Document XRkRBVjbO6OLDbYG89XMdMqy4

3M MEDICAL DEPARTMENT, CORPORATE TOXICOL40GY ProtocolforStudy No. T-6316.17,T-6295.21,T-7132.3;ST-41 FecesMethod Development MetaboFum Study forPerfluorooetanesulfons Derivatives. Study Objecgve: Thisstudyisdesignedtogeneratespecimensforthe3M EnvironmentalLaboratory FluorineAnalyticaClhemistryTeam to usefordevelopmentand validatioan method of fecesmetaboliteanalysis. Research Cient. 3M SpecialtyChemicalsDivision 3M Center,Building236 SaintPaul,MN 55144 Sponsor.* Study Location: 3M SpecialtCyhemicalsDivision 3M Center,Building236 SaintPauL MN 55144 3M StrategiTcoxicologyLaboratory 3M Center,Building270-3S-06room SB314 SaintPauL MN 55144 Study Director. Andrew M. Seacat,PILD. ToxicologySpecialist 3M MedicalDept./CorporateToxicology 3M Center,Building220-2E-02 SaintPauL MN 55144 Ph.:651-575-3161FAX: 651-733-1773 Study Taxicologist:Deanna Luebker,MS Advanced ResearchToxicologist 3M MedicalDept./CorporateToxicology 3M Center,Building220-2E-02 SaintPauL MN 55144 Ph: 651-737-1374FAX: 651-733-1773 Proposed Study Timdine: In-lAfeSftrtDate: Monday November 22, 1999 In-LifeEnd Date: Wednesday November 24, 1999 RegulatopyConrfiance., 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;ST41 TatMatnial-. Dan Hakes,ProductResponsibiliLtiyaison3M ChemicalsDivisionh,aspreviously fivnishedhigh-puritNy-ETFOSE, PFOS and PFOSA to theStrategiTcoxicologyLab. Identification: Name: N-ETFOSE: Narrow Range N-Ethyl perfluorooctanesulfonaraeitdhoanolFM-3923. PFOS: PerfluorooctanseulfonicAcid,PotassiumSalt;CAS 2795-39-3 PFOSA: Perfluotooctanesulfonamide MolecularFormula: N-ETFOSE: C8Fl7SO2N(CH2CH3)CH2CH20H PFOS: CSF17OS02V PFOSA: CsFl7SO2NH2 Lot Number. N-ETFOSE: Lots 30035,30037,30039 mixed and analyzedas one sample. PFOS: Lot # 217 PFOSA: L-10009 Purity: o N ETFOSE: 98 % asdeterminedby GC, GC/MS, '9F-NMR,'H-NMR and DSC technique(s1). PFOS: >Wlo asdeterminebdy '-9F-NMR(2). PFOSA: Analysisby GCMS determinedthatthestarinmgaterialwas over 990/opure(3). Qualitafivaend quantitativceompositionarlesulttshatwere derived from thesinglteria'lIi/'9F-NMRcross-integmtioanalysirsevealed thatthecompositionwas 65.80/CoF3(CF2)xS02-NH2 (Nomal chain)1,8.70/CoF3(CF2)x-CF(CF3)-(CF2)y-SO2-NH2(Internal monomethyl branch)1,1.20/(cCF3)2CF-(CF2)X-SO2-NH2 (Isopropyblran@h)3,.5% CxF2x+I-CF(CF3)-SO2-NH2 (Alpha branch)and 0.280/(oCF3)3C-(CF2),-SOrNH2(t-ButyblranchX4). BPLC/MS characterizatioofnthePFOSA sample revealed9,600 ppm ofPFOS, 1,100 PPM Of C7Fl5SC@NH2, 5 10 ppm of C9Fl9So2NH2, 6,600 ppm of C8Fl6HS02NH2, 24,000 ppm of CISF36IiSO2NH2,1,2oo ppm of C8Fl5H2SO@M2 and lower concentrationosf severalotheramides.Based on thesum of the impuritiest,hepurityofthePFOSA samplewould be approximately 96%(5). 2 T-6316.17;T-6295.21;T-7132.3;ST41 StabilityD:ocumentationwillbe kepton file. StorageConditions: Testmaterialwillbe storedtightlsyealedatroom temperature. Characteristics: informationon synthesimsethods,compositionor othercharacteristitchsatdefine thetestmaterialwillbe kepton file. Aninuits: Species: Rat Strain: Sprague Dawley Source: Harlan Age atinitiatioonftreatment: 9-11 weeks Weight atinitiationftreatment:approximately200-250g Number and sex: 12 males GROUP 1:controtn=3 GROUP 2:N-Et FOSE, ir--3 GROUP 3:PFO,% n--3 GROUP 4:PFOSA, n=3 Identificatiounn:iqueta mark Husban&y.Housing: Allratswillbe individualhloyused inwirebottom metabolismcages to allow forcollectioonfurineand feces. Diet/Water. HarlanTekladIM-485 Mouse/Rat StaiUmableDiet,suppliedby Harlan Teklad,Madison,WL and tapwaterwillbe providedto allratsad libitum throughoutthestudy. Environment: Environmentalcontrolfsortheanimalroom willbe setto maintaina temperatureof 72 30F,humidityof 30-700/oa,minimum of 10 exchanges of room airperhour and a 12 hour light/darckycle. Dose and Dosing Procedw=.* Method of administration/Dosepreparation: Allratswillbe dosed viaoralSavage on day zeroof thestudyusinga volume of 5 nildosingsuspension/kg body weight.Rats ingroup 2 will receivea single400 mglkg dose ofN-Et FOSE, ratsingroup 3 willreceive a single100 mg/kg doseofPFOS and ratsingroup 4 willreceivea single 100 mg/kg dose ofPFOSA. Uniform suspensionosfN-Et FOSE (8*/o8;0 mWmL) and PFOS (20/o2;0 mg/mL) willbe preparedin2*/*Tween 80 usinga 15-raltissuegrinder.Re-ampension of PFOS and N-ETFOSE solids" be performedwith 5 strokesofthetissuegrinderpestelbefore each swnple isdrawn-up inthe*Tinge fordosing. 3 T-6316.17;T-6295.21;T-7132.3;ST41 PFOSA (2%; 20 mg/mL) willbe preparedby dissolvinignacetone,then formingan emulsionin2% Tween 80 to a finacloncentratioonf 1% acetone,20/oTween 80. Re-suspensionofPFOSA solidswillbe performed by pumping theemulsionthroughthesyringeimmediatelybeforedosing. A single5 ml /kg body weightdose ofvehiclewillbe administeredviaoral Savage to ratsingroup I on day zeroofthe study.Two of theratswill receive2% Tween 80 and I willreceivea mixtureof 1% acetone/2% Tween 80. Obsmiwdon ofamimals.ClinicalObservations: Each animalwillbe observeddailyformortalitaynd morbidityand notable findingswillbe recorded.Additionalfindingwsillbe recordedastheyare observed. Body Weights: Each animalwillbe weighed immediatelypriorto treatmentand immediatelypriorto euthanasia. Frequency and Number of Animals: One controland one PFOSA ratwillbe sacrificeodn day I post dose. All remaininganimalswillbe sacrificeodn day 2-postdose. Specimen Coffeefion& Analysis: Method of Specimen Collection: Unne and feceswillbe collectefdrom each metabolismcage on days one and two postdose. The initiavlolume ofurinewillbe recorded,the sides of theurinecollectioanpparatuswillbe washed with5-10 ml deionized water and thefinavlolwne of urinewillbe recorded.Dailyfecesweight willbe recordedforeachmiinw. Animalswillbe euthanizedby C02 and grossnecropsyperformed.During necropsy,blood (::6utW) wiflbe collectevdiatheabdominalaortaand transferretdoblood collectiotnubeswithoutanticoagulanBtl.ood samples willbe allowedto clotfora periodof 15 to 30 minutesatroom temperature,and theclotwillbe spun down ina centrifugaet I100 x g for 5 minutes.The serum willbe transferretdo labeled1.5ml niicrofugteubes and centffigedagainat2000 x g to reinoveany renminingredblood ceus. Each serasamplewiflthen be dividedinto2 afiquotst,ransferretdo a separatelabeledpolypropylenenicrofugetubeand frozenindry ice.One aliquotwill-beused formetaboliteanalysiasnd theotherforbiochemical analysis.Each livewrillbe excisedand weighed.The liverwillbe flash frozeninliquidnitrogenA. smallsection(approximeely3 gmm) willbe removed and storedat-70*C formetabolitaenalysisl.le remainderwill be storedat-700C forbiochemicalanalysisK.idneysfrom animals sacrificeodn day 2-postdose willbe excised,weighed and flashfrozenin liquidnitrogenforfuturebiochemicalanalysis. 4 T-6316.17;T-6295.21;T-7132.3;ST41 Specim.en Handling: Allurineand fecesand thesenun and Everformetaboliteanalysiswillbe temporarilsytoredat-70*C intheStrategiTcoxicologyLaboratory.For analysist,hesesampleswillbe packed indryiceand shippedto: KrisHansen, Ph.D. 3M EnvironmentalTechnology and SafetyServices 935 Bush Avenue St.Paul,l@N 55133-3331 TelephoneNo.: 651-778-6081,FacsimilNeo.:,651-778-6176. Alllddney specimensand theremainderof serum and liverwillbe keptin the StrategiTcoxicologylabforbiochemicalamdysis.These spedmmm willbe storedat-700C. The number,type,and dateof specimensto be generatedfbranalysisareas follows: TABLE I --Specinsens.fEonrWron Specimen Eavironm&wd Lab Spedmens &Pategk T= Lab specimms Urine Feces Sera Liver Sera Liver IKidneys tallab Collectiodnate Day I postdose Day 2 postdose 12 10 12 ID 2 10 2 10 2 10 2 10 1 10 Total 22 22--, 12 12 12 12 10 Data Analysis.AD results will be provided for inclusion in the finalreport. Rmponsibilides. o Deama Luebker and Andrew Seacatwiffbe responsiblfeordosingtheanimals, collectinign-lifsepecimens,perfomiingthenecropsiesand collectinagnd sending tissuespecimens foranalysis. o Andrew Seacatwilldrafta finarleportand ensurethereportreceivesappropriate3M reviewbeforea finalreportisissued. SignoWres: - o@2@A, 9, Dr. Andrew Seacat SeniorResearch To@micologist Study Director Deanna L Luebker,(IS Advanced ToAcologist Study To3dcologist T-6316.17;T-6295.21;T-7132.3;ST41 1/ Date Date 6 T-6316.17;T-6295.21;T-7132.3;ST41 References: 1. Payfer,R- Characterizationf FM-3923, MuctureofLots 30035,30037 & 30039 fbr Two Year FeedingStudy.SMD AnalyticaRlequest#52489. AnalyticaRleport 816,6/23/97.3M SMD Lab Building236-2B-11. 2. Kestner,T. FluorochemicalIsomerDistributiobny L@F-NMR Spectroscopy. SpectroscopyRequest # 53030.3M SpecialtAydhesives& ChemicalsAnalytical Laboratory/SMMD-236-2B-1 1,December 1,1997. 3. Payfer,R- GC/MS analysiosfPFOSA (L-10009).SA&C AnalyticaRlequestNo. 59426.Report 9/24/99.3M SA&C Lab Building236-2B-11. 4. Kestner,T. CheniicalChwWaization ofPFOSA, L-10009,by IH and 19F-NMP, SpectroscopyRequest # 59426.3M SpecialtAydhesives& ChemicalsAnalytical Laboratory/SNEMD-236-2B-1 1,September25, 1999. 5. DeRoos F.ChametedzationofPFOSA Samples,T-7132-1(L-10009)and TN-A1584.Request# A-] 51254. Report 10/7/99,and Letteraddendum to report10/14/99. CorporateAnalyticaTlechnologyCenter,Building201-1-29,CATC Chromatography Group. 7