### PAGE 1 OF 34 PAGES 

## rl is

:~



## REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUM AND ESCHERlCi-IfA COLI

v'



## 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: ' PAGE 2


## QUALITY ASSURANCE REPORT 

## This study type is classed as short-term. The standard test method for this study type ("General Study Plan" in OECD terminology) 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 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 othenvise 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 Standard Test Method Compliance Audit 18, 19 February 2009 Test Material Preparation 16 February 2009 Test System Preparation (reported on 02 March 2009) 18 February 2009 Exposure 19 February 2009 Assessment of Response § 

## 13 March 2009 Draft Report Audit § 

## Date of QA Signature Final Report Audit § 

## Evaluation specific to this study

.................. .......................... DATE: For the Quality Assurance Unit*

*Authorised QA Signatures: Deputy Head of Deparfinent: JM Crowther MIScT MRQA Senior Audit Staff: G Wren ONC MRQA 

# 0 6 APR2009

............... ... ....PROJECT NUMBER:: PAGE3



## GLP C®MPLIANCE STATEMENT The work described was performed in compliance with UK GLP standards (Schedufe 1, Good Laboratory Practice Regulations 1999 (SI 199913106 as amended by SI 200410994)). 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 2004110/EC.
This report fully and accurately reflects the procedures used and data generated.

DATE: 

# ........Q..I..AP.

....

009...... A Bowles BSc (Ho } Study Director

This report may be presented in final fonn as a digital (pdf) document. Such documents are prepared by scanning the paper original, and are considered of equivalent integrity and authenticity to versions producetl by optfcal photocopy. However, in all cases the hantl-signed paper original, held in secure archives, is the defin8ive document

### PROJECT NUMBER: PAGE 4

~~ 

### Z211

~~ 

### 0 

## 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 

# 5. RESULTS 14 5.1 Preliminary Toxicity Test 14 5.2 Mutation Test 14 

## B. GONCLUSION 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 Activation 23 

## Figure 4 Dose Response Cunre: Main Test— With Metabolic Activation 24 Appendix 1 Report of Results in Mutagenicity Test using Micro-organisms 25 Appendix 2 Test Material Certificate af Analysis 31 Appendix 3 History Profile of Vehicle and Positive Control Values 33 Appendix 4 Statement of GLP Compliance in Accordance with Directive 200419/EC 34PROJECT NUMBER: PAG E 5



### REVERSE MUTATION ASSAY "AMES TEST" 

## USING SALMOiVELLA TYPHIMUR/UM AND



## ESCHER/CH/A COLI

SUMMARY
Introduction. 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 Regufation (EC) number 44012008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Methods. 

## Salmonel/a 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 vehicle (ste(le 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 effioacy 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 IJg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No toxicologically 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. A small, statistically significant increase in TA100 revertant colony frequency was observed in the presence of S9 at 500 Ng/plate in the main test. This increase was considered to be of no biological relevance because there was no

### PROJECT NUMBER: PAGE6 

### evidence of a dose-response relationship or reproducibility. Furthermore, the revertant counts at 500 Nglplate were within the in-house historical control range for the tester strain and the fold increase was only 1.13 times the concurrent vehicle control.


## Gonclusfon. The test material was considered to be non-mutagenic under the conditions of this test.

### PROJECT NUMBER: PAGE 7



## REVERSE MUTATION 

## ASSAY "AN1ES TEST" 

## USING SALMONELLATYPHIMUR/UMAND

ESCHERICH/A COLI

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 Gamer et al (4) in whfch mutagenic activity is assessed by exposing histidine auxotrophs of Satmonella typhimurium and tryptophan auxotrophs of Escherichia coli to various concentrations of the test material. 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 B13114 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 mutant strains of Salmonella are incapable of synthesising histidine and are, therefore, dependent on an external source of 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 chemicai mutagen may arise as a result of base-pair substitution in the genetic material (miscoding) or frame-shift mutation in which genetic material is either added or deleted. In order to make the bacteria more sensitive to mutation by chemical and physical agents, several addifional traits have been introduced. These include a deletion through the excision repair gene (uvr8" Salmone/la strains) which renders the organism incapable of DNA excision repair and deep rough mutatfon (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: PAGE 8 

### 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 microsomaf 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 

## Salmonel(a typhimurium TA1535, TA1537, TA98 and TA100 

## E'scherichia coli WP2uvrA The four strains of 

## Salmonel(a 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. AII 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 snd incubated at 37°C for approximately 10 hours. EachPROdECT NUMBER: PAGE 9 

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

12 Preparation of Test and Control Materials The test material 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 material 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 aliow for the stated water/impurity content (79%) 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 determined.
Vehicle and positive controls were used in parallel 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 pg/plate for 

## WP2uvrA

- , 3 pglplate for TA100 and 5 Ng/plate for TA1535 9-Aminoacridine (9AA): 80 pg/plate for TA1537 4-Nitroquinoline-1-oxide (4NQ0): 0.2 pg/plate for TA98In addition, 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP), which are nonmutagenic in the absence of inetabolising 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 Ng/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 male 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 1 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.

89 5.0 ml 1.65 M KCl/0.4 M MgC12 1.0 ml 0.1 M Glucdse-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 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% BD Bacto agar and 0.5% sodium chloride with 5 ml 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 pg/plate. The assay was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA

- ), 0.1 ml of test material formulatfon, 0.5 ml of S9-mix or phosphate buffer and 2 ml of molten, trace histidine or tryptophan suppfemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test material and a

### PROJECT 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. Salmonella 

## 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).
15.2 Mutation Test — Experiment 1(Range-finding Test) Five concentrations of the test material (50, 150, 500, 1500 and 5000 pg/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 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mI 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 p[ates (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.
15.3 Mutation Test — Experiment 2(Main Test)The second experiment was perfonned using methodology as described for the rangefinding test, using fresh bacterial cultures, test material and controi solutions. The test material dose range was the same as the range-finding test (50 to 5000 pg/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. All tester strain cultures 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-nix. 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.

### PROJECTNUMBER: 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 wifl be reported as equivocal.



## 4. ARCHIVES Unless instructed otherwise by the Sponsor, all original data and the final report wilf 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: 

### PAGE 14



### 5. RESULTS
5.1 PrelPminary Toxicity Test The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA ). The test material formulation and S9-mfx used in this experiment were both shown to be sterfle.
The numbers of revertant colonies for the toxicity assay were: With (+) or without (-) S9- mix Strain pose (ug/plate) 0 0.15 0.5 1.5 5 15 50 150 500 1500 5000 TA100 ND

+ 

### TA100 136 106 109 

# 1 104 97 122 109 117 133 141 152

WP2uvrA 35 34 23 22 i 27 22 23 20 24 23
+ 

### WP2uvrA" 35 23 36 24 31 23 29 33 33 30 F 24

Nu No 

### oata available due Yo 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 pertormed 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, vehicle and positive controls both with and without 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 NllP4BER: PAGE 15 

## A history profife 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 Ievel and was, therefore, tested up to the maximum recommended dose level of 5000 IJg/plate. No test material precipitate was observed on the plates at any of the doses tested fn either the presence or absence of S9-mix. No toxicologicalfy significant increases in the frequency of revertant colonies were recorded for any of the bacterial straPns, with any dose of the test material, either with or without metabolic activation. A small, statistically significant increase in TA100 revertant colony frequency was observed in the presence of S9 at 500 ug/plate in the main test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the revertant counts at 500 Ng/plate were within the in-house historical control range for the tester strain and the fold increase was only 1.13 times the concurrent vehicle control. AII of the positive control chemicals used in the test induced marked increases in the frequency of revertant cofonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

6. CONCl.USION 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. Natt. 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-commiftee on Guidelines for Mutagenicity Testing. Report Part fll - Cambridge University Press.PROJECT NUMBER: ' 

### PAGE 16 EVERSE MUTATION 

### ASSAY "AMES TEST" 

### USING 5ALMONELLA TYPHIMURtUM AND ESCHERICHIA COLI

Tabte 4 Spontaneous Mutation Ratess (Concurrent Negative Controls)

Range-finding Test
Number of 

### revertants (mean number of colon ies per plate)

Base-pair substitution type Frameshift type TA100 TA1535t WP2uvrA' TA98 TA1537 120 114 (118) 121 26

20 (19) 10 37

31 (30) 21 16

15 (18) 22 14

13 (13) 13

Main Test Number of revertants (mean number of colonies per plate)

Base-pair substitution type Frameshift 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 89) 

### due to large differences between vehicle revertant control counts in original testPROJECT NUMBER: ' PAGE 17 

### REVER8E MUTATION ASSAY 

### "AMES TEST" USENG 

### SALMONELLA TYPHlMURIUM AND 

### ESCHERICHIA COLf 

## Table 2 Test Results: Range-Finding Test— Without Metabolic Activation

Test Period From: 25January 2009 

# 1 To: 28 January 2009 29 January 2009't 01 February 2009t Test Number of revertants (mean number of colonies per plate) Wthout substance Base-pairsubstitutiontype Frameshifttype S9-Mix concentration

( / late) TA100 TA1535} WP2uvrA" TA98 TA1537 (116) (12) (29) 6 .1# (23) 

# 8 (10) 

# - 0 123 10 1,5 25 27 5"3 24 1'0 1.5 112 13 23 10
50 

# 9 p (109) 14 (14) 32 (31) 23 (23) 

# 13 (10)

119 11.1 14 0.6 36 5.6 23 0.0 7 3.1 123 (112) 14 (14) 33 (30) 18 (22) 14 (11) 

# - 150 102 10.5 12 1.5 15 30 3A 22 4.0 10 2.6 111 27 26 g 102 13 (14) 32 (32) 23 (22) 10 (9) 

# - 500 87 (98 .) 100 15 12 29 30



# . 23 12 

# . 8 12 106 15 35 21 8


# - 1500 118 104 (113) 12 (13)

14 33

32 (33) 21

22 (21) 10

11 (10 118 8.1 12 1.2 33 0.6 21 0.6 10 0.6 107 (116} 15 (13) 31 (31) 23 (22) 10 (11) 

# - 5000 125 9.0 11 2.0 13 32

30 1.0 23 1.2 10 1.7 117 21 13 Positive Name ENNG ENNG ENNG 4NQ0 9AA 3 5 2 02 80 controls Concentration 663 (584) 925 (745} 2044 (2119) 263 (232} 1757 (1224) S9-Mix (pg/plate)

No. calonies 

# _ 553 68.9 661 156,3 2142 66.2 200 31.5 726 516.4

per plate 536 648 2170 232 1190

t Experimental prccedure 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-1-oxide 9AA 9-Aminoacridine # Standard deviation

### PROJECT NUMBER: PAGE 18 

### REVERSE MUTATfON ASSAY "AMES TEST" USINCd SALmurVELLA TYPHIMURIUM AND ESCHERICHIA COLI 

## Table 3 Test Results: Range-Ffnding Test- With Metabolic Activation
Test Per[od From: 25 January 2009 To: 28 January 2009 29 January 2009Y 01 February 2009t Test Number of revertants (mean number of colonies per plate) W(th 59- substance Base-pair substitutlon type Frameshift type Mix concentration TA100 TA1535t 

### WP2uvrA' TA98 TA1537

p / late) 

# g~ (105) (12) (37) (28) (13) + 0 5.6# 11 1.2 37 1.5 27 

### 30 2.1 13



### 11 1.5 106 

### 11 3S (91) (12) 

## ~ (32) (28) 9 (11) + 5D 95 8.7 12 2.5 2.0 25 2.6 14 2.5 81 t5 30 30 (100) (9) (32) (29) (11) + 150 93 6.1 0 1.2 

### 8 31 5.6 30 

### 29 1.5 10



### 8 4.2 104 

### 38
(99) (12) 31 {31) 31 (31) 8 (9) + 500 04 11 0.6 4. 4 0"6 32 1.0 8 1.7 97 12 31 (104) (9) (32} 29 (29) 1 6 (12) + 1500 93 10_5 10 1.2 31 2.1 2.5 4.0 104 10 30 32 13 (1Q4) (9)



### 9 35 (33

) 

### 3 

## 2

~ (30) 

### 15 (12) + 5000 07 7.0 0.0 2.1 2.5 10 2.9 109 9 31 30 Name I 2AA 2AA 2AA BP 2AA Positive Concentration 1 1 2 10 5 2 controls S9-Mix (pg/plate) 3417 

### (3482) 228 (167) 597 (605) 

## ~ 1 (240) 

### 647 (626) + No. colonies 3244 276.3 126 53.7 564 46.1 254 1T1 613 18.6

perplate 3785 148 655 245 617



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

### 2AA 2-Aminoanthracene 

## # Standard deviation

### PROJECT NUMBER: ' 

## PAGE19 REVERSE MUTATION ASSAY "AMES TEST" UsING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI 

## Tabfe 4 Test Results: Main Test Wfthout Metabolic Activatlon
Test Period From: 06 Febmary 2009 1 To: 09 February 2009

Test Number of revertants (mean number of colonies per late Without substance Base-pairsubsiitutiontype Frameshifttype S9-Mix concentration



### (Pglp 

### l TA100 TA1535 WP2wrA" TA98 TA1537



### 66 (78) (22) (32) 

# ~~ (18) 

# ~ s (12) - 0 8.7# 25 22 

### 3.0 37

28 

### 4.5 1.7 3.5 82 17 10 

### (72) (19) 28 (28) (22)



# ~ 7 (13) - 50 64 8.0 

### 16 21 2.9 28 0.6 

### 22 27 8.7 3.8 71 10 

### ( 7 4) 

# ~ 6 (21) (29) (19) (14) - i50 69 4'6 22 5"0 

### 29 29 0.6 

### 16 7.9 

### 20 4.9 77 13 11 

### (91) 27 (27) 

### (30) (22) 

# ~ (8) - 500 85 11.0 26 1.5 

### 30 1.5 

### 25 2.6 1.7 85 32 21 10 

### (84) 

# ~ 20 

### (24) (25) (17) 9 (10) - 1500 89 20 6.4 9. 9



### 28 26 3.6 

### 20 4.4 0.6 73 19 10 

### (95) Z6 (22) 30 (26) 30 

### (25) p (10) - 5000 87 20.2 20 

### 3.2 5.3 5.0 1.5

118 20 24 11
Positive Name ENNG ENNG ENNG 4NQ0 9AA 3 5 2 0.2 80 controls Concentration 

### 963 (890) 1496 {1314) 1630 {162i ) 

### 273 (256) 

### 369 (544) 

### S9-Mix (p9/plate)

No. colonies

_ perplate 917 790 89.6 1110

1336 193.9 1720

15i4 1033 247

249 14.5 367

B97 305.4



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

### PROJECT NUMBER:: 

### REVERSE MUTATION ASSAY "AMES TEST" USING 

### Sacnnonkf.LA TYPHIMURIUM AND 

### ESCHERICHIA COLI 

## Table 5 Test Results: Main Test- With Metabolic Activation
Test Period From: 06 February 2009 1 To: 09 February 2009

Test Number of revertants mean number of colonies per late) With S9- substance Base-pair substitution type Frarneshift type Mix concentration

(p / late TA100 TA1535 WP2uvrA' TA98 TA1537



### + 0 

# ~~~ (105) 

# ~ p (11) 3 

# ~ (32) 

# ~ (34) 9 (9) 4.5# i.2

12 31 2.3 29 5.6 2.5 101 6 (105J 8 (

9 ) 3a (3

1) 

### ~ (26) 9 (12) + 50 101 4.6 1.7 8 4.9 77.4 3.1 104 29 18 t5

+ 150 98 (101) 15 (15) 29 (29) 27 (26) $ (11)



### 107 5.2 20 

### 5.0 30 

### 1.0 25 

### 1.2 g 

### 4.4 124 * 14 (13) 30 (32) 32 (38) 10 (9 ) 108 9.9 10 2.6 38 5.7 44 6.0 7 1.7 

### 111 (105) 

### 11 (11) 32 {30j 14 (29} 13 (10j + 1500 104 5.6 

### 12 0.6 11 

### 26

32 3.5 

### 42 14.0 

### 8 2,6 100 30 9 

### (95) 11 

### (12) (37) (27) (9) + 5000 93 2.0 

### 9 3.1 15 

### 30 1.2 

### 27 

### ~ 3.0 

### 10 1.2 97 30 30 8
Positive Name 2AA 2AA 2AA BP 2AA 1 2 10 5 2 controls Concentration 2308 (2896} 121 (183) 438 {505) 186 {196) 629 (614j S9-Mix No



### ~ 9colonees 

### + 3043 207 555 222 656

per plate 3336 529.6 220 53.8 522 60.3 179 23'1 556 51.7 PAG E 20



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~ a~ RPROJECT NUMBER: PAGE 25 

### REVERSE MUTATION ASSAY "AMES TEST" USINu sacmunrrLLA TYPHIMURIUM AND ESCHERICHIA COLI 

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

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

## unknown)

Lot No. or tne new Purlty of the new chemical substance 21% chemical substance tested tested 

## Name of impurities Ash (predominantly sodium chloride) around 1%, residuaf monomer max 0.2%q and concentration CAS No. Vapour pressure 

## Molecular weight Typically 40,000 Partition coefficient Melting point (°C) Appearance at ordina ry temperature ~ 

### ber coloured iiquid 0 

### Boiling point (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 Waterjt Miscibleat

. DMSOj - Immiscible - in solvent 50 m /ml Acetonet Immiscible - DMFt Immiscible - THFt Immiscible -

j Solubility data provided by the Sponsor tfi Solubility data generated by the testing facility 

## No visible change in test material formulations throughout each experiment, therefore, assumed stable

### PROJECT NUMBER:

_ 

### PAGE 26

--- 

### . . . _ _ _ REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURlUM AND J=SCHERlCH1A COL( 

## Appendix 1 (continued)
2. TESTER STRAINS

Tester strain 

# I Obtained from Date obtained TA100 UCB* 04 August 1995 TA1535 UCB* 04 August 1995 WP2uvrA - BIBRA*" 17 August 1987 TA98 UCB* 04 August 1995 TA1537 UCB* 04 August 1995



## 3. 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 2. Purchase (Supplier) Prepared on 23 November 2008 (range-finding test) 18 May 2008 main test Lot No. (in case of purchase)
- 

### Storage temperature -196 0 C 

## (2) Preparation of S9 Animal used Inducing substance Species strain Rat, Wistar Name Phenobarbitonelp-naphthoflavone Sex Male Administration method Oral Age (in weeks) Weight 6- 8 weeks

— 

### 200g Administration period and amount (mg/kg bod ei htlda 3 days 80l100 mg per kg perday 

## (3) Composition of S9-MPx Constituents Amount in 1 ml S9-Mix Constituents Amount in 1 ml S9-Mix S9 0.1 ml NADP 4.0 Nmol MgC12 8.0 pmol Na-Phosphate buffer 100.0 pmol KCI 33.0 pmol Glucose-6-phosphate 5.0 pmol Glucose-6-phosphate deh dro enase

UCB: University of California at Berkeley BIBRA: British Industrial Biological Research Association

### PROJECT NUMBER:. PAGE 27 :EVERSE MUTATION ASSAY "AMES TEST" USING 

### SALMONELLA TYPHIMURIUM AND 

### ESCHERICHIA COLI Appendix 1 (continued)


## 4. PREPARATION OF THE TEST MATERIAL IN SOLUTION (encircle the applicable response)
Name Supplier Lot No. Grade Purlty (%) Solvent used Sterile distilled AGUETTANT 3003930 12 water LTD Reason for selection of Fully miscible at 50 mg/ml solvent Appearance of the test Suspension Others Solution ( ) 

### material re aration 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 10 minutes room temperature (range-finding test) preparation unfil use 35 minutes room temperature (main test) Conversion of purity Yes No


## 5. CONDITIONS OF PRE-CULTURE ETC. (1) Conditions
Name Manufacturer Lot No. Nutrient Broth Oxoid NB No.2 Oxoid Ltd. 629250 02/13 Period of pre-culture 10 hr Culture Plask (form, size) Costar 75 cm 2 tissue culture flasks Amount of culture medium 5 ml Amount of strain 20p1 inoculated
(2) Cell 

## viability at the end of pre-culture Base-pair substitution type Frameshrft 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 Conversion from O.D. value (encircle the applicable number) 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 test

### PROJECT NUMBER: PAGE 28 

### : REVERSE MUTATION ASSAY "AMES TEST" UsiNe 

### BALMONELLA TYPHIMURIUM AND ESCHERICHIA COL/ 

## 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 07/13 urchase


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

## (1) Test Method and the reason why it was selected

1. Pre-incubation method 

# O2 Plate incorporation method 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 sofution ml 0.1 ml Na-phosphate buffer ml 0.5 ml Composition S9-mix (in case of inetabolic activation

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

### ml Pre-incubation T emperature °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 Colony counter Conduction of correction 1. No OYes (Correction method: Area and miscount correction)

### PROJECT NUMBER: PAGE 29 Hydrotriticum PVP: REVERSE MUTATfON ASSAY "AMES TEST" US1NG 

### SALMONELLA TYPHIMURI UM AN D 

### ESCHERICHIA COLI 

## Appendix 1 (continued)
9. TEST RESULTS (1) The results should be reported on the attached forrrt (2) Judgement of the results Judgement (encircle one) Positive cg ative Reason for judgement: No toxicologically significant increases in the frequency of revertant cofonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. A small, statistically significant increase in TA100 revertant colony frequency was observed in the presence of S9 at 500 μg/plate in the main test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the revertant counts at 500 pglplate were within the in-house historical control range for the tester strain and the fold increase was only 1.13 times the concurrent vehicle control. (when judged as positive, attach a table for relative activity) 

## (3) Referential matters Formulated concentrrations were ad]usted to allow for the stated water/impurity content (79%) of the test material. The test material was fully miscible in sterile distilled water at 50 mg/ml (the most concentrated stock solution) in solubility checks performed 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 Ieast one bacteriat strain with or without metabolic activation. Biological refevance of the results wili be considered frst, 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. 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-naphthoffavone was used to induce higher enzyme levels in the rat liver S9. The 39- 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 sterility during the preliminary toxicity test. The use of approximate half-log dilutions instead of twofold dilutions was agreed with the JMOL in January 9995. [REMARKS] "Referential matters" —(fill in the view etc of the Study Director on the test results)

### PROJECT NUMBER:

~ 

### PAGE 30 ,: REVERSE MUTATION ASSAY "AMES TEST" USING 

### SALMONELLA TYPH(MURIUM AND 

### ESCHERICHIA COLI 

## Appendix 1 (continued) 

# 1®. OTHERS

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

Shardlow, Derbyshire, DE72 2GD, UK Fax No: 00441332 799018 Study Title Genetic Toxicologist Name A J Bowles BSc (Hons) Director Years of experience: 14 

### Study 272410002 number Experiment 1: From: 25 January 200 To: 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 January 2009



## 2. Test Period: To: © 1 APR 2009PROJECT NUMBERc ' 

### PAGE 31 

## : REVERSE MUTATtON ASSAY "AMES TEST" USING SALMOIYELLA TYPHtMUftIUM 

## AND ESCHEI2ICHIA COLI 

## Appendix 2 Test Material Certificate of Analysis

Certiflcate of Analysis

A qualiry menagement system reglstsretl to ihe intemalional standan] ISO ee01 was used ta manufacoore and test Nis matadaL The name Armted at tM1e entl of ih!s da.ument Is an electronlc aignaNre.

Customer details Customer RefInspection Lot 090000083934 C of A Printed. 28.112008 " er No. er. No. Quantlty, 0.000 DA Cantact. Fes No.

Batch Details Product Name: ProductCode: Cust Product Name: Cuat Product Code: ®atch No. Date of Test: wrll.20U$ Specificatlon: REVIEWED 23-JUN-2004 Date ot Manufeature: 09.04.2008 Date of Expiry: 09.04.2010 Quality Control Results Analytical Test Specification Limit Method No. Chamcterlsiic Lower Upper Vafue Unit Status

Addendum 00 Pass or Fail Pass - P G6000052 DP1711 B APPEARANCE YELLOW Pass - P

(COLOUR) G6000052 DP1711B APPEARANCE CLEAR Paes - P

(CLARITY) 66000052 DP1711B APPEARANCE VISCOUS LiQU1D Pass - P

(FORM) G6000054 DP17/3 ODOUR CHARACTERISTIC,FREE Pass - P OF OBJECTIONAL ODOUR G6000171 DP2/1 B ASH 0.0 2.0 1.3 % P G6000173 DPi415 COLOUR 0.0 7.0 4.2 Gatdner P G6003174 DP8/5 PH 4.5 5.5 5.5 P G6000177 DP1/3B TOTAL SOLIDS 20.0 24.0 21.0 % P G6010176 DC2/2 NITROGEN 22 3.4 2.6 % P G6020001 DM712S TOTAL COUNT 100 CFU/G MAX Pass - P

C930°C G6020002 DM211S YEASTS 8 100 CFU/G MAX Pass - P

MOULDS ® 25°C

. G6020011 DM16I1S GRAM ABSENT Pass - P

f'age 1 af 2

### PROJECT NUMBER, PAGE 32 :EVERSE MUTATION ASSAY "ANIES TEST" USING SALMONELLA TYPHIMURIUM 

### AND ESCHERICHIA COLI 

## Appendix 2 Test Material Certificate of Ana[ysis (continued)

Certlficate of Analysis

_
A quelUy management syslem+egisteretl [o 1he Intemallonal stenderct ISO 9001 was usetl to manufaoture and tast thLa matedal. ihe namo printetl at the entl of tNs tlocumenl is an elactronic signature.

Customer detatls Customer flef. Inspection Lot 090000083934 C of A Printed. 28.11.2008

3rder No.

, Jal. No. C]uanflty. 0.000 OA ConYacL Fax No.

NEGATIVE BACILLI 1G G6020012 i71,41112S STAPH.AUREUS ABSEN"r Pass
- P

1G It is recommend e d that this product be used w3thin 6 months of delivery
Batcb Status: Pass
Confifined by

Stephen Hughes

Page2of 2

### PROJECT NUMBER: PAGE 33 

### : REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHfMUR1UM AND ESCHERICH(A COLI 

## Appendix 3 History Profile of Vehicle and Positive Control Values
cnIwRINFn VFNICI F ANn UNTREATED CONTROL VALUES 2007 

# .._- Strain S9-Mix 

# -------------

~ TA100 Sy +59 TA1535

S9 +S9 WP2uvrA S9 +S9 TA102

-39 +S9 TA98

-S9 +59 TA1537

-S9 +S9 WP2uvrA

pKM101 -S9 +S9 WP2pKM101

-S9 +S9
Mean 94 87 21 13 26 28 273 309 21 26 11 13 132 162 100 128 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 27 278 263 136 165 Values 807 629 767 601 541 384 159 107 1281 609 1245 569 1197 128 127 15
Pn3ITIVF CONTROL VALUES 2007

----- Strain 59-Mix --------_

TA100 -59 +S9 . TA1535

-59 +S9 WP2uvrA -

-59 +S9 TA102

-S9 +S9 TA98

-S9 +S9 TA1537

-S9 +S9 WP2°vrA

pKM1 P1 -S9 +59 WP2pKM101

-SB +S8
Mean 558 1231 343 231 652 505 1281 96D 256 308 1697 339 960 1103 2134 997 SD 313.5 651.1 864.2 476.5 707.9 535.2 458.9 303.6 289.7 280.5 1193.6 254.4 1679.6 1010 .6 1047.0 586.4 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 196 197 197 203 159 159 J60 61 419 202 1414 198 1 64 64 9 11


### CoMRINFD VEHICLE AND UNTREATED CONTROL VALUES 2008
Strain S9-Mix TA100

-S9 +59 TA1535

-59 +S9 TA102

-S9 +S9 TA98

-S9 +S9 TA1537

-39 +S9 TA1538

-S9 +S9 WP2uvrA 7

-S9 +S9 WP2uvrApKM101 -S9 +S9 Wp2pKM101

-S9 +S9
Mean 105 101 23 15 279 ' 313 19 26 13 13 11 17 26 29 166 196 83 126 SQ 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 Va{ues 891 688 821 588 137 KLJ 1160 620 1 1139 569 163 4 1620 446 1 145 104 178 44


### POSITIVE CONTROL VALUES 2008

TA10D TA1535 TA102 TA98 TA1537 TA1538 WP2uvrA" WP2uvrA WP2pKM101 Strain pKM101 59-Mix S9 +S9 -S9 +S9 -S9 +59 -S9 +S9 -S9 +59 -59 +39 -69 +59 -S9 +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 2572 70.8 178.2 476.2 101.7 262.4 200.5 231.5 273.9 490.7 781.5 5942 328.5 Min 505 303 79 103 719 654 75 120 78 92 64 112 101 138 432 749 0.31 206
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 1335 237 1338 227 161 42 1177 174 146 25 130 25



### SD = Standard devfation Min = Minimum vatue Max = Maximum value

### PROJECT NUMBER: PAGE 34



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

2004/9/EC



## ~ p~ 

# Department

~ 

# e of Health

THF DFPr1R"i'MFNT OF HEALTH OF THF GO'VFIBNNIIiNT

OI' THE UiVTTED IZSNGD0M GOOD LABORf1TORY PRACTICF;
STATP;A{KNT OF CON'1PL7A.tiCYi Sn ACCORDANCE 19ITH DIAI?.CT[YG'-000-F/9It')C

TGST FilC/llT

- Y TGSTTFPL

Iine9an Lnhoratorios Ltd. .lnalyticnllClintcal Cheniistry SlLardloiv Bnsinessl'artc Gnvirunmental r'ate London @ond Frn•ironmentalToxicology 

### Shardlow NYutugenicity uern} - Phys/Chem DE72 2GD TOZfcolaay

D6TF.0I+iNSPF.CrtON 19l11,4ugu5t 2008 A. general ins'pectian Por coinplienee %vitli the Prinriples oI Good La6oratory Pract'ice wac carried out nt ihe abovc tcst facility as gartof thc UK GLP Complinncc Programmc. qt the tinie af inspeotion no deviations +vere found oCsufPicient maenitude to atTsct the calidity oY non-elinical atudies penrormed at thetie faciGtie.s.

Dr.Antlrca , ;l. Grae Head, OR GLP Nonitoc6rgAut3mritei ~ `♦ 1 i ~ \ i • C. I ~



## REVERSE MUTATION ASSAY `°AMES TEST" USING SALMONELLA TYPH/MUR/UM 

## AND ESGHER/GHIA GOLI



## PROJECT NUMBER:

I verify that this is an exact copy of the original report which is located in the Archives of Harlan Laboratories Ltd, Shardlow, UK.

ABowles BSc (Hons)

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


# DATE

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