Document NeqMeJ810X0O2X69dpxDgDXNw

M u ta g e n ic i ty T e s t 1n g o f 2-[N-ethyl-N-perfruoloalkyl(C=I- B)sulfonylaminolethylacrylate in B a c t e r ia I R e v e r s e M u ta t io n A s s ay s (study number 2862) T- C" B M L, I N C. NOV25 iN6 QUALITY ASSURANCE CERTIFICATE TitkofSft4:Muftgndcftfyttixogf24N-etlail-N-perfmoloafti-(8C)-s1uybnylanduol eftlacrylatienbacteriranlwse nmtationassays StudyNumber: 2 8 6 2 77dsstudywas condww incompliaumwith ' Stosdffdtsobe Observedby muwemfdty Testing '(NodftcatiNmo.74 Mhds" ofLabour,lqwr, Septembe1r,JM) and 4 GLp Standards apphedtoln&wbw Chmdcab " (NofificatNiom.39 of* Plwntbg andCom*n@on Bwr^ onnavdA8mscyN,a 229ofdoe AffabyBwza@ Mims" ofHedtisand welfar&e No.85ofdw Bask bdxs&iesBweam,Mbdmy offtwmtimd Tradeandbs&bft,yjapa,418,I_W). Imtify dW thexwdwds andpnxzdwn usedinthetator desafiwpnx-&elyinthefiuwmpor4 and thatdw reporterdesultmsflectdw raw dataaccuratek 77#ecbcunotanceosfauditsand hopectlemamaofouows. Dateof0, A. Item DateofReportInspection toMaxagmext Mar.23,1991 ReviewofSOP oW pvwwl Apr.4 1991 Apr.10,1991 Gewratinspection Apr.1Z 1991 Apr.18,1991 Revww Of&4t F"Wt and auditofraw data Apn 18,1991 Apr.24,1991 Apr.8,1996 Reviewoffixdmpott (mitteninjapamw) Reviewoffmalreport (wruft inEqiisis) Apr.24,IM Apr.8,1996 QualityAnurm" Unit S*V EvaixotioDxepwbnad BM7,,hr- Masaki*-Kowamocki TitleAfamagff Date: I.-@ QUALITY ASSURANCE STATMEIVT TitloefStWy Muftgmicitfytft of24N-efti-N-pofmoloafi@y8l)(sCu-l1fonylandnol eftlaaylatienbactLWamtvem nmtatioanssays Sb* NxmbL-r2: 8 6 2 Thisreporhtasbem auditebdyQualitAynmwKe Persomwalndn considerteodrepresean&tw andacmatetramiatioofndw origin-narlmt No.2862w,y injaponw,issueodnApri2l4,19K pottauymwndedsbw Afmrh8,1995,bByAH,L-m. Date.: 4.pt,il Mana8er,QualitAysswma Umt,SafetEyvaluatiDoenpoftmt Position:BML I= (ComponjNanwandTitk) Nwm: MasahirKortramocki (TYM) (signahm) STATEMENT OF STUDY DIRECTOR Title of Study : A mutagenicity testing of 2-[N-ethyl-N-perfruoloalkyl (C=l- 8)sulfonylaminolethylacrylatein bacterial reverse mutation assays Study Number : 2862 This is to certify that the above study was conducted in compliance with "Standards to be Observed by Mutagenicity Testing Institutions" (Notification No.76, Ministry of Labour, Japan, September 1, 1988) and "GLP Standards Applied to Industrial Chemicals" (Notification No.39 of the Planning and Coordination Bureau, Environment Agency, No.229 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare & No.85 of the Basic Industries Bureau, Ministry of International Trade and Industry, Japan, November 18, 1988). Date Position Study Director, Mutagenesis Tests Division BM1, INC. (Company Name and Title) Name Yoshihiro Oguma (Type) (Signaturxlr Study number : 2862 Title of Study : A mutagenicitytesting of 2-[N-ethyl-N-perfruoloalkyl (C=l- 8)sulfonylamino]ethylacrylatien bacterial reverse mutation assays Purpose of the test: To assess the effect of the test substance in bacterial reverse mutation assays. This study was performed in accordance with the guidelines set forth in "Standards for Mutagenicity Tests using Microorganisms"(Notification No.77, Ministry of Labour, Japan, September 1, 1988) and "Guidelines for Toxicity Testings of Chemicals" (NotificationNo. 237 of the Planning and Coordination Bureau, Environment Agency, No. 306 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare & 303 of the Basic Industries Bureau, Ministry of International Trade and Industry, Japan, March 31, 1987). And it was conducted in compliance with "Standards to be Observed by Mutagenicity Testing Institutions"-(NotificationNo.76, Ministry of Labour, Japan, September 1, 1988) and "GLP Standards Applied to Industrial Chemicals" (Notification No.39 of the Planning and Coordination Bureau, Environment Agency, No.229 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare & No.85 of the Basic Industries Bureau, Ministry of International Trade and Industry, Japan, November 18, 1988). Sponcer Name Address Sumitomo 3M Limited 3-8-8 Minamihashimoto, Sagamihara-shi, Kanagawa 229, Japan Testing facility Name Address BML,INC. 1361-1 Matoba, Kawagoe-shi, Saitama 350-11, Japan Date of test Initiation March 18, 1991 Completion April 24, 1991 Persons in.charge Study Director Persons in charge of study Yoshihiro Oguma Shigemi Kimura, Youko Hasegawa Name of the person who prepared the final report, and date of preparation Yoshihiro Oguma Title: Study Director Date : April 24, 1991 I certify that this study was conducted under the specified protocol. Munehiro Terasoma Title: Administrator Date : April 24, 1991 -2- 1. Summary Reverse mutation assays using Salmonella typhimurium strains TAIOO, TA1535, TA98 and TA1537 and Escherichia coli WP2 uvra were conducted on 2-[N-ethyl-N-perfruoloalkyl(C=l- 8)sulfonylaminolethylacrylate with and without metabolic activation. As a result,'the test substance did not induce significant revertants to be compared with the solvent controls and dose-dependent increase was not found in any strains with or without metabolic activation. It was concluded that 2-[N-ethyl-N-perfruoloalkyl(C=1-8)sulfonylaminol ethylacrylate was not. mutagenic to bacteria under the experimental conditions. 2. Date of study Initiation of the dose-findingtest : March 28, 1991 Initiationof the main test : April 9, 1991 Completion of the main test : April 12, 1991 3. Test substance (1) Test substance 1) NamelAnother namel 2-[N-ethyl-N-perfruoloalkyl(C=l8-)sulfonyl amino]ethylacrylate (Sample D-11 2) BML-code BML-1107 3) Lot No. 103 4) Purity greater than 99 % wt/wt 5) Name and concentrationof impurities' phenothyadine 60 20ppm hydroquinonemonomethylether 170 35ppm 6) Structuralformula : CzHa 0 1 11 C.F,,.,SO,N-CH,CHzO-C-CH=CH2 n-l- 8 n= 8 (main component) : ca. 78 % n=l - 7 : ca. 21 % (total) 7) Molecular weight 625 8) Appearance at room temperature : amber wax 9) Melting point : 27-42*C 10) Boiling point : ca.150*C(ImmHg) 11) Vapor pressure : information not provided 12) Solubility : Oil soluble 13) Degree of solubility Water : insoluble Dimethylsulfoxide (DMSO) : Insoluble Acetone : over 50 % 14) Partitioncoefficient : information not provided 15) Stability : stable The test substance was analysed and all data are retained by the client. -4- (2) Solvent Name Manufacturer Lot No. Purity : Acetone : Wako Pure Chemical Industries, Ltd. : ECG2489 : greater than 99% (Specialgrade of JIS standard) 4. Controls (1) Negative control Negative controls were treated with Acetone only. (2) Positive control compounds The names and concentrations of these compounds are given in the table below. Strain without S9(og/plate) with S9(mg/plate) TA100 TA1535 TA98 TA1537 WP2 uvra AF-2 NaN3 AF-2 ICR-191 AF-2 (0.01) (0.5) (0.1) (1.0) (0.01) B(a)P 2AA B(a)P B(a)P 2AA (5.0) (2.0) (5.0) (5.0) (10.0) AF-2 NaN3 ICR-191 2AA B(a)P 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (Wako Pure Chemical; Lot No. LAJ1702; Special grade of JIS standard, Purity greater than 95 %) Sodium azide (Wako Pure Chemical; Lot No. DCJ6398; Special grade of JIS standard, Purity greater than 95 %) 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino] acridine -2HC1 (Polysciences,lnc.;Lot No. 52300; Purity greater than 95 %) 2-Aminoanthracene (Wako Pure Chemical; Lot NO. KPQ0892; Special grade of JIS standard, Purity greater than 95 %) Benzo(a)pyrene (Wako Pure Chemical; Lot No. AWK3751; Special grade of JIS standard, Purity greater than 95 %) Sodium azide(NaN3) was dissolved In distilled water(Hikari Pharmacy Co., Ltd.;Lot No. 9003VA), the other compounds were dissolved in DMSO (Wako Pure Chemical Industries Ltd.;Lot No. AWE6768, ECM4254). Compounds mutagenic to each strain were used for positive controls; these were chosen according to the "New Guide Book of Mutagenicity Test Using Microorganisms," the Ministry of Labour, Japan (1986). -5- 5. Materials (1) Tester strains 1) Strains used Base-pair substitutiontype S.typhimurium TA100, TA1535E.coli WP2 uvra Frameshift type S.typhimurium TA98, TA1537 2) Supplier and date obtained S.typhimurium TA100 National Institute of Hygienic Sciences, Japan; April 15, 1982 S.typhimurium, other TA strains Dr. Ames, U.C. Berkeley, CA, U.S.A.; January 20, 1988 E.coli WP2 uvra Department of Molecular Oncology, Institute of Medical Science, University of Tokyo. Japan; November 24, 1987 3) Reason for choice These strains are well established as sensitive to many classes of mutagens and are widely used. 4) Condition of storage The cell suspension was mixed with dimethylsulfoxide(spectrophotometric grade in a ratio of 0.07 to 0.8 volume and stored at -80*C. (2) S9 Mix 1) S9 Name: S9 Supplier: Kikkoman Co. Lot No.: RAA-248 Prepared on: January 11, 1991 Purchased on: January 25, 1991 Species, Strain: Sprague-Dawley Rat Sex: Male Age: 7 weeks Weight: 199-246g Inducing substance: Phenobarbital(PB) and 5,6-benzoflavone(BF), i.p. injection Dose: PB=30+60+60+60 mg/kg (4 days); BF=80 mg/kg(l day) -6- 2) Cofactor Name: Supplier: Lot No.: Cofactor Boehringer Mannheim Yamanouchl K.K. 711 3) Compositionof S9 Mix (per Iml) S9 fraction 0.1 ml MgCl2 8.0 4mol KC1 33.0 gmol G-6-P 5.0 gmol NADPH 4.0 mmol NADH 4.0 Amol Na-Phosphate buffer (pH 7.4) 100.0 Amol (3) Minimum glucose agar plates Name Supplier Lot No. Prepared on Purchased on Storage : Minimal glucose agar plates N : Nisshin Flour Milling Co., Ltd. : BM020CG : March 1, 1991 : March 11, 1991: stored at room temperature 6. Methods (1) Preparation of the test substance By reason of Information from sponsor, the test substance was dissolved and tested in Acetone. The concentration of the substance was doubled(100mg/ml) and half(O.05ml) of this solution was added to reduced the toxicity-ofAcetone. Its solution was stored in refrigeratorJust prior to use. (2) Dose selection The dose range was determined from the dose-finding tests using the following seven dose levels: 1.2, 4.9, 20, 78, 313, 1250 and 5000 ug/plate. In the dose-findingtest, growth inhibitionwas not found in all strains with or without metabolic activation. Accordingly, we selected 5000 ag/plate as the maximum dose for all strains tested with or without metabolic activation. So for all tester strains,we used five doses serially 1 : 2 were generally used. -7- (3) Test procedure (preincubation method) 4),5).6) The cells of each strain were inoculated into a culture tube containing 10 al of Nutrient Broth No.2 (Oxoid)and these were cultured to the early stationary phase of growth, shaking(100rpm)for 8.h at 37*C. For tests without metabolic activation, 0.5ml of O.IM Na-phosphate buffer(PH7.4) was added to each tube containing0.05 al of the test substance, and 0.1 ml of each fresh bacterial medium was added to each test tube. For tests with metabolic activation, 0.5ml of S9 mix Instead of 0.5ml of O.lM Na-phosphatebuffer(pH7.4) was added to each tube containing 0.05 ml of the test substance. The mixture was preincubated for 20 min in a water bath at 37*C. 2 ml of top agar was then added to the mixture, and the contents of each tube were mixed and poured over the surface of the minimum glucose agar plate. The overlay agar was allowed to solidify at room temperature. All plates were incubated for 48 h at 37*C and then the number of revertant colonies were counted. Afterward, bacterial growth inhibition was checked using a stereoscopic microscope. Added 1:10 to the top agar (0.6% Bacto-agar, 0.5% NACI) that were melted by warming just prior to use, were 0.5mM L-histidine and 0.5mM biotin for the.S. typhimuriumstrains, and 0.5mM tryptophan solution for the E. coli strain. All plating were done in duplicate. 7. Criteria of evaluation If a significant, reproducible increase in the number of revertants (twice that of the solvent control) was detected in at least one of the test points, or a reproducible dose-related increase occurred over more than one point, the results would be considered positive. Statistical method was not used as an aid in evaluating the test results. 8. Results and discussion The test results are shown in Tables 1, 2 and Figs.1, 2. The figures were prepared from Table 2. In the dose-findingtests and the main tests, the test substance did not induce revertants more than twice that of the solvent controls and dosedependent increase was not found In any strains with or without metabolic activation. The positive controls showed an Increase in the number of revertants compared to the solvent controls, indicating that this study was performed properly. From these results, it was concluded that this test substance was negative. No growth inhibitionwas found in any strain. Precipitate on the plates was found at over 313 gg/plate without metabolic activation and over 1.2 gg/plate with metabolic activation. The sterile tests performed on both the test substance and the S9 mix indicated no bacterial contamination throughout this experiment. -8- 9. Deviation from protocol,etc. During the study period, there were no environmental factors considered to have affected the reliabilityof the data, and there was no deviation from the protocol. 10. Conclusion From the above results, it was concluded that 2-[N-ethyl-N-perfruoloalkyl (C=l- 8)sulfonylamino]ethylacrylate was non-mutagenic in bacterial mutation assays under the experimental conditions. 11. Storage of the records and samples All records and samples generated during this study (listed below) will be stored in the archival area of BML.,INC.for 10 years after the tests. At the end of the 10-year period, the sponsor will be consulted regarding the disposal or continued storage of the records and samples. 1) Protocol and Amendments 2) Records concerning the test substances 3) Raw data 4) Records on the Activities of Quality Assurance Unit 5) Test substance 6) Draft of the final report 7) The final report 12. References (1)B.N. Ames, F.D. Lee, and W.E. Durston: An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens, Proc. Natl. Acad.Sci., USA, 70, No.3, pp.782-786, March 1973 (2) J. McCann, N.E. Spingarn, J. Kobori, and B.N. Ames: Detection of Carcinogens as Mutagens: Bacterial Tester Strains with R Factor Plasmids, Proc. Natl Acad. Sci., USA, 72, No.3, pp.979-983, 1975 (3)N.H.L. Green and W.J. Muriel: Mutagen Testing using Trp' Reversion in Escherichia coli, Mutation Res., 38, pp.3-32, 1976 (4) D.M. Maron and B.N. Ames: Revised Methods for the Salmonella Mutagenicity Test, Mutation Res., 113, pp.173-215, 1983 (5)Japanese Ministry of Labour, in New Guidebook of Mutagenicity Tests Using Microorganisms, Japan Industrial Safety and Health Association, 1986 (6)Y. Tajima, T. Yoshida, and T. Kada, in Screening for Detection of Chemical Mutagens, Koudansha Co., 1973 -9- T a b Ie I T ab I c o f D o s e F in d in g T e s t R e s u I ts Name of test substance 2-rN-ethyl-H-perrfucloaikyl(Cxl-g)sulfonylasirlolethlyacrylate No.2 8 6 2 With(+)or Test substance without(-)concerltration somix (g &/plate) TAIOO Numberof revertants(numberof colonies/plate) Base-pairsubstitutiontype TA1535 WP2uvrA Frameshift type TA98 TAIS37 Solvent 153 it 25 control 174 164) 21 18) 16 31 10 21) 24 ( 28) 12 iso 18 23 17 14 1. 2 177 169) 19 19) 27 25) 22 ( 20) 14 14) 25 25 21 11 4. 9 11i 15 168) 20 ( 23) 16 21) 18 ( 20) 10 11) 165 22 27 20 8 S 9 M Ix 20 158 162) 24 ( 23) 29 28) 24 22) 9 9) 149 19 27 26 8 78 150 150) 20 ( 20) 33 30) 24 25) 13 11) 313 141 14 t 22 t 26 t 12 It 159 150) 11 13) 23 # ( 23) Z3 * 25) 11 1 12) 1250 141 t 21 9 21 1 17 t 7t 157 1 ( 149) it t Is) 28 t ( 25) 14 t 16) 7 7) @5L a 0 0 Solvent control 1. 2 145 # 190 # ( 163) 145 132 ( 139) 120 s iss $ ( 137) 18 9 15 t 17) 19 21 ( 20) 22 1 10 1 ( 16) 29 s 20 S ( 25) 30 31 ( 31) 29 4 30 t ( 3D) 22 t 24 t ( 23) 28 27 ( 28) Z2 * 26 t ( 24) 10 1 11 t 11) 16 is ( 17) 21 4 21 # ( ZI) 4. 9 180 4 135 # 158) 26 # 13 4 28 @* 20) 20 : ( 24) 19 # 25 # ( 22) 21 # 18 # ( 20) S 9 M ix 20 112 # 20 9 23 9 35 4 20 # .152 9 132) 18 s 19) 24 9 ( 24) 28 t ( 32) 21 t ( 21) 160 20 9 25 # 31 t 20 78 145 1 153) 14 $ ( 17) 31 : ( 28) 31 9 ( 31) 19 # ( 20) 3 13 147 # 13 s 24 31 9 14 t 152 # 150) 22 t ( 18) 28 t ( 26) 28 9 ( 30) 22 4- is) 12 5 0 129 1 16 1 30 1 24 t 16 4 117 9 123) 14 ; ( is) 28 4 ( 29) 32 t ( 28) 21 4 19) 5000 142 22 S 25 # 25 9 17 138 t 140) 20 9 ( 21) 31 4 ( 28) Z5 4 ( 25) 15 16) Positive control not requiring sgmix N am e Concentration (,ag/pIa te) Numboefr colonies/plate A F- 2 0 01 723 694 709) NN 05 294 234 264) A F- 2 0 01 135 123 129) Positive control requiring s9mix N am e Concentration (,ug/plate) Numb e,r of Colo Ie 3/plate I BaP 5. 0 896 789 843) 2 A A as 2A A 2. 0 185 176 10. -3 is,) 5758 0 367) i #1:2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamti2d:eSodium azide $3:2-idetboxy-6-chloro-g-[3-(2-chloroethaymli)nopropylamino]acridine-.ZHCI ,t4:Benzo(alpyrene $5:2-Aminoanthracene AF- 2 01 5TO 568 569) a Ca P 5 212 IT4 0 193) 1cR - 1 9 1 10 9 10199 1059) B [a P 5 99 102 0 101) # :The test substancewas found precipitatingon the plates. No tes:Theaveragenumber of colonies in each concentrationis shown in the T a b Ie 2 T a b I e o t T es t R es u I ts Name of test substance:2-[?(-ethyl-M-perrfuoloaIkyl(C-1-B) suIfonylamino]ethyiacryIate No.2 8 6 2 lith(+)orTest substance without(-)concentration sgmix u &/plate) Number of revertants(number of colonies/plate) Base-pairsubstitutiontype TAIOO TA1535 WP2uvrA Frameshift type TA98 TA1537 Solvent 140 19 23 22 3 control 132 ( 136) Is 19) 35 ( 29) 31 ( 27) 12 8) 313 152 S is s 26 s 26 t 7 130 4 ( 141) 29 t 23) 22 9 ( 24) 34 9 ( 30) 8 138 # 22 # is t 32 s 7 S 9M 1 x 625 147 # ( 143) 17 4 ( 2D) 31 t ( 25) 24 t ( 28) 6 7) 143 4 14 t 28 9 28 4 14 1 250 137 t ( 140) 20 # ( 17) 26 9 ( 27) 23 4 ( 26) 7 11) 136 # 20 t 28 9 so s 5 2 500 137 # ( 137) 25 t ( 23) 31 t ( 30) 26 # 28) 7 6) 112 # 22 9 21 2 28 9 6 5 00 0 95 s ( 104) 20 ( 21) 38 t ( 29) 36 t 32) 7 7) Solvent 146 20 27 38 17 control 145 146) 18 ( 19) 25 26) 41 40) 21 19) 313 133 14 37 9 40 119 126) 16 t ( 15) 27 # 32) 39 19 1 40) 22 21) S 9 M Ix 625 135 11 s 28 t 32 is 146 4 141) 27 S ( 19) 35 S 32) 26 4 29) 15 17) 1 2 50 151 4 144 # 148) 2500 123 # 141 # 132) 5 000 135 # 141 # 138) Positive control not requiring s9mix N am e Concentration (.ag/plate) Number of colonies/plate A F- 2 0 01 623 554 589) Positive control requiring s9mix N am e Concentration (A &/plate) Number of colonies/plate , BaP 5. 0 $67 819 843) is s 13 9 ( 14) 17 : 17 S ( 17) is # 22 t ( 20) NaN 0. 5 336 366 2AA 351) of 2. 0 181 155 168) 28 # 25 : 27) 23 9 39 t 31) 30 t 26 t 28) A F- 2 0. 0 1 139 149 144) 2AA 10. 0 569 535 577) Al 4 44 4 ( 43) 19 4 15 4 ( 17) 39 t 37 9 ( 38) 18 # 17 ( 18) 35 # 10 27 4 ( 31) 4 7) A F- 2 1 C R - 1 9 1 '3 0. 1 479 386 433) B P 1 1057 1138 B 1098) P 5. 0 171 160 166) 5 125 101 0 113) 91:2-(2-Furyl)-3-(5-nitra-2-furyl)acrylam;i2d:eSodium azide lt3:2-Metboxy-6-chloro-g-(3-(2-eblaroetbyl)aainopropylaminalacridine-ZRCL #A:Benzotalpyrene $5:2-Aminoanthracene #:The test substancewas found precipitatingon the plates. Notes:The averagenumber of colonies in each concentrationis shown in the Fig. 1 (Colonies/Plate) 200 - Dose response 180 - 160 - 140 120 100 80 60 40 20 0 0 313 625 1250 curve No-2862 s9 oTA100 sTA1535 OWP2uvrA *'TA 9 8 +TA1537 2500* 5000(g g/plate' Fig. 2 (Colonies/Plate) 200 - Dose response 180 160 - 140 120 - 100 - 80 - 60 40 20 0 0 313 625 1250 cu rv e No.2862 s 9+ OTA100 sTA1535 13WP2uvrA *TA98 +TA1537 2500 5000 u g/plate)