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 



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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 Chemicals, No. 471 

 EPA Health Effects Test Guidelines, OPPTS 870.5100 

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

STUDY PLAN : 25 January 2011 

AMENDMENT 1 TO THE STUDY PLAN : 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 
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RESPONSIBLE PERSONS: 

LAB MANAGEMENT : Christopher Banks, DABT

Managing Director

 Alyson Leyshon, M.Sc.

 Senior Director of Toxicology Operations 

QUALITY ASSURANCE : Szabolcs Gáty, M.Sc.

Head of QAU

Brigitta Barkó, M.Sc.

 Deputy Head of 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.

Krisztina Sipos, M.Sc. 

TECHNICAL LEADER : Tamás Buda

CENTAL DISPENSARY : Tamás Mészáros, PhD

(Technical Team Leader)

TECHNICAL ASSISTANTS : Tímea Kovácsné Böröcz

 Emma Petőné Kohán

Ákos Klaffl
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TABLE OF CONTENTS 

Page

STATEMENT OF THE STUDY DIRECTOR 2

STATEMENT OF THE MANAGEMENT 3

QUALITY ASSURANCE STATEMENT 4

1. SUMMARY 9

2. INTRODUCTION 11

3. OBJECTIVE OF STUDY 11

4. GUIDELINES 12

5. MATERIALS AND METHODS 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 USED IN THE EXPERIMENTS 26

5.10. DEVIATIONS TO THE STUDY PLAN 27 

6. RESULTS AND DISCUSSION 28

6.1. PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY 

TEST) 28

6.2. INITIAL AND CONFIRMATORY MUTATION TESTS 29

6.3. VALIDITY OF THE TESTS 31

7. CONCLUSION 31

8. DISTRIBUTION OF THE FINAL REPORT 32

9. REFERENCES 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 Incorporation Method) 1- 4 of APPENDIX 4
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Complementary Initial Mutation Test 

(Plate Incorporation Method) 1- 1 of APPENDIX 5 

Confirmatory Mutation Test (Pre-Incubation Method) 1- 5 of APPENDIX 6 

Historical Control 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-Specific Positive Control Chemicals 14 

Table 3: Genotypes of the Strains Used for Mutagenicity Testing 16 

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

Table 5: Summary of Controls 24 

Table 6: Chemicals Used in the Experiments 27 

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

Table 8: Summary Table of the Initial Mutation Test

and Complementary Initial Mutation Test 2 of APPENDIX 2

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

The test item Polymer was tested for potential mutagenic activity 

using the Bacterial Reverse Mutation Assay. 

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

Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and 

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

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

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

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

Initial Mutation 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 test, the test item formulations were prepared using 

PEG 400. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were 

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

the test item concentrations in the performed main experiments (Initial Mutation Test, 

Complementary Initial Mutation Test and Confirmatory Mutation Test) were: 5000; 

1581; 500; 158.1; 50 and 15.81 μg/plate. 

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 value. There were no consistent dose-related trends 

and no indication of any treatment effect. 

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

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

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

system. 

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

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

activation, furthermore in Salmonella typhimurium TA100 bacterial strain with and 

without metabolic activation in the Confirmatory Mutation Test. 

Precipitate was observed in the Confirmatory Mutation Test in all of the examined 

bacterial strains at 5000 μg/plate concentration with and without metabolic activation.
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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, the viability of the bacterial cells was checked by a 

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

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

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

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

In conclusion, the test item Polymer had no mutagenic activity 

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

study.
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2. INTRODUCTION

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

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

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

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

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

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

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

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

indicates mutagenic activity of the test item. 

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

reversions. The Salmonella typhimurium strains are constructed to differentiate 

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

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

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

substitutions (AT to GC). 

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

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

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

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

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

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

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

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

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

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

the pre-incubation assay. 

3. OBJECTIVE OF STUDY

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

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 uvrA in the presence and absence of activated rat liver S9

fraction. 
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4. GUIDELINES

This study followed the procedures indicated by the following internationally accepted 

guidelines and recommendations: 

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

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

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

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

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

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

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

Mutation Test Using Bacteria”, 30 May 2008 

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

bacterial mutagenicity testing published by the major Japanese Regulatory Authorities 

including METI, MHLW and MAFF. 

5. MATERIALS AND METHODS 

5.1. TEST ITEM

5.1.1. Name and Data of Test Item* 

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.
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Storage condition: Room temperature (15-30oC)

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

glasses, face mask) for unknown materials were 

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 

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

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

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

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

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.

Because of this 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 homogenized 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

continuously mixed during the treatment. Test formulations 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 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
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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 demonstrated the 

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

summarized in Table 2. 

Table 2: Strain-Specific Positive Control Chemicals

Metabolic 

activation

Strain specific positive chemical

Vehicle Dose 

quantity/plate Strain

Chemical and Supplier Batch number / 

Expiry date

non-activation

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

MERCK 

S5061894 / 

28 Feb 2013 DMSO 4 μg

Salmonella

TA98

non-activation Sodium azide (SAZ)

SIGMA-ALDRICH 

MKAA3815 /

30 Jan 2011

Distilled 

water

 2 μg

Salmonella

TA100

TA1535

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
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5.2.2. Negative Controls (Solvent Controls) 

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

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

chemicals were used for vehicle (solvent) control groups: 

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 2013 

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

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

Salmonella typhimurium TA100 16 November 2010, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA 

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

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

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

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

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

5.3.2. Genotypes 

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

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

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

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

rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for 

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

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

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

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

(WP2 uvrA) is also defective in DNA excision repair. The genotypes of the tester strains 

used for mutagenicity testing are summarized in Table 3. 

Table 3: Genotypes of the Strains Used for Mutagenicity Testing 

Strain

Genotype Mutations Main

DNA

target

Plasmid trp./his 

mutation

type of 

mutation cell wall DNA- repair

S.typhimurium

TA98 hisD3052 Frameshift rfa uvrB GC pKM101

S.typhimurium

TA100 hisG46 Base pair 

substitution rfa uvrB GC pKM101

S.typhimurium

TA1535 hisG46 Base pair 

substitution rfa uvrB GC No 

S.typhimurium. 

TA1537 hisC3076 Frameshift rfa uvrB GC No 

E.coli 

WP2 uvrA trpE Base pair 

substitution + uvrA AT No 

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

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

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

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

cryoprotective agent.

5.3.4. Confirmation of Phenotypes of Tester Strains 

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

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

resistance (amp), as well as spontaneous mutation frequencies 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 measured routinely in mutagenicity experiments and 

expressed as the number of spontaneous revertants per plate. Historical control values 

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

metabolic activation were in the period of 2004 to 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 

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.
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5.3.7. Viability of the Testing Cultures

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

105, 106, 107 and 108 dilutions of cultures on Nutrient Agar (Section 5.4.3.) plates. The 

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

5.4. MEDIA

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

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

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

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

Glucose 20.0 g 

Magnesium sulfate 0.2 g 

Citric acid 2.0 g 

di-Potassium hydrogenphosphate 10.0 g 

Sodium ammonium hydrogenphosphate 3.5 g 

Agar agar 15.0 g 

Distilled water q.s. ad1

 1000 mL

Minimal glucose agar plates [MERCKOPLATE] (Batch Number: 04329, Expiry date: 

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

Mutation Test and Confirmatory Mutation Test; and Batch Number: 04331, Expiry 

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

Certificates of Analysis were obtained from the Supplier. 

5.4.2. Nutrient Broth No.2 

Nutrient Broth No.2. 25.0 g 

Distilled water q.s. ad 1000 mL

Sterilization was performed at 121o

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 Sodium chloride dissolved in 

Distilled water (final volume 1000.0 mL).

 

1 q.s. ad = quantum sufficiat ad (a sufficient quantity to make)
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5.4.3. Nutrient Agar

Nutrient Agar 20.0 g 

Distilled water q.s. ad 1000 mL

Sterilization was performed at 121o

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 performed at 121o

C in an autoclave. 

Histidine – Biotin solution (0.5 mM): 

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

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

Distilled water q.s. ad 1000 mL

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

Complete Top Agar for Salmonella typhimurium strains:

Histidine – Biotin solution (0.5 mM) 100 mL

Agar solution 900 mL

5.4.5. Top Agar for Escherichia coli Strain

Tryptophan solution (2 mg/mL): 

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

Distilled water q.s. ad 1000 mL

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

Complete Top Agar for Escherichia coli strain:

Nutrient Broth by 5.4.2. 50 mL

Tryptophan solution (2 mg/mL) 2.5 mL

Agar solution by 5.4.4. 947.5 mL
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5.5. METABOLIC ACTIVATION SYSTEM

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

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

fraction.

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

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

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

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

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

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

the Experiments). The composition of solution refers to 1000 mL. 

5.5.1. Induction of Liver Enzymes 

Male Wistar rats (275-319 g, animals were 8 weeks old at the initiation) were treated 

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

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

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

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

induction of liver enzymes used for preparation S9 used in this study was 20 September

2010. 

5.5.2. Preparation of Rat Liver Homogenate S9 Fraction

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

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

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

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

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

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

The sterility of the preparation was confirmed. 

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

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

concentration of the S9 fraction used was determined to be 34.2 g/L. The date of 

preparation of S9 fraction for this study was 23 September 2010 (LAB code: E10891).
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The biological activity in the Salmonella assay of S9 was characterized using the two 

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

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

5.5.3. The S9 Mix 

Salt solution for S9 Mix: 

NADP Na 7.66 g 

D-glucose-6 phosphate Na 3.53 g 

MgCl2 x 6 H2O 4.07 g 

KCl 6.15 g 

Distilled water q.s. ad 1000 mL 

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

The complete S9 mix was freshly prepared containing components as follows: 

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

Rat liver homogenate (S9) 100 mL 

Salt solution for S9 Mix (see above) 400 mL 

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

5.5.4. 0.2 M Sodium Phosphate Buffer, pH 7.4 

Solution A: 

Na2HPO4 x 12H2O 71.63 g 

Distilled water q.s. ad 1000 mL

Sterilization was performed at 121 oC in an autoclave. 

Solution B: 

NaH2PO4 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
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5.6. DESCRIPTION OF THE TEST PROCEDURE

The study 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 Initial and Complementary 

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 Range Finding Test 

(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 

homogenized 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 without test bacterium 

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

+ phosphate buffer 500 μL

+ top agar 2 mL)

Test item 

concentration in 

the test tube

μg/tube

100 suspension suspension 5000

5.6.1.2. Concentration Range Finding Test (Informatory Toxicity 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 μg/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 preliminary 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 six dosing solutions. The maximum test concentration was

5000 μg test item/plate.

Examined Polymer concentrations were: 5000; 1581; 500; 158.1; 50 

and 15.81 μg/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 summarized in Table 5. 
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Table 5: Summary of Controls 

Type of control Activation 

with S9 Mix Solvent Top 

Agar 

Strain-Specific 

Positive Chemical 

Solutions (5.2.1.)

Phosphate 

Buffer

Untreated – – + – +

Untreated + – + – –

Solvent – + + – +

Solvent + + + – –

Positive Reference 

Control – – + + + 

Positive Reference 

Control + – + + – 

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

solvents were run in the assay.

5.6.3. Experimental Method

The experimental methods were conducted according to the methods described Ames 

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

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

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

5.6.4. Procedure for Exposure in the Initial Mutation Test and Complementary Initial Mutation 

Test

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

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

described in Section 5.3.6.) were exposed to the test item both in the presence and 

absence of an appropriate metabolic activation system. 

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

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

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

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

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

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

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

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

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

5.6.5. Procedure for Exposure in the Confirmatory Mutation Test

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

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

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

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

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

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

incubator. 

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

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

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

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

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

hours. 

5.7. EVALUATION OF EXPERIMENTAL DATA

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

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

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

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

software.

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

the vehicle control plate. 

Criteria for a Positive Response:

A test item was 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.
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An increase was considered biologically relevant if: 

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

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

control plates, 

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

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

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

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

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

determining factor for a positive response.

Criteria for a Negative Response:

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

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

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

5.8. ARCHIVES

The study documents and samples: 

- study plan and study plan amendment, 

- all raw data,

- retained sample of the test item,

- correspondence, 

- study report and any amendments 

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

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

facility SOPs. 

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

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

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

written consent. 

5.9. CHEMICALS USED IN THE EXPERIMENTS

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

summarized in Table 6.
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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 June 2010

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

L-Histidine HCl x H2O MERCK K37522450 30 June 2012

KCl MERCK K40299136 31 July 2014

KCl MERCK K41042236 31 May 2015

LAB-Lemco powder OXOID 442051 31 March 2011

MgCl2 x 6 H2O SIGMA-ALDRICH 019K0038 January 2014

NaCl Lachner PP/2010/03837 31 March 2013

NADP Na x H2O SIGMA-ALDRICH 077K7000 April 2011

NaH2PO4 REANAL KBM5207601 July 2011

Na2HPO4 x 12H2O REANAL KBM60552 January 2015

Na2HPO4 x 12H2O REANAL KBM61668 August 2015

Nutrient Agar MERCK VM100650 06 October 2014

Nutrient Broth No.: 2 OXOID 907248 30 June 2015

Peptone, Soybean SIGMA-ALDRICH 1420831 30 August 2011

L-Tryptophan SIGMA-ALDRICH 1400132 July 2012

5.10. DEVIATIONS TO THE STUDY PLAN

Concerning: 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 
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Concerning: Study 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: Nutrient 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 water: 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 DISCUSSION

6.1. PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)

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

preliminary test was performed using Salmonella typhimurium TA98 and Salmonella 

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

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

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

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

100, 31.6 and 10 μg/plate. 
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The observed numbers of revertant colonies were mostly in the normal range. Slightly 

higher numbers of revertant colonies compared to the solvent control plates were 

observed in Salmonella typhimurium TA98 and TA100 tester strains in some cases 

with or without metabolic activation. However, those differences were minor, they 

were well below the threshold limit, there was no dose response and the mean 

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

Precipitate was observed in the Preliminary Range Finding Test in both examined 

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

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

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

6.2. INITIAL AND CONFIRMATORY MUTATION TESTS

In the Initial Mutation Test and 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 were carried out 

using Salmonella typhimurium strains (TA98, TA100, TA1535, TA1537) and 

Escherichia coli WP2 uvrA strain. The Complementary Initial Mutation Test* were 

carried out using Salmonella typhimurium TA100 strain. 

*Note: In 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. Therefore, this part of the test was considered invalid. An additional 

experiment (Complementary Initial Mutation Test) was performed in a new experimental 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 TA100 at solvent control

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

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

Initial Mutation Test in Salmonella typhimurium TA1535 tester strain with metabolic 

activation at the concentrations of 500 and 1581 μg/plate. The mutation factor value 

was 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 values were below biologically relevant threshold 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: 1.73) also in 

this experiment.

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

Confirmatory Mutation Test also in Salmonella typhimurium TA1535 bacterial strain 

with metabolic activation at the concentration of 5000 μg/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 the observed mutation factor value remained below the biologically 

relevant threshold value. The number of revertant colonies was 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 numbers of revertant colonies compared to the solvent control 

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

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

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

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

biological variability of the test.

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

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

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

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

variability of the test system. 

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

background lawn development) was observed in Salmonella typhimurium TA98, 

TA1535 and TA1537 strains at 5000 μg/plate without metabolic activation, 

furthermore in Salmonella typhimurium TA100 bacterial strain with and without 

metabolic activation in the Confirmatory Mutation Test. 

Precipitate was observed in the Confirmatory Mutation Test in all of the examined 

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

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

standard deviations) are summarized in Tables 8-9 (Appendix 2) and Appendices 4, 5 

and 6. 
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6.3. VALIDITY OF THE TESTS 

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

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

solvent control plates were within the historical 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 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.

7. CONCLUSION

The test item Polymer was tested for potential mutagenic activity 

using the Bacterial Reverse Mutation Assay.

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

Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and 

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

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

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

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

The study included a Preliminary 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 Initial and Complementary 

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 6) that under the 

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

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

In conclusion, the test item Polymer had no mutagenic activity 

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

study.
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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 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, Bacterial Reverse 

Mutation Test, No.: 471, Paris. Adopted 21st July 1997 

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

Mutation Test Using Bacteria”, 30 May 2008 

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

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

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

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

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

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

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

ENV/MC/CHEM(98)17
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APPENDICES
STUDY CODE: 10/339-007M Page 1 of Appendix 1 

COPY OF THE GLP CERTIFICATE
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SUMMARY TABLES OF THE RESULTS

Table 7: Summary Table of the Range Finding Test 

Concentrations

(μg/plate)

Mean 

values of 

revertants / 

Mutation 

factor (MF)

Salmonella thyphimurium tester 

strains

TA 98 TA 100

-S9 +S9 -S9 +S9

Untreated 

control

Mean 26.0 30.0 128.3 139.0

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 (4μg) Mean 299.7 -- -- --

MF 13.42 -- -- --

2AA (2μg) Mean -- 2321.3 -- 2210.7

MF -- 74.88 -- 14.80

SAZ (2μg) Mean -- -- 1552.0 --

MF -- -- 11.55 --
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Table 8: Summary Table of the Initial Mutation Test and 

Complementary Initial Mutation Test 

Concentrations

(μg/plate)

Mean 

values of 

revertants / 

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 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 0.85 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 (4μg) Mean 262.7 -- -- -- -- -- -- -- -- --

MF 13.13 -- -- -- -- -- -- -- -- --

2AA (2μg) Mean -- 2346.7 -- 3099.3 -- 197.0 -- 200.3 -- --

MF -- 97.78 -- 20.71 -- 15.55 -- 54.64 -- --

2AA (50μg) Mean -- -- -- -- -- -- -- -- -- 236.7

MF -- -- -- -- -- -- -- -- -- 7.89

SAZ (2μg) Mean -- -- 1126.0 -- 1806.7 -- -- -- -- --

MF -- -- 7.63 -- 208.46 -- -- -- -- --

9AA (50μg) Mean -- -- -- -- -- -- 1008.0 -- -- --

MF -- -- -- -- -- -- 378.00 -- -- --

MMS (2μL) Mean -- -- -- -- -- -- -- -- 1041.3 --

MF -- -- -- -- -- -- -- -- 39.54 --
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Table 9: Summary Table of the Confirmatory Mutation Test 

Concentrations

(μg/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 24.3 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 (4μg) Mean 289.0 -- -- -- -- -- -- -- -- --

MF 14.69 -- -- -- -- -- -- -- -- --

2AA (2μg) Mean -- 2705.3 -- 2719.3 -- 188.0 -- 182.0 -- --

MF -- 95.48 -- 16.72 -- 16.11 -- 26.00 -- --

2AA (50μg) Mean -- -- -- -- -- -- -- -- -- 139.7

MF -- -- -- -- -- -- -- -- -- 5.30

SAZ (2μg) Mean -- -- 1193.0 -- 1014.7 -- -- -- -- --

MF -- -- 8.06 -- 121.76 -- -- -- -- --

9AA (50μg) Mean -- -- -- -- -- -- 376.7 -- -- --

MF -- -- -- -- -- -- 66.47 -- -- --

MMS (2μL) Mean -- -- -- -- -- -- -- -- 906.7 --

MF -- -- -- -- -- -- -- -- 36.76 --
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Preliminary Range Finding Test (Informatory Toxicity Test)

Test Item : Polymer 

Date : 26-28 January 2011 

Strain : Salmonella typhimurium TA98

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

Revertant colony number Revertant colony number

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

(μg/plate) (μg/plate)

5000 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 26.0 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 

Mean 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 

100 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 : Polymer 

Date : 26-28 January 2011 

Strain : Salmonella typhimurium TA100 

Cell count (overnight culture): 2.37 x 109 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 

Mean 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 128 164 

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 **Distilled 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 : Polymer 

Date : 08-10 February 2011 

Strain : Salmonella typhimurium TA98

Cell count (overnight culture): 1.55 x 109 CFU/mL

Revertant colony number Revertant colony number

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

(μg/plate) (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

Mean 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 revertants (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 : Polymer 

Date : 08-10 February 2011 

Strain : Salmonella typhimurium TA1535 

 Cell count (overnight culture): 1.46 x 109 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 control** 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 : Polymer 

Date : 08-10 February 2011 

Strain : Salmonella typhimurium TA1537 

 Cell count (overnight culture): 2.09 x 109 CFU/mL

Revertant colony number Revertant colony number

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

(μg/plate) (μg/plate)

5000 3 3 Untreated 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 

500 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 : Polymer 

Date : 08-10 February 2011 

Strain : Escherichia coli WP2 uvrA

 Cell count (overnight culture): 4.13 x 109 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 control** 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 February 2011 

Strain : Salmonella typhimurium TA100 

 Cell count (overnight culture): 2.53 x 109 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 control** 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 (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 1 of Appendix 6 

LAB

Confirmatory Mutation Test (Pre-Incubation Method)

Test Item : Polymer 

Date : 15-17 February 2011 

Strain : Salmonella typhimurium TA98

 Cell count (overnight culture): 1.93 x 109 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 28.3 

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 reduced 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 : Polymer 

Date : 15-17 February 2011 

Strain : Salmonella typhimurium TA100 

 Cell count (overnight culture): 2.53 x 109 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 control** 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 reduced 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 (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 6

LAB

Confirmatory Mutation Test (Pre-Incubation Method)

Test Item : Polymer 

Date : 15-17 February 2011 

Strain : Salmonella typhimurium TA1535 

 Cell count (overnight culture): 3.34 x 109 CFU/mL

Revertant colony number Revertant colony number

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

(μg/plate) (μg/plate)

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 control** 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 reduced 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 revertants (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 : Polymer 

Date : 15-17 February 2011 

Strain : Salmonella typhimurium TA1537 

 Cell count (overnight culture): 2.18 x 109 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 1.79 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 1.33 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 

SD 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 background

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 : Polymer 

Date : 15-17 February 2011 

Strain : Escherichia coli WP2 uvrA

 Cell count (overnight culture): 3.05 x 109 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 control** 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 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 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 TA98 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) with 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