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PB NOTOX REPORT NOTOX Project 115932 x HH } B i er IE =BiTr1 b|l [H1 R pMnpireRECEIVED vous NOV it-9 1994 72 10. 80x 3, Se Pan Hambake ring TheMr C+LO9GG . J. 7-534.) REPORT EVALUATION OF THE MUTAGENIC ACTIVITY OF Tse IN THE AMES SALMONELLA/MICROSOME TEST (VITH TNOEPENDENT REPEAT) NNOOTTOOXX SPursoejceacntce11358313627 page 107 23 - 604473 RECEIVED NOV ana-9 1994 TOAILLLOGY T-5874 NOTOX Project 115932 STATEMENT OF GLP COMPLIANCE 5 ------------------------------------ : NOTOX 8.V., 's-Hertogenbosch, The Netherlands i Tmohset.struedcyentdesecdriitbieodn o1fn: this report was conducted in compliance with the #i The OECD Principles of Good Laboratory Practice # which are essentially in conformity with: 3 RTheegulUantiitoednsStPaarttes58F.ood and Drug Adinistration. Title 21 Code of Federal 3 TohfeFUendiertaeld SRteagtuelsatiEonnvsiroPnamretnt1a6l0. Protection Agency (FIFRA). Title 40 Code ii FTehdeerUanliteRdeguSltaatteisonsEnvPiarrotnm7e9n2t.al Protection Agency (TSCA). Title 40 Code of With the exception that the stability of the test substance in the vehicle vas unknown. . Study Director Ing. .3. van de Waart Yee Date: A 3 004474 page 2 - I T-5674 a. nm NOTOX Project 115932 3 QUALLTY ASSURANCE STATEMENT z -_-- % NOTOX B.V., 's-Hertogenbosch, The Netherlands. Ssttuuddyy psrpoecceidfuircesprwoecreessessubWjeercet taolsoperiinosdpieccteidnsaptectpieornisodaincdingteenerrvaalls.non Tmheitshodrsepaonrdt wraessulatusditaecdcurbaytetlhye NrOeTfOlXectQuatlheityraAwssdautraa.nce Unit and the DATES OFAUQADUI_TSINSPECTIONS! 24-01-1994 26-01-1994 15-03-1994 REPORTING DATES 24-01-1994 26-01-1994 15-03-1594 Quality Assurance Manager .3. Mitchell B.c. 004475 Date: 20-1 -a, - page 3 3 T-5874 3i FReEpPoORRTrAAePePRmOoVGAL ooo NOTOX Project 115932 - STUDY DIRECTOR: Ing. E.3. van de Waart We== ote: /ov(igrt MANAGEMENT: oTre.chnLiCc.al EnDniirnegcator oate: 20/ou l1gg lu 004476 page 4 - T-5874 NOTOX Project 115932 PREFACE n = nn Sponsar 3CanBaedlagsituramat- C11hemcal EBC 5Be:l2g0i7u0nZWIINORECHT Study Honitor Testing Facility Mr. RH. Cox HNOaTnObKake8.n7w.etering 3 5Th2e31N0e0the-rsl-aHnedrstogenboseh Study Director Technical Coordinator Study Plan Ing. E.3. van de Weart 6. van Gort SCotmpalertetd:: JFaenburaurayry251,0, 19199494 TEST SUBSTANCE remem: Igentitication Tose7 Description sate Crean solid 2334 Purity 100% Specific Gravity 17 Instructions for : test substance storage At room temperature in the dark coSntdabiitliiotnys under storage Expiry date stable January 01, 1996 i Stahboluers foirn avtehilcelaest wDaitneerthylsulphoxide:innoot indicated 5 EE VEHICLE ' TQhuealitteyst (sMuebrsctka.nceTewsatssduibssstoalnvceedcoinncednitnreatthiyolnssulpwheorxeidpereopfaresdpedcitrreocsctolpyic #| prior to use. I 004477 pages - 1 T-5874 NOTOX Project 115932 GUIDELINES gThueidesltiundeys:procedures described in this report were based on the following - TGOourxigidacenolilisonagetysi:onfoSraflormToensEetcliolnnagoiotcfypCChhoie-saoutprcieaurlnast,iRoenvGeuriasdneedliMDnueetvaetlmioo.opnme47nA1ts:s(aO"yEG,CeDn)e(,iaidcoOEpCtDed Hay 26, 1983) Toxicity: 8.14: "Other Effects-Motagenicity: Salmonella typhimurivn - - European Economic Community (EEC), Directive 92/69/EEC. Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Reverse Mutation Assay". EEC Publication no. L383 (adopted December, 1992). ARCHIVING pNOrToOtKoco8.lv,. rweiplolrt,arctheistvearthteiclfeollroewfienrgencdeatasamfpolreatandlearsatw d1a0tay.ears: osagcTIvE purpose of the study aThbeiliatbyjecttoivienduofce thriesverssteudymuvtaastiotnos evinalua stgeenetheofhtiessttisduibnset:arnecqeuifroirngits S2Snadlmtoinneltrolhfea apTtrhyeepsshteeinmcRueTrsCiIuoDmf-oabragMcaetnetirasibhaosll.icsTthrseayisniasesmsetyo(Swp9a-rsMo.idnuCyco.enduhitstteiddiine-tihnedespbeenedmeenet Justification for selection of the test systes The Ames test has been shown to be a rapid and inexpensive indicator for aTtnhhdee bmBauastceat-gerpeianaiilrc ssatcurtbaisivtniisttyuteimoofpnloaymeuwdtiadgeweenrrsea.ngceapaobflecheomficdaeltecctomipnogunbdost.h frameshift 0044778 <page 6 - T-5874 NOTOX Project 115932 2 : TEST SYSTEM Test Syste salmonella typhimurium bacteria i: Rationale Rtehceogrneiczoemdmenbdyedthetesitntseyrnsatteimon(ael.g.guiEPdAe,linOeEsCD,as EEC). Source oart. BeBrrkuecleeyN,. AUm.eSs.,A.Un(iTvAeIr5s35i,tyToAf15C3a7liafnodrnTiAaS; 1987 and TAL00; 1993). The characteristics of the individual strains were as follows: strain Histidine mutation Mutation type TA918537 hniissc033007526/R- factorTM FFrraammeesshhiifftt TTAA1L50305 hhiisscGe46s/R-factor BBaassee--ppaaiirr ssuubbssttiittuuttiioonnss *: R-factor =plasaid pHIOL (increases error-prone ONA repair) Each tester strain contained the following additional mutations: rgfaal : dneuetpatiroonughin(tdheefegctailvaecto1siepompeotlaybsoalcicshmaride cellcoat) GbAiTo : mduetfaetcitoinveibniontiitnratseyntrheedsuicstase urs: Troespsairof8thgeenee)xcision repair system (deletion of the ultraviolet- S(t-o1c9k6Cc)u.ltuSrtersaionfs wtehreeforuergusltarraliynschweecrkeedsttoorecdonfiinrm1igtuheiidrnihtisrtoigdeinne- : TrAeLq0u0i)r,eneUnVt-,Secnrsyisttiavlityviaonldetthseensniutsibveirtyo,f aspmopnitcainleloiunsrerseivsetrtaannctes.(TA and CELL CULTURE Preparation of bacterial cultures wSearmepletsraonfsfefrrroezden isnttoockenrciuclhteudresnuotrfiebnatctebrrioath ((307x0C1,d n1o5.0 2s)pm)a,ndunitniclubattheedcuinltauresshakrienagchebdathan 0g.r0o.wnofcu0l.t4ureast 7o0f0eanmch(s1t0raicnelwlse/rmel)u.sedFrefsorhlya test. 004479 - page 7 - i T5874 NOTOK Project 115932 i Agar plates AaTgigataerrr:phlead1it8uensg. p(u2Grliu9tciocesm)de caaggoaanrrtaim(neOedxdoiiudm2,5cmoc1notdeagilnu12ec6do)s.epeirn Voge-Bonner Medium E (*), 10 g glucose, 0.5 ng biotin and 0.6 mg histidine. (*) Vogel, H.J. and Bonner, 0.M., 1956, ApcuertiyfliocrantiiotnhinaansdesomoefpErscohpereicrhtiaice3o.sli::Biopla.rtial Chen., 218, 97-106. Top agar Environmental conditions VpSoaugmrepillf-eiBseodnonfaegra3rMalewdaistuompheaEatgeacdrontwtaoeirndeiinstggroaln0vs.ef6e%rtrh(eewd/va)gianrt.o 1tu0b8e]s 9ve1r8e5saututboecslavweidthfRoret2a0 cmaipns.atTo1p.30ag.ar AT1 incubations were carried out in the dark atthe37exCp.erTihneenttemperature was monitored during Rererence sussTANCES NTVheegeatinve tconoTtfroe)":the test article. Positive controls: WiStahonut metaebnotleiacractivaHtiOonLS(-59-aix atonipiate Solvent TAI535 sodium azide (SA) (Fluka) 1 ug Saline TA1537 Tas 9a(-uaaanmnoismnsyoecanicnrCeihdiin(ntOceMa)y(9(ASCi)gaa) 60 ug 4 ag Saline savine TAO methyldethansulfonate. (wis) 60 ug oud . (Merck) With metabolic activation (s59-n1x): EB, Strain rTTyAa1i5o37 Chemica 2-aminoanthracene (2AR) 2(-saigmmtan)oanthracene (2m) Concentration/plate 5 ug osu = Solvent DMSO oso SpolhvSieTinnyets=sfofr orelferoencge Sisautbcisnteaa.nc(leNsedital Pharma Ned. 5.1.) OMSD' = dinethylsuiphoxide of spectroscopic quality (Herck). : 004450 5 pages - T-5674 NOTOX Project 115932 METABOLIC ACTIVATION SYSTEM Preparation of $9-honogenate: RoratSplriavegruemiDcarwolseoymarlatse,nzywmheischwewreereroobuttaiinneeldy pfrreopmarBeRLd, fSrwoimtzaedrullatndm.ale Wistar T1haeboarnaitmoarlyscownedrietihoonuss,edasatdeNsOTcOrXibeidn ainsptehceiaSlOP'rso.omThuenderratsstawnedraerdinjected wienitgrhatp)eriitnonceoarlnlyoflw.ithFivaesodlayustiolnate(r2,0%twh/evy)woefreArkoicllloerd 1by254dec(a5p0i0tamtgi/okng; body s(atchreiyfiwceer)e. dTehneiedliavcecrsessoftothefoordatsforweraet lreeamsotved12ashoeuprtsicaplrleyc,edianngd washed in cNaogl-dED(T0A.C) Susbtesreiqlueent0l.y1 Mthesodliiuvnersphowserpehatmeincbeudffeirna(pHbl7e.n4d)ercoanntdaihnoimnogge0n.i1zedmM wians'3cveonlturmiefsugeodf pfhorosp1h5atmeinbuaftfe9r000witg.h aThePotstueprernhaotmoagnetni(zSeSr). waTshetrhaonmsofgeernraetde into sterile ampules, which were stored in liquid nitrogen (-196C). Preparation of $9-mix: 5599--mniixx wcaosntparienpeadrepderim10medmli:ate3l0yngbefNoArDeP uasned a1n5d.2kempgtgolnucoiscee-6d-uprihnogsphtahteeteisnt. 5M.g5Clyml soalquutaiobni;des1t;ml 20.ml330.H5KHCI sosdoliuutniopnh,ospahnadte0.b5ufmlfer$9.pHT7h.e4;ab1ovmel 0.08 M sforlaucttiioonn,s wwehriechmiwxasedadadndedfaifltteerr f(i0.l2t2eru-ms)t-esrtielriizlaitzieodn o(faptahret fSr9o-mmixthe $9components) EXPERIMENTAL PROCEDURE selection of dose levels . sweiltehctsitornainofTAaLn00a,deqbuoatthewirtahngeandofwidtohseosutwaSs9-mbiaxs.edEionghta pcroenlciemnitnraartyiontsestwere tFeisrtsetdexinpertirmiepnlticaotfeth(ethimsutapgreenleismiisnarayssatye)s.t wTahse hriegphoersttedcaosncaentpraarttioonf tofhe test article used in the subsequent mutagenesis assay was 5 mg/plate. 004481 - page 9 - 3 T-5874 NOTOX Project 115932 Direct plate incorporation assay *: Five different substance were dtoessetsed (iwnithtriapplpircoaxtiematienlyeachhalfs-trlaoign.steTphse)toefsttsheubstteasntce was texepsetreidmebnottsh. wiTtohp aangdarwiitnhotuotp Sag9a-rmixtubienseawcahs smterlatiend, anidn thweoateidndetopen4d5eCn.t The fforlelshowibnagctesroilaultiocnusltwuerree (1s0u9cceceslslisv/enlly) adofdedoneto o3f mtlhetotpestaegarr:str0a.i1nsm,l of a 0.1 ml ocfasea doifluacttiiovnatoifonthaesstaeysst)suorbst0.a5nceml i0n.1DHSMO,phoasnpdhaetiethebruff0e.r5 m(1inS9c-amseixof(in ncoonn-taecnttivoaftitohne atsospayasg)a.r Tthuebeiwnagsrepdoiuernetds woenrteo maixseeldecotnivaeVoargtarexplaantde.theAfter sdaorlkidiaftic3a7tiConforof48theh. toApfteargart,histheperpiloadtesrewveerretantturn(ehdisatniddiniencuibnadteepdendienntt)he colonies were counted. *) Acmaersc,inoBg.Ne.n,s MancdCanmnu,tagJ.ensandwiYtahmatshaekiS,alnE.o,nel1l9a75/,mamMneatlhioadns mfiorcrodseotmeecting mutagenicity test, Mutation Res., 31, 347-364. *) Mnaurtoang,eni0.cMi.tyantdestA,mesM,itBa.tNi.o,n 1R9e6s3.,, 1R1e3v,ise1d73-m2e1t5h.ods for the Salmonella Erratun, 1583, Mutation Res., 113, 535. Colony counting TwhiethraenveArrttaenkt cmoodleolnie8s80(choilsotniydinceounitnedrepoerndemnatn)ualwleyr,e cifounlteesds athuatnoma4t0ically cporleocniipeistatpeertpolaitnetewrefreereprweistehnta.utPolmaatteesd wciotlhonysufcfoiucniteinntg tweesrte acrotuinctleed manually. ACCEPTABILITY OF ASSAY . Aa)n AThmeesnetgeasttivies ccoonntsriodlereddataacc(enputmabbelreofifspitontmaeneetsoustherefvoelrltoawnitsngpecrritpelraitae:) sfhooruledachreatseosntearblystrbaeinw.ithin the laboratory background historical range b) Tshteraipnossitwihviechcoanltsroolrecahseomniacballysarsehouwlidthipnrodtuhceelarbeosrpaotnosreys ihnistaollrictaelster Fruarntgheerdnoocruem,enttehde for each positive control substance. mean plate count should be at least two times the ) cTohnecusrerleenctt.edvehdiocselercaonngteroslhouglrdoupinmcelaund.e a clearly toxic concentration as dexetmeonndstrtaote5dngb/ypltahtee.preliminary test with strain TAL00 or should 004482 - page 10 - i T5874 NOTOX Project 115932 DATA EVALUATION AND STATISTICAL PROCEDURES ANotefsotrnaslubhsytpaontcheesiisscotnessitdienrgedwasnegdoantei.ve (not mutagenic) in the Ames test ai)f: The total number of revertants in any tester strain at any concentration wisithnoouttgrmeeattaebrolitchanacttiwvoattiionne.s the solvent control value, with or b) Tihnedepneengdaetnitvleyrerseppoenasteedshexopuelrdinbeentr.eproducible in at least one Aa) teIsttisnudbuscteasncaet ilseasctonsaid2e-rfoeldd,posdiotseiverel(amtuetdageinniccr)eiansethien Athneesnutmebsetr ifo:f arneyveorftantthse wtietshterressptreacitnst,o etihtehenrumbweirthionrducweidthobuytthmeetasboollviecntacctoinvtartoiloni.n Hsoiwgenviefri,canatn.y mIfeantheplatteestcosuunbtstaonfcelessshowtehdani2n0thies cfoinrsstidetreesdt toonlybeanot pWoistihtidvoesesresjpuostnsebelaotwonaendorexcteweodicnogncetnhtorsaetisohnosw,ingtheposaistsiayvewaesffercetpseatiend b) Tthhee pFoisrsittivteestr.esponse should be reproducible in at least one independently repeated experinent. Tehneterpreicnetodintghecrfiintaelriaevwaelrueatinoont adebcsiosliuotne. and other modifying factors might 604453 - page 11 - T-5874 ; NOTOX Project 115932 - ] ResuLTS TOXICITY OF THE TEST SUBSTANCE The survival of the TAL0D culture was determined by comparing the nuaber of colonies on the plates containing the test substance with those on the solvent control plates. Both in the absence and presence of S9-mix the survival of strain TA100 was not reduced up to and including test substance concentrations of 5000 up9gr'/eapsnledantceeInoc(flTuadb$3li-en2gi1x)a.. cBoansceedntroantitohneseofda5t0a0,0 utgh/epltaetset sinubstthaencaebsewnacseteasndted up THE AMES SALHONELLA/MLCROSOHE PLATE TEST WTiatbhlesT5187a4n.d 2A11shobwacttehreiarlessulttrsainosf tshheoveSdalnmeognaetlilvae/mircersopsoonmseesploavteer ttheest entire dose range of the test substance, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated Weixtpehriinmeonutrs.TaTbhoeratnoergyatibvaeckgarndounsdtrahiisnt-osrpieccailficrapnogessitiivnediccoanttirnogl thvaatluthees were tfeusntctcioonndeidtipornosperwleyr.e optimal and that the metabolic activation system concuszon Based on the results of this study it is concluded that the test substance is not mutagenic in the Ames Salmonella/microsome assay. 004484 page 12 - i T-5874 " TTT NoTox Project 115932 TT 3 ' - TABLE 1 MUTAGENIC RESPONSE OF T-5874 IN THE AMES SALMONELLA/MICROSOME PLATE 5 TEST. Experinent 1 o(ousge/plate) M3earneplniuacbaetre opflatreesver(tsan5t.0.()Hiws*i)thcodliofnfieerse'nt strains ofS. typhinuriun TALS35 TA1537 TASB TAL00 Without $9-mix 103 10303 1030303 3530300000 SPoolsvietnitve cocnotnrtorlo:l 08::43 180::53 10: 2 2746:+228 87.:23 98::23 8: 3 5777:+ 389 2246::83 223::22 22:3 64230s1542 112289:4 127 11368122322 11398::1l7l 6l:s1u5 1317623s3 1219 103 10303 1030303 353030000 SPoolsvietnitvecocnotnrtorlo:l With 59-mix m9:i22 85::54 7:3 2178+:230 76+:483 663333 7:1 6685+:486 2235::42 2280::35 25:4 22535::37 u43e: 84 11336031+ 107 1107::57 145701:189 61001 s&1311 *10T.e1st.mlsubOsMStOance precipitated slightly on the plates 004485 + page 13 - bi 7.5874 - TTT NoToX Project 115932 TABLE 2 TMEUSTTA.GENIC RESPONSE OF T-5878 IN THE AMES SALMONELLA/MICROSOME PLATE Experinent 2 Dose (ug/plate) Mean number of revertant (His*) colonies/ 3 replicate plates (s 5.0.) with different strains ofS. typhimurium 130303 1303000 50000 SPooslivteinvteccoonnttrrooll TA1535 170+:13 2804:120 17: 4 215373+ 134 TA1537 TA98 TA100 Without 9-mix 88::22 66::13 12315 2308::72 2299%:13 25: 5 3941:1452 42787+5691 112068:4183 11641::106 129% 6 1219223 3+ 597 100 333 1000 530303000 PSooslivteinvte coconnttrrooll* 1s 4 61 4 7+ 3 15:: 32 175431551 With $9-mix 5+ 3 53 4 7:2 673: 1 2 34413 30: 5 36:8 40:12 31: 5 2146:3562 33296s+237 1204 6 11 331 s+1856 114417 143: 2 614194 23+ 563 ** T0e.s1tm)subDsMtSOance precipitated slightly on the plates ? One plate infected with other bacteria 604486 - page 14- T5674 TTT worox project 115932 3 APPENDIX 1 1 Ethveidefnoclelowoifngtesptrecairptiictlaetiopnreccoidpei.tate on the plates is recorded by addition of ; PRECIPITATION CODE Definition Characteristics Slight precipitate Moderate Precipitate Heavy precipitate DHoiwsetvienrg,uisthheedprbyecinpoittiacteeabldeoepsrencoitpiitnaftleueonncethaeutoplmaattee.d cDoiusnttiinngguisohfedthebyplaatmea.rked amount of precipitate on tDhiestiplnagtuei,sherdeqbuyirainglartghee palmaotuenttoofbeprheacnidpitcoautneteodn. the plate, making the required hand count difficult. 004487 - page 15 - 3 T-5874 : TTT NOTOX Project 115932 IAnPdiPviEdNuaDl IpXlate2counts; (following pages) sExtpreariinment TAL5351 : WITHOUT $9-MIX plate 12 3 MEAN SD Dose(ug/plate) 313030 u6 s8 31 180:s 34 13030300 2B93 78 08 :x35 5000" 5 13 9 042 PSooslivteinvtecocnotnrtorlol 2968 283a4 2472 2746 x+282 WITH S9-MIX plate 12 3 MEAN SD Dose(ug/plate) 130303 1n 2 o8 s8 no9:22 13030300 51107 125 85::54 5000 5 51 7:3 sPoolsvietnitvecocnotnrtorlol 1859 2175 2538 287.1324 + Test substance precipitated slightly on the plates 04488 - page 16 - T-5874 2 ESxtpreariinnent 1 TA1537 TT 7 NOTOX Project 115932 TT - WITHOUT $9-MIX plate 12 3 Dose(ug/pate) MEAN So 100 333 1000 3330 5000* 6 10 7 499 10 11 7 9 10 4 61 8 8+2 7+3 9+2 8x3 8+3 SPoolsvietnitve ccoonnttrorlol 6159 51161 5365 5777 +693 WITH $9-MIX plate 123 Dose(ua/plate) MEAN sD 100 333 103530030000:1 5 5 10 692 5370879 4a7 743 6+4 667 ::i3a3 SPoolsvietnitvecocnotnrtorlol 6653 7610 59110 6658 ++ 864 + Test substance precipitated slightly on the plates 004489 page 17 - E T-5874 - TTT NOTOX Project l1s932 z1 sEtxrpearininent hss1 : WITHOUT S9-MIX 3 plate 102 3 MEAN SD Dose(ug/plate) 130303 229 2296 223 22% ++ 43 10330300! 2250 223 223 223:: 22 50004 2 19 23 2: 3 SPoolsvietnitvecocnotnrtorlol 6296 58189 6424 6423 4+ 542 WITH $9-MIX plate. 1 2 3 MEAN SD Dose(ug/plate) 133030 219 227 22 223%.: a2 103303004 3213 2289 2250 228:1 35 50004 27 28 2 2% i 4 sPoolsvietnitvecocnotnrtorlol 22680 22436 22536 2523 +x 37 * Test substance precipitated slightly on the plates 004490 - page 18 - 1-5874 : i 33 Esxtpreariinment TAL001 TTT notox project 115932 - WITHOUT $9-MIX plate 12 3 Dose(ug/plate) 103 112253 111229 113366 10303 115071 115680 112506 1030303 113348 112458 116287 35030300: 114598 115391 11380 SPoolsvietnitvecocnotnrtorlol 1135803 1136389 1319750 MEAN SO 12294 1+ 127 u13s8 2+322 11398 +1117 6f.r+e15 18136732 +42191 WITH S9-MIX plate 12 3 MEAN SD Dose(ug/plate) 103 11438 113436 8138 13145 84 10330 41307 112.82 112218 18360 +x 107 1030303 11560 113398 115336 1107s+ 75 35030300 113309 115515 116488 1750 +2189 : sPoolsvietnitve cocnotnrtorlol 613672 518862 156861 610710 +13111 + Test substance precipitated slightly on the plates 004491 - page 19 - 2: Tse - 3 sEtxrpearinnent hiss2 NOTOX project 115932 WITHOUT 59-MIX plate 12 3 MEAN so Dose(ug/plate) 100 15300330030003:: solvent control Positive control 165 1va 0s B97u11o0u 5 ous 245 256 270 7 +3 120E+iiEhw1 13.4 257 +13 WITH 59H plate vase(ug/prate) 1015003003 ES00o0: 12 3 7siowunns8 [iEsT MEAN SD wHHEoeEs asiisz Solvent control Positive control 45 6 111 215 196 5& 1 174 1 55 * Test substance precipitated slightly on the plates 004492 page 20 - SEtxrpaeirninent 2 TA1537 WITHOUT $9-MIX pate 1 2 3 ean so Dose(ug/plate) 130303 78 71 o6w 103303001 5s 68 73 5000 INF 12 15 88:22 56::13 12 515 PSoolsvietnitvecocnotnrtorlol ay4 3552 3786 14 s+ as2 WITH $9-MIX plate 123 MEAN SD Oose(ug/plate) 130303 57s 22 s3 551.3a 10330300: 5786 73 s73i2 5000 is 8 742 PSooslivteinvtecocnotnrtorlol 2764s 2085s 1652 21641256 I+NFTes=tPlsautbestainncfeectperdeciwpitihtatoetdhers1ibgahcttleyriaon the plates 004493 - page 21 - T-5874 NOTOX Project 115932 sExtpreariinment, h982 WITHOUT $9-MIX plate 12 3 Dose(ug/plate) 130303 00 33 2281 31303000 20% 3225 3209 5000: 31 2 2 PSoolsvietnitvecocnotnrtorlol 42377 55279 43288 MEAN SO 208s: 72 229:+13 5:5 28 + 1 77:69 WITH $9-MIX plate 12 3 MEAN SD Dose(ug/plate) 313030 2259 325 3409 03.:153 31303000 331 3386 5283 036:1+ 28 5000 6 27 30 31:5 PSooslivteinvte cocnotnrtorlol a148 33603 36301 3329 1+ 237 + Test substance precipitated slightly on the plates 004494 - page 22 - 7 T-5674 sExtpreariinment TA1002 NOTOX Project 115932 WITHOUT $9-MIX plate 12 3 MEAN SD Dose(ug/plate) 130303 11203 112246 18298 01286 ++ 183 31303000 11648 112650 113546 1626:: 106 5000 128 135 123 129: 6 sPoolsvietnitvecocnotnrtorlol 1212542 1316209 12u6s5 1219223 +& 579 WITH S9-MIX plate 12 03 MEAN SO oose(ug/plate) 130303 u0s4 u13e8 111257 2u0s + 1s6 13030300 10303 113383 112170 3m3e:: 16 5000: 141 183 14s 3:2 SPoolsvietnitve cocnotnrtorlol 51845 61717 6u8e5 6e19s+ 563 * Test substance precipitated slightly on the plates - page 23 - 004495