Document VGY82mpVD5RbmY3b0XZYzmm7o

STUDYCODE : K01-1815 ARL2G-022| [Receipt No. T96-2503 Report No. T-4663 -3%) FINAL REPORT BACTERIAL REVERSE MUTATION TEST OF v-l October, 1996 Hita Research Laboratories Chemical Biotesting Center Chemicals Inspection & Testing Institute Japan DECEIVE= 000199 I (| moon |i | i QUALITY ASSURANCE STATEMENT HCihteamiRceaslesarIcnhspeLcatbioornato&rieTse,stiCnhgemIincasltatBi,otJeastpianng Center SpSonsoUr MIT3OMLIMMITO ED TSittuldey: coder KOL-18IS Bacterial reverse mutation test of v-1 `This report was audited by the Quality Assurance Section. 1, the undersigned, hereby declare that this report reflects the original Japanese report. Section Chief, Quality Assurance Ono December 17, 1176 (Signature) [ i Mae Keiji Shiraishi, B.S. 600200 Ko1-1815 1, the undersigned, hereby declare that this report provides a correct English translationofthe Final Report. (Study code No. K01-1815 issued on October 30, 1996) ) December 17, 1976 Shozo Ogura Hita Research Laboratories Chemical Biotesting Center Chemicals Inspection & Testing Institute, Japan 600201 - Ko1-1815 GSLTPATEMENT Hita Research Laboratories, Chemical Biotesting Center Chemicals Inspecti&on Testing Institute, Japan SS ponsoU r MITO3MM LIMO ITED Title Bacterial reverse mutation test of v-1 StCudoy deN__oKOI.-18:15 1, the undersigned, hereby declare that this study was conducted in compliances with "Standards to be observed by Testing Institutions for Toxicity Investigations" (Japan's MOL, No.76, September 1, 1988). Management: _S_ iginon rigineal d Shigetaka Yamane, Ph. D. October 30, 1996 G00202 KoL-1815 QUALITY ASSURANCE STATEMENT Hita Research Laboratories, Chemical Biotesting Center Chemicals Inspection & Testing Institute, Japan SpS onsoU r: MITO3MM LIMO ITED Title Bacterial reverse mutation test of v1 `Study CodeNo KOL-1815 `This study was audited by the Quality Assurance Section and the study procedures were inspected on the following dates. Dates of Inspections Datesof Reports to DatesofReports to and Audits Study Director anagement September 12, 1996 September13, 1996 September 17, 1996 October 1, 1996 October 1, 1996 October 1, 1996 October 30, 1996 October 30, 1996 October 30, 1996 1, the undersigned, hereby declare that this report provides an accurate descriptionof the methods and procedures used in this study and that the reported results accurately reflect the raw data obtained, Section Chief, Quality Assurance: Siignoringine al d October 30, 1996 Keiji Shiraishi, B.S. (00203 Ko1-1815 Study code: KO1-1815 Test substance code: HR3291 Sponsor code: 5-030 TITLE Bacterial reverse mutation test of v-1 SPONSOR SUMITOMO 3M LIMITED 8-8, Minami-Hashimoto 3-chome Sagamihara-shi, Kanagawa, 229 Japan TESTING FACILITY Hita Research Laboratories, Chemical Biotesting Center Chemicals Inspection & Testing Institute, Japan 822, 3-chome, Ishii-machi, Hita, Oita 877, Japan PURPOSE OF STUDY The purpose of this study was to determine the mutagenic potential of the test substance using Salmonella typhimurium and Escherichia coli. TESTING METHOD This study was conducted in accordance with the following guidelines: "Standards for `Toxicity Investigations" (Japan's MOL, No.77, September 1, 1988). `GLP COMPLIANCE This study was carried out in compliance with the following GLP requirement: "Standards to be observed by Testing Institutions for Toxicity Investigations" (Japan's MOL, No.76, September 1, 1988). PERIOD OF STUDY Commencementoftest: Dose finding test: Completionofobservation: Presentationoffinal report: September 17, 1996 September 25, 1996 October 14, 1996 October 30, 1996 00204 Kol-1815 LOCATION AND PERIOD FOR RETENTION OF RAW DATA Data and test substance are retained in the archives and the test substance storage roomofHita Research Laboratories for 10 years following the date ofthe notification specified under Item 1 of Article 57-2 of Industrial Safety & Health Law, respectively. After termination of the retention period, any measures taken are done so with the approvalofthe sponsor. PERSON CONCERNED WITH STUDY Study Director --Signedinoriginal October 30, 1996 Shozo Ogura Hita Research Laboratories Mutagenicity Section Study Staff Person in chargeofStorage Tsunehiko Inai, B.S. Shizuka Kouda ANY UNEXPECTED SITUATIONS AND DEVIATIONS FROM PROTOCOL There were no unexpected situations and deviations from protocol which might have affected the test results. C00205 Kol-1815 CONTENTS MATERIALSANDMETHODS 1. TEST SUBAS NDPT OSIA TIVN ECONCTROELS~~ +ssssssessssssss 2 2 BACTERIAL STRAINS erm------ 4 3. MEDIUMAND 9 MIX [ES 4 PRE-CULTURES sree . PREPARATION OF TEST SUBSTANCE AND POSITIVE CONTROLS JE 6 METHODS cnmsserters meres -- 7. MICROSCOPIC OBSERVAANDTCIOLOONNY COUNTING === 8 INTERPRETOFARTESIUOLTNS ++eossssssssssseesssnsssinnsss 8 CONCLUSION serene se---- REFERENCES evens 9 APPENDIX TABLESAND FIGURES ++sreessssssssssssssssssnsssinss 1.6 00206 KoL-1815 SUMMARY The reverse mutation test of v1 was performed on Salmonella typhimurium strains TA100, TAIS3S, TASS, TAIS37 and a Escherichia coli strain WP2 uv using the preincubation method with and without metabolic activation. The results showed that the numbers of their revertant colonies for all strains in groups `which were treated with the test substance were less than twice thatofeach negative control with and without S9 Mix. `The numbers of the revertant colonies in the negative control and the positive controls `were within the background data in our laboratories. Based upon the above results, v1 was judged to have no reverse mutagenic potential under the present test conditions, 600207 --1- Kol-1815 MATERIALS AND METHODS 1. TEST SUBSTANCE AND POSITIVE CONTROLS 11 Test substance (Information provided by the sponsor) 1) Name Potassium saltofN-ethyl-N-perfluorobutylsulfonylglycine Other name: v-1 CAS No.: 67584-51-4 2) LotNo. Lot1 3) Supplier SUMITOMO 3M LIMITED 4) Structural formula or rational formula (Outline of manufacturing method, in case both were unknown) C4FsSONCH,CO0K CoHs (molecular formula CHFKNOLS) 5) Purity 97.3 wink 6) Impurities KCI 2.7 ww 7) Physicochemical properties Appearance at ordinary temperature: light gray powder Molecular weight 42330 Stability stable Melting point - Boiling point ~ Vapor pressure - Partition coefficient - Solubility: - Degreeofsolubility Water: 2S wihi* DMSO: 25wh%* Acetone: < 10 wA% Others: -- * Examined in our laboratories ,. 000208 01-1815 8) Storage conditions room temperature 9) Care on handling Gloves, a mask, 2 head cap and a lab coat were worn when handling. 12 Positive controls 1) 2-2-Furyl)-3-(-nitro-2-furyDacrylamide (AF-2) Manufacturer: Wako Pure Chemical Industries, Ltd Lot No LEN0S71 Properties: reddish-orange crystalline powder Purity 99.5% Grade: special grade 2) Sodium azide (NaNs) Manufacturer: Wako Pure Chemical Industries, Ltd. Lot No. DLP2438 Properties: white crystalline Purity 99.4% Grade special grade 3) 2:Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylaminoJacridine + 2HCI (ICR-191) Manufacturer: Polysciences, Inc. Lot No. 412795 Properties yellow crystalline powder Purity - Grade -- 4) 2-Aminoanthracene (2AA) Manufacturer: Wako Pure Chemical Industries, Ltd. Lot No. DLR7869 Properties: yellowish-green-brown powder Purity. 95.7% Grade: - 5) Storage conditions A cold and dark place 6) Care on handing Gloves, a mask, a head cap and a lab coat were worn when handling. 0G209 a= : Ko1-1815 2. BACTERIAL STRAINS 21 Strains selected Salmonella typhimurium siwains TA100, TAS, TAIS3S and TAIS37 were obtained from Dr. B.N. Ames, UniversityofCalifornia, U.S.A. ,on June 20, 1990. A Escherichia coli strain WP2 uA was obtained from Japan Bioassay Laboratories, on April 6, 1995 S. phimurium strains TA100, TAS3S and a E. coli strain WP2 ur were used for the detection of base-pair substitution mutation, while S. typhimurium strains TA98 and TALS37 were for the detectionofframeshift mutation. 22 Storage The test sulfoxide strains were (DMS0)*/0.5 stored mi of as frozen stock broth culture) at cultures (0.045 ml of dimethyl 80C (ultra-deep freezer MDF- 291, Sanyo). * Purity 2 99.0%,Lot No. CF103, Dojindo Laboratories 23 Characterizationofstrains 1)_ Characteristicsofstrains Mutation on Mutation on Membrane Refactor Strains synthesis of excision mutation (pKM101) amino acid _ repair ps) Salmonella typhimurium TAIS3S hisGd6 Aur fa - TAIS37 HisC3076 Aww fa - TA hisD3052 Aww fa + TAI00 hisG4S Aww tfa + Escherichia coli WP2 urd mw Aud + - `The amino acid requirement for growth was demonstrated by using histidine for S. typhimurium strains and tryptophan for E. coli strain. The presence of Rfactor, membrane mutation and mutation on the ability to repair DNA lesions were confirmed by ampicilin resistance, sensitivity to crystal violet and UV sensitivity, respectively. 000210 a= Kol-1815 2) Dateof characterization Salmonella typhimurium Escherichia coli TAIS3S TAIS37 TA98 TA100 WP2 urd July 18, 1996 June 7, 1996 June 7, 1996 March 6, 1996 April 18, 1996 3. MEDIUMAND $9 MIX 31 Medium 1) Minimal glucose agar plate (prepared in our Laboratories) The medium was prepared as follows, and poured 30 mi into a petri dish Components Amount included in one litre 20 x Vogel-BonneEr 50 ml 40 wiv% Glucose 50 ml Agar 15g (1) Agar: Bacto-Agar (Lot No. 71892AJB or 90800JA, Difco Laboratories) (2) Manufacturing date: dose finding test on September 11, 1996 main test on October 3, 1996 2) Soft agar The solution containing 0.5 mM histidine and 0.5 mM biotin for S. typhimurium strains or 0.5 mM tryptophan for E. coli strain was added to the soft agar solution containing 0.6 W/v% agar (Bacto-Agar, Lot No. 71892AB, Difco Laboratories) and 0.5 w/v% NaCl ina ratio of1 : 10. 32 $9 Mix 1) Ratliver $9 (Kikkoman Co., Ltd) Induction method: SD male rats, 7-week-old (203-254 g), were intraperitoneally administrated phenobarbital (30 mg/kg * 1time, 60 mg/kg 3 times) and 5,6-benzoflavone (80 mg/kg 1 time). Lot No. RAA-350 (manufactured on August 23, 1996, purchased on September 4, 1996) Storage: -80T (ultra-deep freezer MDE-291, Sanyo) 2) Cofactor for 9 Mix (Oriental Yeast Industries, Ltd.) Lot No. 999602 Storage: 20C (bio-freezer GS-2603, Nippon Freezer Ltd) 5 000211 KoL181s 3) Composition of $9 Mix NOAneDPmHlo,f4$u9moMlixNcAoDntHa,ine1d00 mumoll MofgC0l2,M33spodmioulm-KpCh,osphmaotelbuGf-f6e-rP,(54Hja7m.a4l) and 0.1 mi $9. 4 PRECULTURES cFornotmaitnhiengst1o0ckmiculttiureesn,t20bruoltohfNot.h2e b(acLtoetriNaol.su1s9p4en5s6i4o4n3w,aOsXinOoIcuDlaLtted.d) toaLn-dtutbhee. bbaycttehreiaMlocnulotdureshwaakseri(ncMubOatNedOaSt 3I7N1A%-,0.T5aiCtecfoCro.8,hLwtidt)h shTahkeinvgiaabtl5e0ceflilmceosu/nmtisn (cNalocvualsapteecd,frLoKmBthJeapvaalnu)east wthhiecehndweorfiendceutbeartmiionnedaraet s6h6o0wanmbbelyows,pectrophotometry [Tao [tass TASS | TAIS3T [vNioa.bloefcells findg 21 a4 24 10m) eines | 21 | 21 |a0| 23 | 50] 5. 51 PREPARATION OF TEST SUBSTANCE AND POSITIVE CONTROLS Test substance 1) Preparation LTohteNtoes.t Ks6ubBs7t4a,ncOetswuaksadPihsasromlavecdeuitnicdaisltiFlalcedtowrayt)etro(mdiasktiell5edwwiavte%rcfoonrcennetcrtaitoino,n 2) and diluted with the same solvent to give appropriate concentrations. Stabilityof the test solution No denaturation of the test solution was observed exothermic reaction until 2 hours after preparation. for the color and. the 3) Preparation time 52 Prepared immediately before use and used within 0.5h at oom temperature, Positive controls 1) Preparation NaNy was dissolved in distilled water (Lot No. K6B74). 24A were dissolved in DMSO (Lot No. D069), ~AF-2, ICR-191 and o0R12 --6- KOL-1815 2) Preparation time and storage condition SParneypoa)r.ed on every 3 months and stored at -80C (ultra-deep freezer MDF-291, 6 METHODS The test was carried out for S. typhimurium strains TAIS3S, TAIS37, TA9S, TA100 and a E. coli strain WP2 uvrA using the pre-incubation method both with and without metabolic activation system. The plating was done in triplicate for 6.1 the negative control and in duplicate for the test substance and positive controls. Procedures After 0.1 ml of the test substance solution, 0.5 ml of 0.1 M sodium phosphate tbhueffmeirx(tpuHre7s.4w)erore $i9ncMuibxa,teadndfo0r.120mlmiofntahte 3ba7ct+eri0a.l5cCu.ltuTrweowemrle oafddtehde tsoofattaugbea,r `was then added to each tube and poured onto minimal glucose agar plate. After incubation for 48 h at 37 0.5C, the mumber of revertant colonies were counted. As the sterility test, each 0.1 ml of each bacterial suspension, test substance solution, $9 Mix or 0.1 M sodium phosphate buffer (pH 7.4) were smeared on a minimal glucose agar plate and incubated at 37 + 05C for 48 h, and then checked the bacterial contamination. Distilled water was used as a negative control, and the following positive controls were used for each bacterial strains. TAIO0 TAIS3S WP2uwd TASS __ TAISH SOMix(-) __AF-2 NaN; AF-2 AF2 ICR-191 001 0s 001 01 1 SOMix (+) __2AA 24A 244 2AA 244 1 2 10 0s 2 62 Dose selection (ng/plate) 1) Dose finding test The test was carried out at the highest dose of 5,000 ug/plate and 6 doses of 1,000, 500, 100, 50, 10nd S pg/plate. As a result, growth inhibition was observed at 5,000 ug/plate both with and without S9 Mix. 00213 --1- 01-1815 2) Main test Based on the results of the dose finding test, a main test was performed at the highest dose of 5,000 pg/plate and 5 lower doses diluted with a geometric progression of2. 7. MICROSCOPIC OBSERVATION AND COLONY COUNTING 7.1 Microscopic observation The stateofrevertant colonies (size and number of colonies), depositionofthe test substance and the growth inhibition were examined with a stereo microscope. 7.2 Colony counting `The number of colonies were counted with a manual counter or a colony analyzer (CA-7 or CA-9, Toyo-sokki Co., Ltd). Correction for counting errors was made for measurements with the colony analyzer. Each plate was measured three times, and the average of these three measurements was adopted as the number of revertant colonies on the plate. The average for each dose was calculated from the valuesofthe plates used. ~ Decimalsofthe average figures were rounded off. 8. INTERPRETATION OF RESULTS The test substance was judged to be positive, when the number of revertant colonies was twice or moreofthe negative control, and when the dose-relationship and the reproducibility were obtained. Any statistical procedures were not used. RESULTS `The numbers of their revertant colonies for all strains in groups which were treated with the test substance were less than twice that of each negative control with and without $9 Mix. The positive controls showed the distinct increase of revertant colonies, and the positive controls and the negative control were within a range of the background data in our laboratories. 000214 --8- K01-1815 The growth inhibition was observed at more than 2,500 ug/plate both with and without 59 Mix There were no fluctuations which affected the test results since the sterility test confirmed the absenceofany micro-organisms. CONCLUSION In conclusion, v -1 wasjudged to have no reverse mutagenic potential under the present test conditions, REFERENCES I. Ministry of Labor (1991) Guidebook on Mutagenicity Tests using Micro- organisms, New Edition (in Japanese) published by Japan Industrial Safety and Health Association, 2. Green MHL. and W.J. Muriel (1976) Mutagen testing using Trp* reversion in `Escherichia coli, Mutation Res, 38: 3-32. 3. Maron, DM, and BN. Ames (1983) Revised methods for the Salmonella `mutagenicity test, Mutation Res, 113: 173-215. 00215 --9- Dose finding test Test substance: y= ree ST COPY AVAILABLE vit) With(+) or |[cTeosmtesturbasttianocne|BealMsasberuboftireavteritaonntst(emmb| er of colporaiuessefpslhatiet)sve | Shr | Gehlw) w[100 [7h 1685 5 TA 98 [Tsh 1537 Czoo[BEmEfLwESaRlE [osDEo] e]tCn[2iScalct9ya] boa toa] 59 Mix [oo lo[r mei an]25m] on] eo [cw[hn]Sco Sn] oa] Oe oe CeEC[Ew | oe | : `rol | B a (am) u Hi) 08) [308 ETN EEEYHEIEIE ew [SeSaw Sw]] Em] 25 votive |b |wr|wn |wa| wea |ow| requiring i + colmieaplte 410 : : 8 oi Positive -- [wn | wn| wn| sain Cai] +|eTo [oo T+| A Kusber of 13 [ta 3 C0 [35 C0 [6 Co [32 com [190 Soles Pareatbsis shos the se of ah plate, #* : Observed bacterial growth inhibition. "AF-2: 2-(2-Furyl)-3-(S-nitro-2-furyl)acrylanide ET) 146 hk 2-tmimnanthranone "Nay: Sodiua azide 000216 -ICR-191: 2-Methoxy-6-chloro-8-(3-(2-chloroethy1)-asinopropylanino)acridine:2HCI Main test ees Test substance: v1 2 [fleFobhe WithCH) or |Test subatance vithut() | comentraion| Nash of revertants (omber of coloaieplate) Buse-pu mistitation re_|_Pranesbpiet_s| Shs | Gals) T[mmy 100 [tBn 1695|WPzu|Tvae98d| TBho 1637 E ETEE HEFE HE 59 Mix 13u2 s| 1 fw| %Amd FE0) 10 om [en[ECECw EC] Cw] sr am] SeSl 330) Ib am] 2 N HET FTEEE IE `negative |1g cs) H a EE wm or[[Et0ioown[BCoewFE%awlEimnCca]bBoonm]] [mo[ime]Sw]SC]Se] wi) IE FEIEEEI Poe | em | 02 |wn |wz|wz | wm| requiring op S00 |ma| 3 00 | 58Co [52 Co | Co [5 com | Positive ra nii aber platofe) 181 Sr [oi] gi(on[38 om [3 Com [28 Com[5 0| + hsered bacterial growth bition, Notes Parenthesis shows the mean of each plate. "AP-2: 2-(2-Puryl)-3-(5-nitro-2-furyl)acrylanide -Nak;: Sodiua azide -"I2CMR:-1821-:Asi2n-oMaentthohxrya-c6e-ncehloro-9-(3-(2-chloroethy16)-0as0in2op1ro7pylasino) acridine-2HCl Dose 15 finding test 35 10 g Eg I 2 KoL-1815 O:T 100 Da:iT4P21u5r0zs . 50 EE . 50 1000 Fig. 1 Dose-response curve without 9 Mix 5000 (ug/plate) 15 OT 100 CAHTPA2a15r3d5 Ta 2 25 2 50 50 10 501000 Fi.g. 2 Dose-response curve wi. th 9 Mix 600218 5000 (ug/plate) Dose 4 finding test 3 x i 2 01-1815 Oita 8 OTA 1857 . 510 5 10 01000 | Fig. 3 Dose-response curve without $9 Mix 5000 (ug/plate) O:4 8 Oi 1837 a 2 3 2g x 3 21 . 510 50 100 . 50 100 Fig. 4 Dose-response curve with 59 Mix 600219 5000 (ue/plate) Main test 151 5 10 8 3 32 Biissits O:TA 100 O:TA 1535 ANP2uvr 155 ais 625 1250 250 Fig. 5 Dose-response curve wi.thout $9 Nix (we/ite) fa 25 100 8 3 & O:TA 100 D:TA 1535 A:WP2urrd 156 Fi.g. 6 31 Dose-response 825 curve with 1250 $9 Mix 600220 2500 5000 (ug/plate) Main test 4 2a 2 i &u Miitts 0:14 98 O:TA 1537 156 3m 625 1250 2500 Fig. 7 Dose-response curve without 59 Mi. x 000 ) 5 4 2 oy 5 8 i 22 1 om 8 O:TA 1537 156 13 625 1250 Fig. 8 Dose-response curve with 9 Mix 00221 2500 (ug/5p00l0ate)