FINAL REPORT

Study Title

EVALUATION OF THE MUTAGENIC ACTIVITY OF 

IN THE SALMONELLA TYPHIMURIUM

REVERSE MUTATION ASSAY AND THE ESCHERICHIA COLI

REVERSE MUTATION ASSAY (WITH INDEPENDENT REPEAT)

Author

Test Facility

 

Laboratory Project Identification

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1. CONTENTS 

1. CONTENTS ...................................................................................................................................... 2

2. STATEMENT OF GLP COMPLIANCE ............................................................................................. 4

3. QUALITY ASSURANCE STATEMENT ............................................................................................ 5

4. SUMMARY ........................................................................................................................................ 6

5. INTRODUCTION .............................................................................................................................. 7

5.1. Preface ...................................................................................................................................... 7

5.2. Aims of the study ....................................................................................................................... 7

5.3. Guidelines ................................................................................................................................. 7

5.4. Storage and retention of records and materials ........................................................................ 8

6. MATERIALS AND METHODS .......................................................................................................... 8

6.1. Test substance .......................................................................................................................... 8

6.1.1. Test substance information ............................................................................................. 8

6.1.2. Study specific test substance information ...................................................................... 8

6.1.3. Test substance preparation ............................................................................................ 8

6.2. Reference substances .............................................................................................................. 9

6.2.1. Negative control .............................................................................................................. 9

6.2.2. Positive controls .............................................................................................................. 9

6.3. Test system ............................................................................................................................... 9

6.4. Cell culture ..............................................................................................................................10

6.5. Metabolic activation system ....................................................................................................11

6.5.1. Preparation of S9-fraction .............................................................................................11

6.5.2. Preparation of S9-mix ...................................................................................................11

6.6. Study design ...........................................................................................................................11

6.6.1. Dose range finding test .................................................................................................11

6.6.2. Mutation assay ..............................................................................................................11

6.6.3. Colony counting ............................................................................................................12

6.7. Electronic data capture ...........................................................................................................12

6.8. Interpretation ...........................................................................................................................12

6.8.1. Acceptability of the assay .............................................................................................12

6.8.2. Data evaluation and statistical procedures ...................................................................12

6.9. List of deviations .....................................................................................................................13

6.9.1. List of protocol deviations .............................................................................................13

6.9.2. List of standard operating procedures deviations .........................................................13

7. RESULTS .......................................................................................................................................13

7.1. Dose range finding test ...........................................................................................................13

7.2. Mutation assay ........................................................................................................................14

7.2.1. Experiment 1 .................................................................................................................14

7.2.2. Experiment 2 .................................................................................................................15

8. DISCUSSION AND CONCLUSION ................................................................................................16

9. REFERENCES ...............................................................................................................................16

TABLES 

Table 1 Toxicity of in the dose range finding test ......................................14

Table 2 Toxicity of in the first experiment ..................................................15

Table 3 Toxicity of in the second experiment ............................................16

Table 4 Dose range finding study: Mutagenic response of in the 

Salmonella typhimurium reverse mutation assay and in the Escherichia coli 

reverse mutation assay .................................................................................................17

Table 5 Experiment 1: Mutagenic response of in the Salmonella 

typhimurium reverse mutation assay and in the Escherichia coli reverse mutation 

assay .............................................................................................................................18

Table 6 Experiment 2: Mutagenic response of in the Salmonella 

typhimurium reverse mutation assay and in the Escherichia coli reverse mutation 

assay .............................................................................................................................19

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APPENDICES 

APPENDIX 1 SUMMARY TABLES .....................................................................................................17

APPENDIX 2 SUPPORTING MATERIALS AND METHOD ...............................................................20

APPENDIX 3 DETAILED TABLES......................................................................................................21

APPENDIX 4 HISTORICAL CONTROL DATA OF THE SOLVENT CONTROL ................................33

APPENDIX 5 HISTORICAL CONTROL DATA OF THE POSITIVE CONTROL SUBSTANCES .......34

 

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4. SUMMARY 

Evaluation of the mutagenic activity of in the Salmonella typhimurium reverse 

mutation assay and the Escherichia coli reverse mutation assay (with independent repeat). 

was tested in the Salmonella typhimurium reverse mutation assay with four 

histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the 

Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli

(WP2uvrA). The test was performed in two independent experiments in the presence and absence of 

S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). 

The study procedures described in this report were based on the most recent OECD and EC 

guidelines. 

Batch of was a clear colourless liquid with a purity of ≥ 95%. In the dose 

range finding test, was dissolved in dimethyl sulfoxide. However since the stability 

of in dimethyl sulfoxide was unknown and is stable for at least 

24 hours in ethanol, ethanol is the preferred solvent. For this reason was dissolved 

in ethanol in the mutation experiments. 

In the dose range finding test, was tested up to concentrations of 5000 μg/plate in 

the absence and presence of S9-mix in the strains TA100 and WP2uvrA. did not 

precipitate on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels 

of 100 μg/plate in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was 

observed at any of the dose levels tested. 

Based on the results of the dose range finding test, was tested in the first mutation 

assay at a concentration range of 3 to 1000 μg/plate in the absence and presence of 5% (v/v) S9-mix 

in tester strains TA1535, TA1537, TA98 and TA100 and at a concentration range of 100 to 

5000 μg/plate in the absence and presence of S9-mix in tester strain WP2uvrA. 

precipitated on the plates at the top dose of 5000 μg/plate. Toxicity was observed in all tester strains, 

except in tester strain WP2uvrA in the absence and presence of S9-mix and TA98 in the absence of 

S9-mix. 

In an independent repeat of the assay with additional parameters, was tested at a 

concentration range of 0.3 to 100 μg/plate in the absence and presence of 10% (v/v) S9-mix in tester 

strains TA1535, TA1537, TA98 and TA100 and at a concentration range of 100 to 5000 μg/plate in the 

absence and presence of S9-mix in tester strain WP2uvrA. precipitated on the 

plates at the top dose of 5000 μg/plate. Toxicity was observed in all tester strains, except in tester 

strain WP2uvrA in the absence and presence of S9-mix. 

did not induce a significant dose-related increase in the number of revertant (His+) 

colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of 

revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic 

activation. These results were confirmed in an independently repeated experiment. 

In this study, the negative and strain-specific positive control values were within the laboratory 

historical control data ranges indicating that the test conditions were adequate and that the metabolic 

activation system functioned properly. 

Based on the results of this study it is concluded that is not mutagenic in the 

Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. 

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5. INTRODUCTION 

5.1. Preface 

Sponsor

Study Monitor

(on behalf of the sponsor)

Test Facility

Study Director

Study Plan Start : 05 January 2012

Completion : 23 February 2012

5.2. Aims of the study 

The objective of this study was to evaluate the test substance for its ability to induce reverse mutations 

in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidineindependent strains, and in a gene of tryptophan-requiring Escherichia coli bacterial strain resulting in 

a tryptophan-independent strain. 

Background of the test system 

The Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay 

have been shown to be rapid and adequate indicators for the mutagenic activity of a wide range of 

chemical compounds. 

The assay was conducted in the absence and presence of a metabolizing system (S9-mix). 

The Salmonella typhimurium strains used in this study were TA1535, TA1537, TA98 and TA100. The 

Escherichia coli strain used was WP2uvrA. The strains TA1537 and TA98 are capable of detecting 

frameshift mutagens, strains TA1535, TA100 and WP2uvrA are capable of detecting base-pair 

substitution mutagens (1-5). 

5.3. Guidelines 

The study procedures described in this report were based on the following guidelines: 

- Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing 

of Chemicals; Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test" (Adopted 

July 21, 1997). 

- European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the 

Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse 

Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 31 May 2008. 

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5.4. Storage and retention of records and materials 

Records and materials pertaining to the study including protocol, raw data and the final report are 

retained in the archives for a period of at least 2 years after finalization of the report. After this 

period, the sponsor will be contacted to determine how the records and materials should be handled. 

 will retain information concerning decisions made. 

 will retain a test substance sample until the expiry date, but no longer than 10 years after 

finalization of the report. After this period the sample will be destroyed. 

6. MATERIALS AND METHODS 

6.1. Test substance 

6.1.1. Test substance information 

Identification 

Structure

Molecular formula

Molecular weight

CAS Number

Description

Batch

Purity

Test substance storage

Stability under storage conditions

Expiry date 17 October 2012 

6.1.2. Study specific test substance information 

Test substance handling Flush container with nitrogen after handling

Stability at higher temperatures

Stability in vehicle Dimethyl sulfoxide: Not indicated

Ethanol: At least 24 hours

Solubility in vehicle Dimethyl sulfoxide: Not indicated

Ethanol: Not indicated

6.1.3. Test substance preparation 

In the dose range finding test, the test substance was dissolved in dimethyl sulfoxide (SeccoSolv, 

Merck, Darmstadt, Germany). In the mutation experiments, the test substance was dissolved in 

ethanol (pro analyse, Merck, Darmstadt, Germany). Test substance concentrations were used within 

3 hours after preparation. 

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6.2. Reference substances 

6.2.1. Negative control 

The vehicle of the test substance, which was dimethyl sulfoxide (dose range finding test) and ethanol 

(mutation experiments). 

6.2.2. Positive controls 

Without metabolic activation (-S9-mix): 

Strain Chemical Concentration/plate Solvent 

TA1535 sodium azide (SA) 5 μg Saline 

(Sigma Aldrich Chemie, Steinheim, Germany) 

TA1537 ICR-191 (Acros Organics, Geel, Belgium) 2.5 μg DMSO 

TA98 2-nitrofluorene (NF) (Merck) 10 μg DMSO 

TA100 methylmethanesulfonate (MMS) (Sigma) 650 μg DMSO 

WP2uvrA 4-nitroquinoline N-oxide (4-NQO) (Sigma) 10 μg DMSO 

With metabolic activation (+S9-mix): 

The positive control substance used for all tester strains was 2-aminoanthracene (2AA) (Sigma). The 

following doses were used: 

Strain Concentration/plate Amount of S9-mix Solvent 

TA1535 2.5 μg 5 and 10% DMSO 

TA1537 2.5 μg 5% DMSO 

TA1537 5 μg 10% DMSO 

TA98 1 μg 5 and 10% DMSO 

TA100 1 μg 5% DMSO 

TA100 2.5 μg 10% DMSO 

WP2uvrA 10 μg 5 and 10% DMSO 

Solvents for reference substances 

Saline = physiological saline (B. Braun, Melsungen AG, Germany) 

DMSO = dimethyl sulfoxide (Merck) 

6.3. Test system 

Test system Salmonella typhimurium bacteria and Escherichia coli bacteria 

Rationale Recommended test system in international guidelines (e.g. OECD, EC). 

Source Salmonella typhimurium strains: 

Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames): 

TA100 received on 14-06-2006, 

used batch: TA100.241109 and TA100.11-1 (25-Nov-11) 

TA98 received on 14-06-2006, 

used batch: TA98.071210 and TA98.11-1 (02-Dec-11)

TA1537 received on 03-12-2009, 

used batch: TA1537.081210 and TA1537.11-1 (25-Nov-11)

TA1535 received on 14-06-2006, used batch: TA1535.11-1 (25-Nov-11) 

Escherichia coli strain: 

Trinova Biochem GmbH, Germany (Master culture from The National Collections 

of Industrial and Marine Bacteria, Aberdeen, UK) 

WP2uvrA received on 06-02-2008, used batch: EC.110211 (11-Feb-11) 

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The characteristics of the different Salmonella typhimurium strains were as follows: 

Strain Histidine mutation Mutation type 

TA1537 hisC3076 Frameshift

TA98 hisD3052/R-factor* Frameshift 

TA1535 hisG46 Base-pair substitutions 

TA100 hisG46/R-factor* Base-pair substitutions 

*: R-factor = plasmid pKM101 (increases error-prone DNA repair) 

Each tester strain contained the following additional mutations: 

rfa : deep rough (defective lipopolysaccharide cellcoat) 

gal : mutation in the galactose metabolism 

chl : mutation in nitrate reductase 

bio : defective biotin synthesis 

uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene) 

The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, 

crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of 

spontaneous revertants. 

The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair 

system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of 

mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment (Ref.1). 

The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the 

number of spontaneous revertants. 

Stock cultures of the five strains were stored in liquid nitrogen (-196°C). 

6.4. Cell culture 

Preparation of bacterial cultures 

Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, 

Hampshire, England) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached 

an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were 

used for a test. 

Agar plates 

Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 

18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad 

Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also 

contained 12.5 μg/plate biotin (Merck) and 15 μg/plate histidine (Merck) and the agar plates for the 

test with the Escherichia coli strain contained 15 μg/plate tryptophan (Acros Organics). 

Top agar 

Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride 

(Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass 

tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C. 

 

Environmental conditions 

All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual 

range 36.0 – 39.2°C) in the dark. Temporary deviations of maximally 1 hour (in the range of 

38.0– 38.4°C) occurred due to addition of plates (which were at room temperature) to the incubator or 

due to opening and closing the incubator door. Based on laboratory historical data these deviations 

are considered not to affect the study integrity. The temporary deviations from the temperature are 

explained in protocol deviation 3. 

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6.5. Metabolic activation system 

Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were 

obtained from Charles River, Sulzfeld, Germany (see protocol deviation 1). 

6.5.1. Preparation of S9-fraction 

The animals were housed at n a special room under standard laboratory conditions, as 

described in the Standard Operating Procedures. The rats were orally dosed for three consecutive 

days with a suspension of phenobarbital (Bufa B.V., IJsselstein, The Netherlands) (80 mg/kg body 

weight) and ß-naphthoflavone (Sigma Aldrich Chemie) (100 mg/kg body weight) in corn oil (Roth, 

Karlsruhe, Germany) (they were denied access to food for 3 to 4 hours preceding each dosing). One 

day after the final exposure (24 h), the rats were sedated using oxygen/carbon dioxide and then killed 

by decapitation. The rats received a limited quantity of food during the night before sacrifice. The livers 

of the rats were removed aseptically, and washed in cold (0°C) sterile 0.1 M sodium phosphate buffer 

(pH 7.4) containing 0.1 mM Na2-EDTA (Merck). Subsequently the livers were minced in a blender and 

homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was 

centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampoules, which 

were stored in liquid nitrogen (-196°C) for a maximum of 1 year. 

Before use, all S9 batches were characterised with the mutagens Benzo-(a)-pyrene (Sigma) and 

2-aminoanthracene (Sigma), which require metabolic activation, in tester strain TA98 at concentrations 

of 5 μg/plate and 1 μg/plate, respectively. 

6.5.2. Preparation of S9-mix 

S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 ml: 30 mg 

NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate 

(Roche Diagnostics, Mannheim, Germany) in 5.5 ml or 5.0 ml Milli-Q water (first or second experiment 

respectively) (Millipore Corp., Bedford, MA., USA); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 

0.08 M MgCl2 solution (Merck); 1 ml 0.33 M KCl solution (Merck). The above solution was filter 

(0.22 μm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-

fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml 

S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment. The 

S9-batches used were no. 11-1 and 11-3. 

6.6. Study design 

6.6.1. Dose range finding test 

Selection of an adequate range of doses was based on a dose range finding test with the strains 

TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 

1000, 3330 and 5000 μg/plate were tested in triplicate. The highest concentration of Phenyl-tolylethane used in the subsequent mutation assay was 5000 μg/plate or the level at which the test 

substance inhibited bacterial growth. 

6.6.2. Mutation assay 

At least five different doses (increasing with approximately half-log steps) of the test substance were 

tested in triplicate in each strain. In the first experiment was tested both in the 

absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and 

WP2uvrA (see protocol deviation 2). In an independent repeat of the assay with additional parameters, 

the test substance was tested both in the absence and presence of 10% (v/v) S9-mix in all tester 

strains. 

The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in 

the presence and absence of S9-mix. 

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Top agar in top agar tubes was melted by heating to 45°C. The following solutions were successively 

added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109cells/ml) of one of the tester 

strains, 0.1 ml of a dilution of the test substance in dimethyl sulfoxide (dose range finding test) and 

ethanol (mutation experiments) and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M 

phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the 

content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, 

the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period 

revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan 

independent (Trp+) for Escherichia coli were counted. 

6.6.3. Colony counting 

The revertant colonies (histidine independent or tryptophan independent) were counted manually if 

less than 40 colonies per plate were present. If more than 40 colonies were present, these were 

counted automatically with a Biocount 4000 Pro-S-colony counter. Plates with abundant test 

substance precipitate which interfered with automated colony counting were counted manually and the 

evidence of test substance precipitate on the plates was recorded. The condition of the bacterial 

background lawn was evaluated, both macroscopically and microscopically by using a dissecting 

microscope. 

6.7. Electronic data capture 

Observations/measurements in the study were recorded electronically using the following programme: 

REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, 

USA): Temperature. 

6.8. Interpretation 

6.8.1. Acceptability of the assay 

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is 

considered acceptable if it meets the following criteria: 

a) The negative control data (number of spontaneous revertants per plate) should be within the 

laboratory historical range for each tester strain (see APPENDIX 4). 

b) The positive control chemicals should produce responses in all tester strains, which are within the 

laboratory historical range documented for each positive control substance. Furthermore, the mean 

plate count should be at least three times the concurrent vehicle control group mean (see 

APPENDIX 5). 

c) The selected dose range should include a clearly toxic concentration or should exhibit limited 

solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 

5 mg/plate. 

6.8.2. Data evaluation and statistical procedures 

No formal hypothesis testing was done. 

A test substance is considered negative (not mutagenic) in the test if: 

a) The total number of revertants in tester strain TA100 is not greater than two (2) times the 

concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or 

WP2uvrA is not greater than three (3) times the concurrent vehicle control. 

b) The negative response should be reproducible in at least one independently repeated experiment. 

A test substance is considered positive (mutagenic) in the test if:

a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent 

control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is 

greater than three (3) times the concurrent vehicle control. 

b) In case a repeat experiment is performed when a positive response is observed in one of the tester 

strains, the positive response should be reproducible in at least one independently repeated 

experiment. 

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The preceding criteria were not absolute and other modifying factors might enter into the final 

evaluation decision. 

6.9. List of deviations 

6.9.1. List of protocol deviations 

1. The S9-fraction was made in-house. The rat liver microsomal enzymes (S9 homogenate) was 

prepared from adult male Wistar rats that had been orally dosed with a suspension of 

phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight). 

Evaluation: Since rat liver S9-mix induced by a combination of phenobarbital and 

ß-naphthoflavone and by Aroclor are recommended by the OECD guideline and the positive 

control data were within the laboratory historical range for each tester strain, this deviation of the 

S9 homogenate had no effect on the results of the study. 

2. In the first mutation experiment all five tester strains (TA1535, TA1537, TA98, TA100 and 

WP2uvrA) were tested. 

Evaluation: The stability of the the solvent DMSO, used in the dose range finding, was unknown. 

However since the stability of the the solvent ethanol was indicated, ethanol was used in the 

mutation experiments. The performing of the first mutation experiment with all five tester strains 

had no effect on the results of the study. 

3. The temperature was recorded to be outside the range of 37.0 ± 1.0°C specified in the protocol 

for approximately 1.5 hour in the dose range finding test (with a maximum of 39.2°C), in the first 

experiment (tester strain TA98) for approximately 0.5 hour with a maximum of 38.7°C and in the 

second mutation experiment with a maximum of 39.0°C. 

 Evaluation: These short term deviations were observed within three hours after initiation of the 

test and were caused by adjustment of the temperature in the incubator after placing an amount 

of selective agar plates in the incubator. The negative control data (number of spontaneous 

revertants per plate) were within the laboratory historical range for each tester strain, therefore 

these short deviations of the temperature had no effect on the results of the study. 

4. In the second experiment in tester strain TA1535, the lowest dose level could not be evaluated. 

Evaluation: According to the OECD guideline five concentrations of the test substance should be 

used for the experiment and the other five dose levels could be evaluated: therefore this deviation 

had no influence on the study result. 

The study integrity was not adversely affected by the deviations. 

6.9.2. List of standard operating procedures deviations 

Any deviations from standard operating procedures were evaluated and filed in the study file. There 

were no deviations from standard operating procedures that affected the integrity of the study. 

7. RESULTS 

7.1. Dose range finding test 

was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 

33, 100, 333, 1000, 3330 and 5000 μg/plate in the absence and presence of S9-mix. The results are 

shown in Table 4, the individual data are presented in APPENDIX 3.

Precipitate 

Precipitation of on the plates was observed at the start of the incubation period at 

concentrations of 3330 and 5000 μg/plate and no precipitate was observed at the end of the 

incubation period. 

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Toxicity 

To determine the toxicity of the reduction of the bacterial background lawn, the 

increase in the size of the microcolonies and the reduction of the revertant colonies were examined. 

The definitions are stated in APPENDIX 2.

In tester strain WP2uvrA, no reduction of the bacterial background lawn and no biologically relevant 

decrease in the number of revertants were observed. 

The reduction of the bacterial background lawn and the reduction in the number of revertants in tester 

strain TA100 is presented in Table 1. 

Table 1 Toxicity of in the dose range finding test 

 (Reduction of the bacterial background lawn and in the number of revertant colonies) 

Strain Without S9-mix With S9-mix

 Dose Bacterial Revertant 

 (μg/plate) background lawn colonies

Dose Bacterial Revertant 

(μg/plate) background lawn colonies

TA100

 100 slight 1)

 333 moderate 1)

1000-5000 extreme microcolonies

 100 slight 1)

333-5000 extreme microcolonies

-

1

 No reduction in the number of revertant colonies less than the minimal value of the historical 

control data range. 

Mutagenicity 

In the dose range finding test, no increase in the number of revertants was observed upon treatment 

with under all conditions tested. 

7.2. Mutation assay 

7.2.1. Experiment 1 

Based on the results of the dose range finding test, the following dose ranges were selected for the 

first mutation experiment: 

TA1535, TA1537, TA98 and TA100: 

Without and with S9-mix: 3, 10, 33, 100, 333 and 1000 μg/plate 

WP2uvrA: 

Without and with S9-mix: 33, 100, 333, 1000, 3330 and 5000 μg/plate 

The results are shown in Table 5, the individual data are presented in APPENDIX 3.

Precipitate 

Precipitation of on the plates was observed at the start of the incubation period at 

concentrations of 3330 and 5000 μg/plate and at 5000 μg/plate at the end of the incubation period. 

Except in the absence of S9-mix (TA1537, TA98, TA100 and WP2uvrA), where precipitation at the 

start of the incubation period was already observed at 1000 μg/plate. 

Toxicity 

In tester strain WP2uvrA, no reduction of the bacterial background lawn and no biologically relevant 

decrease in the number of revertants were observed. 

The reduction of the bacterial background lawn and the reduction in the number of revertants of the 

Salmonella typhimurium strains are presented in (For definitions see APPENDIX 2).

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Table 2 Toxicity of in the first experiment 

 (Reduction of the bacterial background lawn and in the number of revertant colonies) 

Strain Without S9-mix With S9-mix

 Dose Bacterial Revertant 

 (μg/plate) background lawn colonies

Dose Bacterial Revertant 

(μg/plate) background lawn colonies

TA1535 333 slight 1)

1000 moderate 1)

 33 slight 1)

 100 moderate 1)

 333 moderate microcolonies 

1000 extreme microcolonies

TA1537 333-1000 slight 1)

 33 slight 1)

100 moderate 1)

333 moderate extreme

1000 extreme microcolonies

TA98 100 normal 1) 33-100 slight 1)

333-1000 moderate 1)

TA100 100-333 slight 1)

1000 moderate 1)

33-333 slight 1)

1000 extreme microcolonies

-

1

 No reduction in the number of revertant colonies less than the minimal value of the historical 

control data range. 

Mutagenicity 

In the first mutation experiment, no increase in the number of revertants was observed upon treatment 

with under all conditions tested. 

7.2.2. Experiment 2 

To obtain more information about the possible mutagenicity of a second mutation 

experiment was performed in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based 

on the results of the first mutation assay, the following dose ranges were selected for the first mutation 

experiment: 

TA1535, TA1537, TA98 and TA100: 

Without and with S9-mix: 0.3, 1, 3, 10, 33, 333 and 100 μg/plate 

WP2uvrA: 

Without and with S9-mix: 100, 333, 1000, 3330 and 5000 μg/plate 

The results are shown in Table 6, the individual data are presented in APPENDIX 3.

Precipitate 

Precipitation of on the plates was observed at the start of the incubation period at 

concentrations of 3330 and 5000 μg/plate and at 5000 μg/plate at the end of the incubation period. 

Toxicity 

In tester strain WP2uvrA, no reduction of the bacterial background lawn and no biologically relevant 

decrease in the number of revertants were observed. 

The reduction of the bacterial background lawn and the reduction in the number of revertants of the 

Salmonella typhimurium strains are presented in (For definitions see APPENDIX 2).

Final Report
 

- Page 16 - 

Table 3 Toxicity of in the second experiment 

 (Reduction of the bacterial background lawn and in the number of revertant colonies) 

Strain Without S9-mix With S9-mix

 Dose Bacterial Revertant 

 (μg/plate) background lawn colonies

Dose Bacterial Revertant 

(μg/plate) background lawn colonies

TA1535 33 slight 1)

100 extreme microcolonies

 33 slight 1)

 100 moderate 1)

TA1537 100 slight 1) 100 slight 1)

TA98

 10 slight 1)

33 moderate slight

100 extreme microcolonies

 10 slight 1)

33-100 moderate 1)

TA100 33 slight moderate

100 moderate extreme

 33 slight moderate

100 moderate moderate

-

1

 No reduction in the number of revertant colonies less than the minimal value of the historical 

control data range. 

Mutagenicity 

In the second mutation experiment, no increase in the number of revertants was observed upon 

treatment with under all conditions tested. 

8. DISCUSSION AND CONCLUSION 

All bacterial strains showed negative responses over the entire dose range, i.e. no significant doserelated increase in the number of revertants in two independently repeated experiments. 

The negative and strain-specific positive control values were within the laboratory historical control 

data ranges indicating that the test conditions were adequate and that the metabolic activation system 

functioned properly. 

Based on the results of this study it is concluded that is not mutagenic in the 

Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

9. REFERENCES 

1 Leonardo, J.M., Dornfeld, S.S. and Peak, M.J., 1984, Evaluation of E. coli K12 343 \ 113 and 

derived strains for microbial mutagenicity assays. Mutation Res., 130, 87-95. 

2 Ames, B.N., McCann, J. and Yamasaki, E., 1975, Methods for detecting carcinogens and 

mutagens with the Salmonella/mammalian microsome mutagenicity test, Mutation Res., 31, 347-

364.

3 Maron, D.M. and Ames, B.N., 1983, Revised methods for the Salmonella mutagenicity test, 

Mutation Res., 113, 173-215. Erratum, 1983, Mutation Res., 113, 533. 

4 Green, M.H.L. and Muriel, W.J., 1976, Mutagen testing using Trp+ reversion in Escherichia coli, 

Mutation Res., 38, 3-32.

5 Vogel, H.J. and Bonner, D.M., 1956, Acetylornithinase of Escherichia coli: partial purification and 

some properties. J. Biol. Chem., 218, 97-106. 

Final Report
 

- Page 17 - 

APPENDIX 1 SUMMARY TABLES 

Table 4 Dose range finding study: Mutagenic response of in the 

Salmonella typhimurium reverse mutation assay and in the Escherichia coli 

reverse mutation assay 

Dose Mean number of revertant colonies/3 replicate 

 (μg/plate) plates (± S.D.) with tester strain TA100 

TA100 WP2uvrA 

 Without S9-mix 

 positive control 844 ± 36 1337 ± 54

 solvent control 88 ± 5 14 ± 4 

 3 77 ± 6 12 ± 2 

 10 74 ± 8 14 ± 4 

 33 72 ± 11 10 ± 2 

 100 66 ± 8 s 15 ± 6 

 333 66 ± 24 m 10 ± 4 

 1000 MC e 14 ± 6 

 3330 MC e 11 ± 1 

 5000 MC e 12 ± 6 

------------------------------------------------------------------------------------------------- 

With S9-mix1

 positive control 1214 ± 88 303 ± 45

 solvent control 73 ± 6 13 ± 5 

 3 81 ± 8 17 ± 3 

 10 69 ± 12 16 ± 3 

 33 81 ± 11 18 ± 2 

 100 60 ± 12 s 18 ± 1 

 333 MC e 17 ± 6 

 1000 MC e 11 ± 2 

 3330 MC e 9 ± 4 

 5000 MC e 11 ± 4 

Solvent control: 0.1 ml dimethyl sulfoxide 

1 The S9-mix contained 5% (v/v) S9 fraction 

s Bacterial background lawn slightly reduced 

m Bacterial background lawn moderately reduced 

e Bacterial background lawn extremely reduced 

MC Microcolonies 

Final Report
 

- Page 18 - 

APPENDIX 1 – continued –

Table 5 Experiment 1: Mutagenic response of in the Salmonella 

typhimurium reverse mutation assay and in the Escherichia coli reverse 

mutation assay 

Dose Mean number of revertant colonies/3 replicate plates (± S.D.) with 

(μg/plate) different strains of Salmonella typhimurium and one Escherichia coli strain 

TA1535 TA1537 TA98 TA100 WP2uvrA 

Without S9-mix 

 positive control 790 ± 23 756 ± 15 989 ± 45 932 ± 18 1107 ± 37

 solvent control 9 ± 2 7 ± 2 15 ± 4 92 ± 15 21 ± 2 

 3 8 ± 5 4 ± 1 19 ± 4 100 ± 6 

 10 11 ± 2 4 ± 1 21 ± 3 90 ± 13 

 33 9 ± 2 5 ± 1 18 ± 4 81 ± 10 21 ± 8 

 100 9 ± 2 3 ± 1 15 ± 2 69 ± 5 s 26 ± 2 

 333 10 ± 3 s 3 ± 1 s 13 ± 5 68 ± 12 s 26 ± 7 

 1000 6 ± 1 m 2 ± 1 s 13 ± 4 70 ± 14 m 22 ± 1 

 3330 19 ± 4 

 5000 SP 22 ± 6 

------------------------------------------------------------------------------------------------------------ 

 

With S9-mix1

 positive control 266 ± 12 184 ± 61 469 ± 117 945 ± 63 434 ± 12

 solvent control 6 ± 3 4 ± 1 22 ± 8 76 ± 15 25 ± 2 

 3 5 ± 3 3 ± 2 19 ± 3 77 ± 8 

 10 7 ± 2 3 ± 1 18 ± 3 73 ± 9 

 33 6 ± 3 s 3 ± 1 s 20 ± 1 s 79 ± 6 s 26 ± 2 

 100 11 ± 1 m 2 ± 2 m 20 ± 4 s 67 ± 3 s 26 ± 4 

 333 MC m 1 ± 1 m t 23 ± 1 m 76 ± 8 s 24 ± 3 

 1000 MC e MC e 16 ± 1 m MC e 19 ± 5 

 3330 16 ± 3 

 5000 SP 20 ± 1 

Solvent control: 0.1 ml ethanol 

1 The S9-mix contained 5% (v/v) S9 fraction 

t Toxicity in the number of revertant colonies compared to the solvent control 

s Bacterial background lawn slightly reduced 

m Bacterial background lawn moderately reduced 

e Bacterial background lawn extremely reduced 

SP Slight Precipitate 

MC Microcolonies 

Final Report
 

- Page 19 - 

APPENDIX 1 – continued –

Table 6 Experiment 2: Mutagenic response of in the Salmonella 

typhimurium reverse mutation assay and in the Escherichia coli reverse 

mutation assay 

Dose Mean number of revertant colonies/3 replicate plates (± S.D.) with 

(μg/plate) different strains of Salmonella typhimurium and one Escherichia coli strain 

TA1535 TA1537 TA98 TA100 WP2uvrA 

Without S9-mix 

 positive control 911 ± 17 859 ± 57 1154 ± 40 793 ± 37 1289 ± 45

 solvent control 6 ± 3 6 ± 2 13 ± 5 143 ± 5 17 ± 3 

 0.3 INF 4 ± 1 12 ± 2 103 ± 9 

 1 9 ± 2 7 ± 5 19 ± 2 104 ± 13

 3 9 ± 3 4 ± 2 16 ± 1 93 ± 15

 10 7 ± 2 6 ± 1 12 ± 3 s 95 ± 7 

 33 7 ± 1 s 7 ± 2 10 ± 3 m t 60 ± 3 s t 

 100 MC e 4 ± 1 s MC e 33 ± 20 m t 17 ± 5 

 333 18 ± 5 

 1000 18 ± 4 

 3330 17 ± 2 

 5000 SP 18 ± 6 

------------------------------------------------------------------------------------------------------------ 

 

With S9-mix1

 positive control 172 ± 35 127 ± 37 471 ± 83 645 ± 70 338 ± 20

 solvent control 6 ± 3 4 ± 1 21 ± 3 131 ± 10 21 ± 5 

 0.3 INF 6 ± 2 15 ± 1 62 ± 7 

 1 6 ± 2 4 ± 1 21 ± 2 64 ± 6 

 3 9 ± 3 4 ± 3 18 ± 3 68 ± 12

 10 11 ± 2 5 ± 1 24 ± 5 s 71 ± 7 

 33 7 ± 1 s 6 ± 2 17 ± 2 m 54 ± 5 s t 

 100 5 ± 3 m 5 ± 2 s 17 ± 6 m 57 ± 7 m t 18 ± 1 

 333 19 ± 3 

 1000 23 ± 4 

 3330 15 ± 4 

 5000 SP 23 ± 3 

Solvent control: 0.1 ml ethanol 

1 The S9-mix contained 10% (v/v) S9 fraction 

t Toxicity in the number of revertant colonies compared to the solvent control

s Bacterial background lawn slightly reduced 

m Bacterial background lawn moderately reduced 

e Bacterial background lawn extremely reduced 

SP Slight Precipitate 

MC Microcolonies 

INF Plates infected 

Final Report
 

- Page 20 - 

APPENDIX 2 SUPPORTING MATERIALS AND METHOD 

Bacterial background lawn evaluation 

The condition of the bacterial background lawn is evaluated (if indicated), both macroscopically and 

microscopically by using a dissecting microscope (results are normal unless indicated in tables). 

Definition Characteristics 

Normal Distinguished by a healthy microcolony lawn. 

Slightly reduced Distinguished by a slight thinning of the microcolony lawn. 

Moderately reduced Distinguished by a moderate thinning of the microcolony lawn. 

Extremely reduced Distinguished by an extreme thinning of the microcolony lawn and an 

increase in the size of the microcolonies compared to the solvent control 

plate. 

Absent Distinguished by a complete lack of any microcolony background lawn. 

Precipitation evaluation 

Evidence of test article precipitate on the plates is recorded by addition of the following precipitation 

definition. 

Definition Characteristics 

Slight Precipitate Distinguished by noticeable precipitate on the plate. 

However, the precipitate does not influence automated counting of the plate. 

Moderate Precipitate Distinguished by a marked amount of precipitate on the plate, requiring the 

plate to be hand counted. 

Heavy Precipitate Distinguished by a large amount of precipitate on the plate, making the 

required hand count difficult. 

Evaluation of the reduction in the number of revertants 

The reduction in the number of revertant colonies compared to number of revertants in the solvent 

control is evaluated as follows: 

A reduction of 21-40%: slight reduction. 

A reduction of 41-60%: moderate reduction. 

A reduction of 61-99%: extreme reduction. 

If the size of the microcolonies was increased to small colonies due to an extremely reduced 

background lawn the reduction is evaluated as microcolonies. If no revertant colonies are observed on 

the plates the reduction is evaluated as a complete lack of revertants. 

However, any mean plate count equal to the minimal value of the historical control data range should 

be considered not toxic. 

Final Report
 

- Page 21 - 

APPENDIX 3 DETAILED TABLES 

Individual plate counts; (following pages) 

Dose range finding 

Strain TA100 

----------------------------------------------------------- 

 WITHOUT S9-MIX

 plate 1 2 3 MEAN SD

 dose (μg/plate) 

 positive control 819 827 885 844 ± 36

 solvent control 90 82 91 88 ± 5 

 3 84 73 75 77 ± 6 

 10 71 69 83 74 ± 8 

 33 72 62 83 72 ± 11

 100 s 63 75 61 66 ± 8 

 333 m 93 55 49 66 ± 24

 1000 e MC MC MC MC 

 3330 e MC MC MC MC 

 5000 e MC MC MC MC 

----------------------------------------------------------- 

 WITH S9-MIX

 plate 1 2 3 MEAN SD

 dose (μg/plate) 

 positive control 1253 1113 1275 1214 ± 88

 solvent control 78 74 66 73 ± 6 

 3 80 89 73 81 ± 8 

 10 82 68 58 69 ± 12

 33 92 81 71 81 ± 11

 100 s 71 47 1 61 1 60 ± 12

 333 e MC MC MC MC 

 1000 e MC MC MC MC 

 3330 e MC MC MC MC 

 5000 e MC MC MC MC 

----------------------------------------------------------- 

s: Bacterial background lawn slightly reduced 

m: Bacterial background lawn moderately reduced 

e: Bacterial background lawn extremely reduced 

MC: Microcolonies 

1: Counted by hand 

Final Report
 

- Page 22 - 

APPENDIX 3 – continued –

Dose range finding 

Strain WP2uvrA 

----------------------------------------------------------- 

 WITHOUT S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 1306 1306 1400 1337 ± 54

 solvent control 13 11 19 14 ± 4 

 3 14 10 11 12 ± 2 

 10 12 19 12 14 ± 4 

 33 12 10 9 10 ± 2 

 100 21 13 10 15 ± 6 

 333 7 15 8 10 ± 4 

 1000 10 11 20 14 ± 6 

 3330 12 10 10 11 ± 1 

 5000 5 17 13 12 ± 6 

----------------------------------------------------------- 

 WITH S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 326 251 332 303 ± 45

 solvent control 8 18 12 13 ± 5 

 3 14 17 19 17 ± 3 

 10 19 16 14 16 ± 3 

 33 19 19 16 18 ± 2 

 100 17 17 19 18 ± 1 

 333 11 16 23 17 ± 6 

 1000 11 10 13 11 ± 2 

 3330 6 13 8 9 ± 4 

 5000 7 15 11 11 ± 4 

----------------------------------------------------------- 

Final Report
 

- Page 23 - 

APPENDIX 3 – continued –

Experiment 1 

Strain TA1535 

----------------------------------------------------------- 

 WITHOUT S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 791 766 812 790 ± 23

 solvent control 9 10 7 9 ± 2 

 3 13 4 6 8 ± 5 

 10 9 12 11 11 ± 2 

 33 10 9 7 9 ± 2 

 100 10 10 6 9 ± 2 

 333 s 7 10 12 10 ± 3 

 1000 m 7 5 5 6 ± 1 

----------------------------------------------------------- 

 WITH S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 272 274 252 266 ± 12

 solvent control 4 9 6 6 ± 3 

 3 2 6 7 5 ± 3 

 10 7 5 8 7 ± 2 

 33 s 3 6 8 6 ± 3 

 100 m 11 12 10 11 ± 1 

 333 m MC MC MC MC 

 1000 e MC MC MC MC 

----------------------------------------------------------- 

s: Bacterial background lawn slightly reduced 

m: Bacterial background lawn moderately reduced 

e: Bacterial background lawn extremely reduced 

MC: Microcolonies 

Final Report
 

- Page 24 - 

APPENDIX 3 – continued –

Experiment 1 

Strain TA1537 

----------------------------------------------------------- 

 WITHOUT S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 770 756 741 756 ± 15

 solvent control 7 8 5 7 ± 2 

 3 3 5 5 4 ± 1 

 10 3 5 3 4 ± 1 

 33 5 6 4 5 ± 1 

 100 3 4 3 3 ± 1 

 333 s 4 2 2 3 ± 1 

 1000 s 3 2 1 2 ± 1 

----------------------------------------------------------- 

 WITH S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 114 212 226 184 ± 61

 solvent control 3 4 4 4 ± 1 

 3 0 4 4 3 ± 2 

 10 2 2 4 3 ± 1 

 33 s 2 4 2 3 ± 1 

 100 m 2 0 4 2 ± 2 

 333 m t 0 2 2 1 ± 1 

 1000 e MC MC MC MC 

----------------------------------------------------------- 

s: Bacterial background lawn slightly reduced 

m: Bacterial background lawn moderately reduced 

e: Bacterial background lawn extremely reduced 

MC: Microcolonies 

t: Toxicity in the number of revertant colonies compared to the solvent control 

Final Report
 

- Page 25 - 

APPENDIX 3 – continued –

Experiment 1 

Strain TA98 

----------------------------------------------------------- 

 WITHOUT S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 1036 947 983 989 ± 45

 solvent control 20 12 14 15 ± 4 

 3 22 15 20 19 ± 4 

 10 23 18 23 21 ± 3 

 33 14 19 21 18 ± 4 

 100 15 13 16 15 ± 2 

 333 11 10 19 13 ± 5 

 1000 17 14 9 13 ± 4 

----------------------------------------------------------- 

 WITH S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 582 477 349 469 ± 117 

 solvent control 30 15 21 22 ± 8 

 3 16 19 21 19 ± 3 

 10 16 18 21 18 ± 3 

 33 s 19 20 20 20 ± 1 

 100 s 18 18 25 20 ± 4 

 333 m 24 22 22 23 ± 1 

 1000 m 17 15 17 16 ± 1 

----------------------------------------------------------- 

s: Bacterial background lawn slightly reduced 

m: Bacterial background lawn moderately reduced 

Final Report
 

- Page 26 - 

APPENDIX 3 – continued –

Experiment 1 

Strain TA100 

----------------------------------------------------------- 

 WITHOUT S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 946 938 911 932 ± 18

 solvent control 75 98 103 92 ± 15

 3 106 95 100 100 ± 6 

 10 81 84 105 90 ± 13

 33 73 79 92 81 ± 10

 100 s 74 66 66 69 ± 5 

 333 s 78 72 55 68 ± 12

 1000 m 80 75 54 70 ± 14

----------------------------------------------------------- 

 WITH S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 994 967 874 945 ± 63

 solvent control 60 81 88 76 ± 15

 3 78 69 84 77 ± 8 

 10 76 62 80 73 ± 9 

 33 s 86 76 76 79 ± 6 

 100 s 66 65 70 67 ± 3 

 333 s 78 67 83 76 ± 8 

 1000 e MC MC MC MC 

----------------------------------------------------------- 

s: Bacterial background lawn slightly reduced 

m: Bacterial background lawn moderately reduced 

e: Bacterial background lawn extremely reduced 

MC: Microcolonies 

Final Report
 

- Page 27 - 

APPENDIX 3 – continued –

Experiment 1 

Strain WP2uvrA 

----------------------------------------------------------- 

 WITHOUT S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 1149 1085 1086 1107 ± 37

 solvent control 20 21 23 21 ± 2 

 33 18 14 30 21 ± 8 

 100 25 29 25 26 ± 2 

 333 33 19 25 26 ± 7 

 1000 21 22 23 22 ± 1 

 3330 23 16 18 19 ± 4 

 5000 SP 27 16 24 22 ± 6 

----------------------------------------------------------- 

 WITH S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 441 441 421 434 ± 12

 solvent control 24 27 23 25 ± 2 

 33 28 24 27 26 ± 2 

 100 28 28 21 26 ± 4 

 333 24 27 21 24 ± 3 

 1000 20 23 13 19 ± 5 

 3330 15 14 20 16 ± 3 

 5000 SP 20 19 21 20 ± 1 

----------------------------------------------------------- 

SP: Slight Precipitate 

Final Report
 

- Page 28 - 

APPENDIX 3 – continued –

Experiment 2 

Strain TA1535 

----------------------------------------------------------------------- 

 WITHOUT S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 892 922 920 911 ± 17

 solvent control 8 3 7 6 ± 3 

 0.3 INF INF INF INF 

 1 8 8 12 9 ± 2 

 3 5 11 11 9 ± 3 

 10 7 7 6 7 ± 1 

 33 s 7 10 5 7 ± 3 

 100 e MC MC MC MC 

----------------------------------------------------------------------- 

 WITH S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 212 147 158 172 ± 35

 solvent control 5 10 4 6 ± 3 

 0.3 INF INF INF INF 

 1 6 4 7 6 ± 2 

 3 6 12 8 9 ± 3 

 10 12 8 12 11 ± 2 

 33 s 6 8 8 7 ± 1 

 100 m 2 8 6 5 ± 3 

----------------------------------------------------------------------- 

s: Bacterial background lawn slightly reduced 

m: Bacterial background lawn moderately reduced 

INF: Plate could not be evaluated, due to infection (see protocol deviation 4) 

Final Report
 

- Page 29 - 

APPENDIX 3 – continued –

Experiment 2 

Strain TA1537 

----------------------------------------------------------- 

 WITHOUT S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 843 922 812 859 ± 57

 solvent control 4 6 7 6 ± 2 

 0.3 4 3 4 4 ± 1 

 1 13 6 3 7 ± 5 

 3 5 6 2 4 ± 2 

 10 6 6 5 6 ± 1 

 33 9 7 5 7 ± 2 

 100 s 5 4 3 4 ± 1 

----------------------------------------------------------- 

 WITH S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 126 91 164 127 ± 37

 solvent control 3 5 5 4 ± 1 

 0.3 5 8 5 6 ± 2 

 1 4 3 4 4 ± 1 

 3 1 5 6 4 ± 3 

 10 4 5 5 5 ± 1 

 33 8 7 4 6 ± 2 

 100 s 7 3 5 5 ± 2 

----------------------------------------------------------- 

s: Bacterial background lawn slightly reduced 

Final Report
 

- Page 30 - 

APPENDIX 3 – continued –

Experiment 2 

Strain TA98 

----------------------------------------------------------- 

 WITHOUT S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 1190 1111 1160 1154 ± 40

 solvent control 8 18 12 13 ± 5 

 0.3 13 12 10 12 ± 2 

 1 18 18 21 19 ± 2 

 3 15 17 16 16 ± 1 

 10 s 8 13 14 12 ± 3 

 33 m t 13 9 8 10 ± 3 

 100 e MC MC MC MC 

----------------------------------------------------------- 

 WITH S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 403 447 563 471 ± 83

 solvent control 23 22 18 21 ± 3 

 0.3 16 16 14 15 ± 1 

 1 19 22 21 21 ± 2 

 3 16 21 17 18 ± 3 

 10 s 29 20 22 24 ± 5 

 33 m 19 16 17 17 ± 2 

 100 m 20 21 11 17 ± 6 

----------------------------------------------------------- 

s: Bacterial background lawn slightly reduced 

m: Bacterial background lawn moderately reduced 

e: Bacterial background lawn extremely reduced 

MC: Microcolonies 

t: Toxicity in the number of revertant colonies compared to the solvent control 

Final Report
 

- Page 31 - 

APPENDIX 3 – continued –

Experiment 2 

Strain TA100 

----------------------------------------------------------- 

 WITHOUT S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 803 825 752 793 ± 37

 solvent control 138 148 143 143 ± 5 

 0.3 113 100 97 103 ± 9 

 1 110 113 89 104 ± 13

 3 102 101 76 93 ± 15

 10 95 88 102 95 ± 7 

 33 s t 58 59 64 60 ± 3 

 100 m t 10 43 46 33 ± 20

----------------------------------------------------------- 

 WITH S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 720 580 636 645 ± 70

 solvent control 129 122 141 131 ± 10

 0.3 65 67 54 62 ± 7 

 1 71 60 61 64 ± 6 

 3 77 73 54 68 ± 12

 10 79 68 66 71 ± 7 

 33 s t 59 55 49 54 ± 5 

 100 m t 56 64 51 57 ± 7 

----------------------------------------------------------- 

s: Bacterial background lawn slightly reduced 

m: Bacterial background lawn moderately reduced 

t: Toxicity in the number of revertant colonies compared to the solvent control 

Final Report
 

- Page 32 - 

APPENDIX 3 – continued –

Experiment 2 

Strain WP2uvrA 

----------------------------------------------------------- 

 WITHOUT S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 1241 1330 1296 1289 ± 45

 solvent control 15 20 16 17 ± 3 

 100 15 23 14 17 ± 5 

 333 14 16 24 18 ± 5 

 1000 22 18 14 18 ± 4 

 3330 16 20 16 17 ± 2 

 5000 SP 11 20 23 18 ± 6 

----------------------------------------------------------- 

 WITH S9-MIX

 plate 1 2 3 MEAN SD 

 dose (μg/plate) 

 positive control 360 332 322 338 ± 20

 solvent control 16 26 21 21 ± 5 

 100 19 17 17 18 ± 1 

 333 17 23 17 19 ± 3 

 1000 28 21 21 23 ± 4 

 3330 18 17 11 15 ± 4 

 5000 SP 19 25 25 23 ± 3 

----------------------------------------------------------- 

SP: Slight Precipitate 

Final Report
 

- Page 33 - 

APPENDIX 4 HISTORICAL CONTROL DATA OF THE SOLVENT CONTROL 

TA1535 TA1537 TA98 TA100 WP2uvrA

S9-mix - + - + - + - + - +

Range 3 - 25 3 – 31 2 - 19 2 - 16 11 - 49 12 - 59 61 - 195 58 - 179 8 - 45 6 - 46

Mean 10 8 5 5 19 24 106 88 22 23

SD 4 3 2 2 5 7 22 21 7 7

n 1208 1216 1073 1098 1191 1208 1224 1221 1090 1105

SD = Standard deviation 

n = Number of observations 

Historical control data from experiments performed between July 2008 and June 2011. 

Final Report
 

- Page 34 - 

APPENDIX 5 HISTORICAL CONTROL DATA OF THE POSITIVE CONTROL SUBSTANCES 

TA1535 TA1537 TA98

S9-mix - + - + - +

Range 245 - 1270 60 – 943 89 - 1086 82 - 677 401 - 1342 225 - 1656

Mean 887 265 314 374 1016 792

SD 112 111 146 104 133 286

n 1123 1150 1011 1039 1136 1157

TA100 WP2uvrA

S9-mix - + - +

Range 348 - 1417 229 - 1752 138 - 1479 122 - 1248

Mean 950 1103 1074 372

SD 117 261 212 137

n 1153 1170 1023 1047

SD = Standard deviation 

n = Number of observations 

Historical control data from experiments performed between June 2008 and June 2011. 

Final Report