Document 0JowZNZJwD0eK8ELaJyQQ1dwd
| @HAZLETON
WASHINGTON
AR226-0%5]
GENOTOXICITY TEST ON
T-5710.1
IN THE IN VIVO/IN VITRO UNSCHEDULED DNA SYNTHESIS AND CELL PROLIFERATION IN RAT LIVER CELLS
[EINAL REPORT
AUTHOR
Maria A. Cifone, Ph. D.
PERFORMING LABORATORY
Hazl9e2t0o0n LWeaesshbiunrggtoPni,keInc.
Vienna, Virginia 22182
LABORATORYPROJECT ID HWA Study No.: 15516-0-494
SUBHITIEDTO
3M Corporation
Building 220-2E-02 3M Center
ST. Paul, MN 55144-1000
SCTUODYMPLETIDOATNE September 14, 1993
15516-0-494
1 of 32
601293
2 WASHINGTON
QUALITY ASSURANCE STATEMENT
PROJECT TITLE: DGNeAnoStyonxtihceistiys Taensdt CoenllT-5P7r1o0l.i1feIrnatiVoinvo/iInnRaVtitrLoiveUrnscCheelldsuled
PROJECT NO.: 20991
HHA STUDY NO.: 15516-0-494
PROTOCOL NO.: 494, Modified for 3H Corporation
tQhuealiatbyoveAssruerfaenrceenceidnsppreocjteicotnsweorfethceondsutcutdeydanadccorredviinegw otof tthhee fSitnaanldarrdeport of
gOepneerraatlingreqPuriorceemdeunrtess ooff tthhee QaupaplriotpyriaAtsesurGaonocdeLUanbiotratanodryaPcrcaocrtdiicneg troegutlhaetions.
mFainnadginegmsentfraonmdthtoe tihnespsecttuidoynsdiraendctofrinaoln rtehpeorftollroewviinegw wdaetrees:reported to
jonD
Fini
itor
Perfusion/3-25-93 7D-ra6f,t7,r8e,p9o,r1t2-r9e3view/ F9i-n1a4l-9r3eport review/
3-25-93 7-15-93 9-14-93
K. Newland B. Mullett B. Mullett
15516-0494
uaf%ityad AMssuurlanlce! Un
atientReelseas
2
01294
WASHINGTON
COMPLIANCE AND CERTIFICATION STATEMENT The described study was conducted in compliance with the Good Laboratory Practice Regulations as set forth in the Code of Federal Regulations (21 CFR 58, 40 CFR 792, and 40 CFR 160) and the Organization of Economic Cooperation and Development Principles of Good Laboratory Practice C(81)30 (Final) Annex 2, issued 1979-1980 (effective 1981). To the best of the signers' knowledge, there were no significant deviations from the aforementioned regulations or the signed protocol that would affect the integrity of the study or the interpretation of the test results. The raw data have been reviewed by the Study Director, who certifies that the evaluation of the test article as presented herein represents an appropriate conclusion within the context of the study design and evaluation criteria. All raw data, documentation, records, protocols, specimens and final reports generated as a result of this study will be archived by Hazleton for a period of at least one year following submission of the final report to the sponsor. After the one year period, the sponsor may elect to have these materials retained int the storage facilities of Hazleton for an additional period of time or sent to a storage facility designated by the sponsor. SUBMITTED BY:
dee Z Hon
Andrea L. Ham, B. S. Supervisor
9/4/53
Date
Study Director:
Prona 7 Lone:
Maria A. Cifone,'Ph. D. Study Director 7 Division of Genetic and Cellular Toxicology
9-44-23
Study Completion Date
15516-0-494
6012395
WASHINGTON
TABLE OF CONTENTS
PAGE NUMBER
ABSTRACT . LL . tee
6
L SPONSOR . . vt titties 7
I. MATERIAL TESTED . oo vv ttt tie eae ee eee uu
7
A. Genetics Assay No.
B. Identification
C. Physical Description
D. Date Received
TIL TYPEOF ASSAYS oo vvvit tein. 7 IV. PROTOCOL NUMBER . . . . . o.oo... 7
v. LL
SE
7
AB.. SEtxupderyimIennittailatSitonartDatDeate
Cc. Experimental Termination Date
VI. AS.UPERVsItSuOdRyY DPiErReScOtNoNrEL . . . . . . uo www uuu uuu o
7
B. Scientist
[HN Study Supervisor
VIL OBJECTIVE oo ovt titties 7
VIIL DEFINITION . oo titi tiie tte e eee eee a
8
I MAA.TERIAILnSdic. at. or Ceiilsotitit iit... 8 CB.. MOsemdoitaicForPumUpDsS AasndsayLabel for Cell Proliferation Analysis
D. Control Articles
X. EXPERIMENT DESIGN (UDS ASSAY) . . . .. .............. 10 A. Dosing Procedure B. Dose Selection and Perfusion Time CDc.. UUDDSS AAnsaslayysis
15516-0-494
4
601296
g WASHINGTON
TABLE OF CONTENTS (CONTINUED)
XI. AE.XPERITMrEeNaTtmDeEnStIGNa:ndCEDLoLsePLReOvLeIlFsERATION ANALYSIS . . ........ 12
B. C.
TIimspsluaentaCtoilolnectoifonOsmaondticPrePpuamprsation
D. E.
AsIsmemsunsomheinsttoocfheCmeilclal PSrtoaliinfienrgation Rates
XII. ASSAY ACCEPTANCE AND EVALUATION CRITERIA: UDS . . . . ....... 13
XIII. ASSAY EVALUATION CRITERIA: CELL PROLIFERATION . . . ........ 15
XIV. INTERPRETATION OF UDS RESULTS . . . . ............... 15
XV. INTERPRETATION OF CELL PROLIFERATION RESULTS . .......... 17
XVI. CONCLUSIONS . . . o.oo tive iennnn.... 18
XVII REFERENCES . . . . ........................ 18
XIII. EXPERIDMATEATNATBLAESL. . . . . .. .............. 21
XIV. APPENDIX A: HISTORICAL CONTROLS . . . . . ............ 29
15516-0-494
5
C0129?
@HAZLETON
ABSTRACT The purpose of this study was to determine the hepatotoxicity and/or genotoxicity of the test material by measuring DNA repair as unscheduled DNA synthesis (UDS) and cell proliferation (CP) measured as S-phase induction in rat liver cells after in vivo treatment. The test material was administered mteoasruartsemeanttdoosfesbotohf UaDpSproanxdimacetlellypr81o0l,ife4r0a5t,io2n0.3, and 101 mg/kg for the The test material, T-5710.1 did not induce significant changes in UDS as measured by the nuclear labeling of rat hepatocytes. Primary hepatocyte cultures were prepared at two sacrifice times, approximately 2-3 hours and 15-16 hours after administration of a single oral dose. For each sacrifice time, three male rats were treated with test material suspended in corn oil. The hepatocyte cultures were incubated with 10 uCi/ml *HTdr for about 4 hours and autoradiography was performed the *HTdr-treated cultures. After autoradiography, three treatment groups from each sacrifice time were selected for analysis of nuclear labeling beginning with the highest dose where cellular morphology was adequate for analysis and proceeding to successively Tower doses. None of the criteria used to indicate UDS was approached by the treatments and no dose-related response was observed. The test material, T-5710.1 was therefore evaluated as inactive for the induction of UDS in rat hepatocytes after in vivo treatment and in vitro culture. In the cell proliferation assay, no evidence for an increase in cell proliferation was observed following treatment with the test material. Five animals per condition were labeled with BrdU for 72 hours using ALZET osmotic pumps. Samples of the liver and duodenum were fixed in 10% neutral Formalin and later embedded in paraffin. Samples were also processed for analysis by a pathologist. Sections from the left lateral, right median and anterior lobes of the livers as well as samples from the duodenum were taken and processed for immunohistochemistry. Each slide was prepared with sections from both liver and duodenum. The duodenum (a rapidly proliferating organ) was used as an internal control for delivery of label and immunohistochemical staining. The percentage of nuclei incorporating label in the liver was determined microscopically. Only hepatocyte nuclei were enumerated. No statistically significant increases in cell proliferation were observed. T-5710.1 was therefore considered negative for the induction of both UDS and cell proliferation in rat liver cells.
15516-0-494
6 0i238
2 WASHINGTON
In Vivo/In Vitro GUennsocthoexdiuclietdy DTNeAstSyonnthTe-s5i7s10a.n1d iCnell Proliferation in Rat Liver Cells
1 SPONSOR: 3M Corporation
11. MATERIAL TESTED:
A. Genetics Assay No.: 15516 B. Identification: T-5710.1 CD.. DPahtyesicRaelceiDveesdc:ripFteibornu:aryC2r4e,am19c9o3lored granular material
III. TYPE OF ASSAYS: DNInAVSiyvnot/hIensiVsitrAossayRawtitPhriTmwaoryTHiempeaptooicnyttseanUdnscCehlelduled Proliferation
IV. PROTOCOL NUMBER: 494, Modified for 3MCorporation v. STUDY DATES:
A. Study Initiation Date: February 24, 1993 B. Experimental Start Date: March 4, 1993 C. Experimental Termination Date: May 6, 1993
VI. SUPERVISORY PERSONNEL:
A. Study Director: Maria A. Cifone, Ph. D. BC.. SStcuidenytiSsutp:erviMsaorri:e MAcKnedorne,a LM.. HPahmi,l. B.S.
VII. OBJECTIVE:
The objective of this assay was to detect DNA damage and/or
hepatotoxicity caused by the test material by measuring DNA repair as
Seux-nipsshctahesenedcueliendadnudDcNtAidoengsryenienthoerfastiDsNlAi(vUeDdSra)macgeaelnldwseceraleflteirnpfrieornlrivefdievroaftriotomrneaant(mCePin)ntc.mreeaassTeuhreeidn
as
net
nuclear grain counts in hepatocytes obtained from treated animals when
compared to those from untreated animals. The types of DNA damage are
ruenssupletcifiinedthebutinmcuosrtporbeatiroencogonfiznaebwlebasbeysth(einccelluldiunlgar*Hr-etphayimridsiynset)eminatnod
DNA during a short (4 hours) in vitro culture period (1).
Cel] proliferation measured the fraction of cells undergoing cell replication in rat liver using an immunohistochemical technique (2,3) to
ATdnieivtmeearclstswbewrreoermeodigesioovxleayntuerdiadifsnoielnlgol(weBirnodgrUa)ladidmnoicsnoiersptoorfraatttiehoden tdoeufsrtiBnmrgadtUDeNrAfioarslyn72tahndehsoiutsrh.se in
15516-0-494
7
C01299
@ HAZLETON
WASHINGTON
yivo with an ALZET osmotic pump implanted subcutaneously. Quantification of cells that have incorporated DNA precursors over the 72-hour period measures cell proliferation in the liver (4). VIII. DEFINITION:
The UDS assay is designed to measure unscheduled DNA synthesis (UDS) in rat liver cells (hepatocytes) using the autoradiographic technique described by Williams, 1980 (5). Hepatocytes were isolated from the livers of rats exposed in vivo to the test article. Only a small percentage of the cells enter S-phase (replicative DNA synthesis) during the brief exposure period, so the incorporation of *HTdr into DNA during n vitro culturing, as analyzed by autoradiography, may be used as a measure of the repair of DNA damage caused by treatment with the test wairtthiclken.own ThmiustagUeDnSicmeaosrurceamrecnitnogoefniDcNAarcetipvaiitrieasppeoafrschetmoiccaolrsr.elate well
Hepatotoxicants such as carbon tetrachloride and dinitrotoluene induce
an increase in cell proliferation to replace necrotic tissue (3,6).
These proliferating cells may be detected during S-phase analysis.
Other chemicals may induce S-phase in the absence of hepatotoxicity. It
is not apparent how cell proliferation may act in thecarcinogenic
process but there are numerous mechanisms which can be affected during
replication (7-10). Chemically induced cell proliferation may increase
the probability of spontaneous mutations as well as increase the
probability of converting unrepaired DNA adducts into mutations.
Unscheduled cell proliferation may also play a role in the expansion of
preneoplastic populations leading to the emergence of a fully
transformed clone of cells. Some of these examples act by a non-
genotoxic mechanism and it is theoretically possible to detect
nongenotoxic
technique.
carcinogens
as well
as genotoxic
carcinogens
using
this
IX. MATERIALS:
A. Indicator Cells
Young adult male rats of the Sprague-Dawley strain, 10-12 weeks
old at the time of dosing, were purchased from Charles River
Laboratories, Inc. (Cr1:CDBR). This healthy random bred strain
was selected to maximize genetic heterogeneity and assure access
to a common source. Animals scheduled for this study were housed
according to standard operating procedures and were fed Purina
Certified Rodent Chow (Formula 5002)
Animals were quarantined a minimum of
and water ad
7 days prior
libitum.
to random
assignment to study groups and identification by tail tattoo.
Animals were anesthetized prior to surgery for preparation of cell
cultures, using about 60 mg/kg sodium pentobarbital, and were
exsanguinated during the harvest procedure.
15516-0-494
8 C01300
? WASHINGTON
T3h2e4.5l-i4v3e9r.1ceglrlasmsforforthetheUDSeaarlsysaytiwmeeproeinotbtaaindned343f.r2o-m43r7a.t2s gwreaimgshing tfhoer Ttihveerlsatienr stiitmyepowiintth. a Tchoellacgelelnsasewerseoluotbitoanine(dseebySpecetrifounsioXn.C.of UcaDunSldtuAwrseesrsaey)wu.esreedMmotanhioenltasayaiemnreeddcauylastufrmoeorsnoalwnaeayrleeyrssiesstataobflabitoshuehtedUD3S7inCacctuiinlvtiuatrye. disAlhles humidified atmosphere containing approximately 5% CO. uFsoirngtheMetcoelflaneprol(imfeetrhaotxiyofnluraasnsea,y, Ptihtemana-nHiomoarles, weIrnec.)aneisnthhaeltaitzieodn wasenurebesctuhatneaesnsietaohuesatlniydzeo(dndeorwsiAatLlhZETCsOu,rfpaauncmdep)e.pxesraSneagvnueiimnnatalyt-etwdwaosprahiosouerrpsttioclaartleelmryo,vianalsneiormftaelds t1h0e% NTeiuvterrasl anBduffdeuroeddenuFmorm(acloinnt.rol organ). The organs were fixed in B. Media For UDS Assay mTheentecedllwitchult1u0r%esfetwaelreboevsitnaeblisserhuemd, i2n mWdillLi-agmlsu'tamMiendei,um10E0suugp/pmllewsittrheoputtomysceirnunsuilsfarteef,erraendd t1o50ausg/WmMiEI.genAtfatmiecrinthe(WeMEs+t)a.blisWhHmEesnt p47eriCoid/,mMoclueltu(rWeMsE-twreeraet)r.efed with WME containing 10 uCi/ml HTdr, C. Osmotic Pumps and Label for Cell Proliferation Analysis wAeLrZeETuseods.motiAcsipnugmlpes T(oAtLZA(0C4o22r0p5o)ratwiaosn,usePdalethArlosuog,houCtA),thMeodsetludy2.ML1 TThhee ppuummppshwaesrea p2r0e00-f1illecdapwaictihtyBrwdiUthata apumcponcreantteraotfio1n0 uo1f/hour. 20 ng/ml. D. Control Articles 1. Vehicle control
Aandvehfiicvleeractosntrfoolr cceolnlsisptrionlgifeorfattihorneewerraetstrfeoartetdhebyUDSoraalssay 2g8a2v5a1g7e:5w9i)thwatshepveerhfiocrlmee,d catornalloiltim(e1p0oimnlt/sk.g, DVuekhei,cle control mhaenpiaptuolcayttieosnsorusteidssufeosr twheorseesduebrjievcetdedftroomaitlreaotfedtheanimals. D10osmiln/gkgv.olume of the vehicle control animals did not exceed
15516-0-494
9
01301
WASHINGTON
2. Positive control article
TSFh-oeprhaptshoeesiUtiDniSvertaitcmoenhpteropoialnttoscc,yotmeDpsiomuienntdhvyiiIsvnoik.tnroowsTnahemtionpeoisnid(tDuWicNve,e UCDcASoSntorrol
62-75-9, Sigma Chemical Co., Lot# 29F0679) was dosed at
1105-m16g/khgourfsortitmheepo2i-n3t.hourFsortciemlelpoipnrtoliafnedra1t5iomng,/kg15 fomrg/ktgheof DainMndNtrwafapisevreiutsroeantdesaalsfortihncejeelcpltoisoipntriovfleoirfecoernaatctrhioolnt.imweeprToehirnteter.eartaetds bfyor UDS
E. Test Article
10 mi/kg. For the preparation of the dosing solutions, the test article was
suspended in corn oil at a concentration of 81.1 mg/ml and serial dilutions made prior to dosing for each timepoint and used fresh. The maximum dosing volumes for the test article did notexceed
X. EXPERIMENT DESIGN (UDS ASSAY):
A. Dosing Procedure
For each tinepoint, three rats were treated by oral gavage with
the test article. Delivery volumes were calculated on the basis of the most recent animal weight and the target dose. The maximum volume of the test article solutions administered did not exceed
c1o0nmtlr/oklgs. werFeresuhsedprfeopraraatniyontsesotifngtespturpaorstei.cleCoinnfivremhaitciloen anodf the canodncdeonstirnagtioonf tohfetahessatyeswtasmatneortiadleteurnmdienredconinditcioonnjsuncotfipornepwairtahtion
this study.
B. Dose Selection and Perfusion Time
bThaesedhigohnesLdt,,doisnefoorfma8t1i0onmgs/ukpgplwiaesd sbeyletchteedSpfoonrsorb.othTthirmeeepoints
additional doses of test material were prepared using 2-fold
dilution steps and a minimum of 3 animals per dose. Two
taifmteeproitnhtes awdemrienisetmrpaltoiyoend oral gavage.
offora sascirnigflieced;ose2-3ofhtohuersteasntd
a15r-t1i6clehobuyrs
c.
15516-0-494
UDS Assay
This assay was based on the procedures in rats described by
Williams (1980), Mirsalis, Tyson and Butterworth (1982) and
pBeurtftuesriwoonrthofetlivale.rs (i1n98s7)i.ty Tfhoer habeopuattocfyotuersmwienurteesobwtiatihnedHabnyks'
10
C01302
2 WASHINGTON
bbbuiasflfaen(rcse-daatmisnpaoHletts7h.y2(].CaeTMTthh-eenrM)g-W"NM-,EfIrNe-ceto)enttcraoainantciaenitgniic5n0g-a1c00i.0d5 u(mnEMiGtTesAt/)hm,lylaeonnfdegHlEyPcEoSl-
omcifonlueltxaecgsie.sneadseThTei(vWehHrEeCp)taitsowscausyetepseinrwfeuarseecdulotbtuthrareionuedgdihshbtyhcemoenlctihavaiennriicnfagolrWHd1Ei0Cs.p-er1sT1ihoen
csluusmppesndeadndtdiesbsruies.andThceellcsellwerseuspaelnlsoiwoend cell pellet resuspended in WME+. After
wtaosscetetnlteriftuogerdemoanvde tcehlel obtaining a viable cell
ccoounntta,iniangseraie2s5-momf r3o5u-nmdm, cpullatsutriecdicsohveesrsl(iapt animal to assess attachment efficiency) was
alnedastat 6 lepaesrt an2impaelr inoculated for each
paneirmadlishw.ith Ianpdpivriodxuiamlatecluyltu0r.e5s xwe1r0e tattoo: number.
viidaebnlteifcieeldlswitinh
3thmel
aonfimWalES
Achneulmliadtictfuailcethdumreenasttmpofesorrpihoetrdheeoecfaorn1lt.ia8ei-rn2.i1ntgimhe5op%uorisCnOt,a.twaasbFoourustetdh3e7tolCaetistntearablish the
tthiemepaotitnatc,hmeannt aptetraicohdm,entunaptetraicohdedofce1i.l7s-2w.0erehourresmowvaeds cultures were refed with 2.5 ml WHE-treat. Three of
uasnedd.theAfter the replicate
wrceeurpleltiucrkaeetspetsfrfoowremreaenauacshleydsainsitmoailnaswsteehesrseevauetsntetadchofmfoernttet.chheniAUcnDaySl arspesrmaoaybi;lneimntsgwowciuotlfhtutrhees
acuulttourraedsiogfrraopmhye.achAtatniamcahlmenbty trypan blue dye exclusion to
ienffsiictiuenmciycrwoasscodpeitcermainnaleydsifso,r
two using
determine the viability of the
attached cultures.
twAheferteerirnecafuebldaatboweriltihnfgoWrMpEe1Ir7i.o8cd-o1n8to.af9inaihbnooguurts0.42(52h-om3urMshotulhraysbmeildteiidnmeepcoeailnnldt)crueoltrtuurrn1ee8sd.3-to a1d8d.i8tihoonursof(11%5-1s6odihuomurscittriamteepoitnot)t.he Ncuocvleerisliwpesre(tchoenntaisnwionlglentheby tcehlrleemocnhoalnagyeesrso)f faocreti7c-10acmiidn:uettehsa.nol Th(e1:3c)ellasndwedrreiednexftorfaitxedleaisnt d2i4ppheodursi.n KoTdheakfNiTxBe2d cemouvlesrisolni,psanwderderimeodu.ntedTheonemgullasssionslicdoeast,ed dselviedelsopewdereinsDt1o9r,edfifxoerd,8 daanyds'sattain4edC.witThheheemmualtsoxiyolnisnwearnde etohseinn. D. UDS Analysis
mtTahhegenivcfieidlcelaostiswocenrreeeunenxdaeomrfinaoienldaumitimocmmreaortssiiccoonpicocauanndltleytrh.eatfiUaeDpSlpdrwoawxsaismmaetdaeislsuyprlea1dy5e0db0xyon counting nuclear grains and subtracting the average number of
15516-0-494
1
C01303
WASHINGTON
grains in three nuclear-sized areas adjacent to each nucleus
(cytoplasmic count). This value is referred to as the net nuclear grain count. The coverslips were coded to prevent bias in grain counting.
wTsuehnerelleeescnstseectodortnehcudeec,rlllwesiaasrnedongraianneiyadncihcoacctcoceauodsnv.iteornswalaOlisnplyndue(cnttluehecrrilemeeiinbecldwoaicvtkefheronsrelndiofrpimsfbaytlypegrmrraoairannpndshioommlaltoloy)ggies numerous to'count were excluded as cells in which replicative DNA
synthesis occurred rather than repair synthesis. The average mean
ntaehvteerantgrueicdplleiafcroartgereaacichnovectrroseulanittpmse(n+t(s15ct0oannddtioattriadolnd.neuvicalteiio)n)fowraseacdhetearnimmianled anfdrom
XI. EXPERIMENT DESIGN: CELL PROLIFERATIONANALYSIS
A. Treatment and Dose Levels
AaaTnn1iamlayalznseiafclerslolnweeprarceohlidfdoeosrseaedtialosenvedleastcarn7id2b-ehcodounrtisrnolatfhetgerroUuDpSa
wseercteiouns.ed tFoive single oral dose.
B. Implantation of Osmotic Pumps
A2aL0nZ0eE0sTt4h1etMioozdfeeldBrud2UsMiL1nagt oMasemtcootofinaccneenpturmaapstccioo(rnLdoiotnf#g2004to22m0gs5/t)maln.dwaerrdeThepprroeaclneoidamudareledsswweiartnehd o(ndeorspaulmp suprefracaen)i.mal Twhaes iancsiespitoincalwalsy cilnosseerdtedwistuhbcwuotuanndeoculsilpsy and ttihhmeeprleaanntiwemedarlestnhomreoencilitdnaoiyrcseadlpruinstoiigrlnstotohfesactirinimffeeicctoeif.ons.acrTihfeiceosmtootiecnsupruempsthwaetre
C. Tissue Collection and Preparation
Each aninal was anesthetized prior to removal of organs for
aanndalyfsiixse.d duodenum,
ainTthiensesuttuhreoarlwaictibhcuffhceiagrvheidtyceflwolarsmatluoirpnne.onveedrA,acnrwdoasstshealsselocitvierorenmroevomefodvefdrom
ethaacth tahneimaTlabealndwafsixeadd.miniThseterdeudodeconrurmecwtalsy itnocleuadcehd aasnimaanl.indFiocratotrhe
llaitveerr,al, urigphatrramfeidnianembaenddderdighstectainotnesriowrerelobteask.en Sfirmomilatrhley left
prepared sections of the duodenum were also made and a section of the duodenum was mounted on each slide. Slides were also prepared
atoccdoertdienrgminteo siftanadnayrdabnporromcaeldiutrieess fowrereexparmeisneantti.on by a pathologist
15516-0-494
12
01304
WASHINGTON
D. Immunohistochemical Staining
hematoxylin counterstain. Parallel slides were stained with The slides were deparrafinized and rehydrated prior tostaining
using the BIOGENIX Supersensitive Kit using DAB stain and hematoxylin and eosin for analysis by a pathologist.
E.
Assessment of Cell Proliferation Rates
Tthheat setchteioTnabeolf Tabel delivery
wtahse was
dcporunoofpdieerrnmlueymd,waadssmliinmdiiescstreorsfercdoompitotchaeltlheydifeafnxieamrmaeilnn.tedlOotnboecseenwseurree
eToxbaemsinetdherfeofrorleobcuellalr dciofufnetrienngcewsa.s peLrafboerlmiendg wwiatsh ssiemcitliaornsanofnrgomthtehe
left lateral lobe. The percentage of nuclei incorporating label
in the Tiver was determined microscopically at approximately 400
gteone6r0a0tXedmagbnyifciocmaptuitoenr.. AThe1.a0rmemasstquoarbee cionudnetxeedd woceurlearragnrdiodmly
divided into 10 x 10 squares was used to define the counting area. At Teast 2000 nuclei were examined per animal with a minimum of 3
sections and 8 fields per section.
Any nuclei that were blue were considered unlabeled and any nuclei containing any brown chromogenic hue were considered labeled
unless a clear artifact was present. Only hepatocyte nuclei were enumerated. Fields that contained areas of necrosis were not included in the evaluation. The slides were coded for (blind) evaluation as to treatmentgroup.
S-phase nuclei labeling indices for each animal were calculated as
follows:
Labeled S-phase nuclei (LI)=nh o.oeflp abea ledtocynt ucle ei X 100
total no. of hepatocytes counted
XII. ASSAY ACCEPTANCE AND EVALUATION CRITERIA: ups
Astnahteiasstfseiaseytd.rneosrTumhlaitlsslyloiwnislltylinigbfemalaclyonnosofitdetrehenedcocmrapicatcseesrpitaaalbllleisttefesodtr
beevlaolwuataireon of situations, so
the study director must exercise scientific judgnent in modifying
trheeliacbriiltietryiaanodr cacocnespitdaenrcien.g other causes that might affect assay
1. 15516-0-494
The viability of the hepatocytes collected from theperfusion process normally exceeds 70%. A variety of factors can affect cell yield and viability, so values below 70% are not uncommon nor necessarily detrimental. Toxicity of treatment with test article may be reflected inperfusion viability, therefore no lower limit will be set.
13
C01305
? WASHINGTON
15516-0-494
2. Tahsesayvitarbeialtimteyntosf mtuhsetmboenol7a0%yerorceglrleatceurl.tureNsormuasleldy,fotrhethe viability of attached cells is about 85%.
3. Tphoepulpaotsiiotniveemcpolnotyreodlwaiss ruseesdpontsoivdeemoanndstrtahteemetthhaotdotlheogycelwlas caaduesqiunagtewefaokr torhendoetUeDcStiaoctnivoiftyU,DS.theFoarvertaegset mreastpeornisaelsto the tpoosiitnidviecatceontUrDoSl. trFeoartmteensttsmmatuesrtiaelxsceecdleabroltyh ccaruistienrgiaaudsoesdearbesleantceed oUfDSaacptoisviittiyv,e canontarsoslaylwoisltl fobre ateccchenpitcaabllereiansotnhse.
4. oGfraitnheceouvnatluadtaitoanobitfaoibnteadinpeedr fanriommaltwoisreapclciecpattaebleculasturpeasrt baendaavtaillaebalset 5f0ronnuc2leoif ptheer c3ulatnuirmea.ls Gtrreaaitnedc.ount data should
5. Atimmeipnoiimnutm. ofR3epedaotsetrTieavleslsnewieldl onbleyaanuaglmyeznetd tahteenacuhmber of oafna3lyzdeodsedloesveellseveblust miunsttheincfliurdset attrialleastto oancehideovseea total previously assayed as acceptable.
Sbeavseirsalforcrietvearliuaatihoanveofbeeanteessttambaltiesrhieadl whasicha,ctiivfemeitn, thperoUvDiSde a asstsaatyi.sticTahle canrailtyesriisa foofrthaephoissittoirviecarlesdpaotnaseanadrecbaalsceudlatoinona of the dmeisncirmiubmedinbcyreCaasesciraenqouiraendd fGoayrlaorsi(g11n)i.ficant UDS response as
tThhaettceasutsem:aterial is considered active in the UDS assay at doses
1. fAniveingcrraeiansse pienrtnhuecmleeuasn anbeotvenuctlheearcognrcauirnrenctounntegattoivaet least control average, leading to a positive number and/or
2. Tihnecrepaesrecenatt olfeasntucl1e0i% waibtohveftihvee aorvermaogree oneftthgeraicnosncutrorent negative control animals.
Galesnoerablelym,et.if Htohweevfeirr,stscaotnisdfiatcitoinonisofsatoinslfyieodn,e ctohnedisteicoonndcawnillalso
vianrdiiectayteofUDSnucalcetiavritlya.beliDnigffepraettnetrnDsN,A-daandmagwienagk
aaggeennttss
can may
give a strongly
caofnfdecittioonnslyaraemcionnosriidteyreodf tihneancelelvsa.'luatTihoenr.eforIen,cabsoetsh owhfertehe above
wiinlclreabesesconasreidenroetdobascetrivveedfoirn talhlatthcornedeitainoinmalisf, cetlhlestefsrtommattweorioafl
1
G01306
@ HAZLETON
WASHINGTON
the three animals show increases. show an average Tess than -5.00 or
If the negative control animals more than 1.00 grains per
nucleus, the assay will normally be considered invalid.
The the
test material is above conditions
considered are met in
inactive in this assay if none any of the treated animals.
of
When results the presence
are neither clearly of a dose response,
positive nor clearly negative, the frequency distribution of
cellular responses, and is considered; the test
the reproducibility of data article is then classified
among
slides
"weak positive" or "equivocalTM.
as "negative", A group in which one ofthree
animals case by
shows increases in nuclear Tabeling will be decided on case basis depending on the level of activity in cells
a
from the active animal, the level of activity in cells from the inactive animals and the presence or absence of activity in
surrounding groups.
The positive control nuclear labeling: is not used as areference
point to estimate mutagenic or carcinogenic the UDS activity of the test material. UDS
risk associatedwith elicited by test
agents in this assay is probably more dependent on the type of DNA damage inflicted and the available repair mechanisms than on the
potency of the test agent as a mutagen or carcinogen. Some forms of DNA damage are repaired without the incorporation of new
nucleic acids. Thus, the positive controls are used to demon-
strate that methodology
the was
cell population employed was responsive adequate for the detection of UDS.
and
the
XITI. ASSAY EVALUATION CRITERIA: CELLPROLIFERATION
XIV.
A mean and standard deviation for the percentage terminal body weights and terminal liver weights
of S-phase cells, were calculated for
each treatment Jou Statistical analysis
using the individual animal was performed using one-way
mean values. analysis of variance
techniques (12). Control versus treatment group comparisons were done
with rank
Dunnet's t-test transformations
(13, 14). In the case of variance heterogeneity, of the data were performed prior to analysis of
variance and Dunnet's t-test. An S-phase, terminal body weight or terminal liver weight percentage in a dose group that deviates from the
percentage in the concurrent control group at a significance level of
Pps0.05 was considered significantly different than the control group.
INTERPRETATION OF UDS RESULTS:
At the request of the Sponsor, the test material, T-5710.1 was suspended
ainppecaorrendoitlo aftorma acocnrceeanmtractoiloonredo,f 8o1p.a1quemg/vmils.cousThesutsepsetnsimoanteriinalthe
vehicle. Individual dosing stocks of 81.1, 40.6, 20.3 and 10.1 mg/ml
15516-0-494
15
01307
@ HAZLETON
WASHINGTON
were prepared just prior to dosing. Three rats per dose level were
treated with 810, 405, 203, 16 hours groups, in volumes
and 101 mg/kg which did not
for both the 2-3 exceed 10 ml/kg.
hours
and
15-
For the early timepoint, perfusions were initiated 2.1-2.6 administration of a single dose of the test material. The
hours after hepatocytes
bcloulelecetxecdlusfioorn)thefrUoDmS7a8s.s2a%y-98r.a6n%gedof itnhevitaobtiallitycel(ldsetceorlmliencetdedby intrythpean
perfusate (Table 1). and the viability of
The attachment efficiency varied the attached cells was very good,
from 61.5%-104.1% ranging from
88.9%-97.5%.
The minimum criteria for a UDS response at this timepoint were
determined by comparison to the averages of the control treatments (Table 2). The average mean
concurrent negative net nuclear grain count
for the vehicle control animals was -0.32 cells containing five or more net nuclear
and the average percent of grains was 3.89%. Apositive
response consisted 4.68 (5 net grains
of average mean net nuclear above the control value) or
grain counts exceeding at Teast 13.89% of the
nuclei containing five or more grains (10% above control value). "None of the treatments with the
the average negative test material samples
caused nuclear labeling control. Furthermore,
significantly no dose-related
different trend was
from thenegative evident. Incontrast,
the DMN treatments induced large increases in nuclear labeling that greatly exceeded both criteria used to indicate UDS. Since the positive
control animals were responsive, the test results were considered to provide conclusive evidence for the lack of UDS induction by the test
material samples at the 2-3 hours timepoint.
Heavily-labeled nuclei (blackened with numerous undergoing DNA replication (S-phase) as opposed
grains) represent cells to DNA repair. For each
slide phase
canaallcyuzleadt,ed atandlearsetpor5t0e0d ceflolrs ewaechreansicmaanlnedon antdhe tbhaesiisncoifdenatce loefastS-
s1t5u0d0y cwealsls loowbsearndveddid(Tnaobtlein2)t.erfeTrhee wniutmhbertheparsesseanyt. in the 2-3 hours
aFodrmintihsetrlaattieorntoimfepaoisnitn,glepedrofsuesioonfs twheeretesitnimtaitaetreidal1.5.2T-h1e5.h6ephaotuorcsytaefster
collected for the UDS assay ranged in viability (determined by trypan
blue exclusion) from perfusate (Table 3).
79.5%-92.2% of the total cells collected in the The attachment efficiency varied from 60.9%-91.1%
8a3n.d5%t-h9e7.v9i%a.bility of the attached cells was very good, ranging from
cThaelcumliantiemdumbacsreidteruipaon ftohre aavUDeSragreesopofnsteheatnegtahteivleatceornttriomlepaoniinmtalwsas for
the 15-16 hours timepoint -0.54, average percent of
(Table 4; average mean net nuclear grains cells containing five or more net nuclear
=
grains = 2.50%). exceeding 4.43 or
The criteria were at least 12.5% of
mean net nuclear grain counts the nuclei containing five or
more
15516-0-494
16
.01308
@ HAZLETON WASHINGTON
grains. nuclear
None of labeling
the treatments with the significantly different
test from
material samples caused the negative control and
no dose-related trend was evident. induced large increases in nuclear
The positive control treatments Tabeling exceeding both criteria
used
to indicate UDS. Since results were considered
the positive control animals responded, the to provide conclusive evidence for the lack
test of
UDS induction by the test material samples at the 15-16 hours timepoint.
Huenadveirlgyo-ilnagbeDlNeAd rneupcllieciati(obnlacaskenoepdpowseidthtonuDmeNrAoursepagirra.ins)Therenpurmebseenrt cells
observed for each animal in the interfere with the detection of
15-16 UDS.
hours
study
was
Tow
and
did
not
XV. INTERPRETATION OF CELL PROLIFERATION RESULTS: A. General Observations
Cells stained with the brown DAB chromogen were observed in the
dunusocdheenduumlefdromdeaatllhs ofocctuhrereadn.imalTshe usperdeseinncethoefstluadbye.l
No in all
the
animals indicated proper delivery of the BrdU label andacceptable
immunohistochemical staining. control) was eliminated from
One animal from Group the evaluation because
1 of
(vehicle extremely
high time
labeling than the
indices. This animal other animals and may
was treated at adifferent have been dosed improperly.
One animal from Group 6 (positive control) was also eliminated
from the study because the results demonstrated a lack ofresponse
by the animal. Since DMN is known to induce large cell proliferation (15), this may have represented
increases in a dosing error.
The liver different
weights of the dosed from control animals
animals were not significantly and no obvious changes were
observed at autopsy. The mean liver weight of the positive
control was slightly elevated (p < 0.05) the large increase in DNA synthesis (and
as. expected considering subsequent cell
proliferation) that was occurring. 5) had a mean terminal body weight
The that
high dose animals (Group was less thancontrol
value (p < 0.05) indicating some toxicity.
Slides from treated and control animals gross findings at the time of sacrifice
were were
also examined and generally sporadic
and/or incidental with no apparent relationship to treatment.
15516-0494
17
0L1309
? WASHINGTON
B. Summary of Labeled Cell Counts for the Liver
ATlaabbsleuelmimna5.gryionIfdnedxitvhie(dLuIla)albfeolarneidmeaaclcelhlcgoruconoutupsntsiasrefporsrehsoeewannctheidgnroiTnuapbtlheies6.tshhiorTwdnhe imnean ncTeooclbruemosns.iasndwTathsheereoblswaeabreslvendwo.asapTprhaaernednontmecrwpoirtteihfcienreantrtheieaasllwoebleraseb,elnieonxtgceedpitbnutwahneynroetof the included in the count.
Tsihynendtihmceeasatienss bdtauhcraktignrgoTuentsdhsetlh7aab2ne-lhio1nu%grofilnatbdehelxeinn(guGcrlpoeeuirpioh1d)a.d wausNnode0r.s6gi1ognneiwfhiiDccNahAnt ainndcrealalsesmeawnerelaobeblsienrgvedindiincetshewedroesedbelgorwoup1s% w(hGircohupsis2etxhpreocutgehd 5f)or c1o2n-twreoelk oanlidmamlaslewarsats3.1.51Thewhmicehanislabseilginnigficianndtelxyofeletvhaetepdositive (p 50.01).
dTiohnsecesreeaastreesscuolnitncsenttdhreeamtoLiInosntisrnattfehreomthla1it0v1eTrm-g5i7/n1k0gm.a1lteod8ri1ad0tsnmogta/fktigen.rducaeLasrsigniegglneifiorcaalnt cionnctrreoalsesaniimnaltsh.e lTahbeelimneganinldaebxeliwnegreionbdsexervoefdthien tphoesitpiovseiticovnetrol animals was 31.51.
XVI. CONCLUSIONS
Tnhuecletaerstlmaabteelriinagl,ofTr-a5t710p.r1imadriyd hnoetpationcdyutceessaitgniefiitchaenrtthcehan2g-3eshoiunrsthe
mtgi/mkegpowienrteodreltihever1e5d-16byhooruarls as inactive in the induction
gtaivmaegpeo.int of UDS in
~wTh-e5n71d0o.1seswasoft8h1e0r,efo4r0e5 rat liver cells.
eanvdalu2a0t3ed
TSh-ephatseestcmealtlesrifaollldoiwdingnota were Tabeled for 72 hours
siinndgulcee osriaglnidfoisceantofchT-a5n7g1e0s.1i.n following dosing and all mean
tThheenaunmibmearlsof labeling indices
opifonsaicttthiievveetrcienoanttetrdhoelgrioanundpiusmcatwlisoenrewaosfbe3Dl1No.Aw51s.1"%y.nthTe-Ts5hi7es10m.ei1annwraalstabetTlihivenergrefociernledlesxe.vaolfuattheed as
15516-0-494
18
01310
? WASHINGTON
XVII. REFERENCES
1. rgMieerpnsaoaitlroixsia,cssacJya..rcc.i,nEonTgvyeisnroson'n,.inCM.uKt.th,aegeiannnedsviiBsv.,Eo.4-:B5iu5nt3t-ve5ri6wt2or,roth1h:9e8p2a.tDoectyetcetioDnNAof
2.
Defazio, (1987). vivo. J.
AI.m,unLoehairsyt,ocJh.eAn.i,caHledldeeyt,ectDi.oW.n oafndprToaltitfeersraaltli,ngM.cHe.iNl.s Histochem. Cytochem. 35, 571-577.
in
3. p5Lra-onbilreiorfn,eordaeTt.oiLx.oy,nurisBdteiurdgnieeers,a.ndET.oK-.xt,ihcyoamlniodgdiEisantceho9f,,n 6Pr4..oId.en(t19h8e9p)a.toCcoemlplaurliasron of
4.
fMBoiurrtstaetlrhiweso,ritnhJ,.v,ivBo.PEr.or,basttA,shhebp6y.a,,tocJay.n,tde B6.DeNrAmW-uidlrelezip,aamisrE:.,assACaaysp.crioatnoMocu,otlatDi.a,onnd
guide Res.
189:123-133, 1987.
S. MDuWNitAlalgireaenmpssa,,iVrGo.alMn..d:6m.utTahgeF.endeDesetiescSteriironenslioavfnedrchcAe.umlitcHuaorllelsam,untdaIengr:e,nsC-(hcEeadsmr.ic)ci,anlogPelnesnunby Press, NY, pp. 61-79, 1980.
6. dDMiiNnrAistaslryionstt,ohleusJe.inCse..inanCdraartBcuihtnteoepgraewtnooercstyihts,e,sB3.f:Eo2.l:4l1o-w2iI4nn5gd,uicnt1i98ov2ni.vooftruenastcmheendtulewdith 7. DRMNaeArlsamtsaiynon,nthsehDsi.iSps.,otfoCahtettphlaeetyih,cepapR.teCor.co,axirscCooinmnweoagyep,nrioclJii.Gtf.ye,raotfainodtnhePoapnppde,rorxJei.pAsl.oimcea(t1i3v8e8).
CXpayrn1ociledirifneoRr)eas-t.2o-rps4y8,rdiim6(i72d3-i9en-ty6hl7y4tl4hh.ieox]yl)apcehttihcalaactied an(dWy-[144-,c6h4l3o)ro-in6-(r2at,s3.-
8. WcCaarrnawcdiedcrok,c,k,IGn:.VP..M",.liEv(le1sr9e7v6Ci)ee.lrl, CCeaNlnolcretrhp"r,foiloiClaflmeaernrdaotniB,oionmHe.adMni.dc,aelLxipnPesrreeilsmlse,,ntaCl.A.liavnedr Ansterdan.
9.
CRoelquumibraenmoe,ntA.o,f carcinogenesis
cRealjlalapkrsomlii,ferS.a,tioanndfSoarrmtah,e Di.nSi.tRi.ati(o1n981o)f. as assayed by three different procedures.
liver Cancer
Res. 41, 2079-2083.
10. doGilfaimnloiisnv,oebreAn.rzDee.gn,een.eBruJat.cthieoErxn,p.onNM.eLtd.u.moR9r.,33f,oarn3md1a3tA-iu3bo2,n4.Ji.nC.rat(s195f1e)d. 4-The effect
15516-0-494
19
C0131
2 WASHINGTON
11. DCeNavAsacliuraaentpoia,nigrD.acAsh.seanyia.cnadlsMDu.Wta.astiGpooansyiltoRiersvee(a1ro9cr8h3,)n.e1g2a2tSitv8a1et-i8si5tn.ictahle cherpiatteorciyatefsor
12. DMeisniearn,, BM.Jc.Gra(w1-9H7i1l)l.,StNeawtiYsotrik,calznPdriEndciitpiloens,ipnp.Exp14e9r-i2m2e0ntal
13.
Dcuonmnpeatrti,ng C.sWe.ver(a1l955t)r.eatmAenmtusltiwiptlhe 50, 1086-1121.
caocmopnatrrioslo.n
Jp.roAcme.durSteatf.orAssoc.
14. Dcuonnntertotl,. CB.iWo.met(r1i9c6s4).20, Ne48w2-t4a9b1l.es for multiple comparisons with a
15. Hsaantmdu,dyMoA.lleicvauenldrarCeifMffuoetnceat,gsenMie.nsAd.iusce(d1179(9b11y)9.)a, siU1n6s.geleofdocselel opfroDlMNi.ferEantviiornonmteontal
15516-0-494
20
01312
@ HAZLETON WASHINGTON
XIII. EXPERIMENTAL DATA TABLES
15516-0-494
'
21
01312
WASHINGTON
TABLE 1 SUMMARY OF CULTURE DATA FROM IN VIVO/IN VITRO RAT HEPATOCYTE UDS ASSAY
2-4 HOUR TIMEPOINT
Client: 3M Corporation
Client Code: T-5710.1
-_--
HUA Assay No.: 15516-0-494 Trial Initiation Date: 23-Mar-93
TCeosntdition ANnuimmbaelr DToasregetLevel(1) ---
VPiearbfiulsiitoyn AEtftfaiccihemnecnyt(2)AtVtiaabcihlmietnyt(2)
Vehicle Control - Corn 011 (ml/kg)
34449
10.0
34448
10.0
34447
10.0
Positive Cont3r4o4l43 1 - D1MN0.(m0g/kg)
34442 34441
10.0 10.0
Test Material - (mg/kg)
334444337 881100 34435. 810
34425 34424 34423
405 405 405
34413 34412
34411
203 203 203
3401 101
34400
101
-_--
34399
101
78.2 82.8 86.1
87.6
77.2 93.3
8835..32 8.3
79.4 87.2 90.9
91.3 85.7 98.6
98.0
83.5 87.9
92.3 83.9 67.6
72.5
76.5 78.1
8849..67 743
75.1 75.9 79.6
86.0 62.8
63.3
70.2
61.5 104.1
93.5 94.5 90.4
92.3
92.6 93.0
9952..29 92.8
92.0 88.9 93.1
95.2 95.5 95.3
97.5
2.3 94.6
N(o1)tesT:hree animals per dose level were treated.
(2) Results based on viable counts (trypan blue dye exclusion) of randomly selected areas on two plates.
DMN = DimethylInitrosamine
15516-0-494
:
22
C013ia
@ HAZLETON
WASHINGTON
TABLE 2
SUMMARY OF UDS DATA FROM IN VIVO/IN VITRO RAT HEPATOCYTE UDSASSAY 2-4 HOUR TIMEPOINT
CT lCtentCCToe:-- T510t .1 saaTetau) inis tiatioi nDatee : o3ts arss Client: 3M Corporation
Client Code: T-5710.1
HWA Assay No.: 15516-0-494 Trial Initiation Date: 23-Mar-93
C --_ W mew Test Animal
Cond. Number
Target Dose *
Mean Net Nuclear % Cells
Grains He) w/ 25
+ SI
NNG
Mean Percent Cyto. ~~ S-Phase Grains* Cells
Vehicle Control 34449 34448 34447
- Corn 0i1 10.0 10.0 10.0
(ml/kg) -1.17 + 0.48 0.47 + 0.51 -0.27 + 0.24
2.00 6.67 3.00
6.00 4.61 4.63
0.20 0.47 0.30
Positive Control 1 - DMN (mg/kg)
34443
10.0
16.10 + 0.76
34442
10.0
15.66 + 4.30
34441
10.0
13.13 1.77
96.00 95.33 92.67
8.95 6.41
Nn
0.07 0.87 0.13
Test
Material 34437 34436 34435
34425 34424" 34423
34413 34412* 34411
(mg/kg) 810 810 810
405 405 405
203 203
203
0.17 + 0.41 -0.03 + 0.70 0.24 + 0.00 -0.07 + 0.64 -0.51 + 1.37 -0.89 + 0.81 -0.23 + 2.11 0.62 + 0.03 -0.52 + 0.87
2.00 6.67 4.00
4.00 4.00 2.00
7.00 9.00
6.67
4.86 5.68 4.89
4.39 4.04 6.58 1.72 5.07
7.15
0.13 0.13 0.20
0.40 0.50
0.30 0.50 0.10
0.13
oT a TR --F-- uERiRmEiSL EtTEeTeohsnaeS Slt SrneLinStGy-- SSyceeIootE2c0cCoStrctTotiLsAcaIt(Tt1)6)wvroTtrcytAmr,sSiacinote {Nerv of Sygate grain coms an triplicate covrstpe C150 tat etl.
15516-0-494
23
01315
@ HWAASZHLIENGTTOONN
TABLE 3
SUMMARY OF CULTURE DATA FROM IN VIVO/IN VITRO RAT HEPATOCYTE UDS ASSAY 15-16 HOUR TIMEPOINT
Client: 3M Corporation
Client Code:T9701 Client Code: T-5710.1
HWA Assay No.: 15516-0-494
TrTiriaall InIniittiiaattiioonneDaatetee:: o255-mMaarr--9s3s
JT Test Condition
Animal Number
eeeh Target
Dose Level(1)
MeyErrielns@ aie Perfusion Attachment Attachment
Viability Efficiency(2)Viability(2)
Vehicle
Control 34452
34451 34450
CORN
OIL (ML/KG) 10.0
10.0 10.0
81.6
86.9 88.1
75.1 89.7 66.3
83.5 97.9 92.0
Positive Control 1 - DMN (MG/KG)
34446
15.0
34445 34444
15.0 15.0
80.3
86.2 89.6
79.4
60.5 68.2
88.4
92.5 93.1
Test Material - (MG/KG)
mm gE es 34440 34439
34438
34428
34427
34426 34416
34415 34414
34404 34403 34402
810 810
810
405
405
405 203
203 203
101 101 101
87.4 82.9
80.2
79.7
88.1 92.2
79.5
89.7 85.1 92.1 85.9 87.3
91.1 76.6
66.9
76.6
72.4 79.5 73.5
82.6 60.9 75.0 83.3 85.5
93.9 94.4
86.8
93.7
91.0 97.1 89.7
95.6
89.1 93.9 96.5 95.7
Notes: (1) Three animals per dose level were treated. (2) Results based on viable counts (trypan blue dye exclusion) of
randomly selected areas on two plates. DMN = Dimethylnitrosamine
15516-0-494
24
01316
WASHINGTON
TABLE 4
SUMMARY OF UDS DATA FROM IN VIVO/IN VITRO RAT HEPATOCYTE UDS ASSAY
15-16 HOUR TIMEPOINT
Client: 3M Corporation
HWA Assay No.: 15516-0-494
Client Code: T-5710.1
-
_-- Test
Cond.
Animal Number
Target Dose *
Trial Initiation Date: 25-Mar-93
nnn
Mean Net Nuclear % Cells Mean Percent
Grains (MNNG) Ww 25 Cyto. ~~ S-Phase
+ SD
NNG Grains* Cells
Vehicle Control - CORN OIL (ML/KG)
34452%
10.0
0.04 + 0.37
34451
10.0
-0.73 + 0.68
34450
10.0
-0.73 + 0.40
4.00 2.67
1.33
3.61 6.69
4.55
0.00 0.27
0.00
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 34440
34439
34438 34428
34427
34426
34416
34415
34414
(MG/KG) 810
810
810 405
405
405
203
203
203
-0.29 + 1.40
~0.40 + 0.48
-0.24 + 1.07 -0.10 + 0.23
0.75 0.20
-0.40 + 0.12
0.43 0.40
0.63 + 0.41
0.05 + 0.54
9.33
1.33
6.67
3.33
7.33
2.67
7.33
7.33
7.33
8.54
5.26
5.79
5.47 4.54
6.67
5.21
5.46
1.37
0.07
0.00
0.00
0.00 0.27
0.13
0.27
0.13
0.07
(A-- Fr0eietateanoinFmaCtnoeeverpmaatiio-- er,ne rLeaNvetethlLSsaoaretrcr3uac-- reasar,pOicBaecoRvrietoi-- eraC1s50coaranl pcliee)-- ityh saunrda ddeveiattioen r
ratcies oF cll (Gresko hor con te 3 ot mie arn n Bete vst
2 oimeBeciECo0rda R0yofitSSnoElttaEanrSalatoyciclnsesegwrooarisnrscaasmtemsrioanootnfeispeptwiencraWteeGncncoeveeorsfiiopaen w2(c15o0vSeotrttaeildpacaCt1t0y0.
served shen total celles.
500
cls
par
15516-0-494
25
001317
WASHINGTON
Client: 3M Corporation Client Code: T-5710.1
Table Cell Proliferation Sumary
HWA Assay No.: 15516-0-494 Trial Initiation Date: 5-Mar-93
Group/Sex | Dose Level | Labeling Indexb
(mg/kg)
(%)
Liver Weight | Terminal Body
(9)
Weight (g)
me
od 0.61% 0.18
16.42 1.21 | 421.6 13.6
TM
101
0.49 0.46
17.16 + 1.46
420.0 + 23.7
3M
203
0.26 + 0.26
17.24 + 1.17
407.5 + 20.6
Mm
405
0.50 + 0.38
18.48 + 3.06
389.1 + 40.8
SM
810
0.34 0.25
18.32 1.10
365.1 + 24.6%
6HC
158 | 31.51% 15.86%| 19.48 + 2.06%| 459.8 + 30.1
"`PFievrecenatnaigmealsofpelrabeglreodupheupnaletsoscytiendniuccalteeid per total number of hepatocytes counted "(Faotur aTneiamsatls20p0e0r) group ""VPeohsiictlievecocnotnrtorlo,l, 1015mlm/ak/gkg ofofcDorNn oil *Significant atp < 0.05 *+ *SIingcnrieafsiecanitn tahte pm<ean0.01 4 Decrease in the mean
15516-0-494
2%
01318
WASHINGTON
AnHuimmbaelr
33459 3344446610 34463
GGrroouupp MSeDan
N
3344446645
34466 34467 34468
GGrroouupp MSeDan
N
34469 3344447701 34472 34473
GGrroouupp MSeDan
N
Gr[Soeuxp m TMIMMH
M2M
2M 2M 2M
3M E3dH 3MMn
Cell ProlTiafbelreat6ion Assay Individual Animal Data
MLeabaenl ing ndex_(%)
0.62 00..8612 0.38
0.0.6118
4
01.2149
0.62 0.19 0.19
00..4496
5
0.43 00..0150 00..1607
00..2217
5
TBeordymiWneailght (a
432.0 442092..92 422.2
4211.36%
4
443209..79
425.2 435.8 378.2
42203..07
5
303.0 34921..10 440082..59
40270.056 5
LiTveerrmiWneailght (a)
16.02 1147..8294 17.53
161..4212
4
1167..3646
17.85 18.85 15.10
171..4166
5
16.21 1188..5116 1175..4856
17.24 117
5
15516-0-494
27
01338
WASHINGTON
Animal Group
Mumber ___jsex
34474
aH
34475
an
34476
aM
34477
aM
34478
aM
NGGrroouupp SMDean
34479
SM
34480
5M
34481
5M
34482
5M
34483
5M
Group Mean GNroup SD
34514
oH
34515
6M
3344551176
66MM
Group Mean
Group SD N
CelTlabPlreol6if(eCroanttiionnueAds)say Individual Animal Data
LaMbeaenling
Index ()
0.95
0.19 0.29 0.19 0.86
00..5308 5
0.29
0.24 0.29 0.76 0.10
0.34 05.25
22.14
47.43
1442..2244
31.51
15.86 4
BToedryniWneailght
(a)
401.6 369.9 379.8 342.6 451.4
3809..18 5
346.3
375.2 369.1
398.6 336.2
365.1 245.6
499.3
448.5
442683..03
459.8
30.1 4
LiTveerrniWneailght
qq
20.22
14.87 18.83 16.06 22.42
183..4086 5
17.80
19.22 17.57 19.75 17.26
18.32 51.10
22.09
19.45
1197..3016
19.48
2.06 4
15516-0-494
2
C01320
@ HAZLETON
WASHINGTON
XIV. APPENDIX A: HISTORICAL CONTROLS
15516-0494
29
C0132
? WASHINGTON
HISTORICAL NEGATIVE CONTROLS
IN VIVO/IN VITRO UNSCHEDULED DNA SYNTHESIS ASSAY
Number of data points is 20
Animal # --_--
UDS Grains/ Nucleus + SD
*%
of
Nuclei
with
25Net
Nuclear
GrainsA*v*eGrraagiensCy*t*o
FW Tein Erm
1 2
-0.23 -1.39
0.33 30.49
2.7 0.0
6.17 7.76
3 4
-0.99 0.47 0.12 30.89
1.3 4.7
8.19 7.69
5 6
-1.43 0.55
10.76 10.45
0.7 0.0
10.61 3.55
7 8
0.67 0.32 0.11 20.63
0.0 0.0
3.93 2.66
9 10
-0.11 20.25 -0.05 0.39
0.7 0.0
2.66 4.25
1 12
-1.05 -0.01
0.31 0.89
0.0 6.7
2.79 7.55
13 1
-0.38 -0.27
0.30 20.22
1.3 2.7
8.55 6.58
15 16
-1.38 -0.18
0.31 0.33
0.7 4.0
9.89 8.18
17 18
-0.57 0.34 -0.21 0.47
33 2.0
8.97 7.94
19 20
-0.59 0.38 -0.62 0.52
--_----------
0.0 0.0
7.75 7.20
en
Average:
-0.52
spa
40.50
1.5 11.0
6.56 $2.44
Range:
Low High
-1.74 0.12
0.0 6.7
2.66 10.61
-_--
*
gDaSte = oArvedruapgleicaotfe nceotvenrusclliepasr
g(r1a5i0nceclolusn)ts a+nalsytzaenddarfdor daevisaitnigolne
from triplianimal.
** a
ASDver=agSetanvdaalrudesDefvoirattiroinplicate or duplicate coverslips for a single animal.
15516-0-494
30
COLuRL
2 WASHINGTON
HISTORICAL POSITIVE CONTROLS
IN YIVO/IN VITRO UNSCHEDULED DNA SYNTHESIS ASSAY
4-Hour Timepoint
Number of data points is 9
AT -n_i--mal # NUuDcSleGursai5nsD/ * m %NetofNNuucclleeair Gwriatihns25** GArrvaeirenasgme**Cyto
1 2
22.27 23.25
42.36 1.08
98.0 98.7
7.46 7.01
3 4
22.25 19.44
11.96 20.75
10905..03
5.84 8.43
5 6
24.03 14.22 29.75 47.98
98.7 96.0
4.99 5.26
7 8
19.05 17.47
1.76 1.78
98.0 98.0
6.56 6.65
_F --9 16.83 S 4.00 R 97.3 5.74
Avsepra age:
21.59 43.98
97.8 1.4
6.44 1.10
Range:
Low High
16.83 29.75
_--
10905..03
5.26 8.43
*
UDS
=
Average
of
net
nuclear
grain
counts
+
standard
deviation
EP from tripli-
** Acvaetreagoer dvaulpuleiscatfeorctovreirpslliicpaste(o1r50ducpellilsc)ateancaolvyezresdlifposr aforsinaglseingalneimaaln.imal.
a SD = Standard Deviation
15516-0-494
31
01322
g WASHINGTON
HISTORICAL POSITIVE CONTROLS
IN VIVO/IN VITRO UNSCHEDULED DNA SYNTHESIS ASSAY
15-Hour Timepoint
Number of data points is 12
Animal #
DNuScleGursai5nsD/ *
%NetofNNuucclleeair wGirtahins25++ AGvreariangse**Cyto
21
1123..6758 12..5320
34
172..7273 12..7834
65
98..2867 11..1155
7
9.14 1.48
8 9
65..0616 21..7909
1110
1101..1052 21..1137
12
15.40 1.07
7860..77
69..0954
7662..70
75..7718 7
6696..40
55..050
80.7
a
5406..00
22..5713
6781..73
76..0195
84.7
5.61
Average:
10.16
sRpaange:
2.99
HLiogwh
155..4606
1629..04
25..0669
580..70
92.H4
* UDS = Average of net nuclear grain counts + standard deviation frostripli*x Acavteeragoer vdaulpuleiscatfeorcotvreirpslliicpaste(o15r0ducpelllisc)ateancaolvyezresdlifposr aforsiangsliengalniadnli.mal a SD = Standard Deviation
15516-0-494
&
coL324