Document k6VLqyMzak9gpVnD57y4VboV

TRADE SECRET AR226-3093 DuPont HL-1998-01316 Study Title H-23005: Bacterial Reverse Mutation Test L a b o r a t o r y Project ID: HL-1998-01316 Author: N. Lawrence Gladnick, B.A. Study Completed on: March 13,1998 Performing Laboratory: E, I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Work Request No. 0, CM! T * OB! s ,n l a .D 0 .Sr, Compaq Page 1 of 31 H-23005: Bacterial Reverse Mutation Test DuPont HL-1998-01316 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with TSCA (40 CFR 792) Good Laboratory Practice Standards and OECD Principles of Good Laboratory Practice (C(81)30(Final), Annex 2), with the following exception: 1) Expired Oxoid nutrient plates were used in trial 1 for a portion of the phenotype confirmations. The plates were used a few days beyond their expiration, and all supported growth of viable bacteria, as was necessary for the phenotypic confirmations. 2) Treatment solutions or suspensions were not analyzed for identity, composition, uniformity, or stability-of the test and control articles. The procedures used by trained personnel to prepare the treatment solutions intended to ensure: The accuracy of concentration because control articles were weighed on an analytical balance accurate to 3 decimal places and the test article and the vehicle in which the test and control substances were dissolved were accurately measured with graduated pipettes or flasks; Uniformity because all treatment solutions or suspensions were mixed prior to administration to the test system; and Stability because treatment solutions or suspensions were prepared just prior to administration to the test system. This deviation did not affect the validity or the integrity of the study. Submitter/Sponsor: E. I. du Pont de Nemours and Company Study Director: Toxicology Associate Date not contain TSCA CB1 Company Sanitized. Does H-23005: Bacterial Reverse Mutation Test DuPont HL-1998-01316 QUALITY ASSURANCE DOCUMENTATION Haskell Sample Number(s): 23005 Dates of Inspections: Conduct: January 21, 1998 Records, Reports: February 26-27, and March 2, 1998 Dates Findings Reported to: Study Director: March 2, 1998 Management: March 13, 1998 Reported by: 'AJ <k.P. Jeff Chapman Quality Assurance Auditor Date not contain TSCA CB1 Sanitized. Does C om paq H-23005: Bacterial Reverse Mutation Test DuPont HL-1998-01316 CERTIFICATION I, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. _ Reported Approved and Issued by Study Director: N. Law renciGladnick, B.A. Toxicology Associate M ob? Date Company Sanittead. Dons nol contain TSCA CB1 H-23005: Bacterial Reverse Mutation Test DuPont H L -1998-01316 TABLE OF CONTENTS GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT............................................... 2 QUALITY ASSURANCE DOCUMENTATION.................................................................................. 3 CERTIFICATION.................................................................................................................................. 4 STUDY INFORMATION.......................................................................................................................6 STUDY PERSONNEL..................................:....................................................................................... 7 SUMMARY............................................................................................................................................. 8 INTRODUCTION.................................................................................................................................. 9 MATERIALS AND METHODS............................................................................................................ 9 A. STUDY PROTOCOL..................................................................................................................9 B. TEST MATERIALS.................................................................................................................. 10 1. Test Substance............................................................................................................... 10 2. Negative and Positive Controls....................................................................................... 10 C. TESTER STRAIN CHARACTERIZATION, STORAGE, AND CULTURE.............................10 1. Tester Strain Characterization................. 10 2. Tester Strain Storage and Culture................................................................................... 11 D. METABOLIC ACTIVATION SYSTEM................................................................................... 12 E. CONCENTRATION SELECTION.................................. 12 F. STABILITY AND CONCENTRATION VERIFICATION........................................................12 G. BACTERIAL MUTAGENICITY ASSAYS.............................................................................. 13 H. STATISTICAL ANALYSIS..................................................................................................... 13 I. ACCEPTABILITY CRITERIA...................................................................................................13 1. Tester Strain Phenotypes and Spontaneous Reversion............................................... 13 2. Tester Strain Titer.......................................................................................................... 14 3. Positive Control Values..................................................................................................14 4. Toxicity..........................................................................................................................14 5. Rejection of Plates, Concentration Levels, or Assays..................................................... 14 J. CLASSIFICATION GUIDELINES.................................... 15 RESULTS AND DISCUSSION............................................................................................................ 16 CONCLUSION..................................................................................................................................... 17 RECORDS AND SAMPLE STORAGE...............................................................................................17 REFERENCES........................... 18 TABLES................................................................................................................................................ 19 ABBREVIATIONS FOR TABLES................................................................................................ 20 TABLE 1 - Mutagenic Activity in Salmonella typhimurium TA97a in Trial 1................................. 21 TABLE 2 - Mutagenic Activity in Salmonella typhimurium TA97a in Trial 2 ................................. 22 TABLE 3 - Mutagenic Activity in Salmonella typhimurium TA98 in Trial 1....................................23 TABLE 4 - Mutagenic Activity in Salmonella typhimurium TA98 in Trial 2................. 24 TABLE 5 - Mutagenic Activity in Salmonella typhimurium TA100 in Trial 1................................. 25 TABLE 6 - Mutagenic Activity in Salmonella typhimurium TA100 in Trial 2................................. 26 TABLE 7 - Mutagenic Activity in Salmonella typhimurium TA1535 in Trial 1............................... 27 TABLE 8 - Mutagenic Activity in Salmonella typhimurium TA1535 in Trial 2............................... 28 TABLE 9 - Mutagenic Activity in Escherichia coli WP2 uvrA (pKMIOl) in Trial 1 .......................29 TABLE 10 - Mutagenic Activity in Escherichia coli WP2 uvrA (pKMIOl) in Trial 2 .....................30 TABLE 11 - Historical Control Data............................................................................................... 31 Company Sanitized. Does not contain TSCACBI -5 - H-23005: Bacterial Reverse Mutation Test STUDY INFORMATION DuPont HL-1998-01316 Haskell Number: 23005 Composition: Known Impurities: Physical Characteristics: Tan liquid 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/Completed: January 12, 1998 / (see report cover page) In-Life Initiated/Completed: January 16, 1998 / February 6, 1998 Company S a n te e *. Does no, contain TSCfl C 8| -6- H-23005: Bacterial Reverse Mutation Test STUDY PERSONNEL Management: Matthew S. Bogdanffy, Ph.D. Scott E. Loveless, Ph.D. Study Director: N. Lawrence Gladnick, B.A. Primary Technician: Lisa M. Sulecki, A.S. Technical Publisher: Debra L. Dacey DuPont HL-1998-01316 Company Sanitized. Does not contain TSCA CBI -7 - H-23005: Bacterial Reverse Mutation Test DuPont H L-1998-01316 SUMMARY The active ingredient o f the test substance, H-23005, 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). The test was performed in two phases. The first phase consisted o f a preliminary toxicity assay that was used to establish test substance solubility in the selected solvent and the concentration range for the mutagenicity assay. The second phase, consisting o f the initial and independent repeat assays, evaluated the mutagenic potential o f the test substance. Concentrations o f 10, 50,100, 500,1000, 2500, and 5000 /xg/plate, adjusted for the active ingredient of the test substance, using the Salmonella strains and Escherichia coli strain, were evaluated in comparison to negative (solvent) controls. In a second independent repeat assay, these same concentrations, adjusted for the active ingredient o f the test substance, were tested in comparison to negative (solvent) controls. Under the conditions o f this study, no evidence o f mutagenic activity was detected in either of two independent trials. The test substance was negative. Company Sanitized. Does not contain TSCA CBi -8 - H-23005: Bacterial Reverse Mutation Test DuPont HL-1998-01316 INTRODUCTION This study evaluated the mutagenic potential o f the test substance, H-23005, in Salmonella typhimurium strains TA97a, TA98, TA100, and TA1535 and in Escherichia coli strain WP2 uvrA (pKMIOl). The bacterial reverse mutation test uses amino-acid requiring strains o f Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion o f one or a few DNA base p a ir s /'2,3) The Salmonella tester strains are unable to synthesize histidine because o f specific point mutations in genes coding for histidine biosynthesis. Additional mutation o f 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 (pKMTOl) is sensitive to mutagensliiat 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/ ,5) 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. STUDYPROTOCOL The study was designed to comply with: U.S. EPX", Office o f Prevention, Pesticides and Toxic Substances (OPPTS) Guidelines (Subpart H, 40 CFR Part 799.9510 [1997]); Guidelines o f the Organisation for Economic Cooperation and Development (OECD Guidelines for Testing o f Chemicals, No. 471/472 "Bacterial Reverse Mutation Test" (Draft Updated Guideline, February, 1997)); The study design met or exceeded the above guidelines with the following exceptions: S. typhimurium tester strain TA97a using ICR 191 as a positive control was used as a substitute for strain(s) TA97 or TA1537 (OECD). E. coli WP2 uvrA (pKMIOl) was the only E. coli tester strain used (OECD). These exceptions did not affect study validity for the following reasons: 1) Salmonella typhimurium tester strain TA97a has improved growth properties and has been shown to be more sensitive to frameshift mutagens; 2) In the absence o f clear evidence to suspect that the test substance was a DNA cross-linking agent, E. coli WP2 uvrA (pKMIOl) was used for an assessment o f test substance mutagenicity at AT base pairs. The strains used in this study comply with harmonized guidelines designed to minimize variations among testing procedures worldwide (OECD, OPPTS). Company Sanitized. Does not contain TSCA CBS -9 - H-23005: Bacterial Reverse Mutation Test DuPont HL-1998-01316 B. TEST MATERIALS 1. Test Substance The test substance is H-23005. Additional information regarding the test substance is found on the study information page o f this report. The test substance was assumed stable during this assay and no evidence o f instability was observed. 2. Negative and Positive Controls Based on information supplied by the sponsor and solubility testing, sterile water was chosen as the test substance solvent, diluent, and negative control. The negative control was assumed stable during this study, and no evidence o f instability was observed. Any impurities were not expected to have interfered with the study. Positive controls included the following: Positive Control Substances 2-Nitrofluorene (2NF) 2-Aminoanthracene (2AA) Sodium azide (NAAZ) ICR 191 Acridine mutagen (ICR 191) 9,10-Dimethyl-1,2-benzanthracene (DMBA) . AT-Ethyl-AT-nitro-iV-nitroguanidme (ENNG) Chemical Abstracts Service Registry Number [CAS # 607-57-8] [CAS #613-13-8] [CAS # 26628-22-8] [CAS # 17070-45-0] [CAS # 57-97-6] [CAS # 4245-77-6] Deionized water was the solvent for NAAZ and ICR 191. The solvent for 2AA, 2NF, DMBA, and ENNG was dimethyl sulfoxide (DMSO). The positive controls were assumed stable in this study and no evidence o f instability was observed. Any impurities were not expected to have interfered with the study. C. TESTER STRAIN CHARACTERIZATION, STORAGE, AND CULTURE 1. Tester Strain Characterization S. typhimurium tester strains were obtained from Dr. Bruce Ames, Berkeley, CA, USA. E. coli WP2 uvrA (pKMIOl) was obtained from the National Collection o f Industrial Bacteria, 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: Company Sanitized. Does not contain TSCA CBI H-23005: Bacterial Reverse Mutation Test DuPont HL-1998-01316 Strain Gene Locus Excision Repair LPS S. typhimurium TA97a AM56610 AuvrB rfa S. typhimurium TA98 hisD3Q52 AuvrB rfa S. typhimurium TA100 hisG 46 AuvrB rfa S. typhimurium TA102 S. typhimurium TA1535 hisG428 hisG46 (+) AuvrB rfa rfa E. coli WP2 uvrA (pKMIOl) trpE AuvrA NA LPS=lipopolysaccharide; NA=Not applicable; (+) proficient R-factor (pKMIOl) Plasmid Present Present Present Present Absent Present pAQl Plasmid Absent Absent Absent Present Absent Absent The deletion (A) in uvrB (a gene that codes for one protein involved in DNA excision repair) increases bacterial sensitivity to several mutagens/"' The uvrB trait is confirmed by demonstrating an increased bacterial sensitivity to ultraviolet light. Because this deletion also extends through a gene needed for biotin biosynthesis, the bacteria 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. 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.^ The pA Q l plasmid, conferring tetracycline resistance, is tested at aliquot date for TA102. Although some testing guidelines require S. typhimurium strain TA1537, Salmonella strains TA1537, TA97 or TA97a may be used interchangeably.(3) Salmonella strain TA97 has been demonstrated to be more sensitive to ffameshift mutagens and the reconstructed strain, TA97a, was used in place o f TA97 due to its improved growth properties.(8) Although some testing guidelines require E. coli WP2 uvrA, in the absence o f data indicating a difference in sensitivity between the plasmid and non-plasmid containing strains, E. coli WP2 uvrA (pKMIOl) was used to assess bacterial mutagenicity.(3) E. coli WP2 uvrA (pKMIOl) possess a deletion (A) in uvrA (involved in DNA excision repair), that is confirmed by demonstrating an increased sensitivity to ultraviolet light. 2. Tester Strain Storage and Culture All bacterial strains were stored frozen in approximately 8% (v/v) DMSO in Oxoid Nutrient Broth No. 2 at approximately -70C. Overnight cultures were prepared by inoculating 20 mL of Oxoid Nutrient Broth No. 2 with 0.1 mL o f a thawed bacterial suspension and incubating at ~37C with shaking. Overnight cultures were then stored on ice until used for mutagenesis assays. Bacterial strain phenotypes were confirmed concurrently with each trial. Results for reported trials demonstrated the appropriate responses. Company Sanitized, Does not contain TSCA CBI -11 - H-23005: Bacterial Reverse Mutation Test DuPont HL-1998-01316 D. METABOLIC ACTIVATION SYSTEM Because bacterial strains lack many o f the enzymes required to convert various 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 Maron and Ames (1983). The exogenous metabolic activation system was a cofactor-supplemented post-mitochondrial fraction, (i.e., 9000 x g; homogenate o f 1 g wet liver weight in 3 mL o f an approximately 0.15 M KC1 solution) prepared from the livers o f young male Sprague Dawley rats treated with the enzymeinducing agent Aroclor 1254 (500 mg/kg i.p.) for five 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 systems, the prepared S9 mixture was streaked on nutrient agar capable o f supporting the growth o f viable bacteria. The amount of Aroclor-induced rat liver S9 in the exogenous metabolic activation system was as follows: 4.0 mg Aroclor-induced rat liver S9 protein ~10 % [v/v] / mL exogenous metabolic activation system The component concentrations in the exogenous metabolic activation system were as follows: 8 mM MgCL 33 mM KC1 5 mM Glucose-6-phosphate (as a sodium salt) 4 mM NADP+ (as sodium salt) 100 mM Sodium phosphate buffer pH 7.4 E. CONCENTRATION SELECTION In accordance with testing guidelines, the highest concentration evaluated in this study was approximately 167 mg/mL or 5000 /g/plate o f the active ingredient o f the test substance. F. STABILITY AND CONCENTRATION VERIFICATION Solutions o f the test substance were prepared immediately prior to treatment and were presumed to be stable under the conditions of the study. Treatment and control solutions were not analyzed for concentration, uniformity, or stability. Top agar was not assayed for stability nor were the concentrations o f the test substance or control articles since this assessment was not considered necessary to achieve the objectives o f the study. Test substance sterility was confirmed by - streaking the stock concentration in triplicate onto the surface o f nutrient agar plates capable o f supporting the growth o f viable bacteria. Company Sanitfced. Does not contain TSCA CBI - 12- H-23005: Bacterial Reverse Mutation Test DuPont H L-1998-01316 G. BACTERIAL MUTAGENICITY ASSAYS This study consisted o f two independent trials. For each trial, three replicates were plated for each tester strain in the presence and absence o f the exogenous metabolic activation system (Aroclor-induced rat liver S9) 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 108bacteria to approximately 2 mL o f top agar [0.6-0.7% agar (w/v) and 0.5-0.6% NaCl (w/v)] 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 o f a 0.4% [w/v] glucose with Davis salts, purchased from MOLTOXTM). Treatments in the absence o f the metabolic activation system were as those in the presence o f 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 o f 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. When necessary, plates were refrigerated at approximately 4C prior to evaluation and counting o f revertant colonies. Bacterial background lawns were evaluated for evidence o f test substance toxicity and precipitation. Evidence o f toxicity was scored relative to the concurrent negative control plates and recorded with the mean revertant count for the strain, condition, and concentration. Revertant colonies for a given tester strain and condition were counted entirely by automated colony counter unless otherwise documented in the study records. Plates were also examined for test substance precipitation. Plates with test substance precipitation that interfered with the automated colony counting were counted manually when possible. H. STATISTICAL ANALYSIS Data for each tester strain were evaluated independently. For each tester strain, the average number o f revertants and the standard deviation at each concentration with or without an exogenous metabolic activation system were calculated. I. 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 for reported data was as follows: 1. Tester Strain Phenotypes and Spontaneous Reversion To demonstrate the presence o f the rfa mutation, all S. typhimurium tester strain cultures must have exhibited sensitivity to crystal violet. To demonstrate the presence o f the uvrB mutation, all S. typhimurium tester strain cultures must have exhibited sensitivity to ultraviolet light in ___________________________________________________ C mpany Sanitized Does no, contain TSna o pi - 13- H-23005: Bacterial Reverse Mutation Test DuPont H L-1998-01316 comparison to strain TA102 (which is proficient in the repair o f small amounts o f ultraviolet light-induced DNA damage). All S. typhimurium tester strain cultures must have exhibited L-histidine dependent growth and E. coli WP2 uvrA (pKMIOl) must have exhibited L-tryptophan dependent growth. To demonstrate the presence o f the uvrA mutation, the E, coli tester strain must have exhibited sensitivity to ultraviolet light. Tester strain cultures o f S. typhimurium TA97a, TA98, TA100 and E. coli WP2 uvrA (pKMIOl) must have exhibited resistance to ampicillin. Based on published data and historical data collected at the testing facility, all tester strain cultures must have exhibited a characteristic number o f spontaneous revertants per plate in the absence o f the test substance. The mean revertants per plate in the presence or absence of exogenous metabolic activation systems was 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) (101-227). Historical data collected for the testing facility is presented in Table 11. 2. Tester Strain Titer To ensure that appropriate numbers o f bacteria were plated, all tester strain culture titers must have been approximately 1 x 109cells/mL. 3. Positive Control Values Mean positive control values must have exhibited at least a three-fold increase over the respective mean o f the concurrent negative (solvent) control value for each tester strain and condition. 4. Toxicity A m inim um o f three non-toxic concentration levels was required to classify the test substance. A concentration level was considered toxic if it caused a >50 % reduction in the mean number of revertants per plate relative to the mean o f the concurrent negative control value. 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 two data points, or if variability between replicate plates was judged excessive. Excessive variability was v determined using scientific judgment. An assay for an individual strain was rejected if the negative control was rejected, if mutagenic activity was absent on all positive control plates, or if the tester strain failed to exhibit the appropriate phenotypes. Only those trials that met the criteria o f acceptability were included in this report. All data will be maintained in the study records. Company Sanitized. Does not contain TSCA CBf - 14- H-23005: Bacterial Reverse Mutation Test DuPont HL-1998-01316 J. CLASSIFICATION GUIDELINES A test substance was classified as POSITIVE (i.e., mutagenic) if the average number o f revertants in any strain at any test substance concentration was at least two times greater than the average number o f revertants in the concurrent vehicle control and there was a concentration-related increase in the mean revertants per plate in that same strain. A test substance was classified as NEGATIVE (i.e., not mutagenic) if there were no test substance concentrations with an average number o f revertants that were at least two times greater than the average number o f revertants in the concurrent vehicle control; or there was no positive concentration-related increase in the mean revertants per plate in that same strain. In consultation with the sponsor, negative results may be confirmed. Results not meeting criteria for positive or negative classification were evaluated using scientific judgment and experience. A study conclusion may be reported as EQUIVOCAL. Company Sanitized. Does not contain TSCA CBS H-23005: Bacterial Reverse Mutation Test DuPont H L-1998-01316 RESULTS AND DISCUSSION The active ingredient o f the test substance, H-23005, 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). The test was performed in two phases. The first phase consisted o f a preliminary toxicity assay that was used to establish test substance solubility in the selected solvent and the concentration range for the mutagenicity assay. The second phase, consisting o f the initial and independent repeat assays, evaluated the mutagenic potential o f the test substance, with the test concentrations adjusted so that the active ingredient was assessed at 5000 /xg/plate. Solubility and Preliminary Toxicity Assay Based on information supplied by the sponsor on the test substance evaluation form and preliminary solubility determinations, sterile water was chosen as the test substance solvent, diluent, and negative control. The test substance dissolved completely at 50 mg/mL for the preliminary toxicity assays, and at approximately 167 mg/mL for the two independent trials. The solution appeared off-white and slightly opaque. The preliminary toxicity assay was used to establish a concentration range over which the test substance was assayed. Concentrations o f 10, 50,100,250, 500,1000,2500, and 5000 pg/plate o f the test substance were evaluated, one plate per dose, in comparison to negative (solvent) controls. Salmonella typhimurium strains TA97a and TA100 and the Escherichia coli strain WP2 uvrA (pKMIOl) in the presence and absence o f an exogenous metabolic activation system (Aroclor-induced rat liver S9) were used in the preliminary toxicity assay. Trial 1 and Trial 2 The results from the preliminary toxicity assay showed no significant decrease in the number of colonies at the highest concentration (5000 /g/plate) assessed in comparison to negative solvent controls in the Salmonella strains or Escherichia coli strain. Toxicity was not observed at any concentration, and only microscopic precipitate was observed. The active ingredient o f the test substance was to be evaluated at the highest concentration set forth by applicable test guidelines in comparison to negative controls. Data from both independent trials were consistent with that o f the preliminary toxicity test. No toxicity was observed at any concentration. In the second independent repeat assay, the same concentrations were tested as in the first assay in comparison to negative controls for all bacteria. Precipitate was observed in all assays with all strains, in plated concentrations as low as 100 /xg/plate and up to 5000 /g/plate. Data are presented in Tables 1-10. Company Sanitized. Does not contain TSC CBI -16- H-23005: Bacterial Reverse Mutation Test DuPont HL-1998-01316 CONCLUSION Under the conditions o f this study, no evidence o f mutagenic activity was detected in either o f two independent trials. The test substance was negative. RECORDS AND SAMPLE STORAGE Specimens (if applicable), raw data, and the final report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware S i f t e d . Does rr,,I contain TSCACB1 - 17- H-23005: Bacterial Reverse Mutation Test DuPont HL-1998-01316 REFERENCES 1. Ames, B.N., et al. (1975). Methods for Detecting Carcinogens and Mutagens with the iS/mone/Za/Mammalian-Microsome Mutagenicity Test. Mutation Research, 31, 347-364. 2. Maron, D. M. and B. N. Ames (1983). Revised methods for the Salmonella mutagenicity test. Mutation Research 113,173-215. 3. Gatehouse, D., et al. (1994). Recommendations for the Performance o f Bacterial Mutation Assays. Mutation Research, 312, 217-233. 4. Wilcox, P., et al. (1990). Comparison o f Salmonella typhimurium TA102 with Escherichia coli WP2 Tester strains. M utagenesis, 5 ,285-291. 5. Brusick, D. J., et al. (1980). An evaluation o f the Escherichia coli WP2 and WP2wvrA reverse mutation assay. Mutation Research 76,169-190. 6. Ames, B. N., et al. (1973). An improved bacterial test system for the detection and classification o f mutagens and carcinogens. Proc. Natl. Acad. Sci. USA 70, 782-786. 7. McCann, J., et al. (1975). Detection o f carcinogens as mutagens: bacterial tester strains with R factor plasmids. Proc. Natl. Acad. Sci. USA 72, 979-983. 8. Levin, D. E., et al. (1982). A new Salmonella tester strain, TA97a, for the detection of frameshift mutagens. A run o f cytosines as a mutational hot spot. Mutation Research 94, 315-330. Company Sanitized. Dosa not contain TSCA CB1 - 18- H-23005: Bacterial Reverse Mutation Test DuPont HL-1998-01316 TABLES -19- Company Sanitized. Does not contain TSCA CBI H-23005: Bacterial Reverse Mutation Test DuPont H L-1998-01316 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 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 of a precipitate by the test substance 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 include the following: N Absence o f any noteworthy observation Gotnp3'^ nolcon.ainTSCACBl -20- H-23005: Bacterial Reverse Mutation Test DuPont H L-1998-01316 TABLE 1 MUTAGENIC ACTIVITY OF H-23005 IN SALMONELLA TYPHIMURIUM TA97a IN TRIAL 1 Concentration H-23005 (active ingredient) (fjg/plate) Revertants Plate Plate Plate 12 3 Average (S.D.) Observations A. WITHOUT METABOLIC ACTIVATION 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 126 154 129 130 157 162 137 119 162 144 163 160 154 170 132 151 140 122 141 188 161 118 139 135 136 (15) 150 (17) 139 (22) 156 (10) 152 (19) 138 (15) 163 (24) 131 (11) ICR 191 2 pg/piate 2421 2478 2450 2450 (29) B. WITH METABOLIC ACTIVATION (Aroclor--induced rat liver S9) 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 154 161 143 177 174 147 176 153 145 166 163 159 172 156 174 168 178 166 170 165 156 138 151 134 153 (9) 166 (17) 158 (16) 163 (4) 167 (10) 171 (6) 164 (7) 141 (9) DMBA 20 pg/plate 1720 1703 1742 1722 (20) TO,P0 TO,PO TO,PO TO,PO TO,PI TO,PI T0,P2 T0,P2 N TO,P0 TO,PO TO,PO TO,PO TO,PI TO,PI T0,P2 T0,P3 N Company Sanitized. Does not contain TSCA CBI -21 - H-23005: Bacterial Reverse Mutation Test DuPont HL-1998-01316 TABLE 2 MUTAGENIC ACTIVITY OF H-23005 IN SALMONELLA TYPHIMURIUM TA97a IN TRIAL 2 Concentration H-23005 (active ingredient) (pg/plate) Revertants Plate Plate Plate 12 3 Average (S.D.) Observations A. WITHOUT METABOLIC ACTIVATION 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 111 134 149 177 130 121 137 145 140 139 155 146 151 152 141 164 165 168 124 109 124 130 127 105 131 (19) 143 (30) 141 (4) 147 (8) 148 (6) 166 (2) 119 (9) 121 (14) ICR 191 2 pg/plate 799 852 786 812 (35) B. WITH METABOLIC ACTIVATION (Aroclor-induced rat liver S9) 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 135 154 159 150 147 143 147 165 147 187 178 157 157 153 177 162 138 163 140 141 150 136 125 150 149 (13) 147 (4) 153 (10) 174 (15) 162 (13) 154 (14) 144 (6) 137 (13) DMBA 20 pg/plate 853 928 849 877 (45) TO,P0 TO,PO TO,PO TO,PI TO,PI TO,PI T0,P2 T0,P2 N TO,P0 TO,PO TO,PO TO,PI TO,PI TO,PI T0,P2 T0,P3 N Company Sanitized. Does not contain TSC CBI - 22- H-23005: Bacterial Reverse Mutation Test DuPont HL-1998-01316 TABLE 3 MUTAGENIC ACTIVITY OF H-23005 IN SALM ONELLA TYPHIM URIUM TA98 IN TRIAL 1 Concentration H-23005 (active ingredient) (pg/plate) Revertants Plate Plate Plate 12 3 Average (S.D.) Observations A. WITHOUT METABOLIC ACTIVATION 0 .0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 14 13 15 17 17 24 16 19 17 15 16 12 13 19 13 13 21 18 21 20 20 8 10 11 14 (1) 19 (4) 17 (2) 14 (2) 15 (3) 17 (4) 20 (1) 10 (2) 2NF 25 pg/plate 1046 960 1054 1020 (52) B. WITH METABOLIC ACTIVATION (Aroclor-induced rat liver S9) 0 .0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 29 24 35 32 31 28 29 35 28 29 32 24 25 21 24 28 22 12 20 22 12 16 10 10 29 (6) 30 (2) 31 (4) 28 (4) 23 (2) 21 (8) 18 (5) 12 (3) 2AA 2 pg/plate 972 1012 1050 . 1011 (39) TO,P0 TO,PO TO,PO TO,PO TO,PI TO,PI T0,P2 T1,P2 N TO,P0 TO,PO TO,PO TO,PO TO,PI TO,PI T0,P2 T0,P3 N Company Sanitized. Does n o . contain TSCACBt - 23- H-23005: Bacterial Reverse Mutation Test DuPont H L -1998-01316 TABLE 4 MUTAGENIC ACTIVITY OF H-23005 IN SALMONELLA TYPHIMURIUM TA98 IN TRIAL 2 Concentration H-23005 (active ingredient) (pg/plate) Revertants Plate Plate Plate 12 3 Average (S.D.) Observations A. WITHOUT METABOLIC ACTIVATION 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 25 23 23 20 27 27 19 14 12 19 28 14 19 14 23 20 16 22 20 20 23 20 21 23 24 (1) 25 (4) 15 (4) 20 (7) 19 (5) 19 (3) 21 (2) 21 (2) 2NF 25 pg/plate 1651 1574 1561 1595 (49) B. WITH METABOLIC ACTIVATION (Aroclor -induced rat liver S9) 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 18 13 28 19 14 18 18 14 15 26 25 20 19 13 21 27 17 22 24 23 24 15 17 21 20 (8) 17 (3) 16 (2) 24 (3) 18 (4) 22 (5) 24 (1) 18 (3) 2AA 2 |4g/plate 748 710 794 751 (42) TO,P0 TO,PO TO,PO TO,PO TO,PI TO,PI T0,P2 T0,P2 N TO,P0 TO,PO TO,PO TO,PI TO,PI T0,P2 T0,P2 T0,P3 N Company Sanitized. Does not contain TSCA CBI -24 H-23005: Bacterial Reverse Mutation Test DuPont HL-1998-01316 TABLES MUTAGENIC ACTIVITY OF H-23005 IN SALMONELLA TYPHIMURIUM TA100 IN TRIAL 1 Concentration H-23005 (active ingredient) (pg/plate) Revertants Plate Plate Plate 12 3 Average (S.D.) Observations A. WITHOUT METABOLIC ACTIVATION 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 115 106 100 93 91 95 103 92 86 126 104 104 137 99 91 90 96 106 103 89 87 95 68 76 107 (8) 93 (2) 94 (9) 111 (13) 109 (25) 97 (8) 93 (9) 80 (14) NAAZ 2 pg/plate 617 696 679 664 (42) B. WITH METABOLIC ACTIVATION (Aroclor--induced rat liver S9) 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 122 98 129 124 107 100 121 115 106 107 100 111 102 128 115 101 115 135 110 120 109 80 99 118 116 (16) 110 (12) 114 (8) 106 (6) 115 (13) 117 (17) 113 (6) 99 (19) 2AA 1 pg/plate 362 358 430 383 (40) TO,P0 TO,PO TO,PO TO,PO TO,PI TO,PI T0,P2 T0,P2 N TO,P0 TO,PO TO,PO TO,PI TO,PI T0,P2 T0,P2 T0,P3 N Company Sanitized. Does not contain TSCA CBI H-23005: Bacterial Reverse Mutation Test DuPont H L-1998-01316 TABLE 6 MUTAGENIC ACTIVITY OF H-23005 IN SALMONELLA TYPHIMURIUM TA100 IN TRIAL 2 Concentration H-23005 (active ingredient) (pg/plate) Revertants Plate Plate Plate 12 3 Average (S.D.) Observations A. WITHOUT METABOLIC ACTIVATION 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 102 130 103 125 97 126 118 99 103 128 101 108 143 113 134 135 126 131 123 114 122 101 113 97 112 (16) 116 (16) 107 (10) 112 (14) 130 (15) 131 (5) 120 (5) 104 (8) NAAZ 2 pg/plate 843 854 832 843 (11) B. WITH METABOLIC ACTIVATION (Aroclor--induced rat liver S9) 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 117 136 139 124 111 149 125 145 127 158 135 116 133 117 139 117 129 130 148 106 117 112 96 102 131 (12) 128 (19) 132 (11) 136 (21) 130 (11) 125 (7) 124 (22) 103 (8) 2AA 2 pg/plate 896 914 885 898 (15) TO,P0 TO,PO TO,PO TO,PI TO,PI TO,PI T0,P2 T0,P2 N TO,P0 TO,PO TO,PO TO,PI TO,PI TO,PI T0,P2 T0,P3 N Company Sanitized. Does not contain TSCA CB1 H-23005: Bacterial Reverse Mutation Test DuPont HL-1998-01316 TABLE 7 MUTAGENIC ACTIVITY OF H-23005 IN SALM O NELLA TYPHIM URIUM TA1535 IN TRIAL 1 Concentration H-23005 (active ingredient) (pg/plate) Revertants Plate Plate Plate 12 3 Average (S.D.) Observations A. WITHOUT METABOLIC ACTIVATION 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 12 14 14 20 14 6 9 11 15 10 12 14 10 13 9 16 9 15 11 11 10 9 10 12 13 (1) 13 (7) 12 (3) 12 (2) 11 (2) 13 (4) 11 (1) 10 (2) NAAZ 2 pg/plate 479 561 585 542 (56) B. WITH METABOLIC ACTIVATION (Aroclor--induced rat liver S9) 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 675 4 11 12 12 6 8 13 9 9 8 8 15 10 7 7 12 10 10 988 6 (1) 9 (4) 9 (3) 10 (2) 10 (4) 8 (2) 11 (1) 8 (1) 2AA 2 pg/plate 170 183 223 192 (28) TO,P0 TO,PO TO,PO TO,PO TO,PI TO,PI T0,P2 T0,P2 N TO,P0 TO,PO TO,PO TO,PI TO,PI T0,P2 T0,P2 T0,P3 N Company Sanitized. Does not contain TSCA C - 27- H-23005: Bacterial Reverse Mutation Test DuPont H L-1998-01316 TABLE 8 MUTAGENIC ACTIVITY OF H-23005 IN SALM ONELLA TYPHIM URIUM TA1535 IN TRIAL 2 Concentration H-23005 (active ingredient) (pg/plate) Revertants Plate Plate Plate 12 3 Average (S.D.) Observations A. WITHOUT METABOLIC ACTIVATION 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 9 6 10 9 13 6 10 8 9 9 15 9 13 12 8 988 12 12 10 7 15 13 8 (2) 9 (4) 9 (1) 11 (3) 11 (3) 8 (1) 11 (1) 12 (4) NAAZ 2 p.g/plate 628 571 623 607 (32) B. WITH METABOLIC ACTIVATION (Aroclor--induced rat liver S9) 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 4 9 10 12 16 12 7 11 17 797 14 13 11 11 9 12 10 9 14 8 8 12 8 (3) 13 (2) 12 (5) 8 (1) 13 (2) 11 (2) 11 (3) 9 (2) 2AA 2 (xg/plate 79 96 123 . 99 (22) TO,P0 TO,PO TO,PO TO,PO TO,PI TO,PI T0,P2 T0,P2 N TO,P0 TO,PO TO,PO TO,PI TO,PI TO,PI T0,P2 T0,P3 N Company Sanitized. Does not contain TSC CBS -28- H-23005: Bacterial Reverse Mutation Test DuPont H L-1998-01316 TABLE 9 MUTAGENIC ACTIVITY OF H-23005 IN ESCHERICHIA C O IIW P 2 uvrA (pKMIOl) IN TRIAL 1 Concentration H-23005 (active ingredient) (pg/plate) Revertants Plate Plate Plate 12 3 Average (S.D.) Observations A. WITHOUT METABOLIC ACTIVATION 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 137 168 136 134 126 132 131 151 173 162 142 96 137 158 120 131 118 120 140 114 167 150 164 132 147 (18) 131 (4) 152 (21) 133 (34) 138 (19) 123 (7) 140 (27) 149 (16) ENNG 2 pg/plate 1237 1384 1302 1308 (74) B. WITH METABOLIC ACTIVATION (Aroclor-induced rat liver S9) 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 2AA 25 pg/plate 182 154 185 154 171 214 151 140 146 142 170 156 126 134 146 175 148 150 156 165 153 133 129 130 2222 2276 2172 174 (17) 180 (31) 146 (6) 156 (14) 135 (10) 158 (15) 158 (6) 131 (2) 2223 (52) TO,P0 TO,PO TO,PO TO,PO TO,PI TO,PI T0,P2 T0,P2 N TO,P0 TO,PO TO,PO TO,PI TO,PI T0,P2 T0,P2 T0,P2 N -29- Company Sanilizad. Doas noi contain TSCA CBI H-23005: Bacterial Reverse Mutation Test DuPont HL-1998-01316 TABLE 10 MUTAGENIC ACTIVITY OF H-23005 IN ESCHERICHIA COLIWP2 uvrA (pKMIOl) IN TRIAL 2 Concentration H-23005 (active ingredient) (M-g/plate) Revenants Plate Plate Plate 12 3 Average (S.D.) Observations A. WITHOUT METABOLIC ACTIVATION 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 121 114 106 127 127 137 126 154 131 137 152 137 137 129 128 142 123 137 113 135 116 99 102 101 114 (8) 130 (6) 137 (15) 142 (9) 131 (5) 134 (10) 121 (12) 101 (2) ENNG 2 pg/plate 1342 1296 1330 1323 (24) B. WITH METABOLIC ACTIVATION (ArocIor-induced rat liver S9) 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 147 153 154 178 167 156 187 188 164 180 172 169 171 176 202 220 182 163 137 144 167 153 160 174 151 (4) 167 (11) 180 (14) 174 (6) 183 (17) 188 (29) 149 (16) 162 (11) 2AA 25 pg/plate 1030 1161 1214 1135 (95) TO,P0 TO,PO TO,PO TO,PO TO,PI TO,PI T0,P2 T0,P2 N TO,PO TO,PO TO,PO TO,PI TO,PI T0,P2 T0,P2 T0,P3 N Company Sanitised. Boas net contain tsca CBI -30- H-23005: Bacterial Reverse Mutation Test DuPont H L-1998-01316 TABLE 11 HISTORICAL CONTROL DATA1 Tester Strain Control [Positive Control2] Exogenous Metabolic 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. co li WP2 uvrA (pKMIOlj Negative Negative Positive [MMS-1000] Positive [ENNG-2] Positive [2AA-25] Positive [2AA-250] Absent Absent Present Present Absent Absent Present Present Absent Absent Present Present Present Absent Absent Present Present Absent Absent Absent Present Present Present 121 128 853 1211 (31) (32) (252) (457) 16 (6) 15 (6) 576 (173) 387 (134) 101 117 1747 1357 941 (18) (25) (671) (334) (316) 23 26 1391 1656 (7) (7) (364) (544) 156 172 1656 1662 1592 1682 (27) (28) (446) (155) (487) (372) 54-218 65-253 339-2604 94-2682 7 -4 6 5-39 206-1156 55 - 1323 59-151 67 -1 9 6 476-3209 702 -1 6 8 3 308-2035 - 9 -4 6 11-49 567-2373 437-3114 90-221 93-255 208 - 2453 1353-1918 485-2484 929-2230 'THT-istAorica1l dJ a. ta for it.estA-er straii_n__s___u__s_ ed in the reported study. Data are based on studies reported during the period 1996 up to and including November 26,1997. 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, preincubation/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-1,2benzanthracene), ENNG (A-ethyl-A-nitro-A-nitrosoguanidine). The number following abbreviation is the microgram (pg) amount per plate or vial used for the positive control. Com Pany Sanitized. Does esnot contain TSCACBl -31 -