Document 1vzGd467YnJeDmLzQGex90OZ

3M MEDICAL DEPARTMEENT, CORPORATE TOXICOLOGY ProtocolforStudy No. T-6316.17,T-6295.21,T-7132.3;ST-41 FecesMethod Development Metabolism Study forPerfluorooctanemffonate Derivatives. Study Objective: Thisstudyisdesignedtogeneratespecimensforthe 3M EnvironmentalLaboratory FluorineAnalyticalChemistryTeam to use fordevelopment and validatioanmethod of fecesmetaboliteanalysis. Research C7ient. 3M SpecialtyChemicals Division 3M Center,Building236 SaintPad, MN 55144 sponsor.* 3M SpecialtCyhenficalDsivision 3M Center,Building236 SaintPauL MN 55144 Study Locadon: 3M StrategicToxicologyLaboratory 3M Center,Building270-3S-06 room SB314 SaintPaul,MN 55144 Study Director.- Andrew M. Seacat,Ph.D. ToxicologySpecialist 3M MedicalDept./CorporateTo)iicDlogy 3M Center,Building220-2E-02 SaintPaa MN 55144 Ph.:651-575-3161FAX: 651-733-1773 Study Tmdcologist: Deanna Luebker,MS Advanced Research Toxicologist 3M MedicalDept./CorporateToxicology 3M Center,Building220-2E-02 SaintPatiLMN 55144 Ph: 651-737-1374FAX: 651-733-1773 Proposed Study Timdine. In-LifeStartDate: Monday November 22, 1999 In-LifeEnd Date: Wednesday November 24, 1999 Regulatory Conrliance., Thisstudywillbe performedinthe3M StrategiTcoxicologyLaboratoryunder a defined protocoland classifieads a "ClassB Study"as explainedinTOX SOP 0950, Strategic ToxicologyLab GLP Program Procedure. T-6316.17;T-6295.21;T-7132.3; ST-41 Tat Mate".Dan Hakes,ProductResponsibilLiitayiso3nM ChemicalsDivisionh,aspreviously fiunishedhigh-puritNy-ETFOSE, PFOS and PFOSA totheStrategiTcoxicologyLab. Identification: Name: N-ETFOSE: Narrow Range N-Ethyl perfluorooctanesulfonameitdhoanolFM-3923. PFOS: PerfluorooctanSeulfonicAcid,PotassiumSalt;CAS 2795-39-3 PFOSA: PerfluorooctanesWfonamide MolecularFormula: N-ETFOSE: C8Fl7SO2N(CH2CID)CH2CH20H PFOS: C#F,70S%X' PFOSA. CsFl7SO2NH2 Lot Number. N-ETFOSE: Lots 30035,30037,30039 mixed and analyzedas one sample. PFOS: Lot# 217 PFOSA: L-10009 Purity: N ETFOSE: 98 % as determinedby GC, GC/MS, '9F-NMR,'H-NMR and DSC technique(s1). PFOS: >990/,asdeterminedby '9F-NNM (2). PFOSA: Analysisby GCMS determinedthatthestarinmgaterialwas over Wlo pure(3). Qualitativaend quantitaticvoempositionarlesulttshatwere derived from thesinglteria'lIV'9F-NMR cross-integmfiaonalysirsevealed thatthecompositiownas 65.8% CF3(CF2)x-SO2-NH2 (Normal chain)1,8.70/CoF3(CF2)x-CF(CF3)-(CF2)y-SO2-NH2(Internal monomethylbranch)1,1.2% (CF3)2CF-(CF2)x-SO2-NH2 (Isopropyblranih)3,.5% CxF2x+I-CF(CF3)-SO2-NH2 (Alpha branch)and 0.280/(CcF3)3C-(CF2).-SOrNH2 (t-BUtYblranChX4). BPLC/MS characterizatioofnthePFOSA samplerevealed9,600 ppm ofPFOS, 1,100 ppm ofC7Fl5SO2NH2,510ppmof C9Fl9SO2NH2,6,600 ppm ofCgFl6HS02NH2, 24,000ppm of CISF36HS02NH2. 1,200ppm ofCgFlsH2SO2NH2and lower concentrationosf severalotheramides.Based on the sum ofthe impuritietsh,epurityofthePFOSA swnplewould be approximately 96%(5). 2 T-6316.17;T-6295.21;T-7132.3; ST-41 StabilityD:ocumentationwillbe kepton file. StorageConditions: Testmaterialwillbe storedtightlsyealedatroom temperature. Characteristics: Infor7natioonn synthesimsethods, compositionor othercharacteristitchsatdefine thetestmaterialwillbe kept on file. Animals: Species: Rat Strain: Sprague Dawley Source: Harlan Age at initiatioonftreatment: 9-11 weeks Weight at initiatioonf treatment: approximately200-250g Number and sex: 12 males GROUP 1:controtn=3 GROUP 2:N-Et FOSE, n--3 GROUP 3: PFOS, tv=3 GROUP 4: PFOSA, n7--3 Identificationu:niquetadmark Husban&y.Housing: AD ratswillbe individualhloyused inwire bottom metabolismcages to allowforcollectioonf urineand feces. Diet/Water.HarlanTekladIM-485 Mouse/M StedlizablDeiet,suppliedby Harlan Teklad,Madison,WL and tapwaterwiU be providedto allratsad libitum throughoutthestudy. Environment: Environmentalcontrolfsortheanimalroom willbe setto nwntain a temperatureof 72 30F,humidityof 30-700/oa,minimwn of 10 exchanges of room airperhour and a 12 hour light/darckycle. Dose and Dosing Procedures: Method of administration/Dosepreparation: An ratswillbe dosed viaoralgavage on day zero ofthestudy usinga volume of 5 nildosingsuspension/kg body weight. Rats ingroup 2 will ree6ve a single400 mgtkg dose of N-Et FOSE, ratsingroup 3 willreceive a single100 mg&g dose of PFOS and ratsingroup 4 willreceivea single 100 mg/kg dose of PFOSA- Uniform suspensionsof N-Et FOSE (SO/o8;0 mg/mL) and PFOS (20/o2;0 mghnl) willbe preparedin2% Tween 80 using a 15-rnlfissuegrinder. Re-suspensionof PFOS and N-ETFOSE solidsvhflbe performedwith 5 strokesof thetissuegrinderpestelbefore each sample isdrawn-up inthe*,ringefordosing. 3 T-6316.17;T-6295.21; T-7132.3;ST-41 PFOSA (2%; 20 mg/mL) willbe preparedby dissolviinngacetonet,hen formingan emulsionin2% Tween 80 to a finacloncentratioonf I% acetone,2% Tween 80. Re-suspensionofPFOSA solidwsillbe performed by pumping theemulsionthroughthesyringeimmediatelybeforedosing. A single5 ml /kg body weightdoseofvehiclewillbe administerevdiaoral gavagetoratsingroup I on day zeroofthestudy.Two of theratswill receiverlo Tween 80 and I willreceivea mixtureof 1% acetone/2% Tween 80. Observadon ofanimals: ClinicalObservations: Each animalwillbe observeddailyformortalitaynd morbidityand notable findingwsillbe recorded.Additionaflindingwsillbe recordedastheyare observed. Body Weights: Each animalwillbe weighed immediatelypriorto treatmentand immediatelypriortoeuthanasi& Frequency and Number ofAnimals: One controland onePFOSA ratwin be sacrificeodn day I postdose.AD remaininganimalswillbe sacrificeodn day 2-postdose. Specimen CoffecdonA Analysis. Method ofSpecimen Collection: Urine and feceswillbe collectefdrom eachmetabolismcase on daysone and two postdose. The initivaollume ofurinewillbe recorded,thesides oftheurinecollectioanpparatuswin be washed with5-10 mi deionized waterand thefinavlolume of urinewillbe recorded.Dailyfecesweight willbe recordedfbreachanimal. Animals willbe euthanizedby C02 and grossnecropsyperformed.During necropsy,blood(m 6 iiilw)illbe collectevdiatheabdominalaortaand transferretdobloodcollectiotnubeswithoutanticoagulanBtl.ood swnples willbe allowedtoclotfora periodof 15to 30 minutesatroom temperaturea,nd theclotwillbe spun down ina centfiffiagteI100x g for 5 minutes.The serumwillbe transferretdolabeled1.5 iW niicrofugteubes and centrifugeadgainat2000 x g toremove any remainingred blood ceus. Each serasamplewillthenbe dividedinto2 aliquotst,ransferretdoa separatelabeledpolypropylenemicrofugetubeand frozenindry ice.One aliquotwillbe usedformetaboliteanalysiasnd theotherforbiochemical analysisE.ach liverwillbe excisedand weighed.The liverwillbe flash frozeninliquinditrogenA. smallsection(appro)dmatel3ygrams)win be removed and storedat-70'C formetabolitaenalysisT.he remainderwill be storedat-700C forbiochemicalanalysisK.idneysfrom aniinws sacrificeodn day 2-postdose willbe excisedw,eighedand flashfrozenin liquidnitrogenforfuturebiochemicalanalysis. 4 T-6316.17;T-6295.21;T-7132.3; ST-41 Specimen Handling: Allurineand fecesand thesenunand Everformetabolitaenalysiwsillbe temporarilsytoredat-70*C intheStrategiTco,,dcologLyaboratory.For analysist,hesesampleswillbe packed indryiceand shippedto: KrisHansen,Ph.D. 3M EnvironmentalTechnologyand SafetyServices 935 Bush Avenue St.Paul,MN 55133-3331 TelephoneNo.:651-778-6081,FacsimileNo.:,651-778-6176. AU lddneyspemmens and theremainderof senun and liverwillbe keptin the StrategiTco3dcologylabforbiochemicalanalysisT.hese specimens willbe storedat-700C. The number,t)W, and dateofspecimenstobe generatedforanalysiasreas follows: TABLE I ---SP mens-foren Specimen Environmental Lab Speebum Swuftk Tax Lab speclmem Urine Fem sera Liver Sera Liver 1Kidncys tallab Collectidoante DiLy I VM dose Da-v 2 PW dow 12 10 12 10 2 10 2 10 2 10 2 10 10 Total 22 22 12 12 12 12 10 Data Analysis. Allresultwsillbe providedfbrinclusioinn thefinalreport. ResponsibUides: Deanna Luebker and Andrew Seacatwillbe responsiblfeordosingthe anftnals, collectinign-lifsepecimens,performingthenecropsieasnd collectinagnd sending tissuespecimensforanalysis. Andrew Seacatwilldrafta finarleportand ensurethereportreceiveasppropriat3eM reviewbeforea finarleportisissued. 5 Signatures: Dr. Andrew Seamt SeniorResearch To)dcologist StudyDirector Deanna J.Luebker, as Advanced To3iicologist Study To)dcologist T-6316.17;T-6295-21;T-7132.3; ST41 Date Date 6 T-6316.17;T-6295.21;T-7132.3;ST41 References: 1. PayferR,- CharacterizatoifoFnM-3923,NfmtureofLots30035,30037 & 30039fbr Two Year FeedingStudy.SMD AnalyticaRlequest#52489. AnalyticaRleport9 816,6/23/97.3M SMD Lab Building23&2B-1 1. 2. Kestner,T. FluorochemicaIlsomerDistributiboyn "P-NMR Spectroscopy. SpectroscopyRequest# 53030.3M SpecialtAydhesives& ChemicalsAnalytical Laboratory/ SMMD-236-2B- 11,December 1,1997. 3. Payfer,R. GC/MS analysiosfPFOSA (L-10009).SA&C AnalyticaRlequestNo. 59426.Report 9t24/99.3M SA&C Lab Building236-2B-11. 4. Kestner,T. ChemicalChamcteizafionofPFOSA, L-10009,by IH and 19F-NMRSpectroscopyRequest# 59426.3M SpecialtAydhesives& ChemicalsAnalytical Laboratory/ SA4MD-236-2B- 11,September25, 1999. 5. DeRoos F.CharacterizatioofnPFOSA Samples,T-7132-1(L-10009)and TN-A1594.Request# A-151254. Report 10/7/99a,nd Letteraddendum to report10/14/99. CorporateAnalyticaTlechnologyCenter,Building201-1-29,CATC Chromatography Group. 7