Document pmgnD3gMKb1K0oVxDRNBR7aR7

CORNINGHazleton MUTAGENICITY TEST ON o T6292 ; IN AN IN VIVO MOUSE MICRONUCLEUS ASSAY EINALREPORT AUTHOR Hemalatha Murli, Ph.D. PERFORMINGLABORATORY. Corning Hazleton Inc. (CHV) 9200 Leesburg Pike Vienna, Virginia 22182 LABOPRR OJEACTIT DENTOIFIRCATYION CHV Study No.: 17384-0-455 SUBMITTETDO 3M 3M Center, Building 220-2E-02 St. Paul, Minnesota 55144-1000 SCTUODYMPLETIDOATNE May 2, 1996 a) ota. CHV Study No.: 17384-0-455 1o0f24 01325 CORNING Hazleton QUALITY ASSURANCE STATEMENT Project Title: In Vivo Mouse Micronucleus Assay Project No.: 20996 Assay No.: 17384 Protocol No.: 455 Edition No.: 17 Quality Assurance inspections ofthe study and reviewofthe final reportofthe above referenced `project were conducted according to the Standard Operating Procedures ofthe Quality Assurance `Unit and according to the general requirementsofthe appropriate Good Laboratory Practice regulations. Findings from the inspections and final report. review were reported to management and to the study director on the following dates: `Weighingof Test Article/03/05/1996 DilutionofTest Article/03/05/1996 Draft Report Review/04/24/1996 Final Report Review/05/02/1996 03/05/1996 03/05/1996 04/25/1996 05/02/1996 C. Smith C. Smith C. Orantes C. Orantes `Quality Assurance Unit ate Released CHV Study No.: 17384-0-455 2 01326 `CORNINGHazleton STUDY COMPLIANCE AND CERTIFICATION The described study was conducted in compliance with the Good Laboratory Practice regulations as set forth inthe Food and Drug Administration (FDA) Title 21ofthe U.S. Code of Federal Regulations Part 58, issued December 22, 1978, (effective June 20, 1979) with any applicable amendments. There were no significant deviationsfromthe aforementioned regulationsorthe signed protocol that would affect the integrity ofthe study or the interpretationofthe test results. The raw data have been reviewed by the Study Director, who certifies that the evaluationof the test article as presented herein represents an appropriate conclusion within the context of the study design and evaluation criteria. All testand control results in thisreportare supported by an experimental data record and this arencdoradchoapsyobfeetnherefviniaelwreedpobryttgheenSertautdeydDaisreacrteosru.ltAolfltrhiasw sdtatuad,ydwoiclulmbeentaartcihoinv,edreicnotrhdes,stporroatgoecol facilities of Corning Hazleton Inc. for at least one year following submission ofthe final report to the Sponsor. After the one year period, the Sponsor may elect to have the aforementioned `materials retained in the storage facilities of Corning Hazleton Inc. for an additional period of time, or sent to a storage facility designated by the Sponsor. Submitted By: Study Director: NowatereMout: Hemalatha Murli, Ph.D. Mammalian Cytogenetics Department of Genetic and Cellular Toxicology sales Study Completion Date CHV Study No.: 17384-0-455 3 01327 CORNINGHazleton TAOFBCOL NTEE NTS Page No. SUMMARYee eeeeeaeiaeeinnsnaneeeisee 6 10 SPONSOR c.evveenueeaninainnnaninenisesieeeieenieenieene7n 20 MATE(RTesItAAfclLe) ....onvveeennnnasineninsnnineninenineann? 21 Clients Identification 22 DateReceived, 23 Physical Description 24 Genetics Assay No. 30 TYPEOFASSAY..cuuvvirnnreinnnniinnniesnineninenineninennn 40 PROTOCOLNO.....eeeveneuasninnaninnainssninsainsnninenineand 50 STUDYDATES ....vvorennnmaninnnnsnnnnsnennineniieenineeinneens 7 51 InitiationDate 52 ExperimSetanrttDaatle 53 Experimental Termination Date 60 SUPERVISORYPERSONNEL ........coreoiesninsninenninnaninnnesd 61 StudyDirector 62 Laboratory Supervisor 70 OBIECTIVE ..eveeounneenneaeinneinneinseinseieenineeineen7n 90 SOLUBILITYAND STABILITY:.......ovvresisniireeennninneeneieens8 100 DOSESELECTION STUDY ....oouviieniraninanninaninainnaaeines 101 Dose Selection 102 Dosing Information 103 Results and Interpretation 104 Conclusion CHVStudy No.: 17384-0455 4 01328 CORNINGHazleton 110 MICRONUCLEUS STUDY .......oovvvreesonninneennnnnneennnnnnn1ee1 111 112 Dose Selection Micronucleus Assay Dosing Information 120 BONEMARROWHARVEST, SLIDEPREPARATIONANDANALYSIS .....12 130 EVALUATION CRITERIA ........ccouveresnnnnreenninnenennnin1n3n 131 General 132 DataPreseanndtInatertprietaotinon 140 RESULTSANDINTERPRETATION.....ooenviireennniineennennnnn13n 150 CONCLUSION ...oooi'nnnnnrmnnninniieeesnninnressnnieneeeeninnn1nn5 160 REFERENCES ......cccocuvvmmmaannnnnnnnsnininseesninneeennnnie1n5n 170 DEVFRI OMTA HEST IGNEI DPRO OTON COL ......vovvvreineenn1s5. 180 EXPERIMENTDATATABLES .......oovoeomiinreeniiiinnienenninnn1n6n CHV Study No.: 17384-0455 5 01329 `CORNINGHazleton SUMMARY Mutagenicity Test on T-6292 inan In Vivo Mouse Micronucleus Assay `The objectiveofthis in vivo assay was to evaluate the abilityofthe test article, T-6292, to induce: micronuclei in bone marrow polychromatic erythrocytesofCrl:CD-1%(ICR) BR mice. In the dose selectionstudy,the and dosed by oral gavage at 0, test article was solubilized in acetone:comn 1000, 1400, 1800, 2200, and 2600 mg/kg. oil Six (40%:60%, viv) animals (three `malesand thre females)wereassigned toeachdose group. Animals were observed for three days after dosing for toxic signs and/or mortality. Based on the resultsofthe dose selection study, the maximum tolerated dose was estimated as a(b4o0u%t:26200%0, mvg:v/)kga.ndIdnotsheedmoircarlognauvcalgeeusatas5s5a0y,,t1h1e0t0e,stanadrt2ic2l0e0wmags/skogl.ubTileinzeadniinmaalcset(ofinvee:mcaolmeosil and five females) were randomly assigned to eachdose/harvesttime group. Vehicle and positive c"Tohnetraonligmraoluspdso,sceudthwaintahtitzheedtaesptparrotxiiclmeatweelrye2c4uthhoaunrastiazfetedradpopsrionxgi,mwateerleyi2n4c,lu4d8edanidn t7h2ehaosusarys. after dosing for extractionofthe bone marrow. T`phoelytcehstrommaatteirciaelr,yTth-r6o2c9y2t,esdiudndneort tihneduccoendaitsiiognnisofifcatnhtisinacsrseaayseanindmiisccroonnsuicdleeriedinnbegoanteivmearinrtohwe `mouse bone marrow micronucleus test. CHV Study No.: 17384-0-455 6 COLIGE `CORNING Hazleton Mutagenicity Test on T-6292 in an in vivo Mouse Micronucleus Assay 10 SPONSOR: 3M 20 MATERIAL (Test Article) 21 Clients Identification: T-6292 22 Date Received: January 16,1996 23 Physical Description: Amber, waxy solid 24 Genetics Assay No.: 17384 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 ExperimentalStartDate: February 20, 1996 53 Experimental Termination Date: April 16,199 60 SUPERVISORY PERSONNEL 6.1 Study Director: Hemalatha Murki, Ph.D. 62 Laboratory Supervisor: Monica Vegarra, BS. 70 OBJECTIVE Tinhdeucobejmeicctriovenoufcltehiisininbovniveomaasrsaryowwapsoltyocehvraolmuaattiectehreyatbhirloictyyotfets hofeCtreslt:aCrDti-c1le%,(TC-R6)29B2R, to mice. This study was conducted using modificationsofthe procedures suggested by Heddle etal. (1983). CHV Study No.: 17384-0455 7 01331 CORNINGHazleton 80 MATERIALS RAidvuelrt Lmaabloeraatnodrifeesm,aPloertmaigcee,,MsIt.raiTnhCirslh:eCaDl-t1hy%,(IrCaRn)doBmR,brweedrsetrpauirncwhaassesdelfercotmedCthoarles TmhaexipmrioztoecgoelnfeotricthhiestsetruodgyenweaitsyaapnpdraotvetdhebsyatmheetCimHeVa-sAsuCrUeCacpcreisosrttoo athceoimnimtoiantiosnouorfce. dosing. Animalswere housed up to sevenpercage during quarantine,andhoused up 10 five per cage at randomization. The temperature and relative humidity were maintained at 7246F and 55+15%, respectively, excepton February 17, 18, and 19, 1996, for the dose selection study, when the relative humidity was recorded at 39.1%, 37.3%, and 34.5%, respectively, and on February 24, 24, 25, and March 2, 1996, for the micronucleus assay, rwehsepencttihveelreyl.atAive1h2-uhmoiudritlyigwhat/s12r-ehcoourrdeddarakt 3cy2c.l5e%,wa3s4.m4a%in,t2a0in.e8d%., aAndco3m5m.e5r%c,ial diet (thPeurdiunraatiCoenrotifftihede Lsatbuodyr.atoTrhye Pfeeleldetwsas#a5na0l0y2z)eadnbdywtahteermawneurfeacatvuarilearblfoeracdonlciebnittruamtfioorns ospfsecpiefciiefdienduthrieeanvtys.meTthaelsw,aatfelratwoaxisn,ancahllyorziendaotendahyrdetrroocsaprebcotnisve, boarsgiasnfooprhsopsepchifaiteed, and `umsiecdrfooorrghaonuissimnsg, pesticides, a the animals. lkalinity, heavy metals,and h Personnel handling animals alogens. Sanitized caging was or working within the animal facilities were required to wear suitable protective garments and equipment. Arnainmdaomllsyweasrseigqnueadratnotisnteuddyfogrrosuepvsenanddaywsebreefoirnedibveiidunaglpllyawceeidgohnedstpurdiyo.r tAondiomsailnsg.weArlel 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 termination ofthe study all surviving animals were euthanatized by CO, inhalation followedby penetrationofthe thorax. Any extra animals notusedfor the study were. used for training purposes. 9.0 SOLUBILITY AND STABILITY: The test article, T-6292,was supplied as an amber,waxy solid. The solubilityofthe test article was evaluated in 0.5% high viscosity carboxymethylcellulose (CMC), com oil, `oainlddaicdetnootnep:rcodoumcoeilsui(t4a0b%le:6s0us%p,env:svi)o.nsTohfeTv-e6h2ic9l2e.s,A0n.5a%ccheipgthabvliesceomsuiltsyiConMwCasand com obtained in acetone:comn oil (40%:60%, v:v) at a concentrationof about 422.3 mg/ml. CHV Study No.: 17384-0455 8 01332 ; `CORNINGHazleton `The stabilityofthe test material under the dosing conditionsofthis assay is the responsibilityofthe sponsor. 100 DOSE SELECTION STUDY 10.1 Dose Selection Dose levels of0, 1000, 1400, 1800, 2200, and 2600 mg/kg were administered by oral gavage for the dose selection study. 102 Dosing Information `TThheeawneiimgahltsruasnegde oifntthheeadnoismealsesluescetidoninastshaeydwoesreerdaonsgeedfionndiFnegbrasusaaryy 2w0a,s13909.61.S35o.l1utiaonnds2w1e.r6e-2p6r.e2pgarraedmsj,ufsotrptrhioermtaoldeossianngdafnedmawleerse, rperseppeacrteidveblyy.maDkoisningg2 260 mg/ml stock for the high dose (2600 mg/kg). This was prepared by adding 11.0 ml of40%:60%, v:vofacetone:com oil (Sigma, Lot # 243SKHXG: Duke's ccoolmooreid,eLmoutl#si5oDn t1h7at1s2e:t4t6l)edtdoo3w.n12t0o0agboiflaTy-e6r2w9i2t,hraesfuilnatlinvgoilnaumneopoafq1u2e.,0 mclr.eamAwenreopparqeupea,recdrfeoarmt-hceolo1r0e0d0,em1u4l0s0i,o1n8r0e0sualntded2u2p0o0nmmgi/xkigngd.osDeilleuvteilosn.s Aofltlhdiosssitnogck stocks were placed on magnetic stir plates during the dosing procedure. `Dwoeseiknsganwdaosnaechdiaeyveodldusaitntghea t1i0m.e0omfld/oksgindgo.sinAgnvooultulmien.eofAltlheandiomsailnsgwsecrheeemieghits found in the following table. Atotal of 36animalswas usedinthis assay. DOSE GROUPS TREATMENT MF T6292 0 mg/kg 1000 mg/kg 1400 mg/kg 1800 mg/kg 2200 mg/kg 2600 mg/kg 303 33 33 33 303 33 All doses given were on an acute (one-time only) basis. CHV Study No.: 17384-0455 9 0i322 | `CORNINGHazleton 103 Results and Interpretation Asltludayn(itmharlese wdeayrse)efxoarmtionxeicd eafffteecrtdsoasnidn/goranmodrtdaaliiltyitesh.roAulglhoauntimtahlesduarpapteiaorneodfthe normal immediately and about 4.6 hours afer dosing. 2Ap6p0r0omxigm/aktgedloys2e0l.e3vhelosuraspapfetaerreddosnionrgma,la,lleaxnciempatlfsoartotnheem0a,l1e00(0#,6512440)0,fr1o8m00thaend d2o6s0e0 lmegve/lkagpdpoesaereldevheylpwohacitcihveapwpietahresdquhiynptoeadcteiyvees.anTdhechmarloemsodaatcrthyeor2r4h0e0amagn/dkg the females at the 2400 mg/kg dose level appeared hunched and exceptionally hypoactive with squinted eyes and chromodacryorrhea. Approximately 44hoursaferdosing, theanimalsat the 0 and 1000 mg/kg dose alepvpeelasraepdpheyaproeadctniovrem.alO.nTeh2e2a0n0immagl/skagttdhoese1l4e0v0elafnedma1l8e00(#m6g5/3k1g)dwoasse lfeovuenlds dead. The remaining animals at the 2200 and 2600 mg/kg dose levels appeared hunched and very hypoactive with dyspnea. Approximately 68.4 hours after dosing, the animalsatthe 0and 1000 mg/kg dose. levels appeared normal. The animalsatthe 1400 and 1800 mg/kg dose levels appeared hypoactive with rough haircoats. The animalsatthe 2200 and 2600 mg/kg dose levels appeared hunched and hypoactive with rough haircoats. `The mortality data for this assay are summarized in the following table: SummaryofMortalities Withi3n Days in Mice Dosed Acutely with T-6292 Observations OmgThrkegatment ~~ 1000 mg/kg 1400 mg/kg 1800 mg/kg 2200 mg/kg 2600 mg/kg M0a3le on on 03 3 3 Fem0a3le on on 03 73 3 CHV Study No.: 17384-0-455 10 601334 CORNING Hazleton 104 Conclusion Based on these results, the maximum tolerated dose was estimatedtobe about 2200 mgkg. 110 MICRONUCLEUS STUDY 1L1 Dose Selection Based on results from the dose selection study, dose levelsof 550, 1100, and 2200 mg/kg were selected for testing in this study. 112 Micronucleus Assay Dosing Information `CTyhcelaonpihmoaslpshuasmeiddien(thCeASmi#cr6o0n5u5c-l1e9u-s2;asSsiagymwae,rLeotdo#se4d4oH0n4M8a6r)c,hth5e,p1o9s9i6ti.ve caonndtrwoal,swaadsmisnoilsutbeirleidzebdyionrsatlergialevadgeeioanti8z0e.d0wmagt/ekrg(.LToh#te 1v9e,hipcrleepacroendtraotl,CaHV) 4oi0l,%:Lo6t0%#,SvD:vo1f7 a1c2e:4t6o)n, ew:acsoamdomiiln(isStiegrmead,cLoonctu#rr2e4nt3lSy KwiHtXhGthaentdesDtuakrteicslecaotma volume of 10.0 ml/kg. The weight rangeoftheanimals usedinthe micronucleus raessspaeycwtaivsel2y7..2T-h3e5.d2osgirnagmssoalnudti2o1n.sf7o-2r8t.h1egarsasmasywfeorrethperempaalreesdanbdy fmeamkailnegs,a 220 mg/m stock for the high dose (2200 mg/kg). This was prepared by adding a1n0.d0tmhlenofbrvienhgiicnlgetthoe5s.u5s0p7e0nsgioonf tTo-6a2f9i2n,almvioxliungmethoofr2o5u.g0hlmyl.forAapbaoluety2elmlionwu-tteasn emulsion that became a bilayerofpale tan solution over a heavy yellow solution was obtained. Dilutionsofthis stock were prepared for the remaining dose levels. During formulation and during the dosing procedure, all dosing stocks were pDloasceedGroonumpa)gwnaestiaclssoirapslsaitgense.dtAotsheecosntdudgyroaunpdofwaasnidmoaslesd (wdietshigtnhaetheidgShedcoosnedaorfy the test article. These animals were only usedinthe assayasreplacements for any `which died in the primary dose group. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. Vehicle and positive control groups, cuthanatized adpopsreodxwiimtahtetlhyet2es4thaorutricsalfetweerrdeoseiuntgh,anwaetrizeeidnacplpurdeodxiimnattheelyas2s4a,y.48Tahneda7n2imhaolusrs after dosing for extractionofthe bone marrow. An outlineofthe dosing scheme is found in the following table: CHV Study No.: 17384-0455 n 01335 . `CORNINGHazleton AtDootsailnogfS1c2h0eamneimfaolrsMwiacsruosneudclienutshiAssassasyay NumberofAnimals Assigned P2riHmary D4o8seHGro1u3H SecoGnrdoaurpysD*ose Treatment MF MF MF Male Female T 220e 0 mg er5. 5 55 . 55 . 5 5 1100 mg/kg 55 55 55 - 550 mg/kg 55 55 55 - - Vehicle Control, ace1t0o.ne0:mclo/mkogil, 40%:60%, v:v 55 -- -- -- Positive Control, Cyclophosphamide, 80.0mghkg - - - - - The animals assigned to the secondary dose groups were dosedandwere only used to replace animalswhich died in the primary dose groupatthe high dose level. All extra animals not used as replacements were euthanatized at the completionofthe trial. The ageofthe animals at the timeof dosing was eight weeks and one day. `Volumes dosed were 10.0 ml/kg andwere based upon individual animal weights. 120 BONEMARROW HARVEST, SLIDE PREPARATION AND ANALYSIS At the appropriate harvest time, the animals were cuthanatized with CO, followed by `penetrationofthe thorax and the adhering soft tissue and epiphysesofboth femora were rcoenmtoaviendi.ngT3h-e5mmalrrboowviwnaessfelruusmhe(donferotmubtehefobroenaecahnadnitmraaln)s.ferFroeldltoowcienngtrciefnutgreitfuubgeastitoon pweelrleetstphreeatidssoune,stlihdeessuapnedrnaaitradnrtiewda.sTrheemoslvieddesbwyearsepifriaxteidionnamnedthpaonrtoilo,nsaonfdthsteaipneeldleitn May-Grunwald solution followed by Giemsa (Schmid, 1975).The air-dried slides were coverslipped using Depex mounting medium. The slides were coded for analysis, and scored for micronuclei and the polychromatic: erythrocyte(PCE)to normochromatic erythrocyte (NCE) cell ratio. Standard forms were usedto record these data. One thousand PCEs per animal were scored. The frequency of `micronucleated cells was expressed as percent micronucleated cells based on the total 'PCES present in the scored optic field. The normal frequencyofmicronuclei in this Cr:CD-1%(ICR) BR strain is about 0.0-0.4%. CHV Study No.: 17384-0455 12 01336 ; CORNINGHazleton NThCeESfroebqsueernvceydofinPtCheEopvteircsfuiseNldCsEwhSilweasscdoertienrgmtihneefdirbsyt 1sc0o0r0inegrytthheroncuymtebse.r of PCE and 13.0 EVALUATION CRITERIA: 131 General The criteria for the identificationofmicronuclei were thoseof Schmid (1976). rMiicnrgosnhuacpleedimiwcerroenudcalrekilyocsctaaisnieodnaalnldy ogcecnuerrraeldl.y rMoiucnrdo,naulctlheoiuhgahdalshmaornpdbaonrdders awnadswtehreemigcernoernaulcllyebaettewdeceelnl, 1n/o2t0tahnedmi1c/r5otnhueclseizueso;ftthhues tPhCeEo.ccTahsieonuaniltcoefllscwoirtihng m`moorree)tmhiacnroonnueclmeiic.roTnuhcelsetuasinwiansg pcrooucnetdeudraespoenremimtitcerdotnhuecldeifafteerdenPtCiaEt,ionnobtytwcool(oorr of PCEs and NCES (bluish-grey and red, respectively). 132 Data Presentation and Interpretation IDnadtiavairdeuasluamnmiamraildzaetdabayreseaxlsaondprdeosseentgerd.oupTshefoarntahleysdiisfoffertehnetsteimdaetpaoiwnatss. performed using an analysisofvariance (Winer, 1971) on either untransformed h(ewtheernogveanreioaunsc)espraroepohrotmioongseonfecoeulsl)s waintdhrmainckrtornauncslfeoirpmeerda(nwimhaeln. vaIrftihanecaensaalryesis oufsevdartioadnecetewramsinseigwnhiifcichandtos(ep<g0r.o0u5p)s,,aifaDunnyne,ttw'esret-steisgtni(fDiucnannettlty,di1f9f5e5r;en1t9f6r4o)mwtahse negative control. Analyses were performed separately for each harvest time and ssteaxticsotimcbailnlyatsiiognni.fiTchanet cdroisteer-iraelfaotreddeitnecrrmeiansienignampiocsriotniuvcelreeastpeodnPsCeEiSn,voolrvtehdea detection ofa reproducible and statistically significant positive response for at dleoasset roensepdoonssee nleovearl.stAattiesstitcaarltliycsleigtnhiafticianndtuacnedd rneepirtohdeuacrisbtlaetiisntcicraelalsyesaitgnoinfeicdaontse level was considered negative. In either case, the final decision was based on scientific judgment. 140 RESULTS AND INTERPRETATION: dAlulraatniiomnoalfsthweeraessoabysefrovretdoxiimcmsedyimaptteolmysaaftnedr/doorsmionrgtaalintdiepse.riAodlilcaanlliymatlhsroiungthhoeuvtethhiecle and positive control groups (except one female#, 6846, from the vehicle group, which appeared hypoactive at about 1.4 hours after dosing) appeared normal afer dosing and CHVStudyNo.: 17384-0-455 13 60.1337 CORNING Hazleton rfeemmaaliene(d# h6e8a4l6t)hyfrunotmilththeevaephpircolperigartoeuphaarpvpesetarteidmens.ormBaylaabnodutr2e0m.a4ihnoeudrhseaaflttehryduonstiinlgt,he 24 hour harvest time. aApppperaorxeidmahtyeplosyct1.i4veh,ouwristhafstoermedoaslisnog,shalolw5i5n0g,e1x1c0e0ssainvde s2a2l0iv0amtigo/n.kg dose level animals Anporpmraolx.imAatttehley 2101.040hmogu/rksgafdtoersedolseivnelg,,tthhee25450homugr/hkagrvdeosstemlaelveesl aanpipmeaalrsedapnpoeramraeldand the females appeared hypoactive. The 48 hour harvest animals appeared hypoactive with rough haircoats.The72 hour harvest animals appeared. hypoactive with rough haircoats and some with dyspnea. `Anpoprrmoalx.imAatttehley 4131.040hmogu/rskgafdtoersedolseivnelg,,tthheea5n5i0mamlgs/akpgpdeoasreedlehvyelpoaancitmiavleswaiptpheraroeudgh h2a2i0r0comatgs/kagndddoysseplneevae.l, tShoemaeniamlasolshaapdpseqauriendtehdypeoyaecstaivnedwwietrherhouungchhehdai.rcAoattts,hedyspnea, squinted eyes and chromodacryorrhea. Approximately 47.5 hours after dosing, 1 female (#6916) from the 2200 mg/kg dose level and 48 hour harvest was found dead. nAoprpmraolx.imAattetlhye 6181.040hmogu/rskgafdtorsedolseivnegl,, otnhee5f5e0mamlge/(k#g6d9o2s0e) lweavselfaonuinmdaldseaadppaenadrethde remaining animals appeared hypoactive. At the 2200 mg/kg dose level, the animals appeared hypoactive with rough haircoats and some were hunched. Tpohleyctehsrtoamratticilce,eTr-yt6h2r9o2c,ytiensduocveedr tnhoesliegvneilfsicoabnsteirnvcerdeaisnetshienvmeihcircloenuccolnetartoelsd in either sex otrhaet72anhyooufrthhaervheasrtveasntd t5i5m0e,s.11D0u0,e atondto2x2ic0i0tmy,gt/hkegPdCoEs/eNgCrEourpastiwoesorfetshiegniffeimcaalnetlsyfrom reduced when compared to the vehicle control females. The positive control, CP, induced sciognntirfoilcsa,nwtiitnhcrmeeaasness iannmdisctraonnduacrldeaetrreodrsPoCf3E.in8b6o%th0s.ex4e9s%aasncdo4mpa.re8dt0o4.t7h%4e%vefhoircltehe males and females, respectively.Thedata summarized by dose grouparepresented in Table 1andindividual animaldataare found in Tables 2 through 7. Historical control data are presented in Table 8. CHV Study No.: 17384-0455 14 01338 CORNINGHazleton 150 CONCLUSION: ``Tmhaerrteoswt pmoatleyrciharlo,mTa-t6i2c9e2r,ytdhirdoncoytteisnudnudceerastihgenciofnidciatnitoinnsocrfetahsieisnamsiscaryoannudcliesicoinnsbiodnereed negative in the mouse micronucleus assay. 160 REFERENCES: Dunnett, C.W.: A multiple comparisons procedure for comparing several treatments with acontrol. J. Am. Statist. Assoc., 50:1096-1121, 1955. Dunnett, C.W.: New tables for multiple comparisons with a control. Biometrics, 20:482491, 1964. Heddle, J.A., Hite, M., Kirkhart, B., Larsen, K., MacGregor, 1T., Newell, G.W. and Salamone, M.F.: The inductionofmicronuclei as a measure ofgenotoxicity. Mutation Res., 123:61-118, 1983. Schmid, W.: -Themicronucleus test. Mutation Res., 31:9-15, 1975. Schmid, W.: The micronucleus test for cytogenetic analysis. Chemical Mutagens: Principles and Methods for Their Detection, Vol. 4 (A. Hollander, ed). Plenum, pp.31-53, 1976. Winer, BJ: Statistical Principles in Experimental Design, McGraw-Hill, New York, Second Edition, 1971. 17.0 DEVIATION FROM THE SIGNED PROTOCOL The following deviation was made from the signed protocol. stReuldayt)iaveth3u9m.i1d%i,ty37w.a3s%,reacnodrd3e4d.o5%n,Freebsrpueacrtyiv1e7l,y,1a8,ndanodn 1F9e,br1u9a9r6y,(2f4o,rt2h4,e2d5o,saensdelMeacrticohn 2, 1996, (for the micronucleus assay), whe the relative humidity was recorded at 32.5%, 34.4%, 20.8%, and 35.5%, respectively. CHV Study No.: 17384-0455 15 01339 `CORNINGHazleton 18.0 EXPERIMENT DATA TABLES CHV Study No.: 17384-0-455 16 013%0 CORNINGHazleton SPONSO3RM: TESTARTICLE: T-6292 ASSAY: 17334 TREATMENT DOSE ConTRoLS VEHICLE POSTIVE d0p%GCAocmcOoinld CPiOmghs TABLE1 MICRONUCLEUS DATA SUMMARY TABLE HTAIRMVEEST %M MICE 1R00O0A NPEURCANLNEIMAOATPLCEEF DSSE. (IR) MALES FEMALES TOTAL Mh 0123004 0104004 Mh 38640490 AMAOTS O1+003 | 43504s% | RAMTEIAONPCSEEN.CE. MALES FEMALES 0514008 040400 0722008 0814000 TESTARTICLE Somghs Mh ah nh Noomgks he ah Th 00mgks 2h Gh 7h 0144006 004000 0s0oT 0124006 lsoo 0104005 0184006 R400? 0G6s0 0124005 0164007 018e00 0064002 0100S 0054005 010400 0ISi006 02650 0134004 | OBs0m | 0lsa00t | 0Ba0n | 0164003 | 005400 | 0142004 | 0L408S | 0164005 | 0562005 oman 0e1s012 0474083 0x00 06007 02400 ORaON 0574005 0752008 O@zon 0d8E00et oma0 0etso0m 0esx00 0&0s0Il 0564007 030s0me +* SSiiggnpiifficcaannltlyygroewateertthhaanntthheeccoommeessppoonnddiinnggvevehhiicclleeccoonnttrrooll,, pp<<00..00S5.. CP = Cyclophosphamide CHVStudy No.: 17384-0455 17 01341 `CORNING Hazleton MICRONUCLEUSTESTTaIBNLDEIZVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T4252 ASSAY NO: 7384 a No mo Rea NUMBER fi"CES CENCE p-- ne rose conTRo rerawe VY amawoi1 1s a oo. auomas wa Aanmnu8 ow oe oo: 8 sens w@F ap E23ousu0sa oo Jaao,m.o1a ia ow PaoI oT 1a| mo N$C0oo5=CcFydoanpommsteuanidceyto NTCAE FNOoRmotomsdecyPss CHV Study No.: 17384-0455 18 co134z `CORNINGHazleton TABLE 3 MICRONUCLEUS TEST- INDIVIDUAL ANIMAL DATA SPONSOR: 3M `TEST ARTICLE: T-6292 ASSAY NO. 17384 RSATMEN| aanmwalr NENNOMN orato io NUMBER [0 PCENCE 24 HOUR HARVEST FEMALE VEHICLECONTROL 40% AccloncClom0O%il o69n00 21 [062s9 oonn 20 015009 @s 0 064 POSITIVECONTROL CP800 mpg. aamn 526 0o6s 0 osm3 0o4e1 os a 057 TEST ARTICLE 550 mee. 60089 31 005838 @%01 1i 006386 ost 0 190 1100 mee ooor 11 013070 o 3s o1 00944s ow 0 089 2200 meh oonu 20 0o1m8 oona 21 0oa2t6 oo 087 CP= Cyclophosphamide. MPCNE==MPioclryocnhurcolmeautsierythrocyte # MN PCE = Micronucleated PCEs. `NCE = Normochromatc erythrocyte: CHV Study No.: 17384-0-455 19 601343 CORNINGHazleton MICRONUCLEUSTESTT-AIBNDLI4EVIDUAL ANIMAL DATA sronsoR: TEST ARTICLE: T6252 AAsPsaYONOE; 1,7m a mo TREATMENT Tove ICE ete "arounmawvest ne Teor enrs comme wm 1m a FFoE Ja=@ow 3 si os won aau 33 ouwn F oof E8 F[ perbaP-- C am--a e prose NE emotamaie eyo CHV Study No.: 17384-0-455 20 0i344 `CORNINGHazleton MICRONUCLEUSTESTT-AIBNLDEISVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6292 ASSAY NO: 17384 TREATMENT 48HOURHARVEST TTT FevaLE AaNIeMAL ON mRmAToIO NUMBER CES CENCE TEST ARTICLE 550mune oeosno 033 o00n% e8ns 20 0io0n 1100 mas o@Gni4 202 000856133 6os 23 0o8sss 200mas ieeenzr 3 2 081 0s eenno1 007ss M*ANni=mMailcfroonuuncdldeeuasd PC#MEN=PPCoElysch=rMomiactircoenryutchrlocayPttCeeEdS NCE =Nommochromticecythroeyic CHV Study No.: 17384-0455 21 C01345 CORNINGHazleton MICRONUCLEUS TESTT-AIBNLDEISVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T:6292 AY ASSSAAYNNOO 17384 m nL amo TREATMENT {over JE rcimce ToumARvesT as y-- some ao @aA m s003 mo1a wows aa aaan 5 3011 08oaemm momre Aa aao s11 em oole oon HiCapiPC ecorneso m apsoets NCE Nomottanate cya CHV Study No.: 17384-0-455 2 01346 CORNINGHazleton MICRTOESTTNAIBNDLUIEV?ICDUALLANEIMAULDASTA sponsor: TEST ARTICLE: T6252 A AssavNo; 1m PO oi E mo TREATMENT {vee PCE cine [EP py vom om ome o42 uiom a1 --. ao o a F0woam 0 om wore wm aoa mw o253u wow PcCpEinPmtonpdedostaprons r NE Kaemotv omate c aybrocs CHV Study No.: 17384-0-455 23 01347 `CORNING Hazleton TABLES MOUSE MICRONUCLEUS HISTORICAL CONTROL DATA 7/95 THROUGH 12/95 FOOLEDVEHICLECONTROLSmy MAX AVNG 3 MICRONUCLEATEDPCESPER 1000 PCE. MEANOF 1000PERANIMAL + SE. MALES FEMALES TOTAL RATIO PCENCE MEASNE. MALES FEMALES 000 02 0087a40007 0 024 00BIA0XS wo oor 031 or 085 00M.0W0S | 05020021 " a 024 103 058740025 " PCOyScIloTpIhVoEspChOamNiTdeR,O$L0.0Smpg MAMXN AVG N S26080 3604020 In 150 636 310s025 1 240 ot 538 om la26:0184 | 0STs0m0 1 1 040 079 osssrons 1 P'NCCEE== PNoolrymcohcrhormoatmiactiecryetrhyrtohcryotceyte CHV Study No. 17384-0-455 2 601348 oFRvotoscroLrnor.o. 35. : EBTEION 17 CORNINGHazleton In v0 MOUSE MIGRONTOLEDS ASSAY CC orOnEineg HaPsE eieertoenane Insc.(sauk(asC)iHVto)RhneewgiuSflli6lan0tailcooannrrdseu.pLcootr,rtTihtnwihiisinlsglprHsoatbtesuoldsyasotulboi,njnecccttarocim.tpciolciaaaTlnhucedepihtsvatsibuetyd(hysQ)Gwuoiaollodfiltybe A by Gov oe 5500 Leasbucg Pike. Vienna, Virginia 22162. BART 1. SPONSOR INFORMATION AND APPROVALS I. sponsor momvTIFIGATION Company vemos BM Address: , udm 13. test series wevmeroton, 7 6292 - Inr. gest ARTILE ass SSEeETchereminSaptoinsoonr.of theebtiensettaricoicclhee Os1c7abirleiptuytatainodnsche1s tethset aFretsipcolneeibilicy Iv. NOTIFICATION oF RESTLATORY sTMESSION I IE n orde ereteo BE ScoinTiplLyimweiHstEYhinotsxhe coviour Please +pGamLrPeasoreefrgaulsacsthtieeoddnyusl,e1scofoinassruuelantddiieensgfwolhraibcorhreagtufolaralilteosruynder indicace nich agency. Lf any, might receive rela Tor his study: o-- orwenerrerntn0 -- et -- 3 vow moa A TD masa C--_0-- oreo =0 om -- meacrrmea ass 00o1f34109 PROTOCOL NO. 455, EDITIO. N 17 v. srupy pares Proposed Experimental Start Date: Proposed Experimental Termination Date: VI. APPROVAL OF STUDY PROTOCOL Study Director: Hemalatha Hurli, h.D. Sponsor's Authorized Representative: Gren (iy ~ CORNINGHazleton Date: saves __ 1/1/55 "rss 2 0f 10 001350 PROTOCOL No. 455, EDITION 17 PART 2 - STUDY PROTOCOL CORNINGHazleton IN YIVO HOUSE MICRONUGLEDS ASSAY 1. ommermvE TCEhSelsooeborjdEeeecnmtiaictviecaceoefirvyittthhyirsoscaysdtteuddyisstreiusmptctioeollnesvaoflinuatmtheoeusmaeittoebtsoitnceamrstapirpcarlroeawtuifsonrviinve. 11. pernmrions MHiecrrooensuccmlee(uss:) aandlsomasllofcharcoesatartiicn Coe behind at mitordc anaphase. cbohdryo,moscoomnesisftrianggnenotf(se)n,tiwrehich After telophase. these eESeaeoinnds)mayandfofrrmagsmienngtl(es)ormamyulntoitplebemiicnrcolnuudceldeiininthethedaughter Cytoplasa. III. RATIONALE reZheleomicnrionnd(usccelhsemtuids,artet1si9tc73l;ecsanHwehdsidecrlvheeeixanstaeLar.ferraep1i9dw83i)ts.hcrneMoerinmcarfloornmuicctlloeatisitcoagreecneilcl eSecitte.rea3nn3d75cych.raonmobTsenomstehcsiosreodfassdcauhyrr,ionmgopsoolimynecthefrrropamhgaamtseienctbseecrayluteshfetrocbtyehtheeiysndpe(dr2usCriEiss)ntg (Seionnd e opanveherimalaserizoonmwicfarrroonemucselcreoyirt,ehdroibffloarpsrtetsheetnotp,reersyretenhmcraeoicnyotfienmittchhreeonncuuycctlloeepilu.assai.s DJaeCetteiicttieiisoocnteeddo.fcemlailncdrpotonpiuumclelaetiiinovnoislnvneiodsn-iennluicmslieenaaarttceehddi.ngcelfTloesrstmiseatrattpihuchslaesse spreads cStoepereinnig cspaignednltesficbaenrbefudnectteicotnedortfhorromuagthimonicraosnwuecllleusas induction (schaid, 1975). wv. warms A. pr Animals YOouEng kaedulotiemaalsetahendcifneemaloef mdiocseingo.f wtihlelICbeR spturracihna,sed6- firvoamguCeh-apraiuelseyR.iveIrnc.LaboTrhsitsorsitersa,inIhnac.s, boerenHasrellaencted to 30f 20 001351 PROTOCOL 3. NO. 453, EDITIO|N 17 CORNINGHazleton meanxsiurmeizeaccgeesnsetitco haetceormomgoennesiotuyrcea.nd at the same time Control Articles Ca1n0ydcmllwo/ipklhglo)spbwehialmaliddmeibnei(suCPts,eerde8d0asmbgyt/hkeogr;aplodsoigstaiivnavggee.cvoolnutTmrheoel vofaerhtiiccllee Cuthsoeendtrsoaflmoerartrthoeiuctleteesawts,ilalratnicdcolncesoinsactunrdroewfnitltllhye bwesiotlahvd,emnitntiheosrtetvreeeshdticblye VSaordlenuiimcnelieswtiealnrldedniontotametohxuecneteesdxp2ee0qruiammleln/tktagol tfahoenrimmoaarlxasli.mugmavThvaeogleudmoaesnsidngI? S"deneianyisatrrsatwiaotnesr., 0T.h3e7 avqeuheiocluesscgarebnoexryamleltyhyulsceedlliunlotshee Solution, or com oil. Vv. DempENAL pEston A. snimal Husbandry AwSLieLlv1e'nabpeppleiricsacoballgaeeteddCHuVbryinSgOsPesxq.uawrialAnlntiimbnaeel,sfowlailnlodvlewdib.lelhAobneuismehadolussuepd to SiapefmptoehroauFtsiuevrdee,purnid7oe2rrFttoh+ee6xfFop;lelrhoiuwmmieinndgiticynl,iitmia5at5t1iiconc.o1nsdL;iAtniilominagslh:st C(yPcurre.ina12CheorutrisfieldighLta/bdoarrakt.oryAChcoowmmder#c5i0a02l)diaentd tap wiantaelryzewdillbybetheavmaainluafblaectaurderlibfiotrumc.onceThnetrafteieodnsis of hnSuyptderrcioiecfnaitresbd.onhse,Tahveyormwgeaattnaeolrpsh,oisspahfaalntaaetlsoy,xzienda,ndbcihaslnponeruciainflailteyeddon a ApTneeistmtraiolcssipdeecwsti,ilvlehebaebvasyqiusmaertafanolrtsi,nsepdeaclikffaoliriendaittmyil,ceraosaotnrdg7ahnadilasoymgsse,nbse.fore being placed on study. SAncicmoarldsingwiltlo CbeoniansgsigHnaezdlettoonstSutdayndagrrdoupOspeartatirnagndom wPTerhioegcyhetdwsui.rlels.beAndAionmisameladslswbialswleidllbeupbeounnwieqtiuhgeehleyidndiidpveriindoturiafliteodandiobsmyianlega.r : tLaagb.el/cTarreda.tment groups will be identified by cage ais of 10 001352 ROTOGOL HO. 455, EDITION: 17 `CORNINGHazleton BSSnainanietraisry oorcamgeecsroksiwenisglslwibsetuhiitusnaebdlt.ehe paPrneoirtmseaoclntnievfleacihglaainrtdmileeinsntgswialnld aie 5. Dose selection aTThosemeihiimgnhdiodctoaestreiaosngeednofedroatsloelx,yicwiitTlyhle (hbeei.gghs.eldodeseceattehds,haosudled8p0r2persoosfdiuocntehe SoCehEoktea)to.icomaoOifntey-POhEavsleftuosaneddnoaor5nmeo-ccqhhueraorimtntetirecrmofeerdyittahhtiresocahynitdgehsldoovsedose hTeolraltsk.elTtensspoudctitvhealty.4 weUaske coflaastohgiegnh wdiolsle biencrdeeatseecsted, ind is therefore recomended. Eo1 tneoeaeppFirrnoostpyrisstetadenybreawniglpelecfbfoeirnmd5ei0dn0.g0 mdagTt/hakeg.tsroepTdehovesaeidloastbeelsetl,eedvealisnranttgheeestedodse 13 he foeued as an smendment. Dose saNcEEINOING STUDY rIheudeose Fraanngeifaindtihengtisvteudygrowuiplsl wbiellconcdouncstiesdt uofsin3gmFlieveantdrea3tment Honale mice. Group Designation and Treatment Reginens Group No. tNautmeber ofFeMmilcee Rowe Du(raavtsion : 5 er e5 ere*rmee----5-- ee e2 r5 --s --0--5-- 5 : ----5--er2e0 tres re e--e. ------5 ----5 ----0------ s -- -- se-- ee:e ere e a e v0 e5 e ass PART 5 of 10 001353 FROTOCOL C. 0. 435, EDITI;"ON 17 `CORNINGHazleton TThheat rocuatset Injection aofrtiacdlaeincihsatrraacttieorniswtiilclsbepreocrlauldegavoargael. gavIanget.heIPevent will be cuployed. These routes of Administration have Mbeienninisseclceacttieodnbfecoarustehisthetyestareprothceedumroes,t cTohmemondosroiuntgesvoloufme will nIToootuttreeesmxucseocefudtaad2r0m,imnlis/suktbgr-actfuiotroanncoortuahslatgamadavmayigneibsetarnuadsteidIoPnssardemoifinnitbsrytarvfaeetneiodo.unss,.TheOtehsetr mSTaootlleuvrteiniotenel,:wiwolafltecro`.gremn0e.or5ial7l.lysaAlbLienLes.aonliu0s.ba5i%llsizaweqiduleloiunsbeoncedaorsobfeodsytmhbeeacshefdyollculepolowlniuntgose pIrnoctiovciodluilamebnoddayenwte.ights. Dose levels will be sasigned by a "pB5or1de7ypawrbeeeidgphartnesdpahwrieelldld uabsteeatmpabkeieienonrtprtoitoerdmopsetiornagtd.ousriensDgo.suinntgiDlossdioonlsguitnifgoonrsm(u0w-li2altlionbe PSoiunrgalre..dosAeT.L animals will be cuthaniced 3 days after receiving a TThoer tahneimadlusrawciiloln obfe inhalation followed otbhseersvteeddy.daiAlnyimfaolrs tvoixlilc bseignssucahnadnimzoerdtablyitCyO by penetration of che thorax. V5TEh1eI1L datbiehleynusebodebssevsroaviaegtsniteoindmsatFeoorf thttehoexiMcasxuibsmsyuemmqputeoTnmotslearcanycdte/odogrenDeomtsoierctsa(lHisTtDs)is.easy. Ddoasteas MEcRoCLEDS STUDY Dosing schedule and Rouce of Administration HwNadornrvimenasiltsliywciaaltnlioancb)euteawiplpdlrooswbieingmautsreeeldgyim(2es6ne,s (4Tsai.bnlgel7e2bheoluorws. after Swdpnpirnoixsicmeaatteiloyn26ofhotuhersteasftterartaidcmlien,istfardatiaton of the CCroeRdt.rolEaqrucalitlneusm.bersA ooftamlaleosf a1n1d0 afneimmaalless wwiillll bbee used B"uetnidseoaasiceshdctaor6nesaiacsntesinentcgongrdofoaurpy3.-d1o0sAenmalagedrsdoiutapinowdniat3lh-10gtrheofuepmhailogefhsdomsaey GSFoxitcheitytesist meaxtpeercitaeld. atThtihse hgirgohupdowsiellanbde hdoesedaniimfals aSinenyttohwnihdsaircyhgrodiuoepse wpirglirloorupontwoliylelubethbaeunsaedsdeitaae.srmirnTehepedlacbueysmeetnohtfes tshfteourdy diector. Freshly prepared solutions will be apes . sof 10 001354 PROTOCOL NO. 455, EDITIO. N 17 CORNING Hazleton etmopxlicoyesdi.gnsThaendanmiomratlaslitwyi.ll be observed daily for NUMBER OF ANIMALS USED FOR MICRONUCLEUS ASSAY Group No. Treatment Harve(sMtaleTsimeasndAfFteemraleTsr)eatment 26 Hours 48 Bours 12 Hours 12 VPeohsiictlieveCoCnotnrtorlol~~ 55 ++55 ----- ---- 3 " MLeodwiDuomsDeose 545 545 ses 545 54s 545 s High Dose 545 sts 5+5 TOTAL 25425 15415 15+15 Total 5+ 5+ 0s 5 1155441155 1541s 55435 The the route event of administration that test article vcihlalracbeteroirsalticgsavapgree.cludIen odroaslinggavvaogleu,meIwPililnjenocttieonxcweieldl 20bemle/mkpgl.oyedT.heseTheroutes otfheamdomsitnisctormamtoinonrohuatvees bofeenadmsienliesctterdatibeocnaufsoer they this are test Upsreodcedaurree.intOrtahveenrourso,uteisntorfamuasdcmuilnairs,trastuibo-ncuttahneteomuasy be administrations or by feed. D. Extraction of Bone Marrow Etuhtohraaxn,asiaandwihlilndbeliwmibthboCneOs, wiflolllobweedrebmyovpeedneftorratmiaornroowf the eCxrtarnascfteirorne.d toThecemnatrrriofwugewiltlubebse cfolnutsahiendinfgrom3-5themlbobnoevinaend serum (one tube for each animal). 5. Preparation of Slides Fsoulpleorwniantgantcewnitlrlifubegatrieomnovetdo pbeylalestpirtahetiotnissauned, potrhetions of the pellet will be spread on slides and air-dried. MTahey-GsrluindewsaldwilSlolutthieonnbeandfixGeidemsian,meatnhdanporlo,tecstteadinebdy in msoluindteisngarewitchodceodvefrosrliapnsa.lysiFso.r control of bias, all : ares "as 7 of 10 001355 PROTOCOL NO. 455, EDITI.ON 17 `CORNINGHazleton F. Scoring the Slides Aannimaatlt.emptThweilflreqbeuemnacdyeoftomisccroorneucolneea-ttehdouscaenldls PwCiEsllpebre expressed number of aPsCEspearncaelnytzemdi.cronTuhcelenaotremdalceblalcskgrboasuendd on the frequency of micronuclei in the ICR mouse strain is around 0.0-0.4%. T(hNeCEsf)reqwiulelncybeofdetPeCErsminveedrsubsy mature erythrocytes scoring the number of PsCcEosrinagndthNeCEsfiorbstser1v0e0d0 in the optic erythrocytes fields on the while slide. VI. DATA TShcehmicdrit(e1r9i7a6).forMitchreoniudcelnetiifairceatidaornkloyf micronuclei stained and are those generally of round, occur. aMlitchroounguhclaelimonhdaveandshrairnpg-bsohradepresd micronuclei occasionally and are generally between 1/20 and 1/5 the size of micronucleated cell, not The the mPiCEc.ronuTchleeuusn;it thoufs scthoerinogcciassiotnhael cell with more micronucleated than CE, one mot micronucleus is counted as two (or more) micronuclei. one Tphoelycshtraoimnaitnigc pranodcednuorremocpherrommiattsictheerydtihfrfoecryetnetsiat(ibolnuibsyh-gcroelyoranofd red, respectively). Data Presensation Tnhuembedrataof rmeipcorrotneudcwlielaltedinPcClEusd,e the the npuemrbceerntaogfe PCoEfsmisccorroendu,cletaheted PeCrEyst,hraoncdytetshe forratieoacohf epxopleyrcihmreonmtaatlicanitmoaln.ormochromatic Evaluation Criteria Tshiegnicfriictaenrtia dofsore-raelpaotseidtiviencrreeasspeonsien is a statistically micronucleated PCEs, or the detection of response for aat releparsotducoinbeledosaendlesvtealt.istAicatlelsyt significant positive article that induces nsetiatthiesrticaalsltyatissitginciaflilcyantsiagnndifirceapnrtodduocsiebleresipnocnrseeasenorata one dose level is considered negative. In either case, the final decision is based upon scientific judgement. "Iss 8 of 10 (01356 PROTOCOL NO. 455, EDITIO:N 17 CORNINGHazleton VII. IIENSTERPRETATION vTahreiaanncaelys(iWsineorf, homogeneous) of th1i9s71)datoan weiiltlherbeupnetrrfaonrsmfeodrmeusdin(gvhaenn rank transformed (when variances are avnaarliyssaicsesofare DiTeEtreetsrh:eogea(nnDsauolmuymses)tits,proof1p9o3v5ra;triioa1nn9sc64e)ofiwsiclesllilgsnbeiwfiuitscheadnmtitco(rpod<n0eu.t0ce5lr)em,iinepaerwDuhaminnciehmcatdl'o.sse Gornoturpesl.. AEAnaanyl,yseasrewislilgnibeficpaenrtfloyrmdeidffseerpeanrtatfelryomfothre enaecghathiavrevest tine and sex combination. VIII. REFERENCES Diuenantemtetn,tsC.w.i:th Aa mcuolnttriopll.e coJ.mpaAmr.isoSntsatipsrto.ceAdsusroec.f.or50c:o1m0p9a6r-i1n1g21s,eve1r9a5l5. DBuinonmeetttr,icsC,.W.2:0:48N2e-w45t1a,ble1s964f.or multiple comparisons with a control. NHeewdmedellales,,ureGJ.UAo..f, agneHdintoetS,oaxliMac.mi,otnyeK.,irkMh.MaFur.t:ta,tioTB.hn,eReLisan.rd,suecn1t,2i3o:Kn.6,1o-f1M1am8ci,Gcrreo1gn9ou8r3c,.ieiJ.aTs. Schmid, W.: The micronucleus test. Mutation Res., 3LiS-15, 1975. SCchheamiidc,alW.M:ucagTehnes:micrPorniunccliepulsesteasntd MfeorthocdystogfeonretTihceiranaDleytseicst.ion,In, Vol. 4 (A. Hollaender, ed.). Plenum, pp. 31-53, 1976. WMicnGerra.w-HBi.lJl.,: NeSwtaYtoirskt,icaSlecoPnrdincEidpilteison,in1E37x1p.erimental Design, IX. REPORT FORMAT CfEiVnalemprleopyosrtawisltlandparrodvidreepotrhte ffoorlmlaotwinfgorinefaocrhmaatsisosny.design. The o + o. alss Qsupaolnistoyr Aisdseunrtainfciecatsitoant.ement. Sstiagtneamteunrte ooff Test article GsLtPudyComdpilrieacntcoer.. identification and CHV Study Number. A pfheytseiicpatlwidlelscrbieptiinocnluodfedthien ttehsits asretcitciolne. and date of DTyaptees ofof assstauydy anidniptriosttoicoonlannudmbceorm.pletion. MSettuhdoydsd.irector and senior technician. 001357 5 of 10 . PROTOCOL NO. 455, EDITION 17 `CORNINGHazleton . Evaluation criteria. . Interpretation of results. . Conclusions. e @ References. Test results presented in tabular form. X. CBANGES OR REVISIONS Asniygncehdanbgyestheor StruedvyisiDoinrsectoofr,thidsateadp,praonvdedmapirnottaoicnoeldwiwlilthbethdioscupmreonttoecdo,l. XI. ANIMAL CARE AND USE STATEMENT In the opinion of the Study appropriate, the study does nDoitrecdtuoprl,icantoeaalntyernparteivvieoustewsotirnkgwmietthhotdhsisare material, and protocol will the number and be reviewed by species selected are appropriate. the CHV-IACUC for compliance with This regulatory guidelines concerning the care and compliance, a modification will be required. use Any of animals. If not in changes or revisions of this approved protocol will be sent to the CHV-IACUC for their review. XII. RECORDS TO BE MAINTAINED AgLeLnerraatweddataas, adorceusmuelnttaotfiotnh,is resctourddys,wilplrotboecoalrsc,hivaendd the final report in the storage facilities submission of of Coning Hazleton Inc. for at least the final report to the sponsor. one year following After the one year period, the retained in sponsor may the storage elect to have facilities of the aforementioned materials Corning Hazleton Inc. for an additional period of time or sent to a storage facility designated by the sponsor. 001358 4195 10 of 10 C5o30m0ingishdae:iPnete ToSa Faorasan fox April 25, 1996 ` S3tMevMeendCi.caGlorDdeopna,rtPmhe.Dn.t, DABT `Building 220-2E-02, 3M Center St. Pa MNu 5514l 4-1, 000 CORNING Hazleton SL e230" In Vivo Micronucleus Assay RE: DRAFT REPORT AND PROTOCOLAMENDMENTS `Protocol No.: 455CO, Ed. No.: 4, Modified for Sponsor Genetics Assay No.: 17384, 17384-1 Test Material: T-6292 2 hi / Dear Dr. Gordon: Enclosed please find two (2) copiesofthe above referenced report. The report includes an ubnessiiggnneeddQuupaolintiysAssuasnucreaonfctehSetaFtineamlenRtepaonrdt.Compliance and Certification Statement, which will Also enclosed is a copyof protocol amendments "uTnhdeerfisniaglnreedpowritthwialnl ybequiessstuieodnasf, tcromymoeunrtrse,voirewneacnedssnaotriyfirceavtiisoinotnos uast .yoPulreaeasrelcioensttaccotnvtehneience. "The report will be finalized afte 1 year ifno notification is received. Thank you for giving us this opportunity towork with you. Sincerely, CORNING Hazleton NessMet HMaemmmalMiualni,CyPthogDe.netics Department of Genetic and Cellular Toxicology HM/psj enclosures 001359 AMENDMENT TO THE STUDY PROTOCOL STUDY TITLE: IN VIVO MOUSE MICRONUCLEUS ASSAY PROTOCOL NO.: 455, Edition 17 STUDY NO.: 17384-0-455 Page 1 of1 `Amendment #1 Section 2, Part V.B. The Sponsor has LD data in rats of 1463 mg/kg solubilized in acetone/comn oil mixture. Based on' `this information, the dose selection study will be conducted with acetone/comn oil preparations testing dose groupsof 0, 1000, 1400, 1800, 2200, and 2600 mg/kg. -- STUDY DIRECTOR Noseatothnbunks Hemalatha Murli, Ph.D. `Mammalian Cytogenetics DepartmentofGenetic and Cellular Toxicology _2lajie Date 001360 ee ee STUDY TITLE: AMENDMENT TO THE STUDY PROTOCOL IN VIVO MOUSE MICRONUCLEUS ASSAY Page lof1 PROTOCOL NO.: 455, Edition 17 vee STUDY NO.: 17384-0-455 Amendment #2 Section 2, Part V.C. Based on the resultsofthe resultsofthe selection study, the mouse `micronucleus assay will be conducted testing dose groups of 550, 1100, and 2200 mg/kg. A secondary dose group will be used. --e ---- ee STUDY DIRECTOR Nowatsttn Mots 228496 Riba A ult `Hemalatha Murli, Ph.D. Date Mammalian Cytogenetics Departmentof Genetic and Cellular Toxicology 001361