## FINAL REPORT

Study Title



## EVALUATION OF THE MU TAGENIC ACTIVITY OF COPS_1

IN THE **SALMONELLA TYPHIMURI UM**  REVERSE MUTATION

ASSAY AND THE **ESCHERICHIA COLI** REVERSE MUTATION

ASSAY

Author

C.M. Verspeek

- Rip

Test Facility

WIL Research Europe B.V.

Ham bakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 508230

Substance 204240/B

- Page 1 of 30

-COPS_1 Project 508230

- Page 2 - 1. CONTENTS

1. CONTENTS

................................................................................................ ...................................... 2

2. STATEMENT OF GLP COM PLIANCE

............................................................................................. 3

3. QUALITY ASSURANCE ST ATEMENT

............................................................................................ 4

4. SUMMARY

................................................................................................ ........................................ 5

5. INTRODUCTION

.............................................................................................................................. 6

5.1. Preface

................................................................................................ ...................................... 6

5.2. Aims of the study

....................................................................................................................... 6

5.3. Guidelines

................................................................................................ ................................ . 6

5.4. Storage and retention of records and materials

........................................................................ 7

6. MATERIALS AND METHOD S

.......................................................................................................... 7

6.1. Test substance

.......................................................................................................................... 7

6.1.1. Test substance information

............................................................................................. 7

6.1.2. Study specific test substance information

...................................................................... 7

6.1.3. Test substance preparation

............................................................................................ 7

6.2. Reference substances

.............................................................................................................. 8

6.2.1. Negative control

.............................................................................................................. 8

6.2.2. Positive controls

.............................................................................................................. 8

6.3. Test system

............................................................................................................................... 8

6.4. Cell culture

................................................................................................ ................................ 9

6.5. Metabolic activation system

...................................................................................................... 9

6.5.1. Preparation of S9

- mix

..................................................................................................... 9

6.6. Study design

........................................................................................................................... 10

6.6.1. Dose range finding test

................................................................................................ . 10

6.6.2. Mutation assay

.............................................................................................................. 10

6.6.3. Colony counting

............................................................................................................ 10

6.7. Electronic data capture

........................................................................................................... 10

6.8. Interpretation

........................................................................................................................... 10

6.8.1. Acceptability of the assay

............................................................................................. 10

6.8.2. Data evaluation and statistical procedures

................................................................... 11

6.9. List of deviations

..................................................................................................................... 11

6.9.1. List of protocol d eviations

............................................................................................. 11

6.9.2. List of standard operating procedures deviations

......................................................... 11

7. RESULTS

................................................................................................ ....................................... 11

7.1. Dose range finding test/first mutation experiment

................................................................... 11

7.2. Experiment 2

........................................................................................................................... 12

8. DISCUSSION AND CONCL USIO N

................................................................................................ 12

9. REFERENCES

............................................................................................................................... 12

TABLES

Table 1 Dose range finding test: Mutagenic response of COPS_1 in the Salmonella

typhimurium reverse mutation assay and in the Escherichia coli reverse mutation

assay

............................................................................................................................. 13

Table 2 Experiment 1: Mutagenic response of COPS_1 in the Salmonella typhimurium

reverse mutation assay

................................................................................................ . 14

Table 3 Experiment 2: Mutagenic response of COPS_1 in the Salmonella typhimurium

reverse mutation assay and in the Escherichia coli reverse mutation assay

............... 15

APPENDICES

APPENDIX 1 SUMMARY TABLES

..................................................................................................... 13

APPENDIX 2 SUPPORTING MATERIALS AND METHOD

............................................................... 16

APPENDIX 3 DETAILED TABLES

...................................................................................................... 17

APPENDIX 4 HISTORICAL CONTROL DATA OF THE SOLVENT CONTROL

................................ 28

APPENDIX 5 HISTORICAL CONTROL DATA OF THE POSITIVE CONTROL SUBSTANCES

....... 29

APPENDIX 6 CERTIFICATE OF ANALYSIS

...................................................................................... 30COPS_1 Project 508230

- Page 5 - 4. SUMMARY

Evaluation of the mutagenic activity of COPS_1 in the Salmonella typhimurium reverse mutation assay

and the Escherichia coli reverse mutation assay.

COPS_1 was tested in the Salmonella typhimurium reverse mutation assay with four histidine

requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the

Escherichia coli reverse mutation assay with a tryptophan

- requiring strain of Escher ichia coli

(WP 2 uvrA). The test was performed in two independent experiments in the presence and absence of

S9

- mix (rat liver S9

- mix induced by Aroclor 1254 ).

The study procedures described in this report were based on the most recent OECD , EC and

EPA/TSCA guidelines.

Batch 14L1103 of COPS_1 was a clear colourless liquid. A correction factor of 2.48 was used to

correct for the purity of 40.3%

. The test substance was dissolved in Milli

- Q water

.

In the dose range finding test, COPS_1 was tested up to concentrations of 5000 μg/plate in the

absence and presence of S9

- mix in the strains TA100 and WP 2 uvrA. COPS_1 did not precipitate on

the plates at this dose level. The bacterial background lawn was not reduced at any of the

concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Results of this dose range finding test were reported as part of the first mutation assay.

Based on the results of the dose range finding test, COPS_1 was tested in the first mutation assay at

a concentration range of 52 to 5000 μg/plate in the absence and presence of 5% (v/v) S9

- mix in the

tester strains TA1535, TA1537 and TA98. The bacterial background lawn was not reduced at any of

the concentrations tested and no biologically relevant decrease in the number of revertants was

observed. Results of this dose range finding test were reported as part of the first mutation assay

.

In a follow

- up experiment of the assay with additional parameters, COPS_1 was tested at a

concentration range of 492 to 5000 μg/plate in the absence and presence of 10% (v/v) S9

- mix in the

tester strains TA1535, TA1537, TA98, TA100 and WP 2 uvrA. The bacterial background lawn was not

reduced at any of the concentrations tested and no biologically relevant decrease in the number of

revertants was observed.

COPS_1 did not induce a significant dose

- related increase in the number of revertant (His + ) colonies

in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant

(Trp + ) colonies in the tester strain WP 2 uvrA both in the absence and presence of S9

- metabolic

activation. These results were confirmed in a follow

- up experiment

.

In this study, the negative and strain

- specific positive control values were within the laboratory

historical c ontrol data ranges indicating that the test conditions were adequate and that the metabolic

activation system functioned properly.

Based on the results of this study it is concluded that COPS_1 is not mutagenic in the Salmonella

typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay both in the

absence and presence of S9

- metabolic activation

.COPS_1 Project 508230

- Page 6 - 5. INTRODUCTION

5.1. Preface

Sponsor Society of Chemical Manufacturers and Affiliates

1850 M Street, NW Suite 700

WASHINGTON, DC 20036

USA

Study Monitor Dr. C. T. Helmes

Test Facility WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Study Director C.M. Verspeek

- Rip

ineke.verspeek@wilresearch.co m

Study Plan Start : 23 March 2015

Completion : 13 April 2015

5.2. Aims of the study

The objective of this study was to evaluate the test substance for its ability to induce reverse mutations

in a gene of histidine

- requiring Salmonella typhimurium bact erial strains resulting in histidine

independent strains, and in a gene of tryptophan

- requiring Escherichia coli bacterial strain resulting in

a tryptophan

- independent strain.

Background of the test system

The Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay

have been shown to be rapid and adequate indicators for the mutagenic activity of a wide range of

chemical compounds.

The assay was conducted in the absence and presence of a metabolizing system (S9

- mix).

The Salmonella typhimurium strains used in this study were TA1535, TA1537, TA98 and TA100. The

Escherichia coli strain used was WP 2 uvrA. The strains TA1537 and TA98 are capable of detecting

frameshift mutagens, strains TA1535, TA100 and WP 2 uvrA are capable of detecting base

- pair

substitution mutagens (1

- 5).

5.3. Guidelines

The study procedures described in this report were based on the following guidelines:

- Organisation for Economic Co

- operation and Development (OECD), OECD Guidelines for Testing

of Chemicals; Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test" (Adopted

July 21, 1997).

- European Community (EC). Co mmission regulation (EC) No. 440/2008, Part B: Methods for the

Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse

Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 3 1 May 2008.

- United States Environmental Protection Agency Prevention, Pesticides and Toxic Substances

(7101) EPA 712 -C- 98

- 223 August 1998. Health E ffects Test Guidelines OPPTS 870.5 100 Bacterial

Reverse Mutation Test.COPS_1 Project 508230

- Page 7 - 5.4. Storage and retention of records and materials

Records and materials pertaining to the study including protocol, raw data and the final report are

retained in the WIL Research Europ e archives for a period of at least 2 years after finalization of the

report. After this period, the sponsor will be contacted to determine how the records and materials

should be handled. WIL Research Europe will retain information concerning decisions ma de.

WIL Research Europe will retain a test substance sample until the expiry date, but no longer than

10 years after finalization of the report. After this period the sample will be destroyed.

6. MATERIALS AND METHOD S

6.1. Test substance

6.1.1. Test substance informa tion

Identification COPS_1 (40.3 wt. % in H 2 O)

Appearance Clear colourless liquid

Batch 14L1103

Purity/Composition See Certificate of Analysis

Test substance storage At room temperature

Stable under storage conditions until 21 November 2015 (expiry date)

See APPENDIX 6 ; Certificate of analysis.

6.1.2. Study specific test substance information

Purity/composition correction factor Yes, correction factor 2.48

Test substance handling No specific handling conditions required

Stability at higher temperatures Stable

Chemical name (IUPAC), synonym or

trade name 1

- Propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

- , sodium salt (1:1)

CAS Number 52556

- 42

- 0

Molecular s tructure



### O SO 3



### OH Na + _

Molecular formula C 6 H 11 NaO 5 S

Molecular weight 218

. 22

Specific gravity/density 1.17 at 20°C

Solubility in vehicle Water : 1

- 45%

Stability in vehicle Water: Stable

6.1.3. Test substance preparation

A correction factor of 2.48 was used to correct for the purity of 40.3%.

COPS_1 was diss olved in Milli

- Q water (Millipore Corp., Bedford, MA., USA). Preparation of test

solut ions started with solutions of 124 mg/ml

. The lower test concentrations were prepared by

subsequent dilutions in Milli

- Q water. Test substance concentrations were used wi thin 2 hours of

preparation.COPS_1 Project 508230

- Page 8 - 6.2. Reference substances

6.2.1. Negative control

The vehicle of the test substance

, which was Milli

- Q water

.

6.2.2. Positive controls

Without metabolic activation (

- S9

- mix):

Strain Chemical Concentration/plate Solvent

TA1535 sodium azide (SA) 5 μg Saline

( Sigma Aldrich Chemie, Steinheim, Germ any )

TA1537 ICR

- 191 (Acros Organics, Geel, Belgium ) 2.5 μg DMSO

TA98 2- nitrofluorene (NF) (Merck) 10 μg DMSO

TA100 methylmethanesulfonate (MMS) (Sigma) 650 μg DMSO

WP 2 uvrA 4- nitroquinoline N

- oxide (4

- NQO) (Sigma) 10 μg DMSO

With metabolic activation (+S9

- mix):

The positive control substance used for all tester strains was 2

- aminoanthracene (2AA) (Sigma). The

following concentrations were used:

Strain Concentration/plate Amount of S9

- mix Solvent

TA153 5 2.5 μg 5 and 10% DMSO

TA1537 2.5 μg 5% DMSO

TA1537 5 μg 10% DMSO

TA98 1 μg 5 and 10% DMSO

TA100 1 μg 5% DMSO

TA100 2 μg 10% DMSO

WP 2 uvrA 15 μg 5 and 10% DMSO

Solvents for reference substances

Saline = physiological sali ne (Eurovet Animal Health, Bladel, The Netherlands )

DMSO = dimethyl sulfoxide ( Merck )

6.3. Test system

Test system Salmonella typhimurium bacteria and Escherichia coli bacteria

Rationale Recommended test system in international guidelines (e.g. OECD and EC).

Source Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames

(TA1535: 2006, TA1537: 2009, TA98: 2006, TA100: 2006; and Master culture

from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK

(WP 2 uvrA: 2008)]

The characteristics of the different Salmonella typhimurium strains were as follows:

Strain Histidine mutation Mutation type

TA1537 his C3076 Frameshift

TA98 his D3052/R

- factor* Frameshift

TA1535 his G46 Base

- pair substitutions

TA100 his G46/R

- factor* Base

- pair substitu tions

*: R

- factor = plasmid pKM101 (increases error

- prone DNA repair)

Each tester strain contained the following additional mutations:

rfa : deep rough (defective lipopolysaccharide cellcoat)

gal : mutation in the galactose metabolism

chl : mutation in nitrate reductase

bio : defective biotin synthesis

uvrB : loss of the excision repair system (deletion of the ultraviolet

- repair B gene)COPS_1 Project 508230

- Page 9 - The Salmonella typhimurium strains a re regularly checked to confirm their histidine

- requirement,

crystal violet sensitivi ty, ampicillin resistance (TA98 and TA100), UV

- sensitivity and the number of

spontaneous revertants.

The Escherichia coli WP

2 uvrA strain detects base

- pair substitutions. The strain lacks an excision repair

system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of

mutagens has been enhanced by permeabilization of the strain using Tris

- EDTA treatment (Ref.1).

The strain i s regularly checked to confirm the tryptophan

- requirement, UV

- sensitivity and the number

of spontaneous revertants.

Stock cultures of the five strains were stored in liquid nitrogen (

- 196°C).

6.4. Cell culture

Preparation of bacterial cultures

Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD,

Hampshire, E ngland) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached

an optical density of 1.0 ± 0. 1 at 700 nm (10 9 cells/ml). Freshly grown cultures of each strain were

used for test ing

.

Agar plates

Agar plates (ø 9 cm) contain ing 25 ml glucose agar medium. Glucose agar medium contained per liter:

18 g purified agar ( Merck ) in Vogel

- Bonner Medium E, 20 g glucose (Fres e nius Kabi, Bad Homburg,

Germany ). The agar plates for the test with the Salmonella typhimurium strains also contained

1 2.5 μg/plate biotin ( Merck) and 15 μg/plate histidine ( Sigma ) and the agar plates for the test with the

Escherichia coli strain contained 15 μg/plate tryptophan ( Sigma ).

Top agar

Milli

- Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride

( Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass

tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

Environmental conditions

All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual

range 35.7

– 38.3°C) . The temperature was continuously monitored throughout the experiment. Due to

addition of plates (which were at room temperature) to the incubator or d ue to opening and closing the

incubator door , t emporary deviations from the temperature may occur

. Based on laboratory historical

data these deviations are considered not to affect the study integrity

.

6.5. Metabolic activation system

Rat liver microsomal enzymes (S9 homogenate) wer e obtained from Trinova Biochem GmbH,

Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected

intraperitoneally with Aroclor 1254 (500 mg/kg)

.

Each S9 batch is characterised with the mutagens Benzo

- (a)

- pyren e and 2

- aminoanthracene, which

require metabolic activation, in tester strain TA 100 at concentrations of 5 μg/plate and 2.5 μg/plate,

respectively.

6.5.1. Preparation of S9

- mix

S9

- mix was prepared immediately before use and kept on ice. S9

- mix contained per 10 ml: 30 mg

NADP ( Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose -6- phosphate

(Roche Diagnostics, Mannheim, Germany) in 5.5 ml or 5.0 ml Milli

- Q water (first or second experiment

respectively) (Millipore Corp., Bedford, MA., USA); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml

0.08 M MgCl

2 solution (Merck); 1 ml 0.33 M KCl solution (Merck). The above solution was filter

(0.22 μm)

- sterilized. To 9.5 ml of S9

- mix components 0.5 ml S9

- fraction was added (5% (v/v)COPS_1 Project 508230

- Page 10

- S9

- fraction) to complete the S9

- mix in the first experiment and to 9.0 ml of S9

- mix components 1.0 ml

S9

- fraction was added (10% (v/v) S9

- fraction) to complete the S9

- mix in the second experiment.

6.6. Study design

6.6.1. Dose range finding test

Selection of an adequate range of doses was based on a dose range finding test with the strains

TA100 and WP 2 uvrA, both with and without 5% (v/v) S9

- mix. Eight concentrations, 1.7, 5.4, 17, 52,

164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of COPS_1 used

in t he subsequent mutation assay was 5 000 μ g/plate.

6.6.2. Mutation assay

At least five different doses (increasing with approximately half

- log steps) of the test substance were

tested in triplicate in each strain. The above mentioned dose range finding study with the two tester

strains TA100 and WP 2 uvrA, is reported as a part of the first mutation experiment. In the second part

of this experiment, COPS_1 was tested both in the absence and presence of 5% (v/v) S9

- mix in the

tester strains TA1535, TA1537 and TA98 . In a follow

- up experiment with additional parameters, the

test substance was tested both in the absence and presence of 10% (v/v) S9

- mix in all tester strains.

The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in

the presence and absence of S9

- mix.

Top agar in top agar tubes was melted by heating to 45 ± 2 °C . The following solutions were

successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10 9 cells/ml) of one of

the tester strains, 0.1 ml of a dilution of the test substance in Milli

- Q water and either 0.5 ml S9

- mix (in

case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non

- activation assays). The

ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective

agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at

37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His + ) for

Salmonella typhi murium bacteria and tryptophan independent (Trp + ) for Escherichia coli were counted.

6.6.3. Colony counting

The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with

sufficient test article precipitate to interfere with automated colony counting were counted manually.

Evidence of test article precipitate on the plates and the condition of the bacterial background lawn

were evaluated when considered necessary, macroscopically and/or microscopically by using a

dissecting micros cope.

6.7. Electronic data capture

Observations/measurements in the study were recorded electronically using the following

programme:

- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ,

USA).

- Ames study Manager version 1.23 (Perceptive Instruments Ltd., St Edmunds, Suffolk, United

Kingdom ).

6.8. Interpretation

6.8.1. Acceptability of the assay

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is

considered acceptable if it meets the following criteria:COPS_1 Project 508230

- Page 11

- a) The vehicle control and positive control plates from each tester strain (with or without S9

- mix) must

exhibit a characteristic number of revertant colonies when compared against relevant historical

control data generated at WIL Research Eur ope.

b) The selected dose range should include a clearly toxic concentration or should exhibit limited

solubility as demonstrated by the preliminary toxicity range

- finding test or should extend to

5 mg/plate.

c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If

the results are considered invalid due to contamination, the experiment will be repeated.

6.8.2. Data evaluation and statistical procedures

No formal hypothesis testing was done.

A t est substance is considered negative (not mutagenic) in the test if:

a) The total number of revertants in the tester strain TA100 is not greater than two (2) times the

concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or

WP 2 uvrA is not greater than three (3) times the concurrent vehicle control.

b) The negative response should be reproducible in at least one follow

- up experiment

.

A test substance is considered positive (mutagenic) in the test if:

a) The total number of revertants in the tester strain TA100 is greater than two (2) times the

concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or

WP 2 uvrA is greater than three (3) times the concurrent vehicle control.

b) In case a follow up experiment is performed when a positive response is observed in one of the

tester strains, the positive response should be reproducible in at least one follow up experiment.

6.9. List of deviations

6.9.1. List of protocol deviations

There were no deviations from the protocol.

6.9.2. List of standard operating procedures deviations

Any deviations from standard operating procedures were evaluated and filed in the study file. There

were no deviations from standard operating procedures that affected the integrity of the study.

7. RESULTS

7.1. Dose range finding test /first mutation experiment

COPS_1 was tested in the tester strains TA100 and WP 2 uvrA at concentrations of 1.7, 5.4, 17, 52, 164,

512, 1600 and 5000 μg/plate in the absence and presence of S9

- mix. Based on the results of the dose

range finding test, the following dose range was selected for the mutation assay with the tester strains,

TA1535, TA1537 and TA98 in the absence and presence of S9

- mix: 52, 164, 512, 1600 and

5000 μg/plate.

The results are shown in Table 1 and Table 2

. The individual data are presented in APPENDIX 3

.

Precipitate

Precipitation of COPS_1 on the plates was not observed at the start or at the end of the incubation

period in any tester strain.

Toxicity

To determine the toxicity of COPS_1 , the reduction of the bacterial background lawn, the increase in

the size of the microcolonies and the reduction of the revertant colonies were examined. The

definitions are stated in APPENDIX 2

.COPS_1 Project 508230

- Page 12

- No reduction of the bacterial background lawn and no biologically relevant decrease in the number of

revertants were observed.

Mutagenicity

N o increase in the number of revertants was observed upon treatment with COPS_1 under all

conditions tested.

7.2. E xperiment 2

To obtain more information about the possible mutagenicity of COPS_1 , a second mutation

experiment was performed in the absence of S9

- mix and in the presence of 10% (v/v) S9

- mix. Based

on the results of the first mutation assay, COPS_1 was test ed up to the dose level of 5000 μg/plate in

strains TA1535, TA1537, TA98, TA100 and WP 2 uvrA. The results are shown in Table 3

, the individual

data are presented in APPENDIX 3

.

Precipitate

Precipitation of COPS_1 on the plates was not observed at the start or at the end of the incubation

period.

Toxicity

In the second mutation assay, there was no reduction of the bacterial background lawn and no

biologically relevant decrease in the number of revertants at any of the concentrations tested in all

tester strains in the absence and presence of S9

- mix.

Mutagenicity

In the second mutation assay, no increase in the number of revertants was observed upon treatment

with COPS_1 u nder all conditions tested.

8. DISCUSSION AND CONCL USION

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose

related increase in the number of revertants in two experiments.

The negative and strain

- specific positive control values were within the laboratory historical control

data ranges indicating that the test conditions were adequate and that the metabolic activation system

functioned properly.

Based on the results of this study it is concluded that COPS _1 is not mutagenic in the Salmonella

typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay both in the

absence and presence of S9

- metabolic activation

.

9. REFERENCES

1 Leonardo, J.M., Dornfeld, S.S. and Peak, M.J., 1984, Evaluation of E. coli K12 343 \ 113 and

derived strains for microbial mutagenicity assays. Mutation Res., 130 , 87

- 95.

2 Ames, B.N., McCann, J. and Yamasaki, E., 1975, Methods for detecting carcinogens and

mutagens with the Salmonella/ mammalian microsome mutagenicity test, Mutation Res., 31 , 347

-

364.

3 Maron, D.M. and Ames, B.N., 1983, Revised methods for the Salmonella mutagenicity test,

Mutation Res., 113 , 173

- 215. Erratum, 1983, Mutation Res., 113 , 533.

4 Green, M.H.L. and Muriel, W.J., 1976, Mutagen testing using T rp + reversion in Escherichia coli

, Mutation Res., 38 , 3

- 32.

5 Vogel, H.J. and Bonner, D.M., 1956, Acetylornithinase of Escherichia coli : partial purification and

some properties. J. Biol. Chem., 218 , 97

- 106.COPS_1 Project 508230

- Page 13

- APPENDIX 1 SUMMARY TAB LES

Table 1 Dose range finding test: Mutagenic response of COPS_1 in the **Salmonella**

**typhimurium** reverse mutation assay and in the **Escherichia coli** reverse

mutation assay

Dose

(μg/plate) Mean number of revertant colonies/3 replicate plates ( ± S.D.) with

one strain of Salmonella typhimurium and one Escherichia coli strain.

TA100 WP2uvrA

Without S9

- mix

Positive control 623 ± 71 1455 ± 103

Solvent control 98 ± 5 21 ± 3

1.7 88 ± 13 25 ± 6

5.4 87 ± 1 7 30 ± 6

17 108 ± 12 22 ± 5

52 98 ± 4 24 ± 4

164 91 ± 13 24 ± 10

512 96 ± 2 28 ± 5

1600 97 ± 8 23 ± 7

5000 98 ± 11 n NP 22 ± 3 n NP

With S9

- mix 1

Positive control 888 ± 128 321 ± 7

Solven t control 95 ± 14 28 ± 9

1.7 106 ± 13 39 ± 5

5.4 101 ± 5 28 ± 5

17 110 ± 5 36 ± 11

52 101 ± 1 33 ± 5

164 100 ± 8 31 ± 3

512 109 ± 13 30 ± 2

1600 112 ± 11 28 ± 7

5000 80 ± 12 n NP 36 ± 7 n NP

1 Plate incorporation assay (5% S9)

NP No precipitate

n Normal bacterial background lawnCOPS_1 Project 508230

- Page 14

- APPENDIX 1 

## – continued 

## –

Table 2 E xperiment 1 : Mutagenic response of COPS_1 in the **Salmonella typhimurium**

reverse mutation ass ay

Dose

(μg/plate) Mean number of revertant colonies/3 replicate plates ( ± S.D.) with

different strains of Salmonella typhimurium

.

TA1535 TA1537 TA98

Without S9

- mix

Positive control 797 ± 41 655 ± 60 1000 ± 24

Solvent control 14 ± 4 6 ± 2 16 ± 3

52 13 ± 3 5 ± 4 19 ± 5

164 14 ± 4 4 ± 1 19 ± 3

512 11 ± 7 5 ± 2 15 ± 6

1600 19 ± 3 7 ± 4 16 ± 4

5000 23 ± 4 n NP 8 ± 2 n NP 19 ± 6 n NP

With S9

- mix 1

Positive control 251 ± 12 433 ± 28 1313 ± 45

Solvent c ontrol 10 ± 7 11 ± 4 28 ± 9

52 13 ± 2 5 ± 4 27 ± 9

164 13 ± 8 10 ± 3 22 ± 4

512 11 ± 7 13 ± 5 27 ± 8

1600 18 ± 4 7 ± 3 24 ± 3

5000 20 ± 5 n NP 14 ± 3 n NP 28 ± 2 n NP

1 Plate incorporation assay (5% S9)

NP No precipitat e

n Normal bacterial background lawnCOPS_1 Project 508230

- Page 15

- APPENDIX 1 

## – continued 

## –

Table 3 Experiment 2: Mutagenic response of COPS_1 in the **Salmonella typhimurium**

reverse mutation assay and in the **Escherichia coli** reverse mutation assay

Dose

(μg/plate) Mean number of revertant colonies/3 replicate plates ( ± S.D.) with

different strains of Salmonella typhimurium and one Escherichia coli strain.

TA1535 TA1537 TA98 TA100 WP2uvrA

Without S9

- mix

Positive control 792 ± 3 710 ± 141 97 2 ± 43 842 ± 61 1350 ± 26

Solvent control 17 ± 2 6 ± 1 15 ± 1 99 ± 17 33 ± 12

492 21 ± 2 14 ± 4 19 ± 7 84 ± 8 26 ± 5

878 20 ± 3 7 ± 2 21 ± 4 100 ± 11 32 ± 8

1568 30 ± 12 8 ± 3 24 ± 6 102 ± 9 38 ± 7

2800 28 ± 5 9 ± 2 19 ± 5 81 ± 11 40 ± 3

5000 24 ± 6 n NP 8 ± 3 n NP 15 ± 9 n NP 100 ± 6 n NP 36 ± 2 n NP

With S9

- mix 1

Positive control 204 ± 8 486 ± 12 599 ± 12 1379 ± 4 291 ± 20

Solvent control 12 ± 5 15 ± 4 50 ± 9 93 ± 7 45 ± 12

492 28 ± 9 13 ± 5 34 ± 3 109 ± 20 41 ± 5

878 19 ± 4 15 ± 6 33 ± 13 108 ± 14 36 ± 4

1568 17 ± 3 17 ± 8 42 ± 5 109 ± 8 33 ± 6

2800 26 ± 4 10 ± 7 32 ± 8 101 ± 13 31 ± 3

5000 22 ± 7 n NP 12 ± 9 n NP 52 ± 17 n NP 108 ± 8 n NP 37 ± 3 n NP

1 Plate incorporation assay ( 10% S9)

NP No precipitate

n Normal bacterial background lawnCOPS_1 Project 508230

- Page 16

- APPENDIX 2 SUPPORTING MATERIALS AND METHOD

Bacterial background lawn evaluation

The condition of the bacterial background lawn is evaluated (if indicated), both macroscopically and

microscopically by using a dissecting microscope (results are normal unless indicated in tables).

Definition Characteristics

Normal Distinguished by a healthy microcolony lawn.

Slightly reduced Distinguished by a slight thinning of the microcolony lawn.

Moderately reduced Distinguished by a moderate thinning of the microcolony lawn.

Extremely reduced Distinguished by an extreme thinning of the microcolony lawn and an

increase in the size of the microcolonies compared to the solvent control

plate.

Absent Distinguished by a complete lack of any microcolony background lawn.

Precipitation evaluation

Evidence of test article precipitate on the plates is recorded by addition of the following precipitation

definition.

Definition Char acteristics

Slight Precipitate Distinguished by noticeable precipitate on the plate.

However, the precipitate does not influence automated counting of the plate.

Moderate Precipitate Distinguished by a marked amount of precipitate on the plate, requiring the

plate to be hand counted.

Heavy Precipitate Distinguished by a large amount of precipitate on the plate, making the

required hand count difficult.

Evaluation of the reduction in the number of revertants

The reduction in the number of revertant colonies compared to number of revertants in the solvent

control is evaluated as follows:

A reduction of 21

- 40%: slight reduction.

A reduction of 41

- 60%: moderate reduction.

A reduction of 61

- 99%: extreme reduction.

If the size of the microcolonies was increased to small colonies due to an extremely reduced

background lawn the reduction is evaluated as microcolonies. If no revertant colonies are observed on

the plates the reduction is evaluated as a complete lack of revertants.

However, any mean plate count equal to the minimal value of the historical control data range should

be considered not toxic.COPS_1 Project 508230

- Page 17

- APPENDIX 3 DETAILED TABLES

Individual plate counts; (following pages)

LIST OF ABBREVIATIONS

n Normal bacterial background lawn

NP No precipitateCOPS_1 Project 508230

- Page 18

- APPENDIX 3 

## – continued 

## –

Dose range finding

Strain TA100

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 550 692 626 623 ± 71

solvent control 102 93 98 98 ± 5

1.7 78 83 103 88 ± 13

5.4 79 107 75 87 ± 17

17 113 95 117 108 ± 12

52 102 94 99 98 ± 4

164 91 78 103 91 ± 13

512 95 95 98 96 ± 2

1600 99 88 103 97 ± 8

5000 88 n NP 97 n NP 109 n NP 98 ± 11

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 819 809 1035 888 ± 128

solvent control 110 82 93 95 ± 14

1.7 93 118 106 106 ± 13

5.4 103 95 104 101 ± 5

17 106 115 109 110 ± 5

52 102 100 101 101 ± 1

164 91 106 102 100 ± 8

512 98 124 105 109 ± 13

1600 118 99 118 112 ± 11

5000 71 n NP 94 n NP 76 n NP 80 ± 12COPS_1 Project 508230

- Page 19

- APPENDIX 3 

## – continued 

## –

Dose range finding

Strain WP2uvrA

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 1573 1393 1398 1455 ± 103

solvent control 22 23 18 21 ± 3

1.7 27 18 29 25 ± 6

5.4 24 34 33 30 ± 6

17 16 24 26 22 ± 5

52 24 27 20 24 ± 4

164 35 16 22 24 ± 10

512 23 27 33 28 ± 5

1600 31 1 9 19 23 ± 7

5000 24 n NP 22 n NP 19 n NP 22 ± 3

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 329 317 316 321 ± 7

solvent control 31 18 35 28 ± 9

1.7 44 35 37 39 ± 5

5. 4 33 24 26 28 ± 5

17 26 34 47 36 ± 11

52 38 33 29 33 ± 5

164 29 30 34 31 ± 3

512 31 31 27 30 ± 2

1600 33 20 30 28 ± 7

5000 33 n NP 30 n NP 44 n NP 36 ± 7COPS_1 Project 508230

- Page 20

- APPENDIX 3 

## – continued 

## –

Experiment 1

Strain TA1535

WITHOU T S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 842 760 790 797 ± 41

solvent control 15 10 18 14 ± 4

52 16 14 10 13 ± 3

164 18 10 15 14 ± 4

512 7 19 8 11 ± 7

1600 19 22 16 19 ± 3

5 000 23 n NP 19 n NP 27 n NP 23 ± 4

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 250 263 239 251 ± 12

solvent control 16 3 10 10 ± 7

52 15 11 12 13 ± 2

164 19 4 15 13 ± 8

512 19 5 10 11 ± 7

1600 16 22 15 18 ± 4

5000 14 n NP 23 n NP 23 n NP 20 ± 5COPS_1 Project 508230

- Page 21

- APPENDIX 3 

## – continued 

## –

Experiment 1

Strain TA1537

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 716 653 596 655 ± 60

solvent control 5 8 5 6 ± 2

52 7 7 0 5 ± 4

164 4 5 3 4 ± 1

512 3 7 5 5 ± 2

1600 7 3 11 7 ± 4

5000 10 n NP 8 n NP 7 n NP 8 ± 2

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/pl ate)

positive control 430 463 407 433 ± 28

solvent control 10 16 8 11 ± 4

52 5 1 9 5 ± 4

164 13 11 7 10 ± 3

512 12 18 8 13 ± 5

1600 5 11 6 7 ± 3

5000 11 n NP 15 n NP 17 n NP 14 ± 3COPS_1 Project 508230

- Page 22

- APPENDIX 3 

## – con tinued 

## –

Experiment 1

Strain TA98

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 990 982 1027 1000 ± 24

solvent control 14 20 15 16 ± 3

52 24 19 15 19 ± 5

164 21 19 16 19 ± 3

5 12 11 11 22 15 ± 6

1600 12 15 20 16 ± 4

5000 12 n NP 22 n NP 22 n NP 19 ± 6

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 1268 1314 1358 1313 ± 45

solvent control 39 24 22 28 ± 9

52 37 26 19 27 ± 9

164 20 26 19 22 ± 4

512 30 32 18 27 ± 8

1600 23 27 22 24 ± 3

5000 27 n NP 26 n NP 30 n NP 28 ± 2COPS_1 Project 508230

- Page 23

- APPENDIX 3 

## – continued 

## –

Experiment 2

Strain TA1535

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 794 792 789 792 ± 3

solvent control 18 18 15 17 ± 2

492 19 23 20 21 ± 2

878 19 23 18 20 ± 3

1568 37 16 37 30 ± 12

2800 29 23 33 28 ± 5

5000 18 n NP 30 n NP 23 n NP 24 ± 6

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 195 207 209 204 ± 8

solvent control 18 9 10 12 ± 5

492 37 19 27 28 ± 9

878 23 18 16 19 ± 4

1568 18 14 19 17 ± 3

2800 31 24 24 26 ± 4

5000 14 n NP 25 n NP 27 n NP 22 ± 7COPS_1 Project 508230

- Page 24

- APPENDIX 3 

## – continued 

## –

Experiment 2

Strain TA1537

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 858 696 577 710 ± 141

solvent control 7 7 5 6 ± 1

492 11 18 14 14 ± 4

878 8 5 8 7 ± 2

1568 8 5 11 8 ± 3

2800 8 8 12 9 ± 2

5000 7 n NP 5 n NP 11 n NP 8 ± 3

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 472 494 492 486 ± 12

solvent control 10 16 18 15 ± 4

492 13 8 17 13 ± 5

878 8 18 18 15 ± 6

1568 26 12 13 17 ± 8

2800 17 11 3 10 ± 7

5000 10 n NP 5 n NP 22 n NP 12 ± 9COPS_1 Project 508230

- Page 25

- APPENDIX 3 

## – continued 

## –

E xperiment 2

Strain TA98

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 964 1018 933 972 ± 43

solvent control 14 14 16 15 ± 1

492 23 11 23 19 ± 7

878 22 16 24 21 ± 4

1568 18 30 23 24 ± 6

2800 14 19 23 19 ± 5

5000 8 n NP 26 n NP 12 n NP 15 ± 9

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 599 588 611 599 ± 12

solvent control 60 45 44 50 ± 9

492 35 31 36 34 ± 3

878 18 39 42 33 ± 13

1568 39 48 39 42 ± 5

2800 23 39 35 32 ± 8

5000 35 n NP 53 n NP 69 n NP 52 ± 17COPS_1 Project 508230

- Page 26

- APPENDIX 3 

## – continued 

## –

Experiment 2

Strain TA100

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

d ose (μg/plate)

positive control 775 896 854 842 ± 61

solvent control 118 87 91 99 ± 17

492 84 91 76 84 ± 8

878 98 112 90 100 ± 11

1568 101 112 94 102 ± 9

2800 93 79 72 81 ± 11

5000 105 n NP 94 n NP 102 n NP 100 ± 6

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 1374 1382 1381 1379 ± 4

solvent control 88 101 90 93 ± 7

492 117 124 86 109 ± 20

878 92 118 113 108 ± 14

1568 101 10 9 116 109 ± 8

2800 110 106 86 101 ± 13

5000 116 n NP 106 n NP 101 n NP 108 ± 8COPS_1 Project 508230

- Page 27

- APPENDIX 3 

## – continued 

## –

Experiment 2

Strain WP2uvrA

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 1379 1329 1341 1350 ± 26

solvent control 41 39 19 33 ± 12

492 22 31 26 26 ± 5

878 41 26 30 32 ± 8

1568 42 30 41 38 ± 7

2800 38 38 44 40 ± 3

5000 37 n NP 37 n NP 34 n NP 36 ± 2

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 268 306 298 291 ± 20

solvent control 33 57 46 45 ± 12

492 35 44 44 41 ± 5

878 35 33 41 36 ± 4

1568 35 37 26 33 ± 6

2800 29 34 31 31 ± 3

5000 39 n NP 33 n NP 38 n NP 37 ± 3COPS_1 Project 508230

- Page 28

- APPENDIX 4 HISTORICAL CONTROL DATA OF THE SOLVENT CONTROL

TA1535 TA1537 TA98 TA100 WP2uvrA

S9

- mix

- + - + - + - + - +

Range 6 - 33 3 - 34 3 – 25 3 - 21 7 – 50 10 - 52 69 - 1 53 70 - 1 56 13

– 68 12 – 70

Mean 19 15 7 9 20 27 11 1 111 29 36

SD 5 5 3 3 6 7 16 15 7 7

n 930 984 792 816 1029 1054 990 971 765 786

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between July 2013 and December 2014

.COPS_1 Project 508230

- Page 29

- APPENDIX 5 HISTORICAL CONTROL DATA OF THE POSITIVE CONTROL SUBSTANCES

TA1535 TA1537 TA98

S9

- mix

- + - + - +

Range 78 - 1 932 78 - 1 332 62

– 1125 52

– 1112 347 – 13 32 210 - 1 885

Mean 804 2 38 590 3 83 8 80 7 51

SD 334 105 201 145 1 31 275

n 1012 1017 776 810 1022 1050

TA100 WP 2 uvrA

S9

- mix

- + - +

Range 5 49 – 1 394 6 20

- 2955 123 - 1 958 90 - 1 390

Mean 9 34 14 56 1359 2 50

SD 114 391 314 1 74

n 993 984 779 792

SD = Standard deviation

n = Number of observations

Historical con trol data from experiments performed between July 2013 and December 2014

.COPS_1 Project 508230

- Page 30

- APPENDIX 6 CERTIFICATE OF ANALYSIS