Document zzG0OXNav9wkoEOdgRoVnDD8m
:
CORNING Hazleton
MUTAGENICITY TESTWITH "43%
IN
THE
SALMONELLA
-
ESCHERICHIA
T6342
{ACoie omnes
COLIMAMMALIAN-MICROSONE RESSPhSE
MUTATION ASSAY
Nn a
FINAL REPORT
AUTHOR
Timothy E. Lawlor, MA.
PERFORMINGLABORATORY
Comi9n2g00HaLzeleestbounrgIncP.ik(eCHV) Vienna, Virginia 22182
LABORATORYPROJECTID
CHV Study No. 17073-0409
SUBMITTTEDQ
Building32M20C-o2rEp-or0a2ti3onM Center St.Paul, MN. 55144-1000
STUDYCOMPLETIONDATE
December 14, 1995
00353
CHV StudyNo. 17073-0409
Tof30
CORNINGHazleton QUALITY ASSURANCE STATEMENT
STUDY TITLE: ASsalsmaoynella Escherichia coli Mammalian-Microsome Reverse Mutation
ASSAY NO: 17073-0409
PROTOCOL NO: 409, Edition 4, Modified for 3M Corporation
UPQnruoiautlcicattnydwAeasrcsecuorcraodnnicdneugcittnoesdtpehacctgcieoonrnedsroianflgtrhteoeqtushiterueSdmtyeanantndsdarrdevOipeewroatfitnhgePfrioncaeldruespcosrotofftthheeQaubaloivteyrAesfseurrenacnecde
arengdultaottihoenss.tuFdiynddiirnegcstofrroomn tthhee ifnoslpleocwtiinognsdaatneds:ofiftnahlereappoprrtoprreivaiteewGsoenrge rLeapboorrtaetdortyo mPraancatgiceement
fou
Findings &
;
Plating - 09/19/95 Draft Report Review- 11/0695
Final Report Review - 12/14/95
09/19/95 11/07/95
12/14/95
M. Murphy C. Orantes/ S. Ballenger C. Orantes
ality Assurance Unit
CHV Study No.: 17073-0-409
27 Date Rfeascd
00354 2
`CORNINGHazleton STUDY COMPLIANCE AND CERTIFICATION
The study was conducted in compliance with the Good Laboratory forth in the Food and Drug Administration (FDA)Title21 ofthe
Practiceregulationsas
set
aRpepgluilcaatbiloenas mPeanrtdm5e8n, tiss.sueTdheDreecweemrbeerno22s,ig1n9i7f8i,ca(netfdfeecvtiiavteioJnusnefr2o0Um,.St1.h9e7C9ao)fdoaeronedfmewFniettdihaeornnaeyld
regulations or the ofthe test results.
signed protocol that would affect the integrityofthe The raw data have been reviewed by the Study
study
or
the
interpretation
ceovnatleuxattoifotnohfethsetutdeystdaerstiicglne aasndperveasleunatteidonhecrreitienrira.epresenatns approDpirrieactteorc,ownchlouscieorntifwiietshitnht,atht hee
Study Director:
Is
(----
TimothyE fLawlor, M.A.
Bacterial Nlutagenesis
Genetic and CellularToxicology
(2049s
Study Completion
Date
CHV Study No.: 17073-0-409
00355
3
TAOFBCOL NTEE NTS
CORNINGHazleton PageNo.
I. STUDYINFORMATION ...........oooiiiiiiiiiiiiinnnnnnneei
7
HL MATERIALS ANDMETHODS...........ceeiiniiiinnnii
0
IV. RES ANU DCOL NCLT USIOSNS ..0..........ccceeieiiriineeiiii iii. 23
Vo DATATABLES...........oooooiiiiiiiiiiiiiiiiiiiiiiiiiiiiilll 26
CHV Study No.: 17073-0409
C00356
4
`CORNING Hazleton
SECTION I. SUMMARY INTRODUCTION AND CONCLUSIONS
CHV Study No.: 17073-0-409
00357 5
SUMMARY
CORNINGHazleton
A. Introduction
mutagenic activAitttyhienrtehqeuSeasltmoofne3lMla-CEosrcphoerratiicohni,a Ccoorlnii'nMgamHmaazlleitaonn-MInicc.roinsvoemsetiRgeatveedrsTe-6M3u4ta2tfioorn srAtesrvsaeairynss.eamTnuhdtiaasttaitsohsneastyarteyvptathloeupahhitasentdildtoihcneeutslesoitncuaasrntiiEncsltechhaeenrgdie/conhroiimatescoomflelstpateebscotileifrtisectsrSafaiolnrmbtoohneteihlrlianabittlhiyetpyphrtioemsuiernnidcuuecmeatnesdter daebrsievnecdeoffraomnAerxoocgleonroTMu.sinmdeutcabeodlriactalcitvievra(t$i9o)n.systemof mammalian microsomal enzymes
TrehnegedfoisnedsintgessttedudiynutshienmgutteasgteenriscriatiynsasTsAay10w0eraensdelWePc2teudbvarsAedanodntetnhedoresseuslotfs otefsat darotsiecle ranging from 5,000 to 6.67 pg per plate, one plateperdose, both in the presence and absenceof S9 mix. TTAh9e8t,esTteArLs0t0r,ainTsAuIsSe3d5,inTtAhIeSm3u7t,agaennidcEistcyhaesrsiacyhiwaecroeliSatelsmtoenresltlraaintyWpPh2iumuvrriAu.m tTehsteerassstraayinwsas ccoonncduurcrteendtwviethhicflieveanddospeossoifttiveestcoanrttrioclles iunsibnogththtrheeepprleasteesncpeeranddosae.bseTnhceedoofsSes9 tmeisxteadlwoenrgewith 5.000,3,330, 1,000, 333, and 100 ug per plate in both the presence and absenceof S9 mix,
B. Conclusions
Mutation AssayThinedirceastulettshoaft,tuhnedSearltmhoeneclonldai-tiEosncshoefrtihcihsiasctuodlyi,/3MaMmmCaolrpioarna-tMiionc'rsosteosmtearRteicvleer,se
T-6342, did not strains cither in
tchaeupsreeasepnocseitoirveabisncernecaesoefimnitchreonsuommbaelroefnrzeyvmeerstapnrtespapreerdplfartoemofanyofthe
tester
AroclorTM-induced rat liver (59).
CHV Study No.: 17073-0409
00358
6
CORNING Hazleton
SECTION IL. STUDY INFORMATION
CHV Study No.: 17073-0-409
00359 7
CORNING Hazleton
STUDY INFORMATION
A. Sponsor: 3M Corporation
B. Test Anicle: T-6342
I. Physical Description: clear colorless liquid 2. Date Received: 07/21/95
C.
Typeof
Assay:
Salmonella-Escherichia Mutation Assay
coli/Mammalian-Microsome
Reverse
1. Protocol Number: CHV Protocol 409, Edition 4 Modified for 3M Corporation
2. CHV Study Number: 17073-0409 D. Study Dates
1. Study Initiation Date:
08/28/95
2 Experimental Start Date:
09/12/95
3. Experimental Termination Date: 09/22/95
E. Study Supervisory Personnel
Study Director: Laboratory Supervisor:
Timothy E. Lawlor, M.A. Michael S. Mecchi, B.S.
CHV Study No: 17073-0409
600366
8
`CORNINGHazleton
SECTION Ill. MATERIALS AND METHODS
CHV Study No.: 17073-0-409
G00361 9
CORNING Hazleton
MATERIALS AND METHODS AmesetTahle(e1x9p7e5r)imaenndtaGlremeatnerainadlsM,umrieetlho(d1s97a6)n.d proceduresare based on those described by MATERIALS
A. TesterStrains
I. Salmonella typhimurium sapuexcoitfriocpghesnoTtAySpe,soTfAtLh0Teh0se,estTteArsIateiSrn3ssSat,rraeainnssdhuToswAendISwin3e7rTeaabstlhedeeISsacrlimboendelblyaAtmypehsimefuarliu(m19h7i5s)t.idiTnhee
TABLE TESTERSTRAINGENOTYPES
hisG46 hisC3076 hisD3052
TAIS3S TAIS37
T--A_1--00
TAS
LPS Repair R Factor Ha wrB ofa wrB +R
mInutaadtdiiotnisontwhoicahmuetnahtainocne itnhetihresheinsstiitdiivnietyopteorsoon,methmeutteasgteernisctrcaionmspcoounntadisn. tTwhoeadrdfitwiaonlallmutation drleeisfpuiolcptioselniyncsyatchicenhclaroresiasdoseefsboaprnerreimeeoraftbthiahlteifteyonrztmoysmceetrhstearisenusrpcfolanacsseisobefslteohffeocrhbtaehcmetiecsrayialnltshcseeusliclshowfaalpsla,trhotTsohefetcrohenestualitniinnggcellalrgweall ring systems (i. benzo(a)pyrene) that would otherwise be excluded by a normal intact cell wall,
dsTeyhlseettseiemocnwoehnxidtcemhnudgtsraettaihtorlnoy,ueagdnhehltahenetcieiosonothgfeetnsehe,ensaiultlvirvoBfittyhgoeenfet,tehsertseeesrultstrtsaraiiinnnssactdooenfstiaociimneeinntmguDttNhaigAsedneesxl.ceitsSiioionnncaerlestpohaerieruqvurirBe the vitamin biotin for growth.
tShterasiennssiTtAiv9it8yoafntdhTeAsIe0s0traailnssotcoonstoamien mthuetaRg-efnasc.torTphleasmmeicdh,apnKiMsIm0b1y,wwhhiicchhthfiusrtphlearsimnicdreases
DinNcrAearseepsasiernpsoitliyvmietyratsoemuctoamgpelnesx
has been involved
swuigtghesthteedmitsombaetcbhy-rmeopdaiifryipnrogceasns.existing
bacterial
CHV Study No.: 17073-0409
10
G00362
CORNING Hazleton
TsheuisbststetiiditsnutetriaioinnndsmeupTteAan9gdee8nnacsenadn(pdTrAToLtAoS1t3r07o0pahiyrs)rrebevyveerfrtrteaedmdebsfyhrimofumttmhaiugstetaingdseinnwesh.diecTpheAcnIadSue3sneScebios(traeuvxerotterdobpyhybtao)se
substitutions.
h frameshifs and base
2. Escherichia coli
described by Green andThMeurtieeslter(1s9t7r6a)in. used was the tryptophan auxotroph WP2uwA as
tdIhenefaisdctdirieatniincoytnowthsoihacohmwuetenanhthaianoncnceeisdnitmthsuetsaetnbrsiyilptititvoyiptshyianntcooespteohrmeoenu,mvutrhtAargteeepnsatiiecrr scsytorsmatpieonmucnowdnostu.alidnTsnhaisudAeficDiNenAcyraelplaiorws remove the damaged partofthe DNA molecule and accurately repair it afterwoarrmdasl,ly act to iTnesdteeprenstdreanicneW(Ppr2outortrropihsyr)evbeyrtbeadsefrsoumbsttirtyupttioopnhmauntdaegepnesn.dence (auxotrophy) to tryptophan
3 `So ofTuestrerSc traiens
a Salmonella typhimurium
Bruce Ames, DepartmentofBiTohcheetmeisstetrryst,raUinnisveirnsuisteyoaft CCaHliVfowrneirae,rBeecrekievleedy.directly from Dr. b. Escherichia coli
National Collectionof IndustriTahleBatcetsetrerias,trTaoinr,reWyPR2eusveraAr,chinSutasteiaont,CScHoVtlwanads(rUencietievdedKifnrgodmotmh)e.
4. Storage oftheTester Strains
a Frozen Permanent Stocks
(ov0e5m-i1g5htmlc)ulatturse-s,70adCd.ing
DMFSrOoze(0n.p09ermmiamneintofsctuolctkusrew)earnedprferpeaezriendg
by growing fresh small aliquots
CHV Study No.: 17073-0-409
n
00363
b. Master Plates
CORNING Hazleton
pfrhoizmeunrpieurmma,naenntexscteoscskoofnthoismtiMidanisintmee,arlapnaldgaatbreisoatpwipner,roeaprnpidraetfpeoarlryteesdsutbpeyrplssettrmraeeiannkstieTndgAwei9atc8hhta1en)sfdtoeTrrAsS1ta0rla0mi,onnferlolma a
Easmcphiecriilcihnia(2c5oluig,/amnl)e,xcteosesonsfurterytphteopsthaabnl.e 523C.
maintenance Tester strain
omfatshteerppKlMat1e0s 1weprleassmtoirde;daantd
2)
for
S. PrepoafOrveranigthtiCuloturnes a Inoculation
Ibnyotcrualnastfeedrrfilnagskascwoelroenyplfarcoemd tOihnveeaanspihpgarkhoeptrr/iciauntlcteuumrbaeasstfotorerrwuphslieactheinwtoaalslatlpearsstoiknggcropnartmoacimenetdiuondrgebcseuglwitenurreoepiemrneaodtciiuuolnma.,ted
(shaking, 125 late log phase
+wh2e5nptmur;biidnictuybmatoinoint,or3i7ng+
2C) so began.
thatthe
overnight
cultures
were
in
log
phase
or
b Harvest
woefirnecuhabravteisotnedwaosncdeetaeprrmeidneetderTbmoyisenpneesdcuttrurerobtpihhadotittcoyumlwetatursriercsemwaocenhrieetdoharasirvndegestoteferdcmuiilnnteuldartebetyluroabgipdpeihrtaycs.een,tCtuhleulrecnsgth
Ohtarvavenersgmrrietoatcwahnnecd(esat(ad%teTino)sniartreyyao)dfciunalgttuolrneeassatms0ap.ey5cXterxoh1pi0hboittcoedmlelesctreperea.rsemTdihsiaesnnsdtiattrighveaitttythuterobcisudloittmuyereemsnushtuaarvgeeesnntsoh.attoCcvuuelrtgurreoswn.
were
removed
from
incubation
when
the
target
%T
was
reached
and
were
placed
at
+
ltures 3C.
6. ConfoifTr estm erSa traitnGeinotoypens.
the dayoftheir use in tTheestmeurtsatgreaniinccuilttyuarsessay:were checked for the following genetic markers on
a Salmonella typhimurium
1) fa Wall Mutation
demonstation ofthe sensitivity ofthe`cTuheltpurreesteoncceroysftatlheviorl/eat.walAlnmaultiaqtuiootnofwaans coovnerfniirgmhetd by
CHV Study No.: 17073-0-409
2
00364
CORNING Hazleton icsneuhnlistibutiritevioioftnyoeafdicbsahkctscetorrnaitiaanliwngairnsogwot1vh0eriulnagoiadfzoocnnrtyeositpamllmaetvedisiolacetotenlwtyaaissnuiarndrgdoesuden.ldeicSnteignvsteihtmeievddiitisyka.waansd daenmaonntsibtiroattice.dby
2) pKMIOI Plasmid R-factor oesvteemrigshtrtaicnusltTuAre9o8faenadchTAst1r0ai0n bwyasdeomvToehrnelsatpiardteoisneotnnocoepoflrfaettsishsectoapnnKtcMaei1tn0oi1nagmppsilecalieslcmltiiindv.ewmaAesndciaoalniafqnuiodrtmoaenfdafnor danetmiobnisottircatseendsibtyivbiatcytedriisaklcgornotwatinhiinngt1h0e uzgoonefaimmpmiecdiilaltienlywassurardoduendd.inRgesthiestdainskc:e was
3) Characteristic Numberof Spontaneous Revertants y1e0h0iculleacloinqturootlssoftthahtearceulcthuarreacatleornigstwicio`tTfhhtethhmeeeaarpenpsrpnoeupcrmtiibavetereosvtfeahisinpcsolnewteoarnneesodeuelsemcortneisvvteerrmtaeatndetidsabpyerppllaattiengin the
b. Escherichia coli
1) Characteristic Numberof Spontaneous Revertants 1ye0h0icwlleacloinqturootlsos ftthahtearWePc2huarvarctAerciuslttiucrToehfteahlmoeengarenwsipnteuhcmttibhveeeraosptfprrasionppsroinwatetareneevodeuheismcorlneesvtoerrntaastneetldsepcbetyirvppellaamtteiednigian, the
7. TesterStrainMedia
a Culturing Broth
Nvuotgreile-ntBoBnrnoetrh sNao.l s2ol(udtriyonpo(wVdo"eTgrhe)el.barnodthBuosnendert,o g1r95o6w)osvuepmpilgehmtenctueldtuwreisthof2.th5e%te(swt/evr)sOtraoints was
b. AgarPlates
0B.o2nn%e(rwimvi)nigmlauclosmee.dium E (VBogoetltoamnadgaBron(n2e5r,mi19p5e6r),1s5uxpp1l0e0memnmtedpetwriithdi1sh.)5%wa(swiVso)gaegla-rand
CHV Study No.: 17073-0409
13
00365
`CORNING Hazleton
. Overlay Agar for SelectionofRevertants
110005m%lNaagCalr (fwor/vs)elaencdtwioanosfsuhpipsOtlvieedrmilenaenytree(vdteorpwt)iaatngthsa,1r0owraml2s)op0rf.1e5)pma0r.Me5dmtwriMytphthoi0ps.ht7ai%ndisanogela/urbti(iowotinnvps)eoralun1tdi0o0nmplerof
wsauagapsrpalfdeodmreesdnetlteeocdttithooenpsoaufgptaprrlyepwmtaeosnptuhesadendtroienpveatrhgtaeanrotsv(.e0r.l5Waymh.ieonHfowSwa9etvmeeirrx,pewwrahs2ernmeiqSou9ifrmesidu,xp2pw.la0esmmenlnoottfetrdehqeuired, water
etnhesurreesdultthiantgt2h.e5fmilnaolftdoipluatgeadr asnudppalmeimneontaecdidtospupapglaermweanst cuosnecdefnotrratthieoonvserrleamya.inTehdistthodepislaugaiamrge)nbaontdh
in the presence and absence of $9 mix.
B. Liver
E Reaction Mi 9 Mi
I. 5H9omogenate
BMaotlcehcu0l3a6r9T(o3x8i.c8olmoeggyo,fLpIinrvcoe.tr,eAmininncpareporoslomim)sa,.lMeTDnhzey2h1mo4em0s1o,(gSeB9naahttocemhow0ga5es6n8aptr(ee3p)8a.wr8eemdreefrpooufprmrcmohataleseeidnSfpperrraogmmuie).and 5D0a0wlmegy/kragtsasthdaetshcaridbbeedebnyiAnjmeectsedef(ia.lp,,)1w9i7t5h. AroclorTM 1254 (200 mg per ml in com ofl) at
2. S9Mix
procedure. The S9 mixThcoenSta9imneidxtwhaescoprmeppoanreendtismimneddiicaatteedlyinprTiaobrl1e IiIt.s use in any experimental
HO
TABLEII. S9MIXCOMPONENTS
070ml
IM NaH,PO,/Na,HPO,, pH7.4 0.25M Glucose-6-phosphate:
010ml 0.02mi
0.10M NADP 0.825M KCI/0.2M MgCl,
0.04 ml 0.04 ml
-- $9 Homogenat_ e ---- [21W.1000mmmll
CHV Study No.: 17073-0409
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00356
C. Controls
CORNING Hazleton
1 Vehicle Controls
mabasxenicme uofa$l9iqmuoitxo.ftTehsVetevhaerithciilccelleecdocinoltnurttorilooslnwpwelarasteepdpl)la,attaeedld,onfugosriwanilglthatea5s0t1e0u0lstparllaiiaqnlusioqtbouooftthovifenthihtcheleeapp(rpeerqsouepanlce10atnhde
tester strain and a 500 pl aliquot of$9 mix (when necessary), on selective agar.
riate
2. PositiveControls
strains plated concurren`tTlhye wciotmhbitnhaetaisosnasyofaproesiintdiivcaetceodntirnoTlas,blaectIiIvIation condition and tester
Tester TABLEIIL.POSITIVE CONTROLS Cone
TSAta%in TA9%
S9+Mix Po2s-iatmiivneoaCnotnhtrraoclene - 2nitrofluorene,
pe2rp5luagte 10g
TAI00 TAI0
+
2s-oadmiiunmoaaznitdheracene
25pg 20ug
TAIS3S + TAISSS =
-aminoanthracene sodium azide
251g 204g
TAIS7 + TAISY
2I.CaRm-i1n9o1anthracene
25ug 20pg
--WW_PP--22uurrriAA vo_+- gui42--nnaimtgirnoolqauniitnhonrlaicneee-nNNe-ooxiodex1i2105d.010epggg.
a Source and Grade of Positive Control Articles
(2C9A7S.#52%6622-8n-it2t2o-f8l)u,orSeingem(aCCAhSZe-m#ai6mc0i7an-lo5a7Cn-ot8.h),r,pauAcrleidntreyi>(ch9C8AC%hSe;#m6Ii1Cc3Ra--l113C9-o18.)(,pCuSAriiSgtmy#a1978C0h%7e;.m4si5o.c0da)il,uCmoa,sipduerity
Polysciences purity >99%.
Inc.
purity
>95%;
d-ntroquinoline-N-oxide
(CAS
#56.57-5),
Sigma
Chemical
Co,,
CHV Study No.: 17073-0409
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00367
3
i"
CORNING Hazleton
a TestAnice
by plating a 50 ul aliquot (theTshaememovsotlucmonecuensterdatinedthteesatsasratyi)cloendisleulteicotnivweaasgcahrecked for sterility bo S9Mix
agar.
The $9 mix was checked for sterility by plating 0.5 ml on selective
METHODS
A. Dose RangefindingStudy
determined assay.
in
oTrhdeerg0roawltlhowintkhiebietloercyteifofneotf(pcyptrootponriictitey)doosftehsetotebset
article tested
to n
tthheemteusttsaysgteemnwas
1 Design
aatnodnWePpluasterpAerdbootshe.in TtThhheeeptdreosetsseearnrtcaiecnlgaeenfwdianasdbiscnehgneccsektouedfdy$fwo9ramsciyxpt.eotrofTxoiercnmietdydosuuespsiotn0fg.atteemssattexarirstmtiucrlamei.nwseTrAe1t0o0wed concentration of5 mgperplat.
a Rationale rciesoargdaeipnleayrrdaailtlsvycelerymeephrdiegsfhernontmuatmibveeorfotfhTsahptoenoctbysatenorctvooexuidscoirtneyvetorhfteatonhttehsetpreesttreasprtlteairtcels,etrgoarbiansdsearatvineoddnsbooenfcateueysstteeorotfsotTirAaci1tn0y0Tc'AasIn0b0e enPsdtuahnutsri,cl didoieffsenreonttproasnsrgeoesusotiftncheeyetfxoapteowracilmiletnymtuamtlaatyviarobineattoihobanst.etrhvTeehdSe.alEAmslochnoee,rltilhcahticyayptchooitmfouxtsiescoiuttemyr ssmtadriauinnesdhapvyea mmuixt.ageTnhiecrietfynotarhs,esatphyriesbsawesoneucdleoodnfret$qh9ueipmrrieextshemnaacydeifovfraeraryebnsgterentacetetloyafrfttrihcoelmeStdh9oastmeionrb.asnegrevsedbeintetshteedabisnetnhcee of 0g
CHV Study No.: 17073-0-409
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G00368
CORNING Hazleton
2 EvaluationoftheDose Rangefinding Study
per plate and/or by athiCyntnoitnogxiocridtiysiaspdpeetaercatnacbeloeftashaedbeaccrteearisaelibnatchkegnruomubnderloafwnr.evertant colonies
3
i
.
a No Cytotoxicity Observed
trhaengheifgihnedsitndgossteuodfy.test articleSuisnecdeinnothceytmouttoaxgiecnitiyciwtaysaosbssaeyrwvaesd tihnethseadmoesaesrtahnagtetfeisntdeidnignstthuedy,
-
B. Mutagenic
I. Design
TweAr1e53t7es,teadnadlWonPg2wuivtrhATthhbeeotaaphspsiranoyptrwhieaasptrepeevserehfnioccrelmeaenaddnudasbpisonseginttceiesvteoefrcosSnt9trramoilinsxs..TATF9hi8ev,eddoTosAse1es0oso0f,fteTtseAtsLtaSra3trit5ci,lcele were selected based on the resultsofthe dose rangefinding study.
2. Freandq Rouu te oe f Admn inisc traty ion
(i1n9c8o3r)p.oraTthiiosnmmeetthhooddoollToohggeyytheoasrstiegbrienseatlnrlaysinhdseoswwcenrriteboeeddxeptboeycstAedma etwositdehereratalens(tg1ea9or7tf5ic)clleaansvdsiaeMsotahfre opcnlhaeatmenidcaAlmes mmiutxag(ewnhse.reIanptphreopprliaattee)inwceorrepocroatmiboinnmeedthinodmoollotgeyn,atghaertwehsticarhtiwcales, otvheertleasitderonsttroaianmainndimthaelSa9gar dploastees.ofFotlhleowteisntgaritnicculeb,attihoenveathi3c7le%c2ontCroflosra4n8d+th8ehpro,srietvievretcaonnttcroollosnwieesreweplraetecdouinntetdr.ipliAclalte,
C. PlatingProcedures
mutagenicity
These assay.
procedures
were
used
in
both
the
dose
rangefinding
study
and
the
aEcatcihvaptliaotnecwoandsitliaboenlaenddwidtoshea. cTohdee wSh9imcihxidaenndtidfiileudtitohnesotefstt haretitcelset, atretsitcplheawseer,etepsrteeprasrteradin, immediately prior to their use.
CHV Study No.: 17073-0-409
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00369
CORNING Hazleton
rwWeahqsueianrde8dd,e9d5m0ti00x2uw.l5aosmfln$oo9tfmrmieoqxlu,tire1en0d0,sew1ll0eoc0fttuievlseotfteotrpessattgerarairsn(tarmnaaidinna5tn0adiunl0eodfautvle4oh5fivce+lhe2icCl)e. orWhteestnarSt9icmliexdouses
2mai1dxd5teuxdret100w02a0smvmmolrptoetefmrxieodlditasenh.dn soAevfleiereclrtaiitvdheeotnotpoatghaer.suArffatceeotfh25reqmuliorefdmcionmipmoanloretbneotststthoaarmtdiacbgleaeredncooasndetdaseiadn,eedthjeu
incubated fo4r aliquot.
8hrat 37 + 2C. oPvoesriltaiyvehcaodnstoolildiafriteidc,letshweeprleatpelsawteedreusiinnvgerate5d0apnlpdlating
D. ScotrheiPlnatges ewvearleuahteilodnacto5ul+Pdl3attaekCseupwnlhtaiiclceh.suwcehretinmoettehvatalcuoaltoendyicmomuendtiiantgelayndfoblacltoewriinagl tbhaeckingcruobuantdioanvpneriod
1. BaBcteariacl kgrLaownuEvalnuatdion e10vitdheenvceehoicflceyctoonttorxoilciptlTyahtaeencdaontndedsiwttaairsotniorcelfcetoprhrdeeecdibpaaiclttaoetrneig.alwEibvtaihcdktehgnercoreuevonefdrctlyatanowttnocxowiuacnsittseyvfawolarusaaltlseopdlrafetodersraetlatthiavte Sdeocsteioonn. the data tables using the code system presented at the end ofthe Materials and Methods
2 Counting RevertantColonies
fpolrattehsecpoonstiatiinviencgotnetsrtola`sTcwheelrneuwmceobrueenrctooefudrnetbveyedarutmaatnnoutmaacltolelydo.ncioeTslhoepneynrucpmolubantetereorfo.frrtehveevrethainclceolcoonntireoslps eanpdlaatlle
E. AnalyofsDiatsa
TearbleescaSlceucltaitoendo.FfotrThhialesl rrreeesppuollrittcs.aotfetphleastiengcsa,lctuhleamtieoanns arreeveprrteasnetnstpeedripnltaatbeualnadr ftohermstianntdhaerdDadetvaiation
EVALUATION OF TEST RESULTS Before assay data were evaluated, the criteria foar valid assay had to be met,
CHV Study No.: 17073-0409 18
C00370
CORNING Hazleton
A. CriFotrAeValridiAssaay
The following criteria were used to determine a valid assay:
1 Testor StrainIntegrity : Salmonella typhimurium
a ofa Wall Mutation
cultures exhibited sensitivity t`oTocrdyestmaolnsvitorlaett,e the presence ofthe rf wall mutation, tester strain
b. pKMIOI Plasmid
culturesoftester strains TA98TaonddeTmAo1ns0t0raetxehitbhietepdrerseesnicsteoafnctehteo Ra-mfpaicctiolrlip,lasmid, pkM101,
Characteristic Numberof Spontaneous Revertants
vcuiltthurtehse evxehhiibcilteeudnadecrhasrealcetcetriisvTteioccodnnedumimtoinbosentrsr.oaftseTphtoehneatcraceneqepuotiuarsbelmreeenrvtaenrfgtoearsnhtfisoptreirdtihpnelea,vteethhiwechlteeesnctoeprnltasrttoreladsainlwoanrgs
as follows:
TA TAI00
8-60 60 - 240
TAIS3S TAIS37
4. as 2-25
2 TesterStrainIntegrity : Escherichia coli
a Characteristic Numberof Spontaneous Revertants
icutlhturteheexvheihbiictleeduandcehrarsaeclteecrtiisvt`eiTcocondnuedmmiobtniesorntosrf.atseTpohtnehteaacrnceeeqopuutisarberlmeeevnerrtatnfagonertsftporeryrpWtpolPpaht2aenu,wshtheewnetpeslstaetre1dgsatlraoinng 40 revertants per plate.
5x
in Cultre Denis
oftester strain culturesTwoerdeemgornesattrerattehatnhaotraepqpuraolprtioat0e5nxum1b0erbsaocftebraicatepreiraamlreanpdl/atoerdh,atdheedaenssitty
CHV Study No.: 17073-0409
19
c00371
`CORNINGHazleton 0t.a5rxge1t0lebvaecltoefritauprebrimdilt.y demonstrated to produce cultures with a density greater than or equal to
4. Positive Control Values
a Positive Control Values in the Absenceof S9 Mix
m3-uftoalgdeinn,crtehaesmeeoavnervtahleuemeoafan pTvoaosliudteeiomvfoetnchsotenrtarvtoelehitfcholaretactorhneetsrtpeoeslcttefirovrsetttrheaasittnessrtwrsaetirrna.eincaepxahbilbeitoefdiadtelnetaisftyiang a
b.
Positive (59 Mix
Control Values Integrity)
in
the
Presence
of
S9
Mix
tpersotemrutstargaeinnion tihtse mpurtesaegnecneiocfftTohroem(dSse)9m,omtnihsextrmeaexthaeinbtihvtaaetldtuhaeoteflSet9ahsmteiapxo3sw-ifatosilvdceaicpnoacnrbtelraeosloeffoomrveetararbteohselpimezceitaningveavalue ofthe vehicle control for tha strain.
dAenmoancscterpattabeldebpootshittihveeicnotnetgrroiltyoinftthheepSre9smenicxeaonfdSt9hefaobarilistpyoecfiftihcesttreasitnerwsatsraeinvtaolduaettedecaasthaving mutagen.
2. Cytotoxicity A minimumofthree non-toxic doses were required to evaluate assay data.
B. Criteria For A Positive R
were
evaluated
Once the criteria as follows:
fora
valid
assay
had
been
met,
responses
observed
in
the
assay
1 TesterStrainsTA98TAL00.and WP2urA
2-fold increase in the mFeoanarrteevsetratratnitcslepteor bpleatceonosfiadterleeadstpoosniteiovfe,thiet sheadtetsoteprrostdruaciensaotvleerastthae rmeevaerntarnetvserptearntpslapteerhpaldatteoofbtehaeccaopmprpoapnriieadtebvyehaidcolesecornetsrpolo.nsTehtiosiinnccrreeaassiengincotnhceemnteraantinounsmobfetrhoef test article.
CHV Study No.: 17073-0-409
20
C00372
CORNING Hazleton
2 TesterStrainsTALS3SandTAL537
T3o-feoalndrienvcerretaasntesipnetrheplmaFteoeaornaftrtheeveset raaptrpatrincotlperpietoartpbeeavtceeohinocsfliedaecrloeenadtsrtpoolos.niteToihvfei,tshiitenshceardetaetssoetepirrnsottdhrueacimneseaotvleerastt ea.
Tevertants test article.
per
plate
had
to
be
accompanied
by
a
dose
response
to
increasing
concentraantipounsmboefrtoef
RECORDS TO BE MAINTAINED ftAholillslrosawtiuwnddgyaswtiu,lblmdiobscesuamireocnnhotiafvtteihdoeni,nfitrnheaeclosrrtedopsro,argtthee1f0patcrhioeltioStcpeooslon,sfaornC.dortnAhfitnfegirnHatalzrleeptoortn gInecn.e.rafotredataesaastregsuelyteoafr SSrlpaaozynlseeotlroe.nctntc.o,hafvoeratnheaadfdoitrieomneanltpieornieoddomaftetriimaelsorresteanitnet0d aisnttohreasghteeorfoaancgieelifytaeycaidrleiptseiiregsinooadft,eCtdohrbeSnyipno(ghn.esor
REFERENCES
Ames,
BwNit.h,tJh. eMScaClamnonn,elalnad/ME.amYmaamlaisaank-i.MicMreotshoomdes 31:347-364 (1975).
for detecting Mutagenicity
carcinogens and Test. Mutation
mutagens Reseach
Brusick,MfouDnt.Ja.e,tviaolVnu.aRtF.eisoSneiaomrftcmhho7en6,E:s1Hc.6h9eS-.r1i9cR0hois(ae1n9ck8orl0ia).nWzP,2V.anAd.WRPayZ,uarnd Rr.evSe.rsSteamffuotradt.ion assay.
Green, MMuHtaLt.ionanRdesWe.arJc.hMu3r8i:e3l-.32Mu(t1a97g6e)n. testing using 7p" reversion in Escherichia col,
Maron, DMuMta.tiaonndRBe.seAamrecsh.11R3e:v1i7s3e-d21M5et(h1o98d3s).for the Salmonella Mutagenicity Test. Vogel, HprJop,eratineds.D.JM..BiBooln.nCehre.m.Ac2et1y8l:o9m7i-t1h0i6na(1s9eo56f). coli: Partial purificationand some
CHV Study No.: 17073-0-409
21
00373
CORNING Hazleton
`pTrmheieccircpoiostncadotipetiicaoasnlfoloyfl(tluohsweisn:bgaacdkigsrsoeucntdinbgacmtiecriraolscloapwen)ifoervianlduiactaetdiobnosothfmcayctrootsocxoipciitcyalalnydatnedst article
CODE
1 2
DEFINITION
Normal
RSelidguhctelyd
CHARACTERISTICSOF BACKGROUNDLAWN
A healthy microcolony lawn. ivAenhcnirocetlaiesceecaoibnnltterhoetlhspiilnzanetioen.fgothfethmeicmrioccroolcoonlieosnycolmapwanraenddtaonthe
3
ReModduecreatdely
iAncmraeraskeedintthhiennsiinzgeoofftthhee mmiiccrrooccoolloonniyeslacwonmpaanrdeadnto the
vehicle control plat.
4
REexduuecmeedly
iAnncreexatsereimnethtehisninzienogfotfhtehmeimcircorcoolcoonlioensycloamwpnaarneddatno the
vehicle contol plate.
5
Absent
A complete lackofany microcolony lawn.
6
PrOebcsicpuirteadteby
eTvhaelubaatcekdgdruoeuntdo bmaicctreorsiaclopliacwnancda/nonromtabceroascccoupriatcetleyst
article precipitate.
bLEavlciltdoerewiniacnlegolapfrwenmc.aicpritoastceocpoidceetsottahreticcoledeprnecuimpbitearteusoendtohe epvlaaltueasties trheecocrodnedditbiyonadodfitthieonboafctkhgpeound
PF
PSrleicgihptitate
Nhootwiecveearb,lethmeapcrreocsipciotpaitce dporeecsipniottatienfolnuetnhceeplaautteo,mated
countingofthe plate.
mp PrMeocdiepriattaete
wTohueladmionutnertfoefremawictrhosacuotopmiactperdecciopuinttaitengo,ntthhuesprleaqtueiring the
plate 0 be hand counted.
hp
Heavy
Precipitate
`plTahteelmaragkeeasmtohuenrteoqfuimraecdrhoasncdopciocunptriecnigpiditfaftiecuoltn, the
pwErhxeiacicmpphiltheaat;de.4tmo pbewcoouulndteidndmiacantuealalpyladtueeobtosetrhveemdatrokheadvaemaonuenxtotrfemmealcyrorsecdoupciecd tbeasctkagrrioeulend lawn
CHV Study No.: 17073-0-409
2
00374
`CORNINGHazleton
SECTION IV. RESULTS AND CONCLUSIONS
CHV Study No. 17073-0-409
23 00375
RESULTS
CORNING Hazleton
A. Test ArticleHandling
dLioltut3i2o6n)prweapsaruesTdeh,detathsestttheseatrvtaierchtliiec,clleTe.-f6oA3rt4m2e1,d0aw0amcsgisproe,rrecmdoilao,trwlrheosioscmshowtleuamtsipoenrt.ahteuTmrhoee.stteDctoenicoennitzreadtewdatsepr e(CHV. solution in all succeeding dilutions prepared for the mutagenicity assay. article rem a
B. DoseRangefinding Study
tWhPe2duouserArainngebfoDitnohdsitehnsegtpsorteubsdeeytncecsoetnaeddnudicntaetbdhseeonmncutetoahgfetneSis9tciamtrityxical(seosnauesyipwnlegartteepesseteerlrdescottsreead)in.bsasTTeeAdn1o0n0tahnedresults of
2ta.hrteiTcphlreee,ssfeenodcmaetoa5r,w0ae0br0seetgn0ec6ne.oe6rf7at$ue9gd mpineirxExpalpsaeterev,iimwdeeenrntecet1e7s0t7e3d-aAn1d. tNheorceystulottsoxairceitpyrewsaesntoebdsdienorsvTeeasdbolifestie1sttahned
the numberofrevertants per plate.
d by a normal background lawn and no decrease in
C. MutagenicityAssay.
pdlaattaew+ersetagnednaerrdTahdteeevdimiaunttiaEogxnepnfeiorcriiemtaeycnhatsts1ra7ey0a7tr3emse-unBlt1t.sanfTdorhceTo-ndt6ar3toa4l2agrareroepurppers(eeTsnaetbneltdeeda4s)inmaeTnaadbnlaersseiv3nednaianvndts4.peTrhese
counts (Table 3).
idual plate
TtThhheuetparrgeeessnueilcntiscteoyfaatnshdseaay.dbosseTenhcreeaondfgoesSfe9isntmdeiisxnt.gedstwuedryew5e,r0e00u,se3,d3t3o0,se1l,e0c00f,i3ve33d,osaensdt1o0b0eptgespteerd pilnattheein both
ptIrhneeEsnxeupnmceebrieomrreoafnbrtsee1vn7ecr0et7a3on-ftBs9Ipe(mrTiapxbllaetse3wearned o4)b,sealrlvdeadtawiwtehraenayccofetphtaeblteesatenrdsnroatpaossiediitvheeriincrheeases in
All criteria foar valid study were met,
CHV Study No.: 17073-0409
2
G00376
CONCLUSIONS
`CORNING Hazleton
ciTnahdueicsraaeetseuplotthssaitto,ifuvtenhdieenrSatlhmeocnoenldliat-iEosncshoefrtihcihsiastcuodlyi,/3MMammCoarlpioarna-tiMoinc'rsotseostmearRtiecvlee,rsTe-6MutationAssay the presence or abscernecaesoefimnitcheronsuommbaelreonfzyremveesrtparnetpsapreerdpflratoemofAraoncyloofrtTMh.ientdeuscteerd3s4rta2rta,ilndisivederint(oh8te9r).in
CHV Study No.: 17073-0-409
25 00377
`CORNINGHazleton SECTION V. DATA TABLES
CHV Study No.: 17073-0-409
26 G00378
Tes anrIuE 0: xem: 0
VEMIGLE:
`CORNING Hazleton
ase 1
T-sa4z Dose mGEFIOIG stumy
1707301
DATE PLATED:
Detondzed vases
DATE COUNTED:
12-50-93 15-50-95
M00 REVERTNTS PER PLATE
vaspare i HTAoTnE mvaEcabanszon HTAiTrE mvaLboyszon
0.00ts(avsa1h3icie)
12
3
os
2
Tose Arochicte
103
)
o
:
10.0 ss
100
2
us
2
a
\
7"
1
or 100
120
:
12
)
105
\
os
+
533 or
138
107
2
ns
:
or
+
1000 3330
1s
:
wm
2
7
+
107
3
r _--s000 1r s 3 r 0 :
I
Cents tnt
i, IE Trtoo,n
3 = steraaty cuts
Diet wpim taspeovt
CHV Study No.: 17073-0-409
27
00379
CORNINGHazleton
mst ARezcu 1: [I
VENICLE: --_--
Tass 2
soz aavcsEDDING STUD
1-6342
--
DATE PLATED:
Detonized vases
DATE COUNTED:
12-Sep-95 15-Sep-35
VEsuve RevERTANTS PER PLATE varriaE Te e HTAnTE T maElobmamoe n HFanle vaEosn o
0.00bso(ventere)
16
Test Ar7stcle
un
10.0
1
3
u
0.7
M
100
2
sss
or
.
1000
u
5330
20
as000tr-- 1ecm
2
2
2
2
u
2
3
1
+
2
u
2
:
s
2
+
:
A
:
1s
:
:
1
2
2
n
s
e : e1ee re c3 en
P [VEivi egoCatn ton tlbS,oioagpyI seers I3 = Si,
k sotaraasely trteducese tts
CHVStudy No.: 17073-0-409
28
00350
TEST ARTICLE ID: T-6342 EXPERIMENT ID: 17073-B1 DATE PLATED: 19-Sep-95 DATE COUNTED: 22-Sep-95
MUTAGENICITTYABALSESA3Y INDIVIDUAL PLATE
CRoEuSnUtLsTS
CORNINGHazleton
VEHICLE: Deionized water PLATING ALIQUOT: 50 yi
CKOSNES nat Liver
vena rex rar
ncroron
hwissas rzuven
222aaamnnittnrnooeiaannnttthhhereaaacccuaennnees
1133 23.0
LLoa/sppiieaee Ja/piace
mhsa Vrach
Ldookiennlias denttsouinelinech-oxide
r12.0g6 ebpevu/roppinleeesss
+
BaTckegrrounad Lam Bvaluacion = Gtrianty codvens
Coders = 52
sttaoery
catuens
TOM preisitite ap Setarase peeeipi
3 == Sshoetcaurraacdelbyyspaetuceinpdisace
Cenguires hand' cocuanste) 2 = h(eEavaygaprrechiapnidcanret)
CHV Study No.: 17073-0-409
29
00351
`CORNINGHazleton
TEST ARTICLE ID: EXPERIMENT ID: DATE PLATED: DATE COUNTED:
T-6342 17073-B1 19-Sep-95 22-Sep-95
MUTAGENICITTYABALSESA4Y SUKRIARY
RESULTS
VEHICLE: Deionized vater PLATING ALIQUOT: 50 yl
mw
Iitineiriene 35 purist
Thise awit nite
Bysusie
4T== neexrceraalmaly codueed "SHON precipitate CHV Study No.: 17073-0-409
53= daleiagrnely reduces wp = m(acdaegruaitceesphraencdipsiotuantde)
3 2
===
homeobadevecyruaatpderlaycbtypripetrdaauccceiepditate
(eaqutren hand cone)
30 00382
