Document a1XKYKMdj51mkvjE7NGb6BpbB

M u t a g e n i c i t y T e s t i n g of 2-[N-ethyl-N-perfruoloalkyl(C=l~ 8) sulfonylami.no] ethylacrylate in B a c t e r i a l R e v e r s e M u t a t i o n A s s a y s (study number 2862) T " 32-2.5 pie P' | B M L , INC. QUALITY ASSURANCE CERTIFICATE T itle o f Stu dy : M u tagen icity testing o f2 -[N -e tk y l-N -p e ifru o lo a lk y l(C = l ~8 )sulfonylam ino] ethylacrylate in bacterial reverse m utation assays Study Number : 2 8 6 2 This study w as conducted in com pliance w ith * Standards to be O bserved b y M u ta gen icity Testing Institu tion s "<N o tifica tio n N o . 76, M in istry o f Labour, Japan, Septem ber 1,1988) and * G I F Standards applied to Industrial Chem icals " (N o tifica tio n N o . 39 o f the Planning and Coordination Bureau, Environm ent Agency, N o . 229 o f the Pharm aceutical A ffa irs Bureau, M in istry o f H ea lth an d w elfare & N o.85 o f the B a sic Industries Bureau, M in istry o f international Trade and Industry, Japan, 18,1988). I certify th at tire m ethods and procedures used in the test are described precisely in the fin a l report, and th at the reported results reflect the raw data accurately. The circum stances o f audits and inspections areas fo llo w s. D ate o f Q . A Item D ate o f R eport Inspection to Managem ent M ar. 23,1991 R eview o f S O P and protocol A p r. 4,1991 Apr. 10, 1991 General inspection A p r. 12,1991 A p r. 18,1991 R eview o f draft report and audit o f raw data A p r. 18,1991 A p r. 24,1991 Apr. 8,1996 R eview a ffin a l report (w ritten in Japanese) R eview a ffin a l report (w ritten in English) Apr. 24,1991 A p r. 8,1996 Q u a lity Assurance U n it Safety Evaluation Departm ent B M L , Inc. M asahiro Kuram odti T itle: Manager d p ,:/ S ', / ? 1 005260 QUALITY ASSURANCE STATEMENT T itle o f S tu d y : M utagen icity testing o f 2 -[N -eih yl-N -p erfru o lo a lk yl(C = l ^8) sulfon ylam ino] etkylacrylate in bacterial reverse m utation assays Study Number : 2 8 6 2 This report has been audited by Q u a lity Assurance Personnel and is considered to represent a true and accurate translation o f the original report N o . 2862, w ritten in Japanese, issued on A p r il 24,1996, p a rtia lly amended since M arch 8,1995,by B M L , In c . April . rfU P osition : M anager, Q u a lity Assurance U n it, Safety Evaluation Departm ent BM L, In c (Com pany Nam e and Tide) Name : M asahiro Kuram ochi (Type) (Signature) 005261 S T A T E M E N T OF S T U D Y D I R E C T O R Title of Study : A mutagenicity testing of 2-[N-ethyl-N-perfruoloalkyl (C=l~8)sulfonylamino]ethylacrylate in 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 Study Director, Mutagenesis Tests Division Position : BM1, INC. (Company Name and Title) Name : Yoshihiro Oguma (Type) 005262 Study number : 2862 Title of Study : A mutagenicity testing of 2-[N-ethyl-N-perfruoloalkyl (C=1-- 8)sulfonylamino]ethylacrylate in 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" (Notification No. 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" (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). -1- 0 0 5 2 6 3 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 : Yoshihiro Oguma Persons in charge of study : 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 005264 -2- 1. Summary Reverse mutation assays using S a l m o n e l l a t y p k i m u r i u m strains TA100, TA1535, TA98 and TA1537 and E s c h e r i c h i a c o l i WP2 uvrh. were conducted on 2-[N-ethyl-N-perfruoloalkyl(C=l~ 8)sulf onylamino]ethylacrylate 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)sulfonylamino] ethylacrylate was not mutagenic to bacteria under the experimental conditions. 3 2. Date of study Initiation of Initiation of Completion of the dose-finding test : March 28, 1991 the main test : April 9, 1991 the main test : April 12,1991 3. Test substance (1) Test substance 1) Name{Another name} : 2-[N-ethyl-N-perfruoloalkyl(C=l~8)sulfonyl 2) BML-code amino]ethylacrylate :BML-1107 {Sample D-l} 3) Lot No, :103 4) Purity :greater than 99 % wt/wt 5) Name and concentration of impurities : phenothyadine 60 20ppm hydroquinonemonomethylether 6 ) Structural formula : 170 35ppm C 2H 5 0 CnF z n + i S O z N - C H zC H zO-- C-- C H = C H z n=l~ 8 n = 8 (main component) : ca. 78 % n = 1 ~ 7 : ca. 21 % (total) 7) Molecular weight : 625 8) Appearance at room temperature : amber wax 9) Melting point :27-42C 10) Boiling point :ca.l50C (ImmHg) 11) Vapor pressure : information not provided 12) Solubility :Oil soluble 13) Degree of solubility Water : insoluble Dimethylsulfoxide (DMSO) : insoluble Acetone : over 50 % 14) Partition coefficient : information not provided 15) Stability : stable The test substance was analysed and all data are retained by the client. 005266 -4- (2) Solvent Name Manufacturer Lot No. Purity Acetone Wako Pure Chemical Industries, Ltd. ECG2489 greater than 99% (Special grade 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(ug/plate) with S9(ug/plate) TA100 TA1535 TA98 TA1537 WP2 u v r A AF-2 (0.01) NaNs (0.5 ) AF-2 (0.1 ) ICR-191 (1.0) AF-2 (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,Inc.; 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). 005267 -5- 5. Materials (1) Tester strains 1) Strains used 11 2 ) 3> Base-pair substitution type : S . t y p h i m u r i u m TA100, TA1535 E . c o l i WP2 uorA Frameshift type : S . t y p h i m u r i u m TA98, TA1537 2) Supplier and date obtained S . t y p h i m u r i u m TA100 : National Institute of Hygienic Sciences, Japan; April 15, 1982 S . t y p h i m u r i u m , other TA strains : Dr. Ames, U.C. Berkeley, CA, U.S.A.; January 20, 1988 E . c o l i WP2 uurA : 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 -80C . (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: Phnobarbital(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) 005268 -6- 2) Cofactor Name: Supplier: Lot No.: Cofactor Boehringer Mannheim Yamanouchi K.K. 711 3) Composition of S9 Mix (per 1ml) S9 fraction 0.1 ml MgClg 8.0 jumol KC1 33.0 pmol G-6-P 5.0 umol NADPH 4.0 pmol NADH 4.0 |umol Na-Phosphate buffer (pH 7.4) 100.0 pmol (3) Minimum glucose agar plates Name Supplier Lot No. Prepared on Purchased on Storage : Minimal glucose agar plates M : 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(lOOmg/ml) and half(0.05ml) of this solution was added to reduced the toxicity of Acetone. Its solution was stored in refrigerator just 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-finding test, growth inhibition was not found in all strains with or without metabolic activation. Accordingly, we selected 5000 ug/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. 005269 -7- (3) Test procedure (preincubation method) 4]'55 '65 The cells of each strain were inoculated into a culture tube containing 10 ml of Nutrient Broth No.2 (Oxoid) and these were cultured to the early stationary phase of growth, shaking(lOOrpm) for 8 h at 37C . For tests without metabolic activation, 0.5ml of 0.1M Na-phosphate buffer(pH7.4) was added to each tube containing 0.05 ml 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 0.1M Na-phosphate buffer(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 37C . 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 37C 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% NaCl) that were melted by warming just prior to use, were 0.5mM L-histidine and 0.5mM biotin for the 5. t y p h i m u r i u m strains, and 0.5mM tryptophan solution for the E . c o l i strain. All plating were done in duplicate. 7. Criteria of evaluation 5) 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.l, 2. The figures were prepared from Table 2. In the dose-finding tests 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 inhibition was found in any strain. Precipitate on the plates was found at over 313 \ ig fplate without metabolic activation and over 1.2 pg/plate with metabolic activation. The sterile tests performed on both the test substance and the S9 mix indicated no bacterial contamination throughout this experiment. 005270 9. Deviation from protocol,etc. During the study period, there were no environmental factors considered to have affected the reliability of 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 forthe 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) M.H.L. Green and W.J. Muriel: Mutagen Testing using Trp+ Reversion in E s c h e r i c h i a c o l i , 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, i n New Guidebook of Mutagenicity Tests Using Microorganisms, Japan Industrial Safety and Health Association, 1986 (6) Y. Tajima, T. Yoshida, and T. Kada, i n Screening for Detection of Chemical Mutagens, Koudansha Co., 1973 005271 -9- Table L T a b l e of Dose F i n d i n g T e s t R e s u l t s Name of test substance : 2-fN-ethyl-W-perfruoloalkyl (C*l~8)sulfonylamino1ethylacrylate No. 2 8 6 2 Nith(+)or Test substance without(-) concentration S9Mi jc (it g/plate) Solvent control 1. 2 4. 9 S 9M i x (-) 20 78 Number of revenan ts (number of colonies/plate) Base--pair substitution type Frameshift type TA 100 153 174 ( 164) TA 1 5 3 5 14 21 ( 18) TP 2 u v r A 25 16 ( 21) TA 9 8 31 24 ( 28) TA 1537 10 12 ( 11) 160 177 ( 169) 150 175 ( 168) 18 19 ( 19) 25 20 ( 23) 27 ( 25) 25 16 ( 21) 17 22 ( 20) 21 18 ( 20) 14 14 ( 14) 11 10 ( 11) 165 158 ( 162) 22 24 ( 23) 27 29 ( 28) 20 24 ( 22) 8 9 ( 9) 149 150 ( 150) 19 20 ( 20) 27 33 ( 30) 26 24 ( 25) 8 13 ( 11) 3 13 141 it 159 t ( 150) 14 # 11 if ( 13) 22 t 23 It ( 23) 26 # 23 # ( 25) 12 # U it ( 12) 12 5 0 5000 Solvent control 1. 2 141 # 157 # ( 149) 145 # 180 It ( 163) 145 132 ( 139) 120 # 153 ( 137) 21 # 14 i ( 16 it 15 t ( 19 21 ( 22 t 10 # ( 18) 17) 20) 16) 21 it 28 if ( 29 it 20 t ( 30 31 ( 29 if 30 It ( 25) 25) 31) 30) 17 it 14 it ( 22 it 24 it ( 28 27 ( 22 it 26 # ( 16) 23) 28) 24) 7 it 7 it ( 10 11 it ( 16 18 ( 21 it 21 t ( 7) 11) 17) 21) 4. 9 180 t 135 # ( 158) 26 # 13 if ( 20) 28 it 20 f ( 24) 19 # 25 it ( 22) 21 it 18 if ( 20) S 9M i x 20 112 if 152 if ( 132) 20 if 18 t ( 19) 23 it 24 f ( 24) 35 it 28 it ( 32) 20 it 21 # < 21) (+ ) 160 # 20 if 25 if 31 it 20 # 78 145 if ( 153) 14 t ( 17) 31 t ( 28) 31 it ( 31) 19 # ( 20) 3 13 147 if 152 # ( 150) 13 i 22 if ( 18) 24 it 28 # ( 26) 31 it 28 # ( 30) 14 it 22 if ( 18) 12 5 0 129 it 117 t ( 123) 16 it 14 if ( 15) 30 28 # ( 29) 24 it 32 it ( 28) 16 S 21 if ( 19) 5000 142 if 138 t ( 140) 22 if 20 if ( 21) 25 if 31 it ( 26) 25 it 25 it ( 25) 17 it 15 # ( 16) Positive control not requiring SUMix Name Concentration ( it g/plate) Number of colonies/plate AF- 2 0.01 7 694 ( 709) N a N , *' 0. 5 294 234 ( 264) AF- 2 0.01 135 123 ( 129) AF- 2 ICR-191" 0. 1 570 568 ( 569) 1. 0 1019 1099 ( 1059) Positive control requiring J 9ili1X Name Concentration (it g/piate) Number of colonies/plate B [ a] P " 5. 0 896 789 ( 843) 2 A A ** 2. 0 185 176 ( 181) 2 AA 10.0 355 378 ( 367) B [a] P 5. 0 21 2 174 ( 193) B C'a] P 5, 0 99 102 ( 101) #1:2--(2--Fury1)--3--(5--n i tro--2--furyljaerylamide #2:Sodium azide >3:2-Methoxy-6-chloro-9-[3-(2-chloroe thy Dami nopropy lanino] acri dine -.2HC1 it4:Benzo[a]pyrene #5 : 2-Aminoanthracene #:The test substance was found precipitating on the plates. Notes:The average number of colonies in each concentration is shown in the ( ). 005272 Table 2 T a b l e of T e s t R e s u l t s Maine of test substance : 2-[M-ethyl-M-perfruoloa1kyl(C*l-~8)sulfonylaminoTethylacrylate No. 2 8 6 2 tfith (+)or Test substance without(-) concentration 39Mi x { / i g/plate) Solvent control 3 13 S 9M i x 625 Number of revenants (number of olonies/plate) Base-pair substitution type TA 10 0 TA1535 WP 2 u V r A HO 132 ( 136) 19 18 ( 19) 23 35 ( 29) 152 # 130 ( H I) 16 9 29 9 ( 23) 26 9 22 9 ( 24) 138 9 147 9 ( 143) 22 9 17 9 { 20) 18 9 31 9 ( 25) Frameshift type TA9 8 T A 15 3 7 22 31 ( 27) 3 12 ( 8) 26 34 9 ( 30) 79 8 . (. 8) 32 9 24 9 ( 28) 7* 6 9( 7) (-) 12 5 0 2500 5000 Solvent control 143 9 137 9 ( 140) 136 9 137 9 ( 137) 112 # 95 9 ( 104) 146 145 ( 146) 14 9 20 9 ( 17) 20 9 25 9 ( 23) 22 9 20 9 ( 21) 20 13 ( 19) 28 9 26 9 ( 28 9 31 9 ( 21 9 36 9 ( 27 25 ( 27) 30) 29) 26) 28 9 23 9 ( 30 9 26 9 ( 28 9 36 9 ( 38 41 ( 26) 26) 32) 40) 14 9 7 t{ 54 7 *( 69 7 *( 17 21 ( 11) 6) 7) 19) 3 13 133 9 119 9 ( 126) 14 9 IS 9 ( 15) 37 9 27 9 ( 32) 40 9 39 (4L it 40) 19 9 22 ( 21) S 9M i x 625 135 9 146 9 ( 141) 11 9 27 9 ( 19) 28 9 35 9 ( 32) 32 9 26 9 ( 29) 18 i 15 9 ( 17) (+ ) 12 5 0 151 9 144 9 ( 148) 15 9 13 9 C 14) 28 9 25 9 ( 27) 41 9 44 9 ( 43) 19 9 15 4 ( 17) 2500 123 9 141 9 < 132) 17 9 17 9 ( 17) 23 9 39 9 ( 31) 39 9 37 9 ( 38) 18 9 17 9 ( 18) 5000 Positive control not requiring S 9M1x Name Concentration ( u g/plate) Number of colonies/plate Positive control requiring S9Mix Name Concentration (i g/plate) Humber of colonies/plate 135 # 141 9 ( 138) A F - 2 f1 IS 9 22 9 ( NaN, 20) 1 30 9 26 9 ( 28) . A F -- 2 0. 1 623 554 ( 589) B [ a 1 P *' 05 336 366 ( 351) 2AA 0. 1 139 149 ( 144) 2AA 50 867 819 ( 843) 20 181 155 ( 168) 1 0. 0 569 585 ( 577) 35 9 27 9 ( 31) AF 2 10 94 9( 7) I C R - 1 1 ** 0i 479 366 ( 433) B [ai P 1. 0 1057 1138 ( 1098) B [a] P 50 171 160 ( 166) 5. 0 125 101 ( 113) Jfl:2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide #2:Sodium azide t3:2-Methoxy-6-chloro-9-[3-(2-ebloroethyl)aminopropylaino]acridine'2HCl #4;Benzo[a]pyrene #5:2-Aminoanthracene #:The test substance was found precipitating on the plates. Notes:The average number of colonies in each concentration is shown in the ( ). BEST COPY AVAILABLE 005273 Fig. 1 (Colonies/Plate) 200 P Dose response curve N o .2J362 (S 9 - ) 180 oTAlOO TA1535 160 aWP2uvrA 140 120 100 80 - 60 - 40 20 0 H V ---------- 1------------- 1------------- 1------------- '--------------1 0 313 625 1250 2500 5000 ( i g/plat Fig. 2 (Colonies/Plate) Dose-response curve N o . 2862 (S9+)