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# REPORT

Bacterial Reverse Mutation Test

HLS s tudy number: CEF0007

Version ID: Audited Draft 1 Report

Issue date: 2 May 2013

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# Sponsor and Test Facility Details

Sponsor

Test facility Huntingdon Life Sciences

Eye Research Centre

Eye

Suffolk

IP23 7PX

UK

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# Table of Contents

Sponsor and Test Facility Details

................................ ................................ .......................... 2

Compliance with Good Laboratory Practice

................................ ................................ .......... 5

Quality Assurance Statement

................................ ................................ ................................ 6

Contributing Scientists

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

Summary

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

1. Introduction

................................ ................................ ................................ .................. 9

2. Experimental Procedure

................................ ................................ ............................. 10

2.1 Test substance

................................ ................................ ............................... 10

2.2 Bacterial strains

................................ ................................ .............................. 10

2.3 Positive controls

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

2.3.1 In the absence of S9 mix

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

2.3.2 In the presence of S9 mix

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

2.4 S9 metabolizing system

................................ ................................ .................. 1 1

2.4.1 Preparation of S9 fraction

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

2.4.2 Preparation of S9 m ix

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

2.5 Selection of vehicle and formulation of test substance

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

2.6 Mutation test procedure

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

2.6.1 First test

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

2.6.2 Second test

................................ ................................ ......................... 13

2.7 Stability, homogeneity and formulation analysis

................................ .............. 13

3. Assessment of Results

................................ ................................ ............................... 14

3.1 Acceptance criteria

................................ ................................ ......................... 14

3.2 Analysis of data

................................ ................................ .............................. 14

3.3 Criteria for assessing mutagenic potential

................................ ....................... 14

4. Deviations from Protocol

................................ ................................ ............................ 15

5. Maintenance of Records

................................ ................................ ............................. 16

6. Results

................................ ................................ ................................ ....................... 17

6.1 First test

................................ ................................ ................................ .......... 17

6.2 Second test

................................ ................................ ................................ ..... 17

7. Conclusion

................................ ................................ ................................ ................. 18

8. References

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

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# List of Tables

Table 1 Results obtained in the absence of metabolic activation: test 1

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

Table 2 Results obtained in the presence of metabolic activation: test 1

................... 22

Table 3 Viability counts obtained in test 1

................................ ................................ . 24

Table 4 Results obtained in the absence of metabolic activation: test 2

.................... 25

Table 5 Results obtained in the presence of metabolic activation: test 2

................... 27

Ta ble 6 Viability counts obtained in test 2

................................ ................................ . 29



# List of A ppendices

Appendix 1 Historical Control Data

................................ ................................ ................. 30

Appendix 2 Eye Research Centre GLP Compliance Statement

................................ ...... 31

Appendix 3 S9 Quality Control Certificate

................................ ................................ ....... 32

Appendix 4 Certificate of Analysis

................................ ................................ .................. 33

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# Compliance with Good Laboratory Practice

: Bacterial Reverse Mutation Test

The study described in this report was conducted in compliance with the following Good

Laboratory Practice standards, with the exception stated below, and I consider the data

generated to be valid.

The UK Good Laboratory Practice Regulations (Statutory Instrument 1999

No. 3106, as amended by Statutory Instrument 2004 No. 994).

OECD Principles of Good Laboratory Practice (as revised in 1997),

ENV/MC/CHEM (98) 17.

EC Commission Directive 2004/10/EC of 11 February 2004 (Official Journal

No. L 50/44).

These principles of Good Laboratory Practice are accepted by the regulatory authorities of

the United States of America and Japan on the basis of intergovernmental agreements.

In line with normal practice in this type of short-term study, the protocol did not require

analysis of the dose form.

........................................................................................ ................................................

Kenneth May BSc CBiol M SB Date

Study Director

Huntingdon L ife Sciences

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# Quality Assurance Statement

: Bacterial Reverse Mutation Test

The following inspections and audits have been carried out in rel ation to this study:

Study Phase Date(s) of Inspection Date of Reporting to Study

Director and Management

Protocol Audit 08 Mar 2013 08 Mar 2013

Report Audit 29 Apr 2013

- 02 May 2013 02 May 2013

Process based inspections : At or about the time thi s study was in progress inspections of

procedures employed on this type of study were carried out. These were conducted and

reported to appropriate Company Management as indicated below:

Process Based Inspections Date(s) of Inspection Date of Reportin g to

Management

Study management and

conduct (In Vitro) 27 Nov 2012 27 Nov 2012

Plate reading (In Vitro) 29 Jan 2013 30 Jan 2013

Formulation procedures,

sampling and treatment (In

Vitro) 06 Mar 2013 08 Mar 2013

In addition, an inspection of the facility where this study was conducted was carried out on an

annual basis. These inspections were reported to Company Management.

........................................................................................ ................................................

Tim Whatling MRQA Date

Group Manager

Department of Quality Assurance

Huntingdon Life Sciences

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# Contributing Scientists

: Bacterial Reverse Mutation Test



# Study management

Kenneth May BSc C Biol M SB

Study Director

Lucinda Weston

- Stiff BSc

Assistant Study S cientist

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# Summary

In this in vitro assessment of the mutagenic potential of histidine-dependent

auxotrophic mutants of Salmonella typhimurium , strains TA1535, TA1537, TA98 and

TA100, and a tryptophan-dependent mutant of Escherichia coli , strain WP2 uvr A (pKM101),

were exposed to diluted in dimethyl sulphoxide (DMSO). DMSO was also used

as a vehicl e control.

Two independent mutation tests were performed in the presence and absence of liver

preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first

test was a standard plate incorporation assay; the second included a p re -incubation stage.

Concentrations of up to 5000 μg/plate were tested initially . This is the standard

limit concentration recommended in the regulatory guidelines that this assay follows. The

maximum tested concentration was reduced to 1 500 μg/p late for the second test in the

Salmonella strains because of toxicity observed in the first test. Other concentrations used

were a series of ca half -log 10 

## dilutions of the highest concentration.

Toxicity (observed as a reduction in revertant colony numbe rs) was seen in the Salmonella

strains following exposure to at 1500 and 5000 μg/plate in the first test and at

1500 μg/plate in the second test . No signs of toxicity were observed in the E. coli strain in

the first mutation test following exposur e to , but toxicity (observed as a reduction

in revertant colony numbers) was seen following exposure to at 5000 μg/plate in

the second test.

No evidence of mutagenic activity was seen at any concentration of in either

mutation test.

The concurrent positive controls verified the sensitivity of the assay and the metabolising

activity of the liver preparations. The mean revertant colony counts for the vehicle controls

were within or close to the current historical control range for the laboratory.

It was concluded that showed no evidence of mutagenic activity in this bacterial

system under the test conditions employed.

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# 1. Introduction

This report describes a study designed to assess for its potential to cause point

(gene) m utation in Salmonella typhimurium and Escherichia coli . The study was conducted

in compliance with the following regulations or guidelines:

OECD Guidelines for the Testing of Chemicals (1997). Genetic Toxicology:

Bacterial Reverse Mutation Test, Guideline 471.

EC Commission Regulation No. 440/2008. Method B.13/14: Mutagenicity

- Reverse mutation test using bacteria. OJ L 142/248.

US EPA Health Effects Test Guidelines (1998). OPPTS 870.5100 Bacterial reverse

mutation test. EPA 712-C-98-247.

The in vitro technique described by Ames and his co- workers ( Ames

, McCann and Yamasaki

1975 ; Maron and Ames 1983 ) enables the mutagenic effect of a test substance to be

determined by exposing specially selected strains of S. typhimurium to the test substance.

Wild

- type S. typhimurium is capable of synthesising the essential amino acid, histidine, but

the mutant strains used in this test are incapable of this function. When these strains are

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

in a proportion of the population. These are referred to as revertants, and are readily detected

by their ability to grow and form colonies on a histidine-deficient medium (supplemented

with biotin, since these strains are also incapable of biotin synthesis).

A technique based on similar principl es has also been described by Green (1984) . This

system employs mutant strains of E. coli that are incapable of synthesising the amino acid,

tryptophan, which is required for growth.

The se strains carry additional mutations that render them more sensitive to mutagens

( Gatehouse et al , 1990 & 1994 ). The S. typhimurium strains have a defective cell coat, which

allows greater permeability of test substances into the cell. All the strains are deficient in

normal DNA repair processes. In addition, three of them possess a plasmid (pKM101) which

introduces an error-prone repair process, resulting in increased sensitivity to some mutagens.

Some substances do not exert a mutagenic effect until they have been metabolised by enzyme

systems not available in the bacterial cell. Therefore, the bacteria and test substance are

incubated in both the absence and presence of a supplemented rat liver homogenate fraction

(S9 mix).

The study comprised two indep endent tests to assess the mutagenic potential of the test

substance.

The protocol was approved by Huntingdon Life Sciences Management, by the Study Director

and by the S tudy Monitor on 4 April 2013.

Experimental start date: 9 April 2013.

Experimental com pletion date: 24 April 2013.

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# 2. Experimental Procedure

2.1 Test s ubstance

Identity:

Chemical name:

Batch number: 12342

Expiry: To be advised and included in final report & study file



## Appearance: White crystalline p owder

Storage conditions: Room temperature, dark

Purity/Assay: To be advised and included in final report & study file



## Date received: 20 February 2013

Certificate of analysis: Appendix 4



# 2.2 Bacterial s trains

The genotype of ea ch bacterial strain used in this study is outlined in the following table:

Species Strain Genotype



## Salmonella typhimurium TA1535 his G46 rfa ∆ uvr B

Salmonella typhimurium TA1537 his C3076 rfa ∆ uvr B

Salmonella typhimurium TA98 his D3052 rfa ∆ uvr B pKM101

Salmonella typhimurium TA100 his G46 rfa ∆ uvr B pKM101

Escherichia coli WP2 uvr A (pKM101) trp E ochre uvr A pKM101

Note: The deletion ( ∆ ) through uvr B also affects the nitrate reductase ( chl A) and biotin ( bio ) genes



## These strains were used to detect base changes and frameshift mutations as follows:

base change mutagens: S. typhimurium TA1535 and TA100, and E. coli WP2 uvr A

(pKM101).

frameshift mutagens: S. typhimurium TA1537 and TA98.

The strains of S. typhimurium and E. coli were obtained from Moltox In c.

Batches of the strains were stored at ca -80°C as aliquots of nutrient broth cultures. Dimethyl

sulphoxide (DMSO) was added to the cultures at 8% v/v as a cryopreservative. Each batch of

frozen strain was tested for amino acid requirement and , where applicable, for cell membrane

permeability ( rfa mutation), sensitivity to UV light, and the pKM101 plasmid, which confers

resistance to ampicillin. The responses of the strains to a series of reference mutagens were

also assessed.

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## For use in tests, an aliq uot of frozen culture was added to 25 mL of nutrient broth and

incubated, with shaking, at 37 ° C for 10 hours. These cultures were intended to provide a

viable cell density of at least 10 9 

## per mL, which was confirmed by performing viability

counts, in whic h aliquots (0.1 mL) of a 10

- 6 

## dilution of the 10

- hour cultures were spread on

the surface of plates of nutrient agar. After incubation at 37°C for 24 hours, the total number

of resultant colonies was counted.



# 2.3 Positive c ontrols

2.3.1 In the absence of S9 mix

Ide ntity: Sodium azide

CAS No.: 26628

- 22

- 8

Solvent: DMSO

Concentration: 2 μg/plate for strains TA100 and TA1535

Identity: 9

- Aminoacridine

CAS No.: 90

- 45

- 9

Solvent: DMSO

Concentration: 50 μg/plate for strain TA1537

Identity: 2

- Nitrofluorene

CAS No.: 607

- 57

- 8

S olvent: DMSO

Concentration: 2 μg/plate for strain TA98

Identity: 4

- Nitroquinoline

- 1

- oxide

CAS No.: 56

- 57

- 5

Solvent: DMSO

Concentration: 2 μg/plate for strain WP2 uvr A (pKM101)

2.3.2 In the presence of S9 mix

Identity: 2

- Aminoanthracene

CAS No.: 613

- 13

- 8

Solvent: DMSO

Concentration: 5 μg/plate for strains TA100 and TA1535

10 μg/plate for strain WP2 uvr A (pKM101)

Identity: Benzo[a]pyrene

CAS No.: 50

- 32

- 8

Solvent: DMSO

Concentration: 5 μg/plate for strains TA98 and TA1537



# 2.4 S9 m etabolizing s ystem

2.4.1 Preparation of S9 fr action

S9 fraction, prepared from male Sprague

- Dawley derived rats, dosed i.p. with phenobarbital

sodium (30 mg/kg 4 days before killing and 60 mg/kg 1, 2 & 3 days before killing) and 5,6

-

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## benzoflavone (80 mg/kg 2 days before killing) to stimulate mixed

- fun ction oxidases in the

liver , was purchased from a commercial source and stored at approximately

- 80°C. The quality

control statement relating to each batch of S9 preparation used is included in the raw data and a

copy is included in this report as Appendix 3

.

Lot No.: 3021 (Date of preparation: 25 October 2012)

2.4.2 Preparation of S9 mix

The S9 mix contained: S9 fraction (10% v/v), MgCl 2 

## (8 mM), KCl (33 mM), sodium

phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM) and

NADH (4 mM) in water. All the cofactors were filter

- sterilised before use.



# 2.5 Selection of v ehicle a nd f ormulation of t est s ubstance

The Sponsor indicated that had low solubility in water, but good solubility in

dimethyl sulphoxide ( DMSO ). D MSO ( HPLC grade) was, therefore, used as the vehicle for

this study

.

The highest concentration of tested in this study was 50 mg/mL in the chosen

vehicle, which provided a final concentration of 5000 μg/plate. This is the standard limit

concentrat ion recommended in the regulatory guidelines that this assay follows. The highest

concentration in each test was diluted with DMSO to produce a series of lower

concentrations, separated by approximately half

- log 10 

## intervals.

All concentrations cited in th is report are expressed in terms of the sample as

received.



# 2.6 Mutation t est p rocedure

2.6.1 First test

Aliquots of 0.1 mL of the test substance solutions (seven concentrations up to 5000 μg/plate),

positive control or vehicle control were placed in glass tubes . The vehicle control was

DMSO . S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by

0.1 mL of a 10- hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin

(0.05 mM) and tryptophan (0.05 mM). The mix ture was thoroughly shaken and overlaid onto

previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was

individually labelled with a unique code, identifying the contents of the dish. Three Petri

dishes were used for each treatme nt. Plates were also prepared without the addition of

bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate

buffer. All plates were incubated at approximately 37 ° C for ca 72 hours. After this period,

the appearance of the background bacterial lawn was examined and revertant colonies

counted using an automated colony counter (Perceptive Instruments Sorcerer ).

Any toxic effects of the test substance may be detect ed by a substantial reduction in mean

revertant colony counts, by a sparse or absent background bacterial lawn , or both. In the

absence of any toxic effects, the maximum concentration selected for use in the second test is

the same as that used in the fir st. If toxic effects ar e observed, a lower concentration might

be chosen, ensuring that signs of bacterial inhibition ar e present at this maximum

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## concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If

precipitate is observed on the plates at the end of the incubation period, at least one

precipitating concentration should be included in the second test, unless otherwise justified

by the Study Director.

2.6.2 Second test

As a clear negative response was obtained in the first t est, a variation to the test procedure

was used for the second test. The variation used was the pre-incubation assay in which the

tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were

incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay. The

maximum concentration chosen for the Salmonella strains was 1500 μg/plate, and t he

maximum concentration chosen for the E. coli strain was again 5000 μg/plate . Six

concentrations were used.



# 2.7 Stability, ho mogeneity and f ormulation a nalysis

The stability of and the stability and homogeneity of in the vehicle

were not determined as part of this study. Analysis of achieved concentration was not

performed as part of this study.

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# 3. Assessment of Re sults

3.1 Acceptance criteria

For a test to be considered valid, the mean of the vehicle control revertant colony numbers for

each strain should lie within or close to the current historical control range for the laboratory

unless otherwise justified by the St udy Director. The historical range is maintained as a

rolling record over a maximum of five years. Also, the positive control compounds must

induce an increase in mean revertant colony numbers of at least twice (three times in the case

of strains TA1535 and TA1537, which have relatively low spontaneous reversion rates) that

of the concurrent vehicle controls. Mean viable cell counts in the 10

- hour bacterial cultures

must be at least 10 9 

## /mL.



# 3.2 Analysis of data

The mean number and standard deviation of rever tant colonies were calculated for all groups.

The “fold

- increases” relative to the vehicle controls were calculated in order to compare the

means for all treatment groups with those obtained for the vehicle control groups.



# 3.3 Criteria for assessing mutagenic potential

If exposure to a test substance produces a reproducible increase in revertant colony numbers

of at least twice (three times in the case of strains TA1535 and TA1537) that of the

concurrent vehicle controls, with some evidence of a positive conce ntration

- response

relationship, it is considered to exhibit mutagenic activity in this test system.

If exposure to a test substance does not produce a reproducible increase in revertant colony

numbers, it is considered to show no evidence of mutagenic acti vity in this test system. No

statistical analysis is performed.

If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response,

even after additional testing, the test data may be subjected to analysis to determine the

statistical significance of any increases in revertant colony numbers. The statistical

procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test

followed, if appropriate, by trend analysis. Biological importance will be considered along

with statistical significance. In general, treatment

- associated increases in revertant colony

numbers below two or three times those of the vehicle controls (as described above) are not

considered biologicall y important. It should be noted that it is acceptable to conclude an

equivocal response if no clear results can be obtained.

Occasionally, these criteria may not be appropriate to the test data and, in such cases, the

Study Director would use his/her scie ntific judgement.

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# 4. Deviations from Protocol

There were no deviations from the protocol.

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# 5. Maintenance of Records

Following completion of this study all raw data, specimens and samples, except those

generated or used during any Sponsor’s or supplier’s analys is, were stored in the archives of

Huntingdon Life Sciences. Types of sample and specimen which are unsuitable, by reason of

instability, for long term retention and archiving may be disposed of after the periods stated

in Huntingdon Life Sciences Standar d Operating Procedures.

A copy of the final report will be retained indefinitely and all Quality Assurance inspection

records for a period of 20 years. All other appropriate specimens and records will be retained

for a minimum period of one year from the d ate of issue of the final report. At the end of this

retention period the Sponsor will be contacted and advice sought on future archiving

requirements. Under no circumstances will any item be discarded without the Sponsor’s

knowledge.

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# 6. Results

The result s obtained with , vehicle controls and positive control compounds are

presented in Table 1 and Table 2 (first test) and in Table 4 and Table 5 (s econd test).

The absence of colonies on sterility check plates confirmed the absence of microbial

contamination of the S9 mix, buffer and test substance formulation.

The viability counts (see Table 3 and Table 6 ) confirmed that the viable cell density of the

cultures of the individual organisms exceeded 10 9 

## /mL in all cases, and therefore met the

acceptance criteria.

The mean revertant colony counts for the vehicle controls were within or close to the curr ent

historical control range for the laboratory ( Appendix 1 ). Appropriate positive control

chemicals (with S9 mix where required) induced substantial increases in revertant colony

numbers with all strains in all reported tests, confirming sensitivity of the cultures and

activity of the S9 mix.



# 6.1 First test

Toxicity (observed as a reduction in revertant colony numbers) was seen in the Salmonella

strains following exposure to at 1500 and 5000 μg/plate. No signs of toxicity

we re observed in the E. coli strain following exposure to . The maximum

exposure concentrations selected for use in the second test were 1500 μg/plate ( Salmonella

strains) and 5000 μg/plate ( E. coli strain).

No substantial increases in revertant colo ny numbers over control counts were obtained with

any of the tester strains following exposure to at any concentration tested in either

the presence or absence of S9 mix.



# 6.2 Second test

Toxicity (observed as a reduction in revertant colony numbers) wa s seen in the Salmonella

strains following exposure to at 1500 μg/plate , and in the E. coli strain following

exposure to at 5000 μg/plate

.

No substantial increases in revertant colony numbers over control counts were obtained with

any of th e tester strains following exposure to at any concentration tested in either

the presence or absence of S9 mix.

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# 7. Conclusion

It was concluded that showed no evidence of mutagenic activity in this bacterial

system under the test conditions employed.

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# 8. 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.G., ROWLAND, I.R., WILCOX, P., CALLANDER, R.D. and FORSTER,

R. (1990) Bacterial mutation assays in: KIRKLAND, D.J. (Ed.). UKEMS Sub

- committee on

Guidelines for Mutagenicity Testing. Report. Part I. Basic Mutagenicity Tests , pp.13

- 61.

Cambridge University Press, Cambridge.

GATEHOUSE, D., HAWORTH, S., CEBULA, D., GOCKE, E., KIER, L., MATSUSHIMA,

T., MELCION, C., NOHMI, T., OHTA, T., VENITT, S and ZEIGER, E. (1994) Report from

the working group on bacterial mutation assays: International Workshop on Standardisation

of Genotoxicity Test Procedures. M utation Research, 312, 217

- 233

GREEN, M.H.L. (1984) Mutagen testing using trp + 

## reversion in Escherichia coli in

KILBEY, B.J., LEGATOR, M., NICHOLS, W. and RAMEL, C. (Eds.). Handbook of

Mutagenicity Test Procedures. Second edition , pp.161

- 187. Elsevier Sci ence Publishers BV,

Amsterdam.

MAHON, G.A.T., GREEN, M.H.L., MIDDLETON, B., MITCHELL, I. de G., ROBINSON,

W.D. and TWEATS, D.J. (1989) Analysis of data from microbial colony assays in:

KIRKLAND, D.J. (Ed.). UKEMS Sub

- committee on Guidelines for Mutagenici ty Testing.

Report. Part III. Statistical Evaluation of Mutagenicity Test Data , pp.26

- 65. Cambridge

University Press, Cambridge.

MARON, D.M. and AMES, B.N. (1983) Revised methods for the Salmonella mutagenicity

test. Mutation Res

. 113 , 173

- 215.

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## Table 1 Results obtained in the absence of metabolic activation: test 1

Study number: CEF0007

Experiment: Test 1

Assay conditions: Plate incorporation assay

Without metabolic activation

Strain Addition Concentration

per plate Mean

revertants

per plate Standard

Deviation Fold increase

relative to

vehicle Individual revertant

colony counts (Sorcerer)

TA98 DMSO 34.3 1.2 35, 33, 35

5 μg 39.0 4.0 1.1 35, 43, 39

15 μg 37.0 7.9 1.1 40, 28, 43

50 μg 33.7 4.2 1.0 35, 37, 29

150 μg 34.0 3.0 1.0 34, 37, 31

500 μg 28.0 4.6 0.8 27, 33, 24

1500 μg 3.3 2.5 0.1 3, 1, 6

5000 μg 0.0 0.0 0.0 0, 0, 0

TA100 DMSO 113.0 3.6 117, 110, 112

5 μg 111.3 3.2 1.0 115, 110, 109

15 μg 113.7 14.7 1.0 119, 97, 125

50 μg 108.3 19.9 1.0 131, 94, 100

150 μg 118.0 14.1 1.0 133, 116, 105

500 μg 105.7 7.6 0.9 99, 114, 104

1500 μg 24.0 3.0 0.2 27, 21, 24

5000 μg 0.0 0.0 0.0 0, 0, 0

TA1535 DMSO 26.7 4.5 27, 22, 31

5 μg 15.7 2.3 0.6 13, 17, 17

15 μg 22.0 6.6 0.8 28, 15, 23

50 μg 24.3 3.8 0.9 20, 27, 26

150 μg 19.3 2.1 0.7 17, 21, 20

500 μg 19.7 2.3 0.7 17, 21, 21

1500 μg 3.3 1.5 0.1 2, 3, 5

5000 μg 0.0 0.0 0.0 0, 0, 0

TA1537 DMSO 14.7 2.5 17, 15, 12

5 μg 15.7 5.9 1.1 18, 9, 20

15 μg 18.3 3.2 1.2 16, 22, 17

50 μg 15.7 2.3 1.1 13, 17, 17

150 μg 15.3 0.6 1.0 15, 16, 15

500 μg 15.0 0.0 1.0 15, 15, 15

1500 μg 3.0 1.7 0.2 1, 4, 4

5000 μg 0.0 0.0 0.0 0, 0, 0

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## Table 1 Results obtained in the absence of metabolic activation: test 1

(continued)

Study number: CEF0007

Experiment: Test 1

Assay conditions: Plate incorporation assay

Without metabolic activation

Strain Addition Concentration

per plate Mean

revertants

per plate Standard

Deviation Fold increase

relative to

vehicle Individual revertant

colony counts (Sorcerer)

WP2 uvrA

(pKM101) DMSO 159.0 17.6 172, 139, 166

5 μg 149.0 33.9 0.9 132, 188, 127

15 μg 141.3 2.1 0.9 143 , 142, 139

50 μg 151.3 5.5 1.0 154, 155, 145

150 μg 162.3 11.5 1.0 169, 149, 169

500 μg 162.0 18.5 1.0 163, 180, 143

1500 μg 165.0 23.8 1.0 192, 156, 147

5000 μg 123.0 5.3 0.8 127, 125, 117

TA98 2NF 2 μg 282.3 1.5 8.2 282, 284, 281

TA100 NaN3 2 μg 1450.3 83.1 12.8 1445, 1370, 1536

TA1535 NaN3 2 μg 1236.3 110.1 46.4 1349, 1231, 1129

TA1537 AAC 50 μg 496.7 120.7 33.9 526, 600, 364

WP2 uvrA

(pKM101) NQO 2 μg 1193.0 10.4 7.5 1186, 1205, 1188

Key to Positive Control s

2NF

NaN3

AAC

NQO 2

- Nitrofluorene

Sodium azide

9- Aminoacridine

4

- Nitroquinoline

- 1

- oxide

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## Table 2 Results obtained in the presence of metabolic activation: test 1

Study number: CEF0007

Experiment: Test 1

Assay conditi ons: Plate incorporation assay

With metabolic activation

Strain Addition Concentration

per plate Mean

revertants

per plate Standard

Deviation Fold increase

relative to

vehicle Individual revertant

colony counts (Sorcerer)

TA98 DMSO 52.3 4.2 57, 51, 49

5 μg 54.3 4.2 1.0 53, 51, 59

15 μg 49.7 3.5 0.9 53, 46, 50

50 μg 48.0 1.7 0.9 49, 49, 46

150 μg 36.7 3.2 0.7 33, 38, 39

500 μg 43.0 5.6 0.8 44, 48, 37

1500 μg 16.0 3.6 0.3 20, 13, 15

5000 μg 0.0 0.0 0.0 0, 0, 0

TA100 DMSO 131.3 11.4 144, 128, 122

5 μg 108.3 3.1 0.8 105, 111, 109

15 μg 103.3 11.7 0.8 90, 108, 112

50 μg 133.7 13.9 1.0 130, 122, 149

150 μg 138.0 10.4 1.1 126, 145, 143

500 μg 148.3 12.0 1.1 160, 136, 149

1500 μg 44.0 7.8 0.3 53, 39, 40

5000 μg 0.0 0.0 0.0 0, 0, 0

TA1535 DMSO 28.0 2.6 31, 26, 27

5 μg 19.3 1.2 0.7 20, 18, 20

15 μg 24.3 7.2 0.9 16, 28, 29

50 μg 23.7 4.6 0.8 29, 21, 21

150 μg 23.0 3.5 0.8 27, 21, 21

500 μg 18.0 2.0 0.6 20, 16, 18

1500 μg 1.3 0.6 0.0 2, 1, 1

5000 μg 0.0 0.0 0.0 0, 0, 0

TA1537 DMSO 31.0 4.4 26, 33, 34

5 μg 22.7 1.5 0.7 24, 23, 21

15 μg 29.3 5.5 0.9 23, 33, 32

50 μg 32.7 2.1 1.1 35, 32, 31

150 μg 26 .0 5.2 0.8 20, 29, 29

500 μg 26.7 2.5 0.9 24, 29, 27

1500 μg 1.3 1.2 0.0 2, 2, 0

5000 μg 0.0 0.0 0.0 0, 0, 0

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## Table 2 Results obtained in the presence of metabolic activation: test 1

(continued)

Study number: CEF0007

Experiment: Test 1

Assay conditions: Plate incorporation assay

With metabolic activation

Strain Addition Concentration

per plate Mean

revertants

per plate Standard

Deviation Fold increase

relative to

vehicle Individual revertant

colony counts (Sorcerer)

WP2 u vrA

(pKM101) DMSO 162.7 6.4 159, 170, 159

5 μg 171.0 9.6 1.1 167, 164, 182

15 μg 157.7 9.7 1.0 147, 166, 160

50 μg 169.7 5.9 1.0 163, 174, 172

150 μg 150.0 9.5 0.9 145, 144, 161

500 μg 160.3 14.6 1.0 177, 150, 154

1500 μg 154.7 0 .6 1.0 155, 154, 155

5000 μg 142.0 13.0 0.9 150, 149, 127

TA98 B[a]P 5 μg 214.0 3.5 4.1 210, 216, 216

TA100 AAN 5 μg 988.0 101.8 7.5 878, 1079, 1007

TA1535 AAN 5 μg 374.0 135.8 13.4 515, 363, 244

TA1537 B[a]P 5 μg 165.7 31.2 5.3 158 , 200, 139

WP2 uvrA

(pKM101) AAN 10 μg 1422.3 181.5 8.7 1335, 1301, 1631

Key to Positive Controls

B[a]P

AAN Benzo[a]pyrene

2- Aminoanthracene

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## Table 3 Viability counts obtained in test 1

Study number: CEF0007

Experiment: Test 1

Assay conditions: Plate incorporation assay

Viability plate summary

Strain Mean counts

per plate Standard Deviation Individual colony counts

(100 μL aliquots of 10

- 6

dilution of 10

- hour

culture)

TA98 Viability 246.3 12.5 23 2, 255, 252

TA100 Viability 276.7 3.8 274, 281, 275

TA1535 Viability 281.0 10.5 292, 280, 271

TA1537 Viability 258.7 7.5 259, 266, 251

WP2 uvrA

(pKM101) Viability 307.0 14.2 296, 302, 323

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## Table 4 Results obtained in the absence of metabolic activation: test 2

Study number: CEF0007

Experiment: Test 2

Assay conditions: Pre

- incubation assay

Without metabolic activation

Strain Addition Concentration

per plate Mean

revertants

per plate Standard

Deviation Fold increase

relative to

vehicle Individual revertant

colony counts (Sorcerer)

TA98 DMSO 38.0 5.6 43, 32, 39

5 μg 37.0 5.0 1.0 42, 32, 37

15 μg 43.0 2.6 1.1 44, 40, 45

50 μg 42.3 7.5 1.1 51, 38, 38

150 μg 38.3 1.2 1.0 39, 39, 37

500 μg 41.0 1.7 1.1 43, 40, 40

1500 μg 6.7 0.6 0.2 7, 7, 6

TA100 DMSO 141.0 6.1 138, 148, 137

5 μg 136.3 10.7 1.0 148, 127, 134

15 μg 139.0 7.8 1.0 144, 130, 143

50 μg 149.7 23.5 1.1 131, 142, 176

150 μg 138.3 2.3 1.0 14 1, 137, 137

500 μg 130.3 2.1 0.9 131, 128, 132

1500 μg 14.7 5.7 0.1 10, 13, 21

TA1535 DMSO 24.0 1.7 26, 23, 23

5 μg 26.3 3.8 1.1 22, 28, 29

15 μg 23.3 0.6 1.0 23, 24, 23

50 μg 22.7 5.5 0.9 28, 17, 23

150 μg 26.0 6.1 1.1 22, 33, 23

500 μg 22.7 2.3 0.9 20, 24, 24

1500 μg 0.3 0.6 0.0 1, 0, 0

TA1537 DMSO 9.7 0.6 9, 10, 10

5 μg 8.0 2.6 0.8 10, 9, 5

15 μg 11.7 3.8 1.2 9, 10, 16

50 μg 9.7 2.5 1.0 10, 7, 12

150 μg 8.7 1.5 0.9 7, 9, 10

500 μg 6.7 2.5 0.7 9, 7, 4

1500 μg 0.0 0.0 0.0 0, 0, 0

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## Table 4 Results obtained in the absence of metabolic activation: test 2

(continued)

Study number: CEF0007

Experiment: Test 2

Assay conditions: Pre

- incubation assay

Witho ut metabolic activation

Strain Addition Concentration

per plate Mean

revertants

per plate Standard

Deviation Fold increase

relative to

vehicle Individual revertant

colony counts (Sorcerer)

WP2 uvrA

(pKM101) DMSO 152.7 14.0 139, 167, 152

15 μg 151.0 26.5 1.0 171, 121, 161

50 μg 137.3 13.1 0.9 133, 152, 127

150 μg 139.7 20.6 0.9 138, 120, 161

500 μg 148.0 19.0 1.0 127, 164, 153

1500 μg 150.3 5.5 1.0 145, 156, 150

5000 μg 47.3 15.9 0.3 34, 65, 43

TA98 2NF 2 μg 578.3 108.9 15.2 518, 513, 704

TA100 NaN3 2 μg 963.0 118.0 6.8 1094, 865, 930

TA1535 NaN3 2 μg 1069.0 267.8 44.5 775, 1133, 1299

TA1537 AAC 50 μg 671.7 41.9 69.5 720, 647, 648

WP2 uvrA

(pKM101) NQO 2 μg 1425.7 90.7 9.3 1321, 1475, 1481

Key to Positive Controls

2NF

NaN3

AAC

NQO 2

- Nitrofluorene

Sodium azide

9- Aminoacridine

4

- Nitroquinoline

- 1

- oxide

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## Table 5 Results obtained in the presence of metabolic activation: test 2

Study number: CEF0007

Experiment: Test 2

Assay conditions: Pre

- incubation assay

With metabolic activation

Strain Addition Concentration

per plate Mean

revertants

per plate Standard

Deviation Fold increase

relative to

vehicle Individual revertant

colony counts (Sorcerer)

TA9 8 DMSO 52.7 2.5 55, 53, 50

5 μg 65.0 3.0 1.2 65, 62, 68

15 μg 55.0 6.0 1.0 61, 49, 55

50 μg 47.3 1.2 0.9 48, 46, 48

150 μg 49.7 1.2 0.9 51, 49, 49

500 μg 48.3 6.5 0.9 55, 42, 48

1500 μg 17.0 5.6 0.3 12, 23, 16

T A100 DMSO 164.0 8.5 165, 172, 155

5 μg 182.3 11.8 1.1 175, 196, 176

15 μg 176.0 20.4 1.1 199, 169, 160

50 μg 159.7 10.0 1.0 150, 170, 159

150 μg 187.0 19.1 1.1 207, 169, 185

500 μg 152.7 13.5 0.9 139, 166, 153

1500 μg 7.7 4.9 0.0 11, 2, 10

TA1535 DMSO 28.0 2.6 31, 27, 26

5 μg 30.0 3.6 1.1 27, 29, 34

15 μg 25.7 2.5 0.9 26, 28, 23

50 μg 31.0 2.0 1.1 33, 31, 29

150 μg 29.0 3.6 1.0 26, 33, 28

500 μg 20.0 3.5 0.7 22, 16, 22

1500 μg 0.7 0.6 0 .0 1, 1, 0

TA1537 DMSO 29.0 2.0 27, 29, 31

5 μg 27.7 5.5 1.0 22, 33, 28

15 μg 29.3 5.5 1.0 23, 32, 33

50 μg 24.3 1.5 0.8 23, 26, 24

150 μg 21.0 1.7 0.7 20, 20, 23

500 μg 19.0 1.7 0.7 20, 20, 17

1500 μg 2.0 1.7 0 .1 1, 4, 1

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## Table 5 Results obtained in the presence of metabolic activation: test 2

(continued)

Study number: CEF0007

Experiment: Test 2

Assay conditions: Pre

- incubation assay

With metabolic activation

Strain Addition Concentration

per pl ate Mean

revertants

per plate Standard

Deviation Fold increase

relative to

vehicle Individual revertant

colony counts (Sorcerer)

WP2 uvrA

(pKM101) DMSO 156.7 18.0 165, 169, 136

15 μg 169.3 25.5 1.1 161, 149, 198

50 μg 170.3 5.8 1.1 167, 167, 177

150 μg 168.3 2.3 1.1 167, 167, 171

500 μg 170.0 8.5 1.1 178, 161, 171

1500 μg 160.0 4.6 1.0 164, 161, 155

5000 μg 49.7 9.3 0.3 39, 54, 56

TA98 B[a]P 5 μg 313.3 18.4 5.9 318, 293, 329

TA100 AAN 5 μg 2113.3 225.4 12.9 2352, 2084, 1904

TA1535 AAN 5 μg 612.0 143.7 21.9 621, 464, 751

TA1537 B[a]P 5 μg 130.0 14.7 4.5 147, 121, 122

WP2 uvrA

(pKM101) AAN 10 μg 1252.7 79.5 8.0 1303, 1161, 1294

Key to Positive Controls

B[a]P

AAN Benzo[a]pyrene

2

- Aminoa nthracene

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## Table 6 Viability counts obtained in test 2

Study number: CEF0007

Experiment: Test 2

Assay conditions: Pre

- incubation assay

Viability plate summary

Strain Mean counts

per plate Standard Deviation Individual c olony counts

(100 μL aliquots of 10

- 6

dilution of 10

- hour

culture)

TA98 Viability 267.0 15.6 285, 259, 257

TA100 Viability 254.7 25.7 225, 271, 268

TA1535 Viability 251.0 15.6 260, 260, 233

TA1537 Viability 254.0 38.2 210, 279, 273

WP2 uvrA

(pK M101) Viability 323.7 17.9 328, 339, 304

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## A ppendix 1 Historical Control Data

Presented below are the historical control data (mean revertant colony counts) from the

period 1 November 2007 to 31 October 2012.

DMSO

S9 Mix

- +

- +

- +

- +

- +



### Maximum 227 240 32 35 233 272 51 68 27 50

Minimum 98 108 10 12 90 108 23 32 5 11

Mean 149 168 22 23 142 163 36 50 12 29

No. of values 432 432 394 394 332 332 432 432 394 394

Standard deviation 21 24 4 3 27 29 5 7 3 5 TA100 TA1535 TA98 TA1537 WP2 uvr A

(pKM101)



## Positive Controls

S9 Mix

- +

- +

- +

- +

- +

NaN 3 

### AAN NaN 3 

### AAN NQO AAN 2NF B[a]P AAC B[a]P

(2 μg) (5 μg) (2 μg) (5 μg) (2 μg) (10 μg) (2 μg) (5 μg) (50 μg) (5 μg)

Maximum 2246 4910 2338 1457 4494 2739 791 727 1917 2196

Minimum 367 522 145 119 524 294 114 127 133 68

Mean 911 2078 924 312 1825 747 284 283 622 179

No. of values 775 775 712 713 628 629 775 775 710 710

Standard deviation 254 746 317 114 642 376 91 85 279 107 

## TA100 TA1535 WP2 uvr A

(pKM101) 

## TA98 TA1537

NaN 3 

### Sodium azide

2NF 2-Nitrofluorene

AAC 9-Aminoacridine

B[a]P Benzo[a]pyrene

AAN 2-Aminoanthracene

NQO 4-Nitroquinoline-1-oxide

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## A ppendix 2 Eye Research Centre GLP Compliance Statement

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## A ppendix 3 S9 Quality Control Certificate

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## A ppendix 4 Certificate of Analysis

To be provided by sponsor and included in final report & study file