### 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 Ju ly 2011



## STUDY CODE: 11/076-007M

## STUDY CODE: 11/076-007M Page 5 of 30



# LAB GENERAL INFORMATION

S TUDY 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 PER IOD II. : 07-09 June 2011

DRAFT REPORT : 15 July 2011

FINAL REPORT : 18 July 2011

## STUDY CODE: 11/076-007M Page 6 of 30



# LAB RESPONSIBLE PERSONS:

LAB MANAGEMENT : Christopher Banks, DABT

Managing Director

Alyson Leyshon, M.Sc.

Senior Director of Toxicology Operations

QUALITY ASS URANCE : 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

CENT R AL DISPENSARY : Tamás Mészáros, PhD

. (Technical Team Leader)

TECHNICAL ASSISTANTS : Tímea Kovácsné Böröcz

Emma Petőné Kohán

Ákos Klaffl

## STUDY CODE: 11/076-007M Page 7 of 30



# LAB TABLE OF CONTENTS

Page

STATEMENT OF THE STUDY DIRECTOR 2

S TATEMENT OF THE M ANAGEMENT 3

QUALITY ASSURANCE STATEMENT 4

1. SUMMARY 9

2. INTRODUCTION 10

3. OBJECTIVE OF STUDY 10

4. GUIDELINES 11

5. MATERIALS AND METHOD S 11

5.1. TEST ITEM 11

5.2. POSITIVE AND NEGATIVE CONTROLS 12

5.3. BACTERIAL STRA INS 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 TH E EXPERIMENTS 25

5.10. DEVIATIONS TO THE STUDY PLAN 25

6. RESULTS AND DISCUSSI ON 26

6.1. PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY

TEST) 26

6.2. INITIAL AND CONFIRMA TORY MUTATION TESTS 26

6.3. VALIDITY OF THE TEST S 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 t he 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

## STUDY CODE: 11/076-007M Page 8 of 30



# LAB 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

## STUDY CODE: 11/076-007M Page 9 of 30



# LAB 1. SUMMARY

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

Reverse Mutation Assay.

The ex periments 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 fo r mulated in

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

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

t he 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 biolo gical 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, t he 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 u n der

the experimental conditions applied the test item did not induce gene mut a tions 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.

## STUDY CODE: 11/076-007M Page 10 of 30



# LAB 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 S almonella typhimurium strains are co n structed to differentiate

between base

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

Escherichia coli WP2 uvrA tryptophan (trp) reversion s ystem 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 muta genic metab o lites. In order to overcome this

major drawback, an exogenous metabolic activation sy s tem is added in the form of a

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

activation system uses nicotinamide

- adenine din ucleotide 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 **β**

naphthoflavone, 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 detec ted 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 uvr A in the presence and absence of activated rat liver S9 fraction.

## STUDY CODE: 11/076-007M Page 11 of 30



# LAB 4. GUIDELINES

This study followed the procedures indicated by the following internationally

accepted guidelines and recom mendations:

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

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

## 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 ME THODS

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- 30 o 

## 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 (dat ed 16 May 2011) colourless, viscous liquid was stated. However,

according to the Sponsor, the observed appearance is acceptable.

## STUDY CODE: 11/076-007M Page 12 of 30



# LAB 5.1.2. Identification, Receipt

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

precautions req uired 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 seri al dilution using the selected vehicle

. Continuous stirring was applied during formulation and dosing to ensure the sta b ility

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.

## STUDY CODE: 11/076-007M Page 13 of 30



# LAB 5.2.1. Positive Controls

Strain specific positive controls were included in the assay, which demo n strated 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

- phenylene

diamine (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 / So lvent 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:

Aceton e:

Supplier: REANAL

Batch No.: KB R52522 / KBM62741*

Expiry date: May 2015 / Feb ruary 2016

Grade: Ph.Eur.6

Purity: >99.9 %

*Note: Lot KB R 52522 was used in the preliminary experiment; lot KBM62741 was used in the main

tests.

## STUDY CODE: 11/076-007M Page 14 of 30



# LAB Dimethyl sulfoxide (DMSO):

Suppli er: Sigma -Aldrich Co.

Batch No.: BCBD8784V

Expiry date: December 2 016

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 Toxico

logy Inc., Boone, North Carolina, USA

Salmonella typhimurium TA100 04 December 2008, BioReliance Corporation,

Rockville, Maryland, USA

Salmonella typhimuri um TA1535 16 November 2010, MOLTOX

- Molecular Toxico

logy Inc., Boone, North Carolina, USA

Salmonella typhimurium TA1537 16 November 2010, MOLTOX

- Molecular Toxico

logy Inc., Boone, North Carolina, USA

Escherichia coli WP2 uvrA 16 November 2010 , MOLTOX

- Molecular Toxico

logy 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 -p rone "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.

## STUDY CODE: 11/076-007M Page 15 of 30



# LAB 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

mutati on type of

mutation cell wall DNA - repair

S.typhimurium

TA98 hisD3052 Frameshift rfa uvr B GC pKM101

S.typhimurium

TA100 hisG46 Base pair

substitution rfa uvr B GC pKM101

S.typhimurium

TA1535 hisG46 Base pair

substitution rfa uvr B GC N o

S.typhimurium

. TA1537 hisC3076 Frameshift rfa uvr B GC N o

E.coli

WP2 uvrA trp E Base pair

substitution + uvrA AT N o



### 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 Ph enotypes 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 frequen cies 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.

## STUDY CODE: 11/076-007M Page 16 of 30



# LAB 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 spont a neous 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 d etailed

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 platin g 0.1 mL of the

10 5 

## , 10 6 

## , 10 7 

## and 10 8 

## 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.

## STUDY CODE: 11/076-007M Page 17 of 30



# LAB 5.4.1. The Typical Composition (g/1000 mL) of Minimal Glucose Agar

Glu cose 20.0 g

Magnesium sul fate 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. ad 1 

## 1000 mL

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

16 June 2011 ) were pr ovided 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 121 o 

## C in an autoclave.

5.4.3. Nutrient Agar

Nutrient Ag ar 20.0 g

Distilled water q.s. ad 1000 mL

Sterilization was performed at 121 o 

## C 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 pe rformed at 121 o 

## C in an autoclave.

1 

### q.s. ad = quantum sufficiat ad (a sufficient quantity to make)

## STUDY CODE: 11/076-007M Page 18 of 30



# LAB Histidine – Biotin solution (0.5 mM):

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

L- Histidine x·HCl x H 2 

## O (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 filt er.

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 SYST EM

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 Microbi ological

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 yea rly.

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.

## STUDY CODE: 11/076-007M Page 19 of 30



# LAB 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

CO 2, 

## 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

K Cl. 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 aliq uoted 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 character ized using the two

mutagens 2

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

by microsomal e nzymes. 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- gluc ose-6 phosphate Na 3.53 g

MgCl 2 

### x 

## 6 H 2 

## O 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.

## STUDY CODE: 11/076-007M Page 20 of 30



# LAB The complete S9 mix was freshly prepared containing comp onents 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:

Na 2 

## HPO 4 

## x 12H 2 

## O 71.63 g

Distilled water q.s. ad 1000 mL

Sterilization was performed at 121 o 

## C in an autoclave.

Solution B:

NaH 2 

## PO 4 

## 24.0 g

Distilled water q.s. ad 1000 mL

Sterilization was performed at 121 o 

## C 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.

## STUDY CODE: 11/076-007M Page 21 of 30



# LAB 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 formulatio ns

(emulsion s ) using Acetone or PEG400 a s 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

s olution

(test item solution 50 μL

+ phosphate buffer 500 μL

+ top agar 2 mL) Test item

concentration in

the test tube

**μ** g/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 μ g/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 Te st 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 s ix

steps to obtain seven dosing solutions for lower doses

. Examined concentrations

in the main tests were 5000; 1581; 500; 158.1 ; 5 0; 15.81 and 5 μg/plate.

## STUDY CODE: 11/076-007M Page 22 of 30



# LAB 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

B uffer

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.

Bact eria (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).

## STUDY CODE: 11/076-007M Page 23 of 30



# LAB The conten t 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

a ctivation 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 be tween 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 Excel TM



## software.

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

on the vehicle (solvent) control plate.

## STUDY CODE: 11/076-007M Page 24 of 30



# LAB 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

twic e 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], s tatistical method may be used as an aid in

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

determining factor for a posi tive 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, w ith 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 ar chives 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.

## STUDY CODE: 11/076-007M Page 25 of 30



# LAB 5.9. CHEMICALS USED IN TH E EXPERIMENTS

The chemicals used in the independently perf ormed 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

- pho s phate Na SIGMA

- ALDRICH 079K3788 August 2012

n- Hexane CARLO ERBA P9B025029 C March 2015

L- Histidine HCl x H 2 

### O MERCK K37522450 30 June 2012

KCl MERCK K41042236 31 May 2015

MgCl 2 

### x 6 H 2 

### O SIGMA

- ALDRICH 019K0038 January 2014

NaCl Lachner PP/2010/10084 31 May 2013

NADP Na x H 2 

### O SIGMA

- ALDRICH 020M7009V March 2013

NaH 2 

### PO 4 

### REANAL KBM5207601 Ju ly 2011

Na 2 

### HPO 4 

### x 12H 2 

### O 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 I 504901 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.

## STUDY CODE: 11/076-007M Page 26 of 30



# LAB 6. RESULTS AND DISCUSSI ON

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

syst em (±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.

S lightly 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 relevan t threshold

value and no dose

- response was observed. The numbers of revertant colonies were

within the historical control range in all cases.

Slightly l ower numbers of revertant colonies compared to the solvent control plates

were observed in both tester strain s 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 CONFIRMA TORY MUTATION TESTS

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

Mutation Test, the pre

- in cubation 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 test s were of 5 000; 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.

## STUDY CODE: 11/076-007M Page 27 of 30



# LAB Using the plate incorporation method, the highest revertant rate was observed in the

Initial Mutation Test in Salmonella typhimurium TA 1537 tester strain at

5000 μg/plate concentration without me tabolic 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 bi ologically 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 S almonella typhimurium TA 1537 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 obser ved mutation factor

values were below the biologically relevant threshold value and no dose

- response was

observed. Th e 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

biolog ical 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 n umbers 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 TEST S

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

values of revertant colony numbers of untreated and solvent control plate s 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.

## STUDY CODE: 11/076-007M Page 28 of 30



# LAB 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 TA 98, 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 prep ared

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 Fi nding 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 u n der

the expe rimental conditions applied the test item did not induce gene mut a tions 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 strain s 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

## STUDY CODE: 11/076-007M Page 29 of 30



# LAB 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

Re verse Mutation Test ”, adopted 21 st 

## 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 R e verse

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 Agricu l ture and Regional

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

ENV/MC/CHEM(98)17

## STUDY CODE: 11/076-007M Page 30 of 30



# LAB 

# APPENDICES

## STUDY CODE: 11/076-007M Page 1 

## of Appendix 1



# LAB COPY OF THE CERTIFICATE OF ANALYSIS

## STUDY CODE: 11/076-007M Page 1 of Appendix 2



# LAB SUMMARY TABLES OF TH E RESULTS

Table 7: Summary Table of the Range Finding Test

Concentrations

( 

### μ g/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 0.9 7

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 μ g) Mean 384.0

-- -- --

MF 12.80

-- -- --

2AA (2 μ g) Mean

-- 3203.3

-- 2806.0

MF

-- 74.50

-- 25.51

SAZ (2 μ g) Mean

-- -- 1819.3

--

MF

-- -- 22.55

-- #: undiluted test item was used for dosing

## STUDY CODE: 11/076-007M Page 2 of Appendix 2



# LAB Table 8: Summary Table of the Initial Mutation Test

Concentrations



### μ g/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

D istilled 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 μ g) Mean 304.7

-- -- -- -- -- -- -- -- --

MF 14.06

-- -- -- -- -- -- -- -- --

2AA (2 μ g) Mean

-- 2306.7

-- 2230.7

-- 190.3

-- 184.7

-- --

MF

-- 69.90

-- 19.62

-- 13.60

-- 20.52

-- --

2AA (50 μ g) Mean

-- -- -- -- -- -- -- -- -- 384.7

MF

-- -- -- -- -- -- -- -- -- 9.16

SAZ (2 μ g) Mean

-- -- 1496.7

-- 1304.0

-- -- -- -- --

MF

-- -- 18.18

-- 111.77

-- -- -- -- --

9AA (50 μ g) Mean

-- -- -- -- -- -- 505.3

-- -- --

MF

-- -- -- -- -- -- 94.75

-- -- --

MMS (2 μ L) Mean

-- -- -- -- -- -- -- -- 121 7.3

--

MF

-- -- -- -- -- -- -- -- 45.65

--

## STUDY CODE: 11/076-007M Page 3 of Appendix 2



# LAB Table 9: Summary Table of the Confirmatory Mutation Test

Concentrations



### μ g/plate) 

### Mean

values of

revertants /

Mutation

factor (MF) Salmonella typhimurium tester strains Escherichia

coli

TA98 TA100 TA1 535 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 3 9.0

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 4 2.0 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 μ g) Mean 300.0

-- -- -- -- -- -- -- -- --

MF 10.71

-- -- -- -- -- -- -- -- --

2AA (2 μ g) Mean

-- 2366.7

-- 1769.3

-- 217.0

-- 246.7

-- --

MF

-- 59.17

-- 16.74

-- 15.88

-- 27.41

-- --

2AA (50 μ g) Mean

-- -- -- -- -- -- -- -- -- 255.0

MF

-- -- -- -- -- -- -- -- -- 8.14

SAZ (2 μ g) Mean

-- -- 1772.0

-- 1194.7

-- -- -- -- --

MF

-- -- 25.56

-- 108.61

-- -- -- -- --

9AA (50 μ g) Mean

-- -- -- -- -- -- 513.3

-- -- --

MF

-- -- -- -- -- -- 128.33

-- -- --

MMS (2 μ L) Mean

-- -- -- -- -- -- -- -- 95 3.3

--

MF

-- -- -- -- -- -- -- -- 39.18

--

## STUDY CODE: 11/076-007M Page 1 of Appendix 3



# LAB Preliminary Range Finding Test (Informatory Toxicity Test)

Test Item :

Date : 25-27 May 2011

Strain : Salmonella typhimurium TA98

Cell count (overnight culture): 1.59x 10 9 

## CFU/mL

Revertant colo ny 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 3 5

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 deviatio n

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

directly to the test tube MF 0.85 1.40

## STUD Y CODE: 11/076-007M Page 2 

## of Appendix 3



# LAB 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 10 9 

## 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 1 798 (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

Mut ation 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

Cel l count (overnight culture): 1.79 x 10 9 

## CFU/mL

Revertant colony number Revertant colony number

Concentration:

- S9 +S9 Concentration:

- S9 +S9

(μg/plate) (μg/plate)

5000 40 41 Untreated cont rol 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 Aceton e 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

. 1 7 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 cont rol)

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 10 9 

## 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

Me an 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 c ontrol** 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 Aceton e 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 (solven t 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 10 9 

## CFU/mL

Revertant colony number Revertant colony number

Concentration:

- S9 +S9 Concentration:

- S9 +S9

(μg/plate) (μg/plate)

50 00 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

. 6 6 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 Aceton e 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= mea n 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: w ith 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 Incorp oration Method)

Test Item :

Date : 01-03 June 2011

Strain : Salmonella typhimurium TA1537

Cell count (overnight culture): 1.76 x 10 9 

## CFU/mL

Revertant colony number Revertant colony number

Concentration:

- S9 +S9 Con centration:

- 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 Aceton e 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

M ean 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: Mutatio n 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 10 9 

## 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

2 4 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 Aceton e con trol 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

5 0 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

Cel l count (overnight culture): 2.13 x 10 9 

## CFU/mL

Revertant colony number Revertant colony number

Concentration:

- S9 +S9 Concentration:

- S9 +S9

(μg/plate) (μg/plate)

5000 33 37 Untreated cont rol 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 Aceton e 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 Mutati on Test (Pre

- Incubation Method)

Test Item :

Date : 07-09 June 2011

Strain : Salmonella typhimurium TA100

Cell count (overnight culture): 2.77 x 10 9 

## 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 Aceton e 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 con trol 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 contr ol 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 10 9 

## 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 Distil led 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 Aceton e 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

Cel l count (overnight culture): 2.32 x 10 9 

## CFU/mL

Revertant colony number Revertant colony number

Concentration:

- S9 +S9 Concentration:

- S9 +S9

(μg/plate) (μg/plate)

5000 4 10 Untreated contr ol 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 Aceton e 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 10 9 

## 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

S D 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 Aceton e 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 contro l 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 T A1537 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 activati on (

- 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

r evertants 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,

TA153 5: 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