Document BDXr7dB9z0jMy3EkKQxndba4
CORNING Hazleton
MUTAGENICITY TEST ON
T- 6294
EER
IN AN IN VIVO MOUSE MICRONUCLEUS ASSAY. 225500
| May14 1995
FINALREPORT
A aM a
Qtrc010>
AUTHOR
Hemalatha Murli, Ph.D.
PERFORMINGLABORATORY Corni9n2g00HaLzeleestbounrIgncP.ik(e.CHV)
Vienna, Virginia 22182
LABOPRR OJEA CT ITDENO TIFIRCATY ION
CHV Study No.: 17385-0-455
,
SUBMITTTEOD
3M Center, Bui3lMding 220-2E-02
St. Paul, MN 55144-1000
SCTUODYMPLETIDOATNE
May 10, 1996
CHV Study No.: 17385-0-455
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Co06371
`CORNING Hazleton QUALITY ASSURANCE STATEMENT
Project Title: In Vivo Mouse Micronucleus Assay
Project No.: 20996
Assay No.: 17385
Protocol No.: 455
Edition No.: 17
Quality Assurance inspectionsofthe study and reviewofthe final report ofthe above referenced
U`pnriotjeactndwearcecocrodnidnugcttoedthaecgceonredrianlg rteoqtuhiereSmteanntdsaorfdtOhpeeraaptpirnogprPiraotceeGduoroedsoLfatbhoreaQtuoarlyiPtryacAtsiscuerance
regulations. Findings from the inspections and final report review were reported to management
and to the study director on the following dates:
jo
-
I
Dosing/03/12/1996 Harvest/03/13/1996
Draft Report Review/05/03,06/1996 Final Report Review/05/10/1996
03/12/1996 03/13/1996 05/06/1996 05/10/1996
C. Orantes C. Orantes C. Orantes C. Orantes
,
wha eble ao cofe/n:
Quality Assurance Unit
"Date Released
CHV Study No.: 17385-0-455
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CORNINGHazleton
STUDY COMPLIANCE AND CERTIFICATION
The described study was conducted in compliance with the Good Laboratory Practicreegulations as set forth in the Food and Drug Administration (FDA) Title 21 ofthe U.S. Code of Federal
aRmeegunldamteinotnss.PaTrhte5r8e, wisesrueednoDescigenmibfiecran2t2d,ev1i9a7t8i,o(nesfffercotmivtehJeuanfeor20e,me1n9t7i9o)newditrhegaunlyataipopnlsicoarbtlhee
signed protocol that wouldaffectthe integrityofthe study or the interpretationofthe test results.
The raw data have been reviewed by the Study Director, who certifies that the evaluationof the test article as presented herein represents an appropriate conclusion within the context ofthe
study design and evaluation criteria.
All test and control results in this report are supported by an experimental data record and this record has been reviewed by the Study Director. All raw data, documentation, records,protocol
and a copyofthe final report generataesd a resultofthis study will be archived in the storage
facilities ofCorning Hazleton Inc. for at least one year following submissionofthe finarleportto the Sponsor. After the one year period, the Sponsor may elect to have the aforementioned
t`miamtee,rioarlssernettationeadstinortahgeesftaocrilaigtey fdaecsiiligtniaetseodfbCyomthienSgpoHnaszolre.ton Inc. for an additional period of
Submitted By:
Study Director:
|:
i
Hemalatha Murli, Ph.D. Mammalian Cytogenetics
Department of Genetic and Cellular Toxicology
$- req
Study Completion Date
/
CHV Study No.: 17385-0455
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CORNINGHazleton
TABLE OF CONTENTS
SUMMARY oii 100 SPONSOR iii 20 MATE(TeRstAItlAe) L
21 Clients Identification 22 DateReceived 23 PhysicalDescription 24 Genetics AssayNo.
Page No. 6
............uuuuiieeieaiiiiieaaaeenn 7
30 TYPEOFASSAY ...ooiiiiiinniiiiiiiieie iii
T
40 PROTOCOLNO. oii
50
STUDYDATES 5.1 Initiation Date
.....oovvviiiiiniiiiiiieieeeiiiieieeee eine
T
52 ExperimSetanrttDatle
53 Experimental TerminationDate
60 6S 1 SU tudyP DirePE cEtoRrSR ONNEV L..I ....S ....O ....R vvieY ieeaniiiinnaneeseennil 62 LaboratorySupervisor
70 OBJECTIVE ..oooennviiiiiininiiiiiie eee
T
80' MATERIALS .ovviiiiiieesiseiiiieeeiiiie 8 90 SOLU ANDB STAI BILL ITY:I...T...Y .....eiiiiiiiiiieiieeniinneinn 8
10.0 DOSESELECTION STUDY 10.1 DoseSelection
ovine O
102 Dosing Information
103 ResanduIntelrprtetatsion
104 Conclusion
CHV Study No.: 17385-0455
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CORNING Hazleton
11.0 M11.1IDCosReSOeleNctiUSonTUCDYL.E...U..S....uvuiuiiiinnenniiiiiiininnnnnnnn 1 112 Micronucleus Assay Dosing Information
120 BONE MARROWHARVEST, SLIDE PREPARATION AND ANALYSIS L2
13.0
EVALUATION CRITERIA 13.1 General
...............ouiiiiiiiiinnanaiiiieee1e3e
132 Data Presentation and Interpretation
140 RESULTSANDINTERPRETATION.........uuuuuennnnninnnnnnnnnnn1n.3
15.0 CONCLUSION iii
1S
160 REFERENCES ................oooiiiiiiiiiiiiiiiiiiiiiiiiiniineeeeen 15
17.0 DEVIATION FROM THE SIGNED PROTOCOL
...........cuvvvvivinnnnn... 15
180 EXPERIMENTDATATABLES ..................ooiiiiiiiiiiiiinnn1n6.
'
CHVStudy No.: 17385-0455
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CORNINGHazleton
SUMMARY
Mutagenicity Test on T- 6294 inan In Vivo Mouse Micronucleus Assay
`iTnhdeucobejmeicctrivoenoucfltehiisininbovniveomaasrsrayowwapsol1y0cehvraolmuaattiectehreyatbhirloictyyotefstohfeCtrelst:CarDt-i1cl%e(,ITC-R)62B9R4,mtioce.
aInndthdeodsoesdebsyeloercatligonavsatgudeya,tt1h0e0t0e,st2a0r0t0i,cle30w0a0s, s4u0s0p0e,nadnedd 5i0n 0a0cemtgo/nkee:.coSmixoial n(i4m0a%ls:6(0th%r,eev:mva)l,es
and three females) were assigned to each dose dosing for toxic signs and/or mortality.
group.
Animals
were
observed
for
three
days
after
4B0a0s0edmogn/ktgh.e rIensutlhtesomfitcrhoenudcolseeusselaescstaiyo,ntshteutdeys,ttahrteimclaexwiamsusmustpoelenrdaetdedindoasceewtoanse:ecsotrinmaotiled as `(m4a0l%es:6a0n%d,fivv:ev)feamnadledso)sewderbeyroarnaldogmalvyagaessaitg1n0e0d0t,o2e0a0c0h,daonsde/4h0a0rv0esmtg/tkigm.e gTreonupa.niVmeahliscl(efiavend apsossaiyt.iveThcoentarnoilmaglrosudpos,seedutwhiatnhitzheed taepsptraorxtiicmlaetweelrye2e4uthhoaunriszaefdtearpdporsoixnigm,atweelrye2i4n,cl4u8deadndin7t2he hours after dosing for extractionofthe bone marrow. p`Tohleyctehsrtommaatteirciaelr,yTth-r6o2cy9t4e,sduinddneorttihnedcuocnediastiigonnisofifctahnits ianscsraeaysaenidn imsiccornosniudcelreeidinnebgoanteivmeairnrtohwe `mouse bone marrow micronucleus test
'
CHV Study No.: 17385-0-455
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CORNINGHazleton
Mutagenicity Test on T- 6204 in an in vivo Mouse Micronucleus Assay
10 SPONSOR: 3M
20 MATERIAL (Test Article)
21 Clients Identification: T- 6294
22 Date Received: January 16,1996
23 Physical Description: Wax-like amber colored solid
24 Genetics Assay No.: 17385
30 TYPEOF ASSAY: In Vivo Mouse Micronucleus Assay
40 PROTOCOL NO.: 455, Edition 17
50 STUDY DATES
5.1 Initiation Date: January 18, 1996
52 Experimental Start Date: March 6, 1996
53 Experimental Termination Date: April 1, 1996
60 SUPERVISORY PERSONNEL
61 Study Director: Hemalatha Murli, Ph.D.
#62 Laboratory Supervisor: Monica Vegarra, B.S.
70 OBJECTIVE
tTohienodbujceectmiivceroofnutchliesiininvibvoonaesmsaayrrwoaws ptooleyvcahlruoamtaettihce earbyitlihtryoocfyttehseoftesCtralr:tCicDl-e,1%T(-I6C2R9)4,BR
mice. This study was Heddle etal. (1983).
conducted
using
modifications
of
the
procedures
suggested
by
CHV Study No.: 17385-0455
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CORNING Hazleton
80 MATERIALS
RAidvuelrt Lmaabloeraatnodrifese,maPloertmaigcee,,MsIt.raiTnhCirslh:eCaDl-t1hy%,(IrCaRn)doBmR,brweedrsetrpauirncwhaassesdelfercotmedCthoarles `mTahxeipmrioztoecgoelnfeotricthhiestsetruodgyenweaitsyaanppdraotvetdhebsyatmhee CtiHmVe -asAsCurUeCacpcreisosr ttoo athceoimnimtioantiosnouorfce. dosing.
TAnhiemtaelmspewrearteurheouasneddrefliavteipveerhucmaigedidtuyriwnegrequmaariannttianien,edanadt 7h2o4us6edFfiavneda5t5r+a1n5d%o,mization.
3re8s.p1e%ct.ivAely1,2e-xhcoeuprtliognhtM/a1r2-chhou9r,d1a9r9k6,cywchleenwathsemraeilnattaiivenehdu.miAdictoymwmaesrcrieaclorddieedt a(sPurina
Certified Laboratory durationofthe study.
Pellets # The feed
5002) and water was analyzed by
were available ad the manufacturer
libitum for the for concentrations
of
ssppeecciiffiieedd nhuetarvieyntmse.talTsh,eawfalatteorxiwna,schalnoarliynzaetdedohnyadrroectarrobspoencst,ivoergbaasniosphfoosrpshpaetceisf,ieadnd
umiscerdofoorrghaonuissimnsg, tpehsetainciidmeasl,s.alkPaelrisniotnyn,ehlehaavnydlmientgalasn,iamnadlshaolrowgoenrsk.inSgawniittihziendthceagainnigmawlas
facilities were required to wear suitable protective garments and equipment.
rAannidmoamllsywaesrseigqnueadratnotisnteuddfyogrroseuvpesn anddaywsebreefoirndeibveiidunagllpylawceeidgohnedstpurdiyo.rAtnoidmosailngs.weArlel iadneinmtailfsiewdebryeedaorsteadg.baDsoesdeuoprotnrtehaetmienndtivgirdouuaplsbwoedryewiediegnhttisf.iedAnbiymcaalgsewcearrde/luanbielq.uely
fAotltlhoewetderbmyinpaetnieotnroafttihoneosfttuhdeytahlolrsaux.rviving animals were euthanized by CO, inhalation
90 SOLUBILITY AND STABILITY:
# oTfhtehetesttesatrtairctliec,leT-wa6s29e4v,alwuaastseudpipnl2ie%dahsigah wvaisxc-olsiikteyacmabrbeorxcyomleotrheydlcseollildu.loTshee(sCoMluCb)ilaitnyd (th4i0s%w:a6s0%n,otv:avs)uiatnadblaetrvaenhisclluec.entS,olcurbeialmi-tycowlaosretdheenmuelvsailouantweadsinobatcaeitnoende:actoarn oil o`cfotnicmeen.traSthiaoknionfgatphpirsobxiilmaayteerlryes4u2l1t.ed75inmgth/emlr,e-wemhuilcshiffiocratmieodnoafbtihleayemrixatfutreer.a short period oAfctehteontees:tcmoamteoriila(l4u0n%d:e6r0t%h,e dvoivs)inwgacsotnhdeitvieohniscolfeotfhcihsoaiscseayfiosrtthheisraessspaoyn.sibTihlietysotafbtilhiety sponsor.
CHV Study No.: 17385-0-455
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`CORNING Hazleton
100 DOSE SELECTION STUDY
101 Dose Selection
Dose levels oral gavage
of for
1000, 2000, 3000, the dose selection
4000, study.
and
5000
mg/kg
were
administered
by
102 Dosing Information
``TwheeigahntirmaanlgseoufstedheinatnhiemdaolsseusseeldecitnitohneadsossaeywrearnegedfoisneddinognasMsaarycwha6s,
1996. 26.6-
The 34.7
waenrde23p.r1ep-a2r6e.d7jugsrtamprsi,orfotrotdhoesmianlgeasndawnedrfeemparleepsa,rreedspbeyctmivaekliy.ngDaos5i0n0gmsgo/lmutlions
ascteoctkonfeor(Stihegmhai,ghLodtos#e2(4530S00KHmXg/Gk)g:).coTmhiosilw(aDsukper'espacroedmboiyl,adLdoitn#g 7S.D517m1l2:o4f6),
b(i4l0a%ye:r60(%bo,tVt:ovm)a1n0d5t.o0p0)25thgatobfeTc-a6m2e9a4n, eremsuullstiinogniunpaotnrsanhsalkuicnegntwittahn aanfdinyaelllow
3v0o0l0umaenodf1400.000mmgl./kDgilduotsieonlesovefltsh.iAslsltdoocskiwnegrsetpocrkespawreerdefpolratcheed1o0n00m,ag20n0e0t,ic stir
plates during the dosing procedure.
`Dwoeseiknsgawnadstwacohdiaeyvsedoludsiantgthae1t0i.m0eomlf/kdgosdionsgi.ngAnvooluutmlei.neoAfltlhaenidmoaslisngwesrceheeimgehtis found in the following table.
DOSE GROUPS
TREATMENT
MF
T-6294
'
1000 mg/kg 2000 mg/kg
303 303
3000 mg/kg
3003
4000 mg/kg
303
5000 mg/kg
303
All doses given were used in this assay.
on an
acute
(one-time
only)
basis.
A
totalof
30
animals
was
CHV Study No.: 17385-0455
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CORNING Hazleton 103 Results and Interpretation
sAltludayni(tmharlese wdeayrse)efxoarmtionxeicdeafffteecrtsdoasnidn/goranmodrtdaaliiltyietsh.roAulglhoauntimtahlesduaprpaetairoendo.fthe normal immediately after dosing. hAyppporaocxtiimvaetaenldy h1uhnocuherda.fter dosing, all animals in all dose groups appeared hAyppporaocxtiimvaetaenldy 2t4heho5u0r0s0amftge/rkdgosdionsge,galrlouanpiamlaslosaipnpaelalrdeodsheugnrcohuepds. appeared hAyppporaocxtiimvaetealnyd 4h4unhcohuerds.afStoermdeosainnigm,alaslianntihmeal1s00i0n,al2l0d0o0s,eagnrdo3u0ps00apmpge/akrgeddose 5le0v0e0lsmhga/dkgsqduoisnteeldeveeylesshaanddodtyhseprsnehaaadnddyasllpnheaad.rSouogmhehaainricmoaaltss.inOtnhee 4fe0m0a0laend (#6689) from the 5000 mg/kg dose group was found dead. hAyppporaocxtiimvaetaenldy h7u4nhcohuerds.afTtheer dmoosritnagl,itaylldaantaimfaolrsthiinsaallssdaoyseargersouumpsmaarpipzeeadreidn the following table:
`Summary of Mortalities Within 3 Days in Mice Dosed Acutely with T- 6294
Observations
-- Tr10e0a0tmmge/nktg
'
2000 mg/kg 3000 mg/kg
4000 mg/kg
5000 mg/kg
104 Conclusion
Ma0l3e I 3 3 3
Fem0a3le 3 03 03 73
A
4B0a0se0dmoenkt.hese results, the maximum tolerated dose was estimated to be
CHV Study No.: 17385-0455
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CORNING Hazleton
110 MICRONUCLEUS STUDY
ILI Dose Selection
4B0a0se0dmogn/krgeswuletrsefsreolmecttheeddfoosretessetliencgtiionntshtisudsyt,uddyo.se levels of 1000, 2000, and
112 Micronucleus Assay Dosing Information
C`Tyhcelaonpihmoaslpshuasmeiddein(tCheASmi#cr6o0n5u5c-l1e9u-s2;asSsiagymaw,erLeotdo#se4d4Ho0n48M6a)r,chthe12p,os1i9t9i6v.e `caonndtrwoal,s waadmsisnoilsutbeirleidzebdyionrsatlergialevadgeeioanti8z0e.d0wmagt/ekrg.(LoTth#e 1v9e,hpicrleepacroendtraotl,CHacVe)tone 4(S0i%g:m6a0,%L,otv:#v,2w4a3s5KadHmXiGni)s:tceroemdocioln(cDururkeen'tslycowimthoilt,heLottes#t aSrDti1c7l1e2a:t4a6)v,olume of 2150..03-m3l/5k.g6.anTdhe21w.e4ig-h2t8.r4anggreamofstfhoer atnheimmaallseussaenddinfetmhaelemsi,crroenspuecclteiuvselays.saTyhweas dosing Solutions for the assay were prepared by making a400 mg/ml stock for the ahritgihcldeouspet(o4a00v0olmgu/mkeg)o.f 2T5himslwaansd psrtierprairnegdvibgyoraodudsilnyg wtihtehvaehsipcalteultao. thAe test strhaanksilnugcewnatstaonbtaanidneyde.llDoiwlubtiiloanyseorf(tbhoitstosmtoacnkdwteorpe) ptrhaetpabreecdfaomretahne ermeumlasiinoinngupon pdroespearleavteilosn. aAnldltdhoesdionsgisntgopckrsocweedurreep.lAacesdecoonndmaggrnoeutpiocfsatniripmlaaltses(dduersiinggnated hSiegchonddoasreoyfDotshee tGerstouarpt)icwlea.saTlhseoseasasniigmnaeldstwoetrhee osntluydyuasendd iwnatshedoassesdaywiatsh the replacements for any which died in the primary dose group.
hTaernveasntitmiamles g(friovuep.malVeehsiacnlde afinvde pfoesmiatlievse)cwonetrreolragrnoduopmsl,yeaustshiagnniezdedto each dose/-
`
adpopsreodxwiimtahtetlhye t2e4sthaorutrisclaefwteerrdeoseiuntgh,anwiezreed ainpcplruodxeidmaitnetlhye2a4s,sa4y.8 aTnhde7a2nhiomuarlss
after dosing for extraction of the is found in the following table:
bone
marrow.
An
outlineofthe dosing
scheme
CHV Study No.: 17385-0-455
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(00381
CORNING Hazleton
Dosing Scheme for Micronucleus Assay
Number ofAnimals Assigned
Primary 24H
Dose Groups~~ 48Hr 72Hr
Secondary Groups
Dose
rTraeamtmeentt T-6294
000MMFF MMF F MMFF MaMallee FFeemmaallee
100 mg/kg
55 55 55 - .
200 mgikg
55 55 55 - -
400 mg/kg
55 55 55 5s
Vehicle Control, 40%Fria
Co
ZPoosstiitivvee CCoonnturooll,,CCyycclloopphhoosspphhaammiiddee,,0800.0mmgg/hkggS5 55 _ -- -- >- -- oo-
* Trheeplaanciemaanlismaalsssiwghniecdhtoditehde sinectohnedparriymdaroysedogsreougprsowuepraet dthoesehdigahnddowseerleevoenll.yAulsledexttora `animals not used as replacements were euthanized at the completionofthe tral.
`The
ageofthe animals atthe timeofdosing animals was used in this assay
was
eight
weeks
and
one
day.
A
total
of
120
Volumes dosed were 10.0 ml/kg based upon individual animal weights. 120 BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS
`Aptentehteraatpipornooprfitahteethhaorrvaexs.t Ttihmee,adthheerainnigmsaolfstwteisrseueeuatnhdaneipziepdhywsietshofCbO,o,thfoflelmoowread wbeyre
croenmtoaviendi.ngT3h-e5mamrirboowvwianessfelruusmhe(donferotmubtehefboorneeacahndanitmraaln)s.ferFroeldltoowicenngtrciefnutgreiftuugbaetsion to
"pewlelreetstphreeatidssoune,sltihdeessuapnedrnaaitradnrtiewd.asTrheemoslvieddesbwyerasepifriaxteidoninamnedt`hpaonrotlio,nsanofdtshteaipneeldleitn
May-Grunwald solution followed by Giemsa (Schmid, coverslipped using Depex mounting medium.
1975).
The
air-dried
slides
were
eTrhyethsrloicdeystew(ePrCeEc)odtoednoforrmoancahlryosmisa,tiacndersyctohrreodcyftoer m(iNcCrEo)nuccelleliraatniod.thSetapnodlayrcdhrfoomramtsicwere muiscerdo1n0ucrleecaortdedthceesllesdwataas. eOxpnreestsheodusaasnpderPcCeEntSpmeircraonniumcalleawteerdeceslclosrebda.seTdhoenftrheequtoetnacly of PCEs present in the scored optic field. The normal frequencyofmicronuclei in this
Crl:CD-1*(ICR) BR strain is about 0.0-0.4%.
CHV Study No.: 17385-0455
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600382
CORNING Hazleton
N`TCheESfroebqsueernvceydofinPCthEeSopvteircsfuiselNdCs EwhSilweasscdoertienrgmtihneedfirbsyt s1c0o0r0ienrgytthheroncuytmebse.r of PCE and 13.0 EVALUATION CRITERIA:
131 General
MTihcerocrniutcelrieaifwoerrtehedairdkenltyifsitcaaitnieodnoafndmigcernoenraulcllyeirowuenrde, athlotsheooufghSaclhmmoindd(a19n7d6). arinndgwshearpeegdemniecrarlolnyucbleetiwoececnas1i/o2n0alalnydo1c/c5urtrheeds.izMeoicfrtohneucPlCeEi.haTdhsehaurniptboofrdsecrosring ``wmaosrethtehamnicornoenumcilceraotneudclceeluls, wnoatstchoeumnitcerdoansucolneeusm;ictrhounsutchleeoactceadsPioCnEa,l ncoeltltwwioth(or omforPeC)EmsicarnodnuNcCleEiS. (Tbhlueissht-agirnienygapnrdocreedd,urreesppeecrtmiivtetleyd).the differentiation by color
13.2 Data Presentation and Interpretation
IDnadtiavairdeuasluamnmiamrailzdeadtabyarseeaxlsaondprdeossenetgedr.oupTshefoarntahleysdiisfofferetnhtesteimdeatpaoiwnatss..
(pewrhfeonrmvaerdiuansciensg aarneahnoamloysgiesnoefouvasr)iaanncder(aWniknetrr,an1s9f7o1r)meodn(cwithheenr vuanrtiraanncsefsorarmeed
ofhevtaerrioagnecneeowusa)s psriogpnoifritciaonntso(pf<c0e.l0l5s),wiatDhumnincertotn'usctl-eteistpe(rDuannniemtatl,. I19f5t5h;e1a9n6a4l)yswiass
nuesgeadttiovedecotnetrrmoli.neAwnhailcyhsedsosweergreopuepsr,foirfmaendy,sewpearreatseilgyniffoirccanatclhy hdairfvfeersetnttifmreoamntdhe
ssteaxticsotimcbailnlyatsiiognn.ifiTchanet cdroistee-riraelfaotreddeitnecrrmeiansienignampiocsriotniuvcelreeastpeodnPseCEin,voolrvtehdea
dleeatsetctoinoendoosafe rleepverlo.dAucibtelset aanrtdicslteattihsatticianlldyucsiegdninfeiictahnetr paossitattiivsetircealslpyonsisgeniffoircaatnt
'
dleovseel rweasspocnosnesniodreraedstnaetgiasttiivcea.llyIsnigeniitfhiecracnatsae,ndthreepfrinoadlucdiebcliesiionncrweaassebaatseodneondose
scientific judgment.
140 RESULTS AND INTERPRETATION:
dAlulraatniiomnoafltshweeraessoabysefrovretdoxiimcmseydmiaptteolmysaaftnedr/dorosmionrgtaalintdiepse.rioAdlilcaanlliymatlhsroiungthhoeutvethhiecle aapnpdropporsiitaitveehcaornvtersotltgimreosu.ps appeared normal after dosing and remained healthy until the
CHV Study No.: 17385-0455
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00383
CORNING Hazleton
oAlnletheosturaratfitcelre ddoossiendg,graloluapnsiampaplesaartedallnodromsaelleivmemlesdaipapteealryedaftheyrpodaocstiinvge.. Approximately
aApppperaorxeidmastlieglhytl2y1h.y5phooauctrisvaef,tewritdohssinogm,ealslhaonwiimnaglssiignntsohfed1y0s0p0nemag./kAgldloasneimgarlosupin the
s20o0m0e mfgem/aklgesdoaslesoghraoduproauppgehahraeidr hcyopaotsacatnidvel,acwriitmhinmaotsitons.hoAwllinagnismiaglnss ionftdhyesp4n0e0a0,magn/dkg
adlossoehgardouupngarppoeoamreedd hhyapioraccotaitvse,anwditehxcmeossstivsehloawcirnimginsaitginosno.fdyTshprneeeaf,emaanldesmo(s#t72fe4m3a,le7s2
hour were
harvest group; found dead.
7133,
7214,
secondary
dose
group)
fromthe
4000
mg/kg
dose
group
aApppperaorxeidmastlieglhytl4y5h.y5phooacutrisvaef,tewridtohssinogm,ealhlavaniinmgarlosuignh thhaeir10co0a0tsm. g/Akllg adnoismealgsroiunpthe u20n0g0romogm/ekdgadnodsehugnrcohuepd.appOenaerefdemsalilgeht(l#y72h0yp9o)afctriovme,thaen2d0s0o0mmegf/ekmgaldeossealasnodw7e2rehour shlairgvhetsltyghryopuoapctwiavse,fowuintdh dseeavde.ralAlslhoawniimnaglssiignnstohfed4y0s0p0nemag,/kagnddossoemgerfoeumpalaepspeaalrseodwere hunched and had ungroomed hair coats.
eAxpcperpotxifmoratoenleyf6e9m.a5leho(u#r7s19af2t)erfrdoosmintghe, a1l0l0a0nimmga/lksgindoaslledgorsoeugpraopuppesaraepdpehayrpeodacntoirvmea,l, cold to touch, had dyspnea, and was hunched.
p`Tohleyctehsrtoamratticilce,erTy-th6r2o9c4y,teisndouvceerdtnheo lseivgenlisfiocbasnterivnecdreiansetsheinvemhiiccrloencuocnlteraotlesdin either sex
gorroautpa,n2yo4fantdhe48hahroveusrtmtailmeess.frTohmetPheCE1/00N0CaEndra2ti0o0s0inmtgh/ekmgadloessefgrroomuptsh,e aponsdit4i8vehocuonrtrol
vmeahliecslefrcoonmtrtohle m4a0l0e0s.mgT/hkegpdoossiteigverocuopntwroelr,eCsPi,gniinfidcuacnetdlysihgingihfeircatnhtanintchreeacsoersreinspmoincdrion-g
+ sntuacnldeaartdederPrCorEssofi2n .bo4t2h%sex0es.1as2%coamnpda5r.e1d4t%o %th0e.v6e5hi%clfeorcotnhteromlasl,eswiatnhdmfeeamnalseasn,drespec-
tively. animal
The data
data summarized by dose group are found in Tables 2 through 7.
are presented in Table 1 Historical control data
and individual are presented in
Table 8.
CHV Study No.: 17385-0455
14
6006384
CORNING Hazleton
150 CONCLUSION:
`Tmhaerrteoswt pmoalteyrciharlo,mTa-ti6c29e4r,ytdhirdocnyottesinudnudceerathseigcnoinfdiicatnitonisnocfretahsies ianssmaiycraonnducilsecioninsibdoenreed negative in the mouse micronucleus assay.
160 REFERENCES:
Dunnett, acontrol.
C.W.:. A multiple comparisons procedure for J. Am. Statist. Assoc.,50:1096-1121, 1955.
comparing
several
treatments
with
Dunnett, C.W.: 491, 1964.
New tables
for
multiple
comparisons
with
a
control.
Biometrics,
20:482-
aHnedddSlalea,moJ.nA.e, ,HiMt.eF,.M:.,ThKierkihnadrutc,tiB.o,noLfamrsiecnr,onKu.c,lMeaicaGsreagmoera,suJ.rT.e, oNfegweenlolt,oxGic.iWty.. Mutation Res., 123:61-118, 1983.
Schmid, W.: The micronucleus test. Mutation Res., 31:9-15, 1975.
MSucthamgiedn,s:W.:PrTinhceipmliecsraonnducMleetuhsotdesstffoorrTchyetiorgeDneetteicctiaonna,lyVsoils.. 4Ch(Ae.miHcoalllaender, ed). Plenum, pp. 31-53, 1976.
Winer, B.J.: Statistical York, Second Edition,
Principles 1971.
in
Experimental
Design,
McGraw-Hill,
New
17.0 DEVIATION FROM THE SIGNED PROTOCOL
7 3D8u.e1%to. unTkhinsohwandrneaosoinmsp,acotnoMnarthceha9n,im1a9l9s6,orthtehreeilnatteigvreihtyuomfitdhiteyswtuadsy.recorded as
CHV Study No.: 17385-0455
15
C00385
`CORNINGHazleton
180 EXPERIMENT DATA TABLES '
CHV Study No.: 17385-0455
16
LuL8sE
`CORNINGHazleton
SPONSOR3:M `TEST ARTICLE: T-6294 A-S--_S--AY: 17385 TREATMENT DOSE
conTROLS VEHICLE
POSTIVE
40P%iACcoimooniel/ CPOOOmghe
TABLE1 MICRONUCLEUS DATA SUMMARY TABLE
HTAIRMVEEST MEA%NMOIF C1R00O0NPEURCALENAIPTCMEEADSSEL. (HR) MALES FEMALES TOTAL 24h 0124006 024002 0074003 | 24h 2420.2 SI44065 3784055 |
RATMIEOAPNC:ESNEC.E MALES FEMALES
0594007 0894007
0744010 0914008
TESTARTICLE
1000mghs 24h Wh Th
A0OmpAs he "he
Th AN0mgkg 2b
4h 7M
0082006 0104004 044004 00002 0I24004
004007
014007 0004002 0104000
0042002 0122006 0302038 0065006 0044002
003003
0124006 0082006 0014002
0064003 | 0ns0m | 024013 | 0044003 | 00B400 | 0124005 |
013004 | 006400 | 0074002 |
1054003 0s200r 054008 1022005 0892006 0482009
0674007 0932008 0482007
0844003 1004003 084005 0824000 1002006 0582008
0764005 0804007 0552003
* Significantly greater than the corresponding vehicle control, p<0.05. CP= Cyclophosphamide.
CHV Study No.: 17385-0-455
17 600387
`CORNING Hazleton
TABLE MICRONUCLEUS TEST- INDIVIDUAL ANIMAL DATA SPONSO3RM:
TEST ARTICLE: T6204 A As SSATYRENAOT:ME1N73T85er RUBaEL e |PpryoOrgryeeE pweanmicoe
Semoun maRvesT aie
VEGECNmOL wsemioicomol nM7emo ooo30 oo0d7m
mTeo ooieom
[r-- crmams wwmooemr ooowsw
mWoBon;
estar
0 mpi mwwoooooorso uiisaw
ETAeo om
2000 mete
nwwoeoao.0
aw iw
wooooo a
,
wotmurs amnmooeoor eesw or ie
moor
FP$E==CNFyicakrpohyaprhaenicee ry NFNCE PNCaErNoiormmaapnroCnEes
CHV Study No.: 17385-0-455
18
600388
`CORNING Hazleton
TABLE3
MICRONUCLEUS TEST- INDIVIDUAL ANIMAL DATA
`SPONSOR: 3M
`TEST ARTICLE: T6294
AAsSASvANY oNOo:vm1738S5
TREATMENT
_--
HOURHARVEST
FEMALE
NAUNMIBMAELR | MPTVCE5Y pRCAETNICOE iooopcesTCR
VEHCLECONTROL 40% Accioneli0%eComail ~~ n 7199 o0 o03e%
2710s0 01 003109
[I
oa
POSITIVECONTROL
cPs00 meng
m ns m o10s4
an mnn esoxm%
oomm om
TEST ARTICLE
1000myn. 17s8 |' 0o5s%o
[ [CI 1 nao
oosms 03
2000 meg
mne eo0 oris
7m3s 24
03
oossto om
400 meng
m0l6 ' 00751s
'
wm m 2 o07n6
_-_--
ws 3 08
MCPN==CMyicclroopnhuocslpehuasmide:
#PMCEN=PCPoElsyc=hrMoimcartoincuecrlyethtreodcyPtCeES.
`NCE = Normochromati erythrocyte:
CHV Study No.: 17385-0455
19 606389
`CORNING Hazleton
TABLE 4
MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA
SPONSOR: 3M
`TEST ARTICLE: T-6294
ASSAY NO.: 17385 TREATMENT
aANImMAL NUMBER
,oPqCOcEWeYN|,
PRCAETNICOE
48 HOUR BARVEST
MALE
TEST ARTICLE
1000 mph. 2000 meg 4000 mgr.
m33s 02 008s9 nnsne 21 008510 ns 11s 7as 2 0098%9 nntn 13 oosn ne 2 059 e74d0 00 o0s9t3 7n4al4 1' 018052 760 103
PMCNE==MPioclryocnhurcolmeautsic erythrocyte `#NMCEN=PNCoEmsmo=chMircormoantuiccleeartyetdhrPocCytEe
'
CHV Study No.: 17385-0-455
20 6006990
CORNINGHazleton
TABLES
MICRONUCLEUS TES-T INDIVIDUAL ANIMAL DATA
SPONSOR: 3M
TEST ARTICLE: T-6294
ASSAY NO. 17385 TREATMENT
N NAUNMIs BMAELR |Cpee8s pRcAETR47NICOE
8 HOUR HARVEST
FEMALE
TEST ARTICLE
1000mg 2000 mers. 4000 mae
n a2 es2 oLiso m ano o 0ro m6 ' 038
aJ s LLiots w mso i o05s1 nso 12 m nsoo o0sss m ns 03 o1ss my 0 066
PMCNE==MPioclryocnhurcolmeuasic erythrocyte N# CMEN=PNCoEmso=chMircormoantucieeayttehdroPcCyEtse
'
CHV Study No.: 17385-0455
21 00391
`CORNING Hazleton
TABLE 6
MICRONUCLEUS TES-T INDIVIDUAL ANIMAL DATA
SPONSOR: 3M
`TEST ARTICLE: T-6294
ASSAY NO.: 17385 TREATMENT
aNAUNwMIBMEARL | "CP5CTWE
aRAmTIOe pCENCE
72 HOUR HARVEST
MALE
TEST ARTICLE
1000 mg. 2000 meg 4000 mpg.
n4es 01 0os3s4 nniasst 22 0o4ms ne 2 oa nnaars o1 0o4n7 n13s6 2+ 008340 nse 3 026 nme6 11 ooamr nprt 11 00344s ns 1 03
PMCNE==MPioclryocnhurcolmeautsic erythrocyte
`#NMCEN=PNCorEm=ocMhircormonautcileeartyetdhrPoCcyEtSe
.
'
CHVStudy No.: 17385-0455
22 (00392
CORNING Hazleton
TABLE?
MICRONUCLEUS TEST- INDIVIDUAL ANIMAL DATA SPONSOR: 3M
TEST ARTICLE: T-6294
ASSAY NO: 17385
TREATMENT
--_--
72 HOUR HARVEST
FEMALE
NAUMMBAELR |CPPCMEPNS PgCaETNioC!E ooopsFORNCE
TEST ARTICLE
1000 mgr
2000 mes
000 mes
-_-- M* ANni=mMailcfroounnudcldeeuasd #PCMEN=PPCoElysc=hrMoimcatriocnuecrlyathtreodcyPtCeES `NCE = Normochromati erythrocyte:
a mm oou o03n6
211938 EE
o01
oots os
nnot eo0 0o3s9s
anme 0 30 i
0o3n9
nn o o' 0os a mm i 0o 0o4 s m4 1 059
'
CHV Study No.: 17385-0-455
23 606393
CORNING Hazleton
TABLES
MOUSE MICRONUCLEUS HISTORICAL CONTROL DATA 7/95 THROUGH 12/95
FOOLEDVERICLE CONTROLS MaAX AVG N
MICMREOANNUOCFL1E0A00TPEEDR PACNEIsMFAELR1S0E0.0PCE MALES FEMALES TOTAL
RATIO PCENCE MEA+SNE. MALES FEMALES
0o0x0 00m0007
"
000 00R0I2+40008
"
oor 017
031 oss
00ss+0005 | "
0ssosoca o
024 0sw1s0o2s
a
PCOySIcTlIoVpEChOoNsTpRhO8a0Lm.S0imdkes, Mi AMVAGX N
`PNCCEE == NPoolrymcohcrhormoamtiacticcryehrryothcryotceyte:
200 362s4a020
i
150 3060360s
15
20 S38
os on
34610018 | 0smiaoo
001490 ossisoeoms
' CHV Study No.: 17385-0455
24 (00394
PCHRVOTOSCTOULDYNON.O. 4E35,DTRIO1N7
`CORNINGHaz
NI
eto
IN YIVO HOUSE MICRONUCLEDS ASSAY
LCtoahrebnowironagrtokrHyaiznlPperrtaocogtnriecIsensc.(aGnL(dPG)Vt)hReegwuifllialntailcoonnrsde.upcotrtThtwihisilslprsobtetuodcysoulbi,jneccctormitptoliicaaaunldciepthwasibety(hsq)Guoalooifdty Acsosnudruacntceed biyn aCHcVcoradtan5c2e00wiLtehesbSuOrPgs PatikeC,orViineinnnga,HaVzilregtionniaTnc3.5163T.he stusy will be
PART 1. SPONSOR INFORMATION AND APPROVALS I. sponsor pmvrIFrGATION
Company Meme: _0 _3p0000000
Address:-- St.lFoatd maadl
00000000
II. EST mrrcie roewrrercation: _T = 6294 r--e --------------------------------
01. 3EST ARTICLE ANALYSTS
Dcehtaerramcitenraitsitoincsofasthedeftiensetdaritnictlhee of the Sponsor.
GsLtPabirleigtuylatainodnstheis ttehset
arretsipcolnesibility
IV. NOTIFICATION OF REGULATORY sumMISSION / msIuunsbtnoirsdbseeironnot.toifcioCemHdpVlmyLafwiinattlahlinotsrheapGamLra?tstoreferg4ulsacsthtiueoddnyusl,eisocfoinnstsuetunlddtieiednsgfvolhraibcorhreapftlaoalrclieerswsader
trheegulraetsourlytsreovfietwh.is Pslteuadsye indicate which agency, if any. might receive
= Undetermined J -- C--2 wer C--0 wom
FA L--D Ea-TScA o--d omer 3 -- ome
L--d
EA-FIFRA
ass
1 of 10
600395
PROTOCOL NO. 455, EDITION 17
v. stor pares
Proposed Experimental Start Dates Proposed Experimental Termination Date: VI. APPROVAL OF STUDY ERoTocoL
Study Director:
FomaTatha Varll, FR,
Sponsor's Authorized Representative:
Shc
Gro
`CORNING Hazle
ING
eon
Dates
suse: 1/045
`
ares
2 of 10
000396
PROTOCOL NO. 455, EDITION 17
CORNINGHazleton
PART 2 - STUDY PROTOCOL
IN VIVO MOUSE MICRONUCLEDS ASSAY I. OBJECTIVE
cThleastoobgjeencitciveactoifvitthyisansdtuddyisrisupttioonevaofluatthee maittoetsitcaratpipacrlaetufsorin polychromatic erythrocyte stem cells in mouse bone marrow in vive.
II. pEFDNITIONS Mcihcrroomnouscolmeeu(ss:) aand/somrallofcahcreonmtartiicn bcohdryo,mocsoomnesisftrianggmenotf(se)n,tiwrehich clahgrsobmeohsion(dmse)atamnidtoftriacgmeannta(psh)asem.ay Anfotterbeteilnocplhuadseed,inthtehsee daughter ncuyctloepil,asma.nd say form single or multiple micronuclei in the
II. RATIONALE
aThgeenmtsicraonnductleesuts artteisctlecsanwhsiecrhve inasteraferraepiwditshcrneoernmaflormictloatsitcogecnelilc
dfoirvmiesdionfro(mSchcmhirdo,mos1o9m75e;s oHreddclheroemtosaolm.e. fr19a8g3m)e.ntsMilcefrtonbuechlienidadreuring
e(nSacphhmiads,e a1n9d75)c.an bIen sthciosreadssdauyr,ingpoliynctherropmhaatsiecbeecrayutsheroctyhteeyspe(rFsCiEss)t
in the During
bone marrow are maturation from
esrcyotrehdrobfloarstthetopreersyetnhcreocyotfemitchreonnuucclleeiu.s
is
extruded, Detection
wohfilmeicmriocnruocnluecileiin, noinf-npurcesleenatt,edrceemlalisn
iisn
tthheuscytoplasm.
! finacitlrietaatteedd,celalndptoipmuelatiinovnoslveids ienlimsienaartcehdi.ng Tfeosrtmeatratpihclaesse spreads
calfafsetcotgienngicspaignednltes-ficbaenr befudnectteicotnedor thfroorumgahtimoincraosnuwcellelusas induction
(schnid, 1975).
TV. MATERIALS
A snimels
Y1o0uwngeekasdulotldmaaltethaendtifmeemaloef
mice of dosing,
the ICR will be
strain, purchased
SprroanguCeh-aDralvelseyR,iveIrnc.LaboTrhaitsorsieersa,inIhnacs.,beoernHasrellaencted to
alos
3 of 10
06397
we CUFT AVAIL ABLE
PROTOCOL NO. 455, EDITION 17
CORNING: kion
naxinize genetic heterogeneity and at the same tine
5 contol arsicies a1Cn0ydcnllwoipklhg]o)spbwehialmadiddmeibnei(suCtsPe,crde8da0smbgyt/hkeogr:aplosdiogtasiviavngege.cvoonltuTrmhoeelvoeafhritcircele utCshoeendtrsoaflmoerartrthoieuctleteeasstw.ilalratnidccolcnesoinascntudrrowefinlttllheybwesiotalhdv,meinnttihseotrerrevseetdhicbiye aadrmtiincilsetearnedd into atmhoeuntesxpeerqiuemlenttoa.l tahenimmeaixsi.mumThveoludmoessing SvSaoodrllmeuuiymtnieiosanwtr,ieraltlwoiafotnnecosrot.,rne0xs.cTi3ehl%ee.dvaeq2hu0iecomlules/skgcgaerfnoberorraorlrmlaeylihwgyealevcdaegleiinaalntodhseIP
Vv. pempeow peso A nisl Husbandry "ASLieL1v1e"naEpegpelriicsaocblalageeteddCuArYbiynSgOsPexsq.uawrilaAlnntiiobnaeel,sfowalinlldowlewdib.lelhoAbunesiemhdaolusseepd to 5Gtpe2mptehoroauFtsiuevrdee,upnrdi7eo2rrttoh4e6exF%po;elrliohmvueimnnitgditciyln,iimtai3ta5it1icone:o15n7d:iasniiiomgnahslt:s vc(ayFtcuelz:ei,fwais1l2lCohobreuerisafivealdiilgahLtba/lbdeoarraakdt.oryAAiCbhciootuwmmmser1cT5ih0ae02l)Fedeiadentdisap
` RSSynpdaerlcoyicstaeirdehdobnhyse,atvhyeorgmmeaatnnaoulpfsha,ocstpushriaeltarecsof,moirna,ncdocnshcpleeoncrtiirfnaiatetideodns of pFnieetstetriioecsniptdesec.st,ivehTehaebvaywsaistmeetrfaolriss,sapanelackliaytlsiieendditmbyil,eermoaeonardlglehnyailooongnse.nas. Ebreiinogaisplawcieldl obne sqeueadrya.ntined for at lease 7 dare before APanrciocncoaerlddusirnegwsi.ltlo ACbnoeinniaansigssigBwneiealdllettoobneswtSeutidagynhdegadrrodupprosipoerartattiornagSnodsoimng. wtTeahgie.gyhtwsTi.lrleatbmaeennidtmoaslgesrdouwbpiaslslewdibleulpounnbeiqtuhieedleynitnidifidiveeinddtuiafbliyedcaangbieymalear 1251 cara.
ass
"ot 10
00398
PROTOCOL NO. 455, EDITION 17
CORNINGHaz] cron
Sanainmiatlasryorcawgoerskiwnigllwibtehiusnedt.he Paneirmsaolnneflachialnitdileisngwill beequirpemqeunitr.ed to wear suitable protective garments and
B. Dose Selection
The high dose generally maximum tolerated dose.
will The
be selected as 801 of the high dose should produce
some indication of toxicity (e.g., of ratio of PCEs to normochromatic
death, depression erythrocytes
(NCEs). One-half and will normally be used
one-quarter of this as the intermediate
high dose and low dose
Levels, respectively. the likelihood that a
Use weak
of a high clastogen
dose will
increases be detected,
and is therefore recommended.
If no appropriate finding study can
range finding be performed.
data The
are top
available, a range dose tested in the
dose
rangefinding study will be S000 mg/kg. The dose levels tested
will be issued as an amendment.
DOSE RANGEFINDING STUDY
The dose groups.
rangefinding study will Each of the five groups
be conducted will consist
using five treatment of 3 male and 3
female mice.
Group Designation and Treatment Regimens
Group No.
Number of Mice Male Female
Duration Route Days
1
3
3
ro
3
!
_--
2
3
3
ro
3
3
3
3
20
3
"
3
3
ro
3
s
3
3
20
3
4195
5 of 10
6006399
PROTOCOL
C. / ass
NO. 455, EDITION 17
CORNING
etn
Tibthunheaejtnecrtotsuieetsoletnecwatoirefltdliacbdlembeecianuiccssaheptalrroaatyctheteidyeo.rniasrvetTihilecltsshee.bepnrreeooscuretalleusdcegoamovfomaoegsnsed.cngboaasvlItaneagseet,ohhfetiIobenvehnatve
niaTonoitucatireenasimxsscotcefeudalatadi2ra0o,innmilfss/oukirbgrcatuthftiioaosrnneotoretuashslattsgpmdraanovyicanegdiebusercaerwn,aedceidIoFTnarsceaedoadfiodsnnbitrynsegierFseosesronsiim.oinnmnse.TwheilOltthesetr
mSSaootllevuretniitoasnl:, wiwoalrtlecrog,renne0r.oia5ll7.lysaAlbLienLes,Aonlibum.ba5il%lsiazqoeudieloiunsbeoncedaerrobefedrtyhvmeeechfdyoiloeleaoisnienstoss
pIrnodtiovciedluddasebnoddayonwee.sghis. Dose Levels will be sestpme burs
wBpiorldelypawbzeeeidgpahrntedspawrhieeldlle jbauetst atmpabrkiieeonnrtptrotieomrdopsetironagtd.uorsiensDger.wianegiD.lossaioonbsgiitnifgo.onrs(m0as-i3tiiionne
Tsoiunrgsl)e.dovAeL.L animals Will be suthanised 3 devs afver Ceceicing &
Tf{hooerhaatlhunetaiadolnusrafwtoiillollnowebodef otbbyhseepresvnteeeddtyr.adtaiiolAnyvifosfoaristhtewoixlitlchorbseeirg,nesetshaedninmeodrtabylithye
wWTiAhlLe1LdabteihleynUseobdebssetarosviaegsttniieomdnastef.oorfthttehoexHiacssuibsasyuemmqputeoTnmetsleraeenrcdve/odoprsmDoemrnoeir.ctya(lHiisbto)i.yes. dpaatcaes
vicRowerLsus stony
Dosing Schedule and Route of Adminserstion
HNaaodzrmvmieansiltsltyrwaiatlniloancb)eutwesiplpdlroossbiiensgautsreeeldgyim(2esane,e (a4s8pi.ineg.l2ebehlooewe)s.after
SsCoodnpitrnrosoxlciarsaatrtetiilocylne2s4o.fhothAuer.sortaeafsltteroafrtaid1c1ml0ien:iasnciarmnaadtteiaosnwito)f tbehe
aaUtnseidne.aalcshEqtcuroaenlastimnseutnmitbnegrgsroofuopfs..mLa0lAemnsalaedasndditainFdoenmasoliie1s5srowfiselppleroefbse
used nay
oTbfoexidtcohiesteydtesaitas maeaxtpseeerccitoaenldd.aratyTdtiohsesehgigrgrohouuppdweiwslielthanbaetheathoeemeistahniimdfaoless
aiSnneycotwnhhdisaircyhgrdodouisspeWpAgrLxiLoorupontwloiylelubtehbaeunsaedsseitaea.rsmirnTeehpseiswbceyeemtnhotefn stehonerdy
director, Freshly prepared sotucions wil) bo
oof 10 01000
PROTOCOL NO. 455, EDITION 17
CORNINGHazlefon
etmopxlicoyesdi.gnsThaendamnoirmtaallsitwyi.ll be observed daily for
NQMIER OF ANIMALS USED FOR MICRONUCLEUS ASSAY
Group No.
Irestment
Harve(sttaleTsinaensdAfFteemarleTsr)eatment 26 Hours 48 Hours 12 Hours
Tota
21 vPeohsiictlievecCoonnttrrooll 55 4+53
3 Low bose
545
--ol sis
Ishsess sis 1341s
5 HiMegdhiumDoDsoese
354455 554+s5 sse4ss 11354431ss
ToTAL
25425 15415 1541s ss ss
Tthhee erovuetnet tofhatadmtiesntisatrrtaitciloen wcihlalracbteeroirsatlicgsavapgree.cludIen
odofroaslaindgmgiavnvaiogsletu,mreatIwPiiolnilnhjnaeovctetiobenexecwneieldslel20ebectmlee/dmkpglb.oeyceaduT.sheesetThheeryoutaecse
tphreocmedousrte.comOmtohnerrourtouetsesof ofadamdimniinsitsrtartaitoinonforthatthimsaytebset
ausdeadiniasretraitnitornasvenoorusb,y ifenetdr.amuscular, sub.cutancous
D. Extraction of Bone Marrow
etExhutotrrhaaacxn,taisoianan.d wihliTlhned bmelairwmirbtohwbownCieOls,lwifbolelllfoblweuesdhreebdmyovfperedonmeftotrrhaetmiaboroncneoowfantdhe
.
tsreaznusnfe(rorneedtutobecefnotrriefaucgheantiumbaels).containing 3-5 ml bovine
E. Preparation of Slides
Fsoulpleorwniantgantcewnitlrlifubegatrieomnovetdo pbyellaestpirtahetiotnissauned, potrhetions Of the pellet will be spread on slides and air-dried.
MTahey-GsrluindvesaldwilSlolutthieonnbeandfiGxieedmsian.meatnhdanporlo,tecstteadinebdy in msoluindetsingarweitchodecdovefrosrliapnsa.lysiFso.r control of bias, all
"rss
7 of 20
01001
PROTOCOL NO. 455, EDITION 17
CORNING Hazleton
F. scotrheiSlnidges Aannimaatlt.emptThewilflreqbeuemnacdyeoftomisccroorenuocnlee-attheoduscaenldlsXwCiEsllpebre neuxmpbreersseodf PasCEspearncaelnytzemdi.cronTuhcelenaotmeadlceblalcskgrboausnedd on the arroeuqnudenc0y.0-o0f.4m7i.cronuclei in the ICR mouse strain is
T(hNeCEsf)rewqiulelncybeodfetCeErsminveedrsubsy msactourriengerthyethnruomcbyetresof PsCcEosrinagndthNeCEsfirosbtser1v0e0d0 eirnytthhreocoyptteisc ofnieltdhse wshliildee.
VI. pam
SThcehmicdrit(e1r97i6a).forMitcheroniudcelnetdifairceatidoanrklofy msitcarionneudcalenid agreenertahlolsye of orcocuunrd., aMlitchoruognhuclaellmonhdaveandsharripng-bsohradpeerds maincdroanrueclgeeineroaclclaysiboneatlwleyen m1i/c20roannudcle1a/5tedthecelsli,zenootf Tthhee mBiCcE.ronuTchleeuusn;itthoufsscthoerinogccaissiotnheal mceilclronwuicthleamtoerde BtChEa,n noonte mtiwocro(nourclmeorues)imsiccroounnutceldeia.s one PThoelycshtraoimnaicnigc parnodcenduormeocpherrommiattsictheerydtihfrfoecryetnetsiat(ibolnuibsyh-gceoelyoranodf red. respectively). Date Presentation nThuembedrataofrmeipcorrotneudclweialtledincPClEusd,e tthhee pnuemrbceerntaogfePoCfEsmisccroorendu,cletaheted / eBCrEyst,hroacnydtetsheforratieoachofepxopleyrcihmreonmtaatlicanitmoaln.ormochromatic Evaluation criteria
Tshiegnicfriictaenrtiadofsore-raelpaotseidtiviencrreeasspeonsien miiscraonsutcalteiastteidcalBlCys, or the dreetsepcontsieonfoorf aatrleeparsotducoinebledosaendlesvtealt.istAicatleslty asritgincilfeicatnhtatpoisnidtuicvees nsetiatthiesrticaalsltyatissitginciafliclayntsiagnndifirceapnrtodudcoisbelereisnpcorneseasenorataone dose ilseveblaseids ucpoonnsidsecrieedntinfeigcatijvued.gemeInnt.either case, the final decision
ales
8 of 10
01002
PROTOCOL NO. 455, EDITION 17
VII. IEST INTERPRETATION
CORNINGHazleton to
vTahreiaanncaelys(iWsineorf, th1i97s1)datoan weiiltlherbeupnetrrfaonrsmfeodrmuesdin(gvhaenn homogeneous) or rank transformed (when variances are
vaanrailaynsciess oafre
hItfe-tteetrshteogea(nnDeauolnuynsse)itts,profo19p5ov5ra;triioa1nn9sc64e)ofiwsiclesllilgsnbeiwfiiucsteahdntmitco(rpod<ne0ut.ce0lr5)em,iinep&ewrDhuinacnnehitmatdl'o.sse
gctroionmuetprsoa,ln.difsAenxaanylc,yosmeb`asicneawtisiliolgnn.ibeficpaenrtfloyrmeddiffseerpeanrtateflryomfotrheenaecghathiavrevest
VIII. REFERENCES
Dturnenaettmte,ntsC.w.i:th Aa mcuolnttriopll.e coJ.mpaAmr.isoSntsatipsrto.ceAdsusroec.f.orS0c:o1m0p8a6r-i1n3g21s,eve1r9a55l. DBuinonmeetttr,icsC,.W2.0::48N2e-w49t1a,ble1s964f.or multiple comparisons with a control.
HNeewdedllle,, JG..WA..,anHditeS,alMa.m,oneK,irkMh.aFr.:e, BT.h,e iLanrdsuecnt,ionK.,ofMmaciGcrreognourc,leiJ.Ta.s, 2 measure of genotoxicity. Mutation Res., 123:61-118, 1983.
Schaid, W.: The micronucleus test. Mutation Res., 31:9-15, 1975.
SCVochlhe.mmiidc,al(AW..H:utHaoglTelhnaese:nmdiecrrP,orniuneccdil.)pe.luess PtlaeensndtumMf,eotrhpopc.dysto3g1fe-o5nr3e,tTihce1i97ra6n.aDleytseicst.ion:In.
WMicnGerra,w-HBi.lJl.,: NeSwtaYtoirskt,icaSlecoPnrdincEidiptlieosn,in1E97x1p.erimental Design. ` IX. REPORTFomuAT
CfHiVnalemprleopyosrtawisltlandpraorvdidreepotrhte ffoorlmlaotwinfgorinefaocrhmaatsisoany.design. The
o 419s
QSupaolnistoyr Aisdseunrtainfciecatsitoant.ement. SStiagtneamteunrte ooff GsLt?udyComdpilriescntcoer.. PTehsytsicaarlticdleescriidpetnitoinficofatitohen atnedstCaArVtiSctluedyanNdumbdeart.e oAf Tryepceeipotf waislslaybeandincplruotdoecdolinnutmhbiesr.section. DSattuedsy doifrescttuodry ainnditisaetniioorn atnedchnciocmipalne:tion. ethods.
001003 9 of 10
PROTOCOL NO. 455, EDITION 17
`CORNINGHazleton
Evaluation criteria.
Interpretation Conclusions.
of
results.
References.
Test results presented in tabular form.
X. CHANGES OR REVISTONS
AsniygncehdanbgyestheorSrtuedvyisiDoinrsectoofr,thidsateadp,proavneddmapirnottaoicnoeldwwiillthbethidsocupmreontotceodl,.
XI. ANIMAL CARE AND USE STATEMENT
Ianpprtohperioaptien,iontheof stthuedyStduodeys nDoitrecdtuoprl,icantoe aalntyernparteivivoeustewsotrikngwmietthhotdhsis are
material, and protocol will
the number and be reviewed by
species selected are appropriate. the CHV-IACUC for compliance with
This
croemgpulliaatnocrey, gauimdeoldiinfeiscatcioonncewrniilnlg bethereqcuairreeda.nd
use Any
cofhanagneismalosr. reIvfisniootns in
of this review.
approved
protocol
will
be
sent
to
the
CHV-IACUC
for
their
XII. RECORDS TO BE MAINTAINED
AgLeLnerraatweddataas, adorceusmueltntaotfiotnh,is resctourddys,wilplrotboecoalrsc,hivaendd tinhe thfeinasltorraegpeort
facilities submission
of of
Coning Hazleton Inc. for at least the final report to the sponsor.
one year following After the one year
period, the retained in
sponsor may the storage
elect to have facilities of
the aforementioned materials Corning Hazleton Inc. for an
atdheditsipoonnasolr.period of time or sent to a storage facility designated by
'
01004
4l9s
10 of 10
AMENDMENT TO THE STUDY PROTOCOL
STUDY TITLE:
IN VIVO MOUSE MICRONUCLEUS ASSAY
PROTOCOL NO.: 455, Edition 17
STUDY NO.:
17385-0-455
Page 1 of|
`Amendment #1
Section 2, Part V.B.
The Sponsorhas LDy, data in ratsof 2459 mg/kg in males and 1580 mg/kg
cinonfdeumcalteesd.teBsatsinegddoonsethgirsoiunpfsoromfat1i0o0n0,, t2h0e0d0o,s3e00se0l,e4ct0i0o0n,satnuddy will be
5mi0x0t0umrge/kg. The test article will be solubilized in acetone/corn oil
STUDY DIRECTOR
Nawostatin Facts
Hemalatha Murli, Ph.D.
Mammalian Cytogenetics
Department of Genetic and Cellular Toxicology
,
2).28/96
Date
01005
AMENDMENT TO THE STUDY PROTOCOL
ee STUDY TITLE: IN VIVO MOUSE MICRONUCLEUS ASSAY PROTOCOL NO.: 455, Edition 17 STUDY NO.: 17385-0455
Page 1 of1
Amendment #2 Section 2, Part V.C Based on the resultsofthe dose selection study, dose levels of 1000, 2000,
`and 4000 mg/kg will be tested in the mouse micronucleus assay. A secondary dose group will also be used.
STUDY DIRECTOR | Nowalatis He Hemalatha Murli, Ph.D. Mammalian Cytogenetics Departmentof Genetic and Cellular Toxicology
2 )q Date
01006
