Document x5OMKxbBN1kga1MpeOG2aEKyb

Attachments to Letter to C. Auer dated May 18,2000 Studies and Other Information on Certain Perfluorooctane Sulfonate-Related Compounds 12. N-MeFOSEA N-methylperfluorooctane sulfonamidoethyl acrylate Genotoxicitv 1) Evaluation of the Mutagenic Activity of T-5869 in the Ames Salmonella/Microsome Test (with independent repeat), NOTOX, Project No. 115965, 3M Reference No. T5869.1 (FMZ-3559, Lot 2408, wide-range), April 20, 1994 2) Evaluation of the Ability of T-5869 to Induce Chromosome Aberrations in Cultured Human Lymphocytes (with independent repeat), NOTOX, Project No. 115943, 3M Reference No. T-5869.2 (FMZ-3559, Lot 2408, wide-range), June 5, 1994 3) Evaluation of the Mutagenic Activity of T-5869 in an In Vitro Mammalian Cell Gene Mutation Test with L5178Y Mouse Lymphoma Cells (with independent repeat), NOTOX, Project No. 115954, 3M Reference No. T-5869.3 (FMZ-3559, Lot 2408, wide-range), April 20, 1994 005581 REPORT EVALUATION OF THE MUTAGENIC ACTIVITY OF T -5869 IN THE AMES SALMONELLA/MICROSOME TEST (WITH INDEPENDENT REPEAT) NOTOX Project 115965 NOTOX Substance 38196 - page 1 of 23 - NOV -9 B84 /V i iL.,i^W 5V 005582 "V* T-5869 STATEMENT OF GLP COMPLIANCE NOTOX Project 115965 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 is unknown. Study Director Ing. E.J. van de Waart - page 2 - 005583 T -5869 QUALITY ASSURANCE STATEMENT NOTOX Project 115965 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 REPORTING DATES 24-01-1994 26-01-1994 15-03-1994 24-01-1994 26-01-1994 15-03-1994 Quality Assurance Manager C.J. Mitchell B.Sc. Daterie? -U - i t, - yage 3 - 005584 T -5869 REPORT APPROVAL STUDY DIRECTOR: NOTOX Project 115965 Ing. E.J. van de Waart .../ . ..................... Date: MANAGEMENT: Dr. I.C. Enninga Technical Director OC-M, Date: 2 o / i + / i g g t + ] nnnr^ p a g e 1~ n j - 005585 T-5869 NOTQX Project 115965 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 04, 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-5869 Cream solid 2408 95% 1.5 At room temperature in the dark Stable January 01, 1996 Dimethylsulphoxide: not indicated VEHICLE The test substance was suspended in dimethylsulphoxide of spectroscopic quality (Merck). Test substance concentrations were prepared directly prior to use. - page 5 0055S6 T -5869 NOTOX Project 115965 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 Assav". fadoDted 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. - page 6 - 005587 T-5869 NTOX Project 115965 MATERIALS AND METHODS TEST SYSTEM Test System Rationale Source Salmonella typhimurium bacteria Recognized by the international guidelines as the recommended test system (e.g. EPA, OECD, EEC). 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 TA98 hisD3052/R-factor* TA1535 hisG46 TAIOO hisG46/R-factor* *: R-factor = plasmid pKMIOl (increases Frameshift Frameshift Base-pair substitutions Base-pair substitutions -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 ultravioletrepair 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. - page 7 - 005588 T -5869 NOTOX Project 115965 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 0.63 (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 1metabolic 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/plate 1 vg 60 ug 4 vg 650 vg Solvent Saline Saline Saline DMSO With metabolic activation (+S9-mix): Strain Chemical TA1535, TA1537 2-aminoanthracene (2AA) TA98, TA100 2-ami noanthracene (2AA) (Sigma) Concentrati on/pl ate 5 yg 0.5 pg Solvent DMSO DMSO Solvents for reference substances Saline = physiological saline (Medital Pharma Ned. B. v.) DMSO = dimethylsulphoxide of spectroscopic quality (Merck). - page 8 - 005589 T -5869 NOTOX Project 115965 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 1n 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 NAOP 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. - page 9 - G05590 T-5869 NOTOX Project 115965 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 (1n case of activation assays) or 0.5 ml 0.1 M 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, 3. 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. - page 10 - 005591 T-5869 NOTOX Project 115965 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. - page 11 - 005592 T -5869 NQTOX Project 115965 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 l). 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-5869. 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. - page 12 - 005593 T-5869 NOTOX Project 115965 TABLE 1 MUTAGENIC RESPONSE OF T-5869 IN THE AMES SALMONELLA/MICROSOME PLATE TEST. Experiment 1 Dose (yg/plate) Mean number of revertant (His+ ) colonies/ 3 replicate plates (+ S.D.) with different strains of S.typhimurium TA1535 TA1537 TA98 TA100 Without S9-mix 3 10 33 100 333 1000 33302 50002 Solvent control12 Positive control 6 4 7 3 5 3 8+ 4 5 1 7+ 2 265 + 12 5 2 5+ 2 8 3 6+ 5 7+ 4 6+ 3 359 + 29 16 + 13 + 14 19 + 18 3 1 3 6 4 17 + 2 621 30 136 14 140 + 2 122 + 5 140 + 24 142 + 6 139 + 21 143 + 10 133 + 5 163 + 11 1372 29 3 10 33 100 333 1000 3330 2 5000 2 Solvent control1 Positive control With S9-mix 51 8+ 1 8+ 2 92 5+ 1 8+ 3 240 + 8 5 2 6+ 4 72 6+ 3 5 1 7+ 1 507 + 18 18 + 21 + 16 + 16 + 16 + 5 2 5 5 2 16 + 2 441 + 38 130 + 9 133 + 12 136 + 2 118 + 5 135 + 8 148 5 150 + 14 134 10 170 11 601 31 1 0.1 ml DMSO 2 Test.substance precipitated slightly on the plates page 13 005594 T-5869 NOTOX Project 115965 TABLE 2 MUTAGENIC RESPONSE OF T-5869 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 5000 2 Solvent control12 Positive control 8+ 5+ 10 + 10 + 8 4 1 4 4 2 6+ 2 274 + 28 13 + 9+ 8+ 10 + 10 5 1 3 2 3 7 + 3577 89 19 + 18 + 20 + 29 + 29 + 3 4 2 7 3 21 + 2 643 54 171 + 20 159 + 4 153 + 14 168 + 17 129 + 9 160 + 8 1143 38 100 333 1000 33302 50002 Solvent control1 Positive control 7+ 12 9 8+ 7+ 1 5 4 3 1 7+ 2 218 34 With S9-mix 9 2 5 4 7+ 2 5+ 3 7+ 3 22 + 25 + 26 + 26 + 23 + 4 3 2 5 1 84 665 86 25 + 3 253 + 7 136 13 128 + 14 134 + 7 136 14 137 5 139 + 8 490 100 1 0.1 ml DMSO 2 Test substance precipitated slightly on the plates page 14 00559S T -5869 NOTOX Project 115965 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. - page 15 - 0S596 T-5869 APPENDIX 2 Individual plate counts; (following pages) Strain Experiment TA1535 1 NOTOX Project 115965 WITHOUT S9-MIX plate 123 Dose(ug/plate) 100 333 1000 33301 50001 11 4 3 10 5 5 843 4 7 12 6 45 Solvent control Positive control 857 274 269 251 MEAN SD 6+4 73 5+3 8+4 51 7+2 265 12 WITH SS'-MIX plate 123 MEAN SD Dose(vig/plate) 100 333 1000 33301 50001 Solvent control Positive control 55 88 68 8 11 54 6 7 9 8 5 5 11 9 231 242 246 5+1 8+1 8+2 9+2 51 8+3 240 + 8 1 Test substance precipitated slightly on the plates - page 16 - 005597 T -5869 NOTOX Project 115965 Strain Experiment TA1537 1 WITHOUT S9-MIX plate 123 MEAN SD Dose(yg/plate) 100 333 1000 33301 50001 Solvent control Positive control 653 547 7 5 11 12 3 3 4 11 5 469 326 370 380 5 2 5+2 83 65 74 6+3 359 29 WITH S9-MIX plate 123 MEAN SD Dose(yg/plate) 100 333 1000 33301 50001 5 47 926 759 378 565 5+2 64 7+2 6+3 51 Solvent control Positive control 866 486 513 521 71 507 18 1 Test substance precipitated slightly on the plates page 17 005598 T -5869 NOTOX Project 115965 Strain Experiment TA98 1 WITHOUT S9-MIX plate 123 Dose(yg/plate) 100 333 1000 33301 50001 17 18 12 13 12 13 14 17 12 26 16 15 15 16 22 Solvent control Positive control 18 19 15 637 586 639 MEAN SO 16 13 + 14 + 19 + 18 3 1 3 6 4 17 + 2 62l + 30 WITH S9 -MIX plate 123 MEAN SD Dose(yg/plate) 100 333 1000 33301 50001 14 24 17 20 24 20 21 11 17 20 17 10 18 16 15 18 + 21 f 16 + 16 + 16 + 5 2 5 5 2 Solvent control Positive control 17 13 17 485 419 420 16 + 2 441 + 38 1 Test substance precipitated slightly on the plates page 18 00S599 T-5869 NOTOX Project 115965 Strain Experiment TAIOO 1 WITHOUT S9-MIX plate 123 MEAN SD Dose(yg/plate) 3 10 33 100 333 1000 33301 50001 123 134 151 140 138 142 121 127 117 121 131 167 146 145 136 162 122 132 133 153 144 138 133 128 136 + 14 140 t 2 122 + 5 140 + 24 142 + 6 139 f 21 143 + 10 133 + 5 Solvent control Positive control 150 168 170 1383 1339 1395 163 + 11 1372 + 29 WITH S9-MIX plate 123 MEAN SD Dose(yg/plate) 3 10 33 100 333 1000 33301 50001 140 127 124 120 143 135 137 134 136 113 122 118 138 141 126 142 150 151 135 162 153 129 128 145 130 + 9 133 + 12 136 + 2 118 + 5 135 + 8 148 + 5 150 + 14 134 + 10 Solvent control Positive control 162 182 166 637 586 58! 170 + 11 601 + 31 1 Test substance precipitated slightly on the plates page 19 005600 T-5869 NOTOX Project 115965 Strain Experiment TA1535 2 WITHOUT S9-MIX plate 123 Dose(yg/plate) 100 333 1000 33301 50001 13 5 6 664 7 15 8 5 13 11 6 8 10 Solvent control Positive control 847 296 283 242 MEAN SD 8+4 5+1 10 + 4 10 + 4 8+2 62 274 28 WITH S9-MIX plate 123 MEAN SD Dose(yg/plate) 100 333 1000 33301 50001 Solvent control Positive control 886 9 10 18 4 11 12 10 5 10 867 958 185 217 253 71 12 + 5 9+4 8+3 71 7+2 218 + 34 1 Test substance precipitated slightly on the plates page 20 - 005601 T -5869 NOTOX Project 115965 Strain Experiment TA1537 2 WITHOUT S9-MIX plate 123 Dose(pg/plate) 100 333 1000 33301 50001 13 17 8 8 8 10 6 12 7 9 12 9 8 13 8 Solvent control Positive control 5 11 6 679 516 535 MEAN SD 13 + 5 91 8 f 3 10 + 2 10 3 7+3 577 + 89 WITH S9-MIX plate 123 MEAN SD Dose(ug/plate) 100 333 1000 33301 50001 Solvent control Positive control 11 8 7 10 2 3 8 95 338 749 3 11 10 645 760 591 9+2 5+4 7+2 5+3 73 8+4 665 + 86 1 Test substance precipitated slightly on the plates page 21 005602 T -5869 NOTOX Project 115965 Strain Experiment TA98 2 WITHOUT S9-MIX plate 123 Dose(pg/plate) 100 333 1000 33301 50001 16 20 22 14 22 19 18 22 19 33 21 32 31 26 29 Solvent control Positive control 22 19 21 696 588 644 MEAN SD 19 + 3 18 + 4 20 f 2 29 + 7 29 3 21 + 2 643 54 WITH S9-HIX plate 1 2 3 MEAN SD Dose(iig/plate) 100 333 1000 33301 50001 19 20 27 22 27 26 28 25 25 20 27 30 22 24 24 22 + 4 25 + 3 26 + 2 26 f 5 23 + 1 Solvent control Positive control 28 23 23 260 246 254 25 + 3 253 + 7 1 Test substance precipitated slightly on the plates - page 22 - 00S603 T -5869 NOTOX Project 115965 Strain Experiment TAIOO 2 WITHOUT S9-MIX plate 123 MEAN SD Dose(yg/plate) 100 333 1000 33301 50001 Solvent control Positive control 165 194 155 160 155 163 152 168 140 160 157 188 139 121 127 155 156 170 1124 1118 1186 171 + 20 159 4 153 + 14 168 + 17 129 9 160 8 1143 38 WITH S9-MIX plate 123 MEAN SD Dose(yg/plate) 100 333 1000 33301 50001 125 132 151 141 129 114 141 134 128 119 145 143 141 132 137 136 13 128 + 14 134 + 7 136 + 14 137 + 5 Solvent control Positive control 138 148 132 392 591 488 139 + 8 490 + 100 x Test substance precipitated slightly on the plates page 23 005G04