DuPont-3908

Page 1 of 36 Study Title AAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli
Laboratory Project ID: DuPont-3908

T EST G UIDELINES : U.S. EPA Health Effects Test Guidelines OPPTS 870.5100 (1998) OECD Guidelines for Testing of Chemicals Section 4: Health Effects, No. 471 (1998)
A UTHOR : N. Lawrence Gladnick, B.A.
S TUDY C OMPLETED ON : February 29, 2000
P ERFORMING L ABORATORY : DuPont Pharmaceuticals Company Safety Assessment E. I. du Pont de Nemours and Company P.O. Box 30, Elkton Road Newark, Delaware 19711-3507 for 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
W ORK R EQUEST N UMBER : 13163

S ERVICE C ODE N UMBER : 502
D U P ONT P HARMACEUTICALS C OMPANY S TUDY N O .: THA99-12-20, THA99-12-17, and THA99-12-19AAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 2 - CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study.AAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 3 - TABLE OF CONTENTS Page
CERTIFICATION.................................................................................................................. ......... 2 LIST OF TABLES ................................................................................................................. ......... 4 STUDY INFORMATION.............................................................................................................. . 5 STUDY PERSONNEL ................................................................................................................ ... 7 SUMMARY ........................................................................................................................ ............ 8 INTRODUCTION................................................................................................................... ........ 9 MATERIALS AND METHODS.................................................................................................... 9 A. Study Protocol............................................................................................................... ...... 9 B. Test Materials............................................................................................................... ..... 10 1. Test Substances ........................................................................................................... 10 2. Negative and Positive Controls................................................................................... 10 3. Tester Strain Source, Characterization, Storage, and Culture..................................... 11 4. Metabolic Activation System...................................................................................... 12 C. Test Substances Concentration Selection, Stability, and Verification.............................. 12 D. Test Methods: Bacterial Mutagenicity Assay .................................................................. 12 E. Statistical Analysis ......................................................................................................... ... 13 F. Acceptability Criteria ....................................................................................................... . 13 1. Tester Strain Phenotypes and Spontaneous Reversion ............................................... 13 2. Tester Strain Culture Titer........................................................................................... 14 3. Positive Control Values............................................................................................... 14 4. Non-Toxic Concentration Levels................................................................................ 14 5. Rejection of Plates, Concentration Levels, or Assays................................................. 14 G. Classification Criteria...................................................................................................... .. 15 RESULTS AND DISCUSSION ................................................................................................... 15 CONCLUSIONS.................................................................................................................... ....... 16 RECORDS AND SAMPLE STORAGE....................................................................................... 16 REFERENCES..................................................................................................................... ......... 17 TABLES......................................................................................................................... ............... 18 APPENDIX - HISTORICAL CONTROL DATA....................................................................... 35AAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 4 - LIST OF TABLES Page TABLE 1: Mutagenic Activity In Salmonella typhimurium TA97a For #873 ............................ 20 TABLE 2: Mutagenic Activity In Salmonella typhimurium TA98 For #873.............................. 21 TABLE 3: Mutagenic Activity In Salmonella typhimurium TA100 For #873............................ 22 TABLE 4: Mutagenic Activity In Salmonella typhimurium TA1535 For #873.......................... 23 TABLE 5: Mutagenic Activity In Escherichia coli WP2 uvrA (pKM101) For #873.................. 24 TABLE 6: Mutagenic Activity In Salmonella typhimurium TA97a For #903 ............................ 25 TABLE 7: Mutagenic Activity In Salmonella typhimurium TA98 For #903.............................. 26 TABLE 8: Mutagenic Activity In Salmonella typhimurium TA100 For #903............................ 27 TABLE 9: Mutagenic Activity In Salmonella typhimurium TA1535 For #903.......................... 28 TABLE 10: Mutagenic Activity In Escherichia coli WP2 uvrA (pKM101) For #903................ 29 TABLE 11: Mutagenic Activity In Salmonella typhimurium TA97a For #1116 ........................ 30 TABLE 12: Mutagenic Activity In Salmonella typhimurium TA98 For #1116.......................... 31 TABLE 13: Mutagenic Activity In Salmonella typhimurium TA100 For #1116........................ 32 TABLE 14: Mutagenic Activity In Salmonella typhimurium TA1535 For #1116...................... 33 TABLE 15: Mutagenic Activity In Escherichia coli WP2 uvrA (pKM101) For #1116.............. 34AAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 5 - STUDY INFORMATION 9th Collective Nomenclature: 1-Propene, 2-bromo-3,3,3-trifluoroSynonyms/Codes: • 2-Bromo-3,3,3-trifluoroprop-1-ene

• AAWG #873

• #873

• H-24293

• 2-Bromo-3,3,3-trifluoropropene

• 2-Bromo-1,1,1-trifluoroprop-2-ene

• 1300-B-05

• Q49-28 (SynQuest Laboratories) (Lot No.)
Haskell Number: 24293 Composition: 2-Bromo-3,3,3-trifluoroprop-1-ene Purity: 98% Known Impurities: Not supplied by the sponsor Physical Characteristics: Colorless liquid

9th Collective Nomenclature: 1-Butene, 4-bromo-3,3,4,4-tetrafluoroSynonyms/Codes: • 4-Bromo-3,3,4,4-tetrafluorobut-1-ene

• H-24294

• AAWG #903

• #903

• 4-Bromo-3,3,4,4-tetrafluorobutene

• 1-Bromo-1,1,2,2-tetrafluorobut-3-ene

• BTFB

• 1300-B-11

• Q65-17 (SynQuest Laboratories) (Lot No.)
Haskell Number: 24294 CAS Registry Number: 18599-22-9AAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 6 - STUDY INFORMATION (Continued) Composition: 4-Bromo-3,3,4,4-tetrafluorobut-1-ene Purity: 99.2% Known Impurities: Not supplied by the sponsor Physical Characteristics: Colorless liquid

9th Collective Nomenclature: 1-Butene, 3-bromo-3,3,3,4,4-pentafluoroSynonyms/Codes: • 2-Bromo-3,3,4,4,4-pentafluorobut-1-ene

• AAWG #1116

• #1116

• H-24295

• 2-Bromo-3,3,4,4,4-pentafluorobutene

• 3-Bromo-1,1,1,2,2-pentafluorobut-3-ene

• 1300-B-12

• Q6A-55 (SynQuest Laboratories) (Lot No.)
Haskell Number: 24295 Composition: 2-Bromo-3,3,4,4,4-pentafluorobut-1-ene Purity: 98.2% Known Impurities: Not supplied by the sponsor Physical Characteristics: Colorless liquid Stability: The test substances appeared to be stable under the conditions of the study; no evidence of instability was observed. Sponsor: Advanced Agent Working Group Halon Alternatives Research Corporation 2111 Wilson Boulevard, Suite 850 Arlington, VA 22201 Study Initiated/Completed: December 2, 1999 / (see report cover page) In-Life Initiated/Completed: December 7, 1999/ January 10, 2000AAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 7 - STUDY PERSONNEL DuPont Pharmaceutical Company Study Director: N. Lawrence Gladnick, B.A.

Management: Ronald D. Snyder, Ph.D. Primary Technician: N. Lawrence Gladnick, B.A. E. I. DuPont de Nemours and Company Study Monitor: E. Maria Donner, Ph.D. Report Preparation: Brenda TiffinAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 8 - SUMMARY Three test substances, AAWG #873 (#873), AAWG #903 (#903), and AAWG #1116 (#1116), were evaluated in the bacterial reverse mutation assay using Salmonella typhimurium strains TA97a, TA98, TA100, TA1535, and Escherichia coli strain WP2 uvrA (pKM101) in the presence and absence of an exogenous metabolic activation system (Aroclor ® -induced rat liver S9). For AAWG #903 and AAWG #1116 a single trial assay for each test substance was performed using the plate incorporation method to evaluate the mutagenic potential of the test substances. For AAWG #873, a single trial assay was performed using a preincubation method similar to that described by Westphal et al. ( Arch Toxicol , 68 :79-84). A preincubation procedure was employed for AAWG #873 due to the test substance’s volatility (boiling point 33-35 ° C). The plate incorporation assay utilizes molten agar (45-48 ° C) as the medium for transference of the test substance, bacterial cultures and metabolic activation system (or control) to the agar plate for colony growth. The preincubation assay provided a more intimate involvement of the test substance, bacterial culture(s) and the metabolic activation system (with or without S9) in a closed airtight system with minimal headspace at a much lower temperature. Concentrations of 5, 10, 50, 100, 500, 100, 2500, and 5000 μg/plate per test substance were assessed in comparison to negative (solvent) controls. The solvent, diluent and negative control used in the assays was dimethyl sulfoxide (DMSO). Test substance-related toxicity, evidenced by the reduction of the microcolony background lawns and/or as a concentration-related reduction in the mean number of revertants per plate, was observed with all three test substances with all strains. No test substance-related precipitate was observed at any concentration of #873, #903, and #1116 with any strain. All tester strains exhibited appropriate phenotypic characteristics. For each of the three test substances, the mean number of revertants 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 for any of the three test substances in any of the Salmonella typhimurium strains or the Escherichia coli strain. Based on the findings, #873, #903, and #1116 were concluded to be negative for the induction of mutagenicity in the bacterial reverse mutation test.AAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 9 - INTRODUCTION This study evaluated the mutagenic potential of the test substances, AAWG #873 (#873), AAWG #903 (#903), and AAWG #1116 (#1116) in Salmonella typhimurium strains TA97a, TA98, TA100, and TA1535 and in Escherichia coli strain WP2 uvrA (pKM101). The bacterial reverse mutation test uses amino acid requiring strains of Salmonella typhimurium and Escherichia coli to detect mutations that involve substitution, addition or deletion of one or a few DNA base pairs. (1,2) 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 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 (pKM101) is unable to synthesize tryptophan due to an ochre mutation in the gene required for tryptophan biosynthesis. E. coli WP2 uvrA (pKM101) 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 of chemically induced revertants to the number of spontaneous revertants, the mutagenicity of the test substances can be assessed.

MATERIALS AND METHODS

A. Study Protocol The protocols consisted of the stand-alone Protocol Sheet and the Haskell General Testing Procedure GT008-T-010 (“Bacterial Reverse Mutation Test for Solids, Liquids, and Gases”, effective 06/10/98). The study was designed to comply with:

 U.S. EPA, Office of Prevention, Pesticides and Toxic Substances (OPPTS) Guidelines (Subpart H, 40 CFR Part 870.5100 [1998]);

 Guidelines of the Organisation for Economic Cooperation and Development (OECD Guidelines for Testing of Chemicals, No. 471 [1998]); The study design complied with the testing guidelines cited above 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 and/or TA1537 (EU and Japan).

 E. coli WP2 uvrA (pKM101) was the only E. coli tester strain used (EU and Japan).

 One trial was conducted with the test substances as specified by the sponsor (EU and MAFF).AAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 10 - These exceptions did not affect the study validity for the following reasons:

 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 frameshift mutagens and the reconstructed strain, TA97a, was used due to its improved growth properties. (4)

 Although some testing guidelines require E. coli WP2 uvrA , no significant difference in mutagen detection between the plasmid and non-plasmid containing strains would be expected. Therefore, E. coli WP2 uvrA (pKM101) was used to assess potential mutagenicity. The strains used in this study comply with harmonized guidelines designed to minimize variations among testing procedures worldwide (OECD 1998, OPPTS 1998).

B. Test Materials 1. Test Substances The test substances, #873, #903, and #1116, are colorless liquids that were stored at room temperature. Additional information regarding the test substances can be found on the study information page of this report. The test substances were 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, dimethyl sulfoxide (DMSO) was chosen as the test substances 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 Chemical Abstracts Service (CAS) Registry Number 2-Nitrofluorene (2NF) 607-57-8 N

-Ethyl- N -nitro- N -nitroguanidine (ENNG) 4245-77-6 Sodium azide (NAAZ) 26628-22-8 ICR 191 Acridine mutagen (ICR 191) 17070-45-0 9,10-Dimethyl-1,2-benzanthracene (DMBA) 57-97-6 2-Aminoanthracene (2AA) 613-13-8

Deionized water was the solvent for sodium azide (NAAZ). The solvent for 2AA, 2NF, ICR 191, DMBA, and ENNG was dimethyl sulfoxide (DMSO). The manufacturer, lot number, and purity of the DMSO will be recorded in the study records. Neither the amount and nature of the contaminants nor the use of DMSO is expected to affect the integrity or validity of the study. The positive controls were prepared in advance and maintained frozen at approximately -70 ° C. 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.AAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 11 - 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 (pKM101) was obtained from the National Collection of Industrial Bacteria, Torrey Research Station, Scotland, UK. The characteristics of S. typhimurium tester strains TA97a, TA98, TA100, TA102, TA1535 and E. coli WP2 uvrA (pKM101) are as follows:
Strain Gene Locus Excisio n Repair LPS R-factor (pKM101) pAQ1 Plasmid S. typhimurium TA97a his D6610 ∆ uvrB rfa Present Absent S. typhimurium TA98 his D3052 ∆ uvrB rfa Present Absent S. typhimurium TA100 his G46 ∆ uvrB rfa Present Absent S. typhimurium TA102* his G428 (+) rfa Present Present S. typhimurium TA1535 his G46 ∆ uvrB rfa Absent Absent S. typhimurium TA1537 his C3076 ∆ uvrB rfa Absent Absent E. coli WP2 uvrA (pKM101) trpE ∆ uvrA NA Present Absent *Concurrent control strain used to distinguish differential sensitivity/resistance to ultraviolet light. LPS = lipopolysaccharide NA=Not applicable (+) proficient in excision repair;

 Also his O1242. ∆ = symbol for “deletion” uvrA and uvrB are defective DNA repair genes.

The deletion ( ∆ ) in uvrB (a gene that codes for one protein involved in DNA excision repair) increases the bacterial sensitivity to some mutagens. (5) 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. (6) The presence of the pKM101 or R-factor plasmid, conferring ampicillin resistance, also enhances an error-prone DNA repair system that is endogenous to these bacteria. (7) The pAQ1 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 -70° to -80°C in ~8% (v/v) DMSO in Oxoid Nutrient Broth No. 2. The E. coli WP2 uvrA (pKM101) strain was stored at approximately -70°C in ~30% glycerol in Oxoid Nutrient Broth No. 2. Prior to 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 ~37°C with shaking. Tester strain phenotypes were confirmed on overnight cultures concurrently with each trial and results for all trials demonstrated the appropriate responses.AAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 12 - 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. (1) 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 KCl solution) prepared from the livers of young male Sprague Dawley rats treated with the enzyme-inducing agent Aroclor ® 1254 (500 mg/kg i.p.) as a single dose 5 days prior to sacrifice. The Aroclor ® -induced rat liver S9 (purchased from MOLTOX  ) was characterized for protein content and metabolic activity by the vendor. To confirm the sterility of the exogenous metabolic activation system, an aliquot was plated on nutrient agar capable of supporting the growth of viable bacteria. The amount of Aroclor ® -1254 induced rat liver S9 in the exogenous metabolic activation system was 4.0 mg S9 protein (~10% [v/v]) / mL. The cofactor-supplement concentrations in the exogenous metabolic activation system were 8 mM MgCl 2 , 33 mM KCl, 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 Substances Concentration Selection, Stability, and Verification In accordance with testing guidelines, the highest concentration for each test substance evaluated in this study was 5000 μg/plate. Solubility information was confirmed prior to the start of the study. The stock concentrations were calculated and adjusted for the test substances’ displacement. Solutions of the test substances 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 substances were assessed for sterility by plating a small amount of the highest of each 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 each test substance. Three replicates (per test substance) were plated for each tester strain in the presence and absence of the exogenous metabolic activation system at each respective test substance concentration. For each test substance, positive and negative controls were included for each strain and condition. For AAWG #903 and AAWG #1116 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 10 8 bacteria to approximately 2 mL of 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 MOLTOX  ). Because of the volatility of AAWG #873 (boiling pointAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 13 - 33-35 ° C) at the temperature at which top agar is maintained (45-48°C), a preincubation method similar to that described by Westphal et al. (1994) was utilized. (8) Dilutions of AAWG #873 in DMSO were added to 0.5 mL of exogenous metabolic activation system (or S9 control) and 0.1 mL (containing approximately 1 x 10 8 bacteria) of an overnight bacterial culture. Three replicates were incubated in sealed airtight 2-mL vials for approximately 60 minutes at ~37°C. Following incubation, the entire contents of each vial were removed using a sterile glass pipette and added to 2.0 mL of top agar [0.5 – 0.6% agar (w/v) and 0.6 – 0.7% NaCl (w/v)] containing 0.05 mM L -histidine, D -biotin and L -tryptophan. 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 37°C for about 48 hours. Plates were refrigerated at approximately 4 ° C ( ± 3 ° C) prior to evaluation and counting of revertant colonies. Bacterial background lawns were evaluated for evidence of test substance toxicity and precipitation. Evidence of toxicity was scored relative to the concurrent negative control plates and recorded with the mean revertant count for the strain, condition, and respective test substance 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 and test substance were evaluated independently. For each tester strain, the mean number of revertants and the standard deviation at each concentration in the presence of and absence of the exogenous metabolic activation system were calculated. Data for each of the three test substances are reported separately.

F. Acceptability Criteria For each of the three test substances, an individual trial must have included a negative and positive control and at least 5 concentration levels per 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 Phenotypes and Spontaneous Reversion
All S. typhimurium tester strain cultures were required to exhibit L -histidine dependent growth. To demonstrate the presence of the rfa mutation, all S. typhimurium tester strain cultures were required to exhibit sensitivity to crystal violet. To demonstrate the presence of the uvrB mutation, all S. typhimurium tester strain cultures were required to exhibit sensitivity to ultraviolet light in comparison to strain TA102, which is proficient in the repair of small amounts of ultraviolet light-induced DNA damage. E. coli tester strains were required to exhibit L

tryptophan dependent growth. To demonstrate the presence of the uvrA mutation, the E. coliAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 14 - tester strain were required to exhibit sensitivity to ultraviolet light. Tester strain cultures of S. typhimurium TA97a, TA98, TA100 and E. coli WP2 uvrA (pKM101) were required to exhibit resistance to ampicillin to demonstrate the presence of the pKM101 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 substances. The acceptable mean revertants per plate 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 (pKM101) (90-227). Historical data collected at the testing facility are presented in the appendix at the end of the report.
2. Tester Strain Culture Titer To ensure that appropriate numbers of bacteria were plated, all tester strain culture titers were approximately 1 x 10 9 cells/mL. 3. Positive Control Values All mean positive control values were required to exhibit at least a three-fold increase over the respective mean of the concurrent negative control value for each tester strain and condition.
4. Non-Toxic Concentration Levels A minimum of 5 analyzable (of which 4 must be non-toxic) concentration levels was required to evaluate the test substances. A concentration level was considered toxic and analyzable if the test substance at that specific concentration caused a >50% reduction of 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 was rejected if contamination, test substance precipitation or conditions resulted in a 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 the variability between replicate plates was judged to be excessive. Variability between plates is strain dependent and can be affected by treatment with the test substances; therefore, scientific judgement was used in determining the acceptability of the data. An assay (for an individual strain and condition) was 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.AAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 15 - G. Classification Criteria A test substance was classified as POSITIVE (i.e., mutagenic) if the mean number of revertants in any strain (except Salmonella typhimurium strain TA1535) at any test substance concentration was at least two times greater than the mean number of revertants in the concurrent strain specific 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 typhimurium strain TA1535 must be at least three times greater than the mean number of reverant of its concurrent negative control. A test substance was classified as NEGATIVE (i.e., not mutagenic) if, in all strains except TA1535, there were no test substance concentration 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 Salmonella 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 reverants of their respective concurrent vehicle control and no concentration-related increase in the mean revertants per plate. 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 AAWG #873, #903, and #1116 were evaluated in the bacterial reverse mutation assay using Salmonella typhimurium strains TA97a, TA98, TA100, TA1535, and Escherichia coli strain WP2 uvrA (pKM101) in the presence and absence of an exogenous metabolic activation system (Aroclor ® -induced rat liver S9). The tests for AAWG #903 and #1116 were performed using the plate incorporation method and the test for AAWG #873 was performed using the preincubation method to evaluate the mutagenic potential of the test substances. Dimethyl sulfoxide (DMSO) was chosen as the test substances solvent, diluent, and negative control. In this study, concentrations of 5, 10, 50, 100, 500, 1000, 2500, and 5000 μg/plate per test substance were tested in comparison to negative (solvent) controls. Mean positive control values for all three test substances, 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 1- 15). All tester strains exhibited appropriate phenotypic characteristics. For each of the three test substances, the mean number of revertants observed in the negative control for each strain was within the prescribed acceptable range. No test substance-related precipitate was observed with any of the assays at any concentration level. Test substance-related toxicity, evidenced by the reduction of the microcolony background lawns and/or as a concentration-related reduction in the mean number of revertants per plate, was observed with all three test substances with all strains (Tables 1-15). In tester strains TA97a, TA98, TA100, and E.coli WP2 uvrA (pKm101) there were no test substanceAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 16 - concentrations with a mean number of revertants that were 2 times greater than the mean of the concurrent vehicle control (Tables 1-3, 5-8, 10-13, and 15). In tester strain TA1535, there were no test substance concentrations with a mean number of revertants that were 3 times greater thanthe mean of the concurrent vehicle control (Table 4, 9, and 14). There was no concentrationrelated increase of the mean revertants per plate in any strain with any concentration level of #873, #903, and #1116.

CONCLUSIONS Under the conditions of this study for all three test substances, no evidence of mutagenic activity was detected. Based on the findings, #873, #903, and #1116 were concluded to be negative for the induction of mutagenicity in the bacterial reverse mutation test in Salmonella typhimurium and Escherichia coli

.

RECORDS AND SAMPLE STORAGE At the request of the sponsor, specimens (if applicable), raw data, and the final report will be retained at DuPont Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware.AAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 17 - REFERENCES 1. Maron, D. M. and B. N. Ames (1983). Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113 , 173-215.
2. Green, M.H.L. and W. J. Muriel (1976). Mutagen testing using TRP + reversion in Escherichia coli. Mutat. Res. 38 , 3-32. 3. Gatehouse, D.G., Haworth, S., Cebula, T., Gocke, E., Kier, L., Matsushima, T., Melcion, C., Nohmi, T., Venitt, S. and E. Zeiger (1994). Recommendations for the Performance of Bacterial Mutation Assays. Mutat. Res. 312, 217-233.
4. Levin, D. E., E. Yamasaki, and B. N. Ames (1982). A new Salmonella tester strain, TA97, for the detection of frameshift mutagens. A run of cytosines as a mutational hot spot. Mutat. Res. 94 , 315-330. 5. 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.
6. Ames, B. N., F. D. Lee, and W. E. Durston (1973). An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Natl. Acad. Sci. USA 70 , 782-786.
7. McCann, J., N. E. Springarn, 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. 8. Westphal, G.A., Blaszkewicz, M., Leutbecher, M. Müller, A., Hallier, and H.M. Bolt (1994). Bacterial mutagenicity of 2-chloro-1,3-butadiene (chloroprene) caused by decomposition products. Arch of Toxicology 68 , 79-84.AAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 18 - TABLESAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 19 - 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:
T0 Normal , background microcolony lawn appeared normal. T1 Slightly reduced , background microcolony lawn was noticeably thinner. T2 Moderately reduced , background lawn was markedly thinner resulting in an increase in the size of microcolonies compared to the vehicle control plate(s). T3 Severely reduced , background lawn was distinguished by an extreme thinning resulting in an increase in the size of the microcolonies compared to the vehicle control plate(s). Microcolonies were seen readily by the unaided eye and were greatly enlarged relative to controls. T4 Absent , plate(s) were distinguished by a complete lack of any microcolony lawn over a majority of the area of the plate(s). Formation of a precipitate by the test material was documented using the following key:
P0 No precipitate, no precipitate observed. P1 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 Acridine mutagen DMBA 9,10-Dimethyl-1,2-benzanthracene 2NF 2-Nitrofluorene 2AA 2-Aminoanthracene NAAZ Sodium azide ENNG N -Ethyl- N -nitro- N -nitroguanidineAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 20 - TABLE 1
MUTAGENIC ACTIVITY IN Salmonella typhimurium TA97A FOR #873

Concentration Revertants (μg/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations

A. WITHOUT METABOLIC ACTIVATION 0 145 129 142 139 (9) T0,P0 5 124 144 145 138 (12) T0,P0 10 128 156 127 137 (16) T0,P0 50 118 117 108 114 (6) T0,P0 100 129 123 127 126 (3) T0,P0 500 111 117 115 114 (3) T0,P0 1000 93 96 115 101 (12) T1,P0 2500 82 92 60 78 (16) T1,P0 5000 54 61 84 66 (16) T2,P0
ICR 191 2 μg/plate 2568 2628 2484 2560 (72) N

B. WITH METABOLIC ACTIVATION (Aroclor ® -induced rat liver S9) 0 161 152 145 153 (8) T0,P0 5 148 148 152 149 (2) T0,P0 10 113 109 137 120 (15) T0,P0 50 110 124 153 129 (22) T0,P0 100 148 134 139 140 (7) T0,P0 500 166 148 132 149 (17) T0,P0 1000 138 123 104 122 (17) T0,P0 2500 89 105 114 103 (13) T1,P0 5000 85 90 83 86 (4) T1,P0
DMBA 20 μg/plate 1018 984 912 971 (54) NAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 21 - TABLE 2
MUTAGENIC ACTIVITY IN Salmonella typhimurium TA98 FOR #873

Concentration Revertants (μg/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations

A. WITHOUT METABOLIC ACTIVATION 0 28 17 23 23 (6) T0,P0 5 27 14 20 20 (7) T0,P0 10 29 27 23 26 (3) T0,P0 50 23 15 13 17 (5) T0,P0 100 23 16 14 18 (5) T0,P0 500 23 11 11 15 (7) T0,P0 1000 17 21 16 18 (3) T1,P0 2500 15 16 20 17 (3) T1,P0 5000 12 10 12 11 (1) T2,P0
2NF 25 μg/plate 1710 1844 1551 1702 (147) N

B. WITH METABOLIC ACTIVATION (Aroclor ® -induced rat liver S9) 0 25 32 28 28 (4) T0,P0 5 37 26 18 27 (10) T0,P0 10 30 34 28 31 (3) T0,P0 50 28 22 21 24 (4) T0,P0 100 23 24 32 26 (5) T0,P0 500 27 24 34 28 (5) T0,P0 1000 39 20 31 30 (10) T0,P0 2500 30 24 28 27 (3) T1,P0 5000 26 39 22 29 (9) T1,P0
2AA 2 μg/plate 1115 1119 1327 1187 (121) NAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 22 - TABLE 3
MUTAGENIC ACTIVITY IN Salmonella typhimurium TA100 FOR #873

Concentration Revertants (μg/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations

A. WITHOUT METABOLIC ACTIVATION 0 168 138 144 150 (16) T0,P0 5 155 151 163 156 (6) T0,P0 10 164 155 163 161 (5) T0,P0 50 126 156 124 135 (18) T0,P0 100 151 136 160 149 (12) T0,P0 500 131 129 116 125 (8) T0,P0 1000 106 102 97 102 (5) T0,P0 2500 85 81 67 78 (9) T1,P0 5000 65 67 68 67 (2) T2,P0
NAAZ 2 μg/plate 855 842 809 835 (24) N

B. WITH METABOLIC ACTIVATION (Aroclor ® -induced rat liver S9) 0 154 164 105 141 (32) T0,P0 5 106 132 129 122 (14) T0,P0 10 136 109 107 117 (16) T0,P0 50 124 116 125 122 (5) T0,P0 100 157 136 135 143 (12) T0,P0 500 157 148 152 152 (5) T0,P0 1000 129 158 133 140 (16) T0,P0 2500 126 157 117 133 (21) T0,P0 5000 143 142 143 143 (1) T0,P0
2AA 1 μg/plate 896 862 1085 948 (120) NAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 23 - TABLE 4
MUTAGENIC ACTIVITY IN Salmonella typhimurium TA1535 FOR #873

Concentration Revertants (μg/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations

A. WITHOUT METABOLIC ACTIVATION 0 23 18 14 18 (5) T0,P0 5 18 15 14 16 (2) T0,P0 10 11 7 13 10 (3) T0,P0 50 18 17 14 16 (2) T0,P0 100 15 21 19 18 (3) T0,P0 500 11 16 10 12 (3) T0,P0 1000 12 15 17 15 (3) T0,P0 2500 10 14 11 12 (2) T1,P0 5000 7 10 10 9 (2) T2,P0
NAAZ 2 μg/plate 663 732 524 640 (106) N

B. WITH METABOLIC ACTIVATION (Aroclor ® -induced rat liver S9) 0 14 23 9 15 (7) T0,P0 5 14 11 21 15 (5) T0,P0 10 15 19 14 16 (3) T0,P0 50 20 10 16 15 (5) T0,P0 100 16 11 24 17 (7) T0,P0 500 11 17 21 16 (5) T0,P0 1000 16 12 16 15 (2) T0,P0 2500 20 14 15 16 (3) T1,P0 5000 15 12 16 14 (2) T1,P0
2AA 2 μg/plate 362 406 443 404 (41) NAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 24 - TABLE 5
MUTAGENIC ACTIVITY IN Escherichia coli WP2 uvrA (PKM101) FOR #873

Concentration Revertants (μg/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations

A. WITHOUT METABOLIC ACTIVATION 0 130 121 165 139 (23) T0,P0 5 103 159 157 140 (32) T0,P0 10 133 153 187 158 (27) T0,P0 50 117 126 152 132 (18) T0,P0 100 121 115 117 118 (3) T0,P0 500 142 132 127 134 (8) T0,P0 1000 95 118 107 107 (12) T0,P0 2500 90 97 76 88 (11) T1,P0 5000 84 72 66 74 (9) T1,P0
ENNG 2 μg/plate 1376 1656 1642 1558 (158) N

B. WITH METABOLIC ACTIVATION (Aroclor ® -induced rat liver S9) 0 188 181 202 190 (11) T0,P0 5 203 209 196 203 (7) T0,P0 10 227 220 198 215 (15) T0,P0 50 229 186 171 195 (30) T0,P0 100 237 230 200 222 (20) T0,P0 500 252 220 229 234 (17) T0,P0 1000 239 256 230 242 (13) T0,P0 2500 143 140 167 150 (15) T1,P0 5000 141 163 137 147 (14) T1,P0
2AA 25 μg/plate 902 1049 1025 992 (79) NAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 25 - TABLE 6
MUTAGENIC ACTIVITY IN Salmonella typhimurium TA97A FOR #903

Concentration Revertants (μg/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations

A. WITHOUT METABOLIC ACTIVATION 0 138 130 126 131 (6) T0,P0 5 133 120 141 131 (11) T0,P0 10 148 130 126 135 (12) T0,P0 50 128 116 R* 122 (8) T0,P0 100 154 156 128 146 (16) T0,P0 500 82 79 84 82 (3) T1,P0 1000 52 83 60 65 (16) T1,P0 2500 54 42 39 45 (8) T2,P0 5000 44 56 41 47 (8) T2,P0
ICR 191 2 μg/plate 1133 1494 1658 1428 (269) N

B. WITH METABOLIC ACTIVATION (Aroclor ® -induced rat liver S9) 0 164 164 158 162 (3) T0,P0 5 160 149 166 158 (9) T0,P0 10 157 167 168 164 (6) T0,P0 50 174 153 179 169 (14) T0,P0 100 142 152 160 151 (9) T0,P0 500 172 180 160 171 (10) T0,P0 1000 99 122 132 118 (17) T1,P0 2500 80 79 84 81 (3) T1,P0 5000 67 74 54 65 (10) T2,P0
DMBA 20 μg/plate 1712 1648 1825 1728 (90) N *Plate rejectedAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 26 - TABLE 7
MUTAGENIC ACTIVITY IN Salmonella typhimurium TA98 FOR #903

Concentration Revertants (μg/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations

A. WITHOUT METABOLIC ACTIVATION 0 20 10 16 15 (5) T0,P0 5 25 27 25 26 (1) T0,P0 10 18 16 23 19 (4) T0,P0 50 20 23 26 23 (3) T0,P0 100 13 21 22 19 (5) T0,P0 500 13 12 13 13 (1) T0,P0 1000 13 13 14 13 (1) T1,P0 2500 5 4 2 4 (2) T2,P0 5000 0 0 0 0 (0) T3,P0
2NF 25 μg/plate 1583 1648 1588 1606 (36) N

B. WITH METABOLIC ACTIVATION (Aroclor ® -induced rat liver S9) 0 18 21 30 23 (6) T0,P0 5 21 25 37 28 (8) T0,P0 10 29 27 29 28 (1) T0,P0 50 26 19 24 23 (4) T0,P0 100 31 25 28 28 (3) T0,P0 500 20 24 28 24 (4) T0,P0 1000 18 9 16 14 (5) T1,P0 2500 18 9 12 13 (5) T2,P0 5000 9 13 6 9 (4) T2,P0
2AA 2 μg/plate 716 707 574 666 (80) NAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 27 - TABLE 8
MUTAGENIC ACTIVITY IN Salmonella typhimurium TA100 FOR #903

Concentration Revertants (μg/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations

A. WITHOUT METABOLIC ACTIVATION 0 122 147 157 142 (18) T0,P0 5 125 122 153 133 (17) T0,P0 10 121 119 147 129 (16) T0,P0 50 122 118 136 125 (9) T0,P0 100 118 126 160 135 (22) T0,P0 500 113 107 112 111 (3) T0,P0 1000 108 103 114 108 (6) T0,P0 2500 96 71 68 78 (15) T1,P0 5000 71 70 69 70 (1) T2,P0
NAAZ 2 μg/plate 2007 2116 1930 2018 (93) N

B. WITH METABOLIC ACTIVATION (Aroclor ® -induced rat liver S9) 0 126 147 128 134 (12) T0,P0 5 141 132 123 132 (9) T0,P0 10 134 146 144 141 (6) T0,P0 50 128 127 145 133 (10) T0,P0 100 140 145 112 132 (18) T0,P0 500 132 114 124 123 (9) T0,P0 1000 123 105 127 118 (12) T0,P0 2500 107 106 104 106 (2) T1,P0 5000 100 82 80 87 (11) T2,P0
2AA 1 μg/plate 1327 1271 1357 1318 (44) NAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 28 - TABLE 9
MUTAGENIC ACTIVITY IN Salmonella typhimurium TA1535 FOR #903

Concentration Revertants (μg/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations

A. WITHOUT METABOLIC ACTIVATION 0 19 12 13 15 (4) T0,P0 5 12 15 15 14 (2) T0,P0 10 13 14 12 13 (1) T0,P0 50 16 15 12 14 (2) T0,P0 100 9 11 12 11 (2) T0,P0 500 14 14 16 15 (1) T0,P0 1000 15 20 10 15 (5) T0,P0 2500 11 11 13 12 (1) T1,P0 5000 11 11 5 9 (3) T2,P0
NAAZ 2 μg/plate 2193 1791 2042 2009 (203) N

B. WITH METABOLIC ACTIVATION (Aroclor ® -induced rat liver S9) 0 14 15 9 13 (3) T0,P0 5 12 12 14 13 (1) T0,P0 10 15 17 20 17 (3) T0,P0 50 10 7 14 10 (4) T0,P0 100 17 14 11 14 (3) T0,P0 500 8 17 8 11 (5) T0,P0 1000 11 15 6 11 (5) T0,P0 2500 10 9 4 8 (3) T1,P0 5000 7 4 3 5 (2) T2,P0
2AA 2 μg/plate 253 245 248 249 (4) NAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 29 - TABLE 10
MUTAGENIC ACTIVITY IN Escherichia coli WP2 uvrA (PKM101) FOR #903

Concentration Revertants (μg/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations

A. WITHOUT METABOLIC ACTIVATION 0 190 164 177 177 (13) T0,P0 5 152 160 194 169 (22) T0,P0 10 172 174 152 166 (12) T0,P0 50 182 166 167 172 (9) T0,P0 100 190 173 164 176 (13) T0,P0 500 174 183 166 174 (9) T0,P0 1000 165 174 159 166 (8) T0,P0 2500 109 93 74 92 (18) T1,P0 5000 91 86 106 94 (10) T1,P0
ENNG 2 μg/plate 2129 1995 1817 1980 (157) N

B. WITH METABOLIC ACTIVATION (Aroclor ® -induced rat liver S9) 0 145 148 140 144 (4) T0,P0 5 186 138 157 160 (24) T0,P0 10 169 148 154 157 (11) T0,P0 50 168 137 114 140 (27) T0,P0 100 166 175 143 161 (17) T0,P0 500 177 122 133 144 (29) T0,P0 1000 153 120 138 137 (17) T0,P0 2500 127 119 110 119 (9) T1,P0 5000 115 103 106 108 (6) T1,P0
2AA 25 μg/plate 1633 1611 1750 1665 (75) NAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 30 - TABLE 11
MUTAGENIC ACTIVITY IN Salmonella typhimurium TA97A FOR #1116

Concentration Revertants (μg/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations

A. WITHOUT METABOLIC ACTIVATION 0 95 104 111 103 (8) T0,P0 5 96 84 81 87 (8) T1,P0 10 74 66 67 69 (4) T2,P0 50 74 66 73 71 (4) T2,P0 100 66 70 65 67 (3) T2,P0 500 24 19 33 25 (7) T3,P0 1000 27 19 27 24 (5) T3,P0 2500 35 24 27 29 (6) T3,P0 5000 0 0 0 0 (0) T3,P0
ICR 191 2 μg/plate 1422 1317 1533 1424 (108) N

B. WITH METABOLIC ACTIVATION (Aroclor ® -induced rat liver S9) 0 161 133 151 148 (14) T0,P0 5 156 121 144 140 (18) T0,P0 10 148 133 126 136 (11) T0,P0 50 150 118 124 131 (17) T1,P0 100 104 139 131 125 (18) T1,P0 500 106 77 94 92 (15) T1,P0 1000 71 55 66 64 (8) T3,P0 2500 35 14 0 16 (18) T3,P0 5000 0 0 0 0 (0) T3,P0
DMBA 20 μg/plate 1697 1996 1941 1878 (159) NAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 31 - TABLE 12
MUTAGENIC ACTIVITY IN Salmonella typhimurium TA98 FOR #1116

Concentration Revertants (μg/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations

A. WITHOUT METABOLIC ACTIVATION 0 23 26 21 23 (3) T0,P0 5 15 24 26 22 (6) T0,P0 10 23 14 19 19 (5) T0,P0 50 25 19 26 23 (4) T0,P0 100 24 23 26 24 (2) T0,P0 500 16 21 13 17 (4) T1,P0 1000 8 17 13 13 (5) T1,P0 2500 9 14 11 11 (3) T3,P0 5000 0 0 0 0 (0) T3,P0
2NF 25 μg/plate 1866 1652 1536 1685 (167) N

B. WITH METABOLIC ACTIVATION (Aroclor ® -induced rat liver S9) 0 27 26 25 26 (1) T0,P0 5 23 26 27 25 (2) T0,P0 10 31 21 24 25 (5) T0,P0 50 26 29 28 28 (2) T0,P0 100 33 26 23 27 (5) T0,P0 500 15 25 19 20 (5) T1,P0 1000 20 15 12 16 (4) T1,P0 2500 14 11 17 14 (3) T3,P0 5000 7 9 4 7 (3) T3,P0
2AA 2 μg/plate 290 272 369 310 (52) NAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 32 - TABLE 13
MUTAGENIC ACTIVITY IN Salmonella typhimurium TA100 FOR #1116

Concentration Revertants (μg/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations

A. WITHOUT METABOLIC ACTIVATION 0 110 109 124 114 (8) T0,P0 5 96 111 112 106 (9) T0,P0 10 116 116 133 122 (10) T0,P0 50 114 115 129 119 (8) T0,P0 100 140 139 149 143 (6) T0,P0 500 104 92 85 94 (10) T1,P0 1000 99 81 90 90 (9) T1,P0 2500 49 66 48 54 (10) T2,P0 5000 55 64 56 58 (5) T3,P0
NAAZ 2 μg/plate 1593 1846 1604 1681 (143) N

B. WITH METABOLIC ACTIVATION (Aroclor ® -induced rat liver S9) 0 128 118 141 129 (12) T0,P0 5 134 124 142 133 (9) T0,P0 10 142 130 160 144 (15) T0,P0 50 165 154 155 158 (6) T0,P0 100 129 132 137 133 (4) T0,P0 500 85 98 114 99 (15) T1,P0 1000 93 104 109 102 (8) T1,P0 2500 103 61 83 82 (21) T3,PO 5000 0 0 0 0 (0) T3,PO
2AA 1 μg/plate 1417 1513 1281 1404 (117) NAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 33 - TABLE 14
MUTAGENIC ACTIVITY IN Salmonella typhimurium TA1535 FOR #1116

Concentration Revertants (μg/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations

A. WITHOUT METABOLIC ACTIVATION 0 14 19 16 16 (3) T0,P0 5 14 15 16 15 (1) T0,P0 10 14 19 16 16 (3) T0,P0 50 16 15 16 16 (1) T0,P0 100 13 16 18 16 (3) T0,P0 500 9 12 6 9 (3) T1,P0 1000 8 6 7 7 (1) T2,P0 2500 0 0 0 0 (0) T3,P0 5000 0 0 0 0 (0) T3,P0
NAAZ 2 μg/plate 1660 1653 1734 1682 (45) N

B. WITH METABOLIC ACTIVATION (Aroclor ® -induced rat liver S9) 0 15 14 15 15 (1) T0,P0 5 14 16 15 15 (1) T0,P0 10 23 17 17 19 (3) T0,P0 50 16 15 16 16 (1) T0,P0 100 15 16 15 15 (1) T0,P0 500 6 11 7 8 (3) T1,P0 1000 13 9 12 11 (2) T1,P0 2500 12 17 11 13 (3) T1,P0 5000 7 6 2 5 (3) T3,P0
2AA 2 μg/plate 491 352 385 409 (73) NAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 34 - TABLE 15
MUTAGENIC ACTIVITY IN Escherichia coli WP2 uvrA (PKM101) FOR #1116

Concentration Revertants (μg/plate) Plate 1 Plate 2 Plate 3 Mean (S.D.) Observations

A. WITHOUT METABOLIC ACTIVATION 0 139 122 126 129 (9) T0,P0 5 143 105 134 127 (20) T0,P0 10 127 143 112 127 (16) T0,P0 50 146 166 135 149 (16) T0,P0 100 124 145 125 131 (12) T0,P0 500 149 134 119 134 (15) T0,P0 1000 104 113 98 105 (8) T1,P0 2500 66 80 71 72 (7) T1,P0 5000 55 63 70 63 (8) T2,P0
ENNG 2 μg/plate 1544 1821 1808 1724 (156) N

B. WITH METABOLIC ACTIVATION (Aroclor ® -induced rat liver S9) 0 123 153 154 143 (18) T0,P0 5 166 131 142 146 (18) T0,P0 10 175 177 200 184 (14) T0,P0 50 186 181 176 181 (5) T0,P0 100 187 201 213 200 (13) T0,P0 500 184 174 196 185 (11) T0,P0 1000 199 158 194 184 (22) T0,P0 2500 95 112 128 112 (17) T1,P0 5000 93 95 109 99 (9) T3,P0
2AA 25 μg/plate 720 844 954 839 (117) NAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 35 - APPENDIX - HISTORICAL CONTROL DATAAAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella typhimurium and Escherichia coli DuPont-3908

- 36 - HISTORICAL CONTROL DATA a

Tester Strain Exogenous Metabolic Range Control [Positive Control b ] Activation System Mean (S.D.) Minimum - Maximum
S. typhimurium TA100 Negative Absent 122 (29) 54 - 218 Negative Present 128 (29) 65 - 253 Positive [NAAZ-2] Absent 872 (274) 339 - 2604 Positive [2AA-1] Present 1187 (476) 94 - 2682 S. typhimurium TA 1535 Negative Absent 15 (6) 4 - 46 Negative Present 14 (6) 4 - 39 Positive [NAAZ-2] Absent 618 (199) 127 - 1270 Positive [2AA-2] Present 362 (136) 44 - 1323 S. typhimurium TA97a Negative Absent 103 (18) 59 - 164 Negative Present 124 (26) 67 - 196 Positive [ICR 191-2] Absent 1822 (662) 476 - 3359 Positive [DMBA-20] Present 1534 (559) 563 - 2773 Positive [2AA-1] Present 960 (348) 308 - 2281 S. typhimurium TA98 Negative Absent 22 (7) 7 - 47 Negative Present 26 (7) 11 - 53 Positive [2NF-25] Absent 1403 (372) 567 - 2774 Positive [2AA-2] Present 1552 (598) 250 - 3114 E. coli WP2 uvrA (pKM101 ) Negative Absent 148 (29) 82 - 221 Negative Present 167 (29) 93 - 255 Positive [MMS-1000] Absent 1656 (449) 208 - 2453 Positive [ENNG-2] Absent 1627 (317) 1049 - 2253 Positive [2AA-25] Present 1577 (450) 485 - 2484 Positive [2AA-250] Present 1682 (372) 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 of 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-1,2- benzanthracene), ENNG ( N -ethyl- N -nitro- N -nitrosoguanidine). The number following abbreviation is the microgram (μg) amount per plate or vial used for the positive control.