Document NGodQyMgwX5g36BEyogGnjq7R
,
AR226-0432
CORNINGHazleton
MUTAGENICITY TEST WITH
:
T-6564
IN THE SALMOMNUETLALATI-OENSCAHSESRAICYHWIIATCHOALICMOANMFMIARLMIAATNO-RMYIACSRSOASYOME REVERSE
FINAL REPORT
AUTHOR
mrs
:.
Timothy E. Lawlor, MA.
[F.
dg
Foi ag
:
PERFORMINGLABORATORY Ue= Smrrie
Coming Hazleton Inc. (CHV)
Vi9e2w00,LVeiersgbiunriag P2i1k8e2
oT
LABORATORYPROJECTID
CHV Study No.: 17750-0-409R
SUBMITTTEDO
.
BuiSltdPiangu3lM2,2M0Co.Nr3p5o.5r03a2t1i43o4nM-1C0e0n0ter
STUDYCOMPLETIONDATE
September 13, 1996
i
CHV Study Nos 17750.0-409%
Lof34
i
0232
CORNING Hazleton
:
QUALITY ASSURANCE STATEMENT
STUDY TITLE: S`aAslsmaoynewiltlha-aECsocnhfeirrimcahtioarycoAlssMaaym.malian-Microsome Reverse Mutation
ASSAY NO: 177500-409R
PROTOCOLNO.: 409R, Edition 4, Modified for 3M
Quality Assurance inspections ofthe study and reviewofthe final repor: ofthe above referenced.
:
project were conducted according to the Standard Operating Proceduresofthe Quality Assurance
Unit and according to the general requirementsofthe appropriate Good Laboratory Practice
;
regulations. Findingsfromthe inspections and final report review were reported to management
?
and to the study director on th following dates:
Scoring Plates - 06/28/96
0628/9
Draft Report Review - 08/29/96
0873019
Final Report Review -09/13/96
0911319
P. Conley C. Orantes C.Orantes
{
iy Assurance Unit
Date Refeas3/e51d
i
CHV Study No.: 17750-0-409R
2
C0033
CORNING Hazleton
STUDY COMPLIANCE AND CERTIFICATION
`fTohrethsbtuydtyhweaFsoocdonadnudctDerduignAcdommipnliisatnrcaetiwoint(hFtDhAe)GoiondTitLlaebo2r1aotfotrhyePrUaSct.icCeordeegouflFateidoenrsaals set Regulations Part 55, issued December 22, 1978, (effective June 20, 1975) with any applicable
`amendments. There were no deviations from the aforementioned regulations or the signed
protocol that would affect the integrityofthe study or the interpretationofthe test results. The raw data have been reviewed by the Study Director, who certifies that the evaluationofthe test
adretsiicglen aasnpdreevsaelnutaetdiohnerceriitnerriea.presents an appropriate conclusion within the contextofthe study
:
Study Director:
;! 2 ee
BTaicmtoetrihayl EM.obgLejnwleors,iMs.A. Genetic and Cellular Toricology
-13-7
Study Completion Date
:
CHV Study No.: 17750-0-409R.
1
i
3
.
00234
CORNINGHazleton
;
TAOFBCOLNTENETS
Page No.
Lo SUMMARY Loonie
J}
I STUDYINFORMATION oot
T
.
HL MATERIALS ANDMETHODS oui
ceil 9)
IV. REAS NDU CONCL LUSIT ONSS .....oouniiiiniiiinniiieiiicniin 28
V. DATATABLES ....................... wrreanssvesssrne
27
CHV Study No.: 17750-0409R :
4 C6035
[I CORNING Hazleton
SECTION I. SUMMARY
:
INTRODUCTION AND CONCLUSIONS
x
CHV Study No.: 17750-0409R
i
5 00236
:
SUMMARY
`CORNINGHazleton
A. Introduction
mutagenic sctiAvtihy in trheequSeasltmoofneSlMlaC-orEpxocrhaetriicohni,acCoolmliMnagmmHaazllieatnonMnicc.roinsvoemsteigRaetveedrTs-e6564 for mMeuttaabtoilointeAssfsorytwheiitrhaCiolnyfiteomitndourcye Arsevseyr.se Tmhuitsataisosnasyaetvtahluahtiestditdiene(lcotcuaslinethaengdeonrofmse of
`specific Salmonella typhimurium tester strains and at the tryptophan locus in an Escherichia coli tester strain both in the presence and absenceofan exogenous metabolic activation system of `mammalian microsomal enzymes derived from AroclorTM.induced rat liver (9).
The doses tested inthe mutagenicity assay were selected basedonthe resus of a dose
rangefinding study using tester strains TA100 and WP2uvrA and ten dosesoftest article ranging
from 5,000 to 6.67 ug per plate, one plate per dose, both in the presence and absence of S9 mix.
i
The teste sais used in the mtageicy assy wee Slmonele tphimurm ste sins
ii
ccTooAnn9cd8uu,rcrtTeeAndt1w0vi0eth,hicTilAev1ea5n3ddo5s,peosTsAoi1fi5ve3es7tc,oanatnnridollsEsicsnhibenorgtihhchtiehae cplorlaeitseteesnspcteeerradsntodrsaaeib.nsWeTPnh2ceeudvoorfsASe.s9 emTsihtxeeadalswosenargyewwiatsh
r5e,s0u0l0t,so3f,3t30e, i1,i00l0;m3u3t3a,geannidci1t0y0susgapererpleatceoinnbfoitmhetdhienparnesienndceepeannddenatbseexnpceeriomefn9t.mix. The
B. Conclusions
:
CMuotraptoiroantiAosns'ytTehswteitarhreisaeulClt,osonTff6ti5hm6ea4oS,ardlymdoAnnseostlclyaaui-snEedsaicchpetortishctahitiv,aeuncidnoeclrrie/taMhseaemcimnoanhldeiianonun-msMobifecrrtohosifosmrseuecRvyeev,retrss1e
per plateofanyofthe tester strains either in the presence or absenceof microsomal enzymes
`prepared from AroclorTM-induced rat liver (S9).
.
CHV Study Nos 177500409
J
6 00237
|
CORNINGHazleton
SECTION II. STUDY INFORMATION
;
CHV Study Nos 177500405
:
7 00238
CORNING Hazleton
STUDY INFORMATION A Sponsor: 3M Corporation B. TestAicle: TL6156647 Fet0isx
1
Physical Description: clear colorless liquid
2
Date Received: 05/30/96
3:
c TypeofAssay:MiSatlamtonoenllAas-sEascyhweirtihchCioancfoilrimMaarmomrayliAasns-eMicrosome Reverse
:
1.
Protocol Number: CHV Protocol 409R, Editio4n
2 CHV Suudy Number: 177500-409R
D. SudyDues
1 Study Iniation Date:
o0ams
.
2 Experimental SanDae: ozs
5. Experimental Teminaton Date: 08279
:
E. Study Supervisory Personnel
Study Director:
Timothy E. Lawlor, MA
:
Laboratory Supervisor:
Michael S. Mecchi, B.S.
i
CHY Study No: 1775004058
!
5 00239
4
`CO RNI NG
Hazleton
SECTION IIL. MATERIALS AND METHODS
: CHV Study No.: 17750-0-409R
i
9
00240
'
CORNING Hazleton
i
MATERIALS AND METHODS
Ames efTahle(e1x9p7e5r)iamnedntGarlemeantearniadlsM,umrieetlho1d9s7a6n)d procedures are based on those described by
MATERIALS
A. TesterStraing
1 Solmonella nphimurium
auxotrophs TA98, TAI"0T0h,e tTeAsIteSr3sSt,aiannsduTsAedISw3e7reasthdeeSsaclrimboendelblyaAtmypehsimeurraliu1m97h5is)t.idiTnhee specific genotypesofthese stains are show in Teble
TABLEL TESTERSTRAINGENOTYPES
hisG46 isCI076 hisD30S2 LPS Repair R Factor
TAISIS TALS
da wed .
|
T_A--I00
TASS da wr WR
Imnutaadtdiiotnisonwthoicahmuetnahtainocneithtehir sheinsstiitdiivnietyopoersoon,methmeutteasgteernitcracnomspcoounntdaisn.tTwhoeadrfdaitwoanlallmutation
lreispuolptosliynsatchcehlaorsisdoefboanrreioerftthhae feonrzmysmethserseusrpfoancseibolftehfeobratchteersiyanlthceeslilswolflp,arToheftrheesulting cell wall
!
dienfgicsiyesntceymisnc(irce.asbeesnpzeor(mae)apbyirleintey)ttohacterwtoaiunldcloastsheesrowfiscehbeemeixccallusdseudchbyasatnhoosremaclonitnatiancitncgellawrgaell
Tsyhsetesmecwohnidcmhutgraetaitolny,aednehlaentcieosntohfetsheensiwtvirvBitygoefnet,herseseulsttsraiinnsadefiscoimeentmuDtNagAenesx.cisSiionnceretphaeir1B.
.
dveilteatmiionnbeixotteinndfsorthgrroouwgthhth bio gene,al ofth tester stains containing this deletion require the
:;
thStersaeinnssiTtiAv9i8tyoanfdthTeAs1e0s0taailnsso0constoamien mthuetaRg-efnasc.torTphleasmmeicdh,apnKiMs1m0b1y,wwhhiicchhtfhuisrtphlearsimnicdreases
DinNcrAearseepsasiernpsiotliyvmiteyratsoemcutoamgpelnesxhiansvobleveendswuigtghesthteedmitosmbaetcbhye-mroedpiafiyiprnogceasns.existing bacterial
}
CHV Study No.: 17750-0409R
10
00241
CORNINGHazleton
:
hTiessttiedrinsetrianidnsepTeAn9d8enacned(pTrAo1toSt3r7opahrye)rbeyvefrrtaemdesfhriofmt hmiusttaigdeinnes.depTeAn1dSe3nSceis(aruevxeorttreodphbyy)btaose
ssuubbssttiittuuttiioonn mmuuttaatgieonnss and TAI00 i reverted by mutagens which cause both frameshifis and base
2. Escherichiacoli described by Green andThMeurtieestler(1s9t7r6a)i.n used was the tryptophan auxotroph WP2uvrA as
Idnefaidcdiietnicoynwthoiachmuetnahtainocneisnitthsesetnrsyipttiovipthyantoospoermoen,muthteagteenstiecr sctormapinouconndtsa.inTshaisuvdrefAicDiNenAcyreaplaliorws trheemostvreaitnhteodsahmoawgeednhpaanrtcoefdtmhuetaDbiNliAtymsoilnecceutlheeawnvdraAccruerpaaitrelsyyrsetpeamirwiotualfdtenrwoarrmdasl.ly act 10
:
TiensdteepresntdreanicneW(Pp2ruotvortrAopishyr)evbeyrtbeadsefrsoubmsttrityupttioopnhamuntdaegpenesn.dence (auxotrophy) to tryptophan
.
3. SoofuTesrtercStraeins
a Salmonella typhimurium
Dr.
Bruce
Ames,
`The tester strains Deparmentof Biochemistry,
in use at CHV were received directly UniversityofCalifornia, Berkeley.
from
b. Escherichia coli
National Collection ofIndustriTahleBatcetseterrias,trTaoirn,reWyP2ReusveraAr,chinStuasteioant,CScHoVtlawnads(rUencietievdedKfirnogmdotmhe)
4. StooftherTesatergStraeins
:
a Frozen Permanent Stocks
ovemight cultures, adding DMFSrOoz(e0n.p09ermmUa/nmelnotfstcoulctkusrew)earendprferpeaerziendgbsymaglrlowailinqguoftrsesh (5-1.5 mb at 5-70C.
-
CHV Study No.: 17750-0409R
1 C0042
:
CORNING Hazleton
:
b.
Master Plates
Master plates were prepared by sreaking exch ester sain from a
Ifyrpozheinm
upreirumma,neanntexsctoecsks
onto
ofhis
minimal agar
tidine, and bi
appropriately supplemented with 1)
otin, and for tester strains TA98 and
for Salmonella
TA100,
ampicillin (25 g/ml), to ensure the stable maintenanceofthe pKM101 plasmid; and 2) for
E5s2c3herCi.chia coli, an excessoftryptophan. Tester strain master plates were stored at
5. PrepoafOrveranigthtCiuloturnes
+ Inoculation
by
transferring
a
colony
from
tOhveeranpiprgohptricualtteurmeasstfeorr
pulsaeteintoalal
testing procedures were
flask containing culture
inoculated
medium.
Inoculated flasks were placed in a shakerfincubator which was programmed to begin operation
:
(shaking, 125 25 rpm; incubation, 37 + 2C) so that the overnight cultures were in log phase or
i
late log phase when turbidity monitoring began.
}
b. Harvest
To ensure that cultures were harvested in late log phase, the length of incubation was determined by spectrophotometric monitoringofculture turbidity. Cultures
t`rwearneshmairtvaenscteed(%oTnc)eraeapdriendgeotnermsipneecdtrtuorpbhiodtiotmyewtaers.reTahcihsedtaarsgedtettuerrbmiidniteyd ebnysuarpeesrtcheanttcultures
}
:
O`whevareveregrrreeoamcwohnveed(dsatafdtreionomsniaitrnyyo)cufcbuaaltttiuloreneasswtmh0ae.ny5 Xetxhhe1i0tbairtcgeedltelc%srpTeearswemadlssarenendasictthihevadittatytnhedcwsuelortmueerpemsluahcaaegvdeenatnso.5t&oCvu3elrtCgu.rreoswn.
6. ConfoifTmestmerSaimitnGeinootypnes
Tester sran cultures were checked for the following genetic markers on
*
`the dayoftheir use in the mutagenicity assay:
!
a Salmonella typhimurium
1) ofa Wall Mutation
The presenceofthe 1/2 wall mutation was confirmed bs
demonstrationofthe sensitivity ofthe culture to crystal violet. An aliquotofan overnight
1
CHV Study No.: 17750-0-409R
12
0243
CORNING Hazleton
;
sceunlstiutrieoviftyedaicshksctornatianiwnainsgo1ve0rilsgidofocnrtyostpallavtisolceotnwtaaisniandgdesdel.ecSteinvseitmievditiyawaansd daenmaonntisbuiaottiecd by
inbibitionof bacterial growth in a zone immediately surrounding th disk
2) PKMIO! Plasmid Refactor
tester stains TA98 and TAI100 by dem"oTnhsetprraetsieonnocfeorfestihsetapnKcMeI1to01amppilcaislmliind.wAsncaolnifquiortmeofdafnor aonvteirbniiogthict sceunlstiutrievoiftycdaicshk sctornatianiwnainsgo1v0erilaidofaomnptiocpillaltiesnwcaonstaadindiendg.slReecstiisvteanmceedwiaasand an demonstrated by bacterial growth inthe zone immediately sumounding the disk.
3) Characteristic Number of Spontancous Reverants
!i i
v1e0h0iculleacloinqturootlssotfthahtearceulcthuareraacltonegriwsi"otThfhetthmheeaarpenpsrpnoeupcmrtibisveteresovtfershaipiconlnewteaornneesodeulesemcortneivsveetramtteandetidsab.pyerplplaattiengin the
i
b Escherichia coli
1) Characteristic Number of Spontaneous Revertans
;
vehiclecontrolsthat are characteristic"oTfhtehmeereasnpencutmibveersotfrsaipnosnwtearneeoduesmorenvsetratatnetds bpyerppllaattiengin the 100 ul aliquots ofthe WP2uvrA culture along with the appropriate vehicle on selective media
:
7. TesterStainMedia
a Curing Broth
::
`Vogel-Bonner salt solution(Vo"gTheelabnotdhBuosnender0, g19r5o6w)osvuepmpilgehmtenctueltdurweistohf2t.h5e%t(eswtievr)sOuxsoinisdwas Nutrient Broth No. 2 (dry power).
:
bo AgarPlaes
0Bo.n2n%e(rwmhi)nigmluaclomseediumE (VBoogteltoamndagBaorr(n2e5r,mi19p5e6r),1s5uxpp1l0e0memnetmepdewtiithdis1h.)5%wa(sw/Vvo)gaegla-r and
CHV Study No.: 177500409
I
i
i
00z4a4
CORNINGHazleton
:
. Overlay Agar for Selection of Revertants
0.5% NaCl (wiv) and was sup`pOlveermleanyte(dopw)itahga1r0wmalsopfre1p)a0r.e5dmwiMthhi0s.t7id%inaeg/abrio(twi/nvs)oaluntdion per
100 agar
mflorasgealrecftoirosnoefletcrtyiopntoofphhiasntirdeivenretarnetvse.rtaWnthse, norS29)
0.5 mix
mM was
trreyqupitroepdh,a2n.s0omlultiofotnhpeer
100
ml
of
swuapspaldemdeendtteodttheopsaugpaprlweamesnutseedditonptahgearov(e0r.l5aym.loHfowwaetveerr,pewrh2enmiSo9fsmuipxpwlaesmennotterdeqtuoipreadg,arw)ataenrd
`tehnesruerseudltthiangtt2h.e5fmiinaloftdoiplaugtaesd asunpdpalmeimneontaecdidtospupapglaermweanst ucsoencdefnotrratthieoonvserrleamya.inTehdisthdeilsuatmioenboth
in the presence and absenceof$9 mix.
:
5 Kireelli
jon Misture (SMG
1. SHOomogenate
i;
Molecular Toxicology,LIinvce.s, mAincnarpoosloimsa,lMeDnzy2m14e0s1(,SB9ahtocmho0g6e4n2at(e3)9.w5emregopfurpcrhoatseeidn fperromml) and
: :
DBaatwclhey06r4a6tst(h4a3t.4hamdgobfeepnroitnejienctpeedr(im.pl.)).wiTthhe AhroomcogleonraTMte12w5a4s(p2r0e0pmargepdefrrmolm minalceoSmproaig)uaet-
500 mg/kg as described by Ames ef al, 1975,
2. S9Mix
:
procedure. The S9 mix`TchoenSta9imneidxtwhaescpormeppoanreendtismmineddiicaatteeldyinprTieobrl0e Itls use in any experimental
:
HO
`TABLE
59MIX COMPONENTS 070ml
i
012M5NMaHG,lPucOo,s/eN-a6;-pHhPoOs,p,haptHe 7.4
010m 002ml
:
0.10M NADP 0.825M KCUO.2M MgCl,
004ml 0.04 ml
1 i
59 Homogenate
o1.l00omml
CHV Study No.: 17750-0409.
{
1
!
0024s5
sy
CORNING Hazleton
C Contrals
1. VehicleConyols
absenceof
$9
mix.
Vehicle controls were The vehicle control was
plated for al plated, using
tester strains both in the presence and a 50 ul aliquotofvehicle (equal to the
`temsatexrismruaminalainqduoato5f0t0esutl aarltiiqculoetdiolfuSti9onmiplxat(ewdh),enalnoencgeswsiatrhy)2, 1o0n0swelleacltiiqvueoatgaorft.he appropriate
:
2. PositiveConurals
stains plated concurren"tTlhye cwoimtbhitnhaetaisosnasyofaproesiintdiivceatceodntirnoTlas,blaecLtIivation condition and tester
) i
"TTesAtBerLE
POSITIVECONTROLS Cone
::
STiAmSiSn S9+Mix Po2saimtiinvoeaCnotnhtrraoclene pe2rg5lmatge
TA% ~ 2mnitrofluorene TAI0 + 2-aminoanthracene
100g 25ug
TAO TAIS}S +
sodium azide 2-aminoanthracene
20g 25g
TAIS3S TAIS}7 +
sodiumazde 2-aminoanthracenc
2250p1gg
:.
TAI? - ICRISI
204g
WP2orA + 2-aminoanthracene 250g
WP2ivrA _-_ d-nitroquinoline-N-oxide 1.0 ug
a Source and Grade of Positive Control Articles
purity 2 97.5% 2-nitrofluoren2e-(amCiAnSoa#n6t0h7v-a5c7e-n8e),(AClAdSri#c6h1C3-h1e3m-i8c)a,lSCiog.m,apuCrhietmyi9c8a%l;Csoo,dium
i
aPzoildyesc(iCenAcSes#2I6n6c2,8p-u2r2i-t8y)>,9S5i%gamnadChSeimgimcaalChCeom.icpuarlitCyo>,p9u8r%i;tyIC9R8-%1;914-n(iCtArSoq#ui1n7o0l7i-n4e5--N0-)o.xide
(CAS #56-57-5), Sigma Chemical Co., purity >99%.
:
CHV Study No.: 17750-0-409R.
:
is
00246
CORNING Hazleton
3. Sterilite Conrols
a TestAnicle
:
by plating a 50 ul aliquot (heTshaememovsotlucmoneceunsterdatiendthteesatsasratyi)cloendisleulteicotnivweaasgacrh.ecked for sterility
bo s9Mix
: agar
The $9 mixwascheckedforsterility by plating 0.5 mi on selective
METHODS
;
A. DoseRangefindingStudy
i
determined in o"rTdheergtrooawltlhoiwntkihbisteolreycetfifoenoctf(acpyptrootpoxriicaittey)doofsetshtotebset taetsitledeitnotthheemtuesttagseynsitceimtywas
+
assay.
1. Desi
:
and WP2uvrA both in t`Thheepdroesseenrcaengaenfdinadbisnegncsetoufdy$w9asmipxe.rfToernmeddosuessionfg tteesstearrstticrlaeinwseTrAe1t0es0ted
actonocneentprlaattieopneor dfosem.eg Tphereptaetset.articlewas checked for cytotoxicity up toa maximum
a Rationale
.:
is generally representative of tThhateocbysteortvoxeidciotnytohftohtehetretteasrtteirclsetroabisnseravneddboencatuessteerofsaTAiIn0T0'AsI00
) :
rceoamdpialryadtiivsecleymehdigfhrnomumrboeutrionfesepxopnetrainmeenotuaslrveavreirattainotns.pTerheplEastc,hegrriacdahtiiaocnsoolftecsyttoetsoxaiicnity can be
a`nWdP2tuhuvsr,Aa ddoiefsfenroenttproasnsgeesosftchyet1otaoxwiacliltmyumtaatyiobne tohbastertvheedS.alAmlosnoe,lltahetcyypthoitomxuirciiutmy istnrdauicnsedhabvye&
tmeisxt. anTihelreefiontrhe,eptrhiessewnocueldofre$q9uimriexthmaaydivfafreyregnreeasttlyafrrtioclmetdhoasteorbasnegrevsedbeintetshteedaibnsetnhceeof $9
`mutagenicity assay based on the presence or absenceofthe S9 mix.
CHV Study No.: 17750.0409R
16
00247
CORNING Hazleton
:
2. EvaluationoftheDoseRangefindingStudy
pesplate and/or by a thCiyntnoitnogriocridtiysiaspdpeetaercatnacbeloefathaedbeaccrteearsiaelibnatchekgnruomubnedrloafwnr.everaant colonies
3. SelectionoftheMaximumDosefo theMutagenicityAssy
a NoCyotoxicity Observed
the highest dose level oftest aSriincleunsoecdyitnottohxeicmiuttyagweansicoibtsyerasvseadyiwnathsetdhoessearmaengaesftihnadtitnegssttedudiyn, the rangefinding study.
B. Mutagenicity Assay
:
I. Design
:
`TAIS37, and WP2uvr`AThbeoatshsianywthaepsrepsenecearndusafibnsgeontceseotrefrstSrm9aiminxse.TAFSdiSv,e TdoAs1e0s0o,ftTeAstISa3rtSi,cle
w`weerree tseeslteecdteadlboansgewditohntthheearpepsruolptrioafttehevedhoisceleraanndgepfoisnidtiinvge csotnutdryo.ls.TheThreesduolsseosfofttheesitntairatlicle
mutagenicity assay were confirmed in an independent experiment.
3
2. FreandqRouuteoefAdnminicstatyion
incorporation methodol"TohgeytoerstiegrinsatlrlayindsewscerriebeedxpboyseAdmetostehrael (te1st9a7rt5i)cleanvdiaMtahreopnlaatned Ames
m(1u9t8a3g)e.nsT.hiIsnmtehethpoldatoeloigncyorhpaosrbaetieonnsmheotwhnotdoodeltoecgttyh2,wtiedtearrtaincgleeo, tfhcltaesssteesrosftrcahienmaincdalthe S9
pmliaxt.(wFhoelrleoawpipnrgopirnicautbea)twioenreatc3o7mb+i2nedC ifnormo4l8t+en8ahgra,rrewvheircthanwtacsoloovneirelsaiwderoentcoouanmtiend.imaAlllagar
:
doses ofthe test article, the vehicle controls and the positive controls were pated in triplicate.
i
C. PlatingProcedures
`mutagenicity as`sTahye.se procedures were used in both the dose rangefinding study and the
i
CHV Study No.: 17750-0-409R
17
00248
CORNINGHazleton
Each plate was labeled with a code which identified the test atcle, test phase, ester strain, `iamcmteivdaitaitonelcyonpdriitiooon atnhdeidroussee level. The S9 mix and dilutionsofthe test article were prepared
v`iWahseand5d9edm1i0x.2w.a5smlnoofrmeoqulitreend,se1l0ec0tipvleotfeosptaegrars(trmaaiinntaanidne0d autl4of5v=eh2icCl)e. ofWhteestnaSrt9icmleixdowsaes
required, S00 ul of $9 mix, 100 plof testerstrain and 50 ulofvehicleortestarticle dose was `added t0 2.0 mlofmolten selective top agar. After the required components had been added, the `mixture was vortexed and overlaid onto the surfaceof25 ml ofminimal bottom agar contained in 215 x 100mmpetri dish. After the overlay had solidified, the plates were inverted and
ailnicquuobta.ted for 48 + 8 hr at 37 + 2C. Positive control articles were plated using a 50 pl plating
:
D. ScotrheiPlnatges
Plates which were not evaluated immediately following the incubation period
:
`were held at 5 +
evaluation could
3C until such
take place.
time
that
colony
counting
and
bacterial
background
lawn
1. BaBcteariolckgrLaownuEvalnuatdion
evidenceofcytotoxicit`yThaencdotnedsittairotniocflethpreecbiapcitteartie.al Ebvaicdkegnrcoeuonfdcyltaowtnoxwiacsiteyvawlausatsecdorfoerd relative
10 the vehicle control plate and was recorded along with the revertant counts for all plates at that
i
dMoesteholdevselSeocntitohne. data tables using the code system presented at the endofthe Materials and
2 CountingRevertantColonies
The numberofrevertant colonies per plate for the vehicle controls and all
i
plates containing test article were counted manually. The numberofrevertant colonies per plate
for the positive controls were counted by automated colony counter with the exceptionof the
:
``pwohsiicthivweecroentcrooulnstfeodr mteasntuearlsltryain WP2uvrA in the absence of $9 mix, in Experiment 17750-B1,
E. AnshsisofData
were calculated.FoTrhalelrreespullitcsatoeftphleatsiengcsa,lctuhleamtieoansnarreeveprrieasnetnstpeedripnltaatebualnardfthoermstianntdhaerdDdaetvaiation Tables Sectionoftis report.
i
CHV Study No.: 17750-0-409R.
3
i
18
i
00249
tL
.
CORNING Hazleton
EVALUATION OF TEST RESULTS
Before assay data were evaluated, the criteria for a valid assay had to be met.
A. CriFotrAeVarlidiAssaay
`The following criteriawereused to determine a valid assay:
.
1. TesterSwainlotegrity Salmonella typhimurium
a ofa Wall Mutation
!:
cultures
exhibited
sensitivity
`To demonstrate
to crystal violet
the
presenceofthe
rf
wall
mutation,
tester
strain
}
b. pKMIOI Plasmid
3
culturesofteste srains TA`aTonddeTmAo1ns0t0raetxehitbhietperderseesnicsetoanfctehteo Ra-mfpaiccitlolripnlasmid, pKMI01,
. Characteristic NumberofSpontaneous Reverunts
;
cultures exhibited a characteris`tTiocdneummobnsetroraftestphoenrteaqnueioruesmreenvterftoanhtispteirdipnlea,ttehwehteesnteprlastureadinalong
: }
wwietrhetahsefvoelhliowcsl:e under selective conditions. The acceptable ranges for the mean vehicle controls
)
TASS
8-60
TAI00 60-240
:
TASS 4. 4s TASS? 2.25
!
2. TestorSuainlnvegrity : Escherichia coli
2 Characteristic NumberofSpontancous Reveruants
;
culture
exhibiteda
To characteristic
nduemmobnesrtorfatsepothnetarneeqouuisrermeevnetrtoanrtstrpyeprtpolpahtaen,whtheentepsltaetredstarlaionng
CHV Study No.: 17750-0-409R
19
i
C0250
CORNINGHazleton
`cwointthrotlhse wveahsicletou4n0derrevseerlteacnttisvepceornpdaittei.ons. The acceptable range for the WP2uvrA mean vehicle 3. Tester StusinCultureDensity
To demonstrate that appropriate numbers ofbacteria are plated, the density
`aoftatregsetterlsetvrealioncfutlutrubriedsitwyerdeemgornesattrearttehdantoo rpreoqduuacletcou0l.t5urxes1w0itbhacatdeernisaipteyr
mland/or had
greater than or
reached
equal to
0.5 x 10 bacteria per ml.
4. PositiveControlValues
a Positive Control Values inthe AbsenceofS9 Mix
To demonstrate that the tester strains were capableofidentifying a
i:
3`-mfuotladgeinn,crtehaesmeeoavnervtahleuemeofena pvoasliuteivoeftchoentrvoelhifcolreacorenstpoelctfiovrettheasttesrtasitnr.ain exhibited at least a
:
b. Positive Control Values in the Presenceof S9 Mix
'
(9 Mix Integrity)
To demonstrate thatthe S9 mix was capableofmetabolizing a
:
topefrstothemreutsvateaghiiecnnlietnoctiohtnestmrpourlteasfegonercnteihoacftfttohiremn(.Ss)9,mtihex meexhainbivtaeldueaotfletahsteapo-sfiotlivdeicnocnrteraosleofvoreratrheespmecetainvevalue
mutagen. i
An acceptable positive control in the presence of S9 mix for a specific strain was evaluated as
`having demonstrated both the integrityofthe S9 mix and the abilityofthe tester strain to detect a
Ss. Cooicity
MN
A minimumofthree non-toxic doses were required to evaluate assay data.
B. CrFiorAtPoseitirveReispoanse
were evaluateda`Oncfeoltlhoewsc:riteria foar valid assay had been met, responses observed in the assay
CHV Study No.: 17750-0409R ;
2 00251
CORNINGHazleton
i
1. TesterStrainsTA98,TAL00.andWP2ipvrA
2-fold increase in the mFeoarnarteevsetratnaincislepteor bpeatceoonfsaiderleeadstpoosniteiovfet,hiet sheadtetsoteprrostdruaciensaotvleerastthae
:
mreevaerntarnetvserptearntpslapteerhpaldatteoobftehaeccapopmrpoapnriieatdebvyehaidcloesecornetrsoplo.nsTehtiosiinnccrreeaassiengincotnhceemnteraantinounmsobfetrhoef
test ance.
2. TesorSuainsTALSISandTALSYT 3-fold increase inthe mFeoarnarteevsetratnainctlsepetorbpleacteonosiafderleedastpoosniteiovfet,hiet sheatdesttoeprrsotrdauicnesaotvleerastthae rmeevaerntarnetvserptearntpslapteerhpaldatteoofbtehaeccapopmrpoapnriieatdebvyehaidcloesecornetsroplo.nsTehtiosiinnccrreeaassiengincotnhceemnteraantinounmsobfetrhoef test aricle.
i
RECORDS TO BE MAINTAINED
Athlilsrsatwuddyatwai,ldobceuamrecnhtiavteidoni,n trheecosrtdosr,agtehefparcoitliotceoslo,fanCdortnhienfginHaazrleeptorotngIenn.erfaoteadtalseaastreosnueltyoefar
fmoalyloewliencg tsoubhmaivsestihoenoaffotrheemefnitniaorneepdormtat(e0rtihaelsSproetnasionre.d iAnfttheer tshteoroangee yfaecairlipteiresioodf,CtohemiSpnognsor
HSapzolnestoorn Inc. for an additional periodoftime or sent to a storage faciliy designated by the
REFERENCES
Ames, BwiNth.Jth.eMScaCiamnonn,ealnlda/EM.amYmamaalsiaakni-.MicMreotshoomdes MfuortadgeetneictciintgycTeasrtc.inoMguetnastiaonndRmeusteaagrecnhs
i
1347-364 (1975),
1
Brusick,AnD.eJv,alVu.aFt.ioSnimomfotnhe,EHsSch.erRiocsheinakrcoalniz,WPV.2A.anRdayW,Pa2nudvRr.AS.reSvtearffsoermd.utation assay.
Mutation Research 76:169-190(1980).
3
Green, MMuHtaLt.ioannRdesWe.aJr.cMhur3i8e:l3.-32M.ut(1a9g7e6n).testing using ip" reversion in Escherichia coli
CHV Study No.: 17750-0409R
21
7
C0052
CORNINGHazleton Maron, D.M., and B. Ames. Revised methods for the Salmonella Mutagenicity Test.
Mutation Research 113:173-215 (1983).
Vogel, HprJop.eratnieds.D.JM..BiBooln.neCrh.emA.c2et1y8l:o9m7i-t1h0i6na(s19e5o6f)E . coli: Partialpurificationand some
CHV Study No.: 17750-0-409R
:
2
00253
CORNING Hazleton
i
BB ACTEA RIALCKGLR AWNO EVALU UATN IONCD ODE
"mTihcerocsocnodpiitciaolnolyf(tuhseinbgaackdgirsoseucntdinbgacmtiecriraolscaowpen)ifoervianlduiactaetdiobnosothfmcayctrootsocxopiitcyalalnydatnedst article
i
precipitate as follows:
CODE DEFINITION CHARACTOFEBRACIKGSROTUNIDLCAWSN
1 Nomal Ahealthy microcolony lawn.
2 RSleidguhcleyd Ainncorieacseesibn lthtehsiinzneionfgothfethmeicmrioccroolcoonlioensycolmapwnaraenddoanthe vehicle control plate.
:
3 MRoeddeurcaetdely Aincmraeraskeedintthhiennsiinzgeoofftthhee mmiiccrrooccaolloonniyeslacwonmapnardeadnto the vehicle
contol plate
i::
4 ERexdruecmeedly iAncnreexatsreeimnetthheisninzieongfotfhtehemimcircotcoocloolnioensycloamwpnaarneddaon the velicle contol plate
5 Awsem A complete lackofany microcolony lawn.
6 OPrbescicpuitradteby eTvhaelubaatcekdgdruoeuntdo bmaicctreorsiaclopliacwnancdalnonromtabceroascccoupraitcelteyst article precipitate.
i.
Efvolildoewnicnegopfremcaicpritoastceocpoidcetetsottahreticcoledeprnecuimpbitearteusoendtthoeepvlaaltuesaties trheecocrodnedditbiyonadoditfthionboafctkhgeround
bacterial Taw.
SPriecgihpiistc Nhootwiecveearb,lethmeapcrreocsipciotpaitce pdroeecsipniottatienfolnuetnhceeplaautteo,mated countingof the plate
{
mp MProedceirpiattaete wTohueladmionutnertfeorfemwaictrhoasucotpoimcatperdecciopuintattiengo,ntthhuesprleaqtueiring the plate to be
i
hand counted.
hp HPreeciapitte pTlhaetelamragkeeasmtohuenrteoqufimraecdrhoasncdopciocunptriecnigpidtifaftiecuolnt the Ewxhaimcphleh:ad4tmopbewcoouulndteidndmiacnautaaplllyatdeueobtsoetrhveemdatrokheadvaeamnoeuxnttorefmmealcyrorsecduocpeidc beasctkagrrtioculned lawn precipiae.
:
CHY Study No: 17750-0409R
FS)
.
00254
CORNING Hazleton 1
SECTION IV. RESULTS AND CONCLUSIONS
i
.
CHV Study No.: 17750-0409R
:
:
2
00255
Bi
CORNINGHazleton
RESULTS
A. TestArticle Handling
(CHY Lots 347,Th3e49t,esatnadrt3ic5l2e), Tw6a5s6u4s,edwaasstshteorveehdiactler.ooAmtt1e0m0pemrgaptuerrem.lD,ewihoniiczhewdawsattehre most canoinccleentrreamtaeidnteodacksdoilluuttiioonnipnrealplasreudc,cetehdeitnegstdairltuitciloenfsoprrmeepadraedclfeoarr,thceolmourtleasgsensiocluittiyona.ssTayhe test
B. DRosaengefindiStnudgy
the dose rangefDinodsiensgtsobetutdeysctoenddiucnttehdeomnuttahgetneistciatrtyicalsesauysiwnegrteessteelrescrteadinbsasTeAdIo0n0tahnedresults of
`arWtiPcl2ei, rfAroimn5b,o0t0h0tthoe6p.r6e7seungcepearnpdlaatbes,ewnecereofteSs9temdiaxnd(otnhee prleastuletpsearrdeopser)e.senTteend dinosTeasblofetses1tand
t
2t.heTphreesseendcaetaorwaebrseegnecneeorfatSe9d minixExapseerviimdeenntce1d77b5y0-aA1n.ormNaol bcyatcoktgorxiocuintdy lwaawsnoabnsdernvoeddeicnreeiatsheerin
:
the numberofrevertant per pate.
C. MutagenicityAssay
`These data were"TgheenmeurtaatgeedniinciEtxypaesrsiamyenrtessul1t7s7f5o0r-TB-1,65167475a0r-Cp1r,esaenndte1d7i7n50T-aDbI.le3sThtehdraotuaghr7.e
:
pgrreosuepnt(eTdabalsesme4a,6n,raenvder7t)aansndpears pilnadtievi+dusatlanpdlaarted dceovuinattsi(oTnafbolresea3c,h5,traenadtm7e)nt and control
"`Tmhuetargeesnuilctisotyftashseayd.osTehreandgoesfeisntdeisntgedstwuedryew5e,r0e00u,s3e,d3t3o0,se1l,e0c0t0f,iv3e33d,osaensdt1o0b0eiegspteerd ipnlattheein both the presence and absence of S9 mix,
:
aIsnstahye, iEnxitpiearlimmuetnatge1n7ic7i5t0y-Ca1ss(ayT,abElxepse5rainmednt6),17al7l5d0a-tBaIwe(Traeblaecsce3ptaanbdle4)a, annnddo ipnotshieticvoenfinicrrmeaatsoesr.y
:i
ipnretsheenncuemobrearbosfenrceevoerftaSn9tsmipxe.s pIlnatEexwpeerreimoebnster1v7e7d5w0i-tChl,anaynoofnt-hdeosteesrteesrpSoanisinvsee5i.t0h-efrolidn the
:
`iTncArIeSa3s7e wihnitchehadbisdennoctemoefeS9thmiexc.ritFeroiratfhoirs repaossiotnitveestreerssptornasien wTaAsISo3bs7erwvaesdrweitteshteedstinetshterain
absenceof $9 mix in Experiment 17750-DI.
:
InunmEbexrpoefrriemveentrt1a7n7t5s0-pDeIr l(aTtaeblwee7r)e, aolblsdeartvaedwewriethactceestpetrabslreaiannTd AaLndS?noipnotshietivaebsiennccreeaosfes in the
$9 mix.
i
All criteria for a valid study were met.
1
CHV Study No.: 17750-0-409R
25
|
G00256
1.
`CORNING Hazleton
CONCLUSIONS
.
A`TshsearyeswuiltthsoaftCohnefSiarlmmaotnoerlylAas-sEasycihnedriicchaitathcaotl,iu/nMdaemrmtahleicaonn-dMiiticornossoofmtehiRsevsetrusdye,M3uMt.ation
`pCeorrppolrataetoiofna'snyteosfttarhteicltee,stTe-r6s5t6ra4i,nsdieditnhoetr icnautshee aprpeosseintciveeoirnacbrseaesneceionfthmeicnruomsboemraolfernezvyemrteasnts
prepared fromAroclorTM induced rat liver (59)
i
CHV Study No.: 17750-04098.
i
2%
600257
i. CORNING Hazleton
SECTION V. DATA TABLES
CHV Study No.: 17750-0-409R s
27 00258
`CORNINGHazleton
naa 1
nose avcrFIOIG StoDy
Teor AnriouE 1: T6564
JE
--
Date FATED: 12-30n-96
VENIGLE: Detentzed wares
Dare coma: 18-30n-96
-
Tatoo RpvERTANS IR LATE
vermin i emETorn e Eo I h TeFmAe J mSE e
:]|
Test(sA0rospsel)cie
es
1
106
s
78
1
n
\
10.0
1
s
os
s
:
ww33.3
112220
1A
107s8
1\
100
us
:
"
A
:
ss
16
3
us
s
:
wr
=
:
os
\
1000
so
A
w
:
: e sos0s0 w 1ue2 r .\ r wo .3
]
E ST im BEEOUeSeS. JD IEE,
:
CHV Study No.: 17750-0-409R
:
28
00259
1.
i oz 2 i
CORNING Hazleton
!
.
Teer samscaz 1: Tesen DOSE RANGEFINDING STUDY
memo 0 1730-01
Date ELATED: 12-3ume96
VENIGLE: Detontsed vase _--
Date comzD: 10-sume96
"ruck neveRzaves pn pate paseuae ECTr rine tEie n raTe in v suLaraomse eon--
.
si
10
3
2
:
:i
Tees Aoreic
1s
:
P
:
:
10.0
2
:
u
:
i
wr33.3
16
1
5
3
16
1
=
3
;
100
ws
1
3
w
:
0
:
..
;
w
To
3
2
:
:
1000
s
:
"
:
:
ss0
u
:
.
:
;
_--3000
10
:
s
:
:
CHV Study No.: 17750-0-409R
i
29
00260
1.
CORNINGHazleton
|4
MUTAGENICITnYasAeSSA3Y TNDIVIDUAL PLATE
CREoSnUeLsTS
TEST ARTICLE ID: T-6364
peEneNT 10: 1750-81
DATE PLATED: 20-Jun-96 DATE COUNTED: 28-3un-96 ------------
VEHICLE: Detonized vater PLATING ALIQUOT: 50 41
TE NEJ J SE BEa i a wrmr
pra
_--
FEL)
"1's arson cotucas
3 = mederataly casscnt
i
CHV Study No.: 17750-0-409R
i
:
30
c00261
CORNINGHazleton
i
MUTAGENICITTYanAzSSA4Y RESULTS
`Story
TEST ARTICLE ID: T-6364
:
EXPERIMENT TD: 1775031
DATE PLATED: 20-Jun-96 DATE COUNTED: 28-Jun-96
VEHICLE: Deionized vater PLATING ALIQUOT: 50 pl
eee
eee.
em me
we am oan wen
--_--_----_
7 =5 Gnianyprecipoidseasse CHV Study No.: 17750-0-409R.
wp5= sfatunrate precipitate
[ER sSetadovynpcyaptease 31
:
00262
;
CORNINGHazleton
j
MUTTARGOEINVIICoIUaTAYLoAPeLSASTA5SY CREoSuUvLsTS
Test ARTIcuz To: T-esed
exert 1: 17750-C1
DATE PLATED: 25-3u1-96
VEHICLE: Deionized vater
DATE COUNTED: 30-9u1-96. 31-Jul-56
FLATING ALtauor: 50 41
-- ee ------
san
au
-_
one
_--
1:
Te PSIeIeIr D53 apience
hane L domTaine 1a0 npupmae
BS TR nE ie 3Te1a0niy r eventr 3I= p sR eerse uates.
i
CHV Study No.: 17750-0409R
;
32
00263
:
CORNINGHazleton
:::
MUTAGENICITTYamALSzSA6Y RESULTS StuRy
TEST ARTICLE ID: T-6564
:
EXPERIMENT ID: 17750-C1
DATE PLATED: 25-Jul-96 DATE COUNTED: 30-3u1-96, 31-3u1-96 _--m--
VEHICLE: Defonized vater PLATING ALIQUOT: 50 p1
ram ue
wie a sum wen
i
or arses wow moe wos [a
I nos :
:
_--
ms CII Te
Th sam
ee
Ti
ATi
"1's artery ravens
sL edeeseI pracpitase
3 = woderasaly redsent
2 taryerseMcasiunpe)e
1
CHVStudy No.: 17750-0409R
33
00264
`CORNINGHazleton
none AEAC icshs7 A Efe
ras Anmscas mo: 7-364
.
xen 1: 1775001
DATE PLATED: 25-hug96
VEUICLE: Detontzed vater
ceDAT--E co-- nrTED--: 27-- -hug---96 ----------Fu--t A--uiau-- ors --50 4-- 1 ------
nwa
A
FEE
i
------
SIE
i pes 38 re
CHV Study No. 177500405
34 C0265