Document mq6XrVRVKQpJ8dd6d335b5BKd

AR226-3151 DuPont-3906 TRADE SECRET Study Title H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli Laboratory Project ID: DuPont-3906 Test Guidelines: U.S. EPA Health Effects Test Guidelines OPPTS 870.5100 (1998) OECD Guidelines for Testing o f Chemicals Section 4: Health Effects, No. 471 (Adopted 1997) Author: N. Lawrence Gladnick, B.A. Study Completed on: May 8,2000 Testing Facility: DuPont Pharmaceuticals Company Safety Assessment Section Stine-Haskell Research Center P.O. Box 30, Elkton Road Newark, Delaware 19714 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: Service Code Number DuPont Pharmaceuticals Company Study No.: Page 1 o f 25 Company Sanitized. Does not contain TSCA CBI H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation o f data obtained from this study. Reviewed by Study Monitor: i <x> ----- ------------------------ Maria Donner, Ph.D. Senior Research Scientist Genetic Toxicology DuPont Haskell Laboratory for Toxicology and Industrial Medicine OP K _ uo Date Approved By: ^ t - ------------- j ior Ronald D. Snyder, Ph.D / Director o f Genetic Toxicology DuPont Pharmaceuticals Company Date Issued by Study Director: Staff Scientist DuPont Pharmaceuticals Company of nuty 'g g Date -2Company Sanitized. Does not contain T3GA CBi H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli TABLE OF CONTENTS CERTIFICATION............................................................................................. LIST OF TABLES............................................................................................ STUDY INFORMATION................................................................................. STUDY PERSONNEL..................................................................................... SUMMARY...................................................................................... ................ INTRODUCTION............................................................................................. MATERIALS AND METHODS..................................................................... A. Study Protocol....................................................................................... B. Test Materials........................................................................................ 1. Test Substance...... ......................................................................... 2. Negative and Positive Controls..................................................... 3. Tester Strain Source, Characterization, Storage, and Culture..... 4. Metabolic Activation System...................... ................................. C. Test Substance, Concentration Selection, Stability and Verification D. Test Methods: Bacterial Mutagenicity A ssay.... .............................. E. Statistical Analysis............................................................................... F. Acceptability Criteria...... .................................................................... 1. Tester Strain Integrity.................................................................... 2. Tester Strain Culture Titer............................................................. 3. Positive Control Values................................................................. 4. Revertant Toxicity.......................................................................... 5. Rejection o f Plates, Concentration Levels, or Assays................. G. Classification Guidelines..................................................................... RESULTS AND DISCUSSION...................................................................... CONCLUSIONS............................................................................................... RECORDS AND SAMPLE STORAGE......................................................... REFERENCES.................................................................................................. TABLES........................................................................................................... APPENDIX - HISTORICAL CONTROL DATA........................................ DuPont-3906 Page ..2 ...4 ...5 ...6 ...7 ...8 ...8 ...8 ...9 ...9 ...9 ...9 .10 .11 .11 ,,12 ,,12 ,,12 ,,12 ,,13 ,,13 ,,13 ,,13 ,,14 ,,14 .. 14 ,,15 ,,16 ,,24 -3 - Company Sanitized. Does not contain TSCA Cb> H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli LIST OF TABLES TABLE 1 STRAIN PHENOTYPE CONFIRMATION........ .................... TA BLE2 ' Mutagenic Activity In Salmonella typhimurium TA97a............ TABLE 3 Mutagenic Activity In Salmonella typhimurium TA98.............. TABLE 4 Mutagenic Activity In Salmonella typhimurium TA100........... TABLE 5 Mutagenic Activity In Salmonella typhimurium TA1535......... TABLE 6 Mutagenic Activity In Escherichia Coli WP2 uvrA (pKMIOl) DuPont-3906 Page ....18 ....19 ....20 ....21 ....22 ....23 -4 - Company Sanitized. Does no! contain TSCA Cl H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli STUDY INFORMATION DuPont-3906 Haskell Number: 24256 Submitter's Notebook Number(s):l Purity: 3 Stability: The test substance appeared to be stable under the conditions o f the study; no evidence o f instability was observed. Sponsor: E. I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Comnleted: 10-13-99/ (see report cover page) In-Life Tnitiated/Completed: 10-14-99 / 10-20-99 -5 Company Sanitized. Does not contain TSC CBr H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 STUDY PERSONNEL The following individuals participated in the conduct o f this study: DuPont Pharmaceuticals Company Study Director: N. Lawrence Gladnick, B.A. Technician: N. Lawrence Gladnick, B.A. Director of Genetic Toxicology: Ronald D. Snyder, Ph.D. E. I. DuPont de Nemours and Company Management: Carolyn S. VanPelt, D.V.M., Ph.D. Study Monitor: E. Maria Dormer, Ph.D. Report Preparation: Brenda Tiffin -6Company Sanitized. D oes not contain TSCA CEr H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 SUMMARY H-24256 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 o f an exogenous metabolic activation system (Aroclor -induced rat liver S9). A single trial assay was performed using the plate incorporation method to evaluate the mutagenic potential o f the test substance. All tester strains exhibited appropriate phenotypic characteristics. The mean number o f revertants seen in the negative controls for each strain was within the prescribed acceptable range. Test substance concentrations o f 5,10, 50,100, 500,1000,2500, and 5000 /ig/plate were evaluated in comparison to the negative (solvent) control. The solvent diluent and negative control used in this study was sterile water. Test substance-related toxicity, as evidenced by the reduction of the microcolony background lawns and/or as a concentration-related reduction in the mean number o f 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 o f revertants seen in the negative controls for each strain was within the acceptable historical negative control ranges. Under the conditions o f this study, no evidence of mutagenic activity was detected in any of the Salmonella strains or the Escherichia coli strain. Based on the findings, H-24256 was concluded to be negative for the induction of mutagenicity in the bacterial reverse mutation test. -7 Cmpany Sanitized. Does not contain TSCA CBI H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 INTRODUCTION This study evaluated the mutagenic potential o f the test substance, H-24256, 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 amino acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion o f 1 or a few DNA base pairs. The Salmonella tester strains are unable to synthesize histidine because o f 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/2^ By comparing the number o f chemically induced revertants to the number of spontaneous revertants, the mutagenicity o f the test substance can be assessed. MATERIALS AND METHODS A. Study Protocol The nrotocol consisted o f the stand-alone Protocol and the Haskell General Testing Procedure t f l f l H H B j B a c t e n a l Reverse Mutation Test for Solids, Liquids, and Gases", effective 1 0 6 A 0 /9 8 )^ h e study was designed to comply with: U.S. EPA, Office o f Prevention, Pesticides and Toxic Substances (OPPTS) Guidelines (Subpart H, 40 CFRPart 870.5100 [1998]); - Guidelines of the Organisation for Economic Cooperation and Development (OECD Guidelines for Testing o f 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 o f the study. -8Company Sanitized. Does not contain TSCA CBi H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 B. Test Materials Test Substance The test substance, H-24256, is was stored at room temperature. Additionalinionnation regardingthe test substance canoe found on the study information page o f this report. The test substance was assumed to be stable during the study and no evidence o f 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 o f the study. Positive controls included the following: Positive Controls 2-Nitrofluorene (2NF) iV-Ethyl-iV-nitro-iV-nitroguanidine (ENNG) Sodium azide (NAAZ) ICR 191 Acridine mutagen (ICR 191) 9,10-Dimethyl-1,2-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 The manufacturer lot number, and purity o f the solvent referenced in the study records. Neither the amount and nature o f the contaminants nor the use o f solvent is expected to affect the integrity or validity o f the study. Deionized water was the solvent for sodium azide (NAAZ). The solvent for 2AA, 2NF, ICR 191, DMBA, and ENNG was dimethyl sulfoxide (DMSO). 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 o f 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, -9 - Company Sanitized. Does not contain TSCA CBI H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli Torrey Research Station, Scotland, UK. The characteristics o f S. typhimurium tester strains TA97a, TA98, TA100, TA102, TA1535 and E. coli WP2 uvrA (pKMIOl) are as follows: Strain Gene Locus Excision LPS R-factor pAQl Repair (pKMIOl) Plasmid S. typhimurium TA97a hisD66l09 AuvrB rfa Present Absent S. typhimurium TA98 ' hisD3052 AuvrB rfa Present Absent S. typhimurium TA100 hisG46 AuvrB rfa Present Absent S. typhimurium TA102* S. typhimurium TA1535 hisG428 (+) rfa Present Present hisG46 AuvrB rfa Absent Absent E. coli WP2 uvrA (pKMIOl) trpE AuvrA NA Present Absent Note: uvrA and uvrB are defective DNA repair genes. LPS = lipopolysaccharide RFA = deep rough mutation NA = Not applicable * Concurrent control strain used to distinguish differential sensitivity/resistance to ultraviolet light. * Also hisOMAl. A 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/3^ 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 o f the lipopolysaccharide (LPS) cell wall so that permeability to large molecules is increased/4^ The presence o f the pKMIOl or R-factor plasmid, conferring ampicillin resistance, also enhances an error-prone DNA repair system that is endogenous to these bacteria.(5) The pA Q l 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 approximately -70C in ~8% (v/v) DMSO in Oxoid Nutrient Broth No. 2. The E. cot WP2 uvrA (pKMIOl) strain was stored at approximately -70C in ~30% glycerol in Oxoid Nutrient Broth No. 2. Prior to the mutagenicity assays, overnight cultures were prepared by inoculating 20 mL of Oxoid Nutrient Broth No. 2 with 0.1 mL o f a bacterial stock and incubating at approximately 37C with shaking, appropriate 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 assay was performed in the presence and absence o f an exogenous metabolic activation system similar to that described by Marn and A m es/ ^ The exogenous metabolic activation system was a cofactor-supplemented post-mitochondnal fraction, (i.e., 9000 x g, homogenate of 1 g wet liver weight in 3 mL o f an approximately 0.15 M KC1 solution) prepared from the livers of young male Sprague Dawley rats treated with the enzyme-inducing agent -10Company Sanitized. Does not contain TSCA CB! H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 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 o f the exogenous metabolic activation system, an aliquot was plated on nutrient agar capable of supporting the growth o f 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 MgCb, 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 /xg/plate. Solubility information was confirmed prior to study start. The stock concentration was calculated and adjusted for test substance displacement. Solutions o f the test substance were prepared immediately prior to treatment and were presumed to be stable under the conditions o f 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 o f the study. Solutions o f the test substance were assessed for sterility by plating a small amount o f the highest test substance concentration onto the surface o f agar plates capable o f supporting bacterial growth. D. Test Methods: Bacterial Mutagenicity Assay This study consisted o f a single trial that assessed the mutagenicity o f the 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 o f negative or positive control or test substance solution, 0.5 mL o f metabolic activation system, and 0.1 mL o f an overnight culture containing approximately 1 x 10s bacteria to approximately 2 mL o f 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 o f 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 o f sterile buffer was used as a replacement for the volume o f 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 approximately 4C ( 3C) prior to evaluation and counting o f revertant colonies. Bacterial background lawns were evaluated for evidence o f test substance toxicity and precipitation. Evidence of toxicity observed in the microcolony background lawns, was scored -11 Company Sanitized. Does not contain TSCA CSi H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 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 o f revertants and the standard deviation at each concentration in the presence o f 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 o f 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 o f the rfa mutation, all S. typhimurium tester strain cultures were required to exhibit sensitivity to crystal violet. To demonstrate the presence o f 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 o f ultraviolet light-induced DNA damage. E. coli tester strains were required to exhibit Ltryptophan dependent growth. To demonstrate the presence o f 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 o f the pKMIOl or R-factor plasmid. All tester strain cultures were required to exhibit a characteristic number o f spontaneous revertants per plate in the absence of the test substance. The acceptable mean revertants per plate o f the negative controls in the presence or absence o f the exogenous metabolic activation system were derived from the means o f 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 control data collected at the testing facility are presented in the appendix at the end o f this report. 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. -12Company Sanitized. Does not contain TSCA CB1 H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 3. Positive Control Values Mean positive control values must have exhibited at least a three-fold increase over the respective mean o f the concurrent negative control value for each tester strain and condition. 4. Revertant Toxicity A minimum o f 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 o f the concurrent negative control and was not equal to 0. 5. Rejection o f 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, met acceptability criteria, and are included in this report. G. Classification Guidelines A test substance was classified as POSITIVE (i.e., mutagenic) if the mean number o f revertants in any strain (except Salmonella strain TA1535) at any test substance concentration was at least two times greater than the mean o f the concurrent strain specific concurrent negative control, and there was a concentration-related increase in the mean number o f revertants per plate in that same strain. The mean number of revertants in Salmonella strain TA1535 must be at least three times greater than the mean number o f revertants o f its concurrent negative control. A test substance was classified as NEGATIVE (i.e., not mutagenic) if in all strains, except for TA1535, there were no test substance concentrations with a mean number o f revertants that was at least two times greater than the mean number o f revertants o f the concurrent negative control, and there was no concentration-related increase in the mean number o f revertants per plate in that same strain. For S. typhimurium 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 o f the concurrent negative control, and no concentration-related increase in the mean number of revertants per plate. - 13- Company Sanitized. Does not contain TSCA CBI H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 Results not meeting criteria for positive or negative classification were evaluated using scientific judgment and experience and may have been reported as EQUIVOCAL. RESULTS AND DISCUSSION H-24256 was evaluated in the bacterial reverse mutation test using Salmonella typhimurium strains TA97a, TA98, TA100, TA1535, and Escherichia coli strain WP2 uvrA (pKMIOl) in the presence and absence o f an exogenous metabolic activation system (Aroclor-induced rat liver S9). The test was performed using the plate incorporation method in order to evaluate.the mutagenic potential o f the test substance. Sterile water was chosen as the test substance solvent, diluent, and negative control. In this study, concentrations o f 5, 10, 50,100, 500,1000,2500, and 5000 /g/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 o f 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 wvryl(pKMIOl), there were no test substance concentrations with a mean number o f 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 o f 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 o f this study, no evidence o f mutagenic activity was detected. Based on the findings, H-24256 was concluded to be negative for the induction o f mutagenicity in the bacterial reverse mutation test in Salmonella typhimurium and Escherichia coli. 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- Cpmpany Sanitized. Does not contain TSCA CB) H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 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 IL L , and W. J. Muriel (1976). Mutagen testing using TRP+ reversion in Escherichia coli. M utat Res. 38, 3-32. 3. 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. 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 o f 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 o f carcinogens as mutagens: bacterial tester strains with R factor plasmids. Proc. Natl. Acad. Sci. USA 72, 979-983. -15Company Sanitized. Does not contain TSC CW H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 TABLES -16- Company Sanitized. Does not contain TSCA CBi H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 ABBREVIATIONS FOR TABLES Evidence for test substance toxicity to the bacteria was documented by recording the appearance o f the plates and background lawn using the following key: TO Normal, background microcolony lawn appeared normal. T 1 Slightly reduced, background microcolony lawn was noticeably thinner. T2 M oderately reduced, background lawn was markedly thinner resulting in an increase in the size o f 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 o f 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 o f any microcolony lawn over a majority o f the area o f the plate(s). Formation o f a precipitate by the test material was documented using the following key: PO No precipitate, no precipitate observed. P 1 Microscopic precipitate, precipitate present that did not interfere with background lawn evaluation or automated colony counting. P2 N on-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 o f any noteworthy observation R Plate rejected Positive controls were abbreviated as follows: ICR 191 DMBA 2NF 2AA NAAZ ENNG Acridine mutagen 9,10-Dimethyl-1,2-benzanthracene 2-Nitrofluorene 2-Aminoanthracene Sodium azide V-Ethyl-V-nitro-V-nitroguanidine - 17- Company Sanitized. Does not contain TSCA CBI H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli TABLE 1 STRAIN PHENOTYPE CONFIRMATION DuPont-3906 Strain L-Histidine/L-Tryptophan Dependent Growth3___________ Additional Growth Characteristics In Absence In Presence Crystal UV Ampicillin0 Tetracyclined Violetb Light0 TA97a TA98 TA100 TA1535 . + SS R + SS R + Ss R + ss S S S s S WP2 uvrA - + Rs R S (pKMIOl) TA102e N/Af N/A N/A R N/A N/A "Tested for histidine (S. typhimurium)ltr:yptophm (E. coli) requirement by the ability to grow in the absence and presence o f histidine/tryptophan; + = Growth; - = No growth. bTested 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 o f pKMIOl plasmid by demonstrating resistance to ampicillin and sensitivity to tetracycline; R = Resistant; S = Sensitive. T A 102 used as a control for UV Light sensitivity only. fN/A = Not applicable - 18Company Sanitized. Does not contain TSCA CBI H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 TABLE 2 Mutagenic Activity in Salmonella typhimurium TA97a Concentration Revertants Og/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations A. Without Metabolic Activation 0 5 10 50 100 500 1000 2500 5000 144 129 148 165 138 130 127 132 137 152 140 139 143 155 140 153 134 140 157 134 145 141 144 155 151 142 157 ICR 191 2 jtig/plate. 1384 1659 1962 151 (7) 134 (6) 144 (5) 150 (13) 146 (9) 142 (13) 144 (14) 136 (5) 145 (11) 1668 (289) B. With Metabolic Activation (Aroclor--induced rat liver S9) 0 5 10 50 100 500 1000 2500 5000 157 148 139 153 149 146 146 146 154 156 163 152 161 157 153 151 167 173 181 139 149 172 157 179 139 160 152 159 (4) 154 (7) 150 (9) 157 (9) 168 (17) 145 (5) 158 (3) 155 (21) 155 (4) DMBA 20 /xg/plate 2197 1659 2196 2017 (310) 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 - 19- Company Sanitized. Does not contain TSCA CBi H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 TABLE 3 Mutagenic Activity in Salmonella typhimurium TA98 Concentration Revertants Og/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations A. Without Metabolic Activation 0 5 10 50 100 500 1000 2500 5000 24 23 25 25 24 15 30 27 25 33 29 25 31 28 26 21 24 15 27 28 26 30 29 20 16 25 17 2NF 25 /ig/plate 1262 1450 1544 28 (5) 27 (3) 25 (1) 29 (3) 27 (3) 20 (6) 22 (7) 25 (2) 19 (5) 1419 (144) B. With Metabolic Activation (Aroclor-induced rat liver S9) 0 5 10 50 100 500 1000 2500 5000 27 25 29 27 26 29 31 30 34 30 25 24 25 31 32 26 35 39 29 22 29 27 32 27 29 37 25 27 (3) 25 (1) 31 (2) 29 (5) 31 (7) 27 (4) 30 (3) 29 (2) 32 (6) 2AA 2 /xg/plate 849 866 747 821 (64) 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 -20Company Sanitized. Does not contain TSCA CBl H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 TABLE 4 Mutagenic Activity in Salmonella typhimurium TA100 Concentration Revertants Og/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations A. Without Metabolic Activation 0 5 10 50 100 500 1000 2500 5000 146 146 157 136 155 157 131 127 169 158 165 157 140 156 153 154 145 159 NAAZ 2 /xg/plate 877 790 164 132 163 150 151 137 130 135 150 790 156 (9) 148 (17) 159 (3) 142 (7) 154 (3) 149 (11) 138 (14) 136 (9) 159 (10) 819 (50) 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) 0 171 166 154 5 167 167 153 10 176 162 162 50 163 153 169 100 169 160 178 500 172 163 182 1000 167 172 192 2500 157 184 157 5000 156 131 172 164 (9) 162 (8) 167 (8) 162 (8) 169 (9) 172 (10) 177 (13) 166 (16) 153 (21) 2AA 2 /tg/plate 745 900 901 849 (90) T0,P0 T0,PO T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 N -21 Company Sanitized. Does not contain TSCA CI H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 TABLE 5 Mutagenic Activity In Salmonella typhimurium TA1535 Concentration Revertants (jU.g/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations A. Without Metabolic Activation 0 5 10 50 100 500 1000 2500 5000 21 22 19 22 22 16 23 20 24 21 21 26 24 21 20 16 17 17 21 25 12 28 14 12 24 24 17 21 (0) 24 (2) 20 (1) 18 (3) 20 (3) 18 (7) 22 (7) 19 (6) 22 (4) NAAZ 2 /g/plate 699 760 783 747 (43) B. With Metabolic Activation (Aroclor-induced rat liver S9) 0 5 10 50 100 500 1000 2500 5000 14 13 21 15 17 13 19 16 18 18 12 14 15 12 13 26 14 14 10 17 20 14 13 15 13 11 14 15 (3) 14 (1) 15 (5) 18 (7) 14 (4) 17 (4) 15 (3) 15 (2) 14 (4) 2 /xg/plate 229 218 235 227 (9) T0,P0 T0,P0 T0,P0 T0,P0 TO,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 -22Company Sanitized. Does not contain TSCA CB! H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 TABLE 6 Mutagenic Activity In Escherichia coli WP2 uvrA (pKMIOl) Concentration Revertants (/xg/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations A. Without Metabolic Activation 0 5 10 50 100 500 1000 2500 5000 183 182 196 199 202 206 180 144 148 187 210 214 189 193 195 202 172 187 ENNG 2 jttg/plate 1456 1350 190 211 196 195 208 208 174 173 169 1402 187 (4) 201 (16) 202 (10) 194 (5) 201 (8) 203 (7) 185 (15) 163 (16) 168 (20) 1403 (53) B. With Metabolic Activation (Aroclor-induced rat liver S9) 0 5 10 50 100 500 1000 2500 5000 138 164 153 157 156 188 169 171 166 140 174 178 174 163 167 176 155 166 158 169 168 168 202 163 174 173 192 151 (20) 172 (7) 161 (7) 163 (12) 160 (5) 175 (11) 180 (19) 169 (6) 177 (13) 2AA 25 (Ug/plate 1513 1383 1371 1422 (79) TO,P0 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 - 23Company Sanitized. Does not contain TSCA Ci H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 APPENDIX HISTORICAL CONTROL DATA -24Company Sanitized. Does not contain TSCA Cl. H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli Historical Control Data2 DuPont-3906 Tester Strain Exogenous Metabolic Range Control [Positive Controlb] Activation System Mean (S.D.) Minimum - Maximum S. typhimurium TA100 Negative Negative Positive [NAAZ-2] Positive [2AA-1] S. typhimurium TA 1535 Negative Negative Positive [NAAZ-2] Positive [2AA-2] 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] E. coli WP2 uvrA (pKMlOl) 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 122 (29) 128 (29) 872 (274) 1187 (476) 15 (6) 14 (6) 618 (199) 362 (136) 103 (18) 124 (26) 1822 (662) 1534 (559) 960 (348) 22 (7) 26 (7) 1403 (372) 1552 (598) 148 (29) 167 (29) 1656 (449) 1627 (317) 1577 (450) 1682 (372) 54 - 218 65 - 253 339 - 2604 94 - 2682 4 - 46 4 - 39 127 - 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 o f study modification (e.g., plate incorporation, pre-incubation/gas, waste water). . b 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 (A-ethyl-N-nitro-A-nitrosoguanidine). The number following abbreviation is the microgram (/xg) amount per plate or vial used for the positive control. - 25Company Sanitized. Does not contain TSCA CBS