Document 3e0kMmydN2DJLK23MBwmgY8Va

AR226-3126 DuPont-3220 TRADE SECRET Study Title H-24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli Laboratory Project ID: DuPont-3220 Test Guidelines: U.S. EPA Health Effects Test Guidelines OPPTS 870.5100 (1998) OECD Guidelines for Testing of Chemicals Section 4: Health Effects, No. 471 (1997) Commission Directive 92/69/EEC EEC Methods B.13 and B.14 Author: N. Lawrence Gladnick, B.A. Study Completed on: July 23,1999 Performing Laboratory: E. I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Work Request No.: Service Code No.: Company Sanitized. Does not contain TSCA CBI Page 1 of 24 H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with U.S. EPA FIFRA (40 CFR Part 160) and/or EPA TSCA (40 CFR 792) Good Laboratory Practice Standards, which are consistent with the OECD Principles of Good Laboratory Practice (OCDE/GD(92)32) except for the items documented below. These items did not impact the validity of the study. 1. Treatment solutions were not analyzed for identity, composition, uniformity, or stability of the test and control substances. The procedures used by trained personnel to prepare the treatment solutions were intended to ensure: The accuracy of concentration because the test and control substances were weighed on an analytical balance accurate to 3 decimal places and the dimethyl sulfoxide and water in which the test and control substances were dissolved were accurately measured with graduated pipettes or flasks; Uniformity because all treatment solutions were mixed prior to administration to the test system; and Stability because treatment solutions were prepared just prior to administration to the test system. 2. The test substance was characterized by the sponsor prior to initiation of this study. Although the characterization was not performed under Good Laboratory Practice Standards, the accuracy of the data is considered sufficient for the purposes of this study. 3. The preparation pages for ICR-191 and NAAZ, positive controls used in the study, could not be located as of the date of report issue. Quality Assurance personnel verified the existence of the prepared test materials, and the positive control data met acceptability criteria set forth in the general testing SOP. This does not impact the study integrity or validity. Submitter/Sponsor: E. I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Director: N. Lawrende Gadnick, B.A. Toxicology Associate A3 S'vL Date Company Sanitized. Does not contain TSCA CBI -2- H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 CERTIFICATION I, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Issued by Study Director: A s^ U , N. Lawrende31adnick, B.A. Toxicology Associate 2 3 Tul m i Date Company Sanitized. Does not contain TSCA CB! H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 TABLE OF CONTENTS GOOD LABORATORY PRACTICE COMPLIANCE STATEM ENT.................................... 2 C E R T I F IC A T IO N ..............................................................................................................................3 STUDY INFORM ATION................................................................................................................. 6 STUDY PERSONNEL....................................................................................................................... 7 SUMMARY......................................................................................................................................... 8 IN T R O D U C T IO N ...............................................................................................................................9 MATERIALS AND M ETH O D S......................................................................................................9 A. STUDY PROTOCOL..............................................................................................................9 B. TEST MATERIALS.............................................................................................................. 10 1. Test Substance................................................................................................................................................10 2. Negative and Positive Controls..................................................................................................................... 10 3. Tester Strain Source, Characterization, Storage, and Culture.....................................................................11 4. Metabolic Activation System.........................................................................................................................11 C. CONCENTRATION SELECTION, TEST SUBSTANCE STABILITY AND VERIFICATION.................................................................................................................... 12 D. TEST METHODS: BACTERIAL MUTAGENICITY ASSAY .........................................12 E. STATISTICAL ANALYSIS.................................................................................................13 F. ACCEPTABILITY CRITERIA.................................. 13 1. Tester Strain Phenotypes and Spontaneous Reversion............................................................................... 13 2. Tester Strain Culture Titer............................................................. 13 3. Positive Control Values.................................................................................................................................13 4. Non-Toxic Concentration Levels.................................................................................................................. 13 5. Rejection of Plates, Concentration Levels, or Assays................................................................................. 14 G. CLASSIFICATION GUIDELINES..................................................................................... 14 RESULTS AND DISCUSSION......................................................................................................15 C O N C L U S IO N S ......................................................................................... 15 RECORDS AND SAMPLE STO RA G E................. 15 REFERENCES.................................................................................................................................. 16 ABBREVIATIONS FO R TABLES................................................................................................17 TABLES.......................... ..................................................... ..................................... ................. 18 Company Sanitized. Does not contain TSCA CBI -4 - H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli___________________________ '________________________DuPont-3220 LIST OF TABLES Page 1. TABLE 1 MUTAGENIC ACTIVITY IN SALMONELLA TYPHIMURIUM TA97A...................................... 19 2. TABLE 2 MUTAGENIC ACTIVITY IN SALMONELLA TYPHIMURIUM TA98......................................... 20 3. TABLE 3 MUTAGENIC ACTIVITY IN SALMONELLA TYPHIMURIUM TA100....................................... 21 4. TABLE 4 MUTAGENIC ACTIVITY IN SALMONELLA TYPHIMURIUM TA1535..................................... 22 5. TABLE 5 MUTAGENIC ACTIVITY IN ESCHERICHIA COLI WP2 UVRA (PKM101)................................. 23 6. TABLE HISTORICAL CONTROL DATA.......................................................................................................... 24 Company Sanitized. Does not contain TSCA CBI -5 - H-24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 STUDY INFORMATION 9th Collective Nomenclature: Poly(difluoromethylene),.alpha.-fluoro-.omega.-[2-[(2methyl-1-oxo-2-propenyl)oxy]ethyl]-, polymer with 2Propenoic acid, 2-methyl-, ammonium salt and 2propenoic acid, 2-methyl-, 2-(diethylamino)ethyl ester Haskell Number: 24119 CAS Registry Number: Submitter's Notebook Number(s): E88438-149 Purity: 95% Known Impurities: Physical Characteristics: Opaque, light yellow emulsion (20% solids) Stability: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed. Sponsor: E. I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Completed: June 8, 1999 / (see report cover page) In-Life Initiated/Completed: June 9, 1999 / June 25, 1999 Company Sanitized. Does not contain TSCA CB! -6 - H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 STUDY PERSONNEL Study Director: N. Lawrence Gladnick, B.A. Management: David F. Krahn, Ph.D. / Ronald D. Snyder, Ph.D. Primary Technician: Sharon E. Mania Toxicology Report Preparation: Debra L. Dacey, A.A.S. Company Sanitized. Does not contain TSCACBF -7 - H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 SUMMARY H-24119 was evaluated in the bacterial reverse mutation assay using Salmonella typhimurium strains TA97a, TA98, TA100, TA1535, and Escherichia coli strain WP2 uvrA (pKMIOl) in the presence and absence of an exogenous metabolic activation system (Aroclor -induced rat liver S9). The single trial assay was performed using the plate incorporation method to evaluate the mutagenic potential of the test substance. All tester strains exhibited appropriate phenotypic characteristics. For all tester strains the mean number of revertants scored for the negative controls was within the prescribed acceptable range derived from historical data. Concentrations of 5,10, 50,100, 500,1000, 2500, and 5000 ^g/plate were assessed in comparison to negative (solvent) controls. The solvent control and diluent used in this study was sterile water. No toxicity was observed as evidenced through the reduction of the microcolony background lawns or as a concentration-related reduction in the mean number of revertants per plate. No precipitate was observed at any concentration in any strain. Under the conditions of this study, no evidence of mutagenic activity was detected in any of the Salmonella strains or the Escherichia coli strain. The test substance was negative. Company Sanitized. Does not contain TSCA CBI -8 - H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 INTRODUCTION This study evaluated the mutagenic potential of the test substance, H-24119, in Salmonella typhimurium strains TA97a, TA98, TA100, and TA1535 and in Escherichia coli strain WP2 uvrA (pKMIOl). The bacterial reverse mutation test uses amino acid requiring strains of Salmonella typhimurium and Escherichia coli to detect mutations, that involve substitution, addition or deletion of 1 or a few DNA base pairs/1,2^ The Salmonella tester strains are unable to synthesize histidine because of specific point mutations in genes coding for histidine biosynthesis. Additional mutation of the defective gene specific to the tester strain can result in individual bacteria regaining the ability to synthesize histidine. Tester strains TA97a and TA98 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) primarily by frameshift mutagens. Tester strains TA100 and TA1535 are reverted by mutagens that primarily cause base pair substitutions. E. coli WP2 uvrA (pKMIOl) is unable to synthesize tryptophan due to an ochre mutation in the gene required for tryptophan biosynthesis. E. coli WP2 uvrA (pKMIOl) is primarily sensitive to mutagens that act at AT base pairs within the trpE gene and may also revert to prototrophy from suppressor mutations at a locus in a tRNA gene.(2) By comparing the number of chemically induced revertants to the number of spontaneous revertants, the mutagenicity of the test substance can be assessed. MATERIALS AND METHODS A. STUDY PROTOCOL The protocol consisted of the Short-Term Study Protocol Sheet and the Haskell General Testing Procedure GT008-T-010 ("Bacterial Reverse Mutation Test for Solids, Liquids, and Gases", effective 6/10/98). The study was designed to comply with: U.S. EPA, Office of Prevention, Pesticides and Toxic Substances (OPPTS) Guidelines (Subpart H, 40 CFR Part 870.5100 [1998]); Guidelines of the Organisation for Economic Cooperation and Development (OECD Guidelines for Testing of Chemicals, No. 471 [1997]); The guidelines of the European Union (EEC Commission Directive 92/69/EEC, Methods B.13 and B.14 [1992]); The study design complied with the testing guidelines cited above with the following exceptions: S. typhimurium tester strain TA97a using ICR 191 as a positive control was used as a substitute for strain(s) TA97 and/or TA1537 (EEC and Japan). E. coli WP2 uvrA (pKMIOl) was the only E. coli tester strain used (EEC). One trial was conducted with the test substance as specified by the sponsor (EEC). Company Sanitized. Does not contain TSCA CBI -9 - H-24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 These exceptions did not affect the study validity for the following reasons: According to some testing guidelines S. typhimurium strain TA1537, Salmonella strains TA1537, TA97 or TA97a may be used interchangeably. Salmonella strain TA97 has been demonstrated to be more sensitive to frameshift mutagens and the reconstructed strain, TA97a, was used due to its improved growth properties. Although some testing guidelines require E. coli WP2 uvrA, no significant difference in mutagen detection would be expected between the plasmid and non-plasmid containing strains; therefore, E. coli WP2 uvrA (pKMIOl) was used to assess potential mutagenicity. The strains used in this study comply with harmonized guidelines designed to minimize variations among testing procedures worldwide (OECD, OPPTS). B. TEST MATERIALS 1. Test Substance The test substance H-24119 is an opaque liquid that was stored at room temperature. Additional information regarding the test substance can be found on the study information page of this report. The test substance was assumed to be stable during the study and no evidence of instability was observed. 2. Negative and Positive Controls Based on information supplied by the sponsor and a solubility assessment at the testing facility, sterile water was chosen as the test substance solvent, diluent, and negative control. There were no impurities, known or reasonably anticipated, in the controls that might interfere with the validity of the study. Positive controls included the following: The positive controls were 'prepared in advance and maintained frozen at -70C. Previou^experience with these negative and positive controls indicates that they are stable in this test system and no evidence of instability was observed during the study. . - 10- company Sanitized. Does not contain TSCA CBf H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 3. Tester Strain Source, Characterization, Storage, and Culture S. typhimurium tester strains were obtained from Dr. Bruce Ames, Berkeley, CA, USA. E. coli WP2 uvrA (pKMIOl) was obtained from the National Collection of Industrial Bacteria, Torrey Research Station, Scotland, UK. The characteristics of S. typhimurium tester strains TA97a, TA98, TA100, TA102, TA1535 and E. coli WP2 uvrA (pKMIOl) are as follows: Strain Gene Locus LPS R-factor Reversion DNA (pKMIOl) Target Repair* Plasmid S. typhimurium TA97a S. typhimurium TA98 S. typhimurium TA 100 S. typhimurium TA 102 S. typhimurium TA1535 hisD6610, hisOl242 hisD3052 hisG46 hisG428 hisG46 uvrB uvrB uvrB (+) uvrB rfa Present rfa Present rfa Present rfa Present rfa Absent E. coli WP2 uvrA (pKMIOl) trpE uvrA NA Present LPS=lipopolysaccharide; (+) proficient for excision repair; NA=Not applicable *Defective DNA repair gene. pAQl Plasmid Absent Absent Absent Present Absent Absent The deletion (A) in uvrB (a gene that codes for 1 protein involved in DNA excision repair) increases the bacterial sensitivity to some mutagens.(5) The uvrB and uvrA traits are confirmed by demonstrating an increased bacterial sensitivity to ultraviolet light. Because the uvrB deletion also extends through a gene needed for biotin biosynthesis, the S. typhimurium tester strains require exogenous biotin to be added to culture media or plates for growth. The rfa mutation causes a partial loss in the integrity of the lipopolysaccharide (LPS) cell wall so that permeability to large molecules is increased.(6) The presence of the pKMIOl or R-factor plasmid, conferring ampicillin resistance, also enhances an error-prone DNA repair system that is endogenous to these bacteria.(7) The pAQl plasmid confers tetracycline resistance to S. typhimurium TA102, that was used solely as a control for UV light sensitivity. Salmonella tester strains were stored at -70C in ~8% (v/v) DMSO in Oxoid Nutrient Broth No. 2. The E. coli WP2 uvrA (pKMIOl) strain was stored at -70C in -30% glycerol in Oxoid Nutrient Broth No. 2. Overnight cultures were prepared by inoculating 20 mL of Oxoid Nutrient Broth No. 2 with 0.1 mL of a bacterial stock and incubating at 37C with shaking. Concurrent with the reverse mutation test, tester strain phenotypes were confirmed on overnight cultures. 4. Metabolic Activation System Because the tester strains lack many of the enzymes required to convert some promutagens to a reactive state, the assay was performed in the presence and absence of an exogenous metabolic activation system similar to that described by Marn and A m es/1' The exogenous metabolic activation system was a cofactor-supplemented post-mitochondrial fraction, (i.e., 9000 x g; homogenate of 1 g wet liver weight in 3 mL of an approximately 0.15 M KC1 solution) prepared from the livers of young male Sprague Dawley rats treated with the enzyme-inducing agent Aroclor 1254 (500 mg/kg i.p.) as a single dose 5 days prior to sacrifice. The Aroclor-induced rat liver S9 (purchased from MOLTOXTM) was characterized for protein content and metabolic activity by the vendor. To confirm the sterility of the exogenous metabolic activation system, an _______________________________________________ -11 - C mpany Sanitized. Doss not conten t s c a CBF H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 aliquot was plated on nutrient agar capable of supporting the growth of viable bacteria. The amount of Aroclor-1254 induced rat liver S9 in the exogenous metabolic activation system was 4.0 mg S9 protein (-11% [v/v]) / mL. The cofactor-supplement concentrations in the exogenous metabolic activation system were 8 mM MgCL, 33 mM KC1, 5 mM glucose-6-phosphate (as a sodium salt), 4 mM NADP+(as a sodium salt), and 100 mM sodium phosphate buffer pH 7.4. C. CONCENTRATION SELECTION, TEST SUBSTANCE STABILITY AND VERIFICATION In accordance with testing guidelines, the highest concentration evaluated in this study was 5000 ng/plate. Solubility information was confirmed prior to study start. Solutions of the test substance were prepared immediately prior to treatment and were presumed to be stable under the conditions of the study. Treatment, control solutions, and the S9 mixture were not analyzed for concentration, uniformity, or stability. Top agar was not assayed for stability or concentration of the test or control substances, strain, or S9/PBS, since this assessment was not considered necessary to achieve the objectives of the study. Solutions of the test substance were assessed for sterility by plating a small amount of the highest test substance concentration onto the surface of minimal glucose agar plates. D. TEST METHODS: BACTERIAL MUTAGENICITY ASSAY This study consisted of a single trial that assessed test substance mutagenicity. Three replicates were plated for each tester strain in the presence and absence of the exogenous metabolic activation system at each test substance concentration. Positive and negative controls were included for each strain and condition. Treatments with the exogenous metabolic activation system were conducted by adding 0.1 mL of negative or positive control or test substance solution, 0.5 mL of metabolic activation system, and 0.1 mL of an overnight culture containing approximately 1 x 108 bacteria to approximately 2 mL of top agar (0.6% [w/v] agar and NaCl) containing 0.05 mM L-histidine, D-biotin and L-tryptophan. These components were briefly mixed and poured onto a minimal glucose agar plate (25-30 mL, 0.4% [w/v] glucose with Davis salts, purchased from MOLTOXTM). Treatments in the absence of the metabolic activation system were the same as those in the presence of the exogenous metabolic activation system with the exception that 0.5 mL of sterile buffer was used as a replacement for the volume of the exogenous metabolic activation system. After pouring onto the surface of minimal glucose agar plates, the top agar was allowed time to solidify, and the individually labeled plates were inverted and incubated at 37C for about 48 hours. When necessary, plates were refrigerated at approximately 4C ( 3C) prior to evaluation and counting of revertant colonies. Bacterial background lawns were evaluated for evidence of test substance toxicity and precipitation. Evidence of toxicity was scored relative to the concurrent negative control plates and recorded with the mean revertant count for the strain, condition, and concentration. Revertant colonies for a given tester strain and condition were counted by an automated colony counter. - 12- Company Sanitized. Does net contain TSCA CBI H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 E. STATISTICAL ANALYSIS Data for each tester strain were evaluated independently. For each tester strain, the mean number of revertants and the standard deviation at each concentration in the presence of and absence of the exogenous metabolic activation system were calculated. F. ACCEPTABILITY CRITERIA An individual trial must have included a negative and positive control and at least 5 concentration levels of the test substance for each tester strain and condition. A data point, concentration level or trial was excluded from analysis when acceptability criteria were not met. The acceptability criteria were as follows: 1. Tester Strain Phenotypes and Spontaneous Reversion All S. typhimurium tester strain cultures must have exhibited L-histidine dependent growth. To demonstrate the presence of the rfa mutation, all S. typhimurium tester strain cultures must have exhibited sensitivity to crystal violet. To demonstrate the presence of the uvrB mutation, all S. typhimurium tester strain cultures must have exhibited sensitivity to ultraviolet light in comparison to strain TA102, which is proficient in the repair of small amounts of ultraviolet light-induced DNA damage. E. coli tester strains must have exhibited L-tryptophan dependent growth. To demonstrate the presence of the uvrA mutation, the E. coli tester strain must have exhibited sensitivity to ultraviolet light. Tester strain cultures of S. typhimurium TA97a, TA98, TA100 and E. coli WP2 uvrA (pKMIOl) must have exhibited resistance to ampicillin. All tester strain cultures must have exhibited a characteristic number of spontaneous revertants per plate in the absence of the test substance. The acceptable mean revertants per plate in the presence or absence of the exogenous metabolic activation system were derived from the means of the historical negative control data and were within the following ranges: S. typhimurium strains TA97a (65-163); TA98 (8-40); TA100 (58-192); TA1535 (2-28), E. coli strain WP2 uvrA (pKMIOl) (90-227). Historical data collected at the testing facility are presented in Table 6. 2. Tester Strain Culture Titer To ensure that appropriate numbers of bacteria were plated, all tester strain culture titers were approximately 1 x 109cells/mL. 3. Positive Control Values Mean positive control values must have exhibited at least a three-fold increase over the respective mean of the concurrent negative control value for each tester strain and condition. 4. Non-Toxic Concentration Levels A minimum of 5 analyzable, of which four must be non-toxic, concentration levels was required to classify the test substance. A concentration level was considered toxic and analyzable if it caused a >50% reduction in the mean number of revertants per plate relative to the mean of the concurrent negative control and is not equal to 0. Company Sanitized. Does not contain TSCA CB1 - 13- H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 5. Rejection of Plates, Concentration Levels, or Assays A plate may have been rejected if contamination, test substance precipitation or conditions resulted on a treatment plate that prevented an accurate colony counting. A concentration level (or a negative control) was rejected if there were less than 2 data points or if variability between replicate plates was judged to be excessive. Variability between plates is strain dependent and can be effected by treatment with the test substance; therefore, scientific judgement was used in determining the acceptability of the data. An assay (for an individual strain) was rejected if the negative control was rejected, if the positive control was rejected, or if the tester strain failed to exhibit the appropriate phenotype. All data from the single trial were retained, and data that met acceptability criteria are included in this report. G. CLASSIFICATION GUIDELINES A test substance was classified as POSITIVE (i.e., mutagenic) if the mean number of revertants in any strain at any test substance concentration was at least 2 times greater than the mean of the concurrent vehicle control and there was a concentration-related increase in the mean revertants per plate in that same strain. A test substance was classified as NEGATIVE (i.e., not mutagenic) if there were no test substance concentrations with a mean number of revertants that was at least 2 times greater than the mean of concurrent vehicle control, or there was no concentration-related increase in the mean revertants per plate in that same strain. Results not meeting criteria for positive or negative classification were evaluated using scientific judgment and experience and may have been reported as EQUIVOCAL. - 14- Company Sanitized. Does not contain TSCA CB1 H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 RESULTS AND DISCUSSION H-24119 was evaluated in the bacterial reverse mutation assay using Salmonella typhimurium strains TA97a, TA98, TA100, TA1535, and Escherichia coli strain WP2 uvrA (pKMIOl) in the presence and absence of an exogenous metabolic activation system (Aroclor-induced rat liver S9). The trial was performed using the plate incorporation method to evaluate the mutagenic potential of the test substance. Sterile water was chosen as the test substance solvent, diluent, and negative control. In this study, concentrations of 5,10, 50,100, 500,1000, 2500, and 5000 pg/plate were evaluated in comparison to negative (solvent) controls. Mean positive control values measured as revertants per plate exhibited greater than a three-fold increase over the means of the respective negative control values for each tester strain (Tables 1-5). All tester strains exhibited appropriate phenotypic characteristics. The mean number of revertants observed in the negative control of each strain was within the prescribed acceptable range. No precipitate was observed in any of the Salmonella strains or in the Escherichia coli strain. (Tables 1 - 5). No toxicity was observed as evidenced by a thinning of the microcolony background lawns or as a concentration-related reduction in the mean number of revertant colonies per plate. There were no test substance concentrations with a mean number of revertants that were 2 times greater than the mean of the concurrent vehicle control, nor was there a concentration-related increase in the mean revertants per plate in any strain (Tables 1-5). CONCLUSIONS Under the conditions of this study, no evidence of mutagenic activity was detected. The test substance was negative. RECORDS AND SAMPLE STORAGE Specimens (if applicable), raw data, and the final report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. Company Sanitized. Does no! contain TSCA CBt -15- H-24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 REFERENCES 1. Maron, D. M. and B. N. Ames (1983). Revised methods for the Salmonella mutagenicity test. Mutt. Res. 113,173-215. 2. Green, M.H.L. and W. J. Muriel (1976). Mutagen testing using TRP+reversion in Escherichia coli. Mutt. Res. 38, 3-32. 3. Gatehouse, D.G., Haworth, S., Cebula, T., Gocke, E., Kier, L., Matsushima, T., Melcion, C., Nohmi, T., Venitt, S. and E. Zeiger (1994). Recommendations for the Performance of Bacterial Mutation Assays. Mutt. Res. 312,217-233. 4. Levin, D. E., E. Yamasaki, and B. N. Ames (1982). A new Salmonella tester strain, TA97, for the detection of frameshift mutagens. A run of cytosines as a mutational hot spot. Mutt. Res. 94, 315-330. 5. Claxton, L.D., Allen, J., Auletta, A., Mortelmans, K., Nestmann, E., and E. Zeiger (1987). Guide for the Salmonella typhimurium / mammalian microsome tests for bacterial mutagenicity. Mutt. Res. 189, 83-91. 6. Ames, B. N., F. D. Lee, and W. E. Durston (1973). An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Natl. Acad. Sci. USA 70, 782-786. 7. McCann, J., N. E. Springam, J. Kobori, and B. N. Ames (1975). Detection of carcinogens as mutagens: bacterial tester strains with R factor plasmids. Proc. Natl. Acad. Sci. USA 72, 979-983. - 16- Company Sanitized. Does not contain TSCA CB1 H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 ABBREVIATIONS FOR TABLES Evidence for test substance toxicity to the bacteria was documented by recording the appearance of the plates and background lawn using the following key: TO Normal, background microcolony lawn appeared normal. T1 Slightly reduced, background microcolony lawn was noticeably thinner. T2 Moderately reduced, background lawn was markedly thinner resulting in an increase in the size of microcolonies compared to the vehicle control plate(s). T3 Severely reduced, background lawn was distinguished by an extreme thinning resulting in an increase in the size of the microcolonies compared to the vehicle control plate(s). Microcolonies were seen readily by the unaided eye and were greatly enlarged relative to controls. T4 Absent, plate(s) were distinguished by a complete lack of any microcolony lawn over a majority of the area of the plate(s). Formation of a precipitate by the test material was documented using the following key: PO No precipitate, no precipitate observed. PI Microscopic precipitate, precipitate present that did not interfere with background lawn evaluation or automated colony counting. P2 Non-interfering precipitate, precipitate present that was visible to the naked eye that did not interfere with automated colony counting. P3 Interfering precipitate, precipitate present that required plate to be counted by hand. . P4 Heavy interfering precipitate, precipitate present that prevented accurate colony counting and obscured the background lawn requiring plate rejection (R). Additional abbreviations may include the following: N Absence of any noteworthy observation R Plate rejected Positive controls were abbreviated as follows: - 17- Company Sanitized. Does not contain TSCA CBf H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 TABLES - 18- Company Sanitized. Does not contain TSCA CB1 H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 TABLE 1 MUTAGENIC ACTIVITY IN SALMONELLA TYPHIMURIUM TA97A Concentration H-24119 (pg/plate) Revenants Plate Plate Plate 12 3 Mean (S.D.) Observations A. WITHOUT METABOLIC ACTIVATION 0.0 125 133 132 5.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 141 155 134 142 127 135 131 142 147 134 123 159 120 139 144 139 153 131 162 141 130 161 139 136 2 jj.g/plate 2067 1730 2177 130 (4) 143 ( I D 135 (8) 140 (8) 139 (18) 134 (13) 141 ( I D 144 (16) 145 (14) 1991 (233) T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 N B. WITH METABOLIC ACTIVATION (Aroclor-induced rat liver S9) 0.0 156 152 158 155 (3) 5.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 136 138 131 149 137 120 127 158 148 165 141 153 131 150 169 155 155 163 144 141 158 144 163 125 135 (4) 135 (15) 144 (16) 153 (12) 150 (19) 158 (5) 148 (9) 144 (19) 1985 1610 1721 1772 (193) T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 N - 19- Coniny Sanitized. Boos nal contain TSC A CBT H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 TABLE 2 MUTAGENIC ACTIVITY IN SALMONELLA TYPHIMURIUM TA98 Concentration H-24119 (pg/plate) Revertants Plate Plate Plate 12 3 Mean (S.D.) Observations A. WITHOUT METABOLIC ACTIVATION 0.0 16 24 15 a 5.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 15 pg/plate 14 21 17 16 13 26 16 23 22 14 18 19 24 21 29 22 21 23 18 17 18 17 27 20 1048 1115 1054 18 (5) 17 (4) 18 (7) 20 (4) 17 (3) 25 (4) 22 (1) 18 (1) 21 (5) 1072 (37) TH METABOLIC ACTIVATION (Aroclor--induced rat liver S9) 0.0 34 34 28 32 (3) 5.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 r 33 25 32 30 (4) 40 24 31 32 (8) 24 15 34 24 (10) 27 22 28 - 26 (3) 29 20 26 25 (5) 29 23 27 26 (3) 33 18 18 23 (9) 30 28 29 29 (1) 2 pg/plate 993 1041 972 1002 (35) T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0.P0 T0,P0 T0,P0 T0,P0 N T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 N -20- Company Sanitized. Does not contain TSC A CBI H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 TABLE 3 MUTAGENIC ACTIVITY IN SALMONELLA TYPHIMURIUM TA100 Concentration H-24119 (fxg/plate) Revertants Plate Plate Plate 12 3 Mean (S.D.) Observations A. WITHOUT METABOLIC ACTIVATION 0.0 181 183 176 5.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 1 Txg/plate 166 185 207 179 183 204 173 166 218 178 213 198 195 170 181 182 184 177 188 206 181 203 189 187 1774 1927 1557 180 (4) 186 (21) 189 (13) 186 (28) 196 (18) 182 (13) 181 (4) 192 (13) 193 (9) 1753 (186) T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 N H METABOLIC ACTIVATION (Aroclor--induced rat liver S9) 0.0 179 174 176 176 (3) 5.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 /ap.g/plate 202 180 205 223 211 204 197 170 196 192 193 224 171 205 190 184 193 201 189 184 195 203 173 180 197 (5) 191 ( I D 196 (8) 210 (23) 192 (20) 204 (1) 187 (12) 178 (7) 1294 1523 1432 1416 (115) T0,P0 T0,P0 T0,P0 T0,P0 TO,Pd T0,P0 T0,P0 T0,P0 T0,P0 N -21 - Company Sanitized. Does not contain T SC A CBt H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 TABLE4 W MUTAGENIC ACTIVITY IN SALMONELLA TYPHIMURIUM TA1535 Concentration H-24119 (Hg/plate) Revertants Plate Plate Plate 12 3 Mean (S.D.) Observations A. WITHOUT METABOLIC ACTIVATION 0.0 7 9 10 5.0 7 12 10 10.0 7 13 4 50.0 11 5 13 100.0 9 94 500.0 5 10 11 1000.0 10 13 6 2500.0 9 12 7 h 5000.0 8 8 12 9 (2) 10 (3) 8 (5) 10 (4) 7 (3) 9 (3) 10 (4) 9 (3) 9 (2) T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 ftl C?pg/plate 232 274 253 253 (21) N B. WITH METABOLIC ACTIVATION (Aroclor-induced rat liver S9) 0.0 5.0 - 10.0 50.0 100.0 500.0 1000.0 - 2500.0 5000.0 ms ^ ^ 2 . pg/plate 13 11 13 17 14 12 16 10 9 12 16 12 16 12 9 12 8 16 . 16 12 8 15 12 11 12 16 16 12 (1) 14 (3) 12 (4) 13 (2) 12 (4) 12 (4) 12 (4) 13 (2) 15 (2) 178 189 225 197 (25) T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 TO,P0 N C o m p a n y Sanitized. Does not c o n ta in T S C A CB1 - 22- H -24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 TABLE 5 MUTAGENIC ACTIVITY IN ESCHERICHIA COLI WP2 UVRA (PKM101) Concentration H-24119 (M-g/plate) Revertants Plate Plate Plate 12 3 Mean (S.D.) Observations A. WITHOUT METABOLIC ACTIVATION 0.0 124 103 91 5.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 >) r -- 1 wm 2 ftg/plate 134 70 87 143 118 107 136 104 107 140 136 124 127 109 104 139 88 108 113 101 88 151 84 103 1500 1227 1246 106 (17) 97 (33) 123 (18) 116 (18) 133 (8) 113 (12) 112 (26) 101 (13) 113 (35) T0,P0 T0,P0 T0,P0 ' T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 1324 (152) N B. WITH METABOLIC ACTIVATION (Aroclor-induced rat liver S9) 0.0 156 180 134 157 (23) 5.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 -- n ^ / 5 [ig/plate 137 173 138 147 169 174 168 186 161 160 174 -147 169 147 176 185 142 156 174 190 185 178 175 169 149 (21) 163 (14) 172 (13) 160 (14) 164 (15) 161 (22) 183 (8) 174 (5) 1329 1632 1591 1517 (164) T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 N Company Sanitized. Does not contain TSCA - 23- H-24119: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3220 TABLE HISTORICAL CONTROL DATA3 Tester Strain Control [Positive Control1*] Exogenous Metabolic Activation System Mean (S.D.) S. typhim urium TA100 Negative Negative Positive [NAAZ-2] Positive [2AA-1] S. typh im u riu m TA 1535 Negative Negative Positive [NAAZ-2] Positive [2AA-2] S. typhim urium TA97a Negative Negative Positive [ICR 191-2] Positive [DMBA-20] Positive [2AA-1] S. typhim urium TA98 Negative Negative Positive [2NF-25] Positive [2AA-2] E. c o li WP2 uvrA (pKMIOl) Negative Negative Positive [MMS-1000] Positive [ENNG-2] Positive [2AA-25] Positive [2AA-250] Absent Absent Present Present Absent Absent Present Present Absent Absent Present Present Present Absent Absent Present Present Absent Absent Absent Present Present Present 122 128 872 1187 (29) (29) (274) (476) 15 (6) 14 (6) 618 (199) 362 (136) 103 124 1822 1534 960 (18) (26) (662) (559) (348) 22 26 1403 1552 (7) (7) (372) (598) 148 167 1656 1627 1577 1682 (29) (29) (449) (317) (450) (372) Range Minimum-Maximum 54-218 65 - 253 339-2604 94-2682 4 -4 6 4 -3 9 127 - 1270 44-1323 59-164 67 - 196 476 - 3359 563 - 2773 308-2281 7-47 11-53 567 - 2774 250-3114 82-221 93 - 255 208 - 2453 1049 - 2253 485 - 2484 929 - 2230 a Historical data for tester strains used in the reported study. Data are based on studies reported during the period 1996 to 1998. Data include all control solvents or diluents, metabolic activation systems based on Aroclor -induced rat liver S9, and all forms of study modification (e.g., plate incorporation, pre- Company Sanitized. Does not contain TSCA CBI - 24-