Document N2V55zQqYgymEXDv26QREaE5y

3M MedicalDepartment Study:T-6295.7 Report No. FACT TOX-030 LaboratoryRequest Number-U2279 ANALYTICAL LABORATORY REPORT FROM THE 26-Week Capsule ToxicityStudy with PerfluorooctanesulfoniAccid Potassium Salt (T-6295)inCynomolgus Monkeys ON THE Determinationof the Presence and Concentration of Perfluorooctanesulfonat(ePFOS) in Liverand Serum Samples Projectldentification 3M MedicalDepartment Study:T-6295.7 Covance In-LifSetudy:#6329-223 AnalyticaSltudy:FACT TOX-030 3M LaboratoryRequest No. U2279 Study Completion Date At signing TotalNumber of Pages 2-33 3M EnvironmentalLaboratory Page 1 3m Medical Department Study: T-6295.7 3M MedicaDlepartmenSttudyT:-6295.7 GLP COMPLIANCE STATEMENT Report No. FACT TOX-030 laboratory Request Number-U2279 ReportNo. FACT TOX-030 LaboratoryRequest Number-U2279 Study T-ide: AnalyficaLlaboratoryReportfromthe26-Week Capsule ToxicitSytudywith PerfluorooctanesulfoAnciicd PotassiumSalt(T-6295)inCynomolgus Monkeys on the DeterminationofthePresence and ConcentratioonfPerflucrooctanesulfon(aPtFeOS) inLiverand Serum Samples Study IdentificatiNounmber: FACT TOX-030, T-6295.7,Covance #6329-223 Thisstudywas conductedincompliancewithUnitedStatesEnvironmentalProtectioAngency Good LaboratoryPractice(GLP) Standards40 CFR Part792,withtheexceptionsinthebulletedlisbtelow.AJI raw data and samples forthisstudyareretainedinarchivesatthe3M Lab and wilble retainedfora periodof atleasttenyears.The analyticaplhase completedatthe3M Lab was performedin accordance with3M ET&SS StandardOperatingProcedures. ExceptionstoGLP compliance: There were two studydirectorisnthisstudy.Thisstudywas designedas two separatestudies. The in-lipfhease studywas consideredtoend atthegenerationand shipmentofspecimens. The analyticasltudywas consideredtostarattthereceipotfthesespecimens foranalysis. This resultedinhavingtwo separatestudydirectorosn,e foreach phase ofthesame study, However, sincethetechnicalperformanceofeach phase was entirelsyeparate,no effectis expectedfrom thisexception. On a few occasions,datawere notrecordedor correctedexacflyas requiredby theGLPS. The 3M TOX 030 protocolstatesintheRegulatoryCompliance sectionthat'7hisstudywillbe conducted inaccordancewiththeUnitedStatesEnvironmentalProtectioAngency Good LaboratoryPracticesStandards,40 CFR 792,withtheexceptionthatanalysisofthetest materialmixtureforconcentrations,olubilithyo,mogeneity,and stabiliwtiyllnotbe conducted, and istheresponsibiliotfytheSponsor."Analyseswere,however,completedon the concentratioannd homogeneity ofthetestmaterialmixture,accordingtonon-GLP validated methods, and are incjudedinthisreportA.s pertheprotocols,olubiliatnyd stability determinationwsere notconducted. Study Director Date Study Sponsor Date 3M EnvironmentalLaboratory 3M Env4-ronmental 1T@aboratory Page 2 Page 2 3m Medical DepartmenL- Study: T-629S.7 3M MedicalDepartment Study:T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 ReportNo. FACT TOX-030 LaboratoryRequest Number-U2279 GLP STUDY-QUALiTy AssuRANCE STATEMENT Study Tite: AnalyticaLlaboratoryReportfrom the26-Week Capsule ToxicityStudywith PerfluorooctanesulfoAnciicdPotassiumSalt(T-6295)inCynomoigus Monkeys on the Determinationofthe Presence and ConcentratioonfPerfluorooctanesulfon(aPtFeOS) inLiverand Serum Samples Study IdentificatiNounmber-. FACT TOX-030, T-6295.7,Covance #6329-223 The analyticaplhase ofthisstudyhas been inspectedby the3M Lab QualityAssurance Unit(QAU) as indicatedintheblowing table.The findingwsere reportedtothestudydirectoarnd management. INSPLCTION DATES DecemberOlM PHASE Sample receipt DATE REPORTED TO MAtJAGEI.7EIVr STUOYDIRECTOR 1/17100 1/17/00 March 19,22,23199 Analysis 3/25/99 3/25/99 October 14/99 Extraction i May 3,8-12,15-19,22-26,29-31100, June 1,2,5,7,8/00 Data June 1,5,7,12-16/00 Draftreport 10/20/99 6/14/00 6/16/00 10/20/99 6/14/00 6/16/GO September 14/00 Draftreport 9/14/00 9114/00 QAU Represo@tative Date 3M Environmental Laboratory 3M Environme---i-@alLaboratory Page 3 Page 3 3m Medical Department Study: T-6295.7 3M MedicalDepartment Study:T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 ReportNo. FACT TOX-030 LaboratoryRequest Number-U2279 STUDY PERSONNEL AND CONTRIBUTORS Study Director Andrew M. Seacat Ph.D. 3M MedicalDepartment 3M Center,Building220-2E-02 PO Box 33220 St.Paul,MN 55133-3220 (651)575-3161 Sponsor 3M ToxicologyServices- MedicalDepartment 3M Center,Building220-2E-02 St.Paul,MN 55133-3220 John L.ButenhoffP,h.D.,SponsorRepresentative Analytical Chemistry Laboratory Liverand Serum Analyses 3M EnvironmentalTechnology and SafetyServices(3M ET&SS) 3M EnvironmentalLaboratory(3M Lab) FluorineAnalyticaClhemistryTeam (FACT) 2-3E-09 935 Bush Avenue St.Paul,MN 55106 KristenJ.Hansen, Ph.D.,PrincipaAlnalyticaIlnvestigator Contributing Personnel David R. Bamidge LisaA. Clemen KellyJ.Dorweiler Mark E. Ellefson Sara E. Estes Barb A. Gramenz Sarah A. Heimdal CariS. Hewitt Marlene M. Heyirg Harold0. Johnson KellyJ.Kuehlwein SallyA. Linda MichaelD. Livingston Joseph C. Pilon ScottR. Post [anA. Smith Anh-Dao Vo Bob W. Wynne In-lifeTesting Laboratory Covance LaboratoriesI,nc. 3301 Kinsman Boulevard Madison,Wi 53704-2595 PeterJ.Thomford,Ph.D.,in-LifPehase StudyDirector 3M EnvironmentalLaboratory 3M Env@-ronme---tal Laboratory Page 4 Page 4 3m Medical Department Study: T-6295.7 3M MedicalDepatnent Study:T-6295.7 TABLE OF CONTENTS Report No. FACT TOX-030 laboratory Request Number-U2279 ReportNo. FACT TOX-030 LaboratoryRequest Number-U2279 GLP Compliance Staterr*nt..............................................................................2......... GLP Study-GualityAssuranceStatement...................................................3...................... StudyPersonneland Contributor.s..........................................................4...................... Introductioand Purpose .................................................................6...................... TestSystem ........................................................................6...................... Specimen Collectioannd Analysis......................................................................7.......................... Specimen Receipt.......................................................................7...................... Dose ConfirmationAnalyses.............................................................................8................................... Materialasnd Methods ......................................................................................8....................................... Chemical Characterizabo.n..............................................................8...................... Method SummOes ..........................................................................8................ AnaNbcal Equipment.................................................................9....................... Deviabons .........................................................................1.0...................... Data QualityObjectivesand Data Integrit.y.................................................1.0....................... Data Summary, Analyses,and Results......................................................11....................... Summary ofQual@y ControlAnalyses Results............................................11....................... Summary ofSample Resufts..........................................................1.2................... Statsbr-aMlethods and Calculabons........................................................1.2...................... Statement ofConclusion.........................................................................................@..1.2................. ListofAttachments.........................................................................................1..2........................... AttachmentA: ControlMatrixCharacterizabonand Dose ConfirmationAnalyses..................1.3............... AttachmentB:Protocoland Deviabon Summary .........................................................15................ AttachmentC: Extractioannd AnalyticaMlethods .............................................4.3...................... AttachmentD: Data Summary Tables......................................................1.7.9...................... Attachment E-.Data Spreadsheets...........................................................................1..8..8........................... Attachment F.-Example Calculabons..........................................................2.2.5......... AttachmentG: InterimCerbficatoefAnalyses...............................................2.2.6....................... AttachmentH: ReportSignaturePage ........................................................2.3.3............. 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 5 Page 5 3m Medical Department Study: T-6295.7 3M MedicalDepartmentStidy:T-6295.7 INTRODUCTION AND PURPOSE Report No. FACT TOX-030 laboratory Request Number-U2279 ReportNo. FACT TOX-030 LaboratoryRequest Number-U2279 C8Fl7-S -0- 11 0 Perfluorooctanesuff(oPnFaOtSe) CASNumbe2r759-39-3 Chemical Formula: C,,Fl7SO.3- MolecularWeight-4.98.98 The purpose oftheanalyticaplhase ofthisstudyistodeterminethepresenceand concentratioonf PFOS (CBF17S03-) inlivearnd serum specimens collectedduringthestudyofCynomclgus monkeys oralldyosed withperfluorooctanseulfoniaccidpotassiumsalt(T-6295). Test System The testsystem speciesand strainselectedwas theCynomclgus monkey from Covance Research Products,Inc.i,dentifiuesdinga collatrag.At theinitationftreatmentt,heCynomolgus monkeys were young adulttoadult,and weighed appmximately3-5 kg. Twenty-twomale and 22 female Cynomolgus monkeys were used as the testsystem inthe present study.Four groups oftestanimalswere establisheadccordingtodosage levelsG.roup 1 consistedof controlCynomolgus monkeys thatdidnotreceivethetestsubstance,butreceivedtheequivalent amount oflactoseingelatncapsulesas thatadministeredtotheGroup 4 animals.Groups 2,3,and 4 were administeredailywith0.03(lowdose),0.15 (middose),and 0.75 (highdose)mg respectiveloyf, T-6295 perkg ofbody weight/day(mgikg/day)trituratweidthlactoseingelaticnapsules(seeTable I for Dosage and Group Characteristics). Table 1. Dosage and Group CharacteristicosfTest System inStudy T-6295.7 STUDY GROUP NUMBER OF ANIMALS TOTAL DOSAGE LEVEL DOSAGE RATIO (mgfkglday) (W:W)a Total:Test System 22 males 22 females Group I (Control) 66 mfaelmeasles 12 0 - Group 2 (Low Dose) 44 fmeamlaelses 8 0.03 1@499 Group 3 (MidDose) 66 fmeamlaelses 12 0-15 1i39 Group 4 (HighDose) 66 fmeamlaelses 12 0-75 139 a Testsubstancetrituratweidthlactose . 48 animalswere inrjudedinthebaselineseracollectiobnu,t44 animalswere assignedfortreatment 3M Environmental Laboratcry 3M Eni@-ronmental Laboratory Page 6 Page 6 3m Medi-cal Departmert Szudy: T-6295.7 3M MedicalDepartment Study:T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 ReportNo. FACT TOX-030 LaboratoryRequest Number-U2279 Alltreatmentgroups were dosed fora minimum periodof26 weeks. Sera specimens were collected from alltestanimalsatvar;oustimepointsduringthein-lipfhease ofthe26-week studyand senttothe 3M Lab foranalysis(seeAttachmentD, Tables D-1a,D-1b). Four animalseach from Groups 1,3,and 4 were designatedas recoverygroup animals.Treatment was discontinuedand the animalswere monitoredforeliminationfcompounds forone year posttreatmentT.he recoverygroups were observedafterthecessationoftreatmentuntiFlebruary25,2000 (Week 79)forGroup 1 and Group 4 recoveryanimals,and untiMlarch 7,2000 (Week 80)forGroup 3 recoveryanimals. Specimen Collection and Analysis Inthe analyticaplhase reportedhere,livearnd seraspecimens collectefdrom alltestanimalswere sent to the3M Lab and analyzedforthepresenceofPFOS (some samples were analyzedtodeterminethe presence ofETFOSE, PFOSA, POAA, PFOSEA, M556, PFOSAA, and themonoester-h,owever,these datawere collectefdorinformationaplurposesonly,and arenotreported)S.pecimens otherthanserum and livetrissueswere collecteadnd receivedfrom Covance Laboratorie(s6329-223),butwere notpart ofthecurrentscope ofanalysisdeterminedby thestudydirectoarnd sponsor.Additionaalnalysesof fecesare beingcompletedand willbe issuedas an amendment tothisfinarleport. Bloodspecimens were centrffugewdithinone hourofcollectioTnh.e serum was thenharvestedand storedina freezersettomaintainspecimens at-60to-80*C untislhippedtothe3M Lab.Liver specimens collectefdrom each animalwere flashfrozeninliquinditrogenand thenstoredina freezer settomaintainspecimens at-60to-80'Cuntislhippedtothe3M Lab.Liverand seraspecimens were shippedtothe3M Lab frozenand on dryice.Liverspecimens from Group 3 (3/01/00)and Group 4 (9/22199)recoveryanimalswere collectevdiabiopsy. Sera and livesramples were extractedusingan ion-pairinrgeagentand methyl-tert-buettyhler(MtBE). Liversamples were homogenized priortotheextractiopnrocedure.Sample extractwsere analyzed usinghigh-pressurleiquicdhromatography-electrospray/tandmeamss spectrometry(HPLCES/MS/MS) inthe multiplreesponse mode. PFOS levelswere quantitatebdy externalstandard calibratioAnn.alyticadletailasre includedinthisreport. Specimens Collectedfrom Study Groups I through 4 (through 2J25/99): Serum Specimens-550 specimens:9--14specimensianimal LiverSpecimens--30 specimens Specimens Collectedfrom the Recovery Group from 2127199to 3107100-. Serum Specimens-224 specimens:18-20 specimenslanimal LiverSpecimens-1 2 specimens from Group 3 and Group 4 animals(8viabiopsy) SPECIMEN RECEIPT Specimens were receivedfromCovance Laboratoriepseriodicaldluyr,ingthe in-lipfhease ofthisstudy, from August 1998 throughMarch 2000.Specimens receivedwere frozenand on dryice.Specimens were loggedinwiththe3M Lab and transferretdofreezersforstorageateither-55'ClG-20'C or -20*C 10*C. Controlmatricesused inlivearnd seraanalysesperformedduringTOX-030 were obtainedfrom commercialsourcesand arepresentedinAttachmentA (seeTableA-1).Samples analyzedatthe3M Lab willbe maintainedfor2 periodof 10 yearsand willbe storedatthelaboratorayt-20'C 1O'C. 3M EnvironmentalLaboratory 3M Env4---onmen--al -Liaboratory Page 7 Page 7 3m Medical Department Study: T-6295.7 3M MedicalDepartment Study:T-6295.7 Report No. FACT @@OX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 LaboratoryRequest Number-U2279 Dme Corfflrmation Analyses Dose confirmatioannalyseswere performedon lactosedose samples (1:39and 1:499)collecteodn 8/11/98duringthe in-ifpfhease ofthestudy:theresultasrepresentedinAttachmentA (seeTable A-2, A-3).The dose confirmatiodnatawere collecteadccordingtoa method thatwas notfullvyalidated. Dose confirmatiownas performedby dilutintghelactosedose samples (1:39- 1,000xand 1:4991,000x)withMilli-wOater,then extracteudsingtheion-paiprrocedure,diluted1:50and 1:5respectively intothe linearange oftheinstrumentF.oreach sample (top,middle,bottom),a matrixspikewas prepared(appmximately5000 pg/gand 400 pg/g)by spikintghedose solutioannd thendilutinagnd extracbngas describedabove. Inallcases,samples were analyzedversusan unextractedcurveusing HPLC-ESIMS/MS. The instrumentaplarametersand analyticaClonditiondsescribedinETS-8-5.1were used fordose solutioannalyses.The averagedose levelmeasured was confirmedtobe 99 27% of thetargetconcentrationM.atrixspikeswere recoveredat>60%. MATERIALS AND METHODS Chemical Characterization Table2 presentsinformatiroengardincgharacterizatoiftohnetestsubstancuesed inthein-lipfhease of thisstudy,and the analyticarleferencesubstanceused intheanalyticaplhase ofthisstudy. Table 2. Characterizationof Test and AnalyticalReference Substances inStudy FACT TOX-030 CHEwcAL NAME SOURCE ExpiFzA-noNDATE STORAGE CONE)MONS CHEmicAL LOT NumBER PHysicAL DESCRPNON PURITY TEST SUBSTANCE ANALYTICAL REFERENCE SUBSTANCES KPFOS Potassium KPFOS Potassium Perfluorooctanesuffonate Perfluorooctanesulfonate 3M SpecialtCyhemicalsDiv. 3M Special@CyhemicalsDiv. THPFOS IH.lH,2H,2Hperflucrooctanesulfaodndic ICN BiomedicsI,nc. 8131/2001 8/3112C)01 1/01/2020 Frozen<-10*C Frozen:s-lo'C Ambienttemperature 217 FC-95.Miitecrystalline er 86.90/a 171 Whitecrystallpionweder 86.4% 59909 Brown powder N/A 53406 Brownwaxy sdid N/A Reserve samples oftheanalyticarleferencesubstancewillbe storedatthe3M Lab fora periodof 10 years,as willany reservesamples oftestsubstancereturnedfromthein-lipfhease ofthestudy. Method Summ2ries Followingisa briefdescriptioonfthelatesmtethods used duringtheanalyticaplhase of thisstudyby the3M Lab.Detaileddescriptionosfthemethods used inthisanalyticaplhase arelocatedin AttachmentC. As thepresentanalyticaplhase ofthisstudyprogressed,more advanced methods evolvedand eariiemrethods were used withdeviabonsuntialmendments totheprotocolwere written. Changes tothemethods includedtheuse ofmethyl-tert-buettyhler(MTBE) insteadofethylacetate, curves plottedby linearegressionweightedI/xinsteadofunweightedcurves,a reductioninthe sizeof theanalyticaclolumn from 100mm to50mm, gradientchanges,and fasteHrPLC cycletimes.A summary ofprotocoland method deviationisspresentedinAttachmentB (seeTableB-1)ofthisreport. 3M EnvironmentalLaboratory 3M Env4-ronment-al Laboratory Page 8 Pacre 8 3m Medical DepartTrenL- Study: T-6295.7 3M Medical Department Study:T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 LaboratoryRequest Number-U2279 Preparatory Methods: ETS-8-6.0,"ExtractionofPotassium Perfluorooctanesulfonatoer otherFluorochemicalCompounds from LiverforAnalysisusingHPLC-Electrospray/MassSpectrometr/' Liversamples were homc>genized inwater.An aliquotof each homogenate was spikedwith THPFOS and extractedusing an ion-pairinegxtractiopnrocedure.An ion-pairinrgeagentwas added tothe sample and the analyteionpairwas partitioneidntoMTBE. The extractwas transferredtoa centrifugetube and put onto a nitrogenevaporatoruntidlry.Each extractwas reconstituteidn1.0mL ofmethanol and passed througha 0.2 pm nylonfilteurs,inga 3 CM3 disposable plasticsyringeintoglassautosampler vials. ETS-84.1, "ExtractionofPotassium PerfluorooctanesulfonatoerOther Fluorochemical Compounds from Serum forAnalysisUsing HPLC-Electrospray/Mass Spectrometr,/' Sera samples were spiked withTHPFOS and extractedusing an ion-pairinegxtractiopnrocedure. An ion-pai@ngreagentwas added tothe sample and the analyteionpairwas partitioneidntoMTBE. The MTBE extractwas removed and put onto a nitrogenevaporatoruntidlry.Each extractwas reconstituteidn1.0mL of methanol and passed througha 0.2 pm nylonfilteurs,ing a 3 cm-, disposable plastiscydnge intoglassautosampler vials. Analytir-alMethods: ETS-8-7.0, "AnalysisofPotassium PerfluorooctanesulfonatoerotherFluorochemicalsinLiver ExtractsUsing HPLC-Electrospray/Mass Spectrometr/' ETS-8-5.1, "AnalysisofPotassium Perfluorooctanesulfonatoer Other FluorochemicalinSerum ExtractsUsing HPLC-Electrospray/Mass Spectrometry" The analyses were performed by monitoringone ormore productionsselectedfrom a single primaryioncharacteristiocfa particulafrluaror-hemicaulsing HPLC/ESIMSIMS. For example, molecularion499, selectedas the primaryionforPFOS (C,F,,SO,-)analysisw,as fragmented to produce ion99 (FSO,-).Tne characte@stiicon99 was monitoredforquantitativaenalysis. An2WcW Equipment The actualanalyticaelquipment settingsused inthe presentanalyticaplhase ofthisstudy variedslightly duringactualdata collectionT.he followingisrepresentativoefthe settingussed during',heanalytical phase ofthisstudy. Liquid Chromatograph: Hewlett-PackardoSeries1100 LiquidChromatograph Analyticaclolumn: KeystoneD Betasil'C"ie 2x5O mm (5pm) Column temperature:Ambient Mobile phase components@ Component A: 2mM ammonium acetateinwater Component B: methanol Flow rate:300 pumin Injectiovnolume: 10 pL SolventGradient:10 minutes system 3M Environmental Laboratory 3M En-virormental Laboratory Page 9 Page 9 3m Medical Department Study: T-6295.7 3M MedicalDepartment Study:T-6295.7 Report No. FACR TOX-030 laboratory Request Number-U2279 ReportNo. FACT TOX-030 LaboratoryRequest Number-U2279 Startat 1O%B Hold at10%B for1.0minute Increaseto95%B over4.5minutes Hold at95%B for2.0minutes Return to 1O%B over 0.5minutes Hold at 10%B for2.0minutes Mass Spectrometer MicromassO API/Mass SpectrometerQuattro11"TriplQeuadrupole system Software:Mass Lynx' 3.2 Cone Voltage:60V CollisioGnas Energy:40-6OeV Mode: ElectrospraNyegaWe Source BlockTemperature:1500C lOOC ElectrodeZ:-spray AnalysisType: MultiplReeactionMonitorin(gMRM) Table 3. NegativeIons Monitored inFACT TOX-030 TARGETANALYRE PPJMARYION(amu) PRODUCT [ON (amu) PFOS 499.0 99.0 THPFOS 427.0 80.0 Devi2tiorLs Itshould be noted thatas the analyticaplhase ofthisstudyprogressed,method parameters were evaluatedto improve ana@/ses.Eadiermethods were used withdeviationsuntialmendments tothe protocolwere writtenD.eviationsfromtheoriginaplrotocoland methods aredocumented inthe AttachmentB (seeTableB-1). DATA QUALITY OBJECTIVES AND DATA INTEGRITY The followindgata qualitoybjecbves(DQOS) were indicateidntheprotocolforthisstudy: LinearityT:he coefficieonftdeterminatio(n@)equaltoor greaterthan0.98 Limitsof Quantitation(LOQ): The Method DetectioLnimit(MDL) forPFOS is12 ppb forserum and 15 ppb forliverT.he LOQ isequaltothelowestacceptablestandardinthecalibratiocnurve. Duplicate/AcceptablePrecision:Precisiownas reproduciblteowithin30% Spike/Ar-ceptablReecoveries:70-130% Confirmatory Methods: Indeterminatseamples may be re-@nalyzedusinga confirmatormyethod. Ifa confirmatorymethod isused,an amendment tothisprotocolshouldbe written. Demonstration of SpecificityS:pecificittoybe demonstratedby chromatographicretentiotnime and mass spectraldaughterioncharacterization. 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 10 Page 10 3m Medical Department S-@udy: T-6295.7 3M MedicalDepartmentStudy:T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 ReportNo. FACT TOX-030 LaboratoryRequestNumber-U2279 DATA SUMMARY, ANALYSES, AND RESULTS Dataqualitoybjectivefsortheanalyticpahlase ofthistudyouflineidnthe3M Lab protocoflorFACT TOX-030 (seeAttachmentB)were metwiththeexceptionnsotedinthisreport. Summary of Quality Control Analyses Results LinearityT:he coefficieonftdeterminatio(n@)ofthestandardcurvewas @!0.985. CalibrationStandards: Quartitatioonfthetargetanalyteswas based on linearegression analysis(unweighted- priotro3/5/99u,nweightedorI/xweighted-3/5/9t9o3/19/99a,nd 1/x weighted-3/19/9t9oend ofthestudy)oftwoextractemdatrixcurvesbracketinegach groupof samples,exceptas notedinthedeviatiosnummary, Highor lowpointson thecurvemay have been deactivatetdoprovidea bettelrineafriotverthecurverangemost appropriatteo thedata. Low curvepointswithpeak areaslessthantwo timesthatoftheextractiobnlankswere deactivated todisqualifaydatarangethatmay have been significanatflfyectebdy backgroundlevelsofthe analyteO.ccasionallya,singlemid-rangecurvepointthatwas an obviousoutliewras deactivated. Quantitatioonfeach analytewas based on theresponseofone specifipcroductionusingthe multiplreesponse-monitorimnogde oftheinstnimen(tseeAttachmentC). Limitsof Quantltation(LOQ):The LOQ isequaltothelowestacceptablestandardinthe calibraticounrve(defineads a standardwithin30% ofthetheoreticvaallue)a,nd isatleastwo timestheanalytepeak areadetectedintheextractiobnlanks.Thisvaluedoes notexceed the validatedLOQ ofthemethod fordatathatisaccepted(seeAttachmentD, TableD-6). Table 4. Determinationof PFOS LOQ inTOX-030 Analyses ANALYTE-MATRLX LOQ PFOS-Sera PFOS-Liver 4.39-15.n2g/mL 26.9---60n.g1/g Blanks: AJIblankswere below thelowerlimiotfquantitatifoonrthecompounds ofinteresTto. simplifaynalysesthatwere complicatedby endogenous levelsoffluorochemicailnsunexposed monkey sera,rabbaserawas selectedas a suitablseurrogatematrix. Duplicate/AcceptablPerecision:Precisiownas determinedby analysiosfMS/MSD and was reproduciblteowithin30%. MatrixSpikes: Matrixspikesand matrixspikeduplicatewsere extractewditheach setofsamples and analyzedduringanalyticarlunsatthe3M Lab.AJIseramatrixspikeswere within30% ofthe theoreticacloncentratioMna.trixspikespreparedinlivewrere compliantwithin30%, withthe exceptionofone spikethatwas preparedwithDay-393 samples and had a lowrecovery.The matrixspikewas reextrac*,aendd therecoverywas within30% ofthetheoreticcaolncentration. Spike/AcceptableRecoveries:Spikerecoverieosf30% ofexpectedvalueswereachievedfor allmatrixspikespreparedinsera.Withone exception(notedearlierm)a,trixspikespreparedinliver were within30%. Use ofSurrogates:The surrogate(THPFOS) was added toallsamplesand standardsT.HPFOS was notused forquantitatiobnu,twas used tomonitorforgrossinstrumenftailurAef.ter11/04/99, thesurrogateresponseofeach analyticarlunwas verifietdodeterminethatitdidnotvarymore than:t5O% fromthemean withineach analyticarlun.No problemswere observedwiththesedata. 3M EnvironmentalLaboratory 3M Env'Lronmental Laboratory Page 11 Page 11 3m Medical Department Szudy: T-6295.7 3M MedicalDepartmentStudy:T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 ReporlNo.FACT TOX-030 LaboratorRyequest Number-U2279 Assuming spikerecoverystudiesforma suitabliendicatiofnendogenous analyterecoveryd,ata are quantitatitvoe30%. The validitoyfthisassumptionhas notbeen verifiebdy othertechniques. Summary of Sample Results Samples from ControlAnimals:Low levelosfPFOS wereoftendetectedintheseraand liveorf thecontrolanimals.These levelwsere significanluoywerthanthosefoundinthelow dose test animals. Samples from Dosed Animals:IngeneralP,FOS levelfsoundintheseraand liveorfthetest animalsincreasedwithdose group.PFOS levelsincreasedas dosage increaseds;ignificant dfferencesbetween male and femalePFOS levelwsere notobservedinsera.However,Group 4 males had notablyhigherPFOS levelsinlivesramples thanGroup 4 females.Detailedsample dataispresentedinAttachmentsD and E. STATISTICAL METHODS AND CALCULATIONS Stadsticamlethods were limitetdothecalculatiofnmeans and standarddeviationsS.ee AttachmentF forexample calculationused togeneratethelivearnd serum sample datainTOX-030. STATEMENT OF CONCLUSION Under theconditionosfthepresentanalyticpahlase ofthisstudy,PFOS was detectedintheseraand livesramples of Groups 2,3,and 4 animals.The ControlGroup 1 animalsshowed minimalamounts of PFOS. PFOS levelsincreasedas dosage increaseds;ignificadnitfferencebsetween male and female PFOS levelwsere notobservedinsera.However,Group 4 (highdose)males had notablyhigherPFOS levelsinlivesramples thanGrou@4 females. Data qualitoybjectivefsortheanalyticpahlase ofthisstudyoutlineidnthe 3M Lab protocoflorFACT TOX-030 (seeAttachmentB) were met withtheexceptionnsotedinthisreport. LIST OF ATTACHMENTS AttachmentA: ControlMatrixCharacterizatiaonnd Dose ConfirmatioAnnalyses Attachment B: Protocoland DeviationSummary AttachmentC: Extractioannd AnalyticaMlethods AttachmentD: Data Summary Tables AttachmentE: Data Spreadsheets AttachmentF: Example Calculations Attachment G: InteriCmertificaotfeAnalyses Attachment H: Report SignaturePage 3M EnvironmentalLaboratory 3M Environme7,L--alLaboratory Page 12 Page 12 3m Medical Department Study: T-6295.7 3M MedicaDlepartmenSttudyT:-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 ReportNo. FACT TOX-030 LaboratoryRequest Number-U2279 ATTACHMENT A CONTROL MATRIX CHARACTERIZATION AND DOSE CONFIRMATION ANALYSES TableA-1.Characterizatioonfthe ControlMatricesUsed forLiverand Sera AnalysesInStudy FACT TOX-030 CONTROL MATRD( FZABBrrSERum FZABBrrSERUM MONKEY SERUM MONKEY SERUM MONKEY SERUM Sourr,e ExpiratioDnate StorageConditions Chemical Lot Physical Descdpflon Sigma Chemir-als 01/01/2010 Frozen-20'C 118H8418 RabbitSerum Sigma Chemicals 01/01/2010 Frozen-20'C 47H4641 RabbitSerum LampireBiological SierraBiomedical N/R 01/01/2010 Frozen-50*C Frozen-20*C 111022515 ' #LY2NO Monkey Serum Monkey Serum NIR 01/01/2010 Frozen-20'C N/R Monkey Serum CONTROL MATRIX RABBRT LNER RABBRT LNER PABi3rTLrvER RABBRT LIVER MONKEY LIVER Source ExpiratioDnate StorageConditions Chomir-alLot Physical Description N/R-not recorded Coming Hazleton 12/01/1999 Frozen-20'C F00007 RabbitLiver N/R 12/01/1999 Frozen-20oc N/R RabbitLiver Coming Hazleton 01/01/2010 Frozen-20'C F00005 RabbitLiver Coming Hazleton 01101/2010 Frozen-20*C F00009 RabbitLiver SierraBiomedical 01/01/2010 Frozen-50*C N/R Monkey Liver 3M EnvironmentalL-aboratory 3M Environmental -@aboratory Page A-1 Page 13 3m Medical Department Study: T-6295.7 3M MedicalDepartment Study:T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 ReportNo. FACT TOX-030 LaboratoryRequest Number-U2279 TableA-2.LactoseDose Verificati(oPnFOS) forStudy6329-223-8/21/99 EXPECTED CONC. (ng/mL) MEASURED C40NC. (ng/mL) %REC. FOR ng/mL EXPECTED CALC. CONC. (Pwg) MEASURED CALC. CoNr- (Pwg) AVERAGE Sm DEVLAT$ON %REC. FOR pglg 1:39DOSE(25000Ppm PFOS) Top 580 479 Middle 500 650 Boftom 500 422 1:499DosE (2000Ppm PFOS) Top 411) 305 Middle 404 287 Boftom 400 272 83% 25000 2D647 83% 130% 25000 32537 130% 84% 25000 21108 24764 6736 27% 84% average/std.deviabon= 99% 27% 74% 2000 1490 74% 71% 2000 1425 71% 68% 2000 1361 1425 64.55% 68% average/std.deviaton= 71% 3% ActualMS Concentration-Actuablackgroundconcentrabond,ividedbyexpected,times100 (Spikedtoolow which accountsforthewidedifferenciensrecovery) TableA-3.Lactose Dose Verifir-ati(oPnFOS-Matrix Spikes)forStudy 6329-223--a/21199 EXPECTED ACTUAL %PEC. CONC. CONC. FOR (ng/mL) I (ng/mL) I ng/mL CALCULATED CONC. (pg/g) EXPECTED CONC. (Pgtg) ACTUAL CALC. CONC. (pg/g) AVERAGE sm DEVTATION %REc. FOR pgtg 1:39DOSE (25000 Ppm PFOS) MS Top 604 507 84-/. 21858 10.8 12.1 Middle 524 485 92-/. 24252 12.5 -82.8 112% -664* Bottom 524 438 83% 21889 12.5 7.82 22666 1374 6% 63% 1:499DOSE (2000Ppm PFOS) MS Top 606 475 78% 2320 9.77 8.30 85% Middle 600 447 75% 2217 9.92 7.93 80% Boftom 596 440 74% 2202 10.0 8.41 2246 64.53-/.4 84% Thisvaleuisan outiearndwas notusedinanycalculatons AVERAGE 87% 3M Environmental Laboratory 3M Environmental Laboratory Page A-2 Page 14 3m Medical Department Study: T-6295.7 3M MedicaDlepartmenSttudyT.-6295.7 ATTACHMENT B PROTOCOL AND DEVIATION SUMMARY Reucrt No. FAC-1 TOX-030 laboratcry Request Number-U2279 ReportNo.FACT TOX-030 LaboratoryRequest Number-U2279 Table B-1.DeviationSummary forFACT TOX-030 DEviATioN MTBE was usedas an extacfim solvent insteadofethylacetate. F@pettewas used insteadof Oxford dispenser. Curves plottedby linearegression weightedltxrathetrhanby linear regressionas specifiedinte pmtoool. A second extractedmatrixcurve was not used tobracketsamples. Recorded extractcn method FACT-M-1.0 ratherthan FACT-M-1. 1. Folkwed extractionmethod ETS43-6.0 ratherthan FACT-M- 1.1. Folla.vedanalyticalmethod ETS-8-7.0 ratherthan FACT-M-2. 1. Follov.,aendaly@l ff)etiwETS-&5.0 ratherthanFACT-". 1. Samples extractedusing 0.5 mL rather than 1.0 mL due toinsufficiesnatmple. Folloveedextractionmethod ETS43-4.0 ratherthan FACT-M-3.1. Matrixspikeswere not spiked with st3ndard (Used as blanks). Contruing calibratiosntandards were not spiked wth standard due toanalysterror. Samples extractedus4ng <0.5 mL due to insufficieinntitisaalmple volurm. DATES OF OCCURRENCE 2/5/99,219m,5/18m, 6/11/99 10/14199 2/13/99,3/5%,3/12199,3119/99, 3/20/99,3/21/99,3/233M/.24199, 3/25%. 4r7t99,4111199,4/12199, 4117/99,5/191%, 5r22/99,&S%. 6/14/99 3/5/99,3/gM. 3/1&99. 3/16/99, 5/19/99,5/22199,10126199.1121/00. 3t24/00,4/27100 6/11/99 10/14199,10/25/99.1/191DO.ar22J()O 7/29199,10120/99,lOr22/99.10/26/-09, 10127199, 1128100.3124/00, 3128/00 3io5l99,N0599 10/25/99 3YO2/99, 3103/99 11/3/99 11/3/99 Z-5199,2/9M. Y2/99,MM, 3/10/99, 3/12/99,3/15/99,3/16199,4/6M, 4/8/99, 812599, 11/3/99,4/21/00 IMPACT ON STUDY No negativiempacton the StK"tBE impmved the absolute recoverieasnd shortenedextm(tontime. No negadve impact on the study. No negativiempacton thestxty-l/xweightedcurvesimproved the precisionand accuracy ofanalysis. No negativeimpact on thestudy--The accuracy ofcalibration checks analyzedevery fivetoten samples wasmonitoretdo ensure contnued accuracy oftheanalysis.The OC providedby the calibrabonchecks issufflcienatnd &* data quality@Mllnot be adversely affected. No negativeimpact on the study-New method was followed. even though oldmethod was recorded. No negativeimpacton the sbidy---New validatednied)od providesimprovements in precisionand extractiontime. No negativeimpacton the study--New validatedrnetiod providesimprovements inprecision,accuracy and analysistime. No negativeimpacton the study--New validatedmethod Providesimprovements inprecision.accuracy and analysistime. No negativeimpacton the study-Studies indicatethatdata qualityisnotjeopardizedusing 0.5 mL of sera. No negativeimpacton the sbidy-New validatedmethod providesimprovements inprecisionand extra(:bcntime. Adequate QC was preparedwth thesample set unspiked samp;es pose no negabve impact c)nthe study. Nlid-leveclurve standardswere substituteads QC forthe nonspiked calibraboncheck standards;the unspiked calibration standardspose no negativeimpact to the sbudy. Studies indicatethatdata accuracy and precisionmay be affectedwhen serasamples lessthan0.5 mL were extracted. Data reportedfrom extractioonfsamples lew than 0.5 mL is noted inthedata tables. 3M Environmental Laboratory 3M Er.-v4-ronme--italLabora!:ory Page B-1 Page 15 3m Medical Department Study: 3,1*E1nvironmentalTechnology and Services T-6295.7 Report No. FACT TOX-030 PO Box 33331 laboratory St.Paul,%IN 55133-3331 6127786442 Request Number-U2279 Protocol #FACT-TOX-030 Study Title 26-Week Capsule Toxicity Study with PerfluorooctaneSulfonicAcid Potassium Salt(T-6295)in Cynomolgus Monkeys PROTOCOL Author Lisa Clemen Date: January 25, 1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St.Paul,MN 55106 LaboratoryProjectIdentification FACT-TOX-030 3M EnvironmentaLlaboratory 3M Environmental Laboratory Page1of9 Page 16 3m Medical Department Study: T-6295.7 ReDort No. FACT TOX-030 laboratory Request Number-U2279 Protoco#lFACT-TOX-030 StudyIdentification 26-Week CapsuleToxicityStudywith PerfluorooctanSeulfoniAccid PotassiumSalt(T-6295)in Cynomoigus Monkeys TestMaterial Perfluoroocstualnfeonaiccidpotassisuamlt (T-6295) Sponsor 3M ToxicologSyervice-sMedicalDepartment 3M Center,Building220-2E-02 St.Paul,MN 55144-1000 Sponsor Representative Andrew Seacat,Ph.D. 3M ToxicologyServices Telephone:612-575-3161 Facsimile:612-733-1773 Study Director KristenHansen,Ph.D. 3M EnvironmentalTechnologyand Safety Services Building2-3E-09 651-778-6018 Study Location(s) In vivo TestingFacility AnalyticalTestingLaboratory Covance LaboratoriesI,nc. 3301 Kinsman Boulevard Madison,Wisconsin 53704 3M EnvironmentalLaboratory Buildinc2,-3E-09 935 Busah Avenue St.Paul,MN 55106 Proposed Study Timetable Study InitiatiDoante Study Completion Date January25,1999 January25,2000 3M EnvironmentaLlaboratory 3M Environmental Laboratory Page 2 of9 Pacie 17 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protoco#lFACT-TOX-030 1. STUD Y Twenty-sixweek capsuletoxicitsytudywithpotassiumperfluorooctasnuelfoniaccid(T-6295)in cynomolgus monkeys. 2. PuRposE This analyticalstudy isdesigned to determinelevelsof potassiumperfluorooctanesulfonate (PFOS) in theliverand serum of cynomoigus monkeys. Additionaltissuesor fluidsmay be analyzed. The in-lifpeortionof thisstudywas conducted atCovance Laboratoriess,tudy #6329223. 3. REGuLAToRyCompLiANcE This study willbe conducted in accordance with the United StatesEnvironmental Protection Agency Good Laboratory PracticesStandards,40 CFR 792, with the exceptionthatanalysisof the testmaterialmixture forconcentrations,olubilityh,omogeneity,and stabilitwyillnot be conducted, and isthe responsibilitoyf the Sponsor. 4. QUALITY ASSURANCE The 3M Environmental LaboratoryQualityAssurance Unit willreview the protocoland audit study conduct,data, and finalreportto determinecompliance with Good LaboratoryPractice Standards and with 3M Environmental LaboratoryStandard Operating Procedures. 5. TESTMATERIAL 5.1 Refer to Covance Laboratoryprotocolforstudy#6329-223. 6. COIVTROL MATRICES 6.1 Identification Monkey liverand serum and/orrabbitliverand serum, traceability numbers willbe recorded in the raw data and includedin the finalreport 6.2 Source Covance Research and/orSigma Chemical 6.3 Physical Description Monkey liverand serum and/orrabbitliverand serum 6.4 Purify and Stability Not applicable 6.5 Storage Conditions Frozen at -20 'C 10 OC or-55 OC 10 OC 6.6 Reserve Matrix A portionof the controlmatrix willbe retainedinthe archivesfor as long as the qualityof the preparationaffordsevaluation,but not longerthan ten years followingtheeffectivdeateof thefinaltestrule(ifapplicable). 6.7 Disposition Matriceswillbe retainedper GLP regulation.Certainmatrices(feces, urine,and blood)may be disposedafterQAU verification. 3M EnvironmentalLaboratory 3M Env@-ronmental Laboratory Page 3 of9 Page 18 3m Medical Department St---udy:T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protoco#lFACT-TOX-030 6.8 SafetyPrecautionsRefetroMSDS forchemicaulssedW.earappropriate laboratoryattirea,nd followadequateprecautionfsorhandlingbiologicamlaterialsand preparing samples foranalysis. 7. REFERENC-E MATERIAL 7.1 IdentificatioPnotassiupmerfluorooctanfeosnautle(PFOS),lot#s 171,215,or217 (equivalenltots) 7.2 Source 3M SpecialtCyhemicals 7.3 Physical Description Whitepowder 7.4 Purityand StabilityResponsibiliotfytheSponsor 7.5 Storage Conditions Room temperature 7.6 Reserve Material A reservseamplefromeachbatchofPFOS usedinthisstudywill be retainedas longasthequalityofthepreparatioanffordsevaluationb,utnotlonger thantenyearsfollowingtheeffectivdeateof thefinatlestrule(ifapplicable). 7.7 Disposition Unused referencmeateriawlillbe retainefdoruseby the3M EnvironmentalLaboratoryand willbe discardedwhen thequalitoyf preparationno longeraffordsevaluation. 7.8 Safety Precautions RefertoMSDS forchemicalsused.Wear appropriate laboratoryattirea,nd followadequate precautionsforhandling biologicalmaterialsand preparingsamples foranalysis. S. TEST SYSTEM Cynomolgus monkeys were used as the testsystem, and were maintained and dosed as described inCovance protocol#6329-223. Group I controlanimalsdidnotreceivethetestsubstance. Groups 2,3,and 4 receivedthetestsubstancedailyfor26 weeks,atconcentrationosf0.02,0.5, and 2.0mgfkg/day,respectivelyR.efertoCovance protocol#6329-223 fortabularpresentation of data.Two animalseach from Groups 1,3,and 4 were designatedasrecoveryanimalsand were allowedatleasta 13 week, which may be extended,recoveryperiodaftercessationof treatment. 9. SPECIMEN AND SAMPLE RECEIPT The 3M EnvironmentalLaboratorywillreceivehomogeneitysamplesfordoseanalysisand specimensof thefollowingbody tissueasnd fluidfsrom theindicatepdointsinthestudy.All specimenswillbe packedon dry iceforshipping. 3M EnvironmentaLlaboratory 3M Environmental Laboratory Page 4 of9 Page 19 3m Medi-cal Depar-"men'- Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Numher-U2279 Protoco#lFACT-Tox-030 Body tissue/fluid Collected Expected# of specimens Serum - allanimals Urine and feces- recoveryanimals Liver- allanimals 7 days priorto treatment 7 days posttreatment everytwo weeks duringtreatment and recovery Day zero of recovery 6,30,and 90 (witha potentia1l80) daysof overy Afterterminatioonfthestudy 616 from main study 24 additionaflrom recovery 24 urine 24 feces Totalnumber oftestanimals:32 Totalnumber ofcontrolanimals:12 Specimens sentto3M EnvironmentalLaboratoriewsillbe receivedand trackedaccordingto applicableStandard OperatingProcedures. 10.PREPARATORY METHODS 10.1 FACT-M-1.0, ExtractionofPotassiumPerfluorooctanesulfonaotreOther Anionic FluorochemicalSurfactanftrom LiverforAnalysisUsing HPLC-Electrospray/M ass Spectrometry 10.2 FACT-M-3. 1,Extractionof Potassium Perfluorooctanesulfonaotre Other FluorochemicalCompounds from Serum or Other FluidforAnalysisUsing HPLCElectrospray/MassSpectrometry 10.3 Ifpreparatorymethods otherthan thoselistedabove areused,an amendment to this protocolwillbe written.Any deviationsfrom thesemethods willbe documented and includedwiththestudydata. 11.ANAL Y77CAL METHODS 11.1 FACT-M-2.0, AnalysisofFluorochemicalsinLiverExtractsUsing HPLCElectrospray/MassSpectrometry 11.2 FACT-M-4. 1,Analysisof Potassium Perfluorcoctanesulfonaotre Other FluorochemicalsinSerum orOther FluidExtractsUsing HPLC-Electrospray/M ass Spectrometry 11.3 Ifanalyticamlethods otherthanthoselistedabove areused,an amendment to this protocolwillbe written.Any deviationfsrom thesemethods willbe documented and includedwiththestudydata. 3M EnvironmentaLlaboratofy 3M Env'-ronmental Laboratory Page 5 of9 LDage 20 3m Medical Department Szudy: T-629S.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 Protoco#lFACT-TOX-030 12.DATA QUALITY OBJEC77VES The number of spikes/duplicatuesse,of surrogatesa,nd informationon otherdataquality indicatorsare included in the analyticalmethods. In addition,the followingcriteriawillbe met: 12.1 Linearityr2 >-0.98 12.2 Limits of detection/ quantitation 12.2.1 Method Detection Limit (N4]DL) for PFOS a) Serum: 12 ppb b) Liver: 15 ppb 12.2.2 PracticalQuantitationLimit (PQL) - Equal tothe lowest standardin the calibrationcurve 12.3 Duplicate acceptable precision < 30% for the method 12.4 Spike acceptable recoveries 70% - 130% 12.5 Use of confirmatory methods Indeterminatesamples willbe re-analyzedusing a confirmatory method. Ifa confirmatory method isused,an amendment to this protocol willbe written. 12.6 Demonstration of specificity Chromatographic retentiontime,mass spectral daughter ion characterization. 13. SuB-CoNTRAcTED ANALYSIS 13.1 All analyses as detailedin thisprotocol will be performed at 3M Environmental Laboratories,Building 2-3E-09, 935 Bush Avenue, St.Paul,MN 55106. 13.2 An amendment to thisprotocolwill be writtenifanalysesare performed at laboratoriesother tha' n the 3M Environmental Laboratory. 14. STATISTICAL ANALYSIS Averages and standard deviationswill be calculated.The statisticamlethods thatwill be used are described below: 14.1 14.2 Data transformations and analysis Data willbe reportedas the concentration (weictprht/weightor weight/vol)of PFOS or metaboliteper tissueor fluid. Statistical analysis Statistics used may include regression analysis of concentrations over time, and standard deviations calculatedfor the concentrations within each dose group. If necessary,simple statisticatlests,such as Student's t test, may be applied to evaluate statisticadlifference. 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 6 of 9 Page 21 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protoco#lFACT-TOX-03o 15.REPORT A reportoftheresultosfthestudywillbepreparedby 3M EnvironmentaLlaboratoryT.he report willinclude,but not be limitedto,thefollowing,when applicable: 15.1 Name and addressof thefacilitpyerformingthestudy 15.2 Dates upon which the studywas initiateadnd completed 15.3 15.4 A statementof compliance by theStudy Directoraddressingany exceptionsto Good Laboratory PracticeStandards Objectivesand procedures as statedin the approved protocol,includin-any changes in theoriginalprotocol 15.5 The testsubstanceidentificatiboyn name, chemical abstractsnumber or code number, strength,purity,and compositionor otherappropriatecharacteristicisf,provided by the Sponsor 15.6 Stabilityand the solubilityof the testsubstancesunder the conditionsof administrationi,fprovided by the Sponsor 15.7 A descriptionof the methods used toconduct the test(s) 15.8 A descriptionof the testsystem 15.9 A descriptionof any circumstancesthatmay have affectedthequalityor the integrity of the data 15.10 The name of the Study Directorand the names of otherscientistsp,rofessionalsa,nd supervisorypersonnel involved in the study 15.11 A descriptionof the transformationsc,alculationso,r operationsperformed on the data,a summary and analysisof the analyticaclhemistry data,and a statement of the conclusionsdrawn from the analyses 15.12 Statisticamlethods used to evaluatethedata,ifapplicable 15.13 The signed and dated reportsof each of the individualscientistosr otherprofessionals involved inthestudy,ifapplicable 15.14 The locationwhere raw dataand thefinalreportareto be stored 15.15 A statementpreparedby the QualityAssurance Unit listintghedatesthatstudy inspectionsand auditswere made, and the datesof any findingsreportedto the Study Directorand Management Ifitisnecessaryto make correctionsor additionsto a finadreportafterithas been accepted,the changes willbe made inthe form of an amendment issuedby the Study Director.The amendment willclearlyidentifythe partof thefinalreportthatisbeing amended, the reasons for the amendment, and willbe signed by the Study Director. 3M EnvironmentalLaboratory 3M Environmenzal Laboratory Page 7 of9 Page 22 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protoco#lFACT-TOX-030 16.LocATION OF RAwDArA, RECORDS, AND FiNALREponT Originadlata,orcopiesthereofw,illbeavailablaet3M EnvironmentaLlaboratortyofacilitate auditsof the study during itsprogressand beforeacceptanceof the finalreport.When the final reportiscompleted, alloriginalpaperdata,includingthoseitems listedbelow, willbe retainedin the archivesof 3M Environmental Laboratoryforatleasta periodof time as specifiedby regulation,and as establishedby 3M Environmental LaboratoryStandard Operating Procedures. 16.1 The following raw data and recordswillbe retainedinthe studyfolderin the study/projectarchivesaccordingto3M Environmental LaboratoryStandard Operatinc, 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 following supporting recordswillbe retainedseparatelyfrom the study folderin the archivesaccording to 3M Environmental LaboratoryStandard Operating Procedures: 16.2.1 Trainin- records 16.2.2 Calibrationrecords 16.2.3 Instrument maintenance logs 16.2.4 Standard Operating Procedures,Equipment Procedures,and Methods 17.SPEctmEN RETENTION Specimens willbe maintained in thelaboratoryspecimen archivesfora periodof time as specifiedby regulationor as long as thequalityof the preparationaffordsevaluation,but not longerthan ten yearsfollowingthe effectivdeateof the finaltestrule(ifapplicable)a,nd as establishedby 3M Environmental LaboratoryStandard Operating Procedures. 18. PROTOCOL AMENDMEIVTS AND DEVIATIONS Planned changes to theprotocolwillbe inthe form of writtenamendments signed by the Stud'Y Directorand the Sponsor's RepresentativeA.mendments willbe consideredas partof the protocoland willbe attachedto the finalprotocol.All changes to the protocolwillbe indicatedin the finalreport.Any otherchanges willbe in the form of writtendeviationss,igned by the Study Directorand filedwith the raw data. 3M EnvironmentalLaboratory 3M E-.ivironmer@--al Laboratory Page 8 of9 Page 23 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protoco#lFACT-TOX-030 19.ATTAcHmENTs 19.1 AttachmentA Preparatorayndanalyticmaelthods 20.SIGNATuREs z@z@ Andrew Seacat,Ph.D., Sponsor Representative @7, Date KristenHansen, Ph.D.,3M EnvironmentalLaboratoryStudy Director Date 3M EnvironmentalLaboratory 3M Envi-ronmental Laboratory Page 9 of9 Page 24 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 GLP Study ProtocoAlmendment StudyNumber: FAC-T-TOY,-D3D Lvcnu- (p@aq-2@l StudyTitle@:L L@@u-k Toxici@S6@@ ?FOS i,,C@,'%Dr^clts StudyDirector:krijAi2nia Amendment Date: 03)oq)qq Amendment Number: This amendment modifiesthe followingportionof theprotocol: 10 Mpl@.As anA iiA.I@@-ci@u@@.As F,@CT-Yn-3-t aiJ FACT-m-q.) rAs 4]u, s<fjitfnex-@aAoA -ihe. Yrtl@ods ha,,- 1,>ttr, tApdaQ @D Ers- 8-@.o arLc4 C4r\@-ETS- 3-5.b. @erurn tx4rc-@'onT upjalf-i4r-e@@oAs w-,ii@, LA@e3 (4nA CAnal@ses, @hp- r-p-rna,ri,-Vl Approved by: Stuhy Director Date 3M En-vironmental Laborato--y Page 25 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 StudyTitle 26-Week CapsuleToxicitSytudywithPerfluorooctaSnuelfonicAcid Potassium Salt(PFOS, T-6295) inCynomolgus Monkeys PROTOCOL AMENDMENT NO. 2 Amendment Date: April29, 1999 PerformingLaboratory 3M EnvirorunentalTechnology & SafetyServices 3M EnviroromentalLaboratory 935 Bush Avenue St.Paul,MN 55106 Laboratory Project ldentirication ET&SS FACT-TOX-030 LIRN U2279 3M EnvironmentaLlaboratory 3M Environmental Laboratory Page 26 3m Medical Department Study: T-629S.7 Report No. FACT TOX-030 laboratory Request Number-U2279 PrOtOcOFlACT-TOX-030 Amendment 2 This amendment modifiesthe followingpartion(so)f the protocol: 1. PROTOCOL READS: Inamendment1,sectio1n0PreparatoMreythodsand SectionII AnalyticalMethods were updatedto ETS-8-4.0 and ETS-8-5.0 as therevisedserum extractionand analyticamlethods. ll.siql AMEND TO READ: These methods were revisedon 41XI99 to ETS-8-4.1 and ETS-8-5.1 which willbe used forallfutureanalyses. REASON., The methods were revisedforclarificatiaonnd toincludelinearegression1,/x weightingforinitiaclurves. Amendment Approval Andrew SeacatPh.D.S,ponsorRepresentative 7130 Date K.risJ.Hansen Pfi.D.S,tudy Director 3M EnvironmentalLaboratory 3M Environmental Laboratory Date Page 27 3m Medical Department Study: T-629S.7 Report No. FACT TOX--130 laboratory Request Number-U2279 StudyTitle 26-Week CapsuleToxicitSytudywithPerfluorooctaSnuelfoniAccid Potassium Salt(PFOS, T-6295) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 3 Amendment Date: June 03, 1999 PerformingLaboratory 3M EnvirorunentTaelchnolog&y SafetyServices 3M EnviromnentalLaboratory 935 Bush Avenue St.Paul,MN 55106 Laboratory Project ldentirication ET&SS FACT-TOX-030 LIRN U2279 3M EnvironmentaLlaboratory 3M Environmental Laboratory Page 28 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 ProtocolFACT-TOX-030 Amendment 3 Thisamendment modifietshefollowinpgortion(osf)theprotocol: 1. PROTOCOL. READS: Section10PreparatorMyethodsand SectionIIAnalyticaMlethods list FACT-M-1.0 and FACT-M-2.0 as the liverextractionand analyticamlethods. AMEND TO READ: These methodswere revisedon 06/03/99 toFACT-M- 1.1 and FACT-M- 2.1 which willbe used forallfutureanalyses. REASON: The methods were revisedforclarificatiaond to expand on the listof target aiialytes. Amendment Approval J-4@@ 2:@ol@t Andrew Seacat Ph.D., Sponsor Representative -713D /Ilq Date 411",, KrisJ.Ha@nsenPh.D.,StudyDire--tor 3M Environmental Laboratory 3M Environmental Laboratory Date Page 29 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Recruest Number-U2279 StudyTitle 26-WeekCapsulTeoxiciSttyudywithPerfluorooctane SulfonicAcid PotassiumSalt(T-6295)inCynomolgus Monkeys PROTOCOL AMENDMENT NO. 4 Amendment Date: April25,2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental laboratory 935 Bush Avenue St.Paul,MN 55106 Laboratory Project Identification FACR Tox-030 ET&SS LRN-U2279 3M EnvironmentaLlaboratory 3M Environmertal Laboratory Page 30 3m Medical DepartTrent Study: T-6295.7 Report No. FACT TOX-030 laboratory Request NuTnber-U2279 ProtocolLRN-U2279 Amendment Number 4 This amendment modifies the followingportion(s)of the protocol: 1. PROTOCOL READS: The sponsor forthe presentstudywas identifieads Andrew Seacat, Ph.D. AMEND TO READ: The roleof sponsor forthe presentstudywas reassignedto John L. Butenhoff, Ph.D. as of the date of signatureapproval ofthisprotocolamendment. REASON: To ensure thatthe study directordoes not also carrythe dutiesof study sponsor, the sponsor rolewas reassigned. Inthismanner, personnel responsibilitiaensd workload are more evenly balanced. 2. PROTOCOL READS: On page 2 of the protocol,KrisHansen isidentifieads the study directorforthe analyticalphase of the study.Peter Thomford isalso identifieads a study director, but forthe in-lifpehase of the study (see Covance LaboratoriesProtocol6329-223). AMEND TO READ: On page 2 of the protocol,Andrew Seacat willbe identifieads the study director, KrisHansen willperform the dutiesof the principalanalyticalinvestigatora,nd Peter Thomford willbe identifieidnthe finalreportas the principalin-lifienvestigatoras of the date of signatureapproval of thisprotocolamendment. REASON: The originalstudy design identifietdwo study directors;one forthe in-lifpehase of the study and one forthe analyticalphase ofthe study.The roleof study director has been reassigned inan effortto ensure compliance with Good Laboratory PracticeStandards thatoutlinestudy personnel requirements. 3. PROTOCOL READS: 10. Preparatory Methods: 10.1 FACT-M-1.1, "Extractionof Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from LiverforAnalysis Using HPLCElectrospray/Mass Spectrometry" AMEND TO READ: 10. Preparatory Methods: 10.1 REASON: ETS-8-6.0, "Extractionof Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from LiverforAnalysisUsing HPLCElectrospray/Mass Spectrometry" The method was revisedto includeextractionsof other tissuetypes and the use of methyl-tert-butyelther (MTBE) instead ofethylacetate inthe extractionprocess. 4. PROTOCOL READS: 11. Analytical Methods: 3M EnvironmentaLlaboratory 3M Env4-rcnmental Laboratory Page 31 3m Medical Departmen-@, Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 ProtocolLRN-U227,9 Amendment Number 4 11.1 FACT-M-2. 1,"Analysisof FluorochemicalsinLiverExtractsUsing H PLCElectrospray/Mass Spectrometry' AMEND TO READ: 11. Analytical Methods: 11.1 ETS-8-7.0, "Analysisof Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds inLiverExtractsUsing HPLCElectrospray/Mass Spectrometr/' REASON: The method was revisedto includecurves plottedby linearregressionweighted 1/x and a reduced cycle time from 13.5 minutes to 9.0 minutes. 5. PROTOCOL READS: 8. Test System Refer to the Covance protocol#6329-223 fortabularpresentationof data.Two animals each from Groups 1,3,and 4 were designated as recovery animals and were allowed atleasta 13 week, which may be extended, recovery period after cessation oftreatment. AMEND TO READ: 8. Test System A tabular presentationof the testsystem data willbe included inthe finalreport forthisproject(FACT Tox-030). Four animals each (two/gender)from Groups 1, 3, and 4 were designated as recovery animals and were observed fora recovery period aftercessation of treatment untilstudy cut-offon March 7, 2000 (Week 80).The observation and analysisofthe recovery group IIIanimals beyond the cut-offdate of thisstudy willbe reported ina new long-term_recovery study. REASON: Additionalanimals were assigned to the recovery group followingapproval of the protocolfor FACT Tox-030. A new study willreportthe long-term recovery of the remaining animals from recovery group III(mid-dose group). 6. PROTOCOL READS: 9. Specimen and Sample Receipt: (Sample Receipt Table] Expected # of Specimens: Serum-all animals:616 from main study,24 additionalfrom recovery Urine and feces-recovery animals:24 urine,24 feces Liver-allanimals:44 Collected: Urine and feces-recovery animals: Day 0, 6,30, 90 (potentia1l80) Total number of test animals: 32 3M EnvironmentaLlaboratory 3M Erivironmen--al Laboratory Page 32 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Nurnber-U2279 ProtocolLRN-U2279 Amendment Number 4 AMEND TO READ: 9. Specimen and Sample Receipt: [Sample Receipt Table] # of Specimens: Serum-all animals:516 from main study,280 additionalspecimens from recovery Liver-allanimals: 42 (eightspecimens via biopsy from recovery group) Total number of test animals: 32 Total number of animals: 48 (12 animals incontrolgroup) Note: 4 animals were not assigned to a study group. Day 0 serum specimens were collected.Specimens ofbody tissuesand fluidsother than serum and liver specimens willbe collectedand receivedfrom Covance Laboratories(6329-223, Study T-6295.7). REAsoN: Additionalanimals were assigned tothe recoverygroup followingapproval of the protocolfor FACT Tox-030. The recovery period has been extended for the recovery group. Specimen collectionfiguresshown are through the end of the study 3/07/00. Amendment Approval John L. Butenhoff,Ph.D.,Sponsor Representative C@@ ) //@;- z@Z,4@ Andrew Seacat,Ph.D., Incoming Study Director #2 f KristenJ.Hansen, Ph.D., Outgoing Study Director Date 7.z@z,@ Date IJDate Date L. Bacon, Lal5'oratorMyanager Date 3M EnvironmentalLaboratory 3M Environmental T@aboratory Page 33 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 StudyTitle 26-WeekCapsulTeoxicitSytudywithPerfluoroocStualnfeoniAcidPotassiuSmalt(T-6295)in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 5 Amendment Date: August 14, 2000 Performing Laboratory 3'vfEnvirorunentaTlechnology & SafetyServices- 3M EnvironmentalLaboratory 935 Bush Avenue St.Paul,MN 55106 Laboratory Project Identification ET&SS FACT-TOX-030 Covance #6329-223 LIMS gU2279 3M EnvironmentaLlaboratorl 3M Environmental Laboratory LDage 34 3m Medical Department Szady: T-6295.7 Report No. FACT TOX-030 labora+@-o@-y Request Nurnber-U2279 Protocol TOX-030 Amendment 5 This amendment modifiesthe followingportion(so)f the protocol: 1. PROTOCOL READS: 2. PURPOSE: This analyticalstudy is designed to determine levelsof potassium perfluoroocianesulfonate(PFOS) in the liverand serum of cynomoli-zusmonkeys. Additional tissuesor fuids may be analyzed. AMEND To READ: Add thatselectfecessamples willbe analyzed forPFOS atCentre. REASON: Feces samples were added afterthe originalprotocolwas wn'tten. 2. PROTOCOL READS: STUDY Environmental Laboratory LocATIONS(PAGE 2): Analytical Testing Laboratory:. 3M AMEND TO READ: 3',IvEtnvironmental Laboratory and Centre Analytical Laboraton*es Inc. (Centre),3048 Research Drive, StateCollege,PA 16801 REASON: Analyses of feces are assigned to Centre, in addition to the analyses of other tissuesat the 3@vlEnvircnmental Laboratory. 3. PROTOCOL. READS: SECTIONS 10. PREPARATORY AND 12. DATA QUALITY Oi3JECTIVES METHODS, 11. ANALYTICAL METHODS AMEND TO READ: Add to these sections-the most current version of "Determination of Flucrochemical Residues in Monkey/Rat Feces by LC/.,IvISI.MS,C"entre method number OONI-O?-3-003, with ar. LOQ in feces of 10 ng/g. The method w;lllincorporatean initial homogenization step i-nmediatelyafter adding the extractionsolvent,using a microhomo,zeniz.-r,to allow fcr complete dispersionof the specimen in the solvent. REASON: To specify -,.hevalidated method to be used for feces analyses along .,,Ithits analyticallimits.NOTE: LC/MS/iMS is an abbreviation for "liquidch-romatograph y/mass spectrometry/mass spec--ometry." 4. PROTOCOL METHODS READS: SECT70N 10. PREPARATORY METHODS AND 11. 3M ANALYTICAL- AMEND TO READ: Cha.-,ceto specify that the most current version of the methods listed should be used. REASON: To specify that the most appropriate version of the preparatory and analvtlcal rr,ethcdsshould be used during the course of the study. 3M EnvironmentaiLaboratory 3M Env4-ronmenl--al Laboral-o--y Page 35 3m Medical Department Study: T-6295.7 Report No. FAC--, TOX-C30 laboratory Request Number-U2279 Protocol TOX-030 Amendment 5 5. PROTOCOL READS: The amended protocol(Amendment #4) statesthatK-risJ.Hansen, Ph.D. isthe PrincipalAnalyticalInvestigatorforthe entirestudy. AMEND TO READ: Add Enaksha Wickremesinhe, Ph.D. as the PrincipalLq-@@,lnvestigatorat 6-r1'. Centre for feces analyses. REASON: To specify the PAI at Centre for fecesanalysis. 6. PROTOCOL REPORTS READS: SECTION 16. LOCATION OF RAw DATA, RECORDS AND FINAL AMEND TO READ: Add that Centre will forward alloriginalstudy-specificraw data to 3Nf Environmental Laboratories,togetherwith copies of appropriatefacility-specifircaw data applicable to thisstudy. Centre will maintain a copy of the applicablestudy-specificraw data, protocol and analyticalreportin the Centre archives,as well as all originalfacility records. REASON: Centre. To specify the archival requirement for the portion of the data developed by 7. PROTOCOL READS: SECTION 17. SPECIMEN RETENTION AMEND TO READ: Add that after the analyticalreport on feces is signed by the study director,allfeces specimens of thisstudy will be returnedto 3,N4Environmental Laboratory. These specimens may then be discardedby writtendirectionof the study director.Specimens of serum and urine may also be discardedby writtendirectionof the study directorafterthe analyticalreport for the 3M Environmental Laboratory analyses is signed by the study director. REASON: To specify the handling of allbiologicalfluids,and to definewhen ver1"ficat'ion IisconsiIdered complete. the quality assurance -,M Environmental Laboratory 3M Env-'-ronmental Laboratory Page 36 3m MEW@o@.Dep@gtiiSerit SEP,15,20M ll:33qM WIS49 UG: 31 f eczw-UL9@g: 17 Report No. 2'5---@;9@ 410 894 91?2aboratory cafm FTMYTICAL LA ReWde@i4 EW LA 2-M--Gg -6el4 231 L5W FACI@3.n, NuF.L@er3:-@YU2279 NO. 338 M2 Amendment Approval Ptutocol Tox-cao I Amendment 5 2z JohnI- BUaLbogFbly., SPOMOT'IAMMIUNdve Andow asam MD., Study Dimtw Widamefthe, Pbl).,Pri=iPAIIn-lilfnOv64Ptw Date rl(2 C) Date 3W IMW@mmaM Lspba*c@y 3M Env@-ronmental Laboratory 1 .10 Page 37 3m Medical Department sz:udy: T-6295.7 Report No. FAC'-7TOX-03C laboratory Request Number-U2279 StudyTitle 26-WeekCapsulTeoxiciSttyudywithPerfluoroocStualnfeonAiccidPotassiSuaml(tT-6295i)n Cynomotgus Monkeys PROTOCOL AMENDMENT NO. 6 Amendment Date: September 11,2000 Performing Laboratory 3NI EnvironmentalTechnology& SafetyServices 3M EnvirorunentaLlaboratory 935 Bush Avenue St.Paul,MN 55106 Laboratory Project Identification ET&SS FACT TOX 030 Covance #6329-223 LIMS #U2279 3M EnvironmentaLlaboratory 3M E@-ivironme-ntal Laboratory Page 38 3m Medical Department Study: T-629S.7 Report No. FACT TOX-030 laboratory Request Number-U2279 ProtocoTlOX 030 Amendment 6 Thisamendment modifiesthefollowinpgortion(so)ftheprotocol: PROTOCOL READS: 8. TEST SYSTEM: Groups 2,3,and 4 receivedthetestsubstancedailyfor26 weeks,at concentrations of 0.02, 0.5,and 2.0 mg/kg/day, respectively. AMEND TO READ: Groups 2, 3, and 4 received the testsubstance daily for26 weeks, at concentrationsof 0.03, 0.15, and 0.75 mg/kg/day. REASON: Test substance doses were changed afterprotocol was written.The Covance protocol reflects the actual doses of testsubstance thatwere given. PROTOCOL READS: The amended protocol (Amendment Number 4, section2.)states:On page 2 of the protocol, Andrew Seacat will be identifiedas the study director,Kris Hansen willperform the dutiesof the principalanalyticalinvestigatora,nd PeterThomford willbe identifiedin the Finalreport as the pr-incipatin-lifeinvestigatoras of the date of signatureapproval of thisprotocol amendment. AMEND TO READ: Peter Thomford will remain as the Covance Study Director for the purpose of issuing the Covance finalreport for the in-lifephase of the study. REASON: Study directorshipwas not relinquishedby Covance for the in-lifephase of the study, since the animal experimental phase was completed before Amendment Number 4 was issued. Amendment Approval John L. Butenhoff PH.D., Sponsor Representative Date Andrew Seacat, Ph.D., Study Director Date 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 39 3m Meddwa!l-eoeparo5p&- t S bOyi@B @-:ft2-d9-5471 410 E384 1122 Report No. FACT TOX-030 anoratory Request Numb@U---:U2279P% Study Title Pmtowl TOX-030 Amencheiit7 26-weak Capsule ToxicityStudy with PerfluorooetaneSiilfoniAccid Potassium Salt (T-6295) inCynomoigus Monkey3 ProtocolAmendinent No. 7 Amendment Date-. September 14, 2000 PerformingLaboratory 3M Environmental Technology and Safety Services 3M Fnvironmen.W Laboratory 93 5 Bush Avenuc St.Paul, MN 55106 LaboratoryProjectIdentification F-r&S,9YACT-TOX-030 Covance #6329-223 LIMS #U2279 3M Environmental Laboratory son 3m, medi@@dlis@parvnEs4t stsWrA;qBT2--g-c@._.71 410 Report No. FACT TOX-030 E3841TIRratory Request Numbe@-aTn79POe Amendinent Approval ProtocolTOX-030 Amendmeilt 7 John L. Butenhoff,Ph.D.,Spotiso?sRepresentative Date AndreL'w'-@e@tPh.D., Study Director Date @P4 3M Env-'@ronme@ntal Laboratory 3m Medical Department Study: T-6295.7 3M Medir-alDepartment Study:T-6295.7 ATTACHMENT EXTRACTION C AND ANALYTICAL METHODS Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 LaboratoryRequest Number-U2279 3M EnvironmentaLlaboratory 3M Environmental Laboratory Page C-1 Page 43 3m Med@-cal Department Study: T-629S.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M ENVIRONMENTAL LABORATORY METHOD EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE FLUOROCHE,NIICAL COMPOUNDS FROM LIVER FOR AINALYSIS ELF-CTROSPRAYFMASS SPECTROMETRY OR OTHER USING HPLC- Metfiod Number: ETS-8-6.0 Author: Lisa Clemen, Rober-tWynne Approved By: Adoption Date: 0 Revision Date: Laboratory Nianager &. /-I./-@.Group Leader a,'A @, LL Technical Reviewer Date --?/-/-@ Datc c ill@ lil Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from liver. 1.2 Applicable Compounds: Fluorochemical surfactantsor other fluorinatedcompounds. 1.3 Nlatrices: Rabbit, rat,bovine, and monkey liversor other tissuesas designated in the validationreport. Word 6.0,195 3M Environmencal Laboratory ETS-8-6.0 Exmction ofPFOS from Liver Page I of 14 Page 44 3m Med@-cal Department Study: T-6295.7 -.Report No. FAC- TOX-030 laboratory Request Number-U2279 2.0 SUNINLARY OF METHOD 2.1 This method describesthe procedure for extractingpotassium perfluorooctanesulfonate (PFOS) or other fluorochemicalsurfactantsfrom liver,or othertissues,using an ion painng reagent and methyl-tert-butylether(MtBE). In thismethod, seven fluorochemicalscan be extracted:PFOS, PFOSA, PFOSAA, ETFOSE-OH, PFOSEA, M556, and surrogate standard.An ion pain'ng reagent isadded to the sample and the analyte ion pair is partitionedintoMTBE. The MTBE extractis transferredto a centrifugetube and put onto a nitrogenevaporator untildry. Each extractisreconstitutedin 1.0 mL methanol then Filteredthrough a 3 cc plasticsyringeattachedto a 0.2 um nylon filterintoglass autovials. 2.2 These sample extractsare analyzed following method ETS-8-7.0 or other appropriate methods. 3.0 DEFINITIONS 3.1 PFOS: perfluorooctanesulfonate(anion of potassium salt)C,F,,SO, 3.2 PFOSA: perfluorooctane sulfonylamide C,F,,So2NHI3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetateC,F1,SO,N(CH,CH,)CH,CO, 3.4 ETFOSE-OH: 2(N-ethylperfluorooctanesulfonamido)-ethylalcohol C,F,,SO,N(CH,CH,)CH,CH,OH 3.5 PFOSEA: perfluorooctanesulfonylethylamide C,F,,SO,N(CH,CH3)H 3.6 M556: C,F,,SO,N(H)(CH,COOH) 3.7 Surrogate standard: IH- IH-'@H'-2H perfluorcoctane sulfonic acid 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use universalprecautions,especiallylaboratorycoats,gocp-ies,and gloves when handling animal tissue,which may contain pathogens. 5.0 INTERFERENCFS 5.1 There are no interferences',-,nowant thistime. 6.0 E()UIPMENT 6.1 The following equipment isused while performing thismethod. Equivalentequipment is acceptable. 6.1.1 Ultra-TurraxT25 Grinder forgrinding liversamples 6.1.2 Vortex mixer,VWR, Vortex Genie 2 6.1.3 Centrifuge,Mistral 1000 or IEC 6.1.4 Sliaker,Eberbach or VWR ETS-8-6.0 Extractionof PFOS from l.i%-er Page 2 of 14 3M Environmental Laboratory Page 45 3m Medical Departmen-- Szudy: T-6295.7 Report No. FAC- TOX-030 laboratory Request Number-U2279 6.1.5 Nitrogen Evaporator,Organomation 6.1.6 Balance (sensitivittyo 0.100 g) 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles,capa@oleof holding 250 mL and I L 7.5 Volurnetn'cflasks,glass,type A 7.6 I-CHEM vials,40 mL glass 7.7 Plasticsampule vials,Wheaton, 6 mL (or appropriatesize) 7.8 Centrifuge tubes7,pol.vpropylene, 15 mL 7.9 Labels 7.10 Oxford Dispensor - -'.0 to 10.0 ml 7.11 Syringes, capable of measuring 5 pLL to 50 @iL 7.12 Graduated pipettes 7.13 Syringes, disposable plastic,3 cc 7.14 7.15 7.16 Syringe filtersn,ylon, 0.2 gm, 25 mm Timer Crimp cap autovialsand caps 7.17 Crimpers Note: Priorto using glassware and bottles,rinse3 times with methanol and 3 times with Milli- Q I'mwater. Rinse s@,liincliesa minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 REAGENTS AND STAND.4RDS 8.1 Type I reagent gade water, Milli-QTIlor equivalent;allwater used in thismethod should be Mi lll_QT.wMater and be provided by a Milli-Q TOC plUSTIISystem 8.2 Sodium hydroxide (N'aOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA),Kodak or equivalent 8.4 Sodium carbonate C-Na@CO,),J.T.Baker or equivalent 8.5 Sodium bicarbonate (,NaHCO,), J.T.Baker or equivalent 8.6 Methyl-tert-butylether,Omnisolv, glassdistilleodr HPLC grade 8.7 Methanol, Omnisolv, glassdistilleodr HPLC grade 8.8 Liver,frozen from supplier 8.9 Dry ice from supplier 8.10 Fluorocbemical standards 8.10.1 PFOS (3N[ SpecialtyChemical Division),molecular wei(,'ht 538 3M Envirormental Laboratory ETS-8-6.0 ExtractionofPFOS froniLivcr Pa-c of 1-4 Page 46 3m Medical Department Sz@udy: T-6295.7 Renort No. FAC-@ TOX-030 laboratcry Request Number-U2279 8.11 8.12 8.10.2 PFOSA (3,,lSpecialtyChemical Division),molecular weight = 499 8.10.3 PFOSAA SpecialtyChemical Division),molecular wclcht = 585 8.10.4 ETFOSE-OH (3M SpecialtyChemical Division),molecular weight = 570 8.10.5 PFOSEA (3M SpecialtyChemical Division),molecular weight = 527 8.10.6 M556 (3M SpecialtyChemical Division),molecular weight = 557 8.10.7 Surrogate standard:4-H, perfluorooctanesulfonicacid (I-H,I-H, 2-H, 2-H C,F,,SO,H) molecular weight = 428 8.10.8 Other fluotochemicals, as appropriate Reagent preparation NOTE: When preparing largervolumes than listedin reagent,standard,or sur-rogate preparation, ad@ustaccordingly. 8.11.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NAOH. Pour intoa 1000 mL beaker containing500 mL Milli-Q"w'aterm,ix untialllsolidasre dissolved.Storeina I L Nalgene bottle. 8.11.2 1 N sodium hydroxide(NaOH): Dilute10 N NAOH 1:10. Measure 10 mL of 10 N NAOH solutionintoa 100 mL volumetricflaskand dilutetovolume using Milli_QTIwIater. Storeina 125 mL Nalgene bottle. 8.11.3 0.5 M tetrabutylammonium hydrogen sulfate(TBA): Weigh approximately169 g of TBA intoa I L volumetriccontaining500 mL Milll_Q71water.Adjustto pH 10 usingapproximately44 to54 mL of 10 N NaOF1 (While adding the lastML of NAOH, add slowlybecausethepH changes abruptly).Dilutetovolume with Milli-Q" water.Storeina I L Nalgene bottle. 8.11.3.1TBA requiresa check priortoeach use toensurepH = 10. Adjust as needed using 1 N NAOH solution. 8.11.4 0.25 M sodium carbonate/sodiubmicarbonatebuffer(NaCO,/NaHCO,): Welc,,h approximately26.5g ofsodium carbonate(Na,CO,) and 2 1.0g ofsodium bicarbonate(NaHCO,) intoa I L volumetricflaska-nd brinlt,ovolume with Milli- Q T,%i water.Sloreina I L Nalgene bottle. Standards preparation 8.12.1 PreparePFOS standardsforthestandardcurve. 8.12.2 Prepareotherfluorochemicalstandards,as appropriate.Nluiticomponent fluorochemicalstandardsareacceptable(forexample,one working standard solutioncontaining1.00ppm PFOS, 1.02ppm PFOSA, 0.987ppm PFOSAA, and 1.10 ppm Et.70SE-OH.) 8.12.3 Weigh approximately100 mg of PFOS intoa 100 mL volumetricflaskand record the actualw--Ight. 8.12.4 Bring tovolume withmethanol fora stockstandardofapproximately1000 ppm (4g/mL). 8.12.5 Dilutethestocksolutionwithmethanol fora working standardI solutionof approximately50 ppm. 3M Env4-ronmental Laboratory ETS-8-6.0 ExtractionofPFOS from Livci Page 4 of 14 Page 47 3m Medi-cal De@Dartment Study: T-6295.7 Report No. FAC-@ TOX-C30 laboratory Request Number-U2279 8.13 8.12.6 Dilute the s-,ocksolutionwith methanol for a working standard2 solutionof approx. 5.0 ppm. 8.12.7 Dilutethe stock solutionwith methanol fora working standard ')solutionof approx. O..@Oppm. Surrogate stock standard preparation 8.13.1 Weigh approximately 50-60mg of surrogate standard 1-H,I-H, 2-H, 2-H, C,F,,S0,H intoa 50 ml volumetric flaskand recordthe actualweight. 8.13.2 Bring to volume with methanol for a sur-rogatsetock of approximately 1000-1200 PPM- 8.13.3 Prepare a surrogateworking standard. Transferapproximately 1.0 ml of surrogate stock to a 10 ml volumetn'c flaskand bring to volume with methanol for a work'uig standard of 10-20 ppm. Record the actualvolume transferred. 9.0 SANIPLE HANDLING 9.1 All samples are received frozen and must be kept frozen untilthe extractionisperformed. 10.0 ()UALITY CONTROL 10.1 Matrix blanks and method blanks 10.1.1 An aliquotof 1.0 mL methanol is used as a solventblank. 10.1.2 Extract two 1.0 mL aliquotsOfMi lli_QTII water following thisprocedure and use as method blanks. 10.1.3 Extracttwo 1.0 mL aliquotsof liverhomogenate followitigthisprocedure and use as matrix blanks. Refer to 11.1.6. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicatesamples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst,usuallya control liver received with each sample set. 10.2.3 Expected concentrationswill fallin the mid-range of the initiaclalibrationcurve. Additional spikes may be includedand may fallin the low-range of the initial calibrationcurve. 10.2.4 Prepare one matrix spike and matrix spike duplicateper 40 samples, with a minimum o C 2 matn*x spikes per batch. 10.3 Continuing calibration verirications 10.3.1 Prepare continuing calibrationverificationsamples to ensure the accuracy of the initiaclalibrationcurve. 10.3.2 Prepare,at a minimum, one continuing calibrationverificationsample per group of 10 samples. For example, ifa sample set= 34, folirverificationasre prepared and extracted. 3M Environmental Laboratory ETS-8-6.0 Extractioonf PFOS from Livcr PaE!c5 of 14 Page 48 3m Medical Department S::udy: T-6295.7 Re-@@ort No. FAC'I' TOX-030 laboratory Request Numbe---U2279 10.3.3 Prepare each continuing calibrationverificationfrom the same matnx used to prepare the initialcur-ve. 10.3.4 The expected concentrationswill fallwithinthe mid-ranizeofthe initial calibrationcur-ve.Additionalspikesmay be included thatfallin the low-rang-.of the initiaclalibrationcurve. This isnecessary ifthe analvstmust quantitateusinc, only the low end of the calibrationcurve (forexample, 5 ppb - 100 ppb, rather than 5_ppb - 1000 ppb). 11.0 CALIBRATION AND STANDARDIZATION 11.1 Prepare matrix calibration standards 11.1.1 Weigh approximately 40 g of liverintoa 250 mL Nalgene bottlecontaining 200 mLs Mi lll_QT*wlalter.Grind toa homogeneous solution. 11.1.2 If40 g isnot available,use appropriateamounts of liverand water to ensure a 1:5 ratio. 11.1.3 Refer to 13.0 to calculatethe actualdensity of liverhomogenate and the concentration of solidlivertissuedisoersed in 1.0 mL of homogenate solution. 11.1.5 Add I mL of homogenate to a 15 mL centrifugetube.Re-suspend solutionby shaking between aliquotswhile preparing a totalof eighteen I mL aliquotsof homogeneous solutionin 15 mL centrifugetubes. 11.1.6 Two I mL aliquots,or otherappropriatevolume, ser-veas matrix blanks. 11.1.7 Typically use the standard concentrationsand spiking amounts listedin Table 1,at the end of thissection,to spike,in duplicate,two standard curves,for a totalof eighteen samples, two matrix blanks,and two method blanks. 11.1.8 Refer to validationreportsETS-8-6.0 and ETS-8-7.0-V-1 or Attachment B, which liststhe working ranges and the Linear CalibrationRange (LCR) for calibrationcurves. 11.1.9 Use Attachment C as an aid in calculatingthe concentrations of the working standards. Refer to 13.0 to calculateactualconcentrationsof PFOS in calibration standards. 11.2 To each working standard,blank,or continuing verificationa,dd appropriateamount of surrogate working standard forthe concentrationto fallwithin the calibrationcurve range 5 ppb -1000ppb. 3M Environme-tal Laboratory ETS-8-6.0 Extractionof PFOS from Liver Page 6 of 14 Page 49 3m Medical Department S+--udy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 11.3 Extractspiked liverhomogenates following12.14-12.25of thismethod. Us-.these standardsto establisheach initiaclur-veon themass spectrometer. Table I Approximate Spiking Amounts forCalibration Standards Working Standard (Approx.Conc.) 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 5.0ppm 5.0 ppm 5.0 ppm 50 ppm @ll Approx. finalconc.of PFOS inliver - Blank 2 0.005 ppm 4 0.010 ppm 10 0.025 ppm 20 0.050 ppm 40 0.100 ppm 10 0.250 ppm 20 0.500 ppm 30 0.750 ppm 4 1.00ppm 12.0 PROCEDURE 12.1 Obtain frozenliversamples. 12.2 Cut approximatelyI c,of liverusinga dissectinsgcalpel.This partoftheprocedureisbest performed quickly,not allowingthelivertothaw. 12.3 Weigh the sample directlyintoa taredplastic sampule vial. 12.4 Record theliverweight inthestudynotebook. 12.5 Return unused liverportionsto freezer. 12.6 Add 2.5 mLs ofwater tosampule vial. 12.7 Grind the sample.Put thegrinderprobein thesample and grindforadout2 minutes,or untilthesample ishomogeneous. 1'-1.8 Rinse theprobe intothesample with 2.5mLs waterusinga pipette. 12.9 Take thegrinderapartand cleanitwith methanol aftereach sample.Referto A-iMDT-EP- 22. 12.10 Cap thesample and vortexfor15 seconds.Label thesampule vialwith thestudynumber, weight,liverID, dateand analystinitials. 3M Environmental Laboratory ETS-8-6.0 Extractionof PFOS from Li@,er Page 7 of 14 Page 50 3m Medical Department Study: T-6295.7 Report No. FA--- TOX-030 laboratory Pecruest Number-U2279 12.11 Pipette 1.0 mL, or other appropr-iatveolume, of homogenate into a 15 mL polypropylene centrifugetube. Label the centrifugetube with the identicalinfortnationas the sampule via].Refer to attached worksheet fordocumenting the remaining steps. 12.12 Pipette two I mL aliquotsof Milli-Qw'atetrocentn'futguebesT.hesweilslervaes method blanks. 12.13 Spike all samples, including blanks and standards readv for extraction with surrogate standard as described in section 11.2. 12.14 Spike each matirx with the appropriate amount of standard as described in 11. 1, or Table I of thatsection,forthe calibrationcurve standards. Also prepare matn*x spikes and continuin@:,c,alibration standards. 12.15 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.16 Check to ensure 0.5 M TBA reagent isat pH 10. Ifnot,adjustaccordingly, 12.17 To each sample, add I mL 0.5 M TBA and 2 mL of the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.18 Using an Oxford Dispenser, add 5 mL methyl-tert-butylether. 12.19 Cap each sample and put on the shaker at a settingof 300 rpm, for20 minutes. 12.20 Centrifuge for 20 to 25 minutes at a settinco,f 3500 rpm, or untillaycrsare wcll separated. 12.21 Label a fresh 15 mL centrifugetube with the same information as in 12.10. 12.22 Remove 4.0 mL of the organic layerto the fresh 15 mL centrifugetube. 12.23 Put each sample on the analyticalnitrogenevaporator untildry,approximately I to 2 hours. 12.24 Add 1.0 mL to each centrifugetube using a graduated pi@)ette. 12.25 Vortex mix for 30 seconds. 12.26 Attach a 0.2 @im nylon mesh filtetro a 3 cc syn'nge and transferthe sample to thissyrinee. Filterinto a 1.5 mL glassautovialor low-volume autovialwhen necessary. 12.27 Label the autovialwith the study number, animal number and gender, sample timepoint, matrix, finalsolvent,extractiondate,and analyst(s)performing the extraction. 12.28 Cap and storeextractsat room temperature or at approximately 4 'C untilanalysis. 12.29 Complete the extractionworksheet, attached to thisdocument, and tape in study notebook or include in study binder,as appropn'ate. 3M Environmental Laboratory ETS-8-6.0 ExtractionofPFOS from Liver Page 8 OF 1,4 Page 51 3m Medical Department Study: T-629S.7 Pe-@ort No. FACT TOX-030 laboratory Pequest Numbe---U2279 13.0 DATA ANALYSIS 13.1 Calculations: AND CALCULATIONS 13.1.1 Calculate the average density of the liverhomozenate bv recording each mass of ten separate 1.0 mL aliquotsof homogenate. Average density (mg/mL) = Average mass (mg) of the aliquots 1.0mL aliquot 13.1.2 Calculatethe amount of liver(mg) per 1.0mL homogenate (orconcentrationof dispersed solidtissueper mL of homogenate suspension) using the following equation: g of Li@er x Averae:edensity*of homogenatelmg/mL) (g of Liver + g of Water) referto 13.1.1 for details. 13.1.3 Calculateactualconcentrationsof PFOS and other fluorochemicalsin calibration standards using the following equation: @iL of Standard x Concentration (@ig/mL) = Final Concentration (4g!g or rng!kg) mg Liver/ I mL homogenate* of PFOS in Liver *referto 13.1.2 for details. 14.0 METHOD PERFORNTA*QCE 14.1 The method detectionlimit(MDL) isanalyte and matrix specific.Refer to MDL report for spec'ificN/IDLandlimiotfquantitation(LOvQa) lu(ersetfoeArttachmeanntdscB). 14.2 The followinqgualictoyntrsoalmpleasrextracwtietdehachbatcohfsampletsoevaluate the qualityof theextractionand analysis. 14.2.1 Method blanksand matrixblanks. 14.2.2 Matrix spikeand matrixspikeduplicatesamplestodeterrninaeccuracyand precisionof theextraction. 14.2.3 Continuingcalibratiovnerificatiosnamples todeterminethecontinuedaccuracy ofthe initiaclalibratiocnurve. 14.3 Refer tosection14 of ETS-8-7.0 formethod performancecriteria. 15.0 POLLUTION PREVENTION AND WASTE MANAGErvIENT 15.1 Sample waste isdisposedinbiohazardcontainersf,lammable solventwaste isdisposedin high BTU containersa,nd used glasspipettewaste isdisposedinbroken glasscontainers locatedin thelaboratory. 3M Environmental Laboratory- ETS-8-6.0 Exrractionof PFOS from L'iver Page 9 of 1-4 Page S2 3m Med@-cal Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 16.0 RECORDS 16.1 Complete the extractionworksheetattachedto thismethod, and tapein thestudynotebook or includein the3-rin-s,tudybinder,as appropriate. 17.0 TABLES, DIAGRAms, FLOWCHARTS, AND VALIDATIo,*4 DATA 17.1 Attachment A, F-xtractiownorksheet 17.2 Attachment B, MDLILOQ valuesand summary 17.3 Attachment C, Calibratiosntandardcalculatioannd concentrationworkshect 18.0 REFERENCES 18.1 The validationre-poratssociatedwith thismethod isETS-8-6.0 & 7.0-V-1. 18.2 AMDT-EP-22, "RoutineMaintenanceofUltra-TurraTx-25" 18.3 FACT-M- 1.1,"ExtractionofPFOS orOther AnionicFltiorochemicaSlurfactantfsrom LiverforAnalysisUsing HPLC -ElectrsoprayfmassSpectrometry" 19.0 AFFECTED DocUNIENTS 19.1 ETS-8-7.0, "Analysisof LiverExtractsforFluorochemicalsusin-IHPLC-Electrospray Mass Spectrometry" 20.0 REVTSIONS Revision Number. Reason For Revision Revision Date 3M Env4@--onmental Laboratory ETS-8-6.0 Extractionof PFOS from I-i,,-er Page 10 of 14 Page 53 3m Med@-cal Department S:@-ddy: T-6295.7 Report No. FA-7 TOX-030 laboratory Request @4umber-U2279 Study Matrix Box Vv'k/Day Date Spiked/Analyst ccv Surrogatz Std approx. ppm actual ppm MSD FC Nlix Std approx. (D.5ppm actual ppiii 1 FC,"vllxStd approx. 5 ppm actual PPM FC Nlix Std approx. 50 ppm actual PPM Comments Blank LiverHomo_genate: Sd LiverExtractionMethod Liveramount Spike surrogateand Standardmix, Vortex 15 sec- PipetteImL of LiverSolution PipetleImL of tO.5,M TBA, pH 10. pH Std. Pipett2e mL of0.25Na-)C03/0.25M NaHC03 3uffer Std. Dispense5m[ of Methyl-t-ButyElther Shake 20 min. Ccntrifuge20-25 min. Remove a 4 mL aliquotof organiclaver Puton NitrogenEvaporatortodrvness Add 1.0mL of Methanol TN-AShaker Speed CcntrifugeSpeed EvaporatorTemperature TN-A- 5artex 30 sec. I Filteursinea 3cc B-D svrin@2wcith a0.2um SRI f-iftienrtoautosamplevial Cont. Cal.Verificationsused thesame matT--Iaxs forthestandardcut-ve. AtiactimentB: MDLi'LOQ Values 3M Env@-ronmen-@al Laboratory ETS-8-6.0 Extractionof PFOS from Li%-er Date & In tials Page II of 16 Page S4 3m Medical Department Szady: T-629S.7 Report No. FAC-@ TOX-C30 laboratory Request Number-U2279 MDLILOQ Compound PFOS PFOSA PFOSAA ETFOSE-OH M55 PFOSEA values for rabbit liver NIDL LOQ Linear CalibrationRange (LCR) (p pb) (ppb) Approximate concentrationtsobe used forpreparingthe Standard Calibration Curve 8.45 26.9 30 ppb - 1200 ppb 3.50 11.1 12 ppb - 1200 ppb 24.6 78.3 30 ppb - 1200 ppb 108 345 60 ppb - 900 ppb* 82.3 262 60 ppb - 1200 ppb 33.9 108 30 ppb- 1200 ppb MDL/LOQ values in rat,bovine, and monkey liverwere not statisticalldyetermined. Two curves in each of these-matriceswere extractedand analyzed with the rabbitlivercurves to determine equivalence. Responses in the rat,bovine, and monkey livercurves were equivalent to the rabbitresponses, therefore,their'i\,IDaLnd LOQ will be assumed to be equivalentto those values as determined for the rabbitliver. Refer to LOQ Summary and MDL studyin ETS-8-6.0 & 7.0-V-1forfurtherinformazion * ETFOSE-OH estimatesonly forMDL and LOQ. Did not meet criterifaorvalidation. Compound: PFO S Liver matrix I Prepared Range of LCR from I range of average (pave curve standards curve (ppb)(ng.,-L)(ppb)(ng/mL) pb Range of LCR from Ranizrof I w std low std '@iighstd curve curve (ppb)(ngiml) (pr)(bn)g/mL) (ppbC)U(rn'gv/eLm) Rabbit 6 19 1137 - 12 - 1200 12- 1200 6-300 12-300 60- 1200 LCR from high std (ppcurve b) (ng/mL) 60- 1200 Compound: PFOSA Liver matrix Prepared Range of LCR from Range of I.CR from Range of LCR from range of average ave curve low std low std high std high std standards curve curve curve carve curve (pob)(ng/-L) (ppb)(ngi'rnL()ppb)(ng/mL)(ppb)(ng.,'mL()Dpb)(ng/mL) (;)c)(bn)g!mL) (ppb)(ngiml) [Rabbit 6.19 - 1237 12 - 1200 1 12- 1200 12 - 300 12-300 60- 1200 60- 1200 Compound: PFOSAA Liver matrix Prepared Range of LCR from Range of LCR from Range of LCR from range of average ave curve low std low std high std high std standards curve curve curve cur-ve curve (ppb()rig/-L)(ppb()ng/-L)(ppb)(ng/mL)(ppb)(ng/mL)(P:)(oi)lgiml)@ppb)(ng,'-L) (ppb)(ng/mL) i Rabbit 6.16 - 1232 12 - 1200 30- 1200 30-900 60-900 NI'A NIA L -1 1 Attachment B: I@IDL,LOQ Values 3M Environmental Laboratory ETS-8-6.0 ExtractionofPFOS from Liver Page 12 of 16 Page 55 3m Medical Department Study: T-6295.7 Recort No. FAC7 TCX-030 laboratory Request Number-U2279 Compound: ETFOSE-OH FLiver Prepared range of Range of average matrix standards curve (ppb)(ng/-L) (ppb)(ngiml) LCR from ave curve (ppb)(ng/mL) Range of LCR from low std low std curve curve (ppb)(ng,'mL) (ppb) (ng@mL) F@,b7bit 6.17- 1235 31 -900 31 -900 NiA N/A .1 1 Range of high sid curve (ppb)(ng'mL) N/A LCR from high std curve b) (ng/,@rL) N,A Compound: PFOSEA Liver matrix I Prepared range of standards (ppb)(ng/TnL) Range of LCR from Range of LCR from average ave curve low std low std curve curve curve (ppb)(ng/TnL) (ppb)(ng/rnL) (ppb)(ng,'M[.)(ppb) (ng/mL,l Range of high std curve (ppb)(ngi-L) LCR from high std curve (ppb)(ng/mL) Rabbit 6.17-1235 31 - 1200 31 - 1200 N/A N/A I NI'A N/A Compound: M556 Prepared Liver range of matrix standards (ppb)(ng/-L) Range of averace curve (ppb)(ng/mL) LCR from ave curve (ppb)(ng/mL) Range of LCR from low std curve low std curve (ppb)(ng/mi-) (p,@, Rabbit 6.17 - 1235 31 - 1200 60- 1200 N/A N,/A Range of LCR from high std high std curve curve p,b)(ng/ml,) (ppb)(ng/-L) Nl,'A N/A Attachmen- C: StandardCalculations ETS-8-6.0 Extraction ofPFOS from Li,.,er 3M Environmental Laboratory Pave 13 of 14 Page S6 3m me--@@-cal Departrrent Study: T-6295.7 Report No. FA----TOX-030 la]DoraL'ory Request Number-U2279 Ion PairStandard Curves - Tissue Prep date(s): Analyte(s): S2mple matrix: Method/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Standard number: Equipment number: Finalsolventand TN: Blank liver/identirier: Actual concentrations of standards in the FC mix I PFOS PFOSA PFOSAA ETFOSE PFOSEA Std conc Std conc Std conc Std conc Std conc ug/mL ug/mL ug/mL ug/n-LL ug/niL 0.500 0.500 0,500 0.500 1 0.500 0.50 0.50 0.50 0.50 0.50 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.-500 0.500 0.500 0.500 0.500 1 0.50 5.00 5.00 5,00 500 5.00 5-00 5.00 5.00 5.00 5.00 5.00 1 5.00 5.00 5.00 500 50.0 i 50.0 50.0 50.0 .0 N1556 Std conc ug/mL 0.500 0.50 0.500 0.500 0.50 5.00. 5.00 5.00 50.0 Std conc All Am't spiked mL 0.002 0.04 o@oto 0,020 0.40 0.010 0.020 0.030 0.004 AifDensity 9 0.167 1 0.167 0.167 0.167 0.167 0.167 0.167 i Calculated concentrations of standards in the sample matrix PFOS Final conc ng,,g 5.99 12.0 PFOSA Final I conc ng/g 5.99 12.0 PFOSAA Finalconc ng/g 5-99 12.0 ETFOSE Final conc nglg 5.99 12.0 PFOSFA M556 Final Final Std conc conc I conc ng/a ngjg ng,,c, 5.99 5.99 1 12.0 12.0 29.9 29.9 29.9 29.9 29.9 29.9 59.9 59.9 59.9 59.9 59.9 59-9 120 120 120 120 1 120 120 299 299 299 299 1 299 299 599 599 599 898 898 898 1198 1198 119-8 599 1 599 898 1198 1198 599 898_ 1198 Surrogate Std conc np-/riil. i 100 All A m't spik-ed ML 0,(J05 Surrogate Finalconc ng/mL 0.500 Validated ranges - approximate concentrations I Liver Rabbit ovine Rat PFOS PFOSA 5-1000 ppb 5-1000 ppb Estimatesonly,use rabbitvalues. Estimatesonly,use rabbitvalues. @AA@ 5-1000 ppb Nionkey Fstimatesonly.use rabbitvalues. ETFOSE-OH 1 5-1000 ppb POAA PFOSE,K 5-1000 ppb 1 5-10 pb A,tachment C: StandardCalculations ETS-8-6.0 Extractionof PFOS froniLi%-er 3M Enii4-ronmental Laboratcry Page 14 of 14 Page 57 3m Medical Department Study: T-629S.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 3M ENVIRONMENTAL LABORATORY METHOD A-NALYSIS OF POTASSIUM PERFLUOROOCTAINESULFONATE OR OTHER FLUOROCIMMICALS IN LIVER EXTRACTS USING HPLC-ELF,CTROSPRAY/MASS SPECTROMETRY Nlethod Number: ETS-8-7.0 Author: Lisa Clemen, Glenn Langenburg Approved By: AdoptionDate: 07)Z2-li@ Revision Date: NA Laboratory N@anager - i@@ Group Leader Technical Reviewer Date -@/ Ili Date o(Il. Date 1.0 SCOPE AND APPLICATION I -I Scope: This method is for the analysisof liverextractsfor fluarochemicalsurfactantsusing HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemical surfactantsor other fluorinatedcompounds, or other ionizablecompounds. 1.3 Matrices: Rabbit,rat,bovine, monkey liver,or other tisslieass designated in the ','alidatiori report. Word 6,'9-5 3M Environmental ETS-8-7.0 Analvsisof LiverExtractUsiii,E-S,/'vlS Laboratory Page I of 10 Page 58 3m Medical DeiDartment S@,-,dy: T-6295.7 Report No. FACT TOX-030 laboratc--y Request Number-U2279 2.0 SUM@',IARYOF NIETHOD 2.1 This method descr-ibesthe analysisof fluorochemical surfactantsextractedfrom liverusing HPLC-electrospray/mass spectrometry,or similarsystem as appropriate.The analysisis performed by monitoring a singleion characten*stiocf a particularfluorochemical,such as the per fluorooc tanesuI-,"onat(ePFOS) anion,m/z = 499. Additionally,samples may be analyzed using a tandem mass spectrometer to furtherverifythe identityof a compound by detecting dau2fiterions of the selectedparent ion. 3.0 DFFINITIONS 3.1 Atmospheric Pressure Ionization (A.Pl):The Micromass Quattro 11triplequadr-upole systems allow for variousmethods of ionizationby utilizingvarioussources,probes,and interfaces.These include but arenot limitedto:ElectrosprayIonization(ESI),Atmospheric Pressure chemical ronization(A.Pcl),Thermospray, etc. The ionizationprocess in these techniques occurs at atmospheric pressure(i.e.not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionizationperformed at atmospheric pressure,whereby ions in solutionare transferredto the gas phase via tiny charged droplets. These charged droplets are produced by the applicationof a strong electricalfield. 3.3 Mass Spectrometrv, Mass Spectrometer (iMS),Tandem Mass Spectrometer (MS/MS): The API Quattro 11 triplequadrupole mass spectrometer isequipped with two quadrupole mass selectivedetectorsand a collisioncell. Ions are selectivelydiscriminatedby mass to charge ratio(mlz) and subsequently detected.A singleMS may be employed forion detectionor an ion may be selectedin the firstquadrupole, fragmented in the collisioncell, and these fragments may be analyzed in the second quadrupole. 3.4 Conventional vs.Z-spray probe interface:Th'e'latesmtodels of Micromass Quattro Il triplequadrupole (post 1998) utilizea "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture.In the conventional conformation itisaimed directlvat the cone aperture,afterpassin,,t,hrough a tortuouspathway in the counter electrode.Though the configurationisdifferentt,he methods of operation,clcaning,and maintenance are the same. However, Z-spray components and conventional components arc not compatible with one another,but only with similarsystems (i.e.Z-spray components are compatible with other Z-spray systems,etc.) 3.5 Mass Lynx Software: System software desiped forthe specificoperationof theseQuattro 11triplequadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar.For more detailsreferto the manual specificto the instrument (Micromass Quattro Iltriplequadrupole NlassLynx or MassLynx NT User's Guide). 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use caution with the volta0ge cables for the probe. 1,@'heenngIa'@g1e1:d1 , the probe employs a voltage of approximately 5000 Volts. ETS-8-7.0 Aiial),soifsLiverExtractUsing,ES/i'vlS 3M Env-'@rc)nment@al Laborato--y Page 2 of 10 Page 59 3m Medical Department S-.udy: T-6295.7 Report No. FAC- TOX-030 laboratory Request Number-U2279 4.1.2 Vvlhenhandling samples or solventswear appropriateprotective-,lovese,yewear, and clothinz. 4.2 Cautions: 4.2.1 Operate the solventpumps below a back pressureof 400 bar (5800 psi).Ifthe back pressureexceeds 400 bar,theHPI 100 willinitiataeutomatic shutdown. 4.2.2 Do not run solventpumps to dryness. 5.0 INTERFERENCES 5.1 To minimize interferencewshen analyzingsamples, Teflon shallnot be used forsample storageor any p4rtof instrumentatiotnhatcomes in contactwith the sample or extract. 6.0 EouiPMENT 6.1 Equipment listedbelow may be modifiedin ordertooptimizethe system.Document modificationsin the raw data as method deviations. ally 6.1.1 6.1.2 Micromass Quattro 11triplqeuadrupoleMass Spectrometerequipped with an electrosprayionizationsource. HPI 100 low pulsesolventpumping system, solventdegasser,column compartment, and autosampler 7.0 SUPPLIES AND MATERIALS 7.1 Supplies- 7.1.1 7.1.2 High purityZ-radeairregulatedto approximately 100 psi(house airsystem) HPLC analyticalcolumn, specificsto be determined by the analystand documented in the raw data 7.1.3 Capped autovialsor capped 15 ml centrifugetubes 8.0 REAGENTS AND STAINDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milll_QTIwater (ASTM type1),allwater used in thismethod shouldbe ATSM type 1,or equivalent,and be providedby a Milli-QTOC Plussystem or other vendor 8.1.3 Ammonium acetate,reagentgrade or equivalent 8.1.3.1 When preparingdifferenatmounts than thoselisteda,djustaccordingly. 8.1.3.2 2.0 mM ammonium acetatesolution:Weigh approximately0.300 g animonium acetateP.our intoa 2000 mL @,olumetriccontainercontaiiiicny 2000 mL Milli-Q" water,mix untilallsolidsaredissolved.Store atroo* m temperature. ETS-8-7.0 AnalysisofLiverExtractUsing ES/'YIS Page 3 of 10 3M Environmen+.-al Laboratory Page 60 3m Medical Department Study: T-6295.7 ReTDort No. FACT TOX-030 laboratory @Request Number-U2279 8.2 Standards 8.2.1 Typically two method blanks,two matrix blanks, and eighteen matrix standards are prepared du@ing the extractionprocedure. Ref,-rto ETS-8-6.0. 9.0 SAMPLE HA,4DLING 9.1 Fresh matrix standards are prepared with each analysis. Extractedstandards and samples are stored in capped autovialsor capped 15 ml centrifugetubes untilanalysis. 9.2 Ifanalysis willbe delayed, extractedstandardsand samples may be stored at room temperature, or refrZiP-cratedatapproximately 4' C, untilanalysiscan be performed. 10.0 ()UALITY 10.1 Method CONTROL Blanksand Matrix Blanks 10.1.1 Solvent blanks, method blanks, and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank priorto each calibrationcurve. 10.2 imatrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effecton the recovery efficiency. 10.2.2 Matrix spike duplicatesare prepared and analyzed to measure the precisionand the recovery for each analyte. 10.2.3 Analyze a matrix spikeand matrix spikeduplicateper fortysamples. With a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spikeduplicateconcentrationswillfallinthe mid-range of the initiaclalibrationcurve. Additionalspike concentrationsmay fallin the lowrange of the initiaclalibrationcurve. 10.3 Continuing Calibration Checks 10.3.1 Continuing calibrationverificationasre analyzed to verifythe continued accuracy of the calibrationcurve. 10.3.2 Analyze a mid-rangc calibrationstandardevery tenthsample, with a minimum of one per batch. 11.0 CALIBRATION AND STANDARDIZATION 11.1 A-nalyze the extractedmatrix standardspriorto and following each set of sample extracts. The average of two standardcurves willbe plottedby linearregression(y = mx - b), weighted I/x,not forcedthrough the origin,using MassLynx or other suitablesoftware. 11.2 Ifthe curve does not meet requirements perforrtrioutine maintenance or reextractthe standard cur-ve(ifnecessary) and reanalyze. 3M Environmental ETS-8-7.0 Analysis of Liver ExtTact Using ES/',IvIS Laboratory Page 4 of 10 Page 61 3m Med@-cal Denartrrer@t study: T-6295.7 Report No. FA--T TOX-030 laboratory Request Num-ber-U2279 11.3 For purposes of accuracy when quantitatinlgow levelsof analyte,itmay be necessaryto use the low end of thecalibratiocnurve ratherthan the fullrange of the standardcur-ve. Example: when attemptingto quantitateapproximately 10 ppb of analyte,generatea calibrationcurve consistingof thestandardsfrom 5 ppb to 100 ppb ratherthan the full range of the cur-ve(5 ppb to 1000 ppb). This willreduce inaccuracyattributetdo linear regressionweighting of high concentrationstandards. 12.0 PROCEDURES 12.1 Acquisition Setup 12.1.1 Set up the sample list. 12.1.1.1 AssIL7na sample listfilename using MO-DAY-last digitof year-increasinc, letterof the alphabet startingwith a 12.1.1.2@Lssign a method (MS file)for acquiring 12.1.1.3 Assign an HPLC program (Inletfile) 12.1.1.4Type in sample descriptionsand vialpositionnumbers 12.1.2 To createa method clickon method inthe Acquisitioncontrolpanelthen mass spectrometerheadingsand selectSIR (SingleIon Recording) or MT;LM (Multiple Reaction Monitoring).Set IonizationMode as appropriateand mass to 499 or other appropriatemasses. A fullscan isusuallycollectedalong with the SIRS. Save acquisitionmethod. IfMS/MS instrumentsare employed, additionalproduct ion fragmentationinformationmay be collected.Refer toMicromass MassLynx GUIDE TO DATA ACQUISITION foradditionalinfor-matioannd MRI@VI. 12.1.3 Typically the analyticablatch run sequence begins and ends with a setof extracted matrix standards. 12.1.4 Samples are analyzedwith a continuingcalibratiovnerificatioinnjectedstandard afterevery tenthsample. Solventblanks should be analyzed periodicalltvo monitor possibleanalytecarryoverand arenot consideredsamples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample trayaccordingto the sample listprepared in Section12.1.1. 12.2.2 Set-up the HP II00/autosampleratthe followingconditionsor atconditionsthe analystconsidersappropriateforoptimal response.Record actualconditionsin the instrument logbook: 12.2.2.1Sample size= 10 @iL injection 12.2.2.2Inj'ect/sampl=e 1 12.2.2.3Cycle time = 9 minutes ETS-8-7.0 AnalysisofLiverExtractUsing ES@NTS 3M Environme--i--alLaboratory Pa@!e5 of 10 Page 62 3m Medical Department S::@,-,dTy-:6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.2.2.4Solvent ramp conditions Time MEOH 0.00 min. 40% 1.0 min. 40% 4.5 min. 95% 6.5 min. 95% 7.0min. 40% 9.0 mi. 40% 2.0 mNI Ammonium acetate 60% 60% 5% 5% 60% 60% 12.2.2.5Press the"Start"button. 12.3 Instrument Set--up 12.3.1 Refer to ETS-9-24.0, "Operationand Maintenance of the Micromass Quattro Ii TripleQuadrupole Mass SpectrometerFittedwith an Atmospheric Pressure IonizationSource,"formore details.- 12.3.2 Check the solventlevelinreservoirsand refililfnecessary. 12.3.3 Check thestainlessteelcapillaryatthe end of the probe. Use an eyepiece tocheck thetip.The tipshouldbe flatwith no jagced ed-es. Ifthetipisfound to be unsatisfactor-v,disassemble the probe and replacethestainlesssteelcapillar@'. 12.3.4 Turn on the nitrogen. 12.3.5 Open the tune page. Clicks on operateto initiatseource block and desolvation heaters. 12.3.6 Open the InletEditor. 12.3.6.1Set HPLC pump to "On" 12.3.6.2 Set the flow to 10 -500 uL/min or as appropriate 12.3.6.3 Observe dropletscoming out of the tipof the probe. A finemist should be expelledwithno nitrogenleakingaround thetipof theprobe. Readjust the tipof the probe ifno mist isobserved 12.3.6.4Allow to equilibratfeorapproximately 10 minutes. 12.3.7 The instrumentuses theseparameters atthe followingsettings.These settingsma,, change inorder tooptimize theresponse: 12.3.7.1 Drying gas 250-400 liters/hour 12.3.7.2 ESI nebulizinC>ggas 10-15 liters/hour 12.3.7.3HPLC constantflow mode flow rate10 - 500 @iL/min 12.3.7.4 Pressure<400 bar (Thisparameter isnot set,itisa guide to ensure the HPLC isoperatingcorrectly.) 12.3.7.5Source block temperature150" 12.3.7.6Desolvation temperature2500 3M Environmental ETS-8-7.0 AnalysisofLiverExtractUsing ESii'v[S Laboratory Pa2c 6 of 10 Page 63 3m Medical Department S-.udy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.3.8 Printthe tur;epage, with itsparameters,and storeitin the study binder ,vitha copy taped into the instrument log. 12.3.9 Click on startbutton inthe AcquisitionControl Panel (thismay vary among MassLynx versions,referto appropriateMassLynx User's Guide). Ensure startand end sample number includesallsamples to be analyzed. 13.0 DATA ANALYSFS 13.1 Calculations: AND CALCULATIONS 13.1.4 Calculate matn*x spike percent recoveries using the following equation: % Recovery Observed Result - Background Result x 100 Expected Result 13.1.5 Calculatepercent differenceusing the following equation: % Difference Exi)ectedConc. -Calculated Conc. x 100 Expected Conc. 13.1.6 Calculateactualconcentrationsin matn'x (@tg0/-): (nizof PFOS calc.from std.Curve.x DilutionFactor) x I ag (InitiaWleight of Liver 1000 ng FinalVolume (mL) 14.0 METHOD PERFORMA.NCE 14.1 Method Detection Limit (MDL) and Limit of Quantitation(LOQ) aremethod, analyte,and matrix specific.Refer to ETS-8-6.0, Attachment B fora liiingof currentvalidatedMDL and LOQ values. 14.2 Solvent Blanks, Method Blanks and Matrix Blanks 14.2.1 Solvent blanks, method blanks,and matrix blanks must be below the lowest standard in the calibrationcurve. 14.3 Calibration Curves 14.3.1 The r2value for the calibrationmust be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percentrecoveriesmust be within 30% of the spiked concentration. 14.5 Continuing Calibration Verification 14.5.1 Continuing calibrationverificatiopnercentrecoveriesmust be within +-300/'oof the spiked concentration. 14.6 If cn'terialistedin the method performance sectionare not met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst.Document allactionsin the appropriatelogbook. 3M Environmental ETS-8-7.0 Analysisof LiverExtractUsing ES,"@IS Laboratory Pa_ge7 of 10 Page 64 3m Medical Denartment Study: T-629S.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 14.7 Ifdata areto be reportedwhen performance criterihaave not been met, the data must be footnotedon tablesand discussedinthetextof thereporl. 15.0 POLLUTION PREVENTION AND WASTE MANAGENIENT 15.1 Samole extractwaste and flammable solventisdisposed inhigh BTU containers,and glass pipettewaste isdisposed inbroken glasscontainerslocatedin the laboratory. 16.0 RECORDS 16.1 Each page generated fora studymust have the followinginformationincludedeitherin the header or hand writtenon the page: studyor projectnumber, acquisitiomnethod, integratiomnethod, sample name, extractiodnate,dilutionfactor(Ifapplicable)a,nd analyst. 16.2 Print the tune page, sample lista,nd acquisitionmethod from MassLynx to include in the appropriatestudy folder.Copy thesepages and tape intothe instrumentrunlog. 16.3 Plot the calibratiocnurve by linearregressionw,eighted l,'xt,hen printthesegraphs and storein the study folder. 16.4 Printdata integrationsummary, integratiomnethod, and chiomatograms from MassLynx and storein the study folder. 16.5 Summarize datausinC,)-suitablesoftware(Excel 5.0+)and storeinthe study folder,referto Attachment A for an example ofa summary spreadsheet. 16.6 Back up electronicdata to appropriatemedium. Record in studynotebook the filename and locationof backup electronicdata. 1'10' TABLEs DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA I'iI. Attachment A: ETS-8-7.0 Data summary spreadsheet 18.0 REFERENCES 18.1 FACT-M-2. Compounds 1,"Extractionof Potassium Perfluorooctanesulfonatoer Other Fluorochemical ftom Live,-forAnalysisUsing HPLC-Electrospray/Mass Spectrometry" 18.2 ETS-9-24.0, "Operation and Maintenance of the Micrc)massAtmospheric Pressure Ionization/MassSpectrometer QuattroIItriplequadrupole Systems" 18.3 The validationreportassociatedwith thismethod isETS-8-6.0 & 7.0-V-1 19.0 AFFECTED DOCIJMENTS 19.1 ETS-8-6.0, "Extractionof Potassium Perfluorooctanesulfonatoer Other Fluorochemical Compounds from Liver or Fluid forAnalysisUsing HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 Analysisof LiverExtractt:,sinEuSii%[S 3M Environmental Laboratcry Page 8 of 10 Page 65 3m Medical Department Study: T-629S.7 Report No. FACT TOX-030 laboratory Request Number-U2279 20.0 REVISIONS Revision Number Reason For Revision Revision Date 3M Environmental ETS-8-7.0 Analysisof LiverExtractUsing ESiN/IS Laboratory Pa c!e9 of'10 Page 66 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Laboratory Study Study: Test Material: Matrix,'FinaSlolvent: Method/Revision: AnalyticalEquipment System Number: InstrumentSoftware/Version: Filename: R-Squared Value: Slope: Y Intercept-. Date of Extraction/Analyst@. Date ofAnalysis/Analyst: Group Dose s2mpie# concentration ng/g lniti2Wlt. 9- Dilution Factor FinalConc. ug/g -STo-p-v-.-ra-@-ie-ni-nTeraorm regressionequation. Group/Dose: Taken from thestudyfolder. Sample#: Taken from thestudyfolder. Concentration (ngig):Taken from theMassLynx integratiosnummary. InitiaWlt. (g):Taken from thestudyfolder. DilutionFactor: Taken from thestudyfolder. FinalConc. (tig/g):Calculatedby dividingtheinitiavlolume from theconcentration Attachment A: Sunurary Spreadsheet ETS-8-7.0 Analysisof LiverExtractUsing ES"%IS 3M Environmental Laboratory Page 10 of 10 Page 67 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M ENVIRONME-N'TAL LABORATORY METHOD EXTRACTIOISL FLUOROCHEMICAL OF POTASSIUM PERFLUOROOCTANIESULFONATE COMPOUNDS FROM SERUi'vl FOR ANALYSIS ELECTROSPRAY/MASS SPECTRONIETRY OR OTHER USING HPLC- Method Number: ETS-8-4.1 Adoption Date: 031'01/99 Author: Lisa Clemen, Glenn Langenburg Revision Date: Approved By: Laboratory Manager Datc Group Leader Technical Reviewer Date Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method isforthe extractionof potassium perfluorooctanesulfonate(PFOS) or other fluorochemical compounds from serum. 1.2 Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 Matrices: Rabbit, rat,bovine, monkey, and human serum or other fluids as designated in the validationreport. Word 6/95 3M Environmental Laboratory ETS-8-4.1 ExtTactionof PFOS from Seriini Page I of 14 Page 68 3m Med@-cal idepart-nert Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0SUNINIARY OF METHOD 2.1 This method descr-ibetsheprocedureforextractingpotassiumpertluorooctanesulfonate (PFOS) or otherfluorochemicaslurfactantfsrom serum,or otherfluidsu,singan ion pairingreagentand methyl-tert-buteytlher(MtBE). Inthismethod,seven fluorochemicalswere extractedP:FOS, PFOSA, PFOSAA, ETFOSE-OH, PFOSEA, M556, and surrogatestandard(see3.0Definitions).An ionpairingreagentisadded to the sample and theanalyteion pairispartitioneidntoMTBE. The MTBE extractis removed and put onto a nitrogenevaporatoruntildry. Each extractisreconstituteidn 1.0 mL of methanol,then filteretdhrougha 3 cc plastiscyn'n,-aettachedtoa 0.2 lAm nylon filteirntoglassautovials. 2.2 These sample extractsareanalyzedfollowingmethod ETS-8-5.1 or otherappropn'ate methods. 3.0 DEFINITIONS 3.1 PFOS: perfluorooctanesulfonat(eanionof potassiumsalt)C,F,IS033.2 PFOSA: perfluorooctanesulfonylamideC,F,.,SO.NE,, 3.3 PFOSAA: perfluorooctanseulfonylamido(ethyl)acetaCt,eF,,SO,N(CH,CH3)CH,CO,- 3.4 ETFOSE-OH: 2(N-ethylperfluorooctasnuelfanamido)-ethyallcohol C,F,IS02N(CH2CH3)CH,CH,OH 3.5 PFOSEA-. perfluorooctanseulfonytethylamideCIF,7SO,N(CH,CH,)H 3.6 M556: C,F,,SO,N(H)(CH,COOH) 3.7 Surrogatestandard:IH-IH-2H-2H perfluorooctanseulfonicacid 4.0 WARNINGS AND CAUTIONS 4.1 Health and safetywarnings 4.1.1 Use universalprecautionse,specialllyaboratorycoats,-oggles,and gloves when handlinganimal tissuew,hich may containpathogens. 5.0 INTERFERENCES 5.1 There areno interferenceksnown at thistime. 6.0 EouipNIE,14T 6.1 The followingequipment isusedwhileperformingthismethod. Equivalentequipment is acceptable. 6.1.1 Vortex mixer,VV@R, Vortex Genie 2 6.1.2 CentrifugeM,istral1000 orIEC 6.1.3 Shaker,Eberbach or VWR 3M Env-'-ronmental Laboratory ETS-8-4.1 ExtractionofPFOS from Serum Page 2 of 14 Page 69 3m Medical Department Study: T-629S.7 Report No. FACT TOX-030 laboratory Request Number-U2279 6.1.4 Nitrogen evaporator,Organomation 6.1.5 Balance ( 0.100 g) 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Eppendorf ordisposable pipettes 7.3 Nalgene bottles,capable of holding 250 mL and I L 7.4 Volumetn'c flasks,class,type A 7.5 I-CHEM vials,glass,40 mL glass 7.6 Centrifuge tubes,polypropylene, 15 mL 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0 mL 7.9 Syringes, capable of measuring 5 4L to 50 pLL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic,3 cc 7.12 Syringe filtersn,ylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovialsand caps 7.15 Crimpers Note: Prior to using glassware and bottles,rinse3 times with methanol and 3 times with Milli_QTM water. Rinse syringesa minimum of 9 times with methanol, 3 rinsesfrom 3 separate vials. 8.0 REAGENTS AND STANDARDS 8.1 Type I reagent grade water, Milli-QTIAor equivalent;all,,vateursed in t.@ilmsethod should be Milll_QT',wlater and may be provided by a Milli-Q TOC plUSTm System 8.2 Sodium hydroxide (iNaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA),Kodak or equivalent 8.4 Sodium carbonate (Na@CO,), J.T.Baker or equivalent 8.5 Sodium bicarbonate (NaHCO,), J.T.Baker or equivalent 8.6 Methyl-T-Butyl Ether,Omnisolv, glassdistilleodr HPLC crade 8.7 Methanol, Omnisolv, glassdistilleodr HPLC grade 8.8 Serum or blood, frozen from supplier 8.9 Fluorochemical standards 8.9.1 PFOS (3M SpecialtyChemical Division),molecular weight= 539 8.9.2 PFOSA (3M SpecialtyChemical Division),riioleculawreight = 499 ETS-8-4.1 Ex=ction ofPFOS from Scrum Paae 3 of 14 3M Environmental Laboratory Page 70 3m Medical Departmen@- S--udy: T-6295.7 Report No. FACT TOX-03C laboratory Request Number-U2279 8.10 8.11 8.9.3 8.9.4 PFOSAA (3M SpecialtyChemical Division),molecular weight = 585 ETFOSE-OH (3M SpecialtyChemical Division),molecular wel,-ht= 5'10 8.9.5 8.9.6 8.9.7 PFOSEA (3M SpecialtyChemical Division),molecular we@-,.h=t 527 M556 (3M SpecialtyChemical Division),molecular weight = 55 /' Surrogate standard:4-H, perfluorooctanesulfonicacid (I-H, I-H, 2-H, 2-H C,F,,SO,H) molecular weight = 428 8.9.8 Other fluorochemicals,as appropn'ate Reagent preparation NOTE: When preparing largervolumes than listedin reagent,standard,or surrogate preparation,adjustaccordingly. 8.10.1 10 N sqdium hydroxide (NaOH): Weigh approximately 200 g NAOH. Pour into a 1000 mL beaker containing500 mL Milli-Q" water,mix untilallsolidsare dissolved. Store in a I L Nalgene bottle. 8.10.2 1 N sodium hydroxide (NaOH): Dilute 10 N NAOH 1:10. Measure 10 mL of 10 N NAOH solutionintoa 100 mL vol'umetricflaskand diluteto volume using Milli-Q" water. Store in a 125 mL Nalgene bottle. 8.10.3 0.5 M tetrabutylammonium hydrogen sulfate(TBA): Weigh approximately 169 g of TBA intoa I L volumetriccontaining 500 mL MilliT.-1 Qwater.Adjust to pH 1.0using approximately 44 to 54 mL of 10 N NAOH (While adding the lastmL of NAOH, add slowly because the pH changes abru-otly)D.iluteto volume with MillI_QTM water. Store in a 1 L Nalgene bottle. 8.10.3.1 TBA requiresa check pn'orto each use to ensure pH = 10. Adjust as needed using I N NAOH solution. 8.10.4 0.25 M sodium carbonate/sodium bicarbonatebuffer(Na,CO,,'NaHC03): Weigh approximately 26.5 g of sodium carbonate (NaCO,) and 2 1.0- of sodium bicarbonate(NaHC03) intoa I L volumetric flaskand bring to volume with MilliQTm water. Store in a I L Nalgene bottle. Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards,as appropriate.Multicomponent fluorochemical standards are acceptable (forexample, one working standard solutioncontaining 1.00ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.11.3 Weigh approximately 100 mg of PFOS into a 100 mL volurfletn'fclaskand record the actualweight. 8.11.4 Bring to volume with methanol for a stock standardof approximately 1000 ppm (4g/mL). 8.11.5 Dilute the stock solutionwith methanol fora working standard I solutionof approximately 50 ppm. 8.11.6 Diluteworking standard I with methanol fora working standard2 solutionof approx. 5.0 ppm. 3M Env-. ronmental Laboratory ETS-8-4.1 Extractionof PFOS from Serutii Page 4 of 14 Page 71 3m medical Department Study: T-6295.7 Ret,,ort No. FACT TOX-030 laboratory Request Numbe---U2279 8.12 8.11.7 Dilute working standard I with methanol fora working standard3 solutionof approx. 0.50 ppm. Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 mg of surrogatestandard I-H,I-H, 2-H, 2-H, C,F,3SO,H into a 50 mL volumetric flaskand record the actualweight. 8.12.2 Bn'ng to volume with methanol fora surrogatestock of approximately 1000- 1200 PPM. ' 8.12.3 Prepare a surrogateworking standard. Transferapproximately I mL of surrogate stock to a 10 mL volumetric flaskand bn*ng to volume with methanol for a working standard of 100 ppm. Record the actualvolume transferred. 9.0 SAMPLE HANDLING 9.1 All samples are received frozen and must be kept frozen untilthe extractionis performed. 9.2 Allow samples to thaw to room temperature pn*orto extraction. 10.0 OUALITY CONTROI, 10.1 Solvent Blanks, Method blanks and matrix blanks 10.1.1 An aliquotof 1.0mL methanol isused as a solvent blank. of Milli_QTII 10.1.2 Extracttwo 1.0mL aliquots water followine thisprocedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquotsof the serum following thisprocedure and use as matrix blanks. See 11.1.4. 10.2 Matrix spikes 10.2.1 Prepare and analyze matn'x spike and matrix spike duplicatesamples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst,usuallythe control matrix received with each sample set. 10.2.3 Expected concentrationswillfallin the mid-range of the initiaclalibrationcurve. Additional spikes may be includedand may fallin the low-range of the initial calibrationcurve. 10.2.4 Prepare one matn'x spike and matrix spike duplicateper 40 samples, with a minimum of 2 matrix spikesper batch. 10.3 Continuing calibration checks 10.3.1 Prepare continuing calibrationcheck samples to ensure the accuracy of the initial calibrationcurve. 10.3.2 Prepare, at a minimum, one continuing check per group o@'10 samples. For example, ifa sample set= 34, four checks are prepared and extracted. 10.3.3 Prepare each continuing calibrationcheck from the same rnatrixused to prepare the initiaclur-ve. 3M Environmental Laboratory ETS-8-4.1 Extraction ofPFOS from Serum Page 5 of 14 Page 72 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Rec-ruest Number-U227@-@ 10.3.4 The expected concentrationswillfallwithin the mid-range of the initial calibrationcurve. Additionalspikesmay be included thatfallinthe low-range of the initiaclalibrationcurve. This isnecessary ifthe analystmust quantitateusing only the low end of thecalibrationcurve (forexample, 5 ppb - 100 ppb, rather than 5 ppb - 1000 ppb). 11.0 CALIBPATION AND STANDARDIZATION 11.1 Prepare matrix calibrationstandards 11.1.1 Transfer I mL of serum to a 15 mL centrifugetube. 11.1.2 If most sample volumes are lessthan 1.0 mL, extractstandards with matrix volumes equal to the sample volumes. Do not extractlessthan 0.50 mL of matrix. Rec-ord each sample volume on the extractionsheet. 11.1.3 While preparing a totalof twenty aliquotsin 15 mL centrifugetubes,mix or shake between aliquots. 11.1.4 Two I mL aliquots,or other appropn'atevolume, serve as matrix blanks. Typically use the standard concentrdtionsand spiking amounts listedin Table 1, at the end of thissection,to spike,in duplicate,two standard cur-ves,for a totalof eighteen standards,two matrix blanks,and two method blanks. 11.1.5 Refer to validationreportETS-8-4.0 & ETS-8-5.0-V-1, which liststhe working ranges and the Linear CalibrationRange (LCR) forcalibrationcurves. 11.1.6 Use Attachment D as an aid in calculatingthe concentrationsof the working standards. See Section 13.0 to calculateactual concentrationsof PFOS in calibrationstandards. 11.2 To each standard,blank, or continuing check, add appropriateamount of surrogate working standard for the concentrationto fallwithin the calibrationcurve ran2e 5 ppb 1000 ppb. 11.3 Extract spiked matn*x standards following 12.6-12.16 of thismethod. Use these standards to establisheach initialcurve on the mass spectrometer. 3M Environmental Laboratory ETS-8-4.1 Extractionof PFOS from Serum Page 6 of 14 Page 73 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Table I Approximate spiking amounts for standards and spikes Using 1.0 mL of matrix Working standard (approx.conc.) -- 4L Approx. finalconc. of analytein matrix - Blank 0.500 ppm 10 0.005 ppm 0.500 ppm 20 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 0.050 ppm 5.00 ppm 20 0.100 ppm 5Cr.0ppm 5 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 1.00 ppm 12.0 PROCEDURE 12.1 Obtain frozen samples and allow to thaw atroom temperature or in a lukewan-n waterbath. 12.2 12.3 Vortex mix for 15 seconds,then transfer1.0 mL or other appropriatevolume to a 15 mL polypropylene centrifugetube. Return unused samples to freezerafterextractionamounts have been removed. 12.4 Record the initiavlolume on the extractionworkshect. 12.5 Label the tube with the study number, sample ID, date and analystinitials.See attached worksheet fordocumenting the remaining steps. 12.6 Spike allsamples, includingblanks and standards,ready forextractionwith surrogate standard as described in 11.2. 12.7 12.8 Spike each matrix with the appropriateamount of standard as described in 11.1,or Table I in that section,forthe calibrationcurve standards.Also prepare matrix spikes and continuing calibrationstandards. Vortex mix the standardcurve samples, matrix spikesamples, and continuing calibration samples for 15 seconds. 12.9 Check to ersure the 0.5 M TBA reagentis at pH 10. Ifnot,adjustaccordingly. 12.10 To each sample, add I mL 0.5 M TBA and 2 mL of 0.25M sodium carbonate/sodium bicarbonate buffer. 12.11 Using an Oxford Dlsr)enser,add 5 mL methyl-tert-butylether. 12.12 Cap each sample and put on the shaker at a settingof 300 rpm, for 20 minutes. 12.13 Centrifuge for 20 to 25 minutes at a settingof 3500 rpm, or untillayersare well separated. ETS-8-4.1 Extractionof PFOS from Serum Page 7 of 14 3M Environmental Laboratory Page 74 3m Medical Department S--udy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.14 Label a fresh 15 mL centrifugetube with the same information as in 12.5. 12.15 Remove 4.0 mL o'-@,.hoerganic layerto thisclean 15 mL centrifugetube. 12.16 Put each sample on the analyticalnitrogen e@,,aporatourntildry, approximately I to hours. 12.17 Add 1.0 mL of methanol to each centrifugetube using a graduated pipette. 12.18 Vortex mix for 30 seconds. 12.19 Attach a 0.2 Am nylon mesh filterto a 3 cc syringe and transferthe sample to this syringe. Filterinto a 1.5 mL glassautovialor low-volume autovialwhen necessar-v. 12.20 Label the autovialwith the study number, animal number and gender, sample timepoint, matrix, finalsolvent,extractiondate,and analyst(s)performing the extraction. 12.21 Cap and store extractsat room temperature or at approximately 4 'C untilanalysis. 12.22 Complete the extractionworksheet, attached to thisdocument, and tape in the study notebook or include in study binder,as appropr-iate. 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations 13.1.1 Calculate actual concentrationsof PFOS, or other applicablefluorochemical, 'in calibrationstandards using the following equation: mL of standard x concentrationof standard /mL) mL of standard + mL of surrogatestandard+ initiamlatrix volume (mL) Final Concentration (@ig/mL)_of PFOS in matrix 14.0 METHOD PERFORMANCE 14.1 The method detectionlimit(NIDL) isanalyteand matrix specific.Refer toMDL report forspecificMDL and limitof quantitation(LOQ) values(seeAttachmentsB and C). 14.2 The followingqualitycontroslamplesareextractewditheachbatchof samplesto evaluatethequalityof theextractionand analysis. 14.2.1 Method blanks and matrixblanks. 14.2.2 Matrix spikeand matrixspikeduplicatesamples to determineaccuracyand precisionof theextraction. 14.2.3 Continuingcalibratiocnheck samples to deter-mintehecontinuedaccuracyof the initiaclalibratiocnurve. 14.3 Refer tosection14 of ETS-8-5.1 formethod performancecriteria. 15.0 POLLUTION PREVENTION AND WASTE MANAGEME14T 15.1 Sample waste isdisposedinbiohazardcontainersf,lammable solventwaste isdisposedin high BTU containersa,nd used glasspipettewaste isdisposedinbroken glasscontainers locatedinthelaboratory. ETS-8-4.1 Extractionof PFOS from Serum Page 8 of 14 3M Environmental Laboratory Page 75 3m Medical Department Study: T-6295.7 Reporz No. FACT TOX-C30 laboratory ReQ-uest Number-U2279 16.0 RECORDS 16.1 Complete the extractionworksheet attachedto thismethod, and tape in the study notebook or include in the 3-rin-study binder,as appropriate. 17.0 ATTACHMENTS 17.1 Attachment @, Extractionworksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibrationstandard concentrationworksheet 18.0 RIEFERENCES 18.1 The validation -reportassociated with thismethod is ETS-8-4.0 & 5.0-V-1. 18.2. FACT-M-3. 1, "Analysis of Serum or Other Fluid Extractsfor Flucrochemicals using HPLC-Electrospray Mass Spectrometry" 19.0 AFFECTED DOCUMENTS 19.1 ETS-8-5. 1, "Analysis of Serum or Other Fluid Extractsfor Fluorochemicals usincy HPLC-Electro spray Mass Spectrometry" 20.0 REviSIONS Revision Number I Reason For Revision Section 12.21 Changed to include sample storage atroom temperature. Section 12.13 Added the shaker speed. Section 12.17 Final volume is 1.0mL; not adjustedfor initiavlolumes less than 1.0 mL. Revision Date 04/02/99 3M Environmental Laboratory ETS-9-4.1 Extractionof PFOS from Senini Page 9 of 14 Page 76 3m Med4-cal Department study: T-6295.7 Report No. laboratorv Reauest ExtractioWnorkshect ETS-8-4.1 FACT TOX-030 Number-U2279 Studv Matrix Box @Vk/Day DateSpiked/Analyst ccv \,IS MSD surtogateStd approx. ppm actual ppm FC-Mix approx.0.5 pm actual PPM FC-Mix approx.5 ppm actual ppm F C:@@@ixt-approx.50 ppm actual PPM I Blank Std amount Serum Extr2ctionMethod Vortex IS see. PipetteMatrix Volume mL PipcttIe mL of 0.5M TBA, pH 10. pH Std@ # Pipctic2 mL of0.25Na2CO3/0.25M NaHC03 buffer Std.4 Dispense5 mL of methyl-t-butyelther TN-A- Shake 20 min. Shaker speed: Ccntrifu_g2c0-25 min. Remove a 4 mL aliquotoforganiclaver Ccntrifu_gsedced: Put on Nitro,ycnEvaDoratortodrvncss TempeTature: Add methanol Volume mL T-,N-A- Vortex30 sec. Filteursin,ga 3ec B-D svringewith a 0.24m f-litCTintoa 1.5mL autosamdievial Cont. Cal.Verificationsused same matrixas forstdcurve. mL D2te& Initials Attachment A 3M E-iv-Lronmen-@al Laboratory ETS-8-4.1 Extractionof PFOS from Serum Page 10 of 14 Page 77 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 MDL[LOQ values forrabbitserum Compound MDL LOQ Linear CalibrationRange (LCR) (ppb) (ppb) Approximate concentrationstobe used forpreparing the Standard CalibrationCurve PFOS 1.74 5.55 5 ppb - 1000 ppb PFOSA 1.51 4.79 5 ppb - 1000 ppb PFOSAA 3.46 20.5 5 ppb - 1000 ppb ETFOSE-OH 11.4 36.2 5 ppb - 1000 ppb M556 6.03 19.2 5 ppb - 1000 ppb PFOSEA 5.71 18.2 1 5 ppb - 1000 ppb MDULOQ values in rat,bovine,monkey, and human serum, and monkey plasma were not statistically determined. Two curves in each of thesematriceswere extractedand analyzed with therabbitserum curves to determine equivalence. Responses inthe rat,bovine,monkey, and human were equivalentto the rabbitresponses,therefore,theirMDL and LOQ willbe the same valuesas determined in rabbit serum. Please see LOQ Summary and MDL study inETS-8-4.0 & 5.0-V-I forfurtherinformation. Attachment B: iviDL/LOQ Sununary 3M Env@-ronmen*@-al Laboratory F-TS-8-4.1 Extractionof PFOS from Serum Page II of 14 Page 78 3m Medical Department Sz:udy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Compound: PFOS Prepared range LCR from Rabbit Serum of standards curve (ppb)(ng/r.,-L) (ppb) (ng/mL) FullRange 0.995-973 24.8-978 Low Curve 4.94-248 4.94-248 High curve 97.8-978 97.8-978 I/X 0.995-978 4.94-978 % Recoverv Range 83-108 85-104 85-106 94-111 Compound: PFOSA Prepared range Rabbit Serum of standards (ppb)(ng/mJ-) FullRange 0.993-976 LCR from curve (ppb) (ng/mL) 4.93-976 Low Curve 4.93-97.6 4.93-97.6 High cur-ve 1/X 24.8-976 0.993-976 24.8-978 4.93-976 % Recovery Range 88-103 87-105 93-102 94-103 Compound: PFOSAA Prepared range Rabbit Serum of standards (ppb)(ng/mL) FullRange 0.991-974 LCR from curve (ppb) (ng/mL) 24.7-974 Low Curve High curve 4 92 -247 - 49.2-974 9.74-247 97.4-974 0.991-974 9.74-974 % Recovery Range 81-111 97-107 85-108 95-115 RSD Range 4.67-11.0 5.34-12.0 4.84-9.80 4.60-10.5 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.. RSD Range 4.18-10.6 6.38-21.8 4.33-12.5 4.11-23.2 A-,tachmenBt: MDLil-OQ Summary 3M Environmental Laboratory ETS-8-4.1 Extractioonf PFOS from Serum Page 12 of 14 Page 79 3m Medi-cal Department S--udy: T-629S.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Compound: ETFOSE-OH Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 -976 4.93-97.6 49.3-976 0.993 -493 LCR from curve (ppb) (ng/mL) 49.3 -976 9.76-97.6 97.6-976 9.76-976 % Recovery Range 77-110 97-107 90-109 86-111 Compound: PFOSEA Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve I/X 0.993-976 4.93-248 49.3-976 0.993-976 LCR- from curve (ppb) (ng/mL) 24.8-976 9.76-248 49.3-976 9.76-976 % Recovery Range 96-106 91-110 86-106 95-117 Compound: M556 I Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve i,ix 0.993 -976 4.93-97.6 97,6-976 0.993 -976 LCR from curve (ppb) (ng/mL) 24.8-976 9.76-97.6 97.6-976 9.76-976 % Recovery Range 88-106 100-105 81-111 97-110 RSD Range 11.2-25.5 14.1-21.3 11.5-19.6 11.1-21.2 RSD Range 10.1-16.2 11.8-19.5 10.2-18.2 10.1-19.1 RSD Range 4.82-17.9 5.95-18.2 5.11-9.74 4.77-19.5 Attachrr.e,B-:iMtDL,'LOQ Sun=ry 3M Environmental Laboratory ETS-8-4.1 Extractionof PFOS from Senim Paue 13 of 14 Page 80 3m Medical Departrrent Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Ion PairStandard Curves - Fluids Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Finalsolventand TN: Blank fluid/identirier: Method/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00ppm: FC mix std approx. 50.0ppm: Surrogate std approx. 100 ppm: Actual concentrations of standards in the FC mix PFOS PFOSA PEOSAA ETFOSE PFOSEA Std conc Std conc Std conc Std conc Std conc ug/n-LL ug/mL ug/rnL ug/mL ug/mL 0.500 0.507 i 0.532 0.501 0.521 1 0 0.507 0.532 0.501 05-21 5.00 5.07 532 5.01 5.21 50 5.07 --- 5.01 5.21 5.00 5.07 5.32 5.01 5.21 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 i [__50. 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 1 M556 Std conc ug/mL 0.501 61 5.01 5.01 50.1 50.1---- 5 0. 1 All Am't All Finalvol spikedmL ML 0.010 0.020 1.025 0.005 o-oi,o 1.015 0.020 1.025 0.005 1.010 -6.@-0 if-d 1.020 1.025 Calculated concentrations of standards in the sample matrix PFOS -,-,PFOSA PFOSAA ETFOSE PFOSEA M556 Surrogate Finalconc Finalconc Finalconc Finalconc Finalconc Finalconc Stdconc ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL 4.93 5.00 1 5.24 4.94 5.01 5.13 100 1 9.76 9.89 101.4 9.78 '-T9-93 1-d2----- -@4- 8 49.3 25.1 26@3 50.0 -7---@@ 24.8 25.2 49.4 1 50.1 5.8 51.S ------TSiunr@raolc-og@-tnccll 97.8 i 99.3 102 ng/mL ----i---4@-- 251 263 f4-8 252 258 500 493 500 524 494 501 513 735 746 782 737 749 6 976 989 1038 978 993 i 1017 All Am't spiked mL 0.005 Validated ranges - approximate concentrations Serum PFOS PFOSA PFOSAA ETFOSE-OH Rabbit 5.00-1000 1 5.00-1000 5.00-1000 1 5.00-1000 1 Bovine Estimatesonly. Use valuesforrabbit. Rat Estimatesonly. Use valuesforrabbit. Monkey & Plasma Estimatesonly. Use valuesforrabbit. Human Estimatesonly. Use valuesforrabbit. PFOSEA 5.00-1000 M556 5.00-1000 Attachment C: Ion PairStandardCurves ETS-8-4.1 Extractionof PFOS from Serum 3M En-v4-ronmental Laboratory Page 14 of 14 Page 81 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M ENVIRONN4ENTAL LABORATORY METHOD A-NALYSIS QF POTASSILTM PERFLUOROOCTANESLILFONATE OR FLUOROCHEMICALS IN SERUiNt EXTRACTS USING HPLC-ELECTROSPRAY/MASS SPECTROMETRY OTHER Method Number: ETS-8-5.1 Author: LisaClemen,RobertWynne Approved By: LaboratoryManager Adoption Date: 03/01/99 Revision Date: 2 Date Group Leader ,@& A Technical Reviewer Date . cvz .2@.1 Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method descn*besthe analysisof serum extractsfor fluorochemicalsurfactants usingHPLC-electrospray/massspectrometry. 1.2ApplicableCompounds: Fluorochemicalsurfactantosrotherfluorl*natceodmpounds, or otherionizablecompounds. 1.3Matrices: Rabbit,ratb,ovine,monkey, and human serum,or otherfluidsasdesignatedin thevalidatiorneport. Word 6i95 ETS-8-5.1 AnalysisofSerum ExtractUsinp,ES/NIS Paec I of 9 3M Environmental Laboratory Page 82 3m Medical Department S--udy: T-629S.7 RelDort No. FACT TOX-030 la]3oratory Request Number-U2279 2.0 SUNL@MARY OF METHOD 2.1 This method descn*bes the analysisof fluorochemical surfactantsextractedfrom serum or other fluids,using HPLC-electrospray/mass spectrometry, or similarsystem as appropriate. The analysisisperformed by monitoring a singleion characten'stiocf a particular fluorochemical, such as the perfluorooctanesulfonate(PFOS) anion, n-L/z=499. Additionally,samples may be analyzed using a tandem mass spectrometer to furtherverify the identityof a compound by detectingdaughter ions of the parent ion. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization (API); The Micromass Quattro 11tn'plequadrupole systems allow for various methods of ionizationby utilizingvan*ous sources,probes, and interfaces.These include but arenot limitedto:ElectrosprayIonization(ESI),Atmospheric Pressure chemical Ionization(APcl),Thermospray, etc. The ionizationprocess in these techniques occurs at atmospheric pressure (i.e.n,ot under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionizationperformed at atmospheric pressure,whereby ions in solutionare transferredto the gas phase via tinycharged droplets. These charged droplets are produced by the applicationof a strong electn'calfield. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro 11 triplequadrupole systems are equipped with quadrupole mass selective detectors.Ions arc selectivelydiscriminatedby mass to charge ratio(Mjz) and subsequently detected. A singleMS may be employed forion detectionor a series(MS/MS) formore speci icfragmentat'on information. 3.4 Conventional vs.Z-spray probe interface:The latestmodels of MicromaQsusatt1r1o tnplequadr-upaslyestems(post1998)utiliaze"Z-sprayc"onformationT.he sprayemitted from a probe isorthogonaltothecone aperture.Intheconventionalconformationitisaimed directlyatthecone aperture,afterpassingthrougha tortuouspathway inthecounter electrode.Though theconfiguratioinsdifferentt,hemethods of operationc,leaning,and maintenance arcthesame. However, Z-spraycomponents and conventionalcomponents are not compatiblewithone another,butonlywith similarsystems(i.e.Z,-spraycomponents are compatiblewith some otherZ-spraysystems,etc.) 3.5 Mass Lynx Software: System softwaredesignedforthespecifiocperationoftheseQuattro IItriplequadrupolesystems.CurrentlyMassLynx has Windows 95 and WindowsNT 4.0 versions.Allversionsaresimilar.For more detailseethemanual specifitcotheinstrument (N41cromassQuattroIItriplequadrupoleMassLynx or MassLynx NT User'sGuide). 4.0 WARNINGS AND CAUTIONS 4.1 Health and SafetyWarnings: 4.1.1 Use cautionwith thevoltagecablesfortheprobe. When engaged,theprobe employs a voltageof approximately5000 Volts. 4.1.2 When handlingsamples or solventswear appropriatperotectivegloves,eyewear, and clothing. ETS-8-5.1 Analysis of Serum ExtractUsing ES,@@IS 3M Env4-ronmenta 1 T aboratory Page 2 of 9 Page 83 3m Medical Department S--udy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 4.2 Cautions: 4.2.1 Do not operate solventpumps above capacity of 400 bar (5800 psi)back pressure. Ifthe back pressure exceeds 400 bar,the HP I 100 willinitiateautomatic shutdown. 4.2.2 Do not run solventpumps todryness. 5.0 INTERFERENCES- 5.1 To minimize interferenceswhen analyzing samples, teflonshould not be used for sample storage or any part of instrumentationthatcomes in contact with the sample or extract. 6.0 EouIPMENT 6.1 Equipment listedbelow may be modified in order to optimize the system. Document any modifications in-theraw data as method deviations. 6.1.1 6.1.2 Micromass Quattro IItriplequadrupole Mass Spectrometer equipped with an electrosprayionizationsource HPI 100 low pulse solventpumping system, solventdegasser,column compartment, and autosampler 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.1.1 High pur-itgyrade nitrogengas regulatedto approximately 100 psi (House air system) 7.1.2 HPLC analyticalcolumn, specificsto be determined by the analyst and documented in the raw data. 7.1.3 Capped autovialsor capped 15 mL centrifugetubes 8.0 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 8.1.2 Methanol, HPLC grade or equivalent Mi Il_lQTIL water, allwater used in thismethod should be M 1 11 1 _QTI'water or equivalent,and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate,reagentgrade or equivalent 8.2 Standards 8.2.1 Typically two method blanks,two matrix blanks, and eighteenmatrix standards are prepared dun'ng the extractionprocedure. See ETS-84.1. 9.0 SAIIPLE HANDLIN'G 9.1 Fresh matn'x standards are prepared with each analysis. Extracted standards and samples are stored in capped autovialsor capped 15 mL centrifugetubes untilanalysis. 3M Environmental ETS-8-5.1 Analysisof Serum ExtractUsing ES/i@(IS Laboratory Page 3 of 9 Page 84 3m Medical Department Study: T-6295.7 Rer)ort No. FACT TOX-030 laboratory Request Number-U2279 9.2 If analysis will be delayed, extractedstandards and samples can be refr-igerateadt approximately 4' C, or at room temperature,untilanalysiscan be performed. 10.0 ()UALITY CONTROL 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each bat-ch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank pn'orto each calibrationcurve. 10.2 Matrix Spikes 10.2.1 Matr-ixspikes are prepared and analyzed to determine the matrix effecton the recovery efficiency. 10.2.2 Matn'x spike duplicatesare prepared and analyzed to measure the precisionand the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spikeduplicateper fortysamples, with a minimum of 2 spikes per batch. 10.2.4 Matrix spikeand matrix spike duplicateconcentrationswillfallin themid-range of the initiaclalibrationcurve. Additional spikeconcentrationsmay fallinthe lowrange of the initiaclalibrationcurve. 10.3 Continuing CalibrationVerifications 10.3.1 Continuing calibrationverificationsare analyzed to verifythe continued accuracy of the calibrationcurve. 10.3.2 Analyze a mid-range calibrationstandardafterevery tenthsample, with a minimum of one per batch. 11.0 CALIBRATION AND STANDARDIZATION 11.1 Analyze the extractedmatrix standardspriorto and following each setof extracts.The average of two standardcurves willbe plottedby linearregression(y = my + b),weighted I/x,not forced through zero, using MassLynx or other suitablesoftware. 11.2 If the curve does not meet requirements,perform routinemaintenance or reextractthe standard curve (ifnecessary)and reanalyze. 11.3 For purposes of accuracy when quantitatinglow levelsof analyte,itmay be necessary to use the low end of the calibrationcurve ratherthan the fullrange of the standard curve. Example: when attempting to quantitateapproximately 10 ppb of analyte,generate a calibrationcurve consistingof the standards from 5 ppb to 100 ppb ratherthan the full range of the curve (5ppb to 1000 ppb). This willreduce inaccuracy attributedto linear regession weighting of high concentrationstandards. 3M Environmental ETS-8-5.1 Analysisof Serum ExtractUsing ES@'MS Laboratory Page 4 of 9 Page 85 3m Medical Department S--udy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.0 PROCEDURES 12.1 Acquisition Setup 12.1.1 Click on staxibutton in the AcquisitionControl Panel. Set up a sample list.Assicyn a filename using MO-DAY-last digitof year-sample number, assign a method (i@,IS) for acquiring,and type in sample descriptions. 12.1.2 To create a method clickon scan button in the Acquisition controlpanel and select SIR (SingleIon Recording) or MRM. Set IonizationMode as appropr-iataend mass to 499 or other appropr-iatmeasses. A fullscan Is usually collectedalong with the SIRS. Save acquisitionmethod. IfMS/MS instruments are employed, additional product ion fragmentation informationmay be collected.See Micromass MassLynx GUIDE TO DATA ACQUISITION foradditionalinformationand ,\4RM (MultipleReaction Monitoring). 12.1.3 Typicallythe analyticablatchrun sequence begins with a setof extractedmatrix standards and ends with a set of extractedmatrix standards. 12.1.4 Samples are analyzed with a continuing calibrationcheck injectedafterevery tenth sample. Solvent blanks should be analyzed periodicallyto monitor possibleanalvte carr-yoverand are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample listprepared in Section 12.1.1. 12.2.2 Set-up the HP I I00/autosampler atthe following conditions or atconditions the analystconsiders appropriatefor optimal response. Record actualconditions in the instrument logbook: 12.2.2.1 Sample size= 10 @iLinj*ection 12.2.2.2 Inj'ect'sample= 1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 8.50 min. 11.0 min. 12.0 min. MEOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% 10% 10% 60% 12.2.2.5 Pr-@ssthe "Start"button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0 for more details. 12.3.2 Check the solve-.iltevelin reservoirsand refillifnecessary ETS-8-5.1 Analysisof Serum ExtractUsing ES/i%,IS 3M Environme--ital- Laboratory Page 5 of 9 Page 86 3m Medical Department Study: T-6295.7 ReDorL-- No. FACT TOX-n@3C laboratory Request Number-U2279 12.3.3 Check the stainlessteelcapillaryattheend of theprobe. Use an eyepiece to check the tip.The tipshould be flatwith no jagged edges. Ifthe tipisfound tobe uns atisfactor-vd,isassemble theprobe and replacethestainlesssteelcapillarv. 12.3.4 Set HPLC pump to"On". Set the flow to 10 - 500 uL/min or as appropriate. Obser-vedropletscoming out of the tipof the probe. Allow to equilibratfeor approximately 10 minutes. 12.3.5 Turn ortthe nitrogen.A finemist should be expelledwith no nitrogenleaking around the tipof the probe. Readjustthe tipof the probe ifno mist isobserved. 12.3.6 The instrumentuses theseparametersatthe following settings.These settingsmay change in order to optimizethe response: 12.3.6.1Drying gas 250-400 liters/hour 12.3.6.2FSl nebulizinggas 10-15 liters/hour 12.3.6.3HPLC constantflow mode, flow rate 10 - 500 pL/min 12.3.6.4Pressure<400 bar (This parameter isnot set,itisa guide to ensure the HPLC isoperatingcorrectly.) 12.3.7 Carefullyguide theprobe intotheopening. Insertprobe untilitwillnot go any furtherC.onnect the voltagecables to theprobe. 12.3.8 Printthe tune page, with itsparameters,and storeitin the studybinderwith a copy taped intothe instrumentlog. 12.3.9 Using the cross-flowcounterelectrodeinthe ES/iMS source isrecommended for the analysisof biologicalmatn*ces. 12.3.10CIickon startbutton inthe AcquisitionControl Panel (thismay vary among MassLynx versions,see appropriateMassLynx USER'S GUIDE). Pressthe start button. Ensure startand end sample number includesallsamples to be analyzed. 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.4 Calculatema,,nx spikepercentrecoveriesusing the followingequation: % Recovery Obser-vedResult-Background Result x 100 Expected Result 13.1.5 Calculatepercentdifferenceusing the followingequation: Difference Exi)ectedConc. -CalculatedConc. x 100 Expected Conc. 13.1.6 Calculateactualconcentrationof PFOS, or otherfluorochemical,inmatrix (pg/mL): (ng,of PFOS calc.from std.Curve x DilutionFactor) x (InitiaVlolume ofmatrix(mL) + mL of Surro,izateStandard FinalVolume (mL) I uv 1000 nu 3M Environmenr-al Laboratory ETS-8-5.1 Analysisof Serum ExtractUsing ES/NIS Page 6 of 9 Page 87 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 14.0 METHOD PERFORMA4CE 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, anal@qe,and matrixspecific.Pleasesee ETS-84.1, Attachment B, fora listinogfcurrentvalidated MDL and LOQ values. 14.2 SolventBlanks,Method Blanks,and Matrix Blanks 14.2.1 Solvent blaaks,method blanks,and matrixblanksvaluesaremust be below the loweststandardinthecalibratiocnurve 14.3CalibratiCounrves 14.3.1 The r2 value forthecalibratiocnurvemust be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matn*x spikepercentrecoveriesaremust be within 30% of thespiked concentration. 14.5 Continuing CalibrationVerifleations 14.5.1 Continuingcalibratiovnerificatiopnercentrecoveriesmust be 30% ofthespiked concentration. 14.6 Ifcriterilaistedinthismethod performancesectionisn'tmet, maintenancemay be performed on thesystem and samples reanalyzedor otheractionsas determinedby the analyst.Document allactionsintheappropriatleogbook. 14.7 Ifdata areto be reportedwhen performance criten'haave notbeen met, thedatamust be footnotedon tablesand discussedinthetextof thereport. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample extractwaste and flammable solventisdisposedinhiz-n hBTU containersa,nd C-,lass pipettewaste isdisposedinbroken glasscontainerslocatedin thelaboratory. 16.0 REcoRDs 16.1 Each page generatedfora studymust have thefollowinginformationincludedeitherinthe header or hand w-ritteonn thepage: studyor projectnumber, acquisitiomnethod, integratiomnethod, sample name, extractiodnate,dilutionfactor(ifapplicable)a,nd analyst. 16.2 Pr-intthe tunepage,sample lista,nd acquisitiomnethod from MassLynx toincludeinthe appropriatestudy folder.Copy thesepages and tapeintotheinstrumentr-untog. 16.3 Plotthecalibratiocnur-veby linearegressionw,eighted I/x,thenprintthesegraphs and storeinthestudy folder. 16.4 Printdata integratiosnummary, integratiomnethod, and chromatop-rams,from MassLynx, and storeinthestudy folder. ETS-8-5.1 AnalysisofSerum ExtractUsingESi"vlS 3M Environmental Laboratory Page 7 of 9 Page 86 3m Med@-cal Department S-.udy: T-6295.7 Report No. FAC-,-TOX-030 laboratory Request Nurnber-U2279 16.5 Summarize datausing suitablesoftware(Excel5.0)and storeinthe study folder,see Attachment A foran example of a summary spreadsheet. 16.6 Back up electronicdata to appropriatemedium. Record in study notebook the filename and locationof backup electronicdata. 17.0 TABLES, DIAGRAms. FLOWCHARTS, AND VALIDATION DATA 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet. 18.0 PEFERENCES 18.1 FACT-M-4. compounds 1,"Extractionof Potassium Perfluorooctanesulfonatoer Other Fluorochemical from Serum forAnalysisUsing HPLC-E lectrospray/,MassSpectrometry 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/MassSpectrometerQuattroIItriplequadrupole Systems" 18.3 The validationreportassociatedwith thismethod is ETS-8-4.0 & 5.0-V-1. 19.0 AFFECRED DOCUMENTS 19.1 ETS-8-4. 1,"Extractionof Potassium Perfluorooctanesulfonatoer Other Fluorochemical Compounds from Serum forAnalysis Using HPLC-Electrospray/.%,IasSspectrometry" 20.0 REVISIONS Revision Number. 1 Reason For Revision Section 6.1.2 Clan'Ecationof HPI 100 system components. Section 11.1 Average of two curves,not standardvalues,are used for plottinglinearregressionand added the I/x weightincg:lof thecurve. Section 12.2.2.4Clarificatioonf solventramp. Section 17.1 Changed from attachmentB to A. Revision Date 04/02/199 ETS-8-5.1 AnalysisofSerum ExtTactUsing ES/',MS 3M Environme--ital Laboratory Page 8 of9 Page 89 3m Medi-cal Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Laboratory Study Study: Test Material: Matrix/FinalSolvent: ,Nfethod/Revision: AnalyticalEquipment System Number: InstrumentSoftwarefVersion: Filename: R-Squared Value: Slope: Y Intercept: Date ofExtractiori/AnalystDate ofAnalysis/Analyst: Group Dose S2MP]e# Concentration ug/mL InitiaVlol. mL - Dilution Factor FinalCone. ug/mL Slope: Taken from linearregressionequation. Group/Dose: Taken from thestudyfolder. Sample#: Taken from thestudyfolder. Concentration (ug/mL): Taken from theMassLynx integratiosnummary. InitiaVlolume (mL): Taken from thestudyfolder. DilutionFactor: Taken from thestudyfolder. FinalConc. (ug/mL): Calculatedby dividingtheinitiavlolume from theconcentration Ac-.achmentA: Summary Spreadsheet ETS-8-5.1 Analysisof Serum ExtractU-sin,E,S,'MS 3M Env-'@ronmental Laboratory Page 9 of 9 Page 90 3m Medical Department Study: T-6295.7 Report No. FACF TOX-030 laboratory Request Nun@cer-U2279 3M ENVIRONMENTAL LABORATORY EXTRACTION-OF FLUOROCHEMICAL METHOD POTASSIUM ]?ERFLUOROOCTANESULFONATE COMPOUNDS FROM SERUM FOR ANALYSIS ELECTROSPRAY/MASS SPECTROMETRY OR OTHER USING HPLC- Method Number: ETS-8-4.0 Adoption Date: I/"'I" Author: Lisa Clemen, Glenn Langenburg Revision Date: Approved By: Laboratory Mana-er/ c Date Gro/up Leader 4 T e@c@nical Review-er Date Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method isforthe extractionof potassium perfluorooctanesulfonate(PFOS) or other fluorochemical compounds from serum. 1.2 Applicable compounds: Fluorochemical surfactants or other fluorinatedcompounds. 1.3 Ntatrices: Rabbit,rat,bovine, monkey, and human serum or otherfluidsas designated in the validationreport. Word 97 SR- I 3M Environmental Laboratory ETS-8-4.0 Extractionof PFOS from Serum Page I of 13 Page 91 3m Medical DepartT-,ent S::udy: T-6295.7 Report No. FAC-- TOX-030 laboratory Request Number-U2279 2.0SUNIMARY OF METHOD 2.1 This method describes the procedure for extractingpotassium perfluorooetanesulfonate (PFOS) or other fluorochemical surfactantsfrom serum using an ion pairingreagent and 5.0 niL of methyl-tert-butyelther(MtBE). In thismethod, seven fluorochemicalswere extracted:PFOS, PFOSA, PFOSAA, ETFOSE-OH, PFOSEA, M556, and surrogate standard (see @7.0Deflnitions).An ion pairing reagent isadded to the sample and the analyte ion pair ispartitionedintoMTBE. The MTBE extractisremoved and put onto a nitrogen evaporator untildry. Each extractisreconstitutedin 1.0ml of methanol, then filteredthrough a 3 cc plasticsyringeattached to a 0.2 @im nylon filterinto class autovials. 3.0 DEFINITIONS 3.1 PFOS: pert-luorooctanesulfonat(eanion of potassium salt)CgFl7SO33.2 PFOSA: perfluorooctane sulfonylamide C8Fl7SO2NH, 3.3 PFOSAA: perfluorooctane sulfonylamido (dthyl)acetateC8Fl7SO-,N(CH,CH3)CH2CO'-- 3.4 ETFOSE-OH: 2(N-ethylperfluorooctanesulfonam ido)-ethylalcohol CsF17SO2N(CH2CH3)CH2CH20H 3.5 PFOSEA: perfluorooctanesulfonylethylamide C8Fl7SO,N(CHICH3)H 3.6 M556: C8Fl7SO2N(H)(CH2COOH) 3.7 Surrogate standard: IH- IH-2H-2H perfluorooctanesulfonicacid 4.0 WAR@NINGS AND CAUTIONS 4.1 Health and safety warnings 4.1.1 Use universalprecautions,especiallylaboratorycoats,goggles, and gloves whcn handling animal tissue,which may contain pathogens. 5.0 INTERFERENCES 5.1 There are no interferencesknown at thistime. 6.0 EQUIPMENT 6.1 The following equipment isused while performing thismethod. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge,Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR 6.1.4 Nitrogen evaporator,Organomation 6.1.5 Bala-nce( 0.100 c,) 3M Environme.-.'-al Laboratory ETS-8-4.0 Extractionof PFOS from Serum Pa--,e2 ot'13 Page 92 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Eppendorf or disposablepipettes 7.3 Nalgene bottlesc,apableofholding250 mL and I L 7.4 Volumetric flasks,glass,typeA 7.5 I-CHEM vialsg,lass,40 mL glass 7.6 Centrifugetubes,polypropylene,15 mL 7.7 Labels 7.8 Oxford Dispenser- 3.0to 10.0ml 7.9 Syringes, capable of measuring 5 pL to50 pL 7.10 Graduated pipettes 7.11 Syringes,disposableplastic3,cc 7.12 Syringefilternsy,lon,0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovialsand caps 7.15 Crimpers Note: Priortousingglasswareand bottlesr,inse3 timeswithmethanol and 3 timeswith Milli_QTM water. Rinse syringesa minimum of 9 times with methanol, 3 rinsefsrom 3 separatevials. 8.0 REAGENTS AND STANDARDS 8.1 Type Ireagnt grade water,Milli-QTM or equivalent,allwater used inthismethod should be Milli-(@TIMwater and may be provided by a Milli-Q TOC PlusTNi system 8.2 Sodium hydroxide(NaOH), J.TBaker orequivalent 8.3 Tetrabutyalmmonium hydrogen sulfate(TBA)K,odak orequivalent 8.4 Sodium carbonate (Na2CO3), J.T.Baker or equivalent 8.5 Sodium bicarbonate(NaHC03), J.T. Baker or equivalent 8.6 rvtethyl-T-ButEytlher,Omnisolv, glassdistilleodr HPLC grade 8.7 Methanol,Omnisolv, glassdistilleodrHPLC grade 8.8 Serum or blood,frozenfrom supplier 8.9 Fluorochemicalstandards 8.9.1 PFOS (3M SpecialtCyhemical Division)m,olecularweight= 538 8.9.2 PFOSA (3M SpecialtyChemicalDivision)m,olecularweight= 499 8.9.3 PFOSAA (3M SpecialtyChemicalDivision)m,olecularweight= 585 8.9.4 ETFOSE-OH (3M SpecialtCyhemical Division)m,olecularweight= 570 3M Environmental Laboratory ETS-8-4.0 Extractionof PFOS from Serum Paoe 3 of 13 Page 93 3m Medical Departmen@-- Study: T-6295.7 ReDort No. FACT TOX-030 laboratory Request Number-U2279 8.10 8.11 8.9.5 PFOSEA (3M SpecialtyChemical Division),molecularweight= 527 8.9.6 M556 (3M SpecialtyChemical Division),molecularweight = 557 8.9.7 Surrogate standard:4-H, perfluorooctanesulfonicacid (I-H,I-H, 7--H,2-H C8Fl3SO3H) molecular weight = 428 8.9.8 Other fluorochemicals,as appropriate Reagent preparation 8.10.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NAOH. Pour intoa 1000 mL beaker containing500 niL MillI_QTM water,mix untilallsolidsare dissolved.Store ina I L Nalgene bottle. 8.10.2 1 N sodium hydroxide(NaOH): Dilute10 N NAOH 1:10. Measure 10 mL of 10 N NAOH solutionintoa 100 mL volumetricflaskand diluteto volume using Milli-QTm water. Storeina 125 mL Nalgene bottle. 8.10.3 0.5 M tetrabutyalmmonium hydrogen sulfate(TBA): Wei&@ approximately 169 g of TBA intoa I L volumetriccontaining500 mL Milli-Q' water.Adjustto pH 10 using approximately44 to 54 mL of 10 N NAOH (While adding the lastmL of NAOH, add slowly because thepH changes abruptly).Diluteto volume with Milli-QwTaMter. Storein a I L Nalgene bottle. 8.10.3.1TBA requiresa check priorto each use to ensure pH = 10. Adjust as needed using I N NAOH solution. 8.10.4 0.25 i'vstodium carbonate/sodiumbiIcarbonatebuffer(NA,CO ,/NallC03):Weigh approximately26.5 g of sodium carbonate(Nal-C03)and -2 1.0 of sodium bicarbonate(NaHC03) intoa IL volumetricflaskand brinc,tovolume with MilliQTm water. Storein a I L Nalgene bottle. Standards preparation 8.11.1 Prepare PFOS standardsforthestandard curve. 8.11.2 Prepare otherfluorochemicalstandards,as appropriate.Multicomponent fluorochemicalstandardsare acceptable(forexample, one working standard solutioncontaining1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.11.3 Weigh approximately 100 mg of PFOS intoa 100 ml volumetricflaskand record the actualweight. 8.11.4 Bring to volume with methanol for a stock standardof approximately 1000 ppm (@iglml). 8.11.5 Dilutethe stocksolutionwith methanol fora working standard I solutionof approximately 50 ppm. 8.11.6 Diluteworking standardI with methanol fora working standard2 solutionof approx. 5.0 ppm. 8.11.7 Diluteworking standardI with methanol fora working standard3 solutionof approx. 0.50 ppm. 3M Environmental Laboratory ETS-8-4.0 Extractioonf PFOS from Serum Page 4 of 13 Page 94 3m Med@-cal Department Sz@udy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 8.12 Surrogate stockstandard preparation 8.12.1Weigc@h approximately50-60mg of surrogatsetandardI-H,I-H, 2-H, 2-H, C8Fl3SO3H intoa 50 ml volumetric flaskand record the actualweight. 8.12.2 Bring to volume with methanol for a surrogatestock of approximately 1000- 1200 PPM.- 8.12.3 Prepare a surrogateworking standard. Transfer approximately I ml of surrogate stock to a 10 ml volumetric flaskand brinc,to volume with methanol for a working standard of 100 ppm. Record the actualvolume transferred. 9.0 SAMPLE HANDLING 9.1 All samples are-receivedfrozen and must be kept frozen untilthe extractionisperformed. 10.0 OUALITY CONTROL 10.1 Method blanks and matrix blanks 10.1.1 Extracttwo 1.0ml aliquotsof Milli_QTM water following thisprocedure and use as method blanks. 10.2 10.1.2 Extract two 1.0mL aliquotsof the serum following thisprocedure and use as matrix blanks. See 11.1.4. Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicatesamples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst,usually the control matrix received with each sample set. 10.2.3 Expected concentrationswillfallin the mid-ran0ae of the initiaclalibrationcurve. Additional spikes may be included and may fallin the low-ran-e of the initial calibrationcurve. 10.2.4 Prepare one matrix spike and matrix spike duplicateper 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibrationchecks 10.3.1 Prepare and analyze continuing calibrationcheck samples to ensure the accuracy of the initiaclalibrationcurve. Ifthe percent differencebetween the initiaclurve and the continuinCg) check differby >30%, re-analyzesamples analyzed afterthe lastacceptable check. 10.3.2 Prepare one continuing check per group of 10 samples. For example, ifa sample set= 34, four checks are prepared and extracted. 10.3.3 Prepare each continuing calibrationcheck from the same matrix used to prepare the initiaclurve. 10.3.4 The expected concentrationsfallwithin the mid-ranc0e of the initiaclalibration curve, Additionalspikes may be included thatfallin thelow-range of the initial 3M E.,ivironmertal Laboratory ETS-8-4.0 Extractionof PFOS from Serum Pasic5 of 13 Page 9S 3m Medical Department S@-ady: T-6295.7 Report No. FACT TOX-030 laboratory Request Nurnber-U2279 calibrationcurve. This isnecessaryifthe analystmust quantitateusing only the low end of the calibratiocnurve (forexample, 5 ppb - 100 ppb, ratherthan 5 ppb - 1000 ppb). 11.0 CALIBRATION AND STANDARDIZATION 11.1 Prepare matrix calibrationstandards 11.1.1 TransferI mL of serum toa 15 mL centrifugetube. 11.1.2 Ifmost sample volumes are lessthan 1.0mL, extractstandardswith matrix volumes equal to the sample volumes. Do not extractlessthan 0.50 mL of matrix. Record each sample volume on theextractionsheet. 11.1.3 While preparinga totalof twenty aliquotsin 15 ml centrifugetubes,mix or shake between aliquots. 11.1.4 Two I mL aliquotso,r otherappropriatevolume, serveas matrix blanks. Typicallyuse the standardconcentrationsand spikingamounts listed'inTable 1,at the end of thissection,to spike,in duplicate,two standardcurves,fora totalof eighteenstandards,two matrixblanks,and two method blanks. 11.1.5 Refer to validationreportETS-84.0 & ETS-8-5.0-V-1, which liststhe working ranges and the LinearCalibrationRange (LCR) for calibratiocnurves. 11.1.6 Use Attachment D as an aid incalculati0ngtheconcentrationsof the workingt, standards.See Section13.0 to calculateactualconcentrationsof PFOS in calibrationstandards. 11.2 To each standard,blank,or continuingcheck, add appropriateamount of surrogate working standardfor the concentrationto fallwithin thecalibratiocnurve range 5 ppb 1000 ppb. 11.3 Extractspiked matrixstandardsfollowing 12.6-12.16of thismethod. Use thesestandards toestablisheach initiaclurve on the mass spectrometer. 3M Environmer@tal-I Laboratory ETS-8-4.0 Extr,ictioofnPFOS from Serum Pa2c 6 of 13 Page 96 3m Med@-cal Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Table1 Approximate spikingamounts forstandardsand spikes Usi g 1.0ml ofmatrix Working standard Approx. finalconc. of (approx.conc.) analyteinmatrix Blank 0.500 ppm 10 0.005 ppm 0.500 ppm 20 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 0.050 ppm 5.00 ppm 20 0.100 ppm 5(70 ppm 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm F- 50.p0pm 20 1.00 ppm 12.0 PROCEDURE 12.1 Obtain frozensamples and allow to thaw. 12.2 Vortex mix for 15 seconds,thentransfer1.0mL or otherappropriatevolume to a 15 mL polypropylene centrifugetube. 12.3 Return samples to freezerafterextractionamount has been removed. 12.4 Record the volume on the extractionworksheet. 12.5 Label the tube with the studynumber, sample ED, date and analystinitialsS.ee attached worksheet fordocumenting the remaining steps. 12.6 Spike allsamples, includingblanksand standards,ready forextractionwith surrogate standard as describedin 11.2. 12.7 Spike each matrix with the appropriateamount of standard as described in 11.1,or Table I in thatsection,forthe calibrationcurve standards. Also prepare matrix spikes and continuing calibratiosntandards. 12.8 Vortex mix thestandardcurve samples, matrixspike samples, and continuingcalibration samples for 15 seconds. 12.9 To each sample, add I mL 0.5 M TBA and 2 mL of 0.25,M sodium carbonate/soduim bicarbonatebuffer. 12.10 Using an Oxford Dispenser,add 5 mL methyl-tert-butyelther. 12.11 Cap each sample and put on the shaker for20 minutes. 12.12 Centrifugefor20 to 25 minutes atapproximately3500 rpm, untillayersare well separated. 12.13 Remove 4.0 mL of the or0uaniclayerto a clean 15 mL centrifugetube.Label thisfresh tube with the same informationas in12.5. 3M Env@-ronmental Laboratory ETS-8-4.0 Extractioonf PFOS frc)mSerum Page 7 of 13 Page 97 3m Medical Departmen@-- Study: T-6295.7 Report No. FACT TOX-r'30 laboratory Request Number-U227u9 12.14 Put each sample on the analyticanlitrogenevaporatoruntildry,approximately I to 2 hours. 12.15 Add 1.0mL or otherappropriatevolume of methanol to each centrifugetube usinZgD a graduated pipette.Methanol volume toadd equalsthe initiavlolume of sample used for the extraction. Note: Ifthe initiavlolume islessthan0.500 mL thefinalmethanol volume willequal 1.0mL. 12.16 Vortex mix for30 seconds. 12.17 Attach a 0.2 pm nylon mesh filtetroa 3 cc syringeand transferthe sample to thissyringe. Filterintoa 1.5mL glassautovialor low-volume autovialwhen necessary. 12.18 Label the autovialwith the studynumber, animal number and gender,sample timepoint, matrix,finalsolvent,extractiondate,and analyst(s)performing.t,h.eextraction. 12.19 Cap and store=tracts atapproximately4 *C untilanalysis. 12.20 Complete the extractionworksheet,attachedtothisdocument, and tape inthe study notebook or includein studybinder,as appropriate. 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations 13.1.1 Calculateactualconcentrationsof PFOS, or other applicablefluorochemical,in calibratiosntandardsusing thefollowingequation: mL of standardx concentrationof standard(uLy/mL) mL of standard+ mL of surrogatestandard+ initiamlatrixvolume (rnL) FinalConcentration(@tg/mL)of PFOS inmatrix 14.0 METHOD PERFORMA.NCE 14.1 The method detectionlimit(MDL) isanalyteand matrixspecific.Refer to N4DL report forspecificMDL and limitof quantitatio(nLOQ) values(seeAttachments B and C). 14.2 The followingqualitycontrolsamples areextractedwith each batchof samples to evaluatethe qualityof theextractionand analysis. 14.2.1 Method blanks and matrixblanks. 14.2.2 Matrix spikeand matrixspikeduplicatesamples to determine accuracy and precisionof theextraction. 14.2.3 Continuing calibratiocnheck samples to determine the continued accuracy of the initiaclalibratiocnurve. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sampie waste isdisposed inblohazard containers,flammable solventwaste isdisposed in hi1g. h BTU containersa,nd used glasspipettewaste isdisposed in broken glasscontainers locatedinthe laboratory. 3M Environmental Laboratory ETS-8-4.0 Extractioonf PFOS from Serum Page 8 of 13 Page 98 3m Med4-,cal Department Study: T-6295.7 Report No. FAC- '70X-C3o laboratory Request Numbe---U2279 16.0 RECORDS 16.1 Complete the extractionworksheet attachedto thismethod, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 ATTACHNIENTS 17.1 Attachment A. Extraction worksheet 17.2 Attachment B, MDLJLOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 18.0 REFERENCES 18.1 The validation report associated with thismethod is ETS-8-4.0 & 5.0-V-1. 19.0 AFFECTED DOCUMENTS 19.1 ETS-8-5.0, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-EL ec trospray Mass Spectrometry" 20.0 RF-VISIONS Revision Number Reason For Revision Revision Date 3M Environmental Laboratory ETS-8-4.0 Extractionof PFOS from Scruni Pa-e 9 of*13 Page 99 3m Medical Department S@udy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 ExtractionWorksheet ETS-8-4.0 Studv #/matrix SurrogateStd approx. ppm actual PPM FC-Mix approx.0.5 ppm actual PPM FC-@Mix approx.5 ppm actual PPM FC-Nlix approx.50 ppm actual PPM Date and Initialis*or Std.or Comments '.Studyntimherwhere thenriginnwlorkghe,.fi-gl.ntciteri B13nk Std Serum ExtractionNlethod 3mount mL Date & Initials Vortex 155ec. Pipette.MatriN Volume mi PipettIernlof 0.5M TBA, pH 10. Std.# Pir)ett2emlof0.25Na,CO,/0.25MNAHCO, buffer Std,$t Dispense5 ml of methyl-t-buteytlher TN-A- Shake 20 min. Centrifuiz2e0-25 min. Centrifueser)ced: Remove a 4 mL aliquot of oreanic laver Puton NicroeenEvaporatotrodryness Evaporator#: T-.mverature: Add methanol Volume mi 7,N-A-- Vortex30 sec. Filteursin2a 3ccB-D syrinizweitha 0.2Limfilteirntoa 1.5ml autosamr)lveial .MSIMSD/- Cont. Checks: Spiked - uL of a --- ppm std( .)fora finalconcentratioonf- ppm. NIS/MSD used sample . Cont.Checks used same matri.a-s forsidcurve. S,,jrrogSattaendardS:piked- uL ofa- ppm std( )to allsamples,standards.indbl@inks AttachmentA 3M Environmental Laboratory ETS-8-4.0 Extractioonf PFOS from Scruiii Pi,-,1c0 of 13 Page 100 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 MDLFLOQ alues for rabbitserum Compound MDL (ppb) LO,Qpb, Linear CalibrationRange (LCR) (p Approximate concentrationsto be used forpre@ppaarriitn7ghethe I Standard CalibrationCurve PFOS 4.76 15.2 5 ppb - 1000 ppb P FO SA 2.89 9.1 5 ppb - 10 00 ppb PFOSAA 2.94 9.33 5 ppb - I000_ppb ETFOSE-OH 13.2 41.9 5 ppb - 1000 ppb M556 PFOSEA 12.6 11.1 40.2 5 ppb - 1000 ppb 35.4-"F5-@-pb--1000 ppb MDL/LOQ valuesinrat,bovine,monkey, and human serum were notstatisticaldleytermined.Two curvesin each of thesethreematriceswere extractedand analyzedwith the rabbitserum curves to determine equivalence.Responses intherat,bovine,and monkey were equivalentto therabbit responses,therefore,thei!M.DrL and LOQ willbe the same valuesas determined inrabbitserum. Pleasesee Attachment C (LOQ Summary) and MDL studyinETS-8-4.0 & 5.0-V-I forfurther information. [on Pairing Extraction of Fluorochemicalsfrom Serum and Analysis by APIfL'VIS(MS) Summary Table:Limitsof Quantitation Compound PFOS PFOSA PFOSAA ETFOSE-OH* PFOSEA @M556 NFDL and LOQ I Matrix NIDL All 4.76 All 2.89 All 2.94 All 13.2* All 12.6 All 11.1 areestimatesonly forETFOSE-OH. LOQ Low std 15.2 25.1 1 9.19 25.0 9.33---@ 25.0 41.9* 1 50.0 40.2 25.0 35.4-----F-25.0 itd 1002 1000 998 10 0 1000 10()0 AttachmentB: MDL/LOQ 3M Environmental Laboratory ETS-8-4.0 ExtractioonfPFOS from Seruin PicycII of'13 Page 101 3m Med4@cal Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Compou d:PFOS Prepared 3erum rangeof matrix standards (pb) (ne/mL) @R,bb,t 1 1.00-1002 Range of LCR from Range of LCR from Range of LCR from averaue ave curve low std low std highsid hieyhsid curve curve curve curve curve I (ppb)(ng/mL) (Ppb)(ng/rnL)(ppb)(niz/mL)@pob)(nL/mL) @ppb)(na/mL)..(pob)(nL/inL) ].DO- 1002 25.1- 1002 5.01-250 5.01-250 100-1002 100- 1002 Compound: PFOSA Prepared Serum rangeof matrix standards (i)t()nbW)mL) Range of LCR from Range of LCR from Range of LCR from average ave curve low std low std high std highstd curve curve CUTVE curve curve (ppb)(ng/mL) (ppb)(ng/mL) (oc)b()ne/mL) (Ppb)(ng/mL) (Ppb)(ne/mL) (ppb)(ne/iTiL) Rabbit 1.00-10()0 1.00- 1000 10.0-1000 5.00- 100 5.00- 100 25.0-10()0 25.0 - 1000 Compound: PFOSAA I Prepared Range of LCR from Range of LCR from Range of LCR from Serum rangeof average ave curve low std low std high std high std matrix standards curve curve curve curve curve (ppb)(ng/mL) (ppb)(ngjml) (pob)(nWmL) _(_Mb)(n-g@mL)(ppb)(ng/mL) (pi)(bn)g/mL) @p pb)(nYL/mL) I.C)-O 998 1.()0-998 25.0-998 4.99-250 9.99-250 49.9-998 99.8-998 Compound: ETFOSE-OH Prepared Range of Serum rangeof average matrix standards curve (ppb)(np/mL) (pDb)(ng/mL) Rabbit I 1.00- 1000 1.00- 1000 1 LCR from ave curve (pDb)(ng/mL) 50.0- 1000 Range of low std Curve (ppb)(ng/ML) 5.00- 100 LCR from low std curve (p b) (ne/mL 10.0-loo ' 1 Range of hi-hstd c@-Irve (ppb@ (ne/rnL) I 50.0- 100 - LCR from high std curve (ppb)(nWinL) 10- 100 Compound: PFOSEA Prepared Serum rangeof matrix standards 1 (ppb)(np/mL) Range of LCR from average ave curve curve (ppb)(np,/mL) (ppb)(ng/mL) Rabbit 1.00-loco 1.00-10()0 25.0-1000 Range of LCR from Rancyeof LCR from low std low std high std high std curve curve curve curve 2pb) (ne/TnL) (ppb)(np-,/tnL(),,)p(bn)g/rTiL) b n1me0@L0),0L) 5.00-250 5.00-250 50- 1000 50- Compound: N[556 Prepared Serum rangeof matrix standards (Ppb@ (neJmL) Rabbit 1.00- 1000 Range of average curve (pob)(nz/mL) LCR from ave curve (pob)(nz/mL) Range of LCR from low std low std curve curve (2ph)(nv-/mL) (ppb)(np/mL) Ran-e of hieh std curve fppb)(ng@mL) 1.00- 1000 25.0- 1000 5,00- 100 5.00- 100 foo- 1000 LCR from highstd curve (ppb)(ng/mL) 100- 1000 AttachmentB: %IDL-fLOQ 3M Env@Lronme.-ita7-,Laboratory ETS-8-4.0 Extractioonf PFOS from Serum Pi,-,1c2 of 13 Page 102 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Ion PairStandard Curves - Fluids Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Finalsolventand TN: Blank fluid/identifier: Method/revision: Targetanalyte(s): FC mix std approx. 0.500ppm: FC mix std approx. 5.00ppm: FC mix stdapprox. 50.0ppm: Surrogate std approx. 100 ppm: Actual concentrationsofstandards inthe FC mix PFOS PFOSA PFOSAA ETFOSE PFOSEA Std cone Std conc S@d conc Std conc Std conc ug/mL ug/mL ug/mL ug/mL ug/niL 0.500 0.507 0.532 0.501 0.521 0.5()0 0.507 0.532 0.501 0.521 5.00 5.07 5.32 5.01 5.21 5.00 5.07 5 .32 5.01 5.21 5--.-0--0--------5--.07 5.32 5.01 5.21 50.0 50.1 53.2 50.1 -5'2-1. 50.0 50.1 53.) 50.1 52.1 -.7 50.0 50.1 - ---5-3-.-2---- 50.1 52.1 50.0 50.1 53.2 50.1 1 52.1 1 M556 Std conc ug/mL 0,501 0.501 5.01 5.01 5.01 50.1 50.1 50. 1_ 50.1 All Aml spikedmL 0.010 0.020 0.005--0.010 0-.-0-2-0---0.005 0.010 0.015 0.020 All Finalvol ML 1.015 1.025 1.015 1-..@0-I2.5.--.-@ 1.010 1.015 1.020 1.025 Calculated concentrationsof standards in the sample matrix PFOS PFOSA Finalconc Finalconc ng/mL ng/mL PFOSAA Finalconc nLJmL ETFOSE PFOSEA N1556 Surrogate Finalconc Finalconc Finalconc Sidconc ng/mL ng/mL ne/rn-L niz/mL 4-.-9-3--------5-.-00 9.76 1 9.89 24.8 1 25.1 49 .3 5.24 1 4.94 5.01 10.4 1 9.78 9.93 5.13 100 F- 26.3 1 24.8 25.2 25.8 Surrogate 49.4 50.1 Finalconc 97.6 98.9 104 97.8 99.3 248 251 1 263 248 252 493 500 524 i 494 501 102 ng/mL 258 500 513 735 746 749 98 9 1038 978 993 1017 All Am'[ spiked ML 0.005 Validatedranges approximate concentrations Serum PFOS PFOSA PFOSAA Rabbit 25.1-1002 1 25.0-tOOO 1 25.0-998 1 Bovine Estimatesoniv.Use valuesforrabbie. Rat Estimatesonly.Use valuesforrabbit, \4onkev Estimatesonlv.Use valuesforrabbit. ; Human Estimatesonly.Use valuesforrabbit. ETFOSE-OH 1 50.0-1000 1 PFOSEA 25 0-1000 N1556 -25@0-1000 AttachmentC: IonPairStandardCurves ETS-8-4.0 ExtractioonfPFOS from Serum 3M Environmental Laboratory Page 13 of 13 Page 103 3m Medical Departmer-t Study: T-6295.7 Repor@-- No. FAC-L TOX-030 laboratory Request Number-U2279 3M ENVIRONMENTAL LABORATORY METHOD ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE OR FLUOROCHEMICALS IN SERUM EXTRACTS USING HPLC-ELECTROSPRAY/MASS SPECTROMETRY OTHER Method Number: ETS-8-5.0 Author: Lisa Clemen, RobertWynne Approved By: Adoption Date: Revision Date: Lab&t@t@ry',Man@o,er Date Group Leader . .3-@fi, CLr,,@, Technical Reviewer Date 31 Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method isforthe analysisof extractsfrom serum forfluorochemicalsurfactants usin0-HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizablecompounds. 1.3 Matrices: Rabbit,rat,bovine,monkey, and human serum, or otherfluidsas designatedin the validationreport. Word 97 SR-t 3M Environmental ETS-8-5.0 AnalysisofSerum ExtractUsin- ESA,IS Laboratory Page I of'9 Page 104 3m Medical Department Study: T-629S.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 SumMARY OF METHOD 2.1 This method describesthe analysisof fluorochemicalsurfactantsextractedfrom serum, using HPLC-electrospray/mass spectrometry,or similarsystem as appropriate.The analysisisperformed by monitoringa singleion characteristiocf a particular fluorochemical,such as the potassiumperfluorooctanesulfonat(ePFOS) anion,M/z= 499. Samples may a]so be analyzed usingan API-ESIMS/'IvISsystem tofurtherverify compound iden'tification. 3.0 DFFINITIONS 3.1Atmospheric Pressure Ionization(API):The Micromass QuattroIIsystems allow for variousmethods of ionizationby utilizinvgarioussources,probes,and interfaces.These includebut are not limitedto:ElectrosprayIonization(ESI),Atmospheric Pressurechemical Ionization(APcl),thermospray, etc.The ionizatiopnrocessin thesetechniquesoccursat atmospheric pressure(i.en.ot under a vacuum). 3.2 Electrospray Ionization(ES, ESI): a method of ionizationperformed at atmospheric pressure,whereby ionizationoccursthrough theproductionof tinychar-ed dropletsina strongelectricaflield. 3.3Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/INIS): The API Quattro Ilsystems areequipped with quadrupole mass selectivedetectors.Ions are selectivelydiscriminatedby mass tocharge ratio(nl/z)and subsequentlydetected.A single MS may be employed forion detectionor a series(MS/i'vlSf)ormore specificfracmentation information. 3.4Conventional vs.Z-spray probe interface:The latestmodels of Micromass QuattroH systems (post1998) utilizae "Z-spray"conformation.The spray emittedfrom a probe is orthogonalto the cone aperture.In theconventionalconformation itisaimed directlyat the cone aperture,afterpassingthrough a tortuouspathway in thecounter electrode.Though the configurationisdifferentt,he methods of operation,cleaning,and maintenance are the same. However, Z-spray components and conventionalcomponents are not compatible with one another,but only with similarsystems (i.eZ.-spray components arecompatible with some otherZ-spray systems,etc.) 3.5 Mass Lynx Software: System softwaredesiCg)ned forthe specificoperationof theseQuattro Ilsystems. CurrentlyNlassLynx has Windows 95 and WindowsNT 4.0 versions.All versionsare similar.For more detailsee the manual specificto the instrument(imicromass Quattro H MassLynx or MassLynx NT USER'S GUDDE). 4.0 WAPNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use cautionwith the voltagecablesforthe probe. The probe employs a voltageof approximately5000 Volts. 4.1.2 When handling samples or solventswear appropriateprotectivegloves,eyewear, and clothing. 3M Environmental ETS-8-5.0 AnalysiosfSerum ExtracUtsingES/@'vlS Laboratory Pa!zc'o-f''9 Page 105 3m Medical Departmert sr-udy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 4.2 Cautions: 4.2.1 Do not operatesolventpumps above capacityof 400 bar (5800 psi)back pressure. Ifthe back pressureexceeds 400 bar,the HP I100 willinitiataeutomatic shutdown. 4.2.2 Do not run solventpumps todryness. 5.0 INTERFERENCES 5.1 To minimize interferenceswhen analyzingsamples, teflonshould not be used forsample storageor any partof instrumentationthatcomes incontactwith the sample or extract. 6.0 EOUIPMENT 6.1 Equipment listedbelow may be modified in orderto optimizethe system. 6.1.1 Micromass ElectrosprayMass Spectrometer 6.1.2 HPI 100 low pulse solventpumping system and autosampler 7.0 SUPPLIES AND MATEMALS 7.1 Supplies 7.1.1 High puritygrade nitrogengas regulatedtoapproximately 100 psi(House air system) 7.1.2 HPLC analyticalcolumn, specificstobe deteri-ninebdv the analyst 7.1.3 Capped autovialsor capped 15 ml centrifugetubes 8.0 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC gradeor equivalent Mill_iQ Tm 8.1.2 Milli-QT"'wAater,allwaterused in thismethod should be " water or equivalent,and may be providedby a Milli-Q TOC Plussystem or othervendor 8.1.3 Ammonium acetate,reagentgrade or equivalent 8.2 Standards 8.2.1 Typicallytwo method blanks,two matrixblanks,and eighteenmatrix standardsare prepared during theextractionprocedure. See ETS-8-4.0. 9.0 SAMPLE HANDLING 9.1 Fresh matrix standardsare preparedwith each analysis.Extracted standardsand samples are storedincapped autovialsor capped 15 mi centrifugetubesuntilanalysis. 9.2 Ifanalysiswillbe dela1yed,extractedstandardsand samples can be refr0igerateadt approximately 4' C untilanalysiscan be performed. 3M Environmental ETS-8-5.0 Analysisof Serum ExtractUsing ESIMS Laboratory Pa2c 3 of9 Page 106 3m Medical Decartment- S::udy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 10.0 OUALITY CONTROL 10.1 Method Blanks and Matrix Blanks 10.1.1 Analyze a rrethod blank and a matrix blank priorto each calibrationcurve. 10.2 Matrix Spikes 10.2.1 Analyze'a matrix spikeand matrix spikeduplicateper fortysamples. With a minimum of 2 spikesper batch. 10.2.2 Expected spikeconcentrationswillfallin the mid-range of the initiaclalibration curve. Additionalspikeconcentrationmsay fallin thelow-rangeof the initial calibratiocnurve. 10.2.3 See Secti-on13 tocalculatepercentrecovery. 10.3 Continuing Calibration Checks 10.3.1 Analyze a mid-range calibratiosntandardafterevery tenthsample. Ifa significant change ( 30%) in peak areaoccurs,relativeto the initiasltandardcurve,stop the run.Only those samples analyzed before the lastacceptablecalibrationstandard willbe used. The remaining samples must be reanalyzed. 10.3.2 See Section13 to calculatepercentdifference. 11.0 CALIBPATION A.ND STANDARDIZATION 11.1 Analyze the extractedmatrix standardspriorto and followingeach setof extracts.The mean of two standardvalues,ateach standardconcentration,willbe plottedby linear regression(r2)forthe calibratiocnurve usingMassLynx or othersuitablesoftware. 11.2 The 2 r value forthe data should be 0.990 or greater.Lower valuesmay be acceptableatthe discretionof the analystand approvalof the Pr01jectLead. 11.3 Ifthe curve does not meet requirements,perform routinemaintenance or reextractthe standardcurve (ifnecessary)and reanalyze. 11.4 For purposes of accuracy when quantitatinglow levelsof analyte,itmay be necessary to use the low end of the calibratiocnurve ratherthan the fullrange of the standardcurve. Example: when attemptingtoquantitateapproximately 10 ppb of analyte,generatea calibrationcurve consistingof thestandardsfrom 5 ppb to 100 ppb ratherthan the fulI range of thecurve (5 ppb to 1000 ppb). This willreduce inaccuracyattributetdo linear regressionwellyhtingof high concentrationstandards. 12.0 PROCEDURES 12.1 Acquisition Setup 12.1.1 Click on startbutton in the Acquisition Control Panel. Set up a sample list.Assign a filenameusing MO-DAY-last digitof year-sample number, assigna method (MS) foracquirlnc,a,nd typein sample descriptions. 3M Environmental ETS-8-5.0 AnalysisofSerum ExtractUsine ES/,\IS Labcratory Pa2c 4 of'9 Page 107 3m Medical Departme@-t S::udy: T-629@-;.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.1.2 To createa method clickon scan buttonin the Acquisitioncontrolpanel and select SIR (SingleIon Recording)or MRM. Set IonizationMode as appropriateand mass to 499 or other appropriatemasses. A fullscan isusuallycollectedalong with the SIRS. Save acquisitionmethod. IfMS/'YIS instrumentsareemployed, additional product ion fr-agmentatioinnformationmav be collected.See Micromass MassLynx GUIDE TO DATA ACQUISIT@ON foradditionalinformationand MR.M (MultipleReactionMonitoring). 12.1.3 Typicallythe analyticablatchrun sequence beginswith a setof extractedmatrix standardsand ends with a setof extractedmatrix standards. 12.1.4 Samples are analyzed with a continuingcalibratiocnheck injectedafterevery tenth sample. Solvent blanksshould be analyzed periodicalltyo monitor possibleanalyte carryoverand are not consideredsamples but mav be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample trayaccordingto the sample listprepared in Section12.1.1. 12.2.2 Set-up the HPI 100/autosampleratthefollowinCg, conditionsor atconditionsthe analystconsidersappropriatefor optim'alresponse.Record actualconditionsin the instrur-nenltogbook: 12.2.2.1Sarrplesize= 10 gL injectionwith a sample wash 12.2.2.2Inject/sample= 1 12.2.2.3Cycle time = 13.5 minutes 12.2.2.4Solvent ramp Time 0.00 min. 7.5 min. 11.0 min. 11.5 min. MEOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% 1017c 10% 60% 12.2.2.5Pressthe "Start"button. 12.3 Instrument Set-up 12.3.1 Refer toETS-9-24.0 formore details. 12.3.2 Check the solventlevelin reservoirsand refililfnecessary. 12.3.3 Check the stainlessteelcapillaryatthe end of the probe. Use an eyepiece tocheck the tip.The tipshould be flatwith no jagged edges. Ifthe tipisfound to be unsatisfactoryd,isassemblethe probe and replacethe stainlessteelcapillary. 12.3.4 Set HPLC Pump to "On". Set the flow to 10 - 500 uLJmin or as appropriate. Observe dropletscomingZ2 outof thetipof theprobe. Allow toequilibratfeor approximately 10 minutes. ETS-8-5.0 AnalvsisofSerum ExtractUsine ES/NIS 3M Env4-ronmental Laboratory Pa@!c5 of 9 Page 108 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.3.5 Turn on the nitrogen.A finemistshould be expelledwith no nitrogenleakino, around the tipof the probe. 12.3.6 The instrumentuses theseparametersatthe followingsettinas.These settincymsay change in order to optimize theresponse: 12.3.6.1Drying gas 250-400 liters/hour 12.3.6.ZESI nebulizinggas 10-15 liters/hour 12.3.6.3HPLC constantflow mode flow rate 10 - 500 @il-/m'in 12.3.6.4Pressure<4GO bar (Thisparameter isnot set,itisa guide to ensure the HPLC isoperatingcorrectly.) 12.3.7 Carefullyguide the probe intotheopenina. Insertprobe untilitwillnot go any furtherC.onnect the voltagecablesto the0probe. 12.3.8 Record tune parameters in the instrumentloogl 12.3.9 Using the cross-flowcounterelectrodeinthe ES/MS source isrecommended for theanalysisof biologicalmatrices. 12.3.10CIickon startbutton in theAcquisitionControl Panel (thismay vary among MassLynx versions,see appropriateMassLynx USER'S GUDDE). Pressthe start button. Ensure startand end sample number includesallsamples to be analyzed. 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.4 Calculatematrix spike percentrecoveriesusing the followingequation: % Recovery Observed Result-Background Result x 100 Expected Result 13.1.5 Calculatepercentdifferenceusing thefollowingequation: 7c Difference ExQected Conc. - CalculatedConc. x 100 Expected Conc. 13.1.6 Calculateactualconcentrationof PFOS, or otherfluorochemical,inmatrix (pg/ml): (ng of PFOS calc.from std.Curve.x.DilutionFactor) x (InitiaVlolume of matrix(ml)+ mi of SurrogateStandard) FinalVolume (mL) I g@!Z 1000 n- 14.0 METHOD PERFOR@MANCE 14.1 Method DetectionLimit(MDL) and Limitof Quantitation(LOQ) aremethod, analyte,and matrixspecific.PleaseseeETS-8-4.0,Attachment B, fora listino,f-currentvalidated MDL and LOQ values. 3M Env4@ronmental ETS-8-5.0 Analysis of Serum Extract Using ES/NIS Laboratory Pa2c 6 of 9 Page 109 3m medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 14.2 Method Blanksand MatrixBlanks 14.2.1Method blanksandmatrixblankswillbe analyzedwitheachsamplesetfor possiblecontaminationorcarryover.Values are expected tofallbelow the lowest standard inthe calibratiocnurve. 14.3 Matrix Spikes 14.3.1 Matrix spikes areanalyzedwith each sample setand the percentrecoveriesare expected to fallwithin 30% of the spiked concentration. 14.4 Continuing CalibrationChecks 14.4.1 Continuing calibratiocnhecks are analyzedata minimum of afterevery 10 samples with each sample set.The percentrecoveriesare expected tofallwithin 30% of the spiked concentration. 14.5 Ifany criterialistedin the method performance sectionisn'tmet, maintenance may be per-formedon thesystem and samples reanalyzedor otheractionsas determined by the analyst.All actionswillbe documented in the instrumentrunlog,the maintenance log,or on the summary sheet with the sample results.. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample extractwaste and flammable solventisdisposed in high BTU containers,and glass pipettewaste isdisposed in broken glasscontainerslocatedinthe laboratory. 16.0 RECORDS 16.1 Store chromatograms in the studyor projectfolder.Each chromatogCr)am must have the followinginformationincludedeitherin the header or hand writtenon the chromatogram: study or projectnumber, acquisitiomnethod, integrationmethod, sample name, extraction date,dilutionfactor(ifapplicable)a,nd analyst. 16.2 Plot calibratiocnurve by linearregressionand storein the studyfolder. 16.3 Printsample listfrom MassLynx and tape intothe instrumentruiilolg. 16.4 Printdata integrationsummary from MassLynx and tape intothe instrumentrunlog. 16.5 Copy instrumentrunlogpaaes,includinginstrumentparametersand sample resultsa,nd storein appropriatestudy folder. 16.6 Summarize data usin- suitable software and store in the study folder. 4 16.7 Back up electronicdatatoappropriatemedium. Record instudy notebook the filename and locationof backup electronicdata. 17.0 TABLES, DIAGRAms, FLOWCHARTS, AND VALIDATION DATA 17.1 Attachment B: ETS-8-5.0 Data reportingspreadsheet. 17.2 The validationreportassociatedwith thismethod isETS-8-4.0 & 5.0-V-1. 3M Environmental ETS-8-5.0 AnalysisofSerum ExtractUsiny ES/'%.IS Laboratory Pi-e 7 of 9 Page 110 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 18.0 REFERENCES 18.1 ETS-9-24.0, "Operation and Maintenance of theMicromass Atmospheric Pressure Ionization/NlasSspectrometer QuattroU Systems" 19.0 AFFECTED DOCUMENTS 19.1 ETS-8-4.0, "Extractionof Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Serum forAnalysisUsing HPLC-Electrospray/Mass Spectrometry" 20.0 REvisiONS Revision Number. Reason For Revision Revision Date 3M Environmental ETS-8-5.0 Analysisof Serum ExtractUsin- ES/NIS Laboratory Page 8 of 9 Page 111 3m Medical Departmen@-- S--udy: T-6295.7 Attachment A Report No. laboratory Request FACT TOX-030 Nurnber-U2279 Laboratory Study Study: TestMaterial: Matrix/FinaSlolvent; iMeLhod/Revision: AnalyticalEquipment System Number: InstrumentSoftware[Version: Filename: R-Squared Value: Slope: Y Intercept: Date ofExtraction/Analys@ Date ofAnaivsis/Anaivst: Group Dose Sample# Concentration ue/mL InitiaVlol. mL Dilution Factor FinalCone. UL,/Ml, SIOI)E:Taken from lineareeressioenquation. Grout)/Dose:Taken from thestudyfolder. Samv]L-#: Taken from thestudyfolder. Concentration(up-/mL):Taken fromtheMassLvnx intelratisounmmarv. InitiaVlolume (rnL):Taken from thestudyfolder. DilutionFactor: Taken fromthestudyfoldcr. FinalConc. (upJmL): Calculatebdy dividintlh,einitivaollume fromtheconcentration 3M Environmental ETS-8-5.0 AnalysisofSerum ExtractUsin- ES/NIS Laboratory Page 9 of9 Page 112 3m Med@-cal Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Numbe---U2279 3M ENVIRONMENTAL LABORATORY METHOD EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE OR OTHER A-,NIONIC FLUOROCHF,MICAL SURFACTANTS FROM LIVER FOR ANALYSIS USING HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: FACT-M-1.0 Author: Lisa Clemen Approved By: Adoption Date: 5'1.C2. Revision Date: k) Laboratory Manager Date Group Leader Date Technical Reviewer Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method is for the extractionof Potassium Perfluorooctanesulfonate(PFOS) or otherfluorochemicalsurfactantsfrom liver. 1.2Applicable Compounds: Fluorochemicalsurfactantosr otherfluorinatedcompounds. 1.3Matrices: Rabbit,-at,bovine, and monkey liversor otherliversas designatedinthe validationreport. Microsoft 7.0.1/95 FACT-M-1.0 Extractionof PFOS from Liver 3M Env@@ronmental Laboratory Page I of 8 Page 113 3m Medical E)epartment Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 2.0 SUMMARY OF NIETHOD 2.1 This method describeshow to extractpotassiumperfluorooctanesulfonat(ePFOS) or other fluorochemicalsurfactantsfrom liverusingion pairingreagentand 5.0 mLs of ethyl acetate.An ion pair-inrgeagentisadded to each sample and partitionedintoethylacetate. Four mLs of extractisremoved toa centrifugetube and put onto a nitrogenevaporator untildry. Each extractisreconstituteidn 1.0mL methanol then filteretdhrough a 3 cc plasticsyringeattachedto a 0.2 @im filteirntoglassautovials. 3.0 DEFINITIONS 3.1 None. 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use universalprecautionswhen handlinganimal liverst,hey may contain pathogens. 5.0 INTFRFERENCES 5.1 There are no known interferenceastthistime. 6.0 EQU[PMENT 6.1 The followingequipment isused whilecarryingout thismethod. Equivalentequipment is acceptable. 6.1.1 Ultra-TurraxT25 Grinderforgrindingliversamples 6.1.2 Vortex mixer,VWR, Vortex Genie 2 6.1.3 Centrifuge,Mistral 1000 or IEC 6.1.4 Shaker, Eber*oach or VVY'R 6.1.5 Nitrogen Evaporator,Organomation 6.1.6 Balance 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Dissectingscalpels 7.3 Eppendorf or disposablepipettes 7.4 Nalgene bottlesc,apable of holding250 mL and I L 7.5 Glass,type A, volumetricflasks 7.6 40 mL glass I-CHE,@vtvials 7.7 Plasticsampule vials,Wheaton, 6 mL 7.8 Polypropylene centrifugetubes,15 mL 7.9 '-abeIs FACT-M-1.0 Extractionof PFOS from Liver Page 2 of 8 3M Env4-ronmental Laboratory Page 114 3m Medical IDepartment Study: T-6295.7 Report No. FACT TOX-030 laboratcry Request Number-U2279 7.10 Syringes, capable of measuring 10 @iL to 50 pL 7.11 Glass, type A, volumetric pipettes 7.12 Graduated pipettes 7.13 Electronicpipettor,Eppendorf or equivalent 7.14 Timer 7.15 Disposable plastic3 cc syringes 7.16 Filters,nylon syn'nge filters0,.2 pm, 25 mm 7.17 Cr-imp cap autovials Note: Pr-iorto using glassware and bottles,rinse3 times with methanol and 3 times with Milli- Q' water. Rinse syringesa minimum of 9 times with methanol, 3 rinsesfrom 3 separate vials. 8.0 REAGENTS AND S@TANDAPDS 8.1 Reagents 8.1.1 Sodium Hydroxide (J.TBaker or equivalent),(NAOH) ION: weigh approximately 200 grams NAOH. Pour intoa 1000 mL beaker containing500 liter(sL) Nlilll_QTM water,mix untilallsolidsare dissolved.Store in a I L nalgene bottle. 8.1.2 Sodium Hydroxide (J.TBaker or equivalent),(NAOH) IN. Dilute ION 1:10. Measure 10 mL of the ION NAOH solutioninto a 100 mL volumetric flaskand diluteto volume using Milli-Q"mwater. Store in a 125 mL nalgene bottle. 8.1.3 Tetrabutylammonium hydrogen sulfate(Kodak or equivalent),('FBA) 0.5M: Weigh approximately 169 grams of TBA intoa I L volumetric containing500 L Milli-Q,-" water. Adjust to pH 10 using approximately 64 mL ION NAOH and diluteto volume with Milli-Q'-"water. Add NAOH slowly while adding the lastI mL of NAOH because the pH changes abruptly. Store in a I L nalgene bottle. 8.1.3.1 TBA requiresa check priorto each use to ensure pH = 10. Adjust as needed using IN NAOH solution. 8.1.4 Sodium carbonate/Sodium Bicarbonate Buffer (J.T.Baker or equivalent), (Na,C03NaHC03) 0.25M: Weigh approximately 26.5 g of sodium carbonate (Na2CO3) and 21.0 g of sodium bicarbonate(,NaHCO,) intoa I L volumetric flask and diluteto volume with Milli-Q' water. Store ina I L nalgene bottle. 8.1.5 PFOS (3M SpecialtyChemical Division),molecular weiizht= 538. 8.1.6 Ethyl Acetate,Omnisolv, glassdistilleodr HPLC grade. 8.1.7 Methanol, Omnisolv, glassdistilleodr HPLC grade. 8.1.8 8.1.9 Liver and controlliver,receivedfrozen from testin,l,aboratory. Milli-QT4 water, allwater used in thismethod should be rvl1 111 _QTII water and ma@, be provided by a Milli-Q TOC Plus system. 8.2 Standards 8.2.1 Prepare PFOS standards forthe standard curve. FACT-M-1.0 Extractionof PFOS from Liver PaLe 3 of 8 3M Environmental Laboratory Page 115 3m Medical Departmer@t Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 8.2.2 Weigh approximately 100 mg of PFOS intoa 100 mL volumetricflaskand record the actualweight. 8.2.3 Bring to volume with methanol fora stockstandardof approximatelv 1000 ppm (gg/mL). 8.2.4 Dilutethestocksolutionwith methanol fora working standardI solutionof approximately 50 ppm. 8.2.5 Dilutethe stocksolutionwith methanol fora working standard2 solutionof approx.5.0 ppm. 8.2.6 Dilutethe stocksolutionwith methanol fora working standard3 solutionof approx. 0.50 ppm. 9.0 SAMPLE HANDLIIN(; 9.1 All liversarereceivedfrozenand must be kept frozenuntilthe extractionisperformed. 10.0 QUALITY CONTROL 10.1 Matrix Spikes 10.1.1 Prepare and analyzematrix spike and matrix spike duplicatesamples to deter,-nine the accuracy of the extraction. 10.1.2 Prepare each spikeusingliverchosen by theanalyst,usuallya controlliver. 10.1.3 Expected concentrationswillfallinthe mid-range of the initiaclalibratiocnurve. 10.2 Continuing CalibrationChecks 10.2.1 Prepare and analyzecontinuingcalibrationcheck samples to determine the continuedlinearitoyf the i,-litciaallibratiocnurve. 10.2.2 One check isprepared per group of ten samples. For example, ifa sample set= 34, four checks are prepared and extracted. 10.2.3 Prepare each continuingcalibrationcheck from the same liverhomogeriate used to prep the initiaclur-ve. 10.2.4 The expectedconcentrationwillfallwithin the mid-range of the initiaclalibration curve. 11.0 CALIBRATION AND STANDARDIZATION 11.1 Prepare Liver Homogenate to Use for Standards 11.1.1 Weigh approximately 40 g of liverintoa 250 mL Nalgene bottlecontaining200 mLs Milli-QT'water.Grind to a homogeneous solution. 11.1.2 If40 g isnot availableu,se appropriateamounts of liverand water in keeping with a 1:5ratio. 11.1.3 See section1').0to calculatethe actualdensityof liver. FACT-M-1.0 Extractionof PFOS from Liver Page 4 of 8 3M Environmental Laboratory Page 116 3m Med4-cal Departmenl- Study: T-629S.7 Report No. FACT TOX-030 laboratory Request Numbe---U2279 11.1.4 Add I mL of homogeneous solutionto a 15 mL centrifugetube.Re-suspend homogeneous solutionby shaking between aliquotswhile preparinga totalof sixteenI mL aliquotsof homogeneous solutionin 15 mL centrifugetubes. 11.1.5 Two I mL aliquotsserveas matrixblanks. Use the standardconcentrationsand spikingamounts listedintable1 to spike,in duplicate,two standardcurves fora totalof fourteensamples. Table 1 Approximate Spiking Amounts for Calibration Standards Working Standard (Approx.Conc.) 0.50 ppm 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm 5.0 ppm 50ppm @iL Approx. finalconc.of PFOS in liver Blank 4 0.0 10 ppm 20 0.050 ppm 40 0.100 ppm -1-0 0.250 ppm 20 0.500 ppm 30 0.750 ppm 4 1.000 ppm 11.1.1 See section13.0 to calculateactualconcentrationsof PFOS in calibrationstandards. 11.2 Extractspiked liverhomogenates following 12.14-12.24of thismethod. Use these standardsto establisheach initiaclurve on the mass spectrometer. 12.0 PROCEDURES 12.1 Obtain frozenliversamples. In spenttissue,note thatthe liverhas not been packaged with other tissues. 12.2 Cut approximately I g of liverusinga dissectingscalpel. 12.3 Weigh the sample directlyintoa taredplasticsampule vial. 12.4 Record the liverweight in the studynotebook. 12.5 Label the sampule via]with thestudynumber, weight,liverID, dateand analystinitials. 12.6 Add 2.5 mLs of water to sampule vial. 12.7 Grind the sample.Put the grinderprobe in thesample and grindforabout 2 minutes,or untilthe sample ishomogeneous. 12.8 Rinse the probe intothe sample with 2.5 mLs water using a pipette. 12.9 Take the grinderapartand cleanitwith methanol aftereach sample. Follow AMDT-EP-22. 12.10 Cap the sample and vortex for 15 seconds. FACT-M-1.0 Extractionof PFOS from Li@.-er 3M Envi-ronmental Laboratory Page 5 of 8 Page 117 3m Medical Department S::udy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.11PipetteI mL homoizenate intoa 15 mL polypropylene centrifugetube.Label the centrifuge tube with the identicailnformationas thesampule vial.(See Worksheet fordocumenting the remaining steps.) 12.12Spike liverhomogenates with the appropfiateamount of PFOS standardas describedin section11.1 or Table 1. 12.13 Pipettetwo I mL aliquotsof Milli-Q' water to centrifugetubes. These willserve as instrument blanks. 12.14Add I mL 0.5 M TBA and 2 mL of the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.15Using a volumetricpipette,add 5 mLs ethylacetate. 12.16Cap each sample and put on the shakerfor20 minutes. 12.17Centrifugefor26 to 25 minutes,untillayersare well separated.Set power on the centrifuge to approximately -'1500rpm. 12.18Remove 4 mLs of organiclayer,usinga 5 mL graduated glasspipette,to a clean 15 mL centr-ifugteube.Label thisfreshtube with th6 same informationas in 12.5. 12.19 Put each sample on the analyticalnitrogenevaporatoruntildry,approximately 2 to 3 hours. 12.20Add 1.0mL of methanol toeach centrifugetube using a graduated pipette, 12.21Vortex mix for30 seconds. 12.22Attach a 0.2 pm nylon mesh filtetroa 3 cc syringeand transferthe sample tothissyringe. Filterintoa 1.5mL glassautovial. 12.23 Label the autovialwith the studynumber, animal number and gender,sample tirrepoint, matrix,finalsolvent,extractiondate,and analyst(sw)ho performed the extraction. 12.24Cap and hold forelectrospraymass spectrometry analysis. 12.25 Complete the worksheet and tapeto page of study notebook. 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.1 Calculatethedensityof liver(mg) in 1.0mL homogenate using thefollowing equation: g of Liver x Average weight of ten I mL aliquots(mp(g of Liver+ g of Water) FACT-M-1.0 Extractionof PFOS from Liver 3M Environmental Laboratory Page 6 of 8 Page 118 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 13.1.2CalculataectualconcentratioonfsPFOS incalibratisotnandardussingthe followingequation: @iL of Standard x Concentration(jig/TnL)= FinalConcentration(@Lg/gor mg/'kg) mg Liver'/I mL homogenate of PFOS in Liver *Average weight of liverin solutionas determined in 13.1.1,by weighing ten I mL homogenates of approximately40 mg liverin200 mL of Milli-Qwater. 14.0 METHOD PERFORMANCE 14.1 The method detection limitis equal to half the lowest standard in the calibrationcurve. 15.0 POLLUTION PREVENT TON AND WASTE MANAGEMENT 15.1 Sample waste isdisposed in biohs7.q d containers, flammable solvent waste isdisposed in high BTU containers, and used glass pipette waste isdisposed in broken glass containers located in the laboratory. 16.0 RECORDS 16.1 Complete the extraction worksheet and tape into the study notebook. 17.0 TABLES, DIAGRAms, FLOWCHARTS, AND VALIDATION DATA 17.1 The validation report associated with this-method isFACT-M-1.0 & 2.0-V-1. 18.0 REFERENCES 18.1 AMDT-EP-22, "Routine Maintenance of Ultra-Turrax T-25" 19.0 AFFECTED DocuMENTS 19.1 FACT--,'vf-2,"Analysis of Liver Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 REVISIONS Revision Number. Reason For Revision Revision Date 3M Environmental Laboratory FACT-M-1.0 Extraction of PFOS from Liver Page 7 of 8 Page 119 3m Medical Der)artmert Study: T-629S.7 Report No. 'T -Laboratory Request ExtractionWorksheet for FACT-M- I FACT TOX-030 Number-U2279 Study,; Sample Number set 4 H,O-Blank LiverBlank PFOS approx.0.5 ppm actual ppm 9w - PFOS approx.5 ppm actual ppm 9w PFOS approx. 50 ppm actual ppm 9w -ffac-tan d Initials for Std. Studv number where theori2inawlorksheetislocated, Blank LiverHomogenate: Std Liver Extr2ction Method Vorlex 15 sec. Ltvcramount p Date & lnit;als PipeiteImL ofLiverSolution PipettIemL oftO.5M TBA, pH 10. Std. Pipette2 mL of0.25Na2CO310.25M NaHCO3 Buffer Std. Pipett5e mL ofEthylAcetate TN-A- Shake 20 min. Centrifue2e0-25min. Centrifuee Speed Remove a4 mL aliquootforganiclaver Puton NitrogenEvaporatortodrvness Evaporator Add 1.0mL ofMethanol TN-A- Temperature Vortex30 sec. Filteursinga 3cc B-D svrinlewitha 0.2um SRI filteirntoa 1.5mL autosamplevial NISi'MSD/- Cont.Checks:Spiked- uL ofa ppm std fora finalconcentratioonf ppm. MS/ivISD usedsample Cont.Checks used same homogenate as forstdcurve, FACT-M-1.0 Extractionof PFOS from Liver Pac,,e8 of 8 3M Env4---onme.--talLaboratory Page 120 3m Medical Departmen-. Study: T-629S.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M ENVIRONMENTAL LABORATORY METHOD ANALYSIS OF FLUOROCHEMICALS IN LIVER EXTRACTS USING HPLC-ELF,CTROSPRAY/MASS SPECTROMETRY Method Number: FACT-M-2.0 Author: Lisa Clemen Approved By: AdoptionDate:5/)L.,/qy RevisionDate: A@;i.i LaboratoryManager Date Group Leader A TechnicalReviewer Date ,@-1271(le Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method isfortheanalysiosf extractosfliverorothertissuefsorfluorochemical surfactantussingHPLC-electrospray/masspectrometry. 1.2ApplicableCompounds: Potassiumperfluorooctanesulfonantieo,nicfluorochemical surfactantosr,otherionizablceompounds. 1.3Matrices: Rabbit,ratb,ovine,and monkey liverosr otherliverassdesignatedinthe validatiorneport. Word 7.0.1/95 FACT-M-2.0 Analysisof LiverExtractUsing ES/MS 3M Environmental Laboratory Page] of8 Page 121 3m Medical Departmen-z S--udy: T-6295.7 Report No. FACT TCX-030 laboratory Request Number-U2279 2.0 SUM,-YIARY OF INIETHOD 2.1 This method describesthe analysisof fluorochemical surfactantsextractedfrom liverusing HPLC-electrospray/mass spectrometry.The analysisisperformed by monitoring a sI*n,cle ion characteristicof a particularfluoro'chemical;,uchlas the potassium perfluorooctanesulfonate(PFOS) anion,M/Z= 499. Samples may also be screened to verif@ compound identification. 3.0 DEFINITIONS 3.1 None. 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltagecable for the probe. When the voltage cable isplugged into the probe DO NOT TOUCH THE PROBE, thereisriskof electricaslhock. 4.2 Cautions: 4.2.1 Do not run solventpumps above capacityof 400 bar (5800 psi).Ifpressuregoes over 400 bar,the HP I 100 will initiataeutomatic shutdown. 4.2.2 Do not rlinsolvent pumps to dryness. 5.0 INTERFERENCES 5.1 Teflon should not be used forsample storage or any part of instrumentationthatcomes in contact with the sample or extract. 6.0 EQUIPMENT 6.1 Equipment listedbelow may be changed in order to optimize the system. 6.1.1 Micromass ElectrosprayMass Spectrometer 6.1.2 HPI 100 low pulse solventpumping system and autosampler. 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.1.1 Nitrogen gas,refrigeratedliquid,regulatedto approximately 100 psi. 7.1.2 HPLC column, specificsto be determined by the analyst. 7.1.3 Capped autovialsor capped 15 mL centrifugetubes. 8.0 REAC;ENTS AND STANDARDS 8.1 Reagents 8.1.1 Methanol,HPLC gradeorequivalent. Word 7.0.1/95 FACT-M-2.0 Analysis of Liver ExtractUsing ES/MS 3M Envi---onmental Laboratory Page 2 of 8 Page 122 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03C laboratory Request Number-U2279 8.1.2Milli-QwTat-emr,allwaterused inthismethod shouldbe N11111 _QT"l water and may be provided by a Milli-Q TOC Plus system. 8.1.3 Ammonium acetate,HPLC grade or equivalent. 8.2 Standards 8.2.1 Typically one H20 blank,one liverblank,and seven liverstandardsare prepared during the extractionprocedure. See FACT-M- I 9.0 SAMPLE HANDLING 9.1 Fresh liverstandards are prepared with each analysis.Extracted standards and samples are stored in capped autovialsor capped 15 mL centrifugetubes untilanalysis. 9.2 If analysiswill be delayed, extractedstandardsand samples may be refrigerateduntil analysis can be @erformed. 10.0_ QUALITY CONTROL 10.1 Matrix Blanks and Method Blanks 10.1.1 Analyze a method blank and matrix blank prior to each calibrationcun,e. 10.2 Matrix Spikes 10.2.1 Analyze a matrix spike and matrix spike duplicatewith each analysis. 10.2.2 Expected concentrationswillfallinthe mid-range of the initiaclalibrationcurve. Additional spike concentrationsmay fallin the low-range of the initiaclalibration curve. 10.2.3 See section 13 to calculatepercent recovery. 10.3 Continuing Calibration Checks 10.3.1 Analyze a mid-range calibrationstandard afterevery tenth sample. Ifa significant change ( 30%) in peak area occurs,relativeto the initiasltandard curve,stop the run. Only those samples analyzed before the lastacceptable calibrationstandard will be used. The remaining samples must be reanalyzed. 10.3.2 See section 13 to calculatepercent difference. 10.4 System Suitability 10.4.1 System suitabilit(y-..gp.eak area,retentiontime and peak shape, etc.)@villbe assessed for each run. 11.0 CAL113RATION AND STANDARDIZATION 11.1 Analyze the extractedliverstandardspriorto and following each setof extracts.The mean of two standard values,at each standard concentration,will be plottedby linearregression forthe calibrationcurve using MassLynx or othersuitablesoftware. 3M Environmental FACT-M-2.0 Analysis of Liver Extract Usinc,,ES/i@vIS Laboratory Page 3 of 8 Page 123 3m Medical Deuartment Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 11.2 The r-v'alue forthe data should be 0,98 or greater.Lower valuesmay be acceptableatthe discretionof the analyst. 11.3 Ifthe curve does not meet requirements,perform routinemaintenance or reextractthe standard curve (ifnecessary)and reanalyze. 12.0 PROCEDURES 12.1 Acquisition Set up 12.1.1 Click on startbuttonintheAcquisitionControl Panel.Set up a sample list.Assign a filenameusing letter-MO-DAY-lastdigitof year-samplenumber, assigna method (MS) foracquiring,and typein sample descriptions. 12.1.2 To createa method clickon scan buttonin theAcquisitioncontrolpanel and select SIR. SetlonizationMode as app:ropriataend mass to 499 or otherappropriate masses. A scan isusuallycollectedalong with the SIRS. Save method. 12.1.3 Typicallythe sample listbeginswith the firstsetof liverstandardsand ends with the second setof standards. 12.1.4 Samples are analyzed with a continuingcalibrationcheck injectedafterevery tenth sample. Solvent blanksshould be analyzed periodicalltyo monitor possibleanalyte carryoverand are not consideredsamples but may be includedas such. 12.2 Using the Autosampler 12.2.1 Set up sample trayaccordingtothesample lisptreparedinsection12.1.1. 12.2.2 Set-up the HP II00/autosampleratthe followingcon'ditionsor atconditionsthe analystconsidersappropriateforoptimal response.Record actualconditionsinthe instrumentlogbook: 12.2.2.1Sample size= 10 @tLinjectionwith a sample wash 12.2.2.2Inj'ect/sampl=e 1 12.2.2.3Cycle time = 15 minutes 12.2.2.4Solvent ramp = Time I 0.00 min. 7.5 min. 11.0 min. 1 11.5 min. MEOH 45% 90% 90% 45% 2.0 mM Ammonium acetate 55% 10% 10% 55% Note: In thisinstrumentconfigurationt,he run must be setup on the clectrosprav softwarewith a "Waiting forinletstart"message beforethe"Start"button is pressed on the HP Workstation. 12.2.2.5Press the"Start"button. FACT-M-2.0 Analysisof LiverExtractUsing ES.,i\,IS 3M Env4-ronmental Laboratory Page 4 of 8 Page 124 3m Medical Department S:::udy: T-6295.7 Report No. FAC-- -@OX-030 laboratory Request Numbe---U2279 12.3InstrumenStep-up 12.3.R1efetroA,@,flDT-EfPo-rm3o1redetails. 12.3.C2heckthesolvelnetveilnreservoainrdrseflilfnecessary. 12.3.3 Check the stainlesssteelcapillaryatthe end of the probe. Use an eye piece to check the tip. The tipshould be flatwith nojagged edges. If the tipisfound to be unsatisfactory,disassemble the probe and replace the stainlesssteelcapillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropn-ate. Observe dropletscoming out of the tipof the probe. Allow to equilibratefor approximately 10 minutes. 12.3.5 Turn on the nitrogen.A finemist should be expelledwith no nitrogenleaking around the tip of the probe. l@.3.6 The instrument uses these parameters at the following settings.These settingsmay change in order to optimize the response: 12.3.6.1Drying gas 250-400 liters/hour 12.3.6.2ESI nebulizinggas 10-15 litersihour 12.3.6.3LC constantflow mode flow rate 10 - 500 uL/min 12.3.6.4Pressure <400 bar (This parameter isnot set,itisa guide to ensure the instrument is operatingcorrectly.) 12.3.7 Carefullyguide theprobe intothe opening. Insertprobe untilitwillnot go any further.Connect the voltage cables to the probe. 12.3.8 Record tune -parametersin the instrument loiz. 12.3.9 Using the cross-flow counter electrodein the ES INIS source isrecommended for the analysisof biologicalmatrices. 12.3.10 Click on startbutton in the Acquisition Control Panel. Press the startbutton at top of sample list.Ensure startand end sarnple number includes allsamples to be analyzed. 13.0 DATA ANALYSIS 13.1 Calculations: AND CALCULATIONS 13.1.1 Calculate matrix spike percent recoveries using the following equation: % Recovery Observed Result-Background Result x 100 Expected Result 13.1.2 Calculatepercent differenceusing the following equation: % Difference Expected Conc. -Calculated Conc. x 100 Expected Conc. 3M Environmental FACT-M-2.0 Analysis of Liver Extract Usini2 ES/N/IS Laboratory Page 5 of 8 Page 125 3m Medical Department Study: T-6295.7 Report No. FAC'-R TOX-030 laboratory Request Number-U2279 13.1.3 CalculateactualconcentratioonfPFOS anionintotaliver(mg): ug PFOS anion calc.from std curve g of liverused formialysis 1000 ugl I mg x Total mass of liver 14.0 IMETHOD PERI@OP@MANCE 14.1 The method detectionlimitis equal to at leastthreetimes the baselinenoise in the matrix blank. 14.2 The practicalquantitationlimitisequal to the lowest standard in the calibrationcurve. 15.0 POLLUTION PRF!RVENTION AND WASTE MANAGEMENT 15.1 Sample waste is disposed in biohazard containers,flammable solventwaste is disposed in high BTU containers,and glasspipettewaste isdisposed in broken glass containers. All containers are located in the laboratory. 16.0 RECORDS 16.1 Store chromatograms in the study folder.Each chromatogram should have the following information included eitherin the header or hand writtenon the chromatogram: study number, sample narne,extractiondate,and dilutionfactor(ifapplicable). 16.2 Plot calibrationcurve by linearregressionand store in the study folder. 16.3 Printsample listfrom MassLynx and tape intothe instrumentrunlog. 16.4 Printdata integrationsummary from MassLynx and tape intothe instrument runlog. 16.5 Copy instrument runlog pages, includinginstrument parameters and sample results,and tape intoappropriate study notebook. 16.6 Summarize data using suitablesoftware and storein the study folder. 16.7 Back up electronicdata to appropriatemedia. Record in study notebook the filename and locationof backup electronicdata. 17.0 TABLES, DIAGRAms, FLOWCHARTS, AND VALIDATION DATA 17.1 Attachment A: FACT-M-2 Data reportingspreadsheet 17.2 The validationreportassociatedwith thismethod is FACT-M- 1.0 & 2.0-V-1. 18.0 REFERENCES 18.1 ANIDT-EP-3 1,"Operation of VG Platform ElectrosprayMass Spectrometer" 3M Environmental FACT-M-2.0 Analysis of Liver ExtractUsing ES;'MS Laboratory Page 6 of8 Page 126 3m Medical Department Study: T-629S.7 Report No. FACT TOX-030 laboratory Request Number-U2'L79 19.0 AFFECTED DOCUMENTS 19.1 FACT-M-1.0, "Extractioonf PotassiumPerfluorooctanesulfonfartoem LiverforAnalysis Using HPLC-Electrospray/MassSpectrometry" 20.0 REVISIONS Revision Number. Rmon For Revision Revision Date FACT-M-2.0 Analysisof LiverExtractUsing ES/MS 3M Environmental Laboratory PagC7e 7 of 8 Page 127 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03C laboratory Request Number-U2279 Laboratory Study Study: Test Material: Matrix/FinaSlolvent: Method/Revision: AnalyticalEquipment System Number-. InstrumentSoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept: Dateof Extraction/Analy5t: Dateof Analysis/Analyst: Group Dose Sample# Concentration ug/mL InitiaVlol. mL Dilution Factor FinalConc. ug/mL Slope: Taken ftom linearegressioenquation. Group/Dose: Taken from thestudyfolder. Samplei$:Taken fromthestudyfolder. Concentration(ug/mL): Taken from theMassLynx integratisounmmary. InitiaVlolume (mL): Taken fromthestudyfolder. DilutionFactor:Taken fromthestudyfolder. FinalConc. (ug/mL): Calculatebdy dividintgheinitiavlolume from theconcentration FACT-M-2.0 Analysisof LiverExtractUsing ES/MS 3M Environmental Laboratory Page 8 of 8 Page 128 3m Med@-cal Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M ENVIRONMENTAL LABORATORY METHOD EXTRACTIOP4-OF POTASSIUM PERFLUOROOCRA.NESULFONATE OR OTHER FLUOROCHEMICAL COMPOUNDS FROM Lll,'ER FOR ANALYSIS USING HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: FACT-M-1.1 Author: LisaClemen, Glenn Langenburg Approved By: -7 1)1"14 LaboratoryManager -ILI',v @E7ro@UPLeader @@, ,A Technical Reviewer AdoptionDate:05/26/98 RevisionDate: 06),))Ill Date 6/1 /14@ Date li),i55 Date 1.0 SCOPE AND APPLICATION 1.1Scope: Thismethod isfortheextractionf potassiumperfluorooctanesulfo(nPaFtOeS) or otherfluorochemicalcompounds from liver. 1.2Applicable Compounds: Fluorochemicalsurfactantosr otherfluon*natecdompounds. 1.3Matrices: Rabbit,rat,bovine,andmonkeyliveorrotherlivearsdesignateidnthevalidation report. N,ficroso6f.t0/95 3M Env4.--onmental Laboratory FACT-M- 1.1 ExtractionofPFOS from Liver Page I of 15 Page 129 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0SUMMARY OF METHOD 2.1 This method describestheprocedureforextractinpgotassiumperfluorooctanesulfonate (PFOS) or other fluorochemicalsfrom liverhomogenate using an Ion pairingreagent and 5.0ml of ethvlacetate.Inthismethod,seven fluorochemicalwsere extractedP:FOS, PFOSA,PF6sAA, ETFOSE-OH, POAA, PFOSEA, and FC-807 monoester (see3.0 Definitions).An ionpairingreagentisadded tothesample and theanalyteIon pairis partitioneidntoethylacetate.Four ml ofextractareremoved and putonto a nitrogen evaporatorunt@ldry. Each extractisreconstituteidn 1.0ml of methanol,then filtered through a 3 cc plasticsyringeattachedtoa 0.2 @Lm nylon filteirntoglassautovials. 3.0 DEFINITIONS 3.1 PFOS: perfluorooctanesulfonate(anion of potassium salt)CF17SOl3.2 PFOSA: perfluorooctanseulfonylamideC,F,7SO,NH, 3.3 PFOSAA: perfluorooctanseulfonylamido(ethyl)acetaCt@eF,,SO,N(CH,CH,)CH,CO,3.4 ETFOSE-OH: 2(,N-ethylper-fluorcoestualnfeonwnido)-ethylalcohol C,F17So2N(CH2CH3)CH,CH20H 3.5 POAA: perfluorooctanoat(eanionofammonium salt)C,F,,COO' 3.6 PFOSEA: perfluorooctanseulfonylethylamideC,F,7SO,N(CH,CH3)H 3.7 FC-807 monoesterC,F,,SO,N(CH,CH,)CH2CH,O-PO,H) 3.8 SurrogatestandardIH, IH,2H,2H perfluorooctanseulfonicacid 4.0 WARNINGS AND CAUTIONS 4.1 Health and safetywarnings: 4.1.1 Use universalprecautionse,speciallylaboratorycoats,goggles,and gloveswhen handlinganimal tissuei,tmay containpathogens. 5.0 INTERFERE14CES 5.1 There areno known interferenceastthistime. 6.0 EOUIPMENT 6.1 The followingequipment isusedwhilecarryingout thismethod. Equivalentequipment is acceptable. 6.1.1 6.1.2 6.1.3 6.1.4 Ultra-Turraxwith T25 grinderattachmentforgrinding/dispersing/emulsifying Vortex mixer,VV@'R,Vortex Genie 2 CentrifugeM,istral1000 orIEC Shaker,Eberbach or VWR 6.1.5 NitrogenevaporatorO,rganomation 6.1.6 Balance,( 0.100 g) 3M Environmental Laboratory FAC'T-M- 1.1 ExtractioonfPFOS from Liver Page 2 of 15 Page 130 3m Medical Departmen,-- Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 7.0 SUPPLIES A,*,*MDATERIALS 7.1 Gloves 7.2 Eppendorf or disposablepipettes 7.3 Nalgene bottlesc,apable of holding250 ml and I L 7.4 Wheaton 6 ml PlasticSampule Vials 7.5 Glass,type A, volumetricflasks 7.6 40 ml glassI-CHENF vials 7.7 Polypropylene centrifugetubes,15 ml 7.8 Labels 7.9 Syringes,capable of measuring 5 @iLto50 gL 7.10 Glass,type A, volumetricpipettes 7.11 Graduated pipettes 7.12 ElectronicpipettorE,ppendorf or equivalent 7.13 Timer 7.14 Disposable plastic3 cc syringes 7.15 Filtersn,ylon syringefilters0,.2 um, 25 mm 7.16 Crimp cap autovials Note: Priorto using glasswareand bottlesr,inse3 timeswith methanol and 3 timeswith Milli- Q TM water. Rinse syringesa minimum of 9 timeswith methanol, 3 rinsesfrom 3 separate vials. 8.0 REAGENTS AND STANDARDS 8.1 ASTM Type Ireagentgade water,Milli_QTM or equivalent;allwater used inthismethod should be Milli_QTMwater and may be provided by a Milli-Q TOC plUST,'Slystem. 8.2 Sodium hydroxide (NaOH), J.TBaker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate(NaC03), J.T.Baker or equivalent 8.5 Sodium bicarbonate(NaHC03), J.T.Baker or equivalent 8.6 Ethyl acetate,Omnisolv, glassdistilleodr HPLC grade 8.7 Methanol, Omnisolv, glassdistilleodr HPLC gade 8.8 Liver tissue,frozenfrom supplier 8.9 Control matrix or blank matrix forstandards,QC checks,blanks,etc. 8.10 Fluorochemical standards 8.10.1 PFOS (3M SpecialtyChemical Division),molecular weight= 538 8.10.2 PFOSA (3M SpecialtyChemical Division),molecularweight = 499 8.10.3 PFOS.-@-A(3M SpecialtyChemical Division),molecular weight = 585 3M Env4-ronmental Laboratory FACT-M- 1.1 ExtractioonfPFOS from Liver Page 3 of 15 Page 131 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 8.10.4 ETFOSE-OH (3M SpecialtyChemical Division),molecularweight= 571 8.10.5 POAA (3M SpecialtyChemical Division),molecularweight = 431 8.10.6 PFOSEA (3NI SpecialtyChemical Division),molecularweight = 527 8.10.7 FC-807 monoester (3M SpecialtyChemical Division).FC-807 isa mixture of triesterd,iestera,nd monoester fluorochemicalcomponents. The monoester molecular weight = 650 8.10.8 SurrogateStandard:4-H, perfluorooctanesulfonicacid (1-H,I-H, 2-H,2-H C,F,,SO3H) molecularweight = 428 8.10.9 Other fluorochemicalsa,s appropriate 8.11 Reagent preparation 8.11.1 10 N sodium hydroxide(NaOH): Weigh approximately200g NAOH. Pour intoa 1000 ml beakercontaining500 ml Milli-Q"'water,mix untilallsolidsare dissolved.Storeina I L Nalgene bottle. 8.11.2 1 N sodium hydroxide (NaOH): Dilute ION NAOH 1:10. Measure 10 mi of 1ON NAOH solutionintoa 100 ml volumetricflaskand diluteto volume using Milli-Q"' water. Storein a 125 ml Nalgene bottle. 8.11.3 0.5M tetrabutylammonium hydrogen sulfate(TBA): Weigh approximately169 grams of TBA intoa I L volumetriccontaining500 MI Milll_QTIwIater.Adjust to pH 10 using approximately44 to 54 ml of ION NAOH and diluteto volume with Milll_QTIwater. While adding the lastfew ml's of NAOH, add slowly because the pH changes abruptly.Storein a I L Nalgene bottle. 8.11.3.1TBA requiresa check priorto each use to ensurepH = 10. Adjust as needed using IN NAOH solution. 8.11.4 0.25M Sodium carbonate/sodiumbicarbonatebuffer(Na@CO,/NaHCO,): Weigh approximately26.5g of sodium carbonate(Na.CO,) and 21.0 g of sodium bicarbonate(,N'aHCO,)intoa I L volumetricflaskand bringtovolume with Milli- Q TM water. Storein a I L nalgene bottle. 8.12 Standards 8.12.1 Prepare PFOS standardsforthe standardcurve. 8.12.2 Prepare otherfluorochemicalstandards,as appropriate.Multicomponent fluorochemicalstandardsareacceptable(e.g.one working standardsolution containing1.00 ppm PFOS, 1.02ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.12.3 Weigh approximately100 mg of PFOS intoa 100 ml volumetricflaskand record the actualweight. 8.12.4 Bring to volume with methanol fora stockstandardof approximately1000 ppm (@ig/ml). 8.12.5 Dilutcthestocksolutionwith methanol fora working standardI solutionof approximately 50 ppm. 3M Environmental Laboratory FACT-M- 1.1 ExtractioonfPFOS from Liver PaLe 4 of 15 Page 132. 3m Medical Department S:@udy: T-6295.7 Report Nc. FACT TOX-030 laboratory Request Number-U2279 8.12.6 Dilutethestock solutionwith methanol fora working standard2 solutionof approx. 5.0 ppm. 8.12.7 Dilutethestock solutionwith methanol fora working standard3 solutionof approx. 0.50 ppm. 8.13 Surrogate stock standard preparation 8.13.1 Prepare-asurrogatestockstandard.Weigh approximately 50-60 mg of surrogate standard 1-H,I-H, 2-H,2-H, C,F,3SO3H intoa 50 ml volumetricflaskand record the actualweight. 8.13.2 Br-ingto volume with methanol fora surrogatestockof approximately 1000- 1200 PPM. 8.13.3 Prepare a surrogate working standard. Transfer approximately 0.5 mi of surrogate stockto i 50 ml volumetricflaskand bringto volume with methanol fora working standardof 10-20 ppm. Record theactualvolume transferred. 8.14 Liver homogenate preparation Note.-The followingprocedure willbe much easiertoperform withfrozen liver tissue.Prevent tissuefromthawing; keep storedon iceuntileccisingaportion of it.- 8.14.1 Weigh 40 g of blank or controlliverintoa 250 ml Nalgene bottlecontaining100 mls Milli-Q'Tw"ater. Record theactualweight of liverand totalvolume of water used. Grind the liverintoa finelydispersedhomogenate with an Ultra-TurraxT25 grinder(highspeed forapproximately3 minutes or untilsufficiently homogenized). Rinse grinderwith an additional100 MI Of MilllQTIIwater,to bring the totalvolume of water added to 200 ml. 8.14.2 To determine theconcentrationof the blank liverhomogenate, transferten 1.0 ml aliquotsof the homogenate totaredpolypropylene tubes,and weigh each aliquot on a balance. The averagedensityof thesealiquotsisdetermined and then the concentration(g of liver/mlof homogenate) can be calculatedas follows: 8.14.3 [,zrams(g)of liver]x favizw.eight of 1.0ml of homogenate (density)(,sz/ml)l f[grams (g)of liver]+ [grams (g)of water]@ 8.14.3 Preparesample liversas describedin8.3.1,butweigh out I g of liverh,omogenize with 2.5 ml of MilliQ" water,and n'nsewith another2.5MI Of MIIIIQTI water. Use Wheaton 6 ml plasticsampule vialsor appropriatereceptacle.Rinse grinder unitafterevery sample with water and thenwith methanol. Label vials appropnately includingstudynumber, sample ID, liverweight,date,and analyst. Record allweights and volumes used. (Do notperform 8.3.2forthe sample liver homogenates). 3M Environmer-tal Laboratory FACT-M- 1.1 ExtractioonfPFOS fromLiver Page 5 of 15 Page 133 3m Medical E)epartment Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 9.0 SANIPLE HANDLING 9.1 All liversare received frozenand must be kept frozen untilthe extractionisperformed. 10.0 ()UALITY CONTROL 10.1 Matrix blanks and method blanks 10.1.1 Extract two 1.0 ml aliquots of the liver homogenate (prepared in 8.14.1-2) following thisprocedure and use as matrix blanks. See Section 11.1.2. 10.1.2 Extracttwo 1.0ml aliquotsof Milli-Q' water followingthisprocedure and use as method blanks. 10.2 Matrix spikes 10.2.1 Prepare dnd analyze matrix spike and matrix spike duplicatesamples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using liverchosen by the analyst,usually the control liver received with each sample set. 10.2.3 Expected concentrationsfallin the mid-range of the initiaclalibrationcurve. Additional spikesmay be includedand may fallin the low-range of the initial calibrationcurve. 10.2.4 Prepare one matrix spike and one matrix spike duplicateper 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibrationchecks 10.3.1 Prepare and analyze continuingcalibrationcheck samples to ensure the accuracy of the initiaclalibrationcurve. Ifthe percentdifferencebetween the initiaclurve and the continuing check differby >30%, reanalyze samples analyzed afterthe last acceptable check. 10.3.2 Prepare one check per group of ten samples. For example, ifa sample set = 34, prepare and extractfour checks. 10.3.3 Prepare each continuing calibrationcheck from the same blank liverhomogenate used to prepare the initiaclurve. 10.3.4 The expected concentrationsfallwithin the mid-range of the initiaclalibration curve. Additional spikesmay be included thatfallin the low-range of the initial calibrationcurve. This isnecessary ifthe analystmust quantitateusing only the low end of the calibrationcurve (e.g.10 ppb - 100 ppb, ratherthan 10 ppb - 1000 ppb). 11.0 CALIBRATION AND STANDARDIZATION 11.1 Prepare liverhomogen ate standards 11.1.1 Transfer I ml aliquotsof blank/controlliverhomogenate prepared in 8.14.1-2 to 15 mi centrifugetubes. 3M Environmental Laboratory FACT-M-1.1 Extractionof PFOS from Li@,,er Page 6 of 15 Page 134 3m Medical Departmert Study: T-629S.7 Report No. FACT TOX-030 laboratory Request Number-U2279 11.1.2 Ifthe volumes of sample liverhomogenates are limited,extractstandardswith liver homogenate volumes equalto the sample volumes. Do not extractbelow 0.50 ml of liverhomogenate. Record thesample volume on the extractionsheet. 11.1.3 V;hllepreparinga totalof twenty aliquotsof liverhomogenate in 15 ml centrifuge tubes,mix or shake between aliquots. 11.1.4 Two I mi, or appropriatealiquotss,erveas matrix blanks. Typicallyuse the standaraconcentrationsand spikingamounts listedinTable I (atthe end of this section)to spike,in duplicatet,wo standardcur-ves,fora totalof eighteenstandards and two matrix blanks. 11.1.5 Refer to the validationreportsFACT-M-1.1-V-1 and FACT-M-2.1-V-1 which list theworking rangesand LinearCalibrationRan-0e (LCR) forcalibratiocnurves. 11.1.6 Use Attachment D as an aid in calculatingthe concentrations of the working standards. See SectIon 13.0to calculateactualconcentrationsof PFOS in calibrationstandards. 11.2 To each standard,blank,or QC check,add appropriateamount of surrogateworking standardforthe concentrationto fallwithintfiecalibratiocnurve range 10 ppb - 1000 ppb. 11.3 Extractspiked liverhomogenate standardsfollowing12.6-12.16of thismethod. Use these standardsto establisheach initiaclurve on themass spectrometer. Table I Approximate Spiking Amounts for Standards and Spikes Using 1.0ml of Liver Working Standard @tL Approx. finalconc. of (Approx.Cone.) - - PFOS in liver Blank 0.500 PPM 4 0.012 ppm 0.500 ppm 10 0.030 ppm 0.500 ppm 20 0.060 ppm 0.500 ppm 40 0.120 ppm 5.00 ppm 10 0.300 ppm 5.00 ppm 20 0.600 ppm 5.00 ppm 30 0.900 ppm 50.0 ppm 4 1.20 ppm 50.0 ppm 6 1.80 PPM 12.0 PROCEDURES 12.1 Obtain frozenliversamples and homogenize as describedin 8.14.3. 12.2 Vortex mix homogenate for 15 seconds,then transfe.1-.0ml or otherappropriatevolume to a 15 ml polypropylene centrifugetube. 12.3 Return liverhomogenate samples to freezerafterextractionamount has been removed. 3M Environmental Laboratory FACT-M- 1.1 Extractioonf PFOS froniLiver Page 7 of 15 Page 13S 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.4 Record the liverhomogenate volume on the extractionworksheet. The finalmethanol volume willequal theinitiahlomogenate volume. For example, ifI ml of homogenate is transferredforextractiont,henthe finalreconstitutiomnethanol volume equals I ml. 12.5 Label the tube with *he study number, liverID, date,and analystinitialsS.ee attached worksheet fordocumenting the remaining steps. 12.6 Spike blank liverhomogenate aliquotswith the appropriateamount of standardas described in Section11.1 or Table I in thatsectionforthe calibratiocnurve standards. Also preparematrix spikesand continuingcalibrationstandards. 12.7 Spike allsamples, includingblanksand standards,ready forextractionwith surrogate standardas describedin Section11.2. 12.8 Vortex mix the standardcurve samples,matrix spikesamples,and continuingcalibration samples for15 seconds. 12.9 To each sample, add I ml 0.5 M TBA and 2 ml of the 0.25 M sodium carbonate/sodium bicarbonatebuffer. 12.10Using a volumetricpipette,add 5 ml ethylacttate. 12.11 Cap each sample and put on theshaker for20 minutes. 12.12 Centrifuge for20 to 25 minutes atapproximately 3500 rpm, untillayersare well separated. 12.13Transfer4 ml of organiclayer,usinga 5 ml graduated glasspipettet,o a clean 15 ml centrifugetube.Label thisfreshtube with the same informationas in 12.5. 12.14Put each sample on the analyticanlitrogenevaporatoruntildry,approximately 2 to 3 hours. 12.15Add 1.0ml or appropriatevolume of methanol to each centrifugetube usinga graduated pipette.Methanol volume equalsthe initiavlolume of liverhomocenate used forthe extraction. 12-16Vortex mix for30 seconds. 12.17 Attach a 0.2 @im nylon mesh filtetro a 3 cc syringeand transferthesample to thissyringe. Filterintoa 1.5ml glassautovial(orlow-volume autovialwhen necessary). 12.18Label theautovialwith thestudynumber, animal number and gender,sample timepoint, matrix,finalsolvent,extractiondate,and analyst(sw)ho performed the extraction. 12.19Cap and storeextractsatapproximately4' C untilanalysis. 12.20 Complete the extractionworksheet, attachedto thisdocument, and tape topage of study notebook or includein studybinder,as appropriate. 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.1 Calculateactualconcentrationsof PFOS, or other appropriatefluorochemical,in calibratiosntandardsusingthe followingequation: 3M Environmental Laboratory FACT-M- 1.1 Extractioonf PFOS from Liver Page 8 of 15 Page 136 3m Medical Departmer.L'- S@@udy: T-6295.7 Report No. FAC-L TOX-030 laboratory Request Number-U2279 ml of Standard x Concentrationof Standard(4-g/ml) ConcentrationofBlank LiverHomogenate (g/ml)(see8.14.2) FinalConcentration(@ig/g)of PFOS in L,-.ver See Attachment D fora sample form to calculatethe concentrationsof standards. 14.0 METHOD PERFORMANCE 14.1 The method detectionlimit(MDL) isanalyteand matrixspecific.Refer to MDL reportfor spee'lficMDL andlimiotfquantitat(iLoOnQ)value(sseeAttachmentBs and C). 14.2 The followingqualitycontrolsamples are extractedwith each batch of samples to evaluate the qualityof the extractionand analysis. 14.2.1 Method blanks and matrixblanks 14.2.2 Matrix spikeand matrixspikeduplicatesamples to determine accuracy and precisionof the extraction 14.2.3 Continuing calibratiocnheck samples to detentilnethe continued accuracy of the initiaclalibratiocnurve. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample waste isdisposedinbiohazardcontainersf,lammable solventwaste isdisposed in high BTU containers,and used glasspipettewaste isdisposed in broken glasscontainers locatedin the laboratory. 16.0 REcoRDs 16.1 Complete the extractionworksheet attachedtothismethod, and tape intothe study notebook or includeintostudy binder,as appropriate. 17.0 ATTACHMENTS 17.1 Attachment A, Extractionworksheet 17.2 Attachment B, MDL/LOQ values 17.3 Attachment C, LOQ summary 17.4 Attachment D, Calibrationstandardconcentrationworkshect 18.0 REFERENCES 18.1 The validationreportsassociatedwith thismethod are FACT-M-1.1 and 2.1-V-1. 19.0 AFFECTED DOCUTVIENTS 19.1 FACT-M-2. 1, "Analysisof Liver ExtractsforFluorochemicalsusing HPLC-Electro spray Mass Spectrometry" 3M Environmental Laboratory FACT-M- 1.1 ExtractioonfPFOS from Liver Page 9 of 15 Page 137 3m Medical Departmer-t Study: T-629S.7 Report No. FACT TOX-030 laboratory Request Number-U2279 20.0 REVISIONS Revision Number. 1 Validation systems, e.-ctraction, etc. of method monkey liver MDL study, Reason For Re-visio to include 7fluorochemicals, cross validation, improvements updates in record keeping new APIIMS(MS) .. to ion pai ring and storing policies, Revision Date 08/01/98 3M Environmental Laboratory FACT-M- 1. 1 ExtractioonfPFOS from Liver Page 10 of 15 Page 138 3m Medical Department Study: T-6295.7 Report No. FAC-- TOX-030 laboratory Request Number-U2279 Study# Matrix Boxg Surroga,.eStd. approx. ppm actual PPM FC Mix Std approx.0.5 ppm actual PPM AnalysUDate H,O Blank Blank FC Mix Std approx.5 ppm actual PPM '"W FC Mix Std approx. 50 ppm actual PPM Comments Blank Std ExtractioMnethod/Revision: amount Date& lniti2is Add SurrogateV,ortex ISsec. PipettSeample Volume ml PipettIemi of 0.5M TBA, pH 10. PH Std. Pipett2emi of0.25Na2CO3/0.25M NaHC03 buffer Std. Pipett5emi of ethylacetate Shake 20 min. Centrifu.g2e0-25min. Remove a 4 mi aliquotof organiclayer Puton Nitro_geEnvaporatortodryness Add methanol Volume TN-AShaker Speed -Centrifugsepeed: Temperature: ml TN-A- Vortex30 sec. _Filterusinga 3ccB-D syrineewitha 0.2pm SRI filteirntoa 1.5ml autosamplevial MSfMSD/- Cont.Checks:Spiked- uL ofa ppm std( ) fora finacloncentratioonf ppm. IvISi'NISuDsedsample Cont,Checks used same matrixas forstdciir-@e. AttachmentA: Extractiownorkshect 3M Environmental Laboratory FACT-M- 1.1 ExtractioonfPFOS from Liver PacycII of 15 Page 139 3m Medical Department Szudy: T-6295.7 Report No. FAC'@" TOX-030 laboratory Request Number-U2279 MDL/LOQCompound @PF-OS PFOSA PFOSAA ETFOSE-OH POAA PFOSEA Monoester values for Rabbit Liver: MDL LOQ Linear CalibrationRan-e (LCR) (ppb) (ppb) Approximate Concentrations to be used for preparing the Standard Calibration Curve 11.8 37.4 38 ppb - 1000 ppb 6.06 19.3 120 ppb - 1200 ppb 55.7 177 180 ppb - 1000 ppb 58.7 187 190 ppb - 1800 ppb 23.7 75.5 76 ppb - 1800 ppb 16.2 n/v 51.7 62 ppb - 1200 ppb n/v I Monoester was not detectable/quantifiabaltethespiked concentrations. MDL/LOQ Compound PFOS PFOSA PFOSAA ETFOSE-OH POAA PFOSEA Monoester values for Rat Liver: MDL LOQ LinearCalibratioRnange (LCR) (ppb) @(ppoppbQb))IAALStpipapnIroximate Concentrationstobe used forpreparingthe Standard CalibrationCurve 24.7 78.7 62 ppb - 1200 ppb 20@7 65.8 20 ppb - 1200 ppb n/v n/v _62ppb - 1200 ppb n/v n/v 120 ppb - 1200 ppb n/v n/v 62 ppb - 1200 ppb n/v n/v 120 ppb - 1200 ppb n/v n/v Monoester was not detectable!quantifiaabtlethespikedconcentrations. i'vIDLILOQ valuesforMonkey Liver: Compound MDL LOQ LinearCalibrationRange (LCR) (ppb) (ppb) Approximate Concentrationsto be used forpreparingthe Standard CalibrationCurve PFOS n/v rVv 59 ppb - 1200 ppb PFOSA 27.4 87.1 28 ppb - 1200 ppb PFOSAA nJv n/v 120 ppb - 1200 ppb ETFOSE-OH n/v I n/v 58 ppb - 1200 ppb POAA n/v n/v 120 ppb - 1200 ppb PFOSEA n/v n/v 120 ppb - 1200 ppb Nlonoester niv n/v Monoester was not detectableq,uantifiablaet thespikedconcentrations. niv = Not valid.Upon analyzingthedata,valuedidnotpassthecriterisaetforthischaracterization. Untilfur-thearnalysisiscompleted,use theLCR todeterminetherange ofstandard concentrationfsorcalibratiocnurvepreparation. AttachmentB:MDL/LOQ Values 3M Environmental Laboratory FAC7-M- 1,1 ExtractioonfPFOS from Ll%,er Pa(ye12 of 15 Page 140 3m Medical Department S::udy: T-6295.7 Report No. FACT TOX-030 labcratory Request Number-U2279 Compound PFOS Matrix Rabbit Bovine NIDL LOQ 11.8 ppb 37.4 ppb n/d= not detenttined' Approximate Linear Rang@@ Low Standard 38 ppb i 60 ppb High Standard 1000 ppb 0 ppb PFOSA i Rat --m 0 rikey Rabbit 24.7 ppb 1 78.7ppb n/v= not valid' 1 1 6.06 ppb 19.3ppb 62 ppb 59 ppb 20 ppb 1200 ppb 1200 ppb 1200 ppb Bovine Rat n/d 20.7 ppb a/d 65.8 ppb 30 ppb 6 ppb 1200 ppb 1200 ppb PFOSAA Monkey i Rabbit Bovine 27.4 ppb 87.1 ppb 6 ppb 1200 ppb 55.7 ppb i 177 ppb 1 180 ppb 1 1900 ppb n/d ri/d 120 ppb 1200 ppb ETFOSE-OH Rat Monkey Rabbit n/v n/v riiv I n/v 58.7ppb 187 ppb ---62ppb 120 ppb 190 ppb- 1200 ppb 1200 ppb 1800 ppb nJd n/d 120 ppb 1200 ppb Rat n/v n/v 120 ppb 1200 ppb Monkey n/v n/v 58 ppb 1200 ppb POAA Rabbit Bovine Rat 23.7 pp b n/d niv 75.5ppb n/d n/v 76 ppb 120 ppb 62 ppb 1800 ppb 1206-ppb1200 ppb Monkey YV'V n/v 120 ppb 1200 ppb PFOSEA Rabbit 16.2 ppb 51.7 ppb 62 ppb 1200 ppb Bovine n/d n/d 30 ppb 1200 ppb Rat n/v nJv 120 ppb 1200 ppb Monkey n/v n/v 120 ppb 1200 ppb Monoester Rabbit n/d ri/d n/a nia Bovine ri/d ri/d rt.a, n@a Rat ib'd n/d ri/a n/a Monkey n/d n/d n)a n/a I -Upper Limitchosenwhere thevaduewas withintheLinearCalibratioRnange (LCR) butdid not excessivelwyeight thestandardcurveoraffectRepeatabilit&yReproducibilitvyalues. 2 -Not determinedrefertso no samplewas analyzedforthisdata. 3 -Not validreferstodatafrom the analysisfailetdomeet specificcriterifaora valid@MDL/LOQ determination. Compound Liver intatrix Rabbit Bovine Rat lvlonek..v PFOS Prepared Rangeof Standards Rangeof Average Curve Range of Low Std. Curve LCR from,,,.-Range of -L-o-@w' td. flighStd. Curve (ppb)(ng/g) (ppb)(ngTg)-,.-. (Ppb)(ng/g) 5.95- 1790 1790 @ir4_-ALqCk, 5.95-298 fl-5@@-299 (ppb)(ng/g) 119 - 1790 600. 200 6.00- 1200 n/a 1240 6.2'-- 1240 04-,;.1'24 nia 11 5 93 - I190 5.9--,1-190 nia nla@4,. nia nla nYa iv nla nia i nla Attachment C: LOQ summary 3M Environmental Laboratory FACT-M- 1.1 ExtractioonfPFOS from Liver Page 13 of 15 Page 141 3m Medical Decartment- Sz,-d,/:---0'295.7 Report No. FA---L TOX-030 lahoratory -Request Number-U2279 !Compound@ I PFOSA Liver Prepared Rangeof -.14CR-,frdin"R.a,ngeof 1. LCR from@-@ Range of LCR,-from Matrix Rangeof Average Low Std. LonSid High Std Std" Standards Curve Curve Curve I -vat" (ppb)(ng/g) (ppb)@ngig) (ppb)(ng/g) (PP,@)(4e&"(@PP6-)-(n-g/g) Rabbit 6.04-1810 6-04- 1210 6.04-301. 121- 1210 J7,- Bovine 5.95-1190 @.95 1190 n/a n/a Rat 6.17-1240- 5.17- 1214900 n/a n/a Nion ey 5.88-1180 5.38-1180 n/a n/a @Compound@ Liver Matrix Rabbit Bovine R2t Monkey PFOSAA 1 Prepared Rangeof Range of Standards Average Curv (ppb)(ng/g)(ppb)(ng/g) 6.33 - 1900 12-/-1900 5.99- 1200 120- 1200 6.21 - 1240 62.1-1240 5@92 - I ISO 59.2-1180 IWlt Rangeof Low Std. Curve (ppb)(ng/g) n/a n/a nla n/a Range of High Std. Curve (ppb)(ng/@gf)t- n/a n/a n/a n/a Compound Liver Matrix Rabbit Bovine Rat Monkey ETFOSE-OH Prepared Range f Rangeof Standards Average Cur-ve0 (ppb)(ng/g)(ppb)(ng/g) 5.96- 1790 1]9-1790 5 87- 1170 58.7-1170 6.09- 1220 5.80- II60 122-1220 294-1160 Range of L Low Std. Curve (ppb)(nWg) n/a n/a n/a n/a Rangeof 1 High Std. Curve ;j (ppb)(nWg) n/a n/a M..., n/a n/a Compound Liver Matrix Rabbit Bovine Rat Monkev I POKA Prepared Range of Range of Average Standards Curv (ppb)(ng/g)(ppb)(ng/g) 6.06- 1820 30.3- 1820 5.93 -1190 59.3 -1190 6.15- 1230 61.5- t230 5.86 - I I"@O II-,- 1170 Range of @LC Low Std. Curve e (ppb)(ng/"@) 30.3-606 nla n/a n/a R2ngeof High Std. Curve (ppb)(ng/g) 303 - 1820 n/a n,a nia ,Compound Liver Matrix Rabbit Bovine Rat Monkey PFOSEA Prepared Rangeof R2ngeof Standards ANera,,e Curve (ppb)(ng/g)(ppb)(ng/g) 6.20- 1860 62.0- 1240 i:, Range of Li.@0RLrgm4@t Rangeof L hi@, Low Std- High Std. Curve Curve '.4 (ppb)(ngfgI),(-@pb)(79/-(,p-pObz)r(ng/g) nla nia@ nia 5.92- 1180 6,14- 1230 29,6 -1180 123 - 1230 n/a -!;@Wa n/a r@a- n/a -wi; n/a iVa 5.85 - ll-,O 58 1170 1)7-"l170L- n/a nia n@a n/a kioncester%vasnot detectabic!quantifiaibnl@e,iver.Tatrfioxrthecgncentrat@cnrangeoC4 94 - 14@10ppb Artachment C: LOQ summary 3M Environme.-ital Laboratory FACT-,Nvf-1.1 Extractionof PFOS from Page 14 of 15 Page 142 3m Medical ]Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 ,PrepDate(s): 11112/98 lAnalyst(s): IRWW-IAS ISampleMatrix: Monkey L_ iver I @Method[Revision FACT-M 1.0 Target FC-,Mix lAnalyte(s): Ion Pair Standard Curves--Tissues ;StudyNumber: @CrossValidation Equipment Number: FinalSolvent& TN Number:;,MeOH TN-A-20"76 i Blank Tissue/d]entifier*. 'Liver FC Mix Std Approx.0.500ppm: IW398-1004 @FC Mix Std Approx. 5.00ppm-.' JW399-1003 i IFC Mix Std Approx.50.0ppm: IW398-1002 i ISurrog2teStdApprox. 16.5ppm: IW398-989 !ActualConcentrationosf StandardsintheFC Mix PFOS PFOSA PFOSAA i StdConc. Std Conc. Std Conc. 1 ETFOSE StdConc. ug/mL ug/mL ug/mL I ug/mL 0.501 0.497 0.500 1 0.490 0.501 0.501 0.497 0.497 0.500 0.500 0.490 0.490 0.501 -F-0.497 0.500 0.490 0.501 5.01 1 0.497 1 0.500 4.97 5.00 0.490 4.90 5.01 4.97 5.00 4.90 5.01 4.9-7 5.00 4.90 50.1 49.7 50.0 49.0 POAA PFOSEA StdConc. I Std Conc. ug/mL ug/mL 0.495 0.494 0.495 1 0.494 0.495 1 0.494 0.495 1 0.494 0.495 0.49 4.95 4.94 4.95 4.94 4.95 4.94 49.5 49.4 All Am't Spiked mL 0.002 0.004 0.010 0.020 0.040 0.020 1 0.030 1 0.00.4 All Li coner,7 g/ml 0.169 0.169 0.169 0.169 0 169 o@ 169 0-169 169 C21culated Concentrations of Standards in the S2mple Matrix. PFOS PFOSA PFOSAA ETFOSE FinalConc. FinalConc. FinalConc. FinalConc. POAA I PFOSEA FinalConc. FinalConc. ng/9 ng/g ng/g ng/g ng/g i ng/g 5.93 5.88 5.92 5.80 5.86 5.85 11.9 11.8 11.8 11.6 11.7 11.7 29.6 29.4 29.6 29.0 29.3 29.2 59.3 58,8 59.2 58.0 58.6 58.5 119 118 118 116 11i 117 296 294 296 290 293 292 593 588 592 580 586 585 889 882 888 870 i 879 877 1186 1176 1183 11 0 1172 1 1169 Surrog2tel All Std Conc. Am't Spiked ug/mL rnL 16.50 0.005 Surrogate' FinalConc. ng/g 488 2 ValidatedR2nzes- ApproximateConcentrations Liver PFOS PFOSA PFOSAA Rabbit 40 - 1000ppb 20 - 1200ppb 180 - 1900ppb Bovine 60 - 1200ppb 30 - 1200ppb 120 - 1200ppb Rat 60 - 1200ppb 170 - 1200ppb 60 - 1200ppb Nionkev 60 - 1200ppb 190 - 1200ppb 120 - 1200ppb ETFOSE-OH 190 - 1800ppb 120 - 1200ppb 120 - 1200ppb 60 - 1200 opb POAA 80 - 1800ppb 80 - 1200 ppb 60 - 1200 ppD 120 - 1200 ppb -PFOSEA 60 - 1200 ppb 30 - 1200 ppb 120- 1200 ppb 120- 1250 ppb Attachment D@ Calibratiosntandard concentratio\nvorksheet 3M Env4-ronmental Laboratory FACT-M- t.I ExtractioonfPFOS from Liver Page 15 of 15 Page 143 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M ENVIRONMENTAL LABORATORY METHOD ANALYSIS OF FLUOROCHEMICALS HPLC-ELECTROSPRAYMASS IN LIVER EXTRACTS SPECTROMETRY USING Method Number: FACT-M-2.1 Author: Lisa Clemen Approved By: Adoption Date: 05/26/98 Revision Date: 0& lo3 Ick Laboratory Manager Date Group Leader Date Technical Reviewer Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method is for the analysisof extractsof liveror othertissuesfor flUOTochemical surfactantsusing HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Potassium perfluorooctanesulfonate,anionic fluorochemical surfactants,or other ionizablecompounds. 1.3 Matrices: Rabbit,rat,bovine, and monkey liversor other liversas designated in the validationreport. Word 7.0.1!95 3M Environmental FACT-M-2.1 Analysisof LiverExtractUsing ES/,MS Laboratory Page I of 9 Page 144 3m Medical Department Szudy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 SUMNIARY OF METHOD 2.1 This method describesthe analysisof fluorochemicalsurfactantesxtractedfrom liverusing HPLC-electrospray/tmass spectrometry.The analysisisperformed by monitoringa single ion characteristiocf a particulafrluorochemical,such as the potassium perfluorooctanesulfonat(ePFOS) anion,M/Z= 499. Samples may alsobe screenedto verifycompound identification. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization(API): The Micromass platform systems allow for variousmethods of ionizationby utilizinvgarioussources,probes,and interfaces.These includebut arenot limitedto:ElectrosprayIonization(ESI),Atmospheric Pressurechemical Ionization(A-Pcl),,Thermospray,etc.The ionizationprocess inthesetechniquesoccurs at atmospheric pressure(i.en.ot under a vacuum). 3.2 Electrospray Ionization(ES, ESI): a method of ionizationperfon-nedatatmospheric pressure,whereby ionizationoccursthrough thq productionof tinycharged dropletsin a strongelectricaflield. 3.3 Mass Spectrometry, @NlassSpectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API platformsare equipped with quadrupole mass selectivedetectors.Ions are selectivelydiscriminatedby mass tocharge ratio(m/z) and subsequentlydetected.A single MS may be employed for ion detectionor a series(MSNTS) formore specificftagmentation information. 3.4 Conventional vs.Z-spray probe interface:The latestmodels of Micromass platfon-n svstems (post1998) utilizae "Z-spray"conformation.The sprayemittedfrom a probe is orthogonalto the cone aperture.In theconventionalconformationitisaimed directlyat the cone aperture,afterpassing througha tortuouspathway in the counterelectrode.Though the configurationisdifferentt,he methods of operation,cleaning,and maintenance are the same. However, Z-spray components and conventionalcomponents arenot compatiblewith one another,but only with similarsystems Z-spray components are compatiblewith otherZspray systems, etc.) 3.5 Mass Lynx Software: System softwaredesigned forthe specificoperationof theseplatform systems. CurrentlyMassLynx has Windows 95 and WindowsNT 3.1 versions.All versions are similar.For more detailssee themanual specificto the instrument(Micromass Platform IIor Quattro11MassLynx or MassLynx NT USER'S GUIDE). 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use cautionwith the voltagecablesforthe probe. The probe employs a voltageof approximately5000 Volts. 4.1.2 When handlina samples or solventswear appropriateprotectivegloves,eyewear, and clothing. 1.@*or-d.0.1/95 FACT-M-2.1 Anatysisof LiverExtractUsing ES/'IvIS 3M Env-@ronmen-Lal Laboratory PagC,e 2 of 9 Page 145 3m medical Departmer@t Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 4.2 Cautions: 4.2.1 Do not ran solventpumps above capacityof 400 bar (5800 psi).Ifpressuregoes over 400 bar,the HP I100 willinitiataeutomatic shutdown. 4.2.2 Do not run solventpumps todryness. 5.0 INTERFERENCE& 5.1 To minimize interferencewshen analyzingsamples forperfluorooctanoate(POAA),teflon should not be used forsample storageor any partof instrumentationthatcomes in contact with the sample or extract. 6.0 EouiPMENT 6.1 Equipment listecbrelow may be modified in orderto optimize thesystem. 6.1.1 Micromass ElectrosprayMass Spectrometer 6.1.2 HPI 100 low pulse solventpumping system and autosampler. 7.0 SUPPLIF-S AND MATERIALS 7.1 Supplies 7.1.1 High puritygrade nitrogengas regulatedto approximately 100 psi 7.1.2 HPLC column, specifictsobe detenninedby theanalyst. 7.1.3 Capped autovialsor capped 15 mL centrifugetubes. 8.0 RFAGFNTS AND STANDARDS 8.1 Reagents 8.1.1 Methanol,HPLC grade or equivalent. 8.1.2 ASTM, Type Iwater,Milll_QTMwater,allwater used inthismethod should be Milli-Q' waterand may be providedby a Milli-QTOC Plus system. 8.1.3 Ar=onium acetate,reagentgradeor equivalent. 8.2 Standards 8.2.1 Typically one method blank, one matrix blank, and ten matrix standards are preparedduring the extractionprocedure. See FACT-M-1.1. 9.0 SAMPLE HANDLING 9.1 Fresh matn*x standardsareprepared with each analysis.Extractedstandardsand samples are storedin capped autovialsor capped 15 mL centrifugetubesuntilanalysis. 9.2 Ifanalysiswillbe delayed,extractedstandardsand samples may be storedatroom temperatureor refrigerateadt4' C untilanalysiscan be performed. FACT-M-2.1 Analysisof LiverExtractUsing ES/MS 3M Env4@ronmental Laboratory Page 3 of 9 Page 146 3m Medical Llepartment Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 10.0 OUALITY CONTROL 10.1 Matrix Blanks and Method Blanks 10.1.1 Anaiyze a method blank and matrixblank pn*ortoeach calibratiocnurve. 10.2 Matrix Spikes 10.2.1 Analyze a matrix spikeand matrixspikeduplicatewith each analysis.With a minimum of 2 spikesper batch. 10.2.2 Expected concentrationswillfallin themid-range of theinitiaclalibratiocnurve. Additionalspikeconcentrationmsay fallinthelow-rangeof theinitiaclalibration curve. 10.2.3 See section13 to calculatepercentrecovery. 10.3 Continuing Cafi@brationChecks 10.3.1 Analyze a mid-range calibratiosntandardafterevery tenthsample. Ifa significant change ( 30%) inpeak areaoccurs,relativeto the initiasltandardcurve,stop the run. Only those samples analyzedbefore the lastacceptablecalibrationstandard willbe used. The remaining samples must be reanalyzed. 10.3.2 See section13 to calculatepercentdifference. 11.0 CALIBRATION AND STANDARDIZATION 11.1 Analyze the extractedmatrix standardspriorto and followingeach setof extracts.The average of two standardcurveswillbe plottedby linearregression(y = my b),not forced through zero,usingMassLynx or othersuitablesoftware. 11.2 Ifthe curve does not meet requirements,perform routinemaintenance or reextractthe standardcurve (ifnecessary)and reanalyze. 11.3 For purposes of accuracywhen quantitatinlgow levelsof analyte,itmay be necessaryto use the low end of the calibratiocnurve ratherthan the fullrange of the standardcurve. Example: when attemptingto quantitateapproximately 10 ppb of analyte,generatea calibrationcurve consistingof the standardsfrom 5 ppb to 100 ppb ratherthan the full range of thecurve (5 ppb to 1000 ppb). This willreduce inaccuracyattributetdo linear regressionweighting of high concentrationstandards. 12.0 PROCEDURES 12.1 Acquisition Setup 12.1.1 Click on startbuttonin the AcquisitionControl Panel.Set up a sample list.Assign a filenameusing MO-DAY-last digitof year-samplenumber, assigna method (MS) for acquiring,and type in sample descriptions. 12.1.2 To createa method clickon scan button inthe Acquisitioncontrolpanel and select SIR (SingleIon Recording)or MRM. Set IonizationMode as appropriateand mass to 499 or otherappropriatemasses. A fullscan isusuallycollectedalong with the SIRS. Save acquisitionmethod. IfMS/MS instrumentsare employed, additional product ion fragmentationinformationmay be collected.See i'vflcromass FACT-M-2.1 AnalysisofLiverExtractUsing ES/NIS Page 4 of 9 3M Ervironmental Laboratory Page 147 3m med4;..calDepartment Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 MassLynx GT7-'IDETO DATA ACQUISITION MR.M (MultipleReactionMonitoring). foradditionalinformationand 12.1.3 Typicallythe sample listbegins with the firsstetof liverstandardsand ends with the second setof standards. 12.1.4 Samples are analyzed with a continuingcalibratiocnheck injectedafterevery tenth sample.-Solvent blanksshould be analyzed pen'odicalltyo monitor possibleanalyte carryoverand are not consideredsamples but may be includedas such. 12.2 Using the Autosampler 12.2.1 Set up sample trayaccordingtothe sample listpreparedin section12.1.1. 12.2.2 Set-up theHP II00/autosampleratthe followingconditionsor atconditionsthe analystconsidersappropriateforoptimal response.Record actualconditionsin the instrumehtlogbook: 12.2.2.1Sample size= 10 pL injectiownith a sample wash 12.2.2.2Inject,sample= I 12.2.2.3Cycle time = 13.5minutes 12.2.2.4Solvent ramp Time ivfeOH 0.00 min. 40% 8.0 min. 90% 71-1.0 min. 90% 12.0 m-in. @-40% 2.0 mM Ammonium acetate 60% 10% 10% 60% 12.2.2.5Press the"Start"button. 12.3 Instrument Sep-up 12.3.1 Refer to ETS-9-24.0 formore details. 12.3.2 Check the solventlevelinreservoirsand refililfnecessary. 12.3.3 Check the stainlessteelcapillaryatthe end of theprobe. Use an eye piece to check the tip.The tipshould be flatwith no jagged edges.Ifthetipisfound to be unsatisfactoryd,isassemblethe probe and replacethestainlessteelcapillary. 12.3.4 Set HPLC pump to"On". Set the flow to 10 - 500 uL/min or as appropnate. Obser-vedropletscoming out of the tipof theprobe. Allow to equilibratfeor approximately 10 minutes. 12.3.5 Turn on thenitrogen.A finemist should be expelledwith no nitrogenleaking around the tipof the probe.Readjust the tipof theprobe iI'no mist isobserved. 12.3.6 The instrumentuses theseparametersatthe followingsettings.These settingsmay change inorder to optimizethe response: FACT-M-2.1 A.nalysiosf LiverExtractUsing ES/NIS PaLe 5 o[9 3M Envi-ronmenL-al Laboratory Page 148 3m Medical Departmen@-- S+--udy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.3.6.1Drying gas 250-400 litersihour 12.3.6.2 ESI nebulizinggas 10-15 liters/hour 12.3.6.3 LC constantflow mode flow rate10 - 500 uL/min 12.3.6.4Pressure<400 bar (Thisparameter isnot set,itisa guide to ensure the instr.imenitsoperatingcorrectly.) 12.3.7 Carefullyguide the probe intothe opening. Insertprobe untilitwillnot go any further.Connect the voltagecables to theprobe. 12.3.8 Printthe tune page,with itsparameters,and storeitinthe study binderwith a copy taped intothe instrumentlog. 12.3.9 Usinc,,thecross-flowcounterelectrodeinthe ES/MS source isrecommended for the analysisof biologicalmatrices. 12.3.10 Click on startbuttonin theAcquisitionControl Panel. Pressthe startbutton at top of sample list.Ensure startand end sample number includesallsamples to be analyzed. 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.1 Calculatematrix spikepercentrecoveriesusingthe followingequation: % Recovery Observed Result-Background Result x 100 Expected Result 13.1-.2Calculatepercentdifferenceusing the followingequation: % Difference= Ext)ectedConc. - CalculatedConc. x 100 Expected Conc. 13.1.3 Calculateactualconcentratioonf PFOS anion intotalliver(mg): ug PFOS aruo*n calc.from stdcurve g of liverused foranalysis 1" ug mg x Totalniassofliver(g) 14.0 METHOD PERFORNIANCE 14.1 Method DetectionLimit(MDL) and Limit of Quantitation(LOQ) aremethod, analyte,and matrix specific.Pleasesee ETS-8-1.1,Attachment B, fora listinogf currentvalidated MDL and LOQ values. 14.1 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.1.1 Solvent blanks,method blanks,and matrix blanks values are must be below the lowest standardinthe calibratiocnurve. 14.2 Calibration Curves 14.2.1 The r-v'aluefo,-the calibratiocnurve must be 0.980 or better. FACT-M-2.1 AnalysisofLiverExtractUsing ES/MS Page 6 of 9 3M Environmental '@.Jaboratory Page 149 3m Medical Department Study: T-6295.7 Report No. FACT TOX-@V30 laboratory Reauest Number-U2279 14.3 Matrix Spikes 14.3.1 Matrix spikepercentrecoveriesaremust be within 30% of the spiked concentration. 14.4 Continuing Calibration Verirications 14.4.1 Continuing calibrationverificatiopnercent recoven'esmust be-+ 30% of the spiked concentration. 14.5 If criterialistedin thismethod performance sectionisn'tmet, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst.Document allactionsin the appropriatelogbook. 14.6 If data are to be reported when performance criteriahave not been met, the data must be footnoted on tablesand discussed in the textof the report. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample waste is disposed in biohazard containers,flammable solvent waste isdisposed in hitc@,hBTU containers,and glasspipettewaste'isdisposed in broken glasscontainers. All containers are located in the laboratory. 16.0 RECORDS 16.1 Each pac7egenerated for a study must have the following information included eitherin the header ocr' hand writtenon the page: study or projectnumber, acquisitionmethod, integrationmethod, sample name, extractiondate,dilutionfactor(ifapplicable),and analyst. 16.2 Printthe tune page, sample lista,nd acquisitionmethod from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibrationcur-veby linearregression,weighted I/x,then printthese graphs and store in the study folder. 16.4 Printdata integrationsummary, integrationmethod, and chromatograms, ftom MassLynx, and store in the study folder. 16.5 Summar-ize data using suitablesoftware (Excel 5.0) and store in the study folder,see Attachment A for an example of a summary spreadsheet. 16.6 Back up electronicdata to appropriatemedium. Record in study notebook the filename and locationof backup electronicdata. 17.0 TABLES, DIAGRAms. FLOWCHARTS, AND VALIDATION DATA 17.1 Attachment A: FACT-,M-2.1 Data reportingspreadsheet FACT-M-2.1 Analysis of Liver ExtT-acUtsing ES/MS 3M Env4-ronmental Laboratory Pa,,e7 of 9 Page 150 3m Medical Departmert Study: T-6295.7 ReDcrt No. FACT TOX-030 laboratory Request Number-U2279 18.0REFERENCES 18.1 FACT-M-1.1, "Extractionof Potassium Perfluorooctanesuflonateor Other Fluorochemical compounds from Senim forAnalysisUsing HPLC-ElectrosprayfMass Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance of the Nlicromass Atmospheric Pressure Ionization/MassSpectrometer QuattroIItn'plequadrupole Systems" 18.3 The validationTCportassociatedwith thismethod isFACT-M-1.1-V & 2.1-V-1, 19.0 AFFECTED DocuMENTS 19.1 FACT-M- 1.1,"Extractionof Potassium Perfluorooctanesulfonate from Liver forAnalysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 REV]SIONS Revision Number. I Reason For Revision Section 6.1.2 Clarificatioonf HPI 100 system components. Section 11.1 Average of two curves,not standardvalues,areused for plottinglinearregression. Section 12.2.2.4Clarificatioonf solventramp. Section 17.1Changed from attachmentB toA. Revision Date 05/04/'99 FACT-M-2.1 Analysisof LiverExtractUsing ES/NIS 3M E,-ivironmental Laboratory Page 8 of 9 Page 151 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Laboratory Study Study: Test Material: Matrix/FinaSlolvent: Method/Revision: AnalyticalEquipment System Number: InstrumentSoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analysr-. Date of Analysis/Analyst: Group Dose-- Sample# Concentration ug/mL InitiaVlol. niL Dilution Factor FinalConc. ug/mL Slope: Taken from linearregressionequation. Grc)up/Dose: Taken from thestudyfolder. S2mple#: Taken from thestudyfolder. Concentration (ug/mL): Taken from theMassLynx integratiosnummary. InitiaVlolume (mL): Taken from thestudyfolder. DilutionFactor: Taken from thestudyfolder. FinalConc. (ug/mL): Calculatedby dividingtheinitiavlolume ftom theconcentration Attachment A: Data Sheet FACT-M-2.0 Analysisof LiverExtractUsing ES,,NIS 3M Environmental Laboratory Page 9 of 9 Page 152 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M ENVIRONMENTAL LABORATORY METHOD ExTRAcTioN OF PoTAssruim PERFLUOROOCTANESULFONATE FLUOROCHE@MICAL COMPOUNDS FROM SERUIM OR OTHER FLUID USING HPLC-ELECTROSPRAY/NIASS SPECTROMETRY OR OTHER FOR A.@NALysis Method Number: FACT-M-3.1 Adoption Date: 04/22/98 Author: Lisa Clemen, Glenn Langenburg Revision Date: to Iat(ctS Approved By: Labor4dtoryManager lvt@,--L @L=@ @7ro@UPLeader :ix; @il -4 L4)Y-@K Technical Reviewer Date '? Date C11.@3l@? Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method isfortheextractioonf potassiumperfluorooctanesulfonat(ePFOS) or otherfluorochemicalcompounds from serum or otherfluid. 1.2 Applicable compounds: Fluorochemicalsurfactantosr otherfluorinatecdompounds. 1.3 Matrices: Rabbit,rat,bovine,and monkey serum, ratwhole blood,and ratmilk curd. Word 6/95 3M Environmental FACT-M-3.1 ExtractioonfPFOS ftom Serum and OtherFluids Laboratory Page I of 17 Page 153 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0SUMNIARY OF METHOD 2.1 This method describestheprocedureforextractinpgotassium perfluorooctanesulfonate (PFOS) orotherfluorochemicalfstom serum,blood,or milk curdusingan ion pairing reagentand 5.0ml of ethylacetate.In thismethod, seven fluorochemicalswere extracted:PFOS, PFOSA, PFOSAA, ETFOSE-OH, POAA, PFOSEA, and FC-807 monoester (see3.0 Definitions)A.n ionpairingreagentisadded tothesample and the analyteionp@irispartitioneidntoethylacetate.Four ml of extractareremoved and put onto a nitrogenevaporatoruntildry.Each extractisreconstituteidn 1.0ml of methanol, then filteretdhrough a 3 cc plastiscyringeattachedto a 0.2pLm nylonfilteirntoglass autovials. 3.0 DEFINITIONS 3.1 PFOS: perfluorooctanesulfona(taenionof potassiumsalt)C,F,,SO,3.2 PFOSA: perfluorooctanseulfonylamideC,F,,SO,NH, 3.3 PFOSAA: perfluorooctanesulfonylamido (ethyl)acetatCe,F,.,SO,N(CH2CH,)CH,CO,- 3.4 ETFOSE-OH: 2(N-ethylperfluorooctasnuelfonamido)-ethylalcohol C,F,,SO2N(CH2CH,)CH,CH,OH 3.5 POAA: perfluorooctanoa(taenionof ai=onium saltC),F,,COO- 3.6 PFOSEA: perfluorooctanseulfonylethylamideC,F,,SO,N(CHCH,)H 3.7 FC-807 monoester C,F1,SO,N(CH2CH3)CH2CH20-PO,H) 3.8 Surrogatestandard:IH- IH-2H-2H perfluorooctanseulfonicacid 4.0 WARNINGS AND CAUTIONS 4.1 Health and safetywarnings 4.1.1 Use universalprecautionse,specialllyaboratorycoats,goggles,and gloveswhen handlinganimal tissuew,hich may containpathogens. 5.0 INTERFERENCES 5.1 There areno known interferenceastthistime. 6.0 EOUIPMENT 6.1 The followingequipment isusedwhileperformingthismethod. Equivalentequipment is acceptable. 6.1.1 Vortex mixer,VV@R, Vortex Genie 2 6.1.2 CentrifugeM,istral1000 orIEC 6.1.3 Shaker,Eberbach or VWR 6.1.4 Nitrogenevaporator,Organomation 6.1.5 Balance ( 0.100 g) FACT-M-3.1 Extractionof PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 2 of 17 Page 154 3m medical Department Study: T-6295.7 Report No. FACR TOX-030 laboratory Request Number-U2279 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Eppendorf or disposablepipettes 7.3 ElectronicpipettorE,ppendorf or equivalent 7.4 Graduated pipettes 7.5 Nalgene bottlesc,apable of holding250 mL and I L 7.6 Volumetric flasks,glass,typeA 7.7 Volumetric pipets,glass,typeA 7.8 I-CHEM vials,glass4,0 mL glass 7.9 Crimp cap autovials 7.10 Centrifugetubes,polypropylene,15 niL 7.11 Labels 7.12 Syringes,capable of measuring 5 @iLto 50 gL 7.13 Syringes,disposableplastic3, cc 7.14 Syringe filtersn,ylon,0.2 gm, 25 mm 7.15 Timer Note: Priorto using glassware and bottlesr,-ins3e times with methanol and 3 timeswith Milli-Q' water. l@insesyringesa minimum of 9 timeswith methanol,3 rinsesfrom 3 separatevials. 8.0 REAGENTS AND STANDARDS 8.1 Type Ireagentgrade water,Milli-QT-mor equivalent;allwater used inthismethod should be Milli-Q' water and may be providedby a Milli-TOQC plUSTIISystem 8.2 Sodium hydroxide (NaOH), J.TBaker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA),Kodak or equivalent 8.4 Sodium carbonate (Na@C03), J.T.Baker or equivalent 8.5 Sodium bicarbonate(NaHC03), J.T.Baker or equivalent 8.6 Ethyl acetate,Omnisolv, glassdistilleodr HPLC grade 8.7 Methanol, Oninisolv,glassdistilleodr HPLC grade 8.8 Serum or blood,frozenfrom supplier 8.9 Control matrix or blank matrix forpurpose of standards,QC checks,blanks,etc. 8.10 Fluorochemical standards 8.10.1 PFOS (3M SpecialtyChemical Division),molecular weight= 538 8.10.2 PFOSA (3M SpecialtyChemical Division),molecularweight = 499 3M Environmental FACT-M-3.1 ExtractioonfPFOS from Serum or OtherFluid Laboratory Page 3 of 17 Page 155 3m Medical Der)arL-ment S-@udy: T-6295.7 Renort No. FACT TOX-030 laboratory Pequest Number-U2279 8.11 8.12 8.10.3 PFOSAA (3M SpecialtyChemical Division),molecularweight= 585 8.10.4 ETFOSE-OH (3M SpecialtyChemical Division),molecularweight = 571 8.10.5 POAA (3M SpecialtyChemical Division),molecularweight= 431 8.10.6 PFOSEA (3M SpecialtyChemical Division)m,olecularweight= 527 8.10.7 FC-807 monoester (3M SpecialtyChemical Division).FC-807 isa mixture of triesterd,iestera,nd monoester fluorochemicalcomponents. The monoester molecular weight = 650 8.10.8 Surrogatestandard:4-H, perfluorooctanesulfonicacid(1-H,I-H, 2-H, 2-H C,F13SO,H) molecular weight = 428 8.10.9 Other fluorochemicals,as appropriate Reagent preparation 8.11.1 10 N sodium hydroxide (NaOH): Weigh approximately200 g NAOH. Pour intoa 1000 mL beaker containing500 mL Milll_QTM water,mix untilallsolidsare dissolved.Storeina I L Nalgene bottle. 8.11.2 1 N sodium hydroxide (NaOH): Dilute 10 N NAOH 1:10. Measure 10 mL o f 10 N NAOH solutionintoa 100 niL volumetric flaskand diluteto volume using Milli_QTIwater. Storeina 125 mL Nalgene bottle. 8.11.3 0.5M tetrabutyalmmonium hydrogen sulfate(TBA): Weigh approximately 169 g of TBA intoa I L volumetriccontaining500 mL Milli_QTIwater.Adjust to pH 10 using approximately44 to 54 mL of 10 N NAOH and diluteto volume with Milli_QTIwater. While adding the lastmL of NAOH, add slowly because the pH changes abnlptly.Storeina I L Nalgene bottle. 8.11.3.1 TBA requiresa check priorto each use to ensurepH = 10. Adjust as needed using I N NAOH solution. 8.11.4 0.25 M sodium carbonate/sodiumbicarbonatebuffer(Na2C03/NaHC03): Weigh approximately26.5 g of sodium carbonate(NaC03) and 21.0g of sodium bicarbonate(,NaHC03) intoa I L volumetricflaskand bringtovolume with MilliQ' water. Storeina I L Nalgene bottle. Standards preparation 8.12.1 PreparePFOS standardsforthestandardcurve. 8.12.2 Prepareotherfluorochemicalstandardsa,s appropriateM.ulticomponent fluorochemicalstandardsareacceptable(forexample, one working standard solutioncontaining1.00ppm PFOS, 1.02 ppm PFOSA, 0.997 ppm PFOSA.,k, and 1.10ppm EtFOSE-OH.) 8.12.3 Weigh approximately 100 mg of PFOS intoa 100 ml volumetn'cflaskand record the actualweight. 8.12.4 Bring to volume with methanol fora stockstandardof approximately 1000 ppm (pg/ml). 8.12.5 Dilutethe stocksolutionwith methanol fora working standardI solutionoF approximately 50 ppm. 3M Environmental FACT-M-3.1 ExtractioonfPFOS from Serum orOtherFluid Laboratory Page 4 of 17 Page 156 3m Medical Department S@:udy: T-6295.7 Repor@'- No. FA'-'TTOX-030 laboratory Request Number-U2279 8.13 8.12.6 Dilutethe stock solutionwith methanol fora working standard2 solutionoc approx. 5.0 ppm. 8.12.7 Dilutethestocksolutionwith methanol fora working standard3 solutionof approx. 0.50 ppm. Surrogate stock standard preparation 8.13.1 Weigh approximately50-60 mg of surtogatestandard1-H,I-H,2-H, 2-H, C,F,,SO,H intoa 50 ml volumetricflaskand recordthe actualweight. 8.13.2 Bring to volume with methanol fora surrogatestockof approximately 1000-1200 ppm. 8.13.3 Prepare a surrogateworking standard.Transferapproximately0.5 ml of surrogatestockto a 50 ml volumetricflaskand bringto volume with methanol fora working standardof 10-20ppm. Record the actualvolume transferred. 9.0 SAMPLE HANDLING 9.1 All samples are receivedfrozenand must be kept frozenuntilthe extractionisperfor-rned. 10.0 ()UALITY CONTROL 10.1 Matrix blanks and method blanks 10.1.1 Extract two 1.0 mL aliquotsof the appropriate matrix (serum or blood, with blood samples diluted1:1with Milli-Q""w'ater)followingthisprocedureand use as matrix blanks. See 11.1.4. 10.1.2 Extracttwo 1.0ml aliquotsof Milli-Q' water followingthisprocedureand use as method blanks. 10.2 Matrix spikes 10.2.1 Prepare and analyzematrixspikeand matrix spikeduplicatesamples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by theanalyst,usuallythe control matrix receivedwith each sample set. 10.2.3 Expected concentrationswillfallin the mid-range of the initiaclalibrationcurve. Additionalspikesmay be includedand may fallinthe low-rangeof the initial calibratiocnurve. 10.2.4 Prepare one matrix spikeand matrixspikeduplicateper 40 samples, with a minimum of 2 matrix spikesper batch. 10.3 Continuing calibration checks 10.3.1 Prepare and analyzecontinuingcalibrationcheck samples toensure the accuracy of the initiaclalibratiocnurve. Ifthe percentdifferencebetween the initiaclurve and the continuingcheck differby >30%, re-analyzesamples analyzed afterthe lastacceptablecheck. 10.3.2 Prepare one check per group of ten samples. For example, ifa sample set= 34, prepare and extractfourchecks. 3M Environmental FACT-M-3.1 Extractionof PFOS from Serum or Other Fluid Laboratory Page 5 of 17 Page lS7 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 10.3.3 Prepare each continuingcalibratiocnheck ftom the same matrix used to prepare the initiaclurve. 10-3.4 The expected concentrationwillfallwithinthe mid-range of the initiaclalibration curve. Additionalspikesmay be includedthatfallin the low-range of the initial calibratiocnurve. This isnecessaryiftheanalystmust quantitateusingonly the low end of the calibratiocnurve (forexample, 5 ppb - 100 ppb, ratherthan 5 ppb - 1000 ppb). 11.0 CALIBRATION AND STANDARDIZATION 11.1 Prepare matrix calibrationstandards Note: Blood coagulatesin air;thereforem,inimize aircontactuntildilution.At this point,add TBA and bufferto each centrifugetube as instep 12.9,then add 1.0mL of the dilutedmatrixsample to each tube. 11.1.1 Transfer I n-lLof serum or I mL of blood (blood isdiluted 1:1 with Milli_QT,'vl water) to a 15 mL centrifugetube. The blood is similarin composition to milk curd and can be used inplace of milk curd forstandardcur-veswhen extracting thatmatrix. 11.1.2 Ifmost sample volumes are lessthan 1.0mL, extractstandardswith matrix volumes equal to thesample volumes. Do not extractbelow 0.50 mL of matrix. Record the sample volume on the extractionsheet. 11.1.3 While preparinga totalof twenty aliquotsin 15 ml centrifugetubes,mix or shake between aliquots. 11.1.4 Two I mL aliquotso,r otherappropriatevolume, serve as matrixblanks. Typicallyuse the standardconcentrationsand spikingamounts listedinTable 1, atthe end of thissection,to spike,in duplicate,two standardcurves,fora totalof eighteenstandardsand two matrix blanks. 11.1.5 Referto validationreportsFACT-M-3.1-V-1 and FACT-M-4.1-V-1, which list theworking rangesand theLinearCalibrationRange (LCR) forcalibration curves. 11.1.6 Use Attachment D as an aidin calculatingthe concentrationsof the working standards.See Section 13.0 tocalculateactualconcentrationsof PFOS in calibrationstandards. 11.2 To each standard,blank,or QC check,add appropriateamount of surrogateworking standardforthe concentrationto fallwithinthe calibratiocnurve range 5 ppb -1000 ppb. 11.3 Extract spiked matrix standards following 12.6-12.16 of thismethod. Use these standards to establisheach initiaclurve on themass spectrometer. 3M Environmenk--al FACT-M-3.1 Extractionof PFOS from Serum or Other Fluid Laboratory Page6 of 17 Page 1SB 3m Medical Department St,-,dy:T-6295.7 Report No. FAC-- TOX-030 laboratory Request Number-U2279 TableI Approximate spikingamounts forstandardsand spikes using1.0ml ofmatrix Working standard @iL Approx. finalconc.of (approx.conc.) analytein matrix - - Blank 0.500 ppm 0.500 ppm 5.00 ppm 5.00 ppm 5.00 ppm 50.0 ppm 50.0 ppm 50.0 ppm 10 0.005 ppm 20 0.010 ppm 5 0.025 ppm 10 0.050 ppm 20 0.100 ppm 5 0.250 ppm 10 0.500 ppm 15 0.750 ppm 50.0 ppm 20 1.00 ppm Table 2 Approximate spiking amounts for standards and spikes using 0.5 mi of matrix Working standard @iL Approx. finalconc.of (approx.conc.) analytein matrix - Blank 0.500 ppm 0.500 ppm 5.00 ppm 5.00 PPM 5.00 ppm 50.0 ppm 50.0 ppm 5 0.005 ppm 10 0.010 ppm 2.5 0.025 ppm 5 0.050 ppm 10 0.100 ppm 2.5 0.250 ppm 5 0.500 ppm 50.0 ppm 50.0 ppm 7.5 0.750 ppm 10 1.00ppm 12.0 PROCEDURF, 12.1 Obtain frozensamples and allow to thaw. 12.2 Vortex mix for 15 seconds,thentransfer1.0 mL or otherappropriatevolume to a 15 mL polypropylene centrifugetube. For blood samples,remove 0.5 mL and diluteto 1.0mL with Milli-Q' water.As soon afterdilutingas possiblep,ipetdilutedblood intoTBAbuffermixture shown in step12.9and mix well. 12.3 Retum samples to freezerafterextractionamount has been removed. FACT-M-3.1 Extractioonf PFOS from Serum or OtherFluid 3M Environmental Laboratory Page 7 of 17 Page 159 3m Medical Departmert Study: T-6295.7 Report No. FACF TOX-030 laboratory Request Number-U2279 12.4 Record the volume on the extractionworksheet. The finalmethanol volume equals the volume transferredfrom the sample.For example, if0.5 mL isremoved fora blood sample, the finalmethanol volume willequal0.5 mL. 12.5 Label the tube with the study number, sample UD, dateand analystinitialsS.ee attached worksheet fordocumenting theremaining steps. 12.6 Spike each matrix with the appropriateamount of standardas describedin 11.1 or Table I or 2 in thatsectionforthecalibratiocnurve standards.Also preparematrix spikesand continuing calibratiosntandards. 12.7 Spike allsamples, includingblanksand standards,ready forextractionwith surrogate standardas describedin 11.2. 12.8 Vor-texmix the standardcur-veswnples,matrix spikesamples, and continuingcalibration samples for 15 seconds. 12.9 To each sample, add I mL 0.5 M TBA and 2 mL of 0.25 M sodium carbonate/sodium bicarbonatebuffer. 12.10 Using a volumetricpipette,add 5 mL ethylacetate. 12.11 Cap each sample and put on theshaker for20 minutes. 12.12 Centrifugefor20 to25 minutes atapproximately3500 rpm, untillayersarewell separated. 12.13 Transfer4 mL of organiclayer,usinga 5 mL graduated glasspipettet,o a clean 15 mL centrifugetube.Label thisfreshtube with the same informationas In 12.5. 12.14 Put each sample on the analyticanlitrogenevaporatoruntildry,approximately 2 to 3 hours. 12.15 Add 1.0mL or otherappropriatevolume of methanol to each centrifugetube using a graduatedpipette.Methanol volume to add equalsthe initiavlolume of sample used for the extraction. 12.16 Vortex mix for30 seconds. 12.17 Attach a 0.2 @im nylon mesh filtetro a 3 cc syringeand transferthesample to this synnge. Filterintoa 1.5mL glassautovialor low-volume autovialwhen necessary. 12.18 Label the autovialwith the study number, animal number and gender,sample timepoint, matrix,finalsolvent,extractiondate,and analyst(sp)erforming the extraction. 12.19 Cap and storeextractsatapproximately4 *C untilanalysis. 12.20 Complete the extractionworkshect,attachedtothisdocument, and tapein the study notebook or includein studybinder,as appropriate. FACT-M-3.1 Extractioonf PFOS from Serum orOther Fluid 3M Environmental Laboratory Page 8 of 17 Page 160 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory RequeSt NUmber-U2279 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations 13.1.1 Calculateactualconcentrationsof PFOS, or otherapplicablefluorochemical,in calibratiosntandardsusingthe followingequation: mL of standardx concentrationof standard(ug /mL) mL of standard+ mL of surrogatestandard+ initiamlatrixvolume (mL) FinalConcentration(gg/mL) of PFOS in matrix 14.0 METHOD PERFORMANCE 14.1 The method detectionlimit(MDL) isanalyteand matrixspecific.Refer toMDL report forspecificMDL and limitof quantitatio(nLOQ) values(seeAttachments B and C). 14.2 The following qualitycontrolsamples are extractedwith each batch of samples to ensure the qualityof the extractionand analysis. 14.2.1 Method blanks and matrixblanks 14.2.2 Matrix spikeand matrixspikeduplicatesamples to determine accuracy and precisionof the extraction 14.2.3 Continuing calibratiocnheck samples to determine the continued accuracy of the initiaclalibratiocnurve 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample waste isdisposed inbiohazard containers,flammable solventwaste isdisposed in high BTU containers,and used glasspipettewaste isdisposed in broken glasscontainers locatedin the laboratory. 16.0 RFcoRDs 16.1 Complete the extractionworksheet attachedto thismethod, and tape in thestudy notebook or includein study3-ringbinder,as appropriate. 17.0 ATTACHMENTS 17.1 Attachment A, Extractionworksheet 17.2 Attachment B, MDLILOQ values 17.3 Attachment C, LOQ Summary 17.4 Attachment D, Calibrationstandardconcentrationworksheet 18.0 P.EFERENCES 18.1 The validationreportsassociatedwith thismethod are FACT-NI-3.1 & 4.1-V-1. 3M Environmental FACT-M-3.1 ExtractioonfPFOS from Serum or Other Fluid Laboratory Page 9 of 17 Page 161 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 19.0 AFFECTFD DocuMENTS 19.1 FACT-M-4.1, "Analysisof Senim or Other Fluid ExtractsforFluorochemicalsusing HPLC-Electro sprayMass Spectrometry" 20.0 REvisIONS Revision Number I Reason For Revision Validationof method to include7 fluorochemicalsa,n additionalmatrix, new API/MS(MS) systems,monkey senun crossvalidation, improvements to ion pairingextractionM,DL study,updatesin record keeping and storingpolicies,etc. Revision Date 07/01/98 3M Environmental FACT-M-3.1 ExtractioonfPFOS from Serum orOther Fluid Laboratory Page 10 of 17 Page 162 3m Medical DepartTren-. Study: T-6295.7 Report No. FAC7 TOX-030 laboratory Request Number-U2279 Study Sample Number set ?'I* H,O Blank Blank FC-Mix approx.0.5 ppm actual ppm #W - FC-Mix approx.5 ppm actual ppm #W- FC-;,@lix- approx.50 ppm actual PPM #W Date andInitialfsor Std.or Comments Study number where theori2inawlorksheetislocated, Blank Std SerumExtr2ction Method Vortex 15 sec. amount mL Date & lniti2is Pit)ettMcatrix Volume mi PipetteI ml of 0.5 M TBA, pH 10. Std. Pipette2 ml of 0.25 Na2CO3iO-25.M NaHC03 buffcr Std. Pipette5 ml ofethyl acetate TN-A- Shake 20 min. Centrifu.ge20-25 min. Ccnffifuge speed: Remove a 4 ml aliquot of or@ganiclayer Put on Nitrogen Evaporator to dryness Evaporator Temperature: Add methanol Volume mi TN-A- Vortex 30 sec. Filterusing a 3cc B-D syringcwith a 0.2pm SRI filteirntoa 1.5ml autosamr)levial MS/MSD@'- Cont. Checks: Spiked - uL of a ppm std (_ ) fora finalconcentration of ppm. MS/.-MSD used sample Cont. Checks used same matrix as for std curve, SurrogateStandard:Spiked _ uL of a _ ppm std( -) to allsamples, standards,and blanks Attachment A: Exrraction worksheet FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environme-n-.al Laboratory Page 11 ofl'j Page 163 3m Medical IDepartmert Study: T-6295.7 Report No. FAC7 TOX-030 laboratory Request Number-U2279 MDLILOQ Compound PFOS PFOSA PFOSAA ETFOSE-OH POAA PFOSEA Monoester values forRabbit Serum: MDL (ppb) LOQ Linear CalibrationRange (LCR) (ppb) Approximate concentrationsto be used forpreparing the Standard CalibrationCurve 1.38 4.39 5 ppb - 1000 ppb 2.23 7.09 10 ppb - 1000 ppb 2.84 9.04 10 ppb - 1000 ppb 3.90 12.4 15 ppb - 1000 ppb 4.31 13.7 15 ppb - 750 ppb 1.09 3.48 25 ppb - 1000 ppb 149 248 MDL and LOQ areestimateosnly.No validMDL was determinablefrom MDL study.Any quantitatipoenrformedformonoesterwillbe an estimatoenly.PleaserefertoFACT-M-3.1 & 4.1-V-I forspecifics. MDLILOQ Compound PFOS PFOSA PFOSAA ETFOSE-OH POAA PFOSEA Monoester valuesforRat Serum: IMDL LOQ Linear CalibrationRange (LCR) (p.pb) 1.27 i (ppb) Approximate concentrationsto be used forpreparing the i 4.04 Standard CalibrationCurve 10 ppb - 1000 ppb 2.14 6.81 25 ppb - 1000 ppb 2.32 7.38 10 ppb - 1000 ppb 3.25 10.3 50 ppb - 1000 ppb 1.20 3.81 5 ppb - 1000 ppb 1.84 1 5.86 10 ppb - 1000 ppb 149 248 MDL and LOQ areestimateosnly.No validMDL was determinablefrom MDL study.Any quantitatipoenrformedformonoesterwillbe an I estimateonly.PleaserefertoFACT-NI-3.1 & 4.1-V-I forspecifics. MDL/LOQ Compound PFOS PFOSA PFOSAA ETFOSE-OH j POAA PFOSEA Monoester valuesforBovine Serum: MDL (ppb) LOQ Linear CalibrationRange (LCR) (ppb) Approximate concentrationsto be used forpreparing the Standard CalibrationCurve 2.11 5.04 6.70 16.0 25 ppb - 1000 ppb 25 ppb - 1000 ppb 2.34 7.45 260 ppb - 1000 ppb 11.3 35.8 1 50 ppb - 1000 ppb 4.64 14.8 15 ppb - 1000 ppb 3.71 11.8 15 ppb - 1000 ppb 149 MDL and LOQ areestimateosnly.No validMDL was determinablefrom MDL study.Any quantitatipoenrformedformonoesterwillbe an estimatoenly.PleaserefertoFACT-M-3.1 & 4.1-V-I forspecifics. No dataisavailableforMDL or LOQ inMonkey Ser-um.Use validatedLinearCalibratioRnange instead. Pleasesee Attachment C (LOQ Summary) and @MDL study in FACT-M-3.1 & 4.1-V-1 forspecifics. AttachmentA: Extractiownorksicet FACT-M-3.1 ExtractioonfPFOS from Serum or OtherFluid 3M Environmental Laboratory Page 12 of 17 Page 164 3m Medical Department Sl-udy: T-6295.7 Report No. FAC-@ T--)X-030 laboratory Request Number-U2279 NTDLILOQ ICompound PFOS PFOSA PFOSAA ETFOSE-OH POAA PFOSEA Monoester values for Nlonkey Serum: MDL LOQ Linear CalibrationRange (LCR) (ppb) .(ppb) Approximate concentrations to be used for preparing the 1.38 2.23 2.84 3.90 4.31 1.09 149 i 4.39 7.09 9.04 12.4 13.7 3.48 248 Standard Calibration Curve MDL and LOQ areestimateosnly. No validMDL was deten=able from MDL study.Any quantitatiopnerformed forPFOS willbe an estimate only. PleaserefertoFACT-M-3.1 & 4.I-V-1 forspecifics. MDL and LOQ areestimatesonly. No validMDL was detem-Linablferom MDL study.Any quantitatiopnerformed forPFOSA willbe an estimate only. Pleasereferto FACT-M-3. I & 4.1-V-I forspecifics. N4DL and LOQ areestimatesonly. No validMDL was determinablefrom MDL study.Any quantitatiopnerformed forPFOSAA willbe an estimate only. PleaserefertoFACT-M-3.1 & 4.1-V-1forspecifics. MDL and LOQ areestimatesonly. No validMDL was determinablefrom MDL study.Any quantitatiopnerformed forETFOSE-OH willbe an estimateonly, Pleasereferto FACT-M-3.1 & 4.1-V-1 forspecifics. MDL and LOQ areestimatesonly. No validMDL was determinablefrom N4DL study.Any quantitatiopnerformed forPOAA willbe an estit only. Pleasereferto FACT-M-3.1 & 4.1-V-I forspecifics. MDL and LOQ areestimatesonly. No validMDL was determinablefrom MDL study.Any quantitatiopnerformed forPFOSEA-OH willbe an estimateonly. Pleasereferto FACT-M-3.1 & 4.1-V-I forspecifics. MDL and LOQ areestimatesonly. No validMDL was determinablefrom MDL study.Any quantitatiopnerformedforELFOSE-OH willbe an estimateonly. PleaserefertoFACT-M-3.1 & 4.1-V-I forspecifics. MDL/LOQ Compound PFOS PFOSA PFOSAA ETFOSE-OH POAA PFOSEA Monoester values for Rat Whole Blood: MDL LOQ Linear CalibrationRange (LCR) (ppb) (ppb) Approximate Concentrations to be used for preparing the 1.25 I 3.96 Standard Calibration Curve 5 ppb - 1000 ppb 1.77 5.65 10 --ppb- 1000 ppb 17.3 55.0 55 ppb - 1000 ppb 7.89 25.1 MDL and LOQ areestimatesonly. No validMDL was determinablefrom MDL study.Any quantitatiopnerformedforETFOSE-OH willbe an estimateonly. Pleasereferto FACT-M-3.1 & 4.1-V-I forspecifics. 4.73 15.1 15 ppb - 1000 ppb 24.2 77.1 80 ppb - 1000 ppb 58.0 185 MDL and LOQ areestimatesonly. No validMDL was determinablefrom MDL study.Any quantitatiopnerformed formonoesterwillbe an estimateonly. Pleasereferto FACT-M-3.1 & 4.1-V-1 forspecifics. Pleasesee Attachment C (LOQ Summary) and MDL study inFACT-M-3.1 &- 4.1-V- I forspecifics. Attachment A: Extractionworkshect FACT-M-3.1 Extractionof PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 13 of 17 Page 165 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Reques@-- Number-U2279 Ion Pain'ng Extractionof Fluorochemicals from Serum and Analysisby APIAI4S(MS) Summary Table: LimitosfQuantitation Compound Matrix MDL Approximate linearrange2 LOQ Low std High std PFOS Rabbit 1.38ppb 4.39 ppb 5 ppb 1000 ppb Bovine 2.11 ppb 6.70 ppb 25 ppb 1000 ppb - Rat Monkey 1.27ppb nJd 4.04 ppb n/d 10 ppb 25 ppb 1000 ppb 1000 ppb PFOSA Rabbit Bovine 2.23ppb 5.04ppb 7.09 ppb 16.0 ppb 10 ppb 25 ppb 1000 ppb 1000 ppb F-at 2.14 ppb 6.81 ppb 25 ppb 1000 ppb Monkey n/d n/d 25 ppb 1000 ppb PFOSAA Rabbit Bovine 'kat Monkey 2.84 ppb 2.34 ppb 2.32 ppb n/d 9.04 ppb 7.45 ppb 7.38 ppb n/d 10 ppb 263 ppb 10 ppb 25 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb ETFOSE-OH Rabbit 3.90 ppb 12.4 ppb 15 ppb 1000 ppb Bovine 11.3 ppb 35.8 ppb 50 ppb 1000 ppb POAA Rat Monkey Rabbit 3.25 ppb n/d 4.31ppb 10.3 ppb n/d 1 113.7ppb 50 ppb 10 ppb 15 ppb 1000 ppb I 1000 ppb 750 ppb Bovine 4.64ppb 14.8 ppb 5 ppb 1000 ppb Rat 1.20 ppb 3.81 ppb 5 ppb 1000 ppb Monkev ii/d rL/d 5 ppb 1000 ppb PFOSEA Rabbit 1.03ppb 3.48ppb 25 ppb 1000 ppb Bovine 3.71ppb 11.8ppb 5 ppb 1000 ppb Rat 1.84ppb 5.86 ppb 10 ppb 1000 ppb Monkey n/d n/d 5 ppb 1000 ppb Monoester' Rabbit 149 ppb 474.0 ppb 250 ppb 1000 ppb Bovine 149 ppb 474.0 ppb 250 ppb 1000 ppb Pa t 149 ppb 474.0 ppb 250 ppb 1000 ppb Monkey n/d n/d 100 ppb 1000 ppb I. Values formonoester areestimatesonly. 2. Higheststandard(approx.1500 ppb) was excludedfrom finalLCR and upper LOQ valuesdue topoor R & R valuesand excessiveweightingof thecalibratiocnurve. Compound: PFOS Serum matrix Prepared R-ang of LCR from range of average ave curve standards curve (ppb)(ngi-L) (ppb)(ng/mL) (ppbXng/mL) Range of LCR from low ld low td curve curve (ppb)(ng/-L) (ppb)(nWmL) Range of LCR from high sld high std curve curve (ppb)(ng/-L) (ppb)(np)mL) Rabbit 4.93- 1450 4.93 - 1450 49.3- 1000 49.3-97.6 4.93-97.6 97.6 - 1450 97.6 - 1000 Bovine 4.93 - 1450 4.93- 1450 97.6-1000 4.93-248 24.8 - 248 97.6 - 1450 97.6 - 1000 Rat Monkey 4.93- 1450 4.93 -976 24.8-976 4.93 - 1450 4.93- 1450 24 4.93-248 )3 9.76-248 97.6 - 1450 24.8-493 97.6 - 1450 1 248 - 1000 97.6 1000 Attachment C: LOQ Summary FACT-,M-3.1 Extractionof PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 14 of 17 Page 166 3m Medical DeDartment study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Compound: PFOSA Serum matrix Prepared rangeof standards (ppb)(ng/mL) Range of average curve (ppbXng/mL) Rabbit 5.00- 1470 5.00- 1470 LCR from ave curve (ppbXng/TTiL) 9.89-1000 Range of low std curve (ppb)(ng/mL) LCR from low std curve (ppb)(ng/mL) Range of high std (ppcurve b)(ng/mL) LCR from high std curve (ppb)(ngimL) 5.00-251 n/a 98.9- 1470 98.9- 1000 Bovine 5.00- 1470- 5.00- 1470 25.1-1000 5.00-98.9 nla 98.9- t470 98.9- 1000 Rat 5.00- 1470 5.00- 1470 50.0-1000 9.89-500 25.1-500 98.9- 1470 98.9-1000 5.00- 1470 5.00- 1470 98.9- 1000 25.1-500 25.1-500 98.9- 1470 n/a Compound: PFOSAA Serum Prepared matrix rangeof standards (ppb)(ng/mL) Rang of average curve (ppb)(ng/mL) LCR from ave curve (ppb)(ng/mL) Range of low std curve (ppb)(ng/-L) LCR from low std curve (ppb)(ng/-L) Range of high std curve (ppb)(ng/-L) LCR from high std curve (ppb)(ng/-L) Rabbit 5.20- 1540 5.20- 1540 104-1000 5.20-263 10.4-263 104- 1540 263- 1000 Bovine 5.20- 1540 5.20- 1540 263- 1000 10.4-521 n/a(1) 104- 1540 263- 1000 Rat 5.20- 1540 5.20- 1540 104-1000 5.20-263 10.4-263 104- 1540 263- 1000 Monkey 5.20- 1540 5.20- 1540 52.4-1000 5.20-263 26.3-263 104- 1540 263- 1000 Compound: Serum matrix ETFOSE-OH Prepared range of standards (ppb)(ng/-L) Range of average curve (ppb)@ng/mL) Rabbit 4.94- 1450 4.94- 1450 LCR from ave curve (ppb)(ng/mL) Range of low std curve (ppbXng/mL) 49.4- 1000 4.94-248 LCR from Range of LCR from low std high std high std Curve curve curve (ppb)(ng/rnL) (ppb)(ng/-L) (ppb)(ng/-L) 9.78-248 97.8 1450 rVa Bovine Rat Monkey 4.94- 1450 4.94- 1450 97.8-1000 4.94- 1450 4.94- 1450 494-1000 4.94- 1450 4.94-1450 97.8- 1000 4.94-248 4.94-248 4.94-248 4.94-248 n/a 9.78-248 97.8 1450 97.8 1450 97.8 1450 248 - 1000 97.8 1000 n/a Attachment C: LOQ Summary FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M E@-ivironmental Laboratory Page 15 of 17 Page 167 3m Medical Departmert Study: T-6295.7 Report No. FAC-L TOX-030 laboratory Request Number-U2279 Compound: POAA Serum Prepared mat3@x rangeof standards (ppbXng/mL) Range of LCR from Rangeof LCR from Range of LCR from average ave curve low std low std highstd high std curve curve curve curve curve (ppb)(ng/-L) (ppb)(ng/rnL)(ppb)(ngirnL)(ppb)(ng/rnL)(ppb)(ng/mL) (ppb)(ng/mL) Rabbit 5.01-1480 5.13- 1510 25.8- 1000 5.13-258 n/a 102- 1510 n/a Bovine 5.01- 1480- 5.13- 1510 102-1000 5.13-258 5.13-258 102- 1510 258-1000 5.01- 1480 5.13- 1510 51.3- 1000 5.13- 102 5.13- 102 102- 1510 102- 1000 5.01- 1480 5.13- 1510 102-1000 5.13- 102 5.13-102 102- 1510 258- 1000 Compound: PFOSEA Serum Prepared Range of LCR from matrix rangeof average ave curve standards curve (ppb)(ng/-L) (ppb)(ng/mL) (ppbXng/rnL) Rabbit 5.13- 1510 5.13- 1510 25.8-1000 Range of LCR from Range o I LCR from low std low std high std high std curve curve curve curve (ppb)(ng/mL) (ppb)(ng/TnL) (ppb)(ng/-L) (ppb)(ng/-L) 5.13-258 ri/a 102 -1510 n/a Bovine 5.13- 1510 5.13- 1510 102- 1000 5.13 -258 5.13-258 102 - 151o 258 loOO Rat 5.13- 1510 5.13- 1510 51.3- 1000 5.13 - 102 5.13 -102 102 - 1510 102 1000 Monkey 5.13 - 1510 5.13 - 1510 102- 1000 5.13 - 102 5.13 - 102 102 1510 258 1000 1 Compound: Monoester Serum matrix Prepared rangeof standards Range of average curve LCR from ave curve (ppb)(ng/-L) (ppb)(ng/-L) (ppbXng/-L) Rabbit 4.94- 1450 9.78-978 n/a Bovine 4.94- 1450 97.8- 1450 n/a Rat 4.94- 1450 248- 1450 248-1000 FM-0-1@ly4-.94- 1450 49.4- 1450 97.8-1000 Ingeneral,thechromatographyforthemoncesterwas very poor (broadpeaks,highbaseline). Curves formonoester inrabbitand bovinewere unacceptableA.ny quantitatiopnerformedwith themonoesteris onlyan estimateand shouldnotbe used forreliablea,ccuratedatareporting. AttachmentC: LOQ Summary FACT-M-3.1 ExiTactioonf PFOS from Serum or OtherFluid 3M Environmental Laboratory Pace 16 of 17 Page 168 3m Medical Department S::udy: T-6295.7 Report No. FAC7 TOX-030 laboratory Request Number-U2279 Ion PairStandard Curves - Fluids Prep date(s): Analyte(s): Sample matrix: Standard number: Equipment number: Finalsolventand TN: Blank fluid/identirier: Method/revision: Target analyte(s): FC mix stdapprox. 0.500 ppm: FC mix stdapprox. 5.00ppm: W398-64t W398-640 FC mix std approx. 50.0ppm: W398-639 Surrogate stdapprox. 17.71ppm: W398-605 Actual concentrationsof standardsinthe FC mix PFOS PFOSA PFOSAA ETFOSE- POAA OH Std conc Std conc g/mL ug/mL Std conc ug/TnL Std conc Std conc ug/mL ug/mL PFOSEA Std conc ug/mL Monoeste r Std conc ug/mL 0.500 0.500 5.00 5.00 5.00 50.0 50. 50.0 50.0 0.507 1 0.532 0.507 0.532 5.07 5.32 5.07 5.32 5.07 5.32 50.1 53.2 50.1 53.2 50.1 1 53.2 50.1 1 53.2 0.501 0.501 5.01 5.01 5.01 50.1 50.1 50.1 50.1 0.509 0.509 1 5.09 5.09 5.09 50.9 50.9 50.9 --,50.9 0.521 0.521 5.21 5.21 5.2 52.1 52.1 52.1 52.1 0.501 0.501 5.01 5.01 5.01 50.1 50.1 50@l 50.1 Calculated concentrations of standards PFOS Final conc PFOSA Finalconc ng/mL PFOSAA Finalconc ng/mL ETFOSE Finalconc ng/mL ng/mL 4.93 5.00 5.24 4.94 9.76 9.89 10.4 9.78 24.8 25.1 26.3 24.8 49.3 50.0 52.4 49.4 976 98.9 104 97,8 2@@ 251 263 248 493 500 524 494 735-- -746 782 737 976 989 1038 978 in the sample matrix POAA PFOSEA Monoester Finalconc Finalconc Std conc ng/mL ng/mL ng/mL 5.01 5.1 4.94 9.93 10.2 9.78 25.2 25.8 248 50.1 51.3 49.4 99.3 102 97.8 252 258 248 501 513 494 749 766 737 993 1017 978 All Am't spikedniL 0.010 0.020 0. 0.610 0.020 0.005 0.010 0. 0.020 Surrogate Std conc ng/mL 2.64 Surrogate Finalconc ng/mL 81.0 All Final Volume mL 1.015 1.025 1.010 1.015 1.025 1.010 1 1.015 1.020 I.C)25 All Am'tspiked (mL) 0.005 Validated ranges - approximate concentrations PFOS PFOSA -PFOSAA---- ETFOSE-OH POAA R2bbit 1 5-1000 ppb 10-1000 ppb 10-1000 ppb 10-1000 ppb 10-750ppb Bovine 5-1000ppb 25-1000 ppb 263-1000ppb 5-1000 ppb 1 5-1000 ppb R2t 10-100p0pb 25-1000 ppb 10-1000 ppb 50-500 ppb 1 5-1000ppb Monkey Estimatesonly. Use valuesfor Rabbit PFOSEA 25-1000 ppb 5-1000ppb 5-1000ppb AttaclunenDt: IonPairStandardCurves FACT-M-3, i Extractioonf PFOS from Serum or OtherFluid 3M E-@lv@-ronmental Laboratory Page 17 of 17 Page 169 3m Medical Department Study: T-6295.7 Report No. FACT TO: 030 laborator 9,@ 3M ENVIRONMENTAL LABORATORY METHOD A.,NALysis OF POTASSIUM PERFLUOROOCTANESULFONATE OR OTHER FLUOROCHIE,NUCALS IN SERUM OR OTHER FLUID EXTRACTS USING HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: FACT-M-4.1 Author: Lisa Clemen, Glenn Langenburg Approved By: Adoption Date: 4/22/98 RevisionDate: Laboratory Manager 1/tfl,l@., @Group Leader A Lbp"@, TechnicalReviewer Date @/-1-7/@y Date 9)@gios Date 1.0 SCOPE AND APPLICATION 1.1Scope: This method isforthe analysisof extractsfrom serum or blood forfluorochemical surfactantsusing HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemicalsurfactantosr otherfluorinatecdompounds, or other ionizablecompounds. 1.3Matrices: Rabbit,rat,bovine,or monkey se= and ratwhole blood or milk curd. Word 6.0.1/95 FACT-M-4.1 Analysis of Serum or Fluid ExtractUsing ES/MS 3M Environmental Laboratory Page I of 9 Page 170 3m Medical Department Study: T-6295.7 Renort No. FACT TOX-030 laboratory Request Number-U2279 2.0 SUMMARY OF METHOD 2.1 This method describesthe analysisof fluorochemicalsurfactantesxtractedftom serum, whole blood,or milk curd using HPLC-clectrospray/massspectrometry,or similarsystem as appropriate.The analysisisperformed by monitoring a singleion characteristiocf a particularfluorochemical,such as thepotassium perfluorooctanesulfonat(ePFOS) anion, NI/Z= 499. Samples may alsobe analyzedusingan APUMS/MS system to furtherverify compound idefitification. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization(A-Pl):The Micromass platform systems allow for variousmethods of ionizationby utilizinvgarioussources,probes,and interfaces.These includebut are not limitedto:ElectrosprayIonization(ESI),Atmospheric Pressurechemical Ionization(APcI),7hermospray, etc.The ionizatiopnrocessin thesetechniquesoccursat atmospheric pressure(i.en.ot under a vacuum). 3.2Electrospray Ionization(ES, ESI): a method of ionizatiopnerformed atatmospheric pressure,whereby ionizationoccursthroughth@ productionof tinycharged dropletsin a strongelectricaflield. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MSfiMS): The API platformsare equipped with quadrupole mass selectivedetectors.Ions are selectivelydiscriminatedby mass to chargeratio(ni/z)and subsequentlydetected.A single MS may be employed forion detectionor a series(MS/MS) formore specificfragmentation information. 3.4Conventional vs.Z-spray probe interface:The latestmodels of Micromass platform systems (post1998) utilizae "Z-spray"conformation.The sprayemittedfrom a probe is orthogonal tothe cone aperture.Inthe conventionalconformation itisaimed directlyatthe cone aperture,afterpassing through a tortuouspathway inthe counterelectrode.Thou-h the configurationisdifferentt,he methods of operation,cleaning,and maintenance arethe same. However, Z-spray components and conventionalcomponents arcnot compatiblewith one another,but only with similarsystems (i.eZ.-spray components are compatiblewith otherZspray systems,etc.) 3.5 Mass Lynx Software: System softwaredesigned forthespecificoperationof theseplatform systems. CurrentlyMassLynx has Windows 95 and WindowsNT 3.1 versions.All versions are similar.For more detailssee themanual specifictothe instrument(Micromass Platform IIor Quattro TIMassLynx or MassLynx NT USER'S GUIDE). 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use cautionwith the voltagecablesforthe probe. The probe employs a voltageof approximately 5000 Volts. 4.1.2 When handling samples or solventswear appropn'ateprotectivegloves,eyewear, and clothing. Word 6.0.1/95 FACT-M-4.1 Analysisof Serum orFluidExtractUsing ESIMS 3M Environmental Laboratory Page 2 of 9 Page 171 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 4.2 Cautions: 4.2.1 Do not operatesolventpumps above capacityof 400 bar (5800 psi)back pressure. Ifthe back pressureexceeds 400 bar,the HP 1100 willinitiataeutomatic shutdown. 4.2.2 Do not nin solventpumps todryness. 5.0 INTERFERENCES 5.1 To minimize interferencewshen analyzingsamples forperfluorooctanoate(POAA),teflon should not be used forsample storageor any partof instrumentationthatcomes incontact with the sample or extract. 6.0 EOUIPMENT 6.1 Equipment listedbelow may be modified in ordertooptimize thesystem. 6.1.1 Micromass ElectrosprayMass Spectrometer 6.1.2 HPIIOO low pulsesolventpumping system and autosampler 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.1.1 High puritygrade nitrogengas regulatedto approximately 100 psi 7.1.2 HPLC analyticaclolunin,specifictsobe determined by the analyst 7.1.3 Capped autovialsor capped 15 ml centrifugetubes 8.0 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTmwater,allwater used in thismethod should be Milli-QTmwater and may be provided by a Milli-QTOC Plus system 8.1.3 Ammonium acetate,reagentgrade or equivalent 8.2 Standards 8.2.1 Typically one method blank, one matrix blank, and ten matrix standards are prepared during the extractionprocedure. See FACT-M-3.1. 9.0 SAMPLE HANDLING 9.1 Fresh matrix standardsarepreparedwith each analysis.Extractedstandardsand samples are storedin capped autovialsor capped 15 ml centrifugetubesuntilanalysis. 9.2 Ifanalysiswillbe delayed,extractedstandardsand samples can be refrigerateadt approximately 4* C untilanalysiscan be performed. FACT-M-4.1 Analysisof Serum orFluidExtractUsing ES,'NIS 3M Environmental Laboratory Page 3 of 9 Page 172 3m Medical ]Department S-@udy: T-6295.7 Report No. FACT TOX-03C laboratory Request Numbe---U-2279 10.0 ()UALITYCONTROL 10.1 Method Blanks and Matrix Blanks 10.1.1 Analyze a method blankand a matrixblankpriorto each calibratiocnurve. 10.2 Matrix Spikes 10.2.1 Analyze a matrix spikeand matrixspikeduplicateper fortysamples. With a minimum of 2 spikesper batch. 10.2.2 Expected spike concentrationwsillfallinthe mid-range of the initiaclalibration curve. Additionalspikeconcentrationmsay fallin thelow-rangeof theinitial calibratiqncurve. 10.2.3 See Section 13 to calculatepercentrecovery. 10.3 Continuing Calibration Checks 10.3.1 Analyze a mid-range calibratiosntandardafterevery tenthsample. Ifa significant change ( 30%) in peak areaoccurs,relativeto the initiasltandardcurve,stopthe run.Only those samples analyzedbefore the lastacceptablecalibrationstandard willbe used. The remaining samples must be reanalyzed. 10.3.2 See Section 13 to calculatepercentdifference. 11.0 CALIBRATION AND STANDARD IZATION 11.1 Analyze the extractedmatrix standardspriorto and followingeach setof extracts.The mean of two standardvalues,ateach standardconcentrationw,illbe plottedby linear regression(r)forthe calibratiocnur-veusingMassLynx or othersuitablesoftware. 11.2 The r'value forthe data should be 0.980 or greater.Lower valuesmav be acceptableatthe discretionof the analystand documented approvalof the ProjectLead. 11.3 Ifthe curve does not meet requirements,perform routinemaintenance or reextracthe standardcurve (ifnecessary)and reanalyze. 11.4 For purposes of accur-acywhen quantitatinlgow levelsof analyte,itmay be necessary to use the low end of the calibratiocnurve ratherthan the fullrange of the standardcurve. Example: when attemptingto quantitateapproximately 10 ppb of analyte,generatea calibratiocnurve consistingof the standardsfrom 5 ppb to 100 ppb ratherthan the full range of thecurve (5 ppb to 1000 ppb). This willreduceinaccuracyattributetdo linear regressionweighting of high concentrationstandards. 12.0 PROCEDURES 12.1 Acquisition Setup 12.1.1 Click on startbutton in the Acquisition Control Panel. Set up a sample list.Assign a filena-meusing letter-MO-DAY-last digitof year-samplenumber, assigna method (NIS) foracquir-inga,nd type in sample descriptions. FACT-M-4.1 AnalysisofSerum or FluidExtractUsing ES/NIS Page 4 of 9 3M Environmental Laboratory Page 173 3m Medical Departrrent S@udy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.1.2 To create a method clickon scan button in the Acquisition controlpanel and selec'@ SIR (SingleIon Recording). Set IonizationMode as appropriateand mass to 499 or other appropriatemasses. A fullscan isusually collectedalong with the SIRS. Save acquisitionmethod. IfMS/MS instruments are employed, additionalproduct ion fragmentation informationmay be collected.See Micromass MassLynx GUIDE TO DATA ACQUISITION for additionalinformationand MPIM (Multiple Reaction Monitoring). 12.1.3 Typicafly the analyticalbatch run sequence begins with a set of extractedmatrix standards and ends with a set of extractedmatrix standards. 12.1.4 Samples are analyzed with a continuing calibrationcheck injectedafterevery tenth sample. Solvent blanks should be analyzed periodicallyto monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample listprepared in Section 12.1.1. 12.2.2 Set-up the HP II00/autosampler at the following conditionsor at conditions the analyst considers appropriateforoptimal response.Record actualconditions in the instrument logbook: 12.2.2.1 Sample size= 10 @iL injectionwith a sample wash 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 15 minutes 12.2.2.4Solvent rwnp Time 0.00 min. 7.5 min. 11.0 min. 11.5 min. MEOH 45% 90% 90% 45% 2.0 mM Ammonium acetate 55% 10% 10% 55 % 77:@] Note: In thisinstrument configuration,the run must be set up on the electrospraysoftware with a "Waitinc, for inletstart"message before the "Start"button ispressed on the HP Workstation. 12.2.2.5Press the "Start"button. 12.3 Instrument Set-up 12.3.1 Refer to FACT-EP-3.0 for more details. 12.3.2 Check the solventlevelin reservoirsand refililfnecessary. 12.3.3 Check the stainlesssteelcapillaryat the end of the probe. Use an eyepiece to check the tip.The tipshould be flatwith no jagged edges. Ifthetipisfound to be unsatisfactory,disassemble the probe and replace the stainlesssteelcapillary. 3M Environmental FACT-M-4.1 Analysisof Serum or FluidExtractUsing ESA@IS Laboratory Pace 5 of 9 Page 174 3m Medical Department St,-,dy: T-6295.7 Report No. FACT TOX--@30 laboratory Request Nurnber-U2279 12.3.4 Set HPLC pump to"On". Settheflowto 10 - 500 uL/min oras appropriate. Observe dropletscoming outofthetipoftheprobe. Allow toequilibratfeor approximately 10 minutes. 12.3.5 Turn on thenitrogen.A finemistshouldbe expelledwith no nitrogenleaking around thetipof theprobe. 12.3.6 The instrumentusestheseparametersatthefollowingsettingsT.hese settingsmay changein ordertooptimizetheresponse: 12.3.6.1Drying gas250-400 liters/hour 12.3.6.2ESI nebulizinggas 10-15 liters/hour 12.3.6.3HPLC constantflow mode flow rate10 - 500 @iL/min 12.3.6.4Pressure<400 bar(Thisparameterisnotset,itisa guideto ensurethe HPLC isoperatingcorrectly.) 12.3.7 Carefallyguidetheprobeintotheopening.Insertprobe untilitwillnot go any ftu-theCro.nnect thevoltagecablestotheprobe. 12.3.8 Record tuneparametersintheinstrumentlog. 12.3.9 Using thecross-flowcounterelectrodeintheES/MS sourceisreconunended for the analysisof biologicamlatrices. 12.3.10CIickon startbuttonintheAcquisitionControlPanel (thismay varyamong MassLynx versionss,ee appropriatMeassLynx USER'S GUDE). Pressthe start buttonat topof sample listE.nsure startand end sample number includesall samples to be analyzed. 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.4 Calculatematrix spikepercentrecoveriesusingthe followingequation: % Recovery Observed Result- Background Result x 100 Expected Result 13.1.5 Calculatepercentdifferencuesingthefollowingequation: % Difference= Exr)ectedConc.-CalculatedConc. x 100 Expected Conc. 13.1.6 Calculateactualconcentratioonf PFOS, orotherfluorochemicali,nmatn'x(@tg/ml): (nizof PFOS calc.from std.Curve.x DilutionFactor@ x I jig (InitiVaollume ofmatrix(ml)+ ml ofSurrogateStandard) 1000 ncc,l FinalVolume (mL) 14.0 NIETHOD PERFORMANCE 14.1 Method DetectionLimit(MDL) and Limitof Quantitatio(nLOQ) aremethod,analyte,and matnx specificP.leasesee FACT-M-3.1, Attachment A fora listinogfcur-renvtalidated MDL and LOQ values. FACT-M-4.1 Analysisof Serum or FluidExtractUsing ES/NIS Page 6 of 9 3M Environme--i@--alLaboratory Page 175 3m Medical Departmer--. S::udy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 14.2 Method Blanksand MatrixBlanks 14.2.1Method blanksandmatrixblankswillbe analyzedwitheachsamplesetfor possiblecontaminationor carryover.Values are expected to fallbelow the lowest standardin the calibratiocnurve. 14.3 Matrix Spikes. 14.3.1 Matrix spikesare analyzedwith each sample setand thepercentrecoveriesare expected to fallwithin 30% of the spikedconcentration. 14.4 Continuing Calibration Checks 14.4.1 Continuing calibratiocnhecks areanalyzed ata minimum of afterevery 10 samples with eacb sample set.The percentrecoveriesare expected to fallwithin 30% of the spiked concentration. 14.5 Ifany criterilaistedin themethod performance sectionisn'tmet, maintenance may be performed on thesystem and samples reanalyzedor other actionsas determined by the analyst.All actionswillbe documented inthe instrumentrunlog,the maintenance log,or on the summary sheet with thesample results. 15.0 POLLUTION PREVFN'TION AND WASTE MANAGEMENT 15.1 Sample extractwaste and flanunablesolventisdisposed inhigh BTU containers,and glass pipettewaste isdisposed inbroken glasscontainerslocatedinthe laboratory. 16.0 REcoRDs 16.1 Store chromatogams in thestudyor projectfolder.Each chromatogram must have the followinginfortnatioinncludedeitherintheheader or hand writtenon the chromatogram: study or projectnumber, acquisitiomnethod, integratiomnethod, sample name, extraction date,dilutionfactor(ifapplicable)a,nd analyst. 16.2 Plot calibratiocnurve by linearregressionand storeinthe study folder. 16.3 Printsample listfrom MassLynx and tape intotheinstrumentrunlog. 16.4 Printdata integratiosnummary from MassLynx and tape intothe instrumentrunlog. 16.5 Copy instrumentrunlog pages,includinginstrumentparametersand sample results,and storein appropriatestudyfolder. 16.6 Summarize data using suitablesoftwareand storein thestudy folder. 16.7 Back up electronicdata to appropriatemedium. Record in studynotebook the filename and locationof backup electronicdata. 17.0 TABLES, DIAGRAms, FLOWCHARTS, AND VALIDATION DATA 17.1 Attachment A: FACT-M-4.1 Data reportingspreadsheet 17.2 The validationr-lpora-stsociatedwith thismethod isFACT-INI-3.1 & 4.1-V-1. FACT-M-4.1 AnalysisofSerum or FluidExtractUsing ES/NIS Page 7 of9 3M Environmental Laboratory Page 176 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 18.0 RIEFERENCF-S 18.1 FACT-EP-3.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/MassSpectrometer Platform Systems" 19.0 AFFFCRED DOCUMENTS 19.1 FACT-M-3. 1,'Txtractionof Potassium Perfluorooctanesulfonatoer Other Fluorochemical Compounds from Serum or Fluid forAnalysisUsing HP L C-Electrospray/Mass Spectrometry' 20.0 RFVISIONS Revision Number. I Reason For Revision Validation of method to include 7fluorochemicals addition of whole blood matrix, surrogate standard, new.4PIIMS(MS) systems, monkey sera cross validation,MDL study, updates in record keeping and storing policies, etc. Revision Date 07/01/98 FACT-M-4.1 AnalysisofSerum orFluidExtractUsing ES AIS 3M Environmental Laboratory Page 8 o[9 Page 177 3m Medical Department St@udy: T-6295.7 Attachment A Report No. laboratory Request FACT TOX-030 Number-U2279 Laboratory Study Study: TestMaterial: Matrix/FinaSlolvent: Method/Revision: AnalyticaElquipment System Number: InstrumentSoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept: Dateof Extraction/Analyst: Dateof Analysis/Analyst: Group Dose Sample# Concentration ug/mL InitialVO). niL Dilution Factor FinalConc. ug/mL Slope: Taken from linearegressioenquation. Group/Dose: Taken fromthestudyfolder. Sample#: Taken from thestudyfolder. Concentr2tion(ug/mL): Taken from theMassLynx integratisounnunary. InitiaVlolume (mL): Taken fromthestudyfolder. DilutionFactor: Taken from thestudyfolder. FinalConc. (ug/mL): Calculatebdy dividintgheinitiavlolume from theconcentration FACT-M-4.0 AnalysisofSerum orFluidExtractUsing ESAIS 3M Environmental Laboratory Page 9 of 9 Page 178 3m Medical Department Study: T-6295.7 3M MedicalDepartment Study:T-6295.7 ATTACHMENT D DATA SUMMARY TABLES Report No. FAC7 TOX-030 laboratory Request Number-U2279 ReportNo. FACT TOX-030 LaboratoryRequest Number-U2279 3M EnvironmentaLlaboratory 3M Environmental Laboratory Page D-1 Page 179 3m Medical Department @-:::udy:T-6295.7 3M Medical Department Study:T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 ReportNo. FACT TOX-030 LaboratoryRequest Number-U2279 TableD-la.Sample Intervalasnd Types by IndividuaAlnimalfrom Study6329-223 ANPAAL ID SERUM SAMPLES COLLECTED (CiTY-WEEK) LIVERSAMPLES COLLECTED (DATLE) REcavr-RySUBGROUP SERUM SAMPLES COLLEcrED (Cl7Y-WF-EK) Group 1 (Conb%)I) 105508M 105517M 105519M 105520M 105526M iO5527M 105529F 105530F 105531F 10SM5F 1 '05544F 105549F Group H (LowDo") 151 Samples a Samples 72Samples 12---0,12.,4,6.8,12,16, 20.24,26,27i 2125/99 11--0,1,2,4,6,8,12.16, 20.24,26 2125/99 12-0,1,2,4,6.8,12,16, 20,24,26,2Tj 2/2-r@M 14--0,12,.4,6,8.12,16.Retoned 10ook)fw3107/00 18--27!2t8i,29.30,31,35,39,43.47, 20.24,26, 27,27i,2Tii 51,53,57,61,65,69,73,T7.79 14---o.1,2,4,61,2816' Returned10cojort3y107/00 18-27ii2i8,,29,30,313,5,39,43.47. 20,24,25, 27,2@i,i7ii 51,53,57,61,6,56.9.73.T7,79 12---01.,2,4,6,8,12,16, 20,24,26,27i 2125/99 14-0,1,2,4.6.81'2,16, Retumedtoook)ny3/D7/00-18-27il@1,28,29,30,31,35.39,43,47, 20,24. 25.27,27i.2Tii 51,53,57,61,65.69,73,77,79 12--o.1,2,4.6,8,12,16, 20,24,26,27i 212%9 12--C-.1,2.4.6,8,12.16. 20,24.26,27ii 2126/99 12--0,12,.4.6,8.12,16, 20,24,26.27ii 2125'99 12---0.21,4,5,8,12,16, 20.24.26.2T, 2125199 14-0,1,2,4,6,182,16, ReUned b oolony3107/00 18-27iii2,8,29.30,31,35.39,43,47, 20.24,26.27,27'i,2Tti 51,53,57,61,65.69,73.T7,79 99 Samples 8 Samples - 105514M 105515M 105516M 105521M iO5537F 10&%IF 1--0 B"@@assigned 12--0,1.2,4.6.8,12,15, 20.24,26,27i 12-0,1,2.4,6,8.12,16, 20.24,26,2Tii 12--0-,-1,2.4,6.8,12,16, 20,24,26,27ii 12-0,1,2.4.6,8.12,16. 20,24,26,27ii 2/26/% 2126/99 2/2M9 2/26199 1-0 BaseVAb assvad 11--0.1.2,4,6,8,12,16, 20,24.26 12--0,12,,4,6,8,12.16, 20,24,26,2Tii 2125% 2126199 1--0 isswed Returnedtocolony Returnedtocolony Retumed tocolony 1-0 Returnedb colony 105547F 10555OF 12---0,12.,4,6,8,12,16, 20,24,26,27ii 12--0,12.,4,6,8.12,16, 1 20,24,26.27 2/26Sg 2t26M ....... 27 Day 183(2/23199) 27i Day 184(2125M) 2TiiDay 185 (2/26199) 27iiiDay 187(212&99) **7wo samples2/20/99 79 Sample on 2@2a/00 79i Sampleon 2/25/00 79iiSampleon 2/25/DO Not*:SamplesforWeek 25 and Week 27 (RecoveryGroup)takenon same day(Day183,Z*23/99) 3M Environmental Laboratory 3M Environmeni--al Laboratory Page D-2 Page 180 3m Medical IDepartment Szudy: T-6295.7 3M MedicalDeparbnent Study:T-6295.7 Rer)ort No. FACT TOX-030 laboratory Request Number-U2279 ReportNo. FACT TOX-030 LaboratoryRequest Number-U2279 TableD-ib.Sample Intervalasndtypes by IndividuaAlnimalfrom Study6329-223 ANUAL ID SERUM SAMPLES COLLECTED (CITY-WEEK) LIVER SAMPLES COLLECTED (DATE) RECOVERY SUBGR(XJP SERUM SAMPLES COLLECTED (QTY-WEEK) Group111 (MidDo") 132 Samples 12 Sampim 7 Sampl" 105505M 105510M 10551"M 105523M 105524M 105528M 105532F 105538F 105539F 105545F 105548F 105552F 14-0, 1,2,4,6,8, 12,16, 20.24,26,.27,271,27ii 12---0,12.4,6,8,12,16. 20,24,26,27ii 2--0.1,Z 4.6,8,12,16, 1 20,24,26,27ii 14-0.1,Z 4,6,8,12,16. 20,24,26,27,Z7-i2,7ii 12--0,12,,4,6,8.12.16, 20.24,26,27ii 12--0,1,2.4,6,8,12,16. 20,24,26,27i 12--0,1.2,4.6.8,12,16, 20,24,26,27i 12---C,1,Z 4,6,8,1126,, 20, 24, 26.27i 14-0.1. 2,4, 6.8,12,16, 20,24,26,27,2Ti,27ii 12-0,1, 2,4,6.8,12.16, 20,24,26.27i 12--0. 1.2,4,6. 8,12, 16, 20.24,26,27i '4-0,1.2,4.6,8.12,16. 20,24, 2r.,2-7,27i,2Ti 3101/00Biopsy 2/26/99 2126W 3101100Biopsy 2/26/99 2/26M 2/251% 2125M 3/01/0B0iopsy 2/25199 2125M 3101/00Biopsy 18-27iii,28,29, 30, 31, 35, 39.43, 47, 51,53,57,61,65.69,73.77,79 18-27iii,28, 29,30, 31, 35, 39, 43,47, 51,53,57.616,5,69,73.77.79 18--27iii2,8. 29, 30,31, 35, 39, 43, 47, 51,53,57,61,65,69,73,T7,79 18-27ii2i8,,29,30,313,5,39,43,47. 51, 53,57, 61.65. 69, 73, 77, 79 Group [V (HighDo") 148 SwnpWs 105506M 11-0,1,2.4,6,8,12,16, 20.24,- 105507'M 12--02,01,2,2,44,,266.,82,71i2,16, 105509M 9-0,1.2,4.6,8.12,16,20 10551im iDS512M 1". 1,2,4,6.8,12,16, 20, 24, 215,27, 27i.27ii 12--0.12,,4,6,8,12,16, 20,24,26,27i 105522M 14---0,1.2, 4. 6, 8.12.16, 1 20,24,26,27.2Ti.27ii '4-0.1,2,4,6,8,12,16, '05533F 20,24,26,27,2T.,2Ti 2--0. 1,2,4,6,8,12, 16, 'aSS34F 20,24,26,27.- ioww 2--0,21,,4,6.8,12,16, 20.24.26,27i 10554OF 105542F 12---0,1.2, 4, 6, 8. 12,16, 20. 24, 26. 27ii 14-0.1,2.4.6,8,12.16, 20, 24,26. 27, 27i,27ii 105551F 12-0, 1,2,4, 6,8, 12,16, 20,24,26,27ii 14Samples none 2125/99 none 9r22199 Biopsy, 2/21-@100 2r25M 9/22/99Biopsy, 2125= 9rZ2/9B9iopsy, 2/25100 2/26199 2/25/99 2/26/99 9/22199Biopsy, 2/25100 2r4W9 80 Samples 20-27iii,28,29, 30, 31,35, 39,43, 47, 51,53, 57, 61,65, 69,73, 77, 79, 79i,79ii 20--27iii2,8, 29,30, 31, 35, 39, 43, 47, 51.53,57,61,65,69.73,77.79.79i,79ii 20-27iii2,8,29,30.31.35,39.43,47, 51,53,57.61,65,69,73.77,79,79i.79ii 20-27iii,29.30.31,35.39,43,47, 51,53, 57,61, 65, 69.73, 77. 79, 79i,79ii 27 Day 183=3199) 27i Day 184 (2125/99) 27@ Day 185 (2126M) 27iiiDay 187 (2128199) '*Two samples2r2O/99 79 Sample on 2123/00 79i Sampleon 2/25/00 79iiSamp@e on 2125/00 Note:SarMiesforWeek 26 and Week 27 (ReooveryGroup)takenon same day (Day183,2i23i9g) 3M Environmental Laboratory 3M Environmental Laboratory Page D-3 Page 181 3m Medical Department Study: T-6295.7 3M Medical Department Study:T-6295.7 Report No. FACF TOX-030 laboratory Request Number-@,72279 ReportNo. FACT TOX-030 LaboratoryRequest Number-U2279 TableD-2.Average PFOS ConcentratlonisnSerum by Dosage Group and Week fromStudyFACT Tox-030(BaselineD-ay 185) Time Point AllPFOS values LOQ@o@@ gWmL Group IM Qbnv&gfd Group IF Group 2M d Group 2F Group 3M Group 3F Group 4M Group 4F Baseline LOQ 0.025 Week 1 LOO 0.025 Week 2 LOO 0.025 Week 4 LOO 0,0152 Week 6 LOGO.0152 Week 8 LOO 0.0 152 Week 12 LOO 0.0152 Week 16 LOO 0,0152 Week 20 LOO 0.01-@2 Week 24 LOO 0.0 152 -AveragePFOS StdDeviaftn AveragePFOS StdDeviabm AveragePFOS StdDeviabm -AveragePFOS StdDeviadon AveragePFOS StdDeviabon AveragePFOS StdDeviabm AveragePFOS StdDeviatm AveragePFOS Std DeviaUm AveragePFOS Std Deviabcn -AveragePFOS Std Deviabm <Loa NA <LOQ @LA <LOO NA <LOQ -I Anomaly NA <LOQ NA <LOQ -I Anomaly NA o.ow -1 Arwiaty 0.00811 0.0407 0.0110 0.0400 0.0120 0.0440 0.0101 -@:LOQ NA <LOG NA <LOQ NA <LOQ NA <LOQ NA 0.0226-2 Anomalies O.C)0189 0.0263-2Anomalies 0.00362 O@0432-2Aromalles 0.00851 0.0504 0.0127 0.0426-1 Anomaly 0.00784 <LOQ t'A 0.869 0.147 1.10 0.0835 1 3.20 0.577 3.61 0.430 4.73 0.432 6.69 0.578 11.2 2.44 12.3 1.40 14.5 3.06 <LOQ NA 0.947 0.110 1.10 0.0963 3.40 0.291 3.71 0.417 4.76 0.577 6.31 0.717 10.5 1.90 19.5 14.5 13.0 0.675 -@LC)Q NA 4.60 0.782 5.81 0.933 17.8 1.68 20.4 1.65 2Ei.0 3.30 35.2 5.39 56.2 5.a4 -63.7 6.71 65-9 6.88 <LOQ NA 3.71 0.455 5.39 O.gW 16.5 1.87 18.8 2-15 24.0 3,06 27.8 3.98 42.1 4.04 58.1.-. 7 60.4 7.24 -ELOQ NA 21.0 1.57 26.9 3.54 95.3 70.4 94.5 8.07 ID9 18.3 122 23.9 189 15.9 144 10.9 215 24.9 <LOQ NA 20.4 2.71 22.0 3.25 92.7 39.6 90.1 7.11 107 11.8 117 11.7 162 19.3 156 21.8 174 20.9 Week 26 Average PFOS LOO 0.0152 SbdC)eviadw Week 27 Average PFOS LOO 0.024a Sbd Deviatm Day 183 Average PFOS LOG 0.0 152 [Std Deviabm Day 184 LOG 0.0152 Awa(M PFC)S Std Deviabm Day 185 kveraw PFOS LOO C@o 152 Std Deviabw -CLCOZLessttante Lo&w Lnd of 0.0459 0.0143 0.0529 0.0145 --0.117-0.0764 0.0233-1 Anomaly NA 0.0432 0.00928 0.0506 15.8 13.2 82.6 66.8 173 171 0.0164 1.41 1.42 25.2 10.8 36.5 22.2 0.0416 15.9 11.1 68.1 58.5 194 160 0.0148 1 5.54 1.52 5.75 4.67 8.93 23.9 0.0533 85.0 81.7 249 230 0.0315 14.9 35.1 46.8 40.3 0.0352 0.00911 69.7 78.0 259 245 4.68 12.0 110 29.2 o.ow 3 294 321 0.0306 0.7 23.3 22.0 170 3M Environmental Laboratofy 3M Environmental Laboratory Page D4 Page 182 3m Med4-cal Department Study: T-6295.7 3M MedicalDepartment Study:T-6295.7 Report No. FACT TOX-030 laboratory Pequest Number-U2279 ReportNo. FACT TOX-030 LaboratoryRequest Numt>er-U2279 TableD-3.Average PFOS ConcentrationsinSerum by Dosage Group and Week from Study FACT TOX-030 (RecoveryGroup) Time Point AllPFOS values Group IM GroupIF Group3M I Group3F LOO (ligftl) pg/mL O.Omglkglday 0.15mgfkg/day Day 187 Avemge PFOS 5,otnL) 0.03DO-1Anomaly 0.0457 1 79.1 39.5 LOO 0.0152 Std Deviabon NA 0.0118 4.67 41.4 Week 28 LOO 0.0152 AveragePFOS fi@) StdDeviabon 0.0371 0.0124 0.0430 84.6 86.1 0.00821 1.51 3.59 Week 29 =0.0152 AveragePFOS (poVnL) StdDeviation 0.0665 0.0742 75.8 69.9 0.00362 0.000664 2.18 11.4 Week 30 LOOO.0152 AveragePFOS wo")L) 0.0313 0.0303 65.2 62.0 StdDeviation 0.000165 0.000485 4.60 10.6 Week 31 LOO 0.0152 AveragePFOS WcWnL) StdDeviation 0.0358 0.000567 0.0368 56.6 79.6 O.C)121 4.T7 43.8 Week 35 AveragePFOS ipgtyl) 0.049 0.0723 84.5 74.4 Loa O.co5m Std Deviabon 0.00303 0.00352 12.0 9.53 Week 39 AveragePFOS (po"t.) 0.0391 0.0509 1 50.1 51.7 LOQ 0.00&96 Std DevLation 0.0106 0.000779 3.16 7.29 Week 43 LOO 000555 AveragePFOS StdDevtabw 0.0319 0.00"0 0.0368 0.0000923 45.7 58.1 1.12 1 0.249 Week 47 LOO 0. Week 51 LOO O.DO555 AverigePFOS fmOnL) StdDeviation Aver-agePFOS (po@nL) StdDeviation 0.0355 0.00221 0.0237 0.00333 0.0459 0.00323 0.0341 0.000403 48.3 1 42.6 3.69 6.70 37.9 35.1 2.62 13.2 Week 53 AveragePFOS (.otrc) 0.0331 1 0.0397 46.2 36.7 LOO 0.00555 StdDeviathn 0.0086 O.C)0311 3.30 6.24 Week 57 LOO O.oosm AveragePFOS wo"*-) StdDeviation 0.0327 0.00526 0.0445 30.2 32.3 0.00385 2.36 1.34 Week 61 LOO 0.005M AveragePFOS wotnl) StdDeviation 0.0351 0.00449 0.0448 31.6 0.00210 5.98 38.2 0.283 Week 65 LOG O@00565 Average PFOS 4*t@L) StdDeviatkr 0.0210 0.0360 32.9 37.6 0.00365 1 0.000= 1 0.0269 2.32 Week 69 LOG 0.005M Average PFOS @wornl) Std De-Aation 0.0406 O@00313 0.04()0 26.4 .5 0.00301 2.59 3.46 Week 73 LOOO@00556 Wmk 77 LOQ 0.00&% Average PFOS (Aovr-L) Std Deviabon AveraW PFOS StdDevtabm 0.0350 0.0115 0. 296 0.00535 0.0365 1 27.3 25.8 0.002&4 4.66 2.91 0.0305 22.5 23.0 0.00167 0.632 6.37 Week 79 LOG 0. I AveragePFOS (vo",L) StdDeviation 0.0215 0.00296 0.0243 1 19.1 21.4 0.00355 1 0.805 2.01 Gmup 4M Group 4F 0.75rngtkgtday 267 258 42.0 15.2 249 236 21.7 18.3 223 194 66.9 19.0 143 162 38.0 7.87 161 185 46.1 21.9 181 i 171 19.5 1 io.i 146 1 161 16.1 11.1 78.8 16.8 124 25.9 94.7 38.4 1 BO.8 36.8 159 284 98.3 8.32 91.4 6.07 98.2 0.490 78.0 1-63 100 50.3 106 3.84 109 0.697 91.5 82.8 55.2 9.68 84.0 75.0 52.4 5.25 54.4 147 27.3 131 60.0 57.0 38.3 19.1 41.1 41.4 25.9 1.15 3M Environmental Laboratory 3M Envi-ronmen--al Laboratory Page D-5 Page 183 3m Mel@ical Depart,-Pent- S@-udy: T-6295.7 3M MedicalDepartment Study:T-6295.7 Report No. FAC-- TOX-030 laboratory TZequest Numbe---U2279 ReportNo. FACT TOX-030 LaboratoryRequest Number-U2279 TableD-4a.PFOS Amounts ReportedInSerum by IndMdual Animal(Baseline-Week26) PFOS REPORTED WEEK 0 I WEEK 1 I (ii ) y IEEK 2 WEEK 4 WEEK 6 WEEK 8 1 WEEK 12 WEEK 16 GroLrIp(C-bcQ IOSSOBM IOS51TM 105519M 105520M 105526M <LOQ .<Loo <OQ -<LCO -<LCO <Loa <Oa <LOG <LOC <LOG <LOQ 4LOQ <LOQ <Oa <LOC) <LOQ -<LOQ .<LC)Q -<LCQ -CLOO .<00 -.<Loa <Oo <Loa <LCQ <t-Oo <Loa KLOO <LOQ <LOQ 0.0381 O@D430 @@-0.0423 <LOQ 0.0244 OM" -0,0408 0 03a2 O@0320 105527M -<LOC) <Loa -<CG -<LOQ <OQ 0.03a5 006423 0.0615 105629F -LOO <LOQ -<LCO -<LOQ &<00 0.0214 .<LOQ 0,0438 105&ICF 105531F lossw lossw -LOO -<LOQ .<LOQ <LOG <Loa <Loa <Loa -<OC) .<Loo -<LOQ <LOQ -<LCQ -<LOQ -LOQ -<LOQ <LOQ <Oo <LCO .CLOQ <Oo 0.0244 0.0206 <LOQ <00 0.0243 0.0254 <LOQ 0.0238 -0.0515 0.0314 -Loa -LOQ io&uw <Loa <LOQ <Loa <LOQ -4-OQ 0.0240 000462 WEEK 20 WEEK 24 0.0348 0.0419 -0.0335 0.0300 0.0389 -00631 O@0617 0.(0@m5mm .0.0493 0.0253 .0.05M 0.0570 0,0"9 0.0486 O.D414 O.C)2B3 0.0397 -00613 ..O,D498 -00497 0.0364 -LOO -0,0325 o.ow WEEK 26 00496 -0.0539 00417 0.0254 0.0376 0.0669 o@()613 0.07ii 00431 00266 -0.04M 005% Grow I (L106513M 105514M 105515M 10551&M lowim 105525AA 106537F DO") <OQ <LOD .<()C) <LOQ -<LC)Q -<LOO -LCO 0.793 .0.787 O.M2 109 1.10 -1.02 1.03 -1.19 -1,13 -1.19 -2.87 2.75 3.13 .4.03 -3.73 105541F 105543F -LOO -LOO -0929 1.16 -3.07 io5uw lO5U7F -<LOQ -<LOG O@931 -106 3.55 10555OF .<Loa O.a32 -0.980 #3.27 G-.Jp M (P&d Do-) 105505PA 105510M 10551&M 105623M 105524M 1056am 105&UF 105 Irw 105639F <LOQ -LOO -LOQ -LOO -<LOQ -LOO -<Loa <LOC) <LOG 4.80 -4.19 5.48 3,54 425 5.52 -4.31 4.12 3,31 -6.07 4.82 .701 456 -624 6,15 -461 6.67 .465 .17.0 .17,2 -20.2 176 1-5.4 -19.2 16.8 18.7-.14@3 1 1o&%4w 105WF 105552F .<oo <LCO <Oo -3.69 3.72 3.12 1 528 -6@42 4.73 .18.7 -15.7 .14.9 GROM IV"0 00-) iosm6m 1 1055UTM 105509M im5lim 105512M 105522M 105533F IDS&W 105536F lossw 105542F 10555iE_ <Lco <LOG <00 <Loa -LOO -<LOQ -<LOQ <Oo -LOO -<Loa -<LOQ CLOO 21,3 -220 -18.8 .19.3 228 -21.6 -22.6 -18.1 -21.0 -16.8 2-4.0 -20.1 .29.9 .236 -25.1 -72.6 23.9 -54.8 22.6 "8.4 31.3 T7,4 28.8 .8Z2 249 .141 -26.0 .145 -23.5 .64.1 .19.5 -67.2 .-199-7,784 18.2 1 -60.2 indicatesa saffpe vwdhan exracbon voiume of<0 5 mL ShadW cells=rrcrtxrd .3-67 .3.31 -3.26 .4.19 -4.28 3.66 .3.85 -3.27 21.0 Z2.5 -20.1 .18.6 .21.8 .18.5 .18.4 18.9 .18.4 ZZ7 -18.4_ le.1 -Gi.0 -8Z8 gZ3 917 .105 lC2 93-0 -M3 -82.8 90.8 -102 szo 4.61 4.17 5.05 510 5.2B 4.&5 6@20 648 6.55 -752 6.81 5.57 10.4 .9.47 10.1 -14.8 11.1 -123 12.5 10@7 11.9 14.1 11@5 4.98 3.94 -7,03 1 107 5.83 1 7.85 14.3 41.1 27.9 2Zl 25.1 24.5 31.6 24.6 27.6 24.3 26.5 24,5 22.3 19.1 40.1 1 -50,4 39@2 5zg 337 60.7 -35.3 5Z2 37.8 63.4 25.4 484 25.8 40.4 -32.8 373 278 46.3 32-2 43.1 23.2 470 24.8 38.7 ---62,3 63.3 r,3,1 75,2 64,0 54.2 69.2 474 -78.2 45 .7 65.3 4Z7 125 86.1 103 92.3 118 131 110 1 121 102 105 116 87.3 116 106 123 99.4 167 -121 126 114 -918.0 -112 131 117 162 158 172 .138 197 152 179 126 216 143 155 145 1f>4 1 169 157 143 184 146 173 193 1 12E7 *1'532' -14.7 .10.9 1 14@l -18.4 13,4 13.7 16@@@ 165 138 -169 -13,5 -148 .12.7 122 77, 65.2 58.8 597 696 -648 -63,1 486 -69.9 83.5 55.1 -61@7 198 188 207 247 .233 190 .200 -169 181 -150 130 .11.4 -129 -695 -72.1 -69@5 62 634 590 722 857 -645 556 -182 142 148 221 203 187 -155 177 165 -142 3M EnvironmentaLlaboratory 3M En-:-'-rcnmenl-.aLlaboratory Page D-6 Page 184 3m Medical Department S::,ady:T-6295.7 3M MedicalDepartment Study:T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 ReportNo. FACT TOX-030 LaboratoryRequest Number-U2279 TableD-4b.PFOS Amounts ReportedInSerum by IndividuaAlnimal(Days 183-Day 185 and WGek 27) PFOS I DAY 183 REpoftTED IU9"d4 DAY 184 69wc) DAY 195 44@"ti WEEK 27 (PwrnL) GroupI(Ct.0 105508M 105517M 105519M 105520M 105525M 105527M 105529F 10553OF IOMIF 105535F IGS5411F 105UW 0.171 -0.0620 - 0.0758 - @ <Loa 0.0233 a o.o4ie @ 0.0310 0.0257 - 0.0366 0.0498 - 0.0763 - @ @ - 0.03M GrouIp(LoDwos4) io55l4M - 105515M - @ 10551 sm - @ 105521M - @ 105537F 105541F @ 105547F @ 1055WF @ Group 19(Mid Do") losm5m -@74@.4 66.4 70.3 los5lom - - @ 105518M - - 10SU3M 95.5 73.0 85.4 105U4M - - @ iosuem - @ 10 F - @ 1056UF - a - IOSWGF .107 W.5 101 la554V - 105SUF - IOSMF 56.9 M.5 -678 Group IV (HighDo") 105506M - - 105507M - 105509M - - 105511M -218 181 309 105512M - 105522M 202 336 -278 10553W 258 255 -441 105534F - - 105&W - iosuw - @ lasur 1 x 1 224 -201 105WIF I - I - a C Sanoe mimcbon daim forWeek Z7 data F,.Wplecobactedon Z?OM irccatesa sanoe withan exbacw vokxm of<0.5 rrL 0.04M - 0.0695 - O.OB35 0.0374 0.0318 0.0335 - 23.9 13.0 11,4 15.5 126 953 11.2 68-5 68.7 60.6 74.7 54,3 565 580 196* 184 - 202 11 1- 182 169 154 126 3M Envimnmental Laboratory Page D-7 3M Environmental Laboratory Page 185 3m Medical IDepartmert Szudy: T-6295.7 3M Medical Department Study:T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 LaboratoryRequest Number-U2279 Table D-Sa.PFOS Amounts Reported InSenim by Recovery Group Animal (Day 187- Week 47) PFO S REPORTED I DAYig7 I 'AfEEK28 WEEK29 WEEK30 ULWMW WoNrIL) tughTIL) 4oftc) WEEK31 64ord4 WEEK 35 WEEK 39 UOWL) WEEK 43 WaktIL) WEEK 47 WoknL) Group I(CM&A 105520M I <.OQ 00284 -0.0890 -1055M 1055"F 00300 0.0373 .0@0459 -0@04M 0.06M .0,0738 105-154ww 0 C;-4717 -0@0372 0.0747 Group 11(M;dDws) iosmsm 82-4 -85.7 773 IOSS23M 75@8 -83.5 .74-2 105639F 10.2 -88.7 T7,9 105562F 68.8 .83.6 61.9 Group IV (HighDoes) 105511M 237 -233 175 iosmm 297 264 270 105=F 269 249 207 IO&WF 248 223 180 Indicatesa sample withan extactcn vo@im of<0.5rnL (3.0314 0.0312 0.0299 O.GMG -88.4 61.9 -89.9 54.5 118 170 156 167 O.CW2 0.0354 0.0282 .0.0453 60.0 53.2 -111 -48.6 129 194 201 -170 0.0437 0,0480 0.0748 O.OM8 -93.0 76.1 .81.2 -67.7 167 195 164 178 0.0316 0.04M 0.0503 -0.0614 -62.4 -57.9 96.8 -0.5 .135 -158 -169 --.154 0.0287 0.03W O.OM O-M87 48.5 44,9 58.3 -M.0 68.9 90.7 179 139 0 0339 0,0370 O.Mi 0.04M 50.9 45.7 47.4 1 37.9 105 -142 104 92 Table D-5b.PFOS Amounts Reported InSerum by Recovery Group Animal (Week 51- Week 79) PFOS I WEEK 51 REPORTED I W,,r.) Group I(Contal) 10@M=-() 1055" 105529F 10554OF .0213 0.0261 O.M" 0.0338 Group II(INd Dos*) 1055iD5M 39.8 iOSS23M 36@O 105-1,,QF 44.5 1055W -25.8 Group fV(HighD-) 10551im 105522M -67.5 122 105533F 87.1 IOSWF 95.7 WEEK 53 WEEK 57 Wpl.1) 4*1,44 00269 0ON2 0-0375 0.0419 0.0289 0.0364 j 000.000424!4,7@8782@92O 0.0418 43.9 28.5 48.5 31.8 41,1 33.3 32.3 31.4 548 6a.5 107 89.5 986 103 979__ 109 WEEK 61 COftt) 0,0319 0.0382 o00@04M 0.048 27.4 35.9 38.0 38.4 __64-7 138 108 log WEEK 65 Wo,,L) 0.0184 0.0236 0.0364 0.0357 329_ 329 39.3 30.0 -52.5 .131 .89.7 -76.0 WEEK 69 WEEK 73 (,,%,nL) bo"t.) I WEEK 77 WEEK 79 .0@0428 0,0383 O.D421 0.0379__ -0.0431 0,0269 0.0388 __0.0345 -0.0334 -0.0259 O@0317 -0.0294 0.0194 0.0236 0.0288 0.0218 24.6 240 .22-1 19.7 28,3 w,6 -23,0 185 36,9 279 -275 22,8 32.0 .23.7 -18.5 19.9 47.0 35.1 329 .121 .73.5 87.0 787 54.5 70,5 713 -240 .434 Indicateassaffnaswdh an etacbm vollrm of<0.5rnL Table D-6.LOO Values Used InAnalyzes by Method and Usage Dates METHOD Sera FACT-M-4.1 ETS-8-&O ETS4@-5.1 LJVW -FACT-M-2.0 FACT-M-2.1 ETS41-7.0 EFmcTivE DATE I 10/10/98 3tOl/99 4r26M 5/2&98 6103199 7/2Z% LOQ ngtml 4.39 15.2 5.55 roig 30.0 37.4 26.9 SAGE DATES 1/25M toZ22199 310&% tD4/17/99 5117M throughtm end ofshidy 1/25199to Sre2M 6tO3f99to 6/14199 7/29M tb rough the eridof study 3M Environmental Laboratory Page D-8 3M Environmental Laboratory Page 186 3m Medical Der)art,7.e@7:S:@-..:dyT:-6295.7 3M MedicalDepartment Study-7.-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 ReportNo. FACT TOX-030 LaboratoryRequest Number-U2279 Table D-7. PFOS Concentrations In Uver by Dosage Group DCSAGE GR(>tjp COLLECNDN DATE PFOS CALCULATM -1--A@PNFrOZS@ CONCENMTMN (nwol Group I(ControQ 105508M 105517M 105519M 105=M 105526M 1055Z7M 1055ZF 10553OF IDMIF 105535F 1055"F 105549F 2f25Q9 Z?-rv% 2r25199 2r25M :mdr*-. 2125t% 2/26/99 2r261% 2/25199 Not*@:, Group 11(Lm Ome) 105514M Z26/99 iD5515M 2r2&% IM16M 105521M 2/26% 2/26M 105537F 2/25M 105UlF iC5547F 2/26M 2r26M 1055WF 2/26M Grotip11 (Mid Do*&) io5W5M 3101100Biopuy 105510M 105518M 105523M Z26199 2r26f99 3/01/00 BIOPSY 105524M 2J261% 1055M 2126% 105532F 2r25/99 10553OF 105MF 2125M 3101/00Blop" 105545F 105548F 105552F 2/25199 2125% 3101/00Biopsy Grcxjp IV (High Dose) 105506M ID55OTM 2125199 2 Anorrialies 143 -1 Anomaly 91 129 128 112 87 97 18237 22709 11417 16734 22818 24847 20102 20735 8252 42169 86173 10203 5W73 48203 8N21 49590 24728 66532 81376 1769C) 412474 2 Anomalies 0.143 0.091 0.129 0.128 0.112 0.087 0.097 18.2 22.7 11.4 16,7 22.8 24.8 20.1 20.7 8.25 42.2 86 10.2 58.7 48.2 80.4 49.6 24.7 66.5 81.4 17.7 412 lD5511M w2z% Biop" Z25M 142465 23480 1 D5512M 2r2-riu% 378723 1 D5522M gr22/99Biopsy zr25= 137561 70781 10=F &r22/99Biopsy 1752a3 2t25100 42668 1055W zr2&99 280575 io55W 2r25M 256669 10554OF 2/26M 267328 IOSS42F %2Z99 Blop" 2r25W 421647 57895 105551F 2r26199 r 287223 <LOCT Less twn t@*Lower Lirriotf C)uanftton (26.9-37.4ng/g) 142 23.5 379 138 70.8 175 42.7 281 257 267 422 57.9 287 3M EnvimnmentalLaboratory 3M Environmental Laborat------y Page D-9 Page 187 3m Medical Department Study: T-6295.7 3M MedicalDepartment Study:T-6295.7 ATTACHMENT E DATA SPREADSHEETS Report No. FAC-- TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 LaboratoryRequest Number-U2279 3M EnvironmentaLlaboratory 3M Environmental Laboratory Page E-1 Page 188 3m Medical Depart-,nent Study: T-629A&TOX-030 Report No. FACT TOX-030 CovanceN6329-22l3aboratory Request study ProducNtumbo(TestSubdarice): N(atruL Method(Revnion: AnmiyticEaqlu4mma Systm N=b@ Irmut@t Softwarwv@m: Fd@e: R-Squa-dValue Slop. Y-kdacept@ Dwz ofExuvctmoWAnalylL DateofAnslyan/Analyst DateofDataReduc6orL/Analyvt SALMPle D@sts 26 Week CapsuleToxicitSytudymth PFOS LnCynomclgwMonker T-6295(PFOS) Monkey Sera FACT-M-3,1AtFACT-M-4.1 Soup020199 MmLy- 3 1 Sm Anachm-u Sm Atuch@u SeeAttichmmts SeeAtuLch@nts 07109/99RWW 02113/99MEE 02/16/9K9rH BASEIINE N40NKEY SERA Group S@ple 0 Dme MethodBik M.tm Blk RBS02099-H20BIk-I PJ3Sa2O99.H20 Blk-2 RBS02099-SomBIk-I PFOS Conc mg/=L 000 000 0.00 Couc"tmtiou ofPFOS Z/.LLor% R., <LOQ <LOQ <LOQ %4e.o PFOS ug/mL <LOQ RSD Std.D-. MS/MM RPD NA RBS02099-Som Blk-2 0.00 <LOQ <LOQ NA QC - 250 ppb &0=099-MS 299 121% &0=099-MM 295 115% 119% 5% Group I 1055MM+ 1.28 <LDQ Control 105517M+ 1.32 <LOQ 0.0 nig/kiVday 105519M+ 316 @LOQ 105520M, 1 46 <LOQ 105526m- 1,62 -LOQ Group 2 L@W-DOOC O@03 Tn&Wday 105527M+ 105529f+ 105530F+ 105531F105535FI03544F I05549F 105513M+ 105514M 105515m, 1 48 2.28 l@13 l@73 146 1 60 5-42 2.63 1.59 0.340 <LOQ ].OQ NA <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ @LOQ NA @LOQ <LOQ <LOQ 105516M 265 <LOQ 105521M+ 1 72 <LDQ 105525M. 1.90 <LOQ <LC)Q <LC)Q 105537F+ 1 @33 105541F- 2@ 23 <LOQ <LOQ I05543F- 2.53 <LOQ 105546F+ 2 50 <LOQ rOS547F- 1 69 <LOQ 105550F+ 1.50- <LOQ ILOQ <LOQ Gm@p 3 105505M 3.13 <LDQ Mid-D@e 105510m+ 1.04 <LOQ 0,15 mgwday 105518m+ 105523M+ 105524M+ 105523M+ 105532F+ l@51 <LoQ 0.930 <LOQ 1 92 <LOQ 2.69 <LOQ <LOQ <Loo 3@13 @LOQ 105539F 225 @LOQ 105539F I05545F+ 10554$F 105532F- 215 <LOQ 3 27 <LOQ 3 60 <LOQ 2.02 <LOQ <I.OQ <LOQ Gmup 4 High-Dose 0 75 mgl*cicy I05506M+ 105507M 105509M 2.19 <LOQ 2.79 <LOQ 2.06 <LOQ 10551IM 2,69 <LOQ 105512M+ 2.64 <LOQ 105522M+ 105533F+ 2.73 <LOQ @LOQ <LOQ 2.02 <LOQ 105534F+ 2.54 'LOQ I05536F+ 2.95 <LOQ 105540F+ 3@45 <LOQ I05542F+ 1.94 <LOQ 105551F 3.52 <LOQ <LOQ LOQ L@t ofQu&nLitnum (LOQ): 4.39nwml Date Entemd/By 02117199 LAC Number-U2279 NA -@Joatpplicable PFOS - Pcrfl@ooetw@lf@to Date Veaw,& By 03123199 OMI, 06(07/00 PW Dew purttyooffectad/nnfied09/12100nuidi0,9112/00LAC + S@ initvioall=eIm.tha0n.5m0I,conc=transohnosubledc@,dredtenutmGe4/28/I0.0A(' Exu-actioVnolume Ratio- htud Volurnw'Fmal%'ol=c FACT-M-4 I Exmi 9@ 3M Environmental Laboratory tox-030-sem223.1C 9/12/00 Page 189 3m Med@-cal Department Study: T-629i4C'iTOX-030 Report No. FA-.--TOX-030 Covance#6329-2l2a3boratory Request Nurr@cer-U2279 Study. Product Nwnbet@Test Subst@e) Mat@ M@thod/R@@ ArAlricalEquipmcrd Synm N=b@ Lmu@@.1 Sft@.r./V-=. F.I.R-Squared Value Stope@ Y.UILCmtpt: Dwe ofExvactborVAnalyst Date ofAnalysislAnalyst. Date of Data Reducuort/Amlyst Sample Data WEEK I ?AONKEY SERA Group Dan Sompl* 0 Method Blk - Ma= Blk QC-250p-pb Group I Cznlrol 0.0mga*dny Gmup 2 Low-D@ 0 03 m4'cglday Group 3 M@d-D@ 0 15 mgil*day RBS02059-H20 Blk-1 RBS02059-H20 Blk.2 R.BS02059--c@ Blk-I RBS02059-3@ Blk-2 i.MS02059-MS MXS02059-MSD 105508M 105517m+ 105519@.i 105520.M 105526.M 105527.M I05529F 105530F 105531F 105535F 105544F- 105549F 10551 SM+ 105516@.1 105521%1 105537F 105541F+ 105547F 10555OF 105505M 105510M. 10531 B.M 105523\4 IC5524M 105528M 105532F105538F 26 Week CapsuleToxicityStudy withPFOS inCynomotgus Monkey, T-6295 (PFOS) Moikey S.. FACT-M-3 I & FAC7-,ki-AI Md.l.. 041098 & A-ci,.06219?t lvl-Lr@ 3 1 See Attachment. See Attachments See Attachments See Attachments 02/05/99SAH OZ/09/9,OZ/10199,02/12/99,0211-,,99HOI,DRB 02/10/99.02116/99.02/26199 1101,T)RB PTOS Come. EVML 0.00 0.00 0,00 01)0 305 --304 0,00 000 0.00 0.00 0.00 0.00 000 o@oo 0.00 0.00 0.00 0.00 495 383 519 6",7 684 464 581 519 287 1109 342 221 319 413 161 257 Coacentmtion Mean of PFOS PFOS uy)ml or % Rtc -LC>Q ILOQ ILOQ <LDQ <LOQ @LCQ 123@'i 123% 123% <LOQ <LOQ <LCQ <LC>Q ILOQ ILOQ @LOQ <LC)Q <I,OQ .-[,C)Q <LO(,) <LOQ C)793 o '6- 0832 1 C)9 0869 lic c 929 0931 0832 0947 4 60 419 -5 48 3@%-l 1:5 5 5: 460 4 31 412 RSD Std.D*v. NISF.MSD RPD NA NA_ NA_ NA 169 014, 11 6 0,110 170 0782 105539F 248 105545F@ 184 105548F 232 105552F 195 3 31 3 69 3 '2 123 3 12 3 71 0 455 Grasp 4 I05506,'A 665 Z13 H*h-D@ 10550'7M- 411 2"0 0'75 mglkg/dy [05509m@ 351 18 9 lossi Im- 492 19 3 105512.M 711 229 7 49 105522,M@ 539 @l1 6 21 0 1 57 105533F- 564 22 6 105534F- 451 18 1 105536F- 393 21 () 105540F- 419 i6R 105542F 749 24 0 13 3 10555IF- 377 20 1 204 2 71 Limitof QuantA&t@n (LOQ): 4 39 ng/niL Data Entemd/By 02/10199.03/01;99 1,A- Date Venfiodt By 03/23.199O?@4,L, Date puritycm@udtveri6ed 09112/00ffutth0.9112/00 LAC NA - Not appl=blc - Serum srutwvolumelessdw 0 50 mi.,concentratiosnhsouldbe coruideretdcntatme 0 PFCS - Perfluorooct=@lf@ct Fxtrwum Vol@c P@tio= Init@lVol@tfYmai Val@e FACT-.%1-4 I Excel 97 3M En-,ri-ronmental Laboratory tox-030-sem223.1C 9-:12iOO Page 190 3m Medical De-uartment S--udy: T-624@kC-TTOX-030 ReDort No. FACT TOX-030 Covince#6329-22l3aboratory Request Number-U2279 Study ProductN=ber(T@t Matrix Sutmt@) Method)Rcw.n. Aza"cal Eqwpmcnt Sywtem N=b@ Im@ent Soft@e/V@on F@enmc '6 Week Cap%LdeTomcityStudy mth PFOS in(,%momolgw Monker T-6295 (PFOS) Monkcy 3ers FACT-M-3 I& FACT-M-4.1 M.d@tin. 041093 & A-ba D62498 M..Lym 3 1 See Artachm@tz ,t-SquaredValue Slope See AttLc?unmu See Attachrnents Y-',ntuc.pt Date of Extr=borvAnWyat See AM&chments 02/09/99 JCP Date of Anairm/Aradyst: Date ofd&ta Reductior@tAnalym: 02/15/99. 02122199 HOJ 02/19/99, 02125199 HOJ Simple Data WEEK 2 MONKEY GCGGF SEP-A Sample I PFOS Concentration Mo.. RSD D- C..C. ofPFOS PFOS Std.Do,. ngl=L vgt=L w % R" mg/mL MS/MSD RPD .Mcg@d Blk RS802D99-H20 Bik-I 0.00 <LOQ RBS02099-H20 Bk-2 0,00 @LOQ NA Matu B& RBS02099-S@ Bik-1 000 <LOQ RB302099-Sm Bik-2 00() <LOQ NA QC - 250 ppb MK302099-MS 293 ?AKS020WMSD 300 11ar/. 121% 12(r/. 2% Group 1 Control 0 0 m&4*day 10550am 105517M 105519M. 10352DM- A D9 3 07 4 53 7 oa <LOQ <LOQ <LOQ <LOQ 105526M 2.02 <LOQ 105527M- 1.89 <LOQ NA 105529F- 0.520 <LOQ Group 2 L.--D.@ 0.03 mglkgiday 10553CF10553 1 F105535F 105344F105549F- 105514M105515M105516M, 105521M. 105537F- 1.0. 190 3.12 0380 0810 575 513 492 425 446 <LOQ <LOQ <LQQ <LCIQ <LIOQ 1 02 1 03 l@ 19 1 )3 1 19 <LOQ 1 10 NA 7 63 00835 105541 F 72' ID5547F- 527 105350F+ 550 1,16 1 06 0.980 879 1 10 0.0963 CMUP3 105505m- 227 Mid-Dome 105510M 301 0 11 mg'kwdy 105518M. 330 105523M 2RS 105524M- 195 105528M 394 105532F- 173 105539F 416 6.07 4 92 701 4 56 624 6 15 461 667 161 s al 0933 103339F- 32 4 65 G-.p 4 High-D@ 105545F 329 1055ASF+ 320 105552F 295 105506M- "47 103507\4- 626 5,28 642 4,73 299 25+1 17@2 5 39 0930 0 15 mgkvdy 105509M 746 23.9 105511 m 706 22.6 105512M 976 31.3 13 1 105522M 8w 105533F 778 105534F- 650 28.8 24.9 26.0 269 3 54 105536F- 588 23.J 10554OF- 5A6 19.5 105542F- 377 19.9 14 8 10353 1F 568 13@2 .12 0 3@25 L@t of Quantitaton (LC)Q) 4.39 NWML Date Entemd/By Date VcnficdfBy 02a2o99. 03102/99 LAC 031231" GML Daiep@tyco@clecLsmnfied 09/17=ffui%k 09/112/00LAC NA - Not appbcable PFCS - Perfluorooctanftulfwate - S@ initia@lh@ lew Uw 0 50 mL. concentratons shotiidbe crmidered tenlbtiw D4/,,WOO LAC Extm=on Voi=e Ratio- IjutW Volume/Fmal Volt=c FAL7-M-.l I Excel 97 3M Env@---onmental Laboratory tox-030-wm223-IC 9iI2ioo Page 191 3m Medical Departmerit Study: T-629k4C@iTOX-030 Covince#6329-223 Report No. FACT TOX-030 laboratory Request Number-U2279 sk* 26Week CapsuleToxmty Studywith?FOS inCynmot" Monkeys Pmduct Nwnber(restSub&Wwt) Matrix Method(R@aiL AzgyticalEquipmentSywtcm N=ber lmtn=mt Software/Vmlm Film-.. T-6295(?FOS) Monkey Sm ETS41-40 andETS-9-03 MadelineD41(?91A,m@ho 062499,& %4-Lym 3 1 and3 2 See Anuh@ts Soup 020199 R-Sq=@d V.1Slope Y-[ntacW. Date ofExtmcticr./ArmLyst. Date fAnsfy@WA@Wyt: S@ Aft-h-u See AmtchmmU See AmLehnwnu 03102/99SAI-DRWW O-VD5M,OYOL'99.OV25/" HOJIMEEI)RB DateofData ReducteWAnWW: @3/0&'99O.M6/99 KIWDRB Sample Data WEEK 4 MONKEY SERA Group Do** PFC3 C@ CMM.NFDStl" ofrym PF)OS RSD gtdlD-. IWN&L *V'=L or % Rw or/mL MSIMSD RPD med.d B& R3SO3O79.H20 Bik-I 0,00 <LOQ RBS03029.H70 Btk-2 0,00 <LCQ <LC)Q NA Matrix Elk ILBS03029-Sas Blk-I 000 @LQQ RBW3M9.Sm 3&-2 0,00 <LOQ <LOQ NA Matrix Blk MKS03029LSen BLk-1 7.21 @LCQ MKSO3Cr29-Sm Egk-2 7,17 <LOQ MK503029-Sen B*-3 7,26 @LOQ MKS03079@Sm Btk-A 632 <LOQ ILOQ NA QC - 250ppb MXS03029.MS-1 253 N,(KS03029.MSD-1 250 102% 101% 101-/. 1% MKS03029-MS-2 142 99% MXS03029-MSD-7 235 95% 3% Group 1 Control 0 0 mga*day 105umm 543 105517M- 6.76 105519M- 791 10552om- 6 11 105526M 6-,3 <LOQ <LOQ <LOQ <LOQ <LOQ 105527M, 198 @LCQ <LOQ NA 105529F- 663 <LOQ 10553CF- 611 <LOQ 10553 1F 9 19 <LOQ 105535F- 9 17 <LOQ 105544F 996 <LDQ 105549F- 8 74 <LOQ ILOQ NA Group 2 105514M- log 287 Lo..D@ 105515M 1?2 75 2 O@03mgkwday 103516M 196 101 105537F- 186 313 181 403 320 0 57- 3.73 ICJ541F+ 153 3.07 105547F 721 10555OF- 163 3.53 953 3,27 3,40 0291 1 Cr.* 3 105505M- 63 7 17.0 %d. D@ to55lom+ 646 17.2 0 15 mg/)Wdoy 105518.M- 101 20+1 105523M 110 10552AM 96@3 105529.M- 957 1'6 15 4 9 47 192 179 168 105512F- 41,4 16,6 105539F 7 05539F- 11 5 9 @A3 :05545F103USF, 105557F+ 46.7 Is 3 --559 7 :'-7 11 4 14.9 165 1 1 97 1 G@p 4 lit L16 High-E)ose :01 M 211 776 O@75n4/Wdy 1055cpgm. 273 5484 105511M + 24 2 48 105512M. 290 774 -38 IOSSIIM+ 309 92.2 95,3 ',04 105533 + 352 141 10353,4F- 3-63 145 105536F, 240 64,1 10554CF- 938 67.2 L-.t fQ@nWaom 105WF10555IF(LOQ): PFOS - 152 NWML 294 794 42 7 t5o 60,2 i 92 7 1 396 1 DateEntered/By OYOW", 03126t99LAC DateVi6e& By 0312Y" GMI@ Datepwty co@te&vcrified- D9112MOmmh. 0911I= LAC NA - Not appbcabk - S@ initivaolhzme lessOw D@50mL. wntmtrat3onsshotddbe com@deredimtauve 04,29/00LAC PFCS - PcrG@occtane&Wfamte Extmcbm Voi=c R@tio- IRUW Volume(FmiaVoi=c E'rS-8-50 Excel9-, 3M Environmental Laboratory lox-030-.m223-IC 9112/00 Page 192 3m Medical Department S'-udy: T-62-94C-YTOX.030 Report No. FAC7- TOX-030 Study: ProductNumber(TeitSubsu=): Ma=: @.lethod(R@on A@t.ty@ Eq.p-ent Sytm N-b., Imtr=ent SoftwwefV@im FLIc@e: R-Squared Value: Slope Y-intmcept [).!eof ExtmcbotVAnLlyst Data of Ara?ynWAxA)yst. Date of Data ReductorVAnalyst: Covance#6329-2l2a3boratory 26 Week C.psWe ToxicityShidy w2th PFOS inCynomoigm 1.@ionke@i T-6295(PFOS) Monkey -'era ETS-94 0 and ETS-8-5,0 A@h. 062499 & Sup 020199 ki-Ly= 3 1 and3 See Anachmmts See Attachments See Anschmenti See Attachments 03ilO/99SAH 031l2f99.03121!99MEE.IDRB 03/1&199,03124/99LAC/DRB Request Numher-U2279 Sample Data WEEK 6 MONKEY SERA Group Dfte Sample p PFOS Come. Concestraden ofPFOS M... PFOS RSD Std.Dtv. ngf=L ug/=L w % R" ug/inL MSIMSD RPD Method Eflk PBS03109-H20 EUk-1 000 <LOQ RBS031096H20 8&-2 7 74 <LOQ NA MatrixBlk RBS03109-Sm EUk-1 000 <LOQ RBS03109-Sen Blk@2 4mig <LOQ MatrixBlk MK503109-Sm Bik-1 2.56 <LOQ MK303109-Sm EUk-2 2,55 <LOQ MK303109-Scra Bik-3 1.93 <LOQ MKS03109-Sen Slk-4 o@50() <LOQ N*A QC -250 ppb MXS03109-MS- 1 264 MKS03109LMSD-1 253 106% 101% 103% 4% MKS03109-MS-2 243 MK303109-MSO-2 249 97% 1W/. 98*/. Group I lo550am- 000 <LCQ Control 105517,%4@ 000 <LOQ 0 0 mWkWday 105579m- 000 <LOQ 105520M 1 60 <LOQ 105526M 105527M- 0610 9@ 49 @LCQ <LOQ <[,OQ NA 105529F+ 1.56 <LOQ 103530F- 3,38 <LOQ 105531F 4..4 <LOQ 105535F- 000 <LC>Q 103544F- 0 DO <LC)Q 105549F- 3@54 <LOQ ILOQ NA C-.p 2 [05514M- 366 3 67 L--D- 105315M. 33.1 3,31 0 03 nWkWdy 105516M- 31@5 3.26 9 10552IM- 41 8 4,19 3 61 0 430 10553'?F- 42,7 4@28 103541F 45 7 3.66 10554?F- 3,6-4"- 365 ii 2 10355OF- 31.1 3.27 3 71 a 417 Gmap 3 105505M 87 4 21@o M,d-Do- 105510m@ 561 0 15 Wkg(day 105518M. 669 2..5 201 105523M- 61@9 18@6 105524M+ 7-.7 105528M- 616 21 & a @19 185 20 4 1 5@ 105532F+ 61 2 IS 4 105538F 94 5 189 105539F+ 61 4 184 105545F 94 5 22,7 105549F+ 61 3 194 11 4 105552F 670 16 1 is8 15 G@p 4 105506M- 18, 91@o tiigh-D@ 105507M- 165 97's 0.75 nWkiVday 105509M 230 92 3 10551im 234 93.7 105512m+ 211 105 54 105522M+ 201 102 9.1 5 80, 105533F 232 930 105334F- 166 833 105316F- 179 898 105540F 909 105542F- 203 la555lF 205 107 -90 82.0 90 1 711 LL=t of Quantitgum(LOQ). PFOS - 15.2rWmL Date EntaedfBy 031l9t99,03124/9L9AC Date V@fied/By O&il?f" SAH.05/19@00 PIO;P%V Date pwry c@mted@mnfied 09flZ/00nm%k 09/IZM LAC PFCS - Perfl@t@Wfomte - Serum mbsj @l@ ten dw 0 50 mL, cmcentratiomshciddbe com:deredientat@ 04/28/00LAC NA - Not apph=ble ExtractioVnolume Ratio- liubalVoLume/Fmal %'ol=c ETS-3-5 D Excel 97 3M Environmental T ahcratory lox-030-scm223-IC 9112/00 Page 193 3m Med@-cal Department S@::udy: T-629F@kC-7TOX-030 Report No. CovsnceO#6329-2l2a3boratory Request Study 26W@k CapsWe To@mty StAy 'nthPFOS m CYnomOigw -MOnkeYs ProductNumbert7est Substance): T-6295 (PFOS) M.tr.. @Method(R@.on AnWyncil Eqwpmmt Syt@ Instr=mt Soft-@eV@on @4@b@ Mmkcy sErS-8-40 md M 9-5 0 sup 02at99 Mu,Ly- 3.1-d 3.2 F,I,.-.: S@-Afth.-u R-Squ@d VaJue: Sft Attachmmts Slope* See Aftct@enLs Y-Ent@cept See Artachmwts Dateof ExtractiortAzWyst 03,03@99 RWW/ICP Date ofAnaly%WAra&ISL OV05t". 0,VO9/99,OV10,99,D-V2" DRB Date of Data ReducwrVArQtyu: 03/0&'99.OY15199,03124M,03.r26MORB Sam pie Data %TEK 3 MONYM SMZA Group smpio 0 PFOS M@ RSD Don C@ O(PFM PF03 31.L0". oV=L ViaL w % Rse aWmL MSIMSD RPD Method Bik Matnx Blk RBS03039-K2O EQ. k R.BS03039-H20 Blk-2 000 RBS03039-Sem Blk-1 00 ILBS03039-Sm BM-2 000 LoQ @<LOQ ILOQ NA @LOQ @LOQ ILOQ NA Mabrbe Blk MKS03039-Sen Blk-@ 323 LOQ MXS03039-Sm B&-2 -210 <LOQ QC.250 ppb MXS03039-MS-l 174 69% MXS03039-MSD@l 191 <LDQ NA 72% 9-16 MKS03039-MS-2 205 82% )4KS03039.MSD-2 lss 73% 10% MKS03039.MS-3 111 MXS03039-MSD-3 203 Group 1 10550am 943 Control 105517M 768 72% 91% <LOQ LOQ 76% 11% 0 a mSA4(day 105519M 120 fO552OM 903 105526M 11.0 105527M 78.9 <LOQ <LOQ @LOQ 0<.03&5 @LOQ IA-mly NA 105529F 16,0 00214 tOS53OF 193 00244 105531F ISO 0.0206 105535F 14 5 <LOQ 1035."F 946 105549F 150 <LOQ 0.0240 0 @.26 -2 A@@&l@, 836 000199 Group 2 03514M 34 5 461 I-aw-Dose :05513M 31 2 7 0 03 mgfwdy 103316M "1 5.15 105521m 31 8 -5 10 105537F 46@ L 528 9 14 473 0 432 105541F 36.3 485 105547F 37.3 499 1 10555OF 344 394 4-6 011177 Group 3 105505M 122 279 Md-Dose toi5lom 92 9 22.1 0.15mg/kviy 105518M 941 25@l 105523M 91 6 24.5 101524M III 31.6 12 7 1 105528M 1 10" 24@6 26,0 3 30 105532F 961 276 105538F 106 24 3 105539F 926 265 105545F 122 24.5 10554F 69 4 '2 3 12 105552F 593 191 240 306 Group 4 05506M a122 125 High-D@ ;05507M 38 961 O@75mga*day 105509M 117 103 105511 m 198 92.3 105512M 115 [is 16 7 105522M 127 131 109 18 3 1 105533F 107 ilo 105534F 981 121 101 36F 100 102 1055,W 102 105 [05542F 113 116 11 1 105351 F 1 2 -873 1 107 11 0 L@t of QuRntitanonCLOQ): PFOS - 152 nwml D@te Entmd,By OMS'99,03/24M. 03(26(99i-AC Dste Venfi@,VBy OBIISM SAlt 051)1100 PJO, O5f23100& 05-124/0P0W Date ptmryc@eoe&@fied 09/12,Wmmh, D9/l2jOOLAC PFOS - PerflwrooctRnmuifo@le NA = Not apphcsble Fxtncnon Volume Ratio- [=bidVolume/Fmal Volume FACT TOX-030 Number-U2279 M-9.5 0 E@@i 97 3M Env@-rc)nme.-ita'-Llaboratory tax-030-scm223-IC 9!12too Page 194 3m med@-cal Department Sz:udy: T-6291EA&TOX-030 Report Nc. FAC- TOX-030 Covance#6329-22l3aboratory Request Number-U2279 ShLdy: Product NLunbMTest Matnx Subgwxt): Method/R@ion: ArWybcal Equipment Systm N@b@ I-tr--t s.ft-./V.,.m FJ@c R-Sq@d Value: Slope 26 Week CapsWe T=ctfy T-6291 (PFOS) Monkey Sm ETS-8-4.0and M-9.5 0 Soup 020199 M-Ly- 3 2 S@ Attachrnmt, S" AtUchMMU See AttaclunmU Study with PFOS m Cy@molgLu kionkrys Y-Inl=cpt: Dote of Extriebori/ArWyst Date ofAngy&WAnalystDate of Data ReductolVAnalyst. S" Amelunents 03/03M RWW/JCP 03/05/99,03/09/W.0-VI".0312.999 OPB 03/DM, DVI5/99,M". ON7&'99 I)RB Simple 1),ata WEEK 12MONKEY SERA G-" D.m S@ple 0 PFOS Coo@ ogintl conopmuldim of PFOS vg/=L or% Rw M@ PFOS ut/.L RSD Std. D-. RPD Method Bik Mabrix Blk matrgx EQ QC -250 ppb R.BS03039-H20 aik-1 R.BS03039-H20 EQ.', RBS03039-SRBS03039-Sm MKS03039-Sen MKS03039-Scra Ek- I ELk-2 RI-I B&-2 MKS03039-?AS-1 MKS03039.MSI)@l MKS03039-MS-2 MXS03039-MSr,2 MK503039-MS-3 0 DO 0 DO O.DO 0,00 323 2.10 174 191 203 195 111 <LOQ <LOQ <LOQ <LOQ @LOQ <LOO 69% 76% 92% 73% 72% LOQ NA @I.DQ NA @LDQ NA -2@,. 9% 10% MKS03039.MSD-3 203 81% 76% 11% G-p 1 Conb@i 0 0 mg/Wday G@p 2 Lo--Dom 0 03 mrwd.y Cre" 3 Mid-D0 15 mVWday G-P 4 High-E)OSC 0 75 mg(Wday 10550am 105517M to5si9m 105520M 105526M 105527M 105529F10533OF (05531 F 105535F 105544F 105549F 103514M 105515M 105516M 103505M 1055tom 1035ism 105523M103524M 10552am 105532F 105338F@055539F 0 545F 10554F 105552F 103-1@06M 103507M 05509M @0551 1 m 103512M 105522M05533F 309 269 329 12.7 21 3 30.1 114 16,4 19@o 943 181 233 41 8 443 414 451 579 34,8 36.9 488 143 135 133 Rs 0 ]is 792 1 969 942 101 109 796 774 103 864 16 .13.9 149 R9 0 ID9 0,0381 0.0430 0 OA39 <LOQ D@D2" 00423 <LCQ 0,D243 O@0254 <LOQ 0.0238 0,0316 6,20 649 655 691 5,57 7.03 5,93 401 392 33.7 33.3 37.1 25 4 25: 32 279 322 232 248 116 106 123 99.4 167 121 126 0 0383. 1Anomaly 0 0263 -1 Anomaim 6.9 631 35 1 '.78 122 21 1 000811 010(33)362 963 0518 il4 0-17 1!3 3 39 i43 3 99 196 23 9 1 I L-,t ofq@tubm (IA)Q):PFOS PFOS - Patuoroocttnftulfanote :05534F 105536F10554OF103542F 105351F 15,,'gi.L Date p=ty IDO 114 636 98.0 900 112 128 131 114 1 117 1 D.I.Enic,ed/By 01,09,99.03,124t",O-V26,9L9AC DateVmfied/ By: OIVIM SAH,OY24= PW .nded@vaiS@d 09/12100mnxk 09/12100LAC Se@ mhal "I@ Im dun 0.50mi, cm@tmbom ico 117 sholddbe comidmd t=tative04(7&00 LAC \A - Notapp6c.ble Extmctim Volume RAto - initw VohznejT@W Val=c E7'S-9-50 Excel 97 3M Env4-ronmenL@-a 1 T aboratory tox-030-$=223- IC 9/12100 Page 195 3m Medical Department Study: T-629AC-TTOX.030 Report No. FAC7 i-OX-030 CovanccW6329-2l2a3boratory Request Number-U2279 Study: Prodwt Nmbci(Test Subel@e): main.: M.thod'P--: AnalyticaElqwpnient Syg= N=ber. 26 Wmk Capsule Tomcj?y Study mth T-6795 (PFOS) Moky S,ETS-9-4 0-d ET@-S. 5 0 Soup 020199 PFOS in Cymmclgtu Monkeys lnswm=t Software/Vavm Mu&Lynx 3 2 FLmme: R-Squared Value Slopc Y-Invercept Nte ofExtracti@VAnafyst Date o(AnakyoWArAlyst, Date of Data lUductioWAna)yv, Sample Data See Anschrnents See AtLKh@ts See AV@hmmts See Amuhmuft 3/IV" SAHFRWW OYIYW,03/19/99 DRA(HOJ 03/16(W,03r24199 DR3 WEEK 16 MONKLFY G rwap Dwe SERA SULF40 0 PFOS CML attaLL concomuttkm acrym ugfuLL or% Rw M@ Pros og/nLL RSD STILDWV. Mcdmd Blk Mabu Blk M&hu Blk QC -250 ;Ypb R.BS03129@H20 Bik-1 0.00 RBS031MH20BIk-2 000 RBS03129-Sm Bik-1 0.00 REM3129-Sm Bik-2 0.00 MXS03129-Sm B&-1 150 MKS03129-Sem Blk-2 76 MKS03129-MS-1 ag MKS03129-MSD-1 :99 MKS031MMS-2 " MKS03129-MSD-2 1I35 @LOQ <LOQ <LOQ <LOQ <LOQ @LCO 72% 76% 76% 9(Y% @LOQ @OQ ILOQ "4% 83% NA NA NA -5% 16% G@p 1 Corwol 105508M 21 @5 105517M - 204 0,0344 O.D409 0 0 mg(kwday ID5519M- 21+1 0.0423 10557am 105526M 103527M 20 ' 0+0332 199 (1.032() 394 0,0615 00407 27 1 00110 105529F 355 0.0438 10553OF- 251 00515 105531F 21.5 00314 105535F1035"F105549F 9.37 <LOQ 116 <LOQ 19 29.9 O@0462 0 0432 -2 Anomahn 0 OCS51 G@p 2 105514M 77.7 1 104 Low-Dm 0.03 mgkwday 105515M- 473 105516M 91 7 10552]M+ 931 105537F 764 105541 F- 692 9.47 oll @4.R 11.1 12.3 21 9 11 2 1 2 44 105547F 668 10.7 91 10555OF 539 795 los 90 G-.p 3 Mid-Elmo 0 15 n-*IWday 105505m, 222 594 10551ONi 33.0 52.9 105519.',4 379 607 05523.%1 326 52 2 @ 05524,M 105529M 05532F @05538F 105339F 415, 1 634 36 2!2 404 2.9 37@3 3A7 1 463 10 4 562 5 84 05545F 323 '3.1 @05548F 323 470 959 105552F 241 38+7 1 421 404 Gr"p 4 H*@Dow 0 75mtwday 103506M 105307M 105509-M @005555I:M2m 105522M 05533F 273 182 257 172 196 197 168 179 196 216 1 254 195 45 164 9 42 189 159 :05534F 1I% 137 105536F+ 14 194 10554F 11 73 105541F 3 68 it 9 L@t ofq@maom (LOQ): PFCS 10555[F 15 2 ngiml 1 1@9"'1 DatfEntm&llf. 127 I 162 OVOLV9.OY17199,03124(99 L.AC I 193 Dole Vmfiedl By: WtL,99 SAH, 0511&00,05174/00PW Date punty m@tedivenfiedi 09tt2/OOnunh.09/17,'0L0AC PFOS - Pcrffuoroact@df@te Semm @tW vidi@ Ins thm 0 50 mL, co@natom shotddbe c@id@ed imtnnw. 04/29100LAC NA - Not sppii"blt Extwtm Volme Ratio- InitiVaoltume;FinalVoi=e ETS-9. 5 0 Fxc6 97 3M Environmental Laboratory tox-030-wm223-IC 9112/00 Page 196 3m Medical Department S--udy: T-629iEACITOX-030 Report No. FAC'-7 TOX-030 Covsnce#6329-2l2a3 boratory Request Number-U2279 SDAY Prodwt Nuinber(Te3Stubstan") MotM,OtodfkAmilytiWEqup@t Systm N=b@ imt@t Soft@cfvmlm Fdenarm: R-Sqwed V.1Slope. Y-int,-@pt DikbeofExtu-bort/Analyst: DateOfAAStYaWAnatyst: DateofDaLs Rt&mtioiVAngM 26W"k CaouleTo)dc@StWywithPFOSm Cynoinolgiukionkeys T-6295 CPFOS) kAonky Sm ETS-8.4,0@M E7S-9-5 0 Soup 020199 M-Lynx 3 1 Sm Anachments S@ And=mts SftAftich@U See Attichmmt& 3115M ICP/SAH OYI&199.0312A/99D" OY17199,OYZY" DKB Sample Data V;EEK 20 MONKEY Grw* D@ Method Bik Mao= Blk Mstnx Blk QC -250 ppb SER.A RI=3159-H20 8*-l RBW3159.H20 B&-2 iLBS03li9.SemBD(-] XBS031WSenSIk-2 MKS03159-Scn Sik-1 )AK.S03159,44nBlk.2 RWO3159.ms-l RM3MMSD-1 PFOS C@ ng(ml 0.00 000 0.00 0,00 0.00 o.00 256 726 ofP703 igglaLwL% Rw <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 103% 91% PFOS ug/mL <LOQ <LOQ <LOQ 97-/. RSD Sid. D@. MS/M3D RPD NA NA NA 13% Cmtrot Grmp 2 L@w-Dose 0.03 mwl*dy Crww 3 Mid-D@ 0 15 mglwday Crwp 4 Higt,D@ 0 75 mWI@Wday L@tofQ@tUwn(LOQ) RBS03159-MS-2 RBS03159.MSD-2 105-1.0am 105517M 105519m105520.M 105526.M 105527M105529F la533OF 105531F105535F 105544F105549F 103514M 05515.M ;05516.M 103521.k( 105337F 105541F 1055ATF 10555OF 105505M 105510M :0551sm (05523M 105524M 105328M 105532F 105538F 105539F+ 1055A5F 0554$F 105352F 105506M 10550-,M+ 105509M 10551im io55l2m 105522M 105533F 105534F 105336F105WF 105542F- 10555tf PFOS-152rWmL 219 U% 223 90% 30.4 0.0349 314. 0.06419 19.3 0.0336 23.2 00300 30.0 0.0369 31,5 1 516 0.0631 0.06[7 393 0.0579 29.3 0.0493 tg 4 0-0263 296 0,0505 391 0.0570 88,6 12@5 867 10,7 101 119 98.3 14,1 70@2 il'O 73 2 tt@5 966 t4.3 359 41 1 237 62.3 233 63.3 248 631 319 752 220 640 323 542 135 69 2 201 474 210 79.2 111 45.7 204 65@3 154 42@7 391 158 29, 139 315 152 352 126 309 143 302 1 145 393 169 303 143 214 146 "1 193 302 151 330 112 Dte EnteredfBy:03(OW". OYl7M9 LAC DateVuific&BY: OIVIL'99SAH, 0511'7/0P0W 00400 00504 12.3 12,3 195 631 51 1 144 156 2% 29.9 00120 252 1 00127 11 4 1,40 14 6 179 744 145 105 6 71 25 4 14 762 109 14 0 1 218 PFOS - P@f=oocanesWfonam N A - Not wwyaed, mt appbelblt D.tep@tycoff"ted@veri5ed0:9/12=nunkO9tl2=LAC Calculatemdthmi sam* 10555OF S@=tWmhunelmd@0.50ML,@tmb@shoWdbecomWmdt-tswe 04r2M L-AC E.@.tim V@]@ Ratio- blib@VohimvTiW Volme M-9.5 0 F@mi 97 3M Environmental Laboratory tox-03D-SCM223-IC W 12/00 Page 197 3m Medical E)epartment Study: T-6291FAC@TOX-030 Report No. FACT TOX-030 Covmnce#6329-22l3aboratory Request Number-U22-/9 Study: PToductNmbcr(Ted Subsimm) Ma=: M.th.d(R@@i.i-: AnalytealEqutpnuouSyxu= N@ber 26Week CapsWe To)uctlyS" T-6295 (PFOS) Monkey Sm M-9-4 0 -d ET@-8.50 Aeba 062499& Sop 020199 vathPFOS m C)-romolg@ %lon@eYs huuw@t Softwue/Vas=L Fdename: V@Ly= 3 1 SeeA=KM=ts R-Squared Value: Slope: Y-Intcroe;n: Dateof ExtwwWAnalyst: DateofAnitysWArak"t Sm A=hrnwts SeeAtt.,@"u S@. Aftachmmt, 3116,'9R9WWI-AK 03117/99M,OM, 0312Y" DRBIMEE Dot.ofdats ReducocriAr&4@t O3W2l'99,=4?99.04/02/"DFLB Sample Data WEEK 24 MONXLRY SERA Cr-P sampit N PFOS c om Mmn ITSD C@ ofPFOS PFOS Std.D". UVML . w% Rm @y)-L MSMSD RPO Medwd Blk R.BS03169-H2O Blk-1 000 @LOQ RBS03169.H7O Bik-2 000 <LOQ @LCIQ NA Ma= Blk ILBS03169-Sem Blk.I ooo <LOQ P.BS03169-Sen 82.2 000 @LDQ < LoQ NA QC -250 ppb PBS03169-MS-1 219 ar/. R.BS03169@MSD-1 736 95% 91! i 8% R.BS03169,-MS-2 231 93% PJM3169-MSD-2 96% 99-/. ILBS03169-MS-3 224 90% RSS03169-?ASD-3 220 89% 90-/. 2% Grm* 1 conami 0 0 mWVday 1035mm 36.4 0.0"9 105517M 303 0,0496 105519M 258 D@0414 105570.1@( 194 0.0223 105526M 248 0.0397 105527M- 36.7 (1.0613 105529F 40.4 0.049% 10553OF- 242 0,0497 00440 177 1 00101 105531F 259 00364 105335F- 8.36 <LOQ [055"F- 162 0,0325 A4 105549F 334 0.0446 0 0426 - IAn.@aly ID0794 Grow 2 105314m- 14.7 L@-Dose 105515M- 1I1,4 0,9 0 03 m&Uday 1055t6M 132 :4.1 21 0 105521M, 141 IN@4 306 105537F 13' @,4 1055AIF 133 :3' 105547F, 949 12.7 10555CF 109 122 5 18 13,0 0 675 G@p 3 105505M 251 7114 Mid-E@.se 105510M 196 652 O@15mgA,g/day 10551am 191 588 105523M 164 59@l 105324M 201 69.6 104 105529M, 155 64.8 659 688 105532F105539F 105539F105543F 10554OF 103552F+ :45 63@) 53 496 696 :,6"9 635 196 56 1 139 617 12 0 60 1 7 24 High-Do" 103506M 287 199 105507M 234 in 0 75mga*day 1055D9M m m lo@51 1m 258 207 105512161 309 247 105522m+ 233 233 F05533F 290 190 116 iis 2A9 105534F- 225 200 105536F, 207 169 Lvmt ofQumtutcn O.CQ@. PFOS 10554OF 10554,F 105551F@ 15@2ngirnl 219 151 240 191 1 131 150 174 Die Enined'By 03f24,99L&C D@t@Vmfi@d)By 0811&1"SAM, O-V24,00PW 17 0 1 209 ?FOS - Peduarooctwmulf@te Datep=ry coffwe&venfie& 09112/WmrrLkD9/IVDOLAC - S@ mtialvol@ lessthm 0.50mL, @n@trato" shouldbe c@id@ed W29100 LAC %i-.Monburkd NA - Not app6-ble ExtractonVol@e Rimo - lmtw Volume/FmalVol@c ETS-9-5 0 Exat 97 3M Environmental Laboratory t.x.030-jm223-IC 9111,00 Page 198 3m Medical Department S--udy: T-62StEAC'tTOX-030 Report No. FACT TOX-C30 Covance#6329-2l2a3boratory Request Number-U2279 shwr. .1W6oekCapultToxiciSttyudyw7thPFOSinCynmoigusNionkeys ProductNumba(Tnt Subsmrkce) T-6295(PFCS) Matrix ?.ionkeySem Methad(R@. ETS-8-A0 -d ETS-&S.O AnaiytcaElquipmmt Sytm N@ber Ameba 0-62498,Soup 020199 lmu@=t SoftwarvV@m: 'AsLy- 3.1 Fde@t See AMWILmcnt3 R-Squared Vaiw: Se. Anachm@ts Slope See Attachments Y-lateTcept See Attachments Date orExlwtcWAraL]yst: 3,'16(99PWW/SAN Date ofAn@OWAn*st: 03/17199.03r2l/99DRBIMEE Date ofData ReduftordAna@v 03123/99,03122/99 ORB/MEE Sam pieData WEEK 26 MONKEY SERA Group S.Mpl. a PF03 Comcmtmtion M..n RSD D." Conc. of pros Pros Sta. Be-. og/=L uVmL or% Ret mg/mL M&MSD RPD mee@d B& m.ou Blk P.B303169.H2O Egk-1 000 P.BS03169-Ft2O Bik-2 0.DO RBS03169-S@ Blk-1 0.00 <LOQ <LOQ ll.OQ NA <LOQ RBS03169-S- Bik-2 000 <IDQ NA QC - 250 ppb FLB303169-.MS- 1 210 85% RBS03169.MSr)-i 232 94% 99% 10% RBS03169IMS-, 229 RM3169.MSD-2 193 92% 7rA 95@'. 17% RBS03169-MS-3 211 RBS03169-MSD-3 213 93% 86% 86% 1% Group 1 1055ow 30.9 O@0496 Control 0 0 n*(Wday 105517M- 31.0 105519.M 27.1 0.0539 0.0417 105520M 16,8 0,0254 (05526M 26.7 0.0376 31.3 105327KI 105529F Al.7 t 48.1 0.0669 0.0613 00459 00143 10353OF 597 0.0736 10353 1F 30 1 00431 105531 F 166 00266 105544F@ 216 0.0433 12 5 105549F 34 7 0,0536 00506 0 016A Group2 Lo@.D@ 105514.k4 151 16,, 105515.M 143 16.3 0 03 rrtwkg/day 105516M 131 105521M- )1, 13.8 9.91 16,9 15 8 1 41 105537F, 101 13.3 10554]F- 113 14,8 105547F 140 13.0 108 10555OF- 85 3 11.4 13 1 42 Gmup 3 105505M 253 9r2.9 Mid-Dose 1055]OM- 149 129 0 15 mwvday 1055]BM- 141 69.5 105523M- 176 72@ 1 105524M+ 160 69 3 30 4 105529M 156 62.4 8,'6 ::52 105537F .109 63 4 105538F 150 59 0 105539F '16 72.2 105545F as 7 105548F- 145 64.5 163 105552F 142 55+6 668 1 108 Group 4 105506M m m Hi$h-D@ 105507M- 213 197 0 75 w4A*day 105509M m m 105511 A 248 142 101517.M 285 143 210 105522.M 299 221 173 36 5 105533F 340 203 105534F 308 IV 105536F- 155 135 10554OF 178 17'1 105542F 206 165 130 10555IF- 163 142 171 22@2 L@t ofQuantuwn (LOQ) PFOS 15.2nWmL Date EnieredfBy. 03123t99 LAC M %Amburld On(.Vwfia& By 08/19,99SA)t 05f24iOOPW Datepxmtyc@tc&verLf@d 09112=nwdt.09112= LjkC PFOS - Perfluctoocw=Wfowe Senun Lrutavloi=e lessdm 0.50mL, concentraocnsihowd be c@idered tentatiw 04,2&00 LAC NA = 'Jotappbcable Ex=acton Vohunc Ratio- ImtuaVohunt.'Fmal%'olme ETS-9-5 0 E.@.1 97 3M Environmental Laboratory tox-030@@223-IC 9/12;00 Page 199 3m Medical Departmer-'- Sz:udy: T-629f@-AC-YTOX-030 Report No. Covince#6329-223 laboratory Request FACT TOX-030 Number-TJ2279 Study Pt.dwtN-bf(T.9 S-b@--) met..: A4.th@d/RA,Wyb.W Syvm N@b@ R-SquLmd Valu. SI.P.Y-t.t-.O: Dot :(Fxuwboo/AnskYrt D.t: fAnaly"AA&Iyvt Dateof Deb PjhdnctoniAA&@YA 26Wwk Cap.W.T-ty Study-th Pros T-6295(PFt)S) %4..kySE,"3-9-I-4d M-8-5 I D.-y07G799 MwLynx 3 3 S. Aft-h.-t. SftAttackm@u S. AH.It-@.u St.AMehm@U 06101/DOSALIKJX 06/0&00.06JI4100,0611uwLANMMH 06la7)OO,WI5=.06Jl9fDLDA.VMMH Cy.-ig- kimky. Sample Data WEEK 27 MONKEY Groop Do" ,M.thodBlk M.= EQ QC . 250ppb G-p 1 Conftl 0a Vwdy Grmp 2 l@-D@ 0 03 mWWd-Y Gmep 3 .M.d-D@ 0 13 m&Wdsy Gmup 4 H.Sh-D@ 0,75.&AqVday Lmt ofq@izubm (LOQ) PFOS- p@fl@w@Wfomte NA - Not Vpb"ble SERA se.pi.0 Pros C.2c. V.l D6010-H@O Bik-5 000 I D601C).@MBIk-6 000 RBSM@o-sn :tk'5 0 Oc RBSM o-sm lk-6 000 105319m-ms-5 350 105519u-msrD)-5 353 103509M 53,1 103319%( 49.5 tos527M $().1 1055)OF 73.1 105531F 431 105133F 366 [05544F 39,9 10551AM 172 105515M 187 loi5l6m 165 105 5 111'ki 273 105541F 181 10554-F il7 10555OF 161, 105310M 316 1055]$M 116 105524M 2-,9 io55',BM 344 10533,F ,30 105533F 760 105545F 267 10554$F 300 105506m* (05507M 4.24. 105312.%4 464 1055)AF 419 105536F 389 10554OF 377 10553IF 291 PFC)S 555ng@L Dtt*Eaur-&By: D.i.V-6.&13y Dstepmtyco@tod(vmhed ExtnctonVol@o CamconLr.uos M,sn RSD ofPros W.L .,% R.. PFOS V.L 59& D-. M&(MSD RPD @LOQ @ LOQ @LOQ NA <LOQ @LOQ @LOQ NA 121% 122% 122% 1% 0,0461 0.1)430 27 4 0.0695 00529 00145 0.0633 0.0374 0.0318 0.0339 00416 35 5 1 ____0.0142 239 130 114 34 8 15,5 15 9 5 34 12.6 951 1 --11.2 1 2 68.5 68 7 60@6 8 44 747 681 5 ?5 54 3 56.5 30 7 658.1 59 1 4 67 196 114 461 202 194 993 192 169 164 14 9 126 160 23 9 .... 06/07100,0&]9tOLOAC 06/0&'0P0IW 09112JDOmmh.D9/12(DLOAC Ex",tm %,ol@cRato- lmtsival@&iFoW Ra?3o Voh=c,TmalVol=t Vol@c Excel97 3M Erivironmental Laboratory 1--030@-223-IC 9112too Page 200 3m Medical Departmert-- Study: T-62-c@AC'YTOX-030 Report No. FACT TOX-030 Covance#6329-223 laboratory Request Number-U2279 S" ProducNt=bcr(Tnt SubstAn") Msuix@ Method(R@m: AnilybcalEqLnpmcntSystm N=b@ irsu@mt SoftwucNmim: Fdcnwne R-Scpwcd Value. SkV.Y-inum@PL DateofExawdon/Analyst DateofA..kynVAnalyst Date ofData Pxducncn/Analyst: 26 Week CapsWe Toxmry Study withPFOS inCynomo4w T-6295 (PFOS) Monkey Sera E7S4-4 0 -d ETS-9-50 Ameba 062498,Soup 020199,ALMadeline0410" %4@Lyrix 3.2 See Attachment& %,(OnkeYs Sm AMuhmcnts S" Attachments SeaAuhmenu 04'D6/99RWW 04/07/99,0,VI2199.OA117/99,DS/14199,0511H7O1J9f9E)RJWEE/YIH 04109/99,0411YW,D4nO/99.05/17199,0-Vl&MHOJIDRa/KJfl Sample DaU DAY 193 MONKLRY Group Do" SERA S..pi.0 Lmut Method EiLk R.BS04069-H20 Bai-i RBS04069-H20 B&-2 Mabix Bik RBS04069-Scra H&-l RBS04069-Sem B&-' QC -250 ppb MXS04069-MS-l MKS040691-MSD- 1 MKS04069-US-2 Group 1 MKS04OWMSD-2 105520M Cmtrol -105526M, 0.0 mgAtWday 105529F 105549F Group 3 103505m+ %fid-D@ tO5323M 0.15mg/kWdy 105539F- 105152F Group 4 105511M- High-Dom 105572,\i 0.75 mg/Wdy 103533F 105542F fQ-bubm (LOQ):PFOS 132 rWw.L PFOS - Perfluoroocunaulfonate .4A - Notapplicable Datep@ry PF03 com..rwb" A4@ RSD Coo@ ofPros PFOS StL D4@. agJ=L V&NNL - % Rw ugt=L MSIMSD RPD 0.00 <LOQ 142 <LOQ NA NA O.DO <LOQ 12,1 <LCQ NA NA 254 102% 246 99% 101% 3% 267 lora 228 92% locr/. 16% 129 297-- 0171 00626 0 117 653 0 01!64 660 0.0756 59,2 21 3 00310 00533 00315 371 74.4 176 797 955 85 0 149 479 107 43.0 391 36.9 91.7 35 1 97.1 216 188 194 287 249 468 245 239 175 176 201 230 403 DoteEnLaed(By@ 19/99O.-VI". MOM LAC DateV@Tihe&By =I 9t99SAH, beforOe&OVOC TY P, c@tednoified: 09112100ffurdi0,9/12/00I.AC - Serum cr@tlvaollumeIm dw 0.50mL, concmtntiom shouldbecomickiedteniatme04/28/00LAC Extncton Volume Ratm - imtw VolmefFinalVolume Ers-9-5 0 EXCel 97 3M Environmental Laboratory iox-030-wm223@IC 9111100 Page 201 3m Med@-cal Department Study: T-629@ACiTOX-030 Report No. FACT TOX-030 Covance#6329-223 laboratory Request Number-U2279 Study ProductNumbct(TestSubstance) M.1m@ \4@Eh.d(R-@-: Analytc&ElqmPmmt SYstm N@b@ tnst@mt SoftwxrefV@on -UcnmeR-Sq@d Val@: Slope Y-intacept: DateofExtnctiorJAnatyst Date fA..Iy,@eArAly.t Dateof DataRedu=oYVAA&tyst* 26Week Ca" ToxcrySh* withPFOS inCy-nmolVa Monkeys T-6295(PFOS) Monkey Sen E-@-3-4@OUW M-9.5 0 Ameba 062498.Soup010199.& MBdelme041098 M@Lym 3 2 SeeAnwh@-z See Aft&Chff-ts S@ Attuh@ts SeeAttachmnu 04,061"RWW o4(07199,04/12/99,ovl7199,05/le",05I/]1O7I1/9D9RBIMELICII o4to9t99,04115,99,0412W",05/17t99,051H1O9J(I9D9RB.IKJH Sample Data DAY134 MONKEY Gnmp D@ SERA smpl. 0 PFW Cwc ngtml concumatwo serros upf=L w % R" Mma PF03 oW.L RSD Sid.D-. MW.MSD RPD M.dwd BA PBS04D69-H20 BLk-I O@OD <LOQ RBSOdM.H2O B&-2 142 <LOQ NA '4A Mab-txBlk P.BW4069-S. Ekk-1 000 @LN -RBS04069-Scn Bik-2 12,? <LOQ NA NA QC -250 ppb MKS04069-MS-1 254 102% MKS04069-MSD-1 246 99% 3% MKS04069-MS-2 267 108% MKS44069.MSD-2 229 92% 16% Gn.p 1 control O.DmgAWday G@p 3 MLd-Dme 0 15mg/kg/dy Iiigh-E)@e 0.75mo*day 105510M 105526M 105529F 105549F 105505M 10552.3M 105539F rO5552F105511m 105522M 105333F 103542F 12.3 <LCQ 232 0.0233 0.023.-3IAnmily NA 436 0'0416 259 280 0.0297 0 0352 000911 621 664 6-,2 692 -730 69 7 469 sio 963 54 390 695 @20 192 ist 42 5 264 336 259 110 242 265 11,9 210 7.24 245 292 -=t ofquanotatiCoLnOQ) PFOS 152 neml DateEntend/By 04119/99.05110M,09=99LAC DateVm&& By- Ogn4/99 SAH, befat 06/OgiD7OKR Datepuritmymctedtvmfied: 09/12= mmh. 09112100LAC PFOS - PmtuofooctinesWfonate Sam 4ut&lvol=e lessOw 0.50mL, concentratosnhsowd be cons,&@edient=ve OV2&'DO LAC N A - Not apphcablc ExtractoVnoi=c Ratio- ijutwVoi=e/Fui4%'ol@c ETS-9-5a F.X.,9l7 3M Env4.ronmental Laboratory lox-030-mm223,-lC 9;12-00 Page 202 3m Medical Departrrent Study: T-6291FACYTOX-030 Report No. FAC-- TOX-030 Covancc#6329-22l3aboratory Request Number-U2279 Study: Prodmi N=bcr(ral Substmw@. Armiytic.EJquLpr@t System N=ba lmouawd Scft-./Venwn@ Fo@: R-Squmd Valm: Slope: Y-intactpl: Date of Exu@-bordAnatywL Date o(Analy&WAnatyst Date of Data RaductLoWArAlyst Sample Data DAY125 MONICEY SERA r.,.w D-. Smpieo Mediod Blk Mau= BLk (2C -250 ppb P BS04069-H20 nSO4069-RIO Rsso4o6g-scm Blk-1 EM-7 alk-1 RBS04069-SCM R&-2 MKS04069'.MS-1 MXS04069-MSE)-l MXS04069-MS-2 MXS04069-MSD-2 Grmp 1 Corard O.OmgAqVday 105520#A 105526M (03529F 105-.'49F Cc*" 3 Mid-Dose 0.15 mg4tday 105505M 105523M 105539F 105352F- Grew 4 High-Dosc 0. 75 mglklvd-y 105511 m 105522M. 105533i7. 1035CF- Lmt ofQu-nttation(LOQ): PFOS 15 2 ng/rnL PFOS - P=t@oc-cunenUomu Date p=6y 26 WftkC4psuleTomory Study %ith PFOS mc@momolgw Niankeys T-6295 (PFCS) mm*cy sen M4.4 0 md ETS.9.3,0 Ameba 062M, Smp 020199, &.MadelLne 041098 M-Lym 3@2 See Anschmmts See AtMchnwnts See Aft-hmmts See Attwtwnmts 04AD&99 RWW 04/07199,Ojjl2f99,04/17/".05/IAM,05117.99HOJ.'DRB:%IEE;Klli 04/0919g.uly".04,70/99,05117/99,0511&'99 HOJ,DRB.'@:]H Pros comessuradw M@ RSD C- of PFOS PFOS Sid.D-. mg/=L 000 14,19 egfml w % Rot <LOQ <LOQ mg/mL N-A MSIMSD RFD NA 000 <LOQ 1274 <LOQ NA NA 2N 246 1 102'A 99% 101% 1% 267 lOrA 229 92% 1DO% 16% 32.0 0.0366 215 37.3 0.0498 0,0432 000928 63.1 0.0763 561 23.6 0.0330 00546 00306 553 70.3 13-1 778 95,4 779 107 967 101 2?6 322 67.9 843 233 2116 309 7 49 15 m 294-- 220 220 44 29 11 20: 371 1170- Date Entaed'Br. 04/19/99.05/IG,99LAC Date Vefifi@d/By@ OL125M SAH,befot.0610&,Oo TKR wffecUA4mified: 09/12/00mmh. 09/12/00LAC + So= 6iitWvolume lessthan0,50mL, wncentritons ihowd be comidered tmubw 0412&/00 LAC Ex.,l 9-, 3M Environmental Laboratory t--030--2nlC 9/1voc Page 203 3m Medical Department Study: T-629aCTTOX-030 Report No. FAC-@ TOX-030 Covance#6329-22l3aboratory Request Numbe@--U2279 stwy: Pmduct Numb@r(Test Substance): Matrix: Method/Rcvision: AnalyticalEquipment System N@bcT InstrumenStoffare(Venion: Filenarne; R-Squaied Value: Slope: Y-intercept: Date of F-xtraction/Amlyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 16 Week CapsuleToxicit)'Siudwyith PFOS inC%-namolgusMonkeys T-6295 (PFOS) Monkey Sm ETS-9-4.0and ETS-9-5.0 Amelia 062499.S..p 020199,& M.d@tin@ 04109A MassLy-nx 3.2 See Attachn=Ls See Attachments See Att2ch@ts See Attachments 04.'0&,99RWW 04@07199,04@12.-199.U;17199,05.'14,99.05'17H.O9J9 DRn,.MEF-KIII tiOJI)RB:K)li Sample Data DAY187 MONKEY SERA Group Sampit 0 PFOS Concentration Mean RSD Dose Cone- ofPFOS PFOS Std. Dev. ng/mL up)ml or-% Rec- uWmL NISINISDRPD Method Bik RBSO,4069.H20 B Ik.1 0.0() <LOQ RBS04069-H20 Blk-2 14.2 <LOQ NA NA Matrix Bik RBS04069-Scia Bik@1 0.00 <LOQ P,BS04D69-Scra Blk-2 12.7 ILOQ ',A NA QC -250 ppb MKS04069-MS-1 254 10rI. MKS04069-MSD-1 246 99% 3% MKSD4069-MS-2 267 108% MKS04069.MSD-2 223 92% 100-11. 16-,. Group I 105520M 13.6 -LOQ Control 105526M 21.1 0.0300 G@0300-I Anonuiv NA 0,0mg(kwday 105529F 39.1 0.0373 25.9 105349F, 31.7 0.0541 0.0457 00119 Gmup 3 105305M 699 a2.4 5 91 .'Aid-Dusc 105523M 662 75.8 79,1 467 0.15 mglkg/day 105539F 63.7 10.2 105 105552F 499 68.9 39.5 41.4 Group 4 10551 lm 225 237 15.7 High-Dose 105522M iss 297 2(17 42,0 0.75 mSAg/day 105533F 289 269 105542F 253 248 5.90 258 15.2 Limitofqu@titaiion(LOQ): PFOS 15.2ng/mL Date Entered/By: 04;19i99,05/10199.05@20,9L9AC* DateVcriricd/Bv:08;25.99SAH.Worc06,0900 TKR Datepuritycomctc&ycrirted: 09;12100@h.09,'1200 LAC PFOS - Perfluorooctanesulfonate - Scmin initivaollume lessthan0.50 niL.concenrralinnsshoitlbdc consideredtentitic% 04 28:00 LA( ETS-8-5 0 E.cci97 3M Env4-ronmental Laboratory TOX-03D-sem223-IC 01)13:2f)00 Page 204 3m Medical Department Study: T-6291FACITOX-030 Report No. FACT TOX-@130 Covancc#6329-22l3aboratory Request Number-U2279 study Prod=tNumber(TeSsutbst@): Matnx McthodtRevwort Analyncid Equtpm@ Syst@ N@b@ 1-m@t set...Vm@- Fdtnamc R-Squued V.Iuc: Slope Y-Intacept: Date of ExtacuaWAn&tyst Date ofAnalysWAnalyst Date of l)at.ReductioWAnalyst 26 Week CAPSUIE Tonary Study mth PFOS Ln Cymmolgw T-6295(PPOS) Monkey Sen ErS-9-40 ad E7S.B.50 AmeLm 062498, Soup 020199. & MadeLLne 041099 M.@Ly- 32 SeeAnachmcnts SeeAttachments SeeAnachments See AM&chm=Ls o4tOL*99 RV/W 04111/99,04(17,W.05/14199 DR"EFIK;H 0411&".0&20/".OY17199 HOI/DRBIKJH Mcmkeys Sample Data WEEK 29 MONKEY Gr-p Dwe Method8* Matnx BLk QC - 1,50ppb SERA 3aspie 4 RBS04OMH20 PJLSO4M9-H2O RBS04099-sm Blk.l Bik-2 B&-l RES04099-Se. Blk-2 MKS04089-&iS-1 MKS04U9-.%(SD-1 MKS04099-)AS-2 MXS040W.MSD-2 PFOS CmVb&L 7.90 701 000 0690 260 267 249 264 C.Netwan" (PFOS GV=L w % Rw <LOQ <LOQ <LOQ @LCQ 105,Y. lOrA 101% 106% M@ PFOS ug/mL @LOQ < I,OQ 106% 103% RSD SIL D@. MSIMSD RPD NA NA -3% 6% Gn" 1 Control 0,0 nMyVday cc*" 3 kfid-Dm O@15 mgtkwday Gn.q 4 High-[)o.c 0.75 aMAzp)day Lmi ofqunbtabon (LOQ); PFOS PFOS - PcrgwroocunesWf@te 105520M 19.4 0.0224 33 5 105526161- 26.4 0,0459 00371 0.0124 io5529F- 26.1 0.04M 11 1055OF- 20.0 00372 00430 0.191 105505M. 481 85@7 1-18 105523M- 511 835 846 151 105539F- 299 99.7 4 16 105552F- 355 936 96 1 3@39 103511 m- 536 105522V. 659 233 g@71 264 249 21 i (05533F 366 249 7.76 105542F 423 223 236 IR,3_ 15 2 naiml Date Entemd/By 041191". 05/10/99 LAC Date V-nfiedt By OIV2.V" SA.H, bef.. 0&0&;00 TKR Date pmty coffected4ymficd: 09'IVOD mnik 09tl2/00 LAC Scium @tial whune Ins Om 0 50 mL, concentratom showd be comidefed tentauve OV2R/DO LAC M-9-5 0 Excel 9' 3M Environmental Laboratory tox-030-sm22'-k-IC 9111*0 Page 205 3m Medical Department Study: T-629fDA4Z@iTOX-030 Report No. FAC-- I-OX-030 Covance#6329-223 laboratory Request Number-U2279 Study ProduvN=bcrfrnt Subst@) Mtt=@ .M@thod/R@An.lylacEaql..p.@raSystm Number tmtr@ Soft@cfVer@ FdcnAme: it-SquucdVW=: Slope: Y.lntacqA: DateofExt=6orVAnal@st DateofAnalysis/AnL@/tt DateofdataRedwbcWAngyst 26 W"k CapsuleToxoty S" mth PFOS T-6295(Pros) kionkeySen ETS-9-40 M ETS-9.50 Amelm 062498,Soup 020199.& MadcLintN M@Lym 3 2 S" AMichm=U SeeAtuchmenu SeeAttachm=ts SeeAttachments 0410&" RWW 04/11/99.04117/99.OY141D9R9B/MEEIKJH 04,16t99.04(2&199,05/17H/O9J9IDRAFKJH inCNMOmOlg@ 1098 ?,ionkeys Sampk Data WELRK 29 MONKLRY SERA G-" Do" MettiodBlk Mau= B& QC -250 ppb 0.0mg/Wday Grm* 3 Mid-D@ 0,15 g(Wday FLBS04M-H20 Blk-I RJIS04089-HW M-2 RBS04089-Sen Bik-1 R13SO4019-Sen82-2 MXS04089-m&l MKSG4U9-USD-1 MKS04089.M&2 MKS04089-MSD-2 10552OM105526M 105329F105549F 105505M 105523)A105539F 105552F_ PF C@ nof "7@I, 0,00 0,680 260 267 249 264 42.2 "7 38,7 681 656 417 613 432 A(PTOS -ChaLw % R- <LOQ -LI)Q <LOQ @LOO 105% 109% 01% @OeA 0.0690 0.0639 0.073 007487_ 77-3 747 T79 619 PFOS uwml RSI) SitD-. MSF.MSD RPD @LOQ NA LOQ NA 106','. 3% 103% 00665 00742 ?5 9 1 699 6% 5.45 O@00362 0994 0 OD0664 299 2 18 62 @14 Graw 4 io55lI.M 560 175 300 H4ti-Dou 105522.M 384 770 223 669 0.75m&4*day 105533F 641 207 9:3 105542F 549 Ito 194 10 Lmmiofq@tittwn (LOQ):PFOS - 15.n2giml DartEnL@d/By 04/19199.OSIIO/9L9AC DateVeiifiedBly- 09(2Y" SAK befm 0610&00TKR Dateptmtyc@cte&@fied. 09112= mmk 09/12/DLOAC PFOS - Pag=oocUr@dmile + St= "tW volumelessdm 0 50mL, concmtraf3omshowdbe consideretdentann,O.LrZ&% LAC M-9-5 0 Excel97 1C 3M Environmental Laboratory 9;1.1.100 Page 206 3m Medical Department S:::udy:T-6291FAC;iTOX-030 Report No. FAC'-7TrX-030 Covance#6329-223laboratory Request Number-U2279 Study. ProductN=ber(rest Substmcc). m.tm: %4cthod(R@im: Anaiyt,W Eqwpmenl Sy.tm N=b@. [mmunent Soft-afefV@m FkmmeR-Squazvd VaJw: Slope. Y-1.1m.pt Dateor ExnutotvAn&W D.t. of AA&)yWAn@ty.t Date of Data RadmoorVAnskym: 26Week Capswe Tomoty T-6295 (PFOS) Stidy %vith PFOS inCynmolgmMonkc)rs Monkey sen ETS-9-4 0 .d ETS-0-5 0 Ameha 062499.Soup 020199.& Madelme 041098 MmLynx 3 2 Sft Anadurmts See Anath@ts Set Attechmmu See Anach@nts 041" RWW 04111/99,041171",05/14,"r)Ra(MEE(KIH OVI&M,04(20199,05/17/99 HOI/DRIVKIH Sample Data WEEK 30 MONKEY SERA Grewp D@ Method Rik Mahu BIk QC -250 ppb 3ankpic 4 RBS04CMirO RBS04089-W@O 8&-1 Blk-2 RLBS04099-Sen EM-1 FLBS04099-Sem Blk-2 MKSO*099-MS-1 MK504099.%(SD-1 MKS04099-MS-2 MKSO4M9-MSD-2 P" Cmc V*ML 790 7,01 0.00 O.Ug 260 267 249 264 c of PFOS uwml w % R- @LC)Q <LOQ <LOQ <LOQ to5% 109% jai% 106% Mnn PFOS uW=L ILOQ <LOQ 106% 103v. RSD Std.D-. m3m3D RPO NA NA 3% 6% Gra* 1 Control 0 0 mg@kwday 10552W 20.4 0.0314 105526M 315 0.0312 00313 O@OW165 105529F 325 00299 1.60 iO5WF 291 00306 00303 O@000435 Gr" 3 Mid-Dom 0.15 mglkwday Gr@p 4 High-Dose 0.75mg/W&y 105503m- 419 105523.M 54 105539F- 425 105552F 340 10551I?A 331 1055221%4 105533F - 475 616 105547F 4*6"0 68.4 619 69.6 54.5 116 170 156 167 7.D6 652 4.60 1711 620 106 26.5 143 390 486 162 797 LmitofQumtitabon(LOQ) ?FOS-15,2ngimL Date EntmdfBy Date Verified/By 04419/99,O-Vla(9L9AC OW2Y99 SAM, befon 06/0&100TKR PFOS - PerfluorooctanesWfomte Date punryconwed@vm6cd. 09/12=nunk D9/12JO0LAC Set= ntal vohune lau than0 50 mL. wncentntiom showd be comidaed tentat)vc0.4"2&'00LAC ETS-8-5 0 Exc 197 3M E@v@-ronmental Laboratory lox-O3O-sa223-IC 9112/00 Page 207 3m Medical Department Study: T -62 91E@Ci'TOX-030 Report No. FACT TOX-030 Covance#6329-223 laboratory Request Number-U2279 shutr Pmdmt N=bcr(TcsSiubsb@e) MatrEc: M@th.dfR@ AnalytcalEquMmcnt Systan N@b@ lmtrmmt Sofmare/Vmton 26 W.k C&pgW, TO)d@stud@ythPFOSm CynmoiguMsonkeY, T-6295 (PFOS) Monkey Sm ETS-9-4 0 -d ETS-9-5 0 Amcbx 062498,Soup 020199.& kiadclme04 1098 M@LyrLx 3.2 Fdcnam. R-Squared Value: Slope Y-Iniacqn: DateofExticboiVAnWyst: Date ofAnky&WAnskyst: Date of Dots ReducWWAntlyst See AtUchmenU See Anuhmmts See Attachrnmts See Adhmmts OA/M@" RWW 0,VII,99,04117199.05/14" DRJVMEF,'KJH 04/t6M, "-20t", 05117/99 HOJIDRB(KIH Sample Data WEEK 31 MONKEY Group Don SERA S.Mpk 0 PFoO@0S3 C@ a mL cswm 4(P703 w% R- M@o PF3OS agimi, RSO SIL D@. M&MSD RPD Method BI Me= Blk QC -250 ppb Group 1 conbdl 0 0 mg*Wday Crvw 3 MW-Dow 0.15mg&Wday Creep 4 Hie-O@ 0 75 mg/Wday PFOS - PerOuorooctmesLdfixute NA - Not Lppb@ble ILBS0409%-H2O Blk-1 RBS04099-H20 Blk.2_ RBSOQMSM Blk-I 3LES04M-Sen Blk-2 MXS040".Ms.t MKS04M-MSD-1 MKSO4N9-MS-2 MKS040119-MSD-2 105520M 105526M IDJ529F 1055OF- 105505M 105523M 105539F- 105552F103511m 105522M 105533F 105542F@ Date p@ty 7@8"0g <LOQ 701 ILOQ NA '0CF0_0 0690 <LOQ @LOQ '06@'. NA 260 105% 267 109-A 106@'.S 3@. 749 101% 2M o@26:4 106% 103% 6% 27.1 0.0362 1 59 36@7 -0 0354___ 00359 0 00056? 24.3 0.0292 329 25 5 O.G433 00369 00121 591 60.0 843 398 53.2 566 4-' sm 111 55 c 291 48.6 A3 9 399 "9 669 @94 161 46 1 591 201 399 170 1.5 Date Entmd/By 04/19199LAC Date Veri6edtBy OW23M SAH, bcfm 06 0&00 TKR coffectediwnfied: 09112100 =it, D9111100 LAC S@ @tml vahlme Im d= 0 50 mL, concntranons should be conudaed tmtztive OA/2&00 LAC Fxtmcbon Vokume Rato - ImbiJ Vol@e!Fm&l %'oit@e ETS-1-5 D Excel 9, 3M Environmental Laboratory tox-0304ce23-IC 9JI2/00 Page 208 3m Medical Department Study: T-62SAC-TTOX-030 Report No. FAC'-7TOX-030 Covance#6329-22l3aboratory Request NuTnber-U2279 S-dY, ProductNumbcr(Test Substamce). .Me=* .M.th.d/R@: A,alytc.E]qu*.nmi Sriem N-b@i 26Week Cap,,AtToxicitfytidy ithPFK)SinCynma*u T-6295(PFOS) Moky S@n ETS-8-4I and ETS-9-5.1 Ameba 062498 Monk-@- Soft@tvv@tt F@ename R-Sq@td V" Slop@: Y-IMBFCOPL DateofExuscoon/Anafrt: DateofAnlysWAn&4@r DateofDataReductiorfAralyst M=Lym 33 SeeAttachm=t3 See AnachmwtL See Anschinmts See Anactunents ORt25/99RWW Ogr28l991,0/05/W.06ilAM MEE(MMH D9/29/99l.GtD&199.0&15= MEEAA&MMH Sample Data WEEK 35 MOINXEY SERA Gmep Don Blk .Me= Blk QC - 250 ppb G@p 1 cortrol 0,0ff*icwday G@p 3 M@d-Dome 0 15 mpi*dmy S..Pl.0 H20 Blk-1 H20 Blk-2 RabbitSem Blk-I -R@bbd S@ Bik-2 MKSM59,-MS-1 MK505289-.MSD-1 MK309259@MS-2 MK505289.MSD-2 10552OK4 105526M 105529F 105549F 105505M105523M 103539F105552F- PFOS Conmntmtion co.c. ttpiros ng'ml u"L or% Rec 0,00 <LOQ 000 <LOQ 0,DO <LC)Q 000 <LOQ 227 91% 254 10'2% 256 103% 239 96% 273 0.0437 35 9 0.0490 74.7 0,0148 6100 0.0698 186 930 190 1 76 1 122 812 135- 677 Mean PFOS vg/=L <LOQ @LCQ 97% 100-Y. 00459 00723 84 5 74 4 RSD Std.D". MSIMSD "D NA %A 11% 7% 660 000303 496 000352 14 1 120 i2g 9 53 Cr.up 4 0.75rVkWday 10551 I.M 417 105522.M 496 105533F 409 105542F 444 167 11.8 195 is! 19 5 164 5 94 178 171 101 Lmt ofQ@mbon (LOQ) PFOS 1 33 n6/mL DateEntemd(By: 10105199,10107/99,D&IgiLDAOC PFOS - Pcrfl@t@ulf@le DateVerifieBdy/ betwe 06AM@00 TKR Da:ep=ty cmected@@%@fied 09112/0"0urK 09112/00LAC Se@ @talwl=o leuth=0 50mL. concentratsohmowdbe @:deredtent@bw04t2&00LAC NA - Not umtym@ notappbcable Extmctm Votmc Ratio- IMtiSVlol@c.(FuW Volume ETS-9-5 Excel9- 3M Env@-ronmental Laboratory to..030-.@m223-IC 911zloo Page 209 3m Medical Department Study: T-62-ci;@kC;tTOX-030 Report No. FACT TOX-C3C Covance#6329-22l3aboratory Request Number-U2279 StudyProductN@ber(T"t Substance) 26 Week CzptWe ToxicityStudy %ith PFOS o C5momokq@ Nionkeys T-6295 (PFIOS) Mabu.Method(Re@wm Ana"cal Eqmpment System InstrumentSofcwwefVmim, F,Icnamc: Monkey Scm ETS-9-4 I and ETS-8-5 I Ame4a 062498 M=Lym 3 3 See AnwJumts R-Squared Value Slope Y-intercept. Date of ExtactiorVAnnJvm: See Auach@b See Anachimnts See An@h@l@ Ogt25/99 RWW D@t@ f Arky.WA@,.Iyv: Date of Debt ReduchorVAnalyst 09r2&199.lGtO5/990.6(WOO MEEfM?,4if 09f29/99.IGIM99, 06115/00MEE/IAS/Ni.Mil Sample Data WEEK 39 MONKEY SERA Gmep 3.0pit 0 D"e ?FOS come. ngt=L Co@ntnij.n of Pros agt=L.r % Roe 'Mean PFOS ugiml RSD Std.D". MSI.MSD RPD Method BLk M.= BLk H20 BBi-I H20 Bik-2 kabtntS@ B&- 1 R.bb,tS@ Rik-2 O.DO O.DO 0.DO 0,()0 <LOQ <.OQ <LOQ <LOQ Lc@Q NA NA QC - 230 ppb MKSOB=9-,MS-l 227 MKS05289-.MSD-1 254 91% 102%__ 97% 11% MKSOgngl-.MS-2 256 MKSO5n9.k4SD-2 239 103% 96% 100-1. 7% G-.p 1 Control 0 0 mg/kWday Gmap 3 M@d-D-@ 0,15 u*kgiday 10552cm 237 105526M 291 105529F 31 A 105549F- .157 105505?4- 311 105523N(- 217 105339F- 142 103352F- 174 0.0316 0.0466 0.0503 0.0514 62.4 579 56,8 46.5 00391 00309 60 1 5) 7 271 00106 153 0000779 5.16 3 16 14 1 7 19 G"ap 4 H,gh-Dme 0 75 mpVkg/dy 105511M- 270 10551^.-A- 236 105333F- 338 10554'F- 210 135 110 158 1 16 16 1 169 696 154 1! 1 L=i of Q@titation(LOQ) PFOS - 5 55 ng@ml Dole Entexed/By. 10/05/99.10/07/990.6;19;00 I,AC DateVerdied/By- b@fomO6/0&100 TKR PFOS - P@flwrooctinmulf@it NA - N.t analyzd, notapph..bl@ Datepmrycmwediwrified: S@ mtW wl=e In& than0.30mL, concentrmuons3houldbe consid@edtenuaw. 04,129(00LAC E,xtmcbm Volume Rato - ImbalVol@c,Fin@ @ olt=c ETS-8-5 I Excel 97 3M Environmental Laboratory tox-030-sem223-IC 9/1 :@oo Page 210 3m Medical Department Study: T-62-CACiTOX-030 Report No. FACT TOX-030 Covance#6329-22l3aboratory Request Nurnber-U227-9 Study ProductNumbWTest Ma=. M@th.&'R.-,-. Analy@caJEqwpm=t Substance). System \=ber 26 Week Capmde Tomarf Study @th ?FOS m Cynomoig@.Mor@ker T-6295(Pros) MorikeySm ETS-&A I=d ETS-8-5 I Am." 062498 l@n=-t S.Itwwefvcnlm: Fdename R-Squared Value. M=Lynx 3 3 See Attachments See Anachments slop.. Y-tnimept: See Attachrn=tm S.. Attch@t. Date of ExtmcboWArA?yst: OIV25/99 RWW Date of Anakywo/Analyst Date of Data ReductoWAnityst: SamplDeatiL WEEK 43MONKEY SERA Gromp Due Sampb. 0 09/2&", ICY05/99.06(14/00MEE/MMH 09/29/99,IW6/99,06/15/00 MEE/IAS/MltH Pros Come. L Con"atraUon orpros wWmL or % Rtc mean Pros agiml RSD Std. Dev. MY.MSD RPD Method Blk H20 B&- 1 000 H20 Ba-2 000 @LOQ <LOQ @LO() NA M.= Blk Rabbit S@m Sk-I 000 RabbitBut Blk-2 D 00 <LOQ <LO() <LOQ NA QC - 250 ppb MXSO$259@ms-I 227 MX3052MMSD.l 254 91% 107% 97% 11% MXS09259@MS-2 256 MK305239-MSD-2 239 103% 96% i00*/. 7% Gr*up 1 Conami 0.0 mp-seday 105520M 105526).( 105529F 105549F 21,5 21.3 32.2 22.9 0.0287 00330 0.0369 0.0367 00319 00368 13.8 000440 0251 00000921 G-op 3 M,d- D@ 0,15 m&k*day 1 G@p 4 0 75 mglkg/dy 105505.M 162 fo5523.M 112 105539F 146 10555^@F- -116 10351 IM 167 105 siz@m 226 105513F 447 10554-,F 347 46.5 44 9 59.3 58.0 66.9 90.7 179 139 45 7 58 1 198 1 159 1 2.44 I'll 0429 0249 21 4 16,9 178 28 4 1 L=t ofQ-ttat@on (LOQ) PFC)s - 5.55nWmL Date EntadfBy IQ/05/99, lDiO7/99,0019,'00 I.AC Date Venfied'By befre 0610&00 TKR Pros - Perfi@@wromte Dam ptmty co"ectcd/wrified: D9/12/00mmil@ 09112,'00LAC 4 S@ =tal wi@s leu ti,@ 0,50 mL. concentratonsshouldbe comide@d tentatiw 041@.&tDOLAC NA antlyz@ riott;YpUcitble Fxtract3onVolume Ratio - [niaw Volume!Final Volume ETS-8-5 I Excel 97 3M Environmental Laboratory tox-030-wm223-IC 911zloo Page 211 3m Medical DepartTrer-z S::udy: T-62*kC-YTOX-0.30 Report No. FACT TOX-030 Covance#6329-22l3aboratory Request Numbe---U2279 S": ProductN@ber(Test Subs=") Matrix: 26WeekCapsuTl=eMty Stud@yth PFC)mSC',Iomol'".Ionke5@ T-6295(PFOS) Monkey Sm M@th,,&'R--, AnalybcalEqwp@t System N@ber Imu@cni SoftwwcdrV@m: Fdename: R-squared Vahle: Slop. Y-Int"ccpt D@i. ofExwcbWArA@yn. Date ofAna@y"AAa)yv: Dote ofDel& Reduchon/Anslyst ETS-8-4 I and ETS-8.5 I AM." 062498 M-Lym 3@3 See Anachments See Anwhmente See Anachmmts See Anach@t. O&r23M RWW 0912&W, 10/05/990,6/14/00 MEEFMMH D9[29/99.10/W", 06/15/00 MEEIIASIM@,111 SiLmple Data WEEK 47 NdONKEY Group 0-t Method Bik MatrixBlk QC - 250 ppb SEPA Sampi* 0 H20 EU- 1 H20 Bk-2 R&bbd Sm Blk-1 Rabbri3ers Blk-2 MKS=59.MSl MK3052MLAISD-1 .MX-U=59-L(S-2 MK305n9.LtSD-2 Pros Come. ughal 000 0()D 000 000 227 254 236 239 C.0c.atmu.0 of Pros ughal.r -AR,c <LOQ <LOQ <.OQ <LC)Q 91% 102% --163% 945% %1.2. Pros uWmL <LOQ ILOQ 97% 100-@6 RSD Std.D.,. MS/MSD RPD ',A N !I% 7% Group 1 Control 0,0m&iwdy 105520M 25,4 105526M 27.7 io5s29F 36@O 105549F 32.6 0.0339 00370 0.0431 0.0436 00355 00459 623 0.00221 704 000323 Group J MW-D@ 0 15 n*,kwday 105505M 152 105523M 114 105539F lis 10555'F 114 50,9 7 65 45 7 483 3,69 47 4 15 7 379 426 670 L..t 4 H*-D@ O@-5, n*(Wday fQ@tut@ (LOQ) PFCS 10551 I.M 105522k4:00,1,1,F33 -F - 5 55ngml 263 105 212 142 124 312 104 277 92 1 983 1 Data Entaod/By 1WO5199,10107/99.0&19;00 L-AC Date VerifiedB/y before06109100TKFZ 21 0 759 8 4", 32 PFOS - PerCuorooctn@uLfonate N A - Not awyzed, not apph@ble Oweptmtyco@tedtwnf"* OW121DOnutiko9llZ,'0L0AC S@mbaJ@h@l@thmG5OmLcmcm@tio@shoWdbecomideredicntatiw. Exractim Volume Ratio LnitaVJol=@'Foal Voh=e 04,r2glOLOAC ETS-9-5 I Excel9? 3M Environmental Laboratory tox-030-mm223-IC 91 t21,00 Page 212 3m Medical Department Study: T-629AC-YTOX-030 Report No. FACT TOX-C30 Covance#6329-22l3aboratory Request Numbei--U2279 Study ProductN@ber(Tat Substance) Mabu, M.Lhod(R-.@ Analytcal EquWm=t System N@ber imr@t softwwwven@m F@e@c R-Sq@ed Value slope Y-Intercept: Date of ExwacbofVAnWyxt: Date ofAna"WAnslyst Dole of Dam RoductiorVAjudrt 26 Week CapWe TomcityStudymth PFCS T-6295 (PFOS) Monkey Seri ETS-9-4 I and ETS-9-5 I Ameba 062498 \4usLynx 3 3 See Attachrnmu See AMwhrmnti See An-.hm.nt, See Attachments 09@25/99 RWIW 09,-291", 10/05199,06114)00 MEEFM'AH O9t29199, 10/06,99.0&15100 MEE/lASiMMH C@norneigus MonkeYs SampleDsts WEEK 51 MONKEY Group Dan SERA 3S.Ple 0 PFOS Conc. nV=L Concontmtlon OFFFOS oVmLor%Rec Mean PFOS G"L tRSD d. D"@ MS/MSD RFD K4.d.d Bik H20 Blk-1 H20 Bik-2 0.00 000 <LCQ @LCQ @LOQ NA Maou BIk Rabbt Sm Bik-1 Rxbba Sets Blk-2 0.00 0.00 <LOQ <LOQ @LOO NA QC - 250 ppb Group 1 Control o@o mwlwday Group 3 Mtd-Dm. 0 15 m&Agidy Group 4 HISK-D@ 0 75 rWkg/day MXSM259.m&l MK305289'-MSD-1 MK308259-MS-2 MK305299LMSD-2 105520M 105526M 105529F 105549F 105505M 105523M 105539F 105552F1055 1IM105522M 105533F 105542F 227 254 256 239 13.3 195 343 29.6 119 109 111 52 135 365 261 297 91% 102% 103% 96% 0.0213 o@0261 0.0344 0.0339_ 398 360 44.5 758 675 122 97.1 95.7 97% 1001/. 00237 0 03A I 37 9 35 1 94 7 91 4 11% 7% 0 140 00333 1.18 O@000403 6.92 '.62 37 6 13@2 406 38,4 665 607 @w o(Q@ (LOQ): PFCS - 5.33nlyml Date EntaedfBy: Date V.nfied/ By. tO/05M, 10107/99.06119,'00LA befom 06109100 TKR PFOS - Perfluorooctanerdf@le NA - Not araly=d, notapph=ble Date p=Ty co"ecc&wnhed: 09/12100 mmh. D9/1 Z;00 LAC Semm irubalwhune lessthin 0 50 mL- cencenrmtiom should be comidered tentabn Fxtmction Volume Rj6o - liubalVohimelPmal Volume D4/28100 LAC F-T@-8.5 I Excel 97 3M Environmental Laboratory lox-030-semn3- IC w 12100 Page 213 3m Medical I)epartment Study: T-629t@kC-TTOX-030 Report No. FACT TOX-030 Cov2nce#6329-22l3aboratory Request Number-U2279 study productNumbetiCTfttSubst@) Ma=: Metho&R@on, Analytcal Equipment Symtcm Number lmwament So*wwciVcntm 26 Week Capsule To)aoty Study mth T-6295 (PFOS) Morkey Sm ETS-8.4 I -it ETS-8-5 I PFOS in L@nomolgw S..PO20199 M=Lynx 33 -Monkey$ Fder=c R-Sq@.d Value Slope: Y-intemept: Date of ExuscttoWAna)yst Date or ArmkyoWArWym@ Date or Data ReductbovAnai@@ SampDiaeta WEEK53MOriKEYSERA Group Sompi. 0 D- .Method Bik Mabu Blk QC - 250 ppb Gmep I Control 0.0 ffwwday H20 131k-5 H20 B[k-5 RAbbit Sera Bik-5 RAbbit Sera Blk-5 I I MKSI 1039-MS-6 i MK311039-kiSD-6 MKSI 1039-MS-5 1 MKSI 1039-MSD-5 i 105520,M i 105526M 105529F 1055,99F See Am=@inents See Aftchmenu See Attachments See Artachmmts I IIDY" SAL itioCI99, liiiII99, illl&199 L-@s 11,VL99, llil2/99,llt2V" MMKQAS PFOS C.M. I ng/mL 0,113,0 2.20 2.50 295 29 4 374 380 33.6 49.9 46.83 52 Comcomtmtlon efpyos oVaL or % Re. <LOQ <LCQ <LOQ <LCQ 00237 00236 2% 11114% 00-0.16.9 00375 00419 M.." PFOS uLVmL "W ILOQ 00236 113% 00331 00397 RSD Std. M,. MSI.MSD RPD N-A NA 0 192 00000451 21/. 26.1 00086 0782 00311 G@p 3 Mid-D@ 0. 15 mg4cg/day 105505M 105523M 105539F 105552F- 2-i 103 91 439 41,1 32 3 46 2 36 7 73 3105 17 0 6 24 G-up 4 H%h-Dow 0,75 mg/kWday lossi im 1055221,1- 103533F 105542F 137 267 246 244 54.8 ol 98 6 97 9 9R 2 45 5 368 0499 -0490 Limit arq@tnaoon (LDQ) PFOS - 5 55 nWmL Date Ente@d/By Date Venfiedi By I I/]SM. 11174199 LAC before 06/08/00 TKR PFOS - Pcr(t@w..Wfmatc NA - Nm @ppb"ble Date puntycor@te&wntied. 09/121OOffuTdi,09/lZ/001,%C Although labitd n MSIMSD-6. th@ -r@ not *@d =d %U be umd bl..k. LAC IIIISM Se@ irutiawll=e Was than 0.50 mL, concertntons ihotjd be consid@ed tenmw@ 04@@.&OO LAC Cxm=on Voltane RAtm - lmt)alVolmwYinal %'ol-.c ETS-8.5 I Exccl97 3M Environmental Laboratory lox-030-scm223-IC Page 214 3m Medical Department Study: T-629]EACTTOX-030 Report No. F-z@C-- Covance#6329-22l3aboratory Request NuTioer-U2279 Study ProducNt@ber(TestSubstance) kia=: Method/Re%uion Analytc2ElciiupmeSnyttem N=ber F'de@e: R-SquaredVejue: slope 26WeekCapoWeTmmty Study@th PFOS. C)@o@.1" li--k-r T-6295(PFOS) monkeysra ETS-9-4I and ETS-9-51 soupozOI99 M-Ly- 33 Se,Anaclun=ts SeeAnach@nts SeeAmwhmnt3 Y-Inter@pt DateofExtmetaWAxa@fst DateorAnaly@Anatnt: DateofDataReductioWArtalyst. SeeAftwhm-ts IliG3/9S9AL tliC4t99.11/11I1t9l9t.&19L9AS IItO&'991,1;12199,11/221M9M9H/IAS SampleData WEEK 57 MONKEY Cmup Dee* Method BLk .Ma= Blk QC -250ppb Gmup 1 Control O@OnWkWday GroupJ MA-Dose 0 15mo*day SERA S..Pla. H20 M-5 H20 BM-5 RabtntS@ EUk-5 Rabbit-%enBLk-5 MKSI lC3g'-.M3-6 M"11039-MSD-6 MKSI IC39@MS-5 MK311039-MSD-5 105520.M 105526M 105529F 105549F 105505M 105523.M 105539F 10555!F PFOS C"cI.L ngim 0830 1 07 2.20 2.50 29.5 29 A 374 380 36.1 45 4 59.9 52.1 71 1 79 4 8'3a,1 Concawtmtion ofpfos I upOuLw % Roe <LOQ <LOQ @LOQ <LOO 00237 O@0236 112% 114% 0.0299 0.0364 O.D472 0,0419 29,5 31.9 33.3 31 4 RSD Std.N@. MIS/,MSD RPD <LDO NA I,OQ NA 0 192 00236 00000453 113% 2% 16 0 0327 0005126 963 00445 000385 7'3836 13 32 3 11 34 G@p 4 High-Dme 0 75 mgtkgiday 10351iM 2'_6"6 1055',ZM- 105333F 2' 105341-- 2781 66.5 '08 99 5 -180 !6@ 03 3 53 @09 106 3 S4 Lmt of Quantitaton(LOQ): PFOS 5 55n&@.L DateEntemdri3y 11115/991,lt24/99LAC DateVeri5o&By before06108/00i-KR PFIOS= PerO"@=c$W(Omte NA - Not pph..bi, Datepmry comcteda@fied D9/12/0n0uyik09ilZ'(L)AOC *- Althou&lhabeleudMSWSD-6, thc@werenotsplkeadnd@,lLc usedn bt"l LAC 11115199 + Be@ irutvmolh@e lessthm0.50mL,concmtrano@shoulbdeconsiderteedntauw 0,4179/0L0AC ExtractiVoonlme RAI%O-lnrrjVwol=c@FmalVol=e ETS-8-5I Excel9-, 3M Environmental Laboratory tox-030-wmn3-IC Page 215 3m Medical Department Study: T-629ACYTOX-030 Report No. FACT TOX-030 Covance#6329-22l3aboratory Request Number-U2279 Sh@dyProductNumber(Tftt Substance) Mat@.: Method(R-o. ArWytcal Eqiupment System N=bcr IrubwneritSoftwwcfVemon Fdename: R-Squar.d Val@: Slope, Y-Lntercept Date of ExMzCWWAruLlyst, Date of AnalywA@Ax%alrti Date ofData ReductioWAna"t 26 W"k Capztde Tomaty Study @th PFCS T-6295 (PFOS) ).IonkeySe@ ETS-9-4@ I and ETS-9-3 I souPO20199 @M.sL),nx 3,3 See Attachments See Anctwn-t, See Attachtnmt3 S" Attact.@.nt, 11103/99 SAL ll/04199. 1ltiim, Iitiv" LAS 11/0". 1I/lZ'99,111'22199MMHGAS m (')no@lg@ Monken Sample Data WEEK 61 MONKEY Group D@ Method Blk Ma= Blk SERA QC - 250 ppb Cmup 1 control 0.0 n%Wkglday Group 3 Mid-D-. 0 15 ff%i*day Cr.op 4 H.gh-Dme 0.75 rn&W&y Lumt ofQusnt=on (LOQ) PFOS PFOS NA - Not appb@bic SAMP4. 0 )420 Etlk-5 H20 BM-5 RabbitSm Blk-5 Rabbit S@ Blk.5 MKS[ 1039-MS-d MKSI 1039-MSD-6 MKS] 1039-MS-5 MKSI 1039-MSD-5 105520M. 105526.'A 105529F 105549F io5505M 10557,3M 105539F105552F 05511 M @05522.,i 05533F :0554,F 3 35 NWML Date p@ry PFOS Conc. W@L Conceptraden i Mtan PFOS I R.c ug/=L RSD Std.. MS(MSD RPD 0830 107 2.20 2.50 29.5 29 4 374 380 eLOQ <-Oq cLOQ <LOQ 0,0237 0.0236 112% 114% <LOQ LC)Q 00236 113% -- NA NA 0.192 -00000453 2% 39.9 47.7 54.0 57 7 0.0319 0.0382 0.0433 O.D46 00351 00448 12.3 0,00449 4,69 O.DD210 684 27 4 189 995 35,9 31 6 598 94 390 0.742 95 : 38.4 39 2 0.293 162 64.7 50,2 339 1 271 136 1()0 50.3 08 0640 1 273 1 11 09 -1 109 0697 Date Entemd'By: 11/15/99,11124t99 LAC Date Verified/By: before 0610&100 TKR coffcctedfvenfied 09/12100 nvnh. 09/12;DO 1,%C Aid@ugh labetedu MWMSD.6, that wac not ipikedand wiU be used= biwl'u LACII/15199 Se@ mtial @i=c l@ d= 0.50 mL. concen@bms showd be consideredtmtstw G4/28100 LAC E.xbwoon Vol@e Ratio- ImtW VolumvFinal Voi=c E7S-8-3 I acal 97 3M Environmental Laboratory wx-030-w@223-IC 9/IZIDO Page 216 3m Medical Department Study: T -6 2 9 @A-& TOX-030 Report No. FAC'-" TOX-030 Covance#6329-223 laboratory Request Number-U2279 stay, ProducNtmber(TtriSubmmcr) %(at= M,thod(R@ All@l_'ty@.1 Eqp.-t Sy.- -4--, sftw..V-@ 26 Wk C.p.W. T-.ity Stdy -th PFOS . Cy-@il- T-6295(Pros) M-ky S.. Mg-4.1 -d M-8-5 I Rby 100699 M-Lr@ 3 3 M@.ky. S. Anwlm" S- Aft.W-t. S- An.,h@t. Y-tm-.Pt S" Aftch-b Ehft :f FAVUU../A.4y@ DetofAulyvWAAWy-t Date ofd%ts RdacwwAAalyv Sample Data 04124= SAL'= 04125=,M7W.Un&W.0510M LA-'@IAXIH:AD%' 04,'27/W,05101=.05101100.DSM4/00 W WEEK 65 MONKEY Group D- SERA smpi. 0 Method aLk Mau= ft 0424Q-H2D Blk.3 I 04240-H20 Blk.4 RBSU74()-S- Bik-3 RBS042&&S- Blk-A PT()S C-c. VML 0300 0.390 0930 273 C.--Wsti" (pros sf.Lw % Rec :LOQ LOO @LOQ @LOQ Pros .@O.L LOQ LOQ RSD St&D-. MS/MSD RPO NA IIA mKso4240-sem Bik-3 864 16.9 MKS04140,-Sm Bik-A 110 0.00951 000144 QC -250 ppb MKS04240-Sm-?AS-3 310 1m WKS04240-S--LtSD-3 236 MX304240-Sm-MS4 306 JAXS04240-Saa&MSD-4 326 92% 119% 127% 107% 1 27% 123% 6% Group I C..tml D@O MVWday I(lis2o)A 12,7 M 526.'A 14@l *5529F 31.5 W5549F 24,7 D.0194 0.0236 0.0364 OM03 57 00210 D 0360 174 OLOO365 143 -OOM522 G@p 3 ?4d-D0.15 mSAWdy C@.p 4 HiBb-D- 0.75 mgrxwd.y 05505M 05523M 101119F M5 0151.2IM: @al 5 %1 05533F- 1I091, 1 344 393 261 1 360 1 3 -i 6o 254 52 5 597 131 1 915 4654 !9 7 0.0920 OLD269 6.18 2@32 60.3 55 2 11,7 I2 60 92 9 968 LLma o(Q@t3ta@ (LOQ) PFCS 5.35orml Data pmty Dt. Eb@.A/By@ DataV-F.& By: 05/01=. 05109/00 I.AC'CSH D&WW TYR 09112:= w..k 09112,'DLDAC PFUS - Perfluaroactm"Wf@t. s@ imta vol@ thm 0 50 mL, conc@tntom houJdb* @dnvd tmtab@, NA - Not ppli-bl@ V.I-. R@ - ltd Vol-.T,,.lVt-@ 0417&'00 LNC M-8-5 Excel 97 3M Environmental Laboratory t-.030-.m223-IC 9,1ii0o Page 217 3m Medical Department Study: T -6 2 9 @A& TOX-030 Report No. FACT TOX-030 CovmcOfi329-223 laboratory ReciuestNumber-U2279 Study: Prodmi V=btr(Tm Subewce) mum M@th.d/R-"alyucalrqw,.m SyUm N@b@ lnmwnerd Saft@WN,'@ FJ@: R.Sqmed VWu# Slope: Y-1@.P. D@ of Exu@rdAradyw D," offA@@ly.WA." t@." D" Sample Data WEEK 69 MONKEY SERA Cr..p D- I..Pk x %imhod Bli M@L- BLk QC - 250 ppb Gr.up I C."Lrw 0.0~.y Gm'p 3 N6d.D@ O@ 15 MWkgld-Y c-p 4 High.rkm. 0 75 g/kgldy 0424G.H2o Blk.3 0420-H20 Blk-4 RB3*4240-Sm Blk.3 JLBSOC34&Sm Blk-4 MSU240-Sm Eft-3 ,WS04240.SmBtk4 W--44240-S-US-3 W304240-S-MSD.3 MK304240.3-US4 mx3N240.3-MSD4 10552DM4 105526M losszw 105549F IOS505M 1055zm 105539F 105552F lossilm 105522M. 105533F lG5-U2F L,= ofQ.==.n (LOQ):PTOS - 5,55-&-L PFOS - DK. pvy 26W..k Cpl@k T..my Stdy uh PFOS . Cy....Ig- T-6295 (PFOS) Mo@ky S@m STS." I-d E'rS-&5I R.by 100699 M--Ly.. 3 3 S. Adwhmrrb Sn AaWhmgMs Sn AUWhmco S. AV-h04@24MO SAL4" 04/2YW. 04,77,00Wn=. OSMVW IA&%M'ADV k%a@k,y, pros conc. aV=L C@.i-u.. fPros %ftL . % R" Pros fIL 0.300 0.3" 0,930 2,75 16 @LOQ @LOQ @LOQ @LOQ 000750 -LOQ <LOQ 11.0 000954 000152 310 172% ZU m 107% 306 119% 326 117-A 123%- 22.2 0.0432 26 3 oom 00406 3Z5 O@0421 24,4 0,0379 00400 361 24.6 371 213 264 459 369 406 320 34 S 311 4170 21_ $40 411 73@7 S25 711 ?5 0 DK, E.Un&By: OSMVOO,OYOWW LAR-CSII Do. Vmfl.& By- O&M2= TXR OWI 2= m,@jt DWI ZVC LAC mgW voh@ Un thm 0 50 mL. cmcmm.@. RSD Sid.D-. M&IMSD RPD NA 169 0,001" 2'?*/. G*A 7.71 000313 752 000301 goo 2,19 100 346 624 52 4 700 525 h@Wd b. c ...i&,,dtcmauw 04,2&00 I.AC ETS4.5 I Euti 97 3M Environmental Laboratory lox-030-aa23-IC 9. 1 Page 218 3m Medical Departmerit sz:udy: T - 62 9 @A& TOX-030 cov"ce#6329-223 Report No. FAC7 TOX-030 laboratory Request Number-U2279 3tiWy 26 Wftk Cprik Taxic@ Sludywth PFOS m Cy@mc4gm Moarfs Produa N-umber(TINSubKmcl): T-4203(PFOS) Mwm IA.thodAt@nAAW.,.dcEWqUtp@ Syu @@b@ ]@@ monlgy Sm ETS.".: -d ET3-8-5 1 Rutry 100699 M..Lym 3 3 Fd@: S. Azt.h..u R-Squamd vwu: S. ANWA@ Slope: Y.Lm-": St.Av-h@ S. AM.CH- D.u :(Ea@.t-VAA-tYDes fAR*MWMmiyg 04124@00SALXRK 04/2M. 04M/00. 04t2g=. C".VOO IAS.%t\ffADV D- fD" OAMM. OS.-LO.M.090M QSMLM LAS S@ple Doin WEEK 73 MONKEY SERA Pros C@estrath RSD D.@ C-. I PTO .1d. ,f/,L g,:Lporro%SR uV@L MS/MSD RPD \ikb.d 131k OG4*-H2o aik-3 0.300 @LOQ 042WH20 9&.4 O@3" @LOQ NA Mmm Blk QC -2M ppb RB3O-C24O.3mBA-3 RRSOC240.SMBlk-4 W=4244@.S=Blk-3 WLW42WS-Bik4 mxsD424s@s-US-3 .MKM4200-S@MSD-3 MXSU240-3-MS-4 NOCS04V&S@MSD4 0.930 215 86-4 11.0 ])a 236 306 326 @LOQ LC)Q 000750 0 omu IZZ% "% 1is% 17)-A LOO 000852 -12.3% MA 169 0 001" 6% C-up 1 Control 0.0 &4Wdy M320M. 22.2 0.0431 323 '"326M 17@6 0.0269 o035O 0.oils )05329F 22@6 0.0386 - 76 ms549F U.7 0,0345 0.0365 000284 G ..p 3 @,W.D0.15.9&wd" G-.p 4 *5505M 163 M3523M+ ist W5539F 160 105532F- 131 *4"jllM 403 74,0 17.1 306 213 4" 279 113 23+7 259 2+91 35 1 so 2 Hi&h-Dm 20@322?A- 374 --733 U4 173 0.75 ,gItWdy :35533F 326 54 5 191 !055c-r. =7 240 1.7 131 L.w fQ@ (LOQ)PFOS 5 35 Do. Fjgm&By OSM3MO.OS"= LAC CSII D.u V.,ido&By Ofi'O2tOTOKR Dw. p.,Uy 0*12MG m.@k "112100LAC PFOS - PttlMwrooMW=Wrmgc - S- in" "h@ k. th.0.5m0L. h..Idb@ I.-d-d .4A - @iOtUPPILCtb[C F@ummm Vol=* Rmmo - INXW Vol=&FmW %'oM=,e D4,2&100LAC ETS-8-S.1 3M EE..n19v, ironmental Laboratory lox.4306mra=3-IC 9-1Z= Page 219 3m Medical Department Study: T-629$Ac7rTOX.030 Report No. FAC--R TOX-030 Co,ance#6329-223 laboratory Request Number-U2279 sn-tr Prod= 4-bg,(T.*S.b.M@) ma@- VALh.&%-, A..ryL@*EqLvm@ Sy- - N-b[@mu.matsft-vv-:, 26W-.Ic,qwk T-way &-4 .9h PFC)-S C),...ig.A-kr 7-6293(PFOS) %4-k.y3ETS44 I d ETS-2-5I Ruby 1006" NU.LY- 3.3 Fdo@, S..A=Wn@ms R-Sq.wW VW-: SetAtLKtunents St." Y-1@cpt D@ :fF=.u.WADdyN S..Aawl..," S. Adh@ 04r4l4M SALXJK Diu fAnat)iWAra": D- fD-RW@An.": 04aS'DO OU77MO. OV7M OVOIW IXS'@M"ADN' 04/77= 'OSiOlM.OVOIIOG.OS06M IAS Sample Dots WIEEK 77 MONKEY SERA creep Dose pros C-@ ."L ofprw W.L - % R@ ... Pro$ C-L RSD Std.D.,. %43,?ASD RFD U.th.dBlk Mom Blk 0300 04240-H2OBik4 0390 RSSM240-SM EU-3 0930 RBSU240,S-BIkA 275 <LOQ @LOQ @LOQ <LOQ ILOQ NA QC -2SO ppb MK-gU240-SeMB&-l MKS04240-SeMB&4--MKSN24O.B-M3-3 MK304240.3-MSD-3 MKS04240.5@MSA PAKS04240-s@MSD4 8.64 -11-0 310 236 306 326 0.00750 00095 122% 92% 119-A 127% 1 000952 16.9 000144 21 6% cmup I CordrW 0 0 mWkVday toss2om. 12.7 o,033.4 Ito 10552CA- 11.9 000259 002% 0 DOSIS 1055VF 21,9 0.031, 546 lG5S49Y. Is 2 0.0294 a 030i 000167 C@op 3 Mi&D@ 0 15.&Udxy Cmup 4 H.gb@D@ 0 75 &&Vdy 10550SN4- 12D 105523M, 101 105539F- 101 10555IF- 70,3 10551im 424 105522M Sli 105533F 463 lO5S42F- ZLI 210 2.3,0 1 D 632 17.3 277 125 230 6.37 319 63 8 170 32 3 336 -4'3a45 570 191 L-a .1 O..nWA@ (LOQ)!PFOS PFOS - Pafluwooctan"Wfmmie 5 55 -&'-L Da. E.Urd)By OYOUN. OYD9= LAC@CSII Dde V-&df By 06M2MO TXR DKt pmty comnedN@Agd: OlwI2MO ffvn?L09/1',IDLOAC + S@ LNAW vobm im thm 0 50mL, concntm.@, houldbec.mdcfed lmmve .4A- Not apphcabk E@- Vi- R@@ - T-@-VIh-.1f..lN .1-, 04!23M LAC ETS-4.5 I M Ex,d 91 3 Environmental Laboratory 9;1-% Page 220 3m Medical Department S--udy: T-629$AC7rTOX-030 ReDort No. FACT TOX-030 Covanc@06329-223 laboratory Reques'@ Number-U2279 sludy PmductNumba(Testsubowce) MMU. M.Lh@d'R- A@.IYUW Eq-Pm-W Sy@ l@ S.R-.V- N-b@ u wk cpw. 'foam Uidj @thPFOS T-6295 (PFOS) 14.m.y SETS-2-4,1 wLd ETS-3-5,I R.Ply 1006" Nt..Ly- 3.3 FU@: R.Squw" Vaiw 31-" Y-in@-Pt D@ :rEnruoWA@.iyK Dal FARWYIWAMIYK: D@ OrDes R-40,b.VA.": Sampk Data WEEK 79 MONY@EY SEI;LA a-" D- S..Pi.. S.. AD-hm.-I So ARKNrAw S44Anl@ S. Abtn@ 04,74= SAII= 0412YW.04fZ7MO.OVMMOVQYW 04/77=. 01101=. OVOIM OS@04M IASNO."ADV IAS Pros C... .V L C.Ma.mu.. f PFDB t/.L .,% R., M... PFOS RSD Sid.DM&'MSD RPD L6.LhodIft W..= Elk 0424&H2O Bik-3 04240-H2OBlk4 ItB304240-S.. Blk@3 Rasoduo-s- Eak@ MX3"240.SmM-3 FAK304240.3- Blk4 0300 a 3" 0930 2.75 8.64 110 -LOQ @LOQ <LOQ -LOQ OLOO750 0009S4 <7.(Y@ 000152 NA NA 169 D 001" QC - 250 ppb ?4K3042@W3@MS-3 310 -MX30424(@.-S@M=-3 2m im m 10-/. 27./. MXS"24&S@MS-4 304 MXSCM24&3-MSr)4 326 119% 127% 1ZP,4 6% r-P 1 C.@dfd 0.0 WU4" W5320M id 1 0-0194 133 IOM26M 17.9 O.D236 00,115 0002% 05329F 221 o.out 146 105@49F 13.6 0.(1211 0 D243 0,00355 0.15 g&wdy ivssosm M523M M55'JgF IC3552F 152 ll).7 4.22 III it's !9 i 0905 142 221 940 133 19,9 '1 4 2voi G@p 4 iossii-im 293 22's 63 1 High-D@ 105522. 1M- 321 394 41 1 159 0.75mtwd" 105@23-IF 262 2.79 105542-IF 253 :0@ 414 1.15 Lmm ofQ%==aam (LOQ) PFOS 5 55 gftL DS& E@*My 05103M.O"IOD LACCSH I),"vfflfitB&y. O6m2m TKR Do,p.,Nyc@.dh-Mf.d 09/12'OO-mkOg/12,DOE.Al' PFOS NA - Not WpUcabLe S@ E@- in@ @t@ lm Om 0.50mL conc.@@.V.1- R&U. - U." V.I-.F.W V-i- h.Wd b. c.-dmd (mw@ 04rlt,00 LAC acel 9@ 3M Environmental Laboratory w..030.=.M-IC Page 221 3m Medical IDeT)artment Study: T-6295.7 Report No. FACT-TOX-030 laboratory Covance# 6329-223 Request FACT TOX-030 Number-U2279 Study. PniductNumber(Test Substame): Mauu: Mc&iod?Revision: Analyucal Equipment SysLcm Numi@ ]muu@nt Soft.aie/Venwn Date fEx:racti.VAmly.t: Date of Analysis/ArLal)st. O.I.,,fData Reducu,4VAmly,i Sample Data 26WcckCapsuic ToxicityStudywith PFOS inCymi@-IgusM,,nkc%, T-6295 (PFOS) Monkey I-i%-w f-ticn=c See Allachrr)cnl@ FACT-M-1.0 & FACT-M-2.0. 2. 1.FT@-8.1 R..@quwcd Value. See Artach=nls Ameba 062493. Madclim (M ID". r).,@yO S- Atta@h=ni@ MassLynx @.2 & 3,3 Y-Imm@@pt .4;mALt2ch@n" 0918,99. O&tl 1199.05123/00 SAH/SRP/St-.*FJ%Al. 05119199,05/22M. 06/09M. ON14/99. 07/29/99. O@f25/00 KjHiHOJIMEFJiAWIA.3 05/20&9. 05/25/99,O&ID/99. W22199, 09.104199.10/12199.50,'26M KJHFHOI NEFJMMHIIAS MONKEY LIVER Group Dwe Week 27 Sample 0 Method Blk Method Bik McthM Blk Mwm Blk Matrix Blk Matrix Blk Matnx But QC - 230 pph 05119/99 QC - 250 pph 0& 11/99 RBLDS]89-H20 Blk.3 RBLO5189-H2 RBLD6ll9.H20 BLk- I1 RBL.061 M..-0523G.H20 Blk-5 05230@H20 Blk-6 RBLDS189-LIT RBLOSI89-L@T RBLD6119-L@-r Blk-II RBLO6119o RBLD5230-L@-r BLk-5 RBLO523&L%T MKLDS230-Lvr Blk-6 Blk-.56 M"5230-Lvr 105508m-ms 105508M.MSD 105517M.MS 105517M.MSD QC - 250 ppb OSf23/00 GroupI Control 0.0 mg/kglday Group 2 L@--Dow 0.03 mgkS/day cxt- 5123/00 MKLDS230-MS.5 MKLO5230-MSD-5 105509M 6)11199 105517M 6/11199 105-148M -..- I1 105517M 51i@" 105519M 105527M 5 9199 51 1 &199 105530F 10553]F 105535F 105544F 1(15514M IW575M 105516M lvs2im 105537F 105541F 105547F 105547F 10555OF Gmup 3 Mid-D,nc 0 15 mglkgtda) 105510M 1055]SM [05524M 105529M 105532F 105539F 105545F 1055"F C@.p 4 Higl@D.0.75 mS*4/day 105507M 105512M 105.S34F IQ553,6F IC554OF 105551F L@.1 -f Q@utal@ (LOQ): PR-OS - 30 0 ngig NA - N,x ")zc4 notappl@hi. PPOS - Perflw@=@ulfinate PFOS Cok- c@. offo(a 0.00 0.454 0.1)0 NA concewuudm W.01=.. <LOQ <LOQ <LOQ NA Mmn PFX)S <I.OQ RSD Std.N,. M.VMSD RFD NA NA o@ <LDQ 1.64 <LCQ <1.(X@ NA ,0.4003 o@oo <LOQ <LOQ <LOQ <I,OQ NA NA 1.2.@ 0.391 15.2 NA <LOQ <LOQ <LOQ <LC)Q NA @[.OQ NA 9,7 492 305 3()0 305 <LOQ 174% iim 101% 103% <LDQ NA 1421, 1021, 332 111% 292 98%- 1()4" 13,@ 564 O@564 143 0.143 477 0,477 119 0,119 R 91 O.D91 22.2 129 0.129 0.121 C)0269 128 0.129 112 0.112 87 0.097 169 97 0.097 0.106 00179 19237 182 22709 227 11417 167.14 228 IR 11.4 16.7 22.8 270 17 A 4 24947 248 623 20102 '2'0'735 283 20.1 20.7 9 73 22.1 2.11 42169 42.2 96173 86 59673 59@7 48201 49@2 59.8 195 80421 49590 80.4 49.6 66-532 66.5 214 81376 91.4 69.5 149 4,12474 412 @ 8721 379 280575 291 25(@669 257 267329 267 287223 287 6.01 396 219 S,O() 273 136 Date EaLcraYBy: 05/25199. O6/lOi99. 06122/99. 10@12N9 I.AC Date Verified/By: 0&02tM PJW. OW5/00 TKR [)arePunry coffwtcdvcrirv.&"/12)00 mmh. 091121001,AC' Smpic dcu@mdm ouW= aW wm not u)cludedinany calculaix,m Sample used forMS/MSD appun tobe spikedaccnuntingfm I,iwmo,%ffies. gaznpic105507 & ,S=pic [Of,507M willbe used forthe MS/MSD and 105518NI c,@c. Verified, R=S=plt=xu@iodaWngwiLhMS/MSD. This@alue@@m,iu"- inin@@icutat@,m, S=p@c dctemirmd an )utliearnd wu not imludcd inw@ calculai%,nsW.@ mcxtractcj 19 willberexlncted FAC7. M-2@ 1 Exccl97 3M EnV4 ronmental Laboratory tox-030-h@w223-1 B,xls 9/12100 I." I'm Page 222 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 tr s s r= 0 ct% 7ul ci CA CL lc_ U cn E @To u dd gz 96 < < z z z z z 9L Z ZZ Z Z v cI@.. j , Z < Zz z f-zi 96 - z: -a u -z 2 E -tr E v E-u CM z@ Z3 L5 Z E E of, E cy +> EE ;> -V E z LL) E E mz a CA C) cn r4 4C2, @n 12 LCLL CY CL 7? E cr, E < 4 C- 3M Environmental Laboratory 0ULo LL2 Ln Page 223 3m Medical Department Study: T-6295.7 Report No. FAC'-7TOX-030 laboratory Request Number-U2279 ILO 0 a cAz<zzz<zz< < < r 00 00 !Zl c@, < @4 C%i r4 c Ln or C6 0i -A -3 %D 00 00 vvv Do to oa bo oo 00 IV, "&b -ca)-ob '6b -6b -ob CD CD rq C, r- m 'o w C, w r- c, f-4r-r, cq r- u v v VVVV Ln cq r4 1= Im 0 10 In r, 10 M @4 C @4 @4 ,r m u 00 14 r64 a CL r-4 r,4 C,4 r,4 r4 r4 @4 E P7 --4 4 14 @l-- U: @4 CT 0 o E z E < > r -43 < < z9 0 000 2< 0 c @, 3M Environmental Laboratory 6. E E << CY z wr > C < u C) u z C) 0 C) C@ LL, wx Page 224 3m Medical Department Study: T-6295.7 3M MedicaDlepartmenSttudyT:-6295.7 ATTACHMENT F EXAMPLE CALCULATIONS Report No. FACT TOX-030 laboratory Request Number-U2279 ReportNo.FACT TOX-030 LaboratoRreyquestNumber-U2279 Formula Used for Sera Analyses in Study FACT TOX-030 AR (ng/mL) x DF x SC x FV (mL) )< 1.0jig = pg/mL x PC = Reported Cone (Mg/mL) EV(-m L) 1000 ng Calculation Used for Group 3, Week 1, Animal ID 105505M 287 ng/mL x 10 x 0.9275 x I mL ML x 1.0 gg 1000 ng 5.32 @ig/mL x 0.864 4.60 pg/mL AR- Analyticalresultfrom MassLynx summary DF- Dilutionfactor SC-PFOS saltcorrectionconstant(0.9275) FV-Final extractvolurne(1.0mL unlessotherwisenoted) EV-Volume of sera extracted PC-PFOS puritycorrectionfactor(86.4%) Formula Used for LiverAnalyses inStudy FACT TOX-030 AR (ng/g)x a curve(1)x SC x DF x 1.0pg = pgigx PC = [PFOS] sample(Aglg) -ds-a-mp-Te- 1000 ng a curve isassumed tobe: I g liver 5 mL H20 Calculation Used for Group 3, Week 27, Animal ID 105510M 524 ng/g x I g/ 5 mL x 0.9275 x 100 x 1.0gg = 48.8 gg/g x 0.864 = 42.2 pg/g 0.9963 g/ 5mL 1000 ng AR- Analyticalresultfrom MassLynx sununary a cur-ve-Density of the liverstandardcurve,assumed to be Ig Iiver/5 ml water a sample-Density of the liversample (1 g sample/5 mL H,O) SC-PFOS saltcorrectionconstant(0.9275) DF- Dilutionfactor PC-PFOS puritycorrectionfactor(86.4%) 3M EnvironmentalLaboratory 3M Environmental Laboratory Page F-1 Page 225 3m Medical Department Study: T-6295.7 3M Medical Department Study-T-6295.7 ATTACHMENT G INTERIM CERTIFICATE OF ANALYSES Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 LaboratoryRequest Number-U2279 3M EnvironmentaLlaboratory 3M Environmental Laboratory Page G-1 Page 226 3m Medical ]Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Reques@'- Number-U2279 PuriW INTEPJM CERTIFICATE OF ANALYSIS Revision1(917100) Centre AnalyticalLaboratoriesCOA Reference 023-018A 3M Product: PFOS, Lot 217 Reference #: SD-018 Test Name Purity: 86.9% Specifications Result 86.9% Appearance Identification NMR Metals (ICP/MS) I. Calcium 2. Magnesium 3. Sodium 4. PotasSiUM2 5. Nickel 6. Iron 7. Manganese Total% Impurity(NMR) Total% Impurity (LC/MS) Total % Impurity (GC/MS) Related Compounds POAA Residual Solvents(TGA) Purityby DSC InorganicAnions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate S. Nitrite 6. Phosphate 7. Sulfate4 OrganicAcids' (IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis': I . Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine VA-dteCrystallinePowder I . TheoreticalValue = 17.8% 2. neoretical Value = 0% 3. IleoreticalValue = 0% 4. TheoreticalValue = 5.95% 5. lbeoreticalValue = 60% Conforms Positive 1. 0.005 wtjwt-% 2. 0.001 wt./wt.% 3. 1.439 wt./wt.% 4. 6.849 wt./wt.% 5. <0.001 wt./wt.% 6. 0.005 wt./wt.% 7. <0.001 wtjwt.% 1.93 wt./wt.% 8.41 wt./wt.% None Detected 0.33 wt./wt.% None Detected Not ApplicableT- I. <0.015 wt./wt.% 2. 0.59 wt./wt.% 3. <0.040 wt./wt.% 4. <0.009 wt./wt.% 5. <0.006 wt./wt.% 6. <0.007 wt./wt.% 7. 8.76 wt./wt.% 1. <0.1 wt./wt.% 2. <0.1 wt./wt.% 3. 0.10 wt./wt.% 4. 0.28 wt./wt.% 1 . 12.48 wt./wt.% 2. 0.244 wt./wt.% 3. 1.74 wt./wt.% 4. 8.84 wt./wt.% 5. 54.1 wt./wt.% COA023-018A 3M Env4j-ronmental Laboratory ExactCopy of Orig;,nal LAL In!Llai Date Page I of3 Page 227 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 INTEHM CER TIFICATE OF ANALYSIS Centre AnalyticalLaboratoriesCOA Reference#: 023-OISA Date of Last Analysis: 08/3 1/00 Expiration Date: 08131/01 Storage Conditions: Frozen:5-IO'C Re-assessment Date: 08/31/01 'Purity= 100%.- (sum of metal impurities,1.45% +LC/MS impurities,8.41%+Inorganic Fluoride,0.59%+NNM impurities,1.93%+organic acid impurities,0.38%+POAA, 0.33%) Total impurity from alltests= 13.09% Purity = 100% - 13.09% = 86.9% 2Potassiwn isexpected in thissaltform and is thereforenot considered an impurity. 3Purityby DSC isgenerallynot applicableto materialsof low purity.No endotherm was observed for thissample. 4S ulfur in the sample appears to be'converted to S04 and hence detectedusing the inorganic anion method conditions.The anion resultagreeswell with the sulfur determination in the elemental analysis,lending confidence to thisinterpretation.Based on the results,the S04 isnof considered an impurity. 'TFA BIBA NFPA PFPA Trifluoroaceticacid Heptafluorobutyricacid Nonofluoropcntanoic acid Pentafluoropropanoic acid 6'Meoreticalvalue calculationsbased on the empirical formula, C8F17SO3-K+ (MW=538) This work was conducted under EPA Good Laboratory PracticeStandards (40 CFR 160). COA023-018A 3M Environmental Laboratory Pagc 2 of 3 Page 228 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 INTE)UM CERTIFICATE OF ANAL YSIS CentreAnalyticalLaboratorieCsOA Reference#: 023-018A LC/N4S PurityProfile: Impurity C4 C5 C6 C7 Total wtjwt.% 1.22 1.33 4.72 1.14 8.41 Note: The C4 and C6 values were calculatedusing the C4 and C6 standard calibration curves,respectively.The C5 valuewas calculatedusing the average response factors from the C4 and C6 standardcurves. Likewise,the C7 value was calculatedusing the average response factorsfrom theC6 and C8 standardcurves. Prepared By: Reviewed By: David S. Bell ScientistC,entre AnalyticalLaboratories Date John Flaherty Date LaboratoryManager, Centre AnalyticalLaboratories COA023-018A 3M Environmental Laboratory Page 3 of 3 Page 229 3m Medical Department S--udy: T-6295.7 Report No. FAC-@ TOX-030 laboratory Request NuT,-@Der-U2279 INTE"M CER TIFICATE OF ANAL YSIS Revision1(917100) Centre AnalyticalLaboratoriesCOA Reference 3M Product: PFOS, Lot 171 023-018B Reference #: SD-009 Purity: 86.4% Test Name Specifications Purity' Result 86.4% Appearance Identification NMR Metals (ICP/MS) I . Calcium 2. Magnesium 3. Sodium 4. Potassium' 5. Nickel 6. Iron 7. Manganese Total% Impurity(NMR) Total % Impurity (LC/MS) Total % Impurity (GCIMS) Related Compounds - POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions 1 . Chloride 2. Fluoride 3. Bromide (IC) 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate 4 Organic Acids 5 (IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis': I . Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine WliiteCrystalliPnoewder I. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Tbeorctical Value = 5.95% S. Theoretical Value = 60% Conforms Positive I. 0.017 wt./wt.% 2. 0,007 wt./wt.% 3. 1.355 wtjwt.% 4. 6.552 wt./wt.% 5. 0.003 wt./wt.% 6. 0.004 wt./wt.% 7. <0.001 wt./wt.% 1.00 wt./wt.% 10.60 wt./Wt.% None Detected 0.30 wtjwt.% None Detected Not Applicable' I. <0.015 wt./wt.% 2. 0.27 wt./wt.% 3. <0.040 wt./wt.% 4. <0.009 wt./wt.% 5. <0.006 wt./wt.% 6. <0.007 wt./wt.% 7. 8.82 wt./wt.% 1. <0. I wt./wt.% 2. <0. I wt./wt.% 3. <0. I wt./wt.% 4. <0.25 wt./wt.% 1 . 12.08 wt./wt.% 2. 0.794 wt./wt.% 3. 1.61 wt./wt.% 4. 10.1 wt./wt.% 5. 50.4 wt./wt.% COA023-018B 3M Env@-ronmental Laboratory E)= COPY of Odginal [AL 1 .1 InE;i- L)ato Pagel of3 Page 230 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 INTERIMCERTIFICATEOF ANALYSIS Centre AnalyticalLaboratoriesCOA Reference 023-018B Date ofLast Analysis:08/31/00 ExpirationDate: 08/31/01 StorageConditions:Frozen:S-10'C Re-assessmentDate: 08/31/01 'Purity= 100% -(sum ofmetalimpurities1,.39% +LC/MS impurities, 10.60%+InorganicFluoride,0.27%+NMR impurities1,.00%+ POAA, 0.30%) Totalimpurityfrom alltests= 13.56% Purity= 100% - 13.56% = 86.4% 2Potassiumisexpectedin thissaltform and isthereforenotconsideredan impurity. 3purityby DSC isgenerallynotapplicabletomaterialsoflow purity.No endotherm was observed forthissample. 4Sulfurin thesample appearstobe convettedto S04 and hence detectedusingthe inorganicanionmethod conditions.The anionresultagreeswellwith thesulfur determinationintheelementalanalysisl,endingconfidencetothisinterpreta5onB.ased on theresultst,heS04 isnotconsideredan impurity. 'TFA HFBA NFPA PFPA Trifluoroacetaicid Heptafluorobutyriaccid Nonofluoropentanoicacid Pentafluoropropanoiaccid 6Theoreticalvaluecalculationbsased on theempiricalformula,CsFl7SO3-K+ (MW=538) This work was conductedunder EPA Good LaboratoryPracticeStandards(40 CFR 160). COA023-01813 3M Environmental Laboratory Page 2 of 3 Page 231 3m Medical Department Study: T-6295.7 Report No. FAC'-7 TOX-030 laboratory Request Number-U2279 INTERIM CER TIFICATE OF ANAL YSIS CentreAnalyticaLlaboratorieCsOA Reference#: 023-018B LC/MS PurityProfile: Impurity C4 C5 C6 C7 Total wt./wt.% 1.03 1.56 6.38 1.63 10.60 Note: The C4 and C6 valueswere calculatedusingtheC4 and C6 standardcalibration curves,respectivelyT.he C5 valuewas calculateudsingtheaverageresponsefactors from theC4 and C6 standardcurves.Likewise,theC7 valuewas calculatedusingthe averageresponsefactorsfrom the-C6and C8 standardcurves. PreparedBy: David S.Bell Date ScientistC,cntreAnalyticalLaboratories Reviewed By: John Flaherty Date LaboratoryManager, CentreAnalyticalLaboratories COA023-018B 3M Environmental Laboratory Page 3 of 3 Page 23,2,, 3m Medical Department Study: T-6295.7 3M Medical Department Study-T-6295.7 ATTACHMENT H REPORT SIGNATURE PAGE Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 LaboratoryRequest Number-U2279 ;71 AndrewM. Seacat1,5tl-Stt:@ri,dDyirector Date John L.BOenhoff,Ph.D.,Sponsor Representative Date Dale L.Bacon, LaboratoryManager Date 3M EnvironmentalLaboratory 3M Environmental Laboratory Page H-1 Page 233