Document jyMM38rqmqJXVmLyYD9eDrjgp

QEHR - 0400. 373 (0008117828) 7 5. AR 226-0 IF ANALYTICAL LABORATORY REPORT one Determination of the Presence and Concentration of Perfluorooctanesulfonate (PFOS) (CAS Number: 2759-393) in the Serum of Sprague-Dawley Rats Exposed to Potassium Perfluorooctanesulfonate via Gavage Laboratory Report No. U2779 Requester Project No. 3M Tox 6295.13 ee--------ee-- r ee--e ----ee esete erer met Study Dates Study Initiation: 10 June 1999 Sample Analysis Initiation: 22 June 1999 Sample Analysis Completion: 28 June 1998 `Study Completion: At signing 001172 PCeAruScNruomobcearn2e7n5s6o3r5s3i La ReSaMvREeeb nspvoiuomoNbmoo ee.nrFt(AaRGLaNTbYr oTUro2actT1oT0rSy8 TABLE OF CONTENTS StuyPerso 2dCOMDUDS rr nerd SIOFR COMPE EANGN . rn2 (GLPSHY:d Quality ASSUTNCE SHAEMeNt........ smam------------------3 SATE RECEIR smm------ POCO DOVRIONS...rrrs Data Summary, ATalSes, and RESUS... SUMMBLYOfQUallyCON!ADGYSSSROSES... . RE SEOM fCONE GSION N 1... REMBMECES. rr mm m------ 8 ---- | 9 nmsmm-- 001173 Pagel CPHetSrNsaomcaeo2s7s5t5o3n8s3s `STUDY PERSONNEL AND CONTRIBUTORS Analytical Chemistry Laboratories: `SAedrvuamncAendalBlyosAensalytical Services, Inc. 1It5haCcaat,hNeYrw1o4o8d5R0oad KrJioshtnenR_J.PeHrakninsse,n,PhP.hD.,D,,AsSstiustdaynDtiSrceicetnotrifcDirector AIrngiufseReTseesatricnhgLaLbaorbaotorrayt, oInrcy. 5H0o5rsShhaeme,hPyA.Dri1v9e0,4B4ulding A RaymGo.Ynordk, Ph.D. DABT,StudyDirector S3MpoTannsdcoorlogy Serv-iMecdiecasl Deparment S3tM PCaeunlt,erM,NBu5l5k1i4n4g-212000.02E.02 Marvin T. Case, DM, Ph, SponsorRepresentative MERongevr.eFrACLTaTboorxs1t0r8y LaboratoryRequestNm (RNHIZTTS 001174 Page tt10 CPAeSrtNoarmsoccearn2es7u5t5o3r9a3ts LaboratoryReMqFuaEengsitormrrNmeaon.raFl(LLaANboWTrICosZctT1oTT0rSy8 `STATEMENT OF COMPLIANCE SudyTe: APnayetcarlLafborautoroy Rerp(oPrctFoOnSot)hiencDheteetSrmeiranuamtinoonfoSerfathgsuPeru-eDsaefwnecyeo"anRadntCsonEacxepnoitsreadteitoonof PotassiumPeruorcocanesuvfiaoGnaavalgee Study denticatonNumber: FACTTox-108 R"eTgiuslsattiuodnyswafoscNoonndcuicritcead iLnacobmrpaltiraynSceuwietsh [FDoaodtaanRedqDuriruegmAedrmdin(i)s:tr2a1tiConFGR o(oPdarLta5b8o)r,atwoirytPhaeceixcsa(pGtLiPo)nisn ahsesbuurlalnecteeudinsit.Abeulodw.prIonaceddduorens,anthdepfriensdienngtssourcuhtaspbeerefnoramuedditaetdehoesSpMeEcnviivreoynbmeynatnaliLnadbeopreantdoernyatnudsaktty Tpaabrotriactiopratyiangncdonwtilacbet lraebtoarianteordifeosr aarpeehrousneodtwi0tehxdcoceuemdenfetatyieoanrsp,Terhneeannatly0ttihciaslsptorutdiyoinncaorcmivpesalatettthteee3MIdM ESntvainrdoanrmdeOnptearlaLtaibngwParsocpeedrufroems.edinaccordancewih3MEnvionmentlTechnologyandSafety Services Excato GpLPtcomiplioancne: + Tuoseparatestudydirectorswereassigned oth ne phaseandthe anaytalphase of isstucy + TheABS finalreportdoesnothaveaStatementofComplance. + T2h1oCFQRAUPasrtatt5e8mentintheABS inalreportindicates compiancewihEPA40 CFRPat732 ratherthan FOA + DuolsyveacloindfaitremdationanalyseswerenotconductedaccordingtotheGLregulations;analyticalmethodwasnot + Notallrawdatawereverfedbythegroupleader designee 4 Ze fb StuyDirector I= Feb -00 Cate Sponsor Representative Go 001175 Pase2e1t0 PaCtAuSoNruomocbaenre2s7u5t9o3n9s3ts asoratory ReIqMuEeRmesptiooNrtnummNboe.enrFt(AaRCLNTaMbTUoorZxaT-t1To0rS8y GLP Stupy QUALITY ASSURANCE STATEMENT SudyTile: APnearlfyutiocraoloLcatbaonreastourfyonRaetpeo(rPtoFOnSt)hein DtheetSeerumionfaotSfpitrohaenguPe-OarwieeyansRdalCesonEcnxepntorcsaetdieotno of Potassium Perfucroocianesufonate via Gavage `Study Identification Number: FACT Tox108 "tThheifsosltluodwyihngatsabbleee.Tnihnesfpiendcitnegdsbwyertheer3epMoErntveidrtontmhensttauldLyadbiorreacttoorraynQduamiatnyaAgsesmuernatn.caUnit (QA)as indicated in INSPECTION DATES Ee EET From 1230-99 To | 010600 Draft Final Report I 01-11-00 0107-00 | J QAU|Representative: 2-11-00 Date 001176 Page3otio rpeerpseermpns 1 Y nR a INTRODUCTION Oo CsFar isntSD semen CF Oo pos Perfluorooctanesulfonate (PFOS) Car emisors orneg F S m Purpose. r t my n ein ge sO deme0Sra -- HS ea s ag SHRNEHA ASS S TET f erstT Syse tem r sarE E nT yTe Sy ),E SA e pupT0%T Tra etreT S inTT sA r L S Shir e n Rsem dEeE0r2naes,arIeresheye t bye geenbneertmirmdSrhr), A gn setgeeraesrirtarennfsf0),hecutrdsee rgeswcessryt iie eGrpg oonee Table 1. FO Generation Rat Population Demographicsfor Study T-6295.13 E [oor raonr |arm |rT arer r |-- Gramma | [EoE nSEro|t weroa o| sm oa maaim ro arsss |zsro asnsg nrs| -- 001177 PCeAtSoNrusmobcearn2e7s5u9n3s9t3n MReEpnovritooNmoe.nFtALCaTboTaoixc1r0ay LaboracryRecues Nor(RNFUZTTS T`heCtesht sRiyavsetreLrmabsoipreactieoersiseasndnsctr.aipnesremlaencetnetdlwydaesntiheeCdHuLsCiOn'gEaRMoVnAeFlP"lsueslfiSeprracgnuee-aOratwaige.yF)Oragterneecreatiivoendfraotms wienrdoiicda etanggweitdt,eaiandftaafgesp.adr1agmeenteerrsatwironeaeivsawleuraetedeinteemdsoasfphaertoefrei(thdearmGraonudpi|oerrumbeersr,sbaurtawienrenot wthaecnrhemceenitv,edT(aWbeliegh4)t. dFaOavaigeinfnecmuadledratsAwrgeurseaRetseeaasrtc6h0Ladbaoyrsaotofraigese,aInncd,weinaglhreedpaoprptrfoorxsitmuadtyelTy-26002-6262.51o3n f`aldmaitnitshtee3rMeadrchheitveess).rMiacleeroatcosnosfitdehresedapmaerstoofutrchefaenstdssyisatenwm.ereusedoy sbreedersandwerenot `SampleColleacndtAnialoysins wFeOraencdol1leacnteidmarlosmastsheiFgnOemdatttehdefcemoanlrlapsooanwderFe1cpuullpesdoasnsdiagnyed1 ipotheophsaOtmndaapyc1oa4pksoursibtgpnratoruetpu.umO,snamcmapyl.2e2s poismtmpeairtteulmy,sfraomzpelneosnwderryeccoelalnecdtmeadirnotmaitnhedfreomzaeinnin(g70arCraorlbselnotwh)utnesttsshyisptepme.d tAotcheol3ecMiEendvsiaomnpmleenswtearie Laboratories. FIOn)thaenadntahteyicoaflissprtinygr(egepnoerrtaetdihoenrs,1s)ewrearseaamnpalleyszceodlfloercttheedpfrroemsehnecepoofpulPaFtOiSo.nof exposed amas(generation `TSheereaxstarmapcltsewsewerroeqeuxatnriaicatteedblyyanaialiyzdedquusiidnegxttruarcbtoiioonnpsrporcaeyduur duscihnrgoamnatoongraaiprhiynigmaagsesnstpaencdiertohmyeltayctate A(nLalMytSic)ailnsdeelteactaerd ionncmuondiecdrninigs(SrMep)omrtde,thaenrddetPaFOSaloevaevlaswlearieeevtalhuastteuddyagbaiinndsetremxatirnatcaiiendsetdabnydatrhdes.IM FisorneAnalytical Chemisty Team(FACT),hebinderislocated nthe 3Marchives. AinnacloymspetsaanscseewssiihngGtohoedpLraebosreantcoeraynPdracocnicceenRtergautliaontoifonPs(F2O1S iCnFtRh 56s)eaVaoifdSaptreagdumeetDhaovdseaynadtsstwaendraercdoonpdeurcattiendg procedureswee aloweddungthepreparaton andanalysisofthsamplesassociatedwihthissuc. SAMPLE RECEIPT o`fSasmplet sfwoermeu Nroevcee, imvbeedfro1m96A8r,ghuosuRegsheFeabrrcuhaLrayb,or1a9t6o9r,ieSsamspploersadrieccaayv,eddwuenrgaapnadcaktatgheeedninddoyftichee.iSnp-ecpihmaesnes wereregisteredwithhe 3M EnvormentalLaboratoryandtansieredtoa freezerfor storage. `LSaabmopralteosroifesb(Pordoytoicsoslue4s1a8n-0d14f,SdtoutyheTr6t2h9a5n,r1a3t)s,ebruutmwwereerneocotlplaerctoefdahnedsrceocpeievoefdfanoalmyAsrsguinshReepsroetaorccohl detebyrthmestiudyndireectdor. `StpheceimSeanmsplseeTntrtaockAidnvgaSnycsetdeBmioSatnaanldyatridcOalpeSreavtiicensg,Prnocc.ed(uArBeS)SpoercainmaleynssissweenrtetroeAcBeSivweidlabnedrreatcukmeeddactocohrediMng Eriednpevonrittir.foiSncpamteeincotni,amslteLonarsbawogirea,ltaobnerdymcUahpiaoninancioonfmepdcluesntttioohdneyopMfraonEoanclvoylissriosananmndeddnasttuaablmaLairbseosriaoctnaotorefytdshpeetchsiumbsectnoundarryacbchitinvadebesor.raSotraomnpiyls(esr)reepfcoearitlpt(,FACT Tax-108);thebinderi located the SM archives Page1s0 001178 m terTme woI enrmnmnEeSy ct MoATmEmRiItAm CLrSarAaNcDaiMaaEtTiioHnOnDSus rre heenshar r is ate.Cramton TarTetut ne Cor nd Ref CTomo e vommoenas| ten ECo oTorosermmmo| : re 001179 foOR remrce r bl. Cchacaonnsr ddTTasmie eern rTTeottunc Cotr nd tc TT pr------ ComeTousomat conse Te E hostel prpon ceadurr e in n ABa sSnfdrwa)rLanet podoa sret (Aate scehmesnt7) Cramer Physical Description and Light-colored powder T om ow] IdentityAnalyses SS oman tered serer sots a-- spen ec A eeCty e tte Se WaE nepFo n EbYeSeEeSarfaoenee, T t ethosse ummarien s t T, Trenrw eseay sn Ct 5eterr rirs E sApt IegTtht sc r oonglLeHsTSTg TS) r ten d SASae t oaet e S B a e en ed ee riS e e PaarSsTeeiseoTtnwrioy Es ran 001180 PCoArStNuaromsoca2n7s5o6r.3s8i3s SMREmieomepnFtArlCLTTaoobxo,10s8y LaborRecutMme LANHUTTD `EquiupsemdaetAnBSt: Lamssystem: MaAustsossapmecptlreormWeatetre:rPsE71S7CpIlEusXAPI 365withTubolonSprayTM intaface MoAbilnephaacsoellucmonmy:pKoenyisetnotcn:eBeatasliTM Ci, 2.1mxm(5Sy0m) ``CCoommppoonneenntt8A6--01-01900mmeetthhaanncoil22mmMMaammmmoonniiuummaaccestaattee ICnojelcutminontveomlpuemrea:tu3re5:4Ambient NeCburuaziangrags:asU:HUPHnPinroigreongen Cleocnsusptaayivnogtpoatgeen:ti-al3:00305V1 Nze=gioa4nsmfto9onrivPt8oFrOeeSd.: MH0 Tota/lruznti=me:f4o8r.02tmeirna7uHt.PesF0OS MH ProtocolDeviations "Therewerenodeviationsfom theorginalprotocal. DATA SUMMARY, ANALYSES, AND RESULTS `SummaryofQualityControlAnalyses Resuits(ABS) + Trhaenginetdefro-mass1a.yp5r7e4ci0s1io%n (oerlaFiOveSs,taannddathred dienvlira-tisosna-yRaScDc)udraatcayfr(oelmadivaeeyoqru--ayEc)orianlgesdafmoplmes 7.32-10.1%. Both areacceptable ranges. + aTchceuirnatceya(sRsEa)yfpormecsitsiaonnda(rRcSsDr)anofgesdtfarndoamr-ds5a8n6-g1e1d.f0o%.mAl1.a1r1e-a5c.c8e3p%tabolreFrOaSng.eTsheitr-sssay + Coefoffdetiermicnatiionweerne20t.99s54. + RueafletrytcootnhterAolBpSafrinaalvorelpuomret ((Aotnae-c0h-m4eennt .t,Toanbsl)e.2) orprecisionand accuracydataontheanalysesof FoS llowinu g iionm ffoSrmaatmm iponlreega aRredsinugr lstasm(ply AeBaSn)aysesinhepresentsi: The eSxmcaoslealdmepvdlotelhsuiemnceassiwnbaerlrayettocenaxltuarnnagcuetmaebndedfrouspmpt1eharenisdai3mpwolfeeqrsueaarnenatdsarsteaaoynnea(lbUyeLzCce)ad(u.fRseeearthstesoaTmayebealdsesias8meipndlacetostnrcaeacsnhitmreeadnitoi)nns maetaisauchrmeednctP)o. ncewnithtinrtheacattriatoonrnangse,and hedatavere reported(vert Table6in Originaldataorcopiesthere areavelabioatthe 3M EnvironmentalLaboratory. Paesc1t0 001181 CPAetSoNrmocbaenre2s7u5t5n3s9t3s MERveepoorntmeFrtaAl LaCTboorxaTt1o0r8y LaboratoryRecues Numer(LANHIZTTS STATISTICAL METHODS MaALcAiBnSt,odsahtcaomfrpoumtpee.aFkoalroewianswgepreeakianrteegariantteedgbrayttihoenP,EMaScCCIuEaXnprreosgurasmcaMtaacwQeuraecnoTMn,veverrtseidon1 t1.e4x,tolnesa,movedfo pePnrAo)f.AoBTnShmoeWdfloaetsnesUvonenfrao,nuaannyddseiadsnnaauplmpylbzieeerddsw:iahtwohewtiehvgeeiWrea,dtss(toa1nn)dTMaqsruodafdatrnwadatriqucarpieatgcyrkecsaosgnietorn(ovtldoetasthcandweare,raon5u.31.dn01fdcoeadmlPc0SattSeo,nesnwsce.inWriaefycannet, gurespriorto reporttheidanag. DATA QUALITY OBJECTIVES AND DATA INTEGRITY Nfoolorwuinmgsdtaatnacqeusaeitxyiosbtjeedcdtiuvensg(DhQeGpsre)sweenrtestouldoywtehadtdwuoriungthhaevsetaufdfye:ctedthequality oneg ofthedata.The + Linearity--The coefficientof determination (7) ofthe standardcurve wasequal oor greaterthan 0.98 + LisofQuaitation(LOQ)--Ecualto the owestacceptablestandardnth cabatoncurve Duplicateaccaptaieprecision-- <30% Spike acceptablerecoveies--70% 0 130% Uso ofconfimatorymethods--noconfimatorymethodswereused + Demonstrtonofspeccwaisdeymons--tratsedbypchroematocgrapihiciretenctionyme,massspectal {MK]proon dcharuacecrzattion AQasnsuaulmaysinensgt,ttthhetaaiotms+t3piv0ok%efe.dreTetcehocvteivroaynlt(LeOoDsf)tfifosorPmasFsaOusSmupltsaiabopinpehrioansdsiicnmaoattitoebnleoyef1n.ev7ne5dnrogtg/eenndLob.auysncdathnehreaterlcihtrneoicqfouuveaesnr.ytI,iGasatteiaronuamr(eLOC) 5 005pgimi. PEGS. STATEMENT OF CONCLUSION StatoefCmoncelunsiotn: Uinndfeorpthhaesceonodfitthoenssuocfyt.hAedpdrietsieonntalt,uPdeFsO,SPwFaOsoSbwsaesrovbesdeirnvseerdainStshseuseesrataokfeFnOffoemmaFl1e gratesenxeporspeaudptsdiufroinmgnthe fbaumtanoterxatpsoesxepdosnedu1r0.heestsubstance,andin 1 generation ppsexposed th lstsubstancevia aciaton, Pagesatio 001182 CPAeoSrNsoomcbaenrs2s7s6t9o3n5s3e Lasers WMquEResmtvcemremraFgnA(ClLLTNoaHTboUorZax.Tt1oT0rSy8 REFERENCES Nore. ATTACHMENTS + AtachmentA:SampleReceiptandChainofCustodyDocumentation + Atach8:mPeronlotcs AttachmenCt: ExtarndaAnaclytticailMetohonds + AtachmenDt: Dose ConfimatonAnayses + AtiEa:DactaShummmaryeandnTabtles AttachmenFt:AnalyticalReports fromContiouingLaboratories + AtachRepmoretSingnatturGePa:ge Page 1010 001183 PCoHrStuNormobcenra2s7u5t5o3n5s3s [A`-- RepFAaCTtTooxi.0h Laboratory RequestNumber(LNWIZTTS ATTACHMENT A `SAMPLE RECEIPT AND CHAIN OF CUSTODY DOCUMENTATION `bSianmdpelrerselcoeciapttedanndtchhaiIeMnoafrccuhsitveosd.yinformation is documentedinthe binderfor 1sreport(FACT Tox 108):he. 001184 Pe(tCAuSoNrucmocbaenre2s7ut5o8ns9t3e ATTACHMENT B PRroTOCOL aEniveeon.meniPaAlCaTbToourttch Labiratory RequestNumber(LRNMUZTTS 001185 ! n2a M ESnevrviiccoensmental|Technology S ty ph a, Bek _'-- Protocol HFACT-TOX-108 Study Title Oral (Gavage) Cross-Fostering Study of PFOS in Rats PROTOCOL Author Lisa Clemen Date: June 8, 1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue . St. Paul, MN 55106 Laboratory Project Identification wr FACT-TOX-108 IM Environmental Laboratory page 1010 001186 Protocol HFACT-TOX-108 Study Identification Oral (Gavage) Cross-Fostering Study of PFOS in Rats Test Material Perfluorooctane sulfonic acid potassium salt (T6295) Sponsor 3M Toxicology Services - Medical Department 3M Center, Building 220-2E-02 St. Paul, MN 55144-1000 Sponsor Representative Marvin T. Case, D.V.M., Ph.D. 3M Toxicology Services Telephone: 651-733-5180 Facsimile: 651-733-1773 Study Director Kristen J. Hansen, Ph.D. 3M Environmental Technology and Safety Services Building 2-3E-09 651-778-6018 Study Location(s) In vivo Testing Facility Analytical Testing Laboratory Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, PA 19044 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St.Paul, MN 55106 3MEnvironmentalLaboratory Page2o1l0 009187 Sub-Contract Laboratory Proposed Study Timetable Study Initiation Date Study Completion Date . Protocol #FACT-TOX-108 Advanced Bioanalytical Services, Inc. 15 Catherwood Road Ithaca, NY 14850 Battelle Memorial Institute 505 King Avenue Columbus, Ohio 43201-2693 . June 08, 1999 June 08, 2000 1. Stuoy' Oral (gavage) cross-fostering studyof potassium perfluorooctane sulfonic acid (PFOS) in rats. 2. Purpose "This analytical study is designed to determine levelsofpotassium perfluorooctanesulfonate (PFOS) in specimens ofliver and serumofrats. The in-life portion ofthis study was conducted atArgus Research Laboratories, study #418-014. All serum samples will be extracted and analyzed at Advanced Bioanalytical Services, Inc. and all liver samples extracted and analyzed at `Battelle Memorial Institute. Additional analyses maybe performed at the 3M Environmental Laboratory as methods are developed and validated. If additional analyses are performed an amendment to this protocol will be written. 3. REGULATORY COMPLIANCE : `This study will be conducted in accordance with the United States Food and Drug Athdamtinainsatlrysaitsioofnt,hGeootedstLmaabtoerraitaolrmyiPxrtaucrteicfeosrSctoanncdeanrtdrsa,tiFoinn,alsoRluublieli2t1y,ChFomRog5e8n,ewiittyh,tahnedexstcaebpitliitoyn `will not be conducted, and is the responsibilityofthe Sponsor. 4. QUALITY ASSURANCE The 3M Environmental Laboratory Quality Assurance Unit will review the protocol and audit study conduct, data, and final report to determine compliance with Good Laboratory Practice Standards and with 3M Environmental Laboratory Standard Operating Procedures. The QA Unit at the sub-contract laboratory will audit their study conduct, data, and results report prior to submitting to the 3M Environmental Laboratory. 3M Environmental Laboratory Page3ofto 001188 Protocol HFACT-TOX-108 5. TestMareriaL 51 Referto Argus Research Laboratory protocol for study #418-014. 6. CONTROL MATRICES 6.1 Identification Rat liver and serum and/or rabbit liver and serum, traceability numbers will be recorded in the raw data andincluded in the final report 6.2 Source Argus Research and/or Sigma Chemical 6.3 Physical Description Rat liverand serum and/or rabbit liver and serum 6.4 Purity and Stability Not applicable 65 Storage Conditions Frozen at -20 C 10 C or 50 C: 10C 6.6 Reserve Matrix A portionofthe control matrix will be retained in the 3M archives for as long as the quality of the preparation affords evaluation, but not longer than ten years following the effective dateofthe final test rule(ifapplicable). 6.7 Disposition Matrices will be retained at the 3M Environmental Laboratory per GLP regulation. Certain matrices (feces, urine, and blood) may be disposed after QAU verification. 6.8 Safety PrecautionsReferto MSDS for chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. 7. REFERENCE MATERIAL 7.1 Identification Potassium perfluorooctanesulfonate (PFOS), lot #s 171, 215, or 217 (equivalent lots) i 7.2 Source 3M Specialty Chemicals 7.3 Physical Description White powder 7.4 Purity and Stability Purity ofPFOS is 99% or greater. Stability has not been determined. L 7.5 Storage Conditions Room temperature 7.6 Reserve MaterialA reserve sample from each batch ofPFOS used in this study will be retained as long as the qualityofthe preparation affords evaluation, but not longer than ten years following the effective dateofthe final test rule (ifapplicable). 7.7 Disposition Unused reference material will be retained for use by the 3M Environmental Laboratory and will be discarded when the qualityofpreparation no longer affords evaluation. aM Environmental Laboratory Page 40f 10 001189 Protocol #FAGT-TOX-108 7.8 Safety PrecautionsReferto MSDS for chemicals ised. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. 8. Test System Female rats were used as the test system and were maintained and dosed as described in Argus Research protocol #418-014. Group 1 control animals did notreceivethe test substance while the group 2 treated animals received the test substance at a concentration of 1.6 mg/kg/day for 6 weeks. Refer to Argus Research protocol #418-014 for tabular presentationofdata. Dosing of the female animals in group 2 was continued during mating, gestation, and lactation. Crossfosteringof the pups born to the control dams were randomized,halfof the pups were placed on control dams while the otherhalfwere placed on the PFOS dosed dams. Likewise, the same was done with the group 2 pups. Eachofthese pups will be marked to identify whether they are from control or PFOS dosed dams. 9. SPECIMENAND SAMPLERECEIPT `The 3M Environmental Laboratory will receive homogeneity samples and specimens ofthe following body tissues and fluids from the indicated points in the study. All specimens will be packed on dry ice for shipping. Body tssue/iuid Collected Serum -- Dam and Pup animals Urine and feces - Dam animals Tive-r Dam and Pup animals `Milk Secreting Glands - Dam animals Milk --Damanimals | At lactation day 14 and terminationofthe study | After terminationofthe study | At lactation day 14 and terminationofthe study | At lactation day 14 and terminationofthe study "Atlactatidoayn 14 Expected # of specimens 60 Dam and 34 Pup | 48 Urine and 48 Feces 0 Dam and 34 Pup mo Total numberofexpected specimens: 352 `Total numberoftest animals: 36 `Total numberof control animals: 42 Specimens sent to 3M Environmental Laboratories will be received and tracked according to applicable Standard Operating Procedures. 3M Environmental Laboratory pagesorio 001190 Protocol HFACT-TOX-108 10. PREPARATORY METHODS : 10.1 FACT-M-L.1, Extraction of Potassium Perfluorooctanesulfonaotre Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 10.2 ETS-8-4.1, Extraction of Potassium PerfluorooctanesulfonateorOther Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLCElectrospray/Mass Spectrometry 10.3 Ifpreparatory methods other than those listed above are used, an amendment to this protocol will be written. Any deviations from these methods will be documented and included with the study data. + 10.4 Ifanalyses are sub-contracted to other laboratories, an amendment will be written to include their methods and copiesofeach method will be attached to this protocol. 11. ANALYTICAL METHODS 11.1 FACT-M-2.1, Analysisof Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry 11.2 ETS-8-5.1, Analysisof Potassium Perfluorooctanesulfonate or Other Fluorochemicals in SerumorOther Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry 11.3 Ifanalytical methodsotherthan those listed above are used, an amendment to this protocol will be written. Any deviations from these methods will be documentedand. included with the study data. 11.4 Ifanalyses are sub-contracted to other laboratories, an amendment will be written to include their methods and copiesofeach method will be attached to this protocol. 12.DATA QUALITY OBJECTIVES `The numberofspikes/duplicates, useof surrogates, and information on other data quality indicators are included in the analytical methods. In addition, the following criteria will be met: 12.1 Linearity 2098 12.2 Limits of detection/ quantitation 12.2.1 Method Detection Limit (MDL) for PFOS a) Sem: 1.75ppb b) Liver 15ppb 12.2.2 Limit of Quantitation (LOQ) ~ Equal to the lowest acceptable standard in the calibration curve 3M Environmental Laboratory Page 6af 10 001191 Protocol #FACT-TOX-108 12.3 Duplicate acceptable precision <30% for the method 12.4 Spike acceptable recoveries 70% - 130% 12.5 Useofconfirmatory methods Indeterminate samples will be re-analyzed using a confirmatory method. If a confirmatory method is used, an amendment to this protocol wil be written. 12.6 Demonstration ofspecificity Chromatographic retention time, mass spectral daughter ion characterization. 13. SUB-CONTRACTED ANALYSIS 13.1 All analyses as detailed in this protocol will be performed at 3M Environmental Laboratories, Building 2-3E-09, 935 Bush Avenue, St. Paul, MN 55106, at Advanced Bioanalytical Services, Inc., 15 Catherwood Road, Ithaca, NY 14850, or at Battelle Memorial Institute, 505 King Avenue, Columbus, Ohio 43201-2693. 13.2 An amendment tothisprotocol will be writtenifanalyses are performed at laboratories other than 3M Environmental Laboratories, Advanced Bioanalytical Services, Inc., or Battelle Memorial Institute. 14. STATISTICAL ANALYSIS Averages and standard deviations will be calculated. The statistical methods that will be used are described below: 14.1 Data transformations and analysis Data will be reported as the concentration (weight/weight or weight/vol)ofPFOS or metabolite per tissue or fluid. 14.2 Statistical analysis Statistics used may include regression analysis of concentrations over time, and standard deviations calculated for the concentrations `within each dose group. Ifnecessary, simple statistical tests, such as Student'st test, `may be applied to evaluate statistical difference. 15.RePoRT A report containing all the resultsofthe study will be prepared by the 3M Environmental Laboratory.Ifanalyses are sub-contracted to other laboratories, each laboratory will prepare a report and submit it to the 3M Environmental Laboratory for inclusion in the 3M Environmental Laboratory report. Each report will include, but not be limited to, the following, when applicable: 15.1 Name and addressofthe facility performing the study 15.2 Dates upon which the study was initiatedand completed 15.3 A statementofcompliance by the Study Director addressing any exceptions to Good Laboratory Practice Standards : 3M Environmental Laboratory Page 70110 001197 Protocol #FACT-TOX-108 15.4 Objectives and procedures as stated in the approved protocol, including any changes in the original protocol 15.5 The test substance identification by name, chemical abstracts number or code number, strength, purity, and composition or other appropriate characteristics,if provided by the Sponsor 15.6 Stability and the solubility ofthe test substances under the conditions of `administration, if provided by the Sponsor 15.7 A descriptionofthe methods used to conduct the test(s) 15.8 A description ofthe test system 15.9 Adescriptionofany circumstancesthatmay have affected the quality or the integrity of the data 15.10 TsuhpeernvaimsoeorfytpehresoSntnuedlyiDnivroelcvteodr ianntdhtehsetnuadymesofother scientists, professionals, and 15.11 A description ofthe transformations, calculations, or operations performed on the. data, a summary and analysisofthe analytical chemistry data, anda statementofthe. conclusions drawn from the analyses. 15.12 Statistical methods used to evaluate the data, if applicable 15.13 The signed and dated reports ofeachofthe individual scientists or other professionals involved in the study, ifapplicable 15.14 The location where raw dataand the finalreportare tobe stored 15.15 A statement prepared by the Quality Assurance Unit listing the dates that study inspections and audits were made, and the datesofany findings reported to the Study Director and Management Ifit is necessary to make corrections or additions to areport after it hasbeen accepted, the changes will be made in the formofan amendment issued by the Study Director. The amendment will clearly identify the partofthe report that is being amended, the reasons for the amendment, and will be signed by the Study Director. 16. LOCATIONOFRaWDATA, RECORDS, AND FINAL REPORT Original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate auditsofthe study during its progress and before acceptanceofthe final report. When the final report is completed, all original paper data, including those items listed below, will be retained in the archives of 3M Environmental Laboratory for at leastaperiodoftime as specified bPryorceegduulraetsi.on, and as established by 3M Environmental Laboratory Standard Operating 3M EnvironmentalLabarstory Pagosorro 001193 Protocol HFACT-TOX-108 16.1 The following rawdataand records will be retained in the studyfolderin the study/project archives according to 3M Environmental Laboratory Standard Operating Procedures: 16.1.1 Approved protocol and amendments 16.1.2 Study correspondence 16.1.3 Shipping records 16.1.4 Raw data 16.1.5 Approved final report (original signed copy) 16.1.6 Electronic copies ofdata * 16.2 The following supporting records will be retained separately from the study folder in the archives accordintog 3M Environmental Laboratory Standard Operating Procedures: 16.2.1 Training records 16.2.2 Calibration records 16.2.3 Instrument maintenance logs 16.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 17. SPECIMEN RETENTION Spepreicoidomefntsimweillasbespmeaciinfiteadinbeydriengutlhaet3ioMn oErnvaisrloonnmgenatsatlheLaqbuaolriattyoorfytshepecpriempeanraatricohnivaefsfofrodrsa `evaluation, but not longer than ten years following the effective date ofthe final test rule (if applicable), and as established by 3M Environmental Laboratory Standard Operating Procedures. 18. PROTOCOL AMENDMENTS AND DEVIATIONS | 5 Planned changes to the protocol will be in the formofwritten amendments signed by the Study Director and the Sponsors Representative. Amendments will be considered as partofthe protocol and will be attached to the final protocol. All changes totheprotocol will be indicated in the final report. Any other changes will be in the formofwritten deviations, signed by the Study Director and filed with the raw data. 3M Environmental Laboratory Page 9 of 10 001194 19. ATTACHMENTS E 19.1 AttachmenAt Preparatory and analytical methods 20. SIGNATURES Protocol HEACT-TOX-108 I Cu Marvin T. Case, D.V.M., Ph.D., Sponsor Representative 7 June [772 Date Lew &/19/: Kristen J. Hansen, Ph.D., 3M Environmental Laboratory Study Director Date 3MEnvicnmentalLaboratory Page 1001 10 001195 Study Title Oral (Gavage) Cross-Fostering Study of PFOS in Rats PROTOCOL AMENDMENT NO. 1 Amendment Date: August 12, 1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX108 LRN U2779 3M Environmental Laboratory 001196 ProtocAomleFnAdCmTe-nTtOXNo1.081 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: Section 2.0 states that all liver samples will be extracted and analyzed at Battelle Memorial Institute. AMEND T0 READ: All liver samples will be extracted and analyzed at the 3M Environmental Laboratory. REASON: Due to time constraints, the liver extraction and analysis will be performed at the 3M Environmental Laboratory. 2. PROTOCOL READS: Section 10.0 and 11.0 lst the following methods to use for extraction and analysis: FACT-M-1.1 "Extractionof Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" FACT-M-2.1 "AnalysisofFluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" AMEND TO READ: The extraction and analytical methods to follow at the 3M Environmental Laboratory are: ETS-8-6.0 "Extractionof Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 "Analysisof Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" REASON: The extraction and analytical methods FACT-M-L.1 and FACT-M-2.1; respectively, were updated on 07/22/99 to ETS-8-6.0 and ETS-3-7.0. Thesemethodswere updated to replace the extraction solvent ethyl acetate with a different extraction solvent MTBE (methyl tert butyl ether), POAA and Moncester were removedfromthestandardmix, and MSS6 was added to the standard mix. The analytical method was updated to include linear regression with 1/x weightaindnag few `minor changes inthe HPLC 1100 instrument parameters. aM Environmental Laboratory 001197 Protoc"olaFmAeCnTd-mTeoNXno1t.081 3. PROTOCOL READS: Section 10.4 and 11.4 state thatifanalysesaresub-contracted to other laboratories an amendment will be written to include their methods. AMENDTO READ: The extractionandanalytical methods to follow at Advanced Bioanalytical Services will be attached to the protocol. "prRoEtAoScOolN: The analytical methodsatthe sub-contractlaboratorywerenot included in the original 4. PROTOCOL READS: Section 12.2.1 b) lists the liver method detection limit as 15 ppb. AMEND TO READ: The liver method detection limit is 8.50 ppb (ng/g). REASON: The validation supporting methods ETS-8-6.0 and ETS-8-7.0 includes a lower method detection limit for PFOS. Amendment Approval = Nh frre TG Marvin Case, D.V.M., Ph.D., Sponsor Representative 17 hussnt 1920 Date LL. bo Kris J. Hansen, Ph.D., Study Director 5/15/95 Date aM Environmental Laboratory 001198 Study Title Oral (Gavage) Cross-Fostering StudyofPFOS in Rats PROTOCOL AMENDMENT NO. 2 Amendment Date: September 30, 1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory : 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX108 LIRN U2779 3M Environmental Laboratory 001199 ProtocAomleFnAdCmTe-nTtOXNo1.028 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: Section2.0 statesthatall liversampleswill be extracted and analyzed at the 3M Environmental Laboratory. AMEND 70 READ: No liver samples will be extracted and analyzed. Reason: Liver results are no longer required. 2. PROTOCOL READS: Section 16 statesthatthe original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate auditsofthe study during its progress and before: acceptanceofthe final report. When the final report is completed,all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, electronic copies ofdata, training records, calibration records, instrument maintenance logs and standard operating procedures, equipment procedures, and methods will be retained in the archivesofthe 3M Environmental Laboratory. AMEND TO READ: Section 16 states that the original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate auditsofthe study during its progress and before acceptance of the final report. When the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, and electronic copiesofdata will be retained in the archives of the 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procdures, and `methods will be retained in the archivesofthe facility performing each analysis. REASON: Clarificationofthe dispositionofarchived recordsifanalyses are performed at a sub-contract laboratory. 3M Environmental Laboratory 001200 ProtocaomleFnAdCmTe-nTtOXN1o0.82 3. PROTOCOL READS: Section 17 states that specimens will be maintaiinntehde 3M Environmental Laboratory specimen archives. AMEND TO READ: Specimens will be maintainedinthe 3M Environmental Laboratory specimen archives. All specimens sent to sub-contract laboratories will bereturnedto the 3M Environmental Laboratory upon completion ofanalysis and submissionofthe sub-contract laboratory(s) final report. Thespecimens will be returned with the following documentation: the signed original chain ofcustody and records ofstorage conditions while at the sub-contract facility. REASON: Clarificationofthe dispositionofspecimens and documentation for analyses performed by a `sub-contract laboratory. Amendment Approval Marvin Case, D.V.M., Ph.D., Sponsor Representative fib ff Kristen J. Hansen, Ph.D., Study Director Date: 10/5/99 Date 3M Environmental Laboratory 001201 PCerAtSaNroomcbaorres2s7t6o5n3s5t3s borat ReEenmaovnioueoPsAs(eLGRaNbsToUor2at71t73r9sy T-- AETTTACHeMeENT C -- A ---- ee------ EXTRACTION AND ANALYTICAL METHODS ProprietaryandContent 001202 |L.] ADVANCED BIOANALYTICAL SERVICES, INC. METHOD VALIDATION REPORT TITLE: METHOD VALIDATION FOR THE QUANTITATION OF RPAETRFSLEUROURMOBOYCTTAUNREBSOUILOFNOSNPARTAEY(LPCFO/SM)SIN ! DATE: 26 August 1999 REPORT: 99VDJA0L.MLDOC AUTHORS: PREPARED FOR: NUMBER OF PAGES: David J. Anderson, M.S. Amie J. Prince, B.S. Holly D. Ross, M.S. 3M Environmental Technology and Safety Services St. Paul, MN 55133-3331 50 001203 15 Catherwood Road = Ithaca. New York 14850 (607) 266-0665 Fax (607) 266-0749 AUTHORS: FOR: DATE: TITLE: David J. Anderson, M.S. `Amie J. Prince, B.S. Holly D. Ross, M.S. 3M Environmental Technology and Safety Services 26 August 1999 METHOD VALIDATION FOR THE QUANTITATION `OF PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/MS ABSTRACT A sensitive, specific, accurate, and reproducible analytical method was developed by Advanced BioAnalytical Services, Inc., Ithaca, New York to quantitate perfluorooctancsulfonate (PFOS) in rat serum samples. Serum samples (50 uL) were extracted by a liquid-liquid extraction procedure to isolate the analyte from rat serum. Sample extracts were reduced to dryness, reconstituted, and analyzed by turbo ion spray liquid chromatography/mass spectrometry (LC/MS) in the negative ion mode. The assay demonstrated a lower limit of `quantitation (LLQ)of 0.05 pg/mL using 50-uL sample aliquots. The calibration curves were fit from 0.05 pg/mL to 20 pg/mL for PFOS by a weighted (1/y') quadratic equation. The coefficients of determination ofthe calibration curves ranged from 0.9962 to 0.9965. Precision and accuracy quality control (QC) samples were prepared at concentrations 1 00.2, 6, and 18 pg/mL PFOS. Quality control (QC) samples were prepared at a concentration of 100 g/mL PFOS for partial volume analysis. The intra- and inter- ! assay precision (RSD) results calculated from all QC samples ranged from 1.92% to i 487% for PFOS. The intra- and inter-assay accuracies (RE) calculated from QC ! samples ranged from -3.58% to 5.58% for PFOS. The mean extraction recoveries were from 87.6% to 100% for PFOS and 89.9% for the internal standard as). PFOS was measured as stable inrat serum for up to 24 hours at ambientemperature. PFOS - was measured as stable in rat serum at ~20 C, currentlyviene for up to 33 days, and man ABONvAENCTED SERVICES, INC. Prgezors ssvoimo1 m00C 001204. . , -- rafetienrjetchtreede2f7reheozuer/sthaafwtecryicnlietsi.al Rreelcoinasbtlieturteisounl.ts were obtained for sample extracts IM ASDoViANnCsEDca SERVICES. INC. Page 3of30 001205 99VDIAOLMLDOC QAU STATEMENT Periodic inspectionsofthe method validation for the quantitation of PFOS in rat Sseerrvuimcewser(eABcSo)ndfuocrtceodmbplyitahnecQeuwailtihtyEAPsAsuGraLnPcereUgnuiltatoifonAsdv(4a0ncCeFdRBiPaorAtna7l9y2t)i.cal The study was inspected on the following dates: 13,21 May 1999; 7,8, 14,15 June 1999;1, 2 July 1999;3, 4 August 1999. Resultsofthe inspections were reported to ABS Management on: 13,21 May 1999; 7,8, 14, 15 June 1999;1,2 July 1999; 3, 4 August 1999. Resultsofthe inspections were reported to the Study Director on 5 August 1999, Based on the inspections and the data reviewed, this report is a complete and accurate representationofthe data. Hak Cormack, B.S. Date Quality Auditor . --_-- MW ASov2an5ce0n , SeRvICES. INC Page sorso 001206 OO ssvoIROILMIDOC TITLE: Report Number: Baoedins SIGNATURE PAGE METHOD VALIDATION FOR THE QUANTITATION OF PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/MS 99VDIA0L MLDOC David J. Andegfon, M.S. Research Scientist 24, hutusT 1979 Date Reviewed by: + 1 (2 t ithleen Cormack, B.S. Associate Auditor 2644299 Date Authorized for Release by: ofr-- John R/Perkins, Ph.D. Assistant Scientific Director 2 1g ar.55 Date 001207 s-- ec-- aaovanc-- en ---------- Mm Services,GLIK.E PagSeof 0 99VDIAOLMLDOC TABLE OF CONTENTS QAU STATEMENT wots A SIGNATURE PAGE worsens TAOFBCONLTENETS w.covssssssssnsnsomssssonn | LOIFTAS BLEST ... LIST OF FIGURES..coesrseresnssssssssmssmssssssssmssmssmssssssssd i 1 INTRODUCTION ccna 10 2. EXPERIMENTAL sss 10 2.1. CHEMICALS ANDMATERIALS tse 10 2.2. LC/MS INSTRUMENTATION ccs 10 2.3. SAMPLE PREPARATION AND EXTRACTION PROCEDURE crv | 1 2.4.RATSERUM VALIDDAATTA ION rss11 2.5. ASSAY EVALUATION cocoon 12 | 2.5.1.Intra:andINEr-ASSaY ACCIACY vores 12 i 2.5.2. Intra: ad INMET-AS5aY PIECISION corse 12 2.5.3. Partial VOIMEARBLYSIS costs 12 H 2.5.4. Lower Limit of QUANKation (LLQ) errs 3 2.5.6. CaITYOVEr EVANRHON co. 13 2.6. STABILITY OF PFOS IN QUALITY CONTROL SAMPLES crc 13 26.1. Ambient-Temperature Stability of PFOS in Rat Serum... 13 2.6.2. Freezer Stability of PFOS in Rat Serum at ~20 *Cooercrrrrrcnics 14. 2.6.3.Freeze/Thaw Stability inRat Serum... 1 27. REPRODUCIBILITY OF REINJECTING EXTRACTED SAMPLES... 14 2.8. EXTRACTION RECOVERY consists 14 3. RESULTSAND DISCUSSION wcorrnrnnssnnivses15 LL. [LEE 001208 3.1. ASSAY EVALUATIONRESULTS cosmo 16 3.1.1. Intra: and Inter-ASSay ACGUTRY corre 16 -_-- ABIDOVAANNACLYETDICAL Pagesof 0 99VDIADLMLDOC 3.1.2. Intra and Inter-ASSay PRECISION ccm 16 3.14. Partial VOTE ARBIYSIS....cororssseosssssonronsn 16 3.1.5. Lower Limit ofQUANGatON (LLQ) wrens 17 3.1.7. CAITYOVEr EVAIAUON ccm|] 3.2. STABILITY OF PFOS IN QUALITY CONTROL SAMPLES ccm 17 3.2.1. Ambient Temperature StabilityofPFOS in Rat Serum .........oov......17 3.2.2. Freezer Stability of PFOS in Rat Serum at ~20 C 18 3.2.3. Freeze/Thaw Stability of PFOS in Rat Serum... 18 3.3. REPRODUCIBILITY OF REINJECTING EXTRACTED SAMPLES crc 18 3.4. EXTRACTIONRECOVERY ws18 4: CONCLUSIONS... 19 5. DATARETRIEVAL ums: 19 6. REFERENCES... 19 9. (APPFPOESN)DIINXRAA:TQSUEARNUTMITBAYTTIUONRBOOFIPOENRSFPLRUAOYROLOC/CMTSANr ESUo LFOc NAT. E 37 e--e------------------------------------------ LE ADVANCED Page 050 SHVDIAILMIDC SERVICES. INC. 001209 LIST OF TABLES i Table I: Intra-Assay Accuracy and Precisionofthe PFOS Assay in Rat Serum for i Table 2: IQntCerS-aAmspsalyesAcfcruormaTchyraenedVaPrleicdiastiioonn of the RUDS PFOS Assay in Rat Serum creer for 21 Table 3: ASctcaunrdaarcdysafnrdoPmreTchirseieonVoaflitdhaetiPoFnORSUDASss.a.y.i.n.R.a.t.SeewreurmofroromCanlmibormatsieon 22 Table 4: Calibration Curve Parameters for PFOS in Rat SERUM vcore. 23 Table S: Partial Volume Analysis of POS in Rat SERUM... 26. Table 6: Lower Limit of Quantitation of POS in Rat Serum... 25 Table 7: Ambient-Temperature Stability of PFOS in Rat Serum After 24 Hous........26 Table 8: Freezer Stability of PFOS in Rat Serum at -20 C cvs 27 Table 9: StabilityofPFOS in Rat Serum After Three Freeze/Thaw Cycles ..............28 Table 10: R2e7prHooduurcsibiinliRtEyCoOfNRSeHitnjUeOcNtiSngOEMxtHraOctNed.Sa.m.pl.es.C.onrtaeirnirngrPrFOeSrasftesr n 29 Table 11: Extraction Recovery of PFOS OM Rat SEM... 30. re eertte ememeeeeer eeeeeee tment Mm ADSIVDAANNACLEYDTICAL Page 8 of SO 99VDJAOLMLDOC 001210 SERVICES. INC. LIST OF FIGURES 1 Figure 1: Full-Scan Single MS Mass Spectrum OfPFOS ovr 31 Figur2e: Full-Scan Single MS Mass Spectrumof Tetra-H-PFOS cc. 3 Figure 3: Mass Chromatograms ofPFOS and its Internal Standard in Control Blank Figure 4: MIantsesrnCalhrSotmaandtaorgdraOmnslyof(ZPF70OSSAinDaIRaEt)S.er.u.m Extract Sample Containing 34 Figure 5: MCaosntsaCinhirnogmaPtFoOgSra(m0s.o05fCpagl/imbLr)atainodn Standard 1 the Internal in Rat Serum Extract Standard .....................35 Figure 6: Mass Chromatograms Containing PFOS (20 of Calibration Standard pg/mL) and the Internal 10 in Rat Sandard Serum Extract wr... 36 -_-- am aN ovaS ncenS SERVICES. INC. Page9ofsn 59VDIAGLMLDOC 001211 1. INTRODUCTION The purposeofthis report is to describe the method validation for the quantitative determination ofperfluorooctanesulfonate (PFOS) in rat serum samples after modificationof a previous method (1). The objectives of the method validation were to validate a simple extraction procedure, determine the lower limitof quantitation for routine analysis, determine the extraction recoveries of the analyte and internal standard (IS), determine the analyte stability in rat serum at ambient temperature, long-term freezer storage, and over three freeze/thaw cycles, and to provide specific, accurate, and reproducible quantitative results by turbo ion spray liquid chromatography/mass spectrometry (LC/MS). 2. EXPERIMENTAL 21. CHEMICALS AND MATERIALS Periluorooctanesulfonate (PFOS, Lot# 171) was obtained from 3M, Inc. The internal standard (IS) for PFOS was 1H,1H,2H,2H-perfluorooctane sulfonic acid (Tetra-HPFOS). The intemal standard (Loti $9909) was obtained from 3M, Inc. A detailed listof chemicals and materials is found in Appendix A. Stock and working solutions which were used to prepare the calibration curves for fe the analytes were prepared as described in Appendix A. Stock solutions used in the preparationofquality control (QC) samples were prepared separately from those used in preparationof the calibration curves. Preparationofall solutions used during extraction and analysis are described in Appendix A. 22. LC/MS INSTRUMENTATION `The liquid chromatography/mass spectrometry system consistedof two LC-10AD pumps (Shimadzu, Columbia, MD 21046), a SCL-10A pump controller (Shimadzu, M2 ADV0ANCEDea SERVICES. INC fase 100r0 59VDIAOL MLDOC 001212 Columbia, MD 21046), a WISP 717plus autosampler (Waters Associates, Millipore Corporation, Milford, MA 01757), a Betasil C,q (2 x 50 mm, 5 um) column (Keystone Scientific, Inc., Bellefonte, PA 16823), and a PE SCIEX API 365 mass spectrometer (PE SCIEX, Concord, Ontario). A detailed listof the instrumentation ! and instrument conditions is found in Appendix A. 2.3. SAMPLE PREPARATION AND EXTRACTION PROCEDURE For each analytical run (tray), duplicate 50-uL aliquots of the calibration curve samples were prepared as described in Appendix A. The nominal (theoretical) concentrations of PFOS in the calibration curves were 0.05, 0.1,0.25,0.5,1,2,4,8, 16, and 20 pg/mL. Rat serum quality control samples were prepared in advanceofthe validation study at nominal (theoretical) concentrations of 0.2, 6, and 18 pg/mL for QC1, QC2, and QC3, respectively, as detailed in Appendix A. A dilution QC (QC4, 100 pg/mL) was prepared at a concentration exceeding the upper limit of the calibration curve range (20 pg/mL), and was assayed using a 10-fold dilution for partial volume analysis. Calibration standards and QC samples were extracted by the procedure detailed in Appendix A. 2.4. RAT SERUM VALIDATION DATA "The data were collected using selected ion monitoring (SIM) turbo ion spray LC/MS in the negative ion mode. Peak areas were integrated by the PE SCIEX program MacQuan, version 1.4, residing on a Macintosh computer. Following peak area b integration, the results tables from MacQuan were saved as text files and uploaded to the Advanced BioAnalytical Services (ABS) file server where a weighted an) quadratic regression was performed using the software package Watson v 5.3.1.01 (PSS, Inc., Wayne, PA 19087). All data were rounded to no less than three significant figures by ABS prior to reporting in Tables 1 through 11. The data for em the rat serum validation are stored in ABS Notebook 2304. mn spe gett ot50 Services. nC o0m13| soviAol MLC Al calculations were based on the peak area ratioofthe analyte to the intemal ik standard. Concentrations ofeach analyte in quality control samples were determined by inverse prediction from the calibration curve. 25. ASSAYEVALUATION 251. Intra and Inter-Assay Accuracy The intra- and inter-assay accuracy ofthe method was assessed by determining the relative error observed in the analysisof quality control samples. The mean concentration for each quality control level was divided by the theoretical concentration. One was subtracted from the result, converted to percent and expressed as RE. QC through QC3 were assayed in replicatesoffive. In addition, the RE was reported for standards at all levels over three runs. 252. Intra- and Inter-Assay Precision `The intra- and inter-assay precision of the method was assessed by determining the Relative Standard Deviation (RSD) observed for quality control sample data. The `mean concentration and RSD were calculated for the first run and over three runs for intra-assay and inter-assay precision, respectively. QC1 through QC3 were assayed in replicatesoffive. In addition, the RSD was reported for standards at all levels over thre runs. 2.53. Partial Volume Analysis The effectofdilution on the analysis of PFOS in rat serum was determined by partial volume analysis. QC4 (100 pg/mL) was prepared containing PFOS at approximately five times the upper limit of quantitation (ULQ). Five replicates of QC4 were diluted ten-fold and analyzed. The RSD and RE were reported. - mam s ADVo ANCmED SERVICES. INC Page 120050 002s 99VDIA0LMLDOC | 254. Lower Limit of Quantitation (LLQ) Rat serum control samples from six separate individuals were spiked with PFOS ata concentrationof 0.05 g/mL. The LLQ was determined by obtaining the backcalculated concentrations from these control samples. The overall mean, RSD, and RE were also calculated. 255. Selectivity The selectivity of the assay was determined by LC/MS. To monitor for interference 3 from the biological matrix, rat serum samples containing neither the analyte nor the : internal standard (control blank) were assayed with all experiments. i 256. Carryover Evaluation b `The carryoverofanalyte from one injectionto the next was assessed by analyzing a control blank injected immediately after a high calibration standard (STD10, 20 pg/mL). 2.6. STABILITY OF PFOS IN QUALITY CONTROL SAMPLES QC through QC3 were used to determine the stabilityofthe analyte during sample storage, extraction, and analysis. 261. Ambient-Temperature Stability of PFOS in Rat Serum The stabilityof POS was evaluated by storing QC samples at each concentration level at ambient temperature (ca. 25 C) for nominal timepointsof0, 2.5, 6, and 24 hours after thawing. All QCs were assayed in replicates of five. ue IM 2 ea SERVICES. INC. Page 130150 99VDIAOLMIDOC ! 001215 | | 262. Freezer Stability of PFOS in Rat Serum at -20 C The freezer stabilityof PFOS was evaluated by storing QC samples at each | concentration level at 20 C for 7, 13, and 33 days. Additional freezer stability imepoints wil be reported in an addendum to this report. All QCs were assayed in replicates of four. 263. Freeze/Thaw Stability in Rat Serum The stability of PFOS was evaluated afterthree freeze/thaw cycles. The mean concentrationsofthe QC samples afer three freeze/thaw cycles (ie. atleast 2 hours j frozen storage at the nominal temperatureof ~20 C followed by thawingatambient | temperature for 30 minutes) were compared to the mean concentrations of freshly i thawed QC samples. All QCs were assayed in replicates of five. i4 i 27. REPRODUCIBILITY OF REINJECTING EXTRACTED SAMPLES i The reproducibility of rejecting reconstituted serum extracts was investigated by | reinjecting a set of previously-assayed standards and QC samples which had been stored afte injection at approximately 25 C for 27 hours. The REofthe QC 4 I Samples was used to assess processed sample stability in the reconstitution solution ] ] All QCs were assayed in replicates of five 28. EXTRACTION RECOVERY The extraction recovery ofPOS from rat serum was determined by comparing the p(peoaskt-aerxetararcati)owi(tPhAtRh)eoPfsAaRmopflesasm(p0l2e5s,s4p,iaknedd b1e6foprge/emxLt)rascptiikoend(parfet-eerxetxrtarcatc)t.ioTnhe internal standard was spiked post-extraction for all samples. The recovery of the t25ethmearesfetraenndcaer.d (T4hpege/xmtLra)ctwiaosn raescsoevsseerdy (fo%llRoewcionvgearys)imwialasrdaeptperromaicnhedusbiyngdiPvFidOiSng he pre-cxtract PAR by the post-extract PAR and expressing the result 52 percentage. Five replicates were used at each concentration level. ee MW ASDVoANSCErD ea Page 14 of50 S9VDIAOLMLDOC Services. INC | 001216 | 3. RESULTS AND DISCUSSION A quantitative analytical procedure using turbo ion spray LC/MS in the negative ion mode was developed to meet the high sensitivity, specificity, and reproducibility requirements for the determination of PFOS in rat serum. The full-scan single MS mass spectrum of PFOS showed an abundant [M-K]' ion at m/z = 499 (Figure 1). The full-scan single MS mass spectrumof Tetra-H-PFOS showed an abundant (M-H] ion at m/z = 427 (Figure 2). The following selected ion monitoring (SIM) was used to quantify the analytes in rat serum: PFOS Tetra-H-PFOS (IS) m/z =499.0 mz=427.0 The peak labelingoffull-scan data does not accurately represent the performance of the instrument in SIM mode. The SIM ions were derived from separate experiments using narrow-range scanning, which more accurately depicts the operationofthe instrument in the SIM mode. The mass chromatogramsof a representative control blank rat serum extract are shown iri Figure 3. Mass chromatograms ffom a representative control rat serum extract containing only the intemal standard (zero sample), are shown in Figure 4. Figures and 6 are mass chromatogramsofrepresentative extracts from calibration standards 1 and 10, respectively. . 3 / k ; iji i eeeer----------------------------------001217 MW A2 CvA2 NCEDa sc 15050 ssvDiADLMLDOC Services. INC 31. ASSAY EVALUATION RESULTS 311 Intra- and Inter-Assay Accuracy The intra-assay accuracy (RE) data from QC samples ranged from -2.35 10 5.58% for 5 PFOS (Table 1). The inter-assay accuracy ranged from -3.58 to 4.95% for PFOS Eg (Table 2). These data indicate acceptable intra- and inter-assay accuracy for the i determinationofPFOS in rat serum. H 312. Intra- and Inter-Assay Precision I `The intra-assay precision data (RSD) ranged from 1.99 to 3.28% for PFOS at all QC )1 concentration levels (Table 1). The inter-assay precision results from QC samples li ranged from 1.92 to 4.87% for PFOS (Table 2). The RSD ranged from 0.613 to 4.38% for calibration standards at all levels (Table 3). These data indicate acceptable: 4 intra-and inter-assay precision for the determinationofPFOS in rat serum. I 313. Linearity i} The calibration curves werefit by a weighted (1/y') quadratic regression. B|i Coefficientsof determination (r") were 20.9962 for PFOS in rat serum. The i calibration curve statistics are shown in Table 4. 314. Partial Volume Analysis i; The resultsofpartial volume analysis of QC4 is shown in Table 5. The precision J (RSD) of QC4 samples diluted1 in 10 was 2.71%. The accuracy was 5.89%. These data indicate acceptable accuracy and precision for partial volume analysis for the determinationofPFOS in rat serum. | IM A2DVA2NCEaD SERVICES. INC. Page 160150 | 001218 99VDIADLMIDOC 3.15. Lower Limit of Quantitation (LLQ) `Table 6 shows the lower limitof quantitation (LLQ) data. The serum LLQ H experiment demonstrated that 0.05 pg/mL is an acceptable LLQ for PFOS. The A] precision (RSD) was 4.95% for PFOS. The RE was 4.57%. i it 3.0.6. Selectivity J `The assay was specific for PFOS. No chromatographic interferences were observed in anyofthe control serum samples analyzed (Figure 3). The control blanks and [| zero samples did show some evidence of small chromatographic peaks at the retention times ofthe analyte. These peaks were not quantifiable as they were below the lower limit of quantitation (LLQ). FARA Carryover Evaluation Carryover of PFOS and the IS was evaluated by injectionofan extracted rat serum control blank following an injectionofan extracted high standard (STD 10). The response for the small chromatographic peak at the retention time for PFOS was comparable to the response of a rat serum control blank injected before the high k standard. Thus, carryover is negligible for PFOS. There was no evidence of i carryover for the internal standard. 3 32. STABILITY OF PFOS IN QUALITY CONTROL SAMPLES i 32a `Ambient Temperature Stability of PFOS in Rat Serum i `The resultsof the ambient stability of PFOS in QC samples are shown in Table 7. `The mean predicted concentrations deviated from -4.45 to 10.5% from the 0-hour rvaulumes ffoorrualpl tQC24lehvoeulrssaantdatmibmieenpotinttesm.perBaatsuerde.on these data, PFOS was stable in rat i TmAB DVANS CED AL SFAViCES Me Page 170f SO 001219 99VDIA.OIMLDOC ------ 322. Freezer Stability of PFOS in Rat Serum at 20 C The results of the long-term stability of PFOS in QC samples stored at --20 C are presented in Table 8. The PFOS concentrations deviated from 0-hour values from 2.76 to 12.3% for all QC levels stored up to 33 days. Resultsoffreezer storage after two, four, and eight months will be reported as an addendum to this report. 323. Freeze/Thaw Stability of PFOS in Rat Serum `The resultsofthe freeze/thaw stability of PFOS in QC samples after three freeze/thaw cycles is shown in Table 9. PFOS was stable afer three freeze/thaw cycles with deviations ranging from ~1.38% to 10.1% from the O-cycle samples for all QC levels (Table 9). 3.3. REPRODUCIBILITY OF REINJECTING EXTRACTED SAMPLES `The results of reinjecting reconstituted samples containing PFOS after storage for 27 `hours at approximately 25 C in reconstitution solution are shown in Table 10. Reinjecting processed samples after 27 hours in reconstitution solution was appropriate with RE values ranging from -6.33 to 4.59% at 27 hours for all QC levels. 34. EXTRACTION RECOVERY `The mean recoveries of PFOSandTetra-H-PFOS are shown in Table 11. The mean recoveries ranged from 87.6 to 100% for PFOS. The mean recovery for Tetra-HPFOS was 89.9%. |, AFOvr ANCe ED . SERVICES. INC. Page 18arso 001220 99VDIAOLMLDOC 4. CONCLUSIONS The turbo ion spray LC/MS assay procedure for the determination of PFOS in rat serum has proven to be sensitive, specific, accurate, and reproducible. Its high sensitivity allows reliable and reproducible quantitationofPFOS down to a level of 0.05 pg/mL in rat serum based on 50-L samples. 5. DATA RETRIEVAL `The data for the rat serum validation are stored in ABS Notebook 2304 and in the ABS Archives. 6. REFERENCES | 1. Advanced BioAnalytical Services, Inc. Report 98AGKPO2 MMM. 1 Analytical Report for the DeterminationofPerfluorooctanoate and Perfluorooctanesulfonate in Human Serum by LC/MS. Grace K. Poon, i Ph.D., David Hardwick, B.S., and Ellen Pace, M.A.T., 6 February 1998. i g MAO2 VANCE5 D SERVICES. INC se 190130 | 001221 s3voiao1 Mi00C 7. TABLES Tablel: SIenrturam-AfsosraQy CAccSuarmapclyesand Precision of the PFOS Assay in Rat 3 PFOS Concentration (g/mL) 4 Theoretical Conc. 0Q2C1 QC3 13 i i Rung 0210 652 17.9 0202 631 18.0 fi i 0.206 617 16.7 0.205 632 18.0 1 0.194 635 17.3 Mean 0204 634 17.6 RSD (%) RE (%) 293 L177 199 328 5.58 235 RSD = (SD/Mean) x 100 RE = (Mean/Theoretical)-1] x 100 i g i i 001222 ree ee --eeeesee : MW A2DV5AN0CE5D , Pase20atso s9VoINDLMIDOC SERVICES. INC. Table2: SIenrtuerm-AfsosrayQACccSuarmapclyesanfdroPmreTchirseioenVoaflitdhaetPioFnORSuAnsssay in Rat PFOS Concentration (g/mL) 7 oct C2 C3 : Theoretical Cone.| 02 6 18 3 Rung 0210 652 7.9 0202 631 180 0206 6.17 16.7 l 0205 632 18.0 0.194 635 17.3 Run 10 0187 644 169 0.186 6.44 17.3 i 0.182 632 17.5 t 0.189 619 18.0 f 0.182 631 17.4 i Run 11 0.194 617 182 } 0.197 613 17.1 i 0.184 641 176 : 0.192 61s 17.7 0.184 624 17.0 Mean 0193 630 7.5 RSD (%) 487 192 266 RE (%) 3.58 495 27 RSD RE == (((MSeDan//MTheeaornex)tic1a0l0)-1] x 100 i! 1 mamgrt SERVICES. INC. Page 21 ofSO 001223 99VDIAOI MLDOC Tabled: Accuracy and Precision of the PFOS Assay in Rat Serum for A Calibration Standards from Three Validation Runs | 'PFOS Calibration Standard Concentration (g/mL) Theoretical Conc. STDS 025 STDS 1 ST4D7| ST8DS TO20I Run9 | 0.0483 | 0.0972 0.480| 0942 | 210| 448 | 761 | 158 | 200 | 0.0530] 0.101 0483| 0938 | 213| 455 | 770 | 162 | 109 ! Run 10 | 00.00544921 | [00..1090511 00.447699 || 00..995442 1as 3 | 870857 | [115579|| 210906 i| Run 11 [00..00551100 | {00..00996905|| 00..225512|| 00.447679|| 00.994485|| 222113 || 445430|| 77..6748|| 11558%|| 220030 | Mean |0.0511 | 0.0983| RSD(%) | 438 | 265 | 0.25 | 207 | 0.476 | 115 0.945 [0613 | [ 214 | 150| 430 | 162 | 7.78| 213 | 115282|| 210080 RE(%)| 220| 167 | 0207] 478| 5:53 | 705 | 124| 280| -1.00|0.0764 RRESD==(M(eSaDn//MTheeaonr)etxic1a0l0)-1] x 100 MAS DVANS CEDea SERVICES. INC. Page 20050 001224 99VDIALMLDOC Table4: Calibration Curve Parameters for PFOS in Rat Serum i Run# Quadratic | Coefficient| Intercept | Coefficient of Coefficient (AY| Linear (B) () [Determination (r) i 9 00010769 | 0.13730 | 0.00042009 0.5963 10 00010516 | 0.14535 | 00014910 0.992 : n -00012359 | 0.5173 | 0.00088515 0.9964 i Mean -0.0011215 0.00093206 0.9964 i 2y= AX Bx + C, weighted 1/57 | | i _-- WM S AovaancseD a SERVICES. INC. Pag 230050 001225 ssvDI0LMLDOC Table5: Partial Volume Analysis of PFOS in Rat Serum PFOS Cone. (ug/mL) Qc `Theoretical Conc. 100 Lin 10 dilution) i ; Rung 11 110055 110044 RSMDea(n%) 217016 RE (% 589 RSD = (SD/Mean) x 100 RE = [(Mean/Theoretical)-1] x 100 B i am boson FY SERVICES. INC. Page 24 of50 001226 99VDIA0IMLDOC Table6: Lower LimitofQuantitation of PFOS in Rat Serum Run 11 PFOS i Theoretical Concentration [Cone. Found (ug/mL) [ LQ 0.0489 ; (0.05 pg/mL) 0.0499 0.0554 0.0527 0.0520 Mean 0.0523 RSD (%) 495 RE (%) 457 RSD = (SD/Mean) x 100 RE = [(Mean/Theoretical)-1] x 100 ||| il | iil 001227 m--a----n--A-- DVA--NC--ED -- |------m--ar--io--------------v-- oi--Mi--00C SERVICES. INC. Table7: Ambient-Temperature Stability of PFOS in Rat Serum After 24 Hours Run 11 PFOS (ug/ml) QC (Theoretical Conc.) [00H1o9u4r||2.501H8o7ur|| 60H1o7u7r|[ 24 Hour 0190 Qc 0197 | 0181 | 0179 | 0187 (02 pg/mL) 0184 | 0184 | 0180 | 0191 0192 | 0180 | 0183 | 0182 0184 | 0178 | 0189 | 019% Mean 0190 | 0182 | 0182 | 0.188 RSD (%) 3.03 191 252 201 %DEV from 0 Hour NA 420 | 445 -L16 617 617 6.36 655 Qc? 613 630 638 653 i (6 ugmL) 641 626 635 6385 { 6.15 6.28 622 6.66 1 624 624 6.11 681 | Mean 622 625 628 668 RSD (%) 182 0797 | 186 2.16 %DEV from 0 Hour NA 0525 | 105 7.42 82 80 1 0 Qc3 17.1 18.0 180 194 (18 pg/mL) 17.6 17.7 176 192 17.7 17.9 182 194 17.0 18.0 18.0 19.7 Mean 175 17.9 180 19.4 RSD (%) 284 | 0719 | 126 119 : 9DEYV from 0 Hour NA 233 239 105 NA: Not Applicable RSD = (SD/Mean) x 100 | %DEV from 0 Hour = [(X Hour Conc. - 0 Hour Conc.)/0 Hour Conc.) x 100 FE un sovancen SERVICES. INC. Page 260150 001228 -- $9VDIAOLMIOOC i Table8: Freezer Stability of PFOS in Rat Serum at-20 C : i Qc PFOS (ug/mL) Concentration Run 2)* Run 6)* (Run 3)** Run 16) 0213 0.225 0.211 0245 cl 0.209 (02 ug/mL) 0.208 0222 0218 0210 0217 0234 0228 0208 0219 0.214 0232 0207 Mean RSD (%) 0.209 124 0221 132 0213 155 0235 3.17 %DEV from OHour| NA 572 184 123 Qc? 6.02 5.71 622 6.15 597 584 645 613 (6 ug/ml) 6.00 6.06 639 6.07 627 5.99 6.58 637 Mean 569 589 626 597 638 RSD (%) 3.08 %DEV from O Hour| NA 162 619 138 120 297 828 QC3 202 186 5.0 188 186 195 182 188 (18 ug/mL) 189 182 198 193 18.8 17.9 187 187 Mean 189 189 94 84 87 RSD (%) 3.96 %DEV from O Hour| NA 137 252 242 0.395 2.76 -130 NA: Not Applicable RSD = %DEV (SD/Mean) x 100 from 0 Hou=r [(Conc. - 0 Hour Conc.)0 Hour Conc.] x 100 i *From Protocol FACT-TOX-110 **From Protocol FACT-TOX-111 i ` 001229 ----n--_--_--_--_-- en Aote SERVICES. INC. Page 270f50 99VDIAOIMLDOC Table9: Stability of PFOS in Rat Serum After Three Freeze/Thaw Cycles Run 10 PFOS (ug/ml) QC (Theoretical Conc.) 3 Cycles 0187 0.177 Qc 0.186 0177 (02 pg/mL) 0.182 0.185 0.189 0.185 0.182 0.189 Mean 0.185 0.182 RSD (%) 1.70 312 %DEV from 0 Cycle NA -138 644 6.59 Qc2 6.44 661 (6 pg/mL) 632 6.44 619 639 631 6.40 Mean 634 645 RSD (%) 166 1.63 %DEYV from 0 Cycle NA 2.35 ! 169 2 Qcs 173 19.1 (18 pg/mL) 17.5 192 18.0 19.6 174 18.7 Mean 174 192 RSD (%) 231 180 DEV from 0 Cycle NA 10.1 NA: Not Applicable RSD = (SD/Mean) x 100 %DEV from 0 Cycle = [(3 Cycle - 0 Cycle)/0 Cycle] x 100 mm ive SERVICES. INC. Pac 2tors0 001230 s9vI0LMLDOC -- I Table 10: RPeFpOroSduacfitberil2i7tyHooufrRseiinnjeRcetcioongstEixtturtaicotneSdoSlautmipolnes Containing PFOS (g/mL =0 Hours (Run 10 =27 Hours Run 12) |! Qc 0.187 0.187 0.186 0.182 } (02 pgimL) 0182 0.189 0.188 0.188 Mean 0.182 0.1% 0.185 0.187 RSD (%) RE (%) 1.70 161 7.50 633 Qc2 643 639 644 6.02 (6 ng/mL) 632 619 6.19 6.55 631 622 Mean RSD (%) 634 628 166 323 i RE (%) 562 459 | 169 173 | (18Qucg/3ml) 1177..35 1188.21 i | 18.0 174 173 17.6 | ! Mean RSD (%) 17.4 231 2174.07 !I RE (%) A: Not Applicable 329 173 ! RRESD==(M(eSaDn//TMheeaonrx)etc10a0l-1] x 100 i} 001221 ee ------------------------------------------ man assy Page 29050 99VDIAOLMLDOC SERVICES. INC. Table11: Extraction Recovery of PFOS from Rat Serum Theoretical Conc.| Pre-Extraction Post-Extraction emg spregestp ts i] PFOS Spike | (0.25 pg/mL) Run 14 Mean PFOS Spike 2 (4 pg/mL) Run 14 0.03160 0.02850 0.02930 0.02790 0.03050 0.4597 04751 04776 0.4724 04772 0.02880 110 0.03010 94.7 0.02950 993 0.02930 952 0.02960 103 0.02946 100 05270 872 0.5304 896 0.5335 89.5 0.5298 892 05392 885 New ws PFOS T645 Spike 3 1.679 (16 pg/mL) L719 Run 14 1.535 1612 1874 380 1857 90.4 1.869 920 1.887 814 1.364 86.5 eam 575 Tetra-H-PFOS Spike IS (4 pg/mL) Run13 0.4068 03884 0.4043 03921 0.4078 0.4460 512 0.4423 87.8 0.4445 910 04512 86.9 0.4412 924 Vea 295 "(individual Response forPre-Ext./Individual Response for Post-Ext) x 100 [1]SNC ot SERVICES. INC Page 30of 50 001232 99VDJAOLMILDOC - 8. FIGURES Figure 1: Full-Scan Single MS Mass Spectrum of PFOS Senin rrosar " = " g g EC) 32 ow Po. u u . p MET 1" I" 7 0 449 = -= = ' = 001233 [iS.ooaee s moooo Figure 2: Full-Scan Single MS Mass Spectrumof Tetra-H-PFOS 5 5 " n n gs P e 2Tow 3iw Speci fom TeraH-PFOS QI Losers ar MH] ] 2 i| PM J 0 x i "0 1 s 3% me ) 0 | AF DVAR NCEDR Services. INC. Page 32050 001234 99vDIADLMLDOC Figure3: Mass Chromatograms of PFOS and its Internal Standard in Control Blank Rat Serum Extract me = 270 10763en % 0 n 0 0 2 Pg O) i: 1 23 we = 4590 I3 w E) 0 " 0 0 2 Tin, min 3 : 3stc2cms 1 z Time, min 3 ` All quantitation results and figures were prepared from nom-smoothed data. ABmriosve TeaH.2FOS (15) Iomnz =4990 miz= 4270 R3e1tenionTime 29 001235 ee------------------------------ IM ASDVo ANCsED Rea rages3ar50 59VDIAOLMLOOC SERVICES. INC. Figure 4: Mass Chromatograms of PFOS in a Rat Serum ExtractSample Containing Internal Standard Ouly (Zero Sample) me =an0 23a pn 50 wo o 50 " P) o g w 3 i 7 Tine mia 3 : ]] 3 mz 90 rtezeps HE =0 1 o ] " 5 n2 p 0 } wo ; ] Tie, min 3 : Allquantitation results nd figures were prepared from non-smoothed dats. pTeargaeH.PEOS (15) wa a4m27o0 S25 pel 001236 MMR, ro SERVICES. ING avon sooc FigureS: Mass Chromatograms of Calibration Standard 1 in Rat Serum Extract Containing PFOS (0.05 g/mL) and the Internal Standard me = 4220 sas 0 0 n @ 0 " g : ii i 3 meen i3 w 7 Time, min 3 : nae no 0 " 0 10 i 2 Tire, min 3 : All quantitation results and figures were prepared from non-smoothed data. APnRaOSle lomnz =499.0 R3e1tentionTime. Teaa HPFOS (IS) miz=4270 29 001237 mBo IOANs ALYTICAL SERVICES. INC. Page3sofso 59VDIADLMLDOC T= Figure 6: Mass Chromatograms of Calibration Standard 10 in Rat Serum -- sstess Extract Containing PFOS (20 pg/mL) and the Internal Standard 0 w Cw 2 i ; I 3 T 3 "Time, min 3 i wz =4990 5.14eS cps | HES a | n 60 "0 : w 0 0 PO oTesrme 7F05 19 T 7 Ties min 3 T ---------- =as-iaasn00 Sonion Tine 001238 NN 050, ree [Eo 9. APPENDIAX:QUANTITATION OF PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/MS AUTHORS: David J. Anderson, M.S. Amie J. Prince, B.S. Holly D. Ross, M.S. | TABLE OF CONTENTS ALO CRMC SIUCTUIE(S) rnin:38 i A20 Specimen(s).... om--------" ABO ASSRY PEICIDIE ors:38 FT ------ 38 ASO CHEMIE orcs: 38 A60 MataendrEQiUIaPMlIEsNL.......mrrsssnsssnsssnnsnnns39 ATO PrepOafSrOMaHOtNSion rss A80 Preparationof Quality ControlSAMPIES....vrrsssssrre 8S A90 Liquid-liquid EXIraction PrOCEQUIE vrs 46 A100 INSUUMENE CONBIONS worseT ALLO CalCUlBONS corms 49 001279 Sn --o ----m----B--OW--A--N--CE--D--------p--ea--n------------------so--in--s eotC SERVICES. INC. A1.0 CHEMICAL STRUCTURE(S) i CF-- iok Posium Perfioroosancsulonte i Cf G--G--S--OH FER, i o IH, IH, 2H, 2H-Perfluorooctane Sulfonic Acid MW =428, Tntemal Standard A2.0 SPECIMEN(S) This assay uses S0-uL aliquots of rat serum. Rat serum samples are stored at -20 C. A3.0 ASSAY PRINCIPLE PFOS and its internal standard, Tetra-H-PFOS, are extracted from rat serum samples (50 uL) using a liquid-liquid extraction procedure. The organic layer is evaporated to dryness and then reconstituted. An aliquot is then analyzed by turbo fon spray. liquid chromatography/mass spectrometry (LC/MS) in the negative ion mode. A4.0 COMPOUNDS PFOS: Lot# 171, 99.976% purity, 3M, Inc., Minneapolis, MN. Tetra-H-PFOS: Lot 59909, 90% purity, 3M, Inc., Minneapolis, MN. AS0 CHEMICALS All chemicals may be substituted with thatofan equivalent manufacturer and grade of chemical. 001240 Nm AEDOVAANNACLEYDTICAL SERVICES. INC Page 38of 50 ---- 99VDIAOIMIDOC Acetonitrile Ammonium Acetate Ethyl Acetate Methanol Rhodapex mass calibration solution Sodium Bicarbonate Sodium Carbonate, Annhydrous Tetrabutylammonium Hydrogen Sulfate: Water Rat Serum Rat Serum MCauts#ke0g1o5-n4,,MBIur4d9i4c4k2& Jackson, Cat 24,019-2, Aldrich, Milwaukee, WI 53201 Cat 100-4, Burdick & Jackson, Muskegon, MI 49442 Cat 230-4, Burdick & Jackson, Muskegon, MI 49442 A(LTm.ixBtaukreer,ofPh0i.l1l1ipmsbMurgt,riNflJuor0o8a8c6e5t)ic, a0c.i5d2 m11M0 aRnhdod|ampeMx ACOB-E4X36E,P1-1m2M0 (AalBl tEhXreeEPsCurpapnlbiuerdy,byNJRh0o8n5e1-2P)ouilne1n:c1, Inc. methanol:water. Cat 3509-01, 1.T. Baker, Phillipsburg, NJ 08865 Catt 3604-01, J.T. Baker, Phillipsburg, NJ 08865 HPLC Grade, Cat# 1360-07, J.T. Baker, Phillipsburg, NJ 08865 HMiogdhelPuDr7it3y31(NUAlNtOrpapuurree)W,aBtaemrsStyesatdem, Dubuque, IA 52001 Lampire Biological Laboratories, Piperville, PA 18947 HIandrilaannapBoiloipsr,oIdNuc4t6s2f2o9r Science, Inc., A60 MATERIALS AND EQUIPMENT Mass Spectrometer PtaEnSdeCmIEtrXipAlePqIu3a6dr5uaptomloesmpahesrsicsppercetsrsoumreeteironeiqzuatiipopned with TurbolonSprayTM interface, PE SCIEX, Concord, Ontario L4K 4V8 Data system A1P.I3,SMtaancdDaArdDSvof1t.w3a,rBeu,nMdalecrQvua1n3,vM1u.l4t,iLvCi2eTwuvn1.e3v, 009549 : MW ASDV2AN0CE5D ea SERVICES. INC. Page 39050 99VDIADLMLDOC and Sample Control v 1.3, on a Power Macintosh, PE SCIEX, Concord, Ontario L4K 4V8 HPLC Pump Shimadzu LC-10AD pump, Shimadzu Co., Columbia, MD 21046 : LC Pump Controller Shimadzu SCL-10A, Shimadzu Co., Columbia, MD 21046 Autosampler HPLC Column `Waters 717plus, Waters Associates, Millipore Corporation, Milford, MA 01757 Betasil Cy, 5 um particle size, 2.1 x 50 mm, Cat #852055-701, Keystone Scientific, Inc., Bellefonte, PA 16823 Harvard syringe pump 11 Harvard Apparatus Inc., South Natick, MA 01760 Multi-tube vortexer Cat# 5816-115, VWR Scientific, West Chester, PA 19380 Balance PH Meter Mechanical shaker NANOpure-UV/ swaytsetrempurification Model FX-300, AND Ltd., Tokyo, Japan Model 340, Corning Inc., Coming, NY 14830 Cat# 6000, Eberbach Corp., Ann Arbor, MI 48106 Bamstead, Dubuque, IA 52001 Microbalance Model MT-5, Mettler-Toledo Inc., Hightstown, NJ 08520 Micro weigh boats Cat# 0219-0041, Perkin-Elmer Corp., Norwalk, CT 06859 Weigh boats Cat# 12577025, VWR Scientific, West Chester, PA 19380 Sorvall RT-6000D refrigerated centrifuge Cat# 83071, DuPont Co., Wilmington, DE 19898 Beckman GS6KR Refrigerated centrifuge Beckman, Palo Alto, CA 94304 001242 ----e-- ee------------------ m,nABDIVDAANNACLEYTDICAL Pope soerse 99VDIADLMLOOC SERVICES. NC TurboVap LV Evaporator Pipettes Pipette tips Polypropylene tubes Screw-capped Polypropylene vials Screw-capped Polypropylene vials Plug Tite Multipurpose Caps Pyrex volumetric flask Solvent Filtration Apparatus Nylon Titan Membrane Filters PEEK tubing Liquid Nitrogen Gastight Glass Syringes 0C1a7t4843750, Zymark Instruments, Hopkinton, MA Catt Co., PW-o5b0u00m,,PM-1A0000,18P8-8200, P-100, Rainin Instrument RT-20, Rainin RT-200, C-5000, CP-25, Instrument Co., Wobum, CP-50, CP-250, MA 01888 W13esmtmChxest1e0r0,mPmA', C1a9t3#8015070-574,VWR Scientific, 16.5 x 28658 57 mm, Cat# 60.542, Sarstedt, Inc., Newton, SC 16.5 x 101 SC 28658 mm, Cat# 60.541, Sarstedt, Inc., Newton, Cat 78-127-0019-100, Worcester, MA 01607 Elkay Products, Inc., C10a0t0,2K8i0m1b4lPe-/1K0o,nt2e8s0,14VPi-n2e5l,an2d8,0N1J4P0-813006,0 28014P- 9C5a3t82965-3070010-,0090503,82975-30705010-,00Ki0m0b,l9e5/3Ko7n5t3e0s0,0V0i,neland, NJ 08360 0.45 um Scientific Resources Eatontown, NJ 07724 Inc., Cat# 74547-NN, 0.005" id. x 1/16" 0., Cat# Inc. Oak Harbor, WA 98277 1535, Upchurch Scientific Supplied in-house from bulk Buffalo, NY 14210-2005 tank, BOC Gasses, Cat# 1750, Hamilton Company, Reno, NV 89510 Other general laboratory glassware and supplies were used. 001243 s ee ----e ------------sto eeeeeen eeneret mam ASDVoANaCEDeas Page "iof0 99VDIAOLMIDOC -- A7.0 PREPARATION OF SOLUTIONS NANOpure water (18 megaohm-cm.) or equivalent should be used wherever water is called for. Mix al solutions well. A7.1 PreparationofAnalytical Standard Stock Solutions Analytical Standard Stock Solution, PFOS (1 mg/mL): Weigh approximately 5 mg ofPFOS (after correction for purity) on a microbalance and transfer it to a polypropylene vial. Dilute with the appropriate volumeofmethanol to yield a | mg/mL solution. Prepare a fresh solution every three months. Store the solution at 4C and bring to ambient temperature before use. Standard Spiking Solutions Prepare Standard Working SolutionsA through J in 5-mL class "A" volumetric flasks according to the following dilution scheme. Dilute to the S-mL mark with water to yield the final concentration. Prepare fresh solutions every three months. Store the solutions at 4 C and bring to ambient temperature before use. Standard Volume Spiked Solution Used PFOS Final Conc. x 1000 Stock 200 B 800 Stock 160 c 400 Stock 80 D 200 Stock 40 E 100 Stock 20 F 250 200 pg/mL (A) 10 G 125 200 pg/mL (A) 5 H 625 200 pg/mL (A) 25 1 500 10 pg/mL (F) 10 1 250 10 ug/ml (F) 05 mam Sin rin OY SERVICES. INC. Page 420150 001244 99VDIAOIMIDOC a A-7.2 Preparation of Internal Standard Stock Solutions faternal Standard Stock Solution, Tetra-H-PFOS (I mg/mL): Weigh approximately 5 mgofTetra-H-PFOS on a microbalance and transfer it to a. polypropylene vial. Dilute with an appropriate volumeof methanol to yield a mg/mL solution. Preparea fresh solution every three months. Store th solution a 4C and bring to ambient temperature before use. A-7.3 Preparation of Internal Standard Working Solution ITnatteerrnnalalStSatnadnadradrSdtoWcokrkSoilnugtiSoanltuotiaon10(04-gmL/mclLassTe"tAr"av-oHl-uPmFeOtrSi)c:flAadskd. 4D0i0lupteLtoof the mark with watetro give 4 ug/mL Tetra-H-PFOS solution. Store the solution at C and bring to ambient temperature before use. Prepare fresh solution as needed, A-7.4 PreparationofStandard Curve and Control Blank Prepare fresh calibration standards for each analytical run by combining 360 uL of rat control serum and 40 wLofthe analytical standard working solution (see table below) in labeled 1.7-mL microtubes. Prepare control blanks and zero samples by combining 360 kLofrat control serum with 40 Lofwater. Vortex each for 60 seconds. [am Ng gen SERVICES. INC. Page 430fSO 001245 99VDIAOI MLDOC Analytical Standard Dilution Table: Standar#d Volume Solution to 0 x 9 40 B 8 "0 c 7 "0 D 6 40 E 5 0 F 4 "0 G 3 "0 H 2 40 1 1 "0 1 iL control 360 360 360 360 360 360 360 360 360 360 Final Cone. 0 16 8 4 2 1 0s 025 01 005 A75 Preparation of Other Solutions 0.25 M Sodium Carbonate/0.25 M Sodium Bicarbonate: Weigh 26.5 gofsodium carbonate and 21 gofsodium bicarbonate and dissolve in approximately 900 mL of HPLC-grade water. Transfer the solution to a 1000-mL volumetric flask and dilute to the mark with HPLC-grade water. Mix the solution thoroughly. Store at ambient temperature. Prepare a fresh solution every three months. 1M Ammonium Acetate: Add 7.7 g ofammonium acetate to a 100-mL volumetric flask, dissolve in NANOpure water, and sti until completely dissolved. Dilute to the mark with NANOpure water. Store at ambient temperature. Prepare a fresh solution every three months. 2 mM Ammonium Acetate: Combine 2 mL of 1M ammonium acetate and 900 mL NANOpure water in a 1000-mL volumetric flask; tir until completely mixed. Dilute to the mark with NANOpure water. Store at ambient temperature. Prepare 2 fresh solution every three months. 001246 mn AEE seRvICES. INC Pagessarso 99vDIA0L MLDOC 10 M Sodium Hydroxide: Add 40 gof sodium hydroxide to a 100-mL volumetric flask, dissolve in NANOpure water, and stir until completely dissolved. Dilute to the `mark with NANOpure water. Store at ambient temperature. Prepare a fresh solution every three months. 1M Sodium Hydroxide: Add 10 mL of 10 M sodium hydroxide to a 100-mL volumetric flask. Dilute to the mark with NANOpure water; sti until completely mixed. Store at ambient temperature. Prepare a fresh solution every three months. 0.5 M Tetrabutylammonium Hydrogen Sulfate, pH 10: Weigh 16.98 g of tetrabutylammonium hydrogen sulfate and dissolve in 40 mL ofNANOpure water. Adjusted the pH to 10.0 with 10 M and 1 M sodium hydroxide. Transfer the solution 102 100-mL volumetric flask and dilute to the mark with NANOpure water. 10:90 Methanol:2 mM Ammonium Acetate (Mobile Phase Eluent A): Add 100 mL of methanol and 900 mL of2mM ammonium acetate to a 1000-mL Pyrex bottle. Mix the solution thoroughly and filter the solution through a 0.45 uM filter with a vacuum flask. Store at ambient temperature. Prepare a fresh solution every three months. 90:10 Methanol:2 mM Ammonium Acetate (Mobile Phase Eluent B): Add 900 mLofmethanol and 100 mL of2mM ammonium acetate to a 1000-mL Pyrex bottle. Mix the solution thoroughly and fier the solution through 2 0.45 uM filter with a vacuum flask. Store at ambient temperature. Prepare a fresh solution every three months. A8.0 PREPARATION OF QUALITY CONTROL SAMPLES A8.1 Preparation of Quality Control Stock Solutions QC Stock Solution, PFOS (1 mg/mL): Weigh approximately 5 mgofPFOS (after correction for purity) on a microbalance and transfer it to polypropylene vial. Dilute 001247 mMAEOIVOAANNACLYETDICAL SERVICES. INC Page 45of SO. 99VDI"AOLMLDOC i with the appropriate volumeof methanol to yield a | mg/mL solution. Store the ) solution at 4 C and bring to ambient temperature before use. Prepare a fresh solution every three months. A82 Preparation of Serum QC Samples QC4 (Dilution QC, 100 pg/mL PFOS): Add 1000 uL QC Stock Solution to a 10mL class "A" volumetric flask to yield a 100-ug/mL solution. Dilute to the mark with control serum, cap and mix. Aliquotsofapproximately 0.2 mLareplaced in Polypropylene vials and stored frozen at ~20 C. QC3 (18 pg/mL PFOS): Add 450 uL QC Stock Solution to a 25-mL class "ATM volumetric flaskto yielda 18-ug/mL solution. Dilute to the mark with control serum, cap and mix. Aliquotsofapproximately 0.5 mL are placed in polypropylene vials and stored frozen at ~20 C. QC2 (6 pg/mL PFOS): Add 150 uLofQC Stock Solution to a 25-mL class "A" volumetric flask to yield a 6-:g/mL solution.Diluteto the mark with control serum, cap and mix. Aliquots of approximately 0.5 mL are placed in polypropylene vials and stored frozen at -20 C. QC1 (0.2 ug/mL PFOS): Add 111.1 kL of QC3 to a 10-mL class "A" volumetric flask to yield 2 0.2-ug/mL solution. Dilute to the mark with control serum, cap and mix. Aliquotsofapproximately 0.5 mL are placed in polypropylene vials and stored frozen at 20 C. A9.0 LIQUID-LIQUID EXTRACTION PROCEDURE 21.. TPhreapwarQeCfrsesahmpclaelsi.bration standards for each run according to Section A.7.4. 3. Prepare QC4 samples (diluted1 in 10) in two steps: 2) Add 180uLof control serum and 20 kLofQC# into 1.7-mL microtubes and 001248 mn BR a Page 46.0150 99VDIADI MLDOC vortex b) Aliquot 50 kLofdiluted QC4 into labeled 16 x 100 mm polypropylene tubes. 4. Aliquot 50 uLofeach control blank, zero sample, calibration standard, and quality control samples 1-3 into labeled 16 x 100 mm polypropylene tubes. Standards and blanks are analyzed in duplicate. All QC samples are analyzed in replicates of five. 5. Add I mLof 0.5M tewrabutylammonium hydrogen sulfate, pH 10 to each tube. 6. Add 2mLof0.25 M sodium carbonate/0.25 M sodium bicarbonate to each tube, 7. Add 500 ULofthe Intemal Standard Working Solution to each tube (except control blank, add 500 uL water). 8. AddSmL ethyl acetate to each tube. 9. Shake the tubes on a reciprocal shaker at medium speed for 20 minutes. 10. Centrifuge the tubes at 3000 rpm for 20 minutes at 20 C. 11. Freeze the aqueous phase in an acetone/dry ice bath and keep the tubes in the bath an additional $ minutes. 12. Transfer the ethyl acetate layer to a fresh, labeled 13 x 100 mm polypropylene tube. 13. Evaporate the ethyl acetate layer to dryness in a TurboVapTM at approximately 20C under nitrogen. 14. Reconstitute the dried extracts in two steps: Add 500 uL acetonitrile and vortex for 60 seconds. `Then add 500 uL water and vortex for 60 seconds. 15.Transfer 200 iLof the extract to a labeled polypropylene autosampler vial. A.10.0 INSTRUMENT CONDITIONS HPLC Conditions: Eluent (gradient) [1] I aA SERVICES. INC Mobile Phase A: 10:90 methanol:2 mM `ammonium acetate (v/v) Mobile Phase B: 90:10 methanol:2 mM ammonium acetate (v/v) 001249 Page 470f50 99VDIAOLMI.DOC Eluent Gradient Conditions: o Tow T Cw Flow Rate Autosampler Autosampler Temperature Injection Volume HPLC Column HPLC Column Temperature Typical Initial Column Pressure Autosampler Run Time 300 uL/min. Waters 717plus Ambient : 3t0SpuL Keystone Betasil C,q, 5 um particle size, 2.1 mm x 50 mm Ambient 90 bar 8.0 minutes Analyte RepresentativeRetentionTime(minutes) PFOS 31 Tetra-H-PFOS (IS) 29 Curtain Gas Nebulizer Gas "TurbolonSprayTM Temperature TurbolonSprayTM Auxiliary Gas 'UHP Nitrogen UHP Nitrogen 400C 'UHP Nitrogen at 8 L/min nm EO CAL Page 48 of50. 001250 99VDJA01MI.DOC Tons Monitored: Analvie PFOS Tetra-H-PFOS (IS) IonMonitored mm//zz ==449297..00 Duell Time 550000 mmss Tonization Mode Ton Spray Voltage Declustering Potential MS Acquisition Time Pause Time Negative lon 3000 V 35V 5 minutes 2ms Calibrate the mass axisofthe instrument by infusion of Rhodapex mass calibration solution (Section A.5.0) at a low rate of 10 L/min. Optimize the sensitivityofthe instrument using an infusion ofa 10 pg/mL solutionof the analyte at 10 wL/min into a flow of 190 uL/minofmobile phase using the gradient condition at which the analyte elutes from the LC column (100% eluent B). Mass spectral peak widths should be approximately 0.6 amu athalfheight. One setofcalibration standards was injected at the beginning and one set of calibration standards was injected at the end of each analytical run (tray). A110 CALCULATIONS Calculated concentrations are based on peak area ratiosofPFOS to Tetra-H-PFOS. `The peak area for the analyte ion is divided by the peak area for the corresponding internal standard ion. Data generated from samples in his study were acquired and integrated using PE SCIEX software applications Sample Control (v. 1.3) and MacQuan (v. 1.4). The validation data were formatted in the ABS laboratory information management system (Watson, v.5.3.1.01). All data were rounded to no less than three significant figures by ABS prior to reporting. 001251 MMAD2V3ANSCERDea SERVICES. INC. meses | osvoInmamDOC AccCe ritepriaftor SaampnleAc nalyesis Calibration standards: The coefficientofdetermination (') must be 20.98. At least three-fourths ofthe individual calibration standard replicates will have deviations `within 15% from their nominal concentrations. LLQ acceptance criteria: At least one replicate at the LLQ must exhibit a deviation `within 20% from its nominal concentration.Ifthis criterion is not met, both replicates are rejected and the standards at the next higher level are subjected to the same test. Quality control samples: At least two-thirdsofthe individual QC sample replicates will have deviations within 15% from their nominal concentrations, with at least one replicate at each QC concentration meeting this criterion. ------rr------------------------------001252 IM SAoovaiNcEFDea SERVICES. INC. Pa"s s0t50 VDIVAon DIMLDOC P(eCrAuSoNruomobctearn2e7s5u8t.o3n8ate ATTACHMENT D DOSE CONFIRMATION ANALYSES WMReEpnovirrtoNnome.nFtaAlCTLaTboorxat1o0r8y Laboratory Requesturmoer(SRNH2779 Proprietary and Cantera 001253 Argus a1s014 I" ps enone a AT8n018, Onl Gorn) Cos sig Sly oFPFS is. -- + rE Earato taatn a ppbo raoser -- J ---- TweenDong Cotrmatn rrrm OnEEoTrIA : TorPasr = fh a emir eer|TmA Sa E | sw - rm aann r or snE cto n J-- |8 JweaE. oATh ms R_t CPoArtSuNoraarcoaenrs2s7ut5e9r3a5t3s Artacmente DATA SUMMARY TABLES LaboratoryReWquMReeEstpnNourmfbooerFnA(CRaTNYTUbo2r7s17098 Prpdotany and Content 001255 fPurs eee or[ S-- m Table 1, Concentrations of FOS I at Serum Samples from Study FACT Tox-108 LL: Less than PEee Non theLowerLimiofQuanttation (0.05g/mL) prpeen -- ) os 1 ww er 1 ww es @| [eow n o 1 oom m || [ww | comm] : [ [f me |e s wocoooor irs eon|| ew iw] ee 2m rem 001256 Ccottmrmem r T SoE m Y Table 1. ee Concentrationsof 0SPFOS ftSorSaeme in Rat Serum Samples from Study FACT Tox-108 (continued) Tee mwse = wi we wm | -- ms-- T --w -- I------ w ws we me || I ee-- --T ws-- | [ eeewee wsi | [mewrr| Pp Csi 001257 DPpA Tm R Si e: o n Table 1.e Concene otf PPrEFOaSSiitnRifator SSaonrrasSSamapmrpomSltuedyFAGTTox108 (continued) =-- -- ---- ---- ---- -- eer s w=ee || we[we] ewne ws || eews wese || Cr we Procetay and Canfcensl 001258 fPutiamcearersuyras Cnty ROePS -- coneLT Table 2 RSeopCeGatoAenarlymsais oUf RatmSeruCmuSramplesEf0ro0m )Study FACT Tox-108 Jes PeSEnT t nr llyoull oill ynolleos SSesos lolaT [mI e I 2 [oan28bE asss eE E 3C | E wr E E E5S = wr] S[ esora oe mer TToSiwnnRDrSurarTamsexctesasessa]rmor a|ne =J me| 5Tw 1] SeorReo po nGone: P 1)Fun r RIo E wi aS rn a Tre Table 3S.aPmhaprlmeacsofkrionmeStitcudSyubFgArCoTupT--oCxo1nc0e8ntrations of PFOS in Dam and Liter Serum <1 Lo rar Lover LtSari 05m DF osace S Pros TLC PS SProy s (20) (EL) 1i 1 1w8e91n0 | oom <LLQ[0.0316}<(0.05) || wwe 18910P | om <LLQ0.0382]<(0.05) I 18913 | <LLQ[0.0341]<(0.05) 18913P <LLQ[0.0398}<(0.05) [I Tes 18915 s| Ter | u<<LLiLoQc[l0po.3o022a231o<]<s((0o00.0055s))|)| 7 78938 | <LLQ[0.0311<(0.05) T7 r|_e_i8ssa0 <LLQ(0.03530<(0.05) T ort 38 18915P | <LLQ[0.0242]<(0.05) Ti6ee2e6p || <ALLLO0[0002o29s)<0(00.0)5) 78935P | <LLQ(0.0308]<(0.05) 8E 840P E<LL] Q[0.0231]<(0.05) Tw] A NA-Sampe not assayed. Insuficen sample vue or anayss. Pree srsConte 001259 PeCrRtSusMoaorcoanne2s7t5o5n3s5i3e Lorton RSeMcuERsenspvorootnmmoeoFrrtA(aCATLNTYaoGr3b7o7s3s Table 4<. P4F:OLSesCsoncaennthrLaotvieornLstinoDfuanmitanadtnCr(0o0ss8-Fmo)stered Litter Seru--m SSamaplreosrmbeyleDeosage Group E fy Tprc free py oe Adwmen | oomTeermTs em TAPea ee eeTTR -- [eew e 5 -- [-- A---- ewe---- rs-- wopoues[Ae Cem ewT Taw n Ta aT Teeee w mys] oem ems Se T Ta a weesee o ow we er7gomwoeoos owen sw [AA[Twe erwe a ee ----m--r------ mr e 1 Uniresed Dmamse e 5 Untreated Lie ters [ [wewes TTt<ioione opooovmemicsoooooosss[ T [s 8 s| e | s e T esr | |uoroopoosmnooooss ||| [ew 1 uooosomo0 [se1 ss | uYrCo1o-os2ioos | [O re S m o1e7 monH| N 1 | ew 0| [owe ws w T<iioopooovweooooss1 [8 8 | m1 e TTm--m|| u TreatedDamse. ee --iiooosscoos1sC Treated Litemrs ee ------------| Co o er ese Tem e sTc CT TFeew ee T | s -- omeereT Twi TTc cT Tr e we o ws owe ese m me e Tc c wweereT= =] ] o oww s ee r Twp Tcc 1 mwwee mmss] ows e wenTy c [c eer e ws || e 1 Trea Dae ms n e secET Teee eee =n] e] Foews es T Tie T e o[oweeeo dicoo-se--ncaos | ome es Ts me 10Teeme eT = =] Fe o ow w s ee sT T Te m: o TT & o o 0T T 1eww e w[ T = = wr|| wwe e msei T To o | mmoee m--s | ow T e ees Tse 0TTeemeeo Tr= ] C oesow TT o 01 [r eweww |a wweweroms | Prsnetar nd Corda 001260 Pe(rCtAuSoNruomcoaonre2s7u5t8o3n9a3te shortyReqauMeREsenptvoNirrutomnNbom,eenrFt(AaLlCRTLNaTbMooUrZxaTt1o70rS8y ATTACHMENT F ANALYTICAL REPORTS FROM CONTRIBUTING LABORATORIES Proprietaryand Cantdental 001261 ADVANCED . BIOANALYTICAL SERVICES, INC. " BIOANALYTICAL REPORT Tite: Quantitative DeterminationofPerfluorooctanesulfonate (PFOS) in Rat Serum from Study #FACT-TOX-108 Using Turbo lon Spray LC/MS . Date: Authors: 3 3 27 August 1999 David J. Anderson, M.S. Amie J. Prince, B.S. Holly D. Ross, M.S. J Prepared For: 3M Environmental Technology and Safety Services St. Paul, MN 55133-3331 ABS Report No.: 99ADJA01 MLDOC Study Protocol: FACT-TOX-108 : Number of Pages: 16 . Summary and Conclusions `The concentrationof perfluorooctanesulfonate (PFOS) was determined in rat serum samples collected during the study #FACT-TOX-108 using a sensitive, specific, accurate, and reproducible analytical method developed by Advanced BioAnalytical Services, Inc., Ithaca, NY. RisaotlasteerPuFmOsSamapnldesth(e5i0nutLe)mawlesrteanedxatrrda,ct1eHd,b1yH,a2lHi,q2uHi-dp-leirqfuliudoerxotorcatctainoenspurlofconeidcuraecitdo (anenTgaealttyrizave-edH-iboPynFOtmSuo)rdb.eo.Fioolnlsopwrianygleivqauipdorcahtrioomnaatnodgrraepcohnys/tmiatustsiosnp,esctarmopmleetreyxt(raLcCt/sMwSe)rein the All samples were successfully analyzed within four runs. The lower limit of quantitation was 0.05 ug/mL for PFOS. The precision of this assay (RSD) as determined from the analysis ofquality control samples for PFOS was 4.01%. The precisionofthis assay (RSD), as determined from the calibration standards for PFOS was <5.83%. The relative error (RE)ofthe assay, as determined from the analysis of the quality control samples. . ranged from -7.32 to calibration standards 10.1%. ranged The from REofthe assay, -5.86 to 11.0%. as determined from the analysis of 001262 15 Catherwood Road = Ithaca, New York 14850 (607) 266-0665 m Fax (607) 266-0749 ADVANCED BIOANALYTICAL SERVICES Title: ABS Report No: SIGNATURE PAGE RQuaatntSietrautimvefrDoemteSrtmuidnyat#iFoAnoCfT-PTeOrfXl-u1or0o8ocUtsaniensgulTfuornbaoteo(nPFSOpSr)ayin LCMS 99 MipOc Reported by: DReasveiadrJc.hASncdieerntsiosnt, M.S. Z3Aust 1994 Date Reviewedby: = hd { ) K ithleen Cormack, B.S. Associate Auditor Authorized for Releaseby: 7 John B. Perkins, Ph.D. Assistant Scientific Director zfua Date 27A%.99 Date IMASDVoAlNaCEDrca SERVICES, INC. Page 20116 001263 99ADIAOLMLDOC QAU STATEMENT Periodic inspectionsofthe bioanalytical portionofstudy #FACT-TOX-108 were conducted by the Quality Assurance Unit ofAdvanced BioAnalytical Services (ABS) for compliance with EPA GLP regulations (40 CFR Part 792). `The study was inspected on the following dates: 22 June 1999; 1, 2, 7, July 1999; 12 August 1999. oRne:sults ofthe inspection were reported to ABS Management and Project Team Leader 22 June 1999; 1, 2,7, July 1999; 12 August 1999. Resultsofthe inspections were reported to the Study Director on 12 August 1999. Based on the inspections and the data reviewed, this report is acomplete and accurate representationofthe data. a Assocl iateHArudi4 tor BS. Date 9 | A[ovEancReDY SERVICES. INC. Page3ofis 001264 SADIAOLMLDOC TABLE OF CONTENTS SIGNATURE PAGE wm d TABLE OF CONTENTS mms LI OFTS ABLET S... 1. INTRODUCTION wc 1.1. Study DeSCriptionandObJECHVE curs 2. METHODS coors 2.1. ADBIYHCE] PIOCAUTE(S)crvrvvsrsnsstssrson 2.3. D8 PROCESSING] 3.1. ASS2Y PETROIIANCE 1... ] 3.2. Analytical Results for Study #FACT-TOX-108 rrr 4. SUM ANDMCONA CLUR SIONY S corres 5. DATARETRIEVAL cosines sos 6 REFERENCES worms sms Lc...1] ASDAVANNACETDICAL SERVICES. INC. Pagedof16 001265 99ADIADLMLDOC LIST OF TABLES . Table 1: Summaryof Sample and Assay Information for Protocol FACT-TOX-108.....9 Table 2: Inter-Assay Precision and Accuracy Serum for Study #FACT-TOX-108 for PFOS Quality Control cvs Samples in Rat 10 Table 3: Inter-Assay Precision and Accuracy for PFOS Calibration StandardsinRat Serum for Study AFACT-TOX-108 vrs 11 `Table 4: Calibration Curve Statistics for the DeterminationofPFOS SUdY FFACT-TOX-108 ose in Rat Serum for 12 Table 5: Concentrationsof PFOS in Rat Serum Samples from Study #FACT-TOX-108 Table 6: Repeat Analysis of Rat Serum Samples from Study #FACT-TOX-108 ....... 16 MN ASOAVAANSCERDea SERVICES. INC. Page sof 16 001266 99ADIAOLMIDOC 1. INTRODUCTION 11. Study Description and Objective . p`Tehrefloubcjreocotitvaenoefstuhlifsonsattuedy(PwFaOsS)toidnertaetrsmeirnuemthseamcopnlceesntcroaltlieoctnesdodfuring the study #FACT- TweOrXe-1a0na8l,yz"eOdrauls(inGgavaaglieq)uiCdr-olsisqu-iFdosetxetrraicntgiSontuadnydofturPbFoOSioninspRraatys.l"iqRuaitd serum samples chromatography/mass spectrometry (LC/MS) assay. 2. METHODS 21. Analytical Procedure(s) PmeFtOhSodco(n1c).entSreartuiomnssawmeprleesde(t5e0rumLi)newdeirneraetxtsrearctuemd abcycaordliiqnugidt-oltihqueivdaelxitdraatcetdioLnC/MS p1rHo,c1eHd,u2rHe,2uHs-ipnegreftlhuyolraocoecttataene10suilsofloantiecPaFciOdS(Taentdrath-eH-inPtFeOrSna)lfsrtoamndraartds(e1rSu)m,. Following erveavpeorrsaetdi-opnhatsoedcrhyrnoemsastaongdraspuhbyseoqnuean2t.1recxon5s0timtumtio(n5,usma)mpBleetaesxtirlaTMctCs,wecroelusmepnarated by ((1K0e:y9s0tomneethSacnieonlt:i2fimc,MInca.,mmBeolnleifuonmtea,cePtaAt)e)wiatnhda4n3i%niEtilaulemnotbBil(e9p0h:a10semoefth5a5no%l:E2lumenMt A c`harmommoantiougmraapchetya/tme)a.ssPFspOeSctcroonmceetnrtyra(tLiCon/sMSwe)riendseetleecrtmeidneidonbmyotnuirtbooriinogn s(pSrIaMy)lmioqudied. The negative ions monitored were: m/z = 499.0 for PFOS [M-K]' m/=z 427.0 for Tetra-H-PFOS [M-H]' rSetgurdeysssiaomnoplfepecoankceanrteraatriaotinoss w(epreeakdearteearomfinPeFdOfSr/opmeaa kweairgehatoefdT(e1t/yr?a)-, Hq-uPadFrOaSt)icversus ncaolmiibnraatliocnonscteanntdraartdisonwoefrete0n.0c5a,li0b.1r,at0i.o2n5,st0a.n5d,ar1d,s2., 4T,h8e, n1o6,miannadl2c0onpcgen/tmrLatPiFonOsSo.fthe `PTFhOeSl.owQeuralliimtiytcoofntqruoalnt(itQaCt)iosne(rLuLmQs)amfoprlethsisatatshsraeyewdaifsfedreetnetrcmoinnceednttroatbieo0n.s0(50.g2/,m6L, .and s1a8mppgle/smL(QPCF4O,S1)0w0eurge/amnLa)lywzeerdewpirtehpaeraecdhaatsesaaychbadtilcuhtiinonretphlaitcwataess uosfefdoudr.uriDnigluation QC particular assay and were analyzed with each assay batch in replicates offouras needed. cTohneceanctcreapttiaonncsoe fcraitterlieaasftotrhcraelei-bfroautritohnsosftatnhdearidnsdisvtiidpuualaltceadltihbartattihoenbsatcakn-dcaarldcurleapltiecdates must not deviate more than Atleast one duplicate at the 15% LLQ from must their nominal concentration (except at exhibit a deviation within 20%ofthe the LLQ). nomin42g.f67y7 MW ASDVA2NCED5 SERVICES. INC. Pagesorls $9ADIAOLMLDOC ccrointceerniatrfaotliloonw.edTahree cioneaffciccoirednatoncfedweittehrmAiBnaStiSotnan(d')armduOsptebreat2i0n.g98P.rocTehdeuraecsc.eptance . o`fTtheheacicnedpitvaindcuaelcrQitCersiaamfporlethreepqluiaclaitteyscmountsrtolnostadmepvlieastestmiopruleattehdanth1at5at%lefarstomtwtoh-etihrirds nominal concentrations. deviation within 15%. At least one replicate at each QC concentration must exhibiat A summaryof Table 1. the sample and assay information for study #FACT-TOX-108 is given in 22. AssaySite All samples were analyzed at Advanced BioAnalytical Services, Inc., Ithaca, NY. 23. Data Processing tThheendeagtaatiwveerieocnomlloedcet.edPuesaikngarteuarsbowieornesipnrtaeygrLatCe/dMbSy tsehleePctEedSiCoInEmXonpirtoogrrianmg M(SaIcMQ)uainn, rveesruslitosnta1b.4l,esrefsriodminMgaocnQauMaanciwnetroesshacvoemdpaustetre.xt FfiollelsoawnidnguppleoaakdeadretaoitnhteegArBatSionf,iltehseerver Wwhaetrseona (wvei5g.3h.t1e.d01(1P/Sy'S),qIunacd.r,aWtaicynreeg,rePsAsio1n90w8a7s).pCearlfcourlmaetdiounssiwnegrtehepesrofftowramreed poanckage unrounded numbers. All calibration standard three significant figures before reporting. and QC results were rounded to no less than 3. RESULTS 31. Assay Performance `The performanceofthe assay for `control samples is documented in PFOS Table as 2. determined from the analysisofdaily quality The inter-assay precision (RSD)ofquality icnontthreolressaulmtpslefsrormantgheesdefqruoamlit1y.5c7onttor4o.l 0s1a%mpfloers;POthSe.relTahtievreeewrraosr n(oREm)arrkanegdedinfarccoumracy 13210 10.1%. pTrheecipseirofno(rRmSanDc)eoofftdhaeilsytacnadlairbdrsatriaonngceudrvfersomis 1d.o1c1utmoe5n.t8e3d%infoTrabPlFeOS3.. The inter-assay There was no marked inaccuracy in the between -5.86 to 11.0%. results from these standards; the relative error (RE) ranged 001268 --_----ee IM ASDVOANCNEDcSa Page Tot 16 99ADIA0LMLDOC SERVICES. INC. pTrheesesaltoepde,iny-Tiantbelrece4p.t,Tahnedr*coveaflfuiecsiernatongfeddetferrommin0a.t9i9o5n4 (to)0f.o9r9e8a2cfhoranPalFyOtiScailn rruatnsaerreum, sAlulccAesBsSfulpleyrseoxntrnaecltaqsusailginteydcotontarnoall(yQzCe)sasmapmlpelsesfoarntdhicsalpirborjaetcitownesrteanrdeaqrudisr.edRetsoults of ctrhieteerxitarafcotritohniswaesrseayr.eviReewsueldtbs yarae tmraaiinnetdaianneadlyisnt tahgeaisntsutdythreepcroer-ddseoffinAeBdSa.cceptance 32. Analytical Results for Study #FACT-TOX-108 APlFlOdSatcaownceernetrraotuinodnesdintorantosleerssumthfarnotmhSreteudsyig#niFfAicCaTnt-TfiOgXur-e1s0b8efaorreeprreepsoernttiendg iinn TTaabbllee 5S.. Study Table samples 6 requiring repeat analysis for Study #FACT-TOX-108 are presented in 4. SUMMARY AND CONCLUSIONS `The concentrationofPFOS study #FACT-TOX-108. was determined in rat serum samples collected during the PFOS was Les. isolated from serum bya liquid-liquid extraction procedure and determined by `PTFhOeSq.ualTihtyeopfrtehciesidoentoerfmtihneataisosnasy,waassdseatteisrfmaicnteodryftrhormoutghheoaunta.lyTshiseoLfLqQualwiatsy c0o.n0t5roplg/mL samples was 4.01% for PFOS. 5. DATA RETRIEVAL #TFheACcTal-cTulOaXt-ed10co8ncaernetmraatiinotnaidnaetda ofnrofmilethienatnhaelyasricshoifvresaotftsehreuAmdsvaamnpcleeds fBoiroASntauldyytical Services, Inc., Ithaca, NY in ABS Notebook 2319. 6. REFERENCES 1. MAdevtahnocdeVdalBiidoaAtniaolnytfiocratlheSeQruvaincteistaVtailoindoaftPieornflRueoproorotc9ta9nVeDsJulAfOoLna.tMeL(DPOFCO.S) in Rat Serum by Turbo lon B.S., and Holly D. Ross, SMp.rSa.y2L6CA/uMgSu.stDa1v9i9d9.J. Anderson, M.S. AmiJe. Prince, _-- IM ASCOVAARNSCEFDar SERVICES. INC. Pagesoris 00129 S9ADIAOLMLDOC 7. TABLES Tablel: S10u8mmaryofSample and Assay Information for Protocol FACT-TOX- Species/Matrix: RavSerum Sample Collection and Storage Information: Anticoagulant/Stabilizer: Reported Sample Collection Dates: Dates Received at ABS: Storage TemperaattAuBrSe: None 7 January 1999 to 29 January 1999 16 June 1999 20C Assay Information: Assay Period: 22 June 1999 to 28 June 1999 Analyte: Potassium p(ePrFfOlSu)orooctanesulfonate Analytical Standard: Lot No: PFOS 171 Source: Mm Internal Standard: Lot No: Source: 1H,1H2H2Hperflucrooctane sulfonic acid (Tetra-H-PFOS) 59909 M Calibration Range: Regression Method: Weighting Factor: Lower LimitofQuantitation: Upper Limit of Quantitation: quadratic uy 0.05 pg/mL 20 pg/mL 001270 ---- ee-- e -------------------------- IMASOvSAANCAEFDear Paseois $9ADIAI MLDOC SERVICES. INC. Table2: SIanmtpelr-eAsssianyRPartecSiesriounmafnodr SAtcucduyra#cFyAfCoTr-PTFOOXS-1Qu0a8lity Control . : PFOS Concentration (ug/ml . RunNo. | QCI cz QC3 Qc4 =] 02 6 18 100 100 Lin 10 Dilution) (1 in 20 Dilution) T 0.187 615 73 10 NA 0192 0.191 622 622 173 175 109 110 NA NA : 2 0.182 6.14 173 0.191 613 169 108 NA NA NA 0.191 591 16.8 NA 0.190 620 165 NA NA NA 0.192 619 167 NA NA 3 0.172 6.02 17.0 114 107 0174 622 16.7 nt 0175 5.94 17.0 110 106 100 0177 6.08 17.1 109 103 5 0.184 637 173 109 0.187 636 175 13 NA NA 0.197 639 175 1 NA 0.183 633 17.1 109 NA Mean 0.185 6.18 17.1 110 104 RSD(%)| 401 233 192 157 292 RE 132 3.04 5.00 10.1 3.97 RSD = (SD/Mean) x 100% RE = [(Mean-Nominal)Nominal] x 100% NA: Not Applicable. am ASDOVAANNACLYETDCAL SERVICES. INC. Page 100f 16 001271 99ADIAOLMLDOC = FACT-TOX-108 BF CBErS enee er 2 [+ i mle ee 3 oo | oe] 0237 PROS Concentration (g/mL) EH i 2 0944 452 |] 156 201 00553| 00982 0507 aa | am | ise | a0 :g Tabled: Calibration Curve Statistics for the Determination of PFOS in Rat Serum for Study #FACT-TOX-108 RunNo.|_ Quadratic Linear Coefficient (A)|Coefficient (B) T 0.000727 0.1225 2 -0.0005896 0.1392 3 -0.0006758 0.1401 5 -0.0004928 0.1345 Mean | -0.0006215 |0.1341| y=AX +Bx+C, weighted 1/y* Tntercept 0.001121 0.000944 0.001454 0.001190 0.001178 Coefficient of Determination (r) 0.9959 0.9982 0.9959 0.9954 0.9964 IM ASDoVAANCEPDea SERVICES. INC. Page 120116 001273 99ADIADI MIDOC TableS: Concentrations TOX-108 of PFOS in Rat Serum Samples from Study #FACT- Rar PFOS Concentration Number (ug/ml) 18901 0.0507 18901P <LLQ[0.0323]<(0.05) 18903 480 189037 357 18904 <LLQ[0.0462]<(0.05) 18906 <LLQ[0.0329]<(0.05) 18907 135 18907P 335 . 18908 241 18909 1.06 18910 <LLQ[0.0316]<(0.05) 18910P <LLQ0.0382]<(0.05) 18911 0.0798 18911P 0.123 18912 <LLQ[0.0376]<(0.05) 18913 <LLQ[0.0341]<(0.05) 189137 <LLQ[0.0398]<(0.05) 18915 <LLQ[0.0221]<(0.05) 18915P <LLQ[0.0242]<(0.05) 18917 <LLQ[0.0355]<(0.05) 18917P <LLQ[0.0221]<(0.05) 18918 <LLQ[0.0323]<(0.05) 18918P <LLQ[0.0247)<(0.05) 18919 <LLQ[0.0340]<(0.05) 18920 1.96 18922 <LLQ0.0412]<(0.05) 18922P <LLQ[0.0367]<(0.05) 18923 <LLQ[0.0291]<(0.05) 18923P <LLQ0.0331]<(0.05) 18924 272 18924p 307 18926 <LLQ[0.0323]<(0.05) 18926P <LLQ[0.0229]<(0.05) 18927 <LLQ0.0305]<(0.05) 18927P <LLQI0.0318)<(0.05) 18929 2.40 *Rat Number with a "P" indicates a pup sample. <LLQ: Less than the Lower Limit of Quantitation (0.05 pg/mL) mamm 2 SICANs ALYTAL SERVICES. INC. Page 13016 001274 59ADIADI MIDOC Table5: (Continued) Concentrations of PFOS in Rat Serum Samples from Study #FACT-TOX-108 Rat PFOS Concentration Number* (ug/ml) 18931 <LLQ[0.0382)<(0.05) 18931P <LLQ[0.0196]<(0.05) 18932 <LLQ[0.0399]<(0.05) 18934 125 18934P 34.1 18935 <LLQ[0.0316}<(0.05) 18935P <LLQ[0.0206}<(0.05) 18936 <LLQ[0.0302)<(0.05) 18936P <LLQ[0.0230]<(0.05) 18937 1.80 18938 <LLQ[0.0359)<(0.05) 18939 <LLQ[0.0311]<(0.05) 18939P <LLQ[0.0308]<(0.05) 18940 <LLQ[0.0330]<(0.05) 18940P <LLQ[0.0231]<(0.05) 18941 534 18942 112 18943 65.1 18943P 822 18946 157 18947 86.9 18948 96.1 18950 66.4 18951 724 189517 592 18952 9038 18952P 483 18953 670 18953P 538 18956 975 18956P 893 18957 69.1 18957P 932 18958 683 18958P 562 *Rat Number with a "P" indicates a p<uLpLQs:ampLlees.s than the Lower Limit of Quantitation (0.05 wg/mL) . 001275 Lh.J] ASoAvNanAceToICAL SERVICES. INC Page 140f16 S9ADIADIMIDOC TableS: (Continued) Concentrations of PFOS in Rat Serum Samples from Study #FACT-TOX-108 Rat PFOS Concentration Number* (ug/ml) 18959 592 18960 780 18960P 93.6 18961 69.1 18961P 476 18963 836 18964 856 18964P 929 18965 759 18967 742 18968 809 18969 786 18970 993 18970 969 18971 218 18972 669 189720 582 18973 819 189730 795 18974 172 18976 79 18977 124 *Rat Number with a "P" indicates a pup sample. mn ABIoOvAaNnAcLEYTDICAL SERVICES. INC. Page 15016 001276 S9ADIADLMIDOC B 8o8o8p || -- Table: Repeat Analysis of Rat Serum Tm Te On Samplesfrom Study #FACT-TOX-108 Om Ren ey Remy Rone Rewr 8385E2| amber wCom Niamvbeer Ro"omry Cuommle NCuommeb uCommle Recpoanneed S820F305|| o wWwooarrr w 000 bbu mwi oouuo waossewseiu maaye 3 m 33 m 111 3m7m 7s 555m1ma s y 1 wWw e 10 DDwwuioUvQlaemmesminaym 3121 aus 35 om >i RSUt:NaGmresrirltemUppercLopspfsGuoamiei.tion. 3R1)RRaEanm cAcoFocSnOecRmeORnrmEtiAoNoSnnSAacSYe:adedd ccalrbvoatnon ganee.. 2R15.RRiunEn ccoAnoFeOmnRmSRtcEiPonOOnRwlTiiENinDCccOSlaNbbCert:ioon ganege.. 2323 russe morse ADVANCED BIOANALYTICAL SERVICES. INC. ADDENDUM TO THE BIOANALYTICAL REPORT Title: Addendum - Quantitative Determination of Perfluorooctanesulfonate (PFOS) in Rat Serum from Study #FACT-TOX-108 Using Turbo lon Spray LC/MS Date: 13 October 1999 Addendum Report: 99ADJA0LMLADD.DOC OriginalReport: 99ADJA01.MLDOC Authors: David J. Anderson, M.S. Amie J. Prince, B.S. Holly D. Ross, M.S. Prepared For: 3M Environmental Technology and Safety Services St. Paul, MN 55133-3331 Number of Pages: 3 001278 15 Catherwood Road ithaca. New York 14850 u (607) 266-0665 Fax (607) 266-0749 ADVANCED BIOANALYTICAL SERVICES Title: SIGNATURE PAGE Addendum - Quantitative Determination of Perfluorooctanesulfonate (PFOS) in Rat Serum from Study #FACT-TOX-108 Using Turbo lon Spray LC/MS ABS Report No. Reported by: 99ADJARI.ML4 .DOC DaviJd. Anddgson, M. Research Scientist 13 ocr 29 Date Reviewedby: Hh a rae k athleen Cormack, B.S.. Associate Auditor Authorized for Release by: John R/Perkins, Ph.D. Assistant Scientific Director Accepted by:. YLL. HL Kris Hanson, Ph.D. 3M Study Director 360499 Date 13.00.94 Date H ot 97 Date mam `SSAeoDAAVvNiAAcNLeCYsET.DICIANGL Pagers 001279 9ADIAGIMIADDOOC ADDENDUM SUMMARY ADDENDUM `thAidsdiatdidonenofduSmtuidnydiDciarteecstaocrcseipgtnaantcueroefotnhetheorSiigginnaalturreepoPratge. The signature page of REASON FOR ADDENDUM Original GLP report requires the signatureof the Study Director. NIU AcDoVAlNeCEcD., pases SERVICES. INC. 001280 momo LADDOOC PS etmcca arons tonta ATTACHMENT G REPORT SIGNATURE PAGE Litrato RWeMESanevhveoaomnomeFAAnCabETooseSrsy Affe rl=Dp aFtee os-2000 PHaeinsT.aCaser, DaVMn, PhD. Sponsor Represenap me bep Te n LD7 / 5 Tale Bacon, Laboratory Manager 2Jew/o Tate [------ 001251 Study Title Analytical Laboratory Report on the Determination of the Presence and Concentration of PerfluoErxopooctsaendestuolPfootnaatsesi(uPmFOPeSr)fliunotrhoeocStearneusmoulffoSnpatreagvuiae-GDaavwalgeey Rats REPORT AMENDMENT NO. 1 Amendment Date: 19 April 2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS LRN-U2779 ET&SS FACT Tox-108 3M Environmental Laboratory 001282 ReportAmeFnAdCmeTnTtoxN-o1.081 This amendment modifies the following portion(s) of the final report: 1. FiNAL REPORT: Make the following addition to the final report text. App: Avafltueersthsetaftiendalforreptohretawnaalsytiiscsauledre(fseirgennecde amantderairaclhiavnedd),tesittwsausbsdtiasnccoevweererdetihnaatcctuhreatpeu.riTthyese vtoaldueetsewrmeirneebtahseeadcotunallypounriNtyMofRthaenasleyssessu.bsStuabnsceeqsu(ePntFOcSheLmoitca1l7c1haarnadct2e1r7i)z.aTtihoen ifsinoaclcruerproirntg wainlallybseisawmhenednetdhetyo daerteaiilsstuheedafnoarlyPtFicOaSl rLeostult1s71usainndg t21h7e.purity values from the certificates of REASON: `iTsoisdseusecdrifboertPhFeOcShaLnogtes1t7h1atanwidll21b7e. made to the final report when the certificateofanalysis 2. FiNAL REPORT: Make the following AMEND 70 READ: addition to the exceptions lst in the Statementof Compliance section. * The purity unknown and and wstaasbinloittyodefttehremitensetdspurbisotratnocethaenidniatniaaltyitoinocafltrheifsersteundcye. material are REASON: `The Statementof Compliance section was incomplete. 3. FINAL REPORT: `The purityofthe test 99.49% (Lot 171) as substance is 99.28% listed in Table 2. (Lot 217), and analytical reference material is AMEND TO READ: `The purityofthe unknown. test substance and analytical reference material listed in Table 2 is REASON: The purity values 2 are incorrect. stated for the test substance and analytical reference material listedin Table 3M Environmental Laboratory 001283 Amendment Approval Roport AmeFnAdCmoTnTeonxi1o08| MoenTG Marvin T. Case, D.V.M., Ph.D., Sponsor Representative phe ee Date Hefou Kristen J. Hansen, Ph.D., Study Director 6/19/60 Date aM Environmental Laboratory 0012843