Document byb8ggg5KpOvRxDgOVBGeOQ23
STUDY CODE: KOI-1802
ReceiptNo. T96-2481 Report No. T-4637
FINAL REPORT
BACTERLAL REVERSE MUTANON OF
TEST
September, 1996
letaResearchLaboratories CherticalBiotestinCgenter ChemicalsInspection& TestingInstitute
Japan
14 07
OUALITY ASSURANCE STATENM'NT
Hft Research LaboratoriesC,henucal Biotatmg Center Chmucals Inspection& Testmg b=tate, Japan
SR=or:
SUMITOMO 3M LIMHED-
Title:
Bacteriar]eversemutationtestof r-1
Study c4Dde: KOI-1802
I'hisreportwas auditedby the QualityAssurance Sectim. 1, the undersignedh,ereby declarethatthisreportreflects the originalJapanesereport.
(Date) jo SectionCluef,QualityAssurance (Signature)
r7
KeijiShiraishiB,.S.
KOI-1802
L theundersignedh,erebydeclarethattMs reportprovides a correctEnglishtranslatioofntheFinalReport. (StudycodeNo. KOI-1802 issuedon September25, 1996)
(date)
7, IFY 7
(sign re) J-43& t/
&4v@@
Shozo Ogura
letaResearchLaboratories
ChemicalBiotestinCgenter
ChemicalsInspectio&n
TestingInstitutJea,pan
KOI-1802
GLP STATEN&-NT
IfitaResearchLaboratoriesC,hemicalBiotestingCenter ChemicalsInspection& TestingInstitutJea,pan
S12onsor: Title: StudyCode No.:
SUMITOMO 3M LMTED Bacteriarleversemutationtestof 'r-1 K01-1802
I,the undersigned,hereby declarethatthisstudywas conducted in complianceswith "Standardsto be observedby TestingInstitutiofnosrToxicityInvestigation(sJ"apa:Ws MOL, No.76, September1, 1988).
Management:
Signed inorisdnal SifigetakYaamane, Ph. D.
September 25, 1996
GLP STATENM'NT
IfitRaesearchLaboratoriesC,hemicalBiotestingCenter ChemicalsInspection& TestingInstitutJea,pan
Sponsor: Title: Study Code No.:
SUMITOMO 3M LDAITED Bacteriarleversemutationtestof r-l KOI-1802
I,the undersignedh,ereby declarethatthisstudywas conducted in complianceswith "Standardsto be observedby TestingInstitutiofnosrToxicityInvestigations("Japan!s MOL, No.76, September1,1988).
Thiswas reissuedbecauseofthe finalreportamendment.
Management:
Signedinoriginal ShigetakaYamane, Ph. D.
November 27, 1996
KOI-1802
QILALITY ASSURANCE STATEMENT
FfitRaesearchLaboratoriesC,hemicalBiotestingCenter ChemicalsInspection& TestingInstitutJea,pan
SRonsor: Title: Study Code No.:
SUMITOMO 3M LDAITED Bacteriarleversemutationtestof 'r-I KOI-1802
Thisstudywas auditedby the QualityAssurance Sectionand the studyprocedureswere inspectedon thefollowingdates.
Dates ofInspections
Dates ofReportsto
Dates ofReportsto
and Audits
Study Director
Management
August 15, 1996
August 15,1996
August 15,1996
September 20, 1996
September 24, 1996
September 24, 1996
September 25, 1996
September 25, 1996
September 25, 1996
L the undersignedh,erebydeclarethatthisreportprovidesan accuratedescripfioonf the
methods and proceduresused inthisstudyand thatthe reportedresultsaccuratelyreflect
theraw dataobtained.
SectionChiet QualityAssurance:
Signedinoriginal KeijiShiraishBi.,S.
September 25, 1996
OUALITY ASSURANCE STATEMENT
I-IitRaesearchLaboratoriesC,hemicalBiotestingCenter ChemicalsInspection& TestingInstitutJea,pan
S12onsor: Title: Study Code No.:
SLTNffTOMO 3M LRvtnED Bacteriarleversemutationtestof T'-l KOI-1802
Thisstudywas auditedby theQualityAssuranceSectionon thefollowingdates.
Auditor Kumiko Matsui
Date ofAudit November 27, 1996
Date of Reportto Study Director November 27, 1996
Date ofReport to Management November 27, 1996
This statementwas added to the QualityAssurance Statementissuedon September 25, 1996.
SectionChief,QualityAssurance:
Sip-nedinoriainal KeijiShiraisKB.S.
November 27, 1996
KOI-1802
Study code:
K01-1802
Test substancecode: BR3272
Sponsor code:
S-030
TITLE Bacteriarleversemutationtestof 'r-I
SPONSOR SUMITOMO 3M LRvffIED 8-8,Mnanii-Hashimoto 3-chome Sagamihara-shiK,anagawa, 229 Japan
TESTINGFAC]L= letaResearchLaboratoriesC,hemicalBiotestingCenter ChemicalsInspection& TestingInstitutJea,pan 822,3-chome, Ishii-machil,eta,Oita 977, Japan
PURPOSE OF STUDY The purposeof thisstudywas to determinethe mutagenicpotentiaolf the test substancuesingSalmonellatyphimuriumand Escherichicaoli.
TESTINGNIETHOD Thisstudywas conductedinaccordancewith thefollowingguidelines":Standardsfor ToxicityInvesdgatiori(eJ'apan'sMOL, No.77, September 1,1998).
GLP CONDLLANCE This studywas carriedout in compliancewith the followingGLP requirement: "Standardsto be observedby TestingInstitutiofnosr ToxicityInvestigations" (Japan'sMOL, No.76, September 1,19.99).
PERIOD OF STUDY Commencement of test: Dose findingtest: Completionof observation: Presentatioonf finarleport:
August 21, 1996 September 10, 1996 September 17, 1996 September 25, 1996
KOI-1802
LOCATION AND PERIOD FOR RETENTION OF RAW DATA Data and.testsubstanceareretainedin the archivesand the testsubstancestorage room of letaResearchLaboratoriesfor10 yearsfollowingthedateof thenotification specifieudnderItem I of Articl5e7-2 ofIndustriaSlafety& HealthLaw, respectively.
Afterterminatioonf the retentiopneriod,any measurestaken aredone so with the approvalofthe sponsor.
PERSON CONCERNED StudyDirector:
WITH STUDY
Signed inorigiiw-
September 25, 1996
Shozo Ogura
IfitaResearchLaboratories
MutagenicitySection
Study Staff. Person inchargeof Storage:
Tsunehiko Inai,B.S. ShizukaKouda
ANY UNEXPECTED SITUATIONS AND DEVIATIONS FROM PROTOCOL There were no unexpectedsituationasnd deviafionfsrom protocolwhich might have affectedthetestresults.
KOI-1802
CONTENTS
SLTN4N4ARY
............................I...........
MAT'ERIALS AND METHODS 1.TEST SUBSTANCE AND POSITIVE 2.BACTERIAL STRAINS 3.NIEDIUM AND S9 N4IX 4.PRE-CULTURES
CONTROLS
...............2...
...........................4............
...........................5............
...........................6............
5.PREPARATION CONTROLS
6.METHODS
OF TEST SUBSTANCE AND POSNIVE ....i.........................7......... ............................7 ...........
7.NUCROSCOPIC OBSERVATION AND COLONY COUNTING
...... 8
8.EI=RETATION
OF RESULTS
............................9 ...........
RESULTS
............................9 ...........
CONCLUSION
............................10...........
REFERENCES
............................10...........
APPENDIX TABLES AND FIGURES ..........................1.-6............
KOI-1802
SUN04ARY
The reversemutationtestof 'r-I was performedon Salmonellatyphimuriumstrains TAIOO, TA1535, TA98, TA1537 andFscheilchiacolistraiWnP2 uvra usingthepreincubatiomnethod withand withoutmetabolicactivation. The resultshowed thatthenumbersoftheirrevertanctoloniesforallstrainisngroups which were treatedwith thetestsubstancewere lessthantwicethatof each negative controlw,ithandwithoutS9 Mx. The numbers oftherevertanctolonieisnthenegativecontroland thepositivceontrols were withinthebackgrounddatainour laboratories. Based upon theaboveresults',r-Iwas judgedto have no reversemutagericpotential underthepresenttestconditions.
KOI-1802
MATERLALS AND METHODS
TEST SUBSTANCE AND POSITIVE CONTROLS
I Testsubstance04ormation providedby thesponsor)
1) Name
Reactionproductsof perfluorodimethylcyclohexylsulfolnuyolride,
perfluoroakl(C=0-2) cyclohexylsulfonfylluoridep,otassiumcarbonateand
suffiniaccid
Othername: T'-l
CAS No.:67584-42-3(themain component A(ii7-2))
2) Lot No.
293
3) Supplier SUMITOMO 3M LMTED
4) Structuraflormulaorrationaflormula(Outlineof manufacturingmethod, in case
bothwere unknown)
A CnF2n+
1,@@@S03K
(n=0-2)
76%
B CF3
F
S03K
CF3
C Cn-12mi-lSO3K
(riy=8)
D K2SO4
Odiers (molecularformula 5) Purity 100 W/WO/O 6) Impurities
20% 3%
0.4%
0.6%
7) Physicocheniicaplroperties
Appearanceatordinarytemperature: wlitepowder
Molecularweight:
about 500
-2-
KOI-1802
Stability:
stable
Meltingpoint:
Boilingpoint:
Vapor pressure:
Partitiocnoefficient:
Solubifity:
Degree of solubility:
Water:
-:5o.i%
DMSO:
5 w/vO/o*
Acetone: about 5%
Others:
methanol about 5%
Examined inour laboratories
8) Storageconditions
room temperature
9) Careon handling
Gloves,a nmsk, a head cap and a tabcoat were wom when handling.
1.2 Positivceontrols
1) 2-(2-Furyl)-3-(5-nitro-2-fiiryl)aAcFr-y2l)aniide
Manufacturer: Wako Pure ChemicalIndustriesL,td.
Lot No.-.
LEN0571
Properties: reddish-orangcerystallipnoewder
Purity:
99.5%
Grade:
specialgrade
2) Sodiumazide (NaN3)
Manufacturer: Wako Pure Chemical IndustriesL,td.
Lot No.:
DLP2438
Properties: whitecrystalline
Purity:
99.4%
Grade:
specialgrade
3) 2-Metho)cy-6-chloro-9-[3-(2-chloroethyl)-aminopropylaniino2]HaCcIridine'-
(ICR-191)
Manufacturer: PolysciencesI,nc.
Lot No.:
412795
Properties: yellowcrystallipnoewder
Purity:
Grade:
-3 -
K01-1802
4) 2-Aniinoanthracen(e2AA)
Manufacturer: Wako Pure Chemical IndustriesL,td.
Lot No.:
DLP,7869
Properties: yeflowish-green-brownpowder
Purity: Grade:
95.7% -
5) Storageconditions
A cold and dark place
6) Care on handling
Gloves,a mask a head cap and a labcoat were wom when handling.
2. BACTERL4L SIRAINS 2.1 Strainsselected
Salmonella typhimurium strainsTAIOO, TA98, TA1535 and TA1537 were obtainedfrom Dr. B.N. Ames, Universityof CaliforniaU,.S.A- on June 20, 1990.
A Escherichia coli strain WP2 Laboratorieso,n April6, 1995.
uvra was obtained from Japan Bioassay
S.typhimurium strainTsAI 00, TAl 535 and E. colistrainWP2 uvra were used for the detectionof base-pairsubstitutionmutatioi@while S. I)phimurium strains TA98 and TAI 537 forthedetectionof frameshiftmutation. 2.2 Storage The teststrainswere storedas frozen stock cultures(0.045 ml of dimethyl sulfoxid(eDMSO)*/0.5 rd of brothculture)at-80'C (ultra-deefpreezerMDF291, Sanyo). *Purity 2; 99.0%, Lot No..CF103, Dojindo Laboratories
-4-
KOI-1802
2.3 Characterizationf thestrains
1) Characteristiocfsthestrains
Mutationon
Strains
synthesiosf
amino acid
Mutationon excision repair
Membrane R-factor mutation (pKM 101) (LPS)
Salmonellta>phimurium
TA1535
hisG46
TA1537
hisC3076
TA98
hisD3052
TAIOO
hisG46
Escherichiacoli
WP2 uvra
trp
LuvrB LuvrB LuvrB LuvrB
AuvrA
rfa
r.fa
rfa
+
r.fa
+
+
The amino acidrequirementforgrowth was demonstratedby usinghistidinfeor
S. t)phimiaiumstrainsand tryptophanforF- colistrain.The presenceof R-
factorm,embrane mutationand mutationon theabilittyo repairDNA lesionswere
conflrmedby ampiciuinresistances,ensitivittoycrystavlioletand LTV sensitivity,
respectively.
2) Dateofcharacterization
Salmonellatyphimurium
TA1535
July18, 1996
TA1537
June 7, 1996
TA98
June 7, 1996
TAIOO
March 6, 1996
Escherichiacoli
WP2 uvra
April18, 1996
3. M[EDILTM AND S9 NFIX
3.1 Medium
1) Minimalglucoseagarplate(preparedinourLaboratories)
The medium was preparedas follows,and poured 30 ml intoa petridish.
Components
Amount includedinone htre
20 x Vogel-BonnerE
50 ml
40 w/v/o Glucose
50 rnl
Agar
15 g
(1)Agar:
Bacto-Agar(LotNo. 84707AJA, DifcoLaboratories)
(2)Manufacturingdate: dose findingteston August 29, 1996
main teston September 5, 1996
-5 -
KOI-1802
2) Softagar
The solutioncontaining0.5 mM histidinaend 0.5 mM biotinforS. typhimurium
strainsor 0.5 niM tryptophanf.or E. coli strainwas added to the soft agar
solutioncontaining0.6 w/vO/oagar (Bacto-Agar,Lot No. 71892AJB, Difco
Laboratoriesa)nd 0.5 w/vO/oNaCl ina ratioof I : 10.
3.2 S9 Mx
1) RatEverS9 (KikkomanCo.,Ltd.)
Inductionmethod:
SD male rats,7-week-old(203-254g),were
intraperitoneaaldlmyinistratepdhenobarbita(l30 mg/kg
x Itime,60 mg/kg x 3 times)and 5,6-benzoflavone
(80mg/kg x I time).
Lot No.:
RAA-350 (manufacturedon Au gust23, 1996,
purchasedon September 4, 1996)
Storage:
-80*C (ultra-deefpreezerMDF-29 1,Sanyo)
2) CofactorforS9 Mx (OrientaYleastIndustrieLst,d.)
Lot No.:
999601
Storage:
-20*C (bio-freezGeSr-2603, Nippon FreezerLtd.)
3) Compositionof S9 Mx
One ml ofS9 l@& contained8 JIMOI MgC12, 33 pmol KCI, 5 pmol G-6-P,4 gmol NADPI-L 4pmol NADK 100 pmol of 0.2M sodium-phosphatbeuffer(pH 7.4) and 0.1 ml S9.
4. PRE-CULTURES From the stockcultures2,0 plof bacteriasluspensionwas inoculatetdo L-tube containin1g0 rnlnutrienbtrothNo.2 (Lot No. 194 56443,OXOD:) Ltd.),the bacteriaclulturewas incubatedat37 ::0L.5*C for8 h with shakingat50 timestniin by theMonod shaker(MONOSIN- B A, TaitecCo.,Ltd.) The viablecellcounts calculatefdrom the valueswhich were determinedat660 mn by spectrophotometry (Novaspec,LKB Japan)attheend ofincubationareshown below.
No. of Dose viableccfls findingtest (x109/ml) Main test
TAIOO 1.9 1.9
TA1535 WP2 uvra TA98
2.2
3.4
2.4
2.1
3.6
2.3
TA1537 2.1 2.0
-6-
KOI-1802
5. PREPAPATION OF TEST SU13STANCE AND POSITIVE CONTROLS 5.1 Testsubstance
1) Preparation The testsubstancewas dissolvedinDMSO (LotNo. CF103) to make 5 w/v*/o concentratioannd dilutedwith the same solventto give appropriate concentrations.
2) Stabilitoyfthetestsolution No denaturatioonf the testsolutionwas observedto a colorand exothermic reactionuntil2 hoursafterpreparation.
3) Preparationtime Preparedimmediatelybeforeuse and used within0.5 h atroom temperature.
5.2 Positivceontrols 1) Preparation NaN3 was dissolvedin distillweadter (Distillewdater forinjectionL,ot No. K6B74, Otsuka PharmaceuticaFlactory). AF-2, ICR-191 and '2AA were dissolveidnDMSO (LotNo. CD069). 2) Preparationtimeand storagecondition Preparedon every3 months and storedat -80'C (ultra-deefpreezeMrDF-291, Sanyo).
6. NMTHODS The testwas carriedout for S. typhimurium strainsTA1535, TA1537, TA98, TAIOO and aE colistraiWnP2 uvra usingthepre-incubatiomnethod both with and withoutmetabolicactivatiosnystem. The platingwas done intriplicaftoer the negativecontroland induplicatfeorthetestsubstanceand positivecontrols.
6.1 Procedures After0.1 rnlof the testsubstancesolution0,.5 ml of 0.1 M sodium phosphate buffer(pH 7.4)orS9 W and 0.1 ml ofthebacteriaclulturewere added to a tube, themixtureswere incubatedfor20 min at37 0.5C. Two nd of the softagar was thenadded to each tube and poured onto a minimal glucoseagarplate. Afterincubatiofnor48 h at 37 :L0.5C, the number of revertanctolonieswere counted.
-7-
KOI-1802
As the steriliteyst,each 0.1 ml of each bacteriasluspension,testsubstance solutionS,9 Nfixor 0.1 M sodium phosphatebuffer(pH 7.4)were smeared on a mkAnW glucoseagar plateand incubatedat 37 0.5C for 48 h, and then checkedthebacteriaclontamination.DMSO was used as a negativecontrol,and thefollowingpositivecontrolswere used foreach bacteriasltrains.
S9 Nfix S9 Nfix
TAIOO AF-2 0.01 2AA
1
TA1535 NaN3 0.5 2AA 2
WP2 uvra AF-2 0.01 2AA 10
TA99 AF-2 0.1 2AA 0.5
TA1537 ICR-191
1 2AA
2
(gg/plate) 6.2 Dose selection
1) Dose findingtest The testwas carriedout atthe highestdose of 5,000 gg/plateand 6 doses of 1,000, 500, 100, 50, 10 and 5 gg/plate.As a result,growth inhibition observedatdosesof more the 1,000gg/platfeorS.typhimuriumstrainTsAIOO, TA1535, TA1537 and E. colistrainWP2 uvra and 5,000 gg/platefor S. typhimuriumstraiTnA98 withoutS9 Nfix. Growth inhibitionbservedatdose of5,000 iLg/plaftoerS. typhimuriumstrains TAIOO, TA1535, TA1537 and E. coliWP2 uvra withS9 Mix.
2) Main test Based on the resultsof the dose findingtest,a main testwith S9 NEx was performedatthehighestdose of 5,000 gg/plateand 5 lower doses dilutedwith a geometricprogressioonf 2. Without S9 W a maximum dose was decided 1,250gg/platienthecaseof S. "himurium stranis TAIOO, TA1535, TA1537 and E. colistrainWP2 uvra and.5,000 gg/plateinthe caseof S. typhimurium strainTA98 and 5 lower doseswere decidedineachbacteriasltrainby dilution witha geometricprogressionof 2.
7. NUCROSCOPIC OBSERVATION AND COLONY COLTNTING 7.1 Mcroscopic observation
The stateofrevertanctolonies(sizeand number of colonies)d,epositionof thetest substanceand the growth inhibitiownere examined witha stereomicroscope.
-8-
KOI-1802
7.2 Colony counting The number of colonieswere countedwitha manual counteror a colony analyzer (CA-9, Toyo-sokldCo.,Ltd). Correctionforcountingerrorswas made for measurementswith the colony analyzer.Each platewas measured threetimes, and the averageof thesethreemeasurements was adopted as the number of revertanctolonieson theplate. The averageforeach dose was calculatefdrom thevaluesof theplatesused. Decimalsof theaveragefigureswere rounded off.
8. E4TERPRETATION OF RESULTS The testsubstancewas judged to be positivew,hen the number of revertant colonieswas twiceor more of thenegativecontroland thedose-relationshainpd thereproducibiliwteyre obtained. Any statisticparlocedureswere notused.
RESULTS
The numbers oftheirrevertanctoloniesforallstrainisngroupswhich were treatedwith thetestsubstancewere lessthantwicethatof each negativecontrolwith and without S9 mx.
The growth inhibitiownas observedat more than 1,000 Ag/platein S. typhimurium strainTsAI 00,TAI 535,TAI 537 andE. colistraiWnP2 uvra and at5,000;Lg/platienS. ophimurium straiTnA98 withoutS9 Nfix. The growth inhibitiwoans noted at 5,000 jig/platienS. typhimuriumstrainsTAIOO, TA1535, TA1537 andE. colistraiWnP2 uvr,4withS9-Mx.
The positivceontrolsshowed thedistincitncreaseof revertantcoloniesa,nd the positive controlsand the negativecontrolwere withina range of the background datain our laboratories.
There were no fluctuationwshich affectedthe testresultsincethe steriliteyst confirmedthe absenceofany niicro-organisms.
-9-
KOI-1802
CONCLUSION
Inconclusion, 'r-1 was judged to have no reversemutagenic potentiaulnder the present testconditions.
REFERENCES I . Ministryof Labor (1991) Guidebook on MutagenicityTests using Mcro-
organisms,New Edition(inJapanese)publishedby Japan IndustrialSafetyand Health Association, 2. Green M.RL. and W.J. Muriel (1976) Mutagen testingusing Trp@+reversionin Escherichiacoli,MutationRes.,38: 3-32. 3. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test,Mutation Res., 113: 173-215.
-10-
Dose finding
test
X01-i8D2
Test substance: 1
With(+)or Test substance
inbu ofrevertan(tmsobu ofcolmdes/plate)
vithout(-) ementration Bile-paiorutatitut'type
Bg Nil
(99/P'ate) TA 100 TA 1535 WP2 uvra
Frameshifttype TA 98 TA 1537
S9 Mix
negltive control
5
10
50 100
iu 97 ( 98)
94 -
103 (98) 92
86 (100)
114 103 85 (94) 87 82 85)
9 9 ( 11) 16
6 4 (10)
9 ( 13)
is 9 10 (10) 10 11 11)
19 21 ( 20)
21
25 19 ( 22)
24
16 ( 20)1 21 ( 18) 14 21
20 25 ( 23) 23
18 ( 21) 24
29
22 (26)
2186 (22) 23 19 (ZI)
12 14 ( 13) 13
' ( 8) 9 1110 (11) 12 6 ( 9) 12 9 (U)
5DO
1000
5000
negative control
iu 86 1050 85*
81 @D.*
94 u 107
11 95)
6 95*) 4*
6*
85*) '0**
17 98) 9
7
9) 5*)
0*)
U)
19
24
11
15 ( 17)
( 24) 23
8
22
7*
'14**( 18*) 19 ( 21) 12*
io*
15*
16*)
'0*.
-2- ll*)
29
34
20
24 29) 30 ( 35) 15
33
42
.18
10) 10*)
0*)
18)
S9 Mix
5 10 50
100 500
1000 5000
9
15
95) a
94
95 90) 10
85
8
8818 85) 10 12
106
(106) 8
105
-L(
97 ( 97) 12
96
8
92
10
105 ( 99)
97*
9*
83*( go*) 5*
12) 9)
11)
11)
10)
8) 7*)
19 23)
27
31 ; (29)
30
23 ( 27
28 24 ( 26)
27 23 (25)
20 25)
29 24*
23$)
22*
39 ( 37)
35
21
36 (29)
30 (33)
36
30
28 (29)
23 40
32)
32 34)
36 33
25 29)
19 14 17)
14
23 19) 15
14)
13
.11 13)
15
13 22 18)
9 ( 11)
12
5*( 6*) 6*
Positive cojitranlot
requirin
go Kii
xule
Al-2
Coneeiitratim (pg/plate) 0.01
h@nkr of 344 colonies/plate 356 ( 350)
NLKB 0.5
314 290 ( 302)
AF-2 0.01
136 157 ( 147)
AF-2
IM-191
0.1
1
560
2485
537 ( 549) 12348 (2417)
Positive cmtrol requiir'n
Nne
ZAA
2AA
Conamtrati (09/Pl1a*tRe,) 1 ---1 2
Nmber of 658
154
colonies/pl1ite6l74 ( 666) 132 ( 143)
Notes Parenthesisshows the men of each plate.
2AA 10
577 602)
627
zm 0.5
302 ( 287)
271
zm 2
159
* : Observedbacterialgrowthinhibition.
-AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
-MaN3: Sodium azide
-ICR-191: 2-Methoxy-6-chloro-g-(3-(2-chloroethyl)-aminopropylaminoa)cridine-2HC1 .2AA: 2-Aminoanthracene
Main test
Test substance:-c- 1
Vitb(+o)r Teat nbbtance
Daberofrevertan(tmsoberd colodes/plate)
vithout(-) concentration Date-paiorutititititoynpe
frisesbifttype
89mix (PgiPl"t'T)A 100 TA 1636 WP2 us@ra TA 98 TA 1537
negative control
93
6
n
84 ( 94) U ( 10) 18
105
11
25
26 21) 28
28
9 27) 9 ( 10)
11
39.1 93 (90) 15 ( 14) 20 19)
87
13
18
106
15
24
78.1 117 (112) 1 (15) 21 23)
6 13 (10)
12 7 (10)
S9 Mix
156
101
is
22
(97) IR (17) 21
22) 2211
18 21) 11
15)
313
105
12
21
25
10
92 (99) 7 (10)
20)
27) 2 (11)
-.&iL- -29
625 8926 (89) 10 (9) 2241( 23) 2233 23) 9g (9)
10D* az*
91*)
13*
1"(
16*)
is*
21
17*( 18*)[24
23)
9* 6*(
8*)
m
21 22)
-1@
23
5000
1187: jj*)
lD2 negative
a
31
36
21
control U 98) 8 ( 8) 30 ( 33) 38 37) 20 (22)
109
a
37
38
24
S9 Mix
156 102 ( 103) 13 (10) 42 ( 40) 28 34) 21 (21)
103
-6
38
21
313 11838 ( 101) 71 ( 9) 3217 (29) 3272 (30) '221 (18)
87
9
33
32
14
625 106 (97) la (10) 32 (33) 30 (31) 13 (U)
1250
80 92 (86)
9
11 (10)
29 21
(25)
4358 (42)
1265 (21)
2500 10958 (97) 99 (9) 1 (19) 2T8, ( 30) 1164 (15) 5000 110174**(ill*)97**(8$) ,*( 5*) 123 (23) l'l**(10*)
Positive
contronlot requiring
)im
AF-2
Cuentratim@
(pg/plate) 0.01
Huber of 353 77) colmi!slpla-teiiL1
kgs
AAFF--221E9)) AF-2
ICR-191
0.5
0.01
0.1
1
369( 378) 132 ( 144@ 590(579) 1688(1886)
387
155
567
12084
Positive control requiring 89 mix
law
2m
2m
2JA
2AA
UA
Cmeentratim
(W plate
1
2
10
0.5
2
Huber o@colonif
765 690 (
728)1 113212
(ir)&63847
( 661)
309 264(
154 287)1 153(
154)
Notes Parenthesisshowsthe meanof eachplate.
* : Observebdacteriaglrowthinhibitim.
-AF-2: 2-(2-@FurYl)-3-(S-nitro-2-furyl)acrylimide
-NaN3: Sodium azide
-ICR-191:2-Methoxy-6-cholro-9-(3-(2-cholroethyl-)aminopropylanino)acridine-2HCI
-2A: 2-Amifioanthracene
D o s e f i nd ing 150-
t es t
Cd 1 934 C. 1ON
a)
41
50-
X01-1802
O:TA 100 O:TA 1535 A:wp2uvrd
0-
5 10
50 100
500 1000
Fig. I Dose-response curve without S9 Mix
5000 (Aglp I ate)
150-
O:TA 100
O:TA 1535
,&:WP2avrJ
loo-(
50-
B
5 10
50 100
500 1000
Fig. 2 Dose-response curve with S9 Mix
(gg/plate)
3
D o s e finding 3D-
test
20-
0ci 10-
K01-1802
0: TA 98 0: TA 1537
0 5 10
50 100
500 1000
Fig. 3 Dose-response curve without Sg Mix
5000 (Ag/Plate)
40-
cd 30-
ui
20-
.6-a Id IU
0: TA 98 O:TA 1537
5 10
50 100
500 1000
Fig. 4 Dose-response curve with S9 Mix
5000 (,ug/plate)
-4-
M a n te st
15D-
100cu
U2 w
4 0 ti
50-
KOI-1802
0:TA 100 O:TA 1535 ,&:WP2uvrJ
D-
39.1
78.1
156
313
625
Fig. 5 Dose-response curve without SO Mix
1250 (Ag/plate)
150-
4d 1007
&3 (U
50-
O:TA 100 13:TA 1535 &:WP2vr.4
E3
E3
156
313
625
1250
Fig. 6 Dose-response curve with SO Mix
E3
2500
5000
(,ug/plate)
M a in te st 30-
ow
20-
K01-1802
O:TA 98 O:TA 1537
1013
a
o
39.1 78.1 156
313
625
1250
Fig. 7 Dose-response curve without Sg Mix
2500 5000 @(Ag/plate)
50-
40-
30-
0 I.-$
0 ci
2
ad
10-
0: TA 98 0: TA 1537
156
313
625
1250
Fig. 8 Dose-response curve with Sg Mix
2500
50'00
(Ag/Plate)
6
FINAL REPORT ANENDNMNT
IfitRaesearchLaboratoriesC,hemicalBiotestingCenter ChemicalsInspection& TestingInstitutJea,pan
1. Title(Studycode) Bacteriarleversemutationtestof T'-l(KOl-1802)
2. Amendment (Items) CAS No. and structuraflormulaor rationaflormula(Annex 1)
3. Authorization Study Director
Signedinoriginal Shozo Ogura
November 27, 1996
Annex 1
1.1 TestsubstanceWormation providedby thesponsor)Page 2 Before changes:1) Name
Reaction products of perfluorodimethylcyclohexylsulfonyl fluoride,perfluoroa(lCk=y0l-2) cyclohexylsulfonyfluoride, potassiumcarbonateand sulfuriaccid Other name: 'r-I CAS No.: 67584-42-3
4) Structuraflormulaor rationaflormula(Outlineof manufacturing method,incaseboth were unknown)
CnF2n+ "*@@S03K
CF3
F
S03K
CF3 CniF2m+.lSO3K K2SO4 (molecularformula
(m7-8)
(n=0-2)
-2-
Afterchanges:1)
Name Reaction products of perfluorodirnethylcyclobexylsulfonyl fluoride,perfluoroalk(ylC=0-2) cyclohexylsulfonylfluoride, potassiumcarbonateand suffiniaccid Othername: 'r-I CAS No.: 67584-42-3Lthemain comronent A(n-2
4) Structuraflormulaor rationaflormula(Outlineof manufacturing method, incasebothwere unknown)
A CnF2n+
S03K
(n7-0-2)
B CF3
F
S03K
760/o
CF3
c
CMF2M+IS03K
(M7-8)
D
K2S04
Others (molecularformula
200/o
30/o aAN
0.6%
Reason for changes: To document new informationsuppliedby the sponsor.
Date effective:
November 13, 1996
-3 -