## FINAL REPORT

Test Facility Study No. 787718, Report No. 30768



# Alcohols, C18-22 Distn. Residues Testing for Mutagenic Activity with Salmonella typhimurium TA 1535, TA 100, TA 1537 and TA 98 and Escherichia coli WP2 uvr A

TEST FACILITY: Charles River Tranent Edinburgh EH33 2NE UK

SPONSOR: SASOL Germany GmbH Anckelmannsplatz 1 D-20537 Hamburg Germany

Page 1 of 37

## Page 2 Test Facility Study No. 787718

TABLE OF CONTENTS

1 COMPLIANCE STATEMENT

............................................................................. 7

2 QUALITY ASSURANCE STATEMENT

.............................................................. 8

3 RESPONSIBLE PERSONNEL.............................................................................. 9

4 SUMMARY........................................................................................................... 10

5 INTRODUCTION

................................................................................................ 11

5.1 Regulatory Citations............................................................................................. 11

5.1.1 Test Guideline ....................................................................................................... 11

5.1.2 Quality Compliance .............................................................................................. 11

5.2 Test Facility........................................................................................................... 11

5.3 Key Dates in the Conduct of the Study:............................................................... 11

5.4 Archiving of Data.................................................................................................. 12

5.5 Deviations from the Study Protocol ..................................................................... 12

6 MATERIALS AND METHODS

.......................................................................... 12

6.1 Test Item................................................................................................................ 12

6.2 Dose Formulations ................................................................................................ 12

6.2.1 Preparation of Dose Formulations ....................................................................... 12

6.2.2 Analysis of Dose Formulations ............................................................................. 13

6.3 Test System ........................................................................................................... 13

6.4 Justification of Test System .................................................................................. 13

6.5 Bacteria ................................................................................................................. 14

6.5.1 Bacterial Cultures ................................................................................................. 15

6.6 Experimental Design............................................................................................. 15

## Page 3 Test Facility Study No. 787718

6.7 Justification of Dose Levels .................................................................................. 16

6.8 Administration of Test/Control Items.................................................................. 16

6.8.1 Vehicle Controls.................................................................................................... 16

6.8.2 Positive Controls ................................................................................................... 17

6.8.3 Test Item................................................................................................................ 17

6.9 Experimental Procedure....................................................................................... 17

6.9.1 S9 Mix...................................................................................................................

. 17

6.9.2 Toxicity Tests ........................................................................................................ 18

6.9.3 Mutation Tests ...................................................................................................... 18

6.9.3.1 Treatment Methods .............................................................................................. 19

6.10 Quality Control of Bacterial Strains .................................................................... 19

6.11 Calculations and Data Acceptance

....................................................................... 19

6.11.1 Calculations........................................................................................................... 19

6.11.2 Historical Control Data ........................................................................................ 19

6.11.3 Acceptance Criteria

.............................................................................................. 20

6.11.4 Interpretation of Mutagenicity............................................................................. 20

7 RESULTS.............................................................................................................. 21

7.1 Toxicity Tests ........................................................................................................ 21

7.2 Mutation Tests ...................................................................................................... 21

7.2.1 Quality Control ..................................................................................................... 21

7.2.2 Vehicle Control Groups........................................................................................ 21

7.2.3 Positive Control Groups ....................................................................................... 21

7.2.4 Alcohols, C18-22 Distn. Residues ......................................................................... 22

8 CONCLUSION ..................................................................................................... 23

## Page 4 Test Facility Study No. 787718

9 REFERENCES ..................................................................................................... 24

10 TABLES ................................................................................................................ 25

11 APPENDICES....................................................................................................... 34

Final Page of Report........................................................................................................... 37

## Page 5 Test Facility Study No. 787718

LIST OF TABLES

Table 1 Toxicity Test.................................................................................................. 25 Table 2 First Mutation Assay (Direct Plate Incorporation Method) ............................. 26 Table 3 Second Mutation Assay (Pre-incubation Method) .......................................... 30

## Page 6 Test Facility Study No. 787718

LIST OF APPENDICES

Appendix 1 Certificate of Analysis ................................................................................... 34 Appendix 2 In vitro Activation Preparation Form.............................................................. 35 Appendix 3 Historical Positive and Negative Control Data ............................................... 36 Appendix 4 Quality Control Checks of Bacterial Cultures................................................. 37

## 1 COMPLIANCE STATEMENT Page 7 Test Facility Study No. 787718

I, the undersigned, hereby declare that this study was performed in accordance with the OEeD Principles of Good Laboratory Practice as incorporated into the United Kingdom Statutory Instrument for GLP and as accepted by Regulatory Authorities throughout the European Community, United States of America (FDA and EPA) and Japan (MHL W, MAFF and METI).

The study was conducted according to the procedures herein described and this report represents a true and accurate record of the results obtained.

# COlin%€~ Study irector Date

## 2 QUALITY ASSURANCE STATEMENT Page 8 Test Facility Study No. 787718



## Study title: Alcohols, C18-22 Distn. Residues: Testing for Mutagenic Activity with 

## Salmonella typhimurium TA 1535, TA 100, TA 1537 and TA 98 and 

## Escherichia coli WP2uvrA

The Charles River Quality Assurance Unit conducted a protocol review, process-based inspections and report audits relevant to this short-term study, as detailed below.

Date of QA Activity

05 October 2009 06 October 2009

09 October 2009 07 October 2009 02-03 November 2009 24 November 2009 Activity Date of Report to Management

Protocol Review Dose Preparation/Dosing/ Protocol Compliance Colony Counting QC Checking Draft Report Audit Final Report Audit 05 October 2009 06 October 2009

12 October 2009 08 October 2009 03 November 2009 24 November 2009

The protocol review and report audits were reported to the Study Director on the same date as management. The outcome of each process-based inspection is also reported to the Study Director, where relevant.

Facilities relevant to this study are included in Charles River's annual facility inspection
programme. The outcome of each inspection is reported to Management.

This report is considered to describe accurately and completely the procedures used in the study and the results obtained.



# "st..J Dv 

### e."""bf!!/**'l,..CO~** 

### Date

## Page 9 Test Facility Study No. 787718

3 RESPONSIBLE PERSONNEL

Study Director: Colin G Riach BSc

Report Compilation: Colin G Riach BSc

Technical Assistance: Wanda Zajac Pawel Jurczak

Quality Assurance: Jane P Dunsire CBiol MSB Alison McNaughton HNC

Sponsor Representative (Study Monitor): Hans Certa PhD

## Page 10 Test Facility Study No. 787718

4 SUMMARY

Alcohols, C18-22 Distn. Residues was tested for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in Escherichia coli WP2 uvr A. Alcohols, C18-22 Distn. Residues was formulated in dimethylsulphoxide, in which it gave a part solution/part suspension.

Two independent tests were conducted on agar plates in triplicate in the absence and presenceof an Aroclor 1254-induced rat liver S9 preparation and the co-factors required for mixedfunction oxidase activity (S9 mix). The first test was conducted by the direct plate incorporation method, while the second test was conducted by the pre-incubation method. Alcohols, C18-22 Distn. Residues was dosed at concentrations ranging from 6 to 2000 μg per plate in both assays (2000 μg per plate was the highest practical concentration, as limited by the solubility of the test item).

Concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix.

No evidence of mutagenic activity was obtained with any strain in either test.

No toxicity to the bacteria was observed. Alcohols, C18-22 Distn. Residues precipitated at the highest concentration of 2000 μg per plate.

It is concluded that Alcohols, C18-22 Distn. Residues is not mutagenic in strains of Salmonella typhimurium and Escherichia coli, when tested in the absence and presence of metabolic activation up to and beyond its limit of solubility in the test system.

The study was performed in accordance with the principles of Good Laboratory Practice.

## Page 11 Test Facility Study No. 787718

5 INTRODUCTION

The objective of this study was to assess the mutagenic potential of the test item, Alcohols, C18-22 Distn. Residues, using Salmonella typhimurium and Escherichia coli in a bacterial mutagenicity assay, the Ames test.

5.1 Regulatory Citations

5.1.1 Test Guideline

This study was performed to comply with OECD guideline 471, the European Commission Annex V Test Method B13 and B14, ICH Guidelines CPMP/ICH/141/95 and CPMP/ICH/174/95 and USA EPA 712-C-98-247. The ICH Guidelines are recognised by Regulatory bodies of the European Community, United States of America and Japan.

5.1.2 Quality Compliance

The study was conducted in accordance with the OECD Principles of Good Laboratory Practice as set forth by the United Kingdom Department of Health and as accepted by Regulatory Authorities of the European Community, United States of America (FDA and EPA) and Japan (MHLW, MAFF and METI).

All routine activities conducted during the course of this study are detailed in Charles River’s Standard Operating Procedures.

5.2 Test Facility

This report describes the methods used and the results obtained in tests carried out at the laboratories of:

Charles River Tranent Edinburgh EH33 2NE UK

5.3 Key Dates in the Conduct of the Study:

Study Initiation: 01 October 2009 Experimental Start Date: 02 October 2009 Experimental Completion Date: 12 October 2009 Study Completion Date: See Compliance Statement page for date of Study Director's signature.

## Page 12 Test Facility Study No. 787718

5.4 Archiving of Data

All raw data generated and recorded during this study are stored in the Scientific Archives of Charles River Preclinical Services Edinburgh for 2 years after the issue of the final report. After the 2 year period, the Sponsor will be consulted regarding the disposal, transfer or continued storage of the raw data.

The original signed copy of the final report will be stored indefinitely in the Scientific Archives of Charles River Preclinical Services Edinburgh.

5.5 Deviations from the Study Protocol

There were no deviations from the Study Protocol.

6 MATERIALS AND METHODS

6.1 Test Item

Alcohols, C18-22 Distn. Residues (Batch 03585/MA), a beige waxy solid, was received from Sasol Germany GmbH on 11 September 2009 and was stored in the dark at ambient room temperature. The purity was quoted as 100% (being a complex distn. residue) and an expiry date of 31 August 2013 was supplied by the Sponsor. A copy of the Certificate of Analysis is shown in Appendix 1.

6.2 Dose Formulations

6.2.1 Preparation of Dose Formulations

In preliminary tests, Alcohols, C18-22 Distn. Residues was found to have limited solubility in all solvents compatible with the test system. Dimethylsulphoxide (DMSO) was chosen as the solvent giving the best solubility/dispersal characteristics. A partial solution was obtained using the following technique.

DMSO was added to a pre-weighed sample of Alcohols, C18-22 Distn. Residues to give a concentration of 20 mg/mL. The mixture was vortex-mixed for several minutes, and then placed in an incubator at 37°C for ca 45 min. After this, the mixture was vortex-mixed for a further ca 10 minutes. This process produced a milky suspension with fine particles that could be drawn up a wide-bore pipette tip.

To check whether the DMSO was carrying dissolved test item above the saturation limit in the aqueous test conditions, 0.1 mL of the above 20 mg/mL preparation was added to 2.5 mL samples of water and DMSO and the level of precipitate compared. There was a greater level of precipitate/undissolved material present in the water sample than in the DMSO sample and

## Page 13 Test Facility Study No. 787718

it was therefore concluded that a concentration of 20 mg/mL in DMSO (being greater than the saturation level in aqueous conditions) was a suitable maximum concentration to use in the study.

6.2.2 Analysis of Dose Formulations

The test item formulations were prepared immediately prior to dosing (within 1 h). Detailed records of preparation of the dosing solutions were maintained to allow checking of procedures. Chemical analysis of the test item formulations was not conducted; however Alcohols, C18-22 Distn. Residues was tested up to and beyond its limit of solubility in the test system in all experiments.

6.3 Test System

The bacterial reverse mutation assay uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base-pairs. The principle of this bacterial reverse mutation assay is that it detects reverse mutations in histidine or tryptophan loci of the test strains that restore the functional capability of the bacteria to synthesise an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the essential amino acid required by the parent test strain.

6.4 Justification of Test System

Chemicals that react with DNA may cause different types of mutations and many of these can be detected with the use of different strains of bacteria.

It is well recognised that many chemicals that may be reactive in mammalian cells, following metabolic activation, are quite inactive in bacterial cells. S9 mix (homogenate prepared from liver of Aroclor 1254-treated rats, along with cofactors necessary for enzymic activity) provides an exogenous metabolic activation system. S9 mix is combined with the test item and bacterial cells to increase the relevance of the test in assessing the mutagenicity of chemicals to humans.

The relevant testing guidelines recommend that a concentration limit of 5 mg per plate should be used if no physicochemical property prevents this. (In the event, the limited solubility of the test item restricted the highest concentration to 2 mg per plate.)

## Page 14 Test Facility Study No. 787718

6.5 Bacteria

Four strains of Salmonella typhimurium were used:

S. typhimurium TA 1535 S. typhimurium TA 100 S. typhimurium TA 1537 S. typhimurium TA 98

These S. typhimurium strains were obtained in 1976 from Professor B N Ames, Department of Biochemistry, University of California, USA, and have been maintained in liquid nitrogen storage since receipt.

All these S. typhimurium strains contain mutations in the histidine operon, thereby imposing the essential requirement for histidine in the growth medium. Three mutations in the histidine operon are involved:

his G 46 in TA 1535 and TA 100 his C 3076 in TA 1537 his D 3052 in TA 98

his G 46 is a mis-sense mutation that is reverted to prototrophy by mutagens that cause base-pair substitutions.

his C 3076 contains a frameshift mutation that appears to have added a



## − − − − C

G base-pair,

resulting in

− − − − CCCC

GGGG . This mutation is reverted to prototrophy by 9-aminoacridine,

ICR-191 and epoxides of polycyclic hydrocarbons.

his 

## D 3052 also contains a frameshift mutation with the sequence

− − − − GCGCGC

CGCGCG that is

reverted with the deletion of 2 base-pairs,

− − − − GC

CG . It is readily reverted by aromatic amines

and derivatives.

All 4 strains contain the deep rough (rfa) mutation, which results in deletion of the polysaccharide side chain of the lipopolysaccharide coat of the bacterial cell surface. This deletion increases cell permeability to more hydrophobic substances and, furthermore, greatly decreases the pathogenicity of these organisms. A second deletion, through uvr 

## B, renders the

## Page 15 Test Facility Study No. 787718

organisms incapable of DNA excision repair and thus more susceptible to mutagenicity. These 2 deletions also include the nitrate reductase ( chl ) and biotin ( bio ) genes.

TA 100 and TA 98 strains contain a plasmid (R-Utrecht) that was originally shown to increase the sensitivity of the his G 46 mutation in S. typhimurium to methyl methanesulphonate and trimethyl phosphate. The particular R-factor (pKM101) in TA 100 and TA 98 carries resistance to ampicillin.

One strain of Escherichia coli was used:

Escherichia coli WP2 uvr A

This Escherichia coli strain was obtained in 1976 from the National Collection of Industrial Bacteria, Aberdeen, Scotland, and has been maintained in liquid nitrogen storage since receipt.

The strain contains an ochre mutation in the trp E locus and can be mutated to tryptophan-independence either by a base-pair reversion of an A-T base-pair in the trp E locus, or more commonly, by a base-pair substitution within a number of transfer RNA loci elsewhere in the chromosome. The latter mutation causes the original defect to be suppressed (ochre suppression) and involves only base-pair substitutions at G-C base-pairs.

Thus, while trp + 

## reversion systems can detect mutations resulting from chemical attack at both A-T and G-C base-pairs, it does not detect frameshift mutagens. The uvr A mutation causes this bacterial strain to be deficient in the excision of bulky lesions from the DNA and thus readily mutated by certain agents ( eg ultraviolet radiation, polycyclic hydrocarbons).

6.5.1 Bacterial Cultures

Samples of each bacterial strain were grown up by culturing for 16 h at ca 37 ° C in nutrient broth (25 g Oxoid Nutrient Broth No. 2 per litre). Fresh overnight cultures were used in the tests for this study. Samples from the cultures were kept for up to 7 days at ca 4 ° C to allow relevant checks to be performed.

6.6 Experimental Design

Although some testing guidelines, eg ICH, do not dictate an automatic repetition of experiments, it was considered appropriate to prove the reproducibility of all findings in this assay system. Consequently, 2 experiments were conducted in both the absence and the presence of S9 mix.

## Page 16 Test Facility Study No. 787718

The first test was performed using the Direct Plate Incorporation Method. If the first test was negative, the repeat test was performed using the Pre-incubation Method. If the first test was positive, the second test was also performed using the Direct Plate Incorporation Method to confirm the response.

Triplicate plates were poured for each exposure level (n = 6) and bacterial strain (n = 5) in the absence or presence of the exogenous activation system. Each plate was labelled with a number/colour code to identify the study, bacterial strain, treatment and activation type.

6.7 Justification of Dose Levels

The relevant testing guidelines recommend that a concentration limit of 5000 μg per plate should be used if no physicochemical property prevents this. As described in Section 6.2.1 above, the limited solubility of Alcohols, C18-22 Distn. Residues restricted the maximum concentration to 2000 μg per plate.

A toxicity test was performed to establish the range of concentrations to be used in the first mutation test. The concentrations tested were:

6, 20, 60, 200, 600 and 2000 μg per plate

No toxicity was observed in the preliminary toxicity test, therefore the concentrations tested in the first mutation experiment were as follows for all 5 bacterial strains, in both the absence and the presence of S9 mix:

6, 20, 60, 200, 600 and 2000 μg per plate

As neither toxicity nor mutagenic activity was observed in the first mutation assay, the concentrations tested in the second mutation experiment were as follows for all 5 bacterial strains, in both the absence and the presence of S9 mix:

6, 20, 60, 200, 600 and 2000 μg per plate

6.8 Administration of Test/Control Items

6.8.1 Vehicle Controls

DMSO was used as the vehicle control and was plated in triplicate with each strain used, both in the absence and in the presence of S9 mix.

## Page 17 Test Facility Study No. 787718

6.8.2 Positive Controls

The following positive control concentrations were plated in triplicate:

With S9 mix:

2-Aminoanthracene (2AAN): 2 μg per plate with S. typhimurium TA 1535 and TA 1537, 0.5 μg per plate with S. typhimurium TA 98 and TA 100 and 20 μg per plate with E. coli WP2 uvr A

Without S9 mix:

Sodium azide (NaN 3 

## ): 1 μg per plate with S. typhimurium TA 1535 and TA 100 9-Aminoacridine (9-AA): 80 μg per plate with S. typhimurium TA 1537 2-Nitrofluorene (2-NF): 1 μg per plate with S. typhimurium TA 98 N-Ethyl-N-nitro-N-nitrosoguanidine (ENNG): 2 μg per plate with E. coli WP2 uvr A

6.8.3 Test Item

All test item concentrations were plated in triplicate.

6.9 Experimental Procedure

The procedures used are based on the method of Ames et al (1975). Aseptic techniques, conducted under amber light, were used throughout this study.

All water used in the preparation of reagents was produced (in-house), by reverse osmosis followed by mixed-bed deionisation and sterilisation by autoclaving.

6.9.1 S9 Mix

For a general reference, see McGregor et al (1988).

S9 enzymes (supernatant post-mitochondrial fraction obtained after centrifugation at 9000 g ) were prepared in-house from the livers of Aroclor 1254-treated adult, male Fischer rats, as described by Ames et al (1975).

S9 enzymes were stored in sterile plastic tubes immersed in liquid nitrogen ( ca -196°C) and used within 6 months of preparation.

The enzymic activity of each batch of S9 enzymes was characterised by testing selected premutagens in an Ames test with S. typhimurium TA 1538. (Although strain TA 1538 is not used in the study, a considerable amount of historical data exists on enzymic activity with this

## Page 18 Test Facility Study No. 787718

S. typhimurium strain, and these data were used for comparison with current batches of S9 enzymes.) The results for the characterisation of the batch of S9 enzymes used in the mutation tests of the present study are shown in Appendix 2. S9 enzyme batches used must also have demonstrated, within each test, a satisfactory mutagenic response in cultures treated with 2-aminoanthracene (positive control).

To prepare S9 mix, 0.05 M phosphate buffer pH 7.4 was added to pre-weighed cofactors: nicotinamide adenine dinucleotide phosphate (NADP) disodium salt, glucose-6-phosphate (G-6-P) disodium salt, magnesium ions as MgCl 2 

## .6H 2 

## O and potassium ions as KCl, giving final concentrations in the ‘S9 mix’ of:

NADP N a 2 

## 4 mM (= 3.150 mg/mL) Glucose-6- p hosphate N a 2 

## 25 mM (= 7.605 mg/mL) MgC l 2 

## .6H 2 

## O 8 mM (= 1.626 mg/mL) KCl 33 mM (= 2.460 mg/mL)

This solution was immediately filter sterilised by passage through a 0.45 μm disposable filter assembly and mixed 9:1 (v/v) with the S9 enzymes.

6.9.2 Toxicity Tests

To establish suitable exposure levels for the first mutation test it was necessary to conduct an initial dose-finding test in the presence and absence of S9 mix. A single strain of bacteria, S. typhimurium TA 100 was used, and one plate per exposure level of test item was prepared. The test method was similar to that described below for the direct plate mutation assay (Section 6.9.3.1).

Plates were incubated at ca 37°C for 2 or 3 days. The numbers of revertant colonies were noted and the plates carefully examined, microscopically, for thinning of the background lawn of microcolonies. Condition of background lawn was assessed as normal, slightly thin lawn (ST), thin lawn (TL), very thin lawn (VT) or lawn absent (A). Any precipitation of the test item on the plates was noted.

6.9.3 Mutation Tests

Diluted agar (0.6% Difco Bacto-agar, 0.6% NaCl) was autoclaved and, just before use, supplemented as follows:

For S. typhimurium, sterile 1.0 mM L-histidine.HCl, 1.0 mM biotin solution was added at 50 mL per litre of soft agar. For E. coli , sterile 1.35 mM L-tryptophan solution was added at 10 mL per litre of soft agar. These soft agars were thoroughly mixed and kept in a water bath

at ca 45 ° C.

## Page 19 Test Facility Study No. 787718

6.9.3.1 Treatment Methods

Direct Plate Method: For this assay method, 2 mL of soft agar were dispensed into a small, plastic, sterile tube. S9 mix or 0.05 M phosphate buffer, pH 7.4 (0.5 mL) was added, followed by 0.1 mL of bacteria and, finally, the test item solution (0.1 mL). The tube contents (which were continually cooling) were mixed, then poured on to minimal medium plates prepared in-house. The minimal medium plates contained 20 mL of 1.5% BBL Purified Agar in Vogel-Bonner Medium E (Vogel and Bonner, 1956) with 2% glucose.

Pre-incubation Method: For this assay method, 0.5 mL of S9 mix or 0.05 M phosphate buffer, pH 7.4 was dispensed into a small, plastic, sterile tube followed by 0.1 mL of bacteria and, finally, the test solution (0.1 mL). The tubes were then placed in a shaking incubator at

ca 37 ° C for 20 min. After incubation, 2 mL of soft agar was added to each tube. The tube contents were then mixed and poured onto minimal medium plates (preparation described above).

When the soft agar had set, the plates were inverted and incubated at ca 37 ° C for 2 or 3 days and then examined. The numbers of mutant colonies on each plate were determined using a Sorcerer Colony Counter and captured electronically in a validated software system Ames Study Manager (both from Perceptive Instruments). The plates were also examined microscopically for precipitates and for microcolony growth (condition of background lawn was assessed as described in Section 6.9.2).

6.10 Quality Control of Bacterial Strains

All bacterial strains were plated (one plate per strain) onto complete medium and tested for ampicillin resistance (indicating the presence of pKM101) and crystal violet sensitivity (indicating persistence of the rfa mutation: the E. coli strain typically gives a +/- response to crystal violet). All bacterial strains were also checked for ultraviolet radiation sensitivity (indicating persistence of the uvr B mutation) and for essential amino acid requirement.

6.11 Calculations and Data Acceptance

6.11.1 Calculations

The mean number of mutant colonies, plus standard deviation, was calculated for each set of 3 plates. In addition, the fold-increase over the vehicle control was calculated for all test item and positive control treatments.

6.11.2 Historical Control Data

Historical vehicle and positive control values were used to assess the acceptability of the results. The current historical control data are shown in Appendix 3.

## Page 20 Test Facility Study No. 787718

6.11.3 Acceptance Criteria

A test was acceptable if the following occurred:

Each bacterial strain demonstrated typical responses to crystal violet, ampicillin and ultraviolet radiation.

At least 2 of the 3 vehicle control plates were within the following ranges for mean number of revertant colonies: S. typhimurium TA 1535: 4-30; TA 100: 60-200; TA 1537: 1-30; TA 98: 10-60 and E. coli WP2 uvrA : 1-60.

There were at least 2-fold increases over the mean vehicle control values in at least 2 of the 3 positive control plates for each strain and activation state (in the case of TA 100, at least 1.5-fold was required).

No toxicity or contamination was observed in at least 4 concentration levels.

In cases where a mutagenic response is observed, no more than one exposure level is discarded below the concentration that gives the highest mean colony number.

6.11.4 Interpretation of Mutagenicity

For S. typhimurium strains TA 1535, TA 1537, and TA 98 and for E. coli WP2 uvr A, at least a doubling of the mean concurrent vehicle control value was required before mutagenic activity was suspected. For S. typhimurium strain TA 100, a 1.5-fold increase over the control value was considered indicative of a mutagenic effect.

If the mean colony count on the vehicle control plates was less than 10, then a value of 10 was assumed for assessment purposes. In such cases, a minimum count of 20 (representing a 2-fold increase over 10) was required before a response was registered.

A concentration-related response was also required for identification of a mutagenic effect. At high concentrations, this relationship may be reversed, because of, for example, toxicity of the test item to the bacteria, specific toxicity of the test item to the mutants, or inhibition of S9 enzymes (where a mutagen requires metabolic activation by the S9 mix).

A response should be reproducible in an independent test.

No statistical analysis was performed in this study.

## Page 21 Test Facility Study No. 787718

7 RESULTS

7.1 Toxicity Tests

The results of the toxicity test using Alcohols, C18-22 Distn. Residues are shown in Table 1.

No toxicity to the bacteria was observed at the highest concentration of 2000 μg per plate in either the absence or the presence of S9 mix. Precipitated/undissolved test item was observed on the plates at 2000 μg per plate in both the absence and the presence of S9 mix (see Section 6.2.1).

7.2 Mutation Tests

The average numbers of his + 

## and trp + 

## revertant colonies, the individual plate counts, and the fold-increases over vehicle control value per dose level obtained in the mutation tests (conducted using both direct plate incorporation and pre-incubation methods) are shown in Table 2 and Table 3. Condition of background lawn is also noted.

7.2.1 Quality Control

The results of the Quality Control checks are shown in Appendix 4. All Salmonella strains were sensitive to crystal violet, whereas only the plasmid-containing Salmonella strains, TA 98 and TA 100, were resistant to ampicillin. All strains were also tested for sensitivity to ultraviolet radiation emitted over a period of 5-10 s from a source delivering a wavelength of 254 nm. Increased sensitivity to ultraviolet radiation was demonstrated. These results are consistent with the known properties of these strains of bacteria.

7.2.2 Vehicle Control Groups

The vehicle control values were within the normal/historical ranges recorded in this laboratory (Appendix 3) and reported in the literature with these strains of S. typhimurium and E. coli (Ames et al , 1975; Gatehouse et al , 1994).

7.2.3 Positive Control Groups

The positive control values were within the normal/historical ranges recorded in this laboratory for each bacterial strain and activation condition (Appendix 3).

## Page 22 Test Facility Study No. 787718

7.2.4 Alcohols, C18-22 Distn. Residues

No evidence of mutagenic activity was obtained with any strain in either test.

There was no toxicity to any of the strains of bacteria in either test.

Precipitated/undissolved test item was observed on the plates at 2000 μg per plate in both the absence and the presence of S9 mix in both tests (see Section 6.2.1).

## Page 23 Test Facility Study No. 787718

8 CONCLUSION

It is concluded that Alcohols, C18-22 Distn. Residues is not mutagenic in strains of Salmonella typhimurium and Escherichia coli, when tested in the absence and presence of metabolic activation up to and beyond its limit of solubility in the test system.

## Page 24 Test Facility Study No. 787718

9 REFERENCES

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.

Gatehouse D, Haworth S, Cebula T, Gocke E, Kier L, Matsushima T, Melcion C, Nohmi T, Veritt S and Zeiger E (1994). Recommendations for the performance of bacterial mutation assays. Mutation Res, 312 , 217-233.

Vogel H J and Bonner D M (1956). Acetylornithinase of E. coli : partial purification and some properties. J Biol Chem, 218 , 97-106.

McGregor D B, Edwards I, Riach C G, Cattanach P, Martin R, Mitchell A and Caspary W J (1988). Studies of an S9-based metabolic activation system used in the mouse lymphoma L5178Y cell mutation assay. Mutagenesis, 3 , 485-490.

## Page 25 Test Facility Study No. 787718

10 TABLES

Table 1 Toxicity Test

Without metabolic activation

Strain Compound Dose level per

plate Ratio treated / solvent Individual revertant colony counts

TA 100 6 μg 0.8 65 Alcohols, C18-22 Distn. Residues 20 μg 1.1 95 60 μg 1.4 116 200 μg 1.0 88 600 μg 1.1 95 2000 μg 0.9 79 P DMSO - - 84

Key to Plate Postfix Codes P Precipitate

With metabolic activation

Strain Compound Dose level per

plate Ratio treated / solvent Individual revertant colony counts

TA 100 6 μg 0.8 92 Alcohols, C18-22 Distn. Residues 20 μg 0.9 99 60 μg 0.9 103 200 μg 0.8 87 600 μg 0.8 84 2000 μg 0.9 104 P DMSO - - 110

Key to Plate Postfix Codes P Precipitate

## Page 26 Test Facility Study No. 787718

Table 2 First Mutation Assay (Direct Plate Incorporation Method)

Without metabolic activation

Strain Compound Dose level
per plate Mean
revertants per plate Standard Deviation Ratio treated / solvent Individual revertant colony counts

TA 1535 6 μg 15.0 3.5 0.8 11, 17, 17 20 μg 12.3 3.1 0.7 9, 13, 15 Alcohols, C18-22 Distn. Residues 60 μg 17.7 6.7 1.0 22, 10, 21 200 μg 15.3 2.5 0.8 18, 13, 15 600 μg 23.3 4.0 1.3 28, 21, 21 2000 μg 15.7 2.3 0.9 13 P, 17 P, 17 P DMSO - 18.3 8.1 - 9, 24, 22

TA 1537 6 μg 10.3 5.1 0.7 16, 9, 6 20 μg 11.0 1.0 0.7 10, 11, 12 Alcohols, C18-22 Distn. Residues 60 μg 9.0 2.6 0.6 6, 10, 11 200 μg 13.3 7.5 0.9 9, 22, 9 600 μg 10.0 3.0 0.7 13, 7, 10 2000 μg 12.3 2.3 0.8 15 P, 11 P, 11 P DMSO - 15.3 8.6 - 6, 17, 23

TA 98 6 μg 27.0 5.6 1.0 22, 26, 33 20 μg 22.7 1.5 0.8 23, 24, 21 Alcohols, C18-22 Distn. Residues 60 μg 25.3 5.1 0.9 24, 31, 21 200 μg 27.7 2.9 1.0 31, 26, 26 600 μg 26.0 4.4 0.9 28, 29, 21 2000 μg 23.5 10.6 0.8 16 P, 31 P, P C DMSO - 28.3 11.9 - 23, 20, 42

TA 100 6 μg 95.3 19.6 1.1 77, 93, 116 20 μg 102.0 3.5 1.2 100, 100, 106 Alcohols, C18-22 Distn. Residues 60 μg 103.0 2.0 1.2 101, 103, 105 200 μg 100.7 3.5 1.1 101, 97, 104 600 μg 93.3 7.0 1.1 86, 94, 100 2000 μg 104.0 19.7 1.2 120 P, 82 P, 110 P DMSO - 88.0 11.1 - 76, 90, 98

WP2uvrA 6 μg 10.0 3.0 1.4 13, 7, 10 20 μg 10.3 3.1 1.5 13, 7, 11 Alcohols, C18-22 Distn. Residues 60 μg 6.3 2.5 0.9 9, 4, 6 200 μg 5.3 1.5 0.8 4, 5, 7 600 μg 10.7 4.7 1.5 7, 16, 9 2000 μg 3.3 2.3 0.5 6 P, 2 P, 2 P DMSO - 7.0 3.0 - 10, 4, 7

Key to Plate Postfix Codes

P C Precipitate Contaminated

## Page 27 Test Facility Study No. 787718

Table 2 First Mutation Assay (Direct Plate Incorporation Method) (continued)

Without metabolic activation

Strain Compound Dose level
per plate Mean
revertants per plate Standard Deviation Ratio treated / solvent Individual revertant colony counts

TA 1535 NaN 3 1 μg 554.3 35.0 30.2 594, 541, 528 TA 1537 9AA 80 μg 4833.7 346.1 315.2 4484, 4841, 5176

TA 98 2NF 1 μg 880.0 19.7 31.1 859, 898, 883 TA 100 NaN 3 1 μg 989.7 45.1 11.2 938, 1021, 1010 WP2uvrA ENNG 2 μg 90.3 17.9 12.9 111, 79, 81

Key to Positive Controls

NaN 3 9AA 2NF ENNG Sodium Azide 9-Aminoacridine 2-Nitrofluorene N-Ethyl-N-Nitro-N-nitrosoguanidine

## Page 28 Test Facility Study No. 787718

Table 2 First Mutation Assay (Direct Plate Incorporation Method) (continued)

With metabolic activation

Strain Compound Dose level
per plate Mean
revertants per plate Standard Deviation Ratio treated / solvent Individual revertant colony counts

TA 1535 6 μg 14.0 2.6 0.9 13, 12, 17 20 μg 16.3 6.0 1.0 17, 10, 22 Alcohols, C18-22 Distn. Residues 60 μg 15.7 4.0 1.0 20, 12, 15 200 μg 11.7 6.5 0.7 12, 18, 5 600 μg 18.7 8.1 1.1 10, 26, 20 2000 μg 17.0 2.6 1.0 20 P, 16 P, 15 P DMSO - 16.3 0.6 - 16, 17, 16

TA 1537 6 μg 8.3 1.2 0.5 7, 9, 9 20 μg 17.0 10.0 1.0 27, 17, 7 Alcohols, C18-22 Distn. Residues 60 μg 16.0 5.0 1.0 21, 11, 16 200 μg 15.0 2.0 0.9 15, 17, 13 600 μg 15.7 2.3 0.9 13, 17, 17 2000 μg 13.0 5.2 0.8 19 P, 10 P, 10 P DMSO - 16.7 1.5 - 17, 18, 15

TA 98 6 μg 37.3 11.5 1.2 24, 44, 44 20 μg 37.3 7.8 1.2 46, 31, 35 Alcohols, C18-22 Distn. Residues 60 μg 42.3 10.4 1.3 34, 54, 39 200 μg 33.3 9.5 1.0 43, 24, 33 600 μg 44.7 1.5 1.4 45, 43, 46 2000 μg 41.7 2.1 1.3 44 P, 40 P, 41 P DMSO - 32.0 6.9 - 28, 28, 40

TA 100 6 μg 95.0 9.8 1.0 106, 87, 92 20 μg 102.3 12.5 1.1 108, 88, 111 Alcohols, C18-22 Distn. Residues 60 μg 106.3 12.9 1.1 92, 110, 117 200 μg 103.0 5.3 1.1 101, 99, 109 600 μg 114.3 10.3 1.2 123, 103, 117 2000 μg 104.7 6.4 1.1 101 P, 112 P, 101 P DMSO - 96.7 8.1 - 104, 98, 88

WP2uvrA 6 μg 13.7 2.9 1.0 12, 12, 17 20 μg 5.0 0.0 0.4 5, 5, 5 Alcohols, C18-22 Distn. Residues 60 μg 10.7 2.1 0.8 9, 10, 13 200 μg 7.0 2.0 0.5 7, 9, 5 600 μg 10.0 1.0 0.8 9, 11, 10 2000 μg 6.3 2.3 0.5 5 P, 5 P, 9 P DMSO - 13.3 1.5 - 15, 13, 12

Key to Plate Postfix Codes P Precipitate

## Page 29 Test Facility Study No. 787718

Table 2 First Mutation Assay (Direct Plate Incorporation Method) (continued)

With metabolic activation

Strain Compound Dose level
per plate Mean
revertants per plate Standard Deviation Ratio treated / solvent Individual revertant colony counts

TA 1535 2AAN 2 μg 629.0 28.2 38.5 597, 640, 650 TA 1537 2AAN 2 μg 434.7 45.2 26.1 486, 417, 401

TA 98 2AAN 0.5 μg 509.3 66.5 15.9 563, 530, 435 TA 100 2 AAN 0.5 μg 1093.3 72.2 11.3 1137, 1010, 1133 WP2uvrA 2AAN 20 μg 519.7 13.6 39.0 518, 534, 507

Key to Positive Controls
2AAN 2-Aminoanthracene

## Page 30 Test Facility Study No. 787718

Table 3 Second Mutation Assay (Pre-incubation Method)

Without metabolic activation

Strain Compound Dose level
per plate Mean
revertants per plate Standard Deviation Ratio treated / solvent Individual revertant colony counts

TA 1535 6 μg 12.0 4.6 1.2 16, 7, 13 20 μg 10.3 0.6 1.0 11, 10, 10 Alcohols, C18-22 Distn. Residues 60 μg 13.3 3.2 1.3 12, 11, 17 200 μg 15.7 2.5 1.5 16, 18, 13 600 μg 13.7 2.9 1.3 12, 17, 12 2000 μg 14.0 6.1 1.4 18 P, 7 P, 17 P DMSO - 10.3 2.9 - 12, 12, 7

TA 1537 6 μg 10.3 1.2 0.8 11, 11, 9 20 μg 10.7 4.0 0.8 15, 7, 10 Alcohols, C18-22 Distn. Residues 60 μg 10.3 1.2 0.8 9, 11, 11 200 μg 11.0 2.0 0.9 9, 13, 11 600 μg 12.0 6.6 0.9 13, 5, 18 2000 μg 15.0 3.0 1.2 18 P, 15 P, 12 P DMSO - 12.7 2.5 - 15, 13, 10

TA 98 6 μg 25.3 3.8 1.0 27, 21, 28 20 μg 18.7 4.7 0.7 17, 24, 15 Alcohols, C18-22 Distn. Residues 60 μg 26.7 2.5 1.0 29, 24, 27 200 μg 28.3 8.5 1.1 20, 37, 28 600 μg 22.7 8.3 0.9 20, 32, 16 2000 μg 21.0 2.6 0.8 19 P, 20 P, 24 P DMSO - 25.7 1.5 - 24, 27, 26

TA 100 6 μg 76.3 11.9 0.9 90, 71, 68 20 μg 89.0 19.3 1.0 103, 67, 97 Alcohols, C18-22 Distn. Residues 60 μg 97.3 25.7 1.1 84, 127, 81 200 μg 91.7 5.5 1.0 98, 89, 88 600 μg 101.3 14.6 1.1 117, 88, 99 2000 μg 90.0 9.8 1.0 101 P, 87 P, 82 P DMSO - 88.3 2.1 - 86, 89, 90

WP2uvrA 6 μg 8.3 1.2 1.8 7, 9, 9 20 μg 8.7 3.2 1.9 10, 5, 11 Alcohols, C18-22 Distn. Residues 60 μg 13.3 3.8 2.9 15, 9, 16 200 μg 6.0 2.6 1.3 9, 4, 5 600 μg 6.0 1.0 1.3 6, 7, 5 2000 μg 9.7 0.6 2.1 9 P, 10 P, 10 P DMSO - 4.7 4.0 - 1, 4, 9

Key to Plate Postfix Codes P Precipitate

## Page 31 Test Facility Study No. 787718

Table 3 Second Mutation Assay (Pre-incubation Method) (continued)

Without metabolic activation

Strain Compound Dose level
per plate Mean
revertants per plate Standard Deviation Ratio treated / solvent Individual revertant colony counts

TA 1535 NaN 3 1 μg 513.7 37.8 49.7 555, 481, 505 TA 1537 9AA 80 μg 4223.3 423.4 333.4 4308, 3764, 4598

TA 98 2NF 1 μg 608.0 90.8 23.7 710, 536, 578 TA 100 NaN 3 1 μg 1082.0 55.1 12.2 1139, 1078, 1029 WP2uvrA ENNG 2 μg 153.3 23.0 32.9 176, 154, 130

Key to Positive Controls

NaN 3 9AA 2NF ENNG Sodium Azide 9-Aminoacridine 2-Nitrofluorene N-Ethyl-N-Nitro-N-nitrosoguanidine

## Page 32 Test Facility Study No. 787718

Table 3 Second Mutation Assay (Pre-incubation Method) (continued)

With metabolic activation

Strain Compound Dose level
per plate Mean
revertants per plate Standard Deviation Ratio treated / solvent Individual revertant colony counts

TA 1535 6 μg 10.3 1.5 0.9 9, 10, 12 20 μg 11.7 0.6 1.0 11, 12, 12 Alcohols, C18-22 Distn. Residues 60 μg 18.0 1.7 1.5 20, 17, 17 200 μg 15.7 4.5 1.3 20, 11, 16 600 μg 15.7 6.0 1.3 22, 15, 10 2000 μg 18.0 2.0 1.5 20 P, 18 P, 16 P DMSO - 11.7 1.5 - 12, 10, 13

TA 1537 6 μg 10.0 5.2 1.0 13, 13, 4 20 μg 10.3 4.5 1.0 15, 6, 10 Alcohols, C18-22 Distn. Residues 60 μg 18.0 2.6 1.7 16, 21, 17 200 μg 12.0 5.6 1.2 11, 7, 18 600 μg 10.3 1.5 1.0 10, 9, 12 2000 μg 11.7 5.7 1.1 10 P, 7 P, 18 P DMSO - 10.3 2.3 - 9, 13, 9

TA 98 6 μg 23.7 3.1 0.8 21, 27, 23 20 μg 24.0 2.0 0.8 22, 24, 26 Alcohols, C18-22 Distn. Residues 60 μg 37.7 4.5 1.3 38, 42, 33 200 μg 23.7 2.1 0.8 26, 23, 22 600 μg 32.3 4.6 1.1 27, 35, 35 2000 μg 31.3 2.9 1.0 33 P, 33 P, 28 P DMSO - 30.0 3.6 - 26, 33, 31

TA 100 6 μg 96.0 15.6 0.9 78, 104, 106 20 μg 96.7 8.6 0.9 106, 89, 95 Alcohols, C18-22 Distn. Residues 60 μg 104.0 6.0 1.0 110, 104, 98 200 μg 103.0 6.1 0.9 100, 99, 110 600 μg 100.0 5.6 0.9 94, 101, 105 2000 μg 96.0 3.5 0.9 98 P, 92 P, 98 P DMSO - 109.3 12.0 - 110, 121, 97

WP2uvrA 6 μg 13.7 4.2 1.5 9, 15, 17 20 μg 8.0 6.1 0.9 11, 12, 1 Alcohols, C18-22 Distn. Residues 60 μg 10.3 1.5 1.1 12, 9, 10 200 μg 13.0 2.6 1.4 11, 16, 12 600 μg 15.7 6.0 1.7 22, 10, 15 2000 μg 9.7 1.5 1.0 10 P, 8 P, 11 P DMSO - 9.3 3.1 - 6, 10, 12

Key to Plate Postfix Codes P Precipitate

## Page 33 Test Facility Study No. 787718

Table 3 Second Mutation Assay (Pre-incubation) (continued)

With metabolic activation

Strain Compound Dose level
per plate Mean
revertants per plate Standard Deviation Ratio treated / solvent Individual revertant colony counts

TA 1535 2AAN 2 μg 368.7 25.3 31.6 396, 346, 364 TA 1537 2AAN 2 μg 224.3 40.7 21.7 270, 192, 211

TA 98 2AAN 0.5 μg 697.7 33.3 23.3 676, 681, 736 TA 100 2 AAN 0.5 μg 843.7 67.5 7.7 920, 792, 819 WP2uvrA 2AAN 20 μg 469.3 26.3 50.3 483, 486, 439

Key to Positive Controls
2AAN 2-Aminoanthracene

## Page 34 Test Facility Study No. 787718

11 APPENDICES

Appendix 1 Certificate of Analysis

## Page 35 Test Facility Study No. 787718

Appendix 2 In vitro Activation Preparation Form

In vitro Activation Preparation Form Activation Batch: FLI 112

PREPARATION Date Prepared: 15 July 2009

Operators: Wanda Zajac/Colin Riachl Date of Expiry: 15 January 2010

Pawel lurczak

Animal: Rat Number of animals: 20

Sex: Male Average animal weight (g): 232g

Strain: Fischer 344 Date induced: 10 July 2009
Supplier: Har1an UK Inducer: Aroclor 1254
Organ: Liver Supplier: Monsanto (UK) Limited

Total grams of organ: 194 g Preparation solution: 0.15 M KC1

Details of spin: 9,000 g supernatant Number of vials prepared: 74 x 5 mL

Total volume prepared: 577mL 41x3mL
Storage temperature: -196 c C 42 x2 mL
Sterility check: Sterile
METABOLIC ACTIVATION

Operators: Wanda Zajac/Pawel Jurczak Culture batch: G169 Strain: 

### s. typhimurium TA 1538 Batch No. (plates): 000766
Date plated: 17 July 2009 Date cOlmted: 20 July 2009

Substance Quantity per Revertant Colonies per Plate

Plate Mean
Dimethylsulphoxide 100 ML 21 24 22 22

2-Arninoanthracene 0.5 Mg 931 897 899 909
2-Acetylaminofluorene 10 Mg 1700 1805 1901 1802
4-Acetylaminofluorene 1 mg 116 137 121 125

Benzo( o:)pyrene 5 Mg 457 422 428 436
Dirnethylaminoazobenzene 100 Mg 71 88 83 81
QUALITY ASSURANCE

Auditor: Alison McNaughton HNC
Date Audited: 27 July 2009 Date Signed-off: 04 August 2009

## Page 36 Test Facility Study No. 787718

Appendix 3 Historical Positive and Negative Control Data

Ames Test Historical Vehicle and Positive Control Data 2005-2009

Vehicle Controls (Pooled)

- Presence of S9 Mix

Strain TA 1535 TA 1537 TA98 TA 100 VVP2uvrA

Mean 15 17 32 94 9

Standard Deviation 6 7 9 17 4
Range 

## I Min 4 2 13 59 1 

## I Max 31 35 62 160 24

No. of Plates 227 225 225 264 222

Vehicle Controls (Pooled)

- Absence of S9 Mix

Strain TA 1535 TA 1537 TA 98 TA 100 VVP2uvrA

Mean 15 13 23 92 9

Standard Deviation 5 6 7 19 4
Range 

## I Min 5 1 10 60 1 

## I Max 30 31 44 163 23

No. of Plates 224 222 225 264 222

Positive Controls

- Presence of S9 Mix

Strain TA 1535 TA 1537 TA98 TA 100 VVP2uvrA

Substance 2AAN 2AAN 2AAN 2AAN 2AAN

Concentration ()lg per plate) 2 2 0.5 0.5 20

Mean 428 288 510 735 557

Standard Deviation 133 127 218 337 171
Range 

## I Min 92 66 165 331 125 

## I Max 821 765 1646 2468 1103

No. of Plates 222 222 222 225 216

Positive Controls

- Absence of S9 Mix

Strain TA 1535 TA 1537 TA98 TA 100 WP2uvrA

Substance NaN 3 9AA 2NF NaN 3 ENNG

Concentration ()lg per plate) 1 80 1 1 2

Mean 495 5727 958 1116 227

Standard Deviation 106 2102 474 397 122
Range 

## I Min 263 152 331 551 45 

## I Max 882 10354 7084 3553 673

No. of Plates 222 222 225 225 222

Audited: Alison McNaughton HNC Date audited: 21 April 2009 Date signed off: 21 April 2009

## Page 37 Test Facility Study No. 787718

Appendix 4 Quality Control Checks of Bacterial Cultures

First mutation assay (Direct Plate Incorporation Method) Ultraviolet Sensitivity

Cell Type No. of Bacteria Added per

Plate in Mutation Test (x10 8 ) Treated (Colonies) Untreated (Colonies) Resistant to Ampicillin Zone (mm) Sensitive to Crystal

Violet Zone (mm) Amino Acid Requirement

S. typhimurium TA 1535 3.13 0 313 - 20 + 11 Yes

S. typhimurium TA 1537 2.41 0 241 - 21 + 17 Yes

S. typhimurium TA 98 1.38 0 138 + 0 + 13 Yes

S. typhimurium TA 100 2.04 0 204 + 0 + 16 Yes

E. coli WP2 uvr A 2.14 0 214 - 18 +/- 8 Yes

S. typhimurium (Wild Type) N/A 71 382 N/A N/A N/A N/A N/A

Second mutation assay (Pre-incubation Method) Ultraviolet Sensitivity

Cell Type No. of Bacteria Added per

Plate in Mutation Test (x10 8 ) Treated (Colonies) Untreated (Colonies) Resistant to Ampicillin Zone (mm) Sensitive to Crystal

Violet Zone (mm) Amino Acid Requirement

S. typhimurium TA 1535 2.18 0 214 - 21 + 18 Yes

S. typhimurium TA 1537 1.81 0 181 - 22 + 18 Yes

S. typhimurium TA 98 1.00 0 100 + 0 + 15 Yes

S. typhimurium TA 100 1.56 0 156 + 0 + 15 Yes

E. coli WP2 uvr A 2.30 0 230 - 19 +/- 10 Yes

S. typhimurium (Wild Type) N/A 32 205 N/A N/A N/A N/A N/A

N/A Not Applicable + Positive

- Negative