### PAGE 1 OF 34 PAGES 

## ~~ M
~'~,



## REVERSE MUTATION ASSAY "AMES TEST" USING 

## SALMONELLA TYPH(MURIUM AND ESCHERICHIA COLI PROJECT NUfVIBER:



## AUTHORS A Bowles

P W Thompson



## STUDY SPONSOR: TEST FACILITY: Harlan Laboratories Ltd Shardlow Business Park Shardlow Derbyshire DE72 2GD 

## UK Telephone: +44 (0) 1332 792896 Facsimile: +44 (0) 1332 799018PROJECT NUMBER: ' 

### PAGE2


## QUALITY ASSURANCE REPORT This study type is classed as short-term. The standard test method for this study type ("General Study Plan" in OECD tenninology) was reviewed for compliance once only on initial production. lnspection of the routine and repetitive procedures that constitute the 

## study is carried out as a continuous process designed to encompass the major phases at or about the time this study was in progress. In addition, inspection of general facilities 

## not specifically related to this study are done monthly or annually in accordance with QA Standard Procedure. 

## 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. 

## Unless otherwise indicated, 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:

13 November 2008 18, 19 February 2009 16 February 2009 18 February 2009 19 February 2009 § 13 March 2009 

### § Date of QA Signature Standard Test Method Compliance Audit Test Material Preparation Test System Preparation (reported on 02 March 2009) Exposure Assessment of Response Draft Report Audit Final Report Audit § Evaluation specific to this study

......................... ..:.(..

~ . r. ~ .... 

### For the Quality Assurance Unit* 

# 2 0 APR 2009

..... DATE :

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

"Authorised QA Signatures: Deputy Head of Department: JM Crowther MIScT MRQA 

### Senior Audit Staff: G Wren ONC MRQAPROJECT NUMBER: PAGE 3



## GLP COMPLIANCE STATEMENT The work described was perFormed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by 

## SI 200410994)). These Ftegulations are in accordance with GLP standards pubfished 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 2004/10/EC. This report fully and accuratefy reflects the procedures'used and data generated.



## ........................................ .. ~ 

## .. _...... DATE: .... ......5._7 APR 2009 A Bowles BSc (Hons) Study Director

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 al[ cases the hand-signed paper original, held in secure archives, is the definitive documentPROJECT NUMBER: PAGE 4



### QUALITY ASSURANCE REPORT 2 

### GLP COMPLIANCE STATEMENT 3 CONTENTS 4 SUMMARY 5 1. INTRODUCTION 7 2. TEST MATERIAL 8 3. METHODS 8 3.1 Tester Strains 8 3.2 Preparation of Test and Control Materials 9 3.3 Microsomal Enzyme Fraction 9 3.4 S9-Mix and Agar 10 3.5 Test Procedure 10 3.6 Acceptance Criteria 12 3.7 Evaluation Criteria 12 4. ARCHIVES 13 6. RESULTS 

### 14 5.1 Preliminary Toxicity Test 14 5.2 Mutation Test 14 6. CONCLUSION 15 7. REFERENCES 15 Table 1 Spontaneous Mutation Rates (Concurrent Negative Controls) 16 Table 2 Test Results: Range-Finding Test— Without Metabolic Activation 17 Table 3 Test Results: Range-Finding Test— With Metabolic Activation 18 Table 4 Test Results: Main Test— Without Metabolic Activation 19 Table 5 Test Results: Main Test— With Metabolic Activation 20 Figure 1 Dose Response Curve: Range-Finding Test—

Without Metabolic Activation 21 Figure 2 Dose Response Curve: Range-Finding Test—

With Metabolic Activation 22 Figure 3 Dose Response Curve: Main Test— Without Metabolic Activaticn 23 Figure 4 Dose Response Curve: Main Test— With Metabolic Activation 24 Appendix 1 Report of Results in Mutagenicity Test using Micro-organisms 25 Appendix 2 Test Material Certificate of Analysis 31 Appendix 3 History Profile of Vehicle and Positive Control Values 33 Appendix 4 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 34PROJECT NUMBER: PAGE 5



### REVERSE MUTATION ASSAY "AMES TEST" 

### USING SALMONELlA TYPHIMUR(UM AND



### RSCHERICHIA COtt
SUMMARY lntroduction. The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13114 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Methods. 

### Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA' were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 Ng/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.
Results. 

### The vehicfe (sterile distilled water) control plates gave counts of revertant colonies within the normal range. AII of the positive control chemicals 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 efticacy of the S9-mix were validated. The test material 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 ttg/plate. No test material precipitate was observed on the plates at any oP 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 material, either with or without metabolic activation.PROJECT NUMBER: ~ PAGE 6


### Conclusion. The test material was considered to be non-mutagenic under the conditions of this test.PROJECT NUMBER: : PAGE 7



## REVERSE MUTATION ASSAY "AMES TEST" USING SAI.MOIUELlATYPHIMURIUMAND

ESCHERICHIA COL(


## 1. INTRODUCTION This study was designed to assess the mutagenic potential of the test material using a bacterial/ microsome test system. The study was based on the in vitro technique described by Ames and his co-workers (1, 2, 3) and Garner et al (4) in which mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium and tryptophan auxotrophs of Escherichia 

## coli to various concentrations of the test materlal. The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regufation (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 mutant strains of Salmonella are incapable of synthesising histidine and are, therefore, dependent on an external source o€ this particular amino acid for normal growth. When exposed to a mutagenic agent these bacteria may undergo a reverse mutation to histidine independent forms which are detected by their ability to grow on a histidine deficient medium. Using various strains of this organism, revertants produced after exposure to a chemical mutagen may arise as a result of base-pair substitution in the genetic material (miscoding) or frame-shift mutation in which genetic materiaf is either added or deleted. In order to make the bacteria more sensitive to mutation by chemical and physical agents, several additional traits have been introduced. These include a deletion through the excision repair gene (uvr8' Salmonella strains) which renders the organism incapable of DNA excision repair and deep rough mutation (rFa) which increases the permeability of the cell wall. A mutant strain of Escherichia 

## coli (WP2uvrA

- ); which requires tryptophan and which can be reverse mutated by base substitution to tryptophan independence, was used to complement the Salmonella strains. This strain also has a deletion in an excision repair gene (uvrA").PROJECT NUMBER: PAGE8



## 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 material and the bacteria are also incubated in the presence of a liver microsomal preparation (S9-mix) 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 13 January 2009 and 09 February 2009.
2. TEST MATERIAL Sponsor's identification Description Batch number Date received Amber coloured liquid
11 December 2008 Storage conditions Room temperature in the dark The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor. A copy of the Certificate of Analysis is provided in Appendix 2.
3. METH®DS 3.1 Tester Strains 

## Salmonella typhimurium TA1535, TA1537, TA98 and TA100 Escherichia 

## coli WP2uvrA - The four strains of 

## Salmonella used in the test were obtained from the University of California, Berkeley, on culture discs, on 4 August 1995. 

## E. coli strain 

## WP2uvrA" was obtained from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. Ali of the strains were stored at

- 196°C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of 

## rfa, R factors, 

## uvrB or 

## uvrA mutation and the spontaneous reversion rate (5). In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37°C for approximately 10 hours. EachPROJECT NUMBER: . PAGE9 

### culture was monitored spectrophotometrically for turbidity with titres determined by viab[e count analysis on nutrient agar plates.

3.2 Preparation of Test and Control Materials The test material was fully miscible fn sterile distilled water at 50 mg/ml in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle. The test material was accurately weighed and approximate half-Iog di[utions prepared in steri[e distilled water by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (73.5%) of the test material. Analysis for concentration, homogeneity and stability of the test material formulations is not a requirement of the test guidelines and was, therefore, not detennined. Vehicle and positive controls were used in paral[el with the test material. A solvent treatment group was used as the vehicle control and the positive control materials used in the series of plates without S9-mix, were as follows: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 Ng/p[ate for WP2uvrA

- , 3 pg/piate for TA100 and 5 Nglplate for TA1535 9-Aminoacridine (9AA): 80 pg/p[ate for TA1537 4-Nitroquinoline-1-oxide (4NQ0): 0.2 Ng/plateforTA98In addition, 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP), which are nonmutagenic in the absence of inetabofising enzymes, were used in the series of plates with S9-mix at the following concentrations: 2AA at 1 pg/plate for TA100 2AA at 2 pg/plate for TA1535 and TA1537 2AA at 10 pg/plate for 

### WP2uvrA BP at 5 pg/plate for TA98

3.3 Microsomal Enzyme Fraction S9 was prepared in-house on 23 November 2008 (Preliminary and Range-finding tests) and 18 May 2008 (Main Test) from the livers of rnale rats weighing — 200g. These hadPROJECT NUMBER: , PAGE 10 

### each orally received three consecutive daily doses of phenobarbitone/(3-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on Day 4. Before use, each batch of S9 was assayed for its ability to metabolise appropriate indirect mutagens used in the Ames Test. The S9 was stored at -196°C.

3.4 S9-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.

S9 5.0 ml 1.65 M KCl/0.4 M MgClz 1.0 ml 0.1 M Glucose-6-phosphate 2.5 ml 0.1 M NADP 2.0 ml 0.2 M Sodium phosphate buffer (pH 7.4) 25.0 ml Sterile distilled water 14.5 ml A 0.5 ml afiquot of S9-mix and 2 ml of molten, trace histidine or tryptophan supp[emented, 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% BD Bacto agar and 0.5% sodium chloride with 5 mf of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 ml of top agar. Vogel-Bonner Minimal agar plates were purchased from ILS Ltd.

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 assay was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 Ng/plate. The assay was perFormed by mixing 0.1 ml of bacterial culture (TA100 or 

### WP2uvrA
- ), 0.1 ml of test material formulation, 0.5 ml of S9-mix or phosphate buffer and 2 ml of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test material and aPROJECT NUMBER:. PAGE 11



## vehicle control (sterile distilled water) were tested. In addition, 0.1 ml of the maximum concentration of the test material 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 material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn. A technical error was identified during the scoring of the Preliminary Toxicity Test. Salmone(la 

## strain TA100, dosed in the absence of S9, exhibited no bacterial background lawn growth after the specified incubation period. The reason was not identified, however it was decided that a repeat experiment was not required as there was no toxicity noted to either TA100 (presence of S9) and WP2uvrA (absence and presence of S9). It was therefore, assumed that the test material would also be non toxic to TA100 in the absence of S9 (this assumption was confirmed in both the range-finding and main tests).
3.5.2 Mutation Test — Experiment 1(Range-finding Test) Five concehtrations of the test material (50, 150, 500, 1500 and 5000 Nglplate) 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 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle 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 material both with and without S9-mix. AII of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.
3.5.3 Mutation Test— Experiment 2(Main Test)The second experiment was performed using methodology as described for the rangefinding test, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as the range-finding test (50 to 5000 Ng/plate).PROJECT NUMBER: - PAGE 12



## 3.6 Acceptance Criteria The reverse mutation assay may be considered valid if the following criteria are met: AII tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method section 3 with historical control ranges for 2007 and 2008 in Appendix 3. The appropriate characteristics for each tester strain have been confirmed, eg 

## rfa cell-wall mutation and pKM101 plasmid R-factor etc. AII tester strain cuftures should be in the range of 1 to 9.9 x 10 9 bacteria per ml. Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix. The historical control ranges for 2007 and 2008 are presented in Appendix 3.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.
3.7 Evaluation Criteria There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and(or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (6) can also be used as an aid to evaluation, however, statistical significance will 

## not be the only determining factor for a positive response. A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.PROJECT NUMBER:: PAGE 13 

### Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

4. ARCHIVLS Unless instructed otherwise by the Sponsor, all original data and the final report will be retained in the Harlan Laboratories Ltd, Shardlow, UK archives for five years, after which instructions will be sought as to further retention or disposal.PROJECT NUMBER: PAGE14



## 5. RESULTS 5.1 Preliminary Toxicity Test The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA

- ). The test material 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 withouf (-) 59- mix 

## Strai pose (pgipfate)

0 0.15 0.5 1.5 5 15 

# 1 50 

# 1 150 

# I 500 

# 1 1500 5000 TA100 ND

+ TA100 132 121 119 101 

# 1 106 

# 1 108 

# 1 101 122 

# 1 118 

# 1 129 

# 1 121

- WP2uvrA - 22 30 22 26 23 21 21 

# 1 22 

# 1 23 25 25

+ WP2uvrA - 32 36 37 29 30 36 31 35 31 30 30 ND No 

## data available due to technical error 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). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. 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 material, vehic[e and positive controls both with and withoui metabolic activation, are presented in Table 2 to Table 5 with the results also expressed graphically in Figure 1 to Figure 4. Information regarding the equipment and methods used in these experiments as required by the Japanese Ministry of Economy, Trade and Industry and Japanese Ministry of Health, Labour and Welfare are presented in Appendix 1.PROJECT NUMBER: PAGE 15



## A history profile of vehicle and positive control values is presented in Appendix 3. The test material 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 pglplate. No test material precipitate was observed on the plates of 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, at any dose level either with or without metabolic activation. AII 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 material was considered to be non-mutagenic under the conditions of this test.
7. REFERENCES 1. Ames B N, Durston W E, Yamasaki E and Lee F D(1973b) 

## Proc. IVatl. Acad. Sci. (USA), 70, 2281-2285.
2. Ames 

# B N, McCann J and Yamasaki E(1975b) Mutation Research, 31, 

## 347- 364. 3. Maron D M and Ames B N(1983) 

## Mutation Research, 113, 173 - 215. 4. Garner R C, Miller E C, and Miller J A(1972) CancerRes. 32, 2058 - 2066. 5. Mortelmans K, Zeigler E(2000) 

## Mutation Research, 

## 455, 29 - 60. 6. Kirkland D J, (Ed) (1989) Statistical Evaluation of Mutagenicity Test Data UKEMS sub-committee on Guidelines for Mutagenicity Testing. Report Part III - Cambridge University Press.PROJECT NUMBER: PAGE16 REVERSE MUTATION ASSAY "AMES TEST" USING 

### SALMONELLA TYPHIMURIUM AND 

### ESCHERtCHIA COLt 

## Table 1 Spontaneous Mutation Rates (Concurrent Negative Controls)

Range-finding Test Number of revertants (mean number of colonies per plate)

Base-pair substitution type Frameshift type TA100 TA1535t 

### tNP2uvrA - TA98 TA1537 120 114 (118) 121 26 20 (19) 10 37

31 (30) 21 16

15 (18) 22 14

13 (13) 13

Mafn Test Number of revertants (mean number of colon ies per plate)

Base-pair substitution type Frameshlft type TA100 TA1535 

### WP2uvrA - TA98 TA1537 68 94. (81) 81 14

27 (22) 26 24

22 (25) 28 28

20 (27). 33 10

14 (11) 8

Experimental procedure repeated at later date (absence and presence of S9) due to large differences between vehicle revertant controf counts in original test

### PROJECTNUMBER: PAGE17 REVERSE MUTATION ASSAY "AMES TEST" USfNG 

### SALMONELLA TYPHIMURIUM AND 

### ESCHER(CHIA COLI 

## Table 2 Test Results: Range-Finding Test Without Metabolic Activation From: 25 January 2009 To: 28 January 20D9 Test Period 29Janua 2009t 01 Februaij 2009}

Test Number of revertants (mean number of colonies per ptate) Without substance Base-pair substitution type Frameshifttype 59-Ma concentration TA700 TA1535t WP2uvrA" TA98 TA1537

u /plate) 1007 (103) (17) ( 2 9) 20 (21) 

### ~ d (11) 0 11.2# 1D 6.1 29 3.5 3.2 0.6

111 21 32 19 11 (104) (21} 29 (31) 26 (20) 12 (10)
- 50 99 7.8 25 6.7 34 2.5 24 4.0 10 2.5

113 24
0 15D (105) (23) (31) 

### ~~ (15) 1 2 (9) - 110 4.6 25 2.9 32 1.5 12 4.4 9 2.5 102 20 29 109 (106) 19 (21) 27 (30) 23 (22) 10 (10) - 500 110 6.1 30 8.6 32 2.9 20 23 1.7 10

0 0.6 99 13 32 (109) (17)

27 30 (28) 23 (22) i 0 (10}
- 1500 108 2'1 35 1.7 1.2 10 0.6

107 15 27 21 98 (107) (28) 5000 (27) ?9 (20) (11) - 9.5 30 5.7 30 2.5 1.0 13 2.6 106 22 25 21 8 Name ENNG ENNG ENNG 4NQ0 9AA Positive Concentration 3 5 2 0.2 80 controls (pg/plate) 663 925 20 

### ~ 263 1757 S9-Mix

_ No, colonies 553 (5 

### ~ ) (745) 661 2142 (21i9) 200 (232) 726 (1224) perplate 536 68 ' 9 648 156.3 2170 66.2 232 31.5 1180 516.4

t Experimental procedure repeated at later date due to large differences between vehicle revertant control counts in original test ENNG N-ethyl-N'-nitro-N-nitrosoguanidine 4NQ0 4-Nitroquinoline-l-oxide 9AA 9-Aminoacridine # Standard deviation

### PROJECT NUMBER: f PAGE 18 

### REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COL! 

## Table 3 Test Results: Range-Finding Test With Metabolic Activation

Test Period From: 25 January 2009 

# 1 To: 2B January 2009 

### 29 January 2009t 01 February 2009t Test Number of revertants (mean number of colonies per late) With 59- substance

. Base-pair substitution type rrameshift type Mix concentration TA100 TA1535t WP2uvrA - 

### ~ TA98 TA1537 

### (p lplate) (107) 

### (11) 12 

### (36) (27) 

### ~ (10) + 0 104 4.6# 2.1 

### 32 3.2 

### 27 2.0 2.6 112 13 38 29

~ 12 

### 106 (10D) 

### 12 (11) 31 34 

### (33) 24 (24) 13 7 

### (10) + 50 95 5.6 

### 10 1.0 1.5 

### 27 22 2.5 9 3.1 99 11 33 

### (99) 1 

### 9 (12) 35 (33)



### ~ S (23) 8 (8) + 150 6 4'4 3.6 2.1 25 6.7 7 0.6 97 16 31 

### (99) 

### ~ 4 (15) 37 (33) 25 (24) 

### ~~ (11) + 500 95 6.1 4.2 32 22 1.7 9 2.5 106 20 39 (103) (14) (32) (22) 1 (9) . + 1500 95 7.5 13 0.6 33 0.6 25 3.5 2.0 103 14 32 22 9


### 0a (102) 

### (16) 35 (34) (24) (11) + 5000 4'6 19 

### 2.5 1.0 21 Z7 

### 3.1 10

12 

### 1.0 101 t6 33 Name 2AA 2AA 2AA BP 2AA Positive Concentration 1 2 10 5 2 controls 

### 59-Mix (Ijglplate) 3417 (3482) 228 (167) 597 

### (605) 221 

### (240) 647 

### ~ (626)

No. colonies 3244 276.3 126 564 46"1 254 17'1 613 18.6 + perplate 3785

~ ' 7 148 655 245 617



### t Experimental procedure repeated at later date due to large differences between vehicle revertant control counts in original test BP Benzo(a)pyrene 

### 2AA 2-Aminoanthracene # Standard deviation

### PROJECT NUMBER: PAGE 19 

### REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPH/MURIUM AND ESCHERICHIA COL(


## Table 4 Test Results: Main Test- Without Metabolic Activation
Test Period From: 06 rebruary 2009 1 To: 09 February 2009

Test Number of revertants mean number of colonies per late W ithout substance Base-pairsubstitufiontype Frameshifttype S9-Mix concentration TA100 TA1535 WP2uvrA - TA98 TA1537

(pglp 

### late ) 

### 89 (96) 21 (26) 30 (35) 20 

### (21) 16 (11)
- . 0 

### 9~ 7.5# 2 

### ~ 4.2 39 4.5 24 23 1 0 4.6 

### (86) (25) 38. (39) 

### (16) (15) - 50 89 5.5 

### 21 3.6 47 2.1 

### 28 t1 10.1 

### 11

18 3.5 90 27
8 

### ~ (85) (16) (34) (20) 9 0 (10) - 150 6.4 17 16 1.0 39

33 5.0 28

14 7.4 10 0.6 95 

### (84) (28) (26) .(28) 6 (11) - 500 95 11.5 

### 28 2.5 

### 30 4.0 

### 24 6.1

. 4.2 B4 31 22 35 12 (86) (26) (26) 22 - (23) 12 

### ~ (10) - 1500 89 6.4 22 4.0 27 6.6 22 1.7 9 2.1 79 26 32 (106) 15 (23) 31 (34) 14 (24) 8 (9) - 5000. 26 17.7 7.2 2.5 22 11.7 10 1.2 97 28 3 6 Name ENNG ENNG E NNG 4NQ0 9AA Concentrtlon 3 5 2 02 80

E wtro le5 (pg/plae963 1496 1630 273 369

No. colonies 917 (890) 1110 (1314) 1720 (1621) 

### ~ 247 (256) 367 (5 

### ~ ) . perplate 790 89.6 1336 193.9 1514 103.3 249 14 ' 5 897 305.4

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine 4NQ0 4-Nitroquinoline-1-oxide 9AA 9-Aminoacridine # Standard deviation

### PROJECT NUMBER: REVERSE MUTATtON ASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI 

## Table 5 Test Results: Main Test With Metabolic Activation
Test Period From: 06 Februa 2009 To: 09 February 2009

Test Number of revertants mean number of colonies per fate With S9- substance Base-pair substitution type Frameshift type Mix concentration TA100 TA1535 WP2uorA - TA98 TA1537

(pgip 

### l 129 (122) 

### 13 (12) 37 (39) 28 (28) 22 (16) + 0 116 6.6# 12 0.6 

### 38 2.1 

### 24 4.5 

### 17 3.6 121 12 A1 33 15


### 50. . (109) 

### (13) (35) (28) (16) + 08 10'0 14 2.3 14 

### 28 6.7 

### 30 29 3.2 

### 12 4.7 119 37 14 

### {114) (15) 15 

### 2. 2

1 

### (25)

~ 32 

### (27) 8 (8)
. + 150 123 8"1 3.0 1S 2.9 17 8.7 7 1.0 108

. 27 

### 113 (102j 10 (11) 30 39 

### (33) 39 (36) 13 (11) + 500 96 9.3 10 1.7 4.9 

### 39 5-2 

### 10
- 9 2A

. 98 13 31 30 

### 105 (109) 10 

### (11) 38 (36) 45

~ (32) 

### 25 (20) + 1500 129 18.8 

### 11 0.6 11 

### 35

35 1"7 

### 25

26 11.3 

### 13

21 6.1 92 

### (108) 9 (10) 33 

### (36) 30 (30) 16 

### (16) + 5000 100 9'1 2.3 4-2 33 3.5 17 0.6 118 13 41 Name 2AA 2AA 2AA BP 2AA Positive Concentration 1 2 10 5 2 controls S9-M1x (P9/Plate) 2308 (2896) 121 (183) 4 ~ (505) 186 {196) 629 (614) + No. colonies 3043 207 555 222 656

perplate 3336 529.6 220 53.8 522 60.3 179 23.1 556 513 

### PAGE 20

BP Benzo(a)pyrene 2AA 2-Aminoanthracene # Standard deviation0 O (D U)

O O LO

Q

~

0 LO



### C 0 0 LO



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L"TPROJECT NUMBER: : PAGE 25 

## REVERSE MUTATION ASSAY "AMES TEST° USING SALMONELLA TYPHIMURIUM 

## ANC) ESCHERlCH7A COLI 

## Appendix 1 Report of Results in Mutagenicity Test using Micro-organisms GENERAL ITEMS (to be completed by the sponsor)
Name of the new chemicalsubstance (IUPAC nomenclature) Other name

Structural formula or rational formula (or outline of manufacturing method, in case both are unknown)

Lot No. of the new Purity of the new chemicalsubstance 26.5% chemicalsubstance tested tested Name of impurities Ash (predominantly sodium chlorfde) around 5% and concentration Vapour pressure Typically 3500 Partition coefficient CAS No. Molecular weight Melting point (°C) Appearance at ordinarytemperature Ambercoloured liquid Boiling point ( 0 

## C)

- Stability Stable under normal temperatures and pressure
Solvent Degree of Stability Solvent Degree of Stability

solubility in solvent solubility in solvent Degree of solubility Waterrt Miscible at DMSOt Immiscible

- in solvent 50 m /ml Acetonet Immiscible

- DMFt Immiscible

- THFf Immiscible

-

t Solubifity data provided by the Sponsor tt Solubility data generated by the testing facility No visible change in test material formulations throughout each experiment, therefore, assumed stablePROJECT NUMBER , PAGE 26 I tEVERSE MUTATION ASSAY "AMES TEST" USING 

### SALMONELLA TYPHIMURIUM AND 

### ESCHERICHIA COLI
Appendix 1 (continued)


### 2. TESTER STRAINS

Tester strain Obtained from Date obtained TA100 UCB' 04 August 1995 TA1535 UCB* 04 August 1995 WP2uvrA BIBRA*' 17 August 1987 TA98 UCB* 04 August1995 TA1537 UCB* 04 August 1995



### S-9 MIX (1) Source of S9 (encircle the applicable number and fill in the relevant entries) Made in-house or purchase 1 Made in-house 12. Purchase (Supplier) Prepared on 23 November 2008 (range-finding test) 18 May 2008 main test Lot No. (in case of purchase) Storagetemperature -196 0 C
(2) Preparation 

### of S9 Animal used Inducing substance Species strain Rat, Wistar Name Phenobarbitone/(i-naphthoflavone Sex Male Administration method Oral Age (in weeks) Wei ht 9 6- 8 weeks

— 200 9 Administration period and amount (mg(kg bod ei ht/da 3 days 80/100 mg per kg per day
(3) Composition 

### of S9-Mix Constituents Amount in 1 ml S9-Mix Constituents Amount in 1 ml S9-Mix S9 0.1 ml NADP 4.0 pmol M9Cl 2 8.0 pmol Na-Phosphate buffer 100.0 pmol KCl 33.0 

### Nmol 6lucose-6-phosphate 5.0 pmol Glucose-6-phosphate deh dro enase

* UCB: University of Cafifornia at Berkeley ** BIBRA: British Industrial Biological Research AssociationPROJECT NUMBER: PAGE 27 REVERSE 

### Ml3TATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUM AND ESCHERlCHIA COL! 

## Appendix 1 (continued)
4. PREPARATION OF THE TEST MATERIAL IN SOLUTION (encircle the applicable response)
Name Supplier Lot No. Grade Purdy (%) Solvent used Sterile distilled AGUETTANT 3003930 12 water LTD Reason for selection of Fully miscible at 50 mglml solvent Appearance of the test Sotutton Suspension Others ( ) material preparation Suspension and other methods when test Solution achieved by action on an autovortex mixer substance difficult to dissolve Storage time and temperature of solution from Up to 1 hour 40 minutes room temperature (mnge-finding test) preparation until use 40 minutes room temperature (main test)

. Conversion of purity Yes No


## 5. CONDITIONS OF PRE-CULTURE ETC. (1) Conditions
Name Manufacturer 

# I Lot No. Nutrient Broth Oxoid NB No.2 Oxoid Ltd. 629250 02/13 Period of pre-culture 10 hr Culture ffask (form, size) Costar 75 cm 2 tissue culture flasks Amount of culture medium 5 ml Amount ot strain 20p! inoculated 

## (2) Cell viability at the end of pre-culture Base-pair substitution type Frameshift type TA100 TA1535 WP2uvrA" TA98 TA1537 Cell number Range-finding test 1.9 1.2 t 1.8 1.7 1.6 (x 109/ml) Main test 1.4 2.6 2.6 1.8 1.1 Count method (encircle the applicable number) - Conversion from O.D. value 2 Stepwise dilution method 3. Others ( ) Experimental procedure repeated at later date (absence and presence of S9) due to large differences between vehicle revertant control counts in original testPROJECT NUMBER: `. PAGE 28 1 2EVERSE MUTATION ASSAY "AMES TEST" 

### USING SALMONELLA TYPHIMURIUM AND 

### ESCHERlCHIA COLI 

## Appendix 1 (continued)
6. MINIMUM GLUCOSE AGAR PLATE MEDIUM (encircle the applicable number and 

### fill in the relevant entries)
Made in-house or 1. Made in-house purchase 

# O Purchase (ILS Ltd) Prepared on 15 and 23 January 2009 (range-finding test) 25 January 2009 (main test) Lot No, in case of 1051721-02 07113 urchase


## 7. TEST METHOD (encircle the applicable number and fill in the relevant entries) 

## (1) Test Method and the reason why itwas setected

1. Pre-incubation method 

## O2 Plate incorporation metliod Test method used 3. Others 

# ( ) Reason in case of 

# _ others 

## (2) Test Conditions
Pre-incubation Plate Method

Bacterial suspension ml 0.1 ml Test substance solution m! 0.1 ml Na-phosphate buffer ml 0.5 mI Composition S9-mix (in case of inetabolic activation

method) ml 0.5 ml Top agar solution ml 2.0 ml Others 

# ( ) ml 

# - Pre-incubation Temperature °C

Time min Incubation Temperature "C 37°C

Time hours 48 hours


## 8. COUNT METHOD OF COLONIES (Encircle 1 and 2 if both methods used)
Count method 1. By hand 

# O2 Colony counter Conduction of correction 1. No 

# O2. Yes (Correction method: Area and miscount correction)PRQ.IECT NUMBER: PAGE 29 REYERSE MUTATION ASSAY "AMES TEST" 

### USING SALMONELLA TYPHIMURIUM AND 

### ESCHERICHlA COLI 

### Appendix 1 (continued)
9. TEST RESULTS (1) The results shou[d be reported on the attached form (2) Judgement of the results Judgement (encircle one) Positive Ne ative Rea son for iudgement : No significant increase in the frequency of revertant colonies was recorded for any bacterial strain used with any dose of the test material in two separate experiments either with or without metabolic activation. (when judged as positive, attach a table for relative activity) 

### (3) Referential matters Formu(ated concentrations were adjusted to allow for the stated water/impurity content (73.5%) of the test material. The test material was fully miscible in sterile distiEled water at 50 mg/ml (the most concentrated stock solufion) in solubility checks pertormed in-house. Sterile distilled water was therefore selected as the vehicle. There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strafn with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (6) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response. A test matedal 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 a definitive judgement about the test material activity. Results of this type will be reported as equivocal. Phenobarbitone/(3-naphthoflavone was used to induce higher enzyme levels in the rat Iiver S9. The S9- mix used in each experiment of this study was shown to be sterile. The test material formulations showed no evidence of contamination when tested for steri[ity during the preliminary toxicity test. The use of approximate half-log dilufions instead of twofold dilutions was agreed with the JMOL in January 1995. [REMARKS] "Referential matters" —(fill in the view etc of the Study Director on the test results)- PROJECT NUMBER' 

### PAGE

. REVERSE MUTATION ASSAY "AMES TEST° USING sAi.rtatJNeLLA TYPHIhPURIUM AND ESCHERICHlA COL! 

## Appendix 'f (continued) 10. OTii ERS

Name Harlan Laboratories Ltd.
Address Shardlow Business Park, Tel No: 0044 1332 792896

Shardlow, Derbyshire, DE72 2GD, UK Fax No: 0044 1332 799018 SYudy Title Genetic Toxicologist Name A J Bowles BSc (Hons) 

### Director Years of experience: 14 Study 

### number 2724/0001 Experiment 1: From: 25 January 2009 o: 28 January 2009

1. Each experiment Experiment 1(repeat): From: 29 January 2009 To: 01 February 2009 Test date: Experiment 2: From: 06 February 2009 To: 09 February 2009 dates From: 07 Jay ~u~ 5y..200,9„ 9

2. Test Period: LUU 

### To: i P t t{PROJEOT NUMBER: 

### PAGE 31 

### REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUM 

### AND ESCNERfCHfA COLf 

### Appendix 2 Test Material Certificate of Analysis

CMFiicete of Analysis

" A qealily management system reyistered to Ihe InternaQOnal starzdard

- ISO 9001 was used to menutacture and test ihte materlal. The name printed at Ihe end of lh{s docnmgnt Is an electranlc signatura

Customer details Customer ReL - Inspectfon Lot 090000080106

- . C of A Printed. 28.11,2000 .
®uantity. 100 G CA Cuntact, Fax No.

Batch Details Product Name: ProductCode: CuaL Product Namec Cuat. Produet Code: Batch No. 240074 Date of Test: 30.072008 Speoification: CREATED 15-DEG-2005 Data of Menufacture: 08.11.2007 Date of Expiry: 07.11.2010 Quality Control Results Artalyfical Test Speciffcatlon Limit Method No. Characteristic Lower Upper Value Unit Status

Addenoum 00 PassorFail Pass - P G6000052 DP77/1BAPPEARANCE LIQUID

. Pass - P

(FORM) G6000052 DP1711BAPPEARANCE AMBER Pass - P

(COLOUR) , G600D052 DP17f1BAPPEARANCE CLEAR Pass P

- (CLARiTY)

. G6000171 DP2/1BASH 4.0 6-5 4.8 % P G6000173 DP14/5COLOUR 4.0 10.0 5.7 Gardner P G6000174 DP6/5 PH 4.5 5.5 5.1 P G6000177 DP1/36TOTALSOLIDS 26.0 30.0 26.5 % P G6010097 DP3 ISOIONIC POINT 1D.0 14.0 11.2 P

PH G6010176 DC2/2 NITROGEN 3.0 4.0 3.0 % P G6020001 DM112S TOTAL COUNT 100 OPG MAX Pass - P

0 30°C G6020002 DM211SYEASTS& 1000PGMAX Pass - P

MOULDS ® 25'C G6020011 DM1611SGRAM ABSENT Pass - P

Page 10 2

### PROJECTNUMBER:: PAGE 32 : REVERSE MUTATION ASSAY "AMES TEST" USING 

### SALMONELLA TYPHIMURIUM AND ESCHER/CHPA 

### COLI 

## Appendix 2 Test Material Certificate of Analysis (continued)

Certificate of Analysis

A quality manegement system regtstered m the intematbnal stardard ISO 9001 was used te manuractn.e and tast thls matarlal. The Parne printetl at the end of IDla dowment I. an aleprmla tigneture.

Customer details Customer Hef.

. - Inspection Lat 090000060106 C of A Printed.

~ 28.11.2008

- 664144

- _-___ 80685310 Quantdty. 100 G QA Contact. Fatt No,
NEGATIVE BACILLI 1G G6020012 DM11l2SSTAPH.AUREUS A6SENT Pass - P

1G It Is recommended that thls product be used within 6 months of deGvery. Batch Status: Pass Confirmed by

Stephen Hughes.

Page2o/ 2PROJECT NUMBER: S PAGE 33 REVERSE MUTATION ASSAY "AMES TEST" USING 

### SALMONELLA TYPHIMURlUM AND 

### ESCHERtCHIA COLI 

## Appendix 3 History Profile of Vehicle and Posittve Control Values COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2007 Strain S9-Mix TA700

-S9 +S9 TA1535

-S9 +S9 WP2wrA"

S9 +89 TA102

-S9 +S9 TA98

-59 +S9 TA1537

-59 +S9 WP2°vr,4

pKM701 -59 +S9 WP2pKM101

-S9 +S9 Mean 94 87 21 13 26 28 273 309 21 26 11 13 132 182 100 128 SD 16.0 14.8 5.2 3.6 5.8 6.4 40.4 43.2 5.5 6.0 3.8 4.1 48.5 47.8 21.8 21.4 Min 63 50 9 6 12 11 194 208 7 11 2 4 65 78 67 98 Max 156 152 38 28 46 49 390 395 48 57 28 27 276 263 136 165 Values 807 629 767 601 1541 384 159 107 1281 609 1245 569 197 126 127 15 POSITIVE CONTROL VALUES 2007 Strain S9-Mix TA700

-S9 +$9 TA1535

S9 +S9 WP2wrA"

-S9 +S9 TA102

-89 +S9 TA9B

89 +S9 TA1537

-S9 +S9 WP2wrA

pKM101 -59 +S9 WP2pKM101

-S9 +S9 Mean 556 1231 343 231 652 505 1281 960 256 308 1697 339 960 1103 2134 997 S D 313.5 651.1 864.2 4765 707.9 535.2 458.9 303.6 289,7 280.5 1193.6 254.4 1679.6101D.5 1047.D586.4 Min 258 320 79 73 138 139 567 401 78 106 117 107 332 355 1091 285 Max 1634 3777 2087 661 2023 1185 2422 1441 1483 2055 5618 677 2522 3023 3934 2024 Values 196 197 197 203 1159 159 60 61 419 202 414 198 64 64 9 11 

### COMBINED VEHlCLE AND UNTREATED CONTROL VALUES 2008 Slydin S9-Mix TA100

-S9 +S9 TA1535

~ -S9 +S9 TA102

-S9 +S9 TA98

-59 +S9 TA1537

-59 +S9 TA1538

S9 +59 WP2uvrA"

S9 +S9 WP2wrA

PKM101 -S9 +S9 WP2pKM101

-S9 +S9 Mean 105 101 23 15 279 313 19 26 13 13 11 17 26 29 166 196 83 126 SD 18.7 18.5 5.3 4.8 43.4 42.7 4.6 5.3 3.7 3.8 3.5 5.6 5.9 7.2 40.1 37.7 19.9 28.0 Min 63 62 10 8 181 204 10 11 2 4 5 11 12 12 82 105 51 63 Max 171 184 38 36 363 387 50 44 27 28 20 22 49 55 250 271 147 180 Values 1 891 688 1 821 588 1 137 79 1160 620 1139 569 63 4 620 446 145 104 78 44 POSITIVE CONTROL VALUES 2008 S(rain TA100 TA1535 TA102 TA98 1538 WP2wrA" WP2nvrA Wp2pKM101 TA1T 5Z7 A pKM101 S9-Mix 39 +S9 -S9 +S9 -S9 +59 -S9 +S9 -59 +S9 -59 +S9 -59 +S9 -S9 +S9 Mean 487 1138 210 272 1170 1057 155 247 930 279 199 472 548 426 864 2034 1592 654 SD 184.7 5172 107.7 94.9 328.6 257.3 70.8 178.2 476.2 101.7 262.4 200.5 231.5 273.9 490.7 781.5 594.2 328.5 Min 505 303 79 103 719 654 75 120 78 92 64 112 101 138 432 749 431 208 Max 1329 2854 653 777 2525 1758 703 2177 2917 779 1746 1351 1881 3496 2786 3551 2571 1465 Values 231 232 230 229 54 52 335 237 338 227 61 42 177 174 46 25 30 25

SD = Standard deviation 

### Min = Minimum value Max = Maximum value

### PROJECTNUM8ER: PAGE 34



## Appendix 4 Statement of GLP Compiiance in Accordance with Directive

2004/9/EC

(



## DDepartment j{ 

# ~ of Heafth

THE DEPART:VIENT OT HEALTH OF THE GOC'EiINMENT

O&" TFTE L7NITED IzINC;DOVI GOOD LABE7RATORYPRACPICE
STATP.k16NT OP' C0N91't.lAtiCE 1N .4CCORUANCE PTITH DIftECT1YG 20ov`JJF C

TPS"1.' fACtCrrR Tr35T TYPI
[1nrlan Laboratories Ltd. AmtlyticaVCVinicul Ch¢mis

- try' Shardloic 13nsincss Parlc Enrvironmental Fate London Rond EuvfroutnenlniPozicology Shnrdlow Mueugenicity Uerbe - Yhys/C6em Dli72 ?Gll Toxiculogy

1)AT6 OP INSPF,C YION 19HI AugLLst 2003 Agenera( inspection for compliance with the Pt4nciples of Good Labom,ory Practice )s+as carried ouf at t'he ubovtl testfncilit}• z> 11ar,t of the U(i_GLP Comp[iance Progr

- nmme. At the time oi inspecBon no dcviations ivere found oC sufficient macnStu<Te to aCfect tlte eatiJity of noo-clinicxl stadies perFormed at the.ce facilit'ies.

. .—., . ~

F Dr-.lndrmv.,. (:rap Head, UK GL.P Ivlonitocuiqt Autharitv~ G 

# rl ,,,

PROJECT NUMBER7 1 verify that this is an exact copy of the original report which is located in the Archives of Harlan Laboratories Limited, Derby, UK.

AJ Bowles BSc (Hons)

..................................................................................... Study Director

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