Document 3e3qwmBjadjobnOOvQKY9DR93

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 OPPTS870.5'iOO (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 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 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: HUB } SERVICE CODE NUMBER;|Jd^ D U P O NT PHARMACEUTICALS COMPANY STUDY NO.: Hf_ l_H__ B H H I ^( Page 1 of 25 Company Sanitized. Does not contain T8CA 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 of data obtained from this study. Reviewed by Study Monitor: . k c ?^, Mar i a D ^D"^ onner, PhJ ) . Senior Research Scientist Genetic Toxicology DuPont Haskel] Laboratory for Toxicology and Industrial Medicine _oy_K_o^_2_o u a Date " Approved By: ^--^^ ^gfeag ^. -^ur Ronald D. Snyder, Ph.D/ Director of Genetic Toxicology DuPont Pharmaceuticals Company ^^y 2-0^ Date Issued by Study Director: p^ ^7. /j^t^^---- N. Lawdnce Gladnick, B.A. Staff Scientist DuPont Pharmaceuticals Company <9fWlf/ .2.^0 Date -2- Company Sanitized. Does not eorttain TQCACB! H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 TABLE OF CONTENTS Page CERTIFICATION..........................................................................................................................^ LIST OF TABLES..........................................................................................................................4 STUDY INFORMATION............................................................................................................... 5 STUDY PERSONNEL...................................................................................................................6 SUMMARY....................................................................................................................................? 8 INTRODUCTION........................................................................................................................... MATERIALS AND METHODS.................................................................................................... 8 A. Study Protocol..................................................................................................................... 8 B. TestMatedals......................................................................................................................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 Assay.....;............................................................ 11 E. Statistical Analysis............................................................................................................ 12 F. Acceptability Criteria........................................................................................................ 12 1. Tester Strain Integrity.................................................................................................. 12 2. Tester Strain Culture Titer........................................................................................... 12 3. Positive Control Values............................................................................................... 13 4. Revertant Toxicity....................................................................................................... 13 5. Rejection of Plates, Concentration Levels, or Assays................................................. 13 G. Classification Guidelines................................................................................................... 13 RESULTS AND DISCUSSION................................................................................................. 14 CONCLUSIONS........................................................................................................................... 14 RECORDS AND SAMPLE STORAGE....................................................................................... 14 15 REFERENCES.............................................................................................................................. 16 TABLES........................................................................................................................................ APPENDDC - HISTORICAL CONTROL DATA....................................................................... 24 -3- Company Sanitized. Does not contain TSCA Cb< H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 LIST OF TABLES Page TABLE 1: STRAIN PHENOTYPE CONFIRMATION ............................................................. 18 TABLE 2:' Mutagenic Activity In Salmonella typhimurium TA97a............................................ 19 TABLE 3: Mutagenic Activity In Salmonella typhimurium TAPS.............................................. 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 wrA (pKMIOl) ................................23 -4- Company Sanitized. Offes nai contain TSCA C' H-24256: Bacterial Reverse Mutation Test m Salmonella typhimwium and Escherichia coli STUDY INFORMATION 9th Collective Nomenclature Synonyms/Codes: " H-24256 Haskell Number: 24256 CAS Registry Number: Submitter's Notebook Number(s):jj DuPont-3906 Stability: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed. Sponsor: E. I. du Font de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Completed: 10-13-99 / (see report cover page) In-Life Initiated/Completed: 10-14-99/10-20-99 Company Sanitized. Does not contain TSCA CB' H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 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. 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 -6- Company Sanitized. Does itot contain TSCA cr H-24256: Bacterial Reverse Mutation Test in Salmonella typhimuriwn and Escherichia coli DuPont-3906 SUMMARY H-24256 was evaluated in the bacterial reverse mutation assay using Salmonella typhimuriwn 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). A single trial assay was performed using the plate incorporation method to evaluate the mutagenic potential of the test substance. All tester strains exhibited appropriate phenotypic characteristics. The mean number ofrevertants seen in the negative controls for each strain was within the prescribed acceptable range. Test substance concentrations of 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 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 historical negative control ranges. Under the conditions of 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 ofmutagenicity in the bacterial reverse mutation test. -7- Company Sanitized. Does not contain TSCA CB1 | H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 INTRODUCTION This study evaluated the mutagenic potential of the test substance, H-24256, in the bacterial reverse mutation test using Salmonella typhimurium strains TA97a, TAPS, TA100, and TA1535 and in Escherichia coli strain WP2 wrA (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.0'2) 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 TAPS 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 L imBBfl"^^1^^ TheDmtocolcpnsisted of the stand-alone Protocol and the Haskell General Testing Procedure R^e^s Mutation Test for Solids, Liquids, and Gases", effective 06/10/9S). The study was designed to comply with: U.S. EPA, Office of Prevention, Pesticides and Toxic Substances (OPPTS) Guidelines (Subpart H, 40 CFRPart S70.5100 [199S]); " 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-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 B. Test Materials 1. Test Substance _________ ____________ The test substance, H-24256, is a^ was stored at room temperature. Additional information regarding'the test substance canT>e 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) AT-Ethyl-JV-nitro-^-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 of the solvent referenced in the study records. Neither the amount and nature of the contaminants nor the use of solvent is expected to affect the integrity or validity of the study. Deionized water was the solvent for sodium azide (NAAZ). The solvent for 2AA, 2NF, ICP. 191, DMBA, and ENNG v/as 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 of instability was observed during the study. 3. Tester Strain Source, Characterization, Storage, and Culture 5'. 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, Company Saniiized. Does not contain TSCA CBi H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 Torrey Research Station, Scotland, UK. The characteristics o!S. typhimurium tester strains TA97a, TA98, TA100, TA102, TA1535 and E. coli WP2 uvrA (pKMIOl) are as follows: Strain 5'. typhimurium TA97a 5'. typhimurium TA98 S. typhimurium TA100 S. typhimurium TA102* 5'. typhimurium TA1535 E. coli WP2 uvrA (pKMIOl) (3ene Locus hisDCQW'9 hisD3052 hisG46 hisG428 hisG46 trpE Excision Repair AuvrB AuvrB ^uvrB (+) Ayvrff buvrA LPS rfa rfa rfa rfa rfa NA R-factor (pKMIOl) Present Present Present Present Absent Present Note: LPS RFA NA * li) 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 his01242. symbol for "deletion" proficient in excision repair pAQI Plasmid Absent Absent Absent Present Absent Absent 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 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.^ 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. 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 assays, 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, 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 of an exogenous metabolic activation system similar to that described by Maron and Ames.^ The exogenous metabolic activation system was a cofactor-supplemented post-mitochondrial fraction, (i.e., 9000 x g; homogenate of 1 g wet liver weight in 3 mL of an approximately 0.15 M KC1 solution) prepared from the livers of young male Sprague Dawley rats treated with the enzyme-inducing agent -10Company Sanitized. Does snot contain TSCA C3l 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 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 co factor-supplement concentrations in the exogenous metabolic activation system were 8 mM MgC^, 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 m 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 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 Assay This study consisted of a single trial that assessed the mutagenicity of 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 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 approximately 4C ( 3C) prior to evaluation and counting ofrevertant colonies. Bacterial background lawns were evaluated for evidence of test substance toxicity and precipitation. Evidence of toxicity observed in the microcolony background lawns, was scored -11- Company Sanitized. Does not contain TSCA CBf 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 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 S. typhimurium tester strain cultures were required to exhibit L-histidine dependent growth. To demonstrate the presence of the rfa mutation, all 5'. 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 Ltryptophan 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 control data collected at the testing facility are presented in the appendix at the end of 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 lO^ells/mL. -12- Company Sanitized. Does nol 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 of the concurrent negative control value for each tester strain and condition. 4. Revertant Toxicity 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, 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 of revertants in any strain (except Salmonella strain TA1535) at any test substance concentration was at least two times greater than the mean of the concurrent strain specific concurrent negative control, and there was a concentration-related increase in the mean number of 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 of revertants of 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 of revertants that was at least two times greater than the mean number of revertants of the concurrent negative control, and there was no concentration-related increase in the mean number of 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 of 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 of 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 of the test substance. Sterile water was chosen as the test substance solvent, diluent, and negative control. m this study, concentrations of 5,10, 50,100, 500,1000,2500, and 5000 ^ig/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, or E. coli WP2 iw/i(pKM101), 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-24256 was concluded to be negative for the induction ofmutagenicity 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- fi.ompany 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, MH.L. and W. J. Muriel (1976). Mutagen testing using TRP'1' 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. -15- 'Company Sanitized. Does not contain TSCA CW H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 TABLES -16Company Sanitized. Does not contain TSCA CBl 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 of 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 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. P 1 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-1,2-benzanthracene 2-Nitrofluorene 2-Aminoanthracene Sodium azide A^-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 Dependerit Growtha In Absence In Presence 1^ddition)alCirrowth Characteristics Crysstal UV Ampicillin'1 Tetracycline11 Viollet1' Lighit0 TA97a TAPS 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) TA1026 N/A1' N/A N/A R N/A N/A "Tested for histidine (S. typhimurium)/ts'yptoph!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. ''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. ^/A = Not applicable -18- Company 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 Qxg/plate) Revertant s Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations A. Without Metabolic Activation 0 144 152 5 129 140 10 148 139 50 165 143 100 138 155 500 130 140 1000 127 153 2500 132 134 5000 137 140 ICR 191 2 jug/plate . 1384 1659 157 134 145 141 144 155 151 142 157 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 (Aroclor3^-induced rat liver S9) 0 157 5 148 10 139 50 153 100 149 500 146 1000 146 2500 146 5000 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) 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 -19- Company Sanitized. Does not contain TSCA CE) H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 TABLES Mutagenic Activity in Salmonella typhimurium TA98 Concentration Otg/plate) Elevertant s Plate 1 Plate 2 Plate 3 Meari(S.D.) Observations Without Metabc>lic ActivatiIon 0 24 5 23 10 25 50 25 100 24 500 15 1000 30 2500 27 5000 25 33 27 29 28 25 26 31 30 28 29 26 20 21 16 24 25 15 17 2NFF 25 ^g/plate 1262 1450 1544 28 (5) 27 (3) 25 (1) 29 (3) 27 (3) 20 (6) 22 (7) 25 (2) 19 (5) 1419 (144) With Metabolic Activation (Aroclor-induced rat liver S9) 0 27 5 25 10 29 50 27 100 26 500 29 1000 31 2500 30 5000 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^g/plate 849 866 747 821 (64) 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 CBil H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 TABLE 4 Mutagenic Activity in Salmonella typhimurium TA100 Concentration (jug/plate) Revertants Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations Without Metabolic Activation 0 146 5 146 10 157 50 136 100 155 500 157 1000 131 2500 127 5000 169 158 164 165 132 157 163 140 150 156 151 153 137 154 130 145 135 159 150 AAZ 2 ^g/plate 877 790 790 156 (9) 148 (17) 159 (3) 142 (7) 154 (3) 149 (11) 138 (14) 136 (9) 159 (10) 819 (50) With Metabolic Activation (Aroclor-induced rat liver S9) 0 171 5 167 10 176 50 163 100 169 500 172 1000 167 2500 157 5000 156 166 154 167 153 162 162 153 169 160 178 163 182 172 192 184 157 131 172 164 (9) 162 (8) 167 (8) 162 (8) 169 (9) 172 (10) 177 (13) 166 (16) 153 (21) 2AA 2 /ig/plate 745 900 901 849 (90) 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 Ctfl H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 TABLES Mutagenic Activity In Salmonella typhimurium TA1535 Concentration dug/plate) Revertants Plate 1 Plate 2 Plate 3 Mean(S.D.) Observations A. Without Metsibolic Ac;tivation 0 21 5 22 10 19 50 22 100 22 500 16 1000 23 2500 20 5000 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 /zg/plate 699 760 783 747 (43) B. With Metabolic Activation (Aroclor-induced rat liver S9) 0 14 5 13 10 21 50 15 100 17 500 13 1000 19 2500 16 5000 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) 2AA 2 jug/plate 229 218 235 227 (9) 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-24256: Bacterial Reverse Mutation Test in Salmonella typhimwium and Escherichia coli DuPont-3906 TABLE 6 Mutagenic Activity In Escherichia coli WP2 uw-4 (pKMIOl) Concentration QAg/plate) Revertants Plate 1 Plate 2 Plate 3 Mean(S.D.) Observations A. Without Metabolic Activation 0 183 187 5 182 210 10 196 214 50 199 189 100 202 193 500 206 195 1000 180 202 2500 144 172 5000 148 187 ENNG 2/xg/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) 0 138 5 164 10 153 50 157 100 156 500 188 1000 169 2500 171 5000 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^g/plate 1513 1383 1371 1422 (79) 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 -23- Company Sanitized. Does not contain TSCA CB( H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3906 APPENDIX HISTORICAL CONTROL DATA -24- Company Sanitized. Does not contain TSCA CE H-24256: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli Historical Control Data" DuPont-3906 Tester Strain Exogenous Metabolic Control ^Positive Control1'] Activation System Mean (S.D.) Range 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 (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 122 128 872 1187 (29) (29) (274) (476) 15 (6) 14 (6) 618 (199) 362 (136) 103 124 1822 1534 960 (18) (26) (662) (559) (348) 22 26 1403 1552 (7) (7) (372) (598) 148 167 1656 1627 1577 1682 (29) (29) (449) (317) (450) (372) 54 65 339 94 - 218 253 2604 2682 4 4 127 - 44 - 46 39 1270 1323 59 67 476 563 308 - 164 196 3359 2773 2281 7 11 - 567 250 - 47 53 2774 3114 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 (A^-ethyl-A^-mtro-A^-mtrosoguanidme). The number following abbreviation is the microgram (<xg)amount per plate or vial used for the positive control. -25- Company Sanitized. Does not contain TSCA CBS