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 WP2uvrA 

TEST FACILITY: 

Charles River 

Tranent 

Edinburgh 

EH33 2NE 

UK 

SPONSOR: 

SASOL Germany GmbH 

Anckelmannsplatz 1 

D-20537 Hamburg 

Germany 

Page 1 of 37 
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 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 
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 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 
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 Test Facility Study No. 787718 

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

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

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

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

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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 
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 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 
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 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

WP2uvrA. 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 presence 

of 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. 
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 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. 
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 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 
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 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.) 
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 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 uvrB, renders the 
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 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 WP2uvrA 

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 trpE locus and can be mutated to 

tryptophan-independence either by a base-pair reversion of an A-T base-pair in the trpE 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 uvrA 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. 
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 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. 
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 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 WP2uvrA 

Without S9 mix: 

Sodium azide (NaN3): 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 WP2uvrA 

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 
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 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 MgCl2.6H2O and potassium ions as KCl, giving 

final concentrations in the ‘S9 mix’ of: 

NADP Na2 4 mM (= 3.150 mg/mL) 

Glucose-6-phosphate Na2 25 mM (= 7.605 mg/mL) 

MgCl2.6H2O 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. 
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 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 uvrB 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. 
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 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 WP2uvrA: 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 WP2uvrA, 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. 
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 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). 
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 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). 
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 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. 
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 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 Alcohols, C18-22 6 μg 0.8 65 

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 Alcohols, C18-22 6 μg 0.8 92 

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 NaN3 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 NaN3 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 

NaN3

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 2AAN 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 NaN3 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 NaN3 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 

NaN3

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 2AAN 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: -196cC 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 NaN3 9AA 2NF NaN3 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 

(x108

) 

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 WP2uvrA 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 

(x108

) 

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 WP2uvrA 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