Document jmZyq0O9KRrdJ8LQQMb0pOwK9

3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY ProtocolforStudy No. T-7098.1 PHARMACOKINETIC STUDY OF POSF IN RATS 01/07/99 T-7098.1 POSF PK StudyObjective: The objectivoefthisstudyistoassessthepotentiaflororalabsorptionu,rinaraynd fecal clearancaend biologicaplersistencoefperfluorooctanseulfonyflluorid(ePOSF) inmale SpragueDawley ratsaftera singleoraldose. The POSF compound isthestartinmgaterial forthesynthesiosfa wide varietyofperfluorooctanreulfonat(ePFOS) basedmaterials. The purposeof thisstudyisto understandtherateofmetabolismofPOSF toPFOS by the liver.ThisstudywiU providedataforproperriskcharacterizafioofnPOSF. Research CYient: 3M SpecialtCyhemicalsDivision 3M Center,Building236 SaintPaul,MN 55133-3220 Sponsor.- 3M SpecialtCyhemicalsDivision 3M Center,Building236 SaintPaul,MN 55133-3220 Study Location: 3M StrategiTco)dcologyLaboratory 3M Center,Building270-3S-06room SB314 SaintPauL MN 55133-3220 Study Director. Andrew M. Seacat,Ph.D. Sr. ResearchToxicologist 3M MedicalDept./CorporateToxicology 3M Center,Building220-2E-02 SaintPaul,MN 55133-3220 Ph.:651-575-3161FAX: 651-733-1773 Study Toodcologist:Deanna Nabbefeld,MS Advanced ResearchToxicologist 3M MedicalDept./CorporateToxicology 3M Center,Building220-2E-02 SaintPaul,MN 55133-3220 Ph: 651-737-1374FAX: 651-733-1773 Proposed Study Timeline(AssumingEHS&R approvalon Jan.5Ih In-Life Start Date: Januwy IIth,1999 In-Life End Date: February 9th, 1999 Analytical Completion Date: March 22nd, 1999 Final Report Completion Date: April 19th, 1999 01/07/99 T-7098.1 POSF PK RegulatoryConiptiance., This studywillbe performedinthe3M StrategiTcoxicologyLaboratoryunder a defined protocoland classifiaesda "ClassB Study"as explainedinTOX SOP 0950, Strategic ToxicologyLab GLP Program Procedure. TestMaterial.Dan Hakes,ProductResponsibiliLtiyaison3M ChemicalsDivisionw,illfurnishighpurityPOSF. Identification: Name: PerfluorooctanSeulfonylFluoride Molecular Formula: To be provided. Lot Number: The lotnumbers willbe maintainedintheraw data. Purity: Documentationwillbe kept inon file. Stability: Documentationwillbe kept on file. Storage Conditions: Upon receiptt,estmaterialwillbe storedtightlsyealedatroom temperature. Characteristics: Informationon synthesismethods,compositionor othercharacteristitchsat definethetestmaterialwillbe kept on file. Animals: Species: Rat Strain: Sprague Dawley Source: Harlan Age at initiatioonftreatment: Weight at initiatioonftreatment: Number and sex: 30 males 6-8 weeks approximately150-250g Table I - Dose Groups Group *1 Dose N 0 mg/kg 5 Euthanasia day 1 post dose *2 0 mglkg 5 day 4 post dose *3 0 mgft 5 day 29 post dose 4 5 mg/kg 5 day I post dose 5 5 mg/kg 5 dg 4 postdose 6 5 mgft 5 day 29 post dose * Rats ingroups 1-3willbe used concomitantlyas controlanimalsinstudiesT-7071.2, PharmacokineticStudyofM556 inRats,and T-7099.1,PharmacokineticStudyofFX- 845 inRats. Identification: AUA Number: eartagwith animalnumber or uniquetailmark. 2154 2 01/07/99 T-7098.1 Husbandry: POSFPK Housing: Three specifircatsfrom groups 3 and 6 willbe housed individualliyn metabolismcagesforportionsofthestudy(seeTable2). When not in metabolismcages,theseratswillbe group housed instandardcages.All otherratswillbe group housed instandardcagesthroughoutthestudy. Diet/Water: HarlanTekladLM485 Mouse/Rat StefflizabDlieet,suppliedby Harlan Teklad,Madison,WI, and tapwaterwillbe providedto allratsad libitum throughoutthestudy. Environment: Environmentalcontrolsfortheanimalroom willbe setto maintaina temperatureof 72 3*F,humidityof 30-70%, a minimum of 10 exchanges ofroom airperhour and a 12 hour fight/darckycle. Dose and Dosing Procedures: Method of administration/Dosepreparation: A single5mglkg dose ofPOSF willbe administerevdiaoralgavage to rats ingroups4-6 on day zero ofthe study.The POSF willbe preparedas a 1% (1 mg/ml) uniformsuspensionin2% Tween 80 usinga 15 ml tissue grinder.A volume of 5 ml suspension/ kg body weightwillbe administeretdo each rat.Re-suspensionof solidswillbe performedwith 5 strokesofthetissuegrinderpestelbeforeeach sampleisdrawn-up inthe syringefordosing. A single5 ml / kg body weightdose of2% Tween 80 willbe administered viaoralgavage to ratsingroups 1-3on day zero ofthestudy. Observationofanimals. ClinicalObservations: Each animalwillbe observeddailyformortalitaynd morbidityand notable findingwsillbe recorded.Additionalfindingwsillbe recordedastheyare observed. Body Weights: Each animalwillbe weighed immediatelypriorto dosing,weekly thereafter and immediatelypriorto euthanasia. Specimen Collection: Frequency (seeTable2): Urineand fecescollectionwsiD be made on days 1,2,4, 14 and 29 post dose. Necropsieswillbe performedon days 1,4 and 29 postdose. 3 Table 2 -Schedule Jan 10 Jan 11 day 0 DOSING Jan 12 Jan 13 Jan 14 day 1 PD day 2 PD day 3 PD Collection Collection Switchto Dy I PD Switchto met cages. sac reg cages. Jan 17 day 6 PD Jan 24 day 13 PD Jan 31 day 20 PD Feb 7 day 27 PD Jan 18 Jan 19 Jan 20 day 7 PD da_v8 PD da 9 PD Jan" 25 Jan 26 Jan 27 day 14 PD day 15 PD day 16 PD Switchto Collection met cages. Switchto reg.cages. Feb 1 Feb 2 Feb 3 day 21 PD day 22 PD day 23 PD Feb 8 Feb 9 day 28 PD day 29 PD switchto collection met cages Dy 29 PD I sac Jan 21 day 10 PD Jan 28 day 17 PD Feb 4 day 24 PD Jan 15 Jan 16 day 4 PD day 5 PD Collection Switchto reg cages. Dy 4 PD sac. Jan 22 Jan 23 day 11 PD day 12 PD Jan 29 Jan 30 day 18PD day 19 PD Feb 5 Feb 6 day 25 PD day 26 PD 01/07/99 T-7098.1 POSF PK Method of Specimen Collection: Urine and feceswillbe collectedfrom each metabolism cage atthe designatedtimes.The initiavlolume of urinewillbe recorded,thesidesof theurinecollectioanpparatuswillbe washed with 10-20 ml deionized water and thefinavlolume ofurinewillbe broughtto 45 ml withadditional deionizedwater. Day fecesweightwillbe recordedforeach animal.At thedesignatedtimes,animalswillbe euthanizedby C02 and grossnecropsy performed.During necropsy,blood (@-6-ml) willbe collectevdiathe abdominalaortaand transferretdo blood collectiotnubeswithout anticoagulantB.lood sampleswillbe allowedto clotfora periodof 15 to 30 minutesatroom temperaturea,nd theclotwillbe spundown ina centrifugaet 1100 x g for5 minutes.The serum willbe transferretdo labeled1.5ml niicrofugteubesand centrifugeadgainat2000 x g to remove any remainingredblood cebs. Each serasamplewillthenbe transferretdo a separatelabeledpolypropylenetnicrofugteubeand flashfrozeninliquidnitrogen.Liver,kidneysand subcutaneousfatfrom each animalwillbe removed, weighed,flashfrozeninliquidnitrogenand placed individualliyntolabeledsterilseamplebags. The remainderof each carcass willbe placedina labeledziplockbag and frozen(-70'C) untilanalysis. Specimen Handling: Specimenswilltemporarilybe storedina freezersetto maintain-60 to 80*C. For metaboliteanalysist,hesespecimenswillbe packed indry ice and shippedto: 4 KrisHansen, Ph.D. 3M EnvironmentalTechnology and SafetyServices 935 Bush Avenue St.Paul,MN 55133-3331 Ph: 612-778-6081,FAX: 612-778-6176. 01/07/99 T-7098.1 POSF PK The 3M EnvironmentalLaboratorywillmanage theextractioannd analysis of sera,liveru,rineand fecesfortheparentcompound, POSF, and its presumed metaboliteP,FOS. AU tissuesampleswillbe retainedforpossible futureanalysiosf totalorganicfluorin(eTOF) ifdeemed necessaryby the StudyDirector.3M EnvironmentalLaboratoryor itsdesigneewould performthisanalysisA.llresultwsillbe providedforinclusioinnthefinal report. The number,typeand dateof collectioonf specimensto be generatedfor analysiasre as follows: Table 3 - Specimens Specimen day I post dose Serum 10 Liver 10 Kidneys 10 Subcutaneousfat 10 Carcass 10 Urine 6 Feces 6 day 2 post dose 6 6 day 4 post dose 10 10 10 10 10 6 6 day 15 post dose 6 6 day29 post dose 10 10 10 10 10 6 6 Total 30 30 30 30 30 30 30 Data Analysis: Data collectedon tissuelevelsof parentcompound and identifiabmletaboliteswillbe analyzedfortoxicokinetipcarametersand forstatisticaslilgynificadnitfferencebsetween groups usingStudentsT-testand/orANOVA. Responsibilhiff. Deanna Nabbefeldand Andrew Seacatwillbe responsiblfeordosingtheanimals, collectinign-fifsepecimens,performingthenecropsyand collectinagnd sendingtissue samplesforanalysis. KrisHansen, 3M Environmental,willbe responsiblfeoranalyticaalnalysis. Andrew Seacatwilldrafta finarleportand ensurethereportreceivesappropriat3eM reviewbeforea finalreportisissued. 5 Signatures: Dr7@drer@ew Seacat SeniorResearchToxicologist Study Director L7 Deanna Nabbefei,MS Advanced Toxicologist Study Toxicologist Sponsor Representative f)ate Date Date 01/07/99 T-7098.1 POSF PK 6