Document e5d2jeXXyvD4n5Rn7RbdO22jm
SPiBn :veNmoRTeOchX
i
REPORT
EVALUATION OF THE MUTAGENIC ACTIVITY oF Tsar
IN AN IH VITRO MAMMALIAN CELL GENE MUTATION TEST WITH L5178Y HOUSE LYMPHOMA CELLS (WITH INDEPENDENT REPEAT)
NNOOTTOOXX SPurbosjteacntce11356512617 - page 1 of 22 -
Ee T te S5r
>1
I HH
Ti CETIA
CEIVED
I-Ie eh oe
TOXICGLOGY
- .0. 80xT3o4l760,7532043HDa1mLb9"a+k75eH5newseFetoegurreibrnegoh3.aTyhesNetherands 004523
:
or
T-537M,37
REPORT
EVALUATION OF THE MUTAGENIC ACTIVITY OF T-5874
IN AN IN VITRO MAMMALIAN CELL GENE MUTATION TST WITH L5178Y MOUSE LYMPHOMA CELLS (WITH INDEPENDENT REPEAT)
NNOOTTOOXX SPurbosjteacntce11358912817 - page 1 of 22 -
RECEIVED
Nov -9 1594 004524
21
TOAICULOGY
T5874
NOTOX Project 115921
2 S--T_AT--EMENT OF GLP COMPLIANCE
NOTOX B.V.., *s-Hertogenbosch, The Netherlands
BTohsetstruedcyentdeesdcirtiiboend oifn: this report was conducted in compliance with the
i
The OECD Principles of Good Laboratory Practice
which are essentially in conformity with:
RTehgeulUantiitoendsSPtaarttes58F.ood and Drug Administration. Title 21 Code of Federal
TofheFeUdneirtaeld RSetgautleastiEonnvsiroPnamretnt1a6l0. Protection Agency (FIFRA). Title 40 Code
TFehdeerUanliteRdeguSltaattieosnsEnvPiarrotnm7e5n2.tal Protection Agency (TSCA). Title 40 Code of
vWaisthuntkhenowenxception that the stability of the test substance in thevehicle
Study Director
Ing. E.3. van de Waart
a
Date: Ge
{i
&g - pe 004525
T5674
.
NOTOX Project 115921
Q--UALI_ TY AS--SURAmNCE STATEMENT
8] vorox ev. rsohertogenbosch, The Nethertands.
|g] ssttuudsyy pspreocciefdiucresprovceersesessubgweercet taolsoperiinosdpiecctetdnsapce.cpteiroinasdianng1goeinneeryaalls"non ii]] mTheitshodrsepaonrdt rwaessulatusditaecdcurbaytetlhye NrOeTfOlXectQuatlheitysauAssduarraance Unit and the
:
DATES OFAGWAIUTSINSPECTIONS!
REPORTING OATES
JFFaeenbburruuaaarrryyy1712,36;, 11919999494 March 28, 1594
dFFeaebnbruruauaarrryyy1721,34;, 191199949944
March 23, 1594
Quality Assurance Manager
C3. Mitchel .sc.
vate: 0-4-q,
--
~ page 3 -
004526
T-5874
REPORT
_--_--
APPROVAL
N
NOTOX Project 115921
-
STUDY DIRECTOR: 3
Ing. .3. van de Waart Hex
Date a
MANAGEMENT
oTre.chn1i.cCa.l EDninriengcator vate 20 ou Iigg
- paged
004527
3
.
i T-5874
--
NOTOX Project 115921
: PREFACE _-- sponsor
Study Monitor Testing Facility
Study Director Technical Coordinator Study Plan
00
C3anaBdealsgtiruanat- C11hemical EaC 8-2070 ZHLINDRECHT
-
8elgiun
Mr. RH. Cox
NHOaTnObXakBe.nVw.etering 3 5T2he31Ne00the'rsl-aHnedrstogenbosch
Ing. .3. van de Waart
C.M. Verspeek
sCotmpalertetd:: FJaenburaurayry181,4, 11999544
TEST SUBSTANCE EE
IDdeesnctriifpitciaotnion PBuarticthy Specific Gravity Itenssttruscutbistoannsceforstorage coSntdabiitliiotnys under storage
Expiry date
S4thaobulresfoirn vatehilcelaest
TC-r5e8a7n4solid 2130300% 17
At room temperature in the dark stable January 01, 1996 WDaitneerthylsulphoxide:: nnoot indicated
VEHICLE ---
qpIruhiaeolritteystto (Husesurebc.skt).aTnhceeTefswitnaaslsudcbiossntscaoenlncvteerdactoiinnocnedniotnfreatthtihyoelnssuslopwlhevorexenitdperinepoaftrheesdpceudcltitrruoersecsotpmiecdium amounted to 0.8% (v/v).
i ee------------
- FIs
004528
3
R
3 T-5874
NOTOX Project 115921
)
GUIDELINES --- gIuhiedesltiundeys:procedures described in this report were based on the follotwing - TOGourxigidacenolilisonagetysi:onfIonrfoVrTietsErtcoionngoMmainocmfalCCihoea-mnoipceCaerllaslt,ioGenGnueiadnMedultiDanetevieonlnoo.pTme4es7n6tt:s*(,O"EGC(eDna)ed,toipctOeEdCD April 4, 1983)
- TEEoEuxCriocpDieitaryne;ctEic`vOoetnhoe6mr7i/c5E4fC8fo/emEcmEtuCsn,-iMtuyPtaar(gtEeEnCBi):c,iMtDeyit:rheocIdtnsivfVeoirtr8o7th/eM30a2mD/meEatEleCir.amninACanetnlieloxnGVeonofef the Mutation Test*. EEC Publication no. L133 (adopted May 30, 1988).
ARCHIVING _--
NprOoTtOoXcoBl.V,. data.
rewpilolrt,arctheisvtearthteiclfeollroewfienrgencdeatasanfpolre,atallleasstpec1i0meyneasrsa:nd
raw
0BJECTIVE --_--
Purpose of the study
LTa5hb1ei7l8ioYtbyjmeoctutosieviendlouycfmephtohfmioasrwascretlduldsym.utwaaTtshieotnaossseavaytalwutaahsetetchotynhendiudctitenesedt
skiunbassteanc(eTK)forlociutss in in the absence and
pbarseesenpcaeirofmutaatmieotnasb,olifcramseystsehnift(Sm9u-mtiaxt)i.onsTheandTKsmmaultlatidoenlaeltiosnyss.tem detected
J Justuificsatit onfi foorsf eselleei ccttiic oonnooa ffttht heetetiessttsosyyssn tteemm onLuf5t1am7tu8itYoangmaeolnuiscesysaTtcyetnmipvhioistnyaabcolfeelalstobraodraeedteucsrteadnbgaebseeocafupasciheremtiahcleaytleraarcteliaossnsesen,ss.itfiTrvhaeemeTixsn.hdiifctators Cn(eTulKtl+a/st-iodnetosficaTinKed/n-ts)mailnalrethdyerlneeistdiiisontneasn.tkintaosethe(TKc)y,totdouxeictoeftfheectfsorwoafrdthemutpaytriiomnidine natuhncealleoaogntuaieldoegtsureinfeilenudtooerdotihtfsoyrmpicdheiolnslepuhlo(arTryFTl)am.teetdaTbKodlediresfimivcaitaeirnveteobc(tenlaulicsnleedoctaisndoeln)eo:lty tihfhereoomrpdoerate nroeivdcoihunsyancrteohnetlsaeiitsnh.ialngIntoTFtThtehearpecreemlsluestn.acteeTdho,ufs,TeKi,tcheelTrlFsTspwihosinctchaonnewvoielurlstleydsuorirvnitvobyenutichnelecouatclittdiueorsne, oAtoftbeteshteataperostttiecnlsteuibaswlthaincnchaem,nianlgdiiuavcniesngcealrlpiosesmuitttaiogveean.TrKespdoenfsiecieinntthpihesnoatsyspaey. is presumed
- page 6 -
004529
i RE
NOTOX Project 115921
)
MATERIALS AND METHODS
TEST SYSTEM -_-- Test Systen Rationale Source
SThteockculctuulrteusrewseroef
0_ L5178Y mouse lymphoma cells RtEEheCec)o.grneiczoemdmenbdyedthetesitntseyrsntaetimon(ael.g.guEiPdAe,liOnEeCsD.as
GSOtre.anteetAi.cGU.snAi.avCne.drsiCKtnhyaeampio,cfalDLeeipMdauertnta,mgeennTtehseiosfNetRofhaedritlaheatnidosn oT(ir1ni9e8g,1i)n.ailslToyhliastdeedrmiovubesydeClfliryvomenphot(mh1ea97Fc5ei)lslcheTrineL51wa7s8.Y Ll*)oacbuoCsrlaitvpeou,rtyatD.ipornoascneddautSrpeetchtefoorrT,K asJlso.ce.usSss.i,nign 1c9s7up5le,tcuirfeidc 3L15,1781Y7-2m9o.use lymphoma cells, Mutation Res., tchheescekedcelflosr mwyecroeplsatsomraedcoinntalmiiqnuaitdionnitrogen (-196).
CELL CULTURE _-- cell culture conditions
F10 complete culture medium Selective medium Cloning mediun
FL150178cYompmloeutsee incubated in
cluylmtpuhroemamecdei1ulms. weCrueltucruelsturveedrein a hunid atmosphere containing 5%
C05 in air at 37C. Cell density vas
pwceorrmeepfleeretaxebploycsueldkteuptrtoe btmheeeldoiwtuems7twxistu1hb0osuttacnecsleelrsu/immn,l.F10Cells
buffered with 20 mM HEPES.
FF(11600ibccnooe)md,piluesntuepwpiltcehumoleutntutreetdhymwmeiidtdihiunme10c%oannhsdoirshstyeepdoxseaornfutmhH,ianme'L-s g(l50utaUn/mi1nean(d25m0)uga/nmdl perensipceicltliivne/lsyt)r.eptonycin Sc0ue.ll3te3uc%rteiAgvaemremdeNiodubimluenwhic(coDhnisfcicoso)tnteadainndoefd5,F1u0gi/nmcloamdTpdFliTettieon (Signa).
Ccseluroluntmiu,nrgewmmheiedcdihiuunnw,ascosnutsrpiapsnlstefenodernmtoeefddF1wi0intthocoam1p0g%leelhtoerbyse
addition
- page
of 0.33%
7-
Agar
Noble.
004530
i Environmental conditions aAtNmosipnhceurbeati(o8n0s-95w%e)recocnatrariineidngou5t%iCn0pa hinumaiidr
pienrctehentadgaerkvearte3m7oCn.itoTrheedtdeumrpienrgatutrhee and CO-
experinent.
-
REFERENCE SUBSTANCES
i
ee
TVNreegeatniveTceonotTrroelt:he test articre.
Positive controls:
:
EWitthhouytTmnetsabtolhiac nacetisv(uaEtTNiSopn:hGAa(S-Sn9-hmaei.x)t:3e3-50-0; (2 mM) was used. EMS causes direct alkylation
opufriDNtAy. 98%;
Janssen
Chimica)
OW(i0it.h5ntmeMe)tEawbRaoslyicuTseaadc.rtiEOv(aMrOtHiohoansdCaht(+on5n9bi-eemniax4)ec:t5i.v7a5t-e,d bpyurmiitcyro9s9o%n,alJaennszsyemnesChipmriecsae)nt in ctheelluSl9a-rmixD,RA.resulting in a methyldiazoniun fon which could react with
Hab Snolkv"ea snts l for a Resfean rletnscc eolSuute bisotnad nwcietshout calcium and magnesium. Solutions of reference substances were prepared immediately before use.
METABOLIC ACTIVATION SYSTEM -_-- Preparation of $9-honogenate"
0
oRratSplriavgeuremiDcarwolseoymarlatse,nzwyhmiecshwewreereroobuttaiinneeldy pfrreomparBReLd, fSrwoimtzaedrullatndm.ale Wistar TIlhanebtoraranapitemorarilytsonceowaneldrlieytihowoniusts,hedaasatsodleNusOtTciOrXoinbeidn(20ian%swpt/ehvec)iaSlOoPf'rsAo.roomcTlhuoenrderra1t2s5s4twaen(rd5e0a0rdimngj/ekcgtedbody sw(aetcihrgeiyhfti)vceeri)en. cdoTehrneniedloiilva.ecrcseFsisovfetotdhaeyfsoordaltastfeorrw,eraettherlyeemawsotevreed12aksihelopluteridscapblyrleycd,eedciaanpndigtawtaisohne;d in Nicaongl3dEDv(To0Al.Cu)meSsusbtseoefrqiul`eephnot0sl.py1haMttehesobudTfiifuvemerrsphwiowtsehrpehaatmeiPontcbteuedfrfeirhnoma(opgbHelne7i.nz4de)err.caoTnnhdteaihnhoiomnmogoggeen0ni.az1teedmM wianstocsetnetrriilfeugeadmpufloers,15whmiinchatwer9e00s0tog.redTheinsulpiqeurindatannittro(g5e9)n w(a-s196t9rCa)n.sferred
- page 8 -
004531
iJ ren
NOTOX Project 115921
*) mcAunaetrsac,giennoBigNcein.tsyaMentdesCtna.nunt,aMugte3.antsiaownnidthRVeasmt.sh,seak3Si1a,,in3o4.n,7s-131159a47/5m,annMaeltihaondsnffcorrosdaeetsect_ing Preparation of 9-nix pS$ho9o-:smmpiihxxatcweoa;nsta3pi.rn4eepdnagrpeedNrADiPmm;lm:e4d1i.ma0to2e)lmygHEbPeMEfGSColrpe3.n6duHsp6e0.;5and2ai.4k6eSop.tmgTonhKCeTi;acebo1v.de7urimsngoglagrtlhouencstoessstge.. oMpvfeheritecacheblomllwiiaxcsseudsaapdcaedtnneidsdviaFotainifl.ottenerrwaF(si0i.ta2ec2rh-iusemtv)ee-rdsitlebiryzialatidizdoeindngo(0fa.p2atrhemt1 $f9lr-iomvmiexrthcSeo9m-$pm5o-ifnxreantotc.et)iw.oanc:h m1
EXPERIMENTAL _--
PROCEDURE
Selection of Dose Levels/Cytotoxicity Test
tErrieoantintgo {angHeEPEoSf,
6thex in
t1a0 hcetuaacleblslemsnu,cteagseoufnsipsceeinrtdueymd teiisnnt a6 csmyltteorotifolxeiFc-1i30t0y mmldeadtciaeunmtwrbeiurffeufegorebetdtauibwneietdhwbi3yt0
test substance concent
a
brootthateidn tfhoer 3abshenocneaanrdollperresneinxceerratoifaotnSs93-7miCin,x.appThreoxicneantterliyfughealftubJeosg seterps.
sbAyufpte3errncatethnaetnrtiefxupagonasdutrireoe,nsusstptheeenpsscieol(nl1s15ofweg,rtehe8 scemeiplnal)r,sa,teedatcwhifcreofmolinltohewHeadntkr'ebsyatmbreaenlnotavnacs)eodluostfiaotrones.
escxoopulrnuettseisdoendinaansdantfhi"enInaplrrleoydvuecidtnioFNn-e1ua0bfatmueeerdriuamph.parcomTxohicenyatctaemelelltyser2i.4n compared to nontreated control cells,
Rtehleatfiivnael cystuosrpoewnisciiotny.were h and 48 h growth
nccouotlntcuteronextsircatpiaroennpdaorrreadngdeifforf(i4cmuudltotasgeset)noicodiiftsystohletveesttewisaintnsg.asuqsuuebIedsnotuactsnaocseesdoeltttuhoeetriaboietnnesesatpptahlseiusebudshititagainhnbcelese was
gceonnecreanltrcaotnicoenntwraastidoentsermeixnceededibnygt5hemgs/omllubiwleirtsynoitn ttehsetecdu.lture medion. tin
Cleansing s21fpnroiidnnoatryoapnttieenooruimcsnuulttaamuungtrdeaenn1it.cms6ie,tdxyiufmo1(l0alnc>odowneMtcdayittbnhoyiytnnogiaxidcir10ine-tec3y)(MHtAheTys-ptmoiexndgaf,nut)htihnetoe',cer2ledlusxce1v0e-rt7eheMgarmowonuntfoorf ercxeoptneutrraininenedinntgt.o hnyopromxaalnthmiendeiumandat thlyeamsitdinfeor oon1vleydr.ayy Apfbeterefiroordetohsfista2rptediraniygosd tonhceemlleediuvmere
- page 9 -
004532
T-5874
NOTOX Project 115921
Mutagenicity testr
sfTfeohnrledeecpteteesesntdtdendssotuuesbbxessptteacarnenclcelensewcnwaotesnsrc.etnets6rtaextdi10obnosthceexlwlpisetcht(ea1dn0d%/tmow1i)bt,ehosourttro1Sn23g-laxyix1t0o8ixniccet,lwlosfor(1w0a6c/hm1) 3sb7uusfCpf.eenrsSeidoolnvHseanntwseraenFd-p1pl0oascmieetddieivuiemnexc3wpo0iontsthemrldooulctfseonrswteerr3ruinefh.ugteFoorttuhbetehsitseoshpturaspuorbsoseltlaentrhceemieipnnle]rHePaete. eewevxreperryeswsoaitsohhneerd odftawytihceein mwoiurttdhaenrtHBStpSoh,emnoactioynuptneat.iendThlaeongdcpusihlenatecsudleerudedesgdri.ownwetrAFhef-.t1e0sTruhbcecueulxletptxouusprrurereeedsm.sedaticuemljlessscoer dtphieesrhieotsdestcfoonsrtuabTisFntTia-nnrgceesi1s33tamxlnt20c0lmountciaennlgtlssmewodfaisuena3chtdaoydsod.seeteIrwmmemirenedeiapctleaeltlleydsaufirtnvetirovaPeloxopoi(stpouelntroreniintgo cefofliocniyenccoyu)n.terTheorcetlhle snuarkveidvaelye.was counted after 10-14 days with the aptoy
*) MlCly-inMvpMeh.,o,na0.1,m97u9tJ,aoghneVsnaolnia,dsastaKyi.o0.ns,ysatnSedpme,ccthoMarur,taacttJi.eoFrn.iSz.aR,etsi.oB,nat5so9of,n,th6e1A-.1GL0.531.7a8ndY/TBKrowMno,use
*) RmDAe.mosSau..cs,,heer71l,29y,8m0pD,4h.4Eo7.mP-,ao4i7cnP4etalillmslu.etta,tIIi.oSn.sTCe.sattTvutarhenleirdt,ahtyimGo.iNnd.i,naendRakyii,nnatseVer.Ap.rleotacanutdsioSinan,lsLboMuiurytgaa,ttion
*) Jfootrzw,ardM.muatnadtiMointc.hferlelq,uenAc.Dy.,at19t8h1e, thEyfmfiecdtisneofkin2a0secodleodcuscheinmicLaallsoeyon the
Hcmoaolrulcsaienndol.gyennps.hoaF.Jc.elldse. SeInr:resEvaanlduaJt.ioAnshobfysh(oErdst.-)t,ermElsteesvtiser-oNforth
*)
SVeannotdaexricHioetvyeno,f 136, 9-21.
qJ.uCe.rMc.e,tinBruigngecmualnt,ureId.M.mamamndaliDaenbetcse,llsF,.M.MHu.t,ati1o9n84,Res.,
*) mCGlabimevrmela,yl,ia0n.T,.Jc.eC,ialsp1a9mr8uy7t,,ageWGn.u,iicdieKtiyrf,boyr,MuptePa.rtf.io,ornmiKnRrgeesh.lt,,he1R8.m9,o,usMeo1o4r3le-y,1n5p6Mh..o,naMaaysos,ay3.forand
Hutant selection
sAIeenfrlteeetcretcniotouPhnn9e)t0.eedpxeTptKwrr-ieidtsehsfidiiotcshnhieeepsnne,tarkiemoedaudcthaeaynetct.soontatfClaolironmnnieiudnnmggbemrie1cf5 rfoomiflccoi1l.so5enlfexecst1i0vien c1me0el-dl1is7umwdear(yeFpsI,l.awtheidch sTshueerlveicmvtuiitnoagnntcweeflrrleesq.udeentceyrniwnaesdexaspredsessecdribaesdthaebovneu.mberenocfiemsutaatnttshepetrine105of mutant
- page 10 -
004533
T5874
NOTOX Project 115921
or
$ | sccerraaniary oF assar
-
-_--
Ac6B))rimtTAuethtreaitlaiea;aobsnstolausttshearyeeclwaaonsfinctghoensefiffdoieurcrieeddnocsayecscoefopftatbhtelheesotilefsvteinttsumbecsottnattnrhcoeelsfhoavldalsowa5ing5s0o8s.s-ptable
nnuutmabteirono.f surviving cells (109) analysed for expression of tne oy
) @
TT<ihhn5eeducppseeopdrsoint1sti0iav%gneneiocfulcisoocnnaatmnbrutlotelasn(tcaet(leflrtlseeh.aqysultemne2cty-hafnioenls)utlhfeioncnuranettaerseeaas.ntdedindioratheetshoymllvanecinttsrocsoanntirnoel)
was
frequencies.
-DA_T--A EVALUATION AND STATISTICAL PROCEDURES
NAito:tefsotrmaslubshtyapnocteheswiass ctoensstiidnegrevdaspodosniet.ive (utagenic) in the mutation assay
@) 5)
TIthtoe tihnredeuscuselodtlsveanvtterTeceoansrtterporalod2ui-cn0i1ba0ledoisinnec-rdeaneaspeeinnddienenptethnemdeamnnutntleayrntreanpfderaetqeudentceystc.ompared
Aa@)sstaNeyosnteifs:oufbsttahenceteswtased ccoonnsciednetrreadtinoengsatisvheowe(dnota teice that of the sotvent control.
mutagenic) in the mutation mutant frequency at Teast
5) The results vere confirned in an independently repeated test.
Tehneterpreicnetodintghecrfiintaelriaevawleuraetinoont daebcsiosliuotne. and other modifying factors might
page 11 -
004534
T-5876 RESULTS
-
NOTOX Project 115921
CYTOTOXICITY TEST/D0SAGE SELECTION _--
-
P1323r03elimuggm//immn}larybauntdsolpupurwbeaicrlidipstiytatteedstsinitnhdeicaextpeodsitthiaotnT-m5e8d7i4umwaast csoonlcuebnlteraitnioDnMsSOofat tTIhnaeblteahebs1epnsrcheeoswesonfceth$e9a-ndmriexasbuTsl-et5sn6c7eo4fofithnehaibpmiretetealdbiomlitinhcaeryagrcotcwiytvthaottiooofxnictihsetyystleytmnepsht(oSn9wa-imtichxe)lT.l-s58I7ni4n 3st3hu3espgeurgno/smwiltohnboyaft81at%h.eteIsTntyastphuheobmsatparneccseeelnlcsceonioncfenstuSrs9a-pmetinixsoinotnhoefat1te0s30t33ugs/uumbgls/tlabnycbey584%i4n%ah.nidbitaetd
MUTAGENICITY TEST --- BdWoaissteehdourtoanng$e9t-hmewiaxsr:esse1u0lltesctoteo3df33tfohreug/cmmyulttoactguoelxntiiuccriiettyymettdeeissuttimn(gTable 1), the following With $9-mix : 10 to 333 ug/ml culture medium EaaMcsStsiavy(a2twiinoMtn)h, mwaewsthaebrueosalesidcaDaMsNc,taivp(ao0ts.i5iotnin.Mv)eTacsboelnretvrseodl2asainndat3hpeosshitotewisvtethweciotnphteorurotclenmtefaotgraebsotlheiocf ccToe-lu5l8n7t4ss.udruvIirnvidanilgvidasuunabdlcutlchteoulrominuyntgacnotaurenftrse1iqsuotefendccileoisnninTfgaorblaenvsdars4i-oe9ulsecotfciovntehceepnltaarptapetesin,odnisxandofcell Isilnnicgrthehtae,seablesisnesnctehtehaonmfut2Sa-9nf-tomlidxfretihqneucernetcaeysset insinubesxtthpeaenrcimemuetnaitnntd1u.cferdeIqnuaeenxscplyeirgiwhamtsen(to2b.s23e-rafvoeldd Hsfouowbledsvteairnn,cethtehies sceiocnnocsnrieddaeseerxepdeirnniomttehnetm)umtutaaagnndetnincoftreidnqousteehnecyraeblwaastseendcn.eotoTfhreerSpe9rf-oomdriuexc,ibltehe (tneotst2Iinncrteheasepreinsentchee omuftaSn9t-aifxreqtuheenctyestinsbuobtshtanicnedepienndduecnetd enoxpesriignneinftiscant Tv$h9e-ermeisxpwoitnahtnidanne1o.ou4usr+mh0ui.ts8atnotriincfatrlheeqcuopenrntecrsioeelnscediantoaftheSr9a-nsagoielxv;e(n2.ti0-ntdr+iecaa1tt.e2edd icnaroentthremoelaanbssceu+nlcte5u.r0eo.sf 5ff-uotratnont=47g-ffroaelnqdduen4foc1riersEeMsSpienacdntudicveebdylyb)5y- potosit1i0v-efolcdontfroorlDMcNh.emIitcalwsaswetrheereifnorc,reased by cacotnicvlautdieodnthsaytstetmhe(st9e-smtixc)ondfiutnicotnisonweedreprooppteirnlayl and that the metabolic
CONCLUSION -- ee ------Iu-- nndecro-- nctlhues-- ieoxnp,e-- riTs-e5n8-- t7a4l i-- sconndoit-- timountsagdeensiccriibnedtheinTKthimsutarteipoornt.test sys 04535
F 17-5874
NOTOX Project 115921
TABLE 1 CPERLELLSIMINARY CYTOTOXICITY DETERMINATION IN L5178Y MOUSE LYMPHOMA
--- D(ousge/ml)
--
AftercellAsf/tmelr (x 1A05f)ter oh 2h 8h
OOO Total Suspensio%nofgrcoowntthrol
solvent1 control 56.29
103.3
4681
13030
5429
333
55
Without 65.13 a42s 3al1s 10
metabolic 4a57 sal4a a50s1 37
activation 6558..13 3643..70 267..79 127
(-59-mix) 18090 593% a722 15
solvent1 control 66.45
103.3
6604
10303
6632
333
55
---
With metabolic activation (+59 mix)
66.00
66.74
19070..55
110030
s6l6s
6611
10804..74
18073
6724
55.79
18057..64
51
500
54.8
18180 56
*
SSuoblcvuelntturceont=ro1l.6 = x DM10S5Q No subculture
cells/ml
* Total growth = Cell count after 0 h x (24 h cells/ml) x cells subcultured
c(4e8llhsceslulbsc/umlltured
*
Test
substance
precipitated
slightly
tdrieraetcmtelnyt after in the exposition
after medium
24
h
- poses ees
004536
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NOTOX Project 115921
TABLE 2 ICNYTOTTHEOXMIOCUSAENDLMYUMTPAHOGMEANICL51R7E8SYPOTNESSETOFSYSTT-E5M874
EXPERIMENT 1
-
o(uogS/Ea1)
C(%E.OFACTODNTARYOOL) (CA.BE.SOALTUTEDA%Y)S MMUETAANNTNSO. PEORF
PLATE
coSnoltvreonlt 1303 130303 2 aM EMS
Without metabolic activation (-59-nix)
110020
6601
103
6
22..41 34
5700
762
1488
129
60
14.2
coSnoltvreonlt 1303
130303: 0.5 mM OM -_--
With metabolic activation (+59-mix)
1080
5578
94
59
1138 0's
9621
859
20.56
30
38
u.3
SCo.El.ven=tCcloonntirnogl E=ffDiMcSiOency OEMNNS == DEitmheytlhnyeltnhiatnreossualnpihnoenate * Test substance precipitated slightly in the exposition medium
MFRUETQAUTEINOCNY
x 105
22..63 53.82 1s 15.9
12.51 0160 2.1 19.8
- pogets -
004537
T-5874
NOTOX Project 115921
TABLE 3 CINYTOTTHEOXIMCOUSAENDLYMMUPTHAOGMEANICL51R7E8SYPONTSEESTOFSYSTT-5E8M74
EXPERIMENT --_-- (puogse/m) _--
coSnotlvreonlt 1303
12030 2 ah ems
2
C(%E.OFACTOONATYROL0)
Without metabolic 19050 10569 65 91
C(.AEB. SOALTUTEOA%Y)3
MMEUATNANTNOS. PEORF
PLATE
activation (-$9-mix)
8958
45.82
99
6317
97
5.2
91
23.2
MFRUETQAUTEINOCNY
x 10%
23.03 a235 36 17.1
With metabolic
coSnotlvreonlt 100
1303
8922
10303
9%1
osmom a _--_--
CS.oEl.ven=tClcoonntirnogl Ef=fDicSiOency OEMMNS == DEitnheytlhnyeltnhiatnreossualnpihnoenate * Test substance precipitated
activation (+59-aix)
10738
33.24
10730
2134
101
39
[a
14.9
slightly in the exposition
22.82 112 26 21.6 7
medium
TP.
er----------------------
004538
2? T-5874
-
Sse
~
NOTOX Project 115921
APPENDIX
Individual Colony Counts and Cent Counts during Expression periog FORMULAS ANO CALCULATIONS FOR LS178Y MOUSE LYMPHOMA TEST SYSTEM,
Mutant frequency per 10% survivors
=
100
cloning efficiency
Total number of mutant X colonies on selective plates
nunber of seeded cells
Cloning efficiency = Average No. of colonies on cloning plates X 100% 2
LS176Y MOUSE LMPHOWA TEST SYSTEM + CCleonincgounetfsficdiuernicnyg ienxnpreedsisaitoennypearriieosd exposure
- Mutation experiments, individual colony counts
Tables 4-6 Expertnent 1
Tables 7-9 Experiment 2
Abbreviations used: OMN, dinethyinitrosanine Solvent control: DEiMSm,etheytlhsyulTmpehtohxaindeesulphonate
EE
+ pegs -
004539
H5 orse
.
NOTOX Project 115521
ETAxBpLeErim4enLtS1718Y MOUSE LYMPHOWA TEST SYSTEM - CELL COUNTS AND SUBCULTUR.E DATA
Tr T ! RT frre STwSyn oSr TT mmm --------
dose (ug/al)
f[tcreelaltsaebnteforxe 10|[tcreelalstmeanftterx
| 106]%1)
|x [Total
10 wmount
Cell eral
count] x 10932)
|x [Total
10 amount)
Ice1d [cial
count x 10
| 1x2)
ithe metabolic Hetiuation (59-210
Bw 5 Fsr l FE
iiisI 5
88 BF oa onn E ommm oE wx ox ou B FE m .de
BA Son mon on nonoan
we 5 For FHpd itn h HBFeatoeEsdsT eEsActm ivatioo n m (59ao) mar Bm0o8w
EBIs
B35H BBao oaowsfongo uon mnnoooounR
@ SsetHts EancRe tress + 1008
@ cont crouena x 1000
3) cell density 0.4 x 10% c/ml (4) cell density 1.6 x 10% crml
eget A
004540
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NOTOX Project 115921
TABLE 5 L5178Y MOUSE LYMPHOMA
-Ex_p--eRIHENT 1
D(ousge/ml)
_
TER cNol.onionfgcoplloantiees/
Solvent
control 10
10303 3i3n3s
109 ; 105 ; 108 111 ; 113 ; 104 106 ; 112 ; 112
1o97i26:60095 98; 68; 60
TEST SYSTEM - CLONING EFFICIENCY ORY 0
MceoalnoniNoe.s/polfate
Without 9-aix
OOo Calbsoonliuntgeeff(rieocliafteinvcceoyntrol) 0TeTemen
107
109 110
a5g6 &i 2%5 75
54
100
55
102
55
103
38
70
Vith s9-mix
cSo1on0ltvreonlt 1 168;91714 ; :16 a16o8
700 1a00
_3o30n330i3 01156069209;10101555029)55; 011s653s3 dl1so5oos8 ov 725789 3a2904
* plots .
004541
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NOTOX Project 115921
TABLE 6 LESF1F7I8CYIENMCOYUSE LYMPHONA TEST SYSTEM - SELECTION DATA AND CLONING
EXPERIMENT 1
Dose ug/ml
_--
Jtunber
Ifetezs
of
3
colonies/Selection plate|Total|No. of colonies|Mean|MF 1I
as 78se ao Ken L [/clonie ng platr e | ] | |
| Without Metabolic Activation (-s9-aix)
|
Solvent |
I|
control | 0 3 10
34
30
22
13
21
120 123 116
| 120 2.3]
0Bo ldz BBISZ
144
128
os42s48
400523
552461
11217 01059002119 0261
633330 a23m%0 24 18
1We4oo1y2n2 s1h2a8 1s ous i
11i12ag1 a21.8n64
_--
51T 2 wz T 105 115 154
119 ioe) es
| With Metabolic Activation (+59-mix)
i
oSolvvent 1|
I
|
cBoBnJtrol 0BIE
1 |J0o1 1r0 0 1 2i 123 0 1 124002320 10 2003 0 000 213221
115385
o 1L8oin 1s7 1ae 4y i1e6s i2oe.1a] nuim soe
_BM-- 1201l3 131501017131210 1558 1023 l7s5 13933 1ar le36 o1aa
TFT Pe1
MF = Mutant frequency per 105 survivors
-pages -
004542
H Tse74
~
NOTOK Project 115921
TExApBerLi7EmenLtSL278Y MOUSE LYMPHOMA TEST SYSTEM - CELL COUNTS AND SUBCULTURE OATA
T iE Tw r p a
Cdoosere [ceelslstabenforesar[caellts aafterx 061| 20 |tockx 10mone)[Iceek) ScouSngOi ) r| oelx 108 et IS5c2e3 cSouOntE |Loy
Vithou etabetsc Activation -59-ebe
w w5 woo f d 8
5o33 b5d
B aB@ odod 3e0oe nwmroa moakwen oma
ncaoooomn oa
wo
="
eBoosd Rin oghuoun Sooon
itn etanalsc Activation (oronte)
ofForI Bwoo
555u3l
e eB a io o se sa o n sa n m ywe uiae Boose
38 go8 ona 8a
md
io a sh awm lowhou B50a3
sSotEatRten tress 1 100m
@ eE on cor m r 1008
&(3) amy cell density neon 0.4 x 10% crm)
-porter esect ece
004543
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NOTOX Project 115921
TABLE 8 LS176Y MOUSE LYMPHOMA TEST SYSTEM - CLONING EFFICIENCY ony o EXPERIMENT 2
fost)
(ug/ml)
Mos,of colonies cloningEPR plate
Mean No. of
colonies/plate
Cloning efficiency absolute (r%eloafticveontro)
Without $9-mix
Scoonltvreonlt 10
113487;| 114374;} 11431
11357
7] 19050
33
3e130s30
155 ; 147 ; 158
153
1983734!}; 1385552; :I19N3F 189m5
77
106
46a3
55&9
With S9-aix
cSoonltvreonlt 168 ; 149 ; 189 169
as 100
1103003 333
11113660671;}011116234579};;
I11N46F9 156
111l635o394g
on
750 ew
7a780
829%2
7
1
&
3s
a
INF = Plate infected with fungi
_
- Pago
oo
004544
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NOTOX Project 115921
TABLE 9 ELF5F17I8CYIENMCOYUSE LYMPHOMA TEST SYSTEM - SELECTION DATA AND CLONING
EXPERIMENT 2
IDoAseT
[e Number of cs oloniN es/Selee ction plate|ToNtoa,l|[N/oc.onoifngcoplolnaitese||Mean|HF
|| |
--- 11 23 45 6 78910 [1 2 3 | | |
| - -_--
Without Metabolic Activation (-59-mix)
|
Solvent |
| I
c1o0ntrol 33
|160 34 127
4483 41
5572 23
5875 85
6675 32
5
11590
21827
117823
191750
3.3|I a0
3 189 19% 203 197 2.5]
133030 EY
112085 12027
4254 2624
5476 1615
5623 2825
6753 2324
56&21 232
210809 180
l19e5 180
11920 11987 4336 184 101 17.1
---
TT
| _--
With Metabolic Activation (+59-mix)
|
i seaomltvreonlt || 33
10 133
43.84
1452
4423
2404
3 32
200 26 19 206 2.2| 14 150 171 15 2.
10303 333
[l'4190 136
2110 75
3146 21
6130 43
2200 53
2130 21060 2000 113984 214060 11.a6 39 180 223 200 200 2.6
oN ---
11215 1218 1716 1813 1216 19 77 105 9 9221.6]
TT |
MF = Mutant frequency per 105 survivors
- pages-
004535
TA