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ORIGINAL ~~ A#R226-0953
COVANCE.
FINAL REPORT L5178Y TK ** MOUSE LYMPHOMA FORWARD MUTATION ASSAY
`WITH A CONFIRMATORY ASSAY `WITH N-EtFOSE T-6316
AUTHOR
Maria A. Cifone, PhD
PERFORMING LABORATORY
Covance Laboratories Inc (Covance) 9200 Leesburg Pike
Vienna, Virginia 22182
LABORATORY PROJECT IDENTIFICATION
Covance Study No.: 20785-0-431 ICH 3M Study No.: T-6316.12
SUBMITTED TO
3M Corporate Toxicology
3M Center
`Building 220-2E-02
St. Paul, Minnesota 55133-3220
STUDY COMPLETION DATE September 05, 2000
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Covance 20785-0-431 ICH 3M Study No.: T-6316.12 QUALITY ASSURANCE STATEMENT
L5178Y TK * Mouse LymphomwaitFhoNr-wEa(rdFOMSuEtaTt-io6n31A6ssay with a Confirmatory Assay The report has been reviewed by the Quality Assurance UnitofCovance Laboratories Inc., in accordance with the Good Laboratory Practice regulations as set forth in the Food and Drug Administration (FDA) Title 21 of the U.S. CodeofFederal Regulations Part 58, and with any applicable amendments. The following inspections were conducted and the findings reported to the Study Director and study director management. Written status reports of inspections and findings are issued to Covance management according to standard operating procedures.
Inspection Dates
May 31,2000 July19.20,2000 August30,2000
Phase
Scoring Draft Report Review Final Report Review
Dates Reported to Study Director and Study Director Management May 31,2000 July 20,2000 August 30,2000
Auditor
S. Ballinger P. Caceres P. Caceres
OO) LD Representative, Quality Assurance Unit
9lsleo Date
-
245
Covance 20785-0-431 ICH STUDY COMPLIANCE AND CERTIFICATION TfohrethsitnudtyhewaFosocdonadnudctDerduginAcdmoimnpilsitarnacteiownit(hFtDhAe)GToiotlde L2a1boofrtahteoryU.PS.racCtoidceeorfeFgeuldaetriaonls as set RreegguullaattiioonnssoPrartthe5s8i,gannedd wpirtothoaconlytahpaptlwicoaubllde aafmfeencdtmtehnetisn.tegTrhiteyroefwtehreesntouddyevoiratthieonisntferrpormettahteion oevfatlhueattiesotnorefstulhtes,teTstheartriacwledaastaprheasveentbeedenherreevinierweepdrebseyntthseaSntaupdpyroDpirriecattoerc,ownhclouscierotnifwiietshtihnatthtehe Csounptpeoxrtteodfbtyheasnteudxypedreismiegnntaalnddaetvaalrueactoirodnacnridtetrhiias.reAclolrtdeshtasanbdeeconntrreovlierweseudltbsyitnhtehiSstruedpyort are Director.
Study Director:
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Genetic
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Cifone, PAD
nd Molecular
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Date
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Sponsor TAessstayArItnifcloermation Study Dates Supervisory Personnel
TABLE OF CONTENTS
Covance 20785-0-431 ICH 3M Study No.: T-6316.12
Page No.
`Test System Test Article Control Articles
D$o9sMeetRaabnogleifcinAdcitnigvaAtsiosnaySystem Mouse Lymphoma Mutagenicity Assay
DAAasststaaeyPrAEevcsacelenuptatattaiinoocnneCCrrtiteenrsia
TDeossteARratincgleefiHnadnidnlginAgssay Mutation Assays.
CONCLUSION.........
c----------------------------
RECORDS TO BE MAINTAINED
--
reresscane
vnreremmons 11 wenn 17
ELSTORICAL CONTROL DATA sivsssusismsmsasssso otomtot reshapes
smsosmomesmiosspss sts 29
-
2467
Y 20.31% ABSTRACT
Covance 20785-0-431 ICH 3M Study No.: T-6316.12
`The obje
T-6316,
ct
to
iveofthis in vitro assay was
induce forward mutations at
to e
the
valuate the
thymidine
ability
kinase
of th
(TK)
e test
locus
article,
in the
N-EtFOSE
mouse lymphoma,
L5178Y cell line. The test article was soluble in the vehicle (dimethylsulfoxide, DMSO) at
125 mg/mL, the highest concentration prepared.
Aredaotsmeenrtanpgeerfiionddoifngapapsrsaoyxiwmaastepleyr4fohromuersd.wiItnhaadndidtiwoint,hoaudtomseetraabnogleifciancdtiinvgataisosnayuswiintghoaut
`dmoesteabroalnigceafcitnidviantgioanssuasyisngweatrreeiantimtieanttedpewriitohdcoofnacpepnrtoraxtiimoantseflryo2m42h.o47ur1s0 w1a2s50peprgf/omrLme.d.InTthhee
presence and absence of rat liver $9 metabolic activity with a 4-hour treatment period,
N-EtFOSE T-6316 was noncytotoxic up to 19.7 pg/mL. Moderate cytotoxicity was induced
from 39.3 to 157 pg/mL and higher concentrations were excessively cytotoxic or lethal. In the
nhooncaycttoitvoaxtiicointydoast e2.r4a7ngpegf/imndLi,nwg eaasskaycyutsoitnogxiaci2t4y-ahto4u.r9t3repagt/mmenLt,:
period, the test article
and moderately high
induced
cytotoxicity at 9.85 pg/mL. Higher concentrations were excessively cytotoxic or lethal. Dose
levels for the mutation assays were based on these results.
In the nonactivation mutation assay with a treatment period of approximatel4y hours, six doses
ranging from 25.3 to 201 pg/mL were analyzed for mutant induction and `weak cytotoxicity to
high cytotoxicity was induced.
exceeded the minimum criteria
None: `of the analyzed treatments induced amutant
foar positive response. A confirmatory assay was
frequencythat
performed.
tIrnetahtemecnotnfpierrmiaodt,orsyevneonnatcrteiavtamteinotnsmfurtoamtio1.n25as1s0ay1,5w.0hipcgh/wmaLswpeerrefaonramleydzewditahnad 2n4o-chyotuortoxicity to high cytotoxicity was induced. None of the treatments induced a mutant frequency that
exceeded the minimum criteria foar positive response. The test article was, therefore, evaluated
as negative without metabolic activation.
I2n5.t3hetoin1it0i1alpmgu/tmatLiowneraessaanyalinyztehde apnredsweencaekofcytSo9tomxeitcaibtoyltiocvaecrtyivhaitgiohnc,yftooutroxtirceiattymwenatssifnrdoumced.
`None of the analyzed minimum criteria for
treatments in either a positive response.
assay induced a A confirmatory
mutant frequency that exceeded mutation assay was performed.
the
Iannatlhyezceodnafinrdmwaetoarkycaycttoitvoaxtiicointmyuttoahtiigohn caystsoatyo,xeiicgihtty twraesatimnednutcsedf.roNmo2n0e.0otfoth1e50trpegat/mmeLntwsere
induced a mutant frequency that
article was, therefore, evaluated
exceeded the minimum criteria for apositive
as negative with metabolic activation.
response.
The test
"The test article,
the TK locus in
NL-5E1t7F8OYSmEoTu-s6e31l6y,mpwahsomeavacleulalsteudndaserneagcattiivvateifoonrainndduncoinancgtifvoartwiaonr.d
mutations
at
,
my
Covance 20785-0431 ICH 3M Study No.: T-6316.12
STUDY INFORMATION
Sponsor 3M Corporate Toxicology
Test Article Sponsor Identification: N-E(FOSE T-6316 Lot 30035, 30037, 30039 Purity 98.1% (August 19 sample) Dates Received: August T19M, 1#:99910a8nd4AJ3un(eAu07g,us2t00109 sample) Physical Description: white to beige solid (August 19 sample) beige chunky solid (June 07 sample) Storage Conditions: room temperature:
Assay Information
Type of Assay:
WLi5t1h7A8YCTonKf"irmMaotuosrey
Lymphoma
Assay
Forward
Mutation
Assay
Protocol No.: 431 ICH Edition 2
Covance Study No.: 20785-0-431 ICH
Study Dates Initiation Date: May 08, 2000 Experimental Start Date: May 10, 2000 Experimental Termination Date: June 22, 2000
Supervisory Personnel Study Director: Maria A. Cifone, PhD Laboratory Supervisor: Kathryn Flanders, MS
OBJECTIVE `The objective of this study was to evaluate the ability of the test article to induce forward `mutations at the thymidine kinase (TK) locusof L5178Y mouse lymphoma cels as assayed by colony growth in the presenceofS-trifluorothymidine (TFT). The assay design is based on OECD Guideline 476, updated and adopted July 21, 1997, and the ICH S2B Guideline, "Genotoxicity: Standard Battery for Genotoxicity TestingofPharmaceuticals"
2-49
Covance 20785-0-431 ICH 3M Study No.: T-6316.12
TEST SYSTEM RATIONALE
Thymidine kinase medium for DNA
(TK) is an synthesis.
eIfntzhyemtehythmaitdianleloawnsacleolglsTtFoTsailsviangcelutdheydmiidnitnhee
from the culture: growth medium,
the
analog will be phosphorylated via the TK pathway and will cause cell death by inhibiting DNA
safyronertwhvaeisraidbsl.meuitCnaetnlilosornmwahtloictchuhletaTruKerTMeh*metegedernioouztymygpboeeucsianautwshtehiedcehTnKliotvtllooecoDursNn(AoTKTs*yK"n)tahmcetasiiyvsitudynodreeesmrnagoiotnsars.eiqnSugiulrceeh-semtxueotpgaenntosus
ttaohrxeyimtcihdaeirneefn.oreToaafKs"tlshuymmmuoeitddanigttnose,haihnvotewoemDvuNetraA,t.eadr,CeeerlieltsshieswrthasinpctohtnotgaTrnFoewToutbsoelcfyaoourrsmeicntodhlueocyneicdeasbninynottthheientcpeorstrepsaorertniaccteleeo,tfhaitTstFhTe
TK" locus.
MATERIALS AND METHODS
Test Test
System Cells. The
mouse
lymphoma
LS178Y
cell
line,
heterozygous
at
the
TK
locus
and
dDre.siDgonantaelddaCslcilvoenaen3d.7a.re2Cs,towraesd
used for in liquid
this assay. nitrogen.
LSatboocrkatcourlytucruelstuwreersefoorbttahienmeudtfartoiomn
assays
wreeprleacmeadinbtyaicnelelds ifnrloomgtahreitfhrmoizcegnrsotwoctkh.bCyulsetruiraelssuwbecrueltgurroiwngn fionraupshtaoke4rmionnctuhbastoarndatwere then
approximately 37C. A continuous cell log was kept to record growth, doubling times, and
subculture operations. To reduce the frequencyofspontaneous TK" mutants prior to use in the
amuntdatthieonn raestsuaryn,ecdeltlo cnuolrtmuarelsgwreorwethexmpeodsiedumtofcoron3dittoi8ondsaythsa.t selected against the TKTM phenotype
Media and Cell (Amacher et a,
C19u8l0t;urCeliCvoenedritail,on1s9.87T)hseumppeldeimuemntuesdedwiftohr
this study was RPMI horse serum (10% by
1640 volume),
PwthlaaustrotFnhieiscchhoerFrs6'e8s,smeLer-dguilmuutcmaomnwicinetenht,trshaeotidsoianumwmeapmsyerrudevidauutceme,dspuetpnoip5cli%belmleiynntasvnodulsusemtder.eipntCtohlmeoyncciiunln.tgumrTeeredmaietudmmieuncmtonmesxeicsdetipeutdmof
the RPMI 1640 culture medium
addition of 0.24% BBL? agar to
with up
achieve
to
a
20% horse serum, without
semisolid state. Selection
Pluronic" F68 and with
medium was cloning
the
`medium that contained 3 pg/mLofTFT (Clive et al, 1987).
TleabsotraftoorrMyyocnosptloacksmcualtCuornestparmiionrattoiopnre.paMryicngopsltoacskmsafotresftrienegziwnags. pSetrofcokrmcueldtubryesa fcoormtmheercial Mmuytcaotpilonasamsasatyesstwienrgewmaasinaltsaoinpeedrfuoprtmoed4 omnonctulhtsuraefstetrhtahwaewdinfgoraussaemipnlaessfaryosmpraiofrrtozoendistsoccakr.ding tchueltsutroicnkg.mTehtihsodvesriafnidedthtehaitndtihreercetwHeoreechnsotmsytcaoinpilnagsmmeathduordiwngerteheusleifde. of the culture. Both direct
z
250
Covance 20785-0-431 ICH 3M Study No.: T-6316.12
Karyotype Stability. Karyotype stability, as measured by mean chromosomal number, was routinely performed on stock cultures used for assays. Mean chromosomal number was
dKeatreyromtiynpeedapnrailoyrstto,diinsccahridinnggbtahnedsitnogc,kwteosenpseurrfeorthmaetdcohnancgalelssianftehrerceuplatruraetihoandonfota orcccuhrrefdr.ozen
stock culture.
Test Article
`The sponsor was responsible for the determinationofthe test article stability and the test article
Control Articles characteristics as defined in the GLP regulations.
Vehicle Control Article. The vehicleofchoice for the test article was DMSO (Sigma Chemical
Co. Lot No. 9H0020, CAS #67-68-5). Concurrent vehicle controls were performed for each portionofthe assay by exposing the cells to DMSO in culture medium. In the activation portion ofthe assays, the vehicle controls were also exposed to the S9 metabolic activation mix. Single
vehicle control cultures were initiated in the dose rangefinding assays and three vehicle control cultures were initiated in the mutation assays.
Positive Control Articles. The positive control articles listed below were chosen becauseofthe
large data base available and because both chemicals induce small and large colonies (Clive
etal., 1987, Younget al., 1991).
Methyl methanesulfonate (MMS) is a direct acting mutagen that is highly mutagenic to
Lin5d1u7p8liYcaTtKe "at c1e3llps.gMm.MSfor(AtlhdermiocnhaCchtevmaitcioanl Csoe.,yLowtitNho.a 40-8h1o0u9rBwUe,stCmAenSt prod, dn # 66 27-3) was used
duplicate at a concentration of 6.5 pg/mL for the nonactivation assay with a 24-hour treatment period.
Methylcholanthrene (MCA) requires metabolic activation by microsomal enzymes to become `mutagenic to L5178Y TK" cells. MCA (Sigma Chemical Co., Lot No. 77H2515, CAS # 5649-5) was used at 2 pg/mL and 4 pg/mL as a positive control for assays performed
with S9 metabolic activation.
S9 Metabolic Activation System "The in vitro metabolic activation system was comprisedofrat liver enzymes (S9 fraction) and an
energy producing system (CORE) comprisedofnicotinamide adenine dinucleotide phosphate
(NADP, sodium salt) and isocitrate. The S9 enzymes were prepared commercially (Molecular
Toxicology, Inc., Boone, NC) and were tested for sterility and enzyme activity. The enzymes
were obtained from the 9000 x g supernatant (S9)ofliver homogenate from male Sprague Dawley rats treated with Aroclor 1254, which induces mixed function oxidase enzymes capable of transforming chemicals to more active forms. Rats were treated once with 500 mg/kg of
Aroclor 1254, and the S9 was prepared approximately 5 days later. The $9 and reaction mixture
(CORE) were retained frozen at about -80C until used.
art
as/
Covance 20785-0-431 ICH 3M Study No.: T-6316.12
S9 Component
Metabolic |
Activation System Final Conceniration
in
Cultures
NADPTocGiotrdautme[ sal) |3oM tsm_M __||
Dose Rangefinding Assay A preliminary dose rangefinding
cytotoxicity
experiment
was
performed
witha
treatment
period
`oocfcauprprfoorxitmhaettewloy t4eshtoucornsdwitiitohnsa.ndIwniatdhdoiuttioSn,9aacntoinvaactitoinv,atsiionnceassusbasytawnittihalasthriefattmiennttopxeicriitoydoofften
faoprprcyotxoitmoaxtiecliyty2s4tahrotuirnsg awta1s2p5e0rfpogr/mmeLd,. aAndwifodlelrowaendgeobyfnteisnt alrotwicelreccoonncceennttrraattiioonnsspwraespatreesdteidn
2-fold dilution steps. 24 hours at 35-38C
After an exposure in an orbital shaker
attim8eo0fa1p0porrobxiitmsapteerlmyi4nuhtoeu,rsthoercealplpsrwoexriemawtaeslyhed
twice
itrtehatcmuelntturweermeedcoiuunmteadndanrdessuusbpceulntduerdedi,n acuslnteucreesmseardyi.umF.olTlhoweicneglltsheretceerimviinnagtitohne o24f-thhoeur
mruetaattmieonntepxepreiroidmse,ntthse. ceAllscewllerceouinntcuwbaasteddetoeverrmniingehdtauftseirngthpergorcoedwutrhepseirdieondtitcoalmteo astuhroseethfoer
dreodsuectriaonngeifnicnedlilnggrionwftohrmraetliaotinvewatso tthheencounsceudrrteonstelveechticdloesecolnetvreollscfeolrl tchueltmuuretsa.tiTohneaspsraeyl.iminary,
Taepsptaraerntticeleffceocntcoenntgrraotiwotnhscaormepcahroesdentotothceovveerhiaclteoxciocnittryolr.anIgfeltftrloemor1n0o%ttooxi2c0it%y issurovbisvearlvteodnaond S5omlugbi/lmitLyoirs m1a0inmtaMin(ewdh,itchheemvuetratisiolnoweexspte).rimIfenprteicsipiintiattiiaotnedowfitthhe2temstaxaritimculemoccocnucresnitnratthieon of culture medium, the maximum applied dose is at least twice the solubility limit in culture medium. In this assay, the high dose limited by cytotoxicity.
Mouse Lymphoma The assay procedure
Mutagenicity Assay used was based on that
reported
by
Clive
and
Spector
(1975),
Clive
et
al.
(se1l9e7c9t)e,dAtmoaicnihteiarteetaaml.ut(a1t9i8o0n)eaxnpderCilmievnet,eftahle. o(b1j9e8c7t)i.veAwltahsotougchatrerny oatrleelaesvtefnodurosdeossewsertehrough
htheelpeendtirteo eansssauyr.e tThheischporiocceeodufraet cleoamsptefnosuartdeodsefsoronveorrmaalwivdaericayttiootnosxiincicteyllrualnagret.oxicity and
Initial Nonactivation Assay. Logarithmically growing laboratory stock cultures were seeded ciuntlotuaresemreiedsoiufmturbeemsovated6,xan10dtcheellcselplesrrteusbues.peTnhdeedcetlolsawfeirnaelpveollleutmeedobfy 1c0enmtLri.ofufgtarteioant,mtehnet m3e0d+iu1m0.orTbihtes tpuebremsinwuetree. plAafcteedr ainnaenxoprobsiutrale psehraikoedroifnacpubpartooxriamta3t5e-3l48yhCouarnsd,rtohteatceeldlastwere
7
95%
Covance 20785-0-431 ICH 3M Study No.: T-6316.12 centrifuged and the treatment medium removed. The cells were then washed, resuspended in 20 mLof culture medium and returned to the orbital shaker incubator.
The assay conditions consistedofvehicle controls in triplicate, two positive controls and eleven different test article dose levels using one culture per dose level. Treated cultures may be eliminated during the expression period as long as four dose levels are left for analysis of mutant induction. `The appearanceofthe treated cultures was recorded both at the timeoftreatment and afte the treatment period. When the pH indicator in the medium had a color change, the pH was. `measured using pH indicator strips.
A standard expression periodof 2 days was used to allow for mutant recovery, growth and 3exxpr1e0ssicoenllosf/tmhLeiTn K20TMmphLenooftgyrpeo.wthCemlelddieunms.itieIsf wtheerceeldlesteinramicnueldtuorne Dfaaiyled1 taondmuwletreipaltdjyousated to
density o4f x 10 cells/mL on the first day after treatment, the culture was not subcultured. On
Day 2, cell counts were again determined, and appropriate cultures were selected for cloning and `mutant selection
Six doses were selected for mutant analysis. Ifpossible, doses were selected to include nontoxic 10 highly toxic treatment conditions. Although relative growth gives an indicationofwhethear test article is highly or weakly cytotoxic, specific ranges for weak, moderate, highly toxic and excessively toxic are not specified. Several factors go into the calculation and the Study Director `makes a decision concerning the levelofcytotoxicity that has occurred. Cultures with cell densities less than approximately 3 x 10 cells/mL were not considered for selection.
Atotorteaclovoerf3TFxT-1r0esicesltlasntfrmoumtaenatcsh. sTehleicstesdamtpulbee wwaass sdiusstpreinbudteedd iinntsoeltehcrteieon10m0edmimumdiisnhesso.ftTahgaer absolute selection cloning efficiency was determined by seedingthree dishes with a total of approximately 600 cells in agar cloning medium. All dishes were placed in an incubator at 3538C with 4-6% CO:95% humidified air. Aer 13 days in the incubator, the colonies were counted with the Loats Associates, Inc. (LAI) High Resolution Colony Counter (HRCC) System for the Mouse Lymphoma Assay, Version 1.05b04c.
`The mutant frequency was calculated as the ratioofthe total number ofmutant colonies found in each setofthree mutant selection dishes to the total numberofcells seeded, adjusted by the absolute selection cloning efficiency. If one dish in either set was lost due to contamination or other cause, the colony countofthe missing dish was determined by a proportion equation based upon the weightsofthe three dishes of the set and the colony counts in the two acceptable dishes. Ifa lost plate was not available for weighing, the colony count of the lost plate was determined from the average of the two remaining acceptable plates.
`The measurementofthe cytotoxicityofeach treatment was the relative suspension growthofthe. cells over the 2-day expression period (for the 4-hour treatment) or the relative suspension `growth over the 3-day treatment and expression period (for the 24-hour treatment) multiplied by
nr
R53
Covance 20785-0-431 ICH 3M Study No.: T-6316.12
tshuervrievlaalt,ivtehicslpoanrianmgeetfefric(iceanlclyedatretlhaetitviemgeorfoswetlhecotriroenl.atiAvletthootaulghgrnoowtths,triRcTtlGy)a pmreoavsiudreesoafmceealslure of the effectivenessoftreatment and was used as the basis for selecting doses for any necessary subsequent trials.
CasosnafyiursmiantgoarytrNeoantamcenttivpaetriioondoAfsaspapyr.oxTihmeatceolnyfi4rhaotuorrsyeaxscseapyt ptrhaotcetdhuereexwpoassusriemitliamretwoatshe irnacnrgeeafsiendditnogaapspsraoyxiwmiatthealytr2e4athmoeunrts.peTrihoedaosfsaayppwraosxicmoantdeulcyt2ed4 uhosuirnsg.reTseulntstrferaotmmetnhtesdwoesree. initiated, and seven were analyzed for mutant induction.
The cells were obtained from logarithmically growing laboratory stock cultures and were seeded icnutlotuaresemrieedsioufmturbeemsoavted6,xan10dtcheellcselplesrrteusbue.speTnhdeedceilnlsawfeirnael pveollleutmeedobfy2c0enmtrLiofufgtarteioant,mtehnet `3m8edCiuamt.80Th+e1d0oorsbeidtstpubeers mwienruetec.losAefdt,ervoarttreexaetdmeanntdppelraicoeddoifnaapnprorobxiitmaaltsehlayke2r4ihnocuurbsa,ttohreatce3l5lsWreesruescpeenntdreidfuigne2d0amndLthoeftgrreoawttmhenmtemdeiduimuamt r3exmo1v0edc.ellTshpeercemllLs.weTrheetchuelntuwraesshwede,reatnhden returned to the orbital shaker.
A standard expression periodof2 days following terminationoftreatment was used to allow
recovery, growth and expressionofthe TK" phenotype. Cell densities were determined the day after treatment termination, and each culture was adjusted to 3 x 10 cells/mL in 20 mL of
gdraoywatfhtemretdrieuamt.menItfttehremicnealltsioinn,atchuelctuulrteurfeaiwleadstroemtuulrtniepdltyottohae dienncsuibtaytoorf4witxho1u0t/bmeLinogn the first SSeulbeccutletdurfeodr.clTohneinngexatnddamyutcaelnltcsoeulnetctsioang.aiCnuwletrureedsewteerrmeinceodu,ntaendd1a2ppdraoypsriaaftteerccullotnuirnegs.were
Ifnoirttiahle aAdcdtiitviaotnioonf tAhsesaSy9.frTahcetiaocnotifvaattioinvaesrshayomwoagseindaetnetiacanld1n0etcheessnaornyacctoifvaacttioornsa(sCsOaRyEe)xcept
during the `activation
mtirxe,atwmheintchpewraisodoprfepaaprperdoixmimmeadtieatl4eylhyopurrsi.or
The treatment medium to use and kept chilled.
included this S9 Each batch of $9
w`maustatgeesntiecditfoyraascstaiyv.atTihoneoSft9hheomMoCgeAnaptoseitwiavse ccoonmtmreorlcairatlicllyepbreefpoarreeidt (wMaosleucsueldafroTrotxhiecology,
Ilinvce.rsBoofonAer,ocNl.oCr.,12L5o4t-Nion.du9c5e5d)aadnudltcomnasliestSepdroafguteheDa9w0l00eyxrgatss.upCeumlattuarnets wfreorme tchoeunhtoemdog1e3ndiazyesd
after cloning. Eleven treatments were initiated, and four were cloned for mutant analysis.
wCeornefiirdemnattiocarlytAoctthievaintiitoianl Aasctsiavya.tioTnheaspsrayo.ceEdulreevsenusterdeaitnmetnhtescwonefrierminaittoiratyeda,ctainvdateiiognhtaswsearye cloned for mutant analysis. Cultures were counted 13 days after cloning.
SsmiazlilngmAuntaalnytsicosl.onTichse. L5Th1e78oYrigTiKno"ftmhuetabtiimoondaaslsdaiystprriobduutcioensoafbmiumtoadnatlcdoilstorniybustiizoensoifs largeand
A
54
Covance 20785-0-431 ICH 3M Study No.: T-6316.12
considered to reflect the types of genetic damage, with the large colonies derived from cells with intragenic mutations that affect only the TK gene and the small colonies the resultoflarger mutations that affect cell growth as well a the TK gene (Hozier et al, 1985).
aBnodthsmtahlelsmaanldllaanrgdelcaorlgoencioelsoanrieesquwaenrtietqautaendtbifyietdhefoarreaalsl cuunldtuerrest.heAcurbviems.odaTlhceulravregeiscgoelnoenriaetsed
presumably arise from point mutations and LAI Automated Colony Counter was used.
the small colonies from chromosome changes. This counter sizes all analyzed cultures.
The
DATA
Data Presentation The historical control data are presented after the data tables. This report includes the following. information:
dDeonsseitiReasnogfetfhiendiinndigviDdautaal.cuTlhteusreesdaantda tihneclcuedleldd,enfosriteiaecshretlraetaitvmeetnottchoendviethiiocnl,e tchoentdraoillsy cell
Cell Growth Characteristics. This included the number of cells seeded for the analysis of
mInutaadndittiforne,qudeanicliyesc,eltlhdeenpshietnioetsyipnictheexipnrdeisvsiidounalticmuel,tuarneds ctahrersieeldectthirvoeugahgetnhteaenxdprceosnscieonntrpaetriioond.
and cloning were included. The suspension growth for vehicle and positive controls, for the
o4r-hDoauyr t1rdeeantsmietnytisftnuodtiessplciatlcbualcakt)eddeasscr(iDbaeys tIhceelglrdoewntshiotfy/t3hxe 1c0o%n)trxol(cDualytu2recseldludreinnsgittyh/e3 x 10
egxrporwetshsifoonr vpeerhiiocdl.e aFnodrptohseitniovneacctoinvtraotliso,ncaaslscauylawtietdhaas 2[4T-rheoautrmetnrteattemremnitnapteiroinod(,Dtahye 1s)uscpelelnsion
density/3
density/3
x
x
10] x
10 or
[DDaayy22dceenlslidteyinfsintoyt/3spxli1t0baocrk]D,adyes1crdiebnessittyhiefgnrootwstphliotfbtahcek]coxnt[rDolaycu3ltcuerlels.
during the expression period. were included and calculated
For the test-anticle as above, and were
treated cultures, the relative suspension expressed as percentagesofthe average
growths vehicle
control suspension growth. The relative cloning efficienciesoftest anicle-treated cultures were
expressed as percentages of the average vehicle control cloning efficiency. The expression of
RTG was the parameter that was used to assess treatment cytotoxicity and was obtained by
multiplying the relative suspension growth by the relative cloning efficiency/100.
Mutant Selection Data. The mutant selection data includes the total mutant colonies (ic. total
`numberofmutant colony counts obtained
into selective medium and divided among
from
three
3 x10cells sampled from one culture, seeded
culture dishes) and the total viable colonies
(ic. the total number of colony counts obtained from 600 cells sampled from one culture, seeded
into nonselective medium and divided among three culture dishes). These were used to calculate:
stheeedmeudtfaonrtcflroenqiunegncefyffiocriecnaccyhwcauslt0u.re5.xT1h0e. raTthieoroeffcoerlel,stsheeemduetdanfotrfmruetqaunetncsyelweacst:io(ntottoalcemlulstant
-
255
Covance 20785-0431 ICH 3M Study No.: T-6316.12
cionlcolnuideesdftiontatlhivsisaebclteicoonlwonaisest)hexc2loxni1n0g,efMfiuctiaenntcyforfeqvueheincclyewaansd gpiovseintiivne ucnointtsroolfs1c0al.culAaltseod as (total number of viable colonies/600) x 100%.
Assay Acceptance Criteria
Aginveanssbaeylowwaswecronessiadteisrfeideda.ccAecpttiavbalteifoonroervnaolnuaatcitoinvoatfiotnheastessatysrewsuelrtes
only if allofthe criteria repeated independently,
as
needed, to satisfy acceptance criteria. The Study Director reviewed each assay in accordance:
with the following criteria:
Acceptable Controls. The between 60% and 130%. A
average absolute cloning value greater than 100%
efficiencyofthe vehicle controls should be is possible becauseoferrors in cell counts
(usually + 10%) and variations in cell division during unavoidable delays between counting and
cloningofmany cultures
A minimum 2 days is an
acceptable value 8.0-fold increase
ofvorerthteheavoerirgaignealsucselplencsonicoenngtrraotwitohnosf.thLoewveerhivcalleuecsornternodlesrfaonr
assay
unacceptable becauseof poor cell growth.
Tcohnecubrarcekngtraocutnidvamtuiotnanatndfrneoqnuacetnicvya(taivoenraasgseaoyfsv,eehviecnlethcoountgrholthveasluaemse) piospcuallactuilaotnoedfsceeplalrsaitseluysefdor for each condition. For both conditions, the normal rangeofbackground frequencies for assays ptheirsfroarnmgeed awrictnhodtifnfeecreesnstarciellly sitnovaclkisdibsu3t0wxill10be teova1l2u0atxed10wi.thAcsasuatiyosn.with backgrounds outside
Atleast one of the positive control cultures in each trial should induce a mutant frequency of at
least 200 x 10. A trial may be acceptable without a positive control (due to contamination or
technical error) onlyifthe test article is positive.
AincccleupdteaabplpeliHeidgchonDcoesnet.raFtioorntsetsthaatrtriecdluescewitthhe RweTaGk toor 1no0%muotra2ge0n%icofactthieviatvy,eraangeasvseahyicslheould creopnrtersoelnotrs raecaaclhcutlheatmioanxoifmsuumrviavpapllitehdatcoisncbeansterdatoinonbsotghivreenlaitnivtehseuesvpaelnusaitoinongcrroiwtetrhiad.urRiTngGthe efxrperqeusensciioenspienrciroedasaendasrealaftuinvceticolnoonfinlgetehfafliictiye,nacny aattttehmepttitmoeoobftapliantitnrge.atBmeenctasusinetmhuetraanntge of w1a0s%wtaoi2ve0d%if tRhTeG cmounsctenbteramtaiodneofitnohredehritghoesctonassisdaeyredthdeoassesawyasasatcolnecalsuts7iv5e%.ofThaishirgehqeuri,rement etexscteasrstiivcelleyincyctuolttouxriecmdeodsieuml.evelThoerireftihs enohimghaexsit mduosme twoaxisciattylreaesqtuitrweicmeentthefosroltuebstilairttyiclliemsitwhoifcthhe clearly show mutagenic activity.
AonclcyepitfatbhelereNlautmivbeecrloonfinDgoseefsf.iciMeuntcyanist 1fr0e%quoerngcryeawtielrl baendcotnhseitdoetarlednuamccbeeprtaobflevifaobrleevcallounaetsion exceeds about 60. These limits avoid problems with the statistical distribution of scorable. colonies among dishes.
sb
Covance 20785-0-431 ICH 3M Study No.: T-6316.12 Mutant frequencies for at least four different test article dos levels are normally determined in each assay; a minimumoffour analyzed cultures is considered necessary to accept a single assay for evaluation of the test article.
aMnudtavnitabflreecqoulenocniyescoaurnet.noIrnmaolrldyerdetoriavleldowfrfoomr lsoestssoesfdtuhereteodciosnhteasmfionrabtoitohn omrutoatnhtercroelaosnoynsc,ouannt acceptable mutant frequency can be calculated from a minimum of two dishes per set.
Assay Evaluation Criteria The test article was evaluated as positive, negative, or equivocal in this assay. A positive evaluation indicates that the test article is a mutagen (induces gene/chromosomal mutations) in Sthyisstteemstasnydstceamu.sesAnnoegraetsipvoenseevsaltuhaatticoanninbdeiciantteesrptrheetetdesatsarptoiscilteivies.noHnomwuetvaegre,nitcheirnetahries test substances for which the results remain questionable; any test article in this category will be evaluated as equivocal in this test system.
Einvcarleuaastesioonf2o-ffaolPdosoirtgirveeaRteerspinonmsuet.antThferetqesutenarctyicalreeiosbetvaailnueadteodvears tphoesictoinvceiurfrdeonsteb-adcekpgernodeunntd omfuttahentvefhriecqlueenccoyn.troTlhceulbtaurceksg.roTuhned 2m-uftoalndtorfrgerqeuaetenrcyinicsrdeeafsieniesdbaasstehdeoanveerxatgeensmiuvteaenxtpefrrieeqnuceency which indicates such responses are repeatable in additional trials. It is desirable to obtain this relationship for at least three doses, but this goal depends on the dose steps chosen for the assay and toxicity at which mutagenic activity appears.
Thihgehedro)seis-odbetpaeinndeedntforreaqusiirnegmleendtosisewaatiovrendieafrathlearhgiegihnecsrtetaessetaibnlmeuttoaxinctitfyr.eqHuoewnecvye(r4,-ffoolrdtohre test article to be evaluated as positive, any increases must be repeatable in a second trial.
Evaluation of a Negative Response. The test article was evaluated as negativeif a 2-fold increase in mutant frequency was not observed for (1) a rangeofdoses that extended to toxicities causing 10% to 20% RTG, or (2) for relatively nontoxic test articles, a rangeof doses extending, {0 the maximum concentration of $ mg/mL or 10 mM (whichever is lower), or (3) a range of doses that extended to a level approximately twice the solubility limit in culture medium, or (4) the increase(s) are not repeatable in a confirmatory trial.
Esyvsatleumaitfiotnheorfe awnasEqnuoicvooncsailstReentspeovnisdee.nceThfeorteeistthaerrtiaclpeoswiatsiveevoarlunaetgeadtiavseeeqvuailvuoactiaoln.in this test
Other Considerations. Treatments with occur in an assay, but the results obtained
test articles at this high
that reduced the RTG to toxicity were considered
less than 10% biologically
may
irrelevant and were not used to evaluate the test article.
For some test articles, the correlation between toxicity and applied concentration is poor. The interactionofthe test article with the cells is not always repeatable or controlled by
--
257
Covance 20785-0-431 ICH 3M Study No. T-6316.12 dcoonsceenrterlaattieodn.resIpnontshiestchaasne,atphpeliienddcuocnecdenttorxiactiitoyn.asTmheearesfuorreed, binydtehteeRrmTiGniinsgaifbeattdeorsem-easure ofa relationship is present, either test article concentration or induced toxicity (RTG) may be used.
DTihriescptroersmenatyathiaonveofustehde eovtahleuractriiotnerpiarotcoeasrsrimvaeyatnaotn eevnacloumaptiaosns. alTlhteesrtespiotrutatpiroonvsi,daensdthtehe Study reasoning involved when departures from the above description occur.
RESULTS
Test The
Article Handling test article formed
unworkable,
non-homogeneous
suspensions
in
water
at
concentrations
of
4t9h8s,r3e0a0s,on1,0D0 ManSdO50(.C0AmSg#/m6L7-.68I-n5,DMSiSgOm,a tChheetmeisctaalrtiCcol.e, fLoortmNeod.a9sHol0u0ti2o0n)atw5a0s0umsge/d maLs.theFor
vehicle.
Npr-eEp(aFreOdSfEorT-us6e31i6n twhaesasssoalyu.bleThien tshteocvkehuiscelde (foDrMdSosOi)ngatth1e25himgghe/smtLc,ontcheenthriagthieosnt coofntcheenttreasttion aSrttoiccklse wwaersepprreeppaarreeddinbyDpMeSrfOoramti1n0g0s-etriiamlesditlhuetihoingsheisnttdheesvierheidclter.eaDtomesnitngcowncaesntirnaittiioant.ed bFyurther performing 1:100 dilutionsofthe stocks into medium containing the cells. All dosing was. performed with freshly prepared test article stocks.
Dose Rangefinding Assay Ttrheeattmesetntarpteicrlieo,dNo-fEatpFpOrSoExiTm-a63t1e6l4,ywhaosurtsesbtoetdhiwniathpraenldimwiintahroyutdoSs9e mreatnagbeofliincdiancgtiavsastaiyonwiatnhdaa p2r4ehloiumrisn.arTyednosdeosreanlgeevfeilnsdwienrgenounsaecdtiivnaetaicohn acsassaeythwaittrhaangterdeaftmreonmt2p.e4r7iotdoof1a2p50prpogx/immLa;tealy Vehicle control was included under each condition.
(InTatbhleep1r)e,sNen-cEeFaOndSEabTs-en6c3e1o6fwraats nliovneerySt9otmoextiacbuolpitcoa1c9t.iv7itpyg/wmitLh.aMdo-dheoruartterecayttmoetnoxtipceirtyiowdas
iInndtuhceendonfarcotmiv3a9t.i3ontodo1s5e7 rpagn/gmefLinadnidnhgiagshsearycuosnicnegntara2t4i-ohnosurwetrreeaetxmceenstsipveerliyodcy(tToatbolxeic2)o, thleettheaslt.
ahritgihclceyitontdouxciecditnyoacty9l.o8t5oxpigci/tmyLa.t
2H.i4g7heprg/cmonLc,enwteraakticoyntsotwoexriecietxycaetss4i.v9e3lypgc/ytmoLt,oxaincd
moderately or lethal.
Dose levels for the mutation assays were based on these results.
Mutation Assays
I5n0i.t5i,al75N.o5,na1c0t1i,va1t51i,on20M1u,t2a5t1i,o3n01A,ss4a0y1.,
Eleven concentrations of 501 and 601 pg/mL were
N-EtFOSE T-6316 at initiated. Treatments
25.3, at and
atrbeoavteme2n5t1s pingd/umcLedwewreeaktecrymtiontaotxeidcibteyctaouhsiegohfceyxtcoetosxsiicvietcyy(to6t7o.xi6c%ittyo. 14T.h4e%rreemlaaitinvienggrsoiwxths). In
5
Covance 20785-0-431 ICH 3M Study No.: T-6316.12 order for a treatment 10 be evaluated as mutagenic in the initial nonactivation assay, a mutant afvreerqaugeencmyugtraenattefrrethqaunen1c2y7.o1f txhe10conwcaursrerenqtuivreehdi.cleThciosnttrhorlessh(obladckvaglruoeunwdamsuetqaunatl ftroetquweincceyt)h.e Noneof the analyzed treatment induced this level of mutant action (Table 3). Sizing data is shown in Table 4. A confirmatory assay was initiated.
Confirmatory Nonactivation Mutation Assay. In the confirmatory nonactivation assay, the treatment period was approximately 24 hours and results are shown in Table 5. Ten treatments at 1.25,2.50,5.00,7.50, 10.0, 12.5, 15.0, 20.0, 30.0 and 40.0 pg/mL were initiated. Treatments at 20.0, 30.0 and 40.0 pg/mL were terminated becauseof excessive cytotoxicity. The remaining seven treatments were selected for mutant analysis and induced no cytotoxicity to high cytotoxicity (94.2% to 18.7% relative growths). None of the analyzed treatments induced a t`hmeurteafnotref,recqounesnicdyertehdatneexgacteievdeedwitthheoumtinmeitmaubmolcircitaecrtiiovnatoifon1.11S.i2zixng10da.taTfhoer ttehset caortnifcilremiast,ory assay is shown in Table 6.
Initial Activation Mutation Assay. Eleven treatments at 25.3, 50.5, 75.5, 101, 151, 201, 251, 301, 401, 501 and 601 pg/mL were initiated, and treatments at and above 151 pg/mL. were terminated becauseofexcessive cytotoxicity. The remaining four doses were cloned for mutant analysis and induced moderate to very high cytotoxicity (60.0% to 9.4% relative growths). The t`rmeiantimmenutmscirnitdeurcieodn fthoirsalpeovselitoifvmeurteasnptonascetiinonth(iTsatbrlieal7w).asA15c6o.n7fixrm1a0to.ryNaosnseaoyfwtahsepaenrafloyrzmeedd. Sizing data for the initial assay is shown in Table 8.
Confirmatory Activation Mutation Assay. In the confirmatory assay with metabolic activation (Table 9), eleven treatments at 5.00, 10.0, 20.0, 30.0, 40.0, 50.0, 60.0, 80.0, 100, 150 and 200 pg/mL were initiated. Treatments at 5.00 and 10.0 pg/mL were terminated because there were sufficient higher doses available for analysis and the 200 pg/mL treatment was terminated because of excessive cytotoxicity. The remaining eight doses induced weak cytotoxicity to high cytotoxicity (70.4% to 15.4% relative growths). Noneofthe analyzed treatments induced a `emvuatlaunattefdreaqsuneengcaytitvhaetweixtchemeedteadbtohleicmiacntiivmautmioncriintetrhiiasoafss1a2y2..2Sxiz1i0ng.daTthaefotresttheartciocnlfeiwramsa.tory assay is shown in Table 10.
Control Values. The average cloning efficiencies for the vehicle controls were 88.6% and 83.4% without activation and 84.1% and 77.3% with S9 metabolic activation, which demonstrated acceptable cloning conditions for the assays. The positive control cultures, MMS (nonactivation) and MCA (activation) induced large increases in mutant frequency that were greatly in excessofthe minimum criteria
sSmiazlilngmuAntaalnytsicosl.onTiehse. LT5h1e78orYigTiKno"f tmhuetabtiimoondaaslsadiysptrriobduutcioens oafbmiumtoadnatlcdoilsotrniybustiizoens oisf large and considered to reflect the types of genetic damage, with the large colonies derived from cells with intragenic mutations that affectonlythe TK gene and the small colonies the resultof larger mutations that affect cell growth as well as the TK gene. Colony sizing was performed on all
~
25
Covance 20785-0-431 ICH 3M Study No.: T-6316.12 cbuilmtoudraesl (dTiasbtrliebsut4i,o6n,andanmdut1a0)n.t cMoultoannitescoflroonmieMsMfrSomanaldlMthCe Aculttrueraetsedschuolwtuerdesthseheoxwpeedctbeodth small and large colonies.
CONCLUSION TthheeTtKestlaorctuisclei,n NL-5E1X7F8OYSmEoTu-s6e31l6y,mpwhaosmeavacleulalsteudnadserneagcattiivvateifoonrainndduncoinnagctfiovratwiaornd cmountdaittiioonnssat used in this study. Treatments up to the testing limit for this assay were analyzed.
RECORDS TO BE MAINTAINED oAfllthriaswstdautday,wdiolclubmeenatracthiiovne,d rinectohredss,toprraogteocfoalcialintdieas ocfopCyoovfantchee-Vfiineanlnrae,pofrotrgaetnleeraastte1d yaesaar result fmoalyloewlienctg tsoubhmaivsesithoenoaffotrheemefinntailorneepdormtatteortihaelsSproentsaoinre.d Ainfttehretshteor1a-gyeeafracpielirtiioedso, fthCeoSvpaonncseo-r Vienna for an additional periodoftime,or sent to a storage facility designated by the Sponsor.
REFERENCES Aatmathcehethry,mDidEi,nePkaiilnlaeste, Sl.oCc.u,sTinumLeSr,17GN8.m,oRuasye, lVy.Am.p,haonmdaScaellslbsuIrIg.,TDe.sSt. v(a1l9id8a0t)ioPnoianntdmutations interpretation. Mutation Research, 72: 447-474.
Catlitvhee,TD.KalnodcuSspeinctcourl,tuLrFeSd.L5(1197785Y) mLaobuosreatloyrmypphroocmeaducerlels.forMaustsaetsisoinngResspeecairfcihc,l3o1c:u1s7m-u2t9a.tions
Cliv
and
e, D., Johnson,
characterizatio
K.0,, S
n of the
pector, JIF.S.,
LS178Y TK"
Batson, A.G., and
mouse lymphoma
Brown, MMM.
mutagen assay s
(1979)
ystem.
Validatio
Mutation
n
Research, 59:61-108.
GCluiivdee, Df.o,r pCaesrpfaorrym,inWg.,thKeimrobyu,seP.El.y,mKprheholm,aR.a, ssMaoyofroer,mMa.m,mMaalyioa,n Jc.e,lalnmdutOabgeernlicyi,tTy..J. M(u1t9a8t7i)on Research, /89:143-156.
Hcoozlioenry,LJ.,5S1a7w8YyeTrK," J, mCultiaven,tsD.e,aralnydiMnotoherier,clMonMa.l h(i1st9o8r5y). CMhurtoamtioosnomReese1a1rcahb,er/ra4t7i:o2n3s7i-n24s2mall
Young, R., Oveisitork, F., Harrington-Brock, K., Schalkowsky, S., Moore, M., and Myhr, B. c(o1m9p9a1r)isQouna.ntaEtnivvierosinz.eMaonlaelcys.isMuotfaLgeSn1e7s8isT,K1"7, m(uSutpapnlt.c1o9l)o,n7i9e.s in soft agar; an interlaboratory
a
260
Covance 20785-0431 ICH 3M Study No.: T-6316.12 DATA TABLES a 206)
Covance 20785-0431 ICH 3M Study No.: T-6316.12 TABLE 1: CYTOTOXICITY ASSAY WITH N-E(FOSET-6316 (4-HOUR)
`TEST ARTICLE: N-ECFOSE T-6316 STUDY NUMBER: 20785-0431 ICH
`TEST DATE: 05/10/2000
VEHICLE: DMSO
COMMENTS ON TREATMENT: ~4Hous Treatment Period
----ACOPW NCPEpLNgI T/EmRlADTH IONOCEU LL DxT E1N0SI"S TV/mLAC %CVOENT HTIRCO-- LLI E" CEV LWLI(DIA EXNN1SS0IITT AYC/mTlII .VA%CTO VOIENOHTNIRN CO~LLE~
--&
es
eo
10s
100
247
92
920
108
1038
493
108
1080
94
904
085
ns
180
nz
nas
197
91
910
92
885
393
47
470
51
90
785
33
30
28
29
157
35
350
24
21
313
04
a0
03
29
62s
00
00
00
00
1250
00
00
00
00
"RelaGtivee tyo vS ehiclee controy l cell deao sity for all reatments --_ VC = Vehicle control, 1% DMSO
o
el
`Covance 20785-0431 ICH 3M Study No.: T-6316.12
TABLE 2:`CWYITTOHTOOUXTI$C9IATCYTAISVSAATYIOWNI(T2H4-NH.OEU(RF)OSE T-6316
TEST ARTICLE: N-EFOSE T-6316
STUDY NUMBER: 20765-0431 1CH
TEST DATE: 05/10/00
VEHICLE: DMSO
`COMMENTS ON TREATMENT: ~24-Hour Treatment Period
CE AEL D LDBNSIYVA ERL E
`CONCmENgTmR.ATION ve
x10)"
DAY1
Dpav2
To
70
`CONTROL*
TW00
247
1
64
1006
493
72
3
3
085
57
33
24
17
03
10
39
3
oor
00
00
75
oo
00
00
157
NTC
NTC
--
si
NtC
NTC
--
as
NTC
NTC
--
1250
NTC
NTC
--
Cell density determined by hemocytometer
NPVRTeClCa=t=iVvNeeohttiocclvoeeuhncitocenldet,oclco,yntt1root%loxDciecMllSdOensity for all treatments. "Not subculured
~=6=
263
Covance 20785-0431 ICH 3M Study No.: T-6316.12
TABLE 3: INITIAL MUTATION ASSAY WITHOUT ACTIVATION
AB. TGEENSETTAIRCTSICALSE:SANY-NEOF:OS20E78T5-.06-341361 ICH DC._ SVEEHLIECCLTEI:VEDMAGSEONT: TFT3.0 ug/ml.
EF..TCERLELASTAMNEANLTYZDEATDE.: 30x51/01"6/2000 GH_. TERXPERAETSMSEINOTN PPEERIRODI:~2dO4ahyDous:rs
Test Condition CouDnatlsy(CCleilmL, CuRmuSlGsive MTutoalnt VTiosublle EfCfliocniienncgy' RGerlaovwe FrMeuqumemncy
1085 Unis)
Colonies Colonies
00 (1086 Unity
-- Nonsetivation Contrals mr wr Nd ve
avvee
VVeehhiiccllee CCoonnwtreo]l 110202 1114S2 115586
1m7osse 980s7
91101ss i57)5
Vehicle Contol 117 [22 159 157 160 se9 91s' 886 1041 582
MMSIBpgmL MMSI3pgmL
69 124 95 82 120 109
wha asi ws ms ws
08 wre me 08
"Test pCogmmp.ound.
VReehlitcvleeto
ReVleahtiicvleeto
Control
Conmol
23
2s 2 2 as as s)d 16 8
75055 101
24060 19076 04ss 012 adme
910 883
313 11
702 0s
Wf os2 pam ses 81S 152 m7
151 0
210 8s 20080
8609
aums aasss
solss 114644
9m80
R"CEloGningBEafyficTiCenocuyn=tT)ot(a ViDs2biCaCoolwyonsy C(orunatyNu|mCboemrmoifCTrlonlswSbeeendteodre*d1)00
""RMeultaatnitveFGrroewqt=uh=e(Rn(eTlcoattayilveMSuutsapnetnCsoiloonniGersoTwotthal* RVeilaabtlieveCoClloonniiensg) E*f2fxi1c0iEe-nc4y) 100
"VehDieccliemaClointsrmol o= 1vt%oeDexMprSedOs the frequency in units of 10E:6
MutPaogseintiicv.e CEonxtcreoel:dsMMiMnSim=uMmetChryitlermieotnhoafn1c2s7.f1onXate108:6
ot Subeultured "Oneplatecontaminated.
Tota
counts
calculated by using
aweight
proportion
2
Covance 20785-0-431 ICH 3M Study No.: T-6316.12
TABLE4: SIZING DATA FOR INITIAL MUTATION ASSAY WITHOUT ACTIVATION
AB TGEESNTETAIRCTISCALES:SANY-NEOFO2S0E78T5.603-41361 ICH CD. VSEEHLIECCLTEI:VEDMAGSEONT: TFT3.0yim. TREATMENT DATE: 0516/2000
CmRSGGR EfCflocneinncg? GRerlsoivwe Mutant1F0re%quency
TexContiton Cone ToyT DI Abe Re% 0) Tow Smal Taw
Va
ms a2 Es os 94s Te a3 7
Bo 0I8n0s 1007 o%u0s7 sdss llasl $$8s2 4M8s0 3laas
MVS yn) L 1B sme eess dwss asse Bsa am3e08 a1u69n6 1m8s12
TestArice(ugim) 33 eo m2 6 ss 6 8 409 90
5 ss
wws7 dwess
sms2 sslso
M1831
32 0os 2254
o IS ns186 m18s0 780s5 m9ss 16s42 m9a8 M38e1 1247
Ci RSG = Cumulativew Suspensm ion Growth6R5ive h7e1AveragemsVeils ConiuoalaSpinmoGrow09 1
"Cloning Efficiency = Ta Vible ColonyCountNumberofCells Seeded * 100
"Relave Growth=(RelativeSuspension Growth * Relative Cloning Efciency) 100
aan Freque=(nTcoys Mutant ColonesTotal Viable Colonies) * 2105-4
DEexpcriemsaseldssms TootatvoMeuxetparnedtsFrheqeueenqcyu,eSnmaeilynCuonlitosnoyfMuIt0aEn-t6 Frequency and Large Colony Mutant Frequency
"Vehicle Conol = DMSO
positive Conrol: M=MMetShyl metbancsufonstc
Colony Courts incessedby9.099% to compensatefoarofdaish no scanned
=
R6S
Covance 20785-0-431 ICH 3M Study No.: T-6316.12
TABLE5: CONFIRMATORY MUTATION ASSAY WITHOUT ACTIVATION
AB. TGEESNTETAIRTCISCALSES:ANY-NEOW.OS20E78T5.-06.341361 ICH. CD. VSEEHLIECCLTEI:VEDMAGSEONT: TFT30 sgn.
E.F. TCRELELASTMAENNATLYDZAETDE::30x61100"712000 GH._ ETXRPERAETSMSEIONNTPPEERRIIOODD:: ~2d2a4hyosurs
TeCondiion (CDaeilylC1el0lBlSmCoUuLnmis,t) CumRuSlaGt'ive TMouualnt VTioaublle EfCfliocniienncgy|' RGerloawtvhe FrMeuqtuaenntcy
Colonies Colonies
or (U1n0i8gg
Nonactivation Controls" Biy1 Dayz Day Ave ve
AvVeG
Vehicle Conrol Vehicle Conval
120 143
79 62
111240
433714
3issde 7903s
9a9 5si4s
VehcleCowrol 147 61 164 S45 447 128 M1 TS $4 004 519
MMSGSugmL MMSGSupmL
95 49 63 117 96 45 52 83
smo
as
sm 39
no sad 8 wu
"Test wCogmp.ound
RelaVteihviecleto Conol
RVeelhiiclveteo Control
12s
00 75 106 65)9 moose sone 79 60
255000
2930s616 12622 a8l24s 1I5s6s a43 osaac 796422 $M910
715000
9946 7650 115078 6ms2 s 17 san 1s0s3 e6s8ds 8680
E RSG = T1rT 25eas0tment T rminat66i36onT(De3e$y99T%1y)cel21la32d5eensi 31U2o01ax2) T[aDya2Tyew3ece3lolddeennssitasyreT100""oor7DDaasyysss3||dGeennstHnB1ooett8s7pRit7GbR6ac1k5]
"Cloning[EDfafyic3iceenlclyd=enTsoittya3Vxi10seorCDoaloyn2ydCeonusniutNyuirfminbotesrpoliftCbealclks]Seeded * 100
"Relative Growth = (Relive Suspension Growth * Relative Cloning Efficiency/) 100
"MuDteacnitmaFlrisemqovue=ed(nTtooectxayplreMsusttahnetfrCeolqouneinecsyToitnanlitVsiaobfle10CEol-o6nies) * 2108-4
"Vehicle Control = 1% DMSO.
MutPaogseintiicv.e CEonxtcreoel:dsMMiniM =muMmeS tChryitlermieotnhoafnes11u2 onXate108:6
Not subelured
2
Covance 20785-0431 ICH 3M Study No.: T-6316.12
TABLE 6: SIZING DATA FOR CONFIRMATORY MUTATION ASSAY WITHOUT ACTIVATION
BA GTEESNTETAIRCTSICALSE:SANY-BNIOF.OS2E078T5-.60341361. ICH C. VSEEHLIECCLTEI:VEDVASGEONT:TFT3.0 pg/ml. E TREATMENT DATE: 06072000
Cum RSG (09
TetConditon Vehicle Control"
Cone Day) Day? Dayd wWo 10m4s l97l4 3BI wm 076 9s 119
EfCfliocniienngc.y" Abs% Rel% 9m53 1S9273 BS sl
ReGlraotwitvhet 8949 wis
Mutant(xF1r0e%q*uency Toul Small_Lage 5S1146 24ss 22091 sty m2 27
MMS' ug/ml) 66s5 e2s seua 2s6s2 s38l9 a426)7 H87o soeu2s0 3366ss 21075s2
Test Arile(u/mL) 1235 m2 me ess 947 136 9 25 680 4 ms 70 924 762
65900 22089 330011
S2o5o0 s6ess 1e2109 MWse m062 886466
242 ass
ua 680
am 343
we ns
"
0 2s
702 483
MS a8
62 94
912007 1s1s03
66344
150 461 08 22 me 883 187
$158 3320 221s8 %5 395 310
TClooninRgSEGf=fCiumcuilae=tTniovcelySuVsipseblneioCnolGonryoChouRnetlaNiuvmebtehoefCAlvelraSgeeeVdeehdic*le10C0ontrol Suspension Growth
MReulaatnisveFGreqruenoc=yw(=Re(tlTaottihavleMSuutsapnetnCsoiloonniGersoTwohta*ReVliaatblieveCColloonniiensg) E*f2f1ic0i8e-n4cy/) 100
DEexcipmarleisasmsToeotadtvoMueetxaprndetsFsrteqhuefnrceyq,ueSnmcyalinCuonliotsnoyfIM0utEa:n6t Frequency and Large Colony Mutant Frequency
"Vehicle Control = DMSO
CPoosliotniyveCCoounnttrosli: nMcrM=ebMyae9S t.sh0y9el9d%mithoancesoumlpfeonnsstacforare ofdish not scanned
2h 267
Covance 20785-0-431 ICH 3M Study No.: T-6316.12
TABLE 7: INITIAL MUTATION ASSAY WITH ACTIVATION
A GTEENSETTAIRCTSICALSE:SANY-NEOFO2S0E78T5.603-41361 ICH CD. VSEEHLIECCLTEI:VEDMAGSEONT. TFT 30 ug/ml.
EF TCERLELASTAMNEANLTYDZAETDE::501501"62000 HG. TERXPERAETSMSEINOTN PPEERRIIOODD:: 24dhaoyusrs
Test Condition CowDisl(yCCeellimL, CumuRlSaGt'ive TMouualm VTiobule EfCfliocniienncgy'
10ES Units)
Colonies Colonies
$9-Ackvaiion Convo" BT DT AVG
Ave
59 Baich Number: 955
ve
ve
VeVheihciclleeCCoonnvraall VehiceConvol
11121 7171530 214845
mmw smer nmes
108 113 136 188 221 SB sss si
MCAZugmL MCAsugmL
76 1S1 128 62 156 107
@ wm es
@
est
Tespt Cromipound
RVeelhatiicvleeto
VehRiecllaeiCvoen0trol
Control
520553
s8i8e 0ms 62s0 lM e 4a0 994731
718051
0tset en6s2 B1s0 ier aewn 985791
R"SClGoni=ng(DEafyf1iCci=oeTnocuuyl(DVnaiyb2le3CCooul)noyn3y CooruindyNu1mCboermtorfoCevlslsiSbeaeudoerde*d1y00
Relative Growth= (Relative Suspension Grow * Relative Cloning Efiiency)/ 100
"Mutant Frequ=en(Tcotayl Mutat Clonies/Totl Viable Colonics) * 210E-4
"VehDieccliemCaolntisromlov=e1d%0DeMxpSrOess the frequency in unitsof 105-6
MutPaogseintiicv.e CEonxtcreoel:dsMMiCnAim=uMmeCtrhityekrhioonlaoft1h5en6e.X7 108.6
No subcultured
"One plate contained. Totalcountcalculatedbyusiawenigght proportion
ReGluoiwvhe FreMquuaenntcy [CUT nie)
nss0 m7es ss 73 su a wr we
a@7 aa2s noss mmi6
Covance 20785-0-431 ICH 3M Study No.: T-6316.12
TABLE 8: IZING DATA FOR INITIAL MUTATION ASSAY WITH ACTIVATION
5A. TGEENSETTAIRCTSICALSE:SANY-NEOF.OS20E78T5-.06.341361 ICH
. SVEEHLIECCLTEI:VEDMASGEONT. TFT3.0 ug/ml.
E. TREATMENT DATE: 05162000
CmRSGEH EfCfliocniienngcy RGersoivwe MuniF0re9quency
retCondion Cone. DiyT DZ Roa Re% (6) Tow Smal Tue
Teme Ww nor m7 sea 93
BmEe s9e7r
n8s0
WTSe $39 2a9
me mo sss ale ms Ta 49 0S
MCN(upimL) 42 essmos s@n1 e61s 1msa 4su2 a30s6 11560615 116h7)9
Tes Aricle(ug/ml) 13 Ss
s3s emso
mm3s osauy
027
@az8 m8Ss 2me
mors n4b3s Bnyb msi2s sssuo n94s 7m16 2ss8a m278
"CCiloninSgGEf= Ccumeul=naTicvoeylSoVpiesneaiCnolGornaynChoRuelnayisNtuohmfbAeevlersraSgeeeVdeehdic*le10C0ontrol Suspension Grows
R"MeultiavneFGrerqouwentch=y =(R(elTaottiaveMuSutsapnetnCsiloonniGerso/wTtohal* VReilaabtlieveCoClloonniiensg) E*f5f1ic0i6e-n4cy) 100
`EDxepcrimeasledis m5oovredltto eaxnprtesFsrtehqeueqnucye,nScmyalinCuonliotsnyofM0o8t-a5: Frequencyand Large Colony Mutat Frequency
"Vehicle Control = DMSO
pCoosliotniyveCCoounnttrsoli:nMcCasAe=bMye9t.h0y9c9%htaolncioemnpeensate for araofdish nok sand
2
265
Covance 20785-0431 ICH 3M Study No.: T-6316.12
TABLE 5: CONFIRMATORY MUTATION ASSAY WITH ACTIVATION
A5C.TGVEEESHNIETCTLAIERC:TSIDCALMSES:SOANY-NEO.OS20E78T5.603.41361 ICH . SELECTIVE AGENT:TFT30 gm.
EGF.CTTERRLEELAASTTMMAEENANNLTTYDPZAEETRDIEO::Du:0i60--0*72ho0u0r0s H. EXPRESSION PERIOD. 2 ays
TetCondtion _ComDss(iCyeCleilmL, CoRmSiGave MTouula VTiobse ECflfocniinegn RGeulawivhe FrMeqouemncy
OES Unis)
Colones Colonies
[a Unie
$9-ActivatonComo" Bayi DwE 59 Batch Number:955
avvea
avvee
VVeehiicceeCCoownaall 9914 116599 116717
mwo ama 7m9s
sB9s5 5w3s
VeideCowal 143 D9 21 186 19 e283 m3 Mss 62
MMooAAZspsmmLL 7913 B12671127 wmowws emss
s7o amnss
Test Cromspound
CRoeVelmhiiavcleleto
RVeeliiiveteo Convo
10
so we 2i) 7m sm mooe qa M3
"000
7sss ooom sss ssmosasm amm3a os6s 5m093
500 1$20 iwsxs w2n8 o0w wsss ess e au37 4s5o2
1$0000 a2e0 n0es awes m Bs uae wwss uas3s sseoa
RSG
=
B1o5y01
Cow)
Ddaye2
Co2sm
nem se Gray Commented)
ner
usa
si
R"CelloaniivngeEGfrfoiwctihe=nc(=yReTlaotilveVSiusspeenCsoiloonnyGrCoowutnhd*NRuelmabteirveofClCoenlingSeEfecdie*n1c0y0) 100
MutDaenctimFarleqsumenocvye=d (TeoxaprMeostthaeCfroelquiencyTointaulniVitaobfle0CEl-oGnes) * 2108.4
tPiocseiCeonCronotlro=l: 1M9CDAMS= OMetylholntvene
Mutagenic. ExceedsMinimum Crerin of 122.X2 1086
ot Sbculured
270
Covance 20785-0431 ICH 3M Study No.: T-6316.12
TABLE 10: SIZING DATA FOR CONFIRMATORY MUTATION ASSAY WITH ACTIVATION
AB.. TGEENSETTAIRCTSICALSES:AYN-NEOW:FO2S0E78T5.-06-361361 ICH
C. D.
VEHICLE: DMSO SELECTIVE AGENT:
TFT3.0
pg/mL.
E TREATMENT DATE: 061072000
Cum RSG (4) CElffoinciinegnc.y" GRerlaoivwe Mutant(1Fr0e%quency
TetCondiion Cone Dal Daya Abs% Rel% 9 Tow small Luge
Tad W860
ss
M8 102 979
$53 27 296
wWo 10B82 usssr 775639 99193s
8n5s9s
648 652
284 266
364 36
MCA(ug/ml) 2 68 7 mE 955 so 233 se2 121 + B2 @s es 92 51 ms 90 1067
Test Aricle(ug/mL) 20 m2 ow1 sw7 79 4 %3 ns as
0Jo seesss sssss ms2s 93 5821 s50839 22176 21827
sG0o0 3a16 2a518
77420 995579
273
si9 $2
294 26
255 216
500 wo
192 1
209 ass
535 999796
21838
s54a0 230814 225852
TC
RSG
=
0 256 14 Corulaive SuspGernowsRieloatinve
1
ss 147 he Average Vehiels
154 Contral
514 ws Suspension Growth
29
*RCelloantiinvge GErfofwtihc=i=(eReTnloacttayiveViSaubslpeenCsoiloonnGyCroowtuh n* RNelautimvoefbCCleeolnrlisnSgeEeideide*nc10y0) 100
"Mutant Frequency = (Total Mutant Colonies Total Viable Colonies) 2x10B-4
DEexcipmarleissamssoovereaddltMouetxapnrteFsrheqeuefnrceyq,uSemnacyllinCoulniosnyofMu1t0aEn-t6 FrequencyandLargeColony Mutant Frequency
"Vehicle Control = DMSO
pCoosliotniyveCCoounnttrosl:incMreCsAsed=bMyet9h.y0l9c9h%oltaonchormepneensate foraeofdaish no scanned
8 27/
Covance 20785-0-431 ICH 3M Study No.: T-6316.12
HISTORICAL CONTROL DATA
Nonactivation Studies Pooled negative and vehicle control mutant frequencies
Mean (+ SD)
53.04220x10
RNaunmgbeerofexperiments Numberof controls
20510 51214.8x10 156
Positive control mutant frequencies (6.5 pg/mL methyl methanesulfonate)
Mean ( SD)
27271357x 10
RNaunmgbeer of experiments Number of controls
115.910 65132.1x 10 51
Positive control mutant frequencies (13 pg/mL. methyl methanesulfonate)
Mean (= SD)
4839431x5120
RNaunmgbeerofexperiments Numberofcontrols
176.110 512996.x4 10 52
Activation Studies Pooled negative and vehicle control mutant frequencies
Mean (+ SD)
653%27.1x 10
RNaunmgbeerofexperiments Numberof controls
27.610 51450x.103 162
Positive control mutant frequencies (2.0 pg/mL 3-methylcholanthrene)
RaMenagne(= SD) Number of experiments Numberofcontrols
245044.851+0176867.28xx 1100 54 54
Positive control mutant frequencies (4.0 pg/mL 3-methylcholanthrene)
Mean (+ SD) Range
Numberofexperiments Number ofcontrols
567.0+2483 x 10 218910 1414x 1.09
54 54
Because some experiments contained multiple controls, the numberof independent control cultures exceeded the number of experiments.
1 ON mine
2.
272