Page 1 of 48 

### Study Title

Bacterial Reverse Mu tation Test

T EST 

### G UIDELINES 

### : U.S. EPA Health Effects Test Guidelines, OPPTS 870.5100 (1998)

OECD Guidelines for the Te sting of Chemicals, No. 471 (1998)

EC Commission Directive 2000/32/EC Annex 4D–B.13/14 No. L 136

A UTHOR 

### :

S TUDY 

### C OMPLETED ON 

### : November 9, 2006

P ERFORMING 

### L ABORATORY 

### :

L ABORATORY 

### P ROJECT 

### ID:

W ORK 

### R EQUEST 

### N UMBER 

### :

S ERVICE 

### C ODE 

### N UMBER 

### :

S PONSOR 

### :Bacter ial Reverse Mutation Test

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### GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

This study was conducted in compliance with U.S. EPA TSCA (40 CFR part 792) Good Laboratory Practice Standards, which are comp atible with current OECD and MAFF (Japan) Good Laboratory Practices, except for the items documented below. None of the items listed impact the validity of the study.

1. The test substance was characterized by the spon sor prior to the initiation of this study. Although the characterization wa s not performed under Good Labor atory Practice Standards, the accuracy of the data is considered sufficient for the purposes of this study.

2. Neither the vehicle nor the positiv e controls were characterized by the testing facility or the sponsor. However, the vehicle (house-line air) was filtered before use and positive controls were purchased from a reputable vendor and show ed results consistent with historical control data.

3. The concentrations of the posit ive controls were not confir med analytically; however, the solutions were prepared by trained personnel to ensure the accuracy of the concentrations.Bacter ial Reverse Mutation Test

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### QUALITY ASSURANCE STATEMENT

Work Request Number: Study Code Number:

Phase Audited Audit Dates Date Reported to Study Director Date Reported to Management

Protocol: 8-9-Aug-2006 9-Aug-2006 10-Aug-2006

Conduct: 11-Aug-2006 11-Aug-2006 11-Aug-2006

Report/Records: 26-27, 30-31-OCT-2006 1-2-NOV-2006 7-NOV-2006 8-NOV-2006Bacter ial Reverse Mutation Test

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### CERTIFICATION

We, the undersigned, declare that this report prov ides an accurate eval uation of data obtained from this study.Bacter ial Reverse Mutation Test

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### TABLE OF CONTENTS

Page

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT...................................2 QUALITY ASSURANCE STATEMENT ...................................................................................3 CERTIFICATION.........................................................................................................................4 STUDY INFORMATION .............................................................................................................7 SUMMARY ....................................................................................................................................8 INTRODUCTION................................................................................................................... .....10 MATERIALS AND METHODS ................................................................................................10 A. Testing Guidelines .......................................................................................................... ...10 B. Test Substance and Controls..............................................................................................10 C. Test System................................................................................................................. .......11 D. Preparation and Stor age of Tester Strain ...........................................................................12 E. Confirmation of Tester Strain Genotype............................................................................12 F. Experimental Design and Methodology ............................................................................13 G. Analytical Methods.......................................................................................................... ..15 H. Criteria for Determination of a Valid Test.........................................................................16 I. Evaluation of Test Results .................................................................................................18 J. Data Presentation ........................................................................................................... ....18 RESULTS AND DISCUSSION ..................................................................................................18 A. Analytical Results .......................................................................................................... ....18 B. Solubility.................................................................................................................. ..........19 C. Sterility Controls.......................................................................................................... ......19 D. Toxicity-Mutation Test ...................................................................................................... 19 E. Mutagenicity Test ........................................................................................................... ...19 CONCLUSIONS ..........................................................................................................................20 RECORDS AND SAMPLE STORAGE ....................................................................................20 REFERENCES.............................................................................................................................20 TABLES......................................................................................................................... ...............21 Table 1: Summary of analytical concentrations...................................................................23 Table 2: Toxicity-mutation test in Salmonella typhimurium TA98 without S9 ..................24 Table 3: Toxicity-mutation test in Salmonella typhimurium TA100 without S9 ................25 Table 4: Toxicity-mutation test in Salmonella typhimurium TA1535 without S9 ..............26 Table 5: Toxicity-mutation test in Salmonella typhimurium TA1537 without S9 ..............27 Table 6: Toxicity-mutation test in Escherichia coli WP2uvrA without S9 .........................28 Table 7: Toxicity-mutation test in Salmonella typhimurium TA98 with S9 .......................29 Table 8: Toxicity-mutation test in Salmonella typhimurium TA100 with S9 .....................30 Table 9: Toxicity-mutation test in Salmonella typhimurium TA1535 with S9 ...................31 Table 10: Toxicity-mutation test in Salmonella typhimurium TA1537 with S9 ...................32 Table 11: Toxicity-mutation test in Escherichia coli WP2uvrA with S9...............................33 Table 12: Mutagenicity test in Sa lmonella typhimurium TA98 without S9..........................34 Table 13: Mutagenicity test in Sa lmonella typhimurium TA100 without S9........................35 Table 14: Mutagenicity test in Sa lmonella typhimurium TA1535 without S9......................36Bacter ial Reverse Mutation Test

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### Table 15: Mutagenicity test in Sa lmonella typhimurium TA1537 without S9......................37 Table 16: Mutagenicity test in Es cherichia coli WP2uvrA without S9 .................................38 Table 17: Mutagenicity test in Sa lmonella typhimurium TA98 with S9...............................39 Table 18: Mutagenicity test in Sa lmonella typhimurium TA100 with S9.............................40 Table 19: Mutagenicity test in Sa lmonella typhimurium TA1535 with S9...........................41 Table 20: Mutagenicity test in Sa lmonella typhimurium TA1537 with S9...........................42 Table 21: Mutagenicity test in Escherichia coli WP2uvrA with S9 ......................................43 Table 22: Summary of the toxicity-m utation test wit hout rat liver S9 ..................................44 Table 23: Summary of the toxicity-m utation test with rat liver S9........................................44 Table 24: Summary of the mutageni city test without rat liver S9 .........................................45 Table 25: Summary of the mutageni city test with rat liver S9 ..............................................45 APPENDICES..................................................................................................................... .........46 Appendix A: Historic al Control Data ......................................................................................47Bacter ial Reverse Mutation Test

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### STUDY INFORMATION

Substance Tested: •

•

•

•

Number:

Composition:

Purity: 100%

Physical Characteristics: Gas

Stability: The test substance appear ed to be stable under the conditions of the study; no evidence of instability was observed.
Study Initiated/Completed: July 18, 2006 / (see report cover page)

Experimental Start/Termination: July 26, 2006 / August 16, 2006Bacter ial Reverse Mutation Test

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### SUMMARY

The test substance, was a gas and was evaluated for mutagenicity in the Bacterial Reverse Mutation Test using the plate in corporation and gas testing methods. Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2 uvr A were tested in the presence and absence of an exogenous metabolic activation system (Aroclorinduced rat liver S9).

The test was performed in 2 phases. The firs t phase was the toxicity-mutation test, which established the dose range for the mutagenicity test and provided a pr eliminary mutagenicity evaluation. The second phase was the mutagenic ity test, which evaluated and confirmed the mutagenic potential of the test substance.

Dry, filtered air was chosen as the dosing vehi cle based on the compatibility with the test substance and with the target cells.

In the toxicity-mutation test, th e maximum dose evaluated was target ed at 100% for tester strains TA98, TA100, TA1535, TA1537, and WP2 uvr A in the presence and absence of S9. Additional dose levels targeted in the test were 5, 10, 30, a nd 60%. The toxicity-mutation test consisted of trial one (T-1) under the metabolic activation condition a nd trial two (T-2) without an activation system.

In the presence of S9, a 50% or greater reduc tion in cell growth was seen at the top concentrations in TA98, TA1535 and TA1537 accompanied by a dose response in TA98 and TA1535. However, the effect was not reproducible in the mutagenicity tr ial in strains TA 1535 and TA 1537. In TA98 with S9 the toxicity effect was reproducible in th e main assay. Without S9 metabolic activation, there was an elevated numb er of revertants per pl ate in test strain TA98 at low, intermediate and high concentrations and in strains TA100, TA 1535, and TA1537 at the intermediate concentrations; howev er no dose response occurred in any case nor were the effects reproducible in the mutagenicity test. Precip itation was not observed in any strain at any concentration under either test condition.

Based on the toxicity-mutation test , the maximum target dose evalua ted in the mutagenicity test (E-1) was 100% test gas for tester strains TA98, TA100, TA1535, TA1537, and WP2 uvr A in the presence and absence of S9. Additional target concentrations were 5, 10, 30, and 60% for all tester strains.

No positive mutagenic responses were observed in th e mutagenicity test at any concentration or with any tester strain in either the presence or the absence of S9. There was a dose-related decrease in cell growth and a 50% reduction in cell growth seen at the highest concentration in TA98 in the presence of S9. No precipitation was observed at any dose level with any tester strain in either the presence or the absence of S9.

Each chamber was placed under negative pressure a nd pre-heated. As the pressure was released, test substance and/or dry-filtered dilution air wa s drawn into the chamber. The vapor/vapor dose method was employed during toxicity-mutation T-1 yielding actual mean concentrations of 0,Bacter ial Reverse Mutation Test

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### 55.8, 74.0, 76.1, and 100%. The liquid/vapor dose method was used for T-2 and the mutagenicity test. The actual mean expos ure concentrations for T-2 were 0, 20.7, 41.9, 57.8, 70.8, and 92.2%. The actual mean exposure concen trations in the muta genicity test with activation and without activ ation were 0, 23.3, 46.8, 68.5, 71.3 and 88.9%, and 0, 55.1, 83.7, and 90.1%, respectively.

All criteria for a valid study were met. Unde r the conditions of this study, showed no evidence of mutagenicity in the Bacterial Reve rse Mutation Test either in the presence or absence of Aroclor-induced rat liver S9. The test substance was c oncluded to be negative in this study.Bacter ial Reverse Mutation Test

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### INTRODUCTION

The objective of this study was to evaluate the test substance, for its ability to induce reverse mutations at the histidine locus in the genome of Salmonella typhimurium (strains TA98, TA100, TA1535, and TA1537), and at th e tryptophan locus of the Escherichia coli strain WP2 uvr A. The assay was conducted with and without an exogenous S9 metabolic activation system.

MATERIALS AND METHODS

A. Testing Guidelines

This study was conducted in complian ce with the following guidelines:

• U.S. Environmental Protection Agency (EPA), Health Effects Test Guidelines, OPPTS 870.5100, Bacterial Reverse Mutation Test (1998).

• Ninth Addendum to the OECD (Organisation for Economic Cooperation and Development) Guidelines for the Testing of Chemicals, Bacterial Reverse Mutation Test, No. 471 (February, 1998).

• European Commission Directive 2000/ 32/EC of May 19, 2000, Annex 4D-B13/14. Mutagenicity - Reverse Muta tion Test Bacteria. No. L 136.

B. Test Substance and Controls

1. Identification

The test substance, was a gas. The test batch used for this study was assigned identification number Additional informati on regarding the test s ubstance is found on the study information page of this report.

2. Characterization

The sponsor was responsible for the determina tion and documentation of the analytical purity and composition of the test substance. Th e characterization was not performed under Good Laboratory Practice Standards. Th e stability and strength of the test substance were determined under Good Laboratory Practice Standards over a pproximately 48 hr at by visual examination and by analytical procedures.

3. Sample Preparation, and Anal ytical Verification of Test Substance Concentrations

Based on information received from the sponsor, the test substance was considered 100% active ingredient. Therefore, no co rrection factor for purity was applied for The test substance was tested as a gas. Prior to a nd after a 48-72 hour incubation period, a minimum of 2 gas samples were analyzed from each exposure chamber using a gas chromatograph except forBacter ial Reverse Mutation Test

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### the 100% concentration that was determined nominally at treatment -start and confirmed analytically at the end of treatment during the first toxicity-mutation trial T-1.

4. Controls

Negative: Dry-filtered air

Positive (Moltox Inc.): benzo[a]pyrene [CAS# 50-32-8] 4-nitroquinoline N-oxide [CAS# 56-57-5] acridine mutagen ICR-191 [CAS# 17070-45-0] sodium azide [CAS# 26628-22-8] 2-aminoanthracene [CAS# 613-13-8] 2-nitrofluorene [CAS# 607-57-8]

The positive controls were dissolved in di methyl sulfoxide (DMSO, CAS# 67-68-6, 99.9% purity, EMD), except for sodium azide and ICR-191, wh ich were dissolved in sterile water. The positive controls were assumed to be stable during this test and no evidence of instability was observed.

C. Test System

The tester strains were the Salmonella typhimurium histidine auxotroph tester strains TA98, TA100, TA1535, and TA1537, and the Escherichia coli tryptophan auxotroph WP2 uvr A. (1,2,3)


### All tester strains were obtained from Moltox Inc. (Boone, NC). The specific genotypes and phenotypic characterization of th ese strains were as follows:

Additional Mutations Tester Strain HIS/Trp Mutation Repair LPS Plasmid

S. typhimurium TA98 his D3052 ∆ uvr B rfa pkM101 S. typhimurium TA100 his G46 ∆ uvr B rfa pkM101 S. typhimurium TA1535 his G46 ∆ uvr B rfa -- S. typhimurium TA1537 his C3076 ∆ uvr B rfa -- Escherichia coli WP2 uvr A trp E ∆ uvr A - --

In addition to a mutation in either the histidine or tryptophan operons, the tester strains contain additional mutations that enhance their sensitivit y to some mutagens. A mutation of either the

uvr A or uvr B gene results in a deficient DNA ex cision repair system. Since the uvr B deletion extends through the bio gene, the Salmonella typhimurium tester strains also require the vitamin biotin for growth.

The Salmonella typhimurium tester strains also contain the rfa wall mutation which results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide (LPS) barrier that forms the surface of the bacterial cell wall. The resulting cell wall deficiency increases permeability to certain classes of chemicals such as those containing large ring systems that would otherwise be exclude d by a normal intact cell wall.Bacter ial Reverse Mutation Test

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### Tester strains TA98 and TA100 also contain th e pKM101 plasmid, which further increases the sensitivity of these strains to some mutagens.

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independent (prototrophy) by frameshift mutagens. Test er strain TA100 is reverted by both frame shift and base substitution mutagens. Tester strains TA1535 and WP2 uvr A are reverted from auxotrophy to protot rophy by base substitution mutagens.

D. Preparation and Storage of Tester Strain

Frozen permanent stocks of all tester strains were prepared by growing fresh overnight cultures with the addition of 0.09 mL DMSO per milliliter of cultu re. Aliquots were frozen in dry ice and stored at ≤ -70 ° C.

Master plates were prepared by streaking each tester strain fro m a frozen permanent stock onto either nutrient agar plates or minimal glucose agar plates. The minimal glucose agar plates were supplemented with either his tidine and biotin or tryptophan, and for strains containing the pKM101 plasmid, ampicillin. Tester stra in master plates were stored at 5 ± 3 ° C.

Overnight cultures for use in the study were inoc ulated from the appropriate master plates. Cultures were placed in a shaker /incubator for overnight at 150 ± 50 rpm and 37 ± 2 ° C. To ensure that appropriate numbers of bacteria ar e plated, the length of incubation was determined by spectrophotometric monito ring of culture density.

E. Confirmation of Tester Strain Genotype

Tester strain cultures were checked for th e following genetic markers on the day of the preparation of master plates.

The histidine requirement was tested by comparing the growth of each Salmonella tester strainon a histidine/biotin-supplemented minimum glucose agar plate with their growth on a biotinonly minimum glucose agar plate.

The tryptophan requirement was tested by comparing the growth of WP2 uvr A strain on a tryptophan-supplemented minimum gl ucose agar plate with their growth on a minimum glucose agar plate.

For the Salmonella tester strains the presence of the rfa wall mutation was confirmed by demonstration of the sensitivity of the cultures to crystal violet.

The presence of uvr A and uvr B mutation was demonstrated by th eir sensitivity to ultraviolet light of the tester strains.

The presence of the pKM101 plasmid was confir med for cultures of tester strains TA98 and TA100 by demonstration of resistance to ampicillin.Bacter ial Reverse Mutation Test

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### F. Experimental Design and Methodology

1. Exposure Method

The test substance was a gas; therefore, a gas exposure method was employed. Gas exposure chambers were placed under a vacuum. A reservoir containing a prescribed amount of test gas was connected to the chamber in such a manner that the test gas could be drawn into the chamber as the vacuum was released.

2. Exogenous Metabolic Activation and Sham Mix

Liver homogenate (S9, average protein concentration: 36.1 ± 0 mg/mL) prepared from male Sprague-Dawley rats induced with Aroclor 125 4 was purchased commercially (Moltox Inc., Boone, North Carolina).

The S9 was thawed and the 10% S9 mix prepared immediately prior to its use. The S9 mix was held on ice at all times. The S9 mix contai ned proportionate volumes of the following components:

HPLC-grade water 2.4 mL 0.825 M KCl/0.2 M MgCl 2 

### 0.4 mL 0.2 M phosphate buffer, pH 7.4 5.0 mL 0.25 M glucose-6-phosphate 0.2 mL 0.04 M NADP 1.0 mL S9 1.0 mL Total Volume 10 mL

The sham mix was 100 mM phosphate buffer at pH 7.4.

3. Controls

a. Negative Controls

The negative control was dry-filtered air. Negative controls were plated for each tester strain with and without S9 activation.Bacter ial Reverse Mutation Test

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### b. Positive Controls

Tester Strain S9 Mix Positive Control μg per plate

TA98 + benzo[a]pyrene 2.5 TA98 - 2-nitrofluorene 1.0 TA100 + 2-aminoanthracene 2.5 TA100 - sodium azide 2.0 TA1535 + 2-aminoanthracene 2.5 TA1535 - sodium azide 2.0 TA1537 + 2-aminoanthracene 2.5 TA1537 - Acridine mutagen ICR-191 2.0 WP2 uvr A + 2-aminoanthracene 25.0 WP2 uvr A - 4-nitroquinoline-N-oxide 1.0

c. Sterility Controls

Sterility control plates were placed in gas exposur e chambers targeted to receive 100% of the test substance. Other sterility control plates included 0.5 mL sham mix and 0.5 mL S-9 mix.

4. Plate Identification, Frequency, and Route of Administration

Each plate was labeled with the work request number, service code, number, treatment date, and plate number. The plate number corres ponds to a plate code wh ich signifies a positive control, a negative control or a sample plate, a nd tester strain, the pres ence or absence of S9 metabolic activation, dose level, and replicate.

In the non-activated assays, 0.5 mL of sham mix, or sham plus positive controls were added to pre-heated (45–48 ° C) glass culture tubes containing 2 mL of selective top agar, followed by 100 μL of tester strain.

In the S9-activated assays, pos itive controls were added to the appropriate pre-heated (45–48 ° C) glass culture tubes containing 2 mL of selective top agar, followed by 100 μL of tester strain and 0.5 mL of S9 mix.

All mixtures were vortexed and overlaid onto th e surface of minimum glucose agar plates. Using sterile technique, the solidified agar plates were segregated by conc entrations onto sterile stainless steel racks. The stainless steel racks co ntaining the plates are placed in sterile glass gas exposure chambers (estimated 6 liter-sized chambers with Teflon ® 

### stopcocks, Viton ® 

### O-ring gaskets and septums to facilitate sampling) whic h are then sealed. Following the gas exposure, chambers containing the plated bacteria were in cubated for 48-72 hours (the treatment period) at approximately 37oC. Each chamber atmosphere during trials T-1 and T- 2 was circulated while sampling and during the treatment pe riod using a stir bar and stir plate. Heat generated by the stirplates created less than optimal temperatur e control and was theref ore discontinued during mutagenicity trial E-1. To accommodate potential concentration gradation within chambers a representative culture from each strain was pla ced at the bottom, middle and top areas of each chamber. Plates were stored at approximately 4oC before being counted. All toxicity-mutationBacter ial Reverse Mutation Test

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### test preparations of negative (veh icle) controls, test s ubstance, and positive controls were plated in triplicate. All mutagenicity test preparations of negative (vehicle) controls, test substance, and positive controls were plated in triplicate.

5. Dose Level Determination

The targeted dose levels for the toxicity-mutation test were 5, 10, 30, 60, and 100% test gas. The targeted dose levels for the mutagenicity test were 5, 10, 30, 60, and 100%.

6. Toxicity-Mutation Test, Mutageni city Test, and Test Method

The test substance was evaluated along with nega tive and positive controls using tester strains TA98, TA100, TA1535, TA1537, and WP2 uvr A with and without S9 activation. The plate incorporation method was employed. Dose levels fo r the mutagenicity test were chosen from the toxicity-mutation test results and were listed in the study records and the final report. The toxicity-mutation and mutation tests used triplicate plates for each dose level.

7. Scoring

Revertant colonies were counted with either an automated colony counter (Sorcerer, Perceptive Instruments Ltd., Suffold, United Kingdom), or by hand. The appearance of the bacterial background lawn was assessed for test substance toxicity and precipitatio n. Precipitation was assessed by visual examination.

G. Analytical Methods

1. Chromatographic Conditions

a. Toxicity-Mutation Trial T-1

Instrument: Agilent Technologies Gas Chromatograph Model 6890 Column: Agilent Technologies 19091J-413, HP-5 5% Phenyl Methyl Siloxane, 30m x 320μm (Serial No. 3322681H) Column Flow Rate: 13.2 mL/min Injection Volume: 20 μL Oven Temperature: 40°C Detector: Flame Ionization Curve Type: LinearBacter ial Reverse Mutation Test

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### b. Toxicity-Mutation Trial T-2 and Mutagenicity Trial E-1

Instrument: Agilent Technologies Gas Chromatograph Model 6890 Column: GasPro GSC, 30m x 32 0μm (Serial No. GSC 9602-10) Column Flow Rate: 3.2 mL/min Injection Volume: 10μL Oven Temperature: 190°C Detector: Thermal Conductivity Curve Type: Linear

2. Dose Administration and Chamber Concentrations

Dose administration was accomplished by placing each chamber under negative pressure and drawing a prescribed amount of te st substance and/or dry-filtered air from a gas-tight reservoir into each chamber as the pressure was released. The gas-tight reservoir was either a gas sample bag or a syringe. Using a gas tight syringe 20 μL chamber samples were taken before and after trial T-1 and analyzed using a flame ionization de tector. Using a gastight syringe 10μL chamber samples were taken before and after trials T-2 and E-1 and analyzed using a thermal conductivity detector. Atmospheric concentrations were determined using a linear regression standard curve prepared the same day except at the end of tr ial E-1 treatment when no standard curve was prepared due to limited test s ubstance availability. There were no changes in GC conditions and the sample volumes were consistent with the star t of treatment; therefore, the standard curve prepared the day trial E-1 treatment started was applied to the samples drawn at the end of
treatment.

Actual chamber concentrations equaled the av erage of the samples drawn before and after treatment. Standard concentrations ranged from 2.5 to 100% (Trial T-1) or 2.0 to 100% (Trials T-2 and E-1) and were prepared using ga s sample bags and gastight syringes.

H. Criteria for Determination of a Valid Test

1. Tester Strain Integrity

To demonstrate the presence of the rfa mutation, all S. typhimurium tester strain cultures must exhibit sensitivity to crystal violet. To demonstrate the presence of the uvr A and uvr B mutations, all tester strains cultures must exhibit sensitivity to ultraviole t light. To demonstrate the presence of the pKM101 plasmid, tester st rain cultures of TA98 and TA100 must exhibit resistance to ampicillin.

2. Tester Strain Culture Density

To ensure that appropriate numbers of bacteria ar e plated, all tester strain culture densities must be equal to or greater than 0.3 × 10 9 

### cells per milliliter.Bacter ial Reverse Mutation Test

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### 3. Negative Control Values

The tester strain cultures must exhibit a characteristic mean numb er of spontaneous revertants per plate when plated along with the negative (vehicle ) control under selectiv e conditions. The acceptable ranges for the mean values of negative controls are as follows:

Tester Strain Negative Control Range

TA98 8-60 TA100 60-240 TA1535 4-45 TA1537 2-25 WP2 uvr A 5-60

4. Positive Control Values

Each mean positive control value must exhibit at least a 3.0-fold increase over the respective mean negative (vehicle) control value for each tester strain.

5. Toxicity

A minimum of three non-toxic scor able dose levels are required to validate the study. A dose level is considered toxic if it causes:

• A >50% reduction in the mean number of reve rtants per plate relative to the mean negative control value and exhibits a dose-depe ndent drop in the revertant count, or

• A reduction in the background lawn.

In the event that less than 3 non- toxic dose levels are achieved, th e affected portion of the test will be repeated with an appropriate change in dose levels.

6. Data Point Rejection

• A single data point may have been rejected if contamination or excessive toxicity was seen on a treatment plate. A single data point may also have been rejected if excessive precipitate on the plate prevented accurate colony counting.

• A negative control data point may have been rejected if it fell outside the acceptable spontaneous mutation range.Bacter ial Reverse Mutation Test

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### I. Evaluation of Test Results

Criteria for a positive response:

1. Strains TA1535 and TA1537

Data will be judged positive if the increase in mean revertants at the highest numerical dose response is ≥ 3.0-fold the mean concurrent negative c ontrol value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test substance.

2. Strains TA98, TA100 and WP2 uvr A

Data sets will be judged positive if the increase in mean revertants at the highest numerical dose response is ≥ 2.0-fold the mean concurrent negative c ontrol value (vehicle control). This increase in the mean number of revertants pe r plate must be accompanied by a dose response to increasing concentrations of the test substance.

J. Data Presentation

For each tester strain, the mean of the number of revertants and the standard deviations were calculated.

RESULTS AND DISCUSSION

A. Analytical Results

(Table 1)

Analytical results include the average chamber concentration (%) and the standard deviation (±) of all samples from each chamber. Most chamber concentrations were actually >20% higher than the target concentrations. In transitioning from the vapor/v apor dose administration method to the liquid/vapor method and to ensure that th e 100% concentration was ac hieved, 2 rather than 1 chamber was targeted for 100% of the test su bstance during trial T-1. Also in the same instance, the 100% concentration was determined based on nominal calcu lations at treatment start and on analyzed chamber samples at th e end of the treatment. Heavy condensate accumulated in the chambers in 2 trials.

Each chamber was placed under negative pressure a nd pre-heated. As the pressure was released, test substance and/or dry-filtered dilution air wa s drawn into the chamber. While administering the 100% dose during the first tr ial (T-1) with metabolic activa tion there was a change in the dose administration process from the vapor/vap or phase method to th e liquid/vapor phase method. Concurrently, there was de letion of the carrier gas flow; th erefore, the toxicity-mutation trial without activation was conduc ted as a second trial (T-2).

The actual mean exposure concentrations from th e toxicity-mutation test T-1 with activation were: 0, 55.8, 74.0, 76.1 and 100% (2 chambers). Th e actual mean exposure concentrations fromBacter ial Reverse Mutation Test

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### the toxicity-mutation test T-2 without metabolic activation were: 0, 20.7, 41.9, 57.8, 70.8, and 92.2%. The actual mean exposure concentrations fr om the mutagenicity te st E-1 with activation and without activation were: 0, 23.3, 46.8, 68.5, 71.3 and 88.9%, and 0, 55.1, 83.7, and 90.1%, respectively. Due to limited test substance availa bility lower concentratio ns were not evaluated in Trial E-1.

B. Solubility

Dry-filtered air was chosen as the dosing vehicle based on the compatibility of the test substance and the target cells. The highest target concen tration that was tested in the study was 100%.

C. Sterility Controls

No contaminant colonies were observed on the sterility plates for the most concentrated test substance dilution (100%) and the S9 and sham mixes.

D. Toxicity-Mutation Test

(Tables 2-11 and 22-23)

In the toxicity-mutation test, th e maximum dose evaluated was target ed at 100% for tester strains TA98, TA100, TA1535, TA1537, and WP2 uvr A in the presence and absence of S9. Additional dose levels targeted in the test were 5, 10, 30, a nd 60%. The toxicity-mutation test consisted of a trial (T-1) under the metabolic activation conditio n and a second trial (T-2) without an activation system.

In the presence of S9, a 50% or greater reduc tion in cell growth was seen at the top concentrations in TA98, TA1535 and TA1537 accompanied by a dose response in TA98 and TA1535. However, the effect was not reproducibl e in the mutagenicity trial in strains TA1535, and TA1537. In TA98 with S9 the toxicity effect was reproducible in the main assay. Without S9 metabolic activation, there was an elevated numb er of revertants per pl ate in test strain TA98 at low, intermediate and high concentrations and in strains TA100, TA1535 and TA1537 at the intermediate concentrations; howev er no dose response occurred in any case nor were the effects reproducible in the mutagenicity test. Precip itation was not observed in any strain at any concentration under either test condition.

E. Mutagenicity Test

(Tables 12-21 and 24-25)

Based on the toxicity-mutation test , the maximum target dose evalua ted in the mutagenicity test (E-1) was 100% test gas for tester strains TA98, TA100, TA1535, TA1537, and WP2 uvr A in the presence and absence of S9. Additional target concentrations were 5, 10, 30, and 60% for all tester strains; however due to limited test subs tance availability only th e top 3 concentrations were used in the E-1 trail without metabolic activation. The mutagenicity test employed the same exposure method as the t oxicity-mutation test T-2.Bacter ial Reverse Mutation Test

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### No positive mutagenic responses were observed at a ny concentration or with any tester strain in either the presence or the absence of S9. There was a dose-related decrease in cell growth and a 50% reduction in cell growth seen at the highest concentration in TA98 in the presence of S9. No precipitation was observed at an y dose level with any te ster strain in either the presence or the absence of S9.

CONCLUSIONS

All criteria for a valid study were met. Unde r the conditions of this study, showed no evidence of mutagenicity in the Bacterial Reve rse Mutation Test either in the presence or absence of Aroclor-induced rat liver S9. The test substance was c oncluded to be negative in this study.

RECORDS AND SAMPLE STORAGE

Specimens (if applicable), raw data, the protocol , amendments (if any), and the final report will be retained at

REFERENCES

1. Ames, B.N., McCann, J., and Yamasaki, E. (197 5). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian- microsome mutagenicity test. Mutation Research 31, 347-364.

2. Maron, D.M., and Ames, B. (1983). Revised Met hods for the Salmonella Mutagenicity Test. Mutation Research 113, 173-215.

3. Wilcox, P., Naidoo, A., Wedd, D.J., and Gatehouse , D.G. (1990). Comparison of Salmonella typhimurium TA102 with Escherichia coli WP 2 tester strains. Mutagenesis 5, 285-291.Bacter ial Reverse Mutation Test

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### TABLESBacter ial Reverse Mutation Test

- 22 - 

### TABLES

EXPLANATORY NOTES

ABBREVIATIONS :

C.V. coefficient of variation SD standard deviation

Bacterial Background Lawn Evaluation Code – Evidence for test substance toxicity to the bacteria will be documented by recording the appearance of the background lawn using the following code:

T0 Normal , background microcolony lawn appears normal. T1 Slightly reduced , background microcolony lawn is noticeably thinner. T2 Moderately reduced , background lawn is markedly thinner resulting in an increase in the size of microcolonies compared to the vehicle control plate(s). T3 Severely reduced , background lawn is distinguished by an extreme thinning resulting in an increase in the size of the microcolonies co mpared to the vehicle control plate(s). Microcolonies may be seen readily by the unaid ed eye and are greatly enlarged relative to controls. T4 Absent , plate(s) are distinguished by a complete l ack of any microcolony lawn over a majority of the area of the plate(s).

Test Substance Precipitation Code – Formation of a precipitate by the test substance will be documented using the following code:

P0 No precipitate, no precipitate observed. P1 Microscopic precipitate , precipitate present which does not interfere with background lawn evaluation or automated colony counting. P2 Non-interfering precipitate, precipitate present that is visibl e to the naked eye that does not interfere with automated colony counting. P3 Interfering precipitate, precipitate present that requires plate to be counted by hand. P4 Heavy interfering precipitate , precipitate present that preven ts accurate colony counting and obscures the background lawn re quiring plate rejection (R).

Lost Plate Just ification Code:

L0 The loss of this test substance-treated plate does not invalidate the results since the remaining plate at this dose level and the remaining trea ted plates are also comparable to the negative control. L1 The loss of this vehicle control plate does no t invalidate the results since the remaining vehicle control plate is consistent with the historical negative control value for this condition. L2 The loss of this positive control plate does not invalidate the results since the remaining positive control plate is consistent with the historical positive control value for this condition. L3 The loss of this test substance-treated plate does not invalidate the results since the remaining plate is consistent with the remaining treated plates. L4 The loss of this test substance-treated plate does not invalidate the results since the remaining plate at this dose level is comparable to the negative control. L5 The loss of this untreated cont rol plate does not invalidate the results since the remaining plate at the dose level is comparable to the vehicle control and is consistent with the historical negative control value for this condition.Bacter ial Reverse Mutation Test

- 23 - 

### Table 1: Summary of analytical concentrations

Concentration (%) Trial/Condition Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

T-1 With S9 0 0 55.8 42.1 74.0 31.3 76.1 22.0 100 a 0 100 a 0

T-2 Without S9 0 0 20.7 2.1 41.9 1.1 57.8 4.3 70.8 4.7 92.2 0.6

E-1 With S9 0 0 23.3 1.1 46.8 7.0 68.5 0.7 71.3 5.3 88.9 4.2 Without S9 0 0 -- b

-- -- b

-- 55.1 7.0 83.7 2.9 90.1 2.5

a 2 separate chambers.
b Due to limited test substance availability lower concentrations were not tested.Bacter ial Reverse Mutation Test

- 24 - 

### Table 2: Toxicity-mutation test in Salmonella typhimurium TA98 without S9

Strain: TA98 Experiment No: T-2

Rat Liver S9: Absent Cell Titer (cells/mL): 1.32×10 9 Plating Aliquot: 100 μL Date Plated: 4-Aug-06 Actual Concentration Revertants Background (%) Plate Number Per Plate Code Mean SD

Vehicle a 4 1 T0,P0 2 2 5 1 T0,P0 6 4 T0,P0

Positive Control b 94 181 T0,P0 194 14 95 194 T0,P0 96 208 T0,P0

20.7 79 18 T0,P0 17 2

80 18 T0,P0 81 15 T0,P0

41.9 64 1 T0,P0 5 4

65 6 T0,P0 66 9 T0,P0

57.8 49 19 T0,P0 14 5

50 14 T0,P0 51 10 T0,P0

70.8 34 16 T0,P0 13 4

35 14 T0,P0 36 9 T0,P0

92.2 19 0 T0,P0 5 5

20 6 T0,P0 21 10 T0,P0

a dry, filtered air

b 1.0 μg/plate 2-nitrofloureneBacter ial Reverse Mutation Test

- 25 - 

### Table 3: Toxicity-mutation test in Salmonella typhimurium TA100 without S9

Strain: TA100 Experiment No: T-2

Rat Liver S9: Absent Cell Titer (cells/mL): 1.07×10 9 Plating Aliquot: 100 μL Date Plated: 4-Aug-06 Actual Concentration Revertants Background (%) Plate Number Per Plate Code Mean SD

Vehicle a 7 5 T0,P0 39 34 8 38 T0,P0 9 73 T0,P0

Positive Control b 97 1258 T0,P0 1232 33 98 1195 T0,P0 99 1243 T0,P0

20.7 82 106 T0,P0 88 20

83 90 T0,P0 84 67 T0,P0

41.9 67 35 T0,P0 49 12

68 58 T0,P0 69 54 T0,P0

57.8 52 77 T0,P0 66 10

53 59 T0,P0 54 61 T0,P0

70.8 37 78 T0,P0 91 17

38 110 T0,P0 39 85 T0,P0

92.2 22 49 T0,P0 53 9

23 63 T0,P0 24 47 T0,P0

a dry, filtered air

b 2.0 μg/plate sodium azideBacter ial Reverse Mutation Test

- 26 - 

### Table 4: Toxicity-mutation test in Salmonella typhimurium TA1535 without S9

Strain: TA1535 Experiment No: T-2

Rat Liver S9: Absent Cell Titer (cells/mL): 1.13×10 9 Plating Aliquot: 100 μL Date Plated: 4-Aug-06 Actual Concentration Revertants Background (%) Plate Number Per Plate Code Mean SD

Vehicle a 10 1 T0,P0 4 3 11 6 T0,P0 12 5 T0,P0

Positive Control b 100 1078 T0,P0 1059 34 101 1019 T0,P0 102 1079 T0,P0

20.7 85 10 T0,P0 9 3

86 5 T0,P0 87 11 T0,P0

41.9 70 4 T0,P0 7 5

71 13 T0,P0 72 4 T0,P0

57.8 55 6 T0,P0 6 0

56 6 T0,P0 57 6 T0,P0

70.8 40 10 T0,P0 13 5

41 18 T0,P0 42 10 T0,P0

92.2 25 8 T0,P0 6 2

26 5 T0,P0 27 6 T0,P0

a dry, filtered air

b 2.0 μg/plate sodium azideBacter ial Reverse Mutation Test

- 27 - 

### Table 5: Toxicity-mutation test in Salmonella typhimurium TA1537 without S9

Strain: TA1537 Experiment No: T-2

Rat Liver S9: Absent Cell Titer (cells/mL): 0.91×10 9 Plating Aliquot: 100 μL Date Plated: 4-Aug-06 Actual Concentration Revertants Background (%) Plate Number Per Plate Code Mean SD

Vehicle a 13 3 T0,P0 2 1 14 3 T0,P0 15 1 T0,P0

Positive Control b 103 1448 T0,P0 1369 134 104 1214 T0,P0 105 1445 T0,P0

20.7 88 5 T0,P0 6 3

89 4 T0,P0 90 10 T0,P0

41.9 73 0 T0,P0 3 3

74 4 T0,P0 75 5 T0,P0

57.8 58 6 T0,P0 6 1

59 6 T0,P0 60 5 T0,P0

70.8 43 9 T0,P0 8 4

44 4 T0,P0 45 11 T0,P0

92.2 28 1 T0,P0 5 4

29 4 T0,P0 30 9 T0,P0

a dry, filtered air

b 2.0 μg/plate ICR-191Bacter ial Reverse Mutation Test

- 28 - 

### Table 6: Toxicity-mutation test in Escherichia coli WP2 uvr A without S9

Strain: WP2 uvr A Experiment No: T-2 Rat Liver S9: Absent Cell Titer (cells/mL): 2.21×10 9 Plating Aliquot: 100 μL Date Plated: 4-Aug-06 Actual Concentration Revertants Background (%) Plate Number Per Plate Code Mean SD

Vehicle a 1 19 T0,P0 28 8 2 34 T0,P0 3 30 T0,P0

Positive Control b 91 672 T0,P0 640 60 92 571 T0,P0 93 677 T0,P0

20.7 76 48 T0,P0 39 13

77 46 T0,P0 78 24 T0,P0

41.9 61 11 T0,P0 20 9

62 20 T0,P0 63 28 T0,P0

57.8 46 27 T0,P0 28 1

47 29 T0,P0 48 28 T0,P0

70.8 31 37 T0,P0 30 8

32 22 T0,P0 33 30 T0,P0

92.2 16 29 T0,P0 27 3

17 24 T0,P0 18 28 T0,P0

a dry, filtered air

b 1.0 μg/plate 4-nitroquinoline-N-oxideBacter ial Reverse Mutation Test

- 29 - 

### Table 7: Toxicity-mutation test in Salmonella typhimurium TA98 with S9

Strain: TA98 Experiment No: T-1

Rat Liver S9: Present Cell Titer (cells/mL): 0.96×10 9 Plating Aliquot: 100 μL Date Plated: 26-Jul-06 Actual Concentration a Revertants Background (%) Plate Number Per Plate Code Mean SD

Vehicle b 4 9 T0,P0 13 5 5 19 T0,P0 6 11 T0,P0

Positive Control c 94 305 T0,P0 353 48 95 401 T0,P0 96 352 T0,P0

55.8 19 23 T0,P0 19 5

20 20 T0,P0 21 14 T0,P0

74.0 34 20 T0,P0 12 7

35 8 T0,P0 36 9 T0,P0

76.1 49 14 T0,P0 14 5

50 10 T0,P0 51 19 T0,P0

100 d 64 0 T0,P0 3 2

65 0 T0,P0 66 4 T0,P0 79 4 T0,P0 80 5 T0,P0
81 3 T0,P0

a Lower concentrations were exceeded during dose administration and were not tested.

b dry, filtered air

c 2.5 μg/plate benzo(a)pyrene

d The results of 2 chambers tested at 100% are averaged together.Bacter ial Reverse Mutation Test

- 30 - 

### Table 8: Toxicity-mutation test in Salmonella typhimurium TA100 with S9

Strain: TA100 Experiment No: T-1

Rat Liver S9: Present Cell Titer (cells/mL): 0.88×10 9 Plating Aliquot: 100 μL Date Plated: 26-Jul-06 Actual Concentration a Revertants Background (%) Plate Number Per Plate Code Mean SD

Vehicle b 7 90 T0,P0 89 3 8 85 T0,P0 9 91 T0,P0

Positive Control c 97 2812 T0,P0 2747 100 98 2631 T0,P0 99 2797 T0,P0

55.8 22 104 T0,P0 102 16

23 85 T0,P0 24 116 T0,P0

74.0 37 80 T0,P0 87 8

38 95 T0,P0 39 86 T0,P0

76.1 52 104 T0,P0 92 10

53 87 T0,P0 54 85 T0,P0

100 d 67 56 T0,P0 71 14

68 57 T0,P0 69 65 T0,P0 82 91 T0,P0 83 82 T0,P0
84 75 T0,P0

a Lower concentrations were exceeded during dose administration and were not tested.

b dry, filtered air

c 2.5 μg/plate 2-aminoanthracene

d The results of 2 chambers tested at 100% are averaged together.Bacter ial Reverse Mutation Test

- 31 - 

### Table 9: Toxicity-mutation test in Salmonella typhimurium TA1535 with S9

Strain: TA1535 Experiment No: T-1

Rat Liver S9: Present Cell Titer (cells/mL): 0.95×10 9 Plating Aliquot: 100 μL Date Plated: 26-Jul-06 Actual Concentration a Revertants Background (%) Plate Number Per Plate Code Mean SD

Vehicle b 10 5 T0,P0 6 1 11 6 T0,P0 12 6 T0,P0

Positive Control c 100 225 T0,P0 229 18 101 214 T0,P0 102 249 T0,P0

55.8 25 8 T0,P0 11 4

26 16 T0,P0 27 10 T0,P0

74.0 40 13 T0,P0 10 3

41 9 T0,P0 42 8 T0,P0

76.1 55 8 T0,P0 9 2

56 11 T0,P0 57 9 T0,P0

100 70 4 T0,P0 3 2

71 2 T0,P0 72 0 T0,P0 85 5 T0,P0 86 4 T0,P0 87 1 T0,P0

a Lower concentrations were exceeded during dose administration and were not tested.

b dry, filtered air

c 2.5 μg/plate 2-aminoanthracene

d The results of 2 chambers tested at 100% are averaged together.Bacter ial Reverse Mutation Test

- 32 - 

### Table 10: Toxicity-mutation test in Salmonella typhimurium TA1537 with S9

Strain: TA1537 Experiment No: T-1

Rat Liver S9: Present Cell Titer (cells/mL): 0.78×10 9 Plating Aliquot: 100 μL Date Plated: 26-Jul-06 Actual Concentration a Revertants Background (%) Plate Number Per Plate Code Mean SD

Vehicle b 13 1 T0,P0 3 2 14 4 T0,P0 15 5 T0,P0

Positive Control c 103 171 T0,P0 149 24 104 151 T0,P0 105 124 T0,P0

55.8 28 5 T0,P0 5 2

29 6 T0,P0 30 3 T0,P0

74.0 43 6 T0,P0 6 3

44 8 T0,P0 45 3 T0,P0

76.1 58 3 T0,P0 5 2

59 5 T0,P0 60 6 T0,P0

100 73 0 T0,P0 1 2

74 1 T0,P0 75 0 T0,P0 88 1 T0,P0 89 4 T0,P0 90 0 T0,P0

a Lower concentrations were exceeded during dose administration and were not tested.

b dry, filtered air

c 2.5 μg/plate 2-aminoanthracene

d The results of 2 chambers tested at 100% are averaged together.Bacter ial Reverse Mutation Test

- 33 - 

### Table 11: Toxicity-mutation test in Escherichia coli WP2 uvr A with S9

Strain: WP2 uvr A Experiment No: T-1 Rat Liver S9: Present Cell Titer (cells/mL): 1.59×10 9 Plating Aliquot: 100 μL Date Plated: 26-Jul-06 Actual Concentration a Revertants Background (%) Plate Number Per Plate Code Mean SD

Vehicle b 1 28 T0,P0 37 13 2 30 T0,P0 3 52 T0,P0

Positive Control c 91 504 T0,P0 489 13 92 480 T0,P0 93 483 T0,P0

55.8 16 46 T0,P0 40 9

17 30 T0,P0 18 43 T0,P0

74.0 31 39 T0,P0 37 2

32 38 T0,P0 33 35 T0,P0

76.1 46 51 T0,P0 55 4

47 59 T0,P0 48 54 T0,P0

100 61 32 T0,P0 47 12

62 54 T0,P0 63 61 T0,P0 76 57 T0,P0 77 46 T0,P0 78 34 T0,P0

a Lower concentrations were exceeded during dose administration and were not tested.

b dry, filtered air

c 25 μg/plate 2-aminoanthracene

d The results of 2 chambers tested at 100% are averaged together.Bacter ial Reverse Mutation Test

- 34 - 

### Table 12: Mutagenicity test in Salmonella typhimurium TA98 without S9

Strain: TA98 Experiment No: E-1

Rat Liver S9: Absent Cell Titer (cells/mL): 0.97×10 9 Plating Aliquot: 100 μL Date Plated: 9-Aug-06 Actual Concentration a Revertants Background % Plate Number Per Plate Code Mean SD

Vehicle b 109 18 T0,P0 18 4 110 14 T0,P0 111 22 T0,P0

Positive Control c 199 118 T0,P0 129 11 200 130 T0,P0 201 140 T0,P0

55.1 154 15 T0,P0 14 1

155 14 T0,P0 156 14 T0,P0

83.7 124 5 T0,P0 9 3

125 11 T0,P0 126 11 T0,P0

90.1 139 6 T0,P0 10 4

140 13 T0,P0 141 11 T0,P0

a Lower concentrations were not tested due to limited test substance availability.

b dry, filtered air

c 1.0 μg/plate 2-nitrofloureneBacter ial Reverse Mutation Test

- 35 - 

### Table 13: Mutagenicity test in Salmonella typhimurium TA100 without S9

Strain: TA100 Experiment No: E-1

Rat Liver S9: Absent Cell Titer (cells/mL): 0.92×10 9 Plating Aliquot: 100 μL Date Plated: 9-Aug-06 Actual Concentration a Revertants Background % Plate Number Per Plate Code Mean SD

Vehicle b 112 94 T0,P0 97 3 113 99 T0,P0 114 99 T0,P0

Positive Control c 202 652 T0,P0 634 15 203 625 T0,P0 204 626 T0,P0

55.1 157 78 T0,P0 86 9

158 85 T0,P0 159 96 T0,P0

83.7 127 82 T0,P0 85 8

128 94 T0,P0 129 78 T0,P0

90.1 142 66 T0,P0 60 12

143 68 T0,P0 144 47 T0,P0

a Lower concentrations were not tested due to limited test substance availability

b dry, filtered air

c 2.0 μg/plate sodium azideBacter ial Reverse Mutation Test

- 36 - 

### Table 14: Mutagenicity test in Salmonella typhimurium TA1535 without S9

Strain: TA1535 Experiment No: E-1

Rat Liver S9: Absent Cell Titer (cells/mL): 0.86×10 9 Plating Aliquot: 100 μL Date Plated: 9-Aug-06 Actual Concentration a Revertants Background % Plate Number Per Plate Code Mean SD

Vehicle b 115 9 T0,P0 11 2 116 11 T0,P0 117 13 T0,P0

Positive Control c 205 633 T0,P0 628 24 206 602 T0,P0 207 650 T0,P0

55.1 160 13 T0,P0 11 2

161 11 T0,P0 162 10 T0,P0

83.7 130 9 T0,P0 7 2

131 5 T0,P0 132 8 T0,P0

90.1 145 14 T0,P0 10 3

146 8 T0,P0 147 8 T0,P0

a Lower concentrations were not tested due to limited test substance availability

b dry, filtered air

c 2.0 μg/plate sodium azideBacter ial Reverse Mutation Test

- 37 - 

### Table 15: Mutagenicity test in Salmonella typhimurium TA1537 without S9

Strain: TA1537 Experiment No: E-1

Rat Liver S9: Absent Cell Titer (cells/mL): 0.69×10 9 Plating Aliquot: 100 μL Date Plated: 9-Aug-06 Actual Concentration a Revertants Background % Plate Number Per Plate Code Mean SD

Vehicle b 118 3 T0,P0 5 2 119 6 T0,P0 120 6 T0,P0

Positive Control c 208 1061 T0,P0 1188 110 209 1262 T0,P0 210 1241 T0,P0

55.1 163 4 T0,P0 4 1

164 5 T0,P0 165 4 T0,P0

83.7 133 5 T0,P0 3 2

134 4 T0,P0 135 1 T0,P0

90.1 148 4 T0,P0 3 2

149 3 T0,P0 150 1 T0,P0

a Lower concentrations were not tested due to limited test substance availability

b dry, filtered air

c 2.0 μg/plate ICR-191Bacter ial Reverse Mutation Test

- 38 - 

### Table 16: Mutagenicity test in Escherichia coli WP2 uvr A without S9

Strain: WP2 uvr A Experiment No: E-1 Rat Liver S9: Absent Cell Titer (cells/mL): 1.44×10 9 Plating Aliquot: 100 μL Date Plated: 9-Aug-06 Actual Concentration a Revertants Background % Plate Number Per Plate Code Mean SD

Vehicle b 106 46 T0,P0 40 7 107 32 T0,P0 108 43 T0,P0

Positive Control c 196 497 T0,P0 557 55 197 604 T0,P0 198 569 T0,P0

55.1 151 40 T0,P0 34 8

152 25 T0,P0 153 37 T0,P0

83.7 121 34 T0,P0 29 8

122 33 T0,P0 123 19 T0,P0

90.1 136 25 T0,P0 29 5

137 35 T0,P0 138 27 T0,P0

a Lower concentrations were not tested due to limited test substance availability

b dry, filtered air

c 1.0 μg/plate 4-nitroquinoline-N-oxideBacter ial Reverse Mutation Test

- 39 - 

### Table 17: Mutagenicity test in Salmonella typhimurium TA98 with S9

Strain: TA98 Experiment No: E-1

Rat Liver S9: Present Cell Titer (cells/mL): 0.97×10 9 Plating Aliquot: 100 μL Date Plated: 9-Aug-06 Actual Concentration Revertants Background % Plate Number Per Plate Code Mean SD

Vehicle a 4 23 T0,P0 24 4 5 20 T0,P0 6 28 T0,P0

Positive Control b 94 356 T0,P0 368 10 95 371 T0,P0 96 376 T0,P0

23.3 79 25 T0,P0 22 4

80 18 T0,P0 81 23 T0,P0

46.8 64 28 T0,P0 22 7

65 15 T0,P0 66 22 T0,P0

68.5 49 13 T0,P0 16 3

50 18 T0,P0 51 18 T0,P0

71.3 34 16 T0,P0 14 2

35 13 T0,P0 36 13 T0,P0

88.9 19 13 T0,P0 12 1

20 11 T0,P0 21 13 T0,P0

a dry, filtered air

b 2.5 μg/plate benzo(a)pyreneBacter ial Reverse Mutation Test

- 40 - 

### Table 18: Mutagenicity test in Salmonella typhimurium TA100 with S9

Strain: TA100 Experiment No: E-1

Rat Liver S9: Present Cell Titer (cells/mL): 0.92×10 9 Plating Aliquot: 100 μL Date Plated: 9-Aug-06 Actual Concentration Revertants Background % Plate Number Per Plate Code Mean SD

Vehicle a 7 20 T0,P0 76 49 8 111 T0,P0 9 96 T0,P0

Positive Control b 97 2079 T0,P0 2527 388 98 2771 T0,P0 99 2731 T0,P0

23.3 82 99 T0,P0 90 16

83 99 T0,P0 84 71 T0,P0

46.8 67 78 T0,P0 93 20

68 115 T0,P0 69 85 T0,P0

68.5 52 87 T0,P0 90 10

53 82 T0,P0 54 101 T0,P0

71.3 37 97 T0,P0 99 9

38 92 T0,P0 39 109 T0,P0

88.9 22 99 T0,P0 94 5

23 90 T0,P0 24 92 T0,P0

a dry, filtered air

b 2.5 μg/plate 2-aminoanthraceneBacter ial Reverse Mutation Test

- 41 - 

### Table 19: Mutagenicity test in Salmonella typhimurium TA1535 with S9

Strain: TA1535 Experiment No: E-1

Rat Liver S9: Present Cell Titer (cells/mL): 0.86×10 9 Plating Aliquot: 100 μL Date Plated: 9-Aug-06 Actual Concentration Revertants Background % Plate Number Per Plate Code Mean SD

Vehicle a 10 11 T0,P0 11 3 11 8 T0,P0 12 14 T0,P0

Positive Control b 100 228 T0,P0 215 14 101 201 T0,P0 102 216 T0,P0

23.3 85 22 T0,P0 14 7

86 13 T0,P0 87 8 T0,P0

46.8 70 18 T0,P0 17 7

71 24 T0,P0 72 10 T0,P0

68.5 55 22 T0,P0 11 10

56 4 T0,P0 57 6 T0,P0

71.3 40 8 T0,P0 10 3

41 13 T0,P0 42 10 T0,P0

88.9 25 9 T0,P0 10 1

26 9 T0,P0 27 11 T0,P0

a dry, filtered air

b 2.5 μg/plate 2-aminoanthraceneBacter ial Reverse Mutation Test

- 42 - 

### Table 20: Mutagenicity test in Salmonella typhimurium TA1537 with S9

Strain: TA1537 Experiment No: E-1

Rat Liver S9: Present Cell Titer (cells/mL): 0.69×10 9 Plating Aliquot: 100 μL Date Plated: 9-Aug-06 Actual Concentration Revertants Background % Plate Number Per Plate Code Mean SD

Vehicle a 13 6 T0,P0 7 2 14 5 T0,P0 15 9 T0,P0

Positive Control b 103 197 T0,P0 215 72 104 295 T0,P0 105 154 T0,P0

23.3 88 5 T0,P0 7 2

89 6 T0,P0 90 9 T0,P0

46.8 73 4 T0,P0 5 1

74 5 T0,P0 75 6 T0,P0

68.5 58 8 T0,P0 5 4

59 0 T0,P0 60 6 T0,P0

71.3 43 10 T0,P0 6 4

44 6 T0,P0 45 3 T0,P0

88.9 28 6 T0,P0 6 2

29 4 T0,P0 30 8 T0,P0

a dry, filtered air

b 2.5 μg/plate 2-aminoanthraceneBacter ial Reverse Mutation Test

- 43 - 

### Table 21: Mutagenicity test in Escherichia coli WP2 uvr A with S9

Strain: WP2 uvr A Experiment No: E-1 Rat Liver S9: Present Cell Titer (cells/mL): 1.44×10 9 Plating Aliquot: 100 μL Date Plated: 9-Aug-06 Actual Concentration Revertants Background % Plate Number Per Plate Code Mean SD

Vehicle a 1 38 T0,P0 46 7 2 52 T0,P0 3 49 T0,P0

Positive Control b 91 348 T0,P0 327 22 92 304 T0,P0 93 330 T0,P0

23.3 76 51 T0,P0 44 15

77 54 T0,P0 78 27 T0,P0

46.8 61 32 T0,P0 30 5

62 25 T0,P0 63 34 T0,P0

68.5 46 28 T0,P0 37 10

47 48 T0,P0 48 35 T0,P0

71.3 31 38 T0,P0 41 4

32 46 T0,P0 33 40 T0,P0

88.9 16 38 T0,P0 46 8

17 54 T0,P0 18 46 T0,P0

a dry, filtered air

b 25 μg/plate 2-aminoanthraceneBacterial Reverse Mutation Test- 44 -Table 22:Summary of the toxicity-mutation test without rat liver S9ActualNumber of Revertants Per PlateConcentration TA98 TA100 TA1535 TA1537 WP2uvrA(%) Mean SD Mean SD MeanSD Mean SD Mean SDvehicle 2 2 39 34 4 3 2 1 28 8positive control 194 14 1232 33 1059 34 1369 134 640 6020.7 17 2 88 20 9 3 6 3 39 1341.9 5 4 49 12 7 5 3 3 20 957.8 14 5 66 10 6 0 6 1 28 170.8 13 4 91 17 13 5 8 4 30 892.2 5 5 53 9 6 2 5 4 27 3Experiment No: T-2PlateAliquot:100μLTable 23:Summary of the toxicity-mutation test with rat liver S9ActualNumber of Revertants Per PlateConcentrationaTA98 TA100 TA1535 TA1537 WP2uvrA(%) Mean SD Mean SD MeanSD Mean SD Mean SDvehicle 13 5 89 3 6 1 3 2 37 13positive control 353 48 2747 100 229 18 149 24 489 1355.8 19 5 102 16 11 4 5 2 40 974.0 12 7 87 8 10 3 6 3 37 276.1 14 5 92 10 9 2 5 2 55 4100b3 2 71 14 3 2 1 2 47 12Experiment No: T-1Plate Aliquot: 100 μLaLower concentrations were exceeded duringdose administration and were not tested.bThe results of 2 chambers tested at 100% are averaged together.Bacterial Reverse Mutation Test- 45 -Table 24:Summary of the mutagenicitytest without rat liver S9ActualNumber of Revertants Per PlateConcentrationaTA98 TA100 TA1535 TA1537 WP2uvrA% Mean SD Mean SD MeanSD Mean SD Mean SDvehicle 18 4 97 3 11 2 5 2 40 7positive control 129 11 634 15 628 24 1188 110 557 5555.1 14 1 86 9 11 2 4 1 34 883.7 9 3 85 8 7 2 3 2 29 890.1 10 4 60 12 10 3 3 2 29 5Experiment No: E-1PlateAliquot:100μLaLower concentrations were not tested due to limited test substance availabilityTable 25:Summary of the mutagenicity test with rat liver S9ActualNumber of Revertants Per PlateConcentration TA98 TA100 TA1535 TA1537 WP2uvrA% Mean SD Mean SD MeanSD Mean SD Mean SDvehicle 24 4 76 49 11 3 7 2 46 7positive control 368 10 2527 388 215 14 215 72 327 2223.3 22 4 90 16 14 7 7 2 44 1546.8 22 7 93 20 17 7 5 1 30 568.5 16 3 90 10 11 10 5 4 37 1071.3 14 2 99 9 10 3 6 4 41 488.9 12 1 94 5 10 1 6 2 46 8Experiment No: E-1PlateAliquot:100μLBacter ial Reverse Mutation Test

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### APPENDICESBacter ial Reverse Mutation Test

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### Appendix A:

Historical Control DataBacter ial Reverse Mutation Test

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### HISTORICAL CONTROL DATA a

Tester Strain Exogenous Metabolic Range Control [Positive Control b ] Activation System Mean (SD) c Minimum - Maximum

TA98 Negative Absent 22 (8) 6 - 47 Negative Present 29 (9) 10 - 54 Positive [2NF-1] Absent 190 (81) 49 - 361 Positive [2NF-25] Absent 1403 (372) 567 - 2774 Positive [BAP-2.5] Present 359 (65) 250 - 490 Positive [2AA-2] Present 1552 (598) 250 - 3114 TA100 Negative Absent 126 (40) 54 - 253 Negative Present 131 (32) 65 - 253 Positive [SA-2] Absent 960 (219) 339 - 2604 Positive [2AA-1] Present 1187 (476) 94 - 2682 Positive [2AA-2.5] Present 2097 (418) 1525 - 3018 TA1535 Negative Absent 16 (7) 4 - 46 Negative Present 14 (5) 4 - 39 Positive [SA-2] Absent 752 (192) 127 - 1270 Positive [2AA-2.5] Present 191 (72) 96 - 384 TA1537 Negative Absent 8 (3) 3 - 16 Negative Present 11 (7) 3 - 29 Positive [ICR 191-2] Absent 1649 (399) 1188 - 2573 Positive [2AA-2.5] Present 100 (30) 48 - 170 WP2 uvr A Negative Absent 44 (11) 21 - 64 Negative Present 39 (16) 14 - 68 Positive [4NQO-1] Absent 476 (95) 303 - 656 Positive [2AA-25] Present 514 (107) 278 - 669

a Historical data for tester strains used in the reported study. Data are based on studies reported since 1996. Data include all control solvents or diluents, metabolic activ ation 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: SA (sodium azide); 2AA (2-aminoanthracene); 2NF (2-nitrofluorene); ICR 191 (ICR 191 Acridine mutagen); 4NQO (4-nitroquinoline-N-oxide); BAP (benzo[a]pyrene). The number following abbreviation is the microgram (μg) amount per plate or vial used for the positive control. c SD = standard deviation