Page 1 of 48

Study Title 

Bacterial Reverse Mutation Test 

TEST GUIDELINES: U.S. EPA Health Effects Test Guidelines, OPPTS 870.5100 

(1998) 

OECD Guidelines for the Testing of Chemicals, No. 471 

(1998) 

EC Commission Directive 2000/32/EC Annex 4D–B.13/14 

No. L 136 

AUTHOR:

STUDY COMPLETED ON: November 9, 2006 

PERFORMING LABORATORY: 

LABORATORY PROJECT ID: 

WORK REQUEST NUMBER: 

SERVICE CODE NUMBER: 

SPONSOR: 
Bacterial 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 compatible 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 sponsor prior to the initiation of this study. 

Although the characterization was not performed under Good Laboratory Practice Standards, 

the accuracy of the data is considered sufficient for the purposes of this study. 

2. Neither the vehicle nor the positive 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 showed results consistent with historical control 

data. 

3. The concentrations of the positive controls were not confirmed analytically; however, the 

solutions were prepared by trained personnel to ensure the accuracy of the concentrations. 
Bacterial 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-2006 
Bacterial Reverse Mutation Test 

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CERTIFICATION 

We, the undersigned, declare that this report provides an accurate evaluation of data obtained 

from this study. 
Bacterial 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 Storage 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 Salmonella typhimurium TA98 without S9..........................34 

Table 13: Mutagenicity test in Salmonella typhimurium TA100 without S9........................35 

Table 14: Mutagenicity test in Salmonella typhimurium TA1535 without S9......................36 
Bacterial Reverse Mutation Test 

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Table 15: Mutagenicity test in Salmonella typhimurium TA1537 without S9......................37 

Table 16: Mutagenicity test in Escherichia coli WP2uvrA without S9 .................................38 

Table 17: Mutagenicity test in Salmonella typhimurium TA98 with S9...............................39 

Table 18: Mutagenicity test in Salmonella typhimurium TA100 with S9.............................40 

Table 19: Mutagenicity test in Salmonella typhimurium TA1535 with S9...........................41 

Table 20: Mutagenicity test in Salmonella typhimurium TA1537 with S9...........................42 

Table 21: Mutagenicity test in Escherichia coli WP2uvrA with S9 ......................................43 

Table 22: Summary of the toxicity-mutation test without rat liver S9 ..................................44 

Table 23: Summary of the toxicity-mutation test with rat liver S9........................................44 

Table 24: Summary of the mutagenicity test without rat liver S9 .........................................45 

Table 25: Summary of the mutagenicity test with rat liver S9 ..............................................45 

APPENDICES..............................................................................................................................46 

Appendix A: Historical Control Data ......................................................................................47 
Bacterial Reverse Mutation Test 

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STUDY INFORMATION 

Substance Tested: •

•

•

•

 

 Number: 

 

Composition: 

 

Purity: 100% 

 

Physical Characteristics: Gas 

 

Stability: The test substance appeared 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, 2006 
Bacterial 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 incorporation and gas testing methods. Salmonella 

typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2uvrA 

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 first phase was the toxicity-mutation test, which 

established the dose range for the mutagenicity test and provided a preliminary mutagenicity 

evaluation. The second phase was the mutagenicity test, which evaluated and confirmed the 

mutagenic potential of the test substance. 

Dry, filtered air was chosen as the dosing vehicle based on the compatibility with the test 

substance and with the target cells. 

In the toxicity-mutation test, the maximum dose evaluated was targeted at 100% for tester strains 

TA98, TA100, TA1535, TA1537, and WP2uvrA in the presence and absence of S9. Additional 

dose levels targeted in the test were 5, 10, 30, and 60%. The toxicity-mutation test consisted of 

trial one (T-1) under the metabolic activation condition and trial two (T-2) without an activation 

system. 

In the presence of S9, a 50% or greater reduction 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 trial in strains TA 1535 

and TA 1537. In TA98 with S9 the toxicity effect was reproducible in the main assay. Without 

S9 metabolic activation, there was an elevated number of revertants per plate in test strain TA98 

at low, intermediate and high concentrations and in strains TA100, TA 1535, and TA1537 at the 

intermediate concentrations; however no dose response occurred in any case nor were the effects 

reproducible in the mutagenicity test. Precipitation was not observed in any strain at any 

concentration under either test condition. 

Based on the toxicity-mutation test, the maximum target dose evaluated in the mutagenicity test 

(E-1) was 100% test gas for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA 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 the 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 and pre-heated. As the pressure was released, 

test substance and/or dry-filtered dilution air was drawn into the chamber. The vapor/vapor dose 

method was employed during toxicity-mutation T-1 yielding actual mean concentrations of 0, 
Bacterial 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 exposure concentrations for T-2 were 0, 20.7, 41.9, 57.8, 

70.8, and 92.2%. The actual mean exposure concentrations in the mutagenicity test 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. 

All criteria for a valid study were met. Under the conditions of this study, showed no 

evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the presence or 

absence of Aroclor-induced rat liver S9. The test substance was concluded to be negative in this 

study. 
Bacterial 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 the tryptophan locus of the Escherichia coli strain 

WP2 uvrA. The assay was conducted with and without an exogenous S9 metabolic activation 

system. 

MATERIALS AND METHODS 

A. Testing Guidelines 

This study was conducted in compliance 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 Mutation 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 information regarding the test substance is found on the 

study information page of this report. 

2. Characterization 

The sponsor was responsible for the determination and documentation of the analytical purity 

and composition of the test substance. The characterization was not performed under Good 

Laboratory Practice Standards. The stability and strength of the test substance were determined 

under Good Laboratory Practice Standards over approximately 48 hr at by 

visual examination and by analytical procedures. 

3. Sample Preparation, and Analytical Verification of Test Substance Concentrations 

Based on information received from the sponsor, the test substance was considered 100% active 

ingredient. Therefore, no correction factor for purity was applied for The test 

substance was tested as a gas. Prior to and after a 48-72 hour incubation period, a minimum of 

2 gas samples were analyzed from each exposure chamber using a gas chromatograph except for 
Bacterial 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 dimethyl sulfoxide (DMSO, CAS# 67-68-6, 99.9% 

purity, EMD), except for sodium azide and ICR-191, which 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 WP2uvrA.(1,2,3) 

All tester strains were obtained from Moltox Inc. (Boone, NC). The specific genotypes and 

phenotypic characterization of these strains were as follows: 

 Additional Mutations 

Tester Strain HIS/Trp Mutation Repair LPS Plasmid 

 

S. typhimurium TA98 hisD3052 ∆uvrB rfa pkM101 

S. typhimurium TA100 hisG46 ∆uvrB rfa pkM101 

S. typhimurium TA1535 hisG46 ∆uvrB rfa -- 

S. typhimurium TA1537 hisC3076 ∆uvrB rfa -- 

Escherichia coli WP2uvrA trpE ∆uvrA - -- 

 

In addition to a mutation in either the histidine or tryptophan operons, the tester strains contain 

additional mutations that enhance their sensitivity to some mutagens. A mutation of either the 

uvrA or uvrB gene results in a deficient DNA excision repair system. Since the uvrB 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 excluded by a normal intact cell wall. 
Bacterial Reverse Mutation Test 

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Tester strains TA98 and TA100 also contain the 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. Tester strain TA100 is reverted by 

both frame shift and base substitution mutagens. Tester strains TA1535 and WP2uvrA are 

reverted from auxotrophy to prototrophy 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 culture. Aliquots were frozen in dry ice and 

stored at ≤-70°C. 

Master plates were prepared by streaking each tester strain from a frozen permanent stock onto 

either nutrient agar plates or minimal glucose agar plates. The minimal glucose agar plates were 

supplemented with either histidine and biotin or tryptophan, and for strains containing the 

pKM101 plasmid, ampicillin. Tester strain master plates were stored at 5 ± 3°C. 

Overnight cultures for use in the study were inoculated 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 are plated, the length of incubation was determined 

by spectrophotometric monitoring of culture density. 

E. Confirmation of Tester Strain Genotype 

Tester strain cultures were checked for the 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 strain 

on 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 WP2uvrA strain on a 

tryptophan-supplemented minimum glucose 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 uvrA and uvrB mutation was demonstrated by their sensitivity to ultraviolet 

light of the tester strains. 

The presence of the pKM101 plasmid was confirmed for cultures of tester strains TA98 and 

TA100 by demonstration of resistance to ampicillin. 
Bacterial 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 1254 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 contained proportionate volumes of the following 

components: 

HPLC-grade water 2.4 mL

0.825 M KCl/0.2 M MgCl2 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. 
Bacterial 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 

WP2uvrA + 2-aminoanthracene 25.0 

WP2uvrA - 4-nitroquinoline-N-oxide 1.0 

 

c. Sterility Controls 

Sterility control plates were placed in gas exposure 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 corresponds to a plate code which signifies a positive 

control, a negative control or a sample plate, and tester strain, the presence 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, positive 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 the surface of minimum glucose agar plates. 

Using sterile technique, the solidified agar plates were segregated by concentrations onto sterile 

stainless steel racks. The stainless steel racks containing 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) which are then sealed. Following the gas exposure, 

chambers containing the plated bacteria were incubated 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 period using a stir bar and stir plate. Heat generated by the 

stirplates created less than optimal temperature control and was therefore discontinued during 

mutagenicity trial E-1. To accommodate potential concentration gradation within chambers a 

representative culture from each strain was placed at the bottom, middle and top areas of each 

chamber. Plates were stored at approximately 4oC before being counted. All toxicity-mutation 
Bacterial Reverse Mutation Test 

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test preparations of negative (vehicle) controls, test substance, 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, Mutagenicity Test, and Test Method 

The test substance was evaluated along with negative and positive controls using tester strains 

TA98, TA100, TA1535, TA1537, and WP2uvrA with and without S9 activation. The plate 

incorporation method was employed. Dose levels for 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 precipitation. 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: Linear 
Bacterial 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 320μm (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 test 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 detector. 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 trial E-1 treatment when no standard curve was 

prepared due to limited test substance availability. There were no changes in GC conditions and 

the sample volumes were consistent with the start 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 average 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 gas 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 uvrA and uvrB 

mutations, all tester strains cultures must exhibit sensitivity to ultraviolet light. To demonstrate 

the presence of the pKM101 plasmid, tester strain cultures of TA98 and TA100 must exhibit 

resistance to ampicillin. 

2. Tester Strain Culture Density 

To ensure that appropriate numbers of bacteria are plated, all tester strain culture densities must 

be equal to or greater than 0.3 × 109

 cells per milliliter. 
Bacterial Reverse Mutation Test 

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3. Negative Control Values 

The tester strain cultures must exhibit a characteristic mean number of spontaneous revertants 

per plate when plated along with the negative (vehicle) control under selective 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 

WP2uvrA 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 scorable 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 revertants per plate relative to the mean negative 

control value and exhibits a dose-dependent 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, the 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. 
Bacterial 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 control 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 WP2uvrA 

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 control value (vehicle control). This 

increase in the mean number of revertants per 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/vapor dose administration method 

to the liquid/vapor method and to ensure that the 100% concentration was achieved, 2 rather than 

1 chamber was targeted for 100% of the test substance during trial T-1. Also in the same 

instance, the 100% concentration was determined based on nominal calculations at treatment 

start and on analyzed chamber samples at the end of the treatment. Heavy condensate 

accumulated in the chambers in 2 trials. 

Each chamber was placed under negative pressure and pre-heated. As the pressure was released, 

test substance and/or dry-filtered dilution air was drawn into the chamber. While administering 

the 100% dose during the first trial (T-1) with metabolic activation there was a change in the 

dose administration process from the vapor/vapor phase method to the liquid/vapor phase 

method. Concurrently, there was deletion of the carrier gas flow; therefore, the toxicity-mutation 

trial without activation was conducted as a second trial (T-2). 

The actual mean exposure concentrations from the toxicity-mutation test T-1 with activation 

were: 0, 55.8, 74.0, 76.1 and 100% (2 chambers). The actual mean exposure concentrations from 
Bacterial 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 from the mutagenicity test 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 availability lower concentrations 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 concentration 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, the maximum dose evaluated was targeted at 100% for tester strains 

TA98, TA100, TA1535, TA1537, and WP2uvrA in the presence and absence of S9. Additional 

dose levels targeted in the test were 5, 10, 30, and 60%. The toxicity-mutation test consisted of a 

trial (T-1) under the metabolic activation condition and a second trial (T-2) without an activation 

system. 

In the presence of S9, a 50% or greater reduction 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 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 number of revertants per plate in test strain TA98 

at low, intermediate and high concentrations and in strains TA100, TA1535 and TA1537 at the 

intermediate concentrations; however no dose response occurred in any case nor were the effects 

reproducible in the mutagenicity test. Precipitation 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 evaluated in the mutagenicity test 

(E-1) was 100% test gas for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA 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 substance availability only the top 3 concentrations 

were used in the E-1 trail without metabolic activation. The mutagenicity test employed the 

same exposure method as the toxicity-mutation test T-2. 
Bacterial Reverse Mutation Test 

- 20 - 

No positive mutagenic responses were observed 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. 

CONCLUSIONS 

All criteria for a valid study were met. Under the conditions of this study, showed no 

evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the presence or 

absence of Aroclor-induced rat liver S9. The test substance was concluded 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. (1975). 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 Methods 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 WP2 tester strains. Mutagenesis 5, 285-291. 
Bacterial Reverse Mutation Test 

- 21 - 

TABLES 
Bacterial 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 compared to the vehicle control plate(s). 

Microcolonies may be seen readily by the unaided eye and are greatly enlarged relative to 

controls. 

T4 Absent, plate(s) are distinguished by a complete lack 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 visible 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 prevents accurate colony counting and 

obscures the background lawn requiring plate rejection (R). 

Lost Plate Justification 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 treated plates are also comparable to the negative 

control. 

L1 The loss of this vehicle control plate does not 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 control 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. 
Bacterial 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 100a

 0 100a 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. 
Bacterial 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): 1.32×109

 

Plating Aliquot: 100 μL Date Plated: 4-Aug-06 

Actual 

Concentration Revertants Background 

(%) Plate Number Per Plate Code Mean SD 

 

Vehiclea 4 1 T0,P0 2 2 

 5 1 T0,P0 

 6 4 T0,P0 

 

Positive Controlb

 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-nitroflourene 
Bacterial 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): 1.07×109

 

Plating Aliquot: 100 μL Date Plated: 4-Aug-06 

Actual 

Concentration Revertants Background 

(%) Plate Number Per Plate Code Mean SD 

 

Vehiclea 7 5 T0,P0 39 34 

 8 38 T0,P0 

 9 73 T0,P0 

 

Positive Controlb

 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 azide 
Bacterial 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): 1.13×109

 

Plating Aliquot: 100 μL Date Plated: 4-Aug-06 

Actual 

Concentration Revertants Background 

(%) Plate Number Per Plate Code Mean SD 

 

Vehiclea 10 1 T0,P0 4 3 

 11 6 T0,P0 

 12 5 T0,P0 

 

Positive Controlb

 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 azide 
Bacterial 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): 0.91×109

 

Plating Aliquot: 100 μL Date Plated: 4-Aug-06 

Actual 

Concentration Revertants Background 

(%) Plate Number Per Plate Code Mean SD 

 

Vehiclea 13 3 T0,P0 2 1 

 14 3 T0,P0 

 15 1 T0,P0 

 

Positive Controlb

 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-191 
Bacterial Reverse Mutation Test 

- 28 - 

Table 6: Toxicity-mutation test in Escherichia coli WP2uvrA without S9 

Strain: WP2uvrA Experiment No: T-2 

Rat Liver S9: Absent Cell Titer (cells/mL): 2.21×109

 

Plating Aliquot: 100 μL Date Plated: 4-Aug-06 

Actual 

Concentration Revertants Background 

(%) Plate Number Per Plate Code Mean SD 

 

Vehiclea 1 19 T0,P0 28 8 

 2 34 T0,P0 

 3 30 T0,P0 

 

Positive Controlb

 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-oxide 
Bacterial 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): 0.96×109

 

Plating Aliquot: 100 μL Date Plated: 26-Jul-06 

Actual 

Concentrationa

 Revertants Background 

(%) Plate Number Per Plate Code Mean SD 

 

Vehicleb 4 9 T0,P0 13 5 

 5 19 T0,P0 

 6 11 T0,P0 

 

Positive Controlc

 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 

 

100d

 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.
Bacterial 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): 0.88×109

 

Plating Aliquot: 100 μL Date Plated: 26-Jul-06 

Actual 

Concentrationa

 Revertants Background 

(%) Plate Number Per Plate Code Mean SD 

 

Vehicleb 7 90 T0,P0 89 3 

 8 85 T0,P0 

 9 91 T0,P0 

 

Positive Controlc

 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.
Bacterial 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): 0.95×109

 

Plating Aliquot: 100 μL Date Plated: 26-Jul-06 

Actual 

Concentration a Revertants Background 

(%) Plate Number Per Plate Code Mean SD 

 

Vehicleb 10 5 T0,P0 6 1 

 11 6 T0,P0 

 12 6 T0,P0 

 

Positive Controlc

 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.
Bacterial 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): 0.78×109

 

Plating Aliquot: 100 μL Date Plated: 26-Jul-06 

Actual 

Concentration a Revertants Background 

(%) Plate Number Per Plate Code Mean SD 

 

Vehicleb 13 1 T0,P0 3 2 

 14 4 T0,P0 

 15 5 T0,P0 

 

Positive Controlc

 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.
Bacterial Reverse Mutation Test 

- 33 - 

Table 11: Toxicity-mutation test in Escherichia coli WP2uvrA with S9 

Strain: WP2uvrA Experiment No: T-1 

Rat Liver S9: Present Cell Titer (cells/mL): 1.59×109

 

Plating Aliquot: 100 μL Date Plated: 26-Jul-06 

Actual 

Concentration a Revertants Background 

(%) Plate Number Per Plate Code Mean SD 

 

Vehicleb 1 28 T0,P0 37 13 

 2 30 T0,P0 

 3 52 T0,P0 

 

Positive Controlc

 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.
Bacterial 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): 0.97×109

 

Plating Aliquot: 100 μL Date Plated: 9-Aug-06 

Actual 

Concentrationa

 Revertants Background 

% Plate Number Per Plate Code Mean SD 

 

Vehicleb 109 18 T0,P0 18 4 

 110 14 T0,P0 

 111 22 T0,P0 

 

Positive Controlc

 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-nitroflourene 
Bacterial 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): 0.92×109

 

Plating Aliquot: 100 μL Date Plated: 9-Aug-06 

Actual 

Concentrationa

 Revertants Background 

% Plate Number Per Plate Code Mean SD 

 

Vehicleb 112 94 T0,P0 97 3 

 113 99 T0,P0 

 114 99 T0,P0 

 

Positive Controlc

 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 azide
Bacterial 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): 0.86×109

 

Plating Aliquot: 100 μL Date Plated: 9-Aug-06 

Actual 

Concentrationa

 Revertants Background 

% Plate Number Per Plate Code Mean SD 

 

Vehicleb 115 9 T0,P0 11 2 

 116 11 T0,P0 

 117 13 T0,P0 

 

Positive Controlc

 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 azide
Bacterial 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): 0.69×109

 

Plating Aliquot: 100 μL Date Plated: 9-Aug-06 

Actual 

Concentrationa

 Revertants Background 

% Plate Number Per Plate Code Mean SD 

 

Vehicleb 118 3 T0,P0 5 2 

 119 6 T0,P0 

 120 6 T0,P0 

 

Positive Controlc

 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-191
Bacterial Reverse Mutation Test 

- 38 - 

Table 16: Mutagenicity test in Escherichia coli WP2uvrA without S9 

Strain: WP2uvrA Experiment No: E-1 

Rat Liver S9: Absent Cell Titer (cells/mL): 1.44×109

 

Plating Aliquot: 100 μL Date Plated: 9-Aug-06 

Actual 

Concentrationa

 Revertants Background 

% Plate Number Per Plate Code Mean SD 

 

Vehicleb 106 46 T0,P0 40 7 

 107 32 T0,P0 

 108 43 T0,P0 

 

Positive Controlc

 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-oxide
Bacterial 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): 0.97×109

 

Plating Aliquot: 100 μL Date Plated: 9-Aug-06 

Actual 

Concentration Revertants Background 

% Plate Number Per Plate Code Mean SD 

 

Vehiclea 4 23 T0,P0 24 4 

 5 20 T0,P0 

 6 28 T0,P0 

 

Positive Controlb

 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)pyrene 
Bacterial 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): 0.92×109

 

Plating Aliquot: 100 μL Date Plated: 9-Aug-06 

Actual 

Concentration Revertants Background 

% Plate Number Per Plate Code Mean SD 

 

Vehiclea 7 20 T0,P0 76 49 

 8 111 T0,P0 

 9 96 T0,P0 

 

Positive Controlb

 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-aminoanthracene 
Bacterial 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): 0.86×109

 

Plating Aliquot: 100 μL Date Plated: 9-Aug-06 

Actual 

Concentration Revertants Background 

% Plate Number Per Plate Code Mean SD 

 

Vehiclea 10 11 T0,P0 11 3 

 11 8 T0,P0 

 12 14 T0,P0 

 

Positive Controlb

 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-aminoanthracene 
Bacterial 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): 0.69×109

 

Plating Aliquot: 100 μL Date Plated: 9-Aug-06 

Actual 

Concentration Revertants Background 

% Plate Number Per Plate Code Mean SD 

 

Vehiclea 13 6 T0,P0 7 2 

 14 5 T0,P0 

 15 9 T0,P0 

 

Positive Controlb

 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-aminoanthracene 
Bacterial Reverse Mutation Test 

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Table 21: Mutagenicity test in Escherichia coli WP2uvrA with S9 

Strain: WP2uvrA Experiment No: E-1 

Rat Liver S9: Present Cell Titer (cells/mL): 1.44×109

 

Plating Aliquot: 100 μL Date Plated: 9-Aug-06 

Actual 

Concentration Revertants Background 

% Plate Number Per Plate Code Mean SD 

 

Vehiclea 1 38 T0,P0 46 7 

 2 52 T0,P0 

 3 49 T0,P0 

 

Positive Controlb

 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-aminoanthracene 
Bacterial Reverse Mutation Test 

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Table 22: Summary of the toxicity-mutation test without rat liver S9 

Actual Number of Revertants Per Plate 

Concentration TA98 TA100 TA1535 TA1537 

(%) Mean SD Mean SD Mean SD Mean SD 

vehicle 2 2 39 34 4 3 2 1 

positive control 194 14 1232 33 1059 34 1369 134 

20.7 17 2 88 20 9 3 6 3 41.9 5 4 49 12 7 5 3 3 57.8 14 5 66 10 6 0 6 1 70.8 13 4 91 17 13 5 8 4 92.2 5 5 53 9 6 2 5 4 

Experiment No: T-2 

Plate Aliquot: 100 μL Table 23: Summary of the toxicity-mutation test with rat liver S9 

Actual Number of Revertants Per Plate 

Concentration a TA98 TA100 TA1535 TA1537 

(%) Mean SD Mean SD Mean SD Mean SD 

vehicle 13 5 89 3 6 1 3 2 

positive control 353 48 2747 100 229 18 149 24 

55.8 19 5 102 16 11 4 5 2 74.0 12 7 87 8 10 3 6 3 76.1 14 5 92 10 9 2 5 2 

100 b 3 2 71 14 3 2 1 2 

Experiment No: T-1 Plate Aliquot: 100 μL 

a Lower concentrations were exceeded during dose administration and were not tested. b The results of 2 chambers tested at 100% are averaged together.
Bacterial Reverse Mutation Test 

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Table 24: Summary of the mutagenicity test without rat liver S9 

Actual Number of Revertants Per Plate 

Concentration a TA98 TA100 TA1535 TA1537 

% Mean SD Mean SD Mean SD Mean SD 

vehicle 18 4 97 3 11 2 5 2 

positive control 129 11 634 15 628 24 1188 110 

55.1 14 1 86 9 11 2 4 1 83.7 9 3 85 8 7 2 3 2 90.1 10 4 60 12 10 3 3 2 

Experiment No: E-1 

Plate Aliquot: 100 μL a Lower concentrations were not tested due to limited test substance availability Table 25: Summary of the mutagenicity test with rat liver S9 

Actual Number of Revertants Per Plate 

Concentration TA98 TA100 TA1535 TA1537 

% Mean SD Mean SD Mean SD Mean SD 

vehicle 24 4 76 49 11 3 7 2 

positive control 368 10 2527 388 215 14 215 72 

23.3 22 4 90 16 14 7 7 2 46.8 22 7 93 20 17 7 5 1 68.5 16 3 90 10 11 10 5 4 71.3 14 2 99 9 10 3 6 4 88.9 12 1 94 5 10 1 6 2 

Experiment No: E-1 

Plate Aliquot: 100 μL 
 

Bacterial Reverse Mutation Test 

- 46 - 

APPENDICES 
 

Bacterial Reverse Mutation Test 

- 47 - 

Appendix A: 

Historical Control Data 
 

Bacterial Reverse Mutation Test 

- 48 - 

HISTORICAL CONTROL DATAa 

 

Tester Strain Exogenous Metabolic Range 

Control [Positive Controlb

] 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 uvrA 

 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 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: 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