Document nmYebv6xOw1oYaYKO2mp4MLJ1

CcoONVAANNSCCES. Jy 1.1999 Scoommmser fgVeembaecissntacnestnTdoes Paul Lier, PhD. S3MionSToEsicslos St. Paul, MN 55144-1000 Dose Dr. Lider The expert iw desert nthis ete, ofthe mous lymphoma ll tationsingperformed by 'NOTOX on 3M product, T-6906,wasconductedatmy request by Dr. Brian Myhr, AssociateDirectorof Genetic andeCelrluolearnTosoxaitecoolnogtywaitaCnoovtaenhceSo'smtViNeonitnas, VLA55foaceiliotyt. aoDrv.aoM.ydhDrhoaedshfeaodmpoveodro2o0fryai enagrs odfe, Bo wy `performanceofthe mutation assay, werereviewedas reported in a draft report signed April 25,1998, by hBEe est aril, T-69a0a6sr,oveasvovieias5ie5ssBebyaatnhet.NOOcToo OaXsevseuodviodonirooctnhortsspoyopnroitenethtnetthce aaebpln of eeaekssnsensTRotaspeydShelttabegpeeaenimniofS16e90n8 sep "The specific deficienciesofthe study (or reporting) arelistedas follows: 1. Th mouse phon asa as bnvldtod spfforclan 372C of SITSY cel, The report does not specify the clone. and the issue is raised ifNOTOX has any karyotypic documentation that Sor imine 3.7.2C cells were used. This is very important to establishbecausethe mutagenic responseof a different 2. The $9 was prepared in-house from Wistar rats. S9 is usually prepared fromFischer 344 or Sprague- vDawolaety riaSt3es,SnssoontoncerawatotnodeSnrsw5eakebdovuvtit t5h5ue (e3ff0ispctatchyyocrftwhmae eSw9accsphrepvarbaattiaoasn.anSdcdweasinytcdWoodcnhuoamensnDtetaVthdiNonhmoaeofehhittsns orf2oo00 Te ee os ee a oe nit, d5. EeeMSeshosuldonomtewble tstdosPhoeioessionnmdecson4tr0oplNfoIoMSod:nawcahtiivesahctidaounrcssetaocotmndhiSiornbsl.orThios coomnbpiocu,ndeoett currently highly recommended. (Note: the concentrationof EMS in thecultureswas not reported.) 4. The aeborsston aipporesttotahve3eo0psrs0odtmptnoiosrnTsofostlWltmuotantalooinos,goimec i msoortahss erseociflsackof frequencies was givenas 7 to 38 x 10%, which is much too low, especially for the microwell method. ena on -- nes 004393 eInxtphoendeanttsarattahbleerst,hDanMnNegsahtoiuvledexbpeosnheonwtisnfgormmauntyanmtofrreqsumeanlcliecso.l)onies than large colonies. In Table 3, in pnaornteiciunlatrh,eDcoMncAurirsesnthonwegnatiinvdeuccionntgroplr.imaTrhiilsy dleasrcgreicboelsonpioeso.r eTxhpeerriemaernetavlercyonfdeiwtisonmsalforcodleotneicetsinagndinadlumcoesdt mutants 5a.rtTichlee sdhoosuelrdabnegem-feiansduirnegdtebsyt umuslitnigp3l-yhiongurthteretawtomecnotlsuimsndsoesfcrreibsepdoinnseTsabglieve1n. T(hReCtC oanxd RioCcfEti-hstteeeysotur nSuortvaitviaolnsidencrTeaabslees s1t)etaodciallycwuiltahteitnhcerea"ssiunrgvidvoasle" (dRoSw)nrteol2a3t%iavtoett3h3e3soulgv/emnlt cwointthroolu.t $W9.he(nThtheisinicsrdeoansce,inthe spurrevciivpailtaattetdhmeatteorpiadlo)seOonfe10ne0e0dpsgo/nmlly itsotsatkraentgheeanasdssauygtgoes2t0s%sosmureviavdaslo,rp5titohnoerfesjoulsutbwlaestensot anreteidclteotoshift ttohe24re-phorotu.r tFrueratthmeernmtosrfeo,rtthheemmousttatcioonmamsosnaymaesasiunrdeiocfatteodxiinctihtey (raRnTgGe)fiinndcilnugdersestuhletsdescercetaiosneionngpraogweth14raotef: o`wvoeurldthbeeextprureesfsoirotnhepe+ri5o9d.spoorttihoenotfotxihceittyewstoiungl,dsboeetvhaetntghreeamtuetrattihoannaisnsdaiycawtietdhiSn 9Tawbalse v1.eryThliekeslaymcearried 0altohgiegthheerr.test article concentrations than needed. The precipitating range could probably havebeen avoided TRhTerGeowraRsScosnhsoeunlsdubseaugsreedefmoerntthaemtoonxigcimtoyumseeasluyrmempehnotm.a aWsosraykeixspoenrgtosiantgtthoec1h9o9o9seIoWnGeTmPeawsourrek,shop that pcornocbeanbtlryatRiToGn.ranTgheeacnhdoitcheusoftototxhieceivtyalmueaatisounroefmetnestticocmriptoicuanldtso tinhethcihsoaiscseayo.f pTrhopeemretteshtoadrtuisceled in the. NOTOX study will lead to inappropriately high concentrationsoftest article being tested. 6w.ithIn$b9otmhetmaubtoaltiiconactaisvsaatyiotnr.ialTs,hitsheisejxupsrtesnsoitonacpceeprtiaobdlew.asIt3 idsawyeslalfkenrotwhne t3h-ahtotuhretmraeaxtimmenutmwmituhtaTn-t6906 ddaeytesc.titohne osecncsuirtisvi2tydaoyfts eaftaesrstaryeaitsmesnetr,ioausnldytchoemmpurtoamnitsefdr.eqTuhenecdyetfealcltsioranpoifdlymutthaergeeanftiecr.actBiyvitwyaiotfinTg-6f9o0r63. (ifreal. and I doubt tis) would become much enhanced at 2 daysofexpression. 7. The conditions for determining mutant frequencies were not optimal for controlling variability. Forthe 4mipclraotweesltlomgeitvheo0d..7i6t8isxr1e0cocmlmelnfdoerdantaolyussies.1.6 ThceelNs/OsTelOXforsCtuEdyaunsde2d00| 0ceclellwleslweflorfCorEmuatnadn2t0s0e0lection x c1e0l%lschevlelllfor3muptlaanttss.forTohnelysm0a.l5l7e6r nxum1b0ecrlslussfeodrbaynalNysOisT.OXThaeresodfettarigmaerntmaelthtoodproefctihsieoans,saanydacnoanlsyezqeuse3ntx aesspseacyiafllluyctwuhateinonrselcaotuivledtwoelllowbenergeastpiovnesicbolnetrfoolr msupturainotusfr2e-qfueonlcdieasn.d 3T-hfiolsdisinlcirkeelaysewshiantmouctcaunrtrefdrfeoqrutenhcey, Table 3 results with S9 that were evaluated as showing mutagenicity. m8.utaInttss.nobtutcltehaer Phooiwsstohnecmaultcaulnattiforneqsuheonuclydwcaosmecaflcruolmattehde. prTohpeorttaibolneosfswheolwstowtailthnunmobmeurtoanftws.ellTshweittehxt eivndailcuaatteesdtahinsdwtaotsaldoenmeptcyorwreecltllsy,shbouutldnobevegriivfiecnatfioorneiascphocsuslitbulree,from the data provided. Total wells 9r.esuIlntadsdhiotuilodn nteovtehre hraevaesobnesegnievveanlauabtoevde afsorponsoittievveenfomramkuitanggenaincietvya.luaTtaibolneat2 trheissuplotinatr,etjhuestS9noatctivation ifnrteeqruperentcayblies.sTyhmpetotmesattimacteorfisalomperetceicphintiactaeld partob1l0e0m0.pgT/hmelr,eawnadsthneossuudbdsetanntainadl lianrcgreeaisnecrieassemailnlmmuuttanatn.t ctooxliocniiteys. rifelmaetiavseutroedlabrygeRmTuGta.ntwsoucloldonpireosb,abwlhyichhavisewbheaetn oenxecewssoiuvled(elexspsectthannea1r0ly%alRlTtGh)e.timIe.FRATnGd the measurements had been used. the highest selected dose probably would not have exceeded about 300 ng 001394 Table 3 results with $9 activation there was avery toxic response at a1r7e%prsoubravibvlayl,jtushterdeewmoansstnroatiinncgreaassseayin vmaruitaabniltitfyr.eqAuetnc1y7.5 gTh/ims ,wawshere aSop-pcaarlelnetdlyretshpeonmsoessattox2i5c0tarenadtm7e5n0tgint/hme taarbeltehaenredfosrheounlodt hbaelvieevsahbolwenwitthheoluatrgceosntfimrumtaatnitonfruenqdueenrcyb.etteTrhe assay conditions. These were probably excessively toxic treatments by the RTG measurementof toxicity. JAuslya,n1a9d9d7e,dtonoptee,rftohremre2i4s-nhoourreatrseoantmbeyntusp.daStheodrtO-EteCrDm tgrueiadtemleinntessoffo3r-m5ouhsoeurlsyamrpehsoumffaicaisesnatwy,itadhoapntded in `hwoiutrhoturteaSt9meancttisvaitnioan.secIofnndegtraitailvweitrheosuultts9aaeroebtcauirnreendtalnydusieftdhteo tmeesettmaItCerHiagluiidsealipnehsafromraFcDeAutictahle,n 24smautbemriisasliso,n.wilOtfhoelrloswubtmheisuspidoantsedtoOFEDCA,DtgouiCdFelSiAneN; ftohrereexaismpnloe,nefeodrffooor d2a4d-dhiotuirvetsreoatrmefnotosd. coTnhtiasct iinnftohremFatDioAnRweadsbojoukst.confirmed by an FDA staffer involvedwiththe revisionofthe genotoxicity guidelines Tnohtahneksiytoauteftoorctohnetoapcptourst.unity to review this work. Should you have any additional questions,pleasedo Sincerely, Steve R. Haworth, Ph.D. VGeinceetPirceasinddenCtellular Toxicology 001395