Document OzaxLoN8gD3wKNY7dnrowB8Nw

08/13/99 T-6316.15;ST-30 3M MEEDICAL DEPARTMEENT, CORPORATE N-ETFOSE Bile TOXICOLOGY ProtocolforStudy No. T-6316.15;ST-30 N-ETFOSE BfleMethod DeveloRment inRats Study Objeedve.* The objectivoefthisstudyisto collecutrineand bilespecimensfrom controland NETFOSE treatedratsformethod development.ThisstudywillsupporttheN-ETFOSE ADNM studyinCynomologus monkeys currentluynderway atCovance Madison. Research Client. 3M SpecialtCyhemicalsDivision BiodegradableSurfactantTseam 3M Center,Budding 236 SaintPa4 MN 55133-3220 sponsor.. SVudy Locadon: 3M SpecialtCyhemicalsDivision 3M Center,Building236 SaintPauL MN 55133-3220 3M StrategiTcoxicologyLaboratory 3M Center,Building270-3S-06room SB314 SaintPaul,MN 55133-3220 Study Diredor.- Andrew M. Seacat,Ph.D. Sr. ResearchToxicologist 3M MedicalDept./CorporateToxicology 3M Center,Building220-2E-02 SaintP" MN 55133-3220 Ph.:651-575-3161FAX: 651-733-1773 Study Ta@dcokgist Deanna Luebker,MS Advanced ResearchToxicologist 3M MedicalDept./CorporateToxicology 3M Center,Building220-2E-02 SaintP" MN 55133-3220 Ph: 651-737-1374FAX: 651-733-1773 Proposed Study 7-imaUne. b-Life StartDate: 8/12/1999 In-lAfeEnd Date: 8/20/1999 Regul&M Conrfiance.* Thisstudywillbe performedinthe3M StrategiTcoxicologyLaboratoryunder a defmed protocoland cw"ed as a "ClassB Study"as explainedinTOX SOP 0950,Strategic Tomeology Lab GLP Program Procedure. 09/13199 T-6316.15;ST-30 N-ETFOSE Bile TestMaterial: Dan Hakes, ProductResponsibiliLtiyaison3M ChemicalsDivisionw,illfumish highpurityN-ETFOSE. Identification: Name: Narrow Range N-EthylperfluorooctanestffonamiedtohanolF,M3923. MolecularFormW&: C8Fl7SO2N(CH2CH3)CH2CH20H Lot Number.- Lots 30035,30037, 30039 mixed and analyzedas one sample. Purity: 98.14% Stability: Documentationwillbe kepton file. StorageConditions: Upon receiptt,estmaterialwillbe storedtightlsyealedatroom temperature. Characteristics: Informafionon synthesimsethods,compositionor othercharacteristitchsat definethetestmaterialwillbe kepton file. Animals. Species: Rat Strain: Sprague Dawley Source: Harlan SpecificationsB:ileCannulated- Free End Age atinitiatioonftreatment: 6-8 weeks Weight atinitiationftreatment: approximatel1y50-250g Number and sex: 20 males,20 females Table I -Dose Groups -Group Dose N 1 0 mgft 5 2 20 in 5 13 0 meft 5 14 120 mwkg 15 Sex Male Male Female Female Identification: cartagwithanimw number or uniquetaimlark. AUA Number.- 2246 Husban&y.Housing: Upon arrivaalt3M allanimalswillbe individualhloyused instandard cages. AR ratsfrom groups2 and 4 and I eachfrom groups I and 3 willbe transferretdoindividuamletabolismcagesfollowingdosingand housed 2 08/13/99 T-6316,15;ST-30 N-ETFOSE Bile therethroughday 4-postdose.Allotherratswillremainindividually housed instandardcagesthroughoutthestudy. Diet/Water: HarlanTekladLM-485 Mouse/Rat StedlizablDeiet,suppliedby Harlan Teklad,Madison,WL and tapwater willbe providedto allratsad libitum throughoutthestudy. Environment: Environmentalcontrolfsorthe mfimalroom willbe se to maintaina temperatureof 72 3*F,humidityof 3 0-700/ca,minimum of 10 exchanges of room airperhourand a 12 hour fight/darckycle. Dose and Dosing Procedures.Method of administration/Dosepreparation: A single20mg/kg dose ofN-Et FOSE willbe administerevdiaoralgavage to ratsingroups2 and 4 on day zero ofthestudy.The dosingsolutionwill be preparedas a 0.040/(o4m&d) uniformsolutioinn20/cTween 80 usinga 15 mi tissuegrinder.A volume of 5 irAsuspension/ kg body weightwillbe administeredto eachrat.Re-suspensionof solidswillbe performedwith5 strokesof thetime grinderpestelbeforeeachsampleisdrawn-up inthe syringefordosing.A single5 rnl/kg body weightdoseof rlo Tween 80 willbe administerevdiaoralgavage to ratsingroups I and 3 on day zero ofthe study(seetable2). Observadon ofanimals.ClinicalObservations: Each animalwillbe observeddailyformortalitaynd morbidityand notable findingswillbe recorded.Additionalfindingwsillbe recordedas theyare observed. Body Weights: Each wfimalwillbe weighed immediatelypriortodosingand immediately priorto euthanasia. Specimen Cogection: Frequency (Seetable2): Bilewillbe collecteddailyupon arrivaalt3NL (AU bflecollectepdriorto dosingwillserveascontrolbile.)Urinecollectiownisllbe made on days I 4 post dose. NecropsieswiU be performedon day 4-postdose. 3 Table 2 - Schedule Sun Mon Aug 15 Dy-1 BilecoU Augl6 DyO DOSE Bilecoll Tues Augl7 Dyl postdose Bilecoll Urine coll 08113/99 T-6316.15;ST-30 N-ETFOSE Bile Wed Aug 18 Dy2 postdose BilecoH. Urine coll Thurs Augl2 Dy-4 Arrival@ 3M Bilecoll Aug 19 Dy3 postdow BUe coll Urine com Fri Augl3 Dy-3 Bilecoll Aug 20 Dy4 poodose Bilecoll Urinecoll Sat Augl4 Dy-2 Bilecoll Method of Specimen Collection: Bilewillbe collectefdrom eachratdailyupon arrivaalt3Ni The exteriorizepdortionofthecatheterwillbe removed from the pouch of the protectivjeacketandthewireplugintheFreeEnd cathetewrillbe removed. A newlyopened syringewillbe used towithdrawnbilefrom the catheterof eachrat.The bflefrom eachmt willbe equallyspliatnd placed into2 labeledtubes.The wireplug willbe insertebdackintothecatheter followingbflecollectioannd theexteriorizepdortionofthecatheterplace inthepouch oftheprotectivjeacketto preventcatheterchewing.Each tubeofbilewillbe frozen(-70*C) untilanalysisU.rinewillbe collected from each metabolismcage on days 1-4post dose. For each urine specimen,theinitiavlolume willbe recordedand thesidesof theurine collectioanpparatuswillbe washed with 10-20raldeionizedwater. The urineand wash from each cage willbe collecteidna 50 rnltube and the finalvolume willbe broughtto 45 mi withadditionadleionizedwater. Each urinespecimenwillbe frozen(-70*C) untilanalysisO.n day 4 post dose animalswillbe euthanizedby C02 and grossnecropsyperformed. During necropsy,blood(z 6 n-d)willbe collectevdiatheabdominalaorta and transfeffetdobloodcollectiotnubeswithoutandcoagulant.Blood sampleswillbe allowedto clotfora periodof 15 to30 minutesatroom temperature,and theclotwillbe spun down ina cenu*ge at I100 x g for 5 minutes.The serum willbe transferretdo labeled1.5 ml microfugetubes and centrifugedagainat2000 x g to remove any remainingredblood cens. Each sera swnple willthen be transferredto a separatelabeled polypropylenenicrofugetubeand (-70*C) untilanalysisE.ach livewrill be removed, rinsedinsalinew@eighed,placedintoa labeledsterilseample bag and frozen(-70*C) untilanalysis. Specimen Handling and Analysis: Specimens willtemporarilbye storedina freezersetto maintain-60 to 800C intheStrategiTcoxicologyLaboratory. 4 09113/99 T-6316.15;ST-30 N-ETFOSE Bile For metaboliteanalysisu,rine,liverand serawillbe packed indryiceand shippedto: KrisHansen,Ph.D. 3M EnvironmentalTechnologyand SafetyServices 935 Bush Avenue St.PauL MN 55133-3331 Ph: 612-778-6081,]FAX: 612-778-6176. For analyticamlethod developmentpurposes,bilewillbe packed indry icedand shippedto: KarstenLevsen FraunhoferInstituftuerTo)dkologieund Aerosofforschung N'AolaiFuchsstr1.D-30625 Hannover GERMANY The StrategiTcoxicologylaboratorwyillretainone halfof allthebfle specimenscollectefdorin-housemethod development,verificatioanalysis and/oras a retainforpossiblefutureanalysis. Allresultswillbe providedforinclusioinnthefinarleport. The number, typeand dateof collectioonf specimensto be generatedfor analysisareas follows: Table 3 -Speci ens Sp day -4 Bilefor 20 Fraunhofer Bileforin- 20 house analysis Urine Serum Liver day day day day0 dayI day2 day3 day4 Total -3 -2 -1 PD PD PD PD 20 20 20 20 20 20 20 20 ISO I I 20 120 20 20 20 20 20 20 180 I 12 12 12 12 48 - 20 20 20 20 Data Analysis. Data coffectedon N-ETFOSE metaboliteconcentrationisnserun,4liverand urineusing validatedmethods wiU be analyzedforto)dcoldnefipcarmeten and forstafisficeuy significadnitfferencebsetween groupsusingStudentsT-testand/orANOVA- Data collecteduringthemethod developmentand validatiopnhasesof bileand unne metaboliteanalysiswillbe analyzedforprecisioannd accuracyasnecessaryto developa validatedmethod. Providedthatenough oftheretainedbileand urinesamplesare 5 W13/99 T-6316.15;ST-30 N-ETFOSE Bile availablfeollowingthedevelopmentofvalidatemdethod theymay thenbe analyzedforNETFOSE metabolitelevelsunder thevalidatedmethods to generatedataforuse ina definitivreport. Responsibilhies. Deanna Luebker and Andrew Seacatwillbe responsiblfeordosingthewiinws, couft-tinign-fifsepecimens,performingthenecropsiesc,ollectinagnd sending specimensforanalysiasnd may performadditionavlqmiments on thecollectetdissues formethod developmentor verificatio'n. KrisHansen,3M EnviromnentaLwillbe responsiblfeoranalysiosf serum and liver tissuesa,nd theurinesamplesfollowingdevelopmentof a validatemdethod forurinary metaboliteosfN-Et FOSE ifpossible. KarstenLevun, Fmunhofer Insdw fuerToxikologieund Aerosolforschunwgillbe responsiblfeorthedevelopmentofa validatemdethod forbiharymetabolitesand the subsequentan*= oftheretainebdflesampim providedthereisa mfficienstupply. Andrew Seacatand Deanna Luebker" drafta finarleportand ensurethereport receivesappropriate3M reviewbeforea finarleportisissued. 6 Signatures: Dr. Andrew Seacat SeniorResearchToxicologist Study Director Deanna Luebker,MV' Advanced Toxicologist Study Toxicologist Sponsor Representative O8tl3/99 T-6316.15;ST-30 N-ETFOSE Bile 7@7 /5Iq Date Date 4t2 /L4i5/)-7 Date I,?A I I -&I 7