Document kmRjQ9ZyB10gQRZz9L5Jd3QzO

Pathology Associates International A Company of ScienceApplicationsInternationaClorporation AnErobrmk)wmdCoMmy Sponsor: 3M St.Paul,Minnesota PROTOCOL Study Title: CellProliferatiSotnudy withN-EthylPerfluorooctanesulfonamEitdhoanol(N-EtFOSE; 3M T-6316.11), PerfluorooctanSeulfonicAcid PotassiumSalt(PFOS; 3M T-6295.16)a,nd N-EthylPerfluoroctanesulfonamid (PFOSA 3M T-7091.1)inRats Date: January12, 1999 Performing Laboratory R.O.W. Sciences 15 FirstrieRlodad GaithersburgM,aryland 20878 Laboratory Study Identification: Study Number: (1132-100) PAI ProjectNumber: (Histolognyumber tobe assignedby PAI by protocolamendment) iS Worman's Mill Court, Suite I Frederick, Maryland 21701 * (301) 663-1644 (301) 663-8994 FAX Study Number 1132-100 Page 2 Study CellProliferatiSotnudy withN-EthylPerfluorooctanesulfonarnEitdhoanol(N-EtFOSE; 3M T6316.11),PerfluorooctanSeulfonicAcid PotassiumSalt(PFOS; 3M T-6295.16),and N-Ethyl Perfluoroctanesulfonani(iPdFeOSA 3M T-7091.1)inRats Purpose To assesscellproliferatiaonnd peroxisomeproliferatiionnratsadministeretdestmaterialinthe diet. Sponsor 3M CorporateToxicology Building220-2E-02,3M Center St.Paul,NN 55144-1000 Study Representative Marvin T. Case,D.V.M, Ph.D. 3M CorporateToxicology Phone No.: 651733-5180 Fax No.:651733-1773 Email:mtcase@numn.com AlternativeStudy Representative Andrew M. Seacat,Ph.D. 3M CorporateToxicology Phone No.: 651575-3161 Fax No.:651733-1773 Email:amseacat@mnun.com Study Location R.O.W. Sciences 15 FirstfielRdoad GaithersburgM,aryland 20878 Study Number 1132-100 Page 3 Study Monitor Sandra R. Eldridge,Ph.D. PathologyAssociatesInternational Phone No. 301624-2036 Fax No. 301663-8994 Email:srepaisaic@aol.com Study Director Gary W. Wolfe,Ph.D.,D.A.B.T. R.O.W. Sciences Phone No.: 301330-3723 Fax. No.: 301330-3738 Email:gwolfe@lab.row-com Principal Investigator San&a R. Eldridge,Ph.D. PathologyAssociatesInternational Phone No. 301624-2036 Fax No. 301663-8994 Email:SREPAISAIC@aOl.cOm Study Pathologist Carolyn Moyer, D.V.M., Diplomate,A.C.V.P. PathologyAssociatesinternational Phone No. 301624-2928 Fax No. 301663-8994 Proposed Study Timetable In-lifSetartDate:To be addedby protocoalmendment; Day 0 In lifeEnd Date:To be added by protocolamendment AuditedDraftReportDate:To be added by protocolamendment Regulatory Compliance This studywillbe conductedinthespiriotfGood LaboratoryPractice(GLP) regulations. S tudy Number 1132-100 Page 4 Animal Care and Use Statement AllproceduresinthisprotocolareincompliancewiththeAnimal WelfareAct Regulations9, CFR 1-4.In theopinionof theSponsorand studydirectort,hestudydoes notunnecessarildyuplicate any previouswork. Quality Assurance Not applicable. Test Materials TestMaterial: N-hWubr, (completedby 3K -IdenEfic-ati'o-nN:-btkL)br, Lot Number: FM 3929 Purity. -- -9-@-2- 1Vo Stability: > 5 years Storage room temp. Conditions: Characteristics:waxy solid Y=SOI( PFOS (completedby 3W PFOS 217 99% > 5 years room temp. whitepowder PFOSA (tobe added by protocol khendment) -FF-OSA Wy-144,o76l463437] (tobe added by proetnodcmoelnt) ametnodcment) Wy room temp. amber waxy solid Reserve (Archive)Samples A reservesample (approximately5 g)of eachlotwillbe takenand stored.artoom temperature. These samples willbe transferretdotheSponsoraftercompletionof thein-lifpehase tobe retained in accordancewith 40 CFR 792.195. Dispositionof Test Material Afterauthorizatiofnrom theSponsor,any remainingtestmaterialwillbe returnedto: Marvin Case,D.V.M., Ph.D. 3M CorporateToxicology Building220-2E-02,3M Center St.Paul,Minnesota 55144-1000 Phone No.:651-733-5180 Fax No.: 651-733-1773 Study Number 1132-100 Page 5 Animals pecies: s-EF-::ni@n:e_s:@ Source: Age atInitiatiofnTreatment: Crl:CD'(SD)IGS BR CharlesRivFr-L-aboraton@eI-n-cs.,,Raleigh,NC Preferabfe-6weeks of age,but not more than 8 @Week-sof age Weight atInitiatioofnTreatment: 150 to300 9 umber and Gendern males identificatiboyn-fn-@viduaclartagsand cage cards Study Number 1132-100 Page 6 Husbandry Housing: Diet: Sin0alehoused inhana0ing stainlesssteelwireca0oes 1 Teklad 7012 CertifieRdodent Diet.Freshfoodwillbe providedweekly. Feed isanalyzedby themanufacturerforconcentrationosf specifiedheavy metals,aflatoxinc,hlorinatheydrocarbons,organophosphatesa,nd specified nutrientsS.pecifiednutrientasnalysesareon fileatR.O.W. Sciences. Water- Tap water,providedaA libitumviaan automaticwateringsystem or water bottles.The waterisanalyzedatleastwo timesperyearforcontaminantsand specifimcicrobes.The resultosftheseanalysesareon fileatR.O.W. Sciences. Contaminants: The studydirectoarnd/ortheSponsor have consideredpossibleinterfering substancespotentiallpyresentinaniinalfeedand water,includingthetest materialitselofrpossiblestructuralrleylatedmaterialasswellas theitems listeidn(2)and(3) above. None ofthesecontaminantsarereasonably expectedtobe presentinanimalfeed orwateratlevelsufficientto interfere with thisstudy. Environment: The targetetdemperaturesarebetween 64 and 79F witha relativheumidity between 30% and 70%. Temperatureand humidityaremonitored continuouslyA. 12-hourlight/12-houdrarkcyclewillbe maintained.Ten or greaterairchanges/hourwillbe maintained. Acclimation: Animalswillbe acclimatedtothefacilitfyora minimum of 7 days priortothe starotf dosing.Animals willbe observedforgeneralhealthand suitabiliftoyr testingduringthisperiod.Animals thatarediseasedorunsuitablefortesting willbe removed from thestudy. Randomization: Usina computer-oeneratedrandom numbers witass0i-nmenttogroups,At the time0of randomizca'tiont,heweightvariatioonf theanimalsof each sexused shouldnotexceed 2 S.D.of themean weight,and themean body weights foreach group ofeach sex willnotbe statisticadlilfyferent. Justification: Ratswillbe used becauseof theextensivehistoricdaaltabase,and theFDA requirementsfora rodentspecies. Study Number 1132-100 Page 7 Group Designations, Dietary Levels and Scheduled Sacrifice Time Points NumbeFofM-ale Rats Group Number Control N-ETFOSE (TimePoint) (0pprq)--300 100 30 ppm 1 10 10 10 10 (48hrs) 2 10 55 5 (7d s) 3 10 55 5 (14days) - 4 (Iwk 10 55 5 10 l@twk recovery' TToottaalNo. of Animals 50 55 5 30 30 30 PFOS 10 5 5 5 5 30 PFOSA Wy-14,643 TotalNo. --IOOPPM--Iooo m ofanimals 10 5 65 5 5 40 5 5 40 5 5 40 5 5 40 30 25 225 Dosing Procedures Method of Administration Dietary.AnimalsinGroupsI through3 willreceivteestdietfor48 hours,7 days,and 14 days,respectively. Animals inGroups 4 and 5 willreceivetestdietfor14 daysfollowedby a I or 4 week recoveryperiod,respectively. Reason for Dosing Route The potentiahluman exposureisby theoralroute. Dose Preparation Before initiatiofn treatmentd,osepreparatioonfeach testmaterialwillbe mixed. Alldose preparationwsW be storedatroom temperature.Dose preparatiownillbe documented and reported.See AttachmentI fortestdietpreparatiopnrocedures. Study Number 1132-100 Page 8 Retention Sample Samples (approximately100 g) willbe taken from thedose preparatioannd storedatroom temperature.Unless used foranalysest,hesesampleswillbe discardedatleast1 month aftercompletionof thein-lifpehase. Observation of Animals Clinical Observations Each animalwillbe observedtwicedaily(am. and p.m.)formortalitaynd moribundity; findingswillbe recordedas theyareobserved. Body Weights Priortotreatment(atrandomization)w,eekly forWeek I through4 weeks ofrecovery. Food Consumption Weekly forWeek I through4 weeks ofrecovery. ClinicalChemistry Animals willbe fastedovernightbeforeanimal'sschedulednecropsy;bloodwillbe collectefdrom a jugularveinintoan EDTA-coated tube.Serum enzyme levelsof alaninean-dnotransfera(sAeLT), alkalinpehosphatase,aspartataeminotransfemse(AST),cholesteroalnd triglyceridweisllbe determined. Termination Unscheduled Sacriflces and Deaths Necropsieswillbe done. Animals tobe sacrificewdillbe anesthetizewdithCO,, weighed,and exsanguinated. Study Number 1132-100 Page 9 Scheduled Sacrifices Interim Sacrifices At 48 hrs,7 days,and 14 days,animalswillbe fastedovernight,bled forserum samples, anesthetizewdithC021weighed, and exsanguinated. NOTE: Two serum sampleswillbe needed,(1)a 0.5ml sampleforclinicaclhemistryand (2)a 1.5ml sample forcompound levelanalysis. The abdominalcavityof eachanimalwillbe opened,theliverwillbe removed and weighed,and liversampleswillbe collectedA.nimals willbe discardedafterliver collection. Terminal Sacrifices After1 and 4 weeks ofrecovery,animalswillbe fastedovernightb,ledforserum samples, anesthetizewdithC02,weighed, exsanguinated,and necropsied. NOTE: Two serum sampleswillbe needed,(1)a 0.5ml sampleforclinicaclhemistryand (2)a 1.5ml sampleforcompound levelanalysis. Postmortem Procedures Necropsy The necropsywillincludeanexaminationoftheexternafleatureosfthecarcass;allexternal body orificest;heabdominal,thoracica,nd cranialcavitieso;rgans;and tissues. Cell Proliferation Tissue Collection and Immunohistochemical Evaluation Representativseamples of theleftlaterallobeof theliverand any macroscopic lesionsof the liverwillbe collecteadnd preservedinzincformalin. Afterfixatione,ach sampleof Everwillbe deliveredto: Sandra R. Eldridge,Ph.D. PathologyAssociatesInternational 15 Worman's MillCourt,SuiteI FrederickM,aryland 21701 Study Number 1132-100 Page 10 Proliferaticoenllnuclearantigen(PCNA) evaluatiownillbe done on thesamples. In addition, liversectionpsreparedfrom thesame tissueblockwillbe stainedwith hematoxylinand eosin and examined microscopically. Palmitoyl-CoA Oxidase Tissue Collection and Analyses A sample (approximately500 mg) of therightlaterallobeof theliverwillalsobe collectefdrom selectanimalsand flash-frozeinliquidnitrogen.See AttachmentIIfIorprocedure. 71beliver tissuewillbe storedina freezersettomaintain-60 to-80*C untilanalyzedby Covance for palmitoyl-CoAOxidaseactivity.The liversamplesto be analyzedwillincludeallstudy animals,EXCEPT forthe Wy-14,643 animalsand allanimalsfrom the 4-week recovery groups. in additiotno thisstudy,samplesfrom a previous3M studywillbe analyzedfor palmitoyl-CoAOxidaseactivittyh;esesamples consistof liversamples from 35 ratsand 35 guinea pigs. Tissue Collection for Electron Microscopic Evaluation Sectionsof liverfrom allanimalswillbe collectedn,iincedto approximatelyone millimeter cubes and placedina fixativaeppropriatfeorelectronmicroscopy.The containersand fixative willbe providedby PAI. Electromnicroscopywillbe performedon one miinw perteatmnt group exhibitintghehighestcellproliferatirveesponseas wellas one controlmih@ atthe discretioonftheSponsor,from one timepointaswellasthe4-week recovery.Thus, EM will be performedon one animalfrom thecontrolN,-ETFOSE (one dose only to be determined), PFOS, PFOSA, and Wy groupsatone of thetimepoints,aswellas the4 week recovery,for a totalof 10 animals. Remaining Liver Tissue The remaininglivertissuewillbe frozenand storedat-60 to -80'C for possiblefuture analysis. Organ Weights At thescheduledsacrificetsh,eliverwillbe weighed. Histopathology Liverfrom each animalthatisexaminedforcellproliferatiwoinllbe stainedwith hematoxylin and eosin,and examined microscopicallfyorhistopathologicchanges. Study Number 1132-100 Page II Reports One copy of thedraftreportwillbe sentto the Sponsor. The reportwillincludethe following information: Experimental Design and Methods Results dose analyses mortality clinicaolbservations body weights body weight changes food consumption testmaterialconsumption clinicaplathologyresults palmitoyl-CoA oxidaseactivities macroscopic observations microscopicobservations ultrastrucmraolbservations cellproliferatioanssessments Record Retention AR raw data,documentation, records,protocol,specimens, and finalreport generated as a resultof thisstudy willbe archivedin the storagefacilitioefsPAI for a period of 1 year followin-submissionofthefinalreporttotheSponsor. One yearaftersubmission of thefinal report,allof the aforementionedmaterialswillbe senttothe Sponsor and a returnfee willbe charged. All raw datastoredon magneticmedia willbe retainby PAI. Study Number 1132-100 Page 12 PROTOCOL APPROVAL marvin*ase, 2D@-V Study Representative 3M CorporateToxicology 04 UEY- W. e, h.D., StudyD' t r R.O.W. sSv-*Inrces A. T Eldridge. Ph.D. PrincipleInvestigator Pathology AssociatesInternational / liz Date 99 Date Study Number 1132-100 Page 13 Attachment: I Test Diet Preparation Procedures 1) Determine theamount of testdiet(feed)thatisto be preparedand weigh out thatamount of feed. 2) Calculatethe amount of testarticltehatisneeded to preparethetestdietat the desired concentration. 3) Accuratelyweigh outthenecessaryamount of testarticle. 4) Transfertheweighed testarticlteoa containerand add a smallvolume of acetoneto container. Manuallymix to dissolvethetestmaterw addingacetoneas necessary(typicarlatioof test materialto acetoneis. 1 g- 15-20 ml acetone).Visuallyinspecttestmaterial/acetofrolre I solubilitoyf testmaterial. 5) Preparea pre-mixby transferritnhgedissolvedtestmateriailnto4 kg of feed in a.Hobart mixing bowl. Mix for 10 minutes.Transferthepremixto a largermixer,add remaining amount of weighed dietm,ix for30 n-dnutes. Study Number 1132-100 Page 14 Attachment: II Collection of Tissue Samples for Biochemical and Molecular Analysis Because of the extreme instabilitoyf certainenzymes and biomolecules,itisessentialthattissues be harvestedas soon afterdeath as possibleand flashfrozen inunediateliyn liquidnitrogen. Failureto followtheseproceduresmay leadto lossof theentiresample and allthe energiesand resourcesthatwere investedintogeneratingthe samples. Therefore,make every effortto comply with thefollowing: 1) Harvest the tissuesamples as soon as possibleafterdeath. Delays may allow for biodegradationand/orinactivatioonf thedesiredendpoint. 2) lirunediatesluybmerse the tissuesample directlyinto liquidnitrogen. Dry ice or other alternativewsillnot suffice.Itis importantthatthe tissuebe immersed directliyn liquid nitrogenTransferringitto a dryvessel(orsample containers)uspended in liquidnitrogenwill not suffice.The tissuemay freezeto thevesselwalland willthenbe impossibleto remove withoutcompletelydestroyingthevessel(orsample container). 3) Be absolutelsyureto maintainthetissuefrozen. Itshouldbe storedin a sealedcontainerat700C and shipped or transfeffeodn dry ice. If needed, the frozensample can be fractured (brokenintoportionsfor differenatpplicationbsy) placingin a cruciblewhich containsliquid nitrogentokeep thesample frozenwhile grinding/fracturing.