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H- NOTOX Safety & Environmental Research B.V. REPORT MW - m T EVALUATION OF THE MUTAGENIC ACTIVITY OF T-5874 IN THE AMES SALMONELLA/MICROSOME TEST (WITH INDEPENDENT REPEAT) NOTOX Project 115932 NOTOX Substance 38187 - page 1 of 23 - 004472 r e c e iv e d NOV - 9 1994 Hambakenwetering 3 TO AICO LO G Y P.O. Box 3476, S23 DL 's-Hertogenbosch. The Netherlarrs Tel.: 073 - 41 95 75 Fax: 073 - 41 85 43 7^.1 REPORT EVALUATION OF THE MUTAGENIC ACTIVITY OF T-5874 IN THE AMES SALMONELLA/MICROSOME TEST (WITH INDEPENDENT REPEAT) NOTOX Project 115932 NOTOX Substance 38187 - page 1 of 23 - 004473 RECEIVED mov'-9 1894 TOa iUu LOGY T -5874 STATEMENT OF GLP COMPLIANCE NOTOX Project 115932 NOTOX B.V., 's-Hertogenbosch, The Netherlands The study described in this report was conducted in compliance with the most recent edition of: The OECD Principles of Good Laboratory Practice which are essentially in conformity with: The United States Food and Drug Administration. Title 21 Code of Federal Regulations Part 58. The United States Environmental Protection Agency (FIFRA). Title 40 Code of Federal Regulations Part 160. The United States Environmental Protection Agency (TSCA). Title 40 Code of Federal Regulations Part 792. With the exception that the stability of the test substance in the vehicle was unknown. Study Director Ing. E.J. van de Waart 004474 - page 2 - T -5874 QUALITY ASSURANCE STATEMENT NOTOX Project 115932 NOTOX B.V., 's-Hertogenbosch, The Netherlands. Study procedures were subject to periodic inspections and general non study specific processes were also inspected at periodic intervals. This report was audited by the NOTOX Quality Assurance Unit and the methods and results accurately reflect the raw data. DATES OF QAU INSPECTIONS/ AUDITS 24-01-1994 26-01-1994 15-03-1994 REPORTING DATES 24-01-1994 26-01-1994 15-03-1994 Quality Assurance Manager C.J. Mitchell B.Sc. 00447S Date: i n -u . - page 3 - T -5874 REPORT APPROVAL STUDY DIRECTOR: NOTOX Project 115932 Ing. E.J. van de Waart Date: MANAGEMENT: Dr. I.C. Enninga Technical Director OJ 004476 - page 4 - T -5874 NOTOX Project 115932 PREFACE Sponsor Study Monitor Testing Facility Study Director Technical Coordinator Study Plan 3M Belgium - Chemical EBC Canadastraat 11 B-2070 ZWIJNDRECHT Belgium Mr. R.H. Cox NOTOX B.V. Hambakenwetering 3 5231 DD 's-Hertogenbosch The Netherlands Ing. E.J. van de Waart G. van Oort Start : January 25, 1994 Completed : February 10, 1994 TEST SUBSTANCE Identification Description Batch Purity Specific Gravity Instructions for test substance storage Stability under storage conditions Expiry date Stable for at least 4 hours in vehicle T -5874 Cream solid 2334 100% 1.7 At room temperature in the dark Stable January 01, 1996 Water : no Dimethylsulphoxide: not indicated VEHICLE The test substance was dissolved in dimethylsulphoxide of spectroscopic quality (Merck). Test substance concentrations were prepared directly prior to use. 0 0 4 4 7 7 - page 5 - T-5874 NOTOX Project 115932 GUIDELINES The study procedures described in this report were based on the following guidelines: - Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 471: "Genetic Toxicoloqy: Salmonella typhimurium Reverse Mutation Assay", (adopted May 26, 1983). - European Economic Community (EEC), Directive 92/69/EEC. Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; B.14: "Other Effects-Mutagenicity: Salmonella typhimurium Reverse Mutation Assay". EEC Publication no. L383 (adopted December, 1992). ARCHIVING NOTOX B.V. will archive the following data for at least 10 years: protocol, report, test article reference sample and raw data. OBJECTIVE Purpose of the study The objective of this study was to evaluate the test substance for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains to produce histidine-independent strains of these micro-organisms. The assay was conducted in the absence and in the presence of a metabolic system (S9-mix). Justification for selection of the test system The Ames test has been shown to be a rapid and inexpensive indicator for the mutagenic activity of a wide range of chemical compounds. The bacterial strains employed were capable of detecting both frameshift and base-pair substitution mutagens. 004478 - page 6 - T -5874 NOTOX Project 115932 MATERIALS AND METHODS TEST SYSTEM Test System Salmonella typhimurium bacteria Rationale Recognized by the international guidelines as the recommended test system (e.g. EPA, OECD, EEC). Source Dr. Bruce N. Ames, University of California at Berkeley, U.S.A. (TA1535, TA1537 and TA98; 1987 and TAIOO; 1993). The characteristics of the individual strains were as follows: Strain Histidine mutation Mutation type TA1537 hisC3076 Frameshift TA98 hiSD3052/R-factor* Frameshift TA1535 hisG46 Base-pair substitutions TAIOO hiSG46/R-factor* Base-pair substitutions *: R-factor = plasmid pKMIOl (increases error-prone DNA repair) Each tester strain contained the following additional mutations: rfa : deep rough (defective lipopolysaccharide cellcoat) gal : mutation in the galactose metabolism chi : mutation in nitrate reductase bio : defective biotin synthesis uvrB: loss of the excision repair system (deletion of the ultraviolet- repair B gene) Stock cultures of the four strains were stored in liquid nitrogen (-196C). Strains were regularly checked to confirm their histidinerequirement, crystal violet sensitivity, ampicillin resistance (TA98 and TAIOO), UV-sensitivity and the number of spontaneous revertants. CELL CULTURE Preparation of bacterial cultures Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no. 2) and incubated in a shaking bath (37C, 150 spm), until the cultures reached an O.D. of 0.4 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test. 004479 - page 7 - T-5874 NOTOX Project 115932 Agar plates Top agar Environmental conditions Agar plates (0 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid, code L28) in Vogel-Bonner Medium E (*), 10 g glucose, 0.5 mg biotin and 0.6 mg histidine. (*) Vogel, H.J. and Bonner, D.M., 1956, Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem., 218, 97-106. Vogel-Bonner Medium E containing 0.655 (w/v) purified agar was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 120C. All incubations were carried out in the dark at 37C. The temperature was monitored during the experiment. REFERENCE SUBSTANCES Negative control : The vehicle of the test article. Positive controls: Without metabolic activation (-S9-mix): Strain Chemical TA1535 sodium azide (SA) (Fluka) TA1537 9-aminoacridine (9AC) (Janssen Chimica) TA98 daunomycine (DM) (Sigma) TA100 methylmethanesulfonate (MMS) (Merck) Concentration/piate 1 yg 60 yg 4 yg 650 yg Solvent Saline Saline Saline DMSO With metabolic activation (+S9-mix): Strain Chemical TA1535, TA1537 2-aminoanthracene (2AA) TA98, TA100 2-aminoanthracene (2AA) (Sigma) Concentration/piate 5 yg 0.5 yg Solvent DMSO DMSO Solvents for reference substances Saline = physiological saline (Medital Pharma Ned. B.V.) DMSO = dimethylsulphoxide of spectroscopic quality (Merck). 04480 - page 8 - T-5874 NOTOX Project 115932 METABOLIC ACTIVATION SYSTEM Preparation of S9-homogenate: Rat liver microsomal enzymes were routinely prepared from adult male Wistar or Sprague Dawley rats, which were obtained from BRL, Switzerland. The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the SOP's. The rats were injected intraperitoneally with a solution (20% w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later, they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (0C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2 -EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196C). Preparation of S9-mix: S9-mix was prepared immediately before use and kept on ice during the test. S9-mix contained per 10 ml: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 ml aqua bidest; 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution; 1 ml 0.33 M KC1 solution, and 0.5 ml S9. The above solutions were mixed and filter (0.22 ym)-sterilized (apart from the 59fraction, which was added after filter-sterilization of the S9-mix components). EXPERIMENTAL PROCEDURE Selection of dose levels Selection of an adequate range of doses was based on a preliminary test with strain TA100, both with and without S9-mix. Eight concentrations were tested in triplicate (this preliminary test was reported as a part of the first experiment of the mutagenesis assay). The highest concentration of test article used in the subsequent mutagenesis assay was 5 mg/plate. 004481 - page 9 - T-5874 NOTOX Project 115932 Direct plate incorporation assay *: Five different doses (with approximately half-log steps) of the test substance were tested in triplicate in each strain. The test substance was tested both with and without S9-mix in each strain, in two independent experiments. Top agar in top agar tubes was melted and heated to 45C. The following solutions were successively added to 3 ml top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in DMSO, and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 H phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37C for 48 h. After this period revertant (histidine independent) colonies were counted. *) Ames, B.N., McCann, J. and Yamasaki, E., 1975, Methods for detecting carcinogens and mutagens with the Sal monella/mammalian microsome mutagenicity test, Mutation Res., 31, 347-364. *) Maron, D.M. and Ames, B.N., 1983, Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Erratum, 1983, Mutation Res., 113, 533. Colony counting The revertant colonies (histidine independent) were counted automatically with an Artek model 880 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually. ACCEPTABILITY OF ASSAY An Ames test is considered acceptable if it meets the following criteria: a) The negative control data (number of spontaneous revertants per plate) should reasonably be within the laboratory background historical range for each tester strain. b) The positive control chemicals should produce responses in all tester strains which also reasonably are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times the concurrent vehicle control group mean. c) The selected dose range should include a clearly toxic concentration as demonstrated by the preliminary test with strain TA100 or should extend to 5 mg/plate. 004482 - page 10 - T -5874 NOTOX Project 115932 DATA EVALUATION AND STATISTICAL PROCEDURES No formal hypothesis testing was done. A test substance is considered negative (not mutagenic) in the Ames test if: a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation. b) The negative response should be reproducible in at least one independently repeated experiment. A test substance is considered positive (mutagenic)in the Ames test if: a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant. If the test substance showed in the first test only a positive response at one or two concentrations, the assay was repeated with doses just below and exceeding those showing positive effects in the first test. b) The positive response should be reproducible in at least one independently repeated experiment. The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision. ^4483 T -5874 NOTOX Project 115932 RESULTS TOXICITY OF THE TEST SUBSTANCE The survival of the TAIOO culture was determined by comparing the number of colonies on the plates containing the test substance with those on the solvent control plates. Both in the absence and presence of S9-mix the survival of strain TAIOO was not reduced up to and including test substance concentrations of 5000 yg/plate (Table 1). Based on these data, the test substance was tested up to and including a concentration of 5000 yg/plate in the absence and presence of S9-mix. THE AMES SALMONELLA/MICROSOME PLATE TEST Tables 1 and 2 show the results of the Salmonella/microsome plate test with T-5874. All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within our laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly. CONCLUSION Based on the results of this study it is concluded that the test substance is not mutagenic in the Ames Salmonella/microsome assay. 044S4 - page 12 - T-5874 NOTOX Project 115932 TABLE 1 MUTAGENIC RESPONSE OF T-5874 IN THE AMES SALMONELLA/MICROSOME PLATE TEST. Experiment 1 Dose (pg/plate) Mean number of revertant (His+ ) colonies/ 3 replicate plates (+ S.D.) with different strains of S.typhimurium TA1535 TA1537 TA98 TAIOO Without S9-mix 3 10 33 100 333 1000 33302 50002 Solvent control1 Positive control 10 + 8+ 8 10 10 + 4 3 5 3 2 6+ 2 274 28 8+ 2 7 3 9 2 8 3 8+ 3 7+ 3 577 + 89 24 26 + 23 + 22 t 22 4 3 2 2 3 21 2 643 54 124 12 129 7 146 + 22 138 32 149 17 138 11 146 + 15 144 14 163 + 11 1372 + 29 3 10 33 100 333 1000 33302 50002 Solvent control1 Positive control With S9-mix 9+ 11 + 8+ 5+ 7 2 2 4 5 3 7+ 2 218 + 34 7+ 3 6+ 4 6 f 3 6+ 3 7 1 8+ 4 665 + 86 23 + 25 + 28 + 24 + 25 + 4 2 3 5 4 25 + 3 253 + 7 141 + 8 143 + 4 130 + 10 136 + 7 147 + 7 140 + 5 150 + 19 147 8 170 11 601 + 31 1 0.1 ml DMSO 2 Test,substance precipitated slightly on the plates 004485 page 13 T -5874 NOTOX Project 115932 TABLE 2 MUTAGENIC RESPONSE OF T-5874 IN THE AMES SALMONELLA/MICROSOME PLATE TEST. Experiment 2 Dose (yg/plate) Mean number of revertant (His+ ) colonies/ 3 replicate plates (+ S.D.) with different strains of S.typhimurium TA1535 TA1537 TA98 TAIOO 100 333 1000 33302 50002 Solvent control1 Positive control 7 3 10 1 8 2 21 10 17 4 13 4 257 + 13 Without S9-mix 8 2 8 2 6 3 6 1 12 9 15 3 30 + 28 + 29 + 29 + 25 2 7 1 3 5 108 + 18 126 + 3 124 + 10 161 + 6 129 + 6 4 2 391 + 45 28 + 1 123 + 7 477 + 69 1292 + 59 100 333 1000 33302 50002 Solvent control1 Positive control 11 + 6+ 7 11 + 5+ 4 4 3 3 2 5+ 1 174 + 55 With S9-mix 5+ 3 5 4 7+ 2 6+ 1 7+ 2 34 + 13 30+5 36+8 40 + 12 31+5 4+ 2 216 + 56 26+7 339 + 23 120 + 6 118 + 15 133 + 6 114 + 17 143 + 2 114 + 3 649 56 1 0.1 ml DMSO 2 Test substance precipitated slightly on the plates 3 One plate infected with other bacteria 004486 page 14 T-5874 NOTOX Project 115932 APPENDIX 1 Evidence of test article precipitate on the plates is recorded by addition of the following precipitation code. PRECIPITATION CODE Definition Characteristics Slight Precipitate Moderate Precipitate Heavy Precipitate Distinguished by noticeable precipitate on the plate. However, the precipitate does not influence automated counting of the plate. Distinguished by a marked amount of precipitate on the plate, requiring the plate to be hand counted. Distinguished by a large amount of precipitate on the plate, making the required hand count difficult. 004487 - page 15 - T-5874 APPENDIX 2 Individual plate counts; (following pages) Strain Experiment TA1535 1 NOTOX Project 115932 WITHOUT S9-MIX plate 123 Dose(yg/plate) 100 333 1000 33301 50001 11 5 13 6 8 11 12 3 8 13 9 7 9 13 9 Solvent control Positive control 847 296 283 242 MEAN SD 10 4 8+3 8+5 10 3 10 + 2 62 274 + 28 WITH S9 -MIX plate 123 MEAN SD Dose(yg/plate) 100 333 1000 33301 50001 12 8 8 11 13 9 5 7 12 1 10 5 5 5 11 9+2 11 + 2 8+4 5+5 7+3 Solvent control Positive control 958 185 217 253 7+2 218 + 34 1 Test substance precipitated slightly on the plates 004488 page 16 T-5874 NOTOX Project 115932 Strain Experiment TA1537 1 WITHOUT S9-MIX plate 123 Dose(yg/plate) 100 333 1000 33301 50001 6 10 49 10 11 9 10 6 11 7 9 7 4 8 Solvent control Positive control 5 11 6 679 516 535 MEAN SO 8+2 7+3 9+2 8+3 83 7+3 577 + 89 WITH S9-MIX plate 12 3 MEAN SD Dose(yg/plate) 100 333 1000 33301 50001 5 5 10 692 594 378 787 7+3 6+4 6+3 6+3 7+ 1 Solvent control Positive control 3 11 10 645 760 591 8+4 665 + 86 1 Test substance precipitated slightly on the plates 004489 page 17 T-5874 NOTOX Project 115932 Strain Experiment TA98 1 WITHOUT S9-MIX plate 12 3 Dose(yg/plate) 100 333 1000 33301 50001 22 29 21 29 26 23 25 23 22 20 22 23 24 19 23 Solvent control Positive control 22 19 21 696 588 644 MEAN SD 24 + 26 + 23 + 22 + 22 + 4 3 2 2 3 21 + 2 643 + 54 WITH S9 -MIX plate. 12 3 MEAN Dose(yg/plate) 100 333 1000 33301 50001 Solvent control Positive control 19 27 24 26 22 26 31 28 25 23 29 20 27 28 21 28 23 23 260 246 254 23 25 + 28 24 25 25 253 + 1 Test substance precipitated slightly on the plates SD 4 2 3 5 4 3 7 004490 page 18 T-5874 NOTOX Project 115932 Strain Experiment TA100 1 WITHOUT S9-MIX plate 123 MEAN SO Dose(yg/plate) 3 10 33 100 333 1000 33301 50001 125 112 136 123 129 136 157 160 120 101 158 154 134 145 168 138 148 127 149 159 130 158 131 144 124 + 12 129 7 146 + 22 138 + 32 149 4* 17 138 + 11 146 15 144 + 14 Solvent control Positive control 150 168 170 1383 1339 1395 163 + 11 1372 29 WITH S9-MIX plate 12 3 MEAN SD Dose(yg/plate) 3 10 33 100 333 1000 33301 50001 143 133 148 144 146 138 140 128 121 137 142 128 150 139 153 146 138 136 130 151 168 139 155 148 141 + 8 143 + 4 130 10 136 + 7 147 + 7 140 + 5 150 + 19 147 8 Solvent control Positive control 162 182 166 637 586 581 170 + 11 601 31 1 Test substance precipitated slightly on the plates 004491 page 19 3.m T-5874 NOTOX Project 115932 Strain Experiment TA1535 2 WITHOUT S9-MIX plate 12 3 Dose(yg/plate) 100 333 1000 33301 50001 11 6 5 10 9 10 8 7 10 17 33 14 21 16 14 Solvent control Positive control 13 17 9 245 256 270 MEAN SD 7+3 10 + 1 8+2 21 + 10 17 4 13 + 4 257 + 13 WITH S9 -MIX plate 12 3 MEAN SD Dose(yg/plate) 100 333 1000 33301 50001 7 14 11 3 11 5 4 10 8 8 11 13 45 7 11 + 4 6+4 73 11 + 3 5+2 Solvent control Positive control 45 6 111 215 196 5+1 174 + 55 1 Test substance precipitated slightly on the plates 004492 - page 20 - I T-5874 NOTOX Project 115932 Strain Experiment TA1537 2 WITHOUT S9-MIX plate 123 Dose(yg/plate) 100 333 1000 33301 50001 8 10 6 7 7 10 683 567 INF 12 15 Solvent control Positive control 426 441 355 378 MEAN SD 8+2 8+2 63 61 12 > 15 42 391 45 WITH S9 -MIX plate 123 MEAN SD Dose(ug/plate) 100 333 1000 33301 50001 725 925 785 567 488 53 54 7+2 6+1 7+2 Solvent control Positive control 462 276 208 165 42 216 56 1 Test substance precipitated slightly on the plates INF = Plate infected with other bacteria G04493 T-5874 NOTOX Project 115932 Strain Experiment TA98 2 WITHOUT S9-MIX plate 12 3 Dose(yg/plate) 100 333 1000 33301 50001 30 31 28 30 34 21 30 29 29 26 32 30 31 24 21 Solvent control Positive control 27 29 28 437 557 438 MEAN SD 30 + 2 28 + 7 29 + 1 29 3 25 5 28 1 477 + 69 WITH S9 -MIX plate 12 3 MEAN SD Dose(pg/plate) 100 333 1000 33301 50001 Solvent control Positive control 29 25 49 25 35 30 43 38 28 31 36 53 36 27 30 18 30 31 314 343 360 34 -f 13 30 + 5 36 8 40 + 12 31 5 26 + 7 339 + 23 1 Test substance precipitated slightly on the plates 004494 page 22 T -5874 NOTOX Project 115932 Strain Experiment TA1QO 2 WITHOUT S9-MIX plate 123 MEAN SD Dose(yg/plate) 100 333 1000 33301 50001 Solvent control Positive control 110 124 89 123 126 128 114 125 134 168 160 156 128 135 123 124 129 115 1252 1360 1265 108 + 18 126 3 124 + 10 161 + 6 129 6 123 7 1292 59 WITH S9-MIX plate 12 3 MEAN SD Dose(yg/plate) 100 333 1000 333Q1 50001 Solvent control Positive control 118 116 127 104 134 115 133 138 127 100 133 110 141 143 144 114 111 116 585 677 685 120 + 6 118 + 15 133 + 6 114 + 17 143 2 114 + 3 649 + 56 1 Test substance precipitated slightly on the plates page 23 004495