Document 82g8q0ZexnYrzrq8yZrajQKoa

3M EnvironmentalTechnology and Services PO Box 33331 St.Paul,MN 55133-3331 6127786442 Protocol #FACT-TOX-1 11 Study Title Oral (Gavage)PharmacokineticRecovery Study ofPFOS inRats PROTOCOL Author LisaClemen Date: June8,1999 Performing Laboratory 3M EnviromnentaTlechnology& SafetyServices 3M EnvirorunentaLlaboratory 935 Bush Avenue St.Paul,NIN 55106 Laboratory Project Identification FACT-TOX- III U2994 Alt (:E !=-f 3M EnvironmentLaalboratory Page 1of10 Protoco#lFACT-TOX-111 StudyIdentification Oral(GavageP)harmacokinetRieccovery StudyofPFOS inRats TestMaterial Sponsor Sponsor Representative Study Director Study Location(s) In vivo Testing Facility AnalyticalTesting Laboratory Perfluorooseutlafnoenaiccipdotassisuamlt (T-6295) 3M ToxicoloSgeyrvic-eMsedicaDlepartment 3M Center,Building220-2E-02 St.Paul,NfN 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 Argus Research Laboratories,Inc. 905 Sheehy Drive,BuildingA Horsham, PA 19044 3M Envirom-nentalLaboratory Building2-3E-09 935 Bush Avenue St.Paul,NIN 55106 3M EnvironmentaLlaboratory Page 2 of10 Sub-ContracLtaboratofies Proposed Study Timetable Study InitiatioDnate Study Completion Date Protoco#lFACT-TOX-1II AdvancedBioaiialytSiecravliceIsn,c. 15 Catherwood Road IthacaN,Y 14850 BattelleMemorial Institute 505 King Avenue Colwnbus Ohio 43201-2693 June 8,1999 August 8,2000 1. STUDY Oral (gavage)pharmacokineticrecoverystudyofpotassiumperfluorooctanseulfonicacid (PFOS) inrats. 2. PURPOSE This analyticasltudyisdesignedtodeterminelevelsofpotassiumperfluorooctanesulfonate (PFOS) in specimens of liverand serum of rats.The in-lifpeortionofthisstudywas conducted atArgu-sResearch Laboratoriess,tudy#418-015. All serum samples willbe extractedand analyzed atAdvanced BioanalyticalServices,Inc.and allliversamples extractedand analyzed at BattelleMemorial InstituteA.dditionalanalysesmay be performed atthe3M Environmental Laboratory as methods aredeveloped and validated. Ifadditionalanalysesareperformed an amendment tothisprotocolwillbe written. 3. REGULATORY COMPLIANCE This studywillbe conductedin accordancewith theUnited StatesFood and Drug Administration,Good LaboratoryPracticesStandards,FinalRule 21 CFR 58,with the exception thatanalysisof the testmaterialmixtureforconcentrations,olubilityh,omogeneity,and stability willnot be conducted,and istheresponsibilitoyfthe Sponsor. 4. QUALITY ASSURANCE The 3M EnvironmentalLaboratoryQualityAssurance Unitwillreview theprotocoland audit studyconduct,data,and finalreportto determinecompliancewith Good LaboratoryPractice Standards and with 3M EnvironmentalLaboratoryStandardOperatingProcedures. The QA Unit atthe sub-contracltaboratorywillaudittheirstudyconduct,data,and resultsreport priorto submittingto the3M EnvironmentalLaboratory. 3M EnvironmentaLlaboratory Page 3 of10 Protoco#lFACT-TOX-Ill 5. TESTMATERIAL 5.1 RefertoArgus ResearchLaboratoryprotocolforstudy#418-015. 6. CONTROL MATRICES 6.1 ldentiricatioRnat liverand senun,and/orrabbitliverand serumtraceabilintuymbers willbe recorded in the raw data and included in the finalreport 6.2 Source Argus Research and/or Sigina Chemical 6.3 Physical Description Rat liverand serum, and/or rabbitliverand serum 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 will be retainedin the 3M archives for as long as the qualityof the preparationaffordsevaluation,but not longer than ten years followingtheeffectivedateof the finaltestrule(ifapplicable). 6.7 Disposition Matriceswillbe retainedatthe 3M EnvirorunentalLaboratoryper GLP regulation.Certainmatrices(feces,urine,and blood) may be disposed afterQAU verification. 6.8 Safety Precautions Refer to MSDS forchemicals used. Wear appropriate laboratoryattirea,nd follow adequate precautionsforhandling biologicalmaterialsand preparingsamples foranalysis. 7. REFERENCE MATERIAL 7.1 Identification 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 StabilityPurityof PFOS is99% or greater.Stabilithyas notbeen 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 the preparationaffordsevaluation,but not longer than ten years followingtheeffectivedate of the finaltestrule(ifapplicable). 7.7 Disposition Unused referencematerialwillbe retainedforuse by the 3M m Environmental Laboratory and willbe discardedwhen the qualityof preparationno longer affordsevaluation. CD 3M Environmental Laboratory Page 4 of 10 Protoco#lFACT-TOX-1II 7.8 SafetyPrecautionsRefertoMSDS forchemicaulsed.Wearappropriate laboratorayttirea,nd followadequateprecautionfsorhandlingbiologicamlaterialsand preparingsamples foranalysis. TEST SYSTEM Rats were used asthetestsystemand were maintainedand dosed as describedinArgus Research protocol#418-015. The femaleratswillbe giventhetestmaterialor controlonce daily beginning42 days priorto cohabitatiounntilday 0 ofpresumed gestation.See tableI formore dosage information. Dosage Group 1 2 3 Table 1 Dosage Levels,Concentration,and Volumes Number of female mts 8 8 8 (D Dosage mg/kg/day 0 0.1 I.6 @- k ql7-2-lll Concentration mg/kg/day 0 0.02 0-.-23 Dosage volume mL/kg 5 5 5 9. SPECIMEN AND SAMPLE RECEIPT The 3M Environmental Laboratory will receive homogeneity samples and specimens of the followingbody tissuesand fluidfsrom theindicatepdointsinthe study.Allspecimenswillbe packed-ondry iceforshipping.See table2 formore specimeninformation. Body tissue/fluid Serum - Dam and Pup animals Urine and Feces - Dam and Pup animals Liver- Dam and Pup animals Table 2 Specimen Information Collected Dain-Predose,Days 7, 14,15,2 1, and 22 Pup-Day 21 Predose,Days 7, 15,21,and 22 Dam-At theterminatioonf the study Pup-Day2l Expected # of specimens 144 Dam 24 Pup (pooled) 120 Urine and 120 Feces 24 Dam 24 Pup (pooled) Totalnumber of expectedspecimens:456 Totalnumber of testanimals:16 Totalnumber of controlanimals:8 3M Environmental Laboratory Page 5 of 10 Protoco#lFAC T-TOX-I11 Specimens sentto3M EnvironmentalLaboratoriewsillbe receivedand trackedaccordingto applicableStandardOperatingProcedures. 10. PREPARATORY METHODS 10.1 FACT-M- 1.1,Extraction of Potassium Perfluorooctanesulfonate or Other Anionic FluorochemicalSurfactanftrom LiverforAnalysisUsing BPLC-Electrospray/Mass Spectrometry 10.2 ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Sermn or Other Fluid forAnalysisUsing BPLCElectrospray/Mass Spectrometry 10.3 Ifpreparatorymethods otherthanthose listedabove are used, an amendment to this protocolwillbe written.Any deviationsfrom thesemethods willbe documented and includedwith the study data. 10.4 Ifanalysesare sub-contractedto otherlaboratoriesa,n amendment willbe written to includetheirmethods and copiesof each method willbe attachedto thisprotocol. 11.ANALYTICAL METHODS 11.1 FACT-M-2. 1,Analysisof Fluorochemicalsin Liver ExtractsUsing HPLCElectrospray/N4assSpectrometry 11.2 ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicals in Serum or Other Fluid ExtractsUsing BPLC-Electrospray/Mass Spectrometry 11.3 Ifanalyticalmethods otherthan those listedabove areused, an amendment to this protocolwillbe written.Any deviationsfrom thesemethods willbe documented and included with the study data. 11.4 If analysesare sub-contractedto otherlaboratoriesa,n amendment willbe written to includetheirmethods and copiesof each method willbe attachedto thisprotocol. 12.DATA QUALITY OBJECTIVES The number of spikes/duplicatesu,se of surrogates,and infortnationon otherdata quality indicatorsareincludedin theanalyticamlethods. In addition,the followingcriteriwaillbe met: 12.1 Linearity r'@:0.98 rxn lz2 Limitsof detectionIquantitation 12.2.1 Method DetectioLnimit(MDL) forPFOS a) Serum: 1.75ppb b) Liver: 15 ppb 3M EnvironmentaLlaboratory Page 6 of10 Protoco#lFACT-TOX-111 12.Z2 LimiotfQuantita(tLiOoQn)- Equatlothelowesatcceptasbtlaendairnd thecalibratiocnurve 12.3 Duplicate acceptable precision < 30% forthemethod 12.4 Spike acceptable recoveries 70% - 130% 12.5 Use of conrirrnatorymethods Indeterminatseampleswillbe re-analyzeudsinga confirmatormyethod. Ifa confirmatormyethod isused,an amendment tothis protocowlillbe written. 12.6 Demonstration of speciricityChromatographircetentiotnime,mass spectral daughterioncharacterization. 13. SuB-CoNTRAcTED ANALYSIS 13.1 Allanalysesasdetaileidnthisprotocolwillbe performedat3M Envirorunental LaboratorieBsu,ilding2-3E-09,935 Bush Avenue,St.Paul,MN 55106,atAdvanced BioanalyticaSlervicesI,nc.1,5 CatherwoodRoad,IthacaN,Y 14850,oratBattelle Memorial Institut5e0,5 King Avenue,Columbus,Ohio 43201-2693. 13.2 An amendment tothisprotocolwillbe writteinfanalysesareperformedat laboratorioetsherthanthe3M EnvirorunentaLlaboratorieAsd,vanced Bioanalytical ServicesI,nc.o,rBattellMeemorial Institute. 14. STATISTICAL ANALYSIS Averagesand standarddeviationwsillbe calculateTdh.e statistimceatlhodsthatwillbe usedare describedbelow: 14.1 Data transformations and analysis Datawillbe reportedastheconcentration (weight/weighotrweight/volo)fPFOS ormetabolitpeertissuoerfluid. 14.2 Statisticaalnalysis Statistiucssedmay includeregressioannalysiosf concentrationosvertime,and standarddeviationcsalculatefdortheconcentrations withineach dose group.Ifnecessarys,implestatistictaelstss,uch asStudent'sttest, may be appliedtoevaluatestatisticdailfference. 15.REPORT A reportcontainingalltheresultosfthestudywillbe preparedby the3M Environmental LaboratoryI.fanalysesaresub-contractetdootherlaboratorieesa,ch laboratorwyillpreparea reportand submitittothe3M EnvirorunentaLlaboratoryforinclusioinnthe3M Environmental >< Laboratoryreport.Each reportwillincludeb,utnotbe limitetdo,thefollowingw,hen applicable: 15.1 Name and addressofthefacilipteyrformingthestudy 15.2 Datesupon which thestudywas initiataendd completed CD 3M Environmental Laboratory Page 7 of 10 Protoco#lFAC!*T-OX-111 15.3 A statemeonftcomplianbcyetheStudyDirectaodrdressianngyexceptiotnosGood LaboratoryPracticeStandards 15.4 Objectivesand proceduresas statedintheapprovedprotocoli,ncludingany changes intheoriginaplrotocol' 15.5 The testsubstanceidentificatiboynname, chemicalabstractnsumber orcode number, strengthp,urity,and compositionor otherappropriatceharacteristiicfsp,rovidedby theSponsor 15.6 Stabilitaynd thesolubilitoyf thetestsubstancesunder theconditionsof administrationi,fprovidedby theSponsor 15.7 A descriptioonf themethods usedto conductthetest(s) 15.8 A descriptioonf thetestsystem 15.9 A descriptioonf any circumstancesthatmay have affectedthequalityor theintegrity of thedata 15.10 The name of theStudy Directorand thenames of otherscientistpsr,ofessionalasn,d supervisorypersonnelinvolvedinthestudy 15.11 A descriptioonf thetransformationcsa,lculationosr,operationpserformedon the data,a summary and analysisof theanalyticaclhemistrydata,and a statementofthe conclusionsdrawn from theanalyses 15.12 Statisticmaelthods used toevaluatethedata,ifapplicable 15.13 The signedand datedreportsof eachof theindividualscientisotrs otherprofessionals involvedin thestudy,ifapplicable 15.14 The locationwhere raw dataand thefinalreportaretobe stored 15.15 A statementpreparedby theQualityAssuranceUnit listintghedatesthatstudy inspectionsand auditswere made, and thedatesof any findingsreportedto theStudy Directorand Management Ifitisnecessarytomake correctionosr additionstoa reportafterithas been accepted,the changeswillbe made intheform of an amendment issuedby theStudy DirectorT.he amendment willclearlyidentiftyhepartofthereportthatisbeing amended, thereasonsforthe amendment, and willbe signedby theStudy Director. 16.LocA TION OF PA w DATA, RECORDS, AND FINAL REPORT Originaldata,or copiesthereofw,illbe availablaetthe3M EnvironmentalLaboratoryto m facilitaatueditsof thestudyduringitsprogressand beforeacceptanceofthefinalreportW.hen thefinalreportiscompleted,alloriginaplaper data,includingthoseitemslistebdelow,willbe retainedin thearchivesof 3M EnvironmentalLaboratoryforatleasta periodof timeas specified by regulationa,nd as establishebdy 3M EnvironmentalLaboratoryStandardOperating Procedures. CD @2. 3M Environmental Laboratory Page 8 of 10 Protoco#lFACT-TOX-1II 16.1 Thefollowirnagwdataandrecordwsilbleretainiendthestudyfoldeirnthe study/projecatrchivesa-ccordingto3M EnvironmentalLaboratoryStandardOperating Procedures: 16.1.1 Approved protocoland amendments 16.1.2 Study correspondence 16.1.3 Shipping records 16.1.4 Raw data 16.1.5 Approved finalreport(originaslignedcopy) 16.1.6 Electroniccopiesof data 16.2 The followingsupportingrecordswillbe retainedseparatelfyrom thestudyfolderin thearchivesaccordingto3M EnvirorunentaLlaboratoryStandardOperating Procedures: 16.Zl Trainingrecords 16.2.2 Calibrationrecords 16.2.3 Instrumentmaintenance logs 16.2.4 StandardOperatingProcedures,Equipment Procedures,and Methods 17. SpEcimEN RETENTION Specimens willbe maintainedin the3M Environmental Laboratory specimen archivesfora periodof timeas specifiebdy regulatioonr aslong asthequalityof thepreparatioanffords evaluationb,utnot longerthantenyearsfollowingtheeffectivdeateofthefinaltestrule(if applicable)a,nd as establishebdy 3M EnvironmentalLaboratoryStandardOperatingProcedures. 18.PROTOCOL AMENDMENTS AND DEVIATIONS Plannedchanges totheprotocolwillbe intheform ofwrittenamendments signedby theStudy Directorand theSponsor'sRepresentativeA.mendments willbe consideredas partof the protocoland willbe attachedto thefinalprotocolA.ll changesto theprotocolwillbe indicated in thefinalreportA.ny otherchangeswillbe intheform of writtendeviationss,ignedby the StudyDirectorand filedwiththeraw data. 19.ATTAcHmENTs 19.1 AttachmentA Preparatoryand analyticamlethods 3M EnvironmentaLlaboratory Page 9oflO 20.SiGNATuREs Protoco#lFAC T-TOX-111 9&W.4,764-t- MarvinT.Case,D.V.M.P,h.D.S,ponsoRrepreseniative '7 Date Krtsten1.Hansei,Ph.D.,3M EnviromnentalLaboratoryStudy Director Date 3M Environmental Laboratory Page 10 of10 StudyTitle Oral(GavageP)harmacokinReetciocverSytudoyfPFOSinRats PROTOCOL AMENDMENT NO. 1 Amendment Date: August 12,1999 Performing Laboratory 3M EnviromnentaTlechnology& SafetyServices 3M EnviromnentalLaboratory 935 Bush Avenue St.Paul,MN 55106 Laboratory Project ldentirication ET&SS FACT-TOXI 11 LIRN U2994 3M EnvironmentLaalboratory ProtocoFlACT-TOXI 11 Amendment No.I Thisamendment modifiesthefollowinpgortion(so)ftheprotocol: 1 PROTOCOL READS: Section10.0and 11.0listthefollowingmethodstouse forextraction and analysis: FACT-M-1.1 "Extractionof Potassium Perfluorooctanesulfonatoer Other Anionic Fluorochemical Surfactantfrom Liver forAnalysisUsing BPLC-Electrospray/N4ass Spectrometry" FACT-M-2.1 "Analysisof Fluorochemicalsin Liver ExtractsUsing BPLC-Electrospray/Mass Spectrometry" AMEND TO READ: The extractionand analyticalmethods FACT-M- 1.1 and FACT-M-2. 1, respectivelyw,ere updated on 07/22/99 to: ETS-8-6.0 "Extractionof Potassium Perfluorooctanesulfonatoer Other Fluorochemical Compounds from Liver forAnalysisUsing BPLC-Electrospray/Mass SpectromeW/' ETS-8-7.0 "Analysis of Potassium Perfluorooctanesulfonatoer Other Fluorochemical Compounds in Liver ExtractsUsing BPLC-Electrospray/N4ass 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 differentextractionsolventMTBE (methyl tertbutyl ether),POAA and Monoester were removed from thestandardmix, and M556 was added to the standardmix. The analyticalmethod was updated to includelinearregressionwith 1/xweighting and a few minor changes in the BPLC I100 instrumentparameters. 2. PROTOCOL READS: Section10.4 and 11.4statethatifthe analysesaresub-contractedto other laboratoriesan amendment willbe writtento includethesemethods. AMEND TO READ: The extractionand analyticamlethods to follow atAdvanced Bioanalytical Serviceswillbe attachedto theprotocol. The extractionand analyticalmethod to followatBattelleMemorial Instituties: "Method forAnalysisof PerfluorooctaneSulfonate(PFOS) in Rat Ser*by LC/MS/MS, Versionj" I.D (9L;vt,@. REASON: The analytical methods at the sub-contract laboratories were not included in the originalprotocol. LA C- CD 3M Environmental Laboratory ProtocoFlACT-TOX1 11 Amendment No. 1 3. PROTOCOL READS: Section12.2.1b) listlsivemrethoddetectiolnimitas 15ppb. AMEND TO READ: Thelivemrethoddetectiloinmiits8.50ppb(ng/g). REASON: ThevalidatisounpportimnegthodEsTS-8-6.a0ndETS-8-7.i0ncludaedlower method detectionlimitforPFOS. Amendment Approval l@far4nCase,D.V.M., Ph.D.,Sponsor Representative 17A Date Kris J.Hansen, Ph.D.,Study Director Datb rn mx 3M EnvironmenLtaabloratory StudyTide Oral(GavagPeh)armacoldnReetciocverSytudoyfPFOSinRats PROTOCOL AMENDMENT NO. 2 Amendment Date: September30,1999 Performing Laboratory 3M EnvironmentaTlechnology& SafetyServices 3M EnvironmentaLlaboratory 935 Bush Avenue St.Paul,NfN 55106 Laboratory Project ldentirication ET&SS FACT-TOX1 11 LIRN U2994 3M EnvironmentLaalboratory ProtocolFACT-ToXiii Amendment No.2 Thisamendment modifiesthefollowinpgortion(so)ftheprotocol: 1. PROTOCOL READS: Sectio2nstatetshestudiysdesignetdodeten-nilneevelosfpotassium perfluorooctanesulfon(aPtFeOS) inspecimensof liverand serain rats. AMEND TO READ: The studyisdesignedtodeterminelevelsof potassiumperfluorooctanesulfona(tPeFOS) in specimensof livers,era,a.nd urine in rats. REASON: The urineanalyticamlethods were validatedand approved afterapprovalof theoriginal protocol. 2. PROTOCOL READS: Section10.0and 11.0listhefollowingmethods touse forextractioannd analysis: ETS-8-4.1 "ExtractioonfPotassium PerfluorooctanesulfonaotreOther Fluorochemical Compounds from Senan forAnalysisUsing BPLC-Electrospray/MassSpectromety" ETS-8-6.0 "ExtractioonfPotassium PerfluorooctanesulfonaotreOther Fluorochemical Compounds from LiverforAnalysisUsing BPLC-Electrospray/MassSpectrometry" ETS-8-5.1"Analysisof PotassiumPerfluorooctanesulfonaotreOtherFluorochemicalCompounds in Serum ExtractsUsing BPLC-Electrospray/MassSpectrometry" ETS-8-7.0"Analysisof PotassiumPerfluorooctanesulfonaotreOtherFluorochemical Compounds inLiverExtractsUsing BPLC-Electrospray/MassSpectrometry" m co AMEND TO READ:: ETS-8-4.1"Extractioonf PotassiumPerfluorooctanesulfonaotreOtherFluorochemical Compounds from Serum forAnalysisUsing BPLC-Electrospray/MassSpectrometry" ETS-8-6.0"Extractioonf PotassiumPerfluorooctanesulfonaotreOtherFluorochemical Compounds from LiverforAnalysisUsing BPLC-Electrospray/MassSpectrometry" ETS-8-96.0"Extractioonf PotassiumPerfluorooctanesulfonaotreOtherFluorochemical Compounds from UrineforAnalysisUsing BPLC-Electrospray/MassSpectrometry/Mass Spectromew/' ETS-8-5.1 "AnalysisofPotassium PerfluorooctanesulfonaotreOther Fluorochemical Compounds inSerum ExtractsUsing BPLC-Electrospray/MassSpectrometry" ETS-8-7.0 "AnalysisofPotassium PerfluorooctanesulfonaotreOther Fluorochemical Compounds inLiverExtractsUsing BPLC-Electrospray/MassSpectrometry" ETS-8-97.0"Analysisof PotassiumPerfluorooctanesulfonaotreOtherFluorochemical Compounds inUrineExtractsUsing HPLC-Electrospray/MassSpectrometry/Mass Spectrometry" REASON: The extractioannd analyticamlethods ETS78-96.0 and ETS-8-97.0,were approved afterapprovalof theoriginaplrotocol. 3M Environmental Laboratory ProtocoFlACT-TOXIII Amendment No.2 3. PROTOCOL READS: Section6.1listcsontromlatricaesratlivearndserumorrabbiltivearndserum.Source Argus and/orSiginaChemical. AMEND TO READ: Controlmatricesidentificatiroantliverand serum,rabbitliverand serum,human urine and/orraturine.SourceArgus,SiginaChemical,Lampire BiologicalsB,iologicalSpecialty Corp. and/orGolden West Biologicals. REASON: Additionof controlurinespecifications. 4. PROTOCOL READS: Section12.2.1a)listseramethod detectionlimitas 1.75ppb and b) listlsivermethod detectionEmit as 15 ppb. AMEND TO READ: The method detectionlimitsforallcompounds and matriceswillbe takenfrom themethods used forextractioannd analysis. REASON: The method detectionlimitslistedarespecifitco the3M EnvironmentalLaboratory. Statementwas added to allowforsub-contracteadnalysesand/orrevisedmethods. m Cl) CD 3M Environmental Laboratory ProtocoiFACT-TOXIII Amendment No.2 5. PROTOCOL. READS: Sectio1n6statetshatheorigindaaltao,rcopietshereowfi,lbleavailabalttehe3M EnvironmentalLaboratoryto facilitaatueditsof thestudyduringitsprogressand before acceptanceof thefinalreport.When thefinalreportiscompleted,alloriginaplaperdata, including:approvedprotocoland amendments, study @orrespondences,hippingrecords,raw data,approved finalrepom electroniccopiesof data,traininrgecordsc,alibratiornecords, instrumentmaintenance logsand standardoperatingprocedures,equipmentprocedures,and methods willbe retainedinthearchivesof the3M EnvirorumentaLlaboratory. AMEND TO READ: Section16 statesthattheoriginadlata,orcopiesthereofw,illbe availablaetthe3M EnvironmentalLaboratoryto facilitaatueditsof thestudyduringitsprogressand before acceptanceof thefinalreport.When thefinalreportiscompleted,alloriginaplaper data, including:approved protocoland amendments, studycorrespondences,hippingrecords,raw data,approved finalreport,and electroniccopiesofdatawillbe retainedinthearchivesof the3M EnvironmentalLaboratory.Allcorrespondingtraininrgecordsc,alibratiornecords, instrumentmaintenancelogs,standardoperatingprocedures,equipmentprocedures,and methods willbe retainedinthearchivesof thefacilitpyerformingeachanalysis. REASON: Clarificationf thedispositioonf archivedrecordsifanalysesareperformedata sub& contractlaboratory. 6. PROTOCOL READS: Section17 statesthatspecimenswillbe maintainedinthe3M EnvironmentalLaboratory specimen archives. AMEND TO READ: Specimens willbe maintainedin the3M EnvironmentalLaboratoryspecimen archives.All specimenssentto sub-contracltaboratoriewsillbe returnedtothe3M Envirorunental Laboratoryupon completionof analysisand submissionof thesub-contracltaboratory(s) finalreport.The specimenswillbe returnedwiththefollowingdocumentation:thesigned originalchainof custodyand recordsof storageconditionswhileatthesub-contracftacility. REASON: Clarificatiofnthedispositioonf specimensand documentationforanalysesperformedby a sub-contracltaboratory. FL El EL CD 3M Environmental Laboratory Amendment Approval ProtocolFACT-TOXIII Amendment No.2 Marvin Case, D.V.M., Ph.D.,Sponsor Representative Date'* 4an en,Ph.D.,Study Director Date 3M EnvironmentalLaboratory Study Title AnalyticLaalboratoRreyporotntheDeterminatiooftnhePresencaendConcentratioofn PotassiumPerfluorooctanesulfon(aCtAeS Number 2759-39-3)inthe Serum, Liver,and Urine of Crl:CD'BR VAF/Plus" RatsExposed toPFOS Via Gavage PROTOCOL AMENDMENT NO. 3 Amendment Date: 20 January2000 Performing Laboratories UrineAnalyses 3M EnvironimentaTlechnologyand SafetyServices FluorineAnalyticaClhemistryTeam Buildin2g-3E-09 935 Bush Avenue St.Paul,N[N 55106 Performing Laboratories LiverAnalyses BattellMemorialInstitute 505 King Avenue Columbus,OH 43201-2693 Serum Analyses Advanced BioanalyticSaelrvices, Inc. 15 Catherwood Road IthacaN,Y 14850 m C7 E; 12. Laboratory Project ldendrication ET&SS LRN-U2994 FACT TOX- I11 Argus Study: 418-015 3M MedicalDepartmentStudy: T-6295.14 3M EnvironmentaLlaboratory ProtocolLRN-U2994 Amendment Number 3 Thisamendment modifiesthefollowinpgortion(so)ftheprotocol: 1. PROTOCOL READS: The studydirectofrorthepresensttudywas identifientdheprotocoals KristeJn. Hansen, Ph.D. AMEND TO READ: The roleofstudy directorforthe presentstudy was reassignedto Marvin T. Case, D.V.M.,Ph.D.,as of 20 January 2000. The previousstudy directorK,ristenJ. Hansen, has been reassignedto the roleofPrinciplAenalyticalInvestigator. REASON: The roleof study directorwas reassignedinan efforto ensure compliance with Good LaboratoryPracticeStandards thatoutlinestudypersonnelrequirements (referto 21 CFR Part58). 2. PROTOCOL READS: The sponsor forthepresentstudywas identifiaesd MarvinT. Case, D.V.M.,Ph.D. AMEND TO READ: The roleof sponsor forthe presentstudywas reassignedto John L. Butenhoff, Ph.D.,as of20 January 2000. REASON: To ensure thatthe study directordoes not alsocarrythe dutiesof study sponsor,the sponsor rolewas reassigned.Inthismanner, personnelresponsibilitiaensd workload are more evenly balanced. 3M EnvironmentalLaboratory Amendment Approval ProtocolLRN-U2994 Amendment Number 3 John L.ButenhoffP,h.D.,SponsorRepresentative Date IA@ Kristen J Hansen, PhD., Outgoing Study Director oxlb-Avo Date Marvin T. Case, D. V M, Ph.D., Incoming Study Director -Date =4 C:) CD 3M Environmental Laboratory Study Title Oral(GavageP)harmacokineRteiccoverSytudyofPFOS inRats_ PROTOCOL AMENDMENT NO. 4 Amendment Date: 20 April2000 Performing Laboratories 3M Environmental Technology and Safety Services FluorineAnalyticalChemistry Team Building2-3E-09, 935 Bush Avenue St.Paul,MiN 55106 Centre AnalyticalLaboratories,Inc. 3048 Research Drive StateColle-e,PA 16801 BattelleMemorial Institute ,-;0K5ing Avenue Columbus, OH 43201-2693 Advanced BioanalyticalServices,Inc. 15 Catherwood Road Ithaca,NY 14850 Laboratory Projectldentification ET&SS LRN-U2994 FACT TOX- III Argus Study: 418-015 -'Iii''vvtiedicDaelpartment Study: T-6295.14 3M Environmental Laboratory ProtocolLRN-U2994 Amendment Number 4 Thisamendment modifiesthefollowinpgortion(so)ftheprotocol: 1. PROTOCOL READS: The amended section2.0textstatetshatthisstudyisdesignedtodeterminePFOS in specimens,of ratliver,serum, and urine. AMEND TO READ: This study isdesigned to determine PFOS in specimens of ratliver,serum, feces,and urine. REASON: I The analysisof fecaltissueforthe targetchemical and/oritsanalyteswas added to the'scope of the study followingthe issuanceof theprotocol.Feces extractionand analyticalmethods were not validatedand approved priorto protocolapproval. 2. PROTOCOL READS: The amended section6.0listsrator rabbitliver,serum, and urine. AMEND TO READ: Add: rator rabbitfeceswith a physicaldescriptionof rator rabbitfeces. REASON: Analysis of fecaltissueforthe targetchemical and/oritsanalyteswas added to the scope of the study following the issuanceof the originalprotocol. 3. PROTOCOL READS: Section I')1. listsallof the laboratorietshatwillbe conducting analysesforthisstudy. AMEND TO READ: Add: Centre AnaIN-ticaLlaboratories,Inc.,3048 Research Drive, StateCollege,PA 16801 REASON: Feces analvseswere added to the scope of thisstudy.The sub-contractlaboratoryperforming analyses was not Ln the originalprotocol. 4. PROTOCOL READS: Sections 10.4 and 11.4statethatifthe analysesaresub-contractedto otherlaboratoriesan amendment willbe writtento includethese methods. AMEND TO READ: The fecesextractionand analyticalmethod used by Centre AnalyticalLaboratorieswill be; OOM-02'J-00'i (Revision2),"Determinationof FluorochemicalResidues inMonkey/Rat Feces by LCAIS,.NIS.'@ REASON: The sub-contract laboratory performing feces analyses was added to the scope of thisstudy; thismethod was not validatedand approved priorto protocolapproval. 3M EnvironmentalLaboratory ProtocoLlRN-U2994 Amendment Number 4 Thisamendment modifiesthefollowinpgortion(so)ftheprotocol: 1. PROTOCOL READS: The amended section2.0textstatesthatthisstudyisdesignedtodeterminePFOS in specimens of ratliver,serum, and urine. AMEND TO READ: This study isdesigned to determine PFOS in specimens of ratliver,serum, feces,and urine. REASON.The analysisof fecaltissueforthetargetchemical ancuor itsanalyteswas added to thescope of thestudy followingthe issuance of theprotocol.Feces extractionand analyticamlethods were not validatedand approved priorto protocolapproval. 2. PROTOCOL READS: The amended section6.0 listsrator rabbitliver,serum, and urine. AMEND TO READ: Add: rator rabbitfeceswith a physicaldescriptionof rator rabbitfeces. REASON: Analysis of fecaltissueforthetaroetchemical and/oritsanalyteswas added to the scope of the study followine theissuanceof the oriainalprotocol. 3. PROTOCOL READS: Section 13.1 listsallof the laboratoriesthatwillbe conducting analysesforthisstudy. AMEND TO READ: Add: Centre AnaiN.-ticLaalboratories,Inc.,3048 Research Drive, StateCollege,PA 16801 REASON: Feces analyseswere added to the scope of thisstudy.The sub-contractlaboratoryperforming analyses -v%-ansot in the originalprotocol. 4. PROTOCOL READS: Sections 10.4 and 11.4 statethat ifthe analyses are sub-contractedto other laboratoriesan amendment willbe writtento includethese methods. AMEND TO READ: The fecesextractionand analyticalmethod used by Centre AnalyticalLaboratorieswillbe; OOM-023-OO'l (Revision2),"Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/'-\ISAIS." REASON: .r,," The sub-contractlaboratoryperforminc,feces analyseswas added to the scope of thisstudy; thismethod was not validatedand approved priorto protocolapproval. CD 3M Environmental Laboratory Amendment Approval APmreotnodcmolenLtRNNu-mUb2e9 2. John L. Butenhoff,Ph.D., Sponsor Representative 9r z.:@o,e Date TUa@@Ti@nCaseD,.V.M.P,h.DS.t,udDyirector /q ,I Date m 3M EnvironmentalLaboratoty