Document gbXZ4odqONgrjbvQmjg1XGz2Q

EVALUATION OF MUTAGENICITY STUDIES DEVELOPED ON (PFOS) PEP.FLUOP.OOCTANE SULFONATE PREPARE[) BY PROF. NICOLA LOPPIENO EmeritusProf.of Genetics Universityof Pisa,I,taly January 2000 EVALUATION OF MUTAGENICITY-doc INTP-0t)UCTION Perfluoruoctanesulfonat(ePFOS) is one of the degradation products of Perfluoruoctanesulfonylfluoraicdoem,ponentof severalproductlinesby 3M, widely used for severalpurposes,such as Surface Treatments,Surfactantsand others. These degradationproductscan be found inthe environmentduringseveralstagesof a product'lsifecycles,uch asduringthe industriaslynthesist,he incorporationfthe fluorochernicailnstoa finishedproduct,duringthe distributioonf the productto users,duringthe use of the productby consumers,and duringdisposaalt allthese stages(3M, August31st1,999). Becauseof possiblheuman exposureto thesechemicalsby differentroutes,director indirect,3M has developed a set o toxicologicasltudieson PFOS and other degradationproducts. In the present Report we evaluatea set of mutaqenicity/!genotoxicsittuydies performed on PFOS, in order to express an opinion on the potential mutagenic/genotoxirciskposedby thischemical. The set of mutagenicity/genotoxicsittuydiessubmittedtoourevaluatiocnonsistosf: .1.Salmonell-aEscherichicao/i/MammalianMicrosomeReverseMutationAssay with. PFOS. CovanceStudy No. 20784-0-409,completedon November 5,1999. 2. Chromosome AberrationsinHuman Whole BloodLymphocyteswithPFOS. Covande Study 20784-0-449,completedon October 25th,1999. 3. Unscheduled DNA Synthesisin Pat LiverPrimary CellCultureswith PFOS. CovanceStudy No. 20784-0-447,completedon November 9,1999. 4. rn VivoMouse MicronucleuAss-say on T-6295.CorningHazletonInc.,No. 174030-455,completedon May 23rd'1996. EVALUATION 1. Salmonella- Escherichiaco/i/MammallanMicrosome Reverse Mutation Assay with PFOS 1.1 Identificationf the testchemical The test chemicalevaluatedin thisstudy has been indicatedas PFOS, FC-95, Lot 217,as white crystallinpeowder.No other identificatipornopertiesare reportedin the study.Purityof the testchemicalhas non been indicated. The test chemicalwas dissolvedin T)MSO: the 2.00 pg/ml solutionused for the dilutionesmployed inthe assaywas stable. 1.2 Testerstrains The strainsused in the assay were Styphimurium strainsTA98, TA100, TA1535, TA1537 and Ecoli strainWP2 uvra. 1.3 Metabolicactivation The assaywas performed inthe absence and inthe presenceof a metabolicactivation system.This was provided by S9 mix, liverhomogenate,purchased by Molecular ToxicologyI,nc. Batch 0872 (42.8 m.9protein/ml)T.he hornogenatewas prepared frornrnaleSprcigue-bawleryatsinjected(i.p.w)ithAroclor1254 (200 mg/ml) at 500 mg/kg. Data on compositionof 59 are includedinthe Report. 1.4 t)osesof the chemical The tested doses of the chemicalunder evaluatiownere determinedafter a dose range findingstudy. Tested doses were between 5,000 and 33.3 pg/ml (6 doses)inthe presenceof $9 mix and between 5,000 and 0.333 pg/plate(9 doses) inthe absence of S9 for S.typhimuriu4m strainsA.s regardsEcoliWP2 uvrathe dosestestedwere between 5,000 and 33.3gg/plate(6 doses)both inthe presenceand absenceof S9 mix.I exp. with 3 plates/dosewas performed. 1.5.Positivecontrols Positive-controlisn the presenceof S9 mix were benzo(a)pyrenefor TA98; 2Aminoanthracenefor TA100, TA1535, TA1537 and WP2 uvra.In the presence of S9 mix the positivecontrolswere 2-nitrofluorenfeor TA98; Sodium Azide for TA100 and TA1535; ICR-191for TA1537, 4-nitroquinoline-N-oxifdoer WP2 uvra 1.6.Results Resultsindicatethat PFOS did not cause a positiveincreaseinthe mean number of revertants/lpateinalltested strainsand allconditions. Allpositivceontrolswere found mutagenicand comparablewith literaturdeata. 1.7.Comments The study was performed inaccordancewith Good LaboratoryPracticeregulations laiddown by the US-EPA. The protocolemployed in the assay was that one recommended by OECD, the "BacteriaRleverse Mutation Assay",referredto the test conditionsand positive controlsH.owever,itshouldbe noticedthatdue to the negativeresultsobserved,the experimentneeded to be repeatedfor a confirmation. The test reportdoes not includethe identificatidoanta and the CAS no.,as requeste' dby the OECD protocol. 2. Chromosomal Aberrations in Human Whole Blood Lymphocytes with PFOS 2.1.Identificatioonf the test chemical The testchemicalevaluatedinthisstudy was indicatedas PFOS, FC-95,Lot #217, as white crystallinpeowder. No other identificatipornopertiesare includedinthe report.The purityof the test chemicalwas not indicated. The testchemicalwas dissolvedin[)MSO:a solutionwas obtainedwitha 1.0% dilution of the E)MSO stocksolutio(n401 mg/ml). A 2.2.Biologimcatlerial Lymphocytewsereobtainefdrom humanvenousbloodprovidebdy a healthym,ale aduldtonor. For chromosomeaberrationassay,replicatceultureswere used at each concentrativoenh,iclceontroalndpositicvoentrol. In thepresencaend intheabsenceof a metaboliscystem,thetreatmenwtiththe chemicallasted3 hours;the culturewsere laterincubatedfor 22 hours, corresponditnog1.5timesthecelclyclteime(15hrs.). 2.3M.etaboliacctivation The assaywasperformedinthepresencaendintheabsenceofa metaboliscystem. Thiswas providebdy S9 mix,livehrom.0genatpeu,rchasebdy MoleculaTroxicology Inc.LotNo.929.The homogenatewas preparedfrom maleSprague-bawleryats injecte(di.pw.i)thAroclo1r254at500-mg/k5g daysbeforesacriifce. r)atoanthecompositioofntheS9 areincludeidntheReport. 2.4. I)oseosfthechemical Concentratioonfs the test chemicalwere between 27.2and 400 gg/ml (15 concentratioinntsh)efirstexperiment. The assaywas repeatedwithconcentratiobnestween12.5and 559 gg/ml(11 concentratiownist)houtmetaboliacctivatiaonnd between12.5and 449 pg/ml(10 -concentrations)wmiethabolicactivatio1n00 cells/replicaftleask for 4 concentratiofntshetreatedculturetsh,enegativceontroaln,donepositicvoentrol wereanalyseTdh.e mitotiicndexwasevaluateidn1000cells. 2.5Positiavnednegativceontrols MitomyciCn (MMC) was usedas positivceontroalt 3 concentratio(n1s-01,-5,.2.0 pg/ml)for the non-activatceudlturesw,hereas Cyclophosphami(dCeP)at 3 concentrati(o2n5sm50,75 pg/mlw)asusedforactivatecdultures. Forthechromosomaeberratieovnaluatioonnlyoneconcentration/posciotnitvreohlas beenevaluated. E)MSOat10gL/mlwasusedasa negativceontrol. 2.6 Results The toxicitye,valuatedby means of the mitoticindex,was around the order of 30% inthe presenceof metabolicactivatioannd 8-92% inthe absence of 59 mix. The test articledid not induce chromosomal aberrations,polyploidyor endoreduplicatiofnrequencieshigherthan the negativecontrolculturesw,hereas the positivecontroltreated culturesdemonstrated'a higher frequencyof induced chromosomal aberrationsin the absence or presence of metabolicactivation conditions. 2.7.Comments The study was performed in accordancewith the Good LaboratoryPractice regulationslaiddown by the US-EPA. The testreportdoes not includethe identificatidoantaand the CAS No. 3. Unscheduled DNA SynthesisinRat LiverPrimary CellCultureswith PFOS 3.1Identificcitiofn the testchemical The test chemicalevaluatedinthisstudy has been indicatedas PFOS,,FC-95, Lot #217, as white crystallinpeowder. No other identificatipornopertiesare includedinthe report.The purityof the test chemicalhas not been indicated. The test chemicalwas dissolvedin I)MSO: a suspension/translucent liquidwas achievedat 4010 pg/ml. 3.2 Biologicamlaterial Rat hepatocyteswere obtainedby perfusionof livert;he exposure periodwas of 19.6 - 20 hours;23 hoursafterthe beginningof treatments,cytotoxicitwyas assessedas cellulamrorphologyand cellsurvival. Triplicatceellcultureswere used for UDS analysism,ade by means of thymidine incorporation. 3.3Metabolicactivation Hepatocytesare ableto undergo metabolicactivatiobny themselves. A 3.4.Doses of the chemical Fifteenconcentrationosf PFOS were applied,rangingfrom 4000 gg/mi to 0.250 MG/ml; the chemicalwas cytotoxicdown from 25 gg/ml. 3.5.Positiveand Negative controls As positivecontrol,2-AcetylAmino Fluorene(2-AAF) was used at a finalconcentration of 0.10pg/ml.Negativecontrolwas providedby DMSO at a concentrationof 1%. 3.6.Results The survivaolf the concentrationasnalysedof the test chemicalfrom 25 pg/rnlto 0.5 pg/mi was between 65 and 100%; the one of the 2-AAF was 88.5% at a concentrationof 0.1gg/mi. The Mean Net NuclearGrams (MNNG) of the treatedcultureswere not different from the vehiclecontrol(DMSO = 1% survivalw)hile 2-AAF treatedcultures(0.1 gg/ml)presenteda M.N.N.G.of 19.35. In thisstudy,PFOS was considerednot able to induceUDS in rat hepatocytes treatedinvitro. 3.7.Comments The study was performed in accordancewith the Good LaboratoryPractice regulationslaiddown by the US-EPA. The protocolissimilarto the OECT) Guideline482. The Report does not includeidentificatidoanta and CAS No. of the testchemical. 4. Mutagenicitytest on T-6295n in an in vivomouse micronucieusAssay 4.1.Identificatioonf the testchemical The testchemicalevaluatedinthisstudy has been indicatedas T-6295, as off-white mixture of powder and fliakes, There are no indicationrseferringto the PFOS chemical. The chemicalwas dissolvedincorn oil,carboxymethyl cellulosaend acetone. 4.2 Biologicamlaterial 7 Adultmcilaendfemalemices,traiCnrlC:D-1(ICR)BR wereusedforthisassay. After a preliminaryrange findingassay,3 doses were employed inthe main assay, namely 237.5 mg/kg - 475 mg/kg - 950 mg/kg. 5 males and 5 females were used for each dose and for treatment (24 - 48 -72 hours).In the highestdose (950 mg/kg) a few animalsdied and were replacedby others treated inthe same way. Treatment was by oralgavage. 4.3.Positiveand Negative Controls Positivecontrolwas provided by 10 animals(5M and 5F) treated with Cyclophosphamide,80 m.9/kg and analysed 24 hours after treatment. Negative controlwas providedby 10 animals(5M and 5 F) treated with deionisedwater and analysed 24 hours after treatment. Bone marrow cellswere analysedfor the presence of micronuclei,the polychromaticerythrocyte (PCE) and the normochromatic erythocyte(NCE). 1000 PCE per animalwere scored and the no. of micronucleiwas recorded. The spontaneousmicronucleifrequency for thisstrainisabout 0-0.4%. 4.4.Results The chemicaltested did not inducea significantincreasein micronucleiinduction frequency inallconditionsand concentrations. There was indicatioonf some toxicityat 48 and 72 hours treatment,indicatedby the ratioPCE*NCE. The positivecontrol(Cyclophosphamide)induced a significantincrease in the percentage of micronucleatedcells. 4.5.Comments The study was performed in accordance with the Good Laboratory Practice regulationslaiddown by the Food and Drug Administration(FDA). The protocolwas similarto the OECD Guideline474. The Report does not includethe informationon the identificatiodnata and CAS no., as requested by OECD Guideline474. GENERAL CONSIBEP.ATIONS 1. Perfluoruoctanesulfonate (PFOS) was tested for the evaluation of its mutc.qenicity/_qenotoxicitiyn three in vitrosystems, namely (i)the induction of gene mutations in bacterialcells,(ii)the inductionof chromosome aberrations in human lymphocytes, (iiit)he inductionof UDS in rat primary livercells,and inone in vivo system, namely (iv)the induction of micronuclei in bone marrow cells of mice. Besides some inadequacies present inthe reports which could be corrected, due to the existence of data required, PFOS resulted a chemical classifiableas a non-mutagenic compound, thus representing no mutagenic risk.Data presented for this chemical allow its classificationas a "probable non-mutagen/non-genotoxic in vivo",according to IPC5 classificatioannd proposal (1). 2. Mutageni city/.genotoxicity tests are useful predictive systems for both carcinogenicityand mutagenicity; they have been extensivelyvalidated in a set of internationalprogrammes during the last30 years. Several strategies have been proposed to use a small battery of test methods with the highest probabilityto produce positiveand easilyinterpretableresults.In 1995, IPCS (ASHBY, J. et al) proposed the scheme reported in Fig.l.IPCS' strategy is based on the use as a firsttier of the two most employed in vitrotest methods for analysing the potential for the induction of gene mutation and of chromosome aberrations,namely (1)the Reverse Mutation Assay using Bacteria (OECD Guidelines 471 and 472) and (2) In Vitro Mammalian Chromosome Aberration Test (OECD Guideline 473). OECD Guidelinesfor performing these two in vitrotests have been revised several times by the group of experts on the basis of development of techniques and methodologies, as well as on the basis of experience developed during the years on the use of the two methodologies. The present estimate of the amount of chemicals tested by these two methodologies amounts to more than 10,000 assays. Several in vitrogenotoxicitytests are currently available.The combination of two in vitrotests,. - bacterialreverse mutation test (or n vitromammalian cellgene mutation test for specificchemicals,for which a scientificjustificationmust be provided) - in vitromammalian cellchromosome aberration test provide ingeneral,sufficientevidence of mutagenic and/or genotoxic potential.(2) Depending on the results,other in vitroor in vivotests may be required. Use of in vivotests islimitedto confirmation of a muta_qenicactivityalready observed in vitro. The scheme of testing chemicals of different use for their mutagenic/genotoxic potentialproposed by several different agencies bases itsscientificrationaleon the schemeproposedby ICPS,as the lattearllowisdentificatOifotnhe two typesof mutagenic/genotohxaizcardsof the chemicalnsa,melycarcinogenoircsomaticell mutagenandgenetiocrgermcelilnvivronutagen. The sameschemehasbeenacceptedby a setofnationahlealtphrotectiaognencies, amongwhichtheDepartmenotfHealthoftheUnitedKingdomw,hoseCommitteeon MutageniciotfyChemicalisnFood,ConsumerProductasndtheEnvironmenitn,1989 statedasfollows-. "Wherea chemicailsnotproposefdorhumaningestiaonndwhereitisexpectedthat itsusewilble limiteadn,dthedegreeof humanexposuriessmallorcontainabiltei,s acceptablteo limitinvestigattiontwo tests,one to detectgene mutationin bacteriaan,dthesecondtodetectclastogeneiffcectsinmarnmalicaenlls("3.) IPCS'schemefor testingfor mutagenciity/genotcoixtiyis exemplifieidn Fig.2 developeidnthosecasesinwhichtheresultosfthefirstwoinvitrtoestsproduced both or onlyone positivreesultt,hus indicatiangcertainpotentiatlo induce mutageniecffects. It shouldalsobe consideretdhatthe resultosf the mutageniciatryenotvaliadt 100% levelt;hesamecanbe saidforallthetoxicologisctauldiesw,hendefinint_hqe toxicologihcaazlardandthepossiblreiskforconsumers. However,inordertoillustrtahtevaluoefsuchtestsi,nthesectorofmutagenicity, itispossiblteo evaluatteheprobabiliotfyonechemicatlo be carcinogenoircnoncarcinogenoincthebasisof theresultosfmutagenicitteystsT.he otherpredictive valuoefthemutagenicitteystsn,amelytheprobabiliotftyhepredictioofnthegerm, cellrsnutoqenisatpresenntotpossibldeuetoscarcitoyfdata. N.LOPRIENO et al,haveinvestigattehde relationshbieptsweenthe mutagenicity testinrgesultosf the two ih vitraossayso,ne in vivoassayand the resultosf carcinogenicaistsyaysT.he resultasrereporteidnFig.w3hichshowstheaccuracoyf thecorrecitdentificaotficohnemicalfsora reliabnluemberofassaysd,erivefdrom theanalysoifsatotaolf3762 chemical(sseeFi.9.(44)) Sincefluorochemicarlesidualosf differencthemicalstructur(eandbiological reactivitayr?e)knownto be presentina fluorochemicparloductandsincesome of them have been foundin planteffluentor in groundwateart differenltevel concenttratiointss,eems reasonablteo developa fullprogrammeof testinfgor rnutagenicity/genotpooxtiecnitiyoaflallpossiblde rivativwehsichhavebeenfound inthe environmenotr whichcouldalsobe predictetdo produceto some extenta specifihcumanexposure. Among them,forinstancNe-ETFOSA andN-MEFOSA, PFHS,N-ETFOSEalcohoalnd N-MEFOSE alcohoelt,c. in The mutageincity/genoctiotxyitestinpgrogrammeshouldincludfeo,reachchemical, thetwobasicinvitrcoomplementartyestsn,amely: - theinductioofngenemutatioinnbacteriaclell(s4SalmonelsltarainasndIEcoli strain); - theinductiofnchromosomeaberratioinnsa mammaliacnellin(eHamsterovary Cellosrhumanlymphocytes. The resultosf thesetwo testsi,fnegativoen the basisof PFOS'resultsc,ould reinforctehe conceptof theabsenceof a mutagenic/genotorxiisckinconnection witha potentiahlulmyanexposurteothesechemicals. Alltypesofresultosbtainewditha testinogfmanyfluorochemidcearlivativsehsould moreovercontributteo the establishmeonft a data-basseystemfor further consideratioofntshetoxicologipcraolfiloefthesespecifcihcemicals. P.EFEP-ENCES 1. ASHBY, J.WATERS MO, PRESTON J., ADLER I.b. et al, IPCS Harmonization of Methods for the Predictino and Quantification of Human Carcinogenic/MutageniHcazard and for Indicatingthe ProbableMechanisms of Actionof CarcinogensM.utationResearch 2. EUROPEAN COMMISSION (Report EUP. 15945 EN): Methods and Testing Strategiesfor Evaluatingthe GenotoxicPropertiesof Chemicals,Luxembourg, 1995; 3. COMMITTEE PRODUCTS ON MUTAGENICITY OF CHEMICALS IN FOOD, CONSUMER AND THE ENVIRONMENT, Guidelinesfor the Testing of Chemicalsfor Mutagenicity.Her Majesty'sStationaryOffice,Londo,1989; 4. LOPRIENO. G.BONCRISTIANI, G.LOPRIENO and M.TESORO, Data Selection and Treatment of Chemicals Tested for Genotoxicityand Carcinogenicity, EnvirnomentalHealth Perspectives,96, 121-126,1991 (and other unpublished data).