TS-CKHI01

Attachment #13

Health Effects Data on the New Chemical Substance 

Bacterial Reverse Mutation Assay (Ames Test) 
LAB Research Ltd.

Address: 8200 Veszprém, Szabadságpuszta 

Phone: +36 88 545 300 

Fax: +36 88 545 301 

€ 

FINAL REPORT

THE TESTING OF

WITH BACTERIAL

REVERSE MUTATION ASSAY

Date of Final Report: 18 July 2011

STUDY CODE: 11/076-007M 



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GENERAL INFORMATION 

STUDY TITLE : The Testing of with Bacterial Reverse 

Mutation Assay 

TEST ITEM : 

SPONSOR

TEST FACILITY : LAB Research Ltd. 

 Address: H-8200 Veszprém, 

 Szabadságpuszta, Hungary 

 Phone: +36 88 545 300 

Fax: +36 88 545 301 

BASIS OF STUDY : OECD Guidelines for Testing of Chemicals, No. 471 

EPA Health Effects Test Guidelines, OPPTS 870.5100 

 Commission Regulation (EC) No. 440/2008, B.13/14. 

STUDY PLAN : 23 May 2011 

AMENDMENT 1 TO THE STUDY PLAN : 01 June 2011 

RANGE FINDING TEST : 25-27 May 2011 

EXPERIMENTAL PERIOD I. : 01-03 June 2011 

EXPERIMENTAL PERIOD II. : 07-09 June 2011 

DRAFT REPORT : 15 July 2011 

FINAL REPORT : 18 July 2011 
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RESPONSIBLE PERSONS:

LAB MANAGEMENT : Christopher Banks, DABT 

Managing Director

 Alyson Leyshon, M.Sc. 

 Senior Director of Toxicology Operations 

QUALITY ASSURANCE : Szabolcs Gáty, M.Sc.

Head of QAU

 Brigitta Barkó, M.Sc. 

Deputy Head of QAU

 Istvánné Kiss, M.Sc. 

QA Inspector

STUDY DIRECTOR : Judit Hargitai, PhD. 

 Phone: +36 88 545 273 

 E-mail: judit.hargitai@labresearch.hu

CENTRAL DISPENSARY : Tamás Mészáros, PhD. 

(Technical Team Leader)

TECHNICAL ASSISTANTS : Tímea Kovácsné Böröcz

Emma Petőné Kohán

Ákos Klaffl
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TABLE OF CONTENTS 

Page

STATEMENT OF THE STUDY DIRECTOR 2

STATEMENT OF THE MANAGEMENT 3

QUALITY ASSURANCE STATEMENT 4

1. SUMMARY 9

2. INTRODUCTION 10

3. OBJECTIVE OF STUDY 10

4. GUIDELINES 11

5. MATERIALS AND METHODS 11

5.1. TEST ITEM 11

5.2. POSITIVE AND NEGATIVE CONTROLS 12

5.3. BACTERIAL STRAINS 14

5.4. MEDIA 16

5.5. METABOLIC ACTIVATION SYSTEM 18

5.6. DESCRIPTION OF THE TEST PROCEDURE 20

5.7. EVALUATION OF EXPERIMENTAL DATA 23

5.8. ARCHIVES 24

5.9. CHEMICALS USED IN THE EXPERIMENTS 25

5.10.DEVIATIONS TO THE STUDY PLAN 25

6. RESULTS AND DISCUSSION 26

6.1. PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY 

TEST) 26

6.2. INITIAL AND CONFIRMATORY MUTATION TESTS 26

6.3. VALIDITY OF THE TESTS 27

7. CONCLUSION 28

8. DISTRIBUTION OF THE FINAL REPORT 28

9. REFERENCES 29

APPENDICES 30 

Copy of the Certificate of Analysis 1- 1 of APPENDIX 1

Summary Tables of the Results 1- 3 of APPENDIX 2

Preliminary Range Finding Test (Informatory Toxicity Test) 1- 2 of APPENDIX 3

Initial Mutation Test (Plate Incorporation Method) 1- 5 of APPENDIX 4

Confirmatory Mutation Test (Pre-Incubation Method) 1- 5 of APPENDIX 5

Historical Control Data 1- 2 of APPENDIX 6

Copy of the GLP Certificate 1- 1 of APPENDIX 7
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LIST OF TABLES

Page

Table 1: Formulation of the Test Item for the Main Experiments 12 

Table 2: Strain-Specific Positive Control Chemicals 13 

Table 3: Genotypes of the Strains Used for Mutagenicity Testing 15 

Table 4: The Solubility of the Test Item in Acetone 21 

Table 5: Summary of Controls 22 

Table 6: Chemicals Used in the Experiments 25 

Table 7: Summary Table of the Range Finding Test 1 of APPENDIX 2

Table 8: Summary Table of the Initial Mutation Test 2 of APPENDIX 2

Table 9: Summary Table of the Confirmatory Mutation Test 3 of APPENDIX 2
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1. SUMMARY

The test item was tested for potential mutagenic activity using the Bacterial 

Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of 

Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and 

TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli 

(Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial 

supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavoneinduced rats. 

The study included a Preliminary Solubility Test, a Preliminary Range Finding Tests 

(Informatory Toxicity Tests), an Initial Mutation Test (Plate Incorporation Method) 

and a Confirmatory Mutation Test (Pre-Incubation Method). 

Based on the results of the preliminary solubility tests, the test item was formulated in 

Acetone. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were 

examined in the Range Finding Test. Based on the results of the Range Finding Test, 

the test item concentrations of 5000; 1581; 500; 158.1; 50; 15.81 and 5 μg/plate were 

examined in the main tests. 

In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed 

revertant colony numbers were above the respective biological threshold value. There 

were no consistent dose-related trends and no indication of any treatment effect. In all 

test item treated groups, the numbers of revertant colonies were below the biological 

relevance when compared with the solvent controls and were within the historical 

control range and were within the normal biological variability of the test system. 

The mean values of revertant colonies of the solvent control plates were within the 

acceptable control data range, the reference mutagens showed the expected increase in 

the number of revertant colonies, the viability of the bacterial cells was checked by a 

plating experiment in each test. The tests were considered to be valid.

The reported data of this mutagenicity assay show (see Appendix 2 to 5) that under 

the experimental conditions applied the test item did not induce gene mutations by 

base pair changes or frameshifts in the genome of the strains used. 

In conclusion, the test item had no mutagenic activity on the growth of 

the bacterium tester strains under the test conditions used in this study.
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2. INTRODUCTION

The bacterial reverse mutation test is a microbial assay which detects point mutations 

induced by chemicals causing base changes or frameshift mutations in the genome of 

amino-acid requiring strains of Salmonella typhimurium and Escherichia coli. The 

auxotrophic S. typhimurium and E. coli strains are unable to grow on minimal medium 

- containing inorganic salts and glucose as a carbon source - except for spontaneous 

revertants. But in the presence of a mutagenic agent, some of them can be converted to 

prototrophs after a reverse mutation to the wild type. These revertants can grow and 

form colonies in minimal medium. An increased number of the revertant colonies 

indicates mutagenic activity of the test item. 

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ 

reversions. The Salmonella typhimurium strains are constructed to differentiate 

between base-pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The 

Escherichia coli WP2 uvrA tryptophan (trp) reversion system measures trp- → trp+ 

reversions. The Escherichia coli WP2 uvrA detects mutagens that cause base-pair 

substitutions (AT to GC). 

The bacteria used in this assay did not possess the mammalian enzyme system that is 

known to convert promutagens into mutagenic metabolites. In order to overcome this 

major drawback, an exogenous metabolic activation system is added in the form of a 

mammalian microsomal enzyme activation mixture (liver extract, S9 fraction). The 

activation system uses nicotinamide-adenine dinucleotide phosphate (NADP+)-

cytochrome P450 dependent mixed function oxidase enzymes of the liver. The liver 

extract was obtained from rats, which were pre-treated with phenobarbital and βnaphthoflavone, two inducers of several drug-metabolizing enzymes.

The bacterial mutagenicity test, along with other short-term assays, is used extensively 

to evaluate substances for mutagenic activity. Some mutagens (e.g.: nitrosamines) are 

poorly detected in the standard plate incorporation assay, but they can be detected by 

the pre-incubation assay. 

3. OBJECTIVE OF STUDY

The purpose of this study was to evaluate the mutagenic potential of test item 

by measuring its ability to induce reverse mutations at selected loci of several strains 

of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain 

WP2 uvrA in the presence and absence of activated rat liver S9 fraction.
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4. GUIDELINES

This study followed the procedures indicated by the following internationally 

accepted guidelines and recommendations: 

– Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 

471, "Bacterial Reverse Mutation Test", adopted 21st July, 1997 

– EPA Health Effects Test Guidelines, OPPTS 870.5100 "Bacterial Reverse 

Mutation Test", EPA 712-C-98-247, August 1998 

– EPA Health Effects Test Guidelines, OPPTS 870.5100 "Escherichia coli WP2 and 

WP2 uvrA Reverse Mutation Assays", EPA 712-C-96-247, June 1996 (Public Draft)

– Commission Regulation (EC) No. 440/2008, B.13/14. "Mutagenicity: Reverse 

Mutation Test Using Bacteria", 30 May 2008 

The method described the above mentioned guidelines conforms to the guidelines for 

bacterial mutagenicity testing published by the major Japanese Regulatory Authorities 

including METI, MHLW and MAFF. 

5. MATERIALS AND METHODS

5.1. TEST ITEM

5.1.1. Name and Data of Test Item

Name:

Synonym: Polymer

Chemical Name:

CAS Number:

Lot Number: 

Molecular weight: >2000 Da 

Description: Colourless liquid* 

Purity: 97.4 % 

Manufacture Date: 10 May 2010 

Expiry Date: 24 October 2012 

Storage conditions: Room temperature (15-30o

C)

Safety Precautions: Routine safety precautions (lab coat, gloves, safety 

glasses, face mask) for unknown materials were 

applied to assure personnel health and safety. 

*On the Test Item Data Sheet (dated 16 May 2011) colourless, viscous liquid was stated. However, 

according to the Sponsor, the observed appearance is acceptable.
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5.1.2. Identification, Receipt

The test item of a suitable chemical purity was supplied by the Sponsor. All 

precautions required in the handling and disposal of the test item were outlined by the 

Sponsor. These documents are part of the raw data. Identification of the test item was 

performed in the Central Dispensary of LAB Research Ltd. on the basis of the 

information provided by the Sponsor (name, batch number, appearance and colour).

Copy of the Certificate of Analysis is shown in Appendix 1. 

5.1.3. Formulation

The behaviour of the test item solutions with the solution of top agar (Sections 5.4.4.) 

and phosphate buffer (Section 5.5.4.) was examined in a preliminary solubility test. 

Acetone was used as vehicle to prepare the stock formulation of the test material. Test 

formulations were freshly prepared at the beginning of the experiments in the testing 

laboratory from the stock formulation by serial dilution using the selected vehicle. 

Continuous stirring was applied during formulation and dosing to ensure the stability 

of the formulation. 

Analytical determination of the test item concentration, stability and homogeneity was 

not performed because of the character and the short period of study. 

Table 1: Formulation of the Test Item for the Main Experiments

No. of concentration

Concentration

of the test item

Concentration 

(μg/plate)

1 100 mg/mL 5000

2 31.62 mg/mL 1581

3 10 mg/mL 500

4 3.162 mg/mL 158.1

5 1 mg/mL 50

6 0.3162 mg/mL 15.81

7 0.1 mg/mL 5 

5.2. POSITIVE AND NEGATIVE CONTROLS

Positive and negative (solvent) were included in the experiments. Routine safety 

precautions (lab coat, gloves, safety glasses and face mask) were applied to assure 

personnel health and safety. 
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5.2.1. Positive Controls 

Strain specific positive controls were included in the assay, which demonstrated the 

effective performance of the test. The strain specific positive control chemicals are 

summarized in Table 2. 

Table 2: Strain-Specific Positive Control Chemicals

Metabolic 

activation

Strain specific positive chemical

Vehicle Dose 

quantity/plate Strain

Chemical and Supplier Batch number / 

Expiry date

non-activation

4-nitro-1,2-phenylenediamine (NPD)

MERCK 

S5061894 /

28 Feb 2013 DMSO 4 μg

Salmonella

TA98

non-activation Sodium azide (SAZ)

SIGMA-ALDRICH 

MKBF0623 /

02 Feb 2012

Distilled 

water 2 μg

Salmonella

TA100

TA1535

non-activation 9-aminoacridine (9AA)

MERCK 

S03761 / 

31 Dec 2012 DMSO 50 μg

Salmonella

TA1537

non-activation

Methyl-methanesulfonate 

(MMS) 

SIGMA-ALDRICH 

MKBD8856V /

30 Dec 2011

Distilled 

water 2 μL E.coli 

WP2 uvrA

activation

2-aminoanthracene 

(2AA)

SIGMA-ALDRICH 

STBB1901 / 

31 Oct 2011 DMSO 2 μg

all 

Salmonella

strains

activation

2-aminoanthracene 

(2AA)

SIGMA-ALDRICH 

STBB1901 / 

31 Oct 2011 DMSO 50 μg

E.coli WP2 

uvrA

Abbreviation for tester strains: Salmonella: Salmonella typhimurium, E.coli: Escherichia coli

5.2.2. Negative Controls (Vehicle / Solvent Controls) 

In the study three vehicle control groups were used depending on the solubility of the 

test item and the solubility of strain specific positive chemicals. The following 

chemicals were used for vehicle (solvent) control groups:

Acetone:

Supplier: REANAL

Batch No.: KBR52522 / KBM62741* 

Expiry date: May 2015 / February 2016

Grade: Ph.Eur.6 

Purity: >99.9 % 

*Note: Lot KBR52522 was used in the preliminary experiment; lot KBM62741 was used in the main 

tests.
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Dimethyl sulfoxide (DMSO):

Supplier: Sigma-Aldrich Co. 

Batch No.: BCBD8784V 

Expiry date: December 2016

Grade: puriss, p.a., ACS reagent 

Purity: 99.96% 

Distilled water: 

Produced by: TEVA Hungary Co.

Batch No.: 8490910 

Expiry date: September 2013

5.3. BACTERIAL STRAINS

5.3.1. Origin

Date of arrival and origin: 

Salmonella typhimurium TA98 16 November 2010, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA 

Salmonella typhimurium TA100 04 December 2008, BioReliance Corporation, 

Rockville, Maryland, USA

Salmonella typhimurium TA1535 16 November 2010, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA 

Salmonella typhimurium TA1537 16 November 2010, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA 

Escherichia coli WP2 uvrA 16 November 2010, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA 

The true copies of original certificates and other documents of the strains are collected 

and stored in the Microbiological Laboratory of the LAB Research Ltd.

5.3.2. Genotypes

In addition to histidine or tryptophan mutation, each strain has additional mutations, 

which enhances its sensitivity to mutagens. The uvrB (uvrA) strains are defective in 

excision repair, making them more sensitive to the mutagenic and lethal effects of a 

wide variety of mutagens because they cannot repair DNA damages. The presence of 

rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for 

larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which 

participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. 

This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is 

used to identify its presence in the cell. 
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The Escherichia coli strain used in this test (WP2 uvrA) is also defective in DNA 

excision repair. The genotypes of the tester strains used for mutagenicity testing are 

summarized in Table 3. 

Table 3: Genotypes of the Strains Used for Mutagenicity Testing 

Strain

Genotype Mutations Main

DNA

target

Plasmid trp./his 

mutation

type of 

mutation cell wall DNA- repair

S.typhimurium

TA98 hisD3052 Frameshift rfa uvrB GC pKM101

S.typhimurium

TA100 hisG46 Base pair 

substitution rfa uvrB GC pKM101

S.typhimurium

TA1535 hisG46 Base pair 

substitution rfa uvrB GC No 

S.typhimurium. 

TA1537 hisC3076 Frameshift rfa uvrB GC No 

E.coli 

WP2 uvrA trpE Base pair 

substitution + uvrA AT No 

Abbreviations of tester strains: S.typhimurium.: Salmonella typhimurium; E.coli: Escherichia coli

5.3.3. Storage

The strains are stored at -80 ± 10oC in the Culture Collection of the Microbiological 

Laboratory of the LAB Research Ltd. Frozen permanent cultures of the tester strains 

were prepared from fresh, overnight cultures to which DMSO was added as a 

cryoprotective agent. 

5.3.4. Confirmation of Phenotypes of Tester Strains

The phenotypes of the tester strains used in the bacterial reverse mutation assays with 

regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin 

resistance (amp), as well as spontaneous mutation frequencies were checked regularly 

according to Ames et al. [1] and Maron and Ames [2]. 

Established procedures (Standard Operating Procedures) for the preparations of each 

batch of frozen stock culture, raw data and reports of phenotype confirmation are 

stored in the Microbiological Laboratory of LAB Research Ltd. 
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5.3.5. Spontaneous Reversion of Tester Strains 

Each test strain reverts spontaneously at a frequency that is characteristic of the strain. 

Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) 

or tryptophan (in Escherichia coli strain) independence is measured routinely in 

mutagenicity experiments and expressed as the number of spontaneous revertants per 

plate. Historical control values for spontaneous revertants (revertants/plate) for 

untreated control sample without metabolic activation were in the period of 2004 to 

2010 were (as guide) as follows: Salmonella typhimurium TA98: 8-54, TA100: 58-

211, TA1535: 2-31, TA1537: 1-24, Escherichia coli WP2 uvrA: 9-86. More detailed 

historical control data are shown in Appendix 6. 

5.3.6. Procedure for Growing Cultures 

The frozen bacterial cultures were thawed at room temperature and 200 μL inoculum 

were used to inoculate each 50 mL of Nutrient Broth No. 2 (Section 5.4.2.) for the 

overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37°C 

in a Gyrotory water bath shaker. 

5.3.7. Viability of the Testing Cultures

The viability of each testing culture was determined by plating 0.1 mL of the

105

, 106, 107 and 108 dilutions of cultures on Nutrient Agar (Section 5.4.3.) plates. The 

viable cell number of the cultures was determined by manual counting. 

5.4. MEDIA

The supplier, batch number and expiry date of the used chemicals described in 

sections 5.4.1.-5.4.5. are summarized in Table 6 (Section 5.9. Chemicals Used in the 

Experiments). The composition of media refers to 1000 mL. 
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5.4.1. The Typical Composition (g/1000 mL) of Minimal Glucose Agar 

Glucose 20.0 g 

Magnesium sulfate 0.2 g 

Citric acid 2.0 g 

di-Potassium hydrogenphosphate 10.0 g 

Sodium ammonium hydrogenphosphate 3.5 g 

Agar agar 15.0 g 

Distilled water q.s. ad1

 1000 mL

Minimal glucose agar plates [MERCKOPLATE] (Batch Number: 04416, Expiry date: 

16 June 2011) were provided by Merck. Certificates of Analysis were obtained from 

the Supplier. 

5.4.2. Nutrient Broth No.2 

Nutrient Broth No.2 25.0 g 

Distilled water q.s. ad 1000 mL

Sterilization was performed at 121oC in an autoclave. 

5.4.3. Nutrient Agar

Nutrient Agar 20.0 g 

Distilled water q.s. ad 1000 mL

Sterilization was performed at 121oC in an autoclave. 

5.4.4. Top Agar for Salmonella typhimurium Strains

Agar solution: 

Agar Bacteriological 4.0 g 

NaCl 5.0 g 

Distilled water q.s. ad 1000 mL

Sterilization was performed at 121o

C in an autoclave. 

 

1 q.s. ad = quantum sufficiat ad (a sufficient quantity to make)
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Histidine – Biotin solution (0.5 mM): 

D-Biotin (F.W. 244.3) 122.2 mg

L-Histidine x·HCl x H2O (F.W. 209.63) 104.8 mg

Distilled water q.s. ad 1000 mL

Sterilization was performed by filtration using a 0.22 μm membrane filter.

Complete Top Agar for Salmonella typhimurium strains:

Histidine – Biotin solution (0.5 mM) 100 mL

Agar solution 900 mL

5.4.5. Top Agar for Escherichia coli Strain

Tryptophan solution (2 mg/mL): 

L-Tryptophan (F.W. 204.23) 2000 mg

Distilled water q.s. ad 1000 mL

Sterilization was performed by filtration using a 0.22 μm membrane filter.

Complete Top Agar for Escherichia coli strain:

Nutrient Broth by 5.4.2. 50 mL

Tryptophan solution (2 mg/mL) 2.5 mL

Agar solution by 5.4.4. 947.5 mL

5.5. METABOLIC ACTIVATION SYSTEM

Test bacteria were also exposed to the test item in the presence of an appropriate 

metabolic activation system, which is a cofactor-supplemented post-mitochondrial S9 

fraction.

The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological 

Laboratory of LAB Research Ltd. according to Ames et al. [1] and Maron and Ames 

[2]. The documentation of the preparation of this post-mitochondrial fraction is stored 

in the reagent notebook in the Microbiological Laboratory which is archived yearly.

The supplier, batch number and expiry date of the used chemicals described in 

sections 5.5.3. and 5.5.4. are summarized in Table 6 (Section 5.9. Chemicals Used in 

the Experiments). The composition of solution refers to 1000 mL. 
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5.5.1. Induction of Liver Enzymes

Male Wistar rats (257-305 g, animals were 7-8 weeks old at the initiation) were treated 

with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage 

for three consecutive days. Rats were given drinking water and food ad libitum until 12 

h before sacrifice when food was removed. Sacrifice was by ascending concentration of 

CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the 

induction of liver enzymes used for preparation S9 used in this study was 07 March 

2011. 

5.5.2. Preparation of Rat Liver Homogenate S9 Fraction

On Day 4, the rats were euthanized and the livers were removed aseptically using sterile 

surgical tools. After excision, livers were weighed and washed several times in 0.15 M 

KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl 

per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 

9000 g and the supernatant was decanted and retained. The freshly prepared S9 

fraction was aliquoted into 1-3 mL portions, frozen quickly and stored at -80 ± 10oC.

The sterility of the preparation was confirmed. 

The protein concentration of the preparation was determined by a chemical analyzer at 

540 nm in the Clinical Chemistry Laboratory of LAB Research Ltd. The protein 

concentration of the S9 fraction used was determined to be 35.3 g/L. The date of 

preparation of S9 fraction for this study was 10 March 2011 (LAB code: E11041). 

The biological activity in the Salmonella assay of S9 was characterized using the two 

mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation 

by microsomal enzymes. The batch of S9 used in this study functioned appropriately. 

5.5.3. The S9 Mix 

Salt solution for S9 mix: 

NADP Na 7.66 g 

D-glucose-6 phosphate Na 3.53 g 

MgCl2 x 6 H2O 4.07 g 

KCl 6.15 g 

Distilled water q.s. ad 1000 mL 

Sterilization was performed by filtration through a 0.22 μm membrane filter.
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The complete S9 mix was freshly prepared containing components as follows: 

Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 mL 

Rat liver homogenate (S9) 100 mL 

Salt solution for S9 Mix (see above) 400 mL 

Prior to addition to the culture medium the S9 mix was kept in an ice bath. 

5.5.4. 0.2 M Sodium Phosphate Buffer, pH 7.4 

Solution A:

Na2HPO4 x 12H2O 71.63 g 

Distilled water q.s. ad 1000 mL

Sterilization was performed at 121oC in an autoclave. 

Solution B: 

NaH2PO4 24.0 g 

Distilled water q.s. ad 1000 mL

Sterilization was performed at 121oC in an autoclave. 

Sodium phosphate buffer pH 7.4: 

Solution A 880 mL

Solution B 120 mL

5.6. DESCRIPTION OF THE TEST PROCEDURE

The study included a Preliminary Solubility Test, a Preliminary Range Finding Tests 

(Informatory Toxicity Tests), an Initial Mutation Test and a Confirmatory Mutation 

Test. In the Range Finding Test as well as in the Initial Mutation Test, the plate 

incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation 

method was used. 

5.6.1. Concentrations 

Concentrations were selected on the basis of the Preliminary Solubility Test and 

Preliminary Range Finding Tests (Informatory Toxicity Tests). In the Initial Mutation 

Test and Confirmatory Mutation Test seven concentrations were used.
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5.6.1.1. Preliminary Solubility Test

The solubility of the test item was examined in a preliminary solubility test. Based on 

the available information, the test item is insoluble in water. The solubility of the test 

item was examined using Dimethyl sulfoxide (DMSO), N,N-Dimethylformamide 

(DMF), Acetone, Tetrahydrofuran, n-Hexane, Cyclohexane and PEG 400 as solvent at 

100, 50 and 25 mg/mL concentrations. No complete dissolution was observed at these 

concentrations. However, if continuous stirring was applied, the resulted formulations 

(emulsions) using Acetone or PEG400 as vehicle were suitable for the test. Due to the 

better biocompatibility to the test system, Acetone was chosen for vehicle of the study. 

The obtained stock solution (50 μL) with the solution of top agar (5.4.4.) and 

phosphate buffer (5.5.4.) was examined in a test tube without test bacterium 

suspension. The results of the Solubility Test are summarized in Table 4. 

Table 4: The Solubility of the Test Item in Acetone

Concentration 

of test item

in Acetone

(mg/mL)

Solubility

in Acetone

Solubility in the top 

solution

(test item solution 50 μL

+ phosphate buffer 500 μL

+ top agar 2 mL)

Test item 

concentration in 

the test tube

μg/tube

100 emulsion clear solution 5000

5.6.1.2. Concentration Range Finding Tests (Informatory Toxicity Tests) 

Based on the solubility test, 100 mg/mL stock formulation was prepared in Acetone, 

which was diluted in 6 steps by factors of 2, 2.5 and approximately √10. The revertant 

colony numbers and the inhibition of the background lawn of auxotrophic cells of two 

of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at 

the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test 

item.

Note: The highest concentration was tested without solvent as well (the equivalent amount of test item 

was added directly to the test tube).

5.6.1.3. Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test and 

Confirmatory Mutation Test)

Based on the results of the preliminary tests, 100 mg/mL stock formulation was 

prepared from the test item with Acetone, which was diluted by serial dilutions in six 

steps to obtain seven dosing solutions for lower doses. Examined concentrations 

in the main tests were 5000; 1581; 500; 158.1; 50; 15.81 and 5 μg/plate.
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5.6.2. Control Groups Used in the Tests 

Strain-specific positive and negative (vehicle/solvent) controls, both with and without 

metabolic activation were included in each test. In addition, untreated control was 

used demonstrating that the chosen vehicle induced no deleterious or mutagenic 

effects. The control groups are summarized in Table 5. 

Table 5: Summary of Controls 

Type of control Activation 

with S9 mix Solvent Top 

Agar

Strain-Specific 

Positive Chemical 

Solutions (5.2.1.)

Phosphate 

Buffer

Untreated – – + – +

Untreated + – + – –

Solvent – + + – +

Solvent + + + – –

Positive Reference 

Control – – + + + 

Positive Reference 

Control + – + + – 

Note: if the solvent of the positive control substance differed from the solvent of the test item, both 

solvents were run in the assay.

5.6.3. Experimental Method

The experimental methods were conducted according to the methods described Ames 

et al. [1] and Maron and Ames [2], Kier et al. [3], Venitt and Parry [4], OECD 

Guideline No 471 1997 [5], Commission Regulation (EC) No. 440/2008 [6] and EPA 

Guidelines, OPPTS 870.5100, 1998, 1996 [7][8]. 

5.6.4. Procedure for Exposure in the Initial Mutation Test 

A standard plate incorporation procedure was performed, as an Initial Mutation Test. 

Bacteria (cultured in Nutrient Broth No.2 as described in Section 5.3.6.) were exposed 

to the test item both in the presence and absence of an appropriate metabolic activation 

system. 

Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into 

individual test tubes (3 tubes per control or concentration level). The equivalent 

number of minimal glucose agar plates was properly labelled. The test item and other 

components were prepared freshly and added to the overlay (45°C). 
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The content of the tubes: 

top agar 2000 μL

solvent or test item solution (or reference controls) 50 μL

overnight culture of test strain 100 μL

phosphate buffer (pH 7.4) or S9 mix 500 μL

This solution was mixed and poured on the surface of minimal agar plates. For 

activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to 

each overlay tube. The entire test consisted of non-activated and activated test 

conditions, with the addition of untreated, negative and positive controls. After 

preparation, the plates were incubated at 37°C for 48 hours. 

5.6.5. Procedure for Exposure in the Confirmatory Mutation Test 

A pre-incubation procedure [1][2][5][6][7] was performed as a Confirmatory Mutation 

Test since the result of the Initial Mutation Test was negative (see details in Section 6.).

Before the overlaying, the test item solution (or solvent or reference control), the 

bacterial culture (Section 5.3.6.) and the S9 mix or phosphate buffer was added into 

appropriate tubes to provide direct contact between bacteria and the test item (in its 

solvent). The tubes were gently mixed and incubated for 20 min at 37oC in a shaking 

incubator. 

After the incubation period, 2 mL of molten top agar was added to the tubes; the 

content was mixed up and poured onto minimal glucose agar plates as described for 

the standard plate incorporation method (Section 5.6.4.). The entire test consisted of 

non-activated and activated test conditions, with the addition of untreated, negative 

and positive controls. After preparation, the plates were incubated at 37°C for 48 

hours. 

5.7. EVALUATION OF EXPERIMENTAL DATA

The colony numbers on the untreated / negative / positive control and test item treated 

plates were determined by manual counting. The mean number of revertants per plate, 

the standard deviation and the mutation factor* values were calculated for each 

concentration level of the test item and for the controls using Microsoft ExcelTM

software.

* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants 

on the vehicle (solvent) control plate.
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Criteria for a Positive Response: 

A test item was considered mutagenic if:

- a dose–related increase in the number of revertants occurs and/or;

- a reproducible biologically relevant positive response for at least one of the dose 

groups occurs in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:

- if in Salmonella typhiumurium TA100 strain the number of reversion is more than 

twice higher when compared to the spontaneous reversion rate of the solvent 

control plates, 

- if in Salmonella typhiumurium TA98, TA1535, TA1537 and Escherichia coli

WP2 uvrA strains the number of reversions is more than three times higher when 

compared to the spontaneous reversion rate of the solvent control plates. 

According to the guidelines [5][6][7], statistical method may be used as an aid in 

evaluating the test results. However, statistical significance should not be the only 

determining factor for a positive response.

Criteria for a Negative Response:

A test article was considered non-mutagenic if it produces neither a dose-related 

increase in the number of revertants nor a reproducible biologically relevant positive 

response at any of the dose groups, with or without metabolic activation.

5.8. ARCHIVES

The study documents and samples: 

- study plan and study plan amendment, 

- all raw data,

- retained sample of the test item,

- correspondence,

- study report and any amendments 

will be stored in the archives of LAB Research Ltd. (H-8200 Veszprém, 

Szabadságpuszta, Hungary) according to the Hungarian GLP regulation [9] and to test 

facility SOPs. 

After the retention time has elapsed, all the archived materials listed above would be 

offered to the Sponsor or retained for a further period if agreed by a contract. No raw 

data or material relating to the study will be discarded without the Sponsor's prior 

written consent. 
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5.9. CHEMICALS USED IN THE EXPERIMENTS

The chemicals used in the independently performed experiments of the study are 

summarized in Table 6. 

Table 6: Chemicals Used in the Experiments 

Chemical Supplier Batch Number Expiry date

Agar Bacteriological OXOID 1124998-02 23 March 2015

D-Biotin SIGMA-ALDRICH 036K1504V October 2014

Cyclohexane Lachner PP/2009/00776 31 January 2014

N,N,-Dimethylformamide CARLO ERBA P0C02490C March 2016

Distilled water HungaroPharma 805 0411 05 October 2011

D-Glucose 6-phosphate Na SIGMA-ALDRICH 079K3788 August 2012

n-Hexane CARLO ERBA P9B025029C March 2015

L-Histidine HCl x H2O MERCK K37522450 30 June 2012

KCl MERCK K41042236 31 May 2015

MgCl2 x 6 H2O SIGMA-ALDRICH 019K0038 January 2014

NaCl Lachner PP/2010/10084 31 May 2013

NADP Na x H2O SIGMA-ALDRICH 020M7009V March 2013

NaH2PO4 REANAL KBM5207601 July 2011

Na2HPO4 x 12H2O REANAL KBM61668 August 2015

Nutrient Agar MERCK VM100650 06 October 2014

Nutrient Broth No.2 OXOID 987078 31 January 2016

PEG400 SIGMA-ALDRICH BCBD1911 July 2012

Tetrahydrofuran MERCK I504901 31 October 2012

L-Tryptophan SIGMA-ALDRICH 1400132 July 2012

NADP: β-Nicotinamide adenine dinucleotide phosphate

5.10. DEVIATIONS TO THE STUDY PLAN

There was no deviation to the Study Plan. 
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6. RESULTS AND DISCUSSION

6.1. PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)

In the Preliminary Range Finding Test, the plate incorporation method was used. The 

preliminary test was performed using Salmonella typhimurium TA98 and Salmonella 

typhimurium TA100 tester strains in the presence and absence of metabolic activation 

system (±S9 Mix) with appropriate untreated, negative (vehicle/solvent) and positive 

controls. In the test each samples (including the controls) were tested in triplicate.

In the Range Finding Tests the concentrations examined were: 5000, 2500, 1000, 316, 

100, 31.6 and 10 μg/plate.

Slightly higher numbers of revertant colonies were observed in some cases in 

Salmonella typhimurium TA98 tester strain with metabolic activation. However, the 

observed mutation factor values were well below the biologically relevant threshold 

value and no dose-response was observed. The numbers of revertant colonies were 

within the historical control range in all cases.

Slightly lower numbers of revertant colonies compared to the solvent control plates 

were observed in both tester strains in some cases with or without metabolic 

activation. However, the observed numbers of revertant colonies had no biological 

relevance and were in the historical control range in all cases. 

The experimental results (revertant colony numbers per plate, mutation factors and 

standard deviations) are detailed in Table 7 (Appendix 2) and in Appendix 3. 

6.2. INITIAL AND CONFIRMATORY MUTATION TESTS

In the Initial Mutation Test, the plate incorporation method; in the Confirmatory 

Mutation Test, the pre-incubation method was used. The Initial Mutation Test and 

Confirmatory Mutation Test were carried out using Salmonella typhimurium strains 

(TA98, TA100, TA1535, TA1537) and Escherichia coli WP2 uvrA strain. Each test 

was performed in the presence and absence of metabolic activation system (±S9 Mix) 

with appropriate untreated, negative (vehicle/solvent) and positive controls. In the 

main test each sample (including the controls) were tested in triplicate.

The examined test item concentrations in the main tests were of 5000; 1581; 500; 

158.1; 50; 15.81 and 5 μg/plate. 

In the Initial Mutation Test and Confirmatory Mutation Tests, none of the observed 

revertant colony numbers were above the respective biological threshold value. There 

were no reproducible dose-related trends and no indication of any treatment effect. 
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Using the plate incorporation method, the highest revertant rate was observed in the 

Initial Mutation Test in Salmonella typhimurium TA1537 tester strain at 

5000 μg/plate concentration without metabolic activation. The mutation factor value 

was 1.86. Higher revertant counts compared to the solvent control plates were 

detected at some other tested concentrations in this experiment. However, the 

observed mutation factor values were well below the biologically relevant threshold 

value. The numbers of revertant colonies were within the historical control range in 

all cases.

Using the pre-incubation method, highest revertant rate was observed in the 

Confirmatory Mutation Test in Salmonella typhimurium TA1537 tester strain at

15.81 μg/plate with metabolic concentration. The mutation factor value was 2.00. 

Higher revertant counts compared to the solvent control plates were detected at some 

other tested concentrations in this experiment. However, the observed mutation factor 

values were below the biologically relevant threshold value and no dose-response was 

observed. The numbers of revertant colonies were within the historical control range.

Furthermore, higher number of revertant colonies compared to the Acetone solvent 

control was observed for DMSO solvent control (MF: 1.80) also in this experiment. 

Sporadically, higher numbers of revertant colonies compared to the solvent control 

were detected in the Initial Mutation Test and Confirmatory Mutation Test in some 

cases. However, no dose-dependence was observed and they were below the 

biologically relevant threshold value. The numbers of revertant colonies were within 

the historical control range in all cases, so they were considered as reflecting the 

biological variability of the test.

Sporadically, lower revertant counts compared to the solvent control were observed in 

the Initial Mutation Test and Confirmatory Mutation Test at some concentrations. 

However, the differences were usually minor and the mean numbers of revertant 

colonies were in the historical control range in all cases, thus they were considered as 

biological variability of the test system. 

The experimental results (revertant colony numbers per plate, mutation factors, and 

standard deviations) are summarized in Table 8-9 (Appendix 2) and Appendices 4 

and 5. 

6.3. VALIDITY OF THE TESTS 

Untreated, negative (solvent) and positive controls were run concurrently. The mean 

values of revertant colony numbers of untreated and solvent control plates were within 

the historical control data range. The reference mutagens showed a distinct increase of 

induced revertant colonies. The viability of the bacterial cells was checked by a plating 

experiment in each test. The tests were considered to be valid.
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7. CONCLUSION

The test item was tested for potential mutagenic activity using the Bacterial 

Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of 

Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and 

TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli 

(Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation 

system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared 

from the livers of phenobarbital/β-naphthoflavone-induced rats. 

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test 

(Informatory Toxicity Test), an Initial Mutation Test and a Confirmatory Mutation 

Test. In the Range Finding Test as well as in the Initial Mutation Test, the plate 

incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation 

method was used. 

The reported data of this mutagenicity assay show (see Appendix 2 to 5) that under 

the experimental conditions applied the test item did not induce gene mutations by 

base pair changes or frameshifts in the genome of the strains used. 

In conclusion, the test item had no mutagenic activity on the growth of 

the applied bacterium tester strains under the test conditions used in this study.

8. DISTRIBUTION OF THE FINAL REPORT

Sponsor: 1x copy, bound 

 1x copy, unbound 

 1x PDF file

Archive: 1x original, bound 
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9. REFERENCES

1. BRUCE N. AMES, JOYCE MCCANN and EDITH YAMASAKI:

Methods for Detecting Carcinogens and Mutagens with the Salmonella

/Mammalian-Microsome Mutagenicity Test. Mutation Research,

 31: 347-364, 1975 

2. DOROTHY M. MARON and BRUCE N. AMES:

 Revised Method for the Salmonella Mutagenicity Test. Mutation Research,

 113: 173-215, 1983 

3. L.D. KIER., Brusick D.J., Auletta, A.E., Von Halle, E.S., Brown, M.M., Simmon, 

V.F., Dunkel, V., McCann, J., Mortelmans, K., Prival, M., Rao, T.K. and Ray, V: 

The Salmonella Typhimurium / Mammalian Microsomal Assay. A Report of the 

U.S. Environmental Protection Agency Gene-Tox Program. Mutation Research, 

168: 69-240, 1986 

4. S.VENITT and J.M.PARRY: 

Mutagenicity Testing, a Practical Approach IRL Press Limited, Eynsham, Oxford 

England, 1984 

5. OECD Guidelines for testing of Chemicals, Section 4, No. 471 “Bacterial 

Reverse Mutation Test”, adopted 21st July 1997 

6. Commission Regulation (EC) No. 440/2008, B.13/14. “Mutagenicity: Reverse 

Mutation Test Using Bacteria”, 30 May 2008 

7. EPA Health Effects Test Guidelines, OPPTS 870.5100 “Bacterial Reverse 

Mutation Test”, EPA 712-C-98-247, August 1998 

8. EPA Health Effects Test Guidelines, OPPTS 870.5100 “Escherichia coli WP2 and 

WP2 uvrA Reverse Mutation Assays“, EPA 712-C-96-247, June 1996 (Public Draft)

9. Hungarian Good Laboratory Practice Regulations: 9/2001 (III. 30.) EüM-FVM joint 

decree of the Minister of Health and the Minister of the Agriculture and Regional 

Development (in Hungarian) which corresponds to the OECD GLP, 

ENV/MC/CHEM(98)17
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APPENDICES
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COPY OF THE CERTIFICATE OF ANALYSIS
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SUMMARY TABLES OF THE RESULTS

Table 7: Summary Table of the Range Finding Test 

Concentrations

(μg/plate)

Mean 

values of 

revertants / 

Mutation 

factor (MF)

Salmonella typhimurium tester strains

TA98 TA100

-S9 +S9 -S9 +S9

Untreated 

control

Mean 32.7 37.3 86.0 107.7

MF 0.91 1.12 0.99 0.93

Distilled water 

control

Mean -- -- 80.7 --

MF -- -- 0.93 --

DMSO 

control

Mean 30.0 43.0 - 110.0

MF 0.83 1.29 - 0.95

Acetone 

control

Mean 36.0 33.3 86.7 115.3

MF 1.00 1.00 1.00 1.00

5000# Mean 29.0 40.0 72.7 129.7

MF 0.81 1.20 0.84 1.12

5000 Mean 34.3 35.3 79.3 112.0

MF 0.95 1.06 0.92 0.97

2500 Mean 34.3 40.7 83.7 86.3

MF 0.95 1.22 0.97 0.75

1000 Mean 36.0 38.3 78.3 119.3

MF 1.00 1.15 0.90 1.03

316 Mean 28.3 37.3 86.7 115.7

MF 0.79 1.12 1.00 1.00

100 Mean 25.7 44.0 90.3 121.3

MF 0.71 1.32 1.04 1.05

31.6 Mean 27.3 42.3 84.7 131.7

MF 0.76 1.27 0.98 1.14

10 Mean 30.7 46.7 83.3 119.0

MF 0.85 1.40 0.96 1.03

NPD (4 μg) Mean 384.0 -- -- --

MF 12.80 -- -- --

2AA (2 μg) Mean -- 3203.3 -- 2806.0

MF -- 74.50 -- 25.51

SAZ (2 μg) Mean -- -- 1819.3 --

MF -- -- 22.55 --

#: undiluted test item was used for dosing
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Table 8: Summary Table of the Initial Mutation Test 

Concentrations

μg/plate)

Mean 

values of 

revertants / 

Mutation 

factor (MF)

Salmonella typhimurium tester strains Escherichia 

coli

TA98 TA100 TA1535 TA1537 WP2 uvrA

-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9

Untreated 

control

Mean 25.0 33.0 93.3 110.7 11.3 8.0 3.0 4.0 25.3 34.3

MF 1.07 1.11 1.02 1.10 0.92 0.51 0.64 0.60 0.81 0.88

DMSO 

control

Mean 21.7 33.0 -- 113.7 -- 14.0 5.3 9.0 -- 42.0

MF 0.93 1.11 -- 1.13 -- 0.89 1.14 1.35 -- 1.08

Distilled water

control

Mean -- -- 82.3 -- 11.7 -- -- -- 26.7 --

MF -- -- 0.90 -- 0.95 -- -- -- 0.85 --

Acetone 

control

Mean 23.3 29.7 91.7 101.0 12.3 15.7 4.7 6.7 31.3 39.0

MF 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

5000 Mean 28.7 39.0 72.0 108.3 14.7 12.3 8.7 7.7 29.0 31.7

MF 1.23 1.31 0.79 1.07 1.19 0.79 1.86 1.15 0.93 0.81

1581 Mean 24.0 27.0 81.7 94.7 13.3 10.3 7.3 5.7 26.0 39.7

MF 1.03 0.91 0.89 0.94 1.08 0.66 1.57 0.85 0.83 1.02

500 Mean 27.3 29.3 81.7 104.0 13.3 11.3 5.3 6.3 22.7 34.0

MF 1.17 0.99 0.89 1.03 1.08 0.72 1.14 0.95 0.72 0.87

158.1 Mean 25.3 32.7 84.7 112.3 14.7 11.3 6.3 6.3 27.7 37.0

MF 1.09 1.10 0.92 1.11 1.19 0.72 1.36 0.95 0.88 0.95

50 Mean 24.7 31.0 90.0 107.7 11.7 15.3 4.3 5.7 25.0 35.0

MF 1.06 1.04 0.98 1.07 0.95 0.98 0.93 0.85 0.80 0.90

15.81 Mean 25.7 35.7 89.0 108.3 9.0 12.0 4.3 6.3 27.0 38.7

MF 1.10 1.20 0.97 1.07 0.73 0.77 0.93 0.95 0.86 0.99

5 Mean 23.0 36.3 79.3 120.0 15.0 13.7 5.0 7.3 28.7 32.0

MF 0.99 1.22 0.87 1.19 1.22 0.87 1.07 1.10 0.91 0.82

NPD (4 μg) Mean 304.7 -- -- -- -- -- -- -- -- --

MF 14.06 -- -- -- -- -- -- -- -- --

2AA (2 μg) Mean -- 2306.7 -- 2230.7 -- 190.3 -- 184.7 -- --

MF -- 69.90 -- 19.62 -- 13.60 -- 20.52 -- --

2AA (50 μg) Mean -- -- -- -- -- -- -- -- -- 384.7

MF -- -- -- -- -- -- -- -- -- 9.16

SAZ (2 μg) Mean -- -- 1496.7 -- 1304.0 -- -- -- -- --

MF -- -- 18.18 -- 111.77 -- -- -- -- --

9AA (50 μg) Mean -- -- -- -- -- -- 505.3 -- -- --

MF -- -- -- -- -- -- 94.75 -- -- --

MMS (2 μL) Mean -- -- -- -- -- -- -- -- 1217.3 --

MF -- -- -- -- -- -- -- -- 45.65 --
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Table 9: Summary Table of the Confirmatory Mutation Test 

Concentrations

μg/plate)

Mean 

values of 

revertants / 

Mutation 

factor (MF)

Salmonella typhimurium tester strains Escherichia 

coli

TA98 TA100 TA1535 TA1537 WP2 uvrA

-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9

Untreated 

control

Mean 29.3 37.3 78.0 100.0 9.3 12.7 3.0 3.7 24.3 31.3

MF 0.99 0.95 1.08 0.95 0.88 0.81 0.50 0.73 1.20 0.99

DMSO 

control

Mean 28.0 40.0 - 105.7 - 13.7 4.0 9.0 - 31.3

MF 0.94 1.02 - 1.01 - 0.87 0.67 1.80 - 0.99

Distilled water

control

Mean - - 69.3 - 11.0 - - - 24.3 -

MF - - 0.96 - 1.03 - - - 1.20 -

Acetone 

control

Mean 29.7 39.3 72.3 105.0 10.7 15.7 6.0 5.0 20.3 31.7

MF 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

5000 Mean 26.3 39.3 71.7 108.0 10.3 12.7 5.3 7.7 22.0 38.0

MF 0.89 1.00 0.99 1.03 0.97 0.81 0.89 1.53 1.08 1.20

1581 Mean 28.0 39.7 74.0 103.7 8.0 12.0 6.7 7.7 19.3 39.0

MF 0.94 1.01 1.02 0.99 0.75 0.77 1.11 1.53 0.95 1.23

500 Mean 29.3 42.0 74.3 121.7 8.0 12.0 5.7 7.3 20.3 34.7

MF 0.99 1.07 1.03 1.16 0.75 0.77 0.94 1.47 1.00 1.09

158.1 Mean 31.7 38.7 72.3 107.7 8.0 10.0 4.0 7.0 21.3 31.7

MF 1.07 0.98 1.00 1.03 0.75 0.64 0.67 1.40 1.05 1.00

50 Mean 30.3 38.3 73.7 106.7 5.3 8.3 4.3 5.7 24.0 35.7

MF 1.02 0.97 1.02 1.02 0.50 0.53 0.72 1.13 1.18 1.13

15.81 Mean 26.3 41.7 79.7 116.0 10.0 10.7 4.0 10.0 21.3 33.7

MF 0.89 1.06 1.10 1.10 0.94 0.68 0.67 2.00 1.05 1.06

5 Mean 27.0 40.7 75.3 114.7 8.3 15.0 5.3 8.3 14.3 33.0

MF 0.91 1.03 1.04 1.09 0.78 0.96 0.89 1.67 0.70 1.04

NPD (4 μg) Mean 300.0 -- -- -- -- -- -- -- -- --

MF 10.71 -- -- -- -- -- -- -- -- --

2AA (2 μg) Mean -- 2366.7 -- 1769.3 -- 217.0 -- 246.7 -- --

MF -- 59.17 -- 16.74 -- 15.88 -- 27.41 -- --

2AA (50 μg) Mean -- -- -- -- -- -- -- -- -- 255.0

MF -- -- -- -- -- -- -- -- -- 8.14

SAZ (2 μg) Mean -- -- 1772.0 -- 1194.7 -- -- -- -- --

MF -- -- 25.56 -- 108.61 -- -- -- -- --

9AA (50 μg) Mean -- -- -- -- -- -- 513.3 -- -- --

MF -- -- -- -- -- -- 128.33 -- -- --

MMS (2 μL) Mean -- -- -- -- -- -- -- -- 953.3 --

MF -- -- -- -- -- -- -- -- 39.18 --
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Preliminary Range Finding Test (Informatory Toxicity Test)

Test Item : 

Date : 25-27 May 2011 

Strain : Salmonella typhimurium TA98

Cell count (overnight culture): 1.59x 109 CFU/mL

Revertant colony number Revertant colony number

Concentration: -S9 +S9 Concentration: -S9 +S9 

(μg/plate) (μg/plate)

5000 30 33# 37 34# Untreated control 33 35 

30 32# 30 41# 34 35 

43 22# 39 45# 31 42 

Mean 34.3 29.0# 35.3 40.0# Mean 32.7 37.3 

SD 7.51 6.08# 4.73 5.57# SD 1.53 4.04 

MF 0.95 0.81# 1.06 1.20# MF 0.91 1.12 

2500 34 40 DMSO control* 25 42 

36 54 30 45 

33 28 35 42 

Mean 34.3 40.7 Mean 30.0 43.0 

SD 1.53 13.01 SD 5.00 1.73 

MF 0.95 1.22 MF 0.83 1.29 

1000 46 34 Acetone control 41 42 

32 41 36 35 

30 40 31 23 

Mean 36.0 38.3 Mean 36.0 33.3 

SD 8.72 3.79 SD 5.00 9.61 

MF 1.00 1.15 MF 1.00 1.00 

316 32 29 Positive control 312 2AA 3424 

26 38 NPD 424 (2 μg) 3216

27 45 (4 μg) 416 2968 

Mean 28.3 37.3 Mean 384.0 3203.3 

SD 3.21 8.02 SD 62.48 228.35 

MF 0.79 1.12 MF 12.80 74.50 

100 20 42 

29 53 

28 37 MF: Mutation factor

Mean 25.7 44.0 SD: Standard deviation

SD 4.93 8.19 +S9: with S9 mix

MF 0.71 1.32 -S9: without S9 mix

31.6 35 50 

26 35 

21 42 

Mean 27.3 42.3 Mutation factor= mean revertants (test item)

SD 7.09 7.51 mean revertants (solvent control)

MF 0.76 1.27 

10 28 50 

26 50 

38 40 *: DMSO control was used because of NPD and 2AA

Mean 30.7 46.7 

SD 6.43 5.77 #: the equivalent amount of test item was added 

MF 0.85 1.40 directly to the test tube
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Preliminary Range Finding Test (Informatory Toxicity Test) 

Test Item : 

Date : 25-27 May 2011 

Strain : Salmonella typhimurium TA100 

Cell count (overnight culture): 1.60 x 109 CFU/mL

Revertant colony number Revertant colony number

Concentration: -S9 +S9 Concentration: -S9 +S9 

(μg/plate) (μg/plate)

5000 67 79# 116 131# Untreated control 81 113 

87 63# 107 128# 88 100 

84 76# 113 130# 89 110 

Mean 79.3 72.7# 112.0 129.7# Mean 86.0 107.7 

SD 10.79 8.50# 4.58 1.53# SD 4.36 6.81 

MF 0.92 0.84# 0.97 1.12# MF 0.99 0.93 

2500 71 86 DMSO control* 83 101 

105 95 88 111 

75 78 82 118 

Mean 83.7 86.3 Mean 84.3 110.0 

SD 18.58 8.50 SD 3.21 8.54 

MF 0.97 0.75 MF 0.97 0.95 

1000 91 101 Acetone control 84 114 

64 133 87 105 

80 124 89 127 

Mean 78.3 119.3 Mean 86.7 115.3 

SD 13.58 16.50 SD 2.52 11.06 

MF 0.90 1.03 MF 1.00 1.00 

316 75 107 Distilled water 84 78 

90 117 control** 78 124 

95 123 80 118 

Mean 86.7 115.7 Mean 80.7 106.7 

SD 10.41 8.08 SD 3.06 25.01 

MF 1.00 1.00 MF 0.93 0.92 

100 88 113 Positive control 1826 2AA 2828 

90 118 SAZ 1798 (2 μg) 2960

93 133 (2 μg) 1834 2654 

Mean 90.3 121.3 Mean 1819.3 2806.0 

SD 2.52 10.41 SD 18.90 142.28 

MF 1.04 1.05 MF 22.55 25.51 

31.6 76 125 

94 133 MF: Mutation factor

84 137 SD: Standard deviation

Mean 84.7 131.7 +S9: with S9 mix 

SD 9.02 6.11 -S9: without S9 mix

MF 0.98 1.14 

10 85 122 

89 122 

Mutation factor= mean revertants (test item)

76 113 mean revertants (solvent control)

Mean 83.3 119.0 

SD 6.66 5.20 *: DMSO control was used because of 2AA

MF 0.96 1.03 **: Distilled water control was used because of SAZ

#: the equivalent amount of test item was added 

directly to the test tube
STUDY CODE: 11/076-007M Page 1 of Appendix 4 

LAB

Initial Mutation Test (Plate Incorporation Method)

Test Item : 

Date : 01-03 June 2011 

Strain : Salmonella typhimurium TA98

Cell count (overnight culture): 1.79 x 109 CFU/mL

Revertant colony number Revertant colony number

Concentration: -S9 +S9 Concentration: -S9 +S9 

(μg/plate) (μg/plate)

5000 40 41 Untreated control 27 36 

25 44 29 31 

21 32 19 32 

Mean 28.7 39.0 Mean 25.0 33.0 

SD 10.02 6.24 SD 5.29 2.65 

MF 1.23 1.31 MF 1.07 1.11 

1581 24 29 DMSO control* 23 29 

21 22 19 36 

27 30 23 34 

Mean 24.0 27.0 Mean 21.7 33.0 

SD 3.00 4.36 SD 2.31 3.61 

MF 1.03 0.91 MF 0.93 1.11 

500 27 28 Acetone control 24 30 

34 25 23 34 

21 35 23 25 

Mean 27.3 29.3 Mean 23.3 29.7 

SD 6.51 5.13 SD 0.58 4.51 

MF 1.17 0.99 MF 1.00 1.00 

158.1 28 35 Positive control 298 2AA 2324 

24 34 NPD 304 (2 μg) 2280 

24 29 (4 μg) 312 2316 

Mean 25.3 32.7 Mean 304.7 2306.7 

SD 2.31 3.21 SD 7.02 23.44 

MF 1.09 1.10 MF 14.06 69.90 

50 28 36 

26 25 

20 32 

Mean 24.7 31.0 MF: Mutation factor

SD 4.16 5.57 SD: Standard deviation

MF 1.06 1.04 +S9: with S9 mix 

15.81 29 35 -S9: without S9 mix

24 33 

24 39 Mutation factor= mean revertants (test item)

Mean 25.7 35.7 mean revertants (solvent control)

SD 2.89 3.06 

MF 1.10 1.20 

5 26 36 

20 42 

23 31 

Mean 23.0 36.3 *: DMSO control was used because of NPD and 2AA

SD 3.00 5.51 

MF 0.99 1.22 
STUDY CODE: 11/076-007M Page 2 of Appendix 4

LAB

Initial Mutation Test (Plate Incorporation Method)

Test Item : 

Date : 01-03 June 2011 

Strain : Salmonella typhimurium TA100 

 Cell count (overnight culture): 2.04 x 109 CFU/mL

Revertant colony number Revertant colony number

Concentration: -S9 +S9 Concentration: -S9 +S9 

(μg/plate) (μg/plate)

5000 79 94 Untreated control 101 102 

69 105 85 122 

68 126 94 108 

Mean 72.0 108.3 Mean 93.3 110.7 

SD 6.08 16.26 SD 8.02 10.26 

MF 0.79 1.07 MF 1.02 1.10 

1581 83 91 DMSO 86 124 

83 98 control* 78 99 

79 95 84 118 

Mean 81.7 94.7 Mean 82.7 113.7 

SD 2.31 3.51 SD 4.16 13.05 

MF 0.89 0.94 MF 0.90 1.13 

500 82 98 Distilled water 86 136 

91 97 control** 74 123 

72 117 87 109 

Mean 81.7 104.0 Mean 82.3 122.7 

SD 9.50 11.27 SD 7.23 13.50 

MF 0.89 1.03 MF 0.90 1.21 

158.1 84 100 Acetone control 97 91 

98 105 87 106 

72 132 91 106 

Mean 84.7 112.3 Mean 91.7 101.0 

SD 13.01 17.21 SD 5.03 8.66 

MF 0.92 1.11 MF 1.00 1.00 

50 88 103 Positive control 1440 2AA 2200 

89 94 SAZ 1486 (2 μg) 2284 

93 126 (2 μg) 1564 2208 

Mean 90.0 107.7 Mean 1496.7 2230.7 

SD 2.65 16.50 SD 62.68 46.36 

MF 0.98 1.07 MF 18.18 19.62 

15.81 91 113 

86 101 

90 111 Mutation factor= mean revertants (test item)

Mean 89.0 108.3 mean revertants (solvent control)

SD 2.65 6.43 

MF 0.97 1.07 

5 82 120 MF: Mutation factor

73 133 SD: Standard deviation

83 107 +S9: with S9 mix

Mean 79.3 120.0 -S9: without S9 mix 

SD 5.51 13.00 

MF 0.87 1.19 *: DMSO control was used because of 2AA

**: Distilled water control was used because of SAZ
STUDY CODE: 11/076-007M Page 3 of Appendix 4

LAB

Initial Mutation Test (Plate Incorporation Method) 

Test Item : 

Date : 01-03 June 2011 

Strain : Salmonella typhimurium TA1535 

 Cell count (overnight culture): 1.72 x 109 CFU/mL

Revertant colony number Revertant colony number

Concentration: -S9 +S9 Concentration: -S9 +S9 

(μg/plate) (μg/plate)

5000 12 11 Untreated control 16 6 

13 16 9 7 

19 10 9 11 

Mean 14.7 12.3 Mean 11.3 8.0 

SD 3.79 3.21 SD 4.04 2.65 

MF 1.19 0.79 MF 0.92 0.51 

1581 18 9 DMSO control* 7 9 

10 13 7 15 

12 9 9 18 

Mean 13.3 10.3 Mean 7.7 14.0 

SD 4.16 2.31 SD 1.15 4.58 

MF 1.08 0.66 MF 0.62 0.89 

500 12 7 Distilled water 15 18 

16 12 control** 12 10 

12 15 8 11 

Mean 13.3 11.3 Mean 11.7 13.0 

SD 2.31 4.04 SD 3.51 4.36 

MF 1.08 0.72 MF 0.95 0.83 

158.1 15 9 Acetone control 13 15 

19 12 13 18 

10 13 11 14 

Mean 14.7 11.3 Mean 12.3 15.7 

SD 4.51 2.08 SD 1.15 2.08 

MF 1.19 0.72 MF 1.00 1.00 

50 10 10 Positive control 1212 2AA 197 

10 19 SAZ 1280 (2 μg) 183 

15 17 (2 μg) 1420 191 

Mean 11.7 15.3 Mean 1304.0 190.3 

SD 2.89 4.73 SD 106.06 7.02 

MF 0.95 0.98 MF 111.77 13.60 

15.81 8 13 

9 13 

10 10 Mutation factor= mean revertants (test item)

Mean 9.0 12.0 mean revertants (solvent control)

SD 1.00 1.73 

MF 0.73 0.77 

5 16 17 MF: Mutation factor

14 11 SD: Standard deviation

15 13 +S9: with S9 mix

Mean 15.0 13.7 -S9: without S9 mix 

SD 1.00 3.06 

MF 1.22 0.87 *: DMSO control was used because of 2AA

**: Distilled water control was used because of SAZ
STUDY CODE: 11/076-007M Page 4 of Appendix 4

LAB

Initial Mutation Test (Plate Incorporation Method)

Test Item : 

Date : 01-03 June 2011 

Strain : Salmonella typhimurium TA1537 

 Cell count (overnight culture): 1.76 x 109 CFU/mL

Revertant colony number Revertant colony number

Concentration: -S9 +S9 Concentration: -S9 +S9 

(μg/plate) (μg/plate)

5000 8 9 Untreated control 4 5 

9 7 3 4 

9 7 2 3 

Mean 8.7 7.7 Mean 3.0 4.0 

SD 0.58 1.15 SD 1.00 1.00 

MF 1.86 1.15 MF 0.64 0.60 

1581 6 6 DMSO control* 7 9 

8 6 4 8 

8 5 5 10 

Mean 7.3 5.7 Mean 5.3 9.0 

SD 1.15 0.58 SD 1.53 1.00 

MF 1.57 0.85 MF 1.14 1.35 

500 5 5 Acetone control 5 7 

6 6 3 5 

5 8 6 8 

Mean 5.3 6.3 Mean 4.7 6.7 

SD 0.58 1.53 SD 1.53 1.53 

MF 1.14 0.95 MF 1.00 1.00 

158.1 8 5 Positive control 524 2AA 192 

5 8 9AA 516 (2 μg) 183 

6 6 (50 μg) 476 179 

Mean 6.3 6.3 Mean 505.3 184.7 

SD 1.53 1.53 SD 25.72 6.66 

MF 1.36 0.95 MF 94.75 20.52 

50 4 6 

5 6 

4 5 

Mean 4.3 5.7 MF: Mutation factor

SD 0.58 0.58 SD: Standard deviation

MF 0.93 0.85 +S9: with S9 mix 

15.81 4 7 -S9: without S9 mix

3 6 

6 6 Mutation factor= mean revertants (test item)

Mean 4.3 6.3 mean revertants (solvent control)

SD 1.53 0.58 

MF 0.93 0.95 

5 6 8 

6 8 

3 6 

Mean 5.0 7.3 *: DMSO control was used because of 9AA and 2AA

SD 1.73 1.15 

MF 1.07 1.10 
STUDY CODE: 11/076-007M Page 5 of Appendix 4

LAB

Initial Mutation Test (Plate Incorporation Method)

Test Item : 

Date : 01-03 June 2011 

Strain : Escherichia coli WP2 uvrA

 Cell count (overnight culture): 3.66 x 109 FU/mL

Revertant colony number Revertant colony number

Concentration: -S9 +S9 Concentration: -S9 +S9 

(μg/plate) (μg/plate)

5000 27 34 Untreated control 24 41 

27 32 27 32 

33 29 25 30 

Mean 29.0 31.7 Mean 25.3 34.3 

SD 3.46 2.52 SD 1.53 5.86 

MF 0.93 0.81 MF 0.81 0.88 

1581 28 48 DMSO control* 28 40 

26 34 21 41 

24 37 28 45 

Mean 26.0 39.7 Mean 25.7 42.0 

SD 2.00 7.37 SD 4.04 2.65 

MF 0.83 1.02 MF 0.82 1.08 

500 21 37 Distilled water 21 44 

20 34 control** 25 38 

27 31 34 34 

Mean 22.7 34.0 Mean 26.7 38.7 

SD 3.79 3.00 SD 6.66 5.03 

MF 0.72 0.87 MF 0.85 0.99 

158.1 29 30 Acetone control 27 36 

31 40 32 33 

23 41 35 48 

Mean 27.7 37.0 Mean 31.3 39.0 

SD 4.16 6.08 SD 4.04 7.94 

MF 0.88 0.95 MF 1.00 1.00 

50 22 36 Positive control 1220 2AA 442 

24 29 MMS 1148 (50 μg) 324 

29 40 (2 μL) 1284 388 

Mean 25.0 35.0 Mean 1217.3 384.7 

SD 3.61 5.57 SD 68.04 59.07 

MF 0.80 0.90 MF 45.65 9.16 

15.81 31 39 

20 39 

30 38 Mutation factor= mean revertants (test item)

Mean 27.0 38.7 mean revertants (solvent control)

SD 6.08 0.58 

MF 0.86 0.99 

5 36 37 MF: Mutation factor

20 32 SD: Standard deviation

30 27 +S9: with S9 mix

Mean 28.7 32.0 -S9: without S9 mix

SD 8.08 5.00 

MF 0.91 0.82 *: DMSO control was used because of 2AA

**: Distilled water control was used because of MMS
STUDY CODE: 11/076-007M Page 1 of Appendix 5 

LAB

Confirmatory Mutation Test (Pre-Incubation Method)

Test Item : 

Date : 07-09 June 2011 

Strain : Salmonella typhimurium TA98

Cell count (overnight culture): 2.13 x 109 CFU/mL

Revertant colony number Revertant colony number

Concentration: -S9 +S9 Concentration: -S9 +S9 

(μg/plate) (μg/plate)

5000 33 37 Untreated control 31 34 

26 40 30 39 

20 41 27 39 

Mean 26.3 39.3 Mean 29.3 37.3 

SD 6.51 2.08 SD 2.08 2.89 

MF 0.89 1.00 MF 0.99 0.95 

1581 27 35 DMSO control* 39 40 

25 45 21 41 

32 39 24 39 

Mean 28.0 39.7 Mean 28.0 40.0 

SD 3.61 5.03 SD 9.64 1.00 

MF 0.94 1.01 MF 0.94 1.02 

500 30 34 Acetone control 29 33 

30 52 32 42 

28 40 28 43 

Mean 29.3 42.0 Mean 29.7 39.3 

SD 1.15 9.17 SD 2.08 5.51 

MF 0.99 1.07 MF 1.00 1.00 

158.1 28 46 Positive control 302 2AA 2320 

32 38 NPD 278 (2 μg) 2364 

35 32 (4 μg) 320 2416 

Mean 31.7 38.7 Mean 300.0 2366.7 

SD 3.51 7.02 SD 21.07 48.06 

MF 1.07 0.98 MF 10.71 59.17 

50 29 36 

32 39 

30 40 

Mean 30.3 38.3 MF: Mutation factor

SD 1.53 2.08 SD: Standard deviation

MF 1.02 0.97 +S9: with S9 mix 

15.81 33 45 -S9: without S9 mix

25 43 

21 37 Mutation factor= mean revertants (test item)

Mean 26.3 41.7 mean revertants (solvent control)

SD 6.11 4.16 

MF 0.89 1.06 

5 33 43 

24 36 

24 43 

Mean 27.0 40.7 *: DMSO control was used because of NPD and 2AA

SD 5.20 4.04 

MF 0.91 1.03 
STUDY CODE: 11/076-007M Page 2 of Appendix 5

LAB

Confirmatory Mutation Test (Pre-Incubation Method)

Test Item : 

Date : 07-09 June 2011 

Strain : Salmonella typhimurium TA100 

 Cell count (overnight culture): 2.77 x 109 CFU/mL

Revertant colony number Revertant colony number

Concentration: -S9 +S9 Concentration: -S9 +S9 

(μg/plate) (μg/plate)

5000 68 101 Untreated control 73 95 

79 112 79 103 

68 111 82 102 

Mean 71.7 108.0 Mean 78.0 100.0 

SD 6.35 6.08 SD 4.58 4.36 

MF 0.99 1.03 MF 1.08 0.95 

1581 77 97 DMSO 74 114 

76 118 control* 76 94 

69 96 81 109 

Mean 74.0 103.7 Mean 77.0 105.7 

SD 4.36 12.42 SD 3.61 10.41 

MF 1.02 0.99 MF 1.06 1.01 

500 74 125 Distilled water 70 87 

71 119 control** 77 117 

78 121 61 108 

Mean 74.3 121.7 Mean 69.3 104.0 

SD 3.51 3.06 SD 8.02 15.39 

MF 1.03 1.16 MF 0.96 0.99 

158.1 72 104 Acetone control 62 104 

71 109 66 103 

74 110 89 108 

Mean 72.3 107.7 Mean 72.3 105.0 

SD 1.53 3.21 SD 14.57 2.65 

MF 1.00 1.03 MF 1.00 1.00 

50 71 121 Positive control 1836 2AA 1908 

73 90 SAZ 1896 (2 μg) 1808 

77 109 (2 μg) 1584 1592 

Mean 73.7 106.7 Mean 1772.0 1769.3 

SD 3.06 15.63 SD 165.55 161.51 

MF 1.02 1.02 MF 25.56 16.74 

15.81 89 121 

68 113 

82 114 Mutation factor= mean revertants (test item)

Mean 79.7 116.0 mean revertants (solvent control)

SD 10.69 4.36 

MF 1.10 1.10 

5 88 103 MF: Mutation factor

69 121 SD: Standard deviation

69 120 +S9: with S9 mix

Mean 75.3 114.7 -S9: without S9 mix 

SD 10.97 10.12 

MF 1.04 1.09 *: DMSO control was used because of 2AA

**: Distilled water control was used because of SAZ
STUDY CODE: 11/076-007M Page 3 of Appendix 5

LAB

Confirmatory Mutation Test (Pre-Incubation Method)

Test Item : 

Date : 07-09 June 2011 

Strain : Salmonella typhimurium TA1535 

 Cell count (overnight culture): 2.17 x 109 CFU/mL

Revertant colony number Revertant colony number

Concentration: -S9 +S9 Concentration: -S9 +S9 

(μg/plate) (μg/plate)

5000 15 14 Untreated control 8 11 

9 16 7 11 

7 8 13 16 

Mean 10.3 12.7 Mean 9.3 12.7 

SD 4.16 4.16 SD 3.21 2.89 

MF 0.97 0.81 MF 0.88 0.81 

1581 6 17 DMSO control* 12 13 

10 10 17 13 

8 9 12 15 

Mean 8.0 12.0 Mean 13.7 13.7 

SD 2.00 4.36 SD 2.89 1.15 

MF 0.75 0.77 MF 1.28 0.87 

500 7 14 Distilled water 8 10 

10 10 control** 14 7 

7 12 11 15 

Mean 8.0 12.0 Mean 11.0 10.7 

SD 1.73 2.00 SD 3.00 4.04 

MF 0.75 0.77 MF 1.03 0.68 

158.1 8 8 Acetone control 12 11 

8 13 7 16 

8 9 13 20 

Mean 8.0 10.0 Mean 10.7 15.7 

SD 0.00 2.65 SD 3.21 4.51 

MF 0.75 0.64 MF 1.00 1.00 

50 4 7 Positive control 1276 2AA 252 

5 4 SAZ 1036 (2 μg) 199 

7 14 (2 μg) 1272 200 

Mean 5.3 8.3 Mean 1194.7 217.0 

SD 1.53 5.13 SD 137.42 30.32 

MF 0.50 0.53 MF 108.61 15.88 

15.81 8 12 

10 8 

12 12 Mutation factor= mean revertants (test item)

Mean 10.0 10.7 mean revertants (solvent control)

SD 2.00 2.31 

MF 0.94 0.68 

5 13 14 MF: Mutation factor

6 14 SD: Standard deviation

6 17 +S9: with S9 mix

Mean 8.3 15.0 -S9: without S9 mix 

SD 4.04 1.73 

MF 0.78 0.96 *: DMSO control was used because of 2AA

**: Distilled water control was used because of SAZ
STUDY CODE: 11/076-007M Page 4 of Appendix 5

LAB

Confirmatory Mutation Test (Pre-Incubation Method)

Test Item : 

Date : 07-09 June 2011 

Strain : Salmonella typhimurium TA1537 

 Cell count (overnight culture): 2.32 x 109 CFU/mL

Revertant colony number Revertant colony number

Concentration: -S9 +S9 Concentration: -S9 +S9 

(μg/plate) (μg/plate)

5000 4 10 Untreated control 3 5 

3 5 3 2 

9 8 3 4 

Mean 5.3 7.7 Mean 3.0 3.7 

SD 3.21 2.52 SD 0.00 1.53 

MF 0.89 1.53 MF 0.50 0.73 

1581 6 15 DMSO control* 7 7 

5 5 2 13 

9 3 3 7 

Mean 6.7 7.7 Mean 4.0 9.0 

SD 2.08 6.43 SD 2.65 3.46 

MF 1.11 1.53 MF 0.67 1.80 

500 7 8 Acetone control 5 6 

5 8 4 5 

5 6 9 4 

Mean 5.7 7.3 Mean 6.0 5.0 

SD 1.15 1.15 SD 2.65 1.00 

MF 0.94 1.47 MF 1.00 1.00 

158.1 5 4 Positive control 467 2AA 215 

3 9 9AA 577 (2 μg) 276 

4 8 (50 μg) 496 249 

Mean 4.0 7.0 Mean 513.3 246.7 

SD 1.00 2.65 SD 57.01 30.57 

MF 0.67 1.40 MF 128.33 27.41 

50 5 7 

6 6 

2 4 

Mean 4.3 5.7 MF: Mutation factor

SD 2.08 1.53 SD: Standard deviation

MF 0.72 1.13 +S9: with S9 mix 

15.81 4 9 -S9: without S9 mix

4 11 

4 10 Mutation factor= mean revertants (test item)

Mean 4.0 10.0 mean revertants (solvent control)

SD 0.00 1.00 

MF 0.67 2.00 

5 6 11 

6 8 

4 6 

Mean 5.3 8.3 *: DMSO control was used because of 9AA and 2AA

SD 1.15 2.52 

MF 0.89 1.67 
STUDY CODE: 11/076-007M Page 5 of Appendix 5

LAB

Confirmatory Mutation Test (Pre-Incubation Method)

Test Item : 

Date : 07-09 June 2011 

Strain : Escherichia coli WP2 uvrA

 Cell count (overnight culture): 3.08 x 109 FU/mL

Revertant colony number Revertant colony number

Concentration: -S9 +S9 Concentration: -S9 +S9 

(μg/plate) (μg/plate)

5000 21 39 Untreated control 28 24 

25 37 23 35 

20 38 22 35 

Mean 22.0 38.0 Mean 24.3 31.3 

SD 2.65 1.00 SD 3.21 6.35 

MF 1.08 1.20 MF 1.20 0.99 

1581 17 36 DMSO control* 24 30 

21 39 26 33 

20 42 21 31 

Mean 19.3 39.0 Mean 23.7 31.3 

SD 2.08 3.00 SD 2.52 1.53 

MF 0.95 1.23 MF 1.16 0.99 

500 21 35 Distilled water 26 44 

24 32 control** 23 31 

16 37 24 35 

Mean 20.3 34.7 Mean 24.3 36.7 

SD 4.04 2.52 SD 1.53 6.66 

MF 1.00 1.09 MF 1.20 1.16 

158.1 28 38 Acetone control 21 31 

20 26 21 31 

16 31 19 33 

Mean 21.3 31.7 Mean 20.3 31.7 

SD 6.11 6.03 SD 1.15 1.15 

MF 1.05 1.00 MF 1.00 1.00 

50 22 31 Positive control 988 2AA 214 

23 33 MMS 852 (50 μg) 240 

27 43 (2 μL) 1020 311 

Mean 24.0 35.7 Mean 953.3 255.0 

SD 2.65 6.43 SD 89.20 50.21 

MF 1.18 1.13 MF 39.18 8.14 

15.81 20 33 

23 35 

21 33 Mutation factor= mean revertants (test item)

Mean 21.3 33.7 mean revertants (solvent control)

SD 1.53 1.15 

MF 1.05 1.06 

5 16 23 MF: Mutation factor

12 35 SD: Standard deviation

15 41 +S9: with S9 mix

Mean 14.3 33.0 -S9: without S9 mix

SD 2.08 9.17 

MF 0.70 1.04 *: DMSO control was used because of 2AA

**: Distilled water control was used because of MMS
STUDY CODE: 11/076-007M Page 1 of Appendix 6 

LAB

HISTORICAL CONTROL DATA

(Period of 2004-2010) 

Untreated control data

without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)

TA98 TA100 TA1535 TA1537 E.coli TA98 TA100 TA1535 TA1537 E.coli

Mean value of 

revertants 24.1 118.8 11.6 6.9 31.8 29.0 124.8 13.0 8.5 39.0

Minimum 8 58 2 1 9 9 62 3 1 12

Maximum 54 211 31 24 86 52 200 36 26 79

DMSO control data

without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)

TA98 TA100 TA1535 TA1537 E.coli TA98 TA100 TA1535 TA1537 E.coli

Mean value of 

revertants 23.5 116.8 11.5 6.4 31.1 28.2 123.0 12.8 7.7 38.7

Minimum 9 54 2 1 10 10 61 2 0 12

Maximum 54 200 35 27 69 52 199 31 27 78

Distilled water control data

without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)

TA98 TA100 TA1535 TA1537 E.coli TA98 TA100 TA1535 TA1537 E.coli

Mean value of 

revertants 24.6 119.4 11.8 7.2 33.5 29.9 125.6 13.1 8.7 40.1

Minimum 9 51 2 1 9 12 69 3 2 11

Maximum 46 208 34 23 65 61 223 32 23 74

TA98: Salmonella typhimurium TA98, TA100: Salmonella typhimurium TA100, 

TA1535: Salmonella typhimurium TA1535, TA1537: Salmonella typhimurium TA1537,

E.coli: Escherichia coli WP2 uvrA
STUDY CODE: 11/076-007M Page 2 of Appendix 6 

LAB

HISTORICAL CONTROL DATA (continued)

(Period of 2004-2010) 

Acetone control data 

without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)

TA98 TA100 TA1535 TA1537 E.coli TA98 TA100 TA1535 TA1537 E.coli

Mean value of 

revertants 23.5 109.0 10.7 6.2 30.6 29.5 119.8 11.1 8.0 37.7

Minimum 9 77 5 2 13 14 79 4 2 23

Maximum 43 184 20 14 56 47 188 20 22 60

Positive reference control data

without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)

TA98 TA100 TA1535 TA1537 E.coli TA98 TA100 TA1535 TA1537 E.coli

Mean value of 

revertants 474.9 938.7 817.0 552.4 754.1 1841.7 1870.7 249.4 260.0 344.8

Minimum 102 385 167 41 146 236 649 78 66 91

Maximum 1920 2525 2625 2748 1953 4390 4288 2250 1023 2010

TA98: Salmonella typhimurium TA98, TA100: Salmonella typhimurium TA100, 

TA1535: Salmonella typhimurium TA1535, TA1537: Salmonella typhimurium TA1537,

E.coli: Escherichia coli WP2 uvrA
STUDY CODE: 11/076-007M Page 1 of Appendix 7 

LAB

COPY OF THE GLP CERTIFICATE