Document DGeGJ6KjLz1OQMZVVQJk63OMB

AR226-3101 ]- Wit/VFJ Huntingdon i= CONFIDENTIAL DPT434/984093 BACTERIAL M UTATION ASSAY Sponsor DuPont Specialty Chemicals, JCahcakmsobnerLsaWboorraktos,ry, NDUSeJeA0p8w02at3e,r, Research Laboratory Huntingdon Life Sciences Ltd., ESuyfef,olk, IEPN2G3 L7APXN,D Final report issued 1 October 1998 _ y Company Sanitized. Does not contain TSCA CB1 CONTENTS Page COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS ............................... 3 QUALITY ASSURANCE STATEMENT.......................................................................................... 4 SUMMARY ........................;....................... ......................................................................................... 5 INTRODUCTION ............................................................................................................................... 6 TEST SUBSTANCE...........................................................................................!............................... 8 EXPERIMENTAL PROCEDURE...................................................................................................... 9 ASSESSMENT OF RESULTS........................................................................................................... 13 MAINTENANCE OF RECORDS...................................................................................................... 13 RESULTS .............................................................................................................................................. 14 CONCLUSION............................... 14 REFERENCES ........................ 15 TABLES 21.. RReessuullttss oobbttaaiinneedd wwiitthh SS.. ttyypphhiimmuurriiuumm TTAA91800foflollolwowinigngexexppoosusureretc lH H jjH H K ............ 1168 453... RRReeesssuuullltttsss ooobbbtttaaaiiinnneeeddd wwwiiittthhh SSE... ttcyyopplhhiiiCmmMuurr8iiuu91mmfTToAAllo11w5533in57gffeooxllllpooowwsiiunnrggeeexxppoossuurree 222420 APPENDIX 1. Histo.4rical control data................................................................................................................... 26 Company Sanitised. Does not contain TSCA Ct COMPLIANCE W ITH GOOD LABORATORY PRACTICE STANDARDS TPrhaectsitcuedsytadnedsacrrdibse, dwiinthththise reexpcoerpttwioanssctaotneddubcetleodwi,nacnodmI pcloiannscideewr tihthe tdhaetafoglelonweriantgedGtooobde Lvaalbido.ratory The United Kingdom Good Laboratory Practice Regulations 1997 (Statutory Instrument No .654). EC Council Directive 87/18/EEC of 18 December 1986 (Official Journal No L 15/29). OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17. In line with normal practice in this type of short-term study, the protocol did not require analysis of the dose form. KHSteuunndntyienDdgdiroenct,,Lo.ri,fe . Sciences . Ltd. \ Date Company Sanitized. Doss not contain TSCA CBI QUALITY ASSURANCE STATEMENT The following have been inspected or audited in relation to this study: Study Phases Inspected Protocol ' FAPTPolrruaeordtmaceitetmuSslsceaontBirtoiannsged Inspections R epo rt Date ofInspection 3 August 1998 681154AJaJApnupruliyrlailr11y19999919889898 2 September 1998 Date ofReporting 3 August 1998 681154AJaAJpnupruliyrlairl11y91999918989898 2 September 1998 Protocol: An audit of the protocol for this study was conducted and reported to the Study Director and Company Management as indicated above. Process based inspections: At or about the time this study was in progress inspections and audits of rcoountdinuectaenddarnedpreetpitoivretepdrtooceadpuprreospreimatpeloCyoemdpoannythMis atynpaegeomf estnutdays winedriecactaerdriaebdoovuet.. These were RcoenpdourctteAduadnidt:reTphoisrtreedptoortthheasSbtuedeynDauirdeictetodrbayntdhCe oQmupaalintyyAMsasnuaragnecmeeDnteapsaritnmdeicnatt.edThabisoavued. it was rTehseulmtsettohoredfsl,epcrtothceedruawresdaantad. observations were found to be accurately described and the reported DHAGueuGpndatoiitrndotmgdr,daerodnnt LofifQe uSacliietyncAesssLurtdan. ce, Date Company Sanitized. Does not contain TSCA CBP *SUMMARY. . . . . . . In this in vitro assessment ofthe mutagenic potential ofZonyl FS-62, histidine dependent auxotrophic mdsueubptesatnnadntsecneotdfmiSluuattleamdnotinnoefplluEarsitcfyihepedhriwimchautireairu,cmwo,lhi,sictsrhatrwianiasnsTaCAlMs1o58u39s5Ie,d(TWaAsPa125nu3ev7gr,AatT/ipvAKe9cM8on1at0nro1dl).,TwAe1r0e0e,xapnodsead ttroyptthoephteasnt TAinwvroooclvlionerdde1ap2ep5nr4ed--eiinnntdcmuucbueatdtai-otrinaotnsstta(eSgse9ts. mwiexr)e. pHerfieormfiresdt winatsh-.eapsrteasnednacredapnldataebsinecnocrepoofraltiivoenr pasresapyar,atthioenssefcroonmd cCoonncceennttrraattiioonnsreocofmump etond5ed00in0 tphge/prelagtuelawtoerryegtuesidteedlinienstthheismausstaatyiofnoltleoswtss.. TOhthiseriscodniceesnttarantdioanrds ulismedit wtoewraerad-ssethrieesteostfecrasthraalifn-sloignioeidthileurtimonustaotifotnhetehsti.ghest concentration. No signs of toxicity were observed Ntesot.evidence of mutagenic activity was seen at any concentration ol either mutation Tofhitehceolnivceurrprerenptaproastiiotivnes.controls demonstrated the sensitivity of the assay and the metabolising activity aItctiisvictyonincltuhdiesdbathctaetr,iawl hsyenstetmes.ted in purified water, hows no evidence of mutagenic Company Sanitized. Does not contain TSCA CBI INTRODUCTION TsyTsitiesmre.poTrhtedesstucdriybewsaas sctounddyudcetesdiginnecdotmo palsisaenscsetwheithmtuhteagfoenlliocwpiontgengtuiaild elin e s^ ^ ^ ^ ^ a bacterial BOaEcCteDrialGRueidveelrisneesMfuotartioTnesTtiensgt. of Chemicals. (1997) No. 471: Genetic Toxicology: TOEoE.JxC.icNiAtoy.n,LnBe3.x1833to. AOD,ti1hr5ee7crt.eivffeec9ts2/-6M9/EuEtaCge.n(ic1i9ty9:2E) sPcharetricBhia: Mtoeltih-oRdsevfeorrseDMetuetramtiionnatAiosnsaovf ATEoEssxCaicyiA.tynO,nB.eJ.x1N4to.o.ODLtih3ree8cr3teivAffee,c19t6s20-/6.M9/EutEagCe.ni(c1it9y9:2S)alPmaortneBlla:tyMpheitmhoudrsiumfor- RDeevteeirmseinMafuiotantioonf US EPA 40 CFR Part 799 (1997) Toxic Substances Control Act Test Guidelines NSuob- -1se5c8t.ion 799.9510, .TSCA bacterial reverse mutation test;. F,ede..r.a.l Register' Vol` 62' The method described was also designed to comply with ICH (1996 & 1997), and followed the recommendations ofthe United Kingdom Environmental Mutagen Society (Gatehouse e/ al 1990). sMTpheaecroiiannllayvnistderolAectmetecedhsns1tiqr9au8ien3s)doeesnfcaSrbaibllemesdothnbeeylmlAaumttyaepgsheianmnicdurehifufisemccttooo-wfthaoertkteeessrttss,suu(bAbssmttaaennscc,eeM.toNcCboearnmdneatlaelnyrmdSi.Ynteaydmphbaiysmaekuxirpiuo1m9si7n5igs maoicnsraecipgrdaeaiipnuvbamaelbelrlth(eoasinfusottpssifydp, nitlanhetnmheidseiesnafniurdsteeinendpcrgeetwinaotdhdintieeh.lnyebtsdWifoseoetthrinemnect,nitaestlaidtnhkacbeemeysseitnpthholseaetsrcaieareciasinditbnsr,ialahiiantirypssetritaodoerpixengoperaroo,tlisswobeonudaitnonttcodfhatepfhaoaemrbmmplueoutcapotaonufgltlobeasninttoiir,oetainsrnie.novssnTyeurnhassteehehsdeeimsstiiaisnudr)te.iatnhtreiieosfdneteerftrsioectdieathntroeet mrAequteutacirnhetndisqftouraerignbrsaoswoedtfhoE.nscshimeriilcahriparincocilpi lewshhicahs aalrseo ibneceanpdabeslecriobfedsybnythGesreiseinng(1t9h8e4)a.mTinhoisascyisdtemtryepmtopplhoayns psSiTneo.htnsotesysiepttshsihtvseriimataciynuetplsrolli.uaussmosAmemldsildetrtcmha(aiepnurKrtssaytMgrhaeaaiInvdnOseds.li)ataiorwdenehadfileeccfhmtiicviuineettnarcttoieodilnlnusccneoowsarmthaiwnaclhheDicrrrhNeonArad-lpleorrreowptnhsaeeigrmrrepepramoateciorrerseppseresorsemc.neessIaisntbi,vialreidetydstuoitolitoifmnntgeutsthaitnrgeseeuinnbsocs.rfteatanhTsceehemdse Company Sanitized. Does not contain TSCA CBf nMaboastenanyvcaseiulaabnbsdtlaenpicnreessthedneocbenaoocttfeearixaselurptcpealllem.muTetanhgteeerdnefilcoivreeefrftehfcreatcbutainoctintlert(ihSae9yanmhdaixvte)esptbreseeupnbasrmetadentcfareboomalrisereadintsbcuypbreeanvtezidoyumisnelybsoytrtsehtaettmehdes with a substance (Aroclor 1254) known to induce a high level of enzymic activity. * The protocol was approved by Huntingdon Life Sciences Management on 7 July 1998, the Sponsor on 17 July 1998 and by the Study Director on 31 July 1998. The experimental phase ofdie study was conducted between 3 and 17 August 1998. Company Sanitized. Does not contain TSCA CBI Identity: Chemical name: i Appearance: Storage conditions: Lot number: Expiry date: Purity: Date received: TEST SUBSTANCE Room temperature 2 Years from date ofreceipt 23 June 1998 Company Sanitized. Does not contain TSCA CBf EXPERIMENTAL PROCEDURE BACTERIAL STRAINS The following strains were used: :S typhimurium ................. ..... TA . 1535: tpcDhoaNenirtAcaseiulnrlbesswpataiatiulhrltissi(yotrisnfdatsein,mmebuum(ttaaviitssirsoBnenno))st.aesneImdnt suhiistataisvrtieeaovntedore(tfhferediacsmGtbivye4es6mhl)iipfabtnoumyptouailtsgyaesgaanelcstnsocsh.cdaaeruifdsicieniegcnotbaaitnsoena S. typhimurium TA100: JapsmKthpMeicIisOlalimln).ereaIssnisTtaaAdndc1ie5ti3oa5nndbtouintbccaroesneat-sapiniangisrsaseunrbessisttiiisvttuiattnyioctenost,sraointmsiesfemarlufsaotacgtaoebrnlescot(onpfladesrermtienicdgt certain frameshift mutagens. .. S. typhimurium TA1537: ddsbeeeenflaeesrtcisitotiivanveeohtfiionsatisadaiginneDgenlNetsfrbAaacmsareuee-ssppihnaaiiigfrrt. fsmryasumtteaemtsiohniafnt(d/mnsluiCptao3tpi0oo7nl6ys)s.aincvcLohilakvreiindTgeAcino5sae3tr.5ti,onIitt oiissr S. typhimurium TA98: tacdcwlooekfnnoyettcalaaatiidinntvijssnaegcathenaDenogtNptehbKnAeatMrsshecr1-iaesp0upt1aisadiiripinrsnlgae(ssdbfmyorasausitdmeeb.m-lepesIahtfiairiianfssmtdurmebevssulhitetpiaritfotutetiptodimoonblnuy(ys/ts.iaaawtcgioDcehnn3ast0)sr,i5dc2bea)uu. tsciAnonoaggttadibnebylueittstiihomaanlpssoloaef E(W. cPo2lui vCrMA/8p9KIM: 101) contains an ochre mutation. It is reverted by many agents causing A-T ibnastrea-npsafierrsRubNsAtituloticoineslsaetwthheerterpinEthloeccuhsroomr boysoGm-eC. bItasise-aplsaoirdsuefbicstiietnuttiionnas mDNutAagernespatihransysittsempa(ruenvtrAs)t,raminidWiPs 2m. orIet arelsaodilcyonrteavienrstetdhebypKcMertIaOinl plasmid. The strains of S. typhimurium were obtained from Professor B.N. Ames, University of California, Berkeley, California, USA. The strain of E. co'li was obtained from the National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland. Batches of the strains were obtained from master stocks held in liquid nitrogen. The test batches were ptaaoplliapqstluhmicoeiatdsbcluwoelf,htunifcruoehtrsrciceaoenntltlf8ebmr%rsoetmrhvebs/cvirusatlnaatuesnrcepaesetrcmomryiaedomawpbprieeliircsteyeilrlsiv(tnora.frtaievdTem,h.auet t-aEr8eta0isocpnhCo)n.,bssaeDetscinmhosieftoittvhhfiyetlyfrssottuzrolaepinUnhsoVsxttriodaliieganh(sDtweaMrainesdSsOttoeh)sfetwedpdiaaK,sgMwnadohIdseOteridcel mutagens was also assessed. For use in tests an aliquot of frozen culture was added to 25 ml of nutrient broth (Merck No. 2) and incubated, with shakings at 37C for 10 hours. These cultures provided at least 109 cells per ml which were measured by spreading aliquots (0.1 ml) of a 1O'6 dilution of the overnight cultures on the surface of plates of nutrient agar. Company Sanitized. Does not contain TSCA fRl POSITIVE CONTROLS In the absence of S9 mix Identity: Supplier: PLuortintyu:mber: Appearance: Solvent: Concentration: N2S0i-gFEm-t0ha2yC3l-5hNe-mniitcraol-Atnitrosoguanidme (ENNG) >97% Pale yellow crystalline powder 5DMpgS/pOla(tAe fldorricshtr,aAin.CT.AS.1-s5p3e5ctrophotometric grade, >99.9% pure) 3 pg/plate for strain TA100 2 pg/plate for strain CM891 PBISduuaerptnietpthyilt:inyeur::mber: ' Appearance: Solvent: Concentration: Identity: PBSuuarpticpthyli:neur:mber: . ASoplpveeanrta: nce: Concentration: 9S9I99-igA8%mCm-aQinC3o5ha8ecmridicinale Yellow powder DMSO (Aldrich, A.C.S. spectrophotometric grade, >99.9% pure) 30 pg/plate for strain TA1537 , , 2-Nitrofiuorene 90A81l%2d8ri6c7h Chemical Company Beige powder DI MpgS/pOla(tAe fldorricshtr,aAin.CT.AS.98spectrophotometric grade, >99.9% pure) In th presence of S9 mix SIduepnptiltiyer:PBuarticthy:number: Appearance: Solvent: Concentration: A59226-lA2%d3rmi4ci-hn0oC2a4hnethmraiccaelnCe ompany . Green powder 2D10MppgSg/pO/plal(atAetelfdofroricrshtsr,tarAiani.nCT.CASM.1s58p39e51ctrophotom*etric grade, >99.9% pure) Identity; PBSuuarpticpthyli:enru-m ber ASoplpveenart-ance: Concentration: 90AB87el%7dn7rzi8co-hj1aC0jp5hyermeniecal Co' mp.a..n..y................. Yellow powder 5DMpgS/pOla(tAe lfdorricshtr,aAin.sCT.SA. 1sp5e3c7t,roTpAh9o8toamnedtrTicAgIr0a0de, >99.9% pure) Company Sanitized. Does not contain TSCA CEI PREPARATION OF S9 FRACTION SSepxe:cies: SSotruarince: : AWgeei:ght: RMaatle HSparralagnueO-DlaacwLltedy derived 7-8 weeks <300 g lSiv9efrrsawcteioren swtimasuplaretepdarbeydAfrrocmeloar g1r2o5u4p, aodfmciani1st0eraendimasaals.sinMgliexeindtrfau-npcetriiotonnoexalidinasjeecstiyosnteimn sAirnacthhies roailt at a dosage of 500 mg/kg- bodyweight. On the fifth day after injection, following an overnight starvation, the nits wiere lolled and their livers aseptically removed. The following steps were carried out at Q-4C under aseptic conditions. The livers were placed in pd(o0Sr.if1me95pSefarMt9rhaacytKftiliorboCanenc1n,t)zi(tow3ahnneamtshwhlrd;aoaiKcsmspeConet!ngee:sesa1etnnedagddtien2lii-tvwnoaemara)sailnibqcoebueafaonnoctttrsrfteeiafarrbunaiaegcdlieensdngmteotarburetaetdanf9tosi0aorf0teen0-ru8res0ageds.sCtfaoToyurhanenw1tis0Ulittrelhmetrrqialiutin-htiTuyreetuwedrsmra..asuTxatThhalsgeohoemeencfoihfsciguececpapnkecrierysendeca.orut.farsneFoatorcfslhrloab7wcat,1iitonc2ghn- Date ofpreparation:'9 June 1998 PREPARATION OF S9 MIX S9 mix contained: S9 fraction (10% v/v), MgCl2(8 mM), KC1 (33 mM), sodium orthophosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filter-sterilised before use. SELECTION OF SOLVENT AND FORMULATION OF TEST SUBSTANCE Tfohlleowsoilnugbiwliatyrmoifntghetote3s5t-4su0bCstawncitehwgaesntalesssetsirsreidnga.t T50hemregf/omrel pinurpifuireidfiewdawterat(eorb, tianinwedhibchy itthedirsesovlevresde osmosis of tap water) was used as the solvent forthis study. All concentrations cited in thisj-eport are expressed in terms of p u r e ^ ||0 M f l i.e . correction was made for the purity o ^ P J jt otcontain TSCACBl MUTATION TEST PROCEDURE First test The test substance was added to cultures of the five tester strains at seven concentrations separated by ca half-logio intervals. The highest concentration o f j m m P e s t e d was 50 mg/ml in the chosen solvent, which provided a final concentration o f5000 pg/plate. This is the standard limit concentration rseoclvoemnmt, epnudrif!ieidnwthaeterr.egTuhlaetoapryprgoupirdiaeltienpesostihtiivseacsosnatyrofloslwloewres.alsTohiencnluedgeadti.ve control was the chosen Ai(mpnHmael7idq.4iua)toewtlyoefrbOey.pl2lammclelodofifnm1g0olalhtsesonuturabgbeaascr.:tceorAinantlacianuliilnqtuguroe0t.a5onmfdM00.1.5himmstllidoSin9f etmh/beiixotteoinsrt/0trs.yo5plumtotilpo0hn.a1nw.MasHpahireiortsmrpajhixatftuoerlbelouwwffeaedsr ttEdhhiaseochrhoaVducpdgmeihttrirloiyitnedshihotssahf.kbewTnaahcsarteenierndidapoievivtnirediruoldaariidsldlheyeroslntaowtboeealrpsleesredeuvsswsieodituthhfseolyarseptUearrcneihiplqiatucyreoendoccofpedtneehtterrciaottdieroirssneths.epssuoPbnlcasdottienansntgacwientoe,inreSag9as2lhs5moeeimxpt,rleaimpdnaedinrnetipimdHfyawnilncipgtahhgotaahturPe.t cbbopalfcoikfnegyfr.ocuoAnudlnltbepral.acttee,sriwalerlae.winncwubaasteedxaamt i3n7edCanfodr rceave7r2tahhtoucorslo. iuAesftecrouthnitsedpeursiiondgthaeDapmpeinaoranaucetomofattehde Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony mtctcohooixennuicintcmoetsenpuftmofrcearoctoibntosfcyntewhtnhwreteerraeaesatniboocosbhnenson-enstcrooevenxremo,idcfsaaicalgloynicnosocuwmesoenefprdtrlbecaiatotneicnottbcenhearseincasktslhregaoicrtnuiooohlundninbdbdimtteieobasonyatbcwbtataeeroireniucaehpllddor.lesabseweennn.tth, eIa'titnssathhtmhoeeuetloadabpsbstecehnoaecnntecsueuosnrefterdadantiitynohnattoh.txeiifIcfdaieresalftolf.lewyceItasrf Second test tAhse aseccloenadr.neTghaetivvearrieastpioonnsuesewdaws aosbttahienepdrei-nintchuebfairtisotnteasst,saayvianriwathioicnhttohediteubteesst wpreorceeidnucruebwateads uaste3d7foCr tfhoer t3o0pmcoinnucteenstrwatitihons,hbakuitnognlbye.ffoivree tchoencaedndtirtiaotinonosfwtheereaguasredo.verlay. 5000 pg/plate was again chosen as STABILITY AND FORMULATION ANALYSIS ' sdTteuhtdeeryms.tianbeidlitays opfartthoefttehsits sstuubdsyta.nAcenaalnydsisthoef asctahbieilvietyd coofntcheenttreastitonsuwbsatsanncoet pinerftohremesodlvaesnptawrteoref tnhoist Company Sanitized. Doss not contain TSCA CBI ASSESSMENT OF RESULTS Fstorarina stheostultdo lbieeincodniseidraenregde'vstaaltieddthinethmeeaapnporofptrhieatesoSlvtaenndt acrodnOtroplerraetvinegrtaPnrot cceodlounrey. nAulmsob,etrhsefpoorseitaicvhe ccoonnttrrooll.compounds, must cause at least a doubling of mean rev!e-r.ta^nt colo..ny n.umbers over the negative tfThohelleoswmolievnaegnnctnrucitomenrbitareo:rlogfrroeuvpesr.tanTthceolmonuiteasgfeonricalalcttrievaittymeonftagrteosutpssuwbsetraenccoemwpaasreadssweistshedthobsyeaopbptalyininegd tfhoer a) tscpIweofeprnitfcrasoeeirrdaamttethmereeedeed.cnxotptoneWcrsiuihmtrhorewenanttset&,vsowidtlivetsnheucnbeatsnctoyaofnnbcmtaerouclpttsear,rgoiewadnluiitcschetrsaascoiatnminveeiitinytehcTverinerdaeitsnnhecitsehinetoerfspetarvesespyreostnatsecnimtetiv.oceorlNdoaobnossyees-ntnraceutelimasttobiiocfenarSsls9haoinpmfa,ailxyitn,slii^tstwaiisosst b) mcIfountrtcaeugartemrneinecntatcswotilvivthietynatintectsohtnissturotbelsssttasinnycseteeidmtoh.eesrNnmootusttpaarttoiisodtnuiccatelesraetnpairltoydsiusiscicibsolpneesiirndfocerrremeadesdets.oosfhaotwleansot 1e.v5idtiemnecse thoef c) tmsIihnufeubtpnhtsaaetgrbaaernegencsroieaucb'plsasthsecmsrtoivuavbe)titdatayaginwneidennhdiibtcchf)haoi,scialsttdiaetvdotsiiitsttsyifsoaiyeintsnisastefltpmhytaeis.trshattiNegenrscogatrpsimsthtyeaasrty(tiieasambt)ifecot.ahprleSeaarhsfncouoalubrelmlysadtsreaiadn"snpciinsoeinspociisetrridrevfceaeoors"rnemtoosiienrdrdee"r.srneeoevdlgevaretttoaitvhnseeth"coisorwselouspneevoyoindnfseutehnmgeciebvteeeorsnstf dIfetneromcinleeatrhe"psotsaittiisvteic"alressipgonnifsiceancacne obfeaonbytaoibnseedr,vethdeintecrsetadseastainmraeyvebretanstubcojelcotneyd ntuomabnearlsy.sisThtoe astnaatilystsiicsaol fpvraorcieadnucreefsolulosewdedwbilyl Dbeunthnoetste's dteesstc.ribed by Mahon et al (1989) and will usually be MAINTENANCE OF RECORDS mAlalteerxiaplesr;imcoelnletcatlivdealytadeafriinseindgasfrtohme "mtheatesrtiuadlsy")(winiclllurdeimngaindothceumpreonptearrtyy orafwthedSatpao,nrseocro. rds and other tHohfeuinsrtseiuntuegrdnoo,fndthLiseipffeoisnSaacllieornerpcfeousrrtst.hheaArllfrtreeetrteansiunticothnhetoimmf eath,teetrhimealasStpeinroiniatslsso.arrcwIhfiilvlreebqefuoecrsotanedtpa,ecrHtieouddnatoinnfdgfditvhoeeniyrLeaaidfresviSfcrceoimesnoctuhegeshrtwlnoitlngl continue to retain the materials, subject to a reasonable fee being agreed with the Sponsor. Huntingdon Life Sciences shall also retain a copy of the final report in its archive indefinitely. Company Sanitized. Doas net contain TSCA CBf RESULTS THhiee mreesuanltsvaolbuteasinqeudoted have been corrected tpootshiteivneeacroensttrowlhcoolme npuomunbdesr.are presented in Tables 1 to 5. The absence ofcolonies on sterility check plates confirmed the absence o f microbial contamination. Ho fiitehteo-ctaulltcuorleosnoyfctohuenintsdoi-vnidnuuatlrioerngtaangisamr ps.lates (see ' Tables) confirmed the viability add high ' cell density .. nTacopuhnmpetrrboomeplrresicaawhnteeimtrhSeitvacaelanlrldstsaatnrr(adwtinciOtsoh,ploceSnoray9nt.fi,mincrgmoixuPinnrwgtoshcseeefrdnoeusrirtredieviqoeituryisqrooeudlfov)ttehenedintcdbucuyolctnGuetdrraeotseslshuanob.wdustseaaernceettiitvawalitiyltihn(oi1cnf9rde,9taih0ese)e.Ss9rAaimnnpgipxerre.sovpesrtriataattenedtpcoinosliotdinviyee FIRST TEST NtoefsoStes9rumsbtsirxtaai.nntsiafl.o..il.nl.ocwreiansgeesxipn- or.s.e.uv..r.ee.r..tt.a.o.n..t..Hc..o..l.o..Bn..y...n.luBmbe^rs aonv.y.ercocnocnetrnotlractoiounntisn weiethreerotbhteaipnreedsewnciteh oarnaybosefnthcee _Nsec^ovnisdibtelsetd. unninAg toopf thexepboascukrgerocounncdelnatwrantioonf noofn5-0re0v0eprtgan/ptlcaetellswwasasthoebrteafionreedsfeolellcotwedinfgorexupsoesuinrethtoe SECOND TEST tNoefsoStes9rumsbtsritxaai.nntsiafloilnlocwreiansgeesxipnorseuvreertant colony numbers overcocnocnetrnotlractoiounntisn weiethreerotbhteaipnreedsewncitehoarnaybosefnthcee No visible dunning of the background lawn of non-revertant cells was obtained following exposure to CONCLUSION It is concluded that, when tested in purified water, activity in this bacterial system. ;hows no evidence of mutagenic Rnmnanv Sanitized. Does not contain TSCA CBl REFERENCES AMES, B.N., McCANN, J. and YAMASAKI, E. (1975) Methods for detecting carcinogens and miitagftns with the Salmonella/mammalian microsome mutagenicity test. Mutation Res. 31, 347. GATEHOUSE, D.G., ROWLAND, I.R., WILCOX, P., CALLANDER, RD. and FORSTER R f(o1r99M0u)tBagaectneirciiat!ymTeustatitniogn. aRsesapyosrt.in:PKarIRt /KrLeAvisNeDd., DB.aJ.si(cEMd.u).tagUeKnEicMitySTSeusbts-c:oUmKmEitMteSe RoencoGmumideenlidneeds Procedures, p.13. Cambridge University Press, Cambridge. LGERGEAETNO, RM,.MH..L, N. (I1C9H84O)LMS,uWtag. eanndteRstAinMg EuLsi,nCg.ir(pE*dsr.e).veHrsaionnd.binooEksochfeMriucthaigae,cnaicliityinTKesItLPBrEocYed, uBr.eJs.., Second edition, p.161. Elsevier Science Publishers BV, Amsterdam. ICH (19^Gteffi)ldSd^^GuKlairce on Specific Aspects of Regulatory Genotoxicity Tests. ICH (1997) Genotoxicity: A Standard Battery of Genotoxicity Testing of Pharmaceuticals. aMUnKAdEHTMWOSNE,ASGuTb.SA-,c.DoTm..,Jm.G(iIRt9teE8eE9)NoAn, nMaGl.Hyusi.idLse.ol,infMedsIaDtafDofrLroEmMTOmutNiacg,roeBbn.ii,acliMtcyIoTlToCneHsytiEanLsgs.La,yRsIe.Dipno:ErKtG.IR.,PKRaLrOtABNIIINDI.S, ODStN.aJ.t,i(sWEtidc.D.a)..l Evaluation o fMutagenicity Test Data, p.26. Cambridge University Press, Cambridge. MARON, D.M. and AMES, B.N. (1983) Revised methods for the Salmonella mutagenicity test. Mutation Res.113, 173. Company Sanitized. Doss not contain TSCA CBi TABLE 1 Results.obtained with Salmonella typhimurium -TA98 following exposure to Addition (Mg) 1{None; sterility check |?000); [sterility check None; IQ-6dilution of overnight culture, plated on nutrient agar (total counts) Standard deviation S9 mix + present - absent; Revertant colony counts and means Fest I B Mean sd + 0 45 + 44 + + + 31 31 38 + + 32 41 0 37 35 37 34 38 27 32 0 44 43 42 36 31 34 48 -42 4 41 5 37 6 34 3 36 4 31 4 40 8 38 43 43 41 41 34 42 39 4 24 41 39 35 9 34 34 45 38 6 37 38 35 37 2 44 36 35 38 5 41 50 34 42 8 45 30 31 35 8 43 39 34 39 554 184 580 284 562 A.V 565 13 229 119 143 129 130 iZ Company Sanitized. Dess not contain TSCA CBl TABLE 1 - continued Results obtained with Salmonella typhimurium TA98 following-exposure:tq Plate Addition No. (l*g) 1 None; sterility check 2 ZonyId>FS*62(5000); sterility check 5000) 1500) 500) 150) 50) I t ^rifLsd wst0r (O:l ml) 15 Benzo[a]pyrene (5) 16 2-Nitrofluorene (1) None; 10 dilution of 17 oyernjj^c& iue, plated onnutrient.agar (total counts) sd Standard deviation S9m ix + present A -absent______ +0 Revertant colony counts and means Test 2 (with pre-incubation) B C Mean 00 0 sd 0 _0_ _0_ __0_ + 34 38 37 + 37 42 34 + 37 42 27 0 _0_ 36 2 38 4 35 8 + 39 + 37 + 36 42 20 37 42 39 36 41 2 .32 JO 36 J_ 35 38 29 34 5 28 41 38 36 7 32 37 37 35 3 30 44 37 37 7 35 37 39 __ 37_ _2_ 43 32 41 .....39 _6_ + 478 471 484 478 1_ 246 306 304 285 34 no116 112 113 Company Samiized. Boss not'contain TSCA CB1 TABLE 2 Results obtained with Salmonella tyghimurium TA100 following exposure to Plate Addition No. (fig) None; sterility check 5000); 18 Purifiedwaiter (0.1 ml) 19 Benzo[a]pyrene (5) 20 ENNG (3) None; 10 dilution of 21 overnight culture, plated on nutrient agar (total counts) sd Standard deviation S9 mix + present -absent Revertantcolonyeountsand means Test I B Mean sd + + + + + Iiwn0o 100 106 100 101 93 90 96 109 100 -0 H89- -9 7 11 94 95 5 85 96 11 102 104 5 103 101 2 + 86 87 + 110 94 92 102 100 114 103 101 96 88 94 9 97 8 102 99 14 99 100 86 95 8 106 92 90 11 104. 100 9 92 98 10 106 108 106 107 1 109 67 82 86 21 108 94 94 99 8 118 107 96 107 11 412 377 426 405 25 370 386 390 22 135 130 130 132 Company Sanitized. Does not certain TSCACBI TABLE 2 - continued Results obtained with Salmonella tyjMmurium TA100 following exposure to Plate Addition No. (gg) None; sterility check '5000); sterility check 5000) 1500) 500) 150) 10) 14 Purified water1(0:1 ml) 15 Benzo[a]pyrene (5) 16 ENNG (3) None; 10 dilution of 17 overnight culture, plated on nutrient agar (total counts) sd Standard deviation S9m ix + present -absent Revenant colony counts and means Test 2 (with pre-incubation) B Mean sd 00 + + + + + 99 97 99 87 85 94 407 400- 7 101 99 99 2 97 102 99 3 106 102 98 10 94 112 97 14 104 115 89 103 13 109 104 89 101 10 100 99 108 102 5 95 88 96 93 4 95 109 102 102 7 95 90 88 91 4 95 103 92 97 391 480 414 428 46 384 387 395 389 106 107 107 1G7 Company Sanitized. Dess not contain t sc a CBl TABLE3 Results obtained ynjh Salmonella typhimurium TA1535 following exposure to Addition (fig) 1 None; sterility check V H B f c 5000); jsterilitycheck PurffiedwaterX0.1 ml) 2-Aminoantfaracene (2) None; 10 dilution of overnight culture, plated on nutrient agar (total counts) Standard deviation S9 mix + present -absent Revenant colony counts and means Test I B Mean sd 0 00 + 19 W -T7- 2 + 15 20 23 19 4 + 15 19 16 17 2 + 22 16 16 + 14 12 14 18 3 13 1 + 21 + 21 17 16 20 21 18 3 21 1 16 17 23 19 12 19 27 20 12 15 19 8 16 4 19 15 28 17 16 15 21 6 16 1 19 15 9 14 5 19 21 34 25 8 19 19 15 18 2 17 22 19 19 129 119 132 127 100 117 129 115 141 129 139 136 Doss not contain TSCA CBl TABLE 3 - continued Results obtained with Salmonella tyjjhimurium TA1535 following exposure tol None; 10 dilution of overnight culture,"plated on nutrientagar (total counts) Standard deviation S9 mix +:present - absent Revertant colony counts and means Test 2 (with pre-incubation) B Mean sd O 00 0 20 20 21 22 21 14 21 14 22 14 44- 20 14 14 20 46- 3 20 1 16 4 19 5 IS 4 20 23 19 21 16 21 13 17 4 14 13 19 15 3 23 23 22 23 1 20 22 14 19 4 20 20 23 17 22 19 2119 2 125 118 112 118 90 123 117 110 18 121 111 117 116 TABLE4 Results obtained with Salmonella typhimurium TA1537 following exposure to! Company Sanitized. Doss not contain TSCA CB1 TABLE 4 - continued Results obtained with Salmonella typhimurium TA1537 following exposure to Plate Addition No. (Pg) 1 None; sterility check 5000); sterility,check 5000) 1500) |500) 150) [50) 14 Purified:water;(0;l ml) 15 Benzo[a]pyrene (5) 16 9-Aminoacridine (30) None; 10 dilution of 17 overnight culture;plated on nutrient agar (total counts) si Standard deviation S9 mix + present A - absent______ +0 Revenant colony counts and means Test 2 (with pre-incubation) B C Mean 00 0 sd 0 0 0 __0_ _ 0 __ _0_ +9 + 10 +6 + + 9 12 +7 12 10 16 9 10 12 8 15 14 ___ 12_ 8 12 10 12 9 11 13 _9__ ; 2 4 3 4 J_ _3_ 12 8 7 8 12 8 99 3 2 8 14 8 12 13 12 10 7 12 + 10 174 7 176 10 203 10 3 12 1 10 3_ 9 ~" _2_ 184 16 143 141 136 140 4 93 115 108 105 11 Company Sanitized. Dona noi contain TSCACBI TABLE5 Results obtained with Escherichia coli CM891 following exposure to Revertant colony counts and means Test i Plate Addition S9 mix + present B Mean sd - absent 1* Purified waterfO.1 ml) 2. 2-Aminoanthracene (10) 20 ENNG (2) None; 10"*dilution of 2l overnight culture, piated on nutrient agar (total counts) sd Standard deviation 0 0 __0_ _0__ 0 + -96 + 100 + + 119 108 + 106 4- too + 115 + 96 99 - :--99 111 94 108 90 107 99 96 97 102 103 101 95 107 110 --98-- 102 106 105 100 12 104 104 2- 9 15 5 6 2 10 7 - 102 100 109 104 5 - 119 108 86 104 17 - 108 109 00 106 5 - 99 87 92 93 6 - 81 112 109 101 17 -- n10o4 93 88 _88___ 96 95 8 98 11 - 107 95........ 12 105 9 + 435 474 415 441 30 ~ 768 758 740 755 14 123 inI IS 117 117 Company Sanitised. Dos; contain TSC CBl TABLE 5 - continued Results obtained with Escherichia coli CM891 following exposure to] Addition (Pg) 1 None; sterility check 5000); {sterility check 1000) 1500) 500) 150) 50) 14 Burified water <0:1 ml) 2-Aminoanthracene (10) None; 10 dilution of overnight culture, plated on nutrient agar (total counts) Standard deviation S9 mix + present - absent Revertant colony counts and means Test 2 (with pre-incubation) B Mean sd 000 0 + 87 96 -96 -93 5 + 100 93 103 99 5 + 94 96 101 97 4 + 115 125 114 118 6 + 102 114 115 110 7 102 100 109 104 102 100 78 93 13 107 118 128 118 11 101 90 107 99 9 97 104 101 101 4 115 102 114 110 7 112 131 93 112 19 443 402 465 437 32 842 886 900 876 30 123 124 111 119 Company APPENDIX 1 Historical control data Presented below are the historical control data from the period 1 April 1997 to 30 June 1998. Purified w ater solvent controls Sterno . S9 mix TA10Q TA1535 - : + - + -CM-891+ Minimum 78 8] MMeaaxnimum No. of values m 99.5 99 ; 123 100.4 99 12 33 18.7 95 10 29 18.8 95 79 164 112.0 31 73 211 121.3 31 -TA98+ 26. 2745 47 36.1 38.1 98 98 -TA1537+ 77 21 12.1 96 22 ____129.62 Positive controls Strain S9 mix Minimum Maximum Mean No. of values TA 100 -_ + 190 1477 240 1231 377.3 510.8 274 273 TA - 153+5 37 80 1243 533 193.8 268 233.3 267 CM891 -+ 294 231 2312 2097 1296.7 723.1 89 89 -TA98+ - TA-1-537 117 649 243.7 271 200 823 488.8 270 (80 ug) 562 4532 1695.4 266 (30 pg) 169 384 251.5 6 + 94 396 210.2 271 Company san iltad . Does noi contain TSCA OBI