Document dQyJkoXKg9EeLNzQ5rvnq71d0
gr 25, 1399812:41PK POREDICAL 220 ZF 02c@oucuTess + E7zi77s %0. 0773 5253
'
REPORT
EVALUATION OF THE MUTAGENIC ACTIVITY OF
1.6506 IN AN JVNITRO MAMMALIAN CELL GENE MUTATION TEST
WITH L5178Y MOUSE LYMPHOMA CELLS
(WITH INDEPENDENT REPEAT)
NNOOTTOOXX SPurbosjteacntce22738475481
* page 1 of 31 -
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i 001362
UN 25, 1398912: 41PM POHEDouICAL 220 JFE Q2ENMOUTERSTERS+ 67331773 Ho.0mE3 E3
T-6906
S-T_ATEMENT OF GLP CoupLIANCE
NOTOX Project 223458
!
NOTOX B.V., 's.Hertogenbosch, The Nstherlands
The study
conducted
described in this report has been correctly
in compliance with the most recent edition
reported
and
was
of:
The OECD Principles of Good Laboratory practice Which are essentially in conformity with: RTheegulUantiitoendsStPaarttes58F.ood and Drug Administration. Title 21 Code of Fedora) FTehdeerUanliteRdeguSltaatteisonsEnPvairrtanm16e0n.ta) Protection Agency (FIFRA). Title 40 Goge of FTheedrUanlitaRseguSltaattieosnsEnvPairreonm29e3n.tal Protection Agency (TSCA). Title 40 Code of
Stuy Director: CM. Verspeek-R1p
OVop=--=""
date: 271 Oecede ww
Manageneat EHIncegoa.dtoxoifc3.oGlaonvgeaytnicge8waart
ym
te
- page 2
:i
}
001363
25/108 25, 1399912:41Pi PCEDICAL 220 ZF 024@N.Lo+u67T3E31R77S8 HI NH oe
.
7.6306
NOTOX Project 223458
_-- QUALITY ASSURANCE STATEMENT
|
NOTOX B.V., 's-Hertogenbosch, The Netherlands
Tthheismertehpoodrst awnads raeusduilttesd abycctuhreateNlOyTOXrefQlueacltitytheAssruarwandactea.Unit to ensure that
DwTeuhrreeindgaitnetssphoecotofendQ-.suiatleityInAsspseucrtiaonncse pirnoscpeedcutrieosnsapanpdlicaaubdiltes toa.rothigsivetnypebelofowstudy
DATES OF QAU INSPECTIONS/ AUpITS
:
on-site inspections
1627-.0011-.11999988 1911-900-.03023.-.111599599888 protocol tnspect fan 112.1997
report audit
22-04-1998
REPORTING DATES
2176.:0011-11999988 211025-..000233-..113993993888 31.12.1557
22-04-1998
Head of Quality Assurance
C.J. Mitchell B.Sc
Date: 22-14 - 9%
- page 3
!
001364
Loon BEST COAVAPR AY BLE TESA, 25. 1309312: 417K POFUEDICAL 220 2E Q2ENiuouTERS e733t7TS
0.0773 NP. 35 pea
T6906
NOTOX Project 223458
_ SUURY --m----
ocTeuhltilsastiroeinpn sortthaet dpetrhseecsreitnbhceyesmidatnihdnee aeKbfisfneeancsctees
olfocuTs-69(0T6K-Tooncutsh)e of S5-mix.
iinndLusct1i7o8nY
moofusfeorwIayrmdphona
f1S$r05a5c9mCt0oi6nocnewi.nantsrhianteteitsohftneeisdrFsitourfpsatn1td0o00sceoacnnocdnedn7t5re0axtpuiegor/nimsmlenotffn r4et2shepaencpdtre4is5veelnuycg.e/m)Tof-6i5on%06th(ewviavsa)bstSeeons.cteed oifp
and secons sxper fnentrespoct ively.
AU these dose Tavole appropriate cytotoxicity was observed.
.
STIdon8oswUeteknhrieadhujperlrsoietnns.-gepnrctpaehrceeicopefiixptpiaSrtt9ea-ismtnitgixin,ogcnonciconenncbteorntahttriamotunitsoa.ntiTo1hn3eaTtseosssxaiycssietvtyheer'eobtstoehxraivnceidttyhenottfohTx-i6c5ier0ci6yt iinn
scoufxbpsettrhiaemnecinentisrt.eilaaTlthleyedresfeuforfrveeicvtitnhigasndcrepalnerlorseti.ofadniTnhfdsesicsncengiodtoebinnsstebertilvoeaxrftiviicenoiddntisyinngw1.satrheperoccbalanobfnliiyrnagessetfefjsnitcioeennccy
TfTIrhoneteqtuhsieeenncdcouayncbdesaetneaxctpesheiergoinfmTieKf$-ni9Ttco,amcneutTst.-a6bi9on0lc6irceasshaeocwteiidvnaatthieo1n.n7u-ftfnaolntdthefirfneicrqrsueteasneceyxipnaatrtihnteehenmTtuKtT.a-n6ls9a0t6es digin
SoLrueyrxvieiisveecddonectmehEneatrrtaertediaottnmo.entthT.eheTshorilesvlHeaonmtwtaiavvneecero,ncttlrhatoanhltii,sn`gtwhiheenefcrafrecieatcasuissee)n4c4w%yasooffobttshheeervecradenmiaosinnliyinganticieooilntlieys
cflSooehssnesesarirvdmteeehdrna.tend twtihwseo-feoofnlfldeyctc1o0mwpasa(r2ea2db%satoorfvet4nd4ek)o.snollySvienincnte,ocnoennotsrdxopoles,re sttmurehrenivstsipvoanannlsoderoetrfnhesetiehaeintneiscyaoolnmtiessnpanwfoetteer
mutagenic fnnotthebiaalbosgeinccaelloyf r$5e.lmeivxant and T-6906 1s concidered ey be sot
bom3pie7snoreapretveaeadoBrqoaiunsnelecyrnneccaaeytseosaft EftnKhet(hvTe/Kv-F)TiorScs9utsm.aentdIanbostlehiceconfdaicrtesxitpvearteixiopnenar,ntnTe.rn6ets9,p0e6chtes.hjaov{eenysore2n22s.ethanands
. Corlfeabnjsceutusircouenlitounfree81cc%oomnWpcaaaensrenetdarvbaeittreoiyrovnttehodexoficco1l0cl0o0ngcrueognw/ttmrhlatiwooafns.thceAbsessroevlvveeedrnetafdtceeleranyt4e8irnanneogpse752sern)aurs
TLS8uoe1ln8ai"nngRaheeofqrfueivecenriceynhocwyadsoosfeGDdrGaeaysrpy3o.endsIeanitnrtehttleehasettsfecsocaunolsbnlhsdsitpaepnxlcwpaaeetsrecdiosnnbeTscnoeterr,nvtterwhdaeh.teidoojnreseerroonfrienn3n5e0ioianochnof75t0he
mTwPuoattnraaeeggeetbnnaoetirenecsydTyiTnantdotthabIeexn1pcbtrloeaasnledoengxcipneeeoarnlilntyeonxtrisecleacvnoadnnctet.nhterTahitenircoernfesos,rsee,sth7ei.rn6e9t0hs6encr7sseeSccieconeancnsdes eetsxhncpereerssiocmocnesrss
of 59-mix
;
thee
001365
25/JUN. 25. 1399812: 42PK PCFHEDICAL 220 2% UZGEN.WOUTERS + "BESTcop UI13 NE Sb ces
T-6906
NOTOX Project 223458
In the first experiment,
exper inant, were increased compared
both ta the
the number of small and solvent contro) values.
large mutant colonies In the second
mAInTy the large mutant olonies were increases sorperey to the isonldvuecnttioncoonftroclhrovmalouseosm.al Thmeutastmiaolnls.coTlhoenielsargcean cobelonaisessocmipapteeadrewdithto trheesult from Sfrecting the TX gens. mutants with single gene mutations (substitutiens, deletions of base-pairs)
#00 that the metabolic act vation system (39-mix) Funct sansa prasersc .
1aMnu0dt.a5n1t5a.nf7dr-efqo1ul1ed.n6cf-ioaerlsdEM{SfnodruincOeWdtNh,ebyffniprossttihetiavfneidrsscteocnaotnnrddolesxecpcheoernmdiicmeaexlntsperrwieenrseepnetcitnircvereselpayes,cetdainbvdyelyb8y.6
It was therefore concluded that the test presence of S9-mix, were appropriate for
conditions, both the detection of
fn the sbsence and a mutagenic response
SIytst1semcuonndcelrudetdhe theaxtperT-f6n9o0n6ta)is comnudtiatgieannisc diensctrhiebemd.ousien TlhyimsphocmoanoLr5s178Y test
.
pages -
601366
CU 3 IYOLLALN MOMUIUAL LLU 2 UENATERS + 87531773 NLU NE 41 sos
Co
BEST OPT AVALLABLE
T6906
NOTOX Project 223458
PREFACE
Sponsor .
Study Monitor
Testing Factiity Study Director Technical Coordinator Study Plan
SUMtu.ssaPkaul, MINESOTA 55133-3220
MMCeoW.nBadeRaiHsg.tiruanCaotxNV11- Chemical Enc B8e2l0g7i0un201NORECHT H5N2Oa3Tm1ObX0a0BkVe".nHwaerttenrogge3nbesch The Nether Tangs CM. Verspesk-Rip G. van Cort SCtoamrptleted : 1142 AJparniu)ary19518988
TEST SUBSTANCE
: DB2aedtosnccthrifpitciaanton
0CTr46e05n00s166coloured sorta
PuTSretsaitbtiyltiutbystaunndceerstsotroargaege Astmroom temperature fn the dark
coESntxapdbiiirtlyiiotdnyistein ventete
Ds3i1tmaeDbtalhceyelnsvuelrph1o5x9i8de: at Teast 56 h
TTeessttosnusbsetranc1es craetseo.naible fur the completeness and GLP Complfance of a11
ARCHIVING
)
M80dENraDYdatautIT1oraratchiTvoeastthe10pryoetsoscol, report, tost articte referencesample
"pages
-
001367
TNE 1308 ISR MEDICAL 220 2E 02
ym ng
T-6906
NOTOX Project 223458
GUIDELINES
gThueidedlesiundeys:procedures described in this report were based on the following
+ GOuorixgdiacenolilisonagetysi:onFIonrfoVrTietsErtcoionngMomainocmfaiCCihoae-nmoipcCeaerllaslt,iGoenGnueiadneMdlutiDanetevfeolnnao.pmTee4n7s6tt:s(,O`EGC(eDna)ed,toipctDeEdCD
+
AEpurriolpea4,n 1Ec9o8n4o)ndc Community S7/48IEEC, Part B: Methods
f(oErEC)t,heAnDneetxernVinoaftitohne
oEfECToDxiirceicttyi:ve
1aDH9niu9grt6ea)cDg.tierinevicectii8tv7ye/:3906I2/n5/4VE/iEEtCrC,oE,EMCaEmEPCmuablPliuicabalntiicCoaenltliononG. ennoeL. 1M3L3u2t4a(8taidoo(npatdeTodepstteM:daySe30p,tem1b9e8r8) 20,
O-B_J--ECTIVE Objective fTTehosectusionbgjinecttLsi5v1ea7b8iYolfmiottyuhsiestosTtyinunpddhyuocnewaa.sfocretwlolasredvamluutsatteiotnhse matutatgheenitchympiadtiennecykionfasTe69(0T6K) by
Background of the test systen
ssoLmfy5as1ltm7leu8ntYdaegn1leo5enutisaicebolnealscyttnoipvhidotneyatecoctfellbasasberaoraepdaiurrseadanlgtebeercoaaftuiscoehnesnt,ihceafylraamrceleassssheeisnf,stitmTiuhvteeatTiiKnodnmisuctaaattnogirosnal
fCoreilflJlsKuCoIdore)oftihcayirneeindtriensieinst(taThnFyTtn)i.dtioTnKethedkeifnciaycstieoetno(txTiK)cc,elelfdsfueecctatsno Into ite phosphorylated
otfhetfheorwpayrrdimimduitnaetioannaj(oTguKeI not incorporate the analogue
CpcreeellslieuslnacrTe uomfsetaTbKc,oelllTisFonTwhaiisrcehccdoWnbeivtrleailrivtnaeetsddiuvrevisinovtl(eoenluyncinulceflorcteouionldtteiud)dre:eesn,tohvweehisncyuhnctlhaeerosetiisdl.eesthIannleesdthseedthfeor
p[Tiritosaceteedtduo.rea eTiitKtheidrsefpisocpsioesnnittbalnepehoautnosoltydyipseoc.rribynFiunrtathhteeerancboteriteow,enenobyftmhaetepdhpeiltuwymtoienscgtoinfsttfhuaeebirsnetTianFnntTgcesT,F1Togmiatvriesnagg
s{ofuebpsrTteFasTne-cnrete.sitshtoandtiffneurteanntts ty(psemsallof anldesiloanrsge incdoulcoendiesi)n
which are believceisaseer the DNA by the test
Atotbeestaaprottiecnlteiawlhicmhammianldiuacnesceallpomsuittaigveen.response in this assay 1s presuned
+ page 7
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001368
TOR 25.0309 12:35P NEDICAL 220 ZE 02
Hams zoe
T-6906
NOTOX Project 223458
MATERIALS AND METHODS
TEST SYSTEM
Test Systen Rationale Source
TShheockculctuulrteusresweroef CELL CULTURE
cthheecskeedceflolrs
LS178Y mouse Yymphona cents Rgueicdoemlmiennedsed(et.egs.t EsFyAs,temOECiDn, iEnEtCe)rnational GUDenr.nievteAir.Gcs.siAt.yaCn.dofCKLnheaeaimpdi,ecna,lDeMTphuaetratgmNeeenntethseiroslfanoRdfasditah(et1i98oS5nt)ate TdheirsiveRdoufsreonTytnhgehanFeisccheelrl LT5i1n7e8YwasTinoer,1giinsao1llayted
by Clive (1975)
mwyacroeplsatsomraedcfonnt1iaqnuitdmfaonsnitrogen (-196C)
F10 complete culture medtun FF1100 mceodmipulmetewitchuolutturethmyemdiidiunmecaonndsihstyepdoxaonfthHianme's
rsp(eeeGsrnipuibmecc,coit)li,lLvi-engsl/luysup)ttp.ralemepimtneaenntye(cd2inmwMi)t(h50an1dU0/%ml(vainvd)
horse
50 ug/ml
Cell culture conditions
Exposition medium
SeTective nediun Non-seleicvte nediun Environmental conditions
FpL1r50e1f7ec8roYambpmlloyeutseekepcltuylmbtpeuhlrooemwame7cdeixlulms1.05w.eCrceee11llcsdu/elmntlsuirteyd wians
FC1e0llscuwletrureeemxepdoisuemd, tboufTf-e6r9e0d6 wfiotrh320honuirsHEiPnES
or for 24 medfum,
hours
fn
F10
complete
culture
Selective nedfuncansisted of F10 complete hcourlsteuresermeudniuamn,d 5suupgp/lmelmeTntFeTd (wSiitghna)10% (viv) CsNeuorlnutnu-.rseelneeidcivuten,medsiuupnplceannesnitsetdewditohf F1100% hcoormcpelete
aAintImotshiepnhcedurabreakti(oa8tn0s-9575w%eC)r.ecTocnnaetrariitenedtmnpgeoru5tat%uiCrneOZaanhidnuncatoiyr. expperecrenftneangte. were monitored during the
pag9e 8
.001369
TIN 250399 1236P MEDICAL 220 JE 02
nam oz
T6506
NOTOX Project 223458
T-RE--ATMENT OF THE TEST SUBSTANCE TJtouhseluisteteyst (Mseurbcskt)a.nceTeswtassudbisstsaonlcveedcoinncednitmreatthiyolnssulpwehroexidperepoafresdpecdtirroescceorypicprior
R_EF--ERENCE SUBSTANCES
NegatRiEvVeERScoEntFro)e. tent articie, beingdinethylsulphoxide.
Positive controls: Solvents for Reference Substances
Hank"s balanced salt solution without calcium and magnes fum. Solutions of reference substances were prepared immediately before use.
WitthyhoIuntetmheatnaebsoullipchonaactteiva(tEiHSo;n CA(S-$9no-.nx)6:2-50-0; purity 98%; Janssen Chimica)
(1 7M) was used. EMS causes direct alkylation of DNA.
Mit metabolic activation (+59-mix): D(i0m.e5th1yH)Iniwatsrosusaemdi.neDM(NOWNh:adCAtSo-nboe. a6c2t-i7v5a-t9e,d bpyurmiitcyro9s9o%,malJanesnszeynnesChipmriecsae)nt in tcheellu$l9a-rmixD,l.resulting in a methyldiazontum fon which could react with
M-ET--ABOLIC ACTIVATION SYSTEM
Preparation of $5-fraction
Rraatts,Tiwvheirchmiwcerroesanoas)taiennezdynefsromwerCeharrloeustiRnievleyrpWriegpaa,redSuifzrfoemlda.dulGtermmaanlye Wistar
Tlhaeboraantiomraylscwonedrietihoonuss,edasatdNeOscTrOiXbe1dn ia nstpheeciaSlOPrso.omThuenderratsstwaenrdeardinjected
intraper itoneally with a
bdPeorcdeaycpeidwtieaintggihotsn)a;criin[ftihcceoeyr)n.weorfle.
soluti
dFeinvieed
on (20% (w/v)) of Aroclor 1254
daaycscesTsatetro, fotohdeyfworereat killelasetd b1y3
(500mg/kg
hours
- page 9 -
001370
THEE 39 136A
T6306
MGEEDDIICCAALL 22200 3ZEF 202
BR
2
NOOK Project 223458
cvSTSoheuhlenbeutsrnredleiqlisfuevueegno0retfs:dl1ypo1ffhootrshstepoheh1d5atTtuireavsaietnrbspsuhfawoftxeseerprre5he0a0tWr0emietimhnob3.cvueefaddTfhePeroa1t1stse(eupprptheibrc7Nina.oeal4nnt)ladaygen,erctnonaastnn(eadodrp:wnnriaonTsnignhtneeotdo0m.no1eiinrrnsenncyiceioorleLnnsdgitss(e0ovcca)sh
transferred (-196C)
into
sterile
ampules,
which
were
stored
in
liquid
nitrogen
Preparation of So-ntx scSho9on:stmpaihixanteweda;sp3epr.r4emplam:gredN1A.D6iP3m:mneg4diMuaotnCeoll1py.H6EbPHeEyfS0o.;re2T.hu4es6eabmaognvdeKCkTes;p3t1u1t.a7nfomnfgcm.ggsl$u51-c1noeiesx6re. (0.22 pOmF)-9s8t-e1r)ilviazseda.ddeTdo 0(.550ml(v$I9v)-miSx3-fcroamcptonieonn)tsto0.5comm]plec9e-ftrhaecttioonrm(ibnatch 97.8 Mseutsapbeonlsiiocn.acTthievatcioonncewnatsrataicohnievaerdthbey Sa9d-dTirnagct0i.o2nmlInSt5h-enixsxpteatietaicohn a1meccoilnl
was 8% (v/v)
--_-- EXPERIMENTAL PROCEDURE Cleansing
Fceedllesr SwoeredosgerorwangfeorPid"idanyginang71m0ictoanggeltectieeycutlteusrte.inm,edihuem mcaouneteainIiynogno0nts,
period of 2 days on medium containing Mypoxanthine ine ihymiding one hypoxanthine, 2 x
medium) to reduce
10" the
M aminopterin and 1.6 x 10-5 M thymidine (HATamount of spontaneous mutants, followed by a recovery
Abfetfeorre tshitsartpienrgiodthecelelxsporwiemreentreturned to normal medium at least for 1 day
Duse range ringing t
cIyntodteoxricittoysedlaetactwearpearaopbrtiasinteeddboysetrTeeawteilsngforxsu10r6 gsseenriica.tyssteepsetirnegs,d in 6
Tctm-1ee6sn9tte0rx6ispfuoubwsgasiesttainottcnueebsetmseecddowineucfmrne,enttrhrieoanttaiaatobenssdsetnecfrieoinrlcearnad3a0shoiiunmnrlgstwhceioentnhtparrieasfrpeupangrtceoeesrithu9mab-etif,srrlaeywcitstbithaoinpe.a inTnTuohpmebeerrecotf
tTehset hsiugbhsetsatncet.estiendthceonccuelntturraetioRnacwians determined by the solubility of the BTsAefaontlloeauvrntacileo3dnsohsfoaublryttshe3cseoesxlupnuopttseriurornineaf,tiaagnnattdthieofannicdnaalslltrsleeypsswuesirnp(ee1Fn1-s5s1ieo0p9:narcao5otfrmepildhnea)tf'.ercaecnsluailctethh,ueroeotlvripetaoaoseteianoegonnn.tsyLaTnakk's "SeIlnlpsrovfendtNheeuFbianuae)r shuasupseonesiyosnanewseorre counted wich 4 miososcone oti an
`page to
601371
INES 398 136K KEDIICALCAL 22020 238 02
10 0.0773 21 !
T-6906
NOTOX Project 223458
Tor
For
GEIS
that,
yore
seeded
for
deternination
of
the
cloning
effictency
(cE)
mTfSoaoonrcndrceoeettnmiitpotrtneay:rtpiwtlohe3anel)tcleecssleillnwlfeewnr.aosetnuswspasecedelandllresesedcidtoinpiwvsneiertwwhhwmeieerdctleih)huedn.ni(ol2u3vAtfietsd3ei6rb-alwnee5dT]cdsaoeytmesoidncetrdteonstefnebpoowurretpeleilsaostreconefarttbaeorr9c6r.orsed
magnification of 4x
naked eye or with a microrceen tat 1
pTuhaebecralocfulaetmipotny
P
woeflltshebyclotnheingToteaflficniumebnecry
woafswedieltse.rmiTnhiesd
vbyalodeevsiodringSaltlheey
P((00))', thneumbzeerroofteermmptoyf wtehlelsP/otiostsao)n ndiusstbreirbuotfiowna.its
TGhEe=P(I0n)Pa(n0d) tIhneusbCeEraroef ecxeplrlessspeldateads ppeerrcewnetlalg.e.
Mutagenicity test
T IreatmenaE tE Tof Ethe ER celTlhsin the absence ant 1g the presence of $5.mix in to
(105/m) were used per culture. teInsdtepesnudbesnttanceexpecroinmceenntts.ratiPoerns cwuelrteureexp8ecXte10d
cells (10%/m1) to be toxic, 16
were used. If x 105 cells
gtCTeeehTlsellt ccscouuunllbcttseuutnrrateanrsscaetiifonninnttehhoeexfpopatsrbhiesetseien$onc9nce-efmroefaofdcitSuiS9no3-n.nni(ixFix1nw0etrcheeompeelxxepptooesseidctuilfotonrurme2e4dmieuhdmaiuurwmsa)stoenth(etvhiev)
HfEePsEtS)s.ubsThteancceellinceuxltpuorseistiwoenrenepdliaucned(F1in0w3ec0ruelmt)uerxecpeosnmetedrdiifufumogreb3uffpeorseeds vtieteon3t0
mixer at 37C.
Solvent
and
positive
controls
were
tubes
included
on
a
roller
sSACuofepltneuetrtrrniiaofstnuaapgnaoatnstudiroaefnn,idnrsaetclesolpulyssspf(enw1ne1sFr5e-f1a9,0nsec8opofammrpitalnhte)eet,de.ceeflarlcoshm, ftothlwelioceewxepdofsnibtytiaovnnekmsnoespd)ailuoonrrcboyInhe3sare
tu ture cegim
T"hIemprcoevlelds Nineutbhaeuefrinahlassnuoscpyetnosnieotnerere counted with a microscope with an
pGlroansicnigateelfyficaifetenrcyexdpiorseucrtelyToafttehre ttersetstoseunbtstance 2 x 56-well microtiter
ooPfflattdehasey C0wE.i.thThe1 pclealtle/swelwlerewersecorpeldateadftefror9eadcayhs diosnecubtaotidoentefromrinedettheermisnuartviiovanl
BFEo5xrr7pi3re,egrsrs7eaispion3snesiasocfenetrleilrooedfthoahefnedt2hm4deuethtacoenaurtlrnlsinpaechtexuinplooottnsuyurpreoeesf: ttihtneheemrfuoetrmaantitmhiefnrgedeqsuG]eenicesyelvteerreescourlttbunrceed
tSrReEsStrontthse
3[1houBrosseiebxlpeo)suwreeretinseu.bcuDlutruirnegd:withtihTshr5ec.eulntdiauxyrseveparofereaocrodltbhuoerressseeoroecrth
3 orys of che
otfretahtmeenCtE
w(iCtEgh) thaned tetshte
msuutbasnttancfer~eqtpuhaeegneccye1(l1MlFs+).were
plated
for determination
-
001372
JME AICI RENICAL 2 2 02
ws nn
T-6506
NOTOX Project 223458
aAssaeya,stbotfhourindtohseesabosfentchee atnedstprseusbesntcaenceofweSroenise.lected for the mutation
mfdoinrcrwoedtleilttseerrnofinpalatait9oe6sn-/wceoolflnctedhnietshrC.agti1otnch)eel)fcnewlalnsonsausdsedpeledencstipioervneswmewleeldriei(nd2ilxutSeednea]n]d seeded
PccFloooarnntcteeadsnietntrifeoanrrtgniioC2nnE0ag0t0iwaoenrncdeeloWlpfFslatwthieenerdesWFeinleactthtiroveteealm9e6nd-uwimeublmelr (mToiFfTc.r5so.et7li6etcextrio1np0)l5.atceesTl,hles/emaicchrowselilver
with 5% Co,
fncubated for 12 days at 87C in huni? ied air
SfAofartheer2nythl.htiesbtyriaanzdcodulibinaugtni0o.nb5rapnneigrd/iemo,ld MStiThgTee(ap3)l-a(tt4eo,s'5e-afDcoihrnewttahhlely.TtFhTTih-aeszeapl)le-ac2tt-eiyso1n|fow3rer5ethestcaiened
and HF were scored with the naked eye or with the microscope.
MTFhe=ca{l-1cnulPat(i0on)/o2f 0t0IhCe0E)mutation frequency The mutant frequency was expressed as the
was determined, as follows nunber of mutants per 10!s
surviving cells.
-DET--ERMINATION OF THE MUTANT COLONIES
The colontes were divided in:
Small colonies Large colonies
MMaonordrpphwhoioltlohoggiiaccaadlliamdleeetsnsesredeclnoeslsesonicteohslaonwniiatehsquwaairttsehhrarapofhaczaoynwteolulr.
ocfonatowuerila.nd with a diameter Targer than a quarter
AcolYoenlyl. containing mare than one snail colony was classified as one small
AAcxeoyloenly.lcoconnttaaiinnitng
Targe colony
more than
ene x11
one
and
large colony was
ne Tar cate
classified
vas elseif
es
re
one
sn
large
ane
TThheeetainognes mTuhteatsimoanlsl.
cc(ooslluoobnnsitteeisstutcaiaponpnesba,eredadeslsteootciiroaentsseudltowfiftbrhaosmet-mhpeuatiairnnetd)usctawfioftnehctosifinngcghlretohomgoesnToXemeg,ene
page 12
001373
Se Li RIL LEU
LE
T6506
NOTOX Project 223458
A-C_C--EPTABILITY OF ASSAY cArimtuetraitai:on assay was considered acceptable if ft met the following b&)) AfTthuenbTeaerabsstoofluattseurtvchliroeveniinngogfcteehlflefsifcoi(ue1rn05c)ydoscoefosultdohfebetshoealnvaetnletsytsecdsounfbtosrrtoalnsecxepw,rasesasnioa5nc0c%eofptatbhele ) <TTKhe1m0usptpeaortntia1o0nn%gaucslamnuatbalnetceflrlesq.uency in the untreated ar solvent control wes 4) fTIhrneedquucpeeondsciitseiisvgenifciocnatnrtols(at(etlehaysitnstthhraenee-sfuollfdo)natiencraendasedsimeinthtyhneitmruotsaanntine) ) cIlneatrhleyabtsoexinccecoofnce$n9t-rmaitxiotnh.e selected dose range had to include a
D_AT--A EVALUATION AND STATISTICAL PROCEDURES
ANo tefsotrnaslubshtyapnoctehewsaiss it:
tceosntsiindgerewdaspodsonietive
(nutagenic)
in
the
mutation
assay
a) )
TI0hte tihrneedsuucsleotdlsveantwterleceoansrtterporalod3u.fcnfioblalddeoisinenc.rdaeenapseeinnddienenptetnhdmeeanmntunlteyarntraenpfgeraetqeudenctyestc.ompared
aAsstaeystifsubstance was considered negative (not mutagenic) in the mutation
8) b)
TNthohenreeer-eofsfoullttdhsecwtoeenrspeatreedcdoncfotionrcmteehndetras`itonlivoaennnstisnchdooenpwteerndodlea.ntnluytanrtepefsrteeqduentcesyt.of
at
least
Tehneterpreicnatodintghecfriintaelriaevawlerueatinoont adebcsiosliuotne. and other modifying factors might
- page 13 -
001374
T5030 IIE
oy MEDICAL 220 2 02
Yo. m113 r
T-6906
RESULTS
DOSE RANGE FINOING TEST
NOTOX Project 223458
TCco-on6nc9ce0an6itrrpeariteicotinhp.iietatceodnceinntrtahteioenxpvoassitiuosnedmessdiutmhe athigahecsotncesnectsratsiuosnmeiorfel1000 ug/m1
thIehnertephsreaesndeoansscee rnoaefngeS9s.sfneinincxdeinn(tgTraabttlieeosnt,1)raLnsg1e7ayofmousteo 1l0y0m0phwoamranscelnltshew'ereehetmmreesattdedwitTHh)
STCEhxaeeptpopoepneradgrr"aeenttcneaonnreyee,J[o0aFirrratIehfEeouScscoolctnuhotmenr,cireenctlcorunaltttaiuiornness) tohofef
rcuelltautrievse the test
sGcuyeplrlsatcatcnnotyuen.tsshseep(eeoxrpEreeasiLsered:d
os
tchoeuntpsercaerentaTgiestecdlonininAgppeefnfdiicxie1n.caytTivoafetetche5le)l resmuarivniivnagl
dirsetty afin Sosointe
cells (individual plate
as
PCvoasmmpaTrebeddueooftereandcsheteescoCfoonuSsn5tt:eaidSxu,abtstttahhneecencuocnnobcneercnetnortfartaciteoilnolnsofocfo1u30n30t30edpLaaaifintn]erwbiy3s Ghoroneu,druswnmhtyrnetaeeotcmtesinttte
cCoompmarpedrstaao mstahee
compa
ustoelvdentshocvoendtr
onlo.'clTehaer
iTneidtLieatlilyn
suSrnviShveingC
l
ocneilnlgs
red to t
aSt5ra-inency
he
Fenaqnilo aanse
rem
ocfloSniwnigx,effniocireendcuyctoifonthweassolovbesnetrvceodntirnolthe
cell
count
of
the
9 Calienairngrecdeulclstiodnireicntltyhe acfltoenrintgreaetfmfeincti.encTyhecoimnpiatrieadllyto stuhrevivviapntgercsell1sontstheorweed
J
TIhnoctehpeeraabtsieanncewiotfht9o-mxiixcittyheupsetloect5e0d%
There
cdoosmeparraengeto
dtihde
nosteleinnec.ludceontaror
Ccoommppllefenotsreee,ceutl.cteuflrolen cm2ue4ldtiuMuromeu)sr.e ftno ththea'muttesatgensuibcsittayncet.esti,h einpotnhsetiaobnsemnecsetoonf S(9e-imnix,
MUTAGENICITY TEST
CFrroanmciannicaansd,for srhoixouthse
clo
pceorncceennttargaetsionosf
ocfe1)T8s5u0r5v.ivaTlnhaunigditnhse
mTuotraennst
counts
of
in Tnaibnlgesand5-1s2eleofctAipvpeenpdliaxtes3,. and cell counts during subculturing are listed
SopExeFhp5ree5irimpmFiaaeglnthntieahe1diersesesu,elttisLvaantogifeont:whaes1d0aS.seel3e3cr.taendgloeor.aFri1n7md5ui,tnigg53it5ne,isctisktapyndaetgxhieiondseoslunbh/nilcAi]LtYy SteHsEt,L: PeReUxtLpagIoeGsnEiNotTniEioAntLymiendtieeusmrt.intgeH,sotwitenhvgee:r,exspitenhrceeimdenonostte welanesovuepglhsapd1ee0s0ye'duggr/on]upeancgouutpwaLrgdls HwEeTre too
-.
+ page 14 -
.
001375
JR 350308 ZIRE MEDICALL20 220 3EE0202
RRaEmIs
T6906
NOTOX Project 223458
The following dose range was selected for mutagenicity testing: WWiitthhouSt9.m$9i-xnix:: 1303,, 1180,0, 333.3342a,hd561,00075uag/nmdl 10e0xpeusgi/mtlionexmpeodsiituimon medium Evaluation of the toxteity
aInndthuepwaarbdssencweereoft5oo9 mteotxaicbolfiocr faucrttihvaetriotne,stitnhge. doIsne thlee.vpelrseseonfce56oufgS9T-6906/m
meevtaalbuaotleidc. activation, no severe toxicity was observed and a1) dose levels were TWhiethoduotseS9l-enviexl:s s10e,lec1t8,ed33toanmdeas4u2reyg/mnu)taenxtpofsretqiuoenncimeesdioant the TK-Tocus were:
With 9-mix : 33, 100, 333 and 1000 ug/a1 exposition medium
comparable to the solvent control even at the highest tested dose. In the absence of S5-mix (Table 2), the cell count of all concentrations
tafetsetred24shohwoeudrs notreraetdmuecntti.onThceompcalroendingto etfhfeiciceelnlcycoofunttheofrtehmeainsionlgvenctellcsonwtarsol
In the presence of $9-mix, substance concentration of
after 3 hours 1000 ug/plate
treatment, the cell showed no reduction
count of compared
the test to the
ccscouoenbnltlscrteoanclnto.crueanttTihcoeoonnfcceetonflhlte3ra3cst3ooiulnovutnegsn/tamfotfcesor3nh5to3r4wo8elda,nhdoauwrhr1se0er0d0euoacfsutgis/otunnhblecuolfschteou4lwrl4ees%drc(ceoTouadmnbputlaceertded6l)aottnoeatthtoefhtees6t1s%olvent
uctcghlo/eompnpliaranretgemedaeifntwfioasnfgcthreeecndeculcylcesleld(cbTyoaubn4lt4e%,2o)f,thtehset ottshhoeelrvetnedtsotsecosnuTtbersvotella.snceTshheocwoencdcleonnnoitnrgraetdieuofcnftiiocoifnen3c5iy5n
of
the
Fasuelrlrteehcaetdreydpiranesvceishpitigithgaeasttteidondofsnsehtohwieendethxtephaotsaibtsaietnocnaemceaodnnidcuenpn.rtersaTethniecorneefdoo&frS7i95t-0hmiiuexg,-/dlsae1e )wrs-ai6s3h0e6r
concentrations did not precipitate in the exposition medium
Experinant 2
Based on the results of experiment 1 and the solubility test, the following
dose levels were selected for mutagenicitytesting
Without S9-mix: 10, 20, 30, 40, 45, With $9-mix: 10, 33, 100, 175, 250,
50 and 55 4g/m) exposition medium 375, 500 and 750 pg/ml exposition medium
Inatlhueatiaobnsenocfethofe t5o9ximce:tabolic activation, the dose level of 10 uglml was not ascdcoeotslnieesvciatdTteeeidrvoeendf,sornotonhfmeut5ta0odnoxtsaiencdfarlnee5vdq5eulenunog/tcomyflrme1we0leaeusrvuegarnetTtm-oe6sfn5ot0rt.6ox/minusclt.iannwctaeIsnftrnhteoihtqseuespcneroclenyescceetnmneectadersautofrfoieromn5e9mn.tuw.tamsaenttaTbhoelic ffrreeqquueennccyy mneeaassuurreenmeenntt., Tshiencedostehislevceolnscenotfra3t7i5onanwdas500natg/rmellevvaenrts fnoor msuetjaenrtted
for mutant frequency measurement, since these concentrations were too toxic.
- page 15 -
001376
JIG. 030 138
T6506
MEDICAL 20200 2228 0202
Yomms 2 216 NOTOK Progect 22318
WTWIihEteNhoduSot3s-em$i5lx-enviexl;s 23s03e,;lec310t0,0e,d40t17o53,mndea245s05urennoCrmmu)t75a0entxppf/ursmei)qiuaesnnhcpineaestsiaontan tmheediTuKm-locus were StIhhnee thtceeelsltabssCoeuubnnsctteanoocffeSth9ce-omnicsxeonltv(reTnaattbiloecnon3t)o,rfa.&a5fteFurhg/em2l4clahwnaosiunrgsredetufrcfeeiadctimeebnynctyS6thhoef cctoehmlamlarrecesotunnottinogf
cells directly after treatment of 45 #g/m1 showed a reduction of 78%.
oIfn t7h5e0 upgriepsleantcee osfhow5e9d-minox', retdheuctcieolnl ccoomunptareodf ttohethteestcelslubsctouanntceofconthcee.ntorraitioonn rcstehohdenoutwrcerotdelin,oarnienwdiwhuanecsgrteioaocnsebslsletsrhovefeodf6c2e,ltclhose4m9actraeoesnudtdntes4todu0b%stathtraeensccpcoeelncoctneciinonvtngerclaeytne.itforfnaiIstcniioetonnhfceyo1f0c0ol,f7o9n0ti1hn7og5sgiaeapsnflidfeaiccc2rai5te0nnceuyg/mo)f Schoonwterdo,& wrheedruecatsiontheafc6l6a%n.ing efficiency of the concentration of 5 yin
coItJfonhouewnetttrhhi,eegdhunpierronsienitns-t,giepanrlcepthlecreyie'pcosifieutpxraip$vttr9ii-aevmntsiigsixnin,gogcnoncciecnpoleenlnrbsct.ioerotnadhttTrihma1oesutnstiseaon.tnotioboT1snhsaeebrsaJveetsasrosstvxa.aiydcssietvt1yohhreeathtbeostxeharcinvlceoidnttiyhneignofttsoahxTeri-ie6i9sna0eyl6nlc11yn0
Wnexueptreaergiennnoeitnctasacccte(ipTtvaaibbtllyees ofa1n3Td-166t9)h0.e6,Heoxwptehevereirmi,enndtitsvhiedwuerarelesulcntoastloanounyfssedtcwohueetnortemuetovcfaaoglnifdeaaniytirecii0sttysheeidntetsshttseacy
gteh1iTs craoruentFidnudriinngg not an accidental
ftiihnnedtioenxxgip.crietsysioins
pperroiboadblyarea
treesptortseudbstianncAeppernedjiastes3.
eTrrheecrtefoorneg
Evaluatioon of putagenteity
yooteowo STaeeostel ery
--3
The the
spontaneous mutant frequency absence of 89-mix (first apd
An the sefond
solvent treated experiment) and
control cultures in in the presence of
89-mix (first experiment) wep
ahbissetnocreicaolf c$9o-nmtirxo)l adnedta o ranu ge
outside the maximum value of Ire
(0.7)
and
x 10%
38(mexan10%2.5(mxean1052]
0
x
in
t1h0e) priensetnhcee
of 10
$9-mix}; for nz66 ands mutants per 10 clonable
respectively). But these cells, which was defined
values in the
were below the Acceptability
of 1s
assay' as an adequate and
acceptable considered
background for this to be acceptable.
assay.
Therefore, this test
The spantaneous mutant frequency in the solvent-treated cantrol culture, in
ta(hnmedeamnsaex2ci.o9nnudnx evx1a0p%je)ureimienonftthtehienprtheihsseetnopcrreiecsaeolnfce$c9o-nomtfirxoS)l9;-mdifaxot,ra nwr=aa6sn3g)eb.et(w(e1.e0n- tahnedmi.n6imuxm105
j8u1t5anatnafr1e5q7u-en0c1i0esfoirndEuWcSedfnbytpheosftiirvset acnodntrsoelconcdhemexipcearlsimwenetreroisnpcurcetasoetdsb!y
fsargoynoersdnstdpeitebmthcyietoi1(nvd0Ssee.5,l5t--yeicbxtoa()itnTohdanfbul1inoen1cf'.t26tiah-oefanmnoeudladtdbas3)gef.eonnrcieIctDMaNnwr,diessppiorntnehsseteerhneecafenofdrierofstthcaoStnoae-nmtdyihuxess.eemscwoeanttrdhasatbeaxoppnilpeenrrosiomTneeinomntteesion
property.
~ Fapasge 16 -
601377
TIES 0 IEJEPEY
Tessas
MHEDICALEDICAL222200 Z2F0022
DBeERaTr0 EhAaPYyAsn2n0aname
NOTOX project 220438
inshe
T-6906
sheence
of
59
netabotic
activation
in
the
First
experiment
(Table
2
tT$SnhKhIe-eeerlTeorccVseauEslsmSle.disinadiiniIrnnnnygitothttieachtaeeivlmnllausdsytneuaeccnSweotaunSsrdoaucfi2trsevc2eixe%qgpduCneeocirnnoficcnimeyscenuanrtntserttsa(ttTio1hanraebc,lreiTeaKhTs3h)eJ,esoocriruTnese-ie6ten9hts0eoi6vvmeeeuosrthnoasoirnwrrrteiedn]fcihramhieetq1reu.te7TeFno-rsfceoylidSdayete0ts,vhees
ecsdxouoprsnevetrirvroane,lesnptaoEnfhisnsetdhreeitnlhccaeretelaliassnnecarahvefiaatpsseercvwiotasnrtssehiaesdtThemoteresreetnedtrstasnwnn:aea'rnstt.thoeenelFyoSfr1oeF1re0sc%mtac(cso2eam2ks%aorosiufpnse44hre%eiet)n.ndinLeSSiuCnrlcEcet,iyn none
1a considered ta be nat mutagenic nthe aobeloonicedgtceeat1y)reer1meanent ness Lent
abserved only atane-very toxic congentrarisnon sevens sere io er ehE In the presence of
3.2-fo1d increases
8% in
t(hveiv)fir$s9t maentdabosleiccondaectxipveartiimoen,nt Tr-e69s0p6ectsihvoewleyd
22.
and
mutant
frequency
at the TK-locus--In-the first
experiment,
the
in the increase was
Ios
Jul
$5(7 SFrgthreeodwustccuhtlboicnouainltntguotfrh`eeeiccomokuarsnedocbsteearvtmeohdeftcn`re1t0lha 00egptrgac/f nemtillhsswoahfpsitshtoobesseeFraavtrederrhaefptceer ro48ssaned t7t27n2 ofhours
CHT eamanrxceepreermcsiuotn0nteesasninatdtlevrrweteerre,drtefeaflesiabhcnotitdteeyhrnevceyn11sa'stoietfnooxdbditsaocuybegrau3n.ercaaaegsIenanprotontnnehestesnottxrasieccsCaoonbsnasotnetN cenhxeetppeetrnriieaamrcerenesmtns,asevrmtvthereiraraasitgonncSemreSeearErseieocwnion,red)
mutagenic in thetoprbeesebnicoelogoifca8l9lmyinrelevant. Torefore. esos je enc Cw OLE!e
e
rene Spares In the first experiment, obSath enter ofsa nd the number of small and args man large mutant colonfes
o
\ seoxlpveernitmencta,nrmaaiTnlvyalutehse Tlahregedmiuftfaenrtencceoltooninecsaenwertehe innucmrbeearssedofcomspmaalrled atnod the
aculrhsaeruroagnelcalosmympouanttrahaalenbtlsesu.ncfaoaultnToithnoeineosssf.masilTtnlhaeltlcheollaaonrfngdiieresTstcaorlgatoaennndiceassseteecnSoaipenepsdsesescxrihpemeespressIiommoemtntetoerusisoernntatr(rnmoacaarcreeecprftiIinnoEedniiTo,LnSgd,
wWFiftehctsiinngglethegeTnHe gone: mutations (substitutions, deletions of base-pairs)
2Do:
_concLusion sinxpceornncelnutsailon.coTn-as6e9t0a6ns1sdemsuetrasgoenndi"c1xinthtihee TTHopmustation test system under tne
page 17
601378
UES 399 12:38
T-6s06
BEDICAL 220 ZE 02
Yo.
1g
NOTOX Project 223458
REFERENCES
oAalcehsenaaeDtEs,inPLHSIILTBSY.aSi.sCe. 1Ryamyp.homVa. cPeollisn.t m1utaaptgioinsatationthevo' mmiimcese
TrRLtooe7axisiicenomolshoaigiycntegteme.istttaiitnTgfT,aTln"eget,MSautttaSt.vthCiaeolntGtuhTRtyueirmsonienedarir,ncahen,gGkNi5in4ni:kse3er.B9pa1ry.fet4ot0ceu6UtsiA(oi1nn9,a79L)e.rtSaoatmtutbauoorisnd,ss.0.5.
$DSe UatracgiennoBigAcein.tsyMaetnedsCtm.aumt.aMguet3n.astiaownndithRYaemtsah.seak3Si1u..in54.a3.n.191l09sa75/,niMmetejhseo1ndsmifcorrosdeestsecting 72,
"RiTSHcoeuxlhbleesrP:ymrao,nag.rrWaEOen..v,*ie"MwMuButahtaniatdygiaeonnanenasBR.li,eyssseibFsayrracnochcfh,iestn,hiceaWllcia,tgeeranLttausnrgeei:nnbaaVc-k7r,5epGRe.hr,ineoPcfestetrhpesoonns,eteepA..,
Gaeve. Di Caspary, ., Kirby, BEB,7:8K1r1a4hl3, (R1.9,81,Moore, N.. Mayo, J. ang
DLnGOuabtaneTaretglaayelv,nieDaeniTis.dssJcaga.etiy,iinloss1ny9mos8unt7at.e,namdg,KeG.ncu0Miih.ucda,tiertaaytScf,iptooerecnrMtiupozteraar,ttfiioooFrnnmiSnoRfge.stt.hheBea1t6mLs9o5.ou1ns7.e8SY4I3Al'7GyKsm.opHnhaiovnmedasBarrsooswmanmy,hofrMosrMM.
7 LsClypimevpcehi,ofmiacD..cleoScluplsesc,*mtuoNtruattJaiFto.inSos.naRtLeastbehoaerrRaceTshtK..orT3ya1sc.u`b1spr7o.i6c9n1e5-d1cu6ur{3ie1.st5u5rf5eodr aLsasiessesyimnoguse
8 cSACeaoltlcleaos,ym,ianJrgM.i,ustcoaeAntlr)iloeontsftu,rrtvheiesCv.;aFal.g1,a1ra1nG,drCel3seo7nun1,tian3tgM8i.goHa.nnLd.f,raeiaLcuorswneo,ctyiJt.inraaon(tnd51MS7ue8riremiloc,uese.W.', 1rpia1sp98h3o,na
5 EaCodtt.ue.rVee3s.nitma,nudnaAarilaentt.celCiFa..) "I1n:98W4,itaTgheenidceitteycttieosntionfg.gesnebrmaucteatjiunonssninmreach
10 Jotz, M. and S.MitacnhdelPla,rryA..D.J.+ M.1,981I,RLEfpfreescsts limites
HnSSfgeeoigrrryrweeeaghosoroxrmdisaeHnmioducetetlvyaJl.eten,s,AosnhJIbnf.:yreCqE(,uvEedasnl.Bcu)rya,itgiagotEenlnstaehonvef,siehtrTho-HyrN.mtoirdttaiehnnrdesHooDkfleitlvne2aeas0nrtsdss.ec.oodlFfeoMcdu.esacHhr.eic.nmiin1Lco9a5m8l1n6s7,e8,YonFmootuuhseege
sai
of abercetin in cultired amma tian cai1e, dition Res., 156,
page 1s -
001379
TIN ZS 399 12GB MEDICAL 220 2 02
fom 21s
T-6906
NOTOX Project 223458
TALE 1 RDeolsaetiAvGeEsurFvLivIaNl,G 5Tesoft twionmdesL)otro vous roi ces
ose
ca cour
(
soe crcines
ug/m)
EXAF0TsEURRE
AFExTeEoRstRe
Solvent 3
10
10303
333
1000"
control
RWeitehout metabolic activation RCE
100 101
100
120
84 70
9875
3
866
7
48
13
&s = ReFFx BoC
doy 12 i"
3
[Ap
Eula
Hith metabolic activation
`
Solvent contro} 100
1a0s3
911506
1 ws 100
tes
132
54
11030030031
111047 50
1120 0
wwsa to
136
we
H- W
+31
Solvent "T6906
control = dimethylsulphoxide precipitated in the exposition
medium
<page 15 -
001380
TRZ59 12:30
KEDICALL222200ZZFF 0202
"owmmac -
T6506
NOTOX Project 223458
ABER CTIOTOXC AND WTAGEIC RESPONSE OF T.6306 14 THE MOUSE LWPho Leu Test sve
semen ;
welllso
ooGsaeta)
STTREEARTNENTT 8TSGREFOTNCHOTNNTTRROL * OF CONTROL
AWCECLLTLOUSTC STERI
DACBiSEvOsLNUTE 5 WoTIfoTHameWTrsoA.NTS
wXR1Ea0GrEeNCsY
OF conTRoL
#ithout metabolfe activation J
N18Scioonlotvreenlt 015030
110w00
11350700
3 wsesam)n 5es h
" S32e3 dwiet33t
5
7@
wBmoo owoamsaaimnn ibb)yy
i8 43
ws ior
"
2
716 (0s) ss
With metabolic activation
Scoo5ln3vternat 15020 Nm 15
10I50]3
10wo0
S $3 ass an uoay) 4gpa
a
333
56
56
1000"
121
108.
318 2
wo
9%,
wan ay
32 (8s 141)
38
63
pn
ow
102
o
1274
@ 181 (72s 891) 16000 -- pre
6
3 ess mI) rss
5GSaE'l.ver=CttChlaannntieentsgh)aEn<fefsdiuicniIeeotnhochryyaltseutphoxtee
O} MtTh6e=50CDE 6iEgeptrE huetyctIienpriiT tktarrtoeesadatnioiiaTnnnetctehle(lXseoxfp8osciofnttieoCanonr)kmteodTiiuSmte cell cout afte reatoent (4 of contro) x
oT== samgie ccoolloonniteess
- page 20
001381
IH26 0968 1:09
MEDIICAL 200 2EE0022
Yo. mam ie a! s
-
T-6906
NOTOX Project 223458
TABLES CYTOTOXIC AD WTAGENIC REGKONSE OF T6306 IN THE MOUSE LYWPKOMA Ls1781 Tes sysron EXPERIMENT 2
DGoasela)
ATSFERTLEELRCAOTNT 4TC0EE7AEACOFNNTTTERROL * OF cool
CWAEACiLTLUSAOLFTSHEVE 0 covteaL
AGCBaeSvO'LsUATTE 5 WToIFoTrHnaeWlTmsoANTS
XwT3aR9iEoE
WoScoolnvtreonlt
Hithout metabolic activation
-
i1m00 10204
100
$5 woz) 70
M
"30
"7a
So hehe
2na
75i%4 0
a I lam m(i6e oaB15y) g7ad3 6 mia oa ad
os
n
10
s
18 % (12 82"1) 1090
ith setaboric activation
ae 3cs3netvreenl 1o00
111030
Wf 10
a
[i]
1500}|
F Wo ALwom) es
A buch 350
7oo~ 25000
st
=
u
9i5d
T 2 heeie
1050711
m14o7
3 (4s 3aB)a
02 (G5e5 a91))
alge
a149
son
om
"
a
2 8 (1% 70) seg
OCSBMSaE)"l.ve2nE=DttiChsclyaootnnnhiatyntrIghenaiEntf=refosdisucaipinehtneocmneyslieseuTtpnhoxiyde
1 3
htTh6ees5m0CELaE6l Epcraaelfcotirneptriietsaattrleadataiafnnttcheel(lxeeoxfp[8ocsoionfttirocononn)nteraattiunnthe
cen
count
after
trantment
(%
of
contre)
x
72 Sarge colonies
+ page 21 -
001382
T0036 309 1:30 MMEEDDIICCAALL 222200 Z2E 202
Nam 22
T-6906 NOTOX Project 222458
APPENDIX 1
INDIVIDUAL COLONY COINTS AND CELL COUNTS DURING EXPRESSION PERIOD
:+2 -
MEGGuelltlooalnntiiinncoggonuneeetffxsffpiiecdcriueiernnnieccnnyygtseoi,xfmpmeritdenhcaeasitvdoeionldsyseaplaerrfatcn1eag9rrgeomfe'xipnodssieunnrgtestest
TTTaaabbbllleeess455:-612 ExEDpoxspeeerririnmaeennngtse 2f1inding test
Abbreviations used: Solvent control:
dDEWX,,iedntiThneyieatIhntpeytlhhnhaiontyetrsospahnoinneate
BLE4 DoseRE FINDING TEST -_-- tGaGalasalenmD fEcinlmgopntiyngSwierpleate T amyS we ane A SeenE ,frriceta. nyey
saolnvednt 2
thous eetabieteactivation
na8: gdow
io
w 6 aa
wmw2 eoo 4aB4io8 4a0
1301 5
5aiin
a5is 5
a
@ an
swets uiant onwgo vo owiok
th setaberte sctivntion
756
5 8 mmw
wmw aeo db3ulo3a8@
iisa" 1
a n8o in 3 50
=
sa
T6906 precttates 1 the expesstion medron
+ page 22
001383
JIN.Z5. 0399 12:30PK T-6506
MEDICAL 22020 2E 0202
Foamam ku NOTOX Project 22345
APPENDIX 1cont tnued
EXPERIMENT 1
HOLES WCEALLRC E caOMb0TsST ecUacLtTS iIvaRtEionan
T TT wrs
b(opnaatlym)y J{TSe iTreLatneBnbsetRaxre1C0SitcraeTnltyaeeantceer 10o61T58)T TC1 otaSRsEmeunt?) (1SimT) TSo40TT8 | ya)
sGB aevann|td|e 8JH 003
io-- Id
@B200333 7 0 113 s m mom
a. 1 6B5a0 ax
[PoI 1 boiy]
a@ o 3n 3o M 1|
E10I0 5) |g Pi od4 5 40 1 a0 om
0) B sells arte N tremmont x 1008
2 r con com e +100
a (33)) na coetnviaen nsetTtoyr su0t.a0ti2o1n05asseaym
OLE Wciatuh mcetaro weincoasn cuesecntrsoiane oak
Boseaarernen
Te| eveSnE
we ToTTer
renin 3soi iaioins
1S08Tc0)hyrTScEax)eTOE
TT TartTo cylTTT S SEE e TT ww --
mune 501 Brad gen) S508)
sae= tnveinslt ) 4g wos
iigg
EJ TE
E4400wo)1osasmeoimy uwyo H | eor wgm
N mesCafI +E2
Z@ of4 eooze 00onmF odmo ooodfuy
frgoosnm ago
0) cS elle aH tte renR tannt 5 1008
E gan couR x 10m
(3)
((#8))
1csoo-ll6)l90dqeepnnrsseitcttiyypit0 1a6te. xdx14 i200n58 thiee/mmsyxgenttion medn
+ page 23 -
001384
FIR Z5 399 12:40
T-6906
MKEEDDIICCAALL 22200 %PEE 0202
APPENDIX 1 continues
EXPERIMENT 1
LOR
EAT]
'
NOTOX Project 223458
IMLEY Clon SFICIBNGY nay o
DGoasein Remptby werner id]
TI otalnEa, or
Cloning feaciien!s
5% Sonron)
cSoowlovnansc hy
W4sl;
a aKithout setabolteactuation
8
5
now
"wei]
.
ia2 7
B : H8 :
1i&d}
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6100 ":i a
Pl
P"
wSwon=lt"vfeenlt
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BEH1th potatoe sctsuntion
i55
a
w5m
owamoo aablig oas
iioi5
a75 a
o55u i
T + CEN dnkth directlyTafeE Ter trr eseme ny ma T
+ page 24 -
001385
IR 0309 120M MMEEDDIICCAALL 22020 2ZEE 0202
fboo..37 2 2
-
T-6906
NOTOX Project 223458
APPENDIX 1 continued
EXPERINEN1T
TALES SELECTION OATA M0 clo Errcrency o Tm awe ra TT WA hpFeaErTselection 7ayeea ITNo.aeonf tr1 o Se ClSonTm ing y piweace' NNTeoooorlrtccee || wr
s-- otvent |fo ea Witohouta metabo1lic, ]activation ro
SG 8 U1iR80f537D1orsYfe:ipiirerzr $
Iil o meo m Eon
oxoonm
o(pllo mygogomhmoeode1s,
orm oN
d We wdee agmads dTasEoR 0oH F 8OB EjEeomM m ooonas
soownee |1
0 settle activtion TT
WWG@osooAofTEf:EdLosgffgrrodm2gds
I Plo xERow L DhooaR
Ewllowmnolee Eiwom
oiluoass,
MwmyE womwa womda I dalRoRn B uEgyuMw opa aed
4T86=50Mu6tapnrtecfir5eiqcuaetnecdy pInurth1e05exspuorsviitviaorns medtm
+ page25 -
601386
IONE 0308 12400 WEDIGAL 220 25E 02
Soamorn
oT
T-6506
NOTOX Project 223458
APPENDIX
EXPERIMENT 2
1
continues
BLES WCEiLSLholctonestsbweoTtesauctisvaetioncara
T TT wrw
gDueotie ) TIelrTeeiYsstnernbetftaxre10S614r"1T11esstare nee108180) rm ock mhoone?) TS--5eo0n)--rSco0o--un8]--| 2--)
saB euwn|ld 0 .8 "oo
PPo _o--h
8a$403oii 1 aoazow 8om
E8BE.oT1d3 [[I[[III
" B Bo oF 3 o oo 3 n n8on
osss |510g
1 E1H7 1"1@ 3 17 a [a]
(1 i sale at fterrvruatne x 10m
@ Sconhcoim re x 1000
(Gf11 a7cnaallvddeodmaatPotry
0.4 x 108 crm mutation assay
"Pee 28
001387
TIN.26. 1309 12:40
m
KEDICAL 220 ZE 02
my oso --
To. am
T6906
APPENDIX 1cant fnued EXPERIMEN2T
NOTOX Project 223458
BLE JO WcHtEuRcmoettsalwsoasctuioen cura
dueose
wn {ceEttLe Fbeftore TELEV)TSrSeeS
ale restart soratne
T: R
een
eaungl T| eT SeET r
Le]T
n 20606) Tech `aunt 65m) 6160 rots} net) S01 SO30EaLy
saoBo llveln0t o8rssi w os
@8 004f8 0 oooddae mmomowoLae | ae om
Bmaolor4 om3on BB oosouso0aoEoRmoho o4h n i | ggeee mow
5a0s0 $6111 1156
7Ww7)lsge
b a4s3
b[EeX
aa
4"77 11
B58 OW80 L1
00.22 4
M35BE3
33))
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110022
55
a 1aon3
[AShERTa,
@ a eon comeE x 100
((@(633))) 0)
acon)siueadeetnensuiitryny eth T60 precs
0$.46x51005 imp tactuetdeeisnonthanennyjonstion
aedtun
* page27 -
:
601388
JIE 1308 12:40R MEDICAL 22300 23FE 0207
F001 228
T-6906
NOTOX Project 223458
APPENDIX 1 continued
EXPERIMENT 2
aE1 conti erricimer yo
f Galen) cr lang 7
asieirvaene EF@& 5i8 os
ithe a"w0i80gh8 sBai8in8 wsiina
asienvtenes fii soFasI ozno
8 2gai 88s8 #HiI0o0n8 8aHio8aa
"THESE pact taced in the
lI SlCeloeninggert5tiecotnenoey)
metabolic station
15n74 5
[L[n II Ew]
ii1t8 isiln
g EIi7 oi7
sw
0 essere activation
57o
w n58 o aow
uitie in
g 8 i 2 d u:
io
WE o Ka
apueieron msten
- Page 28 .
-
001389
IO 75. 0398 12:400 WSEEDDIICCAALL 222200 278 02
m0.077o3 nn
-
T-6906
NOTOX Project 223458
APPENDIX 1 continued
EXPERIMENT 2
TALE 12 SELECTION okra Mo Lowi EFFicENGY
T wim Fpre seTlT ecmtionee pwhatmeT Me. or 1mmT Crloning pa|tNeeaara re) c--ET--|| --
i
7
ly [Lo SL moryl ce | ue
I solvent |fTEv
Nithout metssorie peetvarion ea ToEn B 17 e
I]
TR RAoiTdeEy $$d dir
r1bdr ore
3rpsar ad
I E i l io E aB E wE opo3w o mbi[I wnoilmw e o|orla.1e
LEfEik ebwg siws W lwoR o@F&4 I lmeesdoie aa ins
soewwveent |1
0 erator activator T
E W%oI ogfR iaed FE
o1drmnm oaiemnm EEA
IlPmomdoR pwd og
BOoBw oo[ dlIu4 ohmhee |t,e
M wWmo oof1m 3isE
sI3mRp
adyEmm
I ElaE moA E oBA soon
ER2B ohlnAeEonpmfe onEluEedE
WF = Mutant fraquency per 105 survivars
hess.
001390
JN.26.0380 12:40 KVEDEICNATL E9292A00L428 0200
0.92 77330
T-6906 -
NOTOX Project 223458
APPENDIX 2
ADIVIOUAL COLONY COUNTS 0 CELL COUNTS 0F Nor epoRren exeerments
EXPERIMENT
TABL1E3 WCHEELRL Cmeoaubres1iwceacatsicatroang cara
bCosecoe rsI[!cT eiTtstOTebaSngatryxsoSfiTCt10eO)stdnwerSneeterxTofix) E to r g eleno colcog] TPTERET TT
ST olvent)EL
u Smee Ein) 30820 rece} Smt) S508 a1
MmBaoloodr81pm of r on23
HsdOrl do
Mosuemmm aw
oW wmr oileeos wow
W 7505)1e.
v[I w
k5s d 38 1 oa s a 6s 1 [a]38H2E
(4) sE ell ste tursro, noune x 1008
@ S eo coun r fr
fd Re (3)
[4]
con cel
density density
0.4 1.6
x x
105 105
es) /m1
(8) T-6906 precipitated in the exposition medium
ee 14 cone rman ou0
GGoorerm BclonRfigoNtate Py
rGu h ve or allCelennitgsetrriiccttney
5H Sanreny
saienrwvtalnt mo
w B Hl o w
Yh sutspette activation
r1d
7 a 8
fe, 4008
5i
on7501) 85700%a
i1a02
8 noa on
8a
82
(1) T6906 srecpisates 0 the eaporseron meer
+ page 20 -
. 601391
TUE I309 124
T-6906 +
MVEERDTI,CAL 200 2SEE 0202
w0o..2773 2 31
NOTOX Project 223458
APPENDIX
EXPERIMENT 4
2
continued
BLE 35 FGEERmectoaunrestwicoacstsicatironng cura
oE Lmeaeraer l!Ie caita Srbetarys 1S10sTare 2 s g pTeeor om] UP S O TTwT
eaten x abitetoen's sof) och SeeneS E00 5comp8l r1on EmS EL)e eeio sa)
sW B Z eBtveno Ot)E o o DLsLo E Bm R B H 48 oHasww oW we o| o ee o
50 75
1
16
0d
1 9.3
58
Bo ons
a
opldag8
05); LU
16 1
Io 1 47
8 5
1 38
i 1
14 40 oa
8 a
28 1 mom
1 a0 Ios
a 5
0) T sale atT ter co, um x 1008
6
11a
2
@ E con countr x 100
8[3) ancFeonlslnmdddbernseyiicthys t0 vite. x1n4 i0n5ocheemmgasteson seston
ME Le clone ericioey oa0r
BGoasleen ecxlpitngwenptiearynr ITotaRna. Ior E,SClloniengseettteeseney
55 Enron)
saiotwmverns mo
m8 Bs8 Rm
E----
101
a2
ionw
mwoo on750 1)
8 88 3 w5:in4s
iii
ai 8 u5
5104
56
107
11 T6906 precipitated 0 the exponteron meron
- page a1
|
001392
