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AR226-3154 TRADE SECRET Study Title H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli Laboratory Project ID: DuPont-3828 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 (Adopted 1997) AUTHOR: N. Lawrence Gladnick, B.A. STUDY COMPLETED ON: May 23, 2000 TESTING FACILITY: DuPont Pharmaceuticals Company Safety Assessment Section Stine-Haskell Research Center P.O. Box 30, Elkton Road Newark, Delaware 19714-0030 SPONSOR: 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 NUMBER:V|||U J SERVICE CODE NUMBERi^BBiJ DUPONT PHARMACEUTICALS^*- & Page 1 of 25 Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3828 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Reviewed by Study Monitor: ly\\.0^i ^-. '^<~^-Js-SV^ '- --Maria Donner, PhA Senior Research Scientist Genetic Toxicology DuPont Haskell Laboratory for Toxicology and Industrial Medicine _[i^/,__,^<_o^^\_2-0 & o Date ApprovedB,: Q ^ ^ g^_ Ronald D. Snyder, Ph.D. Director of Genetic Toxicology DuPont Phannaceuticals Company H ^ ^ , Date "-"as % ^ ^4. A^u^ "^ N. Lawrea^Gladnick, B.A.Date Staff Scientist DuPont Phannaceuticals Company -2- Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3828 TABLE OF CONTENTS CERTIFICATION...........................................................................................................................2 LIST OF TABLES..........................................................................................................................4 STUDY INFORMATION............................................................................................................... 5 STUDY PERSONNEL................................................................................................................... 6 SUMMARY............:.......................................................................................................................? INTRODUCTION........................................................................................................................... 8 MATERIALS AND METHODS................................................................................................... 8 A. Study 8 Protocol..................................................................................................................... B. Test Materials...................................................................................................................... 9 1. Test Substance............................................................................................................... 9 2. Negative and Positive Controls..................................................................................... 9 3. Tester Strain Source, Characterization, Storage, and Culture....................................... 9 4. Metabolic Activation System...................................................................................... 10 C. Test Substance Concentration Selection, Stability, and Verification................................ 11 D. Test Methods: Bacterial Mutagenicity Test..................................................................... 11 E. Statistical Analysis............................................................................................................ 12 F. Acceptability Criteria........................................................................................................ 12 1. Tester Strain Integrity.................................................................................................. 12 2. Tester Strain Titer........................................................................................................ 12 3. Positive Control Values............................................................................................... 13 4. Non-Toxic Concentration Levels................................................................................ 13 5. Rejection of Plates, Concentration Levels, or Assays................................................. 13- G. Classification Criteria........................................................................................................ 13 RESULTS AND DISCUSSION................................................................................................... 14 14 CONCLUSIONS........................................................................................................................... RECORDS AND SAMPLE STORAGE....................................................................................... 14 14 REFERENCES.............................................................................................................................. 16 TABLES........................................................................................................................................ APPENDIX - Historical Control Data........................................................................................ 24 -3- Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3828 LIST OF TABLES Page Table 1: Table 2: Table 3: Table 4: Table 5: Table 6: Strain Phenotype Confirmation...................................................................................... 18 Mutagenic Activity in Salmonella typhimurium TA97a................................................ 19 Mutagenic Activity in Salmonella typhimurium TA98.................................................. 20 Mutagenic Activity in Salmonella typhimurium TA100................................................ 21 Mutagenic Activity in Salmonella typhimurium TA1535..............................................22 Mutagenic Activity in Escherichia coli WP2 uvrA (pKMIOl) .....................................23 -4- Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli STUDY INFORMATION Substance Tested: Synonyms/Codes, Haskell Number: 23960 ... /L DuPont-3828 Physical Characteristics: 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: February 2, 2000 / (see report cover page) In-Life Initiated/Completed: February 11, 2000 / February 21, 2000 -5. Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli STUDY PERSONNEL The following individuals participated in the conduct of this study: DuPont Pharmaceuticals Company Study Director: Technicians: Director of Genetic Toxicology: N. Lawrence Gladnick, B.A. Dwayne A. Lavoie, B.S. James C. Finer N. Lawrence Gladnick, B.A. Ronald D. Snyder, Ph.D. E. I. du Font de Nemours and Company Management: Carolyn S. VanPelt. D.V.M., Ph.D. Study Monitor: E. Maria Donner, Ph.D. Report Preparation: Brenda Tiffin DuPont-3828 -6- Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3828 SUMMARY H-23960 was evaluated in the bacterial reverse mutation test using Salmonella typhimurium strains TA97a, TAPS, TA100, TA1535, and Escherichia coli strain WP2 uvrA (pKMIOl) in the presence and absence of an exogenous metabolic activation system (Aroclor-inducedrat liver S9). A single trial test 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. Test substance concentrations of 5,10, 50,100, 500,100, 2500, and 5000 /ig/plate were evaluated in comparison to the negative (solvent) controls. Test substance-related toxicity, evidenced by the reduction of the microcolony background lawns and/or as a concentration- related reduction in the mean number ofrevertants per plate, was not observed. No precipitate was observed at any concentration with any strain. All tester strains exhibited appropriate phenotypic characteristics. The mean number ofrevertants seen in the negative controls for each strain was within the acceptable prescribed negative control ranges. Under the conditions in this study, no evidence of mutagenic activity was detected in any of the Salmonella strains or the Escherichia coli strain in the presence or absence of the exogenous S9 metabolic activation system. Based on the findings, H-23960 was concluded to be negative for the induction ofmutagenicity in the bacterial reverse mutation test. 7- Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3828 INTRODUCTION This study evaluated the mutagenic potential of the test substance, H-23960, in the bacterial reverse mutation test using Salmonella typhimurium strains TA97a, TA98, TA100, and TA1535 and in Escherichia coli strain WP2 uvrA (pKMIOl). The bacterial reverse mutation test uses ammo acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition, or deletion of 1 or a few DNA base pairs.012) The Salmonella tester strains are unable to synthesize histidine because of specific point mutations in genes coding for histidine biosynthesis. Additional mutations in 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.^ 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 B""' ' ' 06/10/98). ' nsisted of the stand-alone Protocol and the Haskell General Testing Procedure 'Bacterial Reverse Mutation Test for Solids, Liquids, and Gases", effective 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 [Adopted 1997]); The study design complied with the testing guidelines cited above with the following exception: " One trial was conducted with the test substance as specified by the sponsor. This exception did not affect the study validity as a second trial was not considered necessary by the sponsor to meet the objective of the study. -8- Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3828 B. Test Materials 1. Test Substance ajlJBIHIIIIlUB^ The test'substance, H-23960, is was storeci at room temperature. Additional information regardingthe 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 on 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: Chemical Abstracts Service (CAS) Positive Controls 2-Nitrofluorene (2NF) A^-Ethyl-^-mtro-A^-nitroguanidine (ENNG) _____Registry Number_____ 607-57-8 4245-77-6 Sodium azide (NAAZ) ICR 191 Acridine mutagen (ICR 191) 9,10-Dimethyl-l,2-benzanthracene (DMBA) 26628-22-8 17070-45-0 57-97-6 2-Aminoanthracene (2AA)_________________613-13-8. Deionized water was the solvent for sodium azide (NAAZ). The solvent for 2AA, 2NF, ICR 191, DMBA, and ENNG was dimethyl sulfoxide (DMSO). The manufacturer, lot number, and purity of the solvent was recorded in the study records. Neither the amount and nature of the contaminants nor the use of solvent was expected to affect the integrity or validity of the study. The positive controls were prepared in advance and maintained frozen at approximately -70C. Previous 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. 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: -9- Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3828 Strain S. typhimurium TA97a S. typhimurium TA98 S. typhimurium TA100 S. typhimurium TA102* 5'. typhimurium TA1535 S. typhimurium TA1537 E. coli WP2 uvrA (pKMIOl) Gene LOCU!3 Reversion Target hisD66l(f hisD3052 hisG46 A^G428 hisG46 hisC3076 TrpE DNA Repair* AywZ? AiwB ^uvrB (+) &uvrB &uvrB AuvrA LPS rfa rfa rfa rfa rfa rfa NA R-factor (pKMIOl) Plasmid Present Present Present Present Absent Absent Present pAQI Plasmid Absent Absent Absent Present Absent Absent Absent Note: LPS rfa NA * * A (+) uvrA and uvrB are defective DNA repair genes. = lipopolysaccharide = deep rough mutation = Not applicable Concurrent control strain used to distinguish differential sensitivity/resistance to ultraviolet light. Also /KS01242. symbol for "deletion" proficient in excision repair The deletion (A) in uvrB (a gene that codes for a protein involved in DNA excision repair) increases the bacterial sensitivity to some mutagens.05 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/45 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/55 The pAQI plasmid confers tetracycline resistance to S. typhimurium TA102, that was used solely as a control for UV light sensitivity. The Salmonella tester strains were stored at approximately -70C in -8% (v/v) DMSO in Oxoid Nutrient Broth No. 2. The E. coli WP2 uvrA (pKMIOl) strain was stored at approximately -70C in -30% glycerol in Oxoid Nutrient Broth No. 2. Prior to the mutagenicity test, 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 approximately 37C with shaking. Tester strain phenotypes were confirmed on overnight cultures concurrently with the single trial. 4. Metabolic Activation System Because the tester strains lack many of the enzymes required to convert some promutagens to a reactive state, the test was performed in the presence and absence of an exogenous metabolic activation system similar to that described by Maron and Ames/0 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 10Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3828 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 aliquot was plated on nutrient agar capable of supporting the growth of viable bacteria. The amount ofAroclor-1254 induced rat liver S9 in the exogenous metabolic activation system was 4.0 mg S9 protein (-10% [v/v]) / mL. The cofactor-supplement concentrations in the exogenous metabolic activation system were 8 mM MgClz, 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. Test Substance Concentration Selection, Stability, and Verification In accordance with testing guidelines, the highest concentration evaluated in this study was 5000 jUg/plate. Solubility information was confirmed prior to that start of the study. The stock concentrations were calculated and adjusted for test substance displacement. 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 agar plates capable of supporting bacterial growth. D. Test Methods: Bacterial Mutagenicity Test This study consisted of a single trial that assessed the mutagenicity of the test substance. 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 NaCI) 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 approximately 37C for about 48 hours. Plates were refrigerated at 4C ( 3C) prior to evaluation and counting of revertant colonies. -11- Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3828 Bacterial background lawns were evaluated for evidence of test substance toxicity and precipitation. Evidence of toxicity observed in the microcolony background lawns, 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. E. Statistical Analysis Data for each tester strain were evaluated independently. For each tester strain, the mean number ofrevertants 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 Integrity All & typhimurium tester strain cultures were required to exhibit L-histidine dependent growth. To demonstrate the presence of the rfa mutation, all S. typhimurium tester strain cultures were required to exhibit sensitivity to crystal violet. To demonstrate the presence of the uvrB mutation, all S. typhimurium tester strain cultures were required to exhibit 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 were required to exhibit L-tryptophan dependent growth. To demonstrate the presence of the uvrA mutation, the E. coli tester strain was required to exhibit 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, to demonstrate the presence of the pKMIOl or R-factor plasmid. All tester strain cultures were required to exhibit a characteristic number of spontaneous revertants per plate in the absence of the test substance. The acceptable mean revertants per plate of the negative controls 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 the appendix of this report. 2. Tester Strain Titer To ensure that appropriate numbers of bacteria were plated, all tester strain culture liters were approximately 1 x 109 cells/mL. -12- Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3828 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. is 4. Non-Toxic Concentration Levels A minimum of 5 analyzable (of which 4 must be non-toxic) concentration levels were required to classify the test substance. A concentration level was considered toxic and analyzable if the test substance at that specific concentration caused a 50% reduction in the mean number ofrevertants per plate relative to the mean of the concurrent negative control and was not equal to 0. 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. Scientific judgement was used in determining the acceptability of the data. An assay (for an individual strain) would have been 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 study were retained, and data that met acceptability criteria are included in this report. G. Classification Criteria A test substance was classified as POSITIVE (i.e., mutagenic) if the mean number ofrevertants in any strain (except S. tymphimurium strain TA1535) at any test substance concentration was at least two times greater than the mean number ofrevertants of the concurrent negative control, and there was a concentration-related increase in the mean number ofrevertants per plate in that same strain. For S. tymphimurium strain TA1535 there must be no test substance concentration with a mean number ofrevertants that is at least three times greater than the mean number of revertants of its concurrent negative control and a concentration-related increase in the mean number ofrevertants per plate. A test substance was classified as NEGATIVE (i.e., not mutagenic) if all positive classification criteria for all strains are not met. Results not meeting criteria for either positive or negative classification were evaluated using scientific judgment and experience and may have been reported as EQUIVOCAL. -13- Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3828 RESULTS AND DISCUSSION H-23960 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 test 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, test substance concentrations of 5,-10, 50,100, 500,1000, 2500, and 5000 /zg/plate were tested 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 2-6). All tester strains exhibited appropriate phenotypic characteristics (Table 1). The mean number of revertants observed in the negative control for each strain was within the prescribed acceptable range. No test substance-related precipitate or evidence oftoxicity was observed in any of the Salmonella typhimurium strains or in the Escherichia coli strain (Tables 2-6). In tester strains TA97a, TA98, TA100, orE. coli WP2 uvrA (pKmIOl), there were no test substance concentrations with a mean number of revertants that were two times greater than the mean of the concurrent vehicle control (Tables 2, 3, 4, and 6). m tester strain TA1535, there were no test substance concentrations with a mean number of revertants three times greater than the mean of the concurrent vehicle control (Table 5). There was no concentration-related increase in the mean revertants per plate in any strain. CONCLUSIONS Under the conditions of this study, no evidence of mutagenic activity was detected. Based on the findings, H-23960 was concluded to be negative for the induction ofmutagenicity in the bacterial reverse mutation test in Salmonella typhimurium and Escherichia coli in the presence or absence of an exogenous metabolic activation system. RECORDS AND SAMPLE STORAGE Laboratory-specific or site-specific data, such as personnel files and equipment records will be retained by the facility where the work was done. At the request of the sponsor, raw data and the final report will be retained at DuPont Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. REFERENCES 1. Maron, D. M. and B. N. Ames (1983). Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113, 173-215. -14- Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coll DuPont-3828 2. Green, M.H.L. and W. J. Muriel (1976). Mutagen testing using TIU^ reversion in Escherichia coli. Mutat. Res. 38, 3-32. 3. Claxton, L.D., Alien, J., Auletta, A., Mortelmans, K., Nestmann, E., and E. Zeiger (1987). Guide for the Salmonella typhimurium I mammalian microsome tests for bacterial mutagenicity. Mutat. Res. 189, 83-91. 4. Ames, B. N., F. D. Lee, and W. E. Durston (1973). An improved bacterial test system for the detection and classification ofmutagens and carcinogens. Proc. Natl. Acad. Sci. USA 70, 782-786. 5. 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. 15Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3828 TABLES -16- Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3828 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. Tl Slightly reduced, background microcolony lawn was noticeably thinner. T2 Moderately reduced, background lawn was markedly thinner resulting in an increase in the size ofmicrocolonies 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: ICR 191 DMBA 2NF 2AA NAAZ ENNG Acridine mutagen 9,10-Dimethyl-l,2-benzanthracene 2-Nitrofluorene 2-Aminoanthracene Sodium azide A^-Ethyl-iV-nitro-A^-nitroguanidine -17- Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli Table 1 Strain Phenotype Confirmation DuPont-3828 Strain L-Histidine/L-Tryptophan Dependent Growtha In Absence In Presence Additional Growth Characteristics Crystal UV Arnpicillin" Tetracycline" Violet1' Light0 TA97a TA98 TA100 TA1535 + S S R S + S S R S + S S R S . + S S S S WP2 uvrA. + R S R S (pKMIOl) N ^ TA102e N/A N/A R N/A N/A 'Tested for histidine (S. typhimurium)/tryptoph'm (E. coli) requirement by the ability to grow in the absence and presence ofhistidine/tryptophan; + = Growth; - = No growth. ''Tested for rfa deletion by demonstrating sensitivity to crystal violet; S = Sensitive; R = Resistant. 'Tested for uvrA and uvrB deletion by demonstrating sensitivity to UV light; S = Sensitive; R = Resistant. rested for presence ofpKMIOl plasmid by demonstrating resistance to ampicillin and sensitivity to tetracycline; R = Resistant; S = Sensitive. ^AlOl used as a control for UV Light sensitivity only. ^/A = Not applicable 18Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli Table 2 Mutagenic Activity in Salmonella typhimurium TA97a DuPont-3828 Concentration (^g/plate) Plate 1 Revertants Plate 2 Plate 3 Mean (S.D.) A. WITHOUT METABOLIC ACTIVATION 0 5 10 50 100 500 1000 2500 5000 112 142 117 97 129 137 135 154 143 119 127 150 161 157 128 126 148 126 133 143 122 133 126 153 147 148 142 ICR 191 2 /Ag/plate 1254 1053 1324 124 (16) 121 (21) 144 (10) 132 (16) 149 (18) 133 (13) 133 (11) 137 (14) 146 (3) 1210 (141) B. WITH METABOLIC ACTIVATION (Aroclor-induced rat liver S9) 0 5 10 50 100 500 1000 2500 5000 162 170 142 149 170 156 162 145 163 149 169 177 162 162 182 154 174 160 162 172 157 160 197 183 171 166 149 158 (14) 158 (11) 157 (10) 165 (14) 169 (12) 163 (10) 164 (8) 180 (19) 162 (12) DMBA 20 /xg/plate 1077 1102 882 1020 (120) Observations TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO.PO N TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO.PO N 19Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli Table 3 Mutagenic Activity in Salmonella typhimurium TA98 DuPont-3828 Concentration (^g/plate) Plate 1 Revertants Plate 2 Plate 3 Mean (S.D.) A. WITHOUT METABOL,IC ACTIV.ATION 0 5 10 50 100 500 1000 2500 5000 25 18 16 23 21 23 18 24 18 21 19 19 20 23 25 13 21 19 16 25 13 20 10 15 19 22 19 2NF 25 ^g/plate 1867 1707 1610 20 (5) 22 (1) 20 (3) 20 (1) 23 (3) 18 (4) 18 (6) 15 (5) 20 (2) 1728 (130) B. WITH METABOLIC ACTIVATION (Aroclor-mduced rat liver S9) 0 5 10 50 100 500 1000 2500 5000 18 22 26 29 22 27 24 25 30 31 17 18 29 30 23 28 29 18 26 27 28 19 22 19 15 24 29 22 (4) 26 (4) 26 (3) 22 (8) 27 (4) 25 (6) 27 (1) 20 (2) 23 (7) 2AA 2 /xg/plate 2162 1941 2195 2099 (138) Observations TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO N TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO.PO N -20- Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli Table 4 Mutagenic Activity in Salmonella typhimurium TA100 DuPont-3828 Concentration (^g/plate) Plate 1 Revertants Plate 2 Plate 3 Mean (S.D.) A. WITHOUT METABOLIC ACTIVATION 0 5 10 50 100 500 1000 2500 5000 147 143 136 151 141 151 149 163 153 163 154 148 151 148 137 136 153 144 150 160 159 134 138 142 154 150 137 NAAZ 2 /ig/plate 689 808 837 142 (6) 148 (6) 155 (7) 155 (8) 145 (7) 144 (9) 156 (6) 138 (4) 147 (9) 778 (78) B. WITH METABOLIC ACTIVATION (Aroclor-inducedrat liver S9) 0 5 10 50 100 500 1000 2500 5000 185 176 170 186 139 164 182 170 168 153 151 149 159 173 154 166 162 189 160 160 150 159 139 163 155 155 152 177 (8) 163 (24) 173 (8) 151 (2) 162 (10) 172 (15) 157 (6) 154 (13) 154 (2) 2AA 2 /Ag/plate 2240 2244 2150 2211 (53) Observations TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO N TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO.PO N -21- Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli Table 5 Mutagenic Activity in Salmonella typhimurium TA1535 DuPont-3828 Concentration Oxg/plate) Plate 1 Revertants Plate 2 Plate 3 Mean (S.D.) A. WITHOUT METABOLIC ACTIVATION 0 5 10 50 100 500 1000 2500 5000 17 15 9 16 17 8 13 19 14 12 17 13 10 13 11 7 10 18 14 14 19 12 9 15 17 13 11 NAAZ 2 fig/plate 745 779 798 14 (4) 14 (5) 15 (3) 14 (3) 11 (2) 12 (6) 16 (3) 12 (3) 14 (3) 774 (27) B. WITH METABOLIC ACTIVATION (Aroclor-inducedrat liver S9) 0 5 10 50 100 500 1000 2500 5000 19 11 11 8 9 14 16 19 10 11 12 13 18 13 6 11 14 13 13 10 13 12 12 9 14 14 16 14 (5) 10 (3) 15 (5) 12 (1) 12 (6) 13 (2) 12 (2) 11 (2) 15 (1) 2AA 2 jug/plate 342 300 392 345.(46) Observations TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO.PO N TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO.PO N -22- Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3828 Table 6 Mutagenic Activity in Escherichia coli WP2 uvrA (pKMIOl) Concentration (^g/plate) Plate 1 Revertants Plate 2 Plate 3 Mean (S.D.) Observations A. WITHOUT METABOLIC ACTIVATION 0 5 10 50 100 500 1000 2500 5000 133 150 144 154 151 147 183 139 154 174 149 141 187 159 160 171 173 154 175 126 107 169 153 125 138 134 136 tl^ EmN-MNINGU 2/xg/plate 1846 1758 1727 142 (9) 151 (4) 159 (22) 155 (17) 169 (16) 166 (10) 136 (35) 149 (22) 136 (2) 1777 (62) TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO.PO N B. WITH METABOLIC ACTIVATION (Aroclor-inducedrat liver S9) 0 5 10 50 100 500 1000 2500 5000 152 149 134 169 182 146 183 199 171 207 229 160 197 189 168 130 210 135 169 212 182 205 177 167 177 185 143 145 (10) 166 (18) 184 (14) 199 (35) 185 (15) 158 (45) 188 (22) 183 (20) 168 (22) 2AA 25^g/plate 1459 1768 1694 1640(161) TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO.PO N -23- Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3828 APPENDIXHISTORICAL CONTROL DATA -24- Company Sanitized. Does not contain TSCA CBI H-23960: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3828 HISTORICAL CONTROL DATA" Tester Strain Exogenous Metabolic Control [Positive Control1'] Activation System Mean (S.D.) Range Minimum - Maximum S. typhimurium TA97a Negative Negative Positive [ICR 191-2] Positive [DMBA-20] Positive [2AA-1] S. typhimurium TA98 Negative Negative Positive [2NF-25] Positive [2AA-2] 5'. typhimurium TA100 Negative Negative Positive [NAAZ-2] Positive [2AA-1] S. typhimurium TA 1535 Negative Negative Positive [NAAZ-2] Positive [2AA-2] E. coli WP2 uvrA (pKMIOl) Negative Negative Positive [MMS-1000] Positive [ENNG-2] Positive [2AA-25] Positive [2AA-250] Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Present Present Present Present Present Present Present Present Present Present Present Present 103 124 1822 1534 960 (18) (26) (662) (559) (348) 22 26 1403 1552 (7) (7) (372) (598) 122 128 872 1187 (29) (29) (274) (476) 15 (6) 14 (6) 618 (199) 362 (136) 148 167 1656 1627 1577 1682 (29) (29) (449) (317) (450) (372) 59 67 476 563 308 - 164 196 3359 2773 2281 7 11 567 - 250 - 47 53 2774 3114 54 65 339 94 - 218 253 2604 2682 4 4 127 - 44 - 46 39 1270 1323 82 93 208 1049 485 929 - 221 255 2453 2253 2484 2230 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-incubation/gas, waste water). Abbreviations for positive controls: NAAZ (sodium azide); 2AA (2-aminoanthracene); 2NF (2-nitrofluorene); MMS (methyl methanesulfonate); ICR 191 (ICR 191 Acridine mutagen); DMBA (9,10-dimethyl-l,2benzanthracene), ENNG (/V-ethyl-A^-mtro-A^-nitrosoguanidme). The number following abbreviation is the microgram (^g) amount per plate or vial used for the positive control. -25- Company Sanitized. Does not contain TSCA CBI