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AR226-3072 Final Report DuPont HL.O-1 TRADE SECRET Study Title ESCHEINRITHCH-H2EI2A0S5AC1LO:MLMOI UPNLTEALALTGAEEN1IN7ICPCIHOTIRYMPT(OIERRSAITITMINIOGANNADSSAY Laboratory Project ID Haskell Laboratory Repor* No. 1997-00035 Authors ValenJtoinhenOD. WRaegecnee,r,BIISI, M S Study Completed on 04-02-97 Performing Laboratory Haskell LEaboIradtuoENrlPyekowtfonoantrrkdRT,eooDNaxdeiecl,maoPwloo.augOrryesaBan1nod9dx7IC1n54od0musptrainayl Medicine Microbiological Associates. Inc. Study No. G96CF43.5020! I Medical Research Project No. Page I of 54 Company Sanitized. Does not contain TSCA CBI SHa-l2m2o0n51el:laMtyuplahgimenuicriitu>m- Taensdtirnlgschinertihcehia coli Plate Incorporation Assay__________ DuPont HLO-1997-00035 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with EPA FIFRA (40 CFR 160) EPA TSCA (40 CFR 792) Good Laboratory Practice Standards. OECD Principles of Good Laboratory Practice (C(81)30(Final), Annex 2), and MAFF Japan Good Laboratory Practice Standards (59 NohSan No. 3850) with the following exceptions: The iodrecnotintytr,osltraerntigctlhe,wpuerreitynoatnddecteormmpionseidtiobny tohreottehsetirncghfaarcaiclitteyr.istics to define the test mAnixatluyrseesswtoerdeetneormt pineerfothremuendifboyrmthietyt,ecstoinncgefnatcrialittiyo.n, or stability ofthe test or control The sbtyabtihleityteostfinthgeftaecsitlitoyr. control article under the test conditions was not determined Treatment solutions or suspensions were not analyzed for identity, composition, uniformity, or tshtaebtirleitaytmofetnhtesotelusttiaonnds caorentirnotlenadrteicdletos. enTshuerep:rocedures used by trained personnel to prepare A. The accuracy of concentration because the test and control articles were weighed ptoeinspteatansnaodnrcaflolyantstikcroas;ll bsuablasntacnecaecscwurearteedtoiss3odlveecdimwaalspalaccceusraatnedlythmeevaeshuircelde winitwhhgircahdtuhaeted B. Uniformity because all treatment solutions or suspensions were mixed prior to administration to the test system; and C. aSdtambiinliitsytrbaeticoanustoe tthreeattmesetnstysstoelmut.ions or suspensions were prepared just prior to These deviations did not affect the validity or the integrity of the study. Submitter. E. I. du Pont de Nemours and Company Sponsor. DuPont Specialty Chemicals Study Director: VjjjtvivA L>. MViaclreonbtiinoeloOgi.cWal aAgsnseorc,iIaItIe,sM, InSc.. Study Monitor: MA Study o. G96CF43.5020 II Tatohxisioconl,oPghis.Dt . -2- 2. Afn't t<h7 Date Company Sanitized. Does not contain TSCA CBI SHa-l2m2o0n5e1l:laMivupthiiigmeunrieiuihmTaensdtiKngschinenthcehu, a>ti Plate Incorporation Assay GENERAL INFORMATION DuPont HLO-1997-00025 Stability. Test substance appeared to be stable under the condition of the study; no evidence of instability was observed. Composition: Physical State Liquid MA Study No G%C'F it.502011 Company Sanitized. Does not contain TSCA CBI SHa-l2m2o05n1ellaMtyuptahgnennuincw)nTaensdtinEgschincnthcehia o li Plate Incorporation Assay DuPont HLO-IW7-00035 Major Impurities: . Unknown, none considered to be of toxicological significance at this time. I Sponsor: ED.u1P.odnut PSopnetcidaeltyNCemheomuricsaalsnd Company ' Wilmington, Delaware 19898 Study Initiated/Completed. j November 27, 1996 / April 2, 1997 .4 In-Life Initiated/Completed. 'December 3, 1996/January6, 1997 1 a. lAAolcrlacrthaeiwdveidnsaRtao,caknvdilrlee,poMrtasrywlailnldb.e$Amasianmtapinleedofinthtehete-asrtcshuivbestsaoncfeMwicilrlobbeioalrocghici,vaeldAastsoEc.iaIi.tedsu, IPnocn.,t* de Nemours and Company. Haskell Laboratory' for Toxicolo^ and Industrial Medicine, Newark, Delaware. Vr MA Study No GV6CF4.V5020 II -4 - Company Sanitized. Does not contain TSCA CBI Study Title: Study Number: Study Director: QUALITY ASSURANCE STATEMENT HTY-P2H20IM51U:RIUMMUTAANGDENIECSICTHYERTIECSHTIIANGCOILNI THE SAIMOHELLA PLATE INCORPORATION ASSAY G96CF43.502011 Valentine 0. Wagner, III, M.S. rhi, study as been divided o f s t u d i e s .i random sampling aPp^a^ ' 9 " procedures, documentation, equipment phases over a series Proceaures^ thft stutJy is records, etc., are examined Good Laboratory Practice p5e8r?foWrmed in k ' S "the% U4S EP^A GLhPbse -<p40a CnF.R 792 anSdt ^4d0a Cr jFR Operating Procedures. rhe following are the inspection dates, phases inspected, and report iates of QA inspections of this study. INSPECT ON 02 DEC 96, TO STUDY DIR 02 DEC 96. TO MGMT 02 DEC 96 PHASE: Protocol Review INSPECT ON 03 DEC 96. TO STUDY DIR 06 DEC 96. TO MGMT 06 DEC 96 PHASE: Strain characterization INSPECT ON 12 DEC 96. TO STUDY DIR 12 DEC 96, TO MGMT 13 DEC 96 PHASE: Preparation of S9 mixture INSPECT ON 13 FEB 97, TO STUDY DIR 13 FEB 97. TO MGMT 20 FEB 97 I PHASE: Draft Report INSPECT ON 04 APR 97. TO STUDY DIR 04 APR 97. TO MGMT 04 APR 97 PHASE: Draft to Final Report data of the study. fU ouuu- ^ ktu C iu d M Claire L. Courtemanche, B.S. w QUALITY ASSURANCE MA Study No. G96CF43.502011 -5- Q fyu %149? DATE CompanySanitized^Dogjyjgt^QBigi^Uifi^^fi I Sponsor-supplied Information Summary ..................................................... P u rp o s e ........................................................ Characterization of Test and Control Articles MTMSPMCPEaorelvtraeeuesilatultttrtiliaeainbumaSbrgaigiloyleiastininlstaoiaytaficnecnonrdmTyidArtoySaTceMf.ctAoVsiRo.vxetrasae.i.itl.cntshis.i.id.gao.ut.o.y.ny..TldP..t...AS.ss.r..e..o...y.......ss..cs........tst..e...e......a.d..m.........y...u................r........e...............s................................................................................................................................................... RSPMesorueullulttiabsmgialeiinnntyadersTyDisTeiAsoscxstusi..csa.is.yt.iy.o.s..n.A..........s......s.....a........y.......................................................................................... Conclusion .................................................. References .................................................. Data Tables ................................................... Appendix I: Historical Control D ata........... Appendix II: Study Protocol........................ Page .. 2 .7 .8 .. 8 . . .... . . . ..111111999200020 . . . . ...11113333 . 13 14 13 42 44 SHa-l2m2o05n1e:llaMtuytpahgiemnuicriituymTaenstdinEgsicnhethriechia coli Plate Incorporation Assay DuPont HLO-1997*00035 SUMMARY aattWyhsspessPhaaT2pyyimhr.iweieuvlTiarrmtihsAeuesipmntes(apearrtfcKryeotosirMnctmteoldeIrxeOp,dishclHt)iairtna-syi2eintn2,aws0stthhos5Tae1pyA,mph,9rawuew8stase,aaessgsn,Tetceuunesssit1cieenai0ddtngy0dt,itaonhasTesbetAahsspytee1lana5(bbtic3elnaei5sicithonitaaecflnrtohiaAdarenplrdToodrrcAeoianlvsot9dieero7er-nsarpinaeenmdanmngudedecetuhentfdEoatotd.rirreo.acpntthoTeelalahiistvemsateaesfruysisrttaSaseuy9gtrss.epi)nsnh,tgiTrawcasihSiaetnye.s, used to evaluate the mutagenic potential of the test article. coofmaWpppaartotiebxriilmiwtyaatswel'iyftfhW5t0h0Hemtaagrsg/metthl,ceethslloesl.vmeTanxthiemoftuecmshtocaiorctneicclbeenastwreadatsioosnnoltuseobstllueedbi.inlitwy aotefrthaet atecsotnacretinctlreataionnd padsorsesaceIyinpw, ittathahseteeapcmnrheoailexrimvimaeipdnupamurresyicdniotagosbxealiecpcilottayontxecaidescnsiitatnyrya,ttwhitoehansemoomufbtaas1xge0ire0mvnemuicdmgi.t/ymdoBalssaasesnaetyddesawtoe5ands0tw5hp0ea\0spf05ilna0ptd0igin0ngpg/exasrglioqppfuleaotrthetp.e.laNttoeex,iitcthhietiysr apprIenctiahbelemtuotxagiceintyiciwtyasasosbasye,rvneodp. osTithiveeorveesrpaolnl seevawluasatioobnsearnvdedd.osNeeriathnegresprteecsitpeditaaterenaosr follows: -=neglive, + positive (maximum fold increase) in thUenBdearcttehreiacloRndeivtieornses oMf tuhtaistisotnudAy,sstaeystwaritthiclaenHIn-2d2e0p5e1nwdeanstcRonecplueadtedAstosabye. negative MA Study No. C96CF43.50201I -7 - SHa-l2m2o05n1e:llaMtuyptahglemnuicriituymTaenstdinEgsicnhethriechia coll Plate Incorporation Assay DuPont HLO-1997-00035 PURPOSE o(ofrseiTtvsheemrapel utsatrbrpaooilnsietseoos)ff StbhayilsmmsoetunadesyullrawintaygspihttoismeauvbraiilluiutmyatteaontdihneodnumecuesttarregaveinenrioscefpEmo.tuceontalttiiiaionlnotshfaetthpsereetlseeescnttecadertialconlade absence of S9 activation. CHARACTERIZATION OF TEST AND CONTROL ARTICLES wbs1ty1aos/rtT2hen2dheo/e9taSp6tptreoroosnvtosimodawrertdataiesc.smlaUaesp,spyeieHogrlannl-ot2eurw2der0cetl5eh,iqi1epp,urtcio,wodttdheateceshtnaetretudesmsctfheraboiorveutmeirlcdd9lee6bxbCwepyFoas4ssMtuo3dr.rieeecTsrdctohorbaiebitloitege<ldohs3gtta.0iascraCatilyc. leAeAllswosnwoaecslviicaqihtrueaasirdt,aicoaItnnnecdr.diwzaoeatdens The vehicle used to deliver H-22051 to the test system was sterile distilled water, (CAS# 7732-18-5), obtained from Life Technologies, Inc. Positive controls plated concurrently with the mutagenicity assayjirejistedbelow Strain S9 Activation Positive Control TA98, TA100, TA1535 TA97a WP2 uvrA (pK M IO l) TA98 TA100, TA1535 TA97a 2-aminoanthracene (Sigma Chemical Co.) 2-nitrofluorcnc (Aldrich Chemical Co^ Inc.) sodium azide (Signa Chemical Co.) 9-aminoacridine (Sigma Chemical Co.) To determine the sterility of the test article, the highest test article dose level used in the mutagenicity assay was plated on selective agar with an aliquot volume equal to that used in the assay. . ! I MA Study No. G96CF43.502011 SHa-l2m20oSn1e:llaMtuyptahgiemnuicriituymTaenstdinEgscinhetrhiechia coll Plate Incorporation Assay DuPont HLO-1997-00035 MATERIALS AND METHODS Test System TfD(ar1nAor9d.m71HB60thti)0rhe.ue. ectTSEeeaNAs.lAtm1ace5tmoroi3olnsel5ntesrta,laeallaiUsnntCdtesneoriTusvltsslAeeeetrrrc9dsati7siitwtoynarnaeaoWirsnoef sfPCdthw2IeanesleudciSfrvrueoairsbrAlrtnmeeridic(aoaep,lbniKBvyealenMelAaddrkmItoOyMenpellesha)1yirem1.aitns/uEa2erd6i.i.Beu/c9(smaa1c0c9lJrltiaj7ebkwn5retid)aiadds,a1ibnArn1eyde/bc1aGeLe0uirevr/dxe9veeeoi2denntnidroeo,iantrpSena0hccdtl7oslyMt/(Tl01'aA19un/8rd98i2.8e7),l Erti(soeG.vhcrreTeeoirsveelttieenisdidrtsetaienrbsndeesydntirbmsnMaiydituniuevtmsrpaeiTgeeutnelAto,and91gsbe89enat7nhasc6senea;-dtp(tBhpTcarraaiAuortust9ssoiceuc7takrbauboseasoptetrithtehauybrlt)f.eia,robvsa1neyem9prmf8teare0asiudr)hmt.afisfretutoisobmahnsnistfhdit,tiumrsbattiaiutodhstnieaensgpre.eatndihrseTa.psneueTsfbntreesdastremittneusecrttsreishaot(iirnanfatiimwnmTouATuttrAta1aot01tipio05ohn3nysi5ss). yociips1nnnuirr2ecoellufthgcOadurbotroirrvanaueomrmegterpimrsoshnmttanehoiibgnedetwedohgituifamtatomstecpsoreruhcpe.tolrarbtioTonkuctefphteroagrerreloialsai/nyailpsnlwtasepescufndrehuoortrfaiebarexrcknoaiitpitmpdzhtnuoreaagreamrnbtttpeeiacoaddpalutnyriteetliraytttdumiorp1morr0pbaeaeo9eyrnsdnmod.ewcixnoneiFeowmflttlreocseasamulhtsltohloapeapcrawlehtyvekirranirvenragms1eivgtnt2su.efitti5rlrsoenloetdiorelm.paiEdcitmneuavtrlhcale.aahtesattisteoaeaTcnpl3luo,hpp7clgeerteouoarpn2crcpahtehearcaCinitafnsuwtletiaaena,astlpgsrtkmhaptwne~riamtossael5mxtoesre0insnpimrtgimtpltwaatoahllntraeeecootrlddeyeeff determined by viable count aw 'S on nutrient agar plates. Metabolic Activation System Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The Sipb7n9u,r1jele2wkpc-aadtpirsioremenpdperoaoethrpfnayaAt0lriboe8reodn/n0czfol7or(f/oar9Sm)6a19n2,m0wt5h94aar/,sla2e5ca50esS/sn09paer6ymategaogdun/fekdfoo-grDr1m, 1aiftswi/sv2mlea5ebu/yd9itlaa6riyatgysatesnntpoidinncmsdottuoorecrtSteaeoadbdlosmwaalitociztnrehief-2lia7lc-aea0i^tymCpHnhuoiimneatnuiSltnh9uursawmecadels.nAebElaUaatnccUhh. gamitnlsuidxucT)o3s,he3sc.eeom-nST6tM9-oapimhncKooiinxsnOgpfwihri1aamn0st0eapt,h4m1ree0Mmp0satMmeprerh/Mi3dloi-tsnipympihchomooafstteitepnhdhabeiaamuSttfeei9fdlebyearu-nbaafdefdtefepSornHhraieantm7epit.d4Hsmm,uw7isux.ea4ces.lsae,pnToardtehi0dpec.e5aoSprnhemhtdaaolmisniap^leSihdqd9auti1meoa0,ti8%txoetmfuleyrMeacb(M5ShehfgmwoaCMrmales2 plated on selective agar. MA Study N o. G96CF43.502011 Company Sanitized Does not contain TSCA CBI SHa-l2m2o05n1e:llaMtuyptahgiemnuicriituymTaenstdinEgsicnhetrhiechia colt Plate Incorporation Assay DuPont HLO-1997-00035 Solubility Test oneAorsomluobreiliotyf ttheestfowllaoswcionngdsuocltveednttsoinsetlheectotrhdeervoehfipcrleef.erTehneceteasstlwisatesdc:opnudruifcieteddwuastienrg, vdeimhiectlhey, slseulelfcotesiddein, eotrhdaenroolfapnredfearceentcoen,et.haTthpeertmesittteadrtpicrleepwaraastitoenstocfdthtoe hdiegtheermstisnoelutbhlee or worhable stock concentration, up to 500 mg/mL Preliminary Toxicity Assay tp(epesKrtTMadhroteIsiOcepl:ler)enwoldinmothusinloedlaverbecyemtitvaoiesgxshimactyitieycnduima.lstusaTarleeyansgwdaoarofssiTeunsAlbeeModvteht,losTtoheAseft1taph0bre0lei,stseTehsnAttch1aee5rta3idnc5oldeseTaw-bAreas9rene7ngac(edeaoaotv^efeWdrr,awotKnhliieuvchpevrlrtahSAtee9 activation. Mutagenicity Assay TtalihlvAoeen1Trmg0hS0wue9,tiamTtaghcAuetaint1pvai5gcap3terpi5onoo,ipnTctre.iAitnayAt9tieaa7lslvlasdeoaohayfnsitc(edhilneelWeiattvienPaesdll2tspaauonorvtfdsriticAteiilnvsed(te.peacKAproteMinmnctldrIieoOne,linslmv)tweruihneemiprctelehoepaefltcfapoaitvrsneeesdtasrdoeywonlssis)ctehaewnltaaeedsnvseduptelossraesbsodittsifetvetaoenemscectsevoaTaonnlAtfurca9orlat8leest, were plated in triplicate. Plating and Scoring Procedures metThhodeoltoegsyt osryigstienmallywdaessierixbpeodsebdy Atometsheetatle.s(t19a7r5ti)calendvuiapdtahteed pbylaMte arinoncoarnpdorAatmioens (1983). fwipwbmsBWurofaroieDto/nsidVtpOtoiioshuma)mnnlurcewaapmetwTlthpadaiemoelgasensanabdmVetryeadomdfoeywiifugtnnehowmametaleseftlliee-dtiBhVEdtcdMswoiooa2u(ninaig.Vlsat5cnlneheineode-l,%-ndQgr2Bmte5srsrolaieua(RnmnWatplaiitengomnpl/aeesVndlogoaern)oflemlBtmusntOfwo,toet5iiaxnponna0loWitntnlameeimedgrrdsarMeaauftlfNrpeowe1,merprru9ciaeltt5eoeShecnr6adnimhycec)it.sLeunahetcsemi-Ttnnhmot1tBooiei0nnsE.pdr0wtgoitadcwtm0aagBhiot.niia8leonteNrnhrott,%taniioof2mionD.nm.ma5tpi2g-gnulb%yiaga(s(imrgdeoWs1d(trat(WemyWm.r5/wn/Vp/wiV%aiVeaothal)l.n)wtsadd(oSOadWne9VgpexdiMoa/oroaoV)r0mirg-.^o)d5eSz.j^lpFNeNNh-%dBtaououumowttpNrtnrnnthiamaehnewtaCnneeienxrtt.l Broth No. 2 (dry powder). Eoh niate was 2lab2el?ed w2i?th aamcoaddeosyus,te.m thdeastcirdiebnetdifiteod tdheetateilsttoartMicliec,rotebsiotlpohgaicsea,l Associates, lnc.'s Standard Operating Procedures. of SZT9eoosLrt arTticmlemdXiilxu,nti1o0sn0eslfewiclteoirvfeetpetrsoetpepraasrgetradariinmatamn4ed5d5ia02tedlCyobfeAvfoefhrteeicrulevseoo.rrtOetxentsaetg-ah,rattilhcfel(e0n.w5u)xemrteuriaelbddwjteeadrs onvnetrrlnaiid othnetotethset asrutricfaleceaolifau2o5tmwlaosfrmepilnaicmeadlbbyotato5m0 oagra1r0. 0W/tlhaenhqpulaottinogf athpeprpoopsriitaivtee positivecontfol. After the Plates ^ wwe'noT cot^^toraedtotoly 2-" -H i tony coaming could be conducted. . aexrtaiTmclheinetarctaioroincnditwivtiiobthnvoouufstitnmhgeagabnadicfiitscesareticiaotlinnb.gacTmkogicxrrioocuistncydoaplnead.wndPewrgearaespeeivtoaaftleuparwetecadsipfeiitvvaatilounaUtweledenrcbeeys<ovcfiotserusmt relative to the vehicle control plate using the codes shown below. Normal puringi.ictnjd by a healthy microcolony lawn P frtinp.uh.rf hy a noticeable thinning o f the microcolony lawn and Slightly Reduced possM y a slight increase in the size of the microcolomes compared to the vehicle control plate. Distinguished by a marked thinning of the microcolony lawn resultingin Moderately a pronounced increase in the size of the microcolomes compared to the Reduced vehicle control plate.__________ ' ------------ n hihynMmd by an extreme thinning of the microcolony lawn resulting Severely Reduced in an b a ea se in the size of the microcolomes compared^to the vehicle control plate such that the microcolony lawn is visible to the unaided eye as isolated colonies. ^ ^ _ r ^ r i - g n i c t hy a complete lack of any microcolony lawn over >90% of Absent the plate.___________________ ______________ ______________------------- Obscured by Precipitate ThP tarifgrr>nnil hacterial lawn cannot be accurately evaluated due to microscopic test article precipitate. Distinguished by precipitate on the plate that .s wsible to the nakedl eye NP Non-Interfering Preapitate bm anyprecipitate particles detected by the automnted cobnyrounter total less than 10% of the revertant colony count (e.g., <3 parades on a plate with 30 revertants.) Distinguished by precipitate on the plate that is vuuble to the naked eye IP Interfering Precipitate mid amTpredpitate partides detected by the automated colony counter exceed Y10% of the revertant colony count (e.g., >3 particles on a plate with 30 revertants.) Company Sanitized. Does not contain_TSgASi Reppnrreeetvilcriemieprttyiiatnanabttreyyctotaooluoxitnniocitmeietsyraffateoesrrdseaawycgooilltrrohentnhayuetetcposolmtauetrnaettseeetdrrxahoicniorblaioetnennddytitarcoecoxltyuiivcniabttytiyin.ogbnPawclnaoetdernesduwicntoilioeuthnsnswsteutedfhfreiemcicaaeosnnsuutanatyetlelsywdt.aaesrittihtcheleer Evaluation of Results For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported. TItprheotAevsgIeiFOtrmrioeOtvaareentateTtihnsfrAettrachh9teteoea7vstnnaehtincrteaath^canrrtptrnderieectaaesWaltseiketpoiPtmenoio2nrsfebusmtpevohltreafeheAatveetndea(moslporusteKefeavaatMaerenrettrdivtsIcalOppeelneohoalt).nissscitswtlaeieDvoteecinrtaseh,oetieanejtutqtpermdsuoseegalutateelsvskdrttafooclpsouaftororeutsa.rhsisitenetgiDrvaardweeamodatiiasosttfheesstrTehera-eAttesrhse1miapfln5oanomc3rtnre5tn^ewdsnaewos^ueairtnsueeinn^jaouemfsdsegtewtQiehatndone pipan vehicle control value. Criteria for a Valid Test # ATmpsTthplAlAaueolIsTtnOiSmaunthatOavniielodnrmAnTeMaRoonAn(u-gorfe9seiwalb7n2lcar)ai4eteno0.atvag-ernne.sTdcrdAttAAreaWlirilnt1lhelP5stcerstW32iura5aindlunPtimueuvn25mlrrtueheAcu-stbsueivt4(oelrbpt5mvrnAuKe!:eruThoiem(MnspisAftceKbIltm9tOdehaM7feeuSloa^m)sI,reutOmr8ortvdhi0olnura)eeBss-mckattmr2urdgaoe4leutenta0emutnpssa;reltgotWear.MheafsnoentPsemodCit2crc,wtuauihuhtttslyeseevattusrradtptarAsheercsaeresa(tnespsyperotoKrirntfesaomMctsiiteenbcenInesOoccimtucfeeeloe)lrtAto,^hnuMf1esre5tiehdUndeetmiu-erte^et3e^dr8usUrAveoom.anurl^9igufidtm8hso.f't beotcaboxesryfihtetceheiaobrernvemiitaraSeapatasebatprrcrreleueetcdZphamttasitvtnoeldetyaofotvhsrdte(eeh1he-ard)miqeceeuAlepe-aafeAlocn>noltddo5vdne0eoti0nrhsno.%te3iclcx.rdll1eeerrA0aevocsdepmeoulcnciieiisnntntlrilioocmsttolhn/hmuneveimsanlni.lrduuoetehTemvfr.ehteehbdremTertaerthmeoneioaxstennfiacrcornenoneiduufv-outmnoeofctrnxe.bttieiaaeocncrno(dht2osrom)fpbsoeouroAvesstlevhtiertrveibevotreehdteflaseuatnchaccomttrecsineooefptnmroareeonllrpqlionvaupwmmailrtlaiuehnuetsddeeget background lawn. MA Study No. G96CF43.502011 Company Sanitized. Does not contain TSCA CBI SHa-l2m2o05n1e:llaMtuyptahgiemnuicriituymTaenstdinEgsicnhethriechia coll Plate Incorporati.on AAssay DuPont Hi 0-1997-00035 RESULTS AND DISCUSSION Solubility Test ccoomnWcpeanatttierbaritlwiitoyans wsoefilteahpctpetrhdoexaismtathragteeelstyol5cve0el0nlstm.ogf /cTthnhole,ictehteebsamtseaadxrtioimcnluesmowlucaobsnilcisteyonloutrfbatltheioenitnetisetswteatdtie.dreaatnda Preliminary Toxicity Assay pLthraeecTyoi;hrpeeitlthiarmeetesimnunaaltjxisiylioptmofpuxtrhmiedaecdtpyioaroasebneslliscempaeyltiiao,^ntxtaaehirtdceiyiotimtynnoawxtoxhiafdiemst1y0omu0bmaussmtsedaarggovyes/emanedri.tlceeiasBtpyntreadeadssseaeswdan5ayot0senwd5,,10taihs0npe0T5lfa0fiaitn0gibnd0lpgieapesaggrl1spipqloeuafrtotelptK,.ltahtNtoiesxe.5tid'tcohtsteyer Mutagenicity Assay sumTmhaerirzeesduilntsToafbtlehse2m6 auntadg2en7i.cTityheassesadyataarwe epreregseenneteradteidn iTnaEbxlepser6imthenrotsuBgh1 and B2. Neither predpitate nor appreriable toxidty was observed. withInaEnyxpoefrtihmeentetsBtelr, tshtreaiinnistiianl mthuetpagreesneindctyeaasnsadya, bnsoepncoesio^feSr9esapcounvsaetsiowaere observed In Experiment B2, the independent repeat assay, no positive responses were observed with any of the tester strains in the presence and absence of S9 activation. CONCLUSION touhf^edtAhBeMeMactcetherscirttiereaarrlinsRatdrefuaoviInreorsanseifvnoaMftlhitduhetisasptturisoedtsnuyedAnwycs,eesHraeay-nMmwd0eitaSthb1asasdenndidIcenesndcooerifp.beAendurdosiecnenlott.hrR-epineoppdreu^oactcteodAscopsrlsa.oatynTliivhnse*derircSnea9o*t.e"th1t?aQntf,, SHa-l2m2o05n1e:llaMtuyptahgiemnuicriiutymTaenstdinEgsicnhethriechia coil Plate Incorporati_on Assay DuPont HLO-1997-0003S REFERENCES AmMes.uBta.gNe.nJs.wMitchCthanenSaon/md oE/.iWYa/am/Masaamkim(1a9li7a5n)MMicerthoosodms feoMr DuetategcetninicgitCy aTrecsint,oMgeuntsatainodn Research, 31:347-364. Brapsi^ck..,.DrioJ,nSiomfmtooen,EVsc.hFe.,rRichoisaenckorlainWz,PU2 San. dRaWy,PWA Awr,AanRd eRv&ersSetaMffuotradti(o1n98A0s)sAayn. Mutation Research, 76:169-190. GreEesnc,heMric.Hhi.aL.coalni,dMWutaJt.ioMn Rureiesela(r1c9h7368):3M-32u.tagen testing using trp* reversion in Levfion,rDthe.,dYeatemctaisoankio, Ef .fraanmdeBs.hNif.tAmmuetsag(1e9n8s.2) AA nreawn Soaflmqortnoeslmlaetsesatesrastrmainu,taTtiAo9n7al, hot-spot, Mutation Research, 94:315-330. Maron, D.M. and B.N. Ames (1983) Revised Methods for the Salmonella Mutagenicity Test, Mutation Research, 113:173-215. Vogel, HJ. and D.M. Bonner (1956) Acetylomithinase ofE.coli: Partial Purification and Some Properties, J. Biol. Chem., 218:97-106. MA Study No. G96CF43.502011 - 14 H-22051: M(uytpahgiernuirciiutymTaenstdinEgscinhethriechia coti Plate Incorporation Assay DuPont HLO-1997-00035 Salmonella Mutagenicity Assay Preliminary Toxicity Assay Table 1 Test Article Id Study Number Experiment No. Date Plated Counted by Vehicle Plating Aliquot H-22051 G96CF43.502011 Al 12/03/96 hand water 50 /il Test Article Concentration /ig per plate Vehicle _____________________ TA98 m r h S9 Activation-- _ without Activation---- Revertants Background Revertants Background per plate Code* per plate Code* 22 1 11 1 6.7 10 33 67 100 333 667 1000 3333 5000 16 . 16 19 14 12 23 20 16 8 17 1 1 1 1 1 1 1 1 1 1 10 1 15 1 91 15 1 81 10 1 10 1 13 1 12 1 14 1 `Baeksrouad bacterial valuation coda 1-Normal 2-Sllhtly raducad i-Cztraoaly raducad 5-Ab>nt NP-RAn-Interfaring Precipitate Mfedarataly raducad 6-0bscured by praclpitata IP-Interfering Fracipltata MA Study,No. G96CF43.50211 15 - Company Sanitized. Does not contain TSCA CBI SHa-i2m2o0n51tU: aMtuyptahgiemnuiciiiutymTaenstdinEgscinhethriechia eoU Plate Incorporation Assay DuPont HLQ-1997-00035 Salmonella Mutagenicity Assay . Preliminary Toxicity Assay Table 2 Test Article Id Study Number Experiment No. Date Plated Counted by Vehicle Plating Aliquot H-22051 G96CF43.502011 A1 12/03/96 machine water 50 pi Test Article Concentration Mg Pr plate TA1Q0 With S9 Activation Revertants Background per plate Code* ut chaut Activation Revertants Background per plate Code* Vehicle 6.7 10 33 67 100 333 6160700 3333 5000 167 162 198 162 185 191 169 154 172 162 134 1111111111 165 143 149 167 147 163 156 165 155 160 159 1111111111 "Background bacterial evaluation coda _ lMoraal J-SUgbtly reduced Extremely reduced S"Absent HP-Son-Interferlng Precipitata 3Moderately reduced 6e0beeured by precipitate IP-Intertering Precipitate . MA Study No. G96CF43.502011 -16- Companv Sanitized. Salmonella Mutagenicity Assay Preliminary Toxicity Assay Table 3 Test Article Id Study Number Experiment No. Date Plated Counted by Vehicle H-22051 G96CF43.502011 Al 12/03/96 hand ws oateurl Test Article Concentration MS Pe r p la t e Revertants Background per plate Code* Vehicle 6.7 10 33 61700 333 6136037030^ 5000 12 11 10888 11700 8 1111111111 f r e u n d b e c t e r m ^ u e t l od. 2 < .U i h t l y rwlueKl ( b t r a i l T reduced 3^*bant HP-Hon-Interfarine Precipitai Revertants Background per plate Code* 11 1 71 12 1 71 78 11 141 11 10 1 91 3 4 federately reduced Cbecured bp precipitate IP-Interterin Precipitate MA Study No. G96CF43.502011 17 Salmonella Mutagenicity Assay Preliminary Toxicity Assay Table 4 Test Article Id Study Number Experiment No. Date Plated Counted by Vehicle Plating Aliquot H-22051 G96CF43.502011 Al 12/03/96 machine water 50 nl Test Article Concentration Hg per plate Vehicle 6.7 10 33 67 100 333 667 1000 3333 5000 TA97a tflth S9 Activation Revertants Background per plate Code* Without Activation Revertants Background per pla. Code" 179 1 130 1 137 1 150 1 148 1 151 1 168 1 152 1 172 1 149 1 160 1 121 1 131 109 .121 131 104 127 104 124 132 110 1111111111 1`Background bacterial valuation cod * -Hoto.l 2-Sllghtly reduced Extremely reduced 5"Abeant BMton-Interfering Precipitate 3<Moderately raducad 6Obaeurad by praelpltata IP-Interferlng Praelpltata MA Study No. G96CF43.5020I I 18 ) SHa-l2m2o05n1e:llaMtuytpahgiemnuicriituymTaenstdinEgscinhetrhiechia coil Plate Incorporation Assay D uPont H LO -1997-00035 Salmonella Mutagenicity Asaay Preliminary Toxicity Asaay . Table S Test Article Id Study Number Experiment No. Date Plated Counted by Vehicle Plating Aliquot H-22051 G96CF43.502011 Al . 12/03/96 machine water 50 pi ' Test Article Concentration pg per plate WP2 uvrA (pKMlOl) VIth S9 Activation Without Activation____ Revertahts Background Revertants Background per plate Code* per plate Code* Vehicle 160 1 135 1 6.7 10 33 67 100 333 667 1000 3333 5000 140 1 153 1 148 1 155 1 113 1 168 1 153 1 173 1 136 1 1 144 1 152 1 152 1 170 1 135 1 117 1 171 1 145 1 160 1 170 1 140 1 `Background bacterial evaluation coda 1-lfonsal 2-Slltbtly raducad *-Ixtraoaly raducad W b a a n t HP-Hon-Intarfarin* Praclpitata ' Mfodarataiy raducod 6"Cbaeurad by praclpitata . IF-Xntarfarinc Praclpitata .jji 4i! i MA Study No. C96CF43.502011 - 19- Company Sanitized. Does not contain TSCA CBI SHa-l2m2o05n1e:llaMtuyptahgiemnuicriituymTaenstdinEgscinhetrhiechia coli Plate Incorporation Assay DuPont HUM997-00035 t Salmonella MutaganiclCy Assay Table 6 Test Article Id : H-22051 Study Number : G96CF43.502011 Strain : TA98 Liver Hlcrosomes : None Vehicle : water Plating Aliquot : 50 M1 Experiment No : B1 Cells Seeded : 1.0 X 10* Date Plated : 12/12/96 Counted by : Concentration Plate Reyertante Background Average Standard MB per Plate Number per plate Code* Revertants Deviation Vehicle 01 02 03 27 1 29 1 18 1 25 6 100 01 26 1 02 32 1 03 46 . 1 35 . 10 333 01 16 1 02 20 1 03 12 1 16 4 1000 01 25 1 02 22 1 03 15 1 21 5 3333 01 20 1 02 12 1 03 20 1 17 5 5000 01 20 1 02 20 1 03 19 1 20 Positive Control 2-nitrofluorene 1.0 Mg Per plateb 01 100 1 02 123 1 03 172 1 132 1 37 'Background bacterial valuation coda - 1-Normal Z-Slightly raducad b t r a n l y raducad S-Abaant HP-Non-Interfering Precipitate ^Positive control platas vara machina countad 34todarataly raducad 6K}bscurad by pracipitata IP-Irtarferin* Precipitata m MA Study No. G96CF43.502011 20 Salmonella Mutagenicity Assay Table 7 Teat Article Id : H-22051 Study Number : G96CF43.S02011 Strain : TA98 Liver Mlcrosomes : Rat liver S9 Vehicle : water Plating Aliquot : 50 ftl Experiment No : B1 . Cells Seeded : 1.0 X 10' Date Plated : 12/12/96 Counted by : hand Concentration Plate Revertanta Background Average Standard /ig per plate Number per plate Code* Revertanta Deviation Vehicle 01 02 03 25 1 17 1 15 1 19 5 100 01 28 1 02 24 1 03 27 1 26 2 333 01 26 l 02 27 1 03 29 1 27 2 1000 01 33 1 02 29 1 03 35 1 32 3 3333 01 33 1 02 25 1 03 39 1 32 7 5000 01 24 1 02 31 1 03 25 1 27 4 Positive Control 2-aminoanthracene 1.0 /ig per plateb 01 719 1 02 702 1 03 782 1 734 42 `Background bactarlal valuation coda l'Normal 2"Sllghtly raducad t-Extramaly raducad 5-Abaar.t NP-Hon-Intarfaring Praclpltata bPoaltlva control plataa vara machin countad 3*Mcdarataly raducad S^Otacurad by praclpltata IPalntarfaring Praclpltata MA Study No. G96CF43.S02011 - 21 Company Sanitized. Does not contain TSCA CBI SHa-l2m2o0n51tU: aMtuyptahgiemnuicriituymTaenstdinEgsicnhetrhiechia coil Plate Incorpor-at_ion A m y DuPont HLO-1997-00035 Sslnonella Mutagenicity Asaay Table 8 Teat Article Id : H-22051 Study Number : G96CF43.502011 Strain : TA100 Liver Microsomes : None Vehicle : water Plating Aliquot : 50 pi Experiment No : B1 Cells Seeded : 0.8 X 10* Date Plated : 12/12/96 Counted by : machine Concentration Plate Revertants Background Average Standard /ig per plate Number por plate Code* Revertants Deviation Vehicle 01 02 03 144 1 159 1 131 1 145 14 100 01 116 1 02 156 1 03 127 1 133 21 333 01 128 1 02 150 1 03 124 1 134 14 1000 01 147 1 02 154 1 03 126 1 142 15 3333 01 121 1 02 128 1 03 138 1 129 9 5000 01 114 1 02 135 1 03 118 1 Positive Control sodium azide 1.0 pg per plate 01 736 1 02 762 1 03 743 1 122 747 11 13 `Background bacterial evaluation coda 1-Horaal 2-Slightly reduced 4-Estraoely reduced 5-Abaent HP-Bon-Interfering Precipitate 3*tederately reduced 6-Cbaeured by precipitate IP"Interfering Precipitate MA Study No. G96CF43.502011 u - 22 - S an itized. Does nnt r.nntain TRPA P.Rl SHa-l2m2o05n1e:llaMtuyptahgienniuicriituymTeasntdinEgsicnhethriechia coU Plate Incorporation Assay DuPont HLO-1997-00035 Salmonella Mutagenicity Assay Table 9 Test Article Id H-22051 ! Study Number Strain G96CF43.502011 TA100 Experiment No : B1 Cells Seeded : 0.8 X 10s Liver Mlcrosomes Rat liver S9 Date Plated : 12/12/96 Vehicle Plating Aliquot water 50 pi Counted by : machine Concentration Mg per plate Vehicle 100 333 Plate Revertants Number per plate 01 153 02 124 03 148 01 167 02 129 03 142 01 160 02 141 03 154 Background Code" 11 1 11 1 11 1 Average Revertants 142 146 152 Standard Deviation 16 19 10 1000 01 148 1 02 166 1 03 141 1 152 13 3333 01 136 1 02 122 1 03 150 1 136 14 5000 01 148 1 02 161 1 03 147 1 152 8 Positive Control 2-aminoanthracene 1.0 ug per plate 01 761 1 02 778 1 03 806 1 "Background bacterial evaluation coda 1-Hrrmal 2-SlightLy raducad -Extremely raducad 5-Abeent HP-Non-Interfering Precipitate 3-Moderately raducad ' 6-Obacured ay precipitata IP-Interfering Precipitate MA Study No. G96CF43.502011 Company Sanitized. Does not contain TSCA CBI SHa-l2m2o05n1e:llaMluyptahgiemnuictiiutymTaenttdinEgscinhetrhiechia coJi Plate Incorporat<ion Atsay D uPont HLO-1997-00035 Salmonella Mutagenicity Assay Table 10 Test Article Id : H-22051 Study Number : G96CF43.502011 Strain : TA1535 Liver Microsomes : None Vehicle : water Plating Aliquot : 50 /il Experiment No : B1 Cells Seeded : 3.0 X 10* Date Plated : 12/12/96 Counted by : hand Concentration ' Plate Revertants Background Average Standard /ig per plate Number per plate Code* Revertants Deviation Vehicle 01 02 03 81 13 1 11 1 11 3 100 01 12 1 02 11 1 03 6 1 10 3 333 01 17 1 02 6 1 03 11 1 11 6 1000 01 13 1 02 10 1 03 8 1 10 3 3333 01 11 1 02 11 1 03 12 1 11 1 5000 01 11 1 02 13 1 03 11 1 12 1 Positive Control sodium azide 1.0 /<g per plateb 01 556 1 02 535 1 03 559 1 550 13 `Background bacterial valuation coda 1-Normal Z-Slifthtly rduead 4-Extraoaly radiicad S-Abaent NP-Non-Intarfarln* Pracipitata ''Positive control plataa ara machina countad 3*tadratly raducad 60bacurad by pracipitata IF-Intariarin Pracipitata MA Study No. G96CF43.S02011 - 24 - Company Sanitized. Does not contain TSCA CBI SHa-l2m2o0n51el:laMtuyptahgiemnuicriituymTeasntdinEgsicnhethriechia . DuPont HLQ-1997-00035 Salmonella Mutagenicity Assay Table 11 Test Article Id Study Number Strain Liver Mlcrosomes Vehicle Plating Aliquot H-22051 G96CF43.502011 TA1535 Rat liver S9 water 50 pi Concentration Plate Revertants pg per plate Number per plate Experiment No : B1 Cells Seeded : 3.0 X 10s Date Plated : 12/12/96 Counted by : hand Background Average Standard Code* Revertanta Deviation Vehicle 01 02 03 14 1 13 1 12 1 13 1 100 01 17 1 02 10 1 03 11 1 13 4 333 1000 01 21 1 02 16 1 03 14 1 01 17 1 02 25 1 03 20 1 17 4 21 4 3333 01 13 1 02 11 1 03 12 1 12 1 5000 01 16 1 02 14 1 03 16 1 15 1 Positive Control 2 -amlnoanthracene 1.0 pg per plateb 101 91 102 116 1 11203 128 19 background bacterial evaluation coda 41-Honaal -Zxtraoaly reduced 2-SUghtly reduced 5*Abent HP*lon-Intorfarlng Precipitate hpoaltlva control platee were Bachine counted 3 4 fodarately reduced gaObaeured by precipitate IP>tntarfering Precipitate MA Study. No. G96CF43.502011 Company Sanitized. D oesjiaL cafliaiaJS fi^fiB l SHa-l2m2Qon51e:llaMtuyptahgiemnuicriituymTaenstdinEgscinhetrhiechia eoU . Plate Incorporation Assay DuPont H LO -1997-00035 Salmonella Mutagenicity Assay Table 12 Test Article Id : H-22051 Study Number : G96CF43.502011 Strain TA97a Liver Microsomes : None . Vehicle : water Plating Aliquot : 50 ill Experiment No : B1 Cells Seeded : 3.1 X 10s Date Plated : 12/12/96 Counted by : machine Concentration Plate Revertants Background Average Standard lig per plate Number per plate Code* Revertants Deviation Vehicle 01 02 03 135 1 150 1 157 1 147 11 100 01 135 1 02 136 1 03 129 1 133 4 333 -i,:. 01 190 1 02 139 1 03 163 1 164 26 1000 01 162 1 02 166 1 03 173 1 167 6 3333 01 196 1 02 160 1 03 192 1 183 20 5000 01 180 1 02 188 1 03 193 1 187 7 Positive Control 9-aminoacridine 100 pg per plate 01 1934 1 02 2118 1 03 1682 1 1911 219 Background bacterial valuation coda 1-Normal 2-Sligtatly raducad 4-Extramely raducad 5-Abiant HP-Non-Intarfarlng Precipitata 3-Modarataly raducad 6-Obacurad by precipitata IP-Interfering Precipitata MA Study No. G96CF43.502011 -26Company Sanitized. Does not contain TSCA CBI SHa-l2m2o05n1e:llaMtuyptahgiemnuicriituymTaenstdinEgicinhttrhicehla eoli Plate Incorporation Assay DuPont HLO-1997-00035 Salnonella Mutagenicity Asaay Table 13 Test Article Id Study Number Strain Liver Mlcrosomes Vehicle Plating Aliquot H-22051 G96CF43.502011 TA97a Rat liver S9 water 50 pi Experiment No Cells Seeded Date Plated Counted by B1 3.1 X 10 12/12/96 machine Concentration Plate Reverenti Background Average Standard /ig per plate Number per plate Code* Revartants Deviation Vehicle 100 333 1000 3333 01 02 03 01 02 03 01 02 03 01 02 03 01 02 03 235 1 230 1 205 l 220 l 240 1 254 1 204 1 233 1 146 1 215 1 213 1 198 1 192 1 221 1 219 1 223 16 238 17 194 44 209 9 211 16 5000 01 217 1 02 229 1 03 234 1 227 Positive Control 2- aminoanthracene 2.0 pg per plate 01 1230 1 02 1481 l 03 1662 1 1458 9 217 `Background bactarlal evaluation coda 1-Horaal 2-Sllghtly raducad t-Bxtraaaly raducad 5Absent HF-tlon-Intarfaring Precipitate 3-tfederately raducad 6-Obscured by precipitate IP-Interfering Precipitate MA Study No. G96CF43.5020I I - 27 - Company Sanitized. Does not contain TSCA CBI SHa-l2m2Qon51e:llaMtuyptahglemnuicriituymTaenstdinEgscinhetrhiechia coil Plate Incorporation Assay DuPont HLO-1997-00035 E. coll Mutagenicity Assay Tabla 14 Test Article Id : H-22051 Study Nuaiber : G96CF43.502011 Strain : WP2 uvrA (pKMIOl) Liver Mlcrosomes : None Vehicle : water Plating Aliquot : 50 pi Experiment No : B1 Cells Seeded : 7.6 X 10s Date Plated : 12/12/96 Counted by : machine Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code* Revertants Deviation Vehicle 01 02 03 232 1 201 1 194 . 1 209 20 100 01 258 1 02 235 1 03 212 1 235 23 333 01 267 1 02 250 1 03 251 1 256 10 1000 01 236 1 02 279 1 03 271 1 262 23 3333 01 244 1 02 256 1 03 305 1 268 32 5000 01 273 1 02 313 1 03 272 1 286 23 Positive Control methyl methanesulfonate 1000 pg per plate 01 1829 1 02 1771 1 03 1792 1 1797 29 a8ack(round bacterial valuation cod l"Norval 2-311(htly reduced {Extremely reduced 5-Ahient HP^Hon-Interferlnt Precipitata . S^loderetely reduced MJbacurad by preciptete IP>Interferln( Preciptete MA Study No. G96CF43.502011 -28 - SHa-l2m2Qon5e1l:laMtuyptahgiemnuicriituymTaenstdinEgscinhetrhiechia coll Plate Incorporation Assay DuPont HLO-1997-00035 E. coll Mutagenicity Assay Table 15 Test Article Id Study {lumber Strain Liver Mlcrosomes Vehicle Plating Aliquot H-22051 696CF43.502011 WP2 uvrA (pKMIOl) Rat liver S9 water 50 pi Experiment No Cells Seeded Date Plated B1 7.6 X 10# 12/12/96 Counted by : machine Concentration Plate Revertants Background Average Standard fig per plate Number per plate Code* Revertants Deviation Vehicle 01 02 03 242 1 276 1 222 1 247 27 100 01 254 1 02 294 1 03 263 1 270 21 333 01 315 1 02 268 1 03 265 1 283 28 1000 01 305 1 02 286 1 03 310 1 300 13 3333 01 . 02 03 285 303 313 1 1 1 300 14 5000 01 296 1 02 303 1 03 293 1 297 5 Positive Control 2-aminoanthracene 10 fig per plate 01 1664 1 02 1591 1 03 1410 1 1555 131 background bactarial valuation coda l"Normal Z-Slightly raducad 4*Extranly raducad SAbaant NPHon-Intrfring Pracipltata 344odarataly raducad 6*Obacurad by pracipltata IP*Intrfaring Pracipltata MA Study No. G96CF43.502011 - 29 Company Sanitized. Does not contain TSCA CBI SHa-2lm20o5n1e:llaMuttyapgheimniucriituymTeasntdinEgsicnhetrhiechia coti Plate Incorporation Assay_______DuPont HLO-1997-00035 Salmonella Kutagenlcity Assay Table 16 Test Article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-22051 G96CF43.502011 TA98 None water 50 M1 Experiment No : B2 Cells Seeded : 1.1 X 10s Date Plated : 01/03/97 Counted by : hand Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code* Revertants Deviation Vehicle 01 02 03 14 1 15 1 16 1 15 1 100 01 20 1 02 18 1 03 16 1 18 2 333 01 11 1 02 11 1 03 5 1 93 1000 01 7 1 02 10 1 03 16 1 11 5 3333 01 7 1 02 6 1 03 11 1 83 5000 01 4 1 02 2 1 03 11 1 65 Positive Control 2-nitrofluorene 1.0 pg per plateb 01 138 1 02 152 1 03 122 1 137 15 background bactarial avaluatlon coda 1-Hormal 2-Slightly raducad 4-Extramaly caducad S-Abient HP-Hon-Interfacing Precipitate ^Positive control plates wars machine counted - 3^toderately raducad 6-Obscursd by precipitate IP-Interfering Precipitate f) MA Study No. G96CF43.S02011 30- aCompany Sanitized. Does not contain TSCA CBI SHa-l2m2Qon5e1U: aMluyptahgimenuicniutymTaenstdinEgsicnhetrhiechia coll Plate Incorporat_ion Assay DuPont HLO-1997-00035 Salmonella Mutagenicity Assay Table 17 Test Article Id : H-22051 Study Number : G96CF43.502011 Strain : TA98 Liver Microsomes : Rat liver S9 Vehicle : water Plating Aliquot : 50 pi Experiment No : B2 Cells Seeded : 1.1 X 10 Date Plated : 01/03/97 Counted by : haind Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code" Revertants Deviation Vehicle 01 02 03 19 1 16 1 25 1 20 5 100 01 18 1 02 25 1 03 21 1 21 4 333 01 26 1 02 19 1 03 35 1 27 8 1000 01 20 1 02 13 1 03 16 1 16 4 3333 01 18 1 02 13 1 03 23 1 18 5 5000 01 6 1 02 7 1 03 14 1 9 Positive Control 2-aminoanthracene 1.0 pg per plateb 01 746 1 02 824 1 03 656 1 742 4 84 `Background bacterial evaluation coda 1-Kotmal 2-Slishtly rtducad Extremely raducad 3-Abaant KP-Hon-Interfarlnt Precipitate bPoiltlva control pletaa were machine counted 3^todarataly raducad 6-Obecured by precipitate IP-Interfering Precipitate MA Study No. G96CF43.5020I I - 31 - Salmonella Mutagenicity Assay Table 18 Test Article Id Study Number Strain Liver Mlcrosomes Vehicle H-22051 G96CF43.502011 TA100 None water 50 pi Concentration .Plate Revertants Mg per plate Number per plate Bacii C Experiment No Cells Seeded Date Plated B2 1.5 X 10 01/03/97 Counted by machine Average Standard Revertants Deviation Vehicle 01 02 03 134 1 134 1 93 1 120 24 100 01 121 1 02 131 1 03 82 1 333 01 121 1 02 116 1 03 112 1 111 26 116 1000 01 104 1 02 100 1 03 107 1 104 3333 01 134 1 02 117 1 03 125 1 125 5000 01 103 1 02 87 1 03 99 1 96 Positive Control sodium azide 1.0 Mg 1 01 699 1 02 696 1 03 660 1 `Background bactarial avaluation code 1-Normal 2-Sllghtly reduced Extremely reduced 5Absent NP-Hon-Interierlng Precipitate 685 22 3Htoderately reduced 6-0bscured by precipitate IP-Interferlns Precipitate MA Study No. G96CF43.502011 Company Sanitized. Does not contain TSCA CBI SHa-l2m20o5n1e:llaMtuyptahgimenuicniutymTaenstdinEgsicnhethriechia coli Plate Incorporation Assay DuPont HLO-1997-OOQ35 Salmonella Mutagenicity Assay Table 19 Test Article Id : H-22051 Study Number : G96CF43.502011 Strain : TA100 Liver Microsomes : Rat liver S9 Vehicle : water Plating Aliquot : 50 /ti Experiment No : B2 Cells Seeded : 1.5 X 10* Date Plated : 01/03/97 Counted by : machine Concentration Plate Revertants Background Average Standard Mg per plate Number per plate Code* Revertants Deviation Vehicle 01 02 03 139 1 117 1 121 1 126 12 100 01 129 1 02 142 1 03 114 1 128 14 333 01 150 1 02 117 1 03 132 1 133 17 1000 3333 01 147 l 02 133 1 03 136 1 01 141 1 02 132 1 03 127 1 139 7 133 7 5000 01 134 1 02 137 1 03 156 1 142 12 Positive Control 2-aminoanthracene 1.0 Mg per plate 01 1183 1 02 1112 1 03 1019 1 1105 82 "Background bacterial avaluatlon coda l*Honnal 2aSllfthtly raducad 4*Extremely raducad 5"Absant HP-Hon-Intarfaring Precipitata 3*Moderately raducad 6-Obscurad by precipitate IP-Interferin* Pracipitat* m MA Study No. G96CF43.5020I l - 33 - Company Sanitized. Does not contain TSCA CBI SHa-l2m20o5n1e:llaMtuyptahgimenuic/iiutymTaenstdinEgsicnhetrhiechia coll Plate Incorporation Assay DuPont HLCM997-00035 Salmonella HutagenlciCy Assay Table 20 Test Article Id Study Humber Strain Liver Microsomes Vehicle Plating Aliquot H-22051 G96CF43.502011 TA1535 Hone water 50 /il Experiment No Cells Seeded Date Plated Counted by B2 2.8 X 10B 01/03/97 hand Concentration ftg per plate Vehicle Plate Number 01 02 03 Revertants per plate 11 15 14 Background Average Code* Revertants 1 1 1 13 .Standard Deviation 100 01 11 1 02 18 1 03 14 1 14 333 01 8 1 02 8 1 03 11 1 1000 01 8 1 02 9 1 03 10 1 3333 01 11 1 02 2 1 03 6 1 5000 01 11 1 02 9 1 03 6 1 Positive Control sodium azide 1.0 Mg 1 01 552 1 02 549 1 03 524 1 542 15 background bacterial evaluation coda 1-Honaal 2-Slihtly reduced Extremely reduced 5-Abeent HP-Hon-Interfering Precipitate bPosltlve control platea were machine counted 3Hoderately reduced 6-Obtcurad by precipitate IP-Interfering Precipitate MA Study No. G96CF43.502011 34 Company Sanitized. Does not contain TSCA CBI SHa-l2m20o5n1e:llaMtuyptahgimenuicriiutymTaenstdinEgsicnhetrhiechia coli Plate Incorporation Assay DuPont HUM997-00035 Salmonella Mutagenicity Assay Table 21 Test Article Id Study Number Strain Liver Microsomes Vehicle H-22051 G96CF43.502011 TA1535 Rat liver S9 water 50 jil Experiment No Cells Seeded Date Plated Counted by B2 2.8 X 10" 01/03/97 hand Concentrtion Plate Revertants Background Average Standard jig per plate Number per plate Code* Revertants Deviation Vehicle 01 02 03 12 1 11 1 91 11 2 100 01 15 1 02 11 1 03 9 1 12 3 333 01 12 1 02 20 1 03 17 1 16 4 1000 01 18 1 02 10 1 03 13 1 14 4 3333 01 11 1 02 8 1 03 16 1 12 4 5000 01 12 1 02 18 1 03 10 1 1.3 4 Positive Control 2-aminoanthracene 1.0 jig Per plateb 01 97 1 02 102 1 03 103 1 101 3 `Background bactarlal valuation cod 1-Normal Z-Sligbtly rducd 4-Eztramaly raducad 5-Abaant NP-Non-Intarlarins Pracipitata bPoaltiva control plataa ara machina counbad 3-Modarataly raducad 6-Obicurad by pracipitata IP-Intarfaring Pracipitata MA Study No. G96CF43.502011 35 Company Sanitized. Does not contain TSCA CBI Salmonella Mutagenicity Assay Table 22 Test Article Id Study Number Strain Liver Microsomes Vehicle H-22051 C96CF43.502011 TA97a None water Experiment No Cells Seeded Date Plated B1021. 3/ 0 X3 / 9170 machine Plating Aliquot Standard Concentration Deviation Hi per plate Vehicle 000123 11109648 111 106 100 000123 111421664 111 129 16 333 000123 111251542 111 130 22 1000 000123 111232721 111 127 3333 000123 111033920 111. 124 13 5000 000123 111234702 111 133 }10001293-aminoa22c128r387I036dine 100111/ig 1763 772 14Background bacterial valuation coda -Hormal -Eatraoaly raducad j-Slightly raducad 5-Abaant HPHon-Xntarfaring Pracipltata 3-ttoderataly raducad g^jbecured by precipitata IP-Interfarlng Precipitata SHa-l2m20o5n1e:llaMtuyptahgimenuicriituymTaesntdinEgsicnhethriechia coll Plate Incorporation Assay DuPont HLO-1997-00035 m Salmonella Mutagenicity Assay Table 23 Test Article Id Study Number Strain Liver Microsomes Vehicle H-22051 G96CF43.502011 TA97a Rat liver S9 water 50 pi Experiment No Cells Seeded Date Plated Counted by B2 1.3 X 106 01/03/97 machine Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code* Revertants Deviation Vehicle 01 02 03 147 1 155 1 142 1 148 7 100 01 146 1 02 146 1 03 159 1 150 8 333 1000 3333 01 140 1 02 168 1 03 162 1 01 163 1 02 163 1 03 180 1 01 154 1 02 155 1 03 160 1 157 15 169 10 156 3 5000 01 145 1 02 146 1 03 165 1 152 Positive Control 2-amlnoanthracene 2.0 pg per plate 01 2068 1 02 2052 1 03 2126 1 2082 11 39 `Background baetarlal valuation coda 1-Hormal 2-Slightly reduced Extremely reduced 5"Abiant HP-Hon-Intarterlnx Precipitata 3-Hodarataly reduced g-Obicured by precipitata IP-Interferin Precipitata MA Study No. G96CF43.502011 37 - Company Sanitized. Does not contain TSCA CBI SHa-l2m20o5n1e:llaMtuyptahgimenuicriiutymTaenstdinEgsicnhetrhiechia coll Plate Incorporation Assay DuPont HLO-1997-00035 E. coll Mutagenicity Assay Table 24 Test Article Id H-22051 Study Number G96CF43.502011 Strain VP2 uvrA (pKMIOl) Liver Microsomes None Vehicle : water Plating Aliquot : 50 jil Experiment No : B2 Celia Seeded : 4.5 X 10a Date Plated : 01/03/97 Counted by : machine Concentration Plate Revertants Background Average Standard /ig per plate Number per plate Code* Revertants Deviation Vehicle 01 02 03 164 1 145 1 150 1 153 10 100 01 192 1 02 144 1 03 175 1 170 24 333 01 182 1 02 194 1 03 193 1 190 7 1000 01 169 1 02 158 1 03 199 1 175 21 3333 01 158 1 02 180 1 03 212 1 183 27 5000 01 205 1 02 146 1 03 170 1 174 Positive Control methyl methanesulfonate 1000 fig per plate 01 1501 1 02 1585 1 03 1500 1 1529 30 49 `Background bacterial valuation cod 1-Boraal 2-Sllahtly rducd Extremely raducad S-Abaant SP-Bon-Interferii!* Precipitata J-Modarately reduced 6-Obaeured by praclpltata IP-Intarfaring Precipitata MA Study No. G96CF43.502011 - 38 - Company Sanitized. Does not contain TSCA CBI ! SHa-l2m2o05n1e:llaMtyuptahgimenuiHcituymTaenstdinEgsicnhetrhiechia coti Plate Incorporation Assay DuPont HLO-1997-00035 E. coll Mutagenicity Assay Table 25 Test Article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot : H-22051 : G96CF43.502011 : WP2 uvrA (pKMIOl) : Rat liver S9 : water : 50 m 1 Experiment No : B2 Cells Seeded : 4.5 X 10e Date Plated : 01/03/97 Counted by : machine Concentration Plate Revertants Background Average Standard Mg per plate Number per plate Code* Revertants Deviation Vehicle 01 02 03 164 1 171 1 160 1 165 6 100 01 182 1 02 165 1 03 171 1 333 01 203 1 02 185 1 03 229 1 173 9 206 22 1000 01 174 1 02 173 1 03 207 1 185 19 3333 01 187 1 02 199 1 03 212 1 199 13 5000 01 251 1 02 202 1 03 192 1 215 32 Positive Control 2-aminoanthracene 10 Mg per plate 01 1668 1 02 1690 1 03 1714 1 1691 23 `Background bactorltl valuation coda 1-Sonnai 2-Sllghtly reduced 4-Extremely raducad 5-Abaant NF-Hon-Intarfarlng Praclpltote 3-Modarately reduced 6-Obaeurad by precipitate IP-Interfering Praelpitata MA Study No. G96CF43.502011 39Company Sanitized. Does not contain TSCA CBI SHa-l2m20o5n1e:llaMluyptahgimenuicriiutymTaenstdinEgsicnhethriechia coli Mate Incorporation Assay DuPont HLO-1997-00035 Salmonella/E. coli Mutagenicity Asaay Summary of Results Table 26 Test Article Id : H-22051 Study Number ; G96CF43.502011 Experiment No : B1 Average Revertants Per Plate standard Deviation Liver Microsomes: None UP2 uvrA Dose (/tg) TA98 TA100 TA1535 TA97a (pKMlOl) 0.0 100 333 1000 3333 5000 Pos 25 6 145 14 11 3 147 11 209 20 35 10 133 21 10 3 133 4 235 23 16 A 134 14 11 6 164 26 256 10 21 5 142 15 JO 3 167 6 262 23 17 5 129 9 11 1 183 20 268 32 20 1 122 11 12 1 187 7 286 23 132 37 747 13 550 13 1911 219 1797 29 Liver Microsomes: Rat liver S9 Dose (jig) TA98 TA100 TA1535 TA97a VP2 uvrA (pKMlOl) 0.0 100 333 1000 3333 5000 Pos 19 5 142 16 13 1 223 16 247 27 26 2 146 19 13 4 238 17 270 21 27 2 152 10 17 4 194 44 283 28 32 3 152 13 21 4 209 9 300 13 32 7 136 14 12 1 211 16 300 14 27 4 152 8 15 1 227 9 297 5 734 42 782 23 112 19 1458 217 1555 131 0.0 - Valitela platina aliquot of 50 <*1 Poa - Poaltiv# Control eoneantrationa ai apaclfiad In Matonaia and Mathods aaetlon MA Study No. G96CF43.502011 Company Sanitized. Does not contain TSCA CBI Saloonella/E. coll Mutagenicity *ssay Summary of Results TaKI A 27 Tes. Article Id : H-22051 Study Number : G96CF43.502011 Experiment No : B2 Average Revertants Per Plate Standard Deviation Liver Microsomes: None Dose (jig) TA98 010. 00 3130300 3253 5000 Pos 15 18 119 8 6 137 1 2 35 3 5 15 TA100 120 111 116 104 125 96 685 24 26 5 4 9 8 22 TA1535 TA97a po (pKMIOl) 13 14 9 9 6 9 542 2 106 9 153 + 4 129 16 170 2 130 22 190 + 1 127 1 6 175 5 124 13 183 + 3 133 t 8 174 + 15 1763 772 1529 10 24 7 21 27 30 49 Liver Microsomes:: Rat liver S9 Dose (jig) 010. 00 3130300 3333 5000 Pos TA98 TA100 20 + 21 + 27 + 16 + 18 + 9+ 742 j 126 + 4 128 8 133 + 4 139 + 5 133 + 4 142 + 84 1105 + 12 14 17 7 7 12 82 TA1535 TA97a WP2 uvrA (pKMIOl) 11 12 16 14 12 13 101 2 148 3 150 4 157 4 169 4 156 4 15? + 3 2082 7 165 8 173 15 206 10 185 3 199 11 215 39 1691 9 22 19 13 32 23 0.0 - Vahicle plating aUq-iot of 50 |tl Poa Poaltlva Control concantratlona aa apaclfiod in Matarlala and Hathoda aaction. ! MA Study No. G96CF43.50201. Company Sanitized. Does not contain TSCA CBI SHa-l2m20o5n1e:llaMmutladgmenuinciutymTeasntdinEgsicnh the erich ia coll Plate Incorporattion Assay DuPont HLO-1997-00035 appendix I Historical Control Data M A Study No. G 96CF43.50r.01i Company Sanitized. Does not contain TSCA CBI SHa-l2m20o5n1e:llaMtuytpahgiemnuicriituymTeasntdinEgsicnhethriechia coll Plate Incorporation Assay DuPont HLO-1997-00035 Historical Negative and Positive Control Values 1993 - 1995 * revertants per plate Activation Strain Control None Rat Liver Mean SD Min Max Mean SD Min Max TA98 Nsg 20 7 4 52 27 8 4 65 Pos 316 185 17 3045 1091 538 94 3506 TA100 TA1535 Neg 137 25 67 268 156 27 50 323 Pos 672 194 100 2054 1133 510 136 3682 Neg 12 5 1 53 13 5 0 46 Pos 503 206 17 3704 128 130 18 2153 TA97 and TA97a Neg Pos 131 24 67 176 174 35 101 2<>2 1019 534 382 2541 1024 406 376 1S42 WP2 uvrA (pKMIOl) Neg 242 57 91 420 268 61 113 425 Pos 1918 506 557 3072 1469 469 540 2668 SD =standard deviat (including but not ion; Min limited = minimum value; Max= maximum value; Neg = negative control to deionized water, dimethylsulfoxidc, ethanol and acetone), Postpositive contro M A Study No. G96CF43.502011 ' 43 Company Sanitized. Does not contain TSCA CBI 11:2051: Mtil.iiU'im-ily Testing in llie S a lm o n e lla typ ltim uriuin ;tnd E scherichia coli Piale Incorporano Assay DuPont lll.O -l,,07-tllltH5 A PPEN D IX II Study Protocol M \ Sl..J> N'o COM I n.50:ol I 44 - Company Sanitized. Does not contain TSCA CBI SHa-l2m20o5n1e:llaMtuyptahgimenuicriituymTeasntdinEgsicnhetrhiechia eoU Plate Incorporat.ion A. ssay DuPont HLO-1997-OOU35 iiMW" a> . .- MA Siudy Number: Ct Ql mCF- H 3-^> o 2 0 1 1 Bacterial He* erse Mutatimi Away dh an Independent Repeal Away 10 lU RPOSli The purpose o f this stuJv is to evaluate the mutagenic potential o f the test article by ^ a s S T l s ahili.v to mduce reverse mutations a. selected loe, ol several str.nn. ol Salm onella iyph.no,num and at the tryptophan locus of Eschcnchui io h mr (pKMIOl) in the presence and absence o f SO activation. 2.0 SPONSOR 2.1 Name: 2.2 Address: E.I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine PNOewBaorkx,SDOE, El1k9t7o1n4Road . 2.3 Representative: Brian H. Mathison, Ph.D. 2.4 Sponsor Project : MR-10850 3 0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 3.1 Test Article: 3.2 Controls: Ngative: Positive: To Test an<clc vehicle 9-aminoacridine 2-aminoanthracene methyl methancsulfonatc 2 -nitrofluorene sodium azide V* 3.3 Determination of Strength. Purity, etc. The Sponsor will he directly responsible Tor determination and documentation o f the anal) t!cal purity and composition of the test article and the stability and strength of k* <4nctn> cnliitkins. 3 4 Test Article Retention Sample The retention of a reserve sample of the test article will be the responsibility of the Sponsor. rrolocnl SFGTMi:oi I 11 *'-* yI of 10 AMSICSOROCBIAIOTLEOSG, IINCCA.L MA Study No. G96CF43.502011 Company Sanitized. Does not contain TSCA CBI I SHa-t2m2o0n5e1U: aMtuyptahgimenuicriiutymTaenstdinEgsicnhetrhiechia coB Plate Incorporation Assay DuPont HLO-1997-00035 4.0 I LSTING FACILITY AND KEY PERSONNEL 4.1 Name: Toxicology Testing Facility Microbiological Associates. Inc. 4.: Address: 4630 Medical Center Drive Rockville. MD 20850 4.3 Study Director: Valentine O. Wagner III. M S. TEST SCHEDULE 5.1 Proposed Experimental Initiation Date: i x | 3 | ^ 5.2 Proposed Experimental Completion Date: 5.3 Proposed Report Date: l U i V n * li"t\9"7 6.0 TEST SYSTEM The tester strains will include the S. typhimurium histidine auxotrophs TA98. TAIOO. TAcI5o 3l i5teasntedr TA97A as described by Ames et at. (1975) and Lev,n strain WP2 uvrA (pKMIOl) as described by Green and [f Muriel (1976). h Gtnoivpc of the Strain. Uwd for Mutagen Tcsimb_ _ _ _ ^ _ _ _ _ _ Histidine Mutation <iuG46 TA 1535 TA 100 AuD3052 AuOMU TAPS TA<>7A TrMyputtaotpiohnan IrpC (WpPK2MwIOtAl) Additional Mutations LPS Repair R-tacior r/a Amr6 rfu IbirB Aui-rA l-ach S txphim urium tester strain contains, in addition to a mutationtn the histidine operon additional mutations that enhance sensitivity to some mutagens. The r/o mutation results in a cell wall deficiency that increases the permeability of the cell to certain classes o f chemicals such as those containing large ring systems that would otherwise be cxc'ud" The deletion in the mrB gene results in a deficient DNA excision-repair system. Tester strains TA98. TA100 and TA97A also contain the pKM 101plasmid (carrying the R/faetor). I, has been suegested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process. | .\i)g and TA97A are reverted from histidine dependence (auxotrophv) to histidine independence (protoirophv. by frameshill mutagens. TAI00 is reverted by both framcsh.lt ITnlncnl SI*C;T50:OII I 1114 ft : of io y AMSICSOROCBIAIOTLEOS.GIINCCA.L MA Study No. G96CF43.5020I I -46 - Company Sanitized. Does not contain TSCA CBI B H-22051: Mutagenicity Testing in the Salm onella typliimurium and Escherichia colj Plate Incorporation Assay DuPont HLO-1997-00035 .inJ base >nb->tiiiii>n mutagens and TAI535 is reverted iml> h> mutagens that cause base Mihsinulion.s I he E coli tester s lu m has an A I base pair al the critical mutation site within the n r f eene itt ileus .7 . Il>,)t)i Tester strain U P2 /.irA (pKMIOl I hits a deletion in the mrA eene resulting in a Jelieient DNA eseision-repair system. Tester strain \\p ; Mir.\ (pKMIOl 1 also contains the pKMIOl plasmid described above Trsptophan revertants can arise due to a base dunce at the originally mutated sue or by a base change elsewhere in the chromosome causing the original mutation to be suppressed. Thus, the specificity of the resersion mechanism is sensitive to base-pair substitution mutations, rather than Irameshilt mutations (Green and Muriel. 1976) The X tvphimurium tester strains were received directly from Dr. Bruce Ames. University of California. Berkeley The E coltester strain was received from the National Collection of Industrial and Marine Bacteria. Aberdeen. Scotland (United Kingdom). 7.0 EXPERIMENTAL DESIGN AND METHODOLOGY The test article will be tested at a minimum of five dose levels along with appropriate neeativc and positive controls with tester strains TA98. TA100. TAI535. TA97A and P2 i/irA tpKMIOl) with and without S9 activation. All dose levels of test article, negative controls and positive controls will be plated in triplicate. Solubility* Determination 1 Unless the Sponsot has indicated the test article vehicle, a solubility determination will be conducted to determine the maximum soluble concentration or workablesuspension up to a maximum of 500 mg ml. Vehicles compatible with this test Svstem, in order of preference, include but arc not limited to: deionized water (CAS 7732-18-51. Jimethylsullbxidc (CAS 67-68-5). ethanol (CAS 6-4-17-5) and acetone (CAS 67-64-1) The vehicle of choice will be the solvent, selected in order of preference, that permits preparation of the highest vvorkable'solubli- slock concentration, up to 500 ntg ml 3 Preliminary Toxicity Assay to Select Dose Levels Selection of dose levels for the mutagenicity assay will be based upon the toxicity and precipitation profile of the lest article assessed in a preliminary toxicity assay Ibis preliminarv assay will be conducted by exposing TA98. TAIOO. TAI-VP. UV'-A and WP2 air A (pKMIOl) to negative controls and to at least eight concentrations of lest article, one plate per dose level, in both the presence and absence of S*>activation Unless indicated o.herwisc by the Sponsor, the highest dose will be the highest workable concentration in the vehicle of choice but not to exceed ' ini' plate loxicilv will be evaluated as a decrease in the number of revertanl colonies per plate and or a thinning or disappearance ol the bacterial background lawn Precipitation will be evaluated following the incubation period In the event that the test article cannot be delivered at a high enough concentration in an appropriate vehule to he toxic or if test a.-iclc precipitate is present on the plates is. .,,.,isi'(.Tfii:iiii 111101 -'"Mu ^ M IAC SRSOOBCIIOALTOESG. IICNAC.L I MA Study No. G96CF43.502011 - 47 - Company Sanitized. Does not contain TSCA CBI SHa-l2m20o5n1e:llaMtuyptahgimenuicriiutymTaenstdinEgsicnhetrhiechia coll Plate Incorporation Assay DuPontHLO-1997-00035 after incubation, the Sponsor will be consulted prior tu selection of dose levels lo r' the mutaecnicitv assav. In selecting dose levels for the mutagenicity assay ^ e following'uuideiines will be employed. Whenever possible, the highest dose lorihe ; muiagenicftv assav will be selected to give some indication or toxicity vvirtiput exceedine 5 mg plate. For freely soluble, nontoxic test articles, the highest dose lve will be s' mg'plate. For precipitating, nontoxic test articles, the highest dose level will be selected in an attempt to yield precipitate at only the top one or two dose . levels The precipitate will be evaluated after the incubation period by visual examination without magnification. Doses will be selected such that precipitate does not interfere with scoring. '. . ; > :, ;V; 7.3 Frequency and Route of Administration :' The test svstem will be exposed to the test article via the plate incorporation. methodology originally described by Ames el ul. (1975) and updated by Maron and Ames (1983). This methodology has been shown to; detect a wide range o f classes of chemical mutagens (McCann el a i . 1975; McCann and Ames. 1976). Awfitlel rbetheredpaetaalegde.neTrahteeddoinsethleevfeirlsstuassesdayinhathveesbceceonnedvaaslsuaaytevdv, itlhl ebemthuetasgaemnieciatysathsossaey ubseecdhianntgheed fidrsute assay unless the Study Director determines that to such parameters as exccessive cytotoxicity the * or pi. range should ipuate. : r ;, . ; :; ^ :; 7.4 Controls ;\ v 7.4.1 Positive Controls , .7 All combinations of positive controls and tester strains plated concurrently with the assay are listed below: Positive Controls : -- Strain ActiSv9ation Positive Control Co(nce'pnltarateti)on ; TA98. TA too. TAI53S TA97A U p: mrA (pKMIOl) TA98 TA100. TAI35 TATA WP: mrA (pKMIOl) * '. - 2*aimnoanthracene :-nitrol1uorene sodium azide 9'iminoacridine methyl meihanesulfonate . 1.0 j 20 to io 10 7} 1.000 7.4 1 Secalivc Controls i \ppropriatc negative controls will be plated for each tester strain with Profumi and without SI'CTMIMI I 11'04 'IIS S9 activation. 4 The of 1(1 negative control :will MAheSICSthORe COveIBhAiIcOTleLEOS,GIINCCA.L MA Study No. C96CF43.502011 48 - Company Sanitized. Does not contain TSCA CBI ^ Htfirtr aii 3 1 SHa-l2m20o5n1e:llaMtuyptahgimenuicriiutymTaesntdinEgsicnhetrhiechia coll Plate Incorporat.ion Assay DuPont HLO-1997-00035 alone, unless .here is no historical basis for use of the selected vehicle. In the latter case, both untreated and vehicle controls wil. be used. 7.4..' Sterility Controls The most concentrated test article dilution and the Sham and S`) mixes will be checked for sterility. Exogenous Metabolic Activation Aroclor P54-induced rat liver S9 will be used as the metabolic aclivat.onsystenv T S O homogenate will be P arcel a single prepared ind st'ored frozen at approximately -70C S . u" d will be assayed for its ability to metabo., 2 -aminoanthracene and 7 .1 2 -dimethylbcnzanthracene to forms mutagen.c o . lyphimuriiim TAIOO. . , IIC1. <he S9 will be thawed and mixed w-ith a cofactor pool to adenine dinucleotide phosphate. 8 mM MgCls a" d " KCI "J ^ phosphate buffer at pH 7.4. This mixture ts referred to as S9 mix. Sham m.x v .it he 100 mM ohosphate buffer at pH 7.4. 6 Preparation of Tester Strain Ouvvcerrnniifgchmt ccuulutuuries will be inoculated fromcultthuereaspaprreophrairavteestmedasitnerlaptleatleogorphfraosme, tthhee of harvest. . All cultures will be harvested by spectrophotomclric monitoring of culture turbidity ol approximately 10 cells/ml. . 7 l est System Identification .. i. i .. ,.,ti hr libeled with a code S'stem that identifies the test article, test * . . . . , . ____On.*rntmit Proc*ed*ures.* |*rtno*l Sl'iiTSO^OII H 5of Ml y MICROBIOLOGICAL ASSOCIATES, INC. -1 1 j i I i j Company Sanitized. Does not contain TSCA CBI 7.8 Test Anide Preparation Unless specillai otherwise, test anide dilutions will be prepared immediately prior to use. All test article dosine will he at room temperature under yellow liuht. 7.9 Treatment of Test System One half milliliter (0 5 ml) of S9 mix or Sham mix. 100 pi of tester strain and 50 pi ol vehicle, test article dilution or positive control will be added to 2.0 ml of molten selective lop agar at A52C. When necessary to achieve the target concentration or eliminate toxic vehicle effects, aliquots of other than 50 pi of test anide/vehicle/positive control will be plated. The mixture will be vortex mixed and ,hc surf"ce r 25 ml o f minimal bottom agar. After the overlay has solidified, the plates will be inverted and incubated for approximately 48 to 72 hours al.!>.7.^C' Pla,cs ,hat 3:6 no1 coun,ed immediately following the incubation period will be stored at 42C. 7.10 Colony Counting The condition of the bacterial background lawn will be evaluated lor evidence of test article toxicity and precipitate. Evidence of toxicity will be scored relative to the negative control plate and recorded along with the revcriant count for that plate. 7.11 Tester Strain Verification On the day of use in the mutagenicity assay, all S. n p h im u m m , tester strain cultures will be checked for the following genetic markers: The presence o f the rfu wall mutation will be confirmed for all tester strains bv demonstrating sensitivity to crystal violet. The presence o f the mrB mutation will be confirmed for tester strains TA98. TAIOO. TAI535 and TA97A by demonstrating sensitivity to ultraviolet light. The presence of the pKMIOl plasmid will be confirmed for tester strains TA98. TAI00 and TA97A bv demonstrating resistance to ampictllin. On the day of use in the mutagenicity assay, the E coli tester strain cultures will be checked lor the presence of the rnrA mutation by demonstrating sensitivity to ultraviolet light. The presence of the pKMIOl plasmid will be confirmed for tester strain WP2 inrA (pKMIOl) by demonstrating resistance to ampicillin. s o criteria for determination of a valid test I he following criteria must he met for the mutagenicity assay to be considered valid: I'rniiici.isi'C fsiirnii MA Sludy No. G96CF43.502011 sotto 6of, -50- M ICROBIOLOGICAL > ASSOCIATES. INC. Company Sanitized. Does not contain TSCA CBI inm m m SHa-l2m20o5n1e:llaMtuytpahgiemnuicriituymTeasntdinEgsicnhethriechia ' " Piate Incorporation Assay________DuPont HLO-1997-00035 8.1 Tester Strain Integrity To demonstrate the presence ol the r/n mutation, all S. lyphim uriunt tester strain cultures must exhibit sensitivity to crystal violct. To demonstrate the presence of the rrvrB mutation, all .S' ryphinuirium tester strain cultures must exhibit sensitivity to ultraviolet light. To demonstrate the presence ot the irvrA mutation, nil E. coli tester strain cultures must exhibit sensitivity to ultraviolet light. To demonstrate the presence o f the pKMIOl plasmid R-factor. tester strain cultures o f TA98. TAIOO. TA97A and WP2 nvrA (pKMIOl) must exhibit resistance to ampicillin. 8.2 Spontaneous Revertant Background Frequency- Based on historical control data, all tester strain cultures must exhibit characteristic number of spontaneous revertants per plate in the negative controls (vehicle). Thc mean revertants per plate must be within the following ranges (inclusive): TA98, 10 - 50; TAIOO, 80 - 240; TAI535.5 - 45; TA97A. 80 - 240; WP2 rA (pKMIOl). 150 . 380. 8.3 Tester Strain Titers To ensure that appropriate numbers o f bacteria are plated, all tester strain culture liters must be equal to or greater than 0.3x10* cells per milliliter. 8.4 Fositive Control Values Each mean positive control value must exhibit at least a three fold increase over the respective mean negative control value (vehicle) for each tester strain. 8.5 Toxicity A minimum of three non-toxic dose levels will be required to evaluate assay data. A dose level is considered toxic if it causes a >50% reduction in the mean number of revertants per plate relative to the mean negative control value (this reduction must be accompanied by an abrupt dose-dependent drop in the revertant count) or a reduction in the background lawn. In the event that fewer than three non-toxic dose levels are achieved, the affected portion of the assay will be repeated with an appropriate change in dose levels. 9.0 EVALUATION OF TEST RESULTS For a test article to be evaluated positive, it must cause a dose-related increase in the mean creovnecretnatnrtastiopnesr opflatteest oafrtiactleleaassst poenciefietdesbteerloswtr:ain over a minimum o f two increasing Protocol SPGT50IOII 11/04 6 * ot 10 TAMSICSOROCBIAIOTLEOS,GIINCCA.L h MA Study,No. G96CF43.502011 - 51 - Company Sanitized. Does not contain TSCA CBI SHa-l2m2o05n1e:llaMtuytpahgiemnuicriituymTeasntdinEgsicnhetrhiechia coli Plate Incorporation Assa.y DuPont HLO-1997-00035 0.1 Strain TA1535 Data sets will be judged positive if the increase in mean revertants at the peak o f the dose response is equal to or greater than three times the mean negative control value (vehicle). 9.2 Strains TA9S. TAIOC. TA97A and WP2 mrA (pKM101) Datasets will bejudged positive i f the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean negative control value (vehicle). In consultation with the Sponsor, negative results may be confirmed as needed and equivocal results may be clarified by further testing using modified experimental conditions. 10.0 REPORT A report o f the results o f this study will be prepared by the Testing Laboratory and. will accurately describe all methods used for generation and analysis of the data. The report will include: Test substance: identification and CAS no., i f known; physical nature and purity, if known; physicochemical properties relevant to the conduct of the study, i f known; stability of test article, if known. Solvent/Vehide: justification for choice of vehicle; solubility and stability of test article in solvent/vchicle. if known. Strains: strains used; number o f cells/ml per culture; strain characteristics. Test conditions: amount of test substance per plate with rationale for dose selection and number of plates per concentration: media used: type and composition o f metabolic activation system, including acceptability criteria; treatment procedures. Results: signs of toxicity; signs of precipitation; individual plate counts, the mean number of revertant colonies per plate and standard deviation; dose-response relationship, where possible; statistical analysis, if any: concurrent negative and positive control data means and standard deviations: historical negative and positive control data with ranges, means and standard deviation. Discussion of results Conclusion l,riilM nlSI,C.TiO:OII 11'lit'1ft MA Study No. G96CF43.502011 8 nr It) ^XM' IACSRSOOBCIIOATLEOSG. IICNAC.L - 52. - Company Sanitized. Does not contain TSCA CBI SHa-l2m2o05n1e:llaMtuytpahgiemnuicriiutymTaenstdinEgscinhetrhiechia coli Plate Incorporation Assay DuPont HLO-1997-00035 11.0 RECORDS AND ARCHIVES Upon completion of the final report, all raw data and reports will be maintained by the Quality Assurance Unit of Microbiological Associates. Rockville. MD in accordance with the relevant Good Laboratory Practices Regulations. 110 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE This protocol has been written to comply with OECD Guidelines 471 and 472 (Genetic Toxicology: Bacterial Reverse Mutation Assay). Revised Draft Document. September 1995 and with the International Conference on Harmonisation o f Technical Requirements for Registration of Pharmaceuticals for Human Use. Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. Step 4 Final Draft Julv 18 1995. ' This study will be performed in compliance with the provisions of the Good Laboratory Practice Regulations for Nonclinical Laboratory Studies. Will this study be submined to a regulatory agency? ves If so. to which agency or agencies? EPA/TSCA. OECD: MAFF Unless arrangements are made to the contrary, unused dosing solutions will be disposed o f following administration to the test system and all residual test article will be disposed o f following finalization of the report. 13.0 REFERENCES Ames. B.N., McCann. J. and Yamasaki. E. (1975). Methods for detecting carcinogens and mutagens with the iW/nonW/a/mammaliammicrosome mutagenicity lest. Mutation Research J/:347-364. EGsrceheenr.icMhi.aHc.Lo.li.. and Muriel. '.V.J. (1975). Mutation Research Jtf:3-32. Mutagen testing using trp" reversion in International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. Step 4 Final Draft. July 18. 1995. Levin. D O.. Yamasaki. E. and Ames. B.N. ( 1982). n new Salmonella tester strain. TA97. for the detection of frameshift mutagens. A run of cytosines as a mutational hot-spot. Mutation Research 94:315-330. McCann. J. and Ames. B.N. (1976). Detection of carcinogens as mutagens in the Salmonella'micTosomt test: assay of 300 chemicals: discussion. Proc. Natl. Acad Sci USA 73:950-954 Pminroispmsnrnii irtM'ift MA Study No. G96CF43 502011 9 of in C^ AMSISCORCOIBAIOTLEOS,GIINCCA.L - 53 - Company Sanitized. Does not contain TSCA CBI SHa-l2m2o05n1e:llaMtuytpahgiemniucriituym. TaenstdinEgscinhetrhiechia coll Plate Incorporation Assay DuPont HLO-1997-00035 McCann. J.. Choi. E.. Yamasaki. E. and Ames. B.N. (1975). Detection o f carcinogens as mutagens in the Salnionella'm'KTOsomc test: assay of 500 chemicals. Proc. Natl. Acad. Sci. USA 72:5135-5139. ' Maron. D.M. and Ames. B.N. (1983). Revised Methods lor the Salm onella Mutagenicity Test. Mutation Research 113:173-215. OECD Guidelines 471 and 472 (Genetic Toxicology: Bacterial Reverse Mutation Assay). Revised Draft Document. September 1995. Wilcox. P.. Naidoo. A.. Wedd. DJ. and Gatehouse. D.G. (1990). Comparison of Salm onella typhimurium TAI02 with Escherichia coli WP2 tester strains. Mutagenesis 5:285-29). ' 14.0 APPROVAL (Print or Type Name) STUDY DIRECTOR nln/ffa DATE T ro ttim i S r C T 5 ( i: m i 11/OJ'06 MA Study No. G96CF43.5020I I 10of 10 -54 - ">T AMSISCORCOBIAIOTLEOS,GIINCCA.L Company Sanitized. Does not contain TSCA CBI
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