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3M Medical Department Study: T6316-S Analytical Report: FACT TOX-013 LRN-U2095 Study Title AnalyticSatludy2(N-Ethylperfluoroocstualnfeonamido)-ethainnol Two Generation Rat Reproduction AnaVical Laboratory Report Title Determinationofthe Presence and ConcentrationofPFOS, M556, PFOSAA, and PFOSA inthe Liverand PFOS, M556, PFOSAA, PFOSA, and ETFOS&OH inthe Sera ofCri:CCPBR VAF/Plue Rats Exposed to ETFOSE-OH Data Requirement Not Applicable Author 3M Environmental Laboratory Study Completion Date At signing Performing Sera Anatyses 3M EnvironmentalLaboratory Building2-3E-09, 935 Bush Avenue St.Paul, MN 55106 Laboratories Uver Analyses BattellMeemodal Institute 505 King Avenue Columbus, OH 43201-2693 Project ldentirication 3M Medical Department Study: T-6316.5 Argus In-LifSetudy:418-009 Analytical Report: FACT TOX-01 3 3M Laboratory Request No. U2095 TotalNumber of Pages 135 3M Environmental Laboratory Page 1 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Thispage has been reservedforspecificountryrequirements. 3M Environmental Laboratory Page 2 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 GLP Compliance Statement AnalyticalLaboratory Report Title: Determinationof the Presence and Concentration ofPFOS, M556, PFOSAA, and PFOSA inthe Liverand PFOS, M556, PFOSAA, PFOSA, and ETFOSE-OH inthe Sera of Crl:CCPBR VAF/Plus* Rats Exposed to ETFOSE-OH Study IdentificatioNnumber: T-6316.5, FACT TOX-01 3, LRN-U2095 This study was conducted incompliance withUnited StatesFood and Drug Administration(FDA) Good LaboratoryPractice(GLP) Regulations21 CFR Part58, withthe exceptionsinthe buileted lisbtelow.Allraw data,protocol,analyticarleportand.samples forthisstudy are retainedin archivesat the 3M Environmental Laboratoryand willbe retainedfora periodof at leastten years. The analyticaplhase completed at the 3M Environmental Laboratorywas performed in accordance with3M ET&SS Standard Operating Procedures. Exceptions to GLP compliance: There were two study directorsinthisstudy.This study was designed as two separate studies.The in-lifpehase was considered to end atthe generationand shipment of specimens. The analyticasltudy was considered to startat the receiptofthese specimens foranalysis.This resultedin having two separate study directorso,ne foreach phase of the same study.However, since the technicalperformance of each phase was entirely separate,no effectisexpected from thisexception. Some changes made inthe standard preparationlogs obscured the originaelntry,did not document the reason forthe change and/orwere not initialeadnd dated by the person making the change. The samples thatwere analyzed on 3/16/00 utilizesdtandards thathad an expirationdate of 2/00. Livervalues generated at contractlaboratorieswere correctedby 3M Environmental Laboratoryto reflecthe officiaplurityvalues from the COA. Revised finalreportswillbe solicitefdrom the contractlaboratoryand willbe added as a reportamendment at a later date. * Expirationdates on some reagents and solutionswere missing. Study Director @@ @?@ 3 -// ..- SponsorRepres-entative Date OCA 6-et . Date 3M EnvironmentaLlaboratory Page 3 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 GLP Study-Quality Assurance Statement AnalyticalLaboratoryReport Title: Determinationof the Presence and Concentration of PFOS, M556, PFOSAA, and PFOSA inthe Liverand PFOS, M556, PFOSAA, PFOSA, and ETFOSE-OH inthe Sera of Crl:C(DPBRVAF/PlusO Rats Exposed to ETFOSE-OH Study IdentificatiNounmber T-6316.5,FACT TOX-01 3, LRN-U2095 This study has been inspectedby the 3M Environmental LaboratoryQualityAssurance Unit (OAU) as indicatedinthefollowingtable.The findingswere reportedto the study directorand laboratorymanagement. Inspection Dates Phase Date Reported to Management Study Director 10/12199 Extraction 10/26/99 10/26/99 6/5/00- 6/14/00 Data 6/16/00 6/16/00 9/11/00- 9/13/()0 Draftreport 9/14/00 9/14/00 t: QAURep@rne@stative@ 3M Environmental Laboratory Page 4 3M Medical Department Study: T6316.S Analytical Report: FACT TOX-013 LRN-U2095 Tableof Contents GLP ComplianceStatement.....................................................3.......................... GLP Study--QualityAssurance Statement ........................................4.......................... Study Personneland Contjibutm ................................................7.......................... Introductioannd Purpose ........................................................a.......................... Test System ...............................................................8.......................... Specimen CoUecfionand Analysis..............................................9.......................... Specimen Receiptand Maintenance ..............................................9.......................... Chemical Characterizatio.n......................................................1.0........................ Dose ConfirmationAna@y@ ..................................................1.0........................ Method Summaries ............................................................I..I........................ 3M EnvironmentalLaboratory..................................................1.1........................ PreparatoryMethod .......................................................1.1........................ AnalyticaMlethod .........................................................1.1........................ AreVical Equipment......................................................1.1........................ Devietk)m..................................................................12......................... Data QualityObjectivesand Data Integrit.y........................................1.2........................ Data Summary, Analyses,and Results...........................................1.3........................ Summary ofQuafityControlAnalyses Results...................................1.3........................ Summary ofSample Results..................................................1.4........................ StatisticMaelthods and Calculations..............................................1.4........................ Statement ofConclusion................................................................................1.4..................... Appendix A: Chemical CharacterizationC,ontrolMatricesand Dose ConfirmationAnalyses.............................................................1.5........................ Appendix B: Protocol............................................................18........................ AppeMix C: Extracbm and AnalyticaMlethods .....................................3.7........................ ETS43-4.1,ExtractioonfPotassium Perfluorooctanesulfonatoer other Fluorochemical Compounds from Serum forAnalysisUsing HPLC-Electrospray/Mass Spectrometry. (14pages)....................................................................3.8....................... ETS-8-5.1,AnalysisofPotassium Perfluorooctanesulfonatoer Other FluorochemicaLs inSerum ExtractsUsing HPLC-Electrospray/MassSpectrometry,(9 pages)............5.2........ Appendix D: Data Summary Tables ..............................................6.1........................ Appendix E: Data Spreadsheets ..................................................6.4....................... Appendix F: Example Calculadons................................................7.0....................... Appendix G: ContractLab Report.................................................7.1....................... Appendix H: lntedm CertificatoefAnalysis.........................................1.3.1...................... 3M Environmental Lahoratory Page 5 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix 1:Report Skjnature Page ..................................................1.3.5.......................... Listof Tables Table 1. Test System Popula6on Demographics and Dosage Levels for S" (418-009.)..........................................................8........................... Table 2. Characterizationofthe Test Ar6c]einStudy FACT TOX-01 3 ...................1.0............. Table 3. Negafive Ions Monitored in3M LaboratoryAnalyses ..........................1.2................... Table 4. DeviationSummary forFACT TOX-013 .....................................1.2........................... Table 5. Determinationsof the LOO inthe Analyses of Serum Extracts..................13............ Table 6. Characterizatioonf the ControlMatricesUsed forSera Analyses In S" FACT TOX-01 3 .....................................................1.5.......................... Table 7. Characterizatioonf the ControlMatrices Used forLiverAnalyses in S" FACT TOX-0 13 .....................................................1.5.......................... Table 8. Characterizatioonf the AnalyticalReference MaterialsUsed forSera Analy@ inStudy FACT TOX-0 13 ...................................................1.6.......................... Table 9. CharactednOon of the AnalyticalReference MaWals Used forLiverAnaty@ InS" FACT TOX-01 3 ...................................................1.6.......................... Table 10. Tween Dosing ConfirmationforStudy In-ffe#418-009 ........................1.7................ Table 11.Tween Dosing Confirmaton--Matrix Spikes forStudy ln4ife#418-009 ..........1.7..... Table 12. Reported Fiuorochemical Levels In Sera Analyses inStudy FACT TOX-0 13 ....6.1. Table 12. Reported FluorochemicalLevels in Sera Analyses inStudy FACT TOX-013 (continued.)..............................................................6.2.......................... Table 13. Reported FluorochemicalLevels in LiverAnalyses inStudy FACT TOX-013 ....6.2. Table 13. Reported FluorochemicalLevels inLiverAnalyses inStudy FACT TOX-01 3 (continued.)..............................................................6.3.......................... 3M Environmental Laboratory Page 6 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Study Personnel and Contributors Study Director Marvin T. Case, D.V.M.,Ph.D, Study Director 3M Corporate Toxicology- MedicalDepartment 3M Center, Building220-2E-02 St.Paul,MN, 55144-1000 651-733-5180 Sponsor John L. Butenhoff,Ph.D.,Sponsor Representative 3M CorporateToxicology- Medical Department 3M Center, Building220-2E-02 St. Paul,MN 55144-1000 Ana"cal Chemistry Laboratories Sera Analyses 3M EnvironmentalLaboratory(3M Lab) KristenJ.Hansen Ph.D..AnalyticaIlnvestigator LiverAnalyses BattellMeemorial Institute ion C. Andre, Ph.D.,AnalyticaIlnvestigator 3M Lab ContributingPersonnel David R. Bamidge. Ph.D. LisaA. Clemen Lisa Dick,Ph.D. KellyJ. Dorweiler Mark E. Ellefson Sara E. Estes Barb A. Gramenz Sarah A. Heimdal Cari S. Hewitt Marlene M. Heying Harold 0. Johnson KellyJ. Kuehlwein SallyA. Linda Joseph C. Pilon Scott R. Post 12n A. Smith Kathy M. Stock Anh-Dao Vo Bob W. Wynne Location of Archives Alloriginalraw data,protocol,and analyticarleporthave been archivedat the 3M Environmental Laboratory.The testsubstance and analyticarleferencestandard reservesamples, as wellas the specimens pertainintgo the analyticaplhase ofthisstudy,are archived atthe 3M Environmental Laboratory.Controlsera and liverwillbe maintained at the contractlabalong withthe test substance. 3M Environmental Laboratory Page 7 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Introductioannd Purpose The purpose of the study is to determine the presence and concentration of PFOS, PFOSA PFOSAA, and M556 in liversamples and PFOS, PFOSA, sera samples collectedfrom rats exposed to ETFOSE-OH. PFOSAA, ETFOSE-OH, and M559 in This study was initiatedon 1 October 1998. Test Systwn Five groups of FO generation male and female rats and 3 groups of Fl generation male and female rats were used as the testsystem. Table I outlines the rat population demographics and dosage levels for study 418-009. On day 4 of lactation,lifterwsere culled to four male and four female pups, where possible. On day 21 of lactation,25 male and 25 female pups in Groups 1,11,and IIIwere selected for continued evaluation. Fl generation male and female rats were given appropriate dosages of the test article via gavage beginning on day 22 of lactationor postpartum through the day before sacrifice. The testsystem species and strainselected was the Crl:CMR VAF/Plus* (Sprague-Dawley) rat received from Charles River Laboratorias, Inc.,and assigned temporary numbers untilassigned to the study. Rats were permanently identifiedusing Moner self-piercingear tags when assigned to the study. FO generation ratswere identifiedwith ear tags. Pups were not identifiedduring lactation,as parameters were evaluated in terms of the lifterA.t weaning, each Fl generation rat selected for continued observation was identifiedwith a Monei*self-piercing ear tag. FO female rats were approximately 65 days of age and weighed approximately 179-22gg when received. FO male rats were approximately 58-67 days of age and weighed approximately 223-331 g when received. Weight data are included inArgus Research Laboratories, Inc.finalreport (study number 418-009). Table 1. Test System Population Demographir-s and Dosage Levels for Study (418-009) Populaflon Number of FO Generation Rats per Sex Number of Fl Generation Rats per Sex Dosage (mgfkgiday) Dosage Group 1 35 (ConbDo Dosage Group 11 35 Dosage Group 111 35 Dosage Group IV 35 Dosage Gmup V 35 25 0 (vehide) 25 1 25 5 - 10 15 3M Environmental Laboratory Page 8 3m Medical Department Study: T6316.S Analytical Report: FACT TOX-013 LRN-U2095 Specimen Calkteflonand Analysts Sample specimenswere collectebdy Argus(study418-009)and senttothe3M Environmental LaboratoryforanalysisL.iverand sera specimens were collectedfrom FO male ratsatthe completionofthe cohabitationperiodand FO female ratson day 21 postpartum.Liverspecimens were collectedfrom FO generationlitterasn,d stomach contentspecimens were collectedfrom the FO and F2 generationlitterTsh.e analysisofthe stomach contentswere not partofthe scope of analysisdetermined by the study directorT.he number and type ofspecimens collectedfor analysesinthe analyticaplhase ofthisstudy are presented below. Specimens Collectedfrom Study Groups Ithrough V (through 11/30/98): Serum Specimens-45 specimens LiverSpecimen&-65 specimens Blood specimens were centrifugedaftercollectionS.erum was then harvestedand immediately frozenon dry iceand maintainedfrozenat-70*C untilshipped tothe 3M Environmental Laboratory.Liverspecimens collectedfrom each animalwere frozenand retainedat-70*C until shipped tothe 3M EnvironmentalLaboratory.Stomach contentspecimens were frozenat-20*C untilshipped tothe 3M EnvironmentalLaboratory.Liver,sera,and stomach contentspecimens were shipped tothe 3M EnvironmentalLaboratoryfrozenand on dry ice. Sera and liversamples were extractedbeginningon October 11, 1999 usingan ionpairing reagentand methyl-tert-buteytlher(MTBE) forthe sera and ethylacetateforthe liversamples. Liversamples were homogenized priortothe extractionprocedure.Sample extractswere analyzed usinghigh-performanceliquidchromatography-electrospray/tandemass spectrometry (HPLC-ESMSMS) inthe multiplreesponse monitoringmode. PFOS, PFOSA, PFOSAA, ETFOSE-OH, and M556 levelswere quantitatedby externalcalibrationP.FOSEA was not analyzed due toinconsistenatnalysisand failedQC. Analyticaldetailsare includedinthisreport. Specimen Receipt and Maintenance The 3M EnvironmentalLaboratoryreceivedfrom Argus, serum, liverand stomach content specimens collecteadtpredeterminedtime pointsduringand atthe end ofthein-iffpehase of Argus study 418-009 on 8-4-98,10-1-98 and 1-29-99.Allspecimens were receivedfrozenan dry iceand were immediatelytransferretdostorageat-200C IOOC. Specimens thatwere analyzed at Battellweere shipped frozenon dry ice. Controlmatricesused inliverand sera analyseswere obtainedfrom commercial sources and are presented inTable 6 and 7.Samples analyzed atthe 3M EnvironmentalLaboratorywillbe maintainedfora periodof10 years and willbe storedatthe laboratoryat-20"C l OOC. 3M Environmental Laboratory Page 9 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Chemical Characterization ETFOSE-OH CAS Number: 1691-99-2 Chemical Formula:C,F,7SO,N(CH,CH,)CH2CH,OH MolecularWeight:571.0 Chemical characterizatioinformationon the testarticliespresented intabularform below. Chemical characterizatioinformationon the analyticarleferencematerialsused inthisstudyis presentedintabularform inAppendix A (see Tables 8 and 9)and the interimCertificatoef AnalysisavailableinAppendix 1. Table 2.Characterizationof the Test Articlein Study FACT TOX-OI 3 Test Article Chemical Name Soume ETFOSE-OH FM-3929 2(N-Ethylperfluorooctanesulfc>namido)-ethanol 3M ExpirationDate 05/2000 Storage Conditions Ambient temperature Chemical Lot 30035,30037,30039 Physical DescripUon Waxy Solid Purity To be determined* *Thepuritoyfthetestarticdleeterminerdoominalb@y NMR onslys@S.ubsequentchemical charactermation isoocumng and th-tsanaV@--al report wil be amended oedfficatoof ana"is isLssued. to lndkee the puritywhen a Dose Confinmaton Azmlyses The dose confirmation data were collected according to a method that was notfullvyalidated. Dose confirmatioannalyseswere performed on testarticlseamples taken atthe startofdosage, at6 weeks, and atthe end ofdosage duringthe in-lifpehase ofthe study. Dose confirmationanalyseswere performed on 3 dose levelscollectedduringthe in-lifpehase of the study:the resultsare presentedinAppendix A (see Tables 10 and 11). Dose confirmatiownas performed by dilutintghe Tween dose samples withMilli-wOater intothe linearange ofthe instrumentF.or each sample, a matrixspikewas prepared (atapproximately 50-100% ofthe expected dose level). In all cases, samples were analyzed versus an unextracted curve using HPLC-ES/MS/MS. The instrumental parameters and analyticalconditions described inETS-8-5.1 were used fordose solutionanalyses. 3M Environmental Laboratory Page 10 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Method Summaries Following is a brief description of the methods used during this analytical study by the 3M Environmental Laboratory. Detailed descriptions of the methods used are located in Appendix C. The methods and analyticalequipment settingsused by Battelleare presented in the Battellefinal report (see Appendix G). 3M EnArorwnerdal Laboratory PREPARATORY METHOD ETS-8-4.1, "Extractionof Potassium Perfluorooctanes ulfonate or Other Fluorochemical Compounds from Serum forAnalysis using H PLC-Electrospray/M ass Spectrometry' Sera samples were extracted using an ion-pairingextraction procedure. An ion pairing reagent was added to the sample and the analyte ion-pairwas partitionedinto MTBE. The MTBE extract was transferred to a centrifuge tube and put onto a nitrogen evaporator untildry. Each extract was reconstituted in 1.0 mL of methanol, then filteredthrough a 3cc plasticsyringe attached to a 0.2@im nylon filteirnto a glass autovial. At4ALYnCAL METHOD ETS-8-S.i, 'Analysis of Potassium Perfluoroocta nesulfon ate or Other Fluorochemicals in Serum Extracts Using H PLC-Electrospray/M ass Spectrometry' The analyses were performed by monitoring one or more product ions selected from a slingle primary ion characteristicof a particular@uorochemical using HPLC-ESMSMS. For example, molecular ion 499, selected as the primary-ion for PFOS (QF,7S03-) analysis, was fragmented furtherto produce ion 99 (FSO,-). The characteristicproduct-ion 99 was monitored forquantitative analysis. ANALY-ncAL EOUIPMENT The followingequipment phase of this study. and parameters are representative of those used during the analytical Liquid Chromatograph: Hewlett-Packard* Series 1100 Liquid Chromatograph Analyticalcolumn: Keystone BetasilTm Cle 2x5O mm (5 pm) Column temperature: Ambient Mobile phase components: Component A: 2mM aqueous ammonium acetate Component B: methanol Flow rate:300 pumin injectionvolume: 10 pL Solvent Gradient: 10 minutes Start at 40%B Hold at 40%B for I minute Increase to 95%B over 3.5 minutes system 3M Environmencal Laboratory Page 11 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Hold at95%B for2 minutes Return to40%8 over 0.5 minutes Hold at40%B for3 minutes Mass Spectrometer: Micromass* API/Mass Spectrometer Quattro11-TriplQeuadrupole system Software:Mass Lynx@ 3.2 Cone Voltage:20-60 V CollisioEnnergy: 25-45 eV Mode: ElectrosprayNegative Source Block Temperature: 1500C l OOC Z-spray source AnalysisType: MultipleReactionMonitoring(MRM) Table3. NegativeIons Monitored In3M LaboratoryAnalyses TargetAnalyto PrimaryIon(AMU) Product Ion(AMU) PFOS PFOSA PFOSAA 499.0 498.0 584.0 99.0 78.0 169.0 ETFOSE-OH M556 THPFOS 630.0 556.0 427.0 59.0 78.0,169.0 80.0 oeviatiom Deviationsfrom the originaplrotocoland methods are documented inthe tablebelow: Table 4. DeviationSummary forFACT TOX-013 Deviatton Date(s)ofOccurrence Pk>ettewas used instead Oxford dispenser IQ/12/99 02-I.OmL ofsample was used forex"ction insteadof l.OmL. Milkcurd umples were not anawed. I- 10/12/99 Entirestudy Impact on Study Standards and samples were prepared identkmltyN.o adverse knpact on swdy. Cunvnt work IndicatesOW volumes kO.5 mL provideresub equivalentto 1 mL extmctionvolumes. Resub ofsample volumes <0.5 mL have not been valklated and wig be marked inft date taus. No milkcurd data isavailableforft final report Data QualityObjectives and Data Inte-grity The followingdata qualitoybjectives(DQOS) were indicatedinthe method performancesectionof ETS-8-5.1,'AnalysisofPotassium Perfluorooctanesulfonatoer Other FluorochemicalsinSerum ExtractsUsing H P LC-Electrospray/Mass Spectrometry*: 3M Environmental Laboratory Page 12 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 0 LinearityT:he coefficieonftdeterminatio(nr2) equaltoorgreaterthan 0.980 0 Limitsof Quantitation(LOQ):The LOQ forPFOS is5.55ppb, PFOSA is4.79 ppb, PFOSAA is20.5 ppb, ETFOSE-OH is36.2 ppb, and M556 is 19.2ppb. 0 Acceptable Spike Recoveries,70-130% Data Summary, Analyses, and Results Withthe exceptionsnotedinthisreportd,ataqualityobjectivefsortheanalyticaplhase ofthis studyoutlinedinthe 3M EnvironmentalLaboratorymethod ETS-8-5.1 (seeAppendix C) and the Battellefinalreport(seeAppendix G) were met. Although extractioannd analysiswere initiateidn September 1998,the study was reprioritizaendd put on hold.Upon restartintghe study,the decisionwas made toreextractand analyze the specimens. No datafrom the originaalnalysisare includedinthisreportT.he datainthisreportreflecotnlythatobtainedfrom specimens extracted on, orafterOctober 11,1999. Sumrnary of Quality Control Analymm Results Linearity:The coefficienotfdetermination(r2)ofthe standardcurveswere'->0.980. CalibrationStandards: Quantitatioonfthe targetanalyteswas based on linearegression analysis(1/xweighted)oftwo extractedmatrixcurves bracketingeach group ofsamples. High or low pointson the curve may have been deactivatedtoprovidea betterlinearfitover the concentrationrange most appropriatetothe data.Allactivecurve pointsare accuratetowithin 70% oftheoreticavlalue.Low curve pointswithpeak areas lessthan two times thatofthe extractionblankswere deactivatedtodisqualifay data range thatmay have been significantly affectedby background levelsofthe analyte.Occasionally,a singleoutliecrurve pointmay have been deactivatedQ.uantitationofeach analytewas based on the response ofone or more specifipcroduction(s)usingthe multipleresponse-monitoringmode ofthe instrument (see Appendix C). Limitsof Quantitation(LOQ):The LOQ isequaltothe lowestacceptedstandardinthe calibratiocnurve (definedas a standardwitha concentrationthatiswithin30% ofthe theoreticavlalue,and which has atleasttwo times the analytepeak area detectedinthe extractiobnlanks). Table S.Determinationsof the LOQ inthe Analyses ofSerum Extmcts Analyto Method LOQ PFOS 5.55ppb PFOSA 4.79 ppb PFOSAA ETFOSE-OH 20.5 ppb 36.2ppb M556 24.9 ppb 3M Environmental Laboratory Page 13 3M Medical DeDartment Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Blanks:Allblankswere below thelowerlimiotfquantitatifoonrthe compounds of interesTto, simplifyanalyses thatwere complicated by endogenous levelsoffluorochemicalsin unexposed ratsera,rabbitsera was selectedas a suitablesurrogatematrixforstandard curves. Precision: Precisionwas determined by analysisof MS/MSD 10%. and was reproducibleto within a Matrix Spikes: Matrix spikes and matrix spike duplicates were extracted with each set of samples and analyzed duringanalyticarluns.With the exceptionof M556, allsera matrix spikeswere within30% ofthe theoreticacloncentration.Both matrixspikesshowed a recovery of 69% forthe M556. These resultswere verifiedD.ata qualityobjectiveswillbe adjusted to reflecthisrecovery. Surrogates: The surrogate(THPFOS) was added to allsamples and standards.THPFOS was not used forquantitationb,utwas used to monitorforgross instrumentfailureT.he surrogateresponse of each analyticarlun was verifiedto determine thatitdidnot vary more than:t50% from the mean withineach analyticarlun. Assuming spikerecovery studiesform a suitableindicatioonf endogenous analyterecovery,sera data are quantitativteo 30% forallanalysisbut M556; M556 data isquantitatedto 31%. The validitoyf thisassumption has not been verifiebdy othertechniques. Sumnwy of Sample Results Samples from Control Animals: Low levelsof PFOS, PFOSA, PFOSAA, ETFOSE-OH, and M556 were oftendetected inthe sera and liverof the controlanimals.These levelswere significantloywer than those found inthe low dose testanimals. Samples from Dosed Animals: In general,PFOS, PFOSA, PFOSAA, ETFOSE-OH, and M556 levelsfound inthe sera and liverofthe testanimals increased withdose group. Detailed sample data tablesare presented inAppendices D and E. StatisticaMlethods and Calculations Statisticamlethods were limitedto the calculationof means and standard deviations.See Appendix F forexample calculationsused to generate the liverand serum sample data inFACT TOX-0 13. Statement of Conclusion Under the conditionsof the present studies,PFOS, PFOSA, PFOSAA, ETFOSE-OH, and M556 were observed inthe sera and liverof ratsdosed with ETFOSE-OH duringthe in-lifpehase of the study. 3M Environmental Laboratory Page 14 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix A: Chemical CharacterizationC,ontrolMatricesand Dose ConfirmationAnalyses Table 6.Characterizationof the Control Matrices Used for Sera Analyses in Study FACT TOX-01 3 Location 3M Lab ControlMatrix Rat Serum (TN-A-2001) Rabbit Serum (TN-A-2573) Source Expirabon Date Stor-agCeonditons FChemicalLot # PhysicalDescription NIR-not recorded sigma 20io Ambient 17H93O6 Rat Serum Sigma 2010 Ambient 11BH8418 RabbitSerum Table 7.Characterizationof the Control Matrices Used forLiverAnalyses in Study FACT TOX-Oi3 Location BattelleMemorial Institute ControlMatrix Source ExpiratonDate StorageConditions ChemicalLot PhysicalDescription N/R--notrecorded Rat Liver Harlan N/R N/R N/R Rat Liver 3M Environmental Laboratory Page 15 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Table 8.Characteftationofthe AnalyticalReference MaterialsUsed forSera Analyses InStudy FACT TOX013 Lor-ation 3M Lab PFOS C-pFir%Os. PFOS@K C$FgSO@4Hs PFOSAA CF,TSOIN((PHXH&) (CHaCO014)) ET.FOSE-OH CgF,rSON(CHXH2) ..CHICHOH M556 CFOSCX(H) (CHICHs)) THPF.O$* CMgF,;NO33 Source Expira*m Ods storage Coridftkxis 3m Spedwty Ch&rAcais 06131)01 AmbWwA mnv-kire NR oijoir2olo AmbientWnpwat" NIR 01101/2010 Affi.4oltempwawra Chemical Lot 171 Number L-i5709 NS ll29Wg9 Physical D"crfptlan Purky V*Ae ayswpowder 88.4% Lvt yeb- waxy soM TBD Tan waxy sold TBD *Surrogatestandard- 1H,lH,2H,2H-TetrahydropernuorooctanesuKonaiccid KIR-not recorded TBD--to be determred NA--not appkicable 3M CP/PCP DWow ai/01120io AmbientWyVersture 935 Amber waxy soW TBD 31A alloir4lo Ambwt WMwft" NB 113047-M v@%itsPC-d- TOD ICN Bkxnedicals 01/2010 AmbietA*nveratum 53405 Brown waxy sofid NA Table S.Characterizatioonfthe AnalyticalReference Materials Used forLiverAnalyses inStudy FACT TOX-01 3 LocaUon BattelleMemorial Institute Materials Source PFOS 3M MS,@S@6 3M P FOSAAI,,-.P,.FOSA 3m THPFCW r-4 Expiration Date storage CondlOons 00/31/01 Arnbiard wrvmln 0110112010 AnibWd *nverst" 2010 Oliol/2010 I Amberit re AnVYAnt mffversure MIR AfftmWnpwah" Chemical Lot Number 171 NB 113047-80 617 Physical VJhftcrystawine WvW powder NIR Douripdon pw4der L-15709 59M LghtyeM- NIR %OOXY Purity I 8&4% TBD I :@;D TW NA *Sum)gate standard- 1H,IH,2H,2H-Tatrahydroperftuorooctanesulfaocniidc NIR-not remrded TBD--to be dateitnited NA--not appricabic 3M Environmental Laboratory Page 16 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Table 10.Tween Dosing ConfiffnationforStudy In-lif#e4ig-009 Group Dose Sample Number Expected Conc. Measured Conc. ETFOSE (ng/mL) ETFOSE (ng/mL) Group 1--Contro0lmg/mL Group 2-0.2 mg/mL Group 3-1.0 mg/mL Group 4-2.0 mg/mL Group 5-3.0 mg/mL Honwgeneity Sampk@&3.0 mglml B418-00@A, 06M= - B418-(X@A, 07/15M8 B41 "09-8, 06=/98 B-41S-Wg-B, 07/15/98 B41 SM9-C, 06A)8/98 B-418M9-C, 07/15198 B-41 8-009-D,06JO8/98 B418-009-D, 07/15/98 B-41B-00@E, 06=/98 B41 8-(X&E. 07/15/98 B418-OMA, 05108/98 1 of6T B-416-009-A,06/08198 3 of6 M B-418-009-A,MM&W 5 of6 0.00 NA 200000 200000 1000000 100000 2000000 2000000 3000000 3000000 3000000 3000000 3000000 0.00 NA NA NA 1020000 9.42000 2190000 2750000 3060000 3640000 3250000 3690000 3790000 NA Not applicable ETFOSE % Recovery Accuracy NA NA NA NA 102 94 110 138 102 121 108 123 126 Table 11. Tween Dosing Confirmation-Matrix Spikes for Study ln41fe*41"09 Sample Number Expected Conc. ETFOSE (ng/mL) Measured Cone. ETFOSE (ng/mL) ETFOSE % MS Recovery Accuracy B-418-009-B,06A)8A)8-MS 1200 NA B-418-009-B,07/15198-MS 1200 NA B-418-009-C0,6/08A"S 900 818 B-419-009-C,0711&S"S 900 826 B-418-009-D,06108/WMS 900 9io 8-41&409-D,07/1-%"S 900 733 B-418-009-E,06A)8198-Ms 1100 973 B418-009-E, 071ISM8-MS 1100 1089 NA NA 91 1 92 101 al as I 99 B-418-009-A,05A)8198 1100 949 86 1 of 6 T-MS B-418-009-A.W08/98 1100 1053 96 3 of6 M-MS B41 B-009-A,06/OMB 5 of6 B-MS T 1100 944 86 NA n Not appkable 3M Environmental Laboratory Page 17 I 3M Medical Department Study: T6316.5 I Appendix B: Protocol I I I I I I I I I I I I I I I I 3M Environmental Laboratory 1 Analytical Report: FACT TOX-013 LRN-U2095 Page 18 3M Medical Department Sl@udy: T6316.5 Analytical Rep 3_ To 5 3M ENVIRONTNMNTAL LABORATORY PROTOCOL- ANALYTICAL STUDY 2(N-Ethylperfluorooetanesulfonamidion)-ethanol Two Generation Rat Reproduction In-vivostudyreferencneumber: Argus418-009 Studynumber:FACT 060998.1 Testsubstance2:(N-Ethylperflucrooctanesulfonamido()N--eEtthFaOnSoEl-OH) Name and addressofSponsor: MarvinCase 3M Toxicology Services 3M Center Building220-2E-02 St.Paul,NlN 55144 Name and address of testingfacility: 3M EnvironmentalTechnology and Services 935 Bush Avenue, Building2-3E-09 St.Paul,MN 55106 Experimental startdate: Expected terminationdate:December 31, 1998 Method numbers and revisions: FACT-M-1.0, ExtractionofPotassiumPerfluorooctanesulfonaotreOther Anionic Surfactantfsrom LiverforAnalysisUsing HPLC-Elrctrospray/Mass Spectrometr@ FACT-M-2.0, Analysisof Fluorochen-iicalisn Liver ExtractsUsing HPLCElectrospray/MassSpectrometry FACT-M-3.0, Extractionof Potassium Perfluorooctanesulfonatoer Other Anionic Surfactantsfrom Serum forAnalysisUsing BPLC-Electrospray/Mass Spectrometry FACT-M-4.0, Analysisof Fluorochernicalisn Serum ExtractsUsing BIPLCElectrospray/MassSpectrometry Author:LisaClemen KrisHansen Study Director 1&4e@1.0oc Date Marvin Case Date Sponsor Representative 3M@Environmental Laboratory Page 19 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 1.0 PURPOSE The analyticalportionof thisdosing study isdesigned evaluatethelevelsof perfluorooctane sulfonate(PFOS), or anothermetaboliteof 2(N-ethylperfluorooctanesulfonamido)-etha(nNo-l ]@tFOSE-OH) designatedby the study director,in the liverof theparentand subsequent generationsof the testsystem,or inthe serum as necessary. The in lifeportionof thisstudywas conducted atArgus Research Laboratories. 2.0 REGULATORY COMPILIANCE This study isconducted in compliance with the Food and Drug AdministrationGood Laboratory Practicesregulationas statedin 21 CFR 58. Any exceptionswillbe noted in the finalreport. 3.0 TFSR MATERIAM 3.1 Test, control,and referencesubstances and matrices 3.1.1 Analyticalreferencesubstance: Potassium perfluorooctanesulfona(tPeFOS), lot # 217 3.1.2 Amalyticalreferencesubstance matrix: Rat liverand serum 3.1.3 Analyticalcontrolsubstance: None 3.1.4 Analyticalcontrolsubstance matrix: Rat liverand serum 3.2 Source of materials 3.2.1 Analyticalreferencesubstance: 3M SpecialtyChemical Division;traceability informationwillbe includedin the finalreport 3.2.2 Analyticalreferencesubstance matrix: Argus ResearchLaboratories; traceabilitiynformationwillbe includedin thefinalreport 3.2.3 Analyticalcontrolmatrix: 3.2.3.1 Rat liver- Argus Research Laboratories;traceabiliitnyformationwillbe includedin thefinalreport;or Rabbitliver- Covance Laboratories;traceabilitiynformationwillbe includedin thefinalreport 3.2.3.2 Rat serum - Sigma Chemical Company; traceabiliitnyformationwillbe includedin thefinalreport 33 Number of testand control samples. Liver samples fortestingwere receivedfrom 40 testanimals and 10 controlanimals. Serum samples willbe testedatthe discretionof the Study Director. 3.4 Identilricatiofn testand control samples: The samples areidentifieudsing theArgus Research Laboratoriesidentifiersw,hich consistof a letterfollowedby the Argus project number, the animal number, the group designation,and the draw date. 3M Environmental Laboratory 2 Page 20 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 3.5 Purityand strengthofmaterialsC:haracterizatoifotnhepuritayndidentitoyfthe referencematerialistheresponsibilitoyf theSponsor. 3.6 Stabilitoyf testmaterial:Characterizatiofn thestabiliotfy thetestmaterialisthe responsibiliotfy theSponsor. 3.7 Storage conditionsfor testmaterials:Testmaterialasrestoredatroom temperature. Samples arestoredat-20 10 *C. 3.8 Dispositionof testand/or controlsubstances: Biologicaltissuesand fluidsareretained per GLP regulation. 3.9 Safetyprecautions: Refer tothematerialsafetydatasheetsofchemicalsused. Wear appropriatelaboratoryattirea,nd followadequateprecautionsforhandlingbiological materialsand preparingsamples foranalysis. 4.0 ExPERrv[ENTAL - Overview Tissuesfrom animalsdosed as describedin Argus Research LaboratoriesProtocol#418-009 are receivedforanalysisof fluorinecompounds. At thediscretioonftheStudy Director,a seriesof analyticatlestswillbe performed on selecttissues. Initiallayl,lliversamples willbe analyzedforPFOS by electrospray/massspectrometry (ES/MS). On thebasisof findingsfrom theseanalyses,additionaslample matricesmay be evaluatedor othermetabolitesmay be targeted.Ifadditionaalnalysisisperformed,a protocol amendment willbe written. 5.0 EXPERTMFNTAL -AnalyticalMethods 5.1 FACT-M-1.0, Extractionof PotassiumPerfluorooctanesulfonaotreOther Anionic Surfactantfsrom LiverforAnalysisUsing BPLC-Electrospray/MassSpectrometry 5.2 FACT-M-2.0, AnalysisofFluorochemicalsinLiverExtractsUsing BPLCElectrospray/MassSpectrometry 5.3 FACT-M-3.0, ExtractionofPotassium PerfluorooctanesulfonaotreOther Anionic Surfactantfsrom Serum forAnalysisUsing HPLC-Electrospray/MassSpectrometry 5.4 FACr-M4.0, AnalysisofFluorochcrrlicailnsSerum Extrwts Using HPLCElectrospray/MassSpectrometry 6.0 DATA ANALYSIS 6.1 Data transformationsand analysis:Data willbe reportedastheconcentration (weight/weighto)f fluorideper tissueorsample, orofPFOS per unitoftissueorfluid. 6.2 Statisticaalnalysis:Statistiucsed may includeregressionanalysisoftheserum concentrationosvertime,and standarddeviationscalculatedfortheconcentrationwsithin each dose group. Ifnecessary,simple statistictaelstss,uch as Student'sttest,may be appliedtoevaluatestatisticdailfference. 3M Environmencal Laboratory 3 Page 21 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 7.0 MAP4TENANCF, oF RAw DATA AND REcoRDs 7.1 The following raw data and recordswill be retainedin the study folderin the archives according toAMDT-S-8: 7.1.1 Approved protocol and amendments 7.1.2 Study correspondence 7.1.3 Shipping records 7.1.4 Raw data 7.1.5 Electroniccopies of data 7.2 Supporting records to be retainedseparatelyfrom the study folder in the archives according toAMDT-S-8 willincludeatleastthe fohowing: 7.2.1 Training records 7.2.2 Calibrationrecords 7.2-3 Instrument maintenance logs 7.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 7.2.5 Appropriate specimens. 8.0 REFERENcFs 8.1 3M Environmental Uboratory QualitySystem Chapters 1,5 and 6 8.2 Other applicable3M Environmental Laboratory Quality System Standard Operating Procedures 9.0 AYRACHMENTS 9.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactantsfrom Liver for Analysis Using EPLC-Electrospray/Mass Spectrometry 9.2 FACT-M-2.0, Analysis of Fluorocheniicalsin Liver ExtractsUsing BPLC- Electrospray/Mass Spectrometry 9.3 FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonateor Other Anionic Surfactantsfrom Serum for Analysis Using BPLC-Electrospray/M ass Spectrometry 9.4 FACT-M4.0, Analysis of Fluorochemicals in Serum ExtractsUsing I-IPLC- Electrospray/Mass Spectrometry 3M Environmental Laboratory 4 Page 22 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Study Title Combined Oral(Gavage)FertiliDteyvelopment and-Perinatal/Postnatal ReproductionToxicityStudy ofN-ETFOSE inRats PROTOCOL AMENDMENT NO. 1 Amendment Date: July28,1999 Performing Laboratofy 3M EnvironmentalTechnology& SafetyServices 3M EnvironmentalLaboratory 935 Bush Avenue St Paul,NlN 55106 Laboratory Project ldentfrication ET&SS FACT-TOX-013 LLPN U2095 3M EnvironmentaLlaboratory 3M Environmental Laboratory Page 23 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 ProtocoFlACT-TOX-013 Amendment 1 Thisamendment modifiesthefollowingportion(so)ftheprotocol: 1. PROTOCOL READS: The proposedstudycompletiondateislisteads 12/31/98. AMEND TO READ: The proposedstudycompletiondatais6/30/00. REASON: The proposed completion datewas changed to allow time for analyzingall matricesof interest. Amendment Approval Marvin Case Ph.D., Sponsor Representative !)at KrisJ.gansen Ph.D.,Study Director Date 3M Envirt)nmentaLlaboratory 3M Environmental Laboratory Page 24 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 ProtocoFlACT Tox-013 Amendment Number 3 3. PROTOCOL READS: DispositioonftestandcontroslubstanceBsi:ologicatlissueasnd fluidasreretainepderGLP regulation. AMEND TO READ: Specimens willbe maintainedinthe3M EnvironmentalLaboratoryspecimen archives.All specimens senttosub-contracltaboratoriewsillbe returnedtothe3M Environmental Laboratoryupon completionof analysisand submissionofthe sub-contracltaboratory(s) finalreport.The specimenswillbe returnedwith thefollowingdocumentation:thesigned originalchainof custodyand recordsof storageconditionswhile atthesub-contractfacility. REASON., To defineindetailtheappropriatedispositioonf specimens analyzedatsubcontract laboratories. Amendment Approval Marv Case,D.V.M., Ph.D., Sponsor Representative Date @-arK. Lundlbbe@rgg,,-PSPhth.u.D@D.i.,r,Setcuto irector Kristen1.Hansen, Ph.D., PreviousStudy Director Date /0/i5115 Date Dale L.Bacon, Ph-B'@'3M Environmental Laboratory Management Dite 3M EnvirDnmental Laboratoiy 3M Environmental Laboratory Page 30 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Study Title Analytical Study of 2(N-Ethylperfluorooctanesulfonamido)-ed=ol in Two Generation Rat Reproduction PROTOCOL AMENDMENT NO. 4 Amendment Date: 20 January 2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St.Paul, MN 55106 Laboratory Project Identification ET&SS LRN-U2095 FACT TOX-0 13 Argus Study: 418-009 3M Medical Department Study: T-6316.5 3M EnvironrwntalLaboratofy 3M Environmental Laboratory Page 31 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Pr,otocoLlRN-U2095 Amendment Number 4 This amendment Modifiesthefollowingportion(so)ftheprotocol: 1. PROTOCOL READS: The studydirectofrorthepresentstudywas identifiientdheprotocoals James K. Lundburg, Ph.D. AMEND TO READ: The roleofstudydirectofrorthe presentstudywas reassignedto MarvinT. Case, D.V.M.,Ph.D.,as of20 January 2000. The previousstudydirectorJ,ames K. Lundburg, has been reassignedtothe roleofPrinciplAenalyticaIlnvestigator. REASON: The roleofstudydirectowras reassignedinan efforttoensure compliance with Good LaboratoryPracticeStandards thatoutlinestudypersonnelrequirements (referto21 CFR Part58). 2. PROTOCOL READS: The sponsorforthe presentstudywas identifieads MarvinT. Case, D.V.M.,Ph.D. AMEND TO READ: The roleofsponsorforthe presentstudywas reassignedto John L.Butenhoff, Ph.D.,as of20 January 2000. REASON: To ensure thatthestudydirectodroes notalsocarrythe dutiesofstudy sponsor,the sponsor rolewas reassigned.Inthismanner, personnelresponsibilitiaensd workload are more evenlybalanced. 3M EnvironmentalLaboratoty 3M Environmental Laboratory Page 32 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Protocol LRN-U2095 Amendment Number 4 Amendment Approval John L ButenhoffPhD., Sponsor Representative /0,, Date Jaf K Lundberg, PhD., 2!@lging Study Director .0..- JA q)ate I Marvin T.Case,b. VM, Ph.D.,IncomingStudyDirector Date 3M Environmental Laboratory Im 2nvironmental Laboratory Page 33 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Study Title AnalyticaSltudyof2(N-Ethylperfluorooctanesulfonamido)i-nethanol Two GenerationRat Reproduction PROTOCOL AMENDMENT NO. 5 Amendment Date: August 31,2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M EnvironmentalLaboratory 935 Bush Avenue St.Paul,NIN 55106 Laboratory Project Identification FACT-TOX-013 ET&SS LRN U2095 Argus Study: 418-009 3M Medical Department Study: T6316.5 3M Environmental Laboratory 3M Environmental Laboratory Page 34 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 ProtocoFlACT TOX-013 Amendment No. 5 This amendment modifiesthe followingportion(s)of the protocol: 1. PROTOCOL REAos: The PrinciplAenalyticaIlnvestigatfoorrthepresensttudywas identified as James K. Lundberg, Ph.D. 2. AMEND TO READ: The roleof PrincipleAnalyticalInvestigatofrorthepresentstudywas reassignedto KristenJ.Hansen Ph.D. REASON: The roleof PrincipleAnalyticalInvestigatowras reassigneddue to availabilitoyf resources. 3M Environmental Laboratory 3M Environmental Laboratory Page 35 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 ProtocoFlACT TC)X-013 Amendment No. 5 Amendment Approval -2 . JohnL.ButenhoffP,h.D.,SponsorRepresentative ZC@,z@ Date Ma@vin T. Case, D. V.M., PILD.,Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 36 3M Medical Department Study: T6316-S Analytical Report: FACT TOX-013 LRN-U2095 Appendix C: Extractionand AnalyticalMethods This appendix includesthe followingmethods: ETS-8-4.1,Extractionof Potassium Perfluoroactanesulfonatoer Other Fluorochemical Compounds from Serum forAnalysisUsing HPLC-Electrospray/Mass Spectrometry, (14 pages) ETS-8-5.1,Analysisof Potassium Perfluorooctanesuffonatoer Other Fluorochemicalsin Serum ExtractsUsing HPLC-Electrospray/Mass Spectrometry,(9 pages) 3M Environmental Laboratory Page 37 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LM-U2095 3M ENVIRONMENTAL LABORATORY METHOD EXTRACTIONFLUOROCREMICAL OF POTASSIUM PERFLUOROOCTANESULFONATE COMPOUNDS FROM SERUM FOR ANALYSIS ELEC'fROSPRAY/MASS SPECTROMETRY OR OTE[ER USING HPLC- Method Number: ETS-8-4.1 Author: Lisa Clemen, Glenn Langenburg Adoption Date: 03/01/99 Revision Date: 711?1 Approved By: Laboratory Manager Date Group Leader C,4.,A uilrltll Technical Reviewer Date olzai,/99 Date 1.0 SCOPE AND APPLICATIOrq 1.1 Scope: This method is for the extraction of potassium pertluorooctanesulfonate (PFOS) or otherfluorochemicalcompounds from serum. 1.2 Applicable compounds: Fluorochemical surfactantsor other fluorinatedcompounds. 1.3 Matrices: Rabbit,rat,bovine,monkey, and human serum or otherfluidsas designatedin the validationreport. Word 6/95 3M Environmental Laboratory ETS-9-4.1 Extractionof PFOS from Senirn Page I of 14 Page 38 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 2.0 SUMMARY OF MFTHOD 2.1 This method describes the procedure for extractingpotassium perfluorooetanesulfonate (PFOS) or other fluorochemical surfactantsfrom serum, or other fluids,using an ion pairingreagent and methyl-tert-butylether(MtBE). In thismethocl seven fluorochemicals were extracted:PFOS, PFOSA, PFOSAA, ETFOSE-OH, PFOSEA, M556, and surrogatestandard (see 3.0 Definitions).An ion pairingreagent is added to the sample and the analyte ion pair is partitionedintoMTBE. The MTBE extractis removed and put onto a nitrogenevaporator untildry. Each extractisreconstitutedin 1.0 mL of methanol, then filteredthrough a 3 cc plasticsyringe attachedto a 0.2 pm nylon filteirnto glass autovials. 2.2 These sample extractsare analyzed following method ETS-8-5.1 or other appropriate methods. 3.0 DEFINITIONS 3.1 PFOS: perflucrooctanesulfonate(anion of p otassium salt)C,F,IS03- 3.2 PFOSA: perfluorooctanesulfonylamide C,F17So2NH, 3.3 PFOSAA: perfluorooctanesulfonylamido (ethyl)acetatCe@F17SON(CH2CH,)CH,C02- 3.4 ETFOSE-OH: 2(N-ethylperfluorooctanesulfonainido)-ethyallcohol C8Fl7SO2N(CH,CH,)CH2CH20H 3.5 PFOSEA: perfluorooetanesulfonylethylamide C8Fl7SO2N(CH2CH3)H 3.6 M556: C,F,7SON(H)(CH2COOH) 3.7 Surrogate standard:1H-IH-2H-2H perfluorooctanesulfonicacid 4.0 WARNINGS AND CAUTIONS 4.1 Health and safety warnings 4.1.1 Use universal precautions,especially laboratory coats,goggles, and gloves when handling animal tissue,which may contain pathogens. 5.0 INTERFERENCES 5.1 There are no interferencesknown at thistime. 6.0 EQUIPMENT 6.1 The following equipment isused while performing thismethod. acceptable. Equivalent equipment is 6.1.1 Vortex mixer, VV;R, Vortex Genie 2 6.1.2 Centrifuge,Mistral1000 or IEC 6.1.3 Shaker, Eberbach or VVvrR 3M Environmental Laboratory ETS-8-4.1 Extractionof PFOS from Serum Page 2 of 14 Page 39 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 6.1.4 NitrogenevaporatorO,rganomation 6.1.5 Balance ( 0.100 g) 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Eppendorf or disposablepipettes 7.3 Nalgene bottles,capable of holding 250 mL and 1 L 7.4 Volumetricflasksg,lass,typeA 7.5 I-CHEM vials,glass,40 niL glass 7.6 Centrifugetubes,polypropylene,15 mL 7.7 Labels 7.8 Oxford Dispenser- 3.0to 10.0mL 7.9 Syringes, capable of measuring 5 @LLto50 gL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic,3 cc 7.12 Syringe filtersn,ylon, 0.2 gm, 25 rnm 7.13 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers Note: Prior to using glassware and bottles,rinse 3 times with methanol and 3 times with Milli-(@Tmwater. Rinse syringes a minimum separate vials. of 9 times with methanol, 3 rinses from 3 8.0 REAGENTS AND STANDARDS 8.1 Type I realent grade water, Milli-QTM or equivalent; allwater used in thismethod be Milli-Q water and may be provided by a Milli-Q TOC PlusT' system should 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylanunonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (NaC03), J.T.Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T.Baker or equivalent 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilledor HPLC grade 8.7 Methanol, Omnisolv, glass distilledor BPLC grade 8.8 Serum or blood, frozen from supplier 8.9 Fluorochemical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 ETS-8-4.1 Extractionof PFOS from Senun Page 3 of 14 3M Environmental Laboratory Page 40 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 8.10 8.11 8.9.3 PFOSAA (3M SpecialtyChemical Division)m,olecularweight = 585 8.9.4 ETFOSE-OH (3M Specialty Chemical Division),molecular weight = 570 8.9.5 PFOSEA (3M Specialty Chemical Division),molecular weight = 527 8.9.6 M556 (3M SpecialtyChemical Division)m,olecularweight= 557 8.9.7 Surrogatestandard:4-H,perfluorooctanseulfonicacid(1-H,1-H,2-H, 2-H C,F,3So3H) molecularweight= 429 8.9.8 Other fluorochemicalsa,s appropriate Reagent preparation NOTE: When preparinglargervolumes thanlisteidnreagent,standard,or surrogate preparationa,djustaccordingly. 8.10.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NAOH. Pour into a 1000 niL beakercontaining500 mL Milli-Q' water,mix untilallsolidsare dissolved.Storeina I L Nalgene bottle. 8.10.2 IN sodium hydroxide (NaOH): Dilute ION NAOH 1:10. MeasurelOmLoflO N NAOH solution into a 100 m-L volumetric flask and diluteto volume using Milli-(@" water. Store in a 125 mL Nalgene bottle. 8.10.3 0.5 M tetrabutylammonium hydrogen sulfate(TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 mL Milli_QTI water. Adjust to pH 1.0using approximately 44 to 54 mL of 10 N NAOH (While adding the lastniL of NAOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-Q' water. Store in a 1 L Nalgene bottle. 8.10.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using I N NAOH solution. 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer (NaCO,/NaHCO,): Weigh approximately 26.5 g of sodium carbonate (Na@C03) and 21.0 g of sodium bicarbonate (NaHCO,) into a 1 L volumetric flask and bring to volume with Milli- Q TM water. Store in a I L Nalgene bottle. Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.11.3 Weigh approximately 100 mg of PFOS the actual weight. into a 100 ML volumetric flask and record 8.11.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (@Lg/mL). 8.11.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.11.6 Dilute working standard 1 with methanol for a working standard 2 solution of approx. 5.0 ppm. ETS-8-4.1 FxtractionofPFOS from Serum Page 4 of 14 3M Environmental Laboratory Page 41 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 8.12 8.11.7Diluteworking standard I with methanol for a working standard 3 solution of approx. 0.50 ppm. Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 mg of surrogatestandard 1-H,1-H, 2-H, 2-H, C,F,,SO,H into a 50 mL volumetric flaskand recordthe actualweight. 8.12.2 Bring to volume with methanol for a surrogatestock of approximately 1000- 1200 PPM. 8.12.3 Prepare a surrogateworking standard. Transfer approximately I mL of surrogate stock to a 10 mL volumetric flaskand bring to volume with methanol for a working standard of 100 ppm. Record the actualvolume transferred. 9.0 SAMPLE HANDLING 9.1 All samples are received frozen and must be kept frozen untilthe extractionisperformed. 9.2 Allow samples to thaw to room temperature priorto extraction. 10.0 ()UALITY CONTROL 10.1 Solvent Blanks, Method blanks and matrix blanks 10.1.1 An aliquot of 1.0 ni-Lmethanol is used as a solvent blank. 10.1.2 Extracttwo 1.0mL aliquotsof Milli_QTIIwater following thisprocedure and use as method blanks. 10.1.3 Extracttwo 1.0 mL aliquotsof the serum following thisprocedure and use as matrix blanks. See 11.1.4. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst,usually the control matrix received with each sample set. 10.2.3 Expected concentrationswill fallin themid-range of the initiaclalibrationcurve. Additional spikesmay be included and may fallin the low-rarigeof the initial calibrationcurve. 10.2.4 Prepare one matrix spike and matrix spike duplicateper 40 swnples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibrationchecks 10.3.1 Prepare continuing calibrationcheck samples to ensure the accuracy of the initial calibrationcurve. 10.3.2 Prepare, at a minimum, one continuing check per group of 10 samples. For example, ifa sample set = 34, four checks are prepared and extracted. 10.3.3 Prepare each continuing calibrationcheck from the same matrix used to prepare the initiaclurve. 3M Environmental Laboratory ETS-84.1 Extractionof PFOS from Serum Page 5 of 14 Page 42 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 10.3.4 The expectedconcentrationwsillfallwithinthemid-rangeof theinitial calibratiocnurve.Additionalspikesmay be includedthatfallinthe low-range of theinitiaclalibratiocnurve.This isnecessaryiftheanalystmust quantitateusing only the low end of the calibrationcurve (forexample, 5 ppb - 100 ppb, rather than 5 ppb - 1000 ppb). 11.0 CALIBRATION AND STANDARDTZATION 11.1 Prepare- matrix calibration standards 11.1.1 Transfer 1 mL of serum to a 15 mL centrifuge tube. 11.1.2 Ifmost sample volumes are less than 1.0 mL, extractstandards with matrix volumes equal to the sample volumes. Do not extractless than 0.50 mL of matrix. Record each sample volume on the extraction sheet. 11.1.3 Vaiile preparing a totalof twenty aliquotsin 15 mL centrifuge tubes, mix or shake between aliquots. 11.1.4 Two I mL aliquots,or other appropriate volume, serve as matrix blanks. Typically use the standard concentrzitionsand spiking amounts listedin Table 1, at the end of this section, to spike, in duplicate,two standard curves, for a total of eighteen standards, two matrix blanks, and two method blanks. 11.1.5 Refer to validation report ETS-8-4.0 & ETS-8-5.0-V-1, which liststhe working ranges and the Linear Calibration Range (LCR) for calibrationcurves. 11.2 11.3 11.1.6 Use Attachment D as an aid in calculating the concentrations of the working standards. See Section 13.0 to calculate actual concentrations of PFOS in calibrationstandards. To each standard, blank, or continuing check, add appropriate amount of surrogate working standard for the concentration to fallwithin the calibrationcurve range 5 ppb 1000 ppb. Extract spiked matrix standatds following 12.6-12.16 of thismethod. Use these standards to establisheach initialcurve on the mass spectrometer. 3M Environmental Laboratory ETS-84.1 Extractionof PFOS from Senan Page 6 of 14 Page 43 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Table I Approximate spiking amounts for standards and spikes Using 1.0 mL of matrix Working standard (approx. conc.) @iL Approx. fmal conc. of analyte in matrix Blank 0.500 ppm 10 0.005 ppm 0.500 ppm 5.00 ppm 20 O.OIOPPM 5 0.025 ppm 5.00 ppm 5.00 ppm 50.0 ppm 50.0 ppm 50.0 ppm 10 0.050 ppm 20 0. 100 ppm 5 0.250 ppm 10 0.500 ppm 15 0.750 ppm 50.0 ppm 20 1.00 ppm 12.0 PROCEDURE 12.1 Obtain frozen samples and allow to thaw at room temperature or in a lukewarm waterbath. 12.2 Vortex mix for 15 seconds, then transfer1.0 mL or other appropriate volume to a 15 niL polypropylene centrifuge tube. 12.3 Return unused samples to freezer after extraction amounts have been removed. 12.4 Record the initialvolume on the extraction worksheet. 12.5 Label the tube with the study number, sample DD, date and analyst initials.See attached worksheet for documenting the remaining steps. 12.6 Spike allsamples, including blanks and standards, ready for extractionwith surrogate standard as described in 11.2. 12.7 Spike each matrix with the appropriate amount of standard as described in 11.1, or Table 1 in that section,for the calibrationcurve standards. Also prepare matrix spikes and continuing calibrationstandards. 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 12.10 Check to ensure the 0.5 M TBA reagent is at pH 10. Ifnot, adjust accordingly. To each sample, add I mL 0.5 M TBA bicarbonate buffer. and 2 mL of 0.25M sodium carbonate/sodium 12.11 Using an Oxford Dispenser, add 5 mL methyl-tert-butyl ether. 12.12 Cap each swnple and put on the shaker at a settingof 300 rpm, for 20 minutes. 12.13 Centrifuge for20 to 25 minutes at a settingof 3500 rpm, or untillayers are well separated. ETS-8-4.1 Extractionof PFOS from Senun Page 7 of 14 3M Environmental Laboratory Page 44 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 12.14 12.15 12.16 12.17 Label a fresh 15 mL centrifuge tube with the same information as in 12.5. Remove 4.0 mL of the organic layer to this clean 15 mL centrifuge tube. Put each sample on the analyticalnitrogen evaporator untildry, approximately hours. Add 1.0mL of methanol to each centrifuge tube using a graduated pipette. I to 2 12.18 Vortex mix for 30 seconds. 12.19 Attach a 0.2 gm nylon mesh filterto a 3 cc syringe and transferthe sample to this syringe. Filterinto a 1.5 mL glass autovial or low-volume autovialwhen necessary. 12.20 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, finalsolvent, extractiondate, and analyst(s)performing the extraction. 12.21 12.22 Cap and store extracts at room temperature or atapproximately 4 *C until analysis. Complete the extraction worksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. 13.0 DATA ANALYSIS AN]) CALCULATIONS 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS, or other applicable fluorochemical, in calibrationstandards using the following equation: mL of standard x concentration of standard (4g /mL) mL of standard + mL of surrogate standard + initiamlatrix volume (mL) Final Concentration (@Lg/mL) of PFOS in matrix 14.0 METHOD PFRFORMANCE 14.1 The method detection limit (MDL) is analyte and matrix specific.Refer to MDL report for specificMDL and limitof quantitation(LOQ) values (see Attachments B and C). 14.2 The following quality control samples are extracted with each batch of samples to evaluate the quality of the extractionand 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 the extraction. 14.2.3 Continuing calibrationcheck samples to determine the continued accuracy of the initialcalibrationcurve. 14.3 Refer to section 14 of ETS-8-5.1 formethod performance criteria. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample waste is disposed in biohazard containers, fla=able solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. ETS-8-4.1 Extractionof PFOS from Serum Page 8 of 14 3M Environmental Laboratory Page 45 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 16.0 RECORDS 16.1 Complete the extractionworksheet attachedto thismethod, and tape in the study notebook or includein the 3-ringstudybinder,as appropriate. 17.0 ATTACHMENTS 17.1 Attachment A, Extractionworkshect 17.2 Attachment B, NMIJLOQ valuesand summary 17.3 Attachment C, Calibrationstandardconcentrationworksheet 18.0 REFERIENCES 18.1 The validationreportassociated with thismethod isETS-84.0 & 5.0-V-1. 18.2 FACT-M-3.1, "Analysis of Serum or Other Fluid Extracts forFluorochemicals using HPLC-Electrospray Mass Spectrometry" 19.0 AFFECTED DOCUMENTS 19.1 ETS-8-5. 1, "Analysisof Senun or Other FluidExtractsforFluorochemicalsusing HPLC-Electrospray Mass Spectrometry" 20.0 REviS[ONS Revision Number I Reason For Revision Section 12.21 Changed to includesample storageatroom temperature. Section 12.13 Added the shaker speed. Section12.17 Finalvolume is 1.0mL; not adjustedforinitiavlolumes lessthan 1.0mL. Revision Date 04/02/99 3M Environmental Laboratory ETS-8-4.1 ExtractionofPFOS from Serum Page 9 of 14 Page 46 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LP,N-U2095 ExtractionWorksheet ETS-8-4.1 Study Matrix Box # Wk/Da DateSpiked/Analyst cc ms MSD Surrogate Std approx. ppm actual ppm FC-Mix approx. 0.5 pm actual PPM FC-Mix approx. 5 ppm actual PPM FC-Mix approx. 50 ppm actual PPM Comments Blank Std Serum ExtractionMethod amount Vortex 15 sec. PipetteMatn,x Volume mL PipetteI mL of 0.5M TBA, pH 10. pH Std.# Pipette2 mL of 0.25NayCO3/0.25M NaHC03 buffer Std.# Dispense 5 mL ofmethyl-t-butyelther TN-A- Sbake 20 min. Shaker speed: Centrifuge20-25 min. Centrifugespeed: Remove a 4 mL aliquotoforganiclayer Put on Nitrogen Evaporatorto dryness Temperature: Add methanol Volume ML TN-A- Vortex 30 sec. Filterusinga 3cc B-D syr-ingweith a 0.2pm filteirntoa 1.5mL autosamplevial Cont. Cal. Verificationsused same matrix as for std curve. Attachment A 3M Environmental Laboratory ETS-8-4.1 Extractionof PFOS from Serum mL Date & Initials Page 10 of 14 Page 47 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 MDL/LOQ valuesforrabbit serum Compound MDL LOQ Linear Calibration Range (LCR) (ppb) (ppb) Approximate concentrations to be used for preparing the PFOS PFOSA PFOSAA 1.74 1.51 3.46 I 5.5 4.79 20.5 Standard Calibration 5 ppb - 1000 ppb 1 5 ppb -1000 ppb 5 ppb - 1000 ppb Curve ETFOSE-OH M556 11.4 6.03 36.2 19.2 5 ppb - 1000 ppb 5 ppb - 1000 ppb IPFOSEA 1 5.71 18.2 5 ppb - 1000 ppb NL]DIILOQ values in rat,bovine, monkey, and human serum, and monkey plasma were not statistically determined. Two curves in each of these matrices were extracted and analyzed with the rabbitserum curves to determine equivalence. Responses in the rat,bovine, monkey, and human were equivalent to the rabbitresponses, therefore,theirMDL and LOQ willbe the same values as determined in rabbit serum. Please see LOQ Summary and NML study in ETS-8-4.0 & 5.0-V- I for furtherinformation. Attachment B: MDL/LOQ Sunmiary 3M Environmental Laboratory ETS-".l Extractionof PFOS from Ser= Page II of 14 Page 48 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Compound: PFOS Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range 0.995-978 Low Curve 4.94-248 High curve 97.8-978 1/X 0.995-978 1 Compound: PFOSA Rabbit Serum Prepared range of standards (ppb) (ng/mL) Full Range Low Curve High curve I/X 0.993-976 4.93-97.6 24.8-976 0.993-976 LCR from curve (ppb) (ng/mL) 24.8-978 4.94-248 97.8-978 4.94-978 LCR from curve (ppb) (ng/mL) 4.93-976 4.93-97.6 24.8-978 4.93- 976 Compound: PFOSAA Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve ri/X- 0.991-974 4.92-247 49.2-974 0.991-974 LCR from curve (ppb) (ng/mL) 24.7-974 9.74-247 97.4-974 9.74-974 Recovery Range 83-109 85-104 85-106 94-111 % Recovery Range 88-103 87-105 93-102 94-103 % Recovery Range 81-111 97-107 85-108 95-115 RSD Range 4.67-11.0 5.34-12.0 4.84-9.80 4.60-10.5 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 RSD Range 4.18-10.6 6.38-21.8 4.33-12.5 4.11-23.2 AttaclunentB: MDLRLOQ Sununary 3M Environmental Laboratory ETS-8-4.1 Extractionof PFOS from Serum Page 12 of 14 Page 49 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Compound: ETFOSE-OH Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve I/X 0.993-976 4.93-97.6 49.3-976 0.993-493 LCR from curve (ppb) (ng/mL) 49.3-976 9.76-97.6 97.6-976 9.76-976 % Recovery Range 77-110 97-107 90-109 86-111 Compound: PFOSEA Prepared range Rabbit Senim of standards (ppb)(ng/mL) Full Range Low Curve High curve 1/X 0.993-976 4.93-248 49.3-976 0.993-976 LCR from curve (ppb) (ng/mL) 24.8-976 9.76-248 49.3-976 9.76-976 % Recovery Range 96-106 91-110 86-106 95-117 Compound: Rabbit Semm FullRange Low Curve High curve I/X 556 Prepared range of standards (ppb)(ng/mL) 0.993-976 4.93-97.6 97.6-976 0.993-976 LCR from curve (ppb) (ng/mL) 24.8-976 9.76-97.6 97.6-976 9.76-976 % Recovery Range 88-106 100-105 81-111 97-110 RSD Range 11.2-25.5 14.1-21.3 11.5-19.6 11.1-21.2 RSD Range 10.1-16.2 11.8-19.5 10.2-18.2 10.1-19.1 RSD Range 4.82-17.9 5.95-18.2 5.11-9.74 4.77-19.5 Attachment B-.MDLFLOQ Summary 3M Environmental Laboratory ETS-8-4.1 Extractionof PFOS from Serum Page 13 of 14 Page 50 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Ion PairStandard Curves - Fluids Prep date(s): Analyte(s): Sample matrix: Standard number: Equipment number: Final solvent and TN: Blank fluid/identifier: Method/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Actual concentrations of standards in the FC mix PFOS PFOSA PFOSAA ETFOSE PFOSEA M556 Std conc Std conc Std conc Std conc Std conc Std conc ug/mL 0.500 ug/mL 0.507 ug/mL 0.532 ug/mL 1 0.501 ug/mL 0.521 ug/mL 0.501 0.500 0.507 0.532 0.501 0.521 0.501 5.00 5.07 5.32 5.01 5.21 5.01 5.00 5.07 5.32 5.01 5.21 -5.01 5.00 5.07 5.32 5.01 5.21 5.01 50.0 50.1 53.2 50.1 52.1 1 50. 1_ 50.0 50.1 53.2 50.1 52.1 50.1 50.0 50.1 53.2 50.1 52.1 50.1 50.0 50.1 531 50.1 52.1 50.1 All Am't spiked mL 0.010 0.020 0.005 0.010 0.020 0-005 0.010 0.015 0.020 All Finalvol ML 1.015 1.025 1.010 1.015 1.025 L 1-010 1.015 1.020 1.025 Calculated concentrations of standards PFOS Finalconc ng/mL PFOSA Finalconc ng/mL PFOSAA Finalconc ng/rnL ETFOSE Finalconc ng/mL 4.93 5.00 1 5.24 4.94 9.76 9.89 10.4 9.78 24.8 25.1 49.3 50.0 26.3 52.4 24.8 49.4 97.6 98.9 104 97.8 248 251 263 248 493 500 524 494 735 746 1 782 737 976 989 1 1038 978 in the sample matrix PFOSEA M556 Surrogate Finalconc Finalconc ng/mL ng/mL Std conc ng/mL 5.01- 5.13 100 9.93 25.2 50.1 10.2 25.8 51.3 Surrogate Final conc 99.3 102 ng/mL 1 252 ___258 500 501 513 749 766 993 1017 All Am't spiked niL 0.005 Validated ranges - approximate concentrations Serum -Rabbit Bovine PFOS PFOSA PFOSAA ETFOSE-OH 5.00-1000 1 5.00-166".. 5.00-1000 1 5.00-1000 Estirmtesonly. Use valuesforrabbit. Rat Estiffiatoensly. Use valuesforrabbit. Monkey & Plasma Estiniateosnly. Use valuesforrabbit. uman Estiniateosnly. Use valuesforrabbit. PFOSEA 5.00-1000 M556 5.00-1000 Attachment C: Ion Pair Standard Curves ETS-8-4.1 Extractionof PFOS from Serum 3M Environmental Laboratory Page 14 of 14 Page 51 3M Medical Department Study: T6316.S Analytical Report: FACT TOX-013 LM-U2095 3M ENVIRONMENTAL LABOF.ATORY METHOD ANALYSIS QF POTASSIUM PERFLUOROOCTANEST-TLFONATE OR FLUOROCHEAUCALS IN SERUM ExTRAcTs USING HPLC-ELEcTRospRAY/MAss SPEcrROMETRY OTHER Method Number: ETS-8-5.1 Author: Lisa Clemen, Robert Wynne Approved By: LaboratoryManager Adoption Date: 03/01/99 Revision Date: qlo@i@M 2.c@/ Date Group Leader @L A Technical Reviewer Date oyLa4,@l Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method describesthe analysisof serum extractsforfluorochemicalsurfactants using B?LC-clectrospray/mass spectrometry. 1.2AppUcable Compounds: Fluorochemical surfactantsor other fluorinatecdompounds, or otherionizablecompounds. 1.3Matrices: Rabbit,rat,bovine,monkey, and human serum, or otherfluidsas designateciin the validationreport. Word 6/95 ETS-8-5.1 Analysis of Seniin ExtractUsing ES/MS Page I of 9 3M Environmental Laboratory Page 52 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 2.0 SUMMARY OF METHOD 2.1 This method describesthe analysisof fluorochemicalsurfactantesxtractedfrom serum or other fluids,using BPLC-electrospray/rnassspectrometry,or similarsystem as appropriate. The analysisisperformed by monitoring a singleion characteristiocf a particular fluorochemical,such as theperfluorooctanesulfonat(ePFOS) anion,M/z= 499. Additionally,swnples may be analyzedusing a tandem mass spectrometerto furtherverify the identityof a compound by detectingdaughter ions of the parention. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization (API): The Micromass QuattroIltriplequadrupole systems allow forvariousmethods of ionizationby utilizinvgarioussources,probes,and interfaces-.These includebut arenot limitedto:ElectrosprayIonization(ESI),Atmospheric Pressure chemical Ionization(APcl),Thermospray, etc.The ionizatiopnrocessin these techniquesoccurs at atmosphericpressure(i.e.n,ot under a vacuum). 3.2 Electrospray Ionization(ES, ESI): a method of ionizatiopnerformed at atmospheric pressure,whereby ions in solutionaretransferredtothe gas phase viatinycharged droplets. These charged dropletsare produced by the applicationof a strongelectricaflield. 3.3Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro11triplequadrupole systems areequipped with quadrupolemass selective detectors.Ions areselectiveldyiscriminatedby mass to charge ratio(m/z)and subsequently detected.A singleMS may be employed forion detectionor a series(MS/MS) formore specificfragmentationinformation. 3.4 Conventional vs.Z-spray probe interface:The latesmtodels of Micromass QuattroII triplequadrupolesystems (post1998) utilizea "Z-spray"conformation.The spray emitted from a probe isorthogonalto the cone aperture.In the conventionalconformation itisaimed directlyatthe cone aperture,afterpassing through a tortuouspathway inthe counter electrode.Though the configurationisdifferentt,he methods of operation,cleaning,and maintenance are the same. However, Z-spray components and conventionalcomponents are not compatible with one another,but only with similarsystems (i.e.Z,-spraycomponents are compatible with some otherZ-spray systems,etc.) 3.5 Mass Lynx Software: System softwaredesigned forthespecificoperationof theseQuattro IItriplequadrupole systems. CurrentlyMassLynx has Windows 95 and WindowsNT 4.0 versions.All versionsare similar.For more detailssee themanual specificto theinstrument (Micromass QuattroIItriplqeuadrupole MassLynx or MassLynx NT User'sGuide). 4.0 WARNINGS AND CAUTIONS 4.1 Health and SafetyWarnings: 4.1.1 Use cautionwith thevoltagecablesfortheprobe. NVhen engagecltheprobe employs a voltageof approximately 5000 Volts. 4.1.2 When handling samples or solventswear appropriateprotectivegloves,eyewcar, and clothing. 3M Environmen,.al Laboratory ETS-8-5.1 AnalysisofSenim ExtractUsing ES/MS Page 2 of 9 Page 53 3M Medical Department Study: T6316.S Analytical Report: FACT TOX-013 LRN-U2095 4.2 Cautions: 4.2.1 DO Dot operatseolvenptumps abovecapacitoyf400 bar(5800psi)backpressure. If the back pressure exceeds 400 bar, the HP 1100 will initiateautomatic shutdown. 4.2.2 Do not run solventpumps todryness. 5.0 INTFRFERENCES- 5.1 To minimize interferences when analyzing samples, teflon should not be used for sample storageorany partofinstrumentatiotnhatcomes incontactwiththesample orextract. 6.0 EouipmENT 6.1 Equipment listebdelow may be modified inordertooptimizethesystem. Document any modificationsintheraw dataasmethod deviations. 6.1.1 6.1.2 Micromass QuattroH triplqeuadrupoleMass Spectrometerequippedwith an electrospraiyonizatiosnource B?l 100 low pulse solvent pumping system, solvent degasser, colurrm compartment, and autosampler 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.1.1 High purity grade nitrogen'gas regulated to approximately 100 psi (House air system) 7.1.2 HPLC analyticalcolunin, specifics to be determined by the analyst and documented in the raw data. 7.1.3 Capped autovials or capped 15 mL centrifage tubes 8.0 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 Methanol, BPLC grade or equivalent 8.1.2 NElli-QTwamter, allwater used in thismethod should be Milli-Q' water or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Arnmoaium acetate,reagent grade or equivalent 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. See ETS-84.1. 9.0 SAMPLE HANDLING 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL cenhifuge tubes untilanalysis. 3M Environmental Laboratory ETS-8-5.1 Analysisof Serum ExtractUsing ES/MS Page 3 of 9 Page 54 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 9.2 If analysiswill be delayed, extractedstandards and samples can be refrigeratedat approximately 4' C, or at room temperature,untilanalysiscan be performed. 10.0 OUALITY CONTROL 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks,method blanks and matrix blanks are prepared and analyzed with each baich to determine contamination or cariyover. 10.1.2 Analyze a method blank and a matrix blank priorto each calibrationcurve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effecton the recovery efficiency. 10.2.2 Matrix spike duplicatesare prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spikeduplicateper fortysamples, with a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicateconcentrationswillfallin then3id-range of the initiaclalibrationcurve. Additional spike concentrationsmay fallin the lowrange of theinitialcalibrationcurve. 10-3 Continuing Calibration Verifications 10.3.1 Continuing calibrationverificationsareanalyzed toverifythe continued accuracy of the calibrationcurve. 10.3.2 Analyze a mid-range calibrationstandard afterevery tenthsample, with a minimum of one per batch. 11.0 CALIBRATION AND STANDARDIZATION 11.1 Analyze the extractedmatrix standardspriorto and following each set of extracts.The average of two standard curves willbe plottedby linearregression(y = my + b),weighted I/x,not forced through zero,using MassLynx or other suitablesoftware. 11.2 If the curve does not meet requirements, perform routinemaintenance or reextractthe standard curve (ifnecessary) and reanalyze. 11.3 For purposes of accuracy when quantitatinglow levelsof analyte,itmay be necessary to use the low end of the calibrationcurve ratherthan the fallrange of the standard curve. Example: when attempting to quantitateapproximately 10 ppb of analyte,generate a calibrationcurve consistingof the standards from 5 ppb to 100 ppb ratherthan the fan range of the curve (5 ppb to 1000 ppb). This willreduce inaccuracyattributedto linear regressionweighting of high concentration standards. 3M Environmental Laboratory ETS-8-5.1 AnalysisofSerum ExtractUsing ES/MS Page 4 of 9 Page 55 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 12.0 PROCEDURES 12.1 Acquisition Set up 12.1.1 Click on startbutton in the Acquisition Control Panel.Set up a sample list.Assign a filename using MO-DAY-last digitof year-sample number, assign a method (MS) for acquiring,and type in sample descriptions. 12.1.2 To createa method clickon scan button in the Acquisition controlpanel and select SIR (SingleIon Recording) or MRM. Set IonizationMode as appropriateand mass to 499 or other appropriatemasses. A fullscan is usuallycollectedalong with the SIRS. Save acquisitionmethod. IfMS/MS instrumentsareemployed, additional product ion fragmentation information may be collected.See Micromass MassLynx GUIDE TO DATA ACQUISITION foradditionalinformation and NUZM (MultipleReaction Monitoring). 12.1.3 Typically the analyticalbatch run sequence begins with a setof extractedmatrix standards and ends with a set of extractedmatrix standards. 12.1.4 Samples are analyzed with a continuing calibrationcheck injectedafterevery tenth sample. Solvent blanks should be analyzed periodicallyto monitor possible analyte canyover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample listprepared in Section 12.1.1. 12.2.2 S et-up the HP 11 00/autosampler atthe following conditions or atconditions the analystconsiders appropriatefor optimal response.Record actualconditions in the inst-urnentlogbook: 12.2.2.1 Sample size = 10 @iL injection 12.2.2.2 Inject/sample= 1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent rwnp = Time 0.00 min. 8.50 min. 11.0 min. 1 .0 min. MEOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% 10% 16% 60% 12.2.2.5Press 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 solventlevelin reservoirsand refillifnecessary. 3M Environmental Laboratory ETS-8-5.1 Analysisof Semm ExtractUsing ES/MS Page 5 of 9 Page 56 3M Medical Departmen@: Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 12.3.3 Check thestainlessteelcapillaryattheend of theprobe. Use an eyepiece to check thetip.The tipshouldbe flatwithno jagged edges.Ifthetipisfound to be unsatisfactoryd,isassemblethe probe and replacethestainlesssteelcapillary. 12.3.4 Set EPLC pump to "On". Set theflow to 10 - 500 uumin or as appropriate. Observe dropletscoming out ofthetipof theprobe.Allow to equilibratfeor approximately10 minutes. 12.3.5 Turn orrthenitrogen.A finemistshouldbe expelledwithno nitrogenleaking around thetipof theprobe. Readjustthetipoftheprobe ifno mistisobserved. 12.3.6 The instrumentusestheseparametersatthe followingsettingsT.hese settingsmay change in orderto optimizethe response: 12.3.6.1Drying gas 250-400 liters/hour 12.3.6.2ESI nebulizinggas 10-15 liters/hour 12.3.6.3HPLC constantflow mode, flowrate10 - 500 @iljmin 12.3.6.4Pressure<400 bar (Thisparameterisnotset,itisa guideto ensure the HPLC isoperatingcorrectl) y. 12.3.7 Carefullyguide theprobe intotheopening.Insertprobe untilitwillnot go any furtherC.onnect thevoltagecablesto theprobe. 12.3.8 Printthetunepage,with itsparameters,and storeitinthestudybinderwith a copy tapedintotheinstrumentlog. 12.3.9 Using thecross-flowcounterelectrodeintheES/MS sourceisrecommended for theanalysisof biologicalmatrices. 12.3.10CIickon startbuttonintheAcquisitionControlPanel(thismay vary among MassLynx versions,see appropriatMeassLynx USER'S GUI]DE). Pressthe start button.Ensure startand end sample number includesallsamples tobe analyzed. 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.4 Calculatematrix spikepercentrecoveriesusingthefollowingequation: % Recovery Observed Result- Backzround Result x 100 Expected Result 13.1.5 Calculatepercentdifferencuesing the followingequation: % Difference Expected Conc. - CalculatedConc. x 100 Expected Conc. 13.1.6Calculateactualconcentratioonf PFOS, or otherfluorochemicali,nmatrix (ng ofPFOS calc.from std.Curve x DilutioFnactor) x I Lig (InitiVaollume ofmatrix(mL) + mL of SurrogateStandard) 1000 ng FinalVolume (mL) 3M Environmental Laboratory ETS-8-5.1 Analysis of Sennu ExtractUsing ESINIS Page 6 of 9 Page 57 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 14.0 METHOD PERFORMANCE 14.1 Method DetectionLimit(MDL) and Limitof Quantitatio(nLOQ) aremethod, analyte,and matrix specificP.leaseseeETS-8-4.1,Attachment B, fora listinogfcurrentvalidated MDL and LOQ values. 14.2 SolventBlanks,Method Blanks, and Matrix Blanks - 14.2.1 Solventblanks,method blanks,and matrixblanksvaluesaremust be below the loweststandardinthe calibratiocnurve 14.3 Calibration Curves 14.3.1 The ? value forthe calibratiocnurve must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spikepercentrecoveriesare must be within 30% of the spiked concentration. 14.5 Continuing CalibrationVerifications 14.5.1 Continuingcalibratiovnerificatiopnercentrecoveriesmust be 30% ofthe spiked concentration. 14.6 Ifcriterilaistedinthismethod performance sectionisn'tmet,maintenancemay be performed on the system and swnples reanalyzedor otheractionsas determinedby the analyst.Document allactionsinthe appropriatelogbook. 14.7 Ifdata areto be reportedwhen performance criterihaave notbeen met, thedatamust be footnotedon tablesand discussedin thetextof thereport. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample extractwaste and flammable solventisdisposedinhighBTU containersa,nd glass pipettewaste isdisposedin broken glasscontainerslocatedinthelaboratory. 16.0 REcoRDs 16.1 Each page generatedfora studymust have thefollowinginformationincludedeitherinthe header or hand writtenon thepage: study orprojectnumber, acquisitiomnethod, integratiomnethod, sample name, extractiondate,dilutionfactor(ifapplicable)a,nd analyst. 16.2 Printthetunepage,sample lista,nd acquisitiomnethod from MassLynx toincludeinthe appropriatestudyfolder.Copy thesepages and tapeintotheinstrumentrunlog. 16.3 Plotthe calibratiocnurveby linearregression,weighted I/x,thenprintthesegraphs and storein the study folder. 16.4 Printdataintegratiosnummary, integratiomnethod, and chromatograms,from MassLynx, and storeinthestudyfolder. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum ExtractUsing ES/MS Page 7 of 9 Page 58 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 16.5 Summarize data using suitablesoftware (Excel 5.0) and storein the study folder,see Attachment A for an example of a sun-Lmary spreadsheet. 16.6 Back up electronicdata to appropriatemedium. Record in studynotebook the filename and locationof backup electronicdata. 17.0 TABLES, DIAGRAms, FLOWCHARTS, AND VALIDATION DATA 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet. 18.0 REFERFNCES 18.1 FACT-M-4. compounds 1,"Extwtion of Potassium Perfluorooctanesulfonateor Other Fluorochemical from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro U triplequadrupole Systems" 18.3 The validationreportassociatedwith thismethod isETS-8-4.0 & 5.(@-V-1. 19.0 AFFECTED DocuMFNTS 19.1 ETS-8-4. 1, "Extraction of Potassium P erfluorooctanesulfonateor Other Fluorc>chernical Compounds from Serum forAnalysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 P.EVISIONS Revision Number. 1 Reason For Revision Section6.1.2 ClarificationofBP1100 system components. Section 11.1 Average of two curves, not standard values,are used for plottinglinearregressionand added the I/x weighting of the curve. Section 12.2.2.4Clarificationof solventratnp. Section 17.1 Changed from attachment B to A. Revision Date 04/02/99 3M Environmental Laboratory ETS-8-5.1 Analysisof Serum ExtractUsing ESIMS Page 8 of 9 Page 59 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Laboratory Study Study: Test Material: Matix/Final Solvent: Method/Revision: AnalyticalEquipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y InterceptDate of Extraction/Analyst: Date of Amalysis/Analyst: Group Dose Sample# Concentration up,/mL InitialVol. niL - Dilution Factor Final Cone. urtnil Slope: Taken from linearregressionequatiorl Group/Dose: Taken from thestudy folder. S2mple#: Taken from the study folder. Concentration (ug/niL): Taken from theMassLynx integratiosnummary. InitialVolume (niL): Taken from the studyfolder. DilutionFactor: Taken from thestudy folder. Final Conc. (ug/niL): Calculatedby dividingtheinitiavlolume from theconcentration Attachment A: Sununary Spreadsheet ETS-8-5.1 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory Page 9 of 9 Page 60 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U209S Appendix D: Data Summary Tables Table 12. Reported Fluorochomical Levels in Sera Analyses in Study FACT TOX-013 Dosage Group Specimen 10 PFOS WWML) 10097F 0.0394 10105F 0.0181 10106F 0.0258 10107F 0.0343 ioioaf 0.0253 9922M* 0.0115 9930M* 0.0134 9931 M 0.00725 rill 9932M 9933M* 0.0162 0.0156 10121F 9.82 10126F 19.8 10136F 5.96 10140F 6.27 10142F 13.1 9961m* 34.8 11 9964M 30.4 11 9965m* 74.9 11 9967M 25.1 11 9970M* 38.9 10155F* 87.8 10 156F 76.1 10164F 49.6 III 10172F 68.4 III 10177F 42.2 III 9997M 108 III 9999m, 178 III 10()OIM 94.9 III 10002M 113 III 10004M 130 IV 10187F rv 10194F 89.5 73.4 rv 10203F 126 IV 10211 F 99.7 IV i0214F 98.3 IV iooigm 302 rv 10024M* 477 rv 10029M* 296 rv 10033M 272 i,r@vIIrvVVvv 10034M 249 Tentativevalues,initivaolluffmw" <0.5 mL PFOSA 441knL) PFOSAA UotrnL) <LOQ (4.79ppb) <LOQ (4.79ppb) <LOQ (4.79ppb) <LOO (4.79ppb) <LOQ (4.79ppb)- <LOQ (4.79ppb) <LOQ (.4.7p9pb) <LOQ (4.79ppb) <LOQ (4.79pp@L <LOG (4.79ppb) 0.0682 0.112 0.0663 0.0507 0.0665 0.0962 0.188 0.114 0.147 0.165 0.328 0.352 0.265 0.325 0.335 0.574 0.579 0.480 0.393 0.465 0.461 0.576 0.651 0.670 0.569 0,613 0.553 0.610 1 0.804 0.637 <LOQ (20.5ppb) -CLOO (20.5ppb) <LOQ (20.5ppb) <LOO (.20.p5pb) <LOO (20.5ppb) <LOG (20.5ppb) cLOQ (20.5ppb) <LOQ (20.5ppb) -CLOO (20.5ppb) <LOQ (20.5ppb) 1.59 4.55 1.18 0.690 2.09 1.40 5.86 1.96 1.26 5.55 9.9 6.91 4.66 8.17 4.58 11.8 18.7 12.1 10.4 14.9 8.00 10.6 19.0 10.2 12.3 22.5 40.5 28.8 24.5 31.4 ETFOSE-OH (pWmL) U&N (pWmL) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (38.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb)_ <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <.LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) 1 <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (24.9ppb) <LOQ (24.9ppb) <LOQ (24.9ppb) <LOQ (24.9ppb) <LOQ (24.9ppb) -CLOQ (24.9ppb) <LOQ (24.9ppb) <LOQ (24.9ppb) <LOQ (24.9ppb) <LOQ (24.9ppb) I.W 4.19 0.952 1.15 2.45 4.69 5.18 4.54 3.44 6.11 43.3 22.3 18.0 17.0 17.9 29.1 1 73.6 25.1 38.1 37.8 39.0 25.6 39.6 28.8 33.8 71.3 102 94.9 90.9 1 56.7 3M Environmental Laboratory Page 61 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Table 12.Reported Fluorochemical Levels in Sera Analyses in Study FACT TOX-013 (continued) D---" Group Spednwn ID PFOS (pgiml) v NR NR v NR NR v NR NR v NR NR v NR NR v- 10042M 238 v 10044M 235 v iOO45M 326 loosim 162 v 10054M 182 NR - Swnpis notrecok-edormported Tent&We vok", lnitkviokww ww <0.5mL PFOSA (pWmL) NR NR NR NR NR 0.791 0.972 0.897 0.574 0.669 PFOSAA (pg/mL) NR NR NR NR NR 25.2 20.7 26.8 14.0 15.8 ETFOSE-OH "ftL) NR NR NR NR NR <LOQ (36.2ppb) cLOO (36.2ppb) <LOQ (36.2ppb) <LOQ (36.2ppb) <LOQ (36-2ppb) M556 (pWmL) NR NR NR NR NR 62.4 55.6 93.8 30 7 55.'5 Table 13.Reported Fluorochemical Levels InLiverAnalyses In Study FACT TOX-013 Do"" Group I I I I I I I I I I I 11 11 11 11 it 11 11 It 11 If I) it 11 Spocknon ID 10097F 10105F 10106F 10107F 10108F 9922M 9930M 9931M 9932M 9933M iOO97M 10105M 10106M 10107M iolo8m 10121F 10126F 10136F 10140F 10142F 996im 9964M 9965M 9967M 9970M 10121M 10126M 10,36M iOl4OM 10142M PFOS (pglg) 0.149 <LOQ 0.121 <LOQ <LOQ 0.585 0,816 o.a36 1.04 1.01 0.281 0.242 0.226 0.221 0.251 25.1 22.9 39.8 23.7 22.1 116 102 89.9 80.7 87.3 54.7 67.8 53.7 28.0 71.5 PFOSA(pWg) <LOG <LOQ <LOQ <LOQ <LOQ <LOQ cLOQ <LOQ <LOQ <LOQ <Loa <LO-Q <LOQ <LOG <LOQ 0.514 0.708 1.40 0.601 0.508 4.49 4.10 2.88 3.88 5.42 1.90 2.65 2.35 1.22 2.06 PFOSAA(pWg) <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <Loa -CLOO <LOQ 2.68 4.34 5.01 2.67 2.57 11.0 15.6 9.79 8.62 10.4 5.87 14.6 9." 2.82 6.55 mu b") -CLOO <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOG <LOQ <LOG <LOG <LOQ <LOQ 1.19 1.75 2.88 1.55 1.41 12.8 10.2 11.6 8.95 11.6 5.39 7.75 4.64 3.27 4.58 3M Environmental Laboratory Page 62 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Table 13.Reported FluorochemicalLevelsInLiverAnalyses InStudy FACT TOX-01 3 (continued) Do"" GroW III III III III III III III III III III III III III rv rv rv r-v rv rv IV IV rv IV IV [V rv rv IV v v v v -V Spacimen 10 10155F 10156F 101r>4F 10172F 1017'7F 9997M 9999M ioooim --10002M 10004M 10155M loi58m 10164M 10172M 10177M iOI87F 10194F 10203F 10211 F 10214F IGOISM ID024M 10029M 10033M 1D034M -10187M 10194M 10203M 102liM 10214M 10042M 10044M IOG45M 1005im 10054M PFOS (pg/g) 102 130 179 119 105 415 234 498 257 386 89.0 219 203 188 153 164 240 344 255 264 831 791 556 751 556 226 277 448 457 3" 1218 1356 1132 1063 1054 PFOSA(pWg) PFOSAA(pog) 2.22 2.24 1.94 2.17 2.88 10.8 9.41 8.67 8.29 8.41 5.11 6.14 6.26 6.20 6.91 2.80 4.06 3.14 3.39 4.56 12.8 11.0 11.2 12.6 11.0 6.36 9.96 9.93 11.4 8.11 16.4 13.0 10.9 9.80 io.8 15.6 20.2 22.7 11.9 20.0 73.5 28.6 85.2 34.2 64.9 12.0 27.3 29.4 --33.7 17.1 31.1 51.5 49.5 46.6 51.4 122 148 86.2 129 135 27.4 45.5 76.. 56.2 -60.0 lea 206 150 1 157 165 Us ojwg) 7.17 7.64 8.41 5.87 8.16 53.5 39.3 66.4 38.0 54.6 11.5 33.3 43.1 29.9 31.4 19.8 26.8 26.6 27.5 29.3 84.5 97.5 78.2 117 82.2 39.9 39.5 -@7.8 65.4 64w5 128 152 133 118 161 3M Environmental Laboratory Page 63 3M Medical Department Study: T6316.5 Appendix E: Data Spreadsheets Analytical Report: FACT TOX-013 LRN-U2095 3M Environmental Laboratory Page 64 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 3M Environmencal Laboratory Page 65 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 zz z 99 ;21 ZO Odd d @dd @d dd6d..dd.Zd d.dilad- 6,d 9 !22 -Lill, 3M Environmental Laboratory Page 66 3M Medical Departmenr- Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 r sz . .. .. . . . . . 0 ggq@g 3M Environmental Laboratory Page 67 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 zz zz zz iz Z. @l g@ zt a P 2 st 4 q ga P. ii3 33i I 3M Environmental Laboratory Page 68 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 z It .............. g 14 8 Sk $I Es 3M Environmental Laboratory Page 69 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix F:Example Calculations Formula Used for Sera Analyses in Study FACT TOX-Oi3 AR (ng/mL) x DF x SC x FV (mL) x 1.0 gg EV (ML) TUUO-ng- x PC = Reported Concentration (jig/mL) Calculation Used for Group 4, Animal ID 10033M 340 ng/mL x 500 x 0.9275 x I mL 0.5 mL x 1.0 jig TMU-ng- x 0.864 = 272 ILg/mL AR-Analytical resultfrom MassLynx summary DF-Dilution factor SC-PFOS saltcorrection constant (0.9275) FV-Final extractvolume (1.0 mL unless otherwise noted) EV-Volume of sera extracted PC-PFOS purity correction factor (86.4%) 3M Environmental Laboratory Page 70 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix G: Contract Lab Report This appendix includesthe followingcontractlaboratoryreport: BattelleMemorial InstitutSet,udy Number N003604-D, 2 (N-Ethylfiuorooctanesulfonamido)-ethanionlTwo Generation Rat Reproduction, (59 pages) 3M Environmental Laboratory Page 71 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BIOLOGICAL SANDIE ANALYSIS BatteUe Study Number: N003604-D 3M EnvironmentalLaboratoryStudy Number: FACT 060998.1 Balffeiie ... Putdng Twhnalogy To Kt,* FINAL REPORT 2 (N-Ethylfluorooctinesulfonamido)-ethainnol Two GenerationRat Reproduction SPONS 3M To)dcology Scrvice3 3M Center Building 220-2E-02 St Paul,UN 55144 StudyInitiatiomSeptember 15, 1999 Testins!Fie'ditv BatteUt 14emoriallustibge 505 Ying Avenue Call bilq.Ohio 43201-2693 Prepared PatrickL South,B.S. 3M Environmental Laboratory Page 72 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BatteUeSwAy Number N003604-D 3M F-nvkonmenW LaboratoryShidyN=bcr. FACR 060998.1 FINALREPORT 2 (N-'Ethyinuoroactinesulfonamido)@-ethiLnolin Two Genemtion Rat Reproduction Joa C. Andre, Ph.D. BattellPrmclpalhnwuge= RichardW. SlautcrP,h.D.,D-kB.T. BatteUeSemor Prog= Dn=tor Date Date 3M Environmental,.Laboratory Page 73 3M Medical Department Study: TG31G.5 Analytical Report: FACT TOX-013 LRN-U2Q95 3M EnNiro@ BattelleSb* I-T=bcr N003 604-D Labamtory Study Numbw. FACT 06o998.i 2 (N-Eth-ylfluorooctanesulfonmnido)-ethanol in Two Generation Rat Reproduction EXECUTMSUbD4ARY Rat liversamplw scat to BamUe by 3M Environm--ntal Technology and Senices vmm analyzed by ffie previously validatedmethod "Mzthod forAnalysis of Potassium Perfluorooctanesulfonate(PFOS) inRat Livw by LC/MS/MS". Samples we= cctwted and analyzed by I-ligh-Performanceliquid ChromatogWby Mass Spectroscopy (LC/MS/MS) for PFOS, M-556, PFOSAA, and PFOSA content only. Related fluorocbemic-A-.q mentioned in the analytical=thod were not investigated. The resultsfor the conc=t-adon determinationsinthe liversamples from thissb* uc attached as appendices to thisreport. Conc=bmfions are reported as rnit-gosf anabytr.(gg) per gram of liver6&sm waractc& 3M Environmental Laboratory Page 74 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 3M Eav:rO@ BatteUeSb* Number. N003604-D Lab"A)tY StIdY Number. ]FACT 060998.1 QUALrrY ASSURANCE STATEMMNT Ttis study was inspected by the QualiV Assurance Unit and reports were submitted to the task leader, study director,and associated management as follows: Phase Inspected Inspection Date Date Reported to BattelleTask Leader/ Battelle Management Date Reported to Offsite Study Director/ Management Sample weights Sample hamogerization Extraction Sample anabrsis 10/12/1999 10/12/1999 10/13/1999 10/13/1999 QuaUVAssurance Unit Date ]3atteUeMemorial Institute iv 3M Environmental Laboratory Page 75 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 3M Envio=enW BatteUtStudyNumber No03604-D LabaVory Sb* Number. FACT 060998.1 G4DOD IABOIRATORY PRACTICES CONTLIANCE STATENIENT StudyTitle: 2 (N-EthylfluorooctanesulfoniLmido)-ethiannTowlo GenerationRat Reproduction Thisstudywas conductedincompliancewiththe Food and Drug Adniir@st-atiotGeosod Laboratory PracticeRegulations(21 CFR 58),with theexceptionthatthemass spectrometrydataforthe liver sarnpleswas collectedand processed.with theMassLynx sofcwaresystem (version3.1),which was not funyvalidated.The studywas fistedon Battelle'Msaster I:isotf regulatedstudies. Jon C. Andre, Ph-D. Battelle Principal Inve3tigator Kris Hamen, PhM. Study Director v 3M Environmental.Laboratory Date Date Page 76 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 3M Envi@enW BattelleSb* Number. N003604-D LabmVOry StudyNumber. FACT 060998.1 Table of Contents Lage Executive summary .......................................................................................................m........................... QualiAtsysura= Statemen.t...................................................................i.v.............. ComplianceStatemenL....:....................................................................v............. Tableof Contents..........................................................................v.i............. 1.0 lutxkfucdon ........................................................................................I................ 2.o Rdcre= Substzwes.................................................................1............. 3.0 ReceiptofSamples.. ................................................................................1........................... 4.0 Analysis of Samples ...........................................................................1............... 4.1 Summary of Medwd .......................................................................1................ 4.2 lkjmdts...............................................................................................................2............... 4.2.1 QualityControl....*.......................................................................2................... 4.2.2 Sample Results ...............................................................3............... 5.0 Conclusions ...........................................................................................................3.............. 6.0 Aclmowledgements ...........................:.................................................4.............. 7.0 Specimen Storage and Record Archives .........................................................4............... Table I. Example of hmuu=t List of Tables Parameters UsOd tO Analyze Samples ....................................2............... Summary Appendix A (Results) Results forRat liverSiimple Analysis ..................................................A............ DailyAccepm= Appendix B (DailyAcceptance CriteriaSummaU) CriteriSaummary .....................................................B.-.1......... Appendix C (Sample Inventory List) Sample InventoryList...............................................................C.-.1......... Appendix D (Chiomatogram3) RepresentativeChrom,,tgrams .................................................................. Appendix E (Protocol,Amendments, and Deviations) Prot=L Am=dments, and Deviation.s...................................................E.-.1........ Appendix F (PFOS PurityReport) PFOS PurityReport .............................................................................. vi 3M Environmental Laboratory Page 77 3M medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 3M Envko=enW BatteUeStidy Number N0036o4-D Laboratmy Sbidy Number FACT 060998.1 1.0 Introduction This report presents a dmcription of the method used to analyze PFOS, M-556, PFOSAA, and PFOSA inratliversamples from 3M Study Number FACT 060998.1 (TOX-013) and the resultsfi-omUs analysis.See Appendix E for a copy of the study protoco4 amendnxnts, and protocol deviationreports (sigaed deviationreportswere not availablefor inclusionhem). 2.0 ReferenceSubstances The analyticalreferencesubstancw for thissti4 were supplied by 3NL The following lot==ber or b=king number designationsapply: PFOS (lot17 1),M-556 (TN-A-2203), PFOSAA (TN-A-1293) and PFOSA (L-15709). Note thatbased on information supplied to'BatteUefrom 3K PFOS has two equivalent names. The nami- appearing on the Matmial Safety Data Sheet and bottlelabelis potassium perfluaroalkyl sulfonate. The name more commonly used by 3M in analyticalmcdwds and correspondence ispotassium perfluorooctanesulfonate.The lattername willbe used in thisreporl See Appendix F for puritydata supplied by 3M to BatteUe. The referencesubstances were stored at room tempo-at=. The surrogate standard was 11-L1H,2K2H-Perfluorooctane sulfonicacid,lottrumber 59909, supplied by ICN. The surrogate standard was stored at room temperature. 3.0 ReceiptofSamples Samples were received frozen and intactat Battelle,from 3M Environmental Technology and Services,in one batch on October 6, 1999. Samples were generated by Argm Research under protocol mnnber 418-009. See Appendix C for a copy of the inventory list.The samples were storedat appmximately -20'C. 4.0 Analysis of Samples 4.1 Summary of Method Samplesw= analy@ by a previousvlaylidatemdethod(BattelslueidynumberN003604-A).The currentversionof themethod isattachedto thisreportin Appendix E. S amples we= analyzedby LC/MS/MS, and an example oftheinstrumentparametersislistediaTable 1.Note thatonlyPFOS, M-556, PFOSAA, and PFOSA (acdthesur-rogatew)ere quantitatedThe otherrelatefdlucrochemicalsa,lthc)tigh presentinthestocksolutionsw,ere not monitored. QuadraticTcg=sions weighted 1/xwere used to constructthecalibratiocnurves. 3M Environmental Laboratory Page 78 4e(i-ca-- :@enar@@,Tenr-Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BateUt StidyNumber N003604-D 3M Fnviro=mtal Labontory ShidyNumber FACT 060998.1 TiLble 1. Example of Instrument Parameters Used to Analyze Samples Aut(o-4aiiijiler T[PLC pumis,; IV[iL%S.p%Letriiiiieter Analvticalcciluitiii Nltktlllpelia-ic i.ompt)ncnt-i Gr.idientpr(orile Make: Gilson Make: Gilson Make: Maromass )Aodel:234 MO&13:305 and 306 Model: QuattroLC with Z-wroy sour--e Ycyswnc BetasU C 18, 5 gtn, 2 x 50 mm, Part No. 055-701-2 Component A. Ammonium Component 13:Ammonium a=ta*2mK:metht=L 60-40.v-.v aceute(2mK-rnethanol, 5:95,vv Ttwe. min 0 1 4.5 6 6.1 8.5 9 11 %B 0 0 100 100 100 100 0 0 Flaw. mIJmln 0.3 0.3 0.3 0.3 0.6 0.6 0.3 0.3 [njecti4in Vottillic Flo,kv column tellel) UPLC prt--,%ure &%IS I(Iurce Dt-%tilvaiiii-i-iit% Neljuliirer-.,it% S4iurce hlfvcli leiviii DL-utl%aiiiilneiiii) Cone V4141;t-_-e Conisitm cncr,-@ CibiliSitift-@A% 10 JIL LC column flow atswt splito 20 plhnin intotheNO Ambicia Approximately 840 psi atgradientsUd Elech-m@, Negative Ion Nitrogenat-575 llhr Nitmgen at-80 L/hr 1400C 2500C 70 V for S-S.PFOS 20 V forM-556, PFOSAA, PFOSA 40 eV Argor@ Bas ceU,at-2.5xl 0-3 mb l@luttiplier Rc%tituti4in Ions m4oniforeti 650 V 12.0for MSI -,10.0 for MS2 427>8 1 MRM tmL,6tionfor SS 499>99 NIRM bvnsitionforPFOS 556>78 NMM transition for M-556 584>1 69 NIRM bwlsitionforPFOSAA 498:>78 URM tansitionforPFOSA Total run lime II minutes Appnk%im;ltc SS: 4 min retenti4in time%: PFOS: 4.2 min M-556: 4.4 PEOSAA: 4.5 PFOSA: 5 2 --aboratory Page 79 3M Medical Department Study: T6316-S Analytical Report: FACT TOX-013 LRN-U2095 4.2 Results BattelleSbi4 Number. N003604-D 3M aviro=cntal LaboratoryStWy Number FACT 060998.1 4.2.1 QualityControl Sysu= suitabilitaycceptancecriteriwaere establisheduringthemehod vnliintionand arc included inAppendix C, SectionIX Acceptmee Criteria.Relevant statistifcrsom each sample set areprovided inAppendix B. Representativechromatograms aregiven inAppendix D. 4.2.2 Sample Results The resultsof the sample analysesaswell as a method detectionlimitdeterminationam presentedinAppendix A Allliversamples were initialelxytractedas undilutedhomogenates. After the data were reviewed,dilutionosf the homogenatcs were performed inorderto bring analyu concentrationswithinthe calibratiornange. The firstanalysis-thaptrovided acceptabledata foran analytcwas used inreporting Four extractionsetswere requiredtoprovide data for each analyte. The limitof qt=titationisdcfmod as theconcentrationof thelowest standardwhich mcets acceptance criterifaor accuracy(25% RE). The notationBLOQ denotes"Below Limit of Quantitatioe forsamples thathad concentrationslower than thetheoreticacloncentrationforthe 0.13 lxgtgcalibratiosntandard.The notationALOQ denotes"Above Limit of Quantitation!f'or samples thathad ccncentrafionshigherthan the theoreticacloncentrationfor the 13 pgtg calibration stnnii.c,L Samples dia were initiallAyLOQ were dilutedwith blank liverhomogenate and reextracted.Samples thatwere expected to be ALOQ were firstdilutedwith blank liverhomogenate bcforccxtraction.T@c"Corrccted PFOS Conc" presentedinthe resultstablesisthcconccnu-ation found forthedilutedsample multipliedby itsdilutionfactor(finalvolume + sample hamogenatp volume). The method detectionlimit(MDL) of PFOS was calculatedtobe 0.0173 ILglgfrom the analysisof 7 replicatpereparationsof 0.13 liglgcalibratiosntandard.The MDL was calculatedby multiplyingthe standarddeviationof the found concentrationsof the 7 reps by 3.*143;the Signal-toNoisc (S/N) ratiowas caIcuiatedby dividingthe mean found concentrationof the 7 reps by their standard deviation.The 'method of MDL determinationwas provided by theSponsor. 5.0 Conclusions AR analysesmet acceptancecriteriuanlessotherwisenotccl 3 3M Environmental Laboratory Page 80 3M Medical Department Study: T6316.S Analytical Report: FACT TOX-013 LRN-U209S BaftUe Sb34 Number. N003604-D 3M Envkonmental LaboratmySb3dyNumb=- FACT 060998.1 6.0 Acknowledgements Acknowledgement of principalcontributorsparticipatinigntheper:forma=c of d3isStt* atBattelle ispresentedin thefollowinglist. Participant Title Jon C. Andre, PILD. RichardW. SlauterP,h.D.,DA.B.T. PatrickL. South,B.S. Gerke IL van derZwaag, US. Bobby C.Braswell,B.A. Cynthia J.Ryan, B.S. -BattellPerincipalInvestigator SeniorProgram Director Mass spectroscopist Sample preparationchemist Qualityassuranceauditor Lead qualityassuran= auditor 7.0 Specimen Storage and Record Archives A.Uoriginaplq= data,aswellaselectroniccopiesofdata,willbe forwardedto3M forarchival. Copies of both thefinalreportand alloriginalpaper data generatedinconjunctionwith thisstudywillbe maintainedby BattelleA.U residualiversamples,extractsa,nd unused testarticlweillbe disposedof or returnedtothe Sponsor as directedby theSponsor. 4 3M Environmental Laboratory Page 81 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 3M Enviro@t8l BatteUe Study Nwnbw N003604-D LabOntOrY Study Nmnber FACT 060998 1 "FE"IX A -RESULTS 3M Environmental Laboratory Page 82 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BatteUeStudy Number-.N003604-D 3M F_aviro=cntalLaboratoryStudyNumber: FACT 06099S.1 "VS. M-MK PPO&AA. PPO" urrsui sluer. SPREADSHM SoFrA"lt DATA SWrniU LM PAT UVM% tiougo" MANUAUY IKXCEL 97 "Moe I 2 3 4 7 la 11 12 13 14 Is Is ,7 Is Is 21) 21 22 23 24 25 29 27 286 30 31 32 33 34 305 37 38 3,04 41 Q 43 IL5 40 47 40 44 so &1 52 83 54 5u, 37 ou an a2 43 kaom Number Samole Type om watwnim 1 om Waowniw 1 oem mawnid om am Mobf"Q 2 Oadi 2 ow Mauwnid 2 ow matemai 2 ow Ltmom" 2 90" MewrnW 3 900 kisurnw 3 om 3 ioooi wamrmg 3 10002 mabwnw 3 10004 maumm 4 lome Maurnal 4 10024 matwnw 4 10= Mabfnw 4 10= Ab&bN"W 4 10034 10042 wawrmi 10044 WA"ff*A 10043 imi Matwroi WAlbUnW 1OW4 111 10097 F@W 1 10105 FeW 1 10100 F@tW 1 1("07 F*tW 1 10104 Fmol 2 10135 Few 2 10140 F 2 10142 2 lCi2i Few 2 10128 FetW 3 10177 Few 3 loi55 F--' 3 IC"58 F*W 3 10154 F4W 3 10172 7 4 10197 F= A 10194 FWW 4 102M Few 4 IC211 FeW 1=4 FeW 41 low Me%' lk@A 1 lolc6 MT*W.W 1 10100 maw(rdw 1 ICHOT mamcnw 1 1()106 maurnei 2 10130 mah""Nd 2 10140 2 lQi42 k4latmal 2 11)121 ASbfrw 2 10126 mabcnal 3 10177 maumnw 3 10155 14-ftm-' 3 Matpmw 3 i10o1l554 a matemal 3 10172 mabmnw 4 10167 matmal 4 loi94 mautaw 4 IC203 maowffw 4 IC211 Matunal 4 1=4 MaturW "03 M-6" "OS" Pro" &77F-01 IL44E-0 9@ma-m I.ME6w 1.17E+(N) 1-349442 1.180"a 14464= 9.349+0 1.019+M 4JWC+= 2.?iR*= ILTSE@= 2A7*4-0 4A7%40 9.829+0 9.159+0 gA3e$42 SA4E+02 aA394a IAIE+= I.ffm-*= 1.319+0 1.239+03 I-TZE+= 1.72E41 5= 1.AOE.01 BLOC BLOQ 4.$iE+(" Z74E+01 2@M" 2-WE-t*l XS$E+01 IME+02 I.ISE+02. i.50E#42 2An+M 1.34E+C I.SGE+02 2.78E4= 3."E+02 2ME+02 3.0s&02 3.26E4" 2.WE-01 ISIE-CM 15ae4n ZOGE-M L21**-M 3.24LLOO 9.209H-0 L33L401 7 *Nt+Oi 77L4= ii.'03t+= 2-%3e',= 2.3aE+02 2-t@E-6-C 2.629+02 3--1E+02 3,t$ 3129e+n 3.$@ BLOQ W-00 BLOO 9= K= 1.2BE401 I=r;2Z I.leE,@M a-sectm I.laE-401 4-369-+01 &239+*1 L"E+M &WE401 L44EOOI SAZe#Qi L?59+m ?Jd" I.I?E+02 LzLeNn _ue+aa "-ftg+aa 1.33C+02 l.i8E4" .Siff+02 BLOO SLOO BLOC SLOO SLOO 2-aaE-@m I-M 1.41gtM I.IVE-W 1-75E!Z &16E+M 7,17E+M T.54E--M &41E--M S.BTE-W IME@01 2ME+41 2.668-001 2.?$E+01 2.93E+01 BLC)O BLOQ BLOO M= BLOQ 4.DAE-M 3.27E-M 4-5GE-M &39eow T.T5&0 3.148"1 1,15E,-Ol L33L40i 4.31 E491 2@"E-01 3.2SC-@01 3@46LL"l a.71E-01 L34L'+Ol LASE401 P" a. C4 BLOQ BLOQ B= 8= lm 00 1 106-(" I:a4*"l &79E4-(M Lactsm KDQ 4.4@E*W 4.IOE44M ZUM Lml= 1.04" ?-Im"i 2AU"t L32804M 3A2&N GAS" i.22ei-a IAOE"A &82" I.Ve,42 I.Maqa lien+= xwo+oa L4z!sR 1.0matal 5,4194M LMIM 12W;M& LAM i.2meo4n i-lopocn 1,12goOt J.UM 1.IOE*(n I.fdE"t 1,3ogo4n 1.34eK2 ij7E4*a 1.$aE*= al 8= BLOQ LOG :Lccl &01E@.W 207E-W 2JZE=M anctm 4.34E+W i@ooE-@m o.ace4m I.OW-*4n a= MCC 0= 1.4OEtW 0.("941 IOW.&T ILIAE.&T 7,QBE41 2.OOR*M I.SSE+01 M02L4*1 2--rZ+01 I.ISE--(M &11" 3.ISZ+" 4.28e+ot 4."" IS41+01 BLOO BLO(2 BLOO I 8= M= &446+W 2&2M 0 5i5E+W &rreow 1.4$ei-Oi 1.71" 1.20E-ol 2.73**4H 2.246+01 &3764@H 2.74" 4-ME+01 7.$Ce**l IL$21-6-01 &009+01 2.8@EOW ZZMIM 224E-tW 1,94C-@W 2inE!I& 2AQE*W 4.DOL4M 3.t4E4(M &ZOE44M IMM!M SLOO &= K= am 2-'494M laffm ZDW_+% liggo L2" &DIE.W LIIE-#M LL4M 6.20E-.(m SZM OL3aE*00 B.vw;!&m 9-gaE-W 1,1494()t &llr-ow BLC)Q BELOW LMr OF QUANWATMN Anoh'dx dab bW. Nwmw tat - Ocb*w 13.low Undwfbis- Ocb)bw 14,IM N@W - Ccbbw 14,ION BOM undemhm - Ocmbw m Ing AM samples AJIsanVW* Allsampw Allsampi" undiluted unduuted dBuW dRuW A-1 3M Environmental Laboratory Page 83 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BattelleStudy N=bcr- N003604-D 3M EnvirarumentalL'a*boratorSytudyNumber. FACT 060998.1 METHOD DETECTION LIMIT (MDL) STUDY: N003604-D ANALYSIS DATE AND INSTRUMENT DATA ENTERED: SPREADSHEET SOFTWARE: RESULTS ID: 130ct99; 9053 Electronir-allyand manually ExceIS7 All concs in pg/g Calculated Concentration of Replicate 1 Calculated Concenu-ation of Replicate 2 Calculated Conc=b-ation of Replicate3 CalculatedCon-ntrationofReplicate4 CalculatedConcenuafioa of Replicate5 CalculatedConcentrationofReplicate6 CalculatedConcentrationofR@plicate7 Mean Concentration StcLDev. Sp5ceLevel SIN ............ LOQ (detf.rom 10 x stddev.Onaise") LOQ (det.from calm=c low stcl) Curve Coeff ofDeterrninnfian Date analyzed Method 'Kev I - SpikeLeveltoohigh;SpikeLevel must be < lOxMDL 2 - SpflcLeeveltoolovr,SpikeLevelmust be > UDL 3 - SIN toolow-,SIN must be > 5 4 - CoeffofDet ofcahbrationcurveunacceptable M-556 0.1269 0.1220 0.1433 0.1368 0.1670 0.1225 0.1190 0.1339 0.0170 0.1331 7.88 0.17005 0.1331 0.9979 130ct99 LC/MS/NIS PFOSAA 0.1484 o.12al 0.14a4 0.1605 0.1681 0.1513 0.1385 0.1488 0.0128 PFOSA 0.1187 0.1325 0.1521 0.1441 0.1490 0.1382 0.1413 0.1391 0.0119 0.1334 11.66 0.1315 3 11.74 im: @4, 0.12763 0.1334 0.11853 0.1315 0.9937 0.9891 130ct99 LGNiS/MS 130ct99 LC/NISINIS A-2 3M Env4@ronmental Laboratory Page 84 3M medical Department Study: T6316.S Analytica'l Report: FACT TOX-013 LRN-U2095 BaftUe StudyNumber. N003604-D 3M Envio=CnW L&Ore=Y StudyNumber. FACT 060998.1 AIPIPF,NDIX IEI-DAILY ACCFPTANCIE CRITERIA SUMMARY 3M Environmental Laboratory Page 85 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 B alleUcSt* Nl=bw. N003 604-D 3M Eaviro=cntal LabOratOrY StudY Number. FACT 060998.1 PFOS ot &I IN RAT STUDY NUMBER: DATA ENTERED: SOFTWARE: LIVER N003604-D A&ANUALLY EXCEL97 REGRESS*N PARAMETBU APAWYWB OM ockaw 13,100 001*bw lk IGN ottobw 1 k Iwo Octi)bw20,ION PFW b&W PFOSM PFOSA PFOS M-M PFOSAA p@ PFOO LSMS PFOW PFOSA PFC)A PFC)SA )e C"ff -MOIS4 41DOW 001= .155 4).00389 &m4a OL -M CLOD= QLO= a 00= .71.9 ooom5 .153 x CO@ff- I." CLa27 oam 1.50E-+04 l.A2 0.575 O=i 1.73C-+04 2-17 0.312 O.Om 1--Z3E-" 2.07 1.65E+04 1.11 407n 4IM13 -aco3ag --M 4XOOM ACOW 400321 -M -MOW5 -mom= 400= rn oobcw-waanoalftn OA" CLM Orw omp 4 Cal pt I DAM O.M orw mg d Cal ptI CL90 txm CA" oow One rep of oW pt7 ackaded Oro repd cd pt5 ackw*d O.Sm OAS4 am O.We Orw at amipt7 ockmiw One rep d pts1, S.5 uckdsd One repatpto4.7mduded G.9a7 O.We 3M Environmental Laboratory Page 86 3M Medical Department Study-. T6316.S Analytical Report: FACT TOX-013 LRN-U2095 BatteUe Study Number N003604-D 3M Enyimxlmcntal Laboratory Study Number FACT060998.1 PFOS st al IN RAT STUDY NUMBER: DATA ENTERED: SOFTWARE: LIVER N003604-D MANUALLY EXCEL97 QC Resuft AnatysLsDate Ocbober 13,ION An&Mo PFOS M-5m PFOSM PFOSA Octobst 16,1989 PFOS ccb)bw ia,-1989 Octaw 20.1999 PFOSAA PFOSA PFCS M45M PFOSAA PFOSA PFOS PFOSA OC Levok no/mL la 3.3 0.7 0.18 10 33 0.7 0.16 10 &3 CL7 0.16 10 3.3 07 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.18 10 3.3 0.7 1 0.18 10 3.3 0.7 0.15 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.18 10 3.3 0.7 0.18 10 3.3 o.7 %RSD %RE 6.6 -4.0 3.3 .-4.6 7.9 -6.4 3.1 -12.2 4.7 -2.4 11.7 2.5 3.0 -8.8 11.5 -5.1 6.5 -1.7 5.1 -10.4 8.7 43.0 14.5 8.2 16.9 -4.3 2 -2.1 95.a 1.3 1 4.6 1 3.9 5.5 4.5 3.3 9.7 3.4 2.7 8.0 17.7 4.3 2.1 7.4 17.7 11.6 a.i 8.0 8.8 14.5 15.5 8.8 -2-7 -2.a 6.7 -3.7 4.5 -0.8 -2.9 -7.0 15.8 -3.9 5.4 -4.4 1 13.7 1 8.7 11.7 7.0 15.1 14.7 17.0 21.2 25.a 14.7 11.4 15.1 21.3 16.7 11.5 12.6 0.4 -4.5 -1.7 -20.3 -8.1 -2.9 23.4 11.0 15.4 0.8 -1.5 -20.6 -18.5 0.0 13.7 0.0 . 18.0-. 2.4 2.0 3.7 5.3 2.3 4.0 13 3.4 -8.0 -12.0 -21.7 -1&0 -1.6 6.5 1.9 i 11.5 B-2 3M Environmental Laboratory Page 87 3M.Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BattellSetidyNumber N003604-D 3M aviomnental LabomtoryStidyN=2bw. FACT 060998.1 "IFE"IX C-SE"LE UiW?ffORY LIST .3M Environmental Laboratory Page 88 3M Medical Department Studi: T6316.S Analytical Report: FACT TOX-013 LRN-U2095 BattelleStIdY NuMber. N003604-D 3M Environmentil Laboratory Study Number FACT 06099&., ShidyTOX-013.Ar-.LS41&.OCQ.SaffVWlnbm-agonfbrdtnwtb sample I 2 3 4 5 a 7 10 11 12_ 13 14 15 18 17 18 19 20 21_ 22 23 24 25--28 E=Z: 28 1 29 1 -30 31 32 33 34 35 38 37 38 39 40 41 42 43 44 45 4a 47 48 49 50 51 52 53 54 55 5a 57 58 59 60 a2 e3 84 85 Samote Descripdan 3wnple Type F@@-o=-grpi-m MatwnW RatLhw FC@-Mo-gMt-M FO-OW1.7pi-M mabomw Ratuvw Mabamal Rat Uyw FO-O= w MxtwTwd Rat Lkw PO,@w FO-N8l-grpi6M Mabwnal Rat Uvw MntmW RatLKW FO,@w -RstLhw F(@-99&K-opi6m FC-9W-grpiW Mabu. Rg Lhw MatwnW RatLNw FC- li-M Mawnel RatUvw FO-M7-Mill-M MoftrnidRstUvw Mawnid RatUvw FO-10001-CMIII-M Watoo. Rot Uvw FO-IOOC2-wpllW_ Watwnid Rot Uver FO-10004grpllw WatwnW RatUver FO-1001SIPPIV-M FO-IOC24-WpfV-M Moter@ RutLlvw Ma*mal RetLlvw FO-10029-gp(V-M MaUwrmi Rat Ltvw FO-10033-=IV-M wtonmi RatLtvw FO-10034.qprv4A MateffudRatLlvw FO-100642-MV-M MabwTtalRat Llvw FO-10044-qpV-M MaWnal RatLlvw FO-10045-grpV-M M&Wnal Rat Ltvw FO-10051-grpV-M Matemal RatUver FO-10054@V-IA Matwnal RatLhw FO.10097.qpl.F Fobd Uyw F-0-10105-ini-F F-staulv*r FO-10105-gpi-F FetalUvw PC-10107-7pi-F FetW L?@w FO-10108-7pi-FetWLNw FO-10138-grplW FeW LIYW FC-10140.7plw FetalLhw FO-10142-grpit-F F*tW Lhw FO-10121-7pit-F FetalLlvw FO-10125-grpli@- FeW Llvw FO-10IT7-Wpill.-F FetalL)vw FO-10155-qplll-F FetalLKw FO-10158.gTill-F FetalLlvw FO-10184-mil@-F FetW LIVW F(@.lOlT2-grpill-F FetalLIVW FC-10187-MiV-F IFOW FO-'olG4-j;rp(V-F Fetal PO-10203-grpN-F IFetalLtvw FO-10211-gmN-F IFetalLiyw FO-10214-=rV-r- FabiJUvw FO-10097-qpl-F Matwnel RatUver F,0.10105-grpw MatwnW Rat Lhw FO-10106-gvp;-F MatwnidM Llvw FO-10107-grp@-F FO-10105-MI-F Mabwrud Rat Uver matemai Ratuyor F4-10136-=II-F mabon1w Rat uvor F(@-10140-gipiw MabwW RatUvw FC-10142-grpl@-F MaWnal RatLKW FO-10121-grpil-F Matemal RatLNw F(3-1012"rpll-F Matwrwl RatLhw FO-10177-gmllw Mawnid RatLlvw FO-101&5.gplll-F: Lia*n%d RatLKW F@0-10156-9mill-F Matwrmd Rat UVW FC.-lol64-grpill-FfAstwnW RatLlvw 1 FO-10172-gypill-F MaWnal RatLfvw F-C@-10187-71)(V-F MatwW Rat FO-10194-grpN-F mawrnw Rat Llvw FC@-10203-grpN-F twruwRat Lhw 1 FO-102li-grpfv.F MFawnW RatUver 1 FC)-.10214-WpN-F Imauwnai Rat Uver C-1 3M Environmental Laboratory Page 89 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BatteUeStudYNumber N003604-D 3M EnviamnenW Labonimy Soj4 Number FACT 060998.1 APPENDIX D-REPRESENTATIW C]EIROMATOG'RAMS 3M Environmental Laboratory Page 90 3M. Medical Department Study: T6316.5 Analytical Report:-FACT TOX-013 LRN-U2095 BatteUe Study Number N003604-D 3M Enviro=eatal Laboratory Study Number. FACT 06099 8.1 Baftelle QLC 9053 ESI- leOctM Sm (Mn, lx2) 100- 3.44 43*473.47 3_W 3.5a a 100- Blank Uver - Surrogate 10 uL; N003604-ri - 4.43 SURRO0G0AT1E.19 SURROGATE 2: MRM 10:58:1381 OPerator. PPLL o@f@2 Chrhaan0tn0ore,rles 1ES- 4C2277..a0708>Ia81. 181 4..3=2 '3 4.64 4.5833@3.00 5 W5.93 5.1955..334 4@5@5@.64 5-IM5 ......... 420 r(5honneW ES499.00 > 99.00 1.7ge3 PFOS 1 ol IGC@ctbdtm n, 100- 4.44 5.28 i .. a5.07 i - - . .- @:'?AFkM'c;FfChannels ES558.00 > 78.00 317 3.81 01 lei56ti:@S6m'(M.n,. i@2@ ...... loo- PFOSAA 5.07 5.58 5.8a e.ie 3: M M of 3 Channels Ec-'- 584.00 > 169. 6.3 98 ni ie(@@4 100- s"m,(Mn, i)d@ 4.69 ............... &07 PFOSA 3.n 4.37 ...4.bb' 5.5.@77 nelsES498.00 > 78.00 433 0.211 nme D-1 3M Environmental Laboratory Page 91 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 13=eUe Stady Number. N003604-D 3M Environme'ntalLabomtory Shidy Numb= FACT 060998.1 OLC 9063 ESIIeOctO8 Sm 100- (Mn. l)t2) &94 Calibration Point 7-(0.13.ug/g) 10 uL; N003604-D SURROGATE 2: MRM 16-OctII: Operator. PLS of 2 Channels ES427.00 > 81.00 leoctoo Sm (Mn, ixz) 100- 4.20. PFOS 0 16C)@@ m n, 10 4.38 @@nel3 E-, 499.00 > 99. 1.08 i: l@AM j-3TChannWs ES556.00 > 78.00 3.gOe3 0-CM8 sm (m le 8 Sm (Mn,-l@@ ...... 100- PFOSAA ..M. -@56 4.54 16(5&b6 SM'(M 100- PFOSA 5.07 @f @,c@iail@,el3ES584.00 > 189.OC 71@ 0.15 5.55 6.01 i-.l@IW c@f@'d s EtS- 498.00 > 78.m 1.08 Tlme r-r D-2 3M Envirbnmental.Laboratory Page 92 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BattaUeStudY Number. N003604-D 3M ErLYwonimentalLabOrdtOrY StdY Number. FACT 060998.1 lie "O'LCa"O19053 ESI- l6Octl6 Sm (Mn, lx2) ioo- 3.95 QC 4 (0.15U 10 UL; -NO0361 SURROGATE 2:MRM le-oct-199 12:50: OPomtor. PL of2 Channels ES- 427.00 > al. O.Ge nl 1ed@t6it'M'(Mn, ixz) 100- ol 18 ....... 4.22 PFOS 4.42 M-556 100- 4.85 PFOSAA 3.83 -M,C'Mlnx.2) 5.08 PFOSA 3. -4.b-O' As.00 MIW W2"C@ha@nnals 'ES- 499.00 > 99.00 1.55o4 i: mAiA of @'dKa@ isESW6.00 > 78.00 4.97e3 i:MIW;i3Ch@annels.E@-I(X 584.00 > IS 31 .31 511..7711 3-: M M of 3 Channels ES498.00 > 78. 1.71 D-3 3M Environmental Laboratory Page 93 3M Medical Department Study: T6316.5 'Analytical Report: FACT TOX-013 LRN-U2095 BattelleStudy Number N003604-D 3M Environmental Laboratory Study Number. IFACT ()60998.1 Batelle QLC 9063 ESIl3Oct2O Sm 100- (Mn. Ix2) 3.94 Sample 3 10 uL; N003604-D SURROGATE 2: MRM 13-Oct-199 16:01- 'Operator. PL of 2 Channels ES- 427.00 > 81.0 3.O8e4 30ct2O Sm (Mn. ixz) 100- 4.22 PFOS .2: M of2 Channel3-ES499.00 > 99.OC a-0204 nI L 136cdd 8 -m'(M'n,i'x'2') 100- 4.44 M-556 i:'NIW -of-37C--h"an@nsisES-r%58.00> 78.OC 347 136c,@id 100- 3.83 ..1 'm (wIl'n, Ix2) PFOSAA 130 100- 'M n.-ik@2 3.88 4.8e3 PFOSA 5.05 5.27 55..5522 5.M94 6.15 @35 3: M ocff5C3@hCanhna@nenel3 ES564.00 > 189.OC .36 .583 6.14 3: MFW c@ ES- 498.00 > 78.00 875 5.41 5.77 71me D-4 3M Environmental Laboratory Page 94 3M Medical Department Study: T6316.S Analytical Report: FACT TOX-013 LEN-U2095 BattellSetudyNurnber-N003604-D 3M EnvironmentalLaboratoryStudyNumber: FACT 060998.1 Battelle QLC -4063 ESI- i4SOct56Sm (Mn,lx2) 100- 3.92 Sample 37 10 uL; N0036C4-D .i 3.39 18oct58.5m.(M,n, 00- SURROGATE 4.18 PFOS 2:MRM 20:30: OPOrator. PLS of2 ChanneLs ES. 427.00 > 81.OC 6.94e4 notser,469.00 > 99.OC 6.53ea 7 160ct56 m'imn,'@25 ....... 100- 4.40 % M-556 i:'Mik9of3Channel$ES558.00 > 78.00 i.Beas 160ct58Sm (Mn,1)0-) 4.81 PFOSAA Idti@@'nalEsS- 584.00 > 169.00 180Oc-t5La-S-m--r(Mn,-I--x'r2-). 100- 5.03 PFOSA i:'I@Ai;Ai3 ChannelsES498.00 > 78.OC 2.85ea (""@3. 3M Environmental Laboratory ....... D-5 TIMO Page 95 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BaneU SbldyNmbcr. N003604-D 3M Eavhmn=tol LaboratorySbidyNumber. FACT 060998.1 "PE"IX FPROTOCOI.,, AMENDNMNTS, AND DEVIATIONS 3M Environmental-Laboratory Page 96 3M Medical Department Study: T6316.S Analytical Report: FACT TOX-013 LRN-U2095 BattelleStudy Niumber.NO03604-D 3M Environmental Labomtmy Study Number. FACT 06099S.1 7 - 7-.Of- 3M ENviRoNmENTAL LABORATORY PROTOCOL - ANALrncAL STUDY 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction In-vivostudy reference number. Argus 418-009 Study number. FACR 060998.1 Test substance:2(N-Mylpanuorooc=wffoaamido)-Cd=Ol (N-ET<)SE-OH) Name and address of Sponsor: . Marvin case 3M ToxicologyServicc3 3M Center Bilileii2n2g0-2E-02 SL Paul,MN 55144 Nam and address.oftestinsfacmtr. 3M EnvironmrnW Technologyand Senices 935 Bush Avenue, BuUding 2-3E-09 SL Paul,MN 55106 Experimental startdate: Expected ierminationdate:December 31,1998 Method numbers and revisions: FACr-M-LO, Fxtwdon ofPaUmium PerfluorooctantsulfonoartOetherAnionic Surfactantfsrom liverforAnalysisUsing HPLC-Electrospray/Mass Spectrometry FACr-M-2.0, AnalysisofFluorochemicalsinIiverExtractUssing HPLCElec=spray/NlassSpectrometry FACr-MaO, Extrwdon ofPotassiumPerflucrooctanesulfonoartOetherAnionic Surfactantfsrom Scn= forAnalysisUsing MILC-ELec@@/Mam Spectrometry FACr-M-4.0, Analysisof FluorochemicalsinSerum Exbwft Using RPLCElectrosprayJMassSpectrometry Author.LisaClemea KzisHan= Study Direcwr r bam NiarAn Can DaW Sponsor Representadve 3M Environmental Laboratory Page 97 3M Medical Department Study: T6316.S Analytical Report: FACT TOX-013 LRN-U2095 BattelleStudy I;Umber N003604-D 3M Environmental Laboratmy Study Number-. FACT 060999.1 I,()PURPM Ilie anilyticalportionof thisdosing study isdesigned evaluatethelevelsof pertluorooc=e mxcnam (PFOS), or another metaboliteof 2(N-ethylperfluorooctanes&onamido)-etiumol(N. PYOSE-OH) desigaatedby the study dirt=r, in theliverof theparentand mbsequw generations of the testsysterm,or in the serum as necessary. 7be in lifeportionof thisstudy was conducted at Argus Research Laboratories 2.0 REGMATORY COM7LLkNCE This study isconducted in compliance with ft Food and Drug Admini@on Good IAboratory Practicesregulationas statedin 21 CYR 58. Any exceptionswillbe notedin thefinalrepmt 3.0 TEsTMATmaAts 3.1 Test, contro@ and reference substances and matxices 3.1.1 Analydcal reference substance: Potassium peffluorooctanesulfonat(eP.FOS), lot 217 3.1.2 Anadydcal reference substance matrix: Rat liverand serum 3.1.3 Analydad controlsubstance: Ncrne 3.lA Analytical controlsubstance matrix-. Rat liverand serum 3.2 Source of maw:iah 3.2.1 Analytical reference substance: 3M SpecialtyChemical Division;b2cesbUity informationwillbe included in the finalreport 3= Analydcal reference substance matrix: Argm Re3earch LAboraxnim*. traceabilitiynformationwillbe included in thefinalreport 3.2-3 Analy-deal controlmatru: 3.L3.1 .Rat liver- Argus Research Laboratories;bwubility informadon willbe included in the finalreport;or Rabbit liver- Covance Laboratories;U-xzabilityinformationwill be included in thefinalreport 3.M.2 Rat senun - Sigma Chemical Company-, bu=bility informationwM be included in the finalreport 3-3 Number of testand control samples. Liver samples fortestingw= receivedfrom 40 testanimalq and 10 controlanimals. Serum samples willbe testedatthe discretionof the Study Dire=r. 3.4 Identiftcationof testand control samples: ne samples areidentifiedusing theArgas Rrsearcb Laboratoriesidentifiersw,hich consist of a letterfollowed by theArps project number, the anitnnlnumber. the group designation,and thedraw date. 2 3M Environmental Laboratory Page'98 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BattellSetudyNumber. N003604-D 3M EnviromentalLabomtory StudyNumber: FACT 060998.1 3.5 Pmrity and strengthof MaOxials: Characterizationfthepurityand identitoyf the referencematmial istheresponsibiliotfytheSponsor. 3.6 Stabilitoyftestmaterw: chmwftrizadon ofthestabiliotfythetestmatmw isthe respon'mbilityoftheSponsor. 3.7 Storage conditionsfor testmaterials:Test materialasm stomd atroom temperatum Samples am stomd at-20 10 *r-. 3.9 Dispedtlon of testand/or controlsubstances'':Biologicatlissueasnd fluids'amretained per G12 mgdation. 33 Safetyprecautions:Refertothematerialsafetydatasheetsofchemicalsusedl Wear appropriatleaboratoraytdm and followadequateprecautionfsorhandlingbiological rnntezialasnd preparingsamplesforanalysis. 4.0 ExpmumENTAL - Overvievy TLwues from linirnjtldsosed as describedinArgus ResearchLaboratoriePsrotocol#4184M am receivedforanalysiosf fluorinecompounds. At thediscretioonfthe StudyDirectora. seriesof analyticatlestswillbe performed on selectissues. Initiallayl,lliversampleswillbe analyzedforPFOS by electrospray/masssper-rtometry OLSftAS).On thebasisoffindingsfrom theseanalyses,additionaslample matricesmay be evaluatedor othermetabolitemsay be targetraIfadditionaalnalysisisperforn=cla protocol amendment willbe written. 5.0 Emnumm%rrAL -AnalyticalMethods 5.1 FAC17-M-1.0, Extwdon of PotassiumPcxfluorooctanesulfonaotreOtherAaionic Surfactantfsrom LiverforAnalysisUsing BPLC-El==SPMYMW3 SPM=MCtry 5.2 FACr-M-2.0, AnalysisofFluorochemicalisnLiverExtractsUsingBIILCElectwpray/Mass Spectrometry 5-3 FACr-M-3.0, Exuwdou ofPotassiumPeffluorooctanesulfonoartOetherAnionic SurfactantfsrothSerum forAnalysisUsing BPLC-Mer-trospray/Mas4Spw=metry SA FACr-M-4.0, Analysisof FluorochemicalsinSe= Exh-actsUsing EPLCElect,ospray/MassSpectrometry 6.0 DATA &14ALYM 6.1 Data transformntionsand analysis:Data willbe reportedastheconcentration (weight/weighto)f fluoridpeer tissueor sample,or ofPFOS perunitof tissueorfluid. 6.2 Statisticalnalysis:Statistiucsed may hwlude regeseiorainalysisoftheserum concent-ationo3vertime,and standarddeviationscalculatedfortheconcentrationwsithin each dose group. Ifnecessary,simplestatistictaelstss,uch asStudent'sttest@may be appliedtoevaluatestatisticdailff=nce. 3 E-3 3M Environmental Laboratory Page 99 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 IjRN-U2095 BattelleStudy Number. N003 604-D 3M Enviro=ental LabcratOrY StudY Number. FACT 060998.1 7.0 KkDrrEl4ANCE OF RAW DATA A" RECORDS 7.1 Ile fohowing raw data and records willbe retainedinthe studyfolderin the archives accordingto AM=-S-8: 7AA Appmved protocoland an=dments 7.U Study correspondence 7A3 Shipping records 7.lA Raw data 7.LS Electronicc*es ofdata 7.2 Supporting rwxrds tobe retainedsttaratelyfrom the sti@dyfolderin the =chives accordingto AMM-S-8 willinclude atleastthefohowinir. 7.11 Training records 7= Cahl=don records 7.23 7.2A bst-m=t msint=ance logs Standard OperatingProcedures, Equip=ut Pro=&=, and Methods 71.5 Appropriatespwim=. ERENCES 8.1 3M Environmental IAboratory Quality System Chapters 1, 5 and 6 8.2 Other applicable3M Envhv=ental Laboratory QualitySystem Standard Operating Procedures 9.0 ArrACMKE@M 9.1 FACr-M-1.0, Extw-don Of POt333*U= Perfluorooctanesulfonmeor Otber Anionic Surfactant3from liverforAnalysis Using BPLC-ElectmspmyftAm SpectromW-y 9.2 FACr-M-2A, Analysisof Fluorochemicals inliver Exuacts Using IR"ICEectroqxmyn.lm spectrometry 93 FACr-M-3.0, Extw:tion of Potassium Perfluorooctancsulfonatoer Other Anionic Surfactant3from Senun forAnalysis Using I-IPLC-EectrosprayftAms Spc=mctry 9A FACr-M-4.0, Analysisof Fluorochemicals in Sm= Extwu Using BPLC- Eleamspray/Mass Spectro=try E-4 3M Environmental Laboratory 4 Page 100 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BattrllSctudYNumber N003604-D 3M EnviroxumentalLabor=U StudYNumber FACT 060998.1 BWMOD FOR pxRlrLuoRoocrANZSULFONATZ ANALYM OF POTASSIUM RPOS) IN RAT IIVZR NY LCMSM VerAmeLo Shay xw Vrim=by: PutickL Sac& kpC Andre6Ph-D. Z,5009r.@ 3M Environmental Laboratory pap I ofLd C-1 Page 101 3M Medical Department Study: T6316.S 3M Eaviro=cnW Analytical Report: FACT TOX-013 LRN-U209S BatteUeStildyN7xmberNO03604-D Laboratory Study Number. FACT 060998.1 M=MOP FC)R ANALYSIS OF lOTASSrUM P=nrOROOCrAXXSULPONATZ (.FPOS) IN RAT LIV3M BY Lcjbf.%W Ver3L= LS SbulYN&: 'L stnaL4xy S*nm oxadn* ftm "M =ckL Tasc&Ubradw smodwft on *-dhd vitbmm*m mndsri.buM=t4 md Ivft adoiacowl The orgvwcpbom m wap=md to&7=u md ru=nekwma hLmdhwa itx=Wy* by LCACUIDL -ra , =dacgb=potmsxi=pmem Sxad tASPMPWZ>XWI" ZU HVM ===Mm=wAnbftdtmomcbmicai=uV=mk nL SABU.LW tm r%A. ofC=uAy recank V IV. G=a3uL IKSTxucnom CaUbc= aU, balsom toft SCF m bsbm= -F )Ao= A T/bw &a VOW= --6A vada ft=adw vah@ soud IntM method LAbd &U modud uW m matutb= n spoCI2-1ba*2 appmpdm ". IfyM %*-A In Tum ILgoludw kc 2AL= %vim be mv to lobd f-4,@ theprqwadm diftmd 9* tr wbi& ibeso-d- hiddly, SISMan *0 aml pw cctis=mbod to-1 tWt ym bm &Dowcd &a mdfiadn I an -,-i-I jwdmgmm &To andan , *, -benbe= .opafy c&HbtatoLMy= devim ftm ft mcdkpc do-at tb4-N-ct md obtLbL tke zpymvd of ft a* andy dtmct-.CC U* U-A- n Swo as V=xMk Yw.i =d due &a daz -,I- am to pap oaLviblcthheywm nnd& Ifanly@ puma au dam = a shide don uloybe me& Ina AnsW W=dm an Page- If-itip6 mo make ewxiak the-L'Fk4-1 -m@t- muK be bidatedmd daudby the PC== -king dw -07. Lbwmxm orNA dabom -t;mAypHcowe. 0 Tba zwlw Isw In c ttoMalys-* It3;; 1 9 suxkzmm toneedtw The- dganowgkal mtwbot do atopmmWb4k&%Kod =am do atftlbrcmft ac&Mw InwocMW afew soft-- =named cc-I- oabft is conzkiend=Wed e I;oconzcdm wal be -- Ix pwiv ar slitcmumt cfmY UK adclobtiPtPOS" - Use &lawvwumcaic.Eppe-kof vW-t-. - P-hil-41som@ pipcttshrdw=xiag zcdnno0c swodem & CANtact whk Tcom b7 dw text -rdd showd be V. NIATZRLUA SeeTjbleI *r In mqwred cbcad=ls.rccgcnm and 2ohectLUn TabltI lbrdacmnc=bm Cho& &a Ilbel=srt6l4 to-,re dw aU --- bt,an om exphedandthotW m tbsFqW pwdly va& Pap 2 43fid c@2 E-6 3M Environmental-Laboratory Page 102 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-IZ2095 BattelleStudyNumber N003604-D 3M Environmental Laboratory Study Number. FACT 060998.1 bf=OD IPOR ANALYSIS OF POTASSIUM PEP.7L'UOROOCrANNSULFONATZ (PYOS) IN RAT L"ZR Vftdm LO staty BY IriMSM TAW L 3[akrbis t .@t lip PodkornactmeI;T,IH pmdbwcaoctc= Suhgxwf6 Acid m-sm PFCSAA PPOSA PFOSF-A Ru Liva A cot= Sandad S=ropm sft.,6.d Auaydcw Stmdard i AMEIydcd Stmdard AnLbtkaL SbwAitrd A-4ikal SundiLrd A-4*:ML I St=d=d M&bi)C I 16@ Pbase 3m ict4 3m 3m 3M 3m 3m Ekd= Spagoo- ROM Tamp Rmm T-P Rom T-M Room To= Tamp Tamp Tmo ---2(rC RT Sodtiz- Ey&=odc6 R-gcu Pm Nam 3-T TwzbuqW==oni= E==PRV RT CnL*4 SoW= C*bm=M E.%== P"p wftcoj ILT Iticwbc=- F@@ Pftp MaHc% IT Etoi Amum Exta= PrV KT mm-q wow Bvfiw pEE LO P-tff- S-+* ws R-gcm PtcN I Wtobflo'Phan PH @, PH uwtw czhbmdcm A&Mip- AMU TM I XT ILT meam Pa= T=pazmm VL iquwmcrr Sm Tsbk 2 ibran requirem-odjarpie= of cquhmccl U@oft iabletodo=meut tteacOW gim (4L&zakoundcl)afcquipmci&CLharkcaM=WcmctalLoquipm@rcquirb2g-wk-g-(44@ bW==) taemsum itIsc=mnl P&P 3Ciifd C-3 E-7 3M'Environmental Laboratory Page 103 3M Medical Department Study: T6316.5 Analytical Report:' FACT TOX-013 LRN-U2095 BatteUe StudY Numbw. N003 604-D 3M Environmental Laboratory Stuty Number. FACT 060998.1 BCMOD FOR PIWLUOROOCRANESULFONATZ ANALYM OF POTASSIUM (PMS) IN RAT LXY= VU36M LO Stad7lqo4 BY LCJBMM B&bM= we4ft sm Ptpc= 2ipcm Ptpo=. T"2. W.IgSb=,d.* w R-m-a Bahw= PtpetS-W@ks ca=-400 PipctR=g=zM WIMS laX @qgmpl- Rgpcuw LIYW &off. 3t=W en i hcmc Sd E=ad q.ffml Bim TnnspcctWb= mlxnicdc OOAW shakcr Swm QCs S& E=aa SL=qim E=.= ED=y ZYM-K isML pdypmvikska. LS ML 12x7l=* 5 mL SWIS" 20% 2002 127-Tl6O-W T."Lv I GrindUver Vabonowe pa@ A vehudeldcP4m clan A Tmnsft pipcm plutic B%"K1m1rhr Ma" V0o.ln,.."i. Dih - mm vqmnlc= DUudow Trmmft Exbun ccnttfi, 4A MA MA 3M Environmental Laboratory Pap 4of16 C-4 E-8 Page 104 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BaMUe StudY Nu3nber N003604-D 3M E'n"viro=entalLaboratm7 Sbuly Nu3nben FACT 060998.1 M IHOD'FOR p=n-VoRoocrAMSULPONAT3C ANALYSIS Of PMASSMM (PYM IN RAT LJVKR 13YLCJMIM Vcr3im Le vm P.IOC=X= A. rmu-xdon of2 =M Ammahm Aceftto twxk.I)t=WvethesdW in-m md dha Novdom wt& Sotmdm mq be wed IL C. Acmd of AcoW And vah=c Datecepmpumd= su* NO: acoft= prpwti., f-2-9% Sdinui H7droxide Shdion Weigh 2m * 2 g of socgmmhydra@i&bwo abeskw.AM = mL ofUMI-Q W" md mix todinahv. Cod =d U=z&c toa poblxWACM bouk tw IW=p SabldM mq be snumdft 6 -t- atrou- Acmd n@ of sodtarnb7dr=dde Actmi volum MM-Q -am No. preparadon of-"*A Sodlu= U7droxide Soludon AM la =L of-29% Sod= Fvdraxi& sab*i- to a lwul va=xwc An.- tovclume whh Mn-Q vaul Tn=dw to a potypnvyine b*We Saudon amy be =red tr 6 mondo itxmm U=q=wms. nuk nd gm mmo@L Aam, vowm of-29% NaM A@l Jlnd vobm= Dam of pxpm=d= sohniam D. ?Mamtion afTetr2butykm=onhun solution0..5K (PIEi[(i) Rligw r.Hy-,Joe Hydrogenulfxft CLSA) pg buffer 7 yrbmdcr 10 PH r@rnnr. yR -odbg AAd 169 a I S afTBA m -5W inL EUM-Q vuw bi a bed= A4= to pE to IILCO a 0.02uzbms-51-L-MML aflg% so&= By@ saw= om lalow MLVA Mn-q water,and ulbr-Acquatft pH to 10.00 a O.M usbma -2-9% l@b= md nbL T=e= to a potyprwykas bocdo Rw - hr =6 nmoft mnd atro= tmcpanlmo, but the nE mak be to sub m kdjmlo pH 10.0 2 0.02wft 2.9% Sadin- Hydnod& Saiv#6- n 11 pat@ 5 of id 3M Environmental Laboratory C-5 E-9 Page 105 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013' LRN-U2095 BattaUe Study Number. N003604-D 3M Envirorlmenml LabOratory Study Number. FACT 060998.1 B=OD FOR PUnIJOROOCTANESULYONA7Z ANALYSIS OF POTASSRUM VFOS)J:N RAT UVIR VWAM LO shmo IrYLCNVMM Acmd num of7s&. Acowl BAd voh=C A=md Ami pm Dim ofpvpaad= stav No: 3L Preparation ofO-U M Carbonaft Buffer welo 26.3* (Ligofmflx= -tmm md 2LO a 0.1goracdhn Wo&=m md n wdnnmclooo-mLvoh=mctdctLa&Dt=dftdn=dWabkUMK mium tovolumewithIMII-Qvam. W@ and- toa pobVW@ bmdo fw swnp Sol,-Icmnay b-cumd fbrI --,h wbes Mhpmt4 AcuW mm ofso&,- --bons= A=a' @ ofsodum bimrbouatc A--d gn&Lvohmc atprepamdow Sb* No-. F. rnpmmt[on of M*bU* Phase Cauqcm=t A.-Wx uVAbw 600imLct2=M smmmninm md 400=L at =06-@al Sdudon=my bewed be Immtb@it --ad atzciownov-3nm Acmd volume of2 =M wcuw -mL A=aivab=aofnieduL=L_,=L Daw ofpzpazdow Sb* No: C=paDcot B: lgx wgctbor50 mL of2 mM amnwidum ace= md 950 OL Of zwtbawl Soludm way beund farI m=th v atMGM bmupaxbm Acbmivolumeof2 mM -n= mL A=mlwhn=Ofme&anaL -nL Dan ofprqmxd= Sb* NO: G. pmn=tion orstockSurrozab Standard and Warldng Surropft Standard (WSS) 1. SkickS=optA SumdardCMOOO Pohdl. Wei& 25 2 2 mg ofIM M 2K 2M-yagcorooctzn add od vumfa toa loo-OLvmwmctrk tL*. Dumt" In vab@ WLth md niblSton reft!gaamd.;-, t= T.TV Acmd Velgbt: AcftW Dffiuka DateM-PTCPUzdm stD4 NO: pav 6 or16 .3M Environmental Laboratory C-6 E-10 Page 106 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX@013 LRN-U2095 BatteUe StudY Numimr. N003604-D 3M EnvirotumentalLaboratory Study Number. FACT 060999.1 MM720D FOR PERM'COROOCTANESUWOKATE ANALYSIS OF POTASSMM (PFOS) IN RAT LMR Vali= La SeedyNG.: BY LCJBMM IEL 5mck IA smck IB St=k 2A -9@7B $to* 3A Sbxk 3B -Sbxk 4A sbxk 4R Suxk SA, sbxk SB 2. wss (1000avlmlr. DO= loowl ofawk =mgft soodod m 25mL widlma&@l mdzdx_ A=-' Vab=o at3bxkbnc=l Sumbrct: ACUW Doulka Dam ofpmpandw Preparition of CaUbradon Sah-entStwJo aid Working Standuds L Saltonswckc I%roub amaym vcio &a spacm ona@m ofoam--d Oodepeadadi@r vicigheadsA gadB rWOcLta) UstodInTable3 andu=sfcrI= opum voh=etdcl4--u-Dinchv Inmcdmol. dlium$avab=c withxmW=i2t.md mixwel Stcmzcbig@y l@p-P,4*=UVHSbL 2. NflxedSolveotSwd= ptpa theSpedfl-I n%@= ofCU* =LfydoLl@.4@ vqgt- A a lbod b Table3 =d --%- bm a singlevolumcuicZak. Dtnolw inutetad dho tovah=c whh medmal ad mix vmEL Sam reng ' k pectocudtm='UV U&bL Rep= d3apmc= withRgplkaftB nockLVm ; aohwejbdownxx" t*prvwe dw w*Hdmg*wadardl Dam ofpxcpmztt= su* No: 3. Waddug Smndxtds(WS): Dilum the awki avAwarldngsundzrdswith=cdlmal a vp=MW bL Table 3 and =ix weEL Dou afpvcps=dc= cc M" PMSsm M-556 M170 Y-M PFOSAA PFCSAA PFOSA FPCISA FFGSF-K Table:L@CaftradouSaivedSmdamdW=kbegSwzdxrds f.it @--cl %4:111.11.%Ili,.tsell %al.11I[ILAI 1.11 L.Cc t:iil.llV..i I ilt.,tN ..1. .1111.) 50*imff 1 25 2 0-5 MS 16 1 ME on2m... 10 125 2 0.5m 10 1 -502 1 mff wattwwl 10 1 2.5t 0.5MR .10 1 93*lvmg 46 * 0.3Mg So 2 1 MR -:F5t 0-5 ME 50 1 MR 4 2-4MOM 2,vo.ow 5@004,000 IAMOOO 3@.OOMO. 2,'MOOD Pap 7 af 16 C-7 3M Environmental Laboratory Page'107 3M Medical Department Stiidy: T6316.5 Analytical Report: FACT TOX-013 LP-N-U2095 BaMlle StlidyNumber. N003604-D 3M Environmental Laboratory Study Number. FACT 060998.1 FOR PEknUOROOCrANMS=ONATZ ANALYSIS OF POTASSMM (PFOS) IN RAT LTYIER BY LCS@ Vftvim Lo SbbdyNo.: -Sbxk 6B Sbzk A 14med SUDCK3 va I W32 WS 3 -WS 4 W53 W36 -w3 7 PFC97A 1 25 2 0.5MR stoftI thm iRmA i wL each Swxks I dim 3 1INLL 43LWB aschoo b&md Sto& A 34med -S&Xk IMLOO k m@ @ vis 1 WS2 2 ML1 2 ML-- WS3 WS4 WS5 uktim rm =Ibljtl@@m 10 50=mtu=m=n 21 is 10 0 weighau=Iiy@ -A-.dsioalkakthneeamsta.m0&1 UseVOIUZMCCJC ]@M= 300.000 2000 1000 200 1 pmimtion of CaUbration Standards nd BULnks 1. Lhcr hamov=ato Prep- hi-t@Umb=cgcnm tabwk by v4tigYmgapproxbmndj4OgaE blaukUmt=a5WmLNAIB=bOGU=-Ir,'- 2DOmLafMB-Qmm r,r+mtD&bo=gcnemsmisp=Wm ANquatinmir 3GmLyardmstw p (IPP- A-' Mus m'LIvw.A=nd vohl=ofwstcr I)am ofPrep: Stodr. r RM.UCM Det=ninadcmsitoyfcalib@ matcbc mm RomoaDtATz TEmotiGHLY md --=bu the bm=Mtm= of10mvacaw wci&ngx ofI=L pmtow oftbaTMWUGMYM= MM ROMOGENATE D&AMLCMY PRIOR TO larm AIZQUOT RMYOVAT- TableL CAinwationStdi/QC-MMrix D-sky 2. Lt4a C4LU=ti= SaLudards prep= eachDvermmmatim M&rd b? akitnia.45mL ofmanud Uyw bcuiog== (STIRFAohiC)GENATE W13= ALI(ZUOIM4C;)bw a 13 =L cc=acti4mtibesod addtzl5g0 pL OEVIS 4=MoM P..sL- "4pB=@ clt v=du-ds md 6 bl=kl SeeTabW 3 fm wb=cl Tm An,- Am dank In -nm=cdtobeappm;d=xWTL50-04MT- Mlxv*M pw a elf16 C4 3M Environmental Laboratory Page 108 3M Medical Department Study: T6316.5 Analytical Report': FACT TOX-013 LRN-U2095 BatteUe Study Number N003604-D 3M Enviro=ental LaboratoryStudy Number FACT 060999.1 BWMOD FOR ANALYSM OF POTAWRUM pxRyLuoRoocrANESULFONAT:t (.PFOS)IN RAT LXVXR BY LCIM&M Versim LO sbaft C.It TableS. C$Mradon Sbadards a@d Btmb !-i.itice 'FArl-'CtVf)l Actig.ItVt@l rsr.-,Ct .%ttitit %,Iglliit.lt '%..I.l.l..,l L) FiliAlVt.[ I ws I so 2 W32 3 WS3 4 W34 50 3 WS5- 50 6 vis6 so 7 W37 50 BLmmk lAcOR 50 0.5 0.5 0.5 0.5 0.5 R ---Oj 0.5 DOIC of ympacadon ofCalnd&tlaz@c 2m -f3 1000 GA -400 2A 200 L3 too OAd 30 om 20 -0.13- J. PrtpamtiozL of QuaHty ControlIlver SiLmples(QCs) 1. Qmaft C=nml Working Stamiards Du= do lba0wingxri= Vol,- methanolInvahnnevictwdm md iodk well Prepambt&bwh= used.A=nl Yoh== we bapwmftm 7 rrvii wsi wts7WS2 WS 3 2. T Naxed StockA Mbmd StockB ws vvm aulu LS,000 QQCC ZSS I2 ICBM,, ?mt=adm atqaallqCzutrolLhwSu*la P.@pw eachQC Inbulkby I"Itngdinvohunct& Zaakappr=dnsm*baW= withm)autcd UverbamoAL.IQUOMNGL CSTIR EXXVSDGENATE VIE= rim QC VM vnhany #ad All-tit,avdum with=ddutedhwb--gco (TMEEMOCENATRWI= AL.IQLIOTIN(3@bccx THOROUCMY =4 A 2--mL Akpft boo yalypv"l=c tibaandam K qTM4=wzly -2M Tahk 7.QC Prtp=lica . ......... -QC WS 2 QC WS 2 tmmvr-zs 3 --&CW@wS 3 4 WS4 0.16 1)= ofQC PMV. Ympamtion ofMS C2ieckStandard forSystrm Saftabi3ity Ptpct250OALofWS 2 at-10,00n0&lsnalnd2.5=L ciWfSS at-low ashulbt is= thesma5o-mL volumemic'4---uDouto tovolume withMACH nd nLf3L Page 9 of16 3M Environmental Laboratory C-9 E-13 Page*io 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BatteUe Study Nl=bcr. N003604-D 3M F-nvirc)runentLaalboratory study Number. FACT 060998.1 M=OD YOR FEMUOROOCTANMULFONATZ A:NALYM 07 POTASSMX (PFOS) IN RAT LrM Versim LO Stutylqo.: BY LCMSOM I@ prepa=tlon ofhomagemkw retaywy fter umpks TOA"-M tbor=mytmm thebo=gcnizmdmpmmx. unkamogeoindunkuvw vM beZ=tUU4 isdcp"- at3 =ncmwdm levelmsd bomoged=W n 20awL Mft m ba do= av=7 daydm houwgcml=dm ofAU* nzzv- Ispedb=W L ?um VP odmobely0.5S of sahm am bm ewh GE6. is =L t-wyi cmuif= tub= RcmdwdouafUm. 2. Add IW @E.-iWS L.3, d 4(. WS p- tub=)Ispmpm trdaclftm atWprwd=L" 4. 0-3.-d 0.4Wg. 3. N02idpotlwmm atUverInS by 2.3and sk tW mxq =L afvjum 4. Ho pni= ewh Ih-e-r=pi- ad hamoV-1-pmbewitk=Ctbw vot== a( wum usedInxmp 3.addfngA'=-w bonopot=d anpIL CL= bawatcuimwwithMcCE bct =WW- Cap andvam= hamoscma tw md In-c=acd=L bL Prcps=tion of D*auttonCheck Sample L Pin= 235 mL of-ffh-A lycrho-ogm MMECMI)aM4A7ZV=Z AIIQtXnM40) buo a IS mL acuacdm U" zod a& 50 laLofmxod SW* A. 2. Da= 50 PL ofsup I Sol-rt,-(VCR= SoLurmN VIE= AUQUOTING) -with0.45 mL ofi--4fl-neldim bmwg=- CM ROMCGEXXTE WFM.E ALIQUOTING) bi3. 15 =L w=acdca - 3. Ti" lashmilbdcp,-; Ifbr--Acdonady m dapwhm zm* nnrffln wM bedllt==dd ==Wtcd. N. nomotenization ofstudy samples L Pb= apprazimLWy 0.5g afwahanotembied mdy sumpleUmbm a 15 =L hiblR=wdwdou ofltvm 2. )AutdplytbcMR= ofUvw Ing by'7-5=d aM dds n&W mL ofme= 3. Eomagcmi=eachUvcr--pi- wddnml,--g= pmb*wi&mo&w valmm ofvm= uscdtmxtop2.addtmgtinu M UMV=bccd M-04 4. Cnes,bomtemimw withMcOH bdv= soomi- Cap&-Avac=bomge"s tw=sbmexuacdm 0. AnAtyAs StLndards, 13bmk% QCs. and Sa=lples 1. MM LrnM BOMOCENATES THOROUGMY ZMK)IM ALV4UOTM md *a 500;,Lct-ch QC (4r*lc=s p- kwel)-@d oth-s-om b&q c=x=d li=Li-mLpcl)r@@ w=cdm "- The cala& md bh@ am ah-4 allqwtod. 2. To tbABUW= - IS (3fcpsladd100.uLatMeM imd vas 3. To theBlaWn + IS(3 rcps)md tother=Linfnssum- add 100.ULVM =4 vcr=r. 4. Add Q.5mL of0.5M TSA (pFl10) laalltoba md va bddly. S. Add imL ofo.25U -bom- bi md vw bdeft. 6. Add 2.5=L ofahyl accmm Pi= thetnb= Adcwqs an U -thd *Abw ata wmbmgof300ft-20,-;- 7. c=m&V mbcxata af3300rpmtw-20 -t-- tomcpwm BCVCL L Transfe2rmL ofdLetop ' S 1 119-m a A.- pab7mi=4 obi6 pw 10of 16 3M Environmental Laboratory C-10 E- 14 Page 110 3M Medical Department Stud@,,:T6316.5 Analytical Report-.FACT TOX-013 LRN-U2095 BattelleStudy Number: N003604-D 3M Environmental Laboratory Study Number FACT 06099'8.1 METHOD FOR PEItFLUOROOCrAN7SUIXONATIC ANALYSIS OF POTASSMM (PFOS) IN RAT LWM Versim Le StudyNw An&17@ - BY LCMSM 9. Evaparce to&jwm nmder mkmgm ata xvbg af3C'C lbr-M 10. R==gk= 00 Mdd=$ In 300 JALOf MgftrAg WkhVMVAd2& ii. srin@ cmam Iwo vbb ft =WydL Stomvidsnftfauumd (uptoIm=&) tfLCftASINwM nab@;, m-' Idw@&W.Mom3.der Teo=wnqwracucmm= vmsAcmanozomddmb2gvaudffdc&ma. exuz= ofthecd Ak bunksn.d QC&miWbermmd tw iiipo3 fteobw th@@@ pmpmadenIfbddatram ucmemmn *ocxpdwvl*Uffnod&06 Dam cocfaludfalmk wmm= prcr. DIP ofQC -ft= pm: P. LC/MSJM Analysb &LW *FJP$4% 1. Tise-threyn= condiacmspecifitendT".WMW cmdwam an desiva-dmLy be-.dfad byu -RIYU topro&- SC-gah" Pa* AWL TiLb;IA - LCIBMM Ccmdhtms ModcL_- U% Mai= _ ModcL- makr. laodel- M: BccLsilCIS,SA 2 x 30 mzc6?=Nix 055-70 C-MMDoncaLA: Ammocii= acetuu=m:du=L 60:40,rv v-v Time, min .0 2sa Flow,UIJ=tn 0 a.3 a 4.5 100 0.3 6 100 0.3 6.1 100 (L6 iu 100 CL6 0 0.3 0 0.3 10 UL LC colu= Bow splhto*30 PLJmin t ;dWn) bm dw NO atranmft -Ambient 1000yd atip@ mn on) E!o@. Necad"Ion *Nlroltactn575ljbr OMImLcn at30 L/hr L4w) 0140*C -250-C -M v ( 020vt V) Ppcgi.-M-(D 14)PFOSA,PPOSAA, PFOSEA, M-dK W70 040eV L-tv) Ppcs. PPOSAA. I&S-94U.M 300v( o4 PFOSEA An= at@L5 x 10'mb tu ceB .6m v V) inb) 112-0 *r Iwo1 010.0&run( I 4DSMDL4b Aob Al fXr/Zr/9? Pme 11of16 C-1 1 3M Env;-ronmental Laboratory E-15 Page 111 3M Medical Depart@ment Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BatteUe Study Number N003604-D 3M Envirommental Laboratory Study Number. ]FACT 060998.1 NlrMOD FOR ANALYM OF POTASSRUM FEP.MUOROOCRANESULFONATZ (PYOS) IN RAT LRVZR BY LCINMW VersiesLO ar.-Si g 499>" I&M manddomft PFM siom lam -it[.-ftI&M S?M.16U9RM -W- lbrU= SW169WUb=ddantrPPOSAA 498>73IfitM"--M- tc PFCDA loig:-1M6W9 --4Hco fbrPFOSEA =in) PFOS.4.3o3Lm - 41 mim UM. 4.6n%in PFOSAA: 4.1misl PPOSA: 3.1=bm C YFOSE.4- 5.7mtfn lam) uda) Min) mw Mb) win) oPammct= tbk=aybe rb=pdby the=sbst A-lyahmala(l 2. The abo"=mdid= *ouldbeambabkftdiom=== QUIMOLCCW 91053)N.loditcadonsrmq be nc=cwy If Ukr=xm Qmnm Sad" Is=c& SpllthsIcrwpowcobum viaaraystom =04m w devim 3. CAL%rjbmthe=Lu arvm* thatheaLHbrstiIms=imbtobyvLm3ahlmewan (cm&a ton&pW) to a xiitrble nobn-pbanIm IsstM detw-lowRombdoa oW wArAto behisbc&rm thaund lbrandyzin-anvl- 4. 'ro- the7opcrpwtwmmm ofthe Wea do bub@ diak smndard.Tbo resmhzshcuw be ==guxblo loa -b&cdca Ifwnffab?AL S. Use zLn M-md cbromwngmpby 4nowattomaftwon qia= iocoom da oatptigtm d@eaulysil & --Il-gOrditSrm thelcsd qm ftm dieummaw chmnwoxmpby batpidansaftwonriu=L 7. Make dn& Weedo- ofmck ed mandardQ.r- a* -mWm orw-k lhks atk= 4 injccd= ofthebmuum= cbeck L R= sa slz=showd typ&cLIlmyA cwmd 80 lujacdo=dw to%*"omat cs IOUCaeouSielpr@i@m T@ n=Uuqbor A I bmtib@Wpm a riskafyieldLu=Sac=pudao cum VILL CALCULANONS L Sp=dsbee Saftwim Vend= 2. )M AndysisSmbmm vec3diamn 3. C-k:ol- the xvcmp d-lily Of ft Uvctbc-ogcuu (10 reps) b2 -mm@MT 4. Udng&ezycmpdcui7aftoba r-=kulmksdmdmsdq (-g ofUm pa mL ofduubedhmcv=mlo): UmdUntedlhw dcm&y (=S'mL) (9ofUm Iwi-p dcns4afl--secaM>U at Hvrx+ & ciwf=) wh= SofUvcrandgofmam am w&ua =cd top.*- bak U-IfteaNK 6=udryofwi= Is----A tobe Is@ML Dtlutodbyadwmty(mg(mL)-UndMzmdd-dq*DihFm= Pap 12of16 C-12 3M Environmental Laboratory Page 112 .3M Medical IDepartment Study: T6316.S Analytical Report: FACT TOX-013 LRN-U2095 BatteUeStudyNumber N003604-D 3M F-nvironmentaLlabontmy StudyNumber. FACT 060999.1 BWMOD FOR ANALYSIS OF POTA&MM plp_vLUOROoCTANZSULFONATZ (PPOS)IN 3tATLrM Verdan Ll BY LAC/MS/M Wbem dUs '%- - 21LI- Li 111bDac==tfbr 10% dBa ofUvor incdxoimd QC -im ofcaromdmsaodw& md QCsbyufng*A man ofmob md iftdm onlycnohadmsiv cm=d-@ 6. Cakzaafttba-1 cmcmwzdan ofPPOS md a&w ancrachamkak biDvw 2c &o --ds md QC& n taowlc C==WS) - Cc= @m&@ DOoodLhw &=skyOnv@) x10M 6ft x le pgbg 7. Ammm dw thek-vzd- of the peak um cttha Wa -nide=d .1 Fkg@ibnczmtf= wb=pcr&r-A sundanl =Mud m L r.,.-6tmmgmui=eqmd=niadngthapmk- g adoCm Wdd6m Ofauk =I%Csd= =Ddud O-Vds)toun -dda coccotnaimInam tx@) ft PPM 1"56. MM, andPFOSA.A---rd toma x@@ cmwca&adm ImLHverbrPPMA imdPFOSZA. PPOS.MSSCN070.&M PFOSAA we g-g-@ by=Lugibem-pa a-d-d Worad amuhrt PFC)SAwm PM.RPA at umndnted@widxmrtehwmm todidmm=m (aftod SMM&rd oilrgdcnCWWI. U3@ & quarxdc.qp wdzhft2d/zo,d& -do" hr an xujyt=6 9. r.L,.t.adcm=lincadnin@ foreachiniwdcof-1r-,3tiomundud.QC.anduxingdwrqrtuionpmwnm=-dtbsp-kmxpammtimarar=L 10. 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N003604-D 3M F-nv;,ronmentLaalboratoryStudyNumber FACT 060998.1 NM-MOD FOR ANALYSN OF POTASSRUM pIRMUORoocrANFSULPONATZ (FFOS)Ui PAT IZV3CRBY LCIMM Versim Lo Stady Nag AndrdDoc TW& 9.C-Hh--4;,S-undwd A=cpumm lwla PFOS PPOSA PPOSEA 1 20 CIS% &TLCOM 20r25% stLCCn 20 CIRA - LAM 2f)rli*A mrTIt-h lnmq%L@Tren 194otevIL- T rvlm UplaSediradonsundtrdWjad nqboccdndodftmltwcx pravl&ddwaoe kjgcdmrw=insywlcvcLR=wvalafLa, I kvdmqbod@IfWpmvalisabu&wA ]rn -.4mjowllm.umv dn - bradamed%ythet-i-in caubratlm=mpvgbe O-r or C. Qcs Ina===auei=Acvd r-rar orttaiivo-,aatndp,----trebati-msundsrd dayhdon oftLbe*QC-S#---[UAwa*o &aowlng Hnd= PF@7CT" 10.QC 0 20 0 556 20 MS,70 20 SSMF PPOSAA 20 PF SA 20 PFOOSS'EJSI 25 R=O,.g ofbxuvidul-lmk= ftm &c QC bw be dam If&=MvoldA bY a Leccpl-@tkn(z4,b23trame@E-tMbnelinnorDL=WA Q aK rooft). Ifthewymp detcrmhkedconcczbad= forou QC levelcmeadz-tm NMI& 68 tut t-@4- or=w# dim= dwaid'so=)tLaoL Tba r= mri be mpoud or ILPulks Ctdw T= may be cocsw=cd ==publil For=m3ple, If20 low QC &09 dw stftd maybemceptedtbabx" -"-'a= bmduftd bytheULa@ anda wid4avciW cwc=awiccl D. 'Homagenjur3tecoveqand DEntionCheck Ss=ples TM av=p rt=mwyacmm&o 3 kvtbof I C=Vcj amp," 22iMa M *AtCt tbsdoud= Cho* nmpl= sbmll bn witbln&a of70-130%toc@ VA=VD Of =wbodomtfw=mpniodbyanawmbie I.;CwwmioAl-OQS) TmmrxWod U=iuofquan&o@d=ut nominall0y.13;Wg out ibrpFos.)A-M UM md PFO&AA. PW 14ofli C-14 3M'Environmental Laboratory Page 114 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BatteUc Study Number. N003 604-D 3M Eaviro=ental Laboratc)rSytady Number. FACT 060998.1 ba-MOD FOR A,@@YSIS OF POTASSIUM PZPJL170ROOcrANISULPONATZ (PFOS) IN ]tAT LTYM VM-A= Le BY LcjbtwZ A-1yodDam ParPFWA andPYOSB-&%to v&UdMd LOQI - =MBMDY OM PWZ =dL DW ID20 offt ofdw -Ebatim smodudi6In- cwcu=oi= afPPC" md ppoWAra=dnuodwcamcdoL TbeaWymoonowwe=vwnnvMbs wmbntcdvi*cachrmackoduMbeh-h-windw C ' tfdM=ma=*u= crbcdlWtoymobchidcd3to#nml vAicbmq==UmdwUmcmomnxd= bd" tMvWWoodWvoi (wabanyOM Wg),wMbe app 4 &am& ir. srwuscfty -rbgXgdod MXM= b= -A biadkeam atsankamood@ 20% cfLOQ. Tm 11 1 ofthe='Dead-cum xppcwxit**Mtn= c==:dm lbr=V m q=udud- A=WtWba px*- (cam)ofdo Wwca wed mmdard.*waft% vm be=ida-m -@fRe6-ct/id=m tubmdmed st=V am qwuuffledkopuly G, GCU221 Mnabu"acccpu= tnm-duudlismotbodisca* afr I I =aakmd O=n mcd& em Dan=, a=opu= cdbwk ofa vatidee-dbM aM oo=mgy).Whom repucm =ablm kn= ft how- otdmn oc=si=W acdkm hardwo ofspreadsheeotrme Me annetworkddvl )M COMMir?r3 3M Environmental Laboratory ftr 15ofils C-15 E-19 Page 115 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BatteUe StudY-N=ber. 14003604-D 3M Envim=cnt3l LabOratOrY StLIdyNumber. ]FACT 060998.1 ?"MOD FOR ATULYM OlrPOTA-%SMM nM@,'UOROOCrANISUIJOZU'n qM'-'D Df R&T UV= ]BYW,?-MM XCL CoNcLmareto Ted-f-I Review QC R&Vicw Dam Dam I)atc 3M Environmental Laboratory pw idof16 C-16 E-20 Page 116 3M Medical Department Study: T6316.5 Analytical Re'p.ort':FACT TOX-013 LRN-U2095 BattelleStudy Number. 14003604-D 3M Eaviromnental Laboratory Study Number. 'FACR 060998.1 combi=d Stridy Tldo orai (Gavage) FertilitDyevelopment and Pciaaul/PostmaW Reprod=don To@dcity Study ofN-EtFOSE in Rob PROTOCOL AMENDMENT NO. i. Amendment Date: July28, 1999 Pwfonning Labornfory 3M Environmental Tecbnology & SafetyScm'ces 3M Environmental Laboratory 935 Bush Av=ua St PmiL UN 55106 Laborstory.Project ldent67cadon ET&SS FACr-TOX-013 LIRN XJ2095 3Ai Eff&wmontiii Labwatory 3M Environmental Laboratory B-21 Page 117 3M Medi.cal Departmen-t Study: T6316.S 3M Envwomment2Ll Analytical Report: FACT TOX-013 LRN-U2095 BatteUeStudyNumber N003604-D Laborecry Sta4 Number FACT o6o998.1 ProtocolFACT-TOX-013 Amen&rmnt I This amendment modifies the following portion(s)of the protocol: 1. PROTOCOL pzADs: Tbc proposedstudycompledon dateisUsW as 12/31/99. AmEmD To READ: The proposedstudy compledon datais6130/00. REAsom.- nic proposedcompletiondatewas changed toallowtime foranalyzing&U matricesof biteresl Amendmeiit Approval Mwvin Cue Ph.D.,SponsorRepra=talive Kris J.Hansen PILD.,StudyDirector DaW. c 2-/dl Date 3A4Ewkwvn&-M Lab"fofy 3M Environmental Laboratory F-,22 Page 118 3M Medical Department Study: T6316.5 Analytical Report:. FACT TOX-013 -LRN-U2095 BattcUc StLIdyNumber. N003604-D 3M F-nviromnentalLabomu)ry stidy Number FAcro6o998.t C=bined Study TWO Oral (Gavage) F=tUity Dmlopment and Pabatd/Pmbidd P,cproductionTo@dcity StLidyofN-EtFOSE in Rib PROTOCOL AMENDMENT NO. 2 Amendment Dafe: September 10, 1999 Performing Laboratory 3M Enviromn=tal Tacbnology & SafetySavices 3M ExLvim=ental LaborAtOrY 935 Bush Av-, St PauL MN 55106 Laboratory project ldentffcadon ET&SS FACT-TOX-013 LIRNU2095 30W Envkww*ntW Lebomfoly 3M Environmental Laboratory B-23 Page 119 3M Medical Department Study: T6316.5 Analytical Re@ort@: FACT TOX-013 LRN-U2095 BattaUe Study Number. N003604-D 3M Eavirdmnental Laboratory Study Numb er. FACT 06099g.1 Pn7tOcd FACT-TOX-013 Amendment 2 This amendment modifles the following portion(s) of the p.-aboc 1: 1. PROTOCOL REA= The protocolsl-am thatliverwillbe =twted Enviro=ental Labor=r)r.. and anabrmd atthe 3M AmEmD To READ: The liverspecimens willbe ccu=tcd and =a47xd blifitute5,05 King Avenue, Columbus, Ohio 43201-2693. atBaMUe Memorial REASON., The liverspecimens will be sent to Battelk Memorial lnsd=e for axtacdon and analysisdue to time constraintsia the 3M Enviro=ental Laboratmy. 2. PROTOCOL READS: The protocol statesthat swm following methods: specimens willbe cctwted and analy-@AA FACr-M-3.0, "Extwdon of Potassiu= Perfluarooctanesulfonat@o-r Other Anionic Surfwtanb fiom Sermn forAnalysis Using BPLC-Electrospmy/Mass Spectrometry" FACT-M4.0, "Analysisof Fluorochemicalsin Scn= Ext=U Using I]PLC- Elec"raymm Spectrometry" AMEND TO READ: The serum specunenswillbe odractedand analyzedfollowingmethods: ETS-84.1,"Extr=tionofPotasai=PcrfluoroocUnesulfanateor0thcrFluorochemical Compounds from Serum forAnalysis Using HPLC-Electrospray Maw Specftmetry" ETS+5.1,."Analysis of P otassium PerfluorooctanesWfonate or Other Fhwrochemical Compounds in Serum Ext -actsI-IPLC-Mectvspray Mass Spect=etry" REASON.- The extuction and analyticalmethods FACT-M-3.0 and FACT-M-4.0, respectively,were updated on 04/27/99 to ETS-&4.1 and ETS-8-5.1. 3M Envhwmentat Laboratory 3M Environmental*Laboratory E-24 Pa@e 120 3M Medical Department Study: T6316.S 3M Envimmn=tal -Analytical Report: FACT TOX-013 LRN-U2095 BancUe Sbl4 Number. N003604-D Laboratory SttidyNumber. FACT 060998.1 PrOtOcci FACT-TOX-013 Amendmo#7t 2 3. PRoTocoL READS: 7'w protocol stnt thatliverspecimens willbe =twaed followingmedu)ds: and analyzed FACr-bf-1.0, -Extwdcn of Potassium Perflucrooctm=ulforiateor Other Anionic sur&ctwu from Liver foranabisisUsing EPLC-Electroq)ray/Ma3 SpWftMCtCy" FACT-M-2.0, -Analysis ofFiiuorocbemicalsinLiverExt=ts Using IIPLC- Elec"r&YMM SpectromeW Amex To RRAD: no liverqxcimens wiU be cd=ted and analyzedfouowing method.- M@"&@ fbrAnab,3is of Pcriluorwctane Suffonaw (PFOS) in Rat liverby LCftA&?A% Version 1.0 REAsoN.* Since the liver=tracdon and analysiswas mib-m@@ to Bada Mmodal Insttute,tta3=cndinent was wntten to includethgarlivermethods and titIaL Amendmen* t Approval A ma;v@a taw ,Spon=pcprwmtadve I)ke Y,d3ten J.Hansgm Ph.D-, Study Director 3M Env*wmr9w Labmfty 3M Environmental Laboratory E-25 Data Page 121 3M medical Department Study: T6316-S Analytical Report: FACT TOX-013 LRN-U2095 Baft@e StudY N=2bw. N003 604-D 3M ]EavironmentalLabomtory study Number. FAcr o6o998.1 Shidy Tide AnalyticalSbidy 2(N-Ethylpcrfimrooctsaegulfo do)-et@l in Two C7cneratRiaotRnepmduction PROTOCOL AMENDMENT NO. 3 Amendment Date., October 4. 1999 Pwfbnnlng Laboratory 3M Environmental Tecbnology & SafetySwAca 3M Enviro==IW LaliomtcrY 935 Bush Avenue SL PRUL IAN 55106 Laboratory Project ldendffcatfan ET&SS FACr-TOX-013 LIRN TnO9S 3M Environmental Laboi@atory F-,-26 Page 122 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-TJ2095 ]3aMcIleStudy N=ber N0036o4.r) 3M F-nvim=cntal Laboratory Study Number. ]FACT 060998.1 PMtOcOl FAC7- TOX-013 Amendment Number 3 Thl3 amendment modiflas the following portion(s) of tho protocol: 1. PRorocoL READS: Kristen 1.H=wn, PhJ). isttacStudy Director. AMEND To READ: 12m K Lundberg, Pb-D. isthe Study Director. RE"ON.* Original study design has changed due to irv&ilabt'loiftyresources and 1-mes K. Lun&ag win begin serving as the study directorfor'FACr-TOX-013 as of 4 October 1999. I PROTbcoLR.E4m: Secti7o.n1st2tthatthefollowrianwgdataandrecorwdislblercudneidnthestudfyolder in the arrbivesaccording to ANM-S-8: Approved protocoland mnendmaras; study correspondence; shipping records;raw data,and electroniccopies of datl Additionally, Section 7.2 sww thatsupporting recordsto be retainedseparatelyftom the xtudy fold= in the arrbivw according to AUDT-S-8 willinclude at leastthe following: Trainingrecords; cah@on records;instrumentm2fiA=ance lop; Standard Opwating Procedures. Equipmcmt PmcedurM and Medw&; and appropriatespecimens. AMF-ND To READ: Section 7 states:"Me originaldata,or copies th=.wf, wiU be availableatthe 3M Enviro=ental Laboratory to facilitataeuditsof the study during itsProgress and before acceptance of the finalrcporl Whca the firmlreportiscompleted, alloriging paper dati4 including:appi ved protocol and mnemti- study correspondence, dappmg rc=ds. raw data,approved finalreport,and clectmrliccopies of data willbe retainedin the arbives of the 3M EnvironmenW Laboratory. All COMBPOnding trainingrecords,canr@don records, instn=cnt maintenance lop, standardoperatingprocedures,"Luipm=t pmeedmm. and methods willbe retainedin the uchivcs of fficfacilitpyerfo=iug each =abrgs- REASON: To directsubcontractlaboratorieisn the dispositionof the items Usted above. W Envimnimntal LabaWery 3M Environmental Laboratory E-27 Page .123 3M Medical Denartment Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 B=elle Study Number. N003604-D 3M EnvirorumentalL-aboratoryStudy Number. FACT 060998.1 PmtOccl FACT TOX-013 AR*ndment NUmber 3 3. PRorocoL READS.Dispositionof testand controlsubstances:Biological6wues and fluidsam mtgned per GLp regumom A mEmD To RuD.- Specimens wiU be mainbined inthe3M Envuom=LW Laboratoryqmmmm amhrft& An sentto mib-wntract laboratoriewsillbe ratumed tothe 3M r-nmpntal ratoryupon compmon of maalys[sand mibmi&sionofthesub-c@ Wmntory(s) finalreportThe spec=cns willbe returnedwith thefouowmg d- -cmt-Hon- &egvaed of4inalchainof custodyand recordsof swnp conditionwsinleatthesub-cauftsa6=Mty. REASCW.* To defineindetailtheqtr%nixte Evosition of ipwhumd-naly7@ laborztorie& atmibc@ Amendment Approval Marv Casa, D.Vl,,L,PILD., Sponsor Rq=scntative J---lY'-L-=A=& Ph-D.Stutf@r Y,ris@ 1.Hamen, Ph.D., Previous Study Director Date &ct Date Date Dale L Bacon, PbA4, 3M Enviro=crlal LaboratoryManagement 3A4&wtwawnfal Laboratcxy 3M Environmental Laboratory E-29 Dite Page 124 3M Medical Department Study: T6316.5 Analytical Report: FACT'TOX-013 LRN-U-2095 BattelleStudy Number. N003 604-D 3M F-nviro=ental Laboratory Study Number. FACT 060998.1 DEVIATION REPORT Batti-TIStudy Number. N003604-D 3M Eaviro=cntal Technology and ScMce3 Study Number. 1 2 (N-Ethylp@oroocftnesulfonamido@@l Two Gcnemtion Rat P-eprodu@;don FACR in 060998.1 TYPE OF DEVIA71ON&- PROTOCOL DA77ES OF DEVIAMONS: OCUAW is,1999 NARJRE OF DEMAIIONS: Some of theacaydcsl awthod -cc tume critctdwaere wt nut fordo LCJMSMS analysisconductedIgOctW atTt-tteUe7.1am daymem rel&W toprotoool-amenimmt 2. See below for omnmary. PPOS M-556 M-556 M-556 M-SS6 PFOSAA PFOSAA QC3 exceeded 20 % error(-20.3% acomal) OC2 exceeded20% error(23.4% wft4 gg exceeded20% R&D 1.2% wbnb QC4 exceeded 20% RSD M.8% =map Diludm recoveryexceeded 130% (131.5% actmd with21.6% RM) 20 % error(-20.6% actad) QC4 exczoded 20 % RSD (21.3% aebud CAUSE op DEviAmoNs: Sample proparationand/orLCIMS/MS variabiyitiocveer the cuum of the axople *A may have contzi@ to the devi-ti- Do(PACr OF DEVIAMONS ON THE M"UDY: Mm arrantQC valueswere bracketedby acceptrble QC conecubadon levelswhich dc=nsb2im thatthecalibratiocnurvesgenerallyprovidedgood aomn%L7 over thekatod range.The dilutiornecoveryforM-556 was not consideredtobe --adin* bi&bl enough, atonlyapproximately1.5% above thenormal acceptancelevel,tohave sipifi-zdy imp-tad.-thedatl. CORRECTM ACnON:Mds protocoldeviationmmmm=7 was prepared forlndusionb:L&o flnalreporl APMOVED BY: Jon C. Andm Ph.D. Das BanoUs PrincipalInvestigator Kristem1. Hansen. Ph.D. Study Director Data. N003604-D ProtocolDeviation 1, Page 1 of 1 3M Environmental Laboratory F@-29 Page 125 3M Medical Department-Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BaUtUe StudyNumber. No03604-D 3M F-nvirorunentaLlabOmtMy Stidy Number FACT 060998.1 DEM'NON ]REPORT B2tWl StudyNumber- N003604-D 3M Enviro=cnW Technology and ServicesStudyNumber. FACR 060998.1 2 (N-Ethylperfluorooctanemffonamido)-@iln Two GenerationRat lzcpzoducdm riPE OP DEVIAMONS: PRCrrOCOL DATE OF DEVIAMON&- Odober 20, 1999 NA7UU OF DEVIA71ONS: PFOS QC3 exceeded d3o29% RE madiod totdrammt (sewd -@21.7%). 7u daudon recoverycheck standarddidnot meet the70-lM%. rewverf reqabvmmt (-ti-1,5.0% for PFOS and 4.6% fbrPFOSA). 7bom method &vM=s relatbtoamendment 2 of theibidyptab@wl CA'USE OF DEVIAMONS: Sample preputdcm =Wor LCJMSMS variabdidmam dw courseofdw sample ad mq haw ccntn'butedfo &a QC deviation.Sample preparationw.c. appearsto have be= the cum fbrthecmuticn. veryreodtl DdPACr OF DEVIA'NONS ON THE SI'UDY: Mm =ant QC vdm levelwasbmaAMd by a=VW" QC coacmbzdon laveb which demnerates thatthecahlmdion curv= generallypmvkied good &=nwy forsuidyn=plcs overthetestedmass. A comparisonof theresultosbwned fbrthedilutexdu3dynmplas bm 2DOet99andpre%iamresulb thatweresheittyALOQ (130ct9gimd 190et99)demonstrategdoodagrownentbetwom do 2 deu=dnadow. I'hiswould indicattehatthedilutioonfthesbuly==ples was performedwmwdy 200et99 so dmL zw inqmt an the quantitadons occurred. CORRECMVE ACMON-7bin protocodleviatiomnu=&ry -u preparedforInclusioinndw fmalraporl APPROVED BY: Jon C- Andz% Ph.D. Dam Ba-li,,PrincipIanlvewptor Kri*m J.Hmam, Ph.D. StudyDirector Des L N003604-D ProtocolDeviation2,Page I of I E-30 3M Environmental Laboratory Page 126 3M Medical Department Study: T6316.S Analytical Report: FACT TOX-013 LRN-U2095 BaMlle Stuty Number. N003604-D 3M Enviro=ental LabcratOrYStutY Number. FACT 060998.1 DEVIATIK)N REPORT BattelleStudy Number N003604-D 3M Eaviroumeatal Technology and ServicesStudy Number FACR 060998.1 2 (N-Ethy4=fluoxooctan@onamido)-@01 In Two Generation Rat Ileproduction TYPE- OF DEVIATIONS: PROTOCOL DATES OF DEVIATIONS: October 12, 1999 through October 20,1999 NATURE OP DEVIATIONS: 7be lotnumber of PFOS used was not 217 as per section3.1.1 of the protocol 7rbe source of mfere= substm= matrix was not Argus Remmh Laboratoriesas specifiedin section3.2.2 of the protocol Tnitiaalwlyse3 of liverdid not exclusivelytargetPFOS as per section4.0 of protocol. CAUSE OF DEVIATIONS: Only PFOS lotnumber 171 was availableatBatWI TTari-qnwas the suppu= of controlratliversused to prepare bLmk3, s=dards, and QCs for the analyticaplortion of the study. AU 4 analytesof interest(PFOS, M-556, PFOSAA, and PFOSA) were monitored during each analysis. IMPACT OF DEVIATIONS ON THE STTJDY- PFOS lotm=ber 171 and HarL-insuppliedliverwere both used forBaUelle's validationof the analyticalmethod (Battac study number N003604-A). These materialsallowed achievement of the reported method accep=w criteriaso thatthem is no impact on the study. Tbz concurrent analysisof PFOS and metaboliteswas an efeciency improvemenl CORRECTIVE ACMON: This protocoldeviationreport was prepared. APP3LOVED BY. Son C. Andre, Ph.D. BattellePrincipalInvczdptor Date Kristen1.E[==, StLidyDirector Ph.D. N003604-D Proto=l Deviation3, Page I of I 3M Environmental Laboratory E-31 Date Page 127 3M-Medical Department Study: T6316-S Analytical Report: FACT TOX-013 LRN-U2095 BaftUt S" Number Noo3604-1) 3M EnvircmmcntalLabmmtmy Sh* Ni=ba: FACT 060998.1 APPENI)IX F - PFOS PUPJAY ]REPORT 3M Environmental Laboratory Page 128 3M Medical Department Study: T6316.5 ETSS 2 3w 3M Eavim=entd Analytical Report: FACT TOX-013 LPN-U2095 .'-BattellSetudy Number. N003604-D LaboMory StidyNumber. FACT 060998.1 651 778 42.26 04126/99 09-.5,6f5 :W-103 ?0:341 3NI =C"LW kDEM.%Vw A CENUCAM ANiLynCAL L%BORATORY Lin Ckmm -"Sdl) - ET&SS- 24)3-M Rnum S 37M E= Tom Xastma -(3-5633) -SAAC AzWydcal lAb -236-2B- II M=h 24,1999: PmUmlnul MpW forFC-95 MCG). kK 171 SAMPLXDTSCRTFMON-. Tyu sampig vu =bjecmd-to IR-NMR and 4FNNM sp=tralanslyso to determinethspwity of theucmhw prod= and to thar=eri= as nu!nyImpuritycompcncnu as pouibk- A portionof the=mile wu accmuely wclZhe4 spikedwith a known amount of 1.4-bis(wUbcrourtbyf)benzwe (p-Fg:)Q.wA then totallydissolvedin DWO-4 forsubsequ=t analysisby NNM A 400 MHz mpecmam (0 h5783O.40 1) " a 376 MHz I*F-,NMM spe=m (# f5783(i.410)wao wquired usinga Vuixn tnrrypius 4M FR-NMR spw==ter. trzoof theP-BFX internalst=dard vu intendedtopermit the datermiwdcm of the absoluteweigbi pem=t concentrationsof theassignedcotrpomats witbout necetluily needng to Wandf@ or quantityan thec*nvccc= In thAurople mi=m RESULTS-. 71:ccombined NNM umple u romiva spectral d= were used to assign 4 of the wajor and mom of thoiwner componsta in this 7be quxllmtiyc and qm=dtativc =mpositional rcmdts tb2cwere daived ft= the single UW NMR Internal uxadu@db@on analyses an u=nia6zed in TABLE-1 an thoofollowing pall I have mparted both mladve and absolute weight percent conc=tritions. One po3sible mwcn 0= the absoluca wt.% values =W up to O=n d= 10011 may be due to the f=t h" I ","@ 4 of the @omporcns contnl"ed 9 cwlo,,s, 11 lem were any short= chain homclczs Present Ci.c,7, 6, 5, atr-.=batLs), then ft average emug@c='d molecular weight% would have be= somcwhu lem dun thou 4sed in the c4leulaiicns. In pderal. the 'F-NUR tochnlq= is w pwdcul&dy waH suited for Identifying or qmdfying small amunu of various fluorachea@cal homelos hnpuiity commpm=u nnlet-athe c!h2i us vtry shon. A mom conviou cha=teriiadott of any otba ftmm=barmc3l hamolop w=W require "-ysis by clectospmy MS or a sin-ilartech- qmL Additional wer!k wa@ld be required in in effortto positiv'elyverify ft =tadvely Luiped compio=t3 Usted In TABLE- I (dc=ttd by possible). S=U &==a of other unidentiried 4npuddw in alsode,ect-,'bm tl%iN&M specm but additiona work would be required in an effortto identifyor quantity dvw other matulall Copies of the NI@,M sp=tm will be provide@d far yau at 'aIn= dale. If you have any questions about the remalls In this initialreport fcr PC-95, lot 17 1. ple-@l letme know. I apclogiw for the delay In completing this Wtid wc& Tom K&Stm c Rkk Pwftu - SAAC AndrkA Lab - 23&2R-1 I AW ash==& L=7130J=dl 3M Environmental Laboratory Par - tF-I Page 129 3M Medical Department Study: T6316.5 ETSS 2 3W Much 24. 1"9 3MEnviro=ental Analytical Report: FACT TOX-013 LRN-U2095- BancUe Study Number N003604-D LaboratoryStadyN=ben FACTo6oggs.1 651 M 4n6 04126/;9 (*:56 f9 :(3/03 ?0:341 SAA:C Aub-dcd LAb RequM # 37330 Wtw Rwrt forvc.%. lot171 TABLE-1 Sampic PC-95. lot171 OTM TN-A-CM34 Overall(Zuwdtsdve C=posidang R=WU by.@W'$F-N2vM IM=d Sbmduamdw Sauc=r2l Aafpments CF3(cpi)t-SO3(K-()+) LN==d chi= "==a x 07 ft CAICWSAIM papa@") . C:FACT2.@c F3)@(CFUVS03(-)X(+) owomai saaamokyl brwA;b6- Z.*Ya S. JL0 (@A y 0 (Lfeecalm'-f4oaparpom) NMR Almhft Wg&S (lintitsrw mmmmnwt) 70-311 17.7% =mm x US fbrcak:Watkmpapom) (:.Pa..rCF(CF3)@-)X(+) (Aipba bruch. mxmme i a 6 fcccalcdadm pwpxm) PZUMIS F-WrcF@rsc@l(-) K(+) (&so=* I fcwc4cul&dQupapow) CFr(CFACCCCFIMCF2)7-SOI(-) K(+) (Intanalpmdlma&@i bmmt6 Luww x+y a 4 and iLo 0 for 3.3% 0.37% (LI6% Andysm roa iduto va-,&@s (3intiunw 10.2% 3.2% 00.161% Poodble CFrSF4-C.FU-SO3(-) IL(+) (mgume i I forcakuladcapurpom) Probabla IC4+) (HydrocarboAsWfonwA nlr an=* x- I forc&kwul" purpam) eibrancIaLs-s.umex 4 forcalc,-Iarp;ucrnpcs&x) 0.11% O.Ml% 0.=% 0.10% OMO% 0=6% F-2 3M Environmental Laboratory Page 130 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix H: InterimCertificatoefAnalysis 3M Environmental Laboratory Page 131 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 INTE"M CER TIFICA TE OF ANALYSIS Revision ](917100) Centre Analytical Laboratories COA Reference 023-018B 3M Product: PFOS, Lot 171 Reference #: SD-009 Test Name Purity' Purity: 86.4% Specifications Result 86.4% Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. PotassiUM2 5. Nickel 6. Iron 7. Manganese -Total % Impurity (NMR) Total % Impurity (LC/MS) Total % Impurity (GC/MS) Related Compounds POAA Residual Solvents (TGA) Pur-ity -b-y5--s-C InorganicAnions (IC) I . Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate' Organic Acids' (IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis': I. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine White CrystallinePowder I. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Tbeoretical Value = 0% 4. Tbeoretical Value = 5.95% 5. TheoreticalValue = 60% Conforms Positive 1. 0.017 wt./wt.% 2. 0.007 wt.lwt.% 3. 1.355 wt./wt.% 4. 6.552 wtjwt.% 5. 0.003 wt-/wt.% 6. 0.004 wtjwt.% 7. <0.001 wt/wt.% 1.00 wtjwt.% 10.60 wt./wt.% None Detected 0.30 wt./wt.% None Detected Not Applicable' I . <0.015 wt./wt.% 2. 0.27 wt./wt.% 3. <0.040 wt./wt.% 4. <0.009 wt./wt.% 5. <0.006 wt./wt.% 6. <0.007 wt./wt.% 7. 8.82 wtjwt.% 1. <0.1 wt./Wt.% 2. <0.1 wtjwt.% 3. <0. I wt./wt.% 4. <0.25 wtjwt.% 1 . 12.08 wt./wt.% 2. 0.794 wtjwt.% 3. 1.61 wtjwt.% 4. 10.1 wtjwt.% 5. 50.4 wtjwt.% COA023-018B 3M Environmental Laboratory Pagel of3 Page 132 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 INTEMM CER TIFICA TE OF ANAL YSIS CentreAnalyticaLlaboratorieCsOA Reference#: 023-018B DateofLastAnalysis0:8/31/00 ExpirationDate: 08/31/01 Storage Conditions:Frozen:S-IO'C Re-assessment Date: 08/31/01 'Purity= 100% - (sum of metalimpurities1,.39% +LC/MS impurities, 10.60%+InorganicFluoride,0.27%+NMR impurities1,.00%+ POAA, 0.30%) Totalimpurityfrom alltests= 13.56% Pur-ity= 100% - 13.56% = 86.4% 2potassiumisexpectedinthissaltform and isthereforenot consideredan impurity. 3Purityby DSC isgenerallynot applicabletomaterialsof low purity.No endotherm was observed forthissample. 4S ulfurin thesample appearsto be convertedto S04 and hence detected-usintghe inorganicanionmethod conditions.The anionresultagreeswellwiththe sulfur determinationinthe elementalanalysisl,endingconfidenceto thisinterpretatioBna.sed on theresultst,heS04 isnot consideredan impurity. 'TFA HFBA NFPA PFPA Trifluoroacetaicid Heptafluorobutyriaccid Nonofluoropentanoicacid Pentafluoropropanoiaccid 6Theoretical value calculations based on the empirical formula, C8Fl7SO3-K@ (MW=538) This work was conducted under EPA Good LaboratoryPracticeStandards(40 CFR 160). COA023-01 8B 3M Environmental Laboratory Page 2 of 3 Page 133 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LP,N-U2095 INTE"M CER TIFICA TE OF ANAL YSIS Centre Analytical Laboratories COA Reference #: 023-018B LC/MS Purity Profile: Impurity C4 C5 C6 C7 Total WL/wt. % 1.03 1.56 6.38 1.63 10.60 Note: The C4 and C6 values were calculatedusing the C4 and C6 standard calibration curves, respectively.The C5 value was calculatedusing the average response factors from the C4 and C6 standard curves. Likewise, the C7 value was calculatedusing the average response factorsfrom the C6 and C8 standard curves. Prepared By: David S. Bell Scientist,Centre AnalyticalLaboratories Date Reviewed By: John Flaherty Date Laboratory Manager, Centre Analytical Laboratories COA023-018B 3M Environmental Laboratory Page 3 of 3 Page 134 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix 1:Report Signature Page MarvinT. Case D.V.M..Ph.D.,StudyDirector Date John L.ButenhoffP,h.D.,SponsorRepresentative Date Dale L.Bacon,Laborator@manager Date 3M Environmental Laboratory Page 135