### TS-CKHI02



## Attachment #14

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

POLYMER

WITH BACTERIAL

REVERSE MUTATION ASSAY



## Date of Final Report: 15 April 2011



## STUDY CODE: 10/339-007M

## STUDY CODE: 10/339-007M Page 5 of 33



# LAB GENERAL INFORMATION

STUDY TITLE : The Testing of Polymer with

Bacterial Reverse Mutation Assay

TEST ITEM : Polymer

MANUFACTURER

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 Ch emicals, No. 471

EPA Health Effects Test Guidelines, OPPTS 870.5100

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

STUDY PLAN : 25 January 2011

AMENDMENT 1 TO THE S TUDY P LAN : 07 February 2011

RANGE FINDING TEST : 26-28 January 2011

EXPERIMENTAL PERIOD I. : 08-10 February 2011

EXPERIMENTAL PERIOD II

- III . : 15-17 February 2011

DRAFT REPORT : 25 March 2011

FINAL REPORT : 15 April 2011

## STUDY CODE: 10/339-007M Page 6 of 33



# LAB RESPONSIBLE PERSONS :

LAB MANAGEMENT : Christopher Banks, DABT

Managing Director

Alyson Leyshon, M.Sc.

Senior Director of Toxicology Operations

QUALITY ASSURANCE : Szabolcs Gáty , M.S c.

Head of QAU

Brigitta Barkó

, M.Sc.

Deputy Head of QA

Éva Fábiánné Makovi, B.Sc.

QA Inspector

STUDY DIRECTOR : Judit Hargitai , PhD

Phone: +36 88 545 273

E- mail: judit.hargitai@labresearch.hu

SCIENTIFIC ASSISTANT : István Ágh, M.Sc.

Kriszt in a Sipos, M.Sc.

TECHNICAL LEADER : Tamás Buda

CENTAL DISPENSARY : Tamás Mészáros, PhD

(Technical Team Leader)

TECHNICAL ASSISTANTS : Tímea Kovácsné Böröcz

E mma Petőné Kohán

Ákos Klaffl

## STUDY CODE: 10/339-007M Page 7 of 33



# LAB TABLE OF CONTENTS

Page

STATEMENT OF THE STUDY DIRECTOR 2

STATEMENT OF THE MANAGEMENT 3

QUALITY ASSURANCE STATEMENT 4

1. SUMMARY 9

2. INTRODUCTION 11

3. OBJECTIVE OF STUDY 11

4. GUIDELINES 12

5. MATERIALS AND METHOD S 12

5.1. TEST ITEM 12

5.2. POSITIVE AND NEGATIVE CONTROLS 14

5.3. BACTERIAL STRAINS 15

5.4. MEDIA 18

5.5. METABOLIC ACTIVATION SYSTEM 20

5.6. DESCRIPTION OF THE TEST PROCEDURE 22

5.7. EVALUATION OF EXPERIMENTAL DATA 25

5.8. ARCHIVES 26

5.9. CHEMICALS USE D IN THE EXPERIMENTS 26

5.10. DEVIATIONS TO THE STUDY PLAN 27

6. RESULTS AND DISCUSSI ON 28

6.1. PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY

TEST) 28

6.2. INI TIAL AND CONFIRMATORY MUTATION TESTS 29

6.3. VALIDITY OF THE TEST S 31

7. CONCLUSION 31

8. DISTRIBUTION OF THE FINAL REPORT 32

9. REFEREN CES 32

APPENDICES 33

Copy of the GLP Certificate 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 Incorp oration Method) 1- 4 of APPENDIX 4

## STUDY CODE: 10/339-007M Page 8 of 33



# LAB Complementary Initial Mutation Test

(Plate Incorporation Method) 1- 1 of APPENDIX 5

Confirmatory Mutation Test (Pre

- Inc ubation Method) 1- 5 of APPENDIX 6

Historical Cont rol Data 1- 1 of APPENDIX 7

LIST OF TABLES

Page

Table 1: Formulation of the Test Item for the Main Experiments 13

Table 2: Strain

- Specif ic Positive Control Chemicals 14

Table 3: Genotypes of the Strains Used for Mutageni city Testing 16

Table 4: The Solubility of the Test Item in PEG 400 23

Table 5 : Summary of Controls 24

Table 6: Chem icals Used in the Experiments 27

Table 7 : Summary T able of the Ra nge Finding Test 1 of APPENDIX 2

Table 8 : Summary T able of the Initial Mutation Test

and Complementary Initial Mutation Test 2 of APPENDIX 2

Table 9 : Summa ry T able of the Confirmatory Mutation Test 3 of APPENDIX 2

## STUDY CODE: 10/339-007M Page 9 of 33



# LAB 1. SUMMARY

The test item Polymer was tested for potential mutagenic activity

using the Bacterial Reverse Mutation Assay

.

The experiments were carried out using histi dine- 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 mitochond rial

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

The study included a Preliminary Range Finding Test (Informatory Toxicity Test), an

Initial Muta tion Test (Plate Incorporation Method ) , a Complementary Initial Mutation

Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre -Incubation

Method).

Based on the results of a solubility t est, the test item formulation s w ere prepared using

PEG 400 . 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 in the performed main experiments (Initial Mutation Test

, Complementary Initial Mutation Test and Confirmatory Mutati on Test) were: 5000;

1581 ; 5 00; 158.1 ; 50 and 15.81 μg/plat e.

In the Initial Mutation Test , Complementary Initial Mutation Test and Confirmatory

Mutation Test, none of the observed revertant colony numbers were above the

respective biological threshold va lue. 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 with in the

historical control range and were within the normal biological variability of the test

system

.

Slight cytotoxic, inhibitory effect of the test item was observed in Salmonella

typhimurium TA98, TA1535 and TA1537 strains at 5000 μ g/plate without metabolic

activation , furthermore in Salmonella typhimurium TA100 bacterial strain with and

without metabolic activation in the Confirmatory Mutation Test

.

Precipitate was observe d in the Confirmatory Mutation Test in all of the examined

bacterial strains at 5000 μ g/plate concentration with and without metabolic activation.

## STUDY CODE: 10/339-007M Page 10 of 33



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

historical 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 mutagen icity assay show (see Appendix 2 to 5 ) that u n der the

experimental conditions applie d 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 Polymer ha d no mutagenic activity

on the growth of the bacterium tester strains under the test conditions used in this

study.

## STUDY CODE: 10/339-007M Page 11 of 33



# 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 S almonella 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 mu tagenic 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 ite m.

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

E

scherichia coli WP2 uvrA tryptophan (trp) reversion system measures trp - 

## → trp +



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

- pair

subst itutions (AT to GC)

.

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

known to convert promutagens into mutagenic 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

acti vation 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 **β**

naphthoflavone, two indu cers 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

Polymer 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: 10/339-007M Page 12 of 33



# LAB 4. GUIDELINES

This study followed the procedures indicated b y 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 21 st 

## July, 1997

– EPA Health Effects Test Guidelines, OPPT S 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)

– Commis s ion 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 Autho rities

including METI, MHLW and MAFF.

5. MATERIALS AND METHOD S

5.1. TEST ITEM

5.1.1. Name and Data of Test Item *

Substance name: Polymer

Chemical Name:

Batch number:

Molecular weight: Mw = 130000 Da (Mw < 2000 Da below 0.1%)

Purity : 98.6 %

Description of the test item: White waxy solid

Manufacture date: 24 August 2010

Expiry date : 23 August 2012

* Based on the Test Item Data Sheet (dated 17 December 2010) and Certificate of Analysis (dated 10

January 2011) provided by the Sponsor.

## STUDY CODE: 10/339-007M Page 13 of 33



# LAB Storage condition: Room temperature (15-30 o 

## C)

Safety Precautions: Routine safety precautions (lab coa t, gloves, safety

glasses , face mask) for unknown materials w ere

applied to assure personnel health and safety.

5.1.2. Identification, Receipt

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

p recautions required in th e hand ling 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).

5.1.3. Formulation

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

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

Because of thi s test item was not soluble in any physiologically compatible solvents,

therefore a fine suspension was used instead of a solution

. All dilutions of test item were

made in the testing laboratory using PEG 400. For the stock formulations in the main

tests, the appropriate amount of test item was homogeni z ed in a ball mill using the

appropriate amount of vehicle. The resulting suspension was further diluted with the

vehicle to prepare the test solutions. All the stock formulations and test solutions were

cont inuously mixed during the treatment. Test formulation s were freshly prepared at

the beginning of the experiments in the testing laboratory

.

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 50 00

2 31.62 mg/mL 1581

3 10 mg/mL 500

4 3.162 mg/mL 1 58.1

5 1 mg/mL 50

6 0.3162 mg/mL 15.81

## STUDY CODE: 10/339-007M Page 14 of 33



# LAB 5.2. POSITIVE AND NEGATIVE CONTROLS

Positive, negative (solvent) and untreated controls were included in the experiments.

Routine safety precautions (lab coat, gloves, safety glasses and face mask) were

applied to assure personnel health and safety.

5.2.1. Positive Controls

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

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

summari z ed 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 (NP D)

MERCK 

### S5061894 /

28 Feb 2013 DMSO 4 μg Salmonella

TA98

non

- activation Sodium azide (SAZ)

SIGMA -ALDRICH 

### MKAA3815 /

30 Jan 2011 Distilled

water 2 μg Salmonella

TA100

TA1535

non

- activation Sodium azide (SAZ)

SIGMA -ALDRICH 

### MKBF0623 /

02 Feb 201 2 Distil led

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 201 1 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: Salmone lla : Salmonella typhimurium , E.coli: Escherichia coli

## STUDY CODE: 10/339-007M Page 15 of 33



# LAB 5.2.2. Negative Controls (Solvent Controls)

In the study t hree ve hicle 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:

PEG 400:

Supplier: Sigma -Aldrich Co.

Batch No.: 1435799

Expiry date: February 2011

Dimethyl sulfoxide (DMSO) :

Supplier: Sigma -Aldrich Co.

Batch No.: BCBC5888V

Expiry date: May 2016

Distilled water

5.3. BACTERIAL STRAINS :

Produced by: TEVA Hungary Co.

Batch No.: 7530810

Expiry date: August 201 3

5.3.1. Origin

Date of arrival and origin of the strains used in the preliminary experiment:

Salmonella typhimurium TA98 04 December 2008, BioReliance Corporation

, Rockville, Maryland, USA

Salmonella typhimurium TA100 04 December 2008, BioReliance Corporation

, Rockville, Maryland, USA

## STUDY CODE: 10/339-007M Page 16 of 33



# LAB Date of arrival and origin of the strains used in the main tests :

Salmonella typhimurium TA98 16 No vember 2010, MOLTOX

- Molecular Toxico

logy Inc., Boone, North Carolina, USA

Salmonella typhimurium TA100 16 November 2010, MOLTOX

- Molecular Toxico

logy Inc., Boone, North Carolina, USA

Salmonella typhimurium TA1535 16 November 2010, MOLTOX

- Molecular T oxico

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

- prone "SOS" DNA repair pathway induced by DNA dam age.

This plasmid also carries an ampicillin resistance transfer factor (R

- factor) which is

used to identify its presence in the cell. The Escherichia coli strain used in this test

(WP2 uvrA ) is a lso defective in DNA excision repair. The genotypes of the t ester 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.typh imurium

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

## STUDY CODE: 10/339-007M Page 17 of 33



# LAB 5.3.3. Storage

The strains are stored at

- 80 **±** 10oC in the Culture Collection of the Microbiological

Lab oratory of the LAB Research Ltd. Frozen permanent c ultures of the tester strains

wer e prepared from fresh, ov ernight cultures to which DMSO wa s added as a

cryoprotective agent.

5.3.4. Confirmation of Phenotypes of Tester Strains

The phenotypes of the t ester 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 are 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.

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 (at Escherichia coli WP2 uvrA

tryptophan) independence is mea s ured routinely in mutagenicity experiments and

expressed as the number of spont a neous revertants per plate. H istorical control values

for spontaneous revertants (revertants/plate) for untreated control sample without

metabolic activation were in the period of 2004 to 2009 were (as guide) as follows:

Salmonella typhimurium TA98: 9

- 54, TA100: 58 -211, TA1535: 2 -31 , TA1537: 1 -24,

Escherichia coli WP2 uvrA : 9 -66. More detailed historical control data are shown in

Appendix 7.

5.3.6. Procedure for Growing Cultures

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

w ere 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 s haker.

## STUDY CODE: 10/339-007M Page 18 of 33



# LAB 5.3.7. Viability of the Testing Cultures

The viability of each testing culture was determined by plating 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 T able 6 (Se ction 5 .9. Chemicals Us ed in the

Experiments ). The composition of media refers to 1000 mL.

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

Glu cose 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. ad 1 

## 1000 mL

Minimal gl ucose agar plates [MER CKOPLATE] ( Batch Number: 04329 , Expiry date:

29 March 2011 used in the p reliminary Range Finding Test, Complementary Initial

Mutation Test and Confirmatory Mutation Test ; and Batch Number: 04 331 , Expiry

date: 30 March 2011 used in the Initial Mutation Test ) were provided by Merck.

Certificate s of Analysis w ere obtained from the Supplier.

5.4.2. Nutrient Broth No.2

Nutrient Broth No.2. 25.0 g

Distilled wa ter q.s. ad 1000 mL

Sterilization was performed at 121 o 

## C in an autoclave.

Note: Due to technical reason, the Nutrient Broth No.2 was prepared in the following way in the Initial

Mutation Test: 10.0 g of LAB Lemco Powder, 10.0 g of Peptone, 5.0 g of Sodiu m chloride dissolved in

Distilled water (final volume 1000.0 mL).

1 

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

## STUDY CODE: 10/339-007M Page 19 of 33



# LAB 5.4.3. Nutrient Agar

Nutrient Agar 20.0 g

Distilled water q.s. ad 1000 mL

Steriliz ation was performed at 121 o 

## C in an autoclave.

5.4.4. Top Agar for Salmonella typhimurium Strains

Ag ar solution:

Agar Bacteriological 4.0 g

NaCl 5.0 g

Distilled water q.s. ad 1000 mL

Sterili zation was performed at 121 o 

## C in an autoclave.

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

Di stilled water q.s. ad 1000 mL

Steriliz ation was performed by filtration using a 0.22 **μ** m 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

S teriliz ation was performed by filtration using a 0.22 **μ** m membrane filter.

Complete Top Aga r 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

## STUDY CODE: 10/339-007M Page 20 of 33



# LAB 5.5. METABOLIC ACTIVATION SYSTEM

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

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

fraction.

The post

- mitoch ondrial fraction (S9 fraction) wa s 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

section s 5 .5.3. and 5.5.4. are summarized in Table 6 (Section 5.9. Chemicals Used in

the Experiments). The composition of solution refer s to 1000 mL.

5 .5.1

. Induction of Liver Enzymes

Male Wistar rats ( 275

- 319 g, animals were 8 weeks old at the initiation) we re 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 concen tration 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 20 September

2010.

5.5.2. Preparation of Rat Liver Homogenate S9 Fraction

On Day 4, t he 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 3 4.2 g/L. The date of

preparation of S9 fraction for this study was 23 September 2010 (LAB code: E10 891 ).

## STUDY CODE: 10/339-007M Page 21 of 33



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

mutagens 2

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

by microsomal e n zymes. 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

MgCl 2 

### x 

## 6 H 2 

## O 4.07 g

KCl 6.15 g

Distilled water q.s. ad 1 000 mL

Sterilization was performed by filtration through a 0.22 **μ** m membrane filter.

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 homogena te (S9) 100 mL

Salt solution for S9 Mix (see above) 400 mL

Prior to addition to the culture medium the S9 M ix 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

St eriliz ation 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

Steriliz ation was performed at 121 o 

## C in an autoclave.

Sodium phosphate buffer pH 7.4:

Solution A 880 mL

Solution B 120 mL

## STUDY CODE: 10/339-007M Page 22 of 33



# LAB 5.6. DESCRIPTIO N OF THE TEST PROCED URE

The s tudy included a Preliminary Range Finding Test (Informatory Toxicity Test), an

Initial Mutation Test , a Complementary Initial Mutation Test and a Confirmatory

Mutation Test

. In the Range Finding Test as well as in the I nitial and Complementary

Initial Mutation Test

, the p late i ncorporatio n method was used. In the Confirmatory

Mutation Test, the p re -incubation method was used.

5.6.1. Concentrations

C oncentrations w ere selected on the basis of the Preliminary R ange Finding T est

(Informatory Toxicity Test). In the Initial Mutation Test

, Complementary Initial

Mutation Test and Confirmatory Mutation Test, the same concentrations were used

.

5 .6.1.1. Preliminary Solubility Test

The solubility of the test item was examined in a preliminary experiment in the

following solvents (LAB Study code: 10/339-001P):

1. Distilled water

2. PEG 400

3. Dimethyl sulfoxide (DMSO)

4. N,N

- Dimethylformamide (DMF)

5. Acetone

6. Ethanol

7. Glyceryl trioctanoate

8. Sunflower oil

9. Paraffin oil

10. n- Hexane

11. HCl

The test item was insoluble in the examined solvents. The test item was also

homogeniz ed in a ball mill using DMSO or PEG 400. The resulting DMSO

formulation was a sticky solid formulation; however, the use of PEG 400 resulted a

suitable formulation (suspension). Therefore, PEG 400 was chosen for vehicle of the

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

and phosphate buffer ( 5 .5.4.) was examined in a test tube wit hout test bacterium

suspension. The results of this compatibility test are summarized in Table 4.

## STUDY CODE: 10/339-007M Page 23 of 33



# LAB Table 4 : The Solubility of the Test Item in PEG 400

Concentration

of test item

in PEG 400

(mg/mL) Solubility

in PEG 400 Solubility in the top

solution

(test item solution 5 0 μL

+ phosphate buffer 500 μL

+ top agar 2 mL) Test i tem

concentration in

the test tube

**μ** g/tube

100 suspension suspension 5000



## 5 .6.1.2. Concentration Range Finding Test (Informatory Toxi city Test)

Based on the solubility test, 100 mg/mL stock formulation was prepared in PEG 400

, 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.

5 .6.1.3. Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test

, Complementray Initial Mutation Test and Confirmatory Mutation Test)

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

prepared from the test item with PEG 400 , which was diluted by serial dilutions in five

steps to obtain a total of s ix dosing solutions

. The maximum test concentration was

5000 μ g test item/plate.

Examined P olymer con centrations were : 5000 ; 1581 ; 500 ; 158.1 ; 50

and 15.81 μ g/plate

.

5.6.2. Control Groups Used in the Tests

Strain

- specific positive and negative ( 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 summariz ed in Table 5

.

## STUDY CODE: 10/339-007M Page 24 of 33



# LAB Table 5 : Summary of Controls

Type of control Activation

with S9 Mix Solvent Top

Aga r Strain

- Specifi c

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 Expos ure in the Initial Mutation Test and Complementary Initial Mutation

Test

A standard plate incorporation procedure was performed, as an Initial Mutation T est and

Complementary Initial Mutation Test . Bacteria (cultured in Nutrient Broth No.2. as

described in S ection 5 .3.6.) were exposed to the test item both in t he 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 label l ed. The test item and other

components were prepared fresh ly and added to the overlay (45°C).

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

## STUDY CODE: 10/339-007M Page 25 of 33



# LAB 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 enti re 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 Expos ure in the Confirmatory Mutatio n Test

A pre

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

T est 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 en tire 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 o n

the vehicle control plate

.



## Criteria for a Positive Response:

A test item wa s considered mutagenic if:

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

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

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

## STUDY CODE: 10/339-007M Page 26 of 33



# LAB An increase wa s considered biologically relevant if:

- if in Salmonella typhiumurium TA100 strain the number of reversion wa s more

than twice high er when compared to the spontaneous reversion rate of the solvent

control plates,

- if in Salmonella typhiumur ium TA98, TA1535, TA1537 and Escherichia coli

WP2 uvrA strains the number of reversions wa s 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 meth od 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 wa s 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 pl an 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 S ponsor's prior

written consent.

5.9. CHEMICALS USED IN TH E EXPERIMENTS

The chemicals used in the independently pe rformed experiments of the study are

summariz ed in Table 6.

## STUDY CODE: 10/339-007M Page 27 of 33



# LAB 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 119K1533 November 2013

Distilled water HungaroPharma 8000111 10 July 2011

Distilled water HungaroPharma 8141010 26 April 2011

Distilled water HungaroPharma 8161210 07 Jun e 2010

D



### - Glucose 6- pho s phate Na SIGMA

- ALDRICH 079K3788 Aug ust 2012

L- Histidine HCl x H 2 

### O MERCK K37522450 30 June 2012

KCl MERCK K40299136 31 July 2014

KCl MERCK K41042236 31 May 2015

LAB

- Lemco powder OXOID 442051 31 March 2 011

MgCl 2 

### x 6 H 2 

### O SIGMA

- ALDRICH 019K0038 January 2014

NaCl Lachner PP/2010/03837 31 March 2013

NADP Na x H 2 

### O SIGMA

- ALDRICH 077K7000 April 2011

NaH 2 

### PO 4 

### REANAL KBM5207601 July 2011

Na 2 

### HPO 4 

### x 12H 2 

### O REANAL KBM60552 January 2015

Na 2 

### HPO 4 

### x 12H 2 

### O REANAL KBM 61668 August 2015

Nutrient Agar MERCK VM100650 06 October 2014

Nutrient Broth No.: 2 OXOID 907248 30 June 2015

Peptone , S oybean SIGMA

- ALDRICH 1420831 30 August 2011

L- Tryptophan SIGMA

- ALDRICH 1400132 July 2012



## 5.10. DEVIATION S TO THE STUDY PLAN

Co ncerning: Participants of the study

According to the Study Plan: Krisztina Pátkainé Fejes will participate in the study

Deviation: Krisztina Pátkainé Fejes did not participate in the

study

Reason: Technical reason

Presumed effect: None

## STUDY CODE: 10/339-007M Page 28 of 33



# LAB Concerning: St udy schedule

According to the Study Plan: Initial Mutation Test will be ended on 10 February

2011

Deviation: Initial Mutation Test was ended on 17 February 2011

Reason: Experiment in Salmonella typhimurium TA100

strain was invalid (no revertants grew on any of the

plates) and an additional experiment was performed

to complete the data

Presumed effect: Positive (the additional experiment assured the

validity of the study)

Concerning: The composition of Nutrient Broth No.2

According to the Study Plan: N utrient Broth No.2.: 25.0 g

Distilled water: ad 1000.0 mL

Deviation: Nutrient Broth No. 2 was prepared in the

Confirmatory Mutation Test in the following way:

LAB Lemco Powder: 10.0 g

Peptone: 10.0 g

Sodium chloride: 5.0 g

Distilled wate r: ad 1000.0 mL.

Reason: Technical reason

Presumed effect on the study: None (the preparation followed the typical formula

of the Nutrient Broth No.2 given on the container of

the chemical)

These deviations were considered not to adversely affect the results or integrity of the

study.

6. RESULTS AND DISCUSSI ON

6.1. PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)

In the P reliminary Range Finding Test

, the plate incorporation method was used. The

preliminary test was performed using Salm onella typhimurium TA98 and Salmonella

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

system (±S9 Mix ) with appropriate untreated, negative (solvent) and positive controls.

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

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

100, 31.6 and 10 μ g/plate

.

## STUDY CODE: 10/339-007M Page 29 of 33



# LAB The observed numbers of revertant colonies were mostly in the normal range. Slightly

h igher numbers of revertant colonies compared to the solvent control plates were

observed in Salmonella typhimurium TA 98 and TA100 tester strain s in some cases

with or without metabolic activation . However, those differences were minor, th e y

w ere well below the threshold limit, t here was no dose response and the mean

numbers of revertants was in the historical control range in each cases

.

Precipitat e was observe d in the Preliminary Range Finding Test in both examined

strains at 5000 μg/plate concentration with and without metabol ic activation.

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

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

6.2. INITIAL AND CONFIRMATORY MUTATION TESTS

In the Initial Mutation Test an d Complementary 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 w ere carried out

using Salmonella typhimurium strains (TA 98, TA100, TA1535

, TA1537) and

Escherichia coli WP2 uvrA strain

. The Complementary Initial Mutation Test* were

carried out using Salmonella typhimurium TA100 strain.

*Note: I n the Initial Mutation Test, no revertants were observed on the test item treated , untreated,

negative and positive control plates in Salmonella typhimurium TA100 tester strain, thus the evaluation

could not be performed

. Th erefore , this part of the test was considered invalid. An additional

experiment (Complementary Initial Mutation T est) was performed in a new experiment al period to

complete the data.



## Each test was performed in the presence and absence of metabolic activation system

(±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. In the

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

* Note: In Complementary Initial Mutation Test, in Salmonella typhimurium TA1 00 at solvent control

with metabolic activation one of the replicates was infected. Mean values and mutation factors we re

calculated in these cases using the data of the remaining two replicates.



## The examined test item concentrations in the main tests were: 5000; 1581; 500 ; 158.1 ;

50 and 15.81 μg/plate

.

In the Initial Mutation Test , Complementary 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: 10/339-007M Page 30 of 33



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

Initial Mutation Test in Salmonella typhimurium TA 1535 tester strain with metabolic

activation at the concentration s of 50 0 and 158 1 μ g/plate . The mutation factor value

w as 1.73. Higher numbers of revertants were observed at other tested concentrations

in this experiment

. However, no dose

- dependence was observed, the observed

mutation factor value s were below biologically relevant thres hold value and the

numbers of revertant colonies were within the historical control range in all cases

. Furthermore, higher number of revertants compared to the PEG 400 solvent control

was observed for untreated control (MF: 1.36) and DMSO control (MF: 1.7 3) a lso in

this experiment.

Using the pre

- incubation method, highe st revertant rate w as observed in the

Confirmatory Mutation Test also in Salmonella typhimurium TA 1535 bacterial strain

with metabolic activation at the concentration of 5000 μ g/plate . The mutation factor

value was 2.76. Higher numbers of revertants were observed at other tested

concentrations in this experiment . However, no dose

- dependent relationship was

observed, and th e observed mutation factor value remained below the biol ogical ly

relevant threshold value. The number of revertant colonies w as within the historical

control range. Furthermore, higher number of revertants compared to the PEG 400

solvent control was observed for untreated control (MF: 1.52 ) and DMSO control

(MF : 1.40) also in this experiment.

Sporadically, higher number s of revertant colonies compared to the solvent control

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

case s. 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 coun ts compared to the solvent control were observed in

the Initial Mutation Test and Confirmatory Mutation Test in some other cases.

However, the differences very minor and the mean numbers of revertant colonies were

in the historical control range in all cas es , thus they were considered as biological

variability of the test system

.

Slight cytotoxic, inhibitory effect of the test item (reduced / slightly reduced

background lawn development ) was observed in Salmonella typhimurium TA98,

TA1535 and TA1537 strain s at 5000 μ g/plate without metabolic activation

, furthermore in Salmonella typhimurium TA100 bacterial strain with and without

metabolic activation in the Confirmatory Mutation Test.

Precipitate was observe d in the Confirmatory Mutation Test in all of the examined

bacterial strains at 5000 μ g/plate concentration with and without metabolic activation.

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

s tandard deviations) are summarized in Table s 8-9 (Appendix 2) and Append ices 4 , 5

and 6.

## STUDY CODE: 10/339-007M Page 31 of 33



# LAB 6.3. VALIDITY OF THE TEST S

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

values of revertant colony numbers of untreated and DMSO and Distilled water

solvent control plates were within the histori cal control data range. No historical

control database exists for PEG 400; however, the observed data for this solvent were

in the historical control range of other solvents, therefore were considered to be

acceptable.

The reference mutagens showed a dist inct 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.

7. CONCLUSION

The test item P olymer was tested for potential mutag enic 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 auxotrop h strain of Escherichia coli

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

system, which wa s a cofactor

- supplemented post

- mitochondrial S9 fraction prepared

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

The study included a Preliminary Range Finding Test (Informatory Toxicity Test), an

Initial Mutation Test , a Complementary Initial Mutation Test and a Confirmato ry

Mutation T est . In the Range Finding Test as well as in the Initial and Complementary

Initi al 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 6 ) that u n der the

experimental conditions applied the te st 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 Polymer ha d no mutagenic activity

on the growth of the bacterium tester strains under the test conditi ons used in this

study.

## STUDY CODE: 10/339-007M Page 32 of 33



# LAB 8. DISTRIBUTION OF THE FINAL REPORT

Sponsor: 1x copy, bound

1x copy, unbound

1x PDF file

Archive: 1x original, bound

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 Ag ency 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, Bacterial R e verse

Mu tation Test, No.: 471, Paris. 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 Te st ” 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. 3 0) Eü M- 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: 10/339-007M Page 33 of 33



# LAB 

# APPENDICES

## STUDY CODE: 10/339-007M Page 1 of Appendix 1

COPY OF THE GLP CERTIFICATE

## STUDY CODE: 10/339-007M 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 thyphimurium tester

strains

TA 98 TA 100

- S9 +S9

- S9 +S9

Untreated

control Mean 26.0 30.0 128.3 1 39.0

MF 1.11 0.99 1.02 1.07

Distilled water

control Mean

-- -- 134.3

--

MF

-- -- 1.06

--

DMSO

control Mean 22.3 31.0

- 149.3

MF 0.96 1.02

- 1.15

PEG400

control Mean 23.3 30.3 126.3 129.3

MF 1.00 1.00 1.00 1.00

5000 Mean 31.0 30.7 160.7 153. 0

MF 1.33 1.01 1.27 1.18

2500 Mean 32.0 33.3 162.0 177.7

MF 1.37 1.10 1.28 1.37

1000 Mean 22.0 31.3 164.0 149.0

MF 0.94 1.03 1.30 1.15

316 Mean 29.3 29.3 166.0 152.7

MF 1.26 0.97 1.31 1.18

100 Mean 26.7 26.0 164.0 160.3

MF 1.14 0.86 1.30 1.24

31.6 Mean 22.7 30.0 133.0 159.3

MF 0.97 0.99 1.05 1.23

10 Mean 30.0 31.7 150.7 166.3

MF 1.29 1.04 1.19 1.29

NPD (4 **μ** g) Mean 299.7

-- -- --

MF 13.42

-- -- --

2AA (2 **μ** g) Mean

-- 2321.3

-- 2210.7

MF

-- 74.88

-- 14.80

SA Z (2 **μ** g) Mean

-- -- 1552.0

--

MF

-- -- 11.55

--

## STUDY CODE: 10/339-007M Page 2 

## of Appendix 2



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

Complementary Initial Mutation Test

Concentrations

**(μ** g/plate) 

### Mean

values of

revertants /

Mutation

factor (MF) Salmonella thyphimurium tester strains Escherichi a

coli

TA98 TA100 TA1535 TA1537 WP2 uvrA

- S9 +S9

- S9 +S9

- S9 +S9

- S9 +S9

- S9 +S9

Untreated

control Mean 25.3 27.7 137.7 148.0 6.7 10.0 2.7 3.3 17.3 29.7

MF 1.10 0.90 0.98 0.90 0.74 1.36 0.73 0.67 0.96 1.16

DMSO

control Mean 20.0 24.0

-- 149.7

-- 12.7 2.7 3.7

-- 30.0

MF 0.87 0.78

-- 0.91

-- 1.73 0.73 0.73

-- 1.17

Distilled water

control Mean

-- -- 147.7

-- 8.7

-- -- -- 26.3

--

MF

-- -- 1.05

-- 0.96

-- -- -- 1.46

--

PEG 400

control Mean 23.0 30.7 140.3 164.0 9.0 7.3 3.7 5.0 18.0 25.7

MF 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

5000 Mean 27.3 28.0 131.0 160.0 7.0 12.0 5.0 2.3 22.3 25.7

MF 1.19 0.91 0.93 0.98 0.78 1.64 1.36 0.47 1.24 1.00

1581 Mean 26.3 24.7 128.0 152.3 7.7 12.7 3.0 4.3 23.3 29.3

MF 1.14 0.80 0.91 0.93 0.8 5 1.73 0.82 0.87 1.30 1.14

500 Mean 30.3 31.3 140.3 153.0 6.3 12.7 4.7 5.7 25.3 22.0

MF 1.32 1.02 1.00 0.93 0.70 1.73 1.27 1.13 1.41 0.86

158.1 Mean 25.3 35.3 131.7 154.3 8.3 12.0 5.0 4.3 24.3 27.0

MF 1.10 1.15 0.94 0.94 0.93 1.64 1.36 0.87 1.35 1. 05

50 Mean 17.7 30.0 133.0 159.0 12.7 11.3 4.7 6.0 25.0 25.0

MF 0.77 0.98 0.95 0.97 1.41 1.55 1.27 1.20 1.39 0.97

15.81 Mean 20.7 30.0 129.0 136.7 10.3 10.0 3.7 3.7 21.0 32.7

MF 0.90 0.98 0.92 0.83 1.15 1.36 1.00 0.73 1.17 1.27

NPD (4 **μ** g) Mean 262 .7

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

MF 13

. 13

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

2AA (2 **μ** g) Mean

-- 2346.7

-- 3 099

. 3

-- 197.0

-- 200.3

-- --

MF

-- 97

. 78

-- 20.71

-- 15.55

-- 54.64

-- --

2AA (50 **μ** g) Mean

-- -- -- -- -- -- -- -- -- 236.7

MF

-- -- -- -- -- -- -- -- -- 7.89

SAZ (2 **μ** g) Mean

-- -- 1126.0

-- 1806.7

-- -- -- -- --

MF

-- -- 7.63

-- 208.46

-- -- -- -- --

9AA (50 **μ** g) Mean

-- -- -- -- -- -- 1008.0

-- -- --

MF

-- -- -- -- -- -- 378.00

-- -- --

MMS (2 **μ** L) Mean

-- -- -- -- -- -- -- -- 1041.3

--

MF

-- -- -- -- -- -- -- -- 39.54

--

## STUDY CODE: 10/339-007M Page 3 

## of Appendix 2



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

Concentrations

**(μ** g/plate) 

### Mean

values of

revertant /

Mutation

factor

(MF) Salmonella thyphimurium tester strains Escherichia

coli

TA98 TA100 TA1535 TA1537 WP2 uvrA

- S9 +S9

- S9 +S9

- S9 +S9

- S9 +S9

- S9 +S9

Untreated

control Mean 23.7 32.0 136.7 163.3 8.0 12.7 7.3 5.0 19.7 30.3

MF 1.31 1.04 1.63 1.22 1.26 1.52 1.83 1.07 1.26 1.26

DMSO

control Mean 19.7 28.3

- 162.7

- 11.7 5.7 7.0

- 26.3

MF 1.09 0.92

- 1.21

- 1.40 1.42 1.50

- 1.10

Distilled water

control Mean

- - 148.0

- 8.3

- - - 24.7

-

MF

- - 1.77

- 1.32

- - - 1.57

-

PEG 400

control Mean 18.0 30.7 83.7 134.3 6.3 8.3 4.0 4.7 15.7 24.0

MF 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

5000 Mean 2 4.3 31.3 70.0 109.0 7.3 23.0 4.0 8.3 16.7 18.0

MF 1.35 1.02 0.84 0.81 1.16 2.76 1.00 1.79 1.06 0.75

1581 Mean 15.3 46.0 69.3 116.3 6.7 9.3 5.3 11.3 15.0 25.7

MF 0.85 1.50 0.83 0.87 1.05 1.12 1.33 2.43 0.96 1.07

500 Mean 21.3 29.7 73.3 87.7 5.7 9.0 3.7 5.7 12.3 21.7

MF 1.19 0.97 0.88 0.65 0.89 1.08 0.92 1.21 0.79 0.90

158.1 Mean 20.7 34.0 98.7 108.0 6.3 10.3 2.7 8.7 14.0 21.3

MF 1.15 1.11 1.18 0.80 1.00 1.24 0.67 1.86 0.89 0.89

50 Mean 21.3 35.0 93.0 120.7 6.3 11.3 3.3 5.0 19.0 21.7

MF 1.19 1.14 1.11 0.90 1.00 1.36 0.83 1.07 1.21 0.90

15.81 Mean 22.0 33.0 71.7 111.0 5.7 11.3 2.0 7.0 14.7 20.7

MF 1.22 1.08 0.86 0.83 0.89 1.36 0.50 1.50 0.94 0.86

NPD (4 **μ** g) Mean 289

. 0

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

MF 14

. 69

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

2AA ( 2 **μ** g) Mean

-- 2705.3

-- 2719.3

-- 188.0

-- 182.0

-- --

MF

-- 95.48

-- 16.72

-- 16.11

-- 26.00

-- --

2AA (50 **μ** g) Mean

-- -- -- -- -- -- -- -- -- 139.7

MF

-- -- -- -- -- -- -- -- -- 5.30

SAZ (2 **μ** g) Mean

-- -- 1193.0

-- 1014.7

-- -- -- -- --

MF

-- -- 8.06

-- 121.76

-- -- -- -- --

9AA (50 **μ** g) Mean

-- -- -- -- -- -- 376.7

-- -- --

MF

-- -- -- -- -- -- 66.47

-- -- --

MMS (2 **μ** L) Mean

-- -- -- -- -- -- -- -- 906.7

--

MF

-- -- -- -- -- -- -- -- 36.76

--

## STUDY CODE: 10/339-007M Page 1 of Appendix 3



# LAB Preliminary Range Finding Test (Informatory Toxicity Test)

Test Item : Polyme r

Date : 26-28 January 2011

Strain : Salmonella typhimurium TA98

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 33 P 30 P Untreated control 22 36

30 P 33 P 34 27

30 P 29 P 22 27

Mean 31.0 30.7 Mean 2 6.0 30.0

SD 1.73 2.08 SD 6.93 5.20

MF 1.33 1.01 MF 1.11 0.99

2500 37 31 DMSO control* 18 40

25 37 30 27

34 32 19 26

Mea n 32.0 33.3 Mean 22.3 31.0

SD 6.24 3.21 SD 6.66 7.81

MF 1.37 1.10 MF 0.96 1.02

1000 27 30 PEG 400 25 27

20 31 control 24 29

19 33 21 35

Mean 22.0 31.3 Mean 23.3 30.3

SD 4.36 1.53 SD 2.08 4.16

MF 0.94 1.03 MF 1.00 1.00

316 27 28 Positive control 321 2AA 2200

27 32 NPD 295 (2 μg) 2324

34 28 (4 μg) 283 2440

Mean 29.3 29.3 Mean 299.7 2321.3

SD 4.04 2.31 SD 19.43 120.02

MF 1.26 0.97 MF 13.42 74.88

1 00 28 21

22 35

30 22

Mean 26.7 26.0 P: Precipitate

SD 4.16 7.81 MF: Mutation factor

MF 1.14 0.86 SD: Standard deviation

31.6 28 33 +S9: with S9 mix

22 34

- S9: without S9 mix

18 23

Mean 22.7 30.0 Mutation factor= mean revertants (test item)

SD 5.03 6.08 mean revertants (solvent control)

MF 0.97 0.99

10 36 33

25 33

29 29

Mean 30.0 31.7

SD 5.57 2.31

MF 1.29 1.04 *DMSO control was used because of NDP and 2AA

## STUDY CODE: 10/339-007M Page 2 

## of Appendix 3



# LAB Preliminary Range Finding Test (Informatory Toxicity Test)

Test Item : Polyme r

Date : 26-28 January 2011

Strain : Salmonella typhimurium TA100

Cel l count (overnight culture): 2.37 x 10 9 

## CFU/mL

Revertant colony number Revertant colony number

Concentration:

- S9 +S9 Concentration :

- S9 +S9

(μg/plate) (μg/plate)

5000 154 P 145 P Untreated control 120 150

171 P 152 P 133 136

157 P 162 P 132 131

Mean 160.7 153.0 Mean 128.3 139. 0

SD 9.07 8.54 SD 7.23 9.85

MF 1.27 1.18 MF 1.02 1.07

2500 164 194 DMSO control* 121 155

179 169 134 155

143 170 151 138

Me an 162.0 177.7 Mean 135.3 149.3

SD 18.08 14.15 SD 15.04 9.81

MF 1.28 1.37 MF 1.07 1.15

1000 159 159 PEG 400 

### 137 134

166 134 control 

### 121 116

167 154 

### 121 138

Mean 164.0 149.0 Mean 126.3 129.3

SD 4.36 13.23 SD 9.24 11.72

MF 1.30 1.15 MF 1.00 1.00

316 159 155 Distilled water 12 8 164

178 146 control** 147 158

161 157 128 149

Mean 166.0 152.7 Mean 134.3 157.0

SD 10.44 5.86 SD 10.97 7.55

MF 1.31 1.18 MF 1.06 1.21

100 155 165 Positive control 1480 2AA 2312

183 161 SAZ 1512 (2 μg) 2200

154 155 (2 μg) 1664 2120

Mean 164.0 160.3 1552.0 2210.7

SD 16.46 5. 03 98.31 96.44

MF 1.30 1.24 11.55 14.80

31.6 119 160

137 187 P: Precipitate

143 131 MF: Mutation factor

Mean 133.0 159.3 SD: Standard deviation

SD 12.49 28.01 +S9: with S9 mix

MF 1.05 1.23

- S9: without S9 mix

10 152 171

159 165 Mutation factor= mean revertants (test item)

141 163 mean revertants (solvent control)

Mean 150.7 166.3

SD 9.07 4.16 *DMSO control was used because of 2AA

MF 1.19 1.29 * *D istilled water control was used because of SAZ

## STUDY CODE: 10/339-007M Page 1 of Appendix 4



# LAB Initial Mutation Test (Plate Incorporation Method)

Test Item : Polyme r

Date : 08-10 Febr uary 2011

Strain : Salmonella typhimurium TA98

Cel l count (overnight culture): 1.55 x 10 9 

## CFU/mL

Revertant colony number Revertant colony number

Concentration:

- S9 +S9 Concentration:

- S9 +S9

(μg/pla te) (μg/plate)

5000 28 17 Untreated control 27 26

24 30 27 28

30 37 22 29

Mean 27

. 3 28

. 0 Mean 25

. 3 27

. 7

SD 3. 06 10

. 15 SD 2. 89 1. 53

MF 1

. 19 0

. 91 MF 1

. 10 0

. 90

1581 24 26 DMSO control* 20 16

27 23 19 30

28 25 21 26

Mean 26

. 3 24

. 7 Mean 20

. 0 24

. 0

SD 2

. 08 1

. 53 SD 1

. 00 7

. 21

MF 1

. 14 0

. 80 MF 0

. 87 0

. 78

500 33 33 PEG 400 19 34

35 36 control 25 33

23 25 25 25

Mean 30

. 3 31

. 3 Mean 23

. 0 30

. 7

SD 6

. 43 5

. 69 SD 3

. 46 4

. 93

MF 1. 32 1. 02 MF 1. 00 1. 00

158.1 32 39 Positive control 246 2AA 2312

19 33 NPD 264 (2 μg) 2480

25 34 (4 μg) 278 2248

Mean 25

. 3 35

. 3 262

. 7 2346

. 7

SD 6. 51 3. 21 16

. 04 119

. 82

MF 1

. 10 1

. 15 13

. 13 97

. 78

50 17 33

24 32

12 25 MF: Mutation factor

Mea n 17

. 7 30

. 0 SD: Standard deviation

SD 6

. 03 4

. 36 +S9: with S9 mix

MF 0

. 77 0

. 98

- S9: without S9 mix

15.81 19 30

23 29 Mutation factor= mean revertants (test item)

20 31 mean r evertants (solvent control)

Mean 20

. 7 30

. 0

SD 2

. 08 1

. 00

MF 0

. 90 0

. 98 *DMSO control was used because of NPD and 2AA

## STUDY CODE: 10/339-007M Page 2 

## of Appendix 4



# LAB Initial Mutation Test (Plate Incorporation Method)

Test Item : Polym er

Date : 08-10 Febr uary 2011

Strain : Salmonella typhimurium TA1535

Cel l count (overnight culture): 1.46 x 10 9 

## CFU/mL

Revertant colony number Revertant colony number

Concentration:

- S9 +S9 Concentration:

- S9 +S9

(μg/plate) (μg/plate)

5000 7 15 Untreated control 7 10

7 10 5 12

7 11 8 8

Mean 7.0 12.0 Mean 6.7 10.0

SD 0.00 2.65 SD 1.53 2.00

MF 0.78 1.64 MF 0.74 1.36

1581 6 12 DMSO control * 11 6

10 14 8 19

7 12 3 13

Mean 7.7 12.7 Mean 7.3 12.7

SD 2.08 1.15 SD 4.04 6.51

MF 0.85 1.73 MF 0.81 1.73

500 3 11 Distilled water 10 12

7 14 c ontrol ** 12 12

9 13 4 11

Mean 6.3 12.7 Mean 8.7 11.7

SD 3.06 1.53 SD 4.16 0.58

MF 0.70 1.73 MF 0.96 1.59

158.1 9 15 PEG 400 9 5

5 11 control 10 7

11 10 8 10

Mean 8.3 12.0 Mean 9.0 7.3

SD 3.06 2.65 SD 1.00 2.52

MF 0.93 1.64 MF 1.00 1.00

50 19 8 Positive control 1824 2AA 183

9 13 SAZ 1756 (2 μg) 208

10 13 (2 μg) 1840 200

Mean 12.7 11.3 Mean 1806.7 197.0

SD 5.51 2.89 SD 44.60 12.77

MF 1.41 1.55 MF 208.46 15.55

15.81 10 10

9 12 MF: Mutation factor

12 8 SD: Standard deviation

Mean 10.3 10.0 +S9: with S9 mix

SD 1.53 2.00

- S9: without S9 mix

MF 1.15 1.36

Mutation factor= mean revertants (test item)

mean revertants (solvent control)

*DMSO control was used because of 2AA

**Distilled water control was used because of SAZ

## STUDY CODE: 10/339-007M Page 3 

## of Appendix 4



# LAB Initial Mutation Test (Plate Incorporation Method)

Test Item : Polyme r

Date : 08-10 Febr uary 2011

Strain : Salmonella typhimurium TA1537

Cel l count (overnight culture): 2.09 x 10 9 

## CFU/mL

Revertant colony number Revertant colony number

Concentration:

- S9 +S9 Concentration:

- S9 +S9

(μg/plate) (μg/plate)

5000 3 3 Unt reated control 2 3

6 3 4 2

6 1 2 5

Mean 5.0 2.3 Mean 2.7 3.3

SD 1.73 1.15 SD 1.15 1.53

MF 1.36 0.47 MF 0.73 0.67

1581 2 2 DMSO control* 2 3

5 5 4 4

2 6 2 4

Mean 3.0 4.3 Mean 2.7 3.7

SD 1.73 2.08 SD 1.15 0.58

MF 0.82 0.87 MF 0.73 0.73

50 0 5 6 PEG 400 2 3

5 5 control 6 6

4 6 3 6

Mean 4.7 5.7 Mean 3.7 5.0

SD 0.58 0.58 SD 2.08 1.73

MF 1.27 1.13 MF 1.00 1 .00

158.1 6 5 Positive control 984 2AA 224

2 6 9AA 992 (2 μg) 196

7 2 (50 μg) 1048 181

Mean 5.0 4.3 1008.0 200.3

SD 2.65 2.08 34.87 21.83

MF 1.36 0.87 378.00 54.64

50 4 7

5 8

5 3

Mean 4.7 6.0

SD 0.58 2.65 MF: Mutation factor

MF 1.27 1.20 SD: Standard deviation

15.81 5 4 +S9: with S9 mix

4 2

- S9: without S9 mix

2 5

Mean 3.7 3.7

SD 1.53 1.53 Mutation factor= mean revertants (test item)

MF 1.00 0.73 mean revertants (solvent control)

*DMSO control was used because of 9AA and 2AA

## STUDY CODE: 10/339-007M Page 4 

## of Appendix 4



# LAB Initial Mutation Test (Plate Incorporation Method)

Test Item : Polyme r

Date : 08-10 Febr uary 2011

Strain : Escherichia coli WP2 uvrA

Cell count (overnight c ulture): 4.13 x 10 9 

## FU/mL

Revertant colony number Revertant colony number

Concentration:

- S9 +S9 Concentration:

- S9 +S9

(μg/plate) (μg/plate)

5000 29 29 Untreated control 17 24

20 20 17 29

18 28 18 36

Mean 22.3 25.7 Mean 17.3 29.7

SD 5.86 4.93 SD 0.58 6.03

MF 1.24 1.00 MF 0.96 1.16

1581 23 31 DMSO control * 20 36

26 31 16 33

21 26 30 21

Mean 23.3 29.3 Mean 22.0 30.0

SD 2.52 2.89 SD 7.21 7.94

MF 1.30 1.14 MF 1.22 1.17

500 20 20 Distilled water 21 26

25 19 c ontrol ** 26 26

31 27 32 42

Mean 25.3 22.0 Mean 26.3 31.3

SD 5.51 4.36 SD 5.51 9.24

MF 1.41 0.86 MF 1.46 1.22

158.1 21 26 PEG 400 18 27

25 25 control 18 24

27 30 18 26

Mean 24.3 27.0 Mean 18.0 25.7

SD 3.06 2.65 SD 0.00 1.53

MF 1.35 1.05 MF 1.00 1.00

50 28 20 Positive control 1200 2AA 208

25 22 MMS 908 ( 50 μg) 248

22 33 (2 μL ) 1016 254

Mean 25.0 25.0 Mean 1041.3 236.7

SD 3.00 7.00 SD 147.64 25.01

MF 1.39 0.97 MF 39.54 7.89

15.81 19 29

24 32 MF: Mutation factor

20 37 SD: Standard deviation

Mean 21.0 32.7 +S9: with S9 mix

SD 2.65 4.04

- S9: without S9 mix

MF 1.17 1.27

Mutation factor= mean revertants (test item)

mean revertants (solvent control)

*DMSO control was used because of 2AA

**Distilled water control was used because of MMS

## STUDY CODE: 10/339-007M Page 1 of Appendix 5



# LAB Complementary Initial Mutation Test (Plate Incorporation Method)

Test Item : Polymer

Date : 15-17 Febr uary 2011

Strain : Salmonella typhimuri um TA100

Cell count (overnight culture): 2.53 x 10 9 

## CFU/mL

Revertant colony number Revertant colony number

Concentration:

- S9 +S9 Concentration:

- S9 +S9

(μg/plate) (μg/plate)

5000 115 150 Untreated control 127 151

149 176 138 133

129 154 148 160

Mean 131.0 160.0 Mean 137.7 148.0

SD 17.09 14.00 SD 10.50 13.75

MF 0.93 0.98 MF 0.98 0.90

1581 131 128 DMSO control * 141 155

131 150 128 137

122 179 134 157

Mean 128.0 152.3 Mean 134.3 149.7

SD 5.20 25.58 SD 6.51 11.02

MF 0.91 0.93 MF 0.96 0.91

500 120 161 Distilled water 160 162

159 139 c ontrol ** 129 132

142 159 154 146

Mean 140.3 153.0 Mean 147.7 146.7

SD 19.55 12.17 SD 16.44 15.01

MF 1.00 0.93 MF 1.05 0.89

158.1 120 144 PEG 400 128 169

155 150 control 131 n.d.

120 169 162 159

Mean 131.7 154.3 Mean 140. 3 164.0

SD 20.21 13.05 SD 18.82 7.07

MF 0.94 0.94 MF 1.00 1.00

50 131 160 Positive control 1128 2AA 2906

140 173 SAZ 1066 (2 μg) 3158

128 144 (2 μg) 1184 3234

Mean 133.0 159.0 Mean 1126.0 3099.3

SD 6.24 14.53 SD 59.03 171.69

MF 0.95 0.97 MF 7.63 20.71

15.81 107 129

157 139

123 142 MF: Mutation factor

Mean 129.0 136.7 SD: Standard deviation

SD 25.53 6.81 +S9: with S9 mix

MF 0.92 0.83

- S9: without S9 mix

n.d.: no data (plate was infected)

Mutation factor= mean revertants (t est item)

mean revertants (solvent control)

*DMSO control was used because of 2AA

**Distilled water control was used because of SAZ

## STUDY CODE: 10/339-007M Page 1 of Appendix 6



# LAB Confirmatory Mutation Test (Pre- Incubation Method)

Test Item : Polyme r

Date : 15-17 Febr uary 2011

Strain : Salmonella typhimurium TA98

Cell count (overnight culture): 1.93 x 10 9 

## CFU/mL

Revertant colony number Revertant colony number

Concentration:

- S9 +S9 Concentration:

- S9 +S9

(μg/plate) (μg/plate)

5000 29 SR , P 34 P Untreated control 21 31

19 SR , P 28 P 21 32

25 SR , P 32 P 29 33

Mean 24.3 31.3 Mean 23.7 32.0

SD 5.03 3.06 SD 4.62 1.00

MF 1.35 1.02 MF 1.31 1.04

1581 14 45 DMSO control* 21 26

13 51 19 26

19 42 19 33

Mean 15.3 46.0 Mean 19.7 2 8.3

SD 3.21 4.58 SD 1.15 4.04

MF 0.85 1.50 MF 1.09 0.92

500 21 30 PEG 400 22 25

14 32 control 17 35

29 27 15 32

Mean 21.3 29.7 Mean 18.0 30.7

SD 7.51 2.52 SD 3.61 5.13

MF 1.19 0.97 MF 1.00 1.00

158.1 17 24 Positive control 317 2AA 2668

22 30 NPD 264 (2 μg) 2676

23 48 (4 μg) 286 2772

Mean 20.7 34.0 289.0 2705.3

SD 3.21 12.49 26.63 57.87

MF 1.15 1.11 14.69 95.48

50 27 33

22 27

15 45 P: Precipitate

Mean 21.3 35.0 SR: Slightly r educed background lawn

SD 6.03 9.17 MF: Mutation factor

MF 1.19 1.14 SD: Standard deviation

15.81 22 39 +S9: with S9 mix

20 26

- S9: without S9 mix

24 34

Mean 22.0 33.0

SD 2.00 6.56 Mutation factor= mean revertants (test item)

MF 1.22 1.08 mean revertants (solvent control)

*DMSO control was used because of NPD and 2AA

## STUDY CODE: 10/339-007M Page 2 

## of Appendix 6



# LAB Confirmatory Mutation Test (Pre- Incubation Method)

Test Item : Polyme r

Date : 15-17 Febr uary 2011

Strain : Salmonella typhimurium TA100

Cell count (overnight culture): 2.53 x 10 9 

## CFU/mL

Revertant colony number Revertant colony number

Concentration:

- S9 +S9 Concentration:

- S9 +S9

(μg/plate) (μg/plate)

5000 67 SR , P 103 SR , P Untreated control 126 172

68 SR , P 105 SR , P 151 171

75 SR , P 119 SR , P 133 147

Mean 70

. 0 109

. 0 Mean 136

. 7 163

. 3

SD 4

. 36 8

. 72 SD 12

. 90 14

. 15

MF 0

. 84 0

. 81 MF 1

. 63 1

. 22

1581 48 141 DMSO control * 146 165

69 90 137 167

91 118 147 156

Mean 69

. 3 116

. 3 Mean 143

. 3 162

. 7

SD 21

. 50 25

. 54 SD 5

. 51 5

. 86

MF 0. 83 0. 87 MF 1. 71 1. 21

500 81 75 Distilled water 160 171

70 76 c ontrol ** 148 186

69 112 136 172

Mean 73

. 3 87

. 7 Mean 148

. 0 176

. 3

SD 6. 66 21

. 08 SD 12

. 00 8. 39

MF 0

. 88 0

. 65 MF 1

. 77 1

. 31

158.1 85 119 PEG 400 87 127

105 88 control 78 131

106 117 86 145

Mean 98

. 7 108

. 0 Mean 83

. 7 134

. 3

SD 11

. 85 17

. 35 SD 4. 93 9. 45

MF 1

. 18 0

. 80 MF 1

. 00 1

. 00

50 86 112 Positive control 1164 2AA 2634

101 123 SAZ 1231 (2 μg) 2748

92 127 (2 μg) 1184 2776

Mean 93

. 0 120

. 7 Mean 1193

. 0 2719

. 3

SD 7

. 55 7

. 77 SD 34

. 39 75

. 22

MF 1

. 11 0

. 90 MF 8

. 06 16

. 72

15.81 77 111 P: Precipitate

69 101 SR: Slightly r educed background lawn

69 121 MF: Mutation factor

Mean 71

. 7 111

. 0 SD: Standard deviation

SD 4

. 62 10

. 00 +S9: with S9 mix

MF 0. 86 0. 83

- S9: without S9 mix

Mutation factor= mean revertants (test item)

mean revertants (so lvent control)

*DMSO control was used because of 2AA

**Distilled water control was used because of SAZ

## STUDY CODE: 10/339-007M Page 3 

## of Appendix 6



# LAB Confirmatory Mutation Test (Pre- Incubation Method)

Test Item : Polyme r

Date : 15-17 Febr uary 2011

Strain : Salmonella typhimurium TA1535

Cell count (overnight culture): 3.34 x 10 9 

## CFU/mL

Revertant colony number Revertant colony number

Concentration:

- S9 +S9 Concentration:

- S9 +S9

(μg/plate) (μg/pl ate)

5000 8 SR , P 31 P Untreated control 7 8

4

SR , P 12 P 8 15

10 SR , P 26 P 9 15

Mean 7.3 23.0 Mean 8.0 12.7

SD 3.06 9.85 SD 1.00 4.04

MF 1.16 2.76 MF 1.26 1.52

1581 4 5 DMSO control * 7 13

8 11 7 8

8 12 10 14

Mean 6.7 9.3 Mean 8.0 11.7

SD 2.31 3.79 SD 1.73 3.21

MF 1.05 1.12 MF 1.26 1.40

500 9 11 Distilled water 10 8

2 9 c ontrol ** 6 6

6 7 9 10

Mean 5.7 9.0 Mean 8.3 8.0

SD 3.51 2.00 SD 2.08 2.00

MF 0.89 1.08 MF 1.32 0.96

158.1 6 11 PEG 400 6 11

6 13 control 7 8

7 7 6 6

Mean 6.3 10.3 Mean 6.3 8.3

SD 0.58 3.06 SD 0.58 2.52

MF 1.00 1.24 MF 1.00 1.00

50 4 11 Positive control 1076 2AA 187

9 13 SAZ 1084 (2 μg) 178

6 10 (2 μg) 884 199

Mean 6.3 11.3 Mean 1014.7 188.0

SD 2.52 1.53 SD 113.23 10.54

MF 1.00 1.36 MF 121.76 16.11

15.81 4 8 P: Precipitate

7 10 SR: Slightly r educed background lawn

6 16 MF: Mutation factor

Mean 5.7 11.3 SD: Standard deviation

SD 1.53 4.16 +S9: with S9 mix

MF 0.89 1.36

- S9: without S9 mix

Mutation factor= mean revertants (test item)

mean revertan ts (solvent control)

*DMSO control was used because of 2AA

**Distilled water control was used because of SAZ

## STUDY CODE: 10/339-007M Page 4 

## of Appendix 6



# LAB Confirmatory Mutation Test (Pre- Incubation Method)

Test Item : Polyme r

Date : 15-17 Febr uary 2011

S train : Salmonella typhimurium TA1537

Cell count (overnight culture): 2.18 x 10 9 

## CFU/mL

Revertant colony number Revertant colony number

Concentration:

- S9 +S9 Concentration:

- S9 +S9

(μg/plate) (μg/plate)

5000 2 R , P 7 P Untreated control 4 6

5

SR , P 10 P 9 5

5

R , P 8 P 9 4

Mean 4. 0 8. 3 Mean 7. 3 5. 0

SD 1

. 73 1

. 53 SD 2

. 89 1

. 00

MF 1

. 00 1

. 7 9 MF 1

. 83 1

. 07

1581 2 12 DMSO control* 3 5

8 8 5 8

6 14 9 8

Mean 5

. 3 11

. 3 Mean 5

. 7 7

. 0

SD 3

. 06 3

. 06 SD 3

. 06 1

. 73

MF 1.3 3 2. 43 MF 1. 42 1. 50

500 3 4 PEG 400 4 4

5 9 control 2 6

3 4 6 4

Mean 3

. 7 5

. 7 Mean 4

. 0 4

. 7

SD 1. 15 2. 89 SD 2. 00 1. 15

MF 0

. 92 1

. 21 MF 1

. 00 1

. 00

158.1 2 12 Positive control 391 2AA 172

4 8 9AA 385 (2 μg) 180

2 6 (50 μg) 354 194

Mean 2. 7 8. 7 376

. 7 182

. 0

S D 1

. 15 3

. 06 19

. 86 11

. 14

MF 0

. 67 1

. 86 66

. 47 26

. 00

50 5 4

2 4 P: Precipitate

3 7 R: Reduced background law development

Mean 3

. 3 5

. 0 SR: Slightly Reduced ba ckground

SD 1

. 53 1

. 73 MF: Mutation factor

MF 0. 83 1. 07 SD: Standard deviation

15.81 1* 6 +S9: with S9 mix

2 10

- S9: without S9 mix

3 5

Mean 2

. 0 7

. 0

SD 1. 00 2. 65 Mutation factor= mean revertants (test item)

MF 0

. 50 1

. 50 mean revertants (solvent control)

*plate was partially dried out

*DMSO control was used because of 9AA and 2AA

## STUDY CODE: 10/339-007M Page 5 

## of Appendix 6



# LAB Confirmatory Mutation Test (Pre- Incubation Method)

Test Item : Polyme r

Date : 15-17 Febr uary 2011

Strain : Escherichia coli WP2 uvrA

Cell count (overnight culture): 3.05 x 10 9 

## FU/mL

Revertant colony number Revertant colony number

Concentration:

- S9 +S9 Concentration:

- S9 +S9

(μg/plate) (μg/plate)

5000 22 P 19 P Untreated control 26 32

20 P 21 P 20 30

8

P 14 P 13 29

Mean 16.7 18.0 Mean 19.7 30.3

SD 7.57 3.61 SD 6.51 1.53

MF 1.06 0.75 MF 1.26 1.26

1581 17 23 DMSO control * 19 25

17 23 7 35

11 31 18 19

Mean 15.0 25.7 Mean 14.7 26.3

SD 3.46 4.62 SD 6.66 8.08

MF 0.96 1.07 MF 0.94 1.10

500 16 20 Distilled water 20 40

12 25 c ontrol ** 31 33

9 20 23 32

Mean 12.3 21.7 Mean 24.7 35.0

SD 3.51 2.89 SD 5.69 4.36

MF 0.79 0.90 MF 1.57 1.46

158.1 18 26 PEG 400 15 30

18 20 control 12 19

6 18 20 23

Mean 14.0 21.3 Mean 15.7 24.0

SD 6.93 4.16 SD 4.04 5.57

MF 0.89 0.89 MF 1.00 1.00

50 28 21 Positive control 910 2AA 125

8 22 MMS 912 ( 50 μg) 147

21 22 (2 μL ) 898 147

Mean 19.0 21.7 Mean 906.7 139.7

SD 10.15 0.58 SD 7.57 12.70

MF 1.21 0.90 MF 36.76 5.30

15.81 13 19 P: Precipitate

15 16 MF: Mutation factor

16 27 SD: Standard deviation

Mean 14

. 7 20

. 7 +S9: with S9 mix

SD 1

. 53 5

. 69

- S9: without S9 mix

MF 0. 94 0. 86

Mutation fact or= mean revertants (test item)

mean revertants (solvent control)

*DMSO control was used because of 2AA

**Distilled water control was used because of MMS

## STUDY CODE: 10/339-007M Page 1 

## of Appendix 7



# LAB HISTORICAL CONTROL DATA

(Period of 2004-2009)

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 24.7 122.8 12.0 6.5 31.8 29.3 127.2 13.2 8.2 38.9

Minimum 9 58 2 1 9 9 62 3 1 12

Maximum 54 211 31 24 66 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 24.2 121.0 11.8 5.8 31.0 28.5 125.7 13.0 7.3 38.5

Minimum 10 54 2 1 10 11 64 2 0 12

Maximum 54 200 35 27 69 52 199 31 25 70



## Distilled water control data

without metabolic activation (

- S9 Mix) with metabolic activation (+S9 Mix)

TA98 TA100 TA1535 TA1537 E.coli TA9 8 TA100 TA1535 TA1537 E.coli

Mean value of

revertants 25.3 123.9 12.2 6.8 33.5 30.5 128.1 13.2 8.4 40.0

Minimum 9 51 2 1 9 12 69 3 2 11

Maximum 46 208 34 21 65 61 223 32 23 74



## Positive reference control data

without metabolic activation (

- S9 Mix) wi th metabolic activation (+S9 Mix)

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

Mean value of

revertants 507.3 916.2 795.8 508.7 726.6 1750.4 1889.3 258.8 274.1 364.4

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