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 799018 
PROJECT 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 MRQA 
PROJECT 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 document 
PROJECT 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 34 
PROJECT 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. Each 
PROJECT 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/plateforTA98 

In 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 had 
PROJECT 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 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. 

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 21 122 123 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) (29) 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 

15D 0

(105) (23) (31) ~~ (15) 12 (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) 27 (17) 30 (28) 23 (22) i0 (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) 19 (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 148 ~'7 655 245 617 

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 10 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) 90 (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) 

- 1500 89 22 - (23) 12 ~ (10) 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 

Ewtro

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 14 2.3 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 1S 3.0 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 98 13 31 30 - 9 2A . 

105 (109) 10 (11) 38 (36) 45 ~ (32) 25 (20) + 1500 129 18.8 11 11 0.6 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 deviation 
0 

O 

(D 

U) 

O 

O 

LO 

Q 

~ 

0 

LO 

C 

0 

0 

LO 

N N 

Q 

~ 

y 

C LO 

O 

m a 

U 

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O 

O ~ 4 ~ 

.~ 

N 

7 Q O ~ 

Ly_ N 

7 ~ 

Y ~ ~ r ~ LC) 

c ~ 

c ~ 

rL ~ ~ rn c 

cu o 

a ~ T 

m r 

V 

~ 

N 

~ O 

~ 

N 

N 

~ 

N 

N 

O 

❑ 

O O o O O O o 

N O OJ CO V N 

T C 

r a}e ~d~s}ue}~ana~ 

~ 

L 

.~. 

~ 

u. 
0 0 0 0 0 0 0 

N O 00 CD V N 

r ~ 

. 

m 

L 

3 

~ 

a;ejd/s;ue}Jana ~{ 

0 

0 

0 

LO 

0 

0 

LO 

~ 

C? O LO 

aI 

m w 

fl- m 

z 

FU 

O w 

~ O 

a 

~ 

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c 

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a=~ N 

y 

~ 

U 

~ 

U_ 

O 

~ 

N 

~ 

N 

~ 

s 

~ 

a+ 

N 

d 

H 

O) 

C 

a 

c 

~ 

N 

~ 

C N 

~ 

Gi 

~ 

7 

v 

d 

N 

C 

O 

C 

N 

N 

~ 

N 

N 

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0 

12 
C 

O 

r 

N 

9 

~ 

U 

Q 

U 

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B 

~ 

d 

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3 

O 

L e-~ 

~ 

rt~+ 

N 

N 

h 

r 

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7 

U 

N 

N 

C 

O 

Q 

N 

N 

~ 

m 

N 

O 

0 

0 

O 

O 

LO 

O 

O 

~ 

r 

O 

O 

LO 

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~ 

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LO 

0 

N 

~ 

a -,. m =L 

O o 0 0 o O o 

N O OD (O V N 

r r 

~ M a}ejdfs4uejJanaH 

d 

L 

.~. {

~ 
~ 

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(~ 

Q 

a 

0 

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O 

LO 

O 

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~ ~ 

0 O LO 

N 

Q W 

m 

~ 5 z 

r 

U 

o ~ tO O 

a 

O 

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0 

.~ O 

U Q_ _ U Q: 

w 

x 

U 

C/) 

W 

O 

z 

d Sv ~ 

a 

~ 

~ 

~ 0- 

ti 

Q 

J 

J 

w 

z 

O 

~ 

J 

Q 

h 

(J 

z 

N 

~ 

IO 

W 

r 

(!7 

W 

~ 

d 

} 

Q 

N 

N 

d 

z 

O 

d 

r 

D 

2 

W 

U) 

x 

w 

~ ~ _ 

c O 

~ 

~ 

> 

~ 

U 

~ 

t~ 

O 

.II 

t'G w 

N 

~ 

t w_ 

~ 

w 

N 

N 

F 

C 

.~ 

~ Ny 

L 

7 

U 

d 

N 

C 

O Q 

d 

d 

N O 

® 

v 

~ 

~ 

~ 

a> 

° ° o °

~ 

coo° oNO 

~ r c 

aleld/s}ue}iana~j 

L"T 
PROJECT 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) 

Purity of the new Lot No. 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 

stable 
PROJECT 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 -1960C 

(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 

M9Cl2 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 Association 
PROJECT 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 2tissue 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 test 
PROJECT 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 

Reason 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: To: i P t t{ LUU 
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/ 2 
PROJECT 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 

SD 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 TA1T 5Z7A Wp2pKM101 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 ..................................................:...........