Document DvGRqjege7aZRmgKnqE9wGmG5
ARI26-0232
MUTAGENICITY TEST WITH
T-6357
CORNINGHazleton SLELRN
flan 5p
-
hh
Sol
IN THE SALMONELLA - ESCHERICHIA COLIMAMMALIAN-MICROSOME REVERSE MUTATION ASSAY
FINAL REPORT
AUTHOR
Timothy E. Lawlor, MAA.
PERFORMINGLABORATORY
Corning Hazleton Inc. (CHV) 9200 Leesburg Pike
Vienna, Virginia 22182
LABORATORYPROJECTID
CHV Study No.: 17387-0-409
SUBMITTETDO Building 220-32EM-02 3M Center
St. Paul, MN 55144-1000
SCTUODYMPLETIDOATNE April 1,199
CHV Study No.: 17387-0409
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QUALITY ASSURANCE STATEMENT
STUDY TITLE:
Salmonella - Escherichia coli/Mammalian-Microsome Reverse Mutation Assay
ASSAY NO.:
17387-0-409
PROTOCOL NO.: 409, Editio4n
`Quality Assurance inspectionsofthe study and reviewofthe final reportofthe above referenced
project were conducted according to the Standard Operating Proceduresofthe Quality Assurance
Unit and accorditnog the general requirementsofthe appropriate Good Laboratory Practice
regulations. Findings from the inspections and final report reviewwerereportedto management
and to the study director on the following dates:
Characterization of `Tester Strains - 02/14/96
Draft Report Review - 03/16/96
Final Report Review - 04/01/96
02/14/96 03/18/96
04/01/96
C. Orantes S. Ballenger S. Ballenger
wu Jbl,
Quality Assurance Unit
CHV Study No.: 17387-0-409
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#/ fre
Date Released
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STUDY COMPLIANCE AND CERTIFICATION
`The study was conducted in compliance with the Good Laboratory Practice regulations as set
forth by the Food and Drug Administration (FDA)inTitle 21of theU.S. Code of Federal Regulations Part 58, issued December 22, 1978, (effective June 20, 1979) with any applicable
`amendments. There were no deviations from the aforementioned regulations or the signed
protocol that wouldaffectthe integrity ofthe study or the interpretationofthe test results. The
raw data have been reviewed by the Study Director, who certifiesthatthe evaluationofthe test
article as presented herein represents an appropriate conclusion within the contextofthe study
design and evaluation criteria.
Study Director:
sl Timothy E. Jawlor, M.A. Bacterial Mitagenesis Genetic and Cellular Toxicology
4196 Study Completion Date
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TABLE OF CONTENTS
`CORNINGHazleton Page No.
IL STUDYINFORMATION..........oiiniiiniiiiiiiiiniiiinennninn7n TL MATERIALS ANDMETHODS.........uuiiiniiiniiniiiianiaiiiinnn9. IV. RESULTS AND CONCLUSIONS.........ccouiiniinnniiniininnn.. 23 Vo DATATABLES ........cccuiiiieiineinnmiiiniiniiiieiiiieiiaenennn 26
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SECTION I. SUMMARY INTRODUCTION AND CONCLUSIONS
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SUMMARY A. Introduction At the request of 3M, Corning Hazleton Inc. investigated T-6357 for mutagenic
activity in the Salmonella-Escherichia colilMammalian-Microsome Reverse Mutation Assay. This assay evaluated the test article and/or its metabolites for their ability to induce reverse mutations at the histidine locus in the genomeof specific Salmonella typhimurium tester strains and atthetryptophan locus in an Escherichia coli tester strain both in the presence and absence ofan exogenous metabolic activation system ofmammalian microsomal enzymes derived from AroclorTM-induced rat liver (59). `Thedosestested in the mutagenicity assay were selected based onthe results of adose rangefinding study using tester strains TA100 and WP2uvrA and ten dosesoftest article ranging. from 5,000 10 6.67 pg per plate, one plateper dose,bothin the presence and absence of S9 mix. The tester strains usedinthe mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TAIS35, TAIS37, and Escherichia coli tester strain WP2uvrA. The assay was conducted with five dosesoftest articleinboth the presence and absence of S9 mix along with `concurrent vehicle and positive controls using three plates per dose. The doses tested were 5,000, 3,330, 1,000, 333, and 100 pgper plate in both the presence and absence of S9 mix.
B. Conclusions
`The resultsofthe Salmonella - Escherichia coliMammalian-Microsome Reverse Mutation Assay indicate that, under the conditionsofthis study, 3M's test article, T-6357, did not cause apositive increase in the numberof revertants per plateofanyofthe tester strains cither in the presence or absenceofmicrosomal enzymes prepared from AroclorTM-induced rat liver (39).
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`CORNINGHazleton SECTION IL. STUDY INFORMATION
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STUDY INFORMATION
A. Sponsor: 3M
B. Test Article: T-6357
1. Physical Description: clear amber liquid
2. Date Received: 01/16/96
C. TypeofAssay: Salmonella - Escherichia coli/Mammalian-Microsome Reverse Mutation Assay
1. Protocol Number: CHV Protocol 409, Edition 4
2. CHVStudy Number: 17387-0-409
D. Study Dates
1. Study Initiation Date:
01/3019
2. Experimental Start Date:
02/0496
3. Experimental Termination Date: 02/21/96
E. Study Supervisory Personnel
Study Director:
Timothy E. Lawlor, M.A.
Laboratory Supervisor: ~~ Michael S. Mecchi, B.S.
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SECTION III. MATERIALS AND METHODS
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MATERIALS AND METHODS `The experimental materials, methods and procedures are based on those described by Ames et al (1975) and Green and Muriel (1976).
MATERIALS
A. TesterStrains
1. Salmonella typhimurium
`auxotrophs TAS, TA1T0h0e, tTeAsLteSr3s5t,raainnsduTsAed15w3e7reasthdeeSsaclrmiobendelblyaAtmypehsimeturailu(m19h7i5s)t.idiTnhee specific genotypesofthese strains are shown in Table I
--_---- - TABLE" I.TESTERSTRAINGENOTYPE. S
hisGd6 hisC3076 hisD30S2
TAIS3S TAI1537
_TA--100
TA9
LPS Repair a wrB fa wrB
R Factor . +R
In addition to a mutation in the histidine operon, the tester strains contain two additional r`emsuutlattsioinnstwhehilcoshsoenfhoanneceoftthehier esennzsyitmievsityretsopsonosmieblmeuftoargtehneicsycnothmepsoiusnodfsp.arTthoeftrhfea wall mutation dleifpiocpioelnycsyacicnhcarreiadseesbaprerrimeeratbhialtiftyortmosctehrteasinurcflaacsesoefstofhcehebamcitcerailasl cseulclhwaalsl.thoTsheecroenstualitniinnggcellalrgweall ring systems (i.e. benzo(a)pyrene) that would otherwise be excluded by a normal intact cell wall.
`The second mutation, a deletionofthe uvrB gene, results in a deficient DNA excision repair dseylsetteimonwehxitcehndgsretahtlryouegnhhathnecebsiothgeensee,nsailtliovfittyhoeftthesetseer ssttrraaiinnssctoonstaoimneinmgutthaigsednesl.etSiionncaelstohere1quiBre: the vitamin biotin for growth.
tShterasiennssiTtAiv9i8tyoafntdhTeAse10s0traailnssotcoonstoamien tmhuetaRg-efnasc.torTphleasmmeicdh,apnKiMs1m0b1y,wwhhiicchhthfiusrtphlearsimnicdreases DinNcrAearseepsasiernpsoitliyvmietyratsoemuctoamgpelnesxhiansvobleveendswuigtghesthteedmitsombaetcbhy-rmeopdaiifryiprnogceasns.existing bacterial
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hTiessttiedrinsetrianindsepTeAndSenacned(pTrAo1to5t3r7opahrye)rbeyvefrrtaemdesfhriofmt hmiusttaigdiennes.deTpeAn1dSe3n5ceis(aruevxeorttreodphbyy)btaose ssuubbssttiittuuttiioonns.mutagens and TA100 is reverted by mutagens which cause both frameshifis and base
2. Escherichia coli
described
by
Green
`The tester strain and Muriel (1976).
used
was
the
tryptophan
auxotroph
WP2uvrA
as
dIenfaidcdiietnicoynwthoiachmuetnahtainocniesntithsesetnrsyipttivoipthyantoospeormoen,mutthaetgeestneircsctoramipnoucnondtsa.inTshaisuvderfAicDiNenAcyraelpaliorws the strain to show enhanced mutability since the uvrA repair system would normally act to remove the damaged partofthe DNA molecule and accurately repair it afterwards.
Testerstrain WP2uvrA is reverted from tryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) by base substitution mutagens.
3. Soou f Tesr terc Strae ins
a Salmonella typhimurium
Bruce
Ames,
Department
of
`The tester strains in use at CHV were received directly Biochemistry, UniversityofCalifornia, Berkeley.
from
Dr.
b. Escherichia coli
National Collection of Industri`aTlheBatcetseterrias,trTaoirn,reWyP2ReusveraAr,chinStuasteioant,CScHoVtlwanads(rUencietievdedKifnrgodmotmh)e.
4. Stoo fthr eTesa terg Strae ins
a. Frozen Permanent Stocks ovemight cultures, adding DMFSrOoz(e0n.p0e9rmmla/nmelnotfsctuolctkusrew)eraendprferpeaezriendgbsymaglrlowaliinqguoftrsesh (0.51.5 ml) at <- 70C.
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s. `PreopfaOvermiaghttCiultournes
a Inoculation Overnight cultures for use in al testing procedures were inoculated
by transferring a colony from the appropriate master plate to a flask containing culture medium. Inoculated flasks were placed in a shaker/incubator which was programmed to begin operation (shaking, 125 = 25 rpm; incubation, 37 2C) so thatthe overnight cultures were in log phase or late log phase when turbidity monitoring began.
b. Harvest To ensure that cultures were harvested in late log phase, the length
of incubatwiaosn determined by spectrophotometric monitoring of culture turbidity. Cultures were harvested once a predetermined turbidity was reached as determined by a percent transmittance (%T) reading on a spectrophotometer. This target turbidity ensures that cultures have reached a densityofat least 0.5 X 10 cells per ml andthatthe cultures have not overgrown. `Owveerergrreomwonve(sdtaftrioomnairny)cucbualttiuorneswmhaeny tehxehitbairtgedtec%rTeasweadssreenasicthievditayntdowseormeepmluatcaegdeants5. &Cu3ltCu.res
6. ConfoifTrestm erSa trat inGi enoo typn es
the dayoftheir use in tTheestmeurtastgreaniincciutlytuarsessayw:ere checked for the following genetic markers on a Salmonella typhimurium 1) rfa Wall Mutation
demonstrationofthe sensitivity ofthe`Tchueltpurreesteoncceryostfatlheviorlfeat.walAlnmaultiaqtuiootnofwaasncoovnefrinirgmhetd by
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2) pKMI01 Plasmid R-factor tester strains TA98 and TA100 by demTohnestprraetsieonncoeforfestihsetapnKcMe1t0o1amppilcaislmliind.waAsncaolnifqiuortmeodfafnor `anotviebrinoitgihct sceunlstiutrievoiftcy adicshk cstornatianiwnainsgov1e0rulagiodfaomnptiocpillaltiesncwoanstaaidndiendg.seRleescitsitvaenmceedwiaasand an demonstrated by bacterial growth in the zone immediately surrounding the disk.
3) Characteristic Numberof Spontaneous Revertants vehicle controls that are characteristic`oTfhtehmeearenspneucmtibveerostfrasipnsonwtearneeoduesmornesvterrtaatnetdsbpyerppllaattiengin the 100 pl aliquotsofthe culture along with the appropriate vehicle on selective media.
b. Escherichia coli 1) Characteristic Number ofSpontaneous Revertants
`vehicle controls that are characteristic`oThfetmheearenspneucmtibveerostfrasipnosnwtearneeoduesmornesvterrtaatnetds bpyerppllaattiengin the 100 ul aliquotsofthe WP2uvrA culture along with the appropriate vehicle on selective media.
7. TesterStrainMedia
a Culturing Broth Vogel-Bonner salt solution (Vo`TgheelbraontdhBuosnendert,o g19r5o6w)osvueprpnliegmhtenctueltduwreistohft2.h5e%t(eswt/evr)sOtrxaoinisdwas Nutrient Broth No. 2 (dry powder).
b. AgarPlates Bonner minimal medium E (VBogoetltoamndagBaornn(2e5r,ml19p5e6r),1s5uxpp1l0e0memnmtepdetwriithdis1h.)5w%a(sw/Vvo)gealga-r and 0.2% (Wh) glucose.
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c. Overlay Agar for Selectionof Revertants
Overlay (top) agar was prepared with 0.7% agar (w/v) and 0.5% NaCl (w/v) and was supplemented with 10 ml of 1) 0.5 mM histidine/biotin Solution per 100 ml agar for selectionofhistidine revertants, or 2) 0.5 mM tryptophan solution per 100 ml of agar for selection oftryptophan revertants. When S9 mix was required, 2.0 mlofthe supplemented top agar was used in the overlay. However, when S9 mix was not required, water `was added to the supplemented top agar (0.5 mlofwater per 2 ml of supplemented top agar) and the resulting 2.5 mlofdillted supplemented top agar was used for the overlay. This dilution ensured that the final top agar and amino acid supplement concentrations remained the same both in the presence and absence of S9 mix.
B LivrMi
E Reaction Mi
"
1. SH9omogenate
Liver microsomal enzymes (9 homogenate) were purchased from Molecular Toxicology, Inc., Annapolis, MD 21401, Batch 0623 (42.4 mgofprotein per ml). `The homogenate was prepared from male Sprague-Dawley rats that had been injected (i.p.) with AroclorTM 1254 (200 mg per ml in com oil) at S00 mg/kgasdescribed by Ames ef al, 1975.
2 59Mix
`The S9 mix was prepared immediately prior to its use in any experimental procedure. The S9 mix contained the components indicated in Table I1.
HO TABLE II. $9MIXCOMPONENTS070ml
IM NaH,PO/Na;HPO,, pH7.4
010ml
0.25M Glucose-6-phosphate
0.02ml
0.10M NADP
0.04ml
0.825M KCl/0.2M MgCl,
0.04ml
$9 Homogenate
010ml
------------------------------1--.00 --ml
C. Controls
1. VehicleControls
absence of $9 mix.
Vehicle controls were plated for all tester strains both in the presence and The vehicle control was plated, using a 50 pl aliquotofvehicle (equal to the
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`CORNING Hazleton
maximum aliquotoftest article dilution plated), along with a 100 pl aliquotofthe appropriate.
tester strain and a 500 pl aliquot of S9 mix (when necessary), on selective agar. 2. PositiveControls
`The combinationsofpositive controls, activation condition and tester strains plated concurrentlywiththe assay are indicated in Table Ill
Tester TABLEIIL POSITIVE CONTROLS Cone
TStAai%n S9+Mix TA98 TAI0 + TAI0 TAISIS + TAS} TAIS)? + TAS}? WP2orA + WP2wrA
Po2s-aimtiinvoeaCnotnhtrraoclene pe2rp5lpagte
Z2nitrofluorene
10g
2-aminoanthracene
25pg
sodium azide
20ug
2-aminoanthracene
25ug
sodium azide
204g
2-aminoanthracene
25g
ICR-191
20 ug
2-aminoanthracene 250g
4-nitroguinoline-N-oxide 1.0 pg.
a Source and GradeofPositive Control Articles
2-aminoanthracene (CAS #613-13-8), Sigma Chemical Co., purity > 97.5%; 2-nitrofluorene (CAS #607-57-8), Aldrich Chemical Co., purity 98%; sodium azide P(oClAySsci#e2n6c6e2s8-I2n2c-.8)p,urSitiygm>a95C%h;em4i-cnailtrCooq.,uinpoulriintey->N9-o8x%i;deI(CRC-A1S91#5(6-C5A7S-5#)1,7S07i-g4m5a-0C)h,emical Co., purity >99%.
3. SterilityControls
a Test Anicle by plating a 50 jl aliquot (the`sTahmeemovsotlucomneceunsterdatinedthteesatsasratyi)cloendisleulteicotnivweaasgacrh.ecked for sterility
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bo S9Mix
agar.
`The S9 mix was checked for sterility by plating 0.5 mi on selective
METHODS A. DRosaengefindSitnudgy
determined in o`rTdheertgoroawltlhowinthhiebisteolreycetfifoenoctfa(cpyptrootporxiicaittey)doosfetshetotebset taretsitceldeitnotthheemtuesttagseynsitceimtywas assay.
1. Design
and WP2usrA both in `tThheepdroesseenrcaengaenfdinadbisnegncsetuodfywSa9smipxe.rfToernmeddosuessionfgtteessttearrsttircaleinwseTrAe1t0es0ted
at one plateperdose. concentration of 5 me
The per
ptleasttea.rticlewas
checked
for
cytotoxicity
up
to
amaximum
a Rationale
is generally representativeofth"TahteocbysteortovxeidciotnyothfetohteheterstteasrtteirctleraoibnsseravneddboencatuessteerofstTrAaiLn0T0A's100 rceoamdpialryadtiisvceelrynhedigfhrnoummrboeutroinfe esxppoenrtiamneenotuaslrveavreirattainotns. pTerheplEastec,hegrriacdhaitiaocnosliofesctyetortsotxriaciinty can be ``aWnPd2tuhuvsr,Aaddiofefserneonttproasnsgeesosftchyetroftaoxwiacliltmyumtaatyiobne tohbastertvheedS.alAmlosnoe,llthaetcyypthoitmoxuirciiutmy istnrdauicnsedhabvyea mteisxt.arTtihcelreefinorteh,etphriessweonucelodfreSq9uimriexthmatadyifvfaerryengtretaesttlyarftricolmetdhoasteorbasnegrevsedbeinttehseteadbsinenthcee of S9 mutagenicity assay based on the presence or absenceofthe S9 mix.
2. EvaluationoftheDoseRangefindingStudy
per plate and/or by a thCiyntnoitnogxiocridtiysiaspdpeetaercatnacbeloefatshaedbeaccrteearsiaelibnatchkegnruomubnderlaowfnr.evertant colonies
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3. SelectionoftheMaximumDosefor theMutagenicityAssay
a No Cytotoxicity Observed the highest doseoftest articleSuisnecdeinnothceytmouttoaxgiecintiyciwtaysasosbasyerwvaesditnhtehseadmoesaesrtahnagtetfeisntdeidnignstthuedy, rangefinding study.
B. MutagenicityAssay
1. Design
`The assay was performed using tester strains TA98, TA100, TAIS35, `TA1537 and WP2uvrA both in the presence and absence of S9 mix. Five dosesoftest article were tested along with the appropriate vehicle and positive controls. The dosesof test article were selected based on the resultsofthe dose rangefinding study.
5
--
`The tester strains were exposed to the test articleviathe plate incorporation methodology originally described by Ames ef al (1975) and Maron and Ames (1983). This methodology has been shown to detect a wide rangeofclassesofchemical ``mmiuxta(gwehnes.reIanpptrhoepprliaattee)inwceorrepocroamtiboinnemdetihnodmoollotgeyn,atghaertwehsticarhtiwclaes, othveertleasitderosnttroaianmainnditmhaelSa9gar plate. Following incubation at 37 + 2C for 48 + 8 hr, revertant colonies were counted. All dosesofthe test article, the vehicle controls and the positive controls were plated in triplicate.
C. PlatingProcedures
`mutagenicity
`These assay.
procedures
were
used
in
both
the
dose
rangefinding
studyand
the
Eacatcihvaptliaotnecwoansdiltaiboenlaenddwidtohsea.cTodhee w$h9imcihxidaenndtidfiileudttihoensotefsttharetitcelset, taerstticplheawseer,etepsrteerpasrteradin, immediately prior to their use.
`When S9 mix was not required, 100 uloftester strain and 50 pl of vehicle or test article dose r`weaqsuiardedd,ed50t0o 2p.l5omfl$o9fmmioxl,te1n00seullecotfitveestteorpsatgraairn(amnaidn5t0aipnleodfavte4hi5c2leCor).testWahretinclSe 9domsiexwwaass added to 2.0 ml ofmolten selective top agar. After the required components had been added, the
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`mixture was vortexed and overlaid onto the surfaceof25 mlof minimal bottom agar contained in 215 x 100 mm petri dish. After the overlay had solidified, the plates were inverted and incubated for 48 & hr at 37 2C. Positive control articleswereplated usinag 50 pl plating aliquot.
D. ScotrheiPlnatges
Plates which were not evaluated immediately following the incubation period
were held at 5 + evaluation could
3C take
until such place.
time
that
colony
counting
and
bacterial
background
lawn
1. BaB ctea rialckgrLao wnu Evaln uatd ion
`The conditionofthe bacterial background lawn was evaluated for
1e0vitdheenvceehoifcclyetocotnotxriocliptlyataendantdeswtaarstircelceoprrdeecdipaitlaotneg. wEivtihdtehnecreoevferctyatnottocxoiucnittsyfworasallscpolraetdesraetlatthiavte
dose on Section.
the
data
tables
using
the
code
system
presented
at
the
endofthe
Materials
and
Methods
2 ComtingR
Coloni
`The numberofrevertant colonies per plate for the vehicle controls and all plates containing test article were counted manually. The numberofrevertant colonies per plate for the positive controls were counted by automated colony counter.
E. AnalysisofData `were calculatedF.orThalel rreespullitcsaotfetphleatsiengcsa,lctuhleamtieoanns arreeveprrteasnetnstpeedripnlattaebualnadr fthoermstianntdhaerdDadetvaiation `Tables Sectionofthis report.
EVALUATION OF TEST RESULTS Before assay data were evaluated, the criteriafor a valid assay had tobemet.
A Criteria For A Vali
The following criteria were used to determine avalid assay:
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1. TesterStrainIntegrity : Salmonella typhimurium
a rfa Wall Mutation
cultures
exhibited
sensitivity
`To demonstrate to crystal violet.
the
presenceofthe
rfa
wall
mutation,
tester
strain
b. pKMIOI Plasmid
culturesoftester strains TA98TaonddeTmAo1ns0t0raetxehitbhietpedrerseesnicsetoanfctehteo Ra-mfpiaccitlolrinp.lasmid, pKM101,
. Characteristic Number of Spontaneous Revertants
cultures exhibited a characteris`tTiocdneummonbsetrroaftespthoentraenqeuoiursemreenvterftoarnthsispteirdipnlea,ttehwehteesnteprlsattreadinalong
with the vehicle as follows:
under selective conditions.
The
acceptable
ranges
for
the
vehicle
controls
were
TA9
8- 60
TAL00 60 - 240
TAIS3S 4-45
TAI537 2-25
2. Tester Strain Integrity : Escherichia coli
a. Characteristic Numberof Spontaneous Revertants
culture exhibited a characterist`iTco nduemmobnesrtorfastpeotnhtearneeqouuisrermeevnetrtfaonrtstrpyeprtpolpahtaen,whtheentpesltaetredstarlaoinng.
with the controls
vehicle under selective conditions. was $ to 40 revertants per plate.
The
acceptable
range
for
the
WP2uvrA
vehicle
3. TesterStrainCultureDensity oftester strain cultures`wToerdeemgornesattrearttehatnhoatraepqpuraolprtioat0e.5nuxm1b0erbsaocftebraicatpeerriamalreanpdl/aoterdh,atdheredaecnhsietdy 0a.t5arxge1t0lebvaecltoefritaurpbeirdimtly. demonstrated to produce cultures with a density greater than or equal to
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4. PositiveControlValues a Positive Control Values in the Absence of S9 Mix
mutagen, the mean valueof a pToosidteimvoencsotnrtartole tfhoartatrheestpeesctteirvsetrtaeisntserwsetrreaincaepxahbilbeiotefdiadtenletaisftyiang a 3-fold increase over the mean value of the vehicle controlforthat strain.
b. Positive Control Values in the Presence of $9 Mix (59 Mix Integrity) To demonstrate that the S9 mix was capable of metabolizing a
tpersotmerutstargaeinnitno tihtes mpurteasgenecneiocftfohrem(Ss)9,mtihexmeexhainbivtaeldueaot fletahsteapo3s-iftoilvdeicnocnrteraoslefoovrearrtehsepemcetainvevalue ofthe vehicle control for that strain. dAenmoancscterptaatbeldebpootshittihveeicnotnetgrroiltyoifntthheepSr9esmenicxeaondf$9thefoarbialistpyeocfiftihcesttersatienrwsatsraeivnatlouadetteedctasahaving mutagen.
5. Cutotoxicity A minimumofthree non-toxic doses were required to evaluate assay data.
B. Criteria For A Pesitive R
were
evaluated
Once the criteria as follows
for
a
valid
assay
had
been
met,
responses
observed
in
the
assay
1 TesterStrains TA98 TA100,andWP2uvrA
For atest article to be considered positive, it had to produce at least a 2m-efaonldrienvcerretaasnetsinpetrheplmateeaonftrheveeratpapnrtosppreiartpelavteehoifclaetcloenatsrtolo.neThoifsthiensceretaesseteirnstthreaimnseoavnernutmheber of revertants per plate had to be accompanied by a dose response to increasing concentrationsofthe test article.
CHV Study No.: 17387-0409
20
C0592
CORNING Hazleton
2. TesterStrainsTA1S35and TA1537
For atest article to be considered positive, it had to produce at leasat
m3-efaonldrienvcerretaasntespinetrhpelamteeaonftrheveeratpapnrtosppreiraptleavteehiocfleatcloenatsrtolo.neTohfitsheinsceretaesseteirnstthreaimnseaovnenrutmheber of
revertants test article.
per
plate
had
to
be
accompanied
by
a
dose
response
to
increasing
concenirationsofthe
RECORDS TO BE MAINTAINED
All raw data, documentation, records, the protocol, and the final report generated as aresult of
tfhoilsloswtiundgy wsiulblmibsesairocnohifvtedheinfitnhaelsrteoproargtetfoactihleitSipesonosfoCro.rnAiftnegrHtahzeloetnoenyIenacr.pfeorrioatd,letahsetSopnoenyseoarr
`may elect to have the aforementioned materials retained in the storage facilities of Corning
Hazleton Sponsor.
Inc.
for
an
additional
period
of
time
or
sent
to
a
storage
facility
designated
by
the
REFERENCES
Ames, BN., J. McCann, and E. Yamasaki. Methods for detecting carcinogens and mutagens `with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Research 31:347-364 (1975).
Brusick, DJ, V. F. Simmon, H. S. Rosenkranz, V. A. Ray, and R. S. Stafford.
An evaluationofthe Escherichia coli WP2 Mutation Research 76:169-190 (1980).
and
WP2uvrA
reverse
mutation
assay.
Green, MH.L. and W. J. Muriel. Mutagen testing using rp" reversion in Escherichia coli. Mutation Research 38:3-32 (1976).
Maron, D.M., and B. Ames. Revised Methods for the Salmonella Mutagenicity Test. Mutation Research 113:173-215 (1983).
Vogel,
HJ, and D.M. Bonner. AcetylomithinaseofE. properties. J. Biol. Chem. 218:97-106 (1956).
coli:
Partial purification and some
CHV Study No.: 17387-0409
21
C00593
CORNING Hazleton
BB ACTEA RIALCKGLR AWNO EVALU UATIN ON CD ODE ``Tmhiecrcoosncdoiptiicoanlolyft(uhseinbgacakdgirsosuecntdinbgacmtiecriraolscloapwen)ifsoervianlduiactaetdiobnostohfcmyatcortoosxciocpiitcyalalnydatnedst article precipitate as follows:
CODEDEFINITION 1 Nomal
CHARACTOEF BRACIKGSROTUNID LCAWSN A healthy microcolony lawn.
2 ReSldiughcteldy
Aincnroetaisceeaibnltehtehsiinzneionfgtofhtehmeicmrioccroolcoonlioesnycolamwpnaraenddtaonthe vehicle: control plate.
3 ReModduecreadtely
Aincmraeraskeedintthhiennsiinzgeoofftthhee mmiiccrrooccoolloonniyeslacwonmpaanrdeadnto the vehicle control plate.
4 REexdturceemdely
Ainncreexastereimnethtehisninzienogofftthheemimcircorcoocloolnioensy cloamwpnaarneddatno the vehicle control plate.
5 Absent
A complete lack ofany microcolony lawn.
6 PrOebcsicpuitraetdeby
Tevhaelubaatcekdgdruoeuntdo bmaicctreorsiaclopliacwnancda/nonromtabceroascccoupraitcelteyst article precipitate.
Efovlildoewnicnegopfrmecaicpriotastceocpoidcetetsottahreticcoledeprnecuimpbitearteusoendtthoeepvlaaltuesaties trheecocrodnedditbiyonaodfdtithieonboafctkhgeround bacterial lawn.
sp PrSelcigihptitate
Nhootwiecveearb,lethmeapcrreocsicpiotpaitce pdroeecsipniottatienfolnuetnhceeplaautteo,mated countingofthe plate.
mp PrMeocdiepriatattee
``Twhoeuladmionutnertfeorfemwaictrhoasuctoopmiactperdecciopuinttatiengo,ntthhuesprleaqtueiring the plate to be hand counted.
hp PrHeecaivpyitate pTlhaetelmaragkeeasmtohuenrteoqfuimraecdrhoasncdopciocunptriecnigpidtifaftiecuolnt.the
wExhaimcphlhea:d4tmopbewcoouulndteinddmicaantueaallpyladtueeobtosetrhveemdatrokheadvaemaonuenxttorfemmealcyrorsedcuocpeidc bteasctkagrrtioculned lawn precipitate.
CHV Study No.: 17387-0-409
2
00594
`CORNING Hazleton
SECTION IV. RESULTS AND CONCLUSIONS
CHYV Study No.: 17387-0-409
23
C00595
CORNINGHazleton
RESULTS
A. TestArticleHandling
lots
336
and
The 337)
test article, T-6357, was was used as the vehicle.
stored at room temperature. Deionized At 100mgper ml, which was the most
water (CHV concentrated
rsteomcakindeildutaisoonlpurteipoanriend,altlhseutcecseteadritincglediflourtmioendsapcrleepaarrecdolfoorrletshse smoultuatgieonn.iciTthyeatsessatya.rticle
B. Dose RangefindingStudy
thedose
Doses to rangefinding
be tested inthe mutagenicity assay were study conducted onthetest article using
selected based on the results tester strains TA100 and
of
aWrtPic2leu,rfAroimn5b,o0t0h0tthoe6p.r6e7seingcpeearnpdlaatbe,sewnceereofteSs9temdainxd(tohnee prleastuletpsearrdeosper)e.senTteend dinosTeasbolfeste1stand
t2.heTphreesseendcaetaorwaebrseegnecneeroatf$e9d minixExapseerviimdeenntce1d73b8y7-aA1n.ormNaol bcyatcoktgorxoicuintdylwaawsnoabnsdernvoeddeicnreeiatsheerin
the numberofrevertants per plate.
C. MutagenicityAssay
data were gen`eTrhaetemdutiangeEnxipceirtiymeanssta1y7r3e8s7ul-tBs1f.orTTh-e6d3a5t7aaarree pprreesseenntteeddiansTmaebalensr3evaenrdta4n.sTpheerse cploautnets (sTtaabnldear3d),deviation for each treatment and control group (Table 4) and as individual plate
m`Tuhteagreesnuilctistoyftahsseayd.osTehreandgoesfeisntdeisntgedstwuedryew5e,r0e00u,se3,d3t3o0,se1l,e0c0t0f,iv3e3d3o,saensdto10b0e
tested pg per
in the plate in
both
the presence and absenceof S9 mix.
tIhneEnxupmerbiemreonftre1v7e3r8t7a-nBts1p(eTrabpllaetse3wearned o4)b,saelrlvdeadtawiwtehraenaycocfetphtaeblteesatenrdsntroaipnossietiitvheerinincrtehaeses in presence or absenceof S9 mix.
All criteria for a valid study were met.
CHV Study No.: 17387-0409
2
00596
`CORNING Hazleton CONCLUSIONS A`TshsearyesuilntdsiocfattehtehaSta,lumnodneerlltahe-cEosncdhietriiocnhsoiaftchoilsi/sMtuadmym,a3lMi'asn-tMesitcarrotiscolme,eTR-e6v3e5r7s,edMiudtnaottiocnause a pporseisteinvceeionrcraebasseenicnetohfemniucmrboesromoaflreenvzerytmaenstsppreerpaprleatdeofrfoamnyAorofctlhoerTMte-sitnerdusctreadinrsateiltihveerr i(nS9t)h.e
CHV Study No.: 17387-0409
25
00597
`CORNINGHazleton SECTION V. DATA TABLES
CHV Study No.: 17387-0-409
26
COCH98
`CORNING Hazleton
is 1
PT --
Test ARTIOLE TD: T-6357
Exes 1730471
DATE PLATED: 04-Fab-36
VEHICLE: Detontzed vacer
DATE count: 07-7ab-96
--------------------------------
TAL00 REVERTAVES PER PLATE
variate SRERVEaRTCtTansGRsOaLolRnooDn AREVTEARTTTiens yomLREaaRnoonn
(50 p1)
Sout daoriegin
97
1
1s
2
102
1
as
1
10.0
o
2
us
1
53.3
12
s
o
3
66.7
108
'
a
+
100
108
2
os
1
533
"
1
102
1
or
12s
)
06
2
1000
156
:
0
:
3330
1a
1
10
:
e s000 m 155 r 1 e 10s 1
"quant so se iy
ementrs srotae--nt -- To Ai as psr istts 0 totetvae377rmpereenettsette
CHV Study No.: 17387-0-409
27
00599
`CORNINGHazleton
mos 2
DosE RANGEFTOTNG STUDY
Test ARTIS 1D: 7-357
[Er ----
DATE PLATED: 04-Fab-56
------VE-- NICLB--: -- Deton-- ized -- vater----------DAT-- E co-- ur:--07---eb---96 --
Vesuve RaVERTANS PER PLATE
papas E soitne a svAELbnAmIOlN e sTaetne saEtaan
.00Ga(vsehh)icle
1
3
u
3
Test Arotiecle
1
'
2
+
10.0
26
1
1
'
3.3
n
'
1s
1
c6.7
n
)
1s
3
100
1
2
2
:
333
2
:
2
1
cr
n
:
is
3
1000
n
\
n
3
3330
1
2
1
3
e s000 r 1 : r ws :
Tr Stn Sas nity soto
Jotun T--N--N --
3 = mederacely reduced
TES
a
CHV Study No.: 17387-0-409
28
00600
CORNINGHazleton
TEST ARTICLE ID: EXPERIMENT ID: DATE PLATED: DATE COUNTED:
T-6357 17387-B1 14-Fab-96 21-Feb-96
MUTAGENICITTYABALSESA3Y INDIVIDUAL PLATE
CROEUSNUTLSTS
VEHICLE: Deionized water PLATING ALIQUOT: 50 yl
-- comes
mee
awe
wasn
muon woe
----------------eeeeeeee
TAIOO TASS
2-Iu-saisntonmonmcchheraacceennes
23..53 ppaa//ppllaattee
eee
TTAAIOLOS ssooddiiuumm aasritddee
22..00 ppalpalatcee
*
Ba+ c=kgrnoournmdalLawn
Evaluation
Codest 2=
altanely
reduced
= alighe precipitate wp = m(ordaeqruaitzeasprhaencdipciotuantte)
3 = moderated reduced Pe = h(eeaovqyutrpernechtaincdacceoune)
CHV Study No.: 17387-0-409
29
0050
CORNINGHazleton
MUTAGENICITTYABLAESSA4Y RESULTS SUMMARY
TEST ARTICLE ID: T-6357
EXPERIMENT ID: 17387-B1
DATE PLATED: 14-Feb-96
VEHICLE: Deionized vater
DATE COUNTED: 21-Feb-96
PLATING ALIQUOT: 30 pl
mum me
ie mon mw wen
_----
W41030335 a3snmiimnoossnntthhrraacceanne 13..53 ppaallppaattee "THB125u3v7eh 23aanniimmoossnnthcrharcaannae 352..03 paallpptieantee
TTHhI6s0 saaottitoonn assuiidtee
2+.o0 fwapripeieseee
fWPaeuvek benteeoquinobine Nooxtda 21..00 w1a/rpriaeceee
A[=pxtcreeney ceded or = lhe peecipiente
Tpe ayttr cats up = sCoedaeurarteesprhaacdtpistoamsse)
5 == nCohdeuccuarteadlbyycpadrveccaidpice [SstoGnosnpeobseihvaindincyoun)
CHV Study No.: 17387-0-409
30
C0502