Document yr7NYG3p15eRROGLxMXZBNvmr

STUDY CODE: KOI-1815 ReceiptNo. T96-2503 Report No. T4663 FINAL REPORT BACTERIAL REVERSE MUTATION OF v-1 TEST October,1996 I-litRaesearch Laboratories Chemical BiotestingCenter ChemicalsInspectio&n TestingInstitute Japan R- 0 -\v 1-77y07 OUALITY ASSURANCE STATENENT Hita Research LaboratoriesC,hemical BiotestmgCenter Chemicals Inspection& TestingInstitutJea,pan S12onsor: SUMITOMO 3M LUAMD Title: Bacterialreversemutationtestof v -1 Study code: KOI-1815 This reportwas auditedby the QualityAssuranceSection. I,the undersigned,hereby declarethatthisreportreflects the originalJapanese report. (Date) oca.,@ 1 SectionChief,QualityAssurance (Signature) KeijiShiraishiB,.S. KOI-1815 I,the undersigned,hereby declarethatthisreportprovides a correctEnglishtranslatioonftheFinalReport. (Study code No. KO I-1815 issuedon October 30, 1996) (date) (signature) Shozo Ogura Mta Research Laboratories Chemical BiotestingCenter Chemicals Inspection& TestingInstituteJ,apan KOI-1815 GLP STATENIENT Bta ResearchLaboratoriesC,hemicalBiotestingCenter ChemicalsInspection& TestingInstitutJea,pan S12onsor: Title: StudyCode No.: SUlyffTOMO 3M LMTED Bacteriarleversemutationtestof v -1 KOI-1815 I,the undersigned,herebydeclarethatthisstudywas conductedin complianceswith "Standardsto be observedby TestingInstitutiofnosrTo)dcityInvestigation(sJ"apares MOL, No.76, September1,1988). Management: - Signedinorip-inal ShigetakaYamane, Ph. D. October30, 1996 K01-1915 QUALITY ASSURANCE STATEMENT Mta ResearchLaboratoriesC,hemicalBiotestingCenter ChemicalsInspectio&n TestingInstitutJea,pan S12onsor: Title: Study Code No.: SUNUTOMO 3M LD4MD Bacteriarleversemutationtestof v -1 KOI-1815 This studywas auditedby theQualityAssuranceSectionand thestudyprocedureswere inspectedon thefollowingdates. Dates of Inspections Dates of Reportsto Dates ofReportsto and Audits Study Director Management September 12, 1996 September 13,1996 September 17,1996 October 1,1996 October 1,1996 October 1,1996 October 30, 1996 October30, 1996 October 30, 1996 1,theundersigned,herebydeclarethatthisreportprovidesan accuratedescriptioonf the methods and proceduresused inthisstudyand thatthereportedresultasccuratelryeflect theraw dataobtained. SectionChief,QualityAssurance: Signedinoriginal KeijiShiraishBi.,S. October 30, 1996 KOI-1815 Study code: K01-1815 Testsubstancecode: BR3291 Sponsor code: S-030 TITLE Bacteriarleversemutationtestof v -I SPONSOR SUMITOMO 3M LIMITED 8-8,NEnami-Hashimoto 3-chome Sagamihara-shiK,anagawa, 229 Japan TESTING FACILITY Mta ResearchLaboratoriesC,hemicalBiotestingCenter ChemicalsInspection& TestingInstitutJea,pan 822,3-chome, Ishii-machiI,-IitOai,ta877,Japan PURPOSE OF STUDY The purpose of thisstudywas to determinethe mutagenicpotentiaolf the test substanceusingSalmonellatyphimuriumand Escherichicaoli. TESTING METHOD Thisstudywas conductedinaccordancewiththefollowingguidelines":Standardsfor ToxicityInvestigation(sJ"apan'sMOL, No.77, September1,1988). GLP COMPLIANCE This study was carriedout in compliancewith the followingGLP requirement: "Standardsto be observedby TestingInstitutiofnosr ToxicityInvestigafione.' (Japan'sMOL, No.76, September1,1988). PERIOD OF STUDY Commencement of test: Dose findingtest: Completion of observation: Presentatioonf finarleport: September 17,1996 September 25, 1996 October 14,1996 October 30, 1996 K01-1815 LOCATION AND PERIOD FOR RETENTION OF RAW DATA Data and testsubstanceareretainedin the archivesand the testsubstancestorage room of Mta ResearchLaboratoriefsor10 yearsfollowingthedateof thenotification specifieudnder Item 1 of Article57-2of IndustriaSlafety& HealthLaw, respectively. Afterterminatioonf the retentiopneriod,any measurestakenare done so with the approvalof the sponsor. PERSON CONCERNED Study Director: WITH STUDY Signed inoriszinal October30, 1996 Shozo Ogura letaResearchLaboratories MutagenicitySection Study StaffPerson inchargeof Storage: Tsunehiko InaiB,.S. ShizukaKouda ANY UNEXPECTED SITUATIONS AND DEVIATIONS FROM PROTOCOL There were no unexpectedsituationasnd deviationfsrom protocolwhich might have affectedthe testresults. KOI-1815 CONTENTS SUMMARY ............................1............ MATERIALS AND METHODS 1.TEST SUBSTANCE AND POSITIVE 2.BACTERIAL STRAINS 3.MEDIUM AND S9 NHX 4.PRE-CULTURES CONTROLS ...............2... ............................4........... ............................5........... ...o ..........................6......... 5.PREPARATION CONTROLS 6.METHODS OF TEST SUBSTANCE AND POSITIVE ............................6........... ............................7........... 7.MICROSCOPIC OBSERVATION AND COLONY COUNTING ...... 8 8.INTERPRETATION OF RESULTS ............................8........... RESULTS ............................8........... CONCLUSION ............................9........... REFERENCES ............................9........... APPENDIX TABLES AND FIGURES ........o ...................1-.6...... KOI-1815 SUNRVIARY The reverse mutation test of v -1 was performed on Salmonella typhimurium strains TAIOO, TA1535, TA98, TA1537 and aescherichia colistrainWP2 uvra using the preincubationmethod with and without metabolicactivation. The resultsshowed thatthe numbers of theirrevertantcoloniesfor allstrainsin groups which were treatedwith the testsubstancewere lessthan twice thatof each negative controlwith and withoutS9 N4ix. The numbers of the revertantcoloniesinthe negafivecontroland the posifivecontrols were withinthebackground datainour laboratories. Based upon the above results,v -I was judged tohave no reversemutagenic potential under the presenttestconditions. KOI-1815 MATERIALS AND NETHODS TEST SUBSTANCE AND POSITIVE CONTROLS 1.1 Test substance(Informatiopnrovidedby thesponsor) 1) Name Potassiumsaltof N-ethyl-N-perfluorobutylsulfonylglycine Othername: v -I CAS No.: 67584-51-4 2) Lot No. Lot 1 3) Supplier SUNflTOMO 3M LIMITED 4) Structuraflormulaor rationaflormula(Outlineof manufacturingmethod,incase both were unknown) C4F9SO2NCH2COOK I U2ki5 (molecularfomiula CgH7F9RNO4S) 5) Purity 97.3 w/wO/o 6) Impurities KCI 2.7 w/wO/o 7) Physicochemicalproperties Appearance atordinarytemperature: lightgraypowder Molecularweight: 423.30 Stability: Meltingpoint: stable - Boilingpoint: - Vapor pressure: - Partitiocnoefficient: - Solubility: - Degree of solubility: Water: 5 W/vo/o* DMSO: 5 W/vo/o* Acetone: < 10 w/vO/o Others: Examined inour laboratories -2- KOI-1815 8) Storageconditions room temperature 9) Careon handling Gloves,a mask, a head cap and a labcoatwere wom when handling. 1.2 Positivecontrols 1) 2-(2-Furyl)-3-(S-nitro-2-furyl)a(cArFy-l2a)mide Manufacturer: Wako Pure ChemicalIndustrieLst,d. Lot No.: LEN0571 Properties: reddish-orangecrystallipnoewder Purity: 99.5% Grade: specialgrade 2) Sodium azide (NaN3) Manufacturer: Wako Pure ChemicalIndustriesL,td. Lot No.: DLP2438 Properties: white crystalline Purity: 99.4% Grade: specialgrade 3) 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]a2cHrCiIdine (ICR-191) Manufacturer: PolysciencesI,nc. Lot No. -. 412795 Properties: yellowcrystallipnoewder Purity: Grade: 4) 2-An@noanthracene(2AA) Manufacturer: Wako Pure Chemical IndustrieLst,d. Lot No.: DLR7869 Properties: yellowish-green-browpnowder Purity: Grade: 95.7% - 5) Storageconditions A coldand dark place 6) Care on handling Gloves,a mask, a head cap and a labcoatwere wom when handling. 3- KOI-1815 2. BACTERIAL STRAINS 2.1 Strainsselected Salmonella typhimurium strainsTAIOO, TA98, TA1535 and TA1537 were obtainedfrom Dr. B.N. Ames, UniversityofCaliforniaU,.S.A. on June 20, 1990. A Escherichia coli strainWP2 Laboratorieso,n April6, 1995. uvra was obtained from Japan Bioassay S. typhimurium strainTsAIOO, TA1535 and a E. colistrainWP2 uvra were used for the detectionof base-pairsubstitutiomnutation,while S. typhimurium strains TA98 and TAI 537 were forthedetectionofframeshifmtutation. 2.2 Storage The teststrainswere storedas frozen stock cultures(0.045 ml of dimethyl sulfoxid(eDMSO)*/0.5 ml of brothculturea)t -80'C (ultra-deefpreezerMDF291, Sanyo). * Purity '2-9:9.0%, Lot No. CF103, Dojindo Laboratories 2.3 Characterizatioonf strains 1) Characteristiocfsstrains Strains Mutationon synthesiosf amino acid Mutationon excision repair Membrane R-factor mutation (PKMIOI) (LPS) Salmonelltayphimurium TA1535 hisG46 TA1537 hisC3076 TA98 hisD3052 TAIOO hisG46 Escherichiacoli WP2 uvra trp LuvrB LuvrB LuvrB LuvrB Z@uvrA rfa rfa rfa + rfa + + The amino acidrequirementforgrowth was demonstratedby usinghistidinfeor S. typhimurium strainsand tryptophanforE coli strain. The presence of Rfactor,membrane mutationand mutationon the abilittyo repairDNA lesionswere confirmedby ampiciuinresistances,ensitivitoy crystavlioletand UV sensitivity, respectively. -4- KO 1-1815 2) Date ofcharacterization Salmonellatyphimurium Escherichiacoli TA1535 TA1537 TA98 TAIOO WP2 uvra July18, 1996 June 7, 1996 June 7, 1996 March 6, 1996 April18,1996 3. NEDIUM AND S9 NIIX 3.1 Medium 1) Minimalglucoseagarplate(preparedinourLaboratories) The medium was preparedasfollows,and poured30 ml intoa petridish. Components Amount includedinone litre 20 x Vogel-BonnerE 50 mi 40 w/vO/oGlucose 50 mi Agar 15 g (1)Agar: Bacto-Agar(LotNo. 71892AJB or90800JA, DifcoLaboratories) (2)Manufacturingdate: dose findingteston September 11,1996 main teston October3, 1996 2) Softagar The solutioncontaining0.5 mM histidinaend 0.5 mM biotinforS. typhimurium strainsor 0.5 mM tryptophanfor E. coli strainwas added to the soft agar solutioncontainin0g.6 w/vl/oagar (Bacto-AgarL,ot No. 71892AJB, Difco Laboratoriesa)nd 0.5w/v*/oNaCl ina ratioof I :10. 3.2 S9 Nfix 1) Rat liveSr9 (KikkomanCo.,Ltd.) Inductionmethod: SD male.rats7,-week-old(203-254g),were intraperitoneaaldlmyinistratepdhenobarbita(l30 mglkg x I time,60 mg/kg x 3 times)and 5,6-benzoflavone (80mg/kg x Itime). Lot No.: RAA-3 50 (manufacturedon August 23, 1996, purchasedon September4, 1996) Storage: -809C (ultra-deefpreezerMDF-29 1,Sanyo) 2) CofactorforS9 Mx (OrientaYleastIndustrieLst,d.) Lot No.: 999602 Storage: -209C (bio-freezGeSr-2603,Nippon FreezerLtd.) 5- KO I-1815 3) Compositionof S9 Nfix One n@ of S9 NEx contained8 gmol MgCI2, 33 gmol KCI, 5 gmol G-6-P, 4 gmol NADPK 4gmol NADK 100 gmol of 0.2M sodium-phosphatebuffer(pH 7.4) and 0.1 ml S9. 4. PRE-CULTURES From the stockcultures2,0 glof thebacteriasluspensionwas inoculatedto L-tube containing10 n-dnutrienbtrothNo.2 (LotNo. 194 56443, OXOID Ltd.)and the bacteriaclulturewas incubatedat37 0.5*C for8 h with shakingat50 times/min by theMonod shaker(MONOSIN- H A, TaitecCo.,Ltd.) The viablecellcounts calculatedfrom the valueswhich were detern-dnedat660 mn by spectrophotometry (Novaspec,LKB Japan)atthe end ofincubationare shown below, No. of Dose viablecells findingtest Ux 109/n-@) Main test 1 TA100 2.1 2.1 TA1535 WP2 uvra 2.1 4.4 1 2.1 1 .0 1 TA98 2.4 2.3 TA1537 2.1 1__ 2.0 5. PREPARATION 5.1 Test substance 1) Preparation OF TEST SUBSTANCE AND POSITIVE CONTROLS The testsubstancewas dissolvedin distillewdater (distillweadter forinjection, Lot No. K6B74, Otsuka PharmaceuticalFactory)to make 5 w/vO/oconcentration and dilutedwith the same solventto giveappropriateconcentrations. 2) Stabilitoyf the testsolution No denaturationof the testsolutionwas observedfor the color and the exothermicreactionuntil2 hours afterpreparation. 3) Preparationtime Prepared immediatelybeforeuse and used within0.5 h atroom temperature. 5.2 Positivecontrols 1) Preparation NaN3 was dissolvedin distillweadter (Lot No. K6B74). AF-2, ICR-191 and 2AA were dissolvedinDMSO (LotNo. CD069). -6 - KOI-1815 2) Preparationtimeand storagecondition Prepared on every3 months and storedat-809C (ultra-deefpreezerNMF-291, Sanyo). 6. METHODS The testwas carriedout for S. typhimurium strainsTAIS35, TA1537, TA98, TAIOO and a E. colistrainWP2 uvra usingthe pre-incubatiomnethod both with and without metabolicactivatiosnystem. The platingwas done in triplicatfeor the negativecontroland induplicateforthe testsubstanceand positivecontrols. 6.1 Procedures After 0.1 ml of the testsubstancesolution,0.5 ml of 0.1 M sodium phosphate buffer(pH 7.4)or S9 Nlix,and 0.1 ml ofthe bacteriaclulturewere added to a tube, the mixtureswere incubatedfor20 min at 37 0.5C. Two ml of the softagar was then added to each tube and poured onto.a minimal glucoseagar plate. Afterincubationfor 48 h at 37 0.5C, the number of revertanctolonieswere counted. As the sterilityest,each 0.1 rnlof each bacterialsuspension,testsubstance solution,S9 Nfix or 0.1 M sodium phosphatebuffer(pH 7.4)were smeared on a minimal glucoseagar plateand incubatedat 37 0.5@c for 48 h, and then checked the bacterialcontamination.Distillewdater was used as a negative control,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 TA98 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 at the highestdose of 5,000 gg/plateand 6 doses of 1,000, 500, 100, 50, 10 and 5 gg/plate. As a result,growth inhibitiownas observed at 5,000 gg/plateboth 'Withand withoutS9 Mix. 7- KOI-1815 2) Main test Based on theresultsof the dose findingtest,a main testwas performedat the highestdose of 5,000 gg/plateand 5 lower doses dilutedwith a geometric progressionof2. 7. MICROSCOPIC OBSERVATION AND COLONY COUNTING 7.1 Microscopicobservation The stateof revertanctolonies(sizeand number of colonies)d,epositionofthetest substanceand thegrowth inhibitiownere exan-dnedwith a stereomicroscope. 7.2 Colony counting The number of colonieswere counted witha manual counteror a colonyanalyzer (CA-7 orCA-9, Toyo-sokkiCo.,Ltd). Correctiofnorcountingerrorswas made formeasurementswiththecolonyanalyzer. Each platewas measuredthreetimes, and the averageof thesethreemeasurementswas adopted as the number of revertanctolonieson the plate. The averageforeach dose was calculatefdrom thevaluesof theplatesused. Decimalsof theaveragefigureswere rounded off 8. INTERPRETATION OF RESULTS The testsubstancewas judged to be positivew,hen the number of revertant colonieswas twiceor more ofthenegativecontrola,nd when thedose-relationship and thereproducibiliwteyre obtained. Any statistipcraolcedureswere notused. RESULTS The numbers of theirrevertanctoloniesforallstrainisngroupswhich were treatedwith the testsubstancewere lessthantwicethatofeach negativecontrolwith and without S9 Nfix. The positivecontrolshowed the distincitncreaseof revertanctoloniesa,nd thepositive controlsand the negativecontrolwere withina rangeof the backgrounddatain our laboratories. 8- K01-1915 The growth inhibitiwoans observedatmore than 2,500 gg/platbeoth with and without S9 NEX. There were no fluctuatiownhsich affectedthe testresultsincethe sterilitteyst confirmedtheabsenceof any micro-organisms. CONCLUSION Inconclusion,v -1was judgedto have no reversemutagenicpotentiaulnder thepresent testconditions. REFERENCES 1 Ministryof Labor (1991) Guidebook on MutagenicityTests using Micro- organisms,New Edition(inJapanese)publishedby Japan IndustriaSlafetyand HealthAssociation, 2. Green M.H.L. and W.J. Muriel(1976)Mutagen testinugsingTrp+ reversioinn EscherichicaoliM,utationRes.,38: 3-32. 3. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test,Mutation Res., 113: 173-215. -9 - Dose finding test K01-1815 Test- substance* v - 1 With(+)or Test substance without(-) concentration Numberof revertmts(mnberof colonies/pltte) Base-p&irsubstitutiotnype Frame3hifttype S9 mix (Ag/Pl"t') TA 100 TA 1535 WP2 uvr.4 TA 98 TA 1537 S9 Mix S9 Mix negative control 5 10 50 100 500 1000 5000 negative control 5 10 50 100 500 1000 5000 90 101 ( 101) ill 118 ( 114) -109 99 126 ( 113) 101 ( 104) 107 98 116 (107) 109 (114) 118 104 (100) 95 80* 76*( 78*) 110 95 95) 91 91 96 94) 104 102) 99 101 (96) go 105 (98) 91 100 (98) 96 93 (102) 110 120* 102*( 13 33 30 9 10 ( 12) 41 ( 37) 33 ( 31) 6 7) 14 37 1 31 7 1 1141 ( 13) 3445 ( 40) 3253 (29-) a9 -9) 11 (12) 19 (421 12 44 is (13) 39 (36) 1 8 32 12 (14) 16 16 7 (12) 33 (34) 34 48 (43) 38 8 30 (12) 34) 15 38 3* 33* ( 4*) 27*) 5* 20* 10 41 15 (12) 40 3B) 12 32 8 (10) 37 38) 12 39 11 (13) 35 38) 14 40 12 35 8 (10) 39 37) 15 33 8 (12) 35 34) 12 36 7 (10) 41 39) 9 32 (11) 32) 12 32 6* 8*) 25* 27*) 10*( 28* 1297 (23) 95 7) - 23 (25) 12 (11) 26 9 3316 (34 23 (24) 25 10 (10) 10 8 9 ( 9) 27 (Z7) 7 (-9) 27 10 io* 13*) 85**( 7*) 15* 34 23 36 34) 18 19) 33 16 34 15 34) is) 34 17 30 (-32) 2F35 24) 34 31 ( 27) 17 16) 23 14 43 (44) 'o (22) 45 23 30 (37) 16 (19) 43 21 - 31 (35) 19 (20) 39 20 25* 24*) 0* ( 7*) 23*(_ _ 14$ Positive controlnot reqmrng So Mix Name AF-2 Concentration (gg/plate) 0.01 Nober of 351 ( 381) colonitalplate410 N&Ns 0.5 382 ( 384) 385 AF-2 AF-2 ICR-191 0.01 0.1 1 - 179 ( 184) 487 ( 527) 1734 (1851) 188 566 1968 Positive control requiring S9 Nil Name 2M 2AA concentration (N@plate) I 1I 2 Nmber of 727 133 leolonies/pla1ti621 ( 674) 1 158 ( 146) Notes Parenthesis shows the meam of each plate. * : Observedbacterialgrowthinhibition. 2AA 10 I 650 ( 647) 644 2AA 0.5 304 ( 289) ZM 2 149 -AF-2:2-(2-FurYl)-3-(5-nitro-2-furyl)acrylamide -NaN3: Sodium azide -ICR-191: 2-Methoxy-6-chloro-9-(3-(2-chloroethy1)-aminopropylamina) acridine-2HCI .IIA- 9.-Iminnmn+hrApano Main test K01-1815 Test substance:v - 1 With(+)or Test substance Rtmberof revertimt(smoberof colonies/plate) without(-) concentration Base-piirsubstitutiotnype Frameshifttype 89 mix (Aglp"t') TA 100 TA 1535 WP2 uvr.4 TA 98 TA 1537 negative control 156 113 112 ( 110) 106 100 117 ( 109) 18 39 10 ( 15) 36 ( 37) 17 36 1 12 27 11 ( 13) ( 30) 33 28 25 ( 30) 38 28 ( 31) 34 _ 13 10 ( 14) 19 8 ( 11) 1 S9 Mix 313 625 1250 2500 5000 negative control 156 132 (116) 100 119 (117) 114 104 103 (104) 82*(87*) 91* 79*(84*) 88* 1DB 117 (115) 120 103 (110) 116 11 15 ( 13) 10 (11) 11 12P 12 (12) 7* 8*( 8*) 0* 0*( 0*) 9 9 (11) 14 6 ( 7) 8 24 30 33 ( 29) 34 ( 32) 30 32 (31) 3334 (34) 36 50 (43) 32D9 (30) i 35* 21* 3 *( 33*) 3* 22*) 36* l@188**( 35* (21*) 33* ) 24* 38 42 29 34) 46 (42) 34 39 4398 44) 3450 (38) 20 8 ( 14) 18 (21) 18 (23) 27 3* 9*( 6*) 0* 6*( 3*) 24 28 (26) 26 23 (29) 35 S9 Mix 313 97 (93) 9 (10) 12 47) 35 (41) 31 (29) 89 10 _u 46 26 625 109 (112) 11 (12) 35 36) 31 (34) 26 (26) 115 13 36 37 26 1250 ',',0( 2 111) 38 7-(-10) 41 (43) 26 2500 106* 121*) 8* 6*( 33* 7*) 38*( 36*) 37* 31*( 34*) 7* 1 136*( 14*( 103* 3$c 41* 24* 0* 5000 118*(ill*)1*( 2*) 33*(37*) (24*) 24* -- 7*( 4*) Positive controlnot requrin S9 mix Name AP-2 Concentration (R/Plate) 0.01 Number of 356 colonies/plate337 347) NaNa AF-2 AF-2 ICR-191 0.5 0.01 0.1 1 328 ( 312) 133 ( 123) 456 201;; ( 463) (2068) 295 113 470 2081 Positive control requiring S9 Mix Name 2AA Concentration- (g@plate) 1 Nmber of 804 coloniies/plateA74 ( 839) ZAA 21 191 ( 177) 163 2AA 10 582 ( 572) 561 2AA 0.5 1 276 ( 266) 255 2AA 2 171 159 (1765)] Notes Parenthesisshowsthe mean of eachplate. * : Ob5ervedbacterialgrowthinhibition. -AF-2:2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide -NaN3: Sodium azide -ICR-191: 2-Methoxy-6-chloro-9-(3-(2-chloroethy1)-aminopropylamino) acridine.2HC1 -2AA: 2-Aminoanthracene D o s e finding 150- test ed 100-4 cu 4 0 ci 50- K01-1815 0: TA 100 0: TA 1535 WP 2 vr.4 B- 0-= 5 10 50 100 B 500 1000 Fig. I Dose-response curve without S9 Mix 5000 g/plate) 150- 4c-u) co 10@ cn ci Cd 50- 0: TA 100 O:TA 1535 ,&:WP2uvr4 0 5 10 50 100 500 1000 Fig. 2 Dose-response curve with Sg Mix 5000 (,ai/1pat e) D o s e finding 40- test 30@ U3 20- Cd K01-1815 0: TA 98 13:TA 1537 10 0- 5 10 50 ioo 500 1000 Fig. 3 Dose-response curve without S9 Mix 5000 (gg/plate) 50- 40- Cd 4 30- 20-E 0: TA 98 0: TA 1537 10- 0 5 10 50 100 500 1000 Fig. 4 Dose-response curve with S9 Mix -5000 (gg/plate) Main 150- t e st ed 4 100- cn KOI-1815 0: TA 100 13:TA 1535 &:WP2Lrvr,4 Cd 50cg E3 B- 0- 156 313 625 1250 2500 Fig. 5 Dose-response curve without S9 Mix 5000 (gg/plate) 150- cu cd C. 100rn cu 50ce. 0: TA 100 0: TA 1535 A: WP2irvrj 0 156 313 625 1250 Fig. 6 Dose-response curve with Sg Mix 2500 5000 (gg/plate) i 40- 3N cu 0 20ci 10- K01-1815 0: TA 98 0: TA 1537 0- 156 313 625 1250 2500 Fig. 7 Dose-response curve without Sg Mix 5000 (gg/plate) 50- 40- Cd 4 300 "-4 0 2D- cu 10- 0: TA 98 0: TA 1537 0156 313 625 1250 Fig. 8 Dose-response curve with S9 Mix 2500 5000 (9g/pIate)