Document MGzqgy9GEjokDzjNq8RDVoJzL
@ HAZLETON
CWEARSHINGTnOeN
om
SEEP 2T0 S1393 AM
1001.02
GENOTOXICITY TEST oN
T-5711.1
IN THE IN AVNIDVOC/EILNL'vP.RORLOIFUENRSACTHIEODNUALED DONA SYNTHESIS
IN
RAT
LIVER
SSAYS CELLS
EINAL_REPOR'
AUTHOR
Maria A. Cifone, Ph. D.
Hazleton Washington, Inc. 9200 Leesburg Pike
Vienna, Virginia 22182
LABORATORY PROJECT 1p
HWA Study No.: 15515-0-494
. SUBMITTEDTo
Buil3MdiCnogrp2o2r0a-t2io-n02 3 M Center
St. Paul, MN55144-1000
15515-0-494
September 14, 1993
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QUALITY ASSURANCE STATEMENT
PROJECT TITLE:
UGnesncohteodxuilceidtyDNTAestSynotnheTs-i5s711a.n1d Rat Liver Cells
CienllInPVriovloi/fIernatViiotnroAssays
in
PROJECT NO.: 20991 PROTOCOL NO.: 494
HWA STUDY NO.: 15515 EDITION NO.: Modified for 3H Corporation
QoOfpuearltaihettiynagAbsosPvurerocarenedcfueerreeisnncseopdfectptrhioeojnesQcutaolfwitetyrheeAscssotunurddauyncctaeendd/UoanrictcorraednvidinegwaccotoofrdttihhneegSfttionaanldtahrerdeport
Fgeinnedrianlgsrefqruoimretmheentisnsopfecttihoensapapnrdoprfiinaatle management and to the study director on
GroeopdortLabroervaiteowrywerPeracrteipcoerterdegutloations. the following dates:
Inspection/Date
S4c-o2r8i-n9g3of slides/
7D-r1af3t+1r6e,p2o6r,t27r-e8v3iew/
EindingsReported
4-28-93 7-27-93
Auditor
K. Newland B. Mullett
9F-in1a4l-9r3eport review/
9-14-93
B. Mullett
aTity Assurance UATE
at9/e14/4 Release
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COMPLIANCE AND CERTIFICATION STATEMENT
The described study was Practice Regulations as
conducted set forth
in in
compliance with the the Code of Federal
Good Laboratory Regulations
58, 40 CFR 792, and 40 CFR 160).
(21 CFR To the best of the signers' knowledge, there
were no significant deviations from the aforementioned signed protocol that would affect the integrity of the
regulations or study or the
the
interpretation of the test results. The raw data Study Director, who certifies that the evaluation
have been reviewed by of the test article
the
presented
herein
represents
an
appropriate
conclusion
within
the
as context
of
the study design and evaluationcriteria.
Agelnerraatweddataas,daocruesmuelnttaotfiotnh,is resctourddys,wilplrotboecoalrsc,hivsepdecibmyenHsazlaentdonfinfaolrraeppoerrtisod
of at After
Teast one year following the one year period, the
submission of the sponsor may elect
final report to the sponsor. to have these materials
retained in the storage facilities of Hazleton for an additional time or sent to a storage facility designated by the sponsor.
period
of
SUBMITTED BY:
Lhe 2 Hew
SAnudpreeravisL.orHam, B.S.
Study Director:
7
Ida C foe
Marfra A. Cifone,/Ph. D.
SGetnuedtyicDiarnedctoCrelular Toxicology
9/4/93
Date
9-14-23
Study Completion Date
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TABLE OF CONTENTS
PAGE NUMBER
ABSTRACT ow wet
6
L SPONSOR . owe
7
II. MATERIAL TESTED . . . oo A. Genetics Assay No.
v vii.
7
B. Identification
Cc. Physical Description
0. Date Received
III. TY OFP ASSE AYS FHL sss rbd ara t tbr ree rr nn 7
wv. PROTOCOL NUMBER . . . . ...................... 7
Vo STWOVDATES . ........................... 37 A. Study Initiation Date B. Experimental Start Date Cc. Experimental Termination Date
VI. SUA.PERVISStOuRdYyPEDiRrSeOcNtNoErL . . .. ................... 7 c. Study Supervisor
VIL OBJECTIVE...
7
VIII. DEFINITION
rr eet ttt a tre a eee. 8
IX MAA.TERIAILnSdic. at.or..CelL ls LL... 9
B. Media For UDS Assay
CDc.. OCosnmtortoilc APrutmipcsleasnd Label for Cell ProliferationAnalysis
X. AE.XPERIDMoEsNiTngDEPSrIoGNced(uWrSe ASSAY) . . . . ............... 10 B. Dose Selection and Perfusion Time C. UDS Assay D0. UDS Analysis
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TABLE OF CONTENTS (CONTINUED) XI. AE.XPERIMTErNeTatmDeEnStIGNa:ndCEDLoLsePLReOvLeIlFsERATION ANALYSIS . .. ....... 1p
CB.. TIimspsluaentaCtoilolnectoifonOsmanodticPrePpuamprsation DE.. AsIsmeusnsomheinsttocohfemCiecllal PrSotaliinfienrgation Rates XIL. ASSAY ACCEPTANCE AND EVALUATION CRITERIA: UDS . . . . ....... 14 XIII. ASSAY EVALUATION CRITERIA: CELL PROLIFERATION . . . . ....... 1g XIV. INTERPRETATION OF UDS RESULTS . . . . ............... 15 XV. INTERPRETATION OF CELL PROLIFERATION RESULTS .. ......... 17 XVI CONCLUSIONS . o.o LLo L 1s KIL REFERENCES... ......................... 2 XVITI.EXPERIMENTAL DATA TABLES . . . . ................ . 2 XIX. APPENDIX A: HISTORICAL CONTROLS . . ............... 39
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ABSTRACT
gTehneotpouxripcoisteyoofftthhies Jypthests (Us) and
tcseetlsultdypmawrtaoesTriiftaeolradtebityoenrmmeia(nsCPeu)ritnmhgeeasNhueArpeadrtoetpoexricioctyunasncdh/eodruled
DNA
TIntducaeveSrigncieflilcsanatftcehranignesvivfno Gooadferation were observed.
UtDreSatinnenrt.a,i heTphaetotceystesmahatueetrS-iiparreascTn-e5n7it1n1d.iu1nctcideoilndl The test material was
niont
odcdubolpstresuorxeiosnfatfaeoplrpyroa2nx-ai4lmyashtioesulrysof1a25nU,dBS125w5-0e,1r6e 5h0po0r,uerpsaarneaddft1ea0rt00atwmdsmg.i/nsgia"scartdirnapiPnrn|ismaitrsiyemeeshse,psaotorcayttse at
mateerialafosruscpaechndepderfiunsiCMoCn. tinepoint, three male rats womtiornecotrecdstesisntgle oral
ASA1fp%theournresapuatatonordcaydtaieuotgocrruaalpdthiuyor,egsratphfhroyerewUatDsrSeapwteemrreefnotrimngecrduobuoapntsedtfherwointHhTcdar1c0-htueccir/raildc"uHlrtdurfeosr. about
Iwhoeelprerecotseccdehlelf'uoylrarlaonvmaeolrrypshdioossl'eoosgf.y nwuNacoslneeaardoefqluatabhteeelicnfrgoirtbeaerngiaialnynsuiisnsegd mwtiiothptrsiomnceepehodiiignnhtgeswtteoredose
oS$feE0tiUomSaacthieenrdiraablty, htSehpe7att1orce1yattemsweanststhaenrdefnooredoseev-arluealtaetdedasr. esipnaocntseivnewdaiss.coarstpessoiUrntsienrdwsauscTthioen
wIenrethesbsceelrlvedprfoollilfoewriantgiontreaaasftstameyer,ntidnwoisvteih-vroeltarteeadtmeinntcreaansdesininvictorlolpcruolltuirfee.ration
gSSroabnmedpdildteeidonOfiwnetrpheaerallfaifbvieenlr.edandwSiatdmhpuloedBserndwuUenrfewoerrael7s2oFtihhxoeeudrtsesintusi1mn0ag%tenAreLiuZatElrTal bFobrveacweciainni"pmuamlapsnsd.pTearter
pathologist.
processed for. snelymon bya
:
I3S5emcumtneioloh]niss2t5forcoshnaemnptilhseetsrTyfe.rfotnElatachtheerasdl1u,ioddeemnewudamisanwperraeenpdatrarekidegnhwtiatnlhdateSprreaolcceeslseoobsesfoonf the Tivers
TPIinitntcterremoarunnocadlaegnecuonno.tfromluTchlefeoirduidonedcleoinrvupenorry(aatoifrnagplaibldeallbyelapnrdoinliitfmhmeeurnaolthiiivnsegtroocwrhagesannt)dcesntwsierosntmasiobngoitnrhgs.aTinvTehre
ijnnccrreesaassseeels iinicnalclceeyll;ll" pprOronollliyiffehererapatatitioooncnytwewaesrneuicnoldbeuisceerdwveeradendaetsnut5ma0et0riastntegidyc:sagllyi`Ansgidogornasiriferirccycaantted
Pgoosuitlidv;e
wfaosr
tthheereifnodruecticoonnsoidferceeldl
nunmg/g. npergaotiifveeraftoirontheinirnadtuctIivoenrof Ups and
roses,
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In Vivo/In Vitro GUennsocthoexdiuclietdy DTNeAstSyonnthTe-s5i7s11a.n1d iCnell Proliferation in Rat Liver Cells
I. SPONSOR: 3M Corporation
II. MATERIAL TESTED: A. Genetics Assay No.: 15515 B. Identification: T-5711.1, L-1276
c. Physical Description: cream-colored granular material
D. Date Received: February 24, 1993
III. TYPE OF ASSAYS: IDnNAViSvyon/tIhensViistrAsosayRatwitPhrimTawroyTiHmeeppaotionctysteaUndnscCehlelduled Proliferation
IV. PROTOCOL NUMBER: 494, Modified for 3H Corporation
v. STUDY DATES:
A. Study Initiation Date: February 24, 1993 B. Experimental Start Date: March 11, 1993 C. Experimental Termination Date: April 29, 1993
VI. SUPERVISORY PERSONNEL:
A. Study Director: Maria A. Cifone, Ph. D.
B. Scientist: Marie E. McKeon, M. Phil.
C. Study Supervisor: Andrea L. Ham, B.S.
VIL. OBJECTIVE:
The objective of this assay was to detect DNA damage and/or
hunespcahteodtuolxeidcitDyNA csayunstehdesbiys th(eUDSt)estandmatceerllialproblyifmeeraastuiroinng (DCNP)A mreeapsauirredas as
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TSh-ephaesxeistienndcuectiaonnd net nuclear grain
cdioenugdnrutecseedoinfin DhrNeAaptatdoalcmiyavtgeeers wceeorlbeltsaiinaneffdteerrrferdoimn fvtrriomevaotaentdreiaantncmiremenaatls.se
in
waahnrdeenruencssoupmleptcariefidinedttohebutthionsnceuosrtpforrobaemtiruonentcroegoanftiezndaebwlaebnaismbeayslst.he(icnTechlleludutilynapgres*rHeo-pftahiDyrNnAisddyiasnmteae)gme
into DNA during a short (4 hours) in vitro culture period (1).
drCeeeltplelcitcpartobilroionfmeordianetoixroyatnurimTdeiiavnseerureu(dsBridntUgh)eanifrnacicomtrmipuoonnroahtioesfdtoccdehulerlmisincguanlDdNeArtgesocyhinnntighqeucseeilsl(.2,3) to
vAlinivivomerawslistwhewreeraneiAgsLioZvlEeaTntedaosfsmioonltglilocewinpogurmalpaddmioimsnpeilsatnotrfeadttihoesnubtceousfttaBnrmeadotUuesrflioyar.l 72andhoutrhse in
7Q2u-ahnotuirficpaetriioodn
oifndiccealtlessthiantcrehaasveed icneclolrpporroaltiefderDaNtAiopnrecinurstohresliovveerr
the (4).
VIII. DEFINITION:
drTeahstecrlLiiDbvSeedrascsbyealylWsiilsl(idhaeemspsia,gtnoec1yd9t8e0tso)(m5)eu.assiunrgHeeptahutenosccayhutetedosurlawedediroegDrNAaipshosilycanttethdeecshifnsrioqm(uUetDhSe) in
tplheiervcerbesrntiaeogffeeraoxtfpsostuherexepocspeeelrdlisodein,ntvesiorvotSh-tepohatishneecotr(eprsoetrpaltiaicroatnitcilvoeef.
DNAOnlsyyntahessmiasll) *HTdr into DNA
dduurriinngg
maiernatisvcuilrteer.oofcTuhltithseuriUrnDegSp,amieraassoufarneDamNleAynzteddamoafbgyeDNaAcuatruoserepaddaiiorbgyraaptpprheeyaa,rtsmemntatoy
wbiethustehdeastesat correlate well
with known mutagenic or carcinogenic activities of chemicals.
TaHnheepsaietnoctrpoeraxosileciafneitrnsatcieslnulgchpceralosllsicfamerarbyaontiboetnedterttoaecchrtleeopdrliaddcueerinanengcdroSdt-iipnchiatstreiostsoaulneauleyn(se3i,s6i.)n.duce
pOisrtohcenerostschaebpumptiacraetlnhsteremhaoyawrecienlndluucmeeprrooSul-sipfhemaresacethiaionnnistmmhsaeywahbaicsctehncicenanothfebehceaaprfacftioentcootxegidecniidtcayr.ing It
ptrhreeopblaipbcriaoltbiiatobynilio(tf7y-c10oo)nf.vesrptoCinhntegamniecuoanulrsleypamiuirtneaddtuicoDenNdsA ceaalsdldwuecpltrlsoliiasfnetoriantmciurotenaatsiemoanytsh.eincrease
tpUrrnaesnncsehfoeopdrlumalesedtdicccleloplnoepuplorafotlicioefnlelsrsa.tlieoandSiomnmegaytoofalsttohheespeelmaeyerxgaamepnrlcoeelseoaficntatbhfyeulaleyxpnaonn-sion of
tngeeocnnhognteionqxouiteco.ximcechcaarnciisnmogaennds itas isweltlheoasregteincoatlolxyicpocsasricbilneogteonsdetuescitng this
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IX. MATERIALS: A. Indicator Cells
B. C. 15515-0-494
pY1o4ungat adtuhlettimmaeleofratdsosionfg,thweeSrperapguurec-hDaasweldeyfrosmtraCihna,rle1s0-1R2ovewceeks LwLoaasbaorsoaetlmoeracitneesd,sotuoIrncmce.a.xi(mCiArzn1ei:mCaOglesBnRe)ts.icchedhTuehltieesdrohgeefaonlretihtthyiysraasnntdduodmayssbwurreeerdeascrtcoreeassissnd ACanecircmtoairlfdsiiendgwerteRooqdsuetanartnandtaCirhndoewd(opFeorarammtuiilnnaignu5pm0r0o2oc)fedu7arneddsaywsaatnepdrriwaoedrre1tiofbeirdteuPsnue.rmina CAaunslistmiuagrlnesmse,nwteruestionagnsetasubtdoyhuettgirz6oe0udpsngp/rakingdorsitododeinustmuirfpgieecnratytoibofanorrbbipytraetlpa.ialraatrtdiaotnwtoeoeo.rf cell
exsanguinated during the harvest procedure.
3Th1e3.0l-i3v8e9r.6celglrsamsfofrorthetheUDSearalsysaytiwmeerpeoinotbtaaindned324f.r4o-m40r4a.ts weighing
for the
the later timepoint. livers in sity with a
The cells were obtained by collagenase solution (see
6 grams perfusion of
aUnDdS wAsesraey)u.sed Mtohneolsaaymeer cdauyltufroers anwaelryesiesstaofblitshehedUDSinaccStueilcvttiuitroyen.dXi.sCah.yeys
hcuumlitduirfeisedweraetnomsapihnetraeinecdontaasimnoinnoglaaypeprrsoxiatnataebloyut5%37C0C,.in a
For the cell proliferation assay, 408.4-483.2 grams. Approximately
rats were used 24 hours after
that ranged dosing, the
from
animals were Moore, Inc.)
anesthetized using Metofane inhalation anesthesia and one
(methoxyflurane, ALZET' Pump
Pitman-
tfwoao-svreenantsoyev-pattluiocoaflholuytrhseinTlsaietvreetrre,sd
aasnnudibncadulutsoadneweneouruneslya(nceo(nsdttorhroselatlizoersdguarnwf)ai.che)p.Ceor,
animal
prior
Media For UDS Assay
TTehnetecdellwitchult1u0%resfetwaelreboevsitnaeblisserhuend, f2n mWHi11L1-agnlsu'tamMiendei.um10E0spuappilne-
wsittrheoputtomysceirnunsuilsfarteef,erraendd
pe
t1o50a#sg/WmHlEL.gentAfatmeircinthe(eWsMEt+a)b.] isWhMoEe+nt
47riCoid/,mMoclueltu(rWeMsE-twreeraet)r.efed with WMEI containing 10 KCi/ml *HTdr,
Osmotic Pumps and Label for Cell Proliferation Analysis
TTTAhhLheeZeETppuupummsppoesds:hmwaoestrAie2c.Sp2ip0rnu0egm0-lpfesui]lT(loAetcLdaZpA(wa#ic0Cti4oht2ry2p0Bo5rwr)diaUtthiwoaantsa,'4puusPmecapdolnocrteaAhntlretsooru,gohfoCuA1t)0,
thMeodesltus2yM-L1 1 hace.
20 ng/ml.
atioy Sf
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D. Control Articles
1. Vehicle control
A vehicle UDS assay
negative and five
control consisting of three rats for cell proliferation
rats for the treatedby
oral gavage with the vehicle, CMC carboxymethylcellulose, 9004-32-4,
(high viscosity Sigma Lot # 121F0644),
hwaespatpoecryftoersmeodrattisaslluestimweeproeinstusb.jectVeedhictloe alclontorfolthe
manipulations Dosing volume
used for those of the vehicle
derived control
from treated animals. animals did not exceed
10 ml/kg.
2. Positive control article
and Five rats for cell proliferation were treated by The positive control compound
S-phase in rat hepatocytes in
is known to induce UDS or vivo. The positive control
for the UDS timepoints, 62-75-9, Sigma Chemical
Dimethylnitrosamine (DMN, CAS# Co., Lot# 29F0679) was dosed at
1150-1m6g/hkogurfsortitmheepo2i-n3t.hourFsor tciemlelpoipnrtoliafnedra1t5iomng,/kg15.f0omrgt/hkeg
of DMN was used as the positive control. Three rats forUps
intraperitoneal injection for each timepoint.
E. Test Article
0F.o5r%thCeMC pwraespamraadteionby oafddtihengdoCsMCingpowsdoelrutitoonsdeioofnitzheed tewsattearrtwiictlhe,
csotnicrreinntgr.atioTnhse otfest12.a5r,ti2c5l.e0,was50.s0usapnednde1d00 inmg0/.m5l% pCMrCiorat to dosing
for each timepoint. The maximum dosing article did not exceed 10 ml/kg.
volumes
for
the
test
X. EXPERIMENT DESIGN (UDS ASSAY) : A. Dosing Procedure
For the
each test
timepoint, three rats were article. Delivery volumes
treated by oral were calculated
gavage on the
with basis
of the volume
most recent of the test
aanritmiacllewesiugshptensainodnstheadmtianrigestterdeodse.did Thneotmeaxxciemeudm
c1o0ntmrlo/lkgs. werFeresuhsedprfeopraraatniyontsestoifngtespturpaorstei.cleCoinnfivremhaitciloen anodf the
concentration and dosing of
of the
tahessatyeswtasmatneortiadleteurnmdienredconinditcioonnjsuncotfiopnrewpiatrhation
this study.
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B. Dose Selection and PerfusionTime
The for
highest dose of both timepoints
b1a0s0e0d mgon/kginwfaosrmasteiloenctesduppalciceodrding
to protocol,
dTihlrueteioanddisttieopnsalanddosaesminoifmutmestofma3tearniiamlalswerpeer pdroespea.rbeyd thuesinSgpon2s-ofro.ld
timepoints were employed after the administration
for
sacrifice;
2-4
hours
Two and 15-16
of a single dose of the test
hours
oral gavage.
article by
Cc. UDS Assay
WTihlisliaamsssay(1w98a0s),baMsierdsaolnist,heTypsrooncedaunrdesButitnerrwaotrsthdes(c1r98i2b)edbanyd
Butterworth et al. (1987). perfusion of livers in situ
The for
hepatocytes were obtained about four minutes with
by
bbiaslan(c8e-damisnaolettshy(lCaeTMth-eMrg)~--Nf, reN-et)etcroanatcaeitniicngac0i.d5
Hanks" mM ethyleneglycol(EGTA),
buffer at pH 7.2. collagenase (WMEC)
Then WMEI containing was perfused through
and 50-100 units/ml of the liver
HEPES
moifnuetxecsi.sedThleivehrepattioscsyuteesin waereculotbutraeindeidshbycmoencthaainniicnfagolrdi1s0p-e1r1sion
csluusmppesndeadnd tdiesbsruies.andThceellcsellwerseuspaelnlsoiwoend
WMEC. The to settle to removecell was centrifug
cell pellet resuspended count, a series of 35-mm
in WME+. culture
After dishes
obtaining (at least
a 6
ed and the viable cell per animal
canoinmtaalinitnog asase2s5s-mamttraocuhndm,entplaesftfiicciecnocvye)rslwaisp
and at least inoculated
2
per
anima] with approximately 0.5 x 10 per dish. Individual cultures were
viable cells in identified with
3
foreach ml of WME+
eartag number.
the animal
.
Ahnumaidtitfaicehdmenattmopseprhieorde ofcon1t.a7i-n2i.n0g ho5%ursC0,atwaasbouusted37toC
in a establish
the
cell cultures timepoint, an
aftotracthhmeenetarpleireirodtimoefpo1i.n7t-.2.0 Fhoorurtshewalsatutseerd.
After
ctuhletuartetsacwhemreent repfeeridodw,ithun2a.t5tamclheWdHEc-etlrlesatw.ere Thrreemeoveodf
andthe thereplicate
cultures from each animal were replicates were used to assess
used for the attachment.
UDS Any
assay; two remaining
ofthe
wauetroerakdeipotgrfaoprhya.nalAytstiaschimnenttheeefvfeinctienocfy tweacshnidceatlermpirnoebdlems
cultures with
cultures from each animal trypan blue dye exclusion
by to
in
situ
microscopic
for analysis,
two using
determine the viability of the
attached cultures.
15515-0-494
AwefrteerreafeldabweiltihngWMpEeIriocdontoafinaibnogut 0.425houmrMsthlyambiedliende cealnld
cultures returned
t18h.e2 ihnocuurbsato(r15-f1o6r h1o8u.r7s-19t.i2mephooiunrts).(2-N4uchloeuirswetriemeptohiennt)swoolrle1n7.8b-yto
es
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cafeidlxdleidtimooinnnoltoahfyree1re%s)cshoadfniogurems10coifmtirnaautcteeetsitcomiatnchuietde:cseo.tvhearnTsohlleipcs(e1l:(l3c)sonawtneadriondirnnigoexdtthefor casotlaitdleeesda,stsdlii2pd4peeshdowuerisrn.eKsotd`ToahrkeedNfTiBxf2eodrem7cuoldvsaeiyrsosnl,aitpsa4ndwCedrreiinemdol.uinghtteTdhteiognehmtiglleaBjesvosens cfainodxnetdea,oisniiannndgprDsortciaeeidrnuiertdee.?w.ithTWhielleimaunlss'iomnsodiwfeirceattihoenn odfeavelohpeemdatoixnyl5i1n9. D. UDS Analysis mTtahhgeenivcfieidleclosatiwsocenrreeeunenxdoeafrminoaineldamuitimocmmreaortssiiccoonpciocauanndltleytrh.eat fUiaDepSlpdrwoawxsaismmaetdaeislsuyprlea1dy5e0db0yxon cg{rocauyintntosipnlgainsmnituchcrlceeoeaurnntgu)rc.alienasrT-hisasinzdevdasluubaerteraaissctriaendfgjearctrehenedt atvtooeraaesagcehthneunmubcneeltreusnoufclear gcoruanitnincgo.unt. The Coverslips were coded to prevent bias. in grain
Tsunheleleescnsteetdotnhcueecrlllwesiasreongriaenaidcnihcactoceuodnv.terswalOsinplyde(tntuehcrrlmeeieinecdwoivtefhrosrlnoifrpimsfatlypemrroarnapndhioonmallloy)gies wnseuymnreterhoesuscsiosretdoo,cccuoarunrndetdawneyrraetohceceraxsciltouhndaaenld ranesupcalicereillssybnltaihnceksweihnsie.cdh bTyrheegprlaaivicenarstaigvteeosmDeNaAn tanvheeetratngrueicdplleiafcorratgeeraacichnovtecrroseulanittpmse(n+t(1sc5to0annddtiaottraidlond.envuicalteiio)n)fowraseacdhetearnimmianledanfdrom
XL. EXPERIMENT DESIGN: CELL PROLIFERATION ANALYSIS
.
A. Treatment and Dose Levels
AaannNailnyaazlnesimcgaerllsalmwpeerraocelhidfodesoresaedtilaoesnvedlaefstcaernirdbeacdonstiirnnoglltehgeorroUauDlpS
wseercetiouns.ed dose.
Ftoive
B. Implantation of Osmotic Pumps
A2aL0nZ0eE0sTt4h1etMioozdfeeldBrud2sUMLiInagt oMsa emtocotofinaccneenpturmapatscicoo(nrLdooitnfg#200t4o22m0gs5/t)maln.dwaerrdeThepprroeaclneoidanudareledsswwieatrnhed a(ndeorspaulmpsuprefracaen)inaalppwraosximaasteepltyica24llyhouirnsseratfetder sduobsciuntga.neouTshley
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incision was closed with wound until the time of sacrifice to
clips and the animals monitored ensure that there were no clinical
signs of infection. prior to sacrifice.
The osmotic pumps were implanted three days
Cc. Tissue Collection and Preparation
Each aninal was anesthetized prior to removal of organs for
analysis. and fixed
iTnheneutthroarlacibcuffcearveidtyfowarsmaloipne.nedAancdrostsheselcitvieorn reomfoved
duodenum, a each animal
tissue with and fixed.
high cell turnover, was also The duodenum was included as
removed from an indicator
that label liver, 5
was administered correctly paraffin embedded sections
to each animal. were taken from
For the the left
lateral, prepared
right median and right anterior lobes. Similarly sections of the duodenum were also made and a section
of
:
the duodenum according to
was mounted on each standard procedures
slide. Slides were also prepared for examination by a pathologist
to determine if any abnormalities werepresent.
0. Imnunohistochemical Staining
The slides were using first the
deparaffinized and rehydrated prior to staining BIOGENIX Supersensitive Kit using DAB stain and
hematoxylin hematoxylin
counterstain. and eosin for
Parallel slides were stained analysis by apathologist.
with
E. Assessment of Cell Proliferation Rates
The section of that the label
the was
duodenum properly
was microscopically administered to the
examined to ensure animal. Once
eTxabaemlineddelifvoerrylowbauslarcondfiifrfmeeredn,cessl.idesLafbreolmingthweasdisfifmeirleanrt
Tobes among
were the
.
lobes left
therefore cell lateral lobe.
counting was The percentage
performed of nuclei
with sections incorporating
from the label
in the liver was determined microscopically. counted were randomly generated by computer.
The areas to be A 1.0 mm square
indexed ocular grid define the counting
divided into 10 area. At least
x 10 2000
squares was nuclei were
used to examined
per
animal with a minimum of 3 sections and 6 fields per section.
Any nuclei containing
that were any brown
blue were considered chromogenic hue were
unlabeled and any nuclei considered labeled
eunnulmeesrsataedc.learFiealrdtsifatchtatwacsontpareisneendt.areaOsnlyofhneepcartoosciystewenruecleniowt ere
included in the evaluation. The slides evaluation as to treatmentgroup.
were
coded
for
(blind)
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Sfo-lplhoawsse: nuclei Tabeling indices for each animal were calculated as
Labeled S-phase nuclei (LI)=no,totoalfnol.abeolf hepatocytest countedi X 100
XIL. ASSAY ACCEPTANCE AND EVALUATION CRITERIA: UDS
Atsnhaetiastsfesisaetyd.rneosruTmlhatilsslyolinwislltylinigbfemaalclyonnosofitdteherenecdocmrapicatcseesrpitaaalbllleistetfseotdr
ebveallouwatairoen situations,
of so
ttrhheeelicasrbtiiutldeiyrtiydaiarneodcrtocacrocnesmpiutdsatenrcieenx.gercoitsheer sccaiuesnetsifitchatjudmgingehnttafifnecmtodiafseyaiyng
1. cfTahunesioavnfifaebpcirtloiccteeyslslonfyoirtemhleadllhyaenpdeaxtcvoeiceaydbtsielsi7t0cy%o,.llseoActvevadalruifeerstoymbeotlfhoewfapc7et0ro%-rsare
ntvoirtaebaitulmniectnoytm,mwointthhenroertfeosnrteeceansrostaicrllioewleyrmdayeltirmbiietmernwetiflallle.ctbeedTsoexitni.cipteyrfuosfion
2. vTaihsaesbaiyvliitatrbyeialtoimfteynatotsftamctuhhseetdmcobeenloll7sa0%yeirosrcaebglorluetatce8ur5l.%t.ureNsormuasleldy,forthethe
3. paTdohepequulpaaottseiiotnfivoerempctlhoenotyredodeltweacitssiournseesodpfontUsoDiSvd.eemoannFdsortrtahtteeestmetmthaahttoedrotilhaoelgsycelwlas
pcoasuistiinvgewecoanktroorl to indicate WS.
notreUDaStmeancttsivmiutys,t etxhceeeadverbaotghe For test materials clearly
rcersiptoenrisae utsoedthe causing a dose-
raeblsaetnecde oUfDSaacptoisviittiyv,e caonntarsoslaylowisltl fober ateccchenpitcaabllereiansotnhse.
4. GoanfrdaitnhateceoTevuaanstltuadta5i0toannuocbiltfeaioibnpteaedirnpecedurltfaunrrioemm.altwoGirsariaencpclceiopcutanattbeledcautlaatsurpSeahsrotuld
be available from 2 of the 3 animais treated.
5. Atainmamelipynoziiemndutm.doosfeR3epleedavotseelstrlieinavlesltshenweiefldilrsotnbleytrainaaaullgymzetoendtactahhtieeevnaecuhmbaertotoafl
opfre3vidoousselylaesvsealysedbutasmuasctcepitnacbllued.e at least one dose
abSsaessvaieysr.alforcThreeivtaecrlriuiaatteirhioaanvefoofbreeanateepsoststiamtbailtvieesrhieradelswphoaisncshea,ctair1vefembeaitsn,edthpeornovUiDaSde a
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dmseitsnacitrniisubtneidcianlbcyreaCanasaselcyisraiensqouiorfaenddthGefaoyrhliosartosri(i1gc1na)il.ficdaantta UaDnSd rceaslpcounlsaetiogne of the
tThhaettecasutsem:aterial is considered active in the UDS assay at doses
1. cAfoninvteirnogclrraeiaansvseerapigener,thneulcelamedeuiasnnganebtotoveanuptcohlseeiatcriovngecruanrirnuemnbcteorunntaengdat/tooirvaet least
2.
Tihnecrepaesrecenatt loefasntucl1e0i% awbitohvefitvhee negative control animals.
aovrermaogree
onfetthgeraicnosncutrorent
IaGlensndoeircaablteleym,eUtD.SifacHttohiweveivfteiyrr.,st sDacitofinfsdefiratecintotinonDiNsAo-fsdaaotmnialsgfyiineogdn,e agctehonentdsistecicaoonnndgciawvniellala so
cavofanfrdeicietttiyoonnoslfyanrauecmlcieonanorsrii1tdayebreelodifngitnhepaantcetleelrvsna.s'l,uatTainhodenr.ewfeoarkIne',acgaebsnoettssh
moafythsetroabnogvley where
wtIihnleclrtehabrseeeescoanansriiemdaenlrosetd sohabocswteirvvieendcrfeoiarnsetashl.lat tchIofrnedetihteainoninemaglaistf,ivceetlhelcsonttefrsrotolmmaattnweiornigoalfls
nsuhcolweuasn, atvheeraagsesaTyeswsiltlhannor-m5a.l00lyorbemocroensitdhearned1.0i0nvaglriadi.ns per
tThhee atebsotvemactoenrdiiatlioniss acroensmiedtereidn ainnyactofivetheintrtehiastedassaanyimailfs.none of
ctWehhleenluplrraeerssuerlnectsespooanfrseeasn,deoisatenhderrtehsceploenarsreelp,yrodtpuhocesiibtfiirlveieqtuyennocoryfcddlaietsaatrrliyabmuontneiggoantsi1vioedf,es
.
I"sweackonspiodseirteidv;e" tohre t"eesqtuivaorctailc"l.e iAs animals shows increases in nuciear
gtrhoeunpclinaswshiifcihedonaes o"fnagtharteiev.er, labeling will be decided on a
Ifcanrsaoecmtitbvyheecaaancsitemiavbleassiasannidmdaelpt,heendtiphnregesleoenvneclethooerf laaevbcestleinvcioetfyoaficntaiccvetilitlvysitiyfn roicnmelltshe
surrounding groups.
tpThoheientUpDoSstoiatcietvsietviimctaotynetroomfluttahneguecntlieecsatromrlaatcbeaerrlicianilgn.ogiesnUDinScoterliuissckeidtaesadssocbaiyartteeefdsetrweintche dpaaogmteaengntecsy iionnfflttihhciestedtaesssatanydagtiehsentparvaosabialabalbmyluetmaogrreeenpdaoierrpencmdaeerccnhitannoiogsnemnst.hethaStnoympeoen footfrhmeDsNA
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sontfurcalDteNeiAcdthaaacmtiadgset.hearceTehlurlse,p`apitohrpeeudlpawotisiotinhtoiuvetemplctoohyneetdrionlwcsaosrparroeersaptuiosonendsivoteof
dneemwonand the
methodology was adequate for the detection of UDS.
XIII. ASSAY EVALUATION CRITERIA: CELL PROLIFERATION
AgSoa.plMhceauaslneataenvddalusfetosar.ndeaarcSdhtatdtiresevtaiitacmtaeilnotnangfaroloryusptihsuesiopnfegrcleatnbhteealgiinengdiovfiinddSue-axplhwaaassneimpceee]rlflomsremnwesedre
ftuhrseeiangtcmaesonenteo-gfwraoyvuaprainacanolcmyepsairshiestooefnrsovgaewrneieraientcyde,onteercahnwnikitqhtureaDsnusmfn(eo1tr2'm).satito-Cntosnetsrtoofl(1t3hv,eerd1ea4u)t.a In
wSpee-rrpecheanspteeargpfeoerrmcienednttahpgereiocroinnctuoarrdaeonnsatelyscgiorsnoturpooflthvagatrroiudapenvciaeattaenasdsiDfgurnnoinmeftit'chsaencSte--pthelsaetvse.el
An of
P<0.05 was considered significantly different than the control group
XIV. INTERPRETATION OF UDS RESULTS:
Aitn 0t.h5e%rCeNqCuesatt dilutions were
oafcotnhceenStproanstoiro,n used to prepare
otfhe 10t0esmtg/mmalterainadl,serTi-a5l711t.w1o-wfoalsd suspended suspensions of 50.0, 25.0 and 12.5 mg/l.
TIhnedivtiedsutalmatdeorsiianlg astpopcekasrewderteo dosing o Three rats per dose
pfroerpmaraedunfiofrormeacshustpiemnespiooinntinjutshte level per treatment were treated
pvreihoicriet.o with 125,
250, 800, and 1000 mg/kg with volumes which did not exceed 10 ml/kg.
gaFodolmrlientcihtseetdreaatfrioloryn tthioemfepUaoDiSsnitan,sgslaepyedrorfsauensgieoodnfs itnhweevrietaebsiitnliimttaiytaetre(iddaelt2.e.r3m-i2Tn.he6ed hhobeyuprasttroycapyfattneesr
.
bpleurefuseaxtcelus(iToanb)le fr1)o.m and the viability of
6thT8eh.e34a-ta9tt4at.ca2hc%ehdmeoncfteltlheseffwitacositaelnvceyrcyelvlgasoroidec,dollrfearcnotgmeidn3g9.i0nf%r.otm8h7e.1%
74.3%-95.9%.
Tdcohenetterrmomilinniemtdurmeabtycmreicntotemsrpaira(iTsafobonlre tao2)U.DtShe Trheaevseproaanvgseeresagaetofmtehtaihnse
ctoinmceuproriennttwenreegative net nuclear grain count
cyfeeolsrlpsotnhsceeonncteoagniasntiiisnvtgeedcfoionvfterooalvrermaaongrieemalmnseeatnwansnuectl-e0na.ru37clgeraaanirdnsgtrhaewiansav3ce.or6ua%ng,tes p`eeAxrccpeeoensdtiitniogvfe
c4nou.nc6tl3reoi(l5cvonanelttuaeig)nr.ianignsNofniaevbeovooefrttmhhoeeretcrogenrtaratoimlnesntvsa(l1uw0ei%)thabotrohveeattthelesetasatmvaetr1ea3rg.ie6a%lnoefsgaamttpihlveees
ctcohaneutsreDodHlN.nturceFlauetramtrehneltrasmboerliein,ndgucneosdidgoTnsaierfg-iercealniatntlceyrdeadsitefrsfeenrdiennwtansucflerevoainrdetnhltea.benleigInangticvotnehtartast,
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gcornetartel]y provide
ceaoxnncicemleaudlsesidvweecrreeivtirederesinpaconeussiefvodre',tothtehienldaticecksattoefrUeDUsSDu.SitsinSwdieunrcceteioctnohnesbiypdoetsrhieetditveessot
material samples at the 2-4 hours timepoint under these test condsyjens.
suHlneidadeverigloyai-nnlaglaybzeOelNdeA,dreantpulcilTeceiaatsit(obn5l0a0c(kSce-enplheladssew)wietrhaes nosupcmpaeonrnsoeeudds phase
tgoraiDnNsA) rerpeapirre.sentForcelelasch and the incidence of a
s1t8u0d0ycwecalasllscuTlooawbtseaednrdveaddnidd(Trnaeobptloertien2)dt.erffoeTrrheeeawcnihutmhbaenrtimhaeplreassosenanytt.heinbtahseis2-o4f hasoulrost
gaFoodlrmlientcihtseetdrlaatftioeorrn tthioemfepaUoDiSsnitna,gslseapyedrofrsuaesnigoeondfs tiwnheevreitaebsitinliimttaityaetre(iddaelt1.e4r.m9Ti-hn1ee5d.h5beyphaottuorrcysypteaesfster
pbleurefuseaxtcelus(iToanb)le f3r)o.m and the Viability of
t7hT3eh.e4a%ta-tt9at6ca.hc9eh%dm%encoteflletsfhfewiactsioteanvlceyrycvegalorloisde,dcorlfalrneogcmit6ne0gd.2fi%nr'-o0mp8on91%
85.04-97.0%%.
0Tct.hha4eel8c,mu1li5an-t1ie6ndunhobuacrsrseidtteirunipeoanpofitonhrteaa(vUTeDarSbalgeree4s;pofoanvsteehreaagtneegtmaheetainvleantecetorntntruiocmlleepaoarinnigtmraalwisanseforn egrxacienesdian=vge0r.4a6.g75e%2)p.oerrcTeathnetlecoarfsittceerl1il0as.8%wceoroneftatmiheneainngnuncefltievienuccoolrnetamaroirnegirnangientficnvoueucnltoesrarmore gnnoruacidlnoessa.er-rleaNlboaentleeidnogftrtsehinegdntiwrfaeisactaemnvetinldtyesndtiw.iftfherTehtnehtepotfsreiostmtivmtehaetecroninetagrlaotlisvaetmrpecloaentstmreconaltussesdud riiennsdduuilccteasdtewieUnrDcSer.ecaosneSssiindcieenrentdhuecltoepaorpsritolivavibedeelicnocgnotnrceolxlucseieavdneiinmgealvsitdheernecsceprointfdeoerrdi.atheuthseeJdactketoctof uDnSderIntdhuecsteiontesbty ctohneditteisotnsm.aterial samples at the 15-16 hours tinspoint
2
Hgubnesdaeevrrivgleoydi-nlgafobreDlNeAeadcrhenupclalnieicinaatli(obnilnaacstkheeonpepd1o5s-we1id6thhtoonuurmDseNrAosutrsuedpaygirra(.iTnasb)lTeher4e)pnurmwebasesern.tlowcelanlds
did not interfere with the detection of UDS.
XV. INTERPRETATION OF CELL PROLIFERATION RESULTS: A. General Observations dCeuloldsenusmtaifnreodm awlilthofthethebroawnnimaDlAsB cusherdomoignenthewersetudoyb.servNoed in the
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unscheduled deaths occurred. The animals indicated proper delivery
presence of of the BrdU
label label
in all the and acceptable
immunohistochemical staining. control) was eliminated from
One animal from the study because
Group 6 (positive the results
dtoemoinnsdtucreateldargae laicnkcroeafsersespion nsceellby ptrhoelifaenriamatli.on
Since (15),
DMN isknown this may
have represented a dosing error.
The livers of the treated aninals showed a dose-related increase
in weight induced a
compared to significant
control animals. The 5000 mg/kg dose increase (p0.01). The mean liver weight
of
tThaergeposiintcirveaesecsontirnolDNwAassynntothessiisgni(fanidcanStulbyseqeuleenvtatecdel]even though
2prmoelainfertaetrmiionna)l wbeordey iwnediugchedt. thaTthewahsighlesdsosethaannimcoanltsro(lGrvoaujpue5) had
(p< 0.01) indicating some related increases that did
toxicity. not reach
Lower doses showed significant levels.
dose-
STides fron treated and control animals were also examined and
gross findings at and/or incidental
the time of sacrifice were generally sporadic with no apparent relationship to treatment.
B. Summary of Labeled Cell Counts for the Liver
A summary Table 5."
of the labeled cell counts for each Individual animal counts are shown
group is in Table
shown 6. The
in mean
labeling column.
index There
(LI) for each group is presented in the third was no apparent preferential labeling in any of
the
Tobes and the label was random within the Tobes.
8 to 9-fold increase over background. The mean labeling index of The mean background
indicates that less
labeling index than 1% of the
(Group nuclei
1) was 0.95 which had undergone DNA
synthesis increases
during in the
the 72-hour labeling period. Dose-related labeling index were induced by T-5711.1 with
.
significant increases (p0.01) observed in the dosed Groups 4 and
5 (500 and 1000 mg/kg
1000 were
mg/kg). The labeling indices at 500 and 9.06% and 8.13% respectively which represents
an
the positive control elevated (p < 0.01).
animals
was
31.51
which
is
significantly
Tihnecsreeasreessulitns tdheemoLInstirnattehe tlhiavterT-5i7n1m1.a1le irnadtuscedaftseirgnaifiscianngtle oral
dose at concentrations of increases in the labeling
2500 mg/kg index were
and 5000 mg/kg. also observed in
Large the positive
control animals
animals. The was 31.51.
mean
labeling
index
of
the
positive
control
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XVI. CONCLUSIONS
The test material, T-5711.1 did nuclear labeling of rat primary
not induce significant changes in the hepatocytes at either the 2-4hours
timepoint or the 15-16 hours 1000 mg/kg were delivered by
timepoint when oral gavage.
doses of 250, T-5711.1
500
and
evaluated
as
inactive
in
the
induction
of UDS
in
rat
was.therefore liver cells.
SI-nphcaosnetracsetl,ls thfeolltoewsitngmataersiianlgleinodruacleddossiegnoiffiTc-a5n7t11c.h1a.nges in the number
were
labeled
for
72
hours
and
dose-related
increases
in
Theanimals the mean
labeling index were therefore evaluated
observed in the treated groups. T-5711.1 was as active in the induction of DNA synthesis
Tiver cells.
in rat
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XVII. REFERENCES
1. rgMieerpnsaoatilorixsia,cssac3y.a.Cr.c,inEonTvgyiesrnoonsn,.inCM.uKt.th,aegeiannnedsviiBsv.,Eo.4-:B5iu5nt3t-ve5ir6wt2or,roth1h:9e8p2a,DtoectyetcetioDnNAof
2.
D(e1F9a87z)i.o, vive.'J.
HAI.im,sntuonLceohaherimys.,tocCJhy.etAno.i,cchaeHlme.dldeey3t,5e,ctDi.5Wo7.1n-5oa7fn7d.pTraotltiefresraaltli,ngM.cHe.iNl.s
in
3. pSLra-onbilreiorfm,eordaeTt.oiLx.oy,nurisBdteiurdngieeers,a.ndET.oX*.xH,i-ctohalynmodigdiEisantceho9,i,n 6Pr4.o.Id.en(t19h8e9p)a.toCcoemlplaurliasron of
4. fMBiourrtstaetlrhiewso,ritnhJ,.v.ivBo.PEr.or,basttA,shhebpGy.a,,tocJay.n,tdeBG.eOrNmWAui-dlrelezip,aamisrE:.,assACaaysp.crioatnoMocu,otlatDi.a,onnd fglueisd..e
189:123-133, 1987.
S. MDMuNitAlalgireaennsps.a,irVGo.alMn..d:m6.utTahegF.endeDesetiescSteriironensliovfaenrdcheAc.muilctHauolrlelsam,euntdaIegnr:e,nsC-(hcEeadmrsic.ci)naolgPelnesnumby Press, NY, pp. 61-79, 1980.
6. dDMiiNnrAistasrlyointsto,hleusJei.nsCe..ianndCraatrBcuithnteoepgraewtnooercstyihts,e,sB3.f:Eo2.l4:l1o-w2iI4nn3gd,uicnt1i98vo7ni.voofturenastcmheendtulewdith 7. DMRNaeAlrasstmyinno,tnhsehDs.iiSp.s,otfocahtetthpleaetyih,cepapRte.oCr.co,axricsCioonnmwoeagyep,nrioclJii.tGf.ye,raotfainotdnhePoapnppde,rorxJei.pAsl.oimcea(t1i98v8e).
CXpayrnlociledirifneorR)eas-t.2o-rps48y,rdiim6(i72d3-i9en-ty6hl7y4tl4hh.ieox]yl)apchettihcalaatceid an(dWy-[144-,c6h4l3o)roisn6-r(a2t,s3.-
8. WcCaarrnawcdiedcrok.c,k,IGn:.VP..M",.liEv(le1sr9e7v6Ci)ee.lrl, CCNeaolnlrcterhp"r,FoollCialfmaeenrrdaotniB,oinomHe.adMni.d,caelxLipnePsrreielmsles,ntaCl.A.livanedr Ansterdan.
9.
RCoelquumibraenmoe,ntA.o,f carcinogenesis
acResaljlaaslsapakrysoemldii,febyrSa.,ttihoranenedfdoSirafrfmtaeh,reenDit.nSi.ptRri.oactei(do1un9r8e1os)f.. 1Ciavnercer
Res. 41, 2079-2083.
10. 4oG-lfdiinToaisnv,ienroAb.reDen.gz,eenneBeru.attcJhi.eorn,'EoxpnN..Lt.MuendRo..r, f93oa3rn,mdatA3iu1bo3,n-32Ji4.n.C.rat(s195f1e)d. The effect
1515-0-494
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004404
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11. gDCNuaAaslciuraaentpoia,nigrD.caAsh.seamyia.cnadlsMDu.Wta.astiGpooansyiltoRiervsee(a1ro9cr8h3,)n.e1g2a2tSitv8a1et-i8si5tn.ictahle cherpiatteorciyatefsor
12. DWeisniegrn,, BM.Jc.Gra(w1-9H7i1l)l., StNeawtiYsotrikc,alznPdriEndciitpilone,sipnp.Ex1p4e9r-i2m2e0ntal
13.
Dcuonmnpeatrti,ng C.sWe.ver(a1l955t)r.eatmAenmtusltiwpilteh 50, 1096-1121.
acomcponatrrioslo.n
Jp.roAcme.durSteatf.orAssoc.
14. Dcuonnntertotl,' CB.iWo.met(r1i9c6s4).20, Ne48w2-t4a9b1l.es for multiple comparisons with a
15. Hsaantmdu,dyMAo.lTeicvaeunrdlarCeifMffuoentceat,gsenMie.nsAdi.usce(d1179(9b11y9)).a, sUi16sn.egleofdocselelofprDoHlNi.feErnavtiiroonnmteontal
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-
XVIII. EXPERIMENTAL DATA TABLES
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TABLE 1
SUMMARY OF CULTURE DATA FRO2M-.4-IHNOUVRIVOT/IIMNEPOVIINTTRO RAT HEPATOCYTE UDS ASSAY
CClliieenntt: Co3dMe:CoTr-p5o7r1a1tion
HIWnAitSitautdiyonNoD.a:te: 15165-1M5a-r0--94394
TCeonditoionn Niummabler DoTasregetLevel(1) VPiearbfiulsiitoyn m _--
EffiAtcEaicehmneRcty(2) Viab--ATlEacihatniy-- 2)
E
Vehicle Contr3o4l389- CC 1(0ml./0kg) 34388 10.0
6894..18%%
7802..42%%
9940..88%%
34387 10.0
94.2%
82.3%
95.9%
.
Positive Con3t4r4o4l3 - DH1N0(.n0g/kg) 34442 10.0
8777..62%%
7726..55%%
9922..63%%
344e1 10.0
93.3%
78.1%
93.0%
Test Material - (mg/kg)
334443310 11000000 3429 1000
3364441198 550000 34417 500
)
3344440067 225500 3405 250
'
33433954 110000
-_--
34393 100
Notes:
6872..72%% 6755..38%% 83.2% 63.6%
899920...105%%%
8781..99%% 6972..54%%
79.3%
39.2%
9951..01%% 74.3%
6888..36%% 5700..50%%
8925..66%%
92.2%
87.1%
94.3%
8587..35%% 7683..15%% 9817..30%%
81.2%
six
89.8%
((21)) rTRaehnsrdueloetnslaynibnasaselelsdectpoenedrviadarobeslaeesloecnvoeulnttwsowerp(letarttyeprsae.natebdl.ue dye exclusion) of CDHHC == D0.i5m%ethHyiglhnitVriosscaonsiintey Carboxymethylcellulose
15515-0-494
004407
23
------------
? WASHINGTON
TABLE 2
SUMMARY OF UDS DATA FROM IN2-V4I-VHOO/UIRN TVIITMREOPOIRNATT HEPATOCYTE UDS ASSAY
CClliieenntt: "C3oMde:CoTr-p5o7r1a1tion
HIWnAitiSattuidoynNoD.a:te: 151561-5M-a0r--49934
Test ConnSd.
Animal SNDumeberr
Target DDoossee*
MeGarnaiNnest N(uMNcNlGe)ar w% /C>el5ls CMyetaon. SP-ePrhcaesnet #+S5OD C MNNNGG'' GrGraaiinss''CCeelllss
Vehicle 3Co4n3t8r9ol - 1CH0C.0(ml/kg) -0.92 0.41
34388 10.0
0.11 0.20
44..0000 47.9445 017037
34387 10.0
0.29 + 0.53 2.67 568 0.87
.
Positive34C4o4n3trol
110- .D0HN
(mg/kg) 17.9822.06
34442 10.0
8.09+7.98
9556..3030
64..5277
00..1333
34441 10.0
10.450.60 89.33 5552 0.00
Test Mate3r4i4a3l1 - 1(0n0g0/kg) 3430 1000
00..1053 + 00..3355 32..3637 445159 o0l.a2y7
(a) 34429 1000
0.08 0.20 1.00 335 1loo
3344441198 550000 (@ 34417 500
-00..6213 ++ 00..2508 0.06 + 0.45
32..3030 44.5013 o04r0 3.00 340 010
(a) (a)
38407 34406
250 250
34405 250
0.36 0.06
+ +
00..2531
-0.49 + 0.39
12..0000 33.4509 00..6100 0.67 4.49 0.67
/:
Notes:
(21)2 JCATyovhtveroirepaaeltgaieasonmnoiifmca(l5n0seg)trapcbienarltecwdeoaaesrtne.cgLorevaveeirlnalcwiopeeur.ne.tscrNeoeanttetdnr,uicplleiacratearacionvsers(lWiGp)s =(1N50oclteotoarlgrcaailnlssyowuisth-shcoonmdeandd
32 ((54))
DAAcevovteveererraramgsgiieeniepopdeferoccCney1nt5t0orpailgtpaoetsamcolifactclegalrlcassoi)vn:ewrcisotluhinptgssreaoasntertthretihppalenirccaoetrneteaqcguoeavleorfsttoihep5aevnie(lt1y5.m0(catlobeteaatrlad 300 cells par slide
grains cecolllsL)s.
on triplicate cbserved shen
GO(HaMC2
=1Dislmisdtehyniontitanraolsyszaeidn;g(11500S0 =toatvaleracgoelleo)f mweiwrne WsxGemicoansm:s = 0.5% High ViscosityCorborymethytcellulose
on
2
coverslips
C100
total
cells.
15515-0-494
0044408
2
--------------------
WASHINGTON
TABLE 3
SUMMARY OF CULTURE DATA FROM IN VIVO/IN VITRO RAT HEPATOCYTE UDS ASSAY 15 to 16-HOUR TIMEPOINT
Client: 3M Corporation Client Code: T-5711
HWA Study No.: 15515-1-494 Initiation Date: 18-Mar-93
Test
_Co--ndition
Animal
Number
TaDorsgeeLtevel(1)7 PVeirafbuisliitoyn T AtEtffaricchime eencnytm(2) hAVtitaeabcihlmmiteyn(et2--)
Vehicle Control - CMC (ml/kg)
34392
10.0
34391
10.0
34390
10.0
94.4% 88.8% 73.4%
75.7% 66.1% 67.6%
90.6% 97.1% 87.0%
Positive Control 1 - DMN (mg/kg)
34446
15.0
34445
15.0
34444
15.0
80.3% 86.2%
89.6%
78.4% 60.5% 68.2%
88.4 92.5%
93.1%
Test Material - (mg/kg)
34434 34433 34432
1000 1000 1000
82.1% 79.8% 96.9%
98.9% 60.2%
78.1%
90.0% 85.0%
95.2%
34422 34421 34420
500 500
500
85.6% 88.7% 91.1%
78.2% 87.5% 79.0%
94.0% 95.6% 95.8%
34410 34409 34408
250
250 250
84.1%
79.7% 92.5%
92.5%
73.3% 81.6%
94.1% 89.9% 92.0%
T -_-- P 34398
125
34397
125
34396
125
89.1% 85.2%
84.8%
91.7% 96.8% 78.0%
89.8% 90.6% 93.5%
Notes:
(1) (2)
Three animals Results based
opnervidaosbele lcevoeulntswer(etrytpraenatebdl.ue
randonly selected areas on two plates.
dye
exclusion)
of
DMN = Dimethylnitrosamine
CMC = 0.5% High Viscosity Carboxymethylcellulose
15515-0-494
004409
25
----
WASHINGTON
TABLE 4
SUMMARY OF UDS DATA FROM IN VIVO/IN VITRO RAT HEPATOCYTE UDS ASSAY 15 to 16-HOUR TIMEPOINT
Client: 3M Corporation
HWA Study No.: 15515-1-494
Client Code: T-5711
CCTooennsdtd..
Number Animal Number
_TDaDorogsseeet'*
Vehicle Control - CMC (ml/kg)
34392
10.0
34391
10.0
(a)
34390
10.0
+0 ME Grains'celle Initiation Date:
Mean Net Nuclear Grains (MNNG)
+ SD'
% Cells
w 25 NNG
18-Mar-93
Mean
Cyto. Grains'
Percent
S-Phase Cells
=0.17 + 0.15 -0.79 + 0.57 -0.49 + 0.41
1.34 0.67 0.00
2.79 2.53 2.06
0.33 0.20 0.40
Positive Control 1 - DMN (mg/kg)
34446
15.0
3.95 + 2.86
34445
15.0
4.05 + 3.38
34444
15.0
5.01 2.62
38.67 38.00
48.00
2.59 4.58
3.32
0.00 0.07
0.07
Test Material - (mg/kg)
34434 34433 34432
1000 1000 1000
-0.17 + 0.15 -0.89 + 0.01 -0.37 + 0.48
1.33 0.00 0.67
2.47 2.18 2.85
0.33 0.13 0.47
34422 34421
34420
500 500 500
-0.52 + 0.05 -0.91 + 0.22 -1.21 + 0.10
0.00 0.00 0.00
3.33 2.07
2.33
0.27 0.07
0.20
_-- TT 7Rm 34410 250
(a)
34409
250
-0.58 + 0.36 0.02 + 0.14
0.00 1.00
2.22 2.30
0.33
.
34408 250
-0.98 + 0.64
0.00
2.56
0.00 0.13
@NotoAeTvhsver:raeagteaenosfa5tcs3loDrioadnnoee Crime pain
SKLoraveitrSwarone ocrnhesotevkd,teenatarcovrsttiapg
EC150 oota
lclee)tweif
sstranrd
@62 dSRBC0ea0oarrvrteieknCtsdFphaSheriolSrattenialofCcrcSiaieioTsryreitcttohuTnhtnasursa2anteorthietphfparcnuaaotrrecsscaueonve0hr5oser1tA 50restoonutr saotrteay,noneErvretheen
5S0k0
24T0mTS sRVsoaotvros
8 eae sen corsocymethytcntutone
esis an 2 covers C00 oct soon,
15515-0494
004419
26
_
WASHINGTON
Client: 3M Corporation Client Code: T-5711.1
Table 5 Cell Proliferation Summary
HWA Assay No.: 15515-0-494 Trial Initiation Date: ~Mar-93
Group/Sex? | Dose Level | Labeling Indexb
(mg/kg)
(%)
Liver Weight (9)
|
Terminal Body Weight (g)
w
2M 3M
aM SMC
6M
od
625 1250 2500 5000 15
0.95% 0.73
1.06 + 0.56 2.39% 2.25 9.06 + 3.13**1| 8.13% 3.15%%t| 31.51 + 15.86%*t|
18.95 + 2.64
19.70 + 4.49 21.82 + 1.55 23.75 + 2.49 25.73 + 2.93% 19.48 + 2.06
491.8 % 41.8
458.4 + 22.1 477.9 + 16.8 455.1 + 23.1 425.4 + 23.9%, 459.8 + 30.1
JFive animals per group unless indicated
"Percentage of labeled hepatocyte
F(oatur aTneiamsatls20p0e0r) group
nuclei
per
total
number
of
hepatocytes
counted
"*"PVSoeishgiintciilfveieccaoconnntttrroaoltf,,p1<0105.m0lm1/g/kkggofofcOorNn oil
4tDIenccrreeaassee iinn tthhee mmeeaann
15515-0-494
O04414
27
----------
WASHINGTON
Table 6
Cell Proliferation Assay Individual Animal Data
Animal
Group
Mean Labeling
Terminal Body Weight
Terminal Liver Weight
34489
H
3348449901
jimM
34492
LIM
34493
1m
0.95 00..7164
0.76
2.14
Group Mean Group SD N
0.95 0.73
5
34494
TM
34495
M
34496
2M
34497
2M
34498
M
1.14 1.67 1.24 0.14 1.10
Group Mean Group SD N
1.06 0.56
5
34499
3M
34500
3M
34501
3M
34502
3M
.
34503
3M
Group Mean
Group SD N
6.24 2.19 1.62 0.38 1.52
2.39
2.25 5
521.3 4510990.67
492.3 516.1
491.8 41.8
5
492.2 468.8
440.9 450.0 440.3
458.4 22.1 5
456.2 500.7 486.3 476.4
470.1
477.9
16.8 5
19.17 2151..1225
17.74 21.47
18.95 2.64 5
27.31 19.84 15.80 17.77 17.76
19.70 4.49 5
21.98 24.28 20.22 20.84 21.79
21.82 1.55 5
15515-0-494
004412
28
ee
? WASHINGTON
Table 6 (Con't) CeIlnldivPirdoulailfeArnaitmiaoln ADsastaay
Animal Group LMaebaenling
Terminal Body Weight
LivTeerrmiWneailght
3344550045
aaMM
191..2841
34506 34507
aN aM
10.85 3.76
34508
aH
9.62
Group Group
Mean SO
9.06 313
N
5
34509 3344551102 34513
5M 5M
6.86 12.14
5M 5M
8.81 an
Group Group
Mean SD
8.13 315
N
4
34514 34515
M 6M
22.14 47.43
34516 34517
6M 6M
42.24 14.24
;
GGrroouupp MSDean
N
1531..8561 4
443327..45 446.6 484.3 474.5 455.1 2301
5 421.5 455.8 397.9 42005 425.4 23.9
4 499.3 448.5 428.0 463.3 459.8 3001
4
24.69 26.99 22.45 20.35 24.26 23.75 25 .49 28.37 28.17 23.22 23.16 25.73 42.93 22.09 19.45 17.06 19.31 19.48 24.06
15515-0-494
004413
29
eee
@ HAZLETON
-
WASHINGTON
XIX. APPENDIX A: HISTORICAL CONTROLS (us)
ee 15515-0494
004414
30
@HWAAZSHLIENTGTOONN
-
HISTORICAL NEGATIVE CONTROLS
IN VIVO/IN VITRO UNSCHEDULED DNA SYNTHESIS ASSAY Number of data points is 20
Data Point.
font Mele Sr Net acterGrainsee 1 2 3 4 5 6 7 8 9 10
11 12 13 14 15 16 17 18 19
owww0 eo 20
UDS Grains/ Nucleus + SD *
-0.23 0.33 -1.39 0.49 ~0.99 0.47
0.12 0.89 -1.43 0.76 -0.55 0.45 -0.67 0.32 0.11 0.63 -0.11 0.25 -0.05 0.39 -1.05 0.31 ~0.01 0.89 ~0.38 0.30 0.27 0.22 -1.38 0.31 -0.18 0.33 ~-0.57 0.34 ~0.21 $0.47 -0.59 0.38 -0.62 0.52
% of Nuclei with 25 Net Nuclear Grains **
2.7 0.0 1.3
4.7 0.7 0.0 0.0 0.0 0.7 0.0 0.0 6.7 1.3 2.7 0.7 4.0 3.3 2.0 0.0 0.0
Grains Average Cyto Grains *
6.17 1.76 8.19
7.69 10.61 3.55
3.93 2.66 2.66 4.25 2.719
7.55 8.55 6.58 9.89 8.18 8.97 7.94
Tw7.75 7.20
ane: Average:
spa
-0.52 0.50
1.5 11.9
6.64 2.47
TSTver eemTTETee Low High
-1.43 0.12
0.0 6.7
2.66 10.61
*
DS = Morass of net nuclear cate or duplicate coverslips
grain counts + standard deviation (150 cells) analyzed for a single
from triplianimal.
** a
Average values for triplicate SD = Standard Deviation
or
duplicate
coverslips
for
a
single
animal.
ee 15515-0-494
004415
31
WASHINGTON
HISTORICAL POSITIVE CONTROLS
IN VIVO/IN VITRO UNSCHEDULED DNA SYNTHESIS ASSAY
4-Hour Timepoint
Nunber of data points is 9
PDoaitnat
NUuDcSleGursai5nsD/ *
%NetofNuNcuclleeair wGirtahins25** AGrvaeirnasge+Cyto
1 2 3 4 5 6 7 8 9
S. ult A SN . A Average: Range: spa
22.27 23.25 22.25 19.44 24.03
29.75 19.05 17.47 16.83
2.36 $1.08 1.96 0.75 4.22
17.98 1.76 1.78 4.00
21.59 3.98
98.0 98.7 95.3 100.0 98.7 96.0 98.0 98.0 97.3
97.8 1.4
7.46 7.01
5.84 8.43 4.99 5.26 6.56 6.65 5.74
6.44 +1.10
Low High
16.83 29.75
95.3 100.0
5.26 8.43
*
UDS = Average of net nuclear cate or duplicate coverslips
grain counts + standard deviation (150 cells) analyzed for a single
fromtriplianimal.
** a
Average values for triplicate SD = Standard Deviation
or duplicate
coverslips
for
a
single
animal.
15515-0-494
00441
32
--i ----------------
2 WASHINGTON
HISTORICAL POSITIVE CONTROLS
IN VIVO/IN VITRO UNSCHEDULED DNA SYNTHESIS ASSAY
15-Hour Timepoint
Number of data points is 12
Data Point
NDuScleGursai5nsD/ *
%NetofNNuucclleeair Gwirtahins25** AGrvaeirnasge*+Cyto
1 2
13.78 12.65
21..3502
3
12.23 2.84
80.7 76.7
9.94 6.05
4 5
7.77 1.73 9.26 1.15
6
7662..70
57..7718
69.4
5.00
7
89..8174 211..4185
8
6.01 2.70
8606..70
5.52 4.17
50.0
9 10
105..1656 221..1939
11
11.02 1.17
46.0
2.73 2.51
68.7
7.15
12
15.40 1.07
8741..73
6.09 5.61
Asvpearage: Range:
10.16 2.99
1629..04
425..0669
:
HLiogwh
_--
155..6406
50.0 84.7
2.51 9.94
* 1"
AUgvaSetreagoeArvevdraualpugleeiscaotffeornecttorvienprulscillcieapatsre g(or1ra50idncuepclloliuscn)attsea+naclosyvtzeaerndsdlairfpdosrdeafvorisaiatnigolsneingaflnreiommaalnt.irmiapll.i-
a SD = Standard Deviation
15515-5-0--2494
O0447
33
ee EEE------
WASHINGTON
HA
Seudy No. ModtEted
forPr3otHocCoolrpFoor.ati4o3n%
IN VIVO/IN VITRO UNSCHEDIUNLEBDATDNLAIVSEYRNTCHEELSLISS AND CELLPROLIFERATION
QPaHuhnaaadzlsleieFsttDyoAonfAGWsoatsohsudehriavnLnogactrbeokonrf,ianntoIaprncrycco.ogPrrrGdeaaNscnAstc)ieacnwevdiiltc(lhhGeLcPSo)FOniPdnsGuauclitadtrehelHpiiaonszreltsse.ttWuoLdnLyLTHhiabinesshciS`onupmgbepefcloeeincma.ssnectreo
woirtihniEiPhA erates
study WELL be conducted by HA at 9200 Lassbucg Fike. Viess, Simptessasc2210o0n)l
FART 1. SPONSOR INFORMATION AND APPROVALS
I. SPONSOR IDENTIFICATION
Company Name:--MCorporation
000000
Address: ~Bullding20:28:02]3 Center. St.Paul MN55144-1000
-_-- II.
ESTARTICLE IDENTIFICATION: --_--m
III.
IESTARTICLEANALYSIS
cDheatrearcatienraitsitoincsofasthdeeftiensetd airntitchlee GsLtPabirleigtiyatainodnsthoef tFeosrt (ahrrticcloen
t5h8e.10r5e)s,ponEsPiAb-iTlSiCtAy (o4f0 CtFheR S79p2o.n1s0o5r), and EPA-FIFRA (40 CFR 160.105) is
:
1. NOTIFICATIONOFREGULATORYSUBMISSION
f5I8on.r1eo0ir;gdneErPaAgt-eoTnScCicAeo,sm,pl4y0coGwnRisRcuhl7t9iU2n..gS1.0l;afbeEodPreAar-taFolIrFiRreAesLguml4ua0sttGioFbnRe 1cn6oo0dt.ei1sf0i)(FwDeAd, c2o1rteCrFsR
iBPnaadsrittcearotfescawhheisdctuhuldeaygoefLnscsytt,uodLibEeesanwsyuh,bimcBihitgtfhaetldlrteuocneditevhree rtaehgmeeinlrceeyss.oeriyveHrAoo-fveidstcahetSinEeceLSsinienroesaneo:nr
OT vnsecerntned
m EJ mare EJ sare
Our Own Cow Comm
February 1993
Page 1
004418
wAsSHIN GT oN
v. STUDYDATES
Proposed Experimental Stare date: Proposed Experimental Toratnation Date:
Modified forPro3t%ocCoolrpoNor.at4i9o4n -
VI.
APPOFR STO UDYV PROA TOCL OL
Study Director:
Varia A Cifons. PhD.
ee
Sponsor's Authorized Representative:
`
-------- DAE}
Februazy 1993
Page 2
004419
WASHINGTON
Modified forPr3oHtoCcoolrpoNor.ati4o9n4 PART 2. STUDY PROTOCOL.
IN VIVO/IN VITRO UNSCHEDIUNLERADTDNLAIVSEYRNTCEHLELSSIS AND CELL PROLIFERATION
I.
OBJECTIVE
DcTNihAteyoscbyajnuetschetedisvibesyo(tfUhDetSh)tiassatnadsmsaactyoelrilisalptroboyldiemfteeearcsatutriDioNnnAg d(DaCNPmA)agreempeaaanisdru/roaeryd whaeespraetSroeotgcohexestci-es
induction induced in rat liver cells after in vivo treatment.
rTTIhehnececorgeetnaxyisipzeseatsbielnneocnfeetbyadnenutdcetlhcedetaeargbrlcgeeerelaliDuonNlfAacroDduNanArmteaspgdaea(imUraDgaSer)seycvsoiutmlneplsmapreebcdeiftiioendufnetrbrrueetdatewfdursorsmacena"an
sihnocrotrpo(4rahtoiuorns)ofinnevwitbraesecsul(tiunrcelupdeirnigod*H(-1T)d.R)
and reseie a' coe
into the DNA, during a
atuCneendcldhlenrigpqotruhioeenlgi(f2ce,elr3lia)lv.teirroseAnpnliimiacsaraletsidoeansriiesigonngleiadrvtaeetndtolaivmseeirangsuluserienogrtaahlne dfofmsrmeaecontfcihoitnhseteoccfhheencmteiolonlr)s
pbiurnmocpmoordpieomorpxalytauenrdtiedDdiNnAespurb(ecBcurutdarUns)eoorfusosrloyv7.e2r
hoQuurasntfiionflilvcoiawvtieinogwnitohsfdaancienlAilsLstTraEtchiaeorenmhoetseoerr the 72-hour period hes been shou re
pberoulsieffeurlatfioorn tihne tehvealluiavteiron(4o)f. chemicals that may cause noreased ces)
Ir.
DEFINITION
dzTeahscecUrlDiiSbveeadrsbsyacyeVli1llssldiae(nshsie,gpnaet1do9c8yt0ote(ms5e))a.suusHrieenpgautnostcchyheteedasuultveoidrlalDdNibAoegsrtyaenpethlhiaeccs.eidstar(ceUhmDuSmi)qweigsn
.
OcaLuaficuvosereWradsdTbioyoRfgrtrraaeiptanhstcyoz,eexnwptDiolsAwleidbhdoiuntruhivseniegvdcoaasisttnoaatrvmhtieeiccarlsteeue.rsetcuaoTlrfhttiuitsrchileUneDg.Sr,empTeahaisesrsuoicafnsncmaDoelNrryAptsoedrodaa.tmtnaibgbonsyn
aZcetpiavlisciaepspeaorfschteoaiccoarlrse.late well with known mutagenic of oareinegents
OTaHthnehepesairetnocctrpooermxaopisloceiuafnneidtrnssatmcsieaunlycglhincapdserilocclleasirfSben-orapnayhtatibseoeetnrasdtcyeohntlteohrcreetipelddaecdeaunrdniendcgfrnoitSti-rpchoattsoeil.suscusnemeai(iy3nodoiuysc..e
actfaofzxeeicictinteoydg.enidcuIrtipnrgoLcsesrnseo,ptlibacupatptaitrohenenrte(h7ao-rw1e0)c.meulmlseirsopCurhsioenlmipitfrcheoaerclaelatsybissoeensnciaentdcheaeostsfe.d.maiepnssroiorhn:yee
proliferation may increase the probability of spontansous mutations es
February 1993
Page 3
004429
@ HAZLETON
:
WASHINGTON
We11 as frcrense
the
probability
of
Nedtttea comercing
for i DIA
Gomis ators ton
he erence ox tly rameters Char op aselecellsleading to
an
III.
MATERIALS
A. InTdheiciantotcrsCceolrlscelle for his assey VELL be Liver celle beatmed from BTeaeondceduileStmn,E,aTmSlaolier,ao1nFdi)sDFcvbhateeiresnr3e4GT4ondrrceaatteEpsie,corso(rsweneBsiogooheioenSg mm1e5e0seta o 030a0,ng)2,tSpsugPrccdhhpaaeesssoeecldidefurrreo,sm aSedclinbitauan. TS LA ar They will be quarantined a smtiantiemunooffsseevveennddaayysspprriioorr ttoo TeHpoles,crUiIeEs pBreisoeed taompevmeinotahceriioond Sheonpac nnaahrl,0b2sahount 60 TSohleiScpeelnleeedVEsLlLubcoiocnhe(aotesneSdecbtyiopnecTkustEoenpeovfincehneeesLipveerLi3in sSi5c3a wich SUTRuRs ithe om weed She oem ta daoekS Speen 13
EuiSFnsio0eibr1tnetguththaeernMiaeac.otonoulefrdaaslmnytapeer(ooilln(rieomoferdetrpehaaiotnxuihyorfnkpleo6uarcrsaesnaarepyn,e,itmeetarPhietevvmnaaonntT-enMaoeeloesrmeHSv,ialenlIepnbcdIe.amt)GessenaYiensnhhcttahoeloseacenttrlizceeoesdnd endo fone ue
B. Mefd ori UDSu Assm ay
iB2c8Eh1L31003AyfFiogereelSubnoovvaismnaietcaisnneimo(ahHnaeen5ns)ohpELeegLleownsheotemn EtonaLheelSBerECuEPEPLSeme2nc2ed
WET containing 10 uCi/ml *H-Tdr (40-60 Ci/mMole) (WME-treat).
February 1593
rage 6
004421
ey
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Modifled forPro3MtoCcoolrpNoor.ati49o4n
c. Osmotic Pumps andLabelfor CellProliferation Analysis
VALIZLELTbe osusmeodt.ic pAumspisngl(eALlZoAt Cwoirlplorbaetiouns,ed PtahlroouAglhtoou,t CtAhe)', sMtouddye.l 2aMpLr1 aAotLfZEa1T0c4oMlno/cdheeznl.tr2aMTtLhi1eonopsuommfopts2i0cwaipglu/lmaplb.ehas1lal2e0d00vi#t1h cbarpoamcoidteyeswyiutrhiaainpeumpForraatye
D. Control Articles
1. Vehicle control
mbAfaeotvreetrhtrihieaecalltU.eeDdSnTeahwgseiasttahsiyavmetaehnceddoonfsvtiiervnhoegilcamlcceeotsnhosofridossrtsio(cnluegvsleulonaftlplrayoslemilifeencricametdaioonff.orthTtreneeye
nraitts bik
ectxohcneteretodels.atboWmuhatetree1r0ipamolls/skitgrbelbaeocdadyeonswtiesniggwhivtlo,llumbees
emplooyreadl for oral
gSaovragier)e savage
wseeedrifeos witj wo
2. Positivecontrolarticle
The positive control articles used are known to induce UDS or US(0D-DSpM2h)5taismvaeBie/lpiKlongtbnroetafstdDo(hs2eHe-pd4avtihaolcocluyr1tb0seesatunoisdne2d10v5iamvsgto/o.ktg1h.6eThhpoeoFusopirrotssci,ievtledilivcmpeoerntceohoriyonfirtea,rtroeclraeaefrcomcTreaetatnrahysge tchrreeaetedrabtys ifnotrraUpDeSriatnodnefailveinrjaetcstifoonr fcoerlleapcrholniifpeoraitoieon wilt oe
E. TestArticle
.
2Ust3pneolsgnetrsseiosdnrdasisppnregsic,uoiprpfelixtieotedrd.at"bchyteiAoitnnhny,eitooisprapetorsinaoostlnoivroeo,nnfts.ttepheexesrctfhitoanergnsm.gteedamroucnsitctlhbeee twseipsletlcainfvoireemdratlblyyhnubnne
on the cost article vill be described in theAfLiLneospesraecpioovnes pertoren
w. [EXPERIMENTAL DESIGN :UDS ASSAY
A. DosingProcedure
mbAseyetlphertycheltelciieomlnSilpnuofalnorosrysoert,.theecsattrMebaswottixelyralmireatbtliehscyllpewceheruilfnclolhuerlsmosemsdeeya,tvobceeohrdienscteloelefreslmcsiotonlerevdeanvnitesnhtciihlceulsdese/sousotwoceltecveemeionnorsty
averhtiiccllee/sionlvveonltu.mes Ratthsat wwiillll benottreexacteeeddbaybsourtal 10gavmalgiekgwbiotdhytmheimnoen.e
February 1993
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004425
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Nodes or Se eos
Kisarmaceicoostens of exiporu,ce "aaWy obeacneaquresecded bbyr tSheeSpsonpsoor,n.botm
rTeevptainnigcipooncpeosne.~dosSienugbiHiiiTt)y boee the SToeneeyemnaitcelrei,alMLLundberecounsaetdcfioonrs amy
B. DUCSTS onosotreeenee rresaasnl TtpaenEcttte eNEstEepadac onpSopdeeet Pahemeecmrrhtavfi aiuaispsoaeir,ro "oytncsTheohin emmereheriigeyohledstLadydSaiolyastsloetneasllseFotcceotcpueosddadnwowivailellsmuivunesiumuauallslllyoy fbbbeee TEEorTaoeed tToonre0m3a5,teornei pTE GpTt1nr3esS 5G2y1B6eoiunrespaerltnetss VIsLiLngbleedpoeare
C. ThPori2r aesae ahTtsoasp sudaya nyetoSopar iafnnbra eHaocsenaoeppt nsdeadeti wonouneco swtytitehin eHehcsepaoresosceeennduiEreeotsesd3eeb7saypcreabirnabfddeudBsbuibytyotWneNiroililfle1irtaeaLimnmvessceb,,e119198o80l00,a1((i955o6),,7 SCoovnetiaeinaicntga0.(53whh eenydt30 oenmeBokngyeiemieyooethhyol ssiehery),ELE2c0oc0a): TS1i0hn0oeutusnaTiytos"S/omla1oYSfeivmceaodelelsnaygenD5aespeeeneiwniinlsgl,obne fhpheeerrSfouosnned,diScthihroreouonsufhgththhteheMLvielvrie,vrertfhfoioxsr Crnraining the HEL oupmuny enchant Hover clastisn culcureata ProallaanbTees.iepeTnhtee"dctonkWsBespenRmeannewBnoe tanlp2re aemtove 8collSheclcuemlp]s Cand To 0.5 x 10tvhieaOb0l5e assy cells WiEnLL3 comet and ml of WME+. on Culture Sdiisshheesetthhaattvilwillbbee Celtures wilt be doom ir ioloney, Willhovenoc GAESrneaEareasimtitaciahtchehhsaocc3noo.tln5lteapaialvnriitHinekged t5tor%hfeooanto1by5evmi"toPornbovne2a,BhoooyrnuonnrdesA,t0ssatSeEc37anSCntinc8eLra)YhueSmmtt+aatteoitoeemdd nreeldyatamTenene a event CocheeaaaiSniansga,Sltourees vvTeiplisabilees kweLipltl be Actachment efficiency vill be determined for two exicuses from each
----animal using trypan blue dye exclusion and in situ analysis. vogn 6
004423
:
-
ASHINGTON
.
w
a
e
Modified forPro3tMoCcoolrpoNor.at4i9o4n
abAidefndtcieurtrbeiafoteanodrlvaoibffetolhri1nW11g6ELsptoeodrciiou2onm0dcaholcofniuitranrsbga.otu0et.2T4t5hoehmoimuturhecseh,lyeacllluadlwbitienullerleedsatnchdee(lnsrloembctec.uealmtstneeuincrolegisrsonwiinliylt
KcamooocnvdeoeatlrkiascylNeiTarpScss?i)dwaiinfeldoclrhvaban8etselmr-ou(a1n1n2t:d3ea)didnrouianecndedg,sl.adsrTsihNeesedlxitfdoecrsh,eaAccleplleetasssewini2re8nhmoeeosrbseseiocnmreionLee)h
sDsrttiaoeirrneiedtde.fwoirthT7hWeiltelomlua1ln0ssi'doanmysosdwiifalciledthhCeenmaitbnoeexmTydiileigivhsnettlotaonnipdecgdehsstotfsnethdnoDvipeeorr,rotacmEsmsermseee,rsatiyaotnoydF
D. DSAnalysis
mdaFieposaprpsrluotarxhyeieedndaUbtySeolncyaotnuh1ane5lt0yi0svnxiigsd,emnouacgtlnsheiceafrriecceeganlrclaiosiofnnwsaiunlanlndadeubrtseoumobeaftxltraiamccfitmnismenoedgrmsmbtiiehncresr.oaesvecesVo'rDpaEigocreisalilSiyloLraeyc gO(rfcayicgnorpaclioanussnmti.cin coTtuhhnerte)ce.ovneruTschlliiespasrvaswliiuzleeldibsearrceeeafdseerdraedtdjoatspoerretavsenrttoheB.eismesotr inmmooiecoimneebsotnte Counting.
bnT5loh0arecmxkaaelnnneldteyodnlabmypyupcesglaererlaaieirncntgsegdntruoaccoielnlenliusnceworoiunolnultseabctewhoilscclcooouvrneebtrdes,WliiraplonudatbnieaanmlesyylxsyeoelcdacdtafeeotsnreirmoBrmniosn.emdoOrnfseo]sr chTohivecehrmserlaeinpplsniec(ta1t5ni0uvcetloeDtaNarAl gsnyrunactilhneels)ciosufnootrccewuairclrhledtbrereaatdtheeecdreratnnhiienmneadlp.efpesoimercitssoyiibeiooommnmsiiiiicoiieest":sh isatnceoccrthoehnvdeie,cricsaatlnhli.ecpualvametaSriiyaongncbeeeooffartetdhchieoefusfmneeetraerendenptfeaoacrtceeedalalcclohaputonaepcnrueileasfdttoa.ritoe5n0wWceEeLi}blysgoxanresrrestireresuieibrsen
V. EXPERIMENTALMETHODS:CELLPROLIFERATIONANALYSIS
A. Treaant d Dm ose e Lenvet ls
caAenllillaaalpnsriomlarilofemerweaiatlcilhondboaested7Lohesoveedulrasasn.ddceosnctrriobledgrionuptwheillUSbe useecdtioonh.anaiFoimvse
B. Implaonft Osa mot tii cPo umpns
aALtZEaTcMoondceelnt2rMaLt1ioonsmoofti2c0 pummgp/salw.illTbhee parneilnoaaldsedviwlilthb2e00s0ntslocohfecBrridoU
aunsiensgtheMseitaofaannde on(emeptuhsopxyfpleurranaen,inalPitwmialnl-Moboeres,sepItnicc.o)llyinshaarleartiicoyn
February 1993
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004424
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WASHINGTON
Hodieted fPorro3tocCoolrNpoor.ati49o4n
wPseonuausbpuncsrduetWcaLlntLiehLpoasutbselatynhdkeer(ptedthoeraIsranaenlttnahsoeaulrcsrflaaitmcnaoein)cif.aotlrorTesthdhieegeunensitnidcoalflysstiihonpenfreitwcoiirtnlieloosnobfesas`creTlraeorirsfteeipdenosrrwiitcehh
C. TiC sso ue llec ant dPri epao ratn ion
Baananadclhyfsiatxnset.dnaIlnThnweeiulctlhroarblaecbiuacfnfecesartvehidettyfiozvreimdlallipnrb,ieoroApetcnoreodsrseasnsodevcattlhieoonAftovfeorrstguarnmesmaonrfco:hr &at5hneiCalpalulaasrbuaaeenlfdewiwinatishxseusdhbd.ieagdthdn"eiTdhscecesalderluceotddtieuocmrnosnsrnorvveweicirltl,lllywbbieeclolitnathckaolel.unsdoaenEdirbmeeaassl..rtaehnmeoFLvoSecee.ddtlesofomeamenamrtieimtsrhoe!etht o5SE1lfi1ig4dhte0ths3emead`dnuVidIoaLdLane,nsu`aaammlnpsdwolierlbilEegchaotlpnsraoecnhptbeaearrdeimudoaorddefel.onorubmeTsawh.niealllsSyeisbcmiotisilfoannbrcsllyyowedipelrdleppoeeencevheeossniotnbmtemcieeonrssoeien"sss deteraine Lf any sbuoraulicies are shaerrad.
D. Immunchistochemical Staining
The slides will be deparaffinized and rehydrated prior to staining uhesmiantgox1y)linthceounBtIeOrGsEtNaIiXn,Suapnedrs2e)nshietmisvteoryKliitn unsdinogsiDnA.B stain and
E. Assoe f Ces ll Ps rolim fere ation n Rat tes
aTedhneesquursaeetcetthilaoatnbetolhfiengtlhaiebselinnowttaesscthipanrserovpeewdri,llyls1abidedaetsmniifscrtroeonrsecdtohpeitoc.paaltrlhycioreixraemsiinseid mttsoo WdseIatLmeLprlnniionntgeboofifLaidnviaeflryfzeserdle.indceessOnfcirenonllaatbbheeelliddnigifsfaterrreiebnbutstioLoranvveehrda.svihleIlefnbaocScoimrertmmitirieeidmc'saccsht dSL1ia1fbfeeltrehedencheleopbaietnsoclyawtbieelsllinfbgne itnchdoeeuxnltoe(fdLc.1)loabmIeonwgmioltlhediblfeifvedereretnelcroebmseisn.serd1es cs`opamereemmsvseeacsn!t. so2te0hc0et0rimouncclaeenldallvyiztleyldp.ebseCeosxuuancnthiinnegdaswpielrlinabfneliancmaomlnaftvioinrteyhd atcoealilhsne.ipamtmmoacoyyftebemFoicesiieodssmmbpeoosre c(osdeepdarattoelpyr)eveLnft tbhieasdatcne caopmpeeianrgs. relevanc, Tha comselies wir ae
VI. DATAPRESENTATION
Tahnoearleyaz1cenhdalttirrmeeeapptoomrtetnnttw,!ilflortntchleudneegtahteivfeolcloonvtirnogl,inpfoosrimtaitvieonsoimnsretra.bulsaarg rfoormm
February 1993
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004425
WASHINGTON
For ws:
Modified forPro3tocCoolrpoNor.ati4o9n4
Toetalmeoafn ws:
1n5e0t
cnoulcllse)ar+
gsrtaainndarcodudnecvifaotriotnrifpolricsaatceh
caunlttournesan(suisyuoaelslyJoan
Thoer mterainplpiceartceentculotfurceesllsforconcatcahinainnigmafliveanaolrymsoersefnoeetUaneu:clear grains aThnealyazveerdagfeorcyUtpso,plasatc count for triplicate cultures for each animal Tcehlelal)evfeolr ocfachscahneidmualle.d DNA synthesis (percent of heavily lebeled + Perfuston and culture data for {ndividual animals. For Cll Proliferation: TFhoer ccaaclhcualnaitmeadl aSn-aplhyasseed +fosrca5n-dpahradsed.eviation among the three slides
VII. ASSAY ACCEPTANCE ANDEVALUATIONCRITERIA: UDS
ALeTnxieessartusclisitnassgyemonansoylcryimneaonlLttfliyefnaiwclciollmlpJoaufbsdegstmcheaoenlntlscirtdiieentaretemrdosidiaaitcuflcayietispinttogeandbslt,hbeeesflooocrwrtihevemveearssliutsaeotriioSnnifmaoedfelstohre
btoeostt ot
other causes that might affect Tellability and aeseperncse.br sesciiemin
1. TdnTiehosetrbmrivaililmialetbynyit,leaixlccseyoeTdohsfveatl7th0ueo7e.xshiecpbAiaettvlyoaocorwyfite1ett0hsy1ecotofarlrelfeaeatccnattsoeonrdtsfuvsrniuoctnmohmatmfthoehfneepctemrosfrouismlieolgnslepirraeorceiesnos:n
.
bseec,reflected in perfusion viability, therefore no TevetesstitmeetecrrsiLeol)
2. BTCheeenltlvssiamibsuisltaibtobyueto7f805%c%h.eormognroelaatyeerr. celNlorcmualltluyr,esthuesevdifnoirlitehey aosfsaSyecrrreaatt.a
3. DTedhemSetpelacopctyotiesiodivnciiwtvoayefs, UcrDtoheSnestpraaoonvnldesriSiav-sgepehuasarseneedds.ptotonhFseoderemmtoteoentsshtttohrdeamotalpetooesgriytithaiawltvasestchcoaesnudtscerieqenleyglr.cvspoepobulnaortiimooenn
msccuohsnettdrouellxecdefeoDdrIA`Ses-iyptnhhcaehrseesctrsmi.utsetrFioahravuteessetdgrmefaaotteerrIinadltishcaanctele1a%UrDlGyo.fcotmThmheienrgecpaeortctormsmeonrsreb:sse
February 1993
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00442
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_
WASHINGTON
Modiled fPorro3tocCoolrpNoor.ati4o9n4 !
Sabesleantceed UoDfSaoprosSi-tpihvaesecoancttriovlic1y0,stanfoarsstaeychvniilclalbereaacscoenpstable in the
4.
DLaEtaobotbatianiendedfrpoemr culture for WS.
atwnDotaataarlepsvlhiiolculaltdbeebecauclactveuapritelasabbllaeendasfrapotamrlt2eaosoftf
t5h6e meuvcatleuratisoonn hes mesmbe
treated.
5. ARtehempeianftiirmtsartiaotlfrsi3anledeodsteoonlalecyvheialeusvgeaweinaltlttboheteaalmnuasolbfyezre3docffoonarcneabmlosytrshsecdiUodSnusr:teimmbeeaprroeisnmntusii.ra include ac least one dose previously assayed as sccepeamic. bTSaheseviesrcarlfiotcrerrieitvaearlifuoaarthiaoavnepoobfseieatnivCeoessttarbelasriptsoihcenldsswhaairsceha,cbtaiisfveedmeiton,n twahielslUtBpaSrcolaveiscdsieye.s analysts of the historical date as descrived by Gaselamo sad opior
an.
a1.pplTiheed ccoenscenatrrcaitciloenswitlhlatbecacusoen:sidered active n the UDS assay at aAfvinveezianggcreraeilanessaedpiionngr tnthouecalmeeupaosnsiatnbieovtveenmtuhucelbeecaror.ncgaurnrargieennrt;coAuengtatitvoe.atcolmetaestt + Agcnrualitinunsrcersseuscsihesthi1an0t2tthhaeeboamvvueesrbaetghree opfeprecrmecinectnlategalegewoiftfhtnhefnsieevgeaptuiocvrle.elmoc1roaemmtomeneastr Cultures.
WGailelsnloeraialnlldsyio,cabt1eef mUteDhtSe. afcitHriosvwtietvcyeo.rn,diDtsiiafotfniesrfiesanctstaiDtoNinAs-fdoiafendao,gniltoyhgeoansgeeecncotonendccicooominedgsiitviceoonnn vaabaforfvieeecttcyoonnodlfiytniauocnlssemaavrlillllaabfbeneolcrioinntgsyipdaoetfrteetdrhneIsn,cealanlnsde.vawleuaTakhteiraoegnfe.onrceeT,nmoacycashesscoobsohnogoraledy Vtihn1rcereebaeasnecisonnaaslrisedesnrhoeotdw oaibcnstceirrveveaesdefso.rinthaIalftltchtoenhdrnieetgiaaotnniivmieaflcsco,enltltrshoelfrtsooemsleisuvmseeastoefrsithshoie Va0ILavenroargmeallleyssbethcaonns-i5deorredmotroevatlhiadn: one grain por mucous, the erey 2c.ritTehreia taerset meatr.ticle is considered negative Lf none of the above
VPTerhseepsnoennrsceeessu,lotfasnadardteohseneeriertpehrseoprdonuscceil,beialrtilhtyey pFoorfesqdtuaietvnaecsynnoedrnissccalrneiiabmruatlliysonnLeegoacftoinsveeeri,dieuritenhsee
February 1993
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Modified forPro3tMocCoolrpNoor.ati4o9n
diiennpcertnehadesienesgvaolinunatthileoanb.leelvienGlgroouwfpislalcitnibvewihtidycehciindoencdeelolousnt foarfomctahtsrheeeebaycatniicvmaeaslesanibsmahasoliw,ss
ptrheeselnecveelorofabsaecntcievitofy aicnticveiltlys infrsoumrrtohuendiinngactgriovuepsa.nimals and the
UpTDohSienatpcotstiiovtiietvsyetiocmfoantttehreomluttenasugtcelnaeiractriocllraeb.cealriUcnDigSnowegilelinlciicntoertdisbbkey autssesesodtciaaasgteeandtrsewfiietnrhetnhsciheye
aansdsaytheLs agent as
apvraoiblaabbllye mroerpeaidrepmeencdheanntisomns a mutagen or carcinogen.
tthheantyopne
tohfe
DpNoAtednacmyagoef
Itnhfelicetsees
ncSuhocamlteetifhocermarscaitdossf.wDeNrTAehdusra,emsapgtoehenasrpieovseriaetpniavdietrhceeodnmwteirttohhloodwuoitlltolhgebyeviuanssceoadrdpteooqruadatetimeoonnfsootrfratntheeew detection of UDS.
VIII.ASSAYEVALUATIONCRITERIA : CELLPROLIFERATION PcAhamalseceaunlvaaatlneuddessf.toarndeSaatrcadhtidstetrvieicaaattlmieonantnaflgoyrrsoiutspheuosfpienrlgcaebnettlhaeigneginodifnidvSei-xdpuhwaailslelancibemelallpsemrwefiaolnrlseSb.ed tUChsreienagctamseoennteo-fgwravoyaurpianacanolcmyepsaihrseitseoornfosgvewanererfieatnydc,oenreatnewkcihtnthirqaDunuesmsfnoert(m1'a2st)i.to-nteosfCtotnh(te1r3o,dl1a4t)av.ewrisleTlsn pbIhenastpeheerpfceoorrncmceeundrtrapegrneitoirncoantotdrooaslneaglgryrosouiupsp aottfhaatvasdrieigvaniniacfteiecsaannfdcreoDmulnetvnheeeltS'-ospfhtap-s<te0e.sp0te5.rcweiAnlntlabg5ee. considered significantly different than the control group.
IX. REFERENCES
.
1. Magisersnasoyat.loixsi,cEnvcJia.rrCoc.ni,.nogTMeyunstosang,einneCst.ihKse.,,in4:av5nid5v3o-B5.6-E2.i,n B1vu9it8tt2re,orwhoerptaht:ocytDeetDeNcAtiroenpaiorf
2. HIDiemsFatauoznciocoh,hiesnAt..o,chCeLyentaiorccyha,elmJ..dAe3.t5,e,cHt5ei7do1ln-e5y7,o7f.D.pVr.olainfderTaattitenrgsaclell,lM.iHn.N.viv(e1.987)J..
3.
Lbarnoimeord,eoxTy.uLr.i,diBneergera,ndE.K,H-tanhdymiEadcihnoe, proliferation studies. Toxicologist 9,
P.I. 64i.n
(r1o9d8e9n)t Comhpeapraitsoocnelolfula5-r
4. JB.uttPerrovbosrte,h,G.B,.Ea.n,d AG.shbWyi,lliJa.m,s:BerAmupdreozt,ocoE.l, aCnadsgcuiiadneo,foDr.,thMeirisnalviisv,e
February 1993
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WASHINGTON r1a98t7.hepatocyte DNA repair assay.
Modified forPro3tocCoolrpNoor.at4io9n Mutation Research, 189:123-133,
5. Wrielplaiiarmas,ndGm.uMt.a:geTnheesidsetienctliivoenrocfuclhteumriecsa.l mIun,taCgheenmsi-ccaalrcMiuntoaggeennss.bVyoDlN.A
. Pp.
F. De 61-79.
Serres
and A.
Hollaender,
(Eds.),
Plenum
Press,
NY,
1980.
6. Msiyrnstahleissi,s Ji.nC. raantd hBeuptatteorcwoyrttehs, fBo.lEl.o:wiInngduictniovnivoof utnrsecahtemdeunltedwiDtNhA dinitrotoluene. Carcinogenesis, 3:241-245, 1982.
7. sRMayernlstamhtaeinso,insshDti.opS.to,hfe Chhaeetpptaaltteioycc,apreRcr.iCon.xo,igseonCmioecnivptaryyo,olfiJf.teChr.eatpiaeonrndoxaiPnsodpopm,reeppJrl.oiAlc.laftei(rv1ae9t8o8Dr)N.sA pdy1r(i2n-iedtihnyyllhtehxiyolj)apchetthiaclaactied (Wya-1n4d,643)[4i-ncrhaltosr.o-C6a-n(c2e,r3R-exsy.li4d8i,no6)7-329-6764.
8. cCarnacdedro.ck, IVn.:M. "L(i1v9e7r6).CellCeClalncperro"l,ifeCraamteiroonn,Ha.nMd.,exLpienrsiemleln,tCa.lA.liavnerd Warwick,G.P., Elsevier, North Holland Biomedical Press, Amsterdam.
9. oCoflucseblalnop,roAl.i,feRraajtailoanksfmoir, tSh.e ainnditSlaartmiao,n Do.fS.Rl.ive(r19c81a)r.cinoRgeeqnueisriesmenast assayed by three different procedures. Cancer Res. 41, 2079-2083,
10.
GLliivneors,regA.eDn.e,ratBiuotnchoenr,tuNm.oLr.R.foramnadtiAounb,inJ.rCa.ts 3. Exp. Med. 933, 313-324.
(f1e9d51)4-diTahmeinoebfefnezcetnso.f
11. cCahsecaliacnaol,s aDs.A.posaintdivDe.V.or Gnaeyglaotri:ve SitnattihsetihceaplatcorciytteeriDaNAfrorepeaviarlausastaiyn.g Mutation Research, 122:81-86, 1983.
12. VMicnGerra,w-HBi.lJl., (N1e9w71Y)o.rkS,ta2tnidstEidcitailoPnr,inppc.ip1l4e9s-i22n0,ExperimeDnestiagnl,
13. Dsuenvneertatl,`tCr.eWa.tme(n1t9s55v)i.thAamcuolnttirpolle. cJo.mpAanr.isSotnatp.roAcsesodcu.re50f,or10c9o6m-p1a1r2i1n;g 16. cDounmtertoel,! BCi.om.etr(1i9c6s4)2.0, 4N8e2w-4t9a1b.les for multiple comparisons with a
X. REPORTFORMAT The final report will provide the following information.
February 1993
004429
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Modified "foPrro3tocCoolrpNoor.a4ti9o%n
++ TSepsotnsmoarteirdieanltiifdiecnattiifoinc.ation and Assay Number. A physical description osefcttihoen.test material and date of receipt will be included in this
+ TDyapteesooffassstauydyanidniptrioattoicoonlamnudsbceorm.pletion. +- NIanmteesrporfetaSttiuodny Doifrercetsourl,ts.Senior Technician, Scientist ++ CHoinsctloursiicoanls.control data for negative and positive control cultures. ++ STiegsntatruerseusltsofprSetsuednyteSdupeirnvitsaobrulaanrdfoSrtmusd.y Director. ++ MEevtahloudast.ion criteria. + References. Quality Assurance statement.
XI. CHAONRREGVIESIOSNS Asniygnecdhabnygestheorstruedvyisdiiornesctoofr,thdiasteda,ppraonvdedmapirnottaoicnoeld wviiltlh btehisdocpurmoetnotceodl,, The sponsor will be notified of any change or revisions.
XII. REC TOO BEMR AIND TAINSED Aglelnerraatwed daasta,a droecsuumletntoaftiotnh,is rsetcuodryds,wilplrobteocoalrsc,hivaendd infintahle srtepoorratgse ffiancaillitrieepsortoftHoaztlheetosnponfsoorr.at Alfetaesrt otnhee yomeearyefaorllopewriinogd,subthmeisssipoonnsoorf mtahey efalceicltittioes haovfe Hatzhleetoanforfeomrenatnionaeddditmiaotnearliaplesriordetaoifnetdimeinorthesensttotroagea storage facility designated by the sponsor.
February 1993
004430
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