Document rB88d7pgqeVyzM1LVb4DGJV6E

3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY ProtocoflorStudyNo.T-6316.17T,-6295.21T,-7132.3S;T-41 FecesMethod Develot)menMtetabolismStudyforPerfluorooetanesulfonate Derivatives. StudyObjective: Thisstudyisdesignetdogeneratsepecimenfsorthe3M EnvironmentLaalboratory FluorineAnalyticalChemistry Team touse fordevelopment and validatioan method of fecesmetabofiteanalysis. Research Client. 3M SpecialtyChemicals Division 3M Center,Building236 SaintPaul,MN 55144 Sponsor. 3M SpecialtyChemicals Division 3M Center,Building236 SaintPaul,MN 55144 Study Location: 3M StrategiTcoxicology Laboratory 3M Center,Building270-3S-06 room SB314 SaintPaul,MN 55144 Study Director. Andrew M. Seacat,Ph.D. Toxicology Specialist 3M MedicalDept./ CorporateToxicology 3M Center,Building220-2E-02 SaintPauL MN 55144 Ph.:651-575-3161 FAX: 651-733-1773 Study Taxicologist: Deanna Luebker,MS Advanced Research Toxicologist 3M MedicalDept./ CorporateToxicology 3M Center,Building220-2E-02 SaintPauL MN 55144 Ph: 651-737-1374 FAX: 651-733-1773 Proposed Study Timeline: In-LifeStart Date: Monday November 22, 1999 In-LifeEnd Date: Wednesday November 24, 1999 Regulatmy ConWhance. This studywillbe performed inthe3M StrategicToxicology Laboratoryunder a defined protocoland classifieads a "ClassB Study" asexplainedinTOX SOP 0950, Strategic Toxicology Lab GLP Program Procedure. T-63 16.17-,T-6295.2 I-@T-7132.3; ST-41 TestMaterial: Dan Hakes,ProductResponsibiliLtiyaison3M ChemicalsDivisionh,aspreviously ftimishedhigh-puritNy-ETFOSE, PFOS and PFOSA to theStrategiTcoxicologyLab. Identirication: Name: N-ETFOSE: Narrow Range N-Ethyl perfluorooctanesulfonarniedtohano@FM-3923. PFOS: PerfluorooctanSeulfonicAcid,PotassiumSalt;CAS 2795-39-3 PFOSA: Perfluorooctanesulfonamide Molecular Formula: N-ETFOSE: C8Fl7SO2N(CH2CH3)CH2CH20H PFOS: C&Fj7OSO2-K' 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 % asdeterminedby GC, GC/MS, '9F-NMP,'H-NMR and DSC technique(s1). PFOS: >991/oas determinedby 'T-NMR (2). PFOSA: Analysisby GCMS determinedthatthestarinmgaterialwas over 990/opure (3). Qualitativaend quantitativceompositionarlesulttshatwere derived from thesingletria'lW'9]F-NMR cross-integratiaonnalysirsevealed thatthecompositionwas 65.8% CF3(CF2)x-SO2-NH2 (Normal chain)1,8.7%CF3(CF2)x-CF(CF3)-(CF2)y-SO2-NH2(Internal monomethyl branch)1,1.2% (CF3)2CF-(CF2)x-SO2-NH2 (Isopropyblranch)3,.5% CxF2x+I-CF(CF3)-SO2-NH2 (Alpha branch)and 0.28% (CF3)3C-(CF2),-SO2-NH2 (t-ButyblranchX4). HPLC/MS characterizatioofnthePFOSA samplerevealed9,600 ppm ofPFOS, 1,100 ppm ofC7Fl5SO2NH2, 510 PPM Of C@Fl9SO2NH2,6,600 ppm ofCgFl6HS02NH2, 24,000 ppm of CISF36HSo@NH2, 1,200 ppm of CgF,_,H2SO2NH2and lower concentrationosf severalotheramides.Based on the sum of the impuritiest,hepurityof thePFOSA samplewould be approximately 96%(5). 2 T-63 16.17;T-6295.2 1-1T-713 2.3-,ST-41 Stability:Documentation wfl]be kepton file. StorageConditions: Testmaterialwillbe stored6ghtlysealedatroom temperature. Characteristics: Informationon synthesimsethods,Compositionor othercharacteristitchsatdefine thetestmaterialwillbe kepton fde. Animals: Species: Rat Strain: Sprague Dawley Source: Harlan Age atinitiatioonftreatment: 9-11 weeks Weight at initiatioonf treatment: approximately200-250g Number and sex: 12 males GROUP 1:controkn=3 GROUP 2:N-Et FOSE, n=3 GROUP 3: PFOS, n=3 GROUP 4:PFOSA, n=3 Identificatiounn:iquetafmlark Husbandry: Housing: AR ratswillbe individua.Uhyoused inwirebottom metabolismcages to allow forcollectioonfurineand feces. Diet/Water. Harlan TekladLM485 Mouse/Rat SterilizabDlieet,suppliedby Harlan Teklad,Madison,WL and tapwaterwillbe providedto allratsad libitum throughoutthestudy. Environment: Environmentalcontrolsfortheanimalroom willbe setto maintaina temperatureof 72 3'F,humidityof 30-700/oa,minimum of 10 exchanges of room airperhour and a 12 hourfight/darckycle. Dose and Dosing P@,ocedura: Method of administration/Dosepreparation: An ratswiU be dosed viaoralgavage on day zeroof thestudyusinga volume of 5 H dosingsuspension/kg body weight.Rats ingroup 2 will receivea single400 mglkg doseof N-Et FOSE, ratsingroup 3 willreceive a single100 mg/kg dose ofPFOS and ratsingroup 4 willreceivea single 100 mg/kg dose ofPFOSA- Uniform suspensionosfN-Et FOSE (8*/o8;0 mg/mL) and PFOS (2%; 20 mg/mL) willbe preparedin2% Tween 80 usinga 15-rnltissuegrinder.Re-suspensionofPFOS and N-ETFOSE solidswillbe performedwith 5 strokesof thefime grinderpestelbefore each sampleisdrawn-up inthesyringefordosing. 3 T-6316.17-1T-6295.2 I"T-7132.3; ST41 PFOSA (2%; 20 mg/mL) willbe preparedby dissolvinignacetone,then forming an emulsion in2% Tween 80 to a fina@concentrationof 1% acetone,2% Tween 80. Re-suspensionofPFOSA sofidswillbe performed by pumping the emulsionthrough the syringeimmediatelybefore dosing. A single5 ml / kg body weight dose ofvehiclewillbe administeredviaoral gavage to ratsingroup I on day zero ofthe study.Two ofthe ratswill receive2% Tween 80 and I willreceivea mbctureof 1% acetone@2% Tween 80, Observation ofanimals: ClinicalObservations: Each animalwillbe observedday formortalityand morbidityand notable findingswiflbe recorded.Additionalfindingswillbe recorded as theyare observed. Body Weights: Each animal willbe weighed immediately priorto treatmentand immediatelypriorto euthanasiaFrequency and Number ofAnimahOne controland one PFOSA ratwillbe sacrificeodn day I post dose. All remaininganimalswillbe sacrificeodn day 2-postdose. Specimen Collection& Analysis: Method of Specimen Collection: Urine and feceswillbe collectedfrom each metabolism cage on days one and two postdose. The initiavlolume ofurinewillbe recorded,the sides of the urinecollectioanpparatuswillbe washed with 5-10 ml deionized water and the finalvolume ofurinewillbe recorded.Daily fer-ewseight willbe recordedforeach animal. Animals willbe euthanizedby C02 and grossnecropsyperformed.During necropsy,blood (gz6 nil)willbe cobectedviathe abdominal aortaand transferretdo blood collectiotnubeswithout anticoagulantB.lood samples wiflbe allowedto clotfora periodof 15 to 30 minutesatroom temperature,and theclotwillbe spun down ina centrifugeat I100 x g for 5 minutes.The serum wiflbe transferretdo labeled1.5nilrnicrofugetubes and centrifugedagainat2000 x g to remove any remainingred blood ceus. Each serasample willthenbe dividedinto2 afiquotst,ransferredto a separatelabeledpolypropylenemicrofugetube and frozenindry ice.One aliquotwillbe used formetaboliteanalysisand theotherforbiochemical analysis.Each liverwillbe excisedand weighed,The liverwillbe flash fi-ozeninfiquidnitrogen.A smallsection(approximately3 grams) willbe removed and storedat-70'C formetaboliteanalysis.The remainderwill be storedat -70'C forbiochen-@calanalysisK.idneys from animals sacrificeodn day 2-postdose willbe excised,weighed and flashfrozenin liquidnitrogenforfuturebiochemicalanalysis. 4 T-6316.17; T-6295.21-,T-7132.3; ST-41 Specimen Handling: AD urineandfecesandtheserumand livefrormetabolitaenalysivsdlibe temporarilsytoredat-700C inthe StrategiTcoxicologyLaboratory.For analysist,hesesampleswillbe packed indry iceand shippedto: Kris Hansen,Ph.D. 3M EnvironmentalTechnology and SafetyServices 935 Bush Avenue St.Paul,NIN 55133-3331 TelephoneNo.:651-778-6081,FacsimilNeo.: 651-77M 176. AR kidneyspecimensand theremainderof serum and liverwillbe kept in the StrategiTcoxicologylabforbiochemicalanalysisT.hese specimens wiflbe storedat-700C. The number, type,and dateof specimensto be generatedforanalysiasreas foflows: TABLE I -Sp Environmental Lab Specimens Straegic Tar- Lab Specimens mens.for EnWronmentalLab Specimen Collection date Day I postdow Day 2 post dow Urine 12 .10 Feces 12 10 Sera 2 10 Liver 2 10 Sera 2 10 Liver 2 10 , Kidncys 10 Total 22 22 12 12 12 12 10 Data Analysis: AD resultswiU be provided forinclusioinnthefinalreport. Responsibilities: Deanna Luebker and Andrew Seacatwillbe responsiblfeordosingtheanimals, coflectinign-lifsepecimens,perfomiingthenecropsiesand cohectingand sending tissuespecimens foranalysis. Andrew Seacatwilldrafta finarleportand ensurethereportreceivesappropriate3M reviewbeforea finalreportisissued. 5 T-6316,17; T-6295,21;T-7132.3; ST-41 Signatures: Dr.Andrew Seacat g' @@@Dat@e SenioRresearcThoxicologist Study Director A40 Deama 11_.LLUueebbkk@eerr., Date Advww.ed Toiiicologist Study Toxicologist T-6316.17;T-6295-21; T-7132.3; ST-41 References: 1. Payfer,R- CharacterizatioonfFM-3923, Nlb=e ofLots 30035, 30037 & 30039 for Two Year Feeding Study. SMD AnalyficaRlequest 952489. AnalyticaRleport 9 816,6/23/97.3M SMD Lab Building236-2B-1 1. 2. Kestner,T. FluorochemicalIsomer Distributiobny'9F-NMR Spectroscopy. SpectroscopyRequest A 53030. 3M SpecialtyAdhesives& Chemicals Analytical Laboratory f SNUvID-236-2B-1 1,December 1,1997. 3. Payfer,R- GC/MS analysiosfPFOSA (L-10009).SA&C AnalyticaRlequest No. 59426. Report 9/24/99.3M SA&C Lab Building236-2B-1 1. 4. Kestner,T. Chemical CharacterizatioonfPFOSA, L-10009, by IH and 19F-NMRSpectroscopyRequest # 59426. 3M SpecialtyAdhesives& Chemicals Analyfical Laboratory/ SNB4D-236-2B- 11,September 25, 1999. 5. DeRoos F. CharacterizatioonfPFOSA Samples,T-7132-1 (L-10009) and TN-A1584. Request # A- 151254. Report 10/7/99,and Letteraddendum to report 10/14/99. Corporate AnalyticalTechnology Center,Building201-1-29,CATC Chromatography Group. 7