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AR226-3153 TRADE SECRET Study Title H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli Laboratory Project ID: DuPont-3827 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 Font de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 WORK REQUEST NUMBER:)!----| j ^^^^---- v SERVICE CODE NUMBER:|pBJ| DUPONT PHARMACEUTICALS r" ^ COMPANY STUDY NO.: flfl^HH-------- I Page 1 of 25 Company Sanitized. Does not contain TSCA CBI H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli CERTIFICATION DuPont-3827 We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. I by Study Monitor: &, . ^~7\ '" G^-e.-~- -^-J^\-" _ Maria Donner, PhJD, Senior Research Scientist Genetic Toxicology DuPontHaskell Laboratory for Toxicology and Industrial Medicine 1^ Ktf^ 2.oo o Date0 Approved By: (ps n, o i \6(^J>^--________ RonaldD.Snyder.PhJ3. Director of Genetic Toricology DuPont Pfaarmaceuticals Company d^Date tm Issued by Study Dfrector: ^.C^^JS^^^ ^ <j> &/?/) ^r -, 0 3 ^ ^ N. Iawnce Gladnick, B.A.Date Staff Scientist DuPont Pharmaceuticals Company Comoany Sanrtized. Doss nofconfam TSCA CBI -2- H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3827 TABLE OF CONTENTS Page 2 CERTIFICATION........................................................................................................................... LISTOFTABLES...........................................................................................................................4 STUDY INFORMATION...............................................................................................................5 STUDY PERSONNEL ....................................................................................................................6 SUMMARY.....................................................................................................................................? INTRODUCTION...........................................................................................................................8 MATERIALS AND METHODS.....................................................................................................8 A. Study Protocol......................................................................................................................8 B. Test Materials.......................................................................................................................9 1. Test Substance ...............................................................................................................9 2. Negative and Positive Controls.....................................................................................^ 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 CONCLUSIONS............................................................................................................................14 RECORDS AND SAMPLE STORAGE .......................................................................................14 REFERENCES..............................................................................................................................^ TABLES ........................................................................................................................................16 APPENDIX - Historical Control Data.........................................................................................24 Company Sanitized. Does not contain TSCA CB1 -^_ H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli LIST OF TABLES DuPont-3827 Page Table 1: Strain Phenotype Confirmation.................................................................................... 18 Table 2: Mutagenic Activity in Salmonella typhimurium TA97a................................................ 19 Table 3: Mutagenic Activity in Salmonella typhimurium TA98................................................. 20 Table 4: Mutagenic Activity in Salmonella typhimurium TA100............................................... 21 Table 5: Mutagenic Activity in Salmonella typhimurium TA1535............................................. 22 Table 6: Mutagenic Activity in Escherichia coli WP2 uvrA (pKMIOl)..................................... 23 Company Sanitized. Does not contain TSCA CBI -4- H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli STUDY INFORMATION Substance Tested:^B|UU|j| Synonyms/Codes:-,* H-24335 Haskell Number: 24335 Composition, DuPont-3827 Known Impurities 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, 20UO / (see report cover page) In-Life Initiated/Completed: February 11, 2000 / February 22,2000 Company Sanitized. Does not contain TSCA CBI .5- H-24335: 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: Technician: Director of Genetic Toxicology: N. Lawrence Gladnick, B.A. 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-3827 Company Sanitized. Does not contain TSCA CB1 -6- H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3827 SUMMARY H-24335 was evaluated in the bacterial reverse mutation test 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-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 /xg/plate were evaluated in comparison to negative (solvent) controls. Test substance-related toxicity, evidenced by the reduction of the microcolony background lawns and/or as a concentrationrelated reduction in the mean number of revertants 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 of revertants seen in the negative controls for each strain was within the acceptable prescribed negative control ranges. Under the conditions of this study, no evidence of mutagenic activity was detected in any of the Salmonella strains or in the Escherichia coli strain in the presence or absence of the exogenous S9 metabolic activation system. Based on the findings, H-24335 was concluded to be negative for the induction of mutagenicity in the bacterial reverse mutation test. Company SanitSzed. Does not contain TSCA CBI -7- H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3827 INTRODUCTION This study evaluated the mutagenic potential of the test substance, H-24335, in the bacterial reverse mutation test using Salmonella typhimurium strains TA97a, TAPS, 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 point mutations, which involve substitution, addition, or deletion of 1 or a few DNA base pairs.0'^ 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 The protocol consisted of the stand-alone Protocol and the Haskell General Testing Procedure ||BHBjl|t Bacterial Reverse Mutation Test for Solids, Liquids, and Gases", effective 06/10/98). 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 [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. company Sanitized. Does not contain TSCA CBI -8- H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3827 B. Test Materials 1. Test Substance The test substance, H-24335, is q^lHBBIBBHIiBBHwhat 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 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: Positive Controls 2-Nitrofluorene (2NF) A^-Ethyl-^-nitro-^-nitroguanidine (ENNG) Sodium azide (NAAZ) ICR 191 Acridine mutagen (ICR 191) 9,10-Dimethyl-l,2-benzanthracene(DMBA) 2-Aminoanthracene (2AA)_________ Chemical Abstracts Service (CAS) _____Registry Number_____ 607-57-8 4245-77-6 26628-22-8 17070-45-0 57-97-6 613-13-8 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 the 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: Company Sanitized. Does not contain TSCA CBS -9- H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3827 Strain S. typhimurium TA97a S. typhimurium TA98 S. typhimurium TA100 S. typhimurium TA102* S. typhimurium TA1535 S. typhimurium TA1537 E. co^WP2MvrA (pKMIOl) Gene Locus Reversion Target hisD661(f hisD3Q52 hisG46 hisG42S hisG46 hisC30'76 TrpE DNA Repair* AuvrB AuvrB AuvrB (+) AuvrB AuvrB 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 his0l242. 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.^ 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 5. 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.^ 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.^ 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 Company Sanffeed. Does not contain TSCA CBI -10- H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3827 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 of Aroclor-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'1" (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 j^g/plate. Solubility information was confirmed prior to that start of the study. The stock concentration 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. Company Sanitized. Does not contain TSCA CBI -^- H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3827 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 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 Integrity All S. 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 5. 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- ^ompany SanSfeed. Does not contain TSCA CBS H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3827 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 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 of revertants 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 of revertants in any strain (except S. tymphimurium strain TA1535) at any test substance concentration was at least two times greater than the mean number of revertants of the concurrent negative control, and there was a concentration-related increase in the mean number of revertants per plate in that same strain. For S. tymphimurium strain TA1535 there must be no test substance concentration with a mean number of revertants 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 of revertants 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- ompany Sanitized. Does not contain TSCA CB1 H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3827 RESULTS AND DISCUSSION H-24335 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-inducedrat 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 jug/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 of toxicity was observed in any of the Salmonella typhimurium strains or in the Escherichia coli strain (Tables 2-6). In tester strains TA97a, TA98, TA100, or E. 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). In 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-24335 was concluded to be negative for the induction of mutagenicity 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. -14- Company Sanitized. Does not contain TSCA CB3 H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3827 REFERENCES 1. Maron, D. M. and B. N. Ames (1983). Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113,173-215. 2. Green, M.H.L. and W. J. Muriel (1976). Mutagen testing using TRP4" reversion in Escherichia coli. Mutat. Res. 38,3-32. 3. Claxton, L.D., Alien, J., Auletta, A., Mortehnans, 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 of mutagens 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. Company Sanitized. Does not contain TSCA CB3 15- H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3827 TABLES Company Sanffized. Does not contain TSCA CBI 16- H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3827 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 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: ICR 191 DMBA 2NP 2AA NAAZ ENNG Acridine mutagen 9,10-Dimethyl-1,2-benzanthracene 2-Nitrofluorene 2-Aminoanthracene Sodium azide ^-Ethyl-TV-nitro-JV-nitroguanidine -17- Company Sanitized. Does not contain TSCA CB8 H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli Table 1 Strain Phenotype Confirmation DuPont-3827 Strain L-Histidine/lL-Tryptophan Dependerit Growth' In Absence In Presence Additional Growth Characiteristics Crystal UV Ampicillin" Tetracycline0 Violet" Light6 TA97a TA98 TA100 TA1535 + S S R S + S S R S + S S R S + S S S S WP2 im-A + R S R S (pKMIOl) TA102e WAf N/A N/A R N/A N/A Tested for histidine (S. <ypftiWMriMm)/tryptophan (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. Tested for presence ofpKMIOl plasmid by demonstrating resistance to ampicillin and sensitivity to tetracycline; R = Resistant; S = Sensitive. TA102 used as a control for UV Light sensitivity only. 'N/A = Not applicable -18- Company Sanitized. Does not contain TSCA CB3 H-24335: Bacterial Reverse Mutation Test in Salmonella typhimuriwn and Escherichia coli Table 2 Mutagenic Activity in Salmonella typhimurium TA97a DuPont-3827 Concentration Qig/plate) Plate 1 Revertants Plate 2 I late 3 Mean (S.D.) A. WrTHOUT METABC)LIC ACTIVAITON 0 5 10 50 100 500 1000 2500 5000 100 115 148 114 145 150 125 125 137 123 137 130 134 152 134 133 134 119 138 142 141 122 123 128 94 89 97 ICR 191 2 j^g/plate 2034 2319 1858 121 (25) 136 (20) 129 (7) 130 (7) 140 (10) 129 (8) 140 (2) 124 (3) 93 (4) , 2070 (233) B. WITH METABOLIC ACTIVATION (Aroclor6^induced rat liver S9) 0 5 10 50 100 500 1000 2500 5000 142 145 156 142 159 165 151 162 149 134 143 138 152 174 161 170 174 176 153 140 136 108 125 144 116 147 130 148 (7) 155 (12) 154 (7) 138 (5) 162 (11) 173 (3) 143 (9) 126 (18) 131 (16) DMBA 20 /zg/plate 880 855 1222 986 (205) 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 TOJPO TO,PO TO,PO N Company Sanitized. Does not contain TSCA CBS 19- H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli TableS Mutagenic Activity in Salmonella typhimurium TA98 DuPont-3827 Concentration (pig/plate) Plate 1 Revertants Plate 2 Plate 3 Mean (S.D.) A. WITHOUT METABOLIC ACTIVATION 0 5 10 50 100 500 1000 2500 5000 28 14 14 27 17 24 15 18 17 23 32 13 20 13 23 18 17 23 24 12 16 14 9 18 14 13 10 2NF 25 pig/plate 1985 1509 148: 19 (8) 23 (5) 17 (2) 23 (10) 19 (5) 19 (3) 17 (6) 14 (5) 12 (2) 1659 (283) B. WITH METABOLIC ACTIVATION i(An 0 5 10 50 100 500 1000 2500 5000 20 20 36 30 24 23 24 19 20 14 21 26 20 25 25 19 22 28 20 20 26 20 23 12 18 21 26 2AA 2 pig/plate 1544 1518 :159' ^-induced rat liver S9) 25 (9) 26 (4) 21 (3) 20 (6) 23 (3) 23 (5) 22 (3) 18 (6) 22 (4) 1552 (39) 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 Cotftpany Sanitized. Does no{ contain TSCA CB1 -20- H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli Table 4 Mutagenic Activity in Salmonella typhimurium TA100 DuPont-3827 Concentration (^ig/plate) Plate 1 Revertants Plate 2 Plate 3 Mean (S.D.) A. WITHOUT METABOLIC ACTIVATION 0 5 10 50 100 500 1000 2500 5000 160 140 145 146 162 185 143 136 154 155 132 139 126 136 118 151 156 151 103 105 116 63 68 64 52 59 61 NAAZ 2 ^g/plate 665 759 720 148 (10) 164 (20) 144 (9) 142 (12) 127 (9) 153 (3) 108 (7) 65 (3) 57 (5) 715 (47) B. WITH METABOLIC ACTIVATION (Aroi r-ihduced rat liver S9) 0 5 10 50 100 500 1000 2500 5000 161 178 178 151 144 174 159 149 155 153 138 158 124 152 159 176 169 176 162 162 164 91 121 127 117 95 107 172 (10) 156 (16) 154 (5) 150 (10) 145 (19) 174 (4) 163 (1) 113 (19) 106 (11) 2AA 2 ^g/plate 1846 2213 1697 1919 (266) Observations TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO TO,PO T1,PO T2.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 CBS H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli Table 5 Mutagenic Activity in Salmonella typhimurium TA1535 DuPont-3827 Concentration (^ig/plate) Plate 1 Revertants Plate 2 Plate 3 Mean (S.D.) A. WITHOUT METABOLIC ACTIVATION 0 5 10 50 100 500 1000 2500 5000 13 12 16 9 14 13 10 10 11 11 17 18 16 14 13 13 16 10 8 14 20 8 6 10 14 8 8 NAAZ 2 ^.g/plate 931 1020 796 14 (2) 12 (3) 10 (1) 15 (4) 14 (2) 13 (3) 14 (6) 8 (2) 10 (3) 916 (113) B. WITH METABOLIC ACTIVATION (Aro 0 5 10 50 100 500 1000 2500 5000 16 18 13 14 23 20 14 7 9 15 17 14 11 15 16 10 8 15 9 14 10 15 14 8 13 16 13 2AA 2 jug/plate 350 323 334 "-induced rat liver S9) 16 (3) 19 (5) 10 (4) 15 (2) 14 (3) 11 (4) 11 (3) 12 (4) 14 (2) 336 (14) 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-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3827 Table 6 Mutagenic Activity in Escherichia coli WP2 wrA (pKMIOl) Concentration ______Revertants_____ (^g/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) A. WITHOUT METABOLIC ACTIVATION 0 5 10 50 100 500 1000 2500 5000 107 120 89 117 132 97 129 130 97 115 118 96 111 116 110 120 129 115 103 120 95 121 139 107 106 91 89 ENNG 2 pig/plate 1081 1280 1191 105 (16) 115 (18) 119 (19) 110 (12) 112 (3) 121 (7) 106 (13) 122 (16) 95 (9) 1184 (100) B. WITH METABOLIC ACTIVATION (Aroclor-induced rat liver S9) 0 5 10 50 100 500 1000 2500 5000 161 160 166 164 174 176 152 176 168 173 177 147 200 169 199 207 213 150 184 157 186 179 175 164 143 148 149 162 (3) 171 (6) 165 (12) 166 (16) 189 (18) 190 (35) 176 (16) 173 (8) 147 (3) 2AA 25 pig/plate 1583 1224 1313 1373(187) 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 Company Sanitized. Does not contain TSCA CB1 -23- H-24335: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3827 APPENDIX HISTORICAL CONTROL DATA -24- Company Sanitized. Doss not contain TSCA CBi H-24335: Bacterial Reverse Mutation Test in Salmonella typhlmurium and Escherichia coli DuPont-3827 mSTORICAL CONTROL DATA8 Tester Strain Exogenous Metabolic Control [Positive Control ] Activation System Mean (S.D.) Range Minimum - Maximum 5. 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] S. 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 Aroclorinduced 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^-nitro-^V-nitrosoguanidine). The number following abbreviation is the microgram (/(g) amount per plate or vial used for the positive control. 25- Company Sanfffeed. Does no? contain TSCA CBS