Document 1mZ9mX7RzMNdEGw820pqjMNE
REPORT
EVALUATION OF THE MUTAGENIC ACTIVITY OF T5869
IH AN IN VITRO MANHALTAN CELL GENE MUTATION TEST WITH LSI76Y HOUSE LYNPHOMA CELLS (ITH INDEPENDENT REPEAT)
NNOoTTOOXX SPurbosjteacntce11358915946 - page 1 of 22 -
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RECEIVED
NOV -9 1004
TOXICMOeLOGY
T5069 STATEMENT OF GLP COMPLIANCE
NOOK Project 115954
\OTOK 6... *s-Hertogenbosch, The Netherlands
nTeetstTuedcykdntesscaritbieodn fofn:ths report, ves conducted in compliance ith the
ThhiechOECaDrePreisnsceinptlieaslloyf iGnoocdonLfaobroartattyorwyithP:ractice United States Food and Drug Administration.
Title
21
Code
of
Feder!
nReegulations Part 58. Ine United States Environsental
Protection
Agency
(FIFA).
Title
40
Code
Re ederel Reguations Fart 160. TIonCeerUanlitRedeguSltaattieosnsEnvPiarrotns7e5n2t.al
Protection
Agency
(SCR).
Title
40
Cade
of
rYatshuntkhneowenx.ception that the stability of the test substance in the vehicle
Study Director
Ing. .3. van de Vaart
wate: splot
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005632
T-5869 QUALITY ASSURANCE STATEMENT
NOTOK Project 115954
NOTOK 8... *s-Hertogenbosch, The Netherlands.
Ssttuuddyy psrpoecceidfuircesprvoecreesssesubjweecrte taolsoperifnosdpieccteidnspatectpieornisodiacndignetneerrvaalls.non Tmheitshodrsepoarntd wraessualutsditaecdcurbaytetlhye NrOeTfOlXectQuatlhietyraAvssduatraa.nce Unit and the
DATES OFAUQDAIUT_SINSPECTIONS/ JFaenburauraryy241,0, 1199994 March 28, 1994
REPORTING DATES JFaenburaurayry241,4, 1199994 March 28, 1594
Quality Assurance Manager
.3. Mitchell B.Sc.
Da2t0LeAL:, |
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REPORT APPROVAL STUDY DIRECTOR:
MANAGEMENT:
NOTOX Project 115954
-
Ing. .2. van de Naart
Het
outsHHT
oTre.chn1i.cC.al EDninriengcstor
nate 20lou hogy
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NOTOX Project 115954
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eereaece .
sponsar
SCHangadea1sgtiruanst- C11heical 8
-
B5e-l0g7i0unVISNORECHT
Study Wontar
We. RA. Cox
Testing Factlity
5NNoa3Tm5Ob1Ka.k0Be0n.w.e5t-eHreirtnagge3nbosch
The Netherlands
Study Director
Ing. E.3. van de Vaart
Technical Coordinator CaM. Verspeek
study Plan
CSotmpalertetd: FFeebbrruuaarryy 2012,, 1195994
r TEST SUBSTNANECE ~l e m re
Identification
T-s869
description
Crean soltd
saten
2108
Purity
Pe
specitic Gravity
15
TInosottruScotbisotnasnceforstorage At roo temperature in the dark
CStoanndti1tfitoynsunder storage stable
expiry date
January o1, 199
Sthaobuersfoirn vatenTiecaest
Dimethylsutphoxtdes not indicated
TveEw--tcete eieeomesmmrnseeermseeeeree----
RTTheeeceterttaetssyctonpstrueib.osrtaqnutecoseufasvyea..s (TsMhueesrpcefkni:ndaeldTescotronsdcuiebsnssttoralanvtceiedonciononfdcietsnhteertahstyoillovsneusnltphwoeirxneidtephreeopfared
Culture. meciun amounted 0 0.8% (IV).
Cows
005635
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NOTOX Project 115954
=
-cuo- ELINr ES
The study procedures described in this report were based on the following
guidelines:
~ oCOorevgTteaeentsttstoaonteysi:anToIrnforTVietsErtcaionnogNsafoncfsalCCihoea-mnoipceCarellalst,ioGnGeunieadnedMluitDnaeetvieolnnao.psTee4ns7t6t:s(,G"EGCe(Dna)ed,toipcOtEeCdD
hort 4, 1968)
CEEEutDrootpeieeaycnetEic"voonntohemar7ic5edCf/ofEmemcEutCns,iMtuyFtaai(gtEeEnCBi))c,iMteDyti:hroedcIstnivVfeiotr8r7ot/h3eN0a2mD/aeEatEleCir.amniAnCametelixlonVGeoonffe the
Fotation eetro EEL Publication no. L133(adopted May 30, 1988).
ARCHIVING
----
NDrOaTtOaXcaBl.,. rweiplolrt.arctheisvteatrhteiclfeollroewfienrgencdeatasamfpolre,atallleasstpe1c0isyeenasrsa:nd raw
Gata.
--opI s~eceTIvrE mmmisometererereemeermestersr Purpose of the stusy T1Eh51eT76oYHb"jemocSutesievfendyooafrpehtohTniosspvscaetrlduldsyh:utvaaTsthieotnosseseaavytalutwaheestetcthohynemdiudctitenesedt skiuinbnsattshaeenca(ebTKs)efnorcleociautsnsdin bDireessenpcaierofmutaatpieocnssb,otifcranseystsehaift(s9m-uatfaxt)i.onsTheandTKsmmaultlatidoenlaeltiosnyss.ten detected
Justification for selection of the test systen
LoSFl5t1a7`t6miYtoamgsaeonnuisceSysIatytentmpihvoi1nt5ayacobeflellastboraorGeaedteucsretadngbeebseecoafupscaehrentihicetayelraarcteliaossnsesen,ss.itfiTrvhaemeeTIKsnhdiifctators
RTTuittastS iodnesfiTaatndoTntseailna!retdheytlseeistdiiisontneasn.tkintaostehe(1Kc)y,lodtuoexicto etfhfeactfsorwofardthemutpaytriiomnidine
sSPhoTecoiqeauaesta.loaggtsure)neTIneucndore.adtshtfysosripdchieon1es.ipuhlo(arTrFyTl)am.teetdaTbKodediresifnviactiaiervenetobc(tenaluilcsnleedoctainsdoenlo)et;l.ytihnfecroomrpdoerate
PTHoeveod.ninsAyrcneatnhtTeaestiihn.ailngTntToFtThteheaprreceelsaleusnt.acteeTdho,ufs,TEki,tcheelTlrFsT siwpshoinccthoannvweieolrultseldysuriorvntiovbeynuitnchleecouatlcittduierosen,
o3tfTo4beteheetatserpseotitcernsteuibaswnthaincnchaem,sianlggfiuavcniensgcaolrlpiossemiutttiaovgeean.TrKesdpeofnisceieinnttphihsenoatsyspaey. is presumed
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MATERIALS AND METHODS
TEST SYSTEM
}
Test systen Rationale
Ls178Y mouse 1ysphosa cells Rtheecogrneiczoemdenbdyedthetesitntesrynsatteinon(acl.g.guiEdPeA,linOeECsD,as 0).
Source ]
GDre.netAi.cG.sA.aCn.d KCnhaesapi,calDeMpuatratgmeenntesiosf Roafditahteion S(t1a98t1e).UniTvhiesrsimtoyuseoflLyemipdheonm,a Tcehlel NeTtihneerlwaansds oTirnieg,inailsloylatdeerdivbeydClfirvoem t(h1e97F5i)scher LS178Y +L)abColriavteo,ry0.proancdeduSrpeectfoorr, aJs.sFe.sSs.i,ng19s7p5e,cific LT5o1cu7s8YmmuotuasteionTsyapathomtahecTeKllsl,ocMuustaftniocnultRuerse.,d 31, 17-29.
SThteockcuclutlutruersesweoref tchheescekedcelflosr wmeyrceopsltaosrmeadcoinntalmiiqnuaitdionni.trogen (196C).
CELL CULTURE
Cell culture conditions
F10 complete culture mediua Selective sediun Cloning mediun
L511078cYommpoluestee cluylmtpuhromeamecdeilulms. weCrueltucruelsturveedrefn Ci0n,cubinateadfrinata3h7uCa,fdCaeltlaosdepnhseirteycownatsaining 5% pwerraefereaxbploysedkeptto btheelowtes7txs1u0b5stcaenlcles/imnl.F10Cells Cboumfpfleerteed cwiutlhtur2e0 mmiediHEuPaES.ithout serum, FF1100 cnoemdpiluenteitchuolutturethymneiddiiunmecoanndsihsytpeodxaonfthHianne's g(lGuibtcaom)i,nesu(p2plnie)menatneddpweintihcil1l0i%nh/osrtsreeptsoernuyac,inL{50 U/ml and 50 ug/ml respectively). Scuellteucrteivemedmieudniunwhiccohnsicsotnetdainoefd,F10incaodmdpliettieon 0(.S3ig3n%a)A.gar Noble (Difco) and 5 ug/ml TFT cCluolntiunrge nneeddiiuuaa, cosnuspipslteemdentoefdFw1i0thcom1p0l%etheorse saedrduint,ionwhiocfh0.w3as3%tArgaanrsfNoorbmleed. into a gel by
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Environmental conditions
ASIlnnlotshipenhceduraberakti(os8nt0s-9357w%e)Cr.e ccToahnretraitienedimnpgoeurt5a%tuinrC0epa ahniudnmiaCdiOrppEexrpecrennteangte. vere monitored during the -
REFERENCE SUBSTANCES -------
--
TVNheegeatinve ecoonrtfroelT:he test article.
Posttive controls:
YWi(i@mthmooyuue twsmaeesutse aubbseoedll.iiccEsssnMgcStteeicvaauets(EieHnosS:ndG CiAr(Se5c9S tn-o.1al)6k:2y:l5a0t:i0o;n poufriDtNAy.98%; Janssen Chiaica)
LWDiittRrhh ermeettaabteboooerlleicicvacess_aitcsn.te.itvOnaW(etNDliWioNn;nnad(C(AtsoSo5-9nb-loem.ixas)6c:2t.i7v5a:t9e,d Xe resuniing in a methyldiazoniua
pbfuyorniitcwyhrio9cs9ho%R,caolJualndesnszreyensaecsCthipwniritechsae)nt
in
cellular OA.
SoolveSntseTfoarrcReedfeSarltencesoSulbusttai(ncoBenSsS) without calcium and magnesium.
Solutions of reference substances were prepared imsediately before use.
METABOLIC ACTIVATION SYSTEM
ee
preparation of 9-honogenate" RBaatpirvageuremDiacrrolseoymarlatse,nzwyheischvewreereroobuttaiinneeldy
pfrroenparBeRLd, fSrwointzaedrullatndm.ale
Wistar
TTTheeeovaantiosraylosncewoaenrrdlieytihoovnuisst,ehFdiavaseatsdodNelaOsuyTctsOriKioTnbaetiedn(r2,a0in%stptwheh/ecveyi)aSwlOoePfr'ersoA.orknoicTlulhlnoeedrderrba1yt2ss5t4davenec(dra5aep0r0idtiuangtji/eokcngt;edbody
VFeeInysGeveerien Oe)
dcsoeOtrneantreid1lYei1av.c0ec.re1sssNoftsotohdeifuoomrdatpsfhoorvseprhaeattelreeabssuotfvfeed1i2rnahsa(opeiupbdrtlsi7ec.na4pdl)relerycc,eoadnniatdnnagdihnowioangsgehne0td.z1eidna
FNAeEctemToaeorissueegbesaodqfmqpuupfelhoenorsts,ip1yh5auttnmheiiecn.hbua1ftivfveee9rrr0es00isvttegoh.rreeadTihPenionctsetuldepireqruhinodamtoangniettnrio(zg5e9er)n. v(aT1sh9e6thrCoa)mn.osgfeenrarteed
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*) Acnaersc,inoBg.Ne.n,s HacndCmaumt,age3.nsawnidthYamtahseakSia,imEo.,n119l7e5/,mMaemtnihaonldsmifcorrosdoesteecting nutagenicity test. Mutation Res., 31, 347-364.
Preparation of 59-nix
-
5S9o--maiixx wcaosntapirneepadrepder{ualm:ed1i.a0t2ely0gMbeIfCo1rze uHsge;and2.k4e6ptmgonKCTf;ce1d.u7rimnggutchoe stees-ts. Vpheorsephnaitxee;d a3n.d4 fngiltNeArDP;(0.422umoulm)-HsEtPeErSilainzded 0.(5ampiart59.froTmhethaeboSv9e-fsroalcuttiioonns,. Mwehtiacbholwiacs aadcdteidvataifotnerwfaisitaecrhisetveerdilbiyzaatdidoinngof0.2the1 S9l-imviexr cSo9m-pioxnenttos)e.ach ml of cenl suspension.
EXPERIMENTAL PROCEDURE
Selection of Dose Levels/Cytotoxicity Test tprrieoartintgo 6thex1a0c8tuacle1lmsu,tasgeunsipceintdyedteisnt6cym1tatoofxiFc-l1t0ymdeadituamvbeureffeorbetdainietdhb2y0 8r4anHgEePEoSf,te`sint.tshuebsatbasnecnececoonfcseenrturmatiionnsa sitneraiplperox3i0maa1telcyenthraliffugleogtsutbeepsw,ith a brootthateidn ftohre 3abhseoncneaanrdolplerresmeinxceerofatS3-7aiCx.. The centrifuge tubes were Abyfte3rcetnhetriefxupogsautrieo,n tshteepscel(l1s15egr,e8smeipna)r,ateedachfrfoonlltohweedtrebyatmreemnotvaslolouftiotnhse SsuopleurtniaotnanatndanfdinarlelsyusipnenFs-i1o0n moefdi`uthne. ceTlhles,eltlwsiceininthHeanfki'naslbasluasnpceensdiosnaltwere ceoxupnrteesdsedinasanth"eInprreodvuecdtioNneuabfatueerraphparcmooxciymtaotmeeltyer2.4 Rhelanadtiv4e8 chytgortoowxtihcity, ccoonmcpeanrterdattioonnonrtarnegaete(d4 dcoonsterso)lofceltlhse,tewsats suusbesdtatnocedettoermbienesppaliseuditatbole cnoutltutroexsicparnedpardeidffifocrultmuttoagdeinsiscoiltvyeteisntianqgu.eouBescasuosleutitohnestethset hsuibgshteasntce vas concentration was determined by the solubility in the culture medium.
Cleansing rior to mutagenotty (and ytotorielty) testing, the cells vers grom for a1nidnaoyptienricnultaunrde1.m6edixun10>conHtatinhiynmgidi1n0%e (MHAThy-pmoexdainutsh)intoe', r2eduxce10t7heMamount of cspoonnttaainneionugshmyuptoaxnatnst,hinfeolalnodwedthybymidainreecoonvleyr.y Apfetreirodtohfis2pedraiyosdoncelmlesdiuvmere erxepteurrinmeedntt.o norsal mediua at least for 1 day before starting the
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NOTOX Project 115954
Mutagenicity teste Tihndeepteensdt.enst.ubsetxpaenrcenevnetss.te6stexd1b0othcelwlisth(a1n0d5/aw1i)t,hoourt S12onx n10gncteulols (10/a1) sfeorlectteesdt sdousbestacneclelscvoenrcsenterxaptosieodnsfeorxpe3cthedtotothebetsetstronsgulbystatnocxiec,infHorEPeEaSch bSuusfpfeenrseidonHsanwsereF-p1l0acmeeddiuinn w3i0tha1outcensterruisf.ugeFortubtehsisonpuraporsoelletrhemicxelelr at 3V7e5r%e. wa`Ssohlevdenttwiacned wpiotshitiHvEeSS,cocnoturnotlesdwearned sioneclduedded1. FA-f1t0erceuxlptousruerem,edicuemllsfor eevxeprryessoitohnerofdaythefnmourtdaenrt pthoenmoatiynptea.inThJeogcpuhlatsueresgrowwetrhe. sTuhbecuelxtuprreedssiaotnTeast pEehreiotdestforsubTsFtTa-nrceests3taxnt200autcaenltlss woafse3achdaydso.seImwmeerediaptleatleydafitnetro PexOpopseutrreito edfifsihceisenccoyn)t:aiTnhiengce1l5lm1surcvliovnailngvamsedicuonuntteoddeatfetremrin1e0-1c4elldaysusrvwiivtahl th(eclAorntienkg Colony counter or the naked eye. ") CWlMivHe,.0.1,979J,ohnVsaolni,datK.i0o.n,'anSdpeccthoarr,actJe.rF.iSz.a,tioBnatsoofn,theA.GL.517a6ndY/TBrKowHno,use ") TAnyamcphheork,a Dm.utt.a.genPaialsiseaty, sSy.sCt.e,m,TMurunteart,ionG.NR.e,s.,Ray5,9,V.6A1.-10a8n.d Salsburg,
n0:o5u.s, 1T9y8m0p,hoFmaainctellmsu.tatIIi.onsTeastt tvhaelidtahtyimoindinaendkiinnatseerprfeotcautsioinn,5M1u7t8a1tion *) FRJeoostr.zw,,ar7dH2.,mua4tnd4a7t-Hi4io7tn4c.hferlelq,uenAc.yD.,&t1t9h8e1',thEyfefiecdtisneofkin2a0secodleodcuscheinmicLa1l7s8Yon the
BCaorucsienoTgyennpsh.onaF.Jc.elldse. SIenr:resEvaalnudat3.ionAshobfysh(ordst.-)t,ernEltseesvtiser-ofNorth *) HVaonladnedr: Hoeven, 3.C.H., Bruggenan, LN. and Debets, F.M.H., 1984,
G13e6n,ot9o-x2i1c.ity of quercetin in cultured mammalian ceils, Mutation fes., *) CGlbievrel,y,0.1..3C;asp1a9r6y7,, G.u,ideKirfboyr,pePr.Ef.o,rmiKnrgehlt,heR.m,ousMeoorley,spMh.o,naMaaysos,ayJ.foarnd
manna fan 611 mutagenicity, Mutation Res., 189, 143-156.
Mutant selection AifntetrentPhSeOepxperterssidoinshepse,rfeoadcha tcootnatlainniunsgber15ofm11.s5elexct1i0v6ceemleldsiuwner(eTFpT-lated SWseeellreeeccttciiooounnn)t.evderTewKi-tddheeftitechriemeinnnteadkmeudstsaendyete.sscCrflioobrnemiedndgabmoievcferf.oiccoileonnciieess aitn t1h0e-14timdeayso,f wmhuitcanht sThuervimvuitnagntceflrlesq.uency Nas expressed as. the nusber of mutants per 10
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As CCEPTABILITY OF ASSAY e
crAoi)tmueTthreaitaia:obnsoalsustaeycwlaosnincgonseifdfeirceidencacyceopftatbhleesoiflveinttmectontthreolsfolwlasowi>ng50%.-
5) AAtusbleerastofthsruerevivoifngthecelflousr(d10o8s)esanoaflytsheedtefsotr seuxbpsrteasnscieonhaodf atnheacTckeptable
cy
Tmhuetatsipoonn.tapeous mutant frequency in the untreated or solvent control vas <5 ppeorsit1i0veclcoonnatbrollesce(leltsh.ylnethanesulfonate and disethylnitrosanine)
4) TIhneduced significant (at least 2-fold) increases in the mutant
frequencies.
DATA EVALUATION AND STATISTICAL PROCEDURES a Notfhoortmaslubhsytpaontcheeswiass tceosntsiindgerveadspdoosniet.ive (mutagenic) in the mutation assay i2bry':1TtohetihnredeuscuserodtlsvaetnwterTeecaosnrtterpoarlo2d-uifncoilbadledoisniecn-redaaenspeeinnddienenpttehnemdaennmntuetlray;ntraenfpdreeaqtueednctyestc.ompared A test substance was considered negative (not mutagenic) in the mutation ba23)ssaNTtyohwneiecifer:oefstuhtalthtesowtfeesrtteheedcsoconolfnvicerenmntetdrcaoitnntiroaonnls.isnhdoevpeedndeanmtultyanrtepferateeqduentecsyt.at Teast eThneterpreicnetdointghecrfiintaelriaevvaelrueatniootn adebcsiosliuotne.and other modifying factors might
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RESULTS
CYTOTOXICITY TEST/0SAGE SELECTION
.
ehPrxeplooismiitnniaornymaessdodiliuuumbnilaOitrtaycbtosfneaecnsectneostfroatifthnideooincbpsareteteoladfibnoi1tlnh0iaa0ctryyaTgc/-tca5iyl8vt6ao9attniodpoxrnieuccpiswityepysritdttesaea.tset(dSw9ii-tnhmtbh1.e.58I6n9 TSeeee e noeteoeaanotmeemsIttn.xst1uhbe.tsh3tep0ar6nelcsyeeeinpnCchnoeoinsbcaiotefencdtSerola-tltahseiixoignnrtoohsvefutsh3pt3eenosfstyioo/tsnahu.lebstblyyaang2cle2i%odsaianddcenloaltts in I Ole! 4 Granth of
MUTAGENICITY TEST
BSSatvereodurtoonngSe5t-hmewiaxtr:e1:ssu3e3lltettscooteo11df0000tfhyopergg//cmamyuttoacctguuoellxnttiiuuccrriieettyymmeettddeeiissuuttsmin(gT:able 1), the following
ih S5ax (E46 2(2 )i) vvaaseunsesd
aDsWaNc,ativp(ao0ts.i5iotnim.hv)eTcasobenlrtveresodl2aasdnndathp3eosstihteoiswvtetwhiectohnpoteruratclemnetftoaragbeostlhieocf
y E TEn ae taivtvuabebcoittcuhc.etaaemnuuhytrganctaoruenftrseHqHuoseftnecdcileoisnninTfgoarblavenasdris4oe-ul9secoctfoinvtceheenptlaraptapetesni,doinxsa.ndofcell
BsoEth Filnotahte pnreerseeanscee ainndtahbesemnucteantoffSr9e-qmuiexncytheintebsotthsuibnsdteapnecnedenitnduced no
experinents. TIhheese pontae neous
mrutianct fartehqeuceopnnrtcerisoeelsnceIdnatoaftheSr9a-snaogilexv;e(n1t.i.9ntdr+ieca1at.te2eddicnaornetthremoelaabnscseunl+tcue5r.e0os.f
I eTorrnedd ande4nR5aatrereseEsyspaenctdatnunidcveedblyyb)y2.1.postoiti2v3e-foclondtrfoolr DcMhNe.aicIatlswasverteherienfcorreeased by
T vatE ioinlnsaytstetnhe(tSe9s-ta.txc)ondfiutnicotnisonveedreprooppetrilnya.l and that the metabolic
CONCLUSION
I Tn cone clusioni,nde1r-58t6h9e ewxapserfomuenndtatlo bceondnoittiomnustagdeensiccribiendthien TtKhismutraetpoirotn.
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146LE 1 PCREEsL.IMINARY CYTOTOXICITY DETERMINATION IN LSLTBY MOUSE LYKPHONA
ee
Dose
(ug/ml)
oh MR wn cells/ml (x 10%)
After After After
Suspension growth
Total
% of control
Without metabolic activation (-59-81x)
solventi control 3
S6e1ia
4sa9is
37 53
6555 5s
n7a6zs7 1
111010068 5
10500
5So 2a7s a2 xEE0
%2
vith metabolic activation (459 1x)
solvent1 control 3
7c.e9 So
6573 ea
Gh 77
5cs so
101559...066
Se my
1700 54
10=000
eBsH 9 aek 010253
51
SSiobveeunntturrceoent=ro116<x1041055Cgoucntenasf/taelr 0 h x h(24eh ceells/lal) )_ x (E49Rhscuelrlts/ialnes + Test substance precipitated sTightly inGtrtiehresetsaetexynptoasfitteiron|sediaufater 240
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TABLE 2 CLoY1T7O6TYOXITCESTANDSYMSUTTEAMGENIC RESFONSE OF T-5669. IN THE HOUSE LYMPHOMA
EXPERIMENT 1
-
oGosey
CGErAoTOwR)O CGEieAlTOeAnYI
WFWAETAIENSO. POF
MFURTcAaTjIenOcNe a
Without setaboric activation (55-21)
scoont3tvreonlt 250%0
o7s
10130 102&1
@8%
wos 104
7
2i.o0 12
1o'6s To
iiss
iHse
1.
we
With metabolic activation (+59-26)
sGootmvreonlt 100
3
a
8&
13 100:
576 H
8& &
os mom 4
1100 os
00.8 oi
1i1s
o1l4
1.6
8.5
CSE4.o5l".ve=ntECtlcnooynnItinrneogtlnEaf=nfeiNscSutDlepnhcoynte DUeN e=tDistnbeetthiynieneerporseacniipnietated sTightly in the exposition medium
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ABLE 3 CLY5T1O7T8OYXITCESTANDSYMSUTTEAMGENIC RESPONSE OF T5869 IN THE HOUSE LYKPHOMA
EXPERIMENT 2
-
(DuOgSE/m)
C(%.EOLFACTOONTARYOOL) (CA.EB.SOALUTTOEA%Y)3
MMEUATNANNTOS.PEOFR MFURTEAQTUIEONNCY
PLATE
x 10
Without metabolic activation (-9-mix)
cSoonltvreonlt 3
10wm0
886
3130
15082
1008
a
78 95
2 aM ENS 8
81
11.12
00.89
02..64
01.85
2.9
2.0
9.3
7.7
With metabolic activation (+59-mix)
csoonltvreonlt 100
3
9
78
1303
791
1000
82
8881 7
0.5m OM 33
a
00..67
00.55
00..96
00..75
0.7
0.6
7.9
10.7
CSE.oMESl.ve=n=tECtlchooynnltinrneogtlhEafn=feisDcuHilSeOpnhcoynate DOMMNat= Dismuebtshtyalnncietrporseacniipnietated slightly in the exposition mediun
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APPENDIX
Individual Colony Counts and Cell Counts during Expression Period FORMULAS AND CALCULATIONS FOR LS176Y MOUSE LYMPHOMA TEST SYSTEM.
Total number of mutant Mpuetran10t%fsruerqvuievnocrys = clonin1ge00fficiency X cs olone ieseroe noF seld ectie CveATpd slates
Cloning efficiency = fAuveerraaggeelNoo..ooffccoorlposnieess eonn cclloonniinngd plpelatteesd X 100%
L5178Y MOUSE LYMPHOMA TEST SYSTEM -- cCellolnincgounetfsfidcuireinncgy eixmpmreedsisaitoenlypearftieord exposure - Mutation experiments, individual colony counts TTaabblleess 47--69 EExxppeerriimmeenntt 12 Abbreviations used: DEMNS,, deitnheytlhmyeltnhiatnreossualnpihnoenate Solvent control: Dimethylsulphoxide
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NOTOX Project 115954
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TABLE 4 LS176Y MOUSE LYMPHOMA TEST SYSTEM - CELL COUNTS AND SUBCULTURE DATA
Experiment 1
_
-- rErTTESTSTEw S e yore sre T TT1 wrPwETeET e T w e
doopseen f[ecreeilastaebnefoxre106(elcaealltseeanttter 106130) othriSone) [S1ak c5ou1n65199) rotelsnof out {c-- red x 308 (42)
Without Metabolic Activation (-59-aix)
w1o5 H6` w E3d a`` o1s3 $` 1500 `H on `
5s5i23s
P8nREo T3) o56s5s aB s0 luoao
ppxpee
n a
e35oh
55a22 s m Ea o o
ith Netabelic Activation (s53-ate)
pewds pe}
a 330 #30
w 71awm aae 3 uo
o5s 3
# % 333h G aan1 amn % oaa
ss 0aimw S &5o5wmam 3ss5 ai0e a s5 s m . Low
(1) Sseelllles baetaererrstrtoruetautemnetnt x 100% @ ScoEnIt ccooumne or camera x 1005 3) cceonlt ddeuntetttyy 10.64 xx 11003 ccrrmm
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TABLE 5 L5178Y MOUSE LYNPHONA TEST SYSTEM - CLONING EFFICIENCY DAY 0
EXPERIMENT 1
D(ousge/a1) Mcol.onionfgcoplloantfees/ 12 3
cMeoalnonNioe.s/poflate
aCblsoonliuntgeeffriecliaetnicvye (% of control)
--------------------------
Without $9-aix
csoonltvreonlt 154 ; 151; 169 3 161} 19 3 INF
151558
3130 112423} 117385;| I1N5F9 151299
1E0n0s 116637} 116259 ;; I1N26F 16114
"7 15080 &8 18021 872 18049
With 59-mix
csoonltvreonlt 187 ; 188 ; 169 181 103 11255 }111607 ; 1I4N5F 113576 10303 8163}1 116477;; 113888 134a on 80; 76 84 2) INF = Plate infected with fungi
a7 1800
&&
%7%
07
a8
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Tose
\orox project sss --
LABLE 6 LEoF1F7IOCYIEHNOCUYSE LINPHOWA TEST SYSTEN - SELECTION ORTA AD CLOVING
EXPERIMEN1T
Tusee pF rboemr ooffr ccloloannteey siE ssytecetie oen E pamtel Totafla.rotfe scolmone tiess|Heanh? 1|
Comper rth' out ? Metab Activation (-$9-1x)
1|
ssotevetnt
||0,2
y8p2
pFz11213
2211
m ow oomeo m ebe
=igg1i| bBr0i3d
B3mol o11129|293r00830343b9ii1 W102 22 30
3a0ks sak
0i ss2aww am1moo mwew w ma n mww
ea Beiy nds
|
_tin Hetaboltc Activation (459-8) |1|
ssSove--ve--tnt mol
|||
-
19
8o0y3
33s00l
z2oa1r oz1i1l
1owpeolemmomuos
iHSe o0R81| 60d
dod
BB 2 1 11 10 938 392 3333E89a505aab70a u02e32 omueou mviaoges15 UBdw
|
Us e -- = vParteatnetifnrfeeqcuteendc_ywipcehrf1u0ng3tsurvivars
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TABLE 7 L5176Y MOUSE LYHPHOWA TEST SYSTEM - CELL COUNTS AND SUBCULTURE DATA Expertuen2t
-- T (rrEETETTwRyEw T TIT i S TT w
ayEET ehEREso) roth Samant? fern S152 rota sun) rad 5 360 152)
witha watabolsc Activation (-59-xb0)
wo 6 wn 5af Gt wt
us nose es wm ao
b33g
asS00 oa3
fm am
s n hon uu
3 a se 3 dW
wien wataseisc Activation (o53-abe)
se wo no woonnw uw
wnvo ofo w w no f f
s3ie 5
oG Ss 33830 BFE oeom we soa moun
w Siawma 56m
3a a53% m mn oi ne n BNoom
1 ScenHtsRarEteRr tEreNntmNenSe x 1008
@ cence
x soon
(3) Scelladennsiity 0T.4exn1e03 aelnm
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WoraK Project 115954
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LAGLE 8 L178Y MOUSE LYWPHOWA TEST SYSTEN - CLONING EFFICIENCY ORY 0
expeRTHENT 2 Soe No. of colonies! (ug/m) gcloonninggPpleate
yr
SSoalbvreen!t 2
1B0i;s1e00l; 19
13BoD70i :o60ls } em2
Be miwiue
SSoatbvernet Te
2S0R0;i1u0o31i9m8
5 Bo
MmDpiiiwilmseiiusme
w omoeer se
Mean No. of
colonies/plate
Without s9-aix 1113 S7o wo With s9-aix 1mu9s% 1am wv7
Cloning efficiency absolute reloaftivseantron)
o7 &
1m0n0 102
% 5
%a 8
727 10>50
aa 2
a@ 3
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ABLE 9 LE5F1F7I6CYIEHNCOYUSE LYHPHOMA TEST SYSTEM - SELECTION DATA AND CLONING
EXPERINEN2T
e Joss Number of calonfes/Selectione plate Total [o. of calonies|Hean|hF |1
wg/m IETeS S el [rnreemmmzmmnmanzennzmnezesseesese | No. des |/cloning plate | |1i
rer Vithout Metafbo Activation (-59-a1x)
|
ee
Ssoltrolvoente|||n00tg0 5 100
8147 0%
o1z1 00
4021 20
2120 22
116
168 1u0s 15s
1Umz ies
2wll1 1984482354351S 21w 8 35
222 mn
mamu o1w w2 o11 6 umam3
Be toc:With Metabolic Activation (+59-81x)
IM 0s|| uubs 008s wd1e0o27e0)0
1|
a
1
SSolnolovenlt |e|2n0t0 Ss 199
g89o22020002201120 000010
20 12
576
1u wa 1s u9 e sllee
l1e0350)s| usoll
& 1a 1810 12 05
wB eoo O11E 9288950058
310% 6s
0e1sa1s0
m7m 1m9u % se is
17 oe
J WF = Mutant frequency per 10 survivors,
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