Document YGVKvkpq5dYZeKZoXmBZKgy7O

SPONSOR ElfAtochemSA. Direction Securite Environnement La Defense 10 - Cedex 42 92091 Paris-la-Defense France Produit AR226-3012 STUDY TITLE REVERSE MUTATION ASSAY ON BACTERIA SALMONELLA TYPHIMURIUM TEST SUBSTANCE STUDY DIRECTOR Brigitte Molinier STUDY COMPLETION DATE 24th March 1995 PERFORMING LABORATORY Centre International de Toxicologie (C.I.T.) Miserey - 27005 Evreux - France LABORATORY STUDY NUMBER 12373 MMO "Company Sanitized. DOBS not contain T8C& f^t CONTENTS STATEMENT OF THE STUDY DIRECTOR STATEMENT OF QUALITY ASSURANCE UNIT SUMMARY . 1. INTRODUCTION 2. MATERIALS AND METHODS 2.1. TEST AND CONTROL SUBSTANCES 2.1.1 Identification 2.1.1.1 Test substance 2.1.1.2 Vehicle 2.1.2 Preparation 2.1.3 Positive controls 2.2. TEST SYSTEM 2.2.1 Bacterial strains 2.2.2 Metabolic activation system: S9 mix 2.3. EXPERIMENTAL DESIGN 2.3.1 Preliminary toxicity test 2.3.2 Mutagenicity tests 2.4. ASSESSMENT OF DATA 2.5. ARCHIVES 2.6. CHRONOLOGY OF THE STUDY 3. RESULTS 3.1. PRELIMINARY TOXICITY TEST (table 1) 3.2. MUTAGENICITY TESTS (tables 2 to 5) 4. CONCLUSION 5. REFERENCES Tables 1 to 5 6. TEST ARTICLE DESCRIPTION AND TEST ARTICLE ANALYSIS 7. HISTORICAL DATA 3 4 5 6 6 6 6 6 6 7 7 7 8 8 8 9 9 10 10 11 11 11 11 11 12 to 16 17 to 19 20 and 21 IWfeeiiOf^'e ""/--s~s- -'*'" - company Sanitized. Does no! contain TSCA CBI STATEMENT OF THE STUDY DIRECTOR RTehgeuslatutidoynsw: as performed in compliance with the principles of Good Laboratory Practice . O.E.C.D. Principles of Good Laboratory Practice, C(81)30(final) Annex 2. May 12,1981. I declare that this report constitutes a true and faithful record of the and the results obtained during the performance of the study. procedures undertaken 2T7h0is05stEuvdryeuwxa, sFrpaenrcfeo.rmed at the Centre International de Toxicologie (C.I.T.), Miserey, B. Molinier Study Director Date: 24.3.95 Doctor of Toxicology Head of Genetic Toxicology 5any5ariSiz's'd. Does nof coniaw TP;<"" ~"' f- STATEMENT OF QUALITY ASSURANCE UNIT I. Specific study inspections Type of inspections Inspections Protocol Report 14.10.94 27.2.95 Dates (day/month/year) Report to Study Director (*) Report to Management (*) 24.10.94 28.2.95 25.10.94 7.3.95 2. Routine inspections performed on other studies of the same type according to a frequency defined in Q.A.U. procedures Inspected phase Dates (day/month/year) Inspections R" eport to Study Director (*) Report to Management (*) Treatment/test substance (incorporation) Treatment/test substance (preincubation) Test substance/preparation 10.1.95 26.1.95 6.1.95 13.1.95. 7.2.95 6.1.95 16.1.95 7.2.95 10.1.95 The inspections were performed in compliance with C.I.T. Quality Assurance Unit procedures and the Good Laboratory Practice Regulations. (*) The dates mentioned correspond to the dates of signature of audit reports by Study Director and Management. M. Labiche Date: 24.3.95 Pharmacist Head of Quality Assurance Unit and Scientific Archives .CompanySanitized. Does hot contain TSCA W? CIT/Study No. 12373 MMOJIHHBBRB^ Atochem 5 SUMMARY The-objective 4R'F induce of this study was to evaluate the potential of the test substance--^----^^R a reverse mutation in bacteria Salmonella typhimurium (Ames test). This test enables the detection of base-pair substitution or frameshift mutagens. Methods A preliminary toxicity test was performed to define the doses to be used for the mutagenicity study. The test substance was then tested in two independent tests, with or without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9) of rats induced with Aroclor 1254. The tests were performed according to the direct plate incorporation method except the second test with S9 mix, according to the preincubation method (1 hour, 37C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to 5 doses of the test substance (3 plates/dose). After 48 to 72 hours of incubation at 37C. the revertant colonies were scored. The test substanceUm|--------^vdaisssolved in distilled water. The doses of thaffffflffffQv/e-re: 312.5, 625, 1250, 2500 and 5000 ug/plate, 5000 ug/plate beingthetopdoserecommended by international regulations. The doses of the positive controls were as follows: without S9 mix: . 1 ug/plate of sodium azide (NaNg): TA 1535 and TA 100 strains, . 50 ug/plate of9-Aminoacridine (9AA): TA 1537 strain, . 0.5 ug/plate of2-Nitrofluorene (2NF): TA 98 strain, . 0.5 ug/plate ofMitomycin C (MMC): TA 102 strain. with S9 mix: . 2 |.ig/plate of2-Anthramine (2AM): TA 1535, TA 1537, TA 98 and TA 100 strains, . 30 ug/plate ofDanthron (DTH): TA 102 strain. Results For both tests, the control results were equivalent to those usually obtained in our Laboratory. The number ofrevertants induced by the positive controls was higher than the controls, indicating the sensitivity of the test system. The test substmceHIHflSfffffdid not induce any significant increase in the number ofrevertants, with or without S9 mix, in any of the 5 strains, for both tests. Conclusion Under our experimental conditions, the test substan^^c^e--J--J^^J^|^|^|^J^^H^^H^^J^^H^^jjdidnot show mutagenic activity in the reverse mutation assay on Salmonella typhimurium' Does wl contain TSCA fmjL^ii .n.uA.ucul ,; 1. INTRODUCTION This study was performed at the request ofelfAtochem S.A., Paris-la-Defense, France. Bno The^)bj'ectiveof this study was to evaluate the potential of the test substance|UBH induce a reverse mutation in bacteria Salmonella typhimurium (Ames test). This test (Ames substitution et al., 1975; Maron and Ames, 1983) enables the detection of base-pair or frameshift mutagens. Mutagenic substances can induce reversion in lhimisittidedinme-eddeifuicmie,nwt hsitlreainnosnw-rhevicehrteadresttrhaeinns acbalnentoot. grow and form colonies in a histidine- This study was designed in accordance with the following guidelines: .. BO..CE..CD.iDre.cgtiuviedeNlion.e9N2o/6. 94/7E1.E,2.C6t.,hAMnanyex1V98,3Ba^n,d3r1esvtisJeudlyd1ra9f9t2d. ocument of May 1994. 2. MATERIALS AND METHODS 2.1. TEST AND CONTROL SUBSTANCES 2.1.1 Identification Jim|Uj| 2.1.1.1 Test substance________ The test substance, used in the study was supplied by Elf Atochem Documentation supplied by the Sponsor identified the test substance as follows: . batch number: ^ .. dqautaenotiftyreacnedipct:on2t.a1i1n.e9r4: 50 g in a plastic flask wrapped in an aluminium foil . storage conditions: at room temperature, protected from light. Data relating description to the characterization of the test substance are documented in a test article and a test article analysis (presented in paragraph 6) provided by the Sponsor. 2.1.1.2 Vehicle The vehicle was distilled water. 2.1.2 Preparation tTehstes. test substance was dissolved in the vehicle at a concentration of 50 mg/ml for both The preparations were made immediately before use. 'ISompanySahffize'd. Does moi eontem TSftA fiw yi Atocnem 2.1.3 Positive controls Six known mutagens, dissolved in dimethylsulfoxide (except for Mitomycin C in distilled water), were used to check the sensitivity of the test system: Dose (ug/plate) Strains Without S9 mix . Sodium azide (NaNg) . 9-Aminoacridme (9AA) . 2-Nitrofluorene (2NF) . Mitomycin C (MMC) With S9 mix . 2-Anthramine (2AM) . Danthron (DTH) 1 50 0.5 0.5 2 30 TA 1535 - TA 100 TA 1537 TA98 TA102 TA 1535 - TA 1537 - TA 98 - TA 10( TA102 2.2. TEST SYSTEM 2.2.1 Bacterial strains The 5 strains (Ames et aL, 1975; Maron and Ames, 1983): TA 1535, TA 1537, TA 98, TA 100 and TA 102, are supplied by B.N. Ames' Laboratory (University of California, Berkeley, U.S.A.). They are stored in a cryoprotective medium containing 1 ml nutrient broth and 0.09 ml dimethylsulfoxide in a liquid nitrogen container. Each strain derived from Salmonella typhimurium LT 1 contains one mutation in the histidine operon, resulting in a requirement for histidine. In addition, to increase their sensitivity to mutagenic substances, additional mutations have been added: . the rfa mutation causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria and increases permeability to large molecules that do not penetrate the normal bacteria cell wall, . the uvr B mutation is a deletion of a gene code for the DNA excision repair system, which renders the bacteria unable to use this repair mechanism to remove the damaged DNA, . the addition of the pKM 101 ampicillin resistant plasmidic R-factor in the strains TA 98, TA 100 and TA 102 enhances then" detection sensitivity to some mutagens. . in addition, the pAQI tetracycline resistant plasmidic factor has been added to the TA102 strain.' Genotype of the bacterial strains Strains Histidine mutation Additional mutations TA 1535 TA100 TA102 TA 1537 TA98 His G 46 His G 46 HisG428(pAQl) His C 3076 -His D 3052 rfa uvrB rfa uvrB Factor R rfa - Factor R rfa uvrB rfa uvrB Factor R The TA 1535, TA 100 and TA 102 strains are reverted by base-pair substitution mutagens and the TA 1537 and TA 98 strains by frameshift mutagens. In addition, the TA 102 strain detects oxidative mutagens. company Sanltizea. Does nol contain TfWA t^ x ^n.i.ui^uciil 2.2.2 Metabolic activation system: S9 mix The S9 mix consists fractions (S9) of induced enzymatic systems contained in rat liver microsomal and the cofactors necessary for their function. S9 was performed at C.I.T. iancdcuocreddinwg ittoh AAmroecsloert 1a2l.54(1(957030).mTgh/ekgS)9bfyraincttrioanpecroitmoneesaflrom liver homogenates from rats The S9 fraction was preserved in sterile tubes within a route. liquid nitrogen The S9 mix was used at 10% S9 in the S9 mix. The S9 mix was container. prepared immediately before use at +4C and maintained at this temperature throughout the experiment. The S9 mix contained per ml: . 5 umoles Glucose-6-Phosphate, . 4 umoles NADP, . 33 umoles KC1, . 8 umoles MgCI^, . 100 umoles sodium phosphate pH 7.4, . 100 ul S9 (batch No. 38, protein concentration: 27 g/l), . sterile distilled water q.s.p. 1 ml. 2.3. EXPERIMENTALDESIGN The day sampled before treatment, cultures were inoculated from frozen permanents: a crystal under sterile conditions and put into approximately 6 nil of nutrient broth. was The Tnuhteriteensttsbwroetrhewpaesrftohremnepdlaaccecdorudnindgertoa:gitation in an incubator at 37C for about 14 hours. . direct test plate incorporation method (both tests without S9 mix, first test with S9 mix): the substance solution, added to 2 ml molten 0.5 ml of S9 mix containing (when required) and 0.1 ml of the strain were agar traces of histidine and biotin and maintained at m45inCim. uAmftemr eradpiuidmh. omogenization, the mixture was spread out on a Petri plate containing . preincubation method (second test with S9 mix and 0.1 ml of the S9 mix): the test substance solution, 0.5 ml of strain were incubated for 60 minutes at 37C prior adding the overlay After 48 agar and to 72 pouring onto the surface of a minimum agar plate. hours of incubation at 37C, revertants were scored with an automatic counter (Artek Counter, model 880, O.S.I., 75015 Paris, France). 2.3.1 Preliminary toxicity test To assess the toxicity of the test substance to the bacteria, 6 doses (1 plate/dose) tested in the TA 98, TA determined according 100 and TA 102 strains, with or without S9 mix. The top were dose was to the international regulations, i.e. not exceeding 5000 ug/plate. nInumthbeerevoefnctoolofntioexsicisitdye, ctrheeasbeadc.terial lawn is sparse compared to control plates and/or the tTohrey. sterility of the test substance was checked during this test and was found to be satisfac .Company Sanitized. DOBS not contain TSCA CBi v-x.i/omuy .iw. x^/:) MMLflplHBHHHBBBlfAtochem 2.3.2 Mutagenicity tests Rationale for dose selection tThheeracwhodicaetaotfhtrhoeugdohsoeust wthiellsbtuedpye. rformed by the Study Director and will be documented in tThheefotollpowdoinseg wcriiltlebriea:selected according to the results of the preliminary toxicity test and to . iFnoterrnnoanti-otnoaxlicr,egfruelaetliyonsso.luble test substances, the top dose is 5000 ug/plate, according to . dFoosre.non-toxic, poorly soluble test substances, the top dose is the lowest precipitating . For toxic test substances, irrespective toxicity: moderately to markedly of solubility, the top dose is based on the level of 50% of the number of revertants sparse bacterial lawn and/or when compared to the decrease by approximately should not interfere with the scoring of the test. controls. However, precipitation In two independent tests, 5 doses of the test substance (3 plates/dose) strain, with or without S9 mix. were tested on each During each test, the following controls were made using triplicate plates: . vehicle control: strain treated with the vehicle, . positive control: strain treated with the known mutagens mentioned in 2.1.3. The sterility of the S9 mix was checked during each test (before the beginning end of the experiment) and was found to be satisfactory. and at the 2.4. ASSESSMENT OF DATA Treatment of results During each plate test, for each strain and for each experimental point, the number of per was scored. The individual results and the mean number of revertants revertants, with the corresponding standard substance/mutants deviation and ratio (mutants obtained in the presence of the test obtained in the presence of the vehicle), are presented in a table. Acceptance criteria This the study was considered valid because the following criteria were fully met: . number of revertants of the controls was within the range of our historical data, . wthiethninumthbeerraonfgereovefrotaunr thsisotof rtihcealpositive controls was higher than that of the controls and data. Evaluation criteria The following criteria were used as an aid for determining a positive . a reproducible and significant dose relationship. response: and/or . a reproducible and least one of the significant increase (i.e. a doubling in the number of revertants for at doses. tested strains when compared to that of the controls) for at least one of the aAretensottsfuublslytamnceet.is considered as non-mutagenic in this test system if the above two criteria Biological and statistical significances were considered during the evaluation. not contain TSCA'" '^'-'''^^------^--rn Alocnem ,Q 2.5. ARCHIVES The study archives: . protocol and possible amendments , raw data . correspondence . final report and possible amendments are stored on the premises of C.LT., 27005 Miserey, Evreux, France, for 5 years after the end of the experimental study. At the end of this period, the study archives will be returned to the Sponsor. 2.6. CHRONOLOGY OF THE STUDY The chronology of the study is summarized as follows: Procedure Date Protocol approved by: . Study Director . Sponsor 12.10.94 3.10.94 Preliminary toxicity test . treatment First mutagenicity test . treatment 17.1.95 20.1.95 Second mutagenicity test . treatment 26.1.95 ICeTOpaRySanil^ed. Does not contain TSC^ "^ 3. RESULTS i-LO^llCULA 11 3.1. PRELIMINARY TOXICITY TEST (table 1) l^^^^^^^^^l 5T0hemtge/smt ls. ubstance|lJJHBIIftm.vasfreely soluble in the vehicle (distilled water) at When 0.1 ml of this of 5000 ug/plate, solution was added to 2 ml molten agar, corresponding to the top no precipitate was observed in the Petri plate when scoring the dose revertants. Consequently, the doses were: 10,100,500, 1000,2500 and 5000 ug/plate. The test substance was not toxic at the doses used. 3.2. MUTAGENICITY TESTS (tables 2 to 5) The control number of results were equivalent to those usually obtained in our Laboratory. The revertants induced by indicating the sensitivity of the test the positive controls was higher than the controls, system. tThheetospeldeocsteedredcoosmesmweenrdee:d3b1y2.i5n,te6r2n5a,tio1n2a5l0r, e2g5u0la0tioannsd. 5000 pg/plate, 5000 ug/plate being The test substance^BHHHHRj^ number of revertants, with or without S9 mix, innoat niyndoufcthee a5nystrasiingsn.ificant increase in the 4. CONCLUSION Under our experimental conditions, the test substancejflHHHimUJdid mutagenic activity in the reverse mutation assay on Salmonella typhimurium. not show 5. REFERENCES Ames, simple B. N.; Durston, W. E.; test system combining Yamasaki, E. and Lee, F. D.: Carcinogens are mutagens: liver homogenates for activation and bacteria for a detection. Proc. Nat. Acad. Sci. U.S.A., 70.2281'-2285 (1973). Ames, B. mutagens N.; Me Cann, with the D. and Yamasaki, E.: Methods for detecting carcinogens and Salmonella Research. 31, 347-364 (1975). Mammalian-microsome mutagenicity test. Mutation MMuatraotnio, nDR.Mes.eaarncdh. A1m13e.s 1B73. -2N1.:5 R(1e9v8is3e).d methods for the Salmonella mutagenicity test. Wot CQ-fltyr, TSCA CRi w.^ f ^f ATAJ-TAV/a ^ it Alocnem 12 Table 1: Preliminary test of toxicity without S9 mix strains doses T P revertants (/ig/plate) 0 - - TA98 10 100 500 1000 2500 5000 0 - - - - - - - - - - - - - - 10 - - TA100 100 500 1000 2500 5000 0 - - - - - - - - - - - - 10 - - 100 - - TA102 500 - - 1000 - - 2500 - - 5000 - - 0 : vehicle control (sterile distilled water) per plate 22 17 21 33 30 21 21 80 76 76 77 94 90 82 247 241 216 267 285 215 248 with S9ma T P revertants - - - - - - - - - - - - - - - - - - - - . - - - - - - - - - - - - - - - - - - - - ( - - per plate 23 38 24 30 19 25 21 101 96 81 132 90 90 108 .306 297 322 310 327 365 343 T : toxicity : - : no + : slight + + ++ + : moderate : considerable to total P : precipitate of the test substance : - : no + : slight + + : moderate + + + : strong SEompanySanitized. Does noS contain TSCA CB! 13 a Table 2: First mutagenicity test without metabolic activation strains doses dig/plate) 0 T P - - TA 1535 312.5 625 1250 2500 5000 NaN3(l) 0 - - - - - - - - - - - - - - TA 1537 312.5 625 1250 2500 5000 9AA(50) 0 - - - - - - - - - - - - - - 312.5 625 - - - - TA98 1250 2500 - - - - 5000 - - 2NF(0.5) - - 0 - - TA100 312.5 625 1250 2500 5000 NaN3(l) 0 - - - - - - - - - - - - - - 312.5 625 - - - - TA102 1250 - - 2500 - - 5000 - - MMC(0.5) - - 0 : vehicle control (sterile distilled water) revertants per plate 9 6 12 9 16 11 13 13 13 10 8 8 11 6 8 11 10 24 441 416 432 967 5 10 9 7 10 15 5 11 5 15 16 11 12 10 9 429 416 379 32 45 21 38 31 35 41 41 34 47 49 28 30 30 31 36 35 32 155 149 142 149 159 148 144 143 161 153 155 131 138 152 146 124 144 150 134 138 142 592 582 551 297 302 289 299 301 306 274 298 272 269 277 316 273 281 301 237 '258 252 1170 1371 1356 ratio number of revertants with the test substance, number of revertants with the vehicle mean 9 standard deviation 3 ratio - 12 4 1.3 13 0 1.4 9 1 1.0 8 3 0.9 15 8 1.7 430 13 47.7 7 2 - 8 3 1.1 11 4 1.5 7 3 1.0 14 3 1.9 10 2 1.4 408 26 55.6 33 12 - 35 4 1.1 39 4 1.2 41 12 1.3 30 1 0.9 34 2 1.1 149 7 4.6 152 6 - 149 10 1.0 146 13 1.0 145 7 1.0 139 14 0.9 138 4 0.9 575 21 3.8 296 7 - 302 , 4 1.0 281 14 1.0 287 25 1.0 285 14 1.0 249 11 0.8 - 1299 112 4.4 T : toxicify (cf. Table 1) P : precipitate (cf. Table 1) ;3. Ooestso! cor-iwn T<?^ ^" -f^X ~H\J\^Ll^iil 14 Table 3: Second mutagenicity test without metabolic activation strains doses T P (.fig/plate) 0 - - TA 1535 312.5 625 1250 2500 5000 NaN3(l) 0 - - - - - - - - - - - - - - TA 1537 312.5 625 1250 2500 5000 9AA(50) 0 - - - - - - - - - - - - - - 312.5 625 - - - - TA98 1250 2500 - - - - 5000 - - 2NF(0.5) - - 0 - - TA100 312.5 625 1250 2500 5000 NaN3(l) 0 - - - - - - - - - - - - - - 312.5 625 - - - - TA102 1250 - - 2500 - - 5000 - - MMC(0.5) - - 0 : vehicle control (sterile distilled water) revertants per plate 555 8 10 10 2 2 2 10 4 12 3 4 6 401 416 381 3 5 5 11 3 5 11 4 9 3 5 9 10 14 8 7 5 7 13 6 8 365 397 260 24 28 23 18 16 14 44 31 36 25 22 23 37 41 56 33 44 19 177 149 126 103 108 89 90 86 79 99 86 82 84 80 109 94 89 92 86 91 95 658 608 549 230 271 332 254 223 202 265 294 294 275 269 288 300 289 289 247 275 283 1507 1656 1388 ratio number of revertants with the test substance . number of revertants with the vehicle mean 5 standard deviation 0 ratio - 2 0 0.4 9 1 1.9 4 2 0.9 9 4 1.7 4 1 0.9 399 18 79.9 64 - 6 3 0.9 8 4 1.3 6 1 1.0 11 3 1.7 9 4 1.4 341 72 53.8 25 3 - 16 , . 37 2 0.6 7 1.5 23 2 0.9 45- 10 1.8 32 13 1.3 151 26 6.0 100 10 - 85 6 0.9 89 9 0.9 91 16 0.9 92 3 0.9 91 5 0.9 605 55 6.1 278 51 - 226 26 0.8 284 17 1.0 277 10 1.0 293 6 1.1 268. 19 1.0 1517 134 5.5 T: toxicity (cf. Table 1) P : precipitate (cf. Table 1) -""1. -t-l.UA/llL/.UJ, 15 Table 4: First mutagenicity test with metabolic activation strains doses T P (/(g/plate) 0 - - TA 1535 312.5 625 1250 2500 5000 2AM(2) 0 - - - - - - - - - - - - - - TA 1537 312.5 625 1250 2500 5000 2AM(2) 0 - - - - - - - - - - - - - - TAPS 312.5 625 1250 2500 5000 2AM(2) 0 - - - - - - - - - - - - - - TA100 312.5 625 1250 2500 5000 2AM(2) 0 - - - - - - - - - - - - - - 312.5 - - 625 - - TA102 1250 - - 2500 - - ' 5000 - - DTH(30) - - 0 : vehicle control (sterile distilled water) revertants per plate 11 19 15 19 9 18 15 17 15 12 15 20 15 17 15 15 21 22 355 320 410 10 7 8 11 12 12 5 11 7 12 7 9 4 12 6 7 4 8 131 134 84 45 50 34 41 42 37 52 40 41 39 62 40 37 42 62 57 49 49 2648 1532 1200 150 146 123 154 138 150 179 146 148 130 146 145 157 134 140 145 137 145 1501 839 1153 355 386 334 338 320 356 387 368 383 343 313 383 354 339 338 313 351 380 . 711 739 754 mean standard ratio deviation 15 4 - 15 6 1.0 16 1 1.0 16 4 1.0 16 1 1.0 19 4 1.3 362 45 24.1 82 - 12 1 1.4 8 3 0.9 9 3 1.1 7 4 0.9 6 2 0.8 116 28 14.0 43 8 - 40 ' 3 0.9 44 7 1.0 47 13 1.1 47 13 1.1 52 5 1.2 1793 759 41.7 140 15 - 147 8 1.1 158 19 1.1 140 9 1.0 144 12 1.0 142 5 1.0 1164 331 8.3 358 26 - 338 18 0.9 379 10 1.1 346 35 1.0 344 9 1.0 348 34 1.0 735 22 2.1 T: toxicity (cf. Table 1) P : precipitate (cf. Table 1) ratio number of,revertants with the test substance number of revertants with the vehicle Company SanStizeff. Does rib! contain TSCA Cl AUJUUCUl 16 Table 5: Second mutagenicity test with metabolic activation : Preincubation method strains doses T P Qtg/plate) 0 - - TA 1535 312.5 625 1250 2500 5000 2AM(2) 0 - - - - - - - + + ++ - - - - TA 1537 312.5 625 1250 2500 5000 2AM(2) 0 - - - - - + + + ++ - - - - TA98 312.5 625 1250 2500 5000 2AM(2) 0 - - - - - - - + + ++ - - - - TA100 312.5 625 1250 2500 5000 2AM(2) 0 - - - - - - - + - ++ - - - - 312.5 - - 625 - - TA102 1250 - - 2500 - + 5000 DTH(30) - ++ - - 0 : vehicle control (sterile distilled water) revertants per plate 10 8 9 mean 9 standard deviation 1 ratio - 8 10 6 8 2 0.9 7 9 11 9 2 1.0 8 12 8 9 2 1.0 17 10 11 13 4 1.4 7 14 12 11 4 1.2 198 285 253 245 44 27.3 10 6 10 9 2 - 5 6 564 5 856 7 12 7 9 646 7 225 334 323 294 6 5 9 21 32 22 25 1 0.6 2 0.7 1 0.6 3 1.0 2 0.8 60 33.9 6 - 16 21 18 25 20 2449 114 95 105 102 106 98 1862 263 193 329 301 372 297 765 19 22 20 21 24 2385 111 86 89 100 102 104 2540 315 249 376 230 320 307 830 22 17 23 20 25 2629 84 90 85 92 96 95 2297 207 216 296 276 343 279 987 19 3 ' " 20 3 20 3 22 3 23 3 2488 127 103 17 90 5 93 11 98 5 101 5 99 5 2233 344 262 54 219 28 334 40 269 36 345 26 | 294 14. 861 114 T:toxicity(cf.Tablel) P : precipitate (cf. Table I) 0.8 0.8 0.8 0.9 0.9 99.5 - 0.9 0.9 1.0 1.0 1.0 21.7 - 0.8 1.3 1.0 1.3 1.1 3.3 ratio number of revertants with the test substance number of revertants with the vehicle Sott)pa?i.y'Samtl.Aeu. D&ss not contain 1 SCA "^ ^j.'ijiuuj i^u. i^^/j lyjLLviv^HHH^^^jBjHHHiBiiAiocneni 17 6. TEST ARTICLE DESCRIPTION AND TEST ARTICLE ANALYSIS pp--w-^ww-----"- . , te^^-^A- - ^ - . _,,,.,. ', . ,..,.' ,^ ; ^ ^ut .'^ t^ $2 '-.i.i/Di.uuy i-tu. 1^3/3 JVUVll. SERVICE DE TOXICOLOGDE CONFDDENTEEL 3 octobre 1994 alf Atochem elf atochem sa La Defense 10, cedex42 92091 Paris-La Defense France FICHE D 'IN FORMATIONS C IDENTIFICATION Norn commun Nom chimique Origine; n de lot N d'archjvage (CAL) : Elf Atochem, CRRA, Operation Industrielle 88/94 PROPRIETES PHYSICO-CBEMIQUES Apparence Viscosite Temperature ebullition pH Densite Point d'eclair Solubilite INFORMATIONS TOXICOLOGIQUES ET PRECAUTION D'EMPLOl DLO / rat / oral > 2000 mg/kg. Non irritant pour la peau et les yeux. Non sensibilisant. CONDITIONS DE CONSERVATION ET DE DESTRUCTION Conservation Stabilite Destruction : A 1'obscurite et a I'abri de la chaleur. : Stable jusqu'en Septembre 1995 dans ces conditions de stockage : Incineration. Sanitized. Does not oos Company ^Al./Ul.UU.^ 11U. l^J I 3 IVUViV^j elF atochem Ipn Arocnem 19 Centre de Recherche Rhone-AIpes Destinataires : Lo r^Tn<Ac^al(y'iOLoie - MM. CHAIZE / PIED LA1GNEL MONTAGNON GROGNET SiflnaTure : ny Sanifesd Doasnbf contain TSCA MMO/MMJ30.1.95 controls temoins iidil.7 2NF 9AA 2AM 2AM(P) 0-S9mix 0+S9mix TA 1535 mean (SD) mini-maxi Nb values mean (SD) TA 1537 mini-maxi Nb values TA98 mean (SD) mini-maxi Nb values moyenne Nombre moyenne (ecart-type) de valeurs 371(101) 149-564 105 ^-^^'^i'-"''''5E'"^'^J'''^'Sa5'JL!'L'"S.'''"'*i5S::a;-''' '^'f'-"i''"*^"t -'-~'3!yEi^"'WS!'i' (ecart-type) 276(52) 253(43) 10 (3) 12(3) 131-409 66 111-333 54 4- 20 132 4-20 135 189(83) 174(65) 336(71) 9 (4) 9 (3) Nombre de valeurs moyenne (ecart-type) Nombre de valeurs 60-499 81-439 189-457 4 - 21 4 - 20 141(32) 86-331 108 111 72 1495(345) 936-2975 75 54 2633(327) 1685-3250 " 60 138 29(9) 11-55 135 150 26(7) 14-47 159 controls temoins mean (SD) TA 100 mini-maxi Nb values mean (SD) TA 102 mini-maxi Nb values WP2 uvrA mean (SD) mini-maxi Nb values moyenne (ecart-type) Nombre de valeurs moyenne (ecart-type) Nombre de valeurs moyenne (ecart-type) Nombre de valeurs NaN3: l^ig/plate/boite 2NF: 0.5/.ig/plate/boite 9AA: 50/tg/plate/boite MMC: 0.5/ig/plate/boite ENNG: 2/xg/plate/boite 2AM: 2 or lO^ig/plate for Salmonella typhimurium or for WP2uvrA, respectively. 2AM: 2 ou 10/ig/boite pour Salmonella typhimurium ou pour WP2uvrA, respecrivement. DTH: 30/ig/plate/boite (P): preincubarion method/methode par preincubation. company Sanitized. Does noteontain TSCA CBf