PAGE 1 OF 23 PAGES 

REVERSE MUTATION ASSAY "AMES TEST" 

USING SALMONELLA TYPHIMURIUM AND 

ESCHERICHIA COLI 

STUDY SPONSOR: 

41204331.doc/p 

PROJECT NUMBER: 41204331 

AUTHOR: P W Thompson 

TEST FACILITY: 

Harlan Laboratories Ltd 

Shardlow Business Park 

Shard low 

Derbyshire 

DE72 2GD 

UK 

Telephone: +44 (0) 1332 792896 

Facsimile: +44 (0) 1332 799018 
PROJECT NUMBER: 41204331 PAGE2 

QUALITY ASSURANCE REPORT 

This study type is classed as short-term. The General Study Plan for this study type was 

reviewed for compliance once only on initial production. Inspection of the routine and 

repetitive procedures that constitute the study is carried out as a continuous process 

designed to encompass the major phases of this study type at least once every three 

months. 

In addition, general facilities are inspected at least once a year and the results are 

reported to management. 

This report has been audited by the Quality Assurance Unit, and is considered to be an 

accurate account of the data generated and of the procedures followed. 

In each case, the outcome of QA evaluation is reported to the Study Director and 

Management on the day of evaluation. Audits of study documentation, and process 

inspections appropriate to the type and schedule of this study were as follows: 

§ 28 August 2012 

15 October 2012 

10 October 2012 

22 October 2012 

15 October 2012 

§ 21 November 2012 

§ Date of QA Signature 

Study Plan Compliance Audit 

Test Item Preparation 

Test System Preparation 

Exposure 

Assessment of Response 

Draft Report Audit 

Final Report Audit 

§ Evaluation specific to this study 

. 0 4 DEC 2012 DATE ................................................ . 

For the Quality Assurance Unit* 

*Authorised QA Signatures: 

Senior Audit Staff: J G Riley BSc (Hons) MRQA, J M Crowther MIScT MRQA, 

G Wren ONC MRQA, S Bevan BSc (Hons) MRQA, L Blaney MRQA 
PROJECT NUMBER: 41204331 PAGE3 

GLP COMPLIANCE STATEMENT 

With the exception noted below the work described was performed in compliance with 

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 

1999/3106 as amended by Sl 2004/0994 )). These Regulations are in accordance with 

GLP standards published as OECD Principles on Good Laboratory Practice (revised 

1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the 

requirements of Directives 2004/9/EC and 2004/1 0/EC. 

No analysis was carried out to determine the homogeneity, concentration or stability of 

the test item formulation. The test item was formulated within four hours of it being 

applied to the test system. It is assumed that the formulation was stable for this duration. 

This exception is considered not to affect the purpose or integrity of the study. 

This report fully and accurately reflects the procedures used and data generated. 

P W Thompson HNC 

Study Director 

0 3 DEC 2012 

DATE: .................................................. . 

This report may be presented in final form as a digital (pdf) document. Such documents are prepared by scanning the paper original, 

and are considered of equivalent integrity and authenticity to versions produced by optical photocopy. However, in all cases the 

hand-signed paper original, held in secure archives, is the definitive document. 
PROJECT NUMBER: 41204331 

CONTENTS 

QUALITY ASSURANCE REPORT 

GLP COMPLIANCE STATEMENT 

CONTENTS 

SUMMARY 

1. INTRODUCTION 

2. 

3. 

4. 

5. 

6. 

7. 

TEST ITEM 

METHODS 

3.1 Tester Strains 

3.2 Preparation of Test and Reference Items 

3.3 Microsomal Enzyme Fraction 

3.4 S9-Mix and Agar 

3.5 Test Procedure 

3.6 Acceptance Criteria 

3. 7 Evaluation Criteria 

ARCHIVES 

RESULTS 

5.1 Preliminary Toxicity Test 

5.2 Mutation Test 

CONCLUSION 

REFERENCES 

Spontaneous Mutation Rates (Concurrent Negative Controls) 

Test Results: Experiment 1 -Without Metabolic Activation 

Test Results: Experiment 1 -With Metabolic Activation 

Test Results: Experiment 2- Without Metabolic Activation 

Test Results: Experiment 2- With Metabolic Activation 

History Profile of Vehicle and Positive Control Values 

PAGE4 

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5 

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Table 1 

Table 2 

Table 3 

Table 4 

Table 5 

Appendix 1 

Appendix 2 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 23 
PROJECT NUMBER: 41204331 

REVERSE MUTATION ASSAY "AMES TEST" 

USING SALMONELLA TYPHIMURIUM AND 

ESCHERICHIA COLI 

SUMMARY 

PAGES 

Introduction. The test method was designed to be compatible with the guidelines for 

bacterial mutagenicity testing published by the major Japanese Regulatory Authorities 

including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 

471 "Bacterial Reverse Mutation Test", Method 813/14 of Commission Regulation (EC) 

number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised 

guidelines. 

Methods. Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA 100 and 

Escherichia coli strain WP2uvrA were treated with the test item, , using both the 

Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, 

both with and without the addition of a rat liver homogenate metabolising system (1 0°/o 

liver S9 in standard co-factors). The dose range for the first experiment was determined 

in a preliminary toxicity assay and was 50 to 5000 J..Jg/plate. The experiment was 

repeated on a separate day (pre-incubation method) using the same dose range as 

Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations. 

Results. The vehicle (sterile distilled water) control plates gave counts of revertant 

colonies within the normal range. All of the positive controls used in the test induced 

marked increases in the frequency of revertant colonies, both with or without metabolic 

activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were 

validated. 

The test item caused no visible reduction in the growth of the bacterial background lawn 

at any dose level and was, therefore, tested up to the maximum recommended dose 

level of 5000 J..Jg/plate. No test item precipitate was observed on the plates at any of the 

doses tested in either the presence or absence of S9-mix. 

No significant increases in the frequency of revertant colonies were recorded for any of 

the bacterial strains, with any dose of the test item, either with or without metabolic 

activation or exposure method. 

Conclusion. The test item, , was considered to be non-mutagenic under the 

conditions of this test. 
PROJECT NUMBER: 41204331 

REVERSE MUTATION ASSAY "AMES TEST" 

USING SALMONELLA TYPHIMURIUM AND 

ESCHERICHIA COLI 

1. INTRODUCTION 

PAGE6 

The purpose of the study was to evaluate the test item, , for the ability to induce 

reverse mutations, either directly or after metabolic activation, at the histidine or 

tryptophan locus in the genome of five strains of bacteria. 

The test method was designed to be compatible with the guidelines for bacterial 

mutagenicity testing published by the major Japanese Regulatory Authorities including 

METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 

"Bacterial Reverse Mutation Test", Method 813/14 of Commission Regulation (EC) 

number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised 

guidelines. A copy of the Certificate of Compliance with GLP, issued by the UK 

Department of Health, is included. 

The study was based on the in vitro technique described by Ames eta/ (1975), Maron 

and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are 

determined by exposing mutant strains of Salmonella typhimurium to various 

concentrations of the test item. These strains have a deleted excision repair mechanism 

which makes them more sensitive to various mutagens and they will not grow on media 

which does not contain histidine. When large numbers of these organisms are exposed 

to a mutagen, reverse mutation to the original histidine independent form takes place. 

These are readily detectable due to their ability to grow on a histidine deficient medium. 

Using these strains of Salmonella typhimurium revertants may be produced after 

exposure to a chemical mutagen, which have arisen as a result of a base-pair 

substitution in the genetic material (miscoding) or as a frameshift mutation in which 

genetic material is either added or deleted. Additionally, a mutant strain of Escherichia 

coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base 

substitution to tryptophan independence (Green and Muriel (1976)) is used to 

complement the Salmonella strains. 

Since many compounds do not exert a mutagenic effect until they have been 

metabolised by enzyme systems not available in the bacterial cell, the test item and the 

bacteria are also incubated in the presence of a liver microsomal preparation (S9-mix) 
PROJECT NUMBER: 41204331 PAGE 7 

prepared from rats pre-treated with a mixture known to induce an elevated level of these 

enzymes. 

The experimental phase of this study was performed between 22 August 2012 and 

18 October 2012. 

2. TEST ITEM 

Sponsor's identification 

Description 

Purity 

Batch number 

Date received 

Expiry date 

Storage conditions 

The integrity of supplied data relating to the identity, purity and stability of the test item is 

the responsibility of the Sponsor. 

3. METHODS 

3.1 Tester Strains 

The five strains of bacteria used, and their mutations, are as follows: 

Salmonella typhimurium 

Strains Genotype 

TA 1537 his C 3076; rfa-; uvra-: 

TA98 his D 3052; rfa-; uvrB-;R-factor 

TA 1535 his G 46; rfa-; uvra-: 

TA100 his G 46; rfa-; uvrB-;R-factor 

Escherichia coli 

Strain Genotype 

WP2uvrA trp-; uvrA-: 

Type of mutations indicated 

frame shift mutations 

base-pair substitutions 

Type of mutations indicated 

base-pair substitution 

All of the Salmonella strains are histidine dependent by virtue of a mutation through the 

histidine operon and are derived from S. typhimurium strain L T2 through mutations in the 

histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty 

lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability 

to larger molecules. A further mutation, through the deletion of the uvrB-bio gene, 
PROJECT NUMBER: 41204331 PAGES 

causes an inactivation of the excision repair system and a dependence on exogenous 

biotin. In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical 

and UV-induced mutagenesis via an increase in the error-prone repair pathway. The 

plasmid also confers ampicillin resistance which acts as a convenient marker 

(Mortlemans and Zeiger (2000)). In addition to a mutation in the tryptophan operon, the 

E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity 

to some mutagenic compounds. This deficiency allows the strain to show enhanced 

mutability as the uvrA repair system would normally act to remove and repair the 

damaged section of the DNA molecule (Green and Muriel (1976)). 

The strains of bacteria used in the test were obtained from the University of California, 

Berkeley, on culture discs, on 04 August 1995 and the British Industrial Biological 

Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains 

were stored at approximately -196°C in a Statebourne liquid nitrogen freezer, model 

SXR 34. 

In this assay, overnight sub-cultures of the appropriate coded stock cultures were 

prepared in nutrient broth (Oxoid Limited; lot numbers 1014570 03/16 and 1078364 

08/16) and incubated at 37°C for approximately 10 hours. Each culture was monitored 

spectrophotometrically for turbidity with titres determined by viable count analysis on 

nutrient agar plates. 

3.2 Preparation of Test and Reference Items 

The test item was fully miscible in sterile distilled water at 50 mg/ml in solubility checks 

performed in-house. Sterile distilled water was therefore selected as the vehicle. 

The test item was accurately weighed and approximate half-log dilutions prepared in 

sterile distilled water by mixing on a vortex mixer on the day of each experiment. 

Formulated concentrations were adjusted to allow for the stated active ingredient 

(60°/o w/v) of the test item. All formulations were used within four hours of preparation 

and were assumed to be stable for this period. Analysis for concentration, homogeneity 

and stability of the test item formulations is not a requirement of the test guidelines and 

was, therefore, not determined. This is an exception with regard to GLP and has been 

reflected in the GLP compliance statement. 
PROJECT NUMBER: 41204331 PAGE9 

A solvent treatment group was used as the vehicle control and the positive controls used 

in the series of plates without S9-mix were as follows: 

N-ethyi-N'-nitro-N-nitrosoguanidine (ENNG): 

N-ethyi-N'-nitro-N-nitrosoguanidine (ENNG): 

N-ethyi-N'-nitro-N-nitrosoguanidine (ENNG): 

9-Aminoacridine (9AA): 

4-Nitroquinoline-1-oxide (4NQO): 

2 J.,Jg/plate for WP2uvrA 

3 J.,Jg/plate forT A 1 00 

5 J.,Jg/plate for TA1535 

80 J.,Jg/plate forT A 1537 

0.2 J.,Jg/plate for TA98 

In addition, 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP), which are 

non-mutagenic in the absence of metabolising enzymes, were used in the series of 

plates with S9-mix at the following concentrations: 

2-Aminoanthracene (2AA): 

2-Aminoanthracene (2AA): 

2-Aminoanthracene (2AA): 

Benzo(a)pyrene (BP): 

3.3 Microsomal Enzyme Fraction 

1 J.,Jg/plate for T A 1 00 

2 J.,Jg/plate for TA 1535 and TA 1537 

1 0 J.,Jg/plate for WP2uvrA 

5 J.,Jg/plate for TA98 

The S9 Microsomal fraction was prepared in-house (01 July 2012 (preliminary toxicity 

test and Experiment 1) and 16 September 2012 (Experiment 2)) from rats induced with 

Phenobarbitone/~-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to 

preparation on day 4. The S9 homogenate was produced by homogenising the liver in a 

0.15M KCI solution (1 g liver to 3 ml KCI) followed by centrifugation at 9000 g. The 

protein content of the resultant supernatant was adjusted to 20 mg/ml. Aliquots of the 

supernatant were frozen and stored at approximately -196°C. Prior to use, each batch of 

S9 was tested for its capability to activate known mutagens in the Ames test. 

3.4 59-Mix and Agar 

The S9-mix was prepared immediately before use using sterilised co-factors and 

maintained on ice for the duration of the test. 
PROJECT NUMBER: 41204331 

S9 

1.65 M KCI/0.4 M MgCb 

0.1 M Glucose-6-phosphate 

0.1 M NADP 

0.2 M Sodium phosphate buffer (pH 7.4) 

Sterile distilled water 

5.0 ml 

1.0 ml 

2.5 ml 

2.0 ml 

25.0 ml 

14.5 ml 

PAGE10 

A 0.5 ml aliquot of S9-mix and 2 ml of molten, trace histidine or tryptophan 

supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in 

order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on 

the day of each experiment. 

Top agar was prepared using 0.6°/o Bacto agar (lot number 1297425 09/16) and 0.5°/o 

sodium chloride with 5 ml of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan 

solution added to each 1 00 ml of top agar. Vogel-Bonner Minimal agar plates were 

purchased SGL Ltd (lot numbers 32699 09/12, 32841 10/12 and 32927 11/12). 

3.5 Test Procedure 

3.5.1 Preliminary Toxicity Test 

In order to select appropriate dose levels for use in the main test, a preliminary test was 

carried out to determine the toxicity of the test item. The concentrations tested were 

0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 j..Jg/plate. The test was performed 

by mixing 0.1 ml of bacterial culture (TA 100 or WP2uvrA), 2 ml of molten, trace histidine 

or tryptophan supplemented, top agar, 0.1 ml of test item formulation and 0.5 ml of 

S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal 

agar (30 ml/plate). Ten concentrations of the test item formulation and a vehicle control 

(sterile distilled water) were tested. In addition, 0.1 ml of the maximum concentration of 

the test item and 2 ml of molten, trace histidine or tryptophan supplemented, top agar 

were overlaid onto a sterile nutrient agar plate in order to assess the sterility of the test 

item. After approximately 48 hours incubation at 37°C the plates were assessed for 

numbers of revertant colonies using a colony counter and examined for effects on the 

growth of the bacterial background lawn. 
PROJECT NUMBER: 41204331 PAGE 11 

3.5.2 Mutation Test - Experiment 1 

Five concentrations of the test item (50, 150, 500, 1500, 5000 jJg/plate) were assayed in 

triplicate against each tester strain, using the direct plate incorporation method. 

Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of 

test tubes followed by 2 ml of molten, trace histidine or tryptophan supplemented, top 

agar, 0.1 ml of the vehicle, test item formulation or positive control and either 0.5 ml of 

S9-mix or phosphate buffer. The contents of each test tube were mixed and equally 

distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). 

This procedure was repeated, in triplicate, for each bacterial strain and for each 

concentration of test item both with and without S9-mix. 

All of the plates were incubated at 37°C for approximately 48 hours and the frequency of 

revertant colonies assessed using a colony counter. 

3.5.3 Mutation Test .. Experiment 2 

The second experiment was performed using fresh bacterial cultures, test item and 

control solutions. The test item dose range was the same as Experiment 1 (50 to 

5000 jJg/plate). 

As it is good scientific practice to alter one condition in the replicate assay, the exposure 

condition was changed from plate incorporation to pre-incubation. The test item 

formulations and vehicle control were therefore dosed as follows: 

Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of 

test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.1 ml of the vehicle or 

test item formulation and incubated for 20 minutes at 37°C with shaking at approximately 

130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan 

supplemented, top agar. The contents of the tube were then mixed and equally 

distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). 

This procedure was repeated, in triplicate, for each bacterial strain and for each 

concentration of test item both with and without S9-mix. The positive and untreated 

controls were dosed using the standard plate incorporation method described in Section 

3.5.2. 
PROJECT NUMBER: 41204331 PAGE12 

All of the plates were incubated at 37°C for approximately 48 hours and the frequency of 

revertant colonies assessed using a colony counter. 

3.6 Acceptance Criteria 

The reverse mutation assay may be considered valid if the following criteria are met: 

All bacterial strains must have demonstrated the required characteristics as determined 

by their respective strain checks according to Ames et a/ (1975), Maron and Ames 

(1983) and Mortelmans and Zeiger (2000). 

All tester strain cultures should exhibit a characteristic number of spontaneous revertants 

per plate in the vehicle and untreated controls. Acceptable ranges are presented in the 

General Study Plan, Section 4 (negative controls). Combined historical negative and 

solvent control ranges for 2010 and 2011 are presented in Appendix 1. 

All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mi. 

Diagnostic mutagens (positive control chemicals) must be included to demonstrate both 

the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the 

S9-mix. All of the positive control chemicals used in the study should induce marked 

increases in the frequency of revertant colonies, both with or without metabolic 

activation. The historical ranges of the positive controls for 2010 and 2011 are 

presented in Appendix 1. 

There should be a minimum of four non-toxic test item dose levels. 

There should be no evidence of excessive contamination. 

3. 7 Evaluation Criteria 

There are several criteria for determining a positive result. Any, one, or all of the 

following can be used to determine the overall result of the study: 

1. A dose-related increase in mutant frequency over the dose range tested (De 

Serres and Shelby (1979)). 
PROJECT NUMBER: 41204331 PAGE13 

2. A reproducible increase at one or more concentrations. 

3. Biological relevance against in-house historical control ranges. 

4. Statistical analysis of data as determined by UKEMS (Mahon eta/ (1989)). 

5. Fold increase greater than two times the concurrent solvent control for any tester 

strain (especially if accompanied by an out-of-historical range response). 

A test item will be considered non-mutagenic (negative) in the test system if the above 

criteria are not met. 

Although most experiments will give clear positive or negative results, in some instances 

the data generated will prohibit making a definite judgement about test item activity. 

Results of this type will be reported as equivocal. 

4. ARCHIVES 

Unless instructed otherwise by the Sponsor, all original data and the final report will be 

retained in the Harlan Laboratories Ltd., Shard low, UK archives for five years, after which 

instructions will be sought as to further retention or disposal. 
PROJECT NUMBER: 41204331 PAGE14 

5. RESULTS 

5.1 Preliminary Toxicity Test 

The test item was non-toxic to the strains of bacteria used (TA 100 and WP2uvrA). The 

test item formulation and S9-mix used in this experiment were both shown to be sterile. 

The numbers of revertant colonies for the toxicity assay were: 

With(+) or Dose (!Jg/plate) without(-) Strain 

S9-mix 0 0.15 0.5 1.5 5 15 50 150 500 1500 5000 

- TA100 96 99 89 90 73 107 87 91 92 96 117 

+ TA100 118 137 112 111 123 101 95 104 103 100 101 

- WP2uvrA 23 31 21 24 30 34 30 20 32 26 25 

+ WP2uvrA 30 33 30 34 30 22 33 36 26 25 25 

5.2 Mutation Test 

Prior to use, the master strains were checked for characteristics, viability and 

spontaneous reversion rate (all were found to be satisfactory). The amino acid 

supplemented top agar and S9-mix used in both experiments was shown to be sterile. 

The culture density for each bacterial strain was also checked and considered 

acceptable. These data are not given in the report. 

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 

and were considered to be acceptable. These data are for concurrent untreated control 

plates performed on the same day as the Mutation Test. 

The individual plate counts, the mean number of revertant colonies and the standard 

deviations, for the test item, positive controls and vehicle controls, both with and without 

metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 

and Table 5 for Experiment 2. 

A history profile of vehicle/untreated and positive control values (reference items) for 

2010 and 2011 are presented in Appendix 1. 

The test item caused no visible reduction in the growth of the bacterial background lawn 

at any dose level and was, therefore, tested up to the maximum recommended dose 
PROJECT NUMBER: 41204331 PAGE15 

level of 5000 J,.Jg/plate. No test item precipitate was observed on the plates at any of the 

doses tested in either the presence or absence of S9-mix. 

No significant increases in the frequency of revertant colonies were recorded for any of 

the bacterial strains, with any dose of the test item, either with or without metabolic 

activation or exposure method. 

All of the positive control chemicals used in the test induced marked increases in the 

frequency of revertant colonies thus confirming the activity of the S9-mix and the 

sensitivity of the bacterial strains. 

6. CONCLUSION 

The test item, , was considered to be non-mutagenic under the conditions of this 

test. 
PROJECT NUMBER: 41204331 PAGE16 

7. REFERENCES 

Ames B N, McCann J, and Yamasaki E (1975), Methods for detecting carcinogens and 

mutagens with the Sa/monel/a/mammalian microsome mutagenicity test, Mutation 

Research, 31, 347-364. 

Maron D M and Ames B N (1983), Revised Methods for the Salmonella mutagenicity 

test, Mutation Research, 113, 173- 215. 

Mortelmans K and Zeiger E (2000), The Ames Sa/monel/a/microsome mutagenicity 

assay, Mutation Research, 455, 29-60. 

Green, M H L. and Muriel W J (1976), Mutagen Testing Using TRP+ Reversion in 

Escherichia coli, Mutation Research, 38, 3- 32. 

De Serres F J and Shelby M D (1979), Recommendations on data production and 

analysis using the Sa/monel/a/microsome mutagenicity assay, Environmental 

Mutagenesis, 1, 87-92. 

Mahon G A T et a/ (1989) Analysis of data from microbial colony assays. In: Statistical 

Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for 

mutagenicity testing, (Kirkland D J Ed.), Cambridge University Press Report, 26-65. 
Table 1 

EXPERIMENT 1 

TA100 

104 

92 (96) 

93 

EXPERIMENT 2 

TA100 

105 

90 (98) 

99 

PROJECT NUMBER: 41204331 

REVERSE MUTATION ASSAY "AMES TEST" 

USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI 

Spontaneous Mutation Rates (Concurrent Negative Controls) 

Number of revertants (mean number of colonies per plate) 

Base-pair substitution type Frameshift type 

TA1535 WP2uvrA TA98 TA1537 

19 30 18 13 

PAGE17 

18 (15) 30 (28) 29 (24) 10 ( 11) 

8 25 25 10 

Number of revertants (mean number of colonies per plate) 

Base-pair substitution type Frameshift type 

TA1535 WP2uvrA TA98 TA1537 

15 26 32 7 

14 (14) 21 (28) 17 (24) 10 (8) 

14 36 24 7 
PROJECT NUMBER: 41204331 PAGE18 

REVERSE MUTATION ASSAY "AMES TEST" 

USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI 

Table 2 Test Results: Experiment 1 -Without Metabolic Activation 

Test Period From: 24 September 2012 I 

Number of revertants (mean Dose Level Base-pair substitution strains Per Plate TA100 

Solvent Control 100 (108) 105 (Water) 120 10.4# 

102 (91) 501-19 74 

97 14.9 

S9-Mix 109 (103) 1501-19 96 (-) 104 6.6 

90 (96) 5001-19 97 

102 6.0 

112 (106) 1500 1-19 115 

90 13.7 

99 (94) 5000 1-19 91 

93 4.2 

Positive Name 

ENNG 

controls Dose Level 31J9 

S9-Mix No. of 619 (628) 

(-) Revertants 649 18.2 616 

ENNG N-ethyi-N'-nitro-N-nitrosoguanidine 

4NQO 4-Nitroquinoline-1-oxide 

9AA 9-Aminoacridine 

# Standard deviation 

TA1535 WP2uvrA 

15 (15) 35 (26) 15 25 

16 0.6 19 8.1 

12 (16) 32 (26) 19 21 

16 3.5 25 5.6 

10 (14) 36 (29) 19 30 

13 4.6 20 8.1 

16 (14) 33 (29) 13 34 

13 1.7 20 7.8 

19 (17) 33 (31) 18 25 

15 2.1 34 4.9 

16 (15) 32 (30) 14 30 

14 1.2 29 1.5 

ENNG ENNG 

51J9 21-19 

904 (814) 1145 (1 077) 757 79.1 1033 59.9 780 1052 

To: 27 September 2012 

+/-so 

Frameshift strains 

TA98 TA1537 

29 (22) 19 (11) 20 6 

18 5.9 7 7.2 

28 (25) 8 (11) 27 9 

21 3.8 17 4.9 

21 (21) 9 (8) 25 10 

17 4.0 5 2.6 

21 (19) 9 (13) 20 17 

16 2.6 13 4.0 

19 (17) 15 (11) 16 9 

16 1.7 8 3.8 

19 (22) 12 (12) 19 19 

27 4.6 6 6.5 

4NQO 9AA 

0.21J9 801-19 

198 (216) 1112 (928) 247 27.0 1132 335.6 203 541 
Table 3 

PROJECT NUMBER: 41204331 

REVERSE MUTATION ASSAY "AMES TEST" 

USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI 

Test Results: Experiment 1 -With Metabolic Activation 

PAGE19 

Test Period From: 24 September 2012 I To: 27 September 2012 

Dose Level 

Number of revertants (mean +/-so 

Base-pair substitution strains Frameshift strains Per Plate TA100 TA1535 WP2uvrA TA98 TA1537 

Solvent Control 96 (106) 18 (16) 37 (37) 34 (31) 14 ( 11) 106 18 39 40 13 (Water) 116 10.0# 11 4.0 36 1.5 20 10.3 6 4.4 

121 (111) 17 (16) 35 (34) 21 (24) 7 (7) 501-19 111 12 27 27 6 

100 10.5 18 3.2 40 6.6 25 3.1 9 1.5 

S9-Mix 115 (1 09) 9 (14) 36 (37) 34 (31) 13 ( 11) 

(+) 1501-19 103 6.0 17 4.6 36 1.2 25 4.9 15 4.7 108 17 38 33 6 

108 (1 05) 14 (13) 39 (37) 28 (24) 13 (12) 5001-19 122 13 44 21 12 

86 18.1 12 1.0 28 8.2 24 3.5 12 0.6 

121 (108) 19 (17) 29 (32) 28 (28) 8 (12) 1500 1-19 100 17 35 27 12 

104 11.2 16 1.5 33 3.1 29 1.0 16 4.0 

94 (96) 13 (13) 32 (30) 37 (30) 14 (13) 5000 1-19 92 12 26 32 9 

101 4.7 15 1.5 32 3.5 20 8.7 16 3.6 

Positive Name 

2AA 2AA 2AA BP 2AA 

controls Dose Level 1 1-19 21-19 10 1-19 51-19 21-19 

S9-Mix No. of 1253 (1103) 218 (333) 316 (308) 297 (292) 240 (234) 

(+) Revertants 703 350.0 527 168.7 300 8.0 279 11.0 212 19.3 1353 255 308 299 249 

2AA 2-Aminoanthracene 

BP Benzo(a)pyrene 

# Standard deviation 
PROJECT NUMBER: 41204331 

REVERSE MUTATION ASSAY "AMES TEST" 

USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI 

PAGE 20 

Table 4 Test Results: Experiment 2 -Without Metabolic Activation 

Test Period From: 15 October 2012 I To: 18 October 2012 

Dose Level 

Number of revertants (mean)+/- SO 

Per Plate Base-pair substitution strains 

TA100 

Solvent Control 98 (95) 84 (Water) 104 10.3# 

86 (95) 50 j..Jg 105 

94 9.5 

S9-Mix 73 (80) 150 l-l9 80 ( -) 88 7.5 

103 (93) 500 l-l9 77 

98 13.8 

97 (88) 1500 l-l9 85 

81 8.3 

81 (79) 5000 l-l9 67 

88 10.7 

Positive Name 

ENNG 

controls Dose Level 3 l-l9 

S9-Mix No. of 819 (764) 

( -) Revertants 796 76.2 677 

ENNG N-ethyi-N'-nitro-N-nitrosoguanidine 

4NQO 4-Nitroquinoline-1-oxide 

9AA 9-Aminoacridine 

# Standard deviation 

TA1535 WP2uvrA 

16 (15) 37 (35) 16 34 

14 1.2 33 2.1 

11 (15) 21 (28) 22 24 

11 6.4 39 9.6 

14 (15) 29 (31) 14 31 

17 1.7 34 2.5 

14 (13) 34 (31) 13 29 

11 1.5 30 2.6 

11 (14) 33 (36) 16 38 

16 2.9 38 2.9 

10 (1 0) 27 (29) 10 29 

10 0.0 30 1.5 

ENNG ENNG 

5 l-l9 2 l-l9 

555 (615) 1112 (1019) 589 77.0 964 81.0 702 981 

Frameshift strains 

TA98 TA1537 

34 (24) 16 (1 0) 16 6 

22 9.2 9 5.1 

26 (19) 7 (9) 12 10 

18 7.0 9 1.5 

19 (22) 6 (10) 27 11 

20 4.4 14 4.0 

20 (22) 9 (1 0) 20 8 

27 4.0 12 2.1 

29 (27) 8 (9) 35 14 

17 9.2 4 5.0 

16 (18) 9 (7) 17 7 

21 2.6 5 2.0 

4NQO 9AA 

0.2 l-l9 80 l-l9 

196 (195) 1307 (1111) 198 4.2 741 320.9 190 1286 
Table 5 

PROJECT NUMBER: 41204331 

REVERSE MUTATION ASSAY "AMES TEST" 

USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI 

Test Results: Experiment 2 -With Metabolic Activation 

PAGE 21 

Test Period From: 15 October 2012 I To: 18 October 2012 

Dose Level 

Number of revertants (mean +/-so 

Base-pair substitution strains Frameshift strains Per Plate TA100 TA1535 WP2uvrA TA98 TA1537 

Solvent Control 121 (111) 13 (11) 25 (28) 26 (27) 9 (1 0) 105 9 29 26 12 (Water) 106 9.0# 10 2.1 30 2.6 29 1.7 10 1.5 

110 (108) 9 (11) 22 (28) 21 (24) 13 (12) 501-19 120 12 30 27 12 

94 13.1 12 1.7 31 4.9 24 3.0 12 0.6 

S9-Mix 113 (95) 12 (12) 27 (25) 30 (27) 14 (12) 1501-19 86 14 18 26 10 (+) 87 15.3 11 1.5 30 6.2 25 2.6 12 2.0 

99 (100) 11 ( 11) 22 (25) 31 (27) 13 (1 0) 5001-19 93 11 34 29 4 

109 8.1 12 0.6 20 7.6 22 4.7 12 4.9 

88 (98) 9 (10) 32 (26) 22 (25) 7 (6) 1500 1-19 101 9 26 17 8 

104 8.5 12 1.7 19 6.5 35 9.3 3 2.6 

107 (1 00) 16 (13) 31 (28) 25 (30) 15 (9) 5000 1-19 88 10 27 35 7 

106 10.7 12 3.1 27 2.3 31 5.0 6 4.9 

Positive Name 

2AA 2AA 2AA BP 2AA 

controls Dose Level 1 1-19 21-19 10 1-19 51-19 21-19 

S9-Mix No. of 2237 (2142) 263 (260) 455 (400) 254 (267) 356 (278) (+) Revertants 2415 330.9 260 3.5 362 48.8 270 11.8 244 67.7 1774 256 383 277 234 

2M 2-Aminoanthracene 

BP Benzo(a)pyrene 

# Standard deviation 
PROJECT NUMBER: 41204331 

REVERSE MUTATION ASSAY "AMES TEST" 

USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI 

Appendix 1 History Profile of Vehicle and Positive Control Values 

COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2010 

Strain TA100 TA1535 TA102 WP2uvrA TA98 TA1537 

S9-Mix -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 

Mean 104 98 19 15 266 318 29 33 21 25 12 13 

SD 14.7 13.8 4.7 3.3 35.4 31.8 9.1 9.2 3.9 4.3 2.4 2.4 

Min 71 62 8 7 206 234 14 16 11 14 6 7 

Max 171 145 34 28 359 346 54 55 42 42 26 23 

Values 604 441 504 345 31 13 512 369 590 366 566 335 

POSITIVE CONTROL VALUES 2010 

Strain TA100 TA1535 TA102 WP2uvrA TA98 TA1537 

S9-Mix -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 

Mean 459 1091 353 218 1163 1031 395 282 155 249 1285 202 

SD 131.9 452.8 389.4 77.4 385.1 423.0 200.1 81.0 53.5 134.7 833.7 72.4 

Min 228 302 87 103 717 575 129 130 69 84 128 83 

Max 1216 3777 2003 726 1836 2392 978 661 488 1146 4743 530 

Values 159 167 156 160 18 18 144 144 236 169 229 158 

COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2011 

Strain TA100 TA1535 TA102 

S9-Mix -S9 +S9 -S9 +S9 -S9 +S9 

Mean 108 101 22 15 256 298 

SD 3.7 5.1 0.8 1.0 5.7 3.2 

Min 58 60 10 8 198 209 

Max 171 168 34 35 337 385 

Values 1090 716 810 425 165 66 

POSITIVE CONTROL VALUES 2011 

Strain TA100 TA1535 

S9-Mix -S9 +S9 -S9 

Mean 528 888 307 

SD 223.9 398.3 254.4 

Min 249 346 87 

Max 2282 3072 1773 

Values 191 202 192 

SO = Standard deviation 

Min = Minimum value 

Max = Maximum value 

+S9 

304 

122.9 

126 

1150 

196 

TA102 

-S9 +S9 

986 875 

284.1 209.5 

622 628 

2151 1780 

51 51 

WP2uvrA TA98 TA1537 

-S9 +S9 -S9 +S9 -S9 +S9 

30 35 23 25 11 12 

0.9 0.8 0.7 1.5 0.5 1.4 

11 10 11 10 4 5 

59 55 43 43 23 25 

851 495 1167 462 1124 409 

WP2uvrA TA98 TA1537 

-S9 +S9 -S9 +S9 -S9 +S9 

643 283 194 249 946 235 

213.2 83.1 115.2 166.7 640.7 72.9 

198 121 69 81 103 86 

1719 504 1631 2115 5224 491 

177 177 255 204 254 193 

WP2uvrA 

pKM101 

-S9 +S9 

141 169 

47.1 44.3 

75 101 

235 274 

40 32 

WP2uvrA 

pKM101 

-S9 +S9 

811 1833 

335.6 695.6 

446 799 

1660 3255 

11 17 

WP2uvrA 

pKM101 

-S9 +S9 

121 163 

5.3 10.7 

85 108 

168 223 

37 19 

WP2uvrA 

pKM101 

-S9 +S9 

1034 1583 

296.6 881.5 

692 331 

1600 2916 

7 12 

PAGE 22 

WP2pKM101 

-S9 +S9 

90 117 

21.9 30.5 

46 48 

130 166 

29 20 

WP2pKM101 

-S9 +S9 

1443 537 

603.8 207.0 

476 321 

2323 1028 

12 18 

WP2pKM101 

-S9 +S9 

93 115 

2.2 25.9 

69 81 

125 154 

27 11 

WP2pKM101 

-S9 +S9 

1384 628 

1054.4 196.4 

233 432 

2798 1150 

5 12 
PROJECT NUMBER: 41204331 PAGE 23 

Appendix 2 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 

Department 

of Health 

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT 

OF THE UNITED KINGDOM 

GOOD LABORATORY PRACTICE 

STATEMENT OF COMPLIANCE 

IN ACCORDANCE WITH DIRECTIVE 2004/9/EC 

TEST FACILITY 

Harlan Laboratories Ltd 

Shardlow Business Park 

London Road 

Shard low 

Derby, DE27 2GD 

DATE OF INSPECTION 

10 July 2012 

TEST TYPE(S) 

Analytical/Clinical 

Chemistry 

Environmental Toxicity 

Environmental Fate 

Mutagenicity 

Phys/Chem. Tests 

Toxicology 

An inspection for compliance with the Principles of Good Laboratory Practice was 

carried out at the above test facility as part of the UK Good laboratory Practice 

Compliance Monitoring Programme. 

This statement confirms that, on the date of issue, the UK Good laboratory 

Practice Monitoring Authority were satisfied that the above test facility was 

operating in compliance with the OECD Principles of Good laboratory Practice. 

This statement constitutes a Good Laboratory Practice Instrument (as defined in the 

UK Good laboratory Practice Regulations 1999). 

Dr. Andrew J. Gray 

Head, UK GLP Monitoring Authority