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Pathology Review of Reported Tumorigenesis in a Two Year Study ofFM-3924 in Rats November 25,1998 BY Pathology AssociatesInternational 6217 Centre Park Drive West Chester,Ohio 45069 Telephone (513)779-9600 Fax (513)779-9603 PathologyReview ofTumors ReportedinRatsGivenFM-3924 Introduction:Thisrepoprrtesenatnsindependaesnstessmeonftumorigenedsaitsa obtainedfrom a studytodeterminethechronictoxicitaynd carcinogenipcotentiaolfthe fluorochemicaFlM-3924 in ratsT.he study,entitle"dTwo Year (Diet)Toxicity/ CarcinogenicitSytudyof FluorochemicaFlM-3924 inRats,"was sponsoredby the3M Company, St. Paul, Minnesota. Biophase procedureswere conductedat Riker LaboratoriesI,nc.,betweenApril,198,1and May, 1983 and were in compliancewith FDA Good LaboratoryPractic(eGLP) guidelinesA.t RikerLaboratorieLse,onardJ. SibinskiB,A, servedas the StudyDirector.Followingin-lifperoceduresa,llmajor organsand tumors were processedintomicroslideasnd examined by Robert G. Geil, DVM, DACVP. Dr.Geil'sfindingasnd interpretatiwoenrseincorporateidntothefinal reportforthestudy. For thetumorigenesirseview,thecompletestudyreporti,ncludinagllrelevantpathology datawas forwardedtoPathologyAssociateIsnternation(aPlAI),West Chester,Ohio for examinationby RichardH. Bniner,DVM, DACVP. The sponsor(3M) regardedoriginal pathologyinterpretatiobnysDr.Geilas adequatea,nd examinationofmicroscopictissue. sectionwsas notincludedinthereviewprocess.Specifiocbjectiveosfthereviewwere to evaluatetumor dataand toprovidean opinionrelativteo thepotentiarlelationshoifp reportedneoplasmswiththetestmateriablasedupon: 1.Ile incidenceand morphology of observedtumors and associatepdroliferatiavned non-proliferatlievseions.2. A literaturreeviewto examinethebiologibcehaviorand carcinogenetpioctentiaolf similar fluorochemicals3,. Contemporaryknowledgeoftumormechanistidcata,especiallwyith respectto possibleepigenetipcathways, 4. Consideratioonf chronictoxicity, immunosuppression,hormonalmodulationor ancillarbyiochemicalinteractionwshich may serveas modulatingfactorisn thedevelopmentof tumorsin thisstudy,and 5.Personalexperienceinevaluatinrgodentcarcinogenesibsioassays. Review Procedures and Findings 1. Reported tumorigenesis and ancillarypathologic changes for FM-3924: Based upon reportedpathology findings,the *reviewpathologistconcurred that treatment-relatcehdangeswere presentat both the 1-Year(interima)nd 2-Year (terminals)acrificeas wellas insome unscheduledeaths.At the 1-Yearinterim sacrificter,eatment-relatcehdangeswere generallyrestrictteod the liverand were characterizebdy a dose-dependenitncreaseinhepatocellulcayrtomegaly,vacuolation and necrosiaslongwith slightliyncreaseidnflammatorycellinfiltratCeyst.omegaly I and vacuolationwere generallyconsistentwith treatment-inducepderturbationof livercellmetabolism resultinignhypertrophyviaproliferationf peroxisomes and/or smooth endoplasmic reticulum(P-450 enzyme induction).Hepatocellularnecrosis was largelyattributetdo chroniclivercellswellingwith compromised metabolism and/orreduced blood perfusion(hypoxia). Treatment-relatemdicroscopicfindingsin unscheduled deathsand animals continued untilthe terminal(2-Year)sacrificiencludedpersistenhtepatocellulacrytomegaly and-vacuolationin both sexes. Additionallyc,ystoiddegeneration("spongiosishepatis") of the liverwas significantliyncreasedin high dose males. Most notably, hyperplastincodules were increasedin the liverof both sexes and hepatocellular adenomas and carcinomas were increasedinfemales. Based upon reportedpathologydata,dietarylevelsof 100 ppm FM-3924 for two years resultedin unequivocalhepatocellulacrytomegaly,vacuolation and cystoid degenerationas well as increasedhyperplasticnodules in both sexes and increased adenomas and carcinomas in high dose females. Although increasedhepatocelludar neoplasms were not reportedin males, itis likelythatsome of the "hyperplastic nodules" (in both sexes) would be regardedas hepatocellulaardenomas if contemporarydiagnosticcriteriwaere applied(1).Itisnoteworthy,also,thathepatic cylomegaly was observed'inmales given 10 and 30 ppm of the testmatereial suggestingthata NOEL forhepatocytomegalywas not achievedformales assignedto thisinvestigation 2. Literaturereview ofthe biologicbehavior and carcinogenicpotentialof fluorochemicals The reviewerregardedthetestmaterial(N-ethylperfluorooctanesulfonamiedtohanol) as a unique xenobioticwith unknown structure-activrietlyationshipsA. limited literaturreeview was conductedto determineifthe biologicablehavior,including tumorigenesis,of similarfluorochemicalhsad been reported.The literaturreeview includeda survey of rodent carcinogenesibsioassayscompleted by the National Toxicology Program (NTP) and selectscientifijcoumals and biologicalextracts. Although no carcinogenesisbioassayswith "complex" fluorochemicalswere discovered,severalrelevantreportsconcerning subchronic investigationwsere located.Additionallys,everalreports,includingtheNTP 2-yearbioassayof sodium fluoridewere availableto provideperspectiveson thebiologicaelffectsof long-term fluorideexposure. Resultsof thesestudiesarebrieflysummarized as follows: 1. Subchronictoxicitystudiesin ratswith "complex" fluorochemicalhsave identifietdreatment-relatheedpatocellulahrypertrophy(cytomegaly)similarto findingswith FM-3924. In studiesconducted by Van Rafelghem et al,a single intraperitoneianljectioonf perfluoro-n-decanoiacid (PFDA) in severalrodent 2 speciesresultedinpersistenhtepatocellulasrwellingwhich, ultrastructuralwlays, characterizebdy peroxisomalproliferatio(n12). In subchronicstudiessponsoredby the 3M Company, Griffithand Long administeredammonium perfluorooctanoatteo rats,mice and monkeys via oral routes (3).At doses of 30 ppm or greater,ratsdisplayedhepatocellular hypertrophy.This change was more prevalentand severeinmales. Noteworthy was the obser-vatiotnhatallmice given doses of 1000 ppm ad libidum and all monkeys given 100 mg/kg/day (gavage)died preterniinallAyd.ditionallya,ll monkeys given 30 mg/kg/day displayedanorexia,emesis,black stools,facial pallor,and prostrationa;nd one monkey given 10 mg/kg/day displayedanorexia, black stoolsand facialpallor.- Livereffectsh,owever, were not observedin monkeys assignedto these studies.Furthermore,microbialassays using five Salmonella stainsand one Saccharomyces strain,with and without metabolic activationd,id not revealmutagenic activitfyorthetestmaterial. 2. Review of studiesrelatintgo the chronictoxicityof fluorideand fluoridecontainingcompounds was limitedto theNT? 2-yearcarcinogenesisbioassayof sodium fluorideand selectenvironmentalstudiesof fluoridein cattle.In theNTP study,treatment-relatpeadthologicchanges were not observed in the liverof rats given dietaryconcentrationosf up to 175 ppm sodium fluoridein the drinking waterfortwo years(11).Furthermore,in-an extensivesarveyof cattlexposed to high environmentalfluorideconcentrationfsor lifetimeperiods,increasedliver diseaseor neoplasiawas not reportedalthoughmany animalsexhibiteddentaland skeletaclhanges typicalofadvanced fluorosi(s9). 3. Mechanisms resultingin increasedhepatocellularnodular hyperplasia and neoplasiain ratsgiven FM-3924: Resultsof possiblegenotoxicitystudieswith FM-3924 were not provided to the review pathologist.Itwas reasonedthatthiscomplex fluorochemicalprobablywas not strongly mutagenic and that most biologiceffectswere due to perturbationof livercell metabolism,with peroxisomalproliferatioand generaldisruptionof multiplemetabolic pathways. Accordingly,itislikelythatthedevelopment of livercelltumors (and nodular hyerplasia)was associatedwith epigeneticmechanisms, possiblyincludingperoxisomal proliferatioannd geneticdamage (ploidy)associatedwith oxidativestressand/oraltered regulationof the cellcycle. Followingpublicatioonf the finalreportfor thisstudy (1988), numerous journalarticlehsave been publishedwhich identifycarcinogenesiisn rodentsfollowingexposure to non-genotoxictestmaterials.Subsetsof thesechemicals which inducelivercelltumors inrodentsare characterizebdy antecendenthepatocytomegaly and peroxisomalproliferati(o2n,5,6,8). 3 4. Effectsofancillarybiochemicalinteractionwshich may have served as tumor promoters: Comments relativeto allmetabolicaberrationwshich may have influencedlivercell tumorigenesisin thisstudy would be largelyspeculative.Itshould be noted,however, thathepatictoxicitywas unequivocallylinkedwith ingestionof FM-3924, and at high dose levelslivercellalterationhsad resultedin necrosis.Correspondingly,accelerated proliferatioonf hepatocyteswould be expected to repairdamaged tissue,and may have servedas a tinnorpromoter (10).Although itshould be emphasized thatincreasedliver cellproliferatiwohnich may occurinrodentsfollowingexposuretomany toxicmaterials does not invariablyresultinincreasedturnorformation,most pathologistasgreethatcell damage which promotes in increasedmitoticactivitymay contributteo tumor formation (4,10,13). 5. Personalopinionrelativteoa relationshibpetween proliferatilvesionsand FM-3924 observedinthisstudy: Itismy opinionthatdistincitncreaseisnhepatocellulnaeroplasmsinhighdose females, combined with increasedhyperplastincodules in both sexes are clearindicatorsthatthe testmaterialshould be regardedas a livercarcinogenin Spmgue Dawley rats. Other proliferativleesionsand neoplasms were consideredto be spontaneousalterationosr secondaryto the systemiceffectsof alteredhepatocellulamretabolism (7). Based upon histomorphologicchanges observed in thisstudy,itislikelythatepigeneticmechanisms (especiallpyeroxisomalproliferatioonx,idativestressand otherfactorswhich deregulate the cellcycle)were key factorsinthe development of hepatocellulaprroliferativleesions. Ancillarydatawhich would supportepigeneticpathways fortumorigenesisin thisstudy would provide a rationaleforselectioonf exposurethresholdsin humans. Based upon referenceliteraturaevailableforthepreparationof thisreview,liverdamage, including carcinogenesish,as not been reportedin humans exposed to testmaterialsthatpromote hepatocytomegaly and neoplasiain rodentsvia peroxisomalproliferatioannd associated metabolicperturbations Additional Information which might Contribute to the Safety Assessment of FM-3924 I. Mutagenesis assaysor ancillarpyroceduresto establishthe genotoxicpotentialof the testmaterial. 2. Cell proliferatiosntudiesto provide an index treatment-relatiendcreasesin the cell cycle. 3. Uluwtrucwral analysesor contemporary analyticalproceduresto confirm the possibleperoxisomalproliferatioand ancillarymetabolicperturbations. 4. Recovery studiestoestablisthhepersistencoef livercelleffectsfollowingsubchronic exposuresto FM-3924. 4 Conclusions: Baseduponrevieowftheinformatiaovnailabilntehestudyreporitt,is my opinionthatdietary FM-3924 for 2 yearsresultedin chronicliverchanges (megalocytosisi)n malesatalldoselevels(10,30 and 100 ppm) and forfemalesatthe high dose concentratio(n100 ppm). At thehigh dose levelh,yperplastincoduleswere increasedin both sexesand hepatocellulatrumors (adenomas and carcinomas)were increasedin females.Incidencevaluesforliverproliferatilveesionsindicate-dthatFM- 3924 should be regardedas a livercarcinogenfor Sprague Dawley mts under the conditionsof thisstudy.The presenceof persistentd,ose-dependentlivercellcytomegaly suggestedthatepigeneticmechanisms were causativefortumorigenesisin thisstudy,and thatsafe exposurethresholdmsay be establishepdrovidingthattoxicokinetiacnd ancillardyata do not indicateadditionaaldverseeffectsuch as reducedexcretionand bioaccumulation. RichardH. Bruner,DVM, DACVP Date References 1. Goodman, D.G.,Maronpot R.R.,Newbeme, P. M., Popp,J.A.and Squire,R.A. Proliferatiavned SelectedOtherLesionsof theLiverinRats(DraftProposal). SSNDC Guidesfor ToxicologiPcathology.STP/ARP/AFIP, Wash.,D.C. (1993). 2. Green,S. Receptor-MediatedNon-GenotoxicCarcinogenesisN:ew Models forRisk Assesment? Human Exp.Tox.(10):390-395 (1991). 3. GriffithF,.D.and Long,J.E. Animal ToxicityStudieswithAmmonium PerfluorooctanoatAem.. Ind Hyg.Assoc.J.(41):576-583(1980). 4. Huff,J Absence ofMorphologicCorrelatioBnetween ChemicalToxicityand ChemicalCarcinogenesisE.nviro?LHealthPerspect1.01 (Suppl.5):45-54(1993). 5. Lalwani,N.D.,DethloffL,.A.,Haskins,J.R.,Robertson,D.G.,and De La Iglesia, F.A. IncreasedNuclearPloidy,Not CellProliferatiIosnSustainedinthePeroxisome Proliferator-TreaRtaetd Liver.Tox.Path.(25)No.2. 165-176(1997). 6. McDonald, J.S.,Lankas,G.R. and Morrissey,R.E. 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Pathological and Hepatic LTItrastrucftnE-faflectsof a SingleDose of Perfluoro-n-decanoiAccid in the Rat,Hamster, Mouse and Guinea Pig.Fund & AppL Tox.(9),522-540 (1987). 13.Ward, J.M.,Uno, H.,Kurata,Y.,Weghorst, C.M. and Jang,J. CellProliferatioNnot Associatedwith CarcinogenesisinRodents and Humans, Enviro?LHealth Perspect.101 (Suppl.5):125-136 (1993). 6