Document 2qnYpLpxZrGyK42ZNnOGOMLV7
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FINAL REPORT
Study Title
H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes
Authors
Ramadevi Gudi, Ph.D. Meena Rao, B.S.
Study Completion Date
24 November 2003
Testing Facility
BioReliance
~~
9630 Medical Center Drive
Rockville, MD 20850
for
E. 1. duPont de Nemours and Company
DuPont Haskell Laboratory
P.O. Box 50
1090 Elkton Road
Newark, DE 19714-0050
`BioReliance Study Number
AAT9YX.341.BTL
~. Page 1 of39 Company Sanitiad. Doss not conain TSCA CBI
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2H4o7m68a:nIPnerViipvhoeMramBmlaoloidaLnyCmhphroocmyotsesome Aberation Study
Dupont13562
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
This study was conducted in compliance with US. EPA TSCA (40 CFR part 792) Good
LaboratoryPracticeStandardsexceptfortheitemdocumenbetleowd. The itemslisteddonot
impact the validofitthye study.
`Thetestsubstancewascharacterizedby the Sponsorpriortothe initiation of this study. Although characterization was not performed under Good LaboratoryPractice Standards, the accuracyofthe data is considered sufficient for the purposes of this study.
Analysesto determinetheuniformityor concentorfattheitoesnt `mixturesand their stabilitywerenotperformedbythetesting
facility or the Sponsor.
PN
BioReSltuidyaDinrecctoer: `BioReliance Study Management
Kata pplbten!,Ged
'Ramadevi Gudi, Ph.D.
ndJ ely
2 pou203
Date
24 Novzoos
Date
~~
BStiuodRyelNion.neAeATOYX 341 BTL ---
2 Company Saniized. Doss not contain TSCA CBI
~
Hin2H4u7n68a:nIPnerViipthreoraMlazBzlmoaoldiLaynmCphhroocymtoessome Aberration Study
Dupont.13542
Quality Assurance Statement
Study Title: SH-T2U47D6Y8:IINNHVUIMTARONMPAERMIMPAHLEIRAALNBCLHOROODMLOYSMOPMHEOACBYETRERSATION Study Number: AAT9YX.341 BTL Study Director: Ramadevi Gudi, Ph.D. a`pTphirsoascthu,dQyuahlasitybAesesnurdaivnicdeemdoniinttoorasesaecriheosfotfheisne-pphroacseesssovphearseass.erUiseisnogfs2turdiaensd.oPmrocseadmuprleisn,g pdoceumrentfatiionoanrc,comerqdueainpdcmeewnitthrtechoerdUs.,S.etFc,DAarGeooedxLaambionreadtoirnyoPrrdaecrtictoeReagsusulraetitohnast (t2h1eCsFtRu5dy8)i,s tShteanUd.aSr.d,EaPnAd GtLhePsOE(4C0DCFPrRin7c9ip2leasnodf4G0oCoFdRL1a6b0o)r,atthoeryUPKracGtLicPeRaengdultaotiaosnssu,rethtehaJtaptahneessteuGdyLPis `acroentdhuectiendspeeccctoirodnidnagtetso, tphheaspersotioncsoplecatnedd,raenldevraenptorSttadnadtaersodfOpQerAatiinsnpgecPtrioocnesdoufresh.isTshteudfyo.llowing
~~
PIhnasspeec:tPOrnot:oc1o8l-R5e6v-i03ew- 18-Sep-03 ToStudy Dir 18-Sep-03 To Mgmt 18-Sep-03
IPnhsapseec:tCOond:in1g6o-f0sclt-i0d3es- 16-0ct-03 To Study Dir 16-Oct-03 To Memt 16-0ct-03
PIhnasspee:ctDOrna:ft0R9e-pNoorvt-a0n3d-d0a9t-aNaouvdi-t03 To Study Dir 10-Nov-03 To Mgmt 11-Nov-03
IPnhsapseec:tDOrna:ft1t7o-FNionva-l0R3ep-o2r1t-Nov-03 To Study Dir 21-Nov-03 To Mgmt 24-Nov-03
"aTchciusrraetpeolrytdreefslcercitbtehsetrhaewmedatthaoodftshanedsptruodsc.eduresusedinth studyand thereportedresults
boty
BeckyIf
DSchuseleng,
Schreckengost,
Ss
QUALITY ASSURANCE
24 Nov
DATE
2005
--_ BSitouRdeyliNaon.ceAATOYX341 BTL
3
n
--
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Company Samitized. Does not contain TSCA CBI
_
F-24768: In Vitro Memmalisn Chromosome Aberration Study
nHumen PeriphersiBloodLymphoevies __________
CERTIFICATION
Dwone13se
f`Wreo,mtthheisunsdteudrys.igned, declare that this report provides an accurate evaluation of data obtained
IssuedbyStudyDirector:
Prunecbn] Zed.
Ramadevi Gudi, Ph.D. BioRelianceStudyDirector
U- Wov-2028
Date
suAdpypMroonvieodrb,y _E- Hoi Boner.
13epov-acen
--~
`Sen`Ei.orMaRreisaeaDrocnhnTeorx,iPcho.lDo.gist
Date
--_
E-- BioT Ra-- tane ------eeoeeeenmryeey
StudyNo.AATOYX.341BTL
4 Company Saniized. Dos not contain TSCA CBI
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iBnL2H4o76m8a:PnIenriVpihreoraMlamBmloaoldisInChromosome Aberstion Study
1356)
TABLE OF CONTENTS
PAGE
`GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT ...o.ovsmssmsomssmssssssmsmmsnsd.
QUALITY ASSURANCE STATEMENTc..cssnsssesnsd
SE SE STUDYINFORMATION oss] BONARconsmmmmmmmssssimsnsmssissmommmmmssiimsmmmond
PURPOSE ccnaJ
CHARACTERIZATION OFTESTAND CONTROLSUBSTANCES...
10
MATERIALSANDMETHODS...
0
(COLOL FMEETACPHT ASEICEO LLSN , ......c.cocsvmsssssssss RA--------------
SELECTION OFDOSELEVELSFORANALYSE...
13
CRITERIAFORDETERMMATAVIAOLNGF TES...
15
RESULTSANDDISCUSSIONvss
CONCLUSION css] REFERENCES cssssssssemresssmssssamssssndd
Be Study No. AAT9YX341.BTL
$ Company Santzed. Doss not contain TSCA CBI
~
H-24768: In Vitro Mammalian Chromosome Aberration Study
inHumanPeripheral Blood Lymphocytes
Dupont13542
DATATHSEE mummiesty
`TABLE 1 PRELIMINARY TOXICITY TESTUSINGH-24768 IN THE ABSENCE OF
EXOGENOUS METABOLIC ACTIVATION 4 HOURTREATMENT...
19
TABLE 2 PRELIMINARY TOXICITYTEST USING H-24768 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION 4HOURTREATMENT... minal
`TABLE 3 PRELIMINARYTOXICITYTESTUSINGH-24768 IN THE ABSENCE OF
EXOGENOUS METABOLIC ACTIVATION...
EL
``TTRAEBALT4EEDCWYTIOTGHEHN.E24T7I6C8AINNATLHYESAIBSSOEFNHCUEMOAFNEXPOERGIEPNHOEURSAMLEBTLAOBOODLILCYAMCPTHIOVCAYTTIEOSN
DEFINITIVEASSAY: 4HOURTREATMENT,20HOUR
BARVEST.....crrsnsscrnen 22
`TABLE 5 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH H.24768 IN THEPRESENCE OFEXOGENOUS METABOLIC ACTIVATION DEFINITIVE ASSAY: 4 HOURTREATMENT, 20 HOURHARVEST ....o.occovssrsns33
`TABLE 6 CYTOGENETICANALYSIS OF HUMAN PERIPHERALBLOOD LYMPHOCYTES
`TREATED WITH H-24768 IN THE ABSENCE OF EXOGENOUSMETABOLIC ACTIVATION.
~~
"DEFINITIVE ASSAY: 20 HOURTREATMENT, 20 HOURHARVEST...
24
APPENDIX A HISTORICAL CONTROLDATAcsrssmssmmssmmssmsmsssnss26 APPENDIBX STUDY PROTOCOL ercersrmsnssssesessmemssssmensinsnd
-- BSitouRdeylNioa.ncAeATOYX341 BTL
6 `Company Sanu.zed. oes not contain TSCA CBI
~~
Jn Human Peripheral Blood Lymphocytes
STUDY INFORMATION
Dupont-13542
a H-24768
Haskell Number: 24768
CAS Registry
|
Composition:
a --~ oon pi TR
Physical Characteristics: Tan to light brown paste
Stability: The test substance appeared to be stable under the conditions of the study; no evidenceofinstability was observed.
Sponsor: E. I. du Pont de Nemours and Company `Wilmington, Delaware 19898 USA.
Study Initiated/Completed: September 15, 200/3 (see report cover page)
---In-Life Initiated/Completed: September 17, 2003 / October 26, 2003
Sauls No AABVEHIBTL
Z
Company Samuzea. Does not contain 1 SCA CBI
--_
i2n4H7u6m8a:nPInerViipthreorMalaBmlmoaoldoLnyCmhphroocmyotsesomeAberration Study
Dupont 13542
SUMMARY
The test substance, H-24768, was tested in the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes (PBL) in both the ebsence and presence of an Aroclor-induced S9 activation system. A preliminary toxicity test was `performed 10 establish the dose range for testing in the cytogenetic test. The chromosome aberrationassaywas used to evaluate the clastogenic potentialofthe test substance.
Dimethyl sulfoxide (DMSO) was determined to be the solvent of choice based on the solubility ofthe test substance and compatibilitywiththe target cells. The test substance was tshoeluabslseayi.n DMSO at a concentration of 100 mg/mL, the maximum concentration prepared in
In the preliminary toxicityassay,the maximum dose tested was 1000 pg/mL. Human
`peripheral blood lymphocytes were trated in the absence and presence ofan Aroclor-induced
89 activation system for 4 hours, and continuously for 20 hours in the absence of S9
activation. The test substance was soluble in treatment medium at all concentrations tested.
Selectionofdose levels for the chromosome aberration assay was based on a reduction in the
mitotic index relative to the solvent control. Substantial toxicity, ie, at least a
--
50% reduction in mitotic index, was observed at doses of 30 pg/mL in all three exposure
`groups. Based on these findings, the doses chosen for the chromosome aberration assay
rangedfrom 2.5 to 100 pg/mLforallthreeexposuregroups.
Inthechromosomeaberrationassay,thecellsweretreatedfor 4and 20hoursinthe
non-activatedtestsystem andfor 4 hoursinthe S9activatedtestsystem. Allcellswero
harvested at 20 hours after treatment initiation. The test substance was soluble in treatment
`mediumatall concentrations tested. Selectionofdoses for microscopic analysis wasbasedon
`mitotic inhibition (the lowest dose with at least 50% reduction in mitotic index, relative to the
solvent control and two in the following table:
lower
doses)
in
al
harvests.
The
resultsofthe
assay
are
summarized
_ | reament| Recovery| Harvest
(Toiumse) | (Triowmse) || (Thiomew
RMeidtoutcitcioInndexa.t| 10r| LEDfor
higcheosrteddose | Aberratiy ons| Aberrati` ons
os agmp | GED) | GgmD) |
7116 T 30 [-] 5% | Wow | Wow |
200150 1-1""58% | Nome | Nome |
L* rea lative 10 1solven1t c6ontr1 ol a h2ig0h do[se e+val1uated5foor%chro| mosomeNoabnererat| ions Nome J
--_
LED = Lowest Effective Dose
SBitouRdeylNiaoncAeATOYX.341 BTL
5 Company Sanitized. Does not contain TSCA CBI
~
iHn-H2u47m6a8n: IPnerViipihreoraMlaBmlmoaoldiaLnymCphhroocmyotsesome Aberration Study
Dupont-13542
Based on the findings induction of structural and
of this study, H-24768 numerical chromosome
was concluded to be negative aberrations in the non-activated
for and
the S9
activated test systems in peripheral lymphocytes.
the
in
vitro
mammalian
chromosome
aberration
test
using
human
--~
--~ BioRelince Spe Averisisn
I------ " Company Saniized. Does not contain TSCA CBI
H.24768: In Vitro MammalianChromosomeAbeation Study
~~
in Human Peripheral Blood Lymphocytes
Dupont-13542
PURPOSE
based`Tuhpeopnurtpsoasbeiloiftytthoisinsdtuucdey cwharsotmoosevoamleuaatbeertrhaeticolnasstionghenuimcapnotpeenrtiipahleroalfalytmepsthoscuybtsetsa.ncAe copyof the study protocoli includedin Appendix B.
CHARACTERIZATION OF TEST AND CONTROL SUBSTANCES
was Tahsseigtnesetd stuhbestacnocdee, nHu-2m4b7e6r8,AAwaTsOYrXec.eiveTdhebyteBsitoRseublsitaanncceeownaSsepcthaermabcetrer9i,ze2d00b3y atnhde Sponsor as a tan to light brown paste that should be stored af room temperature. Upon rteecmepieprta,ttuhreet,esptrsotuebcstteadncferwoamsedxepsocsruirbeedtaosliagbhrt.owTnhpeasStpoysnusborstahanscedaentderwmaisnedstotrheedaitderntoiotym, strength, purty composition or other characteristics to define the fest substance and the
stabilityofthe test substance.
The solvent used to deliver H-24768 to the test system was dimethyl sulfoxide (DMSO, (CAS No.: 67-68-5) obtained from Fisher Scientific.
Mitomycin C (MMC; CAS No.: 50-07-7), was obtained from the Sigma Chemical
~
Company, and was dissolved and diluted in sterile distilled water to stock concentrations of
30 and 60 pg/mL for use as the positive control in the non-activated test system.
CCyocmlpoapnhyo,spahnadmwiadse d(iCssPo;lvCedAaSndNdoi.l:ute6d05i5n-s1t9e-r2il)e, diwstaislleodbwtaatienredtofsrtoomck ScoingcmeantrCahteiomniscaolf
p2osaintdive4 cmongt/romlL, ofnore udsoeseaswitthhespuofsfiictiievnet csocnotrraoblleinmetthaepSh9asaecctailvlastewdastesstclsoycstteedmf.or Faonarlyesaicsh.
The solvent for the test substance was used as the solvent control at the same concentration as
that found in the test substance-treated groups.
The negative and positive control substances have been characterized as per the
pCoesrititfiivceatceosnotrfolAnsaulbysstiansceosn afnilde twhietihr mtihxettuersetsinwgafsacidleimtyo.nsTthreatsetdabbilyitayccoeftpthaeblenergeastuilvtse tahnadt
`met the criteria for a valid test.
:
MATERIALS AND METHODS
`Testing Guidelines
`This study was conducted in compliance with OECD Guideline 473 (In Vitro Mammalian
Chromosome Aberration Test), Ninth Addendum to the OECD Guidelines for the Testing of
Chemicals, published by OECD, Paris, February 1998 and with the International Conference
on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human
~~
Use (1996 and 1997).
StBuodykNeo.rAATOYX.341 BTL
E 0 E
--
Company Saniized. Does not contain TSCA CBI
~
HH-u24m76a8:nIPerViipthreoraMlaBmlmoaoldiaLnymCphhorcoymtoessomeAberration Study
Dupont-13542
Test System
adultPemrailpeheoranl1b5loSoedptleymmbpehroc2y0t0e3s fwoerrteheobptraeilniemdinfarroymtoaxihceiatlythayssanyo,n-asnmdooknin2g92S7eypetaermboelrd o2r0t0h3efaodrmtihneisdterfaintiitoinovefadsrsuayg.s.ThTehidsotneosrt shyasdtneomrehcasenbteheinstdoermyoonfsrtardaitoetdhetroabpey,sevnisrialtiivnefetcottihoen clastogenic activityof avarietyofchemicals (Preston et al, 1981).
Activation System
was pArreopcalroerd1f2r5o4m-imnadlueceSdprraatgluiev-erDa$w9lweyasrautsseidndausctehdewmietthabaolsiincglaectiinvtartaipoenristyosnteeaml.inTjhecetiSo9n of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The SO was batch prepared and st`omerteadbaoltize< 27-0amiCnuonatnitlhursaecden.eEaancdh7b,u12l~kdipmreetphayrla-tbieonnzo(fo)San9thwraasceansestaoyefdofromsrimtustaabgielniitcyttoo Salmonella typhimurium TA100.
2
Immediately prior to use, mM magnesium chloride, 6
tmheMS9powtaassstihuamwechdloarniddem,ix1emd Mwitghluacocsoef-a6c-tporhopsopohlatteo,
contain 1 mM
~~
nicotinamide (RPMI 1640
adenine dinucleotide serum-free medium
pshuopspplheamteen(teNdADwPi)than1d0020unpiLts$p9enpiecrilmliilnlilaintedr
medium 100 pg
streptomycin/mL, and 2 mM L-glutamine).
Solubility Test
or
moAresoloufbitlhietyftoelsltowwiansgcsoonldvuecnttseditnotsheeleocrtdtehreofsoplrveefnte.reTnhcee
test was conduoted as listed: dimethyl
using one sulfoxide
(seDlMecStOe)d,ineotrhdaneorol,fparnedfearceentcoen,et.hatThpeermtietsttesdubpsretpaanrcaetiwoansoftetshteedhitgohedsettseorlmuibnlee
the solvent, or workable
stock conceniration, up to 500 mg/mL.
`Preliminary Toxicity Assay
'
chroTmhoesotmoexicaibteyrrtaetsitonwaassspaeyrfaonrdmecdonosirstetdheofpuarnposevealoufatsieolnecotfingfesctonscuebnsttraantcieonesfffeocrt tohne -
c`mointtoatiicniinngdex.9.A4ppromxLimatRelPyM0L.I66m4L0hepcaormipnilzeetdeblomoeddwiausminocsuulpaptleedmiennttoecdentrwiifutghe tub1e%s
phytohaemoagglutinin (PHA). atmosphere of 51% CO; in air
The tubes for 44-48
were hows.
incubated The pH
at and
37+1C in osmolality
a of
humidified the highest
`tmraeianttmaeinntacnoenudritailonpHweirnethmeetarseuartemde,ntamneddithuem.pHAtwtahse taidjmuesotfetde,stifsnuebcsetsasnacrey,trienatomrednetrthteo
~~
icnulteuirtehetrub1e0s wmerLe coefntfrriefsuhgedR,PtMhIe-s1u6p4er0nactaonmtplweatse amspeidraituedm, acnodnttahienicnelgls1w%erePHreAsusfpoerndtehed
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1 `Company Sanilized. Does not contain TSCA CBI
~
2Jn4H7o6m8a:nIPnerVpihreoraM!aBmlmoaoldiaLynmCphhrocoymtoessome Abemstion Study
Dupont 13542
PnoHn-Aac+ti2vmatLedofstSud9ycoorfa1c0tomr LpooSl9),retaocwthioinchmwiaxtsuarded(e8dm0L.1 smerLumtesftreseubmsetdaincuemdcoosnitnaginsionlguti1o%n
in solvent or solvent alone.
fo4r`hToheurcselilnsbwoetrhetheexpporseesdentocesoalnvdenatbsaelnocneeoafndS9toancitinveatcioonnc,enatnrdaftoiorn2s0ofhotuhrestecsotnstuibnsutoaunscley
in the absence of S9 activation. The cells were incubated at 37+1C in a humidified
tartemaotsmpehnetremeodfi5u1m %waCsO;reimnovaeird., Atthethceellcsomwpalesthieodn woifththeca4lchiouumr eaxnpdomsaugrneepseiruiomd.,frtehee
phosphate buffered saline (CMF-PBS), refed with RPMI-1640 complete medium and
rehatruvrensetd,tCooltcheemiincdubwataosrfaoddreadnatoddtihteiocunlatlu1r6eshaoturasf.i"naTlwcoohncoeunrtsraptriioonrotfo0t.h1espgc/hmeLdaunldceetlhdel
cultures were rettotuheminceubatdor uniil cel collection.
Cells were collected by centrifugation, treated with hypotonic potassium chloride
(de0t.e0r7m5iMneKdCiIn),orfidxeetdro, setvaailnueadteatnedst tshuebsntuamncbeereffoefctceolnlsmiitnotmiictoisnidsexp.er 500 cells scored was
Chromosome Aberration Assay
~~
1976;ThEevacnhsraonmdosO'oRmieoradbaenr,ra1t9i7o5n)absysaeyxpwoassingpedrufpolricmaetde ucsulitnugresstaonfdhaurdmapnropceerdiuprheesral(Ebvlaonosd,
lymphocytes (HPBL) to at least 4 concentrationsofthe test substance as well as positive and
isnoiltvieantitoncoofnttrroelast.menTth.e dividing cells were harvested at approximately 20 hours from the
For the chromosome aberration assays, 0.6 mL heparinized blood was inoculated into centrifuge tubes containing 9.4 mL complete medium supplemented with 1% PHA. The
4t4ub-e4s8 wheourres.incTurbeaattemdenatt w3a7s1caCrriiend aouhtumbiydirfeifeededaitngmowsipthhereapporfox5ima1t%elyCO1;0 imnLairfrefsohr complete medium or $9 reaction mixture to which was added 0.1 mL of dosing solution of
test or control substance in solvent or solvent alone.
In the non-activated study, the cells were exposed for 4 or 20 hours at 37+1C in a
pheurmiioddi,fitheed atrtemaotsmpehnetrmeoefd5iu1m%waCs Oreminovaier., Itnhethceel4lshwoausrheexdpowsiutrhecgarlocuipu,mafatnedrthmeaegxnpeossiuurme..
free phosphate buffered saline (CMF-PBS), refed with complete medium containing 1% PHA.
and
cell
returned to the incubator
harvest at 20 hours after
for an additional 16
treatment initiation,
hours. Two hours prior
Colcemid was added to
to the scheduled
the cultures at a
final concentration of 0.1 pg/mL. In the 20 hour exposure group treatment was continuous
until
after
tthreeattimemnetoifnicteilalticoonl,leCctoilocn.emiTdwowhaosurasddperidortotothtehecuslcthuerdeuslaetd
cell harvest at 20 hours
a final concentration of
~
0.1 pg/mL.
BStuodyrNdo.eAATSYX 41BTL
2 Company Sandized. Does not contain TSCA CBI
--
HL2H4u7m68a:nIPnerViipthreoraMlaBmlmoaodliLeynmCphhrocoymtoessomeAberration Study
Dupont13542
atmosIpnhtehreeSo9fac5ti1va%tedCOst;udiyn, taihre. celAlfstewrerteheexepxopsoesdurfeorpe4rihoodu,rsthaet 3tr7ea1tmCenitn maehduimuidmifwiaesd
removed, the cells washed with calcium and magnesium-free phosphate buffered saline
(CMF-PBS), refed with complete medium containing 1% PHA and returned to the incubator
tfroeraatnmeandtdiitniiotniaaltio1n6,
hCouorlsc.emTiwdo
whoausrsapdrdieodr
to
to
thtehescchueldtuulreesd
caetllahafrivneaslt
caotn2c0enhtorautrisonaftoefr
0.1 pg/mL.
Collection of Metaphase Cells
`Twohoursafterthe addition of Coloemid,metaphasecellswere harvfeorsbotthethde
activated and non-activated studies by centrifugation. The cells were collected by
centrifugation at approximately 1200 pm for about 5 minutes. The cell pellet was
resuspended in 5 mL 0.075 M KCI and incubated at 3741C for 20 minutes. At the end of the KCI treatment and immediately prior to centrifuging, the cells were gently mixed and
approximately 0.5 mL of fixative (methanol:glacial acetic acid, 3:1 v/v) was added to each
tube. The cells were collected by centrifugation, the supernatant aspirated, and the cells were
fixed with two washes with approximately 3-5 mLoffixative and stored in fixative overnight
`or longer at approximately 2-8C.
~
Slide Preparation
To prepare slides, the fixed cells were centrifuged at approximately 1200 rpm for
5minutes
fixative.
, the
The
supernatantwasaspirated, and thecellswerere
cells were collected by centrifugation and the
suspended in 1 mLcoldfresh
supernatant aspirated, leaving
0.1 to 0.3 mL fixative above the cell pellet. An aliquotofthe cell suspension was dropped
onto a glass slide and allowed to air dry overnight. Slides were identified by the study
number, dose level, activation condition, harvest time, replicate tube designation and date
pmroeupnatreedd.. The dried slides were stained with 5% Giemsa, air dried and permanently
SelectionofDoseLevelsfor Analysis
+
`The selectionofdose levels for analysis of chromosome aberrationsin HPBL was based
duopsoen wthoxiicchityi,ndiunctehde aftollelaoswtin5g0o%rdteorx.icTihtye, haisghmeesatsudorseed lbeyvemlisteolteicctiendhifboirtieovna,luraetliatoinvewatso tthhee
solvent control, with a sufficient numberofscorable metaphase cells. At least two additional
lowerdose levels were included in the evaluation.
BSitouRdeyliNaon.cAeTOYX.341BT-- L
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Company Saniized. Does not ontain TSCA
~~
inH-2H4u7m68a:nIPnerViipthreoraMlaBmlmoaoldiaLnyCmhphroocmyotsesomeAberration Study
Dupont 13542
EvaluationofMetaphase Cells
`proceSslsi.deIsniwteiarlleyc, otdheednouns-iancgtirvaatneddomannd uS9mabctbeiyvraatsend i4ndhiovuirdueaxlpnoosturienvgorlovuepdswwitehretheevaslcuoartiendg
efxorpocshurroemgorsooump,e atbheerrnaotni-oancstiavnadtiefd a20pohsiotuirvecroenstuilntuwoauss eoxbptaoisnuerdeignrtohuepnwona-sacntoitvanteecdes4sahroiulry
evaluated under oil
fiomrmcerhsrioomnoswoimtheouabterprraitoironksn.oMwelteadpgheasoef
cells with treatment
46 centromeres were examined groups. Whenever possible, a
a`nmdinsicmourmedoffo2r00chmreotmaatpihda-steypsepraenaddsc(h1r0o0mpoesromdeup-ltiycpaeteatbreerraattmieonntsc(oSncdoitttiocnt) awle.,re19e9x0a)m.iTnheed
i`futmhbeepreorfcemnettaagepohfasaebesrprraenatdscetlhlastrweearceheedxaamsitnateidstaincadllsycsoirgendifpiecrandtulpelvieclatbeeffloarsek 1w0a0scerleldsucaerde
scored. figures
Cshurcohmaastiqdu-atdyrpieraadbiearlrsat(isoynmsmientcrliucdaeclharnodmaatsiydmamentdriicsaolchirnotmeartcihdanbgreesa)k,s
tarinrdadeiaxlcsh,aanngde
ecxocmhpalnegxerfeiagrurreasngseumcehntass.dicCehntrroimcossaonmder-itnygsp.e Farbaegrrmaetnitosns(icnchludreocohmrraaocmetnotsiroicmd)e obbrseearkvsedanidn
Ftrhaegmaebnsetnscoebsoefravneyd weixtchhaanngeexfcihgaunregewfeirgeursecworeerde ansota sbcroeraekd a(csharonmaabteirdraotriocnhrboumtoisnostmeea)d.
--
were cells
acnodnssiedveerreedlypadratmaofgetdheceilnlcso(m>pl1e0teabeexrrcahtainognes.)
alPsuolvweerriezerdeccohrrdoedm.osCohmreo(msa)t,idpuglaveprsiz(eadn
wailidgtnhedoafchthreomcahtriocmraetgiido)n ainndoniescohcrhormoamtaitdi,d tghaepssize(oanf walhiigcnhedi,s aecqhuarlomtaotoirc srmeagliloenr tihnanbotthhe
rcehcroomradteiddsb,utthenostizeinocflwuhdeidchiins
equal to or smaller the analysis. The
than XY
the widthofthe chromatids) coordinates for each cell
were with
icnhdreoxmowsaosmraelcoarbdeerdraatsiotnhsewpeerrceernetcaogredoefdcuesllisnginamictaolsibirsapteedr m5i0c0roceslclospceousnttageed..
The mitotic The percent
polyploidandendoreduplicated cells was evaluated per 100 cells.
Controls
003MaMnCd 0w.a6spgu/smedL.asCtPhewpaossituisveedcaosnttrhoelpionsitthievneocno-natcrtoilvaitnetdhsetSu9dyaacttifviantaledcosntcuednytraattfiionnasl fscuoofrnfctiehcneitetrneatsttniosunumsbbseotrfaon2cf0esawcnaodrsa4bu0ls,eeudmgea/tsmaLtph.heassFoeolrcveeblnolttshcwopanotssriostleilvaetecttcehodentfsroaorlmasenaoclnoynesciesd.notsreTahtleieovsneolalsveextnhhtiabtvietfhioinucgnldea in the testsubstance-treated groups.
EvaluationofTest Results
solvenTth-etretaotxeidccoenftfreocltsanodfarterepartemseennttedarfeorbtahseepdreluipmoinnarmyittootxiiccitiynhteisbtitainodn trheelcathirveomotosotmhee
~
aberration assay. and numerically
dTahmeanguemdbecrellasnd(pteyrpceesnotfaabbeerrrraatnitoncesllpse)r,
cell and
the percentageof structurally the frequency of structural
_------
Study No. AAT9YX 341. BTL
1
`Company Sanitized. Does not contain TSCA CBI
--~
H3H4u76m8a:nIPnerViiphreorMalamBmlaolodiaLnyCmhphroocmyotsesome Abestion Study
Dupont 13542
prcaeabslecerurnlatateitdoeindsnatpnehdrercdeealpltoar(tbmeuedatanforarebecnraorcathitinotcnrlseuapdteemrdeinctenlltg)rhoeiuntpot.thaelCptohetrraoclmepanottpaiugdleaoatifnodncieolsflosccehwlrliostmheaxtoiandmeiognraepmdsowraarese
aberrationsorin the frequencyofstructural aberrations per cell.
Statistical analysisofthe percent aberrant cells was performed using the Fisher's exact attterseta.atnmyeFinsttheegsrrt'osuspeuxbwasictttahntcteehsattdowfoatsseheulsesevodellv,teonttchoceomnptCarorocle.hrpaIanni-rtAwhmiesmeeivtetanhgteeopfetreacstepnotswiatasibveerurFasinestdhecre'flsolseoxmaefcaetsautercseht
dose-responsiveness.
`inwtheernAptrlhelteapctoeionrnccolefunsttiaohgneessdoawtfea,rceetlhlbesawtseiesdtthsonaubbesrosruatntiadonwnsscacwiseeenrtceifoiincnscijrdueedargseeeddmetnoitni;andhduoocwseeev-areperos,spiotanissv'ieavregemusiapdnoennestreo
with one or more concentrations being statistically elevated relative to the solvent control
group (p<0.05). A reproducible significant increase at the high dose only with no dose
n`oredsopsonesreoersaproepnwrisoldeulcbieblceonsisgindiefriecadnptoisnictrievaes.eHaotwoenveedro,sveallueevseltohtahtaerrtehsatanttishteichalilgyhdsiogsniefwiicatnht
but do not exceed the range of historic solvent controls may be judged as not biologically
~
siignnificcawnrat.seTohbaseestrevseetdsurbesltaatnicevtoetwhaessoclovnecnltudceodnttrool.be `negative if no statistically significant
Criteria for Determinationof a Valid Test
The frequency of cells with structural chromosome aberrations in the untreated and bcsoeonltsvtreaontltiss.tciocnTathlrleoylipsnecmrruceesantsteabdgee(npo<of0.gcr0ee5a,ltFewirsihttehhra'cnsh6erx%oamcwotisfteohsmitn)ertaehbleearthirivasettiotrooincstahlienrstaohlnevgeepnotfsoctroinnvteergoaclto.invteroslolmvuesntt
Deviations
No known deviations from the protocol or assay-method SOPs occurred during the conduct ofthis study.
Archives
All rawdata,theprotocol,all reports,andstained andcodedslideswillbemaintained
according to Standard Operating Procedure OPQP3040 by the BioReliance RAQA unit
`headquartered at: BioReliance, 14920 Broschart Road, Rockville, MD 20850. Paper records
willberetainedforatleastthreeyears afterwhichtimetheSponsorwillbecontactedfor a
decision as to the final disposition ofthe materials. All study materials returned to the Sponsor
7
or destroyed
`minimun of
willfirst
10 years.
be
copied and the copy will be retained in the BioReliance archives fora
BStuedylNoe.AsNT9YX HLETL
1s Company Sanitized. Doo not contain TSCA CBI
~ p2068a1 irtisenCCeheorAsrionsy
mn
Solubility Test
RESULTS AND DISCUSSION
DMSO was determined to be the solvent of choice based on the solubility of the test substance, and compatibility withthetarget cells. The test substance was soluble in DMSO at a concentration of 100 mg/mL, the maximum concentration prepared in the assay.
Preliminary Toxicity Assay
Dose levels for the chromosome aberration assay were selected following a preliminary
toxicitytest and were based upon areduction in mitotic index relative to the solvent control. `The results ofthe evaluationofmitotic inhibition are presented in Tables 1, 2, and 3. HPBL
cells were first exposed to nine concentrations of H-24768 ranging from 0.1 pg/mL to 1000 pg/mL, as well assolventcontrols, inboth the absenceandpresenceofan Aroclor-
induced S9 activation system for 4 hours, or continuously for 20 hours in the absence of S9
activation. The test substance was soluble in treatment medium at all concentrations tested.
`The osmolality in treatment mediofuthme `highest concentration tested, 1000 ng/mL, was
407 mmol/kg. The osmolality of the solvent (DMSO) in treatment medium `was
~~
404mmol/kg.
approximately
The
7.0.
pHofthe highestconcentrationof
testarticlein treatmentmediumwas
obserTvoexdicaitty30(mpigt/otmiLcininhailbltithiroene)trienaetxmceensts
of 50%, relative
groups. Based on
to
the
the solvent control, was
resultsofthepreliminary
tofxoillcoiwtsy:test, the dose levels selected for testing in the chromosome aberration assay were as
`Treatment Condition
`Treatment `Time
Recovery Time
1
_
1
tr)
Dose levels
(ng/mL)
|
_
_
_|
[%[ov Non-activated
25,5, 10,25, 50, 75, 100
[ssnsanm
| .
Chromosome Aberration Assay
tsa, B Sy SoHnoOARSArTeEOeRCSE ~~
In the chromosome aberration assay, the test substance was solubleintreatmentmedium at all concentrations tested. The osmolality in treatment mediumofthe `highest concentration tested, 100 pg/mL, was 417 mmol/kg. The osmolality of the solvent (DMSO) in treatment
--
HE4u7m68a:nIPnerVipihreoraMlamBmlaoloidaLnyCmhphroocmyotseosme Aberrtion Study
Dupont 13542
`medium `medium
was was
403 mmol/kg. approximately
7.T0h. e
pH
of
the
highest
concentration
of
test
article
in
treatment
`The findings ofthe cytogenetic analysisofthe non-activated 4 hour exposure group are
`presented by treatment flask in Table 4 and summarized by group in Table 7. At the highest
tienshtibciotnicoenntwraasti5o2n%e,varleulaattievdemtiocrtohsecsooplivceanltlycofnotrrocl.hroTmhoesdoomseealbeevrerlastisoenlse,c2te5d ufgo/rmaLn,almyistiostoicf chromosome aberrations were S, 10 and 25 pg/mL. The percentage of cells with structural
and numerical aberrations in the test substance-treated groups was not significantly increased
astbrouvcteurtahlaltyofdtahmeagseodlvcehnrtocmoonstroomles(pi>n0.t0h5e, MFiMshCer'(speoxsatcitvetecsot)n.trTohl)e tpreeractemnetnatggeroofupcel(l2s9.w0i%t)h
`was statistically significant.
The findingsof the cytogenetic analysis of the S9 activated group are presentedby
tcornecaetnmternattifolnaskevainluaTtaebdlemi5craosncdopsiucamlmlayrifzoerdchbryomgroosuopmeinabTearbralteio7n.s, A2t5 tphge/mhiLg,hemsittotteisct
inhibition was 52%, relative to the solvent control. The dose levels selected for analysis of
chromosome aberrations were 5, 10 and 25 pg/mL. The percentage of cells with structural
--_
aanbdovneumtehratiocfalthaebersroaltvieonnts cionntthreolte(spt>s0u.b0s5t,anFcies-hterre'sateexdagcrtotuepsst).waTshenoptersicgennitfaigceanotlfycienlclrsewaistehd
structurally damaged chromosomesinthe CP (positive control) treatment group (15.5%) was
statistically significant.
`The findingsofthe cytogenetic analysisofthe non-activated 20 hour exposure group are
`tepsrtesceonntceedbntyrattrieoantmeevnaltufaltaesdkmiincTraoblseco6paincdaflsoulrmymcahrriozmedobsyomgeroaubeprriantiToanbsl,e275. pAgt/mtLh,e hmiigthoteisct
inhibition was 58%, relative to the solvent control. chromosome aberrations were 5, 10 and 25 pg/mL.
The dose levels The percentage
selected of cells
for analysis of with structural
aabnodvneumtehraticoafltahbeersroaltvieonnts cionntthreolte(spt>s0u.b0s5t,anFcies-hterresateexdagcrtotuepsst).waTshenoptersicgennitfaigceantolfycienllcsrewaistehd
structurally damaged chromosomes was statistically significant.
in
the
MMC
(positive
control)
treatment
group
(33.0%)
CONCLUSION
`The positive and solvent controls fulfilled the requirements for a valid test.
-- Under the conditions of the assay described in this report, H-24768 was concluded to be
negative for the induction of structural and numerical chromosome aberrations in the
non-activated and S9 activated test systems in
`aberration test using human peripheral lymphocytes.
the
in
vitro
mammalian
chromosome
BSitouRdeylNuon.eAeATOYX.341 BTL
---1"
Company Sanitized. Does not contain TSCACBE
~~
iHnL2H4u7m6a8n: 1PerViipthreoraMlazBolnoaoldiLaynmCphhroocymtoessome Aberration Study
Dupont 13542
REFERENCES
Evans, HJ. (1976) (8d), Chemical
Cytological Mutagens,
PmreitnhcoipdlsesfoarnddetMeecttihnogdschfeomrictahleirmuDteatgeecntiso,n,in:voAl.
Hollaender 4, Plenum
Press, New York.
Evans, HJ. and M.L. ORiordan. of chromosome sberrations in
1975. Human mutagen tests.
MpeurtiapthieornalRebsl.oo3d1:l1y3m5p-h1o4c8y.tesforthe
analysis
GallaonwdayT,.$SMo.f,unMi.J(.1A9a9r4)deRmeap,oMrt. IfsrhoimdatweoJrrk,inJLg. gIreotutp, Do.nJ.iKnirvkiltarnod,teTs.tsMofroriac,hPr.oMmoossoemsaslo aberrations, MutationResearch 312(3)241-261.
InteornfatPihoanamlacCeountfiecraelnscefoonr HHaurmmoannizUastei.on (GIenCoHt)ooxficTietcyh:niGcuaildaRnecqeuiorenmeSnptecsiffiocr RAesgpiestcrtastioofn
Regulatory adoption at
sGteenpo4tooxfitchietyITCeHstpsrofcoerssPhoanrmJaucleyut1i9,ca1l9s9.5.
SF2eAd.erdaolcRuemgeisntterre61c:o1m8m1e9n5d-1e8d20f2o,r
April 24, 1996.
--_
InteorfnaPthiaornmalacCeountfiecraelnscefoornHHuamramonniUssaet.ionGe(nIoCtoHx)icoiftyT:ecAh.niSctaalndRaerqduiBraetmteenrytsfofrorGReengoitsotxriactiitoyn
pTroceessoosnfPJhtualrymai1c6e,un1t9i9c7ag.ls.FedSe2raBldRoecguimsetenrt6r2:1e60c26o-16m03m0f,oeNraodnvoepdmtbieoenrad2t1,st1e9p974o.f the ICH
OECCDhroGmuoidseolmieneAbfeorrrattiheon TTeesstt)i,ngReovfiseCdheDmriacfatlsD,ocuGumiednetl,inNeint4h73Ad(dIenndViutmotoMtahmemOalEiCaDn GuideforlthieTnesetisng ofChemicals, publishedby OECD, Paris, February 1998.
Pre`sWtoonl, fRaJn,dWJ..S.AWue,sMs.oAm.(1Be9n8d1e)r,MaJm.Gm.alBrieawneni,n vAi.vVo.aCnadmiannvoi,trJo.cAy.toHgeedndeltei,c AasFs.aysM:caFreesp,orSt. ofthe Gene-Tox Program, Mutation Research, 7:143-188,
.
Scott, D., ND. Danford, Aberration. Assays In
BJ. Dean, Vitro. In:
and D.J. Kirkland. 1990. Basic Mutagenicity Tests:
Metaphase Chromosome UKEMS Recommended
.
`Procedures. D.J. Kirkland (ed.) CambridgeUniversityPress,New York,NY.
SwieErRe.ngNaeSstHmHan,nL(A1.99H1e)ddRleec,oEm.mAe.nSdiegadl,pJr.otPo-cWo.lsGiblamsaend,oRn.La. sBurrilvienyogefr,cGurRr.entDopurgalctaisceanidn
genotoxicity exchange in
testing Chinese
laboratories, IV. Chromosome hamster ovary, V79 Chinese lung
aberration and human
and sister-chromatid lymphocyte cultures,
Mutation Research 246:301-322.
SBtuodyrNdo.eAATOYX-- I4LBTL ----15---------- Company Saniized. Daes not contain' scacat
~
iHn.2B4u76n8:niPneVspihoeMraBroetdoLn Cyomrrees Aberation Sty
Dupont13502
THE PARBESLEINMCIENAORFYETXOOXGIECTNIAOTBYULTSEMEE1STTAUBSOINLGIC24AC7T6I8V1A8TION 4HOUR TREATMENT
TREATMENT
(-uSg9/ml)
DMSO
MITOTIC
) INDEX
8.8
PERCENT
2) CHANGE
H-24768
0.1
8.0
9
03
82
-7
1
76
-14
3
72
-18
10
74
-16
~~
30
42
-52
100
28
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3100000
o1s6
%-82
CFocElolgeecntonslnorvicnogfm1e6ahbootlsresccovtevrtyipoenroiord4:hos 571--C. M--etaph--ase c--t wre PMecrxicreeoniteCdnhdtaeAngp=eere=al(isnrgee. sosemsi'nt5ot0ticedlesss-ecroent)oxl 1i0t.no des)omnircondls,
PS BSudeydNo AATSYX 341511.
'
1
possnotoonTtSCaAiCEn!
Gompany Saeed:
--~
J24Ho7m6a8nI:PneriirphoeaM]aBmlmoaoldiLnymCphhroocmyotseosmsAbertion Study
Dupont13562
TABLE THE PPRERLIMEINSOARFYEETXONOXIGCECINTOYEUTSEMSETTUASBIONLGIHC-2A4C7T6I8VIANTION
e ree4e HOUR TREATMENTe
TREATMENT +89
(ng/mL)
MITOTIC
INDEX
(%)
PERCENT CHANGE
*%)
DMSO
92
H-24768
01
8.4
9
03
82
-11
1
8.0
-13
3
7.8
-15
10
7.8
-15
~~
13000
3444
-52
1300000
|
2120
8-778
------ pt ------------
"ceTolrlexeacttmeoedfnotgs:looHuewuricmnenaognfapoem1re6ituphahboeosrulairlebaclocovteordiylvpyaemtrpfihioooordc4n.ytheoucrlslawe7r4e1Ce.sMdetapthheaspreelsesncweeorfean
`eMPxieporrtceeisnctsoICn1d5ehpxe=rac=ceenn(lvaegsigetn.omeentimtiottsicinedlsessx/c-ocSroen0dt)o0lxm1i00t.t index) controlmitotic index,
Pa BStiuodRyeNloa.mAeATOYX.I4LBTL -
2
--
--
Company Savitized. Donotecontsain TSCA CBI
--~
iHnLH2u47m6a6n: IPnerViipthreoraMlaBmlmoaoldiaLynmCphhroocymteessome Aberstion Study
Dupont13542
TABLES `THE PARBESLEINMCIENOAFRYETXOOXGIECNIOTUYSTEMSETTUASBIONLGICH-A2C47T6I8VIANTION
20HOUR TREATMENT
TREATMENT 9
(ng/mL)
MITOTIC ~~ PERCENT
INDEX ~~ CHANGE
[0]
%)
DMSO
30
H24768
ol 03
80
0
76
I]
1 3
74
3
60
25
10
42
48
~
30
30
pl
100
20
75
300 1000
14
83
04
95
`eTxroegaetnmoeunst:soHuurcmeaonfpmeertgahbeorlsicbalctoiovdatliyomnpfhoorc2y0tehcoulrls awter3e7t1esCt.eMdietnaphshaasbesceenlclesowfearne McoiltloetcitceIdnfdoellxow=i(neglal1si6aomuitrorsesc/o5v0e0rcyepelrlisodc.ored) x 100. ePxeprrceesnstedChasanagpeer=cen(traegaet,ment mitotic index- control motiindex) control mitotic index,
SBtiouRdeylNioa.ncAeAT9YX341 BTL
2 tebe
--------
Company Sasitized. Does not contain TSCA CBI
~~
iHnLH2u47m6a8n: IPnerViipthreoraMlamBimoaoldiaLynmCphhroocymtoessome Aberration Study
Dupont 13542
`CYTO"GWEINTHEHT-I2C47A6N8ALINYSTIHSEOAFBHSUENMCAENOPFTEAERBXILOPEHG4EERNAOLUBSLMOEOTDABLOYLMIPHCOACCYTTIEVSATTIROENATED
-- "DEFINITTIVTEASASAY:I4THOOUURRTTRREEAATTMMEENNTT,,200HHOOUURRBHAARRVVEESSTT
Tama FiskbMiisce Cas 35Avera cats
To NumberofSct Aberin os _Clromaid Chromosome
DSaemvacgleyd
AboArvtnigoens
Gomi) 0) Sor Noma! Scat Br Bc BebeReg Cll Pada
EO ABosa W b0 00 30 0TT0 o00e 0oooo aomm
5naa ABoBsommW 0oo0 00 00 00 0000 00 00 oomm
0 BA 7862mMm 00 00 00 00 00 0000 00 oo0 oomm
--~
BAB d4omw o0 01 00 01 00 o0o0 00 0o oomm
M0_M6--C_-- ABos72 oot 00mm3ommoos4 0000 0oooo = oomme
"sTo*rueNrautmmeorefincmtae:laHbauboelmriarcantapiecorvniasptahriesoronal.utAoonfadd1d0t0ciepolnlahslsdyoosrseew.vr!eorfes2t.e5dpfoLr.& hwoausrstetct3ed715aCsiunlbtghuradbasgeanicnesotfcaxcesxsovgesnous twoxeircintyotaatnhailghyezredddoo leoveexlcsebsustivweatsonxoitirye.quire formicroscopicexamination.Dose eves 50,734nd 100pg. aM3n%iyAtbtgeaerper.adnetsCe=llusm:bneumreriicoalcelilgsrnecudx e1p0o0l5y0p0lociedlalnsdcoeunndtoerd.edupiatd el; suc els excl cllwith i`nCchlruodmeaqtaddbsrieraadkissi,ncrluadedchsroamnadticdomnpdliesorcetsrmoamnsgteimdebnrse.aksa fragments(3)chromatid exchangefigures(Bx) `SseCbvheerrromemilooynsDso.ammeaBgreedaCesllinscilnucdleubdeesseklslasnwdiatcheonsnico mnorgespul(ve1ri)zeDic, doicemn ocahnrdomcoesllosmwei.th 10ormaestructural AverageAberrationsPerCll: severelydamagedclsandpverizationswer counted1s 10shertions.
--~ SBtiuodRyelNson.cAeAT9YX 341.BTL
2 Company Sasized. Doesnot containinTSCACBI
~
iHnLH2u47n6a8n:PIeriVpihterroaMlamBmlaooldiLaynmCpthroocmyotseosrmeAberration Ssdy
Dupont 13542
TABLES `CYT`OWGIETNHEHT-I2C7A6N8AILNYTSHIES OPRFEHSUEMNACEN OPFEERXIPOHGEERNALOUBSLMOEOTDALBYOMLPIHCOACCYTTIEVSATTIROENATED
DEFINITIVE ASSAY: 4 HOURTREATMENT, 20 HOURHARVEST
Tamer
Fk Mdiekxe Cote
ssmmcas
ontNone Svctn bess Gps _Clromsit__ Chromosome
DSuorvalyl
AbAovrisns
Gort)
00 Sores Norrie Scr BB BDRg Ci Poca
BEXBos%s W 0 o0 00 T0 T0 50 T0T0 00 o1 wamo
a5 ne BAosa o 00 00 00 00 00 0s0o 00 o0 oomm
0 ABoaomwm 00 01 01 01 00 000000 0 oomm
--_
x ABoa4omw 0oo0 01 90 00 000000 00 oamm
a Aose owmm 0o 165 3oomBw os5S 010000 o oamm
f"ooTurneetaktymoeafnmthe:itgaHhboeorrlaidncopsceireaivptheieolrnab.luAbtwlnoaaosidnlioytmapruehqoluciydrteoedesfveoevrrm]eicorfoes2sc.toe5pdgifoirne4x.ahwmoiaunraestaitton3d.7D1o1.sCefivneelaher$0p,rae7sg5eainncdeoe1fx0ec0enpsscigxvoe.gaous
wMeirtetneotadnealxyz=endudmubefromxicsosiivegtroexicx 100500clscounted,
%a6lAybgeprrsa.ntCel: numericalels nl polyploid ndendoredoplictedcl;social llexcludecls with
iCnhcrloumdaetqiduBaredasks,inciludliromnd comapdl rcehrosmaetirdbr.eaksand fragments(0;chromatidexchingefires(5)
.
SCehverroemloysDoammeBargeeaksCeilncilndcehberseackls sndwsithaoniorgmonpulv(e3r4i;zDedic,hdrocmoasorcersonmascolnlswe.ith 0 ormre src
.
sAvhearamgaeAnb.errationsPer Cel:severly dansged elsmdplviztonswere cated 10sbestions.
--_ BSitouRdeylNioa.nAcsRT9YX 341. B- TL 2 Company Sasitized. Does not contain TSCA Cal
~~
iFn-H24u7m6a8n:PIenriVpihieroraMlaBmlmoaoldiLaynmpChhorcoymtoessomeAberration Stady
Dupont 13542
TABLE CYTO`GWEINTEHHT-I2C47A6N8AILNYSTIHSEAOBFHSUEMNCAENOPFEERXIOPGHEERNAOLUBSLMOEOTDABLOYLMIPCHOACCTYITVEASTTIROENATED
"DEFINITIVE ASSAY: 20 HOURTREATMENT, 20 HOURHARVEST
Team
FakMInideix Calle 56Aber ct
TolNumberofSnesAbsations Soely Avenge Cops_Crromaid__Cromssone Daaged Abeations
Geni)
06) Scored Nemerical Sct
Br BC BrDeRm Cli facdl
RO BA os82 mmWw 00 o 0 z0 00 00 0900 00 00 oemm
sBas A 82m 0 B oso mw o
01 01 01 00 0000 00 00 ooomo
0 BA s7 wmww 00 oo0 02 00 00 o0o0 00 0 oomx
= BA 34s mmww o0 11 01 11 00 0000 00 oo0 oooooo
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2 Positive control for non-activated studies is mitomycin C (MMC).
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--
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APPENDIX B Study Protocol
Dupont-13542
B--io_R--eliance StudyNo. AAT9YX.341.BTL
2 Company Sawi:zed. oes not contain TSCA CBI
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Sponsor Project Number: _DuPont=13542 _
3
BioRelisneStudy Number: __AATOYK34LBTL
34 Test SubstanceCharcterizaion
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41 Name:
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42 Address:
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43 SudyDirctor: PRhaomnaed:ev3i0G1u.6d1,0.P2h1D6.5
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) 50 PROPOSEDSTUDYDATES
51 ExpeimSeunnDaatle: 52 Experimental Termination Date: 53 DrtRepontDue:
54 FnalRepor: 60 TESTSYSTEM
1756p2003 31 Oct2003 14 Nov 2003 2weeksaftr Sponsorapprovesdrat
Peripheralbloodlymphocyteswillb binedfromhealthy adlswithout arecenthisory deoamfLoetn1hs9e8i0rt)rt.aeddio1tbhesrnasvpiiryav,lein0fetchtiocnlssoorgtehneiacdacmtiivnitiy osftrovaafrtdireiutogyosn.fTchhiesmsiycasltse(mPhraostboenen
70 EXPERIMENTALDESIGN AND METHODOLOGY
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Protocol No. SPGT341 043m2003 Page2010
BIORELIANCE
BioRelanes
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~~
iHn2H4u7m6a8n: IPnerViipthreorMalaBmlmoaoldiaLynCmphhroocmyotseosmeAberaton Study
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70 SolventSelection
711 SolubilityDeteaninaton
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ProNtoo.ScEGoT3l41 04Jun 2003 Page 3of 10
BIORELIANCE
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~~
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3
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74 MetabolicActivation system
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ProtocolNo.SPGTS1 043m2003 Page Gof 10
BIORELIANCE.
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50 CRITERIAFOR DETERMIOFNAAVATLIIDTOESNT
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130 REFERENCES
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chromosomalaberations,MataionResearch 31203)241.261.
IRnetgeimsattaitoinoanl"oCfoPnfheraenrcemoancHaemfouorrHtiusimaacionanUl(sIesC.H)GouifdTaneccehnoincaSl RpeqeuicrAesimpeenfcttsisfoocfr aRdeogpailoanoartyGseincot4ooxfitihteyITCeHsiprfoocersPsohanmaJuclcyti19c,al1s9.95. FSe2dAe.rdaolcRumeentgre6ci1o:m1sm81e9tn8d-e1ed820rf7o,r Apel 24,1996.
D GIReengtnieosmtartaitoinoanloTfsCPoihnnafrgemraoefcnecPuethiocaanlrsmHfaoamrocHsiu.smaSat2ni5onUdseo(.ImCEGe)enoortfoseiTceicych:nioAcalSmtaRnemddqauripedreoBmnaentnetsdrsyspffoorr
419f97t.he ICH proces on July 16, 1997. Federal Register 62:16026-160%0, November 21.
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