Document 6RLdrOjv1xjDe0qojMBp4Yj3g

CORNING Hazleton MUTAGENICITY TEST ON T6357 JCiEECR E, | 47225 195 IN AN IN VIVO MOUSE MICRONUCLEUS ASSAY "2, *TM af FINALREPORT AUTHOR Hemalatha Murli, Ph.D. PERFORMINGLABORATORY Corning Hazleton Inc. (CHV) 9200 Leesburg Pike Vienna, Virginia 22182 LABOPRORJEA CTIDTENTOIFIR CATY ION CHV Study No.: 17387-0-455 SUBMITTTEDO Mm 3M Center, Building 220-2E-02 St. Paul, Minnesota 55144-1000 SCTUODYMPLETIDOATNE April 23, 1996 CHV Study No.: 17387-0455 COGG03 1of26 `CORNING Hazleton QUALITY ASSURANCE STATEMENT Project Title: Jn Vivo Mouse Micronucleus Assay Project No.: 20996 Assay No.: 17387 Protocol No.: 455 Edition No.: 17 pQruoajleicttywAesrseurcaonncdeucitnesdpeacctcioornsdoifntghtoetshteudSytaannddarrdevOipeewraotfinthgePfrioncaledruerpeosrotoffththeeQaubaloivteyrAesfseurrenacnecde rUengiutlaatnidonasc.coFridnidnigngtso fthreomgetnheerailnsrpeeqcutiiroenmseanntdsoffintahlereappoprrtoprreivaiteewGwoeroedrLeapboorrtaetdorfyo mPraancatigceement and to the study director on the following dates: Harvest/02/28/1996 02/28/1996 Draft Report Review/04/18,19/1996 04/19/1996 Final Report Review/04/23/1996 04/23/1996 C. Orantes C. Orantes C. Orantes alElme Ss fbr Quality Assurance Unit Date'Reles CHV Study No.: 17387-0-455 2 C06504 CORNING Hazleton STUDY COMPLIANCE AND CERTIFICATION `The described study was conducted in compliance with the Good Laboratory Practicreegulations as set forth in the Food and Drug Administration (FDA) Title 21oftheU.S. Code ofFederal Regulations Part 58, issued December 22, 1978, (effective June 20, 1979) with any applicable saimgenneddmpernottso.colThtehratewwoeurlednaoffseicgtnitfhiecainnttedgerviityaotfitonhsefsrtoumdythoeratfhoerienmteenrtpiroetnaetdiorneogfutlhateiotnesstorretsuhlets. The raw data have been reviewed by the Study Director, who certifies that the evaluation ofthe test article as presented herein represents study design and evaluation criteria. an appropriate conclusion within the contextof the All test and control results in this report are supported by an experimental data record and this record has been reviewed by and a copyofthe final report the Study generated Director. All raw as a resultofthis data, study documentation, records, protocol will be archived in the storage facilities of Corning Hazleton Inc. for at least one year following submission of the final report to the Sponsor. After the one year period, the Sponsor may electto have the aforementioned `materials retained in the storage facilities of Corning Hazleton Inc.foran additional period of time, or sent to a storage facility designated by the Sponsor. Submitted By: Study Director: Puy `Hemalatha Murli, Ph.D. Mammalian Cytogenetics Department of Genetic and Cellular Toxicology _afes)os Study Completion Date CHVStudyNo.: 17387-0-455 3 00G05 TAOFBCOL NTEE NTS `CORNINGHazleton PageNo. LO SPONSOR ....oeiiiiiiiiiiiiiiiii eee eee 20 2M1 ATECl(RiTeensIttsAIMAdiecnltLief)icationo.oo 7 22 23 Date Received Physical Description 24 Genetics Assay No. 30 TYPEOFASSAY.....ooooiiiiiiiiiiiiiiiiiiiiieeeei 7 40 PROTOCOLNO........oomiiiiiiiiiiiiiiiiieeeee cca 50 5S1TUDYDAITniEtSiati.on.D.a.t.e.cooiiiiiiiiiiiiiiiiieiii eee] 52 53 ExperimSetanrttDaatle Experimental Termination Date 60 SUPERVISORY PERSONNEL 6.1 Study Director ................oooeeeeeeiii tiene] 62 Laboratory Supervisor 70 OBJECTIVE o.oo 7 80 MATERIALS ...........ccooiiiiiiiiiiiiiiiieeeiii ieee8 90 SOLUBANIDSLTAIBILTITYY: .............cc.ccrrrniii iii8 100 DOSESELESCTUTDYION ............ccccocrrurreiiiiiiiiiineni 9 I1R01IALT Dose Selection 102 Dosing Information 103 Results and Interpretation 104 Conclusion CHV Study No.: 17387-0455 4 C00606 `CORNINGHazleton I10R.I5ALII Dose Selection 106 107 Dosing Information Results and Interpretation 108 Conclusion 110 MICRONUCLEUS STUDY ILL Dose Selection .........oooiuuuiiiiiieaasiiiiiee see 13 112 Micronucleus Assay Dosing Information 120 BONEMARROWHARVEST, SLIDE PREPARATIONANDANALYSIS .....15 130 E13V1ALUATIGeOnNerCaRlITERIA ...............evviieeeeniiiiieeeeee 1S 132 Data Presentation and Interpretation 140 RESULTSANDINTERPRETATION ....................................16 150 CONCLUSION .......ovviiiiiiiiiiiiiiiieeeee cee 16 160 REFERENCES .............coooiiiiiiiiiiiiiiiaaeiiiiiiee se 17 170 DEVFI ROMA THETSII GNEO DPRONTOCS OL .........................17 180 EXPERIMENTDATATABLES ........................................18 CHVStudy No.: 17387-0-455 5 06507 CORNINGHazleton SUMMARY Mutagenicity Test on T-6357 inan Jn Vivo Mouse Micronucleus Assay mTihceroonbujceclteiivieonfbtohniesmianrvriovowapsoslayychwraosmattoiecvaelruyatthertohceytaebsioliftCyrolf:tChDe-t1es(tIaCrtRi)cleB,RT-m6i3c5e7., to induce. gInavtahgeedoatse20s0e,le4c0t0i,on6s1t0u,d8y,10t,heantedst1a0r1t0icmlegw/aksg isnolTurbiialliz1eadnidnndoeitoonxiizcesdiwgnastweerarnedobdsoesrevdedbyinoraanly o3f0t0h0e magn/ikmga.ls.SiIxnaTnriimaallIs,(athnriemealmsalweesraenddostherdeebyfeomraallegsa)vwaegreeaat s1s5i0g0n,ed2t3o7e5a, c3h25d0o,s4e1g2r5ouapn.d Animals were observed for three days after dosing for toxic signs and/or mortality. 2Ba0s0e0dmog/nktgh.e rIensutlhtesomfictrhoenudcolseeusselaescstaiyo,ntshteutdeys,ttahretimclaexwiamsusmoltuoblielriazteeddidnodseeiownaiszeedstwiamtaetredanads wdeorseedroarnadlogmalvyaagsesaitgn5e0d0,to1e00a0c,hadnodse2/h0a0r0vemsgt/ktgi.me Tgreonupa.nimVaelhsic(lfeivaenmdalpoessitainvdefciovnetrfoelmaglreosu)ps weuitthhatnhaetitzesetdaarptipcrloexwiemarteeeluyt2ha4nahtoiuzresdaaftpeprrodxoismiantgewleyr2e4,in4c8luadnedd7in2thhoeurasssaafyt.erTdhoesianngimfaorls dosed extractionofthe bone marrow. pTohleyctehsrtommaatteirciaelr,yTth-r6o3c5y7t,esdiudndneort tihneduccoendaitsiiognnisfoifctahnitsinacsrseaayseanindmiisccroonnsuicdleeriedinnbegoanteivmearinrtohwe. mouse bone marrow micronucleus test. CHV Study No.: 17387-0-455 6 06508 CORNING Hazleton Mutagenicity Test on T-6357 in an in vivo Mouse Micronucleus Assay 10 SPONSOR: 3M 20 MATERIAL (Test Article) 21 Client's Identification: T-6357 22 Date Received: January 16, 1996 23 Physical Description: Clear, amber liquid 24 Genetics Assay No.: 17387 30 TYPE OF ASSAY: In Vivo Mouse Micronucleus Assay 40 PROTOCOL NO.: 455, Edition 17 50 STUDY DATES 5.1 Initiation Date: January 18, 1996 52 Experimental Start Date: February 14,1996 53 Experimental Termination Date: March 22, 1996 60 SUPERVISORY PERSONNEL 61 Study Director: Hemalatha Murli, Ph.D. 62 Laboratory Supervisor: Monica Vegarra, B.S. 70 OBJECTIVE iTnhdeucobejmeictcirvoenoufctlheiisininbovnievomaasrsrayowwapsoltyocehvraolmuaattiectehreyatbhirloictyytoefsthofeCtrelst:CarDt-i1cl%e(,ITC-R6)35B7R,. to mice. This study was Heddle et al. (1983). conducted using modificationsofthe procedures suggested by CHV Study No.: 17387-0455 7 COCG09 `CORNINGHazleton 80 MATERIALS ARidvuelrt Lmaabloeraatnodrifees,maPloertmaigcee,MLs.traiTnhiCsrhle:aClDth-y1,%r(aCnRd)oBmRb,rewdesrterapiunrcwhaassesdelferctoemdCthoarles `maximize genetic heterogeneity and at the same time assure access to acommon source. `The protocol dosing. for this study was approved by the CHV-ACUC prior to the initiation of Animals were housed sevenpercage during quarantine, and housed five at randomization. The temperature and relative humidity were maintained at 7246F and 5515%, respectively, except on February 19, 1996, for the dose selection studies, when tmhiecrroelnautcilveeuhsumasisdaiyt:yFweabsruraercyor1d7e,d18a,t 3191,.124%,a2n4d, oanndth2e5,fo1l9l9o6wiwnhgednattehseforerltahtieve humidity was recorded light/12-hour at 39.1%, 37.3%, 34.5%, 32.5, dark cycle was maintained. A 34.4%, and commercial 20.8%, respectively. A diet (Purina Certified 12-hour Laboratory Pellets # 5002) and water were availableadlibitumforthe durationof the study. The feed was analyzed by the manufacturer for concentrationsofspecified heavy ``mTehteawlast, earflwaatosxiann,aclhylzoerdinoanteadrehtyrdorsopceacrtbiovnes,baosrigsafnoorpshpoescpihfaiteedsm,iacrnodosrpgeacniifsimeds,nuptersiteinctis.des, alkalinity, heavy metals, and halogens. Sanitized caging was used for housing the raenqiumiarlesd. tPoewresaornnseulithaabnldelpirnogteacntiimvaelgsaorrmewnotrskianngdweiqtuhiipnmethnet.animal facilities were `Awneirmearlasndwoemrelyquaasrsaingtniendetdofosrtaudtylgerasotupssevaennddwaeyrsebienfdoirveidbueailnlgypwleaicgehdeodnpsrtiuodryt.o Adonsiimnagl.s All animals were dosed based upon the individual body weights. Animals were uniquely identified by ear tag. Dose or treatment groups were identified by cage card/label. At the terminationofthe study all surviving animals were euthanatized by CO, followed by penetrationofthe thorax. Any extra animals not used for the study were used for training purposes. 90 SOLUBILITY AND STABILITY: `The test article, T-6357, was supplied as a clear amber liquid. The solubilityofthe test . carotniccelnetwraatsieovnoalfuaabtoedutin3d9e1i.o6nmigz/emdlw.ateDre.ioAniczleedarw,altieghrtwyaeslltohew svoelhuitciloeonfwcahsooibcteaifnoerdthaitsa assay. The stability responsibilityofthe of the test sponsor. material under the dosing conditionsof this assay is the CHV Study No.: 17387-0455 8 06510 100 DOSE SELECTION STUDY CORNINGHazleton IRIALT 10.1 Dose Selection Dose levels of 200, 400,610,810and gavage for the dose selection study. 1010 mg/kg were administered by oral 102 Dosing Information ``TThhee waneiimgahltsruasnegdeoifntthhee adnoismeaslesleucsteidonianstshaeydwoesreerdanogseedfionndiFnegbarsusaaryy w1a4,s 12989.65.- s3o6l.u6tiaonnds2w2e.r9e-2p7r.e6pagrreadmsj,usftporritohretmoaldeossianngdafnedmawleers,e rperseppeacrteidveblyy.maDkoisningga 1100.09m8gm/lmolf dsteoicoknifzoerdtwheatheirgh(dLootse# (191,0p0r0empga/rkegd).at CThHiVs)watos 1p.r2e0p8a1regdobfyTa-d6d3i5n7g, resultingin a clear pale yellow solution with a final volume ofthis stock were prepared for the remaining dose levels. of 12.0 ml. Dilutions `Dwoeseiknsgolwdasatatchheiteivmeedoufsdionsginag1.0.0Anmlo/uktglidnoesoifntghveolduomsein.g Aslclhaenmiemaislsfowuenrdeinnitnhee following table. A total of 30 animals was used inthisassay. DOSE GROUPS TREATMENT MF T-6357 200 mg/kg 400 mg/kg 610 mg/kg 810 mg/kg 1010 mg/kg 303 3003 303 3003 303 All doses given were on an acute (one-time only) basis. CHV Study No.: 17387-0455 9 00511 103 Results and Interpretation CORNINGHazleton All animals were examined after dosing and daily throughout the durationofthe dstousdiyng(tahllreaendiamyasl)s faoprpteoaxriecdenffoercmtaslanadn/dorremmoartianleidtihese.altIhmymeudntiialttehleyfeonldloofwtihneg observation period. 104 Conclusion Based on these results, the maximum tolerated dose could not be determined. IRIALT 105 Dose Selection Dose levels of 1500, 2375, 3250, 4125 and 5000 mg/kg were administered by oral gavageforthe dose selection study. 10.6 Dosing Information ``TThhee awneiimgahltsruasnegde oinf tthhee danoismeaslesleucsteidoninastshaeydwoesreerdanogseedfionndiFnegbrasusaaryy w1a5,s 12989.65.s3o6l.u4taionnds2w3e.r6e-2p7r.e5pagrreadmsj,usftorprtihoer mtoaldeossianngdafnedmawleerse, prerseppeacrteidveblyy.maDkoisnignga 500 mg/ml stock forthe high dose (5000 mg/kg). This was prepared by adding deionized water (Lo#t 19, prepared at CHV) to 7.4993 g of T-6357 uptoa svtoolcukmweoerfe 1p5r.e0pamrle,drfeosrultthieng15in00a,c2l3ea7r5,pa3l2e5y0e,lalnodw 4so1l2u5timong./kDgilduotsieonlseovfetlsh.is wDeoesiknsgawndasonaechdiaeyveodldusaitntghea t1i0m.e0 omfl/dkosgidngo.sinAgnvooultulmien.eofAltlheandiomsailnsgwsecrheenmieneis found in the following table. CHV Study No.: 17387-0455 10 00G12 `CORNING Hazleton A total of 30 animals was used in this assay. DOSE GROUPS TREATMENT MF T-6357 1500 mg/kg 2375 mg/kg 3250 mg/kg 4125 mg/kg 5000 mg/kg 303 3003 303 303 303 All doses given were on an acute (one-time only) basis. 107 Results and Interpretation sAltludayni(tmharlese wdeayrse)efxoarmtionxeicdeafffteecrtdsoasnidn/goranmodrtdaallityietsh.roughout the durationofthe hIympmoeadcitaitveel,yaanfdtetrhedoostinhge,2rinwethree1n5o0r0mamlg./kIgnthdoes2e3g7r5oumpg,/4kgandiomsaelsgrwoeurpe, 2 males t`wheer3e2l5a0ngmugi/dkagnddoastaexigcr,ouapn,d2thmealreesmaainndin1gfaenmiamlaelswewreerelannogrumiadlanadndathaexailct,hya.ndIn the remaining animals were normal and healthy. Inthe 4125 male was prostrate with labored breathing, and the remaining mg/kg dose group, animals were: 1 whyiptohalcatbivoer.edIbnretahtehi5n0g0,0amngd/tkhge rdeomsaeignrionugp,an1immaallsewaenrde1slfiegmhatllyehwyeproeacptriovset.rate `Awpeprreonxoirmmaatlelaynd1 hheoaultrhya.ferIndotshien2g3,7a5llmagn/imkaglsdofsreogmrtohuep,15| 0m0amleg/akngd d1ofseemgarloeup. `3w2e5r0e hmygp/okagctdiovse,e agnrodutph,e1remmaalienianngd a2nfiemmaallsewsewreerneohrympaolacatnidveheaalntdhyt.heIrnemthaeining waneirmeahlyspwoaecrteivneo.rmIanltahned5h0e0a0ltmhgy./kAglldoasneimgarlosupf,ro1 mmathlee4a1n2d52mfge/mkagledsosweergeroup prostrate, and the remaining animals were hypoactive. `Awpeprreonxoirmmaatlelaynd17hehaoluthrys.afAtlelr daonsiimnagl,safllroanmitmhaels23f7r5ommgth/ekg15w0e0remgh/ykpogadcotisveegarnodup shtuanicnhiendg,oannedye2s.maAllels3(2#'5s06m3g3/6,kg63d3o4s)e allesvoelhaandismqaulisnwteerdeeyheyspowaictthivbeloaonddcolored CHV Study No.: 17387-0455 1 00513 `CORNINGHazleton Ahlulnc4h1e2d5, magn/dk2gmaanliemsal(s#'wse6r3e33h,yp6o3a3c7t)ivaelswoithhaddybslponoedacoalnodrebdlosotdaicnoilnogroend esytealiindisn.g hoandeyterleimdosr.s,Alclhr5o0m0o0damcgr/yokrgrhaneiam,aalnsdwewrereehcyoplodacttoitvheewtiotuhchd.ysTphneeiar;caallgemaallesos haalsdo pservoefroaulnbdlfoooodt csoplloayr,edanspdotwsasinciot.ldOtnoethfeemtoaulceh.(#A63s4e0c)ohnaddfuermianleecaollsoorheaddstaaibnlso, od colored glaze on the eyelids and was cold to the touch. gArpopurpoaxinmdaatlellfye4m2a.l5eshofurrosmatfhteer2d3o7s5inmgg,/akllgaannidma3l2s5f0rmogm/tkhge d1o5s0e0gmrgo/ukpgs dwoesree a`nnodrmtahle arnedmahienailnthgym.alOensew2e3r7e5hmypgo/akcgtidvoes.eOgnroeu3p2m5a0lmeg(/#k6g33d6o)sewgarsofuopumnadldeead 4(1#26533m7g)/wkagsdfoosuengdroduepa,d2anmdaltehse r(e#ma63i3n2i,ng63m3a8l)easnwdeorneehyfpeomaactliev(e#.6I3n55t)hewere rfeomuanidndienagdf;etmhaelreewmaasinhiynpgoamcatlievew.erIen htyhpeo5ac0t0i0vem,g/hukngcdhoesdewgirtohupd,ys1pnmeaal;e the `(h#y6p3oa4c0t)ivaen;dth1efreemmaalieni(#ng63f6e0m)alweesrweefroeuhnydpdoeaacdt;ivteheanrdemhaaidnitnrgemmoarlse,ssqwueirneted eyes and dyspnea. sAuprpvriovxiinmgaatneilmyal6s6.f5rohomutrhsea2ft3e7r5domsgi/nkgg, aalnldan3i2m5a0lsmgf/rokmg tdhoese15g0r0oumpgs/wkeg,reanndoramlall haynpdoahecatlitvhey.wiAtlhldsyusrpvnievai.ngIanntihmeal5s00in0tmhge/4k1g2d5omsge/gkrgoudpo,s2e gfermoaulpewse(r#e's 6346, 6352) were found had dyspnea. dead and the surviving males were hypoactive, hunched and Approximately 71.5 hours (#6342) was found dead. after dosing, 1 male from the 5000 mg/kg dose group gArpopurpo,xi1mmaatleelyfr9o1mhtohuers32af5t0ermdgo/skigngd,o|semaglreou(p#a6n3d44|)mfarloem(t#he6323397)5fmrgo/mktghedose 4125 2375 mg/kg dose group were found dead. ~All and 3250 mg/kg and 4125 mg/kg female remaining animals from dose groups were normal the 1500, and healthy. The surviving male (# pale, hunched and hypoactive. 6343) from the 5000 mg/kg dose group appeared CHV Study No.: 17387-0455 2 C0014 CORNING Hazleton `The mortality data for this assay are summarized in the following table: `Summary of Mortalities Within 3 Days in Mice Dosed Acutely with T-6357 Observations --1500Tmrge/akgtment 2375 mg/kg 3250 mg/kg 4125 mg/kg 5000 mg/kg M0a3le 23 2 33 2 Femoa3le os on 173 3 108 Conclusion Based on these results, the maximum tolerated dose was estimated to be 2000 mg/kg. 110 MICRONUCLEUS STUDY 111 Dose Selection Based on results from the dose selection 2,000 mg/kg were selected for testing in study, dose this study. levels of 500, 1,000, and 11.2 Micronucleus Assay Dosing Information C`Tyhcelaonpihmoaslpshuasmeiddein(tCheASmi#cr6o0n5u5c-l1e9u-s2;asSsiagymaw,erLeod#tose4d4Ho0n4F8e6b)r,utahreyp2o7s,it1i9v9e6. acodnmtirnoils,tweraesdsboyluobriallizgeadviangedeaito8n0i.z0edmgw/aktge.r (TLohte#ve1h9i,cplreecpoanrterodla,tdCeHioVn)izaenddwawtaesr 2(Lvootl#u1m9e,opfre1p0.a0remdl/aktgC.HVT)h,e wweaisgahdtmriannigsetoefrtedhecoannciumrarelnstulsyewdiitnhtthhee mteisctraor-ticle at rneuscpleecutsivaeslsya.y Twhaes d2o9s.i2n-g39s.o5luatnidon2s3.fo0r-3t2h.e2agsrsaaymswefroretphreepmaarleeds bayndmafekmianlgesa, d2e0i0onmigze/dmwsattoecrktofo5r.t0h0e88higghofdoTs-e63(52700u0pmtgo/kagv)o.luTmhiesofwa2s5 pmrl.epaArecdlebayrapdadlieng CHV Study No.: 17387-0-455 13 CO0G15 CORNINGHazleton ysteolclkowwesroleuptrioenpawraesd ofbortatihneerdeamtaaincionngcednotsreatlieovenlosf.20A0semcgo/nmdl.grDoiulputoifoannsoimfatlhsis a(tdteshieghniatgehddSoesecosnedlaecrtyedD.osTeheGsreouapni)mwaalss walesroeaosnsliygnuesdedtoitnhtehsetausdsyayanads was dosed replacements for any which died in the primary dose group. h`Taernveasntimtailmse (gfriovuep.malVeehsiacnlde fainvde pfoesmiatlievse)cwonetrreolragnroduopmsl,ycautshsainagtnitezoeddeach dose/- adpopsreodxwiimtahtetlhye 2te4sthaorutrisclaefwteerrdeoesuitngh,anwaetrizeeidnacplpurdoexdiimnattheelyas2s4a,y.48Tahneda7n2imhaolusrs afier dosing for extractionofthe is found in the following table: bone marrow. An outlineofthe dosing scheme Dosing Scheme for Micronucleus Assay A totalof 120 animals was used in this assay Number ofAnimals Assigned Primary Dose Groups ~~ Treatment 2M4HFr 4M8FH TM2HFr Tes500 mg/kg 55 55 55 1000 mg/kg 55 55 55 2000mye, 55 55 55 Vehicle Control, deionized water, 100 mkg ~~ 5 5 - = - - Positive Control, Cyclophosphamide, 80.0 mghkg 5 5 - - - - SecoGnrdoaurpysD*ose Male Female -55 --- * Trehpelaacneimaanlismaalsssiwghniecdhtdoitehdeisnetchoendparriymadroysedogsreougprsouwperatetdhoesheidghanddosweerleevoeln.lyAullseedxttzoa animals not used as replacements were euthanatized at the completionofthe trial. The ageofthe animals at the timeof dosing was nine weeks and one day. Volumes dosed were 10.0 ml/kg and were based upon individual animal weights. CHV Study No.: 17387-0455 14 C0516 `CORNINGHazleton 120 BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS pAetnetthreaatpipornoopfrtiahteethhaorrvaexstantdimteh,etahdehaenriimnaglssofwtertiesseuuethaanndateipziepdhywsietshoCfOb,otfholfleomwoerdabwyere croenmtoaviendi.ngT3h-e5mmalrrboowviwnaessfelruusmhe(donferotmubtehefobroenaechanadnitmraaln)s.ferFroeldltoowicenngtrciefnutrgieftuugbaetsion to pwelelreettshpreeatdisosune,sltihdeessuapnedrnaaitradnrtiewda.sTrheemoslvieddesbwyearsepifriaxteidoninamnedtphoarntoilo,nsaonfdthsteaipneeldleitn May-Grunwald solution followed by Giemsa (Schmid, coverslipped using Depex mounting medium. 1975). The air-dried slides were e`Trhyethsrloicdyetsew(ePrCeEc)odtoednofomroacnharlyosmias,tiacndersyctohrreodcyftoer (miNcCrEo)nuccelleliraatniod. thSetapnodlayrcdhrfoomramtsicwere muiscerdotnoucrleecaortdedthceesllesdwataas. eOxpnreestsheodusaasnpderPcCeEntSpmeicrraonniumcalleawteerdeceslclosrebda.seTdhoenftrheequtoetnacly of CPrCElS:sCDp-r1e%se(nItCRin)tBheRssctorraeind iospatibcouftiel0d..0-T0h.e4%n.ormal frequency of micronuclei in this N`TChEe SfroebqsueerncvyedofinPtChEeSopvteircsfuiselNdsCwEhilweasscdoertienrgmtihneedfirbsyt 1sc0o0r0ienrgytthheroncuytmebse.r of PCEs and 13.0 EVALUATION CRITERIA: 13.1 General `MTihcerocrniutcelrieaifworertehedairdeknltyifsitcaaitnieodnaonfmdicgernoenruaclllyeirowuenrde, athlotsheooufghSaclhmmoindd(a1n97d6). arnindgwsehrapeegdemniercarlolnyucbleetiwoececnas1i/o2n0alalnydo1c/c5urtrheeds.izMeoifctrohneucPlCeEi.haTdhsehaunriptobofrsdceorrsing. ``wmaosrethtehamnicornoenumcilceraotneudclceeluls, nwoatsthceoumnitcerdonauscolneeusm;ictrhousnutchleeaotcceadsPioCnEa,lncoetlltwwioth(or omforPeC)EmsicarnodnuNcCleEiS. (Tbhlueissht-agirnienygapnrdocreedd,urreesppeecrtmiivtetleyd).the differentiation by color 132 Data Presentation and Interpretation Data are summarized by sex and dose groups forthedifferent time points. pInedrifvoirdmueald aunsiimngalandaatnaalayrseiaslosfo pvraersieanntceed(.WiTnheer,an1a9l7y1si)soofntehiethseer duanttarawnassformed (when variances are homogeneous) and rank transformed (when variances are CHV Study No.: 17387-0455 15 O0G17 CORNING Hazleton ohfetvearroigaennceeowusa)s psriogpnoifritciaonnts(opf<0c.e0ll5s),wiatDhumnincertotn'usctl-eteistpe(rDuannniemtatl,. I19f5t5h;e 1a9n6a4l)yswias s nuesgeadttiovedecotnetrrmoiln.e Awnhailcyhsedsosweergeropupesr,fioframneyd,sewpearreatseilgyniffoirceanatclhy hdairfvfeesretnttifmreoamntdhe ssteaxticsotimcbailnlyatsiiognni.fiTcahnet cdroisteer-iraelfaotreddeitnecrrmeiansienignampiocsriotniuvcelreeastpeodnPsCeEiSn,voolrvethdea dleeatsetctoinoen doosfae rleepvrelo.dAucibtelset aanrtdicslteattihsatticianlldyucseigdnnifeiictahnetr paosstiattiivsetircealslpyonsisgeniffoircaatnt ldeovseel rweasspocnosnesindoerreadstnaetgiasttiicvael.lyIsnigeniitfhiecracnatsae,ndthreepfrinoadlucdiebcliesiionncrweaassebaatseodneondose scientific judgment. 140 RESULTS AND INTERPRETATION: dAlulraatniiomnoalfsthweeraessoabysefrovretdoxiimcmseydimaptteolmysafatnedr/doorsmionrgtaalintdiepse.riAoldlicaanlliymatlhsroiungthhoeuvtethhiecle aapnpdropporsiiatitveehcaornvtersotltgirmoesu.psAalplpteeasrteadrtnicolremdaolsaefdtegrrdoouspisnagpapnedarreedmnaoirnmeadlheiamlmtehdyiuantteillythe hafotuerr mdoasliengatatnhde r1e0m0a0imnged/khgeadlotshey ulnetviell t(h#e6a5p6p2r)o,prwihaitcehhawravsesftotuinmdesd,eaexdcaebpotuftor72onheou7r2s post dosing. pTohleyctehsrtoamratticilce,erTy-t6h3r5o7c,ytiensduovceerd tnhoesliegvneilfsicoabnsteirnvcerdeaisnetsheinvmeihiccrloenuccolnetarotlesdin either sex PorCaEt/aNnCyoEfrtahtieoshoafrvtehset tmiamleess. fDruome tthoet5ox0i0c,it1y0,0a0,siagnnidfi2c0a0n0t rmegd/ukctgidonosweagsroobuspesravtetdhien7t2he `hnouucrlehaatrevdePstCEtismei.n bTohtehspeoxsietsiavse ccoonmtrpoalr,eCdP,toitnhdeuvceehdicsilgenciofnitcraonltsi,nwcirteahsmeseainnsmiacnrdo- tsitvaenldy.ardTheerrdorastoafs7u.m9m8ar%ize1d.b3y0%doasnedg4r.o1u6p%are0p.re9s2e%ntfeodr tinheTamballees1aannddfienmdailveisd,uarlespec- animal data Table 8. are found in Tables 2 through 7. Historical control data are presented in 150 CONCLUSION: m`Tahre rtoeswt pmoalteyreiharlo,mTa-t6i3c5e7r,ytdhirdoncoytteisnudnucdeera tShiegnciofnidciatnitoinnscorefatsheisinasmsiacyroannudcliseicoinnsbiodnereed negative in the mouse micronucleus assay. CHV Study No.: 17387-0455 16 00618 CORNING Hazleton 160 REFERENCES: aDcuonnntertotl,. C.J.WA.:m.AStmautlitsti.plAesscoocm.p,a5r0i:s1o0n9s6-p1r1o2c1ed,ur19e5f5o.r comparing several treatments with Dunnett, C.W.: 491, 1964. New tables for multiple comparisons with a control. Biometrics, 20:482- aHendddSlae,laJm.oA.n,e,HiMteF,.:M,ThKierkihnadrutc,tiBo.,n oLafrmsiecnr,onKu.,clMeaicaGsreagmoera,sJu.rTe,oNfegweenlolt,oxGic.iWt.y. MutationRes,123:61-118, 1983. Schmid, W.: The micronucleus test. Mutation Res., 31:9-15, 1975. Schmid, W.: The micronucleus test Mutagens: Principles and Methods for cytogenetic analysis. for Their Detection, Vol. Chemical 4 (A. Hollaender, ed). Plenum, pp. 31-53, 1976. Winer, B.J.: Statistical York, Second Edition, Principles 1971. in Experimental Design, McGraw-Hill, New 17.0 DEVIATIONS FROM THE SIGNED PROTOCOL `The following deviations were made from the signed protocol. 1. rOencoFredberduaatry311.91,%1.9F9o6,rtfohrethmeicdroosneucsleleeucstiaosnsasyt:udoiens,Fetbhreuraerlyat1i7v,e h1u8,mi1d9i,t2y4,wa24s, 3a4n.d42%5,, a1n9d962,0t.h8e%,rerlaetsipveecthiuvmeliyd.itTyhwisasdirdecnoortdaefdfeactt3t9h.e1a%n,im3a7l.s3%a,nd34t.he5r%e,w3a2s.5n%o, impact on the integrityofthis study. 2. sDeuleecttoioantsetcuhdnyi,catlheerarcotruailndtohseeprleepvaerlsatuisoendofwdeorsei6n1g0,st8o1c0k,safnodr T1r0i1al0Imogf/tmhle. dTohsee difference is <2% and had no impact on the integrityofthis study. CHV Study No.: 17387-0455 17 [hE CORNINGHazleton 180 EXPERIMENT DATA TABLES CHV Study No.: 17387-0455 18 COCGR0 `CORNINGHazleton SPONSOR3:M TESTARTICLE: T-6357 A_S--S_A--Y: 17387 TREATMENT DOSE coNTROLS VEHICLE POSTIVE Water CPAOOmghe TABL1E MICRONUCLEUS DATA SUMMARY TABLE HTAIRMVEEST ~~MEA%NMIOFCR10O00NPUERCALNEAIPTMCEEADSsEL. (HR) MALES FEMALES TOTAL Wh 0@402 0045002 03002| 20h 798130 4164052 6074098 | RATMIEOAPNCSEEN.CE MALES FEMALES 0672005 0784008 0714008 0714005 TESTARTICLE Smeg 24h 4h Th 1000mgke 26h 4h Th omy 2h 4h Th 0024002 0065004 004000 0104003 0104005 0034003 0064006 024002 0@E002 010003 0025002 002002 0044002 0044002 0004000 0101004 0082002 004002 0064002 | 0032002 | 0014001 | 0072002 | 0074003 | 0014001 | O0E004 | 0034002 | 003002 | 0m40 OS4:012 043s006 0634005 0652006 0294004 0614007 0672004 036+003c 073400 0624003 0522003 0774008 0564003 058006 0704007 0694006 0534004 ***SiSginginfiifciacnatnltylgyrleoawteerrtthhaann tthh ccoorrrreessppoonnddiinngg vveehhiicclee ccoonnttrrooll,, pp<<00..0055.. CP=Cyclophosphamide CHV Study No.: 17387-0455 19 C0062 `CORNINGHazleton MICRONUCLEUS TESTT-AIBNLDEIV2IDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6357 ASSAAyY NO: 17387 EAT _-- UHOURHARVEST MALE _-- VEHICLECONTROL ~~ Water POSITIVECONTROL ~~ CP800 mphe. TEST ARTICLE 500 maf 1000 meke 2000 moe -_-- MCPN==CyMcilcorpohnouscplheaumside. #PCMEN=PPCoElysc=hrMoimcatriocnuecrlyathtrcodcyPtCeEs `NCE = Normochromaic erythrocyte ANIMAL TOMN RATIO NUMBERaTwCe TpeET NCE 0 ess 00 006457 o65s1s 01 oonnt so om o6s5m n6s o1s0s6 o onma0 o0r6s sn 50 os essni 00 015041 65s5i7s 01 005876 om 0 08s asest so1 0o6n6 o65n65 11 o05m6 6574 2 oar oe5o47 30 0087 oomm 00 005450 ozo on CHV Study No.: 17387-0-455 20 GO0GR2 `CORNING MICRONUCLEUS TESTTAIBNDLIEVIDUAL ANIMAL DATA SPONSOR3M: TESTARTICLE: T6357 a ASSATYRs ENAOT:ME1a N73T87 vwouRor mpAeLr m TJoCBvwpachr noee _m--omma rons aw [EP -- roveCONTROL CPao mus wanes mm we wo me F@wAr 50 ow " @ a1 0 " @@wo 4x@3 oooamo @ oa 08 G "w@ 121%oonm ""1 0oBm w@w b 11 oox "Gwo00 on a@@ @ 3001 o0oomwk "1 ou FCOVoECircPltooptrmhpotuniederos NFCAEN=PNCEommeu chrnovniave PetEses Hazleton CHV Study No.: 17387-0-455 21 00623 `CORNING MICRTOESTNaInNUDeIsVICDUALLANEIMAULDASTA sronsoRsw: TESTARTICLE T4357 e Assave NO: 1137 TREAT foan mo JJ -i_--mewew-- s wwe ey Tamar wwe @@osxa o +0 uooazum wn wm FA soso 0 om owao 1 uw wo wm mFwFnAoAs1 on - w 000c @ 1 RAr 0s N Pii cee oetomcron o ccre Hazleton CHV Study No.: 17387-0-455 2 COCGRY `CORNINGHazleton MICRTOESTNT-aImNUDeIsVICDUALL ANEIMAULDASTA SPONSOR:3M TEST ARTICLE: T6357 AmsseAY vo. 1317 TREATMENT Nooim PCE pTCaErNGoe e P-- T_ ---- --e mm-- e wm y T-- TT mamas ow eee " @w@n o000 o2uomm "@ +o on wo as no -@6639 5o1r 23047 wo ws @@@wm 0y or oooom. --e----------@----Fo ---- P[CE PPvORo M--opno raonscteEs HO---- CHV Study No.: 17387-0455 23 CO0GRS `CORNING Hazleton 'MICRONUCLEUS TABLE& TEST - INDIVIDUAL ANIMAL DATA `SPONSO3RM: `TEST ARTICLE: T-6357 A ASSA AY NO: 17387 TREATMENT 7HOURHARVEST MALE ANIMAL NOMN RATIO NUMBER P1C00B0S) pcence TEST ARTICLE, 0 ng a an no o 003350 e ano o 0os3t9 ws 0 oa 1000 me ea s 0 001219 eass 0 031 ao ow 000 mek s ao o 003379 ean1o 00335 ono ox *Animal found dead MPCNE ==MPioclryocnhurcolmeautsiccoythroeyte : N#CMEN=PNCoEmsmo=chMriocmartoincuercyltharPotcCeyEtdes CHV Study No.: 17387-0-455 2 C00626 `CORNING Hazleton TABLE 7 MICRONUCLEUS TEST- INDIVIDUAL ANIMAL DATA SPONSOR: 3M `TEST ARTICLE: T-6357 ASSAY NO. 17387 TREATMENT THOURMARVEST FEMALE TAaNNUIMaMBAElLR N"OCMESN pRoAETnICOE (1000) TESTARTICLE 0 mokg 6e83z1 00 003386 66 657 00 0045s3 6659 1 046 1000 mks "wiwen 00 o0s7t6 @a s 00 o0s5t9 6s 0 oat 200 mois 62635 01 o08r6 66665s5t 01 005374 saz o ous PMCNE==MPioclryocnhurcoimeautsic erythrocyte `#NMCEN=PCNoErsmo=cMhircormoantuiccleerayttehdrPocCyEtse CHYV Study No.: 17387-0-455 25 00GR7 CORNINGHazleton TABLES MOUSE MICRONUCLEUS HISTORICAL CONTROL DATA 7/95 THROUGH 12/95 FOOLED VEWICLE CONTROLS. My MAX AVG N %MICRONUCLEATED PCEs PER 1000PCE MEANOF 1000PER ANIMAL 4 SE. MALES FEMALES TOTAL RATIO PCENCE| MEASNE. MALES FEMALES 000 oz 008740007 a 000 024 00810008 oot 017 031 oss 00M:0005 | 0350%0021 a a 024 103 054740025 a" CPyOcSlIoTpIhVosEpChaOmNiTdRe,OL8S0..0 mg. My MAX AVG N NPCCEE == PNoolrymcohrcohmraotmicateiryetrhyrtohcryotceyte 200 ses 36240240 19 150 636 317040245 1 240 oar 538 342640184 | om 0sme0m0 1 1 040 019 o0sssso0zs 19 CHV Study No.: 17387-0-455 26 C0G3R8 pGroriitaiieettc April 19, 1996 Steven C. Gordon, Ph.D., DABT 3M Medical Department Building 220-2E-02, 3M Center St. Paul, MN 55144-1000 CORNING Hazleton RE: DRAFT REPORT AND PROTOCOL AMENDMENTS In Vivo Micronucleus Assay Protocol No.: 455CO, Ed. No.: 4, Modified for Sponsor Genetics Assay No.: 17387, 17387-1 `Test Material: T-6357 Dear Dr. Gordon: Enclosed please find two (2) copies of the above referenced report. The report includes an unsigned Quality Assurance Statement and Compliance and Certification Statement, which will be signed upon issuanceof the Final Report. "The final report will be issued after your review and notification to us. Please contact the undersigned with any questions, comments, or necessary revisions at your earliest convenience. `The report will be finalized after | yearif no notification is received. Thank you for giving us this opportunity to work with you. Sincerely, CORNING Hazleton News[7 Hema Murli, Ph.D. Mammlian Cytogenetics Departmentof Genetic and Cellular Toxicology HM/paj enclosures 000629