## FINAL REPORT

Study Title



## EVALUATION OF THE MU TAGENIC ACTIVITY OF

IN THE SALMONELLA TYPHIMURI UM

REVERSE MUTATION ASS AY AND THE ESCHERICHIA COLI

REVERSE MUTATION ASS AY (WITH INDEPENDENT REPEAT)

Author

Test Facility

Laboratory Project Identification

- Page 1 of 34

-- Page 2 -

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- Page 3 -

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- Page 6 -

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 (WP 2 uvrA). The test was performed in two independent experiments in the presence and absence of
S9 -mix (ra t 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 WP 2 uvrA. 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 WP 2 uvrA, 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 WP 2 uvrA

. precipitated on the plates at the top dose of 5000 μg/plate. Toxicity was observed in all tester strains, except in tester strain WP 2 uvrA 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 WP 2 uvrA

. precipitated on the plates at the top dose of 5000 μg/plate. Toxicity was observed in all tester strains, except in tester
strain WP 2 uvrA 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 WP 2 uvrA 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.- Page 7 -

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 typhimuriumbacterial strains resulting in histidineindependent strains, and in a gene of tryptophan-requiring Escherichia coli bacterial strain resulting in a tryptoph an -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 WP 2 uvrA. The strains TA1537 and TA98 are capable of detecting frameshift mutagens, strains TA1535, TA100 and WP 2 uvrA 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, 3 1 May 2008.- Page 8 -

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 Nu mber 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 sulfoxid e: 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.- Page 9 -

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

WP 2 uvrA 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
WP 2 uvrA 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)
WP 2 uvrA received on 06

- 02 -2008, used batch: EC. 1102 11 (11-Feb-11)- Page 10 -

The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 his C3076 Frameshi ft
TA98 his D3052/R-factor* Frameshift
TA1535 his G46 Base-pair substitutions
TA100 his G46/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 WP 2 uvrA 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 (10 9 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 (Fr es enius 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.- Page 11 -

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 Na 2 -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 MgCl 2 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 -batch es 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 WP 2 uvrA, 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 5 000 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

WP 2 uvrA (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.- Page 12 -

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 (10 9 cells/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

WP 2 uvrA 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 i f: 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 WP 2 uvrA 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.- Page 13 -

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

WP 2 uvrA) 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 WP 2 uvrA 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.- Page 14 -

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 WP 2 uvrA, no reduction of the bacterial background lawn and no biologically relevan t
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 microcoloni es

- 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

WP 2 uvrA:
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- mi x (TA1537, TA98, TA100 and WP 2 uvrA), where precipitation at the start of the incubation period was already observed at 1000 μg/plate.

Toxicity
In tester strain WP 2 uvrA, 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 )

.- Page 15 -

Tab le 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 e xtreme 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

WP 2 uvrA: 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 WP 2 uvrA, 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 )

.- 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.- 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 WP 2 uvrA

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-mix 1

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 Ba cterial background lawn extremely reduced

MC Microcolonies- 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 WP 2 uvrA

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-mix 1

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

33 3 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- 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 WP 2 uvrA

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-mix 1

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 contr ol 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- 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.- 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- Page 22 -

APPENDIX 3



## – continued



## –

Dose range finding

Strain WP 2 uvrA

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

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

------------------------------------------------------------ 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- 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- 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- 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- Page 27 -

APPENDIX 3



## – continued



## –

Experiment 1

Strain WP 2 uvrA

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

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- 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)- 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

10 0 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- 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- 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- Page 32 -

APPENDIX 3



## – continued



## –

Experiment 2

Strain WP 2 uvrA

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

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- Page 33 -

APPENDIX 4 HISTORICAL CONTROL DATA OF THE SOLVENT CONTROL

TA1535 TA1537 TA98 TA100 WP 2 uvrA

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.- 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 WP 2 uvrA

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.