 269|ig/L ......................................... 12 Figure 2. PFOS Calibration Standard (C100500-2) at 539pg/L ...................'..................... 13 Figure 3. PFOS (TCR-00065-022) at 250|ig/L, ESI Negative Ion M ode.......................14 Appendix A: Study Protocol 00P-023-045: PURITY DETER]VONATION OF SA M PL E LOTS OF PFOS, Sample # T C R -00065-022, Including A m endm ent 1 ............. 15 BY----- -------------OATEi j y d Centre Analytical Laboratories, Inc. 3MEnvironmental Laboratory Page 7 o f 15 Page 202 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 1.0 SUMMARY The purity of PFOS from sample # TCR-00065-022, was determined from the LC/MS test data to be 88.0% pure as compared with the PFOS control (TCR-00017-046,97.9% purity). 2.0 INTRODUCTION This report details the results of the analysis of PFOS, Test Control Reference# TCR00065-022. The test was analysis by liquid chromatography - mass spectrometry (LC/MS). The LC/MS was conducted at Centre. The study was initiated on September 28,2000 when the Study Director signed protocol number OOP-023-045. The analytical start date was October 5,2000, and the experimental termination date was October 9,2000. 3.0 TEST SYSTEM There is no test system associated with this characterization study, therefore the GLP requirement for test system description, justification, and identifi cation do hot apply. The route of administration, levels and frequency of administration also do not apply to characterization studies. 4.0 TEST ARTICLE 'TH IS IS AN EXACT COPY OF THE ORIGINAL DOCUMENT" BV---S T t___ DATE*.eja*/<pc The test article was PFOS, Test Control Reference# TCR-00065-022. 3M Environmental Laboratory supplied the test article and it was logged at Centre Analytical Laboratories, Inc. as follows: Compound PFOS TCR-00065-022 Lot or NB Number 193 Centre Control No. 00-023-068 Purity TBD Date Received 10/05/00 The sample received was a white, crystalline solid. It was; received under frozen conditions and was stored in a temperature-monitored freezer kept at <-10C. Centre Analytical Laboratories, Inc. 3MEnvironmental Laboratory Page 8 of 15 Page 203 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 The analytical/control standard PFOS was: Control Article Lot Number PFOS TCR-00017-046 Centre Control No. 00-023-042 Expiration Purity Date 97.9% 8/31/2001 The chemical and physical data for PFOS is as follows. Common Name: Molecular weight: CAS Number Structure: PFOS 499 (CgFnS03') 2795-39-3 O II CgFpS O--O" K+ O Note: the neutral molecule and standard form that the PFOS (anion) is derived from is potassium perfluorooctane sulfonate (CgFnS03K), molecular weight 538. 5.0 EXPERIMENTAL PROCEDUIOES LC/MS Spectral Analysis PFOS sample TCR-00065-022 was analyzed for purity using liquid chromatography/mass spectrometry (LC/MS). The sample was analyzed according to the following procedure. The sample was prepared at about 250 pg/mL in methanol and analyzed by LC/MS using a Hewlett-Packard 1100 HPLC system interfaced with a Hewlett-Packard 1100 mass selective detector. The mass spectrometer was run in negative ionization mode using electrospray ionization (ESI) using Selective Ionization Monitoring for ion m/z 499. The liquid chromatography system was operated in reversed-phaae mode using a C l 8 silica-based column. Each resulting chromatogram was calculated against the analytical control article (TCR00017-046) calibration curve. "THIS IS AN EXACT COPY OF THE OFllGINAL DOCUMENT Centre Analytical Laboratories, Inc. 3MEnvironmental Laboratory Page 9 of 15 Page 204 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 6.0 RESULTS AND DISCUSSION LC/MS Spectra] Analysis Responses were observed for the PFOS sample in negative ion mode using an ESI interface. Quantitation was performed using SIM mode, m/z 499, and PFOS was detected at 7.8 minutes. TH-PFOS was used as the internal standard, m/z 427, and was detected at 7.5 minutes. The total percent purity, calculated against the analytical control article (TCR-00017-046) calibration curve was determined to be 88.0% from the average of three replicate analyses. 8.0 CIRCUMSTANCES THAT MAY HAVE AFFECTED THE DATA Electronic records are not fully compliant with 21 CFR 11, "Electronic records; Electronic Signature." However, approved SOPs were in place and all instrumentation used in this study was fully calibrated and operational. All original raw data were printed as hard copies and fully audited by quality assurance. Verified exact copies and the electronic data will be stored in the archives at Centre Analytical Laboratories. Original raw data will be returned to the Sponsor. 9.0 RETENTION OF DATAAND SAMPLES When the final report is final, all original paper data generated by Centre Analytical Laboratories, Inc. will be shipped to the sponsor. This does not include facility-specific raw data such as instrument logs, however exact copies of temperature logs will be submitted. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, w ill be retained in the Centre A nalytical Laboratories, Inc. archives for the period of time specified in 40 CFR Part 160. Retained samples of reference substances are archived by the sponsor. THE ORIGINAL DOCUMENT" BY. ____ d a t e J o l<3 S~/cr \ Centre Analytical Laboratories, Inc. 3MEnvironmental Laboratory Page 10 of 15 Page 205 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 Table I. Results From LC/MS Of TCR-00065-022, ESI Negative mode 3M ID TCR-00065-022 TCR-00065-022 TCR-00065-022 Standard Standard Standard Standard Standard Standard Standard Standard Standard CCV Retention Time (min) 7.79 7.80 7.80 7.69 7.73 7.77 7.79 7.79 7.77 7.78 7.78 7.78 7.81 Mass 499 499 499 499 499 499 499 499 499 499 499 499 499 Value Determined Pg/L 250 pg/L 219 250 2 2 0 250 2 2 0 Average = *1 1 1 0 0 1 1 17* 54 60 54 50 108 1 0 1 269 284 539 562 808 831 1078 1205 269 278 Standards Average = Std Dev = % Recovery 8 8 .0 8 8 .0 8 8 .0 88.0 * * 110 93 94 105 104 103 112 103 103 7 * First two injections during instrument warm up, not reported. CCV = standard in run used to check calibration (C100500-4 at 269|ig/L) "THIS IS AN EXACT COJ^ OF the origina*- document BY. SSL-- DATE Centre Analytical Laboratories, Inc. 3MEnvironmental Laboratory Page 11 of 15 Page 206 3M Medical Department Study: _T_~6316_1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 Figure 1. PFOS Calibration Standard (C100500-4) at 269ig/L j Sam ple Name:C100500-4 : Sam ple I n fo ; D ate f i l e :C :\HPCHEM\1\DATA\100600\ 10060022. D In je c tio n Date f^ A c q O perator Instrum ent D ilu tio n ' 10/ 6/2000 ` 3 : 11:22 PM OS tV V43-* Instrum ent 1 1 Seq L in e V ia l No. I n j. No. I n j. V o l. A cq. Method A n a ly s is .M ethod L a s t Changed PFOS.M C :\H PC H EM \l\M ET H 0D S \1 0 0 6 0 0 .M 4,0/ 2 3 /2 0 0 0 1 1 : 4 4 :2 6 am A nalysis fo r PFOS -------------- n w r w . b i w g r o : ) n o u w w u w m a a ) w * . w a . m m . r a a r v i r W OW - roooo: r 60000> 50000 40000: 30000zoooo- 10000: * o: ' " 1 i " 1 r * ---------'USUI4 W.'BC*W .T Mg.'<'(TUI8BMUWJUZlUr *AHKS.Ttfft'S m mflTVP 120000 100000 80000 60000 i h- 40000 20000 26 14 1 $ Al 0-> Compound Name TH-PFOS PFOS Amount 500.0 261.5 R .T . 7.58 7.81 ' Area 785730 1078963 A s i g n a l . _ ; . .. MSD1 4 2 7 , S lC t a f t a :42 MSD1 4 9 9 , E lC 4'9 ^7 9 ....................... *** End o f R e p o rt *** Instrum ent 1 Mon, 2 3 . O c t. 2000 0 0 : 1 2 :0 6 pm Page 1 o f 1 "THIS IS ANEXACT COPY OF THE ORIGINAL DOCUMENT" 3Y ----- ^ ^ -- r t X T F lo/atsyoc) Centre Analytical Laboratories, Inc. 3MEnvironmental Laboratory Page 12 of 15 Page 207 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 Figure 2. PFOS Calibration Standard (C100500-3) at 539pg/L Sample Name:C100500-3 Sample Info: ' Data file :C:\HPCHEM\l\DATA\100600\10060015.D n Injection Date Acq Operator Instrument Dilution : 10/6/2000 :GS s :Instrument 1 :1 1:13:21 PM Seq Line Vial No. Inj.No. I n j .V o l . Acq. Method :PFOS.M Analysis Method : C:\HPCHEM\l\METHODS\100600.M Last Changed :10/9/2000 01:45:43pm (modified after loading) Analysis for PFOS 1 13 IS 1 5 ;xl TH-PFOS PFOS 500.0 499.8 7.57 7.80 770327 2238071 MSDl 427, EIC-426.7:42 MSDl 499, EIC-49B.7:49 r' *** End of Report *** Instrument 1 Mon, 9. Oct. 2000 01:46:00 pm Page 1 of 1 Centre Analytical Laboratories, Inc. 3MEnvironmental Laboratory Page 13 of 15 Page 208 3M .M edicaLD ePrtm enLStudy^_T-6^^ Analytical Report: FACT-TOX-003 Lr n -U2104 Centre Study No.: 023-045 Figure 3. PFOS (TCR-00065-022) at 250ig/L, ESI Negative Ion Mode Sample Name:L29092-1 Sample Ino:C100500-8 Data file :C:\HPCHEM\l\DATA\100600\10060019.D ^ I n j e c t i o n Date ( xAcq Operator Instrument Dilution 10/6/2000 GS 6b u -- Instrument 1 1 2:20:49 PM ' Seq Line Vial N o . Inj . N o . Inj. Voi. : : : : Acq. Method Analysis Method Last Changed PFOS.M C:\HPCHEM\1\METHODS\100600.M do/23/2000 11:44:26 am Analysis for PFOS -> i 17 21 1 5 Ml TH-PFOS PFOS 500.0 218.7 7.57 7.79 776592 894026 HSIJ1 427, EIC426.7:42 MSIJ1 499, EIC-498.7:49 n ............................................................................. *** End of Report *** Instrument 1 Mon, 23. Oct. 2000 00:10:19 pm Page 1 Of 1 Centre Analytical Laboratories, Inc. 3MEnvironmental Laboratory THE ORIGINAL DOCUMENT" BY__ -DATE ioO Page 14 of 15 Page 209 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 APPENDIX A Study Protocol Purity Determination of Sample Lots of PFOS Including Amendment 1 THE ORIGINAL DOCUMENT" 8Y. - 2 3 ___ -- DATE Centre Analytical Laboratories, Inc. 3MEnvironmental Laboratory Page 15 of 15 Page 210 3M M edical D epartm ent Study: T-6316.1 3M Medical Department Study: T-6316.1 Appendix H: Report Signature Page Analytical Report: FACT-TOX-003 L R N -U 2104 Analytical Report: FACT TOX-003 LRN-U2104 John L. Butenhoff, Ph.D., Ph.D., Study Director Date / (n 3^-41 Marvin T. Case, D.V.M., Ph.D., Sponsor Representative Date Kristen J. Hansen, Ph.D., Principal Analytical Investigator OS~j3t/0f ' Date William K. Reagen, Ph.D., Laboratory Manager Date 3MEnvironmental Laboratory Page 211