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ANALYSIS OF T-5877
IN A CELL PROLIFERATION ASSAY IN RAT LIVER CELLS
EINALREPORT
AUTHOR
Maria A. Cifone, Ph.D.
PERFORMINGLABORATORY
Hazleton Washington, Inc. 9200 Leesburg Pike
Vienna, Virginia 22182
LABORATORYPROJECT Ip
HWA Study No.: 154-208
SUBMITTTEDo
3M Corporation Building 220-2-02
3M Center St. Paul, MN 55144-1000
SCTUODYMPLETIDOATNE
November 1, 1994
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QUALITY ASSURANCE STATEMENT
PROJECT TITLE: ALniavleyrsiCsellosf T-5877 in a Cell Proliferation Assay in Rat
PROJECT NO.: 20991 PROTOCOL NO.: 493
HUA STUDY NO.: 154-208 EDITION NO.: 1, Modified for 3M Corporation
OQtphueearlaaitbtioynvgAessrPurerofacenercdeuenrceeisdnspopfreocjttehiceotn(Qswu)earleiotfycotAhnsedsuucsrttaeunddcyeacaUncndoirtdrieanvngidewtaococftohretdhieSntgafnidntaaolrdthreeport of
gFeinnedrianlgs refrqoumiretmheentisnsopfecttihoensappanrdoprfiinaatle management and to the study director on
GreopoodrtLabroervaietworwyerPeracrteipcoerterdegtuolations. the following dates:
Inspection/Date
(Dsosuirnggerayn)d pump implantation 7D-ra2f5t,2r6e-p9o4rt review/ Fi1n1a-l1-9r4eport review/
EindingsReported
2-7-9 7-26-94 11-1-94
Auditor
B. Mullett B. Mullett B. Mullett
154-208
JuaTity Assurance Un
COC52E
//1 ate Release
2
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(COMPLIANCE AND CERTIFICATION STATEMENT
The described study was conducted in compliance with the Good Laboratory
Practice Regulations as set forth 58, 40 CFR 792, and 40 CFR 160).
in the Code of Federal Regulations (21 CFR There were no significant deviations from
the aforementioned regulations or the signed integrity of the study or the interpretation
protocol that would affect the of the test results. The raw
data have been reviewed by the Study Director, who certifies that the
evaluation conclusion
of the within
test article as presented herein represents an the context of the study design and evaluation
appropriate criteria.
Al
gen
raw data,
erated as
a
documentation,
result of thi
s
resctourddys,wilplrotboecoalrsc,hivsepdecibmyenHsazlaentd
final
on for
reports
a period
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 time or sent to
satosrtaogreagefacfialciitliietsy doefsiHganzalteetdonbyfotrhean spaodndsiotri.onal
period
of
SUBMITTED BY:
Avice Z Hon
Andrea L. an, HS.
Associate Scientist
u/i/94
Date
Study Director:
wa (1 Stidya DA.irecCtiofron.e, Ph. D.
Genetic and CellularToxicology
//-/- 24
Study Completion Date
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TABLE OF CONTENTS
PAGE NUMBER
I. SPONSOR vv ttt te
ee. 7
II. MATERIAL TESTED . . . ov vv vv ttn teens. 7
AB.. GIedneenttiicfsicAastsiaoyn No.
C. Physical Description 0. Date Received
IIL. TYPEOF ASSAYS . LL iti i ieee eee
nnn
7
IV. PROTOCOL NUMBER . . . . . .. .........uuuuuunn.. 7
Vo STUDYODATES . ........................... 7 A. Study Initiation Date B. Experimental Start Date Cc. Experimental Termination Date
VI. SUPERVISORY PERSONNEL . . . . . . . . ............... 7 A. Study Director
B. Associate Scientist
VIL OBJECTIVE . . . ttt itt tte ieee ee eee, 7
VIII. DEFINITION Crete it at aaa aaa 8
TGOMATERIALS
. o.oo ovvtit
LLL. 8
AB.. OIsnmdoitciactorPumCpesllsand Label for Cell Proliferation Analysis
Cc. Control Articles
X. EXPERIMENT DESIGN . . . . . . . .................. 9
AB.. DIomspilnagntaPtrioocnedoufreOsmotic Pumps
C. Tissue Collection and Preparation 0. Immunohistochemical Staining
E. Assessment of Cell Proliferation
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TABLE OF CONTENTS (CONTINUED)
XI. ASSAY EVALUATION CRITERIA . . . . . . ............... 11
XII. INTERPREOTFARETSUILOTSN. . . . . . ............... 12 A. General Observations
B. Summary of Labeled Cell Counts for the Liver
XIIT. CONCLUSIONS . . . . . ov ov vit i ie tiene inn... 13
XIV. REFERENCES . . ..............00tuninnaa.. 14
XV. EXPEDAR TATI ABLM E .E ..N ..T . AL ................ 15
APPENDIX A APPENDIX B
APPENDIX C APPENDIX D
Individual Anima] and Slide Labeling Indices . ....... 17 Individual Animal Body and Liver Weights and Liver to Body Weight Ratios . . . .................2.% Histopathology Report . . . ................. 29 Statistical Analysis of Labeling Indices . ......... 32
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ABSTRACT
The purpose of this study was to determine the hepatotoxicity of T-5877 by measuring cell proliferation (CP) assayed as S-phase induction in rat liver cells after in vivo treatment. The doses chosen for the study were 100, 200, 400 and 800 mg/kg. Dimethylnitrosamine (DMN) at 15 mg/kg was included as a positive control.
In the cell proliferation assay, a single oral dose of the test material was
administered and five animals per condition were labeled with BrdU for 72
hours using ALZET osmotic pumps. No histomorphological alterations were
observed at 100 and 200 mg/kg but minimal to moderate vacuolization was
observed at 400 and 800 mg/kg. Treatment-related
in the dimethylnitrosamine (DMN) positive control
changes were
animals.
also
observed
Following determination that there were no treatment-related lobular differences in the labeling indices, sections from the left lateral lobe of the livers, as well as samples from the duodenum, were 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. The control animals
had a labeling index of 1.34 and treated animals had labeling indices that ranged from 1.40 to 4.42. When comparing vehicle and treated groups, there were no significant increases in the labeling index in any of the treated groups and no positive trend was observed. The high dose (Group 5) animals did have an elevated mean labeling index but it was not significant because the average value showed heterogeneous variance. Significant increases in cell proliferation were observed in the positive control animals.
pT-r5o8l7i7ferwaatsiotnherienforraet Tciovnesridceerlelds.negative for the induction of cell
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Analysis of T-5877 in a Cell Proliferation Assay
in Rat Liver Cells I. SPONSOR: 3M Corporation
II. MATERIAL TESTED: A. Genetics Assay No.: 154-208 B. Identification: T-5877 C. Physical Description: waxy amber solid D. Date Received: January 19, 1994
III. TYPE OF ASSAYS: Analysis of Cell Proliferation in Rat Liver Cells
IV. PROTOCOL NUMBER: 493, Edition 3, Modified for 3H Corporation
V. STUDY DATES: A. Study Initiation Date: January 10, 1994 B. Experimental Start Date: February 7, 1994 C. Experimental Termination Date: June 14, 1994
VI. SUPERVISORY PERSONNEL: A. Study Director: Maria A. Cifone, Ph.D. B. Associate Scientist: Andrea L. Ham, M.S.
VII. OBJECTIVE: TTh-e587o7bjbeyctmieveasuorfintghiscelalssapyrowlaisfertaotmieoansu(rCeP)hmeepaastuorteodxicaistyS-cpahuasseed by induction induced in rat liver cells after in vivo treatment.
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Cell proliferation measured the fraction of cells undergoing cell replication in rat liver using an immunohistochemical technique (1,2) to detect bromodeoxyuridine (BrdU) incorporated during DNA synthesis. Animals were given a single oral dose of the test material and the Tivers were isolated following administration of BrdU for 72 hours in vivo with an ALZET osmotic pump implanted subcutaneously. Quantification of cells that have incorporated DNA precursors over the
72-hour period indicates increased cell proliferation in the liver (3).
VIII. DEFINITION:
Hepatotoxicants such as carbon tetrachloride
an increase in cell proliferation to replace
and dinitrotoluene induce
necrotic tissue (2,4).
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 the carcinogenic
process but there are numerous mechanisms which can be affected during
replication (5-8). 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 carcinogens as well as genotoxic carcinogens using this
technique.
IX. MATERIALS:
A. Indicator Cells
Young adult male rats of the Sprague-Dawley strain, 10-12 weeks 01d at the time of dosing, were purchased from Charles River Laboratories, Raleigh, NC (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) and water ad libitum. Animals were quarantined a minimum of 7 days prior to random assignment to study groups and identification by implantable microidentification device for the cell proliferation assay. 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. The cell proliferation assay was initiated with rats that ranged from 306 to 355 grams. Approximately 2 hours after dosing, the animals were anesthetized using Metofane (methoxyflurane, PitmanMoore, Inc.) inhalation anesthesia and one ALZET pump per animal was aseptically inserted subcutaneously (dorsal surface).
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Steovernetmoyv-atlwoofhoutrhse lTaitveerr,s aanndimadlusodewneurme. anesthetized with CO, prior
B. Osmotic Pumps and Label for Cell Proliferation Analysis
AweLrZeETuseods.motAicsipnugnlpes l(oAtLZA(#C0o42r3p0o1r)atiwoans,usPeadlothAlrsoou,ghoCuA)t, thMeodesltud2yM.L1 TThhee ppuummppshwaesrea 2p0r0e0-f4i1llecdapawictihtyBwridtUhata paumcponcreatnetraotfio1n0 uolf/hour. 20 mg/ml.
C. Control Articles
1. Vehicle control
Agavvaegheicl(eP.0c.o)ntrwoilthctohnesivsethiincglef,ivecorrnatsoilwas(Dduoksee'ds
by oral Corn Oil,
Lsoutbje2c1t2e9d083t3o)'.iheTissanseuesmanfirpoumlavteihoincsleusceondtrfoolr athneimatlisssuweesre
dveerhiivcelde cfornotnrotlreaatneimdalasnimdailds.notTheexcedeodsin1g0 mvlo/lkugm.e of the
2. Positive control article
Trhaet hpoespiattiovceytecsontirnolvivcoo.mpoTuhned piossiktnoiwvne ctoontirnodlu,ce S-phase in dLionte#t8h2yHl0n3i6t5r)oswaansindeos(eDdMN,atCA1S5#.06m2g7/5k-g3., SFiigvmearaCthsemwicearle Co., treated P.0..
D. Test Article
tFhoer ttehsetpraertpiacrlaetiwoansosfustpheenddeodsining scoorlnutioiolnsatofcothnecenttesrtatiaorntsicloef, v1o0,lum2e0,s 4f0oratnhde80temsgt/malrtipcrlieorditdo dnootsinegx.ceedThe10mamlx/ikmgu.m dosing
X. EXPERIMENT DESIGN:
A. Dosing Procedure
Ffiovrethreatscelplerprcoolnidfietriaotniwonereasstarye.ateDdelbiyvoerraylvoglauvmaegse wweirteh T-5877
tcaarlgceutlatdeosde.on tThhee mbaaxsiismumofvothleunemosotf trheecenttestaniamratlicwleeigshutspeanndsiotnhse
aardtmiicnliesteinredvedhiidclenotwereexcueseedd 1f0omrl/akngy.tesFtriensgh
preparations purpose.
of
test
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D. E.
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Confirmation of the concentration of the test material under conditions of preparation and dosing of the assay was not determined in conjunction with this study.
Implantation of Osmotic Pumps
F(oLrot t#h0e42c3e0ll1) pwreorleifeprraetliooandedasswaiyt,h A20L0Z0ETul MoodfelBrd2UML1at aosmotic pumps
concentration of 20 mg/ml. The animals were anesthetized using
Metofane
was asepti
caaclcloyrd
ing
ins
ertote
dstasnudbacrudtanperooucseldyure(sdo
rasnadl
onseurfpaucmep)
per
animal
approximately 2 hours after dosing. The incision was closed with
wound clips and the animals monitored until the time of sacrifice
to ensure that there were no clinical signs of infection. The
osmotic pumps were implanted three days prior to sacrifice.
Tissue Collection and Preparation
Each aninal was anesthetized prior to removal of organs for
aanndalyfsiixse.d duodenum,
ainTthineseusttuhreoarlwaictibhcuffhceiargvheidtcyeflowlramsatluoirpnne.onveedrA,acnrwdoasstshealsselocitvireoernmorveoefmdovefdron
each aninal and fixed. The duodenum was included as an indicator
tTihvaetrslabferlomwahsighaddmoisneist(eGrreodupco5)rreacntilmaylst,o e5a4chpaanriamfafli.n emFboerddtehde
sections were taken from the left lateral, median and right
anterior Tobes. Once it was determined that no treatment-related
lobular differences were present, slides from the left lateral
Tobe were prepared from each animal. Sections of the duodenum
were also made and a section of the duodenum was mounted on each
slide containing a liver section. Slides were also prepared
according to standard procedures for examination by a pathologist
to determine if any abnormalities were present.
Immunohistochemical Staining
mTTehhaeesussrlieidddeessbywweeirrneecorsdpteoapriaanretadifofnifnooirfzdeBedrtdeaUrnmdiinnratetohiyodDnrNAatoefudsicnepglrliBoiprorgotleoinfisextraaitniinogn. as 3a,n3t-idbioadimeisnobweitnhzidpienreoxitdeatsrea-hcyodnrjoucghaltoerdidestr(eDApBt)avcihdrionmoagnedna and
hematoxylin counterstain.
Assessment of Cell Proliferation
The section of the duodenum was microscopically examined to ensure
ttThahbeaet]hitdghehelildvaobseeerlyanwwaiasmsalpcsornowfpeierrrmeleyde,xaadmmsilininedidesstefroferrdomlotobtuheltahdreifadfnieifrmfaeelnr.tencleoOsbn.ecse of Labeling was similar among the lobes therefore cell counting was
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paneirmfaolrsm.ed wTihtehpesreccetinotnasgefroofmnutchleeilefitncloartpeorraaltinlgobelabferlominallthe wTeirveerrawansdondleytergmeinneerdatemdicrboysccoopmipcuatlelry.. ATh1e.0amrmeassqutaorebeincdoeuxnetded occouulnatringgriadread.ividAetdleianstto 120000x n1u0clseqiuarweesrewaesxamuisneeddtoperdefainnimealthweith a minimum of 3 sections and 6 fields per section. cAonnytaniuncilenig atnhyatbwreorwen cblhureomowgeerenicconhsuiedewreerde cuonnlsaibdeelreeddanldabealneydnuclei eunnulneesrsataedc.learFiealrdtsifatchtatwacsontpariesneendt.areaOsnlyofhenepcartoosciystewenruecleniotwere eivnaclluuadtedioninasthetoevtarleuaattmieonnt. groTuhpe. slides were coded for (blind) Sfo-lplhoawsse:nuclei labeling indices for each animal were calculated as Labeled S-phase nuclei (LI)=nh too.toaelflnpao.beaolfedthepaotoccyteys cntouucnle teeid X 100 XI. ASSAY EVALUATION CRITERIA Tchoeuntperdopwoerrteionasnaloyfzetdhebynumrebpeeratoefdcemlelassurleasbelaendalytosistheofnuvmabreirancoef c(eAlNlOsVA) tsepchhenriiqcuietsy ttoestdewtaesrmianlesoanuytilsilzieded,toandtesrtelvataerdianicneterhaocmtoigoenneietfyf.ects. The cAadldciutlaitoendaltoltyhce.ondauvcetragoene-vwaalyueANfOrVoAm, thDuenntehtrtee's stl-itdeesst,ofTeearcphsa-aJniomnaklheewraes dtaetsat., anSdeerAepgpreensdsiixonCtefsortsstfaotristtriecnadl uasnianglysbiosthofuntlarbaenlsifnogrmeidndiacneds.ranked tFeorrmitnhael tebromdiynawleigwhhtoleratbioodsy,weaigmhetasn,anldivesrtawnediagrhdtsd,eviaandtiolnivweerreto Sc-aplhcausleatevdalufeosr.eacShtattirsetaitcmaelntangaroluypsisusionfglathbeeliinngdiviinddueaxl waasnimpaelrfomremaend gursoiunpg coonmep-awraiysoannsalwyesries doofnevawriitahnceDuntneecth'nsiqute-st.estCoanntdroclontvreorlsusvertsruesatment cpaosseitiovfevagrroiuapncecomhpeatreriosgoennseiwteyr,e droannek utsrianngsfotrhematSitoundsento'fsthte-tedsatt.a weIrnethe performed prior to analysis of variance and Dunnet's t-test. Atolbaobdelyinwgeigihntdexr,atitoermiinnaal dobsoedygrwoeuigphtt,hatterdmeivniaaltesTivferromwetihgehtvalaunedsliinver cthoensicdoenrceudrresnitgnicofnitcraonltlygrdoiufpfearteantstihgannifitchaencceontlreovlelgroofupp.s0.05 was
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XII. INTERPRETATION OF RESULTS A. General Observations 1wereaonbismaelrsvedsuratviv1e0d0 tanrdeat2m0e0ntn.g/kgNobuhtismtionmiomraplholtoogimcoadleraatleterations Varceluaotleidzacthiaongneswasweroebsearlvseodobatser4v0e0danidn 8t0h0e mdgi/mkegt.hylTnrietartomseanmti-ne (inDNA)pppeonsdiitxivA.e control animals. Details of the histopathology are dCeuloldsenusmtaifnreodm waillthofthtehebroawnnimaDlAsB cusherdomoignenthewersetudoyb.servTehde pirnestehnece oTafbe1labealnd ianccalelptatbhlee aniimmmaulnsohiisntdoicchaetmeidcaplropsetraindienlgi.verTyheorfe twhaes BrnodU waapsparreanntdonprweiftehreinntitahle lToabbeesl.ing in any of the lobes and the label Nionncereaosfethien wTeiivgehrts ocofmptahreedtretaotecdontarnoilmalasnimsahlosw.ed aThedosmeea-nrellaitveedr wtehioguhgth olfargteheipnocsrietaisvees cionntDrNoAl swyansthensoitssi(ganndifiscuabnstelqyuenetlevcealtled even p8r0o0limfge/rkagti(oGnr)ouwper5)e ainnidmuacelds. hadHowmeevaenr,tetrhmeina4l00bnogd/ykgwei(gGhrtosupth4)atand wTeirveerletsosbotdhyanwetihgehtGroruaptio1scownetrreoldetvearlmuiene(dp,<th0e.0r1e).wereWhen the wsiitghnif2icpanvtaluiencorfeasbeestweiennth0e.01livanedr t0o.05terfmoirnaGlroubpody3 w(e2i0g0htmg/rkagt)ios a5)n(dp<i0n.c0r1e)a.ses at 400 mg/kg (Group 4) and 800 mg/kg (Group B. Summary of Labeled Cell Counts for the Liver ATabsluema1r."y oIfndtivhieduTaalbelaenidmaclellcoucnotusntsareforshoewanchignroAupppenisdixshAo.wn Tihne mCeoalnumnTabienliTnagbleind1.ex (LI) for each group is presented in the third Tihnedimceaatnesbathcaktgroluensds Tthaabneli2nXgofintdheex n(uGcrloeuip h1)adwausnde1r.g3o4newhiDcNhA siynndticheessiosfdutrhiengtestthe ar72t-ihcoluer-trleaabteeldingcelplesriorda.ngedThefrolanbe1l.i4n0g to 4.42. None were considered significantly elevated and there was
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Tnhoeinhidgihcatdioosne o(f800a msgi/gkngi)fichaandtthpeosihtiigvheesttrelnadbeldiunegtoindterxeatomfen4t..42, bsuitgnibfeiccaaunscee.of aIfhethteerorgeesnuelotsusfrroemspoinnsdei,vidtuhaelreawnaismalaslaacrke coofmpared i(nsdeeiceAsppeonfdi8x.00A),andtwo9.0o5f, twhheilfeivetheanirmeamlasinirnegspotnhdreede,anwiitmhalslabhaedling 1waebaeklirnegspoinnsdei,cesbutcloaslesotomaybabcekgrthoeundresluevletlso.f aTnihmiaslmavyariianbdiilciattye.a oSefethAeppeDnHdNixposDitfiovrestcoanttirsotlicaalnimaanlaslyswiass.34.T9h6e mwehainchlaibseling index significantly elevated (p $ 0.01). Tdhoessee-rerleastueldtsidnecmroeanssetsratien tthheatLIT-5i8n77thedidlivnoetr iinndmuacleesriagtnsifaifctaenrt 8t0r0eamtgm/ekngt.witLhargseingilnecreoraaslesdoisnestheatlacboenlcienngtraitnidoenxsweorfe10o0bsemrgv/ekdg tion tDhHeN-tDrHNeatpeodsitpiovseiticovnetrcoolntraonlimaalnsi.malsThweasmea3n4.9l6abe(lpi<ng0.i0n1d)e.x in the
XIII. CONCLUSIONS TnhuembetrestS-pmhaatsereiacle,ilTs-5f8o7l7l,owidnigd naotsinignldeuceorasligdnoisfeicoafnt10c0hamngg/eksgintothe 8d0o0se-mrge/lkga.tedThterenadnimianlsthweemreeanlablealbeedlinfgorin72dexhowuarss oabndsernvoedsiginniftihceant tirnedautcetdiongrooufpsD.NA Ts-y5n8t7h7eswiassitnherratefolrieverevacellulast.ed as negative for the
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XIV. REFERENCES
1. D(e1F9a87z)i.o, AI.m,munLceahriys,tocJh.eAm.,icaHledldeeyt,ectDi.Wo.n oafndprTaotltiefresraaltli,ngM.cHe.iNl.s in vivo. J. Histochem. Cytochem. 35, 571-577.
2. 5L-abnrioemrodeToxLyuriBdeirngeer,andEX"H.-thaynndidfiancehoi,n prIo.den(t19h8e9p)a.toCcoemlplaurliasron of proliferation studies. Toxicologist 9, 64.
3.
MBiurtstaelriwso,rthJ,., B.PEr.o,bstA,shbGy.,,
J., and
Bermudez, E., G. Williams:'
Casciano, D., A protocol and
guide
1f8o9r:1t2h3e-1i3n3,viv1o987r.at hepatocyte DNA-repair assay. Mutation Res.,
4. DMiNrAsaslyinst,hesJi.Cs. inandratButhteeprawtoorctyht,esB.fEo.l:lowiInngduicntivonivooftruenastcmheendtulweidth dinitrotoluene. Carcinogenesis, 3:241-245, 1982.
5. RMaerlsamtaino,nshD.iSp.,ofCahtetplaetyi,c pR.eCr.o,xisCoomnewayp,rolJ.iGf.e,ratainodnPoapnpd, reJ.pAl.ica(t1i9v88e). pDNrAolisfyenrtahteosriss dtio(2t-heethhyelphaetxoycla)rpchitnhoagleantiecitanydof[4t-hcehlopreor-o6x-i(s2o,m3eCxya1nicdeirnoR)es-.2-p4y8r,im6i7d3i9n-y6l7t4h4i.o] acetic acid (Wy-14,643) in rats.
6. cCarnacdedro.ck,In:V.M".liv(e1r976C)e.ll CCealnlcerp"r,oliCfaemerraotni,onH.aMn.d, exLpinesreilmle,ntaCl.A.Tiavnedr AWnasrtweircdk,an.G.P., Elsevier, North Holland Biomedical Press,
7. RCoelquumibraenmoe,ntA.o,f cRealjlalapkrsomlii,ferS.a,tioanndfoSrarmtah,e Di.nSi.tRi.ati(o1n981o)f. liver Rceasr.ci4n1o,gen2e0s7i9s-20a8s3.assayed by three different procedures. Cancer
8.
oGlfinloisv,erA.rDe.g,eneBruattciheorn, onN.Lt.unoR.r, foarnmdatAiubo,n
J.C. (1951). in rats fed
The
effect
4-diaminobenzene. J. Exp. Med. 933, 313-324.
9. Hsatmu,dyA.Tivaenrd eCifffoencet,s Mi.nAd.uce(d199b1y).a siUnsegleofdocselel ofprDoHlNi.ferEantviiornonmteontal and Molecular Mutagenesis 17(19), 16.
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Client: 3M Corporation
Client Code: T-5877
Table 1
Cell Proliferation Summary
HUA Assay No.: 154-208
Trial Initiation Date: February 7, 1994
Group/Sex3, DLoevseel Labelin(gx) Index?
(mg/kg)
Liv(egrraWnse)ight || WTeeirgmihntal(grBaomdsy)| WLeiivgehrt/Bo(d)y
1m
oc
1.34 + 0.66
13.58 + 1.09 [344.2 + 12.9
3.94 + 0.20
Mn 100 | 2.98% 1.61 [14.3218 [38.117.3 | 4.112036
3M
200
1.40 + 0.92
15.00 + 2.24 |335.6 + 20.5
4.45 + 0.38%
"Mm
400
2.26 + 1.26
14.76 + 1.02 303.6 + 13.0%%1| 4.86 + 0.25%*t
SM
800
4.42 + 3.60
15.38 + 1.05 [300.0 + 15.0%*4| 5.13 + 0.19%*t
oud
159 | 34.96 + 14.86%*1| 12.73 + 1.61 [319.6 + 22.5 | 3.97 + 0.29
Fbpievrecenatnaigmealsofpelrabeglreoduphepatocyte nuclei per total number of hepatocytes
counted (at least 2000)
pCvoeshiitcilveecocnotnrtorlo,l, Co1r5nmogi/lkg of DMN
* Significant at 0.01 < p < 0.05 ** Significant at p < 0.01
4t DIenccrreeaassee iinn tthhee mmeeaann
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APPENDIX A Individual Animal and Slide Labeling Indices
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@HWAAZSHLIENTGTOONN
Slid#e AnimalID 16 47733 17 47733
18 47733
Group #Labeled
TM
10
TM
21
TM
5
#Counted %Labeled
700
1.43
700
3.00
700
0.71
Mean
nn
SD
1.17
19 47734
TM
20 47734
mm
21 47734
TM
8
700
1.14
9
700
1.29
7
700
1.00
Mean SD
1.14 0.14
22 47735
TM
23 47735
wm
24 47735
mm
5
700
0.71
4
700
0.57
4
700
0.57
Mean
0.62
sD
0.08
25 47736
TM
1
700
1.57
26 47736
1M
10
700
1.43
27 47736
TM
15
700
2.14
Mean
Ln
SD
0.38
28 477371
wm
29 47737
TM
30 47737
m
7
700
1.00
13
700
1.86
12
700
Ll
Mean
1.52
so
0.46
GROUP MEAN GROUP SD
1.34 0.66
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Slid#e AnimalID Group #Labeled #Counted %Labeled
4457 4477773388 2oHH 1261
48 47738 ou
29
770000
23..2090
700
414
MesaDn 30..9144
5409 4477713399 MMuu 1292 51 47739 M 16
770000
23.714
700
2.29
Me0an 20..7413
5532 4477774400 22MM
21
770000
32..7010
54 47740 ou
8
700
114
MesaDn 21.3219
5556 4477774411 57 41741
M1u] 3272 49
770000
35..1289
700
7.00
MesaOn 51..1934
5589 4477774422 oMuu
124
60 47782 M 18
770000
01.7517
700
2.57
MesaDn 11..6020
GGRROOUUPP MSEDAN
21..6918
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Slid#e AnimalID Group #Labeled #Counted %Labeled
6612 4477774433 3MM 63 47743 M
62
770000
00..8269
6
700
0.85
Mesaon 00..3637
64 47744 M
1
65 47744 3M
9
66 47744 3M
1
700
2.00
700
1.29
700
1.57
Mean
1.62
sD
0.36
67 47745 M 16
68 47745 M 11
69 47745 M
27
700
2.29
700
1.57
700
3.86
Mean 2.57
sD
117
70 47745 71 47746
3M 3M
72 47746 3M
4 6
700
0.57
700
0.86
7
700
1.00
Mean 0.81
SD
0.22
73 41747 74 47747
3MM
39
770000
01..4239
75 47747 3M 16
700
2.29
Mesaon 10..3933
GGRROOUUPP SMDEAN
10..4902
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Slid#e AnimalID Group Labeled #Counted %Labeled
31 ares a
11
32 47748 a
14
3 44s aM 15
700
1.57
700
2.00
700
2.14
MesaDn 01..3900
u a71789 a
7
3 47749 a 19
3% 41749 ao 10
700
1.00
700
2.71
700
1.43
Mean 1.71
sD
0.89
37 47750 a 19
38 47750 an
39
39 47750 aM 16
700
2.71
700
5.57
700
2.29
Mean 3.52
sD
1.79
40 47751 Eo
4
41 47751 a 13
42 47781 aM
9
700
0.57
700
1.86
700
1.29
Mean
1.24
sD
0.64
43 ar752 4a 47752
a aM
1 17
45 47152 aM
30
700 700
2.00 2.43
700
4.29
Mean 2.90
sD
121
GGRROOUUPP SMEDAN
21..2266
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@ HWAASZHLIENGTTOONN
Slid#e AnimalID
] 47753 2 47753 3 47753
Group #Labeled
SM
23
SM
8
5M
18
#Counted %Labeled
700
3.29
700
1.14
700
2.57
Mean
2.33
SD
1.09
4 47754
5M
62
5 47754
SM
60
6 47754
SM
46
700 700 700
Mean SD
8.86 8.57 6.57 8.00 1.25
7
47755
5M
8 47755
SM
9 47755
SM
12
700
17m
5
700
0.71
5
700
0.71
Mean
1.05
SD
0.58
10 47756
5M
63
11 47756
SM
56
12 47756
SM
ks
700 700 700
9.00 8.00 , lo.1s
Mean
9.05
SD
1.07
13 477571
5M
15
14 47757
5M
10
15 477571
SM
10
700
2.14
700
1.43
700
1.43
Mean
1.67
SD
0.41
GROUP MEAN
4.42
GROUP SD
3.60
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Slid#e AnimalID Group #Labeled
776 a4r7s7es o6HH 326640 8 47758 oH 435
#Counted %Labeled
770000 5327..0104 700 62.14 Me0an 1520..5473
8709 4a7r7s5e9 81 47759
ooHH 225615 oH m
770000 3357..8866 700 24.43 Me0an 327..7214
8832 4477776600 84 47760
ooHH 12565 oH 369
770000 3252..1144 700 52.71 Me$an 3165..6374
88 4477776611 o6HH 229720 770000 a38.757
8 47761 oH 285
700 0.71
e0an 410.6.133
8898 4477776622 o6H 16556
90 47762 oH
8
707000 237..7517 700 12.11 M0ean 81.41.167
GGRROOUUPP SKDEAN
3144..896
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@ HWAASZHLIENGTTOONN
APPENDIX B Individual Animal Body and Liver Weights and Liver to Body Weight Ratios
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reo 5 AMALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS
SSTOUROYAFNTBEARBS:OL1U5T4E20O8RGAN VEIGHTS (5) *ORAFT* ORGAN ABBREVIATION: LI = LIVER SEX RDOOSEP ANNBNEAR TBOEDRYNVATL(5) LI WWooL1ommmmu s3we dneoa WKo ol ommmmss d3a0o niss Kol wmmy mo wn MBER IH NGeROwU:P: ezs 13ss STANDARD DEV: ws 1
WWoozzoemmamsm g3o 3 63s WWoozz ssmaa sMo0 nisiee Nz mma ss zs MNGER 18 NGwo:p: sel5 ex5
STANOMRD OEY: v3 ve
wNo os mmmaa s3m0o wwaa WWo o3 mmese saoo ww NI mng woo wn NBER I WGeROaU:P: ases 15.s00
STANONRD DEV: ws 2a
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orcnoix 8 MALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS
S"TDURDAYFTMSgEARB:SOL1U5T4E20O8RGAN VEIGHTS (3) *ORAFTTM
GROAN ABBREVIATION: L1 - LIVER Sex DSOSoEw MWaGMER TBEUOIMYAL 0) LI
WWoo4e ssoomnee 0w0o 1wka WWoo44 emmss qalos e13n% Wo4 mmm ao am nSeTnAR1A8WGDrEaoV:w: aBmoe5 6T5e
wHooss mmoss smuioo 1l6a2 wHooss omosms asmise a 15m Nos mmm we 16 WASGTERANIDNGARDOEVU:P: 310.050 1L5o3ss8
Tr
WWWoooeee
mBme Bm
awmoeo 30
2 wlise
NNo os6 Bmoem mamsoe ninae
eR INaG:e: aE5 275 somo bv: 22s Let
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APPENDIX 8 ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS
ORAFT*_STOURDGYAKK-UTNOB-ERT:ERN15I4N2A0L8 BODY EIGHT RATICS (X) "DRAFT
ORGAN ABBREVIATION: LI = LIVER SEX GDOOSWE ANNMIBMEARL TBEORDMYINWATL(g) RATIO
WWo o1oBoBTesmt 0 30 43a3m WMooLl BBes a32o0 d4e0rk M1 emmy 30 3m NUMBER INWEGARKO:UP: Hz5 395m
STANDARD DEV: 2s oss
WWoozz oBosmsa 3a0m0o aAowE KKoozz Bsuaa 33000 a44s7e0 Woz mma aes 39% NONBER IHNEGARO:UP: uel5 41506
STANOMRD DEV: 73 036
WWo o3 mBaa 33000 540372 WNoo33 ssomuss a3m0o aAmm W300 as NOMBER INeGARO:UP: 3565 1s) e
STANOMRD DEV: 25 03m
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seros AUATSE OF CELL PROLIFERATION I RAT LIVGR 4S RAFTSiGROAW-oTOe-TERTMIrAoL ED VEIGHT RATGS (3) "TEATS
RAN ABEVIATION: LI- LIVER sox Dosee Mmsw B TERe wm
PPWoooyllosemse H BoE LoE wE wseosnmsaa.r
mmwSSo uislww Es im wRsessaedea
N 4 wosm oom ownssemseee Istimmm H VoieEme um osuwowwne'ees: weeesssasiism
NVWo ooolonmmmsem P BoEwse wseosnmoaa.e
eoammeeo duaaumms BBSS iimB uHsEssasin
05381
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APPENDIX C Histopathology Report
154-208
05582
2
DiazLETN
Analysis of Cell PPartohloilfoegryatRieopnoritn Rat Liver Cells Project No. 154-208
General Protoco Thirty, young adult, male Sprague-Dawley rats were placed in six groups of five rats/group. Group 1 served as the vehicle control. Groups 2, 3, 4, and 5 served as the low-, low-mid-, high-id-, and high-dose groups, respectively, receiving 100, 200, 400, and 800 mg/kg of the test material, T-5877, via oral gavage. Group 6 served as the positive control, receiving 15 mg/kg of dimethyInitrosanine (DMN) via oral gavage. After dosing, an ALZET Model 2HLI osmotic pump containing 20 mg/mL of bromodeoxyuridine (BrdU) was implanted subcutaneously in each rat while it was under Metofane anesthesia. Seventy-two hours after pump implantation, all rats were anesthetized, exsanguinated, and necropsied. Liver, duodenum, and all gross lesions from each rat were placed in 10% neutral-buffered formalin and processed as per HHA SOPs. These tissues from all rats were evaluated microscopically by a boardcertified veterinary pathologist.
Histopathology
Treatment-related change in the liver consisted of minimal to moderate vacuolization in Group 4 (400 mg/kg) and Group (800 mg/kg) rats. Group 6 (DMN treated) rats had varying severities of centrilobular necrosis,
hepatocellular hypertrophy, chronic inflammation, peliosis, and increased
mitoses. The single cases of pelvic dilatation in the kidney and degeneration with mineralization of the testis in Group are considered to be spontaneous and without relation to treatment.
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The test material, T-5877, when administered to male Sprague-Dawley rats in single oral gavage doses of 100, 200, 400, and 800 mg/kg, produced vacuolization in the liver of rats dosed at 400 and 800 mg/kg. No treatmentrelated histomorphologic changes were noted in the liver of rats dosed at 100 and 200 mg/kg.
Pathologist:
Samuel V. Machotka, D.V.M., D.A.B.T., Diplomate, American College of Veterinary
Pathologists Department of Pathology
Date
tang
005564
a
@ HAZLETON WASHINGTON
APPENDIX D Statistical Analysis of Labeling Indices
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Methods: countedThewerperopaonratliyoznesd obyf trheepeantuemdbermeaosfurceesllsanallaybseilsedoftovathreianncuembe(rANOoVfA)cells tefefcehcntisq.ues_Tthoe dseptheerrmiicnietyantyestsiwgansifiaclasnotutdoislei,zedslitdoe,tesatndvarreilaantceed hionmtoegreancetiitoyn. The model used was:
proportion = 4 + dose + slide + slidexdose + .
vAadrdiiattiioonna,llyt,he asvienrcaegethvealusetufdryom dtihde 3nostlidsehsowof aenaych sainginmiaflicwaanstthbeentcwaelecnu-lsaltiedde ttorecnodnduuscitngonbeo-twhayunAtNrOaVnAs,foTremrepdstarnad-Jroannckkehdeedraetat.est [1], and regression tests for
ResultsSi:nce the sphericity tests rejected variance homogeneity for both vehicle vs positive control and vehicle vs treated groups (p = 0.0215 and 0.0000, rseisgpneicftiicvaenlcye),evaltuhaetioGnr.eenAhsousTee-xGteiTsasbelre 1prionbdaibcialtietsi,esthewreereis unsoedsigfnoirficatnhte sfilniddien-gto-isn1icdoempvaarriinagtivoenhsiwcelree wniotthsitgrneiaftiecdangtroiunpse.itheWritchaisne-. aOnndlybetthweeepno-sgirtoiuvpe contro]Theshodwaetda hibgahsleyd soingnitfhiecaanvterealgeevatviaolnueisn slhaobweeldlinegxtorveemrelvyehihcelteerocognetnreoolu.s tvarrainasnfcoerma(tpio=n 0.w0a0s00u)seidn tcoompcaornidnugctveohniec-lweayvsANtOrVeAa.ted AgsroTuepxst soTabtlheast t2heanrdan3k fionrdicbaotteh, tuhnetrreanasrfeornmoesdigannidfictarnatnsdfiofrfmeerdencceassebsetw(peen= ve0h.i0c3l5e3 aannddtrpeat=ed0.g0r6o9u2p,s rtersapnescftoirvmeeldy)d.ata Fduirdtnhoetrmsohroew, aTneyrpssitgrnai-fJiocnacnkthepeosrietitveesttraenndd arsegirnedsisciaotnedofinrTaenxkt. mTaarbgliena4llyevesnigntihfoiucgahntthterenrdegorveesrsidoonsesbas(ped= o0n.02t4h0e).untTrhaenrsefowramsedno dsaitganifsihcoawnetd aTraeckuosfedfitto fdoernobtoethdirreegcrteisosnioannsd (spta=t0i.s0t7i5calandsi0g.n3i7f8i)c.ancTeh:e following notations
*+ ==sisginginfiifciacnatntatatpp<$0.00.105 t= effect in the positive direction
Text Table 1 - Univariate ANOVA
Vehicle vs Positive Control Vehicle vs Treated Groups
DSolsiedpe p
0.0.20206094 ** -
00..04936230
Slidexdose p
0.2499
0.1360
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Text Table 2 -
Dose(mg/kg) Mean UntransSfDormed
VeVehhiiccllee
vs
Positive Control 1.3400 0.4649
Positive 34.9620 13.1132
Vehicle Vehicle
vs
Tr1ea.t3e4d00Groups
0.4649
210000
1.2.49080000 01..73531838
400
2.2540 0.9313
800
4.4200 3.7928
The Descriptive Statistics Median MReaannk-TransforSmDed
316..65720000 83..00000000 11..55841111
1.5200 21..73130000 1.9000 2.3300
187..07000000 157..29000000 16.2000
5.1962 65..24603710 6.3008 8.8713
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Text Table 3 - One-Hay ANOVA _____ UVnethriacnlsefvosrmPeodsitiTvreaCnosnftorromledUntransVforemedh vs~TirTreaac ntsefdol Grrmoeuedps
Treatment .0004**
.0010** .0953 ~~ .0692
Text Table 4 - Test for Trend for Vehicle vs Treated Groups
TReergprsetsrsiao-nJonocfkhUenetrreansTfeosrtned Data
[100284605 *11
Regression of Rank Transforned Data 11870 1
Discussion:
The results of the present study indicate that there was no statistically significant increase in cell proliferation over control due to treatment by the chemical. Moreover, there was no indication of a significant positive trend due to treatment. References: [1] Ajit K. Thakur, A Fortran Program to Perform the Nonparametric TerpstraJonckheere Test, Computer Programs in Bionedicine 18: 235-240, 1984. [2] SAS (Statistical Analysis System), SAS Institute, Cary, NC, 1991.
154-208
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ES
WASHINGTON
iasetou.[d SHEyOR Protocol No. 433, Edicion
ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS
aHnadzleFcDAonGoWaosdhiLnagbtoorna,torIync.Pra(cHtUAi)cewi(lGlLE)coRnedguucltatcihoinss.stuTdhyisinprcootmopcloila,ncecrwiiticchalEPA bPhyasQeu(asl)ityofAstshuerawnocrek iinn aprcocgorredsasnceanwditthheSOfPisnalacrHeapzolrettowinllHasbheinsgutbojme,ctTnteo. audit 2Th1i6s2,study will be conducted by HUA ac 9200 Leesburg Pike, Vienna, Virginie FART 1. SPONSOR INFORMATION AND APPROVALS I. SPONSORIDENITFIGATION
Company Name: __ MCorporation Address: _Bullding 220:26:02. 3 Gener. St. Paul. HY 55164-1000
In. mestarmies pevmipreation: 1-987
III. IESTARTICLEANALYSIS
cDheatrearcnttenraitsitoincsofasthedeftienstedaritnicclhee GsLtPabrielgiutlyatainodnstheoftFeDstA a(r2t1icClFeR t5h8e.10r5e)s,ponEsPiAb-iTlSiCtAy (o4f0 CtFhRe S7p9o2n.s1o0r5.), and EPA-FIFRA (40 CFR 160.105) is
IV. NOTIFOFRIEGUCLATAORTY SUIBMOISSNION $I8n.1o0r;derEPAt-oTScCoAm,pl4y0wCiFtRh U7.9S2..10f;edeErPaAl-FIrFeEgAu,la4t0ioCnFRcod1e6s0.1(0F)DA,and21cCorFtRatn pFaorreeigonf aagesntcuideys,iscotonsbueltisnugbmiltatbeodrattooritehse amgiesntcyb.e nHoVtAifmiaeidntIafinasllaor IBensdtiecratescwhheiduclhe agoefncsyt,udiLeEsanwyh,icRhIGfHaTllTeucnedievre rtehgeulTaetsourlytsrevoifewt.his Pslteuadsye:
C3 vodecernine
ma CJ seams C3 eenrrema
Owe CJ wow J ceo C3 ome
10/93
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V. STUDYDATES
Beoposnt Sxpertuensal Sore Tans: Proposed Experiaencal Teratnacion Dace:
VI. APPOFRSTO UDYV PRA OTOL COL
Seudy Director:
Sara A Gifone. PhD.
Sponsors Authorized Representative:
/
\
Qa Dubin
Protocol No. 493, Edition 3
Dace:
vi
1083 CoasT0 rage 2
WASHINGTON
Protocol No. 493, Edition 3
PART 2. STUDY PROTOCOL ANALYSIINS ROAFT CLEILVLERPRCOELLILFSERATION 1. OBJECTIVE Ttheestombajteecrtiiavle boyf mtehiassuraisnsgayceilsl tpordoeltiefcetrathieopnato(tCPo)ximceiatsyurceaduseasd Sb-yphtahsee
induction induced in rat liver cells after in vivo treatment.
uCenldlergporlonlgifecrelaltiorneplisicadteisoingneidn rtaocmelaisvuerreustihengfarnacltimoanunoofhiscteollcsheaical atnedchntihequeliv(e1r,s2).areAniismoallastedarefolgliovweinnga asidnagilneistorraaltiodnoseofof the chemical pburommpodaepolxaynutreddinseubc(uBtradnU)eoufsolry.72 QhouuarnstifiincvaitvioonwiotfhcealnlsALZtEhTa hoasvmeotic uIsnecfourlporfoartedtheDNeAvaprleucautrisoonrsofocvheermitchaels72-thhoaturmapyerciaoudsehasinbcereenasesdhocwenllto be proliferation in the liver (3).
II. DEFINITION
aHnepaitnoctroexaisceanitnsceslulchprasolicfaerrbaotniotnettroacrhelpolraicdee maencdrodtiincitrtoitssouleuen(e2,4i)n.duce OTthheesre cpormoploiufnedrsatimanyg cienldluscemaSy-pbheasedestyencttheedsidsuriinngtShe-pahbasseencaenaloyfsihse.patoccoaxriceiintoyg.enicIcprioscensost, abpuptaretnhterehowarecenlulmeprrooulsifperroacteisosnesacttshatincatnhebe parfofleicfteedratdiuorninmgayrepilncirceaatsieonth(e5-p8)r.obabCihleimtiycalolfyspionndcuacnecdoucselmlutations as mwuetlaltiaosnsinpcrrieoarsetothea rperpoabiarbiplrioccyesosf. coUnnvsecrhteidnugleDdNAcealdldupctrsoliifnetroation may ealmseorgepnlcaey oafraolefulilnythteranesxfpoarnmseidoncloofnepreonfeoceplllass.ticSocmeellsofltehaedsiengextoamptlhees nacotngebnyotaoxniocngecnaortcoixniocgenmsechaasniwseml.l asItgeinsottohxeircefocraercipnoosgseinbsleustiongdettehicst technique. II. MATERIALS
A. Animals
aYtounthgeadtuilnee omafledosriancgs, ovfiltlhebeSppruargcueh-aDsaeudlefyromscrHaairnl,an1S0p-r12aguweeekDsaloelyd, IInncc.. ((CHrSlD::CSDpOrBaRg)u.e-DaTwhiiesyh(eSaDlOth)EyR)ranodromChabrrleedssRtirvaeirn hLaasborbaeteonriseesl,ected t0o amacxoimmmiozne sgoeunrectei.c heterogeneity and ac the same time assure access
. 10/93
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WASHINGTON
Protocol No. 493, Edition 3
The animals will be housed according to standard operating proce-
dures and will be fed Purina Certified Rodent Chow (formula 5002)
Tdahaneydswraapttrseirortaodtboeliubsuiesteudamn.dfovriTlhtleheybewaislsrlaayndbwoeimllqlyuabaresasnaitngiennseecddhetacoimzisentdiudmbyuemfgororofuepsse.ven surgery.
TPhietnaann-iNmoaolrse,wilIlnc.b)e ainnehsatlhaettiioznedanuessitnhgesiMaetoafndanoene (pmuemtphopxeyrfluarnainmea,l wSielvlentbey-tawsoephtoiucrasllylatienrs,eracneidmaslusbcwuitlalneboeuslaynes(tdhoertsailzedsuwrifatche)C.O prior to removal of the livers and duodenua (control organ). B. QumoticPumpsandLabelforGellProliferationAnalysis
AwAiLLlZZlEETTbeMooussdmeeodlt.ic24LApLumspiosnsmgol(teAiLcZlAoptuCwaoiprlphloarsabteiaoun2s,0e0d0PatulhloroAculgathpooa,uctitCAyt)h,ewiMcsothuddeayl.pu2mMpTLhLe r(aBrtaeu)ofat10aulc/ohnrc.entrThaetiopnuposf w2i0lmlg/bael.illed with bromodeoxyuridine . ControlArticles 1. vehiclecontrol
Afovrehcieellleprnoelgiafteirvaeticoonn.trolThecyonwsiilsltibneg torfeaatemdinwiimtuhm tohfefvievheicrlaets moerthsoodlsven(tussueallleycteodralforgavtahgee)teusstedmatfeorriatlh.e tTehset msaatmeeridaolsing ptorsesaitbmleentsdoswiilnlg vbeoluemmepslofyoerd ofroarltghaevavgeehicvliellconnottroelx.ceWehdersebout
10 ml/kg body weight.
2. Bositivecontrolarticle The postive concrol articles used are known to induce S-phase bpiernoltrirafeteathreeadptaibtoyoncypvteierlslosbien v1i5vog./kgTheofpDoMsNi.tivAeccolnetarsotl fifvoer rcaetlsl will
D. TestArticle
Utnelsetsesd asspescuipfpileidedb.y tAhenyspoopnesroart,iontshepetrefsotrmaerdticolnethweilltesntoramratlilcylebe bsyuchtheasspgroinnsdoirngp,rioerxtrtaocttihoen,inoiftiastoilovneno-fextcehsatnignge. muAsltlbeopesrpaetciiofnised pexforaed on the test article will be described in the final report.
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CO357R
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Protocol No. 493, Edition 3
IV. EXPERIMENTALDESIGN
A. DosingProcedure
Aseplercotliionninafroyr ctheesctweisltlarbeticpleerfounrlmeesds toa by the Sponsor. Materials which may be
dveetheircmlien/esolvveehnitcleL/esoslpveceinftied selected include water,
msthueeicthaytblelcsetellvauerlhtoiisccell,ee/scionalrvvbeoonlxcuy.smeesthRyaclthcsaetlwliwuillllolsbeen,ottcreoexractneeedodilbyboorourtaaln1o0tghamvelarg/ekgwbiotdhy
PwSrepeiognphsator.ra.tioAnlDstMeNronwfaitltelhebretoeustdteisssarootlfivceeldxepoiasnnudrsetepromisaliyetibvedeeircoeonqniutzeresodtlesdwatbieynr.tthheeFresh
osfolvtehnettoerstvemhaitcelreialwilunldebre ucosnedditfioornsanoyftpersetpianrgaptuiropnosaen.d doSstianbgilwiitlyl
be the responsibility of the Sponsor.
B. DoseSelection
Ubenle1ssg/ksgpecoirfiheadlfotthheerwLibsse,, wthhiechheivgehresits dloessse. selFeocutreddowsielsl wuislulalbley sfreolnectceadchudsoisneg alpepverloxainmdatceolnytrtowlo-gfrooludpdwiillultioben sutseepds.to aFniavleyzaenimceallls proliferation at 72-hours.
. ImplaonftOsapottiicPoumpns
ABLrdZUETatMoadeclonc2eHnL1traotsmiootnicofpu20mpsmg/wmill.l beFopllroevlionagdeddowsiicnhg w2i0t0h0 utlheofCesc mp(uammtepetrhipoaexlry,flauntrihamenaela,nwiimPlailltsmbaenw-illaolsoerbpeet,icaanIlnelcs.yt)heitniisnzeheradtleadtuissoinunbgcauMnceeastntochfoeausnsielayan(ddozosnael saunrifnaacles).moniTtheoreidncuinstiioln wtihelltibemecloofsesdacwriitfhicewoutnodencsluirpes athnadtchtehere airnetnhoe rcaltisnicfaolr stihgrnese doafysinpfrecitoiront.o saTchreifoiscmeo.tic pumps will be kept
D. TiC sso ue llec ant dPri epao ratn ion
aEnaaclhysainsi.malThweillthboeracaincestcahveittiyzedwilplriober otpoenreedmovaanld otfheorlgiavnesr froermoved adunoddefniuxme,d aininsesuucerawlitbhuffhiegrhedceflorlmatluirnn.overA, cwriolsls salescotiboen orfemoved
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@HAZLETON WASHINGTON
Protocol No. 493, Edition 3
E. Imnunohistochemical Staining F. Assoe fCes llPs rolim fere ation nRat tes
ws
ge 6
Coase
WASHINGTON
Protocol No. 493, Edition 3
V. DAPTRAESENTATION
The for
efaicnhal tirmeeppooritntw,illforinctlheudenegtahteivfeolcloonwtirnogl,inpfoosrimtaitvieoncoinntrtoalb,ulaarndfeoarcmh
analyzed treatment:
+
The for
ecaaclhculaantiemdalXSa-npahlayszeed*fosrtanS-dpahradsed.eviation
among
the
three
slides
VI. ASSAYEVALUATIONCRITERIA
Abemceaalncualndatesdtanfdoarrdeacdhevitarteiaotnmenftorgrtohueppuesricnegntatghee
of S-phase individual
cells will animal mean
S-phase values. performed using
onSet-awtaiystiacnaallysainsalyosfisvaroifanlcaebeltienchgniiqnudeesx
will (9).
be Control
versus treatment (10,11). In the
cgarsoeupofcovmaprairainscoenshewtielrlogbeenediotnye, wirtanhkDutnrnaents'fsormta-ttiesotn
of
the data t-test.
Switluldenbte'spert-ftoersmtedwiplrliobre tuoseadnalfyosriscomopfarviasroinancoef
and the
Dunnet's positive
cgornoturpolthvaetrsduesviattheesvefhriocmlethecoSnt-rpohla.se
An S-phase percentage
percentage in a dose in the concurrent
control group significantly
aditffaerseingtniftihcaannctehe lceovnetlroolf
ps0.05 group.
will
be
considered
VII. REFERENCES
1.
DeFazio, (1987).
AI.m,muLneoahriys,tocJ.hAe.m,icaHeldldeeyt,ectDi.Vo.n
aofndpTraotltiefresraaltli,ngM.cHe.lNl. in
vivo. J. Histochem. Cytochem. 35, 571-577.
2.
Lbarnoimeord,eoxTy.Lu.r,idiBneergearn,d
E.K. and Eacho, J%-thynidine in
rPo.dIe.nt(1h9e8p9a)toCcoemlplaurliasron
of
5-
proliferation studies. Toxicologist 9, 64.
3. JB.u,ttePrrwoobrstt,h, G.B,.E.a,ndAsGh.byW,illJi.a,msB:ermuAdepzr,otoEc.o,l Caasncdiagnuoi,deD.f,orMitrhsealilns,
vive rat hepatocyte DNA repair assay. 133, 1987.
Mutation Research, 189:123-
4.
Mirsalis, synthesis
Ji.nC.ratanhdepBauttotceyrtweorsthf,ollB.oEw.i:ngIinnduvcitvioontorfeautmnesncthedwuiltehd
DNA
dinitrotoluene. Carcinogenesis, 3:241-245, 1982.
5. MRaerlsamtaino,nshDi.pS.,ofCahtecplaetyi,c pRe.Cr.o,xisCoomnewayp,roJl.iCf.eraatnidonPopapn,d Jr.eAp.lic(a1t9i8v8e). DNA spyrnotlhiefseriastotros tdhei(h2e-peatthoyclahrecxiynlo)gpehntihcailtayteofandthe(4p-ecrholxoirso-o6m-e(2,3XReysl.idi4n8,o) 6-723-9p-y6r7i4n4i.dinylthio)acetic acid (Wy-14,663) in rats. Cancer
10/93
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C3575
WASHINGTON
Protocol No. 493, Edition 3
6.
Craddock, cancer.
IVn:.M."Li(v1e9r76)C.ell
CCealnlceprr"o,liCfaemreartoino,nH.aMn.d,
eLxipnesreilmle,nCt.aAl.
lainvder
Warwick,G.P., Elsevier, North Holland Biomedical Press, Amsterdam.
7.
Cooflucseblalnop,roAl.i,ferRaajtailoanksfnoir, thS.e
and Sarma, initiation
Do.fS.lRi.ver(1c98a1r)c.inogReenqeusiirsemeanst
assayed by three different procedures. Cancer Res. 41, 2079-2083.
8. GLliivneors,reAg.eDn.e,ratBiutocnheorn, tNu.mLo.rR.foramnadtAiuobn, iJn.Cr.ats(1f95e1d)4-dTihaemienfofbeecntzeonfe. J. Exp. Med. 933, 313-324.
9. MWciGnerra,w-HBi.lJl., (N1e9w71)Y.orkS,ta2tnidstEidciatiloPnr,inpcpi.pl1e4s9-i2n20E. xperimeDnestiagnl,
10.
Dsuenvneertatl, trC.eWa.tme(n1t9s55)w.ithAamcuolnttirpolle. cJo.mpAamr.isoStnatp.roAcsesdoucr.e
for. 50,
comparing 1096-
121.
11.
Dunnett, control.
BCi.Wo.met(r1i9c6s4).20, Ne48w2-t4a9b1l.es
for
multiple
comparisons
with
a
VIII. REPORTFORMAT
The final report will provide the following information.
*
STepsotnsmoarteirdieanltifiidceantciiofni.cation
and
Assay
Number.
A physical
description included in
of the test material this section.
and
date
of
receipt
will
be
* *
TDyapteesofofassstauydyanidnitpiraottioocnolanndumbceorm.pletion.
* *
Names of Study Interpretation
Doifrercetsourl,ts.Senior
Technician,
Scientist
* Conclusions.
* *
Historical Signatures
ocofntSrtouldydSautaperfvoirsonergaatnidveStuadnyd
positive control Director.
cultures.
** MTeetshtodrse.sults presented in tabular forms
* *
Evaluation References.
criteria
* Quality Assurance statement.
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AS RENTON
Protocol No. 493, Edition 3
IX. CHAORNREGVIESIOSNS Asniygnecdhabngyestheor struedvyisidoinresctoofr,thdisateadp,proavneddmaprionttoacionledwwililthbethidsocupmreonttoecdo,l. The sponsor will be notified of any change or revisions.
X. RETCOBOEMR AIND TAIS NED
ALL raw daca, documentation, records, protocols, and final reports
generated as a result of this facilities of Hazleton for at
study least
ionlelyebearafrcohlilvoewdinginsutbhemissstioroangeof
the
final elect
rteophoarvtetothetheafosrpoenmseonrt.ionAefdtemratetrhiealosnereyteaairnepedriiond,thethestosrpaognesor
may
facilities of Hazleton for an additional period of time or sent to a
storage facility designated by the sponsor.
10/93
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z WASHINGTON
AMENDNENT TO STUDY PROTOCOL rr--------------------------------------------------
STUDY TITLE: _--
ANALYSIS
OF
CELL
PROLIFERATION
IN
RAT
LIVER
CELLS
HUA PROTOCOL: _--
493,
EDITION
4
EFFECTIVE DATE: FEBRUARY 6, 199 _--
ASSAY NO.: 154-208 =
AMENDMENT RO.: 1
The following changes are made to the study protocol:
The second paragraph of Section ITI. A. Animals is changed from:
W"iTlhle They
wbaienlilmfaebldesPwquiurlailrnaanbtCeienhreocduisfeiademdiancRicomoduremdnitonfgChsteoovwesnt(afdnoadryamsrudlparoi5po0er0r2a)ttoianugnsdepwraoatcneedrdwuiarldelsLibabenidtus.
arnanedsotnhleytizaesdsibgenfeodretosusrtguedryy.g"roups. The rats to be used for surgery will be
to:
w"iTlhle baenimfaeldsPwuirlilnabeCehrotuisfeidedacRcoodrednitngChtoowst(afnodramrudlaop50e0r2a)tianngdpvroacteedruraedsLiabnidtus. Nmoighctontianmtienrafnetrse wairethknthoewnsttuodyb.e pThreeseanntimailnstwhielldibeet qourawraatnteirneadt laemvienlismuwmhicohf aRscaencvdeoonrmddiiaznyagstitoopnrisootfrandttoaherdusaenoipmeaarnladstiwniaglnldprabeonicneradalunrdeoisadleoynftaitsfhsieicgaMntaeimdomnatlwoiilaslntubTdeoyxipgceroorulfpoosgr.ymed Sseucrtigoen.r'*y The rats to be used for surgery will be anesthetized before
.
Tihsechfainrsgtedtfwrooms:entences of Section IV. D. TG issouellecandtPriepao ratinon
T"hEeacthhoarnaicmiacl wcialvlitbyewialnlesbteheotpiezneeddpraindorthteo buffered formalin."
rlievmeorvalreomofvoerdgaannds
ffoirxeadnailnysniesu.tral
to:
a"nEaalcyhsiasn.imalThewiltlhorbaecwiecigchaevdityanwdilalnebsethoepteinzeedd panrdiorthetolirveemrovarlemoovfedo,rgawnesighfeodr and fixed in neutral buffered formalin."
Reason: Information inadvertently left out of protocol.
CO3578
t WASHINGTON -PAGE 2-
AMENDMENT TO STUDY PROTOCOL --e -- c ------------------ STUDY TITLE: ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS
AMENDMENT NO.: 1
ASSAY NO.: 154-208
STUDY DIRECTOR'S 2
SIGNATURE:
Opps fore -
N
DATE: 2 "f=
C3579
3M Internal Correspondence
ce:
To:
From:
Subject:
Date:
R. G. Perkins - 220-2E-10
B.C. Copley - 53-35-02
R. D. Howells - 53-35-02
Key to FC Alcohol Tox Samples
September 14, 1995
T5877
T5710
T5711
T5794
T5795
TS878
Wide Range E(FOSE
Analytical Request 41220
FM 3924 Lot 547
Retain from 2 year feeding study ~~ L-13203
Narrow Range EtFOSE Lot 884 Typical Raw Material for FC-807
Wide Range MeFOSE
Lot 555
Typical Raw Material
for FX-845
Analytical Request 41220 L-10059
Analytical Request 41220 L-1276
Narrow Range MeFOSE Notebook 97900-107-2 Lab Prepared Sample
Wide Range EXFOSE
Notebook 97900-112-2
Lab Prepared Sample
`Wide Range MeFOSE Lab Prepared from
`Washed POSF
Analytical Request 41343 L-13097
Analytical Request 41343
L-13098 Analytical Request 42607
PHD tls
Autachments
05580
REQ 41220
R. Howells
GC/MS analysesofthese samples were accomplished usinag 25 m X 0.32 mm HP-1 GCcolumn to
introduce the samples into
using chemical ionization
theFinnigan
with methane
SSQ-70 mass
as the reagent
spectrometer.
gas. The GC
The sample
column was
components were
operatedfrom 40
ionized
to 300 C
at
a
rateof 10 degrees per minute. The resouflthtesse analyses combined with their corresponding GC/FID area
`percents show the following:
on [m [omar | NR. N-EtFOSE
'W.R. N-EtFOSE
oman Component LD. ceconem
oLotns ies Lot 547 |
omosamie omsonen
ows ons |
lorsonaeonns cJzonoe n|
[------ FE ----
lomsonmelon us cwioow n|
lomsomee
ows lows |
lonsosmo lomns e|wiuox n|
lorsonaocapes | monn| lorsosmenwaanroc[ooansmen| ons
(05581
Component ID.
11276
N-McFOSE
Natty atone GecO.MO
Lot
lomsonowonLcowmeon|
lorsonomenenon| isa
epsonpueernon
J
Lis|
lonsonpeo| noeson|
lcrusoonoruocmscrnoon nseenlaonene|
N-McFOSE_ CsFi7SON(Me)CHz2CH20H
8383%
FE -- CoF19SO2N(Me)CH2CH20H
087%
omsono[ou us n|
Other High Boilers
155%
Ftruirftlhueorrowaocertkichaanshbyederniddeo(neTFbAyAG)Canodnatghaeisne wsiatmhplBeSsAwh(itochgiivnevothlevetsridmeertihvyaltsiizlaytlioetnhoerfst)h.e Tahlicsohwoolsrkwiwtahs Pdreesliigmniendartoy irnevsuelsttsigsahteowthtehpaottaenntailaylzipnrgobtlheemEsttFhOaSt EcouunldderbievaotviezreldoockoeudldbyhiadneyaosniegnmifeitchaontdaomfoaunnaltyosfisN.-EtFOS dAemriivdaetiuzneddewritthheTCF-3AAalcsohhoowlspetahaktiannywiEdXeFOrSaEn-gcehmlaotreriidael.thaHtowisepvreers,enatnianlytzhiensgatmhpelseiasmenomawtecroimaplltehtaetlhyamsabsekeend ebxypethcetedderbievcataiuzseedoCft-8healncuohmoble.rTohfediBfSfeArednterciovamtpiovneehnatssnionttbheeesnamepvlael.uatTehdeyesta,mbeutsosritmiolfaprrporbolbelmesmswialrle:most likely exist with MeFOSE and will be even more complicatiend the analysis of MeFOSEA.
71293
(03582
REQ 41343
J. Grant
introducGeCt/heMsSamanpalleysseisnotoftthheesFeisnnaimgpalneSsSwQe-r7e0amcacsomspslpieschterdomuestienrg. aTh2e5 smamXpl0e.3c2ommpmonHePn-t1s wGeCrecoiolnuimzaedto ruastienogf c1h0emdiecgraeleisonpiezramtiinonutwei.thTmheetrheasnuletsaosftthheerseeagaennatlygsaes.s cTohmebiGnCedcowiltuhmnthweiarscoorprereastpeodndfirnogmG4C0/tFoI3D0a0reCa.at a percents show the following:
`Componeat ID.
PrWecRutNEFOSE
|WR N-EFOSE `main cut
97900-112-1
97900-1122
lorwonen Linx
crsonEceron
le
orsosan
Joe
ER --
leas
lomsone
[un
crsonguamon
lion
[----
oss
Jon|e Jo | lee |
ume | me | fons |
---- SE ----
l|ionus [J ws uma|
Component ID.
NR. N-McFOSE BP. 132 97900-107-2
03582
lorsonmorcnloownon|
ooo
Je |
lowempnJos ma n|
Amide
---------- PY -- 97900-111
lorsomm
fue |
lomsonen
fons |
lorsones
laws |
Em |= [c novosc memsonelom ws n|
lomsonen
Jue |
that include hydrides, chlorine in the backbone, and
unidentified highboilers
03534
To:
From:
Subject: Date:
1. Muggli
5365-02
R. M. Payfer
236-2B-11 (612)733-4212
SARC Analytical Request No. 42607
Dec. 21, 1993
GC/MS analysis of this sample was accomplished using a 25 m X 0.32 mm HP-1 GC column to introduce the sample into the Finnigan SSQ-70 mass spectrometer. The sample components were ionized using chemical ionization with methane as the reagent gas. The GC column was operated from 40 to 300 C at a rate of 10 degrees per minute. GC analysis with flame ionization detection was also done, and the area percent values from this work were applied to the peak identities from the mass spec work. The results of these analyses (which are not necessarily quantitative) show the following:
Mol. Weight |Component LD.
13202 N-MeFOSE
N-Methyl-carboxamides C7F1s-C(OINMe)H| 0.06 %
|ser |owrusoonmer
lose|s
[es Jcurosona
ozs|
C4FsSO2N(Me)CH2CH2OH
003%
C5F11S02N(Me)CHzCH2OH
052%
CoF13S02N(Me)CH2CH20H
338%
CrF15S02N(Me)CHCH2OH
|sr [nwerose crosomumecuoreon
| sto |cmusommacrecnon
|er |orusomumonoron
[ose]
CeF16CISO2N(Me)CHCH2OH
| ees |CaF16SF5-SO2N(Me)CH2CHzOH
029%
03585
[ loerrmmo[sroesws| [oor] CaF17S02N(Me)(CH2CH20)2H
(03586
