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 799018 
PROJECT 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 34 
PROJECT 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 WP2uvrAThe 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. Each 
PROdECT 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 TA98 

In 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 WP2uvrABP 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 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 -1961C. 

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 9bacteria 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 

F24 

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 test 
PROJECT 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) 

.1# 6

(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 15 1.5 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 13 2.0 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 S9-Mix (1224) 

(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 8 1.2 31 5.6 30 29 1.5 10 8 4.2 104 38 

(99) (12) 31 {31) 31 (31) 8 (9) 

+ 500 04 11 4. 0.6 0"6 32 1.0 4 8 1.7 97 12 31 

(104) (9) (32} 29 (29) 16 (12) + 1500 93 10_5 10 1.2 31 2.1 2.5 4.0 104 10 30 32 13 

(1Q4) 9 (9) 35 ) 3 (33 ~ 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 

(74) ~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 9. 20 6.4 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 S9-Mix (544) 

(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# 12 i.2 31 2.3 29 5.6 2.5 101 6 

(105J 8 9 ( ) 3a 1) (3 ~ (26) 9 (12) + 50 101 4.6 8 1.7 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 11 0.6 26 32 3.5 42 14.0 8 2,6 100 30 9 

(95) 11 

(12) (37) (27) + 5000 93 (9) 2.0 9 15 3.1 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 S9-Mix (614j + 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 

BP Benzo(a)pyrene 

2AA 2-Aminoanthracene 

" p50.05 

# Standard deviation 
N 

W 

~ 

Q 

a 

0 0 0 0 0 0 0 0 

~t N o cp co It N 

O 

0 

0 

LO 

O 

0 

uO 

T 

O 

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Q R ~ w 

m 

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p 

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7 

Y 

~ 

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N .`.. 

N 

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7 

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= 

~ 

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N 

N 

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Ol 

s 

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m 

rn 

s 

N 

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ni 

~ 

~ 

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d 

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~ 

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c 

0 

w 

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tC Y 

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G 

~ W_ ~ 

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N 

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~ C 

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d 

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C 

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~ 

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7 

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❑ 

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~ 

P. 

O ICJ I 

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0 

~ 

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~ _Q ~ ~ 

0 0 0 0 0 0 0 

CV O CO CO d' N 

cv a}ejd/sjue}lanab 

d L ~ ~ 

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~ N 

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(D 

Q a 

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m 

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tll 

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rn 

O O O O O O o O O O o 

O 6) 00 I~ CO LO CY M N T 

T 

a}eld/s}uepana}j 

a. 
O O o O O O O O 

V ~ O 00 Cf? V N 

~ 

v a}ejd/s;ue}Janab 

~ 

N 

W 

~ 

Q a 

~ s 

0 0 LO 

0 

0 

~ 

~ 

m ~ 

Q 

~ 

~ m 

rn ~ 

=L ~ 

z 

fti 

o -Wi 

LO O 

:i 

P, 

~ 

~ 

~ 

~ 

a~ 

R 
PROJECT 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) 

Purlty of the new Lot No. or tne 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 

ordinary 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 -1960C 

(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 

material Solution Suspension Others ( ) 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 cm2tissue 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 2009 
PROJECT 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 recommended 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 

WP2uvrAS9 +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 

WP2uvrA7 

-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 Nonitoc6rgAut3mrite 
i ~ ` ♦ 1i ~ \ 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 .....................................................................................