Document zo0o3Voe5p4EdRD1jJnvv6nZB
CORNING Hazleton
EER
MUTAGENICITY TEST ON
7
{
NOY
C5
153
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T6342 No, #4 sf JcoL
MEASURING CHROMOSOME ABERRATIONS IN HUMAN WHOLE BLOOD LYMPHOCYTES
WITH A CONFIRMATORY ASSAY `WITH MULTIPLE HARVESTS
EINALREPORT
AUTHOR
Hemalatha Murli, Ph.D.
PERFORMINGLABORATORY
Corning Hazleton Inc. (CHV)
9200 Leesburg Pike
Vienna, Virginia 22182
CHV Study No.: 17073-0-449CO
SUBMITTTEOD 3M Corporation
Building 220-2E-02 3M Center
St. Paul, Minnesota 55144-1000
STCUODYMPLETIDOATNE
November 1, 1996
CHV Study No.: 17073-0-449CO
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CORNINGHazleton QUALITY ASSURANCE STATEMENT
Project
Title:
Chromosome Confirmatory
Aberrations Assay With
in Human Whole Blood Multiple Harvests
Lymphocytes
With
a
Project No.: 20990
Assay No.: 17073
Protocol No.: 449CO
Edition No.: 2, Modified for 3M Corporation
UpQrunoaijlteicattnydwAeasrcsecuorcraodnnicdneugcittnoesdptehaecctgcieoonrnedsoriafnltghrteoeqtushiteruedSmyteaannntdsdaorfrdtevOhipeeerwoaaptfpirtnohgperPifraiotncaeleGdrouerpoeodsrotLofafbttohrheaetQaoubarlyoivPteryarcAetsfisecureernacnecde raengdultaotitohnes.stuFdiynddiirnegcstofrroomn tthhee ifnoslpleocwtiinognsdaatnesd:final report review were reported to management
Inspection/Date
AdditionofColcemid/08/15/1996 Draft Report Review/09/18-20/1996 Final Report Review/1 1/01/1996
FindingsReported
0815/1996 097201996 11/01/1996
Auditor
C. Orantes C. Orantes C. Smith
=
-
ity Assurance Unit
CHV Study No.: 17073-0-449C0
2/1/54 Date Released
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STUDY COMPLIANCE AND CERTIFICATION CiTsohsoeupeeddreas1ct9ri7io9bn-e1ad9ns8dt0uDd(eyevfewfelacostpicmvoeennd1tu9c8P1tr)eidnwciiitnphlceaosonmpyflaGipaopnolcidecawLbailbteohartmaheteonrOdyrmgePanrntaicszt.aitcieTohCne(rf8eo1r)wE3ec0roe(nFnoinmailc) Annex 2, dinetveigartiitoynosfftrhoemstthuedyafoorrtehmeeninttieornperdetrateigounloaftitohnes otrestthreessuiltgsn.edTphreotroacowldathtaathwaovuelbdeaefonfesrcietgvntiihfeeiwceadntby cTtrehipetreSeritsaeu.ndtys Dainreacptporro,prwihaotececrotnicflieussithoantwtihtehienvatlhueatcioonntoefxttohfethteestsatrutdiycldeeassipgrneasnendteevdahleuraetiinon
arAnellcdotaredscthoaapsnydboecfoetnnhterroeflviniraeelswuerledtpsobriytn tgtheheinseSrrtaeutpdoeyrdtDaiasrreearcsetousrpu.plotAroltlfedtrhabswy sdatantuade,yxpdweoircliummbeeennttaaaritcihdoianvt,eadrreeicncootrrhddes,aSntpdrotthoicsol ftahceilSiptoinessoorf.CoAmfiienrgthHeazolneetoyneaIrncp.erfioord,attlheeasStpoonnesoyreamrafyolelleocwtintgo hsauvbemitshseiaofnoorfetmheentfiionanleodrreapgoert to mtiamtee,rioarlssernettationaedstinortahgeesftaocrilaigteyfdaecsiilginteastoedfCbyortnhienSgpoHnaszolre.ton Inc. for an additional period of Submitted By: Study Director
Moet, Mis HMeammamlaaltihaanMuCryltio,gePnhe.tDi.cs Department of Genetic and Cellular Toxicology
ifi)ae Study Completion
Date
CHV Study No.: 17073-0-449CO
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TABLE OF CONTENTS
ABSTRACT...
10 SPONSOR.....ooooviiiiiiiii
20 M2A1TERIALCl(ieTnEtSsTIAdeRnTtiIfCicLaEti)on
22 DateReceived
23 24
GPehnyseitciacls DAessscaryipNtoi.on
Page No.
eee
eee 6
eee7
...................._.....__
30 TYPEOFASSAY ......coooooiiiiiiiii
40 PROTOCOLNO...........oooouiiiiiiiiieeeeee eo 7
SO 5S.T1 UDYDAITniEtSiati.on D.ate..............o..
52 53
Experimental Start Date Experimental Termination Date
60 6S.U1 PERVISSOtRudYy PDiErRecStOorNNEL ..........................._. 7 62 Laboratory Supervisor
70 OBIECTIVE o.oo
7
80 RATIONALE...........
8
90 EXPERIMENTALDESIGN ...............
-
renee
100 M 10.1 ATCelElAsNR UDseMdIETHAODL S S .........
. J)
102 103
CNeelglatCiuvletuarnedMSeodlvieunmt Controls
104 10.5 106
DPoossietiRvaenCgoenftirnodlinAggeAnstssay for Mitotic Suppression and Dose Determination Aberrations Assay Without Metabolic Activation
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10.7 108
Aberrations Assay Harvest Procedure
With
Metabolic
Activation
109 10.10
Slide Preparation and Aberrations Analysis
Staining and Assay
Evaluation
HO RESULTS oii 1.1 Solubility and Dose Determination
eee
1S
12 13
DDoossee RRaannggeeffiinnddiinngg AAssssaayy WWiitthhoMuettaMbeotlaibcolAicctiAvcattiivoantion
114 11s
CChhrroommoossoommaall AAbbeerrrraattiioonnss AAssssaayy WWiitthhoMuettaMbeotlaibcolAicctiAvcattiivoantion
120 CONCLUSION ...........cocooiiiiiiiiiiiine ee
16
130 REFERENCES ............c.c.ccccoovviiiiiaiiiiaii
140 EXPERIMENTAL DATATABLES ..............._...... 1s
130
DEFINITIONS CELLS ooo
OF
CHROMOSOME
ABERRT ATIONS FOR GIET MSA STAINED
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ABSTRACT aTbheerroabtjieocntsivien coufltthuirseidnwvhiotrloeabslsoaoydwhausmtaonevlaylmupahtoectyhteeasbiwliitthy oafnTd-w6i3t4h2outtomientdaubcoelicchracotmiovastoimoan.l aT1nh%iec(t1le0sstfoalrumttii)colnesfwoaranstdhditsihsaessosvlaeyvh.eidcliencdoenitoonli,zeddeiwoantiezreadtwaatceorn,cewnetrreatdioosneodfw5i0t0h amgd/omsli,ngTvhoelutemset of 98F%.o5r00t3h.8e%1d.,o67s1,e55%r0,a0n,g1e81f%6i,.n7d5,i7n5%g0.,a0s,asna1dy67w1,i05t00h%0a,wne1d6rw7e0i,tohbaosnuedtrv5me0edt0a0ibnoutlghi/ecmmlaictwtoeiviracetitionends,ieccdeo.snocReendturcattiioonnssooff03,156%7., rweistphec0t.i5v0e0l,y,1.a6s7,co5m.p00a,re16d.7w,it5h00t,he16so7l0v,eanntdco5n0t0ro0lgc/ulmtlurew.itRheoduutcmteitoanbsoolfi8c%afc,tthi5ve%at,ciuo4ln%,tu,re1s%d,oasnedd 3100000%pwge/rmel owbistehrmveetdaibnoltihce amcittiovtaitcioinn,dirceesspoefcttihveelyc,ulatsurceosmdpoasreeddwwiitthh1t.h6e7,So5l0v.0e,nt5c0o0n,tr1o6l70c,ulatunrde. acBonandscoeefdnt2or5na3t,tiho5en0sse5,odfa1a012,170t,,h2e155i31n,i0t,5ia02l50,r1i01,a01o20f5t,1h01,e513c00h,1r02o,0m1ao0ns,doam4na0dl120a5bi1eg0r/rimatgli/owmnilsthwasimsteahtyoaubwtoalmsiectcaoabncotdliuivccteadcteisvattiinogn a2c2t.i0vahtoiuorn aasnsdayws.ithCu5l0t5u,re1s01d0o,se1d51w0itahnd25230,15005p,g/1m0l10w,itahndme1t5a1b0olpigc/amcltiwviatthioountwmeerteabeaovtlaiiloucnatined pfoorlycphlroiodmyo,soormaelndaobreerdruaptliiocnas.tioNnowsaisgnoibfsicearnvtedinactretahseecionncceelnltsrawtiitohnschanraolmyozsedo.mal aberrations, 2aIn0ndt0h02e0pcg0o/0nmfuligr/imnmalato4irn6y.20a2sh2so.au1yr,haroesupsrlaiyacsaustnaedyceuarlntnduornweaiscttwhie6vr2ae.t5ii,onnc1uc2bo5n,adt2ie5tdi0o,wnis5t.0h0,R12e95p0,l0i,2c5a10t2,e050c0,u0l1,tu690000,,an12d00, 1600, 4ii6nt.ch0ubhmaoetuteradbnowolinitachcta2ic5tv0ia,vtai5to0ino0n,a.s1s0aC0yu0sl,,uw1r5ie0ts0h,d2o25s00e,0d0,wi2t5h001,25a,nd2530,005000u,g/amnld i9n0202p.1g/amnldf4r6or.me0sthwhoeeurr2e3a.s1saaypsd wacetrievaetviaolnuaastseady.foarncdhwriotmhos50o0m,al10a0b0e,rr1at5i0o0n.5s0.a0n.Nd1o2000s00i.g0naigfnid/cam1n5t0fi0rnocgmr/etamhslee 4if6nr.oc0emlhltoshuewri2at2ch.t1civhharotouirmonosaossmaayl feabxrecoremrpattthifeoon4rs5,a.9wpoehlaoykuprlionaiccdtryie,vaasoteriioennndeaonsrsdaeoydr.uepdluipclaitciaotniownaast o2b0s0e0rvuegd/malt,thaen ceoxntcreenmterlaytitoonxsicandaolsyeselde,vel, wcTuehlreteutrevesedtriawfrhiteoidclleei,nbTil-no6do3ed4p2eh,nudmweaanntslclyoycnmospnihddoueccryettdeednsecwgoianttfihivraemnafdtoorwriiytnhdtoruiucatlis,nmgetcahbroolmiocsaocmtiavlataiboenr.raTthioensse irnesults
CHV Study No: 17073-0-449C0
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ChromoWsiotmhaalCAobnefrirramtaitoonrsyinAsHsuamyaWnitWhhoMulletiBplloeodHaLryvemspthsocytes With T-6342
1.0 SPONSOR: 3M Corporation 20 MATERIAL (TEST ARTICLE)
21 Clients Identification: T-6342 22 Date Received: July 21, 1995 23 Physical Description: Clear, colorless liquid 24 Genetics Assay No.: 17073 30 TYPE OF ASSAY: WCihtrhomaoCsoonmfairlmAabteorrryatAisosnasyiWniHtuhmMaunltWiphloelHearBvleosotdsLymphocytes 40 PROTOCOL NO: 449CO, Edition 2, Modified for 3M Corporation 50 STUDY DATES:
5.1 Initiation Date: July 9, 1996 52 Experimental Start Date:July31, 1996 5.3 Experimental Termination Date: August 26, 1996 60 SUPERVISORY PERSONNEL:
6.1 Study Director: Hemalatha Murli, Ph.D. 62 Laboratory Supervisor: Carol S. Spicer, B.S. 7.0 OBJECTIVE:
TwThi6et3ho4ba2jn,edcttwoiivitenhodofuucttehmiceshtaribonomvloiistcrooamcaatslisvaaaybteiwroanrastitoonesvainlucautletutrheedabhiulmitayonfwthhoeletebstloarotdiclley,mphocytes,
CHV Study No.: 17073-0-449C0O
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80 RATIONALE:
Tihtehacsesllasy tios idnedsuicgenecdhtrooemsotsabolmieshbwrheaektsh.erCthheemitecsatlalryticilneduocreidtslmeestiaobnoslmiateys rceasnulitnitneract opbrrroercaeekssussltiensicnshuvrcioshmibatlhteaitndbatrmheaaatgkeas.rdeoAebinteohrterrraetrjieopoiannisroaerrderbeajyocitonhneisnceeaqllbuneionnrcsmeuacolhfcfaoanwilfauirygeuarosarttmioiobsntsea(ukEnevdsaentinse,crteapbalier 1962).
Inorderto detect chromosomes are
cvhisrioblmeo.soTmhaellaybmeprhraotciyotnes,s
cells must be in in blood do not
metaphase so usually divide
that and
are
sattitmhuelaaptperdotporidaitveidsetaignec(ulmteutreabpyhapshbyety)ohCeomlacgegmliudti,nin (PHA). The cells are later arrested
90 EXPERIMENTAL DESIGN:
uwRseeesrduelithnsartfvhreeosctmehtdrhoe2m0do.o1ssoehmoarualrnsgaeabfeftriernradtiiinnoigtnisaastasisoasnayoy.fwetIrrneeatuthsmeeedndott.soedMrieattnoegtreimfciininendditinhcgeeasdosofssateyh,retahnceguelctutulorteubsrees wpreerleimeivnaalruyatteedstfiosrgeivviednenbceeloowf.toxicity. A summaryofthe treatment schedule for the
----TT=eestt TTTeeetstaAAimrcicllee WvWaaesshh -- Co_l_Ca colecme-- miidd--_FiF-- xiaxatti-- ioonn
- 59.
0
19.5
201 21
=+=89
00
33 w2o01 22211
aaIsnnsdtahynesegcwahetriroveem,coossnoodlmuvacelntteadbceowrnitrtrahotliaso,n2sa2n.a0sdshafoyosur,rearhceaphrlvidecosastteetocifumltethuierneptsohsewietirniievteiuaclsoentdrtiraoaltl.aenadcThhweidtoahsbee2r2lreavteil,ons
4a6n.a0lyhzoeudrfhraormvetshtetciumletsuriens tthreeactoendfaitrmfaotuorrdyostreiallse.velCsharnodmofsroommaolneaobfetrrhaetipoonssiwteivree.c1onatrnodl
doses. given
beAloswu. mmaryof
the
treatment
schedule
for
the
chromosomal
aberrations
assays
is
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Test _--
Culture Initiation
Chemical
Wash
Coloemid Fixation
InitialTrial
0000
-59 48
0
192 200
20
+59 8
0
30
200
220
Confimmatory Trial
-59 48
0
193 20.1
21
+59 48
0
30 201
21
-s9 "8
0
433 44.0
46.0
-- +59 5 8 00 3300 444.00 446600
100 MATERIALS AND METHODS:
10.01 Cells Used:
sHtuermilae,nhveepnaroiunsizbeldooVdacfurtaoimneasrisn.glCeu,lntourremsalw,ehreealintihtyiamtaedlewidtohno0.r3wmals odfrbalwonodinpteor c$ulmtlurceulftourrtehfeorchtrheomdoossoemraalngaebfeirnrdaitnigonassasasysaaynsdin0.165mmilocfebntlroiofdugpeertu1b0e.s0. ml 102 Cell Culture Medium:
aTthmeosceplhlesrweeorfeaibnocuutbate%d
aCtOa,boiuntai3r.7TCheoncualtsulroepem,ewdiithumlouosseedcawpass,
in an RPMI
1640
(LJoRtHESB3i3os1c,iednocsees)rasnugpepfliendmienngteadsswaiy;thLo1t5N%o.fe:tTal06b0o2v4i,nechsreorummos(oFmBaSl; aBbierorcahteiomnesd.,
aQsusaalyi)t,y 1Bi%olpohgyitcoahles)maagngdlusttirneipnto(mPycHiAn-(M1;00Gipbgc/om)l,:pQenuiacliiltlyinBi(o1l0o0giucnailtss)/,mla;nd
2mM L-glutamine (Quality Biologicals).
103 Negative and Solvent Controls:
cIenlltsheanndoncauclttiuvraetimoendiaussma.ys,Sonlevgeatnitvceonctornotlrsolwsewreerceulctuulrteusrecsownthaiicnhincgondteaiionniozneldy water at the highest concentration used in test cultures (1% or 10.0 ml). In the
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athcetinvoantiaocntiavsastaiyosn,atshseanyesgbauttivweiathndthseolSv9enatctciovnattrioolnsmwiexreintchleudsead.me as described in 10.4 Positive Control Agents:
aT(chMteiMvpCaot)siiotfniorsveetrhiceeosn.ntornMoalictatogimevanyttcisionwnhCsiecr(ihCeswAeaSrn#ed5uc0sy-ec0d7l-oi7np,htoShseipgahmsaasm,aiyLdsoewte(#rCPe4)0mHiin2t5oth0me8y)cmieintsaaCbolic c6l0a3s5t-o1g9e-n2,thSaitgdmoae,sLnoott #re4q3uiHr0e2m6e9t)abdooleiscnaocttiavcattidoinr.ectClyycbluotphmousspthbaemicdonev(erCtAeSd t#o aascstaiyvse,itnhtreeremecdoinacteenstrbaytimoincsroofsoMmaMlCen(z0.y1m,e0s2.,Ianntdhe0.c3hgr/ommols)oamnadl CabPer(r2a0t,i3o0n,s alenvdel5s0wiags/maln)alwyezreed uinseedacthooifntdhueceacbehrrroamtoiosnomasaslayasb.errBaottiohnsM.MOCneaonfdtChPedwoesree dissolved in water.
10.5 Dose Rangefinding Assay:
Iwnerteheisnectuebsatts,edcuflotrurtewsowdearyesipnriitoiratteod wtrietahtm0e.n3t.ml of blood per 5 ml culture and 10.5.1 Test Without Metabolic Activation:
T2h3e7lCy.mpThhoecny,tetshewteersteairntciculbeatweadswwiatshhtehde ftersotmartthieclceelflosr w1i9t.h5 phhoousrpshaatte cbounfcfeenrterdastailoinne0.a1ndg/fmreis)h cwoamspladedteed.meTdhieumcuclotnutraesinwienrgeCtohlecnehmairdves(tfiendal2.0 hours later. (See Sections on Harvest and Slide Preparation and Staining). 10.5.2 Test With Metabolic Activation:
CHV Study No.:
Ihnoutrhsisattes=t,37theCliynmtphheopcryetseesnwceeroefainrcautblaitveedr StoOtrheeactetsitonarmtiicslteufroer(t5hr9ee: f1r5aclt/imonl,(MNoAleDcPula1r.5Tmoxgi/cmoll,oagyn,d Iinsc.o,citLroitc a#c0i6d627.)7wmags/mdle)r.iveTdhefr$o9m the Alirvoecrloofrma12l5e4 S0priangduuec-eDtahwelmeiyxreadtsfwuhnictcihonhaodxibdeaesne pernezvyiomuesslywhtirecahteadrewith cinacpuabbalteioofnmteitmaebowlaisziunsgedchbeemciacuaslesptroomloonrgeedacetxipveosfuorremst.o tThheeSt9hrmeiexthuoruer might be toxic o the cells and the enzyme activityof S9 is lost rapidly at
17073-0-449CO
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paavrboooiuddtucp3eo7dssCbi.yblteThheineSac9mteiedvnaiztuyiommneodsifdbsynhoobtritnhdlaiivvneegdFtBaonSsdedrhuiurgmihnlpgyrotrtheeeaicnetsx.ipvoesiunrteerpmeerdiioadtetos
sAafltineer.thCeoemxpploestuereRpPeMriIodcutlhteurceelmlsedweiruemwwaassheadddtewdicteo wtihtehcublutfufreersewdhich 0w-e1repgt/hmeln)iandcduebadtefodrftohre1l8a.s5t 2h.0ohuorsurwisthtoCcoollcleecmtimdet(afpihnaalsceocneclelns,traTthieon acsulwtuarsesdewsecrreibtehdenfohratrhveesntoenda,ctfiixveadt,ioannddossleidreasnwgeerfeinpdrienpgaarsesdaayn.d stained 10.53 Assay Evaluation:
tMhietontuimcbienrdoexfmweatsaapnhaalsyezsedprfersoemnttihne s1u0r0v0ivcionngsedcousteivleevceellslsb.y analyzing 10.6 Aberrations Assay Without Metabolic Activation:
cC1eu0ll.l0tsumwrelerscewulettrrueeraetienidintwi1ia5ttehmditthwceeontetdsratiyafsrutgpierciltoeurbaettsop.trreTedwaettomeerdnmatiynsweiadtfhtceo0rn.c6ceumnlttluroraetfiwionhniotsilafetoiobnl,ootdheper c19o.3mplaentde4R3P.3MIhou1r6s4.0Tmheedicuulmtucroenstwaeirneintgh0e.n1 wuags/hmeldCwoiltchebmuifdferweadssaplliancereadanbbdoauctk cmohandtreoo.mtohesTohcemelalssl.liadbTeeswrrwoaetrihoeonustr.hsenlatsetra,itnheed cienll5s%weGrieehmasravessotleudtiaonndfaoirrtdhreieadnasllyisdiess owfere
10.7 Aberrations Assay With Metabolic Activation:
cC1uu0ll.ttu0urmrelesscwuwelertreuerieinnicinutbi1aa5tteemdditawcteon=td3ra7iyfsuCgperfiotorurbtethsor.eterTehawotuomresdnatiynswtiahtefhtp0rr.e6csuemlntiucoreoeffiwnthihotielaettieobsnl,ootdheper
wanadshtehde Stw9icreeawctiitohnbmuifxfteurreed. saAlfitneeratnhde tthhereceelhlosuwreerxeporesfuerdewpietrhiocdo,mtphleetceellRtsPawrMetIircele
it1ni6cmu4eb0oatfmieohdnai.ruvmeT.shtewTimhteehtac0ep.l1hlasuswgee/rmceellliCsnocwluecbreaemtietddhefnoprhraetsrhevenertsetsdetudroiafnntgdhetphrceeulpltaasurtree2d.p0feorhrioocudyrtsuopofto the
analysis.
genetic
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10.8 Harvest Procedure
ttTrrheeeaattcemedellnwtistuhhseplhpeysnpsotiotoosnnwiweclalsKtCchIeent(cer0li.lf0su7ga5endMd.)tthhfeuosrsaduippsepprrenoraxstieamnatthteewlacyshr1do0imsmocisanroudmteeedss..anTdAhfciteselrls were cfreensthrliyfupgraetpiaorneadndfixraetmioveva(laobsfotlhueteKmCeIt,hatnhoel-cgelllasciwaelreacwetaischaecdidt,hr3:e1e,tviimve)s. wAiitrh. dried slides were prepared from the harvested cells. 10.9 Slide Preparation and Staining;
STlhiedessliwdeesreweprreepsatraeidnebdywdirtohpp5%ingGitheemshaarsvoelsutteidoncuflotrurtehseoannacllyesains solfides. amintdotciocveirndselixppaendd. chromosomal aberrations. All slides were then air-dried
10.10 Aberrations Analysis and Assay Evaluation:
cCeenlltsrowmeerreesseelqeucatledtofotrhegomooddamlornpuhmobleorgy46anwderoenlaynacleyllzsedw.ith the number of
tfOohnreetthehseutndaidrftrfieecdrlee,cnettlhtley,ipnefesoagvafaiivcleha,brloseom,lovfsernootm.maalenaadcbheprorrseaipttliiivocenastceo(cnEutvlratonulsr,ceua1lt9u6fr2oe;usrSwdeeoersSeeeaclnetavielolyszeodf
c1u3l.0t)u.resAtwiltehas>t 2255%ceclellslwsewriethancahlryozmeodsfoomraclharboemrorastoimoansl.abFeorrractoinotnrsolforfobmiatsh,onsaell
slides were data sheets
coded by the
prior to analysis. microscope stage
lCoeclaltsiowni.thMiatboetrriactiionndsexwewraes
recorded assessed
on by
the
aanpaelryczeinntgagteheonf miutotmiocfbcmeileltsotric cells in 1000 cells and the ratio was expressed as
cThhreofmoolsloomwainlgafbaecrtroartsiownesredattaaken into account in the evaluationof the
1. 2.
TThhee ppeerrcceennttaaggeeooffcceellllss wwiitthh amnoyreabtehrarnatoinones.aberration.
3. dAonsye,eiv.ic.d,eancpeosfiotriviencdroesaesirnegspaomnosuen.ts ofdamage with increasing
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GQOR77
CORNING Hazleton
awCbehirrlieotmytaaotbfuildtahtaeendt.desitTsahorcethiycrlowemeatroteiidnnodgtua,cpheso,cwifheorvboesrme,orscvooemndas,ildwaeebrreeerdrnaiotntiteohndesiesnviatnlhceuearttahieowynodmaftathaend Preerpcreesnetnptotlryupelcohidryomaonsdoemnadlorberdeuapklsicaantdiomnawyeproessainballyyzbeedianndducreedsulbtys twoexriceaiytynot tabulated. Historical controldataare presented in Table . tpAhuarctpeloiltsiecsslaabsmustiinafii>mae"udsms"nGauTlms"boeiisrn.ccolnusdiedderinedthteo tcaobnlteasinfor10thaibsecrlraastsiiofniscaftoironsttaotiisntidciaclate
EStxaatcisttiTceaslta(nTalhyaskiusreemfpl.l,oy1e9d8a5)Cotochcroamnp-aArremithteagpeertceesnttfaogreolifnceearlltsrewnidtahnd Fisher's aabbeerrraattiioonn)s,(paonldy,pilfaoipdpyl,icaanbdlee,ndtohreedpueprcleinctaatgieoonficnetlrlesatweidtchelmlosrweitthharnesounlet:s from fvaechtiocrlseacsonsttraotlesd. pTreesvtioaurstilcylewseirgenitfaikceanncientwoaascceosutanbtlaisnhdedthwehfeirnaelpe<v0a.l0u1a.tioAnlolfthe test article was based upon scientific judgement. 110 RESULTS:
TLL Solubility and Dose Determination
aDsesiayo.nizTe-d63wa4t2erwa(sPrdeipsasroeldveadt iCnHdVe,ioLnoitze#d2w0a)tewraasttahecosnoclevnetnrtaotfiocnhoofi5ce00formtgh/imsl dfeoriotnhiezdedosweatrearn,gewfeirnedidnogsaesdsawyi.th Tahdeostiesntgavrtoilcluemseoolufti1o%ns(1a0ndlithmeiv)ehfiorcltehicsontrol, 5as0s0a0y.g/Comnlcewnetrraettieosntseodfi0nt.h1e6d7,os0e.5r0a0,nge1.f6i7n,di5.n0g0a,ss16a.y7,wi5t0h.0a,nd167w,it5h0o0u,t 1m6e7t0a,boalnidc cacotnidviattiioonn.s uTsheed sintatbhiilsitayossfaythies ttehsetraerstipcolnesiubnldietryothfethpreepsaproantsioorn.and dosing
11.2 Dose Rangefinding Assay Without Metabolic Activation
MHietmootliycsiinsdiwcaess woebrseeravneadlyprzieodrftroowmasthheciunltthuerecsudltousreesddwoistehd0w.i1t6h7,500.05000p,g/1m.l67., c65u.%l0,t0u,3re81s6%.d,7o,s15e50d%.0,w,i1t18h6%70,,.55500700%,,,11a.6n677d0,,15a.0n000d,%51w06e0.r70,eg5o0/b0s,mer1v(6eT7da0b,ilnaenthd1)e.5m0Ri0et0odtuipccgt/iimonlnd.iscoeBsfaosfe3d5th%oe,n these results. the initial trial of the aberrations assay without metabolic activation
CHV Study No.: 17073-0-449C0
13
CO0278
CORNING Hazleton
w10a1s0.co1ndu5ct1ed0wi.tahn2ad22025.1010hpo0gu/r,mhalrvest testing concentrations of 127, 253, 505,
11.3 Dose Rangefinding Assay With Metabolic Activation 5MHi0et0mo.otli1yc6s.ii7n,sdi5wc0ae.ss0,wobe16sr7ee,rav5ne0ad0pl,ryiz1oe6rd7t0f,oraownmadtsh5he0i0cnu0ltthpuegr/cemuslldtou(rsTeeasbdldweoist1e)h.d0Rw.ie1td67hu,c5t00i.0o50n0s0p,g/1m.l6.7, w5i%t,h 41%.6,7,1%50,.0a,nd5010,001%67w0e,raendob5s0e0rv0egd /inmt,hae smcitoomtpicarineddicweistohftthheesocluvlteunortfecsodn%ot,rsoeld tmciuelottnausbroeof.l2i5cB3aa,scte5id0v5aot,nio1tn0h1ew0sa,esr1ce5so1un0ld,tu,2c0tt1eh0d,iwn2ii5tt1iha0l,at23ri20a1.l00o,fhtaohunerd ah4ba0er1rv0reasgtti/toemnss.tiansgsacyonwcietnht"ra-
11.4 Chromosomal Aberrations Assay Without Metabolic Activation
INITIALTRIAL
RHeedmuocltyisoinssowfa3s8o%bs,er1v8e%d,p1ri8o%r,to50w%a,sh55in%,thaencdul9t5ur%esindotsheedmiwtiotthic25i1nd0icge/smals,
w`ictohmp1a2r7e,d2w5i3t.h5t0h5e,so1l0v1e0n,t1c5o1n0t,roalncdul2t0u1r0esuwge/mrle,orbesseprevcetidveinlyt.heCchurlotumroessotmraealted
a1b5e1r0raptgi/onmslw(eTraeblaena2)l.yzNeod fsrigonmiftihceanctulitnucrreesastereiantecdelwlistwhi2t5h3c,h5r0o5m,os10o1m0a,land
aberrations, analyzed.
polyploidy,
or
endoreduplicationwas
observed
at
the
concentrations
wcBioatnshdeudac2to2en.d1ththeeassrteivnreegsstduloatsnse,d lt6eh2ve.e5cl,son1of2fi5r,1m22a55,t0o2,r5y500,t0i,5a0l900u,0n9,d0e01r,2n0o01,n2a0c10t6,i0v01a,6t0ai0no,ndac2nod0n0d20i0t0i0ongs/wmelre
with a 46.0 harvest
ug/ml .
CONFIRMATORY TRIAL
ecIvunilttdhueernet2s2ad.to1hs-aherodvuewrsitctoohnfft1ih6re0m0acatunlotrduyr2et0rsi0adl0,osuheged/mmowlli,ytshains1da6w0a0sslpioggb/hsmteler.vvieRddeepdnrucioceortitfoonhsewomaofslh2yos%if,sthweas S11o24l0%v0,e,n6t146c%0o0n,,tr7ao4nl%d,cu2l90t30u%0r,epsagwn/emdrle9, 3orbe%sspeienrcvtteihvdeelmiyin.ttohtCeihccruiolntmduiorcseesosmdaaosslceadobmewpriartrahteid1o2nw5s,itw5he0r0te,he9a0n0a.,
CHV Study No: 17073-0-449C0
1
C00Z79
`CORNING Hazleton
sIiygzneidfifcranotm itnhcerceualsteurinescetlrlesatweidthwicthhro12m5o,s2o5m0a.l5a0b0e.rraantdio9ns0,0ppogl/ympllo(iTday,bloer3). No endoreduplication was observed at the concentrations analyzed.
cIunltthuere4s6.d0o-sheoduwrictohnf1i6r0m0ataonrdy2t0ia0l0, phge/mmoll.ysRiesdwuacstioobnssoerfve5d9p%,ri9o3t%ro,w9as8h%ofantdhe
9ob8s%erivnedthienmtihteotciucltiunrdeiscedsosaesdcowimtphar9e0d0,wi1t20h0t,he16s0o0l,veanntdco2n0t0r0olpgcu/lmtlu,res were.
wriestphec1t2i5v,el2y.50,C5h0r0o,maonsdom9a0l0 gab/ermrat(iToanbslwee4r).e aNnoalsyizgendiffircaonmttihnecrceualsteurienscterlelastewdith
chromosomal aberrations, concentrations analyzed.
polyploidy,
or
endoreduplication
was
observed
at
the.
Tshheowsnenbsiytitvhietyionfctrehaesecdelflrceuqluteunrceyofofr aibnedrurcattiioonnosfinchtrheomcoelslosmeaxlpoasbeerdratotiMonMsCi,s the pcohsriotmivoesocmoantloalbeargernatt.ionTs,hepotleysptlaoritdicyl,eains dcoennsdiodreerdeudplniecgaattiiovne ufnodreirndnuocniancgtivation conditions.
115 Chromosomal Aberrations Assay With Metabolic Activation
INITIALTRIAL
H30e1m0o,lyasnids4w0a1s0 oubg/smelr.vedNoprcieolltsowwearsehviisnitbhlee pcurlituortroestdhoesaedddiwtiitohno20f1C0,ol25c1e0,midto atdhdeictuilotnuorfesCodlosceedmiwdithi4n0t1h0e cuugl/tmulr,esadnodsheedmwoiltyhsi3s0w1a0sigo/bmsle.rveRdedpurcitoirotnostohfe s1o5l%v,en2t0c%o,ntr1o5l%c,ul4t3u%r,es7w7e%r,e aonbdse9r5ve%d iinntthhee mciutlottuircesintdriecaetsedaswictohm2p5a3r,e5d05w,it1h0t1h0e, I1y5z1e0d, f2r0o1m0,thaendcu2lt5u1r0esugt/remalt,edrewsiptecht5iv0e5l,y.101C0h,ro15m1o0s,oamnadl2a0b1er0rautgi/omnls w(eTarbelaen5a)-. Nenodosriegdnuipfliciacnattiionncrweaasseoibnsceerlvlesdwiatththcehcroonmcoesnotrmaatlioanbserarnaatliyoznesd,.polyploidy, or cBoansdeudctoendttheessteinrgesduoltsse, ltehveeclsoonffi2r5m0a,to5r0y0,tri1a0l0u0n,de1r50a0c,ti2v0a0t0i,on2c5o0n0d,itainodns were 3000 g/m with 22.1 and 46.0 hour harvests
CHV Study No.: 17073-0-449C0
15
Go0oZ80
CORNINGHazleton
CONFIRMATORY TRIAL
RpIrneidtouhrcet(2i2oh.na1sr-ovhfeos1ut5or%fco,tnhf5ei%rc,umla6tt9uo9rr%ey,s td8ro2ia9sl%e,,dh9ew7mi%otl,hy2as0ni0ds0w,1a02s500%o0b.isneartnhvdeed3m0ipt0roi0toirgc/tominlwd.aicsehs aansd
acwbioetmrhpraa5tr0i0eo,dns1w0wi0te0hr,eth1ae5n0sa0ol,lyv2ze0en0dt0f,cro2on5mt0r0tohl,ecacunuldlttu3urr0ees0s0wteprrgeea/tmoeldb,sweriertsvhpeed2c5tii0nv,etl5hy0e.0c,uCl1ht0ur0ro0e,msoatsnrdoe.amtaeld
a1b5e0rr0atpigo/nmsl, p(oTlaybplleoi6)d.y,Noor esnigdnoirfeidcuapnltiicnactrieoansewainscoebllsserwvietdh acthtrhoemocosnocmeanltrations
analyzed
-
pIrnitohret4o6h.a0r-vheosutorfcotnhfeicrumlattuorreystdroiasle,dhewmiotlhy2s0i0s0w,a2s50o0b,searnvded30p0ri0orpgt/omlw.ash and
Rwietdhuctthieosnoslovfent15c%o,ntr8o0l%c,ul1t0u0re%s, waenrde 1ob0s0e%rviendthientmhiteoctuilctuirnedsicterseaastedcowmiptahr1e5d0,
Dl2y0uz0ee0d,tof2tr5oo0xm0i,ctiahteyn,dcou3ln0tlu0yr0e5sg2/tmrmeelatt,aepdrhewasispteehcst5iw0ve0er,ley.1a0v0Ca0ih,lrao1bl5me0o0sfooarmnaadnla2la0yb0se0irsrgaitn/ioomnnse(owTefarbtelhaeen7a.-
ccuhlrtoumreossodomsaeldawbeirtrhat2i0o0n0s,upgo/lmylp.loiNdoy,soirgneinfdiocarnetduipnlcirceaatseioinnwcealslsobwsietrhved at the
2co0n0c0enptgr/amtli,onasn
aenxatlryezmeedl,yextocxeipct
for a dose
weak level,
increase
in
endoreduplication
at
Tinhceidseunccceeosfsfcuelllasctwiivtathiconhroofmtohseommeatlabaobleircrastyisotnesministhiellcuustlrtautreeds biyndtuhceeidncwrietahsed nceygcaltoipvheosfoprhaimndiudcei,ntghcehproosimtoisvoemcaolntarboelrargaetnito.nsT,hpeoltyepsltoairdtyi,cleani dconsidered einndcroeraesdeupilniecnadtoiroenduupnldiecratcoinodniattioannsoexftmreetmealbyoltiocxiacctdiovsaetiolne,veelxicneptthef4o6ar.0wheoaukr assay.
120 CONCLUSION:
TiTnhhecesutleetsruterseaurdlttiwcshlewo,elreTe-b6vl3eo4ro2id,fiwheaudsmicnaoninnsldiyedmpepernhdeoedcnynttelegysactcoievnleldsufcwotirteihdnadcunocdnifnwigirtmchahotruotormymoetstrioaamblosallicabaecrtriavtaitoinosn,
CHV Study No.: 17073-0-449CO
16
C00281
`CORNING Hazleton
13.0 REFERENCES:
Evans, HJ. (1962) Chromosomal International ReviewofCytology,
aberrations produced 13, 221-321.
by
ionizingradiation,
tTrheankdura,ndA.hJo.,moBgeerrnye,itK.yJ.i,n apnrodpoMriteiloknes,, PC.oWm.puJrte(1r9P8r5o)grAamFsOiRnTBiRoAmeNdipcrionger,am19f,o2r2t9e-s2t3i3n,g
CHV Study No.: 17073-0-449CO
17 00282
`CORNING Hazleton 140 EXPERIMENTAL DATA TABLES
CHV Study No.: 17073-0-449CO
18 00283
CORNING Hazleton
CHV Study No.:
TABLE 1
DOSE RANGEFINDING ASSAY
AssayNo: 17073 Compound: T-6342
Trial No:
Date: 07/31/96 Lab No: CY7316
Metabolic Activation: -59
Treatment
Index.
NEGATIVECONTROL Row 1620
5
SOLVENTCONTROL Waser
ou x
TEST ARTICLE
oun 74
[ET
am es
som a2
67pm sy
snow 72
CI
Metabolic Activation; +59
som se 10ugm 29 soo pgm 00
Treatment NEGATIVECONTROL Revi 1510 SOLVENTCONTROL war TEST ARTICLE
wowm 016m osoupm 6pm
s0ugel 6Tugm
"Toxic dosc level
soum 6pm wou Wougm 000 pm RPMI 1640 = Cullure mom
%IMnidtexo.tic Ta
83) 0 ss 76 13 se 79
xe so 12 00
17073-0-44900
00284 19
5 2 em ? comaron |t i
Sco
ant pi th on wore CHROMOSOME ABERRATIONSIN HUMANLYMPHOCYTES
Simin .
85F5loec r] oo-- oon [oo or ei r| ih, AL F dE a ie a
won "1
%
a
S KIMI 1640 =Cuim medion M hea itM =MismnC iysin omstems
Bow owaonon
w2
oman tse,
iiF
z32
CHROMOSOMCEeAlDiEkRR2AT1TIOH aNSsAfps eun nLv TES
2 wean Tans common
ou ura
Lon cvaes
Mettt Acvaon: 59
Ee ----------
3iL
sognu]s1c0lo[lsyainIu.c]RImLrocee onme [oro oonrl|T2OE0ESAi SaY S aSY E asr. aE h i
ee seme
TL
+ sam
ams
52
an 3
&$
Spee s hve
Cecme NCMic +Sent
'
pos rhea
wow momo omon @ oe wow oe wos mom onow wow mom on ow
oes eo ew eo i"
25 3
2 tic om
J g:i
mouosonS :seaTORSR so crs
ana
--
Lacy
.
Mette Acar 1
a
omTeanoo r[oer |iaCEe,c"ilee S he Fa oGo"k, Sr
dl FEE
3g
E im nas
ip EE Frnt1040 Ca tons items
wou osom ono
.2
Hil
merei tnt
F
: Pam | i z
ER eee
mostsearSE enum
en
ones eros
onto 0
earl e[ealralele]
a i SE EA le worn og on
2
or
wohnung
$
a
amos2
wow wow ow
:3
E on E EALTtess eren sesemt ma
iE
22
scm
CHROMOSCOMeEAFBaEcRhRA3T1TIOfaNeSemAunbaevroc TES
naa
J
worms
.
2ET Ce ont vara TTTTTw
[--
| zIg
aus wor
"Coat 10[so[uc
ne]
com
e
JS uba ciH s cis w nikoe
oj onoTo uTar | df "tat enes Rims"cums ri
TT memo,
rae
my
ml,
wom ow op
morons ow ow
snows ow on
g&z2
Sfsroninres
awe nt
oug2
FS
HE]
ecm. RT
5g
|5 f:3 [-- c i m re i: 5
como
CHRONOsouaE ArERTi AaeTsIaONsLysocrTEs
.
Twrs
J--
Lance
Me Aca 1
TT meme
po.rm 15555 sc]
i
nonmon[oe
Tr
Looee |2
agltee ie
adles booed i ER
ie
=
---r,
won onononow
--y, m--
Soon now
.
wma aa
mx omos now won nw hn
g88
irrotm
pg ano
cTtE pat
tr |
x
ow :9
u
ee TAA s
9
TABLES
CONTROL DATA OF CHROMO4S9O5MTEHARBOEURGRHAT1I2:O9N5S IN HUMAN LYMPHOCYTES
Activation
AverFrOsLtons %OWfCiels
e TC Parcel Abemions
%WoirbC>ills Abersion
Pol%y. ploidy
dupElincdaioe.d Cals
Mi5o%tic inden
Wiser MN MAX
0o0oso
AG oon
e 2050 00s0 po0100d Oa1ho0 dn0t4e on 0% om om
Salven: Consol
i
"
w " 3 sy
Widow MN ooo MAAGX o0o0ns
00 o2r5
00 0s
00 oo
ao 20
os
00s ou on am
MPioosmiyeCcnoCnt
N Wide MN on
is
3s
00 00 oo or
Negaive Coto
MAAGX o0zg 1
ans 10
s15e0 o0w0 oam0 a6u6 1 n a BR
won mx
oomm
AG oo
0200 07s
00s0 0100 ooso 90s 0% ou om 4a
SolventControl
N"
" "z "
wa MMXN oomm AG oom
2000 08
000s 2000 o00o os7 00s es oan
CProscioipnheoCnohanmtide
Win
N MN
am
n 27
x " z FY 00 00 0 o
-- X A[NCGE v wooerw swuunaos w R wIo eo:v150 x oo2we a9ays
CHV Study No.: 17073-0-449C0
2
G00291
CORNING Hazleton
150
DEFINITIONS CELLS
OF
CHROMOSOME
ABERRATIONS
FOR
GIEMSA
STAINED
SIMPLE
TB Chromatid Break: SB Chromosome Break:
DM. "Double Minute" Fragment:
COMPLEX
ID Interstitial Deletion: TR Triradial: QR Quadriradial: CR Complex Rearrangement: D Dicentric:
DF
WAindtahchorfoamacthircomraetgiido.n iTnhoenaescsohcrioamtaetdidf,ralgarmgeenrttmhaanytbhee `partially or completely displaced.
(Cdherleotmedo)scohmreohmaossaomcleeawritbhreaank,acfeonrtmriincgfraangambennotrtmhaalt is dfirsalgomceaatetd.(AFT)h.isAcnlasAsFifiicsadtiifofnerneontwfirnoclmuadeSsBthoenalcyenitnric: that it can not be related to any specific chromosome. Ttehremsienaarleorsmiantlelrsdtiotuiballededloettsi,onwsh,iocrhemvaeyn srmeaplrlesreinntgs. These possible origins are not distinguishable.
eLhernogmtahioifd,crhersoumlattinigdi"ncuatsomuatl"l ffrroamgmmeindtroergriionngolfyaing beside a shortened chromatidor a gap in the chromatid.
An exchange chromosome
abnedtwaeneancetnwtoricchfrroamgomseontm,esw,hoirchonreesults
in
a three-armed configuration.
Aarnmeedxcchoannfgiegulriakteiaontriradial, but resulting in a four-
Afrnagemxecnhtsa,ngceauasmeodnbgy mtohre eintdhuacntitownoofcshervoemroaslobmreesakos.r
Ainnaecxhcrhoamnogesobmeetwweietnh ttwwoo ccehnrtormomoesroems.esTwhhiischisroefstuelnts
associated with an classified as DF.
acentric
fragment
in
which
case
it
is
Dicentric with fragment
CHV Study No.: 17073-0-449CO
:
27
C0022
COMPLEX (Continued) TC Tricentrc
QC PC
PQeunatdarciecnetnitcr:ic:
HC Hexacentric:
R Ring:
RC Ring Chromatid: RF Cl Chromosome Invachange:
T Translocation:
AB Abnormal:
`CORNINGHazleton
iAnan ecxhcrhoamnogseoimnevowlivtihngthtrhereeececnhtrroommeorseosm.esOfatnednresulting aisnsvooclivaetmedanwiythchtrwoomtoostohmreese AaFn.d aSreucnhaemxecdhaasngfeosllcowasn: ffiovuercceennttrroommeerreess,, uupp ttoo ffiovuerAAFF six centromeres, upto six AF cAencthrromoemroes.omTheiwshiiscohftfeonramsssaocciiartcelde wcointthaainniancgena.tric fragment, in which case it is classed as RF. Single chromatid ring (acentri). Ring with associated acentric fragment. `Enxotchinacnlguedewitthheienntaircehcrhormoomsoosmoem;e. . a ring that does r`eOsbuvlitoiunsg tirntanwsoferaobfnmoartmearliaclhrboemtowseoemnets.wo cWhhreonmosomes identifiable, scored as "T" not "AB". Acbhnroormmoaslommeonwohcoesnetrmiocrcphhroolmoogsyoimsea.bnTohrimsalisfaor the pKearriycoetnytprei,c ainnvderosfitoenn,tehtec.reCslualtssoiffiacattriaonnsluosceatdioinf, tarbannosrlmoacaltiitoyn.cannotbeascribed 10, .g.,a reciprocal
CHV Study No.: 17073-0-449c0
28
60293
OTHER
GT Greater than Ten
PP Polyploid Cell: E Endoreduplication:
NOTCOMPUTED
TG Chromatid Gap:
SG Chromosome Gap: UC Uncoiled Chromosome:
CORNING Hazleton
Gthraenat1e0r atbhearnra1t0ioanbse.rrHateiaovnisl:y Adacmeallgewdhiccehllcsownitlalinbse more amunlatliypzleed ftroagidmeennttisf,y stuhcehtyapsetsohofseabfeorurnadtiaosnssocbieactaeudsewith a trcentridco, notcountas independent aberrations, (An)coefllcchornotamionsionmgemsu.ltiple copiesofthe haploid number cAellf.ailureofchromosomes to separate, resulting in a 4n
c(h"triodmgaatpi"d),.thAensiazcehorfowmhatiicch(uisnsetqauianledt)o roergsimoanllineronthean tushueawlildytihnocfluadecdhrionmfaitniadl.toTtahlessoefaabreernroatteidonbsutasnotthey may not all be true breaks, t(h`eissoacmherolmoacuisdignapb,ot1hG"s)i.steSracmheroamsatcihdrso.matid gap but at . aFbaeirlruarteoiofn.chromatin packing. Probably not a true
CHV Study No.: 17073-0-449C0
29
C0094