Document QXVz5NR8yO7XaBYjgKwYkK6z7

ANALYTICAL ILABORAToRy REPORT ON THE Determinationofthe Presence and Concentration of Perfluorooctanesulfonat(ePFOS) (CAS Number: 2759-39-3) inthe Serum of Sprague-Dawley@' Rats Exposed to Potassium Perfluorooctanesulfonatveia Gavage LaboratoryReport No. U2779 Requester ProjectNo. 3M Tox 6295.13 Study Dates Study Initiati1o0n:June 1999 Sample AnalysisInitiati2o2n:June 1999 Sample AnalysisCompletion:28 June 1999 Study Completion:At signing Perfluoroodariesuffonate CAS Number-2759-39-3 3M EnvirorunentaLlaboratory ReportNo. FACT Tox-loa LaboratoryRequest Number (U:@N)-U2779 TABLE OF CONTENTS Study Personneland Contributo.r.s...................................................1 ....................... StatementofCompliance...........................................................2........................ GLP Study.......................................................................3....................... QualityAssuranceStatement........................................................3....................... Introducbon......................................................................4....................... Purpose...................................................................................4................................... TestSystem .....................................................................4....................... Sample Collectfoannd Ana"is .....................................................5........................ Sample Receipt..................................................................5........................ Materialasnd Methods .............................................................6........................ Chemical Characterizati.o.n.......................................................6........................ Method Summaries ...............................................................7....................... ProtocolDeviatfon.s..............................................................8........................ DataSummary, Analysesa,nd Resub ................................................8........................ Summary of QualityControlAnalyses Results............................................8.......................... Summary of Sample Results...........................................................8.......................... StatistiMceatlhods................................................................9........................ DataQualityObjectvesand DataIntegrf.t.y...........................................9........................ StatementofConclusion............................................................9....................... References......................................................................1.0....................... Attachments....................................................................1.0........................ Page i Peffluomodanesuftnate CAS Number-2759-39-3 STUDY PERSONNEL AND CONTRIBUTORS AnalyticalChemistry Laboratories: SerumAnalyses AdvancedBioAna"calServiceIsn,c. 15 CatherwoodRoad IthacaN,Y 14850 KristeJn.Hansen,Ph.D.,StudyDirector John R. PerkinsP,h.D.,AssistanStcien6fiDcirector In-lifeTesting Laboratory Argus Research LaboratoryI,nc. 905 Sheehy Drive,BuildinAg Horsham,PA 19044 Raymond G. York,Ph.D.DABT, StudyDirector Sponsor 3M ToxicologyServices-MedicalDepartment 3M Center,Buildin2g20-2E-02 St Paul,MN 55144-1000 MarvinT.Case,D.V.M.,Ph.D.,SponsorRepresentative 3M Enviror,,,-ieLnatbaolratory ReportNo. FACT Tox-I08 LaboratoryRequest Number (LRN)-U2779 Page 1 of 10 Perfluomoctanesutfbnate CAS Number-2759-39-3 3M EnvironmentalLaboratory ReportNo.FACT Tox-108 LaboratorFy*uest Number (LRN)-UZR9 STATEMENT OF COMPLIANCE Study Title: AnalyticaLlaboratoryReporton the Determinationofthe Presence and Concentrationof Perfluomoctanesulfonat(ePFOS) intheSerum of Sprague-DawleyoRats Exposed to Potassium PerfluomoctanesuffonatveiaGavage Study ldentificaboNnumber FACT Tox-I08 This studywas conducted incompliancewithFood and Drug AdministratioGnood LaboratoryPractice(GLP) RegulationsforNonclinicaLlaboratoryStudies[DataRequirement(s):21 CFR ( Part58)],withtheexceptionsin the builetedlisbtelow.Inadditiont,he presentstudyhas been auditedretrospectiveblyyan independentquality assurance unit.Auditproceduresand findingsforauditsperformed atthe3M EnvironmentalLaboratoryand at participaticnogntractlaborato@esare housed withdocumentation perfinentothisstudy inarchivesatthe3M laboratoryand vwllbe retainedfora periodnottoexceed tenyears.The analyticaplorbon completed atthe 3M EnvironmentalLab was performed inaccordancewith3M EnvironmentalTechnology and SafetyServices Standard OperafingProcedures. ExceptionstoGLP compliance: Two separatestudydirectorwsere assignedto thein-Ifpfhease and theanalyficaplhase ofthisstudy The ABS finalreportdoes nothave a Statementof Compliance The QAU statementintheABS finalreportindicatecsompliancewithEPA 40 CFR Part792, ratherthan FDA 21 CFR Pan 58 Dose confirmatonanalyseswere notconducted accordingtothe GLP regulationsa;nalyticamlethod was not fullyvalidated Not allraw datawere vefifiebdy thegroup leaderor designee StudyDirector ao Date &.@@ tr4lil- SponsorRepresentative Dag Page 2 of10 Perfluomoctanesutfonate CAS Number-2759-39-3 3M Envimnmentai Laboratory ReportNo. FACT Tox-108 LaboratoryRequest Number (U:ZNYU2779 GLP STUDY QUALiTy AssuRANCE STATEMENT Study Tifle: AnalyticalLaboratoryReport on the Determinationofthe Presence and Concentrationof Perfluorooctanesulfonat(ePFOS) inthe Serum ofSprague-Dawleyo Rats Exposed to Potassium PerfluorooctanesulfonatveiaGavage Study IdentificaboNnumber FACT Tox-1 08 This studyhas been inspectedby the 3M EnvironmentalLaboratoryQualityAssurance Unit(QAU) as indicatedin the followingtable.The findingswere reportedtothe study directorand management. 12-30-99 01-06-00 DraftFinalReport 01-11-00 01-07-00 6'QA Representative Date Page 3 of 10 Perfluorooctanesulfonate CAS Number-2759-39-3 3M EnvironmentalLaboratory Report No. FACT Tox-108 LaboratoryRequest Number (LRN)-U2779 INTRODUCTION 0 11 C8Fl7-6 -0 - 11 0 Perfluorooctanesuff(oPnFaOtSe) CAS Number:2759-39-3 Chemical Formula: C8Fl7SO3- MolecularWeight: 499 Purpose The purpose ofthe study istodetermine the presence and concentrationofPFOS (C8Fl7SO3-) inratserum samples collecteddudng the study of Fl offspringof FO generationfemale ratsexposed to potassium perfluorooctanesuIfonate viagavage. . Data qualityobjectivesforthisanalyticasltudy (outlinedinthe 3M EnvironmentalTechnology and SafetyServices protocolforFACT Tox-1 08) were met. Test System Inthe present study,two groups offemale ratswere used as the testsystem. Group IconsistedofcontrolFO female ratsthatwere administeredonly Tween 80 (vehicle)G.roup 11FO female ratswere administered1.6 mg of potassium perfluorooctanesulfonatpeer kg ofbody weight/day in0.5% Tween 80 (testarticleT)h.e dose corresponds to a concentrationof0.32 mg/mL. Table 1 outlinesthe FO generationfemale ratpopulation demographics forstudy T-6295.13. Group 11FO female ratswere treatedwiththe testarticl4e2 days pnor to cohabitationa,nd through day 14 (pharmacokineticsubgroup) or day 21 postpartum (lactatioannd delivery). Fl male and female ratpups were not directlaydministeredthe testarticleb,utwere exposed to the chemical through matemai gestabon (inuteraexposure) and duringlac;tabonF.l pups were identifieads being from Group I or 11C.ross fosteringwas randomized; halfofthe pups from each group were cross fosteredwith Group Ior 11 females creatingfourFl animal subgroups (A-D).The identificatiaonnd cross-fostehngprocedure isdocumented inthe in-lifsetudy report(FinalReport 418-014 forstudy T-6295.13 page 11-10)V.adous physicaland biological parameters were monitored intestanimals. Table 1. FO Generation Rat Population Demographics for Study T-6295.13 InitiaQluantity Acclimated Control(Group 1) Exposed orTreated (Group 11) Pharmacokinetic Sample Subgroup 86 virginfemale rats 42 female rats 36 female rats 10 female rats 78 virginfemale rats 42 mated female rats 36 mated female rats 10 mated females (8 control2, treatedrats) 25 mated femalearartaltss 25 mated female rats None Page 4 of 10 Perfluorooctanesulfonate CAS Number-2759-39-3 3M EnvimrrentalLaboratory ReportNo. FACT Tox-108 LaboratoryRequest NLwnber(LRNYU2779 The testsystem species and strainselectedwas theCD:CDOBR VAF/PluO (Sprague-Dawley)ratreceivedfrom Charles RiverLaboratoriesI,na,permanentlyidentifieudsing a MoneP sell@piercienagrtag.FO generationrats were identifiewditheartags.Fl generationratswere identifiaesd partof eitherGroup Ior 11lifterbsu,twere not individualtlaygged,and allparameterswere evaluatedintermsofthe lifte(rdam and littenrumbers are in Attachment E, Table 4).FO virgifnemale ratswere at least60 days of age and weighed approximately200-225 g when received(Weightdataare includedinArgus Research Laboratodes,lnr-f,inarleportforstudyT-6295.13on filaetthe 3M archives)M.ale ratsofthesame source and straiwnere used onlyas breedersand were not administeredthetestarticloer consideredpartofthetestsystem. Smimple Collection and Anahmis FO and Fl animals assignedtothecontrolpoolwere culledon day 1 postpartum.On day 14 postpartum,samples were collectedfrom theFO mated female rcftand Fl pups assignedto thephartnacokinefiscubgroup.On day 22 postpartum,samples were collectefdrom theremaininganimals inthetestsystem.Allcollectedsamples were immediatelyfrozenon dryiceand maintainedfrozen(-70*Cor below)untislhippedtothe3M Environmental Laboratodes. Inthe analyticasltudyreportedhere,sera samples collectedfromthe populationofexposed animals (generation FO) and theiorftpring(generatioFnl) were analyzedforthepresenceof PFOS. Sera samples were extractedby a liquid-liqeuxitdractiopnrocedureusingan ionpairingagentand ethylacetate. The extractswere quantitativealnyalyzedusingturboionsprayliquicdhromatography/mass spectrometry (LC/MS) inselectedionmonftodng (SIM)mode, and PFOS levelswere evaluatedagainstextractedstandards. Analyticadletailsare includedinthisreportf;urthedretailsare availablienthestudybindermaintainedby the 3M FluodneAnalyticaClhemistryTeam (FACT);thebinderislocatedinthe3M archives. Analyses assessing thepresence and concentrarfionfPFOS inthe seraofSprague-Dawley ratswere conducted incompliance withGood LaboratoryPracticeRegulations(21CFR 58).Validatedmethods and standardoper-afing procedureswere followedduringthepreparationand analysisofthesamples associatedwiththisstudy. SAMPLERECEIPT Samples were receivedfrom Argus Research Laboratodessporadicallyd,uringand atthe end ofthein-lipfhease ofstudy,from November 1998,throughFebruary,1999. Samples receivedwere packaged indryice.Specimens were registeredwiththe3M EnvironmentalLaboratoryand transferretdo a freezerforstorage. Samples ofbody tissuesand fluidostherthan ratserum were collectedand receivedfrom Argus Research Laboratories(Protocol418-014,Study T-6295.13),butwere notpartofthescope ofanalysisintheprotocol determinedby thestudydirector. Specimens sentto Advanced BioAnalybcalServices,Inc.(,ABS) foranalysiswere receivedand trackedaccording tothe Sample TrackingSystem StandardOperatingProcedure.Specimens senttoABS willbe returnedto the3M EnvironmentalLaboratoryupon completionofanalysisand submissionofthesubcontractlaboratory(fsi)nal reportS-pecimens willbe maintainedinthe3M EnvironmentalLaboratoryspecimen archives.Sample receipt, identificatisotno,rage,and chainofcustody protocolsand data arelocatedinthestudybinderforthisreport(FACT Tox-108);the binderislocatedinthe3M archives. Page 5 of10 Perfluorooctanesutfonate CAS Number-2759-39-3 3M Enviioi-.i,,@-L,a,broarlatory Report No. FACT Tox-1 08 LaboratoryRequest Number (LRN)-U2779 MATERIALS AND METHODS Chemical Characterization Table2 presentsinformatiornegardingcharacterizatoifotnhetestc,ontrola,nd referencseubstancesused inthe in-Iffpehase ofthisstudy. Table2.Characterizatioannd Treatmentofthe TestSubstance,Control,and Reference MaterialsinStudy FACT Tox-108 TestSubstance Source Preparation Maximum StorageT-ime StorageConditions Chemical Lot Number PhysicalDescriptioannd IdentitAynalyses Puhty Stability ControlMaterial Source Preparation Maximum StorageTime Storage Condidons Chemical Lot Number PhysicalDescdpton and IdentitAynalyses Pudty Stability PotassiumPerfluorooctanesulfonate 3M SpecialtCyhemicals Daiiy-Records maintained intheraw data ExpiratioMnay 2000 Room temperature 217 FC-95,Whitepowder 99.28% 48-hrdataofpreparedformulationosn filweiththesponsor DeionizedWater J.T.Baker,PhillipsbuNrJg Suspensionswere prepareddaily Unknown Room temperature Tweens80--M03HO5, L06662, AND M29477 0.5% Tweenz) 80 inReverse Osmosis DeionizedWater There are no known contaminants inthe dietthatwould interferweith thisstudy. 48-hrdata of prepared formulationson filewiththe sponsor Page 6 of 10 Perfluorooctanesuttonate CAS Number-2759-39-3 3M EnvironmentalLaboratory Report No. FACT Tox-1 08 LaboratoryRequest Number (LRN)-U2779 Table 2. Characterizationand Treatment of the Test Substance, Control,and Reference Materialsin Study FACT Tox-108 (continued) AnalyticalReference Material PFOS (Perfluorooctanesulfonate) Source 3M SpecialtyChemicals Preparabon Analybcalprocedureinthe ABS finalreport(AttachmentF) Maximum Storage Time Unused referencematedalwillbe retainedforuse by the 3M EnvironmentalLaboratoryand willbe discardedwhen thequalitoyf preparationo longeraffordsevaluation Storage Conditons Room temperature Chemical Lot Number 171 PhysicalDescdptionand IdentitAynalyses Light-colorepdowder Pudty 99.49% Stability Undetermined Dose analyseswere performedon testsubstancesamplestakenatvadous timesduringthe in-lipfhease ofthe study:dataare locatedinthe studybinderforthisreport(FACT Tcx-108) and arepresentedinAttachmentD. Reserve samples ofcontrolmatedalsfromthe3M EnvironmentalLaboratoryanalyseswillbe storedatthe3M EnvironmentalLaboratoryfora periodnottoexceed 10 yearsfollowintghe effectivdeateofthefinatlestruleo,r untitlhe qualitoyfthepreparationno longeraffordsevaluationa,s willany reservesamples oftestsubstance retumed fromthe in-lipfhease ofthestudy.Reserve samplesofcontromlatedalfromABS willbe storedatthe subcontractlaboratodesinsuch a manner thatthecontromlate@alswillbe preservedforuse inensuingstudies. Method Summaries Followingisa bdefdesc@ptionofthemethods used du@ng thisanalyticasltudyby ABS. Detailedeschptionsof thesemethods are presentedinAttachmentC. ABS Preparatoryand AnalyticaMlethod:Method Validatiofnorthe QuantitationfPerfluorooctanesulfonate (PFOS) inRat Serum by Tuto IonSprayLC/MS. Analyticasltandardstocksolutions,tandardspikingsolutionsi,ntemalstandardstocksolutionsi,ntemal standardworkingsolutionsc,ontrolblanks,qualitcyontrolstocksolutionsq,ualitcyontrolsamples,and study samples are preparedindilutiownisthappropdatevolumes ofmethanolorpudfiedwaterinpreparatiofnor extractioTne.trabutyalmmonium hydrogensulfat(e1mL of0.5molarsolutioni)sadded toallqualitcyontrol and studysamples,alongwith2mL of0.25molarsodium carbonate/0.25molarsodium bicarbonateW,ater (500pL)isadded tothe controbllank,and 5OOpL oftheintemalstandardworkingsolutioinsadded toallother tubes.FivemL ofethylacetateisthenadded toeach tube,and thetubesare placedon a reciprocaslhakerat medium speed for20 minutes.The tubesarethencentdfugeda,nd theorganiclayeristransferretdoa clean polypropylenteube.The organiclayerisevaporatedtodrynessina TurboVapTm atabout20*C under nftgen. The dded extractasrethenreconstitutweidthacetonitdiaend water,and 200pL oftheextracits transferretdoan autosamplertubeforanalysis. Page 7 of10 Perfluomoctonesuffbnate CAS Number-2759-39-3 3M EnvimnrrientaLlaboratory ReportNo. FACT Tox-108 LaboratoryRequest Number (LRN)-U2779 Equipment used atASS: LC/MS/MS System: Autosampler Waters 717plus Mass spectrometerPE SCIEX API 365 withTurbolonSprayT"interface Analyticaclolumn:KeystoneBetasHT"Cie, 2.lx5Omm (5pm) Mobilephase components: Component A-1 0:90medmol:2 mM ammonium acetate Component B--W:10 methanol:2mM ammonium acetate Injecbonvolume: 3-5pL Column temperature:Ambient Curtaingas: UHP nitrogen Nebulizergas: UHP nitrogen Ionsprayvoltage:-3000V DedusteOng potential-:35V Negativeionsmonitored: m/z--499.0forPFOS [M-KT m/z=427.0 forteb-a-H-PFOS[M-H]Totalrun time:8.0minutes Protocol Deviations There were no deviationfsromtheoriginaplrotocol. DATA SUMMARY, ANALYSES, AND RESULTS Summary of Quality Control Analyses Resufts (ABS) The inter-assapyrecisio(nrelatisvteandarddeviatiort-RSDd)atafromdailyqualitcyontroslamples rangedfrom 1.57-4.01%forPFOS, and theinter-assayccuracy(relatievreror-RE)rangedfrom -7.32-10.%1. Bothare acceptableranges. The inter-assapyrecisio(nRSD) ofstandardsrangedfrom1.11-5.83%forPFOS. The inter-assay accuracy(RE) fromstandardsrangedfrom-5.86-11.0%.Allareacceptableranges. Coefficientosfdeterminabonwere ->0.9954. RefertotheABS finarleport(AttachmentF,Tabie2)forprecisioannd accuracydataon theanalysesof qualitcyontroplartalvolume(one-to-tednilubons). Summary of Sample Results (ASS) FollovAngisinfbrrnabornegardingsampleanalysesinthepresentstudy: The samples inanalyticarlunnumbers 1 and 3 were reassayedbecause themeasured concentrations exceeded thecalibratiroanngeand upper limiotfquantitabo(nULO) (refetroTable6 inattachmentF). Smallervolumeswere reextractefdromthesamples and reana@zed.Reassayed samples resulteidn measured concentrationwsithinthecalibratiroannge,and thedatawere reported(refetroTable6 in attachmentF). Odginaldataorcopiesthereofareavailablaetthe3M EnvironmentalLaboratory. Page 8 of10 Perfluorooctanesutfonate CAS Nuffiber-2759-39-3 3M Envimnmental Laboratory ReportNo. FACT Tox-I08 LaboratoryRequest Number (LRN)-U2779 STATISTICAL METHODS At ABS,datafrompeakareaswereintegrabtyetdhePE SCIEXprograMmacQuanTIA,versio1.n4, on a Macintosh computer. Folloviinpgeak area integrationM,acQuan resultsdata were converted totextfilesm,oved to the ABS fileserver,and analyzed withthe Watson7m software package (version5.3.1.01 from PSS, Inc.,Wayne, PA). The Watson analysisapplieda weighted (1/y)quadraticregressiontothe data,and calculationwsere performed on unmunded numbers; however, standard and qualitycontroldata were rounded tothree significant figurespriorto reportingthe data. DATA QUALITY OBJECTIVES AND DATA INTEGRITY No circumstances existedduringthe present study thatwould have affectedthequalityor integritoyfthe data.The followingdata qualityobjecfives(DQOS) were followedduringthe study: Linearity-The coefficienotfdetermination(e)of the standardcurve was equal toor greaterthan 0.98 Limitsof Quantitabon(LOQ)-Equal to the lowestacceptablestandardinthe calibratiocnurve 0 Duplicateacceptable precision- <30% Spike acceptable recoveries- 70% to 130% Use ofconfirmatorymethods-no confirmatorymethods were used Derrkonstrabonof specificity-specificwiatsy demonstrated by chromatographic retentiontime,mass spectral [N&KI product ioncharacterization Assuming thatspike recovery studiesform a suitableindicatioonfendogenous analyterecovery,data are quantitativteo:t30%. The validitoyfthisassumption has not been verifiedby othertechniques.Inserum analyses,the limiotfdetection(LOD) forPFOS isapproximately1.75ng/mL and the limiotfquanttabon (LOQ) is 0.05pg/mL PFOS. STATEMENT OF CONCLUSION Statement of Conclusion: Under the conditionsofthe present studies,PFOS was observed inthe sera of FO female ratsexposed during the in4ifephase ofthe study.AdditionallyP,FOS was observed insera tissuestaken from Fl generationpups from female ratsexposed to the testsubstance,and inFl generationpups exposed tothe testsubstance vialactation, but not exposed intgefo. Page 9 of10 Perfluomoctanesuffonate CAS Number-2759-39-3 REFERENCES None. 3M EnvironmentaLlaboratory ReportNo.FACT Tox-108 LaboratoryRequestNumber (LRN)-U2779 ATTACHMENTS AttachmentA: Sample Receiptand Chain of Custody Docurrientabon Attachment8: Protocol Attachment C: Extractioannd AnalyticaMlethods Attachment D: Dose ConfimiationAnalyses Attachment E: Data Summary and Tables Attachment F: AnalyticaRleports from ContribOng Laboratories Attachment G: Report SignaturePage Page 10 of10 Perfluorooctariesuffbnate CAS Number-2759-39-3 3M EnvironmentalLaboratory Report No. FACT Tox-108 LaboratoryRequest Number (LRN)-U2779 ATTACHMENT A SAMPLE RECEIPT AND CHAIN OF CUSTODY DOCUMENTATION Sample receiptand chain ofcustody informationisdocumented inthe binderforthisreport(FACT Tox-108);@'le binderislocatedinthe 3M archives. Perftwmoctanesulfonate CAS Nuniber-2759-39-3 ATTACHMENT B PROTOCOL 3M EnvironmentalLaboratory ReportNo. FACT Tox-I08 ratdrRyequestNumber (LRN)-U2779 3M Environmental Technol*Lv and Services PO Box 33331 St.Paul.MN 55133-3331 6127786442 Protocol #FACT-TOX-108 Study Title Oral (Gavage) Cross-FosteringStudy ofPFOS inRats PROTOCOL Author Lisa Clemen Date: June 8, 1999 Perfortning Laboratory 3M EnvirorunentalTechnology & SafetyServices 3M EnvirorunentaLlaboratory 935 Bush Avenue St.Paul,MN 55106 Laboratory Project ldentirication FACT-TOX-108 U2779 3M Environmental Laboratory Page 1 of 10 Protoco#lFACT-TOX-108 StudyIdentification Oral(Gavage)Cross-FosterinSgtudyofPFOS InRats TestMaterial Sponsor Sponsor Representadve Study Director Perfluoroocstualnfeonaiccidpotassiusmalt (T-6295) 3M ToxicologyService-sMedicalDepartment 3M Center,Building220-2E-02 St.Paul,MN 55144-1000 Marvin T.Case,D.V.M., Ph.D. 3M ToxicologyServices Telephone:651-733-5180 Facsimile:651-733-1773 KristenJ.Hansen,Ph.D. 3M EnvironmentalTechnology and SafetyServices Building2-3E-09 651-778-6018 Study Location(s) In vivo Testing Facility AnalyticalTestingLaboratory Argus Research LaboratoriesI,nc. 905 Sheehy Drive,BuildingA Horsham, PA 19044 3M EnvironmentalLaboratory Building2-3E-09 935 Bush Avenue St.Paul,MN 55106 3M Environmental Laboratory Page 2 of 10 Sub-ContracLtaboratory Proposed Study Timetable Study InitiatioDnate Study Completion Date Protoco#lFAC T-TOX-108 AdvanceBdioanalytiScearlviceIsn,c. 15 Cathetwood Road IthacaN,Y 14850 BattellMeemorial Institute 505 King Avenue Colwnbus, Ohio 43201-2693 June 08,1999 June 08,2000 1. STUDY Oral (gavage)cross-fosterisntgudyof potassiumperfluorooctanseulfonicacid(PFOS) inrats. Z PURPOSE Thisanalyticasltudyisdesignedtodeterminelevelsof potassiumperfluorooctanesulfonate (PFOS) inspecimensofliverand senun ofrats.The in-lifpeortionofthisstudywas conducted atArgus ResearchLaboratoriess,tudy#418-014.Allserum sampleswillbe extracteadnd analyzedatAdvanced BioanalyticaSlervicesI,nc.and allliversamplesextracteadnd analyzedat BattellMeemorial InstituteA.dditionalanalysesmay be performedatthe3M Environmental Laboratoryas methods aredevelopedand validated.Ifadditionaalnalysesareperformedan amendment tothisprotocolwillbe written. 3. REGULATORY COMPLIANCE This studywillbe conductedinaccordancewiththeUnitedStatesFood and Drug AdministrationG,ood LaboratoryPracticeSstandards,FinalRule 21 CFR 58,withtheexception thatanalysisof thetestmaterialmixtureforconcentrations,olubilithyo,mogeneity,and stability willnotbe conducted,and istheresponsibiliotfytheSponsor. 4. QuAuTyAssuR.4NcE The 3M EnvirorunentaLlaboratoryQualityAssuranceUnitwillreviewtheprotocoland audit studyconduct,data,and finalreporttodeterminecompliancewithGood LaboratoryPractice Standardsand with 3M EnvirorunentaLlaboratoryStandardOperatingProcedures. The QA Unit atthesub-contracltaboratorwyillaudittheirstudyconduct,data,and resultrseport priortosubmittingtothe3M EnvironmentalLaboratory. 3M Environmental Laboratory Page 3 of 10 Protocci#FACT-TOX-108 5. TESTMATERIAL 5.1 RefertoArgus ResearchLaboratoryprotocolforstudy#418-014. 6. CONTROL MATRICES 6.1 ldentiricationRat liverand serum and/orrabbitliverand senun,traceabilintuymbers willbe recorded in theraw data and included in thefinalreport 6.2 Source Argus Research and/or Sigma Chemical 6.3 Physical Description Rat liverand serum and/orrabbitliverand senan 6.4 Purity and Stability Not applicable 6.5 Storage Conditions Frozen at -20 *C 10 *C or -50 *C 10 *C 6.6 Reserve Matrix A portionof the controlmatrix willbe retainedin the 3M archives foras long as the qualityof the preparationaffordsevaluation,but not longer than ten years followingthe effectivedateof the finaltestrule(ifapplicable). 6.7 Disposition Matrices willbe retainedat the 3M Environmental Laboratory per GLP regulation.Certainmatrices(feces,urine,and blood) may be disposed afterQAU verification. 6.8 Safety Precautions Refer toMSDS forchemicalsused. Wear appropriate laboratoryattirea,nd follow adequate precautionsforhandling biologicalmaterialsand preparing samples foranalysis. 7. REFERENCE MATERIAL 7.1 ldentirication Potassium perfluorooctanesulfonate(PFOS), lot#s 171, 215, or 217 (equivalentlots) 7.2 Source 3M SpecialtyChemicals 7.3 Physical Description White powder 7.4 Purity and Stability Purityof PFOS is99% or greater.Stabilithyas not been determined. 7.5 Storage Conditions Room temperature 7.6 Reserve Material A reservesample from each batch of PFOS used in thisstudy will be retainedas long as thequalityof thepreparationaffordsevaluation,but not longer than ten yearsfollowingtheeffectivedateof the finaltestrule(ifapplicable). 7.7 Disposition Unused referencematerialwillbe retainedforuse by the3M EnvirorunentalLaboratory and willbe discardedwhen the qualityof preparationno longer affordsevaluation. 3M EnvironmentalLaboratory Page 4 of 10 Protoco#lFACT-7'OX-108 7.8 SafetyPrecautionsRefertoMSDS forchemica'lussedW.earappropriate laboratoryattirea,nd followadequateprecautionfsorhandlingbiologicamlaterialsand preparingsamples foranalysis. TESTSYSTEM Female mts were used asthetestsystemand were maintainedand dosed as describedinArgus Researchprotocol#418-014. Group I controlanimalsdidnotreceivethetestsubstancewhile thegroup 2 treatedanimalsreceivedthetestsubstanceata concentratioonf 1.6mg/kgtday for6 weeks. RefertoArgus Researchprotocol#418-014 fortabularpresentatioonf data.Dosing of thefemale animalsingroup 2 was continuedduringmating,gestationa,nd lactationC.rossfosterinogf thepups bom tothecontroldams were randomized,halfof thepups were placedon controldams whiletheotherhalfwere placedon thePFOS dosed dams. Likewise,thesame was done with thegroup 2 pups. Each of thesepups willbe marked to identifwyhethertheyarefrom controlor PFOS dosed dams. 9. SPECIMEN AND SAMPLE RECEIPT The 3M EnvironmentalLaboratorywillreceivehomogeneity samplesand specimensof the followingbody tissuesand fluidsfrom theindicatepdointsinthestudy.Allspecimenswillbe packed on dry iceforshipping. Body tissue/fluid Collected Serum - Dam and Pup animals Urine and feces- Dam animals Liver- Dam and Pup animals Milk SecretingGlands - Dam animals Milk - Dam animals At lactatiodnay 14 and terminatioonfthestudy Afterterminatioonf thestudy At lactatiodnay 14 and tenmnationof thestudy At lactatiodnay 14 and terminatioonfthestudy At lactatiodnay 14 Expected # of specimens 60 Dam and 34 Pup 48 Urine and 48 Feces 60 Dain and 34 Pup 58 10 Totalnumber of expectedspecimens:352 Totalnumber of testanimals:36 Totalnumber ofcontrolanimals:42 Specimens sentto3M EnvirorunentaLlaboratoriewsillbe receivedand trackedaccordingto applicableStandardOperatingProcedures. 3M Environmental Laboratory Page 5oflO Protoco#lFAC T-TOX-108 10.PREPARATORY METHODS 10.1 FACT-M- I-1,Extractionof PotassiumPerfluorooctanesulfonaotreOtherAnionic FluorochemicalSurfactanftrom LiverforAnalysisUsing HPLC-Electrospray/Mass Spectrometry 10.2 ETS-8-4.1,Extractionof PotassiumPerfluorooctanesulfonaotreOther FluorochemicalCompounds from Serum or OtherFluidforAnalysisUsing BPLCElectrospray/MasSspectrometry 10.3 Ifpreparatorymethods otherthanthoselistedabove areused,an amendment to this protocolwillbe written.Any deviationfsrom thesemethods willbe documented and includedwith thestudydata. 10.4 Ifanalysesaresub-contractetdootherlaboratorieasn,amendment willbe writtento includetheirmethods and copiesof eachmethod willbe attachedtothisprotocol. II.ANALY77CAL METHODS 11.1 FACT-M-2. 1,AnalysisofFluorochemicalisnLiverExtractsUsing HPLCElectrospray/MasSspectrometry 11.2 ETS-8-5.1,AnalysisofPotassiumPerfluorooctanesulfonaotreOtherFluorochemicals inSerum or OtherFluidExtractsUsing HPLC-Electrospray/MassSpectrometry 11.3 Ifanalyticamlethods otherthanthoselisteadbove areused,an amendment tothis protocolwillbe written.Any deviationfsrom thesemethods willbe documented and includedwith thestudydata. 11.4 Ifanalysesaresub-contractetdootherlaboratorieasn,amendment willbe writtento includetheirmethods and copiesof eachmethod willbe attachedtothisprotocol. 12.DATA QUALITY OBJEC77VES The number of spikes/duplicatuesse,of surrogatesa,nd informationon otherdataquality indicatorasreincludedintheanalyticamlethods. In additiont,hefollowingcriteriwaillbe met: 12.1 Linearity r'@:0.98 12.2 Limits of detection / quantitatfon 12.2.1 Method DetectionLimit(MDL) forPFOS a) Serum: 1.75ppb 12.Z2 b) Liver:15ppb LimitofQuantitatio(nLOQ) - Equaltothelowestacceptablsetandardin thecalibratiocnurve 3M Environmental Laboratory Page 6 of 10 Protoco#lFAC7-TOX-108 IZ3 Duplicateacceptablperecision< 30% forthemethod IZ4 Spikeacceptablerecoveries70% -130% lZ5 Use ofconrirmatormyethods Indeterminsaatmeplewsilblere-analyzuesdinga confirmatorymethod. Ifa confirmatorymethod isused,an amendment to this protocolwillbe wiitten. 12.6 Demonstration of speciricityChromatographicretentiotnime,mass spectral daughterioncharacterization. 13. SuB-CoNTRAcTED ANALYSIS 13.1 Allanalysesasdetailedin thisprotocolwillbe performedat3M Environmental LaboratoriesB,uilding2-3E-09,935 Bush Avenue, St.Paul,h4N 55106,atAdvanced BioanalyticaSlervicesI,nc.,15 Catherwood Road, Ithaca,NY 14850,or atBattelle Memorial Institut5e0,5 King Avenue, Columbus, Ohio 43201-2693. 13.2 An amendment tothisprotocolwillbe writtenifanalysesareperformedat laboratorieostherthan3M EnvirorunentaLlaboratoriesA,dvanced Bioanalytical Services,Inc.,orBattellMeemorial Institute. 14. STATIS77CAL ANALYSIS Averages and standarddeviationwsillbe calculatedT.he statisticmaelthods thatwillbe used are describedbelow: 14.1 Data transformations and analysis Data willbe reportedas theconcentration (weight/weighotrweight/volo)fPFOS ormetabolitpeertissueorfluid. 14.2 Statisticalanalysis Statistiucssedmay includeregressioannalysisof concentrationosvertime,and standarddeviationcsalculatefdortheconcentrations withineach dosegroup. Ifnecessary,simplestatistictaelstss,uchas Student'st test, may be appliedtoevaluatestatisticdailfference. 15.REPORT A reportcontainingalltheresultosf thestudywillbe preparedby the3M Environmental Laboratory.Ifanalysesare sub-contractetdootherlaboratorieesa,ch laboratorywillpreparea reportand submitittothe3M EnvironmentalLaboratoryforinclusioinnthe3M Environmental Laboratoryreport.Each reportwillincludeb,utnotbe limitedto,thefollowingw,hen applicable: 15.1 Name and addressofthefacilitpyerformingthestudy 15.2 Datesupon which thestudywas initiateadnd completed 15.3 A statementofcomplianceby theStudy Directoraddressinagny exceptiontsoGood LaboratoryPracticeStandards 3.M Environmental Laboratory Page 7 of 10 Protoco#lFACT-TOX-108 15.4 Objectivaensdproceduraessstateidntheapprovepdrotocoiln,cludianngychanges intheoriginaplrotocol 15.5 The testsubstanceidentificatiboynname, chemicalabstractnsumber orcode number, strengthp,uritya,nd compositionor otherappropriatceharacteristiicfsp,rovidedby theSponsor 15.6 Stabilitaynd thesolubilitoyfthetestsubstancesunder theconditionsof administratioinf,providedby theSponsor 15.7 A descriptioonfthemethods used toconductthetest(s) 15.8 A descriptioonf thetestsystem 15.9 A descriptioonf any circumstancesthatmay have affectedthequalityor theintegrity of thedata 15.10 The name oftheStudy Directorand thenames of otherscientistpsr,ofessionalsa,nd supervisorypersonnelinvolvedinthestudy 15.11 A descriptioonf thetransformationsc,alculationosr,operationsperformedon the data,a summary and analysisof theanalyticaclhemistrydata,and a statementof the conclusionsdrawn from theanalyses 15.12 Statisticmaelthods used toevaluatethedata,ifapplicable 15.13 The signedand datedreportsof eachof theindividuaslcientisotrs otherprofessionals involvedinthestudy,ifapplicable 15.14 The locationwhere raw dataand thefinalreportaretobe stored 15.15 A statementpreparedby theQualityAssuranceUnitlistintghedatesthatstudy inspectionsand auditswere made, and thedatesof any findingsreportedtotheStudy Directorand Management Ifitisnecessarytomake correctionosr additiontso a reportafterithas been accepted,the changeswillbe made in theform of an amendment issuedby theStudy DirectorT.he amendment willclearlyidentiftyhepartof thereportthatisbeingamended, thereasonsforthe amendment, and willbe signedby theStudy Director. 16.LocATioN oFRAwDATA, RECORDS, AND FINAL REPORT Originaldata,or copiesthereofw,illbe availablaetthe3M EnvirorunentaLlaboratoryto facilitaatueditsof thestudyduringitsprogressand beforeacceptanceofthefinalreport.When the finalreportiscompleted,alloriginaplaper data,includingthoseitemslistebdelow,willbe retainedinthearchivesof 3M EnvironmentalLaboratoryforatleasta periodof timeas specified by regulationa,nd as establishebdy 3M EnvironmentalLaboratoryStandardOperating Procedures. 3M Environmental Laboratory Page 8 of 10 Protoco#lFACT-TOX-108 16.1 Thefollowirnagwdataandrecordwsilbleretainiendthestudyfoldeirnthe study/projecatrchivesaccordingto 3M EnvironmentalLaboratoryStandardOperating Procedures: 16.1.1 Approved protocoland amendments 16.1.2 Study correspondence 16.1.3 Shippingrecords 16.1.4 Raw data 16.1.5 Approved finalreport(originaslignedcopy) 16.1.6 Electroniccopiesof data ' 16.2 The followingsupportingrecordswillbe retainedseparatelfyrom thestudyfolderin thearchivesaccordingto3M EnvironmentalLaboratoryStandardOperating Procedures: 16.2.1 Trainingrecords 16.Z2 Calibratiornecords 16.Z3 Instr=ent maintenance logs 16.Z4 StandardOperatingProcedures,Equipment Procedures,and Methods 17.SpEcimEN RETEN77ON Specimens willbe maintainedinthe3M EnvironmentalLaboratoryspecimenarchivesfora periodof time as specifiebdy regulatioonras longas thequalityof thepreparatioanffords evaluationb,ut notlongerthantenyearsfollowingtheeffectivdeateofthefinatlestrule(if applicable)a,nd as establishebdy 3M EnvironmentalLaboratoryStandardOperatingProcedures. 18.PROTocoL AMENDMENTS AND DEVIA77ONS Planned changesto theprotocolwillbe intheform ofwrittenamendments signedby theStudy Directorand theSponsor'sRepresentativeA.mendments willbe consideredaspartof the protocoland willbe attachedto thefinalprotocolA.llchangestotheprotocolwillbe indicated inthefinalreportA.ny otherchangeswillbe intheform of writtendeviationss,ignedby the Study Directorand filedwiththeraw data. 3M Environmental Laboratory Page 9oflO lg.ArrAcHmENTs 19.1AffachmentA Preparatoarnydanalytimceatlhods 20.SiGNATuREs Protoco#lFAC T-TOX-108 MarvinT.Case,D.V.M.P,h.D.S,ponsoRrepresentative Date i@-ristenH'aJn.sen,Ph.D.,3M EnvironmentalLaboratoryStudyDirector Date 3M EnvironmentaLlaboratory Page 10 of10 StudyTide Oral(GavageC)ross-FosterSitnugdyofPFOS inRats PROTOCOL AMENDMENT NO. I Amendment Date: August 12,1999 Performing Laboratory 3M EnviromnentaTlechnology& SafetyServices 3M EnviromnenW Laboratory 935 Bush Avenue St.Paul,MN 55106 Laboratory Project ldentfrication ET&SS FACT-TOX108 LIRN U2779 3M EnvironmentaLlaboratoty ProtocoFlACT-TOX108 AmendmentNo.I Thisamendment modifiesthefollowinpgortio'n(osf)theprotocol: 1. PROTOCOL READS: Section2.0statetshatallliversampleswillbe extracteadnd analyzedat BattelleMemorial Institute. AMEND TO READ: All liversamples willbe extractedand analyzed atthe3M Environmental Laboratory. REASON.- Due to time constraintst,heliverextractionand analysiswillbe performed atthe 3M Environmental Laboratory. 2. PROTOCOL READS: Section 10.0 and 11.0 listthefollowingmethods to use forextraction and analysis: FACT-M- 1.1 "Extracfionof Potassium Perfluorooctanesulfonatoer Other Anionic Fluorochemical Surfactantfrom Liver forAnalysisUsing BPLC-Electrospmy/Mass Spectrometry" FACT-M-2.1 "Analysis of Fluorochemicals in Liver ExtractsUsing HPLC-Electrospray/Mass Spectrometry" AMEND TO READ: The extractionand analyticalmethods to followatthe3M Environmental Laboratory are: ETS-8-6.0 "Extracfionof Potassium Perfluorooctanesulfonatoer Other Fluorochemical Compounds from Liver forAnalysis Using BPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 "Analysis of Potassium Perfluorooctanesulfonatoer Other Fluorochemical Compounds in Liver ExtractsUsing BPLC-Electrospray/Mass Spectrometry" REASON: The extractionand analyticalmethods FACT-M- 1.1 and FACT-M-2. 1.,respectively, were updated on 07/22/99 to ETS-8-6.0 and ETS-8-7.0. These methods were updated to replace the extractionsolventethylacetatewith a differentextracfionsolventMTBE (methyl tertbutyl ether),POAA and Monoester were removed from the standardmix, and M556 was added to the standardmix. The analyticalmethod was updated to includelinearregressionwith 1/xweighting and a few minor changes in theEPLC 1100 instrumentparameters. 3M EnvironmentalLaboratory ProtocoFlACT-TOX108 AmendmentNo.I 3. PROTOCOLREADS:Sectio1n0.4and11.4stattehaitfanalysaersesub-contratcotoetdher laboratorieasn amendment willbe writtentoincludetheirmethods. AMEND TO READ: The extractioannd analyticamlethods to followatAdvanced Bioanalytical Serviceswillbe attachedto theprotocol. REASON: The analyticamlethods atthesub-contracltaboratorwyere notincludedintheoriginal protocol. 4. PROTOCOL READS: Section12.2.1b) listtshelivermethod detectiolnimitas 15 ppb. AMEND TO READ: The livermethod detectiolnimitis8.50ppb (ng/g). REASON: The validatiosnupportingmethods ETS-8-6.0and ETS-8-7.0includesa lowermethod detectionlimitforPFOS. Amendment Approval ar@vm Case,D.V.M., Ph.D.,SponsorRepresentative Date L KrisJ.Hansen, Ph.D.,S'tudy Director Date 3M EnvironmentaLlaboratory StudyTide Oral(GavageC)ross-FosteSrtiundgoyfPFOSinRats PROTOCOL AMENDMENT NO. 2 Amendment Date: September30,1999 Pertbrming Laboratory 3M EnvirorunentTaelchnology& SafetyServices 3M EnvironmentalLaboratory 935 Bush Avenue St.Paul,MN 55106 Laboratory Project ldendflcadon ET&SS FACT-TOX108 LIRN U2779 3M EnvironmentLaalboratory ProtocolFACT-TOX108 AmendmentNo.2 Thisamendmentmodifietshefollowinpgortion(osf)theprotocol: 1. PROTOCOL READS.' Section2.0statesthatallliversampleswillbe extracteadnd analyzedatthe3M Environmental Laboratory. AMEND TO READ: No liversamples willbe extractedand analyzed. REASON: Liver resultsareno longerrequired. 2. PROTOCOL READS: Section 16 statesthat the originaldata, or copies thereof,will be availableat the 3M Environmental Laboratory to facilitataeuditsof the study during itsprogressand before acceptance of the finalreport.When the finalreportiscompleted, alloriginalpaper data, including:approved protocol and amendments, study correspondence,shipping records,raw data,approved finalreport,electroniccopiesof data,trainingrecords,calibratiornecords, instrument maintenance logs and standard operatingprocedures,equipment procedures,and methods willbe retainedin the archivesof the 3M Environmental Laboratory. AMEND TO READ: Section 16 statesthatthe originaldata,or copiesthereof,willbe availableat the3M Environmental Laboratory to facilitataeuditsof the study during itsprogressand before acceptance of the finalreport.When the finalreportiscompleted, alloriginalpaper data, including:approved protocoland amendments, study correspondence,shipping records,raw data,approved finalreport,and electroniccopiesof datawillbe retainedin the archivesof the 3M Environmental Laboratory. All correspondingtrainingrecords,calibratiornecords, instrument maintenance logs,standard operatingprocedures,equipment proc6dures,and methods willbe retainedin the archivesof the facilitpyerforming each analysis. REASON. Clarificatioonf the dispositionof archived recordsifanalysesareperformed ata sub-contract laboratory. 3M EnvironmentalLaboratory ProtocoFlACT-TOX108 Amendment No.2 3. PROTOCOL READS.' Sectio1n7statetshastpecimenwsilblemaintainiendthe3M EnvironmentLalboratory specimen archives. AMEND TO READ: Specimens willbe maintainedinthe3M EnviroranentaLlaboratoryspecimen archives.All specimens sentto sub-contracltaboratoriewsillbe returnedtothe3M Environmental Laboratoryupon completionof analysisand submissionof thesub-contract'labomtory(s) finalreport.The specimenswillbe returnedwiththefollowingdocumentation:thesigned originaclhain of custodyand recordsof storageconditionswhileatthesub-contracftacility. REASON: Clarificationf thedispositioonf specimensand documentationforanalysesperformedby a sub-contracltaboratory. Amendment Approval Marvin Case,D.V.M., Ph.D.,Sponsor Representative Date' KristenJ.Hansen, Ph.D.,StudyDirector /0 Date 3M Environmental Laboratoty Pedkwooctariesuffonate CAS Nuniber-2759-39-3 ATTACHMENT C EXTRACTION AND ANALYTICAL METHODS 3M EnvironmentalLaboratory Report No. FACT Tox-108 LaboratoryRequest Number (LRN)@U2779 Proprietaraynd Confidental AOVANCEO m SIOANALYTICAL SERVICES, INC. r METHOD VALIDATION REPORT TITLE: METHOD VALIDATION FOR QUANTITATION OF PERFLUOROOCTANESULFONATE RAT SERUM BY TURBO ION THE SPRAY (PFOS) IN LC/MS DATE: 26 August 1999 REPORT: 99VDJA01.Ml.DOC AUTHORS: PREPARED FOR: NUMBER OF PAGES: David J.Anderson, M.S. Amie J.Prince,B.S. Holly D. Ross, M.S. 3M Environmental Technology and Safety Services St.Paul,MN 55133-3331 50 15 Catherwood Road s Ithaca,New York 14850 s (607) 266-0665 a Fax (607)266-0749 AUTHORS: FOR: DATE: TITLE: DavidJ.Anderson,M.S. Amie J.Prince,B.S. HollyD. Ross,M.S. 3M EnvironmentalTechnologyand SafetyServices 26 August1999 METHOD VALIDATION FOR THE QUANTITATION OF,PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/MS ABSTRACT A sensitivsep,ecifiacc,curataen,dreproducibalnealyticmaelthodwas developebdy Advanced BioAnalyticalServices,Inc.,Ithaca,New York toquantitate perfluorooctanesulfona(tPeFOS) inratserum samples. Serum samples (50 pL) were extractedby a liquid-liquiedxtractionprocedureto isolatetheanalytefrom rat serum. Sample extractswere reduced todryness,reconstituteda,nd analyzed by turbo ionsprayliquidchromatography/mass spectrometry(LC/MS) inthenegativeion mode. The assaydemonstrateda lower limitof quantitatio(nLLQ) of 0.05 jig/mL. using50-jiLsample aliquots.The calibratiocnurveswere fitfrom 0.05 gg/mL to 20 gg/mL forPFOS by a weighted (I/Y')quadraticequation.The coefficientosf determinationofthe calibratiocnurves ranged from 0.9962 to0.9965. Precision*andaccuracyqualitycontrol(QC) samples were preparedatconcentrations of 0.2,6,and 18 jig/mL PFOS. Qualitycontrol(QC) samples were preparedata concentratioonf 100 gg/mL PFOS forpartiavlolume analysis.The intra-and interassayprecision(RSD) resultscalculatedfrom allQC samples ranged from 1.92% to 4.87% forPFOS. The intraa-nd inter-assaayccuracies(RE) calculatedfrom QC samples ranged from -3.58% to 5.58% forPFOS. The mean extractionrecoveries were from 87.6% to 100% forPFOS and 89.9% forthe internasltandard(IS). PFOS was measured as stableinratserum forup to24 hours atambient temperature. PFOS was measured asstableinratserum at-20 *C, currentlyforup to 33 days,and M AOVANCED M BIOANALYTICAL SERVICES, INC. Page 2 of 50 99VDJAOI.Mi.DOC afterthreefreeze/thawcycles.Reliableresultwsere obtainedforsample extracts reinjecte2d7 hoursafterinitiarleconstitution. MMM AOVANCED BIOANALYTICAL SERVICES. INC. Page 3 of 50 99VDJAOI.Ml.DOC QAUSTATEMENT PeriodincspectionftshemethodvalidatifoonrthequantitatoifoPnFOS inrat serum were conductedby theQualityAssuranceUnitof Advanced BioAnalytical Services(ABS) forcompliancewithEPA GLP regulation(s40CFR Part792). The studywas inspectedon thefollowingdates: 13,21 May 1999; 7,8, 14,15 June 1999; 1,2 July 1999;3,4 August 1999. Resultsof theinspectionwsere reportedto ABS Management on: 13,21 May 1999;7, 8,14,15 June 1999; 1,2 July 1999;3,4 August 1999. Resultsof theinspectionwsere reportedto theStudy Directoron 5 August 1999. Based on theinspectionsand thedatareviewed,thisreportisa complete and accuraterepresentatioonf thedata. KhthleenCorm'ack,B.S. QualityAuditor Lie? Date MMM AOVANCEO SIOANALYTICAL SERVICES, INC. Page 4 of 50 99VDJAOI.Mi.DOC TITLE: ReportNumber: Reportedby: SIGNATURE PAGE METHOD VALIDATION FOR THE QUANTITATION OF PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/MS 99VD 0 Ml.DO David J.AndeVon, M.S. Research Scientist Z@, kh Lf.57- q Date Reviewed by: 4-4 KkthleenCor-mack,B.S. AssociateAuditor ALtn qg Date -J Authorizedfor C4, Releaseby: John R.!PerkinsP,h.D. Date AssistanStcientifiDcirector AOVANCED BIOANALYTICAL SERVICES. INC. Page5 of50 99VDJAOI.Ml.DOC TABLE OF CONTENTS ABSTRACT ...............................................................2................... QAU STATEMENT .........................................................4................... SIGNATURE PAGE ........................................................5.................... TABLE OF CONTENTS .....................................................6................... LIST OF TABLES ..........................................................8................... LIST OF FIGURES .........................................................9................... 1. INTRODUCTION ......................................................1.0.................... 2. EXPERIMENTAL .....................................................1.0................... 2.I.CHEMICALS AND MATERIALS ..........................................1.0.................... 2.2.LC/MS INSTRUMENTATION ............................................1.0................... 2.3.SAMPLE PREPARATION AND EXTRACTION PROCEDURE .....................11................. 2.4.RAT SERUM VALIDATION DATA ........................................i.I.................... 2.5.ASSAY EVALUATION .................................................1.2................... 2.5.1.Intra-and Inter-AssayAccuracy ....................................1.2.................... 2.5.2.Intra-and Inter-AssayPrecision....................................1.2.................... 2.5.3.PartiaVlolume Analysis...........................................12.................... 2.5.4.Lower LimitofQuantitatio(nLLQ) .................................1.3.................... 2.5.5.Selectivit.y.....................................................1.3.................... 2.5.6.CarryoverEvaluation.............................................1.3.................... 2.6.STABILITY OF PFOS IN QUALITY CONTROL SAMPLES ......................13.................. 2.6.1.Ambient-TemperatureStabilitoyf PFOS inRat Serum ..................13.............. 2.6.2.FreezerStabilitoyf PFOS inRat Serum at-20 *C .....................1.4.................. 2.6.3.Freeze/ThawStabilitiyn Rat Serum .................................1.4.................... 2.7.REPRODUCIBILITY OF RErNiECTING EXTRACTED SAMPLES ..................14.............. 2.8.EXTRACTION RECOVERY ..............................................1.4.................... 3. RESULTS AND DISCUSSION ...........................................1.5.................... 3.1.ASSAY EVALUATION RESULTS .........................................1.6.................... 3.1.1.Intra-and Inter-AssayAccuracy ....................................1.6.................... AOVANCEO SIOANALYTICAL SERVICES, INC. Page6 of50 99VDJAOI.MI.DOC 3.1-2I.ntra-ndInter-AssParyecisi.o.n......................................... 16 3.1.3L.ineari.t.y........................................................ 16 3.1-4P.artiVaollumeAnalysi.s............................................... 16 3.1-5L.owerLimitofQuantitat(iLoLnQ) ..........................1.7............. 3.1-6S.electiv.i.t.y...................................................... 17 3.1-7C.arryoveErvaluati.o.n.....................................1.7............... 3.2S.TABILITYOF PF,OSINQUALITY CONTROL SAMPLES ....................1.7................. 3.2.1.Ambient TemperatureStabilitoyfPFOS inRat Serum ................1.7............... 3.2.2F.reezeSrtabiloiftPyFOS inRatSerumat-20*C.................1.8............. 3.2.3.Freeze/ThawStabilitoyfPFOS inRat Serum ........................1.8................ 3.3.REPRODUCIBILITY OF RErNiEcTiNG ExTRAcTED SAMPLES ................1.8............... 3.4.ExTRAcTiON RECOVERY .................................................1.8................... 4. CONCLUSIONS ...........................................................1.9................... 5. DATA RETRIEVAL ........................................................1.9................... 6. REFERENCES .............................................................1.9................... 7. TABLES ..................................................................2.0................... 8. FIGURES .................................................................3.1................... 9. APPENDIX A: QUANTITATION OF PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/MS ...................3.7.............. AOVANCEO SIOANALYTICAL SERVICES. INC. Page 7 of 50 99VDJAOI.Mi.DOC LIST OF TABLES Table1: Intra-AssayAccuracy and Precisionof thePFOS Assay inRat Serum for QC Samples ........................................................................... 20 Table2: Inter-AssayAccuracy and Precisionof thePFOS Assay in Rat Serum for QC Samples from Three ValidationRuns ..............................2.1.................... Table 3: Accuracy and Precisionof thePFOS Assay inRat Serum forCalibration Standardsfrom Three ValidationRuns ................................2.2.................... Table 4: CalibratioCnurve ParametersforPFOS inRat Serum ....................2.3................ Table 5: PartiaVlolume Analysisof PFOS inRat Serum .........................2.4.................... Table 6: Lower LimitofQuantitatioonfPFOS inRat Serum ......................2.5.................. Table 7: Ambient-TemperatureStabilitoyf PFOS inRat Serum After24 Hours......2.6.... Table 8: FreezerStabilitoyfPFOS inRat Serum at-20 *C .......................2.7.................... Table 9: Stabilitoyf PFOS inRat Serum AfterThree Freeze/Thaw Cycles..........2.8........ Table 10:ReproducibilitoyfReinjectinEgxtractedSamples ContainingPFOS after 27 Hours inReconstitutioSnolution..................................2.9..................... Table 11:ExtractionRecovery of PFOS from Rat Serum ..........................3.0..................... MMM AOVANCEO BIOANALYTICAL SERVICES. INC. Page 8 of 50 99VDJAOI.Mi.DOC LIST OF FIGURES Figur1e:Full-ScSainnglMeS MassSpectruomfPFOS ............................................. 31 Figure2: Full-ScanSingleMS Mass Spectrum of Tetra-H-PFOS ................................... 32 Figure3: Mass Chromatograms of PFOS and itsInternaSltandardinControlBlank Rat Serum Extract..................................................................... 33 Figure4: Mass Chromatograms of PFOS in a Rat Serum ExtractSample Containing InternaSltandardOnly (ZeroSample).................................3.4.................. Figure5: Mass Chromatograms of CalibratioSntandardI inRat Serum Extract ContainingPFOS (0.05pLg/mL)and theInternaSltandard................3.5............. Figure6: Mass Chromatogrmns of CalibratioSntandard10 inRat Serum Extract ContainingPFOS (20 Ag/mL) and theInternaSltandard..................3.6.............. AOVANCEO BIOANALYTICAL SERVICES, INC. Page 9 of50 99VDJAOI.Ml.DOC 1. INTRODUCTION Thepurposoefthirseporitstodescritbheemethodvalidatifonrthequantitative determinatioonf perfluorooctanesulfon(aPtFeOS) inratserum samplesafter modificatioonfa previousmethod (1).The objectiveosfthemethod validatiownere tovalidatea simpleextractiopnrocedure,determinethelower limitof quantitation forroutineanalysisd,eterminetheextractiornecoveriesof theanalyteand internal standard(IS),determinetheanalytestabilitiynratserum atambienttemperature, long-termfreezerstorage,and overthreefreeze/thawcycles,and toprovidespecific, accuratea,nd reproduciblqeuantitativreesultbsy turboionsprayliquid chromatography/massspectrometry(LC/MS). 2. EXPERIMENTAL 2.1. CHEMICALS AND MATERIALS Perfluorooctanesulfon(aPtFeOS, Lot# 171)was obtainedfrom 3M, Inc. The internal standard(IS)forPFOS was IH,IH,2H,2H-perfluorooctanseulfonicacid(Tetra-HPFOS). The internasltandard(Lot#59909) was obtainedfrom 3M, Inc.A detailed lisotf chemicalsand materialissfound inAppendix A. Stockand working solutionwshich were used topreparethecalibratiocnurvesfor theanalyteswere preparedas describedinAppendix A. Stock solutionussed inthe preparatioonf qualitycontrol(QC) sampleswere preparedseparatelfyrom those used inpreparatioonf thecalibratiocnurves. Preparationof allsolutionussed duringextractioannd analysisaredescribedin Appendix A. 2.2. LC/MS INSTRUMENTATION The liquidchromatography/massspectrometrysystem consistedof two LC- IOAD pumps (Shimadzu,Columbia,MD 21046),a SCL- IOA pump controlle(rShimadzu, MMM AOVANCEO SIOAN LYTICAL SERVICES. INC. Pagc 10of50 99VDJAOI.Ml.Doc Columbia,MD 21046), a WISP 717plus autosmnpler(Waters AssociatesM,illipore Corporation,Milford,MA 01757),a BetasilC,,(2 x 50 mm, 5 gm) column (KeystoneScientificI,nc.,BellefonteP,A 16823),and a PE SCIEX API 365 mass spectrometer(PE SCIEX, Concord,Ontario).A detailedlisotf theinstrumentation and instrumentconditionsisfound inAppendix A. 2.3. SAMPLE PREPARATION AND EXTRACTION PROCEDURE For each analyticarlun (tray)d,uplicate50-gL aliquotsof thecalibratiocnurve swnpleswere preparedas describedinAppendix A. The nominal (theoretical) concentrationosf PFOS inthecalibratiocnurveswere 0.05,0.1,0.25,0.5,1,2,4,8, 16,and 20 gg/mL. Rat serum qualitycontrolsamples were preparedin advance of thevalidationstudy atnominal (theoreticaclo)ncentrationosf0.2,6,and 18 gg/mL forQC I,QC2, and QC3, respectivelya,s detaileidnAppendix A. A dilutionQC (QC4, 100 gg/mL) was preparedata concentrationexceedingthe upper limitof the calibratiocnurve range (20 gg/mL), and was assayedusinga 10-folddilutionforpartiavlolume analysis. Calibrationstandardsand QC samples were extractedby the proceduredetailedin Appendix A. 2.4. RAT SERUM VALIDATION DATA The datawere collectedusingselectedion monitoring(SIM) turboionsprayLC/MS inthe negativeion mode. Peak areaswere integratedby the PE SCIEX program MacQuan, version1.4,residingon a Macintosh computer. Following peak area integrationt,heresultstablesfrom MacQuan were saved as textfilesand uploadedto theAdvanced BioAnalyticalServices(ABS) fileserverwhere a weighted(1/y') quadraticregressionwas performed using the softwarepackage Watson v 5.3.1.01 (PSS,Inc.,Wayne, PA 19087).All datawere rounded to no lessthan three significanftiguresby ABS priortoreportinginTables I through11. The datafor the ratserum validationarestoredin ABS Notebook 2304. AOVANCEE:) BIOANALYTICAL SERVICES. INC. Page II of 50 99VDJAOI.Mi.DOC Allcalculationwsere based on thepeak arearatioof theanalytetotheinternal standard.Concentrationsof eachanalyteinqualitycontrolsampleswere determined by inversepredictiofnrom thecalibratiocnurve. 2.5. ASSAY EVALUATION 2.5.1. Intra-and Inter-AssayAccuracy The intraa-nd inter-assaayccuracyof themethod was assessedby determiningthe relativeerrorobservedintheanalysisof qualitycontrolsamples. The mean concentratiofnoreach qualitycontrolevelwas dividedby thetheoretical concentrationO.ne was subtractedfrom theresultc,onvertedtopercentand expressedasRE. QC I throughQC3 were assayedinreplicateosf five.Inaddition, theRE was reportedforstandardsatalllevelsover threeruns. 2.5.2. Intra-and Inter-AssayPrecision The intraa-nd inter-assapyrecisionofthemethod was assessedby determiningthe RelativeStandardDeviation(RSD) observedforqualitycontrolsample data.The mean concentratioannd RSD were calculatefdorthefirsrtun and overthreerunsfor intra-assaaynd inter-assapyrecisionr,espectivelyQ.C I throughQC3 were assayed inreplicateosf five.In additiont,heRSD was reportedforstandardsatalllevels overthreeruns. 2.5.3. PartialVolume Analysis The effectof dilutionon the analysisof PFOS inratserum was determinedby partial volume analysis.QC4 (100 jig/mL)was preparedcontainingPFOS atapproximately fivetimestheupperlimitofquantitatio(nULQ). FivereplicateosfQC4 were dilutedten-foldand analyzed.The RSD and RE were reported. AOVANCEO BIOANALYTICAL SERVICES, INC. Page 12 of 50 99VDIAOI.Mi-DOC 2.5-4. Lower Limit of Quantitation(LLQ) Rat serum controlsamples from six separateindividualswere spiked with PFOS ata concentratioonf 0.05 @Lg/mL.The LLQ was determinedby obtainingthebackcalculatecdoncentrationfsrom thesecontrolsamples. The overallmean, RSD, and RE were alsocalculated. 2.5.5. Selectivity The selectiviotfytheassaywas determinedby LC/MS. To monitorforinterference from thebiologicamlatrix,ratserum samplescontainingneithertheanalytenorthe internasltandard(controlblank)were assayedwithallexperiments. 2.5.6. Carryover Evaluation The carryoverofanalytefrom one injectiotrofthenextwas assessedby analyzinga controlblankinjecteidmmediatelyaftera highcalibratiosntandard(STDIO, 20 Ag/mL). 2.6. STABILITY OF PFOS INQUALITY CONTROL SAMPLES QC I throughQC3 were usedtodeterminethestabilitoyf theanalyteduringsample storage,extractiona,nd analysis. 2.6.1. Ambient-Temperature StabilitoyfPFOS inRat Serum The stabiliotfyPFOS was evaluatedby storingQC samples ateach concentration levelatambienttemperature(ca.25 *C) fornominaltimepointsof 0,2.5,6,and 24 hoursafterthawing.All QCs were assayedinreplicateosf five. AOVANCEO SIOANALYTICAL SERVICES, INC. Page 13of50 99VDJAOI.Ml.DOC 2.6.2. FreezeSrtabiliotfyPFOS inRatSerumat-20*C ThefreezsetrabiloiftPyFOS wasevaluatbeydstorinQgC sampleasteach concentratiolnevelat-20 *C for7, 13,and 33 days.Additionalfreezerstability timepointswillbe reportedinan addendum to thisreport.AllQCs were assayedin replicateosf four. 2.6.3. Freeze/Thaw Stabilitiyn Rat Serum The stabilitoyf PFOS was evaluatedafterthreefreeze/thawcycles.The mean concentrationosf theQC samplesafterthreefreeze/thawcycles(i.e.a,tleast2 hours frozenstorageatthenominaltemperatureof-20 *C followedby thawingatambient temperaturefor30 minutes)were compared to themean concentrationosf freshly thawed QC samples.AllQCs were assayedin replicateosf five. 2.7. REPRODUCIBILITY OF REINJECTING EXTRACTED SAMPLES The reproducibiliotfyreinjectinrgeconstitutesderum extractwsas investigatebdy reinjectinagsetof previously-assayesdtandardsand QC sampleswhich had been storedafterinjectioantapproximately25 *C for27 hours.The RE of theQC sampleswas used to assessprocessedsample stabilitiyn thereconstitutiosnolution. AllQCs were assayedinreplicateosf five. 2.8. EXTRACTION REcovF.Ry The extractiornecoveryof PFOS from ratserum was determinedby comparingthe peak arearatio(PAR) ofsamples(0.25,4,and 16 gg/mL) spikedafterextraction (post-extracwti)ththePAR ofsamplesspikedbeforeextractio(npre-extractT)h.e internasltandardwas spikedpost-extractiofnorallsamples.The recoveryofthe internasltandard(4 gg/mL) was assessedfollowinga similarapproachusingPFOS as thereferenceT.he extractiornecovery(% Recovery)was determinedby dividing thepre-extracPtAR by thepost-extracPtAR and expressingtheresultas a percentage.Fivereplicatewsere used ateach concentratiolnevel. AOVANCEO SIOANALYTICAL Page14of50 MMM SERVICES, INC. 99VDJAOI.-MI.DOC 3. RESULTS AND DISCUSSION A quantitatiavnealyticparloceduruesingturboionsprayLC/MS inthenegativieon mode was developed to meet thehigh sensitivitsyp,ecificitayn,d reproducibility requirementsforthe determinationof PFOS inratserum. The full-scasningleMS mass spectrumof PFOS showed an abundant (M-K]' ionatm/z = 499 (Figure1). The full-scasningleMS mass spectrum of Tetra-H-PFOS showed an abundant [M-H]- ionatm/z = 427 (Figure2). The followingselectedionmonitoring(SIM) was used toquantifytheanalytesinrat serum: PFOS Tetra-H-PFOS (IS) m/z = 499.0 m/z = 427.0 The peak labelingof fall-scadnatadoes not accuratelyrepresentthe performanceof theinstrumentinSIM mode. The SIM ionswere derivedfrom separateexperiments usingnarrow-rangescanning,which more accuratelydepictstheoperationof the instrumentinthe SIM mode. The mass chromatograms of a representativceontrol blank ratserum extractareshown in'Figure 3. Mass chromatograms f@om a representativceontrolratserum extractcontainingonly theinternalstandard(zero sample),are shown inFigure4. Figures5 and 6 aremass chromatograms ofrepresentativextractsfrom calibration standardsI and 10,respectively. AOVANCED BIOANALYTir-AL SERVICES, INC. Page IS of 50 99VDJAOI.Mi.DOC 3.1. ASSAY EVALUATION RESULTS 3.1.1. Intra-and Inter-AssayAccuracy The intra-assaayccuracy(RE) datafrom QC samplesrangedftom -2.35to5.58% for PFOS (Table1).The inter-assaayccuracyrangedfrom -3.58to4.95% forPFOS (Table2).These dataindicataecceptableintra-and inter-assaayccuracyforthe determinatioonf PFOS inratsenim. 3.1.2. Intra-and Inter-AssayPrecision The intra-assapyrecisiondata(RSD) ranged ftom 1.99to 3.28% forPFOS atallQC concentratiolnevels(Table1).The inter-assapyrecisionresultsfrom QC samples rangedfrom 1.92to4.87% forPFOS (Table2).The RSD rangedfrom 0.613to 4.38% forcalibratiosntandardsatalllevels(Table3).These dataindicataecceptable intra-anidnter-assapyrecisionforthedeterminationof PFOS inratserum. 3.1.3. Linearity The calibratiocnurveswere fitby a weighted(1/y2)quadraticregression. Coefficientosfdeterminatio(nr2 )were 2:0.9962forPFOS inratserum. The calibratiocnurvestatistiacrseshown inTable 4. 3.1.4. PartialVolume Analysis The resultsof partiavlolume analysisof QC4 isshown inTable 5. The precision (RSD) ofQC4 samplesdilutedIin10 was 2.71%. The accuracywas 5.89%. These dataindicataecceptableaccuracyand precisionforpartiavlolume analysisforthe determinatioonfPFOS inratserum. ADVANCEO SIOANALYTICAL SERVICES, INC. Page 16 of 50 99VDJAOI.Mi.DOC 3.1-5. Lower Limit of Quantitation(LLQ) Table 6 shows the lower limitof quantitation(LLQ) data.The serum LLQ experimentdemonstratedthat0.05pg/mL isan acceptableLLQ forPFOS. The precisio(nRSD) was 4.95% forPFOS. The RE was 4.57%. 3.1.6. Selectivity The assaywas specififcorPFOS. No chromatographicinterferencewsere observed inany of thecontrolserum samplesanalyzed(Figure3). The controlblanksand zerosamplesdidshow some evidenceof smallchromatographicpeaks atthe retentiotnimesof theanalyte.These peaks were not quantifiablaes theywere below thelowerlimitofquantitati(oLnLQ). 3.1.7. Carryover Evaluation CarryoverofPFOS and theIS was evaluatedby injectioonfan extracterdatserum controlblankfollowingan injectioonf an extractehdigh standard(STD 10).The responseforthesmallchromatographicpeak attheretentiotnime forPFOS was comparabletotheresponseof a ratserum controlblank injectebdeforethehigh standardT.hus,carryoverisnegligiblfeorPFOS. There was no evidenceof carryoverfortheinternasltandard. 3.2. STABILITY OF PFOS IN QUALITY CONTROL SAMPLES 3.2.1. Ambient Temperature Stabilityof PFOS in Rat Serum The resultosf theambient stabilitoyf PFOS inQC samplesareshown inTable 7. The mean predictedconcentrationdseviatedfrom 4.45 to 10.5% from the0-hour valuesforallQC levelsand timepoints.Based on thesedata,PFOS was stableinrat serum forup to24 hoursatambienttemperature. AOVANCEE) SIOANALYTICAL SERVICES. INC. Page 17 of 50 99VDJAOI.Mi.DOC 3.2.2. Freezer Stabilityof PFOS in Rat Serum at-20 *C The resultosf thelong-termstabilitoyfPFOS inQC samples storedat-20 OC are presentedinTable 8. The PFOS concentrationdseviatedfrom 0-hourvaluesfrom -2.76to 12.3% forallQC levelsstoredup to33 days. Resultsof freezerstorageaftertwo,four,and eightmonths willbe reportedas an addendum to thisreport. 3.2.3. Freeze/Thaw Stabilityof PFOS in Rat Serum The resultsof thefreeze/thawstabilitoyf PFOS in QC samples afterthree freeze/thawcyclesisshown inTable 9. PFOS was stableafterthreefreeze/thaw cycleswithdeviationsrangingfrom -1.38% to 10.1% from the0-cyclesamplesfor allQC level(sTable9). 3.3. REPRODUCIBILITY OF REINJECTING EXTRACTED SAMPLES The resultsofreinjectinrgeconstitutesdamplescontainingPFOS afterstoragefor27 hoursatapproximately25 *C inreconstitutisoonlutionareshown inTable 10. Reinjectinpgrocessedsamplesafter27 hours inreconstitutisoonlutionwas appropriatweithRE valuesrangingfrom -6.33to 4.59% at27 hoursforallQC levels. 3.4. EXTRACTION REcovERY The mean recoveriesof PFOS and Tetra-H-PFOS areshown in Table 11. The mean recoveriesrangedfrom 87.6to 100% forPFOS The mean recoveryforTetra-HPFOS was 89.9%. AOVANCEO E310ANAt-YTICAL Page 18of50 MMM SERVICES, INC. 99VDIAOI.Mi.DOC 4. CONCLUSIONS TheturbioonsprayLC/MS assapyrocedurfeorthedeterminatoifoPnFOS inrat serum has proven tobe sensitives,pecifica,ccuratea,nd reproducibleI.tshigh sensitiviatlylowsreliablaend reproduciblqeuantitatioonf PFOS down toa levelof 0.05 gg/mL inratseruxnbased on 50-gL samples. 5. DATA RETRIEVAL The datafortheratserum validatioanrestoredinA13S Notebook 2304 and inthe ABS Archives. 6. REFERENCES 1. Advanced BioAnalyticaSlervicesI,nc.Report98AGKPO2.MMM. AnalyticaRleportfortheDeterminationof Perfluorooctanoataend PerfluorooctanesulfoniantHeuman Serum by LC/MS. GraceK. Poon, Ph.D.,David Hardwick,B.S.,and Ellen'PaceM,.A.T.,6 February1998. AOVANCEO BIOANALYTICAL SERVICES, INC. Page 19 of 50 99VDIAOI.Ml.DOC 7. TABLES Table 1: Intra-Assay Accuracy and Precisionof the PFOS Assay in Rat Serum forQC Samples TheoreticalConc.- PFOS Concentration(gg/ML) QCI QC2 QC3 0.2 6 18 Run 9 0.210 6.52 17.9 0.202 6.31 18.0 0.206 6.17 16.7 0.205 6.32 18.0 0.194 6.35 17.3 Mean RSD RE 0.204 6.34 17.6 2.93 1.99 3.28 1.77 5.58 -2.35 RSD = (SD/Mean) x 100 RE [(Mean/TheoreticalI)]-x 100 AOVANCED SIOANALYTICAL SERVICES. INC. Page 20 of50 99VDJAOI.MI.DOC Table 2: Inter-AssayAccuracy and Precisionof the PFOS Assay in Rat Serum forQC Samples from Three ValidationRuns TheoreticalConc. Run 9 PFOS QCI 0.2 0.210 0.202 0.206 0.205 0.194 Concentration(gg/mL) QC2 QC3 6 18 6.52 17.9 6.31 18.0 6.17 16.7 6.32 18.0 6.35 17.3 Run 10 0.187 6.44 16.9 0.186 6.44 17.3 0.182 6.32 17.5 0.189 6.19 18.0 0.182 6.31 17.4 Run 11 0.194 6.17 18.2 0.197 6.13 17.1 0.184 6.41 17.6 0.192 6.15 17.7 0.184 6.24 17.0 Mean 0.193 6.30 17.5 RSD 4.87 1.92 2.66 RE -3.58 4.95 -2.73 RSD = (SD/Mean) x 100 RE= [(Mean/TheoreticalI)]-x 100 AOVANCEO BIOANALYTICAL SERVICES, INC. Page 21 of 50 99VDJAOI.Mi.DOC Table3: AccuracyandPrecisiofnthePFOS AssayinRatSerumfor CalibratiSotnandardfsromThreeValidatiRounns PFOSCalibratSitoanndaCrodncentrat(igogn/mL) TheoreticalSTDI@ STD2 STD3 STD4 STD5 STD6 STD7 STD8 STD9 sT-DIO Conc. 0.05 0.1 0.25 O.S 1 2 4 8 16 20 Run 9 0.0483 0.0972 0.251 0.480 0.942 2.10 0.0530 0.101 0.252 0.483 0.938 2.13 Run 10 0.0491 0.0951 0.239 0.469 0.954 2.16 0.0542 0.101 0.251 1 0.479 0.942 2.11 Run 11 0.0510 0.0960 0.251 0.469 0.945 2.21 0.0510 0.0995 0.252 0.477 0.948 2.13 4.48 7.61 15.8 20.0 4.5.5 7.70 16.2 19.9 4.58 8.05 19.6 4.43 7.87 155-97 20.0 4.40 7.64 15.8 20.3 4.53 7.78 15.6 20.0 Mean 0.0511 0.0983 0.249 0.476 0.945.- 2.14 4.50 7.78 15.8 20.0 RSD (%) 4.38 2.65 2.07 1.15 0.613 1.90 1.62 2.13 1.22 1.08 RE (%) 2.20 1 -1.67 1 -0.207 1 -4.78 1 -5.53 1 7.05 f 12.4 1 -2-80 1 -1.00 -0.0764 RSD = (SD/Mean)x 100 RE= [(Mean/TheoreticaIl])x-100 AOVANCED SIOANALYTICAL SERVICES, INC. Page22 of50 99VDJAOI.Ml.DOC Table 4: CalibrationCurve Parameters forPFOS inRat Serum Run Quadratic Coefficient Intercept Coefficienotf Coefficien(tA)" Linear(B) (C) Determinatio(nr') 9 -0.0010769 0.13730 0.00042009 0.9965 10 -0.0010516 0.14535 0.0014910 0.9962 11 -0.0012359 0.15173 0.00088515 0.9964 @ll I Mean 0.14479 0.00093206 MoM-um=-M=w 0.9964 I a:y Ax' +Bx + C, weighted I/Yl AOVANCEO BIOANALYTICAL SERVICES. INC. Page 23 of 50 99VDJAOI.Mi.DOC Table 5: PartialVolume AnalysisofPFOS inRat Serum PFOS Conc.(gg/mL) QC4 TheoreticaClonc. 100 Run 9 (I in10dilution) 111 105 105 104 104 Mean 106 RSD 2.71 RE (%) 5.89 D = (SD/Mean) x 100 RE [(Mean/TheoreticaIl])x- 100 . ........................ AOVANCE[) SICANALYTICAL SERVICES. INC. Page 24 of 50 99VDJAOI.mi-DOC' Table 6: Lower Limit ofQuantitationofPFOS inRat Serum Run 11 PFOS 'McoreticaCloncentrationConc.Found (gg/mL) 0.0548 LLQ 0.0489 (0.05gg/mL) 0.0499 0.0554 0.0527 0.0520 Mean 0.0523 RSD (%) 4.95 RE 4.57 RSD (SD/Mean)x 100 RE [(Mean/Theoretical)x-11]00 AOVANCEID BIOANALYTICAL SERVICES. INC. Page 25 of50 99VDJAOI.Mi-DOC p Table 7: Ambient-Temperature Hours Stability of PFOS in Rat Serum After 24 Run I I PFOS (gg/mL) 0--NM-m@ QC (TheoreticaClonc.) QC 1 (0.2gg/mL) Mean RSD (%) %DEV from 0 Hour 0 Hour 0.194 0.197 0.184 0.192 0.1840.190 3.03 NA 2.5 Hour 0.187 0.181 0.184 0.180 0.178 0.182 1.91 -4.20 6 Hour 0.177 0.179 0.180 0.183 0.189 0.182 2.52 -4.45 24 Hour 0.190 0.187 0.191 0.182 0.190 0.188 2.01 -1.16 6.17 6.17 6.36 6.55 QC 2 (6gg/mL) 6.13 6.30 6.38 6.53 6.41 6.26 6.35 6.85 6.15 6.28 6.22 6.66 6.24 6.24 6.11 6.81 Mean RSD (%) %DEV from 0 Hour 6.22 6.25 6.28 6.68 1.82 0.797 1.86 2.16 NA 0.525 1.05 7.42 QC 3 (18gg/mL) 18.2 18.0 18.1 19.1 17.1 18.0 18.0 19.4 17.6 17.7 17.6 19.2 17.7 17.9 18.2 19.4 17.0 18.0 18.0 19.7 Mean RSD (%) O/oDEV from 0 Hour 17.5 17.9 18.0 19.4 2.84 0.719 1.26 1.19 NA 2.33 2.39 10.5 NA: Not Applicable RSD = (SD/Mean) x 100 %DEV from 0 Hour [(X Hour Conc. - 0 Hour Conc.)/OHour Conc.]x 100 AOVANCED BIOANALYTICAL SERVICES, INC. Page 26 of 50 99VDJAOI.Ml.DOC Table 8: Freezer Stabilityof PFOS in Rat Serum at -20 OC QC PFOS g/mL) Nominal Concentration 0 Hour (Run 2)* 7 Days 6)* 13 Days (Run 3)** QC 1 (0.2ug/mL) 0.213 0.209 0.208 0.208 0.207 0.225 0.222 0.218 0.219 0.211 0.210 0.217 0.214 Mean RSD (%) %DEV from 0 Hour 0.209 1.24 NA 0.221 1.32 5.72 0.213 1.55 1.84 6.02 6.22 5.97 QC 2 5.71 6.15 5.84 (6ug/mL) 6.00 6.39 6.07 6.06 6.27 5.99 5.69 Mean RSD (%) %DEV from 0 Hour 5.89 3.08 NA 6.26 1.62 6.19 5.97 1.58 1.20 QC 3 (18ug/mL) Mean RSD (%) %DEV from 0 Hour 20.2 18.6 18.9 18.2 18.9 18.9 3.96 NA 19.1 19.5 19.8 19.3 19.4 1.37 2.52 18.8 18.2 18.8 17.9 18.4 2.42 -2.76 NA: Not Applicable RSD = (SD/Mean) x 100 %DEV from 0 Hour = [(Conc.- 0 Hour Conc.)/OHour Conc.] x 100 *From ProtocolFACT-TOX- I10 **From ProtocolFACT-TOX-1 11 33 Days (Run 16) 0.245 0.234 0.228 0.232 0.235 3.17 12.3 6.45 6.13 6.58 6.37 6.38 2.97 8.28 18.6 18.8 18.7 18.7 18.7 0.395 -1.30 AOVANCEED SICANALYTICAL SERVICES, INC. Page 27 of 50 99VDJAOI.Mi.DOC Table 9: StabilitoyfPFOS inRat Serum AfterThree Freeze/Thaw Cycles Run 10 QC (TheoreticaClonc.) QC 1 (0.2gg/mL) Mean RSD (%) %DEV from 0 Cycle PFOS (gg/mL) 0 Cycle 3 Cycles 0.187 0.177 0.186 0.177 0.182 0.185 0.189 0.185 0.182 0.189 0.185 0.182 1.70 3.12 NA -1.38 6.44 QC 2 6.44 (6jig/mL) 6.32 6.19 6.31 Mean 6.34 RSD (%) 1.66 %DEV from 0 Cycle NA 6.59 6.61 6.44 6.39 6.40 6.49 1.63 2.35 16.9 19.2 QC 3 17.3 19.1 (18gg/mL) 17.5 19.2 18.0 19.6 17.4 18.7 Mean 17.4 19.2 RSD (%) 2.31 1.80 %DEV from 0 Cycle NA 10.1 NA: Not Applicable RSD = (SD/Mean) x 100 %DEV from 0 Cycle= [(3Cycle-0 Cycle)/OCycle]x 100 MM M AOVANCEE) BIOANALYTICAL SERVICES. INC. Page 28 of 50 99VDIAOI-MI-DOC Table 10: ReproducibilitoyfReinjectingExtractedSamples Containing PFOS after27 Hours inReconstitutioSnolution QC 1; (0.2gg/mL) Mean RSD (%) RE QC 2 (6gg/mL) Mean RSD RE QC 3 (18gg/mL) Mean RSD RE (%) NA: NotApplicable RSD = (SD/Mean)x 100 RE = [(MearvneoreticaIl]x)-100 PFOS t=o Hours (R n 10) 0.187 0.186 0.182 0.189 0.182 0.185 1.70 -7.50 6.44 6.44 6.32 6.19 6.31 6.34 1.66 5.62 16.9 17.3 17.5 18.0 17.4 17.4 2.31 -3.29 W'mL) t--27Hours (Run 12) 0.187 0.182 0.188 0.188 0.190 0.187 1.61 -6.33 6.39 6.02 6.19 6.55 6.22 6.28 3.23 4.59 17.3 18.2 18.1 17.3 17.6 17.7 2.40 -1.73 AOVANCED SIOANALYTICAL SERVICES. INC. Page29 of50 99VDJAOI.Ml.DOC Table 11: Extraction Recovery of PFOS from Rat Serum TheoreticalConc. Pre-Extraction Post-Extraction inserum (gg/mL) PFOS Spike 1 (0.25pg/mL) Run 14 Mean PFOS Spike 2 (4gg/mL) Run 14 Mean PFOS Spike 3 (16gg/mL) Run 14 Mean Tetra-H-PFOS Spike IS (4;lg/mL) Run 13 Mean Response (AreaRatio)Response (AreaRatio) 0.03160 0.02850 0.02930 0.02790 0.03050 0.02880 0.03010 0.02950 0.02930 0.02960 0.02956 0.02946 0.4597 0.4751 0.4776 0.4724 0.4772 0.5270 0.5304 0.5335 0.5298 0.5392 0.4724 0.5320 1.649 1.874 1.679 1.857 1.719 1.535 1.612 1.869 1.887 1.864 1.639 1.870 0.4068 0.4460 0.3884 0.4043 0.3921 0.4078 0.4423 0.4445 0.4512 0.4412 0.3999 0.4450 O/oRecovery* 110 94.7 99.3 95.2 103 100 87.2 89.6 89.5 89.2 88.5 88.8 88.0 90.4 92.0 81.4 86.5 87.6 91.2 87.8 91.0 86.9 92.4 *(IndividuaRlesponse forPre-Ext./IndividuRaelsponse forPost-Ext.x) 100 AOVANCEO E310ANALYTICAL SERVICES, INC. Page 30 of 50 99VDJAOI.Mi.DOC 8. FIGURES Figure 1: Full-ScanSingleMS Mass Spectrum ofPFOS Spectrumfrom PROS Q I 98 91 84 77 70 63 56 49 42 35 28 21 14 - 7. 349 360 3.19e7 cps 499 [M-Kl- 399 449 L 420 480 540 ff,/Z AOVANCEE) SIOANALYTICAL SERVICES. INC. Page 31 of 50 99VDJAOI.Mi.DOC Figure2: Full-ScanSingleMS Mass Spectrum ofTetra-H-PFOS Spectrum from Tetm-H-PFOS Q] 96 90 84 78 72 66 60 54 48 42 36 30 24 18 12 6 350 407 L 400 nl/z 1.94e7 cps 427 [M-H]- 450 AOVANCEO E310ANALYTICAL SERVICES. INC. Page 32 of50 99VDJAOI.Ml.DOC Figure3: Mass Chromatograms ofPFOS and itsInternalStandard in ControlBlank Rat Serum Extract rrvz 427.0 I.O7c3cps 90 . so 70 60 so 40 30 20 10 - ni/z 499.0 2 3 Time, min 4 3.54e2 cps 90 so 70 60 50 40 . 30 20 - 10 1 2 3 4 Time, min Allquantitationresultsand figureswere prepared from non-smoothed data. AnaMe PFOS Tetra-H-PFOS (IS) Ion ni/z= 499.0 mlz = 427.0 RetentionTime 3.1 2.9 AOVANCED BIOANALYTICAL SERVICES. INC. Page 33 of 50 99VDIAOI.Mi.DOC Figure 4: Mass Chromatograms ofPFOS ina Rat Serum ExtractSample ContainingInternalStandard Only (ZeroSample) mlz 427.0 2.34e5 cps 9080 70 60 50 40 30 20 10 ffi/z 499.0 90 80 70 60 50 40 30 20 10 - 2 Time, min 4 3.78e2 cps 1 2 3 Time, min Allquantitationresultsand flgureswere prepared from non-smoothed d2t2. Anal= PFOS Tetra-H-PFOS (IS) lm mlz = 499.0 m/z - 427.0 RetentionTime 3.1 2.9 AOVANCEO BIOANALYTICAL SERVICES, INC. Page 34 of 50 99VDJAOI.Ml.DOC Figure5: Mass Chromatograms ofCalibrationStandard I inRat Serum ExtractContainingPFOS (0.05gg/mL) and theInternal Standard ni/z 427.0 3.82e5 cps 90 80 70 60 so 4030 20 10 rn/z 499.0 2 3 Time. min 4 2.1lc3 cps z 90 80 70 60 so 40 30 20 10 1 2 3 4 Time,min Allquantitationresultsand flgureswere prepared from non-smoothed data. AnaL= PFOS Tetra-H-PFOS (IS) im rn/z= 499.0 m/z = 427.0 RetentionTime 3.1 2.9 AOVANCEO BIOANALYTICAL SERVICES. INC. Page 35 of 50 99VDJAOI.Mi.DOC Figure6: Mass Chromatograms ofCalibrationStandard 10 inRat Serum ExtractContainingPFOS (20gg/mL) and theInternalStandard m/z 427.0 3.52e5 cps 9080 70 60 50 40 30 20 10 1 m/z 499.0 90 80 70 60 50 40 30 20 10 2 3 Time, min 4 5.l4e5cps 1 2 3 4 Time, min Allquantitationresultsand flgureswere prepared from non-smoothed data. AnalZe PFOS Tetm-H-PFOS (IS) L9jl m/z = 499.0 ni/z= 427.0 RetentionTime 3.1 2.9 AOVANCED SIOANALYTICAL SERVICES, INC. Page 36 of 50 99VDJAOI.Mi.DOC 9. APPENDIX A: QUANTITATION PERFLUOROOCTANESULFONATE TURBO ION SPRAY LC/MS OF (PFOS) IN RAT SERUM BY AUTHORS: David J. Anderson, M.S. Amie J.Prince,B.S. HollyD. Ross,M.S. TABLE OF CONTENTS A.1.0 A.2.0 A.3.0 A.4.0 A.5.0 A.6.0 A.7.0 A.8.0 A.9.0 A.10.0 A. 11.0 Chemical Structure(s.)...............................................3.8.................... Specimen(s)........................................................3.8.................... Assay Principle.....................................................3.8..................... Compounds ........................................................3.8..................... Chemicals .........................................................3.8..................... Materialsand Equipment .............................................3.9..................... Preparationof Solutions..............................................4.2.................... Preparationof QualityControlSamples .................................4.5..................... Liquid-liquiEdxtractioPnrocedure.....................................4.6..................... InstrumentConditions...............................................4.7..................... Calculations........................................................4.9.................... MMM AOVANCEED SICANALYTICAL SERVICES, INC. Page 37 of 50 99VDJAOI.Mi.DOC A.1.0CHEMICAL STRUCTURE(S) 0 11 CgF,7-S-0-K+ 11 0 C6FI3-C H2 0 11 C-S-OH H2 11 0 PotassiumPerfluorooctanesulfonate MW = 538 1H, IH, 2H, 2H-PerfluorooctanSeulfonicAcid MW = 428, InternaSltandard A.2.0 SPECIMEN(S) This assayuses 50-@LLaliquotsofratserum. Rat serum samples arestoredat-20*C. A.3.0 ASSAY PRINCIPLE PFOS and itsinternalstandardT,etra-H-PFOS, areextractedfrom ratserum samples (50 gL) usinga liquid-liqueixdtractiopnrocedure.The organiclayerisevaporated todrynessand thenreconstitutedA.n aliquotisthenanalyzedby turboionspray liquidchromatography/massspectrometry(LC/MS) inthenegativeionmode. A.4.0 COMPOUNDS PFOS: Lot# 171,99.976% purity3,M, Inc.,Minneapolis,NfN. Tetra-H-PFOS: Lot# 59909,90% purity,3M, Inc.,Minneapolis,N4N. A.5.0 CHEMICALS Allchemicalsmay be substitutewdiththatof an equivalentmanufacturerand grade ofchemical. MMM AOVANCEO BIOANALYTICAL SERVICES. INC. Page 38 of50 99VDJAOI.Ml.DOC Acetonitrile Ammonium Acetate EthylAcetate Methanol Rhodapex mass calibration solution Sodium Bicarbonate Sodium Carbonate,Annhydrous Tetrabutylammonium Hydrogen Sulfate Water Rat Serum Rat Serum Cat# 0154, Burdick& Jackson, Muskegon, MI 49442 Cat# 24,019-2,Aldrich,Milwaukee,Wi 53201 Cat# 100-4,Burdick& Jackson, Muskegon, MI 49442 Cat# 230-4,Burdick& Jackson, Muskegon, MI 49442 A mixtureof 0.11 mM trifluoroacetaiccid (J.T.Baker,PhillipsburNgJ, 08865),0.52 mM Rhodapex CO-436, I mM ABEX EPI10 and I mM ABEX EP- 120 (allthree suppliedby Rhone-Poulenc,Inc., Cranbury,NJ 08512) in 1:1 methanol:water. Cat# 3509-01,J.T.Baker,PhillipsburgN,J 08865 Cat# 3604-01,J.T.Baker,PhillipsburgN,J 08865 HPLC Grade,Cat# J360-07,J.T.Baker, PhillipsburNgJ, 08865 High Purity(NANOpure), Bamstead Model D7331 UltrapureWater System, Dubuque, IA 52001 Lampire BiologicaLlaboratories, PipervillPeA, 18947 HarlanBioproductsforScience,Inc., IndianapolisI,N 46229 A.6.0 MATERIALS AND EQUIPMENT Mass Spectrometer PE SCIEX API 365 atmosphericpressureionization tandem triplqeuadrupolemass spectrometerequipped withTurbolonSprayTMinterfaceP,E SCIEX, Concord, OntarioL4K 4V8 Data system API StandardSoftware,MacQuan v 1.4,LC2Tune v 1.3,MacDAD v 1.3,Bundlerv 1.3,Multiviewv 1.3, AOVANCEO SICANALYTICAL Page 39 of 50 MMM SERVICES. INC. 99VDJAOI.Ml.DOC HPLC Pump and Sample Controlv 1.3,on a Power Macintosh,PE SCIEX, Concord,OntarioL4K 4V8 Shimadzu LC- IOAD pump, Shimadzu Co.,Columbia, MD 21046 LC Pump Controll,-.r Shimadzu SCL- IOA, Shimadzu Co.,Columbia, MD 21046 Autosampler Waters717plus,Waters AssociatesM,illipore CorporationM,ilford,MA 01757 HPLC Column BetasilCl,,5 gm particlseize,2.1x 50 mm, Cat #852055-701,Keystone ScientifiIcn,c.,BellefonteP,A 16823 Harvardsyringe pump II HarvardApparatusInc.,South Natick,MA 01760 Multi-tubveortexer Balance pH Meter Mechanicalshaker NANOpure-LTV waterpurification system Cat# 58816-115,VV*RR ScientificW,est Chester,PA 19380 Model FX-300, AND Ltd.,Tokyo, Japan Model 340,Coming Inc.,Coming, NY 14830 Cat# 6000,Eberbach Corp.,Ann Arbor,MI 48106 Bamstead,Dubuque, IA 52001 Microbalance Model MT-5, Mettler-ToledIonc.,Hightstown,NJ 08520 Micro weigh boats Weigh boats SorvallRT-6000D refrigerated centrifuge Cat# 0219-0041,Perkin-ElmerCorp.,Norwalk, CT 06859 Cat# 12577-025,VVRR ScientificW,est Chester,PA 19380 Cat# 83071,DuPont Co.,Wilmington,DE 19898 Beckman GS6KR Refrigerated centrifuge Beckman, PaloAlto,CA 94304 AOVANCEE:) BIOANALYTICAL SERVICES, INC. Page 40 of50 99VDJAOI.M[.DOC Turbovap LV Evaporator Pipettes Pipettetips Polypropylenetubes Screw-capped Polypropylenevials Screw-capped Polypropylenevials PlugTite MultipurposeCaps Pyrex volumetric flask SolventFiltration Apparatus Nylon Titan Membrane Filters PEEK tubing LiquidNitrogen GastightGlass Syringes Cat#43750,Zymark InstrumentsH,opkinton,MA 01748 Cat# P-5000,P-1000,P-200,P-100,RaininInstrument Co., Wobum,MA01888 RT-20, RT-200, C-5000, CP-25, CP-50, CP-250, RaininInstrumentCo.,Woburn, MA 01888 13 mm x 100 mm, Cat# 15070-574,VWR Scientific, West Chester,PA 19380 16.5x 57 mm, Cat# 60.542,SarstedtI,nc.,Newton, SC 28658 16.5x 101 mm, Cat# 60.541,SarstedtI,nc.,Newton, SC 28658 Cat# 78-127-0019-100,Elkay Products,Inc., Worcester,MA 0 1607 Cat# 28014P-10,28014P-25, 28014P-100,28014P1000,Kimble/Kontes,Vineland,NJ 08360 Cat# 953781-0000,953751-0000,953753-0000, 953826-0000,953827-0000, Kimble/Kontes,Vineland, NJ 08360 0.45@Lm ScientifiRcesources Inc.,Cat# 74547-NN, Eatontown,NJ 07724 0.005"i.d.x 1/16"o.d.,Cat# 1535,Upchurch Scientific Inc.O,ak Harbor,WA 98277 Suppliedin-housefrom bulk tank,BOC Gasses, Buffalo,NY 14210-2005 Cat# 1750,Hamilton Company, Reno, NV 895 10 Othergenerallaboratoryglasswareand supplieswere used. AOVANCED BIOANALYTICAL SEPV'r'F=c-, IN'r' Page 41 of 50 99VDJAOI.MI.DOC A.7.0PREPARATION OF SOLUTIONS NANOpurewater(18megaohm-cm.o)requivalesnhtoulbdeusedwherevewrateirs calledfor.Mix allsolutionwsell. A-7,1 PreparationofAnalyticalStandard Stock Solutions AnalyticalStandard Stock Solution,PFOS (I mg/mL): Weigh approximatel5y mg of PFOS (aftecrorrectiofnorpurity)on a microbalanceand transfeirttoa polypropylenevial.Dilutewiththeappropriatveolume ofmethanoltoyielda I mg/mL solutionP.reparea freshsolutioneverythreemonths. Storethesolutioant 4 *C and bringtoambient temperaturebeforeuse. Standard Spiking Solutions PrepareStandardWorking SolutionsA throughJ in 5-mL class"A" volumetric flasksaccordingtothefollowingdilutiosncheme. Dilutetothe5-mL mark with watertoyieldthefinalconcentrationP.reparefreshsolutionseverythreemonths. Storethesolutionast4 'C and bringto ambienttemperaturebeforeuse. Standard Working A B c D E F G H 1 Volume Spiked SolutionUsed (gL) 1000 Stock 800 Stock 400 Stock 200 Stock 100 Stock 250 200 gg/mL (A) 125 200 gg/mL (A) 62.5 200 gg/mL (A) 500 10 gg/mL (F) 250 10 gg/mL (F) PFOS Final Conc. .(@tg/mL) 200 160 80 40 20 10 5 2.5 1.0 0.5 AOVANCEO IBIOANALYTICAL SERVICES, INC. Page 42 of 50 99VDJAOI.Mi.DOC A.7.2 PreparatioonfInternaSltandardStockSolutions InternaSltandardStockSolutionT,etra-H-PFOS(Img/mL): Weigh approximately5 mg of Tetra-H-PFOS on a microbalanceand transferitto a polypropylenevial.Dilutewith an appropriatevolume of methanol toyielda I mg/mL solutionP.reparea freshsolutionevery threemonths. StoretheSo lutionat 4 *C and bringto ambient temperaturebeforeuse. A.7.3 Preparation ofInternalStandard Working Solution InternalStandard Working Solution (4 jig/mL Tetra-H-PFOS): Add 400 iLLof InternaSltandardStock Solutionto a I00-mL class"A" volumetricflask.Diluteto themark with water togivea 4 gg/mL Tetra-H-PFOS solution.Storethe solutionat 4 *C and bringto ambient temperaturebeforeuse. Preparefreshsolutionas needed. A.7.4 Preparation of Standard Curve and Control Blank Preparefreshcalibratiosntandardsforeach analyticalrun by combining 360 p.Lof ratcontrolserum and 40 gL ofthe analyticasltandardworking solution(seetable below) inlabeled1.7-mL microtubes.Preparecontrolblanksand zero samplesby combining 360 gL of ratcontrolserum with 40 pLLof water. Vortex each for60 seconds. AOVANCEE) BICANALYTICAL SERVICES. INC. Page43of50 99VDJAOI.mi.DOC AnalyticaSltandardDilutioTnable: @Stan,1dnd=arrdd 10 9 8 7 6 5 4 3 2 1 Volume SSppiVikoke'edu:dm((@pL) 40 40 40 40 40 40 40 40 40 40 Solutiotno use A B c D E F G H 1 1 gL control serum 360 360 360 360 360 360 360 360 360 360 MalConc. (jig/mL) 20n@oLn)c* 16 8 4 2 1 0.5 0.25 0.1 0.05 A.7.5 Preparation of Other Solutions 0.25 M Sodium Carbonate/0.25 M Sodium Bicarbonate: Weigh 26.5 g of sodium carbonateand 21 g of sodium bicarbonateand dissolvein approximately900 mL of HPLC-grade water. Transferthesolutionto a 1000-mL volumetricflaskand dilute tothemark with HPLC-grade water. Mix thesolutionthoroughly.Storeatambient temperature.Preparea freshsolutionevery threemonths. I M Ammonium Acetate: Add 7.7 g of ammonium acetateto a 100-mL volumetric flask,dissolveinNANOpure water,and stiruntilcompletelydissolved.Dilutetothe mark with NANOpure water. Storeatambient temperature.Preparea freshsolution every threemonths. 2 mM Ammonium Acetate: Combine 2 mL of IM ammonium acetateand 900 mL NANOpure water ina I000-mL volumetricflask;stiruntilcompletelymixed. Dilute tothe mark with NANOpure water.Store atambient temperature.Prepare a fresh solutionevery threemonths. am= AOVANCEC:) BIOANALYTICAL SERVICES. INC. Page 44 of 50 99VDJAOI.Mi.DOC 10 M SodiumHydroxide:Add 40gofsodiumhydroxidteoa 100-mLvolumetric flaskd,issolveinNANOpure water,and stiruntilcompletelydissolved.Dilutetothe mark withNANOpure water.Storeatambienttemperature.Preparea freshsolution everythreemonths. 1 M Sodium Hydroxide: Add 10 mL of 10 M sodium hydroxidetoa 100-mL volumetricflask.Dilutetothemark with NANOpure water;stiruntilcompletely mixed. Storeatambienttemperature.Preparea freshsolutioneverythreemonths. 0.5M Tetrabutylammonium Hydrogen Sulfate,pH 10: Weigh 16.98g of tetrabutylammoniumhydrogen sulfateand dissolvein40 mL of NANOpure water. AdjustedthepH to 10.0with 10 M and I M sodium hydroxide.Transferthesolution to a I00-mL volumetricflaskand dilutetothemark withNANOpure water. 10:90Methanol:2 mM Ammonium Acetate(Mobile Phase EluentA): Add 100 mL of methanol and 900 mL of 2 mM ammonium acetateto a 1000-mL Pyrex bottle. Mix thesolutionthoroughlyand filtetrhesolutiotnhrougha 0.45gM filtewritha vacuum flaskS.toreatambienttemperature.Preparea freshsolutioneverythree months. 90:10 Methanol:2 mM Ammonium Acetate (Mobile Phase Eluent B): Add 900 mL of methanoland 100 mL of 2 mM ammonium acetatetoa I000-mL Pyrex bottle. Mix thesolutionthoroughlyand filtetrhesolutionthrougha 0.45PLM filtewritha vacuum flask.Storeatambienttemperature.Preparea freshsolutioneverythree months. A.S.0 PREPARATION OF QUALITY CONTROL SAMPLES A.8.1 Preparation of QualityControl Stock Solutions QC Stock Solution,PFOS (I mg/mL): Weigh approximately5 mg of PFOS (after correctiofnorpurityo)n a microbalanceand transfeirttopolypropylenevial.Dilute AOVANCE[) BIOANALYTICAL SERVICES. INC. Page 45 of 50 99VDJAOI.Mi.DOC withtheappropriatveolume ofniethanoltoyielda I mg/mL solution.Storethe solutionat4 OC and bringtoambienttemperaturebeforeuse. Preparea fresh solutionevery threemonths. A-8.2 Preparation of Serum QC Samples QC4 (DilutionQC, 100 gg/mL PFOS): Add 1000;iL QC Stock Solutiontoa 10mL class"A" volumetricflasktoyielda 100-lig/mLsolutionD.ilutetothemark withcontrolserum,cap and mix.Aliquotsof approximately0.2mL areplacedin polypropylenevialsand storedfrozenat-20 *C. QC3 (18gg/mL PFOS): Add 450 gL QC StockSolutiontoa 25-mL class"A" volumetricflasktoyielda 18-gg/mL solution.Dilutetothemark withcontrol serum,cap and mix.Aliquotsof approximately0.5mL areplacedinpolypropylene vialsand storedfrozenat-20 'C. QC2 (6jig/mLPFOS): Add 150 liLof QC StockSolutiontoa 25-mL class"A" volumetricflasktoyielda 6-gg/mL solution.Dilutetothemark withcontrolserum, cap and mix.Aliquotsof approximately0.5mL areplacedinpolypropylenevials and storedfrozenat-20 *C. QCI (0.2@Lg/mL PFOS): Add I11.1 pL ofQC3 toa I0-mL class"A" volumetric flasktoyielda 0.2-gg/mL solutionD.ilutetothemark withcontrolserum,cap and mix.Aliquotsof approximately0.5mL areplacedinpolypropylenevialsand stored frozenat-20 *C. A.9.0 LIQUID-LIQUID EXTRACTION PROCEDURE 1. Preparefreshcalibratiosntandardsforeach run accordingtoSectionA.7.4. 2. Thaw QC samples. 3. PrepareQC4 samples(diluteIdin 10)intwo steps: a)Add 180 liLofcontrolserum and 20 gL ofQC4 into1.7-mL microtubesand AOVANCEO BIOANALYTICAL SERVICES. INC. Page 40 of 50 99VDJAOI.Mi.DOC vortex. b)Aliquot50 liLofdilutedQC4 intolabeled16 x 100 mm Polypropylenetubes. 4. Aliquot50 liLof each controlblank,zerosample,calibratiosntandarda,nd qualitycontrolsamples 1-3intolabeled16 x 100 mm polypropylenetubes. Standardsand blanksare-.analyzeidnduplicate.AllQC samplesareanalyzedin replicateosf five. 5. Add ImL of 0.5M tetrabutylammoniumhydrogen sulfatep,H 10 to eachtube. 6. Add 2 mL of0.25 M sodium carbonate/0-2M5 sodium bicarbonatetoeach tube. 7. Add 500 liLof theInternaSltandardWorking Solutionto eachtube(except controlblank,add 500 gL water). 8. Add 5 mL ethylacetatetoeachtube. 9. Shake thetubeson a reciprocaslhakeratmedium speed for20 minutes. 10.Centrifugethetubesat3000 rpm for20 minutesat20 *C. 11.Freezetheaqueous phase inan acetone/dryicebath and keep thetubesinthe bathan additiona5l minutes. 12.Transfertheethylacetatelayertoa fresh,labeled13 x 100 mm polypropylene tube. 13.Evaporatetheethylacetatelayertodrynessina TurboVapTm atapproximately 20 *C undernitrogen. 14.Reconstituttehedriedextractsintwo steps: Add 500 ;iLacetonitrialned vortexfor60 seconds. Then add 500 pL waterand vortexfor60 seconds. 15.Transfer200 gL of theextractoa labeledpolypropyleneautosamplervial. A.10.0INSTRUMENT CONDITIONS HPLC Conditions: Eluent(gradient) Mobile PhaseA: 10:90methanol:2mM ammonium acetate(v/v) Mobile Phase B: 90:10 methanol:2mM ammonium acetate(v/v) AOVANCEO BIOANALYTICAL SERVICES, INC. Page 47 of 50 99VDIAOI.Mi.DOC EluentGradientConditions: Time (min) 0 1.0 5 5.5 8 Flow Rate Autosampler AutosamplerTemperature InjectioVnolume HPLC Colunm HPLC Column Temperature TypicalInitiaClolumn Pressure AutosamplerRun Time %B 45 100 100 45% Stop 300 gL/min. Waters 717plus Ambient 3 to5 gL Keystone BetasilC,,,5 gm particlseize2,.1 mm x 50 mm Ambient 90 bar 8.0minutes Anal3l RepresentativReetentionTime (minutes) PFOS 3.1 Tetra-H-PFOS(IS) 2.9 Mass S.pectrometerConditions: CurtainGas NebulizerGas TurboIonSprayTm Temperature TurbolonSprayTmAuxiliaryGas UHP Nitrogen UHP Nitrogen 400 *C LJHP Nitrogenat8 L/min AOVANCE(D BIOANALYTICAL SERVICES. INC. Page 48 of 50 99VDIAOI.Mi.DOC IonsMonitored: Analyte PFOS Tetra-H-PFOS (IS) IonizatioMnode Ion Spray Voltage DeclusteringPotential MS AcquisitioTnime Pause Time Ion Monitored m/z = 499.0 m/z = 427.0 NegativeIon -3000 V -35 V 5 minutes 2 ms Dwell Time 500 ms 500 ms Calibrattehemass axisof theinstrumentby infusionof Rhodapex mass calibration solutio(nSectionA.5.0)ata flowrateof 10 liL/min.Optimizethesensitiviotfythe instrumentusingan infusionof a 10 jig/mL solutionof theanalyteat 10 AL/min into a flowof 190 pL/min ofmobilephase usingthegradientconditionatwhich the analyteelutesfrom theLC column (100% eluentB). Mass spectrapleak widths shouldbe approximately0.6arnuathalf-height. One setof calibratiosntandardswas injectedatthebeginningand one setof calibratiosntandardswas injecteadttheend of eachanalyticarlun (tray). A.11.0 CALCULATIONS Calculatedconcentrationasrebasedon peak arearatioosf PFOS toTetra-H-PFOS. The peak areafortheanalyteionisdividedby thepeak areaforthecorresponding internalstandardion. Data generatedfrom samplesinthisstudywere acquiredand integrateudsing PE SCIEX softwareapplicationSsample Control(v.1.3)and MacQuan (v.1.4).The validatiodnatawere formattedintheABS laboratoriynformationmanagement system (Watson,v.5.31..01).Alldatawere rounded tono lessthanthreesignificant figuresby ABS priortoreporting. AOVANCED BIOANALYTICAL SERVICES. INC. Page 49 of 50 99VDIAOI.Mi.DOC AcceptanceCr teriaforSampleAnalysis CalibratisotnandardTsh:e coefficieonftdetertninat(iro)nmustbe @:0.98A.tleast three-fourthosf theindividualcalibratiosntandardreplicatewsillhave deviations within 15% from theirnominal concentrations. LLQ acceptancecriteriaA:t leastone replicataetthe LLQ must exhibita deviation within20% from itsnominal concentrationI.fthiscriterioinsnotmet, both replicateasrerejectedand thestandardsatthe nexthigher levelare subjectedtothe same test. Qualitycontrolsamples:At leasttwo-thirdsof theindividualQC sample replicates willhave deviationswithin 15% from theirnominal concentrationsw,ithatleast one replicateateach QC concentrationmeeting thiscriterion. AC:)VANCEO BIOANALYTICAL SERVICES. INC. Page 50 of 50 99VDJAOI.Mi.DOC Perfluorooctariesutfonate CAS Number-2750@30-3 ATTACHMENT D DOSE CONFIRMATION ANALYSES 3M EnvironmentalLaboratory Report No. FACR Tox-108 LaboratoryRequest Number (LRN)-U2779 Propnetaryand Confidential lox-iuo Argus 418-014 Study: ProductNumber(Test Substance): Matrix: Method/Revision: AnalyticalEquipment System Number: InstrumentSoflwareNersion: Date ofExtraction/Analyst: Date ofAnalysis/Analyst: Date ofData Reduction/Analyst: Argus 418-014, Oral (Gavage) Cross-FosterinSgtudy of PFOS PFOS (T-6295.9) Twccn Dosing Vehicle See listori ETS-8-4.1& ETS-8-5.1 Madeline04lO98 MassLynx 3.2 9/13/99[AS See Attachm See Attachm See Allachm 9/15/99[AS 9/16/99IAS Sample Data Tween Dosing Confirmation Group Dose Sample N QC B-418-014-B,11/1/98(302ppbMS) B-418-014-B,1/27/99(302ppbMS) Group I Control 0.0mgikg/day B-418-014-A(11/01/98) Diluted1/1 B-418-014-A(1/27/99) Diluted1/1 Group 2 1.6mg/kg/day 0.32mg/ml B-418-014-B(11/01/98) Diluted1/808 B-418-014-B(1/27/99) Diluted1/808 LimitofQuantitatioLnimit(LOQ) = PFOS = 30 nglg Method DetectionLimit(MDL): PFOS = 15 ng/g PFOS = Pcrfluorooctanesulfonate ExpectedConc. PFOS ng/mi 201 201 0 0 320000 320000 PFOS Cone. ng/mi 199 141 0 0 252516 276069 PFOS % Recovery Accuracy 99% 70% <LOD <LOD 79% 86% NR = Sample notreceivednor reported. NA = Not Applicable Date Entercd/Analyst: DateVeriflcd/Analyst: 9/27/99GML ETS-8-4.1 Excel97 VersionSR2 AttachmentD FACT Tox-108doseconfirm.xis Perfluorooctanesutfonate CAS Nuniber-2759-39-3 ATTACHMENT E DATA SUMMARY TABLES 3M EnvimnrrientaLlaboratory ReportNo. FACT Tox-I08 LaboratoryRequest Number (LRN)-U2779 Proprietaraynd Confidential Perfluorooctanesuffonate CAS Number-2750-30-3 3M EnvironmentalLaboratory ReportNo. FACT Tox-108 LaboratoryRequest Number (LRN)-U2779 Table 1.Concentrations of PFOS in Rat Serum Samples from Study FACT Tox-I08 <LLQ: Less thanthe Lower LimitofQuantitabon(0.05pg/mL) 118899l00011@p R p118901 p 18903 18903P 18904 18906 18907 18907P 18908 18909 18910 18910P 18911 18911 P 18912 18913 18913P 18915 18915P 18917 18917P 18918 18918P 18919 18920 18922 18922P 18923 18923P 18924 18924P 18926 18926P 18927 18927P 0.0507 <0LL3LL2QQ3R[@Ol0<.(0O323]<(00.5015) 4.80 35.7 <LLQ[0.04621<(0.05) <LLQ[0.03291<(0.05) 1.35 33.5 2.41 1.06 <LLQ[0.0316]<(0.05) <LLQ[0.03821<(0.05) 0.0798 0.123 <LLQ[0.0376]<(0.05) <LLQ[0.0341]<(0.05) <LLQ[0.0398]<(0.05) <LLQ[0.0221]<(0.05) <LLQ[0.0242]<(0.05) <LLQ[0.0355]<(0.05) <LLQ[0.02211<(0.05) <LLQ[0.0323]<(0.05) <LLQ[0.0247]<(0.05) <LLQ[0.03401<(0.05) 1.96 <LLQ[0.04121<(0.05) <LLQ[0.0367]<(0.05) <LLQ[0.0291]<(0.05) <LLQ[0.0331]<(0.05) 2.72 30.7 <LLQ[0.0323]<(0.05) <LLQ[0.02291<(0.05) <LLQ[0.03051<(0.05) <LLQ[0.0318]<(0.05) Proprietaraynd Confidential Perfluorooctanesutfonate CAS Number-2759-39-3 3M EnvironmentalLaboratory Report No. FACT Tox-108 LaboratoryRequest Number (LRN@-U2779 Table 1.Concentrationsof PFOS inRat Serum Samples from Study FACT Tox-108 (continued) <LLQ: Less than the Lower LimitofQuantdabon (0.05pg/mL) lwmi. 18929 18931 18931 P 18932 18934 18934P 18935 18935P 18936 18936P 18937 18938 18939 18939P 18940 18940P 18941 18942 18943 18943P 18946 18947 18948 18950 18951 18951 P 18952 18952P 18953 18953P 18956 18956P 18957 18957P 18958 18958P 2.40 <LLQ[0.0382]<(0.05) <LLQ[0.0196]<(0.05) <LLQ[0.03991<(0.05) 1.25 34.1 <LLQ[0.0316]<(0.05) <LLQ[0.0206]<(0.05) <LLQ[0.0302]<(0.05) <LLQ[0.02301<(0.05) 1.80 <LLQ[0.0359]<(0.05) <LLQ[0.0311)<(0.05) <LLQ[0.03081<(0.05) <LLQ[0.0330]<(0.05) <LLQ[0.02311<(0.05) 5.34 1.12 65.1 82.2 157 86.9 96.1 66.4 72.4 59.2 90.8 48.3 67.0 53.8 97.5 89.3 69.1 93.2 68.3 56.2 Propnetaryand Confidential Perfluorooctanesutfonate CAS Number-2759-39-3 3M EnvircT@-,antaLlaboratory ReportNo. FACT Tox-108 LaboratoryRequest Number (LRN@-U2T79 Table 1.Concentrations of PFOS in Rat Serum Samples from Study FACT Tox-108 (continued) <LLQ. Less thanthe Lower Limd ofQuantitabon(0.05pg/mL) 18959 18960 18960P 18961 18961 P 18963 18964 18964P 18965 18967 18968 18969 18970 18970P 18971 18972 18972P 18973 18973P 18974 18976 18977 59.2 78.0 93.6 69.1 47.6 83.6 85.6 92.9 75.9 74.2 80.9 78.6 99.3 96.9 218 66.9 58.2 81.9 79.5 172 77.9 124 Proprietaraynd Confidential Perfluorooctanesutfonate CAS Number-2759-39-3 3M EnvironmentalLaboratory Report No. FACT Tox-108 LaboratoryRequest Number (LRN)-U2779 Table 2. Repeat Analysis of Rat Serum Samples from Study FACT Tox-1 08 <ULQ: Greaterthan theUpper Umft ofQuantitatio(n20.Opg/mL) I 18903P 18907P 0 Day 14 >ULQ-28.5284>(20) 3 0 Day 14 >ULQ-30.8172>(20) 3 1 35.7 5 35.7 1 1 33.5 5 33.5 1 18924P 0 Day 14 >ULO-27.5496>(20) 3 1 30.7 5 30.7 1 18934P 0 Day 14 >ULQ-36.8737>(20) 3 1 34.1 5 34.1 1 18971 1 Day 14 >ULQ-219.5172>(200) 1 2 218 3 218 2 L Reasons for Reassay: 1) Run 3 concentration exceeded calibrationrange 2)Run 1 concentratioenxceeded calibratiornange Reasons forReported Conc: 1)Run 5 concentrabonwithincalibratiornange 2) Run 3 concentratiownithincalibratiornange Table 3. Pharmacokinetic Subgroup--Concentrations of PFOS Samples from Study FACT Tox-108 <LLO: Less thanthe Lower LimitofQuantftatio(n0.05pg/mL) in Dam and LitterSerum 10 - 1 18910 <LLQ[0.03161<(0.05) 1891 OP 1 18911 0.0798 18911 P 1 18913 <LLO[O.0341]<(0.05) 18913P 1 18915 <LLQ[0.0221]<(0.05) 18915P 1 18926 <LLQ[0.0323]<(0.05) 18926P 1 18927 <LLQ[0.0305]<(0.05) 18927P 1 18939 <LLQ[0.031 1]<(0.05) 18939P 1 18940 <LLQ[0.0330]<(0.05) 1894op 11 1895@= 97.5 11 18971 1 218 18956P 18971 P NA-Sample notassayed. Insufficiesnatmple volume foranalysis. <LLQ[0.03821<(0.05) 0.123 <LLQ[0.03981<(0.05) <LLQ[0.0242]<(0.05) <LLQ[0.0229]<(0.05) <LLQ[0.0318]<(0.05) <LLQ[0.0308]<(0.05) <LLQ[0.02311<(0.05) 89.3 NA Propnetaryand Confidential Perfluorooctanesutfonate CAS Number-2759-39-3 3M EnvironmentalLaboratory ReportNo. FACT Tox-I08 LaboratoryRequest Number (LRN@-U2779 Table 4.PFOS Concentrations in Dam and Cross-Fostered LifterSerum Samples by Dosage Group <LLQ: Less thanthe Lower LimitofQuantitabon(0.05pg/mL) Sample notreceived 1 1 liurA-@Fl 'i@ 1 18903 18907 18908 18909 18918 18920 18924 18929 18934 18936 18937 18941 18942 18901 18904 18906 18912 18917 18919 18922 18923 18931 18932 18935 18938 1 18943 1 18947 1 18948 If 18957 I1 18960 I1 18964 I1 18967 18968 18969 18970 11 18973 11 -1-8974 18946 18950 18951 18952 18953 18958 11 18959 11 18961 11 18963 I1 18965 11 18972 11 18976 18977 4.80 1.35 2.41 1.06 <LLQ[0.0323]<(0.05) 1.96 2.72 2.40 1.25 <LLQ[0.0302]<(0.05) 1.80 5.34 1.12 0.0507 <LLQ[0.04621<(0.05) <LLO[O.03291<(0.05) <LLQ[0.03761<(0.05) <LLO[O.03551<(0.05) <LLO[O.03401<(0.05) <LLQ[0.0412]<(0.05) <LLQ[0.0291]<(0.05) <LLQ[0.03821<(0.05) <LLQ[0.03991<(0.05) <LLQ[0.03161<(0.05) <LLO(O.03591<(O@05) 65.1 86.9 96@ 1 69.1 78.0 85@6 74.2 80-9 78.6 99.3 81.9 172 157 66.4 72.4 90.8 67.0 68.3 59.2 69.1 83.6 75.9 66.9 77.9 124 .11ni A 18976P A 18963P A 18953P A 18961 P A 18946P A 18952P A 18972P A 18950P A 18959P A 18977P A 18958P A 18951 P A 18965P B B B B B B B B B B B 8 1 18931 P 18932P 18938P 18935P 18922P 18923P 18917P 18-9-19P18901 P 189NP 18912P 18906P c 18968P c -18973P c 18970P c 18969P c 18974P c 18967P c 18964P c 18943P C 18957P c 18948P C 18947P c 18960P D 18918P D 18929P D 18941 P D 18920P D 18908P D 18937P D 18934P D 18909P D 18907P D 18942P D 18924P D 18903P D 18936P 53.8 47.6 48.3 58.2 - 56.2 59.2 <LLQ(0.01961<(0.05) <LLQ[0.02061<(0.05) <LLQ[0.03671<(0.05) <LLQ(0.03311<(0.05) <LLQ[0.0221]<(0-05) <LLQ(0.03231<(0.05) 79.5 96.9 - 92.9 82.2 93.2 93.6 <LLQ(0.0247]<(0.05) 34.1 33.5 30.7 35.7 <LLQ[0.02301<(0.05) Propnetaryand Confidential PerfluoroocUmsulfonate CAS Number-2759-39-3 3M EnvironmentalLaboratory ReportNo. FACT Tox-108 LaboratoryRequest Number (LRN)-U2779 ATTACHMENT ANALYTICAL F REPORTS FROM CONTRIBUTING LABORATORIES Pmprietaryand Confxienhal AOVANCED BIOANALYTICAL SERVICES, INC. BIOANALYTICAL REPORT Title: QuantitativeDeterminationof Perfluorooctanesulfonate (PFOS) inRat Serum from Study #FACT-TOX-108 Using Turbo Ion Spray LC/MS Date: 27 August 1999 Authors: David J.Anderson, M.S. Amie J.Prince,B.S. Holly D. Ross,M.S. Prepared For: 3M EnvironmentalTechnology and Safety Services St.Paul,MN 55133-3331 ABS Report No.: 99ADJAOI.MI.DOC Study Protocol: FACT-TOX-108 Number ofPages: 16 Summary and Conclusions The concentrationofperfluorooctanesulfona(tPeFOS) was determinedinratserum samples collectedduringthe study #FACT-TOX- 108 usinga sensitives,pecifica,ccurate, and reproducibleanalyticamlethod developed by Advanced BioAnalyticalServices,Inc., Ithaca,NY. Rat serum samples (50 gL) were extractedby a liquid-liquiedxtractiopnrocedureto isolatePFOS and the internasltandard,1H,IH,2H,2H-perfluorooctanesulfonicacid (Tetra-H-PFOS).Followingevaporationand reconstitutiosna,mple extractswere analyzedby turboion sprayliquidchromatography/mass spectrometry(LC/MS) inthe negativeion mode. All samples were successfullaynalyzed withinfourruns.The lower limitof quantitation was 0.05 gg/mL forPFOS. The precisionofthisassay(RSD) as deter-minefdrom the analysisof qualitycontrolsamples forPFOS was:54.01%. The precisionof thisassay (RSD), asdeterminedfrom thecalibratiosntandardsforPFOS was:55.83%. The relative error(RE) of the assay,as determined from the analysisof thequalitycontrolsamples ranged from -7.32to 10.1%. The RE of the assay,as determinedfrom the analysisof calibratiosntandardsranged from -5.86to 11.0%. 15 Catherwood Road n Ithaca,New York 14850 s (607) 266-0665 n Fax (607) 266-0749 Title: ADVANCED BIOANALYTICAL SERVICES SIGNATURE PAGE QuantitativDeetenninationofPerfluorooctanesulfon(aPtFeOS) in Rat Serum from Study #FACT-TOX-108 Using Turbo Ion Spray LC/MS ABS ReportNo.: 99 mi c Reportedby: MI C David J.Andafson,M.S. Research Scientist *ZiAt4us-T Date Reviewed by: Authorizedfor Releaseby: ;tzkdo,P.A-4,-mact- en Cormack, B.S. AssociateAuditor John 1'.P.erkins,Ph.D. AssistanStcientifiDcirector 7-7 41 Cak Date @7.AV$. Date ADVANCED BIOANALYTICAL SERVICES, INC. Page 2 of 16 99ADJAOI.Mi.DOC QAUSTATEMENT Periodincspectioofntshebioanalytipcoarltioonfstudy#FACT-TOX-108were conductedby theQualityAssuranceUnit of Advanced BioAnalyticalServices(AJ3S)for compliancewithEPA GLP regulation(s40 CFR Part792). The studywas inspectedon thefollowingdates: 22 June 1999; 1,2,7,July1999; 12 August 1999. Resultsof theinspectiownere reportedto ABS Management and PrqjectTeam Leader on: 22 June 1999; 1,2,7,July1999; 12 August 1999. Resultsof theinspectionwsere reportedtotheStudy Directoron 12 August 1999. Based on theinspectionasnd thedatareviewed,thisreportisa complete and accurate representatioonf thedata. i)iGc thleleene@nCCoo ack,.BI.S. AssociateAuditor 2Da2tzeL-o ell-:m AOVANCEED SIOANALYTICAL SERVICES. INC. Page 3 of 16 99ADJAOI.Ml.DOC TABLE OF CONTENTS SIGNATURE PAGE ........................................................2.................. QAU STATEMENT .........................................................3................. TABLE OF CONTENTS .....................................................4................. LIST OF TABLES ...........................................................5................. I. INTRODUCTION ......................................................6................. 1.1.Study Descriptionand Objective.........................................6................. 2. N4ETHODS ............................................................6................. 2.1.AnalyticaPlrocedure(s.)...............................................6................. 2.2.Assay Site...........................................................7................. 2.3.Data Processing.......................................................7................. 3. RESULTS .............................................................7................. 3.1.Assay Performance ....................................................7................ 3.2.AnalyticaRlesultsforStudy #FACT-TOX-108 ............................8................. 4. SUMMARY AND CONCLUSIONS .......................................8................. 5. DATA RETRIEVAL ....................................................8................. 6. REFERENCES .........................................................8................ 7. TABLES ..............................................................9................ AOVANCED BIOANALYTICAL SERVICES, INC. Page 4 of 16 99ADJAOI.Ml.DOC LIST OF TABLES Tablel: Summary ofSample and Assay InformationforProtocolFACT-TOX-108 ....9.. Table 2: Inter-AssayPrecisionand Accuracy forPFOS QualityControlSamples inRat Serum forStudy#FACT-TOX-108 ...................................1.0................. Table3: Inter-AssayPrecisionand Accuracy forPFOS CalibratioSntandardsin Rat Serum forStudy#FACT-TOX- 108 ...................................I.I................. Table 4: CalibratioCnurve StatistifcosrtheDeterminationof PFOS inRat Serum for Study #FACT-TOX- 108 ............................................1.2................. Table 5: ConcentrationosfPFOS inRat Serum Samples from Study #FACT-TOX-108 ............. ....................................................1.3 ................. Table 6: RepeatAnalysisofRat Senun Samples from Study #FACT-TOX-108 .......16... AOVANCED IBIOANALYTICAL SERVICES. INC. Page 5 of 16 99ADJAOI.Mi.DOC 1. INTRODUCTION 1.1. StudyDescriptiaonndObjective Theobjectiovfethistudwyastodeterminteheconcentratioofns perfluoroo'ctanesulfo(nPaFtOeS) inratserum samplescollecteduringthestudy#FACTTOX-108, "Oral(Gavage)Cross-FosterinSgtudy of PFOS inRats."Rat serum samples were analyzedusinga liquid-liqueixdtractioannd turboionsprayliquid chromatography/massspectrometr(yLC/MS) assay. 2. METHODS 2.1. AnalyticalProcedure(s) PFOS concentrationwsere determinedin ratserum accordingtothevalidateLdC/MS method (1).Serum samples(50gL) were extractebdy a liquid-liqueixdtraction procedureusingethylacetatetoisolatPeFOS and theinternasltandard(IS), IH,lH,2H,2H-perfluorooctasnuelfonicacid(Tetra-H-PFOS)from ratserum.Following evaporationtodrynessand subsequentreconstitutiosna,mple extractswere separatedby reversed-phascehromatographyon a 2.1x 50 nun (5 @im)BetasilTmC,,column (KeystoneScientifiIcn,c.B,ellefonteP,A) with an initiamlobilephaseof55% EluentA (10:90methanol:2mM ammonium acetatea)nd 45% EluentB (90:10methanol:2mM ammonium acetate)P.FOS concentrationwsere determinedby turboionsprayliquid chromatography/massspectrometry(LC/MS) inselecteidonmonitoring(SIM) mode. The negativeionsmonitoredwere: m/z = 499.0forPFOS [M-Klm/z = 427.0forTetra-H-PFOS [M-H]- Study sampleconcentrationwsere determinedfrom a weighted(I/Y')q,uadratic regressioonf peak arearatios(peakareaof PFOS/peak areaof Tetra-H-PFOS)versus nominal concentratioonftencalibratiosntandards.The nominalconcentrationosf the calibratiosntandardswere 0.05,0.1,0.25,0.5,1,2,4,8, 16,and 20 gg/mL PFOS. The lowerlimitofquantitati(oLnLQ) forthisassaywas determinedtobe 0.054g/mL PFOS. Qualitycontrol(QC) serum samplesatthreedifferenctoncentration(s0.2,6,and 18 ;ig/mLPFOS) were analyzedwith each assaybatchinreplicateosf four.DilutionQC samples(QC4, 100 jig/mL)were preparedateach dilutiotnhatwas used duringa particulaarssayand were analyzedwith each assaybatchinreplicateosf fourasneeded. The acceptancecriterifaorcalibratiosntandardsstipulatetdhattheback-calculated concent' rationosf atleasthree-fourthosf theindividuaclalibratiosntandardreplicates must not deviatemore than 15% from theirnominal concentratio(nexceptattheLLQ). At leastone duplicateattheLLQ must exhibita deviationwithin20% of thenominal MMM ADVANCEO BIOANALYTICAL SERVICES, INC. Page 6 of 16 99ADIAOI.MI.DOC concentration.The coefficieonftdetermination(r')must be @tO.98.The acceptance criterifaollowedareinaccordancewithABS StandardOperatingProcedures. The acceptancecriterifaorthequalitycontrolsamplesstipulatetdhatatleastwo-thirds of theindividuaQlC sample replicatemsust notdeviatemore than 15% from their nominal concentrationsA.t leastone replicataeteach QC concentratiomnust exhibita deviationwithinl 5%. A summary of thesample and assayinformationforstudy#FACT-TOX- 108 isgivenin Table 1. 2.2. Assay Site Allsampleswere analyzedatAdvanced BioAnalyticalServices,Inc.,IthacaN,Y. 2.3. Data Processing The datawere collecteudsingturboion sprayLC/MS selectedionmonitoring(SIM) in thenegativeionmode. Peak areaswere integratebdy thePE SCIEX program MacQuan, version1.4,residingon a Macintoshcomputer. Followingpeak areaintegratiotnh,e resulttsablesfrom MacQuan were saved as textfileasnd uploadedtotheABS fileserver where a weighted(1/Y'')quadraticregressiownas performedusingthesoftwarepackage Watson (v 5.3.1.01PSS, Inc.,Wayne, PA 19087).Calculationwsere performedon unroundednumbers.Allcalibratiosntandardand QC resultwsere rounded tono lessthan threesignificanftiguresbeforereporting. 3. RESULTS 3.1. Assay Performance The performanceof theassayforPFOS as determinedfrom theanalysisofdailyquality controlsamplesisdocumented inTable 2. The inter-assapyrecision(RSD) ofquality controlsamplesrangedfrom 1.57to 4.01% forPFOS. There was no marked inaccuracy intheresultfsrom thesequalitycontrolsamples;therelativeerror(R.E)rangedfrom -7.32to 10.1%. The performanceofdailycalibratiocnurvesisdocumented inTable 3. The inter-assay precision(RSD) of thestandardsranged from 1.11 to 5.83% forPFOS. There was no marked inaccuracyintheresultsfrom thesestandardst;herelativeerror(RE) ranged between -5.86to 11.0%. AOVANCED In BIOANALYTICAL SERVICES, INC. Page7of16 99ADJAOI.Mi-DOC The slope,y-intercepatn,d coefficienotf determination(r')foreach analyticarlun are presentedinTable 4. The r'valuesranged from 0.9954 to0.9982 forPFOS inmt serum. AllABS personnelassignedto analyzesamples forthisprojectwere requiredto successfulleyxtractqualitycontrol(QC) samplesand calibratiosntandards.Resultsof theextractiownere reviewedby a trainedanalystagainstthepre-definedacceptance criterifaorthisassay. Resultsaremaintainedinthestudyrecordsof ABS. 3.2. AnalyticalResultsforStudy #FACT-TOX-108 PFOS concentrationisnratserum from Study #FACT-TOX- 108 arepresentedinTableS. Alldatawere rounded tono lessthanthreesignificanftiguresbeforereportinginTableS. Study samplesrequirinrgepeatanalysisforStudy #FACT-TOX- 108 arepresentedin Table 6. 4. SUMMARY AND CONCLUSIONS The concentratioonf PFOS was determinedinratserum samplescollecteduringthe study#FACT-TOX- 108. PFOS was isolatefdrom serum by a liquid-liqueixdtractiopnrocedureand determinedby LC/MS. The qualityofthedeterminationwsas satisfactotrhyroughout.The LLQ was 0.05gg/mL PFOS. The precisionoftheassay,asdeterminedfrom theanalysisof qualitycontrol samples was -<4.01% forPFOS. 5. DATA RETRIEVAL The calculatecdoncentratiodnatafrom theanalysisof ratserum samplesforStudy #FACT-TOX-108 aremaintainedon fileinthearchivesof theAdvanced BioAnalytical ServicesI,nc.I,thaca,NY inABS Notebook 2319. 6. REFERENCES 1. Advanced BioAnalyticaSlervicesValidationReport99VDJAOI.MI.DOC. Method ValidatiofnortheQuantitatioonfPerfluorooctanesulfon(aPtFeOS) in Rat Serum by Turbo Ion Spray LC/MS. David J.Anderson,M.S. Amie J.Prince, B.S.,and HollyD. Ross,M.S. 26 August 1999. MMM AOVANCEO E310ANALYTICAL SERVICES. INC. Pag8eof16 99ADJAOI.Ml.DOC 7. TABLES Table1: Summary ofSampleand AssayInformatiofnorProtocoFlACT-TOX108 Species/Matrix: Rat/Serum SampleCollectiaonndStoragIenformation: Anticoagulant/Stabilizer: ReportedSample CollectionDates: Dates Received atA13S: StorageTemperature atABS: None 7 January 1999 to 29 January 1999 16 June 1999 -20 OC Assay Information: Assay Period: 22 June 1999 to 28 June 1999 Analyte: Potassium perfluorooctanesulfonate (PFOS) AnalyticalStandard: PFOS Lot No: 171 Source: 3M InternaSltandard: Lot No: Source: IH, IH,2H,2Hperfluorooctanseulfonicacid (Tetra-H-PFOS) 59909 3M CalibratioRnange: RegressionMethod: WeightingFactor: Lower LimitofQuantitation: Upper LimitofQuantitation: quadratic 1/y2 0.05gg/mL 20 pg/mL AOVANCEO SIOANALYTICAL SERVICES. INC. Page 9 of 16 99ADJAOI.Ml.DOC Table2: Inter-AssParyecisiaondAccuracyforPFOS QualitCyontrol SamplesinRatSerum forStudy#FACT-TOX-108 Run No. QCI 0.2 PFOS Concentrat(igogn/mL) QC2 QC3 QC4 QC4 6 18 100 100 (Iin10Dilution()Iin20 Dilution) 1 0.187 6.19 17.5 110 NA 0.192' 6.22 17.3 109 NA 0.191 6.22 17.5 110 NA 0.182 6.14 17.3 108 NA 2 0.191 6.13 16.9 NA NA 0.191 5.91 16.8 NA NA 0.190 6.20 16.5 NA NA 0.192 6.19 16.7 NA NA 3 0.172 6.02 17.0 114 107 0.174 6.22 16.7 ill 106 0.175 5.94 17.0 110 100 0.177 6.08 17.1 109 103 5 0.184 6.37 17.3 109 NA 0.187 6.36 17.5 113 NA 0.197 6.39 17.5 ill NA 0.183 6.33 17.1 109 NA Mean 0.185 6.18 17.1 110 104 RSD (%) 4.01 2.33 1.92 1.57 2.92 RE -7.32 3.04 -5.00 10.1 3.97 RSD = (SD/Mean) x 100% RE = [(Mean-Nominal)/Nominalx]100% NA: Not Applicable. AOVANCEO BIOANALYTICAL SERVICES. INC. Page10 of 16 99ADJAOI.Mi.DOC cnW)> m C)0 < < z >z C,3>n mm zn Table3: Inter-AssaPyrecisioannd AccuracyforPFOS CalibratioSntandardsinRat Serum forStu NFACT-TOX-108 Run No. 1 STDI 0.05 0.0479 0.0490 STD2 0.1 0.105 0.112 STD3 0.25 0.240 0.258 2 0.0508 0.102 0.245 0.0503 0.0985 0.240 3 0.0474 0.105 0.237 0.0513 0.109 0.245 5 0.0498 0.0936 0.231 0.0553 0.0982 0.244 Mean 0.0502 0.103 0.242 RSD (%) 4.90 5.83 3.20 RE 0.450 2.82 -3.04 RSD = (SD/Mean) x 100% RE = [(Mean-Nominal)/Nominal]x 100% PFOS Concentratio(nigimL) STD4 STD5 STD6 STD7 0.5 1 2 4 0.488 0.891 2.03 4.24 0.509 0.935 2.12 4.51 0.488 0.973 2.07 4.37 0.483 0.970 2.05 4.41 0.476 0.944 2.12 4.52 0.479 0.934 2.07 4.54 0.483 0.952 2.19 4.45 0.507 0.933 2.21 4.47 0.489 0.941 2.11 4.44 2.54 2.71 3.09 2.23 -2.21 -5.86 5.34 11.0 STD8 s 8 7.64 1 8.03 1 7.97 1 7.93 1 7.91 1 7.84 1 7.79 1 7.89 1 7.88 1 1.52 1 -1.55 Page II of 16 I Table 4: CalibrationCurve StatisticfsortheDeterminationofPFOS inRat Serum forStudy #FACT-TOX-109 Run No. Quadratic Linear Coefficie(nAt) Coefficie(nBt) 1 -0.0007277 0.1225 2 -0.0005896 0.1392 3 -0.0006758 0.1401 5 -0.0004928 0.1345 Mean -0.0006215--@- 0.1341 y = Ax' + Bx + C, weightedI/y' Intercept (C) 0.001121 0.0009484 0.001454 0.001190 0.001178 Coefficienotf Determina(tri2)on 0.9959 0.9982 0.9959 0.9954 0.9964 ,.mom ACVANCED SIOANALYTICAL SERVICES. INC. Page 12 of 16 99ADJAOI.Ml.DOC Table5: ConcentrationfPsFOS inRatSerumSamplesfromStudy#FACTTOX-108 Rat PFOS Concentration Number* (gg/ML) 18901 0.0507 18901P <LLQ[0.0323]<(0.05) 18903 4.80 18903P 35.7 18904 <LLQ[0.0462]<(0.05) 18906 <LLQ[0.0329]<(0.05) 18907 1.35 18907P 33.5 18908 2.41 18909 1.06 18910 <LLQ[0.0316]<(0.05) 1891 OP <LLQ[0.0382]<(0.05) 18911 0.0798 1891 1P 0.123 18912 <LLQ[0.0376]<(0.05) 18913 <LLQ[0.0341]<(0.05) 18913P <LLQ[0.0398]<(0.05) 18915 <LLQ[0.0221]<(0.05) 18915P <LLQ[0.0242]<(0.05) 18917 <LLQ[0.0355]<(0.05) 18917P <LLQ[0.0221]<(0.05) 18918 <LLQ[0.0323]<(0.05) 18918P <LLQ[0.0247]<(0.05) 18919 <LLQ[0.0340]<(0.05) 18920 1.96 18922 <LLQ[0.0412]<(0.05) 18922P <LLQ[0.03671<(0.05) 18923 <LLQ[0.0291]<(0.05) 18923P <LLQ[0.0331]<(0.05) 18924 2.72 18924P 30.7 18926 <LLQ[0.0323]<(0.05) 18926P <LLQ[0.0229]<(0.05) 18927 <LLQ[0.0305]<(0.05) 18927P <LLQ[0.0318]<(0.05) 18929 2.40 *Rat Number with a "P" indicateas pup sample. <LLQ: LessthantheLower Limitof Quantitatio(n0.05gg/mL) AOVANCED BIOANALYTICAL SERVICES, INC. Page 13 of 16 99ADIAOI.Mi.DOC Table5: (ContinueCdo)ncentratioonfsPFOS inRatSerumSamplesfrom Study#FACT-TOX-108 Rat PFOS Concentration Number* (gg/ML) 18931 <LLQ[0.0382]<(0.05) 1893 1P <LLQ[0.0196]<(0.05) 18932 <LLQ[0.0399]<(0.05) 18934 1.25 18934P 34.1 18935 <LLQ[0.0316]<(0.05) 18935P <LLQ[0.0206]<(0.05) 18936 <LLQ[0.0302]<(0.05) 18936P <LLQ[0.0230]<(0.05) 18937 1.80 18938 <LLQ[0.0359]<(0.05) 18939 <LLQ[0.031 1]<(0.05) 18939P <LLQ[0.0308]<(0.05) 18940 <LLQ[0.0330]<(0.05) 18940P <LLQ[0.0231]<(0.05) 18941 5.34 18942 1.12 18943 65.1 18943P 82.2 18946 157 18947 86.9 18948 96.1 18950 66.4 18951 72.4 18951P 59.2 18952 90.8 18952P 48.3 18953 67.0 18953P 53.8 18956 97.5 18956P 89.3 18957 69.1 18957P 93.2 18958 68.3 18958P 56.2 *Rat Number with a "P" indicateas pup sample. <LLQ: Less thantheLower Limitof Quantitatio(n0.05@Lg/mL) AOVANCEI) BIOANALYTICAL SERVICES, INC. Page 14 of16 99ADJAOI.Ml.DOC Table 5: (Continued)ConcentrationsofPFOS inRat Serum Samples from Study #FACT-TOX-108 Rat PFOS Concentration Number* (;Ig/ML) 18959 59.2 18960 78.0 18960P 93.6 18961 69.1 18961P 47.6 18963 83.6 18964 85.6 18964P 92.9 18965 75.9 18967 74.2 18968 80.9 18969 78.6 18970 99.3 18970P 96.9 18971 218 18972 66.9 18972P 58.2 18973 81.9 18973P 79.5 18974 172 18976 77.9 18977 124 *RatNumber witha "P" indicateas pup sample. AOVANCEED BIOANALYTICAL SERVICES. INC. Page 15 of 16 99ADJAOI.Mi.DOC m 1> mbo 11)>< <Z> Cm))>znm !n 0 zn Table 6: Repeat Analysisof Rat Serum Samples from Study #FACT-TOX-108 Rat Treatment Time Number* Original Conc. ug/ml Original Curve Number 18903P 18907P 18924P 18934P 18971 0 Day 14 >ULQ-28.5284>(20) 3 0 Day 14 >ULQ-30.8172>(20) 3 0 Day 14 >ULQ-27.5496>(20) 3 0 Day 14 >ULQ-36.8737>(20) 3 1 Day 14 >ULQ-219.5172>(200) 1 >ULQ: Greaterthanthe Upper Limitof Quantitation. *Rat Number with a "P" indicatesa pup sample. Reason for Reassay 1 1 1 1 2 Reassay Conc. ug/mi 35.7 33.5 30.7 34.1 218 Reassay Curve Number 5 5 5 5 3 REASONS FOR REASSAY: 1).Run 3 concentratioenxceeded calibratiornange. 2).Run I concentrationexceeded calibratiornange. REASONS FOR REPORTED CONC: 1).Run 5 concentratiownithincalibratiornange. 2).Run 3 concentratiownithincalibratiornange. Reported Conc. ug/mi 35.7 33.5 30.7 34.1 218 Page 16 of 16 AOVANCEO SIOANALYTICAL MMM SERVICES, INC. ADDENDUM TO THE BIOANALYTICAL REPORT Title: Addendum - QuantitativeDeterminationof PerfluorooctanesuIfonate(PFOS) in Rat Serum from Study #FACT-TOX-108 Using Turbo Ion Spray LC/MS Date: 13 October 1999 Addendum Report: 99ADJAOI.MI.ADD.DOC OriginalReport: 99ADJA01.MI.DOC Authors: David J. Anderson, M.S. Amie J.Prince,B.S. Holly D. Ross, M.S. Prepared For: 3M Environmental Technology and SafetyServices St.Paul, MN 55133-3331 Number of Pages: 3 15 Catherwood Road a Ithaca,New York 14850 a (607) 266-0665 o Fax (607) 266-0749 ADVANCED BIOANALYTICAL SERVICES Title: SIGNATURE PAGE Addendum - QuantitativeDeterminationof Perfluorooctanesulfona(tPeFOS) inRat Serum from Study #FACT-TOX- 108 Using Turbo Ion Spray LC/MS ABS Report No.: 99AD I.Mi. DOC Reported by: 3 David J.Andddso-n,M.!@. Date Research Scientist Reviewed by: .*eathllee"nCSo@ ack,B AssociateAuditor Authorizedfor Release by: John RIPerkins,Ph.D. AssistahtScientifiDcirector Accepted by: I /,@ KrisHanson,Ph.D. 3M Study Director Date Date 4 Date AOVANCEO SIOANALYTICAL SERVICES, INC. Page 2 of 3 99ADJAOI.Ml.ADD.DOC ADDENDUM SUMMARY ADDENDUM Additionof Study Directorsignatureon the SignaturePage. The signaturepage of thisaddendum indicateascceptanceof the originalreport. REASON FOR ADDENDUM OriginalGLP reportrequiresthe signatureof the Study Director. AOVANCEO SIOANALYTICAL SERVICES. INC. Page 3 of 3 99ADJAOI.Mi.ADD.DOC Perfluomoctariesuffonate CAS Number-2759-39-3 ATTACHMENT G REPORT SIGNATURE PAGE 3M EnvironmentalLaboratory ReportNo. FACT Tox-108 LaboratoryRequest Number (LRN)-U2779 KrifteJ-.n'HansenP,h.D.S,tudyDirector Date MarvinT Case,D.V.M.,Ph.D.,Sponsor Representabve gate DaleL.Bacon,LaboratorMyanager Date Propnetaryand Confidenbal StudyTitle AnalyticLaalboratoRreyporotntheDeterminatoifotnhePresencaendConcentratoifon Perfluorooctanesulfona(tPeFOS) intheSerum of Sprague-Dawley"Rats Exposed toPotassiumPerfluorooctanesulfonavtieaGavage REPORT AMENDMENT NO. I Amendment Date: 19 April2000 Petforming Laboratory 3M EnvironmentalTechnology & SafetyServices 3M EnvironmentalLaboratory 935 Bush Avenue St.Paul,NlN 55106 Laboratory Project Identification ET&SS LRN-U2779 ET&SS FACT Tox-108 3M EnvironmentaLlaboratofy FACT Tox-108 ReportAmendment No.1 Thisamendment modifiesthefollowinpgortion(so)fthefinalreport: 1. FINAL REPORT. Make thefollowiandgdititoonthefinarleporttext. ADD: Aftetrhefinarleporwtasissue(dsigneadndarchiveidt)w,asdiscovertehdathepurity valuesstatedfortheanalyticarleferencematerialand testsubstancewere inaccurateT.hese valueswere based onlyon NMR analysesS.ubsequentchemicalcharacterizatiiosnoccurring to determinetheactualpurityofthesessubstances(PFOS Lot 171 and 217).The finalreport willbe amended to detaitlheanalyticarlesultussingthepurityvaluesfrom thecertificatoefs analysiswhen theyareissuedforPFOS Lot 171 and 217. REASON: To describethechangesthatwillbe made to thefinalreportwhen thecertificaotfeanalysis isissuedforPFOS Lot 171 and 217. 2. FI)VAL REPORT. Make thefollowingadditiontotheexceptionslisitntheStatementof Compliance section. AMEND To READ: The purityand stabilitoyf thetestsubstanceand analyticarleferencematerialare unknown and was notdeterminedpriortotheinitiatiofnthisstudy. REASON: The Statementof Compliance sectionwas incomplete. 3. FINAL REPORT. The purityof thetestsubstanceis99.28% (Lot217),and analyticarleferencematerialis 99.49% (Lot171)aslisteidnTable2. AMEND TO READ: The purityofthetestsubstanceand analyticarleferencmeaterialisteidnTable2 is unknown. REASON: The purityvaluesstatedforthetestsubstanceand analyticarleferencemateriallisteidnTable 2 areincorrect. 3M EnvironmentaLlaboratory FACT Tox-108 ReportAmendment No.I Amendment Approval T-Ca,4, M@@in T.Case,D. V.M, Ph.D.,SponsorRepresentative Date Kristen J Hansen, Ph.D., Study Director Date 3M EnvironmentalLaboratory