PAGE 1OF 23 PAGES

REVERSE MUTATIONASSAY"AMESTEST"

USING SALMONELLA TYPHIMURIUMAND ESCHERICHIA COLI

STUDYSPONSOR:

41204331.doc/p PROJECT NUMBER:41204331

AUTHOR: P W Thompson

TEST FACILITY: Harlan Laboratories Ltd Shardlow Business Park Shardlow Derbyshire DE72 2GD UK Telephone: +44 (0) 1332792896 Facsimile: +44 (0) 1332799018PROJECT NUMBER:41204331 PAGE2


### QUALITYASSURANCE REPORT This studytype is classed as short-term. The General Study Plan forthis studytype 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 of this study type at least once every three months.
In addition, general facilities are inspected at least once a year and the results are reported to management.
This report has been audited by the Quality Assurance Unit, and is considered to be an accurate accountofthe data generated and ofthe proceduresfollowed.
In each case, 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 appropriateto thetype and schedule ofthis studywere as follows: 

### § 28 August2012 15 October2012 10 October2012 22 October2012 15 October2012 

### § 21 November2012 

### § Date ofQASignature Study Plan ComplianceAudit Test Item Preparation Test System Preparation Exposure Assessmentof Response Draft ReportAudit Final ReportAudit 

### § Evaluation specificto this study



## . 04DEC 2012 DATE

................................................. Forthe QualityAssurance Unit*

*AuthorisedQASignatures: SeniorAudit Staff: J G Riley BSc (Hons) MRQA, J MCrowther MIScT MRQA, GWren ONC MRQA, S Bevan BSc (Hons) MRQA, L Blaney MRQAPROJECT NUMBER: 41204331 PAGE3

GLP COMPLIANCE STATEMENT With the exception noted below the work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by Sl 2004/0994)). 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 ofDirectives2004/9/EC and 2004/10/EC.
No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within four hours of it being appliedto the test system. It is assumed thatthe formulation was stableforthis duration. This exception is considered notto affectthe purpose orintegrity ofthe study.
This reportfully and accurately reflects the procedures used and data generated.

P W Thompson HNC Study Director 

## 03DEC 

## 2012 DATE:

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

This report may be presented in final form as adigital (pdf) document. Such documents are prepared by scanningthe paper original,

and are considered ofequivalent integrityand authenticityto versions produced by optical photocopy. However, in all cases the hand-signed paperoriginal, held in secure archives, is the definitivedocument.PROJECT NUMBER: 41204331

CONTENTS QUALITYASSURANCE REPORT GLP COMPLIANCE STATEMENT CONTENTS SUMMARY 1. INTRODUCTION 

### 2. 3.

4. 5.

6. 7. TEST ITEM METHODS 3.1 TesterStrains 3.2 Preparation ofTestand Reference Items 3.3 Microsomal Enzyme Fraction 3.4 S9-Mixand Agar 3.5 Test Procedure 3.6 Acceptance Criteria 3.7 Evaluation Criteria ARCHIVES RESULTS 5.1 PreliminaryToxicityTest 5.2 MutationTest CONCLUSION REFERENCES Spontaneous Mutation Rates (Concurrent Negative Controls) Test Results: Experiment 1-WithoutMetabolicActivation Test Results: Experiment 1-With MetabolicActivation Test Results: Experiment2-WithoutMetabolicActivation Test Results: Experiment2-With MetabolicActivation History Profile ofVehicle and Positive ControlValues PAGE4

2 

### 3 4 

### 5 6 

### 7 7 7 

### 8 9 9 10 12 12 

### 13 14 14 14 

### 15 16 17 18 19 20 21 22 Table 1 Table2 Table 3 Table 4 Table 5 Appendix 1 Appendix2 StatementofGLP Compliance in Accordancewith Directive 2004/9/EC 23PROJECT NUMBER:41204331

REVERSE MUTATIONASSAY"AMESTEST"

USING 

### SALMONELLA TYPHIMURIUMAND 

### ESCHERICHIA COLI
SUMMARY PAGES



### Introduction. The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method 813/14 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, TA100 and 

### Escherichia colistrain 

### WP2uvrA were treated with the test item, , using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition ofa rat liver homogenate metabolising system (10°/o liver S9 in standard co-factors). The dose range forthe first experimentwas determined in a preliminary toxicity assay and was 50 to 5000 J..Jg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures ofthe bacterial strains and fresh test item formulations. 

### Results. The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All ofthe positive controls used in the test induced marked increases in the frequency of revertant colonies, both with orwithout metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item caused no visible reduction in the growth ofthe bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of5000 J..Jg/plate. No test item precipitatewas observed on the plates at any ofthe dosestested in eitherthe presence orabsence ofS9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation orexposure method. 

### Conclusion. The test item, , was considered to be non-mutagenic under the conditions ofthis test.PROJECT NUMBER: 41204331



### REVERSE MUTATIONASSAY"AMESTEST"

USING SALMONELLA TYPHIMURIUM

### AND ESCHERICHIA COLI


### 1. INTRODUCTION PAGE6



### The purpose ofthe study was to evaluate the test item, , for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome offive strains ofbacteria.
The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method 813/14 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 DepartmentofHealth, is included. The study was based on the in vitro 

### technique described by Ames eta/

### (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium 

### to various concentrations ofthe test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers ofthese organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium 

### revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) 

### which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence (Green and Muriel (1976)) is used to complementthe Salmonella 

### strains. 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 item and the bacteria are also incubated in the presence of a liver microsomal preparation (S9-mix)PROJECT NUMBER:41204331 PAGE 7 prepared from rats pre-treated with a mixture known to induce an elevated level ofthese enzymes. The experimental phase of this study was performed between 22 August 2012 and 18 October2012.


### 2. TEST ITEM Sponsor's identification Description Purity Batch number Date received Expiry date Storage conditions The integrity ofsupplied data relating to the identity, purity and stability ofthe test item is the responsibility ofthe Sponsor.


### 3. METHODS 3.1 TesterStrains The five strains ofbacteria used, and theirmutations, are as follows: Salmonella typhimurium Strains Genotype TA1537 hisC 3076; rfa-; uvra-: TA98 his D 3052; rfa-; uvrB-;R-factor TA1535 hisG46; rfa-; uvra-: TA100 hisG46; rfa-; uvrB-;R-factor Escherichia coli Strain Genotype WP2uvrA trp-; uvrA-: Type ofmutations indicated frame shift mutations base-pairsubstitutions
Type ofmutations indicated base-pairsubstitution All ofthe Salmonella strains are histidine dependent by virtue ofa mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess afaulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB-bio gene,PROJECT NUMBER:41204331 PAGES



### causes an inactivation ofthe excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortlemans and Zeiger (2000)). In addition to a mutation in the tryptophan operon, the E. coli

### tester strain contains a uvrA-

### DNA repair deficiencywhich enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA 

### repair system would normally act to remove and repair the damaged section ofthe DNA molecule (Green and Muriel (1976)).
The strains of bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 and the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately -196°C in a Statebourne liquid nitrogen freezer, model SXR 34.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot numbers 1014570 03/16 and 1078364 08/16) and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrientagar plates.
3.2 Preparation ofTestand Reference Items 

### The test item was fully miscible in sterile distilled water at 50 mg/ml in solubility checks performed in-house. Sterile distilledwaterwas therefore selected as the vehicle.
The test item 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 allow for the stated active ingredient (60°/o w/v) ofthe test item. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability ofthe test item formulations is not a requirement ofthe test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.PROJECT NUMBER: 41204331 PAGE9
A solventtreatmentgroupwas used as the vehicle control and the positive controls used in the series ofplateswithoutS9-mixwere as follows: N-ethyi-N'-nitro-N-nitrosoguanidine (ENNG): N-ethyi-N'-nitro-N-nitrosoguanidine (ENNG): N-ethyi-N'-nitro-N-nitrosoguanidine (ENNG): 9-Aminoacridine (9AA): 4-Nitroquinoline-1-oxide (4NQO): 2 J.,Jg/plate forWP2uvrA 3 J.,Jg/plate forT A 100 5 J.,Jg/plate forTA1535 80 J.,Jg/plate forT A1537 0.2 J.,Jg/plate forTA98
In addition, 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP), which are non-mutagenic in the absence of metabolising enzymes, were used in the series of plateswith S9-mixatthe following concentrations:
2-Aminoanthracene (2AA): 2-Aminoanthracene (2AA): 2-Aminoanthracene (2AA): Benzo(a)pyrene (BP):
3.3 Microsomal Enzyme Fraction 1J.,Jg/plate forT A 100 2 J.,Jg/plate forTA1535and TA1537 10 J.,Jg/plate forWP2uvrA 5 J.,Jg/plate forTA98

The S9 Microsomal fraction was prepared in-house (01 July 2012 (preliminary toxicity test and Experiment 1) and 16 September 2012 (Experiment 2)) from rats induced with**Phenobarbitone/~-Naphthoflavone** at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day4. The S9 homogenatewas produced by homogenising the liver in a 0.15M KCI solution (1g liver to 3 ml KCI) followed by centrifugation at 9000

### g. The protein content of the resultant supernatant was adjusted to 20 mg/ml. Aliquots of the supernatantwerefrozen and stored atapproximately-196°C. Priorto use, each batch of S9 wastested for its capabilityto activate known mutagens in theAmestest.
3.4 59-MixandAgar The S9-mix was prepared immediately before use using sterilised co-factors and maintained on iceforthe duration ofthetest.PROJECT NUMBER: 41204331 

### S9 1.65 M KCI/0.4 M MgCb 0.1 MGlucose-6-phosphate 0.1 M NADP 0.2 MSodium phosphate buffer (pH 7.4) Sterile distilled water 5.0 ml 1.0 ml 2.5 ml 2.0 ml 25.0 ml 14.5 ml PAGE10



### A 0.5 ml aliquot of S9-mix and 2 ml of molten, trace histidine or tryptophan supplemented, top agarwere overlaid onto a sterile Vogel-Bonner Minimal agar plate in orderto assess the sterility ofthe S9-mix. This procedure was repeated, in triplicate, on the dayofeach experiment. Top agar was prepared using 0.6°/o Bacto agar (lot number 1297425 09/16) and 0.5°/o 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 SGL Ltd (lot numbers 32699 09/12, 32841 10/12 and 32927 11/12).
3.5 Test Procedure 3.5.1 PreliminaryToxicityTest In orderto select appropriate dose levels for use in the main test, a preliminary testwas carried out to determine the toxicity of the test item. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 j..Jg/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100 orWP2uvrA), 2 ml ofmolten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of test item formulation and 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates ofVogel-Bonner Minimal agar (30 ml/plate). Ten concentrations ofthe test item formulation and avehicle control (sterile distilled water) were tested. In addition, 0.1 ml ofthe maximum concentration of the test item 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 ofthe test item. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a colony counter and examined for effects on the growth ofthe bacterial background lawn.PROJECT NUMBER: 41204331 PAGE 11


### 3.5.2 Mutation Test
- Experiment1 Five concentrations ofthe test item (50, 150, 500, 1500, 5000 jJg/plate) were assayed in triplicate againsteach testerstrain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one ofthe bacterial cultures were dispensed into sets of test tubes followed by 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml ofthe vehicle, test item formulation 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 ofVogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration oftest item both with and without S9-mix.
All ofthe plateswere incubated at 37°C for approximately48 hours and the frequency of revertantcolonies assessed using acolony counter.


### 3.5.3 MutationTest

.. Experiment2 The second experiment was performed using fresh bacterial cultures, test item and control solutions. The test item dose range was the same as Experiment 1 (50 to 5000 jJg/plate).
As it is good scientific practice to alter one condition in the replicate assay, the exposure condition was changed from plate incorporation to pre-incubation. The test item formulations and vehicle controlweretherefore dosed as follows:
Measured aliquots (0.1 ml) of one ofthe bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.1 ml ofthe vehicle or test item formulation and incubated for20 minutes at 37°Cwith shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on 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 item both with and without S9-mix. The positive and untreated controls were dosed using the standard plate incorporation method described in Section 3.5.2.PROJECT NUMBER: 41204331 PAGE12


### All ofthe plateswere incubated at 37°C for approximately48 hours and the frequency of revertant colonies assessed using acolony counter.
3.6 AcceptanceCriteria 

### The reverse mutation assay may be considered valid ifthe following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et 

### a/ (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All testerstrain cultures should exhibit acharacteristic numberofspontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the General Study Plan, Section 4 (negative controls). Combined historical negative and solvent control ranges for2010 and 2011 are presented in Appendix 1.
All testerstrain cultures should be in the range of0.9 to 9 x 109 

### bacteria permi. Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity ofthe tester strains to mutagen exposure and the integrity ofthe S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. The historical ranges of the positive controls for 2010 and 2011 are presented in Appendix 1.
There should be a minimum offour non-toxictest item dose levels.
There should be no evidence ofexcessive contamination.
3.7 Evaluation Criteria 

### There are several criteria for determining a positive result. Any, one, or all of the following can be used to determinethe overall result ofthe study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).PROJECT NUMBER: 41204331 PAGE13


### 2. A reproducible increase atone or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis ofdata as determined by UKEMS (Mahon eta/

### (1989)).
5. Fold increase greaterthan two times the concurrent solvent control for any tester strain (especially ifaccompanied by an out-of-historical range response).
A test item will be considered non-mutagenic (negative) in the test system ifthe above criteria are not met. Although mostexperiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results ofthis type will be reported as equivocal.
4. ARCHIVES Unless instructed otherwise by the Sponsor, all original data and the final report will be retained in the Harlan Laboratories Ltd., Shardlow, UKarchivesforfive years, afterwhich instructionswill be soughtas to further retention ordisposal.PROJECT NUMBER:41204331 PAGE14



### 5. RESULTS 5.1 PreliminaryToxicityTest The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experimentwere both shown to be sterile.
The numbers ofrevertant coloniesforthe toxicity assaywere:

With(+) or Dose (!Jg/plate) without(-) Strain

S9-mix 0 0.15 0.5 1.5 5 15 50 150 500 1500 5000

- TA100 96 99 89 90 73 107 87 91 92 96 117 + TA100 118 137 112 111 123 101 95 104 103 100 101

- WP2uvrA 23 31 21 24 30 34 30 20 32 26 25 + WP2uvrA 30 33 30 34 30 22 33 36 26 25 25 

### 5.2 MutationTest Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are notgiven in the report.
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 dayas the Mutation Test.
The individual plate counts, the mean number of revertant colonies and the standard deviations, forthe test item, positive controls and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3for Experiment 1and Table 4 and Table 5for Experiment2.
A history profile of vehicle/untreated and positive control values (reference items) for 2010 and 2011 are presented in Appendix 1. The test item caused no visible reduction in the growth ofthe bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dosePROJECT NUMBER: 41204331 PAGE15 

### level of5000 J,.Jg/plate. No test item precipitatewas observed on the plates at any ofthe dosestested in eitherthe presence orabsence ofS9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation orexposure method.
All 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 sensitivityofthe bacterial strains.
6. CONCLUSION 

### The test item, , was considered to be non-mutagenic underthe conditions ofthis test.PROJECT NUMBER:41204331 PAGE16

7. REFERENCES Ames B N, McCann J, and Yamasaki E (1975), Methods for detecting carcinogens and mutagens with the Sa/monel/a/mammalian microsome mutagenicity test, Mutation Research, 31, 347-364.
Maron D M and Ames B N (1983), Revised Methods for the Salmonella mutagenicity test, Mutation Research, 113, 173-215.
Mortelmans K and Zeiger E (2000), The Ames Sa/monel/a/microsome mutagenicity assay, Mutation Research, 455, 29-60. Green, M H 

### L. and Muriel W 

### J (1976), Mutagen Testing Using TRP+ Reversion in Escherichia coli, Mutation Research, 38, 3- 32.
De Serres F 

### J and Shelby M D (1979), Recommendations on data production and analysis using the Sa/monel/a/microsome mutagenicity assay, Environmental Mutagenesis, 1, 87-92. Mahon G A T eta/ (1989) Analysis ofdata from microbial colony assays. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicitytesting, (Kirkland D

### J Ed.), Cambridge UniversityPress Report, 26-65.Table 1

EXPERIMENT 1

TA100 104

92 (96) 93

EXPERIMENT 2

TA100 105

90 (98) 99 PROJECT NUMBER: 41204331 REVERSE MUTATIONASSAY "AMESTEST" USING SALMONELLA TYPHIMURIUMAND ESCHERICHIA COLI Spontaneous Mutation Rates (ConcurrentNegativeControls)

Numberofrevertants (mean numberofcolonies perplate) Base-pairsubstitution type Frameshifttype TA1535 WP2uvrA TA98 TA1537 19 30 18 13 PAGE17

18 (15) 30 (28) 29 (24) 10 (11)

8 25 25 10

Numberofrevertants (mean numberofcolonies per plate) Base-pairsubstitution type Frameshifttype TA1535 WP2uvrA TA98 TA1537 15 26 32 7 14 (14) 21 (28) 17 (24) 10 (8) 14 36 24 7PROJECT NUMBER: 41204331 PAGE18 REVERSE MUTATIONASSAY "AMESTEST" USING SALMONELLA TYPHIMURIUMAND ESCHERICHIA COLI 

### Table 2 TestResults: Experiment1-WithoutMetabolicActivation
Test Period From: 24 September2012 

### I Numberofrevertants (mean Dose Level Base-pairsubstitution strains Per Plate TA100 SolventControl 100 (108) 105 (Water) 120 10.4# 102 (91) 501-19 74 97 14.9 S9-Mix 109 (103) 1501-19 96 (-) 104 6.6 90 (96) 5001-19 97 102 6.0 112 (106) 1500 1-19 115 90 13.7 99 (94) 5000 1-19 91 93 4.2 Positive Name ENNG controls Dose Level 31J9 S9-Mix No. of 619 (628)

(-) Revertants 649 18.2 616

ENNG N-ethyi-N'-nitro-N-nitrosoguanidine 4NQO 4-Nitroquinoline-1-oxide 9AA 9-Aminoacridine # Standard deviation TA1535 WP2uvrA 15 (15) 35 (26) 15 25 16 0.6 19 8.1 12 (16) 32 (26) 19 21 16 3.5 25 5.6 10 (14) 36 (29) 19 30 13 4.6 20 8.1 16 (14) 33 (29) 13 34 13 1.7 20 7.8 19 (17) 33 (31) 18 25 15 2.1 34 4.9 16 (15) 32 (30) 14 30 14 1.2 29 1.5 ENNG ENNG 51J9 21-19 904 (814) 1145 (1077) 757 79.1 1033 59.9 780 1052 To: 27 September2012 +/-so Frameshiftstrains TA98 TA1537 29 (22) 19 (11) 20 6 18 5.9 7 7.2 28 (25) 8 (11) 27 9 21 3.8 17 4.9 21 (21) 9 (8) 25 10 17 4.0 5 2.6 21 (19) 9 (13) 20 17 16 2.6 13 4.0 19 (17) 15 (11) 16 9 16 1.7 8 3.8 19 (22) 12 (12) 19 19 27 4.6 6 6.5 4NQO 9AA 0.21J9 801-19 198 (216) 1112 (928) 247 27.0 1132 335.6 203 541

### Table 3 PROJECT NUMBER:41204331 REVERSE MUTATIONASSAY "AMESTEST" USING SALMONELLA TYPHIMURIUMAND ESCHERICHIA COLI 

### Test Results: Experiment1-With MetabolicActivation PAGE19

Test Period From: 24 September2012 I To: 27 September2012 Dose Level Numberofrevertants (mean +/-so Base-pairsubstitution strains Frameshiftstrains Per Plate TA100 TA1535 WP2uvrA TA98 TA1537 SolventControl 96 (106) 18 (16) 37 (37) 34 (31) 14 (11) 106 18 39 40 13 (Water) 116 10.0# 11 4.0 36 1.5 20 10.3 6 4.4 121 (111) 17 (16) 35 (34) 21 (24) 7 (7) 501-19 111 12 27 27 6 100 10.5 18 3.2 40 6.6 25 3.1 9 1.5 S9-Mix 115 (109) 9 (14) 36 (37) 34 (31) 13 (11)

(+) 1501-19 103 6.0 17 4.6 36 1.2 25 4.9 15 4.7 108 17 38 33 6 108 (105) 14 (13) 39 (37) 28 (24) 13 (12) 5001-19 122 13 44 21 12 86 18.1 12 1.0 28 8.2 24 3.5 12 0.6 121 (108) 19 (17) 29 (32) 28 (28) 8 (12) 1500 1-19 100 17 35 27 12 104 11.2 16 1.5 33 3.1 29 1.0 16 4.0 94 (96) 13 (13) 32 (30) 37 (30) 14 (13) 5000 1-19 92 12 26 32 9 101 4.7 15 1.5 32 3.5 20 8.7 16 3.6 Positive Name 2AA 2AA 2AA BP 2AA controls Dose Level 11-19 21-19 10 1-19 51-19 21-19 S9-Mix No. of 1253 (1103) 218 (333) 316 (308) 297 (292) 240 (234)

(+) Revertants 703 350.0 527 168.7 300 8.0 279 11.0 212 19.3 1353 255 308 299 249

2AA 2-Aminoanthracene BP Benzo(a)pyrene # Standard deviationPROJECT NUMBER: 41204331 REVERSE MUTATIONASSAY "AMESTEST" USING SALMONELLA TYPHIMURIUMAND ESCHERICHIA COLI PAGE20

Table4 TestResults: Experiment2-WithoutMetabolicActivation
Test Period From: 15October2012 

### I To: 18October2012 Dose Level Numberofrevertants (mean)+/- SO Per Plate Base-pairsubstitution strains TA100 SolventControl 98 (95) 84 (Water) 104 10.3# 86 (95) 50 j..Jg 105 94 9.5 S9-Mix 73 (80) 150 l-l9 80 (-) 88 7.5 103 (93) 500 l-l9 77 98 13.8 97 (88) 1500 l-l9 85 81 8.3 81 (79) 5000 l-l9 67 88 10.7 Positive Name ENNG controls Dose Level 3 l-l9 S9-Mix No. of 819 (764)

(-) Revertants 796 76.2 677

ENNG N-ethyi-N'-nitro-N-nitrosoguanidine 4NQO 4-Nitroquinoline-1-oxide 9AA 9-Aminoacridine # Standard deviation TA1535 WP2uvrA 16 (15) 37 (35) 16 34 14 1.2 33 2.1 11 (15) 21 (28) 22 24 11 6.4 39 9.6 14 (15) 29 (31) 14 31 17 1.7 34 2.5 14 (13) 34 (31) 13 29 11 1.5 30 2.6 11 (14) 33 (36) 16 38 16 2.9 38 2.9 10 (10) 27 (29) 10 29 10 0.0 30 1.5 ENNG ENNG 5 l-l9 2 l-l9 555 (615) 1112 (1019) 589 77.0 964 81.0 702 981 Frameshiftstrains TA98 TA1537 34 (24) 16 (10) 16 6 22 9.2 9 5.1 26 (19) 7 (9) 12 10 18 7.0 9 1.5 19 (22) 6 (10) 27 11 20 4.4 14 4.0 20 (22) 9 (10) 20 8 27 4.0 12 2.1 29 (27) 8 (9) 35 14 17 9.2 4 5.0 16 (18) 9 (7) 17 7 21 2.6 5 2.0

4NQO 9AA 0.2 l-l9 80 l-l9 196 (195) 1307 (1111) 198 4.2 741 320.9 190 1286Table 5 PROJECT NUMBER:41204331 REVERSE MUTATIONASSAY "AMESTEST" USING SALMONELLA TYPHIMURIUMAND ESCHERICHIA COLI TestResults: Experiment2-With MetabolicActivation PAGE 21

Test Period From: 15 October2012 

### I To: 18October2012 Dose Level Numberofrevertants (mean +/-so Base-pairsubstitution strains Frameshiftstrains Per Plate TA100 TA1535 WP2uvrA TA98 TA1537 SolventControl 121 (111) 13 (11) 25 (28) 26 (27) 9 (10) 105 9 29 26 12 (Water) 106 9.0# 10 2.1 30 2.6 29 1.7 10 1.5 110 (108) 9 (11) 22 (28) 21 (24) 13 (12) 501-19 120 12 30 27 12 94 13.1 12 1.7 31 4.9 24 3.0 12 0.6 S9-Mix 113 (95) 12 (12) 27 (25) 30 (27) 14 (12) 1501-19 86 14 18 26 10 (+) 87 15.3 11 1.5 30 6.2 25 2.6 12 2.0 99 (100) 11 (11) 22 (25) 31 (27) 13 (10) 5001-19 93 11 34 29 4 109 8.1 12 0.6 20 7.6 22 4.7 12 4.9 88 (98) 9 (10) 32 (26) 22 (25) 7 (6) 1500 1-19 101 9 26 17 8 104 8.5 12 1.7 19 6.5 35 9.3 3 2.6 107 (100) 16 (13) 31 (28) 25 (30) 15 (9) 5000 1-19 88 10 27 35 7 106 10.7 12 3.1 27 2.3 31 5.0 6 4.9 Positive Name 2AA 2AA 2AA BP 2AA controls Dose Level 1 1-19 21-19 10 1-19 51-19 21-19 S9-Mix No. of 2237 (2142) 263 (260) 455 (400) 254 (267) 356 (278)

(+) Revertants 2415 330.9 260 3.5 362 48.8 270 11.8 244 67.7 1774 256 383 277 234

2M 2-Aminoanthracene BP Benzo(a)pyrene # Standard deviationPROJECT NUMBER: 41204331 REVERSE MUTATIONASSAY "AMESTEST" USING SALMONELLA TYPHIMURIUMAND ESCHERICHIA COLI Appendix 1 History ProfileofVehicleand PositiveControlValues COMBINEDVEHICLEAND UNTREATED CONTROLVALUES 2010 Strain TA100 TA1535 TA102 WP2uvrA TA98 TA1537 S9-Mix -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 Mean 104 98 19 15 266 318 29 33 21 25 12 13 SD 14.7 13.8 4.7 3.3 35.4 31.8 9.1 9.2 3.9 4.3 2.4 2.4 Min 71 62 8 7 206 234 14 16 11 14 6 7 Max 171 145 34 28 359 346 54 55 42 42 26 23 Values 604 441 504 345 31 13 512 369 590 366 566 335 POSITIVE CONTROLVALUES 2010

Strain TA100 TA1535 TA102 WP2uvrA TA98 TA1537 S9-Mix -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 Mean 459 1091 353 218 1163 1031 395 282 155 249 1285 202 SD 131.9 452.8 389.4 77.4 385.1 423.0 200.1 81.0 53.5 134.7 833.7 72.4 Min 228 302 87 103 717 575 129 130 69 84 128 83 Max 1216 3777 2003 726 1836 2392 978 661 488 1146 4743 530 Values 159 167 156 160 18 18 144 144 236 169 229 158 COMBINEDVEHICLEAND UNTREATED CONTROLVALUES2011 Strain TA100 TA1535 TA102 S9-Mix -S9 +S9 -S9 +S9 -S9 +S9 Mean 108 101 22 15 256 298 SD 3.7 5.1 0.8 1.0 5.7 3.2 Min 58 60 10 8 198 209 Max 171 168 34 35 337 385 Values 1090 716 810 425 165 66 POSITIVE CONTROLVALUES 2011

Strain TA100 TA1535 S9-Mix -S9 +S9 -S9 Mean 528 888 307 SD 223.9 398.3 254.4 Min 249 346 87 Max 2282 3072 1773 Values 191 202 192

SO = Standard deviation Min = Minimumvalue Max = Maximumvalue +S9 304 122.9 126 1150 196 TA102 -S9 +S9 986 875 284.1 209.5 622 628 2151 1780 51 51 WP2uvrA TA98 TA1537 -S9 +S9 -S9 +S9 -S9 +S9 30 35 23 25 11 12 0.9 0.8 0.7 1.5 0.5 1.4 11 10 11 10 4 5 59 55 43 43 23 25 851 495 1167 462 1124 409

WP2uvrA TA98 TA1537 -S9 +S9 -S9 +S9 -S9 +S9 643 283 194 249 946 235 213.2 83.1 115.2 166.7 640.7 72.9 198 121 69 81 103 86 1719 504 1631 2115 5224 491 177 177 255 204 254 193 WP2uvrA pKM101 -S9 +S9 141 169 47.1 44.3 75 101 235 274 40 32

WP2uvrA pKM101 -S9 +S9 811 1833 335.6 695.6 446 799 1660 3255 11 17

WP2uvrA pKM101 -S9 +S9 121 163 5.3 10.7 85 108 168 223 37 19

WP2uvrA pKM101 -S9 +S9 1034 1583 296.6 881.5 692 331 1600 2916 7 12 PAGE 22

WP2pKM101 -S9 +S9 90 117 21.9 30.5 46 48 130 166 29 20

WP2pKM101 -S9 +S9 1443 537 603.8 207.0 476 321 2323 1028 12 18

WP2pKM101 -S9 +S9 93 115 2.2 25.9 69 81 125 154 27 11

WP2pKM101 -S9 +S9 1384 628 1054.4 196.4 233 432 2798 1150 5 12PROJECT NUMBER: 41204331 PAGE23


### Appendix2 StatementofGLPCompliance inAccordancewith Directive2004/9/EC


### Department ofHealth

THE DEPARTMENT OF HEALTH OFTHE GOVERNMENT OF THE UNITED KINGDOM GOOD LABORATORY PRACTICE

STATEMENTOF COMPLIANCE IN ACCORDANCEWITH DIRECTIVE 2004/9/EC
TEST FACILITY Harlan Laboratories Ltd Shardlow Business Park London Road Shardlow Derby, DE27 2GD

DATE OF INSPECTION

10 July2012 TESTTYPE(S) Analytical/Clinical Chemistry Environmental Toxicity Environmental Fate Mutagenicity Phys/Chem. Tests Toxicology

An inspection for compliance with the Principles of Good Laboratory Practice was carried out at the above test facility as part of the UK Good laboratory Practice Compliance Monitoring Programme. This statement confirms that, on the date of issue, the UK Good laboratory Practice Monitoring Authority were satisfied that the above test facility was operating in compliancewith the OECD Principles of Good laboratory Practice. Thisstatementconstitutesa Good Laboratory Practice Instrument(as defined inthe UK Good laboratory Practice Regulations 1999).

Dr. AndrewJ. Gray Head, UKGLP Monitoring Authority