Document XROJGvNVwV9Jv2bK25Kmr6p1w

Pathology Associates International A Company of ScienceApplicationIsnternationaClorporation An&WbYe&Owmd Canvany Sponsor: 3M St.Paul,Minnesota PROTOCOL Study Title: CellProliferatiSotnudywithN-EthylPerfluorooctanesulfonamiEdtohanol(N-EtFOSE; 3M T-6316.11), PerfluorooctanSeulfonicAcid PotassiumSalt(PFOS; 3M T-6295.16),and N-EthylPerfluoroctanesulfonamide (PFOSA 3M T-7091.1)inRats Date: January 12,1999 Performing Laboratory R.O.W. Sciences 15 FirstfielRdoad 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 CellProliferatioSntudy with N-Ethyl PerfluorooctanesulfonamidoEthanol(N-EtFOSE; 3M T6316.11),PerfluorooctanSeulfonicAcid PotassiumSalt(PFOS; 3M T-6295.16),and N-Ethyl Perfluoroctanesulfonamid(ePFOSA 3M T-709 1.1)inRats Purpose To assessceH proliferatioand peroxisomeproliferatioinratsadministeredtestmaterialin the diet. Sponsor 3M CorporateToxicology Building220-2E-02,3M Center St.Paul,NIN 55144-1000 Study Representative Marvin T. Case, D.V.M, Ph.D. 3M CorporateToxicology Phone No.: 651733-5180 Fax No.: 651733-1773 Email:mtcase@mmm.com Alternative Study Representative Andrew M. Seacat,Ph.D. 3M CorporateToxicology Phone No.:651575-3161 Fax No.: 651733-1773 Email: amseacat@mmm.com Study Location R.O.W. Sciences 15 FirstfielRdoad Gaithersburg,Maryland 20878 Study Number 1132-100 Page 3 Study Monitor Sandra R. Eldridge,Ph.D. Pathology AssociatesInternational Phone No. 301 624-2036 Fax No. 301663-8994 Email:srepaisaic@aOI.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 PrincipalInvestigator Sandra R. Eldridge,Ph.D. pathologyAssociatesInternational Phone No. 301 624-2036 Fax No. 301 663-8994 Email: SREPAISAIC@AOI.COM Study Pathologist CarolynMoyer, D.V.M., Diplomate,A.C.V.P. PathologyAssociatesInternational Phone No. 301 624-2928 Fax No. 301663-8994 Proposed Study Timetable In-lifSetartDate:To be added by protocolamendment; Day 0 In lifeEnd Date:To be added by protocolamendment AuditedDraftReportDate:To be added by protocolamendment Regulatory Compliance This study willbe conducted inthespiriotf Good LaboratoryPractice(GLP) regulations. Study Number 1132-100 Page 4 Animal Care and Use Statement Allprocedures inthisprotocolareincompliance with the Animal Welfare Act Regulations,9 CFR 1-4.In theopinionof theSponsor and studydirectort,hestudydoes notunnecessarilyduplicate any previouswork. Quality Assurance Not applicable. Test Materials TestMaterial: N-ETFOSE (completedby 3K Identificatio'n:N-ETFOSE Lot Number: -F&f 93-29 Purity: 99.2% Stability: > 5 years Storage room temp. Conditions: CharacterisiCt!. -@Wax-y solid PFOS (completedby 3M) PFOS 217 99% > 5 years room temp. w hitepowder PFOSA (tobe added by protocol ir@endment) PFOSA Wy-14,643 (tobe added by Protocol amendment) Wy > 5 years room temp. amber waxy solid Reserve (Archive) Samples A reservesample (approximately5 g) of each lotwillbe takenand stored.artoom temperature. These samples willbe transferretdotheSponsor aftercompletionof 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 Species: Rat Crl:CD"(SD)IGS BR Source: Age atInitiatiofnTreatment: CharlesRiver LaboratoriesI,nc.,Raleigh,NC ferable6 weeks of age,but notmore than8 weeks of age Weight atInitiatioofnTreatment: 150 to 300 g Number and Gender: Identification: males uniqueidentificatiboyn individuaclartagsand cage cards Study Number 1132-100 Page 6 Husbandry Housing: Diet: Sing0lehoused inhanging stainlesssteelwire cacg;es Teklad 7012 CertifieRdodent Diet.Freshfood willbe providedweekly. Feed isanalyzedby themanufacturerforconcentrationosf specifiedheavy metals,aflatoxinc,hlorinatheydrocarbons,organophosphates,and specified nutrientsS.pecifiednutrientasnalysesareon fileatR.O.W. Sciences. Water- Tap water,providedad_libitumviaan automaticwateringsystem or water bottles.ne waterisanalyzedatleasttwo timesperyearforcontaminantsand specificmicrobes.The resultsof theseanalysesareon fileatR.O.W. Sciences. Contaminants: The studydirectoarnd/ortheSponsor have consideredpossibleinterfering substancespotentiallpyresentinanimal feedand water,includingthetest materialitselofr possiblestructurayrelatedmaterialass wellastheitems listedin(2)and(3) above.None ofthesecontaminantsarereasonably expectedto be presentinanimalfeed or wateratlevelssufficientto interfere with thisstudy. Environment: Acclimation: The targetedtemperaturesarebetween 64 and 79'F witha relativheumidity between 30% and 70%. Temperatureand humidityaremonitored continuouslyA. 12-hourlight/12-houdrarkcyclewillbe maintained.Ten or greaterairchanges/hourwillbe maintained. Animals willbe acclimatedtothefacilitfyora minimum of 7 days priortothe startofdosing.Animals willbe observedforgeneralhealthand suitabiliftoyr testingduringthisperiod.Animals thatarediseasedor unsuitablefortesting willbe removed from thestudy. Randomization: Usina computer-a0eneratedrandom numbers witassi0anmenttogroups,At the timeco'f randorr@zationt,heweight variatioonf theanimalsof each sex used should notexceed 2 S.D.of themean weight,and themean body weights foreach group of each sex willnot be statisticadlilfyferent. Justification: Rats willbe used because oftheextensivehistoricadlatabase,and theFDA requirementsfora rodentspecies. Study Number 1132-100 Page 7 Group Designations, Dietary Levels and Scheduled Sacrifice Time Number of Male Rats Points Group Number Control N-ETFOSE PFOS (TimePoint) (0ppm) 300 100 30ppm 20ppm PFOSA Wy-14,643 TotalNo. 100ppm_1222_p2m ofanimals 1 10 10 10 10 10 10 5 (48hrs) 2 10 55 5 5 5 5 (7days) 1 3 10 55 5 5 5 5 (14days) 4 10 55 5 5 5 5 (1wk recoIS!:L 1 5 10 55 5 5 5 5 (4wk recovery TotalNo. Of Animals 50 __22_L30 --2L-L 30 30 25 65 40 40 40 40 2ZL__Jl Dosing Procedures Method of Administration Dietary.Animals inGroups I through3 willreceivetestdietfor48 hours,7 days,and 14 days,respectively. Animals inGroups 4 and 5 willreceivetestdietfor14 days followedby a 1 or4 week recoveryperiod,respectively. Reason for Dosing Route The potentialhuman exposureisby theoralroute. Dose Preparation Before initiationf treatmentd,ose preparationof each testmaterialwillbe mixed. Alldose preparationwsillbe storedatroom temperature.Dose preparatiownillbe documented and reported.See Attachment Ifortestdietpreparationprocedures. Study Number 1132-100 Page 8 Retention Sample Samples (approximately100 g) willbe taken from the dose preparationand storedatroom temperature.Unless used foranalyses,thesesamples willbe discardedatleastI month aftercompletionof thein-lifpehase. Observation of Animals Clinical Observations Each animalwW be observedtwicedaily(a.m.and p.m.)formortalitaynd moribundity; findingswillbe recordedas theyareobserved. Body Weights Priortotreatment(atrandomization)w,eekly forWeek I through4 weeks of recovery. Food Consumption Weekly forWeek 1 through4 weeks ofrecovery. Clinical Chemistry Animals willbe fastedovernightbeforeanimal'sschedulednecropsy;bloodwillbe collectedfrom ajugularveinintoan EDTA-coated tube.Serum enzyme levelsof alaninean-dnotransfera(sAeLT), alkalinephosphatase,aspartataeminotransferas(eAST), cholesteroalnd triglyceridweisllbe determin*ed. Termination Unscheduled Sacrifices and Deaths Necropsies willbe done. Animals to be sacrificedwillbe anesthetizedwith C02, exsanguinated. weighed, and Study Number 1132-100 Page 9 Scheduled Sacrifices Interim Sacrifices At 48 hrs,7 days, and 14 days,animals willbe fastedovernight,bled forserum samples, anesthetizedwith CO,, weighed, and exsanguinated. NOTE: Two serum sampleswillbe needed,(1)a 0.5 n-islample forclinicaclhemistryand (2)a 1.5ml sample forcompound levelanalysis. The abdominal cavityofeach mainw willbe opened,theEver willbe removed and weighed,and liversampleswillbe collectedA.nimals win be discardedafterliver collection. Terminal Sacrifices After1 and 4 weeks of recovery,animalswillbe fastedovernight,bled forserum samples, anesthetizedwith C021 weighed,exsanguinated,and necropsied. NOTE: Two serum sampleswillbe needed,(1)a 0.5 ml sampleforclinicaclhemistryand (2)a 1.5ml sample forcompound levelanalysis. Postmortem Procedures Necropsy The necropsywillincludean examinationof theexternalfeaturesof t@ecarcass;allextemal body orificest;heabdominal,thoracica,nd cranialcavitieso;rgans;and tissues. Cell ProliferationTissue Collectionand Immunohistochemical Evaluation Representativseamples of theleftlaterallobeof theliverand any macroscopic lesionsof the liverwillbe couectedand preservedinzincfonnalin. Afterfixatione,ach sample of liverwillbe deliveredto: Sandra R. Eldridge,Ph.D. Pathology AssociatesInternational 15Worman's MillCourt,SuiteI Frederick,Maryland 21701 Study Number 1132-100 Page 10 Proliferaticoenllnuclearantigen(PCNA) evaluatiownillbe done on thesamples. In addition, liversectionspreparedfrom thesame tissueblock willbe stainedwithhematoxylinand eosin and examined microscopically. Paimitoyl-CoA Oxidase Tissue Collection and Analyses A sample (approximately500 mg) of therightlaterallobeof theliverwillalsobe collectedfrom selectanimalsand flash-frozeinnliquidnitrogen.See Attachment]Iforprocedure. The liver tissuewillbe storedin a freezersettomaintain-60 to -80*C untilanalyzedby Covance for palmitoyl-CoAOxidase activity.The liversamples to be analyzedwillincludeallstudy animals,EXCEPT for the Wy-14,643 animalsand allanimalsfrom the 4-week recovery groups. In additionto thisstudy,samplesfrom a previous3M studywillbe analyzedfor palmitoyl-CoAOxidase activittyh;esesamples consistof liversamples from 35 ratsand 35 guinea pigs. Tissue Collection for Electron Microscopic Evaluation Sectionsof liverfrom allanimalswiU be collectedn,iincedto approximatelyone millimeter cubes and placedina fixativaeppropriatfeorelectronmicroscopy. The containersand fixative willbe providedby PAI. Electronmicroscopywillbe performedon one animalper treatment group exhibitingthe highestcellproliferatirveesponseas well as one controlanimal atthe discretioonftheSponsor,from one timepointaswellasthe4-week recovery. Thus, EM will be performed on one animalfrom thecontrolN,-ETFOSE (one dose only to be determined), PFOS, PFOSA, and Wy groups atone of thetime points,as wellas the4 week recovery,fora totalof 10 animals. Remaining Liver Tissue The remaining livertissuewillbe frozen and stored at -60 to -80' C for possiblefuture analysis. Organ Weights At thescheduledsacrificetsh,eliverwillbe weighed. Histopathology Liverfrom each animalthatisexamined forcellproliferatiownillbe stainedwith hematoxylin and eosin,and exan-dnedmicroscopicallfyorhistopathologicchanges. Study Number 1132-100 Page II Reports One copy of thedraftreportwillbe senttothe Sponsor. The reportwillincludethe following information: Experimental Design and Methods Results dose analyses mortality clinicaolbservations body weights body weight changes food consumption testmaterialconsumption clinicaplathologyresults palmitoyl-CoAoxidaseactivities macroscopic observations microscopicobservations ultrastructuroablservations cellproliferatioanssessments Record Retention AB raw data,documentation,records,protocol,specimens, and finalreportgeneratedas a resultof thisstudy willbe archivedin thestoragefacilitioefsPAI for a periodof 1 year followingsubmissionofthefinalreportotheSponsor.One yearaftersubmissionof thefinal reporta,lloftheaforementionemdaterialwsillbe senttotheSponsorand a returnfeewillbe charged. Allraw datastoredon magneticmedia willbe retainby PAI. Study Number 1132-100 Page 12 PROTOCOL APPROVAL 7Ma-rvin'CasDe.,V.M., Pti.D. Study Representative 3M CorporateToxicology aA,q,_ti. G . fe,Ph.D.,D.A T SirdyWD - t r R.O.W. SSV.*tnrces /iA2!=/?=fv Date iz /gy Date Sandra R. Eld-ridgeP,h.D. Date PrinciplIenvestigator Pathology AssociatesInternational Study Number 1132-100 Page 13 Attachment: I Test Diet Preparation Procedures 1) Determine the amount of testdiet(feed)thatisto be prepared and wei-h out thatamount of feed. 2) Calculatethe amount of testarticltehatis needed to preparethetestdiet at the desired concentration. 3) Accuratelyweigh outthenecessaryamount of testarticle. 4) Transfertheweighed testarticlteoa containerand add a smallvolume of acetoneto container. Manually mix to dissolvethe testmaterw adding acetoneas necessary(typicarlatioof test materialto acetoneis I g- 15-20 ml acetone).Visuallyinspecttestmaterial/acetofnoer solubilitoyf testmaterial. 5) Preparea pre-mix by transferrintghedissolvedtestmaterialinto4 kg of feed in a.Hobart mixing bowl. @&x for 10 minutes.Transferthepremix to a largermixer,add remaining amount ofweighed diet,mix for30 minutes. 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 afterdeathas possibleand flashfrozen inunediateliyn liquidnitrogen. Failureto followtheseproceduresmay leadtolossof 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 biodegradatioannd/orinactivationf thedesiredendpoinl 2) Inunediatelsyubmerse the tissuesample directliyntoliquidnitrogen. Dry ice or other alternativweisllnot suffice.Itisimportantthatthe tissuebe innnerseddirectliyn liquid nitrogenTransferrinigttoa dryvessel(orsample containers)uspendedin liquidnitrogenwill not suffice.The tissuemay freezetothe vesselwalland willthenbe impossibleto remove withoutcompletelydestroyingthevessel(orsample container). 3) Be absolutelsyuretomaintainthetissuefrozen. Itshouldbe storedin a sealedcontainerat70'C and shippedor transferreodn dry ice. Ifneeded,the frozensample can be fractured (brokenintoportionsfordifferenatpplicationbsy) placingina cruciblwehich containsliquid nitrogento keep thesample frozenwhilegrinding/fracturing.