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~ ~~ ~ ~~~ BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U2636 Study Title Determinationof the Presence and Concentration of PFOS, PFOSA, M556, andM570 in the 13-Week Dietary Study of Crl:CD@(!SD) IGS BR Rats Analytical LaboratoryReporrC Title Determination of the Presence and Concentration ofPerfluorooctanesulfonate, Perfluorooctanesulfonylamide,M556 and M570 in Liver and Sera Samples Data Requirement Not Applicable Author 3M Environmental Laboratory Study Completion Date FebrAutasriygn2in3g, 2001 Performing Laboratories Liver and SeraAnalyses Liver and Sera Extractions 3M Environmental Laboratory Building 2-3E-09,935 Bush Avenue St. Paul, MN55106 Pace Analytical TierII Laboratory, Inc. 1700 Elm Street, Suite100 Minneapolis, MN 55414 Project Identification 3M Study: T-6314.1 Covance In-Life Study: 6329-225 Analytical Report: FACT-TOX-028 3M Laboratory Request No. U2636 Total Numberof Page!; 130 3M Environmental Laboratory Page 1 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 This page has been reservedfor specific country requirements. 3M Environmental Laboratory Page 2 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U2636 . .. GLP Compliance Statement Analytical Laboratory Report Title: Determinationof the Presence and Concentration of.PFOS, PFOSA, M556, and M570 in the13-Week Dietary Study of Crl:CD@(SD) IGS BR Rats Study Identification Numbers: T-6314.1, FACT-TOX-028, LRN-U2636 The determination oMf 570 in liver and sera specimens will be conducted according to documented, but notvalidated methods. This study wasconducted in compliance with United States Foodand Drug Administration(FDA) Good Laboratory Practice (GLP) Regulations for NonclinicaLl aboratory Studies,Final Rule 21 CFR Part 58, with the exceptions in.the bulleted lisbt elow. Exceptions to GLP compliance: 0 Analytical standard stability was not yet determined. 0 Purity of the following analytical reference materials have not been completed at this time;' PFOSA, M556 and M570. 0 Occasionally, correctionswere not made accordling to 21 CFR 58.130 ( e ) . 0 The electronic data systems have not been validated and there is not an electronic audit trail ofcorrectionscurrently available. Authenticated hard copies of chromatography and associated documents will be considered as the original raw data. 0 Results of purity and stability analysis of the test material have not been completed by thesponsor. This informationwill be included in the final toxicology report. '/M :. w SeAancdartew Ph.D., Study Director Date * g A A L & I 7. Johh Butenhoff Ph.D., SponsorRephsentative P* F**r3, Date . ZLWf KristeHnansen Ph.D., PrincipAanlalytical lnvestigator Date 3M Environmental Laboratory Page 3 BACK TO MAIN 3M.Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U2636 GLP Study-Quality Assurance Statemerrt Analytical Laboratory Report Title: Determinationof the Presence and Concentration of PFOS, PFOSA, M556, and M570 in the 13-Week.Dietary Study of Crl:CD@ (SD)IGS BR Rats Study Identification Numbers: T-6314.1, FACT-TOX-028, LRN-U2636 This study has beeninspected by the 3M EnvironmentalLaboratory Quality Assurance Unit (QAU) as indicated in the following table.The findings were reported to thestudy director and laboratorymanagement. I . Inspection Dates' '1 Phase I .0I/30/00 I Analysis Date Reported to Management Study Director 10/31/00 I0/3I/oo 1011o/oo . 1V13-17/00, 11/20/00, I 11127-28/00 la19/00, 12/21/00 12/27-12/28/01 I 02/05-02/06/01 Extraction I I i . . .. Data Draf?report #1 1 Draftrepoi##2 10111/oo 11/28/00 ~01/02/01 02/07/01 10/11/oo I1/28/00 01/02/01 02/07/01 QAU Refmsentative I Date 3M Environmental Laboratory Page 4 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN42636 Table of Contents GLP Compliance Statement.............................................................................................. 3 GLP Study-Quality Assurance Statement ....................................................................... 4 Study Personnel and Contributors..................................................................................... 8 Introduction and Purpose .................................................................................................. 9 Test System ................................................................................................................. 9 Specimen Collection and Analyses............................................................................... 10 Specimen Receipt and Maintenance................................................................................. 10 Chemical Characterizationof Reference Material.s........................................................... 11 Method Summaries ........................................................................................................... 12 Preparatory Methods ............................................................................................... 12 Analytical Methods................................................................................................... 12 Analytical Equipment ............................................................................................... 13 Data Quality Objectives and DataIntegrity........................................................................ 14 Data Summary. Analyses. and Results............................................................................. 14 Summary of Quality Control Analyses Resu.lt.s............................................................ 14 Statement of Data Quality ............................................................................................ 16 Summary of Analytical Sample Results........................................................................ 16 Statistical Methods and Calculations................................................................................. 17 Statement of Conclusion ................................................................................................... 17 Appendix A: Control Matrices and TesSt ubstance Characterization................................. 18 Test Substance Characterization.................................................................................. 18 Appendix B: Protocol. Amendments and Deviation..s........................................................ 19 Appendix C: Extraction and Analytical Methods................................................................ 45 ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfcmateor Other Fluorochernical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometr"y. (14 pages)......................................................................................................................... 46 ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfcmateor Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometr'.y(.14 pages)............................................................................................................................... 60 . ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicals in Serum Using HPLC-Electrospray/Mass Spectrometry.(9 pages)................................. 74 . ETS-8-7.0, "Analysis of PotassiumPerfluorooctanesulfonateor Other Fluorochernicals in LiverusingHPLC-ElectrospraylMass Spectrometry.(10 pages)................................... 85 Appendix D: Data Tables .................................................................................................. 97 Appendix E: Data Spreadsheets ....................................................................................... 105 Appendix F: Example Calculations.................................................................................... 124 Appendix G: Interim Certificate(s) of Analysis ................................................................... 126 3M Environmental Laboratory Page 5 BACK TO MAIN 3M Medical DepartmentStudy: T-6314.1 Analytical Report: FACT TOX-,028 LRN-U263f3 Appendix H: Report Signature Page ................................................................................. 130 3M Environmental Laboratory Page 6 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U2636 List of Tables Table 1. Dosage Levels. Dose Groups andGroup Composition for Study 6329-225 .......9 Table 2. Characterizationof the Analytical Reference Materialsin Study FACT-TOX-02811 Table 3. Negative Ions Monitored in 3M Laboratory Analyses.......................................... 13 Table 4. Matrix SpikeIMatrix Spike Duplicateresults for MI556 in Sera.............................. 15 Table 5. Matrix Spike/Matrix Spike Duplicateresults for PFOSA and M556in Liver..........16 Table 6. Characterizationof the Control Matrices Usedfor Liver and Sera Analysesin Study FACT-TOX-028........................ :................................................................. 18 Table 7. Characterizationof Test Substance in Study FACT-TOX-028............................. 18 Table 8. Rat Serum Week 5 Data for FACT-TOX-028...................................................... 97 Table 9. Rat Serum Week 14 Datafor FACT-TOX-028 .................................................... 98 Table I O. Rat Liver Week 5 Data for FACT-TOX-028....................................................... 99 Table 1 1. Rat Liver Week14 Data for FACT-TOX-028..................................................... 100 Table 12. Data Summary of Average Week5 Rat Serum Concentration(pg/mL) and Standard Deviation ( S D ) for FACT-TOX-028 ..................................................... 101 Table 13. Data Summary of Average Week 14 Rat Serum Concentration (pg/mL) and Standard Deviation (kSD)for FACT-TOX-028 ..................................................... 101 Table 14. Data Summary of Average Week5 Rat Liver Concentration(pg/g) and Standard Deviation ( S D ) for FACT-TOX-028 ..................................................... 102 Table 15. Data Summary of Average Week14 Rat Liver Concentration(pg/g) and Standard Deviation (*SD) for FACT-TOX-028 ..................................................... 102 Table 16. Approximate LOQ Values Usedin FACT-TOX-028 Analyses byMatrix, Compound and Usage Dates............................................................................... 103 Table 17. FACT-TOX-028 Rat LiverWeek 5 Precision Datafor PFOS and PFOSA ......... 103 Table 18. FACT-TOX-028 Rat Liver Week5 Precision Datafor M556 and M570.............104 3M Environmental Laboratory Page 7 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U263tj Study Personnel and Contributors Study Director Andrew M. Seacat, Ph.D. 3M Toxicology Services Building 220-2E-02, 3M Center St. Paul, MN 55144-1000 Sponsor 3M ToxicologyServices Building 220-2E-02 St. Paul, MPJ 55144-1000 John L. Butcsnhoff, Ph.D., Sponsor Representailive Analytical Chemistry Laboratories Liver and Sera Analyses 3M Environmental Laboratory Kristen J. Hansen, Ph.D., Principal Analytical Investigator Liver and Sera Extractions Pace Ana1ytic:al TierI1 Laboratory Inc. 3M Laboratory Contributing Personnel Lisa A. Clemen Kelly J. Dorweiler* Barb A. Gramem* Sarah A. Heimdal* Marlene M. Heying* Harold 0. Johnson *Contract lab professional service employees Kelly J. Kuehlwein* Sally A. Linda* Joe Pilon* Thomas P. wilgner* Bob W. Wynne* Richard D. Youngblom* Location of Archives All original raw data, protocol, and analytical reporht ave been archived atthe 3M Environmental Laboratory. The test substance and analytical reference standardreserve samples, as well as the specimens pertaining to the analytical phaseof this study are archived atthe 3M Environmental Laboratory for a minimum of 10 years. 3M Environmental Laboratory Page 8 BACK TO MAIN 3M Medical DepartmentStudy: T-6314.1 Analytical Report: FACT TOX-028 LRN-U2636 Introduction and Purpose The purpose of the analytical phaseof this studyis to evaluate levelsof PFOS, PFOSA, M556, and M570in liver and sera specimens ofthe rats exposed to N-MeFOSE-OH (T-6314)T. he in-life phase of this study was conducted at Covance Laboratories, Inc., (Covance Study Number 6329225). The in-life data, dose preparation and analysisare reported in Covance Study Number Report 6329-225. The analytical portion ofthe study wasinitiated on 23 August 2000. Test System The test system species andstrain selected wasthe Crl:CD@(SD)IGS BR rats received from Charles River Laboratories, lnc. Four dose groups comprised of 20 male and 20 female rats each (80 males and 80 females total) were fed daily nominal doses of O-(control), 3-(low dose), 30-(mid dose), or IOO-(high dose) ppm of N-MeFOSE-OH (T-6314)in their feed for at least 13 weeks. After 4 weeksli,ver and sera specimens were collected from five-animals/sex/dose group. Athe end of the study (after week 13), liver and sera specimens were collectedfrom all remaining animalsin the test system. All liver and sera specimens were sent to the 3M Environmental Laboratoryfor analysis. Table 1. Dosage Levels, Dose Groups and Group Comipositifoonr Study 6329-225 I Dose Group I Numberof Animals, I Dietaly Levelsa I 4 I(Control 20 male/ 20 female 2 (Low Dose) 20 male/ 20 female 3 (Mid Dose) 20 male/ 20 fema3l0e QQm I (DHoigs4he) 20 malefpe/p1m2m0a00le a Dose Levels are expressed as pprn of N-MeFOSEi-OH. b Control Group animals receiveda basal diet only. 3M Environmental Laboratory Page 9 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U2636 Specimen Collection and Analyses In the analytical study reported here, 160 liver (80 male ant3d0 female) and 80(40 male and 40 female) sera specimens were collected during the coursethoef in-life phase of Covance Study 6329-225 and sentto the3M Environmental Laboratoryto be extracted and analyzed for PFOS, PFOSA, M556 and M570.The study director determined thaitt was appropriate onlyto analyze the liver samples that have corresponding sera samples. There wastoatal of 80 liver analyses and atotal of 80 sera analysesfor each of the four compounds. Blood specimenswere allowed to clot at room temperaturea17d centrifuged. Serum was then harvested and stored in a freezer set to maintain specimens -60C to -80C until shipped to the 3M Environmental Laboratory. Liver specimens collected from each animal weflraesh frozen in liquid nitrogen and then stored in a freezer seto maintain :specimens at -60Cto -80C until shipped to the 3M Environmental Laboratory. Liver and sera specimens were shippteodthe 3M Environmental Laboratory frozen and on dry ic2e2 December 1998 and 13 October 1998, respectively. Liver and Sera samples were sentto Pace AnalyticalTier II Laboratory, Inc. on 21 September 2000. The sera and livers were extracted using3M Environmental Laboratory analytical methods beginning on 25 September 2000 and10 October 2000, respectively. Analytical samples were extracted using anion-pairing procedure. An ion-pairing reagenwtas added to the laboratory sample andthe analyte ion pair was partitioned into methyl-tert-butyl ether (MtBET)h. e MtBE extract was removed and put onto a nitrogen evaporatourntil dry. Each extracted laboratory sample was then reconstituted in 1.OmL of methanol andfiltered through a 3 cc plastic syringe attached toa 0.2 pm nylon filter into a glass autovial. Liver samples were homogenized prtioor the extraction procedure. The extracts were returned11 October 2000to the 3M Environmental Laboratory after the extractions were completed. Sample analyseswere performedby monitoring one product ion selectefdrom a single primary ion characteristic of a particular fluorochemical using HPLC-ES/MS/MiSn the multiple reaction monitoring. Analytical details are includedin this report. The PFOS data reported has been adjusted to reflect the purity determinfeodr the analytical reference material.(See table 2). ~ ~~~ Specimen Receipt and Maintenance The 3M Environmental Laboratory received liver and sera specimens o1n3 October 1998 and 22 December 1998 that were collected during and tahte end of the in-life phase of Covance Study 6329-225. All specimens were received frozen on dry ice and were immediatelytransferred to storage at -20C &0IC. 3M Environmental Laboratory Page 10 3M Medical DepartmentStudy: T-6314.1 Chemical Characterizationof ReferenceMaterials BACK TO MAIN Analytical Report: FACT TOX-028 LRN42636 Table 2. Characterization of the Analytical Reference Materialsin Study FACT-TOX-028 Material I Identification KPFOS" I Potassium Pemuoroodanesulfonate PFOSA Pertluoroodanesufongamide I M556 M556 M570 M570 I source I 3M SpedaltyChemicals I 3M Spedalty Chemicals 1 3M SrledaltyChemicals I 3M SpeciallyChemicals Expiration Date Storage Conditions Chemical Lot Number Physical Description o1mwoio Room Tempemturn L d 171 LgMd o r e d powder Om l nl OO l Room Temperalure Lightyellowwaxy soli o1mnoIo Ream Temperature 01/01/2010 Room Temperahre 31# 113047-80 L-15709 3M# 118506-26 Whte powder Whitepowder Purity 86.4% TBD~ TBD - To be determined a The target analyte is PFO(SPerfluorooctanesulfonate),CBFI~SC); The punty of the substances listed above were nominally determinedNMbyR analyses. Subsequent chemical characterization are occurring and this analytical repowritll be amended to indicate the punotyf these substances when a certificate of analysis are issued. 3M Environmental Laboratory Page 11 BACK TO MAIN 3M Medical DepartmentStudy: T-6314.1 Analytical Report: FACT TOX-,028 LRN-U2636 Method Summaries Following is a brief description of the methods usedfor extraction and analyses during this analytical study by the 3M Environmental Laboratory.Detailelcl descriptions of the methods used in this study are located in Appendix C. The preparatory methods used bythe technicians at the Pace Analytical Tier I I Laboratory, Inc. are methodsfrom and developed bythe 3M Environmental Laboratory. PREPARATORY METHODS ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonateor other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/MassSpectrometry" 0 ETS-8-6.0, "Extraction of Potassium Perfluorooctane-sulfonate or other Fluorochemical Compounds from Liver for Analysis using HPLC-EIectrospray/MassSpectrometry" Both extraction methods use an ion-pairing procedure.An ion-pairing reagent was added to the laboratory sample and the analyte ion pair waspartitioned into methyl-tert-butyl ether (MtBE). The MtBE extract was removed and put onto anitrogen evaporator until dry. Each extracted laboratory sample was then reconstitutedin 1.O mL of methanol and filtered through a 3 cc plastic syringe attached to a0.2 vm nylonfilter intal a glass autovial. Liver samples were homogenized prior to the extraction procedure. ANALYTICAL METHODS ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonateor other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" 0 ETS-8-7.0, "Analysis of Potassium Perfluorooctanesulfonateor other Fluorochemicalsin Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" These methods describe the analysis of fluorochemicasl urfactants from liver and serum extracts using HPLC-ES/MS/MS. The analysis wasperformed by monitoring a product ion selected from a single primary ion characteristic of a parti'cular fluorochemical. For example, molecular ion499, selected as the primary ion for PFOS (C,F,,SO,-) analysis, was fragmented to produce ion 99 (FS033.The characteristic ion 99 was monitoredfor quantitative analysis. 3M Environmental Laboratory Page 12 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U263'6 ANALYTICAL EQUIPMENT The analytical equipment settings usedin the present analyticalphase of this study may have varied slightly during data collection. The following is representatiovfethe settings used during the analytical phase of this study. Liquid Chromatograph: Hewlett-Packard@ Series1100 Liquid Analytical column: Keystone@ Betasil"CI8 2x50 mm (5 pm) Column temperature: Ambient Mobile phase components: Component A: 2mM ammonium acetate Component 6: methanol Flow rate: 300 pUmin Injection volume: 10 yL Solvent Gradient: 10.0 minutes Chromatograph system Time (minutes) %B 0.0 10% 1.o 10% 5.5 95% 7.5 95% 8.0 10% Mass Spectrometer: Micromass@ APVMass Spectrometer Software: Mass Lynxw3.4 Cone Voltage: 30-60 V Collision Gas Energy: 25-45 eV Mode: Electrospray Negative Source Block Temperature:150C *I0C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring(MRM) Q;uattlrlow Triple Quadrupole system Table 3. Negative Ions Monitored in 3M Laboratory Analyses I TargAetnalyte I Primary Ion(mu) I Product Ion (mu) I PFOS 499 PFOSA 498 I I I I M556 556 169 I M570 I 570 I 169 I I THPFOS" I 427 I 80 I *Internal Standard 3M Environmental Laboratory Page 13 BACK TO MAIN 3M Medical DepartmentStudy: T-6314.1 Analytical Report: FACT TOX-028 LRN-U2636 Data Quality Objectives and Data Integrity The following data quality objectives (DQOs) were indicatedin the protocol for this study: 0 Linearity: The coefficient of determination (r2) mustbe equal to or greater than 0.985 using either no weighting or l / x weighting as appropriate. Limits Of Quantitation (LOQ):The LOQ was equal to the lowest acceptable standard in the calibration curve. Acceptable Precision: Precision should be better than 30% for the method. Acceptable Spike Recoveries: 70-130% unless otherwise documented. Demonstration of Specificity: Compound identification will be substantiated by chromatographic retention time (within 5%) and by selection of a characteristic product ion from a characteristic primary ion. See table 3. Data Summary, Analyses, and Results Data quality objectives for the analytical phaseof this studyoutlined in the 3M Environmental Laboratory protocol for FACT-TOX-028 weremet with the exceptions noted in this report (see Appendix B). Summary of Quality Control Analyses Results Linearity: The coefficient of determination (r2) ofthe standard curves was 20.985. Calibration Standards:Calibration curves wereprepared at concentrations within the linear range of the instrument and were not dilutedQ. uantitation of the target analyte was based on linear regression analysis (l/x weighting) of one otrwo extracted matrix curves bracketing each group of samples. High or low points on the curve may havebeen deactivatedto provide a better linear fit over the curve range most appropriateto the data. Lowcurve points with peak areas less thantwo times that of the extraction blanks were deactivatedto disqualify a data range that may have been significantlyaffected by backgroundlevels of the analyte. Occasionally, a single mid-range curve pointthat was an obvious outliermay have been deactivated. Quantitation of each analyte was baseodn the response of onespecific product ion using the multiple response-monitoring mode of the instrument (see Appendix C, Analytical Methods). Limits of Quantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibration curve (defined as a standard within*30% of thie theoretical value), and is at least two times the analyte peak area detectedin the extraction blanks. (AppendixD, Table 16). Blanks: All blanks were below the lower limit of quantitation for the compounds of interest. To simplify analyses that were complicated by endogenoulsevels of fluorochemicalsin unexposed rat sera and liver, rabbit sera and livewrere selected as a suitablesurrogate matrices for the preparation of calibration curves and continuing calibratiovenrification standards. Non-exposed rat sera was used athse control matrix for matrix spike studies. 3M Environmental Laboratory Page 14 BACK TO MAIN 3M Medical DepartmentStudy: T-6314.1 Analytical Report: FACT TOX-028 LRN-U2636 Precision: Precisionwas determined by atriplicate analysis of one extract and was reproducible to within 5%for all analytes. Reproducibility: At the request of the study sponsor, data was collected to assess the reproducibilityof extraction/homogeneity of distributionof the target analytes in liver of dosed test animals. This was conducted by extracting duplicate liver samplefosr each of six, group 2 animals; aseparate homogenatewas preparedfor each of the 12 extractions. The samples were evaluated against an extracted curve antdhe percelnt difference in the levels of each analyte was calculated. Results ofthis analysis are presented in Appendix D, tables 17 and 18. Note that the samples were not spiked. Reproducibility was assessed baseodn the nonspiked levels of analytes. Matrix Spikes: Matrix spike studies were prepared to approximate concentrations in Group I samples. Matrix spikes and matrix spike duplicates (MSIMSDw) ere extracted with each set of samples and analyzed during analytical runs at the 3M Environmental Laboratory. Sera SpikeRecoveries: Two setsof MS/MSD were preparedwith the sera samples.All matrix spikes were within acceptance criteria, withthe! exception of RTS09250-250 ppbMS-5 and -MSD-5 for M556. These spikes were evalluatedat 135 and 140% recovery. Because all the other analyte spikes were within acceptanceriteria, it is unlikely that these high recoveries are a resultof spiking error.The high recovery results were confirmed, but cannot be explained. An additionalfour matrix spikes were prepared and analyzed. These four matrix spikes were within acceptance criteria(S. ee table 4). Table 4. Matrix SpikelMatrix Spike Duplicate resulftosr M556 in Sera Original SeralM556 116 128 Liver SpikeRecoveries: Two sets of MS/MSD were preparewdith the liver samples. Of the four spikes, a single spike, C95991F/Gl/wk5-MS[1-250 ppb was lofwor PFOSA and M556. An additional four matrix spikes were prepared and analyzedT.hese four matrix spikes were within acceptance criteriafor PFOSA. Some responsesfor M556 were still lower than expected. The average recoveryfor the 8 preparedmatrix spikes was 66%. (See table 5). 3M Environmental Laboratory Pagle 15 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-LJ2636 Table 5. Matrix SpikelMatrix Spike Duplicate results for PFOSA aMn5d56 in Liver Original I LiverlPFOSA I+-t+l I 58 II I 79 I 42 j 1071 Internal Standards:The internal standard(IS),THPFOS was added to all samples and standards. THPFOS was not used for quantitation, but was utsoedmonitor for gross inSt~Ument failureT. he 1s response of each analytical run was verifietdo determine that it did not Vary More thanf50% from the mean within each analytical run.IS responses deviatedby > f 50% in several sera and liver analyses. These responses were confirmbeudt cannot be attributed to a specific problem. This datais flagged in the raw data table but nofurther action is beingtaken, as moresuitable internal standards (e.g. radio labeled material) arneot available. Statement of Data Quality It is not possible to verify true recovery of endogenous analytefrom tissues without radio labeled reference material. The only measurement of accuracy available atthis time, matrix spike studies, indicatesthe data are quantitativeto f30%. Determination ofM556 levels in liver maybe biased low and should be considered quantitativeto f 50%. Summary of Analytical Sample Results 0 Samples from Control Animals: Low levels of some analytes were often detected in the sera and liver of some the control animals. These levels were significantly lower than those found in the low dose test animals. 0 Samples from Dosed Animals: In general, analyte level:; found in the sera and liver of the test animals increased with dose group. Analyte levels increased as dosage increased. Detailed sample data tables are presentedin Appendices D and E. 3M Environmental Laboratory Page! 16 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U2638 Statistical Methods and Calculations Statistical methods were limited to the calculation of means andstandard deviations. See Appendix Ffor example calculations used to generate the liver and serumsample data in FACT- TOX-028. When one or more sample results within a group was belothwe LOQ,calculations for average were done using the LOQ value in the calculation Statement of Conclusion Under the conditions of the present studies, the fluorochemicals PFOS, PFOSA, M556 and M570 were observed in the liver and seraof all rats dosed withthe IN-MeFOSE-OHduring the in-life phase of the study. 3M Environmental Laboratory Page 17 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 Appendix A: Control Matrices and Testsubstance Characterization Table 6. Characterization of the Control Matrices Usedfor Liver and Sera Analyseisn Study FACT-TOX-028 ControMlatrix I RabbLitiver Source ExpDiraatteion II Laboratow I 01/01/2010 Storage Conditions -20C *10"c I I I RaSterum RabbSiterum I I I SigmaChernicals SigmaChernicals I I I I oI/o1/2a1o 01/01/2010 -2*0lCC)"C -20C *10"c Lot # F04051-Female Chemic1a9lH89232 PhysicalDescription I RabLbiviter I Rat Serum I RabSbeitrum I ~ ~~ Test Substance Characterization N-Methyl PerfluorooctanesulfonamidoEthanol (N-MeFOSE-OH) CAS Number: 24448-09-7 ChemicaFlormula: CeF&02N ((CH~)(CH~CHZOH)) MoleculaWr eight: 557.0 Table 7. Characterizationof Test Substance in Study FACT-TOX-028 I Test Substance I ChemNicaaml e I Perfiuorooctanle~ulfonamido ethanol Source 3M Specialty Chemicals Expiration Date unknown Storage Conditions Ambient temperature Chemical Lot# T-6314 Based oninformationobtainedfrom the in-life protocol. 3M Environmental Laboratory Page 18 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 Appendix B: Protocol, Amendments and Deviations 3M Environmental Laboratory Page 19 Dep3aMrtmMeendtical Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LtllV-uLDdF Study Title Determination of the Presenceand Concentrationof PFOS,PFOSA, M556,and M570 in the 13-Week DietaryStudy of Crl:CD@(SD)IGSBR Rats ANALYTICAL PHASE PROTOCOL An FDA Good Laboratory Practice (GLP) Compliant Study Date: 15 August 2000 Performing Laboratories 3M Environmental Technology& Safe:@ Services 3M Environmental Laboratory 935 Bush Avenue St. Paul,MN 55106 Pace Analytical Services, Ino. 1700 Elm StreeStE, Suite 100 Minneapolis, MN 55414 Laboratory Project ldentifkation FACT Tox Number: Tox-028 LRN Number: U2636 3M Environmental Laboratory 3M Environmental Laboratory Page 1 of 1 I Page 20 BACK TO MAIN 3M Medical DeDartment Study: T-6314.1 Analytical TROepXo-0rt2: 8FACT LI 11'1 ULVLnJ Protocol Number:FACT-Tox-028 Lab Request Number (LRN): U2636 Study Identification Determination of the Presence and Concentratioonf PFOS, PFOSA, M556, andM570 in the 13-Week DietaryStudy of Cr1:CD" (SD)IGS BR Rats I Test Article N-Methyl PerfluorooctanesulfonamidoEthanol (N-MeFOSE, T-63 14) Sponsor 3M Toxicology Services-Medical Department 3M Center, 13uilding 220-2E-02 St. Paul,MN 55144-1000 Sponsor Representative John L. Butmhoff, Ph.D. 3M Toxicology Services Telephone: (612) 733-1962 Study Director Sfudy Location(s) In vivo Testing FaciIity Andrew M. Seacat, Ph.D. 3M Toxicology Services Telephone: (612) 575-3161 Facsimile: (612) 733-1773 Covance Lal~oratoriesInc. 3301 Kinsman Boulevard Madison, WI 53704 Proposed Study Timetable In-Life Experimental Start Date In-Life Termination Date Analytical Start Date Analytical Termination Date Study Completion Date Pace Analytical Services, Inc. 1700 Elm Skeet SE, Suite 100 Minneapolis;,MN 55414 01 September 1998 03 December 1998 1 Septembe]:2000 1December2000 . 1 March 2001 3M Environmental Laboratory 3M Environmental Laboratory Page 2 of 11 Page 21 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LfilY-ULDJIP Protocol Number: FACT-Tox-028 Lab Request Number (LRN): U2636 1. STUDY Groups of male and femaleCr1:CD (SD) IGS BR rats were exposed daily to basal dioert, N-MeFOSE (T-63 14) in feed for 13 weAetkst.he end of the in-life study phase, liver and sera specimens were sent to th3Me Environmental Laboratory.foranalyses to evaluate the presence and concentration of T-63 14 metabolites; specifically, PFOS, PFOSA, M556, and M570 (see Table1).The determination of M570 in saenrda liver specimens wilble conducted accordingto documented, but not validated, melhods. I I COMPOUND I I II I 1I I ldentificatlon PFOS PFOSA PerfsluuolfmonoacttPeaenfefluomsoucltfaonney-lamide M556 M556 M570 570.97 1 I N/CAAS NumNbIAer 754-912-7695-393 556.96 Molecu4l9a8r.9 Weight (amu) 498.9 I I Table I.Metabolitesof N-MeFOSE (TesAt rticle) to be Analyzedin the Present Study 2. PURPOSE The analytical phase othfis study is designed to evaluate levelosf PFOS, PFOSA,M556, and M570 in liver and sera specimens of the rats expoTs-e,6d31to4.The in-life phase of the study was conducted at Covance LaboratorieIsn,c., (Covance Studynumber 6329-225). All liver and sera specimens willbe extracted by Pace Analytical Services, Inc., and analyzed at the 3M Environmental Laboratory. Specimen matrices fotrhis study willbe serum and liver; d!ata quality objectives ftohris study are indicated in section 12. 3. REGULATORCYOMPL~ANCE With the initial exceptioonf M570 determinationin sera and liver samples, this study will be conducted in accordancweith the United States Food a]nhdug Administration Good Laboratory Practice Regulationsfor Nonclinical Laboratory Studies, Final Ru2l1e CFR Part 58. 4. QUALITY ASSURANCE The 3M Environmental Laboratory Quality Assurance Unit (QAU) will audit the study conduct, raw data, and final reptoordtetermine compliance with Good Laboratory Practice Regulations, this protocol, an3dM Environmental Laboratory Standard Operating Procedures. 3M Environmental Laboratory 3M Environmental Laboratory Page 3 of 17 Pag:e 22 3M Medical Department Study: T-6314.1 -- BACK TO MAIN Analytical Report: FACT TOX-028 Protocol Number:FACT-Tox-028 Lab RequestNumber (LRN): U2636 5. TESTARTELE 5.1 Identification N-Methyl PerfluorooctanesulfonamidoEthanol (N-MeFOSET-6314, CAS 24448-09-7) Note: Acetone was used as the vehicleto dissolve the test article before mixing with the diet. 5.2 PhysicalDescription Amber waxy solid 5.3 Purity and Stability Results of purity and stability analyses completed by ,the sponsor will be preisnenthted final report. 5.4 StorageConditions T-6314 was stored at mom temperaturaet Covance Laboratories during the in-life phase of the study. 5.5 ReserveSamples A reserve sample (approximately5g) fiom each lot was set aside and storaetdroom temperature. Reserve samples were shipped to the Spoantsthoer end of the in-lifpehase of the study. 5.D6 isposal Unused test article was returned to the spoantstohre lend of the in-life phaseof the study. Thetest article will be maintaineidn the 3ME~wironmentaLl aboratory specimen archives for a period of time specified by GLP regulation, aosrlong as the quality of the preparation affords evaluation. 6. CONTROLARTICLE 6.7 ldenfificafion Certified Rodent Die#t5002 meal 6.S2 ource PMI Nutritional International 6.3 PhysicalDescription Meal (ad libitum, unless otherwise specified) 6.4 Purity and Stabiiity There are nolmown contaminants in the diet that would interfere witthhis study. 6.5 Storage Conditions Room temperature 6.6ReserveSamples Approximately lOOg was takenfiom each dose preparation and stored at room temperature. 6.7 Disposal Unless used for dose analyses, samples were discarded one month after the completion of the in-life phaseof the study by Covance Laboratories. 3M Environmental Laboratory 3M Environmental Laboratory Page 4 of 11 Page 23 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LI m!ma Protocol Number: FACT-Tox-028 l a b Request Number(LRN):U2636 7. REFERENCMEATERIALS I PFOS I PFOSA identification Pefluorooctanesulfonate p~~~~~~~ TCR Substance # SD009. SDO18. SE027 or SI3029 I I SourceChemiScaplescialty SD046 (equivalent 3M lots) (equivalent lots) 1 3M (BRu. ckanin) I M556 1 M570 I I SD014 1 I I Che4k:z?!awCnh)emicals SD040 3~ Spe(Gci.aMltyoore) Physical Description powder White powderlchunksl Light-yellow waxy solid White powder White powder Purity Purity of standardsis currently under determinatlon. Purity valuewsill be includedin the final report or Stabillty Storage Conditions Chemical Lot # Resewe Samples Disposal Safety Precautions (Attachment A) unknown I unknown Room temperature RooRmootemmpteemraptuerreatuRroeom temperature 171 or 217 L-15709 Batch 3M# 113047-80 3M# NBI18506-26 A reserve sample from each batcNlot of reference mawteirlilabl e retained for5 years afler submission of the final reporttowor years after completionif not submitted.but not longer than 10 years following the effective date of the final rtuelset(if applicable). Unused reference material will be retainedusfoerby 3M Environmental Laboratory anwdill be discarded when the quality of preparation no longer affords evaluation. Refer to the MSDS for PFOS Refer tothe MSDS for PFOSA the MSDS for Refer to the MSDS Refer to for M570 M556 Table 2. ReferenceMaterials for FACTTox428 8. TESTSYSTEM Four dose groups comprised 2o0f male and20 female Cr1:CD" (SD)IGS BR rats each, were exposed daily to0- (control), 3- (low exposure), 30- (mid exposuorer)1, 00- (high exposure) pg/g N-MeFOSE (T-6314)in their feed for at least 13 weeks (Table 3). Individualtshein control group were identifiedwith an implantable microchipidentification device. AAer 4 weeks, liverand sera specimens were collectefdiom five animals/sex/dose groupA. t the endof the study (after wee1k3), liver and sera specimens were collectefdrom all remaining animalsin the test system. All liver and sera specimens weretostehnet3M Environmental Laboratory for analysis. 3M Environmental Laboratory 3M Environmental Laboratory Page 5 of 7 7 Page 24 ~ ~~~~~ BACK TO MAIN De3paMrtmMeendtical StAudnya:lyTti-c6a3l14.1 Report: FACT TOX-028 c LRN-U26d Protocol Number FACT-TOX-028 Lab Request Number (LRN): U2636 DOSE GROUP NUMBEROF ANIMALS Ih n t d 2 Low Exposure 3 Mid Exposure 4 High Exposure 20 male/ 20 female animals 20 male/ 20 female animals 20 male/ 20 femanlelmals 20 male/ 20 femalneimals 30 ppm 100 ppm a Dose Levels areexpressed as ppm of N-MeFOSE b Controlgroup animalswil receive a basal dieotnly Table 3. Dosage Levels, DoseGroupsand Group Composition in the 13-WeekDietary Study of 1-14 in Rats 9. SPECIMEN RECEIPT Tissue specimens were received from Covance Laboratories duringatatnhde endof the inlife phase of study, on Octoberan1d3,December22,1998. Liver andsera specimens were shipped by overnight courier and were received fiozednryoince. Specimens wereidentified with specimen numbers (lab request numbers or numbers assbigyneCdovance Laboratories) upon receipt, registered with the 3M Environmental Laboratory and transtfoerarefdreezer for storage. All specimens sent tohe 3M Environmental Laboratory were received and tracked according to the Sample Tracking System Standard Operating Procedures. Detoaifls specimen inspection for damage, receipt, storage, identification, chain of cupstroodtoycols, and data will be presented in the ftnal report. 70. PREPARATORY METHODS Method numbers: 70.7 ETS-8-4, "Extraction of Potassium Perfluorooctanewlfonaoter Other Fluorochemical Compounds fromSerum for Analysis Using HPLC-ElectrosprayMaSsps ectrometry" 70.2 ETS-8-6, "Extraction of PotassiumPerfluorooctane:-sulfonateor other Fluorochemical Compounds h m Liver for Analysis using HPLC-EllectrosprayMaSspsectrometry" Analytical samplesare extracted using an ion-paixing extraction procedurAe.n ion pairing reagentis added to a laboratory sample and the analytepiaoinr partitioned into MtBE. The MtBE extract is then removedand put onto a nitrogen evaporatuonr til dry. Each extracted laboratory sampilsereconstituted in,1.O mL of methanol andfiltered through a 3cc plastic syringe attached to 0a.2 pm nylon filter into glass autovials. A modification will be made to the extraction method prior to the commenocfetmhiesnt study; an internal standard (THPFOS) will be added atfltheerextracted laboratory sampleis reconstituted in1.O mL of methanol. 7 7. ANALYTICAL METHODS 77.7 ETS-8-5, "Analysis of Potassium Perfluorooctanewlfonate or OtherFluorochemical in Serum Extracts UsingHPLC-ElectrosprayhlassSpectrometry" 77.2 ETS-8-7, "Analysis of Potassium Perfluorooctane-sulfonateor other Fluorochemicals in Liver Extracts UsingHPLC-ElectrosprayhfassSpectrometry" 3M Environmental Laboratory Page 6of 7 1 3 M Environmental Laboratory Page 25 BACK TO MAIN 3 M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LHN-U2r Protocol NumberF: ACT-Tox-028 Lab Request Number (LRNU):2636 Analyses are performed by monitoring onemore:product ions selected from asingle primary ion characteristic of a particular fluorochexnical usiHngPLC-ESMSMS.For example, molecular ion499, selected as the primary ionfor PFOS (CsF&Oj-) analysis, is fragmented further to produce i9o9n (F'so3-).The characteristic ion99 is monitored for quantitative analysis. The analysisof M570 in sera and liver matrix samples will. be conducted according to the methods used by3M Environmental Laboratoryfor the other target analytes. However, these methods have not been specifically validafoterdM570. 12. DATAQUALIT0Y5JECnVES The number ofspikedduplicates,use of internal standards, and information onther quality indicatorsare included in the analytical methods (AttachmenBt). As there were no sponsor- recommended data quality objectivetsh, e following willbe used: 12.I Linearity The coefficient of determinatio(n2)of the standard curve must be equatol or greater than 0.985 using either no weightingor llx weighting, as appropriate. 12.2 Limits of Quantitation (LOQ) The LOQ may be determined accordintgo 40 CFR Part 136, Appendix B, or alternatively, the LOQ may be equtoalthe lowest acceptable standardin the calibration curve and thleow curve point, whichis within *30% of the calculated calibration curves,as appropriate. 12.3AcceptablePrecision <30% for the method 12.4AcceptableRecoveries 70-130%. Removal of individual outliers fkom cal(:ulations maybe done if it is documented with a reasonable explanation. 12.5 Use of Confirmatory Methods None. If a confirmatory methodis used, an amendment to this protocol wiblle written. 12.6 Demonstration of Specificity,efc. For HPLC/MS/MS, compound identification will be substantiated by chromatographic retention time and by selection of a characteristic proiodnucftiom a characteristic primary ion (seeTable 4). 3M Environmental Laboratory 3 M Environmental Laboratory Page 7 of 7 1 Page 26 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LHN-UZb;Jb- Protocol Number:FACT-Tox-028 Lab Request Number (LRNU): 2636 COMPOUND PRIMARY ION PRODUCT ION APPROXIMATE REENTION TIME (MIN) THPFOS (SS) 427 PFOS M556 556 M570 570 PFOSA 498 99 499 169 169 78 7.2 80 7.3 7.5 7.7 8.0 Table 4. Compound Identification with IiPLCIMSIMS 13. STATISTICAL METHODS AND CALCULATIONS Statistical methods will be limited to the calculation of me:ans and standard deviations. Examples of the calculations used inthis study will be includeidn the final report. 14. SUBCONTRALCATBORATORY Pace Analytical Services, Inc(.Pace-Tier II Facility, Minneapolis,MN) will be performing the extractionof samples for analyses of liver ands e r u m specimens. All extractedsamples will be shippedto the 3M Environmental Laboratory for analysis. Pace will followall applicable 3M Environmental Laboratory Standard Operaling Procedures and the following Pace Standard Operating Procedures: PSS-Admin-01 PSS-DC-05 PSS-OPS-01 PSS-OPS-02 PSS-OPS-03 PSS-OPS-04 PSS-OPS-05 PSS-OPS-06 PSS-OPS-07 PSS-OPS-08 PSS-OPS-13 PSS-OPS-15 PSS-OPS-16 PSS-OPS-3 1 PSS-OPS-32 ETS-9-40 (3M Lab) Quality System LabNotebooksL, ogbooksa, ndPhoneLogs ' Use and Maintenanceof Ultra-Turrax T25 Homogenizer Raw Data Hazardous Waste Disposal Use and Maintenance of N-Evap Analytical Evaporator Use of Compressed Gaseins the Laboratory Use and Maintenance of NANOpuIr[eWater Purification System Cleaning Non-disposable Volumetric :Pipettes Laboratory Calculations Use and Maintenance of Lab Refrigerators, Freezers, and Ovens Glassware Cleaning Use and Maintenance of the Fisher Scientific Isotemp Freezer Use and Maintenance of Shake(rUsse for Shaker Only) Operation, Maintenance and Calibratioonf Laboratory Centrifuges Operation and Maintenance of Shakers and Vortex Mixers (Use for Vortex Mixer Only) 3M Environmental Laboratory Page 8 of 11 3M Environmental Laboratory Page 27 BACK TO MAIN De3paMrtmMeendtical Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U263r PSS-OPS-37 PSS-MTR-01 PSS-MTR-02 PSS-MTR-06 PSS-MTR-08 PSS-MC-01 PSS-mc-02 PSS-DM-03 Protocol NumberF: ACT-Tox-028 Lab Request Number (LRNl)J: 2636 Operation, Maintenance, and Calibratioonf pH Meters and pH Electrodes Calibration of Certified Weight Set Calibration of Certified ThermometerdThennocouples Verification of Calibration of the Eppendorf Pipettor Verification of Calibration and UofsAe nalytical Balances Purchasing of Laboratory Supplies Disposition of Archive Materials Statistical Evaluationof Data 75. REPORT A report of the results of the study will be prepared by 3M :Environmental Laboratory. When analyses are subcontracted to other laboratories, each laboratory will prepare an analytical report and submit a copy of it t3oMthEenvironmental Laboratory for inclusioans an attachment in the 3M Environmental Laboratory final repoxt. Each report will include, but not be limited to, the following, when applicable: 75.7 Name and addressof the facility performing the study. 75.2 Dates upon which the study was initiated acnodmplleted. 75.3 A statement of compliance by the Study Director addressing any excetpotiGonosod Laboratory Practice Standards. 75.4 Objectives and proceduresas stated in the approved protocol, including any amendments to the original protocol. 75.5 The test article identification by name, chemical absltracts numboer code number, strength, purity, and composition or other appropriate characterisftpicrso,vided by the Sponsor. 15.6 Stability and the solubilityof the test articles under the conditions of administration, if provided by the Sponsor. 75.7 A description ofthe methods used to conduct the test(s). 75.8 A descriptionof the test system (not required in the contract lab analyrteicpaolrt). 75.9 A description of any circumstances that may have affected the quality or the integrity of the data. 75.70 The name of the Study Directaonrd the'namesof other scientists, professionals, and supervisory personnel involved in the study. 75.7 7 A description of the transformations, calculations, or operations performeodn the data, a summary and analysis of the analytical chemistry data, aasntdatement of the conclusions drawn from the analyses. 75.72 Statistical methods used to evaluate the data, if applicable. 3M Environmental Laboratory 3 M Environmental Laboratory Page 9 of 11 Page! 28 BACK TO MAIN Dep3aMrtmMeendtical Study: T-6314.1 RepoArt:nalytical - I H N ; U 2 6 3 B FACT TOX-028 Protocol Number: FACT-Tox-028 Lab Request Number (LRN):U2636 75.73 The signed and dated reports of each of the individual scieonrtiostsher professionals involved in the study, if applicable. 75.74 The location where raw data and the final report are to be stored. 75.75 A statement prepared by the quality assurance unit listing the dates that study inspections and audits were made and the daotfesmy findings reportedto the Study Director and Management. 75.76 If it is necessary to make corrections or additiotnclsa final report after it has been accepted, the changes will be made in the fofrman amendment issued by the Study Director, The amendmenwt ill clearly identify thpeart of the final report thiastbeing amended, the reasons for the amendment, and will be signed by the Study Director. 16, LOCATION OFRAWDATA, RECORDSAN, D FINALREPORT The original datao,r copies thereof, will be available at t3hMe Environmental Laboratory to facilitate audits of the study during its progress and before acceptance ofrethpeortf.inal 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 copiesof data will be retained in the archoivf ethse 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methodsb,ewrieltlained in the archiveosf the facility performing each analysis. 76.7 The following raw data and records will be retained in the study folder in the archives according to3M Environmental Laboratory Standard Operating Procedures. 7 6.7.7 Approved protocol and amendments 76.72 Studycorrespondence 76.7.3 Shippingrecords 76.7.4 Rawdata 76.7.5 Approved final report (original signed copy) 76.7.6 Electronic copiesof data 16.2 The following supporting records will be retained separatefiloym the study folder in the archives according to3M Environmental Laboratory Standard Operating Procedures: 76.2.1 Training records 76.2.2 Calibrationrecords 76.23 Instrumentmaintenancelogs 76.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 7 6.2.5 Appropriate specimens 77. DATA/ SPECIMERNETENTION Specimens willbe maintained in the3M Environmental Laboratory specimen archives faor period of time specified by regulation,asorlong as the qualityof the preparation affords evaluation, but not longer than ten years following the effectiveofdtahte final tesrtule (if 3M Environmental Laboratory 3 M Environmental Laboratory Page 10 of 11 Page 29 BACK TO MAIN 3M Medical DepTa-r6tm31e4n.t1Study: Analytical Report: FACT TOX-028 Ltll\l-"Lt)d6 Protocol Number:FACT-Tox-028 Lab Request Numbe(rLRN):U2636 applicable), andas established by3M Environmental Laboratory Standard Operating Procedures. 18. PROTOCOL AMENDMEANDTSDEWATIONS Planned changes to the protocol willinbtehe form of written amendments signed by the Study Director and the Sponsor Representative. Amendmentsbewciollnsidered as part of the protocol and willbe attached to the final protocol. All changes to the protocoblewill indicated in the final report. Any other changes will be infotrhmeof written deviations, signed by the Study Director and filed with the raw data. 19. A~ACHMENTS 79.7 Attachment A:Material Safety Data Sheets(MS1)S) for Reference Materials 79.2 Attachment 6:Extraction and Analytical Methods 20. SIGNATURES John L. ButenhofJ Ph.D., SpoRnseoprresentaDtiavtee Andrew M.Seacat, Ph.D., SDtuirdeyctor 4%?- 3M Environmental Laboratory 3M Environmental Laboratory Page 11 of 11 Page 30 BACK TO MAIN StuDdey3p:MartMmeednitcal T-6314.1 Analvtical ReDort: FACT TO&&8 LRN-U2636 ProtOcOl Number: FACT-TOX-028 Lab Request Number (LRN): U2636 AITACHMENTA MATERIALSAFETY DATASHEETS (MSDS) FOR REFERENCE MATERIALS 3M Environmental Laboratory 3M Environmental Laboratory Attachment A Page 31 3M Medical Department Study: T-6314.1 A ~ A C H M E NBT EXTRACTION ANDANALYTICALMETHODS BACK TO MAIN Analvtical Report: FACT TOX.&28 LRN-U2636 Pm!ocol Number FACT-Tox-028 Lab Request Number(LRN): U2636 3M Environmental Laboratory 3M Environmental Laboratory Attachment B Page 32 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-g28 LRN-U2638 Study Tit/e Covance 6329-225 13-Week DietaryToxicity Study with N-Methyl PerfluorooctanesulfonamidoEthanol (NMeFOSE, T-6314) in Rats PROTOCOL AMENDMENT NO. 4 Amendment Date: August 17,2000 Performing Laboratory 3M Environmental Technology& S a M y Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification Covance 6329-225 ET&SS FACT TOX-028 LRN Number: U2636 3M nvironmenta/Laboratory 3M Environmental Laboratory Page 33 BACK TO MAIN 3M Medical DepTa-r6tm31e4n.t1Study: Analytical Report: FACT TOX-028 LRN-U2638 Covance Protocol6329-225 Amendment 4 This amendment modifies the following portLon(s) of the protocol: 1. PROTOCOL READS: Andrew M. Seacat is assigned to the role of sponsor representative (study monitor). AMENDTO READ: The roleof sponsor representativehas been reassigned toJ ~ h Bn utenhoff. REASON: The role of sponsor representative must be assigned to one person for both the analyticalandin-lifeprotocols.Since 3M is thesponsorforthestudy, the sponsor representative must befkom 3M. 2. PROTOCOL READS: The study director roilseassigned to PeteJr. Thomford f h m Covance Laboratories. AMENDTO READ: The roleof study director has been reassigntoedAndrew Seacat. The role of principal in-life investigator has been reassigned to Peter Thomford. REASON: Per GLP regulations, only one study director may be assignaedstutody. Since 3Mis the sponsor of the study, the study director shoufrlodmb3eM. 3. PROTOCOL READS: The performing laboratoryis listed as Covance Laboratori.es Inc., 3301 Kinsman Boulevard, Madison WI 53704-2595. AMENDTO READ: Change the performing laboratory to 3M Environmental Laboratory, Building 2-3E-09,935 Bush Avenue, St. Paul MN 55106. Selected extractions will bperformed at Pace Analytical Service, Inc.as per thePAI. REASON: All analytical testing will be done3Mat Environmental Laboratory oPr ACE. 4. PROTOCOL READS: The study locationis specified as Covance Laboratories, Ihc., 3301 Kinsman Boulevard, Madison WI 53704-2595. AMENDTO READ: Change the study location 3toM Toxicology Service+Medical Department, 3M Center, Building 220-2E-02,St. Paul MN 55144-1000. 3M Environmental Laboratory 3M Environmental Laboratory Page 34 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LIIN- 7 REASON: 3M Toxicology Serviceiss the sponsorof the study. Covance Protocol6329-225 Amendment 4 5. PROTOCOL READS: There is no principal analytical investigato(rPAI)listed for3M Environmental Laboratory for FACT Tox-028. AMEND TO READ: Kristen J. Hansen has been assignetod the roleof principd analytical investigatorfor the 3M Environmental Laboratory. REASON: No PAI had previously been assigned. 6. PROTOCOL READS: The analytical phase protocol has not wbrietetnen or approved. AMENDTO READ: Add that the analytical phase protocioslattached to thislist of protocol amendments. REASON: There aretwo phases to the study; the in-life phase and the analytical phase. The analytical phase protocol FACTTOX-028 is an amendment to the Covance protocol 6329-225. 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 35 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical ReDort: FACT TOX-028 LRN-U2636 Covance Protocol 6329-225 Amendment 4 Amendment Approval John ButenhogDRaPtehep.rDes.,eSnptaotnivseor Andrew Seacat, Ph.D., Study Director Kristen J; Hansen, Ph.D.. PArinIcanilvpyeatDislctaigtlaetor ' Date 8 / 2 3 m7 3M Environmental Laboratory 3M Environmental Laboratory Page 36 3M Medical Department Study: T-6314.1 3hI Environmental Technology and Services PO Box 3333 I SI. Paul. M N 55133-3331 612 778 6442 BACK TO MAIN Analytical Report: FACT TOX,&28 LRN-U26316 Study Title Determinationof the I :sence and Concentrationof PFOS, M556, and M570 in the 13-Wee Dietary Study of Cr1:CD" (SD) IGS BR Rats PRO )COL AMENDMENT 110.5 Amendment Date: January 05,2001 3M Enviror 31 rrforming Laboratory Lend Technology& Sakty Services Environmental Laboratory 935 Bush Avenue St. Paul, MN 55 106 Laba ttory Project /dentific:afion FACT-TOX-028 LRN Number U2636 3M Environmentel Laboratory 3M Environmental Laboratory Page 37 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U263: FACT-TOX-028 I Amendment 5 This amendment clarifies identification of the test article. 1. PROTOCOL READS: This study uses different abbreviatidns to identify the test article, N-Methyl PerfluoroctanesulfonamidoEthanolj The abbreviations include N-MeFOSE-OH, N-MeFOSE, MeFOSE, MeFOS, and MeFOSE albohol. AMENDTO.' The abbreviations N-MeFOSE-OH,!N-MeFOSE, MeFOSE, MeFOSa,nd MeFOSE alcohol are deemed to be equivalent fotrhis stupy. REASON: To clarifjl the use of these different bbbreviations. I Ahendment Approvial Andrew Seacat, Ph.D., Study Directbr Date 3M Environmental Laboratory 3M Environmental Laboratory Page 38 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 I (Check one) __._ ._____ Record of Deviation I. Identification r .. ........... ._ ................... --. ... - ............... D SOP aMethod IJEQuipmenPtrocedure 0 Protocol 0 Other: ... .................... .,. . . . . - . . . . . . . . . . . . . . . . . . . . - . . ........ Date(s) of cccurrence .................. I--." ........ "........I ....... .- . . . . - . . . . . . . . . . - .... ...- .................................. "_ __ * ...-. .. . - ......__._ .. .. ".. . .- . .- .......... -_._.--.--...... -- ...................... - -........ I.--. ...... ....-- " ... ........... -:.. - ............ ._. . . . . __ ... ._ . I. .................... ..... - _.. . . . . . . . . . _. - .............................. ...................... 1.....-_........~,I..._......_._..__...... _.. .... _ " ._.. ................ ^_.."_. - ........ - ................. _.. . . . . . . . . ..r .. ............. . . . . . . . . . . :.. ......................... ................... ' Date ........ " . i "'.LIa - Spar Rep - Jthr\ DuIcrhoFF Dire&r A,&,l.: &.UP{ i 3M EnvironmentalLaboratory Deviation No. Form ETS-4-8.0 ( a s s i g n e d by Study Director or Project Lead tat the end of study or project) 3M Environmental Laboratory Page 39 3M Medical DepartmentStudy: T-6314.1 Record of Deviati'on I. ldenfificafion BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 (oCnhee) ck o Protocol 6' Other: \ a W locumentNumber- -- .I.-.- Date(s) of ,&%urrence E n - 3- 4, I I II. Description: __ Ill. Actions Taken: (su-c-h as amendment issued,SOP rtwision, etc.) AczumQn-teiTm-.~s_-farr;n.., 3h4Envirohental Laboratow Form ETS-4-8.0 3M Environmental Laboratory Deviation No. d. (assigned by Study Director or Project Lend at the end ofstudy or project) Page 40 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 Record of Deviation 1. Identification 1 .--_I-Study / ProjectNo. + . ' T o . A : m C ' o * = DeviatTioynpe 0-&SOetPhod 32%a- ---_.__-_-.-. tl Espment Procedure (Check one) 0 Protocol 0 Other: I U I -- IV. Impact on Study/ Prqject . I-,-. - - - - I 3M Environmental Laboratory Page 41 3M Medical Department Study: T-6314.1 Record of Deviati'on BACK TO MAIN Analytical Report: FACT TOX-028 LRNlU2636 TOX0028 (LIMS #U2636)-- Deviation type 0 SOP --- X Method 0Equipment Procedure I Required procedurelprocess: ---.- Section 14.2.1: Solvent blanks, method blanks, amndatrixblanks must be belowthe lowest I-- standard on the calibrationcurve. blank iniectedfor a runwai above the LOO. --.- --- In most places a high solvent blank was analyzed, additional solvent bloarnmksethod blanks were analyzed immediately followingthe high blank. This second injectionwas typically w e l l below the LOQ. Occasionally,the first injection of a runis high because it may immediately follow injection of a high standard froma previous run. For this reason, morethan one blank is usually analyzed priorto the start of a calibration curve. These seconadnd third, etc. blanks are below the LOQ and are more representativoef the analytical conditions of the samples; the 4 ,.._sI_t_uI_d__y-_ data is not adversely affected. h lz/oS/d ~ ------__- --- Authorized by lY b& r9qi2 Z@W spCac~rd: , , , h\tnhDCf S)Ud.;I o ; ~ L * ~ &o L b e v i a t i o n NO. (assigned by Stud, Directoror Project Lead Y at the end of studoyr project) Attachment A: Record of Deviation 3M Environmental Laboratoj ETS-4-8.0 Page 1 of 1 Page 42 3M Medical Department Spdy: T-6314.1 Record of Deviation 1. Identification BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 --__.-I_ _._.. - - I__.--. -_-_ - - DocumNeunmt ber: ETS-8-7.0 Date@)of occurre1n0c/e2:3/00 II. Description: Required Procedurehrocess:Analyze a mid-range calibration standard.... Actual Procedurdprocess: A 306 ppb calibration standard was usfeodr sample runDOO1023a. Although 306ppb is in the mid-range of the entire curvem, any of the high curve pointswere deactivated in order to optimize a linear range appropriate tlohe data. As a result, the 306 ppb CCV wasno longer at the mid-rangeb, ut at the same leveasl the highest pointin the calibration curve. ."...-----------I. ----------II_-- -__-~___-____-_-.-- Ill. Actions Taken: (such as amendment issued, SOP revision, etc.) 3M Environmental Laborutoty U F o ET~S-4-8.0 3M Environmental Laboratory Deviation No. 5 (assigned by StudyDirector or Project Lead at the end of study or project) Page 43 3M Medical Department Study: T-6314..1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 Record of Deviation I. Identification ................................................................................................................................................................................................................................................ Study / Project No. TOX028 d IJ B ................................................................................................................................................................................................................................................. DeviaTtiyopne SOP Method EquipmPeronct edure (oCnhe)eck D Protocol d Other: ....................................................................................................................... ....................................................................................... Document Number:ETS-8-5.1 j Date(s) of occurrence: 11/6/00 11. Description: ,, R....e...q...u...i..r..e...d......P...r..o...c...e...d...u...r..e.../.p....r..o...c..e...s...s..:....C.....o...n...t..i.n...u...i..n...g......c...a...l.i..b...r..a...t..i.o...n......v...e...r..i..f..i..c..a...t..i.o....n.....p....e..r..c...e...n...t....r...e..c...o...v...e...r..i..e..s...................... must be w.it.h.i.n. *30% o. f. t.h.e. .s.p.ik. e.d. .c.o.n.c.e.n.tr.a.ti.o.n... . . . . . . . . . . . . . . . . . .......... .......................................................................................................................................................... :.................................................................................... Ac.t.u. .a.l. .P. .r.o. .c. .e.d. .u. .r.d. .process..:...A...c..a..l.i.b..r..a.t..i.o..n.................c..u..r.v..e.............. 5w.saasm,ples,,,a follo,weeddC,,CcalVosing by 400+ .c..a...l..i..b...r..a..t..i..o...n.....c...u...r..v...e........T....h...e..C.....C....V.....w....a...s....a...t...2...5...p...p...b.....a...n...d.....t..h...e....s...a...m....p...l..e...s....w.....e..r..e.a...t.................p...p...b........T....h...e.....l.i..n...e...a..r.................. range of the of the curve optimizedto the data was49.6 to 740 ppb; as a result, the CCV was .o...u...t....o...f...t..h....e....c...a...l..i.b....r..a..t..i..o...n......r..a...n....g...e........S....i.n....c...e.....t..h...e......f..i..v...e......s..a...m.....p....l.e...s......w....e.i.n.r..e...,.t..h...e.....m.....i.d...r..a...n...g...e....o....f...t..h...e.....c..u....r..v...e......a...n...d......were immediately surroutnwdoedcalibrationby ?.t..h..c.e.u....rd..v.a..e.t..sa....i..s....s..u....f..i..c...e...n...t..l..y...'.b....r..a...c...k...e...t..e...d............................... ....................................................................................................................... ............................................................................................................................................................................................................................................. Ill. Actions Taken: Isuch as amenhent issued SOP revision etc) ............................................................. ................................................................................................... ?.........!. .............................................................. A deviation was written. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................................................................................................................................................................................................................................ & L E - & L ........................................................... Recorded By: Harold Johnson -...-....................................................................................................................... CL IV. Impact on Study/ Project ..".................................................... j Date: i November 30,2000 6.3li&?L? -, ............................................................................................................................................................................................................................................... No adversiempact on the data. ................................................................................................................. ..... ,_ ......................................................................................... 3MEnvironntental Laboratory Form ETS-4-8.0 3M Environmental Laboratory Devlation No. G (arsigned by StudyDirectoror Project Leadat the end ofstudy or project) Pag'e 4 4 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U26316 Appendix C: Extraction and Analytical Methods This appendixincludes the following methods: ETS-8-4.1, "Extraction of PotassiumPerfiuorooctanesulfonateor Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Eleci:rospray/Mass Spectrometry," (14 pages) ETS-8-6.0, "Extraction of PotassiumPerfluorooctanesulfonateor Other Fluorochemical Compounds from Livefror Analysis Using HPLC-Electrcspray/Mass Spectrometry," (14 pages) ETS-8-5.1, "Analysis of PotassiumPerfiuorooctanesulfonateor Other Fluorochemicalisn Serum Using HPLC-Electrospray/Mass Spectrometry,("9 pages) ETS-8-7.0, "Analysis of PotassiumPerfluorooctanesulfonateor Other Fluorochemicalisn Liverusing HPLC-Electrospray/Mass Spectrometry,"(10 pages) 3M Environmental Laboratory Page 45 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX,-028 LRN-U26316 3M ENVIRONMENTLAALBORATORY Reviewer METHOD EXTRACTION OF POTASSIUM PERF'LUOROOC'I'ANESUONATEOR OTHER IFLUOROCHEMICAL COMPOUNDSFROM SERUM IFOR ANALYSIS USING HPLC- ELECTROSPRAY/MASS SPECLXOMETRY Method Number: ETS-8-4.1 Adoption Date: 03/01/99 .Author: Lisa Clemen, Glenn Lagenburg Revision Date: q/ a7 m Approved By: ' ' $.*-f ,/.L. Laboratory Manager Date .Group iflZLjY9 Date Technical ' olllaLI4 4 Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method i s for the extraction of potassmiurpnerfluorooctanesulfonate (PFOS) or other fluomchemicalcompounds h m serurn. 1.2 Applicable compounds: Fluorochemicalsurfactantsorother fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey, andhuman senun or other fluids as designated in the validation report. Word 6/95 3M Environmental Laboratory ETS-8-4.1 ExtractionofPFOS fiom Serum Page 1 of 14 Page 46 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 2.0 SUMMARY OF METHOD 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactantsfrom s e ~ mo,r other fluids, usinagn ion pairing reagent and methyl-tert-butyl ether (MBE). In this method, seven fluorochemicalswere extracted PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556,and 'surrogate standard(see 3.0 Defriritions). An ion pairing reagenits added to the sample andthe analyte ion pair is partitioned intoMtBE. The MtBE extract is removed andput onto a nitrogen evaporator untidlry. Each extractis reconstituted in 1.0 mL of methanol, thenfiltered througha 3 cc plastic syringe attachetdo a 0.2 p nylon filter intoglass autovials. 2.2 These sample extracts are analyzed following methodETS-8-5.1 or other appropriate methods. 3.0 DEFINITIONS ' 3.1 ' PFOS: perfluoroo&esulfonate (anion ofpotassiumsalt) C,F,,SO; .. 3.2 PFOSA: periluorooctanesulfonylamide C8F,7SOzN& 3.3, PFOSAA: perfluorooctanesulfonylamido (ethy1)acc:tite C8F,,S0,N(C&CH3)CH2CO; 3.4 EtFOSE-OH 2(Nethylperfluorooctanesulfonamide)-ethyl alcohol C8F,,S02N(CHzCH3)CH2CH20H 3.5 PFOSEA: perfluomoctanesulfonylethylamide C8Fl7SOzN(CHJXI3)H 3.6 M556:C~F~~SO~N(H)(CH~COOH) 3.7 Surrogate standard IH-lH-2H-2H perfluomoctanesulfonic %id 4.0 WARNINGS AND CAUTIONS 4.1 HeaIth and safety warnings. 4.1.1 Use universalprecautions, especially laboratcry coats, goggles,and gloves when handling animal tissue, which may contain pathogens. 5.0 INTERFERENCES 5.1 There are no interfbkces known at thistime. 6.0 EQUIPMENT 6.1 The fobwing equipment is used whileperforming 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 S a m Page 2 of 14 3M Environmental Laboratory Page 47 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U263B 6.1.4 Nitrogenevaporator,Organomation 6.1.5 Balance (It 0.100g) 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Eppendarf or disposable pipettes 7.3 Nalgene bottles, capableof holding 250 mL and 1L, 7.4 Volumetric flasks,glass, type A 7.5 I-CHEM vials, glass4, 0 mL glass 7.6 Centrifugetubes, polypropylene, 15mL 7.7 Labels 7.8 Oxford Dispenser- 3.0 to 10.0 mL 7.9 Syringes, .capabloef 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, rin3setimes with methanol and3 times with Milli-QTMwater. Rinse syringes a minimumof9 times withmethanol, 3 rinses h m 3 separate vials. 8.0 REACENTs AND STANDARDS 8.1 Type I reageit grade water, Milli-Qw or.equivalen~t;,awflater usedin this niethodshould be Milli-Qm water and mabye provided bya It4ilLi-Q TOC Plus- system 8.2 Sodiumhydroxide (NaOH), J.TBaker or equivalent 8.3 Tetrabutylammonium hydrogendfati(TBAj, Kochkor eqhivalent 8.4 Sodium carbonate (Na&O,), J.T. Baker or equivalent 8.5 Sodiumbicarbonate(NfiCO,), J.T. Baker or equivalent 8.6 Methyl-T-Butyl Ether,Omnisolv, glass distilled01: HPLC grade 8.7 Methanol, Omnisolv,glass distilled or HPLC grad.e 8.8 Serum or blood, fi-ozenfrom supplier 8.9 Fluorochemical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weigh=t 538 8.9.2 PFOSA (3M Specialty Chemical Division:), molecular weig=ht499 ETS-8-4.1 Extractionof PFOS from !Serum. Page 3 of 14 3M Environmental Laboratory Page 48 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U26316 8.93 PFOSAA (3M Specialty Chemical,Division):, molecular weigh=t 585 8.9.4 EtFOSE-OH (3MSpecialty Chemical Division), molecular weigh=t 570 8.9.5 PFOSEA (3M Specialty Chemical Division):, molecular weigh=t 527 8.9.6 M556 (3M Specialty Chemical Division), molecular weight=557 8.9.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H,l-H, 2-H, 2-H C$,,SO,H) molecular weight = 428 8.9.8 Other fluorochemicals, as appropriate . 8.10 Reagent preparation NOTE: When preparing larger volumesthanlisted in reagent, standard, or surrogate preparation, adjust accordingly. 8.10.1 10N sodiumhydroxide (NaOH): Weigh approximatelyZOO g NaOH. Pour into a . 1000mLbeak& containing 500 mL Milli-G"water, m i x . until all solids are dissolved. Store in a 1L Nalgene bottle. . 8.10.2 1N sodium hydroxide (NaOH): Dilute 10N NaOH 1:lO. Measure 10 mL of 16 N NaOH solution into a 10m0L volumetric flask and dilute to volumuseing .. .Milli-QTM water. Storein a 125 mL Nalgenc: bottle. ' ' 8.10.3 @ 0.5 M tetrabutylmonium hydrogen sulfate ('IBA): Wei approximately 169 g of TBA into a1L volumetric containing 500mLMilli-Q water. Adjust to pH 10usingapproximately 44to 54 mL of 10N NaOH (While addingthe last mL of ' NaOH, add slowly because the pH changes ;abruptly). Dilute to volume with Milli-Qm water. Store in a 1L Nalgene bottle. 8.10.3.1 TBArequires a check prjor to each use to ensure pH= 10. Adjust as ' needed using 1.NNaOH solution. 8.10.4 0.25 M sodium carbonatdsodiun bicarbonate buffer(Na&!OJNaHCO;): Weigh approximately 26.5 gofsodium carbonate(Na$O,) and 21.0 g of sodim , bicarbonate (NaHCO,) into a 1L volumetric flask and bring to volumewith Milli- Q" water. Store in a 1 L Nalgene bottle. 8.11 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemicalstandards, as itppropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppmPFOS, 1.02 ppm PFOSA, 0.987 ppmPFOSAA, and 1.lo ppm EtFOSE-OH.) 8.11.3 Weigh approximately 100 mg oPf FOS into a 100mL volumetric flaskand record the actual weight. 8.11.4 Bring to volume with methanfoolr a stock standardof approximately 1000 ppm (PdIm 8.11.5 Dilute the stock solution with methanfoolr a working standard 1 solutionof approximately 50 ppm. 8.11.6 Dilute working standard1with methanol $ora working stddard 2 solution of approx. 5.0 ppm. ETS-84.1 Extraction of PFOS from Serum Page 4 of 14 3M Pagle 49 3M Medical DepartmentStudy: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 , 8.12 8.11.7 Dilute workingstandard 1with methanolfor a workingstandard 3 solutionof approx. 0.50ppm. Surrogate stock standardpreparation 8.12.1 Weigh approximately50-60mg of surrogate standardl-Hyl-Hy2-H, 2-H, C$,,SO,H into a 50 mL volumetric flask and record the actual weight.. 8.12.2 8.12.3 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 PPm. Prepare a m g a t e working standard. Transfer approximately1mL ofsurrogate stock to a 10mL volumetric flask and bringto volumewithmethanol for a working standardof 100ppm. ' Record the actual volumtreansferred. 9.0 SAMPLHEANDLING 9.1 All samples are receivedb z e nand must be kept fiozenuntil the extractionis performed.. 9.2 Allow -samplesto thaw to room temperature prior to extraction. 10.0 OUALITY CONTROL 10.1 Solvent Blanks, Methodblanks and matrix blanks . . 10.1.1 An aliquot of 1.0 mL methanol is used as a solventblank ' 10.1.2 Extract two 1.0 mL aliquots of Milli-Qmw;aterfollowing thisprocedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquotsof the serum following this procedureand use as matrix blanks. See 11.1.4. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samptloesdetermine the accuracy of the extraction. 10.2.2 Prepare each spike usinagsample chosen bythe analyst, usuallythe control matrix received with each sample set. 10.23 Expected concentrations wilflall in the mid.-range of the initial calibration curve. Additional spikes may be included and mfaalyl in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicapter 40 samples, witha minimum of2 matrix spikes per batch. 10.3 Continuingcalibration checks 10.3.1 Prepare continuing calibration check samples to ensure the accuracthyeoinf itial calibration curve. 10.3.2 Prepare, at a minimum, one continuing check per groupof 10 samples.For example, ifa sample set= 34, four checks: u e prepared and extracted. 10.3.3 Prepare each continuing calibration checljkrom the same matrix usedto prepare the initial curve. 3M Environmental Laboratory ETS-8-4.1 ExtractionofPFOS from Smum Page 5 of 14 Page 50 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX,-028 LRN-U2638 10.3.4 The expected concentrationswill fall within the mid-rangoef the initial . calibration curve. Additional spikes may be included that infaltlhelow-ringe of the initial calibration curve. This is necessary if the analyst must quantitate using only the low endof the calibration curv(efor example, 5 ppb - 100ppb, rather than 5 ppb - 1000ppb). 11.o CALIBRATION AND STANDARDIZATION 11.1 Prepare matrix calibration standards 11.1.1 Transfer 1mL of serum to a 15mL centrifuge tube. 11.1.2 Ifmost sample volumes areless than 1.0 mid,extract standards withmatrix volumes equal to the sample volumes.Do not extract less than0.50 mL of . matrix. Record each sample volume on the (extraction sheet. 11.1.3 While preparinga total of twenty aliquotsin. 15mL centrifuge tubes,mix or shake between aliquots. 11.1.4 Two 1mL aliquots,or other appropriate volume, servase matrix blanks. Typicallyuse the standard concentrations and spiking amounts lisnteTdable 1, . at the end of this section, to spike, in duplicikte, two standard curves,for a total of . eighteen standards, two matrix blanks, atwndo method blanks. 11.1.5 Refer to validationreportETS-8-4.0 & ETIS-8-5.0-V-1, which listst'he working ranges and the Linear Calibration Ra(LngCeR) for calibration curves. 11.1.6 Use Attachment D as an aid in calculatingthe concentrations of the working standards. See Section 13.0 to calculate actual concentrationsof PFOS in . calibration standards. 11.2 To each standard, blank, or continuingchkk, add aqpropriate amountof surrogate working standardfor the concentrationto fall within the calibration curve rang5eppb,- 1000 ppb. 113 Extract spiked matrix standards followin1g2.6-12.16 of this method.Use these standards to establish each initial curve on themass spectrom.eter. 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from S,erum Page 6 of 14 Page 51 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX,-028 LRN-U2636 Table 1 . I I Approximate spiking amounts for standards and sD- ikes ' Using 1.0 mL of matrix _ ' Workisntagndard PL Appmfixcn.oaonl fc. analyte in matrix 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.OO ppm standards. 12.0 PROCEDURE 12.1 Obtain frozen samplesand allow to thaw at room temperatureinoarlukewarm waterbath. 12.2 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volumtoea 15 mL, polypropylene centrifugetube. 12.3. Return unused samples to fieezer after extraction.arnounts have been removed. 12.A Record the initialvolume on the extraction worksheet. 12.5 Label the tube with the study number, samplIeD, date and analysitnitials. See attached worksheet for documenting the remaining steps. 12.6 Spike all samples, including blanks asntdandards, Iwdy for extraction with su. r.rogate standard as described in 11.2. 12.7 calibration Spike each matrixwith the appropriateamount ofstandardas describedin 11.1, or Table 1in that section, for the calibration curvestandards, Also prepare matrix spikesand continuing L 12.8 Vortex mix the standard curve samples, matrix spike samples, and contincuailnibgration samples for 15 seconds. I 12.9 Check to ensure the0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.10 To each sample, add 1mL 0.5 M TBA and 2 mL of0.25M sodiumcarbonatdsodium bicarbonate b u f k . 12.11. Using an Oxford Dispenser, add5 mL methyl-tert-loutyl ether. 12.12 Cap each sampleand put on theshaker at a setting (of300 rpm,for 20minutes. 12.13 Centrifugefor 20to 25 minutes at a setting of 3500rpm, or h t i l layers are well separated. ETS-84.1 Extraction ofPFOS from S t m Page 7 of 14 3M Environmental Laboratory Pagle 52 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U26316 ' 12.14 Label a fresh 15 mL centrifuge tubewith the same informationas in 12.5. 12.15 Remove 4.0 mL of the organic layetro this clean 15:mL centrifuge tube. 12.1 6 Put each sample on the analytical nitrogen evaporator udnrtiyl,approximately 1 to 2 hours. 12.17 Add 1.0 mL of methanol to each centrifuge tube usinaggraduated pipette. 12.18 Vortex mix far 30 seconds. .. 12.19 Attach a 0.2 pm nylonmesh filter to a 3 cc syringe;andtransferthe sample to this syringe. Filter intoa 1.5 mL glass autovial or low-volume autovial when necessary. 12.20 Label the autovialwiththe study number,animalnumberand gender, sample timepoint, matrix, h a l solvent, extraction date, andanalyst(s) performing the extraction. 12.21 Cap and store extractast room temperatureor at apImximately4 "Cuntil analysis. 12.22 Complete the extraction worksheet, attachedto this document, and tapein the study notebook or include in study binder, as appropriate. 13.0 DATAANALYSIS AND CALCuLAnONS 13.1 Calculations 13.1.1 Calculate actualconcentrations of PFOS, or other applicable fluomchemicali,n - calibration standardsusing the following equation: mL of standardx concentration of standard (ua /mL) . mL of standard +mL of surrogate standar-dI- initial matrix volume(mL) Final Concentration (pg/mL) of PFOS in matrix 14.0 METHODPERFORMANCE 14.1 The method detection limit(MDL)is analyte and m,atrix specific. Refer to MDL report . for specificMOL and'lhitof quantitation(LOQ)values (seeAttachments B 'andC). 14.2 The following quality control samples are extracted with each batch of samples to evaluate the quality of the extraction and analysis. 14.2.1 Methodblanksandmatrixblanks. . 14.2.2. Matrix spike and matrix spike duplicate sainples to determine accuracy and ' precision of the extraction. 14.2.3 Continuing calibration check sampletos determine the continued accuracoyf the initial calibration curve. 14.3 Refer to section 14 of ETS-8-5.1 for method performance criteria. ' 15.0 POLLUTION PREVENTAINODNWASTEMANAGEMENT 15.1 Sample wasteis disposed in biohazard containers, flammable solvent wasties disposed in high BTU containers, and usedglasspipette waste!isdisposed in broken glass containers located in the laboratory. ETS-84.1 Extraction of PFOS from Serum Page 8 of 14 3M Environmental Laboratory Page 53 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 16.0 RECORDS 16.1 Completethe extraction worksheet attached to this methoadn,d tape in the study notebook or includien the 3-ring study bindera,s appropriate. 17.0 ATTACHMENTS 17.1 Attachment A, Extractionworksheet 17.2 Attachment B,MDULOQ values and s u m m a r y 17.3 Attachment C,Calibration standard concentration worksheet 18.0 REFERENCES 18.1 The validation report associated withthismethod isETS-8-4.0 & 5.0-V-1. . 18.2 FACT-M-3.1, "Analysis of Serum or Other Fluid Erctracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" . 19.0 AFF~CTEDDOCUMENTS 19.1 ETS-8-5.1, "Analysis ofSerum or OthkFluid Extrxcts for Fluorochemicalsusing HPLC-Electrospray Mass Spectrometrf` 20.0 REVISIONS . Revision Number 1 Reason For Revisiop Section 12.21 Changedto includesamplestorageartoomtemperature. Section 12-13 Added theshakq speed. Section 12.1.7Final volumeis 1.0mL;not adjusted forinitialvolumes less than 1.O mL. - Revision Date 04/02/99 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 9 of 14 Page 54 3M Medical Department Study: T-6314.1 Extraction Worksheet ETS8-4.1 BACK TO MAIN Analytical Report: FACTTOX-028 LRN-U263'6 3-a-=-- Atsachment A ETS-8-4.1 Extraction of PFOS from ;Serum ___.. ..... Page 10 of 14 Page 55 3M Medical Department Study: T-6314.1 MDLLOQ values for rabbit serum BACK TO MAIN Analytical Report: FACT TOX-028 LRN42636 Ige (LCR) ationsto be used for prepahg'ae hrve . MDULOQ values in rat, bovine, monkey,and human serum, and monkeyplasma Were not statistically determined. Two curvesin each ofthese matrices were extracted and analyzedwith the rabbits e m curves to determine equivalence. Responisnesthe rat, bovinem, mkey, and humanwere equivalent to the rabbit responses, therefore, theMirDL and LOQ will be the same valuesas determined in rabbit serum. Please see LOQ Summary and MDL study in ETS-84.0 & 5.0-'4-1 for furtherinformation. Attachment B:h4DLLOQ Summary ETS-8-4.1 Extraction of PFOS from Strum 31MEnviromrrerrtaktaboratory Page 11 of 14 Page 56 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 Compound: PFOS I Preparreadnge I LCR from I % Recovery I RSD 1 Rabbit Serum of standards CUNe (ppbj ( n w ) (PPb) Full Range LOW Curie High curve 1.R - 0.995 978 - 4.94 248 - 97.8 978 - 0.995 978 24.8 - 978 - 4.94 248 - 97.8 978 - 4.94 978 83-108 5.34-1825.-0104 85-106 94-111 Compaund: PFOSA r Prepared range Rabbit Serum of standards (PPb) (nfM-1 FullRange ' . 0.993-976 Low Curve - 97.6 . Highcurve 24.8 - 976 .1/x 0.993 - 976 LCR fiom curve @pb) (ng/mL) 4.93- 976 ' - 4.93 97.6 - 24.8 978 - 4.93 976 % Recovery Rwze 88-103 4. .9. 3 87-105 93-102 94-103 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 . Compound: PFOSAA ' I I Preparedrange I LCRfrorn I % Recovary I RSD I I I I Rabbit Serum I ofstandards I curve I Range Range Full Range . LOWCWC ' - - 0.991 9724.7 ' . 97841-111 - 4.92-247 9.74 247 97-61.0378-21.8 I 1/x I 0.991 - 974 I 9.749:74 I 95-115 I 4.11-23.2 I Attachment B:MDLnOQ Summary ETS-84.1 ExtractionofPFOS from (Serum 3M Environmental Laboratory * Page 12 of I4 Page 57 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 Compound: EtFOSE-OH Prepared range LCR from Rabbit Serum of standards curve I Full Range (ppb) (ng/mL) bb) I 0.993 -976 I . (nalmL1 49.3 - 976 1 Low Curve 1 4.93 -97.6 I 9.9776.-6 1 I - 1 - Highcurve 493 976 ' 97.6 976 I 1m I 0.993-493 I 9796.76- % Recovery Range . 1 77-110 I 1 97-107 I 1 90-109 I I 86-111 I -- 11.2-2rl 14.1:2a 11.5-19.6 -1 11.1-213 1 ran= Compound:PFOSEA .. I I Prepared I .LCRfrom I %Recovery I RSD I Rabbit Serum of standards Curvt @pb) (nglmL) @pb) Full Range Low c u r v e ' - 976 - 248 High m e 49.3 - 976 l-.' 0.993 -976 24,a - 976 0.993 96-106 9.76 - 241814.8.9-139.5 91-110 - 976 I 9.76 -976 I 86-106 95-117 10.2- 18.2 49.3 I p10.1-19.1 1 - Compound: M556 Prepared range LCR h m % Recovay Rabbit Senun of standards curve . Range @Pb) ( n & a I I - I I I . . . FullRange ~ 1 - I 1 1 I Low Curve 0.993 -976 4.93 97.6 I I I I I I Highcurve ' ' - I I 1/X 97.6-976 0.993 -9.97766 (ppb) (ndmL> 243 976 9.76-97.6 9977.66- 979-71610 88-106 100-105 81-111 4.82-17.9 5.95-18.2 5.11-9.74 4.77-19.5 Attachment B: MDLJLOQ Summary ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory ' Page 13 of 14 Page 58 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACTTOX-028 LRN-U2636 Prep date(s): Andyte(s): Sample ma* Ion Pair Methodrevision: 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: StandardCurves;- Fluids Standard number: Equipment number:. Final solvent andTN: Blank fluid/identifier: Actual concentrationsof standards in the FC mix - - Validated ranees aonroximate concentrations ~~ 1. Serum PFOS PFOSA Rabbit Bovine . 5.00-1000 I 5.00-1000 I Estimatesonly. Usevalues forrabbit Rat Estimates only. Use values forrabbit. Monkey & Plasma Estimates only. Use values for rabbit. Human Estimates only. Use values for rabbit. AttachmentC Ion Pair StandardCurves ETS-8-4.1 ExtractionofPFOS fromSerum 3M Environmental Laboratory Page 14 of 14 Pag'e 59 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 3M ENVIRONMENTLAALBORATORY EXTRACTIOONF POTASSIUM PERJ?LUOROO~~ANESULFoNATENAOTRE OTaER F'LUOROCHEMICAL COMPOUNDS FROM LIVER IPOR ANALYSIS USING HPLC- ELECTROSPRAY/IMASS SPECTROMETRY Method NumbAedro:ptEioTnS-8-6.0 Author: Lisa Clemen, Robert Wynne Approved By: Date: Revision Date: lJ iewer Technical O?/, ll /ss ' 1.0 SCOPE AND APPLICATION 1.1 Scope: Thismethod is forthe extractionof potassium perfluorooctanesulfonate(PFOS)or other fluorochemical compoundsh m liver. 1.2 Applicable Compounds: Fluorochemical surfactantsor other fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, andmonkey livers or other tissues as designatedinthe validation report. Word 6.0195 ETS-8-6.0 Extractionof P.FOSfrom Liver Page 1 of 14 3M Environmental Laboratory Page 60 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX.-028 LRN-U2636 2.0 SUMMARY OF METHOD 2.1 This method describes the procedure for extractingpolassiumperfluorooctanesulfonate (PFOS)or other fluorochemical surfactanftsrom liver, or other tissuesu, sing an ion pairing reagent and methyl-tert-butyl eth(eMr tBE). Inthismethod, seven fluorochemicals can be extracted.:PFOS, PFOSA, PFOSAA, EtFOSE-OH,.P.FOSEA, M556,and surrogate standard. An ion pairing reagentis added to the sample and the analiyoten pair is npiatrrtoitgieonneedvainptoorMatotBr Eu.nTtildheryM. EtBaEchexetxratcratcististrraecnosnfmsti:tdutteodaince1n.OtrimfuLgemetuthbaenanodl tphuetn ontoa filtered througha 3 cc plastic syringe attachetdo a 0.2 pmnylon iilter into glass autovials. 2.2 These sample extracts'&e analyzed following methodETS-8-7.0 or other ,appropriate methods. 3.0 'DEFINITIONS 3.1, PFOS: perfluorooctanesulfonate(anion of potassium !salt) C&?,,SO, 3.2 PFOSA perfluomoctanesulfonylamideC$,,S02NHi! 3.3 PFOSAA:perfluorooctane sulfonylamido(ethy1)acetateC8F,,S0,N(CH$H3)CH2C0, 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethylalcohol C$,,SO~(CH&!H,)CH2C~OH 3.5 PFOSEA perfluomoctane sulfonyl ethylamideC,F,,SOzN(CH2CH3)H 3.6 M556:CJ?,,SOzN(H)(CH.$OOH) 3.7 Surrogate standard lH-lH-2H-2H pduorooctane sulfonic acid 4.0 WARNINGSAND CAUTIONS 4.1 Health and Safety Warnings: . . 4.1.1 Use universal precautions,especially laboratcry coats, goggles,and gloves when . handlinganimal tissue, which may contain pathogens. 5.0 INTERFERENCES 5.1 There areno interferencesknown at this.time. 6.0 EOUIPMENT 6.1 The following equipmentis used while performing this method. Equivalent equipmentis acceptable. 6.1.1 Ultra-Turrax T25 Grinder for grinding liver!samples 6.1.2 Vortex mixer, VWR, Vortex Genie 2 6.1.3 Centrifuge,Mistral 1000 orIEC 6.1.4 Shaker, Eberbach or VWR ETS-8-6.0 Extraction of PFOS from Liver Page 2 of 14 3M Environmental Laboratory Page 61 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 6.1.5 NitrogenEvaporator,Organomation 6.1.6 Balance (sensitivityto 0.100 g) 7.0 SUPPLIJCS AND MATERIALS 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable of holding 250 mL arid 1L 7,s Volumetric flasks,glass, type A 7.6 I-CHEM vials, 40 mC glass 7.7 Plastic sampule vials, Wheaton6, mL (or appropriatesize) 7.8 Centrikge tubes, polypropylene,15 mL 7.9 Labels 7.1 0 Oxford Dispensor- 3.0 to 10.0 ml 7.11 Syringes, capableof measuring 5 pL to 50 pL 7.12 Graduated pipettes 7.13 Syringes, disposable plastic3, cc 7.14 Syringefilters, nylon,0.2 p,25 mm 7.15 Timer 7.16 Crimp cap autovials and caps 7.17 .crimpers Note: Prior to using glassware and bottles, rin3setimes with methanol and3 times with MilliQm water. Rinse syringesa minimumof 9 times with methanol, 3 rinses from 3 separate ' vials. 8.0 REAGENTS AND STANDARDS 8.1 Type I reagent grade water, Milli-Qm or equivalenta;ll waterused'inthis method should be Milli-Qm water and be provided abIyvlilli-Q TOC Plusm system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8;3 Tetrabutylammonium.hydrogensulfate(TBA),Koclakor equivalent 8.4 Sodiumcarbonate (NqCO,), J.T.Baker or equivalent 8.5 Sodium bicarbonate (NaHCO,), J.T. Baker or equivalent 8.6 Methyl-tert-butyl ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol,Omnisolv,glassdistilledor HPLC grade '8.8 Liver,frozen from supplier 8.9 Dry ice from supplier 8.10 Fluorocheniicasltandards 8.10.1 PFOS (3M Specialty Chemical Division), :moleculawr eight = 538 ETS-8-6.0 Extractionof PFOS from Liver Page 3 of 14 3M Environmental Laboratory Page 62 StuDdye:p3aMrtmMeendtical T-6314.1 BACK TO MAIN FACT RepoArt:nalytical TOX-028 LRN-U26316 8.10.2 PFOSA (3M Specialty Chemical Division), molecular weig=h4t 99 8.10.3 PFOSAA (3M Specialty Chemical Division), molecular weig=h5t 85 8.10.4 EtFOSE-OH (3M Specialty Chemical Divisicm), molecular weight= 570 8.10.5 PFOSEA (3M Specialty Chemical Division), molecular wei=gh5t27 8.10.6 M556 (3MSpecialty Chemical Division), mcilecular weig=ht557 8.1 0.7 Surrogate standard: 4-H, perfluorooctane sulionic acid (1-H,l-H, 2-H, 2-H C8F,,S03H)molecular weight=428 8.10.8 Other fluorochemicals,as appropriate 8.11 Reagent preparation NOTE: When preparing larger volumes than listiendreagent, standard, or surrogate preparation, adjust accordingly. 8.11.1, 1O'Nsodium hydroxide (NaOH): Weigh approximately 200 g NaOPHo.ur into a 1000 mLbeaker containing500 mL Milli-Q" water, mix until all solids are , dissolved. Store in a 1L Nalgene bottle. 8.11.2, 1N sodium hydroxide (NaOH): Dilute 10NNaOH 1:lO. Measure 10mL of ' ' 10N NaOH solution into1a00mLvolumetric flask and dilutoe volume using .Milli-QTM waterS. tore in a 125 mL.Nalgenei bottle. 8.11.3 0.5 M tetrabutylaxnmonium hydrogen sulfate (TBA): W.eigh approximately169 g of TBA into a 1L volumetric containing500 mL Milli-QTMwater. Adjustto pH 10 using approximately 44 to 54 mL of 1.0N NaOH (While adding the lasmt L of NaOH, add slowly because thpeH changes abruptly). Dilute to volumewith Milli-QTM water. Store in aL1Nalgene bottle. 8.11.3.1 TBA requires a check prior to each use to ensure=p1H0. Adjust as needed using 1N NaOH solution. 8.11.4 0.25 M sodium carbonatdsodium bicarbonate buffer(NqCO,MaHCO,): Weigh approximately 26.5g of sodium carbonateCNa$OJ and 21.0g.ofsodium bicarbonate (NaHCO,) intoa 1L volumetric flask and bring to volume with Milli- Qm water. Store in a 1 L Nalgene bottle. 8.12 Standards preparation ' 8.12.1. Prepare PFOS standards h r the standard curve. 8.12.2 Prepare other fluorochemical standardsa, s appropriate. Multicomponent fluorochmical standards are acceptable(fo:rexample, one working standard solution c0,ntaining1.00ppm PFOS,1.02 ppmPFOSA, 0.987-ppmPFOSAA, and 1.10 ppm EtFOSE-OH.) 8.12.3 Weigh approximately 100 mgof PFOS into a 100mL volumetric flask and record the actual weight. 8.12.4 Bring to volume withmethanol fora stock standardof approximately 1000ppm oLg/mL). 8.12.5 Dilute the stock solution with methanfolr a working standard1solution of approximately 50 ppm. 3M Environmental Laboratory ETS-8-6.0 Extraction ofPFOS from Liver Page 4 of 14 Page 63 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX.-028 LRN-U26316 8.12.6 Dilute the stock solution with methafnoorla working standard2 solution of approx. 5 .O ppm. 8.12.7 Dilute the stock solution with methanol afowr orking standard3 solution of approx. 0.50 ppm. 8.13 Surrogatestockstandardpreparation 8.13.1 Weigh approximately50-60mg of surrogate standard1-H,l-H, 2-H, 2-H, C8F$03H into a 50 mlvolumetric flask and recorthde actual weight. 8.13.2 Bring to volume withmethanol for a surrogalte stockof approximately1000-1200 PP*. 8.133 hepare a surrogateworking standard. Tramfer approximately 1.0 mlof surrogate stock to a 10ml volumetricflaskand bring to volume with methanoflor a working standard of 10-20ppm. Record theactual volume transferred. 9.0 SAMPLEHANDLING 9.1 Allsamples are receivedh z e n and must be kept froxenuntil the extraction is performed. 10.0 OUALI& CONTROL ' 10.1 Matrix blanks and'methodblanks . 10.1.1 An aliquot of 1.0 mLmethanol is used as a solvent blank. . 10.1.2 Extract two 1.0 mL aliquots ofMilli-QTMwater followingthisprocedure anduse as method blanks. 10.1.3 Extract two 1.0 mL. aliquots of liver homogenatefollowingthisprocedure anduse as matrixRbelafenrks. to 11.1.6. -. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samptloesdetermine the accuracy of the extraction. . 10.2.2 Prepare each spike using a sample chos- by the Analyst,usually a control liver received with each sample set. 10.2.3 Expected conccktrations wilflallin the miti-rangeof the initial calibration curve. Additional spikesmay be included anmday fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spikaendmatrix spike duplicateper 40 simples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibration verification samplteosensurethe accuracy of the initial calibration curve. 10.3.2 Prepare, ata minimum, one continuing calibration verification sample per group of10 samples. For example, if a sample setI.34, four verifications are prepared and extracted. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 5 of 14 PaIgeW 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U26316 1033 Prepare each continuing calibration verificatiofnrom the same matrixused to prepare the initial curve. 103.4 The expected concentrationws ill fall within ,the mid-range of tihneitial calibrationciwe. Additional spikes maybe included that fall inthe low-range of - the initial calibration curve.This is necessary if the analyst must quantitate using only the low end of the calibration curve(for example, 5 ppb 100 ppb, rather than 5 ppb - 1000 ppb). 11.0 CALIBRATION AND STAND~RDIZATION 11.1 Prepare matrix calibration standards 11.1.1 Weigh approximately40 g of liver &toa 250 mL Nalgenebotkcontaining 200 mLs Milli-QTM water. Grind atohomogeneous solution. 11.1.2 If 40 g is not available, use appropriataemounts of liver and watetro ensure a 1 5 ratio. 11.13 Refer to 13.0 to calculate the actual densityo:f liver homogenate andthe concentration of solildiver tissue dispersed in 1.O mL of homogenate solution. 11.1.5 Add 1 mL of homogenate toa 15mL centrifilge tube. Resuspend solutionby shakingbetween aliquots while preparinagtotal of eighteen 1rnL aliquots of homogeneous solutionin 15mL centrifuge tubes. 11.1.6 Two 1mL aliquots, or other appropriate volume, serveas matrix blanks. 11.1.7 Typicdly use the standard concentrations and spiking amounts lisitnedTable 1,at the end of this section, to spike,in duplicate, two standard curves, foar total of eighteen samples, two matrix' blanks,and twomethod blanks. 11.1.8 Refer to validation reportsETS-84.0 and ETS-8-7.0-V-1or Attachment B, which lists the working rangesand the Linear CalibratioRn ange (LCR)for calibration curves. 11.1.9 Use Attachment C as an aidin calculatingthe concentrationsof the working standards. .Referto 13.0 to calculate actualconcentratioris of PFOS in calibration Standards. 11.2 To each working standard,blank, or continuing verification, addappropriateamount of - surrogateworking standardfor the concentrationto fall within the calibration curve ran5ge ppb 1OOOppb. 3M EnvironmentalLaboratory ETS-8-6.0 Extraction of PFOS from Liver Page 6 of 14 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 11.3 Extract soiked liver homogenates followin1g2.14-12.:C5 ofthis method. Use these ~~ standark to establish eachinitial curve onthe mass spectrometer. Approgfmate SpikingAmounts for Calibration Standards - 0.50 ppm 0.50 pprn 0.50 ppm . '0.50 ppm 0.50 ppm 5 .Oppm 5 .O ppm 5.0 ppm 50 ppm I- I 2 4 10 , 20 40 . . to 20 30 ' 4 final conc. of PFOS in liver Blank ~ ~ 0.005 ppm 0.010 ppm 0.025 ppm 0.050 ppm 0.100 ppm 0.250 ppm 0.500 ppm 0.750 ppm 1 .OO ppm 12.0 PROCEDURE 12.1 Obtain frozen liver samples. 12.2 Cut approximately 1g of liverusing a dissecting scabel. This part of the proceduries best performedquickly, not allowingtheliver to thaw. . '12.3 Weigh the sample directly into a tared plastic sampule vial. 12.4 Recordthe liver weight in'thestudy notebook 12.5 Return unused liver portions to fieezer. ' 12.6 Add 2.5 m L s of water to sampulveial. 12.7 Grind the sampleP. ut the grinder probein the samp1.eand grind for about 2 minutes, or until the sampleis homogeneous. 12.8 Rinse the probe intothe sample with 2.5 mLswater using a pipette. 12.9 .Takethe grinder apartand clean itwith methanol after each sample. Refteor AMDT-EP- 22. 12.10 Cap the sample and vortex f1o5r seconds. Labelth.e sampule vialwith the study number, weight, liver ID, date and analyst initials. . 3M Environmental Laboratoty ETS-8-6.0 Extractionof PFOS from Liver Page 7 of 14 Pagle 66 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 12.11 Pipette 1.0 mL, or other appropriate volumeo,f homogenate into a15mL polypropylene centrifuge tube. Label the centrifuge tuwbieth the identical informationas the sampule vial. Referto attached worksheet for documenting the remaining steps. 12.12 Pipette two 1mL aliquots of Milli-Qm water to centrifuge tubes. Thesewill 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 matintwiththe appropriate amounotf standard as described in.11.l,or Table 1 of that section, for the calibration curve 'standardsA.1130 prepare matrix spikes and continuing calibrationstandads. 12.15 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.16 Check to m u r e 0.5 M TBAreagent is at pH 10. If not, adjust accordingly. 12.17 To each sample, add1mL 0.5 M TBA and 2 mL of the 0.25 M sodium carbonatdsodium bicarbonate buffer. 12.18 Using & oxford Dispenser, add5 mLmethyl-terf-butyl ether. 12.19 Cap each sample and puotn the shakerat a settingof300 rpm,for 20minutes. 12.20 Centrifuge for 20 to 25 minutes at a settingof 3500 qpm, or until layers are well separated. 12.21 Label afkesh 15 mL centrifuge tubewith'the same informationas in 12.10. 12.22 Remove 4.0 mL of the organic layerto the fie& 15 nL centrifuge tube. ' ' 12.23 Put each sample on the analytical nitrogen evaporator undtirly,approximately 1to 2 hours. 12.24 Add 1.OmL to each centrifuge tube using a graduated pipette. 12.25 Vortex mix for 30 seconds. .. 12.26 Attach a 0.2 pnnylon mesh filter to a 3 cc syringe andtransfer the sample to this syringe. Filter into a 1.5mL glass autovial or low-volume autovialwhen necessary. 12.27 Label the autovialwith the study number, animal number and gender, sample timepoint, matrix, finalsolvent, extraction date, and analyst(s) performing the extraction. 12.28 Cap and store extractsat room temperature or at approximately4 O C until analysis. 12.29 Complete the extraction worksheet, attached to this document, andintasptuedy notebook or include in study bindears, appropriate. 3M Environmental Laboratory ETS-8-6.0 ' Extraction of PFOS from L,iver Page 8 of 14 Page 67 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations: 13.1.1 Calculate the average density otfhe liver homogenate by recording eamcahss of ten separate1.0 mL aliquots of homogenate. Average density(mg/mL) =Average mass(me) of the aliauots 1.0 ~ alliqulot.~ 13.1.2 Calculate the amount of liver (mg) per1.0 m L , homogenate (or concentrationof dispersed solid tissue pmerL of homogenate r;uspension) usintghe following equation: . g of Liver x Averaged&sitv* ofhomogenate(mdrnT.,) ' (g of Liver + g of Water) * refer to 13.1.1 for details. 13.1.3 Calculate actual concentrationosfPFOS and other fluorochdcals in calibration standards usingthe following equation: pL of Standardx Concentration (ug/ d l=Final Concentration (pgg or mgkg) mg Liver/ 1mL homogenate*of PFOS in Liver *refer to 13.1.2 for details. 14.0 METHOD PERFORMANCE 14.1 Tke method detection limit(MDL)is d y t e and mzltrix specific. Refer to MDL report for , specificMDL and limit of quantitation(LOQ)values (referto Attachments B and C). 14.2 The followihg quality control samples aerxetracted%vith each batchof samplesto evaluate the quality ofthe extraction and analysis. 14.2.1. Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and . precision of theextraction. 14.23 Continuing calibration verification samples to determine the continued accuracy of theinitial calibrationcurve. ' 14.3 Refer to section 14 of ETS-8-7.0 for method perfonnance criteria 15.0 POLLUTlON PREVENTlONAND WASTE MANAGEMIiNT 15.1 Sample wasteis disposedin biohazard containers, flammable solvent wasitsedisposed in high BTU containers, and used glasspipette waste:isdisposed in broken glass containers located inthe laboratory. 3M Environmental Laboratory ETS-8-6.0 Extraction ofPFOS from Liver Page 9 of 14 Palge 68 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACTTOX-028 LRN-U2636 16.0 RECORDS 16.1 Complete the extraction worksheet attachedthtios method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 `TABLEDSIA, GRAMS, FLOWCHARTANSD; VALIDATION DATA 17.1 Attachment A, Extraction worksheet 17.2 AttachmentB,MDLLOQ values and sumniary 17.3 Attachment C,Calibration standard calculationand cancentration worksheet 18.0 REFERENCES 18.1 The validation report associatewd ith this method is ETS-8-6.0 & 7.0-V-1. 18.2 AMDT-EP-22, "RoutineMaintenanceof Ulka-Turrax T-25" 18.3 FACT-M-1.1,"Extraction of PFOS orOther Anionic Fluorochemical Surfactantsfiom Liver for Analysis Using HPLC-ElectrospraylMass Spectrometry" ' 19.6 AFFECTED DOCUMENTS 19.1 ETS-8-7.0, "Analysisof Liver Extractsfor Fluorochmicals using HPLC-Electrospray Mass Spectrometry" 20.0 REVISIONS Revision . . Number. .. Reason For Revisi012 Revision 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 10o f 14 Page 6 9 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 1 Stud.y~ # Matrix I Surrogate Std I FCMix Std I FCMixStd 1 FC Mix Std I Comments rox. 5 ppmapprox. 50 ppm rial ppm actual ppm # I I 4 -I I p: I Date &Initials Attachment B:MDL/LOQ Values 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver . P Page 1 1 of 16 a c W 3M Medical Department Study:T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 MDULOQ values for Compound MDL (ppb) PFOS 8.45 PFOSA 3.50 PFOSAA 78.324.6 EfFOSE-OH345 108 rabbiltiver LOQ LineCaarlibration (ppb)Approximate concentradions to beused Standard Calibration Curve - 26.9 30ppb 1200ppb - 11.1 12ppb 1200ppb - 30ppb 1200ppb 60ppb -900ppb* b for preparing the MDWLOQ values in rat,bovine, and monkeyliva were not statistically determined. Two curves in each of these matrices were extracted and analywziethd the rabbitliver curves to determine equivalence. Responses in the rat, bovine, and monkey liver curves were equivalent to the rabbit responses, therefore, theiMr DL and LOQ will be: assumedto be equivalent to those values as determined for the rabbit liver. Refer to LOQ Summary and MDL study in ETS-84.0 & 7.0-V-1 for furtherinformation * EtFOSE-OH estimates only for MDL and LOQ. Did not meet criteria for validation. Attachment B: MDYLOQ Values 3M Environmental Laboratory ' ETS-8-6.0 Extraction of PFOS from .Liver Page 12 of 16 --ragm 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 Attachment C: Standard Calculations ETS-8-6.0 Exmction of PFOS from Liver 3M Environmental Laboratory Page 13 of 14 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 Ion Pair Standard Curves - Tissue Prep date(s): Analyte(s): Sample matrix. Methodrevision: Target analyte(s): FC mix std approx0..500 ppm: FC mix std approx5.00 ppm: FC mix std approx.50.0ppm: Surrogatestd approx. 100ppm: Standard number: Equipment number:' Final solvent xndTN. Blank liverhdentifier: Actual concentrationsof standards inthe FC mix I 1198 I 1198 I 1198 1 1198 I 1198 I 11!98 Validated - ranges approximate concentrations Rabbit Bovine Attachment C:StandardCalculations 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Surrogate ng/mL All spiked mt Final conc nglmL 0.500 Page 14 of 14 Page 73 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 Reviewer 3M ENVIRONMENTLAALBORATORY METHOD ANALYSISOF POTASSIUM~RFLUORO0CTAE;ESULFONA'IXOR OTHER FLUOROCFIEMICAILNSSERUMEXTRACTS USING HPLC-ELECTROSPRAYMASS SPIECTROMETRY Method Number: ETS-8-5.1 Author: Lisa Clemen, Robert Wynne Approved By: 1 Laboratory Manager Group Leader Technical L ' Adoption Date: 03/01/99 RevisionDate: *'iI fl .. . VZC, Date 4/w /99 Date od2h Date 3WE-oratory 1.0 SCOPEAND APPLICATION 1.1 Scope: This method describes theanalysisof serum extractsfor fluorochemicalsurfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemical surfactantsor c~thefrluorinated compounds,or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkeya,nd human serum, or other fluidass designated in the validation report. Word 6/95 ETS-8-5.1 Analysis of Serum Extract Using ESiMS Page 1 of 9 Page 74 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 2.0 SUMMARY O F METHOD 2.1 This method describes the analysis of fluorochemical surfactants extracted from serum or other fluids, using HPLC-electrospray/mass spectrometry, or similar systemas appropriate. The analysis is performed by monitoring a single ion dharacteristic of a particular fluorochemical, suchas the perfluorooctanesulfonate (IZFOS) anion, m/z= 499. Additionally, samplesmay be analyzed usinga tandem. mass spectrometer to further verify the identityof a compound by detecting daughter ions (of the parent ion. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization(API): The Microma!;s Quattro 11triple quadrupole systems allow for various methoodfsionization by utilizingvarious sources, probes, and interfaces. These include but are not limited to: Electrospray Ionizati(oEnSI), Atmospheric Pressure chemical Ionization (APcI);Thennospray, etc. The ionization procesisn these techniques occurs aat tmospheric pressure (i.e.y not under a vacuum). 3.2 Electrospray Ionization (ES,ESI): a method of ionizationperforined at atmospheric pressure, whereby ionsin solution are transferred to thgaes phase via tiny charged droplets. These charged dropletasre produced by the applicationo:f a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer(MS), Tandem Mass Spectrometer@ISMS): The APIQuattro 11triple quadrupole systemsare equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge (rdatzio)and subsequently detected. A single MS may be employed for ion detectionaosreries ( M S M S ) for more specific fiagmentation information. 3.4 Conventional vs. Zspray probe interface: The latest modelsof Micromass Quattro11 triple quadrupole systems (pos1t 998)utilize a "Z-spray" conformation. The spray emitted from a probeEs orthogonal tothe cone aperture.In the conventional conformationit is aimed directly atthe cone aperture, after passintghrough a tortuous pathwayin the counter electrode. Though the conf$pration is different, the methodsof operation, cleaningy and . maintenance are the same. However, 2-spray components and conventional components are not compatible with onaeno.ther, but only with similar systems(Le., Z-spray componentsare compatible with some 0th- Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operatioonf these Quattro II triple quadrupole systems. CurrentlyMassLynx has Windows95 and WindowsNT 4.0 versions. All versions are similar. For more details see the manual specitfoicthe instrument (Micromass Quattro II triple quadrupoleMassLynx or MzrssLynxNT User's Guide). 4.0 WARNINGS AND CAUTIONS 4.1 Health and SafetyWarnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately5000 Volts. 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. ETS-8-5.1 Analysis of SerumBxtract Using ElSNS Page 2 of 9 3M Enviro-7- Page 75 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 4.2 Cautions: 4.2.1 Do not operate solvent pumps above capacityor400 bar (5800 psi) back pressure. If the back pressure exceeds400 bar, the HPl 100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 INTERFERENCES 5.1 To minimize interferenceswhen analyzing samples, teflon should not be used for sample storage or any partof instrumentation that comes in contact with the samorpelextract. 6.0 EOUIPMENT 6.1 Equipment listed below may.be modifiedin order to.optimize the system. Document any modifications in the raw ,&ta as method deviations. 6.1.1 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an electrospray ionization source 6.1.2 H P l 100low pdse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.1.1 High purity grade nitrogengas regulated to approximately100psi (Houseair system) 7.1.2 HPLC analytical column, specificsto be determlied by the analyst and documented in the raw data. 7.1.3 Capped autovials or capped15 mL centrifuge tubes 8.0 REAGENTS AND STANDARDS .. 8.1' Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Mil1i-Qw water, all water used in this method should bMe iI&Qm water or equivalent, and may be provided by a MiTlliO-QC Plus system or other vehdor 8.1.3 Ammonium acetate, reagent gradeor equivalent 8.2 Standards 8.2.1 Typically two .method blanks, two matrix blanks, and eighteen matrix standards are . prepared during the extraction procedure. SeEe TS-8-4.1, 9.0 SAMPLE HANDLING 9.1 Fresh matrix standards areprepared with each analysis. Extracted standards and samples are stored in capped autovialosr capped 15 mL centrifbge tubes until analysis. ETS-8-5.1 Analysis of Serum Extract Using EE:/MS Page 3 of 9 Page 76 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 9.2 If analysis will be delayed, extracted standards and sarnplecsan be refrigeratedat approximately4" C, or at room temperature,until analysis can be performed. 10.0 OUALITCYONTROL 10.1 Solvent Blanks, Method Blanks andMatrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared aannadlyzed with each batchto determine contaminationor carryover. i0.1.2 Analyze a method blank anda matrix blank prior to each calibration curve. .10.2 Matrix Spikes ,10.2.1 Matrix spikesareprepared and analyzed to determine thme atrix effect on the . recoveryefficiency. 10.2.2 Matrix spike duplicates are prepared and analyzedto measure the precisionand the recovery for each analyte. 10.2.3 Analyze a matrix spikeand matrix spike duplicate per forty samplwesi,th a minimum of 2 spikes perbatch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fainllthe mid-range of the initial calibration curve. Additional spike concentrations mfaalyl in the lowrange ofthe initial calibrationcurve. 10.3 Continuiig Calibration Verifications 10.3.1 Continuing calibration verificationasre analyzed to verifythe continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard after every tenth sample,awmitihnimum of one per batch. 11.0 CALIBRATIANODNSTANDARDIZATION 11.1 Analyze the extracted matrix standards prior to and following eseatchof extracts. The average oftwo standard curves will be plotted by linear regression (y =my + b), weighted l/x, not forced through zero, using MassLynx or other suitablesoftware. 11.2 If the curve does not meet requirements, perform routine mainteonrarneecxetract the standard curve(if necessary) and reanalyze. 11.3 For purposesof accuracy when quantitating low leveolsf analyte, it may benecessary to use the low endof the calibration curve rather than hthl lerange of the standard curve. Example: when attempting to quantitate approximatelyIO ppb of analyte, generatea calibration curve consistingof the standardsfiom 5 ppb to 100ppb rather than the full range of the curve(5 ppb to 1000ppb). This will reduce inaccuracy attributetdo linear regression weightingof high concentration standards. 3~ t n v l r o n m e t y ETS-8-5.1 Analysis of Serum Extract Using E9MS Page 4 of 9 Page 77 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN42636 12.0 PROCEDURES. 12.1Acquisition Set up 12.1.1 Click on start button in the AcquisitionControl Panel. Setup a sample list,Assign a filename using MO-DAY-last digoitf year-sample number,assign a method(MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan buttionnthe 14cquisition control panel and select SIR (Single Ion Recording)or MRM. Set Ionization Modeas appropriate and mass to 499 or other appropriate masses.A full scanis usually collected along with the SIRS. Save acquisition method. If MSiMS inslments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDETO DATAACQWSTTXONfor additional information and ' . M R M (MultipleReactionMonitoring). 12.1.3 ' Typically the analytical batch run sequence begins with a soeftextracted matrix standards and ends with a set of extracted matrix standards. 12.1.4 Samples &-e 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 mbeayincluded as such. 12.2 Using the Autosampler . . . 12.2.1 Set up sample tray according to the sample list prepareidn Section 12.1.1. ' 12.2.2 Set-up the HPllOO/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditiinonthse instrument logbook: 12.2.2.1 Sample size = 10 p.Linjection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time= 13.5 minutes 12.2.2.4 Solvent ramp = 1 Time I MeOH I I 0.00 min. I 40% 8.50 min. 90% 11.0 min. 90% 12.0 min. 40% 2.0mM ' ' I 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up , 12.3.1 Refer to ETS-9-24.0 for more details, 12.3.2 Check the solvent levelin reservoirs and refill ifnecessary. ETS-8-5.1 Analysis of Serum Extract Using E!VTvfS Page 5 of 9 Page 78 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 31vl ntv-ry 12.3.3 12.3.4 Check the stainless steel capillary at the enodf the probe. Use an eyepiece to check the tip. The tip should be flat withnojagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. Set HPLC pump to "On". Set the flow to10 - 500 uUmin or as appropriate. Observe droplets corning out of the otifpthe probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelledwith no nitrogen leaking around the tipof the probe. Readjust the tipof the probeif no mist is observed. 12.3.6 The instrument uses these parameters at the foll.owing settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas250-400literskour 12.3.6.2 ESI nebulizing-gas 10-15 litedhour 12.3.6.3 HPLC constant flow mode, flow rate 10- 500 CLLJmin 12.3.6.4 Pressure 4 0 0bar (This parameter is not set,isita guide to ensure the HPLC is operating correctly.) 123.7 Carefully guide the probeinto the opening. Insert probe untilit will not go any further. Connect the voltage cables to the probe. 12.3.8 Print the tune page, witihts parameters, and store iitn the study binderwith a copy taped intothe instrument log. 12.3.9 Using the cross-flow counter electrodien the ESV" source is recommended for the analysis of biological matrices. 12.3.1OClick on start buttonin the AcquisitionControl Panel (thismay vary among MassLynx versions, see appropriateMassLynx USER`S GUIDE). Press the start button. Ensure start and end sample number includes all samples bteo analyzed. 13.0 DATAANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using lhe following equation: % Recovery = ObservedResult - Backmound Rea& x 100 Expected Result 13.1.5 Calculate percent differenceusing the following equation: - % Difference = ExuectedConc. CalculatedConc. x 100 ExpectCedonc. . 13.1.6 Calculate actual concentratioonf PFOS,or other fluorochemical,in matrix (ClglmL): {nr!of PFOS calc. Erom std.Curve x DilutionFactor) x (Initial Volume.of matrix (mL)+ mL of Surrogate Standard) Final Volume (mL) 1 UE 1000 ng ETS-8-5.I Analysis ofSerum ExtractUsing ES,MS Page 6 of 9 Page 79 3M Medical Department Study: T-6314.1 I BACK TO MAIN Analytical Report: FACTTOX-028 LRN-U2636 14.0 METHODPERFORMANCE 14.1 Method Detection Limit(MDL) and Limit of Quantitertion (LOQ)are method, analyte, and matrix specific. Please seeETS-8-4.1, Attachment El, for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks, and Mat& Blank! 14.2.1 Solvent blanks,method blanks, and matrix blanksvalues are must be belotwhe lowest standard inthe calibration curve 14.3 Calibration Curves 14.3.1 The ? value for the calibration curve must 0b.e980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spikep k e n t recoveries are must bweiihin & 30% of the spiked concentration,' ' 14.5 Continuing Calibration Verifications .. 14.5.1 Continuing calibration verification percent recoveries mustkb3e0% of the spiked concentration. 14.6 If criteria listed in thismethod performance sectionim"tmet, maintenance maybe performed on thesystem and samples reanalyzedor other actionsas determined by,the analyst; Document all actions in the appropriatelogbook. 14.7 If data areto be reported when performancecriteriahave not beenmet,the data mustbe footnoted ontables and discussedin the textof the report. 15.0 POLLUTIONPREVENTIONAND WASTE MANAGEMENT 15.1 Sample extract waste and flammable solvenits disposed inhigh BTU containers, and glass pipette waste is disposed in brokenglass containers locatedin the laboratory. 16.0 RECORDS 16.1 Each page generated for a study must have the following information includedeither in the header or hand written on the page: studyor project number, acquisition method, integrationmethod, samplename, extraction date, dilution factor(if applicable), and anaIyst. 16.2 Print the tune page, sample list, and acquisition method froMmassLynx to include in the appropriatestudy folder. Copy these pages and tape into the instrumednto g . 16.3 Plot the calibration curve by linear regression, weightedl/x,then print these graphs and store in the study folder. 16.4 Print data integrationsummary, integration'method,and chromatograms, from MassLynx, and store in the study folder. ETS-8-5.1 Analysis of Serum Extract Using ESMS ly Page 7 of 9 Page 80 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U26316 16.5 Summarize data.usingsuitable software (Excel5.0) and store in the study folder, see Attachment A for an example of a summary spreadsheet. 16.6 Back up electronic datato appropriatemedium. Record in study notebook the file name and location of backup electronicdata. 17.0 TABLESD. IAGRAMSF.LOWCHARTANSD. VALIDATIOPfDATA . 17.1 Attachment A: ETS-8-5.1 Data s u m m a r y spreadsheet. 18.0 REFERENCES 18.1 FACT-M-4.1, "Extractionof Potassium Perfluorooctanesulfonateor Other Fluorochemical compounds fkom Senun for Analysis Using HPLC-EleotrospraylMasSspectrometry 18.2 ETS-9-24.0, "Operationand Maintenanceof the Micro~nasAs tmospheric Pressure IonizatiodMassSpectrometer Quattro 11triple quadrupole Systems" 18.3 The validation report associatedwith this method is ETS-8-4.0 & 5.0-V-1, 19.0 AFFECTED DOCUMENTS 19.1 ETS-8-4.1, "Extraction of PoWsium Perfluorooctanesul!fonateor Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 REVISIONS Revision NuDmabteer. 1 Revision Reaion For Section 6.1.2 Clarificationof HP1100 system components. Section 11.1 Average of two curves, not standard values, are usefdor plotting linear regression and added thel/x weighting of the curve. Section 12.2.2.4 Clarificationofsolvent ramp. Section 17.1 Changed tom attachment B to A. - Revision 04/02/99 3~ tnvlronmental ~atRsratvry ETS-8-5.1 Analysis of Serum Extract Using ESIMS Page 8 of9 Page 81 3M Medical Department Study:T-6314.1 Laboratory Study # Study: Test Material: M a M i n a l Solvent: MethodRcvision: Analytical Equipment System Number: Instrument S o f t w d e r s i o n : Filename: R-Squared Value: Slope: Y Intercept: Date of J3tractiodAnalyst: Date of AnalysidAdyst BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 GrouplDose: Taken from the study folder. Sample#: Taken from the study folder. Concentration ( u g / d ) : Taken from the MassLynx integration s u r m n a r y . . Initial Volume(mL): Taken from the studyfolder. Dilution Factor: Taken from the study folder. Final Canc. (ugh&): Calculated by dividing the initial volume from the concentration Attachment A: SumSmpraeraydEsThSee-8t -5.1 Analysis of Serum Extract Using ESlEnS Page 9 of 9 Page! 82 BACK TO MAIN 3M Medical Department Study: T-6314.1 Study #: FACT-TOX-028 Analytical Report: FACT TOX,-028 Ll ,BY- 3M Environmental - Lab Method Modification Method: ETS-8-5.1 "Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicals in Sera Extracts Using HPLC-Electrospray/Mass Spectrometry" Section modified: Effective date of modifications: 10.3.2, 14.5.1, add sections 14.3.2-14.3.6 April 26,1999 Section 10.3.2 Method reads: 10.3.2 Analyze a mid-range calibration standardaAer every tenth sample, with aminimumofone per batch. Modify method to read: 10.3.2 Anap l a mid-range calibration standardat least after everyten samples, with a minimumofone per batch. Section 14.5.1 Method reads: 14.5.1 Continuiugcalibration verification percent recoveries must bwicthin f 30%of the spiked concentration. Modifymethod to read: 14.5.1 At least one continuing calibrationverificationpcr ten sampks must show a percent recovery within +/-30% of the spiked concenbation, Section 14.3.2 Method reads: NA Modify method to read: 14.3.2 The second (bracketing) calibrationm e may be deactivated.if instrumental drift affects thedata. The first curve and acceptable calibration checkshall bracket usabledata. 3M Environmental Laboratory Page! 83 BACK TO MAIN 3M Medical Department Study: T-6314.1 Study #: FACT-TOX-028 Analytical Report: FACT TOX-028 LnlV-VLDJB Section 14.3.3 Method reads: NA Modifymethod to red. 14.3.3 Calibration standardswith peak areasless than2 times the culvematrix blank should be deactivatedto disqualify a data range thatmay be affectedby backgroundlevels of the analyte. Section 14.3.4 Method reads: NA Modify methodto read: 14.3.4 Low or high curvepoints may be deactivated tooptimize a linearrbge appropriak to the data. Section 14.3.5 Method reads: NA Modify methodto read 14.3.5 A curve point may be deactivateidf it deviatesmom than 30% from the theoretical valuewhen the curve is evaluated overa linear range appropriatteo the data. Section 14.3.6 Method reads: NA Modify methodto read 14.3.6 A valid calibrationcurve must contain at least 5 active points Signatureof PAI and date q-L 7.7s- Signature of Sponsor and date II/=rIrn 5- Signatur-e of Study Director and date 24995 /Z/Y/a 3M Environmental Laboratory Pa(ge 84 Dep3aMrtMmednitcal Study: T-6314.1 3M ENVIRONMENTLAALBORATORY BACK TO MAIN AnFaAlyCticTal Report: TOX-028 LRN-U2636 hAL,YSIS OF POTASSIUM ~ ~ T J O R O O C T A N J ~ S U L F O N AOTREOTHER FLUOROCHE~CALI~NLIVER EXTRACTSUSING HPLC-ELECTROSPRAYAVLGSSPECTROMETRY Method Number: ETS-8-7.0 Author: LisaClben, GlennLangenburg Approved By: Adoption Date: 0 7/22l'rf RevisionDate: fJA Technical 07hdq7 3lvtmimm- ' -1.0 SCOPE AND APPLICATION 1.1Scope: This methodis for the analysis of liver extracls for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. .1.2Applicable Compounds:Fluorochemical surfactantsor other fluorinated compoundso,r other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey liver, or other tissuasesdesignated in the validation report. Word 6/95 ETS-8-7.0 Analysis of Liver Extract Using ESMS Page 1 of 10 Page 85 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX,-028 LRN-U26315 3-aboratory 2.0 SUMMAROYFMETHOD 2.1 This method describes the analysis of fluorochemical. surfactants extractedfrom fiver using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The anaislysis performed by monitoring a. single ion characteristicalf a particular fluorochemicd, such as the perfluorooctanesulfonate(PFOS) anion,d z = 4919.Additionally, samples maybe analyzed usinga tandem mass spectrometerto furtherverify the identity of a compound by detecting daughter ions of the selected parent ion. 3.0 DEFINITIONS 3.1 Atmospheric PressureIonization (MI):The Micronlass Quattro IItriple qlm&-upole systems allowfor various methodsof ionization by utilizing various sources, probes, and interfaces. These include but are not limittoe:dElectmspray Ionization(ESI),Atmospheric . Pressure chemical Ionization(APcI), Thermospray, etc. The ionization process inthese techniques occursat atmospheric pressure(i.e. not undera vacuum). 3.2 Electrospray Ionization(E$ ESI): a method of ionization performed at atmospheric pressure, whereby ionisn solution are transferretdo the gas phase vitainy charged droplets. These charged droplets are producebdy the applicationof a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectromete(rMS/MS): The API Quattro IItriple quadrupolemass spedrometeais equipped withtwo quadrupole ' mass selectivedetectors anda collision cell. Ionsare selectively discriminatedby mass to charge ratio ( d z )and subsequently detected.A single;MS may be employed for ion detectionor an ion may be selected in the first quadrupole,hgmented in the collision cell, and these fiagments may be analyzed in the second qu:rdrupole. 3.4 Conventional vs.'Z-spray probe interface:The lateslt models of Micromass QuattrIoI triple quadrupole (post1998)utilize a "Z-spray" conformation. The spray emittedfiom a probe is orthogonal to the cone aperture. In the conventional conforrpationit isaimed directly at the cone aperture, after passing throuaghtortuous pathwayin the counter ' electrode. Thoughthe configuration is different, the methods of operation, cleaninga,nd maintenance arethe same. However,Z-spray components and conventional components are not compatiblewith one another,but only with similar systems (i.e. 2-spray componentsare compatible with'otherZ-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for ,the specific operatiofnthese Quam 11triple quadrupole systems. CurrentlyMassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details refteor.themanual specificto the instrument (Micromass QuattroLI triple quadrupoleMassLynx or MassLynx NT User's Guide). 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. When engagedth, e probe employs a voltage of approximately5000 Volts. ETS-8-7.0 Analysis of Liver Extract Using ESlMS Page 2 of 10 Page 86 3M Medical DepartmentStudy: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U26316 4.1.2 When handling samplesor solvents wear appropriate protective gloves, eyewear, and clothing. 4.2 Cautions: 4.2.1 Operate the solvent.pumps below a back pres:^ of400bar (5800 psi). If the back pressure exceeds400 bar, the HP1100 will initiate automatic shutdown. 4.2.2 Do notrun solvent pumpsto dryness. 5.0 INTERFERENCES 5.1 To minimize interferences when analyzing samples, Teflosnhall not be usedfor'sample storageor any part of instrumentation that comeisn contact with the sampleor extract. 6.0 EOUIPMENT 6.1 Equipment listedbelow may be modifiedin order to optimizethe system. Document any . modifications in the raw data as method deviations. 6.1.1 MicromassQ u a m IItriple quadrupole Mass Spectrometer equippedwith an electrospray ionization source. 6.1.2 HP1100 low pulsesolventpumping sysetm,solvent degasser, column compartment, and autosampler 7.0 SUPPLIES AND MATERIALS 7.1 Supplies , 7.1.1 High purity grade air regulated to approximately 100psi (house air system) 7.1.2 HPLC andyticd'column, specifics tobe determined bythe analyst and documented in the raw data 7.1.3 Capped autovials or capped 15 ml centrifkge tubes 8.0 REAGENTS AND STANDARDS .. 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent . ' 8.1.2 Milli-Qm water(ASTMtype I), all water used inthismethod shouldbe ATSM type I, or equivalent, and be providebdy a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent gradeor equivalent 8.1.3.1 When preparing different amountstllan those listed, adjust accordingly. 8.1.3.2 2.0mM ammonium acetate solution: Weigh approximately0.300g ammonium acetate.Pourinto a 2000 mL volumetric container containing 2000 mL Milli-QTM watemr,ix until all solids are dissolved. Store at room temperature. .. ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 3 of 10 - 3 Page 87 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 8.2 Standards 8.2.1 Typically two method blanks,two matrix blanks, and eighteen matrix standardasre prepared during the extraction procedure. Refer EtoTS-8-6.0. 9.0 SAMPLHEANDLING 9.1 Fresh matrix standards are prepared with each analysis. Extracted standardsand sarriples are stored in capped autovialsor capped 15 ml centrifuge tubes until analysis. 9.2 If analysis will be delayed, extractedstandards and siunples may be storedat room temperature, orrefkigerated at approximately4' C, until analysis canbe performed. 10.0 OUALITY CONTROL 10.1 Method Bianks and Matrix Blanks 10.1.1 Solvent blanks, methodbl-, and ma& blanks are prepared and analyzedwith each batch to determinceontaminitionor carryover. 10.1.2 Analyze a method blank anda matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzedd&tomine the matrix effect on the ' recoveryefficiency. ' 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precisiotnheand recovery for each analyte. 10.23 Analyze a matrix spike andmatrix spike duplicate pefrorty samplep. With a minimum of 2 spikesper batch. 10.2.4 Matrix spike and matrix spike duplicateconadrations will fallin the mid-range of the initial calibration curve. Additional spike concentratiomnasy fall in the lowrange of the initialcalibrationcurve. 10.3 Continuing CalibrationChecks ' 10.3.1 Continuing calibrationverifications areanalyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibrationstandard every tenth sample, witahminimumof one per batch. 11.0 CALIBRATION AND STANDARDIZATION 11.1 Analyze the extracted matrix standardsprior to andfollowing each setof sample extracts. , The averageof two standard curves will be plottebd:y linear regression(y =mx +b), weighted l/x,not forcedthrough the origin, using MassLynx or other suitablesoftware. 11.2 Ifthe curve does not meet requirements perform routine mainteonrarneceextractthe standard curve(if necessary) and reanalyze. ETS-8-7.0 Analysis of Liver ExtractUsing;ESiMS Page 4 of 10 - - -3 -. Page 88 3M Medical Department Study:T-6314.1 BACK TO MAIN Analytical Report: FACTTOX-028 LRN-U2636 11.3 For purposes ofaccuracy when quantitating low leveolfsanalyte, it maybe necessary to use the low end of the calibration curve rather ftuhlalnranthge of the standard curve. Example: when attempting to quantitate approximatcly10ppb of analyte, generate a calibration curve consistinogf the standardsfrom 5 ppb to 100ppb ratherthan the full range of the curve(5.ppb.to1000 ppb). This will reduce inaccuracy attributed to Iinear regression weighting ohfigh concentration standards,, 12.0 PROCEDURES 12.1 Acquisition Set up 12.1.1 Set up the sample l.is.t. 12.1.1.1 Assign a samplelist filename using IdO-DAY-last digit of year-increasing letter of the alphabet starting with a 12.1.1.2 Assign a method(h4S file) for acquiring 12.1.13 Assign an HPLC program (Inletfile) 12.1.1.4 Type in sample descriptions and vial position numbers 12.l . 2 To create a method clicokn method in the Acquisition control panel then mass spectrometer headings and seleSctIR (Single Ion Recording) MorRM (Multiple Reaction Monitoring). Set Ionization Modaes appropriate andmass to 499 or other appropriate masses. A full scan is usually collected along with thSIeRS. Save acquisition method. IfMS/MS instruments are employed, additional product ion fragmentation information maybe collected. Refer to MicromassMassLynx GUIDE TO DATA ACQUISITION for additional information anMdRM. 12.1.4 Samples are analyzed witahcontinuing calibrationveriikation injected standard after every tenth sample. Solvent blanks should be analyzed periodically to monitor possibleandyte carryover and are not considered samples but bmeay included as such. ' ' 12.2 Using the'Autosampler 12.2.1 Set up sampletray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HPllOO/autosamplerat the following conditionos r at conditions the analyst considers appropriate for optimal response. Record actual condinititohnes instrument logbook ' .12.2.2.1 Samplesize =,10 injection 12.2.2.2 hjedsample = 1 12.2.2.3 Cycle time=9 minutes ETS-8-7.0 Analysis ofLiver Extract Using ESMS Page 5 of 10 Page 89 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-,028 ' LRN-U2636 12.2.2.4 Solvent rampconditions Time . MeOH .O.OO min. 1.0 min. 40% 4.5 min. 95% 6.5 min. 95% 40% Ammonium acetate 60% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up ., 12.3.1 Refer to ETS-9-24.0, "Operation,and Maintenanceof the MicromassQuattro II Triple Quadrupole Mass Spectrometer Fittweidthan AtmosphericPressure Ionization Source," for more details. 12.3.2 `Check thesolventlevel in reservoirs and refill if necessary. 123.3 Check thestainlesssteel capillary atthe end of the probe. Usean eyepiece to check the tip. The tip should be flat with jnagoged ledges. If the tipis found to be unsatisfactory, disassemble the probe and replace the stainsltesesl capillary, 123.4 Turn on the nitrogen. 123.5 Open the tune page. Clicks on operate to initiatesource block and desolvation heaters. 123.6 Openthe Inlet Editor. - 123.6.1 Set HPLC pump to "On" 123.6.2 Set the flow to 10 500 Uyminor as appropriate 12.3.6.3 Observe droplets coming out of ttihpeof the probe. A fine mist should be evelled with no nitrogen leaking aroundthe tip of the probe. Readjust the tip of the probe if no.mistis observed 12.3.6.4 Allow to equilibrate for approximately10minutes. 12317 The instrumentuses these parametersat the fcdlowing settings. These settings may .changein order to optimize the response: 12.3.7.1 Drying gas 250-400litenhour .. 12.3.7.2 ESI nebulizing gas10-15 literdhow .. 123.7.3 HPLC constantflowmodeflow rate 10-500 pUmin 12.3.7.4Pressure 2400bar (This parameter is not set, it is a guide to ensurtehe HPLC is operating correctly.) 12.3.7.5 Source block temperature150' 12.3.7.6 Desolvation temperature250' En-8-7.0 Analysis ofLiver Extract Using ESlMS Page 6 of 10 Page 90 - ~~~~~~ ~~~ ~~~ 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 12.3.8 Print thetune page, with its parameters, and storeit in the study bindewr ith a copy taped into the instrumenlot g. 123.9 Click onstartbutton in the Acquisition Control Pane(lthismay vary among MassLynx versions, refer to appropriate MassLynx User's Guide). Ensurestart and end sample number includes all samples tobe: analyzed. ' 13.0 DATAANALYSIASND CALCULATIONS i3.i Calculations: 13.1.4.Calculate matrix spike percent recoveriuessing the following equation: %Recovery =. - ObservedResult Backmound R e x 100 Expected Result 13.1.5 Calculatepercent differenceusingthe following equation: - % Difference= Expected Conc. Calculated Cor% x 100 Expected Conc. 13.1.6 Calculate actual concentrationsin matrix (pgr'g): .. inn of PFOS calc. from std. 'Curve x Dilution Factor) (Initial WeidLtiovfer (rz) Final Volume(mL) x ' 1 UE 1000 ng 14.0 METHODPERFORMANCE 14.1 Method Detection Limit (MDL) and Limiotf Quatitation (LOO are method, analyte, and , matrix specific. Referfo ETS-8-60, Attachment B for a listingof current validatedMDL and LOQ values. . 14.2 SolventBlanks, MethodBlanksandMatrix Blanks 14.2.1 Solventblanks, method blanks, and matrix bl,anmksust be below the lowest standard in the calibration curve. 14.3 Calibration Curves 14.3.1 The 3 value for the calibration mustbe 0.980 or better. 14.4Matrix Spikes 14.4.1 Matrix spikepercent recoveries must bwe ithiin 2 30% of the spiked concentration. * 14.5 Continuing Calibration Verification 14.5.1 Continuing calibration verification percent recovermieusst be within spiked concentration. 30% of the 14.6 If criteria listed in the method performance section are not met, maintenance may be performed onthe system and samples reanalyzedor other actionsas determined bythe analyst. Document all actions in the appropriate Iogllook. ETS-8-7.0 Analysis of LiverExtract UsingESMS Page 7 of 10 Page 91 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report:FACT TOX-028 LRN-U2636 14.7 If data are to be reporwtehden performance criteria have not been met, the data must be footnoted on tables and discussedin the textof the report. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample extract wasteand flammable solvenits dispo'sedin high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 RECORDS . 16.1 Each page generatedfor a study must havethe following information includedeither in the . header or hand writtenon the page: study or project:number, acquisition method, integrationmethod, sample name, extraction date, dilution factor (if applicaabnlde), analyst. 16.2 Print the tune page, samplelist, and acquisition method fromMassLynxto includein the appropriatestudy folder. Copy these pagesaqd tape :it0 the instrumentrunlog. 16.3 Plot the calibration curve by linear regression, weighl/texd, then print these graphs and store in the study folder. 16.4 Print data integrationsummary,ktegration method, 2nd chromatograms &om MassLynx and store in the study folder. 16.5 Summarize datausing suitable software (Exce5l.W) and store in the study folder, refer to Attachment A for an example of a summaryspreads:heet. 16.6 Back up electronic datato appropriate medium. Recordin study notebook thefile name and location of backup electronicdata. 17.0 TABLESD,IAGRAMFSL. OWCHARTS. AND VALIDATIONDATA 17.1 AttachmentA ETS-8-7.0 Data s u m m a r y spreadsheet 18.0.REFERENCES 18.1 FACT-M-2.1,"Extraction ofPotassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 18.2 ETS-9-24.0,"Operation andMaintenance of the Mic~.omass Atmospheric Pressure Ionization/Mass SpectrometeQr uattro IItriple quadnipoleSystems" 18.3 The validation report associatedwiththismethod is ETS-8-6.0 & 7.0-V-1 19.0 AFFECIEDDOCUMENTS ' 19.1 ETS-8-6.0, "Extraction of PotassiumPerfluorooctane,wlfonateor Other Fluorochemical Compounds from Liver or Fluid for Analysis Using IIPLC-Electrospray/Mass Spectrometry" ETS-8-1.0 Analysis of Liver.ExtractUsing ESMS Page 8 of 10 Page 92 3M Medical Department StudyT: -6314.1 ~ Revision Number Reason For Revision! BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U26316' Revision &&' 3Fntnvlron- ETS-8-7.0 Analysis of Liver Extnct Using ESMS __ Page 9 of 10 Page 93 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 Laboratory Study f# Study: Test Material: Matriflinal Solvent: MethodIRevision: Analytical Equipment SystemNumber Instrument SoftwareNmion: Filename: .R-Squared Value: Slope: Y Intercept . Date of ExtractiodAnaIyst . Date of Analysidhalyst: Dilution Factor lcinal Conc. udg n o p e : `I'aken from b e a r rcgresslonequabon. CroupfDose: Taken from the study folder. Sample#: Taken fromthe study folder. Concentration (ng/g): Takcp from the MassLytlx integrationsummary. Initial Wt. (g): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Conc. (uglg): Calculatedby dividing theinitial volume from thc: concentration Attachment A. Summary Spreadsheet ETS-8-7.0 Analysis of Liver ExtractUsing :ES/MS Page 10 of 10 Page 94 BACK TO MAIN 3M Medical Department Study: T-6314.1 Shldy #: FACT-TOX-028 d-28 LRN-U2636 3M Environmental - Lab Method Modification Method: ETS-8-7.0 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPILC-Electrospray/Mass Spectrometry" Semctioodnified: Effectivedate ofmodifications: 10.3.2,14.5.1,add stx1ti4o.n3s.2-14.3.6 July 22,1999 Section 10.3.2 Method reads: 10.32 Analyze a mid-range calibrationstandard after e v ~teynth sample, with a minirnm of one p a batch. Modify methodto read: 10.3.2 Analyze a mid-range calibrationstandardat least afterevery ten samples, with a minimumof one per batch. Section 14.5.1 Method reads: 14.5.1 Continuing calibration verification percent recoveriemsust be within f 30% of the spiked concentration. Modify method to read: 14.5.1 At least one continuing calibration verificationper ten sampksmust show a percentrecovery within +/-3o%of the spikcdconcentration. Section 14.3.2 Method reads: NA Modify method to r e a d 14.3.2 The second (bracketing) calibrationcurve may be deactivatedif instrumental drift affects the data. The fmt curve and acceptable calibration checks shall bracket usabdlaeta. 3M Environmental Laboratory Page 95 3M Medical Department Study: T-6314.1 BACK TO MAIN AnFaAlvCticTal Reoort: TOX..028 Study #: FACT-TOX-028 Section 14.3.3 Method reads: NA Modify method to read: 14.3.3 Calibration standards with peak areas letshsan 2 times the curvematrixblank should be deactivated to disqualifaydata range thatm a y be affected by hackground leveolfsthe analyte. Section 14.3.4 Method reads: NA Modify methodto read: 14.3.4 Low or high curve pointsmay be deactivatedto optimizc a lin~xr h g e appropriateto the data. Section 14.3.5 Method reads: NA Modify methodto read: 14.3.5 A c w e point may be deactivatedif itdeviates more than 30% fiom the theoreticalvalue when the curve is evaluated overa linear range appropriatteo the data. Section 14.3.6 Method reads: NA Modify method to read: 14.3.6 A valid calibrationcurve must containat least5 active points. Signathe of PAI and date Signatureof Sponsor anddate t &f/d 5- Signatureof Study Director and date W@U 3M Environmental Laboratory Page 96 3M Medical Department Study: T-6314.1 Appendix D: Data Tables Table 8.Rat Serum Week5 Data for FACT-TOX-028 Group Dose Sample # PFOS PmL PFOSA WmL BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 M556 WmL M570 Irg/mL 3M Environmental Laboratory Page 97 3M Medical Department Study: T-6314.1 Table 9. Rat Serum Week14 Data for FACT-TOX-028 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 reference material. The only measuremeonf taccuracy available at thtishe, matrix spike studies. indicate that the data quantitative*3t0o%. 3M Environmental Laboratory Page 98 3M Medical Department Study: T-6314.1 Table I O . Rat Liver Week 5 Data for FACT-TOX-028 ~~~~ BACK TO MAIN Analytical Report: FACT TOX-,028 LRN-U2636 reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data quantitative*3t0o%. Determination ofM556 levels in liver may be biasedlow and should be considered quantitative *t5o0%. 3M Environmental Laboratory Page 99 3M Medical Department Study: T-6314.1 Table 1I.Rat Liver Week14 Data for FACT-TOX-028 BACK TO MAIN Analytical Report: FACTTOX-028 LRN-U26313 reference material. The only measuremeonf taccuracy available at thtish e , matrix spike studies, indicate that the data quantitative&3t0o%. Determination ofM556 levels in livermay be biasedlow and should be considered quantitative *to50%. 3M Environmental Laboratory Page 1100 BACK TO MAIN 3M Medical DepartmentStudy: T-6314.1 Analytical Report: FACTTOX-028 LRN-U2636 PFOSA Table 12. Data Summaryof Average Week5 Rat Serum Concentration (pg/mLa) nd Standard Deviation (fSD) for FACT-TOX-028 PFGOSroup Dose Average Group 1 0 PPm Sex Average (n-5) Male Female Average iSD 0.00918 f0.00113 0.0405 m.00691 M570 SD CLOQ NA CLOQ NA M556 iSD <LOQ NA cLOQ NA Average iSD f0.00232 0.0109 f0.00430 1 1 I 1 7.4: 1 Group2 3 PPm Male 0F.e0m56a3le 0.021 ~~l~ 2.08 m.232 4.06 m.664 29.0 5 M.00380 f0.0134 0.478 0.665 fO.196 0.486 f0.123 3.85 m.453 3.46 f1.26 42.5 ~~ Gf1ro.6u6p 3 f0.0488 f3.52 a44 30 PPm 0.F7e3m0 ale 44.9 33.9 f61.6284 53.99 *O.157 f7.92 Group 4 100 ppm f3.77 FefmO.a0l8e89 f9.71 ly measurementof accuracy available at this time, mat . indicate that the data quantitative fto30%. NA = Not Applicable Table 13. Data Summary of Average Wee1k4 Rat Serum Concentration (pg/mL) and Standard Deviation (fSD) for FACT-TOX-028 reference material. The only measuremeonf taccuracy available at this time, matrix spike studies, indicate that the data quantitativef3to0%. NA = Not Applicable 3M Environmental Laboratory Page 101 BACK TO MAIN 3M Medical DepartmentStudy: T-6314.1 Analytical Report: FACTTOX-028 LRN-U2636 Table 14. Data Summaryof Average Week5 Rat Liver Concentration (pg/g) and Standard Deviation (+SD) for FACT-TOX-028 M556 Average fSD m570 Average fSD 0.00710 H.00159 0.0227 M.00915 0.00693 kO.00184 k0.521 21.9 0.0233 M.0124 7.20 2.01 k0.664 k0.235 3.72 1.09 f1.47 84.9 k10.4 k0.878 t3.5 36.3 k8.62 k3.08 6.29 153 53.9 k18.2 j without radio-labeled reference material. The only measuremeonf taccuracy available at this time, matrix spike studies, indicate that the data quantitattivoek30%. Determinationof M556 levelsin livermay be biasedlow and should be considered quantitativeto k50%. NA = Not Applicable 128 40.9 +1I.5 k8.39 Table 15. Data Summary of Average We1e4kRat Liver Concentration (pg/ga)nd Standard Deviation ( B D ) for FACT-TOX-028 reference material. The only measuremeonf taccuracy available at this time, matrix spike studies, indicate that the data quantitativteo f30%. Determination of M556 levels in liver may be biasleodw and should be considered quantitativteo f50%. NA = Not Applicable 3M Environmental Laboratory Page 102 BACK TO MAIN 3M MedicalDepartment Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U26316 Table 16. Approximate LOQ Values Usedin FACT-TOX-028 Analyses by Matrix, Compound and Usage Dates Matrix Liver Sera Compound PFOS PFOSA M570 PFOS PFOSA M556 M570 LOCI 0.0122 10.0306 pglg 0.0061 1 10.030!60.0122 pglg 0.0061I10.0306II0.0122 pglg 0.0122 10.0306 0.0084810.0215 pglmL 0.00445I0.00980 pglmL 0.0098010.02,49 VglmL 0.00495IO.00980pg/mL Usage Dates 10123 to 1 /102/00 10123 to 11/02/00 M10515263to 11102100 10123to 11102lOO 10102 to 1 /102/00 10102 to 11102/00 10102to 1 /102/00 10102to 1 1102/00 Table 17. FACT-TOX-028 Rat Liver Week 5 Precision Data for PFOS andPFOSA Group 2 3 PPm 0.732 19.0 0.605 1.01 1.oo 0'764 0.956 05'' 26.2 0.392 0.940 13.5 0.821 0.668 1.oo 0.860 0.452 0.880 0.400 22.3 20.0 3M Environmental Laboratory Page 103 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 Table 18. FACT-TOX-028 RatLiver Week 5 Precision Data for M556 and M570 3M Environmental Laboratory Page 104 3M Medical DepartmentStudy: T-6314.1 Appendix E: Data Spreadsheets BACK TO MAIN Analytical Report: FACT TOX-,028 LRN-U2636 ~~~ 3M Environmental Laboratory Page 1105 BACK TO MAIN acln n m e 0 Q a, CcuO ?W 2x s8 ii 0 Q BACK TO MAIN -I.- 0 aam, (d BACK TO MAIN 2i2 0 Q BACK TO MAIN a, 0m ) a 2 E 0 a s P P 0 c s 0 .Qm -I BACK TO MAIN i M1414 WIRES 4 7 3 7 4 N | EIA T | Edfe dnspennfrnnianed i It pte START gl EHC i mein Try | |NT -- i io i ji - oleLifbtb on]| | Ga issnsss sss sane if dn geoATe TAT i Em gk11 BACK TO MAIN W BACK TO MAIN ..h -0 3 BACK TO MAIN EI ' 1 I ET] ! T if TnA Eb e Eb heobseb SET CoA i MA CT | F|"i Dll l||l pM p lttd mktE ef detodretin] o bl pretilei r g -, il ff b | | } IEEE pb ly J BACK TO MAIN 0 m a m BACK TO MAIN aml m a ii 0 n d BACK TO MAIN aw, d BACK TO MAIN a, m m a E e BACK TO MAIN e 0 Q 2 ag teJ bi CR i pit || bil piri Fold] i BACK TO MAIN | 1 Oo Oo P LIL: | EHf] Hi J Po fend 0gl mHiel I lr lod [ih EH li | mo ; ili poe : i. i JREJ 0 _-- BACK TO MAIN : i ; if 0 BACK TO MAIN a, a0m) e 0 9. a, U BACK TO MAIN t: a 0 2 BACK TO MAIN a, a0 m - 6c B E C a, n 0 E r m a BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U2636 Appendix F: Example Calculations Formula Used for Sera Analyses in Study FACT-TOX-028 AR (ng/mL) x DF x PCxFV (mL) x 1.0 pg = ReportedConcentration (pg/mL) EV (mL) 1000 ng Calculation Used for Group3, Week 5, Animal ID C96031F (PFOS) 475 ng/mL x 100 x 0 . 8 6 4 ~1.0 mL x 1.0 pg = 41.0 pg/mL 1.O mL 1000 ng AR- Analytical result from MassLynx summary DF- Dilution factor FV-Final extract volume (1.O mL unless otherwise noted) EV-Volume of sera extracted PC-Purity Correction (forPFOS, only) Formula Used for Liver Analyses in Study FACT-TOX-028 a AR (ng/g) x curve ( I ) x DFxPC x 1.O pg = Reported concentration (pg/g) a sample 1000 ng (I) a curve is assumed tobe: 1 g liver 5 mLH20 Calculation Used for Group3, Week 5, Animal ID C9603:2 (PFOS) 353 ng/g x 1 g/ 5 mL x 500 x 0.864 x 1.0 pg = 146 pg/g 1.0443 g/ 5mL 1000 ng AR- Analytical result from MassLynx summary a curve-Density of theliver standard curve,assumed tobe lg liver/ 5 ml water a sample-Density of the liver sample(g sample/ 5 mL H20) DF- Dilution factor PC-Purity Correction (for PFOS, only) 3M Environmental Laboratory Page 124 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U2636 Formula Usedfor Liver Precision Analysesin Study FACT-TOX-028 I(Rep1icate l(ng/g) - Replicate 2(ng/g))l x 100% = Relative Percent Difference Average of Replicates 1 and 2(ng/g) Calculation Used for Group2, Week 5, Animal ID C9593'1M (PFOS) l(15.5 ng/g- 15.8ng/g)l x 100% = 1.92% (15.5 ng/g + 15.8 ng/g) / 2 3M Environmental Laboratory Page 125 3M Medical Department Study: T-6314.1 Appendix G: Interim Certificate(s)of Analysis BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 3M Environmental Laboratory Page 126 3 M Medical DepartmentStudy: T-6314.1 ~ ~ ~~ ~~ ~ BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 . L 3048 Research Driie State College, . . . . Phone: (814) 231-8032Fax::(814) 231-1o2r53(814) 231-1580 I N T E R .CERTIFICATE OF.WAI,YSIS Revision 1(9/7/00) Centre Analytical LaboratorieCs OA Refertmce #: 023-018B 3M Product: PFOS,Lot 17.1 Reference #: SD-009 Pnritv: 86.4% Test Name Purity' Result 86.4% Appearance Identification NMR Metals (ICPMS) 1. Calcium M2. agnesium 3. Sodium 4. Potassium2 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity ~~ (LWS) Total % Impurity (GCMS) RelatedCompounds - . POAA .. Conforms Positive I. 0.017 wtJwt.% 2. . 0.007 wt./wt.% 3. 1.355 wtJwt.% 4. 6.552wt./wt.% 5. 0.003 wt./wt.% 6. 0.004 wt./wt.% 7<. 0.001 wt./wt.% 1.00 wt./wt.% 10.60 wt./wt.% None Detected 0.30 wt./wt.% None Detected Not Applicable' 1. Chloride F2.luoride 3. Bromide 4. Nitrate 5. Nitrite P6h. osphate 7. Sulfate' Organic Acids'(IC) 1. P A 2. PFPA 3. HFBA 4. NFPA Elemental Analysis6: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine . .......... .. .. : . .. .' ... .. . . . .. .. ... .. .... . . . .. . .. . . . .. .. .... . .. . . . . .. ... . . . . . . .. .. .. : ... .........,,....,:.:..:.....,.....;....!.. .. .' .. 1. TheoreticaVl alue = 17.8% 2. TheoreticaVl alue = 0% 3. TheoreticaVl alue = 0% 4. TheoreticaVl alue = 5.95% 5. TheoreticaVl alue = 60% 1. <0.015 wt./wt.% 2. 0.27wt./wt.% 3. <0.040 wt./wt.% 4. <0.009 wt./wt.% 5. <0.006wt./wt.% 6. <0.007 wt./wt.% 78.82 wt./wt?h I. <o. 1'wt./wt.% 2. <0.1 wt./wt.% 3. <o. 1wt./wt.% 4. <0.25 wt./wt.% 1. 12.08 wt./wt.% 2. 0.794wt./wt.% 3. 1.61 wt./wt.% 4. 10:l wt./wt.% 5. 50.4 wt./wt.% COAO23-018B Page 1 of 3 -_. ....... Page 127 3M Medical Department Study:T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 L 3048 ResCSDeotaralilrtveceeghe, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF l4NALYSIS Centre Analytical LaboratoriesCOA Reference #: 023-018B Date of Last Analysis: 08131/00 Expiration Date: 08/31/01 storage Conditions: Frozen <-loac Re-assessmentDate: 08131/01 'Purity = 100% - (sum of metal impurities,1.39% +LC/MS impurities, 10.60%+horganicFluoride, 0.27%+NMRimpurities, 1.00%+ POAA,0.30%) - Total impurity fromall tests = 13..56% Purity = 100% 13.56% = 86.4% 'Potassium is expected in this salt form and is therefore not consideredan impurity. 3 P u r i t y by DSC is generallynot applicableto materialsof low purity. No endotherm was t- observed for this sample. \ 4Sulfurin the sample.appearsto be converted to SO4 and hencdeetected using the .inorganicanionmethod conditions.The anion resultagrees well with thes u l h determinationin the elemental analysis, lending confidence tothis interpretation. Based on the results, theSO4 is not considered animpurity, STFA HIBA NFPA PPPA Trifluoraocaicdetic Heptafluorohtyric acid Nonofluoropentanoic acid Pentafluoropropaacniodic '?heoretical value calculationsbased on the empiricalformula, C $ I ~ S O ~ X(+MW=538) This work was conducted undeEr PA Good Laboratory Practice Standard(4s0 CFR 160). COAO23-0 18B 3M Environmental Laboratory Page 2 of 3 ' Page 128 BACK TO MAIN 3M Medical Department Study:T-6314.1 - , Phone: (814) 231 -8032 Analytical Report: FACTTOX-028 LRN-U2636 - State College, PA 16801 Fax: (814) 231-1253 or (814) 231 -1580 I INTERIM CERTIFICATE OF.4NALYSIS Centre Analytical Laboratories COA Reference #: 023-018B LC/MS.PurityProfile: I Imuuritv I wt./wt. Yo c4 c5 C6 6.38 c7 Total 10.60 Note: The C4 and C6 values were calculatedusing the C4 and C6standard calibration cubes, respectively. The C5 value was calculatedusingt h e average response factors fiom the C4and C6 standard curves. Likewise, the C7 valuewas calculated using the averageresponse factorsfrom the C6 andC8 standard curves. Prepared By: cal Laboratories 7Ah Date /ohnFlaherty ' Laboratory Manager, Centre Analytical Date Laboratories COA023-018B 3M Environmental Laboratory Page 3 of 3 Page 129 3M Medical Department Study: 1-6314.1 Appendix H: Report Signature Page BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 m. &f 3 Andrew M. Seacat, Ph.D., DSitruedcytor . Date John L. Butenhoff,Ph.D., SponRseoprresentative Date Kristen J. Hansen, Ph.D., PrincipaAl nalyticalnl vestigator oW2a &I I Date .. , .'. ! William K. Reagen, Ph.D., LaborMataonryager d 1 / 3 d A, Date 3M Environmental Laboratory Page 130