Document RaDbyy1E60d75bRQ77VMnkZ3E
CORNINGHazleton
MUTAGENICITY TEST ON T-6342
MEASURING CHROMOSOMAL ABERRATIONS IN CHINESE HAMSTER OVARY (CHO) CELLS:
`WITH A CONFIRMATORY ASSAY WITH MULTIPLE HARVESTS
EINALREPORT
AUTHOR Hemalatha Murli, Ph.D.
PERFORMINGLABORATORY
Coming Hazleton Inc. (CHV)
9200 Leesburg Pike Vienna, Virginia 22182
Ea wl
errr?
LABOPRR OJEA CT IT DENTOIFIRCATY ION
CHV Study No.: 17073-0-437CO
SUBMITTTEOD
3M Corporation
Building 220-2E-02 3M Center St. Paul, Minnesota 55144-1000
SCTUODYMPLETIDOATNE
September 16, 1996
CHV Study No.: 17073-0-437CO
lof3s
C0029S
CORNING Hazleton
QUALITY ASSURANCE STATEMENT
Project Title: Chromosomal Aberrations in Chinese Hamster Ovary (CHO) Cells: With a Confirmatory Assay With Multiple Harvests
Project No.: 20990
Assay No.: 17073
Protocol No.: 437CO
Edition No.: 4, Modified for 3MCorporation
Quality Assurance inspections of the. `study and reviewofthe final report ofthe above referenced project were conducted according to the Standard Operating Proceduresofthe Quality Assurance Unit and according to the general requirementsofthe appropriate Good Laboratory Practice arengdultaotitohnes.stuFdiynddiirnegcstofrroomn tthhee ifnoslpleocwtiinognsdaatnesd:final report review were reported to management
Dosing-09/06/1995
09/06/1995
Draft Report Review-11/06,07/1995
11/07/1995
Revised Draft Report Review-01/04/1996 01/04/1996
Final Report Review-09/16/1996
09/16/1996
M. Murphy C. Orantes, K. Newland C. Orantes C. Orantes
GBT yy
`Quality Assurance Unit
ite Released
CHV Study No.: 17073-0-437CO
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CORNINGHazleton STUDY COMPLIANCE AND CERTIFICATION
RTaeshgesueldtaeftsoicrortnihsbiePndatrshtteu3Fd,yooiwdsassaunecddonDDdreuuccgetmeAbddemirinn2ci2os,tmrp1al9ti7ia8on,ncee(fFfweDictAth)iTvtiehteJlGuen2oe1od2o0fL,ta1bh9oe7r9aU)t.oSwr.iytChPordaaecotifceFreedgeurlaaltions saimgenneddmpernottosco. lTthheartewoweurled naoffescitgntihfeiciannttegdreivtiyoaftitohnes fsrtoudmytohretahfeorienmteernptrieotnaetidonroefgnutlyhaeatpitopenlssitcorarebsltuehlets stTethsutedayrratdiewcsldieagatnsaaphnradevseeevnbateleuedanthrieeornveiicenriwtreeerdpirab.eysetnhtesSatnudapyprDiorpercitaotre,cwohncolucesritoinfiweistthhiant tthhee ceovnatleuxattioofnthoef.the
aArnlelcdoatcredsothpaaynsodbfecoetnnhterroeflviniraeelswureledtpsobriytntgtheheinseSrrtaeutpdoeyrdtDaaisrreeacstrouesrpu.plotroAltfletdrhaibwsydasanttuaed,xypdeworicilummebenenttaaarltcihdioavnte,adrreeiccoorrdds,anpdrotthoicsol
tfhaeciSlpitoenssoofr.CoAmftienrgthHeazolneetyoenaIrncp.erfioord,attlheeasStpoonnseoyreamrafyolelleocwtintgo hsauvbemitshseiaofnoorftemhenenttfhiieonanSlteodrreapgoer.t to
tmiamtee,rioarlssernettationaedstionrtahgee
storage facility
fdaecsiilgintaestoefd bCyomthienSgpoHnaszolre.ton
Inc.
for
an
additional
period
of
Submitted By:
Study Director:
ei MHaemmamlaaltihaanMuCyltio,gePnhe.tDi.cs Department of Genetic and Cellular Toxicology
o Study LL
Date
CHV Study No.: 17073-0437C0O
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CORNINGHazleton
TAOFB CONL TENE TS
Page No.
ABSTRACT...
cece
teseeses 6
LO SPONSOR .........ooooiiiiiiiiiiiiiiiie
20 M2A1TERIALCl(iTenEtS'TIAdeRnTtiIfCicLaEti)on ...................................... 3
22 23
DateReceived Physical Description
-
24 Genetics Assay No.
30 TYPEOFASSAY ...................cccoo..8
40 PROTOCOLNO.
lB
50 5S.1TUDYDAIniTtiEatSion D.at.e.........ccoooiiiiiiiiiiiiiiiiiieeeii ieee 8
52 53
Experimental Start Date: Experimental Termination Date
60 6.S1UPERVISStOuRdYyDPirEeRcStoOrNNEL ....................c....................8 62 Laboratory Supervisor
70 OBJECTIVE ...............
. tes abptasesttrsne anions
80 RATIONALE.............
2ssamereesnnamse srsseensisd
90 EXPERIMENTALDESIGN ................................... 9
100 M 10.1 ATTeEstACNeRlDlsMI ETHAODL S S .........................
102 103
Cell Culture Medium Negative and Solvent Controls
104 10.5
Positive Control Agents Rangefinding Assays
106 Aberrations Assay Without Metabolic Activation
C10
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`CORNINGHazleton
10.7 108
Abermations Assay HarvestProcedure
With
Metabolic
Activation
109 10.10
ASbleirderaPtrieopnasraAtniaolnysainsdaSntdaiAnsisngay Evaluation
10 RE1S1ULTS.S.o.lu.b.i.li.t.y.a.n.d.D.o.s.e.D.e.t.e.rm.i.n.a.t.io.n.............
cds
12 113
RRaannggeeffiinnddiinngg AAssssaayy WWiitthhoMuettaMbeotlabioclAicctiAvcattiivoantion
114 1s
CChhrroommoossoommaall AAbbeerrrraattiioonnss AAssssaayy WWiitthhoMuettMaebtoalbioclAicctiAvcattiivoantion
120 CONCLUSION ...........ooooiiiiiiiiiieeei
20
130 REFERENCES ......... C20
140 EXPERIMENTALDATATABLES............................... 2 150 DCEEFLILNSITIO.N.S..O.F.CoHooRoOiMiOiSiOiiMiEiA.,BERRATIONtSrrFtsOsRarGeeIsEteMsSssAsaSsTatAaIneNsED dl
CHV Study No.: 17073-0-437CO
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CORNING Hazleton
ABSTRACT
aTbheerroabtjieocntsivienoCfhtinhesseinhvaimtsrtoearssoavyarwya(sCtHoOe)vacleulaltsewtihtehaabinlditwyiotfhoTu-t6m3e4t2abtooliincdauccteivcahtrioonm.osomal
d0F.io4sr9s8to,hlev1ed.d6o6is,ne4sr.tae9nr8ig,leef16id.ne6di,ionn4g9i.za8es,dswa1a6yt6,ewir4at9ht8,aan1dc6o6wn0ic,tehnaotnrudatt4mi9eo8tna0obfuolg4i/9cm8lacmwtgeiv/raemt.itoens,Cteotdnhecientnettshtreaatrditoioscnelse
was of
0.166,
arnnoanndagrceetfdiiuvncadttiiinoognnasasossasfyasy1.,0c0Ao%lmlpinldeottsheeinmcgyittwooattsoixcaicicinhtdiyeexvweaadssucosobimsnpgeaarrveedddosiwininttghhevctouhlelutsmuoerloevefdnot1s%ceodn(tw1ri0otllhci4uml9)t8u.0reisIgn/mtlh,e
2Sb7s%ervaendd6in3t%haesculctoumrpeadroesdedwiwtihthth1e6s6o0lvge/ntmc.ontrIonltchuelatcutrievsawteiorneaosbssaey,rvreedduicnttihoenscoufltu1r9e%s,wdeo3rs3ee%d,
with 166, 498, 1660, and 4980 ug/ml, respectively.
wiBinatcshuebd2a5to0en,dt5wh0ie0t,dha1t12a2550f,,r2o25m500,t0h5,e0d307o,5s70e5,0r,aann1gd0e05f0i0,n0d01i5nu0gg0/,amsaslnaydw,i2tr0hep0ml0eitcpaagtb/eomlcliulcwtiautcrtheiosvuoattfimCoenHtaOibno2cl0eil.cl0sahcwoteiurvreation and
aberrations assays. activation and with
1C2u5l0t,ur2e5s0t0r,ea3t7e5d0w,iatnhd75500,0010u0g0/,ml15w0i0t,hamnedta2b0o0l0icpga/cmtilvawtiitohnouwtermeetaanbaollyizced
ofborsecrhvreodmionsothmealnoanbaecrtriavtaitoinosn. aNssoays.ignAifsiicgannitfiicnacnrteaisnecrienacseellisn cweiltlhs cwhirthomcohsroommaolsoambearlrations was.
aberrations activation
was
observed
in
the
cultures
dosed
with
3750
and
5000
g/ml
with
metabolic
a9In9n5dt,h4e194c79o00n,ufigar/nmmdalt1ow9ri9yt0htrumigael/t,marlbeopwlliiitccahtaoecuttciuvmlaetttuiaroebnsooilnifc2C0a.cH0tOiavnacdteil4ol4ns.aw1enhrdoewuiirtnhacsu2sba4ay9ts,.ed49wC8ui,ltth1u2r15e205s,,t22r4e4a99t0,e,439783,07,46,
f74r6o,m 9t9h5e,441.4190h,oaunrdas1s9a9y0wpiitgho/umtfmreotmabtohlei2c0a.c0tihvoautrioans,saanydanwditwhit1h25204,9,2449908,,3774360,anadnd99d5wgithm
oc4bh9sr7eo0rmvuoegsd/omimlnatlfhreaobnmeorrntahatecit2oin0vs.a.0tiaNonnodas4s4is.ga1nyi.fhioAcuarnstiagsinsnicafryiescaaswneittiihnncmcreeeltalassbeowliinitchceaclchltsriovwaimttoihsonocmhwarelormeaobasenroarmlaatylizoendsfworas
dmaobeestraerbdaotwliiiotcnhsa4cwt9ai7vsa0toigbosn/.ermvAeidsniitgnhneitfh4ie4c.ca1untlhtiuonrucerrseadassosesaeyidnwpwieitrthchem4net9ta7pb0oolluyigpc/lmoalcitdiiyvnawttaihoesn.2o0b.sT0ehrihvsoeupdrosiainststiahveye crwuieltthures
was investigated conditioanss the
under assay
nonactivation conditions by with metabolic activation.
treating
cells
for
3
hours
under
the
sponse same
CHV Study No.: 17073-0437C0O
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`CORNING Hazleton In this repeat trial, replicate cultures of CHO cells were incubated with 250, 500, 1250, 2490, 3740, and 4980 pg/ml without metabolic activation fo3r hours and harvested 20.0 and 44.0 hours after initiationoftreatment. Cultures treated with 250, 500, 1250 and 2490 pg/ml from the 20.0 hour assay and with 500, 1250, 2490, and 3740 ug/ml from the 44.0 hour assay were analyzed for chromosomal aberrations. No significant increase in cells withchromosomal aberrationswas observed in the 20.0 hour assay. A weakly significant increaseincells with chromosomal aberrations was observed in the cultures dosed with 3740 ug/ml in the 44.0 hour
3as7s4ay0. pgA/msliginniftihcean4t4.i0nchroeuarseaisnsapye.rcent polyploidy was observed in the cultures dosed with
`The test article, T-6342, was considered weakly positive for inducing chromosomal aberrations in CHO cells without metabolic activation with athree hour treatment. T-6342 was considered positive for inducing chromosomal aberrations in CHO cells with metabolic activation. T-6342 was considered positive for inducing polyploidy with and without metabolic activation.
CHV Study No.: 17073-0-437CO
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CORNING Hazleton
Chromosomal Aberrations in Confirmatory Assay
WCihtihneMsueltHiapmlsetHearrvOevsatrsyW(iCtHhOT)-6C3el4l2s:
Witah
10 SPONSOR: 3M Corporation
20 MATERIAL (TEST ARTICLE):
21 Client' Identification: T-6342
22 Date Received: July21, 1995
23 Physical Description: Clear, colorless liquid 24 Genetics Assay No.: 17073
30 TYPE OF ASSAY: Chromosomal Aberrations in Chinese Hamster Ovary (CHO) Cells 40 PROTOCOL NO: 437C0, Edition 4, Modified for 3M Corporation 50 STUDY DATES:
5.1 Initiation Date: August 15, 1995
52 Experimental Start Date: August 24, 1995 5.3 Experimental Termination Date: December 04, 1995 60 SUPERVISORY PERSONNEL:
6.1 Study Director: Hemalatha Murli, Ph.D. 62 Laboratory Supervisor: Carol S. Spicer, B.S. 70 OBJECTIVE:
wtToihtiehnoodubutjceemcetctihavrbeooolmfiotcshoiascmtaiilvnavtaiibtoenrr.oraatsisoanyswiansChtoineevsaeluhaatmesttheeraobvilairtyyo(fCtHhOe)tecsetllasr,tiwcilte,h Ta-n6d342,
CHV Study No.: 17073-0-437CO
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80 RATIONALE:
CORNINGHazleton
wTihtehacseslalsy1i0s idnedsuicgenecdhtrooemsotsabolmisehbwrheeaktsh.erCthheemitecsatlalryticilneduocreidtslmeestiaobnsolmiateysrceasnulitnitenract soburrcerhaekstushlatitnibcnrhveriaoskmisabltdeiondnatomhtaagtraeer.jeoienAibtoehrrerrreajtroieiponanisirneaardebbnayocrtomhnaeslceecqloulneifnnicgseuourfcahftaiaiolnwsuary(eEavisanntrsoe,pbae1i9ru6np2dr).eotceecstsaebsle 90 EXPERIMENTAL DESIGN:
Results from the the chromosomal
rangefinding assay aberrations assay.
wInerteheursaendgteofidnedtienrgmiansseaty,hetdhoesceulrtaunrgees
to be were.
used
in
ohfatrhveesctueldt2ur0e.s0wheoruersevafatleuratineidtifaotrioenvoifdetnrceeatomefntto.xicMitiyt.otiAc sinudmemxaarnydofvitshuealtorbesaetrmveanttions
schedule for the rangefinding assay is given below.
----T=EesR` t c TTeespto Aerticli e ~A ~WWaassm hh Ci oCollcceec mmiiddl FeiFxiaxeate tiioonnn
- 59
0
178
179
20
_ +59 0 33 m 179 0 2
paIonnsdtihtneievgceahtcroionvtmerooaslno.dmTasohllevaebanebtrerrcarotanittoirnoolsnssa.sassaSsyiasny,gslreweepclruielctacutroeenscduuwlcetturereedsuwswieetdrhefaour2s0ee.ad0chahotoefuatrcwhhoadrdvooesssetelsteoivefmle,thine
fCthrheormoinmoitonilsalyomtoranialel oaabfnedtrhrweaitptiohosni2st0i.wv0eeracenodnatnr4ao4l.ly1dzohesodeusfr.rhoaAmrvtsehusetmcmtualitmrueyrsoefsitnthrteheaettcerdoenaafttimfreomnuatrtsodrcoyhseetdruliaellvsels and
chromosomal aberrations assays is on the following page.
e for the
CHV Study No: 17073.0437CO
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CORNING Hazleton
-_-- Test
tela
InitialTrial
-s9 +59
TestArticle
0 0
ConfirmatoryTrial
-s9
0
+59
0
-s9
0
+59
0
RepeatTrial
-s9
0
--_---s9 0 0
100 MATERIALS AND METHODS:
Wash
17.8 3
176 3 ag 3
3 33
Colcemid Fixation
18
2
18
20
18
20
179
2
42.1
44.1
42.1
44.1
18
20
R4R_ 4 44
10.1 Test Cells:
TpehremCahniennetsceelhlalmisnteearnodvawreyreceolrlisg(inCalHlOy-oWbtBaLi)nedusferdomin tthheislaasbosraaytowreyreoffDrro.mSa. r`eWocllfofn,eUdntiovemrasiinttyaoifnCkaalriyfootrynpiiac, sStaabnilFirtya.nciTshcios. ceTlhlelicneellhsashaavneasvienrcaegbeeceyncle time of1210 14 hours with a modal chromosome numberof21 102 Cell Culture Medium:
The CHO cells were grown in supplemented with 10 % fetal
bMocvCionye'sserSuamc(ulFtBuSr)e,m1e%diLu-mglwuhtaimcihnew,asand
1%
penicillin and 5% CO, in air.
streptomycin,
at
approximately
37C,
in
an
atmosphereof
about
CHV Study No.: 17073-0-437CO
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00304
103 Negative and Solvent Controls:
CORNING Hazleton
cIenlltsheannodncauclttiuvraetimoendiaussma.ys,Sonlevgeanttivceonctornotlrsolwsewreerceuclutlutruersecsonwthaiicnhincgontthaeisnoeldveonntly faocrttihvaetitoenstaasrstaiycsl,e tahtethneeghaitgihveestancdonscoelnvternatticoonntsreolds winerteestthceulstuarmees.asIndetshceribed in the nonactivation assays but with the S9 activation mix included. 104 Positive Control Agents:
(ThMeMCpo)siftoirvethceonntornoalctaigveanttisownhsiecrhieswearned ucsyecdloinphtohsepahsasmaiydsewe(CrPe)miinttohmeycmietnaCbolic
cacltaisvtaotgieonntsheartiedso.esMintotomryecquiinreCme(tCaAbSolHic50a-c0t7i-va7t,ioSni.gmCa,ycLlootp#ho2s5pHh0a6m1i9d)ei(sCaAS #
a6c0t5i5v-e19in-t2e,rmSeidgimaat,esLboty #mi6c7rFo0s1o5m5a)ldeonezsymneots.actInditrheectclhyrboumtomsuosmtalbeabceornrvaetritonesd to
casosnafyisr,mattwoorycotnrciaelnst;r0a.t5i0onasnodf1M.0MugC/m(l0,.0t8hiarnddri0a.l1)0aungd/mClP,
initial and (5.0 and 10.0
pg/ml)
lweevreelsuwsaeds taonailnydzuecde icnheraocmhoosfotmhael aabbeerrrraattiioonnsasisnaytsh.e CBHotOhcMellMs.COannedoCfPthweerdeose
dissolved in water.
105 Rangefinding Assays:
tIrnetahtemseentasbsyaysse,etdhiengcealplsprwoexriemactuletluyre0.d3fxor1a0ppcreolxlismpaetrel2y5 2c4m'hofulrasskpirnitoor 5tomi of complete McCoy's Sa culture medium.
105.1 Assay Without Metabolic Activation:
TThheen,cutlhteurteesstwaerrteicilencwuabsatweadswhietdh ftrheomtetshtearcteilcllsewfiotrh1p7h.o8shpohuartseabtuf=f3e7reCd. csoanlicneentarnadtifornes0h.1cogm/plme)tewmaesdaidudmedc.onTthaienciunlgtuCroelscweemriedth(efninatlrypsinized Parnedpahraartvieosnteadnd2.S0thaionuirnsg)l.ater. (See Sections on Harvest and Slide
10.52 Assay With Metabolic Activation:
In this assay. the CHO cells were exposed to the test article for three hours
CHV Study No.: 17073-0-437C0
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`CORNING Hazleton
NatA=D3P71C.5inmgt/hemlp,reasnednciesoociftarircatacliidve2r.S79mgr/emalc)t.ionTmhiextSu9refr(aSc9tio1n5 lm, m(MaolleecSuplraarguTeo-xiDcaowlloegyy,ratInsc.whiLcoth h#0a5d8b3e)enwapsredveiroiuvseldyftrroeamtetdhewiltivheArroofclor m1e2t5a4botl0iiznidnugccehtehmeicmailxsedtofmuoncrteioanctoixviedfaosremes.nzTyhmeesthwrheiechhouarreicnacpuabbalteioonf ttoixmiecwtaotshuesceedllbsecaanudstehepreonlzoyngmeedaecxtipvoistuyoreftSo9thisel$o9stmriaxptiudrley amtigabhotubte p3o7ssCi.bleThineacmteidvaituiomnodifdsnhootrthalivveedFaBnSddhuirgihnlgytrheeacetxipvoesiunrteerpmeerdiioadtteosavoid produced by the 9 enzymes by bindingtoserum proteins. sAafltienre.thCeoemxpploestuereMpceCroiyo'dsth5eacmeleldsiwuemrewwaassahdeddedtwtiocethweictuhltbuurfefserwehdich 0w.e1reugt/hmeln)iandcdubeadtfeodrftohre1l6a.st8 2h.o0uhrosuwristthoCcoollcleecmtidmet(afpinhaalsceocneclelns.traTthieon acunldtusrteasinweedraestwhaesn dtersycprsiinbiezdedf,orhtahrevensotneadc,tifvixaetdi,onanrdansgleifdeisndwienrgeaspsraeyp.ared 10.53 Assay Evaluation:
aMintaoltyizcinigndtheexwnausmbaenarloyfzemedtfarphoamstehsephriegsheensttifniv1e0s0u0rvciovnisnegcudtoisveelceevlelsl.s by 10.6 Aberrations Assay Without Metabolic Activation:
aCnuldtu0r.e8s w1er0ecienliltsia(te=d44b.y0sheoeudirnagssaapyp)ropxeirm7a5teclym1f.l2axsk1i0ntoce1l0lsm(l2o0f.c0ohmopulreatsseay) cMocnCfoirym'astoSraymteridailsu,mt.heOcnelelsdwaeyraeftienrccuublattuerdeaitni=ti3a7tioCn,wfiotrhthteheintietsitalaratnicdle at hporuerdse.teTrhmeinceudltduorseesswfeorreabtoheunt w1a7.s7he(d20.w0ithhobuurfafsesraeyd)saalnidne41a.n8d(c4o4.m1plheotuer assay) cMeellCs.oyA'psprSoaxmiemdatieulmyctownotahionuirnsg l0a.t1er,g/thmelceClolslcweemriedharwvaesstpeldacaenddbaaicrkdroinetdostlhiedes owfercehrmoamdoes.omTahleasbleirdreastiwoenrs.e tFhoenr tshteaitnheidrdintr5al%, oGnieedmasyaasfotleurtciuolntufroer tihnietaiantailoyns,is atrhteicclueltiunrMescCwoerye'sinScaubmaetdeid uatm =w3i7thoCutfoFrBtSh.reeAfhtoeurrsthien tthhreeeprheosuernceexopofstuhreetpeestriod cthoempcleleltsewMecrCeowya'ssheSda tmweidcieuwmi.thTbhueffceerlelsd wsaelrieneinacnudbattheedceflolrstwheerreesrteoffetdhweith
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CORNINGHazleton
ptcrhueletpluaarrsetedp=e2fr.oir0ocdhyoutuoprgsteoonfteithnieccutabinamaetloiysofinhs..arvTehsetmweittahph0.a1seg/cemllls Cwoelrceetmhiednhparrevseesnttedduarnidng
107 Aberrations Assay With Metabolic Activation:
aCnuldtu0r.e8sXwe1r0e icneiltlisat(e4d4.b1yhsoeuerdiansgsaayp)ppreorxi7m5atceml?yf1l.a2skxin1t0o c1e0llmsi(2o0fc.0omhpoluertaessay)
tMrceCatoeyd'usndSearmtehdeicuomn.ditOinonesodafymeatfatebrocluilctuarcetiivnaittiiaotniowne,rteheinccuultbuarteesd
that were at =37C
for
wMthecrrCeeoeywh'aossurhSseadinmtewthideciepurmweistwehintbchueofuoftfterFheBedS.staelstAinfaetretarinctldehetahtnehdrceteehelhSowu9errreeexarcpetofiseoudnrwemiiptxehtruicrooedmpitnlheetceells
MupcCto0 yt'hestSiamemoefdhiaurmv.estThweitchel0l.s1wger/emlincCuoblacteemdfiodrtphreesreensttodfutrihnegctuhletulraestpe=r2i.o0d
hoursofincubation. cytogenetic analysis.
The
metaphase
cells
were
then
harvested
and
prepared
for
10.8 Harvest Procedure:
TPrhieosretoobtsheervhaatrivoensstoifntchleudceudltaunreass,sevisssumaelntoobsfetrhvaetpieorncsoenfttcooxnifcliutyenwceeroefmtahdeec.ell
pmroensoelnacyeoerfmwiittohitnicth(elacrugleturroeunfldaesdksc.elTlsh)eocrudlteuardescewlelsreflaolastoinegvainlutahteemdefdoirutmhe..
mTihteotciucltaunrdesinftreormphtahseedcoesllesraanndgewfeirneditnrgeaatsesdawyiwtehr0e.0tr7y5psMiniKzCedLfihrysptottoocnoilclect
solution. This chromosomes.
treatmenthelpsto The cultures were
swell then
the cells and fixed with an
thus disperse the absolute methanol:
glacial
acetic slides
acid were
(3:1, v-v) prepared.
fixative
and
were
washed
several
times
before
air-dried
10.9 Slide Preparation and Staining:
Slides slides
were were
prepared by stained with
5dr%opGpiienmgstahesohlaurtvieosntefodrctuhletuarneaslyosniscolefamnitsolitdiecs.inTdheex
and
cDherpoemxosmoomuanltiabnegrrmaetdiionusm., All slides were then air-dried and cover slipped using
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CORNING Hazleton
10.10 AberrationsAnalysisand Assay Evaluation
cCeelnltsrowmeerreesseelqeucatledtofotrhegomooddamlornpuhmobleorgy21an+d1on(rlayncgeell2s0w-i2t2h) wtheerenuamnableyrzeodf. ftOohnreetthehseutdnaidrftrfieecdrleecneatlnltdsy,pfiefrpsooomsfstichbhelrneoe,mgoaftsrioovmmeaealancadhbesrroerlpavlteiincoatntsceo(cnEutvlrtaounlrsec,ual1tt9uf6ro2eu;srSwdeeoerseeSealcnetvaielolnys,zoefd ct1oh5ne.t0p)r.ooslisAttiwlveeeraecsotcnto2rd5oeldceclpulrlstiwuorreerst.eo aannFaaollryyscizosen.dtrCofelolrolcfsbhiwraiostm,hoaasbloemrsalrialdteiasobneesrxrcwaeetpritoenfrsoerfcrtohoremdepodonseitoivfe adnaatlayszhienegtsthbeyntuhmebmeircorfosmciotpoteisctaceglelsloicnat1io0n0.0 cMeiltlostaincdintdheexrawtaioswaassseesxsperdebsysoend tahse a percentageofmitotic cells.
cThhreofmoolsloomwianlgafbaecrtroartsiownesredattaaken into account in the evaluationofthe
1. The percentageofcells with any aberrations. 2. The percentageofcells with more than one aberration. 3. dAonsye,eiv.ic.d,eancpeosfiotriviencdroesaesirnegspaomnosuen.tsofdamage with increasing wCehrreomtaatbuildataend.d iTshocehyrowmearteidnogta,phso,wifeovbesre,rvcoends,idweerreednionttehdeienvatlhueartaiwondoatfathaend arbeiplrietsyoenfttthreuetceshtraormtiocsloemtoalinbdruecaekscharndommoasyompaolssaibbelryrabteioinnsduscinecdebtyhetyoximcaityy:not tPaebrucleatnetd.polHyipsltooirdiycalwacsonatnraolldyaztedaafrreomprtehseen=t4e4d.0inhToaubrlaess1a0.y and results were pAurceplolsecslasbsuitfaied">a"si"saGlTs"oiisnccolnusdiedderinedthteo tcaobnlteasinfor10thaibsecrlraastsiiofniscaftoironsttaotiistnidciaclate that itis a minimum number.
mStualttiisptliceacloamnpaalyrsiissoensmp(lSooykaeldatnhde FRioshher,'s1E9x8a1c)ttToecsotmwpiatrheatnheadpjeursctemnetnatgefoofr cells cwointthroalsb.errAatiloinnseairnteraencdh ttersetaotfmeinntcrgeraosuipngwnituhmbtheerroefcsuelltlssfrwiotmhtahbeesrorlavteinotns with
CHV Study No.: 17073-0-437C0O
14
60308
`CORNINGHazleton
winacsreeasstianbglidsohseed w(Ahremrietapg<e0.,011.971A)llwafsactaolrssoapsersftoartmededp.revTieosutsalrytwicelreestiagnkiefnicianntcoe
account and judgement.
the
final
evaluationof
the
testarticle
was
based
upon
scientific.
110 RESULTS:
ILI Solubility and Dose Determination
sTt-u6d3y42wawsaesvdaelsucartiebdeidnasstebreiliengdefiroeneilyzesdolwuabtleeri(nLwoatte#r1,7,soprietspasroeludbi0l6i/t1y6/f9or5 tahtis TChHiVs)s.oAluticolneawr,ascodliolrulteesds tsool1ut%io(n10wamslob)taiinneMdcCatoay'csonScaencturlattuiroenomefd4i9u8mmign/tmhle. taebssteanrcteicolefarneymacienllesd. cAomtpalectoenlcyenstorlautbiloenoafnd4a98p0Houfg/ml7.5iwnacsuldteutreermmiendeidu.m,Sttehreile sdteoicokniczoendcewnattreartiwoansotfh4er9e8fomrge /chmosaenndadsialnutiaopnpsrtohperrieaotfewveerheiclperefpoarrtehdisfoarssdayo.sinAg ethaechrasntgoecfkinsodliuntgioans,saaynsd. tAhlelsdoolvseinntgcwoanstraolchciuelvteudrewwitahsado1s%ed(1wi0tuhlm10i)udmolsinogfof s1t6e6r.il4e9d8e,io1n6i6z0e,danwdat4er9.80Cgon/cmelntrwaetrieontseostfed in0.1t6h6,e0r.a4n9g8e,fi1n.d6i6,ng4.a9s8s,ay1s6.w6i,t4h9.a8n,d `without the $9 metabolic activation system.
The this
stability of assay is the
the test article under responsibilityof the
the preparation Sponsor.
and
dosing
conditions
used
in
11.2 Rangefinding Assay Without Metabolic Activation
cIenllthmeocnuolltauryeertrweaatsed<w5i%thco4n9f8l0ueungt,/maln,d nthoevciuslitbuleremictoontsiicstceedllosnwleyorfedoebbsreirsveadn;dthe
mdietaodticcelclesl,lsflwoaatsinogbosreravtetadchiendt.he
A severe reduction culture dosed with
in the number of 1660 pg/ml. The
visible. cell
m1o5n%o;laflyoeartilnogodkeeadducnehlelsalatnhdy daenbdritshewedreegrneoeteodf.conFlfolauteinncgydweabrsisreadluscoewdaasbout
observed similar to
in the culture dosed the negative control.
with
498
pg/ml,
but
the
culture
otherwise
was
aMnitdot1i6c60inudgic/emslw(eTraeblaen1a)lyaznedd cformopmatrheedcutlottuhreessodlovseendt wcointthro1l6..6,At49.186,6016p6g,/m49l8,, the mitotic index was essentially zero. However, ltle or no reduction in mitotic
CHV Study No.: 17073-0-437C0
15
00309
CORNING Hazleton
tihnedefxirswtatsriaolbosfetrhveedchatrothmeosneoxmtalloawbeerrdroasteioonfs 4a9s8sagy/wmilt.houBtamseetdaboonltihceascetrievsautlitosn, `5w0a0s, c7o5n0d,uc1t00e0d, w1i5t0h0a, 2a0ndho2u0r00hagrv/emstlaitntoesrtdearrtitcoleencsounrceenatwraitdieontsoxoifci1t2y5r,a2n5ge0., 11.3 Rangefinding Assay With Metabolic Activation
cWuiltthurSe9domseetdabwoiltihc4a9ct8i0vagti/omn,.anTuhnehecaelllthmyonceolllamyoenrocloanyfelruewnacse owbasserrveedduciendthbey oacpcpurrorxeidma(taebloyut8653%%,,anTadbalese1v).ereArtedtuhcetnieoxntinlotwheernduomsbeeorfo1f6v6i0sipbgle/mmli,totthiec cells wmoansoolnalyyesrocmoenwflhuaetncreedwuacsedth(eabsoaumte2a7s%)t.he sBoalsveedntocnontthreosle,raesnudlttsh,etmhietiontiitcialindex aatbetrerstatairotnisclaescsoanycewnittrhatmieotnasboolfi2c5a0c,t5iv0a0t,io1n25w0a,s2c5o0n0d,u3c7te5d0 waintdh5a02000 huogu/rmlh.arvest 11.4 Chromosomal Aberrations Assay Without Metabolic Activation
InitialTrial
C15h0r0o,maonsdom2a0l00abuegr/rmalti(oTnsabwleer2e). anNaloyzsiegdniffriocmantthienccurletausreess idnocseellds wwiitthh 7c5h0r,om10o0-0, somal aberrations were observed for the cultures analyzed.
Tdheiasd acselslasyacnodvedreberdisa,wsiedveerreanlogsesooffvtiosxiicbiltey.miUtnothiecalcetlhlys,cealnldmaobnooultay2e5rs%,rfeldouacttiinogn 2in00ce0llugm/omnlo.laSyleirghctolnyfulnuheenaclethwyerceellobmsoenrovleadyeirnst,heflcoualttiunrgesdedaodsecdelwlsitahnd15d0eb0riasn,da rmeodnuocltaiyoenrincotnhfelnuuenmcbeerwoefrveisoibbselrevmeidtoitnitchceelclusl,tuarneds adboosuetd 1wi5t%h r1e0d0uc0tpigo/nmiln. ceAltl nthoermlaolw,eswtitahnaolnylzyead sdloisgehtorfe7du5c0tiuogn/miln,tthheencelul mmoonofblvaiesyiebrrlseampipteoatriecdcetlolsbeanadlmost ainbdoeuxtarel1a5ti%veretodutchteiosnolivnentthecodnetgrroeleocufltcuornefsluoecnccuer.redSeavtearlel droedsueclteivoenlss,inegm.,it9ot5i%c, 86%, 67%, and 57% at 2000, 1500. 1000, and 750 jig/ml, respectively.
nBoansaecdtiovnattihoentaobxeircirtaiteisonosbtaasisnaeydwiansthciosnrdiuacl,tetdheatcotensftiarrmtaitcolerycotnricaelnotfratthieons of
125,249, 498, and 44.1 hours,
746,
995,
1490,
and
1990
g/m,
using
two
harvest
times
of
20
CHV Study No: 17073.0-437C0
16
00310
CORNING Hazleton
ConfirmatoryTrial
dFoosretdhewi2t0hh7o4u6r,h9a5rv,es1t4,9c0h,raonmdos1o9m9a0lga/bmerlra(tTiaobnlsew3e)r.e aNnoalysizgendiffircoanmttihnecrceualsteusres
ainnacleylzlesd.witAhschobrtoamionseodmianlthaebefrirrastitorinls,
were observed for the toxicity range
anyofthe was wide,
cultures ranging
from
a74s6evpegr/eml9.5%Threedcuecltlimoonnionlmaiyteorticconifndleuxenacte1w9a9s0 riegd/umcledtob2y5a%boruetdu2c5t%ioantat
c1u9lt9u0regs./mlT,o acnodmpoennlsyat8e3,m1e1t7apmheatsaepshacsoeulsdwbeerescsocroerdedfrformomontehoefrtephleicraetpeliccualtteure,
in orderto score a totalof200 metaphases for this dose.
tFuorresthdeos44e.d1whiothur2h4a9r,ve4s9t8,,c7h4r6o,maonsdom99a5l aubge/rmrlat(iToanbslwee4r)e. anAaglayizne,dnforosimgntihfeiccualn-t
ciunlctrueraessesanianlcyezlelds.wiTthhecpherrocmeontsopmoallypalboeirdryatiisosncsowreedreatotbhsiesrhvaerdvefsotr tainmyeo,ftahned.no
sreilgantiifviecatnottihnecr2e0asheosurwehraervneosttedb.ecTauhseectohnecelnotnrgaetrieoxnspoofstuersettairmteicclaeuwseerdeglreoawteerr
tleotxhiacli.ty.TreTahtemtenwto whiitghhe9s9t5apupgl/imeld dwoassesseovfer1e9l9y0touxgi/c,mlreasnudlti1n4g9i0npagb/omult w4e0r%e
reductions 746 ug/ml.
in monolayer however, the
ctoonxifcliuteynwceasanmduc7h9%lerssed(uacbtoiuotn
in mitotic index. 15% reduction in
At
confluency cultures).
and
18%
reduction
in
mitotic
index
compared
to
the
solvent
control
aTchteivraetsiuolntssoybsttaeimnaedppferaormedthteo tswhoowastshaeyltarcikaolsfainntyhecalabssteongceenoifcaanctiSvi9tymebtyabtohleitcest airntdiculcet.ioHnoofwecvherro,mionsaonmoatlhearbearsrsaatyiohnasviwnagsadnemeoxnpsotsruarteedti(mseeoefSeocntliyo3n h1o1.u6r)s., the. 11.5 Chromosomal Aberrations Assay With Metabolic Activation "
C37h5r0o,maonsdom5a0l00abpegr/rmaltioinnsthweerpereasneanlcyeozfedthferormatthleivcerulSt9urmesetdaobsoeldicwiatchtiv1a2t5i0o,n 2500. swyesrteeombs(eTarbvleed 5i)n. thSeigcnuilftiucraenstdionsceredasweisthin3c7e5l0lsawnidth50c0h0ropmg/omslo.maTlheabterreraattmieonnts wtoixtihe,3c7a5u0sipngg/malbowuats9o5n%lyresldiugchttiloyntionximco,nboultatyheer5c0o0n0flpuge/nmcly tfroeraotnmeenctulwtausrehiagnhdly
CHV Study No.: 17073-0-437C0
1"
$0311
CORNING Hazleton
`gawrbeeorauettear7btu5onx%diacrinettyd,.ucatHinoodwneofnvoleryrt,9h4ethrmeeepmtliaitcpoahttaiescceuislntdwueerxer.ewaaFsnlarolaeytdziuancbgleeddefbarydoomcnetllhyles 1cau3nld%tudcreoebmrwpiisatrhed 0 the solvent control cultures.
sBaasmeedteosnt atrhteiscelerecsounlctesn,ttrhaeticoonnsf,irumsaitngortywtorihalarwvaesstctoinmdeusctoefd2w0itahndes4s4e.n1tiaholulrys,the
ConfirmatoryTrial
tFioornsthwea2s0ohbsoeurrvheadrvoenslt,y afosritgnhiefciuclatnturienscdroeasseediwnictehll4s9w7i0thg/chmrlom(Toasbolmea6l).abeTrhriasitnoxtihcetmrielaotfmiecnitndreexd.uceTdhethterecaotnmfelnutenwciythby37a3b0ougt/7m0l%waansdmcuaucshedleass4t3ox%icr,ecdauuctsiionng tabhoouutgh3t0h%e trreedautcmteinotn wiintchon3f7l3u0engc/ymalndap1p8ea%rereddtuoctbieonsoinmemwithoatticmionrdeext,oxEicvetnhan tThheerseifmoirlea,rodnolsyetihnethhiegihnliytatloxriicalt,retahetrmeenwtaas ntohehdiontsoeflainmiytocfltahsteogaesnsiacyactivity. c(h5r00o0mopsgo/mmall) ianbedrurcaetdioansr.epeIantabbolteh, tbruiatlsl,arbgoet,hinscirmepalseeainndpecrocmepnltecxelalbsewrirtahtions were observed.
tFuorresthdeos44e.d1whiotuhr49ha8r,ve1s2t5,0c,h2r4o9m0o,saonmdal37a3b0ergra/timons(Twaebrleea7n).alyNzoedsifgrniofmictahnetculciunlctrueraessesanianlyczeellds. witAhltchhoruogmhosthoem4a9l7a0beurgr/amtilotnsrewaetrmeenotbwsaersv1e0d0ftoorxaicnytoofytiheel.d psuoflfyipclioeindt cmeeltlaspihnacsreesasfeodrsainganliyfsiicsa,ntiltyw.asAtne3v7e3r0thpegl/esmsl,nottheerdetwhaatstlhietpleerocrennto toxicity evident
iTnhceresausceceisnspfeurlcaecntticvealtlisonwibtyhtchhermoemtoasboolmiaclsaybsetrreamtiwoanss i(altlu2st0rahtoeudrbsyhatrhveelsatr)gein rtehqeuciurletsurmeestaebxoploisceadcttiovcaytciolnopthoobsepchaommiedecl,astthoegpeonsiict.ive control article, which
The initial clastogenic
isnutbesrptraentcaet(iso)noifntthheespererseesunlctesowfatshethSat9
tmheetatebsotlairctiacclteiwvaatsiocnonsvysetretme.d
to
mHeotwaebvoelriz,eadc.coTrhdeinagpptoeairnafnocremoatficolnasptroogveindiecd abcytitvhietySipnontsheora,cttihveattiesotn aprotritclieonisonfot
the assay was notin doubt, but this activity should also have been observed under
CHV Study No.: 17073-0-437CO
18
000312
`CORNINGHazleton
sniotnuaacttiiovnartaiiosnedcotnhdeiptoisosnisbiilfitthyetSha9tsaynstuenmrehcaodgnniozeedffteocxtiocintythienttehset narotniaclcet.ivTathiiosn
causlstauyrewsawsearcetuaanlallyyezleidm.inTahteintgrtehaetmceenltlspweirtiohdisnudnudceerd anboenrarcattiivoantsiobnefcoorneditthieons
rweemreovoendlyafatbeoru3th2ouhrosuresxpleosssurtheafnotrhtehehaarcvteisvtattiiomnesa,sswayhse.reIafsththeectoensttianrutiecdle was
apbreersreantcieoonsf,tthheentetshtearatbiecrlreawtiaosnscasuhsoiunlgdtbheeodbesmeirsveaobflceeifltlhsewittrheaitnmdeunctetdime was
made equivalent to nonactivation assay
the activation was repeated,
assay, using
i, a3
rheoduurceedxptoos3urheoupresr.iodA.ccordingly,
the
11.6
Chromosomal Treatment
Aberrations
Assay
Without
Metabolic
Activation;
3-Hour
iTnhveesptoisgiattievde bryestproenatsiengobcseellrsveudndienrtthheeasssaamyewciotnhdimteitoanbsolwiicthaocuttivmaettiaobnowlaics 3ac0t0i.va1t2i5o0n., 2C4e9l0l,s 3w7e4r0e,tarenadte4d98fo0rg3/hmou,rsusatintgesthaarrvteiscltetciomnecsenotfr2at0iaonnsdo4f425h0ou,rs after the initiationoftreatment.
dFoosretdhewi2t0hh2o5u0r, h5a0r0v,es1t2,5c0h,raonmdos2o4m9a0lga/bmerlra(tTiaobnlsew8e)r.eNaonasliygzneidfifcarnotmitnhcerecausletsuriens tcrelelastmweintthscwherroemonostomvaerlyatboexrircat(iaobnosutwe3r0e%obrseedruvcetdi.onUinnfcorotnufnlauteenlcyy, atthese s2m4a9l0l ungu/mmbl)e,roafndmetthaepnheaxstehsigchoeurlddobseeoexfam3i7n4e0d.pg/Amslfwoaostn5o0tetdoxiinctthheattaobnlel,y7a of T3h8umse,taappphraosxeismaotbetlayin1ed8%forofmthtehecrelelpsliwceatreeciunldtiucraetsehdaads chharvoinmgosaobemrarlataiboenrsr.atiDonuse. w10outhled sheavveerebdeeepnrehsisgihoenraotfmailtoontgiecrihnadrevxe,stthteimpee.rceOnbtvicoelulsslyw,itmhuacbhercrlaotsieornsdoliskeely asstsepasyswaonudldtohcavoempbaereen tnheeceosbssaerryvatbolaecchliaesvteogteonxiiccitrieesspocnosmesp.arable to the activation
tIrtesatimnetnetrepsetirnigodtothnaontewthheatn
the the
test S9
aacrttiicvlaetiwoanssyclsetaermlywmaosrperetsoexnit.c
for the 3 hour Genotoxic
teovxeinctisty(,mauntdattihoenmoertacbhorloimsomsoofmtaelstchaarntigcelse)s taoreclnaescteosgseanriiclysausbssotcainacteesdiwsitthherefore:
oebxspeercvtaetdiotonobfemaocrcoemtpoaxniciietdy ibnytihnecraebasseendcetooxicfi$t9y,wnaostvleersys tsouxgigceistyt.ivTehtuhsa,t tthhee
CHV Study No: 17073-0-437C0
1
600313
CORNING Hazleton
acotnatirnomlelinntgoffaaccteorrtianionbsheirgvhitnogxiccliatsytofgoeanricshaocrttivtiitmyefporertihoids,teasntdarntoitcltehewamsettahbeolic. conversionofthe test article.
Fodorstehdewi4t4hh5o0u0r, h1a2r5v0e,st2,4c9h0r,oamnodso3m7a4l0 uabge/rmrlat(iToanbslwee9r)e. Aanaslmyalzle,dbfurtosmigtnhiefciuclatnutres
w7i.t5h%3of7c4e0llugs/wmilt.h Achsriogmnoifsiocmanatlianbcerreraasteiionnspewracsenotbpsoelryvpeldoiindythaelscoulwtaursesobdsoesrevded
in these cultures. The monolayer confluency
3740 g/ml treatment relative to the solvent
resulted in about controls, and the
55% reduction 2490 pg/ml
in
rterdeautcmeednt(5g7a%v)e faobrotuhte3h0i%ghrdeoduscet.ion. The mitotic index was also significantly
wTihtehsethriesstuelsttsawretircelec,oanpspilsiteedntfwoiarthsthhoertinpteerripordeotfattiiomnet,hawtilalhgiigvhelyritsoextioc tcerlelastment
ctiomnetapienriinogdcahfrteormoinsiotimaatlionaobfetrrraetaitomnesntt.hatTchaen hbaervoebsstertviemde finorthtehe2o0bsoerrv4a4tihoonurof
t`hmeacxuilmtuurmesnusimmbpelrysorfedaubceerdratnhte cteelsltsarwtaisclenottoxdiectiteyr,misnoetdh.atTfhieneradaddijtuisotnmoefntSs9itnothe
bdoesteweleenveelqsutih-taonxaicchtireevaetdmeinnttshiusnsdteurdayctairveatnieocneassnadrnyotnoacmtaikvaeticoonmpaasrsiasyons
ctohnedSit9ioancst.ivaTthioenisnyitsitalemobcsaenrvbaetiionntseorfpcreltaesdttoogebneiaccaoctnisveiqtyueonncley oinfathmeoprreesleinmcieteodf
stotxaincdaarcdtinoonnaocvteirva3thioonurassseaxypso,surraeth,ecrotmhpanarbeedintgo
the due
long to a
exposures for the metabolic effect,
The
fteosrtsahrotritcleeniosutghhereexfoproesuervealpueartieoddsas(ecgl,as3tohgoeunrisc) osnulcyh ftohrathitghhelcyeltlosxicconttraeiantimnegnts
4c4hrhoomuorshoarmveesatbetrirmaetsi.ons do not disintegrate before they can be observed at 20 to
120 CONCLUSION:
Thihgehltyesttoaxritcictlre,eaTt-m6e3n4t2so,fwsahsoretvadluuraatteidonas(ebge.i,n3ghcoluarsst)ogbeontihc itno tchueltpurreesdeCncHeOancdelalbssefnocre. otfreaantm$en9tsm.etabolic activation system. Polyploidy was also induced for the highly toxic
CHV Study No.: 17073-04370
20
00314
`CORNING Hazleton
13.0 REFERENCES:
Armitage, York, NY,
P.19S7t1a.tisticalMethodsinMedical
Research,
John
Wiley&
Sons, Inc., New
Evans, H.J.: Chromosomal aberrations Review of Cytology, 13:221-321, 1962.
produced
by
ionizing
radiation.
Intemational
Sokal, 1981
RR, and
Rohlf, F.J: Biometry, Ed.
2,
W.H.
Freeman and
Company, New York,
CHV Study No.: 17073-0-437CO
21 00315
`CORNINGHazleton 140 EXPERIMENTAL DATA TABLES
CHV Study No.: 17073-0-437CO
2 C00316
`CORNING Hazleton
TABLE1
RANGEFINDING ASSAY FOR ASSESSING TOXICITY `Compound: T-6342
AssayNo: 17073 Trial Noil Date: 082495 Metabolic Activation: 59 Lab No. CY8225
Treament
[[ii] ] CSoanfvleuernc:e
NEGATIVECONTROL Mears
Control
os
00
SOLVENTCONTROL water TEST ARTICLE
00 wm 20 6 semi se
100 0
05 wm oo
100
mor
100
oem 02
0
10 yom 00
=
Metabolic Activation: +59. Lab No. CY8225
Trial Not
Treamen
[5] SCosnSfolluveenncte"
NEGATIVECONTROL _ Mecorssa
Convo
ws
0
SOLVENTCONTROL water 00 we om
100
TEST ARTICLE
8 sem ns
0
em ns
0
- ey ns
100
0 mous
0
oem es
ot*fTahtchihesemednedtcpaeoplihlnatcseisacbelalssnb.eedenAvucaptloouunaaltvteicdse.ulalcAotobsheueravniraetmtineooosnfts twthhkeieccnohnarfnledueamnnacydeehaypbprseierort1vr0oapihhoyen othfhaerhvfeelsatsks
are also evaluatedfor the appearanceoffloating mitotic cellsanddeadcells.
"* Toxic dose level,
CHV Study No.: 17073-0-437C0O
23
00317
f38 3 mm
cpmiron
i
g
CROMOSOMEAEC RATION mhNeCos HsNaLSE AMSTEROVARY E15
oan
Lancs beets
Mee Acs
TTT eee
= Scaausf[e1]u5c]
--
if ole ols
[roo [io[TawexTo To Tar Tor or] ita PER ABEBERRRAKMIAT
-- mer
0
ws
.
ra wow ow on
[a
ome
vo 10Among
.
ERI aw ee a
Q
--
wan
E 22 ape ae e, = =wu i
2&Bg
Cf I 2Erm
E 8
soe Emrt
ns
cenit --
Mee ets
swels elaFcaocEwonnTeoxT Tao on|ZA r Bal 8d8SH5,%
Wn
tn .
SE 2 8
awown 2
aw
------------
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CHROMOSOME ABERRFAoTcION0AhCRmEoSs oHAMeSTEROVARY E115
Tans
J--
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cone [sume comer wits le
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CORNINGHazleton
TABLE 10 CONTROL DATA OF CHROMOSOME69A5BETRHRRAOTUIGOHNS89I5N.CHINESE HAMSTER OVARY CELLS
Activation
Aberrraotirons %OfWCiethlls sWoifthce>l1ls
-- eC------ wos --wn--ape-- rmCel -- C Aberr0-- a0tin ons __-- _AAbbeermr0ia-- 0itioonnss |
Saver Cont
aMvAoX oanss
~
3s
1as0 3
0o0s7 3
woos wi Max
onosm
Av aso
0200 on
00s0 on
PoMsiioinmeyCionnetrol
~
3
3
x
Wow wv ou
wo
00
Negaive Control
MAaGx os2m
~
x
sai o x
1"0 2
woo aw Max
o00n0
ava aon
0500 10
0100 06
SolventContol
x
2
a
we Maawx 0o0n0
7a0
<000
Positive ont
AxG o2a
1"
0
Cyclomapmanide
win Mwanx Ia
2i0o0
050
_--
N avxG w 1a299 w sxix w 30240
CONTROL DATA OF POLYPLOIDY IN CHINESE HAMSTER OVARY CELLS 495 THROUGH 9/95
eC
AcWtiivhaotiwon Aciwvaanion
Msanx omm0
i0o0
Savers Con
A~G o"se
oPse
aix 200000
500
R ANNEGE e o8mSU 0s5SE
CHV Study No.: 17073-0-437CO
32 00326
CORNINGHazleton 15.0 CDEELFILNSITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED
NOT COMPUTED
TG Chromatid gap:
SG Chromosome gap: UC Uncoiled chromosome: PP Polyploid cell: E Endoreduplication: SIMPLE
(o"nteidcgharpo"m)a.tiAd,ntahcehsriozmeaoftiwch(iucnhstaiisneeqdu)alretgoioonr in
nsmoatleldebrutthnaonttuhseuwailldythinocflaudcehdroimnatfiinda.l
These are totals of
aberratioanss they may not all be true breaks.
(bu`tisaotcthhreomsaatmied glaopc,usIGi"n).botShasmiestearscchhrroommaattidisd.gap
aFbaeirlruarteioofn.chromatin packing. Probably nota true
nAucmebllerco(nnt)oaifnicnhgrmoumlotsiopmleesc.opiNeostofctohunetehdapilnoitdhe cells scored for aberrations.
4hanscfealilleidn. whNiocthcsoeupnatreadtiionnothfeccehlrlosmsocsoroemdefpoarirs aberrations,
TB Chromatic break: SB Chromosome break: DM "Double Minute " fragment:
Athnanacthherowmiadttihcorefagicohnroimnaotnide.chrTohmeataisds,ocliaartgeedr dfirsapglmaceendt. may be partially or completely `Cahbrnoormmoalso(mdeelehtaesd)acclheraormborseaokm,efwoirtmhinagn aancentric fragment that is dislocated. tTehremsienaalredeslmeatlilondsoaubnldesdoomtse,isntoemrsetoiftiwahl ideclhetiaornes dainsdtipnrgouibsahbalbyles.mal rings. Their origins are not
CHV Study No.: 17073-0-437CO
33
00327
COMPLEX ID Interstitial deletion:
TR Triradial: QR Quadri radial: D Dicentric:
DF TC Tricentric:
QC PC
Quadricentric: Pentacentric:
HC Hexacentric:
R Ring
RC Ring Chromatid: RF
CHV Study No.: 17073-0437C0
CORNING Hazleton
aLecnhgrtohmoafticdhrroesmualttiindg"icnuta osumta"llfrformagmmiednrteogfiroinnogf clhyriongmabteids.idae shortened chromatidof a gap in the
Acnhrexocmhaonsgeoambnedetwaeneancetnwtoricchfrroamgomseontm.esw,hiocrhone:
results in a tree-armed configuration.
.
cAosnftirgiruardaitali,onbut resulting in a four-armed
Arnesuelxtschian nagcehbreotmwoeseonmtewwoicthhrtowmoocseonmterosmewrheisc.h TinhiwshiischofctaesneasitsioscicaltaesdswiiftihaesadnDaFc.entric fragment
Dicentric with fragment.
rAensuletxscihnanagcehbreotmwoeseonmtehrweiethchtrhroemeocseonmterosmewrheisc,h eOxfctehnanagsessocciaanteidnvwoiltvhetmwaontyo tchhrreoemAoFs.omSeuschand are named as follows:
ffiovuercceennttrroommeerreess,, uuppttooffiovuerAAFF six centromeres, up to six AF
cAencthrromoemroes.omTehiwshiiscohftfeonramsssoacciiartcelde cwointthaainning a
acentric RF.
fragment
in
which
case
iti
classed as
Single chromatid ring (acentric), Ring with associated acentric fragment.
34
0328
CI Chromosome Intrachange: T Translocation: AB
Other GT
CORNING Hazleton
dEoxecshannotgeinwciltuhdientahechenrtoimreoscohmreo;moes.go.m,ea.ring that
cObhvrioomuossotmraenssfreersoulftmiantgeriinatlwboeatbwneoernmatlwo
chromosomes. not "2Ab."
When identifiable, scored as "T"
cAhbrnoomrmoaslommeonwohcoesnetrmiocrcphhroolmoogsyomise.abnTohrimsalis faor wtahnesklaorcyaottiyopne,,paernidceonfttreinctihneverressiulotn,oetfca. aCslcarssiibfeidcatot;ioe.ng.,usaedirefcaipbrnoocramlatlriatnyslcoacantniootn,be
AA hceelalviwlhyicdhamcaognteadincselmlosrheoutlhdanbe10anaableyrzraetdiotnos. aicdteunatilflyy hthaevety>p1e0s,ofea.bge,rmrualttiiopnlseafnrdagmmaeyntsnostuch as those found associated with a tricentric.
CHV Study No.: 17073-0-437C0O
35
00329
