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3M Environmental Technolop_v and Services PO Box 33331 St.Paul,MN 55133-3331 6127786442 Protocol #FA C T- TOX- I 10 Study Title Oral (Gavage) Pharmacokinetic Study of PFOS in Rats PROTOCOL Author LisaClemen Date: June8,1999 Performing Laboratory 3M EnviromnentalTechnology& SafetyServices 3M EnviromnentalLaboratory 935 Bush Avenue St.Paul,MN 55106 Laboratory Project ldentirication FACT-TOX- 110 U2849 3M EnvironmentaLlaboratory Page Iof 10 Protoco#lFACT-TOX-110 StudyIdentification Oral(Gavage)PharmacokinetSitcudyofPFOS inRats TestMaterial Perfluoroocstualnfeonaiccipdotassisuamlt (T-6295) Sponsor 3M ToxicologyService-sMedicalDepartment 3M Center,Building220-2E-02 St.Paul,MN 55144-1000 Sponsor Representative Marvin T. Case,D.V.M., Ph.D. 3M Toxicology Services Telephone: 651-733-5180 Facsimile:651-733-1773 Study Director KristenJ.Hansen, Ph.D. 3M EnvironmentalTechnology and SafetyServices Building2-3E-09 651-778-6018 Study Location(s) In vivo Testing Facility AnalyticalTesting Laboratory Argus Research Laboratories,Inc. 905 Sheehy Drive,BuildingA Horsham, PA 19044 3M Environmental Laboratory Building2-3E-09 935 Bush Avenue St.Paul,MN 55106 3M EnvironmentaLlaboratory Page 2 of10 Sub-ContracLtaboratofy Proposed Study Timetable Study InitiatioDnate Study Completion Date Protoco#lFACT-TOX-110 AdvanceBdioanalytiScearlviceIsn,c. 15 Catherwood Road IthacaN,Y 14850 BattellMeemorial Institute 505 King Avenue Columbus, Ohio 43201-2693 June8, 1999 August 8,2000 1. STUDY Oral(gavage)pharmacokineticstudyofpotassiumperfluorooctanseulfonicacid(PFOS) inrats. 2. PURPOSE This analyticasltudyisdesignedtodeterminelevelsofpotassiumperfluorooctanesulfonate (PFOS) inspecimensof liverand serum ofrats.The in-lifpeortionofthisstudywas condficted atArgus ResearchLaboratoriess,tudy#418-013.All serum sampleswillbe extractedand analyzed at Advanced Bioanalytical Services, Inc. and allliversamples extracted and analyzed at BattelleMemorial InstituteA.dditionalanalysesmay be performed atthe 3M Environmental Laboratory as methods aredeveloped and validated.Ifadditionalanalysesareperformed an amendment to thisprotocolwillbe written. 3. REGULATORY COMPLIANCE This study willbe conducted in accordance with theUnited StatesFood and Drug Administration,Good Laboratory PracticesStandards,FinalRule 21 CFR 58,with the exception thatanalysisof thetestmaterialmixture forconcentrations,olubilityh,omogeneity, and stability willnot be conducted, and istheresponsibilitoyf theSponsor. 4. QUALITY ASSURANCE The 3M Environmental Laboratory QualityAssurance Unit willreview theprotocoland audit study conduct, data,and finalreportto determine compliance with Good Laboratory Practice rn Standards and with 3M Environmental Laboratory Standard OperatingProcedures. The QA Unit atthe sub-contractlaboratorywillaudittheirstudy conduct,data,and resultsreport priorto submittingto the 3M Environmental Laboratory. 3M EnvironmentalLaboratory Page 3 of 10 Protoco#lFACT-TOX-110 5. TESTMATERIAL 5.1 RefertoArgus ResearchLaboratoryprotocolforstudy#418-013. 6. CONTROL MATRICES 6.1 ldentiricationRat liverand seniinand/orrabbitliverand serum,traceabilintuymbers willbe recorded in the raw data and includedin thefinalreport 6.2 Source Argus Research and/orSigma Chemical 6.3 Physical Description Rat liverand serum and/orrabbitliverand serum 6.4 Purity and Stability Not applicable 6.5 Storage Conditions Frozen at -20 OC 10 'C or -50 OC 10 OC 6.6 Reserve Matrix A portion of the controlmatrix willbe retainedin the 3M archives foras long as the qualityof the preparationaffordsevaluation,but not longer than ten years followingtheeffectivedate of thefinaltestrule(ifapplicable). 6.7 Disposition Matriceswillbe retainedatthe 3M EnvironmentalLaboratoryper GLP regulation.Certainmatrices(feces,urine,and blood) may be disposed afterQAU verification. 6.8 Safety Precautions Refer to MSDS forchemicalsused. Wear appropriate laboratoryattirea,nd follow adequate precautionsforhandling biologicalmaterialsand preparing samples for analysis. 7. REFERENCE MATERIAL 7.1 Identification Potassium perfluorooctanesulfonat(ePFOS), 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 not been determined. 7.5 Storage Conditions Room temperature 7.6 Reserve Material A reservesample from each batchof PFOS used in thisstudywill be retainedas long as the qualityof the preparationaffordsevaluation,but not longer rn than tenyears followingthe effectivedateof the finaltestrule(ifapplicable). 7.7 Disposition Unused referencematerialwillbe retainedforuse by the 3M CD Environmental Laboratoryand willbe discardedwhen thequalityof preparationno longer affordsevaluation. 3M EnvironmentalLabor-atory Page 4 of 10 Protoco#lFACT-TOX-110 7.8 SafetyPrecautionsRefertoMSDS forchemicalused.Wearappropriate laboratorayttirea,nd followadequateprecautionsforhandlingbiologicamlaterialsand preparingsamples foranalysis. TEST SysTEm Rats were used asthetestsystem and were maintainedand dosed asdescribedintheArgus Researchprotocol#418-013. The femaleratswillbe giventhetestmaterialor controlonce daily beginning42 days priortocohabitatioannd continuethroughday 14 or 20 of presumed gestationS.ee tableI formore dosage information. Table 1 Dosage Levels, Concentration, and Volumes Dosage Group 1 2 3 4 5 Number of femalerats 16 16 16 16 16 Dosage mg/kg/day 0 0.1 0.4 1.6 3.2 Concentration mg/kg/day 0 0.02 0.08 0.32 0.64 Dosage volume m]LAg 5 5 5 5 5 9. SPECIMEN AND SAMPLE RECEIPT The 3M EnvironmentalLaboratorywillreceivehomogeneity samples fordose analysisand specimens of the followingbody tissuesand fluidsfrom the indicatedpointsin the study.See table2 forspecimen information.All specimens willbe packed on dry iceforshipping. Body tissue/fluid Table 2 Specimen Information Collected Serum - Dam and Fetus animals Urine and feces- Dam animals Liver- Dwn and Fetus aniinals Milk SecretingGlands - Dain animals F7k-n@oticFluid - Dam animals CD Darn-Predose, Days 7, 15, and 21 Fetus-At terminationof the study Predose, Days 7, 15, and 21 Dam and Fetus-At terminationof the study At the terminationof the study Day 15 and Day 21 Expected # of specimens 320 Dam and 320 Fetus (pooled) 320 urine and 320 feces 80 Dain and 80 Fetus (pooled) 80 80 3M Environmental Laboratory Page 5 of 10 Protoco#lFACT-TOX-I10 Table2 Cont SpecimenInformation Body tissue/fluid Collected Expected # of specimens Embryo's with Placentae- Dam Day 15 and Day 21 80 animals Carcass- Fetus animal At theterminationof thestudy 160 Placentae- Fetus animal Day 15 and Day 21 80 Liver/lung- Fetusanimal At theterminationof thestudy 80 Totalnumber of expected specimens:2000 Totalnumber of testanimals:64 Totalnumber of controlanimals:16 Specimens sentto3M EnvironmentalLaboratorieswillbe receivedand trackedaccordingto applicableStandard OperatingProcedures. 10.PREPARATORY METHODS 10.1 FACT-M- 1.1,ExtractionofPotassium Perfluorooctanesulfonaotre Other Anionic FluorochemicalSurfactantfrom LiverforAnalysisUsing HPLC-Electrospray/Mass Spectrometry 10.2 ETS-8-4. 1,Extractionof Potassium Perfluorooctanesulfonatoer Other FluorochemicalCompounds from Serum or Other FluidforAnalysisUsing BPLCElectrospray/MassSpectrometry 10.3 Ifpreparatorymethods otherthan those listedabove areused,an ame ndment tothis protocolwillbe written.Any deviationsfrom thesemethods willbe documented and includedwith thestudy data. 10.4 Ifanalysesaresub-contractedtootherlaboratoriesa,n amendment willbe writtento includetheirmethods and copiesof each method willbe attachedtothisprotocol. 11.ANALYTICAL METHODS 11.1 FACT-M-2. 1,AnalysisofFluorochemicalsin LiverExtractsUsing BPLCElectrospray/MassSpectrometry 11.2 ETS-8-5. 1,Analysisof Potassium Perfluorooctanesulfonatoer Other Fluorochemicals in Serum or Other FluidExtractsUsing HPLC-Electrospray/Mass Spectrometry 11.3 Ifanalyticamlethods otherthanthoselistedabove areused,an amendment to this protocolwillbe written.Any deviationsfrom thesemethods willbe documented and includedwith thestudy data. 11.4 Ifanalysesaresub-contractedtootherlaboratoriesa,n amendment willbe writtento includetheirmethods and copiesof each method willbe attachedtothisprotocol. 3M EnvironmentalLaboratory Page 6 of10 Protoco#lFACT-TOX-110 12.DATA QUALITYOBJECTIVES The numberofspikes/duplicautseeso,fsurrogateasn,dinformation otherdataquality indicatorasreincludedintheanalyticamlethods. In additiont,hefollowingcriteriwaillbe met: IZ I Linearity r2 2:0.98 12.2 Limits of detection / quantitation 12.2.1 Method DetectionLimit (MDL) forPFOS a) Serum: 1.75ppb b) Liver: 15 ppb 12.2.2 Limitof Quantitatio(nLOQ) - Equaltothelowestacceptablsetandardin thecalibratiocnurve 12.3 Duplicate acceptable precision < 30% forthemethod 12.4 12.5 Spike acceptable recoveries 70% - 130% Use of conrirmatory methods Indeterminatesampleswillbe re-analyzedusinga confirmatorymethod. Ifa confirmatorymethod isused,an amendment tothis protocolwillbe written. 12.6 Demonstration of speciricity Chromatographicretentiotnime,mass spectial daughterion characterization. 13. SUB-CONTR,4cTED ANALYSIS 13.1 All analysesas detailedinthisprotocolwillbe performed at3M Environmental LaboratoriesB,uilding2-3E-09,935 Bush Avenue, St.Paul,N4N 55106, atAdvanced BioanalyticalServices,Inc.,15 Catherwood Road, Ithaca,NY 14850, or atBattelle Memorial Institut5e0,5 King Avenue, Columbus, Ohio 43201-2693. 13.2 An amendment tothisprotocolwillbe writtenifanalysesareperformed at laboratorieostherthan 3M EnvironmentalLaboratoriesA,dvanced Bioanalytical Services,Inc.,orBattelleMemorial Institute. 14. STATISTICAL ANALYSIS Averages and standarddeviationswillbe calculatedT.he statisticmaelthods thatwillbe used are rn describedbelow: a 14.1 Data transformationsand analysis Datawillbe reporteadstheconcentration (weight/weightorweight/vol)ofPFOS or metabolitepertissueor fluid. 3M Environmental Laboratory Page 7 of 10 Protoco#lFACT-TOX-110 14.2 StatisticaanlalysisStatistuiscesdmay includreegressiaonnalysoifs concentrationosvertime,and standarddeviationscalculatefdortheconcentrations withineach dosegroup. Ifnecessary,simplestatistictaelstss,uchas Student'st test, may be appliedtoevaluatestatisticdailfference. 15.REPORT A reportcontainingalltheresultosf thestudywillbe preparedby the3M Environmental Laboratory.Ifanalysesaresub-contractetdo otherlaboratorieesa,ch laboratorwyillpreparea reportand submitittothe3M EnvironmentalLaboratoryforinclusioinnthe3M Environmental Laboratoryreport.Each reportwillincludeb,utnotbe limitedto,thefollowingw,hen applicable: 15.1 Name and addressofthefacilitpyerformingthestudy 15.2 Dates upon which thestudywas initiateadnd completed 15.3 A statementof complianceby theStudy Directoraddressingany exceptionsto Good LaboratoryPracticeStandards 15.4 Objectivesand proceduresasstatedintheapproved protocoli,ncludingany changes intheoriginaplrotocol 15.5 The testsubstanceidentificatiboynname, chemicalabstractnsumber orcode number, strengthp,urity,and compositionor otherappropriatceharacteristiicfsp,rovideaby the Sponsor 15.6 Stabilitaynd thesolubilitoyf thetestsubstancesundertheconditionsof administratioinf,providedby theSponsor 15.7 A descriptioonfthemethods usedto conductthetest(s) 15.8 A descriptioonfthetestsystem 15.9 A descriptioonf any circumstancetshatmay have affectedthequalityor theintegrity of thedata 15.10 The name of theStudy 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 each of theindividualscientistosr otherprofessionals involvedin thestudy,ifapplicable 15.14 The locationwhere raw dataand thefinalreportaretobe stored 3M EnvironmentaLlaboratory Page 8 of10 Protoco#lFACT-TOX-ilo 15.15A statemenptreparebdy theQualitAyssurancUenitlistitnhgedatesthatstudy inspectionsand auditswere made, and the datesofany findingsreportedto the Study Directorand Management Ifitisnecessarytomake correctionosr additionstoa reportafterithas been accepted,the changes willbe made inthe form of an amendment issuedby theStudy Director.The amendment willclearlyidentifythe partof the reportthatisbeing amended, thereasons forthe amendment, and willbe signedby the Study Director. 16.LocATION OF RAw DATA, RECORDS, AND FINAL REPORT Originaldata,or copiesthereof,willbe availableatthe 3M Environmental Laboratoryto facilitaatueditsof the studyduringitsprogressand beforeacceptanceof the finalreport.When the finalreportiscompleted,alloriginalpaperdata,includingthoseitemslistedbelow, willbe retainedinthe archivesof 3M EnvironmentalLaboratoryforatleasta periodof time as specified by regulationa,nd asestablishedby 3M EnvironznentalLaboratoryStandardOperating Procedures. 16.1 The followingraw dataand recordswillbe retainedinthe studyfolderin the study/projecatrchivesaccordingto 3M EnvironmentalLaboratoryStandard Operating 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(originalsigned copy) 16.1.6 Electroniccopies of data 16.2 The followingsupportingrecordswillbe retainedseparatelyfrom.thestudy folderin the archivesaccordingto 3M EnvironmentalLaboratoryStandard Operating Procedures: 16.2.1 Trainingrecords 16.2.2 Calibrationrecords 16.2.3 Instrumentmaintenance logs 16.2.4 Standard Operating Procedures,Equipment Procedures,and Methods 3M EnvironmentalLaboratory Page 9 of 10 Protoco#lFACT-TOX-110 17. SpEctmEN RETENTION Specimewnislblemaintainientdhe3M EnvironmenLtaabloratosrpyecimeanrchivfeosra periodoftimeasspecifiebdy regulatioonraslongasthequalityofthepreparatioanffords evaluationb,utnotlongerthantenyearsfollowingtheeffectivdeateofthefinaltestrule(if applicablea)n,d asestablishebdy 3M EnvironmentalLaboratoryStandardOperatingProcedures. 18.PRoTocoL AMENDMENTS AND DEVIATIONS Plannedchangestotheprotocolwillbe intheform ofwrittenamendments signedby theStudy Directoarnd theSponsor'sRepresentativAem.endments willbe consideredaspartofthe protocoland willbe attachetdothefinaplrotocolA.llchangestotheprotocolwillbe indicated inthefinarleportA.ny otherchangeswillbe intheform ofwrittendeviations,ignedby the StudyDirectorand filewdiththeraw data. 19.A TrAcHmENTs 19.1 AttachmentA Preparatoraynd analyticamlethods 20.SIGNATURES Marvin T. Case,D.V.M.,Ph.D.,SponsorRepresentative Date Kristen'H-aJn.sen,Ph.D.,3M EnvironmentalLaboratoryStudyDirector Date 3M Environmental Laboratory Page 10of10 StudyTitle Oral(GavagPeh)armacoldnSettuidcoyfPFOSinRats PROTOCOL AMENDMENT NO. I Amendment Date: August12,1999 Performing Laboratory 3M EnviromnentaTlechnology& SafetyServices 3M EnviromnentaLlaboratory 935 Bush Avenue St.Paul,N4N 55106 Laboratory Project Identirication ET&SS FACT-TOX110 LIRN U2849 m 3M EnvironmentaLlaborstoiy ProtocoFlACT-TOXI10 Amendment No.I Thisamendment modifiesthefollowinpgortion(so)ftheprotocol: 1. PROTOCOL READS: Section2.0statetshistudiysdesignetdodetermipnoetassium perfluorooctanseulfonicacid(PFOS) inspecimensof liverand serum of rats. AMEND TO READ: ThisstudyisdesignedtodeterminePFOS inspecimensof ratlivers,erum, and urine.All Day -1 and GestationDay 21 raturinespecimenswillbe extracteadnd analyzed by the3M EnvironmentalLaboratory. REASON: The urineextractioannd analyticamlethods were not validateadnd approvedpriorto protocolapproval. 2. PROTOCOL READS: Section6.0listrsatorrabbitliverand serum. AMEND TO READ: Rat or rabbiturinefrom 3M Toxicologywitha physicaldescriptioonfrator rabbiturine. REASON: The raturinematrixwas added aftertheprotocolwas approved. 3. PROTOCOL READS: Section10.0and 11.0listhefollowingmethods touse forextraction and analysis: FACT-M-1.1 "ExtractioonfPotassiumPerfluorooctanesulfonoartOetherAnionic FluorochemicalSurfactanftrom LiverforAnalysisUsing BIPLC-Electrospray/Mass Spectrometry" FACT-M-2.1 "AnalysisofFluorochemicalisnLiverExtractsUsing BIPLC-Electrospray/Mass Spectrometry" AMEND TO READ: The extractioannd analyticamlethods tofollowatthe3M Environmental Laboratoryare: ETS-8-6.0 "Extractioonf PotassiumPerfluorooctanesulfonoarteOtherFluorochemical Compounds from LiverforAnalysisUsing BPLC-Electrospray/MassSpectromety" ETS-8-96.0 "Extractioonf PotassiumPerfluorooctanesulfonoarteOtherFluorochemical Compounds from UrineforAnalysisUsing BPLC-Electrospray/MassSpectrometry/Mass Spectrometry" ETS-8-7.0 "Analysisof PotassiumPerfluorooctanesulfonoarteOtherFluorochemical Compounds in LiverExtractsUsing BPLC-Electrospray/MassSpectrometry" 0 ETS-8-97.0 "AnalysisofPotassiumPerfluorooctanesulfonoarteOtherFluorochemical Compounds inUrine ExtractsUsing BI?LC-Electrospray/MasSspectrometry/Mass Spectrometry" 3M EnvironmentaLlaboratory ProtocoFlACT-TOXIIO Amendment No. I REASON: TheextractainodnanalytimceatlhodFsACT-M-1.1andFACT-M-2.1r,espectively, were updatedon 07/22/99toETS-8-6.0and ETS-8-7-0. Methods ETS-8-96.0and ETS-8-97.0were not validatedand approveduntilaftertheprotocol was approved. 4. PROTOCOL READS: Section10.4and 11.4 statethatiftheanalysesaresub-contractedto otherlaboratorieasn amendment willbe writtento includethesemethods. AMEND TO READ: The extractioannd analyticamlethods to followatAdvanced Bioanalytical Serviceswillbe attachedtotheprotocol. The extractioannd analyticamlethod to followatBattellMeemorial Instituties: "Method forAnalysisofPerfluorooctanSeulfonate(PFOS) inRat Ser-aby LC/MS/MS, Version-1"1.D L;ver 0 REASON: The analyticamlethods atthesub-contracltaboratoriewsere notincludedinthe originaplrotocol. 5. PROTOCOL READS: Section12.2.1b) Livermethod detectionlimitis15 ppb. AMEND TO READ: 12.2.1b) Livermethod detectionlimitis8.50ppb (ng/g). 12.2.1c)Urinemethod detectiolnimitis1.5ppb (ng/g). REASON: The validatiosnupportingmethodsETS-8-6.0and ETS-8-7.0includeda lower method detectiolnimitforPFOS inliver.A validatioinnurinewas performed,afterprotocol approval,to supportthismethod detectionlimit. S7 3M EnvironmentaLlaboratory AMendment Approval ProtocolFACT-TOXIIO Amendment No.I T(@@ Marvin Case,D.V.M.,Ph.D.,SponsorRepresentative /7 4A4,@Date Kris J.Han@en,'Ph.D gtudy Director Date 3M EnvironmentalLaboratory StudyTige Oral(GavagPeh)armacoldnSettuidcoyfPFOSinRats PROTOCOL AMENDMENT NO. 2 Amendment Date: September28,1999 Performing Laboratory 3M EnvirorunentTaelchnology& SafetyServices 3M EnvironmentalLaboratory 935 Bush Avenue St.Paul,MN 55106 Laboratory Projectldentiricatton ET&SS FACT-TOX1 10 LIRN U2849 3M EnvironmentLaalboratory ProtocoFlACT-TOXIIO Amendment No.2 Thisamendment modifiesthefollowinpgortion(so)ftheprotocol: 1. PROTOCOL READS: Section2 statetshatallliversampleswillbe extracteadnd analyzedatBattellMeemorial Institute. AMEND TO READ: All dam liverspecimens,(femalemother) and 27 fetus(pooled) liverspecimens willbe analyzed atBattelleMemorial Institute. REASON.The fetalhver/lungisnot a targetmatrix and willnot be extractedor analyzed. 2. PROTOCOL READS: Section 9 statesthat 80 dam liver specimens (female mother) and 80 fetus(pooled) liver specunens willbe sentto theEnvironmental Laboratory. AMEND TO READ: Seventy-threedam liverspecimens (female mother), 27 fetus(pooled)liverspecimens, and 81 fetalliver/lungspecimens (27 liver/lunsgpecimens sampled in triplicatwee)re sentto the Environmental Laboratory. REASON.The number of animals changed during the course of the study. 3. PROTOCOL READS: Section 12.2.1a) serum method detectionlimitis 1.75 ppb b) Liver method detectionlimitis 15 ppb. AMEND TO READ: The method detectionlimitsfor allcompounds used for extractionand analysis. and matrices willbe taken from the methods REASON: T The method detectionlimitslistedare specificto the 3M Environmental Laboratory. Statement was added to allow for sub-contractedanalyses and/or revised methods. CD 3M Environmental Laboratoty ProtocoFlACT-TOXIIO Amendment No.2 4. PROTOCOL READS: Section16 statesthattheoriginadlata,orcopiesthereofw,illbe availablaetthe3M 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,electroniccopiesof data,trainingrecords,calibrationrecords, instrument maintenance logs and standard operatingprocedures,equipment procedures,and methods willbe retainedin the archivesof the3M Environmental Laboratory. AMEND TO READ: Section 16 statesthattheoriginaldata,or copiesthereof,willbe availableat the 3M Environmental Laboratory to facilitataeuditsof the study during itsprogressand before acceptanceof 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,calibrationrecords, instrument maintenance logs,standardoperatingprocedures,equipment procedures,and methods will be retainedin the archivesof thefacilitpyerforming each analysis. REASON: Clarificatioonf the dispositionof archivedrecordsifanalysesare performed ata sub-contract laboratory. 5. PROTOCOL READS: Section 17 statesthatspecimens willbe maintained in the 3M specimen archives. Environmental Laboratory AMEND TO READ: Specimens willbe maintained in the 3M Environmental Laboratory specimen archives. All specimens sentto sub-contractlaboratoriewsillbe returnedto the 3M Environmental Laboratory upon completion of analysisand submission of the sub-contractlaboratory(s) finalreport.The specimens willbe returnedwith the followingdocumentation: the signed originalchain of custody and records of storageconditionswhile at thesub-contractfacility. REASON: Clarificatioonf the dispositionof documentation when shipping specimens foranalyses performed by a sub-contractlaboratory. 3M EnvironmentalLaboratory Amendment Approval I WA" . TQ A Niarvin Case, D.V.M., Ph.D., Sponsor Representative ProtocoFlACT-ToXi lo Amendment No.2 Diti KristenJ.Hansen, Ph.D., Study Dftwtor Date @-2a; 3MEnvironLmaebnotraaltory StudyTitle AnalyticLaalboratoRreyportontheDeterminatioofPnerfluorooctanesul(fPoFnOaSt)e Presenceand ConcentrationinSerum, Liver,and Urine from theGavage Study of T-6295.12 PROTOCOL AMENDMENT NO. 3 Amendment Date: 20 January2000 UrineAnalyses 3M EnvironmentaTlechnologyand SafetyServices FluorineAnalyticaClhemistryTeam Building2-3E-09 935 Bush Avenue St.Paul,MN 55106 Performing Laboratories LiverAnalyses BattellMeemorialInstitute 505 King Avenue Columbus,OH 43201-2693 Serum Analyses Advanced BioanalyticSaelrvices, Inc. 15CatherwoodRoad IthacaN,Y 14850 Laboratory Project ldentirication ET&SS LRN-U2849 FACT TOX-1 10 Argus Study: 418-013 3M Medical DepartmentStudy: T-6295.12 3M EnvironmentaLlaboratoiy ProtocolLRN-U2849 Amendment Number 3 Thisamendment modifiesthefollowinpgortion(so)ftheprotocol: 1. PROTOCOL READS: The studydirectfoorrthepresensttudywas identifinetdheprotocoals KristeJn. Hansen, Ph.D. AMEND TO READ: The roleofstudy directofrorthe presentstudy was reassignedtoMarvin T. Case, D.V.M.,Ph.D.,as of20 January 2000. The previousstudy directorK,ristenJ. Hansen, has been reassignedtothe roleofPrinciplAenalyticaIlnvestigator. REASON., The roleof study directowras reassignedinan efforto ensure compliance with Good LaboratoryPracticeStandards thatoutlinestudy personnelrequirements (refetro 21 CFR Part6'8). 2. PROTOCOL READS: The sponsor forthe presentstudywas identifiaesd Marvin T. Case, D.V.M.,Ph.D. AMEND TO READ: The roleofsponsor forthe presentstudywas reassignedto John L.Butenhoff, Ph.D.,as of 20 January 2000. REASON: To ensure thatthe study directodroes notalsocarrythe dutiesofstudy sponsor,the sponsor rolewas reassigned.Inthismanner, personnelresponsibilitiaensd workload are more evenlybalanced. 3M EnvironmentLaalboratofy Amendment Approval ProtocolLRN-U2849 Amendment Number 3 John L.ButenhoffP,h.D.,SponsorRepresentative &A" Date Kristen J Hansen, PhD., Outgoing Study Director I-Fzb Date Marvin T Case, D. VM, Ph.D., Incoming Study Director &ate 3M EnvironmentalLaboratory Study Title Oral(GavageP)harmacokineSttiucdyofPFOS inRats PROTOCOL AMENDMENT NO. 4 Amendment Date: 20 April2000 Performing Laboratories 3M EnvironmentaTlechnology and SafetyServices FluorineAnalyticaClhemistryTeam Buildinc2y-3E-09,935 Bush Avenue St.Paul,N4N 55106 CentreAnalyticaLlaboratorieIsn,c. 3048 ResearchDrive StateCollegeP,A 16801 BattellMeemorialInstitute --;0K5ing Avenue Columbus,OH 43201-2693 Advanced BioanalyticSaelrvicesI,nc. 15 CatherwoodRoad IthacaN,Y 14850 Laboratory Project Identification ET&SS LRN-U2849 FACT TOX- I10 Arous Study: 418-01@ 'JM MedicalDepartment Study: T-6'-)95.12 CD 3M EnvironmentLalboratory ProtocolLRN-U2849 Amendment Number 4 Thisamendment modifiesthefollowinpgortion(so)ftheprotocol: 1. PROTOCOL READS: Theamendedsectio2n.0textstatetshathistudiysdesignetdodeterminPeFOS in specimens of ratlivers,enim,and urine. AMEND TO READ: This studyisdesignedtodeterminePFOS in specimensof ratlivers,erum,feces,and urine. REASON: The analysisof fecaltissueforthetargetchemicaland/oritsanalyteswas added tothe scope of thestudyfollowingtheissuanceoftheprotocolF.eces extractioannd analyticamlethods were notvalidatedand approvedpriortoprotocolapproval. 2. PROTOCOL READS: The amended section6.0listrsator rabbitlivers,erum,and urine. AMEND TO READ: Add: rator rabbitfeceswitha physicaldescriptioonf rator rabbitfeces. REASON: Analysisof fecaltissueforthetargetchemicaland/oritsanalyteswas added tothescope of the study followinct,heissuanceofthe originaplrotocol. 3. PROTOCOL READS: SectionI')I.listasllof thelaboratorietshatwillbe conductinganalysesforthisstudy. AMEND TO READ: Add: CentreAnal-v-ticLalboratoriesI,nc.,3048 ResearchDrive,StateCollege,PA 16801 REASON: Feces analvseswere added tothescope of thisstudy.The sub-contracltaboratorpyerforming analyseswas not intheoriginaplrotocol. 4. PROTOCOL READS: Sections10.4and 11.4statethatiftheanalysesaresub-contractetdo otherlaboratorieasn amendment willbe writtentoincludethesemethods. AMEND TO READ: The fecesextractioannd analyticamlethod used by CentreAnalytical Laboratoriewsillbe; OOM-02' )-003 (Revision2),"DeterminationofFluorochemicalResiduesinMonkey/Rat Feces by LC/-%IS,,'vlS." REASON: The sub-contractlaboratoryperformingfecesanalyseswas added tothe scope of thisstudy; thismethod was notvalidatedand approved priortoprotocolapproval. CD 3M EnvironmentalLaboratory Amendment Approval ProtocolLRN-U2849 Amendment Number 4 John L.ButenhoffP,h.D.,SponsorRepresentative Date Marvin T Case, D. V.M., Ph.D., Study Director Date 7wt 3M Environmental Laboratofy